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Nanomaterials in
REACH
Project Report
15 August 2011
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Document title: Nanomaterials in REACH project report

Version 15 August 2011 (final)

Authors and Katarzyna Malkiewicz 1, Michala Pettitt 2, Kenneth A. Dawson 3,

affiliations: Arho Toikka 4, Sven Ove Hansson 1, Janne Hukkinen 4, Iseult
Lynch 3, Jamie Lead 2

(1) Royal Institute of Technology (KTH), Department of Philosophy and the History
of Technology, Teknikringen 78B, 10044 Stockholm, Sweden

(2) University of Birmingham (UB), Edgbaston, Birmingham, West Midlands, UK,

B15 2TT

(3) Centre for BioNano Interactions, University College Dublin (UCD), Belfield,
Dublin 4, Ireland

(4) University of Helsinki (UH), Snellmaninkatu 10, 00014 Helsinki, Finland

Project title: Nanomaterials in REACH - evaluation of applicability of existing

procedures for chemical safety assessment to nanomaterials.

Funded by: SKEP ERA-NET (Scientific Knowledge for Environmental Protection)

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31 Contents

32 1 Executive summary ....................................................................................................................... 13

33 2 REACH provisions and ECHA guidelines recommendations - an evaluation of their suitability for
34 NMs 26

35 2.1 Objective................................................................................................................................ 26

36 2.1.1 Introduction to REACH - history, aims ........................................................................... 26

37 2.2 Definition of NMs .................................................................................................................. 27

38 2.3 NMs under REACH and CLP ................................................................................................... 34

39 2.3.1 The main obligations under REACH ............................................................................... 34

40 2.3.2 REACH provisions........................................................................................................... 36

41 2.3.3 REACH associated guidelines......................................................................................... 52

42 2.3.4 Registration of NMs case studies ............................................................................... 65

43 3 Physicochemical properties of NMs .............................................................................................. 76

44 3.1 What is nano? ...................................................................................................................... 76

45 3.1.1 Classifying NMs.............................................................................................................. 77

46 3.2 Physicochemical properties of NMs ...................................................................................... 80

47 3.2.1 Formulation Composition.............................................................................................. 81

48 3.2.2 Core Chemical Composition .......................................................................................... 81

49 3.2.3 Size ................................................................................................................................. 82

50 3.2.4 Morphology ................................................................................................................... 85

51 3.2.5 Surface Properties ......................................................................................................... 86

52 3.2.6 Dissolution Properties ................................................................................................... 89

53 3.2.7 Dispersion Properties .................................................................................................... 89

54 3.3 Relating physicochemical properties to (eco)toxicology ...................................................... 92

55 3.3.1 QSAR models ................................................................................................................. 92

56 3.3.2 Characterising NMs in (eco)toxicity studies. ................................................................. 94

57 3.4 Standards and Prioritisation .................................................................................................. 96

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58 3.5 NMs in a Regulatory Context................................................................................................. 97

59 3.5.1 The Remit of REACH and NMs ....................................................................................... 97

60 3.5.2 Discriminating NMs ....................................................................................................... 98

61 4 Assessment of the potential human toxicity of NMs .................................................................. 118

62 4.1 Interactions of NMs with their surroundings ...................................................................... 118

63 4.1.1 Interaction of NMs with inorganic components ......................................................... 122

64 4.1.2 Interaction of NMs with organic components impact on the size and the identity 123

65 4.1.3 Interaction with organic components impact on biological processes .................... 127

66 4.1.4 Interaction with organic components In vitro in vivo data extrapolation ............. 131

67 4.2 Stability and changes of NMs .............................................................................................. 132

68 4.3 Polymeric NNs ..................................................................................................................... 133

69 4.4 Occupational and consumer exposure to NMs ................................................................... 134

70 4.5 Relating NMs unique properties to risk assessment and regulation .................................. 137

71 4.6 Lessons from asbestos, metals and protein therapeutics ................................................... 142

72 4.7 Conclusions and recommendations regarding human toxicity of NMs .............................. 146

73 5 Assessment of the potential ecotoxicity of NMs......................................................................... 153

74 5.1 REACH Requirements .......................................................................................................... 154

75 5.2 Determining Ecotoxicity ...................................................................................................... 155

76 5.2.1 Dose and Exposure ...................................................................................................... 155

77 5.2.2 Identifying routes of particle toxicity .......................................................................... 158

78 5.2.3 Relevant testing regimes ............................................................................................. 159

79 5.2.4 Species selection ......................................................................................................... 160

80 5.2.5 Endpoint selection ....................................................................................................... 162

81 5.3 Environmental behaviour and fate of NMs ......................................................................... 165

82 5.3.1 Sources of manufactured NMs in the environment .................................................... 165

83 5.3.2 Interactions, transformations and fate. ...................................................................... 170

84 5.4 Regulatory implications of ecotoxicology and environmental fate of NMs ........................ 176

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85 5.5 Exposure Modeling .............................................................................................................. 179

86 6 Roadmapping and Backcasting in a Socio-Ecological System as Risk Governance Tools for NMs
87 and NPs ................................................................................................................................................ 188

88 6.1 Introduction ......................................................................................................................... 188

89 6.1.1 NMs as a governance problem .................................................................................... 188

90 6.1.2 Technology Roadmapping ........................................................................................... 190

91 6.1.3 Technology Forecasting Methods ............................................................................... 191

92 6.1.4 Our roadmapping method........................................................................................... 194

93 6.1.5 Socio-ecological systems ............................................................................................. 196

94 6.2 Nanosilver Roadmap ........................................................................................................... 199

95 6.3 Discussion ............................................................................................................................ 207

96 7 References ................................................................................................................................... 208

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 99 Figures

100 Figure 3.1 The "nano"length scale (as it is currently defined) in context, relative to other common objects.
101 ............................................................................................................................................................................ 112

102 Figure 3.2 Illustration of the exponential increase of surface area as the cube (particle) size decreases.. 113

103 Figure 3.3 Ouput of a typical NM synthesis and size characterisation. ......................................................... 113

104 Figure 3.4 Important physicochemical properties of manufactured NMs in aqueous media...................... 114

105 Figure 3.5 Illustration of the challange in determining the primary particle size for biological / hazard
106 assessment purposes......................................................................................................................................... 115

107 Figure 3.6 A schematic diagram showing the electrical double layer that will develop around a charged
108 particle suspended in aqueous media. ............................................................................................................. 116

109 Figure 3.7 Schematic illustration of proposed multiple upper limits to the definition of NMs. 117

110 Figure 4.1 Zeta potential of copolymer particles of increasing hydrophobicity dispersed in different OECD
111 ecotoxicity testing media. ................................................................................................................................. 149

112 Figure 4.2 Surface chemistry and surface charge of NPs can dramatically effect the nature of the NP
113 dispersion in a biological fluid (e.g. plasma). .................................................................................................. 149

114 Figure 4.3 (a) Crystal structure of the protein apolipoprotein A-1, and schematic of apolipoprotein A-1 in a
115 lipoprotein complex composed of phosholipids. (b) Size comparison of a 70 nm nanoparticle with
116 lipoprotein complexes chylomicrons, very low, low, and high density lipoproteins. ............................... 150

117 Figure 4.4 Comparison of the sub-cellular distribution of (a) molecular yellow-green fluorescence dye
118 versus (b) the same dye constrained within 50nm polystyrene NPs in A549 cells. .................................... 150

119 Figure 4.5 Comparison of the composition of the molecular corona formed at different plasma
120 concentrations around (a) 50nm Sulphoxide-modified polystyrene NPs and (b) 50nm SiO2 NPs. ............ 151

121 Figure 4.6 Risk of accidental exposure to hazardous substances during manufacture of some common NMs
122 compared to other industrial processes such as wine production, petroleum refining or silicon wafer
123 production. ......................................................................................................................................................... 151

124 Figure 5.1 Idealised schematic diagram of nanoparticle entry and transport through the environment. . 185

125 Figure 5.2 Transformation of NMs in the environment. ................................................................................. 186

126 Figure 5.3 Possible interactions between target organisms, NPs (green rods) and a secondary pollutant
127 (red dots)............................................................................................................................................................ 187

128 Figure 6.1 Representation of the roadmap concept........................................................................................ 196

129 Figure 6.2 A schematic of a Social-Ecological System for risk governance of NMs ...................................... 198
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131 Tables

132 Table 1.1 Summary of recommendations ............................................................................................................. 22

133 Table 2.1Summary of testing requirements under REACH ................................................................................... 67

134 Table 2.2 Combined GHS and REACH hazard classification criteria lined up with REACH tonnage bands data
135 requirement suficient for classification endpoits. ................................................................................................ 68

136 Table 3.1 Physicochemical characteristics of NMs and the most appropriate techniques for determining
137 them. ................................................................................................................................................................... 103

138 Table 3.2Common analytical techniques for characterising NMs. 106

139 Table 3.3 Summary of the properties required to characterisze NMs in media proposed by a range of
140 authors. .............................................................................................................................................................. 111

141 Table 4.1 Comparison of the size of a series of copolymer NIPAM:BAM NPs in different OECD
142 recommended ecotoxicity assay media. .......................................................................................................... 148

143 Table 4.2 Comparison of the binding of apolipoproteins from human plasma to polystyrene NPs of
144 different size and surface charge. . .................................................................................................................. 148

145 Table 5.1 Ecotoxicological and environmental fate and behaviour data requirements of REACH regulation
146 by tonnage trigger. ............................................................................................................................................ 183

147 Table 5.2 Criteria for persistence, bioaccumuation and ecotoxicity, complied from REACH Annex XIII. . 184

148 Table 6.1 Interpretation of technology forecasting methods ......................................................................... 193

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150 Appendixes

151 Substance identification according to REACH Annex VI section 2 ........................................................................ 72

152 Reporting requiremnts based on OECD (2010a) guidance document .................................................................. 73

153 Methods to characterise mate rials as "nano" under the SCENIHR (2010) definition ................................. 107

154 The estimated relative presence of NMs used in consumer products on the global market (%) in the near
155 future based on the total amount (tonnes) of NMs currently used in consumer products. ........................ 152
156

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157 Abbreviations and Acronyms

158 1D PAGE 1 Dimensional Polyacrylamide Gel Electrophoresis

159 ACC American Chemistry Council
160 ACHS Advisory Committee on Hazardous Substances
161 ADME Absorption, Distribution, Metabolism, Excretion
162 AF Assessment Factor
163 Ag NPs Silver nanoparticles
164 AFM Atomic Force Microscope
165 ASTM American Society for Testing and Materials
166 AUC Analytical Ultracentrifuge
167 AWQC Ambient Water Quality Criteria
168 BAM N-tert-butylacrylamide (monomer)
169 BET Brunnauer, Emmett and Teller method
170 BLM Biotic Ligand Model
171 BMC Benchmark Concentration
172 BMCL Confidence Limit for BMC
173 BMD Benchmark Dose
174 BMDL Lower confidence limit for BMD
175 BMDS Benchmark dose at a given Standard Deviation
176 BMR Benchmark Response
177 BMR Basal Metabolic Rate
178 BSA Bovine Serum Albumin
179 BSI British Standards Institution
180 CAS Chemical Abstract Services registry number
181 CASG nano REACH Competent Authorities subgroup on NMs
182 CBEN Centre for Biological and Environmental Nanotechnology
183 CEN European Committee for Standardization
184 CEU Council of the European Union
185 CLP Classification, Labeling and Packaging of chemicals
186 CMR Carcinogenic, Mutagenic, and Reprotoxic
187 CMAR Carcinogenetic, Mutagenic, Asthmagenic or Reprotoxic
188 CNS Central Nervous System
189 CNT Carbon Nanotube
190 CPS&Q Consumer Products Safety and Quality Unit (formerly ECB)
191 CSA Chemical Safety Assessment
192 CSL Central Science Laboratory

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193 CSR Chemical Safety Report
194 DCS Differential Centrifugal Sedimentation
195 DEFRA Department for Environment, Food and Rural Affairs
196 DLS Dynamic Light Scattering
197 DNOELs Derived No Observed Effect Level
198 DMELs Derived Minimum Effects Level
199 ECx Effective Concentration to induce a biological effect in x % of the individuals in the
200 test population
201 EC European Commission
202 ECHA European Chemicals Agency
203 ECB European Chemicals Bureau (now CPS&Q Unit)
204 EDx Effective Dose to induce a biological effect in x % of the individuals in the test
205 population
206 ECFA Joint Expert Committee on Food Additives
207 EDL Electric Double Layer
208 EDX-EM Energy Dispersive X-ray Spectroscopy - Electron Microscopy
209 EEA European Environment Agency
210 EINECS European Inventory of Existing Chemical Substances
211 ELA Establishment License Application (manufacturing facilities)
212 ELINCS European List of Notified Chemical Substances
213 EMEA European Medicines Agency
214 ENP Engineered Nanoparticle
215 EPM Electrophoretic Mobility
216 ESD Equivalent Spherical Diameter
217 ESEM Environmental Scanning Electron Microscopy
218 ESIS European Chemical Substances Information System
219 EU-OSHA European Agency for Safety and Health at Work
220 Fl-FFF Flow Field Flow Fractionation
221 FSA UK Food Standards Agency
222 FCS Foetal Calf Serum
223 GHS Globally Harmonized System of classification and labeling of chemicals
224 HPV High Production Volume
225 HQ Hazard Quotient
226 HS Humic Substances
227 HSE UK Health and Safety Executive
228 IARC International Agency for Research on Cancer
229 ICON International Council on Nanotechnology

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230 ICP-MS Inductively Coupled Plasma - Mass Spectroscopy
231 ICP-OES Inductively Coupled Plasma - Optical Emission Spectroscopy
232 IPCS International Programme on Chemical Safety
233 ISO International Organisation for Standardisation
234 IUCLID International Uniform ChemicaL Information Database
235 IUPAC International Union of Pure and Applied Chemistry
236 IRMM Institute for Reference Materials and Measurements (part of JRC)
237 JRC Joint Research Centre (European Commission)
238 JRC-IRMM Joint Research Centre Institute for Reference Materials and Measurements, EC
239 KOW Octanol-water coefficient
240 LC50 Median Concentration lethal to 50 % of a test population
241 LD50 Median Dose lethal to 50 % of a test population
242 LEDx Lowest Effective Dose for a biological effect in x % of the individuals in the test
243 population
244 LOAEL Lowest Observed Adverse Effect Level
245 MDM Minimal Davies Medium
246 MN Manufactured Nanomaterial
247 MOA Mode of Action
248 MS Mass Spectrometry
249 MSDS Material Safety Data Sheet
250 MWNT Multi-walled Nanotube
251 NAS National Academy of Science
252 NGO Non-Governmental Organisation
253 NIA Nanotechnology Industry Association
254 NICNAS National Industrial Chemicals Notification and Assessment Scheme
255 NIPAM N-isopropylacrylamide (monomer)
256 NIPAM/BAM Copoylmer composed of N-isopropylacrylamide and N-tert-butylacrylamide
257 NIOSH National Institute for Occupational Safety and Health
258 NIST National Institute of standards and technology, USA
259 NMs Nanomaterials
260 MWCNT Multi-Walled Carbon Nanotube
261 NNI National Nanotechnology Initiative
262 NOEC No Observed Effect Concentration
263 NOAEL No Observed Adverse Effect Level
264 NOEL No Observed Effect Level
265 NOM Natural Organic Matter
266 NPs Nanoparticles

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267 NSET Nanoscale Science, Engineering and Technology
268 NSP Nanosized Particle
269 OECD Organization for Economic Cooperation and Development
270 OEL Occupational Exposure Limit
271 PBPK Physiologically-based Pharmacokinetic modeling
272 PBT Persistent, Bioaccumulative, Toxic
273 PCS Photon Correlation Spectroscopy
274 PEC Predicted Environmental Concentration
275 PEL Permissible Exposure Limit
276 PEN Project on Emerging Nanotechnologies
277 PIDS Polarisation Intensity Differential Scattering
278 PIP Persistent Inorganic Pollutant
279 PLA Product License Applications
280 PNEC Predicted No-Effect Concentration
281 POP Persistent Organic Pollutant
282 PP Precautionary Principle
283 ppb/ppm Parts per billion/million
284 PPORD Product and Process- Orientated R&D
285 PSD Particle Size Distribution
286 QDs Quantum dots
287 QSAR Quantitative StructureActivity Relationship
288 QSMR Quantitative StructureMetabolism Relationship
289 RCEP UK Royal Commission on Environmental Protection
290 RE Reticuloendothelial
291 REACH Registration, Evaluation, Authorization, and Restriction of Chemicals
292 REACH CASG REACH Competent Authority Subgroup
293 RfD Reference Dose
294 RIPoN REACH Implementation Project on NMs
295 ROS Reactive Oxygen Species
296 SAR StructureActivity Relationship
297 S(S)AR Specific (Standard) Absorption Rate
298 SAS Synthetic Amorphous Silica
299 SAXS Small Angle X-Ray Scattering
300 SCCP EU Scientific Committee on Consumer Products
301 SCENIHR EU Scientific Committee on Emerging and Newly Identified Health Risks
302 SD Standard Deviation

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303 SDS Safety Data Sheet
304 SEC Size Exclusion Chromatography
305 SEM Scanning Electron Microscope
306 SES Socio-Ecological System
307 SIEF Substance Information Exchange Forum
308 SMILES Simplified Molecular Input Line Entry (notation)
309 SRHA Suwanee River Humic Acid
310 SSA Specific Surface Area
311 STEM Scanning Transmission Electron Microscopy
312 SVHC Substances of Very High Concern
313 SWCNT Single-walled Carbon Nanotube
314 SWNT Single-walled Nanotube
315 TEM Transmission Electron Microscopy
316 TGD Technical Guidance Document
317 TLV Threshold Limit Value
318 TR Technology Roadmaping
319 TSCA Toxic Substances Control Act
320 UEL Upper Explosive (flammable) Limit
321 USEPA United States Environment Protection Agency
322 USFDA United States Food and Drug Agency
323 UVCB Substances of Unknown or Variable composition, Complex reaction products or
324 Biological materials
325 vPvB Very Persistent and Very Bioaccumulative
326 VSSA Volume Specific Surface Area
327 WP Work package
328 WPMN Working Party on Manufactured NMs
329 WPN Working Party on Nanotechnology
330 WWC Woodrow Wilson Centre
331 WWTP Waste Water Treatment Plants
332 XPS X-ray Photoelectron Spectroscopy
333 XRD X-ray Diffraction

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335 1 Executive summary

336 Purpose - The purpose of this project was to make an assessment, based on currently available
337 scientific evidence, of the applicability of the current EU regulation on industrial chemicals
338 REGULATION (EC) No 1907/2006 REACH (Registration, Evaluation, Authorisation and
339 Restriction of Chemicals) (EC, 2006), and its associated guidance documents and other
340 guidelines (ECHA, 2007, ECHA, 2008, ECHA, 2010) for ensuring the safety of products containing
341 Nanoparticles (NPs) and Nanomaterials (NMs). A second aim was to determine whether any
342 adjustments to the existing regulations were needed to ensure that the goals of this regulation
343 are also satisfied for NMs, i.e. to improve the protection of human health and the environment
344 from the risks that can be posed by chemicals whilst also enhancing the competitiveness of the
345 EU chemicals industry, a key sector for the economy of the EU. Indeed it is this dual role of
346 protecting both health and safety and industrial competitiveness that is at the heart of much of
347 the debate surrounding the applicability of REACH regulation for NMs, as industry are among
348 the strongest voices saying that current regulations are sufficient to capture any potential risks
349 of NMs, while the scientific community continue to call for additional research to answer this
350 question. The identification and mitigation of potential human and envirnmental risks is vital for
351 consumer confidence and the continued growth of the nanotechnology sector.

352 Several critical differences between the nanoforms of substances and their bulk (not nanoscale)
353 counterparts have been discussed within this project that may necessitate additional
354 considerations for NMs within REACH. These diverging characteristics for NMs in comparison to
355 their micro or macro forms have been grouped according to the following properties:
356 interactions with surroundings, cellular uptake mechanism, cellular exit processes, potential for
357 evolution, and mechanisms of action (see Box 4.1, section 4.1, p.118, and section 4.5, p.137).
358 Specific considerations for regulatory assessment of NMs include: specification of definition and
359 identification of substance, system of triggers for testing requirements, validation of testing
360 methods for risk assessment, and revision of criteria for classification as hazardous and
361 authorisation. Further limitation in the current regulatory framework have been discussed using
362 the life-cycle concepts, including need for a comprehensive inventory on the products
363 containing nanomaterials focus on the variables like NMs functionalisation, NM loadings,
364 different methods for incorporation into products, different product matrices (textiles, plastics,
365 composites), and relies of NPs from those products. Within this project, existing principles,
366 definitions, assumptions, approaches and procedures in REACH and current chemical safety

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367 assessment guidance for human health and environment have been assessed with respect to
368 their applicability to NMs.

369 The project team responded to the SKEP call Impacts of converging technologies for
370 environmental regulation and the presenet project addressed a topic under Pilar II. Reviewing
371 the sustainability of existing legal frameworks with respect to the challenges posed by
372 converging technologies theme 3: Human health and environmental impacts with the main
373 objective of contributing to the delivery of an evidence-base for the effective regulation of new
374 materials during their entire lifecycle according to the precautionary principle. We have
375 addressed this by specifically assessing whether current REACH and related regulations have
376 the capacity to sufficiently protect human and environmental health. The scope of the project
377 did not cover the competitiveness of European industry, and thus we have not necessarily
378 addressed this in all aspects in the report.

379 The report is intended to inform the Member States position in activities within Competent
380 Authorities within REACH, and involvement within OECD Working Party on Nanomaterials.
381 Swedish Chemicals Agency (KemI) and EA in Sweden, HSE and EA in UK, and the environmental
382 agencies in Irland and Finland, representing the Member States authorities involved in those
383 activities have been continuously informed about the project progress and will receive its final
384 report with the aim to be integrated into reference lists for development of their position and
385 action plans.

386 Project expertise and scope of the report - The partners in the project have expertise in
387 regulatory toxicology (KTH), the chemistry of NMs and their behaviour in the environment and
388 ecotoxicity (UB), in mechanisms of NMs interactions with living systems (UCD) and
389 environmental policy (UH). These competences were combined to perform comparative analysis
390 between the properties of conventional chemicals and NMs as they relate to risk assessment and
391 to produce the proposals for adjustments to the REACH framework for safety assessment
392 contained herein. Specifically, chapters 2, 3, 4 and 5 focus on the main objectives of this project
393 by providing an assessment of how the REACH legislation and its guidance documents may need
394 adjustment to ensure that the purpose of REACH is satisfied also for NMs. However, there are
395 borderline areas of regulation where existing evidence justifies public concern over precaution
396 yet regulation is not feasible under the current regime for chemical regulation. Quantitative
397 assessment of potential toxicological and exposure risks, as outlined in chapters 2-5, takes place
398 in the context of numerous uncertain events in the development of nanotechnologies,
399 environmental policy and governance, and public perception of risks all of which in turn

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400 influence the weight and credibility of the evidence. Chapter 6 focuses on these borderline areas
401 and outlines a methodology for mapping out the social, economic and political factors that need
402 to be accounted for, along with the toxicological and ecotoxicological factors, when designing
403 and implementing REACH legislation for NMs.

404 Project outcome - Scope of recommendations - The key recommendations are summarised in
405 Table 1.1, p.22, and have been grouped under the following headings: (I) definitions and naming
406 of NMs for regulation, (II) characterization of NMs, (III) mandatory reporting system for
407 substances in nano form on their own, in preparations and incorporated into products, (IV)
408 systems of triggers for registration and subsequent data obligations, (V) obligations for NMs in
409 articles, (VI) testing and information requirements for NMs, (VII) assessment of potential novel
410 risks from NMs, (VIII) validation of test methods and test conditions, (IX) hazard classification
411 and authorization. The references to the explanatory sections of the report have been provided
412 in Table 1.1, p.22 for each specific recommendation and only brief presentations follow in this
413 section.

414 Recommendations for a definition of Nanomaterials - The definition of NMs for regulatory
415 purposes is still a matter of debate and the REACH regulation and associated guidance
416 documents do not define NMs. According to the EC recommendation on the definition (under
417 public consultation 21/10/2010 19/11/2010) nanomaterial means a material that meets at
418 least one of the following criteria: consists of particles, with one or more external dimensions in
419 the size range 1 nm - 100 nm for more than 1 % of their number size distribution; has internal or
420 surface structures in one or more dimensions in the size range 1 nm 100 nm; has a specific
421 surface area by volume greater than 60 m2/cm3, excluding materials consisting of particles with
422 a size lower than 1 nm (EC, 2010).

423 In this report it is recommended (points 1, 2, 3 in Table 1.1, p.22) that the EC adopts a single
424 overarching definition of NMs, which may be supplemented as required for specific legislation
425 and that this definition includes reference to the following elements: the origin of the
426 nanomaterial, internal structure and/or external dimensions, specific surface area, single lower
427 size limit, multiple upper limits bands, and gaining of biological identity as a result of their
428 interactions with surrounding biomolecules.

429 Recommendations for substance identification and nanomaterials characterisation -

430 REACH defines a substance as a chemical element and its compounds in the natural state or
431 obtained by any manufacturing process, including any additive necessary to preserve its
432 stability and any impurity deriving from the process used, but excluding any solvent which may

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433 be separated without affecting the stability of the substance or changing its composition. The
434 European Commission stated that NMs are covered by this definition (CEC, 2008), and in this
435 sense although NMs are not explicitly mentioned in the regulation all of the REACH provisions
436 apply. It needs to be mentioned however that the convergence of nanotechnology with
437 biotechnology, information and cognitive technologies is predicted to result in new types of
438 products (Roco, 2005) that may require new policies to protect human health and the
439 environment. In this context the analysis of the adequacy of REACH presented in this report is
440 limited to passive NMs.

441 The broad definition of a substance within REACH is based on the chemical element and the
442 list of parameters required for substance identification is neither adequate for distinguishing
443 between the bulk and nanoforms of certain substances, nor for distinguishing between different
444 nanoforms. This results in problems with NMs identification, namely whether NMs are to be
445 considered as equivalent to or different from the bulk material for the purpose of registration. A
446 broader spectrum of properties will be needed to sufficiently characterize a given nanomaterial
447 for the purposes of evaluating hazard and assessing risk. This report (points 4 and 5 in Table 1.1,
448 p.22) recommends extension of the list of parameters required for substance identification
449 under REACH and the establishment of accredited laboratory centers to undertake physico-
450 chemical characterisation of NPs and NMs for registration purposes.

451 Recommendations for REACH provisions for registration and data requirements - The
452 obligation of registration, the registration time and the scope of required data for substances (on
453 their own, in preparations or in articles) under REACH are dependent on the quantities of
454 production or import (tonnage trigger), the phase-in /non phase-in status (see footnote 4, p.34
455 and 8, p. 37) of the substance and the intrinsic properties resulting in classification as hazardous
456 (section 2.3.2.2, p.37 and Table 2.1, p.67).

457 In order to foresee what range of testing and data would be required at present for different
458 NMs, gathering and analysis of the following is essential: 1. information on production / import
459 quantities of different NMs; 2. understanding of status in the categorization for phase-in and
460 non-phase-in substances; 3. assessment of tools/methods available for prioritization and
461 classification for hazard classes of NMs.

462 Only limited and uncertain estimates of the production / import quantities of different NMs and
463 their presence in industrial or consumer products have been reported. The lack of reliable
464 estimates results from the lack of an efficient reporting system for the production and
465 commercial uses of NMs in the EU. Voluntary reporting initiatives have been undertaken in the

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466 UK, France, Germany, Ireland as well as in the US. So far they have failed to fill existing
467 knowledge gaps (Breggin, 2009, Milieu Risk & Policy Analysts, 2010). National mandatory
468 reporting systems initiatives are now being developed in France (according to Art 73 Grenelle
469 law 2, expected to be launched in 2011), Norway (within the Pollution Control Authority (SFT)),
470 US and Australia (Proposal for Regulatory Reform of Industrial NMs). This was also
471 recommended by the Royal Commission on Environmental Pollution in the UK (RCEP, 2008).
472 However the legal status of those national initiatives in European countries has been questioned
473 as they could potentially impinge on REACH (Milieu Risk & Policy Analysts, 2010, Montfort,
474 2010).

475 In this report it is recommended (points 6 and 7 in Table 1.1, p.22) that a mandatory reporting
476 system for substances in nanoforms, on their own, in preparations and incorporated into articles
477 be developed and implemented on the EU level, allowing NM traceability and exposure sources
478 estimation.

479 The assessed within this report is that the current systems of triggers guiding the testing
480 requirements and prioritization for more comprehensive testing would not be efficient for NMs
481 (on account of their low relative mass but extremely high particle numbers) and a list of
482 recommendations regarding more NM-appropriate triggers for registration and the subsequent
483 obligations for NMs has been presented (points 8, 9, 10 in Table 1.1, p.22).

484 Phase-in / non-phase-in criterion - At present the decisive REACH criterion of whether a
485 material is a non-phase-in or a phase-in substance applies also to NMs. NMs will be considered
486 as phase-in (see footnote 4, p.34), if they or their base substance are listed on EINECS
487 (European Inventory of Existing Commercial Chemical Substances) (cf. decision of Member
488 States Competent Authorities), are considered as No-Longer Polymers or have been
489 manufactured in the EU but not placed on the market between 1st of June 1992 and 1st of June
490 2007. The REACH Competent Authorities endorsed a paper (EC, 2008b) prepared by its sub-
491 group on NMs (CASG-Nano) which concluded that nanoforms of existing substances (i.e. those
492 with an EINECS number) would, by default, be treated as phase-in substances. Thus, some NMs
493 are considered as phase-in substances (e.g. gold, TiO2), while others are non-phase-in substances
494 (e.g. fullerenes).

495 It is possible that some substances at the nanoscale, which in the past have been identified by
496 the same EINECS number as their bulk counterparts, may have to be considered as different
497 substances for the purpose of REACH. However, as it currently stands, all substances which were
498 covered by the original EINECS number retain phase-in status (EC, 2008b). This procedure of

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499 retaining phase-in status followed by formation of separate registration if the nanoform(s) are
500 considered as different substances with regard to REACH, will imply data requirements based on
501 the nanoform production volume solely and will therefore result in the minimised data set
502 required. The consequences of this are that the data requirements and registration deadlines for
503 NMs classified as non-phase-in and phase-in are different (Table 2.1, p.67). In practice, due to
504 the as yet quite low production volumes (<1000 tonnes) of some of the nanoforms of substances,
505 categorisation as phase-in would likely lead to a delayed registration (until 2018 for NMs
506 produced at volumes of 1 100 tonnes per year per manufacturer, and until 2013 for NMs
507 produced at volumes of 100 1000 tonnes per year per manufacturer). The categorisation as
508 phase-in also limits the data requirements for substances produced in quantities between 1 and
509 10 tonnes. Data solely on the physico-chemical properties (no toxicity data) for NMs produced
510 up to 10 tonnes would be required (unless prioritized, based on the classification CMR, PBT,
511 vPvT, or other classification together with dispersive use). This differential categorisation of
512 NMs lacks a rational basis. There is no scientific evidence to suggest that those two groups of
513 NMs (phase-in or non-phase-in) represent a different likelihood of causing a concern, and thus
514 no reason to treat them differently.

515 It has therefore been recommended in this report (point 8 in Table 1.1, p.22) that nanoforms of
516 substances should be treated as different substances from their bulk counterparts and that none
517 of the phase-in provisions should apply. Furthermore different nanoforms of chemically same
518 substance may require separate registration and the recommendation on parameters to
519 distinguish between those forms has been included (point 9 in Table 1.1, p.22). However, this
520 recommendation is closely linked to point 10 in Table 1.1, and should not be implemented in
521 isolation.

522 Tonnage thresholds - The limited information that is available on current NMs production
523 volumes indicates that many NMs would not reach the thresholds triggering the requirements
524 for data satisfying meaningful risk assessment, and that a number of them (below 1 tonne)
525 would not be required to be registered (Table 2.1, p.67). The unique properties of substances at
526 the nanoscale that could potentially result in increased health and environmental risks are
527 determined to a great extent by particle size, surface structure and surface activity and to a
528 lesser extent by concentration and tonnage when compared to their micro or macroscale
529 counterparts. Consequently, for the same amount of nano structured and bulk materials
530 determined by mass, there is a much higher number of particles and larger surface with
531 potentially higher activity for nano structured materials than for bulk. The same mass based
532 triggers for nano and bulk forms would result in non-proportional efforts to assess and mitigate

18
533 risks of NMs in relation to the bulk forms. A list of possible alternatives to the current, mass
534 based, tonnage bands for data requirement has been recommended (point 10 in Table 1.1, p.22).
535 Lowering of the existing tonnage thresholds by application of a converging factor e.g. 100 may
536 seem practically attractive; however data requirements for reduced bands after the hypothetical
537 factor of 100 has been applied would still not provide all of the data needed for accurate risk
538 assessments to be performed. With no long-term studies and no toxicokinetics data, no long
539 term hazard assessment could be performed. The extrapolation approaches from short term
540 effects to long effects using assessment factors has been developed solely on the data for
541 conventional chemicals and has not been validated for NMs. Differences in ADME and
542 toxicokinetics between bulk (micro or macro) and nano forms are expected, but are not well
543 investigated as yet. It has therefore been recommended in this report to supplement this
544 approach with nano-specific data requirements necessary for risk assessment.

545 Classification as hazardous - Classification and Labelling (CLP) (Regulation (EC) No

546 1272/2008), the new European Regulation on Classification, Labelling and Packaging of
547 chemical substances and mixtures, introduces a new system for classifying and labelling
548 chemicals, based on the United Nations Globally Harmonised System (UN GHS). By June 2015
549 this regulation will gradually replace the old Directives on classification, labelling and packaging,
550 i.e. Council Dangerous Substances Directive DSD 67/548/EEC and Dangerous Preparations
551 Directive DPD 1999/45/EC. The classification criteria are also relevant for other EU legislation
552 ("downstream legislation"), including REACH.

553 The classification of a substance or preparation as being dangerous is based on one or several
554 endpoints concerning physical-chemical properties, and health or environmental effects.
555 Classification plays a key role in REACH. It must be included in the registration dossier for a
556 substance, and it triggers certain provisions such as prioritization for additional testing in the
557 lower tonnage band (human and environmental effects based, together with the dispersive use
558 provision), the requirement to perform an exposure assessment and risk characterisation as
559 part of the CSA, and the obligation to provide an SDS (for substances above 10 tonnes).
560 Classification of a substance as mutagenic, carcinogenic or toxic to reproduction may lead to an
561 authorisation procedure (IHCP, 2009).

562 Additional to the criteria for CLP classification, REACH sets the criteria for classification as PBT,
563 vPvB (see footnotes 6, p.35 and 7, p.35). Substances fulfilling those criteria can be considered as
564 SVHC and special provisions or obligations may apply to deal with mitigation of these risks.

19
565 However, the data set required by REACH (particularly at the lower tonnages) is not sufficient
566 for classification of substances, including NMs, into the different hazard categories. The different
567 types of hazard identification data that are required under REACH and that can be directly used
568 for classification purposes are presented in Table 2.1, p.67. The classification criteria for the
569 hazard endpoints according to GHS/CLP and REACH aligned with the production volume
570 triggering data sufficient for this classification are presented in Table 2.2, p.68.

571 The applicability and efficiency of the classification system for NMs has been discussed in terms
572 of the criteria for classification (including relevance of dose metrics in the proposed default
573 values, relevance of test species and environmental compartments for which criteria cut-off
574 values are set), the fact that testing for all known hazard classes is not required, and the
575 limitation that the investigation tools have not been validated and possibly lack the required
576 sensitivity for NMs. It is also possible that currently known hazard endpoints will not cover all
577 novel mechanisms of toxicity of NMs. Within this context, recommendations regarding the
578 assessment of long term effects of NMs for human and ecosystems have been included (points
579 12, 13, 14, 15, 16, Table 1.1, p.22) pointing at the appropriate measures of NM degradation,
580 testing of the toxic effects after long term treatment, testing on the environmentally relevant
581 species (e.g. benthic), and determination of biokinetics and ADME processes.

582 Validation of test methods The above data requirement recommendations for NMs closely
583 depend on the validation of test methods and test conditions and specific consideration has been
584 given to the need for development of standardised tests for a range of different in vivo, in vitro
585 and environmental compartments and validation of OECD tests of persistence and
586 bioaccumulation for their applicability to specific NM types (points 19 and 20, Table 1.1, p.22).

587 Authorisation - REACH has established a system under which the use of substances with
588 properties of very high concern and their placing on the market can be made subject to an
589 authorisation. Authorisation is applied in two steps: 1. issuing by ECHA of a Candidate List of
590 substances of very high concern (SVHC), containing substances liable for authorisation; 2.
591 prioritisation of certain substances for Annex XIV, and inclusion of a substance into Annex XIV
592 which are banned unless company-specific authorisation is issued. Authorisation can be
593 obtained if the use of a substance is adequately controlled, or if no substitutes are available and
594 if the economic and social benefits outweigh the risks, in the case of substances for which no
595 safety threshold applies.

596 Authorisation aims to ensure that the risks from the use of such dangerous substances are either
597 adequately controlled or justified on socio-economic grounds, having taken into account the

20
598 available information on alternative substances or processes. For NMs this aim can only be
599 fulfilled to a limited extent at present, due to the limited applicability of existing methods to
600 identify and characterise the hazardous properties of NMs and to classify NMs into hazard
601 classes, a situation that is not improved by the limited data requirements for nanomaterial
602 hazard and exposure assessment set by the system of triggers.

603 Precautionary measures for high aspect ratio / toxic NMs - Until the structure of the REACH
604 regulation is modified and sufficient data are available to conduct meaningful risk assessment of
605 NMs the precautionary principle should be invoked. It is therefore advised that high aspect ratio
606 NPs / NMs are prioritised for evaluation along with NPs / NMs meeting the toxicity criteria
607 (points 21 and 22, Table 1.1, p.22).

608 Obligations for chemicals in articles - The obligations placed on manufactures or importers of
609 articles under REACH include: registering of substances with the ECHA if they are produced or
610 imported in volumes above 1 tonne/year/legal entity and intended to be released during normal
611 use of an article if not already registered for this use; informing the recipient of the article of the
612 presence of a Substance of Very High Concern or SVHC (if concentration >0.1%) and how to use
613 it safely if necessary; notifying ECHA of the presence of a SVHC in an article (if concentration of
614 SVHC in article >0.1% and production volume of the SVHC > 1 tonne per annum) and if exposure
615 to this substance cannot be excluded during normal or reasonably foreseeable conditions of use
616 or disposal (ECHA, 2008).

617 The criteria for the obligations for articles have been developed without any specific
618 consideration of NMs and have been assessed within this project as not being fully satisfactory.
619 The implementation of obligations for NMs in articles would be very limited due to the
620 production volume trigger (1 tonne trigger) which limits registration and notification
621 requirements, the fact that the SVHC criteria are not validated and/or testing is not required
622 limits information and notification obligations, while the 0.1% cut-off of the composition of the
623 whole article does not reflect NMs applications and limits information and notification
624 obligations (Franco, 2007). It is therefore recommended that alternatives are sought to the
625 present provisions for obligations regarding NMs incorporated into articles (point 11, Table 1.1,
626 p.22).

627

21
628 Table 1.1 Summary of recommendations

Section

I Recommendations around nano definitions and naming for regulation

It is recommended that the European Commission adopt a single overarching

definition of NMs for regulatory purposes, which may be supplemented as
required for specific legislation, and that this definition is based on particle size 2.2
1 distribution by particle number, in the appropriate formulation. Details of 3.5.2
delimiters are given in the text.
4.7
Furthermore, it is recommended that account is also taken of the size distribution
of NMs in their definition.

It is recommended that ECHA consider NMs as biological entities and build on

2 4.7
lessons from regulation of protein therapeutics and other biological substances

It is recommended to consider prioritising the debate on naming of NMs to

3 provide clarity around the issue of them being new substances, separate from 4.7
their chemical identity

II Recommendations regarding characterization of NMs

It is recommended that characterisation of the following physico-chemical

parameters be mandatory for all substances defined as 'NMs' with the REACH
framework: size (average and distribution), shape, core chemical composition, 3.2.7
4
specific surface area, surface modification and surface charge. In addition for 3.5.2
oxides of silver and zinc -dissolution, for TiO2 and SiO2 - crystallinity, for oxides of
ceria and iron - oxidation state. The recommendation is explained in the text.

It is recommended that accredited centers be established to undertake physico- 3.3.2

5 chemical characterisation of NMs, and potentially also to prepare regulatory
dossiers in the future.

Recommendations around a mandatory reporting system for substances in

III
nanoforms and incorporated into products

It is recommended that ECHA develop a strategy to collect and disseminate

information on the forms and amounts of NMs imported or produced in the EU,
6 along with information on downstream uses. Such a strategy should include 2.3.2.2
development and implementation of a mandatory EU reporting system for
substances in nanoforms and products containing NMs (a nanoproduct register).

It is recommended that the nanoproduct register collects comprehensive

information on the presence of NMs in articles, thereby allowing traceability and
exposure sources estimation. 2.3.2.5
7
It is recommended that REACH regulations require the disclosure of NMs 5.3.1
incorporated into articles. CLP regulations should require separate labeling of the
nanomaterial content of products.

IV Recommendations regarding triggers for registration (and subsequent

22
 obligations) for nanoforms of substances (and their preparations)

It is recommended that in the registration process nanoforms of materials be

treated as different from their bulk (micro or macro) counterparts and that none
8 2.3.2.2
of the phase-in provisions apply. The provisions applicable to non-phase-in
substances also need revision.

It is recommended that nanoforms with the same core chemistry be

differentiated for separate registration under REACH using differences in the
following physico-chemical parameters: surface modification, size, shape and 3.5.2
surface charge. The recommendation is explained in the text.
9 It is recommended to re-consider the testing approach for NMs from 1st
principles for chemicals the basis of regulation is the chemical nomenclature of
the drug / molecule - for NMs it should include the surface in addition to the 4.7
particle size, shape and composition, as this is what governs all subsequent
interactions and impacts.

It is recommended that an alternative be sought to the tonnage trigger thresholds

used for bulk materials. Possible alternatives described in the text include:
Safety factors. The existing tonnage trigger framework could be used for NMs if
combined with an appropriate safety factor.
Alternative metrics. Existing tonnage trigger thresholds could be used in
10 conjunction with alternative metrics, substituting the mass metric for one more 2.3.2.2
relevant to NMs. Both total particle number, and total surface area per
manufacturer per annum would be good candidates.
Obligatory testing. The most draconian option would be to require all NMs
registered to submit a technical dossier, irrespective of the quantity produced.
The content of this dossier may, but does not necessarily need to, coincide with
that of one of the tonnage trigger bands.

V Recommendations regarding obligations for NMs in articles

It is recommended that alternatives to be sought to the present provisions for

obligations (of registration, notification to ECHA and information of the user)
placed on NMs in articles. The specific recommendations include:
a) Lowering of the volume thresholds for trigger of the obligation of registration
11 and notification for NMs in articles; 2.3.2.5
b) Implementation of nano-specific data requirements as for nano-substances
discussed above (points under IV);
c) Requirement of an information obligation for all (not only known SVHC) NMs
based on the limit applied to the homogenous part of the article.

VI Recommendations regarding testing and information requirements for NMs

It is recommended that alternative data requirements for NPs be developed to

12 replace those linked to the tonnage trigger thresholds (see also recommendation 5.4
10 and section 2.3.2.2).

13 It is recommended that appropriate measures of NM degradation be required for 5.4

all NMs to which REACH regulations are applicable (dissolution in addition to

23
 ready biodegradation for inorganic NMs).

It is recommended that for toxicokinetics / ADME studies are included in the

14 obligatory data requirements for all NMs on the market, irrespective of the 2.3.2.2
volume produced.

As an interim measure it is recommended that a measure of chronic (eco) toxicity

be required for all NMs (to which REACH regulations are applicable).

15 This interim measure could be dropped once sufficient confidence is established 5.1
in safety factors, and it is further recommended that regulatory bodies and the
nanoparticle industry fund the research necessary to demonstrate the linkage
between acute and chronic ecotoxicity.

It is recommended that in addition to the short-term toxicity test on Daphnia that

16 is already required (prioritised samples > 1 tonne and above) short-term toxicity 5.4
tests should also be mandatory for a terrestrial species and a benthic species.

VII Recommendations regarding assessment of potential novel risks from NMs

It is recommended that further high quality research be conducted to

characterize the hazard posed by NMs to at risk consortia (filter-feeding marine 5.2.4
17
bivalves and estuarine benthic communities), and also to assess models for 4.7
vulnerable human populations, such as injured gut and lung models, etc.

It is recommended that research be undertaken to describe novel endpoints

18 arising from exposure to NMs and to assess their relevance in a regulatory 5.2.5
context. Such research should be funded via ERA and SKEP type projects.

VIII Recommendations regarding validation of test methods and test conditions

It is recommended that standardised (eco)toxicity exposure conditions be

5.2.3
established for a range of different in vitro, in vivo and environmental
compartments (freshwater, marine, saturated aquatic sediments, soils).
19
It is further recommended that fundamental research be conducted into the
appropriateness of commonly used culture media and exposure water for NM
4.7
exposures.

It is recommended that, as a matter of urgency, the OECD persistence and

20 bioaccumulation tests detailed in Table 5.2, p.184, be assessed for their 5.4
applicability to specific NM types and their methodologies validated as suitable.

IX Recommendations regarding hazard classification and authorisation

It is recommended that biopersistent (persistent in vivo) NMs with high aspect

ratio (HARNs) be treated within the REACH framework as substances of 2.3.2.4
21 equivalent concern, and their assessment prioritized by inclusion in the SVHC list.
HARN status should be considered for all substances with an aspect ratio of >
1:10 (Tran, 2008) or with one dimension in the nanoscale and one > 5 m.

22 2.3.2.4
It is recommended that where evidence suggests that a nanomaterial would fulfill
the toxicity criteria of the PBT classification it also be treated as a potential

24
 substance of equivalent concern and placed on the SVHC list.

629

630 Recommendation for the governance of nanotechnology - The general conclusion in much of
631 the nanotechnology literature is that more information is needed in order to understand the
632 various uncertainties of novel technologies. However, risk assessment takes place in the context
633 of an uncertain path of events in the evolution of technology, governance and risk perception, all
634 of which influence the weight and credibility of evidence. Thus, while clarifying uncertainties is
635 essential, there is also a need to organize and use the information that is available in a more
636 productive and integrated manner. Chapter 6 discusses and demonstrates a method for doing so,
637 namely, building integrative technology roadmaps for nanotechnology risk governance. The next
638 step with this analysis would be refinement of the methodology through applications.

639 The results of the project have been captured in this report, and the report has undergone a
640 review process prior to publication, whereby opinions were sought from representatives of
641 academica, industry, governemtnal agencies and Non-governmental organisatons who are fully
642 independent of the project. Where relevant, these opinions have been incorporated into the
643 present report. However, the report is the opinion of the authors only, and does not seek to be a
644 consensus document. Upon publication, the report will be disseminated to relevant stakeholders
645 including EC, ECHA, consumer organisations, the scientific community, industry and industry
646 organisations, importers and downstream users, policy oriented organizations and others
647 interested in the safety assessment of NMs.

648

25
649 2 REACH provisions and ECHA guidelines recommendations
650 - an evaluation of their suitability for NMs

651 2.1 Objective

652 In this chapter we present the elements of current EU regulation on industrial chemicals REACH
653 (Registration, Evaluation, Authorisation and Restriction of Chemicals) (EC) No 1907/2006 (EC,
654 2006) and the associated chemical safety assessment (CSA) guidance documents (ECHA, 2007,
655 ECHA, 2008) that have been analysed within the project to assess their adequacy and
656 applicability for risk assessment and regulation of NMs. The overall outcomes, conclusions and
657 recommendation of the project are highlighted, and cross referencing to the specific analysis
658 that lead to these recommendations in other chapters is provided.

659 2.1.1 Introduction to REACH - history, aims

660 The current EU regulation on industrial chemicals REGULATION (EC) No 1907/2006 REACH
661 (Registration, Evaluation, Authorisation and Restriction of Chemicals) (EC, 2006), builds upon
662 four decades of experience with chemicals regulation, with the first regulation adopted in 1967.
663 In the late 1990s it became evident that the existing regulations were not sufficient to protect
664 human health and the environment. To illustrate the problems, examples can be given. Within
665 more than 15 years, out of more than 100 000 registered substances, only 141 substances were
666 identified as being of special concern and the appropriate risk assessment has been performed.
667 Additionally, very limited restriction possibilities existed, because showing evidence of adverse
668 effects in light of practically non-existant data was almost impossible. The initiative to develop
669 new policy was taken in 1998 by 5 Member States who identified key components and drafted a
670 document to the Council of Environment Ministers. Three years later the Commission presented
671 a White Paper drawing up a strategy for a new chemicals policy (EC, 2001) and in 2003 the
672 public version was published. After extensive debates involving different stakeholders the new
673 European chemical legislation, REACH was adopted in December 2006 (EC, 2006) and entered
674 into force on the 1st of June 2007.

675 The aims of REACH are to: 1) Improve the protection of human health and the environment from
676 the risks that can be posed by chemicals; 2) Enhance the competitiveness of the EU chemicals
677 industry, a key sector for the economy of the EU; 3) Promote alternative methods for the
678 assessment of hazards of substances that are less reliant on animal testing; and 4) Ensure the
679 free circulation of substances on the internal market of the European Union. REACH makes
680 industry bear most of the responsibility to manage the risks posed by chemicals that they

26
681 manufacture or import, and to provide appropriate safety information to their users /
682 customers. It foresees that the European Union can take additional measures on highly
683 dangerous substances as needed.

684 In the context of this report, it is important to note that REACH was developed and implemented
685 prior to the potential safety issues around NMs becoming a major source of concern for
686 governments internationally, as the first reports on the potential for nanotoxicity only appeared
687 in the literature around 2003 (Colvin, 2003). Although some discussions on the regulatory
688 implications of nanotechnology were initated at that time (EC, 2004) the knowledge on nano-
689 specific properties was not considered to be sufficient to serve any specific regulatory
690 considerations and consequently the provisions that were developed do not reflect such
691 consideration.

692 In a situation when several NMs have already entered the market and exposure from the
693 environment and consumer products cannot be excluded, adequate methods to ensure safe use
694 of NMs are essential for sustainable development. Several critical differences between the
695 nanoforms of substances and their bulk (micro or macro-scale) counterparts may necessitate
696 additional considerations for REACH regulation and chemicals safety assessement guidance,
697 including clarification of definition and identification of substance, triggers for testing
698 requirements, validation of testing methods, and revision of criteria for authorisation.

699 The purpose of this report is to summarise what is presently known and can be reasonably
700 assumed about the applicability of REACH to NMs and to propose some additional measures that
701 can be taken within the REACH framework to promote safety in the use of NMs.

702 2.2 Definition of NMs

703 The definition of NMs for regulatory purposes is still a matter of debate and the REACH
704 regulation and associated guidance documents do not, as yet, define NMs. The difficuties in
705 developing definitions accepted by different stakeholders arise from very divergent views on: 1)
706 what are the relevant paramenters for distinguishing between the nano and the bulk
707 (micro/macro) - particle size and surface area or nano-specific properties, and 2) what
708 percentage of the nanoscale particles in the material would serve to classify the material to be
709 nano. Different elements like scientific evidence, technical feasibility, economic consequences
710 impact the discussion on whether and how to discriminate between the bulk and nano forms of
711 a material.

27
712 Various proposals for a definition have been developed by international organisations and
713 committees such as the International Organisation for Standardisation (ISO), the European
714 Standardisation Committee (CEN), the Organisation for Economic Co-operation and
715 Development (OECD), EU Scientific Committee on Emerging and Newly Identified Health Risks
716 (SCENIHR), EU Scientific Committee on Consumer Products (SCCP), and the American Chemistry
717 Council. Several national proposals for a definition were also developed including ones from
718 Australia, Canada, Denmark, The United Kingdom and The United States of America, as well as
719 EU definitions developed for specific regulatory purposes such as the Cosmetic Products
720 Regulation, and the Novel Foods Regulation (for a review see (Lvestam, 2010)); examples
721 provided in Box 2.1 (see below).

722 A list of terms and definitions with the focus solely on the nano scale size (1-100nm) and shape
723 have been developed by the European Committee for Standardization (CEN) including the
724 following terms: nanoscale, nano-object, particle, agglomerate, aggregate, NPs, nanoplate,
725 nanofibre, nanotube, nanorod, nanowire, quantum dot (CEN ISO TS 27687, 2008). The size- and
726 shape- related criteria for defining engineered NPs have been broadly accepted (French
727 Academy of Sciences, 2004, Royal Society, 2004, US NSTC, 2004, Hansen, 2007, Lvestam, 2010).
728 The definition of a nanomaterial developed for Regulation of cosmetics products (EC, 2009) is
729 limited to an insoluble or biopersistent and intentionally manufactured material with one or
730 more external dimensions, or an internal structure on the scale from 1 to 100 nm. However the
731 properties insoluble or biopersistent lack explicitly defined criteria in this context.

732 A key feature of NMs, which has driven their development for a wide number of applications, is
733 the fact that quantum effects emerge which introduce new properties that are not shared by
734 non-nanoscale particles with the same chemical composition. Auffan et al. argue that the
735 nanoscale associated properties are more relevant than size when considering the impact of
736 NMs on the environments and human health. They have concluded that particles larger than
737 30nm do not show physico-chemical properties different from their bulk counterparts (Auffan,
738 2009). The analysis however has a number of limitations (discussed in section 3.2.3, p.82) and
739 may not be representative for different classes of NMs. Additionally, it fails to account for the
740 fact that NMs surround themselves with a coating of proteins upon contact with biological
741 systems, which confers a biological identity to them, and thereby grants NMs unique access to,
742 and interaction with, biological machinery and biological barriers, which is entirely
743 unprecedented (Park, 2010b) (discussed in Chapter 4.1, p.118).

28
744 The Council of the European Union (CEU) in the proposal of amendment to Regulation of novel
745 foods, included a definition of NMs with the use of a properties criterion: Properties that are
746 characteristic to the nanoscale include: (i) those related to the large specific surface area of the
747 materials considered; and/or (ii) specific physico-chemical properties that are different from
748 those of the non-nanoform of the same material (CEU, 2009). This proposal was not adopted.
749 Properties-based criteria have been criticised for problems with implementation for regulatory
750 purposes, as difficulties exist to define and measure them routinely (Lvestam, 2010, SCENIHR,
751 2010).

752 SCENIHR (SCENIHR, 2009, SCENIHR, 2010) suggested addition of a surface criteria (i.e. the
753 specific surface area to be above the 60m2 g-1 of material volume) to the dimension-based
754 definition in order to include aggregated and agglomerated NPs with dimensions above 100 nm
755 that retain nano-specific physico-chemical properties due to their specific high surface ratio. In
756 its most recent report SCENIHR concluded that there is no scientific evidence to qualify the
757 appropriateness of the 100 nm size cut-off, and that the whole nanoscale metric (1-999 nm)
758 should be considered. Practically if the median size is < 500 nm a material would be considered
759 as a potential nanomaterial and more detailed characterisation of the number size distribution is
760 required. Any material would be considered as a nanomaterial when > 0.15% (or any specified
761 percentage) of the material, based on number concentration, has a size below the upper limit (of
762 100nm) (SCENIHR, 2010). This approach has been criticised by the Nanotechnology Industry
763 Association (NIA) with the argument that the 0.15% limit is below the detection levels of the
764 currently available methods. Also the statistical approach based on estimation of size
765 distribution by calculation of three standard deviations, has been criticised, as the assumption of
766 the distribution being a normal distribution may not be valid for all manufactured NMs, many of
767 which show Lorenzian, log-normal or other distributions (http://www.nanotechia.org/nia-
768 publications/consultation-responses).

769 According to the EC recommendation on the definition, (under public consultation 21/10/2010
770 19/11/2010) nanomaterial means a material that meets at least one of the following criteria:
771 consists of particles, with one or more external dimensions in the size range 1 nm - 100 nm for
772 more than 1 % of their number size distribution; has internal or surface structures in one or
773 more dimensions in the size range 1 nm 100 nm; has a specific surface area by volume greater
774 than 60 m2/cm3, excluding materials consisting of particles with a size lower than 1 nm (EC,
775 2010).

29
776 The Australian government puts requirements on manufacturers and importers to notify the
777 National Industrial Chemicals Notification and Assessment Scheme (NICNAS) of their intent to
778 manufacture or import nanoscale chemicals and defines NMs as industrial materials
779 intentionally produced, manufactured, or engineered to have unique properties or specific
780 composition at the nanoscale, that is a size range typically between 1 nm and 100 nm, and is
781 either a nano-object (i.e. that is confined in one, two, or three dimensions at the nanoscale) or is
782 structured (i.e. having an internal or surface structure at the nanoscale). Further, the notes to the
783 working definition add that where size distribution shows that 10% or more of a substance
784 (based on number of particles) is at the nanoscale, NICNAS will consider this substance to be a
785 nanomaterial for risk assessment purposes. (NICNAS, 2010).

