Alternative To AnimalTest

DRAFT Overview, 30/03/04
REPORT FOR ESTABLISHING THE TIMETABLE FOR PHASING OUT ANIMAL TESTING FOR THE PURPOSE OF THE COSMETICS DIRECTIVE
OVERVIEW The aim of this report is to provide an objective state of play of the current status of alternative methods/strategies and the prospects for their validation and regulatory acceptance so that they could be used for replacing animal tests in the safety assessment of cosmetic products as required by the EU Cosmetics Directive. This report will serve as basis for the timetable that the Commission needs to prepare in order to estimate the time for phasing out the animal tests as required in the 7th amendment of the cosmetic Directive. An Executive summary is presented at the beginning of the report and covers the main conclusions reached following an expert review of the relevant toxicological areas. Chapter 1 provides background information on the non-animal testing provisions of the 7th Amendment related to the establishment of the Commission Timetable, and describes the procedure established by the Commission in order to meet these provisions. Chapter 2 describes primarily the current safety data requirements for the purpose of the Cosmetics Directive. It emphasizes the need for data on ingredients to allow safety assessment of cosmetic products under relevant conditions of exposure, i.e. more information than what is required for classification & labeling of chemicals. The final part of Chapter 2 provides the reader with the definitions of terms and rationale used by the experts in a consistent way to draw the Tables and conclusions which are included in the following Chapter of the report. Chapter 3 consists of 11 sub-chapters corresponding to the toxicological areas that may need to be addressed for assessing the safety of cosmetic products. Each subchapter provides the outcome of an experts sub-group review which has been coordinated by ECVAM. The main elements of each sub-chapter are: ?? A table summarizing the inventory of the most valuable alternative methods currently known to be available. It includes the status of validation of these methods and the time estimated to achieve ESAC endorsement for individual alternative tests assuming that optimal conditions are met (it does not take into consideration the time needed to achieve regulatory acceptance). ?? the identification of the gaps left by the in vitro methods compared to the animal test; ?? the recommendations for achieving full replacement of animal tests; ?? the time estimated as necessary to achieve full replacement of animal tests assuming that all necessary conditions such as funding, human resources and coordination, are optimally met.
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DRAFT Overview, 30/03/04
Appendix 1 of the report presents the List of Commission services and stakeholders involved in the procedure (appendix 1a) and the list of participating scientific experts (appendix 1b). Appendix 2 provides the Glossary of terms and abbreviations which were used throughout the report. Appendix 3 provides the list of currently available EU adopted methods for testing chemicals (Annex V of 67/548/EEC) Appendix 4 provides the list of currently available OECD guidelines for testing chemicals.
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EXECUTIVE SUMMARY The 7th Amendment to the Cosmetics Directive On 27 February 2003, Directive 2003/15/EC on the approximation of the laws of the EU Member States relating to cosmetics products was adopted as the 7th Amendment to the Cosmetics Directive. It introduces new provisions related to non-animal testing of cosmetic finished products and ingredients. In particular, it establishes a prohibition to test finished cosmetic products and cosmetic ingredients on animals (testing ban), and a prohibition to market in the European Community, finished cosmetic products and ingredients included in cosmetic products which were tested on animals (marketing ban). The testing ban on finished cosmetic products will apply immediately on 11 September 2004, whereas the testing ban on ingredients or combination of ingredients will apply from 11 September 2004 as soon as alternative methods are validated by ECVAM and adopted in EU legislation, but with a maximum cut-off date of 6 years after entry into force of the Directive, i.e., 11 March 2009, irrespective of the availability of alternative non-animal tests. The marketing ban will apply from 11th September 2004 as soon as alternative methods are validated by ECVAM and adopted in EU legislation with due regard to the OECD validation process. This marketing ban will be introduced at the latest 6 years after entry into force of the Directive, i.e., 11 March 2009, for all human health effects with the exception of repeated-dose toxicity, reproductive toxicity and toxicokinetics. For these specific health effects, a deadline of 10 years after entry into force of the Directive is foreseen, i.e., 11 March 2013, irrespective of the availability of alternative non-animal tests, and which could be postponed in case of technical problems in meeting this deadline through Codecision procedure.
The Commission, after consultation of the SCCNFP and ECVAM, and with due regard to the development of validation within the OECD, shall establish timetables for the implementation of the provisions including deadlines for the phasing-out of the various animal tests. In view of establishing these timetables, the Commission decided to set up an ad-hoc Group between Commission services, stakeholder representatives for industry, animal welfare and consumer associations, and the OECD. The participants agreed on nominating experts on the 11 human health effects of concern in order to gain scientific expertise, and ECVAM coordinated and steered the scientific working process.
Safety data requirements for the purpose of the Cosmetics Directive In the EU the Cosmetics Directive (76/768/EEC) imposes that: A cosmetic product put on the market within the Community must not cause damage to human health when applied under normal or reasonably foreseeable conditions of use.... Thus the responsibility for the product safety lies with the cosmetic manufacturer or the person placing a cosmetic product on the Community market, who must be able to demonstrate that this product is safe for the consumer. Animal testing of finished cosmetic products is not explicitly required by the Cosmetics Directive or EU Member States. However, the safety assessment of finished cosmetic products is only possible provided that an adequate toxicological data package on the ingredients is available.
Cosmetic ingredients are substances or mixtures of substances which may be subject to testing according to the methods published in Annex V of the Dangerous Substances Directive (67/548/EEC) or in future in Annexe IX of the Cosmetics Directive (Directive 2003/15/EC). The testing conducted under this legislation depends on tonnage. However, to ensure the safety of cosmetics products put on the EU market as requested by the Cosmetics Directive, all cosmetic ingredients, regardless of tonnage must have appropriate data to permit an adequate safety assessment. Since the Cosmetics Directive does not specify a fixed data set or methods that are needed when assessing the safety of cosmetic ingredients, guidance is given by the SCCNFP with regard to the areas of potential toxicity that need to be addressed. Finally, the safety assessment of a cosmetic product requires taking into account the general profile of its ingredients, i.e. not only their hazard (which is the intrinsic potential of a substance to cause damage to human health and allows for classification and labelling of the substance), but also, and most importantly, the risk to human health (which is the probability of a substance to cause damage under relevant use conditions; this can be evaluated in light of dose-response studies and assessment of the actual exposure).
Experts review and time estimation for phasing-out animal tests Eleven subgroups were formed according to the 11 human health effects of concern. These subgroups comprised experts representing the different stakeholders, i.e. industry, academia, animal welfare associations and governmental bodies. In order to estimate the time necessary to achieve full replacement of animal testing in the field of cosmetics, the participating experts were requested in a first step, to provide an inventory of the most valuable and/or advanced alternative methods currently known to be available in the respective toxicological areas. Based on this inventory, they were requested in a second step to: 2
Estimate the time necessary to achieve ESAC endorsement of the identified methods assuming that optimal conditions are met
Identify the gaps left by the in vitro methods compared to the animal test Give recommendations for achieving full animal replacement Estimate the time necessary, including regulatory acceptance, to achieve full replacement of animal test assuming that all necessary conditions are optimally met (e.g., funding, human resources, etc).
The experts based their judgement on the time necessary to obtain validation and regulatory acceptance of alternative methods using the following time frame (the years given represent durations under optimal conditions, while the plusses indicate that some unforeseeable delays often occur):
Years ? Research & Development 2+ 1+ 1+ Peer review Prevalidation Validation 2+ (1) 5+ (2)
Process:
ESAC endorsement
EEC Regulation OECD Regulation
Status:
optimised
prevalidated
validated
The conclusions reached by the different expert panels are summarized hereafter. It is important to note that the evaluation of the time necessary to achieve full replacement of animal tests often represents a compromise reached by the broad representation of experts rather than full agreement. 1. Acute toxicity Good progress has been made on alternative methods for predicting basal cytotoxicity, which is one component of the acute toxicity potential. Several studies such as the MEIC study have shown that cell culture, and in particular human cells, could be used for the measurement of basal cytotoxicity. Amongst those, the experts identified 8 well-advanced in vitro tests capable of predicting basal cytotoxicity. For some of these validation and ESAC endorsement could be achieved in 2 to 4 years. However, for replacing the whole animal test, other
(1) (2)
For details see Appendix 3 For details see Appendix 4
parameters, such as metabolism, toxicokinetics and target organ toxicity to potentially sensitive target organs, need to be taken into account. Efforts to address these parameters are still at a research level. Once methods are developed and validated, there will still be the need to integrate the individual test components into a validated test battery combined with a prediction model for data extrapolation to define the risk relevant to humans. A proposal for an integrated project (A-Cute-Tox) has been submitted for funding in the context of the ECs 6th Framework Programme in late 2003. This project will be the first attempt to develop a simple and robust in vitro testing strategy for predicting human acute oral systemic toxicity that could totally replace the animal tests currently used. The time necessary to achieve complete animal replacement is strongly dependent on the outcome of this project, and was estimated by the experts at no less than 10 years. 2. Skin Irritation / Corrosion In the field of skin irritation, ECVAM is currently funding a validation study that if successful may result in three validated alternatives (the EpiDermTM human skin model, the EPISKINTM human skin model, and the mouse skin integrity function test (SIFT)) by 2004/2005, and EU regulatory acceptance by 2007/2008. These tests could enable full animal replacement for hazard identification purposes (classification and labelling). With regard to risk assessment (dose-response investigations), these methods might allow only reduction of animal experimentation. Other aspects such as reversibility and dose-response need to be addressed for achieving full animal replacement. It is estimated that this will represent a challenge that will keep scientists occupied beyond 2009.
In the field of skin corrosion, alternative methods have been already validated and accepted for regulatory use in the EU (Annex V B.40) and at OECD level (OECD TG 430 and TG 431) and no animal testing should be performed for this endpoint both for hazard identification and for risk assessment purposes. 3. Eye Irritation Major validation and evaluation studies took place in the 90s to replace the Draize test for eye irritation testing. Although good reproducibility and reliability of the most valuable alternative methods was demonstrated, it was not possible to identify a single method able to replace the Draize rabbit eye test. This is due to different factors including the limited quality of the existing in vivo data, limitations of the animal test method, and the fact that the range of criteria for injury and inflammation covered by the Draize rabbit eye test is unlikely to be 4
replaced by a single in vitro test. The participating experts identified 14 advanced methods in the field, amongst which 6 were considered as most valuable. Four of these most valuable tests (i.e., the Bovine Corneal Opacity and Permeability (BCOP) test, the Isolated Rabbit Eye (IRE) test, the Chicken Enucleated Eye Test (CEET) and the Hens egg test on the chorioallantoic membrane (HET-CAM)) are already accepted by some European Member States Authorities for the classification of severe eye irritants although not formally validated. To achieve full replacement of the Draize rabbit eye test the experts recommended: (1) to assess the existing data by using a weight of evidence approach to validation, (2) to use a high quality in vivo data set, (3) to consider differences in the regulatory classification schemes, and the upcoming implementation of the GHS, (4) to use test strategies, (5) to define strategies for risk assessment purposes, and (6) to support the development of mechanistically-based models to find alternatives for the most difficult endpoints to be replaced such as reversibility or persistence. If these recommendations are followed and the tests prove to be valid, the most valuable methods could be validated and endorsed by ESAC for specific purposes within 4 years. Finally, the participating experts estimated as a compromise that more than 6 years would be necessary for achieving replacement of animal experimentation with regulatory accepted tests/strategies using the current EU classification systems. 4. Skin sensitisation Two different, not mutually exclusive alternative approaches exist to assess the skin sensitising potential of chemicals and products: computer-based expert or QSAR systems and in vitro models. Among these, (Q)SARs systems are the most developed. It is envisaged that further optimisation of such methods could lead to validation and ESAC endorsement within the next 5-6 years. Evaluation criteria and guidance documents on the validation of these in silico systems are being defined by ECVAM and at OECD level. Concerning the cell-based systems, they have in some cases been shown to be capable of distinguishing between sensitisers and non-sensitisers (and could be used for priority setting), however they are still at a basic research level and may not achieve ESAC endorsement within the next 10 years. Further research and refinement activities for these in vitro models are still needed, and good coordination could help focus research on the development of predictive test methods. Due to the complexity of the mechanisms of skin sensitisation, a single test will not be able to replace the currently required animal experiments. Efforts will also be needed to identify the most relevant endpoints and to optimise the existing tests. However, the combination of several in vitro tests, which cover all relevant mechanistic steps of skin sensitisation, into a test battery 5
could lead to the replacement of the animal tests. There will be therefore the need to give guidance for the validation of test batteries. An indicative time foreseen for achieving validation of an alternative test battery for skin sensitisation is 10-12 years, 12-14 years being the time required for regulatory acceptance at the EU level. 5. Skin Absorption / Penetration In the area of skin absorption/penetration, an alternative test is already accepted at the OECD level (TG 428) and by the SCCNFP although it has not passed through a formal prospective validation study. It can be considered as a full alternative to the in vivo test, although only limited experience with repeated-dose testing exists, and that the skin metabolism is not covered by the test. Further specific guidance for cosmetic ingredients is recommended for the standardisation of test protocols. 6. Subacute and subchronic toxicity Chronic toxicity is a consequence of the persistent or progressively deteriorating dysfunction of cells, organs or multiple organ systems, resulting from long-term exposure to a chemical. Since a wide range of endpoints is investigated in the animal chronic toxicity studies, an integrated approach to repeated-dose toxicity testing based on the use of alternative methods with complementary endpoints needs to be developed. The present document presents the main available in vitro models in relation to five of the most common targets for toxicity (liver, kidney, central nervous system, lung and haematopoietic system). The identified tests are all at the research and development level, and none of them is seen today as an ideal method for the evaluation of any of the target organ toxicities. Efforts will be necessary to optimise the existing models, and to search for relevant in vitro models in those cases where fewer models are available (e.g., lung models). Additional basic research is also recommended to better understand the pathogenesis of chronic diseases in general. Finally, additional efforts are necessary to estimate the NOAEL in vitro, and more efforts and funding are needed to evaluate the application of QSAR approaches to predict chronic toxicity and to incorporate these tools into a testing battery and/or tiered strategy. In conclusion, the participating experts estimated that the validation of the existing models would need more than 10 years and that the time necessary to achieve full animal replacement with regulatory accepted tests/strategies will depend on the progress at the research and development level and adequate prioritisation, funding and coordination of efforts.
7. Genotoxicity and Mutagenicity Although several in vitro tests are currently accepted by regulatory authorities to asses the genotoxic and/or mutagenic potential of a chemical, they present some limitations such as the lack of human-like metabolic capacity, toxicokinetics, oversensitivity compared to in vivo situations, or the use of cell lines not relevant to the target organs. For these reasons, the group of experts suggested a completely new test strategy, which is specifically tailored for the cosmetic industry. This test strategy is divided into 4 stages: stage 1 characterises the substance based on existing data and knowledge; stage 2 is a basic in vitro test battery for hazard identification; stage 3 is a follow-up stage using target in vitro model systems and is to be performed only if one or m tests are positive in stage 2; stage 4 uses animal testing and ore is to be performed only if one or more tests are positive in stage 3. Stage 1 is based mainly on existing information on the chemical. Stage 2 comprises regulatory accepted tests (Annex V B.13-B.14 or OECD TG 471; Annex V B.15 or OECD TG4 80; Annex V B.17 or OECD TG 476; Annex V B.10 or OECD TG 473) and/or the optimised micronucleus in vitro test. Stage 3 comprises target in vitro models that still need to be developed and validated, in particular the participating experts recommended the use of skin cells or models as it is the first target for cosmetic products. Finally stage 4 would be performed only when necessary, and would comprise existing in vivo tests. The time estimated for implementation and validation of the building blocks of stage 3 is 8-10 years. To reach full replacement of animal tests, model systems in the area of toxicokinetics and metabolism are required. Moreover the emerging area of toxicogenomics could lead to a better understanding of the process of genotoxicity/mutagenicity which may help to develop the right in vitro models. Taking into account the state of the art in those areas, it was estimated that a total replacement of animal testing for genotoxicity/mutagenicity is not feasible within the next 12 years. The progress will depend on several factors such as the development of in vitro tests in the area of toxicokinetics and metabolism, the development of in vitro tests on target cells relevant for cosmetics, and further progress in the field of toxicogenomics. Importantly, the experts feel that better use of the flexibility provided in the current in vivo guideline approaches could result in a substantial reduction in animal use. 8. UV-induced toxic effects No specific animal test is required for the UV-induced toxic effects. However, for cosmetic ingredients and mixtures of ingredients absorbing UV light (in particular UV filter chemicals used, e.g. to ensure light stability of cosmetics, or used in sun protection products) the acute
photo-toxicity (photo-irritation), photo-sensitisation and photo-genotoxicity potential needs to be assessed according to the SCCNFP Notes for Guidance.
In the area of acute phototoxicity an in vitro test (3T3 Neutral Red Uptake Phototoxicity Test or 3T3 NRU PT) has been already validated and adopted for regulatory use in the EU (Annex V B.41) and at the OECD level (OECD TG 432). It is regarded as a basic screen to identify acute phototoxic potential. Two additional tests, RBC phototoxicity test and human 3-D skin model in vitro phototoxicity test, that underwent validation and prevalidation respectively, are regarded useful and important adjunct tests to overcome some limitations of the 3T3 NRU PT, namely the fairly low UVB tolerance of the 3T3 fibroblasts and the inability to model the bioavailability of test materials topically applied to the skin. In addition, the Photo-RBC allows evaluation of the phototoxic mechanisms involved.
In the area of photo-genotoxicity, almost the whole battery of in vitro genetic toxicity tests has been (or is currently being) converted into test protocols of photo-genotoxicity tests. Currently, two tests (the Photo-Micronucleus Test and the Photo-Comet-Assay test) are being evaluated in a formal validation study. It is expected that these in vitro photogenotoxicity test methods may be validated and adopted at EU level within the next five years.
In the area of photo-allergy (-sensitisation), like in the area of development of predictive in vitro tests for delayed contact sensitisation (Allergenicity) potential without involvement of light, due to a lacking ability to model the complex mechanisms underlying allergy, currently no promising in vitro methods to predict photo-sensitisation potential are in sight (see chapter on skin sensitisation). The only promising alternatives currently under development are in vivo refinements, like the Photo Local Lymph Node Assay (LLNA). Once a reliable and predictive in vitro test battery and strategy for the assessment of dark sensitisation potential will be developed and accepted, adaptation into similar photo-sensitisation tests is considered possible. 9. Toxicokinetics and Metabolism Toxicokinetics describes the kinetic processes of absorption, distribution, metabolism and excretion of a compound in an organism. The participating experts propose a completely new tiered approach based on in vitro and in silico models, comprising three steps: tier 1 assesses the likelihood to have systemic exposure, tier 2 determines the distribution of the compound, and tier 3 determines the potency of a compound. Only when a positive answer is obtained 8
with tier 1, there is the need to proceed to tier 2. The participating experts identified an inventory of the most valuable alternative tests available for tiers 1 and 2, whereas tier 3 comprises the alternative methods identified in the other subchapters. Tier 1 includes a battery of tests assessing dermal, oral and pulmonary absorption. The participating experts recommended further research and development efforts for alternative methods covering the inhalation route, as well as improvements in the intestinal barrier model. Tier 2 includes a battery of tests for the estimation of plasma level, excretion, bioaccumulation and biotransformation (metabolism). Within this tier, the need to further investigate and develop the excretion models (in vitro and in silico) was identified. Furthermore, the lack of appropriate tests to predict or measure the bio-accumulation of compounds was identified, and additional research and development in that area was recommended. The time necessary to achieve validation and ESAC endorsement of the identified methods for use in the context of cosmetics testing was estimated to be approximately 8 years for the completion of all the building blocks of tier 1, and of around 5 years for the relevant building blocks in tier 2. The experts estimated that more than 10 years would be required to achieve full replacement of the animal tests used for the evaluation of toxicokinetics/metabolism, due to the lack of tests for excretion which is however of less relevance for cosmetics as compared to other product classes. Therefore, without considering excretion, the estimated time would be 8 years. The progress that will be made in this area which is regarded as a bottleneck for the development of in vitro test strategies for regulatory use will depend on adequate prioritisation, funding and coordination of efforts. Additional recommendations made by the group are: (1) a follow-up of the discussions by organising an ECVAM workshop (which has taken place on January 2629, 2004); (2) if biotransformation is an issue for a specific compound, this aspect should be considered in all 3 tiers of the test strategy; (3) the validation of the overall test strategy; (4) the importance of the selection of training or validation sets of chemicals; (5) and further research efforts to investigate the pulmonary exposure to particulate compounds. 10. Carcinogenicity The carcinogenicity bioassays are rarely used by the cosmetic industries, but the bioassay is still needed to determine the potency and the target organ(s) of a carcinogenic compound. The process of carcinogenesis is the result from a sequence of stages and complex biological interaction that might be influenced by factors such as age, diet, environment, hormonal balance, etc. It is a long-term process, difficult to mimic with in vitro tests. The participating experts suggested that the carcinogenic potential could be detected by a combination of the identified existing in vitro tests. To achieve validation of these tests 5 years would be 9
necessary for the optimized tests, and more than 10 years for those under R&D. However, these tests present crucial limitations as the absence of metabolic capacity or the use of cell lines not relevant to predict the endpoint at target organ (see point 7). In addition, the identified tests might not predict and detect some non-genotoxic carcinogens since these act through a variety of mechanisms. The participating scientists stated therefore that the modelling of such complex adverse effects cannot be accomplished at present by non-animal tests, and they were unable to suggest a deadline for achieving the full replacement of animal tests. 11. Reproductive and Developmental Toxicity Several in vitro tests were identified covering different biological components of the reproductive and developmental toxicity mammalian cycle. Three of the identified tests are already validated and endorsed by ESAC (the embryonic stem cell test for embryotoxicity, the micromass test for embryotoxicity and the whole embryo culture test for embryotoxicity). For the most optimised, but not yet validated, methods, the time to achieve ESAC endorsement was estimated to be 6 years, and for the tests at research level, the time was estimated to be 10 years. However, due to the complexity of the mammalian reproductive cycle, it is not possible to model the whole cycle with one single in vitro system. In addition, some areas were identified by the experts that cannot be replaced by the currently available in vitro assays such as reproductive behaviour, parturition and post-natal functional development. Finally, several of the proposed tests require the use of donor animals, and currently it cannot be foreseen if and when these tests can be replaced. A series of workshops was recommended to judge the relevance of the in vitro tests currently available and their potential role in a test strategy, to define the gaps of non-covered areas of the mammalian cycle, and to give guidance to the development of new tests that would be required. In addition, there is a proposal for an Integrated Project (ReProTect), which is under consideration in the context of the ECs 6th Framework Program, where the whole reproductive cycle was broken down into work packages and suitable tests were identified. These should now be evaluated, optimised, prevalidated and combined into a testing strategy. In parallel, the conceptual framework to compose and validate test strategies shall be developed.
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Conclusions The main success of the present exercise was to bring together a broad panel of stakeholders and 75 scientific experts representing industries, academia, animal welfare groups and governmental bodies that compiled their respective views on the topics discussed. The participating experts identified a number of tools currently known to be available that might contribute to the replacement of animal tests. Some human health effects can already today be assessed using alternative methods, i.e., skin corrosion, skin absorption and acute phototoxicity. However, for the data requirements in the other toxicological areas further efforts will be needed in order to achieve full replacement of the animal tests. If all the optimal conditions are met, the next areas where replacement of animal tests may occur are skin and eye irritation. In both areas advanced methods are currently available for, or undergoing, validation. Following in time, would be the area of toxicokinetics/metabolism regarded as a bottleneck for the development of in vitro test strategies for systemic toxicity. The gained information would allow further progression of the acute toxicity and genotoxicity/mutagenicity areas. If alternative methods were validated and adopted into regulation in these areas, they would lead already to an important reduction of animal use. Finally, the replacement of animal tests would take the longest in the areas of repeated dose toxicity, i.e., skin sensitisation, subacute/subchronic toxicity, carcinogenicity, and
reproductive/developmental toxicity.
In general, the longer deadlines for phasing-out the animal experimentation were identified in those areas where the alternative methods are still under R&D, or where specific methods under R&D are required to complete the test strategies necessary to achieve full replacement of the animal tests. The necessity to have funds and human resources at R&D level is one of the major bottlenecks for obtaining alternative methods. However, good coordination and prioritisation to focus R&D and test optimisation efforts on alternative methods that are able to predict risks to human health are also crucial. Concerning those areas where the methods are already well advanced and ready for entering the (pre)validation process, the following needs were identified: the assessment of the existing data using weight of evidence approaches, the use of high quality in vivo data, the definition of criteria for validating test strategies as well as in silico models. As for R&D those efforts will only be possible if sufficient financial and human resources are made available. Furthermore, the implementation of the Globally Harmonised System (GHS) for classification could adversely impact timelines for regulatory acceptance.
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The ad-hoc Group concluded that appropriate resources (human and financial) should be allocated to research and development (e.g., Industrial Programmes as well as National and European Funding Schemes). With regard to the validation process, ECVAM will also need to be correctly staffed and funded for being able to face the upcoming challenges. The level of funding and human resources provided at both, R&D and validation process, will affect the time needed to achieve full replacement of animal test. Finally, the improvement and optimisation of the regulatory acceptance procedure, either at Community level, or at OECD level, should be considered in order to make available replacement methods in a timely manner.
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Summary Table Valuable alternative methods identified 7 in vitro tests prevalidated or under (pre)validation for basal cytotoxicity (partial replacement/re duction) QSARs models under R&D 2 Skin corrosion 3 in vitro tests validated and adopted at EU and OECD level 3 in vitro tests under validation, and 1 in vitro test optimised Time necessary to achieve validation (ESAC endorsement)* 2+ years for the 2 methods under validation 4+ years for the method under prevalidation Identified areas or endpoints with no alternative methods Estimated time to achieve full animal replacement with methods adopted at the EU level*
Human health effect
Recommendations
Prospects
Metabolism Toxicokinetics Target organ toxicity for potentially sensitive target organ
Validation of testing battery Development of prediction model(s) Development of complementary endpoints > 10 years
1 Acute toxicity
Progress will depend on the outcome of the A-Cute-Tox proposal for an Integrated Project submitted for funding in the th context of the ECs 6 Framework Programme. It is the first practical attempt to develop a simple and robust in vitro testing strategy for predicting human acute oral systemic toxicity that could totally replace the current animal tests
0 years
Animal testing should not be performed for this endpoint
0 years
2 Skin irritation
1-2 years
For risk assessment: need to address reversibility and dose-response aspects
Develop test strategies for risk assessment purposes
4-5 years for hazard identification > 6 years for risk assessment
Hazard identification: implementation of the GHS could adversely impact timelines Risk assessment: requires further research
Assess the existing data using a weight of evidence validation approach 14 in vitro tests optimised or under (pre)validation, amongst which 6 considered as most promising 1 refinement alternative Obtain a high quality in vivo data set Reversibility or persistence, inflammation, mechanical irritation, effects on conjunctiva Consider differences on the regulatory classification schemes, and the upcoming implementation of GHS Develop test strategies Support the development of mechanistically-based models Define strategies for risk assessment purposes
3 Eye irritation
4 years for the most promising tests
> 6 years
Current activities focus on the retrospective validation of the most promising methods
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Human health effect
Valuable alternative methods identified
Time necessary to achieve validation (ESAC endorsement)*
Identified areas or endpoints with no alternative methods
Recommendations
Estimated time to achieve full animal replacement with methods adopted at the EU level*
Prospects
Further research in the identification of the relevant endpoints and further development-optimisation of existing in vitro tests 4 Skin sensitisation optimised in silico models, several in vitro models under R&D 5-6 years for (Q)SARs 5-12 years for in vitro tests Coordination of EU research activities on the development of predictive test methods to be incorporated in a test battery for the full replacement of the animal test Guidance for the validation of a test battery 1 in vitro test accepted at the OECD level and by the SCCNFP although not formally validated Limited experience with repeated dosing 0 years Combination of skin absorption/penetration with metabolism is not covered by this test Further guidance for cosmetic ingredients testing for a better standardisation of study protocols 6 months 12-14 years
5 Skin absorption / penetration
Need of an integrated approach based on complementary endpoints Several in vitro models under R&D for five of the most common targets for toxicity (liver, kidney, CNS, lung and haematopoietic system) Efforts necessary to optimise the existing models, and to identify adequate in vitro models where fewer robust models are currently available (e.g., lung models) Need of additional basic research to better understand the pathogenesis of chronic diseases Additional efforts necessary to estimate dose-response Evaluation of QSARs approaches to predict chronic toxicity and as a part of a testing battery and/or tiered strategy n.d. The progress made will depend on adequate prioritisation, funding and coordination of efforts
6 Subacute and subchronic toxicity
> 10 years
No generally accepted alternative available for replacing repeated-dose in vivo testing
14
Human health effect
Identified areas or endpoints with no alternative methods
Recommendations
Prospects
7 Genotoxicity and mutagenicity
7 in vitro tests adopted at the EU and OECD level 2 in vitro optimised tests 3 in vitro tests under R&D
1-6 years for optimised tests >6 years for tests under R&D
Human-like metabolic capacity Toxicokinetics Oversensitivity compared to in vivo situations Cell lines not relevant to the target organs
The participating experts developed a new test strategy tailored for the cosmetic industry Development of toxicokinetics and toxicogenomics Development of in vitro genotoxicity tests on target cell models relevant to cosmetics Leverage existing guidelines for in vivo testing to reduce current use of animals > 12 years The experts felt that reduction of animals is an important tool not sufficiently utilized at the moment Full replacement will also depend on the progress in the field of toxicokinetics and toxicogenomics The progress made will depend on availability of funding and mobilisation of resources
8 Acute phototoxicity
1 in vitro test validated and accepted at the EU and OECD level and by the SCCNFP 2 in vitro tests that underwent (pre)validation
Fairly low UVB tolerance 0 years Inability to model the bioavailability of test materials topically applied to the skin Use adjunct tests (that underwent (pre)validation) to overcome limitations 0 years The prediction of acute phototoxic potential is without limitations possible with the in vitro tests currently available
8 Photogenotoxicity
2 in vitro under validation 3 in vitro optimised tests
3 years for the two tests under validation like Skin Sensitisation plus 3 years for adaptation to photo-sensib. 8-15 years for in vitro tests
Toxicokinetics Cell lines not relevant to the target organs
Use 3D human skin models for photogenotoxicity testing to address at the same time toxicokinetics and the use of relevant target cells
5 years
It is hoped that with a sensitive test for photo clastogenicity potential plus the photo-comet test potential photocarcinogens can be detected
8 Photo-allergy (-sensitisation)
1 in vivo refinement method (P-LLNA)
Once a valid in vitro approach (e.g. test battery) for skin sensitisation exists, adapt the tests to be performed with UVA-visible irradiation
> 15 years
Progress will depend on progress in the field of skin sensitisation
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Human health effect
Identified areas or endpoints with no alternative methods Tier 1: further R&D efforts for alternative methods dealing with the inhalation route, and improvements of the intestinal barrier models Tier 2: more R&D to predict or measure the bio-accumulation of compounds Further development of excretion models, although the panel regarded this information of less importance for cosmetics
Recommendations
Prospects
ECVAM workshop which was held on January 26-29, 2004. If biotransformation is an issue for a specific compound this aspect should be considered in all 3 tiers of the test strategy Validation of the overall test strategy The importance of the selection of training or validation sets of chemicals Further research efforts to investigate the pulmonary exposure to particulate compounds The carcinogenicity bioassays are rarely used for cosmetics but the bioassay is still needed to determine potency and target organs of a carcinogenic compound > 12 years (if excretion is included) 10 years (without considering excretion) The participating experts proposed a new tiered approach based on in vitro and in silico models The progress made will depend on adequate prioritisation, funding and coordination of efforts
9 Toxicokinetics and metabolism
Several alternatives identified for tiers 1 and 2, whereas tier 3 comprises alternative methods described in the other subchapters
Tier 1: 3-8 years Tier 2: 3-5 years with one aspect (excretion) > 10 years
10 Carcinogenicity
4 in vitro tests for detecting only the carcinogenic potential of a substance: 3 tests adopted at EU level 1 test under R&D
5 years for optimised tests > 10 years for method under R&D
Long-term process difficult to mimic with short-term in vitro tests
n.d.
In vitro models could detect the carcinogenic potential of a substance, however they present at present crucial limitations in order to achieve full replacement of animal tests In vivo assays with transgenic animals might allow for a reduction/refinement in animal use
16
Human health effect
Time necessary to achieve validation (ESAC endorsement)* Validated tests: 0 years Tests ready for validation: 6 years Tests under R&D: > 10 years Tests to be developed: ? years
Identified areas or endpoints with no alternative methods Some areas identified which cannot be replaced by the currently available in vitro assays such as reproductive behaviour, parturition, post-natal functional development, etc Several of the proposed tests request the use of donor animals, and it is not currently foreseeable when and if these tests can be replaced
Recommendations
Prospects
11 Reproductive and developmental toxicity
3 validated in vitro tests for partial replacement several optimised or under (pre)validation in vitro tests several tests under R&D
Development of test strategies Series of workshops to judge the relevance of different in vitro tests currently available and their potential role in a test strategy, define gaps of non-covered areas of the mammalian cycle, and give guidance to the development of new tests that would be required
n.d.
Progress will depend on the outcome of the workshop series and on the outcome of the ReProTect project which is an Integrated Project under consideration in th the context of he ECs 6 Framework Programme. With this project, the whole reproductive cycle was broken down into work packages and suitable tests were identified that now need to be evaluated, optimised, prevalidated and combined into a testing strategy. In parallel, the conceptual framework to compose and validate test strategies shall be developed
*Assuming optimal conditions
17
CHAPTER 1
Directive 2003/15/EC of the European Parliament and the Council amending Council Directive 76/768/EEC2 on the approximation of the laws of the Member States relating to cosmetic products was adopted on 27 February 2003 and entered into force on 11 March 2003. Directive 2003/15/EC deleted article 4(1)(i) of Council Directive 76/768/EEC that banned the marketing of ingredients and combinations of ingredients tested on animals after 30 June 2000 in order to meet the requirements of Directive 76/768/EEC (this date was postponed until 30 June 2002 by Commission Directive 2000/41/EC dated 19 June 20003). Directive 2003/15/EC introduced new provisions related to non-animal testing, in particular a testing and marketing ban. These provisions came into force on 11 March 2003. Member States have however a deadline to transpose them into national law until 11 September 2004.
The testing ban
1
on finished cosmetic products will thus apply immediately from 11 September 2004. The testing ban on ingredients or combinations of ingredients will apply from 11 September 2004 as soon as alternative methods (including reduction and refinement methods in those cases where full replacement alternatives are not yet available) are validated by the European Centre for the Validation of Alternative Methods (ECVAM) and adopted in EU legislation (Annex V to Council Directive 67/548//EEC5or Annex IX to the Cosmetics Directive). In any case, the maximum cut-off date is 6 years after entry into force of the Directive, which is 11 March 2009. The testing ban will apply for all tests on animals, irrespective of the availability of alternative non-animal tests. The marketing ban6will apply from 11 September 2004 as soon as alternative methods (including reduction and refinement methods in those cases where full replacement alternatives are not yet available) are validated by ECVAM and adopted in EU legislation (Annex V to Council Directive 67/548//EEC or Annex IX to the Cosmetics Directive) with due regard to the OECD validation process. In any case, the marketing ban will be implemented at the latest 6 years after entry into force of the Directive 2003/15/EC, which is 11 March 2009, irrespective of the availability of alternative non-animal tests. A second deadline of 10 years after entry into force of Directive 2003/15/EC exists for animal tests for repeated-dose toxicity, reproductive toxicity and toxicokinetics, which is 11 March 2013. A possibility for a postponement of the marketing ban for those tests is explicitly mentioned in case there are technical problems with their development or their validation
OJ L 66, 11.03.2004,p.26 OJ L 262, 27.09.1976, p. 35 3 OJ L 145, 20.06.2002, p. 25 4 The testing ban is the prohibition by Member States of the performance on their territory of animal testing of ingredients, combination of ingredients or finished products in order to meet the requirements of this Directive.
5 6
OJ L 196,16.08.1967,p.1. The marketing ban is the prohibition by Member States of the marketing of cosmetic product where the final formulation, its ingredients or combination of ingredients, in order to meet the requirements of this Directive, has been subject to animal testing using a method other than an alternative method. .
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before the expiry of the 10 years period and the stipulated deadlines cannot be met. However this will be decided through Codecision procedure. Finally, a re-testing clause for existing ingredients in exceptional circumstances concerning safety is also foreseen. The Commission, after consultation of the SCCNFP and ECVAM with due regard to the development of validation within the OECD, has to establish timetables for the implementation of the provisions, including deadlines for the phasing-out of the various animal tests. The Commission will also present a yearly monitoring of these timetables and decide on possible adaptation of them within the maximum periods (6 or10 years). Following the adoption of the 7th amendment to the Cosmetics Directive 76/768/EEC, the implementation of the provisions related to alternative methods to animal testing has been a priority. In view of the need to establish these timetables, the Commission decided to set up an ad hoc Group between Commission services (DG ENTR., ECVAM, DG SANCO, DG ENV, DG Research) and representatives of stakeholders (consumers, industry and animal welfare Associations). As the 7th amendment foresees a clear reference to the OECD validation process in the context of the establishment of timetables, the Commission invited an OECD representative to participate as well. Scientists from SCCNFP were also invited ad personam to participate in this Group. (see appendix 1a for the list of participants). The first meeting was held on 19 March 2003. The participants supported the approach to have all partners concerned being invited to work together. The aim was to establish an objective state of play for the evaluation of the current status of alternative methods/strategies and the prospects for their validation and regulatory acceptance so that they could be used for replacing animal tests in the safety assessment of cosmetic products as required by the EU Cosmetics Directive. During the second meeting of 20 June 2003, the stakeholders and EC services involved recognised the need for additional scientific expertise and agreed on the nomination of experts on the 11 human health effects of concern (see appendix 1b). ECVAM coordinated and steered the working process. In a first step, the 11 subgroups of experts described the current tests used for safety assessment of cosmetics, provided an inventory of the most valuable alternative tests in the respective fields, and evaluated their status of validation and regulatory acceptance. This information was based on the best of their scientific expertise in the field and on the following background documents: - The ECVAM Report on Alternative (Non-animal) Methods for Chemicals Testing (Worth and Balls, 2002) - The ECVAM SIS Database - COLIPA Reports - AnimAlt-Zebet database 2001 - Other publicly available relevant documents in the respective areas.
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In addition a mini-review on QSARs (Cronin et al., 2003) was distributed to the experts for consideration at the end of the exercise. Discussions within each subgroup took mainly place by electronic mail. The resulting documents were presented to the stakeholders on 10 October, 2003. In order to facilitate discussions and obtain scientific consensus, the different stakeholders involved agreed on financing two series of meetings for the experts, which took place in November and December 2003, respectively. During this second phase, the participating experts identified the gaps left by the alternative methods available with regard to the animal tests, and evaluated the efforts needed to achieve full animal replacement. Finally, they reached compromises on the time estimated as necessary for replacing the given animal test, based on the proposed timescale and assuming that all necessary conditions such as funding, human resources and coordination were met. The final draft documents were presented to the EC services and stakeholders on 9 January, 2004. From a general point of view the result of the exercise was excellent and resulted, in some of the areas, even in the development of building blocks and new testing strategies for replacing the animal tests. A large amount of work has been achieved in a relatively short period of time, specifically the participating experts had to work under important time pressure. This report will serve as basis for the timetable that the Commission needs to prepare in order to estimate the time for phasing out the animal tests. KEY REFERENCES AnimAlt-Zebet database (2001). http://www.bfr.bund.de/cms/detail.php?template=internet_en_index Cronin, M.T.D., Jaworska, J.S., Walker, J.D., Comber, M.H.I, Watts, C.D. & Worth, A.P. (2003). Use of QSARs in international decision-making frameworks to predict health effects of chemical substances. Environmental Health Perspectives 111, 1391-1401. Worth AP; Balls M; Editors. Alternative (non-animal) methods for chemicals testing current status and future prospects. A report prepared by ECVAM working group on chemicals. ALTA 30; suppl.1; 2002
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DRAFT Chapter 2, 07/04/04
REPORT FOR ESTABLISHING THE TIMETABLE FOR PHASING OUT ANIMAL TESTING FOR THE PURPOSE OF THE COSMETICS DIRECTIVE CHAPTER 2
PART 1: SAFETY DATA REQUIREMENTS FOR THE PURPOSE OF THE COSMETICS DIRECTIVE
2.1.
The requirements of the Cosmetics Directive :
In the EU, the Cosmetics Directive (76/768/EEC) imposes the legal obligation that cosmetics which are sold to consumers are safe. This obligation is enshrined in the Article 2, which states: A cosmetic product put on the market within the Community must not cause damage to human health when applied under normal or reasonably foreseeable conditions of use---. Thus, responsibility for product safety lies with the cosmetic manufacturer or the person placing a cosmetic product on the Community market, who must be able to demonstrate that this product is safe for the consumer. Animal testing of finished cosmetic products is not explicitly required by the Cosmetics Directive or EU Member States (Colipa 2003). Both Colipa and the SCCNFP have issued guidelines for the safety assessment of cosmetic products aiming to avoid testing on animals (Colipa 1995, 1997, SCCNFP 1999). In particular, guidance for confirmatory skin compatibility testing of the finished products on human volunteers has been given by the Commissions Scientific Committee on Cosmetics and Non Food Products (SCCNFP/0068/98). It is important to note also that the EU Directive (86/609/EEC), regarding the protection of animals used for experimentation and other scientific purposes, requires that an experiment shall not be performed if another scientifically satisfactory method of obtaining the result sought, not entailing the use of an animal, is reasonably and practicably available. However, the safety assessment of finished cosmetic products without animal testing is only possible provided that an adequate toxicological data package on the ingredients is available. The Cosmetics Directive in Article 7 states: the manufacturer shall take into consideration the general toxicological profile of the ingredient, its chemical structure and its level of exposure.
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The Cosmetics Directive does not specify a fixed data set or methods that are needed when assessing the safety of cosmetic ingredients. However, guidance is given by the SCCNFP regarding the areas of potential toxicity that need to be addressed (SCCNFP/0690/03). 2.2. The toxicological data requirements for cosmetic ingredients based on the SCCNFP guidelines :
1. Acute systemic toxicity (if available) 2. Skin irritation/corrosion 3. Eye irritation 4. Skin sensitisation 5. Skin absorption 6. Subacute and subchronic toxicity 7. Genotoxicity/Mutagenicity 8. UV-induced toxic effects (phototoxicity, photoallergy, photogenotoxicity) 9. Toxicokinetics and metabolism 10. Carcinogenicity 11. Reproductive and developmental toxicity When substantial oral intake can be expected from the product use or when the data on skin absorption indicate a high level of penetration of the ingredients, and if indicated by the toxicological profile of the substance and its chemical structure, information on Carcinogenicity, Reproductive toxicity and Toxicokinetics may be needed, as well as specific additional data on Genotoxicity / Mutagenicity (SCCNFP/0690/03). Assessment of skin corrosion, which is the potential of a substance to cause irreversible damage to the skin, is not generally stipulated by the SCCNFP. On the other hand, there are ingredients which despite having the intrinsic property to be corrosive, can be used safely in cosmetics depending on their concentration in the finished product, the vehicle used, the conditions of use, etc. In such cases, it may be necessary to demonstrate the skin compatibility under use-conditions with an appropriate testing system. Cosmetic ingredients are substances or mixtures of substances. Therefore, the testing methods used to ensure their safety are the same as for substances used in pharmaceuticals, food, paints, agrochemicals etc.1 The protocols recommended for
1
In the case of fragrances, most fragrance suppliers deliver adequate certificates assuring the quality of the fragrance, and indicating the range of safe concentrations per product based on appropriate tests on ingredients. It will be the responsibility of the safety assessor to judge if the information provided by the supplier is adequate for the evaluation of the safety of the finished cosmetic product.
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the different tests listed above are described in the E legislation (Annex V of the U Dangerous Substances Directive (67/548/EEC)] (see Appendix 3) and in the OECD testing guidelines for chemicals (OECD 2002) (see Appendix 4). Among them are scientifically valid alternative methods to animal tests. Consequently, these testing methods for chemicals are also those recommended by the SCCNFP for testing of cosmetic ingredients (SCCNFP/0177/99, SCCNFP/0546/02). However, for the areas where no alternative methods exist, the SCCNFP guidelines assert that the use of animal experiments remains a scientific necessity, and although it needs to be limited to a minimum, this should never be at the expense of consumer safety (SCCNFP/0546/02). Indeed, the SCCNFP acknowledges that in order to meet the fundamental requirements of consumer health protection, they would evaluate studies which were conducted in animals, in accordance with current legal provisions (and preferably under chemical law regulations), only in those cases where no alternative methods are available (SCCNFP/0690/03). The safety assessment of a cosmetic product requires taking into account the general profile of the ingredients, their hazard (which is the intrinsic potential of a substance to cause damage to human health) and the risk (which is the probability of a substance to cause damage under relevant use conditions). Risk to human health is characterized after taking into account dose-response studies, to determine the No Observed Adverse Effect Level (NOAEL), and the exposure assessment. 1) Hazard identification: as defined above, is based on the intrinsic properties of a molecule to damage human health. Information can be obtained by toxicity testing (clinical, epidemiological studies, animal testing, in vitro testing, QSAR studies). 2) Dose-response assessment: provides information with regard to what dose is necessary to cause an effect. The NOAEL (no-observed adverse effect level), resulting from the dose-response relationship, is an important feature for safety assessments and is used to calculate the Margin of Safety (see below). 3) Exposure assessment: the degree of systemic exposure to the compound depends on: ?? Concentration of the ingredient in the finished cosmetic product ?? Quantity of product used (dependent on various factors including: consumer sex, age-group, habits, season, use pattern, frequency, continuous daily use vs. intermittent, sporadic vs. long-term), partition coefficient for rinse-off or leave-on products ?? Body weight of consumer ?? Route of exposure: Percutaneous absorption of the evaluated ingredient, ingestion (for oral hygiene and lipsticks) or inhalation (in case of cosmetics in sprays). 4) Risk characterisation: the probability that the molecule under investigation causes damage to human health and to what extent are examined. The uncertainty factor for cosmetics, called the Margin of Safety (MoS), is calculated according to the formula: NOAEL MoS = where SED represents the Systemic Exposure Dosage SED
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2.3.
The toxicological data requirements for chemicals :
Based on the overall safety requirements of the Cosmetics Directive (Article 2), the cosmetics manufacturer through his safety assessor has the responsibility for the safety of all products put on the EU market. Therefore all cosmetic ingredients, regardless of tonnage must have appropriate data to permit an adequate safety assessment to be done to ensure cosmetics safety. However, the testing conducted under chemical legislation, which is generally considered an important source of safety data, depends on tonnage (Directive 67/548/EEC on the classification, packaging and labeling of dangerous substances). For substances that are produced or imported in amounts below 100 kg per year, the toxicological data requirements of the Directive 67/548/EEC are limited to only: Acute toxicity The toxicological requirements of the same Directive for substances produced / imported at levels between 100 kg and 1 tonne per year include the following basic list: - Acute toxicity (oral, dermal or inhalation) - Skin and eye irritation - Sensitisation - Mutagenicity data For substances produced / imported at levels above 1 tonne and below 1000 tonnes per year, the toxicological requirements of Directive 67/548/EEC may include some or all of the following additional tests ? 1: - Repeated dose (sub-chronic and/or chronic) toxicity data - Reproductive toxicity - Carcinogenicity (screening) - Toxicokinetics When substances are produced / imported at levels above 1000 tonnes per year, Directive 67/548/EEC requires additional data on: - Chronic toxicity - Carcinogenicity - Fertility - Developmental toxicity - Additional toxicokinetics Thus, the fact that most cosmetic ingredients are chemicals means that for some of them the data needed for their safety assessment in cosmetics may be available as a result of compliance with the provisions of the Chemical legislation. However, there are cases when further or new testing (by animal or non-animal tests) of substances used in cosmetics remains a scientific necessity in view of consumer safety (Article 2 of the Cosmetics Directive) and irrespective of the above tonnage-linked restricted requirements under the Dangerous Substances Directive (67/548/EEC).
? 1
For more precise explanation see the requirements laid down in Directive 67/548/EEC
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2.4. Cosmetic safety assessment Specific data demands : 2.4.a. Insufficient data for safety assessment This is the case if the safety assessor working for the cosmetic manufacturer considers that the quality or quantity of available safety data is inadequate to evaluate the safety of the products for which the cosmetic manufacturer will be liable. If, as example a low-volume chemical is intended for use in cosmetics, safety data developed under chemical legislation are unlikely to be sufficient. In particular, if these ingredients are expected to come into contact with the human skin for a long period of time, evaluation of the systemic risk is a key element in evaluating their safety, although not required under the chemicals legislation (67/548/EEC). Indeed, for products applied frequently / repeatedly on the skin (e.g. body lotions, shower gels) or products that can be partly ingested (e.g. oral hygiene products, lipsticks), evaluation of only local tolerance of their ingredients is not sufficient for safety assessment and evaluation of sub-chronic toxicity is of direct relevance. Moreover for UV light-absorbing chemicals in cosmetics that are used in combination with exposure to sunlight, evaluation of photo-induced toxic potential effects is needed, whilst not required by the chemical legislation (67/548/EEC). The hazard classification (presence/absence of effect) provided when testing the chemical in neat form for the hazard identification / classification/ labelling purposes of the Chemical legislation can be used for screening purposes, but cannot always provide all the necessary information needed for evaluating the safety of cosmetic ingredients. For this, dose-response information and testing at relevant-to-use conditions of exposure are needed. 2.4b: Data requests by MS, SCCNFP, experts, Based on the overall safety requirements of the EU Cosmetics Directive, a Member State, the Commission, the SCCNFP scientists or experts may question at any time the safety of an existing ingredient. Unless sufficient safety data is already available, the ingredient will need to be tested (by animal or non-animal tests). For endpoints where no alternative methods are available, animal testing would be used to gather further information. 2.4c: New or existing ingredients of the positive lists Submission of safety data to the SCCNFP is a legal requirement for any new or revised colorants, preservatives and UV-filters. To allow their use in cosmetics, these substances need to be first evaluated and approved by the SCCNFP in order to be included in the positive lists of the Cosmetics Directive (i.e., Annex IV, VI and VII, respectively).
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PART 2: FROM ANIMAL TESTING TO ALTERNATIVE METHODS In toxicological studies, the overall risk assessment for chemicals has been based on the assumption that effects observed in laboratory animals are predictive of effects in humans. However, ethical and animal welfare concerns during the last 20 years, as well as the recently changing EU regulatory environment, in the context of the 7th Amendment to the Cosmetics Directive and the European Commissions development on REACH (Registration, Evaluation and Authorisation of CHemicals), make it obvious that a new approach is needed. The replacement of animal tests for evaluating the safety of chemicals and products is thus a clear necessity. Definitions used with regard to alternative methods An alternative method is any method that can be used to replace, reduce or refine the use of animal experiments in biomedical research and testing for regulatory or educational purposes. The three types of alternative methods, which are referred to collectively as the 3Rs, have been earlier defined by Russell & Burch (Russell & Burch 1959): Reduction alternatives: they provide a comparable level of information from the use of fewer animals, or more information from the same number of animals. These can include the use of a partial replacement test as part of a test strategy. Refinement alternatives: those which alleviate or minimise potential pain, suffering and distress. Replacement alternatives: those which permit a given purpose to be achieved without using living vertebrate animals. As well as the traditional concept of the 3 Rs described above, for reasons of clarity, other terminology has also been used in the present document: Partial replacement: when an alternative method can partially replace an animal experiment but requires the use of additional non-animal alternatives as part of a tiered strategy and/or test battery for achieving full replacement of the animal test. Test battery: a series of tests usually performed at the same time or in close sequence. Each test within the battery is designed to complement the other tests and generally to measure a different component of a multi-factorial toxic effect. Tiered test strategy: tests used in a sequential manner; the tests selected in each succeeding level are determined by the results in the previous level of testing (a stepwise process leading to a decision, consisting of a series of tests performed in a defined sequence). For the moment, the number of validated alternative methods that are available for practical applications in regulatory testing and risk assessment of cosmetic ingredients is limited (SCCNFP/0546/02). Appropriate alternatives exist only for some
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of the toxicological areas relevant for assessing the safety of cosmetic ingredients. For a number of toxicological fields of key importance, appropriately validated alternative tests are lacking. Definitions used with regard to the development, validation and acceptance process of alternative methods There are five stages identified by ECVAM in the evolution of new test methods (Balls et al. 1995, Fentem & Balls 1997): 1. 2. 3. 4. 5. test development; prevalidation; formal validation; independent assessment (peer review); and progression toward regulatory acceptance
The above steps in the validation of a test method have been taken into account by the expert sub-groups when reviewing the existing alternative methods in each of the 11 toxicological areas relevant to cosmetic safety assessment. In drawing up the tables of the report the validation status of the most valuable and currently available non-animal alternative tests has been benchmarked according to these five stages of the validation process. Moreover, the time needed to achieve validation and regulatory acceptance has been estimated in relation to these distinct steps of the process. The following definitions have been considered for the completion of the tables of the report :
R&D
Optimised test
Prevalidation
Prevalidated test
Validated test Validation + Peer-review (ESAC endorsement) Regulatory acceptance
If criteria not met

optimised test
If criteria not met

optimised test
Research & Development: basic research and development of a m ethod and / or test protocol. Could give raise to an optimised test. Optimised test: test system + prediction model (PM) ready to enter the prevalidation process (Curren et al, 1995; cf submission of new test methods to ECVAM at http://ecvam.jrc.it). In the context of this report, the status optimised is attributed also to tests that went through (pre)validation and failed.
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Who may conduct this phase: Universities, National Research Institutes, Industry, others Under prevalidation: Status of method currently undergoing prevalidation. Prevalidation: is a small-scale inter-laboratory study, carried out in 3 phases to ensure that the protocol and prediction model (PM) of a test method is sufficiently optimised and standardised for inclusion in a formal validation study. During phase I, the protocol and PM of a test method are refined in a single laboratory (with prior experience in the use of the test). During phase II, an assessment is made of the transferability of the test protocol to a second laboratory, making any necessary refinements to the protocol and/or PM. During phase III, the relevance and reliability of the test are assessed under blind conditions in three or more laboratories (which generally include the first two laboratories) (Curren et al.1995). (An important outcome from a prevalidation study is that an optimised protocol is identified that could be used in a formal validation study). Who may conduct this phase: ECVAM, Industry, others Prevalidated: a method that has undergone prevalidation and fulfilled the performance criteria on reproducibility and predictive ability. Under validation: Status of method currently undergoing validation Validation: in principle, this is a large-scale inter-laboratory study, performed under blind conditions, and designed to obtain a more definitive assessment of the relevance and reliability of an optimised method for a particular purpose. It can be thought of as a larger-scale version of the phase III stage of prevalidation, in which a larger number of chemicals are tested (although not necessarily in a larger number of laboratories). In general, newly developed test methods enter the prevalidation process and, following successful prevalidation, proceed to formal validation. In addition, weight-of-evidences approaches to validation and retrospective validation are other possible ways of evaluating the relevance and reliability of a method for a particular purpose on the basis of review and analysis of existing data from previously performed individual studies. Who may conduct this phase: ECVAM, Industry, others Under peer review: a critical review conducted by experts who are independent of the experts who performed the original work but who are collectively equivalent to them in technical expertise; it may lead to an endorsement by the ECVAM Scientific Advisory Committee (ESAC endorsement). ESAC endorsement: formal recommendations on the scientific validity of an alternative method and statement on the applicability of the method for a particular purpose. Who may perform this step: ECVAM Scientific Advisory Committee (ESAC) Validated: for the purposes of this report, the term will only mean a method/strategy that has been endorsed by ESAC. Adoption: Regulatory acceptance EC adoption = Annex V of Directive 67/598/EEC or Annex IX of Directive 76/768/EEC (SCCNFP opinion + Standing Committee on Cosmetics)
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International Adoption: acceptance at OECD level
CONSIDERATIONS REGARDING THE ESTIMATED TIMINGS The following average timetable has been agreed by the stakeholders and was used by the experts involved as a reference to estimate the time necessary to achieve full replacement of animal testing:
Years ? Research & Development 2+ 1+ 1+ Peer review Prevalidation Validation 2+ (1) 5+ (2)
Process:
ESAC endorsement
EEC Regulation OECD Regulation
Status:
optimised
prevalidated
validated
The time estimations were made using the above optimal time scale and assuming that all necessary resources (human and financial) are available at any time in the process. The plusses indicate that some unforeseeable delays often occur. It has to be noted that the time estimations were made using the above average time scale and assuming that optimal conditions were met. Optimal conditions means that all necessary resources, for example technical, human, financial and coordination, are met at all times in the process and that the studies undertaken have successful outcomes. It is important to note that the tables presented in the different subchapters summarize the most valuable and / or advanced alternative methods known to the participants in the 11 toxicological areas relevant to cosmetic safety assessment. They estimate the time needed to achieve ESAC endorsement for individual alternative tests (including partial replacement tests). Thus they do not indicate the time necessary to achieve full replacement of animal tests. Nor do they include the time needed to achieve regulatory acceptance. The time necessary for achieving full replacement of animal tests including regulatory acceptance was estimated by the experts in the conclusions of their respective chapters. They take into account other factors such as the need for test strategies, and for the development of new assays in order to overcome the scientific gaps in a particular toxicological area.
(1) (2)
For details see Appendix 3 For details see Appendix 4
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KEY REFERENCES
Balls, M., Blaauboer, B.J., Fentem, J.H., Bruner, L., Combes, R.D., Ekwall, B., Fielder, R.J., Guillouzo, A., Lewis, R.W., Lovell, D.P., Reinhardt, C.A., Repetto,G., Sladowskin, D. Spielmann, H. & Zucco F. 1995. Practical aspects of the validation of toxicity test procedures. The report and recommendations of ECVAM workshop 5. ATLA 23, 1219-147.
Colipa 1995. Cosmetic Product Test Guidelines for the Assessment of Human Skin Compatibility. Brussels, Belgium: Colipa. Colipa 1997. Guidelines for the Safety Assessment of Cosmetic Products. Brussels, Belgium: Colipa. Colipa 2003. Personal communication based on a survey with membership. Council Directive 67/548/EEC of 27 June 1967 on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances. Latest adaptation to technical progress for the 28th time by the Commission Directive 2001/59/EC. Council Directive 76/768/EEC of 27 July 1976 on the approximation of the laws of the Member states relating to cosmetic products. Latest update: 7th amendment by Directive 2003/15/EC of the European Parliament and of the Council of 27 February 2003. Council Directive 86/609/EEC of 28 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes. Latest amendment by Directive 2003/65/EC of the European Parliament and of the Council of 22 July 2003. Curren, R.D., Southee, J.A ., Spielmann, H., Liebsch, M., Fentem, J.H. & Balls, M. 1995. The role of prevalidation in the development, validation and acceptance of alternative methods. ATLA 23, 211-217. Fentem J.H. & Balls, M. 1997. The ECVAM approach to validation. In Developments in Animal and Veterinary Sciences, Volume 27, Animal Alternatives, Welfare and Ethics. Proceedings of the 2nd World Congress on Alternatives and Animal Use in the Life Sciences, Utrecht, The Netherlands, 20-24 October 1996 (ed. L.F.M. van Zutphen & M. Balls), pp. 1083-1089. Amsterdam, The Netherlands: Elsevier. OECD (2002): OECD Guidelines for the Testing of Chemicals. Paris, France: OECD. Russell, W.M.S. & Burch, R.L. 1959. The Principles of Humane Experimental Technique, 238pp. London, UK: Methuen. SCCNFP/0068/98, Final: The Scientific Committee on Cosmetic Products and Non-Food Products Intended for Consumers. Opinion concerning Guidelines on the Use of Human Volunteers in Compatibility Testing of Finished Cosmetic Products. SCCNFP/0177/99, Final: The Scientific Committee on Cosmetic Products and Non-Food Products Intended for Consumers. Opinion concerning Present Development and Validation of Adequate Alternative Methodologies to the Use of Animals in Safety Testing of Cosmetics.
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SCCNFP/0690/03, Final: The Scientific Committee on Cosmetic Products and Non-Food Products Intended for Consumers Notes of Guidance For Testing of Cosmetic Ingredients For Their Safety Evaluation. 5th Revision, adopted by the SCCNFP during the plenary meeting of 20 October 2003. SCCNFP/0546/02, Final: Memorandum concerning the actual status of alternative methods to the use of animals in the safety testing of cosmetic ingredients, adopted by the SCCNFP during the 20th plenary meeting of 4 June 2002. Worth, A.P. & Balls, M. ed. 2002. Alternative (non-animal) methods for chemicals testing: current status and future prospects. ATLA 30, supplement 1: 125
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Acute toxicity
Laura Gribaldo1, Alessandra Gennari1, Karen Blackburn2, Cecilia Clemedson3, Alain Deguercy4, Annarita Meneguz 5, Walter Pfaller6, Irmela Ruhdel7
1
ECVAM, IHCP, JRC, 21020 Ispra (VA), Italy; 2Procter & Gamble, Miami Valley
5
Laboratories, Ross, OH 45061, USA; 3Expertrdet AB, 17264 Sundbyberg, Sweden;

4
BIOalternatives, 75008 Paris, France;
Laboratory of Pharmacology, Istituto
Superiore di Sanita, 00161 Roma, Italy; 6University of Innsbruck, Innsbruck, Austria; 7German Animal Welfare Federation, 85579 Neubiberg, Germany
1. Inventory of methods currently available

From the regulatory point of view, the main objective of acute toxicity testing is basically to classify chemicals according to their intrinsic toxicity as required by the EEC directive on classification, packaging, and labelling of dangerous substances (Council Directive 67/548/EEC and subsequent amendments). This requirement aims to protect public health by regulating exposure to potentially dangerous materials. Classification of chemicals is done on the basis of the medium lethal dose (LD50) value, defined as the statistically derived single dose of a substance that can be expected to cause death in 50% of the animals in an experimental group. The LD50 concept was first introduced in 1927 for establishing the toxic potency of biologically active compounds such as digoxin (1). Since the end of the 1970s the LD50 test has been widely criticised for both scientific and animal welfare reasons (2, 3), and the test procedure has been m odified in various ways to reduce the number of animals required, and to reduce the suffering caused to any animal used (4). This modification to the classical LD50 test includes the fixed-dose procedure, OECD TG 420 (5), the acute-toxic-class method, OECD TG 423 (6), and the up-and-down procedure, OECD TG 425 (7). In 2002 the original LD50 test, OECD 401 (8), has been deleted from the OECD guidelines.
TG 420: Fixed Dose procedure Purpose: the test is of value in minimizing the number of animals required to estimate the acute oral toxicity of a chemical. The method permits estimation of an LD50 with 1
a confidence interval and the results allow a substance to be ranked and classified according to the Globally Harmonised System (GHS) (9). Principle: the test consists in dosing groups of animals (single sex, normally females) in a stepwise procedure using the fixed doses of 5, 50, 300, and 2000 mg/kg (exceptionally an additional dose of 5000 mg/kg may be considered). The initial dose level is selected as the dose expected to produce some signs of toxicity without causing severe toxic effects or mortality based on in vivo/in vitro data (if no information exists the starting dose will be 300 mg/kg). Further groups of animals may be dosed at higher or lower fixed doses, depending on the presence or absence of signs of toxicity. This procedure continues until the dose causing evident toxicity is identified. The preferred rodent species is the rat and the test substance is administered in a single dose by gavage (if not possible the dose may be given in smaller fractions over a period not exceeding 24 hours). Observations: animals are observed daily for a total of 14 days. Observations include changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Attention should be given to tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The principles and criteria summarised in the Humane Endpoints Guidance Document (10) should be taken in consideration. Individual weights of animals have to be determined before and after the substance is administered. All animals should be subjected to gross necropsy and pathological changes should be recorded.
TG 423: Acute Toxic Class Method Purpose: similar to TG 420. Principle: the test consists on a stepwise procedure with the use of three animals of a single sex (normally females) per step. Absence or presence of compound-related mortality of the animals dosed at one step will determine the next step (i.e. no further testing is needed; dosing of three additional animals with the same dose; dosing of three additional animals at the next higher or lower dose level). The starting dose level is selected from one of four fixed levels (5, 50, 300, and 2000) and it should be that which is most likely to produce mortality in some of the dosed animals. When there is
no information on a substance to be tested, it is recommended to use the starting dose of 300 mg/kg. The preferred rodent species is the rat and the test substance is administered in a single dose by gavage (if not possible the dose may be given in smaller fractions over a period not exceeding 24 hours). Observations: similar to TG 420.
TG 425: Up-and-Down Procedure Purpose: similar to TG 420. Principle: the test consists of a single ordered dose progression in which animals of a single sex (normally females) are dosed, one at a time. The first animal receives a dose step below the level of the best estimate of the LD50 (when no information is available to make a preliminary estimate of the LD50, the suggested starting dose is 175 mg/kg). If the animal survives, the dose for the next animal is increased by a factor of 3.2 times the original dose; if it dies, the dose for the next animal is decreased by a similar dose progression. The preferred rodent species is the rat and the test substance is administered in a single dose by gavage (if not possible the dose may be given in smaller fractions over a period not exceeding 24 hours). Observations: similar to TG 420.
Limitations of TG 420, 423, 425 - Validations against actual data and statistical simulations identified areas where all three methods may have outcomes which result in a more or less stringent classification than that based on the true LD50 value (as obtained by the deleted guideline 401) (11). Comparative statistical analysis indicated that all are likely to perform poorly for chemicals with shallow dose-response slopes. For all methods, the study outcome is likely to be influenced by the choice of starting dose level(s), relative to the true LD50 value, especially in the case of shallow slopes. Because Guideline 420 uses evident toxicity as an endpoint instead of death, information on toxic effects seen only at dose levels close to a lethal dose will not always be obtained (12). - Unusually test substances may cause delayed deaths (5 days or more after test substance administration). Substances which cause delayed deaths have an impact on 3
the practicality of conducting a study to Guideline 425 where the duration of testing will be significantly longer compared with other test methods. However, both in Guideline 420 and 423, the finding of a delayed death may require additional lower dose levels to be used or a study to be repeated.
TG 403: Acute Inhalation Toxicity Purpose: the test provides information on health hazards likely to arise from shortterm exposure by the inhalation route. Data from an acute study (such as the estimation of LC50, median lethal concentration) may serve as a basis for classification and labeling. Principle: several groups of animals are exposed for a defined period to the test substance in graduated concentrations (at least three), one concentration being used per group. The preferred species is the rat and at least 10 animals (5 female and 5 male) are used at each concentration level. Animals should be tested with inhalation equipment designed to substain a dynamic airflow of 12 to 15 air changes per hour, ensure adequate oxygen content of 19% and an evenly distributed exposure atmosphere. Where a chamber is used, its design should minimize crowding of the test animals and maximize their exposure to the test substance. Observations: animals are observed daily for a total of 14 days. Observations include changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, and somatomotor activity and behaviour pattern. Attention should be given to tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. Individual weights of animals have to be determined before and after the substance is administered. All animals should be subjected to gross necropsy and pathological changes should be recorded.
Draft TG 433: Acute Inhalation Toxicity Fixed Dose Procedure, as an alternative to TG 403 The purpose of the new suggested draft TG 433 (13) is the same as for TG 403. The revised second version of the draft will be submitted for consideration to OECDs Member Countries during the first quarter of 2004. The main difference between TG 403 and the new draft is that a fixed dose procedure is applied in the latter, similar to the strategy used in TG 420. In combination with he use of evident signs of toxicity as 4
the endpoint rather than death, considerable improvements regarding minimizing animal suffering and reduction of the use of laboratory animals will be achieved.
References 1. Trevan, J.W. (1927). The error of determination of toxicity. Proceedings of the Royal Society (London), Series B 101, 483-514.
2. Zbinden, G. and Flury-Roversi, M. (1981). Significance of the LD50 test for the toxicological evaluation of chemical substances. Archives of Toxicology 47, 77-99.
3. Balls, M. (1991). Why modification of the LD50 test will not be enough. Laboratory Animals 25, 198-206.
4. Balls, M. and Fentem, J.H. (1993). The on-going process to replace the LD50 test. Human Innovations and Alternatives 7, 544-547.
5. OECD (2001). Test Guideline 420. Acute Oral Toxicity Fixed Dose Procedure.
6. OECD (2001). Test Guideline 423. Acute Oral Toxicity Acute Toxic Class Method.
7. OECD (2001). Procedure.
Test Guideline 425. Acute Oral Toxicity Up-and-Down
8. OECD (1987). Test Guideline 401. Acute Oral Toxicity (Deleted in 2002).
9. United Nations-Economic Commission for Europe (UN/ECE) (2003). Globally Harmonised System of Classification and Labelling of Chemicals (GHS). UN, New York and Geneva, 2003.
10. OECD (2000). Guidance Document on the Recognition Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation. Environmental Health and Safety Monograph Series on Testing and Assessment No. 19. 5
11. OECD (2001). Guidance Document on Acute Oral Toxicity Testing. Environmental Health and Safety Monograph Series on Testing and Assessment No. 24.
12. Van der Heuvel, M.J., Clark, D.G., Fielder, R.J., Koundakjian, P.P., Olivier, G.J.A., Pelling, D., Tomlinson, N.J., and Walker, A.P. (1990). The international validation of a fixed-dose procedure as an alternative to the classical LD 50 test. Fd. Chem. Toxicol. 28, 469-482.
13. OECD. Draft Test Guideline on Acute Inhalation Toxicity Fixed Dose Procedure.
2. Inventory of alternatives methods currently available

a. Test HepG2 cell/protein content, 24 h. Short description The Hepatoma cell line Hep G2 is exposed to the test substances. Cytotoxicity is measured as changes in protein content by use of the method described by Lowry et al 1951 (J. biol. Chem. 193, 265). Purpose To be used in a test battery, with test b, c and d (see below) or in combination with only test b. Animal reduction for oral acute toxicity. Developer Described by Dierickx (see references). References Dierickx, P. (1989) Cytotoxicity testing of 114 compounds by the determination of the protein content in HepG2 cell cultures, Toxic. In vitro 3(3), 189-193. Clemedson, C., McFarlane-Abdulla, E., Andersson, M., Barile, F.A., Calleja, M.C., Chesn, C., Clothier, R., Cottin, M., Curren, R., Daniel-Szolgay, E., Dierickx, P., Ferro, M., Fiskesj, G., Garza-Ocanas, L., Gmez-Lechn, M.J., Glden, M., Isomaa, B., Janus, J., Judge, P., Kahru, A., Kemp, R.B.,
Kerszman, G., Kristen, U., Kunimoto, M., Krenlampi, S., Lavrijsen, K., Lewan, L., Lilius, H., Ohno, T., Persoone, G., Roguet, R., Romert, L., Sawyer, T., Seibert, H., Shrivastava, R., Stammati, A., Tanaka, N., Torres Alanis, 0., Voss, J-U., Wakuri, S., Walum, E., Wang, X., Zucco, F and Ekwall. B. (1996) MEIC evaluation of acute systemic toxicity: Part I. Methodology of 68 in vitro toxicity assays used to test the first 30 reference chemicals. ATLA 24, 251272. Known users Both the cell type and the end point measurement are used by several labs. Status of validation Pre-validated in the MEIC programme. The MEIC (Multicenter Evaluation of in vitro cytotoxicity Tests) was a large international evaluation study (19891999) initiated by Bjrn Ekwall. The aim of MEIC was to evaluate the relevance of in vitro cytotoxicity tests for predicting human acute systemic toxicity. The second goal of the programme was to select the best combination of tests (test battery) for use in acute toxicity testing. The MEIC results demonstrated the relevance of using human cell line tests that determine basal cytotoxicity for estimating human acute toxicity. However, the results also indicated that two types of tests, such as in vitro tests relevant for toxicokinetics and in vitro test for target organ toxicity, ought to be included in the test battery in order to improve the predictability. Recommendations The test was developed to estimate lethal human blood concentrations. Tests need to be combined with data on absorption in order to predict administered doses (versus blood concentrations).
b. Test HL-60/ATP content, 24 h. Short description HL-60 cells (human acute promyelocytic leukaemia) is exposed to the test substance. ATP content is measured by means of a Lucifer-LU plus kit as the bioluminescence generated from the enzymatic luciferin-luciferase reaction (Kangas, L. et al, 1984, Bioluminescence of cellular ATP: a new method for evaluating cytotoxicity agents in vitro. Medical Biology 62, 338-343). 7
Purpose To be used in a test battery, with test a, c and d (see below) or in combination with only test a. Animal reduction for oral acute toxicity. Developer References Wakuri, S., Izumi, J., Sasaki, K., Tanaka, N., and Ono, H. (1993) Cytotoxicity study of 32 MEIC chemicals by colony formation and ATP assays, Toxic in vitro 7(4), 517-521. Clemedson, C., McFarlane-Abdulla, E., Andersson, M., Barile, F.A., Calleja, M.C., Chesn, C., Clothier, R., Cottin, M., Curren, R., Daniel-Szolgay, E., Dierickx, P., Ferro, M., Fiskesj, G., Garza-Ocanas, L., Gmez-Lechn, M.J., Glden, M., Isomaa, B., Janus, J., Judge, P., Kahru, A., Kemp, R.B., Kerszman, G., Kristen, U., Kunimoto, M., Krenlampi, S., Lavrijsen, K., Lewan, L., Lilius, H., Ohno, T., Persoone, G., Roguet, R., Romert, L., Sawyer, T., Seibert, H., Shrivastava, R., Stammati, A., Tanaka, N., Torres Alanis, 0., Voss, J-U., Wakuri, S., Walum, E., Wang, X., Zucco, F and Ekwall. B. (1996) MEIC evaluation of acute systemic toxicity: Part I. Methodology of 68 in vitro toxicity assays used to test the first 30 reference chemicals. ATLA 24, 251272. Known users Shinobu Wakuri, Laboratory of Cell Toxicology, Department of Cell biology, Hatano Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano, Kanagawa 257, Japan. Status of validation Pre-validated in the MEIC programme Recommendations The test was developed to estimate lethal human blood concentrations. Tests need to be combined with data on absorption in order to predict administered doses (versus blood concentrations).
c. Test Chang liver cell/morphology, 24 h (the MIT-24 assay). Short description 8
The Chang liver cells are cultured in paraffin-sealed 96-well microtitre plates. Deficient outgrowth of fusiform or spindle-shaped cells is used as a criterion of cyto-inhibition. The cultures are further cultivated for 7 days and used in test d. Purpose To be used in a test battery, with test a, b, and d. Animal reduction for oral acute toxicity. Developer The MIT-24 assay is described by J. Paul, Cell and Tissue Culture, ChurchillLivingstone, Edinburgh, 1975 References Ekwall, B. and Sandstrm, B. 1978, Combined toxicity to HeLa cells of 30 drug pairs, studied by a two-dimentional microtitre method, Toxicol. Lett. 2, 285-292. Clemedson, C., McFarlane-Abdulla, E., Andersson, M., Barile, F.A., Calleja, M.C., Chesn, C., Clothier, R., Cottin, M., Curren, R., Daniel-Szolgay, E., Dierickx, P., Ferro, M., Fiskesj, G., Garza-Ocanas, L., Gmez-Lechn, M.J., Glden, M., Isomaa, B., Janus, J., Judge, P., Kahru, A., Kemp, R.B., Kerszman, G., Kristen, U., Kunimoto, M., Krenlampi, S., Lavrijsen, K., Lewan, L., Lilius, H., Ohno, T., Persoone, G., Roguet, R., Romert, L., Sawyer, T., Seibert, H., Shrivastava, R., Stammati, A., Tanaka, N., Torres Alanis, 0., Voss, J-U., Wakuri, S., Walum, E., Wang, X., Zucco, F and Ekwall. B. (1996) MEIC evaluation of acute systemic toxicity: Part I. Methodology of 68 in vitro toxicity assays used to test the first 30 reference chemicals. ATLA 24, 251272. Known users Dr. Lourdes Garza-Ocanas, Depatment de Farmacologia y Toxicologia, Facultad de Medicina, Universidad Autonoma de Nuevo Leon, Apartado Postal No. 146, Col. Del Valle, Nuevo Leon, Mexico. Status of validation Pre-validated in the MEIC programme. Recommendations
The test was developed to estimate lethal human blood concentrations. Tests need to be combined with data on absorption in order to predict administered doses (versus blood concentrations).
d. Test Chang cell/pH changes, 168 h. Short description The cultures from test c are used. After 168 h the colour of the pH indicator phenol red included in the medium is recorded. Violet colour is judged as total inhibition, while less basic (red) but not normal (orange) colours are considered as partial inhibition. Purpose To be used in a test battery, with test a, b and c. Animal reduction for oral acute toxicity. Developer See test c. References See test c. Known users See test c. Status of validation See test c. Recommendations See test c.
e. Test BALB/c 3T3 Neutral Red Uptake (NRU) Cytotoxicity Assay, 48 h. Short description NRU: cell survival/viability assay based on the ability of viable cells to incorporate and bind neutral red (NR), a supravital dye. NR penetrates cell membranes and accumulates in lysosomes. Alterations of the cell surface result in a decreased uptake and binding of NR. Purpose Animal reduction for oral acute toxicity. 10
Developer Borenfreund E and Puerner J. 1985. Toxicity determination in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24: 119124. References FRAME. 1992. Balb/c 3T3 NRU cytotoxicity test, INVITOX Protocol n. 56, Nottingham. Liebsch M., and Spielmann H. 1995. Balb/c 3T3 cytotoxicity test. Methods and Molecular Biology, vol. 43, In vitro Toxicity testing Protocols. OHare S., Atterwill CK., eds. pp. 177-187. Humana Press, Totowa, NJ. Spielmann H., Liebsch, M., Kalweit S., Moldenhauer F., Wirnsberger, T., Holzhutter HG., Schneider B., Glaser S., Gerner I., Pape W., Kreiling R., Krauser K., Miltenburger HG., Steiling W., Luepke NP., Muller N., Kreuzer H., Murmann P., Spengler J., Bertram-Neis E., Siegemund B., and Wiebel FJ. 1996. Results of a validation study in Germany in two in vitro alternatives to the Draize eye irritation test, the HET-CAM test and the 3T3 NRU cytotoxicity test. ATLA. 24: 741-858.
Spielmann H., Genschow E., Liebsch M., Halle W. 1999. Determination of the starting dose for acute oral toxicity (LD50) testing in the up and down procedure (UDP) from cytotoxicity data. ATLA. 27: 957-966. Known users Extended use. Status of validation Under validation. Recommendations
f. Test Normal human keratinocyte Neutral Red Uptake (NRU) Cytotoxicity Assay, 48 h. Short description The NRU is a cell survival/viability assay based on the ability of viable cells to incorporate and bind neutral red (NR), a supravital dye. NR penetrates cell
11
membranes and accumulates in lysosomes. Alterations of the cell surface result in a decreased uptake and binding of NR. Purpose Animal reduction for oral acute toxicity. Developer Borenfreund E and Puerner J. 1984. A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/NR-90). J. Tissue Culture Meth. 9(1): 7-9. References Heimann R., and Rice RH. 1983. Polycyclic aromatic hydrocarbon toxicity and induction of metabolism in cultivated esophageal and epidermal keratinocytes. Cancer Res. 43: 4856. Spielmann H., Genschow E., Liebsch M., Halle W. 1999. Determination of the starting dose for acute oral toxicity (LD50) testing in the up and down procedure (UDP) from cytotoxicity data. ATLA. 27: 957-966. Known users Extended use. Status of validation Under validation. Recommendations
g. Test In vitro prediction of the maximum tolerated dose (data collected from ECVAM SIS, website: http://ecvam-sis.jrc.it). Short description The minimum in vitro drug concentration which induces changes in cell morphology, LDH release or up to 50% cell mortality (CT50) is assumed to correspond to the drug dose in vivo which gives rise to initial or mild toxic signs, while the minimum in vitro drug concentration which elicits over 90% cell mortality (CT100) is assumed to correspond to the in vivo dose which gives rise to marked clinical signs. CT50 and CT100 values (? g/ml) are transformed into mg/kg/day for the in vivo threshold. Primary cultures of rat hepatocytes are more sensitive and are used to obtain the in vivo values in dogs. MDBK (bovine kidney cells) are less sensitive and are used to obtain the 12
in vivo values in rats. McCoy (human epithelial cells) serve as in vitro control. The following parameters of cell growth and morphology are scored after exposure of the cells (24 h for hepatocytes, 24, 48, 72 h for cell lines) to test compound: surface occupied by growing cells (cell lines only), changes in cell size and shape, presence of cytoplasmic vacuoles, cell detachment, dead and dying cells. Purpose Results of cytotoxicity tests in primary cultures of rat hepatocytes, MDBK and McCoy cells can be used to predict the in vivo 4-wk maximum tolerated dose in rats and dogs. Developer Shrivastava R., John GW., Rispat G., Chevalier A., and Massingham R. 1991. Can the in vivo maximum tolerated dose be predicted using in vitro techniques? A working hypothesis. ATLA. 19: 393-402. References Shrivastava R., Delomenie C., Chevalier A., John G., Ekwall B., Walum E., and Massingham R. 1992. Comparison of in vivo acute lethal potency and in vitro cytotoxicity of 48 chemicals. Cell. Biol. Toxicol. 8: 157-170. Known users
Status of validation In-house development and pre-validated in the MEIC programme. Recommendations
h. Test Transepithelial resistance (TER) and paracellular permeability (PCP) in two renal cell lines (LLC-PK1, epithelial proximal tubular cells and MDCK, epithelial distal cells). Short description TER measurement and the trans-epithelial transport of uncharged small molecules as FITC-inulin (PCP) are reliable parameters for characterising the intactness of an epithelial barrier. Cells were seeded onto 24 well polycarbonate filter plates and then exposed to test substances. Barrier damage
13
is measured by using the REMS autosampler for TER assessment and with fluorescence measurement in the base plate for PCP (Duff et al, 2000). Purpose Measurements of TER and PCP as generalized predictors of nephrotoxicity. Developer Pfaller W., Troppmair E. (2000). Renal transepithelial resistance (TER) and paracellular permeability (PCP) are reliable endpoints to screen for nephrotoxicity. In: Progress in the Reduction Refinement and Replacement of Animal Experimantation. (Ball M., van Zeller A. M., Halder M. eds.) Elsevier Science B. V. Vol. 1, p. 291-304. References Duff T., Carter S., Feldman G., McEwan G., Pfaller W., Rhodes P., Ryan M., Hawksworth G. (2002). Transepithelial resistance and inulin permeability as endpoints in in vitro nephrotoxicity testing. ATLA, 30 Suppl. 2, 53-59. Known users Pfaller W, University of Innsbruck, Austria; Ryan M, Department of Pharmacology, University College Dublin, Ireland; Hawksworth G, University of Aberdeen, Scotland; ECVAM, JRC, Ispra (VA), Italy. Status of validation Under prevalidation Recommendations The test could be used for cell damage assessment in different epithelia.
i.
Test QSARs models. Short description There are a number of software packages for the prediction of human health effects and related toxicities. These systems allow toxicity to be predicted directly from chemical structure and have been used because of their ease of use and rapid application. TOPKAT is a statistically based system that consists of a suite of QSAR models. Models are normally derived after the analysis of large data sets of toxicologic information, usually retrieved from the literature. Molecules are characterized by any of a large number of structural, topologic, and 14
electrotopologic indices. Models are developed using regression analysis for continuous endpoints, and discriminant analysis for categorizing toxicity data. TOPKAT Model Rat Oral LD50: it comprises 19 QSAR models and the data from which these models are derived: experimental acute median lethal dose (LD50) values of approximately 4,000 chemicals from the open literature. Each quantitative structure toxicity relationship (QSTR) model assesses oral LD50 for the rat for a specific class of chemicals. TOPKAT Model for Rat Inhalation Toxicity LC50: it comprises five QSAR models and data from which these models were derived. These multiple regression models were derived from experimental median lethal
concentration (LC50) values on more than 643 chemicals after review of the open literature. Reviewed literature data ranged over various time limits; only exposure times in the range of 0.514 hr were accepted. Endpoints were modeled as log10(1/C) log10(hours of exposure), where C is the concentration in moles/m3. The chemicals are grouped into five class-specific models: single benzenes, heteroaromatics and multiple benzenes, alicyclics, and acyclics with and without halogens. Each QSTR model assesses acute LC50 to rat of a specific class of chemicals in units of moles per cubic meter per hour. Purpose Prediction of toxicity from the chemical structure. Developer References Dearden JC., Barratt MD., Benigni R., Bristol DW., Combes RD., Cronin MTD. (1997). The development and validation of expert systems for predicting toxicitythe report and recommendations of an ECVAM/ECB workshop (ECVAM workshop 24). ATLA, 25: 223252. Accelrys Inc. 2002. TOPKAT. Cambridge, UK: Accelrys Inc. Available: http://www.accelrys.com/products/topkat/ index.html. Cronin MTD., Jaworska JS., Walker JD., Comber MHI., Watts CD., and Worth AP. (2003). Use of QSARs in International Decision-Making Frameworks to Predict Health Effects of Chemical Substances. Environm. Health Perspectives, 111 (10): 1391-1401. Known users 15
Various european regulatory bodies. Status of validation Under research and development. Recommendations
3. Identified steps or tests with no alternative methods available

