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MINUTESPresent:
CONTENTS
ITEM 1: ANNOUNCEMENTS/APOLOGIES FOR ABSENCE 1. The Chairman welcomed two new members Dr Gillian Clare and Dr Elizabeth Parry who had expertise in cytogenetics and aneuploidy. The Chairman also welcomed Dr Claire Cowles (HSE), Mr T Holmes (PSD) and Dr T England (DH Toxicology Unit). 2. An apology for absence for absence was received from Professor Ashby. 3. Members were reminded of the need to declare any interests before discussion of items. ITEM 2: MINUTES OF THE MEETING ON 7TH FEBRUARY 2002 (MUT/MIN/02/2) 4. The minutes were approved with minor amendments. ITEM 3: MATTERS ARISING FROM THE MEETING OF 7TH FEBRUARY 2002
3.1 Dichlorvos 5. The COM was informed that its advice on the mutagenicity of dichlorvos had been agreed and was available on the COM website. Additionally, the COM’s recommendation on a strategy for testing dichlorvos for site of contact mutagenicity had been forwarded to the Advisory Committee on Pesticides (ACP). The ACP had considered this advice. In its role as a regulatory committee, the ACP had requested that an oral gavage study be conducted in the Muta mouse, to investigate the mechanism of forestomach tumours (and to determine whether a genotoxic mechanism could be discounted). Approvals for the advertisement and sale of dichlorvos products had been suspended until these new data become available.
3.2 PAVA 6. Members heard that the COT had agreed a statement on the health effects of PAVA spray, which incorporated the COM advice. The COT had made one small addition to the conclusions regarding genotoxicity, to state that the structure of PAVA suggests the possible formation of reactive oxygen species from the phenol moiety, and other possible active metabolites, which may be mutagenic. The COM Chairman had agreed this change.
3.3 Glossary of terms (MUT/02/14) 7. The committee was reminded that the COM had established a sub-group to help revise the COT/COC/COM glossary of terms related to mutagenicity. Members were informed that one member of the sub-group had taken a fresh look at this issue and had circulated to sub-group members explanations for basic genetic terms, which would then be used as a basis for the other (e.g. test-specific) terms. Members were requested to send any comments they might have to the secretariat. It was hoped that the sub-group could complete its work by e-mail correspondence, with final proposals being presented to the Committee at the next meeting. ITEM 4: MALATHION (MUT/02/11) 8. The minutes of this item are now published as paragraphs 24 to 43. ITEM 5: DIMETRIDAZOLE (MUT/02/12) 9. No interests were declared. 10. Members recalled that Dimetridazole (DMZ) is used in the UK both as a feed additive, for turkeys and guinea fowl, and as in veterinary medicine for game birds. There is controversy about its mutagenicity. The EU Committee on Veterinary Products (CVMP) had concerns regarding the mutagenicity, and felt that further data was needed. This was not forthcoming, and they have recommended that it should not be used in food producing animals. The UK has a derogation allowing use in pheasant and partridges. With regard to food additive use the EU’s Scientific Committee on Animal Nutrition (SCAN) have concluded that there was no genotoxic hazard to consumers. Nevertheless, the European Commission has deleted dimetridazole from the feed additives Directive 70/524 and will no longer be permitted for use as a feed additive after 14 May 2002 (Commission Directive (EC) 2205/2001). 11. DMZ had been considered at the COM meeting in October 2001 and a draft statement agreed. The overall conclusion was that in the absence of data from 2 adequately conducted in-vivo mutagenicity studies it would be prudent to assume that DMZ had mutagenic potential. The statement was sent to the data holders and they immediately offered to provide full reports to 3 in-vivo studies on DMZ, mentioned in earlier documentation. The COM was informed of this at their February 2002 meeting, and agreed that the draft statement could be finalised after consideration of the additional data at the April meeting. The COM was asked to review the full reports of the 3 in-vivo studies, namely, bone marrow micronucleus test in mice, liver UDS assay in rats, and dominant lethal assay in mice. 12. Members reaffirmed that DMZ was mutagenic in-vitro but agreed that the section of the draft statement which referred to in-vivo data should be rewritten to concentrate on DMZ as the full reports of three in-vivo studies were now available. There was no longer any need to refer to limited data on 2-hydroxymethyl-1-methyl-5-nitroimidazole (DMZ-OH, a metabolite of DMZ). 13. With regard to the new in-vivo data, Members agreed that the bone-marrow micronucleus assay in mice was adequate and negative. Members expressed concerns that the in-vivo rat liver UDS assay had not been conducted to modern standards. It was recognised that the study predated the adoption of OECD guideline No 486, but members were concerned that the omission of an early sampling time of 2-4 hours post treatment. Therefore, study would thus have failed to detect the mutagenicity of some compounds such as dimethylnitrosamine. 