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MINUTESPresent:
CONTENTS
ITEM 1: ANNOUNCEMENTS/APOLOGIES FOR ABSENCE 1. The Chairman welcomed Dr R Roberts and Dr S Kennedy members of the COC attending for item 4. The Chairman also welcomed Mr S Samuels (PSD), Dr Stemplewski (MCA) and Dr D Gott (FSA) attending in the place of Dr Benford as FSA secretary to COM. 2. No apologies for absence were received. 3. Members were reminded of the need to declare any interests before discussion of items. ITEM 2: MINUTES OF THE MEETING ON 25TH APRIL 2002 4. The minutes were approved with minor amendments. ITEM 3: MATTERS ARISING FROM THE MEETING ON 25TH APRIL 2002
3.1 DEET 5. The committee had considered the mutagenicity of the insect repellent DEET at the last meeting. The COT would incorporate the advice of the COM into a statement. The COM had no concerns over the mutagenicity of DEET, but had requested to see unpublished in-vitro reports on CHO cells and UDS in hepatocytes that were negative (as assessed by the US EPA). The secretariat had not yet received these reports but would provide them to the COM in due course.
3.2 Revised statement on flunixin-meglumine and meglumine (MUT/02/16) 6. The minutes are now published as paragraphs 31 to 36.
3.3 SHE cell transformation assay: COM statement 7. Members were informed that the COM statement on the Syrian Hamster Embryo Cell Transformation assay had been published in Toxicologic Pathology. ITEM 4: REVIEW OF THE MUTAGENICITY OF MALATHION: ADDITIONAL DATA SUBMITTED POST 25TH APRIL 2002 AND 1ST DRAFT COM/COC STATEMENT (MUT/02/17 & ADDENDUM) 8. The minutes are now published as paragraphs 37 to 50. ITEM 5: SIGNIFICANCE OF IN-VIVO MUTAGENICITY DATA AT HIGH DOSES (MUT/02/19) 9. The COM has previously agreed that it is prudent to assume that there is no threshold for mutagenicity unless appropriate mechanistic data can be provided to identify a threshold related mechanism. In-vivo studies in the bone marrow provide key data in identifying compounds as in-vivo mutagens. In some cases the only data available to indicate such in-vivo activity is from mutagenicity studies using excessively high doses (by current guidelines) associated with severe toxicity/lethality, and there is no evidence from carcinogenicity bioassays to suggest that a compound is a genotoxic carcinogen. Due to the importance of these data in risk assessment, where positive results normally lead to a 'non-threshold' approach being adopted by regulatory agencies, it is important that the observed effects are not secondary to toxicity. 10. The advice of the COM on the interpretation of such 'high dose' positive results in bone marrow mutagenicity assays was requested by DH. In addition the Advisory Committee on Pesticides has asked for generic advice on pesticides which are in-vivo mutagens, only at high does (associated with severe toxicity/lethality), and for which adequate long term carcinogenicity bioassays suggest no evidence of carcinogenicity (or at least do not suggest that the compound is a genotoxic carcinogen). 11. To facilitate discussion in this area paper MUT/02/19 provided three case examples to illustrate these concerns. It was agreed that any COM statement arising from the discussion would provide generic advice only and would not cite the example compounds. Each raised questions regarding the assessment of high dose mutagenicity data and in some cases evidence of a specific mechanistic explanation of the 'high dose' mutagenicity was provided (e.g. hypothermia and/or normal detoxification being overwhelmed). Members noted induction of erythropoiesis was suggested as one further mechanism that may give rise to positive results in the bone marrow micronucleus assay. 12. Members agreed that clear criteria would be needed to define high or excessive doses. The committee was aware that in the past some regulatory guidelines (eg those of the EPA) have defined the top dose (ie the maximum tolerated dose {MTD}) as a high proportion (50-80%) of the LD50. The first UKEMS guidelines had stated that the highest dose level should be 50-80% of the dose that causes lethality. A comprehensive review by a BTS/UKEMS working group in 1992 indicated that the uses of such doses, associated with severe toxicity/lethality at the top dose, were not justified. The working group concluded that about 90% of all tested mutagens would have been identified using a MTD based on non-lethal criteria and that the percentage would have been higher if all the tests considered had been carried out to current protocol standards. That is where the top dose produced some signs of toxicity (but not lethality) such that the next higher dose (not used) could be expected to result in severe toxicity and probable mortality. This had been influential in the updating of the OECD guidelines on mutagenicity testing and the revised texts use this criteria to define the top dose level in the in-vivo mutagenicity assays. For the purposes of this discussion 'high' doses was considered to be when the top dose exceeded the criteria in the current OECD guidelines. 