There are no validated alternative methods available to account for biokinetic factors. These methods will be needed in combination with alternative methods for toxicity endpoints in order to develop alternative methods schemes for systemic endpoints (including acute oral toxicity). There are no alternative methods with fully developed protocols for toxicants that may be preferentially toxic to specific cell types/organ systems such as heart and brain. The greatest strides to date have been made with basal cytotoxicity methods. On the other hand, it has been earlier concluded (Report of the International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity, October 1720, 2000) that for acute systemic toxicity it is probably not needed to routinely test for all possible specific organ effects in different in vitro models. Instead, tests on energy metabolism and tests assessing the ability of a compound to disrupt epithelial barrier functions may be sufficient. In addition, there are no in vitro methods available to predict acute toxicity by the inhalation route. Some models are currently under development.
4. Summary of the alternative methods currently available and foreseeable time to achieve peer reviewed validation
Look at the following table.
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Current endpoints addressed in animal test TG 420 TG 423 TG 425 TG 403 (Changes in skin, eyes, respiratory, circulatory, central nervous system, behaviour pattern, lethargy, sleep, coma, death, necropsy, pathology).
Alternative tests available HepG2 cell/protein content
Endpoints measured
Purpose
Area(s) of application
Validation status
Regulatory acceptance
Comments
Protein content
Partial replacement (tiered strategy/ test battery) Partial replacement (tiered strategy/ test battery) Partial replacement (tiered strategy/ test battery) Partial replacement (tiered strategy/ test battery) Reductiona, partial replacement (tiered strategy/ test battery)
Estimated time to have the method validated (ESAC endorsement)* NDc
All soluble chemicals
Prevalidated (MEIC)
NDc All soluble chemicals Prevalidated (MEIC) _ _
HL60/ATP content
ATP content
NDc All soluble chemicals Prevalidated (MEIC) _ _
Chang liver cell/morphology
Morphology
NDc All soluble chemicals Prevalidated (MEIC) Under validation (ECVAM, ICCVAM) _ _
Chang cell/pH changes
pH changes
BALB/c 3T3 NRU
Cell viability
2+ (for the individual test)
17
Normal human keratinocyte NRU
Cell viability
Reductiona, partial replacement (tiered strategy/ test battery) Reductionb, partial replacement (tiered strategy/ test battery) Partial replacement (tiered strategy/ test battery) Partial replacement (tiered strategy/ test battery)
Under validation (ECVAM, ICCVAM)
2+ (for the individual test) NDc
In vitro prediction of the MTD TER and PCP in two renal cell lines
Cell viability
Prevalidated in the MEIC
Intactness of epithelial barrier
Under prevalidation (ECVAM)
4+ (for the individual test)
QSARs models
Various
R&D
10+
* This table estimates the time needed to achieve ESAC endorsement for individual alternative tests assuming optimal conditions. It does not
indicate the time needed to achieve full replacement of the animal t st, nor does it include the time needed to achieve regulatory acceptance. e Optimal conditions means that all necessary resources, for example technical, human, financial and coordination, are met at all times in the process and that the studies undertaken have successful outcomes.
a
Reduction if the alternative test is considered as single test for the estimation of the starting dose for acute oral toxicity. Reduction since primary cells are used for the in vitro prediction of the maximum tolerated dose.
ND: not defined.
b
c
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5. Conclusion
At the moment there are not validated alternative methods able to completely replace the use of animals in the field of acute toxicity. The list of in vitro tests presented in this chapter cannot be predictive for acute oral toxicity as single methods, but they become a good alternative if integrated in a tiered approach and/or in a test battery. Such a strategy should include a battery of tests covering general cytotoxicity, metabolism, toxicokinetics, and target organ toxicity. While good progress has been made on methods for basal cytotoxicity, methods to address metabolism, toxicokinetics and target organ toxicity for potentially sensitive target organs have barely begun. Once methods for individual endpoints are completed and validated, there will be substantial work required to put the individual components into a validated testing battery combined with a predictive model for data extrapolation to the human situation. It should be noted that alternative methods validated to date are for point of contact effects (e.g. skin corrosivity, eye irritancy) and have not needed to address the complexity of toxicokinetics and multiple target tissues which may have differential sensitivities. Validation of an alternative model for acute oral toxicity is significantly more complex than these previous efforts, and shares many of the complexities and challenges of developing alternative models for other systemic endpoints, such as subchronic toxicity and developmental and reproductive toxicity. Several studies, such as the MEIC study, have shown that cell culture tests give roughly similar results irrespective of the cell type or the growth/viability endpoint measurement used, i.e. almost any cell type and growth/viability endpoint measurement could be used for measurement of basal cytotoxicity. However, the MEIC study also indicated that it is preferably to use human cells for testing. A proposal for an integrated project (A-cute-tox) has been submitted to the 6FP EC in November 2003. The aim of A -cute-tox is to develop a simple and robust in vitro testing strategy for prediction of human acute oral systemic toxicity, which could totally replace the animal acute toxicity tests used today for regulatory purposes. The objectives of the project include: compilation, evaluation and generation of high quality in vitro and in vivo data for comparative analysis; identifying factors (kinetics, metabolism, and organ toxicity) that influence the correlation between in vitro (concentration) and in vivo (dose) toxicity; explore innovative tools and cellular
19
systems to identify new endpoints/strategies to anticipate animal/human toxicity; to design a simple, robust and reliable in vitro test strategy amenable for robotic testing, associated with the prediction model for acute toxicity. The time estimated to achieve complete animal replacement for acute toxicity is strongly dependent on the outcome of this project, which represents the first attempt to create an integrated strategy to be validated with the purpose to predict human systemic toxicity, and it cannot be less than 10 years.
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Subgroup 2. Skin Irritation/Corrosion Valrie Zuang 1 , Marie-Ange Alonso2 , Philip A. Botham3 , Chantra Eskes1, Julia Fentem4 , Manfred Liebsch5 , Johannes J.M. van de Sandt 6 , ECVAM, Institute for Health & Consumer Protection, European Commission Joint Research Centre, 21020 Ispra (VA), Italy; 2 EVIC FRANCE,33290 Blanquefort, France; 3 Syngenta, Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK; 4 SEAC, Unilever Colworth Laboratory, Sharnbrook Bedfordshire MK44 1LQ,,UK; 5 ZEBET BfR, Diedersdorfer Weg 1, 12277 Berlin, Germany; 6TNO Nutrition & Food Research, Department of Explanatory Toxicology, Utrechtseweg 48, 3700 AJ Zeist, The Netherlands. Skin irritation 1. Inventory of methods currently available Since the skin is often exposed, either intentionally or unintentionally, to cosmetic products, it is clear that the potential for a particular product/ingredient to cause skin irritation or for a particular ingredient to cause skin corrosion needs to be carefully evaluated as part of the overall safety assessment process. Dermal irritation is defined as the production of reversible damage of the skin following the application of a test substance for up to 4 hours (OECD TG 404, 2002). It is generally assessed by the potential of a certain substance to cause erythema/eschar and/or oedema after a single topical application on rabbit skin and based on the Draize score (OECD TG 404, 2002). Recently some general refinement provisions were included in the OECD testing guideline 404. This updated version recommends, prior to undertake the described in vivo animal test for corrosion/irritation of the substance, to perform a sequential testing strategy. This strategy is based in a stepwise order on: a weight of evidence analysis, pH considerations, use of validated and accepted in vitro tests, and finally refinement of the animal testing. Some examples of decision-making in the testing strategy are the following: 1) A substance with a pH below 2.0 or above 11.5 should not be tested, due to its suspected corrosivity. A substance found to be corrosive in one of the alternative corrosivity tests (taken up in Annex V of Dir. 67/548/EEC), should not be tested in the Draize test.
1
2)
Although the OECD TG 404 was recently refined, there are to date no validated alternative methods replacing the classical Draize test for predicting the acute skin irritation of cosmetic ingredients or chemicals (Botham et al., 1998).
2. Inventory of alternative methods currently available Whereas relatively few QSAR studies for skin irritation have been reported (see chapter on SAR below), several prevalidation efforts were carried out for in vitro alternative methods, some of which are currently pursued in the on-going ECVAM validation study (Zuang et al., 2002). In vitro alternatives in the field vary from more simple models such as keratinocyte cultures to more complex organotypic cultures and reconstituted human skin models (Botham et al., 1998; van de Sandt et al., 1999). During 1999-2001, some of the most promi sing in vitro methods were evaluated in prevalidation studies (see table 1; Fentem et al., 2001). The five tests included were: EpiDerm, EPISKIN, Prediskin, the non-perfused pig ear model, and the in vitro mouse skin integrity function test (SIFT). The outcome of these studies was that none of the tests was ready for progression to formal validation. Various follow-up activities took place where appropriate modifications were made to enable some test protocols to meet the criteria for inclusion in a formal validation study (Zuang et al., 2002). On the basis of additional work, the EPISKIN, EpiDerm and SIFT test protocols and/or prediction models will be evaluated in a formal validation study funded by ECVAM during 2004 (Fentem and Botham, 2002). The main overall objective is to identify those in vitro alternatives capable of discriminating skin irritants from non-irritants. Several testing strategies for evaluating the skin irritation potential of ingredients and products have been described (Botham et al., 1998; OECD, 2002; Robinson et al., 2002), some of which also involve human volunteer studies (human 4-hour patch test; Basketter et al., 1997). In such strategies human testing is generally used to determine the "relative skin compatibility" and "exposure" parts of the risk assessment of products/formulations (and occasionally ingredients), to confirm that there are no harmful effects when applying a cosmetic product for the first time to human skin or mucous membranes. There are however some specific occasions when cosmetic ingredients can be ethically tested using human volunteers to provide additional data for risk assessment purposes, but this is rather a case-by-case situation where the hazard has been characterised (sensitisation, corrosion, mutagenicity, etc.), than a generically applicable testing approach. In addition for the hazard assessment of single entities, many cosmetics companies conduct in vitro and/or dermatological (human skin compatibility, often following repeated application; COLIPA, 1995; COLIPA, 1997) comparative studies on new formulations. In such studies the results for the new formulation are interpreted relative to those obtained for other similar formulations (typically those with a long history of safe consumer use and satisfaction).
Table 1. Summary of the In Vitro Methods which participated in the ECVAM pre-validation study for Skin Irritation
Method EPISKIN human skin model (commercial system) EpiDerm human skin model (commercial system) PREDISKIN human skin model (commercial system) Pig ear test
Test System reconstructed human epidermal equivalent reconstructed human epidermal equivalent reconstructed human epidermal equivalent pig ear
Endpoint cell viability (MTT reduction assay) cell viability (MTT reduction assay) histology and cell viability (MTT reduction assay) trans-epidermal water loss (TEWL) TEWL and electrical resistance (ER)
Applicability general; a few materials may interfere with MTT reduction general; a few materials may interfere with MTT reduction general; a few materials may interfere with MTT reduction general
Validation Authority ECVAM
Status protocol modification following prevalidation study; revised protocol and prediction model proposed for validation protocol modification following prevalidation did not meet criteria for progression to phase 3 of prevalidation further development; did not meet criteria for progression to phase 3 of prevalidation prediction model modification following prevalidation study; revised protocol and prediction model proposed for validation
References Fentem et al. 2001 Zuang et al. 2002 Portes et al. 2002 Fentem et al. 2001 Zuang et al. 2002 Fentem et al. 2001
ECVAM
ECVAM
ECVAM
Fentem et al. 2001 Zuang et al. 2002 Fentem et al. 2001 Zuang et al. 2002 Heylings et al. 2003
Mouse skin integrity function test (SIFT)
excised mouse skin
general; a few materials may interfere with either TEWL or ER determination
ECVAM
RECONSTRUCTED HUMAN SKIN MODELS Reconstructed human skin models are able to mimic the human skin to a large extend. They are three-dimensional models generated by growing keratinocyte cultures at the airliquid interface on various substrates, and enable the topical application of either neat or diluted test materials (Botham et al., 1998; van de Sandt et al., 1999; Faller et al., 2002). Several human skin models are manufactured commercially, such as the EPISKIN TM model (EPISKIN-SNC, Gerland, France), the EpiDermTM model (MatTek, Ashland, MA, USA) and the SkinEthicTM model (SkinEthic, Nice, France). Moreover, some in-house models were also developed and evaluated for their potential in detecting skin irritation such as the one developed by Cosmital, Wella (Faller et al., 2002), or the one developed by Ponec and co-workers (Ponec and Kempenaar, 1995). 1. The EPISKIN TM model Short description , scientific relevance and purpose EPISKINTM (EPISKIN-SNC, Gerland, France) is a three-dimensional human skin model consisted of a type 1 bovine collagen matrix, representing the dermis, surfaced with a type IV human collagen, upon which is laid after 13 days in culture stratified differentiated epidermis derived from second passage human keratinocytes (Tinois et al., 1991). Its use for skin irritation testing involves topical application of test materials to the surface of the skin, and the subsequent assessment of their effects on cell viability by using the MTT assay. The endpoint used to distinguish between potential skin irritants and non-irritants is the percentage (%) of cell viability (Roguet et al., 1994, 1998). However, the use of other, more mechanistic, endpoints such as Interleukin 1? (IL-1? ) or lactate dehydrogenase (LDH) has also been evaluated (Roguet et al., 1998; Faller et al., 2002). Following results from a prevalidation study, the protocol was refined in order to improve the specificity of the method (Fentem et al., 2001; Zuang et al., 2002). This refinement consisted in reducing the exposure time of epidermis with chemicals. Sensitivity, specificity and accuracy of the new method were improved and the EPISKINTM model is now ready to enter a validation study of in vitro tests for acute skin irritation (Portes et al., 2002). References
Faller C., Bracher M., Dami N., Roguet R. (2002). Predictive ability of reconstructed human epidermis equivalents for the assessment of skin irritation of cosmetics. Toxicology in Vitro 16, 557-572. Fentem, J. H., Briggs, D., Chesn, C., Elliott, G. R., Harbell, J. W., Heylings, J. R., Portes, P., Roguet, R., van de Sandt, J. J. M. & Botham, P. A. (2001). A prevalidation study on in vitro tests for acute skin irritation: results and evaluation by the Management Team. Toxicology in Vitro 15, 57-93. Portes P., Grandidier M.-H., Cohen C., Roguet R. (2002). Refinement of the EPISKIN protocol for the assessment of acute skin irritation of chemicals: follow-up to the ECVAM prevalidation study. Toxicology in Vitro 16, 765-770. Roguet R., Cohen C., Dossou K.G., Rougier A. (1994). EPISKIN, a reconstituted epidermis for assessing in vitro the irritancy of topically applied compounds. Toxicology in Vitro 8, 49-59.
Roguet R., Cohen C., Robles C., Courtellemont P., Tolle M., Guillot J.P., Pouradier Duteil X. (1998). An interlaboratory study of the reproducibility and relevance of EPISKINT M, a reconstructed human epidermis, in the assessment of cosmetics irritancy. Toxicology in Vitro 12, 295-304. Tinois E., Tillier J., Gaucherand M., Dumas H., Tardy M., Thivolet J. (1991). In vitro and posttransplantation differentiation of human keratinocytes grown on the human type IV collagen film of a bilayered dermal substitute. Experimental Cell Research 193, 310-319. Zuang V., Balls M., Botham P.A., Coquette A., Corsini E., Curren R.D., Elliott G.R., Fentem J.H., Heylings J.R., Liebsch M., Medina J., Roguet R., van de Sandt J.J.M., Wiemann C., Worth A.P. (2002). Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation. ATLA 30, 109-129.
Producer EPISKIN-SNC, Gerland, France. Known users Industry and contract testing laboratories use this method. Status of validation and/or standardisation Since the method has already undergone prevalidation and will undergo validation by ECVAM, a defined/optimised protocol is available, as well as data on intra-and interlaboratory variation, protocol transferability and in vitro/in vivo comparisons. Which efforts are needed to complete validation of the method? The method is currently part of the ECVAM validation study of in vitro methods for acute skin irritation, expected to be finished in 2005. The possible use of more mechanistically based endpoints, such as adenylate kinase release and IL-1? will also be evaluated. 2. The EpiDermTM model Short description , scientific relevance and purpose In the EpiDermTM reconstituted human skin model (MatTek, Ashland, MA, USA), human skin-derived keratinocytes are grown on specially prepared Millicell cell culture inserts, forming a multi-layered, differentiated, model of the human epidermis in vitro (Cannon et al., 1994; Earl et al., 1999). The original EpiDermTM test protocol was based on the comparison of the time of exposure required to decrease tissue viability by 50 % (ET50) of the test chemical with the ET50 of a reference standard (Fentem et al., 2001). With this protocol the intralaboratory and interlaboratory reproducibilities were found to be acceptable. On the other hand the test method gave a too high percentage of false negatives during phase III of the prevalidation study. As a follow-up activity to this prevalidation study, the test protocol and prediction model were modified, which improved the predictive ability of the model (Zuang et al., 2002). References
Cannon CL, Neal PJ, Southee JA, Kubilus J, Klausner M. (1994). New epidermal model for dermal irritancy testing. Toxicology in Vitro 8, 889-891.
Earl L.K., Hall-Manning T.J., Holland G.H. (1999). The determination of acute chemically induced ski n irritation using a human skin model. In: Clark D.G., Lisansky S.G., Macmillan R. (Eds.), Alternatives to Animal Testing II. Proceedings of the Second International Scientific Conference Organised by the European Cosmetic Industry. CPL Press, Newbury, p. 227. Fentem J.H., Briggs D., Chesn C., Elliott G.R., Harbell J.W., Heylings J.R., Portes P., Roguet R., van de Sandt J.J.M., Botham P.A. (2001). A prevalidation on in vitro tests for acute skin irritation: results and evaluation by the Management Team. Toxicology in Vitro 15, 57-93. Zuang V., Balls M., Botham P.A., Coquette A., Corsini E., Curren R.D., Elliott G.R., Fentem J.H., Heylings J.R., Liebsch M., Medina J., Roguet R., van de Sandt J.J.M., Wiemann C., Worth A.P. (2002). Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation. ATLA 30, 109-129.
Producer MatTek, Ashland, MA, USA. Known users Industry and contract testing laboratories use this method. Status of validation and/or standardisation As for Episkin, the method had undergone prevalidation by ECVAM and the protocol has been optimised to be included in the ECVAM validation study on acute skin irritation. Thus, data on intra-and inter-laboratory variation, protocol transferability and in vitro/in vivo comparisons are available. Which efforts are needed to complete validation of the method? In order to have one common protocol applicable to different skin models, it was suggested to apply the revised EPISKIN TM protocol (measure of the % of cell viability by using the MTT assay after a reduced time of exposure) to the EpidermTM model. This protocol showed promising results and was chosen to be used for the ECVAM validation study of in vitro methods for acute skin irritation (Zuang et al., 2002). 3. The SkinEthic TM model Short description , scientific relevance and purpose The SkinethicTM cultures are obtained by culturing normal human keratinocytes on a polycarbonate culture inserts lifted to the air-liquid interface, and a stratum corneum forms from growth at the air-liquid interface. The method is based on the model developed by Rosdy and Clauss (1990). Novartis Pharma has proposed a two step acute and cumulative irritation screening system based on this approach (de Brugerolle de Fraissinette et al., 1999). The most used endpoints with this model are the modulation of cell viability, release of IL-1? , and morphological changes. References
de Brugerolle de Fraissinette A., Picarles V., Chibout S., Kolopp M., Medina J., Burtin P., Ebelin M.E., Osborne S., Mayer F.K., Spake A., Rosdy M., De Wever B., Ettlin R.A., Cordier A. (1999). Predictivity of an in vitro model for acute and chronic skin irritation (SkinEthic) applied to the testing of topical vehicles. Cell Biology and Toxicology 15, 121-135. Rosdy M., Clauss LC (1990). Complete human epidermal cell differentiation in chemical defined medium at the air-liquid interface on inert filter substrates. Journal of Investigative Dermatology 95, 409-414.
Producer SkinEthic, Nice, France. Known users Industry and contract testing laboratories use this method. Status of validation and/or standardisation - Is the protocol defined/optimised? YES - Is there data on intra-laboratory variation? YES - Is there data on the transferability? Limited - Is there data on the inter-laboratory variability? Limited - In vivo /in vitro comparisons? Limited - Is the method validated? NO Which efforts are needed to complete validation of the method? There is a need to determine the transferability and inter-laboratory variability of the method before including it in a validation study. 4. Other models Other models exist where the keratinocyte cultures grown at air-liquid interface are cultured in different substrates. Amongst the well-characterised models are: - the Cosmital in-house model (Wella, Switzerland), the Apligraf model (Organogenesis Inc., Novartis Pharmaceuticals Corporation, Canton, MA USA), - the reconstituted epidermis on de-epidermized dermis (RE-DED) used by Ponec and co-workers (Ponec and Kempenaar, 1995; Robinson et al., 2000; Boelsma et al., 1996, 2000). The predictive ability of the cosmital in-house model and the comparison to in vivo data have been assessed (Faller et al., 2002). The use of Apligraf in a tiered strategy for the evaluation of cumulative skin irritation potential of chemicals has also been assessed (Medina et al., 2000). However, none of these models are at the same stage as the previous examples. They would be able to undergo catch-up validation and/or be used within the context of any future test guideline as long as they meet defined performance criteria (as with the skin corrosion TG). References
Boelsma E., Tanojo H., Bodd H.E., Ponec M. (1996). Assessment of the potential irritancy of oleic acid on human skin: evaluation in vitro and in vivo. Toxicology in Vitro 10, 729-742. Boelsma E., Gibbs S., Faller C. Ponec M. (2000). Characterization and comparison of reconstructed skin models: morphological and immunohistochemical evaluation. Acta Derm Venereol 80, 82-88. Faller C., Bracher M., Dami N., Roguet R. (2002). Predictive ability of reconstructed human epidermis equivalents for the assessment of skin irritation of cosmetics. Toxicology in Vitro 16, 557-572. Medina J., de Brugerolle de Fraissinette A., Chibout S.D., Kolopp M., Kammermann R., Burtin P., Ebelin M.E., Cordier A. (2000). Use of human skin equivalent Apligraf for in vitro assessment of cumulative skin irritation potential of topical products. Toxicology and Applied Pharmacology 164, 38-45.
Ponec M. and Kempenaar J. (1995). Use of human skin recombinants as an in vitro model for test the irritation potential of cutaneous irritants. Skin Pharmacol 8, 49-59. Robinson M.K., Cohen C., de Brugerolle de Fraissinette A., Ponec M., Whittle E., Fentem J.H. (2000). Non-animal testing strategies for assessment of the skin corrosion and skin irritation potential of ingredients and finished products. Food and Chemical Toxicology 40, 573-592.
SKIN EXPLANTS AND ORGAN CULTURES 1. The mouse skin integrity function test (SIFT) Short description , scientific relevance and purpose The SIFT (protocol of Syngenta CTL, Macclesfield, UK; Heylings et al., 2001) is based on assessment of mouse skin integrity following exposure to test material.Two methods are used to assess stratum corneum integrity: trans-epidermal water loss (TEWL), and electrical resistance (ER). The SIFT protocol was developed as a prescreen for assessing the skin irritation potential of industrial chemicals. It combines elements of in vitro percutaneous absorption, where skin integrity assessment is fundamental to the method, together with knowledge from models used for the assessment of skin emolliency using intact skin. Mouse skin provides such a model since it is sufficiently thin to use in static glass diffusion cells as intact whole skin, and it displays relevant biochemical activity and highly reproducible and robust barrier properties (Fentem et al., 2001). The basis of the SIFT prediction model is if the ratios of the pre- and post-application values for either TEWL or ER are greater or smaller than five-fold, then the test chemical is deemed irritant or non irritant respectively (Heylings et al., 2003). The prediction model that failed in the prevalidation exercise was modified by using new statistical means of evaluating the data that were more suited for the model. Such modification improved the specificity and sensitivity of the model (Heylings et al., 2003; Zuang et al., 2002). References
Fentem J.H., Briggs D., Chesn C., Elliott G.R., Harbell J.W., Heylings J.R., Portes P., Roguet R., van de Sandt J.J.M., Botham P.A. (2001). A prevalidation on in vitro tests for acute skin irritation: results and evaluation by the Management Team. Toxicology in Vitro 15, 57-93. Heylings, J. R. Clowes, H.M. Hughes L. (2001). Comparison of tissue sources for the skin integrity function test (SIFT). Toxicology in Vitro 13, 597-600. Heylings J.R., Diot S., Esdaile D.J., Fasano W.J., Manning L.A., Owen H.M. (2003). A prevalidation study on the in vitro skin irritation function test (SIFT) for prediction of acute skin irritation in vivo: results and evaluation of ECVAM Phase III. Toxicology in Vitro 17, 123-138. Zuang V., Balls M., Botham P.A., Coquette A., Corsini E., Curren R.D., Elliott G.R., Fentem J.H., Heylings J.R., Liebsch M., Medina J., Roguet R., van de Sandt J.J.M., Wiemann C., Worth A.P. (2002). Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation. ATLA 30, 109-129.
Developer of the method Syngenta Central Toxicology Laboratory, Macclesfield, UK Known users - YES
Status of validation and/or standardisation - Is the protocol defined/optimised? YES - Is there data on intra-laboratory variation? YES - Is there data on the transferability? YES - Is there data on the inter-laboratory variability? YES - In vivo /in vitro comparisons? YES - Is the method validated? NO Which efforts are needed to complete validation of the method? In order to improve the low inter-laboratory reproducibility observed in phase III of the prevalidation study with the revised protocol, the future validation protocol for SIFT will reinforce that the empirical values for TEWL and ER in positive and negative controls must be within certain ranges (Heylings et al., 2003). 2. The PrediskinTM model PrediskinTM (Biopredic, Rennes, France) improved test protocol involves exposure of human skin cultures (obtained from patients undergoing plastic surgery) to test materials for 20 hours, and subsequent assessment of effects on the percentage of cell viability by using the MTT assay (Fentem et al., 2001). In the initial protocol, histology was included if cell viability was greater than 35%. The Prediskin did not perform sufficiently well in phase II for it to progress to phase III; the protocol was overly sensitive, resulting in the prediction of all the non-irritants as irritant (Fentem et al., 2001). In additional studies (a repeat of phase I), the Prediskin protocol was modified by using non-stripped human skin and by increasing the threshold for discriminating between irritants and non-irritants (MTT assay). The change i the prediction model considerably improved the ability of n the test to distinguish irritant from non-irritant chemicals (Fentem et al., 2001). However, further work on PrediskinTM has not been followed up for commercial reasons. 3. The pig ear test The non-perfused pig ear test (protocol of TNO-PML, Rijkswijk, NL) is based on determination of the absolute increase in trans-epidermal water loss (TEWL) from the skin surface, following exposure of the pig ear to test material, as the endpoint to distinguish between irritants and non-irritants. The pig ear test did not perform sufficiently well in phases I and II for it to progress to phase III; the variability in the results obtained was too great, indicating that the test would be of limited predictive value (Fentem et al., 2001). In additional studies (a repeat of phase I), attempts to improve the intralaboratory reproducibility of the pig ear test were unsuccessful. Structure-activity relationships for skin irritation Relatively few QSAR studies for skin irritation have been reported in the literature. The most recent reviews were made by Cronin et al. (in press), Hulzebos et al., (2003) and Patlewicz et al. [in press (b)].
Barratt (1996) reported a QSAR for predicting the primary irritation index (PII) of organic chemicals, but this had little predictive value (r2 =0.42). In the same study, discriminant analysis was shown to discriminate between irritant and non-irritant chemicals, as defined by EU classification criteria, with an accuracy of 67%. Hayashi et al. (1999) reported two QSARs for predicting the molar-weighted PII of phenols. One model, based on absolute hardness, was proposed for chemicals with negative lowest unoccupied molecular orbital (LUMO) energies, whereas the other model, based on the logarithm of the octanol-water partition coefficient (logP), was proposed for chemicals with positive LUMO energies. These models had correlation coefficients of 0.72 and 0.82, respectively (i.e. r2 values of 0.52 and 0.67). Smith et al. (2000) analysed a data set of 42 esters, for which human skin irritation data were available, and for which 19 physicochemical properties had been calculated. Best subsets regression was used to select variables for subsequent inclusion in discriminant models. The best variables were water solubility (lower for irritants than non-irritants), a dispersion parameter (higher for irritants), a hydrogen-bonding parameter (higher for irritants), the sum of partial positive charges (lower for irritants), and density (lower for irritants). A discriminant model based on all five parameters had a sensitivity of 85% and a specificity of 92%. Expert systems such as DEREK for Windows, HazardExpert and TOPKAT have been reviewed in Cronin et al. (2003). The BgVV database has been used to develop specific SAR models for predicting skin irritation/corrosion. These models have been incorporated into a DSS (Gerner et al., 2000a, 2000b; Zinke er al., 2000). The DSS is mainly a rule-based approach, the rules being developed based not only on substructural molecular features but also on physicochemical properties such as molecular weight, acqueous solubility, and log Kow. The rules have been developed and validated on a total of 1508 compounds (of which 199 are classified as being hazardous). The DSS is designed to predict EU risk phrases. (Q)SARs might not enable replacement by themselves, but they are a valuable tool for screening and for prioritisation. Further develop (Q)SARs and/or expert system rulebases for skin irritation is recommended.
References
Barratt, M.D. (1996a). Quantitative structure-activity relationships for skin irritation and corrosivity of neutral and electrophilic organic chemicals. Toxicology in Vitro 10, 247-256. Hayashi, H., Nakamura, Y., Higashi, K., Kato, H., Kishida, F. & Kaneko, H. (1999). A quantitative structure-Activity relationship study of the skin irritation potential of phenols. Toxicology in Vitro 13, 915922.
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Smith, J.S., Macina, O.T., Sussman, N.B., Luster, M.I. & Karol, M.H. (2000). A robust structure-activity relationship (SAR) model for esters that cause skin irritation in humans. Toxicological Sciences 55, 215222. Cronin, M.T.D, Dearden J.C., Worth, A.P., Walker, J.D. In press. QSARs for predicting environmentalhuman health interactions. Environmental & Toxicological Chemistry. Hulzebos, E.M., Maslankiewicz, L., Walker, J.D. (2003) Verification of literature-derived SARs for skin irritation and corrosion. QSAR Comb Sci 22, 351-363. Patlewicz, G., Rodford, R., Walker, J.D. In press. QSARs for predicting skin and eye irritation. Environmental & Toxicological Chemistry. Cronin, M.T.D., Jaworska, J.S., Walker, J.D., Comber, M.H.I., Watts, C.D. & Worth, A.P. (2003) Use of QSARs in International Decision-Making Frameworks to predict health effects of chemical substances. Environmental Health Perspectives 10, 1391-1402. Gerner, I., Graetschel, G., Kahl, J., Schlede, E. (2000a) Development of a decision support system for the introduction of alternative methods into local irritancy/corrositivity testing strtaegies. Development of a relational database. ATLA 28, 11-28. Gerner, I., Zinke, S., Graetschel, G., Schlede, E. (2000b) Development of a decision support system for the introduction of alternative methods into local irritancy/corrosivity testing strategies. Creation of fundamental rules for a decision support system. ATLA 28, 665-698. Zinke, S., Gerner, I., Schlede, E.(2000) Local irritation/corrosion testing strategies: development of a decision support system for the introduction of alternative methods. ATLA 28, 29-40.
3. Identified steps or tests with no alternative methods available The in vitro methods under validation only enable acute (single application) effects to be studied. Models/protocols have yet to be developed for evaluating adverse effects following repeated exposure and for determining the reversibility of these effects. Furthermore, methods/protocols which measure the inflammatory response as well as the time course of an irritant reaction are only at research level. Information on these aspects is key to the overall risk assessment and at present can only be derived from animal experiments, or is based on knowledge about skin irritation in humans which is still rather limited. 4. Summary of alternative methods which are currently available and foreseeable time to achieve peer reviewed validation The human skin model assays (e.g. EpiDerm and EPISKIN) and the mouse SIFT appear to be the most promising in vitro methods for skin irritation testing (Zuang et al., 2002; Portes et al., 2002; Heylings et al., 2003) (see table 2). The forthcoming ECVAM validation study may determine whether any of these methods can adequately distinguish acute skin irritants from non-irritants for classification and labelling purposes (20042005).
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However, there is a need to develop mechanistically based endpoints that are more predictive of skin irritation than are simple cytotoxicity determinations. The existing in vitro models also need to be improved, so that they are better representative of the skin in vivo. For this purpose, more resources should be provided for research aimed at the identification of new markers for skin irritation, and on-going activities should be coordinated, with a view to identify new promising toxicological endpoints and develop new toxicity tests for validation. This research should be undertaken in parallel with the validation of existing test protocols for hazard identification. One approach to this research is through the application of genomics and proteomics which should facilitate the identification of specific biomarkers and the development of new, more predictive, in vitro methodologies. This promising approach is being progressed in the on-going COLIPA skin irritation research programme.
Key references
Basketter, D. A., Chamberlain, M., Griffiths, H. A., Rowson, M., Whittle, E., York, M. (1997). The classification of skin irritants by human patch test. Food and Chemical Toxicology 35, 845-852. Botham, P. A., Earl, L. K., Fentem, J. H., Roguet, R., van de Sandt, J. J. M. (1998) Alternative methods for skin irritation testing: the current status. ECVAM Skin Irritation Task Force Report 1. ATLA 26, 195-211. COLIPA (1995) Cosmetic Product Test Guidelines for the Assessment of Human Skin Compatibility. COLIPA (1997) Test Guidelines for the Assessment of Human Skin Tolerance of Potentially Irritant Cosmetic Ingredients. Faller C., Bracher M., Dami N., Roguet R. (2002). Predictive ability of reconstructed human epidermis equivalents for the assessment of skin irritation of cosmetics. Toxicology in Vitro 16, 557-572. Fentem, J. H., Briggs, D., Chesn , C., Elliott, G. R., Harbell, J. W., Heylings, J. R., Portes, P., Roguet, R., van de Sandt, J. J. M., Botham, P. A. (2001). A prevalidation study on in vitro tests for acute skin irritation: results and evaluation by the Management Team. Toxicology in Vitro 15, 57-93. Fentem, J.H. and Botham, P.A. (2002). ECVAM's activities in validating alternative tests for skin corrosion and irritation. ATLA 30, Suppl. 2, 61-67. Heylings, J. R. Clowes, H.M., Hughes L. (2001). Comparison of tissue sources for the skin integrity function test (SIFT). Toxicology in Vitro 13, 597-600. Heylings J.R., Diot S., Esdaile D.J., Fasano W.J., Manning L.A., Owen H.M. (2003). A prevalidation study on the in vitro skin irritation function test (SIFT) for prediction of acute skin irritation in vivo: results and evaluation of ECVAM Phase III. Toxicology in Vitro 17, 123-138. OECD (2002). OECD test guidelines 404: acute dermal irritation/corrosion. Paris, France: OECD. Ponec M. and Kempenaar J. (1995). Use of human skin recombinants as an in vitro model for test the irritation potential of cutaneous irritants. Skin Pharmacol 8, 49-59. Portes P., Grandidier M.-H., Cohen C., Roguet R. (2002). Refinement of the EPISKIN protocol for the assessment of acute skin irritation of chemicals: follow-up to the ECVAM prevalidation study. Toxicology in Vitro 16, 765-770. Robinson, M. K., Osborne, R., Perkins, M. A. (2000). In vitro and human testing strategies for skin irritation. Annals of the New York Academy of Sciences 919, 192-204. van de Sandt J., Roguet R., Cohen C., Esdaile D., Ponec M., Corsini E., Barker C., Fusenig N., Liebsch M., Benford D., de Brugerolle de Fraissinette A., Fortasch M. (1999). The use of keratinocytes and human skin models for predicting skin irritation. ATLA 27, 723-743.
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Worth, A.P. & Balls, M. (2002). Editors. Alternative (Non-animal) Methods for Chemicals Testing: Current Status and Future Prospects. A report prepared by ECVAM and the ECVAM Working Group on Chemicals. ATLA 30, Suppl. 1. Zuang, V. Balls, M., Botham, P.A., C oquette, A., Corsini, E., Curren, R.D., Elliott, G.R., Fentem, J.H., Heylings, J.R., Liebsch, M., Medina, J., Roguet, R., van de Sandt, H., Wiemann, C., Worth, A.P. (2002). Follow-up to the ECVAM Prevalidation Study on In Vitro Tests for Acute Skin Irritation. ECVAM Skin Irritation Task Force Report 2. ATLA 30, 109-129.
SKIN CORROSION 1. Inventory of methods currently available Skin corrosion tests assess the potential of a substance to cause irreversible damage to skin; namely, visible necrosis through the epidermis and into the dermis, following the application of a test substance for up to four hours. Corrosivity is not a feature one expects to occur with cosmetics, but occasionally could occur after a manufacturing mistake or misuse by the consumer. On the other hand, a cosmetic ingredient that has the intrinsic property to be corrosive, is not necessarily excluded for use in cosmetics. It very much depends on its final concentration in the cosmetic product, the presence of "neutralising" substances, the excipients used, the exposure route, the conditions of use, etc. In the past skin corrosion was assessed using animal studies such as OECD TG 404, but since recently three alternative methods have been included in the Annex V of the Dangerous Substances Directive and are proposed as an OECD draft testing guideline. Moreover, recent refinement provisions included in the OECD testing guideline 404 (OECD, 2002a), recommend, prior to undertake the described in vivo test for corrosion/irritation of the substance, to perform a sequential testing strategy (see point 1 under skin irritation).
2. Alternative methods currently available In vitro methods for skin corrosion The current status of in vitro alternative methods for skin corrosion is summarised in table 1. Three methods were validated (Fentem et al., 1998; Liebsch et al., 2000; ECVAM, 1998, 2000) and included in Annex V of the Dangerous Substances Directive (Directive 67/548/EEC, 2000). These are : - The in vitro skin corrosion rat skin transcutaneous electrical resistance (TER) test, which uses excised rat skin as a test system and its electrical resistance as an endpoint.
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- The human skin model tests such as EPISKIN and EpiDerm , which are reconstructed human epidermal equivalents and use the cell viability (MTT-test) as an endpoint. The CorrositexTM test, which uses penetration of test substances through a hydrogenated collagen matrix (biobarrier) and supporting filter membrane, represents another corrosivity test which was considered to be useful only for acids, bases and their derivates (ECVAM, 2001; NIH, 1999). Although it passed the ECVAM Scientific Advisory Committee (ESAC), it has not been taken up in the EU legislation. It is nevertheless a legal test adopted by the US Department of Transport (US DOT) and a draft Test Guideline In vitro Membrane Barrier Test Method for Skin Corrosion, which is based on the CorrositexTM test method, has been submitted to the OECD for Member Countries considerations. A draft Test Guideline (TG) on in vitro tests for skin corrosion was submitted to the OECD in late 1998, for consideration by the OECD Member Countries. Following a number of commenting rounds, an expert meeting, held on 1-2 November 2001 in Berlin, agreed that the draft TG on in vitro skin corrosion should be divided into two separate TGs: a draft proposal for a new TG 430 on the TER test (not restricted to the rat skin TER test) and a draft proposal for a new TG 431 on the human skin model test. The new TG 430 and TG 431 were accepted by OECD Member Countries in June 2003 and have been sent to the OECD Council for final approval.
TM
TM
Structure-activity relationships for skin corrosion Various SARs for skin corrosion have been reported by Barratt and colleagues (Barratt, 1996a; Barratt, 1996b; Whittle et al., 1996; Barratt et al., 1998). On the whole, the SARs presented in these studies take the form of principal component (PC) plots, which are based on physicochemical properties and show a separation between corrosive (C) and non-corrosive (NC) chemicals. Explicit classification models were not presented. Rather than modelling a heterogeneous group of chemicals, separate analyses were performed for acids, bases, electrophiles and neutral organics (defined as uncharged molecules which lack the potential to react covalently and which do not ionise under biological conditions [Martin Barratt, personal communication]). The most recent presentation of this approach is given in Barratt et al. (1998). In addition to PC analyses, discriminant analysis and neural network analysis were also applied to a group of neutral and electrophilic chemicals (Barratt, 1996a), and to the acids, bases and phenols (Barratt, 1996b). Finally, in another study (Barratt et al., 1996), PC plots for acids were based not only on physicochemical properties, but also on in vitro cytotoxicity measurements in mouse 3T3 cells. More recently, it was shown that a heterogeneous set of organic chemicals could be predicted as C or NC on the basis of melting point (Mpt) and molecular weight (MW) (Worth, 2000), according to the following PM: If Mpt ? 37 ?C and MW ? 123 g/mol, predict as C; otherwise predict as NC.
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Similar rules have been developed by Gerner and colleagues, who have incorporated a system of decision rules into an expert system used by the German BgVV (Gerner et al., 2000a, 2000b; Zinke et al., 2000). An example is the following PM: If MW > 1200 g/mol, then the substance has no local toxic effects.
Tiered testing strategies for skin corrosion In 2002, a supplement to the OECD test guideline 404 for dermal irritation and corrosion was included. In this supplement a sequential (stepwise) testing strategy for hazard identification is recommended which allow for the best practice and ethical benchmark before undertaking in vivo experimentation. Some general refinement provisions have been introduced (OECD, 2002a). The evaluation of a two-step strategy, based on the sequential use of pH measurements and in vitro data, indicated that the use of pH data in addition to TER or EPISKIN data, improves the ability to predict corrosion potential (Worth and Cronin, 2001a). An evaluation of a three-step strategy, based on the sequential use of QSARs, pH measurements and in vitro data, indicated that tiered approaches provide an effective means of classifying chemicals, while at the same time reducing and refining the use of animals (Worth et al., 1998). A study carried out by ECVAM confirmed the usefulness of pH as a predictor of skin corrosion potential, and provided a new PM for identifying chemicals that are corrosive by a pH-dependent mechanism (Worth and Cronin, 2001b). In the EU, a tiered testing strategy for skin corrosion/irritation is being proposed for incorporation into Annex V of Directive 67/548/EEC. This could be achieved by means of the 29 th Adaptation to Technical Progress (ATP) of the directive (Juan Riego-Sintes, personal communication).
3. Future prospects and recommendations Alternative methods for skin corrosion have been validated and accepted for regulatory use in the EU and the OECD Member Countries, so animal testing should not be performed for this endpoint. The hazard identification (classification and labelling) of skin corrosives should be based on the use of a pH test, where appropriate, and an in vitro test (rat skin TER assay, human skin model assay or, for qualifying test chemicals, CORROSITEX). For risk assessment (dose-response investigations, coupled with assessments of skin irritation potential at doses negative in skin corrosion tests), the rat skin TER or a human skin model assay are recommended for use. As a further recommendation is the validation of QSARs and/or expert system rulebases for skin corrosion.
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Key references
Barratt, M.D. (1996a). Quantitative structure-activity relationships for skin irritation and corrosivity of neutral and electrophilic organic chemicals. Toxicology in Vitro 10, 247-256. Barratt, M.D. (1996b). Quantitative structure-activity relationships (QSARs) for skin corrosivity of organic acids, bases and phenols: principal components and neural network analysis of extended datasets. Toxicology in Vitro 10, 85-94. Barratt, M.D., Dixit, M.B., Jones, P.A. (1996). The use of in vitro cytotoxicity measurements in QSAR methods for the prediction of the skin corrosivity potential of acids. Toxicology in Vitro 10, 283-290. Barratt, M.D., Brantom, P.G., Fentem, J.H., Gerner, I., Walker, A.P., Worth, A.P. (1998). The ECVAM international validation study on in vitro tests for skin corrosivity. 1. Selection and distribution of the test chemicals. Toxicology in Vitro 12, 471-482. Directive 67/548/EEC (2000). Annex I to Commission Directive 2000/33/EC adapting to technical progress for the 27th time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labeling of dangerous substances. Official Journal of the European Communities L136, 91-97. EC (2000). Annex I to Commission Directive 2000/33/EC adapting to technical progress for the 27th time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labeling of dangerous substances. Official Journal of the European Communities L136, 91-97. ECVAM (1998). ECVAM News & Views. ATLA 26, 275-280. ECVAM (2000). Statement on the application of the Epiderm human skin model for skin corrosivity testing. ATLA 28, 365-366. ECVAM (2001). Statement on the application of the CORROSITEX assay for skin corrosivity testing. ATLA 29, 96-97. Gerner, I., Graetschel, G., Kahl, J., Schlede, E. (2000a). Development of a decision support system for the introduction of alternative methods into local irritancy/corrosivity testing strategies. Development of a relational database. ATLA 28, 11-28. ICCVAM (2002) OECD Guideline for the Testing of Chemicals. Proposal for a New Guideline. In Vitro Membrane Barrier Test System for Skin Corrosion. 16pp. NC, USA: ICCVAM, NIEHS. Liebsch, M., Traue, D., Barrabas, C., Spielmann, H., Uphill, P., Wilkins, S., Wiemann, C., Kaufmann, T., Remmele, M. & Holzhtter, H. G. (2000). The ECVAM prevalidation study on the use of EpiDerm for skin corrosivity testing. ATLA 28, 371-401. Gerner, I., Zinke, S., Graetschel, G., Schlede, E. (2000b). Development of a decision support system for the introduction of alternative methods into local irritancy/corrosivity testing strategies. Creation of fundamental rules for a decision support system. ATLA 28, 665-98. Fentem, J. H., Archer, G. E. B., Balls, M., Botham, P. A., Curren, R. D., Earl, L. K., Esdaile, D. J., Holzhtter, H. G. & Liebsch, M. (1998). The ECVAM international validation study on in vitro tests for skin corrosivity. 2. Results and evaluation by the Management Team. Toxicology in Vitro 12, 483-524. NIH (1999). Corrositex : an in vitro test method for assessing dermal corrosivity potential of chemicals. NIH Publication No. 99-4495. Research Triangle Park, NC, USA: NIEHS. NIH (2002). ICCVAM evaluation of EPISKIN, EpiDerm (EPI-200), and the rat skin transcutaneous electrical resistance (TER) assay: in vitro test methods for assessing dermal corrosivity potential of chemicals. NIH Publication No. 02-4502. Research Triangle Park, NC, USA: NIEHS. OECD (2002a). OECD test guidelines 404: acute dermal irritation/corrosion. Paris, France: OECD. OECD (2002b) OECD Guideline for the Testing of Chemicals 430. In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER). 11pp. Paris, France: OECD.
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OECD (2002c) OECD Guideline for the Testing of Chemicals 431. In Vitro Skin Corrosion: Human Skin Model Test. 8pp. Paris, France: OECD. Whittle, E.G., Barratt, M.D., Carter, J.A., Basketter, D.A., Chamberlain, M. (1996). The skin corrosivity potential of fatty acids: in vitro rat and human skin testing and QSAR studies. Toxicology in Vitro 10, 95100. Worth A.P., Fenterm J.H., Balls M., Botham P.A., Curren R.D., Earl L.K., Esdaile D.J., Liebsch M. (1998). An evaluation of the proposed OECD testing strategy for skin corrosion. ATLA 26, 709-720. Worth A.P. and Cronin, M.T.D. (2000). Embedded cluster modelling: a novel QSAR method for generating elliptic models of biological activity. In Progress in the Reduction, Refinement and Replacement of Animal Experimentation (eds. M. Balls, A-M. van Zeller, M.E. Halder). pp. 479-491. Elsevier Science, Amsterdam. Worth, A.P. and Cronin, M.T.D. (2001a). The use of bootstrap resampling to assess the uncertainty of Cooper statistics. ATLA 29, 447-459. Worth, A.P. and Cronin, M.T.D. (2001b). The use of pH measurements to predict the potential of chemicals to cause acute dermal and ocular toxicity. Toxicology 169, 119-131. Zinke, S., Gerner, I., Graetschel, G., Schlede, E. (2000). Local irritation/corrosion testing strategies: development of a decision support system for the introduction of alternative methods. ATLA 28, 29-40.
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4. Summary of the currently available alternative methods and foreseeable time to achieve ESAC endorsement Table 2. Summary of validated and most advanced alternative methods for skin corrosion and skin irritation, respectively
Current endpoints addressed in the animal test Alternative tests available In vitro endpoints measured Purpose Area of application Validation Status Regulatory acceptance Comments Estimated time to have the method validated (ESAC endorsement)* 0 years
Skin corrosion: Full thickness destruction of skin tissue
Rat skin transcutaneous electrical resistance (TER) assay
Stratum corneum integrity and barrier function
Replacement
general; additional dye binding step for surfactants and solvents
Validated
EPISKIN human skin model (commercial system)
Cytotoxicity
Replacement
general; a few materials may interfere with MTT reduction
Validated
EpiDerm human skin model (commercial system)
Cytotoxicity
Replacement
general; a few materials may interfere with MTT reduction
Validated
accepted by regulatory authorities as a replacement test in the EU (B.40), but only as a screen for positives in the US; OECD TG 430 accepted by regulatory authorities as a replacement test in the EU (B.40), but only as a screen for positives in the US; OECD TG 431 accepted by regulatory authorities as a replacement test in the EU (B.40), but only as a screen for positives in the US; OECD TG 431
0 years
0 years
19
CORROSITEX (commercial system)
//
Replacement (only for acids and bases and their derivatives)
mainly acids, bases and derivatives
Validated
validated and endorsed (US and EU) method for skin corrosion testing of acids and bases; proposal for a new OECD TG prepared by the US Covers one component of skin irritation (cytotoxicity) Covers one component of skin irritation (cytotoxicity)
0 years
Skin irritation: Erythema/eschar and/or oedema
EPISKIN human skin model (commercial system) EpiDerm human skin model (commercial system) Mouse skin integrity function test (SIFT)
Cytotoxicity
Replacement a/ Partial replacementb Replacement a/ Partial replacementb Replacement a/ Partial replacementb
Cytotoxicity
Stratum corneum barrier function and integrity
general; a few materials may interfere with MTT reduction general; a few materials may interfere with MTT reduction General; a few materials may interfere with the test system
Under validation (ECVAM) Under validation (ECVAM) Under validation (ECVAM)
2 years
2 years
2 years
* This table estimates the time needed to achieve ESAC endorsement for individual alternative tests assuming optimal conditions. It does not indicate the time needed to achieve full replacement of the animal test, nor does it include the time needed to achieve regulatory acceptance. Optimal conditions means that all necessary resources, for example technical, human, financial and coordination, are met at all times in the process and that the studies undertaken have successful outcomes.
a
Replacement for hazard identification (classsification & labelling) Partial replacement (Reduction) for risk assessment purposes,
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Conclusions from Table 2 The ECVAM validation study, if successful, may result in validated alternative methods which will enable partial replacement (reduction) for risk assessment purposes. If the study runs 2004-2005, we may have an international regulatory position by 2007/2008 (based on recent experience of how long these things take). A more technical problem we might face, is the lack of clarity on when the GHS for classification will be introduced and how this could further adversely impact timelines for regulatory acceptance. Moreover, for total replacement, we need to address the reversibility aspect, and doseresponse/risk assessment based solely on in vitro/non-animal methods. The experts are not aware of much research on these elements. The current R&D activities are focused on improving the human skin models (closer to human skin) and identifying better endpoints/predictive markers of human skin irritation using molecular techniques (genomics, proteomics, etc.). These are also important building blocks for a new, integrated approach, to risk assessment of chemicals. This represents a considerable challenge which will keep us occupied well beyond 2009. Finally, we should consider discussing the opportunity side of needing to use alternative techniques for skin irritation. Recently, a lot of thinking has gone into how to improve risk assessments for skin sensitisation by investigating dose-response relationships in hazard tests (e.g. the local lymph node assay) as a measure of potency. The same principle should be considered for irritation, at the moment, we're still working mainly on what happens to a neat test chemical . We should perhaps use the "higher throughput" potential of in vitro assays to do more dose-response studies.
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Subgroup 3. Eye Irritation

Chantra Eskes1, Sandrine Bessou2, Leon Bruner3, Rodger Curren4, John Harbell4, Penny Jones5, Reinhard Kreiling6, Manfred Liebsch7, Pauline McNamee8, Wolfang Pape 9, Menk K. Prinsen10, Troy Seidle 11, Philippe Vanparys12, Andrew Worth1, Valerie Zuang 1
1
ECVAM-JRC, Ispra, Italy; 2IRPF, France; 3Gillette, Needham, USA; 4IIVS, Gaithersburg, USA; 5Unilever, Bedfordshire, UK; 6Clariant, Sulzbach/Ts., Germany; 7 ZEBET, Berlin, Germany; 8P&G, Surrey, UK; 9Beiersdorf, Hamburg, Germany; 10 TNO, Zeist,Holland; 11PETA, Kitchener, Canada; 12J&J, Beerse, Belgium
Contents
1. Inventory of methods currently available 2. In vivo mechanisms of eye irritation 3.Alternative methods currently available to the Draize rabbit eye test 3.1. Isolated organs 3.1.1. Bovine Corneal Opacity and Permeability (BCOP) Test 3.1.2. Isolated Rabbit Eye (IRE) 3.1.3. Chicken Enucleated Eye Test (CEET) 3.2. Organotypic Methods - Chorio-allantoic membrane methods 3.2.1 Hen`s egg test on the chorio-allantoic membrane (HET-CAM Assay) 3.2.2. Chorioallantoic membrane vascular assay (CAMVA) 3.2.3. Chorioallantoic membrane - trypan blue staining (CAM-TB) 3.3. Human corneal epithelium models 3.3.1.The EpiOcularTM assay (ET50-based assay) 3.3.2. The SkinEthic in vitro reconstituted human corneal epithelium (HCE model) 3.3.3. HCE-T Tissue Construct (Gillette) 3.4. Cell based cytotoxicity methods 3.4.1. Neutral Red Uptake 3.4.2. Neutral Red Release Assay 3.4.3. Red blood cell (RBC) haemolysis test 3.5. Cell function based assays 3.5.1. Fluorescein leakage (FL) 3.5.2. Silicon Microphysiometer (SM) or Cytosensor Microphysiometer 3.6. Other assays 3.6.1. Mucosal irritation model: using slugs (e.g. Arion lusitanicus) 3.6.2. The IRRITECTION assay 3.6.3. The Pollen Tube Growth (PTG) assay 3.7. The Low Volume Eye Test (LVET) 3.8. Structure-activity relationships for eye irritation 3.9. The COLIPA Eye Irritation Research Programme
4. Mechanisms of eye irritation occurring in vivo as modelled by in vitro assays 5. Summary of the alternative methods currently available and foreseeable time to achieve validation (ESAC endorsement) 6. Future prospects and recommendations for achieving animal replacement 7.References
1. Inventory of methods currently available The eye can be exposed to cosmetic products and their ingredients either through use of products such as those meant to be used around the eyes (e.g. mascaras, eye creams) or through accidental exposure to products that may enter the eye in diluted form during normal use but are not meant to come into contact with the eye undiluted e.g. shampoos. As such, the evaluation of eye irritation potential for a cosmetic product and its ingredients is essential to provide reassurance that a product is safe for consumers to use through intended and foreseeable uses and accidental exposures to the eye. The conventional test for the irritant and corrosive potential of chemicals is the rabbit eye test which was developed by Draize et al. (1944) and has become the international standard assay for acute eye irritation and corrosion (OECD TG 405, 2002; EC B.5). The test material is applied to the conjunctival sac of the eye of the animal and subsequent physiological responses are classified by careful visual examination of the cornea, iris and conjunctiva according to a numerical scoring system. A variety of different scoring systems assessing the extent of injury to the corneal, the iridial and the conjunctival compartments is currently applied in different regulations ranging from maximum single tissue scores to averaged weighed sum scores of all tissues (cf. Fisher et al., 1995; Chambers et al., 1993; Wilhelmus, 2001). Whereas for the safety assessment of cosmetics the weighed sum scores MAS (maximum average score) and MMAS (modified maximum average score, i.e. not including the results obtained 1 hr after treatment) are still in use, recently the OECD and United Nations together with other international regulatory authorities have agreed on a Globally Harmonised System of Classification and Labelling of Chemicals (GHS) (UN 2003). This is based on averaged single tissue observations taking into account the reversibility of observed effects. The Draize test serves four major goals: a) identification of severely irritant and corrosive substances (according to EU classification R 41), b) testing moderate eye irritation potential (according to EU classification R 36), c) safety assessment of cosmetics, and d) safety assessment of ophthalmic pharmaceutical drugs. The test can be very painful to rabbits and has two other major shortcomings: subjectivity in allocation of the respective scores, in other words low interlaboratory reproducibility (Weil and Scala, 1971; cf. Lordo et al., 1999; cf. Ohno et al., 1999), and differences in sensitivity to tested substances between rabbit and man (e.g. Christian and Diener, 1996). Some modifications have been proposed over the years to reduce the pain to be endured by the animal and achieve higher rates of recovery, such as in the low volume eye test (LVET, Freeberg et al., 1986), where only a tenth of the sample volume used in the standard Draize test is applied to the rabbit's cornea and the eyes not held shut after instillation of the test material. Moreover reduction and refinement approaches are proposed by the OECD sequential testing strategy now included as a supplement to OECD TG 405 (OECD, 2002), where the in vivo rabbit eye test needs only to be performed as a last step, i.e. when the assessment on all other tiers has produced a negative result, to assess mildly to moderately irritant compounds. However this strategy does not eliminate the need for an in vivo test and there is a potential for overclassification of eye irritancy potential of chemicals.
2.
In vivo mechanisms of eye irritation
Maurer, Jester and others (see review in Maurer et al, 2002) have examined the in vivo mechanistic basis for ocular irritation using different chemical classes comprising surfactants (anionic, cationic, and non-ionic) acids, alcohol, aldehyde, alkali and bleaches. They have shown that depth of injury to the cornea, in the early hours after exposure (generally 3 hours), is predictive of the eventual degree and duration of the ocular lesions in the rabbit. Generally slight irritants affect only the superficial corneal epithelium, the mild and moderate irritants affect principally the epithelium and superficial stroma, and the severe irritants act through to deeper parts of the stroma potentially as far as full stromal depth. The reactive chemistries (e.g., bleaches) showed a more delayed onset of toxicity and showed the need for evaluation of depth of injury one day after exposure. The authors also concluded that the depth of injury measurements were more consistent for each test material, over time, than were the macroscopic tissue scores, and that reversibility of the lesions was correlated with the initial depth of the injury. The link between depth of injury (cellular lesions) and macroscopic observations provides a mechanistic basis for the development and validation of ex vivo/in vitro assays for the prediction of depth of injury (depth of cytotoxicity) within the cornea. While conjunctival responses generally precede corneal responses, corneal injury is associated with the moderate and severe irritation responses that require labelling under regulatory guidelines. Amongst the factors that might influence the kinetics of injury to the cornea, the first might be the inherent cytotoxicity potential of the material. For example, those materials that lyse membranes or coagulate proteins should produce immediate cytotoxicity. Surfactants and some organic solvents would be examples of such materials. Those materials that act on cellular energy metabolism may act rapidly but perhaps not as fast as the first group. Materials may also act by alkylation or other action on macromolecules. This type of action would show a delayed cytotoxicity (e.g., delayed necrosis or apoptosis). In the in vivo eye irritation assay, the exposure to the cornea (and conjunctiva) is topical. Actual duration of the exposure is unknown (and probably variable across animals) but might be measured in a few minutes to many hours. Exposure to the deeper cell layers will be dependent on the speed of and ability to penetrate and/or fix to the surface layers before being flushed from the eye. It should also be understood that time required to manifest the cytotoxicity is not the same as the exposure time required for the cells to become injured and thus committed to cell death. Those materials that injury the cells rapidly may be more effective in producing cytotoxicity in the eye.
3. Alternative methods currently available to the Draize rabbit eye test Success in developing and validating alternative tests to replace the Draize rabbit eye irritation test has remained elusive despite major efforts by the European Centre for the Validation of Alternatives (ECVAM), industry trade associations, individual companies and academia. Six major validation or evaluation studies took place between 1991 and 1997. These were the EC/HO study (Balls et al., 1995a), COLIPA study (Brantom et al., 1997), BGA/BMBF study (Spielmann et al., 1993, 1996), CTFA study (Gettings et al., 1991, 1992, 1994, 1996), IRAG study (Bradlaw et al., 1997) and the MHW/JCIA study (Ohno et al., 1999). The outcome of each of these studies was summarised by Balls et al. (1999). No test was found capable of replacing the Draize rabbit eye test, but some of the assays showed considerable promise as screens for ocular irritancy. The main reason for this is the difficulty of comparing in vitro test results with historical animal data where the subjective scoring of tissue lesions in the eye in the Draize test provides variable estimates of eye irritancy. Other possible contributing reasons for the outcomes of recently completed validation studies are: a) the in vitro tests only partially modelled the complex in vivo eye irritation response, b) the protocols and PMs might have been insufficiently developed, and c) the choice of statistical approaches for analysing the data might not have been appropriate (Balls et al., 1999). Though not formally validated, the usefulness of these in vitro/ex vivo methods is well established within some national regulatory agencies and within industry for specific and limited purposes (Worth and Balls, 2002). Confidence for such in-house use of some of the currently available in vitro/ex vivo methods is dependent on the availability of appropriate benchmarks, historical information on similar materials, an understanding of the limitations of the assay(s) and the technical expertise of the user. For these reasons, such use of in vitro/ex vivo methods is often company-specific and for cosmetic products manufacturers more often related to finished product testing. The alternative methods to eye irritation comprise organotypic models such as isolated eyes or components thereof, tissue and cell culture systems and physicochemical tests (see reviews e.g. by Christian and Diener, 1996; Chamberlain et al., 1997; Spielmann, 1997). This document presents those alternative methods that are currently the most developed and the most widely used (for more details see AnimAltZebet database, 2001). 3.1. Isolated organs The basis of these systems is the in vitro/ex vivo exposure to chemical irritants of isolated eyes, isolated corneas or lenses obtained from for instance bovine, porcine, chicken or rabbit origin. Freshly obtained eyes, corneas or lenses obtained from slaughterhouses or from animals used for other toxicological testing are placed in special (superfusion) devises that allow maintenance for a specified period of time which is sufficient for testing of the materials. During testing parameters such as corneal opacity, corneal permeability, corneal hydration, or corneal thickness can be measured with the aid of various apparatuses. The use of isolated corneas or eyes with the corneas in situ have the advantage that all layers of the cornea can be screened, i.e. epithelium, Bowmans membrane, stroma, Descemets membrane and endothelium. This can be useful to determine whether a toxic response is related to a particular
layer of the cornea, e.g. it may be endothelium or epithelium-dependent. The isolated organ (cornea) tests are generally simple techniques (once all necessary equipment has been obtained), with a number of accurate quantifiable endpoints. They employ topical application of the chemical, which theoretically allows for the assessment of a wide range of materials without need for dilution. For some of the methods using isolated eyes or lenses, testing of solids without dilution may practically be difficult. Direct histological evaluation of the tissue lesions in the cornea has been a part of the evaluation of depth and degree of injury in vivo (Maurer et al. 1996). The ex vivo tissues provide the same opportunity to assess specific lesions in the cornea and the overall depth of injury produced. Histological evaluation is now a routine endpoint in some laboratories. Experience from the EC/HO study showed that the opacity and permeability did not predict the degree or depth of injury for certain severely irritating chemicals (for example, sodium oxylate, parafluoranalin, and quinacrine). However, when the treated corneas were evaluated histologically, lesions were clearly observed that would have predicted the severity of the irritation potential (Curren et al, 1999). Certain reactive chemicals have been shown to impact the keratocytes more than the epithelium and lead to a delayed onset of full degree of irritation. Hydrogen peroxide is a good example of such a chemical (Maurer et al, 2001). In the ex vivo cornea, the same kind of lesion is observed (Swanson et al, 2003). Where a test chemical acts directly to induce substantial opacity and/or permeability (or fluorescein staining), the assessment of severe irritation potential may be made directly. However, where only limited opacity or permeability is observed with certain classes of materials (or any unknown class of materials), histology is being used to rule out lesions that do not lead directly to changes in the other endpoints. In current practice, the depth of injury in a test material is compared to that in a reference material. Ultimately, a formal prediction model will be required to link degree and depth of injury measured in the ex vivo cornea (or full thickness corneal construct) with the predicted depth and degree of injury in vivo. Such an effort is underway for a certain chemicals (Cuellar et al., 2003) and an extensive effort will be required to address a broad range of chemical classes.
3.1.1. Bovine Corneal Opacity and Permeability (BCOP) Test 1. Short description, scientific relevance, and purpose of the test By using slaughterhouse material the BCOP assay avoids the keeping and killing of laboratory animals. Freshly isolated cornea is mounted horizontally in a holder which is placed inside a specially modified opacitometer. The mounted cornea divides the test chamber into two compartments with controlled temperature and the test compound is added to the compartment enclosing the epithelial surface of the cornea (Gautheron et al., 1992; Gautheron, INVITTOX protocol no. 98, 1996). After measuring opacity, a fluorescein-containing solution is added to the epithelial side (i.e., the upper compartment) in order to determine the permeability of the cornea by assessment of the optical density (O.D.) of the medium in the lower compartment. The measured numerical values for opacity and permeability can be used to calculate a so-called in vitro score (Gautheron et al., 1994 and Sina et al, 1995). Classification of test materials can be done according to this score (Kay and Calandra, 1962). Better prediction of certain chemical classes is obtained with the addition of histological evaluation of the corneas (Curren et al, 1999).
2. Developer of the method The use of BCOP assay is based on the work of Gautheron and co-workers (Gautheron et al., 1992; Gautheron, 1996; INVITTOX protocol no. 98) and uses a fluorescent dye in an adaptation of the method described by Tchao (1988). 3. Known Users It is routinely used for safety assessment of process intermediates 'in-house' in the cosmetics and drug industries (e.g. Vanparys et al., 1993; Casterton et al., 1996 and Sina et al, 1995). The BCOP is routinely used to test chemicals and formulations associated with highly fragranced products (e.g., air fresheners), household and industrial cleaning products, laundry products, oxidizers (hair dye formulations and cleaning products), insect control (e.g., insect repellents), and personal care products (e.g., shampoos, deodorants). In France, Germany, UK, The Netherlands, Belgium and Ireland the regulatory authorities accept the BCOP assay for identifying severely irritating substances (classified as R41) when the results are positive (Worth and Balls, 2002). The BCOP has been used for registration of insect repellent formulations with the USEPA. 4. Status of validation and/or standardization The BCOP assay performed well in an interlaboratory comparison funded by the EU between 12 laboratories testing 52 substances (EEC contract ref. B91/B43081/013188, 1991; Gautheron et al., 1994): data was found to be reproducible among the participating laboratories. The correlation between the BCOP scores and the Draize MAS values was 0.73. However, four false-negatives (all solid materials) and four false-positives (3 of them liquids) were noted. The BCOP assay was also included in the EC/HO study (Balls et al., 1995a), in which, however, none of the proposed alternatives to the Draize eye test proved sufficient to serve as complete substitute. Several false negative predictions were observed with all of the ex vivo methods in this study. Curren at al, (1999) have reexamined many of these under-predicted chemicals with the addition of the histological endpoint and have shown that these severely irritating chemicals could be identified. At the same time, the BCOP assay has been assessed in the U.S.A. within the CTFA phase III study (Gettings et al., 1996); it was among the group of those 14 endpoints (out of a total of 34 in vitro endpoints investigated) showing the least discordance with results of the Draize test. Moreover, the BCOP assay has been critically appraised by the IRAG Working Group on Organotypic Models evaluating data from eight independent laboratories (Chamberlain et al., 1997). The conclusion was that the BCOP assay overall performed reasonably at identifying moderate and severely irritating materials and performed well for assigning relative potencies. 5. Fields of application and limitations The BCOP assay is amenable to testing a wide range of physical forms and solubility characteristics. It is well suited to identify substances moderately and severely irritating to the eye, but seems to be not as sensitive in distinguishing among mildly irritating materials when applying the standard protocol. Histology can be an aid in good labelling of mild irritants. There seems to be a tendency to underestimate the irritant potential of substances acting more pronounced on the iris or the conjunctivae (Casterton et al., 1996). Recently, the BCOP assay was included in a project which
evaluated several in vitro methods by applying reference standards in order to gain wider acceptance of such assays in the regulatory context (Balls, Brantom et al., 1999). 6. Recommended use in the options of animal replacement The BCOP test is very suitable to select out the moderate, severe and very severe eye irritants since the depth of injury can be measured in this ex vivo tissue model. However, the resolution of mild to very mild levels of irritancy would be better suited to an epithelial tissue construct or cytotoxicity assay. For these reasons it has been suggested to use the BCOP assay in tandem with assays measuring other endpoints such as cytotoxicity (e.g. Rachui et al., 1994; Swanson et al., 1995) or as part of a test battery (e.g. Sina and Gautheron, 1994; Harbell and Curren, 2001). A prevalidation study commissioned by the European Commission, in which three industrial laboratories were involved assessing ten substances that had been tested in the EC/HO study, resulted in a refined assay protocol capable of reducing the variability of the assay (Southee, 1998). Another recent study on shampoos proposed inclusion of histological examination as a further endpoint in order to increase the predictive power of the BCOP assay (Cooper et al., 2001). This approach has been incorporated in the study of Cuellar et al. (2002) where histological changes in the BCOP were compared with histological changes in rabbit eyes treated with the same test materials in vivo. 7. On-going development a) A new opacitometer based on laser light instead of white light has been developed and is in evaluation for its suitability to better rank mild and moderate irritants (Van Goethem, et al., in preparation) b) Improved cornea holders to maintain corneal shape c) Prediction model for histological changes associated with depth of injury in the stroma 8. Efforts needed to complete validation Development of a uniform scoring system for tissue lesions and a training atlas that describes the lesions. Development of the prediction model for depth of injury to supplement the opacity and permeability endpoints. 9. Key references
Balls M; Botham PA; Bruner LH; Spielmann H; The EC/HO international validation study on alternatives to the Draize eye irritation test. Toxicology in vitro; 9(6); 871-929; 1995a Balls M; Brantom PG; Cassidy S; Esdaile D; Fentem J; Liebsch M; McPherson J; Pfannenbecker U; Prinsen M; Preliminary evaluation of the application of reference standards in the prevalidation and validation of in vitro test for eye irritation. In: DG Clark, SG Lisansky and R Macmillan: Alternatives to animal testing II, proceedings, Brussels. CPL Press, Berkshire; 201-204; 1999 Casterton PL; Potts LF; Klein BD; A novel approach to assessing eye irritation potential using the bovine corneal opacity and permeability assay. Journal of toxicology / Cutaneous and ocular toxicology; 15(2); 147-163; 1996 Chamberlain M; Gad SC; Gautheron P; Prinsen MK; IRAG (Interagency Regulatory Alternatives Group) working group 1. Organotypic models for the assessment/prediction of ocular irritation. Food and chemical toxicology; 35(1); 23-37; 1997
Cooper KJ; Earl LK; Harbell J; Raabe H; Prediction of ocular irritancy of prototype shampoo formulations by the isolated rabbit eye (IRE) test and bovine corneal opacity and permeability (BCOP) assay. Toxicology in vitro; 15(2); 95-103; 2001 Cuellar, N., Lloyd, P.H., Swanson, J.E., Merrill, J.C., Clear, M.L., Mun, G., Harbell, J.H., and Bonnette, K.L. (2003) Evaluating the eye irritancy of solvents in a simple fragrance mixture with the bovine corneal opacity and permeability (BCOP) assay. The Toxicologist 72:312. Curren, R., Evans, M., Raabe, H., Dobson, T., and Harbell, J.(1999) Optimization of the bovine corneal opacity and permeability assay: histopathology aids understanding of the EC/HO false negative materials. ATLA 27:344. Gautheron, P., Dukic, M., Alix, D., and Sina, J.F.(1992) Bovine corneal opacity and permeability test: an in vitro assay of ocular irritancy. Fundamental and Applied Toxicology 18:442-449. Gautheron P; Giroux J; Cottin M; Audegond L; Morilla A; Mayordomo-Blanco L; Tortajada A; Haynes G; Vericat JA; Interlaboratory assessment of the bovine corneal opacity and permeability (BCOP) assay. Toxicology in vitro; 8(3); 381-392; 1994 Gautheron P; The bovine corneal opacity and permeability assay. Invittox Protocol; 98; 1996 Gettings SD; Lordo RA; Hintze KL; Bagley DM; Casterton PL; Chudkowski M; Curren RD; Demetrulias JL; DiPasquale LC; Earl LK; Feder PI; Galli CL; Glaza SM; Gordon VC; Janus J; Kurtz PJ; Marenus KD; Moral J; Pape WJW; Renskers KJ; Rheins LA; Roddy MT; Rozen MG; Tedeschi JP; Zyracki J; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (phase III) Surfactant-based formulations. Food and chemical toxicology; 34(1); 79-117; 1996 Harbell, J.W. and Curren, R.D. (2001) In vitro methods for the prediction of ocular and dermal toxicity. In, Handbook of Toxicology, 2 nd Ed. M.J. Derelanko and M.A. Hollinger ED., CRC Press, Boco Raton, pp 835-866. Kay JH; Calendra JC; Interpretation of eye irritation tests. Journal of the Society of Cosmetic Chemists; 13; 281-289; 1962 Maurer, J.K., Parker, R.D., and Jester, J.V. (2002) Extent of initial corneal injury as the mechanistic basis for ocular irritation: key findings and recommendations for the development of alternative assays. Regulatory Toxicology and Pharmacology 36:106-117. Rachui SR; Robertson WD; Duke MA; Paller BS; Ziets GA; Predicting the ocular irritation potential of cosmetics and personal care products using two in vitro models. In vitro toxicology; 7(1); 45-52; 1994 Sina JF; Gautheron PD; A multitest approach to evaluating ocular irritation in vitro. Toxicology methods; 4(1); 41-49; 1994 Sina J.F., D.M. Galer, R.G. Sussman, P.D. Gautheron, E.V. Sargent, B. Leong, P.V. Shah, R.D. Curren, and K. Miller. A Collaborative Evaluation of Seven Alternatives to the Draize Eye Irritation Test Using Pharmaceutical Intermediates. Fundamental and Applied Toxicology 26:20-31, 1995. Southee JA; Evaluation of the prevalidation process. The fluorescein leakage assay. Part 2, final report, European Community contract no. 11279-95-10F1ED ISP GB.; 3; 1998 Swanson JE; Lake LK; Donnelly TA; Harbell JW; Huggins J; Prediction of ocular irritancy of fullstrength cleaners and strippers by tissue equivalent and bovine corneal assays. Journal of toxicology / Cutaneous and ocular toxicology; 14(3); 179-195; 1995 Tchao R; Trans-epithelial permeability of fluorescein in vitro as an assay to determine eye irritants. In: A.M. Goldberg (Ed.): Alternative Methods in Toxicology.Mary Ann Liebert, Inc., New York; 6; 271283; 1988
Van Goethem, F; Hansen, E; Sysmans, M.; Vanparys, Ph. Development of a new opacitometer for the bovine corneal opacity and permeability (BCOP) assay (in preparation). Vanparys P; Deknudt G; Sysmans M; Teuns G; Coussement W; Cautern H van; Evaluation of the bovine corneal opacity permeability assay as an in vitro alternative to the Draize eye irritation test. Toxicology in vitro; 7(4); 471-476; 1993 Worth AP; Balls M; Editors. Alternative (non-animal) methods for chemicals testing current status and future prospects. A report prepared by ECVAM working group on chemicals. ALTA 30; suppl.1; 2002 Zebet103. The use of bovine corneal opacity and permeability as parameters for the eye irritation potential of chemical substances (BCOP assay). AnimAlt-Zebet database; 2001 http://www.bfr.bund.de/cms/detail.php?template=internet_en_index
3.1.2. Isolated Rabbit Eye (IRE) 1. Short description , scientific relevance and purpose The isolated rabbit eye test (IRE test; Burton et al., 1981; Earl, INVITTOX protocol no. 85, 1994) determines the opacification of the cornea and the increase in corneal thickness (corneal swelling) after exposure to irritant substances. Whole eyeballs obtained by immediate dissection from humanely killed laboratory rabbits with healthy eyes are mounted and maintained in a vertical position in a so-called superfusion chamber with controlled temperature and humidity. Pre-warmed saline solution is applied drop by drop directly onto the cornea at regular intervals to keep it moist. Prior to treatment with test sample a visual check for opacity is carried out together with an evaluation of the penetration of fluorescein and the swelling of the cornea, and the damaged tissues are excluded. The eyeball is then either taken out of the chamber or left in situ (depending on the type of test material) and exposed to the test chemical; for example, 10 seconds for identification of severe irritants and 1 minute (or longer) for the ranking of less severely damaging materials/products (Whittle et al., 1992; York et al., 1994). After removal of the chemical, the eye is repositioned in the chamber and the cornea is examined for evidence of opacification along with measuring of corneal thickness. Further assessments are made at 30 min., 1, 2, 3, and 4 hours after dosing. A check on fluorescein penetration is carried out 30 min. and 4 hours after treatment. Scores for corneal opacity (similar to Draize scores) and fluorescein penetration are recorded (qualitative assessments). For each test sample the mean percentage of corneal swelling of three eyes is calculated and compared to an untreated control eye. The preparation and examination of histological sections of the treated corneas can be used to confirm the level and depth of corneal damage. Overall damage is assessed by means of a combination of the different parameters scored, depending on the nature of the effects observed and in-house classification systems may vary (Whittle et al., 1992). Chemical substances causing the cornea to swell by more than 15% have been considered to have the potential to cause severe irritation of the eye in vivo (Lewis et al., 1994), but a more complex classification model combining opacity, corneal swelling and histological observations of the corneal epithelium has been published (Cooper et al., 2001; Jones et al., 2001).
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2. Developer of the method The IRE was developed at the Unilever Environmental Safety Laboratory (now known as the Safety and Environmental Assurance Centre) by Burton and co-workers (Burton et al., 1981; INVITTOX protocol no. 85) based on a modification of previously published apparatus for incubating and examining rabbit corneas (Mishima and Kudo, 1967; Mishima, 1968; Mishima and Hedbys, 1968). 3. Known users The IRE test has been used by several laboratories (e.g. York et al., 1982, 1994; Lewis et al., 1994; Price and Andrews, 1985; Koeter and Prinsen, 1985; Berry and Easty, 1993), and is routinely used for safety assessment of process intermediates and formulations 'in-house' in the cosmetics and drug industries, as part of tiered safety evaluation strategies. In UK, Germany, France and The Netherlands the IRE test is accepted by regulatory agencies for classifying severely irritating substances when the results are positive (Worth and Balls, 2002). 4. Status of validation and/or standardization The IRE initially underwent an interlaboratory trial between three laboratories which used their own criteria to determine the overall in vitro rating of test materials (Whittle et al., 1992). This trial concluded that the predictive ability of the test to separate moderate-severe eye irritants from lesser eye irritants and the consistency of results between laboratories were good. Subsequently the IRE test has been included in the EC/HO (Balls et al., 1995a), in which none of the proposed alternatives to the Draize rabbit eye test proved sufficient as substitute. At the same time the IRE test was assessed in the U.S.A within the CTFA (phase III surfactant-based formulations; Gettings et al., 1996) producing results comparable to the other in vitro assays studied. Moreover, the IRE test has been critically evaluated by the IRAG Working Group on Organotypic Models (Chamberlain et al., 1997), which concluded that the IRE test on its own is of no practical value for predicting irritation potential across the whole irritation range, but it could be used to screen for severely irritating materials. In a more recent investigations concerning the irritation potential of shampoos and conditioners the results obtained with the IRE test (using a more complex classification system and benchmarks) proved consistent with in vivo data distinguishing between mildly and severely irritating formulations (Cooper et al., 2001; Jones et al., 2001). The latter paper also endorses the use of benchmarks in the ranking of materials. 5. Fields and limitations of application The test is probably suitable for testing most types of test material, particularly where severe effects are observed, although physical effects of solids, which may be seen in vivo, are not always apparent in vitro (where the material is static on the treated cornea). There was a wide range of chemistry in the data submitted to IRAG (Chamberlain et al., 1997). In the originating laboratory most experience and usefulness has been obtained with respect to alkaline materials (York et al., 1994) and surfactants (anionic and cationic, Cooper et al., 2001; Jones et al., 2001 and in-house unpublished data). The 10 second application time is most suitable for distinguishing severe eye irritants; longer application times (e.g., 1 minute) are more suitable for the ranking of less
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damaging materials. The IRE test primarily predicts corneal effects and does not provide information on the effects of materials on the conjunctiva of the eye or on the recovery of the cornea from damage (beyond a few hours), which might occur in the eye in vivo. Absence of damage therefore does not suggest that there would be no effects in vivo. 6. Recommendations of use in the view of animal replacement The IRE test is considered to be well suited to identify substances 'severely irritating to the eye' according to EU classification R 41. It is considered to be a valid test for the (pre)screening of severely irritating materials (Chamberlain et al., 1997). The assay is useful for in-house comparative testing of materials/formulations to obviate the need for any animal testing during product development. 7. On-going development Although the original publications on the IRE test (Burton et al., 1981; York et al., 1982) did not include the use of histology as an end point, this is now routinely used at the originating laboratory (Cooper et al., 2001; Jones et al., 2001). 8. Efforts needed to complete validation of the method 9. References
Balls M; Botham PA; Bruner LH; Spielmann H; The EC/HO international validation study on alternatives to the Draize eye irritation test.; Toxicology in vitro; 9(6); 871-929; 1995a Berry M; Easty DL; Isolated human and rabbit eye: models of corneal toxicity.; Toxicology in vitro; 7; 461-464; 1993 Burton ABG; York M; Lawrence RS; The in vitro assessment of severe eye irritants; Food and cosmetics toxicology; 19; 471-480; 1981 Chamberlain M; Gad SC; Gautheron P; Prinsen MK; IRAG (Interagency Regulatory Alternatives Group) working group 1. Organotypic models for the assessment/prediction of ocular irritation.; Food and chemical toxicology; 35(1); 23-37; 1997 Cooper KJ; Earl LK; Harbell J; Raabe H; Prediction of ocular irritancy of prototype shampoo formulations by the isolated rabbit eye (IRE) test and bovine corneal opacity and permeability (BCOP) assay.; Toxicology in vitro; 15(2); 95-103; 2001 Earl L; The rabbit enucleated eye test.; Invi ttox Protocol; 85; 1994 Gettings SD; Lordo RA; Hintze KL; Bagley DM; Casterton PL; Chudkowski M; Curren RD; Demetrulias JL; DiPasquale LC; Earl LK; Feder PI; Galli CL; Glaza SM; Gordon VC; Janus J; Kurtz PJ; Marenus KD; Moral J; Pape WJW; Renskers KJ; Rheins LA; Roddy MT; Rozen MG; Tedeschi JP; Zyracki J; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (phase III) Surfactant-based formulations.; Food and chemical toxicology; 34(1); 79-117; 1996 Jones PA; Budynsky E; Cooper KJ; Decker D; Griffiths HA; Fentem JH; Comparative evaluation of five in vitro tests for assessing the eye irritation potential of hair-care products.; ATLA; 29; 669-692; 2001 Koeter HBWM; Prinsen MK; Comparison of in vivo and in vitro eye irritancy test systems: a study with 34 substances. In: Alternative methods in toxicology.; 3; A9; 1985 Lewis RW; McCall JC; Botham PA; Use of an in vitro test battery as a prescreen in the assessment of ocular irritancy.; Toxicology in Vitro; 8; 75-79; 1994
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Mishima S; Kudo T; In vitro incubation of rabbit cornea.; Investigative Ophthalmology; 6; 329; 1967 Mishima S; Corneal thickness.; Survey of Ophthalmology; 13; 57; 1968 Mishima S; Hedbys BO; Measurement of corneal thickness with a Haag-Streit pachometer.; Archives of Ophthalmology, NY; 80; 710; 1968 Price JB; Andrews IJ; The in vitro assessment of eye irritancy using isolated eyes.; Food and chemical toxicology; 23; 313-315; 1985 York M; Lawrence RS; Gibson GB; An in vitro test for the assessment of eye irritancy in consumer products - preliminary findings.; International journal of cosmetic science; 4; 223-234; 1982 York M; Wilson AP; Newsome CS; The classification of soluble silicates for eye hazard using the enucleated rabbit eye test; Toxicology in vitro; 8 (6); 1265-1268; 1994 Whittle E; Basketter D; York M; Kelly L; McCall J; Botham P; Esdaile D; Gardner J; Findings of an interlaboratory trial of the enucleated eye method as an alternative eye irritation test.; Toxicology Methods; 2; 30-41; 1992 Worth AP; Balls M; Editors. Alternative (non-animal) methods for chemicals testing current status and future prospects. A report prepared by ECVAM working group on chemicals. ALTA 30; suppl.1; 2002 Zebet105. The assessment of the eye irritation potential of chemical substances using the isolated rabbit eye test (IRE test). AnimAlt-Zebet database; 2001 http://www.bfr.bund.de/cms/detail.php?template=internet_en_index
3.1.3. Chicken Enucleated Eye Test (CEET) 1. Short description , scientific relevance and purpose The chicken enucleated eye test (CEET) is prepared by using the whole eyeballs of freshly slaughtered chicken. Prior to dissection, non-damaged corneas are selected by checking fluorescein retention of the corneal epithelium and corneal opacity. Selected intact eyes are mounted and maintained in a vertical position in a so-called superfusion chamber with controlled temperature and humidity. Before applying the test sample, each eye selected is scored for baseline measurement of corneal thickness, corneal opacity and fluorescein retention. Each eyeball is then taken out of the chamber and the cornea is exposed to the test chemical for 10 seconds. After rinsing off the test chemical, the eye is repositioned in the chamber and the cornea is examined for evidence of changes in corneal thickness, corneal opacification and fluorescein retention of damaged epithelium. Morphological effects on the cornea such as loosening of the epithelium are also recorded. The assessments are made at 30, 75, 120, 180 and 240 minutes after treatment. Evidence of fluorescein penetration is checked for only once at 30 minutes after exposure. Eyes are sampled in formaldehyde for possible histopathology of the cornea (depth-of-injury). The mean percentage corneal swelling of three eyes per test sample is calculated; each eye is serving as its own control. Scores for corneal swelling, corneal opacity and fluorescein retention are recorded as toxicological endpoints and mean maximum scores are used to classify materials from non-irritating to severely irritating and to calculate a numerical CEET irritation index, which ranges from 0 to 200 (Prinsen and Koter, 1993; Prinsen 1996). 2. Developer of the method 13
The CEET was developed at the Dutch Organization for Applied Science (TNO) by Prinsen and Koter (1993; Prinsen, INVITTOX protocol no. 80, 1994) and is based on the isolated rabbit eye test described by Burton et al. (1971 and 1981). 3. Known users In France and Germany, the CEET test is accepted for the classification of severely irritating substances. In The Netherlands it is accepted as a (pre)screen to identify substances 'severely irritating to the eye (R41)' but not as a stand-alone test for the classification of irritating and non-irritating substances (Worth and Balls, 2002). The CEET is used in routine by Contract Research Organisations (CROs) and industries including household, toiletries and cosmetic industry. 4. Status of validation and/or standardisation The Enucleated Eye Test was initially validated with rabbit eyes by various users inhouse (Burton, 1971; Burton et al., 1981; Koter and Prinsen, 1985; Price and Andrews, 1985; York et al., 1982) and in a first EC validation program (CEC, 1991). The use of slaughterhouse chicken eyes in the Enucleated Eye Test was first validated by the TNO Toxicology and Nutrition Institute of the Netherlands (Prinsen, 1993). Further in-house validation was performed on the basis of parallel in vivo/in vitro CEET data obtained by routine contract testing of substances from various international sponsors. As recommended by the IRAG (criteria for data submissions by the Interagency Regulatory Alternatives group of the USA, Workshop, Washington DC, November 1993) the critical values of the Draize Eye Test (e.g. individual tissue responses, various indices, days to recover etc.) were compared to the CEET critical scores. Overall correlation coefficients values of 0.82-0.91 were obtained (Prinsen, 1996). International validation was performed in the EC/HO study (Balls et al., 1995a) where interlaboratory variation was satisfactory (0.83-0.85). The general outcome of the validation was disappointing for all alternative methods tested although the organotypic models performed better than other models. For the surfactants, as with most of the alternatives, satisfactory correlations were obtained (0.72-0.83). General factors causing this failure are believed to be the variability of the in vivo data and the MMAS as the single descriptor of eye irritation (Balls et al., 1999). The CEET method is included as a standard method in the INVITTOX databank of In vitro Techniques in Toxicology (Invittox Protocol no. 80, April, 1994, Nottingham, England). 5. Fields and limitations of application Practically, all chemical classes can be assayed by topical application on the cornea. Although the assay predicts corneal effects, it also shows a high correlation with the conjunctival effects (Burton, 1971; Prinsen, 1996). 6. Recommendations of use in the view of animal replacement See point 8. 7. Ongoing development Lately, microscopic measurement of initial injury using ex vivo/ex vitro corneal equivalent systems is recognized as the principal factor determining the outcome of ocular irritation (Maurer, 2002). Since 1999, the corneas used in stand-alone CEETs carried out with predominantly cosmetic ingredients are examined microscopically, the lesions being characterized and the depth-of-injury assessed (Prinsen, unpublished
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data). These findings are taken into account for precision of the irritation potential/classification of the test material. 8. Efforts needed to complete validation In-house validation (parallel testing) has been performed sufficiently enough to justify the use of the CEET as a valid routine screen for eye irritation test in rabbits. Validation programs with existing old in vivo data have proven to be unsuccessful for various reasons mentioned in chapters 1 and 6, but at least the EC/HO validation study proved that the method travels well between laboratories. 9. References
Balls M; Botham PA; Bruner LH; Spielmann H (1995a). The EC/HO International Validation Study on Alternatives to the Draize Eye Irritation Test. Toxicology In Vitro, Volume 9, No. 6, pp. 871-929. Balls M; Berg N; Bruner LH; Curren RD; de Silva O; Earl LK; Esdaile DJ; Fentem JH; Liebsch M; Ohno Y; Prinsen MK; Spielmann H; Worth AP (1999). ECVAM Workshop Report 34, Eye Irritation Testing: The Way Forward, Appendix 1. ATLA 27, pp. 72-77. Burton, A.B.G. (1971) A method for the Food and Cosmetics Toxicology 10, 209-217. Burton, A.B.G., M. York and R.S. Lawrence (1981) The in vitro assessment of severe irritants. Food and Cosmetics Toxicology 19, 471-480. Commission of the European Communities (1991) Collaborative study on the evaluation of alternative methods to the eye irritation test. EC Document XI/632/91, V/E/1/131/91. Koter, H.B.W.M. and M.K. Prinsen (1985) Comparison of in vivo and in vitro eye irritancy test systems: A study with 34 substances, Alternative methods in Toxicology, Volume 3, Chapter A9. Mary Ann Liebert, Inc., publishers. OECD Guideline no. 405 (2002), Acute eye irritation/corrosion, updated version, adopted on 24 April 2002. Price, J.B. and I.J. Andrews (1985) The in vitro assessment of eye irritancy using isolated eyes. Food and Chemical Toxicology 23 (2), 313- 315. Prinsen, M.K and H.B.W.M. Koeter (1993). Justification of the Enucleated Eye Test with eyes of slaughterhouse animals as an alternative to the Draize Eye Irritation Test with rabbits. Food and Chemical Toxicology, Volume 31, no. 1, pp. 69-76. Prinsen M; Chicken enucleated eye test (CEET); Invittox Protocol; 80; 1994. Prinsen, M.K (1996). The Chicken Enucleated Eye Test (CEET): a practical (pre)screen for the assessment of eye irritation/corrosion potential of test materials. Food and Chemical Toxicology, Volume 34, no. 3, pp. 291-296. Worth AP; Balls M; Editors. Alternative (non-animal) methods for chemicals testing current status and future prospects. A report prepared by ECVAM working group on chemicals. ALTA 30; suppl.1; 2002. York, R.S., R.S. Lawrence and G.B. Gibson (1982) An in vitro test for the assessment of eye irritancy in consumer products. International Journal of Cosmetic Science, Volume 4, 223-234. Zebet107. The assessment of the eye irritation potential of chemical substances using the chicken enucleated eye test (CEET). AnimAlt-Zebet database; 2001 http://www.bfr.bund.de/cms/detail.php?template=internet_en_index
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3.2. Organotypic Methods - Chorio-allantoic membrane methods The chorioallantoic membrane (CAM) of the fertilized chicken egg is considered a suitable model for modelling the effects of substances on the conjunctival tissues of the eye (e.g. Christian and Diener, 1996). 3.2.1 Hen`s egg test on the chorio-allantoic membrane (HET-CAM Assay) 1. Short description, scientific relevance and purpose As with some other organotypic models, the HET-CAM test permits the identification of irritative reactions which appear to be similar to those which occur in the eye using the standard Draize rabbit eye test. In the HET-CAM test system, three reactions are determined, namely, haemorrhage, lysis and coagulation (sometimes also hyperemia is used as a parameter) of the chorio-allantoic membrane at the ninth day of embryonation when nerve tissue and pain perception have not yet developed. After placing the test sample directly onto the CAM, an evaluation of the above mentioned parameters over a 5-minute observation period takes place. The most widely used approach is the reaction time method, in which the time until the appearance of each of the three endpoints is determined. Another approach is the irritation threshold method, which determines the concentration of the test material at which effects on these parameters are first observed. Whereas these approaches are mainly used for transparent test materials, a third approach for non-transparent insoluble and solid materials can be used by exposing the CAM to test samples for a fixed time (e.g. 30 seconds or 5 minutes) and examination for a.m. endpoints after careful rinsing to remove the sample. Although different scoring systems have been developed, the original HET-CAM scale, a weighted scale in which coagulation is given a higher weight than haemorrhage and lysis, is still widely used. 2. Developer of the method The original HET-CAM test was developed by Luepke (1985, 1987) and has formed the basis for several modifications to allow testing of materials with different physicochemical properties (e.g. INVITTOX protocol No. 47; Spielmann 1992; de Silva et al. 1992; INVITTOX protocol No. 96; Steiling 1994; Gilleron et al. 1996). 3. Known users The HET-CAM test is already used within industry for identifying potential nonirritating or mildly irritating materials during in-house screening and safety evaluations of formulations and/or raw materials. It is not commonly used for hazard evaluations in the sense of labelling and classification, since it has not yet been recognized as a generally accepted alternative to the Draize rabbit eye in vivo test. However, the HET-CAM assay has been accepted already by the British, French, Dutch and German (in conjunction with the NRU cytotoxicity assay) authorities for the classification of severe irritants (J. Officiel de la Republique Francaise 1996; Schlede and Gerner 1995; Spielmann 1998). 4. Status of validation and/or standardisation The HET-CAM test has been assessed in several validation studies carried out to evaluate the potential of in vitro alternatives as replacements for the Draize rabbit eye test. These include the collaborative studies carried out by cosmetic companies (e.g.
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Bagley et al. 1992; Rougier et al. 1994), and those within the CTFA Program in the US (Gettings et al. 1991, 1994, 1996). Within Europe, independent validation studies were carried out by COLIPA (Brantom et al. 1997; Steiling et al. 1999), the German Ministry for Research and Technology BMFT in conjunction with the German Federal Health Agency BGA (Spielmann et al. 1991, 1993, 1995, 1996), the French association for the welfare of laboratory animals OPAL (Blein et al. 1991), and the interlaboratory study carried out under the auspices of the Japanese Ministry of Health and Welfare together with the Japanese Cosmetic Industry Association (e.g. Hagino et al. 1999). Additionally, the HET-CAM test was included the world-wide EC/HO validation study (Balls et al. 1995a). In the German study, the HET-CAM was evaluated in combination with the neutral red uptake assay. Since various test protocols and/or prediction models have been used in most of these validation studies, standardisation seems to be still an issue. However, this problem also refers to the existing differences in regulatory classification systems used for irritancy in various countries and/or regions. 5. Fields and limitations of application The majority of the validation studies carried out, showed a useful correlation between the HET-CAM test and the Draize rabbit eye test for the assessment of raw materials and cosmetic products. This in vivo versus in vitro correlation revealed good results in the area of mild and non-irritating test materials as well as for surfactants and surfactant-based formulations (e.g. Rougier et al. 1994; Spielmann 1997; Steiling et al. 1999). However, regression analysis showed linearity only in this lower range of irritancy but not over the whole range of Draize MAS scores (Spielmann et al. 1997). The mathematical prediction model used in the COLIPA study also showed certain limitations (Steiling et al. 1999). Although the HET-CAM assay in principle is applicable to all types of chemicals regardless of their physico-chemical properties, measurements on solid and insoluble or sticky materials may cause problems in the reproducibility of test results while pigments and dyes may cause interference by staining the CAM (e.g. Spielmann 1997; Hagino et al. 1999). Additionally, when alcohol/esters and surfactants were tested comparatively using HET-CAM and NRU, a good correlation was observed for surfactants but not for alcohols and esters (Balls, Brantom et al. 1999). Regarding the measured end-points in the in vivo eye irritation test, the HET-CAM assay should mimic the conjunctival response of the Draize rabbit eye test. Although the HET-CAM test is regarded to be an established and reliable test for screening purposes, a further potential limitation can be seen in the absence of the possibility to assess reversibility and/or irreversibility of effects. Severity of effects will be covered by the HET-CAM while for methodological reasons the recovery or persistence of effects is out of the scope of the various HET-CAM test protocols used today. For that reason, the future role of the HET-CAM assay may be restricted to be only one element in a test battery for eye irritation (see chapter 6). 6. Recommendations of use in the view of animal replacement In most cases the acceptance of HET-CAM by national authorities is related to the identification of severe eye irritants. Additionally, the HET-CAM test is an established and reliable test system for screening purposes within the cosmetics industry. As a consequence, the HET-CAM test can be regarded as an alternative which is leading to a reduction of animal experiments already. However, although it is
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widely recognized that the range of evaluation criteria covered by the Draize rabbit eye test can not be replaced by any single in vitro test, further effort to gain more regulatory acceptance of the HET-CAM test should focus on the establishment of a standardized test protocol, as well as standardized criteria against validation should take place. Further recommendations are the inclusion of well known benchmark materials or other reference substances (e.g. Steiling et al. 1999; Hagino et al. 1999), and to develop an objective scoring method, rather to continue using the current subjective one (Christian and Diener 1996). However, it should be noted that the available data base for eye irritation from the Draize rabbit eye test is also based on a subjective scoring of eye irritating effects. 7. On-going development 8. Efforts needed to complete validation of the method Development of a standardized test protocol together with an optimised prediction model, and to establish a more objective scoring system to increase interlaboratory reproducibility of HET-CAM results. 9. Key references
Christian MS; Diener RM; Soaps and detergents - alternatives to animal eye irritation tests.; Journal of the American College of Toxicology; 15(1); 1-44; 1996 Gilleron L; Coecke S; Sysmans M; Hansen E; Oproy S van; Marzin D; Cauteren H van; Vanparys P; Evaluation of a modified HET-CAM assay as a screening test for eye irritancy.; Toxicology in vitro; 10(4); 431-446; 1996 Gilleron L; Coecke S; Sysmans M; Hansen E; Oproy S van; Marzin D; Cauteren H van; Vanparys P; Evaluation of the HET-CAM-TSA method as an alternative to the Draize eye irritation test.; Toxicology in vitro; 11; 641-644; 1997 Hagino S; Kinoshita S; Tani N; Nakamura T; Ono N; Konishi K; Iimura H; Kojima H; Ohno Y;Interlaboratory validation of in vitro eye irritation tests for cosmetic ingredients. (2) Chorioallantoic membrane (CAM) test.; Toxicology in vitro; 13(1); 99-113; 1999 Journal Officiel de la Republique Francaise. Arrete du 29 Novembre 1996 relatif aux methodes officielles d'analyse necessaires aux controles des produits cosmetiques. Journal Officiel de la Republique Francaise; 19137-19138; 1996 Luepke NP; Hen's egg chorioallantoic membrane test for irritation potential.; Food and chemical toxicology; 23(2); 287-291; 1985 Luepke NP; Wallat S; HET-CAM - reproducibility studies. In: AM Goldberg (Ed.): Alternative methods in toxicology. Mary Ann Liebert, Inc., New York; 5; 353-363; 1987 Schlede E; Gerner I; The Draize eye test and progress in development and acceptance of alternatives to this test in Europe. In: Goldberg AM, van Zutphen LFM (Eds): The World Congress on Alternatives and Animals Use in the Life Sciences: Education, Research, Testing. Mary Ann Liebert, Inc., New York; 333-336; 1995 Silva O de; Rougier A; Dossou KG; The HET-CAM test: a study of the irritation potential of chemicals and formulations.; Alternatives to laboratory animals, ATLA; 20; 432-437; 1992 Spielmann H; Kalweit S; Liebsch M; Wirnsberger T; Gerner I; Bertram-Neiss E; Krauser K; Kreiling R; Miltenburger HG; Pape W; Steiling W; Validation study of alternatives to the Draize eye irritation test in Germany: Cytotoxicity testing and HET-CAM test with 136 industrial chemicals.; Toxicology in vitro; 7(4); 505-510; 1993
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Spielmann H; HET-CAM test.; Invittox Protocol; 47; 1992 Spielmann H; Target 2000 - case studies - chemicals testing - reducing animal experiments by 50 %. Proceedings conference 14 and 15 April 1997, Brussels. Ideal Conferences, London, UK; 1-12; 1998 Steiling W; The Hen's EggTest on the Chorioallantoic Membrane (HET-CAM).; INVITTOX Protocol; 96; 1994 Zebet25. The hens egg-chorioallantoic membrane test (HET-CAM test) for the assessment of the eye irritation potential of chemical substances. AnimAlt-Zebet database; 2001 http://www.bfr.bund.de/cms/detail.php?template=internet_en_index
3.2.2. Chorioallantoic membrane vascular assay (CAMVA) 1. Short description, scientific relevance, and purpose of the test The CAMVA assay assesses the possible detrimental effects which potential eye irritants have on the blood vessels of the embryo's CAM. In preparation of the test a small opening is cut into the shell of the egg four days after fertilization and a small amount of albumen is removed to allow optimal growth of the CAM. The opening is resealed and the eggs are incubated for a further 6 days. The age of the CAM used in the test has been limited to 10 days in order to comply with the legislation in the EU countries which prohibits experiments on chick embryos that are older than 10 days (Bagley et al., 1992, 1994). On day 10 the test substance is applied directly onto a small area of the CAM. After exposure for 30 minutes the eggs are examined for any vascular change in the CAM, e.g. haemorrhaging, hyperaemia (capillary injection) or the occurrence of vessels devoid of blood flow (ghost vessels). The concentration of test materials eliciting such damaging effects in 50 % of the eggs is calculated as toxicological endpoint. The test provides a functional vasculature similar to that of the conjunctiva. Vasoactive compounds will act on the smooth muscle cells to dilate or constrict the capillaries. The haemorrhage endpoint indicates cell lysis in the endothelium and surrounding tissues. 2. Developer of the method The CAMVA assay was developed by Leighton et al. (1985) and modified by Bagley et al. (1989, 1991). 3. Known Users The CAMVA is used in the cosmetics industry - mainly in the U.S.A. - for in-house (or contract lab-supported) screening and safety assessment of cosmetics. The human tissue constructs have supplanted the CAM-based assays for the larger companies. 4. Status of validation and/or standardization The CAMVA has been included in several validation studies amongst which are the Soap and Detergent Association Phase III trials (Bagley et al, 1994), the COLIPA validation (Bagley et al, 1999), the US CTFA study (Gettings et al., 1992) and the IRAG working group on CAM-based assays (Spielmann et al., 1997). The study performed under the auspices of the US Soap and Detergent Association (SDA; Bagley et al., 1994) and the collective evaluation of seven Draize test alternatives
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carried out by Sina et al. (1995) found good correlation with the MAS values of the rabbit eye irritation test (correlation coefficient = -0.91 and 75.8 % concordance, respectively). The tested substances were correctly classified as irritants and nonirritants. 5. Fields of application and limitations The CAMVA has been used to determine the dose required to reach a 50% positive response in the 10 eggs tested. Both aqueous and oil soluble materials have been tested. Generally, these have been in the mild to moderate region of toxicity. The study conducted by COLIPA (Brantom et al., 1997; Bagley et al., 1999) concluded that the CAMVA was better suited to assess hydroalcoholic substances than non-hydroalcoholic substances. The CAMVA performed less well with surfactant-based formulations in the Evaluation of Alternatives Program (phase III) conducted by the US CTFA (Gettings et al., 1996) in comparison with the other CAM-based protocols used. A similar observation was made by the IRAG Working Group in its critical appraisal of CAM-based assays; here, the CAMVA provided the best performance with alcohols (Spielmann et al., 1997) whereas HET-CAM assays performed better with surfactants and surfactant-based formulations. The chorioallantoic membrane vascular assay (CAMVA) is considered suitable as a screen for the irritation potential of cosmetic and personal care formulations, alcoholbased materials and surfactants. It has mostly been applied to assess materials in the mild to moderate irritation range (e.g. Cerven and Morena, 1998) but not to classify severely eye-irritating materials. In order to facilitate assessment of the latter, the final report of COLIPA validation study suggested to extend the CAMVA exposure time beyond 30 minutes as well as to improve the mathematical prediction model currently in use (Bagley et al., 1999). 6. Recommended use in the options of animal replacement a) As a screen for vascular effects b) As a screen for ocular effects on the conjunctiva 7. On-going development 8. Efforts needed to complete validation The CAMVA has not been assessed in parallel by more than two or three laboratories in any of the studies mentioned; thus, larger-scale validation is required to firmly establish interlaboratory reproducibility (e.g. Bagley et al., 1992; Brantom et al., 1997; Bagley et al., 1999). Further limitations of the CAMVA are that formulations containing polyethylene glycol are prone to yield false-positive results, and that testing of insoluble materials requires a certain degree of technical skill (Spielmann, 1997). 9. Key references
Bagley DM; Kong BM; Desalva SJ; Assessing the eye irritation potential of surfactant-based materials using the chorioallantoic membrane vascular assay (CAMVA). AM Goldberg (Ed.): Alternative methods in toxicology series - Symposium on in vitro toxicology: new directions. Mary Ann Liebert, Inc., New York; 7; 265-272; 1989
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Bagley DM; Rizvi PY; Kong BM; Salva SJ; Factors affecting use of the hens egg chorioallantoic membrane as a model for predicting eye irritation potential.; Journal of toxicology / Cutaneous and ocular toxicology; 10; 95-104; 1991 Bagley DM; Bruner LH; Silva O de; Cottin M; O'Brien KAF; Uttley M; Walker AP; An evaluation of five potential alternatives in vitro to the rabbit eye irritation test in vivo.; Toxicology in vitro; 6; 136149; 1992 Bagley, D.M., Waters, D., and Kong, B.M. (1994) Development of a 10-day chorioallantoic membrane vascular assay as an alternative to the Draize rabbit eye irritation test. Food and Chemical. Toxicology. 33(12):1155-1160. Bagley D; Booman KA; Bruner LH; Casterton PL; Demetrulias J; Heinze JE; Innis JD; Iii WCM; Neun DJ; The SDA alternatives program phase III: comparison of in vitro data with animal eye irritation data on solvents, surfactants, oxidizing agents, and prototype cleaning products.; Journal of toxicology / Cutaneous and ocular toxicology; 13(2); 127-155; 1994 Bagley, D.M., Cervin, D., and Harbell, J.W. (1999) As sessment of the chorioallantoic membrane vascular assay (CAMVA) in the COLIPA in vitro eye irritation validation study. Toxicology In Vitro 13:285-293. Brantom PG; Bruner LH; Chamberlain M; Desilva O; Dupuis J; Earl LK; Lovell DP; Pape WJW; Uttley M; Bagley DM; Baker FW; Brachter M; Courtellemont P; Declercq L; Freeman S; Steiling W; Walker AP; Carr GJ; Dami N; Thomas G; Harbell J; Jones PA; Pfannenbecker U; Southee JA; Tcheng M; Argembeaux H; Castelli D; Clothier R; Esdaile DJ; Itigaki H; Jung K; Kasai Y; Kojima H; Kristen U; Larnicol M; Lewis RW; Marenus K; Moreno O; Peterson A; Rasmussen ES; Robles C; Stern M; A summary report of the COLIPA international validation study on alternatives to the Draize rabbit eye irritation test.; Toxicology in vitro; 11(N1-2); 141-179; 1997 Cerven D; Moreno O; Bovine corneal opacity and permeability test validation as an alternative to the Draize eye irritation assay. In: H Salem; SA Katz (Eds. ): Adv Anim Altern Saf Efficacy Test. Taylor & Francis, Washington, D.C; 261-267; 1998 Gettings SD; Lordo RA; Hintze KL; Bagley DM; Casterton PL; Chudkowski M; Curren RD; Demetrulias JL; DiPasquale LC; Earl LK; Feder PI; Galli CL; Glaza SM; Gordon VC; Janus J; Kurtz PJ; Marenus KD; Moral J; Pape WJW; Renskers KJ; Rheins LA; Roddy MT; Rozen MG; Tedeschi JP; Zyracki J; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (phase III) Surfactant-based formulations.; Food and chemical toxicology; 34(1); 79-117; 1996 Harbell, J.W. and Curren, R.D. (2001) In vitro methods for the prediction of ocular and dermal toxicity. In, Handbook of Toxicology, 2 nd Ed. M.J. Derelanko and M.A. Hollinger ED., CRC Press, Boco Raton, pp 835-866. Leighton J; Nassauer J; Tchao R; The chick embryo in toxicology: an alternative to the rabbit eye.; Food and chemical toxicology; 23(2); 293-298; 1985 Sina JF; Galer DM; Sussman RG; Gautheron PD; Sargent EV; Leong B; Shah PV; Curren RD; Miller K; A collaborative evaluation of seven alternatives to the Draize eye irritation test using pharmaceutical intermediates.; Fundamental and applied toxicology; 26(1); 20-31; 1995 Spielmann H; Liebsch M; Moldenhauer F; Holzhuetter HG; Bagley DM; Lipman JM; Pape WJW; Miltenburger H; Silva O de; Hofer H; Steiling W; IRAG working group 2. CAM-based assays.; Food and chemical toxicology; 35; 39-66; 1997 Zebet272. The chicken egg chorioallantoic membrane vascular assay (CAMVA) as an alternative to the rabbit eye irritation test. AnimAlt-Zebet database; 2001 http://www.bfr.bund.de/cms/detail.php?template=internet_en_index
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3.2.3. Chorioallantoic membrane - trypan blue staining (CAM-TB) 1. Short description, scientific relevance and purpose The chicken chorioallantoic membrane - trypan blue staining (CAM-TB) method was developed to provide an objective evaluation technique to overcome disadvantages arising from the lack of objectivity and quantitativeness associated with the HETCAM. This method was designed to examine the injurious effect of substances by measuring the amount of trypan blue adsorbed with the CAM as the endpoint of the assay. Trypan blue staining, widely used for measuring cell viability, detects destruction and denaturation of the membrane (Hagino et al., 1999). 2. Developer of the method The CAM-TB test was developed by Hagino et al. (1991, 1993, INVITTOX protocol n. 108, Itagaki et al., 1995). 3. Known users 4. Status of validation and/or standardisation The CAM-TB was part of the Japanese Ministry of Health and Welfare (MHW) project entitled Studies on the test methods to evaluate the safety of new ingredients of cosmetics (Ohno et al., 1999). It was found to have a smaller variability than that of the HET-CAM test, and showed a good correlation with the maximum average Draize (MAS) score when the test chemicals were classified according to their liquid and solid state (Hagino et al., 1999). 5. Fields and limitations of application, i.e., in vivo endpoints and/or chemical classes covered/not covered Coloured substances may interfere with the measurement of endpoint (Ohno et al., 1999). 6. Recommendations of use in the view of animal replacement To be used in a test battery (Ohno et al., 1999). 7. On-going development 8. Efforts needed to complete validation of the method 9. Key references
Hagino S; Itagaki H; Kato S; Kobayashi T. and Tanaka M. (1991) Quantitative evaluation to predict the eye irritancy of chemicals: Modification of chorioallantoic membrane test by using trypan blue. Toxicology in Vitro 5, 301-304. Hagino S; Itagaki H; Kato S and Kobayashi T. (1993) Further evaluation of the quantitative chorioallantoic membrane test using trypan blue stain to predict the eye irritancy of chemicals. Toxicology in Vitro 7, 35-39. Hagino S; Kinoshita S; Tani N; Nakamura T; Ono N; Konishi K; Iimura H; Kojima H; Ohno Y;Interlaboratory validation of in vitro eye irritation tests for cosmetic ingredients. (2) Chorioallantoic membrane (CAM) test.; Toxicology in vitro; 13(1); 99-113; 1999 Itagaki H; Hagino S; Kato S; CAM-TBS test.; Invittox Protocol; 108; 1995 Ohno Y; Kaneko T; Inoue T; Morikawa Y; Yoshida T; Fuji A; Masuda M; Ohno T; Hayashi M; Momma J; Uchiyama T; Chiba K; Ikeda N; Imanashi Y; Itakagaki H; Interlaboratory validation of the
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in vitro eye irritation tests for cosmetic ingredients. (1) Overview of the validation study and Draize scores for the evaluation of the tests.; Toxicology in vitro; 13(1); 73-98; 1999
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3.3. Human corneal epithelium models 3.3.1. The EpiOcularTM assay (ET50-based assay) 1. Short description, scientific relevance, and purpose of the test MatTek's EpiOcular corneal model (MatTek, Ashland, MA, USA) consists of normal, human-derived epidermal keratinocytes that are cultured on specially prepared cell culture inserts using serum free medium, and that differentiate to form a multi-layered structure which closely parallels the corneal epithelium. EpiOcular has been utilized with several common tests of cytotoxicity and irritancy, primarily MTT but also IL-1a, PGE2, LDH, and sodium fluorescein permeability. Comparison with in vivo animal data has been carried out, by using the ET50 value (effective time of exposure to reduce tissue viability to 50%) determined by MTT assay. Using the variable of time rather than dose allows ingredients and formulations to be tested without dilution in medium. Thus both hydrophobic and hydrophilic materials may be tested. As a stratified epithelium, the EpiOcular construct is intended to model damage to the corneal epithelium and conjunctiva (with its very thin epithelium). Therefore, it can be used to resolve degrees of irritancy potential (cellular damage) in the moderate to very mild irritancy range (mild corneal and conjunctival irritation). The tissue construct is capable of also identifying high moderate and severe irritants by their very short ET50 values (see below). However, based on the stromal changes associated with severe irritation, an epithelial construct would not be expected to provide the degree of resolution in the severe range that a full thickness cornea (e.g. ex vivo cornea) would provide. 2. Developer of the method The tissue is produced by MatTek Corporation (Ashland, MS, USA). The test methods were derived from the work conducted by industry using the ZK1200 tissue (see Southee et al, 1999). 3. Known Users The EpiOcular-based assay is used by contract laboratories and industries including cosmetic, personal care, household, and some industrial users. Generally, the goal is to establish a certain limit of toxicity (e.g., prove a certain level of mildness). The EpiOcular test is used as replacement for preclinical safety testing for formulation with mild to moderate irritation potential (Harbell and Curren, 2001). It is also used in formulation optimisation (McCain et al, 2002), and in the safety evaluation of surfactants and surfactant formulations (personal care cosmetics and household products). The Proceter & Gamble Company has developed a prediction model for materials of interest which is based on the prediction model used for the ZK1200 tissue construct. Consistent with their approach to eye irritation testing, the prediction model relates to the low volume eye test (LVET). In particular, the EpiOcular assay has been used to evaluate mild materials for the cosmetic and personal care industry. In this application, exposure times of up to 24 hours are employed. The assay is used to predict success (user acceptance) of repeat
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application products or where mild sting or similar irritation might be expected (McCain et al, 2002, Ghassemi1 et al, 1997). 4. Status of validation and/or standardization Stern et al. (1998) reported that 63% of the tested samples (27 out of 43) were classified correctly within the different ranges of eye irritantcy. Most of the remaining samples were over-predicted by a single irritation class. Overall, 95% (41 of 43 samples) fell within the correct class or one class above the predicted by the in vivo results. No underpredictions of eye irritation were found for any material of this study (Stern et al., 1998). The EpiOcular corneal model has also been included in a project which evaluated several in vitro methods by applying reference standards in order to gain wider acceptance of such assays in the regulatory context (Balls, Brantom et al., 1999). The EpiOcular assay has undergone formal validation study (Blazka et al, 2000 and 2003). The focus of the validation was surfactant ingredients (e.g., individual surfactants) although a limited number of formulations were included. The validation was conducted in two phases. Phase I consisted of 20 test materials tested under code in four laboratories with three valid definitive trials for each test article in each laboratory. Phase II was conducted with 54 test articles in two laboratories with two valid definitive trials for each test article in each laboratory. Both phases were conducted in accordance with the GLP regulations. The detailed submission documents are in preparation. While the model is most often used to identify mild/moderate irritants, it is key that the test be able to identify a material that would fall into the severe category. The prediction models extend into the severe range and existing data show that severe irritants can be identified. These severe irritants can then be tested in in vitro assays such as ex vivo tissue-based assays, that can be focused on the more irritating range of toxicity. 5. Fields of application and limitations The assay is applicable to both hydrophilic and hydrophobic test materials (both formulations and ingredients). Either liquids or solids may be tested. Again, the best resolution has been obtained in the range of the mild to moderate irritants. The EpiOcularTM tissue is overly sensitive to the alcohol and esters group of chemicals (Balls, Brantom et al., 1999). In general, highly volatile liquids, organic solvents, and certain classes of reactive chemicals (e.g., peroxides) may not be appropriate for this model system. 6. Recommended use in the options of animal replacement Based on the depth-of-injury mechanism of eye irritation, the EpiOcular tissue construct can be used to differentiate among mild and moderate irritants and identify potential severe irritants. While the EpiOcular (or other epithelial construct) may not fully differentiate among degrees of severity, it is expected to be able to identify those materials that have more severe irritancy potential. These materials could be more fully characterized with an ex vivo or similar tissue. The combination of the tissue construct and ex vivo cornea could possibly be used to resolve across the full range of irritation potentials.
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7. On-going development The microscopic tissue changes associated with different levels of tissue viability (as measured by MTT) are being evaluated for surfactant ingredients from the corporate validation study. 8. Efforts needed to complete validation Develop data on individual chemicals across classes (that are not necessarily cosmetic ingredients) to understand the strengths and limitation of the EpiOcular assay. 9. Key references
Balls M; Brantom PG; Cassidy S; Esdaile D; Fentem J; Liebsch M; McPherson J; Pfannenbecker U; Prinsen M; Preliminary evaluation of the application of reference standards in the prevalidation and validation of in vitro test for eye irritation. In: DG Clark, SG Lisansky and R Macmillan: Alternatives to animal testing II, proceedings, Brussels. CPL Press, Berkshire; 201-204; 1999. Blazka, M., Harbell, J.W., Klausner, M., Merrill, J.C., Kubilus, J., Kloss C., and Bagley, D.M. (2003) Evaluating the ocular irritation potential of 54 test articles using the EpiOcular human tissue construct model (OCL-200). The Toxicologist 72:221. Blazka, M.E., Harbell, J.W., Klauzner, M., Raabe, H., Kubilus, J., Hsia, F., Minerath, B., Kotler, M., and Bagley, D.M. (2000). Colgate-Palmolive's program to validate the EpiOcular T M human tissue construct model. The Toxicologist 54(1): 188. Ghassemi1, A., Osborne, R., Kohrman, K. A., Roddy, M. T., Harbell, J.W., Kanengiser, B.E. (1997) demonstrating the ocular safety of an eye cosmetic product using alternatives to animal eye irritation tests. The Toxicologist, 36 (1), 42. Harbell, J.W. and Curren, R.D. (2001) In vitro methods for the prediction of ocular and dermal toxicity. In, Handbook of Toxicology, 2 nd Ed. M.J. Derelanko and M.A. Hollinger ED., CRC Press, Boco Raton, pp 835-866. McCain, N.E., Binetti, R.R., Gettings, S.D., Jones, B.C. (2002) Assessment of ocular irritation ranges of market-leading cosmetic and personal-care products using an in vitro tissue equivalent. The Toxicologist, 66 (1-S), 243. Southee, J.A., McPherson, J.P., Osborne, R. Carr, G.J., and Rasmussen, E. (1999) The performance of the tissue equivalent assay using the Skin2TM ZK1200 model in the COLIPA International Validation Study on Alternatives to the Draize Eye Irritation Test. Toxicology In Vitro 13:355-373. Stern M; Klausner M; Alvarado R; Reskers K; Dickens M; Evaluation of EpiOcular tm Tissue model as an alternative to the Draize eye irritation test. Toxicology in Vitro 12:455-461; 1998. Worth AP; Balls M; Editors. Alternative (non-animal) methods for chemicals testing current status and future prospects. A report prepared by ECVAM working group on chemicals. ALTA 30; suppl.1; 2002.
3.3.2. The SkinEthic in vitro reconstituted human corneal epithelium (HCE model) 1. Short description, scientific relevance, and purpose of the test The Skinethic HCE model (SkinEthic, Nice, France) consists of immortalized human corneal epithelial cells (HCE cell line) that are cultivated at the air-liquid interface in a chemically defined medium on a polycarbonate substrate and form an air-epithelial tissue, devoid of stratum corneum, resembling morphologically the corneal mucosa of the human eye. Possible endpoint measurements are: tissue viability using the MTT assay or LDH release, histology, quantification of cytokine release (e.g., IL-1? , IL-6, 26
IL-8, PGE2), and gene expression. In vivo / in vitro comparisons have been made (Nguyens et al, 2003), and interlaboratory reproducibility is currently under evaluation (Van Goethem et al., 2004). 2. Developer of the method The model has been developed at the SkinEthic Laboratories (France). 3. Known users The HCE model is used by several cosmetic, chemical and pharmaceutical companies to test finished products as well as raw materials. 4. Status of validation and/or standardisation The HCE model has been tested in a pre-validation study based on chemical substances (Van Goethem et al., in preparation). 5. Fields and limitations of application, i.e. in vivo endpoints and/or chemical classes covered/not covered Additional testing is needed at lower concentration levels and/or shorter sampling times. 6. Recommendations of use in the topics of alternative replacement Until this test passed formal validation, the test is suited as a pre-screen for eye irritation of chemicals and finished products. 7. On-going development Recently, data were presented on 130 finished products which demonstrated a satisfactory correlation with the in vivo Draize data (Doucet et al, in preparation). The HCE model can be used to demonstrate corneal repair/recovery in vitro (Jones et al., 2001; De Wever et al, 2002). A new model on reconstituted human conjunctival epithelium is currently in development. 8. Efforts needed to complete validation of the method Coordination by ECVAM. 9. References
Nguyen D.H., R.W. Beuerman, B. De Wever and M. Rosdy. Three-dimensional construct of the human corneal epithelium for in vitro toxicology. In: Alternative Methods for the New Millenium. Chapter 14, 145-157. CRC Press. 2003. Brinch D.S., Elvig S.G., Novozymes A/S, Bagsvaert, Denmark. Evaluation of an in vitro human corneal model as alternative to the in vivo eye irritation testing of enzymes. Toxicology Letters, Vol 123, suppl.1, 22, 2001 Courtellemont P, Pannetier M., Perrier P. and Pericoi M. The use of alternative methods and clinical tests in the ocular risk assessment of cosmetic formulations. In: Clarck DG., Lisansky SG; MacMillan R (Eds.), Alternatives to Animal Testing II; CPL Press, 1999. De Wever, Bart; Tornier, C.; Rosdy, M. and Beuereman, R. (2002) In vitro corneal recovery assay using a reconstituted human corneal epithelial model. 4 th World Congress on Alternatives and Animal Use in the Life Sciences, New Orleans, USA.
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Doucet O., Lanvin M., and Zastrow L. Comparison of three in vitro methods for the assessment of the eye irritation potential of formulated products. Lancaster Group-Coty International Research, Monaco. In Vitro and Molecular Toxicology, vol. 12, n2, p. 63-76, 1999. Doucet O., Lanvin M., and Zastrow L. A new in vitro human epithelium model for assessing the eye irritation potential of formulated cosmetic products. Lancaster Group-Coty International Research Center, Monaco. In Vitro and Molecular Toxicology, vol. 11, n4, p. 273-283, 1998. Jones, P., King, A; and Fentem, J (2001) The effect of known eye irritants on the SkinEthic and Epiocular models of corneal epithelium. Poster presented at the European Tissue Culture Society, Granada. Van Goethem F.; Straube F.; Kretz S; Alpe N.; Catoire S.; Romeike A.; Hansen E.; De Wever, B.; Cappadoro M.; Rosdy M.; Adriaens E.; Vanparys P. Prevalidation of a new in vitro reconstituted human corneal epithelial model to assess the eye irritating potential of chemicals. In preparation, 2004.
3.3.3. HCE-T Tissue Construct (Gillette) 1. Short description, scientific relevance, and purpose of the test To develop a reproducible model of the human corneal surface, the Gillette model uses a transfected human corneal epithelial cell line (HCE-T) (Kahn et al., 1993) cultured on collagen-membrane cell culture inserts which, at the air-liquid interface, stratify to form a four- to six-layer epithelium, known as the HCE-T model. Transepithelial permeability to sodium fluorescein (TEP) and transepithelial electrical resistance (TER) have been identified as physiologically relevant parameters for evaluating the barrier function of the corneal epithelium (Maurice, 1967; Tchao, 1988). Cell viability can be determined by the MTT assay, and histomorphology can also be used as an endpoint. 2. Developer of the method Gillette Corporation. 3. Known Users Developers of mild surfactant formulations. 4. Status of validation and/or standardization A full formal validation study has been completed on liquid surfactant-containing formulations. Those data are under preparation as a manuscript. 5. Fields of application and limitations The assay focuses on liquid surfactant-containing formulations. Only limited data are available on other types of materials. Cationic surfactants, which precipitate proteins, tend to be under-predicted with the current prediction model. 6. Recommended use in the options of animal replacement Mild, liquid surfactant-containing formulations where conjunctival and milder corneal lesions are expected. 7. On-going development None
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8. Efforts needed to complete validation The validation study is complete and review by ECVAM would be the next step. However the validation study was restricted to liquid surfactants based formulations. 9. Key references
Kahn CR; Young E; Lee IH; Rhim JS; Human corneal epithelial primary cultures and cell lines with extended life span: in vitro model for ocular studies; Invest Ophthalmol Vis Sci.; 34 (12): 3429-41; 1993 Kruszewski, F.H., Walker, T.L., Ward, S.L., and Dipasquale, L.C.. (1995) Progress in the use of human ocular tissues for in vitro alternative methods. Comments in Toxicology 5: 203-224. Maurice DM; The use of fluorescein in ophthalmological research; Invest Ophthalmo l6(5): 464-77; 1967 Tchao R; Trans-epithelial permeability of fluorescein in vitro as an assay to determine eye irritants. In: A.M. Goldberg (Ed.): Alternative Methods in Toxicology. Mary Ann Liebert, Inc., New York; 6; 271283; 1988
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3.4. Cell based cytotoxicity methods The application of cytotoxicity tests for the assessment of eye irritation potential is based on the observation that some materials which are damaging to the eye can produce cytotoxic effects in various cell types, including those present in ocular epithelial and endothelial tissues.
3.4.1 Neutral Red Uptake 1. Short description, scientific relevance and purpose One of the most widely used cytotoxicity tests is the neutral red uptake assay (NRU assay; Borenfreund and Puerner, 1984, 1985) which measures the ability of a test substance to inhibit the uptake of neutral red dye, a marker of cell viability (Barstadt et al., 1991). The neutral red penetrates cell membranes and accumulates intracellularly in lysosomes. Alterations of the cell surface or sensitive lysosomal membrane, result in a decreased uptake of the neutral red. The NRU assay has been conducted on primary cell cultures as well as on established cell lines, e.g. Chinese hamster V79, CHO, 3T3 Balb/c and rabbit corneal cells (SIRC line). For the test, monolayer cell cultures are grown on microtitre plates and, on having reached nearconfluence, incubated with serial dilutions of test substance yielding a wide range of concentrations. After exposure for usually either 24 h or 48 h, the cells are washed and incubated for three hours with medium containing neutral red dye. Thereafter, the cells are treated with wash/fixation solution and then with appropriate solvent to elute the dye. The optical density of the resulting solution is measured at 540 nm. The concentration of test substance producing a 50 per cent inhibition of neutral red uptake in comparison to control samples, which have not been exposed to test substance, is obtained by extrapolation from the dose-response curve. This so-called NRU50 or IC50 value serves as toxicological endpoint. 2. Developer of the method Standard protocols for the NRU assay (Borenfreund and Puerner, 1984, 1985) have been published for normal human epidermal keratinocytes (NHEK; cf. Harbell et al., 1997), balb/c 3T3 mouse fibroblasts (Clothier, INVITTOX protocol no. 3, 1990; Spielmann, INVITTOX protocol no. 46, 1992; Harbell, INVITTOX protocol no. 100, 1994) and for SIRC cell line derived from rabbit cornea (Blein-Sella and Adolphe, 1995; Adolphe and Blein, INVITTOX protocol no. 40, 1990). 3. Known users The NRU assay is performed in many laboratories of the cosmetics and pharmaceutical industry as an essential part of a test battery or as adjunct assay in a tiered approach of screening for eye irritation potential (e.g. Harbell et al., 1997) and classifying materials according to European classification R41 in combination with organotypical in vitro assays (e.g. Spielmann et al., 1995, 1996; Spielmann, 1997). 4. Status of validation and/or standardisation The NRU assay has been included in all the major validation studies carried out to evaluate the potential of in vitro test alternatives to the Draize rabbit eye test. The NRU assay using NHEK cells was employed in the two studies conducted in the U.S.A. within the CTFAs Alternatives Program (Gettings et al., 1991, 1994, 1996)
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and by the Soap and Detergent Association (e.g. Bagley et al., 1994). Balb/c 3T3 fibroblasts have been used in the NRU assays during the world-wide validation study conducted for the EC/HO study (Balls et al., 1995a), in the studies carried out by COLIPA (Brantom et al., 1997; Jones et al., 1999) and by BMFT/BGA (Kalweit et al., 1990; Spielmann et al., 1991, 1993, 1995, 1996) as well as in the CTFA study (Gettings et al., 1996). The NRU assay with SIRC cells has been assessed in the study by the French association for the welfare of laboratory animals OPAL (Oeuvre Pour l'assistance aux Animaux de Laboratoire; e.g. Blein et al., 1991) and in the interlaboratory study carried out under the auspices of the Japanese Ministry of Health and Welfare and the Japanese Cosmetic Industry Association (e.g. Tani et al., 1999). The performance of NRU assays using NHEK and SIRC cells has also been critically reviewed by the IRAG Working Group on Cell Cytotoxicity Assays (Harbell et al., 1997). In general, it was found that the data obtained with NRU assay were reproducible and correlated well with the results of the in vivo methods, i.e. low volume eye test LVET (Bruner et al., 1991; Blein et al., 1991; Bagley et al., 1992) and Draize MAS values (e.g. Rougier et al., 1992; Gettings et al., 1996; Tani et al., 1999). 5. Fields and limitations of application, i.e., in vivo endpoints and /or chemical classes covered/ not covered The outcome of all comparative studies depended strongly on the class of products and chemicals investigated and a good correlation with Draize MAS proved true notably for surfactants (e.g. Harbell et al., 1997; Jones et al., 1999). When predominantly substances of other classes of chemicals had been included in the study the NRU assay appeared less able to distinguish among the irritants based on Draize eye irritation scores (e.g. Bagley et al., 1994; Doucet et al., 1999). The NRU assay was found to be a particularly useful toxicological endpoint to evaluate and rank products with low eye irritation potential (e.g. Rougier et al., 1992; cf. Christian and Diener, 1996). The NRU assay alone has limited potential to identify moderately and severely irritating substances. A prime factor is the type of product tested: the material should be soluble in or, at least, miscible with water and have only limited acid and alkaline reserve. Extremely high or low pH values and reactivity with the culture medium must be avoided in order to ensure the results obtained are accurate and useful (e.g. Harbell et al., 1997; Spielmann, 1997). Highly soluble products of relatively simple structure appear to be better candidates than more complex or even hydrophobic formulations (e.g. Christian and Diener, 1996). Other compounds that appear to show difficulties to be assessed by the NRU assay are volatile chemicals, explosives, coloured compounds and chemicals that have localised effect upon the lysosomes/endosomes (e.g., cloroquine sulfate which inhibits NRU by alteration of lysosomes/endosomes pH). It is important to assess each individual product class carefully: thus, the NRU assay may be well suited for the assessment of surfactants and surfactant-based formulations such as shampoos whereas evaluating fabric softeners may prove difficult (Harbell et al., 1997). 6. Recommendations of use in the optics of animal replacement It has been shown that, when used in combination with organotypical in vitro assays such as the HET-CAM assay (Spielmann et al., 1995, 1996; Spielmann, 1997) or the IRE test, the NRU assay can be employed successfully as a part of a test battery to identify severely eye irritating materials (and classify them according to European
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classification R41). The neutral red uptake assay in combination with the HET-CAM test, has been included in a project which evaluated several in vitro methods by applying reference standards in order to gain wider acceptance of such assays in the regulatory context (Balls, Brantom et al., 1999). The alcohol/esters and surfactants groups of chemicals were tested with the HET-CAM/NRU assay, which showed a good outcome for surfactants, but not so good for alcohols and esters, although data obtained with HET-CAM/NRU was better than the ones obtained with the EpiOcularTM test. 7. On-going development NICEATM and ECVAM are conducting a collaborative validation: Test method Protocol for Balb/c 3T3 and NHK combined with Neutral Red Uptake Cytotoxicity Assay (Strickland et al., 2003). The focus of this validation study is to assess acute systemic toxicity, however the protocol and reproducibility data could be usefull for the area of eye irritation. 8. Efforts needed to complete validation of the method Many studies and validations were performed with chemical compounds on NRU model. These studies could be completed with formulated products. 9. References
Adolphe M; Blein O; SIRC cytotoxicity test; Invittox protocol; 40; 1990 Bagley DM; Bruner LH; Silva O de; Cottin M; O'Brien KAF; Uttley M; Walker AP; An evaluation of five potential alternatives in vitro to the rabbit eye irritation test in vivo.; Toxicology in vitro; 6; 136149; 1992 Bagley D; Booman KA; Bruner LH; Casterton PL; Demetrulias J; Heinze JE; Innis JD; Iii WCM; Neun DJ; The SDA alternatives program phase III: comparison of in vitro data with animal eye irritation data on solvents, surfactants, oxidizing agents, and prototype cleaning products.; Journal of toxicology / Cutaneous and ocular toxicology; 13(2); 127-155; 1994 Balls M; Botham PA; Bruner LH; Spielmann H; The EC/HO international validation study on alternatives to the Draize eye irritation test.; Toxicology in vitro; 9(6); 871-929; 1995a Balls M; Brantom PG; Cassidy S; Esdaile D; Fentem J; Liebsch M; McPherson J; Pfannenbecker U; Prinsen M; Preliminary evaluation of the application of reference standards in the prevalidation and validation of in vitro test for eye irritation. In: DG Clark, SG Lisansky and R Macmillan: Alternatives to animal testing II, proceedings, Brussels. CPL Press, Berkshire; 201-204; 1999 Barstadt R; Cortesi J; Janus J; Use of Clonetics neutral red bioassay to optimize components of serumfree medium for normal human anchorage-dependent cells.; In vitro cellular & deve lopmental biology; 27; 160-; 1991 Blein O; Adolphe M; Lakhdar B; Cambar J; Gubanski G; Castelli D; Contie C; Hubert F; Latrille F; Masson P; Clouzeau J; Bigot JF Le; Silva O De; Dossou KG; Correlation and validation of alternative methods to the Draize eye irritation test (OPAL project).;Toxicology in vitro; 5(5-6); 555-557; 1991 Blein-Sella O; Adolphe M; SIRC cytotoxicity test. In: OHare S; Atterwill CK (Eds.): Methods in molecular biology. Humana Press, Totowa, New Jersey; 43; 161-167; 1995 Borenfreund E; Puerner JA; A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/ NR-90).; Journal of tissue culture methods; 9(1); 7-9; 1984 Borenfreund E; Puerner JA; Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicology letters; 24; 119-124; 1985
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Brantom PG; Bruner LH; Chamberlain M; Desilva O; Dupuis J; Earl LK; Lovell DP; Pape WJW; Uttley M; Bagley DM; Baker FW; Brachter M; Courtellemont P; Declercq L; Freeman S; Steiling W; Walker AP; Carr GJ; Dami N; Thomas G; Harbell J; Jones PA; Pfannenbecker U; Southee JA; Tcheng M; Argembeaux H; Castelli D; Clothier R; Esdaile DJ; Itigaki H; Jung K; Kasai Y; Kojima H; Kristen U; Larnicol M; Lewis RW; Marenus K; Moreno O; Peterson A; Rasmussen ES; Robles C; Stern M; A summary report of the COLIPA international validation study on alternatives to the Draize rabbit eye irritation test.; Toxicology in vitro; 11(N1-2); 141-179; 1997 Bruner LH; Denver JK; Deirdre AR; Parker RD; Evaluation of seven in vitro alternatives for ocular safety testing.; Fundamental and applied toxicology; 17; 136-149; 1991 Christian MS; Diener RM; Soaps and detergents - alternatives to animal eye irritation tests.; Journal of the American College of Toxicology; 15(1); 1-44; 1996 Clothier RH; The FRAME modified neutral red uptake cytotoxicity test.; Invittox Protocol; 3; 1990 Doucet O; Lanvin M; Zastrow L; Comparison of three in vitro methods for the assessment of the eye irritating potential of formulated cosmetic products.; In vitro and molecular toxicology; 12(2); 63-76; 1999 Gettings SD; Teal JJ; Bagley DM; Demetrulias JL; DiPasquale LC; Hintze KL; Rozen MG; Weise SL; Chudkowski M; Marenus KD; Pape WJW; Roddy M; Schnetzinger R; Silber PM; Glaza SM; Kurtz PJ; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test (phase 1) hydro-alcoholic formulations; (part 2) data analysis and biological significance.; In vitro toxicology; 4(4); 247-288; 1991 Gettings S Dipasquale LC; Bagley DM; Casterton PL; Chudkowski M; Curren RD; The CTFA D; evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase II), oil/water emulsions.; Food and chemical toxicology; 32(10); 943-976; 1994 Gettings SD; Lordo RA; Hintze KL; Bagley DM; Casterton PL; Chudkowski M; Curren RD; Demetrulias JL; DiPasquale LC; Earl LK; Feder PI; Galli CL; Glaza SM; Gordon VC; Janus J; Kurtz PJ; Marenus KD; Moral J; Pape WJW; Renskers KJ; Rheins LA; Roddy MT; Rozen MG; Tedeschi JP; Zyracki J; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (phase III) Surfactant-based formulations.; Food and chemical toxicology; 34(1); 79-117; 1996 Harbell JW; Neutral red bioassay using BALB/c 3T3 cells.; Invittox Protocol; 100; 1994 Harbell JW; Koontz SW; Lewis RW; Lovell D; Acosta D; IRAG Working Group 4. Cell cytotoxicity assays.; Food and chemical toxicology; 35; 79-126; 1997 Jones PA; Bracher M; Marenus K; Kojima H; Performance of the neutral red uptake assay in the COLIPA international validation study on alternatives to the rabbit eye irritation test.; Toxicology in vitro; 13 (2); 325-333; 1999 Kalweit S; Besoke R; Gerner I; Spielmann H; A national validation project of alternative methods to the Draize rabbit eye test.; Toxicology in vitro; 4(4/5); 702-706; 1990 Rougier A; Cottin M; DeSilva O; Roguet R; Catroux P; Tougic A; Dossou K; In vitro methods: their relevance and complementarity in ocular safety assessment.; Lens and eye toxicity research; 9(3-4); 229-245; 1992 Spielmann H; Gerner I; Kalweit S; Moog R; Wirnsberger T; Krauser K; Kreiling R; Kreuzer H; Luepke NP; Miltenburger HG; Mueller N; Muermann P; Pape W; Siegemund B; Spengler J; Steiling W; Wiebel FJ; Interlaboratory assessment of alternatives to the Draize eye irritation test in Germany.; Toxicology in vitro; 5(5/6); 539-542; 1991 Spielmann H; Balb/c 3T3 cytotoxicity test.; Invittox protocol; 46; 1992
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Spielmann H; Kalweit S; Liebsch M; Wirnsberger T; Gerner I; Bertram-Neiss E; Krauser K; Kreiling R; Miltenburger HG; Pape W; Steiling W; Validation study of alternatives to the Draize eye irritation test in Germany: Cytotoxicity testing and HET-CAM test with 136 industrial chemicals.; Toxicology in vitro; 7(4); 505-510; 1993 Spielmann H; Liebsch M; Moldenhauer F; Holzhuetter HG; Silva O de; Modern biostatistical methods for assessing in vitro/in vivo correlation of severely eye irritating chemicals in a validation study of in vitro alternatives to the Draize eye test.; Toxicology in vitro; 9(4); 549-556; 1995 Spielmann H; Liebsch M; Kalweit S; Moldenhauer F; Wirnsberger T; Holzhuetter HG; Schneider B; Glaser S; Gerner I; Pape WJW; Kreiling R; Krauser K; Miltenburger HG; Steiling W; Luepke NP; Mueller N; Kreuzer H; Muermann P; Spengler J; Betram-Neis E; Siegemund B; Wiebel FJ; Results of a validation study in Germany on two in vitro alternatives to the Draize eye irritation test, the HETCAM test and the 3T3 NRU cytotoxicity test.; Alternatives to laboratory animals, ATLA; 24; 741-858; 1996 Spielmann H; Ocular irritation. In: Castell, Gomez-Lechon (Eds.): In vitro methods in pharmaceutical research. Academic Press, London; 265-287; 1997 Strickland, J.A., Stokes, W.S., Casati, S., Paris, M.W., Worth, A., Raabe, H., Cao, C., Clothier, R., Harbell, J., Curren, R., Haseman, J., Tice, R.R. Design of a validation study to evaluate in vitro cytotoxicity assays for predicting rodent and human acute systemic toxicity. Society of Toxicology 42nd Annual Meeting. Salt Lake City UT, 2003. Prepared by the National toxicology Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) and Based on Standard Operating procedure Recommendations from an International Workshop Organised by the ICCVAM. Tani N; Kinoshita S; Okamoto Y; Kotani M; Itagaki H; Murakami N; Sugiura S; Usami M; Kato K; Kojima H; Ohno T; Saijo K; Kato M; Hayashi M; Ohno Y; Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (8) Evaluation of cytotoxicity tests on SIRC cells.; Toxicology in vitro; 13(1); 175-187; 1999 Zebet26. The neutral red uptake (NRU) cytotoxicity assay for the in vitro assessment of the eye irritation potential of chemical substances. AnimAlt-Zebet database; 2001 http://www.bfr.bund.de/cms/detail.php?template=internet_en_index
3.4.2. Neutral Red Release Assay 1. Short description, scientific relevance, and purpose of the test The neutral red release assay (NRR assay) was developed to measure effects of immediate toxicity such as damage to the plasma membrane and loss of lysosomal integrity. The amount of neutral red dye that is taken up has been shown to be directly proportional to the number of viable cells in the culture (Barstadt et al., 1991). The NRR assay has been conducted on primary cell cultures as well as on established cell lines. For the test, monolayer cell cultures that have reached near-confluence or confluence are incubated with neutral red dye for three hours. After the medium has been replaced, the cells are briefly exposed to serial dilutions of test substance for 1 to 5 min. The cells are washed, treated with a wash/fixation solution and then with appropriate solvent to release the dye still retained inside them. The optical density of the resulting solution is measured at 540 nm. The concentration of test substance producing a 50 per cent release of pre-loaded neutral red dye is obtained by extrapolation from the dose-response curve. This so-called NRR50 value serves as
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toxicological endpoint; other endpoints are the concentrations resulting in 20 % or 80 % dye release. 2. Developer of the method The neutral red release assay (NRR assay; Reader et al. 1989, 1990) is a variant of the widely used neutral red uptake assay (Borenfreund and Puerner, 1984, 1985). In the original protocol normal human epidermal keratinocytes (NHEK) have been used (Reader et al., 1989, 1990; cf. Harbell et al., 1997, and Zuang, 2001). Later, a standard protocol with balb/c 3T3 mouse fibroblasts has been developed (INVITTOX protocol no. 54, Clothier, 1992; cf. Zuang, 2001) as well as a commercial assay kit using the SIRC cell line derived from rabbit cornea (PREDISAFETM; cf. Guyomard et al., 1994, and Zuang, 2001). 3. Known Users The NRR has been used primarily by the cosmetic (personal care) industry to evaluate surfactant-based formulations. 4. Status of validation and/or standardization The NRR assay has been included in several interlaboratory studies. The NRR assay using NHEK cells and an exposure time of 5 min. performed well in the studies conducted in the U.S.A. within the CTFAs Alternatives Program. It was one of the six tests that had the best correlation with the Draize test when hydro-alcoholic formulations were evaluated (Gettings et al., 1991). Furthermore, it belonged to the group of those 14 endpoints (out of a total of 34 in vitro assays investigated) showing the least discordance with results of the Draize test when surfactant-based formulations were tested (Gettings et al., 1996). It did, however, not perform so well with oil/water emulsions (Gettings et al., 1994). This conclusion was also reached in the critical appraisal of the NRR assay by the IRAG Working Group on Cell Cytotoxicity Assays (Harbell et al., 1997) which evaluated the outcome of the CTFA study and the data from another study on very mild materials for eye care use. The NRR assay was considered capable for separating non-toxic materials from mild to moderate and more toxic materials and for assessing surfactant-based materials over the range of toxicities normally found in personal care products. However, it should only be applied with caution for the testing of non-surfactant formulations. The influence of product class and class of chemical tested became apparent in the study carried out by the COLIPA study which investigated the effects of 55 different test materials using the commercial PREDISAFETM kit with SIRC cells. The NRR assay did not very accurately predict the Draize MMAS (the correlation coefficient ranged from 0.607 to 0.708), although it performed better than the neutral red uptake assay in that it covered a wider range of eye irritation scores (Brantom et al., 1997). 5. Fields of application and limitations The NRR assay has been shown to be especially effective in the mild to very mild range. It can resolve degrees of mildness where the animal cannot. The assay measures immediate action on the target cells, generally as a function of the concentration of the test material required to reduce the amount of pre-bound dye in the lysosomes of the cell population. Thus, it is amenable to testing surfactants and surfactant formulations.
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The assay would not be appropriate for test materials that act on the cells in ways that do not show an immediate change in membrane integrity. As is the case with most of the cell monolayer-based test systems, it is also limited to water soluble test materials where dilution does not alter the basic toxicity of the materials (e.g., surfactants in contrast to acids or bases). 6. Recommended use in the options of animal replacement The neutral red release assay (NRR assay) is fast to perform and easy to standardize. It is not designed to be a test of general cytotoxicity but has been developed to identify substances potentially capable of causing toxic responses when in brief contact with the eye (such as may occur by accident). The NRR assay is therefore appropriate to evaluate surface-active agents such as surfactants and detergents. Due to the very short exposure time high concentrations of test material need to be tested. The material should be soluble in or, at least, miscible with water and have only limited acid and alkaline reserve. Extremely high or low pH values and reactivity with the culture medium must be avoided in order to ensure the results obtained are accurate and useful (e.g. Harbell et al., 1997; Spielmann, 1997). It is important to assess each individual product class carefully. Highly soluble products of relatively simple structure appear to be better candidates than more complex or even hydrophobic formulations (e.g. Christian and Diener, 1996). 7. On-going development 8. Efforts needed to complete validation In 2001, the European Centre for the Validation of Alternative Methods (ECVAM) has reviewed the status of the NRR assay and recommended to include it in further validation studies for the evaluation of particular product classes such as surfactantbased formulations once the protocol and prediction models have been optimised (Zuang, 2001). 9. Key references
Balls M; Berg N; Bruner LH; Curren R; deSilva O; Earl LK; Esdaile DJ; Fentem JH; Liebsch M; Ohno Y; Prinsen MK; Spielmann H; Worth AP; Eye irritation testing: the way forward. The report and recommendations of ECVAM workshop 34.; Alternatives to laboratory animals, ATLA; 27; 53-77; 1999 Barstadt R; Cortesi J; Janus J; Use of Clonetics neutral red bioassay to optimize components of serumfree medium for normal human anchorage-dependent cells.; In vitro cellular & developmental biology; 27; 160-; 1991 Borenfreund E; Puerner JA; Toxicity determined in vitro by morphological alterations and neutral red absorption.; Toxicology letters; 24; 119-124; 1985 Borenfreund E; Puerner JA; A simple quantitative procedure using monolayer cultures for cytotoxicity assays (HTD/ NR-90).; Journal of tissue culture methods; 9(1); 7-9; 1984 Brantom PG; Bruner LH; Chamberlain M; Desilva O; Dupuis J; Earl LK; Lovell DP; Pape WJW; Uttley M; Bagley DM; Baker FW; Brachter M; Courtellemont P; Declercq L; Freeman S; Steiling W; Walker AP; Carr GJ; Dami N; Thomas G; Harbell J; Jones PA; Pfannenbecker U; Southee JA; Tcheng M; Argembeaux H; Castelli D; Clothier R; Esdaile DJ; Itigaki H; Jung K; Kasai Y; Kojima H; Kristen U; Larnicol M; Lewis RW; Marenus K; Moreno O; Peterson A; Rasmussen ES; Robles C; Stern M; A summary report of the COLIPA international validation study on alternatives to the Draize rabbit eye irritation test.; Toxicology in vitro; 11(N1-2); 141-179; 1997
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Bruner LH; Silva O de; Earl LK; Easty DL; Pape W; Spielmann H; Report on the COLIPA workshop on mechanisms of eye irritation.; Alternatives to laboratory animals, ATLA; 26; 811-820; 1998 Christian MS; Diener RM; Soaps and detergents - alternatives to animal eye irritation tests.; Journal of the American College of Toxicology; 15(1); 1-44; 1996 Clothier RH; The FRAME neutral red release assay.; Invittox Protocol; 54; 1992 Courtellemont P; Hebert P; Biesse JP; Castelli D; Friteau L; Serrano J; Robles C; Relevance and reliability of the PREDISAFE assay in the COLIPA eye irritation validation program (phase 1).; Toxicology in vitro; 13(2); 305-312; 1999 Guyomard C; Bouffechoux J; Bourniche J; Chesne C; Evaluation of PREDISAFE, a cell kit for predicting eye irritancy of cosmetic raw materials and formulations.; Cell biology and toxicology; 10; 375-379; 1994 Gettings, S.D., Lordo, R.A., Hintze, K.L., Bagley, D.M., Casterton, P.L., Chudkowski, M., Curren, R.D., et al. (1996) The CFTA evaluation of alternatives program: An evaluation of in vitro alternatives to the Draize Primary Eye Irritation Test. (Phase III) Surfactant-based Formulations. Food and Chemical Toxicology 34:79-117. Gettings SD; Dipasquale LC; Bagley DM; Casterton PL; Chudkowski M; Curren RD; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase II), oil/water emulsions.; Food and chemical toxicology; 32(10); 943-976; 1994 Gettings SD; Teal JJ; Bagley DM; Demetrulias JL; DiPasquale LC; Hintze KL; Rozen MG; Weise SL; Chudkowski M; Marenus KD; Pape WJW; Roddy M; Schnetzinger R; Silber PM; Glaza SM; Kurtz PJ; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test (phase 1) hydro-alcoholic formulations; (part 2) data analysis and biological significance.; In vitro toxicology; 4(4); 247-288; 1991 Harbell, J.W. and Curren, R.D. (2001) In vitro methods for the prediction of ocular and dermal toxicity. In, Handbook of Toxicology, 2 nd Ed. M.J. Derelanko and M.A. Hollinger ED., CRC Press, Boco Raton, pp 835-866. Harbell, J.W., Koontz, S.W., Lewis, R.W., Lovel, D. and Acosta, D. (1997) IRAG working Group 4: Cell cytotoxicity Assays. Food and Chemical Toxicology 35: 79-126. Reader, S.J., Blackwell, V., OHara, R., Clothier, R.H., Griffin, G., and Balls, M. (1989) A vital dye release method for assessing the short term cytotoxic effects of chemicals and formulations. ATLA. 17: 28-37. Reader SJ; Blackwell V; O'Hara R; Clothier RH; Griffin G; Balls M; Neutral red release from preloaded cells as in vitro approach to testing for eye irritancy potential.; Toxicology in vitro; 4; 264-266; 1990 Schlede E; Gerner I; The Draize eye test and progress in development and acceptance of alternatives to this test in Europe. In: Goldberg AM, van Zutphen LFM (Eds): The World Congress on Alternatives and Animals Use in the Life Sciences: Education, Research, Testing. Mary Ann Liebert, Inc., New York; 333-336; 1995 Silva O de; Cottin M; Dami N; Roguet R; Catroux P; Toufic A; Sicard C; Dossou KG; Gerner I; Schlede E; Spielmann H; Gupta KC; Hill RN; Evaluation of eye irritation potential: statistical analysis and tier testing strategies.; Food and chemical toxicology; 35; 159-164; 1997 Spielmann H; Ocular irritation. In: Castell, Gomez-Lechon (Eds.): In vitro methods in pharmaceutical research. Academic Press, London; 265-287; 1997 Zuang V; The neutral red release assay: a review.; Alternatives to laboratory animals, ATLA; 29; 575599; 2001
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3.4.3. Red blood cell (RBC) haemolysis test 1. Short description , scientific relevance and purpose The red blood cell (lysis) test (or RBC test) is based on the (cytotoxic) potential of a chemical substance to disrupt cell membranes. Membrane damage is assessed by measuring photometrically the leakage of haemoglobin from freshly isolated red blood cells incubated with test materials under standard conditions (e.g. Muir et al., 1983; Pape et al., 1987; Pape and Hoppe, 1990; Lewis et al., 1993; Lewis, 1994). Access to mammalian erythrocytes is easy (e.g. slaughterhouse material). Thus, the RBC test contributes to the reduction of animal numbers used for eye irritation testing. Haemolysis and the denaturation of oxyhaemoglobin (oxyHb) are used as toxicological endpoints in the assay, which is carried out in two steps. In a rangefinding experiment haemolysis and oxyhaemoglobin denaturation (i.e. the change of protein configuration) is determined by measuring the reduction in absorbance at 541 nm (i.e. the one absorption maximum of oxyhaemoglobin) occurring within 60 minutes after exposure to increasing concentrations of test substance. The aim is to find the concentration range where haemolysis occurs. All determinations are made relative to a control sample, in which all erythrocytes were lysed (i.e. defined as 100% haemolysis). The main study serves to establish a more accurate concentration-response curve for haemoglobin leakage in order to calculate the concentration which causes 50 per cent haemolysis (H50 value). OxyHb denaturation determined in the range-finding experiment is expressed as D-max, i.e. the maximum percentage of denaturation seen at any concentration tested, and D-low, i.e. the lowest concentration at which denaturation becomes greater than 10 per cent. The studies by Pape and co-workers (1987, 1990, 1991) showed that any test substance causing haemolysis invariably produced some degree of eye irritation in the Draize rabbit eye test. The endpoints H50 and D-low were found to be inversely correlated with Draize MAS values whereas Dmax was positively correlated with the MAS. The ranking of compounds by both the haemolysis and the oxyhaemoglobin denaturation endpoints, in particular, when employing the lysis/denaturation ratio (L/D ratio), correlated significantly with in vivo eye irritation rankings. 2. Developer of the method The RBC test is based on the (cytotoxic) potential of a chemical substance to disrupt cell membranes (Muir et al., 1983; Pape et al., 1987; Pape and Hoppe, 1990; Pape and Pfannenbecker, 1992; Lewis et al., 1993; Lewis, 1994). One standard protocol of the RBC test was published as INVITTOX protocol no. 37 (Pape and Pfannenbecker 1992) and a second standard protocol derived from the first one was published as INVITTOX protocol no. 99 (Lewis, 1994). 3. Known users The RBC test is used in industry as part of an in vitro test battery which is routinely used as a pre-screen (e.g. Brantom et al., 1997; Harbell et al., 1997).
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4. Status of validation and/or standardisation The RBC test was one of the nine in vitro tests included in the world-wide validation study conducted for the EC/HO study (Balls et al., 1995a; the 60 substances tested included 12 surfactants), in which, however, none of the proposed alternatives to the Draize rabbit eye test proved sufficient to serve as complete substitute. At the same time the RBC test (INVITTOX protocol no. 37) has been assessed in the U.S.A. within the CTFAs Alternatives Program phase III on surfactant-based formulations (Gettings et al., 1996); the endpoints haemolysis, oxyhaemoglobin denaturation as well as L/D ratio were among the group of those 14 in vitro endpoints (out of a total of 34 investigated) with a sensitivity and specificity exceeding 80 % and showing the least discordance with results of the Draize eye test. Moreover, the RBC test has been critically appraised by the IRAG Working Group on Cell Cytotoxicity Assays evaluating data for 14 surfactants. The report pointed out that the RBC test was clearly able to distinguish between very mild and non-mild surfactants but that considerable additional data would be required for a proper assessment (Harbell et al., 1997). The interlaboratory study carried out under the auspices of the Japanese Ministry of Health and Welfare and the Japanese Cosmetic Industry Association involving six to nine laboratories showed acceptable interlaboratory reproducibility (Okamoto et al., 1999; Hatao et al., 1999). Although correlation between haemolysis and Draize MAS was moderate (-0.631), strongly irritating materials could be correctly classified. The study conducted by COLIPA (Brantom et al., 1997; Pape et al., 1999) confirmed that the RBC test was suited to assess acute effects of surfactant based formulations and ingredients. Interlaboratory reproducibility between the seven participating laboratories proved reasonably good. The study also presented several mathematical prediction models for eye irritation potential, one of which, the so-called classification model, has been designed to be used for ingredients (and formulations) other than surfactants (Brantom et al., 1997). 5. Fields and limitations of application, i.e., in vivo endpoints and/or chemical classes covered /not covered Most of the materials used in the studies to establish the RBC test have been surfactants; thus, at present, the assay is specific to this class of chemicals. So far, the RBC test has not been validated for any other classes of chemicals. Importantly, the RBC test can only be used for water-soluble and water-dispersible substances. Coloured substances can be measured using different test parameters for the membrane damage, such as the transmembrane ion exchange (W. Pape, unpublished data) and highly acidic or alkaline solutions may also not be suitable (e.g. Okamoto et al., 1999), which is not of importance since the acidic and alkaline materials are known to be severe eye irritants. 6. Recommendations of use in view of animal replacement Combination of the RBC test with other methods based on different mechanisms to improve prediction of eye irritation potential. 7. On-going development Recently, the RBC Test has been included in a project which evaluated several in vitro methods by applying reference standards in order to gain wider acceptance of such assays in the regulatory context (Balls, Brantom et al., 1999).
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8. Which efforts are needed to complete validation of the method? 9. References