14. FSA asked whether the in-vivo data would cover concerns regarding the possibility that metabolites of DMZ formed by nitroreductase activity in the gastrointestinal tract of animals could carry over to residues in the meat, and also the possibility of nitroreductase activity by the gut flora in consumers. Members felt that any residual concerns regarding the mutagenicity of DMZ and its metabolites would be addressed by a negative result in an in-vivo assay using the oral route in rodents, with the liver as a target organ. Thus it was agreed that a rodent liver UDS assay to modern standards was important. 15. Members agreed that the Dominant lethal assay in mice was adequate and negative. It was noted that a slight reduction in implanations had been observed in this study but this was not indicative of Dominant Lethal mutations. 16. The Committee agreed that further data were needed to provide adequate reassurance that the mutagenic activity seen in-vitro was not expressed in-vivo. This would comprise a liver UDS assay in rats conducted in accordance with the OECD guideline; it was recommended that 4 animals should be analysed at each dose level and time point.The minutes of this item will be published when the COM statement has been finalised. ITEM 6: FLUNIXIN MEGLUMINE AND MEGLUMINE (MUT/02/15 &16) 17. The minutes of this item are now published as paragraphs 44 to 48. ITEM 7: DEET (DIETHYL-M-TOLUAMIDE) (MUT/02/13) 18. No interests were declared. The Department of Health was seeking advice on the health effects of the insect repellent DEET and this had been considered at the last COT meeting with a view to agreeing a statement on this compound. The advice of the COM on the mutagenicity of DEET will be incorporated into the COT statement. The COC have considered the carcinogenicity of data and concluded that there were adequate negative studies in rats and mice and that DEET was not carcinogenic in animals. 19. DEET was cited to give negative results in regulatory studies submitted to the EPA to investigate clastogenicity in CHO cells, and UDS in hepatocytes. No conclusions could be drawn from the oxidative damage mentioned in this paper. The secretariat will continue to try and obtain the full reports of the studies provided to the EPA to allow more definite conclusions to be drawn. 20. Members agreed that DEET did not have any structural alerts for mutagenicity and that it was negative in the Salmonella assay. As DEET also shows no evidence of carcinogenicity in adequate animal studies, it is unlikely to have significant mutagenic potential. 21. Members noted the possibility of high human exposure to DEET. But concluded that 3 well-conducted negative in-vitro tests would be sufficient to adequately assess DEET’s mutagenic potential. In light of the COC's conclusions that there were adequate negative carcinogenicity data in rats and mice, the committee agreed that an additional in-vivo study was not required. The committee agreed that it had no concerns regarding the mutagenicity of DEET, provided that the unpublished in-vitro reports on CHO cells and UDS in hepatocytes were negative (as assessed by the US EPA). ITEM 8: ANY OTHER BUSINESS 22. There was no other business. ITEM 9: DATE OF NEXT MEETING 23. 10th October 2002 ACTIONS
ADDED PARAGRAPHS 24 TO 48 on 28 MARCH 2003 ITEM 4: MALATHION (MUT/02/11) 24. No interests were declared. 25. The Advisory Committee on Pesticides (ACP) has asked the COM for advice on the mutagenicity of malathion (and the COC for advice on carcinogenicity). This will also have implications for the use of malathion in human medicines. 26. A number of impurities in technical malathion are known to have mutagenic potential and knowledge of the purity of the material tested is important when considering the significance of the mutagenicity data (para 16-20 in paper MUT/02/9). 27. Paper MUT/02/9 on the mutagenicity of malathion contained data submitted in test reports to the regulatory agency, additional published studies identified by industry and additional data identified by the Secretariat from the published literature. A number of studies still needed to be obtained by the Secretariat. The committee was asked to initially consider this topic, with a view to finalising a statement on malathion at the next meeting. 28. Members were informed of the details of manufacturer, data holders, pesticide approval holders and companies holding licenses for human medicines. No interests were declared. 29. The COM considered the available data in detail. It was agreed that the primary objective of the COM review was to consider the mutagenic potential of commercially supplied malathion (ie technical grade malathion was reported in the tests of having a purity of between 93-96%). In the COM review purified malathion referred to >96% pure material. In a small number of published studies, the authors had reported using 100% pure malathion. Members noted the role of malathion impurities in the response in tests for mutagenicity, had been cited by the pesticide holder. Members asked a number of questions to be relayed to the pesticide data holder via the Pesticides Safety Directorate (PSD). These related to variation of impurities between different manufacturers and processes, the shelf life of technical grade malathion, and information on the rate and nature of breakdown products during storage. 