Written by: Dr. Danister L.

Perera

Page 1

What is the reason for this write up?

One of the recommendations of a recent progress report of WHO mission (13th Feb. 2012) on CKDu is stated as there are 66 ayurvedic prescriptions that contain aristolochia, increase awareness of ayurvedic practitioners and public of renal toxicity of aristolochia species. If the rationale of this recommendation with regard to the findings of the mission, this statement has indirectly expressed their concern to reconsider the connection between CKD and Aritolochia spp. used for indigenous / traditional medical preparations. From the very beginning of national dialogue to identify the actual cause of CKDu, some biomedical academics were in a suspicion that Sri Lankan CKDu problem may have a connection with a kind of herbal medicine which can produce nephrotoxicity. Then they wanted to lead the whole conversation into a hypothetical conclusion to prove that irrational use of herbal medicines or unsafe of traditional medical preparations can be correlated with CKDu. But this argument was formulated on an evidence-based and a totally unscientific approach which could have led the researchers to a wrong end. What is the history and origin of this hypothesis?

The relationship between aristolochic acid (AA) and nephropathy is not a new hypothesis that is sometimes proven with animal experiments and human case studies. There are many scientific evidences to accept the nephrotoxicity of AA as one of many other active principles available in plants which are having the similar effect on kidneys. Therefore worldwide case reports have demonstrated long term use of the Aritolochia plant species as medicines or foods can produce nephropathy and ultimately lead to CKD. At the same time we must not forget that there are many other environment factors also being scientifically proven to cause CKD and sometimes it is multifactorial as in other countries still remained etiology unknown. According to some nephrologists the progressive renal interstitial fibrosis with urothelial malignancies due to consuming Aritolochia species is called Aristolochic Acid Nephropahty (AAN). This was initially given the name Chinese Herbs Nephropathy (CHN) because renal failure cases reported due to consuming Chinese herbal medicine contained Aristolochia fangchi. However, this could be considered misleading in relation to the hundreds of Chinese medicinal herbs that are safely used throughout the world, including for renal diseases. Thus alternatives such as aristolochic acid-associated nephropathy AA-induced nephrotoxicity and Aristolochia nephropathy are also used. After publishing Belgium outbreak of CHN, mostly connected with weight reducing Chinese herbal medicines, there were more incidents of AA caused nephropathy reported worldwide. Later the hypothesis that AA could be an environmental cause of Balkan-Endemic Nephropathy (BEN) or Danubian endemic familial nephropathy (DEFN) also aristolochia seeds were comingle with wheat used for bread and being baked into bread and therefore consumed on a regular basis in diet. After all the hypothesis of AA and CRF is well-established and supported by many scientific research findings over and over which eventually create an officially accepted skeptic approach to herbal medicine contained Aritolochia spp. I must clearly admit that, in this document I will not attempt to deny the hypothesis of AA causation of CKD which sometimes is claimed to have sufficient scientific evidences but probabaly challenged. My argument is all about the irrational attempt to build up a connection between Aristolochia usage in TRM products and CKDu in NCP or elsewhere in this country. Also this is not a write up against any discipline or professional opinion but to safeguard the uniqueness TRM practices and prevent any negative impact on genuine TRM contribution in beneficiary outcomes in health care in this country. Written by: Dr. Danister L. Perera Page 2

Source: International Society of Nephrology (2008)

What is the status of Aristolochia usage in Sri Lankan traditional medicine (TRM)?

In Sri Lanka, there are three Aritolochia spp. botanically recorded namely; A. indica, A. bracteolata, and A. ringens and A. indica or Indian birthwort is locally known as sassanda or sapsanda and in Ayurvedic terms it is called irwaramli or rudrajata. Mainly the dried roots of the plant are used and sometimes fresh leaves are used to extract juice for medicinal oils. In indigenous medical lore the recipes contained sassanda are prescribed for both internal and external medicine in accordance with the condition of the patient. Comparatively number of recipes contained sassanda in available in published and unpublished TRM sources is higher than Ayurvedic classical literature. Specially sassanda is an common ingredient used in snakebite treatments for controlling acute conditions but not recommended for long term use. In this context we must very carefully asses the current status of usage level and utility range of sassanda in traditional medicine as well as the consumption level sassanda and exposure potentiality to AA in such cases. Therefore following observations must be critically considered before any conclusion with regard to comment on the possibility of sassanda as a cause of renal injury of consumers of TRM. 1. After introducing Ayurveda, TRM practices and practitioners in the country getting diminished and TRM medicines are underutilized. 2. Sometimes most of the TRM preparations are not manufactured due to unavailability of plant raw materials found in natural habitat, one of which can be sassanda. 3. Even though it is widely used in snakebite treatments it is limited only for external applications and it is very rarely prescribed for internal administration. (Also it is statistically evident that most of snakebites are brought to Western hospitals and therefore TRM has a low incidence of utility in snakebite treatments) Written by: Dr. Danister L. Perera Page 3