786 The US-Environment Protection Agency (EPA) outlines the initial proposal of a Stewardship
787 Program for nanoscale materials under the Toxic Substances Control Act (TSCA) introducing the
788 definition of engineered nanoscale material for the purpose of the program. In the paper an
789 engineered nanoscale material is defined as any particle, substance, or material that has been
790 engineered to have one or more dimensions in the nanoscale. The term engineered is
791 intended to mean that the material is 1) purposefully produced and 2) purposefully designed to
792 be a nanoscale material The term nanoscale is generally used to refer to the scale measured
793 in nanometres (1 x 10-9 meters). For the purposes of the Stewardship Program, nanoscale is the
794 size range between the atomic/molecular state and the bulk/macro state. This is generally, but
795 not exclusively, below 100 nm and above 1 nm (EPA, 2010). The recent proposals for the Safe
796 Chemicals Act of 2010 (April 2010) and Toxic Chemicals Safety Act of 2010 (April 2010)
797 intended to address the reform of the Toxic Substances Control Act (TSCA) legislation, do not
798 explicitly address NMs, but both bills consider characteristics of chemical substances and
799 include respectively: chemical structure and composition; size or size distribution; shape;
800 surface structure; reactivity; and other characteristics and properties that may having a bearing
801 on their toxicological properties.

802 The FDA definition of NMs for drug and therapeutic products review, released in June 2010
803 defines NMs as materials with at least one dimension smaller than 1,000 nm (FDA, 2010).

804

30
Nanotechnology:

the research and technology development at the atomic, molecular or macromolecular levels, in
the length scale approximately 1-100 nm; the creation, use of structures, devices and systems
that have novel properties because of their small size; and ability to be controlled or
manipulated on the atomic scale

(US National Nanotechnology Initiative, 2004)

the design, characterisation, production and application of structures, devices and systems by
controlling size and shape at the atomic scale

(Royal Society, 2004)

the ability to work at the atomic, molecular, and supramolecular level (on a scale 1-100 nm) in
order to understand, create and use material structures, devices and systems with
fundamentally new properties and functions resulting from their small size

(Roco, 2003)

NPs:

have three or perhaps two dimensions in the range 1-100 nm

(Borm, 2006)

nano-object with all three external dimensions in the nanoscale (1-100 nm)

(BSI, 2007c)

any intentionally produced particle that has a characteristic dimension from 1 100 nm and has
properties that are not shared by non-nanoscale particles with the same chemical composition

(Auffan, 2009)

NMs:

NMs are materials with more than one external dimension or internal structure at the nanoscale
that could exhibit novel characteristics compared to the same material without nanoscale
features

(SCENIHR, 2007a)

a particulate substance may be regarded as a nanomaterial if the specific surface area exceeds
60 m2 /g (see section 3.2.4).

(SCENIHR, 2009)

When the size (e.g. mean, median, etc.) is <500 nm it is more likely that part of the size
distribution will be lower than 100 nm and that a material may be considered a nanomaterial
() if the characterization demonstrates that >0.15% (or any specified percentage) of the
number size distribution is <100 nm.

31
When the size range (e.g. mean, median, etc.) is 1 nm - 100 nm the material is considered a NM

VSSA above the threshold (e.g. >60 m2/cm3) may be used as an additional qualifier to indicate a
size below 100 nm.

(SCENIHR, 2010)

NMs (for regulatory purposes):

an insoluble or biopersistent and intentionally manufactured material with one or more

external dimensions, or an internal structure on the scale from 1 to 100 nm.

(EC, 2009)

properties that are characteristic to the nanoscale include: (i) those related to the large specific
surface area of the materials considered; and/or (ii) specific physico-chemical properties that
are different from those of the non-nanoform of the same material

(CEU, 2009)

material that meets at least one of the following criteria: consists of particles, with one or more
external dimensions in the size range 1 nm - 100 nm for more than 1 % of their number size
distribution; has internal or surface structures in one or more dimensions in the size range 1 nm
100 nm; has a specific surface area by volume greater than 60 m2/cm3, excluding materials
consisting of particles with a size lower than 1 nm.

(EC, 2010)

industrial materials intentionally produced, manufactured, or engineered to have unique

properties or specific composition at the nanoscale, that is a size range typically between 1 nm
and 100 nm, and is either a nano-object (i.e. that is confined in one, two, or three dimensions at
the nanoscale) or is structured (i.e. having an internal or surface structure at the nanoscale ()
where size distribution shows 10% or more of a substance (based on number of particles) is at
the nanoscale (...) consider this substance to be a nanomaterial for risk assessment purposes

(NICNAS, 2010)

particle, substance, or material that has been engineered to have one or more dimensions in the
nanoscale. The term engineered is intended to mean that the material is 1) purposefully
produced and 2) purposefully designed to be a nanoscale material The term nanoscale is
generally used to refer to the scale measured in nanometres (1 x 10-9 meters). Nanoscale is the
size range between the atomic/molecular state and the bulk/macro state. This is generally, but
not exclusively, below 100 nm and above 1 nm.

(EPA, Stewardship Program under the Toxic Substances Control Act (TSCA), 2010)

materials with at least one dimension smaller than 1,000 nm.

(FDA, 2010)

Box 2.1: Definitions of nano in the scientific and regulatory literature

32
IT IS RECOMMENDED THAT THE EUROPEAN COMMISSION ADOPT A SINGLE
OVERARCHING DEFINITION OF NMs FOR REGULATORY PURPOSES, WHICH MAY BE
SUPPLEMENTED AS REQUIRED FOR SPECIFIC LEGISLATION, AND THAT THE DEFINITION
BE BASED ON PARTICLE SIZE (SEE SECTIONS 3.5.2, P.98 AND 4.7, P.146).

The authors concur with the general approach adopted in SCENIHR 2010 and recommend that
the definition include reference to the following elements1:

1. The origin of the nanomaterial (natural, adventitious or manufactured);

2. Encompasses materials with an internal structure or an external dimension2 within the
nanoscale limits defined. Specific exemptions should be developed for macroscopic
objects with nanoscale features (e.g. membrane filters);
3. Specific Surface Area (SSA) be included as a supplementary parameter, thereby
extending the definition to highly aggregated or agglomerated materials;
4. A single lower size limit of 1 nm be adopted, although it should be acknowledged that
ambiguity will remain due to the overlapping dimensions of molecules, macromolecules
and NMs at this scale.

Multiple upper limits for NMs be adopted. There is currently no scientific evidence that
correlates the onset of nanospecific biological properties with a specific dimension. Rather a
continuum exists of increasing non-bulk properties with decreasing size (Auffan et al., 2009). It
is not therefore possible to set a scientifically meaningful single upper limit and the definition of
nano- as an enforceable term will therefore be driven by regulatory requirements. SCENIHR
(2010) suggests setting multiple upper thresholds for NMs, defining two categories which would
be considered 'nano' in regulatory terms.3

Category 2: Median size <500 nm but >100nm with > 0.15% of the number size distribution
<100 nm.
Category 3: Median size >1 nm but <100 nm with > 0.15% of the number size distribution
<100 nm.

In acknowledgement of the increasing reactivity of NMs as their size decreases it is

recommended that Category 3 be further subdivided as follows:

1
The authors agree with the SCENIHR opinion that all elements of the definition should be applicable to all
possible NMs. As such parameters useful for characterisation, such as dissolution or crystallinity, are not
appropriate for inclusion in the definition as they are only applicable to a subset of particles.
2
External particle size refers specifically to the size of the primary particle, without surface functionalisation.
3
Category 1 is median size < 1000 but > 500 nm, and as such is considered not likely to contain nanoparticles.

33
 Category 3a: Median size >40 nm but <100 nm with a small percentage of the number size
distribution <40 nm.
Category 3b: Median size > 1nm but <40 nm or a small percentage of the number size
distribution <40 nm.

Consider taking further account of the size distribution of NMs in their definition

In its present form, the SCENIHR definition of NMs does not fully account for the size
distribution of NMs in terms of the cut-off and the SSA calculation, as this is based on the
assumption that all particles have the same size, which is not the case. Thus, the theoretical SSA
does not actually account for the potentially significant fraction of particles above and below
this, which will cause the SSA to deviate from the ideal, and may mask the presence of NMs (see
section 4.7, p.146).

805

806 2.3 NMs under REACH and CLP

807 2.3.1 The main obligations under REACH

808 The main obligations under REACH include:

809 (a) Pre-Registration (1st June 2008 - 30th November 2008) of existing substances (so-called
810 phase-in4) that indicates the intention of the manufacturer / importer to register a substance
811 within 11 years. Each company wishing to take advantage of so-called delayed registration
812 deadlines for phase-in substances must pre-register. A substance that is not pre-registered must
813 be registered immediately, if production/import reaches 1 tonne or more per year;

814 (b) Registration - applies to substances manufactured or imported, on their own, in preparations
815 or intentionally released from articles. Other substances in articles are potentially subject to a
816 notification scheme or a full registration on a case-by-case basis. Registration requirements

4
Phase-in substance synonym existing substance, means a substance which meets at least one of the
following criteria: (a) it is listed in the European Inventory of Existing Commercial Chemical Substances
(EINECS), (b) it was manufactured in the Community, or in the countries acceding to the European Union on 1
January 1995 or on 1 May 2004, but not placed on the market by the manufacturer or importer, at least once in
the 15 years before the entry into force of REACH Regulation, provided that the manufacturer or importer has
documentary evidence of this, (c) it was placed on the market in the Community, or in the countries acceding
to the European Union on 1 January 1995 or on 1 May 2004, before entry into force of REACH Regulation by
the manufacturer or importer and was considered as having been notified in accordance with the first indent of
Article 8(1) of Directive 67/548/EEC but does not meet the definition of a polymer as set out in REACH
Regulation, provided that the manufacturer or importer has documentary evidence of this. (REACH Article 3
(20))

34
817 depend on the substance quantity manufactured or imported per legal entity, phase-in (see
818 footnote 4, p.34) or non-phase-in (see footnote 8, p.37) status and intrinsic properties resulting
819 in classification leading to prioritization for testing. This step of the regulations obligations
820 consists of submission of a technical dossier (the content of the technical dossier is dependent
821 on tonnages) and a Chemical Safety Report (for substances above 10 tonnes per year) to ECHA;

822 (c) Evaluation is conducted via reviews of the technical registration dossiers by ECHA and the
823 appropriate Member State Competent Authority(ies); it is separated into dossier evaluation
824 and substance evaluation.

825 (d) Authorisation is applied in two steps: 1. Issuing by ECHA of a Candidate List of substances of
826 very high concern SVHC5, containing substances liable for Authorisation; and 2. Prioritisation
827 by ECHA of certain substances for inclusion in Annex XIV, and inclusion into Annex XIV of
828 substances which are banned unless company-specific authorisation is issued. Authorisation can
829 be obtained if the use of a substance is adequately controlled (i.e. if the substance is contained to
830 achieve an exposure level below a given threshold), or if no substitutes are available and if the
831 economic and social benefits outweigh the risks, in the case of substances for which no safety
832 threshold applies (e.g. PBTs6 or vPvBs7).

833 Exemptions of substances from REACH apply, if any of the following criteria apply to the
834 substance in question: used in human/veterinary medicinal products, used in food/feed, used in

5
Substances of Very High Concern (SVHC) are defined in Article 57 of Regulation (EC) No 1907/2006 (EC, 2006)
and include substances which are: Carcinogenic, Mutagenic or toxic to Reproduction (CMR), meeting the
criteria for classification in category 1 or 2 in accordance with Directive 67/548/EEC (This directive was recently
replaced by the new EU regulation (EC) No 1272/2008 on classification, labelling and packaging of chemical
substances and mixtures, the so-called CLP Regulation. According to the new CLP Regulation these substances
shall be classified as 1a or 1b); Persistent, Bioaccumulative and Toxic (PBT) or very Persistent and very
Bioaccumulative (vPvB) according to the criteria in Annex XIII of the REACH Regulation); or Identified, on a case-
by-case basis, from scientific evidence as causing probable serious effects to human health or the environment
of an equivalent level of concern as those above (e.g. endocrine disrupters).
6
PBT substance - a substance that fulfils all three of the following criteria: 1. Persistence (the half-lives are
higher than: 60 days in marine water, or 40 days in fresh- or estuarine water, or 180 days in marine sediment or
120 days in fresh- or estuarine water sediment or in soil, 2. Bioaccumulation (the bioconcentration factor (BCF)
is higher than 2,000) 3. Toxicity (the long-term no-observed effect concentration (Noec) for marine or
freshwater organisms is less than 0.01 mg/l, or the substance is classified as carcinogenic (category 1 or 2),
mutagenic (category 1 or 2) or toxic for reproduction (category 1, 2, or 3), or there is other evidence of chronic
toxicity, as identified by the classifications: T, R48, or Xn, R48 according to Directive 67/548/EEC). Additional
criteria, based on the case-by-case experts judgment, have been included in the amendment of Annex XIII of
REACH.
7
vPvB substances - a substance that fulfils the criteria of both: 1. Persistence (the half-lives are higher than 60
days in marine, fresh- or estuarine water, 180 days in marine, fresh- or estuarine water sediment or soil; 2.
Bioaccumulation (the bioconcentration factor is greater than 5,000).

35
835 biocides/pesticides (inert (non-active) substances must be registered), non-isolated
836 intermediates that are listed in Annexes IV and V (exempt from registration only). There are
837 specific requirements for polymers: although polymers are exempt from registration, the
838 monomers and other reactants used to produce the polymer need to be registered even if it is
839 only the final polymer is imported. There are reduced requirements for isolated and transported
840 isolated substances if only used under strictly controlled conditions. There are also particular
841 exceptions for R&D and a special notification procedure for uses of substances for Product and
842 Process Orientated R&D (PPORD). Substances used in cosmetics are not exempt from
843 registration, but Chemical Safety Reports on these substances do not require information on
844 direct risks to human health from final product use. Other effects from the ingredients and the
845 actual product, such as occupational health and human health via the environment must be
846 included as part of a registration. Substances used in medical devices are not exempt from
847 registration and may trigger a re-assessment of risks already addressed by specific legislation.
848 Uses of substances in medical devices are not subject to the Authorisation procedure under
849 REACH, but could be subject to restrictions.

850 2.3.2 REACH provisions

851 2.3.2.1 Definitions

852 REACH (Article 3(1)) defines a substance as: a chemical element and its compounds in the
853 natural state or obtained by any manufacturing process, including any additive necessary to
854 preserve its stability and any impurity deriving from the process used, but excluding any solvent
855 which may be separated without affecting the stability of the substance or changing its
856 composition. Article 3(2)) defines a preparation as: a mixture or solution composed of two or
857 more substances; while (Article 3(3)) defines an article as: an object which during production
858 is given a special shape, surface or design which determines its function to a greater degree than
859 does its chemical composition.

860 The European Commission stated that NMs are covered by those definitions (CEC, 2008), and in
861 this sense although NMs are not explicitly mentioned in the regulation all of the REACH
862 provisions apply. There have been several criticisms of this approach, based on analysis of the
863 prospective implementation and limitations of the incremental approach whereby regulation
864 of NMs relies on adaptations of current regulatory frameworks (Franco, 2007, Hansen, 2010).

865 The convergence of nanotechnology with biotechnology, information and cognitive technologies
866 is predicted to result in new types of products that may require new policies to protect human
867 health and environment. The development of NMs is foreseen in terms of successive generations

36
868 of NMs. In addition to the first generation, passive nanostructures (coatings, NPs,
869 nanostructure metals, polymers, ceramics, catalysts, composites, displays) we now see the
870 development of active nanostructures (transistors, amplifiers, targeted drugs and chemicals,
871 actuators, adaptive structures, sensors, diagnostic assays, fuel cells, solar cells, high performance
872 nanocomposites, ceramics, metals etc.). It has been predicted that future generations will consist
873 of three-dimentional nanosystems, assembling techniques, networking and new architectures,
874 biomimetric materials, novel therapeutics and molecular structures by design (US EPA, 2007,
875 Subramanian, 2010). In this context the analysis of the adequacy of REACH presented in this
876 report is limited to passive NMs.

877 2.3.2.2 Registration of a substance (on its own or in a preparation) and data requirements
878 implications for NMs

879 The obligation of registration, the registration time and the scope of required data for
880 substances on its own or in preparation are dependent on the quantities of production or import
881 of the substance (tonnage trigger), the phase-in (see footnote 4)/non phase-in8 status of the
882 substance and the intrinsic properties resulting in classification as hazardous (Table 2.1, p.67).
883 For pre-registered phase-in substances produced or imported into the EU in volumes of more
884 than 1000 tonnes per year per manufacturer (or importer) registration of data shall be
885 completed before 30th November 2010; for substances produced or imported in volumes of
886 more than 100 tonnes per year per manufacturer (or importer) registration will be completed
887 before 31st May 2013, while for substances produced or imported in more than 1 tonne per year
888 per manufacturer (or importer) registration will be completed before 31st May 2018. The non-
889 phase-in substances or the phase-in substances that were not pre-registered shall be registered
890 as soon as the production volume exceeds 1 tonne. Furthermore, significantly different scope of
891 (eco)toxicological data is required for the different tonnage bands (REACH Annex VII, VIII, IX, X).
892 Additionally test requirements for chemicals manufactured or imported in volumes from 1 10
893 tonnes per year are divided into three categories as specified in REACH Annex III, namely: 1.
894 unprioritized phase-in substances9, that were regulated also in the previous legislation and do
895 not qualify for any of the following categories; 2. prioritized phase-in10 substances are

8
Non-phase-in substance: synonym: new substances, a substance that is not a phase-in substance (see
footnote 4).
9
Unprioritized phase-in substances: phase-in substances (see footnote 4) that do not qualify for prioritized
category (see footnote 10).
10
Prioritized phase-in substances: phase-in substances (see footnote 4) that fall into one of the following two
subcategories: a) substances for which it is predicted (by the application of QSAR or other evidence) that they
are likely to meet the criteria for category 1 or 2 classification for carcinogenicity, mutagenicity or reproductive

37
896 substances that were regulated in the previous legislation and in addition fall into one of the
897 following two subcategories (as specified in REACH Annex III): a) substances for which it is
898 predicted (by the application of QSAR or other evidence) that they are likely to meet criteria for
899 category 1 or 2 classification for carcinogenicity, mutagenicity or reproductive toxicity
900 (according to the European classification and labeling directive (EEC 67/548), or the criteria for
901 persistent, bioaccumulating and toxic substances (PBT) (see footnote 6), or the criteria for very
902 persistent and very bioaccumulating (vPvB) (see footnote 7) substances (according to REACH,
903 Annex XIII); b) substances that have dispersive or diffuse consumer use and are predicted to be
904 likely to meet the classification criteria for any human health or environmental effects endpoints
905 under the European classification and labeling directive (EC, 1967); 3. non-phase-in substances
906 (see footnotes 4 and 8). Only very limited information for those 3 categories is requested. Data
907 solely on physico-chemical properties (no toxicity data) for unprioritized phase-in substances;
908 for prioritized and non-phase in substances additional test requirements apply: acute (oral)
909 toxicity, in vivo skin sensitization, one in vitro test for gene mutations in bacteria (further
910 mutagenicity tests can be required in case of positive results), acute toxicity to algae and
911 Daphnia, and biotic degradation (ready biodegradability) REACH, Annex VII). The actual
912 outcome of the test requirements for chemicals in this tonnage band will depend on the
913 prioritization criteria applied. The QSAR models and other methods that will be used to generate
914 data for prioritization purposes have not been defined (Rudn and Hansson, 2010), and
915 certainly no QSARs exist for NMs at present. The summary of testing requirements is presented
916 in Table 2.1. p.67.

917 According to ECHA, chemically surface treated substances11 should not be registered as such
918 under REACH, but the following requirements should be fulfilled: registration of the base

toxicity (according to the European classification and labeling directive (EEC 67/548)) (According to the new CLP
Regulation these substances shall be classified as 1a or 1b), or the criteria for persistent, bioaccumulating and
toxic substances (PBT) (see footnote 6), or the criteria for very persistent and very bioaccumulating (vPvB) (see
footnote 7) substances (Annex XIII of REACH); b) substances that both, have dispersive or diffuse consumer use
and are predicted to be likely to meet the classification criteria for any human health or environmental effects
endpoint under the European classification and labeling directive (EEC 67/548).
11
The surface treatment of a substance is a two dimensional modification of macroscopic particles. A two
dimensional modification means a chemical reaction between the functional groups only on the surface of a
macroscopic particle with a substance which is called a surface treating substance.
By this definition it becomes clear that this kind of modification means a reaction of only a minor part (surface)
of a macroscopic particle with the surface treating substance, i.e. most of the macroscopic particle is
unmodified. Therefore a chemically surface treated substance cannot be regarded as a preparation nor be
defined by the criteria of the Guidance for identification and naming of substances under REACH.
With the same reasoning, a chemically surface treated substance could not be reported for EINECS (European
Inventory of Existing Commercial Chemical Substances) nor be notified according to Directive 67/548/EEC

38
919 substance, registration of the surface treating substance, description of the use of surface
920 treatment in both dossiers, any specific hazards or risks of the surface treated substance
921 covered by classification and labelling and by the chemicals safety assessment and resulting
922 scenarios (ECHA, 2010).

923 The required exposure related information also depends on the volume of production and the
924 intrinsic hazardous properties of the substance. For substances produced (or imported) in
925 quantities of 1 -10 tonnes and substances above 10 tonnes that are not classified as dangerous12
926 or not PBT/vPvB (see footnotes 6 and 7) exposure related information requirements (REACH
927 Article 10 and Annex VI section 6) are limited to the following information: Information on
928 manufacture and use(s) of the substance(s) such as: Overall manufacture, quantities used for
929 production of an article that is subject to registration, and/or imports in tonnes per registrant
930 per year in: the calendar year of the registration (estimated quantity); In the case of a
931 manufacturer or producer of articles: a brief description of the technological process used in
932 manufacture or production of articles; the form and/or physical state under which the substance
933 is made available to downstream users. The concentration or concentration range of the
934 substance in mixtures made available to downstream users and quantities of the substance in
935 articles made available to downstream users; a Brief general description of the identified use(s);
936 Information on waste quantities and composition of waste resulting from manufacture of the
937 substance; the use in articles and identified uses; Uses advised against; Information on exposure
938 including: Main use category: (a) industrial use; and/or (b) professional use; and/or (c)
939 consumer use; Specification for industrial and professional use: (a) used in closed system;
940 and/or (b) use resulting in inclusion into or onto matrix; and/or (c) non-dispersive use; and/or
941 (d) dispersive use; Significant route(s) of exposure: Human exposure: (a) oral; and/or (b)
942 dermal; and/or (c) inhalatory; Environmental exposure: (a) water; and/or (b) air; and/or (c)
943 solid waste; and/or (d) soil; Pattern of exposure: (a) accidental/infrequent; and/or (b)
944 occasional; and/or (c) continuous/frequent.

945 For substances classified as dangerous or PBT/vPvB and produced in quantities above 10 tonnes
946 development of exposure scenarios is required.

because it was covered by the separate EINECS entries of both the base substance (macroscopic particle) and
the surface treating substance (ECHA, 2010).
12
The classification of a substance or preparation as dangerous is based on one or several endpoints concerning
physico-chemical properties, health or environmental effects. Currently, substances and preparations can be
classified according to the following 15 categories: explosive, oxidizing, extremely flammable, highly
flammable, flammable, very toxic, toxic, harmful, corrosive, irritant, sensitizing, carcinogenic, mutagenic, toxic
for reproduction, dangerous for the environment (See also 2.3.2.3.).

39
947 Substances manufactured or imported in amounts below 1 tonne per year per manufacturer or
948 importer do not fall into the registration/data requirements under REACH. However Regulation
949 on classification, labeling and packaging of substances and mixtures, CLP Regulation (EC) No
950 1272/2008 (EC, 2008a) applies to any chemical irrespectable of the production volume.
951 Furthermore different classifications could be obtained for different forms of chemically the
952 same substance (with already existing examples of zinc powder forms, stabilised vs non-
953 stabilised, which received different physico-chemical classification (flammability), or nickel
954 powder, diameter below 1 mm classified for environmental hazard versus the bulk form which
955 is not classified).

956 CLP related provisions; including safety data sheets and classification and labeling requirements
957 already apply. Substances, including NMs, meeting the classification criteria as hazardous must
958 be notified to ECHA by 1 January 2011. However it has to be stressed that the Regulation on CLP
959 does not trigger testing requirements (see section 2.3.2.3, p.47).

960 In order to foresee what range of testing and data would be required at present for different
961 NMs, gathering and analysis of the following is essential: 1. information on production / import
962 quantities of different NMs; 2. understanding of status in the categorization for phase-in and
963 non-phase-in substances; 3. assessment of tools/methods available for prioritization and
964 classification of NMs into the recognised hazard classes.

965 Estimation of NMs production and use. Only limited and uncertain estimates of the production /
966 import quantities of different NMs and their presence in products have been reported. Analysis
967 of the pre-registration database at ECHA by ourselves and others (Milieu Risk & Policy Analysts,
968 2010, Floyd, 2009, Sokull-Klttgen, 2009) indicate that the common parent substances,
969 including cerium oxide, zinc oxide, silicon dioxide, silver, iron, titanium dioxide, aluminium
970 dioxide, gold, that were also chosen for the OECD testing program (OECD, 2008), have been pre-
971 registered with planned registration in 2010, implying production > 1000 tonnes per year.
972 There are also many high volume substances, which are less commonly found in nanoforms
973 (such as aluminium, calcium peroxide, tungsten disulphide). However it needs to be stressed
974 that it remains unclear how different nanoforms are to be dealt with in the registration, and
975 whether during the Substance Information Exchange Forum (SIEFs) formation nanoforms are to
976 be considered as different from the bulk, which would result in nanoform tonnage threshold
977 triggers being applied, leading to delayed registrations with likely less data requirements,
978 thereby perpetuating the current situation whereby insufficient data exists to make scientifically
979 sound risk assessments and regulatory decisions. As an example the dossier already registered

40
 980 for silver does not contain information on nanosilver, and the registration of nanosilver is
981 predicted to be delayed until 2018 despite its widespread use in consumer products (Chemical
982 Watch, 2010).

983 Some rare nano-substances (listed in Nanowerk, see below) such as praseodymium oxide,
984 erbium oxide and gallium antimonide or strontium titanium oxide have been found to be pre-
985 registered. Five entries for substances described specifically as NMs (nano in the name) were
986 found: nanosilver, carbon nanobutes (unspecified, single-walled, multi-walled), and
987 phthalocyanine-fullerene compound, with the registration deadline 2018 implying production
988 volumes up to 100 tonnes.

989 Further attempts have been made to gather and analyse information on the existence of NMs on
990 the market and their production volumes. Those include: inventory of nanotechnology-based
991 consumer products currently on the market which lists 1015 (in 2009) consumer products
992 (www.nanotechproject.org./inventories/); NMs Database from Nanowerk with links to over
993 2000 commercially available NMs (www.nanowerk.com); A to Z Nanotechnology which lists
994 over 1300 NMs (www.azonano.com); and various reports (Milieu Risk & Policy Analysts, 2010,
995 KemI, 2009, Wijnhoven, 2009b) and peer reviewed articles (Aitken, 2006, Aitken, 2008, Holman,
996 2007). Some NMs are produced in rather small quantities, while others are manufactured in
997 significant volumes by international producers. The estimation of the global trend states that
998 nanoscale metal oxides (e.g. TiO2, iron and aluminium oxides), nanoscale polymers and
999 polymeric nanocomposite materials are those being manufactured and applied in the greatest
1000 quantities. Global production of carbon black has been estimated at millions of tonnes (IARC,
1001 2006). Production of carbon nanotubes has been expected to rise from a few hundred tonnes in
1002 2007 to a few thousand tonnes per year in 2010 (Thayer, 2007).

1003 The lack of reliable estimates results from the lack of an efficient reporting system for the
1004 production and commercial uses of NMs in the EU. Voluntary reporting initiatives have been
1005 undertaken in the UK, France, Germany, Ireland as well as in the US. So far they have failed to fill
1006 existing knowledge gaps (Breggin, 2009, Milieu Risk & Policy Analysts, 2010). National
1007 mandatory reporting systems are now being developed in France (according to Art 73 Grenelle
1008 law 2, expected to be launched in 2011), Norway (within the Pollution Control Authority (SFT)),
1009 the US and Australia (Proposal for Regulatory Reform of Industrial NMs). This was also
1010 recommended by the Royal Commission on Environmental Pollution in the UK (RCEP, 2008).
1011 However the legal status of those national initiatives has been questioned as they could
1012 potentially impinge on REACH (Montfort, 2010, Milieu Risk & Policy Analysts, 2010).

41
1013 A mandatory EU reporting system thus appears necessary and indeed a proposal of this type of
1014 system has been drafted outlining a two-stage approach, including a NMs product register that
1015 focuses on collecting information about NMs placed on the market and their uses, and in a
1016 second stage gathering the information needed to accurately assess the potential risks posed by
1017 these nanoproducts (Milieu Risk & Policy Analysts, 2010). This issue has been recently given
1018 high priority within the framework of the Belgian EU Presidency13.

IT IS RECOMMENDED THAT A MANDATORY EU REPORTING SYSTEM, INCLUDING A NMs

PRODUCTS REGISTER, BE DEVELOPED AND IMPLEMENTED.

1019 Applicability of the tonnage triggers. The limited information that is available on current NMs
1020 production volumes indicates that many NMs would not reach the thresholds triggering the
1021 requirements for data enabling meaningful risk assessment, and that a number of them (below 1
1022 tonne) would not be required to be registered. Specifically the data gaps for nanoscale
1023 substances falling within each of the following tonnage bands would include:

1024 < 1 tonne no REACH registration no data

1025 1 10 tonnes (non-phase-in status) scarce data, no fate and behaviour besides the biotic
1026 degradation test which is of unproven applicability for some classes of NMs; no testing for long
1027 term effects in the environment and no short term (acute) tests on fish (only daphnia and alga);
1028 for mammalian toxicity no sub-chronic or chronic tests, in vitro tests only for irritation and
1029 sensitization, and mutagenicity plus acute oral toxicity; very limited exposure related
1030 information.

1031 10 100 tonnes scarce data, limited fate and behaviour testing, no long term effects
1032 assessment in the environment and no sub-chronic or chronic mammalian toxicity tests (28-day
1033 repeated dose mammalian toxicity test or screening for reproductive toxicity may be required,
1034 depending on the outcome of the CSA, but these tests are not mandatory and can be waived
1035 based on the magnitude and nature of exposure); very limited exposure related information
1036 (exposure scenario only for those substances classified as dangerous).

1037 100 1000 tonnes limited data, for environment no long-term toxicity on sediment
1038 organisms, plants and terrestrial organisms, no long-term or reproductive toxicity on birds;

13
http://www.eutrio.be/towards-regulatory-framework-traceability-nanomaterials

42
1039 mammalian toxicity: no chronic toxicity or carcinogenicity; very limited exposure related
1040 information (exposure scenario only for those substances classified as dangerous).

1041 Considering the still low production volumes of NMs, the current tonnage triggers system would
1042 result in an ongoing scarcity of data no assessment of fate in the environment, chronic effects,
1043 or toxicokinetic studies would be required for NMs, thereby perpetuating the current lack of
1044 data for meaningful hazard assessment even for nanoproducts already on the market;
1045 additionally, no sources of exposure could be predicted.

1046 The unique properties of substances at the nanoscale that could potentially result in increased
1047 health and environmental risks are determined to a great extent by particle size, surface
1048 structure and surface activity and to a lesser extent by concentration and tonnage when
1049 compared to their bulk (micro or macro) counterparts. NMs have high surface area per mass.
1050 For the same mass higher activity of nanoforms in comparison to their bulk forms has been
1051 reported and the (specific) surface area, or alternatively the number of particles, have been
1052 discussed as potentially more appropriate metrics of exposure and activity of NMs (Oberdrster,
1053 2005a, Oberdrster, 2005b, Oberdrster, 2007, Sager, 2009, Oberdrster, 2010). In the current
1054 system mass is used as a metric of likelihood of risk, and is thus the rationale behind the
1055 increasing scope of testing requirements with increased production volume. In our opinion, the
1056 same mass based triggers for nano and bulk forms result in non proportional efforts to assess
1057 and mitigate risks of NMs in relation to the bulk forms.

1058 It is becoming widely accepted that the tonnage thresholds used in the REACH regulations to
1059 trigger different levels of data requirements may not be appropriate for NMs (Handy, 2007,
1060 RCEP, 2008, European Parliament, 2008, Hansen, 2009, HM Government, 2010). If the triggering
1061 mechanism was accepted in its current form, REACH would likely fail to provide adequate
1062 protection from the potential risks from NMs for human health and the environment. The
1063 primary concern is that some NMs will simply not come under the scrutiny of the regulations
1064 because they are not produced in sufficient quantities. REACH regulations only apply to
1065 materials produced in excess of 1 tonne per manufacturer / per annum, and a number of
1066 potentially hazardous NMs would not reach that threshold in the near future, but could still be
1067 used in products. An important point to note is that nanomatierals may be only less than 1% of a
1068 product by mass but that could still amount to an enormous number of individul NPs. Given that
1069 even a small mass of NM will contain a large number of NPs and a huge surface area, failure to
1070 capture these materials within the regulatory framework could potentially be an omission with

43
1071 serious health and/or environmental consequences. Thus, we share the opinion that the tonnage
1072 thresholds under REACH are not adequate for NMs.

IT IS RECOMMENDED THAT AN ALTERNATIVE BE SOUGHT TO THE TONNAGE TRIGGER

THRESHOLDS USED FOR BULK MATERIALS (SUBSTANCES).

Alternatives to the existing framework are outlined below, in no particular order:

Conversion factors. The existing tonnage trigger framework could be used for NMs if
combined with an appropriate conversion factor. The > 1 tonne per annum threshold
multiplied by a factor of e. g. 100 would convert to > 10 kg of NM.

Alternative metrics. Existing tonnage trigger thresholds could be used in conjunction

with alternative metrics, substituting the mass metric for one more relevant to NMs.
Both total particle number, and total surface area per manufacturer per annum would be
good candidates.

Obligatory testing. The most draconian option would be to require all NMs registered to
submit a technical dossier, irrespective of the quantity produced. The content of this
dossier may, but does not necessarily need to, coincide with that of one of the tonnage
trigger bands.

1073 Application of conversion factor(s) may seem practically attractive, however data requirements
1074 listed for reduced bands (after the hypothetical factor of 100 has been applied) would still not
1075 address the full set of data needed for accurate risk assessments to be performed. With no long
1076 term studies (below 1 tonne) and no toxicokinetics data no long term hazard assessment could
1077 be performed. The extrapolation approaches from short term effects to long effects using
1078 assessment factors have been developed solely on the data for conventional chemicals and have
1079 not been validated for NMs. Differences in ADME and toxicokinetics between bulk and nano
1080 forms are expected, but are not well investigated as yet, mostly due to difficulties with detecting
1081 NMs in tissue (see point 2.3.3.3, p.57 and chapter 4, p.118).

IT IS RECOMMENDED THAT TOXICOKINETICS/ADME STUDIES FOR HUMAN HAZARD

ASSESSMENT, BE INCLUDED IN THE OBLIGATORY DATA REQUIREMENTS FOR NMs AT ALL
PRODUCTION VOLUMES

44
1082 Phase-in or non-phase-in? At present the decisive REACH criterion of whether a material is a non-
1083 phase-in or a phase-in substance applies also to NMs. NMs will be considered as phase-in (see
1084 footnote 4), if they (or their base substance) are listed on EINECS (European Inventory of
1085 Existing Commercial Chemical Substances) (cf. decision of Member States Competent
1086 Authorities), are considered as No-Longer Polymers or have been manufactured in the EU but
1087 not placed on the market between 1st of June 1992 and 1st of June 2007. The REACH Competent
1088 Authorities endorsed a paper (EC, 2008b) prepared by its sub-group on NMs (CASG-Nano)
1089 which concluded that nanoforms of existing substances (i.e. those with an EINECS number)
1090 would, by default, be treated as phase-in substances. Thus, some NMs are considered as phase-in
1091 substances (e.g. gold, TiO2), while others are non-phase-in substances (e.g. fullerenes).

1092 It is possible that some substances at the nanoscale, which in the past have been identified by
1093 the same EINECS number as their bulk counterparts, may have to be considered as different
1094 substances for the purpose of REACH. In this case, all substances which were covered by the
1095 original EINECS number retain phase-in status (EC, 2008b).

1096 In the European Chemical Industry Council (Cefic) understanding, the phase-in (nano)
1097 substances will be generally pre-registered under the same identity code as the non-nanoscale
1098 equivalent (e.g. EINECS number). This does not imply that they are in all cases the same
1099 substance. During the pre-SIEF (Substance Information Exchange Forum) phase, managed by the
1100 industry, it will be clarified between different registrants if these are the same substances with
1101 regard to REACH regulation, and if they are not several SIEFs will be formed.

1102 This procedure of retaining phase-in status (by pre-registration together with bulk form
1103 followed by formation of separate SIEFs if the nanoform(s) are considered as different
1104 substances with regard to REACH) will imply data requirements based on the nanoform
1105 production volume solely and will therefore result in the minimised data set being required.

1106 The consequences of this are that the data requirements and registration deadlines for NMs
1107 classified as non-phase-in and phase-in are different (Table 2.1, p.67). In practise, due to the as
1108 yet quite low production volumes (<1000 tonnes) of some of the nanoforms of substances,
1109 classification as phase-in would likely lead to a delayed registration (until 2018 for NMs
1110 produced at volumes of 1 100 tonnes per year per manufacturer, and until 2013 for NMs
1111 produced at volumes of 100 1000 tonnes). The classification as phase-in also limits the data
1112 requirements for substances produced in quantities between 1 and 10 tonnes. Data solely on the
1113 physico-chemical properties (no toxicity data) for NMs produced up to 10 tonnes would be
1114 required (unless prioritized, based on the classification CMR, PBT, vPvT, or other classification

45
1115 together with dispersive use). The application of such categorisation to NMs lacks a rational
1116 basis. There is no scientific evidence to suggest that those two groups of NMs (phase-in or non-
1117 phase-in) represent a different likelihood of causing a concern, and thus there is no scientific
1118 reason to treat them differently.

IT IS RECOMMENDED THAT NANOFORMS OF MATERIALS ARE TREATED AS DIFFERENT

FROM THEIR BULK COUNTERPARTS AND THAT NONE OF THE PHASE-IN PROVISIONS
APPLY. HOWEVER THE PROVISIONS APPLICABLE TO NON-PHASE-IN SUBSTANCES ALSO
NEED REVISION TO ADDRES THE POTENTIAL RISKS OF NMs.

IT IS RECOMMENDED (SEE 3.5.2) THAT NANOFORMS WITH THE SAME CORE CHEMISTRY
BE DIFFERENTIATED FOR SEPARATE REGISTRATION UNDER REACH USING DIFFERENCES
IN THE FOLLOWING PHYSICO-CHEMICAL PARAMETERS:

1. Surface modification. Given that the surface chemistry of a nanoparticle will profoundly
influence its behaviour in the environment, the use of different capping agents or surface
functionalisation or stabilisation should always trigger a separate registration.
2. Size. If the multiple size classification of NMs (see section 3.5.2, p.98) is adopted, NMs
falling into different categories should require separate registration.
3. Shape. HARNs should be assessed on a case-by-case basis as substances of equivalent
concern. A substantive difference in circularity should trigger a separate registration.
4. Surface charge. Nanoforms with a substantively different surface charge of should be
treated as separate registrations given the a priori prediction that their environmental
distribution would be substantively different.
5. In addition it is recommended that TiO2 NMs with a marked difference in their crystal
structure (rutile: anatase ratio) be considered distinct nanoforms and be subject to
separate registration. Likewise, preparations of cerium oxide NMs with substantial
differences in their CeO2: Ce2O3 ratios should be considered as separate nanoforms.

1119 Prioritization for further testing. Prioritization for further testing applies to phase-in
1120 substances produced at volumes of 1- 10 tonnes/year/legal entity (l.e). In this tonnage band for
1121 unprioritized substances no testing for environmental and mammalian hazard endpoints is
1122 required. For prioritized substances limited tests for those endpoints are required including:
1123 biotic degradation, acute toxicity on daphnia and algae, skin and eye irritation, skin sensitization,
1124 mutagenicity in vitro, acute oral toxicity. Substances can be prioritized if they are predicted to
1125 likely meet the criteria for category 1 or 2 classification for carcinogenicity, mutagenicity or

46
1126 reproductive toxicity (EC, 1967), or the criteria for persistent, bioaccumulating and toxic
1127 substances (PBT), or the criteria for very persistent and very bioaccumulating (vPvB)
1128 substances (REACH); or that they have both dispersive (or diffuse) use and are predicted as
1129 being likely to meet the classification criteria for any human health or environmental effects
1130 endpoint under the European classification and labelling directive (EC, 1967) or CLP (see section
1131 2.3.2.3, p.47).

1132 QSARs and other non-testing methods that are foreseen as the tools for those predictions have
1133 not yet been validated for a spectrum of chemicals; and more importantly for NMs they are non-
1134 existent at the moment (See section 3.3.1, p.92). Consequently, the provision of prioritization
1135 using QSARs or non-testing methods is not implementable for NMs. The practice of read-across
1136 for NMs from their bulk counterparts should not be a default approach, and should be guided by
1137 emerging evidence that NMs may have to be considered as biological entities (see section 4.1,
1138 118), unless there exists substantive experimental evidence to show that the hazard profile of
1139 different nanoforms of the substance do not differ from the bulk (micro or marcroscale) form.

1140 2.3.2.3 Classification and Labelling

1141 Classification and Labelling (CLP) (Regulation (EC) No 1272/2008), the new European
1142 Regulation on Classification, Labelling and Packaging of chemical substances and mixtures,
1143 introduces a new system for classifying and labelling chemicals, based on the United Nations
1144 Globally Harmonised System (UN GHS). By the June 2015 this regulation will gradually replace
1145 the old Directives on classification, labelling and packaging, i.e. Council Dangerous Substances
1146 Directive DSD 67/548/EEC and Dangerous Preparations Directive DPD 1999/45/EC. The
1147 classification criteria are relevant also for other EU legislation ("downstream legislation"),
1148 including REACH.

1149 The classification of a substance or preparation as being dangerous is based on one or several
1150 endpoints concerning physico-chemical properties, health or environmental effects. DSD
1151 categories of danger for physical, health and environmental hazards include the following 15
1152 categories: explosive, oxidising, extremely flammable, highly flammable, flammable, very toxic,
1153 toxic, harmful, corrosive, irritant, sensitising, carcinogenic, mutagenic, toxic for reproduction,
1154 dangerous for the environment. The UN GHS hazard classes include those which best reflect the
1155 DSD categories of danger but the total number of hazard classes is higher under CLP than the
1156 total number of categories of danger under DSD.

47
1157 Classification and labelling includes evaluation of the hazards of a substance or preparation and
1158 communication of these hazards via the label. This evaluation must be made for any substance
1159 or preparation manufactured, imported or placed on the EU market at any tonnage level. The
1160 classification of a substance should be based on the relevant information already available such
1161 as experimental data generated in tests for physical hazards, toxicological and ecotoxicological
1162 tests, historical human data such as accident records or epidemiological studies, information
1163 generated in in vitro tests, (Quantitative) Structure Activity Relationships ((Q)SAR), read
1164 across, and category approaches. Information may also be available from other EU legislation
1165 for which there are specific requirements for test data to be generated such as Directive
1166 91/414/EEC (Plant Protection Products) and Directive 98/8/EC (Biocidal Products), or from
1167 various non-Community programmes. Testing for physical hazards is required unless adequate
1168 and reliable information is already available. In contrast new toxicological or ecotoxicological
1169 testing is not required. The supplier may decide to conduct new testing in order to fill data gaps,
1170 provided that he has exhausted all other means of generating information. Testing on animals
1171 must be avoided wherever possible and alternative methods (including in vitro testing, the use
1172 of (Q)SARs, read-across and/or category approaches) must always be considered first, provided
1173 that they provide data of adequate quality and reliability.

1174 Classification plays a key role in REACH. It must be included in the registration dossier for a
1175 substance, and it triggers certain provisions such as prioritization for additional testing in the
1176 lower tonnage band (in a case of classification for human and environmental effects together
1177 with the dispersive use), the requirement to perform an exposure assessment and risk
1178 characterisation as part of the CSA, and the obligation to provide an SDS (for substances above
1179 10 tonnes). Classification of a substance as mutagenic, carcinogenic or toxic to reproduction may
1180 lead to an authorisation procedure (IHCP, 2009). Additional to the criteria for CLP classification,
1181 REACH sets the criteria for classification as PBT and vPvB (see footnotes 6 and 7). Substances
1182 fulfilling those criteria can be considered as SVHC and special provisions or obligations may
1183 apply to deal with mitigation of these risks.

1184 However, the data set required by REACH (particularly at the lower tonnages) is not sufficient
1185 for classification of substances (including NMs) into different hazard categories (Rudn C,
1186 Hansson SO., 2010). The different types of hazard identification data that are required under
1187 REACH and that can be directly used for classification purposes are presented in Table 2.1, p.67.
1188 The classification criteria for the hazard endpoints according to GHS/CLP and REACH aligned
1189 with the production volume triggering data sufficient for this classification are presented in
1190 Table 2.2, p.68.

48
1191 The applicability and efficiency of the classification system for NMs can be discussed in terms of
1192 the criteria for classification (including relevance of dose metrics in the proposed default values,
1193 relevance of test species and environmental compartments for which criteria cut-off values are
1194 set), testing for all known hazard classes not being required, the investigation tools being not
1195 validated for NMs and possibly lacking the appropriate sensitivity. Consequently it is being
1196 discussed if the evidence of hazard that according to the current classification scheme and
1197 criteria is not classifiable (e.g. toxicity to soil organisms) should trigger the requirements for
1198 CSA.

1199 It is also possible that known hazard endpoints will not cover all novel mechanisms of toxicity of
1200 NMs (discussed in sections 4.1.3, p.127, 4.6, p.142 and 5.2.5, p.162).

1201 2.3.2.4 Evaluation and authorisation

1202 After registration, ECHA and the relevant Member State Competent Authority conduct the
1203 evaluation via reviews of the registration dossiers, separated into technical dossier evaluation
1204 and substance evaluation.

1205 REACH has established a system under which the use of substances with properties of very high
1206 concern and their placing on the market can be made subject to an authorisation. Authorisation
1207 is applied in two steps: 1. issuing by ECHA of a Candidate List of substances of very high concern
1208 (SVHC), containing substances liable for authorisation; 2. prioritisation of certain substances for
1209 Annex XIV, and inclusion of a substance into Annex XIV (which is then banned unless company-
1210 specific authorisation is issued). Authorisation can be obtained if the use of a substance is
1211 adequately controlled, or if no substitutes are available and if the economic and social benefits
1212 outweigh the risks, in the case of substances for which no safety threshold applies.

1213 Authorisation aims to ensure that the risks from the use of such dangerous substances are either
1214 adequately controlled or justified on socio-economic grounds, having taken into account the
1215 available information on alternative substances or processes. For NMs this aim can only be
1216 fulfilled to a limited extent at present, due to the limited applicability of existing methods to
1217 identify and characterise the hazardous properties of NMs and to classify NMs into hazard
1218 classes, a situation that is not improved by the limited data requirements for NM hazard and
1219 exposure assessment set by the tonnage triggers.

1220 Given the current paucity of information on the (eco)toxicity and environmental behaviour of
1221 NMs there are strong arguments to be made for requiring additional data to be generated on
1222 demographically relevant endpoints and on environmental breakdown (section 5.1, p.154).

49
1223 Until such time as the structure of the REACH regulations are modified and sufficient data is
1224 available to conduct meaningful risk analysis of NMs the precautionary principle should be
1225 invoked, and it is therefore advised that the following categories of NPs are prioritised for
1226 evaluation persistent in vivo (in their physical forms determined by size and shape) with high
1227 aspect ratio and toxic (Tran, 2008).

IT IS RECOMMENDED THAT BIOPERSISTENT (PERSISTENT IN VIVO) NMs WITH HIGH

ASPECT RATIO (HARNS) BE TREATED WITHIN THE REACH FRAMEWORK AS SUBSTANCES
OF EQUIVALENT CONCERN, AND THEIR ASSESSMENT PRIORITISED BY INCLUSION IN THE
SVHC LIST. HARN STATUS SHOULD BE GIVEN TO ALL SUBSTANCES WITH ONE DIMENSION
IN THE NANO SCALE AND ONE > 5 M.

Consideration should not be limited to CNTs, as a range of metals, metal oxides and other
materials can be produced as high aspect ratio NMs. Depending on the biopersistence of these
particles in environmental and physiological matrices they may be more hazardous due to the
toxicity associated with their core chemistry.

IT IS RECOMMENDED THAT WHERE EVIDENCE SUGGESTS THAT A NM WOULD FULFILL

THE TOXICITY CRITERIA OF THE PBT CLASSIFICATION IT ALSO BE TREATED AS A
SUBSTANCE OF EQUIVALENT CONCERN AND PLACED ON THE SVHC LIST. Such evidence
exists for silver NPs in a range of species and exposure media (Choi, 2008, Fabrega, 2009a,
Fabrega, 2009b, Farkas, 2010, Laban, 2010, Morones, 2005, Sotiriou, 2010), and is emerging for
others.

1228 2.3.2.5 Obligations for substances in articles

1229 An article is defined in REACH as an object which during production is given a special shape,
1230 surface or design which determines its function to a greater degree than does its chemical
1231 composition (EC, 2006, REACH, Article 3(3)). The obligations placed on manufactures or
1232 importers of articles include: registering of substances with the ECHA if they are produced or
1233 imported in volumes above 1 ton/year/legal entity and are intended to be released during
1234 normal use of an article if not already registered for this use; informing the recipient of the
1235 article of the presence of a Substance of Very High Concern or SVHC (if the concentration >0.1%)
1236 and how to use it safely if necessary, notifying ECHA of the presence of an SVHC in an article, (if
1237 concentration of SVHC in article >0.1% and production volume of the SVHC > 1 tonne per
1238 annum) and if exposure to this substance cannot be excluded during normal or reasonably
1239 foreseeable conditions of use or disposal (ECHA, 2008).

50
1240 The interpretation concerning the limit concentration of SVHCs in articles for provision of
1241 information to downstream user is controversial. The guidance states that the 0.1%
1242 concentration applies to the total article and not to the individual components. Several member
1243 states argue that those limits should be applied to the constituent parts of articles, in a similar
1244 manner to that implemented in other legislations, such as the Restriction of Hazardous
1245 Substances in electrical and electronic equipment (RoHS) Directive 2002/95/EC (EC, 2003, NCG,
1246 2010).

1247 The current ECHA interpretation implies that different requirements will apply to the same
1248 article when sold separately and when it is incorporated into a more complex article.
1249 Consequently this dilution effect may lead to an NMs-containing article escaping the
1250 information provisions with the consequent loss of relevant health and safety information in the
1251 supply chain.

1252 When the REACH provisions are put into effect for NMs in articles several concerns arise about
1253 potential information gaps.

1254 Within the current approach all NMs in articles that are not intended to be released or are
1255 released unintentionally (through ageing, wear and tear, or accidents) would not be registered,
1256 unless registered by the producer or importer of the substance. In such a case however it is
1257 unlikely that information on all uses, and details of the existence and concentrations of NMs in
1258 articles would be provided.

1259 Registration would not apply even to intentionally released NMs if the production (or import)
1260 (refers to the NMs in the article) does not exceed 1 tonne/year/manufacturer. For NMs
1261 produced at much higher volumes and intentionally released, no comprehensive risk assessment
1262 would be possible due to the limited data required (as for the registration of substances).

1263 The current interpretation of the percentage limit for SVHC, and the obligation to provide
1264 information along the supply chain, increases the likelihood of NMs escaping the information
1265 provisions due to dilution relative to that for conventional chemicals, as the quantities used to
1266 achieve the desired properties, as measured by mass, are much lower. Thus, specific
1267 consideration should be given to unique applications, like coatings, that can result in high
1268 exposures although the total quantity, calculated as part of the whole article, may be well below
1269 the threshold.

1270 Additionally, the current criteria and available methods to identify SVHC NMs have not been
1271 validated for this purpose and may not be satisfactory to identify all possible hazards. At

51
1272 present, there is not a single nanomaterial on the candidate list. This implies a significant
1273 likelihood of some potentially hazardous NMs missing the categorisation as SVHC and escaping
1274 the information and notification requirements.

1275 Finally, even if certain NMs could potentially be categorised as SVHC the existing tonnage trigger
1276 (above 1 tonne/year/legal entity) would limit registration and notification, perpetuating the
1277 data paucity and the limited capacity for risk assessment.

1278 Thus, our assessment suggests that many cases of NMs in articles would not be covered by the
1279 current provisions, leading to them escaping several of the reporting obligations.

IT IS RECOMMENDED THAT AN EU-WIDE REGISTER OF COMPREHENSIVE INFORMATION

ON THE PRESENCE OF NMs IN ARTICLES IS DEVELOPED, ALLOWING TRACEABILITY AND
EXPOSURE SOURCES ESTIMATION.

RECOMMENDATIONS INCLUDE: LOWERING OF THE VOLUME THRESHOLDS FOR TRIGGER

OF THE OBLIGATION OF REGISTRATION AND NOTIFICATION FOR NMs IN ARTICLES;
IMPLEMENTATION OF NANO-SPECIFIC DATA REQUIREMENTS AS FOR NANO-SUBSTANCES;
ESTABLISHEMNT OF AN INFORMATION OBLIGATION FOR ALL (NOT ONLY KNOWN SVHC)
NMs BASED ON THE LIMIT APPLIED TO THE HOMOGENOUS PART OF THE ARTICLE.

1280 2.3.3 REACH associated guidelines

1281 2.3.3.1 Substance identification

1282 There is no consensus as yet regarding whether NMs should be considered as the same or
1283 different substances relative to their bulk counterparts. As a general rule could not be applied, a
1284 case- by-case approach has been taken. The case studies that are being performed within the
1285 RIPoN 1 project (October 2009 September 2010) focus on this issue and the preliminary
1286 results suggested that in some cases the nanoform will be considered and treated as a new
1287 substance (e.g. single walled carbon nanotubes SWCNT) while in other cases this separation is
1288 not obvious (nanosilver, multiwalled carbon nanotubes, nano TiO2) and they could be
1289 considered (at least by some registrants) as the same substances to their bulk counterparts (Dr.
1290 Norah OFarell, teleconference, March 2010).

1291 According to the REACH guidance A substance is completely identified by its qualitative and
1292 quantitative chemical composition, the chemical identity and the content of each constituent in
1293 the substance (Guidance for identification and naming of substances under REACH, June 2007,

52
1294 p.18). When performing a substance identification, the parameters listed in Annex VI, section 2
1295 of REACH (attachment 2.1, p.72) must be used to provide a proper description of the substance.

1296 The broad definition of a substance within REACH is based on the chemical element and the
1297 list of parameters required for substance identification is neither adequate for distinguishing
1298 between the bulk and nanoforms of certain substances, nor for distinguishing between different
1299 nanoforms. This results in problems with NMs identification, namely whether NMs are to be
1300 considered as equivalent to or different from the bulk (not nanoscale) material for the purpose
1301 of registration.

1302 In the REACH guidance on substance identification it is recognised that some substances, which
1303 cannot be identified by their chemical composition alone, need to be further specified by
1304 additional identifiers in order to get their own substance identification. This is laid down in
1305 more detail for UVCB substances (Substances of Unknown or Variable composition, Complex
1306 reaction products or Biological materials). These substances cannot be sufficiently identified by
1307 the above parameters. Additional identifiers might also be needed for well-defined substances if
1308 the properties of the substance differ significantly for reasons other than their chemical
1309 composition. This may be the case for some substances at nanoscale, where for instance
1310 information on characterisation of the substances may be needed for their proper identification,
1311 or indeed their characteristics are dependent on their surroundings (which is likely to be the
1312 case for NMs in biological or environmental matrices).

1313 The Technical Guidance Document (TGD) on substance identification (ECHA, 2007) and
1314 information requirements (ECHA, 2008) considered that The current developments in nano-
1315 technology and insights in related hazard effects may cause the need for additional information
1316 on size of the substances in the future. The current state of development is not mature enough to
1317 include guidance on the identification of substances in the nanoform in this TGD.