Balls M; Botham PA; Bruner LH; Spielmann H; The EC/HO international validation study on alternatives to the Draize eye irritation test.; Toxicology in vitro; 9(6); 871-929; 1995a Balls M; Berg N; Bruner LH; Curren R; deSilva O; Earl LK; Esdaile DJ; Fentem JH; Liebsch M; Ohno Y; Prinsen MK; Spielmann H; Worth AP; Eye irritation testing: the way forward. The report and recommendations of ECVAM workshop 34.; Alternatives to laboratory animals, ATLA; 27; 53-77; 1999 Balls M; Brantom PG; Cassidy S; Esdaile D; Fentem J; Liebsch M; McPherson J; Pfannenbecker U; Prinsen M; Preliminary evaluation of the application of reference standards in the prevalidation and validation of in vitro test for eye irritation. In: DG Clark, SG Lisansky and R Macmillan: Alternatives to animal testing II, proceedings, Brussels. CPL Press, Berkshire; 201-204; 1999 Brantom PG; Bruner LH; Chamberlain M; Desilva O; Dupuis J; Earl LK; Lovell DP; Pape WJW; Uttley M; Bagley DM; Baker FW; Brachter M; Courtellemont P; Declercq L; Freeman S; Steiling W; Walker AP; Carr GJ; Dami N; Thomas G; Harbell J; Jones PA; Pfannenbecker U; Southee JA; Tcheng M; Argembeaux H; Castelli D; Clothier R; Esdaile DJ; Itigaki H; Jung K; Kasai Y; Kojima H; Kristen U; Larnicol M; Lewis RW; Marenus K; Moreno O; Peterson A; Rasmussen ES; Robles C; Stern M; A summary report of the COLIPA international validation study on alternatives to the Draize rabbit eye irritation test.; Toxicology in vitro; 11(N1-2); 141-179; 1997 Bruner LH; Silva O de; Earl LK; Easty DL; Pape W; Spielmann H; Report on the COLIPA workshop on mechanisms of eye irritation.; Alternatives to laboratory animals, ATLA; 26; 811-820; 1998 Harbell JW; Koontz SW; Lewis RW; Lovell D; Acosta D; IRAG Working Group 4. Cell cytotoxicity assays.; Food and chemical toxicology; 35; 79-126; 1997 Hatao M; Murakami N; Sakamoto K; Ohnuma M; Matsushige C; Kakishima H; Ogawa T; Kojima H; Matsukawa K; Masuda K; Chiba K; Yoshizawa K; Kaneko T; Iwabuchi Y; Matsushima Y; Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (4) Haemoglobin denaturation test.; Toxicology in vitro; 13; 125-137; 1999 Lewis RW; McCall JC; Botham PA; A comparison of two cytotoxicity tests for predicting the ocular irritancy of surfactants.; Toxicology in vitro; 7; 155-158; 1993 Lewis W; Red blood cell lysis and protein denaturation.; Invittox Protocol; 99; 1994 Muir CK; Flower C; Abb NJ van; A novel approach to the search for in vitro alternatives to in vivo eye irritancy testing.; Toxicology letters; 18; 1-5; 1983 Okamoto Y; Ohkoshi K; Itagaki H; Tsuda T; Kakishima H; Ogawa T; Kasai Y; Ohuchi J; Kojima H; Kurishita A; Kaneko T; Matsushima Y; Iwabuchi Y; Ohno Y; Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (3) Evaluation of the haemolysis test.; Toxicology in vitro; 13(1); 115-124; 1999 Pape WJW; Pfannenbecker U; Hoppe U; Validation of the red blood cell test system as in vitro assay for the rapid screening of irritation potential of surfactants.; Molecular toxicology; 1(4); 525-536; 1987 Pape WJW; Hoppe U; Standardization of an in vitro red blood cell test for evaluating the acute cytotoxic potential of tensides.; Arzneimittelforschung; 40(I), 4; 498-502; 1990 Pape WJW; Hoppe U; In vitro methods for the assessment of primary local effects of topically applied preparations.; Skin pharmacology; 4; 205-212; 1991 Pape WJW; Pfannenbecker U; Red Blood Cell Test System; Invittox Protocol 37; 1992
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Pape WJW; Pfannenbecker U; Argembeaux H; Bracher M; Esdaile DJ; Hagino S; Kasai Y; Lewis RW; COLIPA validation project on in vitro eye irritation tests for cosmetic ingredients and finished products (phase I): the red blood cell test for the estimation of acute eye irritation potentials. Present status.; Toxicology in vitro; 13(2); 343-354; 1999 Schlede E; Gerner I; The Draize eye test and progress in development and acceptance of alternatives to this test in Europe. In: Goldberg AM, van Zutphen LFM (Eds): The World Congress on Alternatives and Animals Use in the Life Sciences: Education, Research, Testing. Mary Ann Liebert, Inc., New York; 333-336; 1995 Silva O de; Cottin M; Dami N; Roguet R; Catroux P; Toufic A; Sicard C; Dossou KG; Gerner I; Schlede E; Spielmann H; Gupta KC; Hill RN; Evaluation of eye irritation potential: statistical analysis and tier testing strategies.; Food and chemical toxicology; 35; 159-164; 1997 Zebet30. The red blood cell haemolysis and protein denaturation test (RBC test) as an in vitro alternative assay to the in vivo rabbit eye irritation test. AnimAlt-Zebet database; 2001 http://www.bfr.bund.de/cms/detail.php?template=internet_en_index
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3.5. Cell function based assays 3.5.1. Fluorescein leakage (FL) 1. Short description, scientific relevance and purpose The fluorescein leakage test (FL test) is based on the principle that the corneal epithelium has the ability to function as an impermeable barrier to potentially hazardous chemical substances and that eye irritation can ensue when its structure is damaged. In the fluorescein leakage test, the corneal epithelium is mimicked in vitro by adherently growing cells which form tight junctions and desmosomes in culture. The test is performed in a variety of formats using MDCK cells as a rule, although monolayers or multilayers of epidermal keratinocytes (NHEK) or human corneal cells have also been employed (e.g. Cook et al., 1992; Rhoads et al., 1993; Kruszewski et al., 1995). Cells are grown on inserts so that, when confluent, the cell layer separates the medium into two compartments. The cells are exposed to the test substance in the upper compartment for a set period. Then the sample solution is removed and, after a washing step, the cells are incubated with a sodium fluorescein solution (usually for 30 minutes). In the event of (partial) disruption of the cell layer the amount of fluorescein diffusing into the lower compartment is measured spectrophotometrically. The results are expressed relative to the control values obtained with untreated cells (0 % permeation) or unseeded inserts (100 % permeation). Usually the concentrations of test substance causing a leakage of 20 % or 50 % are quoted as toxicological endpoint. Since a large part of the cells remain viable it is also possible to assess recovery after treatment (e.g. Cottin and Zanvit, 1997). 2. Developer of the method The FL test or trans-epithelial permeability assay was established by Tchao (1988) and developed further by Shaw et al. (1990, 1991). The 'Alternatives Laboratory' of the Fund for the Replacement of Animals in Medical Experiments (FRAME, Nottingham, U.K.) and a French cosmetics company have collaborated in working out a standard operational procedure (Clothier, INVITTOX protocol no. 71, 1993; cf. Cottin and Zanvit, 1997). 3. Known users The FL test is performed in several laboratories and is used in the cosmetics industry for in-house screening in a battery of tests (e.g. Cottin et al., 1994; Cottin and Zanvit, 1997; Botham et al., 1997; Drewitt-Barlow et al., 2000). 4. Status of validation and/or standardisation The fluorescein leakage took place in the major validation studies taking place from 1991-1997. The EC/HO validation study concluded that, while none of the nine alternative tests proved suitable to replace the Draize rabbit eye test in all respects, the FL test showed promise when combined with other in vitro tests like the isolated eye tests or uptake of neutral red. The COLIPA study attested the FL test to be reliable as a classification model predicting, in principle, three levels of irritancy, but being more applicable to samples in the mild to moderate irritation range. The report stated also that the reproducibility still had to be confirmed further. In the USA, the FL test (15 min exposure) was evaluated with surfactant-based formulations in Phase III of the Evaluation of Alternatives Program conducted by the CTFA and emerged in the group
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of fourteen well performing alternative assays (out of 34 endpoints investigated; Gettings et al., 1996; Lordo et al. 1999). In 1994, its performance was also critically appraised by the IRAG Working Group 3 on Cell Function Based (Botham et al., 1997). The Working Group considered it suitable as a screen for the eye irritation potential of surfactants, surfactant-based formulations and alcohols, but recommended further standardization and optimisation, e.g. in terms of the properties of the porous culture inserts to be used (cf. Ward et al., 1997), and the replacement of MDCK cells with epithelial cells of corneal origin (e.g. Kruszewski et al., 1995). A refined standard protocol providing solutions to these controversial issues has been proposed in the final report of a prevalidation study of the fluorescein leakage assay commissioned by the European Commission (Southee, 1998). Other workers propose to use the FL test in combination with vital dyes such as Alamar Blue to allow simultaneous measurement of cell layer integrity, cell viability and recovery from damage (e.g. Clothier et al., 1999). 5. Fields of application The fluorescence leakage test is limited to substances that are liquid or can be dissolved in aqueous buffer or light mineral oil. Viscous and solid materials cannot be properly tested as they are difficult to remove after treatment and corrosives are likely to kill the cells when applied within the dose range normally encountered. Thus it is most suited to assess the ocular effects of mild materials. 6. Recommendations of use Main recommendations of use are presented in point 4. 7. On-going development 8. Which efforts are needed to complete validation of the method? 9. References
Balls M; Botham PA; Bruner LH; Spielmann H; The EC/HO international validation study on alternatives to the Draize eye irritation test.; Toxicology in vitro; 9(6); 871-929; 1995a Botham P; Osborne R; Atkinson K; Carr G; Cottin M; Buskirk RG van; IRAG (Interagency Regulatory Alternatives Group) working group 3. Cell function based assays.; Food and chemical toxicology; 35(1); 67-77; 1997 Brantom PG; Bruner LH; Chamberlain M; Desilva O; Dupuis J; Earl LK; Lovell DP; Pape WJW; Uttley M; Bagley DM; Baker FW; Brachter M; Courtellemont P; Declercq L; Freeman S; Steiling W; Walker AP; Carr GJ; Dami N; Thomas G; Harbell J; Jones PA; Pfannenbecker U; Southee JA; Tcheng M; Argembeaux H; Castelli D; Clothier R; Esdaile DJ; Itigaki H; Jung K; Kasai Y; Kojima H; Kristen U; Larnicol M; Lewis RW; Marenus K; Moreno O; Peterson A; Rasmussen ES; Robles C; Stern M; A summary report of the COLIPA international validation study on alternatives to the Draize rabbit eye irritation test.; Toxicology in vitro; 11(N1-2); 141-179; 1997 Clothier R; The fluorescein leakage test.; Invittox Protocol; 71; 1993 Clothier RH; Starzec G; Stipho S; Kwong YC; Assessment of initial damage and recovery following exposure of MDCK cells to an irritant.; Toxicology in vitro; 13; 713-717; 1999 Cook J; Gabriels J; Patrone L; Rhoads L; Buskirk RG van; A human epidermal model that can be used in an automated multiple endpoint assay.; Alternatives to laboratory animals, ATLA; 20; 313-324; 1992
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Cottin M; Dossou KG; Silva O de; Tolle M; Roguet R; Cohen C; Catroux P; Delabarre I; Sicard C; Rougier A; Relevance and reliability of in vitro methods in ocular safety assessment.; In vitro toxicology; 7(3); 277-282; 1994 Cottin M; Zanvit A; Flourescein leakage test: a useful tool in ocular safety assessment.; Toxicology in vitro; 11(4); 399-405; 1997 Drewitt-Barlow B; McPherson F; Broyd F; Williamson P; No tears for baby.; Global cosmetical industry; 167; 16-20; 2000 Gettings SD; Lordo RA; Hintze KL; Bagley DM; Casterton PL; Chudkowski M; Curren RD; Demetrulias JL; DiPasquale LC; Earl LK; Feder PI; Galli CL; Glaza SM; Gordon VC; Janus J; Kurtz PJ; Marenus KD; Moral J; Pape WJW; Renskers KJ; Rheins LA; Roddy MT; Rozen MG; Tedeschi JP; Zyracki J; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (phase III) Surfactant-based formulations.; Food and chemical toxicology; 34(1); 79-117; 1996 Kruszewski FH; Walker TL; Ward SL; DiPasquale LC; Progress in the use of human ocular tissues for in vitro alternative methods.; Comments on toxicology; 5(3); 203-224; 1995 Lordo RA; Feder PI; Gettings SD; Comparing and evaluating alternative (in vitro) tests on their ability to predict the Draize maximum average score.; Toxicology in vitro; 13(1); 45-72; 1999 Rhoads LS; Cook JR; Patrone LM; Buskirk RG van; A human epidermal model can be assayed employing a multiple fluorescent endpoint assay and the CytoFluor 2300.; Journal of toxicology / Cutaneous and ocular toxicology; 12(2); 87-108; 1993 Shaw AJ; Clothier RH; Balls M; Loss of trans-epithelial impermeability of a confluent monolayer of Madin-Darby Canine Kidney (MDCK) cells as a determinant of ocular irritancy potential.; Alternatives to laboratory animals, ATLA; 18; 145-151; 1990 Shaw AJ; Balls M; Clothier RH; Bateman ND; Predicting ocular irritancy and recovery from injury using madin-darby canine kidney cells.; Toxicology in vitro; 5/6; 569-571; 1991 Southee JA; Evaluation of the prevalidation process. The fluorescein leakage assay. Part 2, final report, European Community contract no. 11279-95-10F1ED ISP GB.; 3; 1998 Tchao R; Trans-epithelial permeability of fluorescein in vitro as an assay to determine eye irritants. In: A.M. Goldberg (Ed.): Alternative Methods in Toxicology. Mary Ann Liebert, Inc., New York; 6; 271283; 1988 Ward RK; Mungall S; Carter J; Clothier RH; Evaluation of tissue culture insert membrane compatibility in the fluorescein leakage assay.; Toxicology in vitro; 11(6); 761-768; 1997 Zebet270. The assessment of the eye irritation potential of chemical substances with the fluorescence leakage test. AnimAlt-Zebet database; 2001 http://www.bfr.bund.de/cms/detail.php?template=internet_en_index
3.5.2 Silicon Microphysiometer (SM) or Cytosensor Microphysiometer 1. Short description, scientific relevance and purpose In the assay using the silicon microphysiometer (Bruner, Miller et al., 1991; Bagley et al., 1992, McConnell et al., 1992; Catroux et al., 1993) the changes induced by chemical substances in the metabolic activity of adherent cell cultures, e.g. commercially available normal human epidermal keratinocytes (NHEK) or the fibroblastic L929 mouse cell line, serve as (indirect) measure of irritation potential.
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For the SM assay, cells grown on suitable inserts are placed in a miniature flowthrough chamber which is operated in stopped-flow mode. One of the walls of the chamber is made of coated silicon; and this coating contains pH-sensitive hydroxyl and amino groups. The cultured cells release acidic metabolic products into the medium surrounding them; as a result, the pH of the medium decreases. This produces in turn a change in the surface potential of the coating of the silicon 'sensor chip' or potentiometer which can be correlated to the metabolic state of the cells. At first, the cells' normal metabolic rate (baseline rate) is measured. Cells are then exposed to increasing concentrations of test material and incubated with each dose for different exposure times. After each exposure, the test material is removed and the metabolic rate (i.e. acidification of the medium) is measured again. This is repeated until the cells no longer release any acidic metabolites. The concentration of the test material that inhibits the metabolic rate to 50 per cent (so-called MRD50 value) of control levels is calculated and serves as the toxicological endpoint. 2. Developer of the method Bruner, Miller et al. (1991), McConnell et al. (1992) and Catroux et al. (1993). 3. Known users The assay is used as in-house test in the cosmetics industry as part of a tiered testing procedure (see Botham et al., 1997). It thus helps to reduce the number of animals required for testing eye irritation potential. 4. Status of validation and/or standardisation The assay using the silicon microphysiometer has been evaluated in several validation studies: in the U.S.A. in a comparison of seven methods by Bruner et al. (1991), in a multicentre study (Bagley et al., 1992), and in the Evaluation of Alternatives Program of the CTFA phase III: surfactant-based formulations (Gettings et al., 1996); in Europe in a comparative study of eight methods by Rougier et al. (1992) and in two separate interlaboratory studies, one of which was initiated by the EC/HO study (Balls et al., 1995a) while the other study was conducted by COLIPA (Brantom et al., 1997; Harbell et al., 1999). The performance of the microphysiometer was also critically appraised by IRAG Working Group 3 (Botham et al., 1997). Interlaboratory reproducibility and correlation with Draize MAS scores were good. The COLIPA study noted that the eye irritation potential of acid, alkaline and organic materials was underpredicted by the microphysiometer assay, but that surfactants and surfactantbased formulations were characterized correctly (Harbell et al., 1999). The IRAG Working Group recommended the microphysiometer assay for irritancy screening of liquids, water-soluble substances and surfactant-based products such as personal care and household cleaning products. In order to obtain most reliable data it is recommended that the product to be tested and the in-house benchmark reference share a similar chemical composition (cf. Spielmann, 1997). 5. Fields and limitations of application Overall, the silicon microphysiometer assay is able to classify eye irritating materials into broad categories such as innocuous-mild, mild-moderate and moderate-severe. It is suitable for screening liquid, water-soluble surfactants and surfactant-based formulations that fall into these broad categories of irritancy. The method is noninvasive and the cells are remaining intact; thus, it is also possible to study the recovery of metabolic activity after treatment. However, the method has two
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disadvantages: it can only be used to assess liquid materials, i.e. water-miscible substances or formulations. 6. Recommendations of use in the view of animal replacement For screening of surfactants and surfactant-based formulations that fall into the broad categorisation of innocuous-mild, mild-moderate and moderate to severe (Balls et al., 1995a, Brantom et al, 1997 and Botham et al., 1997). Use as part of a battery of tests in combination with ex vivo methods to enhance the overall predictive capacity of the Cytosensor Microphysiometer for surfactant based materials especially at the sever end of the irritancy spectrum. 7. On-going development No known ongoing development of the Cytosensor Microphysiometer assay system. 8. Efforts are needed to complete validation of the method Evaluation of surfactant materials of even greater irritancy than those typically found in consumer products to confirm the capacity of the Cytosensor Microphysiometer to distinguish severe from moderately irritating surfactant-based materials. 9. References
Bagley DM; Bruner LH; Silva O de; Cottin M; O'Brien KAF; Uttley M; Walker AP; An evaluation of five potential alternatives in vitro to the rabbit eye irritation test in vivo.; Toxicology in vitro; 6; 136149; 1992 Balls M; Botham PA; Bruner LH; Spielmann H; The EC/HO international validation study on alternatives to the Draize eye irritation test.; Toxicology in vitro; 9(6); 871-929; 1995a Botham P; Osborne R; Atkinson K; Carr G; Cottin M; Buskirk RG van; IRAG (Interagency Regulatory Alternatives Group) working group 3. Cell function based assays.; Food and chemical toxicology; 35(1); 67-77; 1997 Brantom PG; Bruner LH; Chamberlain M; Desilva O; Dupuis J; Earl LK; Lovell DP; Pape WJW; Uttley M; Bagley DM; Baker FW; Brachter M; Courtellemont P; Declercq L; Freeman S; Steiling W; Walker AP; Carr GJ; Dami N; Thomas G; Harbell J; Jones PA; Pfannenbecker U; Southee JA; Tcheng M; Argembeaux H; Castelli D; Clothier R; Esdaile DJ; Itigaki H; Jung K; Kasai Y; Kojima H; Kristen U; Larnicol M; Lewis RW; Marenus K; Moreno O; Peterson A; Rasmussen ES; Robles C; Stern M; A summary report of the COLIPA international validation study on alternatives to the Draize rabbit eye irritation test.; Toxicology in vitro; 11(N1-2); 141-179; 1997 Bruner LH; Denver JK; Deirdre AR; Parker RD; Evaluation of seven in vitro alternatives for ocular safety testing.; Fundamental and applied toxicology; 17; 136-149; 1991 Bruner LH; Miller KR; Owicki JC; Parce JW; Muir VC; Testing ocular irritancy in vitro with the silicon microphysiometer.; Toxicology in vitro; 5; 277-284; 1991 Catroux P; Rougier A; Dossou KG; Cottin M; The silicon microphysiometer for testing ocular toxicity in vitro.; Toxicology in vitro; 7; 465-469; 1993 Gettings SD; Lordo RA; Hintze KL; Bagley DM; Casterton PL; Chudkowski M; Curren RD; Demetrulias JL; DiPasquale LC; Earl LK; Feder PI; Galli CL; Glaza SM; Gordon VC; Janus J; Kurtz PJ; Marenus KD; Moral J; Pape WJW; Renskers KJ; Rheins LA; Roddy MT; Rozen MG; Tedeschi JP; Zyracki J; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (phase III) Surfactant-based formulations.; Food and chemical toxicology; 34(1); 79-117; 1996
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Harbell JW; Osborne R; Carr GJ; Peterson A; Assessment of the cytosensor microphysiometer assay in the COLIPA in vitro eye irritation validation study.; Toxicology in vitro; 13(2); 313-323; 1999 McConnell HM; Owicki JC; Parce JW; Miller DL; Baxter GT; Wada HG; Pitchford S; The cytosensor microphysiometer: biological applications of silicon technology.; Science; 257; 1906-1912; 1992 Rougier A; Cottin M; DeSilva O; Roguet R; Catroux P; Tougic A; Dossou K; In vitro methods: their relevance and complementarity in ocular safety assessment.; Lens and eye toxicity research; 9(3-4); 229-245; 1992 Spielmann H; Ocular irritation. In: Castell, Gomez-Lechon (Eds.): In vitro methods in pharmaceutical research. Academic Press, London; 265-287; 1997 Zebet245. The assessment of the eye irritation potential of chemical substances using the silicon microphysiometer. AnimAlt-Zebet database; 2001 http://www.bfr.bund.de/cms/detail.php?template=internet_en_index
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3.6. Other assays 3.6.1. Mucosal irritation model: using slugs (e.g. Arion lusitanicus) 1. Short description , scientific relevance and purpose The eye irritation potential of chemicals can be estimated with the alternative mucosal irritation test using the slug Arion lusitanicus as test organism (Adriaens and Remon, 2002). The irritation effect of the test substances can be estimated by the amount of mucus produced during treatment, and the membrane damage by the release of proteins, LDH and ALP from the mucosal surface of the slug after treatment. Based on these two endpoints the test substances can be classified into the 3 categories corresponding the EU classification (NI, R36 and R41). 2. Developer of the method The mucosal irritation test using slugs has been developed at the laboratory of Pharmaceutical Technology (University of Ghent) and is based on the work of Adriaens and co-workers (Adriaens and Remon, 1999; Adriaens et al., 2001; Adriaens and Remon, 2002). 3. Known users It is routinely used for safety assessment of bioadhesive powder formulations at the laboratory of Pharmaceutical Technology (Ghent University). Occasionally new formulations are tested for their irritation potential for the pharmaceutical industry. 4. Status of validation and/or standardisation The mucosal irritation test did not pass a formal validation study. The model has been optimised by testing 28 reference chemicals from the ECETOC database, and a prediction model has been developed. The reproducibility of the test procedure was evaluated by testing the 28 chemicals 5 times on different occasions. No prevalidation or validation has been set up. 5. Fields and limitations of application, i.e. in vivo endpoints and/or chemical classes covered/not covered Tested chemicals (n=28) covered the whole irritation scale (0-110). Chemical classes that were tested (solids and liquids) are: alcohols (11), surfactants (5), ketones (2), anorganics (2) and miscellaneous (8). 6. Recommendations of use in the topics of alternative replacement Until this test passed formal validation, the test is suited as a pre-screen for eye irritation. 7. On-going development A protocol has been developed to assess local tolerance (nasal, buccal, vaginal mucosa) of pharmaceutical formulations. Until now, more than 70 formulations were tested. 8. Efforts needed to complete validation of the method This method should first be presented to ECVAM in order to decide whether or not it is ready to undergo a pre-validation study.
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9. References
Adriaens, E. and Remon, J.P. (1999). Gastropods as an evaluation tool for screening the irritating potency of absorption enhancers and drugs. Pharm. Res. 16: 1240-1244. Adriaens, E., Dierckens, K., Bauters, T.G.M., Nelis, H.J., Van Goethem, F., Vanparys, P. and Remon, J.P. (2001). The mucosal toxicity of different benzalkonium chloride analogues with an alternative test using slugs. Pharm. Res. 18: 937-942 Callens, C., Adriaens, E., Dierckens, K. and Remon, J.P. (2001). T oxicological evaluation of a bioadhesive nasal powder containing a starch and carbopol 974 p on rabbit nasal mucosa and slug mucosa. J. Contr. Rel. 76: 81-91. Ceulemans J., Vermeire A., Adriaens E., Remon J.P. and Ludwig A. (2001). Evaluation of a mucoadhesive tablet for ocular use. J. Control. Release. 77: 333-344 Adriaens E. and Remon J.P. (2002). The evaluation of an alternative mucosal irritation test using slugs. J. Appl. Tox. Pharm. 182: 169-175 Adriaens, E., Ameye, D., Dhondt, M. M. M., Foreman, P. and Remon, J. P. (2003). Evaluation of the mucosal irritation potency of co-spray dried Amioca/Poly(Acrylic Acid) and Amioca/Carbopol 974P mixtures. J. Control. Release 88:393-399. Dhondt, M.M.M., Adriaens, E. and Remon, J.P.(2003). The Evaluation of the local tolerance of vaginal formulations, with or without nonoxynol-9, using the slug mucosal irritation test. Sex. Trans. Dis. (accepted, October).
3.6.2. The IRRITECTION assay 1. Short description , scientific relevance and purpose The IRRITECTION assay (In Vitro International, Irvine, U.S.A.) is the updated protocol of the former EYTEXTM. This biochemical assay kit is based on the premise that eye irritation and corneal opacity after exposure to irritating chemicals is the result of perturbation or denaturation of corneal proteins (Kelly, 1989). It uses a special macromolecular reagent composed of oligomeric protein, carbohydrates, lipids and low molecular weight components, that when rehydrated, form an ordered macromolecular matrix that mimics the highly ordered structure of the transparent cornea. Test chemicals, when presented gradually to the reagent by utilizing a membrane disc of defined porosity, produce a turbidity of the reagent by changes in conformation and degree of hydration. The turbidity, measured spectrophotometrically, is compared with that produced by eye irritant standards of known Draize score. Whereas chemical irritants typically produce a linear or sigmoidal dose-response, surfactants present a different dose-response curve. 2. Developer of the method In vitro International, Irvine CA, USA. 3. Known users The former EYTEXTM assay has been used for in-house testing world-wide in the chemical, cosmetics and drugs industries and a large database has been built up (cf. Spielmann, 1997). 4. Status of validation and/or standardisation
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The recent IRRITECTION assay did not take part of any major validation study, but the former EYTEXTM assay was tested in the major validation or evaluation studies that took place between 1991-1997. It showed a good correlation with the Draize score test in some studies (Christian and Diener, 1996; Gordon, 1992; Decker and Haper, 1993), and less good or only satisfactory correlation in other studies amongst which the EC/HO study and the Japanese study (Bruner et al., 1991; Balls et al., 1995a; Matsukawa et al., 1999; Sina et al, 1995). At the CTFA program, it performed well with hydroalcoholic formulations but less well with surfactant-based formulations (Gettings et al., 1991; Gettings et al., 1996). The IRAG working group 5 found the EYTEXTM assay suitable for the assessment of shampoos, petrochemical materials and anionic surfactants (Curren et al, 1997). Whereas the COLIPA study concluded that the EYTEXTM assay was not suitable for the assessment of surfactants and formulations based on surfactants (Courtellemont et al., 1999). 5. Fields and limitations of application, i.e., in vivo endpoints and / or chemical classes covered / not covered The former EYTEXTM assay was reported to present limitations in what concerns intensely coloured substances. It underestimated the effects of some cationic surfactants (Matsukawa et al., 1999), whereas those of surfactant formulations containing magnesium and multicarboxylated carbohydrate materials were overestimated (Balls et al., 1995a; cf. Spielmann, 1997). 6. Recommendations of use in the view of animal replacement The COLIPA study suggested that the former EYTEXTM assay be refined and reevaluated in terms of its limitations and well defined mathematical prediction models were required (Brantom et al., 1997; Courtellemont et al., 1999). Furthermore, the former existence of the various different types of EYTEXTM assay protocols also caused difficulties in evaluating the data obtained and in assessing transferability among test laboratories participating in the comparative trials (Curren et al., 1997). Despite these shortcomings the former EYTEXTM was still regarded to have some utility as a primary screen for selected classes of chemicals and certain sets of materials (Curren et al., 1997), or for use in internal comparative studies in combination with specific benchmarks (Courtellemont et al., 1999). The present IRRITECTION assay is the updated protocol of the former EYTEXTM, and additional data is required before any recommendation for validation could occur. 7. On-going development 8. Which efforts are needed to complete validation of the method? 9. References
Balls M; Botham PA; Bruner LH; Spielmann H; The EC/HO international validation study on alternatives to the Draize eye irritation test.; Toxicology in vitro; 9(6); 871-929; 1995a Brantom PG; Bruner LH; Chamberlain M; Desilva O; Dupuis J; Earl LK; Lovell DP; Pape WJW; Uttley M; Bagley DM; Baker FW; Brachter M; Courtellemont P; Declercq L; Freeman S; Steiling W; Walker AP; Carr GJ; Dami N; Thomas G; Harbell J; Jones PA; Pfannenbecker U; Southee JA; Tcheng M; Argembeaux H; Castelli D; Clothier R; Esdaile DJ; Itigaki H; Jung K; Kasai Y; Kojima H; Kristen U; Larnicol M; Lewis RW; Marenus K; Moreno O; Peterson A; Rasmussen ES; Robles C; Stern M; A summary report of the COLIPA international validation study on alternatives to the Draize rabbit eye irritation test.; Toxicology in vitro; 11(N1-2); 141-179; 1997
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Bruner LH; Denver JK; Deirdre AR; Parker RD; Evaluation of seven in vitro alternatives for ocular safety testing.; Fundamental and applied toxicology; 17; 136-149; 1991 Christian MS; Diener RM; Soaps and detergents - alternatives to animal eye irritation tests.; Journal of the American college of toxicology; 15(1); 1-44; 1996 Courtellemont P; Pannetier M; Biesse JP; Larnicol M; Baret JP; Breda B; Evaluation of the EYTEX system in the COLIPA eye irritation program.; Toxicology in vitro; 13(2); 295-304; 1999 Curren RD; Sina FJ; Feder P; Kruszewski FH; Osborne R; Regnier JF; IRAG (Interagency Regulatory Alternatives Group) working group 5 - other assays.; Food and chemical toxicology; 35(1); 127-158; 1997 Decker D; Harper R; Evaluation of the Eytex system for use as a predictor of ocular irritancy of shampoos. In: AM Goldberg (Ed.): Alternative methods in toxicology. Mary Ann Liebert, Inc., New York; 9; 236; 1993 Gettings SD; Teal JJ; Bagley DM; Demetrulias JL; DiPasquale LC; Hintze KL; Rozen MG; Weise SL; Chudkowski M; Marenus KD; Pape WJW; Roddy M; Schnetzinger R; Silber PM; Glaza SM; Kurtz PJ; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test (phase 1) hydro-alcoholic formulations; (part 2) data analysis and biological significance.; In vitro toxicology; 4(4); 247-288; 1991 Gettings SD; Lordo RA; Hintze KL; Bagley DM; Casterton PL; Chudkowski M; Curren RD; Demetrulias JL; DiPasquale LC; Earl LK; Feder PI; Galli CL; Glaza SM; Gordon VC; Janus J; Kurtz PJ; Marenus KD; Moral J; Pape WJW; Renskers KJ; Rheins LA; Roddy MT; Rozen MG; Tedeschi JP; Zyracki J; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (phase III) Surfactant-based formulations.; Food and chemical toxicology; 34(1); 79-117; 1996 Gordon VC; The scientific basis of the EYTEX system.; Alternatives to laboratory animals, ATLA; 20; 537-548; 1992 Kelly CP; EYTEX: an in vitro method of predicting ocular safety.; Pharmacopeial forum; 15; 48155824; 1989 Matsukawa K; Masuda K; Kakishima H; Suzuki K; Nakagawa Y; Matsushige C; Imanishi Y; Nakamura T; Mizutani A; Watanabe R; Shingai T; Kaneko T; Hirose A; Ohno Y; Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients.; Toxicology in vitro; 13(1); 209217; 1999 Sina JF; Galer DM; Sussman RG; Gautheron PD; Sargent EV; Leong B; Shah PV; Curren RD; Miller K; A collaborative evaluation of seven alternatives to the Draize eye irritation test using pharmaceutical intermediates.; Fundamental and applied toxicology; 26(1); 20-31; 1995 Spielmann H; Ocular irritation. In: Castell, Gomez-Lechon (Eds.): In vitro methods in pharmaceutical research. Academic Press, London; 265-287; 1997 Zebet271. The EYTEX assay system as an in vitro alternative to the Draize rabbit eye irritation test. AnimAlt-Zebet database; 2001 http://www.bfr.bund.de/cms/detail.php?template=internet_en_index
3.6.3. The Pollen Tube Growth (PTG) assay 1. Short description , scientific relevance and purpose The toxic effects of chemical substances on the growth of plant pollen tubes have originally been evaluated microscopically by measuring the lengths of several hundred tubes (Gentile et al., 1978; Wolters and Martens, 1987). The pollen tube
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growth test (PTG test) is based on photometric quantification of the production of pollen tube mass under standardised conditions in vitro (Kappler and Kristen, 1987). This is facilitated by the fact that pollen is a non-photosynthetic tissue; hence there is no interference from chlorophylls and other pigments. Pollen from tobacco plants (Nicotiana sylvestris) are suspended at a defined concentration in growth medium and are cultured for 18 hours in the presence of various concentrations of the test substance. The mass of pollen tubes produced during this period is photometrically determined either by measuring the turbidity of the pollen suspension after the incubation with the test substance or by staining the tubes with the dye Alcian blue, which binds specifically to water-insoluble polysaccharides of the pollen tube wall and dissociates after acidification (Kappler and Kristen, 1988). The pollen tubes are precipitated by centrifugation. The extinction values of the redissolved dye in the supernatant, which directly correlate with the pollen tube mass, are determined and the percentages of tube mass increase, i.e. tube growth, are plotted against the test concentration to yield a concentration-response curve. Toxicological endpoint is the median effective ' dose' or ED50, i.e. the concentration of a test substance that reduces pollen tube growth to 50 % of the control. It is calculated from at least five sets of concentration-response curves. 2. Developer of the method Standard protocols for the assay have been published (Kappler and Kristen, INVITTOX protocol no. 55, 1990; Kristen and Kappler, 1995). 3. Known users The PTG test is used in the cosmetics industry for in-house screening and the safety assessment of cosmetics, for the evaluation of pollutants in environmental surveys and for basic research into the biochemical mechanisms of pollen germination. 4. Status of validation and/or standardisation In comparative studies the ED50 values obtained in the PTG test correlated well with the data of other in vitro toxicity assays (Strube et al., 1991) and rat LD50 values (Kristen and Kappler, 1995). The PTG test has therefore been included in several larger validation studies. It performed well in the Evaluation of Alternatives Program conducted by the US CTFA (Feder et al., 1991; Gettings et al., 1991, 1994, 1996; Lordo et al., 1999) and belonged to the group of twelve assays (out of a total of 23 types of in vitro assay investigated) showing the best agreement with results of the Draize test. The CTFA study tested substances with very low to moderate irritation potential: Phase I (1991) evaluated hydroalcoholic formulations, phase II (1994) oil/water emulsions and phase III (1996) surfactant-based formulations. The COLIPA study (Brantom et al., 1997; Kristen et al., 1999) confirmed that the PTG test was suited to assess finished formulations. The prediction model used, however, did not prove satisfactory for individual classes of chemicals such as alcohols, surfactants and strong acids and alkali. Especially, the irritation potential of cationic surfactants was underestimated, while that of anionic surfactants was overestimated. In consequence, there is a need for additional experimental studies to obtain a larger and more relevant database in order to derive a better prediction model (Brantom et al., 1997; Kristen et al., 1999).
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5. Fields and limitations of application, i.e., in vivo endpoints and / or chemical classes covered / not covered The pollen tube growth test seems to be able to predict acute eye irritation potentials for cosmetic formulations. However, the current database does not permit to reliably predict the irritation potential of distinct classes of chemicals and the prediction model currently in use needs to be modified (Brantom et al., 1997; Kristen et al., 1999). The PTG test is not suited for substances insoluble in water or unable to form emulsions. 6. Recommendations of use in the view of animal replacement The pollen tube growth test may contribute to reduce testing on animals as part of a battery of in vitro tests (for pre-screening purposes). However, larger-scale validation is required since the PTG test has not been assessed in parallel by more than two or three laboratories in any of the studies mentioned. The PTG test has been included, nevertheless, in the Multicenter Evaluation of In vitro Cytotoxicity (MEIC) Programme of the Scandinavian Society of Cell Toxicology (Ekwall et al., 1990; Clemedson et al., 1996). 7. On-going development 8. Which efforts are needed to complete validation of the method? 9. References
Brantom PG; Bruner LH; Chamberlain M; Desilva O; Dupuis J; Earl LK; Lovell DP; Pape WJW; Uttley M; Bagley DM; Baker FW; Brachter M; Courtellemont P; Declercq L; Freeman S; Steiling W; Walker AP; Carr GJ; Dami N; Thomas G; Harbell J; Jones PA; Pfannenbecker U; Southee JA; Tcheng M; Argembeaux H; Castelli D; Clothier R; Esdaile DJ; Itigaki H; Jung K; Kasai Y; Kojima H; Kristen U; Larnicol M; Lewis RW; Marenus K; Moreno O; Peterson A; Rasmussen ES; Robles C; Stern M; A summary report of the COLIPA international validation study on alternatives to the Draize rabbit eye irritation test.; Toxicology in vitro; 11(N1-2); 141-179; 1997 Clemedson C; McFarlane-Abdulla E; Andersson M; Barile FA; Calleja MC; Chesne C; Clothier R; Cottin M; Curren R; Dierickx P; Ferro M; Fiskesjoe G; Garza-Ocanas L; Gomez-Lechon MJ; Guelden M; MEIC evaluation of acute systemic toxicity. Part II: In vitro results from 68 toxicity assays used to test the first 30 reference chemicals and a comparative cytotoxicity analysis.; Alternatives to laboratory animals, ATLA; 24; 273-311; 1996 Ekwall B; Barile F; Bjarregaard H; Chesne C; Clothier R; Dierickx P; Preliminary results from the Scandinavian multicentre evaluation of in vitro cytotoxicity (MEIC).; Toxicology in vitro; 4(4-5); 688691; 1990 Feder PI; Lordo RA; Dipasquale LC; Bagley DM; Chudkowski M; Demetrulias JL; Hintze KL; Marenus KD; Pape WJW; Roddy M; Schnetzinger R; Silber PM; Teal JJ; Weise SL; Gettings SD; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test (phase I) hydro-alcoholic formulations; (part I) statistical methods.; In vitro toxicology; 4(4); 231-246; 1991 Gentile AG; Vaughan AW; Pfeiffer DG; Cucumber pollen germination and tube elongation inhibited or reduced by pesticides and adjuvants.; Environmental entomology; 7(5); 689-691; 1978 Gettings SD; Teal JJ; Bagley DM; Demetrulias JL; DiPasquale LC; Hintze KL; Rozen MG; Weise SL; Chudkowski M; Marenus KD; Pape WJW; Roddy M; Schnetzinger R; Silber PM; Glaza SM; Kurtz PJ; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test (phase 1) hydro-alcoholic formulations; (part 2) data analysis and biological significance.; In vitro toxicology; 4(4); 247-288; 1991
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Gettings SD; Dipasquale LC; Bagley DM; Casterton PL; Chudkowski M; Curren RD; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase II), oil/water emulsions.; Food and chemical toxicology; 32(10); 943-976; 1994 Gettings SD; Lordo RA; Hintze KL; Bagley DM; Casterton PL; Chudkowski M; Curren RD; Demetrulias JL; DiPasquale LC; Earl LK; Feder PI; Galli CL; Glaza SM; Gordon VC; Janus J; Kurtz PJ; Marenus KD; Moral J; Pape WJW; Renskers KJ; Rheins LA; Roddy MT; Rozen MG; Tedeschi JP; Zyracki J; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (phase III) Surfactant-based formulations.; Food and chemical toxicology; 34(1); 79-117; 1996 Kappler R; Kristen U; Photometric quantification of in vitro pollen tube growth: a new method suited to determine the cytotoxicity of various environmental substances.; Environmental and experimental botany; 27(3); 305-309; 1987 Kappler R; Kristen U; Photometric quantification of water-insoluble polysaccharides produced by in vitro grown pollen tubes.; Environmental and experimental botany; 28(1); 33-36; 1988 Kappler R; Kristen U; Pollen test system.; Invittox Protocol; 55; 1990 Kristen U; Kappler R; The pollen tube growth test. In: O'Hare S, Atterwill CK (Eds.): Methods in molecular biology, vol. 43: In vitro toxicity testing protocols. Humana Press, Totowa, New Jersey; 189-198; 1995 Kristen U; Jung K; Pape W; Pfannenbecker U; Rensch A; Schell R; Performance of the pollen tube growth test in the COLIPA validation study on alternatives to the rabbit eye irritation test.; Toxicology in vitro; 13; 335-342; 1999 Lordo RA; Feder PI; Gettings SD; Comparing and evaluating alternative (in vitro) tests on their ability to predict the Draize maximum average score.; Toxicology in vitro; 13(1); 45-72; 1999 Strube K; Janke D; Kappler R; Kristen U; Toxicity of some herbicides to in vitro growing tobacco pollen tubes (the pollen test).; Environmental and experimental botany; 31(2); 217-222; 1991 Wolters JHB; Martens MJM; Effects of air pollutants on pollen.; The botanical review; 53(3); 372-414; 1987 Zebet101. The pollen tube growth (PTG) test as a pre-screening alternative for the determination of cytotoxicity and eye irritation potential of chemical substances. AnimAlt-Zebet database; 2001 http://www.bfr.bund.de/cms/detail.php?template=internet_en_index
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3.7. The Low Volume Eye Test (LVET) 1. Short description , scientific relevance and purpose Several refinements of the standard Draize rabbit eye test have been proposed over the years. The low volume eye test (LVET) was developed to investigated the basis for selecting the optimum testing conditions required in order to achieve high accuracy in the prediction of hazard to the human eye (Griffith et al., 1980, 1987; Freeberg et al., 1986). In contrast to the standard Draize eye test procedure, in the LVET only 0.01 ml of the test sample (instead of 0.1 ml or 0.1 mg) is applied directly to the cornea of the eye of the animal and not into the conjunctival sac. Moreover, the eyelid is not held closed forcedly afterwards to retain the sample, but natural mechanisms, i.e. blinking, are allowed to dilute or remove the test material. Numerical scoring of eye irritation is conducted according to the standard Draize system and a LVET-MAS is quoted correspondingly. Since only a tenth of the sample volume used in the Draize eye test is applied less pain has to be endured by the test rabbit. The ensuing irritation of the eye is smaller and the rates of recovery are higher. Like the Draize rabbit eye test protocol the LVET originally required six rabbits per test sample. Statistical studies conducted to determine the effects of reducing the number of animals used showed that a test on three animals would suffice to provide the same information (Bruner et al., 1992). A similar result was obtained in an earlier analysis for the standard Draize test (Talsma et al., 1988) and today the OECD and EU guidelines require only three animals per test (cf. Berdasco et al., 1996). 2. Developer of the method LVET as a refinement method of the Draize rabbit eye test was originally developed by the Procter & Gamble Company. 3. Known users It is used mainly in the United States by the cosmetics and household cleaning products industry (cf. Spielmann, 1997). 4. Status of validation and/or standardisation LVET results have been compared with the literature relating to human exposure, i.e. clinical and consumer eye irritation data (e.g. Allgood, 1989; Cormier et al., 1995) and by comparing the response in trials involving volunteers and rabbits (e.g. Freeberg et al., 1986; Roggeband et al., 2000). These studies indicate that the LVET provides in general a better prediction of the human response to eye irritants than the standard Draize rabbit eye test. The LVET has been evaluated with good reproducibility in several studies by different laboratories, e.g. in the multicentre study involving six different laboratories organized by OPAL (Blein et al., 1991), in the alternatives program of the American Soap and Detergents Association (SDA; Bagley et al., 1994), by Cormier et al. (1996) and in the Evaluation of Alternatives Program of the CTFA in the United States (Gettings et al., 1996, 1998a, 1998b). Linear correlation of data obtained using the LVET compared with the Draize MAS scores was 0.93 (Bagley et al., 1994; Gettings et al., 1996). In contrast, the precision of the LVET-MAS values for oil/water emulsions was found to be relatively poor (Gettings et al., 1998a).
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5. Fields and limitations of application, i.e., in vivo endpoints and / or chemical classes covered / not covered Most of the original datasets generated using LVET were surfactant-based materials or surfactant-based products with some data available on other classes of chemicals. It is currently unknown whether chemical supplier companies hold LVET data on a broader range of materials from chemical classes. More recently, publications such as that by Maurer et al., 2002 show LVET data on a range of materials from different chemical classes. 6. Recommendations of use in the view of animal replacement The US IRAG recommended the LVET to be used to substantiate the irritation potential of suspect severe ocular irritants that have not been unequivocally classified by various in vitro tests (Lambert et al., 1993). The LVET has also been recommended to be used as a reference test for the development of alternative (in vitro) assays to the Draize eye test (e. g. Roggeband et al., 2000). However, up to the present day the LVET has not been included in any guideline by national or international regulatory authorities. 7. On-going development No known ongoing development of the LVET. 8. Which efforts are needed to complete validation of the method? There are current initiatives to request ECVAM the validation of the use of LVET as a refinement in vivo eye irritation methodology for classification and labelling purposes within the Dangerous Substances Directive (Annex VA of the Dir. 67/548/EEC, Council Directive 92/32/EEC). Currently, consumer products subject to the Dangerous Preparations Directive can be tested using ex vivo eye irritation methods to determine if the product is a severe eye irritant meriting R41 classification. If the ex vivo test identifies eye irritancy as less than severe, a standard Draize eye irritancy test is required to further quantify the eye irritancy potential. The current request for LVET validation addresses the use of LVET as a refinement method instead of Draize should an in vivo test be required for classification and labelling purposes. Further validation of LVET beyond the current initiative would require a call of existing data from product manufacturers and chemical supplier companies to determine the extent of materials from different chemical classes evaluated in LVET to support a robust dataset for validation. 9. References
Allgood GS; Use of animal eye test data and human experience for determining the ocular irritation potential of shampoos.; Journal of toxicology / Cutaneous and ocular toxicology; 8(3); 321-326; 1989 Bagley D; Booman KA; Bruner LH; Casterton PL; Demetrulias J; Heinze JE; Innis JD; Iii WCM; Neun DJ; The SDA alternatives program phase III: comparison of in vitro data with animal eye irritation data on solvents, surfactants, oxidizing agents, and prototype cleaning products.; Journal of toxicology / Cutaneous and ocular toxicology; 13(2); 127-155; 1994 Berdasco NAM; Giblert KS; Lacher JW; Mattson JL; Low rate of severe injury from dermal and ocular irritation tests and the validity of using fewer animals.; Journal of the American College of Toxicology; 15(3); 177-193; 1996
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Blein O; Adolphe M; Lakhdar B; Cambar J; Gubanski G; Castelli D; Contie C; Hubert F; Latrille F; Masson P; Clouzeau J; Bigot JF Le; Silva O De; Dossou KG; Correlation and validation of alternative methods to the Draize eye irritation test (OPAL project).; Toxicology in vitro; 5(5-6); 555-557; 1991 Bruner LH; Parker RD; Bruce RD; Reducing the number of rabbits in the low volume eye test.; Fundamental and applied toxicology; 19(3); 330-335; 1992 Cormier EM; Hunter JE; Billheimer W; May J; Farage MA; Use of clinical and consumer eye irritation data to evaluate the low volume eye test.; Journal of toxicology / Cutaneous and ocular toxicology; 14(3); 197-205; 1995 Cormier EM; Parker RD; Henson C; Cruse LW; Merritt AK; Bruce RD; Osborne R; Determination of the intra- and interlaboratory reducibility of the low volume eye test and its statistical relationship to the Draize eye test.; Regulatory toxicology and pharmacology; 23(2); 156-161; 1996 Council Directive 92/32/EEC of 30 April 1992 amending for the seventh time Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances. Official Journal L 154, 05/06/1992 p.1. Freeberg FE; Nixon GA; Reer PJ; Weaver JE; Bruce RD; Griffith JF; Sanders LW; Human and rabbit eye responses to chemical insult.; Fundamental and applied toxicology; 7; 626-634; 1986 Gettings SD; Lordo RA; Demetrulias J; Feder PI; Hintze KL; Comparison of low volume, Draize and in vitro eye irritation test data. I. Hydroalcoholic formulations.; Food and chemical toxicology; 34(8); 737-749; 1996 Gettings SD; Lordo RA; Feder PI; Hintze KL; A comparison of low volume, Draize and in vitro eye irritation test data. III. surfactant-based formulations.; Food and chemical toxicology; 36(3); 209-231; 1998 Gettings SD; Lordo RA; Feder PI; Hintze KL; A comparison of low volume, Draize and in vitro eye irritation test data. II. Oil/water emulsions.; Food and chemical toxicology; 36(1); 47-59; 1998 Griffith JF; Nixon GA; Bruce RD; Reer PJ; Bannan EA; Dose-response studies with chemical irritants in the albino rabbit eye as a basis for selecting optimum testing conditions for predicting hazard to the human eye.; Toxicology and applied pharmacology; 55; 501-513; 1980 Griffith JF; The low volume eye irritation test.; Soap cosmetics chemical specialities; 63(4); 32-63; 1987 Lambert LA; Chambers WA; Green S; Gupta KC; Hill RN; Hurley PM; Lee CC; Lee JK; Liu PT; Lowther DK; The use of low volume dosing in the eye irritation test.; Food and chemical toxicology; 31(2); 99-103; 1993 Maurer J.K., Parker R.D., Jester J.V. (2002) Extent of initial corneal injury as the mechanistic basis for ocular irritation: key findings and recommendations for the development of alternative assays. Regulatory Toxicology and Pharmacology 36, 106-117. Roggeband R; York M; Pericoi M; Braun W; Eye irritation responses in rabbit and man after single applications of equal volumes of undiluted model liquid detergent products.; Food and chemical toxicology; 38(8); 727-734; 2000 Spielmann H; Ocular irritation. In: Castell, Gomez-Lechon (Eds.): In vitro methods in pharmaceutical research. Academic Press, London; 265-287; 1997 Talsma DM; Leach CL; Hatoum NS; Gibbons RD; Roger JC; Garvin PJ; Reducing the number of rabbits in the Draize eye irritancy test: a statistical analysis of 155 studies conducted over 6 years.; Fundamental and applied toxicology; 10; 146-153; 1988
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Zebet236. The in vivo low volume eye test as a refinement of the Draize eye test to assess the eye irritation potential of chemical substances. AnimAlt-Zebet database; 2001 http://www.bfr.bund.de/cms/detail.php?template=internet_en_index
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3.8. Structure-activity relationships for eye irritation Eye irritation is a difficult endpoint to model in silico, because of the complexity of the biological mechanisms that may be involved. Most approaches have therefore modelled eye irritation resulting from physical effects, such as ocular penetration or corrosion (Cronin et al., 2003). The most commonly used approaches have focused on the development of: a) traditional quantitative models (in which a measure of eye irritation potency is predicted); b) classification models (in which a classification of eye irritation potential is predicted); c) the simulation of molecule-membrane interactions; d) neural network models; and e) expert system approaches.. Traditional quantitative models In a study by Cronin et al. (1994), the application of linear regression analysis to a data set of 23 physicochemical properties for 53 organic liquids led to the development of statistically significant QSARs (based on logP) for predicting the molar eye score (MES) of alcohols and acetates. The MES is the modified maximum average score (MMAS) corrected for the number of molecules applied to the rabbit eye. Subsequently, Abraham et al. (1998) used the solvatochromic parameters (molar refraction, polarisability, hydrogen bond acidity and basicity), which are derived from chromatography experiments, to model a data set comprising 38 of the 53 organic liquids previously analysed by Cronin et al. (1994). A QSAR for predicting the MES, based on these parameters and a vapour solubility parameter, had an r2 value of 0.89. In a more recent study by Abraham et al (2003), the MMAS values for 68 pure bulk liquids were adjusted by the liquid-saturated vapour pressure P. It was argued that the adjusted scores, expressed as log (MMAS/P), are equivalent to eye irritation thresholds (EIT) in humans, expressed as log (1/EIT). Analysis of a data set for 91 compounds showed that log (MMAS/P) or log (1/EIT) could be predicted by a fiveparameter solvatochromic equation with an R2 = 0.94. The authors suggested that the mechanism of action in the Draize test and in the human EIT involves passive transfer of the compound to a biophase that does not resemble water or plasma, but resembles an organic solvent, such as N-methylformamide. Classification models In addition to the derivation of regression models, attempts have also been made to develop classification models for eye irritation. For example, Cronin et al. (1994) applied linear discriminant analysis to the data set for 53 organic liquids, but found no linear combination of physicochemical properties capable of discriminating between irritant and non-irritant chemicals (as defined by EU classification criteria). However, in a PC plot based on all 23 variables, the irritant chemicals appeared to form an embedded cluster within the non-irritant chemicals. Similar findings were subsequently reported by Barratt (1995). The phenomenon of embedded clustering of irritant chemicals was investigated further by Cronin (1996), this time by using the technique of cluster significance analysis (CSA) to determine whether the embedded clustering was statistically significant. Out of a total of 23 physicochemical descriptors, it was reported that the five most significant were logP, logP 2, the heat of formation, dipole moment, and a
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topological index. Subsequently, the method of embedded cluster modelling was developed (Worth & Cronin, 1999), which generates elliptic prediction models for the embedded clusters, and was applied to an eye irritation data set (Worth & Cronin, 2000). Simulation of molecule-membrane interactions A different approach to the prediction of the molar eye score was adopted by Kulkarni & Hopfinger (1999). In addition to using parameters based on solute properties, they also used a molecular dynamics method to generate intermolecular membrane-solute interaction properties. QSARs based on these properties were then derived by using a genetic algorithm. A QSAR based on three parameters (two energy terms and a topological index) had an r2 value of 0.92. The 16 chemicals used to derive the model were aliphatic and aromatic hydrocarbons, and aliphatic ketones, alcohols and acetates. Neural network approach In contrast to the studies summarised above, which aimed to develop (Q)SARs for non-surfactant chemicals, an investigation by Patlewicz et al. (2000) focused on surfactants. In this study, neural network analysis indicated that the MAS of cationic surfactants is positively correlated with surfactant concentration and critical micelle concentration, and negatively correlated with logP. Expert system approaches The physicochemical determinants of eye irritation potential were also investigated by Rosenkranz et al. (1998). In comparison with non-irritant chemicals, these workers reported that irritant chemicals have significantly lower molecular weights, higher aqueous solubilities, lower logP values, and greater molecular orbital energy gaps (absolute hardness values). On the basis of the last-named observation, it was concluded that chemical reactivity does not appear to be a requirement for eye irritation. The authors also used the Multi-CASE expert system to identify biophores (substructures which occur with a significantly greater frequency in irritants than in non-irritants) and biophobes (substructures which occur significantly more frequently in non-irritants). The major structural determinants included primary, secondary and tertiary amine groups (i.e. basic groups), as well as carboxylate, organosulphate and sulphonate groups (i.e. acidic groups). In addition to Multi-CASE, other expert systems containing rules for eye irritation include DEREK (which contains 29 structural alerts for eye irritation), HazardExpert, and TOPKAT (which contains 15 chemical class-specific QSARs; Cronin et al, 2003). References
Abraham, M.H., Kumarsingh, R., Cometto-Muniz, J.E. & Cain, W.S (1998). A quantitative structureactivity relationship (QSAR) for a Draize eye irritation database. Toxicology in Vitro 12, 201-207. Abraham, M.H, Hassanisadi, M., Jalali-Heravi, M., Ghafourian, T., Cain, W.S. & Cometto-Muniz, J.E. (2003). Draize rabbit eye test compatibility with eye irritation thresholds in humans: a quantitative structure-activity relationship analysis. Toxicological Sciences 76, 384-392. Barratt, M.D. (1995). A quantitative structure-activity relationship for the eye irritation potential of neutral organic chemicals. Toxicology Letters 80, 69-74.
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Cronin, M.T.D. (1996). The use of cluster significance analysis to identify asymmetric QSAR data sets in toxicology. An example with eye irritation data. SAR and QSAR in Environmental Research 5, 167175. Cronin, M.T.D., Basketter, D.A. & York, M. (1994). A quantitative structure-activity relationship (QSAR) investigation of a Draize eye irritation database. Toxicology in Vitro 8, 21-28. Cronin, M.T.D., Jaworska, J.S., Walker, J.D., Comber, M.H.I, Watts, C.D. & Worth, A.P. (2003). Use of QSARs in international decision-making frameworks to predict health effects of chemical substances. Environmental Health Perspectives 111, 1391-1401. Kulkarni, A.S. & Hopfinger, A.J. (1999). Membrane-interaction QSAR analysis: application to the estimation of eye irritation by organic compounds. Pharmaceutical Research 16, 1245-1253. Patlewicz, G.Y., Rodford, R.A., Ellis, G. & Barratt, M.D. (2000). A QSAR model for the eye irritation of cationic surfactants. Toxicology in Vitro 14, 79-84. Rosenkranz, H.S., Zhang, Y.P. & Klopman, G. (1998). The development and characterisation of a structure-activity relationship model of the Draize eye irritation test. ATLA 26, 779-809. Worth, A.P. & Cronin, M.T.D. (1999). Embedded cluster modelling a novel method for analysing embedded data sets. Quantitative Structure-Activity Relationships 18, 229-235. Worth A.P. & Cronin, M.T.D. (2000). Embedded cluster modelling: a novel QSAR method for generating elliptic models of biological activity. In Progress in the Reduction, Refinement and Replacement of Animal Experimentation (eds. M. Balls, A-M. van Zeller & M.E. Halder). pp. 479-491. Elsevier Science, Amsterdam.
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3.9. The COLIPA Eye Irritation Research Programme Reduction and refinement methods/approaches for the evaluation of eye irritation are available today but validated replacement method(s) have not yet been achieved. There remains a clearly identified need to define alternatives methods that reliably predict the human eye response to chemicals exposure and can replace the in vivo test. As such, a fundamental understanding of what is needed to fill the knowledge gaps is essential to continued progress. As a result of the several reviews that have been conducted to define the way forward for the development and validation of eye irritation alternative methods, the key focus area for future research that emerged is the need for mechanistic understanding of eye injury resulting from chemical exposure. So, for in vitro replacement eye irritation tests to be reliable and predictive of the human response, they will need to be based upon mechanistically relevant biological events. Mechanistically-based in vitro tests for ocular irritation will likely depend upon: 1) well characterised ocular cellular models, 2) assays that measure biochemical endpoints of cellular injury and 3) a database of human responses - all of which cover a wide range of chemical classes and varying degrees of eye irritation. To address development of alternative methods based on mechanistically relevant biological events, the European Cosmetics Industry Trade Association (COLIPA) Steering Committee for Alternatives to Animal Testing (SCAAT) has developed a collaborative research programme with academia. The research programme builds directly on the mechanistic work performed by Maurer et al., 2002 on depth of injury as the basis for predicting initial injury and the dynamics of injury and recovery of the eye from exposure to materials that may or may not be eye irritants. This area of mechanistic understanding has been identified as key for the successful development of animal alternatives methods for eye irritation. There are currently two integrated parts to the COLIPA eye irritation research programme. The first part is focused on the pharmacokinetics of the response of the in vitro perfused cornea to chemical irritants. It is designed to demonstrate whether patterns of change in physiological function and signals of injury released from the cornea can be used to predict a chemicals potential to damage the eye. The time course over which the morphological changes are being studied is up to 22 days. The physiological parameters being measured are primarily: barrier function; corneal thickness and opacity; changes in pH; redox potential and levels of potassium and release of enzymes and inflammatory mediators. The second part of the programme is focused on looking at injury and recovery in human corneal cells, in conventional monolayer cultures and in 3-dimensional models that incorporate the epithelium and stroma or the epithelium, stroma and endothelium. The aim of this part of the programme is to identify endpoints related to the magnitude of injury and the quality of repair in human immortalised cells and 3-dimensional constructs. Also addressed is the characterisation and selection of cell lines. The quality of the constructs is being studied and opacity is measured by confocal microscopy. Keratocyte activation and the release of enzymes and inflammatory mediators is assessed as well as recovery. Net, The COLIPA-funded research in eye irritation is directed towards understanding the mechanism of eye injury and identification of new in vitro endpoints predictive of the in vivo response to chemical injury. The outcome of the research is to have a better understanding of the cellular and molecular mechanisms of chemically induced eye
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irritation. This will hopefully lead to new, appropriate in-vitro end points, more predictive of the in vivo response of the human eye to irritant chemicals, resulting in new or optimised current non-animal methods that would proceed to formal validation. Other areas of potential interest such as changes in gene expression induced by chemicals during injury and recovery are currently being explored by SCAAT to determine the need to extend the current research programme with additional focus areas.
References Balls M; Berg N; Bruner LH; Curren R; deSilva O; Earl LK; Esdaile DJ; Fentem JH; Liebsch M; Ohno Y; Prinsen MK; Spielmann H; Worth AP; Eye irritation testing: the way forward. The report and recommendations of ECVAM workshop 34; Alternatives to laboratory animals, ATLA; 27; 53-77; 1999 Bruner LH; Silva O de; Earl LK; Easty DL; Pape W; Spielmann H; Report on the COLIPA workshop on mechanisms of eye irritation.; Alternatives to laboratory animals, ATLA; 26; 811-820; 1998 de Silva, O. & Basketter, D. (2002) The Research Program of the Steering Committee on Alternatives to Animal Testing (SCAAT). Fourth World Congress on Alternatives and Animal Use in the Life Sciences, New Orleans, USA, 11-15 August 2002 Griffith M, Osborne R, Munger R, Xiong X, Doillon CJ, Laycock NL, Hakim M, Song Y, Watsky MA. Functional human corneal equivalents constructed from cell lines. Science 286(5447): 2169-72, 1999.
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4. Mechanisms of eye irritation occurring in vivo as modelled by in vitro assays Table 1 shows how the currently most advanced in vitro assays for ocular irritation can assess the different mechanisms of injury measured in vivo. Some of the assays do not measure depth of injury but are intended to identify the more severely irritating materials by the speed of their action of the target tissue and/or the dose that is required to product toxicity. Table 1: Mechanisms of eye irritation occurring in vivo as modelled by in vitro assays. Cytotoxicity Assay Category Assay Name Bovine Corneal Opacity and Permeability Isolated organs Isolated Rabbit Eye Chicken Enucleated Eye Test Organotypic Methods ChorioHET-CAM CAMVA Immediate (<5 minutes) Rapid (<30 minutes) Delayed (> 4 hours) Long postexposure with histology Long postexposure with histology* Long postexposure with histology* No No Partial Epithelium (Mild) Depth of Injury Full Upper Epithelium Stroma (Mild(Moderate) Moderate) Yes Yes (better with histology) With histology Yes (better with histology)
Deeper Stroma (Severe) With histology
Yes
Yes
Yes
Yes
Yes
Yes
Yes
With histology
Yes Yes Yes
Yes No Some
Yes Yes Yes
Yes Note 1 Note 1
With histology
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Chorioallantoic membrane methods Human corneal epithelium models
CAMTB EpiOcularTM SkinEthic HCE HCE-TEP (Gillette) Neutral Red Uptake Neutral Red Release Red Blood Cell Haemolysis Transepithelial Passage (MDCK cells) Silicon Microphysio meter
Yes Yes Yes Yes Yes Yes Yes
No Yes Yes Limited Yes No No
No Generally no Generally no No Yes No No
Yes Yes Yes Yes Note 4 Note 5 Note 5
Note 1 Yes Yes Yes Note 2 Note 2 Note 3
Cell based cytotoxicity methods
Cell function based assays
Yes
No
No
Note 5
Yes
Yes
No
Note 6
* The standard protocol extends observations for up to 240 minutes.
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Note 1: Depth of injury is not measured directly in any of the CAM-based assays. Severity of the irritation potential is based on the type of lesion and rapidity of action on the target tissue. Very rapid action on the CAM has been taken to suggest that a test material could act on the stroma as well as the epithelium. Moderate and severe irritants may be identified but the degree of severity might be difficult to determine. Note 2: Depth of injury to the stroma is not measured directly. Very rapid destruction of the epithelial construct is expected to predict rapid destruction of the epithelium in vivo and likely penetration into the stroma. Moderate and severe irritants may be identified but the degree of severity might be difficult to determine. Note 3: The HCE-TEP was developed to predict ocular irritation potential in the mild to moderate range. More irritating materials may be identified by the low concentration required to reduce barrier integrity. Note 4: The long exposure time (generally 24-48 hours) allows detection of all three types of cytotoxicity. However, depth of injury (and thus severity of irritation) is not measured directly. The degree of ocular irritation potential is inferred by the dose required to produce toxicity to the target cells. Note 5: The short exposure and immediate assessment of cell viability (integrity) limits these assays to those materials that act immediately on the cells (e.g., membrane lysis or protein coagulation). However, depth of injury (and thus severity of irritation) is not measured directly. The degree of ocular irritation potential is inferred by the dose required to produce toxicity to the target cells. Note 6: The dosing protocol and endpoint allows for assessment of immediate and some rapid (e.g. metabolic) actions on the cells. However, depth of injury (and thus severity of irritation) is not measured directly. The degree of ocular irritation potential is inferred by the dose required to produce toxicity to the target cells.
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5. Summary of the alternative methods currently available and foreseeable time to achieve ESAC endorsement Table 2: Inventory of the most advanced alternative methods currently available. Tests under development were not mentioned in the table. Current endpoints addressed in animal test
OECD TG 405, EU (B.5) Cornea opacity, iris lesion, redness of conjunctivae, oedema of conjunctivae (chemosis) IRE Opacity, swelling BCOP** Opacity, permeability, depth of injury Alternative tests available Estimated time to In vitro endpoints measured Purpose Area(s) of application Validation status Regulatory acceptance Comments have the method validated (ESAC endorsement)* Partial replacement (a) (tiered strategy and/or test battery) Partial replacement (b) (tiered strategy and/or test battery) Corneal swelling, opacity, CEET** permeability and gross morphological lesions Partial replacement (a) (tiered strategy and/or test battery) Mild, moderate and severe irritants Optimised
(d)
Limited Moderate to Severe irritants Optimised