4. The TRM multi-ingredient recipes contain more than 50-100 ingredients in one mixture and undergo a very complex process of blending to get the final product. 5. With the product diversity and holistic approach patients are always treated with many multiingredient medicines with constant change of preparations. 6. It is very rarely Ayurvedic recipes contained sassanda are popular among Ayurvedic practitioners but not commonly used. 7. Ayurveda pharmacopeia officially published by the GoSL is bearing almost zero recipes contained sassanda. 8. None of the commonly and widely used classical Ayurvedic or proprietary products contains sassanda. 9. There is no history of chronic nephrotoxicity due to ingestion of sassanda in TRM recorded so far from NCP or any area of the island. 10. No sporadic and isolated cases of chronic toxicity of sassanda in TRM indexed from among CKDu victims of NCP.

What is the phytochemical and pharmacological profile of sassanda?

Aristolochia indica L. is one of the plant species belong to Aristolochiaceae family and most of them are used in traditional systems of medicine or folk medicine in any country. It has long been used in Ayurveda, Sri Lankan TRM to treat dysentery, fever, bowel troubles, ulcers, leprosy, skin diseases, menstrual problems, cancer, lung inflammation and snakebites. The plant is also used. In the indigenous system of medicine, the plant was used as emmenagogue, abortifacient, antineoplastic, antiseptic, antiinflammatory, antimicrobial, antifungal, antihelmintic, antiulcer, antiplasmodial, antioxidant, antipyretic, antifertility, anti-spermatogenic, purgative, antipyretic, anti-inflammatory agent. The plant contain Aristolochic acid, has many medicinal properties in various diseasecondition. The Phytochemical screening revealed the presence of alkaloids, triterpenoids, steroids and sterols, flavonoids, tannins and phenolic compounds and cardio glycosides. Some studies have shown the variations of available levels of chemical compounds in different Aristolochia spp. are different. The essential oil of the aerial parts of A. indica is dominated by sesquiterpenes and monoterpenes such as -caryophyllene, -humulene, ishwarone, caryophyllene oxide I, ishwarol, ishwarane, aristolochene, linalool and -terpinolene. The roots of A. indica contain aristolindiquinone, aristololide, 2-hydroxy-1methoxy-4H-dibenzo[de,g]quinoline-4,5-(6H)-dione, cepharadione, aristolactam IIa, -sitosterol--Dglucoside, aristolactam glucoside I, stigmastenones II and III, methyl aristolate, ishwarol, ishwarane and aristolochene. Other components found in A. indica include 12-nonacosenoic acid methyl ester, aristolic acid, (12S)-7,12-secoishwaran-12-ol, (+)-ledol, ishwarone, methyl aristolate, para-coumaric acid, 5H,7,10-selina-4(14),11-diene, isoishwarane, aristolochic acids I, IVa, aristolochic acid IVa methyl ether lactam and aristolactam -D-glucoside.

Written by: Dr. Danister L. Perera

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Aristolochic acid is absorbed from the gastrointestinal tract and distributed unchanged and/or in metabolized form throughout the body. Several structurally defined metabolites were identified following the oral administration of aristolochic acid I and aristolochic acid II to rats and mice. In humans, the reduction products aristolactam I and II are the only metabolites detected in urine although full metabolic profiles have not been reported. Phase II metabolites of aristolochicacids have been identified in the urine of rats, and include N- and O-glucuronides, and acetate and sulfate esters. Synonyms of AA I: Aristolochic acid I

Aristinic acid Aristolochia yellow Aristolochic acid A Aristolochin Aristolochine Descresept Isoaristolochic acid 8-Methoxy-3,4-methylenedioxy-10-nitrophenanthrene-1-carboxylic acid 8-Methoxy-6-nitrophenanthro[3,4-d]-1,3-dioxole-5-carboxylic acid 3,4-Methylenedioxy-8-methoxy-10-nitro-1-phenanthrenecarboxylic acid Tardolyt TR 1736