1318 The diversity and complexity of NMs makes chemical identification and characterization not
1319 only more important but also more difficult. A broader spectrum of properties will be needed to
1320 sufficiently characterize a given NM for the purposes of evaluating hazard and assessing risk.
1321 Chemical properties such as those listed in REACH guidance for substance identification (see
1322 Attachment 2.1, p.72) may be important for some NMs, but other properties are also expected to
1323 be important for the majority of NMs. Discussion regarding the properties of NMs that are
1324 important for their characterisation have taken place in the scientific literature (Bucher, 2004,
1325 Balbus, 2007, Oberdrster, 2005b, Powers, 2006, Powers, 2007, Thomas, 2006, Hansen, 2007,
1326 BSI, 2007b, SCENIHR, 2007a, SCENIHR, 2007b, Crane, 2008, Klaine, 2008, Hassellv, 2008,

53
1327 SCENIHR, 2009, Sayes, 2009). The full list of properties is long and it has been recognised that
1328 such a full characterisation is time consuming, expensive and requires special technical
1329 expertise. A priority list has been suggested including size distribution, agglomeration, surface
1330 area, surface chemistry, shape and morphology, material composition and purity (Stone, 2010).
1331 Additionally, water solubility or dispersibility, crystalline phase, crystallite size, dustiness,
1332 surface charge, surface chemistry, photocatalytic activity, pour density, porosity, octanol-water
1333 partition coefficient, redox potential, and radical formation potential were included in the OECD
1334 guidance document (OECD, 2010, No 14, No 24, See attachment 2.2, p.73). Furthermore some
1335 characteristics of NMs change depending on the medium/surrounding milieu which implies that
1336 these characteristics should be measured under conditions that mimic those of potential human
1337 and environmental exposure, and that multiple measurements may have to be performed during
1338 the course of the experiment, including measuring other biotic and abiotic factors in the media
1339 like pH, Ca2+, presence of natural organic matter and ionic strength would be appropriate (for an
1340 extensive analysis of this topic see sections 3.2, p.80 and 4.1, p.118).

IT IS RECOMMENDED (SEE SECTION 3.2, P.80) THAT CHARACTERISATION OF THE

FOLLOWING PHYSICOCHEMICAL PARAMETERS BE MANDATORY FOR ALL SUBSTANCES
DEFINED AS 'NMs' WITH THE REACH FRAMEWORK:

Size; average and distribution.* Either mean and standard deviation or median and
geometric standard deviation should be reported, depending on the normality of the size
distribution. Guidance on this is required.
Shape. Aspect ratio or defined measure of circularity
Core Chemistry
Specific Surface Area.*
Surface modification, functionalisation and capping agents should be reported.
Surface charge.*
In addition it is recommended that the following specific parameters be characterised for
particular nanoparticle classes:
Zero-valent silver (Ag0) and oxides of zinc: dissolution in distilled H2O.
Oxides of titanium and silicon: TiO2: crystallinity in the form of the rutile: anatase ratio.
SiO2: crystallinity in the form of the amorphous / crystalline nature.
Oxides of cerium and iron: Oxidation state. For ceria this should be required only for
Category 3a (see section 3.5.2, p.98) NMs and should be reported as the CeO2: Ce2O3
ratio.

54
 This list of required specific parameters should not be considered final, it should be augmented
as additional information becomes available in the literature on the (eco)toxicology of particular
classes of NMs and as characterisation methods improve. For example should a suitable routine
methodology become available to measure the degree of entanglement of CNTs, or the rate of
CNT degradation in vitro or in vivo, these should become a requirement.

* Note that parameters indicated with a * are subject to change depending on the nature or their
surroundings, and thus should be reported for a set of representative conditions, which need to
be defined and agreed, which would provide at least a basic consideration of the biological
identity of the NM.

Thus, a further recommendation is that consideration should be given to the state of the
nanoparticle at characterisation. It is recommended that NMs be characterised in a pristine state
(as synthesised / manufactured) and in situ under representative assay conditions. The latter
characterisation is vital if (eco)toxicology results are to be interpreted in a meaningful way.

1341

IT IS RECOMMENDED (SEE SECTION 3.3, P.92) THAT ACCREDITED CENTERS BE

ESTABLISHED TO UNDERTAKE PHYSICO-CHEMICAL CHARACTERISATION OF NMs.

Meaningful characterisation of NMs requires both a range of specialised equipment and a high
degree of technical competence. It is recognised that smaller manufacturers / importers of NMs
may not have recourse to such facilities, and capacity building in this area is required.
Establishing centers of excellence for characterisation would ensure that regulation of NMs is
based on high quality data. Validated, cost-effective techniques should be developed for sample
preparation, technical methodologies and data handling and analysis.

Accredited characterisation centers would also be in a good position to act as 'nanosafety'

centers. The scope of such centers could encompass verification of data supplied under REACH
in registration dossiers, characterisation of NMs after exposure etc. Since the metrology and
analysis performed in situ and in complex media requires different expertise compared to
traditional characterisation, such centres need not always be existing metrology centres, unless
this offers particular advantages.

1342 2.3.3.2 Standardization

1343 The ISO Technical Committee 229Nanotechnologies was created in 2005 and works on various
1344 issues related to standardization and NMs, including terminology and definitions, protocols for

55
1345 toxicity testing and environmental impact studies, measurement techniques, calibration
1346 procedures, and reference materials. It has four subcommittees on (1) terminology and
1347 nomenclature, (2) measurement and characterization, (3) health, safety and environmental
1348 aspects, and (4) material specifications. Three documents were published in 2007/2008: one on
1349 Health and safety practices in occupational settings relevant to nanotechnologies (ISO/TR
1350 12885:2008, 2008), the second one on Terminology and definitions for nano-objects
1351 nanoparticle, nanofibre and nanoplate (CEN ISO TS 27687, 2008), and the third one concerns
1352 inhalation exposure to NPs (ISO/TR 27628:2007, 2007). In 2010 nine new standards were
1353 published concerning vocabulary, classification and methodology (ISO 10801:2010, 2010, ISO
1354 10808:2010, 2010, ISO/TS 10867:2010, 2010, ISO/TS 11251:2010, 2010, ISO/TR 11360:2010,
1355 2010, ISO/TR 12802:2010, 2010d, ISO 29701:2010, 2010c, ISO/TS 80004-1:2010, 2010a, ISO/TS
1356 80004-3:2010, 2010)

1357 The organization for Economic Co-operation and Development (OECD) is leading and
1358 coordinating a project focusing on an initial selection of priority NMs and characterization of
1359 their properties. The list comprises fullerens (e.g. C60), single and multi-walled carbon
1360 nanotubes, carbon black, polystyrene, dendrimers, nanoclays, and nanoformss of Ag, Fe oxides,
1361 TiO2, Al2O3, CeO2, ZnO, SiO2 (OECD, 2008). Various OECD member countries have volunteered to
1362 sponsor the development of the dossiers for most of these materials. A key feature of the OECD
1363 sponsorship programme is that for each nanomaterial a single large industrially relevant batch
1364 has been secured and all testing is being conducted on this single batch, to ensure that the data
1365 produced is comparable. However, in many cases the materials are supplied in powdered form
1366 and cannot be re-dispersed to the primary nanoscale entities (see section 3.2.3, page 90).

1367 The EU Commission has written a draft document giving a mandate to CEN, CENELEC and ETSI
1368 for standardization activities (EU Commission document 58/2009). Thus, work should take into
1369 consideration the following elements: methodologies for NM characterization in the
1370 manufactured form and before toxicity and eco-toxicity testing; sampling and measurement of
1371 workplace, consumer and environmental exposure to NMs; methods to simulate exposure to
1372 NMs.

1373 The development of reference NMs has been discussed within several projects such as
1374 REFNANO (Aitken, 2008) and the ERDC-NIST workshop on nanosilver
1375 (http://nanobiology.ncifcrf.gov/groups/silver/)). Some activities to develop such materials
1376 have started at NIST (National Institute of standards and technology, USA) and at the JRC-IRMM
1377 (Joint Research Centre Institute for Reference Materials and Measurements, EC), although

56
1378 these efforts are currently limited to calibration size standards in aqueous solution for
1379 instrument calibration and/or laboratory accreditation in size determination. The suitability of
1380 different types of NMs as a test/reference materials and their availability today have been
1381 discussed (see also 3.4, p.96)(Stone, 2010). QNano, the FP7 research infrastructure for
1382 nanosafety assessment is developing positive and negative control NMs for biological endpoints
1383 focussing on apoptosis initially, followed by genotoxicity and inflammatory effects.

1384 2.3.3.3 Hazard identification

1385 Revision of OECD protocols. The different types of hazard identification data that are required
1386 under REACH have been presented in Table 2.1., p.67. Some of this data can also be directly used
1387 for classification purposes. The OECD standardized protocols for testing of those endpoints have
1388 been referred to in the OECD draft of the guidance for NMs (OECD, 2009a). However, it needs to
1389 be stressed that these methods have been developed with no specific consideration of the
1390 unique challenges presented by NMs and, as such, that their adequacy for NMs in the current
1391 form needs careful assessment and revision where appropriate.

1392 The list of protocols that have been suggested in the OECD guidance for NMs include methods
1393 for: a) environmental fate assessment including: dispersion stability in water (no standard
1394 method proposed); biotic degradability (possible methods OECD TG301 TG311) and abiotic
1395 degradability (OECD TG111, TG 316); adsorption; bioaccumulation potential (OECD TG305,
1396 OECD TG315); b) ecotoxicological studies: short and long term effects on pelagic species (OECD
1397 TG203, TG204, TG210, TG212, TG215, TG202, TG211, TG201); short and long term effects on
1398 sediment species (OECD TG218, TG219, TG225, TG315); short and long term effects on soil
1399 species (OECD TG207, TG222, TG220); terrestrial/soil (ISO guideline No. 11267, OECD TG207,
1400 TG222, TG220, TG226); effects on microorganisms (OECD TG216, TG217, TG209); c)
1401 mammalian toxicity testing: pharmacokinetics/toxicokinetics (no standard method
1402 recommended); acute toxicity (OECD TG420, TG423, TG425, TG402, TG403); skin corrosion
1403 (OECD TG430, TG431, TG435); skin irritation (OECD TG404); skin sensitization (OECD TG406,
1404 TG429); acute eye irritation (OECD TG405); phototoxicity (OECD TG432); repeated dose
1405 toxicity: oral (OECD TG407, TG409); dermal (OECD TG410); inhalation (OECD TG411, TG412,
1406 TG413); chronic toxicity (OECD TG451, TG452, TG422, TG453); reproductive toxicity (OECD
1407 TG415,TG414,TG416); developmental toxicity (OECD TG414, TG421); and genetic toxicity (in
1408 vitro: OECD TG471, TG473, TG476, in vivo: OECD TG475, TG474, TG486).

1409 The on-going OECD work on validation of existing standard protocols resulted in publication of
1410 the Preliminary guidance notes on sample preparation and dosimetry for the safety testing of

57
1411 manufactured NMs (OECD, 2010a) and Guidance manual for the testing of manufactured NMs:
1412 OECDs sponsorships programme first revision (June 2010) that are living documents to be
1413 updated based on the experience gained with the testing of NMs.

1414 Need for an extended list of hazard identification endpoints in REACH. The list of endpoints to be
1415 studied and reported for NMs in the OECD testing program for NMs (which includes
1416 identification, physico-chemical properties, environmental fate, environmental toxicity, and
1417 mammalian toxicity) has been set (see Attachment 2.2, p.73). Several of those endpoints are not
1418 on the list of REACH requirements. Specific interactions between NMs and biological matrices
1419 and/or formation of a biomolecular corona14 may have important implications for possible
1420 new mechanisms of biological activity and toxicity. The relevance of this type of testing for
1421 hazard assessment is discussed in section 4.1, p.118.

1422 Among the mammalian toxicity tests outlined above pharmacokinetics/toxicokinetics and ADME
1423 studies have been considered as having great value in understanding (nano)material
1424 distribution throughout the body, thereby indicating potential target organs for systemic toxicity
1425 experiments. Kinetic processes including absorption, distribution, metabolism and excretion of
1426 NMs are not well understood and the relation to the classic pharmacokinetics parameters is
1427 not known. Due to the biological nature of NMs, conferred by the bimolecular corona, NMs
1428 have been shown to be actively transported by cells, indicating that in some instances models
1429 based on diffusion and partitioning across membranes will likely not apply (sse also section 4.1,
1430 p.118). In other cases passive diffusion at surfaces and partitioning may still be a valid
1431 mechanism, as in the uptake from water into vertabrates after aggregation at the surface. At this
1432 stage, when the understanding of these processes is weak, the performance of long term studies
1433 along with the short term, involving also the investigation of kinetics and biodistribution are of
1434 particular importance for all NMs, even those produced in lower quantities.

1435 Several additional endpoints have been proposed as a result of RIPoN2 project, including cell
1436 uptake, cell viability, oxidative stress, inflammation, fibrosis, immunotoxicity, cardiovascular
1437 toxicity, ventilation rate, gill pathologies, mucus secretion, brain pathology, animal behaviour
1438 (Hankin, 2011).

14
Biomolecular corona - Surface of NMs in a biological environment are modified by the adsorption of
biomolecules such as proteins and lipids, leading to a biomolecular interface organization that may be loosely
divided into two components named the hard and soft coronas with (respectively) long and short
typical exchange times (Monopoli at al., 2011)MONOPOLI, M. P., WALCZYK, D., LOWRY-CAMPBELL, A., ELIA, G.,
LYNCH, I., BALDELLI BOMBELLI, F., DAWSON, K.A. (2011) Physical-chemical Aspects of Protein Corona:
relevance to in vitro and in vivo biological impacts of nanoparticles. J. Am. Chem. Soc., 133, 2525-2534..

58
1439 Extrapolation assessment factors. The general framework for human health assessment is
1440 based on the derivation of DNELs (Derived No Observed Effect Levels) or DMELs (Derived
1441 Minimum Effects Levels). These calculations are most often based on extrapolation from the
1442 experimentally obtained No Observed Adverse Effect Level (NOAEL) or Lowest Observed
1443 Adverse Level (LOAEL) and the use of assessment factors to account for the variability and
1444 uncertainty associated with the different steps of the extrapolation (duration of exposure, inter-
1445 species, intra-human, routes of exposure, quality of data). In a similar process to that used for
1446 the assessment of environmental hazard, the lowest available No Observed Effect Concentration
1447 (NOEC) is used and assessment factors ranging from 10-1000 are applied in a deterministic
1448 fashion depending on the amount and type of data available. Alternatively the probabilistic
1449 approach introducing species sensitivity distribution in the setting of Predicted No Effect
1450 Concentrations (PNECs) has been recommended (ECHA, 2008).

1451 The values of the assessment factors (AFs) applied for human hazard assessment have been
1452 derived based on an analysis of the historical experimental data of the levels of classical
1453 chemicals causing adverse effects after different durations of treatment/exposure (for the time
1454 extrapolation), and different species (for inter-species factors) and some evidence of differences
1455 in human population sensitivity (for intra-species extrapolation factors). It has previously been
1456 shown that the numerical values of the currently recommended AFs (ECHA, 2008) correspond to
1457 different percentages of the distributions derived from this historical data. Time extrapolation
1458 factors (AF=3 for subacute to subchronic, AF=2 for subchronic to chronic and AF=6 for subacute
1459 to chronic) correspond to the central estimates of the distributions. Inter-species extrapolation
1460 (based on allometric scaling and a use of AF=2.5) corresponds to the 70-80 percentile of the
1461 available distributions. While the factor of 10 for intra human population variability is believed
1462 to cover the 80-90 percentile of the distribution (Malkiewicz, 2009). However, as there is no
1463 historical data related to NMs, the validity of such assessment factors is questionable, but cannot
1464 be assessed with the current paucity of data.

1465 Similarly, the scientific basis for the assessment factors for long term ecotoxicity assessment of
1466 NMs (as discussed in section 5.1, p.154) is limited to non-existent. It is evident that the values of
1467 the extrapolation assessment factors need to be validated for NMs based on the experimental
1468 data with chemicals in the nanoform. To date however, the number of studies satisfying the
1469 criteria of suitability for use in such an analysis is extremely low. No results from studies
1470 designed to investigate these relations are available yet, although the studies performed within
1471 the OECD testing program will likely generate some relevant information. Single publications
1472 suggest high bioaccumulation potential of certain NMs, that can potentially lead to high

59
1473 extrapolation factors in the relation between long and short term toxicity (Zhu, 2010). The case
1474 can therefore be made that, until assessment factors are validated for their applicability to NMs,
1475 REACH should require some measure of chronic toxicity alongside the mandatory acute tests
1476 both for human health hazard assessment and ecotoxicity (See also 5.1, p.154).

1477 QSARs for NMs. QSARs and other non testing methods have gained regulatory acceptance within
1478 REACH as a tool for prioritization of substances for further testing and in some instances as a
1479 valid tool to generate data for risk assessment and hazard classification. In this context QSARs
1480 that are capable of predicting toxic endpoints such as carcinogenicity, mutagenicity,
1481 reproductive toxicity, persistence, bioaccumulation and long term ecotoxicity are of special
1482 regulatory importance. Although the need to develop nano-specific QSAR models has been
1483 recognized by the scientific community and some efforts have been initiated to meet this
1484 challenge, at present no such models are available (Puzyn, 2009), neither are expected in the
1485 short to medium term due to the lack of validated data and methods (See section 3.3.1, p.92).

1486 Physical hazard assessment concerns a sub-set of the properties mentioned above, such as
1487 whether or not the substance is explosive, oxidising, extremely flammable, highly flammable, or
1488 flammable. Very limited work on these aspects of NMs have been conducted to date, as these
1489 have not been considered a priority in terms of understanding the hazards associated with NMs.
1490 As many NMs are prepared, stored and shipped as powders, there may be some associated risks
1491 of explosion of flammability. The oxidising potential of NMs has been assessed, both acellularly
1492 and in cellular systems, with little correlation between physico-chemical properties and
1493 oxidative stress potential of the NMs being observed (Xia, 2006, Duffin, 2007).

1494 One parameter not listed above, but which has very significant impact in terms of hazard
1495 assessment for health and for occupational exposure is dustiness, which is related to the amount
1496 of inhalable particles that are liberated upon pouring. Dustiness is the parameter associated
1497 with dust particles from mining etc. (Tsai, 2009), and has a long association with inhalation
1498 toxicology and the onset of diseases such as asbestosis and silicosis (Kaewamatawong, 2005).
1499 Thus, dustiness is included in the list of physicochemical endpoints for assessment as part of the
1500 OECD sponsorship programme (see Appendix 2.2, p.73). However, once the NMs are dispersed
1501 in a fluid, this parameter is no longer a critical issue, and indeed dispersion is commonly
1502 regarded as a route to mitigating the health risks from powered samples. For the other
1503 physicochemical hazard parameters listed above, some consideration of whether the same sets
1504 of tests are required for NMs that are always maintained in dispersion, and whether these
1505 parameters can/should be assessed in the pristine materials or on the in situ presentation as

60
1506 used in the other (human health and environmental) hazard assessments (so that the data are
1507 comparable) is needed, and guidance documents should be prepared.

1508 Human hazard assessment is discussed in Chapter 4, p.118 and the following
1509 recommendations have been suggested in section 4.7, p.146:

IT IS RECOMMENDED TO CONSIDER NMs AS BIOLOGICAL ENTITIES AND BUILD ON

LESSONS FROM REGULATION OF PROTEIN THERAPEUTICS AND OTHER BIOLOGICALS
(SECTION 4.6-7, P.142)

By considering NMs as biological, focus shifts immediately to assessment of NMs interactions

with surrounding biomolecules and their impacts. It will also remove the issue of legacy NMs
being exempt from re-regulation, including the exemption for polymers, since polymeric NPs
also adsorb a corona of biomolecules to themselves, thereby obtaining a biological identity.

RE-CONSIDER THE TESTING APPROACH FROM 1ST PRINCIPLES FOR CHEMICALS THE
BASIS OF REGULATION IS THE CHEMICAL NOMENCLATURE OF THE DRUG / MOLECULE -
FOR NMs THIS SHOULD INCLUDE THE SURFACE IN ADDITION TO THE PARTICLE SIZE,
SHAPE AND COMPOSITION, AS THIS IS WHAT GOVERNS ALL SUBSEQUENT INTERACTIONS
AND IMPACTS (SECTION 4.1, P.118)

By having the nanomaterial surface and what happens at the nanomaterial-biological interface
as the centre of the regulatory framework, we can make a re-evaluation of the applicability of
the current tier-testing approach for NMs, and where appropriate add additional steps such as
characterisation of the nature of the NPs in situ in the appropriate test medium.

It is essential to focus on addressing issues of nanomaterial reproducibility and surface

composition variability as a priority, as otherwise we will continue to produce large amounts of
safety data on materials that are only representative of themselves. While the approaches
adopted by some of the larger testing programmes of using a single large batch of a specific
nanomaterial from a specific manufacturer are extremely important in terms of validating test
methods and ensuring reproducibility across laboratories, they run the risk of generating large
amounts of safety data that is non-generalisable even to that specific type of nanomaterial, as we
do not yet have any answers as to the variability of synthetic routes and the limits of sameness
in terms of surface properties that we can accept from a safety and regulatory viewpoint.

IT IS RECOMMENDED THAT CLARITY IS PROVIDED FOR THE NAMING OF NMS. A

CLASSIFICATION SHOULD BE DEVELOPED IDENTIFYING THEM AS NEW SUBSTANCES,
SEPARATE FROM THEIR CHEMICAL COMPOSITION. THIS NAMING SYSTEM SHOULD BE
HARMONISED WITH THE, DEVELOPING, REGULATORY DEFINITION OF A NM FOR
COMPATIBLE IMPLEMENTATION OF REACH PROVISIONS

Much of the current uncertainty around regulation of NMs stems from the lack of clarity
regarding the naming of NMs. Addressing this issue will likely speed up the development of an
implementable nano-specific regulatory process, which combines the best elements of existing

61
 regulatory frameworks for biological materials and chemicals.

1510

1511 Ecological hazard assessment is discussed in Chapter 5, p.153, and the following
1512 recommendations have been suggested:

AS AN INTERIM MEASURE IT IS RECOMMENDED THAT A MEASURE OF CHRONIC TOXICITY

BE REQUIRED FOR ALL NMs TO WHICH REACH REGULATIONS ARE APPLICABLE.

See also recommendation on novel endpoints. This interim measure could be dropped once
sufficient confidence is established in safety factors.

IT IS FURTHER RECOMMENDED THAT REGULATORY BODIES AND THE NANOPARTICLE

INDUSTRY FUND THE RESEARCH NECESSARY TO DEMONSTRATE THE LINKAGE BETWEEN
ACUTE AND CHRONIC ECOTOXICITY (SECTION 5.1, P.154)

IT IS RECOMMENDED THAT STANDARDISED ECOTOXICITY EXPOSURE CONDITIONS BE

ESTABLISHED FOR NMs IN A RANGE OF DIFFERENT ENVIRONMENTAL COMPARTMENTS
(FRESHWATER, MARINE, SATURATED AQUATIC SEDIMENTS, SOILS).

These conditions may differ from standardised OECD testing regimes, and should be optimised
to promote NM stability without resulting in adverse effects on test organisms.

IT IS FURTHER RECOMMENDED THAT FUNDAMENTAL RESEARCH BE CONDUCTED INTO

THE APPROPRIATENESS OF COMMONLY USED CULTURE MEDIA AND EXPOSURE WATER.
(SECTION 5.2.3, P.159)

IT IS RECOMMENDED THAT FURTHER HIGH QUALITY RESEARCH BE CONDUCTED TO

CHARACTERISE THE HAZARD POSED BY NMs TO AT RISK CONSORTIA (FILTER-FEEDING
MARINE BIVALVES, ESTUARINE BENTHIC COMMUNITIES AND SOIL DWELLING
ORGANISM) (SECTION 5.2.4, P.160)

IT IS RECOMMENDED THAT RESEARCH BE UNDERTAKEN TO DESCRIBE NOVEL

ENDPOINTS ARISING FROM EXPOSURE TO NMs AND TO ASSESS THEIR RELEVANCE IN A
REGULATORY CONTEXT (SECTION 5.2.5, P.162)

IT IS RECOMMENDED THAT IN ADDITION TO THE SHORT-TERM TOXICITY TEST ON

DAPHNIA THAT IS ALREADY REQUIRED (PRIORITISED SAMPLES > 1 TONNE AND ABOVE)
SHORT-TERM TOXICITY TESTS SHOULD ALSO BE MANDATORY FOR A TERRESTRIAL
SPECIES AND A BENTHIC SPECIES (SECTION 5.4, P.176)

62
 IT IS RECOMMENDED THAT APPROPRIATE MEASURES OF NM DEGRADATION BE
REQUIRED FOR ALL NMs TO WHICH REACH REGULATIONS ARE APPLICABLE.

These will inlude measures of dissolution for some metal and metal oxides in addition to ready
biodegradation for carbonaceous NMs.

Mechanisms which limit or subtanitially reduce the bioavailability of NMs (such as irreversible
adsorption to sediments) should be investigated and incorporated as a required measure if they
prove substantive (Section 5.4, p.176)

IT IS RECOMMENDED THAT, AS A MATTER OF URGENCY, THE OECD PERSISTENCE AND

BIOACCUMULATION TESTS (DETAILED IN TABLE 5.2, P.184) BE ASSESSED FOR THEIR
APPLICABILITY TO SPECIFIC NM TYPES AND THEIR METHODOLOGIES VALIDATED AS
SUITABLE (SECTION 5.4, P.176)

1513

1514 2.3.3.4 Exposure assessment

1515 The applicability of current approaches to the exposure assessment of NMs has been accessed in
1516 the recent SCENIHR (SCENIHR, 2009) report and several problems have been highlighted. It was
1517 concluded that well established knowledge of distribution and fate of chemicals substances, as
1518 it is applied in the current EU guidelines for environmental risk assessment of conventional
1519 chemicals, cannot be used for NMs without modification. Furthermore it has been stressed that
1520 certain methods that are used to measure the rate of dissolution in the environment are unlikely
1521 to be suitable for NMs. Analytical methods to detect and measure the concentration of free NMs
1522 are needed. For human exposure assessment the following conclusion was reached:
1523 improvement is needed to estimate occupational exposure by implementation of more specific
1524 measurement techniques. Additionally, methods to discriminate background versus
1525 manufactured NMs are required, to enable synergistic and/or cumulative exposure effects to be
1526 determined. Exposure assessment from food and consumer products is difficult, because
1527 information on the presence of NMs relies entirely on claims volunterily provided by
1528 manufacturers (SCENIHR, 2009).

1529 Studies on real measurements of NMs in the environment are currently almost non-existent.
1530 Simulations and modelling can be helpful for estimating environmental exposure. Validation of
1531 these models against real measurements is essential however, before their predicative capacity
1532 could be assessed (Mueller, 2008). A recent effort to model the environmental concentrations of
1533 nano TiO2, ZnO, Ag, CNT, Fullerenes from different products like plastics, textiles, cosmetics, food

63
1534 supplements, paints and consumer electronics, along the different phases of life cycle, from
1535 manufacture to disposal or recycling, in air, water, soil sediments and groundwater found that
1536 sludge treated soils and sediments were likely to contain the highest concentrations of NMs (e.g.
1537 up to 0.5 mg of titanium dioxide per kg by 2012) (Gottschalk, 2009). The accuracy of those
1538 model predictions, which are based on assumptions, need to be validated against measured
1539 concentrations in due course.

1540 The current state of knowledge concerning occupational exposure assessment was analyzed
1541 during the OECD workshop on exposure assessment and mitigation for nanotechnology
1542 workplaces and a review has been published (Murashov, 2009).

1543 Within the EU-sponsored NANOSH project, a harmonised approach for a nanomaterial exposure
1544 measurement strategy, data analysis and reporting was developed. The data will be collected
1545 into a database for the future derivation of estimates for exposure scenarios (Brouwer, 2009),
1546 although at the time of publishing this report no further update was available.

1547 For further discussion on environmental exposure see section 3.3-5, p.92 and on human
1548 occupational and consumer exposure see section 4.4, p.134. The specific recommendations are
1549 presented below, with reference to the section containing the detailed discussion.

IT IS RECOMMENDED (SEE SECTION 5.3.1) THAT REACH REGULATIONS REQUIRE THE DISCLOSURE OF
NMS INCORPORATED INTO ARTICLES. DISCLOSURE SHOULD INCLUDE INFORMATION ON LOADING, METHOD
OF INCORPORATION AND DISTRIBUTION WITHIN THE ARTICLE (HOMOGENEOUS / LOCALISED). CLP
REGULATIONS SHOULD REQUIRE SEPARATE LABELING OF THE NM CONTENT OF PRODUCTS. THIS WOULD
ALLOW IDENTIFICATION AND QUANTIFICATION OF THE SOURCES OF NMS BEING RELEASED INTO THE
ENVIRONMENT, AS WELL AS PROMOTING CONSUMER CHOICE.

THE REACH DATABASE IS POTENTIALLY A VALUABLE RESOURCE TO THE RESEARCH COMMUNITY

ALLOWING EXPOSURE MODELS TO INCORPORATE CONCRETE SOURCE DATA FOR THE FIRST TIME. WE
THEREFORE RECOMMEND (SEE SECTION 5.5, P.179) THAT ECHA DEVELOP A STRATEGY TO ALLOW
DISSEMINATION OF INFORMATION ON THE FORM AND AMOUNT OF NM IMPORTED OR PRODUCED IN THE
EU, ALONG WITH INFORMATION ON DOWNSTREAM USES.

1550

64
1551 2.3.4 Registration of NMs case studies

1552 The parent substances of several of NMs have been registered at ECHA by the first registration
1553 deadline of 30 of November 2010. Analysis of those dossiers could potentially provide an
1554 overview on how the nanoforms of those substances have been dealt with in the registration
1555 process. However, this type of analysis was beyond the scope (and timeframe) of this project.

1556 Some specific comments concerning the three cases of nanosubstances that have been
1557 registered, namely carbon black, silicon dioxide and silver, could be included in this report,
1558 based on the publicly available information.

1559 The dossier for carbon black does not contain an exposure assessment because carbon black is
1560 not classified as hazardous. In this case the presence of the nanoform has been discounted in the
1561 explanation provided by the registrant: Carbon Black is a so-called nano-structured material
1562 (), i.e. consisting of primary particles formed in the carbon black process that combine within
1563 milliseconds after formation in the reactor to aggregates and agglomerates. Therefore a
1564 differentiation for Carbon Black between nano-material and bulk/conventional material is not
1565 necessary http://www.cb4reach.eu/index.php?id=faqonreachbyindustry#c31

1566 A similar explanation has been provided by the registrant of silica The registered synthetic
1567 amorphous Silica is a nano-structured material according to ISO TC 229 (Draft CS 27687, draft
1568 TS800004-5), consisting of primary particles forming aggregates and agglomerates within the
1569 silica process. Therefore, a differentiation for synthetic amorphous Silica between nano-material
1570 and bulk/conventional material is not possible (http://www.reach-sas.org/faq.htm, accessed on
1571 the 13 of January 2011).

1572 The understanding gathered so far is that both of those substances are considered by the
1573 registrants as so called nano-structured materials, where the primary particles are forming
1574 aggregates and agglomerates. According to the registrant, differentiation between the
1575 nanoforms and bulk/conventional materials is not possible. Those ststement can be questioned;
1576 although formation of aggregates and agglomerates may apply to some polymorphs of synthetic
1577 amorphous silica (SAS), others like the sol form remain in the formulation as primary particles
1578 in the size range 0.005-0.02 m. Differences in the physical and chemical properties of different
1579 polymorphs of SAS have been reported (ECETOC, 2006). This most likely implies different
1580 toxicities as it has been concluded from inhalation studies that NOAELs for SAS were between
1581 0.5 and 10mg/m3. The difference in values may be explained by different particle sizes; in
1582 general as particle size decreases so does the NOAEL. In this sense, different forms of SAS could

65
1583 present different risk profiles. Although, the relevance of the studies with aerosols of small
1584 particles has been questioned, and they are not considerd as being representative of the
1585 commercial products as only minor amounts (less then 1%) of the commercial products are
1586 respirable (alveolar fraction < 10 microm MMAD) (ECETOC, 2006), the question certainly
1587 remains as to whether exposure to the respirable fraction of particles from the commercial
1588 products remains negligible given the likely increased market penetration of products
1589 containing these materials.

1590 The dossier already registered for silver does not contain information on nanosilver, and the
1591 registration of nanosilver is predicted to be delayed until 2018 despite its widespread use in
1592 consumer products (Chemical Watch, 2010). A case study on a hypothetical registration of
1593 nanosilver under REACH showed that REACH is not sufficiently implementable for this
1594 particular nanomaterial (Pronk, 2009, Wijnhoven, 2009a). Recent analysis (Christensen, 2010)
1595 concluded that no definite conclusions for regulatory decision making could be taken due to lack
1596 of data on exposure and hazard. Based on existing data, concerns were raised that repeated
1597 inhalation in the workplace, possible consumer inhalation and (uncontrolled) nano-silver drug
1598 intake and burn treatment with wound dressings impregnated with Ag NMs may cause risks.

1599 In the absence of sufficient data for risk assessment of NMs, the precautionary principle should
1600 be invoked and complete data requested for NMs in order to build up a database of safety
1601 information.

66
02 Table 2.1Summary of testing requirements under REACH
03 UP unprioritized, P prioritized, Yes 1 depends on the outcome of other test, Yes? case by case consideration, a the test has a direct use for classification purposes, *Registration deadline 30/11/2010 if CMR cat. 1 or 2
04 substances above 1 tonne/year, or an R50-53 substance (PBT/vPvB) above 100 tonnes/year.

Substances

< 1 tonne >1 tonnes

Obligations under REACH dependent on categories

obligation under REACH - NO

Phase-in Non-phase in

CLP obligation - YES

TEST Not-preregistered /
registration as soon as the production volume above 1 tonne / year / manufacture
Pre-registered / delayed registration possible, dependent on tonnage ( *) registration as soon as the production volume above 1 tonne / year / manufacture
31/05/2018 31/05/2013 30/11/2010
1 10 10-100 100-1000 > 1000
1 10 tonnes 10-100 tonnes 100-1000 tonnes > 1000 tonnes
tonnes tonnes tonnes tonnes
UP P UP P UP P UP P
Biotic degradation (Ready biodegradation) a - - Yes Yes Yes Yes Yes Yes Yes Yes
Adsorption/desorption screening - - Yes Yes Yes - Yes Yes Yes
Fate and behavior

Hydrolysis - - Yes Yes Yes - Yes Yes Yes

Activated sludge respiration inhibition Yes Yes Yes Yes Yes Yes
Simulation testing a - - - Yes? Yes? - - Yes? Yes?
Identification of degradation products - - - Yes? Yes? - - Yes? Yes?
Bioaccumulation fish a - - - Yes? Yes? - - Yes? Yes?
Short-term toxicity daphnia a - - Yes Yes Yes Yes Yes Yes Yes Yes
Short-term toxicity algae a - - Yes Yes Yes Yes Yes Yes Yes Yes
Short-term toxicity fish a - - Yes Yes Yes - Yes Yes Yes
Ecotoxicity

Long-term toxicity fish and Daphnia a - - - Yes? Yes? - - Yes? Yes?

Short-term toxicity plants and terrestrial organisms - - - Yes? Yes? - - Yes? Yes?
Long-term toxicity plants and terrestrial organisms - - - - Yes? - - - Yes?
Long-term toxicity sediments organisms - - - - Yes? - - - Yes?
Long-term or reproductive toxicity in birds - - - - Yes? - - - Yes?
Skin + eye irritation (in vitro) - - Yes Yes Yes Yes Yes Yes Yes Yes
Skin sensitization a - - Yes Yes Yes Yes Yes Yes Yes Yes
Mutagenicity (in vitro) - - Yes Yes Yes Yes Yes Yes Yes Yes
Acute toxicity oral route a - - Yes Yes Yes Yes Yes Yes Yes Yes
Additional mutagenicity tests (in vitro) - - Yes1 Yes1 Yes1 Yes1 Yes1 Yes1 Yes1 Yes1
Skin + eye irritation (in vivo) a - - Yes Yes Yes - Yes Yes Yes
Toxicity

Acute toxicity second route a - - Yes Yes Yes - Yes Yes Yes
Subacute (28d) a - - Yes? Yes Yes - Yes? Yes Yes
Screening for reproductive toxicity - - Yes? Yes? Yes? - Yes? Yes? Yes?
Subchronic (90d) a - - - Yes? Yes? - - Yes? Yes?
Reproductive toxicity a - - - Yes? Yes? - - Yes? Yes?
Chronic toxicity and carcinogenicity a - - - - Yes? - - - Yes?
Exposu

Limited, exposure relater information - Yes Yes Yes Yes Yes Yes Yes Yes
re

Exposure scenarios development - - Only classified as dangerous, PBT or vPvB - Only classified as dangerous, PBT or vPvB
Chemical Safety Report - - - Yes - Yes

67
1605 Table 2.2 Combined GHS and REACH hazard classification criteria lined up with REACH tonnage bands
1606 data requirement suficient for classification endpoits.
Classification Testing approaches and classification criteria Tonnage
endpoints trigger with
data
requirements
sufficient for
classification
Acute Four toxicity categories based on acute toxicity by the oral, dermal, or
(mammalian) inhalatory route according to the numeric criteria. Acute toxicity values
toxicity are expressed as LD50 (oral, dermal) or LC50 (inhalation) values or acute
toxicity estimates (ATE)
Exposure route Category 1 Category 2 Category 3 Category 4
Oral (mg/kg ATE 5 5 < ATE 50 50 < ATE 300 300 < ATE 2000
bw)
Dermal (mg/kg ATE 50 50 < ATE 200 < ATE 1000 < ATE 2000
bw) 200 1000
Gases (ppmV) ATE 100 100 < ATE 500 < ATE 2500 < ATE
500 2500 20000
Vapours (mg/l) ATE 0.5 0.5 < ATE 2.0 < ATE 10.0 10.0 < ATE 20.0
2.0
Dusts and Mists ATE 0.05 0.05 < ATE 0.5 < ATE 1.0 1.0 < ATE 5.0
(mg/l) 0.5
Specific target Substances are placed in Cat 1 or 2, depending upon the nature and
organ toxicity severity of the effect(s) observed, by the use of expert judgment on the
single basis of the weight of all evidence available, including the use of
exposure recommended guidance values.

1 tonne (phase-in prioritized or non-phase-in)

Category 1: Substances that have produced significant toxicity in
humans, or that, on the basis of evidence from studies in experimental
animals can be presumed to have the potential to produce significant
toxicity in humans following single exposure;
Category 2: Substances that, on the basis of evidence from studies in
experimental animals can be presumed to have the potential to be
harmful to human health following single exposure;

10 tonnes
Category 3: Transient target organ effects.
The guidance values proposed for single-dose exposure which has
produced a significant non-lethal toxic effect are those applicable to
acute toxicity testing, as follows:
Guidance value ranges for:
Route of Units Category 1 Category 2 Category 3
exposure
Oral (rat) mg/ kg bw C 300 300 < C 2000 Guidance values
Dermal (rat or mg/kg bw C 1000 1000 < C 2000 do not apply,
rabbit) classification
Inhalation (rat) ppmV/4h C 2500 2500 < C primarily based on
gases 20000 human data, and
Inhalation (rat) mg/l/4h C 10 10 < C 20 animal
vapour data in the weight
Inhalation (rat) mg/l/4h C 1.0 1.0 < C 5.0 of
dust/mist/fume evidence
evaluation
Skin Category 1 Skin sensitisers if there is evidence in humans, or there are
sensitization positive results from the animal tests. The OECD test methods include
OECD 406 (the Guinea Pig Maximisation test and the Buehler guinea pig
test) and Guideline 429 (Local Lymp Node Assay). Other methods may
be used like Mouse Ear Swelling Test (MEST). When the adjuvant type
test method is used the response of at least 30% of animals is
considered as posivite, for a non-adjuvant test, a response of at least
15% of animals is positive, and a stimulation index of 3 or more is
considered positive in the local lymph node assay.
Mutagenicity REACH requirements based on tired approach ultimate (in vivo)
test requirements dependent on positive results from initial (in
vitro) testing. The classification, based on in vivo results, depends

68
 on the adequacy of the in vitro method to further prioritize for in
vivo testing.
Category 1 Mutagenicity Substances known to (Cat 1A) or regarded as
if they (Cat 1B) induce inheritable mutations in the germ cells of
humans: Cat 1A positive evidence from human epidemiological
studies, Cat 1B positive results from in vivo inheritable germ cell
mutagenicity tests in mammals, or positive results from in vivo somatic
cell mutagenicity tests in mammals, in combination with some evidence
that the substance has potential to cause mutations to germ cells, or
positive results from tests showing mutagenic effects in the germ cells
of humans without demonstration of transmission to progeny.
Category 2 Mutagenicity substances which cause concern for humans
owing to the possibility that they may induce inheritable mutations in
the germ cells of humans, based on positive results from experiments in
mammals and/or from in vitro experiments obtained from somatic cell
mutagenicity tests in vivo in mammals, or from other in vivo somatic cell
genotoxicity tests which are supported by positive results from in vitro
mutagenicity assays. Note: Substances which are positive in in vitro
mammalian mutagenicity assays and which also show a structure
activity relationship to known germ cell mutagens, should be
considered for classification as Category 2 mutagens.

Ecotoxicity The criteria for classification of a substance in categories Acute 1 to 3

Short term are defined on the basis of the acute toxicity data only (EC 50 or LC50).
aquatic Category Acute 1: 96hr LC50 (for fish), 48 hr EC50 (for crustacean)
and/or 72 or 96 hr ErC50 (for algae or other aquatic plants) 1 mg/l
(can be subdivided to include a lower band L(E)C50 0.1 mg/l.
Category Acute 2: 96hr LC50 (for fish), 48 hr EC50 (for crustacean)
and/or 72 or 96 hr ErC50 (for algae or other aquatic plants) > 1 but 10
mg/l.
Category Acute 3: 96hr LC50 (for fish), 48 hr EC50 (for crustacean)
and/or 72 or 96 hr ErC50 (for algae or other aquatic plants) > 10 but
100 mg/l (can be extended to the range beyond L(E)C50 of 100 mg/l.
Skin and eye Category 1 Corrosive - substance that produces destruction of skin
Corrosion / tissue, namely visible necrosis through the epidermis and into dermis
irritation in at least 1 animal after exposure up to 4 h duration. Three
subcategories are present (1A, 1B, 1C) dependent on the exposure
duration causing the effects, and the observation time.
Category 2 Irritant substance that causes a reaction in at least 2 out of
3 tested animals with a mean score for erythema/eschar or for oedema
between 2,3 4. Supplementary criteria exist.
Category 1 Irreversible effects on the eye / serious damage to the eyes
on the basis of the animal testing with observation of grade 4 cornea
10 tonnes

lesions and other severe reactions. Persistent lesions are those which
are not fully reversible within 21 days.
Category 2 Reversible effects on the eye substances that produce a
reaction in at least in 2 out of 3 tested animals of corneal opacity 1
and/or iritis 1 and/or conjuctival redness 2 and/or conjuctival
oedema 2, calculated as the mean scores following grading at 24, 48
and 72h and which fully reverses within period of 21 days
Ecotoxicity The classification of substance into categories Chronic 1 to 3 follows a
long term tired approach where the first step is to use available information on
chronic toxicity. In the absence of chronic data the combination of the
information about the acute toxicity and the environmental fate
(degradability and bioaccumulation data) is used 15.

15
At present the combination of the information about the acute toxicity and the environmental fate is used
within REACH, as the criteria for chronic data not yet accepted into the regulation.

69
 For non-rapidly degradable substances and chronic data available:
Cat Chronic 1: Chronic NOEC or ECx (for fish and/or for crustacean
and/or for algae or other aquatic plants) 0.1 mg/l;
Cat Chronic 2: Chronic NOEC or ECx (for fish and/or for crustacean
and/or for algae or other aquatic plants) 1 mg/l;
For rapidly degradable substances and chronic data available:
Cat Chronic 1: Chronic NOEC or ECx (for fish and/or for crustacean
and/or for algae or other aquatic plants) 0.01 mg/l;
Cat Chronic 2: Chronic NOEC or ECx (for fish and/or for crustacean
and/or for algae or other aquatic plants) 0.1 mg/l;
Cat Chronic 3: Chronic NOEC or ECx (for fish and/or for crustacean
and/or for algae or other aquatic plants) 1 mg/l;
For substances for which no chronic data is available and is not rapidly
degradable and/or experimentally determined BCF is 500, or if
absent, the log KOW 4:
Category Chronic 1: 96hr LC50 (for fish), 48 hr EC50 (for crustacean)
and/or 72 or 96 hr ErC50 (for algae or other aquatic plants) 1 mg/l;
Category Chronic 2: 96hr LC50 (for fish), 48 hr EC50 (for crustacean)
and/or 72 or 96 hr ErC50 (for algae or other aquatic plants) > 1 but 10
mg/l;
Category Acute 3: 96hr LC50 (for fish), 48 hr EC50 (for crustacean)
and/or 72 or 96 hr ErC50 (for algae or other aquatic plants) > 10 but
100 mg/l.
Subacute Substances are placed in Cat 1 or 2, depending upon the nature and
Specific target severity of the effect(s) observed, by the use of expert judgment on the
organ toxicity basis of the weight of all evidence available, including the use of
repeated recommended guidance values which take into account the duration of
exposure exposure and the dose/concentration which produced the effects.
Category 1: Substances that have produced significant toxicity in
humans, or that, on the basis of evidence from studies in experimental
animals can be presumed to have the potential to produce significant
toxicity in humans following repeated exposure;
Category 2: Substances that, on the basis of evidence from studies in
experimental animals can be presumed to have the potential to be
harmful to human health following repeated exposure. 10 tonnes (possible but can be waived)
The information required to evaluate specific target organ toxicity
comes either from repeated exposure in humans or from studies
conducted in experimental animals. The standard animal studies in rats
or mice that provide this information are 28 day, 90 day or lifetime
100 tonnes

studies that include haematological, clinic-chemical and detailed

macroscopic and microscopic examination to enable the toxic effects on
target tissues/organs to be identified.
The guidance values proposed refer esentially to effects seen in a
standard 90 day toxicity study in rats. They can be used as a basis to
extrapolate equivalent guidance values for toxicity studies of greater or
lesser duration, using dose/exposure time extrapolation similar to
Habers rule for inhalation, which states that the effective dose is
directly proportional to the exposure concentration and the duration of
exposure. The assessment should be done on a case-by-case basis; e.g.
for 28-day study the guidance values below would be increased by the
factor of three. Comment: However, Habers rule was developed for
conventionally chemicals, and simple dose-response relations may
not hold for NMs due to agglomeration / aggregation / dissolution
etc.
Guidance value ranges for:
Route of exposure Units Category 1 Category 2
Oral (rat) mg/ kg bw/d C 10 10 < C 100
Dermal (rat or mg/kg bw/d C 20 20 < C 200
rabbit)

70
 Inhalation (rat) ppmV/6h/d C 50 50 < C 250
gases
Inhalation (rat) mg/l/6h/d C 0.2 0.2< C 1.0
vapour
Inhalation (rat) mg/l/6h/d C 0.02 0.02 < C 0.2
dust/mist/fume
Reprotoxicity Substances are allocated to one of two categories. Within each category,
effects on sexual function and fertility and on development are
considered separately; in addition effects on lactation are allocated to a
separate hazard category.

100 tonnes
Category 1 known or presumed human reproductive toxicant; Cat 1A:
known human reproductive toxicant, based on the evidence from
humans; Cat 1B: presumed human reproductive toxicant, based on
animal studies providing clear evidence.
Category 2 Suspected human reproductive toxicant, based on some
evidence from human or experimental animals, where the evidence is
not sufficiently convincing to place the substance in Category 1.
PBT / vPvB PBT substance - a substance that fulfils all three of the following
criteria: 1. Persistence (the half-lives are higher than: 60 days in marine
water, or 40 days in fresh- or estuarine water, or 180 days in marine
sediment or 120 days in fresh- or estuarine water sediment or in soil).
2. Bioaccumulation (the bioconcentration factor (BCF) is higher than 2
000) 3. Toxicity (the long-term No-Observed Effect Concentration

100 tonnes
(NOEC) for marine or freshwater organisms is less than 0,01 mg/l, or
the substance is classified as carcinogenic (category 1 or 2), mutagenic
(category 1 or 2) or toxic for reproduction (category 1, 2, or 3), or there
is other evidence of chronic toxicity.
vPvB substances - a substance that fulfils the criteria of both: 1.
Persistence (the half-lifes are higher than 60 days in marine, fresh- or
estuarine water, 180 days in marine, fresh- or estuarine water sediment
or soil); 2. Bioaccumulation (the bioconcentration factor is greater than
5,000).
Carcinogenicity For the purpose of classification for carcinogenicity, substances are
allocated to one of two categories based on the strength of evidence and
additional considerations (weight of evidence).
Category 1: Known or presumed human carcinogens, on the basis of 1000 tonnes
epidemiological and/or animal data. Category 1A: known to have
carcinogenic potential for humans, largely based on human evidence; or
Category 1B: presumed to have carcinogenic potential for humans,
based on animal evidence.
Category 2: Suspected human carcinogens, on the basis of evidence
from human and /or animal studies, but which is not sufficiently
convincing to place the substance in Category 1, based on the strength
of evidence together with additional considerations.
1607

71
1608 Attachment 2.1 Substance identification according to REACH Annex VI section 2
1609 For each substance, the information given in this section shall be sufficient to enable each substance to be identified.

1610 If it is not technically possible or if it does not appear scientifically necessary to give information on one or more of
1611 the items below, the reasons shall be clearly stated.

1612 1. Name or other identifier of each substance

1613 1.1. Name(s) in the IUPAC nomenclature or other international chemical name(s)

1614 1.2. Other names (usual name, trade name, abbreviation)

1615 1.3. EINECS or ELINCS number (if available and appropriate)

1616 1.4. CAS name and CAS number (if available)

1617 1.5. Other identity code (if available)

1618 2. Information related to molecular and structural formula of each substance

1619 2.1. Molecular and structural formula (including SMILES notation, if available)

1620 2.2. Information on optical activity and typical ratio of (stereo) isomers (if applicable and appropriate)

1621 2.3. Molecular weight or molecular weight range

1622 3. Composition of each substance

1623 3.1. Degree of purity (%)

1624 3.2. Nature of impurities, including isomers and by-products

1625 3.3. Percentage of (significant) main impurities

1626 3.4. Nature and order of magnitude ( ppm, %) of any additives (e.g. stabilising agents or inhibitors)

1627 3.5. Spectral data (ultra-violet, infra-red, nuclear magnetic resonance or mass spectrum)

1628 3.6. High-pressure liquid chromatogram, gas chromatogram

1629 3.7. Description of the analytical methods or the appropriate bibliographical references for the identification of the
1630 substance and, where appropriate, for the identification of impurities and additives. This information shall be
1631 sufficient to allow the methods to be reproduced.

1632

72
1633 Attachment 2.2 Reporting requiremnts based on OECD (2010a) guidance document
1634
1635 According to the Guidance Manual for the Testing of Manufactured NMs developed within the OECD's Sponsorship
1636 Programme, the following information should be reported for all tested materials and studies (as appropriate):
1637
1638 1 Nanomaterial Information/Identification
1639 The information given should be sufficient to enable each substance to be identified. If it is not technically possible or
1640 if it does not appear scientifically necessary to give information on one or more items below, the reason should be
1641 clearly stated.
1642 Nanomaterial name (chemical name (e.g. IUPAC) and, if different, name from list)
1643 CAS Number.
1644 EC number (if available)
1645 Structural formula/molecular structure.
1646 Composition of nanomaterial being tested, including degree of purity in %, nature of known impurities including
1647 isomers and by-products (in % or ppm) or additive(s) (in ppm or %) (e.g. stabilising agents, surfactants or inhibitors.
1648
1649 As appropriate.
1650 Spectral data (ultra-violet, infra-red, nuclear magnetic resonance or mass spectrum) (If appropriate)
1651 High performance liquid chromatogram, gas chromatogram (If appropriate)
1652 Description of the analytical methods or the appropriate bibliographical references for the identification of the
1653 material and, where appropriate, for the identification of impurities and additives. This information should be
1654 sufficient to allow the methods to be reproduced.
1655 Basic morphology
1656 Description of surface chemistry (e.g., coating or modification)
1657 Major commercial uses
1658 Known catalytic activity
1659 Method of production (e.g., precipitation, gas phase)
1660 Producer/provider
1661 Batch no, and any other information useful to uniquivocally identify the material used
1662 The following properties and characteristics should be reported for all materials and studies (as appropriate)
1663
1664 2 Physical-Chemical Properties and Material Characterisation
1665 The following list is a list of physical-chemical properties that might be relevant to address for NMs, but for the
1666 individual NMs an evaluation of the relevance of each property should take place taking into account also the stated
1667 limits of the applicability of the different tests methods, and the nature of the tested material.
1668
1669 Characterisation of the material as on the shelf:
1670 Appearance (if applicable)
1671 Melting point (if applicable)
1672 Density (if applicable)
1673 Particle size including size distribution
1674 n-octanol-water partition coefficient, where relevant
1675 Water solubility/dispersibility, hydrophilicity
1676 Solubility/dispersability in organic solvents, oleophilicity
1677 Auto flammability (if applicable)
1678 Flammability (if applicable)
1679 Explosiveness (if applicable)
1680 Oxidising properties (if applicable)
1681 Oxidation reduction potential
1682 Stability in organic solvents and identity of relevant degradation products
1683 Storage stability and reactivity towards container material
1684 Stability: thermal, sunlight, metals
1685 pH (if applicable)
1686 Dissociation constant (if applicable)
1687 Other additional relevant physico-chemical information (where available)
1688 Agglomeration/aggregation
1689 Crystalline phase

73
1690 Crystallite and grain size
1691 Aspect ratio, shape (from e.g. TEM, SEM)
1692 Specific surface area
1693 Zeta potential (surface charge)
1694 Surface chemistry (where appropriate)
1695 Stability and homogeneity (on the shelf, in water/organic solvents)
1696 Dustiness
1697 Porosity, pore density
1698 Photocatalytic activity
1699 Pour density
1700 Radical formation potential
1701 Catalytic activity
1702
1703 Characterisation, homogeneity and stability of prepared test items in respective media (as appropriate):
1704 Test item preparation protocol, conditioning (for test media, test matrices as used for toxicological assessment and
1705 fate analysis), homogeneity (minimum sample intake), short term stability
1706 Physical chemical properties:
1707 composition/purity
1708 size and size distribution
1709 agglomeration/ aggregation
1710 Zeta-Potential
1711 Biophysical properties (as appropriate):
1712 protein binding/corona characterisation
1713 residence times
1714 adsorption enthalpy
1715 conformation changes on binding
1716
1717 3 Endpoints
1718 The following endpoints should be addressed for all NMs as appropriate.
1719
1720 Environmental Fate and behaviour
1721 Stability
1722 Phototransformation in air
1723 Hydrolysis
1724 Phototransformation in water
1725 Dispersion stability in water
1726 Abiotic degradability and fate (other)
1727 Biodegradation (as appropriate, depending on the material)
1728 Biodegradation in water: screening tests
1729 Biodegradation in water and sediment: simulation tests
1730 Biodegradation in soil
1731 Identification of degradation product(s)
1732 Sewage treatment simulation testing
1733 Bioaccumulation (as appropriate, depending on the material)
1734 Bioaccumulation: aquatic/sediment
1735 Bioaccumulation: terrestrial
1736 Transport and distribution
1737 Adsorption/desorption
1738 Other distribution data
1739 Other relevant information (when available)
1740
1741 Environmental Toxicity
1742 Aquatic toxicity
1743 Short-term toxicity to fish
1744 Long term toxicity to fish
1745 Short term toxicity to aquatic invertebrates
1746 Long term toxicity to aquatic invertebrates

74
1747 Toxicity to aquatic algae and cyanobacteria
1748 Sediment toxicity
1749 Terrestrial toxicity
1750 Toxicity to soil macroorganisms except arthropods
1751 Toxicity to terrestrial arthropods
1752 Toxicity to soil microorganisms
1753 Additional ecotoxicological information
1754
1755 Mammalian Toxicity
1756 Pharmacokinetics, ADME
1757 Basic toxicokinetics
1758 Dermal absorption
1759 Acute toxicity
1760 Acute toxicity: oral
1761 Acute toxicity: inhalation
1762 Acute toxicity: dermal
1763 Acute toxicity: other routes
1764 Irritation/corrosion
1765 Skin irritation/corrosion
1766 Eye irritation
1767 Sensitisation
1768 Skin sensitisation
1769 Repeated dose toxicity
1770 Repeated dose toxicity: oral
1771 Repeated dose toxicity: inhalation
1772 Repeated dose toxicity: dermal
1773 Genetic toxicity
1774 Genetic toxicity in vitro
1775 Genetic toxicity in vivo
1776 Toxicity to reproduction
1777 Toxicity to reproduction
1778 Developmental toxicity/teratogenicity
1779 Toxicity to reproduction: other tests
1780 Specific investigations:
1781 Neurotoxicity
1782 Immunotoxicity
1783 Other Specific Investigations
1784 Phototoxicity
1785 Blood compatibility/ blood toxicity
1786 Cardiovascular toxicity
1787 Other test data
1788 Chronic Toxicity
1789 Exposure related to humans

1790

75
1791 3 Physicochemical properties of NMs

1792 3.1 What is nano?

1793 Nano specifies a length scale of 10-9 m or a billionth of a metre, and prefixes a variety of words:
1794 nanomaterial, nanotechnology, nanoparticle, denoting their operation at that scale. A billionth of
1795 a metre is difficult to visualise, and Figure 3.1, p.112, shows where nano lies on a length scale of
1796 common objects (Hochella Jr., 2002). Comparatively, NMs have a diameter 10 - 1000 times
1797 larger than the atoms of which they are composed, but ten times smaller than a bacterial cell,
1798 being approximately equivalent in size to viruses.