13. The committee considered that mutagenicity studies producing positive results only at lethal doses could not be interpreted and that a re-test should be undertaked at non-lethal doses. Members were aware that the period of observation following dosing varied between different in-vivo tests and that the minimum lethal dose for the same test chemical could also vary. Where there was evidence that a proposed mechansim of action (eg hypothermia/erythropoesis) could directly induce mutagenicity, members agreed this might help derive an explanation for positive in-vivo results. The committee recommended that that a full evaluation in accordance with the COM guidelines would provide valuable information and that all the available data should be considered. For example structural alerts for mutagenicity and in-vitro mutagenicity data could help interpret in-vivo results. 14. Members agreed that it was important to determine whether chemicals were DNA reactive or aneugenic in-vitro. For chemicals that were not considered DNA reactive or aneugenic but produced in-vivo mutagenicity at only severely toxic/lethal doses it was possible that such mutagenic effects were secondary to toxicity. In situations where there was evidence for in-vitro DNA reactivity or aneugenicity, then the only way to evaluate in-vivo positive results at severely toxic/lethal doses was to repeat the in-vivo test at non-lethal doses. 15. The COM discussed the weight of evidence to be attributed to carcinogenicity studies and felt that negative results might not always outweigh positive findings from 'high dose' in-vivo mutagenicity studies at severely toxic/lethal doses; each instance had to be considered on a case by case basis, taking all the available data in to account. 16. Members considered that a mutagenic effect produced in-vivo in the bone marrow only at a 'high' dose might also be secondary to certain biological or physiological effects. It was agreed that hypothermia and also hyperthermia/fever, which can be produced by some chemicals, could induce micronuclei in bone marrow cells. There was evidence that some chemicals could cause positive results in the bone marrow micronucleus test by excessive stimulation of erythropoiesis. However, the committee noted that erythropoiesis had also been seen at low doses. 17. Members heard that an industrial International Working Group on Gentoxicity testing (IWGT) was intending to consider the significance of positive genotoxicity results at high doses. It was agreed that it would be useful for the COM to be kept informed of the progress of this Working Group on this issue. 18. The secretariat would use the above discussion to draft an initial statement for consideration at the next meeting. [Post meeting note: A member of the COC asked for a generic discussion with COM on the weight of evidence to be attributed to carcinogenicity studies]. ITEM 6: GLOSSARY OF TERMS (MUT/02/14) 19. A sub-group of the COM had produced a glossary of terms related to mutagenicity. This was intended to help revise the COT/COC/COM glossary of terms. The Chairman thanked one member in particular and the sub-group in general for their work and said that it was a very useful document. 20. The committee agreed that the definitions were excellent, but might be too technical for the audience that it was aimed at. It was suggested that a solution would be to use the first line and omit the final paragraph for each definition. Members also suggested that the terms COMET, P53, chromatid gap and CPG should be added to the glossary. ITEM 7: IMPLEMENTATION OF THE OST CODE OF PRACTICE: TEMPLATE FOR COM (MUT/02/18) 21. The COM had discussed the OST code of practice for Scientific Advisory Committees at the 7th February 2002 meeting. The COM complies with most of the requirements of the code. One aspect included a 'Membership Template', which covers the balance of expertise on the committee needed to be considered. A draft COM template and short examples of member profiles used by the sister committee the COT were attached to annex 1 to MUT/02/18 for consideration. The main purpose of the template was to help in arriving at a view on the balance of expertise. 22. Members suggested a number of amendments to the format of the draft template and asked for links with COT to be defined. 