Synonyms of AA II: Aristolochic acid II

Aristolochic acid B 3,4-Methylenedioxy-10-nitrophenanthrene-1-carboxylic acid 6-Nitrophenanthro[3,4-d]-1,3-dioxole-5-carboxylic acid

Several structurally defined metabolites (mainly nitroreduction products) have been reported following oral administration of aristolochic acid I (five metabolites) and aristolochic acid II (three metabolites) to rats and mice. Fewer metabolites were observed in beagle dogs, rabbits, guinea-pigs and humans than in rats and mice. The toxic effects of aristolochic acids I and II have been inferred from effects seen in patients suffering from kidney nephropathy as a result of consuming herbal mixtures containing Aristolochia species, which leads to rapidly progressive fibrosing interstitial nephritis. In experimental animals, high doses of aristolochic acids administered either orally or intravenously caused severe necrosis of the renal tubules, atrophy of the spleen and thymus, and ulceration of the forestomach, followed by hyperplasia and hyperkeratosis of the squamous epithelium. Various constituents of Aristolochia indica including aristolochic acids and aristolic acid (a metabolite) caused termination of pregnancy in female mice, hamsters and rabbits, but not rats. There is sufficient evidence in humans for the carcinogenicity of herbal remedies containing plant species of the genus Aristolochia. There are no data in experimental animals on the carcinogenicity of herbal remedies containing plant species of the genus Aristolochia. There is limited evidence in humans for the carcinogenicity of naturally occurring mixtures of aristolochic acids. There is sufficient evidence in experimental animals for the carcinogenicity of aristolochic acids. Source: International Agency for Research on Cancer (IARC) Written by: Dr. Danister L. Perera Page 5

Plant species known or suspected to contain aristolochic acid and their vernacular names
Botanical name Aristolochia spp. Aristolochia acuminata (Syn. Aristolochia tagala) Aristolochia bracteata Aristolochia clematitis Aristolochia contorta Aristolochia cymbifera Aristolochia debilis (Syn. Aristolochia longa, A. recurvilabra, A. sinarum) Aristolochia fangchi Aristolochia heterophylla Aristolochia indica Aristolochia kaempferi (Syn. Aristolochia chrysops, A. feddei., A. heterophylla, A. mollis, A. setchuenensis, A. shimadai, A. thibetica, Isotrema chrysops, I. heterophylla, I. lasiops) Aristolochia macrophylla (Syn. Aristolochia sipho) Aristolochia manschuriensis (Syn. Hocquartia manshuriensis, Syn. Isotrema manchuriensis) Aristolochia maxima (Syn. Howardia hoffmannii) Aristolochia mollissima Aristolochia moupinensis Aristolochia serpentaria (Syn. Aristolochia serpentaria) Aristolochia triangularis Aristolochia tuberosa Aristolochia tubiflora Aristolochia versicolar Asarum canadense (Syn. Asarum acuminatum, A. ambiguum, A. canadense, A. furcatum, A. medium, A. parvifolium, A. reflexum, A. rubrocinctum) Asarum himalai(y)cum Asarum splendens Common or other names Aristolochia, Guan Mu tong, Guang Mu tong Oval leaf Dutchman's pipe Ukulwe Birthwort Ma Dou Ling (fruit), Bei Ma Dou Ling (root), Tian Xian Teng (herb) Mil homens Ma Dou Ling (fruit); Tian Xian Teng (herb), Qing Mu Xiang (root), Sei-Mokkou (Japanese), Birthwort, Long birthwort, Slender Dutchman's pipe Guang Fang ji (root), Fang ji, Fang chi, Mokuboi (Japanese), Kou-boui (Japanese), Kwangbanggi (Korean) Han Fang Ji Indian birthwort (root), Yin Du Ma Dou Ling Yellowmouth Dutchman's pipe, Zhu Sha Lian Dutchman's-pipe Manchurian birthwort, Manchurian Dutchman's pipe (stem) Guan Mutong (stem), Kan-Mokutsu (Japanese), Mokuboi (Japanese), Kwangbanggi (Korean) Maxima Dutchman's pipe, Da Ma Dou Ling Wooly Dutchman's pipe, Mian Mao Ma Dou Ling Moupin Dutchman's pipe, Huai Tong Virginia snakeroot, Serpentaria, Virginia serpentary Triangular Dutchman's pipe, San Jiao Ma Dou Ling Tuberous Dutchman's pipe, Kuai Jing Ma Dou Ling Tubeflower Dutchman's pipe, Guan Hua Ma Dou Ling Versicolorous Dutchman's pipe, Bian Se Ma Dou Ling Wild ginger, Indian ginger, Canada snakeroot, False coltsfoot, Colic root, Heart snakeroot, Vermont snakeroot, Southern snakeroot, Jia Na Da Xi Xin Tanyou-saishin (Japanese) Do-saishin (Japanese)