1799 There has yet to be international agreement on the specific definition of a nanomaterial16 (NM),
1800 but various suggestions exist in the scientific (Kreyling, 2010b) and regulatory literature as to
1801 what nano- means (see section 2.2, p.27). Mostly these rely on size as the defining principle,
1802 usually with an implication that the fabrication of NMs or structures requires some purposeful
1803 manipulation of their atomic building blocks. For regulatory purposes many of these definitions
1804 are insufficient, as they lack the required specificity to be used in an enforceable manner.

1805 NMs are not a discrete class of substances; they are a heterogeneous group which encompasses
1806 a range of core chemistries, sizes and structures (see below and section 3.2, p.80), yet they have
1807 common characteristics. The most obvious commonality is their size, by consensus with at least
1808 one dimension in the range 1 - 100 nm, but more important are the unique physical and
1809 chemical properties and functionalities that this small size confers (see section 3.2, p.80).
1810 Sometimes specific surface area (SSA) is not included in these unique properties (Auffan, 2009),
1811 but in terms of environmental health, this is likely to be a key novel property. These unique
1812 properties arise primarily from there being a higher proportion of atoms at the surface of the
1813 material / particle compared with the interior; i.e. they have a higher surface area: volume ratio
1814 than larger particles of the same chemistry (Figure 3.2, p.113). This makes NMs particularly
1815 reactive in terms of their catalytic properties and their interaction with other materials. It is this
1816 unique reactivity that industry seeks to exploit in the development of nano-enabled products,
1817 but is also the focus of concern because of its unknown toxicological and ecotoxicological
1818 consequences.

16
The International Organisation for Standardisation (ISO) and the Organisation for Economic Co-operation and
Development (OECD) and the American Society for Testing and Materials (ASTM) continue to work on formal
definitions, but are yet to report definitively. In the European arena SCENIHR has been tasked with defining the
nanoscale for regulatory purposes and have determined size and specific surface area as key parameters..

76
1819 A definition of the nanoscale as being 1-100nm does not take account of the inherent size
1820 distribution of NPs, which means that particles with a mean size significantly larger than 100nm
1821 could potentially have a fraction of particles of 100 nm or less. Thus, all nanoparticle synthesis
1822 routes to date result in a size distribution, which typically falls into one of a set of predictable
1823 profiles, including Gaussian, Lorenzian, log-normal etc. (Grass, 2006). This is a result of the
1824 random nature of the initiation processes, and/or the stochastic nature of the formation of
1825 critical nuclei around which particles precipitate. Controlling and directing nanoparticle
1826 synthesis and the drive to create monodisperse NMs in a controlled manner is an active area of
1827 research. There is still a very considerable way to go in terms of producing particles
1828 reproducibility in terms of their size, size distribution and surface properties, which vary quite
1829 considerable from batch to batch even in commercially available NMs.

1830 A typical NM size distribution curve is given in Figure 3.3, p.113. Thus, at the very least, a NM
1831 definition based on size should refer to the mean particle size, and should give some indication
1832 of the fraction of particles at the mean size as well as the upper and lower bounds for the size
1833 distribution. It is clear from Figure 3.3, p.113, that where the mean particle size is at 100nm or
1834 above, there is likely to be a significant proportion of the particles of sizes < 100 nm, and it is the
1835 (eco)toxicological impact of this fraction of the particles that must be regulated within REACH.
1836 As part of their efforts to define a scientific basis for the definition of NMs for regulatory
1837 purposes, SCENIHR (SCENIHR, 2010) have suggested that a material should be considered as a
1838 nanomaterial if 0.15% of its total particle number is less than 100 nm, coupled with the material
1839 having a specific surface area of > 60 m2/cm3. The European Parliament is considering
1840 implementing a cut-off of 1% of the particle number being < 100 nm.

1841 3.1.1 Classifying NMs

1842 NMs are variously classified according to origin (1), their degree of technological sophistication
1843 (2) and their core chemistry and / or structure (3).

1844 1. NMs may be classified according to how they arise. Natural NMs result from naturally
1845 occurring biogeochemical processes, and include aquatic and terrestrial colloidal materials,
1846 mineral and soil particles, viruses, and ultrafine volcanic ash and dust. Adventitious NMs are
1847 produced unintentionally as a by-product of industrial processes, such as the combustion of
1848 diesel fuel, car tyre or break wear, or as a result of polishing or other abrasive processes.
1849 Manufactured (or engineered) Nanomaterials (NMs) are intentionally produced by industrial
1850 processes and are nanoscale by design. This report is concerned only with the regulation of
1851 the last category of materials. However, it must be bourne in mind that differentiation of
1852 environmental and health effects from manufactured versus adventitious particles is not

77
1853 currently possible, and more like a cumulative type assessment considering total particle
1854 load (irrespective of sourse) will be required. For the sake of clarity, a further subdivision of
1855 manufactured NMs into commercial (i.e. industrially produced) and controlled (i.e. produced
1856 in research laboratories) NMs will be used in the following discussions. These differ in terms
1857 of their quality - how uniform they are, in terms of how well they are characterized and in
1858 their methods of production. Industrial processes often yield nanoparticle cohorts with a
1859 wide size distribution; mean particle size may be larger than 100 nm but a substantial
1860 proportion of the population may be < 100 nm in diameter, although they are often quite
1861 agglomerated / aggregated. This concept is expanded upon in section 3.5.2, p.98 and its
1862 regulatory implications give rise to recommendation 4, Table 1.1, p.22. The implications of
1863 NM size distribution for toxicity testing are discussed in Chapter 4, p.118. In this report we
1864 use the term NMs to include all NMs, including composites and nanostructured materials,
1865 and the term NPs to mean those NMs with two (nanotubes, nanowires etc.) or all three
1866 dimensions in the nanoscale.
1867
1868 2. Advances and innovations in nanotechnology are occurring rapidly. NMs currently in
1869 industrial production, first generation NMs, are largely passive particles, tubes or materials,
1870 which do not intentionally change their chemical or physical form. It is with these current
1871 NMs that this report is primarily concerned. It is anticipated that future (next generation)
1872 NMs will be able to change their physical or chemical form during operation (e.g. during
1873 drug delivery), self-assemble and network, and interface at the molecular level with
1874 biological systems. A widely quoted classification is that of Roco (Roco, 2005), who
1875 anticipates three future generations of materials: active nanostructures such as actuators,
1876 sensors and adaptive structures (2nd generation), three-dimensional systems of nanosystems
1877 (3rd generation) and molecular nanosystems (4th generation). However, as the scope of
1878 nanoscience broadens and materials become more specialized such clear delineations are
1879 likely to blur, and indeed passive NMs take on a biomolecule corona that acts as an interface
1880 with biological systems, as described in Chapter 4, p.118, and picked up again in Chapter 5,
1881 p.153, in terms of NM interactions with components of water and soil. As has been discussed
1882 in the recent Royal Commission on Environmental Pollution report (RCEP, 2008),
1883 governance of such materials may be beyond the scope of the existing regulatory structures
1884 (section 3.5.1, p.97).
1885
1886 3. First generation NMs, those currently in production at an industrial scale, can be grouped
1887 into five broad classes depending on their core chemistry and structure.
1888

78
1889 a. Carbonaceous. Fullerenes are composed of a hexagonal and pentagonal lattice of carbon
1890 atoms and include single and multi-walled carbon nanotubes (CNTs), spherical fullerenes
1891 (buckyballs) and graphene, the flat 2-D lattice monolayer, which is the progenitor for the
1892 other graphitic dimensionalities (Geim, 2007). The high aspect ratio (section 3.2.3, p.82) of
1893 CNTs is of particular toxicological (section 4.6, p.142) and ecotoxicological concern due to
1894 their potential asbestos-like properties (Donaldson, 2010). Research into graphene,
1895 recently produced as a stable individual entity (Novoselov, 2004), is active and extensive
1896 given its novel electronic and thermal properties (See (Geim, 2007, Rao, 2009) for
1897 reviews).
1898
1899 b. Metal oxides. Metal oxide NPs are one of the most commercially important groups of NMs.
1900 Zinc oxide and titanium dioxide are extensively used in cosmetics, amongst other
1901 products, with the later also being used for environmental remediation of waste and
1902 groundwater (Jing, 2009) and removal of air pollutants (Gao, 2008). Cerium oxide is used
1903 primarily as a fuel additive, and is potentially of high concern because of pervasive
1904 environmental release through exhaust emissions.
1905
1906 c. Zero valent metals. Metal NPs have diverse applications. Silver NPs (AgNPs) are potent
1907 bacteriocides and are incorporated into cosmetics and fabrics, finding their way into a vast
1908 variety of products and ultimately into wastewater systems, with as yet unknown
1909 consequences (Blaser, 2008). Nanoscale zerovalent iron (nZVI) has a large capacity for
1910 sorbing common environmental contaminants such as organic solvents, dyes and metals,
1911 and is used in the remediation of contaminated groundwater, particularly in the USA
1912 (Zhang, 2006). This intentional release of NMs into the environment can have other
1913 unintended effects on groundwater chemistry and on biological communities in
1914 wastewater treatment plants (Choi, 2008).
1915
1916 d. Nanopolymers. Dendrimers are highly branched polymers, the structure and
1917 functionalisation of which can be controlled. They are used primarily for drug delivery.
1918 Many other polymers can be made at the nanoscale, including polystyrene and
1919 Poly(methyl methacylate). Historically, polymers have been exempt from REACH
1920 regulations and it follows that nanoscale polymeric materials will also be afforded this
1921 exemption. Section 4.3, p.133, explores the scientific basis of why this exemption should
1922 be revoked for nanopolymers.
1923

79
1924 e. Composites and Semi-conductors. Composite NMs combine several NPs in complex
1925 structures, or embedded within larger materials. QDs are generally a composite of semi-
1926 conductor materials and are used in Information Technology and medical applications for
1927 their unique optical, electronic and catalytic properties. QDs are of (eco)toxicological
1928 concern because many contain heavy metals such as cadmium, a known toxicant, which
1929 can leech out slowly through the (typically) polymeric shell.

1930 This section will focus primarily on reviewing the physicochemical properties of these NMs.

1931 3.2 Physicochemical properties of NMs

1932 Current REACH regulations define substances entirely by their chemical composition:

1933 substance: means a chemical element and its compounds in the natural state or obtained by any
1934 manufacturing process, including any additive necessary to preserve its stability and any impurity
1935 deriving from the process used, but excluding any solvent which may be separated without
1936 affecting the stability of the substance or changing its composition.

1937 This definition is inadequate if regulations are to successfully incorporate NMs. Such a simplistic
1938 definition has no scope to distinguish NMs from the bulk (micro or macro scale) substance, or
1939 different nanoforms from one another. CNTs, fullerenes and graphite are all carbon, but have
1940 very different physicochemical properties. As NMs are often produced for, and defined by, their
1941 novel properties, it could be argued that anything 'nano' is, de facto, a new substance. For
1942 regulatory purposes it is therefore necessary to incorporate physical and further chemical
1943 characterisation into the definition of substance. This precipitates the question as to which
1944 physical parameters are the most meaningful to describe and distinguish NMs, and how should
1945 they be measured? This section gives an overview of the important physicochemical
1946 characteristics of NMs and how these may be determined. Subsequent sections address to what
1947 extent physicochemical properties are predictive of the (eco)toxicological profile of a NM
1948 (section 3.3, p.92) and which parameters are useful and practically implementable in a
1949 regulatory context (section 3.5, p.97).

1950 The most important physical properties of NMs are shown schematically in Figure 3.4, p.114.
1951 They include primary particle characteristics17 such as size and shape, as well as other attributes
1952 such as aggregation kinetics and dissolution, which will be influenced by the interaction of NMs

17
The primary particle size is defined in this report as being the smallest entity biologically available, as this is
the crucial parameter for regulatory purposes. The concept of biological availability is explored in Chapter 4.

80
1953 with each other as well as with their environment. Interactions between physicochemical
1954 parameters are discussed below.

1955 3.2.1 Formulation Composition

1956 The intended chemical composition of the primary particle may not be the only chemical species
1957 present in formulated NPs. Significant impurities may be present due to processing and
1958 treatment and this may influence the behaviour of the NM, including its hazard to environmental
1959 and human health. If commercially produced (as opposed to controlled) NMs are to be used
1960 widely to perform (eco)toxicological studies, it is imperative that researchers have access to
1961 accurate characterisation of the formulation components. Characterisation data provided by
1962 commercial companies may not have the required rigor to allow accurate interpretation of
1963 (eco)toxicological results (Park, 2010a), or may simply have been characterised in inappropriate
1964 media. Independent characterisation in the appropriate physiological or environmental matrices
1965 should therefore be employed to allow meaningful interpretation of results whenever
1966 practicable (see section 3.3.2, p.94).

1967 Residual levels of catalysts and solvents used during manufacture may remain associated with
1968 particles as contaminants (Jurkschat, 2007). This is particularly prevalent in commercially
1969 produced NMs. As these contaminants may themselves be toxic, the purity of a formulation is
1970 especially relevant when performing (eco)toxicological testing (OECD, 2010a) (see section 5.2.2,
1971 p.158). For example, the use of yttrium carbide as a catalyst in the arc-synthesis of SWCNTs has
1972 been shown to impair the function of calcium-ion channels (Jakubek, 2008).

1973 3.2.2 Core Chemical Composition

1974 The core chemical composition of a NM, essentially what the majority of the particle is made of,
1975 will be governed by its formulation and manufacture, and will dictate many aspects of its
1976 behaviour and interactions. The composition of an individual NP is not necessarily uniform, it
1977 may be composed of layers with differing chemistry; a central core, a shell layer and a surface
1978 layer which may or may not be functionalized (Figure 3.6, p.116). As NM/NP size decreases the
1979 distinction of what is core and what is surface becomes blurred, and this perception will also
1980 be influenced by the technique that is used to analyse the particle. By convention it is the
1981 chemistry of the core material that is used as the descriptor (being the origin of the CAS
1982 number), yet it is the outer most layer that will govern the interactions of the material with its
1983 environment. The physicochemistry of the shell and surface layers are covered in sections 3.2.4
1984 and 3.2.5, p.85. In characterizing chemical composition it can therefore be important to analyse

81
1985 both the whole particle and the individual constituent layers. Techniques used to determine
1986 composition include XPS18, XRD and EELS and EDX each of which are attached to EM. Table 3.1,
1987 p.103 and Table 3.2, p.106, and Attachment 3.1, p.107, summarize the techniques available for
1988 nanoparticle characterisation and their relative strengths and weaknesses.

1989 3.2.3 Size

1990 Measuring size. Size is the most often quoted physicochemical property of a NM; it appears to be
1991 a simple and definitive descriptor. This perhaps explains its attraction as a regulatory tool. Yet,
1992 for all but perfectly spherical compact and monodisperse NPs, the size of a particle is not trivial
1993 to describe, or to determine experimentally. Different techniques measure size in different
1994 ways, yielding slightly different estimates. Differences between these estimates can provide
1995 greater insight into the behaviour of the NM, hence the advantage of using multiple methods for
1996 size characterization.

1997 Different techniques measure size based parameters, with different sensitivities and analytical
1998 windows. Non-spherical and porous/permeable NMs are poorly described by current theories;
1999 polydisperse and heterogeneous NMs and media components, such as salts, protein aggregates
2000 or natural organic matter all complicate measurement and data interpretation (Domingos,
2001 2009). The size of irregularly shaped NMs is given as an equivalent spherical diameter (ESD),
2002 often based on the Stokes relationship (Cumberland, 2009), but this can be calculated in a
2003 variety of ways, depending on which measurement equivalency it is based on. Diameter can be
2004 based on equivalent particle volumes, equivalent maximum or minimum dimensions or
2005 equivalent surface areas. It can refer to the core material as measured by EM or on the
2006 hydrodynamic diameter (particle core and associated hydration shell), approximated by DLS.
2007 The methods detailed in Table 3.1, p.103 and Table 3.2, p.106, for determining size all generate
2008 different ESDs, which may not be comparable. An additional level of complexity is that in any
2009 preparation of NPs there will be variation in the size of individual particles, the quoted nominal
2010 size being the average of the population tested. Different characterization methods also yield
2011 different types of average size, depending on whether mass/volume or particle number is used
2012 in the calculation. The quoted size of a NM is therefore intrinsically linked to the method used to
2013 determine it. For regulatory purposes it recommended that multiple methods should be
2014 required to determine size, to provide greater information and reduce bias.

2015 Variation in the size of NPs in a population is described by the particle size distribution (PSD).
2016 The PSD gives an indication of the quality of a particle preparation, as controlled NPs produced

18
A list of acronyms is provided on p. 3-8

82
2017 using well defined protocols have narrower size distributions than those originating from
2018 industrial processes. PSD in itself is an important descriptor of a NM preparation, as a majority
2019 of particles (by number) will have a size lower than the stated average (mean or median). As
2020 changing particle size has been shown to alter toxicity it is important in (eco)toxicological
2021 testing to determine the PSD, and to use preparations with as narrow a PSD as possible.

2022 The size of a NM will affect both its chemical reactivity and its biological availability, although in
2023 complex ways. As NM size decreases the proportion of atoms localized at the surface increases
2024 exponentially; particles with diameters of 50 nm have around 15 % of atoms at the surface, at 10
2025 nm this proportion rises to 40 % (Auffan, 2009, Oberdrster, 2005a). This makes NMs
2026 increasingly reactive as their size decreases. Auffan et al. (Auffan, 2009) argue that there is a
2027 subset of metal and metal oxide NPs with diameters of < 20 30 nm, where unique nanoscale
2028 properties predominate, with larger NPs (> 30 nm) having more bulk-like properties. The
2029 authors argue that only particles that show these unique nanoscale properties should be
2030 classified and regulated separately from their bulk counterparts. A weakness in this paper is that
2031 these distinctions are based on subjective interpretation rather than statistical analysis, a more
2032 conservative reading of their data suggests that 50 nm may be a more appropriate size
2033 distinction. The dataset reviewed is restricted with a heavy bias toward metal and metal oxide
2034 NPs and may not therefore be entirely representative for other classes of NM (carbonaceous,
2035 nanocomposities, etc.). An additional limitation to this approach is that, surface reactivity aside,
2036 NMs may still produce substantial (eco)toxicological consequences through bioaccumulation
2037 leading to increased body burdens.

2038 A simple analysis of particle numbers, based on a mean size of 30 nm, and a log-normal
2039 distribution curve, as shown in Figure 3.3, p.113, suggests that 100 % of the particles are < 100
2040 nm. However, looking at the TEM image it is very unclear as to whether these nominally 30 nm
2041 particles are actually individual particles or whether they are in fact fused together into much
2042 larger aggregates, which would not be biologically available and would in fact behave in a
2043 manner much more akin to micron scale particles. Thus, for many NMs, especially those which
2044 are produced (or purified) as powders and exist as either agglomerated or aggregated particle
2045 clusters, it is difficult to define the actual particle size, and as a consequence of this it can be
2046 very difficult to determine the bioavailable (available for biological uptake) dose which can
2047 interact with living systems as nanoscale particles. This is confounded by the fact that many
2048 particle manufacturers define the primary particle size as the smallest individual entities that
2049 can be detected, even if these entities are clearly fused into much larger particles, and thus are
2050 not bioavailable as discrete particles even following aggressive dispersion procedures. There are
2051 many approaches to dispersing NPs, but in very many cases, even aggressive treatments do not

83
2052 recover the manufacturer-defined primary particle size. This is illustrated in Figure 3.5, p.115,
2053 which shows several TEM images of TiO2 NPs at various stages of dispersion, using a range of
2054 approaches including sonication and capping with ligands. The details of the dispersion
2055 processes are not important in the present context, other than as a means of illustrating that a
2056 revision of the concept of primary particle size is required, whereby the primary particle size
2057 should be the smallest mean cluster size than can be achieved following dispersion efforts19, and
2058 as such is the bioavailable particle or particle cluster size.

2059 This issue of bioavailable particle size is partially addressed within the proposed SCENIHR
2060 definition of NMs, which considers also the (volume) specific surface area ((V)SSA) of the
2061 material, as highly aggregated NMs would have a lower (V)SSA than discrete NPs. This concept
2062 of the SSA of NMs is based on the fact that as the particle size decreases, the surface area
2063 increases dramatically, as shown schematically in Figure 3.2, p.113. In the case of highly
2064 aggregated particles, the (V)SSA is reduced everywhere that particles touch one another.
2065 However, what the SCENIHR approach to SSA does not take account of is the polydispersity of
2066 particulate samples, which will cause the real SSA to deviate quite significantly from the
2067 theoretical SSA of monodisperse particles for the example shown in Figure 3.2, p.113, above,
2068 the theoretical SSA of 30 nm particles would be 4/3r3 = 14,130 m3. However, as can be
2069 observed from Figure 3.3, p.113, only about 10% of the particles are close to 30 nm, with about
2070 25% of the particles being significantly less than 30 nm and 65% of the particles greater than 30
2071 nm, and as such the actual SSA is significantly different from the theoretical SSA.

2072 Further confounding the question of whether it is appropriate to set limits for definition of a NM,
2073 whether based on changes in properties, access to cellular machinery or simply based on an
2074 arbitrary size cut-off is the fact that there is also emerging evidence that passage of NMs across
2075 biological barriers is not necessary in order for the biological impacts of the presence of NMs to
2076 be felt, suggesting signalling from across barriers can also induce effects (Parry, 2010). Other
2077 evidence for non-contact induced impacts relate to signalling impacts induced by particles
2078 localised in lysosomes and impacting on mitochondrial integrity (Bexiga, 2010). This will also
2079 have significant regulatory implications, in terms of how we assess hazard in light of
2080 biodistribution and the current PBPK20 framework.

19
Note that for hazard assessment purposes we are limited to using dispersion approaches that do not
introduce surfactants or other dispersants that are themselves toxic to living systems, as this would only
confound the evaluation of the hazard properties of NMs. This is also not fully appreciated by the entire
community, as many NM synthesis routes still utilise non-biocompatible dispersants.
20
PBK Biodistibution Kinetics - physiologically-based pharmacokinetic (PBPK) modelling is a mathematical
modelling technique for predicting the absorption, distribution, metabolism and excretion (ADME) of a

84
2081 3.2.4 Morphology

2082 Shape. During synthesis the shape of some NMs can be manipulated by selectively adhering
2083 different capping agents. Shape is an important parameter as it can influence how NMs behave,
2084 especially in terms of their aggregation kinetics (section 3.2.7, p.89), which in turn will alter
2085 their environmental distribution and their biological and ecological availability.

2086 NMs with a high aspect ratio (HARNs), ones that have a long thin shape, are of particular
2087 (eco)toxicological concern if they are also bioaccumulative or biopersistent (persistent in vivo).
2088 This concern arises out of mammalian respiratory toxicology and the established link between
2089 the shape of asbestos fibres and lung disease. Obvious comparisons have been drawn with single
2090 and multi-walled carbon nanotubes (CNTs) (Donaldson, 2010, Poland, 2008), the latter of which
2091 has been shown to have pathogenic (asbestos-like) effects in mice when presented as HARNs,
2092 but not when bundled into loose aggregates. Other non-carbonaceous HARNs may have similar
2093 effects if they are also persistent in vivo. Metal oxide HARNs may in fact be of greater concern
2094 due to the intrinsic toxicity of their chemistry. Shape has been shown to alter the toxicity of
2095 silver NPs to a bacterial species (Pal, 2007), but in this study a triangular shape was more toxic
2096 than a rod-shaped particle with a higher aspect ratio. This could be reflective of surface
2097 curvature effects. Characterisation of shape relies heavily on microscopic methods, (TEM, SEM
2098 and AFM) which are time consuming and labour intensive, although information on shape can
2099 also be obtained from DLS and Fl-FFF methods (Table 3.1, p.103 and Table 3.2, p.106). The
2100 appropriate characterisation method depends on the specific NP formulation. BET can only be
2101 applied to dry powders, thus, for NPs supplied as suspensions microscopy methods should be
2102 employed, although the limitations of these methods should be acknowledged.

2103 Crystallinity. Atoms or molecules in a material may be ionically bonded to one another in regular
2104 repeating, symmetrical patterns, known as the crystal structure. In some materials a variety of
2105 different patterns can be obtained, depending on the conditions under which the crystal is
2106 formed. These crystalline polymorphs can have different physicochemical properties and
2107 (eco)toxicological profiles, as illustrated by the different toxicology impacts of crystalline
2108 (quartz) and amorphous (silica) particles. Titanium dioxide (TiO2) has three polymorphs,
2109 anatase, rutile and (the rarer) brookite, the foremost of which is photoactive (able to change its
2110 physical or chemical form in response to light). Most synthesis routes result in a mixture of co-
2111 existing phases and the rutile: anatase ratio of TiO2 NMs is a parameter which should be
2112 mandatory to report under REACH (see recommendation 4 in Table 1.1, p.22; and section 3.2.7,

compound in humans and other animal species. PBPK modelling is used in pharmaceutical research and
development, and in health risk assessment.

85
2113 p.89). Crystal structure can be determined using methods such as Selected Area Electron
2114 Diffraction, X-ray Diffraction and TEM (Table 3.1, p.103 and Table 3.2 p.106).

2115 3.2.5 Surface Properties

2116 Specific Surface Area. Specific surface area (SSA) is the most commonly determined surface
2117 measurement for primary particles. It is defined as the total surface area as a function of mass
2118 and is therefore expressed in units of m2/g or kg. SSA is influenced by particle size (SSA
2119 increases as particle size decreases), morphology, and surface topology i.e. how rough or
2120 porous the surface is. It can be measured as a projected area from an SEM or TEM image,
2121 although this is a 2D representation of a 3D object, or from adsorption of an inert gas (often
2122 nitrogen) in BET analysis (see Table 3.1, p.103 and Table 3.2, p.106). Although a useful measure,
2123 SSA can not give information on the size distribution or degree of aggregation in a nanoparticle
2124 preparation, both of which can be crucial parameters in the biological availability and uptake of
2125 NMs. It is also unable to distinguish between porosity and surface area.

2126 SSA may be a useful metric to describe NMs in a regulatory context as the gas adsorption method
2127 is insensitive to particle agglomeration. In an industrial preparation the majority of NPs may be
2128 present as agglomerates or aggregates with a diameter > 100 nm. This could lead to the
2129 misrepresentation that the material is not nanoscale. SCENIHR (SCENIHR, 2009) have proposed
2130 extending the physical size definition of a nanoparticle by the addition of a SSA limit of > 60
2131 m2/g. This corresponds to the surface area of smooth solid spheres of 100 nm diameter. In the
2132 context of the REACH regulations SSA may offer an alternative, or an adjunct, to the tonnage
2133 trigger mechanism for initiating (eco)toxicological testing (section 2.3.2.2, p.37)

2134 Surface area (or number) concentration may also be more meaningful as a dose metric in
2135 (eco)toxicological testing than particle number or total mass (see section 5.2.1, p.155).

2136 Other concepts of surface area include: Apparent surface area, SSA minus the area lost due to
2137 particle-particle contacts in aggregates (although in some aggregates this loss may effectively be
2138 zero); Active surface area, the area which contains biologically active materials, and
2139 Bioreactive surface area, the area actually accessible to a cell or organism (Handy, 2008, Handy,
2140 2008b). Active and Bioreactive surface areas are also likely to be less than total measured SSA
2141 because of particle aggregation under environmental or assay conditions. Although these latter
2142 concepts are useful in (eco)toxicology they are difficult to define and measure precisely, and as
2143 such are less useful from a regulatory perspective.

2144 Surface Charge. A surface can develop a charge in three ways:

86
2145 Isomorphic substitution is the replacement of one atom by another of similar size
2146 without disturbing the structure of the lattice. If the substituting ion carries a different
2147 charge then the overall charge of the surface will be altered (e.g. substituting a calcium
2148 ion (2+) for an aluminium ion (3+) will result in a -1 charge).
2149 Interaction of the surface with charged solution components. The most important of
2150 these interactions are with hydroxyl ions (OH-) and protons (H+), but interactions also
2151 occur with divalent ions, such as Na2+ and Ca2+ etc.
2152 Interaction of the surface with polyelectrolytes (polymers bearing a chemical group that
2153 will dissociate in water developing a charge). Such polymers include proteins,
2154 polysaccharides and humic substances. Such interactions are often irreversible, due to
2155 the large entropy gain upon release of unimolecular charges (counterions).

2156 The charge a nanoparticle develops when dispersed in water is a key parameter governing its
2157 agglomeration state (section 3.2.7, p.89) and its interaction with other charged ions and
2158 particles in the environment. A schematic of the diffuse electrical double layer (EDL) that forms
2159 upon particle dispersion in aqueous solution is shown in Figure 3.6, p.116. Acid-base titrations
2160 are used to determine the charge at the surface of the particle ( ). Zeta-potentiometry measures
2161 how suspended particles move in water when a charge field is applied, and thus yields a
2162 measure of charge at the hydrodynamic slipping plane. In practice the latter method is more
2163 easily automated and has therefore become popular. The utility and meaningfulness of zeta
2164 potential ( ) for permeable (porous) materials is questionable (Duval, 2005).

2165 As an approximation, particles with a charge of less than -30 mV or over +30 mV would be
2166 considered electrostatically stable in low ionic strength water. As the ionic concentration of
2167 water rises, i.e. as water hardness or salinity increases, additional ions will screen some of the
2168 particle charge and the depth of the EDL will be compressed. This allows particles to approach
2169 each other more closely and will increase the likelihood that a particle collision will lead to
2170 attachment. This is the first step in the process of aggregation. Interaction of NMs with
2171 biomolecules occurring naturally in test matrices and natural waters has the potential to
2172 profoundly influence the surface charge and dispersion stability (see section 5.3.2, p.170).

2173 Surface Speciation. Surface speciation refers to the oxidation state of the surface, or the specific
2174 form a chemical takes on interaction with water. Zero valent iron NPs can oxidize in water so
2175 that the surface is coated with iron oxide; silver nanoparticle surfaces may sorb silver (Ag+) ions
2176 at the surface (see also section 3.2.6, p.89 on dissolution properties); cerium oxide is reduced
2177 from Ce (IV) to Ce (III) due to oxygen deficiencies. (Eco)toxicology may vary markedly with
2178 chemical speciation. This is a significant issue also for NM ageing, as the surface speciation may

87
2179 change with time or during storage or product use, which may result in altered fate and
2180 behavior.

2181 Surface Functionalisation. As production methods become more sophisticated, there is an

2182 increasing capacity to functionalise the surface of NMs with a range of molecular groups. Surface
2183 functionalisation is usually undertaken for one of two reasons. A capping agent, usually a
2184 polymer, can be used during synthesis to control the size or shape of the forming particle,
2185 and/or to confer water dispersibility to an otherwise non-water dispersible NM. Secondly, long,
2186 thin polymers can also be attached to the nanoparticle surface to provide steric stabilization.
2187 Steric stabilization reduces the aggregation of particles through energetic rather than
2188 electrostatic means (cf. surface charge). It can be visualized as physically stopping particles from
2189 making contact with one another by coating them. Surface functionalisation therefore also has
2190 implications for particle aggregation and interaction with other environmental constituents, and
2191 it has been reported to increase the blood circulation time of NPs by helping them to evade
2192 uptake by macrophages (Calvo, 2001).

2193 Hydrophobicity / Lipophilicity. The tendency of a chemical to partition to organic solvents

2194 (hydrophobic / lipophilic chemicals) or to water (hydrophilic chemicals) is described by its
2195 octanol-water partition coefficient (KOW). There are concerns over whether this test is valid for
2196 NMs as some of the assumptions are violated (Crane, 2008, OECD, 2010b). Aggregation of NMs
2197 and their tendency to concentrate at interfaces both violate the assumption that the test
2198 substance has free diffusion through the phases, and make KOW difficult to determine in practice.
2199 This is of note as the KOW is used in environmental risk assessment strategies as a proxy for
2200 bioaccumulation (cell membranes are considered to be more permeable to lipophilic chemicals).
2201 Currently no alternative method for determining hydrophobicity / lipophilicity of chemicals or
2202 NMs is validated. The use of liquid: liquid extraction chromatography has been proposed as an
2203 alternative (Hassellv, 2008), but to date there are no published instances of its use for NM
2204 characterisation and hazard or risk assessment.

2205 KOW is also used as a predictor for the environmental partitioning of a test substance in porous
2206 media (soil and sediment systems), as a good correlation exists between KOW and KOC (the
2207 organic carbon-water partition coefficient) for a number of chemical classes (ECHA, 2008). For
2208 NMs, the predictive value of KOW, in this context is unproven, even if it could be reliably
2209 determined. Under the current REACH framework KOW is unlikely to be used in this manner for
2210 NMs, as adsorption / desorption screening is required only at triggers of >10 tonnes per year.
2211 The distribution coefficient (Kd) and from that KOC would need to be determined empirically if
2212 this measure of environmental partitioning becomes a requirement under any revised

88
2213 framework, and the relevant OECD protocols (OECD TGs 106; -121 and -312) may not be
2214 applicable or implementable for NMs without modification.

2215 3.2.6 Dissolution Properties

2216 Dissolution21. Dissolution, the process of a solid dissolving in a solvent (which may be water), is
2217 particularly relevant for metal NMs. Particle size (Liu, 2009) and SSA will influence the rate at
2218 which dissolution occurs, as will the chemistry of the particle, the volume of the solvent and any
2219 surface coating or functionalisation (Aruguete, 2010). As particulates and free ions may have
2220 different toxicological effects (Blinova, 2010), monitoring dissolution kinetics over timeframes
2221 relevant to (eco)toxicological experiments is important if the source of toxicity is to be correctly
2222 identified. Franklin et al. (Franklin, 2007) demonstrated that the toxicity of zinc oxide NPs to an
2223 algal species could largely be attributed to the rapid dissolution of free zinc ions during the
2224 experiment. Conversely, slow-dissolving NMs may develop a halo of high ion concentrations
2225 near their surface. Organisms coming into intimate contact with such particles would encounter
2226 a much higher dose of dissolved ions than would be predicted by the equilibrium concentration.

2227 Impurities. The synthesis of some NMs requires the use of metal or other catalysts. Established
2228 cleaning methods, such as dialysis, may fail to remove them, leaving the metal ions associated
2229 with the NM as contaminants. Subsequent desorption, once the particle is dispersed in the
2230 environment or has entered a cell, may result from the rich and varying 'combinatorial' dialysis
2231 environment provided by biological conditions (Salvati, 2011). Release of catalyst has been
2232 shown to have toxic effects e.g. through the generation of reactive oxygen species (Limbach,
2233 2005).

2234 3.2.7 Dispersion Properties

2235 NPs have an intrinsically high surface energy, and in accordance with the 2nd law of
2236 thermodynamics, will seek to reduce it.

2237 In biological matrices NMs may reduce their surface energy by selective interaction with their
2238 environment. A biological matrix comprises a range of available proteins, polysaccharides and
2239 other biopolymers, which will diffuse to the surface of the nanoparticle. The selection and
2240 exchange of these molecules at the surface over short timescales ultimately produces a

21
The terms dissolution and dispersion should not be confused. The former denotes the release of ions or
molecules from the surface of a NM and their distribution throughout the available liquid volume as a result of
entropy. Contrastingly, dispersion refers to the distribution of NMs themselves in a volume of liquid (see
section 3.2.6).

89
2241 'biomolecular corona', which passivates the surface and is stable for a given set of conditions
2242 (Monopoli, 2011).

2243 A more general mechanism by which surface energy can be reduced is through agglomeration,
2244 whereby dispersed particles coalesce to form larger clusters. This process is variously referred
2245 to in the literature as: flocculation, aggregation or agglomeration,22 and whilest the nuances may
2246 be different (agglomerates may be thought of as larger and more loosely bound than aggregates)
2247 the basic process is the same.

2248 Dispersed NMs will be in constant motion, fundamentally as a result of Brownian motion, which
2249 brings them into contact. As NMs are inherently unstable due to their high surface energy, such
2250 contact will result in agglomeration, which is an innate property of NMs. Stabilisation of NMs is
2251 achieved electrostatically and/or sterically. Interaction of two identical NPs is the first step in
2252 the formation of a homoaggregate (an aggregate composed of similar core constituents). After
2253 this initial step, particle-particle, particle-cluster and cluster-cluster interactions will all
2254 contribute to the aggregation process.

2255 In the environment and in biological systems the formation of heteroaggregates (aggregates
2256 formed from varying particle types) is a more common process, primarily due to the low
2257 concentrations of individual classes of NMs in ambient conditions. Particularly relevant to the
2258 environmental distribution of NMs is their association with natural organic matter (NOM) and
2259 proteins in aquatic environments and with soil and sediment matrices (section 5.3.2, p.170).
2260 Colloids are naturally arising heteroaggregates with a size range of 1 1000 nm. A large body of
2261 literature exists on their behaviour in environmental systems and this is likely to be predictive
2262 for manufactured NMs.

2263 Agglomeration is often presented as a one-way process, with large agglomerates being lost from
2264 the system through sedimentation. This is an oversimplification. Dispersion of agglomerates can
2265 also occur with a change in conditions of pH, perturbation or water chemistry, resulting in
2266 release of smaller clusters and individual NPs and this has been studied in relation to polymers,
2267 proteins and humic substances (Baalousha, 2009a). The interplay between agglomerates,
2268 dispersed NPs and the media in which they are suspended should therefore be regarded as a
2269 dynamic system. The dynamic nature of this system presents a considerable challenge for
2270 (eco)toxicological studies in terms of the accurate quantification of the bioavailable NM dose and
2271 the nature of the toxicant. This is explored more fully in section 5.2.1, p.155.

22
According to the ISO definitions, aggregates are irreversible and form as a result of fusion of particles during
synthesis, while agglomerates may be reversible.

90
2272 The size and structure of an agglomerate will alter its environmental and biological behaviour
2273 and its potential for chemical interaction. A loosely assembled, porous agglomerate (often
2274 indicative of slow formation) will have a lower effective density and a greater hydrodynamic
2275 drag than a more compact agglomerate. A porous agglomerate is also likely to be more
2276 permeable to diffusion of chemicals and thus to have a greater surface area, which may make it
2277 more active and more prone to dispersion following interaction with proteins or natural organic
2278 matter. Aggregate/agglomerate structure can be characterized using a number of methodologies
2279 including fractal dimension analysis of TEM images (Table 3.1, p.103 and Table 3.2, p.106).

2280 The bioavailability of NMs will be influenced by their dispersion and aggregation/agglomeration
2281 properties. Passive diffusion and receptor-mediated endocytosis of NMs across biological
2282 membranes will be reduced as particles form larger aggregates/agglomerates. Conversely,
2283 presentation as larger clusters may increase ingestion of NMs by higher organisms, and
2284 phagocytosis by single celled organisms evolved to feed on bacteria (which are typically 0.5 1.0
2285 m in diameter). Thus, there are likely to be several size-regimes of interest to regulators, an
2286 issue further explored in section 4.1, p.118.

2287 The dissolution (section 3.2.6, p.89) and dispersion section 3.2.7, p.89) properties of a NM,
2288 which are strongly related to its surface properties, combine to predict its stability in the
2289 environment, which in turn will govern its distribution, its bioavailability and its potential for
2290 bioaccumulation. These concepts are explored more fully in section 5.3.2, p.170.

IT IS RECOMMENDED THAT CHARACTERISATION OF THE FOLLOWING PHYSICOCHEMICAL

PARAMETERS BE MANDATORY FOR ALL SUBSTANCES DEFINED AS 'NMs' WITHIN THE
REACH FRAMEWORK:

Size; average and distribution. Either mean and standard deviation or median and geometric
standard deviation should be reported, depending on normality of distribution. Also, as
methods emerge that are capable of quantifying it, the % particles in an article < 100 nm.
Shape. Aspect ratio or defined measure of circularity
Core Chemistry
Specific Surface Area.
Surface modification, functionalisation and capping agents should be reported.
Surface charge.
In addition it is recommended that the following specific parameters be characterised for
particular NM classes:
Oxides of zinc and zero-valent (Ag0): dissolution in distilled H2O.

91
 TiO2: crystallinity in the form of the rutile: anatase ratio. SiO2: crystallinity in the form of
the amorphous / crystalline nature.
Oxides of cerium and iron: Oxidation state. For ceria this should be required only for
Category 3a (see recommendation 4 in Table 1.1, p.22, section 3.5.2, p.98) NPs and
should be reported as the CeO2: Ce2O3 ratio.

The list of required specific parameters should not be considered final, it should be augmented
as additional information becomes available in the literature on the (eco)toxicology of particular
classes of NMs and as characterisation methods improve. For example should a suitable routine
methodology become available to measure degree of entanglement of CNTs, this should become
a requirement.

Further to this recommendation, consideration should be given to the state of the nanoparticle
at characterisation. It is recommended that NMs be characterised in a pristine state (as
synthesised / manufactured) and in situ under appropriate assay conditions. The latter
characterisation is vital if (eco)toxicology results are to be interpreted in a meaningful way.

2291

2292 3.3 Relating physicochemical properties to (eco)toxicology

2293 A stated aim of the REACH regulations is to limit the requirement for in vivo vertebrate testing
2294 by promoting Quantitative Structure-Activity Relationship (QSAR) modeling and read-across
2295 approaches to generate the (eco)toxicity data required for dossier submissions. QSAR modeling
2296 is an established tool in predicting the (eco)toxicity of molecular chemicals and micro / macro
2297 scale particles, but there is still discussion over whether it is a valid approach for assessing the
2298 risk posed by manufactured NMs, especially given the complexity of the interplay between NMs
2299 and their environment and the current lack of agreed standards and protocols by which to
2300 generate the experimental data required to validate QSAR models.

2301 3.3.1 QSAR models

2302 QSAR modeling is essentially a formalized procedure for relating the chemical structure of a
2303 compound to a defined endpoint via statistical analysis of correlations between the two. The
2304 power of this approach is that high-quality QSAR models should be predictive; the effect on the
2305 endpoint should be able to be derived from a given change in the chemical structure. Potentially
2306 this saves considerable time and resources, as not every variant of a compound will need to have
2307 (eco)toxicity data generated empirically.

92
2308 The fundamental assumption that underpins this approach is that similar molecules will behave
2309 in a similar manner. This principle is termed the Structure-Activity Relationship (S-AR). Is this
2310 true for NMs; will similar NMs behave in a similar manner? There is currently limited evidence
2311 to support such relationships. The literature contains studies where purported similar NMs have
2312 markedly different (eco)toxicological profiles. However, this is often ascribed to systematic
2313 differences in a derived or secondary feature of the test system (for example smaller particles
2314 liberate toxic ions at a greater rate). The issue of batch-to-batch variability is also of
2315 consequence for the development of robust QSARS. How can 'similar' be parametised? How
2316 much variability in, for example, a surface characteristic is permissible for materials to still be
2317 considered 'similar'. This remains a considerable scientific challenge and one that will be
2318 addressed within the EU FP7 Research Infrastructure for nanosafety assessment (QNano), which
2319 will assess the sensitivity of several standard assays to small (systematic) changes in NM
2320 composition (surface charge, crystal phase etc.).

2321 Although QSAR modeling is generally considered as a valid approach to assess the (eco)toxicity
2322 of NMs, development of predictive high quality models is still some way off. Formation of robust
2323 QSAR models has a number of prerequisites (Puzyn, 2009):

2324 As pointed out in section 3.1, NMs are not a discrete class of substances and it is unlikely that a
2325 single QSAR model would be appropriate for all. The first step in developing a QSAR approach
2326 therefore is to develop a conceptual framework for grouping NMs. The established division by
2327 core chemistry may, or may not, be the most appropriate for this. One possibility here could be
2328 the nature of the biomolcule corona surrounding NMs in situ in representative biofluids.

2329 A related step is to develop nanodescriptors, ways of representing and describing the physical
2330 properties of each group of NMs that may be predictive for (eco)toxicological outcomes. These
2331 could be used in conjunction with existing chemical descriptors.

2332 Development and validation of QSAR models requires a high-quality dataset (Schultz and Cronin,
2333 2003). The gold standard for this is a continuous variable, such as LC 50, that has been measured
2334 using a single protocol, in a single laboratory and preferably by the same personnel. Such
2335 protocols must have been shown to be suitable for use with NMs (i.e. the presence of the NMs
2336 does not impact on the assay or the assay read-out) and have been internationally validated, for
2337 example by a round-robin (inter-laboratory) comparison. Such data are absent in the field of
2338 nano-(eco)toxicology; the reasons for which are explored in section 3.3.2, p.94.

2339 For a QSAR to be predictive the relationship between physicochemical characteristics and
2340 (eco)toxicological outcome must be linked in a systematic manner via an understanding of the

93
2341 mode of action. For NMs such systematic understanding is currently lacking. For this field to
2342 progress, greater insights are needed into which physicochemical properties may be
2343 instrumental in governing (eco)toxicology, and this can only be developed with more wide-
2344 ranging and detailed charcaterisation of NMs in situ in (eco)toxicology assays, as well as
2345 improved characterization of the homogenetity / heterogeneity of NM surfaces.

2346 The establishment of robust QSAR models also requires a proven relationship between in vitro
2347 and in vivo toxicity testing, and it is therefore suggested that sufficient invertebrate and
2348 vertebrate testing is necessary to allow such relationships to be quantitatively described.

2349 It is considered that QSAR and read-across approaches are not yet sufficiently mature for them
2350 to be used in a regulatory context for NMs. Research efforts and funding are being directed at
2351 developing models and generating robust datasets with which to validate them. If QSAR models
2352 become sufficiently developed they are potentially useful from a regulators perspective as they
2353 may serve as a rapid screening tool to identify and prioritise new materials of high regulatory
2354 concern. If links between specific physicochemical properties and (eco)toxicity are established
2355 there is also the possibility that QSAR modeling could inform the development of subsequent
2356 generations of NMs, allowing nanoengineers to design away some of the most toxic features of a
2357 material.

2358 3.3.2 Characterising NMs in (eco)toxicity studies.

2359 The lack of high-quality data is the most substantive block to developing meaningful QSAR
2360 models for NMs; the evidence to link physicochemical properties to (eco)toxicological outcomes
2361 is simply lacking. This is largely attributable to nano-(eco)toxicology being a new and
2362 interdisciplinary field, but is exacerbated by the lack of robust NM characterisation in most
2363 (eco)toxicity studies and by the variability23 in laboratory-produced and commercial NMs used to
2364 conduct most studies to date.

2365 A number of reviews and commentaries have called for a minimum standard of NM
2366 characterisation to be required in peer-reviewed nano-(eco)toxicity studies so that the link
2367 between physicochemical characteristics and (eco)toxicity can be investigated in a more
2368 systematic way. Much discussion has ensued as to which characteristics should be reported
2369 routinely and how they should be measured (Powers, 2007, Klaine, 2008, Warheit, 2008). Stone
2370 et al. (Stone, 2010) reviewed current opinion on the subject and concluded that, although

23
Both variation between manufacturers supplying nominally the same material resulting from different
synthesis routes, and batch-to-batch variability within a single manufacturer resulting from poor control and
the random nature of current synthesis routes.

94
2371 differences in emphasis exist, there is a general consensus in the most influential reviews on
2372 what should be measured. Their summary of required properties is reproduced in Table 3.3,
2373 p.111.

2374 Ideally all the physicochemical parameters in Table 3.3, p.111, would be determined for each
2375 test material. This is aspirational and in reality (eco)toxicology laboratories and small producers
2376 of NMs may lack the resources, expertise and access to specialized equipment that is necessary
2377 to undertake these characterizations. As Warheit (Warheit, 2008) points out this laundry list of
2378 physicochemical parameters requires some prioritization, and this may be NM and use specific
2379 to some extent (not feasible for regulatory science). Stone et al. (Stone, 2010) suggest that a
2380 minimum requirement for any NM (eco)toxicology or environmental study should be the
2381 characterization of:

2382 Aggregation / agglomeraton / dispersability

2383 Size
2384 Dissolution
2385 Surface area
2386 Surface charge
2387 Surface composition

2388 Even this minimum requirement would stretch the skills and resources of an average
2389 (eco)toxicology laboratory, and this emphasizes the need for interdisciplinary collaboration in
2390 the research community to generate meaningful studies. The establishment of specialised
2391 centers for the appropriate characterisation of NMs in relevant test media would benefit both
2392 the research community, by the generation of high quality data, and the industrial sector by
2393 making state-of-the-art characterisatiion facilities available to fulfill any future regulatory
2394 requirements. A similar approach has also been suggested by others (Hansen, 2009).

IT IS RECOMMENDED THAT ACCREDITED CENTERS BE ESTABLISHED TO UNDERTAKE

PHYSICOCHEMICAL CHARACTERISATION OF NMs.

Meaningful characterisation of NMs requires both a range of specialised equipment and a high
degree of technical competence. It is recognised that smaller manufacturers / importers of NMs
may not have recourse to such facilities, and capacity building in this area is required.
Establishing centers of excellence for characterisation would ensure that regulation of NMs is
based on high quality data. Validated, cost-effective techniques should be developed for sample
preparation, technical methodologies and data handling and analysis.

95
 Accredited characterisation centers would also be in a good position to act as 'nanosafety'
centers. The scope of such centers could encompass verification of data supplied under REACH
in registration dossiers, characterisation of NMs after exposure, and monitoring of 'at risk'
environmental compartments for NM contamination etc. Since the metrology and analysis
performed in situ and in complex media requires different expertise compared to traditional
characterisation, such centres need not always be existing metrology centres, unless this offers
particular advantages.

2395 3.4 Standards and Prioritisation

2396 The lack of availability of certified standard and reference NMs for a range of different
2397 measurement endpoints is a key issue. In the short-term reference NMs would facilitate method
2398 development and validation and allow studies to be compared in a more rigorous manner. In the
2399 longer term they could act as positive and negative nanoparticle controls for specific
2400 (eco)toxicity endpoints; substances of known toxicity with which to compare test NMs on the
2401 basis of a similar mode of action (MOA). In order for standards to be useful in benchmarking
2402 they must be well characterized and preferably have a known mechanism of toxicity or MOA. On
2403 a practical level, there must be a high degree of inter-batch consistency and sufficient shelf life
2404 so that they can be used with confidence, as well as being dispersible in a range of relevant test
2405 media.

2406 The use of reference standards is widespread in particle toxicology. Carbon black and quartz
2407 particles have been used in rodent respiratory studies. In ecotoxicology, there is a major
2408 knowledge gap surrounding which reference materials may be appropriate, and it is unlikely
2409 that a single nanoparticle type will be suitable for all classes of NMs and for all (eco)toxicity
2410 endpoints. Crane et al. (Crane, 2008) suggest that quartz (sand) particles would not be
2411 appropriate for aquatic and sediment dwelling organisms as they would encounter them in their
2412 natural habitats and may be adapted to dealing with them. NIST have produced citrate stabilized
2413 gold NPs standards, which are currently available in three sizes, for size calibration. IRMM have
2414 developed a colloidal silica reference nanomaterial, again for size calibration. Neither
2415 organisation currently certifies their NMs in (eco)toxicological media.

2416 Given the profusion and diversity of NMs in, or soon to be in, production, and with so many
2417 aspects of the materials to characterize, prioritization is essential. The OECD has produced a
2418 priority list based on materials already in, or close to, production, likely production volumes and
2419 availability of essential information (OECD, 2009b, OECD, 2010a), which includes:

2420 Fullerenes

96
2421 Single-walled carbon nanotubes
2422 Multi-walled carbon nanotubes
2423 Carbon black
2424 Polystyrene
2425 Dendrimers
2426 Nanoclays
2427 Nanoparticulate silver (Ag),
2428 Oxides of iron (Fe2O3 / Fe3O4), titanium (TiO2), aluminium (Al2O3), cerium (CeO2), zinc
2429 (ZnO), and silca (SiO2).

2430 The REFNANO project (Aitken, 2008, Stone, 2010) also considered the issue of reference NM
2431 selection, with greater emphasis on experimental issues and the environmental detection of
2432 NMs. The NMs selected for the priority list, TiO2, silver and polystyrene beads had different
2433 desirable characteristics. TiO2 and silver were particularly relevant because of their high
2434 production volumes. Silver also has proven bactericidal activity, although some concern was
2435 expressed over its dissolution properties. Polystyrene particles have a low solubility, and
2436 fluorescent labeling may facilitate environmental detection, if the dye is stable for extended
2437 periods under cellular conditions, but see work of (Salvati, 2011) for detailed discussion of this
2438 topic and the inherent challenges. Gold NPs were also considered good candidates for inclusion,
2439 due to their low solubility, current availability in a range of sizes, potential for surface
2440 functionalisation and ease of detection in environmental matrices. Both polystyrene and gold
2441 are of low toxicity making them more suitable as negative, rather than positive, controls for
2442 (eco)toxicity studies. The REFNANO group concluded that CNTs were not currently suitable as
2443 reference materials due to inter-batch variability and the consequent difficulty in selecting a
2444 representative CNT from the breadth of products available.

2445 3.5 NMs in a Regulatory Context

2446 This section considers regulatory issues arising from the physicochemistry of NMs, focusing in
2447 particular on the following questions: Does the remit of REACH encompass NMs now, and in the
2448 future? How should NMs be defined within the regulatory context? Is the existing data
2449 requirement framework suitable for NMs, and if not what alternatives exist?

2450 3.5.1 The Remit of REACH and NMs

2451 The REACH regulations are essentially based on substance form. Currently this is chemical form;
2452 a substance is wholly defined within the regulations in terms of its chemical composition or
2453 CAS/ EINECS number. However, there is no theoretical bar in the EC definition of substance to

97
2454 including additional descriptors of physical or physicochemical form. A general consensus exists
2455 that the REACH framework is applicable to NMs currently being manufactured, (RCEP, 2008)
2456 and the European Commission has deemed that all REACH provisions shall apply to NMs (CEC,
2457 2008). Some amendments and additions will be required to the regulations however so that the
2458 specific risks associated with NMs will be effectively captured within the framework. Questions
2459 remain, for example, over the status of polymeric NMs, currently exempted from REACH for
2460 historical reasons, and over how surface-functionalised and surface-coated NMs will be
2461 accommodated when multiple CAS numbers are attached.

2462 Whether the REACH regulations can be stretched to accommodate future generations of NMs is
2463 less certain, and is outside the remit of this report. In general terms however, as NMs become
2464 more sophisticated they will be defined more by their function - what they do, rather than by
2465 their form - what they are composed of. A NM designed for delivering gene therapy to a cell
2466 nucleus for example, is potentially toxic because it can traverse cell and nuclear membranes and
2467 deliver a payload and may then bioaccumulate, rather than necessarily because of its elemental
2468 composition. Therefore, as the field of nanoscience matures it may require discrete regulation
2469 based on the designed function or action of composites. Given the time required to develop
2470 robust legislation this is an issue that should be addressed urgently before these new generation
2471 NMs become prevalent.

2472 3.5.2 Discriminating NMs

2473 The current REACH framework has no mechanism by which chemically identical materials of
2474 different sizes, oxidation states or crystal structures may be distinguished. If regulations are to
2475 be made appropriate for NMs, provision must be made to include physical descriptors that
2476 allow:

2477 NMs to be distinguished from the bulk (micro/macro and dissolved forms of the chemical.

2478 Related nanoforms to be distinguished from one another.

2479 For regulatory purposes the descriptors used must be legally enforceable. As such they need to
2480 be both specific, unambiguous and measureable or quantifiable. The descriptors should allow
2481 the subsequenet data to be generated using validated methodologies, which ideally should be
2482 widely available and cost effective. In itself this list of requirements will be difficult to fulfill, as
2483 many of the physicochemical descriptors discussed in section 3.2, p.80, have no validated
2484 methodologies, or the apparatus or expertise required to determine them is not widely available.
2485 When considering the scientific discussion below it should be borne in mind what is realistic to
2486 achieve and this must be weighed against the precautionary principle; the current lack of

98
2487 appropriate measurement techniques is not a valid reason to allow an unsafe implementation of
2488 nanotechnologies, or to allow a non-science-based regulatory approach to prevail.

2489 Discriminating NMs from Bulk forms. NMs require separate registration from bulk chemicals of
2490 the same composition because there is an a priori assumption that they will behave differently,
2491 i.e. have a different (eco)toxicity profile and occupational / environmental behaviour. Particle
2492 size is one key parameter that will contribute to this behaviour; parameters such as SSA, surface
2493 charge or surface functionalisation may be important and potentially more predictive of
2494 (eco)toxicological outcomes. However size, even at the nanoscale, is a concept that is easily
2495 grasped and its use as a descriptor has become ubiquitous. Size distribution,
2496 aggregation/agglomeration and stability affect a simple interpretation of size, and regulators
2497 should be aware of this; large particles and nanoscale aggregates/agglomerates of equivalent
2498 overall size may show profoundly different behaviour and agglomerates may potentially
2499 disagglomerate under changing conditions. In addition, in any size distribution of NMs the
2500 number of smaller particles may greatly exceed that of larger particles24, and in some
2501 environments these may have a greater (eco)toxicological impact.

2502 If ESD is to be the primary delineating descriptor to distinguish NMs from their bulk form at
2503 what diameter should separate registration start? By convention the nanoform would relate to
2504 any particle with an ESD of 1 - 100 nm. From a scientific perspective this is a somewhat arbitrary
2505 cut-off. There is some evidence that quantum behaviour, which is traditionally associated with
2506 NMs becomes most pronounced at diameters of < 30 nm (Auffan, 2009) or < 50 nm more
2507 conservatively, specifically for metal and metal oxide NMs. However the exclusion of larger NMs
2508 (30 100 nm) implies SSA is not an intrinsic nano-property and it is difficult to justify if a truly
2509 precautionary approach to regulating NMs is to be taken.