(d, e)
acceptance by some member states (i) Limited 3 - 4 years

(j)
Severe irritants
acceptance Optimised
(d)
by some member states (i) Limited acceptance by some member states (i)
(Pre)screening of severe irritants
4 - 5 years
(j)
3 - 4 years
(j)
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Partial Haemorrhage, HET-CAM** lysis, coagulation replacement (c) (tiered strategy and/or test battery) Partial Haemorrhage, CAMVA Hyperaemia, constriction replacement (c) (tiered strategy and/or test battery) Partial replacement (c) CAMTB Cytotoxicity (tiered strategy and/or test battery) Partial replacement EpiOcular** Cytotoxicity (tiered strategy and/or test battery)
Severe irritants Mild/nonirritants for surfactants Optimised

(d)
Limited acceptance by some member states (i) 3-4 years

(j)
Recommended for Moderate to mild Optimised (f) surfactants and surfactant formulations 4-5 years
Optimised
4-6 years
Moderate to mild
Under validation (g)
Validation study on surfactant ingredients
3-4 years (others) (j) 1-2 years (surfactants)
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Partial replacement HCE** (SkinEthic) Cytotoxicity, histology (tiered strategy and/or test battery) Partial replacement HCE-TEP (Gillette) Permeability (tiered strategy and/or test battery) Partial replacement NRU Cytotoxicity (tiered strategy and/or test battery) Partial replacement Neutral Red Release Cytotoxicity (tiered strategy and/or test battery) Moderate to mild surfactants and surfactant formulations Optimised (f) 3-4 years
(j)
Discrimination of irritants vs. non irritants
Under prevalidation
(g)
Prevalidation study on chemicals 3-4 years

(j)
Moderate to mild Under Surfactants and surfactant formulations Severe irritants in combination with HET-CAM Optimised (f) 3-4 years
(j)
Validation study validation

(g)
on liquid surfactants (formulations)
2-4 years
69
Partial Haemolysis, RBC denaturation of oxyhaemoglobin replacement (tiered strategy and/or test battery) Partial replacement FL (MDCK cells) Permeability (tiered strategy and/or test battery) Partial replacement SM Cytotoxicity (tiered strategy and/or test battery) Broad classification for surfactants Optimised (f) 3-4 years(j) Mild to moderate irritants Prevalidated, extensive evaluation studies 3-4 years
(j)
Mild irritants for surfactants
Optimised (f)
3-4 years
(j)
70
Recommended to Cornea opacity, iris lesion, redness of LVET conjunctivae, oedema of conjunctivae (chemosis) Refinement All Optimised (f, h) be used to substantiate the irritation potential of suspect severe ocular irritants and as a reference for alternative tests * This table estimates the time needed to achieve ESAC endorsement for individual alternative tests a ssuming optimal conditions. It does not indicate the time needed to achieve full replacement of the animal test, nor does it include the time needed to achieve regulatory acceptance. Optimal conditions means that all necessary resources, for example technical, human, financial and coordination, are met at all times in the process and that the studies undertaken have successful outcomes. ** Most promising method for achieving animal replacement From slaughterhouses From animals used for other toxicological tests (c) The chicken eggs are used at day 9 of embryogenesis where pain perception have not yet developed (d) Not formally validated according to the current standards (Balls et al., 1995b) but underwent extensive evaluation studies (e) Underwent prevalidation (ECVAM) (f) Underwent extensive evaluation studies (g) Submission for peer-review in preparation (h) Submitted for peer-review (ECVAM) (i) For the classification of severe irritants (j) the estimated time could be shortened by the comparison of the existing data with good in vivo data, and validation could be achieved for some classes of irritants by a weight of evidence approach
(b) (a)
1-2 years
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6. Future prospects and recommendations for achieving animal replacement Validation studies seek to determine the reliability and relevance of a new method(s) compared to the existing method. Most of the previous validation studies on alternatives for eye irritation have succeeded in demonstrating the reproducibility and reliability of promising new methods but have failed to identify a single replacement assay for the in vivo Draize rabbit eye test. It is generally recognised that the range of criteria for injury and inflammation covered by the Draize rabbit eye test is unlikely to be replaced by a single in vitro test. This is due to different factors amongst which are the limited quality of the existing in vivo data (as reflected in its limited consistency), and some methodological limitations of the in vitro systems. The difficulties with existing (available) in vivo data are due mainly to its limited reproducibility across the laboratories from which the reference data have been drawn and the biological variability. The differences in test protocols, subjective scoring and variations in animal responses might all contribute to this low interlaboratory reproducibility (Weil and Scala, 1971 and Cormier et al, 1996). Difficulties are also recognized due to the classification system. First due to the fact that the R41 classification is triggered by two criteria that are not necessarily correlated to each other: the severity of effects on one hand (e.g., tissue scores) and persistence of effects on the other hand. Secondly due to differences in regulatory systems used for decision making regarding classification and labelling in various countries and regions (e.g. Europe, US, Japan). Finally, an additional factor which might hamper the in vitro / in vivo comparison is the comparison to solid materials due to variation in exposure times to solids, which in "old" in vivo studies can vary from a couple of minutes up to 24 hours, to the inflammation process following the mechanical irritation reactions of solids which are identified in vivo but not inevitably in vitro; and the other way round the detection of a strong cytotoxic reaction in vitro which might not become evident in vivo depending on solubility and exposure time. On the other hand, the alternative test systems may present methodological limitations in directly assessing all relevant in vivo parameters in one single method, such as detecting effects on conjunctiva, or assessing the persistence or reversibility of irritating effects. Moreover, some practical limitations might exist in terms of applicability of substances to be studied. In general, companies use in-house benchmark references of same composition than the product to be tested (Zuang, 2001). The use of such reference materials selected at the boundary between classification categories could facilitate the in vitro/in vivo comparison and the final classification of the substances (Balls et al., 1999). To overcome the fact that no single in vitro test system is likely to replace the range of criteria for injury covered by the Draize rabbit eye, several national and international organisations recommend the use of a tiered testing procedure for reducing the number of animals used. Proponents of tiered testing include the OECD (2002), the regulatory authorities in the U.S.A. (e.g. IRAG, Chamberlain et al., 1997; cf. de Silva et al., 1997) and in the EU countries (e.g. Schlede and Gerner, 1995) as well as ECVAM (see ECVAM Workshop 34, Balls et al., 1999). Within the aim of total animal test replacement, these strategies could be designed utilizing the strengths of particular in vitro assay systems to address required ranges of irritation potential and/or chemical classes.
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In order to accelerate the process of validation of the most promising methods for eye irritation, it is also recommended to assess the existing data on the different assays performing a weight of evidence validation approach. An ECVAM workshop will be organised in 2004 to discuss the best criteria to be used in such approach based on gained experience from the passed. However, in order to be successful the validation studies will require the availability of a high quality in vivo data set and a corresponding analysis of these, in order to identify and separate, what is primary injury and what is response to that, e.g. inflammatory process, persistent reactions, and finally which role plays recovery, and to compare with data obtained from in vitro methods. Larger database with reliable in vivo data, preferentially incorporating data such as that generated from the refinement Low-Volume-Eye-Test, on individual chemicals across chemical classes is required. One possible way to solve this issue without using any additional animal experimentation would be to carry parallel in vitro studies on new compounds when in vivo tests are perfomed until the entry into force of the marketing ban. The evaluation/validation could then be made simultaneously on both in vivo and in vitro tests. In addition, it is recommended to support further development of methods that should not only include single aspects of eye irritation (e.g. comparison of cornea, iris and conjunctivae Draize scores) but also the assessment of depth of injury and recovery as potential indicators for irreversibility and reversibility of effects (Maurer et al., 2002). With this purpose COLIPA is currently conducting a mechanistically-based research programme to identify new in vitro endpoints that are more predictive of the in vivo response of the human eye than is the Draize rabbit eye test and which, if successful, will lead to the development of optimised current and/or new animal alternatives methods for the evaluation of eye irritation (see chapter 3.9). Finally, a harmonized optimisation and standardization procedure of the prediction model is further recommended due to the differences in the regulatory classification schemes which had been in place in various countries and regions (e.g. Japan, US, Europe) when most of the existing validation studies took place. This recommendation refers also to already existing validation studies where this was not yet taken into account. With this purpose it may be beneficial, to compare existing data with the recently established OECD and UN GHS for eye irritancy (UN 2003). If the above-mentioned recommendations are followed and the tests prove to be valid, the most promising methods (BCOP, CEET, HET-CAM, EpiOcularTM, and the HCE model) could be validated following an ESAC endorsement for defined applications within 4 years. However, to replace the whole animal experimentation, testing strategies using these methods might be required. Its evaluation and the prediction of the most difficult in vivo endpoints to be replaced such as reversibility, persistence or mechanical irritation could lead to the need of additional years to achieve whole animal replacement. Moreover, a strategy for risk assessment purposes will also need to be defined. The process could be accelerated depending on the progress of the existing alternative tests, and of their prediction models. In addition, the outcome of ongoing research programmes, such as the COLIPA programme, will be instrumental to the progress of mechanistically based animal alternative methods for eye irritation. Taking these considerations into account it is estimated that regulatory acceptance using the current European Classification system could be achieved in excess of 6 years.
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7. References
AnimAlt-Zebet database (2001). http://www.bfr.bund.de/cms/detail.php?template=internet_en_index Balls M; Botham PA; Bruner LH; Spielmann H; The EC/HO international validation study on alternatives to the Draize eye irritation test; Toxicology in vitro; 9(6); 871-929; 1995a Balls, M., Blaauboer, B.J., Fentem, J.H., Bruner, L., Combes, R.D., Ekwall, B., Fielder, R.J., Guillouzo, A., Lewis, R.W., Lovell, D.P., Reinhardt, C.A., Repetto, G., Sladowski, D., Spielmann, H. & Zucco, F. (1995b). Practical aspects of the validation of toxicity test procedures. The report and recommendations of ECVAM workshop 5. ATLA 23, 129-147 Balls M; Berg N; Bruner LH; Curren R; deSilva O; Earl LK; Esdaile DJ; Fentem JH; Liebsch M; Ohno Y; Prinsen MK; Spielmann H; Worth AP; Eye irritation testing: the way forward. The report and recommendations of ECVAM workshop 34; Alternatives to laboratory animals, ATLA; 27; 53-77; 1999 Bradlaw J; Gupta K; Green S; Hill R; Wilcox N; Practical application of non whole animal alternatives: summary IRAG workshop on eye irritation. Food and chemical toxicology; 35;175-178; 1997 Brantom PG; Bruner LH; Chamberlain M; Desilva O; Dupuis J; Earl LK; Lovell DP; Pape WJW; Uttley M; Bagley DM; Baker FW; Brachter M; Courtellemont P; Declercq L; Freeman S; Steiling W; Walker AP; Carr GJ; Dami N; Thomas G; Harbell J; Jones PA; Pfannenbecker U; Southee JA; Tcheng M; Argembeaux H; Castelli D; Clothier R; Esdaile DJ; Itigaki H; Jung K; Kasai Y; Kojima H; Kristen U; Larnicol M; Lewis RW; Marenus K; Moreno O; Peterson A; Rasmussen ES; Robles C; Stern M; A summary report of the COLIPA international validation study on alternatives to the Draize rabbit eye irritation test.; Toxicology in vitro; 11(N1-2); 141-179; 1997 Chamberlain M; Gad SC; Gautheron P; Prinsen MK; IRAG (Interagency Regulatory Alternatives Group) working group 1. Organotypic models for the assessment/prediction of ocular irritation.; Food and chemical toxicology; 35(1); 23-37; 1997 Chambers WA; Green S; Gupta KC; Hill RN; Huntley K; Hurley PM; Lambert LA; Lee CC; Lee JK; Liu PT; Lowther DK; Roberts CD; Seabaugh VM; Springer JA; Wilcox NL; Scoring for eye irritation tests.; Food and chemical toxicology; 31(2); 111-115; 1993 Christian MS; Diener RM; Soaps and detergents - alternatives to animal eye irritation tests.; Journal of the American college of toxicology; 15(1); 1-44; 1996 Cormier, E.M, Parker, R.D., Henson, C., Cruze, L.W., Merritt, A.K., Bruce R.D., and Osborne, R. (1996) Regulatory Toxicology and Pharmacology 23:156-161. Cuellar, N., Lloyd, P.H., Swanson, J.E., Merrill, J.C., Clear, M.L., Mun, G., Harbell, J.H., and Bonnette, K.L. (2003) Evaluating the eye irritancy of solvents in a simple fragrance mixture with the bovine corneal opacity and permeability (BCOP) assay. The Toxicologist 72:312. Curren, R., Evans, M., Raabe, H., Dobson, T., and Harbell, J.(1999) Optimization of the bovine corneal opacity and permeability assay: histopathology aids understanding of the EC/HO false negative materials. ATLA 27:344. de Silva O de; Cottin M; Dami N; Roguet R; Catroux P; Toufic A; Sicard C; Dossou KG; Gerner I; Schlede E; Spielmann H; Gupta KC; Hill RN; Evaluation of eye irritation potential: statistical analysis and tier testing strategies.; Food and chemical toxicology; 35; 159-164; 1997
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Draize JH; Woodard G; Calvery HO; Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes.; The journal of pharmacology and experimental therapeutics; 82; 377-390; 1944 Fisher RG; Paller BS; Ziets GA; Ginn GL; Rachui SR; Predicting in vivo eye irritation potential using a tissue equivalent assay and a bovine corneal opacity and permeability test.; In vitro toxicology; 8(2); 139-147; 1995 Freeberg FE; Nixon GA; Reer PJ; Weaver JE; Bruce RD; Griffith JF; Sanders LW; Human and rabbit eye responses to chemical insult.; Fundamental and applied toxicology; 7; 626-634; 1986 Gettings SD; Dipasquale LC; Bagley DM; Casterton PL; Chudkowski M; Curren RD; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase II), oil/water emulsions.; Food and chemical toxicology; 32(10); 943-976; 1994 Gettings SD; Teal JJ; Bagley DM; Demetrulias JL; DiPasquale LC; Hintze KL; Rozen MG; Weise SL; Chudkowski M; Marenus KD; Pape WJW; Roddy M; Schnetzinger R; Silber PM; Glaza SM; Kurtz PJ; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test (phase 1) hydro-alcoholic formulations; (part 2) data analysis and biological significance.; In vitro toxicology; 4(4); 247-288; 1991 Gettings, S.D., Bagley, D.M., Chudlowski, M., Demetrulias, J.L., Dipasquale, L.C., Galli, C.L., Gay, R., Hintze, K.L., Janus, J., Marenus, K.D., Muscatiello, M.J., Pape, W.J.W., Renskers, K.J., Roddy, M.T. & Schnetzinger, R. (1992). Development of potential alternatives to the Draize eye test. The CTFA evaluation of alternatives program (Phase II). Review of materials and methods. ATLA 20, 164171. Gettings SD; Lordo RA; Hintze KL; Bagley DM; Casterton PL; Chudkowski M; Curren RD; Demetrulias JL; DiPasquale LC; Earl LK; Feder PI; Galli CL; Glaza SM; Gordon VC; Janus J; Kurtz PJ; Marenus KD; Moral J; Pape WJW; Renskers KJ; Rheins LA; Roddy MT; Rozen MG; Tedeschi JP; Zyracki J; The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (phase III) Surfactant-based formulations.; Food and chemical toxicology; 34(1); 79-117; 1996 Lordo RA; Feder PI; Gettings SD; Comparing and evaluating alternative (in vitro) tests on their ability to predict the Draize maximum average score.; Toxicology in vitro; 13(1); 45-72; 1999 Maurer, J.K. and Parker, R.D. (1996) Light microscopic comparison of surfactant-induced eye irritation in rabbits and rats at three hours and recovery/day 35. Toxicologic Pathology 24:403-411. Maurer, J.K., Molai, a., Parker, R.D., Li, L., Carr, G.J., Petroll, M.W., Cavanagh, D.H., and Jester, J.V. (2001) Pathology of ocular irritation with bleaching agents in the rabbit low-volume eye test. Toxicological Pathology 29(3):308-319. Maurer J.K., Parker R.D., Jester J.V. (2002) Extent of initial corneal injury as the mechanistic basis for ocular irritation: key findings and recommendations for the development of alternative assays. Regulatory Toxicology and Pharmacology 36, 106-117. Ohno Y; Kaneko T; Inoue T; Morikawa Y; Yoshida T; Fuji A; Masuda M; Ohno T; Hayashi M; Momma J; Uchiyama T; Chiba K; Ikeda N; Imanashi Y; Itakagaki H; Interlaboratory validation of the in vitro eye irritation tests for cosmetic ingredients. (1) Overview of the validation study and Draize scores for the evaluation of the tests.; Toxicology in vitro; 13(1); 73-98; 1999 Organization for Economic Co-Operation and Development (Ed.); OECD TG (405) for testing of chemicals. Acute eye irritation/corrosion; 2002 Schlede E; Gerner I; The Draize eye test and progress in development and acceptance of alternatives to this test in Europe. In: Goldberg AM, van Zutphen LFM (Eds): The World Congress on Alternatives and Animals Use in the Life Sciences: Education, Research, Testing. Mary Ann Liebert, Inc., New York; 333-336; 1995
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Spielmann, H., Kalweit, S., Liebsch, M., Wirnserberger, T., Gerner, I., Bertram-Neis, E., Krauser, K., Kreiling, R., Miltenburger, H.G., Pape, W. & Steiling, W. (1993). Validation study of alternatives to the Draize eye irritation test in Germany: cytotoxicity testing and HET-CAM test with 136 industrial chemicals Toxicology in Vitro 7, 505-510. Spielmann H; Liebsch M; Kalweit S; Moldenhauer F; Wirnsberger T; Holzhuetter HG; Schneider B; Glaser S; Gerner I; Pape WJW; Kreiling R; Krauser K; Miltenburger HG; Steiling W; Luepke NP; Mueller N; Kreuzer H; Muermann P; Spengler J; Betram-Neis E; Siegemund B; Wiebel FJ; Results of a validation study in Germany on two in vitro alternatives to the Draize eye irritation test, the HETCAM test and the 3T3 NRU cytotoxicity test.; Alternatives to laboratory animals, ATLA; 24; 741-858; 1996 Spielmann H; Ocular irritation. In: Castell, Gomez-Lechon (Eds.): In vitro methods in pharmaceutical research. Academic Press, London; 265-287; 1997 Swanson, J.E., White, B.T., Gran, B.P., Merrill, J.C., and Harbell, J.W. (2003) Evaluating oxidizing/reactive cleaning products in the bovine corneal opacity and permeability (BCOP) assay. The Toxicologist 72:220-221. United Nations-Economic Commission for Europe (UN/ECE) (2003). Globally Harmonised System of Classification and Labelling of Chemicals (GHS). UN, New York and Geneva, 2003. http://www.unece.org/trans/danger/publi/ghs/ghs.html Weil CS; Scala RA; Study of intra- and interlaboratory variability in the results of rabbit eye and skin irritation tests.; Toxicology and applied pharmacology; 19; 276-360; 1971 Wilhelmus KR; The Draize Eye Test.; Survey of ophthalmology; 45(6); 493-515; 2001 Worth AP; Balls M; Editors. Alternative (non-animal) methods for chemicals testing current status and future prospects. A report prepared by ECVAM working group on chemicals. ALTA 30; suppl.1; 2002 Zuang V. (2001) The neutral red release assay: a review. ATLA, 29; 575-599.
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4. Skin Sensitisation
David Basketter1, Silvia Casati2, G. Frank Gerberick3, Peter Griem4, Barry Philips5, Andrew Worth2
1
Unilever, Bedfordshire, UK; 2ECVAM-JRC, Ispra, Italy; 3Procter & Gamble, Cincinnati, USA; 4 Clariant, Sulzbach/Ts., Germany; 5RSPCA, West Sussex, UK.
1. Current data requirements and current tests Rules related to the safety of cosmetic products are imposed by the Council Directive 76/768/EEC (Cosmetics Directive) of 27 July 1976 (with subsequent amendments and adaptations). Article 2 states that: "A cosmetic product put on the market within the Community must not cause damage to human health when applied under normal or reasonably foreseeable conditions of use ..." Article 7 explains that "... the manufacturer shall take into consideration the general toxicological profile of the ingredient, its chemical structure and its level of exposure." In order to help manufacturers in complying with this legal obligation, the Commission's Scientific Committee on Cosmetics and Non-Food Products (SCCNFP) issued a guidance document (SCCNFP, 2000) that has the purpose to provide guidance for testing cosmetic ingredients and for the safety assessment of the finished product, both to the competent monitoring authorities of the Member States, and to persons responsible for putting cosmetics on the market (manufacturers or importers within the European Union). 1.1 For cosmetic ingredients In general, the regulations of the Council Directive 67/548/EEC on the classification, packaging and labelling of hazardous substances apply also to cosmetic ingredients. Some ingredients, e.g. certain plant extracts, do not fall under the directive. Although the SCCNFP pursues the aim of replacing in vivo sensitisation tests with in vitro alternatives, it concedes that no validated alternatives are available to date (SCCNFP, 2000). It therefore requires cosmetic ingredients to be tested in an animal test according to OECD and EU test guidelines and according to Good Laboratory Practice (GLP). The SCCNFP has evaluated the use of human tests for assessing the sensitisation hazard and concluded that: "As a general rule, cosmetic ingredients identified as sensitisers in animal assays or other validated assays when existing should not be studied in humans. The human sensitisation tests are time consuming and very expensive because a large number of volunteers is required in each test (150-200), though, considerable less for the human maximisation test (25) which, as the name says, maximises the response to a certain degree. Further, the selection of human volunteers usually results in the use of an inhomogeneous test group (compared with the more homogeneous group used for animal experiments). The large numbers of participants in most of these tests are necessary in order to reduce the 95% confidence interval for the test result, otherwise the likelihood of unpredicted responses in the consumers increases. If, for instance, no positive reaction occurred in 100 induced test subjects, for statistical reasons up to 36 of 1000 consumers may react. The argument for reducing the number of volunteers in the human maximisation test is the amplifying step
1
introduced by treatment with an irritant test product or sodium lauryl sulphate. In any case, it is scientifically inadequate and unethical to perform predictive tests with a number of subjects insufficient to produce valid data." It explains that: " Predictive human sensitisation tests involve attempts to induce a long lasting or permanent immunologic sensitisation in the individual. T herefore, serious ethical questions arise. In spite of many years of experience with human sensitisation tests, very limited scientific information is available in the literature regarding the consequences involved for the human volunteers who have developed a patch test sensitisation during such a test." and concludes that: " Due to the uncertainties mentioned above it is the opinion of the SCCNFP that predictive human sensitisation tests of potentially cutaneous sensitising cosmetic ingredients or mixtures of ingredients should not be carried out without a better understanding of the immunologic background and mechanisms underlying positive reactions in these kind of tests in human beings. Further, it is questionable whether predictive testing on humans contributes to human safety in comparison with animal testing. At the present, no alternative method for predicting sensitisation has been validated." (SCCNFP, 2000). 1.2 For cosmetic finished products Finished cosmetic products are exempt from Directive 1999/45/EG on the classification, packaging and labelling of hazardous preparations. Since several years, the cosmetic industry carries out the safety evaluations of finished cosmetic products through the use of animal tests on single ingredients, existing data, historic databases and computer expert systems. Finished cosmetic products are not tested on animals any more. With regard to sensitisation testing in human subjects, see opinion of SCCNFP in Section 1.1. 1.3 Available animal tests In the following, those animal tests are reported, for which test guidelines have been developed. Besides these, other protocols for sensitisation tests in guinea pigs (e.g., Draize test, open epicutaneous test, optimisation test, split adjuvant test) are available. 1.3.1 Mouse Local Lymph Node Assay (LLNA) 1.3.1.1 Short description, scientific relevance and purpose In the LLNA, the test substance is applied onto the mouse ear on three consecutive days. On the sixth day, the proliferation of lymphocytes in the draining lymph node, caused by the primary immune reaction, is measured. This is done usually by measurement of the incorporation of H-methyl thymidine into the DNA of proliferating lymphocytes in draining lymph nodes. The LLNA does not include a challenge phase. The endpoint of interest is the stimulation index giving the ratio of thymidine incorporation in lymph nodes from dosed animals compared to the incorporation in lymph nodes from vehicle-treated control animals. The test is positive when the stimulation index ? 3 (SI ? 3). The LLNA has been accepted by the Interagency Co-ordinating Committee on the Validation of Alternative Methods (ICCVAM) in USA as a stand alone alternative to the current guinea pig tests, and as an improvement for animal welfare because it reduces and refines animal use in the hazard identification of skin sensitising substances. The LLNA has been recognised as a
standalone test for skin sensitisation by competent authorities of European Union member states and by the SCCNFP. Another advantage of the LLNA over the guinea pig assays is that the EC3 value, which is the effective concentration of the test substance (percent of substance in vehicle) required to produce a threefold increase in the stimulation index compared to vehicle-treated controls can be used as a quantitative measure of the sensitising potency of the test substance. This might form the basis for a quantitative risk assessment for skin sensitisation in the future. 1.3.1.2 References Kimber et al., 1986; 1989; Kimber et al., 2002 ICCVAM evaluation: Dean et al., 2001; Haneke et al., 2001; Sailstadt et al., 2001 1.3.1.3 Status of validation and/or standardisation A test guideline is available as OECD Test Guideline No. 429. 1.3.2 Mouse Ear Swelling Test (MEST) 1.3.2.1 Short description, scientific relevance and purpose The MEST comprises both the induction phase and the elicitation phase of the immune response. Several weeks prior to and during the test period mice are fed a diet enriched in vitamin A since this has been shown to enhance contact sensitisation . In the protocol which has been used most frequently the induction phase comprises clipping of the fur on the belly region and removal of the outer layers of the epidermis by tape stripping. Freund's Complete Adjuvant (FCA) is injected intradermally before the test substance in vehicle (test mice) or vehicle alone (control mice) is applied topically. The skin is tape stripped each day during the following four days and on days 1, 3 and 5 after FCA injection the same amounts of test substance or vehicle alone are again applied topically. After a rest period of five days the challenge phase begins on day 10 with topical application of test substance at the maximum non-irritating concentration to one ear and of vehicle alone t the other ear of all (test and o control) mice. Ear thickness of test and control ears is measured under ether anaesthesia with a micrometer 24 and 48 hours after application of test substance. Ear swelling is expressed as the difference between test and control ears in percent. The MEST has been evaluated independently by several laboratories and in inter-laboratory studies. It was concluded that the MEST is a useful model for identifying strong contact sensitisers. The Mouse Ear Swelling Assay (MESA) is a variant of the MEST with some modifications in the test protocol. The use of the MESA is very limited. 1.3.1.2 References Asherson and Ptak, 1968; Gad, 1986; 1994 Evaluation: Cornacoff et al., 1988; Descotes, 1988; Dunn et al., 1990; Gad et al., 1987 (Cornacoff et al., 1988; Hignet et al., 1989; Dunn et al., 1990) 1.3.1.3 Status of validation and/or standardisation No standardised test guideline is available for the MEST.
3
1.3.3 Magnusson Kligman Guinea Pig Maximisation Test (GPMT) 1.3.3.1 Short description, scientific relevance and purpose The GPMT is a highly sensitive method using Freunds complete adjuvant as an immune enhancer. It includes both intradermal and topical induction treatment and closed challenge. Three pairs of intradermal injections are given in the shoulder region which is cleared of hair: injection 1 is a 1:1 mixture of Freund's complete adjuvant and physiological saline, injection 2 is the test substance at the selected concentration in an appropriate vehicle and injection 3 is the test substance at the selected concentration formulated in a 1:1 mixture of Freund's complete adjuvant and physiological saline. On days 6-8, a filter paper loaded with test substance in a suitable vehicle is applied to the test area covered by occlusion for 48 hours. Approximately one day before the application, if the substance is not a skin irritant, the test area is treated with 10 % sodium lauryl sulphate in vaseline, in order to create a local irritation. Control animals receive the same treatment using vehicle without test substance. Challenge is carried out on days 20-22 in treated and control animals. A patch loaded with test substance is applied to one flank of the animals, a patch with the vehicle may also be applied to the other flank. The patches are covered occlusively for 24 hours. The skin reaction is evaluated 24 and 48 hours after patch removal. A rechallenge can be done one week after the first one if necessary. According to Directive 67/548/EEC, the test substance is regarded as a sensitiser when at least 30 % of the animals show a positive response. The GPMT is an accepted test for hazard identification of skin sensitising substances. It has been regarded as a more sensitive assay that may also, for certain substances, overestimate the sensitisation hazard for the substance tested. This is partially compensated for by the fact that a positive reaction in 30 % of the animals, compared to 15% in the Buehler test, is required for classifying the test substance as sensitiser. Shortcomings of the test are that the evaluation is not based on objective measuring parameters but on visual inspection of erythema and personal judgement. Evaluation of coloured test substances, e.g. pigments and dyestuffs, is often impossible due to staining of the skin by the test substance. 1.3.3.2 References Magnusson and Kligman, 1969; 1970 1.3.3.3 Status of validation and/or standardisation Test guidelines are available as Number B.06 in Annex V of Directive 67/548/EEC and as OECD Test Guideline No. 406. 1.3.4 Buehler Guinea Pig Test 1.3.4.1 Short description, scientific relevance and purpose The Buehler test uses repeated closed topical applications during induction and closed challenge. One flank is cleared of hair. A filter paper is fully loaded with test substance in a suitable vehicle. The patch is held in place by an occlusive bandage for 6 hours. On days 6-8 and 13-15, the same application is carried out. Control animals are treated similarly with vehicle only. Challenge is performed on day 27-29. The untreated flank is cleared of hair and an occlusive patch of the test substance at the maximum non-irritating concentration is applied to the posterior untreated flank of treated and control animals for 6 hours. Skin
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reactions are evaluated 24 and 48 hours after patch removal. A rechallenge can be done one week after the first one if necessary. According to Directive 67/548/EEC, the test substance is regarded as a sensitiser when at least 15% of the animals show a positive response. The Buehler test is an accepted test for hazard identification of skin sensitising substances. It has been less sensitive and may underestimate the sensitisation potential of a substance. This is partially compensated for by the fact that a positive reaction in only 15% of the animals, compared to 30% in the GMPT, is required for classifying the test substance as sensitiser. Shortcomings of the test are that the evaluation is not based on objective measuring parameters but on visual inspection of erythema and personal judgement. Evaluation of coloured test substances, e.g. pigments and dyestuffs, is often impossible due to staining of the skin by the test substance. 1.3.4.2 References Buehler, 1965 1.3.4.3 Status of validation and/or standardisation Test guidelines are available as Number B.06 in Annex V of Directive 67/548/EEC and as OECD Test Guideline No. 406. 1.4 Available human tests Human skin sensitisation tests have been in use the last 50 years mainly in the United States. There are a number of different human sensitisation tests available. They vary with regard to the number of induction patch tests, the placing of the patches and the use of a maximisation step. However, it is not entirely clear how useful these variations are, because validation of the tests has not kept pace with development of new tests. Also see opinion of the SCCNFP on human sensitisation tests in Section 1.1. 1.4.1 Schwartz-Peck Test 1.4.1.1 Short description, scientific relevance and purpose Subjects receive a single topical patch application of varying dose for 24, 72 or 96 hours during the induction phase and, after a resting period of 10-14 days, are challenged with a challenge patch application for 48 hours. After patch removal, the skin reaction is scored. The Complete Schwartz-Peck test, further comprises a 4 week use test with the product after the challenge patch. The Schwartz-Peck test only detects potent sensitisers and is considered obsolete in comparison with other human sensitisation assays. 1.4.1.2 References Schwartz and Peck (1949) and Schwartz (1951 and 1969) 1.4.1.3 Status of validation and/or standardisation No standardised test guideline is available. The test has not undergone an official validation process.
1.4.2 Human Repeated Insult Patch Tests (HRIPT) 1.4.2.1 Short description, scientific relevance and purpose The HRIPT is performed in at least four different forms. The concentration of material chosen for induction and challenge in the HRIPT is determined by considering the following factors: previous human experience, previous sensitisation tests in guinea pigs and irritation studies in humans. It is common practice to test multiple substances simultaneously, because it saves time and cost, but the scientific basis for multiple simultaneous inductions is not substantiated. a) Draize test: ten consecutive induction patches are applied to new skin sites on the arms or back for 24 hours every other day 3 times a week. Each induction site is evaluated for erythema and edema after removal of the patch. Two weeks after the last induction, a challenge patch is applied for 24 hours and subsequently read. The response after challenge is compared to the responses reported after the early induction patches. b) Shelanski-Shelanski test: it is comparable to the original Draize HRIPT but employs 15 consecutive induction patches to the same site and if erythema and/or edema develops during induction the following patch should be moved to an adjacent untreated area. 2-3 weeks after the last induction a challenge patch is applied for 48 hours and scored. The induction patch responses are also noted and interpreted as evidence of cumulative irritation. c) Voss-Griffith test: it is also like the original Draize HRIPT with nine 24-hour patch tests conducted over a 3 weeks period and challenge is performed 2 weeks later with duplicate patches applied to the induction skin site and to the opposite arm. This assay allowed testing of four materials simultaneously. Repeated challenge is recommended in case of dubious reactions. d) Modified Draize test: it differs from the original Draize test by subjecting the volunteers to a continuous induction period with patch exchange 3 times a week until a total of 10 patches have been applied. The patches are reapplied to the same site, and only if moderate inflammation has developed, the next patch is moved to an adjacent skin site. Challenge is performed on naive skin two weeks later with a 72-hour patch test with a non-irritating concentration of the substance. Nowadays, the HRIPT is usually only performed to confirm the safe use of potentially sensitising substance in consumer products, such as cosmetics or household products. Other substances are not normally tested in the HRIPT. The test concentration is normally at the upper end of the suggested use concentration range and is below concentrations giving positive results in animal tests. 1.4.2.2 References a) Draize et al., 1944, Draize, 1959 b) Shelanski and Shelanski, 1951; Shelanski, 1953 c) Voss, 1958, Griffith and Buehler, 1976 d) Marzulli and Maibach, 1973 1.4.2.3 Status of validation and/or standardisation
No standardised test guideline is available for the HRIPT. The HRIPT has not undergone a formal validation process. 1.4.3 Human Maximisation Test (HMT) 1.4.3.1 Short description, scientific relevance and purpose The HMT includes five repeated 48-hour occlusive patch tests on the same skin site with a 24 hour rest period between removal and reapplication of the patch. Substances with irritating potential are applied in a concentration giving a moderate erythema and for substances that are non-irritating the test site is pretreated with a 24-hour patch of 5% sodium lauryl sulphate before induction. Following a two-week rest period after the last induction patch, the extent of sensitisation is evaluated by a 48-hour occluded patch test with the maximum non-irritating concentration of the substance on a slightly irritated skin site. The challenge site is scored after 24 and 48 hours after patch removal and the sensitisation index is noted. Because the human maximisation test may produce a rather dramatic effect on the skin it may be considered unacceptable today. 1.4.3.2 References Kligman, 1966; Kligman and Epstein, 1975 1.4.3.3 Status of validation and/or standardisation No standardised test guideline is available. The test has not undergone an official validation process.
1.5 References Asherson, G.L., Ptak, W. (1968). Contact and delayed hypersensitivity in the mouse. I. Active sensitization and passive transfer. Immunol. 15, 405-410. Buehler, E.V. (1965). Delayed contact hypersensitivity in the guinea pig. Arch. Dermatol. 91, 171-177. Cornacoff, J.B., House, R.V., Dean, J.H. (1988). Comparison of a radioisotopic method and the mouse ear swelling test (MEST) for contact sensitivity to weak sensitizers. Fund. Appl. Toxicol. 10, 40-44. Dean, J.H., Twerdok, L.E., Tice, R.R., Sailstad, D.M., Hattan, D.G., and Stokes, W.S. (2001). ICCVAM evaluation of the murine local lymph node assay. II. Conclusions and recommendations of an independent scientific peer review panel. Regul. Toxicol. Pharmacol. 34, 258-273, doi:10.1006/rtph.2001.1497. Descotes, J. Identification of contact allergens: the mouse ear sensitization assay. J. Toxicol. / Cutan. Ocular Toxicol. 7, 263-272. Draize, J.H. (1959). Dermal toxicity. Appraisal of the safety of chemicals in foods, drugs and cosmetics. Association of food and drug officials of the United States, Texas State Department of Health, Austin, Texas.
Draize, J.H., Woodard, G., and Calvery, H.D. (1944). Methods for the study of irritation and toxicology of substances applied topically to the skin and mucous membrane. J. Pharmacol. Exp. Ther. 83, 377-90. Dunn, B.J., Rusch, G.M., Siglin, J.C., and Blaszcak, D.L. (1990). Variability of a mouse ear swelling test (MEST) in predicting weak and moderate contact sensitization. Fund. Appl. Toxicol. 15, 242-248. Gad, S.C. (1994). The mouse ear swelling test (MEST) in the 1990s. Toxicol. 93, 33-46. Gad, S.C., Dobbs, D.W., Dunn, B.J., Reilly, C., Walsh, R.D., Auletta, C.S., Hile, R.A., Reagan, E., and Yenser, B. (1987). Interlaboratory validation test (MEST). In: In vitro toxicology - approaches to validation. A.M. Goldberg (Ed.). Liebert, New York; pp. 275-292. Gad, S.C., Dunn, B.J., Dobbs, D., Reilly, C., and Walsh, R.D. (1986). Development and validation of an alternative dermal sensitization test: the mouse ear swelling test (MEST). Toxicol. Appl. Pharmacol. 84, 93-114. Griffith, J.F., and Buehler, E. (1976). Prediction of skin irritancy and sensitization potential by testing with animals and man. In: Cutaneous Toxicity, edited by V. Drill and P. Lazer, pp. 177-173. Academic Press, New York. Haneke, K.E., Tice, R.R., Carson, B.L., Margolin, B.H., and Stokes, W.S. (2001). ICCVAM evaluation of the murine local lymph node assay. III. Data analysis completed by the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods. Regul. Toxicol. Pharmacol. 34, 274-286, doi:10.1006/rtph.2001.1498. Kimber, I., Dearman, R.J., Basketter, D.A., Ryan, C.A., and Gerberick, G.F. (2002). The local lymph node assay: past, present and future. Contact Derm. 47, 315-328. Kimber, I., Hilton, J. and Weisenberger, C. (1989). The murine local lymph node assay for identification of contact allergens: a preliminary evaluation of in situ measurement of lymphocyte proliferation. Contact Dermatitis 21, 215-220. Kimber, I., Mitchell, J.A., and Griffin, A.C. (1986). Development of a murine local lymph node assay for the determination of sensitizing potential. Food Chem. Toxicol. 24, 481-494. Kligman, A.M. (1966). The identification of contact allergens by human assay. III. The Maximisation Test: a procedure for screening and rating contact sensitisers. J. Invest. Dermatol. 47, 393-409. Kligman, A.M., and Epstein, W. (1975). Updating the maximisation test for identifying contact allergens. Contact Dermatitis 1,231-9. Magnusson, B. and Kligman, A.M. (1970). Allergic Contact Dermatitis in the Guinea Pig. 141 pages.: Charles C. Thomas, Springfield, Illinois. Magnusson, B., and Kligman A.M. (1969) The identification of contact allergens by animal assay. The guinea pig maximization test. J. Invest. Dermatol. 52, 268-76. Marzulli, F.N., and Maibach, H.I. (1973). Antimicrobials: Experimental contact sensitisation in man. J. Soc. Cosmet. Chem. 24, 399-421. Sailstad, D.M., Hattan, D., Hill, R.N., and Stokes, W.S. (2001). ICCVAM evaluation of the murine local lymph node assay. I. The ICCVAM review process. Regul. Toxicol. Pharmacol. 34, 249-257, doi:10.1006/rtph.2001.1496. SCCNFP, 2000. The Scientific Committee on Cosmetic Products and Non-Food Products Intended for Consumers Notes of Guidance For Testing of Cosmetic Ingredients For Their
Safety Evaluation. SCCNFP/0321/00 Final, adopted by the SCCNFP during the plenary meeting of 24 October 2000. Schwartz, L. (1951). The skin testing of new chemicals. J. Soc. Cosmet. Chem. 2, 321-4. Schwartz, L. (1969). Twenty-two years' experience in the performance of 200,000 prophetic patch tests. South Med. J. 53, 478-84. Schwartz, L., and Peck, S.M. (1944). The patch test in contact dermatitis. Public Health Rep. 59, 546-57. Shelanski, H.A. (1951). Experience with and considerations of the human patch test method. J. Soc. Cosmet. Chem. 2, 324-31. Shelanski, H.A., and Shelanski, W.V. (1953). A new technique of human patch tests. Proc. Sci. Sekt. Toilet. Goods Assoc. 19, 46-9. Voss, J.G. (1958). Skin sensitization by mercaptans of low molecular weight. J. Invest. Dermatol. 31, 273-279.
2. Inventory of alternative methods currently available 2.1 Alternatives to all p redictive tests for skin sensitisation hazard involving chemical structure activity relationships 2.1.1 State of the art This area of toxicological science has been under sustained investigation for the last two decades. Roberts and Williams (1982) proposed a sophisticated mathematical approach which related the physicochemical properties of a substance, and the dose at which it was applied to skin, to the degree of sensitisation which would occur. Whilst this approach led broadly to the development of quantitative structure activity relationships for several series of closely related chemicals (reviewed in Barratt, Basketter and Roberts, 1999), it also paved the way for the development of structure activity models capable of basic hazard identification. It is these which are discussed below, since traditional approaches to the establishment of QSARs seems to be likely to remain restricted for the reasonably foreseeable future to limited chemical groups. An extensive review of these topics is being published elsewhere (Cronin et al, 2003) Relationships between the structure and biological properties of chemicals can be programmed into knowledge based expert systems. One such expert system is DEREK (Deductive Estimation of Risk from Existing Knowledge; Sanderson & Earnshaw, 1991; Ridings et al., 1996), which is under ongoing development by LHASA Ltd. (School of Chemistry, University of Leeds, UK). DEREK covers a variety of toxicological endpoints (e.g. mutagenicity, carcinogenicity, skin sensitisation), and is in widespread use in the chemical industry. Other expert system approaches to the prediction of skin sensitisation include the TOPKAT (TOxicity Prediction by Komputer Assisted Technology; Enslein et al., 1997) and CASE (Computer Automated Structure Evaluation) systems (Graham et al., 1996). These computer based systems are built upon varying approaches, but all employ physicochemical descriptors of chemical sensitisers and non-sensitisers as a means to provide a more general characterisation of potential allergens. TOPKAT is a suite of two modules, one for non-sensitisers v sensitisers and the other for splitting the latter group into weaker and stronger categories. Its predictions are based on physical and physical chemical properties and not on biological nor on biochemical mechanisms. Unlike DEREK it is not an expert system because it does not use rules or structural alerts. A number of different physical and physical chemical descriptors (shape indices (Kappa), symmetry indices, MW, log P, Kier-Hall electrotopological states (E-States)) are correlated with toxicity data (here skin sensitization data from guinea pig maximisation tests. Query substances are evaluated based on a structural similarity search. These computer systems are used variously by a number of agencies (see Walker et al, 2002; OECD 2002). DEREK embodies both a controlling programme and a chemical rulebase. The chemical rulebase consists of descriptions of molecular substructures called structural alerts, which correlate with specific toxicological endpoints. The user communicates with DEREK by drawing the two-dimensional structure of the chemical under investigation on the screen. The rulebase is then searched against that structure, and any structural alert is highlighted, together with a message indicating the nature of the toxicological hazard. The original skin sensitisation rulebase contained around 40 rules (Barratt et al., 1994), which were derived from an historical database (Cronin & Basketter, 1994) containing data from guinea-pig maximisation tests on 135 chemicals that had been classified as skin sensitisers
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according to EU criteria, as well as a similarly sized group of non-sensitisers. As a result of development of the system over almost 10 years (e.g. Barratt & Langowski, 1999), the number of structural alert rules for skin sensitisation currently stands at 61 in Version 6 of the programme. In addition, DEREK now contains a reasoning engine, which in an integrated manner refines the output with additional information on the ability of the substance to penetrate skin. DEREK will serve as an example of the small number of computer based systems available commercially which are intended to predict whether a chemical possessed the intrinsic hazard of skin sensitisation.
2.1.2.1. DEREK 1. Short description, scientific relevance and purpose The following two step strategy for the assessment of skin sensitisation potential is employed when using the DEREK rulebase: The chemical is processed through the rulebase, to see if it has the potential to react with skin proteins either directly or, in some cases, after metabolism. If no structural alert is triggered, either the chemical does not possess the requisite reactivity, or its reactivity is outside the scope of the current knowledge base. In many cases, absence of chemical reactivity can be confirmed by inspection of the chemical structure. For chemicals that do not possess the appropriate chemical reactivity, no further computational evaluation is performed. However, an evaluation of possible metabolic activation of the compound is considered by expert input. For chemicals or their metabolites that do possess the appropriate chemical reactivity, the second step is to assess their skin permeability/partition parameters. This initially involves using either empirical or calculated values for computation of the log octanol/water partition coefficient (logP) and/or to theoretically predict the log permeability coefficient (logKp) (Barratt, 1995). Molecular weight and melting point values are also used in the prediction algorithms to calculate logKp. Kp values can also be measured by using validated in vitro skin penetration models. Using in vitro models also allows for the visualisation, as well as the quantification, of partitioning of compounds within the skin sub-structures. 2. References See the body of the text 3. Developer of the method LHASA UK in collaboration with Unilever and other groups who participate in the evaluation and evolution of the system. 4. Known users include most of the major cosmetic companies and several agencies. 5. Status of validation and/or standardisation is not straightforward to answer for a computer system, particularly an expert rule based system, which, by definition, evolves as new knowledge is acquired. - Is the protocol defined/optimised? Yes, but optimisation will be ongoing as new knowledge enables the rules to be refined or supplemented. - Is there data on intra-laboratory variation? Not applicable. - Is there data on the transferability? Not applicable. - Is there data on the inter-laboratory variability? Not applicable.
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- In vivo/in vitro comparisons? These have been carried out during model development. Most recently, the BfR (Bundesinstitut fr Risikoforschung Federal Institute for Risk Assessment, http://www.bfr.bund.de/cms/detail.php?template=internet_en_index_js) examined DEREK (Zinke et al, 2002). - Is the method validated? No. 6. What efforts are needed to complete validation of the method? Key to this is a sustained effort to refine DEREK rules with existing knowledge as well as via the acquisition of new knowledge via evaluation of chemistry areas where the system is currently weak. These are identified via simple evaluation with currently available/being prepared datasets. 2.1.2.2 Alternative computer methods, as mentioned in 2.1.1 exist, but generally they are at a similar, or often lower, status of development compared to DEREK. 2.1.3 Future prospects and recommendations 2.1.4 Key references Cronin MT, Jaworska JS, Walker JD, Comber MH, Watts CD, Worth AP. (2003). Use of QSARs in international decision-making frameworks to predict health effects of chemical substances. Environ Health Perspect. 111(10):1391-1401. OECD (2002) OECD database on chemical risk assessment models, Paris, France. Walker JD, Carlsen L, Hulzebos E and Simon-Hettich B (2002) Global government application of analogues, SARs and QSARs to predict aquatic toxicity, chemical or physical poperties, environmental fate parameters and health effects of organic chemicals. SAR QSAR Environ Res, 13, 607 616. Zinke S, Gerner I, Schlede E. Evaluation of a rule base for identifying contact allergens by using a regulatory database: Comparison of data on chemicals notified in the European Union with "structural alerts" used in the DEREK expert system. Altern Lab Anim. 2002 30: 285-98.
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2.2 In vitro tests for skin sensitisation 2.2.1 State of the art Allergic contact dermatitis (ACD) is a cell mediated immune response to small molecular weight chemicals that contact and penetrate the skin. There are a variety of characteristics that determine whether a chemical can function as a contact sensitizer (or allergen) including the ability to penetrate into the skin, react with protein and be recognized as antigenic by immune cells. The ultimate challenge for developing non-animal test methods for skin sensitization testing will be applying our mechanistic understanding of ACD to the design of predictive in vitro alternative test methods (Ryan et al, 2001). Specifically, the in vitro approach should be designed so that a chemicals potential to penetrate the skin, react with protein/peptide (biotransformation may be required) and initiate an antigen-specific immune response is incorporated in the test methods developed. In this section, we review in vitro skin allergy research conducted on single cell cultures and the more complex models such as skin explant and skin equivalent models. 2.2.2.1 Keratinocyte cultures 1. Short description, scientific relevance and pupose Over 90% of the cells present in the epidermis are keratinocytes (KC) and, as such, they are often the first cells in the skin to encounter chemicals which have penetrated through the stratum corneum. KC are able to produce and secrete a number of proinflammatory cytokines, chemokines and growth factors (Matsue et al., 1992), and there is evidence that they play a role in immune-mediated skin diseases including ACD (Schwarz and Luger, 1992). Wilmer et al. (1994) used a commercially available source of human KC to examine the effects of sensitizing and non-sensitizing chemical irritants on selected intracellular and secreted cytokines. The non-sensitizing irritants phenol, sodium lauryl sulfate (SLS), and croton oil induced increases in interleukin-8 (IL-8) production whereas no change or a reduction in the baseline level of IL-8 production were observed following treatment with benzalkonium chloride (BC), CrO3 or the contact allergens 2,4-dinitrofluorobenzene (DNFB) or oxazolone. Croton oil, phenol, SLS, BC and DNFB were all found to stimulate the production and accumulation of intracellular interleukin-1? (IL-1? ). In short, the sensitizing and non-sensitizing irritants tested could not be differentiated by the production of either IL-8 or IL-1? , and the authors concluded that a given pattern of cytokine production is chemicalspecific. The effects of three contact sensitizers on IL-1? mRNA expression in human KC cultured from neonatal foreskins were found also to be varied (Pastore et al., 1995). A dosedependent induction of IL-1? mRNA was seen following treatment with neomycin sulfate. Benzocaine produced no change in its signal, and dinitrobenzene sulfonate (DNBS) suppressed IL-1? mRNA expression. More recently, the in vitro production of IL-1? has been examined using the mouse-derived KC line, HEL30 (Corsini et al., 1998). Five allergens, two irritants and two non-sensitizers were investigated for their ability to induce extracellular (released) and intracellular (cell-associated) IL-1? . Only the contact allergens were found to increase cell-associated IL-1? , in a dose dependent fashion, while both the allergens and irritants induced the release of IL-1? . The non-sensitizing chemicals, ethanol, and glycerol, had no effect on IL-1? production. Researchers have also investigated changes in cell surface antigen expression after allergen or irritant exposure. In examining epidermal cell cultures obtained from the ears and skin of Balb/c mice, Coutant et al. (1999b) reported that the strong contact allergen trinitrobenzene sulfonic acid (TNBS) induced the expression of CD40 on KC (defined as Ia/CD45 negative cells) whereas the irritant SLS did not.
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Recently, Wakem et al. (2000) demonstrated that CD80 transcriptional activity and cell surface expression in cultured human KC were up regulated to similar extents by treatment with either allergens or irritants. They hypothesized that the response of KC to chemical stimuli serves as a signal for activating T cell mediated inflammation for both ACD and irritant contact dermatitis. This suggests that the KC are simply responding to a toxic insult and are not able to distinguish allergens from irritants. Lozsekova et al. (2002) tested the effects of known allergens, namely nickel sulphate, potassium dichromate, cobalt nitrate and cytotoxic cadmium sulphate on the proteins of cellular contacts (vinculin, talin, E-cadherin, desmoplaktin) and actin cytoskeleton (actin filaments) of cultivated human keratinocytes. The localisation of proteins of cellular contacts was detected by means of direct immunofluorescence. The authors reported a decrease in, and destruction of cellular contact proteins and actin cytoskeleton after testing the effect of all allergens, while the most significant changes were detected in E-cadherin, vunkulin and actin filaments. Desmoplaktin and talin were less damaged. 2. 3. 4. 5. 6. References - See body of the text Developer of the method - Not applicable Known users - None Status of validation and/or standardisation - No validation of this method to date. What efforts are needed to complete validation of the method? Not applicable
2.2.2.2. Langerhans cell cultures 1. Short description, scientific relevance and pupose Langerhans cells (LC), considered to be the principle antigen presenting cell (APC) in the skin, play a key role in the development of allergic contact sensitization. LC constitute only 1-3% of all epidermal cells. Many isolation techniques have been developed to obtain purified populations of LC from human and murine sources (Hanau et al., 1988; Koch et al., 1992; Simon et al., 1995; Teunissen et al., 1988), but the numbers of cells obtained are relatively low and, to date, no LC line has been established. Therefore, the availability of sufficient numbers of these cells has been a limiting factor in the development of LC-based in vitro methods. Despite this obstacle, several investigators have focused on events which occur in LC following their exposure to chemical haptens and irritants. Changes which occur in Langerhans cell surface marker expression following in vivo chemical treatment have been explored (Aiba and Katz, 1990; Schwarzenberger and Udey, 1996). Using murine LC and flow cytometry, Herouet et al. (1999) examined the effect of chemical treatment in vitro on the expression of several LC surface markers, including MHC class I and class II, adhesion molecules CD54 and CD11c, co-stimulatory molecules CD80 and CD86, and dendritic cellspecific markers DEC-205, 4F7 and 33D1. They reported that the only surface marker affected by exposure to contact sensitizers was 33D1, a murine specific dendritic cell marker. They observed a consistent, reproducible reduction in 33D1 expression on LC within 30 minutes of exposure to strong (oxazolone, DNBS), moderate (p-paraphenylenediamine) and weak (mercaptobenzothiazole) sensitizers. The irritant SLS also induced a decrease in 33D1 expression. However, SLS similarly affected the other markers examined, presumably due to a surfactant effect on the integrity of the LC cytoplasmic membrane. While the exact role the 33D1 molecule plays in the immune response to contact sensitizers remains to be elucidated,
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the apparently selective down-regulation of this marker by chemical haptens lends itself to further investigation in the development of in vitro tests. Using flow cytometry, Verrier et al. (1999) demonstrated that E-cadherin and HLA-DR expression were altered on the surface of human epidermal Langerhans cells exposed to contact sensitizers (isoeugenol, cinnamaldehyde, TNBS, Bandrowski's base, p-phenylenediamine) for 4 hours at 37?C. A dose-dependent decrease in the mean fluorescence intensity of HLA-DR was reported with no change in the number of HLA-DR positive cells. A decrease in the percentage of E-cadherin positive cells as well as a downregulation of E-cadherin expression was found following treatment with all allergens except isoeugenol. Treatment with SLS did not significantly affect either HLA-DR or E-cadherin expression. Tuschl and Kovac (2001) reported dendritic cells from peripheral mononuclear blood cells responded to NiSO4, dinitrochlorobenzene, 2,4,6-trinitrobenzene sulfonic acid, alpha-hexylcinnamaldehyde and eugenol in regard to their up-regulation of the co-stimulatory molecule CD86, of intercellular adhesion molecule CD54 and of the HLA-DR antigen. The irritant sodium dodecyl sulfate (SDS) and the vehicle dimethyl sulfoxide (DMSO) had no effect. The internalization of surface MHC class II molecules via endocytosis by antigen presenting cells is viewed as an important early step in antigen processing and is one which has been demonstrated in human (Girolomoni et al., 1990) and murine (Becker et al., 1992a,b) LC. Lempertz et al. (1996) explored the possibility of using endocytosis of contact sensitizers by murine LC as the basis for a predictive in vitro assay. Briefly, LC present in epidermal cell suspensions were labeled with an anti-MHC class II monoclonal antibody. By means of a flow cytometric method using second step reagents labeled with pH-sensitive fluorochromes, they found differences in the mean fluorescence intensity of the internalized label which were related to the chemical treatment of the LC. Endocytosis of the MHC complex into acidic compartments resulted in a quenching of fluorescence, whereas internalization into less acidic compartments resulted in a conservation of fluorescence intensity. Cells exposed to the solvent dimethylsulfoxide (DMSO) were used as a reference and their fluorescence intensity was defined as 100%. Treatment with irritants SLS and benzoic acid and nonsensitizing stimulatory materials concanavalin A (Con A) and phorbol 12-myristate 13-acetate (PMA) produced little to no change. Treatment with contact allergens formaldehyde, nickel sulfate, 5-chloro-2-methylisothiazolinone plus 2-methylisothiazolinone (MCI/MI), potassium dichromate, DNFB, dinitrochlorobenzene (DNCB) and trinitrochlorobenzene (TNCB) resulted in a conservation of fluoroescence intensity of 1.7- to 2-fold that of DMSO treated cells. While the authors admit that the influence of hapten on receptor-mediated endocytosis is not proof of the allergenic potential of a chemical, there was a strong correlation between reactivity in their assay and in vivo sensitizing capacity. A similar study exploring the use of MHC-II endocytosis as a means for distinguishing sensitizers from irritants was conducted by Rizova et al. (1999) using human LC. Three different methods were used to evaluate surface expression and internalization of HLA-DR: flow cytometry, laser scanning confocal microscopy (LSCM) and electron microscopy. As observed by flow cytometry, they found that moving freshly isolated LC from 4oC to 37oC produced a decrease in the surface expression of HLA-DR which was linked to the spontaneous internalization of the MHC-II molecule. This spontaneous internalization was increased to a similar extent by preincubation with noncytotoxic concentrations of either sensitizers (DNFB, diphencyprone, oxazolone and 3-n-pentadecylcatechol) or irritants (SLS, BC and benzoic acid). Thus, no clear differences between the two classes of chemicals were detectable by flow cytometry. However, when observed by LSCM, cells which had been treated with the sensitizers internalized the HLA-DR molecules into large vesicles which exhibited a very bright fluorescence in contrast to irritant treated cells which were similar to untreated controls, with HLA-DR molecules in small vesicles showing a diffuse fluorescence. Clear differences were
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also observed using electron microscopy. HLA-DR molecules were found preferentially in lysosomes collected near the nucleus in sensitizer treated LC, while the MHC-II molecules were observed at the cell membrane surface and in prelysosomes in irritant treated and control LC. The induction of tyrosine phosphorylation following stimulation with contact sensitizers has been examined in both human (Khn et al., 1998) and murine (Neisius et al., 1999) LC. Whereas freshly isolated human LC failed to demonstrate changes in phosphotyrosine (p-tyr) following exposure to the strong hapten MCI/MI, 24 hour cultured LC demonstrated a significant increase in p-tyr by flow cytometric quantitation (Khn et al., 1998). Neisius et al. (1999) reported similar increases in p-tyr in murine LC following in vitro stimulation with the strong contact sensitizers TNCB and MCI/MI but not with the irritants SLS or benzoic acid. Although poorly defined, the mechanisms of signaling pathways in LC during haptenmediated activation may serve as a basis for the development of an in vitro test system. Following an encounter with antigen, LC have been shown to migrate out of the skin to regional draining lymph nodes (Kripke et al., 1990; Macatonia et al., 1987). In an attempt to reproduce this event in an in vitro system, Kobayashi et al. (1994) examined the influence of hapten application on LC migration through a reconstituted basement membrane matrix. LCenriched cell suspensions obtained from human skin were treated in vitro with either hapten (trinitrobenzenesulfonic acid (TNBS)), or fluorescein isothiocyanate (FITC)), or irritant (SLS). The treated cells were then seeded into the upper compartment of a modified Boydenchamber over a Matrigel-coated polycarbonate filter underlayed with a cellulose nitrate filter, with fibroblast conditioned medium in the lower compartment to act as a chemoattractant. The number of HLA-DR positive cells on the upper surface of the cellulose nitrate membrane were counted to quantify LC migration. Hapten treated LC were found to exhibit a timedependent increase in migration into the lower filter compared to non-treated cells while the migratory pattern of SLS treated cells did not differ. While donor to donor variability in the absolute extent of LC migration was noted, hapten treatment enhanced the migration compared to untreated cells in every suspension studied. The simplicity of this method lends itself to further investigation using a chemical set with a wider range of allergenic potencies. 2. 3. 4. References - See body of the text Developer of the method - Not applicable Known users - None
5. Status of validation and/or standardisation - No validation of test methods to date. 6. What efforts are needed to complete validation of the method? Not applicable. 2.2.2.3 Peripheral blood derived dendritic cells 1. Short description, scientific relevance and pupose Dendritic cells (DC) are a distinct group of leukocytes characterized by their unique morphology and their ability to initiate immune responses by processing and presenting antigens. As mentioned previously, the ability to obtain large numbers of LC from skin is a limiting factor for their use in the development of in vitro methods to assess contact sensitization potential. Therefore, the development of culture techniques to generate DC from
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CD34+ precursors, either from bone marrow or cord blood, and peripheral blood mononuclear cells has provided a source of LC-like antigen presenting cells for study. In addition to having typical DC morphology, peripheral blood mononuclear cell (PBMC)-derived DC express surface markers consistent with bone marrow derived DC, and are capable of eliciting a primary allogeneic mixed lymphocyte response (Romani et al., 1994). While minor variations in culture techniques and cytokine induction cocktails have been reported, to date there is no one method for generating DC from PBMC that investigators agree upon as standard. In an attempt to develop an in vitro model for contact sensitization, Degwert et al. (1997) examined phenotypical alterations of human blood-derived DC under the influence of subtoxic concentrations of different chemicals and contact sensitizers. They showed that incubation with contact sensitizers urushiol, primin, C10- and C11-primin analogues, alantolactone, isoalantolactone and NiSO4 resulted in a decrease of HLA-DR expression on the surface of the DC, if the incubation period did not exceed 3 hours. Incubation with irritants like SLS and BC did not change DR expression under the same conditions. Adhesion molecule expression (ICAM-1 (CD54)) showed no differences between irritants and contact sensitizers. The authors summarized by saying that this system can be used to discriminate between contact sensitizers and irritants. Using longer incubation times (24 and 48 hr), Aiba et al. (1997) reported that haptens such as NiCl 2 and DNCB cause a significant increase in the surface expression of CD54, CD86 and HLA-DR on monocyte derived DC compared to nontreated controls or to DC treated with irritants (ZnCl 2, SDS, or BC). However, they noted that the changes in expression of these markers were quite variable among the subjects tested, with some subjects having no change at all. With similar findings, Coutant et al. (1999a) illustrated that monocyte derived DC expressed higher surface marker expression of HLADR, CD86, CD40 and CD54 in the presence (48 hrs) of haptens (aminophenol, chlorpromazine hydrochloride, DNCB, nickel sulfate) as compared to irritants (SDS, benzoic acid). After incubation with haptens, but not with irritants, they observed a higher production of TNF-a. In their experience, no release of IL-1, IL-10 or IL-12 was detected. Compared to the activation elicited by haptens, stimulation with staphylococcal enterotoxin B (SEB) induced strongly upregulated HLA-DR and co-stimulatory molecule expression. They concluded that the data underlines the activating potential of haptens versus irritants on DC functions and that the differing levels of DC activation suggest the involvement of distinct cellular events. Using Langerhans cell-like DC generated from CD34+ cord blood cells cultured for 8 days with GM-CSF and TNF-? , Rougier et al. (2000) demonstrated that incubation with hapten for 48 hrs induced DC maturation, based on phenotypic changes such as the expression of CD83, increased expression of HLA-DR and CD86, and a decrease in Ecadherin. All of these changes were observed following treatment with the strong allergen BB, and not with the irritant SDS. However, the weaker allergens tested, PPDA, citronellal and coumarin, mostly induced changes in CD86 expression and not HLA-DR or CD83 which, were observed only in a limited number of subjects (1 or 2 out of 8). Hulette et al. (2002) examined expression of HLA-DR, CD54, CD80, CD86 on DC treated 48 hours with dinitrofluorobenzene (DNFB) and methylchloroisothiazolinone/methylisothiazolinone (MCI/MI), the irritant sodium dodecyl sulfate (SDS), lipopolysaccharide (LPS), and tumor necrosis factor alpha (TNF? ). Treatment of PBMC-DC with either MCI/MI or DNFB induced a slight upregulation of class II major histocompatibility (MHC) expression (HLADR), whereas LPS and TNF? significantly upregulated CD54 and slightly upregulated CD80 and HLA-DR expression. For KG-1 DC (Hulette et al, 2001), only MCI/MI upregulated CD86 expression, whereas TNF? upregulated CD54 and slightly upregulated CD80 and CD86 expression. SDS had no effect on surface marker expression in either PBMC-DC or KG-1 DC. Changes in surface marker expression in PBMC-DC treated with chemical
17
allergens were detected in 2 of 5 donors, suggesting a limited sensitivity of PBMC-DC under these defined isolation and culture conditions. The conclusion was that under these culture and treatment conditions, measurement of surface marker changes in vitro using PBMC-DC or KG-1 DC does not provide a sensitive in vitro method with sufficient dynamic range for assessing the contact sensitization potential of a chemical. Recently, Banerjee et al. (2003) have proposed measurement of IL-1? and IL-1? protein levels to distinguish allergens from irritants. They report that the allergens induced the secretion of IL-1? at concentrations twoto five-fold higher than those of controls, depending on the concentration and the particular irritant. As discussed earlier, the internalization of surface MHC class II molecules via endocytosis by antigen presenting cells is viewed as an important early step in antigen processing and is one which has been demonstrated in human (Girolomoni et al., 1990) and murine (Becker et al., 1992a,b) LC. As a surrogate to LC, Becker et al. (1997) used peripheral blood-derived DC with a flow cytometric method to screen for the modulation of receptor-mediated endocytosis. Briefly, a method was established to monitor the influence of chemicals in the intracellular targeting of antibody-crosslinked MHC class II molecules after their uptake by human DC. The assay is based on the pH-sensitivity of internalized fluorescein-coupled MHC class II specific antibodies. Untreated DC showed quenching of fluorescence intensity due to internalization into acidic intracellular compartments, whereas fluorescence intensity was conserved in DC treated with strong contact sensitizers (DNFB, MCI/MI, imidazol urea). The authors summarize by stating that the data suggest that the capacity of a chemical to modulate endocytotic mechanisms in DC in vitro reflects the probability of that substance acting as a hapten in vivo. IL-1? is the first cytokine which is rapidly produced by LC after topical application of contact sensitizers onto mouse skin (Enk and Katz, 1992). This early up-regulation of IL-1? was specific for contact sensitizers and not irritants. These data strongly suggest that IL-1? plays an essential role for the induction of primary immune reactions like allergic contact dermatitis in the skin (Enk et al., 1993). Therefore, IL-1? expression may provide an alternative approach to the assessment of the sensitizing activity of chemicals. Reutter et al. (1997) investigated the ability of five contact sensitizers and one irritant to induce IL-1? gene expression in vitro in PBMC-DC. DC were cultured in serum free medium supplemented with GM-CSF and IL-4 for 5 days. Then the DC were pulsed for 30 minutes at 37oC with subtoxic concentrations of the contact sensitizers pentadecyl-catechol, TNBS, DNFB, NiSO4, K2Cr2O7 and the irritant SDS. Total RNA was extracted from the DC and IL-1? mRNA expression was detected using reverse transcriptase-polymerase chain reaction (RT-PCR). They reported that all contact sensitizers increased IL-1? gene expression, whereas treatment with the irritant SDS had no significant effect. They concluded that they had developed an in vitro system which may be useful to evaluate the allergic potentials of chemicals and products. De Smedt et al. (2001) reported modulation of phenotype and cytokine production in CD34+ derived DC treated with NiCl 2 and SDS. In a later paper (De Smedt et al, 2002), the authors reported that monocyte-derived dendritic cells were more robust in response to allergens then CD34+ progenitor cells. Pichowski et al. (2000) confirmed that potent contact sensitizers selectively up-regulate IL-1? mRNA expression, but observed this increase from a limited number of donors (4 out of 9) after exposure in vitro to the potent sensitizer DNFB. The variation in DNFB-induced upregulation of IL-1? expression was shown to be donor dependent and not due to inter-PCR variation. They also confirmed that the irritant SLS did not increase IL-1? mRNA levels, even in DC derived from individuals sensitized to DNFB. In a later paper, Pichowski et al. (2001) reported that only modest responses were being
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observed with very strong allergens and that differences in responder/non-responder phenotypes were being detected. They conclusion was the measurement of Il- in human blood-derived DCs was not likely to lend itself to a routine assessment of skin sensitizing activity. Recently it was shown that the use of DC derived from pooled monocytes from different donors reduced the inter-individual and inter-experimental variations of the cell surface expression of CD 86, the upregulation of IL-1? mRNA expression and the downregulation of aquaporin P3 mRNA expression. These parameters showed marked changes in response to TNBS and two aromatic amines used as hair dyes (p-toluylenediamine (PTD) and hydroxyethyl-p-phenylenediamine (HE-PPD)), but no alteration in response to SLS. Both aromatic caused marked DC activation, comparable to the effects of the known skin sensitiser TNBS. The seemingly contradictory results in the LLNA, in which PTD was identified as a skin sensitiser whereas HE-PPD was not sensitising, were only resolved when the skin penetration capacity, which was shown to be about 200-fold lower for HE-PPD compared to PTD, was taken into account (P. Aeby, C. Wyss, H. Beck, P. Griem, H. Scheffler, and Carsten Goebel, J. Invest. Dermatol., accepted for publication). In addition to changes in cell surface marker expression, Aiba et al. (1997) also examined the production of cytokines by monocyte-derived DC. They found that cytokine production was dependent on the chemical treatment. DC exposed to NiCl 2 had a significant increase in the production of IL-1? , IL-6 and TNF-? protein, while DNCB treatment only induced an increased secretion of IL-1? . Lore et al. (1998) developed a direct immunocytochemical method to identify cytokine and chemokine production in peripheral blood-derived DC at the single cell level. This method was used to assess TNF-a, IL-1a, IL-1ra, IL-6, IL-8, IL-10, IL12, GM-CSF, MIP-1a, MIP-1, and RANTES. IL-1ra and IL-1a were expressed in 10-25% of unstimulated cultured DC, while all the other cytokines tested were undetectable. IL-1ra, IL-1a, and IL-1 were expressed in 85% of DC after 3 hr of lipopolysaccharide stimulation. The investigators did not explore DC which had been treated with haptens, but this approach might find utility as a predictor of DC activation. Aiba et al. (1999) showed the activation and apoptosis of peripheral blood derived DC when treated for 24 hrs in culture with various contact sensitizing agents using Annexin V and TUNEL methods. Aiba et al. speculate that it is conceivable that chemicals, more specifically the contact sensitizers, induce a stress response that activates DC. However, because NiCl 2 and DNCB induce DC activation by different mechanisms and because different chemicals induce heterogeneous responses in the augmentation of costimulatory molecules or class II MHC antigen, cytokine secretion and induction of apoptosis, the activation of DC cannot be explained simply as a result of a stress response. Aiba et al. postulate that these chemicals may directly affect the intracellular signal transduction of DC. Although not viewed as an alternative method to predict the skin sensitization potential of chemicals, apoptosis is emerging as a parameter for exploration in hapten/DC interactions. 2. 3. 4. References - See body of the text Developer of the method - Not applicable Known users -None
5. Status of validation and/or standardisation - No validation of test methods to date 6. What efforts are needed to complete validation of the method? Not applicable
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2.2.2.4 Cell lines In addition to using LC or DC, which are difficult to isolate and to get sufficient numbers, investigators have been evaluating various cell lines to use as dendritic cell surrogates. For example, an LC-like murine cell line, XS52, has been developed from the epidermis of newborn BALB/c mice which displays many of the morphologic, phenotypic and functional characteristics of freshly isolated LC (Xu et al., 1995a). XS52 cells are similar to LC in terms of cytokine and cytokine receptor mRNA profiles (Ariizumi et al., 1995; Takashima et al., 1995; Xu et al., 1995b) and they have been shown to up-regulate IL-1? mRNA and secrete relatively large amounts of IL-1? protein upon antigen-dependent interaction with T-cells (Kitajima et al., 1995). Similar to LC, an increase in tyrosine phosphorylation in XS52 cells has been demonstrated following stimulation with strong haptens (Neisius et al., 1999). In addition, it has been shown recently that mature DC can be generated from these cells using soluble factors (Yamada and Katz, 1999). While not available commercially, the XS52 cell line, with its similarities to LC, holds promise for the development of a wholly in vitro method for sensitization testing. Using the THP-1 cell line, Yoshida et al. (2003) have reported recently that one gets augmentation of CD54 and CD86 expression in THP-1 cells treated with allergens. The investigators used flow cytometry and the mean fluorescence intensity to show the changes in the THP-1 cell line. Similarly, Ashikaga et al. (2002) reported an upregulation of CD86 and class II MHC internalization on THP-1 cells treated for 24 hours with DNCB. They stated that the upregulation was enhanced when using gamma interferon along with their allergen treatment. 2. 3. 4. References - See body of the text Developer of the method - Not applicable Known users - None
5. Status of validation and/or standardisation - No validation of test methods to date 6. What efforts are needed to complete validation of the method? Not applicable 2.2.2.5 Co-culture systems Since the induction of contact allergy involves the interaction between LC and T -cells, a culture system which contains both stimulating APC and responding T-cells, would appear to provide the best approach for the development of an in vitro method for predictive sensitization testing. Using hapten treated cultured murine LC, Hauser and Katz (1988) were able to demonstrate primary activation and proliferation of nave T-cells in vitro. LC modified with the chemical allergens TNBS or FITC elicited a strong proliferative response in non-sensitized T helper cells. In addition, these in vitro sensitized T-cells proliferated in response to re-stimulation in vitro with hapten-modified spleen cells. A similar method was used by Moulon et al. (1993) to examine the ability of human LC to sensitize autologous Tcells. They found that 2 day cultured LC, but not freshly isolated LC, treated with TNBS were capable of inducing primary T cell proliferative responses in vitro. The cultured LC were shown to be very potent APC, with T-cell responses observed at a responder to stimulator ratio (R:S) as high as 100:1. The hapten-specificity of the primary T cell response was confirmed by the capacity of the T cells to proliferate in secondary response upon re20
stimulation with TNBS treated LC. Krasteva et al. (1996) utilized this methodology in an attempt to develop a predictive in vitro screening assay for contact sensitizers. T-cell proliferation, as determined by 3H-thymidine incorporation, resulting from stimulation with hapten-modified LC was assessed for strong (TNBS, FITC), moderate (p-phenylenediamine (PPDA) and its oxidation product Bandrowskis base (BB)), and weaker (citronellal, hydroxycitronellal, coumarin) sensitizers as well as the irritant SLS. Only the strong allergens (TNBS, FITC, BB) consistently induced a proliferative response. A co-culture system of Langerhans cells integrated into human reconstructed epidermis has been developed by Rgnier et al. (1997). The authors have recently reported Langerhans cells morphological changes and up regulation of the surface marker CD86 following exposure to the known sensitiser DNFB and NiSO4, and to UV-irradiation. The irritant SLS did not induced the same effects (Facy et al., 2004). While LC are the primary APC in the skin, obtaining sufficient numbers for use is often an issue so the ability of alternative antigen presenting cells to stimulate T cells in culture has been explored. Human peripheral blood monocytes, cultured with GM-CSF alone, or GMCSF and IL-4, have been shown to be capable of inducing in vitro primary sensitization of T cells to trinitrophenyl (TNP; the actual hapten resulting from TNBS treatment of APC), whereas freshly isolated monocytes stimulated either a weak or no response (Kobel et al., 1996). In these studies, the R:S averaged 5:1. Similar attempts to induce primary sensitization to the hapten diphenylcyclopropenone failed. Adherent peripheral blood mononuclear cells cultured in the presence of GM-CSF for 24 hours, that had been derivatized with the hapten DNCB, were shown to be capable of stimulating both proliferation and IFN-? production in nave autologous T cells in vitro (Dai and Streilein, 1998). However, the efficiency of these APC was rather low, as the R:S used for inducing the T cells was 1:1. Using DC derived from CD34+ cord blood cells as APC, Rougier et al. (1998) reported findings similar to those of Krasteva et al. (1996): strong allergens such as TNBS, FITC and BB induced significant proliferation in nave autologous lymphocytes, whereas the irritant SLS, and the less potent allergens (PPDA, citronellal and coumarin) tested, failed to induce a significant T cell response. Overall, these APC were fairly efficient at presenting hapten to nave T cells as they were capable of inducing proliferative responses at R:S of 100:1. Recently, using a modification of their original procedures, Rougier et al. (2000) have been able to produce primary T cell proliferative responses to weaker allergens, PPDA, citronellal and coumarin, in a limited number of subjects. Langerhans-like dendritic cells (LLDC) were generated from CD34+ cord blood progenitors by culture for 8 days in the presence of GMCSF and TNF-? . The LLDC were then incubated for an additional 48 hrs with non-cytotoxic concentrations of the haptens which, presumably, induced maturation of the LLDC and thus increased their immuno-stimulatory capacity. The authors suggest that hapten induced phenotypic and functional maturation of the LLDC may be used as a screening test for strong allergens but detection of weaker allergens would require complementary studies. Rustemeyer et al. (1999) have reported on a method for the in vitro priming of nave T cells using hapten treated autologous DC derived from peripheral blood. Hapten-specific T cells to nickel sulphate and 2-hydroxyethyl-methacrylate (HEMA) were generated by their culture system in which DC, matured by exposure to TNF-? following hapten treatment, were cultured with autologous T cells for 7 days in the presence of a cytokine cocktail consisting of IL1-? , IL-2 and IL-7. The hapten-specificity of the primed T cells was demonstrated by secondary proliferative responses. While the authors did not propose the culture system as a method for predictive sensitization, their protocol does appear to provide a means of priming nave T-cells in vitro.
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An elaborate co-culture system which used hapten-conjugated Pam 212 cells (a murine keratinocyte cell line) to induce a primary sensitization response in T cells from nonsensitized mice was developed by Yozeki et al. (1995). Briefly, mono-layer cultures of Pam 212 cells were incubated with test chemicals, washed, then fixed with 3% paraformaldehyde. T cells from non-sensitized mice which had been depleted of autoreactive cells, along with spleen derived macrophages, were cultured with the chemically-modified Pam 212 cells for 5 days. The T cells were then harvested and re-stimulated with mitomycin c treated, haptenconjugated spleen cells at an R:S of 5:1 for 3 days. A stimulation index (SI) was calculated by dividing the dpm obtained in the presence of hapten modified spleen cells by that obtained in the presence of unmodified spleen cells. A number of chemicals were tested in this culture system including four strong sensitizers (oxazolone, TNP, dinitrophenyl, and FITC), three potent sensitizers (PPDA, nickel chloride and potassium dichromate), two corticosteroids (betamethasone and budesonide) and one irritant (methyl salicylate). The SIs produced by the strong sensitizers were approximately 4.0 while the potent sensitizers SIs were around 2.02.5. The SIs for the corticosteroids and the irritant were less than 2.0. A further examination of this method and a comparison with the guinea pig maximization test with 11 allergens and two irritants was conducted (Arimura et al., 1998). Three of the five chemicals classified as moderate sensitizers and all of the strong sensitizers were positive in both the in vitro test and the guinea pig maximization test. While the performance of this test method warrants further investigation, it is not a non-animal alternative as it relies on mice as a source of cells, though the numbers required are rather few (4 mice to determine the allergenic potential of 13 chemicals). 2. 3. 4. References - See body of the text Developer of the method - Not applicable Known users - None
5. Status of validation and/or standardisation - No validation of test methods to date 6. What efforts are needed to complete validation of the method? Not applicable 2.2.2.6 Human skin equivalent / reconstituted epidermis cultures In vitro three-dimensional cultures of living human skin generally fall into two categories: epidermal or skin equivalents. Epidermal equivalents consist of keratinocytes which, when cultured on a filter or matrix at the air-liquid interface, develop into a fully differentiated epidermis with a stratum corneum. Cell viability, histological changes and the release of IL1? and prostaglandin E2 (PGE2) by the EpiDerm? model following treatment with irritants and allergens were examined by Kubilus et al. (1996). They found that the dose response curves for the release of IL-1? and PGE2 following treatment with contact irritants reflected the cytotoxicity of the dose (i.e. t e higher the cytokine release, the greater the cytotoxicity). h However, following treatment with contact allergens, the amount of cytokine released did not correspond directly to the degree of cytotoxicity as more IL-1? and PGE2 were produced at non-cytotoxic doses of the chemical and varied with the allergen. The SkinEthic model was used by Coquette et al. (1999) to measure IL-1? and IL-8 mRNA and protein levels as well as cytotoxicity following irritant and allergen treatment. More recently, Coquette et al. (2003) have suggested that determination of IL-8, with IL-1? , and MTT conversion shows potential to discriminate and classify irritants and allergens in a single assay. Like Kubilus et al., they report that IL-1? release increases with cytotoxicity after treatment with the irritants Triton
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X100 and BC, but had no effect on IL-8 levels. In contrast, the allergen DNCB did not induce IL-1? but instead, elicited elevated IL-8 levels. However, when mRNA expression was measured, BC, Triton X100 and DNCB all induced an up-regulation in message for IL-8 while only BC up-regulated IL-1? mRNA. The results of a number of studies conducted to assess the ability of irritants and contact allergens to affect cytokine mRNA levels in skin equivalents have been reviewed by Gerberick and Sikorski (1998). Data from those studies indicated that while cytokine message could be modulated by chemical treatment, each chemical appeared to produce changes in cytokine mRNA expression which was unique to the chemical and was also concentration and or time-dependent. No single cytokine or profile of cytokines were identified which were predictive for sensitization potential. However, Corsini et al. (1999) recently reported that interleukin-12 (IL-12) is selectively induced in the Episkin? model by treatment with chemical allergens. Following a 3 hour exposure to test chemicals, mRNA levels for both IL-12 p40 and IL-12 p35 were found to be up-regulated by contact allergens (oxazolone, eugenol and DNCB) but not the irritants (SLS and BC). 2. 3. 4. References - See body of the text Developer of the method - Not applicable Known users - None
5. Status of validation and/or standardisation No validation of test methods to date. 6. What efforts are needed to complete validation of the method? Not applicable 2.2.2.7 Human skin explant cultures Skin explant cultures utilize full thickness human skin specimens typically obtained from breast or abdominal reduction surgery. Like skin equivalent cultures, explants allow for the topical application of test chemicals. However, in addition to keratinocytes and fibroblasts, explants also contain cells of the immune system including LC, as well as other resident skin cells such as melanocytes and endothelial cells. A predictive assay for contact allergens which utilizes human skin explant cultures has been proposed by Pistoor et al. (1996). The assay focuses on changes in the distribution of the LC population following the epicutaneous application of test chemicals at nontoxic concentrations. They observed, in cryostat sections of the explants after 24 hours of culture, that contact allergens, but not irritants or inactive chemicals, induced a dose dependent reduction in the number of LC (HLA-DR+CD1a+ cells) in the epidermis and an accumulation of the remaining LC at the epidermal-dermal junction. However, LC migration in skin explants may not be a reliable endpoint for an in vitro assay for contact allergens, as LC have been shown to spontaneously migrate out of human skin cultured for one to three days (Lukas et al. 1996). Further studies by the same laboratory examined changes in cell surface molecules and cytokine expression by LC in the skin explants following chemical treatment (Rambukkana et al. 1996). They reported that after 24 hours of exposure to contact allergens, the LC which had migrated to the epidermal-dermal junction had a decreased expression of both CD1a and HLA-DR and a significant increase in the expression of ICAM-1 (CD54). The presence of IL-1? was detected in epidermal cells as early as 4 hours following treatment with the contact allergen DNFB, but not with the irritant SDS or the vehicle. After 24 hours of DNFB treatment, IL-1? as well as TNF-? , IL-1? , GM-
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CSF and IL-6 were evident in the epidermal cells. However, these cytokines were also found in the explants treated with SDS. 2. 3. 4. References - See body of the text Developer of the method Not applicable Known users - None
5. Status of validation and/or standardisation No validation of test methods to date. 6. What efforts are needed to complete validation of the method? Not applicable 2.2.3 Future prospects and recommendations The two - not mutually exclusive - approaches for the substitution of in-vivo sensitisation tests are in-silico methods, i.e., computer-based expert or QSAR systems, and in-vitro methods that a mostly based on chemical-induced responses of cell-culture systems. The fundamental difference between the two approaches is that more experience has been gained to date with regard to t e validation of tests and the development of test guidelines h (such as the OECD technical guidelines) for in-vitro alternatives. However, at the moment in-vitro systems for the identification of skin sensitisers are still at basic research level. The advances in basic immunological research continues to put more and more candidates on the list of potential test parameters, i.e. the upregulation or downregulation of the expression of cell membrane proteins and cell-cell signalling molecules, such as interleukins, and changes in the antigen uptake process. Here, a great deal of focused research will be necessary to identify the most relevant parameters and to standardise testing protocols and validate tests. As depicted in the table, every in-vitro test will only be able to assess, at best, one step of the multi-step mechanism of skin sensitisation. This, in turn, means that no single in-vitro test parameter alone will show a sufficient correlation with the sensitising potential in vivo. The validation process has to take this into account: rather than separate validation of one or a few tests a joint validation of an in-vitro test battery is likely to be required. Some more fundamental questions will have to be answered for in-silico systems: How should QSAR systems be validated? How can testing guidelines be developed for QSAR systems? Would such a guideline have to define the data set that is to be used with a certain QSAR system and the prediction space, i.e. prerequisite in terms of certain physico-chemical parameters that a test substance has to fulfil in order to generate meaningful QSAR results. In view of the fact that QSARs currently seem further developed than in-vitro alternatives, it is recommended that discussion of these questions among all stakeholders be initiated and further development supported. The ability of QSAR systems and in-vitro batteries to correctly predict a chemical's property as a human sensitiser or a human non-sensitiser will constitute the decisive measure in identifying the most relevant alternative to animal testing.
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2.2.4 Key references Aiba S. and Katz S. I. (1990) Phenotypical and functional characteristics of in vivo-activated Langerhans cells. Journal of Immunology 145, 2791-2796.
Mechanistic steps in skin sensitisation covered by in vivo sensitisation tests and in vitro alternative methods
First skin contact with sensitiser (induction phase) Skin penetration of the sensitising substance Metabolism of prohapten into hapten (ultimate sensitiser) Binding of hapten to soluble or cell-associated proteins Uptake of hapten-protein adducts by Langerhans cells
GMPT; MEST Buehler test HRIPT; HMT
Alternative in vitro methods