Worldwide, there are an estimated 200 to 350 Aristolochia species, and virtually all of them contain aristolochic acids. Asarum species (wild gingers) also are widely distributed in the United States. Plants of the genus Hexastylis, a group of rare plants endemic to the southeastern United States, were reported to have unexpectedly high levels of aristolochic acids. Concentrations ranged from 3 to 12,980 ppm for AA I and from not detected to 6,325 ppm for AA II. In Asarum species, concentrations of AAs I and II ranged from trace levels to 3,377 ppm. Other studies detected AA IVa at concentrations of 79 to 3,360 ppm of crude drug, aristolactam I at 6 to 358 ppm, and aristolactam II at 14 to 91 ppm. Written by: Dr. Danister L. Perera Page 6

What are the international regulations related to herbal medicine with Aristolochia?

The Therapeutic Goods Administration (TGA) of Australia has issued a Fact Sheet stating that all species of Aristolochia are prohibited for supply, sale or use in therapeutic goods in Australia. The TGA also issued a Practitioner Alert to communicate its concern about traditional Chinese medicine herbal products that are known to contain, or suspected to contain, Aristolochia species, which may contain AA. The TGA has published three lists of botanicals or products at risk of containing AA: botanicals known or suspected to contain AA (Group A); botanicals which may be adulterated with AA (Group B); and products which have Mu Tong and Fang Ji as declared ingredients (Group C). The use of Aristolochia in unlicensed medicines was prohibited by the Medicines Control Agency (2001) in the UK in July 1999, and a further temporary prohibition covering certain herbal ingredients at risk of confusion with Aristolochia came into force in June 2000. The UK Committee on Safety of Medicines (2001) made these prohibitions permanent by issuing a Statutory Instrument to prohibit the sale, supply and importation of any medicinal product consisting of or containing certain plants belonging to a species of the genus Aristolochia or consisting of or containing Guan Mu Tong; or belonging to any of eight specifically listed plants (Akebia quinata, Akebia trifoliata, Clematis armandii, Clematis montana, Cocculus laurifolius, Cocculus orbiculatus, Cocculus trilobus,Stephania tetrandra); or consisting of or containing an extract from such a plant. In addition, the European Agency for the Evaluation of Medicinal Products issued a position paper in October 2000, warning European Union Member States to take steps to ensure that the public is protected from exposure to aristolochic acids arising from the deliberate use of Aristolochia species or as a result of confusion with other botanical ingredients. The European Commission (EC) (2000) has prohibited aristolochic acid and its salts, as well as Aristolochia species, and their preparations in cosmetic products. In 1999, Health Canada issued a warning not to use products containing Aristolochiadue to potential risk of cancer, cell changes and kidney failure. Health Canada issued four additional warnings and advisories in 2001 advising not to use products labeled to contain Aristolochia. The Food and Drug Administration of the USA has issued a Consumer Advisory to communicate its concern about the use and marketing of dietary supplements or other botanical-containing products that may contain aristolochic acids. It has also posted a listing of botanical ingredients of concern, including: botanicals known or suspected to contain aristolochic acids; botanicals which may be adulterated with AA; products in which Mu Tong and Fang Ji are declared ingredients; and botanical products determined by FDA to contain AA. Why multi-ingredient products in TRM?

When many poisonous plant material and toxic metals used in TRM for treating patients, are processed in a proper manner and rationally dispensed it shows efficacy without any side-effect or iatrogenic complication. Almost all of them are multi-ingredient and deliberately mixed in which the chemical profile of final product is not analyzed, screened or identified. These kinds of products demonstrate unique collective activity which is totally different from individual ingredients in primary source. It is evidently proven that according the solvent also the level of solubility and percentage of presence of compounds can vary. In this complex phytochemical mixture many reactions can occur among natural components in both synergetic and antagonistic manners which have not been validated in terms of pharmacodynamics and pharmacokinetics. In this phytochemical complexity there may be antagonistic compounds which can counteract nephrotoxic action of AA and synergetic factors to enhance nephroprotective action of the product. Therefore following observations are very important. Written by: Dr. Danister L. Perera Page 7