2510 If ESD is to be used as the primary method of distinguishing NMs from the bulk an empirical
2511 measure of SSA should also be included. As discussed by SCENIHR (SCENIHR, 2009) this would
2512 prevent the misrepresentation of aggregated or agglomerated materials as not being nanoscale.

IT IS RECOMMENDED THE EUROPEAN COMMISSION ADOPT A SINGLE OVERARCHING

DEFINITION OF NMs FOR REGULATORY PURPOSES, WHICH MAY BE SUPPLEMENTED AS
REQUIRED FOR SPECIFIC LEGISLATION, AND THAT THAT DEFINITION BE BASED ON

24
Although they may contribute proportionally less in terms of mass.

99
PARTICLE SIZE.

The authors concur with the general approach adopted in SCENIHR 2010 and recommend that
the definition include reference to the following elements25:

The origin of the nanomaterial (natural, adventitious or manufactured)

Encompass materials with an internal structure or an external dimension26 within the nanoscale
limits defined. Specific exemptions should be developed for macroscopic objects with nanoscale
features (e.g. membrane filters).

Specific Surface Area (SSA) be included as a supplementary parameter, thereby extending the
definition to highly aggregated or agglomerated materials.

A single lower size limit of 1 nm be adopted, although it should be acknowledged that ambiguity
will remain due to the overlapping dimensions of molecules, macromolecules and NMs at this
scale.

Multiple upper limits for NPs be adopted. There is currently no scientific evidence that
correlates the onset of nanospecific properties with a specific dimension. Rather a continuum
exists of increasing non-bulk properties with decreasing size(Auffan, 2009), and likely there are
several size regimes of biological interest. It is not therefore possible to set a scientifically
meaningful single upper limit, and the definition of 'nano-' as an enforceable term will therefore
be driven by regulatory requirements. SCENIHR (SCENIHR, 2010) suggests setting multiple upper
thresholds for NMs, defining two categories which would be considered 'nano' in regulatory
terms.

Category 2: Median size <500 nm but >100nm with > 0.15% of the number size distribution
of particles <100 nm.
Category 3: Median size >1 nm but <100 nm with > 0.15% of the number size distribution of
particles <100 nm.

In acknowledgement of the increasing reactivity of NMs as their size decreases it is

recommended that Category 3 be subdivided as follows:

Category 3a: Median size >40 nm but <100 nm with a small percentage of the number size
distribution <40 nm.
Category 3b: Median size > 1nm but <40 nm or a small percentage of the number size

25
Authors agree with the SCENIHR opinion that all elements of the definition should be applicable to all
possible NMs. As such parameters useful for characterisation, such as dissolution or crystallinity, are not
appropriate for inclusion in the definition as they are only applicable to a subset of particles.
26
External particle size refers specifically to the size of the primary particle, without surface functionalisation.

100
 distribution <40 nm.
This scheme is illustrated and compared to the SCENIHR proposal in Figure 3.7, p.117. In
acknowledgement of the increased reactivity of NMs as their size decreases, it is expected
that, under any future testing framework, category 3b materials would attract the most
stringent data requirements, with fewer requirements as the size class increases.

2513

2514 Discriminating Nanoforms. This is a particularly difficult issue from a regulatory perspective.
2515 How different do different nanoforms with the same chemical composition have to be to warrant
2516 separate registration?

2517 One approach to differentiating nanoforms is to use arbitrary differences in key physicochemical
2518 descriptors. For example a > 10 nm difference in the ESD of NPs could be used to distinguish
2519 them as distinct nanoforms requiring separate registration. A > 3mV difference in surface
2520 charge, or a > 10% difference in the rate of aggregation or dissolution could be used, either as
2521 stand-alone requirements or in combination to trigger separate registration. Different
2522 descriptors could be given more of less weight depending on the class of nanomaterial; the rate
2523 of dissolution would be given greater importance for metal and metal oxide particles, for
2524 example. Depending on which descriptors are used and how the limits are set, such an approach
2525 could generate a huge number of additional registrations.

2526 Alternatively, an evidence-based approach could be employed. Differences in physicochemical

2527 descriptors, as described above, could be used as an a priori indication that the (eco)toxicity
2528 profile of nanoforms may vary. This would trigger the requirement for basic screening tests to
2529 determine whether or not that was the case. These tests could include, inter alia, acute toxicity
2530 tests on algae or Daphnids, microbial population growth tests, genotoxicity or immunotoxicity
2531 assays or an oxidative stress assay. Only if the resulting profiles of the two nanoforms were
2532 sufficiently similar would a single registration be permissible.

IT IS RECOMMENDED THAT NANOFORMS WITH THE SAME CORE CHEMISTRY BE

DIFFERENTIATED FOR SEPARATE REGISTRATION UNDER REACH USING DIFFERENCES IN
THE FOLLOWING PHYSICOCHEMICAL PARAMETERS:

Surface modification. Given that the surface chemistry of a nanoparticle will profoundly
influence its behaviour in the environment the use of different capping agents or surface
functionalisation or stabilisation should always trigger a separate registration.

101
Size. If the multiple size classification of NMs (as in recommendation above) is adopted, NMs
falling into different categories should require separate registration.

Shape. HARNs should be assessed on a case-by-case basis as substances of equivalent concern. A

substantive difference in circularity should trigger a separate registration.

Surface charge. Nanoforms with a substantively different surface charge of should be treated as
separate registrations given the a priori prediction that their environmental distribution would
be substantively different.

In addition it is recommended that TiO2 NMs with a marked difference in their rutile: anatase
ratio be considered distinct nanoforms and subject to separate registration. Likewise,
preparations of cerium oxide NMs with substantial differences in their CeO2: Ce2O3 ratios should
be considered separate nanoforms.

102
2533

Method Elemental Size Size Shape Crystallinity SSA Surface Dispersion /

Composition Distribution Charge Aggregation

AFM

Analytical
Centrifugation

BET By conversion

DLS

EDX-EM

(E)SEM

Fl-FFF

ICP-MS / ICP-OES In future

PIDS

SACS Limited

SEC

TEM

XRD

Zeta-potentiometry At Stern layer

2534 Table 3.1 Physicochemical characteristics of NMs and the most appropriate techniques for determining them. Details of the techniques
2535 (including their full names), and their strengths and weaknesses are given in Table 3.2.
2536

103
 Method How does it work?
TEM: Imaging technique. An electron
Transmission beam is transmitted through an
Electron ultra-thin sample, interacting
Microscopy with the sample as it passes.
XRD: X-ray X-rays are passed through the
diffraction material producing a diffraction
pattern. Mineralogical
composition, size and structure
can be inferred.
EDX EM: The wavelengths of diffracted or
Energy reflected electromagnetic
dispersive X-ray radiation from the particle
transmission surface reflects the elemental
electron composition of the sample
microscopy
DLS: Dynamic Based on the ability of particles to
light scattering scatter light in a predictable
manner. Measures the fluctuation
of scattered light and correlates
to diffusion coefficient.
PIDS: Combines polarisation effects
Polarisation with wavelength effects and
Intensity multi-angle measurements to
Differential
Advantages Disadvantages Sample Type Size Range Level of Availability
(nm) Perturbation
Very good resolution achievable. Imaging must be performed in Dry Sample 1 - >1000 High High
Can be coupled with elemental vacuum unless ETEM (see below)
analysis. is available. This can cause (usually)
problems and surface artefacts.
Essentially a single particle
technique. Time consuming to get
representative and robust
quantitative population data.
Both chemical composition and Low sensitivity and poor spatial Dry Powder 0.5 - 500 High Medium
internal structure information resolution cf. TEM (a long time
obtained. Bulk method acquisition or synchtron source
complements TEM. Ambient would improve). Large sample
conditions. size required. Complex data
analysis.
Detects elemental composition Tends to work best for heavy Dry Sample - High Medium
and concentration. Is used in elements. Therefore accuracy is
conjunction with TEM or SEM better when these elements >
Excellent spatial resolution (< 10 0.1% of the sample composition.
nm for TEM-X-EDS) Performed under vacuum.
Quantitative data difficult to
obtain, more routinely used for
qualitative description.
Estimates particle sizes in the Not reliable for polydisperse Suspension 0.6 - 6000 Minimal High
whole dispersion in liquid. Best materials: presence of large
used for indication of particles / aggregates /
aggregation. contamination can bias results.
Poorly suitable for complex
biofluids.
Size and size distribution Less well established than DLS. Suspension 10 - >1000 Minimal Medium
calculated from multi-angle Complex calculation relies heavily Complex media
measurements. Good resolution on propriety software.
104
Scattering lower the sensitivity of scattering.
Fl-FFF: Flow Particle separation technique,
Field relying on differential mobility of
Fractionation particles in an applied field.
SEC: Size Suspensions are separated by
Exclusion differential rates of particle
Chromatography passage through a porous
medium column depending on
particle size.
Analytical Relies on differential
Centrifugation / sedimentation in a sucrose
Differential gradient of different sized
sedimentation particles at varying speeds.
centrifugation. Heavier / denser particles
sediment at lower speeds.
SEM: Scanning Imaging technique. An electron
Electron beam scans across the surface of
Microscopy particles in a raster pattern.
AFM: Atomic An ultra-fine tip is scanned across
Force a surface in a raster pattern.
Microscopy Deflections of the tip are
monitored to generate a height
profile and thereby a 3-D image.
ESEM: Imaging technique that can
Environmental operate in humid atmospheres.
Scanning An electron beam scans across
Electron the surface of particles in a raster
Microscopy pattern.
of polydispersed samples.
Provides separation and sizing. Time consuming and difficult to Suspension 1 - 500 Medium Low
Good size resolution. Non- master. High concentrations Complex media
destructive can be used in usually required. Possible losses
conjunction with other due to sample-membrane
techniques. interactions.
Can give high-resolution Risk of sample loss in the column. Suspension Dependent Medium Medium
molecular weight distributions. Non-size interactions with the on pore
Equipment is widely available. No column and molecular mass size. 0.5-10
low size resolution. Suitable for calibration can be problematic. approx
measurements in situ in complex
biofluids.
Broad range of particle sizes can Good size resolution if Suspension 10 - >1000 Low Medium
be accommodated. Familiar appropriate gradient is used.
technique, apparatus relatively Particle shape, density and
widely available. Suitable for NMs suspension media can all
in situ in complex media influence or confound results.
Very good resolution achievable. As for TEM, generally lower Dry Sample 10 - >1000 High Medium
Can provide information on a spatial resolution.
range of parameters including
size and shape.
Imaging wet/dry, forms a 3D Essentially a single particle Dry and Liquid 0.5 - >1000 Low - Medium Medium
image, very high resolution. Tip technique. Time consuming to get Samples
can be functionalised to give representative and robust
information on surface chemistry. quantitative population data.
Lateral sizing inaccurate.
Samples do not need to withstand Essentially a single particle Humid samples 10 - >1000 Medium Low
high vacuum - therefore imaging technique. Time consuming to get
of wet samples is possible causing representative and robust
less perturbation. Very good quantitative population data.
resolution achievable.
105
 SAED: Selected Diffraction pattern is formed as Very high resolution - reliable Only available for TEM (not SEM). Dry Sample - High Medium
Area Electron the electron beam in a TEM method for crystal determination. High degree of expertise
Diffraction passes through a specimen. The required.
crystal lattice pattern can be
determined.

BET-analysis Measures the adsorption of Simple technique. BET analysers Sample drying can introduce Dry Powder 1 - >1000 High High
(Brunnauer, nitrogen gas to a sample which are commercially available with artefacts.
Emmett and correlates to available surface standard protocols.
Teller method) area.

Zeta- Measures the velocity of particle
potentiometry movement in response to an
applied electrical field. This can
be related to the zeta-potential of
the particle.
Well-established technique.
Apparatus is widely available.
Gives average values for the Suspension 3 - >1000 Minimal High
particles in suspension -
information on charge
heterogeneity is limited. Theory
applies to hard spheres - hard to
calculate zeta potential for soft
particles or aggregated samples.
Measures charge at shear plane
rather than at particle surface.

UV-Vis: Absorbance or transmission of
Ultraviolet- light at ultraviolet or visible
visible wavelengths. The peak or profiles
spectroscopy generated can be used to estimate
concentration of soluble or
colloidal organic substances.
Apparatus is widely available. Signal depends on concentration Suspension - Minimal High
Analysis is non-invasive; samples and extinction coefficients.
may be used for further analyses. Turbidity can interfere with
results

2537 Table 3.2Common analytical techniques for characterising NMs. Compiled from Handy et al., (2008), Hassellv et al., (2008), Powers et al.,
2538 (2006) and Tiede et al., (2009) and expanded based on available new knowledge.
2539

106
2540 Attachment 3.1 Methods to characterise materials as "nano" under the SCENIHR (2010)
2541 definition

2542 The basis of the SCENIHR (SCENIHR, 2010) nanodefinition for regulation is that it should be implementable,
2543 not just for simple NMs test samples, but also for complex samples where only a small portion of the
2544 particles present may be in the nanoscale (1 100 nm). Additional complications include the fact that
2545 detection of several NMs types is still very difficult as many methods are based on chemical identification,
2546 which gives no indication of the form of the chemical (e.g. free as ions or molecules, in a nanoparticlulate
2547 state or as a micro or macro scale particle, depending on the nature of the material in question, and the fact
2548 that it can be very difficult to distinguish between anthropogenic forms of materials (e.g. silica and titania)
2549 which have high background levels, and engineered or intentionally added forms in products. Additionally,
2550 few methods are optimized for studying NMs in complex biological milieu or for aggregated / agglomerated
2551 samples (Lynch, 2010). Thus, this section looks at how the SCENIHR and/or European Commission
2552 definitions of nanomateirals can be implemented from a regulatory perspective. Recalling that the
2553 SCENIHR proposed definition is that a material should be considered as a nanomaterial if 0.15% of its total
2554 particle number is less than 100 nm, coupled with the material having a specific surface area of > 60m2/cm3 .
2555 Thus, characterisation of a material or product as being / not being nanoscale will require both
2556 determination of the fraction of particles of size < 100 nm, and determination of the specific surface area of
2557 the material.

2558 Determination of % of particles < 100nm in a particle sample

2559 Scattering techniques: Dynamic Light Scattering (DLS) /photon correlation spectroscopy (PCS) measures the
2560 random fluctuation of scattered light from a suspension of Brownian particles dispersed in a liquid medium
2561 on a timescale of micro-seconds. Intensity fluctuations arise from the size-dependent thermally induced
2562 motion of the particles and the refractive contrast between the particles and medium. DLS can be combined
2563 with Static Light Scattering experiments which allow the determination of particle volume and radius of
2564 gyration. Comparison with DLS experiments can give valuable information about particle shape. DLS is a
2565 weight-averaged technique, and as such large aggregates dominate over smaller particles in scattering and
2566 are therefore over-represented. This also means that DLS is less suitable for size measurement of
2567 polydisperse samples. Additionally, where more than one peak appears in the distribution function, the
2568 applicability of the correlation function fails, and the zeta average size calculated by the software cannot be
2569 taken directly from the measurements. Instead, the Intensity Statistic report, which calculates the mean
2570 and standard deviations of the data for two or more selected records in the records view, must be used to
2571 interpret the results. In this approach, the size distribution consists of 70 size classes on the X-axis with the
2572 relative percentage of particles present in each size class based upon the intensity of scattered light they
2573 have produced plotted on the Y-axis. Thus, as well as the size of the different entities in the sample, we also
2574 get the proportion of each particle size fraction in the sample this is very important in terms of the
2575 SCENIHR definition described above, whereby a sample with > 0.15% of its particles < 100 nm will be
2576 considered to be nanoscale.

107
2577 The limitation of light scattering techniques is that the solutions must be transparent to light and so this
2578 method works only for dilute cases, although more advanced techniques exist which can determine size in
2579 opaque solutions, such as 3D-DLS and Diffusing Wave Spectroscopy which could potentially be used to track
2580 the evolution of NM dispersions, in simple solutions and in biofluids of increasing complexity.

2581 PIDS (Polarization Intensity Differential Scattering): Laser Diffraction Particle Sizing has been developed as
2582 many of the assumptions of conventional diffraction theory lose their applicability in the sub-micron particle
2583 size region, and as such PIDS, which combines polarisation effects with wavelength effects and multi-angle
2584 measurements is used to achieve increased sensitivity of scattering. Using this approach, detailed size and
2585 size distribution is calculated from multi-angle measurements, which also allows improved resolution of
2586 polydisperse samples. Although it is a less well established technique than DLS in the nanosafety
2587 community, PIDS is being assessed for applicability for routine use in the Pharmaceutical industry for drug
2588 particle analysis, and is under consideration as a potential standard approach by the EMEA for
2589 nanomedicines (Gilbert, 2010), and could potentially also be useful for regulation of all NMs within REACH.
2590 The Electrical Sensing Zone Method of determining size distribution is based on the Coulter principle,
2591 whereby particles suspended in a weak electrolyte solution are drawn through a small aperture, separating
2592 two electrodes that have an electric current flowing between them. The voltage applied across the aperture
2593 creates a sensing zone. As NMs pass through the aperture they displace their own volume of electrolyte,
2594 momentarily increasing the impedance of the aperture. This change in impedance produces a tiny but
2595 proportional current flow into an amplifier that converts the current fluctuation into a voltage pulse large
2596 enough to be measured accurately. The Coulter Principle states that the pulse is directly proportional to the
2597 volumetric size of the particle that produced it. Analyzing these pulses enables a size distribution to be
2598 acquired. In addition, a metering device is used to draw a known volume of the particle suspension through
2599 the aperture, a count of the number of pulses can then yield the concentration of particles in the sample. This
2600 method is the basis of an International Standard for size distribution determination, ISO 13319,
2601 Determination of Particle Size Distributions Electrical Sensing Zone Method.

2602 Small Angle X-Ray Scattering (SAXS) determines the electron density scatter when a well-collimated,
2603 monochromatic beam of hard X-rays passes through a material containing nanometer-range
2604 inhomogeneities. It probes inhomogeneities of the electron density on a length scale of typically 1-100 nm. It
2605 is applicable to crystalline and amorphous materials alike. SAXS data contains information regarding the
2606 size, shape, concentration and spatial arrangement of nanoscale inhomogeneities (or particles) present.
2607 SAXS enables determination of nanoparticle and pore size distributions, of specific surface areas and the
2608 structure analysis of inhomogeneous (e.g. core-shell) particles. The technique may also yield information
2609 with respect to the aggregation/agglomeration behaviour of NMs. Under the condition that all particles
2610 essentially have the same size, it is possible to determine their shape and internal structure.

2611 NanoSight Nanoparticle Tracking Analysis: (Brownian motion of particles) this recently developed
2612 technique analyses particles individually (rather than being an ensemble average), and can resolve
2613 polydisperse samples. Once possible limitation is that it needs very dilute samples, which may not be

108
2614 representative of the true sample used in exposure assessment and safety assessment, but clear advantages
2615 include the fact that it can pick up even very small differences in diameter, such as occur upon binding of
2616 proteins into the nanoparticle corona. As with DLS, the size determined from the diffusion length gives no
2617 information about the shape of the particles.

2618 Analytical Ultra Centrifuge: AUC is a fractionating technique such that a distribution of sizes is determined
2619 with high resolution. In contrast to light scattering that measures only mean values, and in contrast to
2620 TEM/SEM, AUC integrates over 1012 1014 particles, such that statistical relevance even of minor fractions is
2621 high. The method is non-destructive, allowing subsequent analyses on the same sample. There are two
2622 different AUC methods- velocity and equilibrium. Using AUC, a sample can be monitored in real time through
2623 an optical detection system using ultraviolet light absorption and/or interference optical refractive index
2624 sensitive systems. Preparative ultracentrifugation has been used for pelleting of fine particulate fractions,
2625 and for harvesting aquatic colloids and NMs onto TEM and AFM substrates (Tiede, 2008).

2626 Differential Centrifugal Sedimentation: DCS is an extremely high resolution method, relative to light
2627 scattering or particle counting, and allows clean separation of narrow peaks that differ in size by as little as
2628 2%, including in complex biological fluids such as plasma (Walczyk, 2010) although some adjustment of the
2629 calculation parameters due to differences in density between the NMs and the adsorbed biomolecules may
2630 be necessary. NMs with a well-defined and narrow size distribution are used as a calibration of the density
2631 gradient prior to measurement. DCs has very high sensitivity, with a minimum detection limit below 10-8
2632 gram active sample for narrow peaks. Analysis of samples of a few micrograms active weight is routine.
2633 However, as with many of the other techniques, supporting evidence from TEM is recommended.

2634 Flow Field-Flow Fractionation (FlFFF): Flow field-flow fractionation is a separation technique that is capable
2635 of determining particle size distributions of colloidal materials <1m in size with the advantage of being
2636 applicable to environmental samples (Gimbert, 2003, Gimbert, 2007), and thus also complex biological
2637 samples. NMs with a well-defined and narrow size distribution are used to calibrate the FlFFF channel prior
2638 to measurement. The mean polydispersity of NM samples over a range of concentrations can easily and
2639 rapidly be determined using FlFFF. The resulting traces can be assessed in terms of peak area, peak width,
2640 and peak maximum to obtain information about the dispersion quality. However, as with many of the other
2641 techniques, supporting evidence from TEM is required.

2642 Determination of the Specific Surface Area (SSA) of a particle sample

2643 BET surface area: Gas sorption (both adsorption and desorption) at the clean surface of dry solid powders is
2644 the most popular method for determining the surface area of powders as well as the pore size distribution of
2645 porous materials. The name for the experiment comes from the most popular equation used to analyse the
2646 data the BET equation (Brunauer, 1938). However, the equations do not take account of whether this
2647 surface area is available for protein and biomolecule binding (these molecules being significantly larger than
2648 gases). They also do not take appropriate account of particle polydispersity, as highlighted for the SCENIHR-
2649 proposed definition of a NM for regulatory purposes. As the technique can only be performed on powders, it

109
2650 cannot report on the SSA of the particles in situ following dispersion or indeed formulation of the particles
2651 into a product. An additional limitation of the technique is that it does not distinguish fully between porosity
2652 and surface area.

110
2653

Property Oberdrster et al., Thomas et al., Powers et al., Warheit (2008) Klaine et al. (2008)
(2005) (2006) 2006,2007)

Size distribution * ** ** ** **

Agglomeration state/dispersion * * ** ** *

Crystal structure * * * **

Chemical composition * * * **

Surface area and porosity * * ** ** **

Surface chemistry * ** ** *

Surface charge * * ** *

Shape and morphology ** ** *

Dissolution/Solubility * * **

Physicochemical properties ** **
(purity)

Method of synthesis **

2654 Table 3.3 Summary of the properties required to characterisze NMs in media proposed by a range of authors. Taken from Stone et al.,
2655 2010. * = Of Importance, ** = Priority.

111
2656

2657 Figure 3.1 The "nano"length scale (as it is currently defined) in context,
2658 relative to other common objects.

2659
2660

112
 2661

2662

2663

2664

2665

2666

2667 Figure 3.2 Illustration of the exponential increase of surface area as the
2668 cube (particle) size decreases. Thus, a 1 cm3 cube has a surface area of 6
2669 cm3, while the same cube divided into 1000 1 mm3 cubes has a surface area
2670 of 60 cm3, and when the 1cm3 cube is divided into 1 nm3 cubes it has a
2671 surface area of 60,000,000 cm3.

2672

2673

2674

2675 Figure 3.3 Ouput of a typical NM synthesis and size characterisation. A.

2676 Transmission Electron Microscopy (TEM) image of the primary particles,
2677 described as being 30nm in diameter on average. B. Size distribution
2678 profile based on an analysis of a statistical number of particles from the
2679 TEM image (adapted from Grass and Stark, 2006). From this distribution, it
2680 is clear that only a portion of the NPs have the mean size (30nm, marked in
2681 red on the curve). However, from an exposure viewpoint, there is an
2682 additional question regarding how strongly agglomerated (aggregated) the
2683 particles are, and what fraction of them is actually available for interaction
2684 with living systems (i.e. biologically available or bioavailable).

2685

113
2686

2687 Figure 3.4 Important physicochemical properties of manufactured NMs in

2688 aqueous media. Adapted from Hassellv and Kaegi (2009).

2689

114
2690

2691 Figure 3.5 Illustration of the challange in determining the primary particle
2692 size for biological / hazard assessment purposes. Each of the four images
2693 are of the same TiO2 sample, treated with different dispersion protocols in
2694 order to produce the smallest particle size possible. Note that the scale bar
2695 represents 100 nm in image A, and 50nm in images B-D. From the
2696 manufacturers viewpoint, the primary particle size is ~3 nm as illustrated
2697 in D, where several individual particles are marked for illustration
2698 purposes on the TEM Images. Clearly the material in A and B is not likely to
2699 be considered nanoscale, and indeed not likely to be taken up into
2700 organisms or cells it is present form. However, the images in C and D show
2701 that these larger aggregates can be broken down into smaller particle
2702 aggregates using an appropriate dispersion protocol (in this case
2703 sonication and use of an appropriate biologiocal dispersant), and that the
2704 size of these smaller particle aggregates is within the nanoscale, meaning
2705 that they could be taken up into organisms or cells by receptor mediated
2706 processes, and could conceivably produce a biological response or toxicity.
2707 Also, it is important to note that there are no individually dispersed NPs in
2708 the size range ~3nm, and as such this size range is not relevant in terms of
2709 defining the material from a hazard assessment viewpoint. Thus, the
2710 primary particle size should be considered as the biologically available
2711 size, which is about 40-50 nms (images C and D).

2712

115
2713

2714 Figure 3.6 A schematic diagram showing the electrical double layer that
2715 will develop around a charged particle suspended in aqueous media.
2716 shows the electrostatic potential at the particle surface, as measured by
2717 acid-base titrations. shows the zeta-potential as determined by the
2718 electrophoretic mobility of the particle. Adapted from Handy et al.,
2719 (2008b).

2720

116
2721

2722 Figure 3.7 Schematic illustration of proposed multiple upper limits to the
2723 definition of NMs. 'A' shows the current SCENIHR scheme (SCENIHR, 2010),
2724 'B' proposed sub-division of category 3 to reflect the increasing reactivity
2725 of NMs with decreasing size, as recommended in this report. The exact
2726 percentage of the number size distribution of particles <40 nm will
2727 determine whether NMs with a median size >40 nm are classified as
2728 category 3a or 3b.

117
2729 4 Assessment of the potential human toxicity of NMs

2730 REACH relies on the OECD test protocols and the tiered testing approach for assessment of the
2731 human hazard associated with substances. As described in Section 3.2, p.80, this definition of
2732 substance is inadequate to successfully assess the potential impacts of NMs, as it does not
2733 distinguish nano from the bulk form of substance, nor does it distinguish between different
2734 nanoforms. Additionally, this definition does not take account of the fact that NMs typically
2735 possess quite a broad distribution of sizes, which may have quite different hazard or exposure
2736 characteristics. Finally, it also fails to account for NMs interactions with their surroundings,
2737 which confers a biological character onto the NMs. These issues are discussed in this chapter.

2738 4.1 Interactions of NMs with their surroundings

2739 Due to their small size and ability to surround themselves with proteins, NMs acquire a
2740 biological identity, and as such have unique access to, and interaction with, biological machinery
2741 and biological barriers, that is entirely unprecedented. Nanoscale materials with their corona
2742 of adsorbed biomolecules are actively taken up into cells, and transported via established
2743 cellular trafficking pathways, such as the endo-lysosomal pathway (Salvati, 2011) (Shapero,
2744 2011). Thus, neither molecular chemicals, which passively diffuse across cellular membranes
2745 based on their partition coefficients, nor bulk materials which are generally too large to cross
2746 cellular membranes and as such can only induce signalling impacts from the cellular surface (e.g.
2747 implants) can be considered as an appropriate reference paradigm for nanoscale materials. A
2748 recent review (Park, 2010b) suggested that as a result of their interaction with the surrounding
2749 proteins, NMs must be treated as biological entities rather than inorganic ones. This has
2750 profound implications for the definition of NMs, and for their characterisation, which currently
2751 focuses on the pristine nanomaterial, rather than the nanomaterial as it exists in situ during the
2752 biological (hazard) assay. Classifying NMs in contact with living systems as biological entities
2753 may also cast some significant doubt on the applicability of traditional ADME (adsorption,
2754 distribution, metabolism and excretion) processes and approaches to assess biokinetics and
2755 biodistribution, as the uptake steps are no longer diffusion limited, but instead are driven by
2756 receptor-mediated processes which can respond and adapt based on demand or other signals.

2757 Related to the unprecedented access of NMs to living systems, is the fact that NMs inside cells
2758 can potentially activate, deactivate or disregulate a far greater range of signalling pathways than
2759 particles of similar composition but larger size (e.g. medical implants, micron-scale particles)
2760 that are confined to interacting from the cellular membrane. An example of this is that
2761 traditionally, apoptosis (programmed cell death) can be triggered via an extrinsic (from the cell
2762 surface) or an intrinsic (via mitochondrial signalling) pathway. For events starting with a

118
2763 stimulus at the level of the plasma membrane, the activation of caspases 8 and 10 leads to
2764 apoptosis via the so-called extrinsic pathway, whereas if the initial stimulus is at the level of the
2765 mitochondria, caspase 9 will be activated via the intrinsic pathway (Ferri, 2001, Riedl, 2004). As
2766 such, NMs which enter cells and localise in the lysosomes can potentially trigger the intrinsic
2767 apoptosis pathways, which would not be available to larger particles which are confined to the
2768 cell surface. Thus, the access of NMs to cellular machinery opens up new mechanisms of
2769 interaction and potential disruption of cellular function. It is natural to assume that in addition
2770 to utilising existing cellular uptake pathways, NMs can potentially also utilise existing cell exit
2771 pathways, such as those mediated by Rab 11 and Rab 4 proteins (Watson, 2005). However,
2772 extensive studies (Salvati, 2011) have shown no evidence of nanoparticle exit from A549 cells
2773 following uptake of a range of NPs of different compositions (polystyrene, silica etc.) and sizes
2774 (50 nm, 100 nm and 200 nm). Similar studies in other cell types (HeLa, 1321N1 astrocytes etc.)
2775 showed similar effects. In all cases, a decrease of fluorescence intensity was observed that
2776 corresponded with cell division and the splitting of the nanoparticle load between the daughter
2777 cells approximately equally. This behaviour could also be modelled successfully using a
2778 nanoparticle flux based approach and assuming cell division (Salvati, 2011). However,
2779 significantly more work is needed to confirm that in rare cases NMs do not pick up protein
2780 signals that enable them to escape the endo-lysosomal pathway and reach sub-cellular locations
2781 other than the lysosomes27. Preliminary evidence from polarised cells suggests that there are
2782 some mechanism(s) for transcellular transport of NPs, and that there may also be some
2783 recycling of NMs back out of the cells, although this is still in early stages of development(Nic
2784 Ragnaill, 2011). Thus, at present it would appear that NMs present a high risk of being
2785 bioaccumulative and biopersistant, and in some cases, this will lead to toxicity, such as onset of
2786 apoptosis or autophagy (Bexiga, 2010).

2787 Some evidence from carbon nanotubes, fullerenes and QDs studies suggests that NMs
2788 disposition depends on the interaction with the reticuloendothelial (RE) cell system: small
2789 water-soluble particles may be excreted by the kidney, while larger particles with specific
2790 surface charge are targeted to RE in the liver, spleen, other organs. The short half-life in the
2791 blood is reflected in redistribution to tissue but no clearance from the body has been observed,
2792 likely resulting in retention of particles in the body. Unlike small organic drugs, NMs are
2793 preferentially trafficking via the lymphatic system (Riviere, 2009). Kreyling has suggested that
2794 based on the current knowledge on systemic translocation of NMs and their accumulation in
2795 secondary organs and tissues, chronic exposures may lead to elevated accumulations of NPs
2796 resulting eventually in adverse effects (Kreyling, 2010a).

27
Note that this refers to non-functionalised nanoparticles only. There are cases where ligands to target the
nucleus have been shown to have some efficacy, although much is not yet understood in this arena.

119
2797 While there is as yet limited evidence of nano-specific endpoints, the SCENIHR report of 2009
2798 cited evidence that NPs were found to promote the formation of protein fibrils in vitro, (Linse,
2799 2007), which combined with the demonstration of translocation of NPs to the brain following
2800 inhalation, gave rise to concern over possible risks of amyloid diseases (SCENIHR, 2009). Other
2801 types of neurotoxicological mechanism cannot be excluded, as experimental evidence of
2802 impairment of blood-brain-barrier function followed by neurotoxicity was observed after
2803 treatment with metal NMs (Al, Ag and Cu) (Sharma, 2009). Similarly the interaction of NMs with
2804 complex biological matrices induced immunological responses and adverse responses to NMs
2805 (Zolnik, 2010). Thus, there is considerable scope for new endpoints for NMs that may not be
2806 investigated within the current standard testing approaches.

2807 A summary of the specific features of NMs that may necessitate additional considerations for
2808 regulatory assessment is presented in Box 4.1 (see below). These are further developed in the
2809 subsequent sections, with a focus on how each feature affects the existing OECD-based testing
2810 approaches, and what additional steps should be included to account for these features, and to
2811 ensure the quality, reproducibility and scientific rationality of the data produced, which forms
2812 the basis of the subsequent risk assessment.

Combined features of NMs that necessitate additional consideration for hazard

assessment for regulatory purposes:

NMs typically present a size distribution, with very few of the particles in a sample
actually corresponding to the mean or average size. This has significant consequences in
terms of a regulatory definition of nano, and makes any arbitrary cut-off value, such as
the 100 nm size first proposed by the Royal Society of Chemistry, somewhat
questionable from a biological interactions viewpoint. SCENIHR (2010) have suggested
that a definition based on 0.15% of the total particle number being <100 nm would
account for this size distribution effect.

Coupled to this size distribution is a significant batch-to-batch variability, affecting both

the size distribution and the NM surface properties, and leading to potentially quite
different biological consequences. The sources of variability in current manufacturing
processes and how significant these are in terms of product quality assurance and
sameness are not yet fully understood.

NMs operate in the size range where endogenous transport pathways are active, and as
such classical ADME approaches may not be applicable. Thus, NMs can gain access to
living systems across barriers in ways that small molecules or larger particles cannot,
and their small size coupled with their biomolecule corona leads to evasion of the
primary immune system, opening up the potential for new biological impacts.

NMs interact with their surroundings in ways that molecular species cannot, as a result of

120
 their extremely large surface area, and as such should be considered as context
dependent rather than substrate specific, as would be more usual for chemicals.28 Such
interactions can lead to a number of different, sometimes contradictory, outcomes,
including:
- changes in particle size distribution (as a result of either particle aggregation or
improved particle dispersion) and thus the available NM dose;
- changes is NM surface properties, including changes in surface molecule
presentation, changes in zeta potential, changes in surface reactivity etc.;
- conference of a biological identity onto the NMs, such that they are recognised
as protein clusters and are actively transported into cells and organisms, unlike
molecular species, which simply diffuse across barriers, based on their octanol-
water partition coefficients;
- potential for secondary toxicity effects resulting from, for example, media
depletion leading to cell or organism starvation, or for interference with assay
read-outs as a result of adsorption of the reporter molecules to the NM surfaces;
- potential for Trojan horse or co-operativity type effects, where species adsorb to
the NM surface and are transported into organisms they would not normally
interact with, and where the resultant toxicity is not related to the nanoscale, but
is a consequence of it.

NMs change in ways that molecular species do not, including:

- changing their size distribution as a function of time as a result of agglomeration /
disagglomeration;
- dissolution of ionic or elemental species from the NMs, again heightened by the
additional surface area presented by NMs. This means that (eco)toxicity
assessment needs to account for both nanoscale and ionic / elemental forms of the
substance, as well impacts from residual impurities resulting from the synthesis
process;
- renewal or exchange of their biomolecule corona as they are actively transported
from one location to another by organisms (e.g. as they pass through biological
membranes via receptor mediated processes);
- biotransformation of NMs and their biomolecule coating following uptake and
sub-cellular localisation, and impact of this on signalling processes.

This is in addition to the usual processes of metabolism and biotransformation that are
associated with degradation of molecular species, and which form the basis of ADME
models.

NMs need to be considered from 1st principles in a regulatory context, and the basis for
regulation of NMs should be their surface characteristics in addition to the particle size,

28
It is worth noting that aspects of this have been dealt with by regulators before (e.g. the regulation of metals
using the biotic ligand model, and biotechnologies (protein-ligand interactions). Thus, the key factor in terms of
regulation of NMs is the convergence of so many variables into a single system, which is truly unique from the
regulatory perspective. This will be addressed further in section 4.1 below.

121
 shape and composition rather than the chemical nomenclature.

By shifting our mind-set to considering the surface as a determinator of nanosafety and

nanoimpacts, we can account for effects such as opsonisation29 of larger particles while
smaller ones manage to evade the immune system.

This also allows us to consider effects of binding to NMs on the protein or biomolecule
functionality, such as changes in presentation and avidity of ligands which can impact on
their specificity and efficiency (Lynch, 2007) or on their aggregation / fibrillation
tendency (Linse, 2007), and to consider the possibility for alterations in the balance or
functionality of proteins via their local concentration being increased at NM surfaces.

Box 4.1: Unique features of NMs that require consideration for regulatory assessment

2813

2814 4.1.1 Interaction of NMs with inorganic components

2815 The size and size distribution of NMs, and thus the biologically available dose presented to the
2816 test organism, cells, and tissues can be significantly different depending on the nature of the
2817 environment. This is illustrated in Table 4.1, p.148, which shows a comparison of the size of a
2818 series of copolymer NPs in different OECD recommended ecotoxicology assay media (Naha,
2819 2009). The salt concentrations induced some significant particle aggregation, especially for the
2820 most hydrophobic NPs (50:50 NIPAM/BAM) which were aggregated in all of the tested media,
2821 likely as a consequence of the salts in the various test media screening the residual surface
2822 charges on the NPs which normally prevent aggregation. Thus, it is difficult to compare the
2823 particle doses across the different NPs and for one specific NP composition across the different
2824 tests, which have different test media.

2825 The same paper also showed that the zeta potential (effective surface charge) of the copolymer
2826 NPs decreased very significantly upon introduction to the various test media, and the decrease
2827 was especially apparent for the most hydrophobic particles, explaining their significant
2828 aggregation in the test media, as shown in Figure 4.1, p.149. It is therefore proposed that the
2829 zeta potential is screened due to interaction with various components present in media. Notably,
2830 a clear correlation between zeta potential and increasing BAM content (which reflects particle
2831 hydrophobicity) is demonstrated in Figure 4.1, p.149, for all growth media.

29
Opsonization is the process by which a foreign organism or particle becomes covered with opsonin proteins,
thereby making it more visible to phagocytic cells. After opsonization, phagocytosis can occur, which is the
engulfing and eventual destruction or removal of foreign materials from the bloodstream by macrophages.
Together these two processes form the main clearance mechanism for the removal of undesirable components
larger than the renal threshold limit from the blood (Owens and Peppas, 2006).

122
2832 This has quite significant consequences for regulation, and implies some type of complex
2833 modelling will be required (such as the biotic ligand model as described in 4.6, p.142), or that a
2834 re-evaluation of the current testing protocols will be required for NMs, to take account of
2835 different dispersion states under different test conditions and to allow comparison of actual
2836 bioavailable dose of a NM across the different test species.

2837 While the example shown here is from ecotoxicity studies, the consequences of interaction with
2838 the surrounding media is also of great importance for in vitro and and in vivo studies. On account
2839 of the fact that size, size distribution and surface charge all change depending on the
2840 characteristics of the dipersion media, characterisation of NMs under pristine conditions (e.g. as
2841 synthesised) or in pure aqueous solution or simple buffer is not sufficient to understand the
2842 nature of the dispersion that is presented during in vitro or in vivo hazard assessment
2843 experiments. Thus, we suggest that in order to characterise NMs for hazard assessment it is
2844 necessary to characterise them under appropriate conditions, such as in the relevant tissue
2845 culture media. To enable in vitro-in vivo correlations and potentially extrapolation to humans,
2846 the nature of the NMs in the different milieu should be determined to allow comparisons to be
2847 made across systems. Thus, research should be conducted into the general applicability of
2848 commonly used culture media and exposure water, e.g. human plasma in tissue culture media
2849 containing 10% Foetal calf serum (FCS), as well as the comparison with the same NMs dispersed
2850 in NOM, for example. Some guidance on a base set of assay media in which all NMs should be
2851 characterised in order to allow read-across between different assays would be valuable and
2852 would progress our understanding of NM interactions with living systems much more rapidly.

2853 4.1.2 Interaction of NMs with organic components impact on the size and the identity

2854 As highlighted in Section 2.2, p.27 and 3.1, p.76, there are many definitions of NMs, and there is
2855 significant debate as to where the cut-off between NMs and macroscale or bulk materials should
2856 be. One of the first reports on the potential health risks of NMs was the 2005 report from the
2857 Royal Society of Chemistry, which first suggested 100 nm as the cut-off. However, there is no
2858 real scientific basis for this choice of cut-off, from a biological or regulatory viewpoint, as it is not
2859 the case that cellular receptors or nanoparticle-protein interactions abruptly switch off at
2860 101nm. However, this nominal cut-off has become commonly accepted, and indeed ISO are also
2861 using this in their definition also (CEN ISO TS 27687, 2008), and the recent European
2862 Commissions Joint Research Centre publication on establishing the basis of a regulatory
2863 definition of suggested that due to difficulties in assessing other properties, nanoscale particle
2864 should be defined in terms of size only at present, and agreed with the size range 1-100 nm (or
2865 greater) as being the nanoscale (Lvestam, 2010). On the other hand, the argument has been
2866 made that there are intrinsic nanoscale properties which result from the confinement of atoms
2867 and electrons within boundaries of a few nanometres, and that these effects are most dominant

123
2868 at sizes less than about 30 nm (Auffan, 2009). These quantum properties can considerably
2869 change fundamental physical material characteristics like the optical, electrical, and magnetic
2870 properties of the nanomaterial. While this is certainly true for inorganic NMs, arguments based
2871 on this completely disregard the fact that NMs take on a biological identity when in contact
2872 with living systems, as a result of their interaction with biomolecules. Indeed there is a mounting
2873 body of evidence that suggests that even much larger NPs enter cells (Shapero, 2011), most
2874 likely as a result of a biomolecule coating and recognition by cellular receptors as biological
2875 entities. Similarly, it has been suggested, based on calculations of the optimal spatial
2876 arrangement of clathrin molecules, that the optimal size for clathrin-mediated uptake is ~27-
2877 30nm (Gao, 2005). However, experimental evidence with a number of different NMs and cell
2878 types has suggested that even much larger particles can be taken up by non-phagocytotic cells,
2879 including 100 and even 200 nm particles albeit with reduced efficiency relative to smaller (e.g.
2880 50 nm) particles (Shapero, 2011, Salvati, 2011). Additionally, there is no scientific basis for using
2881 changes in a series of physico-chemical properties (optical, electrical, and magnetic properties)
2882 which, of themselves, are unlikely to impact on uptake, distribution, bioaccumulation etc., and as
2883 such are not particularly relevant to defining NMs for hazard assessment and regulation.

2884 From a biological viewpoint, it is generally understood that there are two distinct size regimes
2885 on which uptake of cargo, which is loosely defined as nutrients, fluids, pathogens, and micro-
2886 and NPs, which operate on the scale > 500 nm (phagocytosis) and < 500 nm (endocytosis),
2887 although there is still considerable debate as to the size limitations and the mechanisms for both
2888 processes. Ingestion of particles larger than 500 nm size typically occurs via triggered processes
2889 called 'phagocytosis or macropinocytosis' whereas cargo below this size limit is often
2890 internalized by any of the other diverse endocytic processes available at the cell surface
2891 (Kumari, 2010). These include the well-characterized receptor-mediated clathrin-dependent as
2892 well as the less understood panoply of clathrin-independent 'pinocytic' pathways.

2893 A recent study on the size dependence of phagocytosis has shown that the size-dependence
2894 originates from the attachment step and not internalization (Champion, 2008), suggesting that
2895 the characteristic features of membrane ruffles are responsible for particles size recognition.
2896 This conclusion has several implications. First, as a component of innate immunity, macrophages
2897 must recognize the most generic features of foreign targets. Through an array of phagocytic
2898 surface receptors, macrophages recognize a broad range of foreign molecules on targets.
2899 However, the results presented here show that macrophages recognize an even more generic
2900 feature, target size. This is a remarkable example of biology creating a physical structure
2901 imperative to its function (Champion, 2008). Second, the data show that size recognition is
2902 consistently observed for different macrophage receptors and more surprisingly, for
2903 macrophages from three different tissues and species. Conservation of both recognition ability

124
2904 and the size range of recognition (23 m in diameter) could suggest a deeper role for size
2905 recognition in biology (Champion, 2008). Other work confirms the size dependence: the uptake
2906 of 0.58 m polystyrene microspheres by human blood neutrophils and leukocytes illustrated
2907 that phagocytosis increased with increasing particle size (Simon, 1998). Theoretical work has
2908 also been performed to understand the dependence of phagocytosis on particle size (Chen,
2909 1997). These studies predicted that phagocytosis should increase with particle size for
2910 hydrophobic particles and decrease for hydrophilic particles.

2911 Endocytosis, at a scale smaller than 200 nm, poses specific demands on membrane machinery
2912 which needs to bend membranes at this scale (Sarasij, 2007). The eukaryotic cell appears to
2913 have resolved this in many ways; there are several modes of endocytic processes, distinguished
2914 by specific sets of molecular regulators and functional modules that are associated with their
2915 optimal operation, and which operate at different length-scales. Molecules involved in the
2916 generation of a vesicle can be part of a module assisting coat assembly, or involved in the
2917 pinching process, or may represent specific membrane lipids and lipid-modifying proteins, as
2918 described in a detailed review by (Kumari, 2010).

2919 Whether these specific endocytic modes are conserved in their entirety across phyla, and as
2920 such whether similar modes of NM uptake apply across the spectrum of species is not yet
2921 resolved (Dacks, 2008). In plants, taking into account that the cell wall as a physical barrier to
2922 NM accessibility, and the high intracellular vacuolar pressure that builds up turgor pressure, the
2923 very existence of endocytosis was debated for a long time. Now there is accumulating evidence
2924 about the diversity of endosomal pathways and endocytic compartments in plants (Dacks,
2925 2008). Extensive work has been carried out to understand and elucidate the mechanism of
2926 endocytosis in some eukaryotes, and a considerable amount of information is available in some
2927 free-living eukaryotes and pathogens (Dacks, 2008). Thus, evidence is emerging that these
2928 uptake processes are conserved across a wide range of species, suggesting that the size regimes
2929 of biological action could form a sound scientific basis for development of regulatory regimes.

2930 Thus, defining a nanomaterial as having a cut-off 100 nm (as suggested in many of the current
2931 definitions of nanotechnology and NMs, see Section 2.2, p.27) does not have a sound scientific
2932 rationale from a biological perspective, as there is no evidence as yet that suggests that biology
2933 would distinguish an 80 nm particle from a 120 nm in terms of interaction with the endocytotic
2934 machinery. In fact, from the biological viewpoint, between 200-500 nm would seem a much
2935 more appropriate limit as there does appear to be a shift in mechanism in terms of of how living
2936 organisms interact with particles in this size regime, shifting from endocytotic mechanisms to
2937 phagocytotic ones. From a regulatory viewpoint, as there seems to be some lack of information
2938 about which processes particles in the region 200-500 nm fall under (e.g. are they too big for
2939 endocytosis but too small for phagocytosis, or can they utilise both with lower efficiency?), in the

125
2940 absence of data the prudent option would be to define materials with a fraction of particles of
2941 500 nm of less as being potentially NMs.

2942 The conclusion that NMs in a biological media are remarkably strongly associated to a
2943 biomolecular layer (rich in proteins) drawn from their environment shifts the focus of
2944 discussion from the bare material identity to a new more nuanced conception in which particle
2945 size, shape and corona expression are more likely to be the defining features of nanomaterial
2946 biological identity. A recent study (Walczyk, 2010) has shown that NMs (of varying composition,
2947 including polystyrene with various surface functionalities, and silica dioxide NPs) in biofluid
2948 dispersions typically involve a monomeric nanoparticle core, with a strongly associated and
2949 very slowly exchanging protein hard corona (consistent with one or two packed proteins
2950 layers) co-existing with nanoparticle multimer-corona complexes (dimers, trimers etc.) present
2951 in lower quantities. This is shown in Figure 4.2, p.149, where TEM images and data from
2952 differential sedimentation centrifugation show that particle monomer, dimer and trimer
2953 complexes can coexist, maintaining their primary structural properties over many hours,
2954 sometimes slowly reorganizing into different complexes. There are also more rare large particle-
2955 protein complex assemblies which, for one example (sulphonated polystyrene NPs), actually
2956 dominate the dispersion, and in this case it appears that the NPs are essentially subsumed into
2957 existing protein clusters.

2958 The practical implications are that it is possible to study these complexes in isolation, beginning
2959 with their layer composition, size and zeta potential, and studies of molecular corona identity,
2960 already somewhat advanced, will now become of much higher quality as we learn to work with
2961 them in isolation, as well as in situ. The fact that particle-hard corona complexes of several
2962 materials have low zeta potentials suggests a different dispersion stabilization mechanism from
2963 that (such as charge and steric) typical for bare NMs. Likely their stability is conferred by the
2964 specific protein layer characteristics, reminiscent of the design feat by which thousands of
2965 different proteins in plasma are colloidally stable despite their crowded environment.

2966 The presence of particle-protein multimers and the larger complexes will be of high significance,
2967 and (as a specific example) nanomedicine cannot be expected to advance to application until the
2968 immunological consequences of these different co-existing aggregated species are understood
2969 for relevant particles. Furthermore, these same issues may be relevant to NMs with surfaces
2970 modified by antibodies and proteins, for these particles will in general acquire a new molecular
2971 corona in vivo. The consequences are far reaching, suggesting that NMs dispersions for biological
2972 studies must be treated as a single system in which the choice of serum and plasma (and
2973 implicitly thereby, the test organism), and its preparation, may have a significant bearing on the
2974 outcome of experiments. In particular the use of animal (rather than human) derived media
2975 should be re-considered for some applications.

126
2976 The fact that NMs can take on quite different dispersion characteristics in biofluids also has very
2977 significant consequences for how the physico-chemical properties are determined for hazard
2978 assessment purposes, and in particular for determination of the bioavailable dose of NMs. Thus,
2979 a full characterisation of the pristine NMs, or the NMs dispersed in aqueous solution or simple
2980 buffer is not sufficient in order to understand the nature of the dispersion as presented in
2981 hazard assessment assays. Instead, full characterisation of the size, size distribution, zeta
2982 potential etc. in the assay media to be used in the test, and over the duration of the test is
2983 required to fully characterise the nature of the system as presented in the biological assay. The
2984 dispersion characteristics over the time course of exposure in a biological assay can change, and
2985 as such characterisation before, during and after the exposure is recommended, or in the
2986 absence of this, characterisation of the particles under the exposure conditions at several time-
2987 points to provide an informal reference state for the system. In the future, it is likely that
2988 characterisation of the nanoparticle biomolecule corona under the starting exposure conditions
2989 will also be required, including TEM images, DCS curves and proteomic data (1D PAGE gels and /
2990 or mass spectrometric identification / quantification of the nature of the proteins in the corona).

2991 Related to the dynamic size distribution and agglomeration issues that are introduced by the
2992 interaction of NMs with biofluids, is the tendency of NMs to aggregate at higher concentrations,
2993 which can also contribute to non-linear dose-response effects, whereby lower concentrations of
2994 NMs may appear to be less toxic / bioactive than higher doses, when in fact the higher doses
2995 actually have significantly fewer bioavailable NMs due to aggregation of the particles into much
2996 larger clusters which do not interact with living systems in the same way. This also confirms the
2997 need for characterisation data at several time-points corresponding to the duration of the
2998 biological assay or experiment in order to begin to correlate observed impacts with the
2999 dispersion state of the test system. However, one should not completely rule out the potential
3000 effects of agglomerated / aggregated NMs, as these can induce other effects, for example,
3001 adsorption of nutrients leading to starvation or other secondary toxicity effects (Wrle-Knirsch,
3002 2006, Casey, 2008). Thus, there are several potential confounding factors in interpretation of
3003 hazard assessment data from NMs.

3004 4.1.3 Interaction with organic components impact on biological processes

3005 In a typical biological system, limited numbers and types of biomolecules compete for the
3006 nanoparticle surface, the resulting biomolecule corona contains only a few (mostly identifiable)
3007 proteins (Cedervall, 2007a, Cedervall, 2007b, Lundqvist, 2008). Thus, what the cell actually
3008 sees during nanoparticle uptake into the cell, or other biological processes, is the layer of
3009 adsorbed proteins and other biomolecules, and not the underlying chemistry of the
3010 nanomaterial, although this of course influences which of the myriad proteins or other
3011 biomolecules adsorb to the nanomaterial surface(Lynch, 2008, Lundqvist, 2008). For example,

127
3012 there are over 3700 proteins in plasma, and of these, some tens of proteins have been identified
3013 as binding to a range of different NMs. The consequence of this is that in order to correlate the
3014 nature of the nanoparticle protein/biomolecule corona with the nanoparticle fate and
3015 behaviour, and in particular with the biological impacts and potential hazards, we need to know
3016 if those bound proteins stay there for long enough to be biologically significant. For those with
3017 sufficiently slow exchange kinetics, the corona itself constitutes the primary contact to the cells,
3018 and thus, we need to begin to think about NMs in a biological context as biological entities which
3019 are recognized and processed by organs and cells as such. This point has been highlighted in a
3020 recent review, which suggested that NMs must be treated as biological entities rather than
3021 inorganic ones (Park, 2010b).

3022 As a consequence of their large surface area, NMs can be envisaged as scaffolds for protein
3023 adsorption, making the NMs themselves biological entities that are recognised by the body as
3024 protein complexes. This is illustrated in Figure 4.3, p.150, where a comparison is made between
3025 NMs coated with a biomolecule corona of adsorbed proteins, lipids and other biomolecules,
3026 and the nano-scale protein-lipid clusters that are involved in cholesterol transport in the body.

3027 Significant efforts have been made to determine how long the molecular corona remains
3028 associated with different NMs (their residence time) in order to determine whether or not
3029 biological entities ever see the naked NMs under normal exposure / application circumstances.
3030 From these studies it appears that for all tested NMs, the corona forms within minutes to one
3031 hour, and is stable beyond that for periods of up to 24 hours. The in situ corona and the corona
3032 of the particles recovered from the biofluid are remarkably similar, again confirming the
3033 stability of the hard biomolecule corona, once formed (Walczyk, 2010). Lipids and other
3034 biomolecules are also contained within the corona (Hellstrand, 2009).

3035 The consequences of this biomolecule corona is that NMs are recognised by living systems as
3036 biological entities, and as such are processed and trafficked in much the same manner as
3037 biomolecules by the cellular machinery. Thus, NM access to cells should not be confused with the
3038 more common behaviour of small organic molecules such as drugs, which broadly speaking,
3039 partition into organs and cellular compartments according to near-equilibrium physiochemical
3040 principles. In that case, the small molecule structure determines both the bio-distribution and
3041 the functional impact of the molecule. For NMs on the other hand, processing by endogenous
3042 cellular machinery means that NMs are actively taken up by cells, via receptor-mediated
3043 processes, and once taken up are directed within the cell based on existing transport pathways
3044 such as teh endo-lysosomal pathway, allowing for final intracellular and organ level
3045 distributions that are far from equilibrium. This has dramatic consequences for assessment of
3046 uptake and biodistribution of NMs, which will have very different toxicokinetic profiles that
3047 conventional chemicals, and calls into question the relevance of existing assessment approaches

128
3048 based on evaluation of the rates of administration, kinetics of absorption, distribution and
3049 excretion (ADME), much of which is equilibrium based.

3050 As a direct consequence of the active uptake and processing of NMs by cells, the final organ,
3051 cellular and sub-cellular localisation of NMs is dramatically different than that of conventional
3052 chemicals, which partition across cellular membranes based on their hydrophilicity /
3053 hydrophobicity and whose ability to partition can be determined based on simple
3054 measurements of octanol-water partition coefficients (for example). This is also true of passage
3055 across the cellular barriers, such as the blood-brain and blood-foetus barriers which have
3056 evolved to protect delicate organs from unwanted toxicants. Despite years of investment in the
3057 development of drugs to penetrate the blood-brain barrier, very few successes have been
3058 achieved, whereas already several examples of NMs reaching the brain have been reported in
3059 the literature, often with direct involvement of known brain-transporting proteins, such as
3060 albumin or apolipoprotein E (Michaelis, 2006). The outcome of NMs being recognised as
3061 biological entities is that their uptake kinetics and biodistribution must be described based on
3062 energy-dependent fluxes of NMs and that most NMs end up almost entirely in the lysosomes, as
3063 they follow the endo-lysosomal pathway (Shapero, 2011). This can be contrasted sharply with
3064 the uptake and sub-cellular distribution of conventional chemicals, represented for the purpose
3065 of this report by a fluorescent molecular dye, where the dye is very rapidly distributed across
3066 the enter cell, including in the cytoplasm, as shown in Figure 4.4, p.150. A first attempt to
3067 theoretically describe the fluxes of NMs into cells and to model nanoparticle uptake and
3068 distribution has recently been reported, which captures some of these ideas (Salvati, 2011).