LLNA
Skin penetration using ex vivo pig, human or reconstituted skin Cell-free or cell-based protein/peptide binding assays +/- metabolising system
Induction of migration of Langerhans cells (LC) to draining lymph node and induction of maturation of Langerhans cells into antigen-presenting dendritic cells (DC); effects may partly be caused indirectly, e.g. by stimulation of keratinocytes Presentation of haptenated peptide-MHC complexes on dendritic cells in lymph nodes Recognition of presented haptenated peptide-MHC complexes by naive T lymphocytes with suitable T cell receptors Clonal expansion of primed T lymphocytes by proliferation and differentiation into effector and memory T cells and recirculation through blood and lympoid organs Second skin contact with sensitiser (elicitation or challenge phase) Skin penetration of the sensitising substance Metabolism of prohapten into hapten, binding to protein, uptake by Langerhans cells or other cells expressing MHC class II (e.g., keratinocytes) Induction of infiltration of phagocytes and effector T cells from blood into skin site Recognition of presented haptenated peptide-MHC complexes by effector T lymphocytes with subsequent induction of local inflammatory reaction
Cell surface marker and cytokine gene expression in LC, DCand keratinocytes, intracellular signalling; LC migration in ex vivo or reconstituted skin
In vitro priming of naive human or murine T lymphocytes
Computer-based QSAR and expert systems correlate structure with capability to cause skin sensitisation
Aiba S. and Tagami H. (1999) Dendritic cells play a crucial role in innate immunity to simple chemicals. Journal of Investigative Dermatology Symposium Proceedings 4, 158-163. Aiba S., Terunuma A., Manome H. and Tagami H. (1997) Dendritic cells differently respond to haptens and irritants by their production of cytokines and expression of co-stimulatory molecules. European Journal of Immunology 27, 3031-3038. Ariizumi K., Kitajima T., Bergstresser P. R. and Takashima A. (1995) Interleukin-1? converting enzyme in murine Langerhans cells and epidermal-derived dendritic cell lines. European Journal of Immunology 25, 2137-2141. Arimura M., Yokozeki H., Katayama I., Nakamura T., Masuda M. and Nishioka K. (1998) Experimental study for the development of an in vitro test for contact allergens. 2. Comparison of the in vitro sensitization test with the guinea pig maximization test for contact allergens. International Archives of Allergy and Immunology 115, 228-234. Ashikaga T., Hoya M., Itagaki H., Katsumura Y., and Aiba S. (2002) Evaluation of CD86 expression and MHC class II molecule internalization in THP-1 human monocyte cells as predictive endpoints for contact sensitizers. Toxicology In Vitro 16 711-716. Banerjee G., Iyer V. J. and Cherian K. M. (2003) A rapid in vitro method of identifying contact allergens and irritants. Toxicology Mechanisms and Methods 13 103-109.
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Becker D., Mohamadzadeh M., Reske K., and Knop J. (1992a) Increased level of intracellular MHC class II molecules in murine Langerhans cells following in vivo and in vitro administration of contact allergens. Journal of Investigative Dermatology 99, 545-549. Becker D., Nei U., Neis S., Reske K. and Knop J. (1992b) Contact allergens modulate the expression of MHC class II molecules on murine epidermal Langerhans cells by endocytic mechanisms. Journal of Investigative Dermatology 98, 700-705. Becker D., Kuhn U., Lempertz U., Enk A., Saloga J. and Knop J. (1997) Flow-cytometric screening for the modulation of receptor-mediated endocytosis in human dendritic cells: implications for the development of an in vitro technique for predictive testing of contact sensitizers. Journal of Immunological Methods 203, 171-180. Coquette A., Berna N., Vandenbosch A., Rosdy M. and Poumay Y. (1999) Differential expression and release of cytokines by an in vitro reconstructed human epidermis following exposure to skin irritant and sensitizing chemicals. Toxicology In Vitro 13, 867-877. Coquette A., Berna N., Vandenbosch, Rosdy M., De Wever B., and Poumay Y. (2003) Analysis of interleukin-1? (IL-1? ) and interleukin-8 (IL-8) expression and release in in vitro reconstructed human epidermis for the prediction of in vivo skin irritation and/or sesitization. Toxicology in Vitro 17 311-321. Corsini E., Primavera A., Marinovich M. and Galli C. L. (1998) Selective induction of cellassociated interleukin-1? in murine keratinocytes by chemical allergens. Toxicology 129, 193-200. Corsini E., Limiroli E., Marinovich M., Cohen C., Rouget R. and Galli C. L. (1999) Selective induction of interleukin-12 in reconstructed human epidermis by chemical allergens. Alternatives To Laboratory Animals 27, 261-269. Coutant K. D., de Brugerolle de Fraissinette A., Cordier A. and Ulrich P. (1999a) Modulation of the activity of human monocyte-derived dendritic cells by chemical haptens, a metal allergen, and a staphylococcal superantigen. Toxicological Sciences 52, 189-198. Coutant K. D., Ulrich P., Thomas H., Cordier A. and de Brugerolle de Fraissinette A. (1999b) Early changes in murine epidermal cell phenotype by contact sensitizers. Toxicological Sciences 48, 74-81. Cronin, M.T.D., Dearden, J.C., Walker, J.D & Worth, A.P. (2003). Quantitative structureactivity relationships for human health effects: commonalities with other endpoints. Environmental Toxicology and Chemistry 22, 1829-1843. Dai R. and Streilein J. W. (1998) Nave, hapten-specific human T lymphocytes are primed in vitro with derivatized blood mononuclear cells. Journal of Investigative Dermatology 110, 29-33. Degwert J., Steckel F., Hoppe U. and Kligman H. (1997) In vitro model for contact sensitization: I. Stimulatory capacities of human blood-derived dendritic cells and their phenotypical alterations in the presence of contact sensitizers. Toxicology in Vitro 11, 613618.
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De Smedt A. C., Van Den Heuvel R. L., Swi Berneman N., and Schoeters G. E. (2001) Modulation of phenotype, cytokine production and stimulatory function of CD34+-derived DC by NiCl(2) and SDS. Toxicology In Vitro 15 319-325. De Smedt A. C. Van Den Heuvel R. L., Van Tendeloo V. F. Berneman Z. N., Schoeters G. E., Weber E., and Tuschl H. (2002) Phenotypic alterations and IL-1 beta production in CD34(+) progenitor- and monocyte-derived dendritic cells after exposure to allergens: a comparative analysis. Archives of Dermatological Research 294 109-116. Enk A. H., Angeloni V. L., Udey M. C. and Katz S. I. (1993) An essential role or Langerhans cell-derived IL-1? in the initiation of primary immune responses in skin. Journal of Immunology 150, 3698-3704. Enk A. H. and Katz S. I. (1992) Early molecular events in the induction phase of contact sensitivity. Proceedings of the National Acadamy of Science USA 89, 1398-1402. Facy V., Flouret V., Rgnier M. and Schmidt R. (2004) Langerhans cells integrated into human reconstructed epidermis respond to know sensitizers and UV-exposure. Journal of Investigative Dermatology 122, 552-553. Gerberick G. F. and Sikorski E. E. (1998) In vitro and in vivo testing techniques for allergic contact dermatitis. American Journal of Contact Dermatitis 9, 111-118. Girolomoni G., Cruz P. D. and Bergstresser P. R. (1990) Internalization and acidification of surface HLA-DR molecules by epidermal Langerhans cells: a paradigm for antigen processing. Journal of Investigative Dermatology 94, 753-760. Hanau D., Schmitt D. A., Fabre M. and Cazenave J. P. (1988) A method for the rapid isolation of human epidermal Langerhans cells using immunomagnetic microspheres. Journal of Investigative Dermatology 91, 274-279. Hauser C. and Katz S. I. (1988) Activation and expansion of hapten- and protein-specific T helper cells from nonsensitized mice. Proceedings of the National Academy of Sciences of the USA 85, 5625-5628. Hulette B. C., Rowden G., Ryan C. A., Lawson C. M., Dawes S. M., Ridder G. M., and Gerberick G. F. (2001) Cytokine induction of a human acute myelogenous leukemia cell line (KG-1) to a CD1a+ dendritic cell phenotype. Archives of Dermatology Research 293 147158. Hulette B. C., Ryan C. A., and Gerberick G. F. (2002) Elucidating changes in surface marker expression of dendritic cells following chemical allergen treatment. Toxicology and Applied Pharmacology 182 226-233. Herouet C., Cottin M., Galanaud P., Leclaire J. and Rousset F. (1999) Contact sensitizers decrease 33D1 expression on mature Langerhans cells. European Journal of Dermatology 9, 185-190. Kitajima T., Ariizumi K., Mohamadazadeh M., Edelbaum D., Bergstresser P. R. and Takashima A. (1995) T cell-dependent secretion of IL-1? by a dendritic cell line (XS52) derived from murine epidermis. Journal of Immunology 155, 3794-3800.
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Kobayashi Y., Staquet M Dezutter-Dambuyant C. and Schmitt D. (1994) Development of -J., motility of Langerhans cell through extracellular matrix by in vitro hapten contact. European Journal of Immunology 24, 2254-2257. Kobel M. A., Limat A., Mauri D., Braathen L. R. and Hunziker T. (1996) Primary sensitization of Human T lymphocytes by cytokine-cultured, peripheral blood monocytes. European Journal of Dermatology 6, 409-413. Koch F., Kmpgen E., Schuler G. and Romani R. (1992) Effective enrichment of murine epidermal Langerhans cells by a modified (mismatched) panning technique. Journal of Investigative Dermatology 99, 803-807. Krasteva M., Peguet-Navarro J., Moulon C., Courtellemont P., Redziniak G. and Schmitt D. (1996) In vitro primary sensitization of hapten-specific T cells by cultured human epidermal Langerhans cells - a screening predictive assay for contact sensitizers. Clinical and Experimental Allergy 26, 563-570. Kripke M. L., Munn C. G., Jeevan A., Tang J. M. and Bucana C. (1990) Evidence that cutaneous antigen-presenting cells migrate to regional lymph nodes during contact sensitization. Journal of Immunology 145, 2833-2838. Kubilus J., Cannon C., Neal P., Sennott H. and Klausner M. (1996) Response of the EpiDerm skin model to topically applied irritants and allergens. In Vitro Toxicology 9, 157-166. Khn U., Brand P., Willemsen J., Jonuleit H., Enk A. H., van Brandwijk-Petershans R., Saloga J., Knop J. and Becker D. (1998) Induction of tyrosine phosphorylation in human MHC class II-positive antigen-presenting cells by stimulation with contact sensitizers. Journal of Immunology 160, 667-673. Lempertz U., Khn U., Knop J. and Becker D. (1996) An approach to predictive testing of contact sensitizers in vitro by monitoring their influence on endocytic mechanisms. International Archives of Allergy and Immunology 111, 64-70.
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Matsue H., Cruz P. D., Bergstresser P. R. and Takashima A. (1992) Cytokine expression by epidermal cell subpopulations. Journal of Investigative Dermatology 99, 42S-45S. Moulon C., Pguet-Navarro J., Courtellemont P., Redziniak G. and Schmitt D. (1993) In vitro primary sensitization and re-stimulation of hapten-specific T cells by fresh and cultured human epidermal Langerhans cells. Immunology 80, 373-379. Neisius U., Brand P., Plochmann S., Saloga J., Knop J. and Becker D. (1999) Detection of increased tyrosine phosphorylation in murine Langerhans cells after stimulation with contact sensitizers. Archives of Dermatological Research 291, 22-27. Pastore S., Shivji G. M., Kondo S., Kono T., McKenzie R. C., Segal L., Somers D. and Sauder D. N. (1995) Effects of contact sensitizers neomycin sulfate, benzocaine and 2,4dinitrobenzene 1-sulfonate, sodium salt on viability, membrane integrity and IL-1? mRNA expression of cultured normal human keratinocytes. Food and Chemical Toxicology 33, 5768. Pichowski J. S., Cumberbatch M., Dearman R. J., Basketter D. A. and Kimber I. (2000) Investigation of induced changes in interleukin 1? mRNA expression by cultured human dendritic cells as an in vitro approach to skin sensitization testing. Toxicology In Vitro 14, 351-360. Pichowski J. S., Cumberbatch M., Dearman R. J., Basketter D. A and Kimber I. (2001) ., Allergen-induced changes in interleukin 1 beta (IL-1 beta) mRNA expression by human blood-derived dendritic cells: inter-individual differences and relevance for sensitization testing. Journal of Applied Toxicology 21 115-121. Pistoor F. H. M., Rambukkana A., Kroezen M., Lepoittevin J.-P., Bos J. D., Kapsenberg M. L. and Das P. K. (1996) Novel predictive assay for contact allergens using human skin explant cultures. American Journal of Pathology 149, 337-343. Rambukkana A., Pistoor F. H. M., Bos J. D., Kapsenberg M. L. and Das P. K. (1996) Effects of contact allergens on human Langerhans cells in skin organ culture: migration, modulation of cell surface molecules, and early expression of interleukin-1? protein. Laboratory Investigations 74, 442-436. Rgnier M., Staquet M. J., Schmitt D. and Schmidt R. (1997). Integration of Langerhans cells into a pigmented reconstructed human epidermis. Journal of Investigative Dermatology 109, 510-512. Reutter K., Jager D., Degwert J. and Hoppe U. (1997) In vitro model for contact sensitization: II. Induction of IL-1? mRNA in human blood-derived dendritic cells by contact sensitizers. Toxicology in Vitro 11, 619-626. Rizova H., Carayon P., Barbier A., Lacheretz F., Dubertret L. and Michel L. (1999) Contact allergens, but not irritants, alter receptor-mediated endocytosis by human epidermal Langerhans cells. British Journal of Dermatology 140, 200-209.
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Romani N., Gruner S., Brang D., Kampgen E., Lenz A., Trockenbacher B., Konwalinka G., Fritsch P.O., Steinman R. M. and Schuler G. (1994) Proliferating dendritic cell progenitors in human blood. Journal of Experimental Medicine 180, 83-93. Rougier N., Redziniak G., Schmitt D. and Vincent C. (1998) Evaluation of the capacity of dendritic cells derived from cord blood CD34+ precursors to present haptens to unsensitized autologous T cells in vitro. Journal of Investigative Dermatology 110, 348-352. Rougier N., Redziniak G., Mougin D., Schmitt D. and Vincent C. (2000) In vitro evaluation of the sensitization potential of weak contact allergens using Langerhans-like dendritic cells and autologous T cells. Toxicology 145, 73-82. Rustemeyer T., De Ligter S., Von Blomberg M. E., Frosch P. J. and Scheper R. J. (1999) Human T lymphocyte priming in vitro by haptenated autologous dendritic cells. Clinical and Experimental Immunology 117, 209-216. Ryan C.A., Hulette, B.C. and Gerberick G.F. (2001) Approaches for the development of cellbased in vitro methods for contact sensitization. Toxicology In Vitro 15, 43-55. Scholes E. W., Pendlington R. U., Sharma R. K. and Basketter D. A. (1994) Skin metabolism of contact allergens. Toxicology in Vitro 8, 551-553. Schwarz T. and Luger T. A. (1992) Pharmacology of cytokines in the skin. In Pharmacology of the Skin. Edited by H. Mukhtar. pp. 283-313. CRC Press, Boca Raton. Schwarz T. and Luger T.A. (1992) Pharmacology of cytokines in the skin. In: Mukhtar H. (Ed.), Pharmacology of the Skin. CRC Press, Boca Raton, FL, pp. 283-313. Schwarzenberger K. and Udey M. C. (1996) Contact allergens and epidermal proinflammatory cytokines modulate Langerhans cell E-cadherin expression in situ. Journal of Investigative Dermatology 106, 553-558. Simon J. C., Dittmar H. C., de Roche R., Wilting J., Christ B. and Schopf E. (1995) Rapid purification of human Langerhans cells using paramagnetic microbeads. Experimental Dermatology 4, 155-161. Takashima A., Edelbaum D., Kitajima R., Shadduck K., Gilmore G. L., Xu S., Taylor R. S., Bergstresser P. R. and Ariizumi K. (1995) Colony-stimulating factor-1 secreted by fibroblasts promotes the growth of dendritic cell lines (XS series). Journal of Immunology 154, 5128-5135. Teunissen M. B., Wormmeester J., Kapsenberg M. L. and Bos J. D. (1988) Enrichment of unlabeled human Langerhans cells from epidermal cell suspensions by discontinuous density gradient centrifugation. Journal of Investigative Dermatology 91, 358-362. Tuschl H., and Kovac R. (2001) Langerhans cells and immature dendritic cells as model systems for screening of skin sensitizers. Toxicology In Vitro 15 327-331. Verrier A. C., Schmitt D., and Staquet M. J. (1999) Fragrance and contact allergens in vitro modulate the HLA-DR and E-Cadherin expression on human epidermal Langerhans cells. International Archives of Allergy and Immunology 120, 56-62.
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Wakem P., Burns R. P., Ramirez F., Zlotnick D., Ferbel B., Haidaris C. G. and Gaspari A. A. (2000) Allergens and irritants transcriptionally upregulate CD80 gene expression in human keratinocytes. Journal of Investigative Dermatology 114, 1085-1092. Wilmer J. L., Burleson F. G., Kayama F., Kanno J. and Luster M. I. (1994) Cytokine induction in human epidermal keratinocytes exposed to contact irritants and its relation to chemical-induced inflammation in mouse skin. Journal of Investigative Dermatology 102, 915-922. Xu S., Ariizumi K., Caceres-Dittmar G., Edelbaum D., Hashimoto K., Bergstresser P. R. and Takashima A. (1995a) Successive generation of antigen-presenting, dendritic cell lines from murine epidermis. Journal of Immunology 154, 2697-2705. Xu S., Ariizumi K., Edelman D., Bergstresser P. R. and Takashima A. (1995b) Cytokinedependent regulation of growth and maturation in murine epidermal dendritic cell lines. European Journal of Immunology 25, 1018-1024. Yamada N. and Katz S. I. (1999) Generation of mature dendritic cells from a CD14+ cell line (XS52) by IL-4, TNF-? , IL-1? , and agonistic anti-CD40 monoclonal antibody. Journal of Immunology 163, 5331-5337. Yokozeki H., Katayama I. and Nishioka K. (1995) Experimental study for the development of an in vitro test for contact allergens. 1. Primary activation of hapten-specific T cells by hapten-conjugated epidermal cells. International Archives of Allergy and Immunology 106, 394-400. Yoshida Y., Sakaguchi H., Ito Y., Okuda M., and Suzuki H. (2003) Evaluation of the skin sensitization potential of chemicals using expression of co-stimulatory molecules, CD54 and CD86, on the naive THP-1 cell line. Toxicology In Vitro 17, 221-228.
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Summary of the alternative methods currently available and foreseeable time to achieve ESAC endorsement
Current endpoints addressed in animal test
LLNA (OECD TG 429) Lymphocytes proliferation
Alternative tests available
Endpoints measured
Purpose Partial replacement
Validation status
Regulatory acceptance None but limited acceptance by some national authorities
Comments
Estimated time to have the method validated (ESAC endorsement)*
In silico alternatives (Q)SARs
All chemicals (test battery) Partial replacement All chemicals (test battery)
Optimised
5 - 6 years
MEST Ear swelling
Protein / peptide binding
Protein / peptide binding Keratinocyte activation (e.g. production of proinflammatory cytokines) Activation of Langerhans cells and dendritic cells (e.g. migration, cell surface marker expression, cytokine production)
R&D
---
5 - 6 years
GPMT Buehler Test (OECD TG 406 EU B.06) Skin reaction
Keratinocyte cultures
Partial replacement (test battery)
All chemicals
R&D
---
5 - 6 years
Langerhans cell cultures Peripheral blood derived dendritic cells Dendritic cell lines
Partial replacement All chemicals (test battery) R&D ---
These approaches will be evaluated in parallel with only the most successful emerging for formal validation.
5 - 6 years
32
Co-culture systems
Activation of T lymphocytes (e.g. proliferation, cell surface markers expression) Activation of keratinocytes (and skin penetration) Skin penetration and activation of keratinocytes and Langerhans cells Full replacement
All chemicals
R&D
---
10 - 12 years
Reconstituted epidermal cultures
All chemicals
R&D
---
10 - 12 years
Human skin explant cultures
All chemicals
R&D
---
Never, due to insufficient supply of test material
In vitro Test Battery
Various (see above)
To be assembled from individual test methods in the future
All chemicals
---
Guidance for the validation of test batteries has to be developed
10 - 12 years
33
Conclusions Different in silico and in vitro models are being developed to assess the skin sensitising potential of chemicals and products. The two - not mutually exclusive - approaches for the substitution of in-vivo sensitisation comprise computer-based expert or QSAR systems, and in-vitro methods that are mostly based on chemical-induced responses of cell-culture systems. Among these, (Q)SARs systems are the most developed and it is envisaged that further optimisation of such methods, achievable by extending their range of chemical knowledge, will lead to the formal validation within the next few years. The development process of (Q)SARs is mainly carried out by consortia and companies that develop and distribute these programs. An approach to the evaluation of these systems is being defined at OECD level. In November 2002, the 34th Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, Pesticides and Biotechnology held a Special session on QSARs, during which it identified a lack of validated QSARs for use in regulatory assessment and agreed to start an activity aimed at the development of internationally accepted validation principles and procedures, and evaluation of existing and promising QSARs against these principles. Six principles for judging the validity of (Q)SARs were agreed at the International Council of Chemical Associations (ICCA)/European Chemistry Industry Council (CEFIC) Workshop on Regulatory acceptance of QSARs for Human Health and Environmental Endpoints held in Setubal, Portugal on 4-6 March 2002. These were further discussed at the first meeting of the OECD Ad Hoc Expert group on QSARs on 31 March- 2 April 2003. The Expert group developed a draft 2 year work plan (endorsed at the 35th Joint Meeting) which comprises three activities:1) evaluating the so-called Setubal principles for QSARs; 2) developing guidance documents (e.g. on the validation of QSARs); and 3) making validated QSARs available to users. Via the work of ECVAM and the ECB, the European Commission is playing a leading role in the OECD QSAR activity. The validation of (Q)SARs for skin sensitisation is a high priority for ECVAM. Although cell based systems, have in some cases been shown to be capable of distinguishing between sensitisers and non-sensitisers (and so could be used for priority setting), they are still at a basic research level. Advances in basic immunological research continue to increase the number of potential test parameters, eg. the upregulation or downregulation of the expression of cell membrane proteins and cell-cell signalling molecules, such as interleukins, and changes in the antigen uptake process. Further research and refinement activities are needed before a test battery can be set up from the in vitro tests. Research is currently carried out by individual companies and academia, with funding mainly provided by industry and industry associations (i.e. a COLIPA research program). A part of the currently ongoing basic research focuses on the elucidation of the mechanisms underlying skin sensitisation. Therefore, a coordinating advisory body could help to focus research on the development of a predictive test method and, thus, speed up the R&D process. Due to the complexity of the mechanisms of skin sensitisation, a single test will not be able to replace the currently required animal experiments. Efforts are still needed to identify the most relevant endpoints and in the optimisation of existing tests. However, combination of several in vitro tests, covering all relevant mechanistic steps of skin sensitisation, into a test battery can likely lead to replacement of the in vivo tests. The validation process has to take this into account: rather than separate validation of one or a few tests a joint validation of an in-vitro test battery will be required. Currently no guidance for the validation of a test battery is
34
available and development of such guidance would help the development of an animal alternative. It is proposed that such guidance be developed by ECVAM. An indicative time foreseen for the availability of ESAC endorsed validated alternative test battery for skin sensitisation is of ten-twelve years; twelve-fourteen year is the time required for regulatory acceptance at the EU level. This estimated deadline for phasing out the animal experiments can only be met if the necessary resources are made available to progress research, development, and optimisation in this field.
35
Chapter No. 5: Skin absorption / penetration Walter Diembeck1, Chantra Eskes2, Jon R. Heylings3, Gill Langley4, Vera Rogiers 5, Johannes J. M. van de Sandt 6, Valrie Zuang2 Beiersdorf A.G., Hamburg, Germany; 2ECVAM-JRC, Ispra, Italy; 3Syngenta CTL, Alderley Park, Macclesfield, UK; 4Dr Hadwen Trust, Hitchin, Herts, UK; 5SCCNFP, V.U.B., Brussels, Belgium; 6TNO Nutrition and Food Research, Zeist, The Netherlands
1
1. Current data requirements and current tests 1.1. For cosmetic ingredients 1.1.1. Data required from the cosmetic ingredients suppliers: skin absorption testing of chemicals is normally not required by the chemical dangerous substances legislation. For cosmetics, however, companies will require skin absorption data from ingredient suppliers, in particular in the case of actives, e.g. colourants, preservatives, uv filters, substances with limitatations in concentration and site of application. In this case, skin absorption data must be available in the safety dossier (PIR required by the 6th Amendment) presented by the industry to the SCCNFP. 1.1.2. Data required from the cosmetic industry: skin absorption data, in particular of actives, must be provided in the safety dossier (PIR) for all ingredients (6th Amendment). 1.2. For cosmetic finished products: normally no absorption/penetration data required but the above mentioned tests for the ingredients must have been performed using relevant formulations (close to market product). The worst case situation should be taken into consideration (see: updated version of Basic criteria for the in vitro assessment , SCCNFP (2003)). 1.3. Available human tests and/or data: skin absorption studies in human volunteers can be performed in cases of low toxicity of cosmetic ingredient/product, but often no human data are available. In vitro testing is preferably carried out on excised pig or human skin, according to OECD Guideline 428. Only these data are acceptable. In vitro rodent skin absorption data is considered to be irrelevant (see: updated version of Basic criteria for the in vitro assessment , SCCNFP (2003)). For in vivo testing of chemicals in experimental animals, see OECD Guideline 427 Skin absorption: in vivo method (adopted in May 2002). 2. Inventory of alternative methods currently available 2.1 Alternatives to test skin absorption / penetration 2.1.1 State of the art: see ?? OECD Skin Absorption: In vivo Method. Organization for Economic Cooperation and Development (OECD), Environment Directorate, OECD
??
??
??
?? ??
?? ??
??
??
Guideline for the Testing of Chemicals, Guideline 427, Paris (adopted in May 2002, official publication in Febr. 2004)) [rat protocol only] OECD Skin Absorption: In vitro Method. Organization for Economic Cooperation and Development (OECD), Environment Directorate, OECD Guideline for the Testing of Chemicals, Guideline 428, Paris (adopted in May 2002, official publication in Febr. 2004) [either flow-through or static diffusion cells, excised pig or human skin] OECD Guidance Document for the Conduct of Skin Absorption Studies. Organization for Economic Cooperation and Development (OECD), Environment Directorate, OECD Environmental Health and Safety Publications, Series on Testing and Assessment No. 28, Paris (adopted in May 2002, official publication in Febr. 2004) Diembeck W., Beck H., Benech-Kieffer F., Courtellemont P., Dupuis J., Lovell W., Paye M., Spengler J. and Steiling W.. Test guidelines for in vitro assessment of dermal absorption and percutaneous penetration of cosmetic ingredients. Food and Chemical Toxicology, 37, 191-205 (1999) Cosmetic Ingredients: Guidelines for Percutaneous Absorption/Penetration, COLIPA, Brussels (1995) Technical Guidance Document on Risk Assessment in support of Commission Directive 93/67/EEC on Risk Assessment for new notified substances, Commission Regulation (EC) No 1488/94 on Risk Assessment for existing substances and Directive 98/8/EC of the European Parliament and of the Council concerning the placing of biocidal products on the market. Doc. EUR 20418 EN/1, European Communities (2003) ECETOC Percutaneous absorption. European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC), Monograph No 20, Brussels (1993) EPA (United States Environmental Protection Agency) Proposed rule for in vitro dermal absorption rate testing of certain chemicals of interest to occupational safety and health administration. Federal Register, Volume 64, Number 110, June 9 (1999) Howes D., Guy R., Hadgraft J., Heylings J., Hoeck U., Kemper F., Maibach H., Marty J-P., Merk H., Parra J., Rekkas D., Rondelli I., Schaefer H., Tuber U. and Verbiese N. Methods for assessing percutaneous absorption, ECVAM Workshop Report No. 13. Alternatives To Laboratory Animals (ATLA) 24, 81-106 (1996) Regulatory Toxicology and Pharmacology 31, 53-58 (2000). Sanco/222/2000 Guidance Document on Dermal Absorption European Commission, Health and Consumer Protection Directorate-General Doc. Sanco/222/2000 rev 6, of 27 November 2002
?? SCCNFP/0167/99, Final : Basic Criteria for the in vitro assessment of percutaneous absorption of cosmetic ingredients, adopted by the SCCNFP during the 8th plenary meeting of 23 June 1999 ?? Guidelines for in vitro methods to assess percutaneous absorption of cosmetic ingredients in Notes of Guidance for testing of cosmetic ingredients for their safety evaluation, SCCNFP/0321/00 Final (2000) ?? SCCNFP/.../03, Updated basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients,discussed by the SCCNFP at subgroup meeting 10 September 2003, on the agenda of the plenary of 20 October 2003 ?? SCCNFP//03, Final : Notes of Guidance for Testing of Cosmetic Ingredients for Their Safety Evaluation, 5th revision, on the agenda of the plenary meeting of 20 October, 2003 ?? Schaefer H. and Redelmeier T.E. Skin Barrier, Principles of Percutaneous Absorption, Karger, Basel (1996)
3. Identified areas or endpoints with no validated / valid alternative methods available: ?? standardized repeat topical dosing in vitro protocols currently not available. Some experience exists with repeat dosing designs within limited experimental time (24-48 h) ?? skin absorption/penetration in combination with metabolism 4. Conclusions and foreseeable timetable (to achieve complete animal replacement) In vivo endpoint: ?? test substance on (= residue) and within skin layers (stratum corneum, epidermis, dermis), in systemic circulation (= receptor fluid in vitro) after topical application ?? covered by alternative method (except metabolism, repeat dosing) Purpose: ?? reduction (OECD in vivo and in vitro guidelines, together with the guidance document, have been accepted in parallel at the same time) ?? replacement of single dose studies (no metabolism) Status: accepted (by OECD [2002] (in parallel with in vivo test), SCCNFP [2000] Validation authority: no formal validation (sufficient and convincing historical data) Regulatory authority: OECD, EC Area of application: chemicals in general (liquids, solutions, emulsions, no gases)
Recommendations: ?? screening test (= full replacement) ?? part of a tiered strategy (= partial replacement) Estimated time to have the method validated (ESAC endorsement): no peer reviewed validation foreseen (in vitro test already accepted by EC and OECD regulation)
5. References see 2.1.1 Remarks In principal the in vitro test can be seen as a full alternative to the in vivo test in most cases. However, the quality of submitted in vitro data is still not always satisfying with respect to documentation and technical aspects although the SCCNFP has published basic criteria [1999, 2003] and OECD [2000] a guidance document for this kind of study in addition to the respective guidelines. Many labs do still seem to need time (months years) to improve special skills which are a prerequisite for general acceptance of this type of in vitro test. Walter Diembeck, Hamburg, December 15, 2003
3.6. Subacute and subchronic toxicity

Pilar Prieto 1, Cecilia Clemedson2, Annarita Meneguz3, Walter Pfaller4, Ursula G. Sauer5, Carl Westmoreland6
1
ECVAM, Institute for Health & consumer Protection, Joint Research Centre, European Commission, 21020 Ispra (Va) Italy; 2Expertrdet ECB,
Hgklintavgen 7, 17264 Sundbyberg, Sweden; 3Laboratory of Pharmacology, Instituto Superiore di Sanit, Viale Regina Elana, 299, 00161 Roma, Italy;
4
University of Innsbruck, Frtiz-Pregl-Str. 3, Innsbruck, Austria; 5German Animal Welfare Federation - Animal Welfare Academy, Spechtstrasse 1, D-85579 Neubiberg , Germany; 6SEAC - Safety and Environmental Assurance Centre, Unilever Colworth, Sharnbrook, Bedfordshire MK44 1LQ, UK
3.6.1. Inventory of methods currently available Chronic toxicity is a consequence of the persistent or progressively deteriorating dysfunction of cells, organs or multiple organ systems, resulting from long-term exposure to a chemical. Of relevance for cosmetic products are the oral, dermal and inhalation subacute (28 days) and subchronic (90 days) repeated dose studies in rodents: ?? Repeated dose 28-day oral toxicity study in rodents ?? Repeated dose 90-day oral toxicity study in rodents
[OECD 407, EC B.7] [OECD 408, EC B.26]
?? Repeated dose dermal toxicity: 21/28-day study [OECD 410, EC B.9] ?? Subchronic dermal toxicity: 90-day study [OECD 411, EC B.28] ?? Repeated dose inhalation toxicity: 28-day or 14-day study [OECD 412, EC B.8] ?? Subchronic inhalation toxicity: 90-day study [OECD 413, ECB 29] The 28-day or 90-day oral toxicity tests in rodents are the most commonly used long-term toxicity tests. The highest dose administered is designed to cause
some toxicity, but not lethality. Upon completion of the test, a whole host of clinical and histological evaluations are recorded, including experimental
observations and whole body and individual organ analyses. Other subchronic toxicity studies include the combined repeat-dose toxicity and reproductive/developmental toxicity screening test (OECD TG 422).
In the notification process of dangerous substances, long-term toxicity studies are required when the substance under consideration is produced or imported in amounts exceeding 1 ton/year [92/32/EEC]. In the case of the development of cosmetic ingredients which have specific biological properties and which will come into contact with human skin for a long period of time, evaluation of the systemic risk is a key element in evaluating the safety of these new ingredients, irrespective of the tonnage-linked and possibly restricted requirements imposed by the Dangerous Substances Directive [67/548/EEC]. Therefore, in certain cases the use of animal long-term experiments to study one or more potential toxic effects remains to be required by law. The 7th Amendment [2003/15/EC] to the Cosmetic Directive 76/768/EEC allows 10 years from the entry into force date on, to come up with validated alternative tests for repeated exposure.
3.6.2. Inventory of alternative methods currently available 3.6.2.1. Introduction Currently no generally accepted alternative methods are available for replacing repeat-dose in vivo testing. Complete replacement of animal usage in these areas represents an enormous scientific and technical challenge. Since a wide range of endpoints are investigated in in vivo chronic toxicity studies, an integrated approach to repeat-dose toxicity testing based on the use of alternative methods with complementary endpoints will need to be develop. Such an approach must involve a wider consideration of the potential toxicity of a new molecule than considering in vitro toxicity towards target organs alone e.g. the eventual replacement of in vivo repeat-dose toxicity tests must involve some
integration of in vitro data on target organ toxicity with in vitro/in silico data on ADME parameters.
There are a number of questions that need to be answered in the context of replacement of animals in repeated-dose toxicity studies:
1. What organs does the chemical affect? 2. What is the relevance of the quantification of the effect (dosedependence)? 3. What in vitro markers of toxicity are relevant to the chemical? 4. What concentrations of the chemical should be tested? (i.e. what concentrations would be physiologically relevant?) 5. How could data from in vitro target organ toxicity tests be used in Risk Assessment?
The points above illustrate that one of the major challenges in this field will be the ability to identify the testing strategy needed for a new chemical where there is no knowledge on the potential toxicity of the molecule. At present, little research has addressed this problem. In vitro investigations into toxicity in specific target organs/tissues will probably provide important data in the eventual testing strategy. Another important aspect to take into consideration for long-term in vitro testing is the availability of more advance culture techniques that guarantee organ-level culture systems, which stably express as many functional properties of the in vivo counterparts as possible under organotypic conditions.
In addition, repeated-dose toxicity tests in animals have some serious scientific deficiencies due to species differences that reduce the reliability of predicting effects in humans from animal test results. This is reflected by conclusion No. 1 of the ECVAM Workshop 45 (1), which reads: 'The current reliance on chronic tests in laboratory animals is of limited value, because of species differences, general economic and logistical considerations and the high cost of mechanistic
studies.' Worth and Balls (2) point out: '[ ] as in the case of carcinogenicity testing and reproductive toxicity testing, inter-species differences limit the usefulness of animal studies for predicting long-term target-organ and target-system effects in humans.'
3.6.2.2. Available long-term in vitro models - Level - Basic research Only a few attempts have been made to obtain toxicological data from long-term exposure of cell culture or tissue culture systems to toxic chemicals or drugs. Most of these approaches suffer from the fact that they are acute (high-dose) short-term assays (3-8). Recently, attempts have been done to adapt primary cultures and cell lines to allow for the continuous low-dose, long-term administration of known hepatotoxic and nephrotoxic compounds (9).
It is worth mentioning some efforts done to apply QSARs for the prediction of chronic toxicity. In particular, the statistically based system TOPKAT has been proposed as a model for rat chronic lowest observed adverse effect level (10). In the following section the main available in vitro models in relation to five of the most common targets for toxicity (liver, kidney, CNS, lung and haematopoietic system) are included.
? IN VITRO LIVER PREPARATIONS ?
1. Isolated Perfused liver: this is the in vitro model closest to the in vivo situation. The limitations are short lifetime (2-3 hours) and complicated and demanding set-up (11).
2. Liver slices: retain the in vivo tissue organisation. The limitation is short lifetime and less animal reduction than cell based approaches (12, 13).
3. Isolated hepatocytes: it is the most frequently used in vitro model for hepatotoxicity testing. It can produce a metabolite profile of a drug very similar to that found in vivo. The limitations are short lifetime (24 hours) and loss of many liver specific functions. Non-human models are not always predictive of human in vivo situation. The availability of human cells is very limited (14).
4. Collagen sandwich cultures: the structural and functional integrity is retained for up to 15 days. Normal cell shape is kept; intact structure, bile canaliculi are preserved. ALAT and ASAT enzyme release can be studied. It has been successfully used in mimicking the chronic treatment in vivo situation (15). As with isolated hepatocytes, availability of human hepatocytes is an issue. They are promising models for studying human hepatocellular function, including protein synthesis and drug metabolism in vitro.
5. Ito cells and Ito cells/hepatocyte co-cultures: these are useful tools for studying liver fibrogenesis. The life-time is 96 hours. It is not a wellestablished model (16).
6. Genetically engineered cells expressing single human or animal P450 enzymes: There are used as a tool to assess the involvement of certain enzymes in metabolism, metabolite formation, and metabolism-dependent toxicity. The model is well characterised, well documented and easy to use. The limitations are: often only one CYP is expressed and the expression of transfected CYPs is variable and often different form the in vivo situation (17).
7. Cell lines derived from human hepatoma: They express CYP1A1 but there is low expression of other CYPs (17).
8. HepG2 grown under continuous medium supply: This has been used only in a few pilot studies. They are laborious systems for routine use (EU, Standard, Measurment and Testing Programme, SMT4-CT96-2070).
9. Perfusion culture systems: It consists of collagen-coated slides allowing the continuous superfusion of a hepatocyte monolayer with culture medium. This system is stable for at least 2 weeks and guarantees sensitivity to enzyme induction. Different concentrations of the test substance can be perfused. The major application concerns enzyme induction experiments (12)
10. 96-well plate bioreactor: it runs 96 modules in parallel for pharmacokinetic testing under aerobic conditions. The system combines the advantages of a three-dimensional culture system in collagen gel, controlled oxygen supply, and constant culture medium conditions, with the possibility of high throughput and autoimmunization (12).
? IN VITRO KIDNEY PREPARATIONS ?
11. Renal epithelial cells grown under continuous medium supply: Phenotypes with oxidative energy metabolism are available for tubular proximal cells. The morphology is very close to the in vivo parent cell type. Culture periods of up to 6 weeks are easily possible. Human derived proximal tubular cell lines similar in function to parent cells are available. Many functional parameters can be monitored and a number of endpoints can easily be assessed. However, they are laborious systems for routine use. (18).
? CENTRAL NERVOUS SYSTEM ?
12. Primary neuronal cell cultures (rat): They are well characterised. They allow the identification of neurodegenerative compounds. In vitro test systems are available to study biochemical, electrophysiological and morphological
endpoints to assess potential neurotoxicity. Long-term exposure is possible (7-14 days). They are used in industry for screening pharmaceuticals and agrochemicals (19).
13. Permanent neuronal cell lines: There are useful models for detection of delayed neurotoxicity caused by organophosphates because of the presence of target enzymes, neuropathy target esterase and acetylcholinesterase. SHSY5Y (human) and N-18 (mouse) neuroblastoma cell lines have been used for prediction of subchronic neurotoxicity, 96 hours exposure (20, 21).
14. Astrocytes (primary or cell line e.g. C6): The culture system is well developed. Primary astrocytes require long-term culture for differentiation. There is possible to detect reactive astrocytes (glial fibrillary acidic protein expression or cytokine production). Specific cell surface markers can be detected by cell imaging and FACS analysis after a toxic insult (22, 23).
15. Oligodendrocytes: They need long-term culture for maturation and/or testing of toxic compounds. In vitro models are established allowing detailed examination of the mechanisms associated with toxicant-induced
dysmyelination and demyelination. Specific cell surface markers can be detected by cell imaging and FACS analysis after a toxic insult (24).
16. Microglia:
rapid
response
to
neuronal
injury.
Cytokine
production,
morphology, phagocytosis and proliferation are useful endpoints. Cultures require 1-2 weeks for the production of well-characterised microglia. Cell culture models and endpoints measurements are successfully developed. Primary glial cell cultures are widely used in academia and in industry (25, 26).
17. Brain slices from hippocampus: Useful for detecting excitotoxic and/or convulsive properties of drugs (hardly detectable in rodents). They can be
maintained for relatively long periods of time, thus allowing prolonged exposure to assess toxicological effects. They are well-accepted for studying learning and memory deficits (difficult to detect in rodents). Used in agrochemical testing. Less animal reduction than cell based approaches (27).
18. Reaggregating brain cell cultures: It appears to be among the most promising models and reproduce one of the closest in vivo-like complexity (synaptogenesis, myelination). This system comprises an integrated
population of neurons and glial cells 3-dimensionally arranged, which allows histotypical cell-cell interactions to occur similar to those found in the in vivo situation (28-31).
? PULMONARY EPITHELIAL SYSTEMS ?
19. The isolated perfused rat and mouse lung in which the organs architecture and cell-cell interactions are well maintained allow only short-term but no long-term in vitro toxicity testing.
20. Tissue slices from the lung also have retained the in vivo architecture, but again represent only a model for studies on acute toxicity. The lifetime of this model is limited to a few hours.
21. Commercially available human-based respiratory airways models: MatTek's EpiAirway System consisting of
The
human-derived
tracheal/bronchial epithelial cells which form in culture a pseudo-stratified, highly differentiated model with characteristics close to the epithelial tissue of the respiratory tract (http://www.mattek.com/pages/products/epiairway). The reconstituted human alveolar epithelium in vitro from Skinthic consists of human alveolar cells of the immortalised cell line A549 that form an alveolar
epithelial tissue resembling histologically the outer alveolar cell layers of the human lung alveoli (http://www.skinethic.com/).
22. More recently, some successful attempts to develop primary cultures of pneumocytes type II from rat have been performed (32, 33). These cultures still respond to certain signals (ligands, e.g. mechanical stress or ATP) with the release of surfactant (34, 35). In addition, developments are on the way in several laboratories to establish air-liquid interphase models for pulmonary respiratory epithelium under either static or perfusion culture conditions (32, 33). Immortalised alveolar type II cells, still capable of surfactant release are meanwhile available (unpublished results). They respond to defined stimuli with a measurable release of surfactant and appear thus to represent a more reliable model than the human derived lung epithelial cell line A549, which have lost the capability of surfactant release although they contain lipid (mucus?) droplets within their cytoplasm.
? HAEMATOPOIETIC SYSTEM ?
23. Long-term bone marrow culture (LTBMC): A bone marrow derived adherent layer containing a variety of non-haematopoietic cells (fibroblasts, endothelial cells, smooth muscle and macrophages) supports the growth and
development of haematopoietic cells for 8-12 weeks. The system has been adapted for growth of human cells. Both competent bone marrow stromal layers and stromal cell lines that support growth of human cells are the basis for a variety of "stem cells" assays (36).
24. Long-term culture initiating cell (LTC-IC): It is the most frequently used method for assessing the frequency of primitive cells in vitro. LTC-IC are routinely measured at 5 weeks of culture (37).
25. Myeloid-lymphoid initiating cell (ML-IC) assay: This assays allows the in vitro identification of a single human bone marrow progenitor closely related to the haematopoietic stem cell, because it is capable of generating multiple secondary progenitors that can reinitiate long-term myeloid and lymphoid haematopoiesis in vitro (38).
26. Human cobblestone area forming cell (CAF): The CAF method is based on the long-term culture of bone marrow. The method utilises miniaturised LTC established in 96-well plates. The wells can be scored for the presence of cobblestone areas over 2-12 weeks and the frequencies of the cobblestone area-forming cells are calculated using Poisson statistics (39).
27. Blast-CFC: The blast colony-forming cell assay allows formation of colonies with a high secondary re-cloning capacity, self-renewal potential, and the ability to generate lineage-committed progeny in semi-solid culture. The key criterion for blast colony forming cells is the ability to form secondary colonies when replated in clonogenic assays (40).
28. HPP-CFC: Identifies a colony-forming cell with high proliferative potential (HPP-CFC) which generates a macroscopic colony containing about 50000 cells when cultured in a semi-solid medium. It requires up to 28 days to generate typical macroscopic colonies (41)
29. CFU-A: It detects a primitive cell with many properties in common with the murine CFU-S day 12. The assay probably identifies a population of cells containing a proportion of HPP-CFC (42).
? NOVEL CULTURE METHODS ?
30. Hollow fibre bioreactors: cells grow around hollow fibres and through which the culture medium flows (43, 44).
31. Perfusion culture models: combines the advantage of filter inserts with constant replenishment of the medium (15, 45).
3.6.3. Identified steps or tests with no alternative methods available As it was stated before, subacute and subchronic toxicity testing is an area where no alternative methods are yet available. Worth and Balls (2) point out (p. 74) "The readiness of in vitro models for long-term effects to undergo prevalidation and validation will depend on progress made at the research and test development level". A number of recommendations for research and development are also given (2).
3.6.4. Summary of the alternative methods currently available and foreseeable time to achieve ESAC endorsement.
Estimated time Current endpoint addressed in animal test Alternative tests available Endpoints measured Purpose Area of application Validation Regulatory status aceptance Comments to have the method validated (ESAC endorsement)* [OECD 407, EC B.7] [OECD 408, EC B.26] [OECD 410, EC B.9] [OECD EC B.28] [OECD EC B.8] [OECD ECB 29] 413, 412, 411, Perfusion culture systems (collagencoated slides) Various Collagen sandwich cultures Partial replaceme Various nt (tiered strategy and/or test battery) Partial replaceme nt (tiered strategy and/or test battery) Liver toxicants R&D > 10 years Liver toxicants R&D > 10 years
Partial In all these animal tests the biological endpoints evaluated are: Clinical biochemistry, gross necropsy, histological evaluation and target organ Renal epithelial cells grown under continuous medium supply Pneumocyte type II monolayer cultures: liquid/liquid interphase, Air/liquid interphase Surfactant release Various Partial replaceme nt (tiered strategy and/or test battery) Inhalation toxicants R&D > 10 years Various 96-well plate bioreactor (3D culture system in collagen gel) Various replaceme nt (tiered strategy and/or test battery) Partial replaceme nt (tiered strategy and/or test battery) Nephrotoxic ants R&D > 10 years Liver toxicants, pharmacoki netics R&D > 10 years
Partial Reaggregating brain cell cultures replaceme Various nt (tiered strategy and/or test battery) Neuronal and glial cell cultures (primary and cell lines) Various Partial replaceme nt (tiered strategy and/or test battery) CNS neurotoxica nts R&D > 10 years CNS neurotoxica nts R&D > 10 years
3.6.5. Conclusions In vitro subchronic and subacute toxicity testing is one of the challenging areas where a lot of efforts are is still needed in order to achieve animal replacement. All the methods listed in this chapter are at the level of research and development and the progress made will depend on adequate prioritisation, funding and coordination of efforts. These methods are all models to study individual types of target organ toxicity. It is clear that, to adequately replace repeat dose studies in animals, additional target organs from those mentioned (liver, kidney, CNS, lung and haematopoietic system) must also be considered. At the moment the strategy to follow to integrate such models into a testing programme is not defined e.g. the eventual replacement of in vivo repeat-dose toxicity tests will probably involve integration of in vitro data on target organ toxicity with in vitro/in silico data on ADME parameters.
Many of the models discussed for target organ toxicity are currently used to investigate toxicological issues (e.g. mechanisms of toxicity) and not for predictive purposes. Many laboratories from different sectors have experience of using such models for many different purposes. Likewise, ECVAM has already started activities in the area of repeated and chronic toxicity testing by creating a task force. In addition, a workshop is planned by autumn 2004. Furthermore, it will be important to harness the diverse expertise present both in industry and with regulators regarding information currently obtained from repeat dose toxicity studies and from in vitro alternatives. These activities will help to identify priorities and to start defining the strategy. However, to set into practice the recommendations more funding will be needed (e.g. FP6).
None of the in vitro models we currently have is ideal for any of the target organs and therefore some efforts should be put to optimise the models available and to look for good in vitro models in those cases were fewer models are available (e.g. in vitro models to study lung toxicity). More basic research is needed to
better understand the pathogenesis of chronic diseases in general, since it will help to overcome another important gap which is the identification of markers and/or endpoints of toxicity to be used in the corresponding in vitro models. In addition, the estimation of NOAEL in vitro needs further investigation.
Finally, more efforts and more funding are needed to further evaluate the usefulness of QSARs approaches to predict toxicity and to be used as part of a testing battery and/or a tiered strategy.
3.6.6. References
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Establishment of timetables for the phasing out of animal experiments for cosmetics
Genotoxicity/mutagenicity
Daniela Maurici2 , Marilyn Aardema 1, Raffaella Corvi2, Marcus Kleber3, Cyrille Krul4; Christian Laurent5, Nicola Loprieno 6, Markku Pasanen7, Stefan Pfuhler8, Barry Phillips9, Enrico Sabbioni2, Tore Sanner10, Philippe Vanparys11, Procter and Gamble, USA; 2ECVAM-JRC, Italy; 3Cognis Deutschland GmbH & Co. KG, Germany; 4TNO Nutrition and Food Research, The Netherland; 5SCCNFP and EFSA, Bruxelles, Belgium; 6University of Pisa, Italy; 7National Agency for Medicines, Helsinki and Department of Pharmacology and Toxicology, University of Oulu, Finland; 8Wella AG, Germany; 9Research Animals Department RSPCA, UK; 10Institute for Cancer Research, Norway; 11Johnson and Johnson, Belgium.
1
General considerations
Mutagenicity refers to the induction of permanent transmissible changes in the structure of the genetic material of cells or organisms. These changes (mutations) may involve a single gene or a block of genes. Genotoxicity is a broader term that refers to the ability to interact with DNA and/or the cellular apparatus that regulates the fidelity of the genome, such as the spindle apparatus and topoisomerase enzymes. Genotoxicity and mutagenicity testing are an important part of the hazard assessment of chemicals for regulatory purposes. To assess genotoxicity and/or mutagenicity, different endpoints must be taken into considerations: beside point mutations induction, a compound can induce changes in chromosomal number (polyploidy or aneuploidy) or in chromosome structure (breaks, deletions, rearrangements). However, aneuploidy can arise as a result of both genotoxic and non-genotoxic events, since loss of chromosomes can be caused either by direct effects on the chromosome to produce an acentric fragment, or by interference with the site of attachment of the chromosome on the spindle. Due to the diversity of the endpoints, it is then clear that the potential genotoxicity and/or mutagenicity of a compound cannot be assessed by a single assay system. For this reason, the group of experts has attempted to suggest a strategy to better investigate the mutagenic and/or genotoxic potential of the cosmetic products taking into consideration the needs of the cosmetic industry.
1. State of the art in the field of genotoxicity and mutagenicity tests in the view of the 7th Amendment
The cosmetic industry is committed to eliminate animal testing as soon as this is scientifically possible but is also committed to the highest safety standards for its products. It should not be forgotten that mutations and tumour induction are the most severe toxic effects since they are irreversible and very harmful to humans. The in vitro tests determine the potential of a compound to be mutagenic/genotoxic (= hazard identification). There is currently no single validated test that can provide information on all three end-points namely gene mutations, clastogenicity and aneuploidy. As a consequence, a battery of tests is needed to determine the genotoxic and mutagenic profile of a compound. Although several in vitro tests are routinely used and accepted by regulatory authorities, they present crucial limitations which affect the usefulness of the assays to predict mutagenicity/genotoxicity potential of a substance in vivo in mammals and especially in humans. These limitations in general are: - lack of a human like metabolic capacity of the cell lines used - absence of toxicokinetics - oversensitivity compared to in vivo situations low specificity - sometimes the use of cell lines that are not relevant to predict genotoxic endpoints at target organs - in repeated dosing, the target organ of mutagenicity/genotoxicity may be different than the area of application (hair, skin). Due to these limitations, no single in vitro test can fully replace an existing in vivo animal test yet. Therefore, a battery of tests is needed and/or it is necessary to optimise existing in vitro tests and /or develop new tests that focus on target cells. The experts felt the necessity to first establish a strategy that would ultimately lead to partial animal replacement. This led to the identification of the testing gaps, namely new tests that need to be developed to lead to full replacement of animal testing. The focus of this report is on dermally applied cosmetics since this is the largest category, though many of the same considerations would apply to cosmetic products applied via other routes (i.e. orally).
2. Proposed strategy
Strategy is divided in 4 stages: - Stage 1 characterizes the substance based on existing data and knowledge - Stage 2 is a basic in vitro test battery for hazard identification - Stage 3 is a follow up stage in in vitro model systems. This stage is reached if one or more tests are positive in Stage 2 - Stage 4 is in vivo. This stage is reached if one or more tests in Stage 3 are positive
Stage 1 It is important at this stage to collect information about the chemical characteristics of the compound and on its skin absorption using also analysis databases and applying computer based approaches. If it can be proven that there is no dermal absorption and that the compound does not reach the basal cell layer of the skin, mutagenicity/genotoxicity testing is not required. Stage 2 This stage consists of an in vitro test battery for hazard identification. Battery of tests: - Ames test (B13-14/TG471) - Gene mutation test in mammalian cells (preferably mouse lymphoma test) - Micronucleus test and/or chromosomal aberration (preference for micronucleus as it detects not only clastogens but also aneugens more directly than in the metaphase assay) If UV-exposure is expected and the compound can be photoactivated, screening for photogenotoxicity is needed. Photomutagenicity in bacteria or mammalian cells testing is warranted for those chemicals that absorb light in the wavelength of 290 - 700 nm and are used as leave-on products All tests of the basic package should be performed. Although definitive proof of nonmutagenicity/non-genotoxicity is not possible, a compound could be operationally classified as a non-mutagen for human cells if all the tests of the basic battery gave valid negative results. With negative results in the basic battery, further testing may not be requested. Positive results in one or more of the tests trigger further testing to elucidate the mechanism of action in stage 3. Stage 3 At this point, the strategy is to focus on hazard identification in target cells in vitro. Stage 3 is supposed to act as an interme diate step which should, if it can be successfully validated, be able to eliminate "false positive" results from Stage 2. The battery of tests suggested here need to be developed and/or validated Skin cells are the site of the first contact for most cosmetics and are therefore considered to have a high level of exposure. The proposed tests are: - Comet assay in primary skin cells or models - Micronucleus test in primary skin cells or models (if chromosomal aberrations or micronuclei are induced in Stage 2) For photo-mutagenicity/genotoxicity, similar tests can be considered: - Photo-Comet assay on primary skin cells or models - Photo-micronucleus test on primary skin cells or models If the test(s) performed in stage 3 is negative, further testing should not be necessary. For non-dermal cosmetics, new tests such as primary cells or models would need to be developed and validated for the assessment of mutagenicity/genotoxicity.
Stage 4 in vivo if necessary In vivo tests commonly used by cosmetics industries are: - classical in vivo micronucleus test (B12-TG 474) - unscheduled DNA synthesis (UDS) with mammalian liver cells (B39-TG 486) In vivo test that are occasionally used by cosmetics are: - in vivo Comet assay - bone marrow chromosome aberration test (B11-TG 475) - transgenic mutagenicity models (BigBlue, Mutamouse). These models may be appropriate for the determination of genotoxic or mutagenic effects (DNA strand breaks or gene mutations, respectively) in skin cells.
Moreover, some testing strategies have been suggested by SCCNFP: - SCCNFP Recommended strategy for testing hair dyes (SCCNFP/0720/03, 24-25 June 2003) - SCCNFP Notes of Guidance for the testing of cosmetics ingredients and their safety evaluation (SCCNFP/06903, 20 October 2003) - SCCNFP Mutagenicity/genotoxicity tests recommended for the safety testing of Cosmetics Ingredients to be included in the Annexes to Council Directive 76/768/EEC (SCCNFP/0755/03)
References: - Anderson D. and Plewa MJ. (1998). The international Comet assay workshop, Mutagenesis 13,67-73. - Baker RS. et al. (1992). Tumorigenicity of cyclopenta[a]phenanthrene derivatives and micronucleus induction in mouse skin. Carcinogenesis, Mar; 13(3): 329-32, - Criswell KA. et al, (2003). Validation of a flow cytometric acridine orange micronuclei methodology in rats. Mutat Res., 528, 1-18 -Haesen S. et al. (1993). Induction of micronuclei and karyotype aberrations during in vivo mouse skin carcinogenesis. Carcinogenesis, Nov; 14(11): 2319-27. - He SI. and Baker RS. (1989). Initiating carcinogen, triethylenemelamine, induces micronuclei in skin target cells. Environ Mol Mutagen.;14(1):1-5. - Torous DK. et al, (2003). Comparative scoring of micronucleated reticulocytes in rat peripheral blood by flow cytometry and microscopy. Toxicol. Sci. 74 (2): 309-314
3. Inventory of methods currently available

In vitro # Annex V EC B 13-14 B 10 B 17 B 19 B 15 B 16 B 18 In vivo # Annex V EC B 12 #OECD TG 471 473 476 479 480 481 482 #OECD TG 474 Name of the test Bacterial Reverse Mutation test (Ames test) Mammalian chromosome aberration test Mammalian cell gene mutation test (mouse lymphoma test) Sister chromatid exchange assay in mammalian cells (SCE) Saccharomyces cerevisiae gene mutation assay Saccharomyces cerevisiae mitotic recombination assay Unscheduled DNA synthesis (UDS) in mammalian cells Name of the test Mammalian erythrocyte micronucleus test Mammalian bone marrow chromosome aberration test Sex-linked recessive lethal test in Drosophila Melanogaster Rodent dominant lethal test Endpoint Gene mutations in bacteria Chromosome aberrations Gene mutations Mammalian DNA damage Gene mutations in yeast Recombination in yeast Mammalian DNA damage in liver cells Endpoint Structural and numerical chromosome aberrations in somatic cells Chromosome aberrations Gene mutations in germ line Chromosome aberrations and/or gene mutations in germinal tissue Inheritable chromosome aberrations Mutagenicity in foetal cells Heritable chromosome aberrations Mammalian DNA damage in liver cells
B 11 B 20 B 22
475 477 478
B 23 B 24 B 25 B 39
483 484 485 486
Mammalian spermatogonial chromosome aberration test Mouse spot test Mouse heritable translocation assay Unscheduled DNA synthesis (UDS) test with mammalian liver cells
4. Inventory of the alternative methods currently available

Mammalian chromosome aberration test B. 10/OECD TG # 473 Short description, scientific relevance and purpose The purpose of the in vitro chromosomal aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells. In addition, numerical chromosome changes such as polyploidy and duplication can be measured. Structural aberrations may be of two types, chromosome or chromatid. With the majority of chemical mutagens, induced aberrations are of the chromatid type, but chromosome-type aberrations also occur. The in vitro chromosome aberration test may employ cultures of established cell lines, cell strains or primary cell cultures. This test is used to screen for possible mammalian mutagens and carcinogens. Many compounds that are positive in this test are mammalian carcinogens; however, there is not a perfect correlation between this test and carcinogenicity. Developer of the method Evans HJ (1976) Known users Widely used by industry, CROs and academics Status of validation and/or standardization Worldwide accepted by regulatory authorities Field and limitations of applications See general limitations for in vitro tests Recommendations of use in the view of animal replacement As part of an in vitro test battery Ongoing development Ready to use
References - Evans, H.J. (1976). Cytological Methods for Detecting Chemical Mutagens. Chemical mutagens, Principles and Methods for their Detection, Vol. 4, Hollaender, A. (ed) Plenum Press, New York and London, pp. 1-29. -Galloway, S.M., et al, (1978). Chromosome aberration and sister chromatic exchanges in Chinese hamster ovary cells: Evaluation of 108 chemicals. Environs. Molec. Mutagen 10 (suppl.10), 1-175. - Huang, Y., et al, (1983). Aphidicolin - induced endoreduplication in Chinese hamster cells. Cancer Res., 43, 1362-1364. - Locke-Huhle, C. (1983). Endoreduplication in Chinese hamster cells during alpha-radiation induced G2 arrest. Mutation Res., 119, 403-413. - Ishidate, M.Jr. and Sofuni, T. (1985). The in Vitro Chromosomal Aberration Test Using Chinese Hamster Lung (CHL) Fibroblast Cells in Culture. Progress in Mutat. Res, Vol. 5, Ashby, J. et al., (Eds) Elsevier Science Publishers, Amsterdam-New York-Oxford, 427-432.
- Morita, T., et al, (1992). Clastogenicity of low pH to Various Cultured Mammalian Cells. Mutat. Res., 268, 297-305. - Richardson, et al, (1989). Analysis of Data from In Vitro Cytogenetic Assays. In: Statistical Evaluation of Mutagenicity Test Data. Kirkland, D.J., (ed) Cambridge University Press, Cambridge, pp. 141-154. - Scott, D., et al, (1991). Genotoxicity under Extreme Culture Conditions. A report from ICPEMC Task Group 9. Mutat. Res,. 257, 147-204.
Bacterial reverse mutation test B 13-14/OECD TG# 471 Short description, scientific relevance and purpose The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The principle of this bacterial reverse mutation test is that it detects mutations, which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesise an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the aminoacid required by the parent test strain. The bacterial reverse mutation test is rapid, inexpensive and relatively easy to perform. The bacterial reverse mutation test is commonly employed as an initial screening for mutagenic activity. Developer Ames B. (1971) Known users Widely used by industry, CROs and academics Status of validation and standardisation Worldwide accepted by regulatory authorities Field and limitations of application See general limitations for in vitro tests. Recommendations of use in the view of animal replacement As part of an in vitro test battery Ongoing development Ready to use
References - Ames, B.N., et al, (1975). Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Res., 31, 347-364. - Maron, D.M. and Ames, B.N. (1983). Revised Methods for the Salmonella Mutagenicity Test. Mutation Res., 113, 173-215. 7
Saccaromyces Cerevisiae gene mutation assay B. 15/OECD TG # 480 Short description, scientific relevance and purpose A variety of haploid and diploid strains of the yeast Saccharomyces cerevisiae can be used to measure the production of gene mutations induced by chemical agents with and without metabolic activation. Forward mutation systems in haploid strains, such as the measurement of mutation from red, adenine-requiring mutants (ade-1, ade-2) to double adenine-requiring white mutants and selective systems such as the induction of resistance to canavnaine and cycloheximide, have been utilized. The most extensively validated reverse mutation system involves the use of the haploid strain XV 185-14C which carries the ochre nonsense mutations ade 2-1, arg 4-17, lys 1-1 and trp 5-48, which are reversible by base substitution mutagens that induce site specific mutations or ochre suppressor mutations. XV 185-14C also carries the his 1-7 marker, a missense mutation reverted mainly by second site mutations, and the marker hom 3-10 which is reverted by frameshift mutagens. In diploid strains of S. cerevisiae the only extensively used strain is D7 which is homozygous for ilv 1-92. Developer of the method Zimmermann F. (1975) Known users Was used by industry, CRO and academics. Rarely used at present Status of validation and standardisation Accepted by regulatory authorities Field and limitations of application See general limitations for in vitro tests Recommendations of use in the view of animal replacement Not used in a standard battery Ongoing development Ready to use
References - Hannan MA, et al, (1978). Mutagenicity and recombinogenicity of daunomycin in Saccharomyces cerevisiae. Cancer Lett. Dec;5(6):319-24 - Mondon P, Shahin MM. (1992). Protective effect of two sunscreens against lethal and genotoxic effects of UVB in V79 Chinese hamster cells and Saccharomyces cerevisiae strains XV185-14C and D5. Mutat Res. May 16;279(2):121-8. - Sorenson WG, et al, (1981). Comparison of mutagenic and recombinogenic effects of some adenine analogues in Saccharomyces cerevisiae D7. Mutat Res. Jun;82(1):95-100.
Saccaromyces Cerevisiae mitotic recombination assay B. 16/OECD TG # 481 Short description, scientific relevance and purpose Mitotic recombination in Saccharomyces cerevisiae can be detected between genes (or more generally between a gene and its centromere) and within genes. The former event is called mitotic crossing-over and generates reciprocal products whereas the latter event is most frequently non-reciprocal and is called gene conversion. Crossing-over is generally assayed by the production of recessive homozygous colonies or sectors produced in a heterozygous strain, whereas gene conversion is assayed by the production of prototrophic revertants produced in an auxotrophic heteroallelic strain carrying two different defective alleles of the same gene. The most commonly used strains for the detection of mitotic gene conversion are D4 (heteroallelic at ade 2 and trp 5) D7 (heteroallelic at trp 5) BZ34 (heteroallelic at arg 4) and JDl (heteroallelic at his 4 and trp 5). Mitotic crossing-over producing red and pink homozygous sectors can be assayed in D5 or in D7 (which also measures mitotic gene conversion and reverse mutation at ilv 1-92) both strains being heteroallelic for complementing alleles of ade 2. Known users Widely used by industry and academics Status of the validation or standardisation Worldwide accepted by regulatory authorities Field and limitations of application See general limitations for in vitro tests Recommendations of use in the view of animal replacement Not used in a standard battery Ongoing development Ready to use
References - Zimmermann FK, Vig BK (1975). Mutagen specificity in the induction of mitotic crossing-over in Saccharomyces cerevisiae. Mol Gen Genet. Aug 27;139(3):255-68. - Zimmermann FK, et al, (1984). Testing of chemicals for genetic activity with Saccharomyces cerevisiae: a report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutat Res. May;133(3):199-244.
Mammalian cell gene mutation test B. 17/OECD TG # 476 Short description, scientific relevance and purpose The in vitro mammalian cell gene mutation test can be used to detect gene mutations induced by chemical substances. Suitable cell lines include L5178Y mouse lymphoma cells, the CHO, CHO-AS52 and V79 lines of Chinese hamster cells, and TK6 human lymphoblastoid cells. In these cell lines the most commonly used genetic endpoints measure mutation at thymidine kinase (TK) and hypoxanthine-guanine phosphoribosyl transferase (HPRT), and a transgene of xanthine-guanine phosphoribosyl transferase (XPRT). The TK, HPRT and XPRT mutation tests detect different spectra of genetic events. The autosomal location of TK and XPRT may allow the detection of genetic events (e.g. large deletions) not detected at the HPRT locus on X chromosomes. Developer of the method Chu E.H.Y. (for HPRT) and Clive D. (for TK) Known users Widely used by industry, CROs and academics Status of the validation and standardisation Accepted by regulatory authorities Field and limitations of application See general limitations for in vitro tests Recommendations of use in the view of animal replacement As a part of a standard battery Ongoing development Ready to use
References - Aaron, C.S., et al, (1994). Mammalian Cell Gene Mutation Assays Working Group Report. Report of the International Workshop on Standardisation of Genotoxicity Test Procedures. Mutat. Res., 312, 235-239. - Aaron, C.S. and Stankowski, Jr.L.F. (1989). Comparison of the AS52/XPRT and the CHO/HPRT Assays: Evaluation of Six Drug Candidates. Mutation Res., 223, 121-128. - Chu, E.H.Y. and Malling, H.V. (1968). Mammalian Cell Genetics. II. Chemical Induction of Specific Locus Mutations in Chinese Hamster Cells In Vitro, Proc. Natl. Acad. Sci., USA, 61, 1306-1312. - Liber, H.L. and Thilly, W.G. (1982). Mutation Assay at the Thymidine Kinase Locus in Diploid Human Lymphoblasts. Mutat. Res., 94, 467-485. - Moore, M.M., et al, (1987). Banbury Report 28: Mammalian Cell Mutagenesis, Cold Spring Harbor Laboratory, New York. - Moore, M.M., et al, (1989). Differential Mutant Quantitation at the Mouse Lymphoma TK and CHO HGPRT Loci. Mutagenesis, 4, 394-403.
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- Scott, D., et al, (1991). Genotoxicity Under Extreme Culture Conditions. A report from ICPEMC Task Group 9. Mutat. Res., 257, 147-204.
Unscheduled DNA synthesis (UDS) in mammalian cells B. 18/OECD TG # 482 Short description, scientific relevance and purpose The Unscheduled DNA Synthesis (UDS) test measures the DNA repair synthesis after excision and removal of a stretch of DNA containing the region of damage induced by chemical and physical agents. The test is based on the incorporation of tritium labelled thymidine (3H-TdR) into the DNA of mammalian cells which are not in the S phase of the cell cycle. The uptake of 3 H-TdR may be determined by autoradiography or by liquid scintillation counting (LSC) of DNA from the treated cells. Mammalian cells in culture, unless primary rat hepatocytes are used, are treated with the test agent with and without an exogenous metabolic activation system. Developer of the method Williams G. (1976) Known users Widely used by industry, CROs and academics in the past Status of the validation and standardisation Accepted by regulatory authorities Field and limitations of application Used to resolve mechanisms of action. See general limitations for in vitro tests Recommendations of use in the view of animal replacement As a part of a standard battery Ongoing development Ready to use
References - Casciano DA (2000). Development and utilization of primary hepatocyte culture systems to evaluate metabolism, DNA binding, and DNA repair of xenobiotics . Drug Metab Rev. Feb;32(1):1-13. - Williams, G.M (1976). Carcinogen-induced DNA repair in primary rat liver cell cultures: a possible screen for chemical carcinogens. Cancer Letters 1: 231-236 - Williams, G.M. (1977). Detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures. Cancer Res. 37: 1845-1851
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Sister chromatid exchange assay in mammalian cells (SCE) B. 19/OECD TG # 479 Short description, scientific relevance and purpose The Sister Chromatid Exchange (SCE) assay is a short-term test for the detection of reciprocal exchanges of DNA between two sister chromatids of a duplicating chromosome. SCEs represent the interchange of DNA replication products at apparently homologous loci. The exchange process presumably involves DNA breakage and reunion, although little is known about its molecular basis. Detection of SCEs requires some means of differentially labelling sister chromatids and this can be achieved by incorporation of bromodeoxyuridine (BrdU) into chromosomal DNA for two cell cycles. Mammalian cells in vitro are exposed to the test chemical with and without a mammalian exogenous metabolic activation system, if appropriate, and cultured for two rounds of replication in BrdU-containing medium. After treatment with a spindle inhibitor (e.g. colchicine) to accumulate cells in a metaphase-like stage of mitosis (c-metaphase), cells are harvested and chromosome preparations are made. Known users Used by industry, CRO and academics in the past Status of the validation and standardisation Accepted by regulatory authorities Field and limitations of application See general limitations for in vitro tests Recommendations of use in the view of animal replacement Rarely used Ongoing development Ready to use
References - Hagmar L, et al, (2001). The usefulness of cytogenetic biomarkers as intermediate endpoints in carcinogenesis. Int J Hyg Environ Health. Oct; 204(1):43-7. - Russo A. (2000). In vivo cytogenetics: mammalian germ cells. Mutat Res. Nov 20;455(12):167-89.
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In vitro mammalian micronucleus test Alternatives to in vivo micronuclei/ in vivo chromosome aberration test Short description, scientific relevance and purpose The purpose of the in vitro micronucleus assay is to identify agents that cause structural and numerical chromosome changes. The in vitro micronucleus test may employ cultures of established cell lines or primary cell cultures. Developer of the method Evans H. J. (1959) Known users Pharmaceutical, cosmetic industries, CROs and academics Status of validation and/or standardisation Inter-laboratory validation studies include: the Japanese collaborative studies, the European Pharmaceutical industry validation studies and the study coordinated by the French Society of Genetic Toxicology. Fields and limitations of application Micronuclei result from lesions/adducts at the level of DNA or chromosomes, or at the level of proteins directly or indirectly involved in chromosome segregation. Limitations: together with general limitations, apoptosis may interfere with the scoring of micronuclei giving rise to false positives Recommendations of use in the view of animal replacement As part of an in vitro battery Ongoing development A multicentre evaluation study, coordinated by the Institute Pasteur de Lille (France), is ongoing using a new transfected cell line which cannot go into apoptosis. The cell line is CTLL 2 stably transfected with the apoptosis inhibitor gene bcl2 Effort needed to complete validation of the method As many data are already available, the method could be validated by a weight of evidence approach. A draft of the in vitro micronucleus test guideline is expected to be submitted to the OECD in 2004.
References - Aardema, MJ et al (2001). The In Vitro Micronucleus Assay Genetic Toxicology and Cancer Risk Assessment, Ed: W. N. Choy, Marcel Dekker, Basel. - Evans HJ et al. (1959). The relative biological efficiency of single doses of fast neutrons and gamma rays in Vicia faba roots and the effect of oxygen. Part II. Chromosome damage: the production of micronuclei. Intl. J. Rad. Biol. 1, 230-240.
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- Garriott M.L. et al. (2002). A protocol for the in vitro micronucleus test. I. Contributions to the development of a protocol suitable for regulatory submissions from an examination of 16 chemicals with different mechanisms of action and different levels of activity. Mutat Res. 27; 517(1-2): 123-34. - Kirsch-Volders M. et al, (2003). Report from the in vitro micronucleus assay working group. Mutat. Res 540, 153-163. -Meintieres S. et al., (2001). Apoptosis can be a confusing factor in in vitro clastogenic assays. Mutagenesis., 16(3): 243-50. Erratum in: Mutagenesis, 16(5):453. -Meintieres S. et al, (2003). Using CTLL-2 and CTLL-2 bcl2 cells to avoid interference by apoptosis in the in vitro micronucleus test, Environ Mol Mutagen.;41(1):14-27. - Phelps JB et al. (2003). Relative percent cell survival and positive response in the in vitro micronucleus test. Mutat Res 537 115116. - Phelps J.B. et al. (2002). A protocol for the in vitro micronucleus test. II. Contributions to the validation of a protocol suitable for regulatory submissions from an examination of 10 chemicals with different mechanisms of action and different levels of activity. Mutat Res. 26; 521(12):103-12. -Wilhelm von der Hude et al (2000). In vitro micronucleus assay with Chinese hamster V79 cells results of a collaborative study with in situ exposure to 26 chemical substances. Mutat. Res, 468, 137-163.
In vitro Comet assay Alternatives to in vivo test for DNA Damage
Short description, scientific relevance and purpose The Comet assay is a method for measuring DNA strand breaks. DNA strand breaks may be introduced directly by genotoxic compounds or through the interaction with oxygen radicals or other reactive intermediates, or as a consequence of excision repair enzymes. It is highly sensitive and can detect DNA strand breaks in individual cells. The test can be conducted under neutral or alkaline conditions even if the test under alkaline conditions is more common and better standardized. The in vitro Comet assay may employ cultures of established cell lines, cell strains or primary cell cultures. The advantages of the Comet assay include: - DNA strand breaks in individual cells are measured - only small number of cells is necessary - no proliferating cells are required - the assay can be performed on any cell line or tissue Developer of the method Singh N.P. (1988) Known users Pharmaceutical and Cosmetic industries, CROs and academics Status of validation and/or standardisation No ongoing validation studies
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Fields and limitations of application Used for screening purposes and to understand mechanisms of action. Used as a replacement for the in vivo UDS test and to look at genotoxicity in target cells. Limitations: no validation, no official guideline. However, for the in vivo comet assay, recommendations on acceptance criteria and on how to standardise protocols have been recently published (Tice RR et al, 2000). These recommendations and the standardised protocol may be useful also for the in vitro Comet assay. Recommendations of use in the view of animal replacement The Comet assay could replace the in vivo UDS test. This may lead to reduction of animal use. If genotoxicity can be confirmed or ruled out in target tissues, this may lead to a replacement of further animal experiments. Ongoing development No coordinated development is ongoing. Efforts needed to complete validation of the method Efforts are needed to coordinate a formal validation or possibly a weight of evidence validation.
References - Hartmann, A. et al. (2003). Recommendations for conducting the in vivo alkaline Comet assay. Mutagenesis Vol. 18, No. 1, 45-51. -Ostiling O. and Johanson K.J. (1987). Microelectrophoretic study of radiation-induced DNA damage in individual cells, Biochem. Biophy. Res Commun:123, 291-298. - Singh NP et al, (1988): A Simple technique for quantification of low levels of damage in individual cells. Exp Cell Res 175,184-191. - Tice, R.R. et al. (2000). The single cell gel / comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environm. Mol. Mutagen. 35, 206-221.
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In vitro micronucleus test in primary skin cells or models Alternatives to in vivo micronuclei/ in vivo chromosome aberration test Short description, scientific relevance and purpose Primary human keratinocytes Human keratinocytes obtained from foreskin have been shown to contain several isoenzymes of cytochrome P-450. They also continue to express biotransfomation activity in vitro. Primary human keratinocytes have been shown to be sensitive to micronucleus induction by some clastogens and low doses of UVB and UVA. On the other hand, a publication showed that colchicine did not induce micronuclei in human keratinocytes while micronucleus induction was found in other human cells. 3D skin models (three dimensional skin model) There are several 3D skin models which are commercially available. The HCE model (SkinEthic, Nice, France) consists of immortalized human corneal epithelial cells (HCE cell line) that are cultivated at the air-liquid interface in a chemically defined medium on a polycarbonate substrate and form an air-epithelial tissue, devoid of stratum corneum, resembling morphologically the corneal epithelium of the human eye. A possible endpoint measurement may be micronucleus induction. The SkinEthic HCE model has recently been taken up by a few industries to be evaluated for its potential as test model to detect clastogens/aneugens. Some companies considered the potential of the HCE model for photomutagenicity testing. The EpiDerm model (MatTec Corporation, Ashland, MA, USA) consists of normal, humanderived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. This model is already in widespread use for testing of skin irritancy and dermal toxicity. Known users Cosmetic industries and CROs Status of validation and/or standardisation The tests with primary keratinocytes as well as with 3D skin cultures are in a phase of research and development. The development of the in vitro micronucleus test on target cells is not yet coordinated. Fields and limitations of application Both tests must, at this point, be seen as an addition to a standard battery of tests and to be used for mechanistic purposes. Ongoing development No coordinated development is ongoing. Efforts needed to complete the validation of the method Efforts needed to complete the validation of the method are: (1) a protocol needs to be established that leads to reproducible results (2) metabolic capabilities of the primary keratinocytes as well as the keratinocytes growing in 3D cultures should be determined
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(3) the barrier function of the 3D models needs to be assessed to enable a comparison with the in vivo situation (4) the in vitro micronucleus test on human keratinocytes or 3D skin models should be compared with data on in vivo micronucleus test on rodent keratinocytes to assess the predictivity of the test
References - Emri, G. et al. (2000). Low doses of UVB or UVA induce chromosomal aberrations in cultured human skin cells. J Invest Dermat, 115 (3): 435-440. - Heimann R. and R.C. Rice (1983). Polycyclic aromatic hydrocarbon toxicity and induction of metabolism in cultivated esophageal and epidermal keratinocytes. Cancer Res 43, 4856-4862. - Kukkelhoven, M.W.A.C. (1985). Covalent binding of benzo[a]pyrene metabolites to DNA of cultured human hair follicle keratinocytes, Arch. Toxicol., 57, 6-12. - Kuroki, T. et al.(1980). Metabolism of benzo[a]pyrene in human epidermal keratinocytes in culture, Carcinogenesis, 1, 559-565. - Kuroki, T. et al, (1987). Inter-individual variation of arylhydrocarbon-hydroxylase activity in cultured epidermal and dermal cells, Jpn. J. Cancer Res., 78, 45-53. - Lofti, C.F.P. and G.M. Machado-Santelli (1996). Comparative analysis of colchicines induced micronuclei in different cell types in vitro. Mutat Res 349, 77-832. - Nishikawa, T.et al, (1999). Study of a rat skin in vivo micronucleus test: data generated by mitomycin C and methyl methanesulfonate. Mutat Res, 444, 159-166. - Nishikawa, T. et al. (2002). Further evaluation of an in vivo micronucleus test on rat and mouse skin: results with five skin carcinogens. Mutat Res, 513, 93-102. - Van Pelt F.N.A.M. et al, (1990). Immunohistochemical detection of cytochrome P450 isoenzymes in cultured human epidermal cells. Jl Histochem and Cytochem 38, 1847-1851. - Van Pelt, F.N.A.M. et al, (1991) Micronucleus formation in cultured human keratinocytes following exposure to mitomycin C and cyclophosphamide. Mutat Res 252, 45-50. - Van Pelt, F.N.A.M., et al, (1991) Micronucleus formation in cultured human keratinocytes: Involvement of intercellular bioactivation. Toxic. in Vitro, Vol. 5, No. 5/6, pp. 515-518.
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In vitro Comet assay with primary skin cells or models Alternatives to in vivo test for DNA Damage Short description, scientific relevance and purpose Primary keratinocytes and cell lines In the Comet assay, any cell type can theoretically be used for genotoxicity testing. However, only a few publications were found using human keratinocytes as target cell. The Comet assay has been shown to be able to detect genotoxic damage by H2O2 in a human keratinocyte cell line H103 and by carbonyl stress and Photofrin in the human HaCaT keratinocyte cell line. Some companies are exploring the utility of the Comet assay on human keratinocytes. The assay is used more for mechanistic purposes, especially in the field of photogenotoxicity. 3D skin models (three dimensional skin model) There are several models commercially available. The SkinEthic HCE model (SkinEthic, Nice, France) consists of immortalized human corneal epithelial cells (HCE cell line) that are cultivated at the air-liquid interface in a chemically defined medium on a polycarbonate substrate and form an air-epithelial tissue, devoid of stratum corneum, resembling morphologically the corneal mucosa of the human eye. The EpiDerm model (MatTec Corporation, Ashland, MA, USA) consists of normal, humanderived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. This model has already widespread use in the testing of skin irritancy and dermal toxicity. Known users Cosmetic industries and CROs Status of validation and/or standardisation Not yet standardise. The in vitro Comet tests with primary keratinocytes as well as with the 3D skin cultures are in a phase of research and development. Fields and limitations of application Reconstituted skin models are recently taken into consideration to assess photogenotoxicity (Meunier JR et al, 2001). The Comet assay has already been successfully adapted for use with 3D buccal mucosa equivalents (Wolfreys A et al, 1999). Recommendations of use in the view of animal replacement The Comet assay on both models must, at this point, be seen as an addition to a standard battery of tests and for mechanistic purposes. Ongoing development The development of the in vitro Comet tests on target cells is not yet coordinated. Efforts needed to complete the validation of the method Efforts needed to complete the validation of the method are: (1) a protocol needs to be established that leads to reproducible results (2) metabolic capabilities of the primary keratinocytes as well as the keratinocytes growing into 3D cultures should be determined 18
(3) the barrier function of the 3D models needs to be assessed to enable a comparison with the in vivo situation (4) the in vitro micronucleus test on human keratinocytes or 3D skin models should be compared with data on in vivo micronucleus test on rodent keratinocytes to assess predictivity of the test
References - Meunier, J-R et al. (2001). Comet assay on Episkin an in vitro reconstructed skin model: A new tool for the evaluation of (photo)genotoxic potential. Abastract Mutation Res. 483 (Suppl. 1) S168. - Roberts, M.J et al. (2003). DNA damage by carbonyl stress in human skin cells. Mutation Research 522, 45-46. - Thein, N. et al. (2000). A strong genotoxic effect in mouse skin of a single painting of coal tar in hairless mice and in Muta(TM)Mouse. Mutation Research, 468, 117-124. - Wolfreys, A. et al. (1999). Use of a 3D buccal mucosa tissue equivalent to assess DNA damage in the presence and absence of human saliva. Poster EEMS. - Woods, J.A. et al. (2004). The effect of Photofrin on DNA strand breaks and base oxication in HaCaT keratinocytes: A Comet assay study. Photochemistry and Photobiology, 79(1). - Yendle, J. E.et al. (1997). The genetic toxicity of time: importance of DNA-unwinding time to the outcome of single-cell gel electrophoresis assays. Mutation Research, 375, 125-136.
19
In vitro toxicogenomics Short description, scientific relevance and purpose Toxicogenomics is the application of genomics methods to address questions in the field of toxicology. Changes in gene/protein expression as a result of exposure to a toxic chemical or physical agent can be measured in virtually any tissue (in vitro or in vivo). The rapidly developing field of toxicogenomics is expected to have a large impact on both the fields of genetic toxicology and carcinogenicity as a result of increased understanding of these processes. Increased understanding of the biological pathways involved in genotoxicity and carcinogenicity will promote the development of better tools for assessing these endpoints. Initial studies suggest that patterns of induced gene expression changes may be characteristic of specific classes of toxic compounds and identification of these distinctive fingerprints can help classify agents with different mechanisms of action. This has the potential to reduce the amount of testing normally required to define a mechanism or mode of action. Known users Pharmaceutical industries and academics Status of the validation and standardisation Genomics methods are at the stage of research and development. Because the field of toxicogenomics is relatively new, most experimental results are not well enough established to be suitable for regulatory decision-making at this time. Laboratory techniques and test procedures may not be well validated. In addition, test systems may vary so that results may not be consistent or generalized across different platforms. Field and limitation of application They are useful mechanistic tools but the general consensus is they are not suitable at this time for regulatory decision-making. The findings from a specific study often cannot be extrapolated across species or to different study populations (e.g., various human subpopulations with different genetic backgrounds). Ongoing development A move to standardise assays is underway, and much more information should be available within the next several years. References - Aardema M and MacGregor JT (2002). Toxicology and genetic toxicology in the new era of toxicogenomics: impact of omics technologies. Mutat Res, 13-25. - Corton JC and Stauber AJ. (2000). Toward construction of a transcript profile database predictive of chemical toxicity. Toxicol Sci 58, 217-219. - Farr S. and Dunn RT. (1999). Concise review: gene expression applied to toxicology. Toxicol. Sci, 50, 1-9. - Holmes EW et al. (2001). Metabonomic characterisation of genetic variation in toxicological and metabolic responses using probabilistic neural networks. Chem Res Toxicol 14, 182-191. - Nuwayysir et al. (1999). Microarray and toxicology: the advent of toxicogenomics. Mol Carcinogenesis, 153-159. - Pennie WA (2000). Use of cDNA microarray to probe and understand the toxicological consequences of altered gene expression. Toxicol Lett 112/113, 473-477. - Rockett JC and Dix DJ. (1999) Application of DNA arrays to toxicology. Environ Health perspect. 107, 681-685.
20
5. Recommendation for achieving reduction in animal use

General suggestions: - when possible, animals from subchronic and/or chronic toxicity studies should be shared to measure genotoxic effects. By applying flow cytometry or image analysis, blood from rodents could be evaluated for the presence of micronuclei (Criswell KA et al, 2003; Torous DK et al, 2003) - each animal can serve as its own control and the kinetics can be followed in the same animal (sample can be taken over several times). - multiple dosing can be performed on the same animal (micronuclei, comet assay) - no general need for both sexes - instead of two routine in vivo assays, select only one assay (considering genotoxic endpoints)
Specific suggestions: - in classical in vivo micronucleus test (B12- TG 474), a substantial decrease in the number of animals can be obtained by implementing flow cytometry analysis
In vivo tests to be removed from the list because they are not relevant for the purposes of the cosmetic industry: - rodent dominant lethal test , B22-TG 478 - mouse heritable translocation assay, B25-TG 485 - specific locus test - mouse spot test, B24-TG 484 - mammalian spermatagonial chromosome aberration test, B23-TG 483 - sex-linked recessive lethal test in Drosophila Melanogaster, B20-TG 477
21
6. Final Comments
Genotoxicity and mutagenicity testing are an important part of the hazard assessment of chemicals for regulatory purposes. General crucial limitations of in vitro tests are due to the absence of toxicokinetic characteristics and/or to the use of cell lines not relevant to predict genotoxicity at target organs. The current situation is that no single in vitro test can fully replace an existing in vivo animal test. The recommendations provided here are based on a step-wise approach. Stage1 characterises the substance based on existing data and knowledge including data on skin absorption. If systemic exposure of the compound in question cannot be ruled out, a battery of three in vitro tests for hazard identification has to be performed that should cover the endpoints gene mutation, clastogenicity and aneuploidy (stage 2). Stage 3 is a follow up stage in in vitro model systems on target cells (e.g. three dimensional skin models) that has to be performed in case of positive findings in stage 2. Stage 3 is supposed to act as an intermediate step which should, if it can be successfully validated, be able to eliminate "false positive" results from stage 2. If the test(s) performed in stage 3 is negative, further testing should not be necessary. Such tests are not available at the moment and much effort has to be undertaken to develop and validate those tests. Stage 2 of the strategy uses tests that are already adopted by regulatory authorities, whereas the time estimated for implementation and validation of the stage 3 tests is 8-10 years. However, if the outcome of stage 3 still shows a mutagenic or genotoxic potency of the compound tested or if the methods necessary to perform such an intermediate step cannot be successfully validated, confirmatory experiments in vivo will still be required (Stage 4). To overcome the limitations of in vitro testing and reach full animal replacement, model systems in the area of toxicokinetics and metabolism are required that can accurately predict or mirror the in vivo situation. Moreover, the emerging area of toxicogenomics could lead to a better understanding of the process of genotoxicity/mutagenicity which may help to develop the "right" in vitro models. Taking into account the state of the art in those areas, it seems highly unlikely that full replacement in the field of genotoxicity/mutagenicity can be accomplished within the next 12 years. This time estimation is based on the following rationale: (i) model systems in the area of toxicokinetics and metabolism that can accurately predict or mirror the in vivo situation are required and need to be developed, (ii) new in vitro tests on target cells for cosmetics need to be developed and validated against an extended database of reliable in vivo data on target cells which does not exist yet, (iii) various laboratories need to be mobilized to put research in these fields, and (iiii) laboratories and /or organizations need to be found (and founded) to coordinate these research programs. Regarding the reduction of animal use, the experts feel that the flexibility given in the currently used in vivo guideline approaches is not sufficiently utilised at the moment. Improvements in this field could instantly lead to a substantial reduction in the number of animals used within the cosmetic industry. In conclusion, the experts are of the opinion that a total replacement of animal testing in the field of mutagenicity/genotoxicity testing is not feasible within the next 12 years. A total replacement of the in vivo genotoxicity tests will depend, besides the development of in vitro tests on skin models, also on the progress in the fields of toxicokinetics and toxicogenomics.
22
In vitro Endpoints
Purpose
Validation status
Comments
Gene point mutations (B20/TG477; B22/TG478; B24/TG484)
Partial replacement Ames test (tiered Gene point (B13/14strategy mutations TG471) and/or test battery) Partial replacement S. Cerevisiae (tiered Gene point gene mutation strategy mutations (B15-TG 480) and/or test battery) Partial replacement Mammalian cell Gene point (tiered gene mutation mutations strategy test and/or test (B17-TG 476) battery)
Mutagenicity/ genotoxicity
Adopted
EC (Annex V), OECD
--------------
------------------
Adopted
EC (Annex V), OECD
--------------
------------------
Adopted
EC (Annex V), OECD
--------------
------------------
23
Mitotic recombination in S. Cerevisiae (B16-TG 481)
DNA damage
Unscheduled DNA synthesis DNA damage (USD) (B18-TG 482)
DNA damage (B39/TG486)
Sister chromatid exchange (SCE) (B19-TG 479)
DNA damage
Alkaline Comet DNA damage assay
Partial replacement (tiered strategy and/or test battery) Partial replacement (tiered strategy and/or test battery) Partial replacement (tiered strategy and/or test battery) Partial replacement (tiered strategy and/or test battery) Full replacement
Adopted
EC (Annex V), OECD
--------------
------------------
Adopted
EC (Annex V), OECD
---------------
--------------------
Adopted
EC (Annex V), OECD
--------------
------------------
Genotoxicity
Optimised
----------------
Alkaline Comet in skin cells/model
DNA damage
Genotoxicity
R&D
----------------
Coordination for formal or weight of evidence validation Development needs to be coordinated
Formal validat: 4-6 years Weight of evidence validat: 2-3 years 8-10 years
24
Chromosomal aberrations (B11/TG475; B22/TG478; B25/TG485; B23/TG483)
Mammalian chromosomal aberration assay (B10-TG 473)
Chromosomal aberrations
Partial replacement (tiered strategy and/or test battery) Partial replacement (tiered strategy and/or test battery) Full replacement Partial replacement (tiered strategy and/or test battery)
Adopted
EC (Annex V), OECD
--------------
------------------
Detects aneugenes and clastogenes (B12/TG474)
Aneugenes Micronucleus in and cell lines clastogenes Micronucleus in Aneugenes target cells and and clastogenes skin models
Genotoxicity/ mutagenicity
Optimised
OECD test guideline submitted
--------------
Formal valid: 3-4 years Weight of evidence validat: 1-2 years 6-10 years
Genotoxicity/ mutagenicity
R&D
---------------
Development needs to be coordinated Further development and standard needed
Mechanism based
Toxicogenomics
Various
Genotoxicity
R&D
---------------
10 +
25
1/1
Joint DG Enterprise/ECVAM Project

Establishment of timetables for the phasing out of animal experiments for cosmetics:
Subgroup 8
UV-induced effects
by Manfred Liebsch, Horst Spielmann, Wolfgang Pape, Cyrille Krul, Alain Deguercy and Chantra Eskes
2/2
Executive Summary
Regulative Requirements According to the current Notes for Guidance of the SCCNFP, cosmetic ingredients and mixtures of ingredients absorbing UV light (in particular UV filter chemicals used, e.g. to ensure light stability of cosmetics, or used in sun protection products) need to be tested for acute phototoxicity and photo-genotoxicity potential. Testing for photosensitisation potential (immunological photoallergy) is not specifically required, but it is nevertheless often performed. Acute Phototoxicity Due to a thorough multi-stage and multi-centre validation trial (1992-1998) the In Vitro 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU PT) has reached acceptance by the SCCNFP already in 1998. The 3T3NRU PT is recommended by the EMEA/CPMP as basic preclinical test for acute phototoxicity. It was accepted as Annex V Method No. 41 to Dir. 67/548/EEC in the year 2000, and was accepted as new Test Guideline 432 by the OECD in 2002. The 3T3NRU PT is regarded a basic screen to identify acute phototoxic potential. Two additional tests, formally evaluated in controlled blind trials, the RBC Phototoxicity Test (RBC PT) and the Human 3-D Skin Model In Vitro Phototoxicity Test (H3D PT), are regarded useful and important adjunct tests to overcome some limitations of the 3T3-NRU-PT, namely the fairly low UVB tolerance of the 3T3 fibroblasts and the inability to model the bioavailability of test materials topically applied to the skin. In addition, the Photo-RBC allows evaluation of the phototoxic mechanisms involved. In conclusion, identification of acute phototoxic hazards is regarded sufficiently covered by in vitro tests, so that animal testing for that endpoint can be 100% replaced right now.
Photo-genotoxicity In the area of photo-genotoxicity, almost the whole battery of in vitro genetic toxicity tests has been (or is currently being) converted into test protocols of photo-genotoxicity tests. Tests exclusively predictive for gene-mutation, e.g. Photo-Ames-Test and Photo-Thymidine Kinase Test (P TKT) have become less important than tests for clastogenic effects (e.g. PhotoChromosome Aberration Test (P CAT) and Photo-Micronucleus Test (P-MNT). In addition, promising indicator tests like the Photo-Comet-Assay (P COMET) have been developed. Albeit their routine usage, to date none of the new photo-genotoxicity tests has been formally validated. Therefore, the P-MNT and the P-COMET are currently evaluated in a formal interlaboratory validation study. It is expected that these in vitro photo genotoxicity tests methods may be available as accepted methods within the next five years. Photo-Allergy (-Sensitisation) In the area of photo-allergy (-sensitisation), like in the area of development of predictive in vitro tests for delayed contact sensitisation (Allergenicity) potential without involvement of light, due to a lacking ability to model the complex mechanisms underlying allergy, currently no promising in vitro methods to predict photo-sensitisation potential are in sight (see chapter on skin sensitisation). One in vitro screening method, which models covalent binding of a light activated chemical to human serum albumin, may become relevant. However, while binding of an excited chemical to proteins is a prerequisite of photoallergy, it is not a sufficient predictor. The only promising Alternatives currently under development are in vivo refinements, like the Photo Local Lymph Node Assay (LLNA). Once a reliable and predictive in vitro test battery and strategy for the assessment of dark sensitisation potential will be developed and accepted, adaptation of the test battery into similar photo-sensitisation tests is considered possible.
3/3
8.1
ACUTE PHOTOTOXICITY
Chemical phototoxicity is an acute reaction which can be induced by a single treatment with a chemical and ultraviolet or visible radiation. In vivo, the reaction can be evoked in all subjects provided that concentration of chemical and dose of light are appropriate. "Acute" includes both immediate and delayed (e.g. 48 hour) reactions. Since the year 2000, in Europe in vivo testing in animals for acute phototoxic potential is no longer permitted, since a successfully validated alternative method has been accepted for regulatory purposes. Taking into account the scientific evidence from the validation studies as well as in-house data in laboratories of the industry, consensus has been reached internationally that testing for acute phototoxic potential can be covered by in vitro methods. Due to its high sensitivity and specificity, the validated 3T3 Neutral Red Uptake phototoxicity test (3T3NRU-PT) is the core test, which is usually the only phototoxicity test required. Two additional in vitro tests that have provided promising results in blind trials under the auspices of COLIPA and/or ECVAM, the Red-Blood-Cell Phototoxicity Test (Photo RBC) and the Human 3-D Skin Model Phototoxicity Test (H3D PT), they may be used when sufficient information on the phototoxic potential of a chemical cannot be obtained from results obtained in the 3T3NRU-PT. An important advantage of the Photo RBC is that can be used at high doses of UVB in the irradiation spectrum and provides information on the mechanism of phototoxicity. In the H3D PT metabolic competent primary human skin cells are used in an organotypic structure including a stratum corneum barrier and thus allows to model the bioavailability of the test chemical. Although not formally validated for that purpose yet, the H-3D PT is currently used in the cosmetic industry to evaluate the relevance of positive results obtained in the 3T3 NRU PT under aspects of bio-availability in human skin (Spielmann et al. 2000).
8.1.1 The In Vitro 3T3 NRU Phototoxicity Test (3T3 NRU-PT)

8.1.1.1 Short description, scientific relevance and purpose
In the in vitro 3T3 NRU PT the cytotoxicity of a chemical when tested in the dark IC50 (-UV) is compared to the cytotoxicity measured by the activated chemical exposed to a noncytotoxic dose of UV or visible light IC-0 (+UV) at wavelengths > 290 nm. In this test photocytotoxicity is expressed as the concentration related reduction of uptake of the vital dye Neutral Red (Neutral Red Uptake = NRU) 24 hours after exposure of mouse fibroblasts of the cell line Balb/c 3T3 to the test chemical in the presence of UV irradiation in comparison to 3T3 cells that are exposed to the same chemical but not to UV irradiation. The Neutral Red Uptake (NRU) cytotoxicity assay with Balb/c 3T3 fibroblasts (Borenfreund and Puerner, 1985) was adapted for phototoxicity testing. In brief, 3T3 cells are incubated with various dilutions of the test-chemicals in 96 well plates for 1 hour. Thereafter they are exposed to UV/vis light (with an effective UVA radiation of 1.67 mW/cm for 50 minutes, and NRU is determined 24 hours later by measuring the optical density at 540 nm (Spielmann et al. 1994). Concurrently a second set of plates with the same chemicals is kept in the dark and evaluated in parallel. Where possible, the concentration of a test chemical reflecting a 50% inhibition of cell viability (IC 50) is calculated using appropriate non-linear curve fitting models of the concentration-response curve. To discriminate between photo irritant and nonphoto irritant chemicals, the photo irritation factor (PIF) was defined as the ratio of the IC50 values, determined in the absence of UVA and the presence of UVA (PIF = IC50 (-UV) : IC50 (+UV)) (Spielmann et al. 1994, 1996). In a more sophisticated data analysis procedure, the mean photo effect (MPE) is determined, which uses a complete comparison of the area under the curve AUC of the concentration response curves obtained with a chemical in the presence and absence of UV light (Holzhtter, 1997, Peters and Holzhtter 2003).
4/4 Based on the results of the formal validation study of the 3T3 NRU PT (Spielmann et al. 1998), a prediction model (PM) has been accepted at the international level as described in OECD TG 432 "In Vitro 3T3 NRU phototoxicity test" (OECD 2003): A test substance with a PIF < 2 or an MPE < 0.1 predicts: no phototoxicity. A PIF >2 and < 5 or an MPE > 0.1 and < 0.15 predicts: probable phototoxicity and a PIF > 5 or an MPE > 0.15 predicts: phototoxicity. For any laboratory initially establishing this assay, the reference materials listed should be tested prior to testing of new substances for phototoxic assessment. A software package for the calculation of the PIF and MPE is available from COLIPA and from the OECD Secretariat (OECD 2003).
8.1.1.2
Developers of the method
The 3T3 NRU PT was developed by members of the COLIPA Task Force on phototoxicity during a prevalidation study (Spielmann et al., 1994). Test development of an in vitro photocytotoxicity test was initiated at the Battelle laboratory in Germany with human skin cells (Maier et al. 1991). At Beiersdorf in Germany, the Neutral Red uptake (NRU) cytotoxicity test using mouse Balb/c 3T3 fibroblasts (Borenfreund and Puerner, 1985) was adapted for phototoxicity and the prediction model (PM) applying the PIF was developed by statisticians at ZEBET (Berlin, Germany) in 1994 from the data obtained with 20 test chemicals during the prevalidation stage of the ECVAM/COLIPA validation study (Spielmann et al.1994) as described in the previous section. The PM applying the MPE was developed by Holzhtter (Humboldt University, Berlin, Germany) from the data of the formal validation study (Holzhtter 1997) and improved software for data handling and analysis was recently developed by Peters & Holzhtter (2003) and funded by ZEBET at the BfR.
8.1.1.3
Known user
The 3T3 NRU PT is at the world-wide level the established standard test for phototoxic assessment of chemicals in laboratories of the cosmetic, chemical and drug industry in Europe, the USA and Japan.
8.1.1.4
Status of validation and/or standardisation
The 3T3 NRU PT has successfully been experimentally validated from 1992-1998 (Spielmann et al., 1994, 1998a, 1998b) and been accepted for regulatory purposes by the EU in the year 2000 (EC 2000), by the OECD in 2002 (OECD 2002), by the EMEA in 2002 (EMEA 2002) and by the US FDA in 2003 (US FDA 2003).
8.1.1.5
Fields and limitations of application
The 3T3 NRU PT is currently accepted for regulatory purposes for chemicals at the worldwide level (EC 2000; OECD 2002) and for cosmetic ingredients in Europe as outlined by the in the "SCCNFP's Notes of guidance for testing of cosmetic ingredients and their safety evaluation" (SCCNFP 2003). The 3T3 NRU has also been accepted for regulatory purposes by the competent drug authorities in Europe (EMEA 2002), in the USA (US FDA 2003) and in Japan the drug administration of the NIH held a Symposium in December of 2002 on the "Evaluation of in vitro phototoxicity tests" in order to introduce the 3T3 NRU PT for regulatory purposes. The regulatory use of the 3T3 NRU PT in the EU was outlined by ZEBET (BfR, Berlin, Germany) on behalf of ECVAM and the evaluation of the reliability of EU/COLIPA validation studies of this in vitro test was critically assessed during the symposium. The result was favourable for the acceptance of the 3T3 NRU PT as a well validated test that can be used for regulatory purposes in Japan.
5/5 An additional evaluation study has shown that due to their higher potential to absorb UV radiation human keratinocytes are less sensitive in photo-cytotoxicity assays than the mouse fibroblast cell line used in the 3T3 NRU PT assay (Clothier et al., 1999). The 3T3 NRU PT does not provide specific information on the mechanism of phototoxicity and it does not allow to assess the bio-availability of a test compound in the human skin. If this type of information is required, the Photo RBC, applying red blood cells (section 8.1.2), and the H-3-D PT, applying human skin models (section 8.1.3), may be used. The Unilever laboratory in the U.K. has recently reported a step-wise strategy for the use of the 3T3 NRU PT and of human skin models to interpret the results obtained with ingredients of personal care products in in vitro phototoxicity assays (Jones at al., 2003).
8.1.1.6
Recommendations of use in the view of animal replacement
Taking into account EU Directive 86/609/EEC on "The protection of animals used for experimental and other scientific purposes" (EC 1986), safety testing of chemicals and of cosmetic ingredients for phototoxic potential is no longer permitted in EU member countries, since the in vitro 3T3 NRU PT has been accepted as the standard test methods for this endpoint and since two additional in vitro phototoxicity tests complementing the 3T3 NRU PT, the RBC PT and the H3D PT, have undergone pre-validation and are established in laboratories of the cosmetics and chemical industry in the EU as outlined in sections 8.1.2 and 8.1.3.
8.1.1.7
On-going development
Since the validation of the 3T3 NRU PT was successful and regulatory acceptance has been achieved as outlined above, there are currently no on-going validation activities.
8.1.1.8
Efforts needed to complete validation of the method
Since the validation of the 3T3 NRU PT was successful and regulatory acceptance has been achieved as outlined above, there is currently no need to complete any validation activities.
8.1.1.9 Key references a) international test guidelines and regulations
EMEA (European Agency for the Evaluation of Medicinal Products), CPMP (Committee for Proprietary Medicinal Products) (2002) Note for guidance on photosafety testing. CPMP/SWP/398/01, EMEA, London, (http://www.emea.eu.int) 7 pg. European Commission (2000) Test guideline B-41 "phototoxicity - in vitro 3T3 NRU phototoxicity test" of Annex V of the EU Directive86/906/EEC for classification and labelling of hazardous chemicals. O.J. of the European Communities, June 8 2000, L136, 98-107. European Commission (2003) Directive 2003/15/EC amending Council Directive 76/68/EEC on the approximation of the laws of the Member States relating to cosmetic products. O.J. of the European Communities, March 11 2003, L 66, 26-35. OECD (Organisation for Economic Co-operation and Development) Test Guidelines Programme (2002) Draft proposal for a new guideline 432 "In Vitro 3T3 NRU phototoxicity test". OECD Publication Office, Paris, 15 pg. SCCNFP of the European Commission (2003) The SCCNFP's Notes of guidance for testing of cosmetic ingredients and their safety evaluation, 5th Revision, Brussels, 102 pg.
6/6 US FDA (2003) Guidance for industry - Photosafety Testing. US Dept. of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Rockville (USA), (http://www.fda.gov/cder/guidance/index.htm) 22 pg.
b) specific references for the 3T3 NRU PT Balls M; Corcelle G; ECVAM news and views: Statement on the scientific validity of the 3T3 NRU PT test (an in vitro test for phototoxic potential).; Alternatives to laboratory animals, ATLA; 26(1); 7-8; 1998 Borenfreund E; Puerner JA; Toxicity determined in vitro by morphological alterations and neutral red absorption.; Toxicology letters; 24; 119-124; 1985 Clothier R; Willshaw A; Cox M; Garle M; Bowler H; Combes R; The use of human keratinocytes in the EU/COLIPA international in vitro phototoxicity test validation study and the ECVAM/COLIPA study on UV filter chemicals; Alternatives to laboratory animals, ATLA; 27(2); 247-259; 1999 Commission Directive 2000/33/EC of 25 April 2000 adapting to technical progress for the 27th time Council Directive 67/548/EC on the approximation of laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances. Annex V B.41 Phototoxicity - in vitro 3T3 NRU phototoxicity test.; 98-107; 2000 Holzhuetter HG; A general measure of in vitro phototoxicity derived from pairs of doseresponse curves and its use for predicting the in vivo phototoxicity of chemicals.; Alternatives to laboratory animals, ATLA; 25; 445-462; 1997 Jones, PA, King AV, Earl LK, Lawrence RS. An assessment of the phototoxic hazard of a personal product ingredient using in vitro assays. Toxicology in vitro 17, 471-480, 2003 Liebsch, M., Spielmann, H., Balls, M., Brand, M., Dring, B., Dupuis, J., Holzhtter, H.G., Klecak, G., L'Eplattenier, H., Lovell, W.W., Maurer, T., Moldenhauer, F., Moore, L., Pape, W.J.W., Pfannenbecker, U., Potthast, J., De Silva, O., Steiling, W., Willshaw, A. (1994): First results of the EC/COLIPA validation project "in vitro phototoxicity testing".In: Alternative Methods in Toxicology, Vol. 10: In vitro Skin Toxicology - Irritation, Phototoxicity, Sensitization.Eds. A. Rougier, A. Goldberg, H. Maibach;Mary Ann Liebert Publ., New York; pp. 243 - 254 Liebsch M; In vitro 3T3 NRU phototoxicity test. ZEBET/ECVAM/COLIPA Standard Operating Procedure (SOP).; 1-18; 1998, see also INVITTOX Method No. 78, available via ECVAM SIS. Peters B, Holzhtter HG: In Vitro Phototoxicity Testing: Development and validation of a new concentration response analysis software and biostatistical analyses related to the use of various prediction models. ATLA 30, 415.432, 2002 Maier K; Schmitt-Landgraf R; Siegemund B; Development of an in vitro test system with human skin cells for evaluation of phototoxicity.; Toxicology in vitro; 5(6/6); 457-461; 1991 Spielmann, H., Balls, M., Brand, M., Dring, B., Holzhtter, H.G., Kalweit, S., Klecak, G., L`Epattenier, H., Liebsch, M., Lovell, W.W., Maurer, T., Moldenhauer, F., Moore, L., Pape, W.J.W., Pfannenbecker, U., Potthast, J., De Silva, O., Steiling, W. and Willshaw, A. (1994b) EC/COLIPA project on in vitro phototoxicity testing: first results obtained with the Balb/c 3T3 cell phototoxicity assay. Toxicol. in Vitro 8, 793-796 Spielmann H; Lovell WW; Hoelzle E; Johnson BE; Maurer T; Miranda M; In vitro phototoxicity testing. The report and recommendations of ECVAM workshop 2.; Alternatives to laboratory animals, ATLA; 22; 314-348; 1994 Spielmann H; Liebsch M; Doering B; Moldenhauer F; First results of the EU/COLIPA validation trial "In vitro phototoxicity testing".; In vitro toxicology; 9(3); 325-338; 1996
7/7 Spielmann, H., Balls, M., Dupuis, J., Pape, W.J.W., Pechovitch, G. De Silva, O., Holzhtter, H.G., Clothier, R., Desolle, P., Gerberick, F., Liebsch, M., Lovell, W.W., Maurer, T., Pfannenbecker, U., Potthast, J. M., Csato, M., Sladowski, D., Steiling, W., and Brantom, P. (1998). The international EU/COLIPA In vitro phototoxicity validation study: results of phase II (blind trial), part 1: the 3T3 NRU phototoxicity test. Toxic. In Vitro 12, 305-327. Spielmann H; Balls M; Dupuis J; Pape WJW; de Silva O; Holzhuetter HG; Gerberick F; Liebsch M; Lovell WW; Pfannenbecker U; A study on UV filter chemicals from Annex VII of EU Directive 76/768/EEC in the 3T3 NRU phototoxicity test.; Alternatives to laboratory animals, ATLA; 26; 679-708; 1998b Spielmann, H., Mller, L., Averbeck, D., Balls, M., Brendler-Schwaab, S., Castell, J.V., Curren, R., de Silva, O., Gibbs, N.K., Liebsch, M., Lovell, W.W., Merk, H.F., Nash, J.F., Neumann, N.J., Pape, W.J.W., Ulrich, P. and Vohr, H.-W. The Second ECVAM Workshop on Phototoxicity Testing. The Report and Recommendations of ECVAM Workshop 42. ATLA 28, 777-814, 2000 Spielmann H; Acute phototoxicity testing.; Environmental Mutagen Research; 23; 45-56; 2001
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8.1.2 The Red Blood Cell Phototoxicity Test (RBC PT)