1. Final product can have a different phytochemical, pharmacological generated out of blending

and therapeutic effect

2. The intake of single agent is very minute quantity in recommended dosage of final product. It can be always less than recommended safe level of a single ingredient. 3. Most of the studies have shown that AAN or progressive CRF had occurred after ingestion of high dose of AA (active principle) for long time. 4. Sometime this multi-ingredient product is administered with another natural vehicle / adjuvant prepared specially according to the patients condition. 5. Pharmacodynamics of AA and metabolic outcomes can vary due to diversity of phenotypes according to the latest findings. Why research findings of CHN / AAN cannot be applied to TRM products in Sri Lanka? Findings of CHN / AAN researches Arostolochia containing TRM (A) products

Most of the CHN / AAN patients have taken No TRM (A) medicine in the market for selfChinese herbal medicine with AA as a self- medication (OTC) or on medical prescriptions medication (OTC) without medical prescription or advice All most all of CHN / AAN patients have taken TRM (A) products are not prescribed for long term medicine for long time and in normal TRM medications also follow a rational discontinuation manner. Some of them have taken more than one medicine TRM medications are prescribed always in an contained AA in different dosage forms (pills, interchanging consecutive sequence. decoctions etc.) Most of them have taken medicine in high doses Mostly TRM (A) are prescribed in acute or emergency conditions like snakebites and given in small doses which contain very low amount of AA.

Most of proprietary Chinese medicines are not All TRM (A) medicine administered internally has a having high number of ingredients very high number of ingredients that may inhibit direct ingestion of AA and promote pharmacodynamics synergism. In some cases medicines were adulterated with In Sri Lanka Aristolochia spp. are not a source of Aritolochia spp. adulteration in TRM CHN / AAN reported in a specific consumer group Utility of TRM in Sri Lanka has an island-wide or specific geographical area distribution and TRM (AA) products are not geographically-specific Researchers conducted with AA in animal models These data will not be sufficient for validate TRM have proven the causation of progressive products in human nephropathy and renal malignancies. Written by: Dr. Danister L. Perera Page 8

Is there any evidence for utility TRM contained AA in NCP?

No proper study has been done so far for such conclusion in NCP or among the CKD victims. Also it is not rational to assume that CKDu victims were potentially exposed to TRM with AA for any similar medical reason or any geographically specific medicine within last three decades. No medication history of TRM usage of victims was traced in any study pertaining to CKD and no data were collected about usage or availability of sassanda containing recipes in TRM practice within the NCP. Null and void connection between CKDu and Aristolochia spp. in TRM Low availablility of plant Low utility of recipes Phytochemical complexity Multi-ingredient products No specific geographic usage No commonly use products No long term usage No history of chronic AA toxicity

Availability of Aristolochia species in TRM recipes

CKDu in NCP

Different levels of toxicity and clinical features, histo-pathology, renal injury caused by them cannot be compared or synchronized into AAN
Multi-ingredient TRM product or admixture containing Aristolochia indica or sassanda with other medicines or mixed with an adjuvant

T O X I C I T Y

Crude form of the whole plant of Aristolochia indica or sassanda

Water extract of the whole plant of Aristolochia indica or sassanda

Roots of Aristolochia indica or sassanda

S A F E T Y

The ethonolic extract of roots of Aristolochia indica or sassanda

Pure Aristolochic acid

Method of preparation, dosage form, mode of administration, daily intake, period of usage, discontinuity, interactions with other medicines, kidney function, and other pathophysiological factors should critically be evaluated before conclusion. Also we must not forget the pharmacogenomics is an emerging subject in the field of assessing effect of substances within an individual biophysical model. Written by: Dr. Danister L. Perera Page 9

Why this recommendation is WRONG, unjustifiable and prejudiced statement? It is based on technically wrong information generated through a simple and random survey conducted by a group without professional, expert and academic involvement. It implies a wrong assumption that may lead to establish an irrational hypothesis on positive relationship between TRM and CKDu NCP or elsewhere. It reflects a wrong meaning and suspicious concern on TRM products and practices nationally as well as internationally. It has no rational connection with the findings on etiology of CKDu which are obviously related to arsenic and cadmium due to chronic pesticide poisoning. It will have a negative impact unreasonably on TRM with regard to public confidence which prevailed for centuries in the country. It may lead to potential unnecessary regulatory control on TRM in future and encourage detrimental enforcements in export market.