30. Members agreed that malathion was a DNA methylating agent. N7-methyl guanine was the main identified adduct but others cannot be excluded. There were also data to show that iso-malathion (an impurity formed during manufacture and storage) and OOO-trimethylphosphorothioate (a process impurity in technical grade malathion) could alkylate nitro benzyl pyridine in-vitro. Members agreed that there were a number of malathion metabolites identified in metabolism studies, which could theoretically methylate DNA. 31. Members acknowledged that some malathion impurities (eg isomalathion) had the potential to alter the acute toxicity of malathion (due to cholinesterase inhibition) in mammals, by inhibiting metabolism of malathion to non-toxic products. It was feasible that the mutagenicity of malathion could also be altered by the presence of impurities, however, there were no data available to demonstrate such interactions. 32. The Committee agreed that there was no evidence for mutagenicity of technical grade malathion in bacteria. The results in a number of tests in Salmonella typhimurium and Escherichia coli were negative. Details of some studies were still to be obtained. The significance of enhanced SOS activity in Escherichia coli was unclear. Members noted that only brief details of the studies in yeast were available, but considered that further evaluation of the data would be of limited value. 33. Regarding in-vitro assays in mammalian cells, the COM agreed that a cytogenetics assay with technical grade malathion conducted by the pesticide approval holder, was positive in the presence and absence of endogenous metabolic activation at the high dose (which was moderately cytotoxic). Lack of reproducibility in the second trial, may have in part, been due to the dose level selected and also to different exposure conditions used. Thus human lymphocytes were exposed to technical grade malathion for three hours in the first test, then incubated for 17 hours without the test article. In the second test, there was continuous exposure for 20 hours. The highest concentrations analysed for chromosome damage were 450 ug/ml in the first tests and 400 ug/ml in the second test. Members reviewed the available published literature and agreed that details of purity of malathion used was absent in several studies. However, members agreed that positive results had been obtained in published studies in human lymphocytes, where malathion was of technical grade or higher purity had been used. Appropriate references were, Garry V et al. (Teratogenesis, Carcinogenesis, & Mutagenesis 10, 21-29, 1990), Herath J F et al (Cytologia 54, 191-195, 1989), and Walter Z et al (Human Genet. 53, 357-381, 1980). Activity was reported both in the presence and absence of exogenous metabolic activation. Additionally, technical grade and purified malathion (ca 99% pure) had been shown to induce Sister Chromatid Exchanges in CHO cells, in human lymphocytes and fibroblasts and in a human lymphoid cell line. Members also noted that evidence for a gene mutation in the hprt locus in human lymphocytes had been published (Pluth et al. Cancer Research, 56, 2393-2399, 1996). The authors of this study noted that positive results had also been reported for different batches of technical grade malathion, which had contained slightly different levels of impurities. The Committee agreed that the mouse lymphoma assay, recently conducted by the pesticide data holder using technical grade material, had given positive results in the presence and absence of exogenous metabolic activation after a short 4-hour exposure period. But, a second trial using a 24-hour exposure period was negative (Members commented that the control mutation frequency in this study was relatively high). 34. Members agreed that the UDS assay in isolated hepatocytes with technical grade material, which had been submitted by the pesticide data holder, was negative. Members noted that although the top dose level (0.16µl/ml) had produced evidence of cytotoxicity, the dose might have been too low to detect any DNA damage induced by impurities. The Committee noted that a recent COMET assay with malathion (98% purity) and using human peripheral blood lymphocytes had yielded negative results (Blasiak J & Trzeciak A. Polish Journal of Environmental Studies. 7 (4), 189-194, 1998). However, no exogenous metabolic activation had been used and the exposure period (1hour) used in this study was shorter than expected for an adequate study (ca 3 or 24-h). 35. Overall the COM agreed that technical grade malathion was mutagenic in-vitro in human and mammalian cells. 36. The Committee reviewed the available in-vitro mutagenicity data on malaoxon, the principle metabolite of malathion. Malaoxon had mutagenic activity in two mouse lymphoma assays in the absence of exogenous metabolic activation, and induced SCEs in CHO cells. The test material used in these assays was approximately 94-96% pure. Malaoxon (98% pure) had also induced DNA damage in human peripheral blood lymphocytes in one assay (Blasiak J et al. Mut Res 445, 245-283, 1999). 37. The committee considered that the mutagenic activity seen in-vitro with technical grade malathion could, in part, be due to the metabolism of malathion to malaoxon. 38. The Committee agreed that the oral rat bone marrow clastogenicity study, conducted by the pesticide data holder using technical grade material, had been adequately conducted and was negative. Members noted that a number of positive bone marrow studies for both micronuclei and chromosomal aberrations had been published, and that these had used intraperitoneal dosing in mice. The purity of malathion used in these studies varied considerably, but adequate studies had included technical grade material (Giri et al. Mutation Research, 514, 223-231, 2002) and studies where purified malathion (100% pure) had been used (Amer S et al. J Appl. Toxicol. 