8.1.2.1 Short description, scientific relevance and purpose Photohaemolysis is one of the oldest and simplest in vitro techniques for screening of putative photosensitizers (Sacharoff and Sachs 1905). Many different protocols applying various light sources and sources for erythrocytes have been reported in the literature. Taking into account this information, Hetherington and Johnson (1984) published a photohaemolysis method for determining the phototoxic potential of drugs and other chemicals. Additional important endpoints of phototoxicity in red blood cells are free-radical production and the oxidation of haemoglobin. Since methaemoglobin formation is often observed in phototoxicity testing with erythrocytes, both phenomena, photohaemolysis and haemoglobin oxidation, can be tested in a combined RBC (red blood cell) phototoirritation test (RBC PT; Pape et al. 1994, 2001). This approach allows to screen for photosensitizers and to study phototoxic mechanisms at the cellular level in human material. The combined RBC photohaemolysis and photo-haemoglobin oxidation assay is a useful test both for screening and for mechanistic studies. Phototoxic compounds reacting only with DNA will provide negative results in the RBC test, if the generation of active oxygen species (AOS) is not part of the mechanisms of cytotoxicity (photodynamic reactions).
8.1.2.2 Developer of the method Photohaemolysis is one of the oldest and simplest in vitro techniques for screening of putative photosensitizers (Sacharoff and Sachs 1905). In 1984 Hetherington and Johnson (1984) published a photohaemolysis method for determining the phototoxic potential of drugs and other chemicals (Hetherington and Johnson 1984) and the current protocol of the RBC PT was developed by Pape and co-workers at Beiersdorf in Germany (Pape et al. 1994, 2001). The method was transferred to laboratories of the European cosmetic industry during the COLIPA/ ECVAM prevalidation and formal validation studies of in vitro phototoxicity tests conducted from 1992 -1998 (Spielmann et al. 1994, 2000).
8.1.2.3 Known user The RBC PT is established at the laboratory of Beiersdorf AG, Hamburg, Germany (Dr. Wolfgang Pape and Uwe Pfannenbecker) and at the laboratory of Kose Corporation, Tokyo, Japan (Dr. Yuuko Okamoto).
8.1.2.4 Status of validation and/or standardisation Preliminary data obtained with the RBC PT during the COLIPA/ECVAM prevalidation study were very promising (Spielmann et al., 1994). The positive evaluation is supported by resuts obtained under blind conditions in three laboratories with the RBC PT test in the validation stage of the ECVAM/COLIPA in vitro photoirritation validation study (Pape et al. 2001, Spielmann et al., 1998). It was concluded from the results that the RBC PT provides a good overall fit with the in vivo endpoints as well as mechanistic information on two different types of photodynamic reactions (met-Hb formation for type I reactions and photo-haemolytic effects as primary type II reactions). In summary, when conducted according to the SOP of the prevalidation study, the RBC PT can reliably be performed. Moreover, this test provides relevant mechanistic information on photodynamic reactions, which is useful for the evaluation of the photo-safety of chemicals in a testing strategy that starts with the 3T3 NRU PT test, which does not provide information on photodynamic reactions. An additional advantage of red blood cells is their resistance to
9/9 the short-waved UVB-part of sun light, which allows to expose them in the RBC PT to the entire solar spectrum for prolonged periods of exposure (Spielmann et al. 2000).
8.1.2.5 Fields and limitations of application The RBC PT is not a stand alone test to assess the phototoxic potential of chemicals. In contrast to the 3T3 NRU PT, however, the RBC PT can be used at high doses of UVB in the irradiation spectrum and the RBC PT provides information on several mechanisms of phototoxicity, which cannot be assessed in the 3T3 NRU PT. Therefore, the RBC PT is a useful adjunct in vitro test, which should be used complementary to the 3T3 NRU PT, when mechanistic study have to be conducted. (Spielmann et al. 2000).
8.1.2.6 Recommendations of use in the view of animal replacement Taking into account EU Directive 86/609/EEC on "The protection of animals used for experimental and other scientific purposes" (EC 1986), safety testing of chemicals and of cosmetic ingredients for phototoxic potential is no longer permitted in EU member countries, since the in vitro 3T3 NRU PT has been accepted as the standard test methods for this endpoint and since two additional in vitro phototoxicity tests complementing the 3T3 NRU PT, the RBC PT and the H3D PT, have shown promising results in interlaboratory assessments under blind conditions, and are established in laboratories of the cosmetics and chemical industry in the EU as outlined in sections 8.1.1 and 8.1.3.
8.1.2.7 On-going development We are not aware of on-going research or validation activities to improve the RBC PT. However, laboratories that are using the RBC PT in-house may provide information on improvements in the in INVITOX protocol No 81, which was published 10 years ago (Pape et al 1994).
8.1.2.8 Efforts needed to complete validation of the method Since the RBC PT is not the standard in vitro test for acute phototoxicity and since the RBC PT has successfully undergone pre-validation and validation under blind conditions in the COLIPA/ECVAM validation study of in vitro phototoxicity tests, there is currently no need for experimental validation.
8.1.2.9 Key references Pape, W.J.W., Brandt, M. and Pfannenbecker, U.: Combined in vitro assay for photohaemolysis and haemoglobin oxidation as part of a phototoxicity test system assessed with various phototoxic substances. Toxicology in Vitro 8, 755-757 (1994) Pape, W.J.W., Maurer, T., Pfannenbecker, U., Steiling, W.: The Red Blood Cell Test (Photohaemolysis and Haemoglobin Oxidation). EU/COLIPA Validation Programme on Phototoxicity (Phase II) Submitted for publication Pape, W., Pfannenbecker, U.: RBC Photo Assay - Photohaemolysis and Haemoglobin Oxidation. Invittox Protocol No. 81 (ERGATT/FRAME Databank of In Vitro Techniques in Toxicology, ISSN 0960-2194) (1994) Johnson, B.E., Walker, E.M. & Hetherington, A.M.: In vitro models for cutaneous phototoxicity, in: Skin Models, Models to Study Function and Disease of Skin (ed. R. Marks & . Plewig) Berlin, Germany: Springer Verlag (1986) pp. 264-281 Kahn, G. & Fleischaker, B.I.: Red blood cell haemolysis by photosensitizing compounds. Journal of Investigative Dermatology 56, 85-90 (1971)
10 / 10 Traynor NJ, Johnson BE, Gibbs NK.(1996) Photohaemolysis Assay for Drug Phototoxicity Complicated by 'Bleaching of Released Haemoglobin'. Toxicology in Vitro 10, 619-624 Okamoto Y, Ryu A, Ohkoshi K (1999): In vitro alternatives and phototoxicity testing. I. Evaluation of in vitro phototoxicity assays. ATLA 27, 639-644 Winterbourn, C.C.(1986): Free-radical production and oxidative reactions of haemoglobin. Environmental Health Perspectives 64, 321-330 Spielmann, H., Lovell, W. W., Hlzle, E., Johnson, B. E., Maurer, T., Miranda, M. A., Pape, W.J.W., Sapora, O. and Sladowski, D.(1994) In vitro Phototoxicity Testing, ECVAM Workshop Report 2. The Report and Recommendations of ECVAM Workshop 2. ATLA 22, 314-348 Spielmann, H., Balls, M., Dupuis, J., Pape, W.J.W., Pechovitch, G. De Silva, O., Holzhtter, H.G., Clothier, R., Desolle, P., Gerberick, F., Liebsch, M., Lovell, W.W., Maurer, T., Pfannenbecker, U., Potthast, J. M., Csato, M., Sladowski, D., Steiling, W., and Brantom, P. (1998). The international EU/COLIPA In vitro phototoxicity validation study: results of phase II (blind trial), part 1: the 3T3 NRU phototoxicity test. Toxic. In Vitro 12, 305-327. Spielmann, H., Mller, L., Averbeck, D., Balls, M., Brendler-Schwaab, S., Castell, J.V., Curren, R., de Silva, O., Gibbs, N.K., Liebsch, M., Lovell, W.W., Merk, H.F., Nash, J.F., Neumann, N.J., Pape, W.J.W., Ulrich, P. and Vohr, H.-W. (2000) The Second ECVAM Workshop on Phototoxicity Testing. The Report and Recommendations of ECVAM Workshop 42. ATLA 28, 777-814
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8.1.3 Human 3-D Skin Model In Vitro Phototoxicity Test (H3D PT)
8.1.3.1 Short description, scientific relevance and purpose Reconstituted human skin models (3-D Skin Models) are available commercially, or from a few experienced laboratories, in three different types: dermal models (containing skin fibroblasts), epidermal models (containing skin keratinocytes and a stratum corneum), and full skin models (containing skin fibroblasts, keratinocytes and a stratum corneum). Since the latter two types contain viable, metabolising primary skin cells and a skin barrier, both are frequently referred to as "3-D skin models". Human skin models have been used quite successfully in routine laboratory investigations, since they are relevant to the organ of interest. For in vitro toxicity testing, standardisation and control of 3-D skin models needs to be defined clearly in order to assure that reliable and reproducible data are obtained. In contrast to normal cell cultures, e.g. mouse fibroblasts that are used in the NRU PT, human skin models allow for topical application of various types of chemicals and preparations and seem to have less limitation concerning solubility problems. Test materials can be applied to 3-D skin models undiluted, at extreme pH values or even as insoluble materials. The first promising data obtained with a H3D PT were reported 1994-1995 with a full skin model (Edwards et al. 1994; Liebsch et al. 1995), and an epidermal model (Roguet et al. 1994). Since the commercial production of the full skin model Skin2 was stopped in 1996, the test protocol was successfully adapted to the epidermal model EpiDerm (Liebsch et al. 1997). The H3D PT applying EpiDerm was tested in an ECVAM prevalidation study revealing promising results in three laboratories under blind conditions (Liebsch et al. 1999). The test is currently established in several laboratories of the European cosmetics industry (Jones et al. 1999) and has been successfully adopted to the epidermal model SkinEthic (Bernard et al. 1999, Jones et al. 2003). Efforts undertaken to optimise the phototoxicity test protocol and prediction model when transferring it from the full skin model to the epidermal model (Liebsch et al 1997) revealed the basic test protocol and prediction model did not need to be changed. Several studies (Liebsch et al. 1995; Api 1997) reported that in vivo photoallergens that are in vivo not acute photoirritants at the same time (e.g. coumarin, 6-methyl-coumarin, musk ambrette), are classified correct negative by the skin model phototoxicity tests, while they are positive in the 3T3NRU-PT. Reconstituted human skin models are increasingly investigated for their usability in toxicological hazard identification/safety testing, because their organotypic structure with a functional stratum corneum allows for in vitro tests modeling bioavailability of topically applied substances. Because of the stratum corneum barrier, an important role of the H3D-PT test is the verification / falsification of positive results obtained the 3T3 NRU PT. Simple dermal models, which do not contain a skin barrier, show a sensitivity to phototoxic chemicals, which is similar to photo-cytotoxicity tests, as e.g. the 3T3 NRU-PT (Augustin et al. 1997). They are, therefore, not providing any advantage in a phototoxicity testing strategy. 8.1.3.2 Developer of the method Two slightly different approaches for the use of skin models in phototoxicity were developed at about the same time (1994 -1995): Roguet et al (1994) developed a protocol for the human epidermis model EPISKIN, and Edwards et al. (1994) and Liebsch et al. (1995) developed a protocol for the full skin model Skin. The basic test design was similar in both methods: investigation of a test substance dose-response on skin cell viability (MTT reduction) in parallel on tissues non-irradiated and irradiated with the highest non-cytotoxic UVA-vis dose. However, the prediction models used in both tests were different. Roguet et al. (1994) compared IC50 values obtained +UVA and -UVA, and Liebsch et al. (1995, 1997, 1999) determined the lowest dose at which a significant phototoxic effect (LOAEL) was observed. Probably due to the fact that the latter method and prediction model had shown
12 / 12 robustness by the successful transfer from the full skin model Skin to the epidermis model EpiDerm (Liebsch et al. 1997, 1999) without any changes necessary and possible, it is used in several laboratories, also with other human skin / epidermis models (e.g. with SkinEthic: Jones et al. 2003).
8.1.3.3 Known user The H3D PT is established as an adjunct phototoxicity test in some companies of the Cosmetic Industry, e.g. Beiersdorf AG (Hamburg, D), Unilever (Sharnbrook, UK), as well as in contract testing institutes, e.g. Dr. Schrader Institute (Holzminden, D), Institute for In Vitro Sciences (Gaithersburg, USA), MB Research Laboratories (Spinnerstown, USA).
8.1.3.4 Status of validation and/or standardisation After development of the method with the model Skin, the H3D PT was evaluated within phase 1 of the EC/COLIPA phototoxicity validation trial by ZEBET on 20 chemicals in a nonblinded study (Liebsch et al. 1994, Spielmann et al 1994, Liebsch et al. 1995). Later, the H3D PT was again evaluated by ZEBET under blind conditions on 30 test chemicals within phase 2 of the EC/COLIPA validation with promising results. This outcome, however, was never published because the model Skin was not available any more shortly after the end of this study. Later, in an ECVAM prevalidation study, the H3D PT protocol and prediction model developed with Skin was transferred to the epidermis model EpiDerm and revealed excellent results in a blind trial in three laboratories. In this study, attempts to optimise the protocol (Liebsch et al. 1997, 1999) revealed that no optimisation was possible. Even the prediction model developed originally for the Skin H3D PT could not be optimised and predictions achieved with this prediction model were better then those obtained when the PIF or MPE prediction models of the 3T3 NRU PT were applied (Holzhtter, 1998). As an adjunct test to the 3T3 NRU PT the H3D PT can be regarded valid and sufficiently standardised. However, a formal validation study, specifically designed for the role of this test as described in 8.1.3.5. would help establishing its use.
8.1.3.5 Fields and limitations of application Due to the stratum corneum functional barrier of human skin or epidermis models, the H3D PT is not a stand alone test to assess the inherent phototoxic potential of chemicals. In contrast to the 3T3 NRU PT, the H3D PT may miss the correct detection of phototoxins that cannot enter the skin via topical route but may be sufficiently bioavailable in the skin via systemic pathways, e.g. after oral, or parenteral exposure. The H3D PT may also miss the detection of weakly photoreactive chemicals that induce photoallergic reactions only after repeated exposure. However, because the H3D PT models the bioavailability of chemicals and formulations at topical skin exposure, it is qualified as an adjunct test to further investigate chemicals with (probably false) positive outcomes in the 3T3 NRU PT, as specified in paragraph 54 of new draft OECD Test Guideline 432 (OECD 2002). For assessment of phototoxic hazard this approach is conservative, since the penetration barrier of in vitro skin models is known to be weaker compared with the penetration barrier of skin in vivo. If the H3D PT is used as an adjunct to the 3T3 NRU PT as described above (only for chemicals intended for topical use) it has hardly any limitation, because it mimics the in vivo situation and can handle solutions, as well as suspensions. The only limitation known is that a test substance may directly reduce MTT and mimic dehydrogenase activity of the cellular mitochondria. This is only a problem if at the time of the MTT test (24 hours after test substance exposure) still sufficient amounts of the test substance are present on (or in) the
13 / 13 tissues. In this case, the (true) metabolic MTT reduction and the (false) direct MTT reduction can be differentiated. 8.1.3.6 Recommendations of use in the view of animal replacement Taking into account EU Directive 86/609/EEC on "The protection of animals used for experimental and other scientific purposes" (EC 1986), safety testing of chemicals and of cosmetic ingredients for phototoxic potential is no longer permitted in EU member countries, since the in vitro 3T3 NRU PT has been accepted as standard test method for this endpoint and since two additional in vitro phototoxicity tests complementing the 3T3 NRU PT, the RBC PT and the H3D PT, have shown promising results in interlaboratory assessments under blind conditions, and are established in laboratories of the cosmetics and chemical industry in the EU as outlined in sections 8.1.1 and 8.1.2
8.1.3.7 On-going development In an ECVAM funded feasibility study it is currently investigated by ZEBET in co-operation with the Czech Institute for Public Health (SZU) if the H3D PT can be upgraded from a test for phototoxic potential to a test for photo-potency of topically applied substances. In this study, the lowest observed adverse effect levels (LOAEL) determined in the H3D PT will be compared with the LOEAL observed in vivo in human photo-patch tests and data obtained with the 3T3 NRU PT.
8.1.3.8 Efforts needed to complete validation of the method If the feasibility study described in 8.1.3.7 shows promising results, the H3D PT should be formally validated to a test for photo-safety / photo-potency of test chemicals (or formulations thereof) with intended topical use on the skin.
8.1.3.9 Key references
Api, A.M. (1997) In vitro assessment of phototoxicity. In Vitro Toxicol. 10, 339-350. Augustin, C., Collombel, C., Damour, O. (1997) Use of dermal equivalent and skin equivalent models for identifying phototoxic compounds in vitro. Photodermatol. Photoimmunol. Photomed. 13, 27-36. Bernard, F.X., Barrault, C., Deguery, A., de Wever, B., Rosdy, M. (1999) Development of a highly sensitive phototoxicity assay using the reconstructed human epidermis SkinEthik. In Alternatives to Animal Testing II: Proceedings of the second international scientific conference organised by the European Cosmetic Industry, Brussels, Belgium, (eds. D. Clark, S. Lisansky & R. Macmillan), pp 133-137 Edwards, S.M., Donnelly, T.A., Sayre, R.M., Rheins, L.A., Spielmann, H., Liebsch, M. (1994). Quantitative in vitro assessment of phototoxicity using a human skin model: Skin2. Photodermatol. Photoimmunol. Photomed. 10, 111-117 Holzhtter (1998) Annex 4 (Biostatistical Analysis) to Final Report of the ECVAM Prevalidation Project: "Prevalidation of the EpiDerm Phototoxicity Test". Available upon request from ECVAM SIS, Ispra, Italy Jones, P., King, A., Lovell, W., Earl, L. (1999) Phototoxicity testing using 3-D reconstructed human skin models. In Alternatives to Animal Testing II: Proceedings of the second international scientific conference organised by the European Cosmetic Industry, Brussels, Belgium, (eds. D. Clark, S. Lisansky & R. Macmillan), pp 138-141
14 / 14 Jones, PA, King AV, Earl LK, Lawrence RS (2003). An assessment of the phototoxic hazard of a personal product ingredient using in vitro assays. Toxicology in vitro 17, 471-480 Liebsch, M., Spielmann, H., Balls, M., Brand, M., Dring, B., Dupuis, J., Holzhtter, H.G., Klecak, G., L'Eplattenier, H., Lovell, W.W., Maurer, T., Moldenhauer, F., Moore, L., Pape, W.J.W., Pfannenbecker, U., Potthast, J., De Silva, O., Steiling, W., Willshaw, A. (1994): First results of the EC/COLIPA validation project "in vitro phototoxicity testing".In: Alternative Methods in Toxicology, Vol. 10: In vitro Skin Toxicology - Irritation, Phototoxicity, Sensitization.Eds. A. Rougier, A. Goldberg, H. Maibach;Mary Ann Liebert Publ., New York; pp. 243 - 254 Liebsch, M., Dring, B., Donelly, T.A., Logemann, P., Rheins, L.A. and Spielmann, H. (1995) Application of the human dermal model Skin2 ZK 1350 to phototoxicity and skin corrosivity testing. Toxic. in Vitro 9, 557-562 Liebsch, M. Barrabas, C. Traue, T. and Spielmann, H. (1997) Entwicklung eines in vitro Tests auf dermale Phototoxizitt in einem Modell menschlicher Epidermis (EpiDermTM). ALTEX (Alternativen zu Tierexperimenten) 14, 165-174. Liebsch, M., Traue, D., Barrabas, C., Spielmann, H., Gerberick, G.F., Cruse, L., Diembeck, W., Pfannenbecker, U., Spieker, J., Holzhtter, H. G., Brantom, P., Aspin, P., Southee, J. (1999) Prevalidation of the EpiDerm Phototoxicity Test. In Alternatives to Animal Testing II: Proceedings of the second international scientific conference organised by the European Cosmetic Industry, Brussels, Belgium (eds. D. Clark, S. Lisansky & R. Macmillan), pp 160-166 Roguet, R., Cohen, C. Rougier, A. (1994). A reconstituted human epidermis to assess cutaneous irritation, photoirritation and photoprotection in vitro. In Alternative Methods in Toxicology, 10: In vitro Skin Toxicology - Irritation, Phototoxicity, Sensitization (eds. A. Rougier, A. Goldberg & H. Maibach); Mary Ann Libert Publ., New York, pp. 141-149 Spielmann, H., Balls, M., Brand, M., Dring, B., Holzhtter, H.G., Kalweit, S., Klecak, G., L`Epattenier, H., Liebsch, M., Lovell, W.W., Maurer, T., Moldenhauer, F., Moore, L., Pape, W.J.W., Pfannenbecker, U., Potthast, J., De Silva, O., Steiling, W. and Willshaw, A. (1994) EC/COLIPA project on in vitro phototoxicity testing: first results obtained with the Balb/c 3T3 cell phototoxicity assay. Toxicol. in Vitro 8, 793-796
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8. 2
PHOTO-CHEMICAL GENOTOXICITY
In vivo testing for photogenotoxicity is rarely performed, since the skin is not utilised in standard approaches. However, transgenic mutagenicity models (such as lacI or lacZ transgenic mice) may be suited for this purpose, because UV radiation induced mutations in skin cells has been demonstrated (Gorelick 1996). Furthermore, the micronucleus test on rat and mouse skin as target organ has been developed and might be applicable for Photo Micronucleus testing as well (Nishikawa 1999 and 2002). Only a few genotoxicity test have been reported for photochemical genotoxicity in vivo. Satoru Itoh et al have demonstrated in vivo photochemical micronucleus induction by a certain quinolone in skin of mice (2002). Furthermore Positive results have been reported for the fluoroquinolone clinafloxacin in the Comet assay (Bulera et al., 1999). However, if in vivo testing seems at all necessary, these assays need further development and validation. In vivo testing of photogenotoxicity potential is not requested by the SCCNFP. In principle, like with dark genetic toxicity tests, photogenotoxicity testing is a domain of in vitro tests. Based on recommendations of Loprieno (1991), for safety testing of cosmetics the SCCNFP proposes a bacterial test for gene mutation and an in vitro test for chromosomal aberrations in mammalian cells be performed in the presence of UV radiation. So far, none of the many in vitro photogenotoxicity tests currently in use has been formally validated. A crucial point, relevance for in vivo , will not be possible to address in these validation studies, because so far only one photogenotoxic chemical (8-MOP) has proven to be photocarcinogenic in vivo in humans. Many different in vitro photogenotoxicity tests have been adapted for testing the potential of chemicals to damage the DNA after photoactivation. However, it is recommended to focus on those in vitro test systems, which are currently used as standard tests in regulatory testing strategies. Since to date no photogenotoxic chemical is known which exclusively acts through gene mutations, and because the recognised photochemical mechanisms are clastogenic, it is suggested that a test for photochemical clastogenicity (chromosomal aberration or micronucleus test) should have first priority. In addition, the Photo-Comet assay was proposed by an international expert working group as reasonable supplementary test which may give additional information on photogenotoxic properties of a compound (Gocke et al., 2000). The most recent comprehensive review of currently available photogenotoxicity tests has been published by the GUM (German section of the European Environmental Mutagen Society, EEMS) Task Force on photochemical genotoxicity (Brendler-Schwaab et al., 2004). References to introduction. Brendler-Schwaab, S., Czich, A., Epe, B., Gocke, E., Kaina, B., Mller, L.. Pollet, D., Utesch, D. (2004) Photochemical genotoxicity: principles and test methods. Report of a GUM task force. Review . Mutation Res. 566: 6591 Bulera, S.J., Theiss, J.C., Festerling, T.A. & de la Iglesia, F.A. (1999). In vitro photogenotoxic activity of clinafloxacin: A paradigm predicting photocarcinogenicity. Toxicol. Appl. Pharmacol. 156, 222-230. Gocke E, Muller L, Guzzie PJ, Brendler-Schwaab S, Bulera S, Chignell CF, Henderson LM, Jacobs A, Murli H, Snyder RD, Tanaka N. Considerations on photochemical genotoxicity: Report of the IWGPT Working Group. Environ. Molec. Mutagen. 2000: 35, 173-184 Gorelick, N.J. (1996) Validation issues for the use transgenic mouse mutation assays in risk assessment. Prog. Clin. Biol. Res. 395 (1996) 81-108 Itoh, S., Katoh,M. and Furuhama,K. (2002) In vivo photochemical micronucleus induction due to certain quinolone antimicrobial agents in the skin of hairless mice. Mutation Research, 520, 133-139 Loprieno, N. (1991) In vitro Mutagenesis 6: 331 - 333 assay systems for testing photomutagenic chemicals.
16 / 16 Nishikawa, T. et al. (1999) Study of rat skin in vivo micronucleus test: data generated by mitomycin C and methyl methanesulfonate. Mutation Research., 444, 159-166. Nishikawa, T., Haresaku, M., Fukushima, A., Nakamura, T. Adachi, K, Masuda, M., Hayashi, M. (2002) Further evaluation of an in vivo micronucleus test on rat and mouse skin: results of five skin carcinogens. 513, 93-102
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8.2.1
Reverse Gene/Point Mutation: Photo Ames Test (P-AMES)
8.2.1.1 Short description, scientific relevance and purpose Procariontic, bacterial mutation tests are easier and cheaper to perform than any photogenotoxicity tests with eucariontic mammalian cells. Therefore, the P-AMES test was the first in vitro photogenotoxicity test adapted from to the parallel use of light (Jose, 1979) and later proposed for safety testing of cosmetics (Loprieno 1991; COLIPA 1995). In the PAMES test, a dose dependent, test chemical mediated, increase of reverse mutations from histidine dependent strains to histidine independent wild-type is investigated plus and minus a non-mutagenic UV-vis irradiation.
8.2.1.2 Developer of the method The first P-AMES was proposed already six years after the publication of Ames test (Jose, 1979). However, the methodology described by Dean et al. (1991) is more frequently used as it was the methodology and recommend by the SCC (Loprieno, 1991).
8.2.1.3 Known user Several laboratories in Cosmetics and Pharmaceutical Industry, contract laboratories and some academic Institutes are using the P-AMES test.
8.2.1.4 Status of validation and/or standardisation The P-AMES test has never been formally validated. However, attempts to standardise the many existing protocol modifications have been made (Dean et al., 1991; 1992; Chelat et al., 1993; Henderson et al. 1994; COLIPA, 1995). Albeit this status, the P-AMES test has achieved acceptance by the SCCNFP in the field of cosmetic safety testing.
8.2.1.5 Fields and limitations of application According to Brendler-Schwaab et al. (2004) the critical point of all P-AMES test protocols is the UV sensitivity of the Salmonella typhimurium or Escherichia coli strains used. Chemicals often have to be pre-irradiated in the absence of the bacteria to achieve doses of light necessary to activate the photogenotoxins. Moreover, the limited predictivity of the regular "dark" AMES test suggests the use of tests employing mammalian cells rather than bacteria, if DNA point mutation tests are considered as part of a relevant test battery.
8.2.1.6 Recommendations of use in the view of animal replacement While in vivo photogenotoxicity testing is not required for the safety testing of cosmetics, it is hoped hat the current in vitro approaches in photogenotoxicity testing will lead to the definition of an in vitro test battery allowing for an assessment of the likelihood of a compound to turn into a photochemical carcinogen upon excitation and activation with UV or visible radiation. However, while pharmaceuticals with a photocarcinogenic potential may be further investigated in vivo because they can be safely used avoiding any light exposure, chemicals with a photocarcinogenic potential will not be acceptable as cosmetic ingredients, so that in vivo testing will not be required in that area.
18 / 18
8.2.1.7 On-going development Probably because of the decreasing importance of the P-AMES test as a relevant part of sensitive photogenotoxicity testing battery, we are not aware of on-going developments of this test system.
8.2.1.8 Efforts needed to complete validation of the method The P-AMES test is at the state of an optimised test (i.e. ready for prevalidation and validation). Albeit it's acceptance by the SCCNFP a decreasing importance of this test as relevant part of a photogenotoxicity testing battery is obvious, so that it may not be advisable to select this test for entering the expensive procedures of prevalidation and formal validation studies.
8.2.1.9 Key references Brendler-Schwaab, S., Czich, A., Epe, B., Gocke, E., Kaina, B., Mller, L.. Pollet, D., Utesch, D. (2004) Photochemical genotoxicity: principles and test methods. Report of a GUM task force. Review . Mutation Res. 566: 6591 Chtelat A., Albertini S., Dresp J.H., Strobel R., Gocke E. (1993) Photomutagenesis test development. I. 8-Methoxypsoralen, chlorpromazine and sunscreen compounds in bacterial and yeast assays, Mutat. Res. 292, 241250. Dean S.W., Lane M., Dunmore R.H., Ruddock S.P., Martin C.N., Kirkland D.J., Loprieno N. (1991) Development of assays for the detection of photomutagenicity of chemicals during exposure to UV light. I. Assay development, Mutagenesis 6, 335341. Dean S.W., Dunmore R.H., Ruddock S.P., Dean J.C., Martin C.N., Kirkland D.J. (1992) Development of assays for thedetection of photomutagenicity of chemicals during exposure to UV light. II. Results of testing three sunscreen ingredients, Mutagenesis 7, 179182. Henderson L., Fedyk J., Bourner C., Windebank S., Fletcher S., Lovell W. (1994) Photomutagenicity assays in bacteria: factors affecting assay design and assessment of photomutagenic potential of para-aminobenzoic acid, Mutagenesis 9, 459465. Jose J.G. (1979) Photomutagenesis by chlorinated phenothiazine tranquilizers. Proc Natl Acad Sci U S A. 76: 469-72. Loprieno, N. (1991) In vitro assay systems for testing photomutagenic chemicals. Mutagenesis 6: 331 - 333 COLIPA (1995): Photomutagenicity Task Force, Final report, The European Cosmetic Toiletry and Perfumery Association (COLIPA), Brussels, Belgium, August 1995.
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8.2.2
Forward Gene/Point Mutation: Photo-Hypoxanthine-Guanine Phosphoribosyl Transferase Assay (P-HPRT) and Photo Mouse Lymphoma assay (P-MLA)
8.2.2.1 Short description, scientific relevance and purpose In regular ("dark") gentotoxicity testing, in vitro mammalian gene mutation tests are integral part of the regulatory required testing battery, and their predictive value is regarded higher than that of bacterial gene mutation tests. The principle is largely similar as with the Ames test: only mutants survive a selective stress (although reverse mutations are detected with the Ames test and forward mutations are detected with the MLA). The following description shows the test principle for one of the mammalian gene mutation tests, the Mouse Lymphoma Assay (MLA): The L5178YTK+/- mouse lymphoma assay (MLA) detects mutations at the thymidine kinase (TK) locus caused by base pair changes, frameshift and small deletions. Mutant cells, deficient in TK due to the forward mutation in the TK locus (from TK+ to TK-), are resistant to the cytotoxic effect of pyrimidine analogues such as trifluorothymidine (TFT). The mutagenicity of the test agents is indicated by the increase in the number of mutants after treatment. Other assays of this type are using mutations at enzyme loci of hypoxanthine-guanine phosphoribosyl transferase (HPRT), or a transgene of xanthine-guanine phosphoribosyl transferase (XPRT), and are in general performed with Chinese Hamster cell lines like CHO, CHO-AS52 and more frequently, V79. For review of these methods see Nestmann et al. (1991). The in vitro mammalian cell gene photogenotoxicity tests employ the same protocols, but test chemicals are investigated plus and minus a non-or slightly genotoxic UV-vis radiation.
8.2.2.2 Developer of the method The developers of the P-HPRT and P-MLA could not exactly been identified. 8.2.2.3 Known user From the review of Brendler-Schwaab et al. (2004) it is suggested that mammalian cell gene photo mutation tests P-HPRT and P-MLA are established in laboratories Pharmaceutical Industry, Academia, and Public Health Institutes, like the Hadano Research Institute, Food and Drug Safety Centre, Japan (Nakagawa et al., 1997). It is also obvious from the literature that the P-HPRT established in more laboratories than the P-MLA.
8.2.2.4 Status of validation and/or standardisation The P-HPRT and P-MLA have never been formally validated. Whereas the degree of standardisation for the "dark" versions of the two tests is regarded high (as they have become integral part of many safety testing regulations), the photo modifications of these tests still need standardisation, in particular with regard to the UV-vis radiation used. Moreover, since the number of chemical classes tested in these assays is very limited (predominantly psoralenes and other chemicals used in P-UVA therapy, some fluoroquinoloes, and UV filters, Brendler-Schwaab et al., 2004), a thorough data base development phase would be necessary to validate the assays.
8.2.2.5 Fields and limitations of application
20 / 20 The photogenotoxic endpoint covered by the P-HPRT and the P-MLA (gene mutation) is identical with the P-AMES. However, since the mammalian cells are closer to the species if interest (humans), and because the cell lines are more resistant to UV radiation, the likelihood to produce false negative results in the P-HPRT and P-MLA is lower.
8.2.2.6 Recommendations of use in the view of animal replacement While in vivo photogenotoxicity testing is not required for the safety testing of cosmetics, it is hoped hat the current in vitro approaches in photogenotoxicity testing will lead to the definition of an in vitro test battery allowing for an assessment of the likelihood of a compound to turn into a photochemical carcinogen upon excitation and activation with UV or visible radiation. However, while pharmaceuticals with a photocarcinogenic potential may be further investigated in vivo because they can be safely used avoiding any light exposure, chemicals with a photocarcinogenic potential will not be acceptable as cosmetic ingredients, so that in vivo testing will not be required in that area.
8.2.2.7 On-going development The group is not aware of current approaches to further develop, prevalidate, or validate the existing protocols of the P-HPRT and P-MLA. 8.2.2.8 Efforts needed to complete validation of the method The P-HPRT and P-MLA test are in the state of optimised tests (i.e. ready for prevalidation and formal validation studies). However, it is suggested that most regulatory bodies will accept submissions of data from these tests (in place of data from the P-AMES test), provided the protocols are using relevant UV-vis light spectra and doses, and concurrent positive and negative controls. However, if formal validation is anticipated, since the number of chemical classes tested in these assays is very limited (Brendler-Schwaab et al., 2004), a thorough data base development would be necessary to formally validate the assays.
8.2.2.9 Key references Brendler-Schwaab, S., Czich, A., Epe, B., Gocke, E., Kaina, B., Mller, L.. Pollet, D., Utesch, D. (2004) Photochemical genotoxicity: principles and test methods. Report of a GUM task force. Review . Mutation Res. 566: 6591 Nestmann E.R., Brillinger R.L., Gilman J.P., Rudd C.J., Swierenga S.H. (1991) Recomended protocols based on a survey of current practice in genotoxicity testing laboratories. II. Mutation in Chinese hamster ovary, V79 Chinese hamster lung and L5178Y mouse lymphoma cells, Mutation Res. 246, 255284. Nakagawa Y., Wakuri S., Sakamoto K., Tanaka N. (1997) The photogenotoxicity of titanium dioxide particles, Mutation Res. 394, 125132.
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8.2.3
Photo-clastogenicity: Photo Chromosome Aberration Test (P-CAT)
8.2.3.1 Short description, scientific relevance and purpose Tests for clastogenicity (i.e. inducing mutations at the chromosome level) are essential part of the testing strategy for chemicals, cosmetics and pharmaceuticals. Since so far no photogenotoxic substance is known that is exclusively acting at the gene level, the determination of photo-clastogenicity is very important, as it will have relevance for the assessment of a photo-carcinogenic hazard potential of substances (Mller & Kasper, 1998; Gocke et al., 2000; Brendler-Schwaab et al., 2004). The purpose of the in vitro P-CAT is to identify agents that cause structural chromosome aberrations in cultured mammalian cells in the presence of a non clastogenic UV-vis radiation. Structural aberrations may be of two types, chromosome or chromatid. With the majority of chemical mutagens, induced aberrations are of the chromatid type, but chromosome-type aberrations also occur. The in vitro photo chromosome aberration test may employ cultures of established cell lines (mostly Chinese Hamster lines CHO, V79, CHL), or primary cell cultures (e.g. human lymphocytes).
8.2.3.2 Developer of the method A developer of the P-CAT could not exactly been identified.
8.2.3.3 Known user The P-CAT is established in several laboratories pharmaceutical and cosmetics Industry, Academia, and contract testing institutes.
8.2.3.4 Status of validation and/or standardisation The P-CAT has so far not been formally validated. Albeit this, the P-CAT is recommended and accepted by the SCCNFP for safety testing of cosmetic ingredients. Although several attempts have been made to standardise the protocol of the P-CAT over the past 15 years (Gibson et al., 1986; COLIPA, 1995; Murli et al.; 2002), the P-CAT protocols published show differences, in particular with regard to the UV-vis irradiation spectrum used and number of (UV-) dose levels (Brendler-Schwaab et al., 2004).
8.2.3.5 Fields and limitations of application The field of the P-CAT is the detection of photo-chemically induced clastogenicity, the most relevant in vitro endpoint for the assessment of photo-carcinogenic potential. So far, limitations are not known test results seem to independent from the cell lines (or primary cells) used. It may be regarded a limitation that cytogenetic chromosome analysis is the most expensive genotoxic endpoint to investigate, which always requires highly trained personnel and the possibilities for apparatus-supported analysis are fairly low.
8.2.3.6 Recommendations of use in the view of animal replacement While in vivo photogenotoxicity testing is not required for the safety testing of cosmetics, it is hoped hat the current in vitro approaches in photogenotoxicity testing will lead to the definition of an in vitro test battery allowing for an assessment of the likelihood of a compound to turn into a photochemical carcinogen upon excitation and activation with UV or visible radiation. However, while pharmaceuticals with a photocarcinogenic potential may be further investigated in vivo because they can be safely used avoiding any light exposure,
22 / 22 chemicals with a photocarcinogenic potential will not be acceptable as cosmetic ingredients, so that in vivo testing will not be required in that area.
8.2.3.7 On-going development The group is not aware of current approaches to further develop, prevalidate, or validate the existing protocols of the P-CAT.
8.2.3.8 Efforts needed to complete validation of the method The P-CAT is in the state of an optimised test (i.e. ready for prevalidation and formal validation studies). However, it is recognised that regulatory bodies accept submissions of PCAT data, provided the protocols are using relevant UV-vis light spectra and doses, and concurrent positive and negative controls. Instead of planning a formal validation study, existing data should be retrospectively analysed. However, if a formal validation is anticipated irradiation for the P-CAT should be defined and standardised before that study.
8.2.3.9 Key references Brendler-Schwaab, S., Czich, A., Epe, B., Gocke, E., Kaina, B., Mller, L.. Pollet, D., Utesch, D. (2004) Photochemical genotoxicity: principles and test methods. Report of a GUM task force. Review . Mutation Res. 566: 6591 COLIPA Photomutagenicity Task Force (1995) Final report, The European Cosmetic Toiletry and Perfumery Association (COLIPA), Brussels, Belgium, August 1995. Gibson N.W., Hartley J.A., LaFrance R.J., Vaughan K. (1986) Differential cytotoxicity and DNA-damaging effects produced in human cells of the Mer+ and Mer- phenotypes by a series of 1-aryl-3-alkyltriazenes, Cancer Res. 46, 49995003. Gocke E., Mller L., Guzzie P.J., Brendler-Schwaab S., Bulera S.,. Chignell C.F., Henderson L.M., Jacobs A., Murli H., Snyder R.D., Tanaka N. (2000) Considerations on photochemical genotoxicity: report of the IWGTP working group, Environ. Mol. Mutagen. 35, 173184. Mller L., Kasper P. (1998) The relevance of photomutagenicity testing as a predictor of photocarcinogenicity, Int. J. Toxicol. 17, 551558. Murli H., Aardema M., Lawlor T., Spicer C. (2002) Photoclastogenicity -an improved protocol, its validation, and investigation of the photogenotoxicity of DMBA, Environ. Mol. Mutagen. 40, 4149.
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8.2.4
Photo-clastogenicity: The Photo Micronucleus Test (P-MNT)
8.2.4.1 Short description, scientific relevance and purpose Tests for clastogenicity (i.e. inducing mutations at the chromosome level) are essential part of the testing strategy for chemicals, cosmetics and pharmaceuticals. Since so far no photogenotoxic substance is known that is exclusively acting at the gene level, the determination of photo-clastogenicity is very important, as it will have relevance for the assessment of a photo-carcinogenic hazard potential of substances (Mller & Kasper, 1998; Gocke et al., 2000; Brendler-Schwaab et al., 2004). The in vitro micronucleus test (MNT) is a mutagenicity test system for the detection of chemicals which induce the formation of small membrane bound DNA fragments i.e. micronuclei in the cytoplasm of interphase cells. These micronuclei may originate from chromosome fragments lacking a centromere or whole chromosomes which are unable to migrate with the rest of the chromosomes during the anaphase of cell division. Thus, the micronucleus assay is in principle appropriate for the detection of clastogenic and aneugenic effects (the latter is not relevant for photogenotoxicity testing). Of the existing in vitro micronucleus test protocols, the method employing Chinese hamster V79 cells (Kalweit et al.1999), validated in a collaborative study (van der Hude et al. 2000) was successfully adapted to photogenotoxicity testing (Kersten et al. 1999, 2002) by two laboratories. The method is based on concentration-response experiments performed with and without irradiation with a UV-vis sunlight simulation. Currently, the P-MNT is evaluated in a formal interlaboratory validation blind trial, supported by the German Ministry of Research, in five laboratories in Germany and Switzerland.
8.2.4.2 Developer of the method Two modifications of the P-MNT were published in the same year (Snyder & Cooper, 1999 and Kersten et al. 1999). The P-MNT was currently under formal validation was developed (and to a large extent pre-validated) in a collaborative study between Bayer AG, Wuppertal, and the German Federal Institute for Drugs and medical Devices, BfArM, Bonn (Kersten et al. 1999, 2002).
8.2.4.3 Known user The test currently established with a common SOP in the five laboratories participating in the formal validation study: Bayer AG (Wuppertal, D), BfArM (Bonn, D), RCC-Cytotest Cell Research (Rodorf, D), University of Mainz (D), Hoffmann-La-Roche (Basel, CH).
8.2.4.4 Status of validation and/or standardisation The standardised P-MNT protocol of Kersten et al. (1999) is currently under formal validation since early 2003. The study is co-ordinated by the BfArM in Bonn and independently coached by ZEBET at the BfR (chemical coding and distribution and biostatistics). It will be finalised end of 2005. 8.2.4.5 Fields and limitations of application The field of the P-MNT is the detection of photo-chemically induced clastogenicity, the most relevant in vitro endpoint for the assessment of photo-carcinogenic potential. So far, limitations are not known test results seem to independent from the cell lines (or primary cells) used. The limitation of the alternative photo-clastogenicity assay, the P -CAT (high costs of cytogenetic chromosome analysis) is overcome since micronuclei are quicker and
24 / 24 easier to determine as chromosomal aberrations. The test bears possibilities for apparatussupported analysis.
8.2.4.6 Recommendations of use in the view of animal replacement While in vivo photogenotoxicity testing is not required for the safety testing of cosmetics, it is hoped that the current in vitro approaches in photogenotoxicity testing will lead to the definition of an in vitro test battery allowing for an assessment of the likelihood of a compound to turn into a photochemical carcinogen upon excitation and activation with UV or visible radiation. However, while pharmaceuticals with a photocarcinogenic potential may be further investigated in vivo because they can be safely used avoiding any light exposure, chemicals with a photocarcinogenic potential will not be acceptable as cosmetic ingredients, so that in vivo testing will not be required in that area.
8.2.4.7 On-going development Apart from the current formal interlaboratory Validation Study, on-going developments of the P-MNT are not known. However, similar activities like the German/Swiss interlaboratory trial may be under way elsewhere.
8.2.4.8 Efforts needed to complete validation of the method Finalisation of the current formal Validation Study (expected end of 2005).
8.2.4.9 Key references Brendler-Schwaab, S., Czich, A., Epe, B., Gocke, E., Kaina, B., Mller, L.. Pollet, D., Utesch, D. (2004) Photochemical genotoxicity: principles and test methods. Report of a GUM task force. Review . Mutation Res. 566: 6591 Gocke E., Mller L., Guzzie P.J., Brendler-Schwaab S., Bulera S.,. Chignell C.F., Henderson L.M., Jacobs A., Murli H., Snyder R.D., Tanaka N. (2000) Considerations on photochemical genotoxicity: report of the IWGTP working group, Environ. Mol. Mutagen. 35, 173184. Mller L., Kasper P. (1998) The relevance of photomutagenicity testing as a predictor of photocarcinogenicity, Int. J. Toxicol. 17, 551558. Kersten, B., Zhang, J., Brendler-Schwaab, S.Y., Kasper, P. & Mller, L. (1999). The application of the in vitro micronucleus test in Chinese hamster V79 cells to detect druginduced photogenotoxicity. Mutat. Res. 445 (1), 55-71. Kersten, B., Kasper, P., Brendler-Schwaab, S.Y., & Mller, L. (2002) Use of the photomicronucleus assay in Chinese hamster V79 cells to study photochemical genotoxicity. Mutat. Res. 519, 4966 Kalweit S et al. (1999) Chemically induced micronucleus formation in V79 cells comparison of three different test approaches, Mutat Res 439, 183-190. v.d. Hude W et al. (2000) In vitro micronucleus assay with Chinese hamster V79 cells Results of a collaborative study with in situ exposure to 26 chemical substances, Mutat Res 468, 137-163. Snyder R.D., Cooper C.S. (1999) Photogenotoxicity of fluoroquinolones in Chinese hamster V79 cells: dependency on active topoisomerase II, Photochem. Photobiol. 69, 288293.
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8.2.5
DNA damage Indication: The Photo COMET Test (P-COMET)
8.2.5.1 Short description, scientific relevance and purpose The (dark) COMET assay is a method for electrophoretic measurement of DNA strand breaks. The assay can in principle be performed on any eukaryotic cell type and only a small number of cells is necessary. Strand breaks may be introduced directly by genotoxic compounds or through the interaction with reactive oxygen species, or other reactive intermediates. The COMET test is highly sensitive and can detect DNA damage in individual cells. The test is usually performed under alkaline conditions. In the alkaline version of the COMET test, the induction of DNA single strand breaks is measured. The Comet assay was firstly described by Ostling & Johanson (1984), and later further developed by Singh et al. (1988). The protocol of Singh et al. (1988) has been successfully adapted to photo-genotoxicity testing as is described in several publications (Chetelat et al., 1996; Bulera et al., 1999; Nakagawa et al., 1997; Reavy et al., 1997; Marrot et al., 2001), and later during a research project at Bayer AG (Wuppertal, D) supported by the German Ministry of Research and Technology (Brendler-Schwaab et al., 2003). Based on the positive experience gained in this project, the P-COMET was employed in early 2003 in a formal validation study in which six laboratories are evaluating the P-COMET in a blind trial.
8.2.5.2
Developer of the method
Most probably the group of Chelat et al. (1996)
8.2.5.3
Known user
Apart other possible users the test is currently established with a common SOP in the following six laboratories participating in the formal validation study: Bayer AG (Wuppertal, D), BfArM (Bonn, D), RCC-Cytotest Cell Research (Rodorf, D), University of Mainz, Wella Cosmital (CH), Novartis Pharma (Basel, CH).
8.2.5.4
Status of validation and/or standardisation
Even though the P-COMET has recently entered a formal validation study which will under blind conditions produce interlaboratory data with a standard P -COMET protocol, the fact that there are several compounds known for which the P-COMET provides negative predictions, while other phototoxicity or photo-genotoxicity tests are providing positive results, has to be seriously taking into account once the relevance of the P-COMET will be independently assessed. Probably, the P-COMET has entered the formal validation study a bit early, because currently different predictions obtained in the indicator test (P-COMET) and other photogenotoxicity tests (like the P-CA, or P-MNT) are not fully understood.
8.2.5.5
Fields and limitations of application
The field of application of the P-COMET is detection of DNA strand breaks induced by a substance and subsequent/concurrent UV-vis irradiation. The P-COMET is on the one hand an extremely sensitive test, and on the other hand, it has produced negative results with some compounds that are providing positive results in other photogenotoxicity tests like the P-MNT. Such chemicals are for example nalidixic acid and 8-MOP of which only the latter result is understood as DNA-DNA crosslinking under UV light (Brendler-Schwaab et al., 2004).
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8.2.5.6
While in vivo photogenotoxicity testing is not required for the safety testing of cosmetics, it is hoped that the current in vitro approaches in photogenotoxicity testing will lead to the definition of an in vitro test battery allowing for an assessment of the likelihood of a compound to turn into a photochemical carcinogen upon excitation and activation with UV or visible radiation. However, while pharmaceuticals with a photocarcinogenic potential may be further investigated in vivo because they can be safely used avoiding any light exposure, chemicals with a photocarcinogenic potential will not be acceptable as cosmetic ingredients, so that in vivo testing will not be required in that area.
8.2.5.7
On-going development
Apart from the current interlaboratory blind trial, where an agreed P-COMET standard protocol is used, the group is not aware of any other developments.
8.2.5.8
Efforts needed to complete validation of the method
In principle, since the P-COMET is currently evaluated in an inter-laboratory blind trial, the test may, at the expected end of this study (2005) be regarded formally validated. However, before the test can become a recommended part of a photogenotoxicity testing battery, it's relevance and role in a strategy will need further clarification.
8.2.5.9
Key references
Brendler-Schwaab, S., Czich, A., Epe, B., Gocke, E., Kaina, B., Mller, L.. Pollet, D., Utesch, D. (2004) Photochemical genotoxicity: principles and test methods. Report of a GUM task force. Review . Mutation Res. 566: 6591 Bulera, S.J. et al. (1999) In vitro photogenotoxic activity of Clinafloxacin: A paradigm predicting photocarcinogenicity, Tox. Appl. Pharm. 156, 222-230. Chetelat, A-A. et al. (1996) The photomutagenicity of fluoroquinolones in tests for gene mutation, chromosomal aberration, gene conversion and DNA breakage (Comet assay), Mutagenesis 11, 497-504. Gocke E et al. (2000) Considerations on photochemical genotoxicity: Report of the IWGTP working group, Environ Molec Mutagen 35, 173-184. Marrot, L. et al. (2001) Fluoroquinolones as chemical tools to define a strategy for photogenotoxicity in vitro assessment. Toxicology in Vitro 15, 131-142. Nakagawa, Y. et al. (1997) The photogenotoxicity of titanium dioxide particles, Mutation Res. 394, 125-132. Ostling O., Johanson K.J. (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells, Biochem. Biophys. Res. Commun. 123, 291298. Reavy, H.J. et al. (1997) Photogenotoxicity of skin phototumorigenic fluoroquinolone antibiotics detected using the comet assay, Photochem. Photobiol. 66, 368-373. Singh N.P., McCoy M.T., Tice R.R., Schneider E.L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell. Res. 175, 184191. Selvaag, E., Petersen, A. B., Gniadecki, R., Thorn, T. & Wulf, H-C., (2002) Phototoxicity to diuretics and antidiabetics in the cultured keratinocyte cell line HaCaT: evaluation by
27 / 27 clonogenic assay and single cell gel electrophoresis Comet assay). Photodermatol, Photoimmunol & Photomed 18, 90-95.
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8.3
PHOTO-ALLERGY (PHOTO-SENSITISATION)
Testing for photosensitisation potential (immunological photoallergy) is not specifically required for Cosmetic ingredients, but it is nevertheless often performed. For in vivo photoallergy testing, no standard testing protocol exists. Protocol designs frequently used are similar to the Guinea Pig Maximisation Test (GPMT), employing additional UV-vis irradiation (e.g. Guillot & Martini, 1985). In the area of photo-allergy (-sensitisation), like in the area of development of predictive in vitro tests for delayed contact sensitisation (Allergenicity) potential without involvement of light, due to a lacking ability to model the complex mechanisms underlying allergy, currently no promising in vitro methods to predict photo-sensitisation potential are in sight (see chapter on skin sensitisation). One in vitro screening method, which models covalent binding of a light activated chemical to serum albumin, may become relevant. However, while binding of an excited chemical to proteins is a prerequisite of photoallergy, it is not a sufficient standalone predictor, but it can help differentiate photoirritants from photoallergens (Barrat & Brown, 1985; Lovell & Jones 2000). The only promising stand-alone Alternatives currently under development are in vivo refinements, like the Photo Local Lymph Node Assay, P-LLNA (Ulrich et al., 1998; Homey et al., 1998), or a combination of the P-LLNA with the PhotoMouse Ear Swelling test, P-MEST (Vohr et al., 2000). The ECVAM Workshop Report 42 (Spielmann et al., 2000) is still correctly covering the actual status of these methods in detail. Once a reliable and predictive in vitro test battery and strategy for the assessment of dark sensitisation potential will be developed and accepted, adaptation of the test battery into similar photo-sensitisation tests is considered possible. Because at present, serious predictions on the availability of in vitro alternatives for this endpoint cannot be made, and because, formally for cosmetics testing of photoallergenic potential is not required, this chapter is kept as a summary without sub-chapters.
Refernces
Spielmann, H., Mller, L., Averbeck, D., Balls, M., Brendler-Schwaab, S., Castell, J.V., Curren, R., de Silva, O., Gibbs, N.K., Liebsch, M., Lovell, W.W., Merk, H.F., Nash, J.F., Neumann, N.J., Pape, W.J.W., Ulrich, P. and Vohr, H.-W. The Second ECVAM Workshop on Phototoxicity Testing. The Report and Recommendations of ECVAM Workshop 42. ATLA 28, 777-814, 2000 Guillot JP, Martini MC (1985), A new method for the assessment of phototoxic and photoallergic potnetials by topical applications in the albino guinea pig. J. Toxicol. - Cut. & Ocular Toxicol. 4, 117-133. Homey, B., von Schilling, C., Bluemel, J., Schuppe, H.C., Ruzicka, T., Ahr, H.J., Lehmann, P. & Vohr, H.W. (1998). An integrated model for the differentiation of chemical-induced allergic and irritant skin reactions. Toxicology and Applied Pharmacology 153 (1), 83-94. Ulrich, P., Homey, B. & Vohr, H.W. (1998). A modified local lymph node assay for the differentiation of contact photoallergy from phototoxicity by analysis of cytokine expression in skin-draining lymph node cells. Toxicology 125, 149-168. Vohr, H.-W., Blmel, J., Blotz, A., Homey, B. & Ahr, H.J. (2000). An intra-laboratory validation of the Integrated Model for the Differentiation of Skin Reactions (IMDS): discrimination between (photo)allergic and (photo)irritant skin reactions in mice. Archives of Toxicology 73 (10-11), 501-509. Barratt, M.D. & Brown, K.R. (1985). Photochemical binding of photoallergens to human serum albumin: a simple in vitro method for screening potential photoallergens. Toxicology Letters 24, 1-6. Lovell, W.W. (1993). A scheme for in vitro screening of substances for photoallergenic potential. Toxicology in Vitro 7, 95-102.
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Alternative tests available 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU-PT)
In vitro endpoints measured
Purpose
Validation status
Comments
Use adjunct tests to overcome limitations (fairly low UVB tolerance, inability to model the bioavailability of test materials topically applied to the skin)
Photocytotoxicity
Validated (ESAC endorsement ) Validated (no peer review or ESAC statement) prevalidated (ECVAM) Feasibilty study (photopotenc y /photosafety test) under way
EC Annex V B.41, OECD TG 432
0 years
Acute phototoxicity (no specific animal test required)
Red Blood Cell Phototoxicity Test (RBC PT)
Photohaemolysis, haemoglobin oxidation
RBC PT can be used at high doses of UVB
Valid adjunct test to the 3T3 NRU PT Not a stand alone test Valid adjunct test to the 3T3 NRU PT
1 year
Human 3-D skin model in vitro phototoxicity test (H3D PT)
Skin cell viability
Use when 3T3NRU PT is (weakly) positive and bioavailability in the skin is questioned
? Not a stand alone test
Photo-chemical genotoxicity (no specific animal test required)
Photo bacterial reverse mutation test
Gene point mutations
General
Optimised
None, but accepted by some authorities
Use in combination with other endpoints such as chromosomal aberration, as a part of a battery
If at all necessary: Formal validation: 3-5 years
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Photo Mammalian cell gene mutation test (P-HPRT and P-MLA) Photo Chromosome Aberration test (P-CAT)
Gene point mutations
General
Optimised
None, but accepted by some authorities None, but accepted by some authorities
Rarely used in standard battery, additional to bacterial mutations Use in combination with other endpoints such as gene mutations, as a part of a battery Use in combination with other endpoints such as gene mutations, as a part of a battery as replacement of the P-CAT Screening purposes and mechanistic research
If at all necessary Formal validation: 4-6 years
Chromosomal aberrations
General
Optimised
Weight-of evidence validation: 2-4 years
Photo Micronucleus test (P-MNT)
Chromosomal aberrations (including numerical aberrations)
General
Under validation (BfArM and ZEBET)
3 years
Photo Comet assay (P-COMET) Photo-Local Lymph Node Assay (P-LLNA)
DNA damage Photochemically increased proliferation of lymphocytes in draining lymph node
General
Under validation (BfArM and ZEBET)
3 years
Refineme nt
General
Optimised
Photo-allergy (-sensitisation) (no specific animal test required)
In vitro tests
Progress will depend on progress in the field of skin sensitisation
Once a valid in vitro approach (e.g. test battery) for skin sensitisation exists, adapt the tests to be performed with UVAvisible irradiation
like Skin Sensitisation plus 3 years for adaptation to photo-sensib.
8-15 years for in vitro tests
* This table estimates the time needed to achieve ESAC endorsement for individual alternative tests assuming optimal conditions. It does not indicate the time needed to achieve full replacement of the animal test, nor does it include the time needed to achieve regulatory acceptance. Optimal conditions means that all
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necessary resources, for example technical, human, financial and coordination are met at all times in the process and that the studies undertaken have successful outcomes.
Ispra, Italy, 11 March 2004
Final Document
Subgroup of Experts on Toxicokinetics and Metabolism in the context of the follow-up of the 7th Amendment on the Cosmetics Directive
Sandra Coecke1, Bas J. Blaauboer2, Greetje Elaut3, Stuart Freeman 4, Andreas Freidig5, Nigel Gensmantel6, Peter Hoet7, Vassilios M. Kapoulas8, Bernhard Ladstetter9, Gill Langley10, David Leahy11, Geert Mannens12, Annarita Meneguz13, Mario Monshouwer14, Ben Nemery7, Olavi Pelkonen 15, Walter Pfaller16, Pilar Prieto1, Nick Proctor17, Vera Rogiers18, Amin Rostami-Hodjegan 17, Enrico Sabbioni1, Winfried Steiling19, J.J.M. van de Sandt5
ECVAM- JRC, Ispra, Italy; 2Institute for Risk Assessment Sciences (IRAS), Utrecht 3 University, Utrecht, The Netherlands; V.U.B., Brussels, Belgium; 4 GLAXOSMITHKLINE Consumer Healthcare, Weybridge, Surrey, UK; 5TNO Nutrition and Food Research, Zeist, The Netherlands; 6AstraZeneca, Loughborough, Leics, UK; 7KU Leuven, Leuven, Belgium; 8Halandri, Greece; 9Merck KgaA, Grafing, Germany; 10Dr Hadwen Trust, Hitchin, Herts, UK; 11Cyprotex PLC, Macclesfield, Cheshire, UK; 12Janssen Pharmaceutica, Beerse, Belgium; 13Instituto Superiore di Sanit, Roma, Italy; 14Pharmacia, Gruppo Pfizer Inc., Nerviano (MI), Italy; 15 16 University of Oulu, Finland; University of Innsbruck, Innsbruck, Austria; 17 University of Sheffield, Sheffield, England; 18V.U.B., Brussels, Belgium; 19Henkel KgaA, Dusseldorf, Germany
Table of Content
Objectives to be achieved...3 1. Inventory of the methods currently available...3 2. Inventory of the alternative methods currently available6 2a. Introduction.6 2b. Tier 1 and Tier 2 stand-alone methods...10 2c. Tier 1 and Tier 2 integrated in vitro and in silico approaches (some examples)...31 2d. Integrated human volunteer-based approaches....40 3. Identified steps or tests with no alternative methods available...42 3a. Tier 1.42 3b. Tier 2.42 4. Summary of the alternative methods currently available and the foreseeable time to achieve peer reviewed validation....44 5. Recommendations.....50 6. Conclusions ...51
Objectives to be achieved
Amongst the key provision of the 7th Amendment on the Cosmetics Directive, it is stipulated that the commission should provide an estimated timetable for the provision of alternative methods to determine all necessary toxicological endpoints. To deal with the bio-availability and biotransformation of applied chemicals the subgroup on Toxicokinetics and Metabolism was formed with experts nominated by the different stakeholders involved (European Cosmetic Toiletry and Perfumery Association, COLIPA; The European Centre for the Validation of Alternative Methods, ECVAM, European Federation for Cosmetic Ingredients, EFfCI; The European Coalition to End Animal Experiments; The Scientific Committee on Cosmetics and Non-Food Products, The European Organisation of Cosmetic Ingredients Industries and Services, UNITIS - natural origin ingredients, DG ENV, DG RES, DG SANCO, OECD). During the process additional experts were consulted on specific topics essential for the finalisation of the document. Furthermore, a tiered strategy was proposed indicating the necessity to interact with the other expert subgroups involved in this exercise. The overall objective was to produce a document that would be the basis for the establishment of realistic timetable for phasing out animal experimentation in order to comply with the current legislative requirements, but without compromising the level of safety for the consumers of such cosmetic ingredients.
1. Inventory of the methods currently available

The term toxicokinetics is used in a regulatory environment to describe the bio-availability of a substance and its kinetic and metabolic fate within the body under the conditions of a toxicological trial. This includes absorption, distribution, metabolism and/or excretion. NOTE: The term toxicokinetics is used throughout this document as the subgroup understands that this term is used in the regulatory context of the 7th Amendment of the Cosmetics Directive and the future New Chemicals Policy of the European Commission. The term toxicokinetics is used for describing the kinetic processes of absorption, distribution, metabolism and excretion of a compound in an organism in the context of toxicological trials. Some of the experts of the subgroup would preferentially use the terminology "kinetics" or "biokinetics" instead of "toxicokinetics", since the term toxicokinetics" is sometimes also used with a different meaning. However, the subgroup understands that the term toxicokinetics is historically the terminology used in the regulatory context.
The toxicokinetic studies, as described in the different existing internationally accepted test guidelines are designed to obtain species-, dose-, and route-dependent data on the concentration-time course of parent compound and its metabolites, e.g. in blood, urine, faeces and exhaled air. From these data, kinetic parameters can be derived by appropriate techniques. The information that can be taken from the current regulatory in vivo/ex-vivo toxicokinetic studies is: Primary information: ? ? The concentration-time profile of the substance/metabolites in blood (plasma), exhaled air, tissues and other biological fluids, such as urine, bile and the volume of the excreted fluids ? ? If appropriate, protein binding and, if relevant, binding to erythrocytes (in vitro/ex vivo studies).
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Derived information: ? ? Rate and extent of absorption and bioavailability ? ? Distribution of the substance in the body ? ? Biotransformation ? ? Rate and extent of presystemic (first pass) and systemic metabolism after oral, dermal and inhalation exposure. Certain tests for studying toxicokinetics of substances in vivo are described in Annex V to Directive 67/548. As stated in the OECD guideline 417 on Toxicokinetics: "Flexibility, taking into consideration the characteristics of the substance being investigated is needed in the design of toxicokinetic studies." Therefore, the objective of this subgroup on Toxicokinetics and Metabolism is, to list alternative approaches including predictive and modeling approaches, data gathered from in vitro test systems and data gathered from studies in vivo, including humans and to proceed via a tiered test strategy incorporating alternative test systems. With the exception of dermal absorption, detailed data on both, the toxicokinetics as well as on the metabolism of cosmetic ingredients is currently of less importance. Such additional information is only required for cases where specific effects, seen in standard in vivo tests, have to be clarified and their relevance for humans has to be proven. The situation will become quite different, when for example sub-chronic toxicity tests or even reproductive toxicity studies should be replaced by in vitro methods according to the 7th Amendment on the Cosmetic Directive. Due to the complexity of physiological regulations and impact networks within a living animal, it is expected that a battery of different in vitro methods would be necessary to answer all the questions on specific organ toxicity. Under such new circumstances, toxicokinetic and metabolism/biotransformation would become a predominate information for the safety assessments of cosmetic ingredients and vital for designing the most valuable set of in vitro toxicity tests. In this context, it is crucial to mention, that only in cases where a certain cosmetic ingredient becomes bio-available after oral or dermal application or inhalation, further toxicity tests, targeting others then local effects, are officially required. It is currently under discussion to define on an international level the threshold of toxicological concern, probably for specific chemicals. Several in vitro procedures have reached the level of OECD guidelines. Dermal absorption in vitro (OECD 428, draft guideline due for adoption 1 February 2004) where the principles of this method are required and accepted by the SCCNFP (SCCNFP/0750/03, Final); in vitro skin corrosion - transcutaneous electrical resistance test (OECD 430, draft guideline due for adoption 1 February 2004); in vitro skin corrosion - human skin model test (OECD 431, draft guideline due for adoption 1 February 2004); and in vitro 3T3 NRU phototoxicity test (OECD 432, draft guideline due for adoption 1 February 2004). The two in vitro skin corrosion tests are in the EU Annex V (B40), as well as the in vitro phototoxicity test (B41). Additionally, three in vitro embryotoxicity tests have been fully validated by ECVAM but did not arrive to regulatory acceptance due to the absence of a the biotransformation aspect in the test system set-up. Beside the officially accepted test methods, other test systems to measure the bio-availability and the in vitro biotransformation are available and routinely used for specific in-house interests. For some of them, extensive sets of data are available, demonstrating their importance to build up a certain picture of specific qualitative and quantitative information. Regulatory authoritities have recognized that in vitro systems are extremely helpful in addressing potential biotransformation-related issues during the drug development phase. The application of in vitro systems for biotransformation, like e.g. microsomal preparations or isolated hepatocytes, has been described in guidance documents on studies of drug-drug
interaction by US (U.S. FDA-CDER, 1997) and European authorities (EMEA, 1997). The main objective of the guidance documents is to provide suggestions on current approaches to study in vitro interactions and is intended to encourage routine, thorough evaluation of interaction in vitro whenever feasible and appropriate. Absence of a finding that a drug has an important inhibition liability may obviate the need for further clinical investigations or at least help to focus the design of these studies. These in vitro methods from drug-drug interactions have contributed significantly to reduce the risk of a severe side effect due to inhibition. In the document several in silico methods will be described. In general, the kinetics of a compound will be a function of two major sets of determinants: - the physiological properties of the organism in which the kinetics will be studied; - the structural properties of the compound under study. For the latter set of determinants there are possibilities to quantify certain structural properties and relate these to biological activity. This is the basis of the socalled Quantitative Structure-Activity Relationships (QSARs), that are used in describing the biological (or toxicological) activity of a compound in a certain system (e.g. an organism). Likewise, a compound's physico-chemical properties can be used to estimate other properties than biological activity. An example is the use of such properties (e.g. lipophilicity) to estimate blood-tissue partitioning. Thus, here we quantify a physico-chemical property (logPoct) and relate it to a kinetic property: partitioning. On the basis of this a QuantitativeStructure-Property Relationship (QSPR) may be constructed. The main aim of all hazard and risk assessment approaches is to assess human health effects. Ideally, one would say that in silico and in vitro methods should model human toxicokinetic processes and use human cells and/or tissues, respectively. However, due to the limited availability of human cells and tissues, and the ethical concerns which are often raised, other approaches are being developed. In order to avoid any need for species extrapolation, it was highly recommended by the subgroup to use those models which obtain the information relevant for human hazard assessment. In some cases animal derived cells and tissues can comply with this objective, in other cases such as for example Phase I and Phase II biotransformation pathways, species differences are well known and specified. In the latter cases it is essential to build in strategies and approaches that take this aspect into account and use approaches that ultimately be relevant for what is happening in the human body. It has to be noted that some aspects such as human genetic polymorphisms of biotransformation enzymes are not covered in conventional toxicological animal approaches. Research and development efforts based on transgenic cells in vitro are a first step in trying to pick up some well-know genetic polymorphism. This information might be useful for a risk assessor but needs more efforts in order to be ready for incorporation in a toxicokinetics tiered strategy. The issue is of importance for drug development and therapy, but on the role with respect to cosmetics (or chemicals in general), less data are available. This issue is predicted to be taken up rather late in a tiered approach. There is no need to analyse the biotransformation of a certain cosmetic ingredient, if this chemical would not become bio-available or even if there is no toxicological relevance according to the proposed tiered approach. On the other hand it was stated that a detailed understanding of the metabolic competence of the barriers used in Tier 1 and its relevance for the human situation could be a selection criteria to be used in selecting the in vitro models. Special barriers such as the blood-brain barrier, the blood-testis barrier and the placenta barrier were considered to be of minor importance in the context of cosmetics. An interaction with the other subgroups would be helpful for clarifying this matter. In fact, in the reproductive toxicity subgroup there was a clear demand on this matter and future interaction with the subgroup on toxicokinetics and metabolism.
2. Inventory of the alternative methods currently available 2a. Introduction

Today, the following set of toxicological data, conventionally obtained from in vivo experiments, is necessary for the safety assessment of cosmetic ingredients: acute toxicity, local compatibility on skin and eyes, mucosal irritation and skin sensitisation, genotoxicity/mutagenicity, photo-induced toxicity, reproductive toxicity, sub-chronic toxicity, carcinogenicity, dermal absorption, toxicokinetics and human data (SCCNFP 0690/3). To be able to perform a safety assessment including the calculation of the margin of safety (MOS) data on the bioavailability after dermal application is required for cosmetic ingredients mentioned in the Annexes of the Cosmetic Directive. New approaches are based on inclusion of more in silico and in vitro approaches. In the area of genotoxicity/mutagenicity, in vitro test are regularly performed and many of them are officially accepted by the NGOs. To integrate information on the toxic potential of a compound, co-operation with the other subgroups is vital. The contribution of the subgroup on Toxicokinetics and Metabolism is focused on tests included in a tiered-approach concept. Furthermore, this subgroup is cross-cutting since it can provide information for the other subgroups. Data can be provided to indicate if biotransformation has to be considered in the potency test taken up by the other subgroups such as carcinogenicity and reproductive toxicity. An approach based on three Tiers was followed. Tier 1 includes batteries of in vitro and in silico tests which indicate the likelihood of a compound to have systemic exposure. Tier 2 uses test batteries to determine the distribution of a compound after systemic exposure. Tier 3 determines the overall potency of a compound by an integration of potency and toxicokinetic and biotransformation tests. The group listed the stand-alone tests that are the building blocks for each Tier. Furthermore, already existing integrated approaches are described. These integrated in silico and in vitro approaches provide combined information for Tier 1 and Tier 2. A human volunteer-based integrated approach was identified which might lead, in combination with the other in vitro and in silico tools, to a new way of doing human hazard assessment in a regulatory environment.
Tier 1: Likelihood to have systemic exposure Tier 1 is related to the intended use of the product containing the relevant cosmetic ingredient:
?? Dermal absorption (? g/cm as currently recommended by the SCCNFP) after dermal
exposure
?? Resorption from the gastro-intestinal-tract (GI) in case of oral application ?? Bio-availability when inhaled
The overall objective of this Tier is to obtain a universal measure of passage through barriers (e.g. mmol/kg/application), in order to determine the likelihood to have systemic exposure. This first value is a first decision point indicating the need to go further to Tier 2.
Tier 2: Determination of the distribution of the compound The two main objectives of this Tier are (1) to make an estimation of the bioavailability of the parent compound and its relevant metabolites by estimation of plasma level, bio-
accumulation and biotransformation and (2) to generate information on toxicokinetics and biotransformation for use in the potency tests (acute toxicity, skin irritation, eye irritation and mucous membrane irritation, skin sensitisation, subacute and subchronic toxicity, genotoxicity / mutagenicity, UV-induced toxic effects including phototoxicity, photoallergy and photogenotoxicity, carcinogenicity, reproductive and developmental toxicity).At this level in silico and/or in vitro test systems are necessary to determine the likelihood of bioaccumulation in the body. Modelling tools, physico-chemical data, read-across approaches, QS(P/A)R approaches play an important role in this tier. The subgroup recommended to use preferentially in silico approaches in this Tier. Additional in vitro approaches (not listed) can be used to determine active transport following a new decision point (e.g. bood-brain barrier/BBB, placental barrier). In silico and in vitro methods for determining biotransformation-related effects are part of this Tier. At this stage it is not necessary to identify all metabolites of the parent compound. Simple screenings are recommended. The information obtained can be used in order to decide if it is necessary to add or integrate a metabolic competence to the in vitro systems which will be used for the potency testing in Tier 3.
Tier 3: Determination of the potency of a compound This tier will include a base-set of tests including tolerability assays (skin and eye irritation, skin sensitisation, phototoxicity) and the mutagenicity/genotoxicity tests which are mandatory. More specific information on specific target tissue concentration, driven by results obtained from the other toxicity areas can be provided from the results obtained in Tier 1 and Tier 2. In these cases, the method of choice is kinetic-based modelling with/or without inclusion of specific in vitro barrier tests (e.g. BBB).
Overview of the methods in Tier 1, Tier 2 and Tier 3
Tier 1: Likelihood to have systemic exposure

Dermal route
1. Dermal absorption tests 2. QSPR approaches 3. Kinetic modelling
Oral route
4. Cell based assays (Caco-2 and TC-7 cells) and artificial membranes
Inhalation route
5. QSPR approaches/physi cochemical properties Peter Hoet, Sandra Coecke, Nigel Gensmantel 6. Pulmonary epithelium (A549, Calu-3, BEAS-2B , 16HBE14o- and primary cells) Peter Hoet, Sandra Coecke, Olavi Pelkonen
See Chapter 3.5
Sandra Coecke (*IA), Nigel Gensmantel
Sandra Coecke (*IA), Nigel Gensmantel
Pilar Prieto, Mario Monshouwer & Review Bernhard Ladstetter
Tier 2: Determination of the distribution of the compound

Estimation of plasma level
7. QSPR approaches/phy sicochemical properties 8. Plasma protein binding 9. Kinetic modelling
Excretion
No final in vitro test battery available as replacement to the in vivo excretion models
Bio-accumulation
10. QSPR approaches/physicoc hemical properties 11. Blood/tissue partitioning
Biotransformation (metabolism)
12. Metabolic stability & species differences 13. Bioactivatio n assays 14. In silico approaches
Sandra Coecke (*IA), Nigel Gensmantel Standard information from available guidelines (e.g. OECD)
Mario Monshouwer& Sandra Coecke (*IA) & Review Bernhard Ladstetter & Geert Mannens
Gill Langley (*IA) & Sandra Coecke (*IA)
Walter Pfaller, Pilar Prieto, David Leahy, Mario Monshouwer, Geert Mannens
Sandra Coecke (*IA), Nigel Gensmantel,Gill Langley (*IA) Standard information from available guidelines (e.g. OECD)
Sandra Coecke (*IA) & Han van de Sandt & Andreas Freidig
Olavi Pelkonen& Sandra Coecke (*IA) & Review Bernhard Ladstetter & Greetje Elaut & Vera Rogiers & Geert Mannens
Sandra Coecke& Review Greetje Elaut & Vera Rogiers & Geert Mannens
Olavi Pelkonen & Gill Langley (*IA) & Sandra Coecke (*IA) & Nigel Gensmantel & Geert Mannens
Tier 3: Determination of the potency of a compound

acute toxicity skin irritation eye irritation (currently eye irritation is taking similar to irritation on mm) skin sensitisation subacute and subchronic toxicity genotoxicity / mutagenicity, photo-induced toxic effects including phototoxicity, photoallergy and photogenotoxic ity, subgroup UVinduced toxic effects including phototoxicity, photoallergy and photogenotoxic ity,and subgroup toxicokinetics and metabolism carcinogenicity reproductive and developmental toxicity
subgroup acute toxicity and subgroup toxicokinetics and metabolism
subgroup skin irritation and subgroup toxicokinetics and metabolism
subgroup eye irritation and mucous membrane irritation and subgroup toxicokinetics and metabolism
subgroup skin sensitisation and subgroup toxicokinetics and metabolism
subgroup subacute and subchronic toxicity and subgroup toxicokinetics and metabolism
subgroup genotoxicity / mutagenicity,an d subgroup toxicokinetics and metabolism
subgroup carcinogenicity and subgroup toxicokinetics and metabolism
subgroup reproductive and developmental toxicity and subgroup toxicokinetics and metabolism
*IA: integrated approach
2b. Tier 1 and Tier 2 stand-alone methods