23. The secretariat would only place member profiles on the COM website when agreed. It was suggested that it would appropriate to consider this when the new committee was in place in 2003. A finalised document would be placed on the COM website. ITEM 8: IN-VITRO MICRONUCLEUS TESTS (IWGT MEETING) (MUT/02/20) 24. The 3rd International Workshop on Genotoxicity Testing was recently held in Plymouth which included a Working Group on the In-vitro Micronucleus test. There was international consensus that this method was adequately validated, and also on the methodology for a guideline. A member of the COM had attended this meeting and provided the COM with overheads on consensus points reached at the meeting. It was believed that the US could now support the development of an OECD guideline for this test method. 25. The consensus points included that: both human lymphocytes and cell line could be used; cytochalasin-B is only mandatory in human lymphocytes; dose ranging; treatment schedules for cell lines; time point for addition of cytochalasin-B; treatment schedule for human lymphocytes; when to repeat experiments; the experimental unit is the cell and not the culture; number of cells to be scored; recommendations for human lymphocytes; recommendations for cell lines; choice of positive controls; statistics to be used. 26. The COM queried the consensus point that the experimental unit was the cell and not the culture and noted that this did not conform to the approach taken in other previously validated in-vitro tests. The secretariat would raise this aspect with the IWGT on behalf of the committee. ITEM 9: ANY OTHER BUSINESS
9.1 OST code of practice for Scientific Advisory Committees 27. The committee was informed that the chairman had attended a meeting on behalf of the COM to discuss the progress being made by Scientific Advisory Committees in complying with the OST code of practice. The COM conformed to most of the code of practice.
9.2 Meeting of Advisory Committee Chairs and the Chairman of the FSA 28. The COM Chairman informed the committee that two items had specifically been discussed at the dinner with the FSA Chairman, namely openness and horizon scanning. Regarding the latter most committees covered the issue of technical development in their guidance, but a particular difficult issue related to consideration of what agents/compounds would be the next major public health issue leading to media/public concern. It was very difficult to ensure the adequacy of any approach to this problem. The chairman also noted that the COM often responded to requests for advice from other Government committees or organisations. The chairman pointed out that the handling of commercial in-confidence data was a difficult area with regard to holding fully open meetings. The COM had previously held a joint public opening meeting with the COT/COC on genomics and proteomics and was considering holding at least one open session per year in the future to discuss general issues, such as research priorities.
29. The COM heard that advertisements for reappointing three specialist members to the COM from April 2003 would appear in November 2002. ITEM 10: DATE OF NEXT MEETING 30. 6th February 2003 ACTIONS
PARAGRAPHS 31 TO 50 ADDED on 28 MARCH 2003
3.2 Revised statement on flunixin-meglumine and meglumine (MUT/02/16) MINUTES 10 OCTOBER 2002: FLUNIXIN, MEGLUMINE AND FLUNIXIN-MEGLUMINE 31. Flunixin is a non-steroidal anti-inflammatory (NSAID) drug and a non-narcotic analgesic drug with antipyretic activities. Flunixin is mixed with meglumine, an excipient, to give the Flunixin-Meglumine dosage formulation. The relevant EU Committee, CVMP, has concluded that flunixin is not genotoxic in vivo and they have not considered meglumine as it is regarded as an excipient. The Food Standards Agency had concerns at the possible genotoxicity of meglumine and Flunixin- meglumine and Flunixin and therefore requested advice from COM. The COM had considered the available mutagenicity data on these chemicals at its October 2001 meeting, and had reviewed some of the test reports in detail at the February 2002 meeting. The Committee had considered further in-vivo mutagenicity data at the 25 April 2002 meeting and had commented on a 1st draft statement. 32. The Committee had asked for a number of revisions to be undertaken. A 2nd draft statement was submitted to the COM for consideration. 33. Members recalled that a critical result for meglumine was the finding of a positive result in a bone marrow micronucleus test in BS1 mice using intraperitoneal administration. The protocol used had involved two dose of the test material separated by 24 hours and sampling of bone marrow at 6h and 24h after the second dose. The finding of a positive result with meglumine at the 6 h time interval but not at the 24 h interval was considered unusual. 34. The Committee had tried to evaluate this finding by considering some additional in-vivo mutagenicity data which had been undertaken by one COM member. It was noted that the evidence of toxicity seen in BSI mice at 500 and 1000 mg/kg bw had not been observed in Alpk:ApfCD-1 or CBA mice dosed at 1000 mg/kg bw. Members felt that potential strain differences in toxicity could have contributed to the observed mutagenicity. 