What should be done? 1. The flagship stakeholder of the sector, Ministry of Indigenous Medicine and relevant institutions should urgently respond to the WHO and request for justifiable principles based for such recommendation. 2. TRM authorities of should make a request for evidences which should have been based for WHO recommendation and actively analyze the scientific rationale of such data and validate reliability, acceptability, applicability and sufficiency of the same. 3. BMARI should urgently initiate a national research programme to evaluate any probable toxicity or potential chronic poisoning based on pharmaco-epidemiology. 4. Ministry of indigenous medicine must express their commitment for extending safety of TRM products through a national pharmaco-vigilance initiatives and updating ADR database on TRM. 5. Awareness must be raised among professionals, academics and researches pertaining to these issues for encouraging them for further verifications through scientific studies and peer reviews. 6. A national level dialogue must be created to build up a meaningful and productive understanding between biomedical and TRM sectors in a mutually respected manner. 7. Ministry of health and TRM authorities with the support of international agencies WHO must build up a reciprocally co-existing professional culture and introduce pluralistic health care principles into medical education. 8. WHO must consult a wider TRM group to revisit, scrutinize and redraft the above mentioned recommendation and revalidate the TRM practices in encountering such health problem.

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ANNEXURE I: Proposed pathway for metabolic activation and detoxication of aristolochic acid I (AAI), leading to renal injury and urothelial cancer. Aristolochic acid nephropathy (AAN); Balkan endemic nephropathy (BEN); 7(deoxyadenosin-N6-yl)aristolactam I (dA-AAI)

Source: Interdiscip. Toxicol. 2008 June; 1(1): 812

PMCID: PMC2993473

Although hepatic CYP enzymes were found to detoxicate AAI in mice, thus decreasing its renal toxicity individual enzymes, which might metabolize (activate and/or detoxicate) AAI in vivo, and their impact on AAI-mediated nephrotoxicity and carcinogenicity, have not been fully resolved as yet. Therefore, such a subject remains to be investigated. Namely, the evaluation of inter-individual variations in the human enzymes playing a major role in AAI activation and detoxication, including their genetic polymorphisms, remain a major challenge to explain an individual's susceptibility to AAI, and to predict cancer risk among the AAN and BEN patients. Therefore, the study we started in our laboratory addresses still unsettled question whether the metabolism of AAI, and if so, which enzymes participating in this process, determine pathophysiological effects of this compound in development of AAN and BEN diseases. However, some differences between AAN and BEN patients, and rabbits with AAN should be pointed out. Involvement of the columns of Bertin in BEN autopsied patients appears less prominent than in AAN patients. On clinical grounds, four findings in AAN patients differ from BEN: firstly, the sex-ratio in BEN is approximately 1/1, in contrast to the female preponderance in human AAN; this reflects the almost exclusive female attendance of the X clinic. Secondly, AAN patients progress to end-stage renal failure within a few months or years, in contrast with the slow evolution of BEN and of AAN in rabbits over time. This could reflect a higher level of toxic exposure in AAN than in BEN patients and interspecies differences in the susceptibility to the toxic agent as suggested by the differences in the metabolic transformation of AAs between humans and animal species. Written by: Dr. Danister L. Perera Page 11

ANNEXURE II Aristolochic acid I Chem. Abstr. Serv. Reg. No.: 313-67-7 Deleted CAS Nos.: 12770-90-0; 61117-05-3 Chem. Abstr. Serv. Name: 8-Methoxy-6-nitrophenanthro[3,4-d]-1,3-dioxole-5carboxylic acid Synonyms and trade names: Aristinic acid; aristolochia yellow; aristolochic acid A; aristolochin; aristolochine; Descresept; isoaristolochic acid; 8-methoxy-3,4methylenedioxy-10-nitrophenanthrene-1-carboxylic acid; 3,4-methylenedioxy-8methoxy-10-nitro-1-phenanthrenecarboxylic acid; Tardolyt; TR 1736 Aristolochic acid II Chem. Abstr. Serv. Reg. No.: 475-80-9 Deleted CAS No.: 79468-63-6 Chem. Abstr. Serv. Name: 6-Nitrophenanthro[3,4-d]-1,3-dioxole-5-carboxylic acid Synonyms: Aristolochic acid B; 3,4-methylenedioxy-10-nitrophenanthrene-1-carboxylic acid Aristolochic acids (a) Description: Crystalline solid [aristolochic acid I] (Buckingham, 2001) (b) Melting-point: 281286 C, decomposes [aristolochic acid I] (Buckingham, 2001) (c) Solubility: Slightly soluble in water; soluble in acetic acid, acetone, aniline, alkalis, chloroform, diethyl ether and ethanol; practically insoluble in benzene and carbon disulfide (ONeil, 2001) (d) Octanol/water partition coefficient (P): log P, 3.48 Source: IARC Monographs Vol. 82