Tier 1 Oral route 4. Cell based assays (Caco-2 and TC-7 cells) and artificial membranes Responsible: Pilar Prieto, Mario Monshouwer & review Bernhard Ladstetter Short description, scientific relevance and purpose Caco-2 is a human adenocarcinoma cell line widely used as an in vitro model of the intestinal barrier. In culture the cells form monolayers of polarised enterocytes, which present apical and basolateral membranes and well-differentiated brush borders. Differentiation process starts after 7 days of confluence in standard culture conditions and is complete after 14-21 days. They have high electrical resistance. They express typical membranous peptidases and disaccharidases of the small intestine. They express active transporters (e.g. amino acids, sugars, vitamins, hormones), membrane ionic transporters (Na+/K+ ATPases, H+/K+ ATPases, Na+/H+ exchange, Na+/K+/Cl- co-transport, apical Clchannels), membrane non-ionic transporters (permeability-glycoprotein, multidrug resistant associated protein, lung cancer associated resistance protein), and receptors (vitamin B12, vitamin D3, epithermal growth factor, GLUT1, GLUT3, GLUT5, GLUT2, SGLT1). The human origin of this cell line avoids the animal inter-species differences concerning the morphological and physiological features of intestinal cells. It is a relatively fast, simple and flexible method. Permeability data obtained with the Caco-2 cells monolayers are use to predict qualitatively absorption in humans. It is a useful model to rank compounds according to their permeability. Caco-2/TC-7 is a monolayer subclone of the parental Caco-2 cell line. It was isolated from a late passage of Caco-2 cells by the limited dilution technique. Characteristics between both cell lines are comparable, however, combining biochemical analysis and morphological characterization it seems that TC-7 clone is closer to normal enterocytes as compared to parental Caco-2 cells, both for the structure of the brush-border and for the expression level of some hydrolases (sucrose-isomaltase, aminopeptidase N, dipeptidylpeptidase IV) and sugar transporters (SGLT1, GLUT5) associated with the brush-border. As for Caco-2 cells the TC-7 cells also need 14-21 days in standard culture conditions for differentiation.TC-7 cells offer marked advantages over the parental Caco-2 cells, because they express CYP3A4, actively transport taurocolic acid, and have lower levels of Pgp compared with the parental Caco-2 cells. TC-7 transepithelial resistance values are lower than Caco-2 cells and therefore closer to the small intestine. They also exhibit a much higher activity for 1-naphtol glucuronidation than the parental cell line. TC-7 culture model provide qualitative information on compounds interactions involving CYP3A4 and Pgp. This information could be of outmost importance in drug discovery programmes. It is confirmed that the TC-7 clone is a good model for studying P-gp interactions but that is of less value with regard to CYP3A4/5 interactions due to the low expression level. Even for qualitative CYP3A4/5 interactions liver microsomes or supersomes are the better model. Parallel artificial membrane permeation assay (PAMPA) is a relative new in vitro high throughput non cell based assay for measuring drug
10
permeability using artificial membranes. As PAMPA is an application of a filter supported lipid membrane and is completely without pores and active transport systems, this assay can only be used to characterize passive, transcellular permeability properties of chemicals and drugs. Although this might be seen as a disadvantage of PAMPA, it is worth to mention that the majority of drugs (>80%) enter the blood stream by passive diffusion through the intestinal epithelium. Recently, good correlations between artificial membrane permeability values and percentage absorption in humans have been observed for compounds for which the absorption was dictated by permeability. Developer of the method The Caco-2 model was first established by Fogh and co-workers in 1977. Since then, this in vitro model has been characterised and widely used by several research groups. In a personal communication Hans Lennernas said that FDA is interested in the characterisation and standardisation of Caco-2 in vitro models and encourages the production of guidelines on the use of this cell line in different disciplines. The group of Dr. Monique Rousset has obtained Caco-2/TC-7 clone. This research group has a broad experience in the field of intestinal barrier models. Dr. Annalaura Stammati, Dr. Monique Rousset , Dr. Maria Laura Scarino, Dr. Gerard Fabre, Dr. Flavia Zucco, Dr, Gianni dal Negro and Dr. Heide Cross have participated in the ECVAM study in the optimisation of an intestinal in vitro model. All of them have broad expertise in using Caco-2 for many years. PAMPA was first described in the literature by Kansy et al in 1998, from F.Hoffmann-La Roche Ltd. Basel, Switzerland. Since then many others have further used and explored this technology to determine permeability. Known users Pharmaceutical industry, several research laboratories and contract research organisations (CROs). The use of artificial membranes is a rapidly expanding amont all major pharmaceutical industries. Status of validation and/or standardisation Cell based assay ECVAM has funded a study (17299-2000-12 F1ED ISP IT) on the development and refinement of an in vitro Caco-2 cell model of intestinal barrier function. The final report of the study is foreseen in January 2004. The main objectives were a) to define the characteristics of the parenteral Caco-2 cell line and of the several clones (Caco-2/TC-7, Caco-2/AQ, Caco-2/15) and set the minimal requirements for their reliable use for permeability/absorption, metabolism/biotransformation, and toxicity; b) to identify the best strains or clones for specific applications and related parameters; c) to elaborate specific Standard Operating Procedures and d) to establish the relative influence of the tumoral origin of these cells with respect to their capability to differentiate. The model is well standardised and optimised and ready for prevalidation. Artificial membranes
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Pharmaceutical companies are using artificial membranes as a screening tool within drug discovery to measure permeability. The standardisation and validation has been mainly performed internally. Several papers describing the internal validation have been published. The fact that this assay is a non-cell based assay, will facilitates future standardisation and validation procedures. Fields and limitations of application, i.e., in vivo endpoints and/or chemical classes covered/not covered Caco-2 cell line is well accepted for absorption studies. It is suitable for automation and high-throughput systems. The model is also applied in inflammation studies. The in vitro measurements of the trans-epithelial permeability coefficient can be used to predict the oral absorption of compounds. If a sufficient set of compounds with known extents of absorption is used for standardisation of the model at least semi-quantitative figures like high, medium, low absorption can be derived. The Caco-2 and Caco-2/TC7 in vitro models lack globet cells (representing the second most frequent cell type in the intestine) and therefore the mucus layer that in vivo seems to act as a barrier. This can limit absorption of lipophilic compounds. CYP3A4 is very weakly expressed in Caco2 cells. They express P-glycoprotein (Pgp) at levels higher than those found in vivo and therefore the interpretation of transport data are not always in agreement with the in vivo situation. The tightness of the monolayer (high electrical resistance) resembles more to colonic than small intestine tissue, resulting in poor permeabilities for hydrophilic compounds traversing the epithelium via the aqueous paracellular pathway. Another limitation of the model is that permeabilities of compounds that are transported via carrier-mediated absorption are lower in the Caco-2 model as compared to the human small intestine. Redox cycling enzymes are well characterised in Caco-2 cells. Heterogeneity of cell population. Artificial membranes are used to model to address the potential absorption of chemicals. It is extremely suitable for automation and highthroughput (96-well plate format). Different artificial membranes can be used to mimic different barriers (e.g. gut wall, blood brain). The system does not allow to characterize permeability due to other mechanisms, such as active uptake or paracellular transport. The fact that this is a noncell based assay, will avoid the potential problem of cytotoxicity of the chemical under evaluation. Recommendations of use in the optics of animal replacement The described in vitro test systems should be used as part of a tiered testing strategy to predict oral absorption and the likelihood of systemic human exposure. This tiered testing strategy including the in vitro systems described should replace classical bioavailability studies in animal On-going development Further characterisation of transporters and biotransformation in Caco-2 and Caco-2/TC 7 cells. Miniaturisation of transport systems. Evaluation of other membrane compositions to improve predictive capacity Efforts needed to complete validation of the method For both the cell based assays (Caco-2 and Caco.2/TC7 cells) and the artificial membranes, further efforts are needed to assess the predictive capacity of these in vitro systems. Expanding the chemical space by not using solely pharmaceutical classes is recommended. Key references
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1. Bergstrom CA, Strafford M, Lazorova L, Avdeef A, Luthman K and Artursson P (2003) Absorption classification of oral drugs based on molecular surface properties. J Med Chem 46, 558-70. 2. Chantret I., Rodolosse A., Barbat A., Dussaulx E., Brot-Laroche E., Zweibaum A., Rousset M. (1994). Differential expression of sucroseisomaltase in clones isolated from early and late passages of the cell line Caco-2: evidence for glucose-dependent negative regulation. 3. Fogh J., Wright WC., Loveless JD. (1977). Absence of Hela cell contamination in 169 cell lines derived from human tumors. J. Natl. Cancer Inst. 129, 1269-1277. 4. Gres M.C., Julian B., Bourrie M., Meunier V., Roques C., Berger M., Boulnec X., Berger Y., Fabre G. (1998). Correlation between oral drug absorption in humans, and apparent drug permeability in TC7 cells, a human epithelial intestinal cell line: comparison with the parenteral Caco-2 cell line. Pharmaceutical Research 15, 726-733. 5. Kuresh A.Y., avdeef A., Abbott N.J. (2003). In vitro trans-monolayer permeability calculations: often forgotten assumptions. Drug Discovery Today 21, 997-1003. 6. Le Ferrec E., Chesne C., Artursson P., Brayden D., Fabre G., Gires P., Guillou F., Rousset M., Rubas W., Scarino M-L. (2001) In vitro models of the intestinal barrier. The report and recommendations of ECVAM workshop 46. ATLA 29, 649-668. 7. Li A.P. (2001) Screening for human ADME/Tox drug proteins in drug discovery. Drug Discovery Today 6, 357-366. 8. Pelkonen O., Boobis RB., and Gundert-Remy U. (2001) In vitro prediction of gastrointestinal absorption and bioavailability: an experts meeting report, European Journal of Clinical Pharmacology 57, 621-629. 9. Tavelin S, Taipalensuu J, Soderberg L, Morrison R, Chong S and Artursson P (2003) Prediction of the oral absorption of low-permeability drugs using small intestine-like 2/4/A1 cell monolayers. Pharm Res 20, 397-405. 10. Kansy, M., Senner, F., Gubernator, K. (1998) Physicochemical high throughput screening: parallel artificial membrane permeation assay in the description of passive absorption processes. J. Med. Chem. 41(7), 1007-1010. 11. Sugano, K., Takata, N., Machida, M., Saitoh, K., Terada, K. (2002) Prediction of passive intestinal absorption using bio-mimetic artificial membrane permeation assay and the paracellular pathway model. Int. J. Pharm. 241(2), 241-251.
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Tier 1 Inhalation route 5. QSPR approaches/physicochemical properties Responsible: Sandra Coecke (information provided by Prof. Ben Nemery and Dr. Peter Hoet) Short description, scientific relevance and purpose Computational modelling techniques using a systems biology approach are particularly useful in reducing the uncertainties by identifying and studying various physical and biological parameters involved in the exposure-dose-response paradigm. Models to predict gas uptake (and particle deposition) in the upper respiratory tract (URT) and lower respiratory tract (LRT) of humans and laboratory animals. Developer of the method Many general and inhalation specific systems exist. Some ref. are included at the end of section. Known users The pharmaceutical industry has taken up some of the approaches. Status of validation and/or standardisation The authors need more time and input from outside to verify the validation. Most systems underwent self-validation. Fields and limitations of application, i.e., in vivo endpoints and/or chemical classes covered/not covered Most systems are constructed to evaluate some specific groups of products, although by using the right parameters several systems can be adapted towards more general Recommendations of use in the optics of animal replacement The authors question if in view of this Tiered approach it is necessary to develop a separate PQPK-system for inhalation; a good general (integrated) system (including skin, lung and intestines) for chemical compounds will do the job in one go. For particle deposition in the lung probably a separate system is needed. The authors believe that systems for particle deposition must exist. On-going development To improve the QSPR approaches, data input and quality of the data input will be a challenge for the future and can be combined with the advanced made in the cellular systems developed in parallel. Efforts needed to complete validation of the method The major effort to be done is to group different systems (integrate), and validate (and possibly optimising) by comparing to existing data.
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Key references 1. Andersen,M.E., Green,T., Frederick,C.B., & Bogdanffy,M.S. Physiologically based pharmacokinetic (PBPK) models for nasal tissue dosimetry of organic esters: assessing the state-of-knowledge and risk assessment applications with methyl methacrylate and vinyl acetate. Regul. Toxicol. Pharmacol. 36, 234-245 (2002). 2. Blaauboer,B.J. The necessity of biokinetic information in the interpretation of in vitro toxicity data. Altern. Lab Anim 30 Suppl 2, 85-91 (2002). 3. DeJongh,J., Verhaar,H.J., & Hermens,J.L. Role of kinetics in acute lethality of nonreactive volatile organic compounds (VOCs). Toxicol. Sci. 45, 26-32 (1998). 4. Frederick,C.B. Summary of panel discussion on the 'advantages/limitations/uncertainties in the use of physiologically based pharmacokinetic and pharmacodynamic models in hazard identification and risk assessment of toxic substances'. Toxicol. Lett. 79, 201206 (1995). 5. Frederick,C.B., Lomax,L.G., Black,K.A., Finch,L., Scribner,H.E., Kimbell,J.S., Morgan,K.T., Subramaniam,R.P., & Morris,J.B. Use of a hybrid computational fluid dynamics and physiologically based inhalation model for interspecies dosimetry comparisons of ester vapors. Toxicol. Appl. Pharmacol. 183, 23-40 (2002). 6. Vanoirbeek,J.A., Mandervelt,C., Cunningham,A.R., Hoet,P.H., Xu,H., Vanhooren,H.M., & Nemery,B. Validity of methods to predict the respiratory sensitizing potential of chemicals: A study with a piperidinyl chlorotriazine derivative that caused an outbreak of occupational asthma. Toxicol. Sci. 76, 338-346 (2003).
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Tier 1 Inhalation route Inhalation system differences along the tract 6. Pulmonary epithelium (A549, Calu-3, Beas-2B, 16HBE14o-, primary airway and primary alveolar cells) Responsible: Sandra Coecke (information provided by Prof. Ben Nemery and Dr. Peter Hoet) Short description, scientific relevance and purpose In vitro systems are used for cytotoxicity, absorption and transformation of chemical compounds. None of these systems is validated in detail. Cell lines: A549 cell line was initiated through explant culture of lung carcinomatous tissue, depending on the research objectives it is used as an alveolar or an airway epithelial cell. A549 cell are useful in cytotoxicity and show some xenobiotic biotransformation, they show only little or none tight junctions who hampers their usefulness in absorption studies. Calu-3 cells are derived from serous cells of submucosal glands of the airways and are a target for conditions in which muco-ciliary clearance is compromised (not real epithelial cells). Calu-3 cell mainly used in absorption studies (express tight junctions in culture). BEAS-2B cell line derives from normal human bronchial epithelium, and were immortalised by viral infection (adenovirus 12-SV40 virus hybrid (Ad12SV40)). The cells retain squamous differentiation in response to serum (but do not express cilia). Two other cell lines, BBM and BZR, were derived from BEAS-2B. less good tight junctions (compared to Calu-3 and 16HBE14o- cells) 16HBE14o cell line was developed by transformation of cultured bronchial-surface epithelial cells from a one-year old male heartlung patient. Primary cultures: Nasal (human & animal). Airway (human & animal). Alveolar type II pneumocytes (human & animal). These cells are used in different experimental studies (cytotoxicity, biotransformation, and absorption). Not much validation has been carried out. The access to human tissue is for most centres a major problem. In vitro systems used for cytotoxicity, absorption and transformation of particulate compounds. Only a few experimental systems exist, none of these systems is validated. Systems used: Cell lines: Calu-3 and Primary cells, no studies found, we have carried some pilot studies using rat type II pneumocytes. Frequently used models are two human cell lines derived from the respiratory epithelium, A549 and Calu-3
Developer of the method The absorption systems for pulmonary epithelial cells derive from the system used for intestinal cells (Caco-2).
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Some human-based respiratory airways models are commercially available. Known users Drug delivery: Recently the use of pulmonary cells has been used increasingly in pharmaceutical companies because dosing of drugs via inhalation becomes more popular. It is essential to understand the underlying mechanisms to reach effective dosing for a good systemic absorption of the drugs as well as to guarantee safety of the patient. Environment: The study of inhaled pollutant gasses and/or particles is a worldwide topic. Nanotechnology: The implications of pulmonary exposure to nanosized particles will become more common and those newly engineered particles should be tested concerning their safety after inhalation. Cosmetic industry: There is a trend to use sprays and aerosols, thus problems of particle size, inhalation exposure, etc. are highly relevant for cosmetics. Status of validation and/or standardisation All cellular systems described above are used to determine the uptake mechanisms of compounds and particles through the respiratory epithelium. No standardisation of the different techniques exists. However, further development and prevalidation of the methods should be encouraged. Fields and limitations of application, i.e., in vivo endpoints and/or chemical classes covered/not covered Chemical compounds or particles with are hydrophobic are more difficult to test. Particles and chemicals clearly can cause pulmonary inflammation that may lead to further systemic effects such as thrombogenesis and/or allergy. Recommendations of use in the optics of animal replacement Some good (human) airways cell systems (e.g. Calu-3 , 16HBE14o, BEAS-2B, some commercially available models) are available and should be further evaluated. An important question is whether it is possible to use only an airway cell line to study absorption in the whole lung? The use of primary animal cell cultures should be limited. On-going development No consequent standardisation has been proposed until now, different systems are used by different groups. Every group further develops its own system for its own purpose. In view of the developing in silico systems it is essential to investigate more in depth the mechanisms of the pulmonary translocation of particles, this phenomena is not studied in any detail. Efforts needed to complete validation of the method General efforts:
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Although the existing systems, using a human derived airway cell, are very promising, it is still unclear which cell lines (Calu-3, Beas-2B or 16HBE14o-) should be selected to study absorption and toxicity of chemicals or particles. No good cell line exists to test alveolar absorption. The use of human cells, both airway and alveolar, is an option BUT the access to tissue is for most centres a difficult tasks. Specific efforts: - First the different cellular systems have to studied in detail: Different factors to look at: substratum to grow the cells on, the pore size of the substratum, the need to add an artificial surfactant-mucus layer on the apical side of the cells, the type of medium to use, the identification of the minimum TEER value needed to have a good closed monolayer of cells, etc. Only after this phase a more formal validation can be initiated. - Remaining question: Is it possible to use only an airway cell line to study absorption in the whole lung? Key references 1. Agu, R. U., Jorissen, M., Willems, T., Augustijns, P., Kinget, R., en Verbeke, N. (2001). In-vitro nasal drug delivery studies: comparison of derivatised, fibrillar and polymerised collagen matrix-based human nasal primary culture systems for nasal drug delivery studies. J Pharm Pharmacol 53, 1447-1456 2. Foster KA, Yazdanian M, Audus KL. (2001) Microparticulate uptake mechanisms of in-vitro cell culture models of the respiratory epithelium. J Pharm Pharmacol. 53, 57-66. 3. Foster KA., Avery ML., Yazdanian M., Audus KL. (2000) Characterization of the Calu-3 cell line as a tool to screen pulmonary drug delivery. Int J Pharm 208, 1-11. 4. Forbes,I.I. (2000) Human airway epithelial cell lines for in vitro drug transport and metabolism studies . Pharm. Sci. Technol. Today. 3, 1827. 5. Hamilton KO, Backstrom G, Yazdanian MA, Audus KL. (2001) P-glycoprotein efflux pump expression and activity in Calu-3 cells. J Pharm Sci. 90, 647-658. 6. Nemmar A, Hoet PH, Vanquickenborne B, Dinsdale D, Thomeer M, Hoylaerts MF et al. (2002) Passage of inhaled particles into the blood circulation in humans. Circulation 105, 411-414. 7. Xu H, Hoet PH, Nemery B.J (2002) In vitro toxicity assessment of polyvinyl chloride particles and comparison of six cellular systems. Toxicol. Environ. Health A. 65, 1141-1159. 8. Hukkanen J, Lassila A, Pivrinta K, Valanne S, Sarpo S, Hakkola J, Pelkonen O, Raunio H. (2000) Induction and regulation of xenobioticmetabolising cytochrome P450s in human A549 lung adenocarcinoma cells. Am J Respir Cell Mol Biol 22, 360-366. 9. MatTek's EpiAirway (http://www.mattek.com/pages/products/epiairway) 10. Reconstituted human alveolar epithelium in vitro from Skinethic (http://www.skinethic.com/).
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Tier 2 Estimation plasma level 8. Plasma protein binding Responsible: Mario Monshouwer & review Bernhard Ladstetter & Geert Mannens Short description, scientific relevance and purpose The pharmacokinetic and pharmacodynamic properties of chemicals/drugs are largely a function of the reversible binding of chemicals to plasma or serum proteins. Generally, only the unbound fraction of a drug is available for diffusion or transport across cell membranes, and for interaction with a pharmacological/toxicological target. As a result, the extent of plasma protein binding (PPB) of a compound influences its action as well as its distribution and elimination. However, for high extraction ratio compounds, clearance is relatively independent of protein binding. There are three methods generally used for PPB determination: (1) equilibrium dialysis; (2) ultrafiltration and (3) ultracentifugation. Every method has its specific advantages and disadvantages and so far no preferred method has been described. For example, equilibrium dialysis has the advantage that unspecific binding to materials is uncritical which is frequently the case when using ultrafiltration and ultracentrifugation for highly lipophilic compounds. All methods can be automated, are easy to perform and have good precision and reproducibility. To obtain sufficient selectivity and sensitivity, LC/MSMS is an absolute prerequisite unless radiolabelled compounds are available. Developer of the method In general, suppliers of 96-well ultrafiltration devices and equilibrium dialysis equipment in colaboration with pharmaceutical companies have been the drivers for the development of these high-throughput PPB assays. Known users All pharmaceutical companies in their support to drug discovery and development evaluate PPB using their preferred method. Status of validation and/or standardisation Pharmaceutical companies are using equilibrium dialysis, ultrafiltration, and ultracentifugation as a screening tool to determine PPB within drug discovery and development. The standardisation and validation has mainly been performeed internally and occasionally published in peer reviewed scientific journals. Fields and limitations of application, i.e., in vivo endpoints and/or chemical classes covered/not covered Major compound binding proteins in plasma are; albumin, ? 1-acid glycoprotein and lipoproteins and by measuring the plasma protein binding using equilibrium dialysis, ultrafiltration, or ultracentrifugation it is not possible to make and any distinction between these proteins. This might be of interest as the risk for saturation of protein binding is higher for compounds binding to ? 1-acid glycoprotein, due to the relative low plasma concentration of ? 1-acid glycoprotein. Using these high throughput PPB assays it is possible to determine the unbound fraction of a compound. However, in situations of high protein binding a more appropriate parameter could be the equilibrium dissociation constant (Kd). Finally, validation of PPB using either equilibrium dialysis, ultrafiltration, or ultracentrifugation has been performed mainly within pharmaceutical
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companies on potential candidate drugs. How these assays perform outside the chemical space of pharmaceuticals is not known. Recommendations of use in the optics of animal replacement The easy availability of human plasma has made it possible to determine the unbound fraction of compounds by performing in vitro incubations directly in human plasma. It is worth to mention that the same approaches can be used to investigate species differences in PPB. The use of 96-well plate formats has reduced the amount of plasma necessary to determine protein binding. In particular for the evaluation of PPB in small animals like mice, this has reduced the number of animals necessary to obtain sufficient plasma. On-going development Activities are focusing on the improvement of the analytical aspects to the assay and on the risk of non-specific binding. Another area of interest is the Biocore surface plasmon resonance technology. This technology is ideally suited for the analysis of multiple aspects of compound plasma protein interactions. Efforts needed to complete validation of the method As the validation has been performed only of drugs or potential drugs, it would be useful to increase the chemical space representative for the cosmetic industry and evalute PPB. Key references 1. Banker MJ, Clark TH, Williams JA. (2003). Development and validation of a 96-well equilibrium dialysis apparatus for measuring plasma protein binding. J Pharm Sci. 92, 967-974. 2. Fung EN, Chen YH, Lau YY. (2003) Semi-automatic high-throughput determination of plasma protein binding using a 96-well plate filtrate assembly and fast liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 795, 187-94.
3. Kariv I, Cao H, Oldenburg KR. (2001) Development of a high throughput equilibrium dialysis method. J Pharm Sci. 90, 580-587.
4. Pacifici GM, Viani A. (1992) Methods of determining plasma and tissue binding of drugs. Pharmacokinetic consequences. Clin Pharmacokinet.23, 449-468.
5. Rich RL, Day YS, Morton TA, Myszka DG. (2001) High-resolution and high-throughput protocols for measuring drug/human serum albumin
interactions using BIACORE. Anal Biochem. 296, 197-207.
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Tier 2 Bioaccumulation 11. Blood-tissue partitioning Responsible: Han van de Sandt & Andreas Freidig Short description, scientific relevance and purpose The available system is the vial-equilibration technique. A spiked sample of (rat) tissue-buffer homogenate is equilibrated and subsequently, the free (unbound ) concentration of the test chemical is determined. The tissue-blood partition coefficient is calculated using from results from pure buffer, tissue-buffer and blood-buffer incubations. Tissues can be mixed to obtain average values for e.g. richly perfused tissue groups. Olive oil or octanol are often used instead of adipose tissue. The free (unbound) concentration is typically assessed by one of the following techniques: equilibrium dialysis, ultracentrifugation, headspace analysis (for volatiles) or solid phase (micro-) extraction followed by a classical analysis such as HLPC UV or MS. The purpose of this technique is the prediction of the in vivo tissue blood partitioning and the prediction of an in vivo volume of distribution. Developer of the method Headspace equilibration: e.g. Gargas et al. Equilibrium dialysis + ultracentrifugation : several commercial systems are available, typically developed to measure protein binding. Solid phase micro extraction: e.g. Artola-Garicano et al. Known users In vitro tissue - blood/plasma partition coefficient are measured in universities, CRO's and in industrial laboratories. Status of validation and/or standardisation Equilibrium dialysis and ultrafiltration devices are used and validated under GLP. Other techniques are less standardized. Fields and limitations of application, i.e., in vivo endpoints and/or chemical classes covered/not covered To validate the in vitro results, they are compared with in vivo blood tissue partitioning data and in vivo volumes of distribution. Thereby only passive distribution can be modeled by the in vitro system. Active transport across e.g. BBB or extensive metabolism in a tissue is not included. The experimental techniques are generic, but experimental difficulties arise with very hydrophobic compounds (log Kow>4). Measurement of skin-blood partition coefficients are less frequently reported. They may be experimentally more demanding with the vial equilibration techniques due to the stable texture of the skin. Recommendations of use in the optics of animal replacement These tests can help to estimate the plasma level by predicting a volume of distribution. It will also give an estimate of tissue levels, typically assessed in-vivo in a tissue distribution or Whole Body Autoradiography study. Typically, rat tissues are used to predict human values. Data are used as input in PbPk models.
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On-going development Further development of solid phase micro extraction of samples to obtain fast equilibrium and fast measurement of free concentration which are also applicable to very hydrophobic compounds. Efforts needed to complete validation of the method The test has been applied to pharmaceuticals and industrial chemicals. Validation efforts should focus on applications for the prediction of volume of distribution, as this will be an important property to assess systemic exposure. Especially for non-volatile and lipophilic compounds, a systematic approach comparing in vitro predictions and high quality in vivo data is lacking at present. Key references 1. Artola-Garicano E, Vaes WH, Hermens JL. (2000) Validation of negligible depletion solid-phase microextraction as a tool to determine tissue/blood partition coefficients for semivolatile and nonvolatile organic chemicals. Toxicol Appl Pharmacol. 166, 138-144. 2. Gargas ML, Burgess RJ, Voisard DE, Cason GH, Andersen ME. (1989) Partition coefficients of low-molecular-weight volatile chemicals in various liquids and tissues. Toxicol Appl Pharmacol. 98, 87-99. 3. Jepson GW, Hoover DK, Black RK, McCafferty JD, Mahle DA, Gearhart JM. (1994) A partition coefficient determination method for nonvolatile chemicals in biological tissues. Fundam Appl Toxicol. 22, 519-524. 4. Mattie DR, Bates GD Jr, Jepson GW, Fisher JW, McDougal JN. (1994) Determination of skin:air partition coefficients for volatile chemicals experimental method and applications. Fundam Appl Toxicol. 22, 51-57. 5. Poulin P, Theil FP. (2002) Prediction of pharmacokinetics prior to in vivo studies. 1. Mechanism-based prediction of volume of distribution. J Pharm Sci. 91, 129-156.
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Tier 2 Biotransformation (metabolism) 12. Metabolic stability & species differences Responsible: Olavi Pelkonen & review Bernhard Ladstetter & Greetje Elaut & Vera Rogiers & Geert Mannens Short description, scientific relevance and purpose Relatively simple, fast-to-perform and but specialized analytical equipment-based (MS) study to find out whether a compound is metabolically stabile or labile. It consists of a tissue preparation (e.g. human liver preparation, preferably homogenate), appropriate cofactors (e.g. NADPH, UDPGA, PAPS and GSH) incubated together with the compound under study, samples taken at various time points, analyzed for the parent compound (e.g. generic sample preparation and chromatographic and/or LC/MS method). The disappearance of the parent compound gives a measure of metabolic stability. Animal (including human)liver or tissue preparations can be studied under similar incubation conditions for comparative purposes. Developer of the method The method in various forms has been used increasingly in a large number of pharmaceutical companies, CROs and other service companies. Academic groups are focused on further development and refinement of the assay. No single developer can be named. Known users Principally preclinical discovery and development departments of pharmaceutical companies and CROs. Status of validation and/or standardisation Because the test has been used in an early phase in drug discovery as a screening method (and mainly in private industry), standardisation and validation, if done, has mainly been "internal". No formal validation has been undertaken (or at least is known). Some pharmaceutical companies have published their experiences. The stability screening test should be ready for validation after a consensus meeting where common procedures would be decided. Fields and limitations of application, i.e., in vivo endpoints and/or chemical classes covered/not covered Because liver is the main organ for biotransformation, the test fortified with suitable cofactors should give a fairly reliable view of hepatic intrinsic clearance, which is important for many organic chemicals. However, to be able to predict in vivo clearance, a number of assumptions concerning the substance under study, should be made, and consequently, a simple extrapolation model is needed. Naturally, extrahepatic biotransformation is not covered. The methods of metabolic stability using hepatocyte-based assays can be combined with the bioactivation assays (16).
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Recommendations of use in the optics of animal replacement As a part of the in vitro testing strategy, the test should replace animal pharmacokinetic and biotransformation studies to a certain extent. It is worth that animal preparations of different species, including man, when used under comparable condictions, can provide valuable information on species differences. On-going development With the advent of modern MS techniques, the test is suitable for the study of the detailed metabolic profile, both qualitative and quantitative, of a compound under study, in addition to the information about the metabolic stability, calculated intrinsic clearance and the predicted pharmacokinetic values for in vivo condition. The use of recombinant enzymes, transfected cells and metabolically competent human (liver) cell lines are very actively researched at the present moment. Also stem cell -derived liver cells, which retain metabolic competence is one goal. Also species differences can be investigated using the test methods described. Efforts needed to complete validation of the method Because the test has been used almost solely with pharmaceuticals, it would be useful to select a wider assortment of chemicals of interest, e.g. typical cosmetic ingredients with known PK properties, and put them through the test. Key references 1. Bertrand, M.; Jackson, P. and Walther, B. (2000) Rapid assessment of drug metabolism in the drug discovery process. Eur. J. Pharm. Sci., 11(S2), S61-S72. 2. Clarke, S.E. and Jeffrey, P. (2001) Utility of metabolic stability screening: comparison of in vitro and in vivo clearance. Xenobiotica, 31, 591598. 3. Elaut, G.; Papeleu, P.; Rogiers, V. and Vanhaecke, T. (2002) Practical aspects of in vitro biotransformation studies during early drug development. Recent Res. Devel. Drug Metab. Disposition, ed. S.G. Pandalai, Transworld Research Network, Kerala, India, 1, 167-198 4. Gebhardt, R.; Hengstler, J.G.; Mller, D.; Glckner, R.; Buenning, P.; Laube, B.; Schmelzer, E.; Ullrich, M.; Utesch, D.; Hewitt, N.; Ringel, M.; Hilz, B.R.; Bader, A.; Langsch, A.; Koose, T.; Burger, H.J.; Maas, J. and Oesch, F. (2003) New hepatocyte in vitro systems for drug metabolism: metabolic capacity and recommendations for application in basic research and drug development, standard operation procedures 5. Li, A.P. (2001) Screening for ADME/Tox properties in drug discovery. Drug Disc. Today, 6, 357-366 6. Masimirembwa, C.M.; Bredberg, U. and Andersson, T.B. (2003) Metabolic stability for drug discovery and development: pharmacokinetic and biochemical challenges. Clin. Pharmacokinet., 42(6), 515-528. 7. Obach, R.S. (2001) The prediction of human clearance from hepatic microsomal metabolism data. Curr. Opin. Drug Discov. Devel., 4(1), 3644.
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Tier 2 Biotransformation (metabolism) 13. Bioactivation assays Responsible: Sandra Coecke & review Greetje Elaut & Vera Rogiers & Geert Mannens Short description, scientific relevance and purpose Biotransformation can result in a bioactivation , often triggert by phase I enzymes, as well as in a detoxification process, often the outcome of phase II conjugations. The formation of reactive metabolites is relatively frequent and, although biotransformation reactions generally parallel detoxification processes, there are several cases where biotransformation of the compound is the cause of its deleterious effects. Phase I enzymes like cytochrome P450s (CYPs) may generate metabolites capable to react with nucleophiles, covalently bind to macromolecules or initiating radical-chain reactions within cells. Also phase II biotransformation enzymes such as the glutathione S-transferases can be responsible for toxification processes, but often to increase the solubility of the parent substance or its metabolites and therefore increasing their excretion. The identification of biological reactive metabolites may be of importance to estimate possible toxic effects. The estimation of toxic effects due to reactive metabolites from the parent compound requires the access to metabolically competent cells. Primary cultured hepatocytes show the typical phenotype of the adult cells, including drug-metabolising activities and nowadays constitute a valuable in vitro model for the identification of such metabolites. However, species differences as well as differences in the metabolic pattern of specific organs are well-known. Therefore, besides primary cells of liver and other metabolically competent organs, other approaches-based on genetically-engineered cell lines expressing one or more human biotransformation enzymes have been used for this purpose. In all the approaches used, a first point is to ascertain whether toxicity is associated with the biotransformation of a given compound. For that purpose effects are investigated in a metabolic competent model (primary hepatocytes, genetically engineered cell lines, etc.) and in nonmetabolising cells (often cell lines are used). By comparing the concentration-toxicity curves (or IC50 data) of the compound in both models it is possible to get an impression of the likelihood whether the metabolites elicits toxic effects preferentially on hepatocytes or other metabolic competent cells, suggesting that a bioactivation of the xenobiotic is required. Another possibility to explore a possible role of a compounds biotransformation in xenobiotic toxicity is the addition of inhibitors of metabolising enzymes (basically CYP inhibitors) to the incubation media of hepatocytes. Reduction of toxicity effects in the presence of the inhibitor suggests that toxicity could be associated with the biotransformation of tested compound. Cytotoxicity endpoints represent a first approach in toxicity screening. A number of tests, including the MTT assay, the neutral red uptake, the alamar blue assay or enzyme leakage (LDH, transaminases), can be selected. A single parameter is usually sufficient to estimate irreversible injury induced by toxic chemicals. Dose-response curves are calculated and the results are frequently summarised as concentrations of test compounds which produce a 50% effect respect to non-treated cells (IC50). A second set of endpoints measured are related to biotransformation-mediated mutagenic effects. A classical example of this approach is the Ames test (note that S9 mix is derived from treated
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animals and thus not really in vitro) Developer of the method The method in various forms has been used increasingly in most toxicological laboratories as a standard. The refinement of this assay and the adaptation of cells to answer specific questions is an ongoing process.. Known users Well developed standard test in most toxicological laboratories.. Status of validation and/or standardisation A number of in vitro systems are available for studying biotransformation-mediated effects. These include subcellular fractions such as the microsomal fraction, S9 mix, primary cells in suspension, primary cells in culture, continuous cell lines, immortalised primary cells, and genetically-engineered cell lines. To date, no single in vitro methods for determining phase I or phase II biotransformation, or for evaluating biotransformation-dependent toxicity, have been validated according to ECVAMs principles and procedures. The current OECD Test Guideline 417 for assessing the toxicokinetic effects of chemicals is based on in vivo studies. However, many studies provide support for the usefulness of in vitro methods for assessing biotransformation and biotransformation-dependent toxicity. Fields and limitations of application, i.e., in vivo endpoints and/or chemical classes covered/not covered The use of cultured hepatocytes to screen for bioactivation processes associated to xenobiotic toxicity requires a carefully definition of experimental conditions. Primary cultured hepatocytes show a gradual decrease in differentiated hepatic functions, including CYP expression. CYP activity levels are shortly reduced after seeding, probably due to the adaptation of cells to culture conditions. To avoid this drawback, incubations of hepatocytes with the test compounds are performed in short-term cultures. The chemicals are usually added to culture medium one hour after hepatocyte seeding. Information about solubility, volatility and possible interactions with medium components of the compound is previously required. Changes in pH or osmolarity of incubation medium, binding to proteins or formation of precipitates with medium components or toxic effects of the solvent used to dissolve the compound are major factors to be considered. ln the case of genetically-engineered cell lines a drawback is the limited co-expression capacity of biotransformation enzymes, the differences in expression level in comparison with the in vivo situation, the absence of specific co-factors and the normal cellular environment. Even simple parameters for assessing cell cytotoxicity have yielded promising results when comparing in vitro effects with human toxicity as false negatives are infrequent in compounds that are toxic without biotransformation. However, the in vitro models may lack sensitivity for xenobiotics that require a high degree of biotransformation or prolonged exposures to exert their cytotoxic effect. No specific effects, such as genotoxic effects can be identified via a cytotoxicity method. However, examples are published for the determination of biotransformationmediated mutagenic effects.
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Recommendations of use in the optics of animal replacement It is evident that there are species differences in toxicokinetics; this is especially true for the metabolic differences between humans and rodents. Therefore, there is a great need for human-based in vitro models that would offer better predictions of potential hazard to humans than could ever be obtained from laboratory animal studies. On-going development Strategies based on engineered cells with human genes could be valuable contribution to handle the well-known species differences more properly. Stabily transfected cell lines, but also transient cell lines are being created using the knowledge built up in the area of gene therapy. To this end recombinant-defective vectors encoding for major CYP genes involved in foreign compounds biotransformation are being generated and used. Recent advances have been made in the area of computer-based (in silico) biotransformation and toxicity prediction, and comment on the opportunities for prediction of biotransformation-based toxicity. There is a growing interest and importance of in silico prediction of biotransformation and toxicity as tools to assist in library design and lead optimization. There is a potential of in silico models for property prediction, and there is a potential for linkage with vivo models to improve the integration of biotransformation and toxicity into the design of tiered test approaches for regulatory toxicity applications. The future development, integration and application of in silico models will require a balance of local and global model approaches. Efforts needed to complete validation of the method Because variations of the test have been used in different laboratories, it would be essential to gather the information already available in order to proceed and select those approaches were there are already some substantial data sets available. Key references 1. Bull S, Langezaal I, Clothier R, Coecke S. (2001) A Genetically engineered cell-based system for detecting metabolism-mediated toxicity. Altern Lab Anim. 29, 703-716. 2. Busby WF Jr, Penman BW, Crespi CL. (1994) Human cell mutagenicity of mono- and dinitropyrenes in metabolically competent MCL-5 cells. Mutat Res. 322, 233-242. 3. Crespi (2003) The BD Gentest MCL-5 Metabo-Tox Assay Kit allows high throughput screening for potential P450 mediated toxicity in the early stages of drug discovery. http://www.gentest.com/products/HTS_KITS/metabo_tox_assay_kit.shtm 4. Gomez-Lechon MJ, Donato MT, Castell JV, Jover R. (2003) Human hepatocytes as a tool for studying toxicity and drug metabolism. Curr Drug Metab. 4, 292-312. 5. Papeleu, P.; Elaut, G.; Rogiers, V. and Vanhaecke, T. (2002) Cell cultures as in vitro tools for biotransformation studies. Recent Res. Devel. Drug Metab. Disposition, ed. S.G. Pandalai, Transworld Research Network, Kerala, India, 1, 199-233. 6. Peng FC, Tseng HY, Tsai JC, Lin C, Doehmer J. (2003) Role of human hepatic cytochrome P-450s in territrem A metabolism. J Toxicol
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Environ Health A. 66, 1237-1248. 7. Townsend AJ, Kabler SL, Doehmer J, Morrow CS. (2002) Modeling the metabolic competency of glutathione S-transferases using genetically modified cell lines. Toxicology 181-182, 265-269.
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Tier 2 Biotransformation (metabolism) 14. In silico approaches Responsible: Olavi Pelkonen ( additional comments provided by Nigel Gensmantel) & review by Geert Mannens Short description, scientific relevance and purpose Available systems are of three types: 1) expert systems based on a knowledge base of structure-metabolism rules (e.g. Meteor, Metabolexpert), 2) SAR and QSAR modelling, and 3) systems based on pharmacophore or molecular protein modelling (e.g. CYP2C9 or CYP3A4 molecular models). A recent review (van de Waterbeemd and Gifford 2003) lists more than 40 commercial software or data producers of ADMET software, although not all of them are dealing with exactly biotransformation (this list includes SIMCYP, Cloe Screen). These models are in various phases of development. Goals for predictions include possible metabolites, metabolising enzymes, quantitative kinetics etc., although none of these purposes are really achieved to a reliable extent. Developer of the method Many of the individual systems are developed by SMEs and used principally by pharmaceutical companies. Academic groups are usually in collaboration with the companies or transfer their innovations to spinoff companies. Known users Principally preclinical discovery and development departments of pharmaceutical companies. Status of validation and/or standardisation Because the systems have been developed mainly by SMEs and usually used in an early phase in drug development as a screening method (and mainly in private industry), standardisation and validation, if done, has mainly been "internal". There is no consensus as to how in silico systems should be validated, but COST B15 Action is arranging an expert meeting on this subject in April 2004. Fields and limitations of application, i.e., in vivo endpoints and/or chemical classes covered/not covered Data modelling approaches are currently useful in giving a rapid (but not exactly reliable) overview of potential metabolic fate of a compound. Molecular modelling approaches are still at a stage where they can provide a fairly accurate prediction of CYP-isoenzyme selective biotransformation of a compound, but only a at a qualitative level. Usually useful predictions are restricted to a class of chemicals which has been used as a training set. In silico metabolism modelling is clearly an area where improvements are needed. Algorithms are available to identify potential sites of metabolism but what is needed is to have the ability to predict the major metabolic routes when there are a number of potential metabolic pathways. Also the species differene need to be incorporated. It will be necessary to understand more about the enzymology of different species, different organs before meaningful predictions can be made. Extropalation from in vitro pre-clinical species to in vivo pre-clinical
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species is often difficult and prediction to in vivo human from limited in vitro human still represents a major challenge. Recommendations of use in the optics of animal replacement As a part of the in vitro testing strategy, in silico approaches should replace animal toxicokinetic and biotransformation studies to a certain extent. It is worth of stressing that in silico models are (usually) based on existing human data, so predictions are directly applicable to humans. On-going development In silico approaches are developing rapidly, but are still rather crude. The first generation of models is commercially available, but their predictive power and application possibilities vary a lot and are still inadequate for the production of results which are accepted by regulators. It is expected that second-generation and mechanism-based models for predicting biotransformation will be available within a few years. A goal is a combination of models (i.e. meta-models) for partial processes; e.g. biotransformation would include CYPs, phase II enzymes, intrinsic hepatic and extrahepatic clearances and so on. Efforts needed to complete validation of the method A common opinion in the field is that models are only as good as data on which they are based. Consequently, reliable and good-quality data bases are of outmost importance for the development of reliable softwares. Furthermore, because in silico approaches have been used almost solely for pharmaceuticals, it would be useful to select a wider assortment of chemicals of interest, e.g. typical cosmetic ingredients with known PK properties, and put them through the test. Key references 1. Boobis A, Gundert-Remy U, Kremers P, Macheras P, Pelkonen O. (2002) In silico prediction of ADME and pharmacokinetics. Report of an expert meeting organised by COST B15. Eur J Pharm Sci 17: 183-193. 2. Langowski J, Long A. (2002) Computer systems for the prediction of xenobiotic metabolism. Adv Drug Delivery Rev 54: 407-415. 3. van de Waterbeemd H, Gifford E. (2003) ADMET in silico modelling: towards prediction paradise? Nature Reviews Drug Discovery 2: 192204.
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2c. Tier 1 and Tier 2 integrated in vitro and in silico approaches (some examples) Important note Below some examples of integrated approaches. The examples listed are only those were the subgroup could get the information directly from the developers of the integrated approaches. It should be stressed that many other approaches are published in the literature and that the examples below are just a fraction from what is currently available.
Tier 1 Oral absorption 2. QSPR Approaches 4. Caco-2 cell systems
Tier 2 Estimation of plasma level 7. QSPR approaches/physicochemical properties 8. Plasma protein binding Biotransformation 12. Metabolic stability & species differences 14. In silico approaches Responsible: Sandra Coecke (information provided by Dr. David Leahy) Short description, scientific relevance and purpose Cloe Screen is, as claimed by the developer, to be a set of widely accepted in vitro ADME assays optimised for precision, accuracy and high throughput. The assays include metabolic stability using human (and other species) liver microsomes, turbidimetric solubility, Caco-2 permeability (AB & BA), P450 IC50 values in human liver microsomes (3A4, 2C9, 2C19, 1A2, 2D6), plasma protein binding and lipophilicity. The assays are optimised for automated screening using 96 or 384 well plates and internal validation data is available which shows high precision, accurate reproduction of low throughput estimates and can be run at throughputs of up to 200,000 compounds per year, depending on the assay. The developer of the test system aims to use it to build up a large experimental database for the development of QSPR relationships using
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automated QSAR methods available form one or more suppliers such as PharmAlgorithms (Estonia). Developer of the method Cyprotex PLC, was founded in 2001 by Dr David E Leahy,. The company has developed HTADME screening facilities (known as Cloe Screen) for provision of ADME data and Cloe PK software for the simulation of human and animal pharmacokinetics. The company based in Macclesfield in the UK reported to have contracts with major pharmaceutical companies and has specialists in simulation modelling, software development, ADME experimental sciences and automation. Known users The test system mentioned are in common use throughout the global Pharmaceutical industry although there is variation in detailed protocols and variability in results between labs. It is claimed that the throughput achieved by the test system are typically an order of magnitude greater than achieved elsewhere. Status of validation and/or standardisation The developer of the test system reports that Internal validation is carried out routinely and all external clients are also undertaking their own validations. Fields and limitations of application, i.e., in vivo endpoints and/or chemical classes covered/not covered The endpoints covered are: Solubility Intestinal resorption Metabolic stability P450 Inhibition IC50s Caco-2 permeability (AB/BA) Plasma Protein Binding Log P (octanol), pKa Almost all the compounds in the validation sets are drugs or compounds that have been designed to be drugs at some stage of the discovery or development process Recommendations of use in the optics of animal replacement To create a large diverse experimental database with data on representative analogues from multiple chemical series for the extraction of QSPR models which can be used as screening methods for the selection of compounds for further experimental analysis. On-going development Selection of diverse compounds and those of interest to the cosmetic and industrial chemical industry Efforts needed to complete validation of the method The extensive validation data covers drugs and drug-like compounds. Completion requires further independent external validation using
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compounds likely to be of interest to cosmetics and general chemical manufacturing. Key references Yu H. and Adedoyin A. (2003) ADME-Tox in drug discovery: integration of experimental and computational technologies, Drug Discovery Today 8, 854-861.
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Tier 2 Estimation plasma level 9. Kinetic modelling Bioaccumulation 10. QSPR approaches/physicochemical properties Biotransformation 14. In silico approaches Responsible: Gill Langley (information provided by Dr Nick Proctor and Dr Amin Rostami-Hodjegan) Short description, scientific relevance and purpose The developers of the test system Simcyp algorithms claim that have been developed to simulate in vivo human pharmacokinetics from simple and common preclinical data. They further report that the pharmacokinetic output can be in the form of steady-state clearances or plasma/blood concentration profiles which can then be used for kinetic modelling or bioaccumulation predictions. The data required are information regarding systemic availability and elimination which are then combined within the algorithm to provide population estimates of both clearance and potential for drug-drug interactions, as claimed by the developers. The developers obtain commonly in vitro metabolic stability data from recombinantly expressed human cytochromes P450 (rCYPs) or characterised human hepatic microsomes. The population approach of the test system is claimed to allow identification of populations or members of populations most at risk of adverse events. Developer of the method Professor Geoffrey Tucker and Dr Amin Rostami-Hodjegan have been developing Monte Carlo based pharmacokinetic simulation strategies for over 15 years at the University of Sheffield. Their expertise in human pharmacokinetics and examination of drug-drug interactions is reported to be world renowned. Known users The majority of the large global pharmaceutical companies as well as a US regulatory authority hold licences to use the test system and the developers report that several collaborations have taken place and are ongoing with academic researchers at various universities worldwide. Status of validation and/or standardisation Standardisation is currently ongoing by the developers as the algorithm evolves through its development stages. It is reported that internal validation is carried out routinely and that all external clients are also undertaking their own validations for predictions of clearance and druginteractions. Continual optimisation and prevalidation standardisation are being monitored by an internal quality assurance department and overseen by a scientific advisory board consisting of senior academic and industrial experts in the field, as claimed by the developers. It is reported by the developers that the consortium members also provide additional feedback on the performance of the approach and its
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applicability to routine drug development tasks. These consortium members include representatives from industrial and regulatory users who meet at least annually to discuss product development and the results of their independent examinations. Formal validation is planned for the end of Phase II of the software development phase (early 2005). Fields and limitations of application, i.e., in vivo endpoints and/or chemical classes covered/not covered he clinical in vivo endpoints covered by the test system include total and free blood and plasma metabolic, oral and whole body clearances. It is reported that the use of the advanced model also allows the simulation of individual drug concentration-time profiles and interactions between drugs. The drug interactions are quantified by changes in clearance, dynamic plasma drug concentration, or steady-state plasma concentrations following oral or intravenous dosing. All of the above parameters are available for fully customisable individuals sampled from populations ranging from healthy volunteers to patients in specific disease groups and ethnic backgrounds as claimed by the developers. Currently the test system does not incorporate pharmacodynamic or toxicodynamic models, however it is claimed that these can be easily integrated with the data produced using standard simulation techniques and established kinetic relationships. At this time the algorithm also does not predict renal clearance as no suitable in vitro model has been developed, rather it takes estimates of renal clearance and applies known population variabilities to this. The test system has been extensively tested with a diverse range of drug entities and no particular group of compounds has shown consistent inaccuracy in terms of predictions of metabolic clearance, as claimed by the developers. Recommendations of use in the optics of animal replacement The ddevelopers claim that the test system can currently facilitate the replacement of animal experiments for predicting the metabolic clearances of drugs which is commonly performed using interspecies allometric scaling. The developers report that early investigations with 13 extensively metabolised drugs have shown that the test system has a greater accuracy and precision than allometric approaches. Interspecies allometry is still considered the only method for the prediction of renal drug clearance however in vitro systems are in development that have the potential to replace this in the future. On-going development. The developers report that the ongoing development of the test system algorithm is varied and has many long-term goals in addition to maintaining up to date with latest scientific developments and discoveries. Current work is focussing on a paediatric version of the algorithm which is due to be released in 2004. Research is also underway to define a variety of ethnic and disease populations in terms of the required anatomical, physiological, genetic and demographic parameters that enable the accurate simulation of various groups of virtual individuals. Efforts needed to complete validation of the method Once the second development phase is completed the formal validation can be undertaken. This can consist of an extensive comparison with current methods (such as interspecies allometry) and in vivo clinical trial results. The developers indicate that both the central tendency and ranges of the simulations can be used as comparators. In vivo data collection can be based on published literature and collaborations with both external and internal researchers. A full list of source publications will be compiled along with derivations of all equations which form part of the
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algorithm and all sources of data used in the validation exercises, as reported by the developers. Formal external auditing and validation is not planned until the development phase is complete. Key references 1. Lin, J. H. "Applications and limitations of interspecies scaling and in vitro extrapolation in pharmacokinetics." Drug Metab Dispos. 26.12 (1998): 1202-12. 2. Naritomi, Y. et al. "Prediction of human hepatic clearance from in vivo animal experiments and in vitro metabolic studies with liver microsomes from animals and humans." Drug Metab Dispos. 29.10 (2001): 1316-24. 3. Nestorov, I. "Whole body pharmacokinetic models." Clin.Pharmacokinet. 42.10 (2003): 883-908. 4. Proctor, NJ., Rostami-Hodjegan, A. & Tucker, GT (2003) Sources of variability associated with quantitative predictions of drug clearance from recombinant CYP enzymes. Drug Metabolism Reviews 35, 202 Suppl 1. 5. Proctor, NJ., Rostami-Hodjegan, A. & Tucker, GT. (2003) In vitro prediction of population in vivo drug clearance using updated scaling factors. British Journal of Clinical Pharmacology 55, 445. 6. Proctor, NJ., Rostami-Hodjegan, A. & Tucker, GT. (2003) ISEF: an intersystem extrapolation factor for use with recombinant cytochrome P450 expression systems. British Journal of Clinical Pharmacology 55, 437-438. 7. Proctor, NJ., Rostami-Hodjegan, A. & Tucker, GT. (2003) Predicting drug clearance from recombinantly expressed CYPs: intersystem extrapolation factors. Xenobiotica, in press. 8. Sweeney, L. M. "Comparing occupational and environmental risk assessment methodologies using pharmacokinetic modeling." Human and Ecological Risk Assessment 6.6 (2000): 1101-24.
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Tier 1 Oral Absorption 3. Kinetic modelling
Tier 2 Estimation of plasma levels 9. Kinetic modelling Bioaccumulation 11. Blood/Tissue partitioning Responsible: Sandra Coecke (information provided by Dr. David Leahy) Short description, scientific relevance and purpose: Cloe PK is a client-server software implementation of a physiologically based model developed by Cyprotex for the extrapolation and interpretation of in vitro ADME data. The test system has been parameterised for rat and human and the developer claim that it allows the simulation of plasma and tissue (intracellular, interstitial) levels following an oral or an injected dose and that it also simulates fraction unbound as well as total compound levels. Estimation of standard parameters such as Fraction Absorbed, Bioavailability, Clearance and AUC are reported to be obtained via this test system. Given in vitro ADME data the ddeveloper reports that the test system can be used to estimate tissue exposure of a test compound, and provides a way of extrapolating in vitro toxicity estimates, to in vivo risk factors. The test system can also be used in the validation of in vitro toxicity methods against in vivo data, as indicated by the developer. Developer of the method Cyprotex PLC, was founded in 2001 by Dr David E Leahy,. The company has developed HTADME screening facilities (known as Cloe Screen) for provision of ADME data and Cloe PK software for the simulation of human and animal pharmacokinetics. The company based in Macclesfield in the UK reported to have contracts with major pharmaceutical companies and has specialists in simulation modelling, software development, ADME experimental sciences and automation. Known users Physiologically Based Pharmacokinetic modelling methods have a long history of development and use in the environmental health arena, and it is reported that most pharmaceutical companies have one or two specialist modellers looking at application of the models to the design of clinical trials. There are one or two academic groups as well as industry and government organisations active in the field. In recent years, along with Cyprotex, Lion Biosciences (Idea), Bayer Technology (PKSIM) and Simulations Plus (GastroPlus) have launched alternative software products that implement physiologically based pharmacokinetic methods.
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Status of validation and/or standardisation The test system is currently at various stages of evaluation by approximately 20 Pharmaceutical Companies including 8 of the top ten, as claimed by the company. The models have been internally validated for rat and humans using plasma levels from approximately 80 compounds (mainly drugs). Additional internal validation of the intestinal absorption component of the model has been carried out using fraction absorbed data for approximately 100 compounds. External validation studies (carried out as blind trials) have been completed with seven of the companies for 120 compounds in rat and 80 in humans, as reported by the developer. Depending on the parameter, mean error of prediction is between 2 and 5 fold. For example, plasma half-life in rats, 56% of the compounds have half-lives predicted within 2-fold, 84% within 5-fold and 94% within 10-fold. There are few published validation results and they tend to be on very few compounds. There is minimal published information from the commercial companies involved, although the developer claims now to be in a position to make the external and internal validation results available to companies and organizations interested in evaluating the software as a method of predicting human and rat pharmacokinetics. Fields and limitations of application, i.e., in vivo endpoints and/or chemical classes covered/not covered The software can be used to estimate unbound or total plasma levels as well as tissue levels in most principal organs including the gut over time following an oral or intravenous dose, as claimed by the company. The software does not at this stage estimate pharmacokinetics following percutaneous application, although the developers indicates that this can be easily adapted. The developer reports that both internal and external validation compound sets cover a very wide range of physical, structural and ADME properties and there is no evidence of significant class or series-specific systematic errors. However, almost all the compounds in the validation sets are drugs or compounds that have been designed to be drugs at some stage of the discovery or development process. Recommendations of use in the optics of animal replacement The software can be used in combination with experimental or in silico estimates of lipophilicity, in vitro metabolic clearance, permeability, protein binding, solubility. On-going development The developer continues to improve the quality of the plasma level predictions through further development of the existing built-in in silico models for renal clearance, blood-tissue partition coefficients, development of an in silico model for biliary excretion. Extension of the model to the simulation of biological or toxicological effects through linking plasma levels to in vitro concentration-effect relationships is also desirable. A further goal of the developer is the use of the model with the test system generated microsomal P450 inhibition data to estimate compound interactions. Extension of the model to simulate pharmacokinetics from a percutaneous dose is also required. Efforts needed to complete validation of the method The extensive validation data covers drugs and drug-like compounds. Completion requires further external validation using compounds likely to
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be of interest to cosmetics and general chemical manufacturing. The developer indicates that this requires generation of experimental data using the test system (1 month), and simulation work followed by comparison of the results (1-2 months).
Key references 1. Andersen, M. (1995) Development of Physiologically Based Pharmacokinetic and Physiologically Based Pharmacodynamic Models for Application in Toxicology and Risk Assessment. Toxicology Letters 82-83, 341-348. 2. Grass, G. (2002) Physiologically Based Pharmacokinetic Simulation Modelling, Adv. Drug Delivery Reviews 54, 433-451. 3. Leahy, DE. (2003) Progress in Simulation Modelling for Pharmacokinetics, Current Topics in Medicinal Chemistry 3, 1257-1268 4. Yu, L. (1999) An Integrated Model for Determining Causes of Poor Oral Drug Absorption Pharm. Res. 196, 31 19-28.
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2d. Integrated human volunteer-based approaches

Tier 1 Dermal, oral and inhalation routes human in vivo Tier 2 Estimation plasma level, excretion, bioaccumulation, biotransformation (metabolism) human in vivo Responsible: Gill Langley Short description, scientific relevance and purpose Accelerator mass spectrometry (AMS) has been adapted for use in the laboratory to detect extremely low levels of radiolabelled substances in samples of urine, blood, plasma and faeces (and other tissues). In combination with sample separation techniques such as high-pressure liquid chromatography, AMS can quantify levels of drugs, chemicals or biotechnology products and their metabolites at sub-pharmacological and subtoxicological levels (i.e.attomole sensitivity, 10-18 M). Because of its extreme sensitivity and precision, AMS can be used in microdosing studies with human volunteers which are completely safe (both chemically and radiologically), with hardly any preceding animal testing (usually an acute or a 7-day toxicity test in rodents is recommended). Substances can be applied by dermal, oral, inhalational, vaginal and subcutaneous routes. Blood samples can be as small as 5 microlitres. AMS can provide human ADME and detailed human PK data including, importantly, excretion. Developer of the method Professor RC Garner and colleagues, Xceleron Ltd, York, UK. Also the Centre for Accelerator Mass Spectrometry at the Lawrence Livermore National Laboratory, California, USA. Known users There are so far very few AMS facilities working in this area. At present the only UK laboratory is Xceleron, which undertakes AMS analysis for some pharmaceutical companies. The capital cost of the equipment is presently very high (about $1 million, although this can be expected to come down over time and smaller, less costly machines are already becoming available). Status of validation and/or standardisation Developmental. A study has been conducted comparing AMS and liquid scintillation counting in rats which showed good correlations in excretion balance and PK (of fluticasone propionate). A radiolabelled experimental drug administered at microdose levels to human volunteers achieved mass balance between administered and recovered radiation. Fields and limitations of application, i.e., in vivo endpoints and/or chemical classes covered/not covered Data provided: ADME and detailed PK in humans. Any route of administration. At the moment, capital costs are high. Assay costs are about
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$200 per sample. Limitations include the requirement for incorporation of the radiolabel in the test molecule. In many cases, such radio-labelling would be technically impossible, due to principal differences between radio and non-radio synthesis of some chemicals. Nevertheless, the principles of this detection method are perceived by the subgroup as very interesting. The rate-limiting step in sample analysis at the moment is sample preparation, which for radiocarbon involves the technically challenging combustion and graphitization steps. Concerns about saturable metabolism and transport are often cited with microdosing. In humans, using the i.v. route (as well as other routes if desired) minimises bioavailability problems because absolute clearance occurs. Saturable metabolism and transport (at higher doses) can potentially occur but is unlikely. Further human studies on extrapolating from microdoses to higher dose levels should be done as part of the validation process. This can be achieved by using AMS to provide microdose data for substances where human data at higher doses are already available. An criticisms which can be expected from several parties is the application of unknown new chemicals to men. Recommendations of use in the optics of animal replacement When AMS becomes cost-effective and is more routinely used, it could replace all or most toxicokinetic and ADME data normally obtained in animals of different species. On-going development Yes, at Xceleron, York, UK in collaboration with pharmaceutical companies. Efforts needed to complete validation of the method More data on microdose-to-higher dose extrapolation this could be supplied by industry for chemicals, drugs etc for which human data already exists. Key references 1. Barker, J & Garner, RC. (1999) Biomedical applications of accelerator mass spectrometry isotope measurements at the level of the atom. Rapid Commun. Mass Spectrom. 13, 285-293. 2. Combes, RD, Berridge, M, Connelly, J, Eve, MD, Garner, RC, Toon, S & Wilcox, P. (2003) Early microdose studies in human volunteers can minimise animal testing; Proceedings of a workshop organised by Volunteers in Research and Testing. Eur. J. Pharmaceut. Sci. 19, 1-11. 3. Garner, RC, Barker, J, Flavell, C, Garner, JV, Whattam, M, Young, GC, Cussans, N et al. (2000) A validation study comparing accelerator MS and liquid scintillation counting for analysis of 14C-labelled drugs, in plasma, urine ans faecal extracts. J Pharm. Biomed. Anal. 24, 197-209. 4. Garner, RC, Garner, JV, Gregory, S, Whattam, M, Calam, A & Leong, D. (2002) Comparison of absorption of micronized (Daflon 500 mg) and nonmicronized 14C-diosmin tablets after oral administration to healthy volunteers by accelerator mass spectrometry and liquid scintillation counting. J Pharm. Sci. 91, 32-40. 5. Vogel, JS, Grant, PG, Buchholz, BA, Dinglery, K & Turteltaub, KW. (2001) Attomole quantitation of protein separations with accelerator mass spectrometry. Electrophoresis 22, 2037-2045. 6. Young, G, Ellis, W, Ayrton, J, Hussey, E & Adamkiewicz, B. (2001) Accelerator mass spectrometry (AMS): recent experience of its use in a clinical study and the potential future of the technique. Xenobiotica 31, 619-632.
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3. Identified steps or tests with no alternative methods available 3a. Tier 1

There is a lot of effort done for the development of methods looking at passage of compounds through barriers. Most advanced are the methods for dermal absorption (dermal route), followed by methods dealing with passage through the intestinal barrier. However, the methods to investigate the intestinal resorption (Caco-2 and TC7) are generally expressed as Apparent Permeability. Due to the complexity of the absorption barrier (e.g. villi/crypts) and the presence of peristaltic movements, it is not useful to express intestinal absorption data as ? g/cm (as currently recommended by the SCCNFP for dermal absorption). In order to apply intestinal absorption data in the test strategy as identified by this subgroup, a different approach than for skin absorption is needed. Further improvements of the methods have to be carried out to be applicable in a regulatory framework and the specific data requirements related to this. Due to the complexity of the intestinal barrier because of the presence of uptake/efflux transporters and drug metabolizing enzyme systems and the existence of different mechanisms of transport (transcellular and/or paracellular) further improvements of the methods are necessary to allow the applicability within a regulatory framework. Besides the need for method improvement, efforts should also be made to integrate the current available intestinal permeability assays in a more tiered screening strategy. Absorption is a multifactorial problem that is controlled by many variables such as dissolution rate, solubility, lipophylicity and permeability. Therefore, assays measuring permeability should be linked to physico-chemical properties of chemical properties to allow a better evaluation of the likelihood of absorption and/or systemic exposure. More research and developments efforts are recommended for alternative methods dealing with the inhalation route. Important is that in during these efforts methods are developed in line with the data requirements for Tier 1 methods.
3b. Tier 2
The panel of experts concluded a gap of appropriate tests to predict or even measure the bio-accumulation of compounds after application to Men. Very few tests are available to deal with this aspect and therefore more research and development in this area is recommended. The best known alternative approaches to the in vivo excretion models are the conventional ex vivo models (isolated perfused kidney and liver). In the context of animal replacement these methods have obviously drawbacks. However, these exvivo models contribute significantly to the reduction of animals, and are a valuable alternative for in vivo models such as bile cannulated animals. Currently very few research efforts are carried out to deal with the gap of an in vitro model to address excretion. Although it should be mentioned that enormous efforts in the area of drug transporters involved in the renal and or biliary excretion have resulted in several in vitro models measuring whether or not a drug is a potential substrate for a specific transporter. Extrapolation of the in vitro transporter information to a quantitative value for renal or biliary excretion is still not possible, but these in vitro methods contribute to a better understanding of the potential routes of elimination.
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Some efforts are ongoing to deal with biliary excretion via in vitro approaches since this might be linked with toxicity phenomena (transporter problems etc). For biliary excretion sandwich cultures of hepatocytes are being used to study the extent of biliary excretion and to examine the nature of the metabolites in bile. For renal excretion animal kidney derived cell lines can be used (MDCK cells or LLC-PK1 cells). The information required by alternative approaches for predicting renal and/or biliary excretion is highly relevant for chemicals and pharmaceuticals. The panel regarded this information of less importance for cosmetics. In silico models for renal clearance based on a flow/glomerular filtration physiological description are described. The re-uptake rate is a function of lipophilicity parameterized against in vivo renal clearance data for drugs. Furthermore, efforts are undertaken using epithelial and endothelial monolayer cultures or co-cultures of both cell types which may be adopted to study certain aspects of reabsorption/secretion of (toxic) xenobiotics if used in combination with the more advanced perfusion cell culture technologies. Such models, which are applicable to renal, intestinal, pulmonary barriers and the blood brain barrier need to be kept under organotypic culture conditions. These are laborious and not easy to handle experimental systems, which will be restricted to mechanistic investigations or specific questions facilitating in vitro in vivo comparisons.
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4. Summary of the alternative methods currently available and the foreseeable time to achieve ESAC endorsement
Note: The time limes are the opinion of the majority of the group although for some specific areas no full consensus was reached.
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Stand-alone methods
TIER 1: Likelihood to have systemic exposure
Current endpoints addressed in animal test Alternative test available Endpoints measured Purpose Area (s) of application Validation status Regulatory acceptance Comments Estimated time to have the method validated (ESAC endorsement)*
1. Dermal absorption tests in vitro OECD 417: Dermal absorption 1. QSPR approaches/ physicochemical properties
See chapter 3.5
Partial replacement -> Stop criteria for Tier 2 testing
See chapter 3.5
See chapter 3.5
See chapter 3.5
Need for standardisation efforts specifically related to cosmetics
See chapter 3.5
See chapter 3.5
Partial replacement -> Support for cellular tests and part of test strategy
R&D
About 5 years
OECD 417: Oral absorption
2. QSPR approaches/ physicochemical properties
Modelling of intestinal permeability
Mainly pharmaceuticals
R&D
Various computational approaches using SAR/molecular properties are reported. However, they lack active processes (at present). Furthermore, their applicability depends heavily on the quality and the quantity of available data.
3-4 years
3. Kinetic modelling
Kinetic parameters related to intestinal permeability
Partial replacement -> Support for QSPR approaches and part of test strategy
R&D
3-4 years
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4. Cell based assays (Caco-2 and TC-7 cells) and artificial membranes
Intestinal permeability
Partial replacement -> Supported by 2 and 3 stop criteria for Tier 2; decision point if applicable and part of test strategy
Mainly pharmaceuticals, also dietary nutrients
Optimised -> Well standardised, not formally validated, extensive evaluation studies ongoing sponsored by ECVAM
Further improvements of the methods have to be carried out to be applicable in a regulatory framework and the specific data requirements related to this. Permeability and solubility measurements together enable to make predictions on the absorption class of a compound (low, intermediate or high). Urge the development of in vitro tests and QSPR approaches (particle size is important, pressure, etc, blood flow interaction, air exchange).
4-5 years
5. QSPR approaches/ physicochemical properties OECD 417: Pulmonary absorption 6. Pulmonary epithelium (A549, Calu-3, BEAS-2B , 16HBE14o- and primary cells)
Predicting of uptake in upper and lower respiratory tract
R&D
3-5 years
Pulmonary absorption
Partial replacement -> Supported by 5 stop criteria for Tier 2 and part of test strategy
Pharmaceutical, pollutant gasses and/or particles
R&D and optimised models (some commercial models available)
Urge the development of in vitro tests both for chemicals in vapours-gasses and particulate matter.(Water solubility of vapours particles, particle size, , blood flow interaction, air exchange).
7-8 years
TIER 2: Determination of the distribution of the compound

Current endpoints addressed in animal test Endpoints measured Area (s) of application Validation status Regulatory acceptance Estimated time to have the method validated (ESAC endorsement)*
Test available
Purpose
Comments
OECD 417: Estimation of plasma level
7. QSPR approaches/ physicochemical properties
Plasma protein binding
Partial replacement -> Support for kinetic modelling and final estimation of plasma level and part of test strategy Partial replacement -> Data to be used for kinetic modelling and part of test strategy
R&D
3-5 years
8. Plasma protein binding test
Plasma protein binding
Optimised -> n use. Not formally validated but extensive evaluation studies
3-4 years for prevalidation (validation not applicable)
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9. Kinetic modelling
Kinetic parameters relevant in the calculation of plasma level values
OECD 417: Excretion
No final in vitro test battery available as replacement to the in vivo excretion models, conventional Ex vivo models are still in use for isolated cases 10. QSPR approaches/ physicochemical properties
Renal and biliary excretion
OECD 417: Tissue distribution/Bioaccumulation
Calculation of kinetic parameters relevant in the prediction of tissue distribution In vitro tissueblood/plasma partition coefficient
Partial replacement -> Support for QSPR approaches/ physicochemical properties and Protein binding test and part of test strategy Partial replacement -> Less of importance for cosmetics in comparison to chemicals and pharmaceuticals. Part of test strategy: completion of ADME evaluation Partial replacement -> Support for in vitro models and final estimation of tissue distribution /Bioaccumulation and part of test strategy Partial replacement -> Data to be used for kinetic modelling and part of test strategy
R&D
3-5 years
R&D ->Few research carried out for this specific purpose
Urge the development of in vitro tests and QSPR approaches (highly relevant for chemicals and pharmaceuticals; less of importance for cosmetics)
> 10 years
R&D
Urge the development of alternative methods for bioaccumulation
3-5 years
11. Blood/Tissue partitioning
Most chemical classes (Pharmaceuticals, industrial chemicals)
OECD 417: Biotransformation (metabolism)
12. Metabolic stability & species differences
Disappearance parent compound and related species differences
Partial replacement -> Data to be used for kinetic modelling and to select relevant species and part of test strategy
Mainly pharmaceuticals but applications reported for other chemical classes
Optimised ->In use. Not formally validated but extensive evaluation studies Optimised -> Not formally validated but extensive in house evaluation studies. Need for prevalidation including assessment of intra and inter laboratory reproducibility
3-4 years
The stability screening test should be ready for validation after a consensus meeting where common procedures would be decided. General remark: the availability and quality of human derived systems is questionable.
4-5 years
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13. Bioactivation assays
Metabolismmediated toxicity
Partial replacement -> Decision point for introduction of a metabolic competence in the other areas toxicity testing (Tier 3) and part of test strategy
Optimised -> Need for prevalidation including assessment of intra and inter laboratory reproducibility. Development ongoing of throughput screening system (including approaches using models relevant for human)
4-5 Years
14. In silico approaches
Identification possible metabolites, metabolising enzymes, quantitative kinetics, etc..
Partial replacement -> Support for in vitro models and evaluation of the importance of biotransformation in the hazard evaluation and part of test strategy
R&D
4-5 years
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Tier 1 and Tier 2 integrated in vitro and in silico approaches

Current endpoints addressed in animal test OECD 417: some kinetic parameters Test available Endpoints measured Oral absorption, estimation of plasma levels, biotransformation, blood/tissue partitioning Purpose Area (s) of application Validation status Regulatory acceptance Comments Estimated time to have the method validated (ESAC endorsement)* 3-5 years
Integrated in vitro and in silico approaches
Partial replacement -> Support for overall integrated test strategy
R&D
Integrated human volunteer-based approaches

Current endpoints addressed in animal test OECD 417: some kinetic parameters Test available Endpoints measured Quantification parent compound and its metabolites at sub-pharmacological and sub-toxicological levels Purpose Partial replacement > Support for overall integrated test strategy Area (s) of application Validation status Regulatory acceptance Comments Estimated time to have the method validated (ESAC endorsement)* > 10 years
Accelerator mass spectrometry (AMS) method
Mainly pharmaceuticals, also pesticides & dietary carcinogens
R&D
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5. Recommendations
The subgroup strongly recommended to continue to work on a more detailed version of the tiered strategy as described in this document. 1. ECVAM has followed up this recommendation and invited the members of this subgroup to an ECVAM workshop (January 26-29, 2004) to continue the discussion on the strategy for toxicokinetics and metabolism and interchange ideas with experts presence in carcinogenicity, reproductive toxicity, endocrine disruptors etc 2. A detailed understanding of the metabolic competence of the barriers used in Tier 1 and their relevance for the human situation could be a selection criteria to be used in selecting the in vitro models. Therefore, the group suggested to make this information as soon as possible available in a tabular format. Local effects as a result of local bioactivation would be captured this way. The same reasoning can be done when choosing tests for determining the potency of a compound. If biotransformation is an issue for a specific compound, this aspect should be considered in the test strategy related to that specific potency test. 3. It was recommended by the group to consider alternative validation approaches to validate such an overall test strategy. Validation will be referred to as the ECVAM validation process, however, weight of evidence validation should be considered for many of the building blocks in the overall strategy for a toxicokinetic test strategy. A lot of in-house data (in silico, in vitro and in vivo) are available, especially in the area of toxicokinetics. This aspect on validation of a toxicokinetic test strategy will be a logic step that needs to be worked out in detail by the relevant experts. 4. An important topic is the selection of training or validation sets of chemicals, which would cover a much wider assortment of chemicals than what is usual obtained with drugs. A chemical selection committee should be established in order to short list a list of compounds that will be needed. 5. The increasing use of particulate compounds in cosmetics can (maybe) lead to, until now, unknown health risks, certainly when pulmonary exposure is possible. Therefore, further research efforts should be encouraged to investigate this issue more in depth.
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6. Conclusions
Figure 1 represents a flow chart summarising the tiered approach including the different test batteries and the estimated time to have the method validated.
Tier 1: likelihood to have systemic exposure Dermal route

Subgroup dermal absorption 1 year
Oral route
3-4 years
Inhalation route
7-8 years
Tier 2: determination of the distribution of the compound Estimation of plasma level

3-5 years
Excretion?
> 10 years
Tissue distribution Bio-accumulation

3-5 years
Biotransformation /Metabolism
4-5 years
Tier 1 & 2 integrated in vitro and in silico approaches (part of strategy)

3-5 years
Integrated human volunteerbased approaches (part of strategy)

>10 years
Tier 3: determination of the potency of the compound

Integration of toxicokinetics tiered approach and the obtained data with strategies proposed in chapters 3.1-3.8 & 3.10-3.11
Figure 1: Tiered approach for toxicokinetic related data requirements. Note: The time limes are the opinion of the majority of the group although for some specific areas no full consensus was reached. The panel of experts concluded a gap of appropriate tests to predict or even measure the bio-accumulation of compounds after application to humans. Very few tests are available to deal with this aspect and therefore more research and development in this area is recommended. Although excretion models (in vitro and/or in silico) for renal and biliary clearance are described their status is at the level of research and development. Excretion is highly relevant for chemicals and pharmaceuticals. However, the panel regarded this information of less importance for cosmetics. Based on the results as illustrated in Figure 1, replacement can be achieved for Tier 1 only predicting the likelihood to have systemic exposure within a time frame of about 8 years (ESAC statement under optimum condition e.g. in the case their financial and human resources available to coordinate the prevalidation and validation studies). Tier 2 which determines the distribution of the compound could achieve partial replacement within 5 years for the building blocks dealing with the estimation of plasma level, bioaccumulation and biotransformation/metabolism (ESAC statement under optimum condition e.g. in the case their financial and human resources available to coordinate the prevalidation and validation studies).
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However, to have full replacement of the toxicokinetic data requirements more than 10 years was estimated as a result of the lack of tests for excretion. The data obtained in Tier 1 and 2 will be of great value to be incorporated in the test strategies for the safety assessment of cosmetic ingredients especially for the toxicity areas such as acute toxicity, skin sensitisation, genotoxicity/mutagenicity, photoinduced toxicity, reproductive toxicity, sub-chronic toxicity, carcinogenicity and dermal absorption and therefore prevalidation and validation studies for the toxicokinetic area should have a high priority. In conclusion, with the exception of cases having high dermal absorption, detailed data on both, the toxicokinetics as well as on the metabolism of cosmetic ingredients is currently of less importance. It is clear that the situation in the future will become quite different, when sub-chronic and chronic toxicity tests or even reproductive toxicity studies should be replaced by in vitro methods according to the 7th Amendment on the Cosmetic Directive. The complexity of physiological regulations and impact networks within a living animal will require tiered approaches as the Tiered Strategy detailed in this document, combined with decision points and batteries of different in silico and in vitro methods to answer all the questions on systemic toxicities. Under the new circumstances, toxicokinetic and metabolism will become a predominate information for the safety assessments of cosmetic ingredients and vital for designing the most valuable set of in vitro toxicity tests and the progress made will depend on adequate prioritisation, funding and coordination of efforts.
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Establishment of timetables for the phasing out of animal experiments for cosmetics
Carcinogenicity
Daniela Maurici2 , Marilyn Aardema 1, Raffaella Corvi2, Marcus Kleber3, Cyrille Krul4,; Christian Laurent5, Nicola Loprieno 6,; Markku Pasanen7, Stefan Pfuhler8, Barry Phillips9, David Prentice 10, Enrico Sabbioni2, Tore Sanner11, Philippe Vanparys12, Procter and Gamble, USA; 2ECVAM-JRC, Italy; 3Cognis Deutschland GmbH & Co. KG, Germany; 4TNO Nutrition and Food Research, The Netherland; 5 SCCNFP and EFSA, Bruxelles, Belgium; 6University of Pisa, Italy; 7National Agency for Medicines, Helsinki and Department of Pharmacology and Toxicology, University of Oulu, Finland; 8 Wella AG, Germany; 9Research Animals Department RSPCA, UK; 10PCS Consultant Ltd, CH4132 Switzerland, 11Institute for Cancer Research, Norway; 12 Johnson and Johnson, Belgium.
1
General considerations
Substances are defined as carcinogenic if they induce tumours (benign or malignant), increase their incidence or malignancy, or shorten the time of tumour occurrence when they are inhaled, ingested, dermally applied, or injected. The process of carcinogenesis is now recognised as resulting from the transition of normal cells into cancer cells via a sequence of stages and complex biological interactions. The modeling of such complex adverse effects cannot be accomplished at present by the use of non-animal tests. It should be noted that a well performed carcinogenicity bioassay in animals will in many situations also be a chronic toxicity assay, since the two endpoints can be combined in the guidelines for animal testing. Since the induction of cancer involves genetic alterations which can be induced directly or indirectly, carcinogens have conventionally been divided into two categories according to their presumed mode of action: genotoxic carcinogens (= initiators) and non-genotoxic carcinogens (epigenetic carcinogens = promoters). Most potent mutagens are also carcinogens in animal experiments. Information of genotoxic carcinogenic potential of a substance may be obtained from the mutagenicity/genotoxicity studies. Two in vitro tests, cell transformation and gap junction intercellular communication (GJIC) have been proposed as tests that may provide information on possible non-genotoxic, as well as, genotoxic carcinogens. In
2 cases where the in vitro short-term tests suggest possible carcinogenic potential, carcinogenicity bioassay in animals will be useful to determine potency and target organs. It is generally accepted that carcinogenesis is a multi-step process strongly influenced by factors such as age, diet, environment, hormonal balance, etc. The complexity of this process makes it not only difficult to extrapolate findings in animals to humans, but also difficult to develop in vitro alternative test models. Phases which play a role in the development of tumours are initiation, promotion and progression. It seems to be very difficult to build in all these phases a single in vitro alternative model. A large number of systems and models dedicated to the prediction of carcinogenicity have been developed (QSARs and SARs). It has been demonstrated that carcinogenicity is generally only poorly predicted, and the best models tend to be those that can integrate mechanism-based reasoning with biological data (M. Cronin et al, Environ Health Perspective,111, 1392-1402, 2003)
1. Inventory of the alternative methods currently available Cell transformation assay

B21/not formally validated yet Short description, scientific relevance and purpose Mammalian cell culture systems may be used to detect phenotypic changes in vitro induced by chemical substances associated with malignant transformation in vivo. Widely used cells include SHE, C3H10T1/2 and Balb/3T3, and the tests rely on changes in cell colony morphology and monolayer focus formation. Less widely used systems exist which detect other physiological or morphological changes in cells following exposure to carcinogenic chemicals. Cytotoxicity may be determined by measuring the effect of the test material on colony-forming abilities (cloning efficiency) or growth rates of the cultures. Developer of the method SHE: Berwald Y. and Sachs L. (1963, 1965), Di Paolo JA et al (1969) Low pH SHE: LeBoeuf RA (1987) Balb/3T3: Aaronson SA and Todaro GJ. (1968) C3H10T1/2: Mondal S. and Heidelberger C. (1970) Known users Academics. Pharmaceutical and cosmetic industry in screening purposes only. Status of the validation and standardisation It is considered by the European Community, although not formally validated. The OECD is working on the rewriting of a detailed review paper (DRP31) aiming at reviewing all the available data on the 3 main protocols for cell transformation assays. This should be circulating by the first quarter of 2004. Data are available today after different validation studies done in the past. After comments, a decision will be made concerning the writing of a specific OECD Test Guideline. This ongoing work should progress in the coming 3 years. Depending on the results and conclusions of the DRP31 process, the need for specific validation study could be discussed. Field and limitations of application The in vitro test endpoints have been established based on induction of tumorigenicity. Some of the test systems are capable of detecting tumour promoters. Some cell types and substances may require an appropriate external metabolic activation system. When primary cell types are used that possess intrinsic metabolic activity, additional metabolic activation is not used. The scoring of transformed colonies and foci may require some training and experience. Recommendations of use in the view of animal replacement None of the in vitro test endpoints has an established mechanistic link with cancer. Ongoing development Protocol standardisation
References
- Aaronson SA and Todaro GJ. (1968). Development of 3T3-like line from BALB/c mouse embryo culture: transformation susceptibility to SV40. J Cell Physiol. 72:141148. - LeBoeuf RA et al (1987). Enhanced morphological transformation of early passage Syrian hamster embryo cells cultured in medium with a reduced bicabonate concentration and pH. Carcinogenesis 8, 689-697. - Berwald Y and Sachs L. (1963). In vitro transformation of normal cells to tumor cells by carcinogenic hydrocarbons. J. Nat. Cancer Inst. 35: 641-661. - DiPaolo J.A. et al. (1969) Quantitative studies of in vitro transformation by chemical carcinogens. J Ntl Cancer Inst 42, 867-874. - Isfort R.J. et al. (1996). Comparison of the standard and reduced pH Syrian hamster embryo (SHE) cell in vitro transformation assays in predicting the carcinogenic potential of chemicals. Mutat. Res. 356, 11-63. -Mondal S. and Heidelberger C. (1970). In vitro malignant transformation by methylcholanthrene of the progeny of single cells derived from C3H mouse prostate. Proc.Natl.Acad.Sci.U.S.A, 65, 219-225. - Matthews E.J.et al. (1993). Transformation of BALB/c-3T3 cells: V. Transformation responses of 168 chemicals compared with mutagenicity in Salmonella and carcinogenicity in rodent bioassays. Environmental Health Perspectives 101 Suppl 2, 347-482. - Rivedal E. and Sanner T. (1982) Promotional effect of different phorbol esters on morphological transformation of hamster embryo cells. Cancer Letters 17, 1-8.
Gap junction intercellular communication (GJIC)

Short description of the method Gap junction intercellular communication (GJIC) is the intercellular exchange of low molecular weight molecules (less than 1000-1500 D) through gap junction channels between adjacent cells, and has been found to be of importance in regulation of cell growth and differentiation. Dysfunction in this type of communication has been observed to result in abnormal cell growth and behaviour, and associated with several human pathological conditions. Structure activity studies have shown relationship between the ability of substances to inhibit GJIC, and to induce tumours in rodents, but not between inhibition of GJIC and genotoxic activity. This suggests that inhibition of GJIC is involved in non-genotoxic cancer induction, and a candidate endpoint in screening assays for identification of non-genotoxic carcinogens and tumour promoters not detected by conventional genetic toxicology tests. Developer of the method Yotti LP. et al (1979), Murray AW. and Fitzgerald DJ (1979) Known users Academics Status of the validation and standardisation No formal validation of the method has been performed. Several methods exist for the determination of GJIC in different cell types. Field and limitation of application Method for identification and characterization of cancer causing substances without genotoxic activity. Recommendations of use in the view of animal replacement Ongoing development Work is ongoing to determine: ?? Molecular mechanisms involved in regulation of GJIC ?? Role of altered GJIC in human diseases ?? Importance of substances with adverse effect on GJIC in relation to human diseases including cancer ?? Supplemental role of GJIC in addition to other assay end points
References
- Blaha L. et al. (2002). Inhibition of gap-junctional intercellular communication by environmentally occurring polycyclic aromatic hydrocarbons. Toxicol.Sci., 65, 43-51. - Murray A.W. and Fitzgerald D.J. (1979). Tumour promoters inhibit metabolic cooperation in cocultures of epidermal and 3T3 cells. Biochem.Biophys.Res.Commun., 91, 395-401. -Rivedal,E. et al. (2000). Morphological transformation and effect on gap junction intercellular communication in Syrian hamster embryo cells as screening tests for carcinogens devoid of mutagenic activity. Toxicol. In Vitro, 14, 185-192. - Rosenkranz,H.S. et al. (2000). Exploring the relationship between the inhibition of gap junctional intercellular communication and other biological phenomena. Carcinogenesis, 21, 1007-1011. - Yamasaki H. et al. (1996). Nongenotoxic carcinogens: development of detection methods based on mechanisms: a European project. Mutat. Res., 353, 47-63. - Yotti L.P. et al. (1979). Elimination of metabolic cooperation in Chinese hamster cells by a tumour promoter. Science, 206, 1089-1091.
3. Reduction / refinement
Since no replacement methods are to date available in the area of carcinogenicity, we considered also reduction and refinement methods. In all transgenic models described below a limited number of animals 20 25 animals/sex/treatment group can be used. Another advantage is that duration of the experiment in transgenic models is 6 9 months instead of a two years bioassay. However, in addition wild type animals should be included in test battery to demonstrate that no genetic drift may affect interpretation of the results. Transgenic mouse models The transgenic mouse lines were chosen with the objective of developing short-term assay in vivo that could be used to differentiate carcinogenic from non-carcinogenic chemicals and mutagenic from non-mutagenic carcinogens. Mouse with well-defined genetic alteration resulting in over-expression or inactivation of a gene intrinsic to carcinogenesis, but insufficient alone for neoplastic conversion, appear to offer susceptible targets for carcinogenic chemicals. Mainly three mouse models are considered of a greatest potential usefulness: Tg.AC, heterozygous (+/-) p53-deficient, and TgHras 2 lines. Tumour genes altered in these mice have been often reported to be mutated and/or amplified (Ha-ras) or mutated and/or lost (p53) in both human and rodent tumours. As general consideration, it has to be kept in mind that the genetic backgrounds of these 3 models are different, which undoubtedly influences responses to carcinogens. Tg.AC mice These animals carry a v-Ha-ras oncogene fused to the promoter of the zeta globin gene (Leder et al 1990). The v-Ha-ras transgene has point mutations in codon 12 and 59 and the site of integration of the transgene confers on these mice the characteristic of genetically initiated skin as a target for tumorigenesis. The Tg.AC model is thus analogous to the classical, widely studied model for two stage carcinogenicity in mouse skin (Boutwell, 1964). The presence of the v-Ha-ras transgene in Tg.AC mice obviates the obligatory exposure to initiating agents that was necessary in the earlier model of carcinogenesis. The transgene is not expressed constitutively in skin of the Tg.AC mice, so untreated skin is indistinguishable from the skin of the wild-type parent strain. Repetitive dermal applications of recognised mouse skin tumour promoters to untreated skin of Tg.AC mice result in a dose-related development of benign squamous-cell papilloma which can progress to malignancy. The Tg.AC mice line is useful to differentiate carcinogens from non-carcinogens but it cannot be used to distinguish genotoxic complete carcinogenes from agents that have only tumour promoting capability. It has been suggested in the Workshop on the utility of transgenic assay for risk assessment (Washington, DC, February 2003) organised by ILSI-HESI Alternative to Carcinogenicity Testing Committee, that Tg.AC model is the second most
8 commonly requested assay and is considered to be appropriate for evaluation of products intended for dermal application. Heterozygous p53+/- deficient mice Mice that are heterozygous for the suppressor gene p53 are at increased risk for tumour development. The heterozygous p53-deficient mice used in rapid (26-week exposure) studies for the identification of mutagenic carcinogens have one functional wild-type allele and one inactivated null allele and the mice remain generally free of sporadic neoplastic disease during short-term studies. Inactivation of the remaining wild-type p53 allele by mutation or loss due to exposure to a mutagenic carcinogen would be expected to give a selective growth advantage to clonally derived neoplasia and result in shortened tumour latency. Positive controls need to be included in the test battery. It has been reported in the Workshop on the utility of transgenic assay for risk assessment (Washington, DC, February 2003) organised by ILSI-HESI Alternative to Carcinogenicity Testing Committee, that the p53 +/- assay is the most commonly requested assay and it is also considered appropriate in providing data for regulatory purposes.
CBF1-Tg-Hras2 mice CBF1-Tg-Hras2 mice carry five or six copies of a human c-Ha-ras proto-oncogene which is expressed both in spontaneus tumours and normal tissue It has been reported in the Workshop on the utility of transgenic assay for risk assessment (Washington, DC, February 2003) organised By ILSI HESI Alternative to Carcinogenicity Testing Committee, that Tg-H-ras2 model is considered appropriate for evaluation of both genotoxic and non-genotoxic compounds even if further evaluation using additional non-direct acting agents is suggested. Positive controls need to be included in the test battery.