3069 Another consequence of NMs interacting with living systems via active processes, which has
3070 deep significance for regulation of NMs, is that once taken up into cells, NMs do not easily exit
3071 cells. This is most likely a consequence of the fact that their biomolecule coronas do not
3072 naturally contain the necessary protein signalling molecules to activate the exit pathways. Thus,
3073 as described above, all NMs we have tested utilise the endo-lysosomal pathway and thus end up
3074 predominantly in the lysosomes. In several cell lines we have not observed any exit, even where
3075 the particle source is removed and the cells are observed for up to 24 hours, in contrast to
3076 molecular dyes, which diffuse out of cells within minutes once the equilibrium is shifted by
3077 reducing the concentration outside the cells (Salvati, 2011). The decrease of fluorescence
3078 observed at 24 hours in these experiments was confirmed experimentally and theoretically to be
3079 associated with cell division, and the splitting of the nanoparticle load approximately equally
3080 between the daughter cells (Salvati, 2011). Thus, there is very significant potential for
3081 bioaccumulation of NMs in cells, and in particular in the lysosomes, which have been shown to
3082 become very packed with NMs.

129
3083 In addition to the current discussion in the literature regarding nanoparticle exit from cells,
3084 there is also some debate regarding the potential for degradation and/or biotransformation of
3085 NMs in situ in cells, animals or plants. For example, a recent paper has shown that the enzyme
3086 myloperoxidase can induce degradation of carbon nanotubes in cells and in vivo (Kagan et al.,
3087 2010). Another example is the investigation of the biotransformation of Ni(OH)2 NPs by
3088 mesquite plants (Prosopis sp.) where significant differences between coated and uncoated
3089 Ni(OH)2 NPs were observed (Parsons et al., 2010). Likely this will be an arena of increasing
3090 research activity in the near future, as understanding and characterising such changes in the
3091 structure and composition of NMs following their interaction with living systems will be
3092 essential for a complete hazard assessment of different NMs.

3093 A recent review of nanoparticle-protein interactions (Lynch et al., 2008) showed that some
3094 changes in protein conformation may occur upon binding to NMs, depending on the nature of
3095 the interaction, the composition of the NMs, and the stability of the protein in question. Often,
3096 such a change in conformation would be expected to have a negative impact on the protein
3097 activity or functionality, although there are some quite promising uses of such NMs-induced
3098 changes in confirmation /activity, including increasing protein stability towards degradation by
3099 enzymes, and increasing the activity of enzymes via immobilization at surfaces.

3100 Examples of the effects that have been reported to date include: improved activity and
3101 selectivity /enantioselectivity and pH and thermal stability of enzymes such as Candida rugosa
3102 lipase and Pseudomonas cepacia lipase when adsorbed onto nanostructured polystyrene and
3103 polymethylmethacrylate NPs (Palocci, 2007); the half-life of a model enzyme, soybean
3104 peroxidase, adsorbed onto fullerenes at 95 C was ca. 2.5-fold higher than that of the enzyme
3105 adsorbed onto graphite flakes and ca. 13-fold higher than that of the native enzyme (Asuri,
3106 2007); adsorption and subsequent desorption of bovine serum albumin (BSA) from polystyrene
3107 particles caused irreversible changes to the stability and secondary structure of BSA, with the
3108 exchanged proteins having a higher denaturation temperature and a lower enthalpy of
3109 denaturation than native BSA (Norde, 2000).

3110 Since our first report of a NM-induced modulation of the rate of protein fibrillation (polymeric
3111 NPs accelerated the rate of fibrillation of -2-microglobulin (Linse, 2007)) there have been
3112 multiple reports of different NM, including TiO2, gold, QDs, polystyrene, dendrimers and
3113 maghemite NPs, modulating the rate of fibrillation of a range of proteins, including amyloid beta,
3114 alpha-synuclein or islet amyloid polypeptide (Cabaleiro-Lago, 2008, Cabaleiro-Lago, 2010, Wu,
3115 2008, Skaat, 2009, Xiao, 2010). In all cases, the effect is surface modulated, and it appears that
3116 there is more than one mechanism, as some particle-protein pairs result in acceleration of the
3117 protein fibrillation rate, whereas others actually slow (or even reverse) the rate of protein
3118 fibrillation. Depending on the relative affinity of specific NMs for protein monomers, unfolded

130
3119 monomers, oligomers, critical nuclei and other prefibrillar states, the influence of NMs on
3120 protein fibrillation kinetics is likely to be protein dependent (Cabaleiro-Lago, 2008). Quite
3121 different effects may also be anticipated if the protein species are bound in a similar
3122 conformation as in solution or if the binding process alters the conformation of the protein or
3123 causes local or global unfolding. Thus, to further our understanding of protein fibrillation
3124 processes and how these are perturbed by the presence of NMs, it is necessary to collect large
3125 amounts of well-controlled data in a protein and particle dependent manner. However, to date,
3126 the majority of studies have been performed in solution, rather than under more realistic,
3127 competitive-binding conditions, such as would occur in situ in the body. One report of the use of
3128 QDs as ultrasensitive nanoactuators and sensors of amyloid aggregation in live cells has been
3129 published (Roberti, 2009).

3130 The long-term consequences of such changes in protein conformation and/or function and
3131 activity following interaction with NMs are not yet fully understood. However, work is
3132 underway to determine NM interactomes, or the full set of protein-protein interactions that
3133 are affected by interaction with a specific NM, and whether any conformational changes induced
3134 by interaction with the NMs are reversible or irreversible. Such interactomes could be the basis
3135 of future predictive or quantitative structure-activity type relationships (QSARs).

3136 4.1.4 Interaction with organic components In vitro in vivo data extrapolation

3137 An important finding regarding the NM biomolecule corona is that it is concentration (ration of
3138 protein concentration to available NM surface area) dependent for some NPs (Monopoli, 2011).
3139 Thus, the hard corona can evolve quite significantly as one passes from protein concentrations
3140 appropriate to in vitro cell studies to those present in in vivo studies, as shown in Figure 4.5,
3141 p.151, which has deep implications for in vitro-in vivo extrapolations and will require some
3142 consideration in future. Thus, it is a quite general observation that binding leads to relatively
3143 complete surface coverage for even low plasma concentrations. The protein concentration study
3144 also suggests a progressive displacement of proteins with lower affinity in favor of those with
3145 higher (Monopoli, 2011).

3146 Whilst the nature and formation of the NM biomolecular corona is at least developing some
3147 conceptual framework, with experimental methods growing capable of handling the challenge,
3148 the theoretical challenge of understanding why certain proteins are adsorbed in a competitive
3149 manner is as yet unclear. Certainly there are many hints that this is a collective process, and thus
3150 it will be difficult to rationalize on the basis of individual protein binding studies. Thus, whilst
3151 there is growing certainty that the corona is what is seen by the cell, there is as yet relatively
3152 little progress on why a NM chooses those particular proteins from the available set.

131
3153 Turning to some more quantitative issues, in those cases where the system is relatively simple,
3154 one can really begin to frame a study of the adsorption isotherm (by analogy with a long
3155 tradition in physical chemistry) in which the surface coverage can be broken up into its
3156 constituent elements in a relatively complete manner. By applying methods of semi-quantitative
3157 Mass Spectrometry (MS) we can even create the adsorption isotherms of the different
3158 components of the adsorbed layer, and relate the amounts bound from MS to those found from
3159 structural studies. However, there are significant differences compared to the more usual forms
3160 of adsorption, including the fact that, when formed, the protein layer is essentially irreversible
3161 on the time scales of the experiments carried out to assess its structure. We interpret this to
3162 mean that the system seeks to lower its (initially high) surface energy by selecting and
3163 exchanging on shorter timescales from the whole set of proteins that diffuse to the surface.

3164 4.2 Stability and changes of NMs

3165 Dissolution of a range of different engineered NMs, including metal oxides and metals, has been
3166 reported in the literature, as a major contribution to the difficulty of interpreting the toxicity of
3167 NMs, in particular for silver and ZnO NPs. With silver NPs, much of the biological (anti-bacterial
3168 / anti-microbial) effects are not surface effects directly, but are rather an indirect result from
3169 long term release of silver ions (Ag+) by oxidation of metallic silver (Ag0) in contact with water
3170 (Kumar, 2005). A detailed investigation of the effect of storage conditions on the observed
3171 toxicity of citrate-stabilized and poly(vinylpyrrolidone)-stabilized silver NPs in water showed
3172 that the particles were found to slowly dissolve into ions on a time scale of several days, with the
3173 NPs releasing up to 90% of their weight in some cases (Kittler, 2010). The release of Ag+ ions
3174 led to a considerably increased toxicity of the silver NPs which had been stored in dispersion for
3175 several weeks toward human mesenchymal stem cells due to the increased concentration of Ag+
3176 ions. Consequently, aged (i.e., immersed) silver NPs are much more toxic to cells than freshly
3177 prepared silver NPs (Kittler, 2010). A risk assessment by Blaser et al. (Blaser, 2008) using
3178 nanosilver measured classical heavy metal toxicity, via the Ag+ ions, and this may indeed be
3179 relevant for assessment of the toxicity of other heavy metal NPs such as QDs, which are also
3180 known to leach metal ions.

3181 Another aspect that may need consideration is the fact that NMs in formulations or consumer
3182 products will be exposed to a range of storage and use conditions. For example, NMs in food
3183 products may be stored at low temperatures and then heated for consumption. This may also
3184 affect both the NM stability within the food, as well as changing the properties of the
3185 biomolecules in the food, and potentially how these interact with the NMs. To date there is no
3186 literature dealing with this.

132
3187 4.3 Polymeric NNs
3188 Historically, polymers are exempt from REACH regulation, meaning that nanoscale polymeric
3189 materials are also automatically exempt, as the application of REACH regulation to nanoscale
3190 substances is only applicable to materials that fall within the scope of REACH.

3191 Given the discussion presented above, whereby nanoscale materials should be considered as
3192 biological entities, rather than as chemicals, there is no scientific basis or rationale on which
3193 polymeric NMs should be exempt from regulation, as they would not be expected to behave any
3194 differently than other nanoscale particles with regards to interaction with their surroundings to
3195 form a biomolecular corona, and as such, are also recognised by living systems as biological
3196 entities. Indeed, there is significant evidence emerging from ongoing research that polymeric
3197 NMs with cationic charges may be toxic, resulting in apoptosis in cells, whereas negatively
3198 charged NMs of identical composition and size are not (Xia, 2008, Bexiga, 2010). Indeed, studies
3199 of the nanoparticle molecular corona for polystyrene NPs have shown that small changes in the
3200 surface charge of polystyrene NPs can result in significant alternations in the molecular corona,
3201 including binding of quite different proteins into their corona, with implications for uptake and
3202 trafficking. In Table 4.2, p.148, the effect of surface charge and size for the selectivity of the
3203 apolipoprotein family of proteins is shown, and similar selectivity for a range of other protein
3204 families has also been shown (Lundqvist, 2008). Similar differential specificities were observed
3205 for complement pathway proteins, coagulation factors and acute phase proteins, suggesting that
3206 the differential biological impacts of the cationic polystyrene NPs relative to the anionic ones is
3207 related to their surface presentation of proteins, i.e. their biological identity.

3208 The images in Figure 4.4b, p.150, also indicate that polymeric NPs enter cells, localise to the
3209 lysosomes, and do not show any exit potential, thereby suggesting the polymeric NPs may also
3210 bioaccumulate in living systems. Thus, we strongly advise that the current exemptions for
3211 polymeric NMs be reversed, and where a polymeric material contains nanoscale particles, these
3212 should be evaluated in the same manner as all other NMs. Many of the commercial uses of
3213 polymeric NMs will include composite materials composed of NMs dispersed into polymer
3214 matrices, to provide enhanced strength, or for food packaging materials, where for example
3215 nanoclay is used to improve the moisture adsorption of the packaging materials and to reduce
3216 the spoilage of food by oxidation (Chaudhry, 2008). It is believed that NMs contained within
3217 such matrices (composites) will have limited mobility out of the matrix, and are therefore not
3218 likely to pose an exposure or health hazard, although there are still issues related to end-of-life-
3219 cycle and disposal of those composite materials. Another potential use of polymeric NMs is for
3220 controlled drug delivery, although such devices will fall within the remit of the European
3221 Medicines Agency (EMEA) rather than the European Chemicals Agency (ECHA) and REACH

133
3222 regulation. However, it is likely that the EMEA will converge with REACH on many aspects of the
3223 regulation of NMs.

3224 4.4 Occupational and consumer exposure to NMs

3225 The basis of risk assessment is that risk = hazard x exposure, so that if there is no exposure, then
3226 there is no risk even from very hazardous materials. Thus, we have learned to manage our use of
3227 electricity, by limiting exposure, and quality of life in the absence of electricity is almost
3228 unimaginable for the majority of the worlds population. Since nanotechnologies are considered
3229 to be an enabling technology with potential applications and impacts in every technology and
3230 industry sector, managing exposure to NMs will also become a priority for regulators.

3231 There is quite an emerging literature on the topics of occupational and consumer exposure to
3232 NMs, but rather than attempting to summarise that here, which is beyond the scope of the
3233 report, we rather focus on extracting key aspects from two comprehensive reports on these
3234 topics that are of relevance for our purpose of assessing whether current hazard and risk
3235 assessment approaches are suitable for NMs.

3236 Although nanotechnology is a relatively new branch of industry, numerous applications have
3237 already been developed across a range of industry sectors. According to the report
3238 commissioned by the European Agency for Safety and Health at Work on worker exposure to
3239 NMs (Kaluza, 2009), the investment and exposure of workers to related risks in this
3240 technology is concentrated in following fields:

3241 Construction in products improving wear-resistance and rigidity, but also used in
3242 pigments; windows, more efficient insulation materials;
3243 Health care in new drugs and active agents, drug-delivery systems, oral vaccines, tissue
3244 engineering and production of biocompatible materials;
3245 Energy conversion and use increasing efficiency of energy conversion and low-wastage
3246 storage of energy, including new generation photovoltaic cells, more economical lighting,
3247 compact combustion cells;
3248 Automobile (and aerospace) industry reinforced and stronger materials, sensors
3249 optimising engine use, fuel additives, scratch-resistant, and dirt-repellent coatings;
3250 Chemical industry catalysts, adhesiveless bonding techniques, multi-functional and more
3251 efficient ceramics, products used for surface functionalisation and finishing, such as
3252 pigments, corrosion-inhibitors, self-cleaning surfaces, anti-static, functional layers such as
3253 thermal insulation, but also extremely hard and resistant cutting tools;
3254 Electronics and communication optical/optoelectronic components including lasers, high
3255 density memories, pocket electronic libraries, ultra fast compact computers.

134
3256 This list is not all-inclusive. Other areas where nanotechnologies are utilised include
3257 environmental applications, the textile industry, manufacturing of instruments and tools, and
3258 defence. The list of application will grow with the development of nanotechnology.

3259 Sales of products incorporating nanotechnology have been estimated to rise from less than 0.1%
3260 of global manufacturing output in 2006, to 15% in 2014, totalling $2.6 trillion. This is already
3261 being reflected in the number of products available on the market containing (or claiming to
3262 contain) NMs: an analysis of the Woodrow Wilson database on August 21 2008 (containing 803
3263 products), was compared with an analysis of the database on November 26, 2006 (containing
3264 356 products) (database available on www.nanotechproject.org) (Wijinhoven, 2009). It is clear
3265 that as applications and uses of nanotechnology increase, so too will exposure to NMs, both of
3266 workers involved in the manufacture of nano-containing products, and consumers.

3267 In 2009, the Dutch Food and Consumer Product Safety Authority commissioned RIVM (the
3268 National Institute for Public Health & the Environment) to write a report on potential consumer
3269 exposure to NMs in the Netherlands. As part of this process, RIVM undertook an evaluation of
3270 existing nanoproduct inventories and market reports on the nanotechnology industry and
3271 coupled this with an expert consultation in order to perform an analysis of market value, the
3272 amount of NMs currently in existence in consumer products, and the potential exposure of
3273 consumers to NMs (Wijnhoven, 2009b). Attachment 4.1, p.152, contains a table from the RIVM
3274 report showing the estimated relative presence of NMs used in consumer products on the global
3275 market (%) in the near future based on the total amount (tonnes) of NMs used in consumer
3276 products. The expert consultation made a semi-quantitative evaluation of the possible exposure
3277 from the various product categories, without extensive information on the exposure categories.
3278 Despite the lack of information on some characteristics of some products, most experts were
3279 able to categorise most products into high, medium or low possible exposure.

3280 The expert consultation resulted in the conclusion that personal care products and cosmetics
3281 (including sun cosmetics, oral hygiene products, supplements and health products) were
3282 expected by most experts (all but 1) to lead to the highest possible exposure. Fuels for motor
3283 vehicles (after combustion) and do it yourself coatings, adhesives and cleaning products were
3284 expected to lead to high possible exposure by fewer experts.

3285 The general requirements in relation to occupational safety and health of workers at workplaces
3286 are presented in the EU Directive 89/391/EEC. The aim of this framework directive is to ensure
3287 a high level of protection of workers at work including those exposed to NMs - through the
3288 implementation of preventive measures to guard against exposure to risks, and through
3289 provision of information, consultation, balanced participation and training of workers and their
3290 representatives.

135
3291 The European Agency for Safety and Health at Work (EU-OSHA) report indicates that official
3292 occupational exposure limits for NMs have been set only for amorphous silicon dioxide (e.g. in
3293 Germany: (Greim, 1989)). A draft occupational exposure limit for nanoscale titanium dioxide is
3294 also available from the US-American National Institute for Occupational Safety and Health
3295 (NIOSH, 2005). Furthermore, so called benchmark levels that represent a pragmatic guidance
3296 level have been developed for four classes of NMs by the British Standard Institution (BSI,
3297 2007a). For insoluble NMs a general benchmark level of 0.066 occupational exposure limit
3298 (OEL) of the corresponding microsized bulk material (expressed as mass concentration) is
3299 proposed. This factor of 0.066 is in line with the potency difference of microscale and nanoscale
3300 titanium dioxide as described by the US-American National Institute for Occupational Safety and
3301 Health (NIOSH, 2005). For fibrous NMs a benchmark level of 0.01 fibres/ml is proposed. This
3302 level is derived from the current limit value for asbestos removal activities in the UK. For highly
3303 soluble NMs a benchmark of 0.5 OEL is proposed. For CMAR (carcinogenetic, mutagenic,
3304 asthmagenic or reprotoxic) NMs a benchmark level of 0.1 OEL of the corresponding microsized
3305 bulk material (expressed as mass concentration) is suggested.

3306 Studies on the exposure risks from manufacture of NMs have shown that overall, the risk
3307 associated with most NM production processes are comparable to or lower than those of
3308 common non-nano processes (Roberti, 2009), as shown in Figure 4.6, p.151, and discussed by
3309 Wilson in his article on regulation of NMs. Wine production is comparable or slightly more
3310 dangerous than the synthesis of alumoxane particles and single-walled nanotubes. The
3311 production of QDs and nano-titanium dioxide is slightly more dangerous, but still on par with
3312 the manufacturing of silicon wafers, car batteries, aspirin, and petroleum refining. Buckyball
3313 synthesis, the highest risk nanoprocess, did not exceed the danger presented by polyolefin
3314 production. The assessment of normal operations risk presented similar results: polyolefin
3315 production and petroleum refining create a greater threat than all of the NM processes (Watson,
3316 2005).

3317 The EU-OSHA report did identify cleaning operations in laboratories or drilling and cutting
3318 operations of carbon nanotube-doped concrete as potential sources for exposure (Schneider,
3319 2007), and indeed NIOSH has observed that operators do not recognise cleaning processes as a
3320 source of hazard / exposure (Hoover, 2010). However, official data on the number of workers
3321 exposed to NMs are not available.

3322 The nanoproduct register proposed in Chapter 2 offers real potential for generation of exposure
3323 data regarding nanoproducts, based on actual amounts of NMs in products, and the likely uses of
3324 these products. The second phase of the registry proposes collection of hazard data to
3325 complement the exposure data and enable a full risk assessment to be performed. The register
3326 would also provide data to support the classification of widespread NMs as substances that have

136
3327 dispersive or diffuse consumer use, which could provide a route to prioritising NMs for complete
3328 hazard assessment also, in the absence of a priori information regarding their potential for
3329 bioaccumulation, biodegradation or for toxicity.

3330 4.5 Relating NMs unique properties to risk assessment and regulation
3331 The implications of NMs as biological entities for REACH are complex, as it suggests that more
3332 than just the material or chemical description is needed to categorise materials, but also details
3333 on their surface charge, interactions with their environment and their biological identity. The
3334 current REACH guidelines for Physical-Chemical Properties and Material Characterization
3335 (which were not designed with NMs in mind) do not cover interactions with surroundings or
3336 characterization in situ and also do not specify that NMs should (in addition) be characterized in
3337 the form in which they will be presented to the test (i.e. under the in situ conditions, be that in
3338 cell culture media, in river water etc.

3339 A summary of the main differences between conventional chemicals and NMs as they relate to
3340 risk assessment under REACH is presented in Box 4. 2 (see below).

Property Molecular species NMs

Interactions with Limited Extensive take on a biological

surroundings identity

Cellular uptake mechanism Diffusion Actively transported via energy-

dependent, receptor-mediated processes

Cellular exit processes Diffusion None identified as yet

- cell division splits nanoparticle load
equally between daughter cells;
Potential for specific exit signals to be
added to particle surfaces

Potential for evolution Metabolism Dissolution

Aggregation
Metabolism?
Biotransformation?
Degradation
Many of these changes not yet well
understood

Mechanisms of action Mostly known Unknown as yet it is unlikely that NMs,

which engage with the cellular
machinery would have similar

137
 mechanisms of action as chemicals.
Thus, a critical gap at present is the lack
of validated positive and negative
control NPs for a range of biological
endpoints.

Box 4. 2: Comparison between the properties of conventional chemicals and NMs as they relate
to risk assessment

3341 A basic premise in considering the hazard profile of a particular material in nanoscale form is
3342 that it will have at least similar hazards to the same material in a micro or macro form (material
3343 toxicity), although this may not be the case when comparing the hazard of a nanoform of a
3344 chemical with the free form (chemical toxicity including that of ionic species that dissolve from
3345 the NM, e.g. in the case of nanosilver). Thus, if the corresponding micro or macro material has an
3346 innocuous toxicological profile due to biological inertness and biodegradability, the current
3347 approach is to assume that the nanoform may similarly be predicted to be of low hazard.
3348 However, given the increased accessibility of NMs versus their bulk forms, and the fact that they
3349 acquire a biological identity as described in Section 4.1.2-3, p.123, above, this can no longer be
3350 assumed for NMs.

3351 On the other hand, inherently toxic materials such as particulate metal compounds can be
3352 anticipated to show similar or greater toxicity if produced in a nanoform from which they can
3353 solubilise, although evidence for some materials is that the nanoformulation is less toxic than
3354 the chemical itself (e.g. selenium NPs are less toxic to rats than selenite or high-selenium
3355 proteins (Jia, 2005)). A body of evidence built up in in silico and/or in vitro models supports this
3356 premise and additionally indicates that many NMs show greater reactivity in such models
3357 (Meng, 2007, Papageorgiou, 2007). However, in the interpretation of nanosafety, it is important
3358 to consider what the reference state should be the elemental chemical and/or the macroscopic
3359 state of the material.

3360 An alternative route to toxicity from NMs is the elution of residual impurities such as catalysts
3361 from the synthesis process, which may not be removed by clearing processes, but which can
3362 gradually elute during biological exposure. The metal catalyst content in CNTs should also be
3363 considered when the toxicity of carbon nanotubes is investigated (Wang, 2004). This issue is
3364 discussed in detail in Section 5.3.2, p.170. Thus, the main conclusion from Chapter 4 is that NMs
3365 must be thought of, and regulated as, biological entities, rather than as conventional chemicals.
3366 We would suggest that this is even the case for polymeric or polymer-coated NMs, as we have
3367 shown above that polymeric NMs behave in identical manners to inorganic NMs in terms of the
3368 uptake and bioaccumulation behaviour, localising to the lysosomes. Coating of NMs with PEG

138
3369 has long been proposed as a mechanism to reduce non-specific protein binding and to thereby
3370 increase the circulation time of the NMs by helping them to evade uptake by macrophages.
3371 However, the effectiveness of PEG at fully suppressing protein binding is debatable, and since
3372 the PEG-coated NMs will interact with other cells (i.e. the target cells in the case of particles for
3373 nanomedical applications), the same issues of bioaccumulation persist.

3374 In terms of REACH regulation, this means that nanoscale versions of existing materials cannot be
3375 exempt from regulation, as in the nanoscale they become biological entities rather than
3376 chemicals. Coupling this to the SCENIHR definition of a nanomaterial would suggest that
3377 materials with >0.15% particles of < 100nm should be re-tested as NMs. However, this does not
3378 get around the tonnage triggers, which as discussed in Chapter 2, would also make most NMs
3379 exempt from registration. Thus, in terms of assessing the hazard of NMs, our conclusion is that
3380 the existing approaches are not fully sufficient to determine of the potential human hazards
3381 from NMs, and that NMs need to be considered as new substances, ideally as biological identities
3382 rather than as chemicals, and with NM-specific triggers for date requirements. However, it is
3383 worth noting that to date, no specific nanoscale toxicity hazards have been identified. However,
3384 it is widely acknowledged that it is much too early to rule out any potential effects, since there
3385 are no clear toxicity dose-response relationships or thresholds, no agreement on exposure
3386 metrics, and therefore it is not possible to carry out a quantitative risk assessment at present.
3387 While occupational health surveillance has been suggested as an important step, there are many
3388 issues yet to be resolved. The first is the absence of data on informative disease endpoints such
3389 that specific screening cannot be proposed. Of the potential endpoints, effects on the cardio-
3390 vascular and pulmonary systems would be non-specific as they exist at high levels in the general
3391 population and could occur in response to many occupational risk factors, or indeed from
3392 exposure to anthrophgenic pollution particles etc. Biological markers of exposure such as blood
3393 coagulation parameters or levels of pro-inflammatory cytokines, are also non-specific and not
3394 validated (Gibson, 2010).

3395 Progress in determining biological impacts and mechanisms of impact of NMs is severely
3396 hampered by the current lack of agreed nanoscale positive and negative controls for a range of
3397 biological endpoints, including apoptosis, genotoxicity etc. Currently, toxicity assays are
3398 validated using chemicals to show that the tests are working as they should, which does not
3399 account for any potential artefacts from the nanoscale, nor does it account for the potentially
3400 very different mechanisms of action of the chemicals relative to NMs.

3401 Another blockage to rapid progress is the considerable variability between nominally identical
3402 NMs both NMs of similar composition produced by different manufacturers and even batch-to-
3403 batch variability within a single manufacturer. This has particular consequences in terms of
3404 REACH where re-testing is not needed if sameness of a nanomaterial with its conventional

139
3405 counterpart can be demonstrated, however, the joint hazard data set must address the end-
3406 points in Annexes VII X of REACH (as required by the tonnage band). This is picked up in
3407 further detailbelow, where the parameters used to demonstrate samemess of a substance are
3408 listed. Coupled to this is a lack of understanding of how sensitive standard test approaches are
3409 to this variability in NM surface characteristics i.e. graduations in surface charge or surface
3410 chemistry of e.g. 50nm NH2-PS NPs can have very dramatic effects on the endpoint being
3411 studied. Thus, there is quite significant variability in the literature for nominally identical
3412 materials, and at present there is no way to determine if these differences are related to
3413 differences in the NMs themselves, in the protocol used, or are a result of biological variability.
3414 One way to address this, is to determine how sensitive the various assays are to systematic
3415 changes in NM composition, and to establish bandwidths for nanoparticle sameness. This is
3416 one of the issues that the EU Infrastructure for NanoSafety (QNano)30 will address, in its
3417 workpackage dedicated to nanomaterial quality assurance and reproducibility. A series of
3418 assays will be performed using multiple batches of the same nanomaterial, extremely well
3419 characterised, and the physico-chemical differences between the batches will be correlated with
3420 the variation in the biological response (accounting for the limits of sensitivity / detection in the
3421 assays used). This will enable a binning approach to be used for labelling and regulation of
3422 NMs, whereby thresholds within which NMs can be considered the same for a given property
3423 (e.g. size, surface charge, molecular corona etc.) will be established. This is similar to the control-
3424 banding approach used in determining acceptable exposure limits for risk management. The
3425 corollary of this will be that we can also establish the sensitivity of various assays to NM
3426 variability, and as such determine the most sensitive assays that could be used for routine
3427 screening.

3428 In terms of nomenclature for NMs, there are still many quite contentious issues here, regarding
3429 whether size should be indicated in the name for NMs, whether the synthesis route utilised
3430 should be indicated and thereby the potential impurities that may be residual in the NM,
3431 whether all dispersants, coatings or stabilisers should be listed as part of the NM composition,
3432 and so on. This is true even for the so-called 1st generation (passive) NMs, and becomes an
3433 increasingly complex issue as one moves towards core-shell NMs, ligand-functionalised NMs and
3434 other so-called 2nd generation (active) NMs and to 3rd and 4th generation NMs, which are
3435 expected to be systems of nanosystems and molecular nanosystems, respectively (Roco, 2004,
3436 Subramanian, 2010).

30
QNano - A pan-European infrastructure for quality in NMs safety testing. Grant Agreement no: 262163 (EU
FP7 Capacities).

140
3437 Within REACH, to exactly compare the sameness of a substance to an existing substance in the
3438 SIEF directory, the following information is required31:

3439 Name or other identifier of each substance: IUPAC (International Union of Pure and Applied
3440 Chemistry) name, EC and CAS number;

3441 Molecular and structural formula, including SMILES (Simplified Molecular Input Line Entry)
3442 notation, optical activity and molecular weight;

3443 Composition and characterisation including: purity, impurities, additives, spectral and
3444 chromatography data.

3445 The information given should be sufficient to enable each substance to be identified. This data
3446 must be included in section 1 of IUCLID (International Uniform ChemicaL Information Database)
3447 in each separate dossier, in order to demonstrate that their substance has the same identity and
3448 purity as other members of the SIEF32.

3449 Clearly, this does not capture size, shape or surface-related effects, and as such the whole
3450 question of sameness for NMs under REACH should be reconsidered as a matter of priority.
3451 However, there is still a broader research / manufacturing quality assurance issue around the
3452 current batch-to-batch variability of NMs, and the consequences of this for regulation and safety.
3453 One approach to addressing this is to assess the consequences of particle physico-chemical
3454 variability on the biological identity of NMs (their biomolecule corona) and their subsequent
3455 interactions with cells.

3456 Endpoints that are likely to be of particular relevance for NMs, such as neurotoxicity or
3457 immunotoxicity, do not yet have regulatory test methods implemented. However, two methods
3458 related to NM interaction with the immune systems have recently been approved as standard
3459 practices by the American Society for Testing and Materials (ASTM) International (ASTM
3460 International 2008E2524-08, ASTM International 2008E2525-08).

3461 Even the few approved tests do not consider potentially vulnerable populations, such as the
3462 young, the elderly, or those suffering from inflammatory diseases which may enhance the ability
3463 of NMs to cross biological barriers and access the body, such as inflammation of the gut (e.g.
3464 Crohns disease) or of the lung (e.g. cystic fibrosis). Effects of chronic exposure to NMs on these
3465 populations should be investigated as a priority, using models for these diseases.

31
http://www.reach.sgs.com/documents/sgs-safeguards-06810-reach-the-sameness-of-substances-en-10.pdf
32
SIEF substance information exchange forum.

141
3466 4.6 Lessons from asbestos, metals and protein therapeutics
3467 Regulation has a long history, and many of the individual factors mentioned in Box 4.1, section
3468 4.1, p. 118, have been addressed before in a regulatory context. However, the difference here is
3469 that NMs present all of these complexities simultaneously, and as such understanding their
3470 behaviour, and reducing it to simple quantitative structure-activity relationships (QSARs) which
3471 are the basis of regulation of chemicals and pharmaceuticals, is non-trivial. It is worth assessing
3472 what has been done before, and seeing where we can build upon this. This section gives some
3473 insights from ongoing research into the toxicity of asbestos and other relevant models from
3474 which NM-related insights can be drawn for consideration of their applicability to support or
3475 improve REACH.

3476 Asbestos (from Greek asbestinon, meaning "unquenchable" or "inextinguishable") is a set of six
3477 naturally occurring silicate minerals, which have been exploited commercially for their desirable
3478 physical properties for centuries, until serious health consequences emerged (Alleman, 1997).
3479 They all have in common their asbestiform which manifests as long (1:20) thin fibrous crystals.
3480 The inhalation of asbestos fibers can cause serious illnesses, including malignant lung cancer,
3481 mesothelioma (a formerly rare cancer strongly associated with exposure to amphibole
3482 asbestos), and asbestosis (a type of pneumoconiosis) (Alleman, 1997).

3483 The microscopic and macroscopic properties of asbestos fibers stem from their intrinsic, and
3484 sometimes unique, crystalline features. As with all silicate minerals, the basic building blocks of
3485 asbestos fibers are the silicate tetrahedra which may occur as double chains (Si 4O11)-6, as in the
3486 amphiboles, or in sheets (Si4O10)-4, as in chrysotile. In contrast to chrysotile fibers, the atomic
3487 crystal structure of amphiboles does not inherently lead to fiber formation. The formation of
3488 asbestiform amphiboles must result from multiple nucleation and specific growth conditions,
3489 which results in two ribbons of silicate tetrahedra placed back to back. The crystal structure has
3490 sixteen cationic sites of four different types; these sites can host a large variety of metal cations
3491 without substantial disruption of the lattice33. It is this ability to host metal cations that appears
3492 now to be the primary source of the extreme toxicity of the amphibole fibres (Turci, 2011).

3493 Mineral surfaces are dynamic and complex and may be modified in the lung after adsorption of
3494 proteins and other macromolecules or uptake by cells (Mossman, 2008). Elements such as
3495 magnesium from chrysotile (Jaurand, 1984) or iron from crocidolite or amosite asbestos (Hardy,
3496 1995) may be leached or mobilized intracellularly or extracellularly, thus mediating the toxicity
3497 of these fibers through surface charge or redox reactions. Magnesium initially is leached from
3498 chrysotile fibers (Hume, 1992), thus changing their surface charge from positive to negative and

33
Geology of Asbestos: http://www.eoearth.org/article/Geology_of_asbestos

142
3499 reducing their toxic potential (Light, 1977). However, silica release profiles govern and are
3500 predictive of fiber residence times in lung, an estimated 9 months for a chrysotile fiber with a
3501 diameter of 1 m (Hume, 1992). Several studies have shown that size, surface area, surface
3502 chemistry, solubility and possibly shape all play a role in determining the potential for harm of
3503 particles such as asbestos and engineered NMs (Oberdrster, 2005a, Oberdrster, 2007). This
3504 role of shape seems to be dramatically manifested by asbestos: thin, long fibres of the material
3505 can lead to lung disease if inhaled, but grind the fibres down to shorter particles with the same
3506 chemical make-up and the harmfulness is significantly reduced (Davis, 1986). Similar effects
3507 have been shown for carbon nanotubes more recently (Poland, 2008).

3508 A recent study by Turci et al. (Turci, 2011) aimed to identify at the atomic level the source of
3509 toxicity in asbestos, whereby they investigated the effect of progressive iron loading on a
3510 synthetic iron-free model nanofibre previously found non-toxic in cellular tests. A set of five
3511 synthetic chrysotile nanofibres ((Mg,Fe)3(Si2O5)(OH)4) were prepared with Fe content ranging
3512 from 0 to 1.78 wt %. The relationship between fibre-induced free-radical generation and the
3513 physico-chemical characteristics of iron active sites was investigated. The fully iron-free fibre
3514 was inert, whereas radical activity arose with even the smallest amount of iron. Surprisingly,
3515 such activity decreased upon increasing iron loading. Mssbauer and EPR experiments revealed
3516 isolated iron ions in octahedral sites that undergo rhombic distortion at the lowest loadings, but
3517 that increasing the loading reduced the amount of isolated iron. A linear relationship between
3518 the formation of carbon-centred radicals (toxicity) and the amount of (rhombic-distorted)
3519 isolated iron sites was found. The authors suggest that even the smallest iron contamination
3520 imparts radical reactivity, hence toxicity, to any chrysotile outcrop, thereby discouraging the
3521 search for non-toxic chrysotile. However, a deeper message from this is that in the design of
3522 synthetic nanofibres, we need to ensure that residual iron or other cationic metals from the
3523 reaction process are completely removed to ensure that they do not leech from the NMs causing
3524 toxicity. It also offers a potential route to nanosafety by design, whereby reducing the amount of
3525 isolated ions resulting from a synthetic process will lead to reduced toxicity from isolated heavy
3526 metals.

3527 A significant and important conclusion from Turcis study is that the use of model solids that
3528 only differ in one property at a time appears to be the most successful approach for a molecular
3529 understanding of the physico-chemical determinants of toxicity (Turci, 2011). Such findings
3530 could also be useful in the design of safer nanofibres.

3531 The biotic ligand model for metal toxicity to aquatic species. Interaction with surroundings
3532 was identified in Box 4.1, section 4.1, p. 118, as a key difference between NMs and chemicals. It
3533 is worth noting that metals also do this, and that typically, metal toxicity to aquatic organisms is
3534 dependent on the local water chemistry. Variations in pH, concentrations of cations such as

143
3535 calcium, magnesium and sodium, variations in alkalinity and the presence of natural organic
3536 matter can all have a significant effect on the toxicity of metals. As a result, toxicity levels may
3537 vary widely for a given metal. This presents a difficult problem for establishing regulatory
3538 guidelines because in the absence of a way to predict these effects, a conservative regulatory
3539 limit is typically selected. The biotic ligand model (BLM) is a mechanistic approach that greatly
3540 improves the ability to generate site-specific ambient water quality criteria (AWQC) for metals
3541 in the natural environment relative to conventional relationships based only on hardness. The
3542 model is flexible; all aspects of water chemistry that affect toxicity can be included, so the BLM
3543 integrates the concept of bioavailability into AWQC. Using an equilibrium geochemical modeling
3544 framework, the BLM incorporates the competition of the free metal ion with other naturally
3545 occurring cations (e.g., Ca2+, Na+, Mg2+, H+), together with complexation by abiotic ligands (e.g.,
3546 NOM (natural organic matter), chloride, carbonates, sulfide) for binding with the biotic ligand,
3547 the site of toxic action on the organism (Turci, 2011). The BLM quantitatively relates short-term
3548 binding to acute toxicity, with the LA50 (lethal accumulation) being predictive of the LC50
3549 (generally 96 h for fish, 48 h for daphnids although significant adjustments need to be made).

3550 However, even here significant challenges remain (Niyogi, 2004): Most BLMs originate from
3551 tests with fish and have been recalibrated for more sensitive daphnids by adjustment of LA50 to
3552 fit the results of toxicity testing. Issues of concern include the arbitrary nature of LA50
3553 adjustments; possible mechanistic differences between daphnids and fish that may alter log K
3554 values, particularly for hardness cations (Ca2+, Mg2+); assumption of fixed biotic ligand
3555 characteristics in the face of evidence that they may change in response to acclimation and diet;
3556 difficulties in dealing with NOM and incorporating its heterogeneity into the modeling
3557 framework; and the paucity of validation exercises on natural water data sets. Important needs
3558 include characterization of biotic ligand properties at the molecular level; development of in
3559 vitro BLMs, extension of the BLM approach to a wider range of organisms, to the estuarine and
3560 marine environment, and to deal with metal mixtures; and further development of BLM
3561 frameworks to predict chronic toxicity and thereby generate chronic AWQC. In principle this
3562 approach could be further extended to include metal NMs.

3563 Regulation of protein therapeutics. As the biotechnology industry has matured through
3564 various stages of growth, regulatory agencies have evolved in response to the need to define
3565 quality standards (Kozlowski, 2007). Key issues for protein therapeutics, which also have

144
3566 resonances with regulation of NMs, include the potential for immunogenicity 34, and the need to
3567 address issues such as manufacturing quality assurance.

3568 Although the immunogenicity of protein products had been an issue throughout the history of
3569 biotechnology products, the publication in 2002 of information about transfusion-dependent
3570 pure red cell aplasia cases in patients receiving recombinant erythropoietin highlighted the risks
3571 of immunogenicity (Kozlowski, 2007). Increases in the frequency of immunogenic responses
3572 were associated with a manufacturing change of the product distributed in Europe.
3573 Immunogenicity is difficult to predict and can affect both safety and efficacy, with
3574 hypersensitivity, immune suppression, and cytokine release syndrome all being symptoms of
3575 immunologic adverse events in response to biotechnology products.

3576 In biotechnology, these issues were addressed via process control - the biologics mantra is that
3577 "the process is the product" (Kozlowski, 2007). The importance of process for biologics is
3578 codified through separate license applications for products (product license applications or
3579 PLAs) and manufacturing facilities (establishment license applications or ELAs). Because end-
3580 product testing is of very limited utility in assessing clinical performance of complex biological
3581 products, the manufacturing process is critical to assure consistent product quality. The strategy
3582 to deal with this process-dependence was to fix the commercial process to the process used for
3583 the manufacture of clinical lots. Thus, the product was linked to the labeled clinical outcomes
3584 through a highly defined manufacturing process. This suggests that for regulation of NMs too,
3585 addressing the key issues of nanomaterial reproducibility between batches and control of the
3586 surface properties, which determine the biological fate and behaviour, will be the critical step
3587 towards regulation, especially for nanomedicines.

3588 Drug-protein associations can also mis-direct drugs, which is accounted for in the regulation of
3589 pharmaceuticals. A drug in blood exists in two forms: bound and unbound. Depending on a
3590 specific drug's affinity for plasma proteins, a proportion of the drug may become bound to
3591 plasma proteins, with the remainder being unbound. Thus, a drugs efficiency may be affected by
3592 the degree to which it binds to the proteins within blood plasma. The less bound a drug is, the
3593 more efficiently it can traverse cell membranes or diffuse. Notably, it is the unbound fraction
3594 which exhibits pharmacologic effects. It is also the fraction that may be metabolized and/or
3595 excreted. For example, the "fraction bound" of the anticoagulant warfarin is 97%. This means that
3596 of the amount of warfarin in the blood, 97% is bound to plasma proteins. The remaining 3% (the
3597 fraction unbound) is the fraction that is actually active and may be excreted35. However, a recent

34
Immunogenicity is the ability of a particular substance to provoke an immune response in the body of a
human or animal.
35
http://en.wikipedia.org/wiki/Plasma_protein_binding

145
3598 paper suggests that simply using the free fraction is also misleading - because in vivo efficacy is
3599 determined by the free (unbound) drug concentration surrounding the therapeutic target, not by
3600 the free drug fraction (Smith, 2010). Thus, decisions based on free drug fraction could result in
3601 the wrong compounds being advanced through drug discovery programmes. In the case of NMs,
3602 we do not expect a free fraction in the sense that the surface area should in principle be fully
3603 covered with proteins due to their large excess, but there is conceivably the possibility that not
3604 all the NMs in a specific dose have an identical surface coverage of proteins, and therefore they
3605 may not all be processed identically in the body. This is a topic that would need to be addressed
3606 further, as part of a detailed consideration of a regulatory framework that includes NM surface
3607 adsorption effects as one element of the assessment.

3608 4.7 Conclusions and recommendations regarding human toxicity of NMs

3609
CONSIDER TAKING FURTHER ACCOUNT OF THE SIZE DISTRIBUTION OF NMS IN THEIR DEFINITION

In its present form, the SCENIHR definition of NMs does not fully account for the size distribution of
NMs in terms of the cut-off and the SSA calculation, as this is based on the assumption that all
particles have the same size, which is not the case. Thus, the theoretical SSA does not actually
account for the potentially significant fraction of particles above and below this average size, and
thus the actual SSA may differ quite significantly from the theoretical SSA.

CONSIDER PRIORITISING THE DEBATE ON NAMING OF NMS TO PROVIDE CLARITY AROUND THE ISSUE OF
THEM BEING NEW SUBSTANCES, SEPARATE FROM THEIR CHEMICAL IDENTITY

Much of the current uncertainty around regulation of NMs stems from the lack of clarity regarding
the naming of NMs. The issue is complex, even for 1st generation NMs (without complex
functionalities) like titania and silica, where differences in crystal structure, surface stabilisers or
coatings, charge and size all potentially impact on the biological fate & behaviour of the NMs but are
not yet indicated in nomenclature or descriptors of materials (for example, dispersants or coatings
are often buried deep in the MSDS rather than in the product label). Addressing this issue will likely
speed up the development of an implementable nano-specific regulatory process, which combines
the best elements of existing regulatory frameworks for biological materials and chemicals.

CONSIDER NMS AS BIOLOGICAL ENTITIES AND BUILD ON LEARNINGS FROM REGULATION OF PROTEIN
THERAPEUTICS AND OTHER BIOLOGICALS

By considering NMs as biological entities, focus shifts immediately to issues of process quality
assurance (which will in principle address critical issues such as current batch-to-batch variability
which are significantly hampering progress in terms of determining the safety (or otherwise) of
individual NM types) and immunogenicity, and also allow for interactions with sounding
biomolecules and their impacts to be considered. It will also remove the issue of legacy NMs being
exempt from re-regulation, including the exemption for polymers, since polymeric NMs also adsorb a

146
 corona of biomolecules to themselves, thereby obtaining a biological identity.

Building on this, we recommend that fundamental research be conducted into the appropriateness
of commonly used culture media and exposure solutions, including for example human plasma,
tissue culture media containing 10% FCS, aqueous media containing NOM etc., which would allow
cross-comparison of data via these reference states. This would also provide insights as to the
biological identity of NMs under the different conditions.

RE-CONSIDER THE TESTING APPROACH FROM 1ST PRINCIPLES FOR CHEMICALS THE BASIS OF
REGULATION IS THE CHEMICAL NOMENCLATURE OF THE DRUG / MOLECULE - FOR NMS IT SHOULD INCLUDE
THE SURFACE IN ADDITION TO THE PARTICLE SIZE, SHAPE AND COMPOSITION, AS THIS IS WHAT GOVERNS
ALL SUBSEQUENT INTERACTIONS AND IMPACTS.

By having the nanomaterial surface and what happens at the nanomaterial-biological interface as the
centre of the regulatory framework, we can make a re-evaluation of the appropriateness of the
current tier-testing approach for NMs, and where appropriate add additional steps such as
characterisation of the nature of the NPs in situ in the appropriate test medium. This will enable
issues such as opsonisation and immunogenitiy to be addressed as part of the assessment process. It
will also provide an opportunity for a full re-assessment of how we currently perform in vitro in
vivo correlations, to account for the fact that different biomolecules may bind to the NMs under
different concentration regimes, thereby resulting in different biological identities and potentially
different biological consequences.

As part of this process, assessment of methods and tests for other endpoints, not currently tested
for, should also be considered, such as biodistribution kinetics, neurotoxicity, immunogenicity and
especially the effects of chronic exposure on vulnerable human populations, which should be
investigated using models such as the injured gut and injured lung models.

FOCUS ON ADDRESSING ISSUES OF NANOMATERIAL REPRODUCIBILITY AND SURFACE COMPOSITION

VARIABILITY AS A PRIORITY, AS OTHERWISE WE WILL CONTINUE TO PRODUCE LARGE AMOUNTS OF SAFETY

DATA ON MATERIALS THAT ARE ONLY REPRESENTATIVE OF THEMSELVES.

While the approaches adopted by some of the larger testing programmes of using a single large
batch of a specific nanomaterial from a specific manufacturer are extremely important in terms of
validating test methods and ensuring reproducibility across laboratories, they run the risk of
generating large amounts of safety data that is non-generalisable even to that specific type of
nanomaterial, as we do not yet have any answers as to the variability of synthetic routes and the
limits of sameness in terms of surface properties that we can accept from a safety and regulatory
viewpoint. We also need to focus on development of simple methodologies for assessing
nanomaterial surface properties and their evolution with time, and in situ under test conditions, in
order to be able to understand potential impacts from surface ageing.

3610
3611

147
3612 Table 4.1 Comparison of the size of a series of copolymer NIPAM:BAM NPs in
3613 different OECD recommended ecotoxicity assay media. From Naha et al., 2009. The
3614 salt concentrations induced some significant particle aggregation, especially for
3615 the most hydrophobic NPs (50:50 NIPAM/BAM).

3616
3617

3618 Table 4.2 Comparison of the binding of apolipoproteins from human plasma to
3619 polystyrene NPs of different size and surface charge. From Lundqvist et al. 2008.

3620

3621

148
3622

3623 Figure 4.1 Zeta potential of copolymer particles of increasing hydrophobicity

3624 dispersed in different OECD ecotoxicity testing media. (black bars) Milli Q water;
3625 (light grey bars) Algal medium; (white bars) Microtox medium; (dark grey bars)
3626 Daphnia magna media. Data are presented as the mean SD (N=6). From Naha et
3627 al., 2009.

3628

3629

3630

3631

3632

3633

3634

3635

3636

3637

3638 Figure 4.2 Surface chemistry and surface charge of NPs can dramatically effect the
3639 nature of the NP dispersion in a biological fluid (e.g. plasma). (a) Differential
3640 Centrifugal Sedimentation (DCS) spectra of 50nm PS-COOH NPs showing the shift
3641 of the main particle peak following the formation of the biomolecule corona, and
3642 the presence of small numbers of particle dimers and trimers. (b) TEM image of
3643 the molecular corona around a 50nm PS-COOH nanoparticle. (c) Differential
3644 Centrifugal Sedimentation spectra of 50nm PS-OSO3 NPs showing the dramatic
3645 shift of the main particle peak following the formation of the biomolecule corona
3646 in this case very few monomeric, dimeric or trimeric particles remain, and the
3647 majority of the particles appear to be incorporated into protein clusters of higher
3648 molecular weight. This clustering of NPs within a protein coating is shown in the
3649 TEM image (d) of the 50nm PS-OSO3 NPs.

149
3650

3651 Figure 4.3 (a) Crystal structure of the protein apolipoprotein A-1, and schematic of
3652 apolipoprotein A-1 in a lipoprotein complex composed of phosholipids. (Courtesy
3653 of the Theoretical and Biophysical Computational Group, University of Illinois at
3654 Urbana-Champaign.) (b) Size comparison of a 70 nm nanoparticle with
3655 lipoprotein complexes chylomicrons, very low, low, and high density
3656 lipoproteins. These lipoproteins routinely pass into and out of cells, and it is clear
3657 that NMs of a similar size coated with apolipoprotein A-1 could be recognized as
3658 lipoproteins. From Lynch and Dawson, 2008.

3659

3660

3661

3662 Figure 4.4 Comparison of the sub-cellular distribution of (a) molecular yellow-
3663 green fluorescence dye versus (b) the same dye constrained within 50nm
3664 polystyrene NPs in A549 cells. Images from Salvati et al, 2011. Cells were exposed
3665 to nominally identical amount of dye for 24 hours.

150
3666

3667 Figure 4.5 Comparison of the composition of the molecular corona formed at
3668 different plasma concentrations around (a) 50nm Sulphoxide-modified
3669 polystyrene NPs and (b) 50nm SiO2 NPs. In the case of the hydrophobic
3670 Sulphoxide-modified polystyrene NPs, as the plasma concentration increases, the
3671 bands get heavier, indicating that more protein binds with increasing plasma
3672 concentration. On the other hand, with the hydrophilic SiO2 NPs, quite different
3673 patterns of proteins are bound at low and high protein concentrations, suggesting
3674 very different biological identities under the different regimes (low plasma
3675 concentration relating to in vitro concentrations, and high plasma concentrations
3676 relating to in vivo situations). From Monopoli et al., 2011.

3677

3678

3679 Figure 4.6 Risk of accidental exposure to hazardous substances during

3680 manufacture of some common NMs compared to other industrial processes such
3681 as wine production, petroleum refining or silicon wafer production. From this
3682 date, exposure risks from NM manufacture are comparable to or lower than those
3683 from other industrial processes. Data from Wilson, 2006.
3684

151
3685 Attachment 4.1 The estimated relative presence of NMs used in consumer
3686 products on the global market (%) in the near future based on the total amount
3687 (tonnes) of NMs currently used in consumer products.

3688

3689 From: Wijnhoven, S.W.P. , Dekkers , S., Hagens, W. I,. de Jong, W. H. Exposure to NMs in consumer
3690 products. RIVM Letter report 340370001/2009.

152
3691 5 Assessment of the potential ecotoxicity of NMs

3692 Naturally occurring NMs, arising from biogeochemical processes (section 3.1.1, p.77) are
3693 present in all compartments of the biosphere: air, soil and water. They will have occurred in
3694 these environments over geological time (Murr, 2004, Verma, 2002) and at much higher
3695 concentrations than are likely to be attained by anthropogenic manufactured NMs (it is
3696 estimated that a billion metric tonnes of atmospheric dust is produced annually (Kellogg, 2006).
3697 Given that extant organisms will have evolved in these environments why are manufactured
3698 NMs of particular ecotoxicological concern?

3699 Firstly, under the right circumstances naturally occurring NMs can be toxic. For example
3700 volcanic dust and ash, which contain ultrafine particles, have been shown to induce respiratory
3701 dysfunction (Forbes, 2003, Lee, 2004). Similarly, there is ample evidence arising from toxicology
3702 that adventitious NMs in the atmosphere present a human health hazard (Dahl, 2006, Kittelson,
3703 2004) for review see (Handy, 2007). Secondly, manufactured NMs present novel chemical and
3704 structural forms and properties that do not occur naturally. It is therefore possible that
3705 biological detoxifying mechanisms evolved to deal with naturally occurring NMs may be
3706 circumvented or have reduced effectiveness. Thirdly, the rate of innovation and release of these
3707 novel particles will be such that organisms do not have sufficient time to develop an appropriate
3708 response. Lastly, the persistence of NMs in the environment is largely unquantified, but may be
3709 greater than for natural NMs, because of the use of capping agents and surface functionalisation
3710 (Handy, 2008b). Given that some manufactured NMs have been shown to have toxic effects
3711 (Velzeboer, 2008), or are even designed to be toxic to biota (e.g. silver NPs), concern is
3712 warranted.

3713 The ecotoxicology of manufactured NMs is a field defined by uncertainties. As an emerging

3714 discipline there is a paucity of information on almost every aspect required to produce robust
3715 environmental hazard and exposure assessments: toxicological mode of action, species at risk,
3716 bioavailability and bioaccumulation, input sources, routes of release, environmental
3717 concentrations, transport and degradation, and the applicability of current testing strategies and
3718 methodologies. Given the extent of these uncertainties and the diversity of NMs and
3719 nanotechnology applications, there may be a lengthy delay before robust environmental risk
3720 assessments can be completed. Just for NM currently in production, estimates range from 10
3721 (Maynard, 2006b) to 53 years (Choi, 2009). Alternative governance frameworks and risk
3722 assessment methods that would allow policy-makers to make informed decisions in the interim
3723 are currently underdeveloped and hotly debated (Grieger, 2010, Linkov, 2009, RCEP, 2008,
3724 Wiesner, 2009).

153
3725 5.1 REACH Requirements
3726 The REACH requirements for ecotoxicity and environmental behaviour testing are summarized
3727 in Table 5.1, p.183, for increasing tonnage triggers (adapted from (Rudn, 2010)), along with
3728 reference to the appropriate OECD testing methodologies (OECD, 2009a). With the current
3729 tonnage trigger framework (see section 2.3.2.2, p.37 and Table 2.1 p.67), few NMs would be
3730 manufactured in sufficient quantities to initiate the gathering of meaningful ecotoxicity data.
3731 Meaningful data for an ecotoxicologist are data that can predict changes in demographically
3732 important endpoints, endpoints that change population dynamics. These include growth,
3733 reproduction and age-related survival.

3734 For prioritized samples in the > 1 tonne bracket, acute / short-term toxicity studies on algae and
3735 Daphnia are required. These tests routinely generate two measures of toxicity, or endpoints,
3736 the EC50 (median effect concentration) and the LC50 (median lethal concentration). The short-
3737 term, sub-lethal algal study may also be used to calculate alternative measures of toxicity; LOEC
3738 (Lowest Observed Effects Concentration) and NOEC (No Observed Effects Concentration).
3739 Routinely, it is the E(L)C50 measures of toxicity that are combined with arbitrary safety
3740 assessment factors (AFs) (usually x10, x100 or x1,000) to give a Predicted No Effect
3741 Concentration (PNEC) for demographically relevant endpoints. The LOEC/NOEC endpoints from
3742 the short-term algal assay can be used in conjunction with chronic toxicity data from a Daphnid
3743 or fish species to justify the use of less stringent assessment factors (x100, x50, x10). The
3744 scientific basis for these assessment factors is limited to non-existent in the case of NMs, given
3745 that so little reliable chronic data is available and MOAs are so poorly understood. This
3746 introduces a large uncertainty when assessing NM ecotoxicity. To validate assessment factors
3747 having matched acute and chronic data for the same toxicant is ideal, and these studies are just
3748 beginning to emerge (Hall, 2009, Wiench, 2009) see section 5.2.5, p.162. A case can therefore be
3749 made that, until safety factors are validated, REACH should require some measure of chronic
3750 toxicity alongside the mandatory acute tests at the >1 tonne bracket and above.