35. The Committee discussed the most appropriate strategy which could be used to further investigate meglumine. Members had initially considered that a further in-vivo bone marrow assay in mice for chromosomal aberrations would provide additional in-vivo data and would also aid in providing evidence as to whether the micronuclei response in mouse bone marrow was an artefact. In addition members reconsidered the in-vitro mutagenicity data on Flunixin, meglumine and Flunixin-meglumine and agreed there was a need to undertake further studies on meglumine to the current COM guidelines. Members noted that the positive findings in in-vitro studies with Flunixin-meglumine (ie limited evidence for a positive response in three mouse lymphoma assays and an equivocal response in a cytogenetics assay in Chinese Hamster ovary cells) had triggered the further consideration of meglumine. 36. The Committee agreed the most appropriate way forward would be to request additional in-vitro assays with meglumine to properly assess whether meglumine had mutagenic potential in-vitro. Thus data were required from an in-vitro chromosomal aberration assay and a mouse lymphoma assay, which together with the data currently available for a test in bacteria using Salmonella, would complete the in-vitro package to modern standards given in the COM guidance. In this case the weak and inconsistent induction of micronuclei in mice after two treatments with meglumine 24 hours apart with sampling 6 hours after the last treatment would be disregarded as strain- or- system specific effects of no genotoxic relevance if these two additional tests were negative. ITEM 4: REVIEW OF THE MUTAGENICITY OF MALATHION: ADDITIONAL DATA SUBMITTED POST 25TH APRIL 2002 AND 1ST DRAFT COM/COC STATEMENT (MUT/02/17 & ADDENDUM) 37. Members recalled that the Advisory Committee on Pesticides (ACP) asked for advice on the potential mutagenicity and carcinogenicity of malathion (an organophosphorous insecticide) from COM and COC as part of its ongoing review of organophosphorous compounds. Members were reminded there were currently three products with approvals for use in agriculture and horticulture, home garden and use in pigeon lofts. A number of products containing malathion were also licensed as medicines for use in the control of head lice. It was noted that there were a very limited number of alternative head louse treatments available. 38. The COM had an initial consideration of malathion at its April 2002 meeting. The COM reviewed the inconfidence data submitted to the Pesticides Safety Directorate, published studies identified by the pesticide data holder and some additional published studies identified by the secretariat. Further details of test chemical purity had been obtained from some authors of published papers. Additional details of some studies, further published reports and a study being undertaken by the pesticide data holder (in-vivo rat liver UDS) had been submitted for consideration at the October 2002 COM meeting. The secretariat had also obtained further details of impurities present in technical grade malathion distributed by the pesticide data holder and an evaluation by the pesticide data holder of the likely differences in impurities between different manufacturers. 39. The Chairman noted that COC had considered the available carcinogenicity studies at their June 2002 meeting and asked COC members to report on conclusions reached at that meeting. 40. The COM was informed that there were several animal carcinogenicity bioassays available and data were available with malathion (and also its major metabolite malaoxon) in both rats and mice from a range of studies dating from early 1970s' up to the mid 1990s'. However there were three studies undertaken between 1992 and 1996 using dietary administration of malathion to F344 rats and B6C3F1 mice and dietary administration of malaoxon to F344 rats which were considered to be the most informative. An independent peer review of the pathology had been undertaken with the bioassays with malathion. The Maximum Tolerated Dose had been exceeded in the studies using malathion at the two highest dose levels (12000 ppm and 6000 ppm in the diet (except females)) based on decreases in body weight (³ 10% at termination) and treatment related increases in mortality and signs of toxicity. The Maximum Tolerated Dose had not been exceeded in the malaoxon study in F344 rats on the basis of decreased weight gain but there had been clear signs of treatment related toxicity and increased mortality in this study. 