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ANNEXURE II Herbal medicines were first included in the WHO International Conference on Drug Regulatory Authorities in 1986 [30]. In 1991, WHO prepared draft guidelines for assessment of herbal medicines, which were adopted by the 6th International Conference on Drug Regulatory Authorities (ICDRA). This includes basic criteria for quality, safety, and efficacy of herbal medicines. These guidelines provide valuable assistance to national regulatory authorities, scientific organizations, and manufacturers to undertake assessment of documentation of submissions. Warning concerning interstitial renal fibrosis Belgium. On 14 March 2000, the Ministry of Public Health in Belgium issued a public statement addressed to all persons having consumed Chinese plant preparations in the past 10 years, in order to allow these persons to obtain advice on the need for further investigations of their renal function. The reason for this announcement is the observation that urothelioma has been recently discovered in half of the patients having developed end-stage renal failure following the intake of Aristolochia in the early 1990s in herbal slimming agents that were found to contain Aristolochia fangchi instead of Stephania tetrandra or Magnolia officinalis. (1,2). More than 100 patients have been identified in Belgium with interstitial fibrosis of the kidney, a form of nephropathy sometimes referred to as "Chinese herbs nephropathy". At least 70 of these patients have required renal transplant or dialysis. It is estimated that in Belgium about 10,000 persons have used Aristolochia from a contaminated lot in the early 1990s. A "Dear Doctor" letter has been sent to all physicians and pharmacists advising them to refer cases with abnormal serum creatinine levels to a specialised centre. Malaysia. The Drug Control Authority (DCA) decided to cancel the registration of all products containing the herbal ingredient Aristolochia which contains aristolochic acid. This decision was made in the light of reports received from Europe where the use of products containing Aristolochia has been associated with interstitial nephropathy and end-stage renal failure. Aristolochic acid is found in traditional Chinese medicines used as a diuretic and in slimming preparations.(3) United Kingdom. The Committee on Safety of Medicines has banned the import, sale and supply of medicinal products containing aristolochia with effect from 28 July 1999. This action was taken after two reports of end-stage renal failure associated with aristolochia in herbal medicines being taken to treat eczema. As in the cases reported from Belgium, there is evidence that aristolochia was mistakenly used instead of other plants, in these cases clematis, or that the products were contaminated with aristolochia. The Medicines Control Agency is considering making the banning order permanent.(4) United States of America. The Center for Food Safety and Nutrition has issued a "Dear Health Care Professional" letter and a letter to industry warning about nephrotoxicity associated with botanical products found to contain aristolochic acid.(5) Although the FDA has not yet received any reports of such cases, the Center stresses that a thorough history of use of dietary supplements as well as traditional medicines, including Chinese and Ayurvedic (Indian) preparations, should be sought as part of the medical history, particularly in cases of unexplained interstitial renal fibrosis. The Center also sent a letter to industry urging manufacturers to take steps to positively confirm that botanical ingredients are not inadvertently contaminated with plant material containing aristolochic acid, by testing their products to confirm the absence of aristolochic acid, by identifying and reporting adverse events, especially renal system disorders, associated with any product that contains an ingredient that may contain aristolochic acid. The FDA will also issue an import alert to ensure that all products imported into the USA will be examined to ensure that they do not contain aristolochic acid. Written by: Dr. Danister L. Perera Page 13

Note: Preparations containing aristolochic acid were withdrawn in Austria, Germany, Egypt and Venezuela in the early 1980s after the publication of a study in rats that revealed the carcinogenic potential of aristolochic acid. UN Consolidated List of Products whose consumption and/or sale have been banned, withdrawn, severely restricted or not approved by governments. Source: WHO Pharmaceuticals Newsletter No. 2, 2000 China's State FDA (SFDA) has banned two commonly used herbs containing aristolochic acid, a toxin reported to be linked to kidney failure and cancer. Manufacturers have been directed to replace Aristolochiafangchi and Aristolochia debilis with Stephania tetrandra and Inula helenium respectively, in their traditional medicine formulations by 30 September.The Provincial Drug Bureaux has been instructed to carry out inspections to ensure compliance with the ban by 31 October. Medicines found to contain either Aristolochia fangchi or Aristolochia debilis after 30 September will be treated as fake under Chinese law. By a previous order, special restrictions were imposed on four other potentially harmful aristolochic acid-containing herbs (Fructus Aristolochiae, Aristolochia mollissima Hance, Herba Aristolochiae and Aristolochia tuberose) in China; there was no outright ban on these products. Several countries withdrew aristolochic acid containing preparations in 1981 following the demonstration of a carcinogenic potential in a three month toxicity study in rats (see UN Consolidated List of Products whose consumption and/or sale have been banned, withdrawn, severely restricted or not approved by governments, Eighth Issue, Pharmaceuticals, available at
http://www.who.int/medicines/li brary/docseng_from_a_to_z.shtml#p.