p53-deficient, and TgHras have been adopted for the evaluation of carcinogenic potential of pharmaceuticals to replace a mouse life time assay. Evaluation of skin mutation and/or neoplasia in dermally exposed transgenic mice (e.g. Tg.AC, p53) may be relevant after further validation (3-4 years) and can be used in the mechanistic evaluation.
References
] - Botwell RK. (1964). Some biological aspects of skin carcinogenesis. Proc. Exp Tum Res, 4, 207-250, - Dean SW. et al. (1999). Transgenic mouse mutation assay systems can play an important role in regulatory mutagenicity testing in vivo for the detection of site-ofcontact mutagens. Mutagenesis. Jan;14(1):141-51. - Harvey M. et al. (1993). Spontaneous and carcinogen-induced tumorigenesis in p53deficient mice. Nat Genet 5, 225-229. - Leder A, et al. (1990). v-Ha-ras transgene abrogates the initiation step in mouse skin tumorigenesis: effects of phorbol esters and retinoic acid. Proc Natl Acad Sci U S A. Dec; 87(23):9178-82 - Tennant RW. et al. (1999). Genetically altered mouse model for identifying carcinogens. IARC Scientific Pubblication # 146.
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3. Final Comments
The process of carcinogenesis is recognised as resulting from a sequence of stages and complex biological interactions. The modeling of such complex adverse effects cannot be accomplished at present by the use of non-animal tests. The experts suggested that the carcinogenic potential of a substance could be detected by a combination of the existing in vitro genotoxicity tests, cell transformation assays and gap junction intracellular communication assay (GJIC). Although several in vitro tests exist to detect the genotoxic potential of a compound, they present crucial limitations as absence of metabolic capacity, as well as the use of cell lines not relevant to predict the endpoint at target organ (see also chapter on Genotoxicity and Mutagenicity). Moreover, non-genotoxic carcinogens act through a variety of mechanisms that are problematic to predict and to detect. As a general limitation, cancer is a long-term process that is difficult to mimic with relatively short-term in vitro tests. The carcinogenicity bioassays are rarely used for cosmetics but the bioassay is still needed to determine potency and target organ of a carcinogenic compound. The opinion of the experts is that a battery of in vitro short-term tests will not be sufficient to fully replace the carcinogenicity bioassay in rodent in the next ten years. The experts also suggested that in vivo assay with transgenic animals may provide important reduction and refinement in animal use. In conclusion, taking into consideration the present state of the art of the carcinogenicity tests, the experts were unable to suggest a deadline for full replacement.
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Endpoints measured
Purpose
Validation status
Comments
CTA assay (SHE) B21
Transformed colonies
Genotox and nongenotox damage
CTA assay (Balb/3T3) B21
Foci formation
CTA assay (C3H10T) B21
Transformed colonies
Partial replacement (tiered strategy and/or test battery) Partial replacement (tiered strategy and/or test battery) Partial replacement (tiered strategy and/or test battery)
Genotox/carcinogen
Optimised
EC (Annex V)
Develop standard protocol
5 years
Genotox/carcinogen
Optimised
EC (Annex V)
5 years
Genotox/carcinogen
Optimised
EC (Annex V)
5 years
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Non genotox damage
Gap junction intercellular communication (GJIC)
Partial replacement Inhibition of (Tiered GJIC strategy and/or test battery)
Carcinogenicity
R&D
+ 10 years
Document of the subgroup Reproductive and developmental toxicity.
Susanne Bremer1, Rita Cortvrindt2, George Daston3, Brighitta Eletti1, Alberto Mantovani4, Francesca Maranghi4, Olavi Pelkonen5, Irmela Ruhdel6, Horst Spielmann7 ECVAM-JRC, Ispra, Italy; 2Eggcentris NV, Zellik (Brussels), Belgium; 3P&G, Ross OH, USA; 4Laboratorio di Tossicologia Comparata ed Ecotossicologia, Istituto Superiore di Sanit, Roma, Italy; 5Department of Pharmacology and Toxicology, University of Oulu, Finland; 6German Animal Welfare Federation Animal Welfare Academy, Neubiberg, Germany; 7ZEBET, Berlin, Germany
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Chapter: Reproductive and developmental toxicology Introduction: Reproductive toxicity refers to the adverse effects of a substance on any aspect of the reproductive cycle, including the impairment of reproductive function, the induction of adverse effects in the embryo, such as growth retardation, malformations, and death. All these events and interactions are controlled to a large extent by the body's endocrine system. Due to the complexity of the mammalian reproductive cycle it is not possible to model the whole cycle in one in vitro system in order to detect chemical effects on mammalian reproduction. However, the cycle can be broken down in its biological components that can be studied individually or in combination. This has the enormous advantage that the target tissue/organ of an agent can be identified. In certain areas of reproductive toxicity testing, a number of useful and promising in vitro models are already available and need now to be converted into individual tests with a predictive power for safety testing. However it should be clear that most of the single tests cannot be a replacement, only in combination with other tests detailed hazard identification will be possible. The individual tests can then be used as building blocks to compose a tiered testing strategy. It has to be noted that major attention has been devoted to develop and validate assays on some components of reproductive toxicology, in particular the organogenesis of the embryo reflecting important concerns for teratogenic chemicals and the interaction with steroid receptors in order to detect endocrine disrupters. In these areas building blocks for a test strategy exist and have been implemented in several laboratories. Other areas (fertility, implantation, placenta and fetal toxicity) received lower attention. However, as mentioned before the existing in vitro models need to be further developed and the protocols have to be optimised before the relevance and reliability of these methods can be judged. Nevertheless we have to take care not being too reductionist by this approach. Many of the processes we are trying to model (e.g., gametogenesis) are dependent on cell-cell interactions as well as paracrine and endocrine signalling. It's not clear to me how useful cultures of gametes will be in the absence of Sertoli cells, or of Sertoli/germ cell cultures in the absence of endocrine factors. These are questions we don't know the answers yet, but need to be explored if we are to create truly predictive models of reproduction. An inventory of promising tests has been listed under point 3. in point 2 only advanced tests are presented that have been validated or that have been proofed to be transferable from one laboratory to another with the aim to test chemicals. Unfortunately many of the well developed tests rely on tissues derived from intact mammals. This includes micromass, whole embryo culture, sliced testis,
and ovarian follicle culture. These are excellent models, but they still rely on intact rodents as their source. Due to the fact that a revision of the EU Directive 86/609 is planned, it should be mentioned that in future the protection of foetuses could change. This may have consequences for some tests such as the whole embryo culture. 2. Inventory of alternative methods currently available 2.1. State of the art Around 15% of all animals used for toxicological safety testing are employed for the identification of chemical hazards to human reproduction. The complex biology of mammalian reproduction requires the analysis of toxic effects on various aspects of the reproductive cycle such as teratogenicity, endocrine disruption, germ cell mutagenicity, impaired fertility etc. In order to detect chemical effects on different target cells/organs of the reproductive cycle 7 OECD guidelines have been implemented and additional 3 draft guidelines are under discussion. However all guidelines are based on animal tests. ECVAM has recently validated three embryotoxicity tests. Their further use was the topic of an ECVAM workshop in January 2003. This work will be extended to further endpoints, introducing metabolising capabilities and the adaptation from murine to human embryonic stem cells. It has to be stressed that metabolic factors are important not only for test dealing with developmental toxicity but also in all in vitro or ex vivo systems since it is a primary activity in all intact organisms. These aspects should be discussed in connection with all alternatives, either how metabolism (or a lack of it) could affect the outcome of the test, or how and at which later stage interspecies metabolic differences (if the test system is non-human) should be elucidated and taken into assessment of the outcome. Besides metabolism at target (i.e., reproductive/developing) tissues, it might be useful to deal with a specific aspect relevant to risk assessment of cosmetics: percutaneous absorption and metabolism. Far to be a barrier, skin is an actively metabolising tissue in which are recognizable the following enzymes: - The complex of tyrosinase with TRP1 and 2 for the conversion of L-tyrosine to melanine (which is a possible binding sites for xenobiotics); - Some different endonucleases possibly related to skin DNA repair, e.g., after UV damages; - Some cytokines for the local immune response - And, most important, two isoforms of the P450 cytochrome: CYP 1A1 inside the basal layer and CYP1B1 which has aryl hydrocarbon hydroxylase activity in other cutaneous districts, still to be defined.
If deemed appropriate, the message of considering skin-specific metabolism might be conveyed to all in vitro or ex vivo systems dealing with systemic (as opposed to topical) toxicity of cosmetics. Furthermore, the prevalidation of a Leydig cell model (MA 10 cells) originating from a project funded in FP4 by DG Research was initiated. J.Goldmann/EPA also recommended the further test optimisation. Reproductive toxicity was chosen also as the pilot field to develop test strategies for a systemic type of toxicity. By developing an application for an Integrated Project (ReProTect,), which is still under evaluation by DG Research, the whole reproductive cycle was broken down into work packages and suitable tests were identified. These shall now be evaluated, optimised, prevalidated and combined to a testing strategy. In parallel, the conceptual framework to compose and validate test strategies shall be developed. In the following inventory list tests have been classified according to their state of development: 1. Within the first part of the inventory, only test methods that have been successfully validated are listed and their relevance and reliability have been confirmed within a peer review process. 2. The second class of tests is containing very advanced in vitro models; those that are in a pre/validation process, those for which validation studies are planned and those that have the potential to be validated. 3. In the third class of tests promising in vitro models are summarized which need to be further developed. 2.2 Successfully validated tests Alternatives to test for developmental toxicology (OECD TG 414) The thalidomide disaster in the late 50s and early 60s has led to a worldwide concern about the vulnerability of the unborn child. In the field of developmental toxicity, a variety of alternatives to animal testing are available. Within a workshop in 1994 a panel of experts have reviewed the status of alternative methods in this area. The participants of the workshop have recommended the analysis of relevance and reliability of 4 embryotoxicity tests. ECVAM has funded the prevalidation and validation of three recommended embryotoxicity tests: the whole embryo culture (WEC) test, the micromass (MM) test, and embryonic stem cell test (EST). The 4th recommended test, the frog embryo teratogenesis assay has been evaluated by ICCVAM. Within a workshop held in January 2003 a panel of experts have confirmed the transferability and robustness of the tests. However, in order to cover all major manifestations of developmental toxicity the participants of the workshop recommend several improvements and refinements of the different test
concerning the prediction models as well as the integration of metabolic systems etc. (A workshop report is in preparation). The proposed tests can only cover single aspects of developmental toxicity. In order to receive a reliable hazard identification the development of test strategy for developmental toxicity is necessary combining various in silico and in vitro tests. 2.2.1 Alternative method 1: the embryonic stem cell test used as building block in a test strategy in order to replace the OECD guideline 414 (developmental toxicity testing) The EST was performed as described according to an updated version of INVITTOX protocol no. 113. Principally, the test is composed of two procedures, a cytotoxicity test which is conducted with the mouse embryonic stem (ES) cell line D3 and cells of the differentiated mouse fibroblast cell line 3T3, and a differentiation assay using D3 cells. Three toxicological endpoints were identified to classify the embryotoxic potential of chemicals: (i) the inhibition of differentiation of ES cells into cardiomyocytes (ID50) and (ii) the decrease of viability of adult 3T3 cells (IC503T3) and (iii) ES cells (IC50D3) in a MTT cytotoxicity test. The validation of the prediction models provided the following results: EST provides an overall accuracy of 78%. References: 1.Spielmann, H., Pohl, I., Dring, B., Liebsch, M., and Moldenhauer, F. (1997): The embryonic stem cell test (EST), an in vitro embryotoxicity test using two permanent mouse cell lines: 3T3 fibroblasts and embryonic stem cells. In Vitro Toxicology, 10:119-127. 2.Anon. Embryonic Stem Cell Test (EST). INVITTOX protocol no. 113. 2002. The ERGATT/FRAME Data Bank of In Vitro Techniques in Toxicology. ECVAM SIS database http://ecvam-sis.jrc.it/index.html . 3.Scholz G., Genschow E., Pohl I., Bremer S., Paparella M., Raabe H., Moldenhauer F., Southee J.A., Spielmann H. (1999). Prevalidation of the Embryonic Stem Cell Test (EST), a new in vitro Embryotoxicity Test. Toxicology in Vitro 13, 675-681. 4.Genschow, E., Scholz, G., Brown, N., Piersma, A., Brady, M., Clemann, N., Huuskonen, H., Paillard, F., Bremer, S., Becker, K., and Spielmann, H. (2000): Development of prediction models for three in vitro embryotoxicity tests. In Vitro and Molecular Toxicology, 13:51-65. 5. Genschow E., Spielmann H., Scholz G., Seiler, A., Brown N.A., Piersma A, Brady M, Clemann N, Huuskonen H, Paillard F, Bremer B & Becker K. (2002). The ECVAM international validation study on in vitro embryotoxicity tests. Results of the definitive phase and evaluation of prediction models. ATLA 30, 151-176.
6.Elke Genschow, Horst Spielmann, Gabriele Scholz, Ingeborg Pohl, Andrea Seiler, Nicole Clemann, Susanne Bremer and Klaus Becker. Validation of the embryonic stem cell test (EST) in the international ECVAM validation study of three in vitro embryotoxicity tests; ATLA in press Developer of the method: ZEBET Known users: ZEBET (D), Bayer AG (D), Schering AG (D), Altana AG (D), Hoffmann-La Roche (CH), Syngenta (UK), Osaka Dental School (Jap) Status of validation and/or standardisation Is the protocol defined/optimised? INVITTOX protocol, SOP. Is there data on intra-laboratory variation? Yes Is there data on the transferability? Yes Is there data on the inter-laboratory variability? Yes In vivo /in vitro comparisons? Yes Is the method validated? Yes Which efforts are needed to complete validation of the method? Extension of data base- testing of additional chemicals + addition of metabolizing system. 2.2.2 Alternative method 2: The whole embryo culture used as building block in a test strategy in order to replace the OECD guideline 414 (developmental toxicity testing) Mammalian embryo cultures derived from mice, rats or rabbits are used for the detection of developmental toxicants. Head fold or early somite stage embryos are dissected free from maternal tissue, parietal yolk sac and Reicherts membrane, leaving the visceral yolk sac and ectoplacental cone intact. The conceptus is cultured in medium under defined conditions for 24-48 hours. Medium containing a high proportion of serum is usually used and test compounds are added to the cultures for appropriate periods of time. A defined number of endpoints are analysed after the incubation period such as dysmorphogenic effects, embryonic growth, differentiation, yolk sac circulation and vascularisation, effects on the haematopoiesis etc.
References: 1.Rat whole embryo culture (WEC) INVITTOX Protocols No. 68 + 72, 2002 The ERGATT/FRAME Data Bank of In Vitro Techniques in Toxicology. ECVAM SIS database http://ecvam-sis.jrc.it/index.html 2.New D.A.T. (1978). Whole embryo culture and the study of mammalian embryos during organogenesis. Biol. Rev. 53, 81-122.
3.Brown N.A. & Fabro S. (1981). Quantification of rat embryonic development in vitro: a morphological scoring system. Teratology 24, 65-78. 4.Bechter R & Schmid B.P. (1987). Teratogenicity in vitro. A comparative study of four antimycotic drugs using the whole embryo culture system. Toxic. in vitro 1 11-15. 5.Piersma A.H., Bechter R., Krafft N., Schmid B.P., Stadler J., Verhoef A., Verseil C. & Zijlstra J. (1996). An interlaboratory evaluation of five pairs of teratogens and non-teratogens in post-implantation rat embryo culture. ATLA 24, 201-209 6.Sadler TW, Horton WE, Warner CW. Whole embryo culture: a screening technique for teratogensvarious Teratogenesis, Carcinogenesis and Mutagenesis 1982; 2:243-53 7.Genschow E., Spielmann H., Scholz G., Seiler, A., Brown N.A., Piersma A, Brady M, Clemann N, Huuskonen H, Paillard F, Bremer B & Becker K. (2002). The ECVAM international validation study on in vitro embryotoxicity tests. Results of the definitive phase and evaluation of prediction models. ATLA 30, 151-176. 8.Aldert H. Piersma, Elke Genschow, Aart Verhoef, Marielle Q.I. Spanjersberg, Nigel A. Brown, Madeleine Brady, Angie Burns, Nicole Clemann, Andrea Seiler & Horst Spielmann: Validation of the rat postimplantation whole embryo culture (WEC) test in the international ECVAM validation study of three in vitro embryotoxicity tests; ATLA in press.
Developer of the method: Dennis New, Nigel Brown, Tom Sadler Known users: Hoffmann-La Roche (CH), Syngenta (UK), RIVM (NL) Status of validation and/or standardisation Is the protocol defined/optimised? yes Is there data on intra-laboratory variation? yes Is there data on the transferability? yes Is there data on the inter-laboratory variability? yes In vivo /in vitro comparisons? Is the method validated? yes Which efforts are needed to complete validation of the method? Extension of database, inclusion of metabolising system. 2.2.3 Alternative method 3: The micromass culture used as building block in a test strategy in order to replace the OECD guideline 414 (developmental toxicity testing). The MM is making use of cell cultures of the limb bud and/or neuronal cells. The cells are isolated from the limb or the cephalic tissues of a mid-
organogenesis embryos. After preparing a single cell solution the cells are seeded in a high density and undergo differentiation into chondrocytes and neurons without additional stimulation. The differentiation after exposure to test chemicals is analysed by using defined toxicological endpoints. References: 1.The micromass in vitro embryotoxicity test, INVITTOX Protocols No. 114 +122, 2002 The ERGATT/FRAME Data Bank of In Vitro Techniques in Toxicology. ECVAM SIS database http://ecvam-sis.jrc.it/index.html . 2.Brown N.A., Spielmann H., Bechter R., Flint O.P., Freeman S.J., Jelinek R.J., Koch E., Nau H., Newall D.R., Palmer A.K., Renault J.-Y., Repetto M.F., Vogel R., and Wiger R. (1995). Screening chemicals for reproductive toxicity: the current alternatives. The report and recommendations of an ECVAM/ETS workshop (ECVAM Workshop 12) ATLA 23: 869-882. 3.Flint O.P. (1980) The effects of sodium salicylate, cytosine arabinoside and eserine sulphate on rat limb buds in culture. in Teratology of the Limb (eds, H.J. Merker, H.Nau and D. Neubert) Walter de Gruyter and Co.; Berlin, pp. 325-338. 4.Flint O.P. (1983) A micromass culture method for rat embryonic neural cells. J. Cell. Sci. 61: 247-262. 5.Flint O.P. (1987) An in vitro test for teratogens using cultures of rat embryo cells. in In Vitro Methods in Toxicology (eds. C.K. Atterwill and C.E. Steele) Cambridge University Press; Cambridge England, pp. 339-363. 6.Flint O.P., and Orton T.C. (1984) An in vitro assay for teratogens with cultures of rat embryo midbrain and limb bud cells. Toxicol. Appl. Pharmacol. 76: 383-395. 7.Freeman S.J., and Brown N.A. (1987) Sub-mammalian and sub-vertebrate models in teratogenicity screening. in In Vitro Methods in Toxicology (eds. C.K. Atterwill and C.E. Steele) Cambridge University Press; Cambridge England, pp. 391-409. 8.Rockley J., and Richold M. (1990) In vitro micromass teratogen test: interpretation of results from a blind trial of 25 compounds using three separate criteria. Toxicol In Vitro 4: 609-611. 9.Peters P.W.J., and Piersma A.H. (1990) In vitro embryotoxicity and teratogenicity studies. Toxicol In Vitro 4: 570-576. 10.Uphill P.F., Wilkins S., and Allen J.A. (1990) In vitro micromass teratogen test: results from a blind trial of 25 compounds. Toxicol In Vitro 4: 623-626. 11.Whittaker S.G., and Faustman E.M. (1994) In vitro assays for developmental toxicity. in In Vitro Toxicology (ed. S. Cox Gad) Raven Press; New York, pp. 97-122. IP-114 July/96. 12.Genschow E., Spielmann H., Scholz G., Seiler, A., Brown N.A., Piersma A, Brady M, Clemann N, Huuskonen H, Paillard F, Bremer B & Becker K. (2002).
The ECVAM international validation study on in vitro embryotoxicity tests. Results of the definitive phase and evaluation of prediction models. ATLA 30, 151-176. 13.Horst Spielmann, Elke Genschow, Nigel A. Brown, Aldert H. Piersma, Aart Verhoef, Marielle Q.I. Spanjersberg, Hannele Huuskonen, Francoise Paillard , Andrea Seiler:Validation of the rat limb bud micromass (MM) test in the international ECVAM validation study of three in vitro embryotoxicity tests; ATLA in press.
Developer of the method: Oliver Flint, Andreas Kistler Known users: Sanofi-Synthelabo (F), Hoffmann La Ruoche (CH) Status of validation and/or standardisation: Validation (The protocol using micromass cultures of the limb buds has been validated.) Is the protocol defined/optimised? YES Is there data on intra-laboratory variation? YES Is there data on the transferability? YES Is there data on the inter-laboratory variability? YES In vivo /in vitro comparisonsvarious Not sufficient Is the method validated?s Yes Which efforts are needed to complete validation of the method? Extension of the database and inclusion of metabolising systems.
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2.3 Advanced tests 2.3.1 Alternative method 1: Analysis of progesterone produced by a modified Leydig Cell line (cLHMRMA 10) used as building block in a test strategy in order to replace one/two generation studies (OECD TG 415, TG 416) Evidence for the deterioration in mammalian (including human) male reproductive health is accumulating. A wide range of pharmaceutical compounds has been reported to be toxic for one of the pivotal cell types controlling spermatogenesis. Some of these compounds have been demonstrated to have a direct effect on Leydig cells and thus modulate the amount of testosterone formed. It can be anticipated that some compounds affect aspects in the LH/hCG-stimulated steroidogenesis (e.g. influence hCG-stability or hCG receptor interaction). In vitro testing of compounds on their effects on steroidogenesis in Leydig cells requires stimulation with LH(hCG). Therefore, reliability, reproducibility and standardisation of these tests should be improved by the use of Leydig cell lines with an increased basal steroid production. An existing Leydig cell line has been modified to express constitutively active hCG/LH receptors. The cells have high levels of cholesterol sidechain cleavage enzyme activity and progesterone synthesis. The modified Leydig (cLHRMA10) cells are maintained in monolayer culture at 37oC. Potential toxicants are added to the cultures in a suitable solvent and the cultures are maintained for various time periods (eg 2-24h). At the end of the incubation periods the medium is removed for analysis of progesterone. Other tests characterising specific cytotoxic effects on Leydig cells can be carried out. The use of the modified MA10 Leydig cells will enable the effects of potential toxicants to be evaluated that directly affect the process of cAMP controlled steroidogenesis. Also the effects on cytotoxicity, proliferation and apopotosis can be evaluated. It will not test effects of compounds on the LH-receptor-G-protein adenylate cyclase system. It will not predict chemical effects on the hypothalamic-pituitarytesticular axis. References 1. Cooke BA, Lindh LM, van der Molen HJ.1979 The mechanism of action of lutropin on regulator protein(s) involved in Leydig-cell steroidogenesis Biochem J 15;184(1):33-8 . 2. Koizumi T , Li ZG, 1992 The role of oxidative stress in single dose cadmium induced testicular cancer J Toxicol. Envrrron Health 37:25-36. 3.Chaudhary LR, Stocco DM 1989 Inhibition of hCG- and cAMP-stimulated progesterone production in MA-10 mouse Leydig tumor cells by ketoconazole.Biochem Int 18(1):251-62.
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4.English HF, Santer SJ, Levine HB, Santen RJ 1989 Inhibition of testosterone production with ketoconazole alone and in combination with a gonadotrophin relaesing hormone analogue in the rat Cancer Research 46, 38-42 5.Much Product information on web site of the company: www.Monsanto .com 6.Walsh LP, McCormick C, Martin C, Stocco 2000 Roundup inhibits steroidogenesis by disrupting steroidogenic acute regulatory (StAR) protein expression. DM Environ Health Perspect 108(8):769-76 7.Rommerts FFG, Teerds KJ, Hoogerbrugge JW 1988 In vitro effects of ethylene-dimethane sulfonate (EDS) on Leydig cells: Inhibition of steroid production and cytotoxic effects are dependent on species and age of rat. Mol Cell Endocrinol 55:87-94 8.Molenaar R, de Rooij DG, Rommerts FF, Reuvers PJ, van der Molen HJ 1985 Specific destruction of Leydig cells in mature rats after in vivo administration of ethane dimethyl sulfonate. Biol Reprod 33(5):1213-22 9. Gangoli SD 1982Testicular effects of phthalate esters. Environ Hlth Perspect. 45, 77-84 10.Dees JH, Gazouli M, Papadopoulos V 2001 Effect of mono-ethylhexyl phthalate on MA-10 Leydig tumor cells Reprod Toxicol 15(2):171-87/ 11.Sonnenschein C , Soto AM 19898 An updated reviewof environmental estrogen and androgen mimics and antagonists J Steroid Biochem Molec Biol 65:143-150. 12. Nikula H, Talonpoika T, Kaleva M, Toppari J 1999 Inhibition of hCGstimulated steroidogenesis in cultured mouse Leydig tumor cells by bisphenol A and octylphenols Toxicol Appl Pharmacol 157(3):166-73 13.Freeman DA, Goeze PM Porpaczy Z 1993 Finasteride blocks progesterone synthesis in MA-10 Leydig tumor cells Endocrinology 133, 1915-1917 14.Stoner E, 1990 The clinical development of a 5 alpha reductase inhibitor, finasteride. J Steroid Biochem Molec Biol 37, 375-378. 15.Sullivan MH, Cooke BA 1984 The effect of calcium on the potentiation of LH-stimulated steroidogenesis and inhibition of LH-stimulated cyclic AMP production by LHRH agonist (ICI 118630) in rat Leydig cells Mol Cell Endocrinol 34(1):17-22 16.Clegg ED, Cook JC, Chapin RE, Foster PMD, Daston GP 1997 Leydig cell hyperplasia and adenoma formation : mechanisms and relevance to humans Reproductive Toxicology 11,107-121
Developer of the method: Erasmus University, Rotterdam Known users: Erasmus University, Rotterdam, Trinity College Dublin. Status of validation and/or standardisation Is the protocol defined/optimised? In progress.
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Is there data on intra-laboratory variation? Not available, but have been performed during a DG RTD project in the 4th Framework Programme. Is there data on the transferability? Not available, but have been performed during a DG RTD project in the 4th Framework Programme. Is there data on the inter-laboratory variability Not available, but have been performed during a DG RTD project in the 4th Framework Programme In vivo /in vitro comparisons? No Is the method validated? No Which efforts are needed to complete validation of the method? Protocol optimisation and successful prevalidation. 2.3.2 Alternative method 2: Follicle culture bioassay used as building block in a test strategy in order to replace one/two generation studies (OECD TG 415, TG 416). The functional unit within the ovary is the follicle, which is also a morphological unit consists of three basic cell types: theca cells, granulosa cells and the oocyte. The somatic compartment synthesizes and secretes hormones (steroids and growth factors) necessary for the inter-relationship between the other parts of the reproductive tract and the central nervous system. At birth the pool of primordial resting follicles is definitively set; any destruction at this level is definitive and cannot be restored. Each stage during the process of follicular growth exhibits unique patterns of gonadotrophin sensitivity; steroid production and feed back pathways to keep the hypothalamic-pituitary-gonadal axis. The regulation of every component of the follicular unit (Theca cells, granulosa cells and oocyte) can be affected at each specific level and as such disturb the reproductive system at different levels. This creates a situation in which the pattern of infertility induced by a particular agent is dependent on the types of follicle, which are affected. The oocyte is for its growth and maturation dependent on the health and development of the surrounding somatic cells (= process of folliculogenesis). During this long-lasting process, each of these compartments are vulnerable to chemical insults at every developmental stage and insults will be reflected by impaired oocyte quality which, can result in meiotic incompetence, fertilisation failure, or inadequate embryo development. Using the follicle culture bioassay it becomes possible to study low dose / long term effects of chemicals on the somatic compartments of non-luteinised tissue. Steroid production as well as steroid receptor expression can be studied in vitro in relation to oocyte quality. The spent medium from in vitro exposed follicles can be analysed for estrogen, androgen and progestin content (immunoassays). Changes in steroidogenesis and receptor expression profiles under the influence of chemical exposures will be studied in relation to oocyte quality.
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Future prospects and recommendations Developer of the method: Vrije Universiteit Brussel (VUB), Brussels Known users: EggCentris NV, Brussels. Status of validation and/or standardisation. Is the protocol defined/optimised? Sop. Is there data on intra-laboratory variation? yes Is there data on the transferability? No (although used by other lab :Eichenlaub ritter Bielefeld to detect oocyte aneuploidy). Is there data on the inter-laboratory variability? no In vivo /in vitro comparisons? No. Is the method validated ?no. Which efforts are needed to complete validation of the method? (Protocol optimisation ) and successful pre-validation. References 1.Cortvrindt R, Smitz J, Van Steirteghem AC. In-vitro maturation, fertilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system. Hum Reprod 11, 26562666, 1996 2.Cortvrindt R, Smitz J, Van Steirteghem AC. Assessment of the need for follicle stimulating hormone in early pre-antral mouse follicle in-vitro culture. Hum Reprod 12, 759-768, 1997 3.Cortvrindt R and Smitz J. Early preantral mouse follicle in-vitro maturation: oocyte growth, meiotic maturation and granulosa-cell proliferation quantified. Theriogenology 49 (4), 845-859, 1998 4.Cortvrindt R, Hu Y, Liu J, Smitz J. A timed analysis of the nuclear maturation of oocytes in recombinant gonadotropin-supplemented early preantral mouse follicle culture. Fertil Steril 70 (6), 114-1125, 1998 5.R. Cortvrindt, J. Smitz .Follicle culture in reproductive toxicology: a tool for in-vitro testing of ovarian functionvarious Hum Reprod Update, 8, 243-254, 2002. 6.Hu Y, Cortvrindt R, Smitz J. Effects of aromatase inhibition on in vitro follicle and oocyte development analyzed by early preantral mouse follicle culture. Mol Reprod Dev. 2002 Apr;61(4):549-59. 2.3.3 Alternative 3: The human adrenocortical carcinoma cell line H295R The tests could be used as building blocks in order to replace one/two generation studies (OECD TG 415, TG 416). The development of a test protocol for a cell-based test that would address the complete steroidogenesis pathway including aromatase activity from cholesterol
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to 17 ? estradiol is currently ongoing in the US. The test is based on the human adrenocortical carcinoma cell line H295R.
Future prospects and recommendations Developer of the method: Vrije Universiteit Brussel (VUB), Brussels Known users: EggCentris NV, Brussels Status of validation and/or standardisation Is the protocol defined/optimised? sop Is there data on intra-laboratory variation? yes Is there data on the transferability? No (although used by other lab :Eichenlaub ritter Bielefeld to detect oocyte aneuploidy) Is there data on the inter-laboratory variability? no In vivo /in vitro comparisons? no Is the method validated? no Which efforts are needed to complete validation of the method? (Protocol optimisation) and successful prevalidation. References: 1.Sanderson J.T., Seinen W., Giesy J.P., and van den B.M., 2-Chloro-s-triazine herbicides induce aromatase (CYP 19) activity in H295R human adrenocortical carcinoma cells: a novel mechanism for estogenicity. Toxicol. Sci. 54, 121-127, 2000. 2.3.4. Alternative 4: Aromatase tests. The tests could be used as building blocks in order to replace one/two generation studies (OECD TG 415, TG 416). The enzyme aromatase is responsible for the conversion of androgens into estrogens. This biological process normally occurs in several male and female tissues, particularly in the ovary and in the placenta. Several pesticides and flavonoids have been demonstrated to be aromatase inhibitors. An in vitro aromatase assay could be utilized as screening method to assess the potential of various environmental toxicants on aromatase activity. Two study designs are currently under review: 1.The cell lines JEG-3 and JAR choriocarcinoma cell lines have been used as in vitro system. 2.In addition, in vitro subcellular assays have been developed by using human placental microsomes. The microsomal preparation consists of the endoplasmatic reticulum membrane-bound cytochrome P450 aromatase and the NADPH-cytochrome P450 reductase. The test uses a radiometric assay measuring the release of tritiated water from the substrate [1? -3H]-
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andostendione. This test is used in several academic labs and in pharmaceutical companies.
Future prospects and recommendations Developer of the method: Known users: Status of validation and/or standardisation: The US EPA is currently conducting studies to optimise the placental aromatase protocols. This will be compared with the human recombinant aromatase assay Is the protocol defined/optimised? Is there data on intra-laboratory variation? Is there data on the transferability? Is there data on the inter-laboratory variability? Inter-Laboratory validation studies on human recombinant aromatase and/or placental aromatase will be performed. In vivo /in vitro comparisons? Is the method validated? Which efforts are needed to complete validation of the method? References: 1.Pelissero C., Lenczowski M.J. chinzi D., Davail-cussit B. Sumpter J.P. Fostier A. Effects of falvonoids on aromatase activity, an in vitro study. J.Steroid biochem.Mol. 70, 249-256, 1999. 2.Robertson K.M., ODonell, Jones m.E., Meachem S.J., Boon W.C., fisher C.R. Graves K.H., Mclachlan R.I. and simpson E.R. Impairment of spermatogenesis in mice lacking a functional aromatase (Cyp 19) gene. Proc.Natl.Acad.Sci. U.S.A. 96, 7986-7991, 1999. 3.Kellis J.T. and Vickery L.E. Purification and characterization of human placental aromatase cytochroe P-450. J.Biol.chem. 262, 4413-4420, 1987. 4.Means G.D. Kilgore M.W.Mahedroo M.S. Mendelson C.R. and Simpson E.R. tissue-specific promoters regulate aromatase cytochrome P450 gene expression in human ovary and fetal tisuues. Mol.Endocrinology. 5 2005-2013, 1991. 5.Stresser D.M., Turner S.D. McNamara J., Stocker P., Miller V.P., Crespi C.L, and Patten C.J. A high-troughput screen to identify inhibitors of aromatase (CYP 19). Anal. Biochem. 284, 427-430, 2000. 6.Ibrahim A.R., Abul-Haji Y.J. Aromatase inhibition by flavonoids. J.Steroid Biochem. Mol. Biol., 37, 257-260, 1990. 7.Vinggaard A.M., Hnida C., Breinholdt V., larson J.C. Screening of selected pesticides for inhibition of CYP19 in vitro. Toxicol. In Vitro 14, 227-234, 2000
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8.Bueggemeier R.W. and katlic N.E. Aomatase inhibition by an enzymeactivated irreversible inhibitior in human carcinoma cell cultures. Cancer Res. 50, 3652-3656, 1990. 9.Letcher R.J., Drenth H.J., Norstrom R.J., Bergman A., Safe S., Pieters R. Cytotoxicity and aromatase (CYP 19) activity modulation by organochlorines in human placental JEG-3 and JAR choriocarcinoma cells. Toxicol. Appl. Pharmacol. 160, 10-20, 1999. 2.3.5 Alternative 5: Sliced Testis. The tests could be used as building blocks in order to replace one/two generation studies (OECD TG 415, TG 416) Sliced Testis Assay: The US EPA is presently conducting optimization studies of the sliced testes assay including verifying the measurement procedures (Testosterone, RIA, lactate dehydrogenase) and optimization of the experimental factors (incubation media, temperature, volume, atmosphere, etc) A multichemical study will conducted on approximately 15 chemicals. Inter-laboratory validations studies are also planned. This test should be replaced by the H295R assay if the prevalidation of this test is successful. Future prospects and recommendations Developer of the method: Known users: Status of validation and/or standardisation Is the protocol defined/optimised? Currently ongoing conducted by US EPA Is there data on intra-laboratory variation? Is there data on the transferability? Is there data on the inter-laboratory variability? In vivo /in vitro comparisons? Is the method validated? Which efforts are needed to complete validation of the method? Protocol optimisation and successful prevalidation. 2.3.6 Alternative 6: Computer assisted sperm analysis used as building block in a test strategy in order to replace one/two generation studies (OECD TG 415, TG 416) The computer assisted sperm analysis (CASA) allows to monitor adverse effects of chemicals on spermatozoa with possible implications on fertility potential viability, motility, velocity, motion, and morphology of mammalian semen will be analysed in real-time. This allows the detection of reversible and irreversible damages (recovery effect) to the mature sperm as well as repeated dose effects. For reproductive medicine fully automated semen analysers are available.
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Several chemicals have already been tested in different laboratories and an INVITTOX protocol is available.
Developer of the method: Christian-Albrecht-Universitat, Kiel, Germany various Known users: Istituto Sperimentale Italiano Lazzaro Spallanzani, Rome; TNO, Nutrition and Food Research, Zeist Status of validation and/or standardisation Is the protocol defined/optimised? INVITTOX protocol Is there data on intra-laboratory variation? Is there data on the transferability? Is there data on the inter-laboratory variability? In vivo /in vitro comparisons? Is the method validated? Which efforts are needed to complete validation of the method? Protocol optimisation and successful prevalidation. References: 1.Aschmann, C., Stork, T. & Wassermann, O. (1989) Short-term effects of chlorophenols on the function and viability of primary cultured rat hepatocytes. Arch. Toxicol., 63, 121-126. Babich, H. & Borenfreund, E. (1987) in vitro cytotoxicity of organic pollutants to bluegill sunfish (BF-2) cells. Environ. Res., 42, 229-237. 2.Ekwall, B., Selling, J. & Johnels, D. (1987) Toxicity of chlorophenols to HeLA cells as measured in the MIT-24 system. ATLA, 14, 178-181 3.Glden, M. & Burghoff, C. (1990) Effects of membrane directed neurotoxicants on the contractile activity of cultured skeletal muscle cells. ATLA, 17, 215-217. 4.Hong, C.Y., Chaput de Saintonge, D.M. & Turner, P. (1981) A simple method to measure drug effects on human spermatozoa motility. Br. J. Clin. Pharmacol., 11, 385-387. Kolossa, M. & Seibert, H. (1990) A chemically "defined" diluent for cryopreservation of bovine spermatozoa. Andrologia, 22, 445-454. 5.Seibert, H. (1988) Messung der Bewegungsaktivitt der Spermatozen von Mensch und Rind mit Hilfe von Videomikrographie und Computerbildanalyse. Fertilitt, 4, 215-218. 6.Seibert, H., Kolossa, M. & Wasserman, O. (1989) Bovine spermatozoa as an in vitro model for studies on the cytotoxicity of chemicals: Effects of chlorophenols. Cell Biology and Toxicology, 5, 315-330. 7.Seibert, H. & Gosch, U. (1990) A short-term bovine sperm cell assay for the evaluation of the in vitro cytotoxicity of chemicals. ATLA, 17, 228-232.
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8.Tessereaux, I., Glden, M., & Wassermann, O. (1987) Cultured myotubes from skeletal muscle of adult rats. Characterization and action of Anemonia sulcata toxin II. Naunyn Schmiedeberg's Arch. Pharmacol., 336, 232-239. 9.Voss, J.-U. & Seibert, H. (1991) Microcarrier-attached rat hepatocytes as a xenobiotic-metabolizing system in cocultures. Cell. Biol. Toxicol. (Submitted) 10.WHO (1987). WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. Cambridge University Press, Cambridge. 11.Videomicrographic techniques Weiss, D.G. (1989) Videomicroscopic measurements in living cells: dynamic determination of multiple endpoints for in vitro toxicology. Journal of Molecular Toxicology, 1, 465-488. 2.3.7 Alternative 7: Hamster Egg Penetration Test/ Hypoosmotic Swelling Test. The tests could be used as building blocks in order to replace one/two generation studies (OECD TG 415, TG 416) As regards sperm toxicity, a combination of the computer assisted sperm analysis such as HEPT (Hamster Egg Penetration Test) or HOS (Hypoosmotic Swelling Test) would give information about the functional status of the sperm (above all regarding the membrane status for HPO) to assess the sperm quality, to analyse fertilizing capacity and also to detect viable, immotile cells. These endpoints may be highly relevant to such reproductive disorders as delayed conception, and reflect the following events i) sperm capacitation, ii) sperm/oocyte fusion and iii) zona-pellucida binding. Neither sperm count/morphology nor simple zona-pellucida binding appears to assess fertilizing capacity as accurately and efficiently as HEPT. Human spermatozoa (e.g., from semen banks) are used: are there ethical considerations??(Nevertheless, experimental papers using HEPT are currently published by international scientific journals). HEPT is currently used by a number of laboratories working on andrology and reproductive biology (see references); although (to our best knowledge) HEPT is not validated in toxicology, it is described in the WHO Laboratory Manual for the examination of human semen and sperm-cervical mucus interactions (1999), where a protocol is provided.
Future prospects and recommendations Developer of the method: Known users: Status of validation and/or standardisation Is the protocol defined/optimised?sop Is there data on intra-laboratory variation? yes
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Is there data on the transferability? No (although used by other lab :Eichenlaub ritter Bielefeld to detect oocyte aneuploidy) Is there data on the inter-laboratory variability? no In vivo /in vitro comparisons? no Is the method validated? no Which efforts are needed to complete validation of the method?( Protocol optimisation ) and successful prevalidation. References: 1.Francavilla F, Santucci R, Macerola B, Ruvolo G, Romano R. Nitric oxide synthase inhibition in human sperm affects sperm-oocyte fusion but not zona pellucida binding. Biol Reprod. 2000 Aug;63(2):425-9. 2.Francavilla F, Romano R, Santucci R, Macerola B, Ruvolo G, Francavilla S. Effect of human sperm exposure to progesterone on sperm-oocyte fusion and sperm-zona pellucida binding under various experimental conditions. Int J Androl. 2002 Apr;25(2):106-12. 3.Zahalsky MP, Zoltan E, Medley N, Nagler HM. Morphology and the sperm penetration assay. Fertil Steril. 2003 Jan;79(1):39-41. 2.3.8 Alternative 8: Estrogen receptor binding assays. The tests could be used as building blocks in order to replace the uterotrophic test (OECD draft) and one/two generation studies (OECD TG 415, TG 416) The assay is designed to identify chemicals that bind to the estrogen receptor (ER). The ER is a transcriptional regulatory protein belonging to the nuclear hormone receptor superfamily. The receptor plays a major role in controlling the transcriptional activation and/or repression of estrogen-responsive genes. The ER binding assay can detect both estrogen agonists and antagonists but the assay cannot distinguish between the two. An in vitro ER competitive binding assay is generally performed by quantifying the ability of substances to compete with 17? -estradiol for binding. The US EPA is planning to validate the rat uterine cytosol assay. Other binding assays for ER ? and ER? have been established. 948 chemicals have been tested for binding activity on ER ? in Japan (METI study).
Known users: Status of validation and/or standardisation: Is the protocol defined/optimised? yes Is there data on intra-laboratory variation? Is there data on the transferability? Is there data on the inter-laboratory variability?
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In vivo /in vitro comparisons? Is the method validated? Validation planned by US EPA
References: 1.Culig, Z., Hobisch, A., Bartsch, G., and Klocker, H. (2000) Androgen receptor-an update of mechanisms of action in prostate cancer, Urol Res 28: 211-219. 2.Elsby R, Maggs JL, Ashby J, Paton D, Sumpter JP, Park BK. (2001) Assessment of the effects of metabolism on the estrogenic activity of xenoestrogens: a two-stage approach coupling human liver microsomes and a yeast estrogenic assay, J Pharmacol Exp Ther 296:329-37 3.Ramamoorthy K, Wang F, Chen IC, Norris JD, McDonnell DP, Leonard LS, Gaido KW, Bocchinfuso WP, Korach KS, Safe S. (1997) Estrogenic activity of a dieldrin/toxaphene mixture in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based estrogen receptor assays: no apparent synergism. Endocrinology 138:1520-7. 4.Arcaro et al. J Cell Biochem 72:94-102, 1999. 5.Brooks et al. Cancer Res 44:3724-9, 1984. 6.Dodge et al. J Steroid Biochem Mol Biol 59:155-161, 1996. 7.Kramer et al. Toxicol Appl Pharmacol 144:363-76, 1997. 8.Lascombe et al. Environ Health Perpect 108:621-629, 2000. 9.Miodini et al. Br J Cancer 80:1150-5, 1999. 10.Nagel et al. Environ Health Perspect.105:570-6, 1997. 11.Palomino et al. J Steroid Biochem Mol Biol. 50:75-84, 1994. 12.Rijks et al . Eur J Nucl Med. 23:295-307, 1996. 13.Stoessel and Leclercq J Steroid Biochem. 25:677-82, 1986. 14.VanderKuur et al. Biochemistry. 32:7002-8, 1993. 15.Soto et al. Environ Health Perpect 102:380-383, 1995. 16. Arcaro et al. J Toxicol Environ Health A. 59:197-210, 2000. 17.Gaido et al. Endocrinology 140:5446-53, 1999. 18.Klotz et al. J Environ Health Pers. 104:1084-9, 1996. 19.Kraichely et al. Endocrinology 141:3534-453, 2000. 20.Meyers et al J Med Chem. 42:2456-68, 1999. 21.Sun et al. Endocrinology 140:800-4, 1999. 22.Vakharia and Gierthy Toxicol Lett. 114:55-65, 2000. 23.Fertuck et al. Toxicol Lett. 121:167-77, 2001. 24.Bolger et al. Environ Health Pers. 106:551-7, 1998. 24.Hanioka et al. Xenobiotica 29:1213-26, 1999. 25.Hashimoto et al. Dent Mater J. 19:245-62, 2000. 26.Nikov et al. Environ Health Perspect. 108:867-72, 2000.
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27.Nikov et al. J Endocrinol. 170:137-45, 2001. 28.Paker et al. J Biomol Screen 5:77-88, 2000. 29.Saito et al. Toxicol Sci. 57:54-60, 2000. 30. Anon (2002) In Vitro Endocrine Disruptor Final Background Review Documents (BRDs) http://iccvam.niehs.nih.gov/methods/endodocs/ed_brd.htm accessed 25.9.2003. 2.3.9 Alternative 9: Estrogen receptor transcriptional assay. The tests could be used as building blocks in order to replace the uterotrophic test (OECD draft) and one/two generation studies (OECD TG 415, TG 416) A straight-forward screening method for estrogenic compounds is making use of the fact that the receptor for estrogens is a transcription factor that induces transcription of target genes by linking the DNA sequences to the gene of an easily measurable protein (the so-called reporter gene). Introduced in a suitable cell line (HeLa, HepG2, MCF-7, CHO, T47D), an estrogen responsive reporter cell line can be generated allowing large-scale screening of chemicals. The estrogen receptor can be endogenous (MCF-7, T47D cells), transiently or stably transfected. Problems can occur by using the more transient transfected cell lines since they are loosing the vectors after several passages. The in vitro ER TA assays are designed to identify substances that might interfere with normal estrogen activity in vivo by acting as agonist or antagonist. In vitro ER TA assays quantify the induction of reporter gene product or the stimulation of cell growth in response to activation of ER by the test substance or reference estrogen. The antagonism is measured by the inhibition of the reference estrogen induction of the reporter gene, or cell proliferation. Several laboratories are performing ER TA by using the following cell lines: MCF-7 cell lines T47D cell lines MVLN cell lines HeLa cell lines transfected with reporter gene T47D+ pLuc (ER-CALUX ) CHO cell lines transfected with p Luc (Eco SCREEN ) A stable cell line for human ER? (HeLa-cell based) is currently validated in Japan. The outcomes of the validation study will be presented and discussed at the next meeting (early 2004) of the Validation Management Group for NonAnimal Tests within the OECD EDTA.
Developer of the method: Known users: Status of validation and/or standardisation:
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Is the protocol defined/optimised? yes Is there data on intra-laboratory variation? Is there data on the transferability? Is there data on the inter-laboratory variability? In vivo /in vitro comparisons? Is the method validated? Reference: 1.Anon (2002) In Vitro Endocrine Disruptor Final Background Review Documents (BRDs) http://iccvam.niehs.nih.gov/methods/endodocs/ed_brd.htm accessed 25.9.2003. 2.3.10 Alternative 10: Androgen receptor binding assay The tests could be used as building blocks in order to replace the Hershberger test (OECD draft) and one/two generation studies (OECD TG 415, TG 416) The assay is designed to identify substances that bind to the androgen receptor (AR). The AR has a high degree of homology with members of the steroid hormone receptor family based on this, the AR is closely related to the progesterone, glucocorticoid, and mineralcorticoid receptor and this can results in an unspecific bound. The AR binding assay can detect both androgen agonists and antagonists but the assay cannot distinguish between the two. The US EPA is planning a validation study using the rat prostate cytosol assay.
Known users: Status of validation and/or standardisation: Is the protocol defined/optimised? yes Is there data on intra-laboratory variation? Is there data on the transferability? Is there data on the inter-laboratory variability? In vivo /in vitro comparisons? Is the method validated? Validation planned by US EPA
References: 1.Tilley, W.D., Marcelli, M., Wilson, J.D.., and McPhau; M.J. (1989) Characterization and expression of a cDNA encoding the human androgen receptor. Proc Natl Acad Sci USA 86: 327-331.
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2.Wilson VS, Bobseine K, Lambright CR, Gray LE Jr. A novel cell line, MDAkb2, that stably expresses an androgen- and glucocorticoid-responsive reporter for the detection of hormone receptor agonists and antagonists.Toxicol Sci. 2002 Mar;66(1):69-81. PMID: 11861974 3.Culig Z, Hobisch A, Bartsch G, Klocker H. Androgen receptor-an update of mechanisms of action in prostate cancer. Urol Res. 2000 Aug;28(4):211-9. Review. 4.Wilson & French J Biol Chem. 251:5620-9, 1976. 5.Kemppainen & Wilson Urology 48:157-63, 1996. 6.Kemppainenet et al. J Biol Chem. 267:968-74, 1992. 7.Kemppainenet et al. Mol Endocrinol.13:440-54, 1999. 8.Takeo & Yamashito Gen Comp Endocrinol. 117:200-6, 2000. 9.Tilley et al. Proc Natl Acad Sci U S A. 86:327-31, 1989. 10. Brown et al.Steroids. 37:635-48, 1981. 11. Anon (2002) In Vitro Endocrine Disruptor Final Background Review Documents (BRDs) http://iccvam.niehs.nih.gov/methods/endodocs/ed_brd.htm accessed 25.9.2003. 2.3.11 Alternative 11: Androgen receptor transcriptional assay The tests could be used as building blocks in order to replace the Hershberger test (OECD draft) and one/two generation studies (OECD TG 415, TG 416) Similar to the estrogen transcriptional assay the in vitro AR TA assays are designed to identify substances that might interfere with normal androgen activity in vivo by acting as agonist or antagonist. These assays use genetically engineered cell lines for analysing reporter gene products (e.g. luciferase) that is under AR control. Agonisms are generally performed by quantifying the induction of report gene product or the stimulation of cell growth in response to activation of the AR. In case of antagonisms the assay measure the ability of a test substance to inhibit the induction of the reporter gene product or the stimulation of cell growth by a reference androgen. The following cell lines have been manipulated: CV-1 +pLuc CHO-K1 (Eco SCREEN ) MDA-MB-453-kb2 Palm cell lines AR-CALUX variousvarious The US EPA has developed an AR TA assay by using the MDA-kb2 cell line, which expressed both the AR and the glucocorticoid receptor. These cells have the advantage to contain an endogenous androgen receptor avoiding the transfection procedure using a patented human recombinant androgen receptor.
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17 chemicals have been used in the assay development. Currently 10 laboratories in the US are using this cell line.
Developer of the method: Known users: 10 labs in USA Status of validation and/or standardisation at the next Is the protocol defined/optimised? Is there data on intra-laboratory variation? Is there data on the transferabilityvarious Is there data on the inter-laboratory variability? In vivo /in vitro comparisons? Is the method validated? References: 1.Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan.2001 submitted report. 2.Vinggaard AM, Breinholt V, Larsen JC. Screening of selected pesticides for oestrogen receptor activation in vitro. Food Addit Contam. 1999 Dec;16(12):533-42. 3.Vinggaard AM, Hnida C, Larsen JC. Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro. Toxicology. 2000 Apr 14;145(2-3):173-83. 4.Maness SC, McDonnell DP, Gaido KW. Inhibition of androgen receptordependent transcriptional activity by DDT isomers and methoxychlor in HepG2 human hepatoma cells.Toxicol Appl Pharmacol. 1998 Jul;151(1):135-42. 5.Tamura H, Maness SC, Reischmann K, Dorman DC, Gray LE, Gaido KW. Androgen receptor antagonism by the organophosphate insecticide fenitrothion. Toxicol Sci. 2001 Mar;60(1):56-62. 6.Wilson VS, Bobseine K, Lambright CR, Gray LE Jr. A novel cell line, MDAkb2, that stably expresses an androgen- and glucocorticoid-responsive reporter for the detection of hormone receptor agonists and antagonists.Toxicol Sci. 2002 Mar;66(1):69-81. 7.Lambright C, Ostby J, Bobseine K, Wilson V, Hotchkiss AK, Mann PC, Gray LEJr. Cellular and molecular mechanisms of action of linuron: an antiandrogenic herbicide that produces reproductive malformations in male rats.Toxicol Sci. 2000 Aug;56(2):389-99. 8.Hartig PC, Bobseine KL, Britt BH, Cardon MC, Lambright CR, Wilson VS, Gray LE Jr. Development of two androgen receptor assays using adenoviral transduction of MMTV-luc reporter and/or hAR for endocrine screening. Toxicol Sci. 2002 Mar;66(1):82-90. PMID: 11861975.
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9.Kelce WR, Stone CR, Laws SC, Gray LE, Kemppainen JA, Wilson EM. Persistent DDT metabolite p,p'-DDE is a potent androgen receptor antagonist. Nature. 1995 Jun 15;375(6532):581-5. PMID: 7791873. 10.Kemppainen JA, Wilson EM. Agonist and antagonist activities of hydroxyflutamide and Casodex relate to androgen receptor stabilization. Urology. 1996 Jul;48(1):157-63. No abstract available. PMID: 8693644. 11.Kemppainen JA, Lane MV, Sar M, Wilson EM. Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. Specificity for steroids and antihormones.J Biol Chem. 1992 Jan 15;267(2):96874. PMID: 1730684 12.Kemppainen JA, Langley E, Wong CI, Bobseine K, Kelce WR, Wilson EM. Distinguishing androgen receptor agonists and antagonists: distinct mechanisms of activation by medroxyprogesterone acetate and dihydrotestosterone. Mol Endocrinol. 1999 Mar;13(3):440-54. PMID: 10077001. 13.Lambright C, Ostby J, Bobseine K, Wilson V, Hotchkiss AK, Mann PC, Gray LE Jr. Cellular and molecular mechanisms of action of linuron: an antiandrogenic herbicide that produces reproductive malformations in male rats.Toxicol Sci. 2000 Aug;56(2):389-99. PMID: 10910998. 14.Van Dort ME, Robins DM, Wayburn B. Design, synthesis, and pharmacological characterization of 4-[4,4-dimethyl-3-(4-hydroxybutyl)-5-oxo2-thioxo-1-imidazolidinyl]-2-iodobenzonitrile as a high-affinity nonsteroidal androgen receptor ligand.J Med Chem. 2000 Aug 24;43(17):3344-7. PMID: 10966753. 15.Hartig PC, Bobseine KL, Britt BH, Cardon MC, Lambright CR, Wilson VS, Gray LE Jr. Development of two androgen receptor assays using adenoviral transduction of MMTV-luc reporter and/or hAR for endocrine screening. Toxicol Sci. 2002 Mar;66(1):82-90. PMID: 11861975. 16.Sultan C, Balaguer P, Terouanne B, Georget V, Paris F, Jeandel C, Lumbroso S, Nicolas J. Environmental xenoestrogens, antiandrogens and disorders of male sexual differentiation. Mol Cell Endocrinol. 2001 Jun 10;178(1-2):99-105. PMID: 11403899. 17.Terouanne B, Tahiri B, Georget V, Belon C, Poujol N, Avances C, Orio F Jr, Balaguer P, Sultan C. A stable prostatic bioluminescent cell line to investigate androgen and antiandrogen effects. Mol Cell Endocrinol. 2000 Feb 25;160 (12):39-49. PMID: 10715537. 18. Anon (2002) In Vitro Endocrine Disruptor Final Background Review Documents (BRDs) http://iccvam.niehs.nih.gov/methods/endodocs/ed_brd.htm accessed 25.9.2003.
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2.3.12 Alternative chick embryo retina cell culture. The tests could be used as building blocks in order to replace OECD guideline 414 for developmental toxicity testing The screen is a primary culture of chick embryo neural retina cells (CERC) which undergo processes of cell-cell recognition and interaction, growth, and differentiation over a 7-day culture period.
Developer of the method: Known users: Status of validation and/or standardisation at the next Is the protocol defined/optimised? Is there data on intra-laboratory variation? Is there data on the transferability? Is there data on the inter-laboratory variability? In vivo /in vitro comparisons? Is the method validated? References: 1.Daston, G.P., Baines, D., and Yonker, J.E. 1991. Chick embryo neural retina cell culture as a screen for developmental toxicity. Toxicol. Appl. Pharmacol., 109: 352-366. 2.Daston, G. P., Baines, D., Elmore, E., Fitzgerald, M. P., and Sharma, S. 1995. Evaluation of chick embryo neural retina culture as a screen for developmental toxicants. Fund. Appl. Toxicol. 26: 203-210. 2.4 Identified areas or endpoints with no validated alternative methods available In many areas of reproductive toxicology promising in vitro methods are available which have the potential to be converted into a predictive toxicological tests. A series of workshops is necessary in order to agree which sensitive organs and chemicals can affect biological processes of the reproductive cycle. It should be decided which in vitro model has the potential to predict these effects and which efforts are necessary to complete test development. However, all currently available in vitro systems are premature and their reliability and relevance for safety toxicity testing have to be proven in additional studies. These efforts should lead to the identification of the most predictive toxicological endpoints as well as to a preliminary prediction models. The following areas of reproductive toxicology are known to be sensitive to chemical insult and related in vitro systems should be included into test strategies. The selection of the major part of the proposed tests is based on a
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literature review done for the data collection activities of the ECVAM database. Please find also attached the results of the intermediate report in the attachment. A study of updating the literature review is planned. 2.4.1 Germ cell mutagenicity including mutagenic effects on spermatogonial and primordial germ cells (see also subgroup of mutagenicity) The new possibility to culture spermatogonial stem cells allows now to analyse one of the major target of germ cell mutagens in vitro. It needs to be exploited if these cell lines can be used as basis for a germ cell mutagenicity test in vitro. However, the subgroup of mutagenicity stated that germ cell mutagenicity is not relevant for cosmetic testing. Since this issue is controversial discussed this point needs further clarification. References: 1.Feng LX, Chen Y, Dettin L, Pera RA, Herr JC, Goldberg E, Dym M. Generation and in vitro differentiation of a spermatogonial cell line. Science. 2002 Jul 19;297(5580):392-5. 2.van Pelt AM, Roepers-Gajadien HL, Gademan IS, Creemers LB, de Rooij DG, van Dissel-Emiliani FM. Establishment of cell lines with rat spermatogonial stem cell characteristics. Endocrinology. 2002 May;143(5):1845-50. 3.Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, Shinohara T. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod. 2003 Aug;69(2):612-6. Epub 2003 Apr 16. 2.4.2 Germ cell mutagenicity test using permanant embryonic germ cell lines of BALB/cJ mice - an in vitro alternative for in vivo germ cell mutagenicity tests. To offer a sensitive and predictive in vitro method to assess germ cell mutagenicity, we established primordial germ (PG) cell-derived permanent female and male embryonic germ (EG) cell lines of the mouse (strain BALB/cJ). The differences in developmental sensitivity of EG cells and differentiated fibroblast cells of the mouse cell line 3T3 to genotoxicants were tested comparatively under identical test conditions. Cytotoxicity assay was measured by the MTT-test and genotoxic effects were determined by sister chromatid exchanges (SCE) rates induced by standard reference mutagens. Both methods are used to assign the chemicals to two classes of in vivo reproductive toxicity ,non- and strongly genotoxic to germ cells. Applying linear discriminant analysis, a biostatistical prediction model (PM) was developed for the female cell line EG3. This procedure identified a single variable, the Ig(SCE200EG3) as the statistically significant concentration related
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increase of 200% in the mean number of SCEs per metaphase spread after 3 hrs of exposure to be sufficient for separation into the classes: non- and strongly genotoxic to germ cells. Applying this PM to the training set of 5 genotoxic and 3 non genotoxic test chemicals, 100% correct classifications were obtained. References: 1.Klemm, M., Genschow, E., Pohl, I., Barrabas, C., Liebsch M. and Spielmann, H. Permanent embryonic germ cell lines of BALB/cJ mice - an in vitro alternative for in vivo germ cell mutagenicity tests Toxicology in Vitro 15, 447453, 2001. 2.4.3 Germ cell maturation including tests for Spermatogenesis/ Epididymal epithelium, Sperm activation assays, Granulosa tests/Thecal tests, in vitro fertilisation tests 2.4.3.1. Coculture of epididymal spermatozoa and epididymal epithelium. A successful in vitro method of spermatogenesis have been obtained by incubating epididymal spermatozoa with primary cultures of epididymal epithelium. These co-incubation methods promote sperm motility and the capacity of spermatozoa to bind to and fertilize oocytes, and extend the viability of spermatozoa in vitro. 2.4.3.2. Sertoli-Germ Cell Co-cultures. 2.4.3.3. foetal gonad culture (Blanche Capel), allowing to look at male /female development of the gonadal anlage. 2.4.3.4. newborn ovary cultures (rat) ( developed by marc skinner), allows evaluation of effects on the process of follicle formation, the resting follicle pool and follicle growth initiation. Reference Devine PJ, Sipes IG, Skinner MK, Hoyer PB. Characterization of a rat in vitro ovarian culture system to study the ovarian toxicant 4-vinylcyclohexene diepoxide. Toxicol Appl Pharmacol. Oct 15;184(2):107-15, 2002. 2.4.3.5. Seminiferous Tubulus Culture assay. Reference: 1.Staub & Durand P, INRA , France. The whole meiotic process can occur in vitro in untransformed rat spermatogenic cells. Exp cell res 2000, 260 (1): 8595. 2.Development of the meiotic step in testes of of pubertal rats: comparison between the in vivo and under in vitro conditions. Mol Reprod Dev 2003, 65: 86-95. 2.4.3.6.Seminiferous Tubulus Function assay.
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2.4.3.7. Sperm activation assay. 2.4.3.8 In vitro fertilisation sperm toxicity test (INVITTOX No59). 2.4.3.9 In vitro fertilisation for mouse/bovine this technique is well established in many labs working on reproduction. Established endpoints are available. 2.4.3.10 In vitro fertilisation oocyte toxicity test many established techniques are available to evaluate oocyte quality. Eg: spindle staining, chromosome preparations, calcium measurement, ivf, blastocyst analysis. References: See data collection of ECVAM attachment No 1 2.4.4 Cell lines employed in studies of steroidogenesis. Several other cell lines such as R2C (rat Leydig cell tumor line with a high basal P4, high levels of P450 aromatase and 5-? -reductase), H540 (rat Leydig cell tumor line; can produce androgens with db-cAMP pre-treatment, loss of responsiveness to hCG/LH), mLTC-1 (mouse Leydig cell tumor line; P4 and testosterone), KGN (human granulosa-like tumor cell line; relatively high aromatase, P4 secretion responsive to db camp), HO-23 (immortalised human granulosa cell line, P4 secretion), Jc-410 (stable procine granulosa cell line, P4, loss of reponsiveness to gonadotrophins) are available. The usefulness of these cell lines within a test strategy for steroidogenesis needs to be evaluated. Other developed tests are: 2.4.4.1 Porcine Granulosa Cell assay. 2.4.4.2 Human Granulosa Cell assay. 2.4.4.3 Bovine Granulosa Cell Assay. 2.4.4.4 Leydig cell-enriched cultures. 2.4.4.5 The Leydig cell function assay. 2.4.4.6 Pig Leydig cell culture assay. References: 1.Moore HD, Akhondi MA.In vitro maturation of mammalian spermatozoa Rev Reprod. 1996 Jan;1(1):54-60. 2.Laskey J.W., Berman E., Ferrell J.M. The use of cultured ovarian fragments to assess toxicant alterations in steroidogenesis in Sprague-Dawley rat. Reprod. Toxicol, 9, 131-141, 1995. See data collection of ECVAM attachment No 1 2.4.5 Placental toxicity including Placenta barrier (see also subgroup: toxicokinetics and metabolism): 2.4.5.1. Trophoblast cell lines have been established and used to analyse toxic effects.
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References: 1.Ampasavate C, Chandorkar GA, Vande Velde DG, Stobaugh JF, Audus KL.Transport and metabolism of opioid peptides across BeWo cells, an in vitro model of the placental barrier. Int J Pharm. 2002 Feb 21;233(1-2):85-98. 2.McAleer MF, Tuan RS Metallothionein protects against severe oxidative stress-induced apoptosis of human trophoblastic cells.In Vitr Mol Toxicol. 2001 Fall;14(3):219-31. 3.Ma T, Yang ST, Kniss DA Development of an in vitro human placenta model by the cultivation of human trophoblasts in a fiber-based bioreactor system.Tissue Eng. 1999 Apr;5(2):91-102. 2.4.5.2 In addition, the human placental perfusion system focuses on the role of the placenta and its transporters in in utero after exposure of the foetus to toxic compounds and drug from the maternal circulation. It consists of a recirculating double-perfusion system of a single placental cotyledon where maternal and foetal circulations are perfused separately. For the placental perfusion one cotyledon from a placenta without macroscopic tissue trauma is perfused through foetal artery and vein in foetal side, and through the intervillous space in the maternal side. Foetal volume loss (due to leakage of perfusion medium from the high-pressure foetal circulation to the low-pressure maternal circulation) is the best index for membrane integrity and viability of the system. Antipyrine is used as a flow-limited marker for comparing the placental transfer rates of other substances. Prof Vhkangas, University of Kuopio, Finland, is planning to prevalidate the human placental codyleton perfusion system by using some representative chemicals if national resources will fund the study. This system will also provide further information that can be used for the development of a QSAR on the placental barrier. References 1.Ala-Kokko TI, Pienimaki P, Herva R, Hollmen AI, Pelkonen O, Vahakangas K. Transfer of lidocaine and bupivacaine across the isolated perfused human placenta. Pharmacol Toxicol. 1995 Aug;77(2):142-8. 2.4.6 Implantation including attachment of embryos, trophoblast cell lines and placentation) The preparation of the uterus is most important for supporting the pregnancy. The rate of embryo transport and the stage of embryonic development must be synchronized with the preparation of the endometrium to a stage of receptivity for the blastocyts. This preparation is strictly regulated by estrogen and progesterone and provided a narrow window of opportunity for implantation. Chemicals can disturb this process at several levels:
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1) embryo transport can be retarded which will lead to an asynchrony between uterus and conceptus. 2) chemical may affect the preparation of the endometrium. 3) a chemical can interact with the uterus and the embryo and prevent the attachment or invasion. 4) pregnancy cannot be maintained due to disturbance of the hormone balance. Several in vitro models for implantation have been developed in order to study the process of implantation. Cell lines such as JAR cells and RL95-2 cells have been employed as well as primary cell cultures. A broader dicussion is necessary to list additional tests for implantation. References: 1.Kliman HJ, Feinberg RF, Haimowitz JE. Human trophoblast-endometrial interactions in an in vitro suspension. 2.4.7 Uterine function 2.4.7.1 Several endometrial epithelial cell lines are available that can be useful for analysing chemical effects on the uterus function. such as RL95-2 endometrial epithelial cells. 2.4.7.2 In addition, long term cultures of rat vaginal keratinocytes have been established. These cells can be lifted (3D culture) on a membrane, which when exposed to fluctuating steroid levels (estrogen and progesterone) as seen in vivo,re-create seemingly very accurately the structural/histologic changes characteristic of the rat cycle in vivo (Peter Girling, personal communication). References: 1.Li HY, Chang SP, Yuan CC, Chao HT, Ng HT, Sung YJ. Establishment of an efficient method to quantify embryo attachment to endometrial epithelial cell monolayers. In Vitro Cell Dev Biol Anim. 2002 Oct;38(9):505-511. 2.4.8 Blood testis barrier (see also document of toxicokinetics and metabolism subgroup) The analysis of the second cell type of the supporting cells in the male testis is the Sertoli cells. During early sexual maturation, the Sertoli cells develop unique tight junctions in the basolateral aspect of their cytoplasm, which form the basis of the blood-testis barrier and divide the tubular area into adluminal and basal compartments. These compartments are relevant for the successful maturation of sperm cells. In addition, the blood testis barrier will protect the maturing germ cells from chemical insults. Since sertoli cells are a target cell type for chemicals several tests have been developed:
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2.4.8.1 As described for other areas sertoli cell lines have been established without loosing their characteristics. 2.4.8.2 Rat Sertoli Cell-enriched primary culture assay 2.4.8.3 Sertoli cell function assay 2.4.8.4 Sertoli cells Blood Testis Barrier Assay 2.4.8.5 Sertoli cell culture assay References 1.Hofmann MC, Van Der Wee KS, Dargart JL, Dirami G, Dettin L, Dym M. Establishment and characterization of neonatal mouse sertoli cell lines. Androl. 2003 Jan-Feb;24(1):120-30. 2.4.9 Receptor interaction: 2.4.9.1 ER and AR binding studies Additional tests for estrogen binding based on cell lines exist. The use of cell lines will be an advantage in comparison to the rat uterine cytosol assay since these test will not require laboratory animals. The tests are used in several laboratories although some of them have not been standardized jet. 2.4.9.2 Cytosol from MCF7 cell line. 2.4.9.3 Purified recombinant human hER? and hER? hER ? ? ) fluorescent ? An in vitro AR competitive binding assay is generally performed by quantifying the ability of substances to compete with the 5? -dihydrotestosterone (DHT) for binding. As described before the standardisation of studies based on cell lines could replace the rat prostate cytosol assay: 2.4.9.4 COS-1 cell line transfected with human AR. 2.4.9.5 Semi-purified recombinant hAR. 2.4.9.6 In silico models. The US EPA attempted to validate two different QSAR ER binding models, COREPA and CoMFA. 300 chemicals have been tested but the validation failed. EPA has a new computox initiative, which will look at further QSAR model development and refinement. 2.4.10. Thyroid hormones The Thyroid axis interferes with growth, development and reproductive viability, as well as with early stages of embryogenesis, but its complex mode of action is not completely understood and there is evidence that it is involved in a large number of ways in which human and wildlife organs communicate. A range of in vitro techniques has been developed to assess various aspects of the biology of the thyroid cell. The most suitable ones for the detection of chemical effects on the reproductive system need to be defined.
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References 1.Ambesi-impiombato, F.S. et al (1980) Culture of hormone-dependent functional ephitelial cells from rat thyroids. Proc Nat Acad Sci, 77, 3455-3459. 2.Lans MC et al., (1994). Different competition of thyroxin binding to transthyretin and thyroxine/binding globulin by hydroxy-PCBs, PCDDs and PCDFs. Eur J Pharmacol. Apr 4;270(2-3):129-36. 3.Brown, C. G. et al. (1986) Assessment of thyrotoxicity using in vitro cell culture systems. Chem Toxicol, 24, 557-562. 4.Brucker-Davis, R. (1998) Effects of environmental synthetic chemicals on thyroid function. Thyroid, 8, 827-856. 2.4.11 Screening tools for glucocorticoids, mineralocorticoids, FSH, LH, GnRH needs to be further established. FSH, LH can be easily screened in the follicle bioassay (FSH receptor expression on the granulosa cells, LHr on the theca cells and mural granulosa cells in the antral follicles at the end of culture). References: 1.Cortvrindt R, Smitz J, Van Steirteghem AC. Assessment of the need for follicle stimulating hormone in early preantral mouse follicle culture in vitro. Hum Reprod. 1997 Apr;12(4):759-68. 2.Cortvrindt RG, Hu Y, Liu J, Smitz JE. Timed analysis of the nuclear maturation of oocytes in early preantral mouse follicle culture supplemented with recombinant gonadotropin. Fertil Steril. 1998 Dec;70(6):1114-25. 3.Cortvrindt R, Hu Y, Smitz J. Recombinant luteinizing hormone as a survival and differentiation factor increases oocyte maturation in recombinant follicle stimulating hormone-supplemented mouse preantral follicle culture. Hum Reprod. 1998 May;13(5):1292-302. 2.4.12 Hypothalamic-Pituitary-Gonodal Axis: Screening tools are missing. 2.4.13 Alternative tests in the area of development toxicology testing. Beside the validated embryotoxicity tests additional manifestations of developmental toxicology have to be modelled by in vitro/in silico tests such as fetal growth, parturition or functional maturation. Chemical effects on preimplantation embryos can currently be analysed by using bovine and rodent preimplantation embryos. Oocytes can be obtained by the slaughter house and fertilised with bovine spermatozoa, parturition or functional maturation.
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2.4.14 Development and validation of genomics within ex vivo/in vitro tests Through the availability of the DNA- and RNA-microarray approaches, the relative expression of large series of genes can be easily determined. Development, implementation and validation of microarrays within ex vivo-in vitro assays may greatly incresase the predictive value; since effects at molecular level are thought to prelude morphologic effects, subtle, not readily apparent alterations might be targeted. Moreover, the characterization of effects at molecular level will support the interpretation of ex vivo-in vitro assays. Arrays containing genes relevant to reproductive and developmental toxicity are currently available, (e.g., homeobox, apoptosis, genes involved in folate, retinoid or steroid pathways). Moreover, developmental toxicity microarrays are currently being designed and utilized in various laboratories, and they will be applicable to the existing test systems, whenever appropriate. Nevertheless, much has to be done to include genomics within validated alternative test methods, including: Assessing broad sets of either known reprotoxicants and weak/equivocal reprotoxicants; Characterizing the "fingerprint" of relevant mechanisms Assessing dose-effect and time-effect relationships, and last but not least Requirements to standardize the systems in order to enter them into a formal validation process. Some References - Docterman KE, Smith SM. Of meis and men: lessons from a microarray study of teratogen action. Teratology. 2002 Nov;66(5):217-23. - Pennie WD. Custom cDNA microarrays; technologies and applications. Toxicology. 2002 Dec 27;181-182:551-4. - Qin P, Cimildoro R, Kochhar DM, Soprano KJ, Soprano DR. PBX, MEIS, and IGF-I are potential mediators of retinoic acid-induced proximodistal limb reduction defects. Teratology. 2002 Nov;66(5):224-34. Shultz VD, Phillips S, Sar M, Foster PM, Gaido KW. Altered gene profiles in fetal rat testes after in utero exposure to di(n-butyl) phthalate. Toxicol Sci. 2001 Dec;64(2):233-42. 2.4.15 Models used for In Vitro Developmental Neurotoxicity Testing (DNT) The developing nervous system has been recognized as a primary target for a variety of toxicants as immature brain cells (especially neurons) are in general more sensitive to neurotoxicants than differentiated ones. Various models of primary neuronal and glial (or mixed) cultures are used to study the developmental-dependent neurotoxicity as under in vitro conditions
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both type of cells (glial and neuronal) undergo morphological and physiological maturation (including synaptogenesis, myelination, expression of specific neurotransmitters). However the studies are still very much at the developing phase so direct recommendation cannot be advised at this stage. 2.4.15.1 Aggregating cell culture: three-dimensional structure, close to in vivo conditions for cell growth and development (1). 2.4.15.2 Cerebellar and hippocampus slice culture: a system suitable to examine neurotoxicants that selectively induce alterations in CNS myelination by oligodendrocytes (2). 2.4.15.3 Dissociated primary culture of any type of neurons from CNS or PNS e.g. sympathetic neurons: models suitable for neuronal morphogenesis studies (extensive characterization of the endogenous factors that regulate morphogenesis of these neurons both in situ and in vitro has been done) (3). 2.4.15.4 Primary, pure culture of cortical astrocytes or gliotypic cell line, glioma C6: suitable to study toxicants that induce decrease in astrocytes proliferation, maturation (e.g. GFAP expression) (4, 5, 6). 2.4.15.5 C. elegans and zebrafish: well characterized cellular and molecular regulation of neurodevelopment in these two simple organisms, including the fate of individual cells and the identity of genes involved in specific stages of neurodevelopment (7). The entire genomes of C. elegans and zebrafish have been sequenced, enabling integration of developmental neurotoxicity studies at the biochemical, cellular and molecular levels with alternations in structure and function. This allows constructing a genomics/proteomics database for predicting developmental neurotoxicity across species as many of the key events in neurodevelopment are conserved evolutionary. References: 1.Zurich M.G., Eskes Ch., Honegger P., Berode M., Monnet-Zschudi F: Maturation-dependent neurotoxicity of lead acetate in vitro: implication of glial reactions. J. Neurosci. Res., 2002, 70:108-116. 2.Notterpek L.M, Bullock P.N., Maled-Hedayat S., Fisher R., Rome LH:Myelination in cerebellar slice culture: develppment of a system amenable to biochemical analysis. J. Neurosci. Res., 1993, 36;621-634.
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3.Lein PJ, Schuh RA, Jett DA, Bucelli R: Cultured sympathetic neuronsas a model system for investigating the developmental neurotoxicity of organophosphate pesticides. 2003, CAAT 20th Anniversary Symposium, Baltimore, USA. 4.Burry M, Guizzetti M, Oberddoerster J, Costa L.G: Developmental neurotoxicity of toluene: in vivo and in vitro effects on astroglial cells.Developmental Neuroscience, 2003:25, 14-19. 5.Cookson M.R,. McClean R., Williams S.P, Davenport_Jones J., OHare S, Egan, Atterwill C.K, Pentreath V.W: Use of astrocytes for in vitro neurotoxicity testing. Toxic. In Vitro, 1994: 8, 817-819. 6.Developmental neurotoxicity of chlorpyrifos modeled in vitro:comparative effects of metabolites and other cholinesterase inhibitors on DNA synthesis in PC12 and C6 cells. Environmental Health prospectives, 2001, 109: 80-87. 7.Lein P, Goldberg A, Locke P, Silbergeld E: In vitro and alternative approaches to developmental neurotoxicity testing. The 9th meeting of the International Neurotoxicology Association 2003, Dresden Germany. 8.Hill A., Howard CV, Strahle U, Cossins A: Neuro-developmental defects in Zebrafish (danio rerio) at environmental relevant dioxin (TCDD) concentrations. Toxicol.Sci.2003(inpress).
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3. Summary of the currently available alternative methods and foreseeable time to achieve ESAC endorsement Table 1: Alternative tests for implemented OECD guidelines
Current endpoints addressed in animal test Estimated time to have the method validated (ESAC endorsement)*
Endpoints measured
Purpose
Validation status
Comments
Developmental toxicity (OECD TG414, 421)
Embryonic stem cell test
-Inhibition of differentiation of ES cells into cardiomyocyte s (ID50) -IC50 3T3 - IC50 D3
Partial replacement: tiered strategy and/or test battery
Testing structure analogues to Validated developmental (ECVAM toxic compounds; ZEBET) prioritisation
-Inhibition of differentiation Developmental toxicity (OECD TG414, 421) Micromass test
Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery
various Developmental toxicity (OECD TG414,421) Whole embryo culture
The test can only be used in a test strategy that covers main manifestation of developmental toxicity. The test can only be used for a narrow range of chemicals and has to proof its performance in other chemical classes. The test can only be used in a test strategy that covers main manifestation of developmental toxicity The test can only be used in a test strategy that covers main manifestation of developmental toxicity
0 years
0 years
0 years
38
various Developmental toxicity (OECD TG414) chick embryo retina cell culture Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Testing structure analogues to developmental Under R&D toxic compounds; prioritisation The test can only be used in a test strategy that covers main manifestation of developmental toxicity The test can only be used in a test strategy that covers main manifestation of developmental toxicity The test can only be used in a test strategy that covers main manifestation of developmental toxicity The test can only be used in a test strategy that covers main manifestation of developmental toxicity The test can only be used in a test strategy that covers main manifestation of developmental toxicity The test can only be used in a test strategy that covers main manifestation of developmental toxicity
6 years
various Developmental toxicity (OECD TG414,421) foetal culture gonad
Testing structure analogues to developmental Under R&D toxic compounds; prioritisation
> 10 years
various Developmental toxicity (OECD TG414,421) newborn ovary cultures
Testing structure analogues of Under R&D embryotoxic compounds; prioritisation
6 years
various Developmental toxicity (OECD TG414,421) human placental perfusion system
Chemicals passing placenta Under R&D barrier
6 years
various Developmental toxicity (OECD TG414,421) Trophoblast cell lines
Placental toxicity
Under R&D
> 10 years
various Developmental toxicity (OECD TG414,421) JAR cells and RL95-2
Implantation
Under R&D
> 10 years
39
various Developmental toxicity and One and two Generation Reproduction Toxicity study (OECD TG 414, 415,416, 421) bovine and rodent preimplantation embryos -progesterone production One and two Generation Reproduction Toxicity study cLHMR MA 10 (OECD TG 415,416,421) Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Chemical effects on Under R&D preimplantation embryos The test can only be used in a test strategy that covers main manifestation of developmental toxicology The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle
> 10 years
Chemicals Under interfering with prevalidation sterodiogenesis (ECVAM)
6-8 years
One and two Generation sliced Reproduction Toxicity study assay (OECD TG 415,416,421)
-Testosterone production -Leydig cell testis count
Chemicals Under interfering with Prevalidation testosterone (US EPA production EDMVS)
6 years
-Steroid production One and two Generation Reproduction Toxicity study H295R (OECD TG 415,416,421)
Under Chemicals Prevalidation interfering with (US EPA sterodiogenesis EDMVS)
6 years
-Steroid production One and two Generation Reproduction Toxicity study R2C (OECD TG 415,416,421)
Chemicals interfering with Under R&D sterodiogenesis
>10 years
-Steroid production One and two Generation Reproduction Toxicity study (OECD TG415,416,421) H 540
> 10 years
40
-Steroid production One and two Generation Reproduction Toxicity study MLTC-1 (OECD TG415,416,421) Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle
> 10 years
various One and two-Generation Reproduction Toxicity study TM 3 (OECD TG415,416,421)
> 10 years
various One and two-Generation Reproduction Toxicity study I-10 (OECD TG415,416,421)
> 10 years
-Steroid production One and two-Generation KGN (human Reproduction Toxicity study granulosa like (OECD TG415,416,421) cell line)
> 10 years
-Steroid HO-23 production One and two-Generation (immortalised Reproduction Toxicity study human (OECD TG415,416,421) granulosa cell line) -Steroid production
> 10 years
Jc-410 (stable One and two-Generation procine Reproduction Toxicity study granulosa cell (OECD TG415,416,421) line)
> 10 years
41
various One and two-Generation endometrial Reproduction Toxicity study epithelial cell (OECD TG415,416,421) lines (RL95-2) Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle, Currently the endpoint has not proofed its relevance
Uterine function
Under R&D
> 10 years
various One and two-Generation rat vaginal Reproduction Toxicity study keratinocytes (OECD TG 415,416,421)
Uterine function
Under R&D
> 10 years
-aromatase activity One and two-Generation Placental Reproduction Toxicity study microsomal (OECD TG 415,416,421) aromatase assay
Chemicals interacting aromatase activity
Under with Prevalidation (US EPA EDMVS)
6 years
One and two-Generation Follicle Reproduction Toxicity study bioassay (OECD TG415,416,421)
-steroid production -steroid receptor culture expression
Chemical effects on ovarian Under R&D function
6 years
various Partial replacement: tiered strategy and/or test battery
One and two-Generation Computer Reproduction Toxicity study assisted sperm (OECD TG415,416,421) analysis
Chemical effects on haploid Under R&D spermatozoa
>10 years
42
Functional Hamster Egg status of the One and two-Generation Penetration sperm: Reproduction Toxicity study Test/ -sperm quality (OECD TG415,416,421) Hypoosmotic -fertilizing Swelling Test capacity various Coculture of One and two-Generation epididymal Reproduction Toxicity study spermatozoa (OECD TG415,416,421) and epididymal epithelium various One and two-Generation Sertoli-Germ Reproduction Toxicity study Cell Cocultures (OECD TG415,416,421) Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery Detection of the functional status of the sperm cells Under R&D after chemical treatment The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle The test can only be used in a test strategy that covers main manifestation of the reproductive cycle
8 years
Effects on spermatogenesis
Under R&D
>10 years
Germ toxicity
cell
Under R&D
>10 years
various One and two-Generation Seminiferous Reproduction Toxicity study Tubulus Culture (OECD TG415,416,421) assay
Assessing testicular toxicity
Under R&D
>10 years
various One and two-Generation Sperm Reproduction Toxicity study activation assay (OECD TG415,416,421)
effects on sperm function
Under R&D
>10 years
various In vitro One and two-Generation fertilisation Reproduction Toxicity study oocyte toxicity (OECD TG415,416,421) test
oocyte and preimplantation toxicity
Under R&D
>10 years
43
various One and two-Generation Oogonia Reproduction Toxicity study proliferation (OECD TG415,416,421) test Partial replacement: tiered strategy and/or test battery Chemical interference with Under R&D oocyte meiotic process Effects on the blood testis barrier and information if chemicals are able to cross the Under R&D blood testis barrier and can effect the sensitive germ cells The test can only be used in a test strategy that covers main manifestation of the reproductive cycle
10 years
various Partial replacement: tiered strategy and/or test battery
One and two-Generation Reproduction Toxicity study sertoli cell lines (OECD TG415,416,421)
The test can only be used in a test strategy that covers main manifestation of the reproductive cycle
>10 years
Germ cell mutagenicity Rodent dominant lethal test (OECD TG 478)

1
Spermatogonial stem cell lines
Partial replacement: tiered strategy and/or test battery Partial replacement: tiered strategy and/or test battery
Under R&D
various Rodent dominant lethal test (OECD TG 478)

2
preantral follicle culture system
Under R&D
Germ cell mutagenicity.; the relevance of germ cell mutagenicity for cosmetic testing needs further discussion mutagenicity.; The relevance of germ cell mutagenicity for cosmetic testing needs further discussion
>10 years
6 years
The genotox subgroup stated that germ cell mutagenicity is not relevant for cosmetic testing
,The genotox subgroup stated that germ cell mutagenicity is not relevant for cosmetic testing
44
Germ cell Partial mutagenicity. Coculture of replacement: The relevance of epididymal Rodent dominant lethal test tiered germ cell spermatozoa Under R&D >10 years (OECD TG 478) strategy mutagenicity for and epididymal and/or test cosmetic testing epithelium battery needs further discussion Germ cell Germ cell mutagenicity Partial mutagenicity. replacement: The relevance of Mammalian spermatogonial spermatogonial tiered germ cell 3 chromosome test stem cells and Under R&D >10 years strategy mutagenicity for (OECD TG 483) cell lines and/or test cosmetic testing battery needs further discussion various Test strategy for assessing germ cell mutagenicity Partial (Blood testis replacement: Mammalian spermatogonial barrier). The tiered chromosome test sertoli cell lines Under R&D relevance of >10 years strategy (OECD TG 483) germ cell and/or test mutagenicity for battery cosmetic testing needs further discussion various Germ cell Partial mutagenicity. replacement: The relevance of Mouse spot test4 (OECD preimplantation tiered germ cell Under R&D >10 years TG484) embryos strategy mutagenicity for and/or test cosmetic testing battery needs further discussion * This table estimates the time needed to achieve ESAC endorsement for individual alternative tests assuming optimal conditions. It does not indicate the time needed to achieve full replacement of the animal test, nor does it include the time needed to achieve regulatory acceptance. Optimal conditions means that all necessary resources, for example technical, human, financial and coordination, are met at all times in the process and that the studies undertaken have successful outcomes. various
3 4
,The genotox subgroup stated that germ cell mutagenicity is not relevant for cosmetic testing The genotox subgroup stated that germ cell mutagenicity is not relevant for cosmetic testing
45
Table 2: Alternative tests for OECD draft guidelines or planned in vivo tests
Current endpoints addressed in animal test Estimated time to have the method validated (ESAC endorsement)*
Endpoints measured
Purpose
Validation status
Comments
alterations CNS myelination Developmental neurotoxicity (OECD draft 426) Cerebellar and hippocampus slice culture
in Partial replacement: tiered strategy and/or test battery
Dev.neurotoxicants
Under R&D
Developmental neurotoxicity (OECD draft 426)
Dissociated primary culture of any type of neurons from CNS or PNS
neuronal morphogenesis studies
Dev.neurotoxicants
Under R&D
-various Primary, pure culture of cortical astrocytes or gliotypic cell line Partial replacement: tiered strategy and/or test battery
Developmental neurotoxicity (OECD draft 426)
Dev.neurotoxicants
Under R&D
The test can only be used in a test strategy that covers main manifestation of >10 years developmental neurotoxicity. The basis of this tests is still hypothetical The test can only be used in a test strategy that covers main manifestation of >10 years developmental neurotoxicity. The basis of this test is still hypothetical The test can only be used in a test strategy that covers main manifestation of >10 years developmental neurotoxicity. The basis of this test is still hypothetical
46
various Partial replacement: tiered strategy and/or test battery The test can only be used in a test strategy that covers main manifestation of >10 years developmental neurotoxicity. The basis of this test is still hypothetical
Developmental Neurotoxicity (OECD TG426 draft)
Reaggregating brain cultures
Dev.neurotoxicants
Under R&D
Uterotrophic Bioassay for (anti) estrogenic effects; One/two generation studies (OECD TG 415, 416 OECD draft) Uterotrophic Bioassay for (anti) estrogenic effects (One/two generation studies (OECD TG 415, 416 OECD draft) Uterotrophic Bioassay for (anti) estrogenic effects (One/two generation studies (OECD TG 415, 416 OECD draft)
5 6
Oestrogen receptor binding rat uterine cytosol assay
Endocrine Disrupters (EDs)
Under prevalidation (US EPA)
Oestrogen agonists and antagonists
4-6 years5
Oestrogen receptor binding Cytosol from MCF7 cell line
Under R&D
Oestrogen agonists antagonists
and 6 years6
Oestrogen receptor binding Purified recombinant human hER?
Optimised test
and 6 years
Recombinant proteins preferred Recombinant proteins preferred
47
Uterotrophic Bioassay for (anti) estrogenic effects (One/two QSAR ER generation binding models studies (OECD TG 415, 416 OECD draft) Estrogen receptor transcriptional assays (-MCF-7 cell lines Uterotrophic cell Bioassay for -T47D lines (anti) estrogenic -MVLN cell effects lines (One/two -HeLa cell lines generation transfected with studies reporter gene (OECD TG -T47D+ pLuc 415, 416 OECD (ER-CALUX draft) ) -CHO cell lines transfected with p Luc (Eco SCREEN ) Uterotrophic Estrogen Bioassay for receptor (anti) estrogenic transcriptional effects assays based on (One/two human ER generation alpha studies transfected cell (OECD TG line (HeLa-cell 415, 416 OECD based) draft) ER binding affinity Partial replacement: tiered strategy and/or test battery Oestrogen agonists antagonists
Optimised test
and 10 years
-induction/ inhibition of cell proliferation -induction/ inhibition of reporter gene product
Under R&D
and 6-8 years
-induction/ inhibition of cell proliferation -induction/ inhibition of reporter gene product
Under R&D
and 6-8 years
48
Hershberger Bioassay for (anti) androgenic effects (One/two rat prostate generation cytosol assay studies (OECD TG 415, 416 OECD draft) Androgen receptor Hershberger transcriptional Bioassay for assay based on (anti) transfected cell androgenic lines effects (CV-1 +pLuc (One/two -CHO-K1 (Eco generation SCREEN ) studies -MDA-MB(OECD TG 453-kb2 415, 416 OECD -Palm cell lines draft) -AR-CALUX Androgen receptor binding Partial replacement: tiered strategy and/or test battery Under prevalidation (US EPA) Androgen agonists antagonists
and 6 years
-induction/ inhibition of cell proliferation -induction/ inhibition of reporter gene product Partial replacement: tiered strategy and/or test battery Under prevalidation (ECVAMUS EPA) Androgen agonists antagonists
and 6-8 years
recombinant proteins are preferred
50
4. Conclusions For a total replacement of test guidelines assessing the reproductive and developmental hazard of chemicals test strategies have to be developed. Due to the complexity of the reproductive cycle a series of workshops are necessary in which several experts with a complementary expertise will be involved in order to judge the relevance of the different in vitro tests that are currently available and their potential role in a test strategy. Since such test strategies have not been developed yet it was not possible to foresee by the nominated experts if all aspects of developmental and reproductive toxicology have been covered by the proposed tests. It should be stressed that maybe new tests have to be developed since the currently available tests are not sufficient. However, more detailed discussion on specific aspects of reproductive toxicology is required. In addition, it should be pointed out that it is not clear how the tests will perform in test strategies and if other in vitro models with more potential in terms of predictivity have to be developed. The proposed timelines are reflecting time that is needed to develop and validate single tests. Additional time will be necessary to analyse the relevance and reliability of test strategies. Several of the proposed tests are based on primary cell cultures or organ cultures. It is currently not foreseeable when and if these tests can be replaced. In addition, the experts have defined some areas of reproductive/developmental toxicity which are not covered by the proposed tests: reproductive behaviour, parturition, postnatal functional development, gonadotrophine hormones.
Annexe 1 A
Meeting with non animal testing stakeholders and European Commission services
Participation List Stakeholders
ALIROL Josiane AMCOFF Patric AULMANN Walter BANSIL Lee BOURGEOIS Delphine CLEMEDSON Cecilia CORTVRINDT Rita de SILVA Odile DEGUERCY Alain FENTEM Julia H. GRIEM Peter HEINEN Marlou KOETER Herman KREILING Reinhard LAURENT Christian LIEBSCH Manfred MANOU Irne McIVOR Emily RENNER Gerald ROGIERS Vera UNGEHEUER Peter Van de SANDT Jim VANPARYS Philippe UNITIS OECD COGNIS / EFfCI CEFIC Eurogroup for animal welfare Experttrdet EGG Centris L'Oral/Colipa UNITIS/Bioalternatives Unilever/Colipa EFfCI Eurogroup for animal welfare OECD EFfCI SCCNFP Zebet at the BfR Colipa ECEAE Colipa SCCNFP EFFCI TNO Nutrition and Food Research J&JPRD
C:\Documents and Settings\zuang\Local Settings\Temporary Internet Files\Content.IE5\S1A7K1EB\Annexe 1A Liste Participants 4 runions3.doc
Annexe 1 A
Commission services
BONTOUX Laurent CARVALHO Abraao COEKE Sandra ESKES Chantra HARTUNG Thomas JENSEN Charlotte LOUHIMIES Susanna LUCARONI Batrice MENTRE Barbara PEETSO Terje RUET-ROSSIGNOL Martine SANABRIA Arturo SCHUMANN Regina WAGSTAFFE Peter ZUANG Valrie JRC DG ENTR/F/3 ECVAM / DG JRC ECVAM / DG JRC ECVAM / DG JRC SANCO/C/7 DG ENV/C/3 DG RTD/E/4 DG ENTR/F3 SANCO/C/2 DG ENTR/F/3 SANCO/C/2 DG ENTR/F/3 DG SANCO/C/2 ECVAM / DG JRC
C:\Documents and Settings\zuang\Local Settings\Temporary Internet Files\Content.IE5\S1A7K1EB\Annexe 1A Liste Participants 4 runions3.doc
APPENDIX 1B: Participating scientific experts nominated by Stakeholders
CEFIC
Colipa
ECVAM Cecilia Clemedson Laura Gribaldo Alessandra Gennari Annarita Meneguz Walter Pfaller Phil Botham Chantra Eskes Valrie Zuang Chantra Eskes John Harbell Philippe Vanparys Valrie Zuang
EFfCI
Eurogroup and European Coalition *
DG Ent
UNITIS
Additional expertise
1. Acute Toxicity
Karen Blackburn
Irmela Ruhdel
Alain Deguercy
2. Skin Irritation
Julia Fentem
Manfred Liebsch
J.J.M. van der Sandt
Marie-Ange Alonso Leon Bruner Rodger Curren Penny Jones Manfred Liebsch Wolfang Pape Andrew Worth Andrew Worth
3. Eye Irritation
Pauline McNamee
Reinhard Kreiling
Troy Seidle
M.K. Prinsen
Sandrine Bessou
4. Skin Sensitisation 5. Skin Absorption
Frank Gerberick
David Basketter Walter Diembeck
Silvia Casati Chantra Eskes Jon Heylings Valrie Zuang Cecilia Clemedson Annarita Meneguz Walter Pfaller Pilar Prieto Raffaella Corvi Daniela Maurici Enrico Sabbioni Philippe Vanparys Markku Pasanen Chantra Eskes
Peter Griem
Barry Phillips Vera Rogiers JJM van der Sandt
Gill Langley
6. Subacute & subchronic toxicity 7. Genotox & mutagenotox 10.Carcinogenicity 8. UV-induced effects
Carl Westmoreland
Ursula Sauer
Marilyn Aardema
Stefan Pfuhler David Prentice Wolfgang Pape
Marcus Kleber
Barry Phillips
Cyrille Krul Christian Laurent Nicola Loprieno Tore Sanner Cyrille Krul Alain Deguercy Horst Spielmann
Manfred Liebsch
9. Toxicokinetics & Metabolism
Stuart Freeman Winfried Steiling
Bas J. Blaauboer Sandra Coecke Annarita Meneguz Mario Monshower Olavi Pelkonen Enrico Sabbioni Pilar Prieto
Bernhard Ladstetter
Gill Langley
Vassilios Kapoulas Vera Rogiers JJM van der Sandt
Greetje Elaut Andreas Freidig Nigel Gensmantel Peter Hoet David Leahy Geert Mannens Ben Nemery Walter Pfaller Nick Proctor Amin Rostami-Hodjegan Brighitta Eletti Francesca Maranghi
11. Reproductive & developmental toxicity
George Daston
George Daston
Susanne Bremer Rita Cortvrindt Alberto Mantovani Olavi Pelkonen
Horst Spielmann
Irmela Ruhdel
In bold: coordinator of the group * Experts nominated by the European Coalition to End Animal Experiments (ECEAE) and Eurogroup for Animal Welfare wish to note that while they took actively part in the discussions giving rise to the compromise estimates for the time needed to replace animal tests, they believe that in most cases a reliable safety assessment based on testing strategies involving solely alternative non-animal tests could be possible in significantly less time than is envisaged in this report.
APPENDIX II
GLOSSARY Alternative test: A test that reduces the numbers of animals required; refines procedures to lessen or eliminate pain or distress to animals, or enhance animal wellbeing; or replaces animals with non-animal systems or with phylo-genetically lower species. Dose response assessment: The second of four steps in risk assessment consisting in the analysis of the relationship between the total amount of an agent administered to, taken up or absorbed by an organism, system or (sub)population and the changes developed in that organism, system or (sub)population in reaction to that agent, and interferences derived from such an analysis with respect to the entire population. Endpoint: the biological or chemical process, response, or effect, assessed by a test. Hazard: The potential for an adverse health or ecological effect. A hazard potential results only if an exposure occurs that leads to the possibility of an adverse effect being manifested. Hazard identification: the identification of the type and nature of adverse effects that an agent has as inherent capacity to cause in an organism, system or (sub)population. Hazard identification is the first stage in hazard assessment and the first step in the process of Risk Assessment. Hierarchical (tiered) test approach: An approach where a series of tests to measure or elucidate a particular effect are used simultaneously or in an ordered sequence. In a typical approach, one or a few tests are initially used; the results from these tess determine which (if any) subsequent tests are to be used. Lead laboratory: the laboratory selected to perform the initial development of a protocol and to train the other laboratory personnel in the protocol procedures for the performance of an inter-laboratory validation study. This laboratory may also be used to produce the reference data against which the performance of the other laboratories will be evaluated. Mechanistic (based) test: A test that provides a direct relationship between the biological effects observed and the biological effects of interest. Peer review: A documented critical review of a specific scientific work product or products, which is conducted by experts who are independent of the experts who performed the original work but who are collectively equivalent to them in technical expertise. In the context of this report, it may lead to an endorsement by the ECVAM Scientific Advisory Committee (ESAC endorsement). Prediction Model: A formula or algorithm used to convert the results obtained using a test method into a prediction of the toxic effect of interest.
Protocol: The precise, step-by-step description of a test procedure that directs the laboratory as to how to perform the test. The protocol includes the listing and description of all preparations, reagents, supplies, and equipment needed. (Q)SARs (Quantitative Structure-Activity Relationships): Simplified mathematical models of complex chemical-biological interactions. They can be divided into two major types, (Q)SARs and SARs. (Q)SARs are all quantitative models yielding a continuous or categorical result while SARs are qualitative relationships in the form of structural alerts that incorporate molecular substructures or fragments related to the presence or absence of activity. Reference chemicals: Chemicals selected for use in the validation process, for which the anticipated responses are already known. These chemicals should be representative of the classes of chemicals for which the test is expected to be used, and should represent a full range of responses, from strong, to weak, to negative. Different sets of reference chemicals may be required for the different stages of the validation process, and for different tests and test uses. Reference data: An agreed-upon set of values against which the values obtained using the new test will be compared. Relevance: Description of relationship of the test to the effect of interest and whether it is meaningful and useful for a particular purpose. It is the extent to which the test correctly measures or predicts the biological effect and species of interest. Reliability: Extent of reproducibility of results from a test over time within and among laboratories, when performed using the same protocol. Repeatibility: The agreement among test results obtained within a single laboratory when the procedure is performed on the same substance under identical conditions (see Reliability). Replacement test: A test which is designed to substitute for a test that is in routine use and accepted for hazard identification and/or risk assessment, but which has advantages over the accepted test. The endpoint measured may be the same as in the approved test, or it may be different, but equivalent. Reproducibility: The agreement among results obtained from testing the same substance using the same test protocol. (see Reliability) Risk: The probability or degree of concern that exposure to an agent will cause an adverse effect in the species of interest. Risk assessment: A process intended to calculate or estimate the risk to a given target organism, system or (sub)population, including the identification of attendant uncertainties, following exposure to a particular agent, taking into account the inherent characteristics of the agent of concern as well as the characteristics of the specific target system. The Risk Assessment process includes four steps: hazard identification, hazard characterisation (related term: dose-response assessment),
exposure assessment, and risk characterisation. It is the first component in a risk analysis process. Robust(ness): The insensitivity of a test to departures from the specified test conditions. The ability of a test to provide similar results over a range of test conditions under which the test may be used in different laboratories. Screen/Screening test: A rapid, usually simple test conducted for the purpose of general classification of substances according to general categories of hazard. The results of a screen are generally used for preliminary decision making and to set priorities for more definitive tests. Screening tests may be able to identify active substances only, not inactive substances. Test (or assay): An experimental system used to obtain information on the adverse effects of a substance. Used interchangeably with assay. Test battery: A series of tests usually performed at the same time or in close sequence. Each test within the battery is designed to complement the other tests and generally to measure a different component of a multi-factorial toxic effect. Tiered approach: Tests are used in a sequential manner; the tests selected in each suceeding level are determined by the results in the previous level of testing. Transferability: The ability of a test procedure to be accurately and reliably performed in independent, competent laboratories. Valid test: A test determined to be acceptable for a specific purpose and which is based on scientifically sound principles. Validated test: A test for which the reliability and relevance have been established and which is based on scientifically sound principles. In the context of this report, the term will only mean a method/strategy that has been endorsed by ESAC. Validation: The process by which the reliability and relevance of a particular approach, method, process or assessment is established for a defined purpose (cf. chapter 2 for a more detailed definition).
Reference OECD (2003) Draft Guidance Document on the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment. OECD Environment, Health and Safety Publications Series on Testing and Assessment No 34, Paris. Pp. 67.
APPENDIX III 1- THE EU TESTING METHODS ADOPTION PROCESS
In the EU, legally recognised standardised Testing Methods to determine the hazardous properties of chemical substances are contained in Annex V of Dir 67/548/EEC on the Classification, Packaging and Labelling of Dangerous Substances. Once included in Annex V, the results obtained with these methods are recognised by the regulators beyond doubt and with no need of further demonstration. They play a central role for chemicals control and they are referred to in many other pieces of EU legislation (e.g. those related to dangerous preparations, pesticides, cosmetics and biocides also refer to these methods). The standardised Annex V Testing Methods, on the one hand, are the basic tools to generate data needed for hazard and risk assessment of chemicals. These constitute the basis to attain a high degree of protection for human health and the environment in the EU. On the other hand, they are also important for ensuring the Single Market in the EU allowing the free movement of goods between Member States. The Annex V methods are either original or adaptations of internationally recognised standards (e.g. ISO, UN, OECD). When they are adaptations of internationally recognised methods, they also contribute to the free movement of goods at a global level. For this reason, their development is closely linked and co-ordinated, in particular, with the parallel OECD Test Guidelines programme. The Commission (DG Environment) proposes to the Member States the methods which should be introduced into Annex V. This is done by an Adaptation to Technical Progress (ATP) of the mother Directive 67/548/EEC. For such an Adaptation, the Commission prepares a proposal for a Directive that amends Directive 67/548 by introducing new or updating existing methods in its Annex V, and presents it to the relevant Regulatory Committee. In this Committee, Member States delegates possibly discuss to fine-tune the proposal and then vote on it by qualified majority. In case of a favourable vote, the Directive is subsequently adopted by the Commission and soon afterwards published in the Official Journal. All the technical work needed to prepare the Commission proposal for Annex V methods is carried out by the European Chemicals Bureau (ECB) in consultation with the Group of the National Coordinators for Testing Methods, who are experts from the Member States supported by further experts in their respective countries. The National Coordinators represent the Member States Competent Authorities and advise the ECB on any issues related to Testing Methods. Whenever a need for a new or updated method is identified either by the Commission Services or the Member States Authorities, it is discussed by the National Coordinators, who advise, either in a meeting or by written procedure on the proposed testing protocol(s), work plan, priorities and preferred approach.
Generally speaking, three possibilities exist: a) the coordinators may advise to further develop the method by the ECB (in collaboration with other Commission Services, consultants or other experts) and/or by some leading Member State. This may involve the establishment of working groups or task forces as well as organising expert meetings or workshops. Subsequently, the coordinators may advise a1) to introduce the method into Annex V directly or a2) to propose it to the OECD TG Programme. Following adoption in the OECD the method is included in Annex V to Directive 67/548. b) In other cases, the coordinators may advise that any further development of the method is carried out in the frame of the OECD Test Guidelines Programme and, when the corresponding guideline has been adopted there, introduce it in Annex V as well.
The National Coordinators meet normally once a year in May, just before the corresponding OECD WNT (the Working Group of the National Coordinators of the Test Guidelines Programme) meeting, in order to ensure an adequate input and efficient coordination with this programme. In any case, care is taken that Annex V Testing Methods are equivalent in all their technical and scientific parts to the corresponding OECD Test Guidelines or other standards (e.g. ISO, UN) if the methods originated from there. Methods are written in a standard format and additional guidance may be introduced if the National Coordinators so advise. The final version, translated in all the official languages of the EU is then proposed by the Commission in the ATP process, as described above. If the direct introduction into Annex V is followed (path a1 above) the full process from initial proposal to adoption may take 6 months. In other cases, time may be longer, in particular if difficulties are encountered that involve reiterated consultations with experts or the coordinators. 2- TESTING METHODS OF ANNEX V TO DIRECTIVE 67/548/EEC. (Last updated: 5 September 2001). Annex V to Directive 67/548/EEC is divided in three parts which contain Testing Methods for chemicals that address all areas of concern:
?? Part
A contains methods for the determination of PHYSICO-CHEMICAL properties (e.g. melting and boiling point, density, flash point, flammability, explosivity, oxidizing power, etc...). HEALTH (e.g. acute or chronic toxicity, skin sensitisation, irritancy, corrosivity,
?? Part B contains methods for the determination of effects on HUMAN
carcinogenicity, neurotoxicity, etc..., they include also in vitro or alternative methods).

?? Part C contains methods for ENVIRONMENTAL EFFECTS, ecotoxicity and
environmental fate (e.g. toxicity to fish, daphnia or algae, bioconcentration, biodegradability, etc..). The text of the Annex V Testing Methods is available and can be downloaded from the ECB website as well as a list of the Annex V Testing Methods and the corresponding Directives in which they can be found. Only European Union legislation published in the paper editions of the Official Journal of the European Communities is deemed authentic. The Official Journals can be downloaded either from the Eur-Lex site (http://europa.eu.int/eur-lex/) or the EUDOR site (http://eudor.eur-op.eu.int). Taking into account the aim of this report, the following table lists only current methods included in Part B of Annex V (Methods for the determination of effects on Human health) is given below. The following table is intended to identify in which Directive to find the current version of each Annex V Part B method. Care has been taken to ensure that the information is correct, however one cannot exclude completely the possibility of errors. Detailed Table Part B Method Title Introduction Part B Acute toxicity (oral) B.1 DELETED DELETED Acute toxicity (oral) fixed dose method B.1bis B.1 tris B.2
Acute toxicity (oral) Acute toxic class method Acute toxicity (inhalation)
Directive 96/54/EC 92/69/EEC 92/69/EEC 96/54/EC 92/69/EEC 93/21/EEC 92/69/EEC 92/69/EEC 92/69/EEC 96/54/EC 96/54/EC 92/69/EEC 92/69/EEC 2000/32/EC
OJ L 248 1996 L 383 A 1992 L 383 A 1992 L 248 1996 L 383 A 1992 L 110 1993 L 383 A 1992 L 383 A 1992 L 383 A 1992 L 248 1996 L 248 1996
L 383 A 1992 L 383 A 1992
ATP 22nd 17th 17th 22nd 17th 18th 17th 17th 17th 22nd 22nd 17th 17th 26th
Notes
DELETED in 2001/59/EC
B.3 B.4 B.5 B.6 B.7 B.8 B.9 B.10
Acute toxicity (dermal)
Acute toxicity (skin irritation)
Acute toxicity (eye irritation)
Skin sensitization Repeated dose (28 days) toxicity (oral) Repeated dose (28 days) toxicity (inhalation) Repeated dose (28 days) toxicity (dermal) Mutagenicity - In vitro mammalian chromosome aberration test
L 136 2000
B.11 B.12 B.13/14 B.15 B.16 B.17 B.18 B.19 B.20 B.21 B.22 B.23 B.24 B.25 B.26 B.27 B.28 B.29 B.30 B.31 B.32 B.33 B.34 B.35 B.36 B.37 B.38 B.39 B.40 B.41
Mutagenicity - In vivo mammalian bone-marrow chromosome aberration test Mutagenicity - In vivo mammalian erythrocyte micronucleus test Mutagenicity - reverse mutation test using bacteria
2000/32/EC 2000/32/EC 2000/32/EC 88/302/EEC 88/302/EEC 2000/32/EC 88/302/EEC 88/302/EEC 88/302/EEC 88/302/EEC 88/302/EEC 2000/32/EC 88/302/EEC 88/302/EEC 2001/59/EC 2001/59/EC 88/302/EEC 88/302/EEC 88/302/EEC 88/302/EEC 88/302/EEC 88/302/EEC 88/302/EEC 88/302/EEC 88/302/EEC 96/54/EC 96/54/EC 2000/32/EC 2000/33/EC 2000/33/EC
L 136 2000 L 136 2000 L 136 2000 L 133 1988 L 133 1988 L 136 2000 L 133 1988 L 133 1988 L 133 1988 L 133 1988 L 133 1988 L 136 2000 L 133 1988 L 133 1988 L 225 2001 L 225 2001 L 133 1988 L 133 1988 L 133 1988 L 133 1988 L 133 1988 L 133 1988 L 133 1988 L 133 1988 L 133 1988 L 248 1996 L 248 1996 L 136 2000 L 136 2000 L 136 2000
26th 26th 26th 9th 9th 26th 9th 9th 9th 9th 9th 26th 9th 9th 28th 28th 9th 9th 9th 9th 9th 9th 9th 9th 9th 22nd 22nd 26th 27th 27th
French version corrected in 2001/59/EC English version corrected in 2001/59./EC English version Title corrected in 2001/59/EC Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC. Numbering in 96/54/EC.
Gene mutation Saccharomyces cerevisae Mitotic recombination - Saccharomyces cerevisae Mutagenicity - In vitro mammalian cell gene mutation test DNA damage and repair unscheduled DNA synthesis mammalian cells in vitro Sister chromatid exchange assay in vitro Sex-linked recessive lethal test in Drosophila melanogaster In vitro mammalian cell transformation test Rodent dominant lethal test Mammalian spermatogonial chromosome aberration test Mouse spot test Mouse heritable translocation Sub-chronic oral toxicity test. Repeated dose 90 day oral toxicity study in rodents Sub-chronic oral toxicity test. Repeated dose 90 day oral toxicity study in non-rodents Sub-chronic dermal toxicity test: 90-day repeated dermal dose study using rodent species Sub-chronic inhalation toxicity test: 90-day repeated inhalation dose study using rodent species Chronic toxicity test Teratogenicity test rodent and non-rodent Carcinogenicity test Combined chronic toxicity/carcinogenicity test One-generation reproduction toxicity test Two generation reproduction toxicity test Toxicokinetics
Delayed neurotoxicity of organophosphorus substances following acute exposure Delayed neurotoxicity of organophosphorus substances 28 day repeated dose study Unscheduled DNA synthesis (UDS) test with mammalian liver cells in vivo
Skin Corrosion Phototoxicity - In vitro 3T3 NRU Phototoxicity Test
3- PROPOSED T ESTING M ETHODS FOR 29TH ATP The following testing methods are under consideration in the context of the oncoming draft proposal 29th ATP: Draft revised B.1 bis Fixed Dose Procedure (Acute oral toxicity) Draft revised B.1 tris Acute Toxic Class Method (Acute oral toxicity) Draft revised B.4 Skin Irritation/Corrosion Draft revised B.5 Eye Irritation/Corrosion Draft revised B.31 Prenatal Development Study Draft revised B.35 Two Generation Reproduction Toxicity Draft new B.42 Local Lymph Node Assay (LLNA) Draft new B.43 Neurotoxicity study in rodents
APPENDIX IV
OECD GUIDELINES FOR TESTING OF CHEMICALS

1- The OECD Test Guidelines submission and adoption process
The OECD Test Guidelines Programme provides the mechanism for developing new Test Guidelines, and/or updating existing Guidelines. OECD Test G uidelines are broadly accepted by the international scientific community and by appropriate regulatory authorities of OECD Member countries and a number of Non-Member countries. The Joint Meeting of the Chemicals Committee and Working Party on Chemicals, Pesticides and Biotechnology (the Joint Meeting) is the OECD policy body that oversees the implementation of the Test Guidelines Programme. The Joint Meeting reviews and endorses draft Test Guidelines, and builds consensus to overcome policy differences that would otherwise jeopardise progress in Test Guideline development. A pivotal role in this Programme has been assigned to the Working Group of National Co-ordinators of the Test Guidelines Programme (WNT). The European Commission (EC) participates in this group, along with the National Co-ordinators from Member countries as appointed by their respective National Delegations to the Joint Meeting. Other invited experts also participate, as agreed by the Joint Meeting. The WNT considers proposals for new and updated Guidelines for the work programme and prioritises such proposals. The WNT meets yearly, usually in late May or early June. Any proposal to develop a new, or update an existing, Test Guideline should include a critical appraisal concerning its scientific justification, its sensitivity, and its reproducibility. New test methods, and existing Test Guidelines for which major revisions are proposed, should be validated as appropriate prior to their adoption. The process of validation is flexible, but transparency is essential. Proposals to develop new or updated Test Guidelines should be supported by valid arguments, which explain that there is a need for such a new or updated test, supported by regulatory arguments. These should include at least one of the following; ?? That the proposal will achieve further progress in international harmonization of data requirements; ?? That scientific arguments indicate the importance of the test or the modifications; ?? That the proposal addresses issues and/or endpoints which are of major human health or environmental concern. ?? That there are animal welfare advantages to the proposed test/procedure with respect to the 3Rs (replacement, reduction and refinement) without loss of essential information; ?? That the proposed test/procedure will result in reduced cost without loss of essential information. When there is a recognised need for new or updated Test Guidelines, the development/update of these Test Guidelines, as part of the overall work programme approved by the Joint Meeting, can be realised in one of two ways: (i), by beginning with a Detailed Review Paper of the area concerned or, when a particular method has not yet emerged as the preferred one, (ii) by drafting a Test Guideline proposal When a new/revised test is developed sufficiently to be proposed as an OECD Test Guideline, submission to the OECD Secretariat may be done by any of the following processes: ?? a Member country, or the European Commission, through its National Co-ordinator; ?? industry, trade unions, environmental citizen organisations, and animal protection organisations through their representative invited experts on the WNT; 1
??
non-Member countries, through their official observers to the WNT.
Detailed information about the basis for the test and its performance should be documented and available for review, including the validation study reports, any specific data requested by the validation management group, and the peer review report. Details of the process for adopting a test method as an OECD Test Guideline are provided in a separate Guidance Document (OECD (1995), which is currently under review. 1 If the draft Test Guideline proposal meets the OECD criteria, it is submitted to the National Co-ordinators for their review. Through the WNT, National Experts review the proposals for technical content and policy issues such as animal welfare, cost effectiveness and consistency with national regulatory requirements. National Co-ordinators consider the comments from their National Experts and submit National Positions on the proposals. After consensus is reached among Member countries, draft Test Guideline proposals are approved by the WNT, either at the WNT meeting or by written procedure. These proposals are forwarded to the Joint Meeting for their review and endorsement. The Joint Meeting is held about every eight months. A draft Test Guideline rejected by the Joint Meeting will be referred back to the WNT for their reconsideration, together with the reason for its rejection. After the Joint Meeting endorses a new or revised Test Guideline, the Secretariat submits the Test Guideline proposal to the Environment Policy Committee (EPOC). As part of this process, EPOC is invited to review the draft Test Guidelines proposal, within an agreed timeframe (which should be at least six weeks after submission), and, if appropriate, agree to its submission to the Council for formal adoption. The Secretariat can respond to comments from EPOC in a number of ways, ranging from making straightforward edits and corrections to the proposal, through to referring the comments to the WNT, in which case the modified proposal will again be submitted to the Joint Meeting and EPOC for their approval and endorsement. When any EPOC issues have been resolved, the Secretariat submits the draft Test Guideline Proposal, together with a summary of its rationale and political/social implications for Member countries, to the Council with the request to adopt the Test Guideline under the written procedure. Test Guidelines adopted by the Council become effective from the date of Council adoption. The Secretariat then arranges for publication at the earliest possible date. The total time period from submission of a draft Test Guideline proposal to the formal adoption by Council of a new or updated Test Guideline varies. In cases where well-written draft proposals fulfil the general OECD requirements for a new or updated Test Guideline, and are accompanied by the necessary background documentation (including documented validation study, independent peer review), the period from submission to adoption can be within 18 months. Proposals that are less well developed and supported take a longer the time period, dependent upon the number of iterations required by the WNT and/or Joint Meeting, and the complexity of the issues to be resolved.
2- Overview of currently available and draft test guidelines of Section 4- Health Effects
Original adoption date and the most recent updates together with improvements considerin g the 3R: reduction, refinement and replacement. The list also covers the latest drafts and their status and is divided into two sections; short and long-term toxicology testing and genetic toxicology testing.
1
Guidance Document for the Development of OECD Guidelines for Testing of Chemicals. Environment Monographs No. 76, OECD, Paris, 1993, reprinted 1995. 28 pp. [http://www.oecd.org/document/30/0,2340,en_2649_34377_1916638_1_1_1_1,00.html]).
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OECD Guidelines on Short and Long Term Toxicology Testing TG Title No. 401 402 Acute Oral Toxicity Acute Dermal Toxicity Original Adoption 12 May 1981 12 May 1981 Most Recently Updated
Date of Deletion: 20 December 2002 24 February 1987: Animal test (Reduction method) compared to the original TG from 1981, lowering of the dose level Animal test 24 April 2002: Animal test (Refinement/reduction tiered testing strategy regarding the original OECD TG), including in vitro screens 24 April 2002: Animal test; (Refinement/reduction tiered testing strategy regarding the original OECD TG), including in vitro screens, 17 July 1992: Animal test (Reduction method by 50% compared to original OECD TG) 27 July 1995: Animal test (Refinement method compared to original OECD TG) more information on best dosing practice, more information from the same animal. 21 September 1998: Animal test
403 404
Acute Inhalation Toxicity Acute Dermal Irritation/Corrosion
12 May 1981 12 May 1981
405
Acute Eye Irritation/Corrosion
12 May 1981
406
Skin Sensitisation
12 May 1981
407
Repeated Dose 28-Day Oral Toxicity Study in Rodents
12 May 1981
408
Repeated Dose 90-Day Oral Toxicity Study in Rodents Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents Repeated Dose Dermal Toxicity:28-Day Subchronic Dermal Toxicity: 90Day Repeated Dose Inhalation Toxicity: 28/14-Day Subchronic Inhalation Toxicity: 90-Day
12 May 1981
409
12 May 1981
21 September 1998: Animal test
410
12 May 1981
Animal test
411
12 May 1981
Animal test
412
12 May 1981
Animal test
413
12 May 1981
Animal test
90-Day 414 Prenatal Developmental Toxicity Study 12 May 1981 22 January 2001: Animal test (Reduction method regarding the original OECD TG) [use (-20% animals), more information from the same animal] Animal test
415
One-Generation Reproduction Toxicity Two-generation Reproduction Toxicity Study Toxicokinetics Delayed Neurotoxicity of Organophosphorus Substances Following Acute Exposure Delayed Neurotoxicity of Organophosphorus Substances: 28-Day Repeated Dose Study Acute Oral toxicity Fixed Dose Procedure (FDP)
26 May 1983
416
26 May 1983
22 January 2001: Animal test
417 418
4 April 1984 4 April 1984
Animal test. 27 July 1995: Animal test
419
4 April 1984
27 July 1995: Animal test
420
17 July 1992
17 December 2001: Animal test (Reduction, refinement method regarding the conventional TG 401). Less suffering, smaller number of animals Animal test (reduction method compared to original TGs) New Screening test provides essential information with a minimum number of animals. Animal test (reduction method compared to the individual TGs). Combines the new screening test on reproduction toxicity with TG 407 and further reduces the number of animals to an absolute minimum for these combined endpoints. 17 December 2001 Animal test (Reduction method regarding the conventional TG 401). Much smaller number of animals (10% of TG 401) Animal test 17 December 2001: Animal test (Reduction method regarding the conventional TG 401). Smaller number of animals, provides a closer estimate of the
421
Reproduction/Developmental Toxicity Screening Test
27 July 1995
422
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
22 March 1996
423
Acute Oral Toxicity Acute Toxic Class Method (ATC)
22 March 1996
424 425
Neurotoxicity Study in Rodents Acute Oral Toxicity: Up-andDown Procedure
21 July 1997 21 September 1998
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LD50 than TGs 420 and 423. 426 Developmental Neurotoxicity Study Skin Absorption: In vivo method Draft New Guideline (April 2004) Submitted to Animal test Animal test
427
Council
for
adoption.
428
Skin absorption: In vitro method
(April 2004)
Submitted to Council for adoption. True alternative to the in vivo method. Full replacement over the TG 427 Animal test (Reduction / refinement method regarding TG 406) allowing more information and less suffering. Submitted to Council for adoption. Nonanimal test (Replacement method over the corrosion part of TG 404) Submitted to Council for adoption. Nonanimal test (Replacement method over the corrosion part of TG 404) Submitted to Council for adoption. Nonanimal test. Full replacement (no OECD TG existed for animal test) Animal test (Reduction, refinement method regarding TG 403). Uses fewer animals and less suffering. Animal test (Reduction, refinement regarding TG 404). Uses fewer animals and less suffering. Non-animal test (replacement method for the corrosion part of TG 404, for specific applications - only applicable to acids & bases) Animal test Animal test Animal test (the combined TG 453 could offer Reduction compared to individual TG 452 + TG 453)
429
Skin Sensitisation: Local Lymph Node Assay
24 April 2002
430
In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER) In Vitro Skin Corrosion: Human Skin Model Test In Vitro 3T3 NRU phototoxicity test
(April 2004)
431
(April 2004)
432
(April 2004)
433
Acute Inhalation Toxicity: Fixed Dose Procedure (FDP)
Draft New Guideline
434
Acute Dermal Toxixity: Fixed Dose Procedure (FDP)
Draft New Guideline
435
In Vitro Skin Corrosivity
Draft New Guideline
451 452 453
Carcinogenicity Studies Chronic Toxicity Studies Combined Chronic toxicity/Carcinogenicity Studies
12 May 1981 12 May 1981 12 May 1981
OECD Guidelines on Genetic Toxicology Testing. No. Title Original Adoption 26 May 1983 Most Recently Updated
471
Bacterial Reverse Mutation Test
21 July 1997: In vitro test for point mutations *. Non animal test (part of test strategy) Date of deletion: 21 July 1997 (Method merged with TG 471) 21 July 1997: True alternative to the in vivo test. Non animal test (part of test battery, it does not fully replace the in vivo test) 21 July 1997: Animal test (Reduction method compared to 1983 version using a smaller number of animals. 21 July 1997 :Animal test
472
Genetic Toxicology: Escherichia coli, Reverse Assay In Vitro Mammalian Chromosome Aberration Test
26 May 1983
473
26 May 1983
474
Mammalian Erythrocyte Micronucleus Test
26 May 1983
475
Mammalian Bone Marrow Chromosome Aberration Test In Vitro Mammalian Cell Gene Mutation Test
4 April 1984
476
4 April 1984
21 July 1997: In vitro test for gene mutations (genetic toxicity) *. Non animal test (part of test strategy) Non-animal test (flies= non vertebrates) Note: 86/609 defines animals as any live non-human vertebrates
477
Genetic Toxicology: Sex-Linked Recessive Lethal Test in Drosophilia melanogaster
4 April 1984
478
Genetic Toxicology: Rodent dominant Lethal Test Genetic Toxicology: In Vitro Sister Chromatid Exchange assay in Mammalian Cells Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay Genetic Toxicology: Saccharomyces cerevisiae, Mitotic Recombination Assay Genetic Toxicology: DNA Damage and Repair, Unscheduled
4 April 1984
Animal test
479
23 October 1986
In vitro test for DNA exchanges between sister chromatids (genetic toxicity) *. Non animal test (part of test strategy) In vitro test for gene mutations in Saccharomyces (genetic toxicity*. Non animal test (part of test strategy) In vitro test for mitotic recombination in saccharomyces (genetic toxicity*. Non animal test (part of test strategy) True alternative to the in vivo method for DNA damage. Non animal test (part of
480
23 October 1986
481
23 October 1986
482
23 October 1986
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Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro 483 Mammalian Spermatagonial Chromosome Aberration Test Genetic Toxicology: Mouse Spot Test Genetic Toxicology: Mouse Heritable Translocation Assay Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo 23 October 1986
DNA damage. Non animal test (part of test battery, it does not fully replace the in vivo test) 21 July 1997: Animal test
484
23 October 1986
Animal test
485
23 October 1986
Animal test
486
21 July 1997
Animal test
All in vitro tests on Genetic Toxicology Testing are part of a testing strategy which progresses to animal testing only if necessary (in case of equivocal results).

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DRAFT Overview, 30/03/04

DRAFT Overview, 30/03/04

EEC Regulation OECD Regulation

For details see Appendix 3 For details see Appendix 4

Human health effect

Animal testing should not be performed for this endpoint

For risk assessment: need to address reversibility and dose-response aspects

Develop test strategies for risk assessment purposes

4 years for the most promising tests

Human health effect

Valuable alternative methods identified

Time necessary to achieve validation (ESAC endorsement)*

Identified areas or endpoints with no alternative methods

5 Skin absorption / penetration

6 Subacute and subchronic toxicity

No generally accepted alternative available for replacing repeated-dose in vivo testing

Human health effect

Valuable alternative methods identified

Time necessary to achieve validation (ESAC endorsement)*

Identified areas or endpoints with no alternative methods

7 Genotoxicity and mutagenicity

2 in vitro under validation 3 in vitro optimised tests

Toxicokinetics Cell lines not relevant to the target organs

1 in vivo refinement method (P-LLNA)

Progress will depend on progress in the field of skin sensitisation

Human health effect

Valuable alternative methods identified

Time necessary to achieve validation (ESAC endorsement)*

9 Toxicokinetics and metabolism

Long-term process difficult to mimic with short-term in vitro tests

Human health effect

Valuable alternative methods identified

11 Reproductive and developmental toxicity

*Assuming optimal conditions

DRAFT Chapter 2, 07/04/04

The requirements of the Cosmetics Directive :

DRAFT Chapter 2, 07/04/04

DRAFT Chapter 2, 07/04/04

DRAFT Chapter 2, 07/04/04

The toxicological data requirements for chemicals :

DRAFT Chapter 2, 07/04/04

DRAFT Chapter 2, 07/04/04

DRAFT Chapter 2, 07/04/04

Validated test Validation + Peer-review (ESAC endorsement) Regulatory acceptance

If criteria not met

If criteria not met

DRAFT Chapter 2, 07/04/04

DRAFT Chapter 2, 07/04/04

International Adoption: acceptance at OECD level

EEC Regulation OECD Regulation

For details see Appendix 3 For details see Appendix 4

DRAFT Chapter 2, 07/04/04

DRAFT Chapter 2, 07/04/04

Laboratories, Ross, OH 45061, USA; 3Expertrdet AB, 17264 Sundbyberg, Sweden;

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1. Inventory of methods currently available

7. OECD (2001). Procedure.

Test Guideline 425. Acute Oral Toxicity Up-and-Down

2. Inventory of alternatives methods currently available

c. Test Chang liver cell/morphology, 24 h (the MIT-24 assay). Short description 8

3. Identified steps or tests with no alternative methods available