16(1), 1-3, 1996). 39. Overall, the Committee agreed that malathion and technical grade malathion had in-vivo mutagenic activity in mouse bone marrow. Members discussed the possible reasons for a difference between the oral rat study and intraperitoneal mouse studies. It was agreed that first pass liver metabolism following oral dosing in the rat might be important with regard to the negative results in this species. Additionally, members were aware that for some compounds, species specific results in the rodent bone marrow micronucleus test had been reported. 40. The Committee agreed that insufficient data were available to draw conclusions with regard to mutagenicity in a second tissue. 41. The Committee heard that the pesticide data holder had commissioned an in-vivo rat liver UDS assay with chemical grade malathion. Members queried the selection of this assay, as a second tissue assay, particularly in view of negative results being obtained in the in-vitro UDS assay in rat hepatocytes. 42. The Committee discussed the available studies in humans exposed to malathion. It was noted that an increase in the incidence of micronuclei had been reported by the Californian Health Department, in a preliminary study of 13 applicators who used malathion in 1992. This result was not confirmed by a further study undertaken by the same group of researchers in 1993. Overall the Committee felt that no conclusions could be drawn from this study. Members agreed that no conclusions could be derived from the other available published studies relating to monitoring of human exposure for mutagenic effects. The Committee concluded that appropriately designed studies of pesticide applicators using malathion, might be informative with regard to mutagenic activity in humans. 43. The Committee agreed a set of interim conclusions, which would be finalised at the next meeting, when additional, published data and further data from the pesticide approval holder became available. ITEM 6: FLUNIXIN MEGLUMINE AND MEGLUMINE (MUT/02/15 &16) 44. No interests were declared. This topic had been considered at the 11th October 2001 meeting. Most of the data were relatively old and had limitations. Members agreed there was no evidence to suggest that flunixin itself had mutagenic potential in-vivo. However, there was sufficient evidence to conclude that flunixin meglumine was an in-vitro mutagen, which suggested that it was the meglumine component that was responsible for the mutagenic activity. There was a negative in-vivo bone marrow micronucleus test with flunixin meglumine, but this study was considered too limited to allow conclusions to be drawn. Meglumine was not considered to have any structural alerts. 45. A positive result had been reported with meglumine itself in a bone marrow micronucleus test. In this study involving administration of 2 doses 24 hours apart with harvest at 6 and 24 hours later, a positive result was produced only at the 6 hour sampling point. Negative results were also obtained in a second assay using a similar dosing schedule, but with harvest only after 24 and 48 hours. At the meeting in October one member offered to carry out additional testing on meglumine to clarify the position regarding the in-vivo micronucleus test data. Members agreed that this would be helpful. 46. The additional micronucleus data was considered at this meeting and agreed to be inconclusive. One study did indicate activity 6 hours after the 2 doses spaced 24 hours apart. No activity was seen at 24 hours after the second dose. The committee noted that there was much variability in the results with individual animals, and the effects may possibly be due to toxicity. Members agreed that on the basis of the available data, the situation regarding the in-vivo activity of meglumine was not completely resolved. The COM considered that further studies were needed before it could be concluded that the mutagenic activity seen in-vitro with flunixin meglumine (believed to be due to the meglumine component) could be discounted. 47. In accordance with the COM guidelines, it was agreed that in-vivo data from 2 tissues were required. There was a need to resolve the issue relating to the inconsistent data from the in-vivo micronucleus assay in the mouse using the intraperitoneal route. The COM concern related to the possibility of activity being seen 6 hours after 2 doses. Members felt that a metaphase analysis would be appropriate rather than the micronucleus test. It was considered more important to have data on clastogenicity by direct observation of chromosomes rather than by indirect assessment via observed micronuclei. This may also help resolve whether the results obtained in the additional micronucleus test was an artefact due to the high dose or to cytotoxicity. Members agreed that data from a bone marrow chromosome aberration test was needed in mice using 2 daily doses, with a sampling time of 1.5 times the cell cycle (as in the OECD guideline), and with a 6-hour harvest time. 48. Additionally, members considered that data were needed from an assay in a second tissue, and it was agreed that the in-vivo liver UDS assay was appropriate in this regard. Data from further in-vivo studies were needed to provide adequate reassurance that mutagenic activity seen in-vitro is not expressed in-vivo.
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