Source: WHO Pharmaceuticals Newsletter No. 5, 2004 Scientific Committee on Cosmetics and Non-Food Products (SCCNFP) Summary sheet on Commission Review (ref. XXIV/1991/97) Ingredient : Aristolochic acid (I,II,V) CAS n : 313-67-7; 475-80-9; 15918-62-4; 61117-05-3 Function and max in use concentration : botanical Relevant toxicity data : Assessment of acute toxicity (oral, i.v.) : Toxic for kidneys. Assessment of chronic/sub-chronic toxicity : Repeated dose toxicity (oral 21 d., 90 d.): multiple carcinogenic gastrointestinal tract cell metaplasia. Assessment of genotoxicity/carcinogenicity: Mutagenic in vitro, clastogenic in vitro and in vivo. Very potent carcinogen affecting forestomach, kidneys and several other organs. Human data: Causal factor in "Chinese herb nephropathy" (kidney destructive process). Classification: 2a (See Classification of Substances) SCCNFP Opinion Aristolochic acid (A.A.) is mutagenic in bacteria, in mammalian cells and in Drosophila. A.A. binds covalently to DNA inducing similar DNA adducts in vitro and in vivo in rats. A.A. is a very potent carcinogen affecting the forestomach and several other organs including the kidneys in mice and rats after short induction periods with low doses. In rats, the effects were shown to be time and dose dependent. Very recently (1996) A.A. derived DNA adducts biomarkers of A.A. exposure, were found in renal tissues of patients suffering from a rapid kidney destructive fibrotic process designated as Chinese Herbal Nephropathy. Written by: Dr. Danister L. Perera Page 14

These data support SCCNFP opinion that Aristolochic acid and salts must not be used in cosmetic products. Moreover, A.A. being the active constituent of Aristolochia species, the SCC is of the opinion to extend that the ban to Aristolochia species and preparations. Statement on the toxicological evaluation The SCCNFP is the scientific advisory body to the European Commission in matters of consumer protection with respect to cosmetics and non-food products intended for consumers. The Commission's general policy regarding research on animals supports the development of alternative methods to replace or to reduce animal testing when possible. In this context, the SCCNFP has a specific working group on alternatives to animal testing which, in co-operation with other Commission services such as ECVAM (European Centre for Validation of Alternative Methods), evaluates these methods. SCCNFP opinions include evaluations of experiments using laboratory animals; such tests are conducted in accordance with all legal provisions and preferably under chemical law regulations. Only in cases where no alternative method is available will such tests be evaluated and the resulting data accepted, in order to meet the fundamental requirements of the protection of consumer health. Source: Opinion of the intended for Consumers concerning Aristolochic acid and salts adopted by the plenary session of the SCCNFP of 21 January 1998 Regulations: Food and Drug Administration (FDA) Federal Food, Drug, and Cosmetic Act as amended by the Dietary Supplement Health and Education Act Manufacturers and distributors as of 2007 must record adverse events and report to the FDA serious adverse events reported to them about their products. Label requirements for dietary supplements have been established. Manufacturers must establish and meet specifications for identity, purity, strength, and composition and for limits on contamination of dietary supplements under current Good Manufacturing Practices (cGMP) regulations published in 2007. Warnings and Alerts Food and Drug Administration (FDA) Warnings issued in 2000 and 2001 (FDA 2000, 2001a,b) covered botanical products that contain aristolochic acids: Practitioners who prescribe botanical remedies urged to discard those products containing aristolochic acids. Manufacturers and distributors urged to ensure that botanical products are free of aristolochic acids. Consumers urged to immediately discontinue use of botanical products that contain or likely contain aristolochic acids. An import alert issued in 2000 and revised in 2007 provided for the detention of products labeled as Aristolochia or any that could be confused with it unless analytical evidence shows no aristolochic acids. Source: National Toxicology Program, Department of Health and Human Services

Written by: Dr. Danister L. Perera

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ANNEXURE IV

Source: Nephrology Rounds. 2008 Feb. 6(2) Written by: Dr. Danister L. Perera Page 16