AS AN INTERIM MEASURE IT IS RECOMMENDED THAT A MEASURE OF CHRONIC TOXICITY BE REQUIRED FOR

ALL NMS TO WHICH REACH REGULATIONS ARE APPLICABLE.

The OECD's Daphnia magna reproduction test (OECD 211) may be the best available method to
provide such data. The method has a demographically relevant endpoint in terms of
reproductive output, without being unduly onerous to perform. An additional benefit is that the
outputs from OECD 202 (D. magna acute toxicity), which is already mandatory for the prioritised
>1 tonne bracket, and 211 can be used in conjunction to assess the validity of safety factors,
leading to a more robust PNEC.

154
 OECD are currently in the process of assessing all their protocols for suitability for testing NMs
(OECD, 2009b), but it must be stressed that attention must be paid to the details of the
methodology if data generated are to be valid. For instance the use of Elendt M4 or M7 media,
which contains EDTA, may be inappropriate for use with metal NPs while standard media may
require reduced salt levels to limit particle aggregation. It must also be reiterated that NMs
should be characterised under assay conditions at the beginning and end (as a minimum) of
prolonged chronic tests (see section 5.2.1, p.155 on determining dose).

THIS INTERIM MEASURE COULD BE DROPPED ONCE SUFFICIENT CONFIDENCE IS ESTABLISHED IN SAFETY
FACTORS, AND IT IS FURTHER RECOMMENDED THAT REGULATORY BODIES AND THE NANOPARTICLE

INDUSTRY FUND THE RESEARCH NECESSARY TO DEMONSTRATE THE LINKAGE BETWEEN ACUTE AND

CHRONIC ECOTOXICITY ENDPOINTS.

3751 A >1 tonne prioritised classification requires an indication of ready biodegradability. No

3752 measure of abiotic degradation is required (e.g. photolysis, hydrolysis, dissolution) and this may
3753 be the dominated breakdown mechanism for inorganic NMs (section 5.3.2, p.170).

3754 The following sections explore current issues and uncertainties in determining the ecotoxicity of
3755 NMs (section 5.2, p.155), in predicting their environmental behaviour and fate (section 5.3) and
3756 discusses progress towards modeling exposure scenarios (5.5, p.179).

3757 5.2 Determining Ecotoxicity

3758 5.2.1 Dose and Exposure

3759 Relevant metrics. The initial challenge when testing a NM is which metric to choose as a
3760 descriptor of dose. Traditionally, ecotoxicologists think of dose in terms of mass per unit volume
3761 (e.g. mg per litre) and as a static measure. Given that the activity of a NM may be primarily
3762 connected to the available surface area, mass may not be the most relevant metric to use, and
3763 both particle number and SSA per unit volume have been proposed as alternatives. Oberdrster
3764 and co-workers (Oberdrster, 2005b, Oberdrster, 2007) argue that using SSA provides a more
3765 standard measure as it compensates for the effect of varying particle size. The metric chosen
3766 should, to some extent, depend on the test organism, the expected route of exposure and,
3767 perhaps, measurement methods available for particular exposure scenarios. For pelagic species
3768 where exposure to dispersed NMs may predominate, then particle number or SSA may be the
3769 most appropriate dose metric. For benthic or sediment dwelling species, where exposure to
3770 aggregated NMs is likely to be more important (see section 5.3.2, p.170), SSA and particle
3771 number are less meaningful and mass may still be the most appropriate metric. For other
3772 organisms and exposure scenarios, metrics will have to be chosen, as appropriate.

155
3773 Dose metrics can profoundly influence the interpretation of results. A study of the toxicity of
3774 SiO2 NPs to the green micro-alga Pseudokirchneriella subcapitata (Van Hoecke, 2009)
3775 demonstrated that if results were expressed as a mass concentration then 12.5 nm particles
3776 appeared more toxic to the alga than 27.0 nm particles. When particle concentration was
3777 expressed as a surface area however, no such relationship between particle size and toxicity was
3778 found. The measurement of mass concentration is considerably easier than other metrics, and
3779 therefore in practice this is the most likely to be reported. In the absence of a clear consensus,
3780 OECD (2010) recommends recording both mass and surface area as this approach would allow
3781 retrospective analysis of datasets.

3782 Administering and measuring dose. Much of the discussion on administering and measuring dose
3783 relates to dispersion of NMs in an aqueous environment. The complexity of administering dose
3784 in soil and sediment matrices is likely to be greater, but the issues are currently not so well
3785 defined. Only where toxicants are administered orally to vertebrates is the issue of dispersivity
3786 somewhat less relevant.

3787 Ideally a stock suspension of NMs for ecotoxicity tests should be evenly dispersed. Many NMs
3788 are poorly dispersible in aqueous environments and dispersion does not always occur
3789 spontaneously. There are three methods documented in the literature to aid dispersion, all of
3790 which have limitations.

3791 Solvent dispersion. The addition of a solvent or detergent to a NM suspension has been
3792 widely used to aid dispersion of carbonaceous NMs. This has the advantage that it is a
3793 relatively fast preparation method and that particles are likely to stay dispersed as long
3794 as the agent is present. A major drawback is that many of the best dispersing agents are
3795 themselves toxic, either independently or in combination with the nanoparticle
3796 (Andrievsky, 2005, Henry, 2007, Oberdorster, 2004, Zhu, 2006). Dispersants may also
3797 alter the bioavailability or behaviour of a test substance, be it a NM or a conventional
3798 chemical. The use of solvents / dispersants therefore leads to difficulties in data
3799 interpretation and experimental design. The use of naturally occurring organic matter
3800 (NOM) as a dispersant has been proposed as a non-toxic, environmentally relevant
3801 alternative (Chen, 2006, Hyung, 2007, Kennedy, 2009), although difficulties over how to
3802 standardize this approach remain. These and similar materials can be added during
3803 rather than after synthesis in some preparation methods.

3804 Sonication. Sonication is the application of energy to a system, usually as ultrasound

3805 waves. It causes aggregates to disperse by breaking the inter-particle bonds that hold
3806 them together. The principle advantage of this method of dispersion is that it involves no
3807 additional solvents or chemicals and preparation time is still relatively short (hours to

156
3808 days). However, sonication has been shown to damage some NMs; CNTs are cut, altering
3809 their aspect ratio and therefore potentially their toxicity profile (Yu, 2007, Kennedy,
3810 2009). An additional consideration is that NMs re-aggregate once sonication has ceased.
3811 This is a particular problem for chronic toxicity assays with longer durations.

3812 Prolonged stirring. As for sonication, prolonged stirring involves no additional chemicals,
3813 and is again primarily used for carbonaceous NMs. Preparation times can be long (weeks
3814 to months), and although it is a gentler method of adding energy to a system, it still has
3815 the potential to physically damage or chemically alter NMs. Re-aggregation may also
3816 occur once stirring has stopped

3817 For a fuller discussion on the advantages and disadvantages of dispersion methods the reader is
3818 referred to the review of Crane et al. (Crane, 2008). Ultimately the choice of which method, or
3819 combination of methods, is most appropriate will be informed by the dispersive properties of
3820 the NM, the test organism involved (and its tolerance of alternative dispersing agents and
3821 solvents), assay duration over which dispersion must be maintained, and the dosing regime used
3822 (static / semi-static / flow-through).

3823 Even if a homogeneous dispersion of NMs is achieved, in the absence of steric or electrostatic
3824 stabilization (section 3.2.5, p.86), there will be a tendency for particles to re-aggregate over time.
3825 The rate at which this occurs will be dependent on a number of factors including:

3826 Particle concentration. Particle aggregation is a kinetic process, which in part depends on
3827 the number of particles available to collide with each other (section 3.2.7, p.89). In
3828 preparing different dilutions of a stock concentration, not only is the total number of
3829 particles in suspension likely to change, but the proportion of the total present as
3830 aggregates will also vary. If toxicity varies with aggregate size, the dose-response-curve
3831 may reflect the physical availability of the toxicant rather than the concentration of the
3832 colloidal dispersion.

3833 Assay conditions. Organisms must be maintained in conditions appropriate for their
3834 physiology during ecotoxicological testing. Often a synthetic culturing medium is used,
3835 which will contain combinations of inorganic salts, trace elements and perhaps complex
3836 organic molecules. These complex mixtures may have relatively high ionic strengths
3837 (they are high in salts) and this will increase the rate at which particle aggregation
3838 occurs by reducing the effectiveness of electrostatic stabilisation. It may be possible to
3839 dilute culture media, thereby helping to maintain particle dispersion, without causing
3840 physiological stress to the test organism at least over short timescales (Romer, 2011).
3841 The test environment may therefore be a compromise situation, which whilst fulfilling

157
3842 the organisms requirements, reduces aggregation over the relevant timescale for the
3843 assay. OECD (2010) recommends that the assay medium is characterised in terms of
3844 ionic strength, 'hardness' (Ca2+, Mg2+ and total hardness - at least for freshwater media),
3845 pH, dissolved organic matter, alkalinity and the presence of dispersing agents.

3846 Dose when testing NMs should therefore be considered a dynamic variable with the actual dose
3847 received or exposure an organism experiences being altered by the interaction of the particle
3848 with the suspension medium. Thus, there is an overriding need to characterise NMs at the
3849 concentrations used and under identical test conditions (Warheit, 2008). Studies are emerging
3850 where nanoparticle characterization is sufficiently detailed and robust to allow objective
3851 interpretation of results (Fabrega, 2009a, Fabrega, 2009b, Sotiriou, 2010, Rogers, 2009), but
3852 these remain a minority output in the peer-reviewed ecotoxicology literature. This matter needs
3853 to be addressed if progress is to be made toward developing QSAR and read-across approaches
3854 (section 3.3.1, p.92), and many authors have suggested that in peer-reviewed literature a
3855 minimum standard for particle characterization might be established (Stone, 2010).

3856 5.2.2 Identifying routes of particle toxicity

3857 The form a NM adopts on interaction with a test medium or an environmental or physiological
3858 matrix may alter its toxicity. The simplest scenario is that the primary nanoparticle type causes
3859 toxicity to a cell or organism by one of the mechanisms detailed in section 4.1, p.118 As particles
3860 aggregate the toxicity profile may change, either because there is a fundamental alteration in the
3861 mode of action (aggregates exceed the size for membrane permeability for example) or because
3862 the exposure (received dose) to the test organism changes. A study into the acute toxicity of
3863 fullerene (C60) to Daphnia magna concluded that aggregate size was an important factor
3864 (Lovern, 2006). For metal and metal oxide NMs, the rate of dissolution may also alter toxicity if it
3865 is sufficiently fast compared to the duration of the assay, and the dissolved ions are themselves
3866 toxic (Blinova, 2010). Franklin et al. (Franklin, 2007) showed that the toxicity of zinc oxide NPs
3867 could be largely attributed to the zinc ions produced from the nanoparticle during the course of
3868 the assay. Toxicity of dissolved silver ions has also been shown to make an important
3869 contribution to the toxicity of some silver NPs (Lok, 2006, Sotiriou, 2010).

3870 NMs may also act as carriers for other toxicants (Baun, 2008b, Hartmann, 2010, Petersen, 2009).
3871 Due to their high surface reactivity, NMs readily bind other chemical species. This can be during
3872 the course of their manufacture (e.g. the use of metal catalysts in the production of CNTs
3873 (Jakubek, 2008)), or preparation (e.g. the use of tetrahydrofuran (THF) as a chemical dispersant
3874 (Zhu, 2006)) or on release into the natural environment (e.g. binding of lead, cadmium and
3875 copper by MWCNTs (Li, 2003)). Binding of the secondary species to a NM can increase
3876 availability to a cell or organism and thereby its toxicity the NM effectively acts as a Trojan

158
3877 horse. This is well, if not definitively, investigated in the case of THF and related contaminants
3878 and C60 fullerenes (Zhu, 2006, Oberdorster, 2006, Henry, 2007). The use of THF as a dispersing
3879 solvent markedly increases the toxicity of carbon nanotubes and fullerenes to a range of test
3880 organisms, and this may be related to the presence and toxicity of THF oxidation products -
3881 butyrolactone and tetrahydro-2-furanol in the suspension media (Henry, 2007). Conversely,
3882 NMs may sequester contaminants, making them less available to test organisms and thereby
3883 reducing their toxicity (e.g. TiO2 and cadmium (Hartmann, 2010)) see also section 5.3.2, p.170
3884 and Figure 5.3, p.187).

3885 5.2.3 Relevant testing regimes

3886 The discussions above and in chapter 3 illustrate that NMs cannot be thought of in isolation;
3887 their physicochemical form and their toxicity are intrinsically linked to their interactions with
3888 the test medium or the environmental matrix in which they are dispersed. This has led to the
3889 suggestion that ecotoxicological testing and NM characterization should be conducted under
3890 environmentally relevant conditions (Crane, 2008, Klaine, 2008, Gao, 2009). In practice, these
3891 suggestions largely mean that assays should be conducted in solutions with an ionic strength
3892 closer to that of the relevant water, and that natural organic macromolecules (NOM), such as
3893 humic substances (HS) should be included as a dispersion and stabilisation agent (Kennedy,
3894 2009).

3895 Trying to simulate natural conditions in a laboratory assay is open to criticism, both on a
3896 practical and a fundamental level. Practically there is the question of which environment do you
3897 try to simulate, given that resources and expertise are limited? It is often implied in the
3898 literature that NMs are most likely to partition into the aquatic compartment, and therefore
3899 simulating a freshwater may be a good starting point. But what specific ion composition should
3900 be used? What humic substances should be added and in what concentration? Should the assay
3901 receive some level of perturbation, or should it be static? Toxicity assays on algae will require
3902 some level of illumination (e.g. OECD TG 201 requires continuous illumination), what
3903 illumination regime36 should be used (this has the potential to alter toxicity through photolysis
3904 and ROS production)? All these variables may influence NM toxicity and if data are to be
3905 comparable between studies a standard regime would need to be agreed.

IT IS RECOMMENDED THAT STANDARDISED ECOTOXICITY EXPOSURE CONDITIONS BE ESTABLISHED FOR

NMS IN A RANGE OF DIFFERENT ENVIRONMENTAL COMPARTMENTS (FRESHWATER, MARINE, SATURATED
AQUATIC SEDIMENTS, SOILS). THESE CONDITIONS MAY DIFFER FROM STANDARDISED OECD TESTING
REGIMES, AND SHOULD BE OPTIMISED TO PROMOTE NM STABILITY WITHOUT RESULTING IN ADVERSE

36
Both in terms of intensity (lux) and spectrum.

159
 EFFECTS ON TEST ORGANISMS. TEST CONDITIONS SHOULD BE SPECIFIED IN DETAIL AND THE USE OF
NATURAL WATERS AND NON-DEFINED MEDIA INGREDIENTS SHOULD BE MINIMISED. PARTICULAR
ATTENTION SHOULD BE GIVEN TO SPECIFYING EXPOSURE CONDITIONS IN TERMS OF:

DIVALENT ION CONTENT

IONIC STRENGTH

PH

NATURAL ORGANIC MATTER

DOSING REGIME

IT IS FURTHER RECOMMENDED THAT FUNDAMENTAL RESEARCH BE CONDUCTED INTO THE

APPROPRIATENESS OF COMMONLY USED CULTURE MEDIA AND EXPOSURE WATER FOR NM EXPOSURES.
THE BEHAVIOUR OF NMS IN THESE MATRICES SHOULD BE CHARACTERISED (PARTICULARLY IN TERMS OF
AGGREGATION AND DISSOLUTION), AND THE POTENTIAL FOR CONFOUNDING EFFECTS TO BE AMELIORATED

SHOULD BE EXPLORED WITH REFERENCE TO THE APPROPRIATE TEST ORGANISM.

3906 On a more fundamental level it could be argued that trying to simulate environmental conditions
3907 in laboratory assays simply adds extra layers of complexity to interactions that are already
3908 multi-faceted. Whether it is a useful approach or not depends on the purpose of the assay. If, as
3909 would be the case within the REACH regulations, assays are being conducted as part of an
3910 environmental hazard assessment then the approach has some validity. If the purpose of the test
3911 is to determine fundamental mode of action of a nanoparticle against a test species, a more
3912 reductionist approach may yield greater insight into the innate toxicity of the primary particle
3913 type. Ultimately both approaches will be needed, the former in a regulatory context, the latter
3914 for the development of QSAR, read-across and other models (section 3.3.1, p.92).

3915 5.2.4 Species selection

3916 The ecotoxicology literature on manufactured NMs is heavily skewed towards tests on aquatic
3917 pelagic species, particularly the algae Pseudokirchneriella subcapitata, water fleas from the
3918 genus Daphnia and various species of fish. This is understandable, and at one level also
3919 desirable. Traditionally, these groups have been used extensively as model organisms in
3920 ecotoxicology, and much is known about their physiology and ecology. They are the preferred
3921 test organisms in OECD acute toxicity methods 201 - 203 respectively, with the endpoints of
3922 these assays (E(L)C50 and LOEC /NOEC) being considered reliable as input data for wider
3923 environmental hazard assessments. Available nano-ecotoxicology studies are usefully
3924 summarized in the reviews of (Handy, 2008b, Klaine, 2008) for aquatic invertebrates in (Baun,
3925 2008a) and for silver NPs in (Fabrega, 2009a).

160
3926 If the current REACH framework is adopted for NMs, acute aquatic toxicity data are likely to be
3927 the primary, or perhaps only, data available to assess environmental risk. Restricting testing to
3928 such a small group of organisms constitutes a real risk, in that major potential environmental
3929 impacts may be overlooked. As discussed in chapter 3, it is likely that particle number
3930 concentration of dispersed NMs will dominate in surface waters. If NMs aggregate under assay
3931 conditions this will lead to much lower received doses for test organisms compared with
3932 nominal doses, thereby underestimating the likely hazard substantially. Ecotoxicology studies
3933 into the effects of NMs on higher plants (Kumari, 2009, Lopez-Moreno, 2010, Zhu, (2008), soil
3934 and sediment organisms (Coleman, 2010, Hu, 2010, Johansen, 2008, Nyberg, 2008, Roh, 2010,
3935 Roh, 2009, Stanley, 2010, Wang, 2009), and terrestrial invertebrates (Lee, 2009, Lin, 2010a) are
3936 beginning to emerge in the peer-reviewed literature. There remains a paucity of studies on
3937 terrestrial vertebrates, although read-across approaches from human toxicity literature may
3938 give valuable insights into NM effects in this subphylum. Issues arising of particular concern
3939 include the observation that NMs can be adsorbed and translocated by higher plants. Zhu et al.
3940 (Zhu, 2008) demonstrated thatFe3O4 NPs are mobile in plant tissue of hydroponically grown
3941 pumpkin plants, and accumule near the roots and in the leaves, although transfer to the fruit was
3942 not investigated. If this observation was replicated in crop plants it may provide an additional
3943 route for human exposure to NMs.

3944 This study by Zhu et al. (Zhu, 2008) exemplifies the difficulty regulators face when trying to
3945 interpret ecotoxicology studies. Although the paper highlights an additional route of potential
3946 exposure, the study was undertaken using an idealized, non-realistic, system and ascribing risk
3947 therefore becomes virtually impossible. Although pumpkin plants can absorb and translocate
3948 Fe3O4 NPs whether this would in fact occur in a natural system would be dependent on the
3949 bioavailability of the particles in question; how they partition between the liquid and particulate
3950 phases of a natural soil. If nanoparticulate Fe3O4 has a high affinity for soil particles
3951 bioavailability to plants and concomitantly risk, would be low (see section 5.3, p.165).

3952 Conducting ecotoxicity assays on as wide a range of species as possible is, of course desirable,
3953 but impractical given resource restrictions, the additional time delay involved and the lack of
3954 baseline data for some 'at risk' species. There is however, a strong case to be made for research
3955 effort to be concentrated on generating ecotoxicity data on two further groups of organisms,
3956 which may be at high risk from NMs:

3957 Estuarine benthic species. It is predicted that the increasing salt concentration of
3958 estuarine and coastal waters will promote NM agglomeration and sedimentation
3959 (section 5.3.2, p.170). It may therefore be anticipated that estuarine benthic species are a
3960 group at particular risk, although Bradford et al. (Bradford, 2010) showed that a 1000 g
3961 l-1 dose of silver NPs with a nominal diameter of <100nm had no effect on a natural

161
3962 assemblage of estuarine benthic bacteria after 20 days exposure. Generating a more
3963 complete dataset for this community should be given priority. An ASTM method (E2317-
3964 04) already exists for the estuarine copepod Amphiascus tenuiremis and has been used to
3965 assess the toxicity of SWCNTs (Templeton, 2006).

3966 Marine filter-feeding bivalves. Coastal bivalves, such as mussels and oysters, require
3967 particular attention as they are directly consumed by humans. The particle capture
3968 efficiency of bivalves is size dependent, decreasing asymptotically with decreasing size
3969 (Riisgrd, 1988), and they would therefore appear to have a low potential to accumulate
3970 NMs. However in saline coastal waters most NMs will be present as aggregates or
3971 agglomerates in combination with natural organic matter, and this will increase their
3972 availability (Ward, 2009). Uptake of a range of NMs (carbon black, C60, TiO2, SiO2 and Au)
3973 by species of mussel has been shown to be primarily through the digestive gland (Canesi,
3974 2010b, Canesi, 2010a, Tedesco, 2010a, Tedesco, 2010b) triggering toxic effects for a
3975 number of endpoints (lysosomal and oxidative stress biomarkers and lipid
3976 perioxidation). Comparison of Tedesco et al., 2010 a and b, would appear to indicate that
3977 decreasing size of gold NPs increases toxic responses for the digestive uptake route.
3978 Given that bivalves are capable of filtering large volumes of water (3 9 l h-1 per gram of
3979 dry weight (Newell, 2005)) and that NMs may have an increased residence time in
3980 organisms before egestion (Ward, 2009), bivalves show a high potential for
3981 bioconcentration and may be an ideal sentinel or indicator species for nanoparticle
3982 contamination.

IT IS RECOMMENDED THAT FURTHER HIGH QUALITY RESEARCH BE CONDUCTED TO CHARCATERISE THE

HAZARD POSED BY NMS TO AT RISK CONSORTIA. IN PARTICULAR, THE POTENTIAL FOR FILTER-FEEDING
MARINE BIVALVES TO BIOCONCENTRATE NMS SHOULD BE INVESTIGATED WITH A VIEW TO ASSESSING
THEIR POTENTIAL AS SENTINEL SPECIES AND TO DETERMINE WHETHER THEY CONSTITUTE A SIGNIFICANT

TRANSMISSION ROUTE OF NMS TO HUMANS. ESTUARINE BENTHIC ENVIRONMENTS WILL POTENTIALLY

RECEIVE HIGH LOADINGS OF NMS. A MORE COMPLETE UNDERSTANDING IS REQUIRED OF THE IMPACT THIS

MAY HAVE ON INDIVIDUAL SPECIES WITHIN THIS CONSORTIA, AND ON COMMUNITY FUNCTION IN GENERAL.

3983 5.2.5 Endpoint selection

3984 To date, the majority of studies into the ecotoxicity of NMs have looked at short term, acute toxic
3985 effects. In an emerging discipline, such as nano-ecotoxicology, this approach is inevitable. The
3986 use of high concentrations of toxicant to measure gross endpoints, such as mortality,
3987 compresses experimental durations and facilitates detection, providing proof of principle data.

162
3988 Given the reservations over using standard safety factors to calculate PNECs from acute toxicity
3989 data however, there is an urgent need for empirical research into the chronic toxicity of NMs.
3990 Chronic toxicity tests are undertaken over longer durations (typically a minimum of 28 days)
3991 with lower, more environmentally relevant, concentrations of toxicants and use
3992 demographically relevant endpoints such as reproduction, growth and age-related death. Ideally
3993 studies should facilitate direct comparisons by conducting parallel assays addressing acute and
3994 chronic endpoints using the same NMs, and such studies are beginning to emerge (Hall, 2009,
3995 Wiench, 2009).

3996 A number of OECD methodologies exist for demographically relevant endpoints (notably
3997 reproduction: OECD TG 204, -210, -211, -220, -222, -226 and -232) and the organisation
3998 published a preliminary review on their applicability for NMs in support of their ongoing
3999 sponsorship programme (OECD, 2009b). Many of the difficulties discussed in section 5.2.1 on
4000 controlling and measuring received dose are particularly relevant to chronic studies given the
4001 extended duration over which particle aggregation can occur. Two recent research papers show
4002 that reproduction in the soil nematode Caenorhabditis elegans, is adversely impacted by
4003 nanoparticulate silver (Roh, 2009), ZnO, Al2O3 and TiO2 (Wang, 2009). Reproduction also
4004 appears to be a more sensitive endpoint then adult mortality in the water-flea D. magna with a
4005 21 day NOEC of 3 mg l-1 compared to 30 mg l-1 for adult mortality (Wiench, 2009).

4006 Given that NMs are of a size that they may interact directly with DNA or RNA causing strand
4007 breaks (Barillet, 2010), there is the possibility that novel toxic endpoints may arise. Genotoxic or
4008 cytotoxic effects of NMs are widely investigated in human and animal cell lines (see Lewinski, et
4009 al. (Lewinski, 2008) for review), and techniques developed in this arena (see Singh et al. (Singh,
4010 2009) for review) are now being employed to study eco-genotoxicity and eco-cytotoxicity. A
4011 survey of genotoxicity of CeO2, TiO2 and SiO2 NPs by Lee et al. (Lee, 2009) revealed that only CeO2
4012 resulted in DNA damage in D. magna and the aquatic midge Chironomus riparius. CeO2 has also
4013 been shown to induce DNA damage and mutation in soybean plants (Lopez-Moreno, 2010).
4014 Changes in the level of DNA strand breakages have been documented in gill and digestive gland
4015 cells of the freshwater mussel Elliption complanata in response to acute exposure to cadmium-
4016 telluride quantum dots (Gagne, 2008). Disturbance to mitosis has been inferred from
4017 microscopic observation of cell division in onion cells exposed to silver NPs (Kumari, 2009).
4018 Such genotoxic damage, combined with emerging evidence that reproductive processes may be
4019 particular targets for nanoparticle toxicity, may indicate that fecundity or competence of
4020 offspring will be sensitive endpoints. Even small changes in the reproductive success of a species
4021 can have far-reaching consequences for community structure and function in any given habitat.
4022 Such impacts can only be fully described by undertaking multi-generational studies, which are
4023 both costly and time-consuming to perform. OECD protocols exist for inter-generational studies

163
4024 on the reproduction of rats and mice (OECD TG 415, -416) but would have to be adapted and
4025 validated for use with other environmentally relevant species and with NMs.

4026 However, to date there is no proven link between any molecular/cellular biomarker and adverse
4027 outcomes for whole organisms or populations. If 'Omic approaches (gemonics, proteomics,
4028 metabolomics) were able to identify biomarkers / indicators for demographically relevant
4029 endpoints this would offer huge potential for the reduction of vertebrate testing and a major
4030 increase in the capacity to screen novel toxicants.

IT IS RECOMMENDED THAT RESEARCH BE UNDERTAKEN TO DESCRIBE NOVEL ENDPOINTS ARISING FROM

EXPOSURE TO NMS AND TO ASSESS THEIR RELEVANCE IN A REGULATORY CONTEXT. IF GENOTOXIC OR
CYTOTOXIC ENDPOINTS PROVE TO BE PREDICTIVE OF CHANGES IN FECUNDITY OR COMPETENCE OF

OFFSPRING THEIR INCORPORATION INTO THE REGULATORY FRAMEWORK MAY PROVIDE AN IN VITRO

ALTERNATIVE TO IN VIVO STUDIES OF REPRODUCTIVE SUCCESS.

4031 Mesocosm studies and information on trophic transfer of NMs (transfer through the food chain)
4032 is scarce. Such information is vital if uncertainties over the potential for bioaccumulation37 and
4033 biomagnification of NPs are to be reduced. Holbrook et al. (Holbrook, 2008) demonstrated that
4034 QDs, bioconcentrated by the ciliate Tetrahymena pyriformis from aqueous suspension were
4035 transferred to a predator, the rotifer Brachionus calyciflorus. The authors note that although
4036 trophic transfer occurred the potential for biomagnification was small because of the rate at
4037 which the rotifer purged the QDs. A similar scenario was described for the transfer of TiO2 NPs
4038 from D. magna to D. renio, the zebrafish; although transfer was demonstrated, the potential for
4039 biomagnification was low and this was ascribed to the non-lipophilic nature of TiO2 NPs and the
4040 rapid depuration rate of the fish (Zhu, 2010). The bioaccumulation of Zn by the common
4041 woodlouse, Porcellio scaber, was found to be the same irrespective of whether its hazel leaf food
4042 had been laced with ZnCl2 solution, commercial ZnO NPs or ZnO 'macropowder' (Pipan-Tkalec,
4043 2010), and authors therefore conclude that Zn is bioaccumulated in the dissolved, rather than
4044 particulate, form. In a study on the bioavailability of gold NPs to the earthworm, Eisenia fetida,
4045 Unrine et al. (Unrine, 2010) demonstrated, in an environmentally realistic exposure scenario,
4046 that NPs were taken up, accumulated and biodistributed to the tissue of the worm. They

37
The terms bioconcentration, bioaccumulation and biomagnification have specific meaning and should not be
used interchangeably WALKER, C. H., HOPKINS, S. P., ET AL. (2006) Principles of Ecotoxicology, Boca
Raton, Taylor and Francis.. Bioconcentration (and derivative bioconcentration factor - BCF) refers to the
uptake of a pollutant from the ambient medium via any dermal or respiratory tissue exposure and is often used
to describe uptake in aquatic organisms directly from the fresh/seawater environment. Bioaccumulation (and
derivative bioaccumulation factor - BAF) is a more general term refering to the uptake of a compound from
food or water by any mechanism. Biomagnification (syn. bioamplification) is the transfer of a pollutant from
one trophic level to another further up the food chain US EPA (1991) Technical support document for water
quality-based toxics control. EPA 505/2-90-001. Office of Water, Washington DC..

164
4047 conclude that this constitutes a route of entry for NMs into the terrestrial food-web from
4048 contaminated soil.

4049 5.3 Environmental behaviour and fate of NMs

4050 The ecotoxicity of manufactured NMs describes their potential hazard to biota, but this is only
4051 one aspect of an environmental risk assessment. To describe risk fully, hazard must be
4052 combined with a measure of exposure. In an environmental context exposure will be the sum of
4053 the release of NMs into the environment, their transformation and distribution, their persistence
4054 in various habitats, their bioavailability and their ultimate fate.

4055 5.3.1 Sources of manufactured NMs in the environment

4056 Figure 5.1, p.185, shows some of the possible mechanisms of NM release into the environment.
4057 Using 'life-cycle concepts', either formalised life-cycle assessment (LCA)38 or more generic life-
4058 cycle approaches would allow NM sources to be ascertained and quantified. Combining life-cycle
4059 assessment of the nature and magnitude of NM release with exposure modeling (section 5.5,
4060 p.179) may offer a mechanism by which exposure to specific environmental compartments can
4061 be determined with greater accuracy. Life-cycle approaches are particularly powerful when
4062 comparing different options or products with respect to their environmental impact, or when
4063 the interest lies in a tightly defined endpoint such as acidification of freshwaters (Meyer, 2009,
4064 Som, 2010). As the input variables to the model (NM functionalisation, NM loadings, different
4065 methods for incorporation into products, different product matrices (textiles, plastics,
4066 composites)) expand and the output variables and geographical extent become more diffuse
4067 such assessments become concomitantly more complex to perform. A major bar to the
4068 development of life-cycle models is the lack of a comprehensive and reliable inventory of
4069 products containing NMs, their form and their loadings. Compiling such a database is severely
4070 hampered by the lack of any obligation under most legislative frameworks for NMs to be
4071 separately detailed and labeled on products (Som, 2010, WWICS, 2008).

IT IS RECOMMENDED THAT REACH REGULATIONS REQUIRE THE DISCLOSURE OF NMS INCORPORATED

INTO ARTICLES. DISCLOSURE SHOULD INCLUDE INFORMATION ON LOADING, METHOD OF INCORPORATION
AND DISTRIBUTION WITHIN THE ARTICLE (HOMOGENEOUS/ LOCALISED). CLP REGULATIONS SHOULD
REQUIRE SEPARATE LABELING OF THE NANOMATERIAL CONTENT OF PRODUCTS. THIS WOULD ALLOW
IDENTIFICATION AND QUANTIFICATION OF THE SOURCES OF NMS BEING RELEASED INTO THE

38
Life-cycle assessment (LCA) is a formalised methodological framework (e.g. ISO 14040/14044) that
encompasses: goal and scope definition, inventory analysis, impact assessment and interpretation. It is distinct
from more generalised 'life-cycle approaches', which tend to be qualitative or semi-quantitative in nature and
risk- or endpoint specific (Som et al., 2010).

165
 ENVIRONMENT, AS WELL AS PROMOTING CONSUMER CHOICE.

4072 The situation is further exacerbated by a lack of developed modeling approaches to predict the
4073 environmental partition, transformation and degradation of NMs (see section 5.5 on exposure
4074 modeling). A life-cycle approach was used to perform a qualitative analysis of the potential
4075 exposure and hazard arising from the incorporation of ZnO NPs into sunscreens (Osmond,
4076 2010). Khler et al. (Khler, 2008) used lithium-ion batteries and textiles as case studies in
4077 predicting potential release dynamics for CNTs through product life-cycles. Otherwise, the use of
4078 life-cycle approaches for NMs tends to be limited to cradle-to-gate analyses comparing
4079 production methods, or nano-containing and non nano-containing alternatives (see (Meyer,
4080 2009) for discussion). Such studies are of limited use in predicting environmental exposure.

4081 Point source release of free NPs, as a deliberate waste discharge or an accidental spill, can occur
4082 at various stages before their incorporation into manufactured products ('articles' under REACH
4083 regulations). Intentional release of NMs to the environment for in situ remediation of
4084 contaminated groundwater and soil may be an additional source of NMs for some environmental
4085 compartments. Nanosized zero valent iron (nZVI) in particular has been used, primarily in the
4086 USA, for its potential to dechlorinate or oxidise chlorinated solvents in polluted groundwater
4087 (see review of (Karn, 2009)). Unmodified nZVI particles have a strong tendency to aggregate and
4088 adsorb onto mineral surfaces, which limits mobility and therefore environmental distribution
4089 (Phenrat, 2007). To increase mobility through contaminated sites and maintain surface activity
4090 nZVI particles are often modified with polyelectrolyte coatings (Phenrat, 2009, Sirk, 2009). Such
4091 surface modifiers are resistant to desorption, rendering nZVI particles mobile in porous media
4092 (soil and aquifer solids) for extended periods, increasing the likelihood that they may reach
4093 sensitive environments (Kim, 2009). Bimetallic nanoclusters comprising nZVI and nickel (Ni) or
4094 palladium (Pd) have also been used to remediate groundwater contaminated with chlorinated
4095 solvents (Xu, 2009, Zhang, 2009b, Zhang, 2010, Zhan, 2009). As they can be synthesised as high
4096 aspect ratio nanorods (Yoon, 2008), their use is potentially of particular concern, given the
4097 putative link between HARNs and asbestosis-like toxicology (see section 3.2.4, p.85). CNTs and
4098 fullerenes are powerful absorbents for a wide variety of organic compounds including dioxins
4099 (Long, 2001), DDT and metabolites (Zhou, 2006), and polyaromatic hydrocarbons (Gotovac,
4100 2006). Oxidised and hydroxylated CNTs can also be used to sorb metals such as cadmium,
4101 copper and lead (Liang, 2004, Liang, 2006, Li, 2003). The potential therefore exists for CNTs, in
4102 particular, to be used for remediation of contaminated sites, although to date no studies have
4103 been published.

4104 Manufactured NMs incorporated into product matrices constitute a diffuse release source into
4105 the environment. It is predicted that diffuse sources will account for the majority of NMs

166
4106 entering the environment and that they will be the most difficult to control, given the variety of
4107 mechanisms responsible for release and their dispersed distribution (Lowry, 2009, Mueller,
4108 2008). Attempts to quantify release from silver 'nanotextiles' during product use have shown
4109 widely varying results. Silver leaching during simulated washing of textiles varied from 0.3 to
4110 377 g g-1 ((Benn, 2008, Geranio, 2009), with the latter study demonstrating that both ionic and
4111 particulate silver were released. Differences in leaching potential were primarily attributed to
4112 variation in silver loading, textile type and method of NM incorporation, although differences in
4113 methodology between the two studies will also contribute. Kulthong et al. (Kulthong, 2010),
4114 showed similar levels of variation (0 to 322 g g-1 textile) in the release of silver NPs into
4115 artificial sweat. The substance flow analysis by Mueller and Nowack (Mueller, 2008) for
4116 nanoparticulate silver suggests that dissolution will be a major form of release as a result of
4117 washing textiles and cosmetics application.

4118 Nanoparticulate TiO2 is incorporated into a diverse range of articles, from sunscreens, for its
4119 potential to adsorb UV light, to paints and plasters, where it is included as a whitening pigment.
4120 It has been demonstrated that weathering of exterior paints containing TiO2 NPs can result in
4121 direct release of particles into surface waters via storm water drains (Kaegi, 2008)39. Mueller
4122 and Nowack (Mueller, 2008) predict that 60 % of manufactured TiO2 NPs will be incorporated
4123 into cosmetics (predominantly sunscreen) and that 95 % of this will be released to wastewater
4124 treatment. In any assessment of the likely environmental impact of NM release from articles,
4125 careful consideration must be given to how the primary particle type may have been modified
4126 by other components of the preparation, and how this modification may impact on the
4127 environmental distribution and ecotoxicology of the NMs (Auffan, 2009, Osmond, 2010).

4128 Release, will occur during the whole of the product life-cycle as a consequence of matrix
4129 attrition, but may be expected to be at its most extensive during the disposal phase (Royal
4130 Society, 2004). NMs incorporated within products are likely to follow one of four disposal
4131 pathways:

4132

39
A recent publication by the same authors KAEGI, R., SINNET, B., ZULEEG, S., HAGENDORFER, H.,
MUELLER, E., VONBANK, R., BOLLER, M., BURKHARDT, M. (2010) Release of silver NPs from outdoor
facades. Environ. Pollut., 158, 2900-2905., has demonstrated similar release dynamics for nanosilver from
exterior paints.

167
4133

Disposal Route Treatment Primary environmental

compartment/s at risk.

Waste Water Flocculation, sedimentation, filtration Riparian freshwaters, soil, air

Treatment Plants biodegradation.

Landfill None Soil

Incineration Pyrolysis Air

Recycling Mechanical reclamation (milling, Air

shredding), Thermal processing
(pyrolysis, smelting)

4134 In developed countries NMs released to water, both during domestic use and for disposal,
4135 should be directed to wastewater treatment plants (WWTPs). It is estimated however, that
4136 misconnection of the foul-sewerage system (taking domestic effluents from bath, washing
4137 machines and toilets) to surface-water outfalls (directing surface water runoff from roads, roofs
4138 and hard standing) can be as high as 10% (Dunk, 2008). These polluted surface-water outfalls
4139 provide a conduit for direct release of NMs to riparian freshwater systems.

4140 Predictions of how NMs will behave during the staged treatment processes in WWTPs are
4141 difficult to make with any certainty (Brar, 2010). The initial (primary) stage of the process relies
4142 on sedimentation of solids, and for NMs, aggregation will be profoundly influenced by their
4143 original chemistry, but also by the acquisition and loss of surface functionalities arising from
4144 interaction with the surrounding medium and biota. In a simulation, surfactant modified SiO2
4145 NMs were found to flocculate and sediment rapidly, whereas unmodified equivalents remained
4146 in suspension over timescales typical for primary treatment (Jarvie, 2009). The question of
4147 whether NPs will partition to the sludge or remain in the waste-water is not yet fully
4148 investigated. Kiser et al. (Kiser, 2009) showed that the most likely fate of nano- and micro sized
4149 particles of TiO2 during the primary treatment phase was sedimentation followed by
4150 incorporation into the treated sludge ('biosolids'). In the UK biosolids are typically (60 % (Bruce,
4151 2002); >70 % predicted (DEFRA, 2006)) used as agricultural fertilizer, and soil would therefore
4152 be the initial receiving compartment. In other parts of the EU (e.g. Switzerland), where
4153 incineration is the more usual route of disposal, air may be the environmental compartment at
4154 greatest risk of contamination (see below). Secondary and tertiary stages of wastewater
4155 treatment rely on microbial digestion. Aerobic microbial communities are maintained either in

168
4156 suspension or as biofilms in reactors, and concern has been expressed that these communities
4157 may be compromised by exposure to NMs with anti-microbial properties, risking the overall
4158 efficiency of WWTPs (Choi, 2008, Neal, 2008).

4159 The release of NMs from articles disposed of to landfill has not been the subject of
4160 comprehensive study. It could be envisaged that the action of UV light, mechanical fracture and
4161 microbial decomposition (see section 5.3.2, p.170) may all contribute to the release of NMs, and
4162 the current focus on biodegradability of manufactured products may increase the rate or
4163 completeness of that process (Barnard, 2009). It is interesting to note that release from landfill
4164 does not currently feature as an exposure scenario in REACH for conventional chemicals.

4165 The potential for environmental release of NMs from incineration of articles will depend on the
4166 thermal stability of the NM in question and how well the incineration process is controlled in
4167 terms of operating temperature and filtration of gaseous emissions. Conflicting data are
4168 available on the thermal stability of carbonaceous NNs; MWCNTs and SWCNTs have been shown
4169 to be completely vaporised at between 600 and 750 C under oxidative conditions (Wang, 2006,
4170 Yang, 2004). Conversely, Cataldo (Cataldo, 2002) reports that CNTs have a thermal stability
4171 close to that of diamond ( 850 C), although fullerenes are less stable. How NMs will partition
4172 between the slag and combustion gases during incineration is unknown. A consensus exists
4173 however that the multistage flue gas cleaning systems of modern incineration plants should be
4174 sufficient to effectively remove airborne NMs (Mueller, 2008, Som, 2010). In developing
4175 countries incineration processes will be less well controlled, and may amount to burning waste
4176 on open fires. In such circumstances the potential for NM release is sizable, both because
4177 operating temperatures are not sufficiently high to fully vapourise the NMs, and because there is
4178 no filtration of gases to remove the particulates from the released gases.

4179 The imperative to recycle NMs incorporated into products is even more variable by country and
4180 particle type. A best-case scenario would be for discrete products with specific legislative
4181 requirements for recycling. For example, CNTs incorporated into lithium-ion batteries would be
4182 governed in European Union countries by the Waste Electrical and Electronic Equipment
4183 Directive, which requires components to be recycled and disposed of using best available
4184 techniques. In such scenarios recycling rates could reach 90 % (Som, 2010). Recycling processes
4185 generally break down the structure or matrix of a product, either by mechanical shredding /
4186 milling or by heating, which can release component NMs into the dust or gases. Where these
4187 processes are manually operated or only semi-automated there is concern that this may lead to
4188 unacceptable levels of exposure for workers (Khler, 2008).

4189 Controlling the release of NMs will be a key point in any risk mitigation policy; reducing release
4190 will concomitantly reduce exposure and risk. A greater focus is needed to understand how NMs

169
4191 may be prevented from entering the environment, e.g. through the use of nanostructured
4192 sorbents to scavenge particles from gaseous emissions, or through reuse, recycling and
4193 reclamation of NMs from manufactured products. Indeed some nanoengineers believe that if
4194 NMs cannot be made safe by design then eco-responsible life-cycle engineering may constitute
4195 the most feasible mechanism by which to limit environmental and human exposure (see
4196 (Alvarez, 2009) for discussion).

4197 5.3.2 Interactions, transformations and fate.

4198 Predicting the distribution and bioavailability of any NM in the environment is highly
4199 speculative, but may depend on a number of the following variables:

4200 Initial physicochemical characteristics of the particle. Core chemistry, size, particle
4201 charge and surface functionality (Jarvie, 2010) are of particular relevance.
4202 The form in which it is released (free / embedded in a matrix).
4203 The environmental compartment into which it is released (air, soil/sediment matrices,
4204 freshwater, marine) (Navarro, 2008).
4205 The interactions that occur with both abiotic and biotic components of the natural
4206 environment, and how these may transform the NM.

4207 Figure 5.2, p.186, shows possible mechanisms of interactions between an idealized NM and
4208 abiotic and biotic elements of the environment. Interactions that are predicted to be the most
4209 influential in determining NM fate are discussed in more detail below.

4210 Interactions with Secondary Pollutants. The concept of NMs acting as carriers for other toxicants
4211 was discussed for in vitro testing in section 5.2.2, p.158, and similar scenarios exist in an
4212 environmental context. Conversely, the ability of some NMs to sorb and transform pollutants has
4213 been exploited for bioremediation (section 5.3.1, p.165). The possible interactions between a
4214 target organism, a NM and a secondary pollutant are summarized in Figure 5.3, p.187. Whether
4215 the presence of a NM in a polluted environment ameliorates (e.g. influence of CNTs on pyrene
4216 bioaccumulation in earthworms (Petersen, 2009)) or intensifies (e.g. increase in uptake of Cu by
4217 D. magna in the presence of SWCNTs (Kim, 2010)) the toxicity of the secondary compound will
4218 be dependent on the specific form these interactions take. This in turn will depend on the
4219 physicochemical properties of the NMs such as size (Hartmann, 2010, Madden, 2006), crystal
4220 and aggregate structure (Cheng, 2004, Giammar, 2007, Yang, 2007) and chemical composition,
4221 and the properties of the surrounding medium (ionic strength, pH (Liang, 2006, Lu, 2006)).
4222 Baun et al., (Baun, 2008b) observed changes in the mobility of D. magna on exposure to three
4223 organic contaminants (methyl parathion, pentachlorophenol and phenanthrene) in the presence
4224 and absence of nC60 fullerene aggregates. The presence of fullerenes had no impact on the

170
4225 toxicity of the methyl parathion, decreased the toxicity of the pentchlorophenol but increased
4226 the toxicity of the phenanthrene by around 60%. Both faster uptake rates and 1.7 times higher
4227 steady-state concentrations were recorded for phenanthrene in the presence of nC60. This was
4228 correlated with the sorbtion of the compound to the nC60 aggregates (85%).

4229 A scenario can also be envisaged whereby interaction with a NM widens the environmental
4230 distribution of a secondary pollutant, for example the aggregation and sedimentation of a NM
4231 with a secondary pollutant sorbed from the water column.

4232 Interaction with Natural Organic Matter (NOM). The high surface energy of NMs promotes the
4233 adsorption of compounds from the surroundings (section 3.2.7, p.89). Nanoparticle
4234 physicochemistry can be substantially altered by these environmentally acquired coatings,
4235 particularly with respect to surface charge (Keller, 2010, Zhang, 2009a). Conceptually,
4236 environmentally acquired coatings are akin to the biomolecular corona (Chapter 4) acquired by
4237 NMs in physiological matrices such as blood, serum or cell culture media. The most influential of
4238 these interactions for the environmental distribution and bioavailability of NMs is that with
4239 natural organic matter (NOM). NOM comes in many forms; it is an umbrella term for biogenic
4240 and geogenic breakdown products from plants and algae. Primarily composed of carbon-
4241 containing compounds, NOM is present in all aquatic environments at varying concentrations.

4242 The best-studied interactions for NMs are with humic and fulvic acids and with fibrillar
4243 biopolymers such as polysaccharides and glycoproteins. The permutations that these
4244 interactions can take are vast, depending on the specifics of the NM type and abiotic factors such
4245 as pH, ionic strength and NOM concentration. The interaction of NOM with a wide range of NM
4246 types has now been investigated (Deonarine, 2009) (HgS); (Fabrega, 2009a) (Ag); (Hu, 2010)
4247 (Fe3O4); (Keller, 2010) (TiO2, ZnO, CeO2); (Lin, 2010b) (MWCNTs); (Qu, 2010) (nC60); (Quik,
4248 2010) (CeO2); (Xie, 2008) (nC60); (Zhang, 2009a) (ZnO, NiO, TiO2, Fe2O3, SiO2)). There is a
4249 consensus emerging that in general terms humic substances tend to coat NPM, conferring a net
4250 negative charge, and thereby stabilizing the colloidal dispersion of particles in generic
4251 'freshwater' conditions (Baalousha, 2008, Christian, 2008, Hu, 2010). This generalisation is not
4252 true of all NMs however, Zhang et al. (Zhang, 2009a) showed the surface charge and aggregation
4253 of SiO2 NPs to be unaffected by the presence of Suwannee River humic acid (SRHA) possibly
4254 because of the importance of ions such as Ca2+ in causing aggregation. Similarly, stable
4255 dispersions of CdSe/ZnS QDs were unaffected by the presence or absence of SRHA (Slaveykova,
4256 2009).

4257 Any reduction in aggregation is predicted to widen the dispersal of NMs in aquatic environments
4258 and increase their availability to filter feeding organisms within the water column. Conversely,
4259 interaction of NMs with fibrillar NOM, such as polysaccharides and glycoproteins, may facilitate

171
4260 agglomeration via polymer bridging (Buffle, 1998) or entanglement in polymer chains
4261 (Slaveykova, 2009) and favour agglomeration. Agglomeration and subsequent sedimentation
4262 would increase the exposure of benthic communities to aggregated forms of NMs and would
4263 restrict the NM distribution throughout the aquatic compartment.

4264 Aggregation in Aquatic Environments. Electrostatic stabilization of NMs, either by design or via
4265 an environmentally acquired coating, is sensitive to changes in the ion content of the
4266 surrounding liquid (section 3.2.4); as ionic strength increases so will NM agglomeration (Petosa,
4267 2010). The most notable change in the ionic strength of natural waters occurs with the shift
4268 from freshwater to seawater in an estuary (from <0.006M to circa 0.7M), due predominantly to a
4269 rise in the NaCl content. For natural colloids it has been demonstrated that aggregation occurs
4270 with relatively modest increases in ion concentration, the greatest degree of sedimentation
4271 occurring below 2.5 salinity (Stolpe, 2007)40. Colloidal dispersions of a range of NM (nC60,
4272 ZnO, NiO, TiO2, Fe2O3, CeO2) have been shown to aggregate in response to increasing total ionic
4273 strength, or specific ion content of 'freshwaters' (Domingos, 2009, French, 2009, Keller, 2010,
4274 Qu, 2010, Zhang, 2009a). This aggregation response is mediated by the presence or absence of
4275 NOM (Domingos, 2009, Keller, 2010, Qu, 2010, Zhang, 2009a) and by the valency of the ion
4276 (French, 2009). Qu et al. (Qu, 2010) studied the effect of a third environmentally relevant factor,
4277 UV radiation, on nC60 aggregation. In NaCl solutions UV irradiation stabilised this fullerene via
4278 surface oxidation, yet in CaCl2 solutions specific interactions between Ca2+ ions and negatively
4279 charged functionalised groups caused charge neutralisation of the surface and precipitated
4280 aggregation. It is therefore likely that many NMs dispersed in freshwater will rapidly aggregate
4281 in brackish waters and therefore benthic estuarine communities may be at particular risk of
4282 receiving high NM loadings through increased sedimentation (section 5.2.4, 160).

4283 NMs which are sterically stabilised, again either by design or incidentally, will be less prone to
4284 aggregation in the face of rising ion content. Diegoli et al. (Diegoli, 2008) show that NOM
4285 substitutes and/or over-coats the original citrate stabilizer of gold NPs, rendering them less
4286 prone to agglomeration. In this study it was hypothesized that coating NMs with NOM provided
4287 additional steric stabilization of the particles (in addition to imparting a negative charge) and, if
4288 this deduction is substantiated, NMs may pervade the lower reaches of an estuary.
4289 Agglomeration should not be thought of as a one-way process. Disagglomeration of
4290 agglomerated Fe2O3 (Baalousha, 2009a) and nC60 (Xie, 2008) NPs has been shown to occur in
4291 response to an increase in NOM concentration. In dynamic freshwater environments such
4292 temporal and spatial variations in water chemistry and NOM concentration are to be expected.

40
Seawater has a salinity of around 35

172
4293 Sedimentation. Sedimentation of aggregates is also often presented as a one-way process; a loss
4294 process by which a contaminant is removed from the system. This is a flawed representation;
4295 sedimentation to and resuspension from the upper layers of the benthic zone is a two-way
4296 process. The balance of sedimentation to resuspension will depend on the dynamics of the
4297 physical flow environment at the stream bed (Boncangi, 2009), the physical stability of
4298 aggregates, disaggregation and transformation of NMs by benthic organisms and the association
4299 of the aggregates / particles with sediment matrices and microbial biofilms (Battin, 2009).
4300 Information on the specifics of these interactions is not currently fully available for any NM.
4301 Under the correct physical conditions, permanent loss of NMs from the biosphere will occur
4302 through association with sediments and subsequent diagenetic processes, but whether this
4303 constitutes a significant sink for NMs is unknown at present.

4304 The benthic zones of freshwater, estuarine and marine environments are highly productive
4305 microbial habitats. Removal of NMs from the water column through aggregation and
4306 sedimentation simply makes them available to a different community of organisms in, initially,
4307 aggregated form where their effects are not extensively investigated (see section 5.2.4, p.160).
4308 Mhling et al. (Muehling, 2009) show that the antibiotic resistance41 of a naturally occurring
4309 bacterial assemblage is unaffected by exposure to nanoparticulate silver. A report from the same
4310 group revealed that there was also no change in the genetic diversity of the bacterial community
4311 (Bradford, 2010). Microbial, particularly bacterial, communities also exist in sediments at great
4312 depths, and the effect of NMs on these organisms is, as yet, completely uninvestigated.

4313 Interaction with Soil Matrices. It is predicted that aquatic sediments and terrestrial soils may
4314 represent a substantive environmental destination for many NMs (Mueller, 2008). Soils and
4315 sediments are essentially matrices of inorganic and organic particles surrounded by a fluid,
4316 either water or air. The retention of NMs in these environments is dependent on how they
4317 interact with the particulate fraction of the matrix. NMs may be retained by straining, where the
4318 pore size of the matrix is smaller than the nanoparticle, or by filtration where the nanoparticle is
4319 removed from suspension by interception, diffusion or sedimentation on to the surface of the
4320 soil particle (see Christian et al., 2008). NM mobility through a soil has been shown to correlate
4321 with agglomerate size rather than primary particle size (Darlington, 2009), leading to the
4322 conclusion that if NMs are suspended in an aqueous media that is conducive to agglomeration
4323 (e.g. hard freshwater, or seawater) the environmental distribution of the NM will be restricted to
4324 the upper layers of the soil or sediment; the soil will effectively act as a sieve. If primary particle
4325 or agglomerate size is smaller than sediment pore size then mobility will be controlled by
4326 surface interaction between the NP and the particulate fraction of the soil. The effect of surface

41
Used here as a proxy for Ag resistance.

173
4327 charge is likely to predominate and, as soil / sediment particles tend to be negatively charged,
4328 theory would predict that NMs carrying a like charge will be more mobile than positively
4329 charged particles (Loux and Savage, 2008). This has been demonstrated for aluminium NPs
4330 (Darlington, 2009), and has been utilised to increase the mobility of nZVI particles through
4331 surface modification with polyelectrolytes that confer a negative charge (Phenrat, 2009, Sirk,
4332 2009). The retention of NMs in porous matrices is highly dependent on surface functionalisation,
4333 with surfactant stabilized particles having a much higher mobility than would be predicted
4334 theoretically (Lecoanet, 2004). Changes to the fluid component of the system in terms of
4335 saturation, (Chen, 2008a) flow rate or chemical composition (Sen, 2006) can also markedly alter
4336 NM mobility in soil matrices

4337 Physical and Biological Transformations. NMs will be subject to transformation by the physical
4338 environment as well as by the organisms with which they may interact. Physical transformations
4339 will include dissolution (section 3.2.5, p.86), redox reactions (where the oxidation state of the
4340 surface will be changed), phototransformation and photolysis (transformation or degradation
4341 by light) and hydrolysis (degradation by water). These processes are beginning to be studied in
4342 environmentally relevant conditions for some NMs. The agglomeration and dissolution
4343 behaviour of commercial silver NPs was compared to that of micron sized equivalents across a
4344 pH range of 0.5 - 6.5 (Elzey, 2010). Total dissolution of nanoparticulate Ag was reported to have
4345 occurred at pH 0.5, whereas the micron sized particles showed no dissolution, although the
4346 methodology used may have overestimated the ion content. The authors suggested that silver
4347 NPs therefore have a greater potential to contribute to the Ag+ ion content of the environment.
4348 Acidic environments with the pH required for dissolution exist in sulphuric geysers and pools,
4349 and as a result of acid mine drainage. Phototransformation of nC60 by UVA irradiation results in
4350 oxidation and hydroxylation of the surface producing a substance structurally akin to fullerol
4351 (Hwang, 2010). This process is modified by the presence of humic acids, purportedly through
4352 the scavenging of ROS (Hwang, 2010, Qu, 2010). Fullerol has dramatically different properties to
4353 the parent substance; being hydrophilic and negatively charged it is more readily dispersible in
4354 water and will therefore have a different ecotoxicity profile and environmental distribution.
4355 Prolonged exposure (21 days) to UVA may result in the partial dissolution or mineralisation of
4356 the fullerol product (Hwang, 2010). Generating such fundamental knowledge of the processes
4357 that will contribute to the breakdown or persistence of NMs in the environment is vital.

4358 The uptake of NMs by organisms is almost exclusively considered from the perspective of
4359 toxicity to the organism. Yet the NM may also be altered by the encounter. Roberts et al.
4360 (Roberts, 2007) showed that a lysophophatidylcholine coating was stripped from SWCNTs by
4361 passage through the gut of Daphnia, with the water fleas using the phospholipid coating as a
4362 food source. The removal of the coating fundamentally altered the aggregation potential of the

174
4363 SWCNTs. A multitude of other transformations are possible, possibly the most diverse and least
4364 explored are the redox reactions that can be facilitated by bacterial communities in oxic and
4365 anoxic environments (Metz, 2009).

4366 Persistence and Accumulation. A key constituent in any environmental risk assessment is the
4367 length of time a contaminant may remain available to do harm; the longer the residence-time in
4368 the environment the greater the chance that a particular NM will accumulate, perhaps in sink
4369 habitats such as aquatic sediments. Given that possible interactions, transformations and routes
4370 of NM loss and breakdown in the environment are only beginning to be documented and
4371 understood, predicting the stability of any given NM is largely supposition. The physicochemical
4372 behaviour of NMs in vitro can give clues, clusters of C60 fullerenes for example are stable for
4373 months in solutions with an ionic strength of less than 0.05 M, and there is evidence that
4374 fullerenes may persist in the environment over geological timescales (Nowack, 2007b,
4375 Kuzyakov, 2009). Whether microorganisms are able to utilise the carbon in carbonaceous NMs is
4376 key to their environmental breakdown. Schreiner et al. (Schreiner, 2009) demonstrate that
4377 fullerol, a transformation product of nC60 (see above), can be biodegraded by basidiomycetes,
4378 with the carbon being converted to CO2 and incorporated into the lipid of these soil-dwelling
4379 fungi. To date this is the only peer-reviewed demonstration of direct biodegradation of a NM,
4380 although in vitro biomimetic studies give insights into other breakdown pathways ((Allen, 2008,
4381 Metz, 2009). If habitats lack the abiotic or biological means to breakdown NMs, concentrations
4382 may become elevated leading to increased exposure and a greater potential for bioaccumulation
4383 and biomagnification.

4384 Chapter 3 of this report discussed how variable purportedly similar NMs can be in terms of their
4385 physicochemistry, and the profound influence this can have on their behaviour. Chapter 4 and
4386 this chapter examine how this picture is further complicated by the interaction of NMs with
4387 components of physiological or environmental matrices. The adoption of 'molecular coronas' or
4388 'environmentally acquired coatings' may contribute an additional layer of complexity when
4389 predicting how NMs behave in the environment, as the coating acquired will be dependent on
4390 environmental variables. Conversely, some aspects of NP behaviour may be simplified. A
4391 consensus is emerging that NOM, present in all aquatic environments at varying concentrations,
4392 tends to coat pristine NMs (see section 5.3.2, p.170). If further research reveals this to be a
4393 widespread phenomenon, the environmental fate and distribution of NMs, in aquatic
4394 environments at least, may owe more to the interaction of NOM with the surrounding
4395 environment than to the physicochemistry of the NM. This offers the prospect that simplified
4396 models may be able to predict NM aggregation behaviour without undue inaccuracy.

175
4397 5.4 Regulatory implications of ecotoxicology and environmental fate of
4398 NMs
4399 Current requirements under REACH are inadequate to capture the potential complexity of NM
4400 behaviour in the environment; too little information is required, even at the higher tonnage
4401 triggers. It is our opinion that performing robust environmental risk assessments within the
4402 current framework would not be possible.

4403 Definitive recommendations on how the REACH data requirements should be modified to
4404 adequately capture potential environment hazards arising from NMs will only be able to be
4405 made once extensive knowledge gaps have been filled. We have made a number of
4406 recommendations for research areas that should be given priority to facilitate this process. It is
4407 important to stress that REACH regulations must remain responsive, incorporating new
4408 information as it emerges, if a robust governance framework is to be achieved. In the interim a
4409 precautionary approach should be taken.

UNTIL SUCH TIME AS IT IS POSSIBLE TO PREDICT THE ENVIRONMENTAL DISTRIBUTION OF A GIVEN NM

WITH MORE CERTAINTY, ACUTE ECOTOXICITY DATA SHOULD BE REQUIRED FOR MODEL SPECIES FROM

AQUATIC, BENTHIC AND TERRESTRIAL ENVIRONMENTS. IT IS THEREFORE RECOMMENDED THAT IN

ADDITION TO THE SHORT-TERM TOXICITY TESTS ON FRESHWATER ALGAE AND DAPHNIA THAT ARE

ALREADY REQUIRED (PRIORITISED SAMPLES > 1 TONNE AND ABOVE), SHORT-TERM TOXICITY TESTS
SHOULD ALSO BE MANDATORY FOR BOTH TERRESTRIAL AND BENTHIC SPECIES.

Part of OECD 207, earthworm acute toxicity tests, may be appropriate for use with NMs. The
filter paper test is not applicable, as it requires the toxicant to be soluble. In principle however,
the spiked soil test could be used. OECD have not validated an acute toxicity test specifically for a
benthic organism, however an ASTM method (ASTM E2317 - 04) for life-cycle toxicity tests on
the marine copepod Amphiascus tenuiremis may provide a sound base from which to develop an
assay.

ACUTE TOXICITY DATA ON BENTHIC AND TERRESTRIAL SPECIES COULD BE SUPPLEMENTED BY DATA FROM
LONG-TERM STUDIES ON DEMOGRAPHICALLY RELEVANT ENDPOINTS. AN ADDITIONAL REQUIREMENT TO
PROVIDE THESE DATA SHOULD BE CONSIDERED, AS GENERATING CHRONIC DATA WOULD ALLOW BROADER

VALIDATION OF ASSESSMENT FACTORS (SEE RECOMMENDATION FOR PROVISION OF CHRONIC DAPHNID

DATA), AND PROVIDE DATA MORE RELEVANT TO THE NEEDS OF ENVIRONMENTAL RISK ASSESSMENT.

OECD 222, EARTHWORM REPRODUCTION TEST, IS IN PRINCIPLE APPLICABLE TO NMS.

4410 Much of the information required to determine the environmental fate, behaviour and
4411 persistence of a NM will be dependent on the specific characteristics of the NM under
4412 consideration. Regulatory requirements must therefore be tailored to NM type. For example

176
4413 characaterisation of dissolution will be of primary importance for determining the persistence of
4414 metallic NMs (Liu, 2010), but is not applicable to carbonaceous NMs. Biodegradation tests are in
4415 principle appropriate for carbonaceous NMs, although the extent to which carbon in fullerenes
4416 and CNTs is available for biogenic degradation largely remains to be investigated. The potential
4417 for, and relative importance of, biodegradation of inorganic NMs is unknown (cf. dissolution).

IT IS RECOMMENDED THAT APPROPRIATE MEASURES OF NM DEGRADATION BE REQUIRED FOR ALL NMS TO

WHICH REACH REGULATIONS ARE APPLICABLE. THESE WILL INLUDE MEASURES OF DISSOLUTION FOR
SOME METAL AND METAL OXIDES IN ADDITION TO READY BIODEGRADATION FOR CARBONACEOUS NMS.
MECHANISMS WHICH LIMIT OR SUBSTANITIALLY REDUCE THE BIOAVAILABILITY OF NMS (SUCH AS
IRREVERSIBLE ADSORPTION TO SEDIMENTS) SHOULD BE INVESTIGATED AND INCORPORATED AS A

REQUIRED MEASURE IF THEY PROVE SUBSTANTIVE.

See also Below recommendation on applicability of biodegradation methodologies.

4418 More importantly however, significant doubt exists over whether validated methodologies are
4419 appropriate to provide this information. In their preliminary report on the applicability of their
4420 test guidelines to NMs, OECD concludes that their methodologies are largely appropriate (OECD,
4421 2009b). We partly agree but feel that substantial uncertainties still exist, which may require
4422 methodological changes or new methodologies to be introduced. In addition to the problems of
4423 metrology and characterisation discussed in previous sections of this report, the OECD
4424 highlights the challenge of detecting NMs in test systems where they may be bound to sediments
4425 (e.g. in simulation testing - possibly required at tonnage triggers >100 tonne) or incorporated
4426 into biological tissues (e.g. bioaccumulation tests). Although technical solutions are being
4427 developed to address this issue (carbon-14 labeling, neutron activation of metal and metal
4428 oxides etc.), such techniques are not routine or validated for regulatory purposes. Some
4429 methodologies are considered not appropriate for technical reasons. For example, the only
4430 information on environmental fate and behaviour currently required by REACH for prioritised
4431 substances at the >1 tonne trigger is a measure of ready biodegradability. The applicability of
4432 biodegradability tests is not established for inorganic NMs. For carbonaceous NMs, of the six
4433 tests available (OECD 301A-F) to measure ready biodegradability, two are not applicable as they
4434 require the carbon to be dissolved, and three require the test material to be present in relatively
4435 high concentrations (100 mg l-1) which may give rise to issues of aggregation in colloidal
4436 dispersions.

4437 REACH stipulates that information on bioaccumulation is required for substances produced in
4438 quantities over 100 tonnes per annum, however substances with log KOW < 3 do not need to be
4439 considered. Adequate measurements or calculation of KOW for NMs is at present very limited,

177
4440 and lipophilicity may not be a good predictor of uptake of NMs by organisms. The procedure of
4441 the read across from the log KOW of the bulk counterparts (as potentially could be argued by the
4442 registrant) could, for some NMs, result in the registrants escaping the requirement for testing
4443 for bioaccumulation.

4444 Uncertainty over methodological applicability has the potential to cause major regulatory
4445 difficulties. This can be illustrated by looking at the requirements to classify a substance as PBT
4446 / vPvB42. Annex XIII of REACH states that to be classified as PBT a substance must meet criteria
4447 in each of the persistence, bioaccumulation and toxicity categories. These criteria are
4448 summarised in Table 5.2, p.184.

4449 Persistence. The appropriate protocol to determine NM persistence will depend on

4450 particle type, with dissolution being more important for metal NMs and biodegradation
4451 potentially contributing to the breakdown of carbonaceous NMs. As mentioned above,
4452 difficulties in detecting NMs in test systems involving soil or aquatic sediment (OECD
4453 307 and -308 respectively) is a practical obstacle to obtaining robust persistence data.
4454 Bioaccumulation. Two OECD methodologies are appropriate to generate
4455 bioaccumulation data, the flow-through fish test (OECD 305), which gives a measure of
4456 bioconcentration (BCF) and OECD 315, bioaccumulation in sediment-dwelling
4457 oligochaetes that generates a BAF. Both methods rely heavily on KOW to predict non-toxic
4458 experimental concentrations for the test substance. Without KOW to provide a reliable
4459 guide, successful implementation of these tests would require a more iterative approach.
4460 A prerequisite to determining a valid bioconcentration factor (BCF) is free partition of
4461 the test substance between the environment and organism. There is concern that this
4462 condition is violated in the case of NMs as the gill epithelium may limit uptake of
4463 particles >0.5 nm in diameter (OECD, 2009a). There are additional concerns with both
4464 bioaccumulation methodologies in terms of maintaining particle dispersions over 28
4465 days (OECD 305) and detecting (especially non-carbonaceous) NMs in sediment (OECD
4466 315). Methods exist in the peer-reviewed literature for fish dietary bioaccumulation
4467 (Parkerton, 2008), which would circumvent problems of NM dispersion and transport
4468 across the gill epithelium. OECD 305 is currently under review with a modified version
4469 of the Parkerton dietary exposure protocol likely to be incorporated into the revised TG.
4470 Toxicity. Complications of dose and characterisation aside, determining toxicity in terms of
4471 NOEC is relatively straightforward providing that the metrics used correspond to, or are
4472 transosable to, those used in the PBT criteria.

42
Persistent, Bioaccumulative, Toxic / very Persistent very Bioaccumulative

178
4473 With significant uncertainty over the validity of methods to determine persistence and
4474 bioaccumulation, regulators are left without a credible mechanism by which to assign PBT (or
4475 vPvB) status to NMs.

IT IS RECOMMENDED THAT, AS A MATTER OF URGENCY, THE OECD PERSISTENCE AND BIOACCUMULATION

TESTS DETAILED IN TABLE 5.2, P.184 BE ASSESSED FOR THEIR APPLICABILITY TO SPECIFIC NM TYPES AND

THEIR METHODOLOGIES VALIDATED AS SUITABLE.

The precautionary principle would dictate that until such time as robust data are available for
these categories, NMs should be considered as potentially PBT if they fulfill the toxicity criteria
of <0.01 mg l-1 chronic NOEC for an aquatic species, are classified as carcinogenic (I or II),
mutagenic (I or II) or teratogenic (I, II or III) or show other evidence of chronic toxicity, as
detailed in REACH Annex XIII.

4476 In the longer-term development of an alternative 'nano-specific' decision making tree to govern
4477 ecotoxicity and fate and behaviour data requirements may provide a more coherent over-
4478 arching framework for environmental risk assessment. This would require considerable
4479 development and is beyond the scope of this review; however a logical starting point would be
4480 to require simulation testing for the potential of a NM to aggregate in a standard 'realistic'
4481 freshwater. Ecotoxicity and / or bioaccumulation assays could then be performed on the
4482 appropriate biological consortia; pelagic freshwater species or benthic sediment-dwelling
4483 species.

IT IS RECOMMENDED THAT ALTERNATIVE DATA REQUIREMENTS FOR NMS BE DEVELOPED TO REPLACE THE
CURRENT TONNAGE TRIGGER FRAMEWORK. REQUIREMENTS COULD BE BASED ON THE SIMULATED
ENVIRONMENTAL DISTRIBUTION OF THE NM AND INCORPORATE ECOTOXICITY DATA FOR A MODEL
ORGANISM FROM THAT ENVIRONMENT.

4484 5.5 Exposure Modeling

4485 Despite the lack of data on ambient levels, partition coefficients or transport, and significant
4486 other gaps in knowledge, a few attempts have been made to estimate the potential
4487 environmental risk from NMs through exposure modeling. Exposure modeling is, essentially, a
4488 formalised procedure for predicting the concentration of substances in the environment (PEC)
4489 by using mass loading (source) data and flow coefficients to describe how the substance will
4490 move, or 'partition', between environmental compartments.

179
4491 Mueller and Nowack (Mueller, 2008) used a conceptual model to estimate the average mass
4492 loading of three NPs (CNTs, silver (Ag) and titanium dioxide (TiO2)) to air, soil and water
4493 compartments in Switzerland. They compared these Predicted Environmental Concentrations
4494 (PECs) to Predicted No-Effect Concentrations (PNECs) obtained from the literature. The ratio of
4495 PEC to PNEC generates a risk quotient. Should the ratio approach or exceed 1 for any
4496 compartment then a possible risk is indicated. They conclude that significant environmental
4497 risks exist for the water compartment from the release of TiO2 NMs. There are several models
4498 from this group for Ag, TiO2 and ZnO NMs, CNTs and fullerenes. These models relate only to the
4499 nanoparticulate form of the materials, dissolution of ZnO and Ag was modeled as elimination
4500 from compartments. Gottschalk et al. (Gottschalk, 2010a, Gottschalk, 2010b) use probabilistic
4501 material flow analysis in conjunction with Monte Carlo simulations to generate PECs for CNTs,
4502 and NPs of Ag and TiO2. This approach uses probability rather than mass to model inputs and
4503 fluxes in a system, and is particularly useful as a method for reasoning when high levels of
4504 uncertainty are present due to lack of empirical data. They conclude that risk quotients for nano-
4505 Ag and nano-TiO2 in treated wastewater are sufficiently high to cause concern. Blaser et al.
4506 (Blaser, 2008) used the same approach to assess the risk posed to freshwater ecosystems by Ag
4507 NPs incorporated into plastics and textiles. Using the Rhine as a case study, they concluded that
4508 PEC: PNEC ratios greater than 1 could not be ruled out for freshwater ecosystems, in particular
4509 aquatic sediments.

4510 Luoma (Luoma, 2008) uses the source-pathway-receptor-impact framework, also utilised for
4511 NMs by Owen and Handy (Owen, 2007), to assess the potential environmental impact of Ag
4512 nanotechnologies. Scenarios, derived from different levels of consumer uptake of nanosilver
4513 containing products, predict that the environmental concentration of dissolved silver is unlikely
4514 to be elevated above that already seen in natural waters, even with substantial proliferation of
4515 Ag nanotechnologies.

4516 Limitations to Exposure Modeling. The limitation to the usefulness of these models is that
4517 currently, there is little or no empirical data with which to validate them, they are based largely
4518 on generic assumptions, which may or may not be true.

4519 There is no critical inventory of the products that contain NMs, the concentrations used and the
4520 form in which the NMs are incorporated (see section 5.3.1, p.165). Information is required on
4521 production volumes of these products, their end-uses and their routes of disposal if sources of
4522 NM release to the environment are to be identified. Predicting release from these sources will
4523 require some empirical quantification, as products purportedly incorporating the same NMs can
4524 have widely differing release characteristics (see (Benn, 2008, Geranio, 2009, Kulthong, 2010)
4525 for release of Ag from 'nanotextiles'). The REACH regulations with their focus on connectivity in

180
4526 the supply chain and life-cycle assessment of a product may facilitate accurate quantification of
4527 the input sources of NMs to the environment for the first time.

THE REACH DATABASE IS POTENTIALLY A VALUABLE RESOURCE TO THE RESEARCH COMMUNITY

ALLOWING EXPOSURE MODELS TO INCORPORATE CONCRETE SOURCE DATA FOR THE FIRST TIME. WE
THEREFORE RECOMMEND THAT ECHA DEVELOP A STRATEGY TO ALLOW DISSEMINATION OF INFORMATION

ON THE FORM AND AMOUNT OF NM IMPORTED OR PRODUCED IN THE EU, ALONG WITH INFORMATION ON

DOWNSTREAM USES.

4528 Determining environmental concentrations and form (dispersed particulates, agglomerates /

4529 aggregates) of NMs is an emerging discipline, and to date no reliable empirical data is available.
4530 Accurate quantification of NMs in environmental samples faces three challenges:

4531 Mass production of some NMs in manufactured products is still relatively new, and there
4532 will be a lag phase before breakdown products enter the environment. Current
4533 concentrations of NMs in some environmental compartments are predicted to be
4534 extremely low (pg l-1 - g l-1)(Boxall, 2007), below the detection limit of most
4535 characterization methods. Higher concentrations may be found in particularly
4536 susceptible environments, e.g. the solid phase of soils or sediments.

4537 Manufactured NMs will co-exist in the environment with natural NMs such as colloids
4538 and organic carbon, which will be present at much higher concentrations (Wilkinson K.
4539 J., 2007). Similarly, metal and metal oxide NPs will exist in an environment with
4540 background levels of dissolved and particulate trace elements. The challenge therefore is
4541 to be able to detect manufactured NMs, and distinguish elements, of anthropogenic
4542 origin as distinct from those of natural origin. This may ultimately be achieved by
4543 describing a 'signature' of the unusual physicochemical properties of a given NM.
4544 Elements of this signature could include: dispersivity, particle shape, characteristic
4545 chemical composition (e.g. a particle which is 100% CdSe is unlikely to be of natural
4546 origin), separable properties (e.g. magnetism or density) and exploitable labeling
4547 options (fluorescence, radiolabelling) which allow anthropogenic particles to be
4548 distinguished (Howard, 2010). The combination of these elements would give rise to an
4549 unusual, if not unique, 'signature'.

4550 Ideally environmental monitoring of NMs would be done using in situ methods. Each
4551 step of sample preparation risks perturbing the sample further and introducing artifacts.
4552 Sampling, sample handling and separation procedures are therefore critical if
4553 meaningful descriptions are to be made of the environmental distribution of NMs

181
4554 (Howard, 2010, Simonet, 2009). Many of the current methods used to detect, quantify
4555 and characterize NMs depend on the particles being in suspension (EM, XRD see Table
4556 3.1, p.106), or being deposited from suspension (e.g. TEM). Analysing aquatic samples is
4557 therefore relatively straightforward, but determining the concentration of NMs in soil
4558 and sediment matrices will require additional separation steps during sample
4559 preparation. Good reviews of existing characterization methods appropriate for
4560 assessing environmental samples include (Hassellv, 2008, Simonet, 2009, Howard,
4561 2010). Methodological development for NM characterisation is advancing rapidly (e.g.
4562 the use of neutron and synchrotron X-ray scattering techniques (Jarvie, 2010, Jarvie,
4563 2009)) and these techniques may overcome some of the technical challenges of
4564 characterising and quantifying NMs from environmental samples.

4565 Although the uncertainties associated with published exposure models are significant, they do
4566 offer a tool with which to prioritise NMs that may warrant further investigation. Given the
4567 expected proliferation of new NMs, this addresses an urgent need in terms of resource
4568 allocation. Furthermore, the framework of exposure modeling may help focus research on the
4569 remaining areas of uncertainty that are most crucial for development of robust environmental
4570 risk assessments of NMs.

182
 Tonnage Trigger OECD TG recommendation for NM
<1t >1t >10t >100t >1000t
A Bi B ii C
ECOTOXICITY
Long-term or reproductive toxicity in birds no no no no no no no yes?
Long-term toxicity sediment organisms no no no no no no no yes? 218, 219, 225, 315
Long-term toxicity plants and terrestrial organisms no no no no no no no yes? 216, 217, 225, 226
Short-term toxicity plants and terrestrial organisms no no no no no no yes? yes? 207
Long-term toxicity fish and Daphnia no no no no no no yes? yes? 204, 210, 215
Activated sludge respiration inhibition no no no no no yes yes yes 209
Short-term toxicity fish no no no no no yes yes yes 203, 212
Short-term toxicity algae no no yes yes yes yes yes yes 201
Short-term toxicity Daphnia no no yes yes yes yes yes yes 202
FATE AND BEHAVIOUR
Bioaccumulation fish no no no no no no yes? yes? 305
Identification of degradation products no no no no no no yes? yes?
Simulation testing no no no no no no yes? yes? 303
Hydrolysis no no no no no yes yes yes 111
Adsorption / desorption screening no no no no no yes yes yes
Biotic degradation (Ready biodegradation) no no yes yes yes yes yes yes 301, 310

4571 Table 5.1 Ecotoxicological and environmental fate and behaviour data requirements of REACH regulation by tonnage trigger. For >1 tonne
4572 group, A = unprioritised samples, B i - Prioritised phase in likely to be cat. 1 or 2 for CMR or to be PBT or vPvB, B ii - Prioritised phase in which have a dispersive or diffuse consumer use and are likely to
4573 meet the CLP criteria for any human or environmental effect, C - Non phase in - substances introduced to market after REACH implementation. Yes? Denotes data requirements decided on a case-by-case
4574 basis. Adapted from Rudn and Hansson (2010)

183
4575

Persistence (half-life) Bioaccumulation Toxicity

>60 days in seawater BCF> 2,000 in aquatic species 'Long-term' NOEC <0.01 mg/l for aquatic species
>40 days in fresh/estuarine water Classified as Carcinogenic (I or II), mutagenic (I or
II) or teratogenic (I, II or III).
PBT

>180 days in marine sediment Other evidence of chronic toxicity

>120 days in fresh/estuarine sediment
>120 days in soil

>60 days in marine, fresh or estuarine water BCF> 5,000 in aquatic species
>180 days in marine, fresh or estuarine
vPvB

sediment
>180 days in soil

105: Water solubility 305: Bioconcentration: Flow-through Fish Test 204: Prolonged Toxicity Test - Fish
Potential OECD Protocols

111: Hydrolysis 315: Bioaccumulation in Sediment Oligochaetes 211: Reproduction Test - Daphnia magna
306: Biodegradability in Seawater 215: Juvenile growth test - Fish
307: Aerobic and Anaerobic Transformation
in Soil
308: Aerobic/Anaerobic Transformation in
Aquatic Sediment Systems
309: Aerobic Mineralisation in Surface
Water - Simulation Biodegradation Test

4576 Table 5.2 Criteria for persistence, bioaccumuation and ecotoxicity, complied from REACH Annex XIII. For a substance to
4577 qualify as PBT it must fulfil one criterion in each category. Similarly vPvB substances must fulfil both vP and vB criteria.

184
4578

4579 Figure 5.1 Idealised schematic diagram of nanoparticle entry and transport through
4580 the environment. Compiled from (Baalousha, 2009b) and (Baun, 2008a).

185
4581

4582 Figure 5.2 Transformation of NMs in the environment. Modified from (Apte, 2009).

186
4583

4584 Figure 5.3 Possible interactions between target organisms, NPs (green rods) and a
4585 secondary pollutant (red dots). A) Toxicity caused by secondary pollutant in isolation, B)
4586 toxicity cause by nanoparticle in isolation, C) nanoparticle ameliorates the toxicity of the
4587 secondary pollutant and D) nanoparticle increases the toxicity of the secondary pollutant.
4588 Adapted from (Nowack, 2007a).
4589

187
4590 6 Roadmapping and Backcasting in a Socio-Ecological System
4591 as Risk Governance Tools for NMs and NPs

4592 6.1 Introduction

4593 The purpose of this chapter is to discuss situations where broad substance documentation schemes
4594 for managing NMs (including REACH) fall short of accounting for all facets of risk relevant to
4595 societies and to highlight a Socio-Ecological Systems (SES) based Technology Roadmapping (TR)
4596 tool to supplement conventional risk assessment that was developed within the project.

4597 The first part provides a literature review and a synthesis of current literature. In section 6.1.1
4598 governance problems of NMs are identified. There are social, economic and political factors that
4599 need to be accounted for along with the toxicological and ecotoxicological factors when discussing
4600 the workings of REACH. In section 6.1.2 technology roadmapping is introduced while in section
4601 6.1.3 technology forecasting methods are compared. Technology roadmapping in the context of risk
4602 governance of NMs is presented in section 6.1.4, and section 6.1.5 reviews the theory of socio-
4603 ecological systems in the same (risk governance of NM) context . In the second part, section 6.2 the
4604 method is applied a technology roadmap on silver nanoproducts is demonstrated. Conclusions
4605 and suggestions are given in section 6.3.

4606 6.1.1 NMs as a governance problem

4607 There is a regulatory gap in the governance of novel materials and technologies operating at the
4608 nanoscale (Mnyusiwalla, 2003). Novel technologies present novel risks to society. These risks are
4609 not only new: they are qualitatively different from everyday quality control of products and the
4610 more mundane risks that governance regimes are well-equipped to deal with. The problem is
4611 conceptualized through four challenges to risk management posed by NMs: ambiguity of outcomes
4612 and probabilities, distribution of outcomes in society, changes in research & development and the
4613 application of the precautionary principle. These are interrelated and are not specific to NMs alone,
4614 although NMs represent a unique convergence of these factors.

4615 Novel technologies are associated with ambiguity of both the evidence related to probabilities of
4616 events as well as the values associated with the outcomes. Risk is a mental model of causal
4617 connections into the future (Renn, 2008), and novel risks force us to look at novel ways to
4618 conceptualize our mental models and associated responses. As the risks become more complex,

188
4619 simple mental models based on prediction become less and less accurate. In order to respond to
4620 novel technologies, there should be a shift to processes of reflexive anticipation through controlled
4621 speculation based on exploring the underlying dynamics of emergence (Robinson, 2009). Levels of
4622 uncertainty over risk have been classified as simple, complex, uncertain and ambiguous (IRGC,
4623 2008). Simple risks have low levels of uncertainty, whereas complexity refers to difficulties in
4624 identifying and quantifying from the multitude of causal agents, uncertainty due to lack of scientific
4625 data, and ambiguity due to differing valuations on the outcomes. Nanotechnology almost always
4626 carries risks with all these characteristics as the risk is laid on third parties, the valuation of
4627 outcomes will inevitably be contested. The challenge is thus to build methods of identifying levels of
4628 uncertainty and incorporating the best available scientific knowledge into a public participation
4629 process where different stakeholder values are incorporated into policy.

4630 The outcomes of any technology, both negative and positive, are distributed in society in ambiguous
4631 ways. Benefits can lead to a nano-divide, where a gap between those with and without access to
4632 nanotechnology emerges (Roco, 2001). This distribution is not easily accounted for by conventional
4633 risk assessment techniques that operate on the basis of averages and unjustified assumptions on
4634 aggregation of interest between people. Public funding of R&D is also an issue, as spending should
4635 be socially justified and simple reference to GDP growth does not apply when environmental and
4636 health issues are relevant and may disproportionately affect the poor or the sick.

4637 At the same time, nanotechnology also changes the research and development process: many
4638 activities considered R&D in nanotechnology would be basic or applied science in other fields
4639 (Fleischer, 2005). Developers operate together with universities doing basic research, and the
4640 technology cycle is shortened (Lee and Stokes, 2009). This collaboration does not form a wide-
4641 ranging nano-techno-science, but is found in intermittent, point-to-point interactions between
4642 certain aspects of science and technology (Meyer, 2007). Regular governance mechanisms are not
4643 equipped to deal with these quick, unstructured technology developments.

4644 As the saying goes, better safe than sorry. The precautionary principle (PP) is the formalization of
4645 this folk wisdom into regulatory use. The precautionary principle can be interpreted through 4
4646 central components: preventive action in case of uncertainty, burden of proof on proponents of
4647 activity, exploring alternatives to the proposed action, and increased public participation (Kriebel,
4648 2001). A precautionary policy-making method will then have to address each of these issues, along
4649 with a fifth challenge posed by varying degrees of uncertainty. The PP has been taken as a guiding

189
4650 principle in many policy areas and in various national and international policy contexts (Fisher,
4651 2002), but has met with a range of criticisms (Stirling, 2007). A strong precautionary principle
4652 argues that unknown, potential risk should always lead to action, possibly forbidding the actions
4653 leading to risk. This strong version has been characterized as vague, incoherent, antiscientific and
4654 unlawful (Jordan, 1999, Peterson, 2006). The weak version of the precautionary principle
4655 maintains that it is a decision-making rule, i.e. a usable and worthwhile tool for shifting the burden
4656 of proof in situations where probabilities and outcomes are unknown (Peterson, 2006).

4657 REACH interprets the precautionary principle through the no data, no market principle. Producers
4658 are required to prove their products are within the limits of accepted risk, as defined by the various
4659 bands of reporting requirements. REACH implies a strong PP, fulfills the criteria for a weak one, but
4660 does not do much to explicate the precaution. Precaution as preset tonnage bands for future
4661 technologies is not very forward-looking as can be seen in the current debate on whether REACH
4662 in its current format sufficiently addresses the risks potentially posed by NMs. Precaution should be
4663 interpreted as more than a level of proof or a system of warning triggers, and should also include a
4664 system of stakeholder-driven scrutiny of the various pros and cons (Harremos, 2001). Likely these
4665 will be different for different areas of application of nanotechnologies. A method of assessing future
4666 regulatory needs in a way that accounts for these issues is necessary.

4667 6.1.2 Technology Roadmapping

4668 In this section, the use of technology assessment methods for governance purposes is discussed. In
4669 this paper, the focus is on a Socio-Ecological Systems approach to technological roadmapping, along
4670 with a workshop methodology. The method integrates two analytical traditions: Technology
4671 Roadmapping (TR) and Socio-Ecological Systems (SES).

4672 Technology roadmapping is a widely used family of techniques used within industry for strategic
4673 long-term planning, providing structured analyses of the co-evolution of markets, products and
4674 technologies (Phaal, 2004). Technology roadmaps were first used in within-industry analyses, but
4675 as governance and science policy interests grew, the methods were adopted in policy-making as
4676 well. TR is one example of a forward-looking technology assessment adept at analyzing social
4677 consequences; however, it is not very extensive in analyses of ongoing developments between
4678 society and technology. The integration of the SES perspective allows the dynamics in the system to

190
4679 be accounted for. The SES perspective looks explicitly at the interactions of societal and
4680 technological systems and their outcomes.

4681 For governance purposes the definition of a good outcome is more complicated than for a single
4682 company analyzing the potential markets for a product. The risk governance framework needs to
4683 involve the integration of knowledge and values held by a variety of actors and stakeholders (Renn,
4684 2009). The related methodology of backcasting can be used to insert an explicitly normative
4685 approach towards future developments. Backcasting turns the predictive framework upside down
4686 and starts from a desirable future and maps the possible actions that link the current state with the
4687 desired future.

4688 The forecasting in the roadmap and the backcasting from future can be conceptualized as events
4689 between the social, political, economic, and technological subsystems in the framework. Event
4690 chains triggered in any of the systems will have consequences elsewhere, forcing the roadmap to
4691 also include societal effects on the technological systems.

4692 6.1.3 Technology Forecasting Methods

4693 There are two broad classes of methods for technology forecasting: exploratory forecasting and
4694 normative forecasting (Cheng, 2008). Exploratory methods can be seen as methods starting from
4695 the present and aiming to forecast the future and different future scenarios and pathways. Methods
4696 such as growth curves (Ernst, 1997), the Delphi method based on expert opinions (Shin, 1998) and
4697 roadmapping can be seen to some extent as exploratory methods. The growth curve method
4698 consists of fitting a growth curve to a set of data, usually a technologys life cycle curve (Young,
4699 1993). The data used relates to technological performance. After fitting, this curve can be
4700 extrapolated beyond the range of the data, thus obtaining an estimate of future performance. This is
4701 very useful in predicting when the technology will reach a particular stage (Bhargava, 1995, Frank,
4702 2004, Watts, 1997).

4703 Roadmaps are tools for managing the future development of technology. Technology roadmaps
4704 have been widely used since the 1970s. Difficulties arise in maintaining the roadmaps: a roadmap
4705 can become quite large and wide, and it should be maintained over time. In a roadmap, you take a
4706 point in the current time and technology, and draw a map of the future paths and scenarios for that
4707 particular technology. Roadmapping can be seen as a traditional forecasting approach, where
4708 analysis focuses on which possible futures are likely to happen (Phaal, 2004).

191
4709 Technology roadmapping is a technique for long-term strategic planning (Phaal, 2004). It was first
4710 developed for industry use, either at corporate or sectoral level, and most applications and
4711 approaches reflect this (Saritas, 2010). Still, the fundamentals of the technique have been adapted
4712 for science and technology analyses (Kostoff, 2001), as well as to include wider social demands (for
4713 example, see (Kajikawa, 2008) on emerging technologies for cleaner energy).

4714 Normative forecasting methods are methods that aim to forecast a certain, defined and desirable
4715 future. Such methods include backcasting, scenario writing and relevance trees. In essence, we aim
4716 to predict the technological performance depending on future needs. Backcasting is defined as first
4717 creating a desirable future point, and then looking back at how this future could be attained.
4718 Backcasting can be very useful especially with highly complex problems which include a need for
4719 major change, and with wide time-scope and horizon (Quist, 2006). Participatory backcasting
4720 would consist of key elements such as the construction and use of desirable normative scenarios
4721 and goals, broad stakeholder participation, and combining process, participation, analysis and
4722 design with the approach (Quist, 2006).

4723 The scenario writing method takes different future points and elaborates on them, proposing
4724 different scenarios of future technology. These different methods of technology forecasting are
4725 presented in Table 6.1, p.193, with their illustrations and prerequisites.

4726

192
4727 Table 6.1 Interpretation of technology forecasting methods
Method Illustration
Delphi method The method combines expert opinions
concerning the likelihood of realizing the
proposed technology as well as expert
opinions concerning the expected
development time into a single position.
Growth curve The method is based on the parameter
estimation of a technology's life cycle
curve.
Helpful in estimating the upper limit of
the level of technology growth or decline
at each stage of the life cycle.
The case study Predictions regarding the development
method of future technologies are made based
upon an analysis of past developments.
Relevance The method is a normative approach.
trees
The goals and objectives of a proposed
technology are broken down into lower
levels in a tree-like format.
Scenario This method proposes different
writing conceptions of future technology, each
alternative scenario being based on
Prerequisite
All participants should be experts in a
given aspect of the proposed
technology.
Available historical data that covers
extended period of time. If historical
data are not available over a sufficient
time period, only limited information
can be obtained from the data.
The technology's life cycle must be
known.
Complex technology with only a small
number of organizations involved can
be studied.
The hierarchical structure of
technology development must be
known.
Scenario developers must be experts
in all aspects of the proposed
technology.
193
 certain assumptions and conditions.

Backcasting This method first defines a certain,
desirable future point, then looks back to
find pathways and means of attaining
that future.
Construction and use of desirable
normative scenarios and goals.
Broad stakeholder participation.

Combining process, participation,

analysis and design.

Roadmapping Graphic illustrations for managing the Stakeholder interest and

future of technology participation.

4728 Adapted from (Cheng, 2008, Quist, 2006).

4729 6.1.4 Our roadmapping method

4730 In the following section, the methodology for a risk governance roadmapping process is described.
4731 There are some key differences from the prior approaches listed above. This is followed by a
4732 discussion on the integration of the SES framework into roadmapping, as well as a description of
4733 the concrete workshop methods that can be used to produce these Technology Roadmaps in Socio-
4734 Ecological Systems (TR-SES). The TR-SES method responds to the challenges posed by new
4735 technologies by the integration of literature and information from multiple disciplines into a single
4736 roadmap. The intention of the workshop is not to make point predictions on future events, but
4737 rather to articulate unknowns and uncertainties by scenario-building (Hukkinen, 2008). The
4738 roadmap should lead to organized workshops where all participants are given equal access to
4739 discuss the potential for future hazards, while forcing participants to justify their arguments in a
4740 coherent manner, at specified steps in time.

4741 Phaal et al. (Phaal, 2004) identify 8 types of roadmaps for different purposes, as well as 8 formats of
4742 analysis and presentation. The varieties were built for industry purposes, and they need be
4743 amended slightly to fit the governance and regulatory framework. There are similar attempts in the

194
4744 literature. One such approach is the use of visionary socio-technological roadmaps (Ahlqvist, 2007),
4745 where layers of society and technology are combined. Applications have been published on Nordic
4746 information and communication technology developments (ibid.) and Finnish transport system and
4747 services (Tuominen, 2010). Visionary socio-technological roadmaps integrate emerging
4748 technologies with policy developments, as well as connecting the roadmap to the network of actors
4749 involved. This is done by emphasizing application visions and incorporating different social and
4750 technological sectors. These connections can be developed further by focusing on the interactions
4751 between different parts of society through the evolutionary SES perspective.

4752 The use of roadmapping in the context of NMs and nanotechnology is not novel, and indeed has
4753 been the focus of large research projects (NRM project, 2006) and European Technology Platforms.
4754 These roadmaps, available in three different arenas, materials, health and medical systems and
4755 energy, are based on a quantitative Delphi-like approach with online questionnaires. These
4756 roadmaps are exhaustive on the technological aspects, as well as in their market analyses, and map
4757 the current knowledge in the arenas extremely well. The roadmap approach suggested here is
4758 different in two ways: firstly, by focusing on the vision of the future, and secondly, by focusing on
4759 the social and environmental effects and dynamics and how these influence the future. However,
4760 even the Nanoroadmap synthesis concludes with the need for a proactive approach to tackle health
4761 and safety issues (NRM project, 2006) in a timely manner, preferably in parallel with technology
4762 development, so the two roadmapping approaches should be complementary rather than
4763 competing.

4764 The main purpose of all of the techniques described here is to generate a roadmap: (usually) a
4765 graphical representation of possible future paths leading somewhere - often a completed product,
4766 but possibly a wider vision of markets, industries or even society. In the graphic roadmap, time is
4767 usually on the horizontal axis, with a small number of bars or bands on the vertical. Each band
4768 represents a part of the institution in focus, possibly organizational divisions, but often parts of
4769 society, such as markets. Figure 6.1, p.196, shows the generic roadmap structure.

4770

195
4771

4772 Figure 6.1 Representation of the roadmap concept

4773

4774 6.1.5 Socio-ecological systems

4775 The perspective of Social-Ecological Systems has been developed in the context of common pool
4776 resources management (Berkes, 2000), but it aims to be a more general framework. An SES consists
4777 of four main subsystems: the resource system, the resource unit system, the governance system,
4778 and the user system (Ostrom, 2009), interacting with each other and the results or outcomes. This
4779 framework should be considered as a nested conceptual map, with each subsystem unraveling into
4780 a system on its own, with the level of detail growing into second and third tier variables (Ostrom,
4781 2007), as shown schematically in Figure 6.2, p.198.

4782 The system is divided into 4 subsystems, 2 background systems and the interactions between these
4783 systems and their evolution. The aim is to estimate system dynamics by answering three questions:
4784 1) What patterns of interactions and outcomes are likely to result from the set of variables? 2) What
4785 are the likely developments of the systems in the presence of a certain governance system? And 3)
4786 How robust and sustainable is the configuration to disturbances in the systems? (Ostrom, 2007)
4787 The system descriptions also have the benefit of letting the values of the outcomes be determined
4788 simultaneously with the probabilities, building the dialogue into the process, as has been suggested
4789 in policy advice documents (Davis, 2007), instead of separating the science and the dialogue.

196
4790 The background systems are the social setting and all related ecosystems. These systems set the
4791 problem and put it into a more general context. The social setting describes the framing of the
4792 problem, as well as the general background not connected to any individual actor or organization. It
4793 justifies the analysis and makes it understandable to the diverse set of interested parties or
4794 stakeholders. The setting may be called the consensus: the set of issues that are agreeable to all, and
4795 are used to feed the process. These include the governance setting (the set of possible institutional
4796 frameworks that are used), public perception of the issue, media perception of the issue, and the
4797 economic background. The references to a general public or general media opinion are included in
4798 the background when they cannot be appointed to any specific actor.

4799 The related ecosystems include other nanosystems and systems in which the current system is
4800 nested in. The SES is itself an eco(-social) system, and the related systems are similar
4801 conceptualizations. The SES framework is nested by design, and any SES can be conceptualized as
4802 part of a larger system whether defined as geographically larger, or possibly larger in timeframe,
4803 or in some other sense. An nano Risk Governance-SES application on a specific nanomaterial may
4804 relate to the SES for another material, for example if the materials are used in the same products or
4805 if they bioaccumulate in similar ways, adding to the potential risk caused by each. The SES for a
4806 specific material is nested in the SES for a specific production technique, or a specific governance
4807 framework.

4808 For the roadmap scheme, these subsystems are conceptualized as the bands of the roadmap. But
4809 instead of having each band develop independently, the developments are mapped as interactions
4810 between the systems: each action or event in a band will alter the situation in the other bands, and
4811 these developments need to be recorded, as shown in Figure 6.1, p.196.

197
 Interdependencies Social Setting

Multiple nanoparticles
Multiple Governance
in the atmosphere
Frameworks
Regulation and R&D funding enable
new materials
Nanotechnology
system Governance system

Different institutions
have different access
The interplay between R&D mechanisms for the
and production public

Realized and potential
governance decisions affect
NMs production
prouction decisions
system
Medical developments
change life-cycle which in
User groups
turn change demands for
medical developments and citizens

Stakeholder analysis
Social Network
Production system
interactions: developments
enabling new production
methods

Related Ecosystems
4812

Figure 6.2 A schematic of a Social-Ecological System for risk governance of NMs

198
4813 6.2 Nanosilver Roadmap
4814 In this section, a preliminary example of how the nanorisk governance roadmap could
4815 be developed is presented. The roadmap is mainly based on literature with
4816 supplementary material from an intra-project workshop. This roadmap is not a point-
4817 prediction forecast on future developments, but is rather a synthesis of the existing
4818 knowledge in a variety of domains. Technology and society co-evolve, and this roadmap
4819 gives a basis to evaluate the co-evolution of a particular set of technologies. The
4820 roadmap specifies paths into a sustainable situation. The roadmap could be used as an
4821 input into a regulatory discussion or a participatory process it could form the basis for
4822 new stakeholder workshops where the material is discussed and refined, or it could
4823 work as the basis for building indicators for the technology in regulation.

4824 Nanosilver Now

4825

4826
4827
4828
4829

199
4830 Nanosilver Roadmap
4831

4832

4833

4834

4835

4836

4837

4838

200
4839

4840

4841

201
4842 Nanosilver Roadmap Literature

4843 Now

4844 1) Current amount in the environment and realized production rates are not assumed
4845 to be problematic (Mueller, 2008). A hundred-fold change in release rate or
4846 mechanism would be needed in order to increase the ratio of environmental
4847 concentration to no-effect concentration to worrying levels.
4848 2) Most of the literature agrees to a feeling of unease over the current poor
4849 understanding of the potential risks from NMs (Luoma, 2008), but the first
4850 conclusions on the level of risk differ. For example, Asharani et al. (Asharani, 2008)
4851 conclude with a suggestion for restrictions on use, while Mueller and Nowack
4852 (Mueller, 2008) modeled concentrations that give no reason to expect harmful
4853 effects. Ultimately, the evaluation of risk is not a scientific question and what
4854 constitutes high, low, acceptable, and unacceptable cannot be concluded from
4855 the science alone, and must be weighted up against alternate technologies or the
4856 consequences of not developing new products / solutions.
4857 3) Risk research funding for NMs is deemed to be too low (Guzman, 2006) for
4858 example, an analysis of the US National Nanotechnology Initiative funding concluded
4859 that just 1% of research funding was directed towards risk research (Maynard,
4860 2006).
4861 4) Nanosilver is used in a variety of products the PEN inventory43 lists more than 200
4862 consumer products while the Nanowerk database44 lists 40 companies selling 75
4863 different nanosilver formulations as powders or suspensions. Medical products are
4864 the main category, but personal care, cosmetics, and cleaning products as well as
4865 textiles are common too (Chen, 2008b, Luoma, 2008).
4866 5) Nanosilver is mainly used for its antibacterial/antimicrobial properties (Luoma
4867 2008), but other properties are taken advantage of as well, including solar energy
4868 absorption, catalysis, and conductivity (Choi, 2008, Zhao, 2010).
4869 6) The benefits of many nanosilver consumer products as well as the potential hazards
4870 are unknown (Luoma, 2008). Even the evidence for medical products is
4871 unsatisfactory in some cases (Vermeulen, 2007). No cases of environmental or

43
available online at http://www.nanotechproject.org/inventories/consumer/,
44
available online at http://www.nanowerk.com/phpscripts/n_dbsearch.php

202
4872 human harm have realized, but the available data on hazards does not allow for
4873 definite conclusions (Christensen, 2010).
4874 7) There is no nanospecific regulation. The necessity of such a framework is a matter of
4875 dispute (Meili, 2006). Many writers, including Pronk et al. (Pronk, 2009) suggest
4876 amendments to current frameworks to include specific issues related to NMs.
4877 8) There are some sector specific regulatory attempts, the most exhaustive of which is
4878 the new European Union cosmetics legislation, where NMs are defined and
4879 information to authorities is required pre-market entry.45
4880 9) A variety of international and global frameworks are currently discussing
4881 nanotechnology governance. These include the OECD Working Party on
4882 Nanotechnology and the International Organization for Standardization work for
4883 nanostandards, as well as the ECHA RIPoN projects.
4884 10) Currently, it is not entirely clear where regulatory responsibility falls and should fall.
4885 Whether existing sectoral frameworks already cover NMs is one issue (Marchant,
4886 2006), while the range of government nanoscience funding from a variety of sources
4887 (Chau, 2007) demonstrates some lack of coordination in governance.
4888 11) In some product markets, producers choose to self-identify their products as nano
4889 and use it in marketing; in others, the use of nanotechnologies and NMs is not
4890 mentioned (Kimbrell, 2006).
4891 12) No mandatory labeling scheme for nanoproducts is in place (Amoabediny, 2009)but
4892 has been suggested as one tool in managing the risks from nanotechnologies (Davis,
4893 2007), as well as being identified in chapter 5 of this report as a method to collect
4894 accurate data on NM uses in products to begin to predict exposure.
4895 13) First appropriate use guidelines for occupational health and safety suggest
4896 precaution and treatment of NMs as hazardous until more information is available
4897 (Amoabediny, 2009).

4898 Roadmap

4899 14) see 4

4900 15) see 7 + 8
4901 16) The greatest growth (..) is in consumer products utilizing nanosilver to fight
4902 bacterial growth in circumstances where the benefits are less clear (Luoma, 2008).
4903 This concern is also voiced by (Senjen, 2009).

45
The regulation is available at http://eur-
lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:342:0059:0209:en:PDF

203
4904 17) The precise probability of losses cannot be well determined, even if it is likely to be
4905 low, and the possibility of widespread damage is real and something that the
4906 insurance industry must account for (Swiss Re, 2007). Risk assessment and liability
4907 are complicated when faced with high-impact, low-probability events (for example,
4908 (Renn, 2008).
4909 18) Information about products that include nanosilver is not easy to find, and
4910 companies have been known to change their mind about whether a given product
4911 includes or does not include nanosilver (Fauss, 2008).
4912 19) Some claims about nanotechnology in products have been found to be unwarranted
4913 in either misleading claims of effect or about the inclusion of NMs at all; for example,
4914 one of the 6 nanosilver socks tested by (Benn, 2008) was found not to contain any
4915 detectable silver, and some claims of nanoeffects in cosmetics are so far
4916 unquantified (Taylor, 2006).
4917 20) One of the major worries on nanosilver release has been the killing of microbial
4918 communities in wastewater plants, even if the probability is found to be very low, or
4919 no risk at all (Blaser, 2008).
4920 21) Another major worry is the development of resistance to silver in bacteria (Chopra,
4921 2007). Some cases of silver resistance in hospital settings are known, but the
4922 mechanisms are not well-known and quantification is not possible. Some have
4923 expressed concern over daily life and cosmetic products resulting in silver
4924 resistance, too (Senjen, 2009).
4925 22) A range of government agencies (ACHS, 2009, EPA, 2009) and research projects
4926 (NRM project, 2006, Nanologue, 2006) have called for a more comprehensive
4927 research programme on various aspects of nanosilver.
4928 23) Nanosilver exists in a variety of forms and size distributions, potentially critical for
4929 the risk analysis.
4930 24) Usually silver NPs are produced by bottom-up synthesis techniques that differ in
4931 metal salt precursor, solvent and reducing and stabilizing agents used (Tolaymat,
4932 2010).
4933 25) In regulation, a case issue is definition of sameness of NMs. Whether two
4934 formulations made with different methods resulting in different size distributions
4935 can be treated as the same material is crucial for regulation.
4936 26) Intellectual property rights are not well-defined in nanotechnology due to lack of
4937 knowledge at patent agencies and courts and general patent use not well fitted to a
4938 new industry in its infancy (Miller J.C., 2005).

204
4939 27) False advertising claims (Senjen, 2009) make regulation very complicated.
4940 28) The importance of public participation and democratization is emphasized in
4941 virtually all texts on nanotechnology governance and is given priority in various
4942 national and international contexts (Bowman, 2007), even though public awareness
4943 of the issues is low (Cobb, 2004).
4944 29) Release rates of silver ions by nanoproducts contribute to their antibacterial
4945 effectiveness (Luoma, 2008) as well as their risk, and methods to detect, separate
4946 and quantify effects from dissolved and NMs components are needed (Benn, 2008).
4947 30) Reliable estimates of nanosilver production rates and product categories are not
4948 available (Mueller, 2008). As these affect released amounts, our understanding of
4949 the amount of nanosilver in the environment will be vague.
4950 31) The wide range of consumer goods using nanotechnology and including NPs come
4951 with no appropriate use guidelines, as they are not labeled. For example, concerns
4952 have been voiced over increased use of biocidal nanosilver in childrens products
4953 (Senjen, 2009).
4954 32) As public understanding of nanotechnology remains low, there have been calls for
4955 education lest the image of nanotechnology will be left to science fiction writers
4956 (Sheetz, 2005). Consumers and policy-makers are the end-users of scientific data on
4957 risks, and collaboration, coordination and communication mechanisms for risk
4958 research need to be developed (Maynard, 2006b).
4959 33) Detection and monitoring methods as well as better estimates of mobility,
4960 persistence and transformation of NMs are needed (Luoma, 2008).
4961 34) Individual studies have used different estimates of the amounts of nanosized silver
4962 released into the environment, but regulation would benefit from an organized
4963 inventory of products, release rates and exposure pathways (Luoma, 2008, Blaser,
4964 2008).
4965 35) To build these databases, a variety of regulators have suggested a mandatory
4966 nanoproduct or nanotechnology database.
4967 36) A mandatory reporting scheme has been suggested by both regulators, including
4968 Belgium as European Union president46 and researchers (Maynard, 2009).
4969 37) Assessing risks and benefits of nanotechnology is complicated. Current risk
4970 assessment (RA) methods are not easily extended to complex uncertainties that are

46
http://www.eutrio.be/pressrelease/regulation-products-containing-nanomaterial-traceability-pre-
condition-acceptability

205
4971 likely to remain in nanotechnology for the foreseeable future, and a method of
4972 explicating and quantifying the uncertainties is needed (Owen, 2007, Linkov, 2007).
4973 38) In order to understand environmental exposure, releases to environment for
4974 individual products needs to be estimated (Luoma, 2008).
4975 39) Life cycle assessment (LCA) methods are basically applicable to nanotechnology, but
4976 uncertainties in knowledge and data availability need to be addressed as a matter of
4977 priority (Klpffer, 2007).
4978 40) The combination of LCA and RA would enable the assessment of overall impacts and
4979 trade-offs (Klpffer, 2007). Some of these methods are emerging (Dhingra, 2010).
4980 Examples include Nano Life Cycle Risk Assessment (Shatkin, 2008) and
4981 Comprehensive Environmental Assessment (Davis, 2007).
4982 41) The results of product-specific risk estimates need to be applied. This implies rules
4983 based on release-rates, where information comes from analyses accounting for
4984 market rates, consumer behavior and life-cycle analysis as well as toxicology and
4985 ecotoxicology.
4986 42) Simply giving the public access to information is not sufficient (Brown, 2009), but
4987 the inventory needs to be transparent, adaptive and prospective.

4988

4989 Nanosilver roadmap summary

4990 Nanosilver products, markets and knowledge will evolve significantly over the next ten
4991 years. It is common to call for research into some parts of the evolution, but an
4992 assessment of the systemic properties is necessary as well. Organized into a roadmap,
4993 the literature seems to point at three main streams calling for more research: risks,
4994 markets, and consumers. Currently, there is little understanding of NM-containing
4995 products, their use, and the possible risks involved versus the benefits from
4996 incorporation of NMs. Information is especially lacking on the interaction of NM-
4997 containig products, as harms may multiply over different products.

4998 Risk research planning should involve all these areas in directing new research. This
4999 does not imply the need for a grand, centralized research plan, but that any successful
5000 research on risk needs to account for all these factors including product development
5001 projects, if and when they include social and ecological implications in their plan.
5002 Drawing research plans in this manner will emphasize some facets of research and show
5003 gaps in research.

206
5004 Largely, research will continue within current disciplinary paths, but with more
5005 information on the systemic, dynamic effects on other parts of the system. A mandatory
5006 reporting system for nanoproducers seems likely, and research should aim at generating
5007 knowledge that enables this system and the use of the system when it is in place.

5008 Assuming that there will be an inventory of nanoproducts, the application still remains
5009 complicated. The complexity of the system and the interactions in it make simple
5010 categorizations into safety bands hard, and the risk information needs to be available to
5011 the general public, as well as for researchers monitoring exposures and such. The
5012 relationship between intellectual property rights and regulation is just one of the
5013 complicated issues for NMs.

5014 6.3 Discussion

5015 In 2020, products with nanosilver will be even more common in the market than they
5016 are today. Nanosilver is foreseen to be covered by REACH even as it stands today with
5017 the registration deadline expected by 2018. Silver ions release is likely to be lower than
5018 in previous years (due to the reduction in the use of silver as a result of digital
5019 photography). Nanosilver risks are researched and if some are found, it will probably
5020 lead to early response from regulators. Still, the need to assess future developments
5021 remains. Roadmapping when extended to cover all societal facets will help maintain
5022 risk balance / governance in situations where industry, consumers, research and
5023 governance can and does change quickly. This report discusses and demonstrates a
5024 method of building technology roadmaps for nanotechnology risk governance. In the
5025 nanotechnology literature the general conclusion seems to be that more information is
5026 needed to answer the various questions society poses to producers of novel
5027 technologies. While clarifying uncertainties is essential, there is also a need to organize
5028 and use the information that is available in a more productive and integrated manner.
5029 How to operate in the face of uncertainty is not a scientific question, but a value-based
5030 one. Thus, models where scientific risk assessments are carried out in a separate
5031 process from discussions on societal effects are not sufficient. Our method tries to
5032 integrate all of the important facets into one package. The next step with this analysis
5033 would be refinement of the methodology through applications. The method should
5034 integrate the concept of path dependency and enable conceptualization of indicators to
5035 evaluate the roadmap.

5036

207
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