41. Members heard that there was a sustained inflammation of the respiratory tract and in particular nasal tissue in all three studies. This was considered to be in part due to inhalation of the diet and also to localised metabolism of malathion to its diacids in nasal tissue. The subsequent irritation of the nasal tissue was evident through a range of pathological lesions including inflammation and degeneration of the respiratory and olfactory epithelium. The COC had considered that the degree of inflammation exceeded that seen in long term inhalation studies using known irritant materials and was therefore a remarkable finding in these bioassays. It had also been noted that wheezing had been reported as a possible sign of toxicity in one of the earlier bioassay in rats. 42. After peer review of the pathology, a consensus agreement was reached that there were three nasal respiratory adenomas (two (one male/one female) at the high dose of 12000 ppm and one female at 6000 ppm) and olfactory adenoma in one male at 6000 ppm. It was noted that the reported increased incidence of nasal tumours was statistically significant but there were difficulties in assessing the significance of the observed increase given the inadequate and developing knowledge of the historical control incidence of nasal tumours. The COM was informed that histopathology of the nasal tissue was unlikely to be routinely undertaken before the 1990s and there had been advances in sectioning of nasal which also affected the interpretation of the true historical control incidence of nasal tumours. 43. The COC had considered a submission from the pesticide data holder and had agreed that the evidence was consistent with the non-genotoxic induction of nasal tumours in rats by malathion secondary to prolonged severe irritation of nasal tissue. It was noted that there was no evidence for a multiorgan carcinogenic response with malathion. The finding of benign liver tumours in rats and mice at high doses was not considered by COC to be relevant to human health assessment. It was noted that such tumours were only seen in animals which had evident increase in liver weight and hepatocellular hypertrophy. 44. The Chairman thanked COC members for the report the COC evaluation. He asked the Committee to consider the new data before reconsidering its conclusions. 45. Members considered the data on impurities provided by the pesticide data holder and confirmed that several of the impurities were potential alkylating agents. It was noted that the level of isomalathion increased during storage. Members agreed that the identity and levels of impurities in malathion produced by the pesticide data holder and sold within the UK had been adequately determined. However malathion from other sources was likely to be qualitatively similar with regard to impurities, but it was not possible to predict the levels of impurities in these materials. Members considered that the mutagenic activity of technical grade malathion seen in in-vitro experiments was due to malathion (or its conversion to malaoxon) with a contribution made by the presence of impurities but the relative contribution of each of the components was unknown. The secretariat confirmed that the material used in the key carcinogenicity bioassay in F344 rats had been manufactured and supplied to the contract laboratory by the pesticide data holder. 46. Members discussed the in-vivo liver UDS assay in rats submitted by the pesticide data holder. It was noted that cell viability was somewhat low but within acceptable levels for in-vivo UDS assays. Members were concerned with regard to the apparent positive response in animals 24 and 25 in the assay. The Committee did not accept the rationale provided by the contract laboratory that these positive responses were caused by technical errors (staining errors, the timing of dosing relative to other rats and/or use of control rats sampled 2-4 hours after dosing as controls for treated rats sampled 12-16 hours after dosing). Members also considered it feasible that a positive control substance had been dosed to these animals by mistake or there had been a mislabelling of the animals. Whilst such explanations may be plausible, there was a lack of direct evidence linking the positive effects in the two rats to any experimental error. However, it was noted that no evidence of mutagenicity was found in a repeated 12-16 hour sampling time experiment. Overall the COM agreed that no weight could be attributed to the study and that it should be repeated. The Committee concluded there would be no concerns regarding the oral route in rats if a further oral UDS assay using material as supplied in the UK gave negative results. 47. The Committee considered the study by Dulout et al (Mutation Research 105, 413-416, 1982) which had used both the dermal and intraperitoneal routes of dosing in bone-marrow micronucleus assays in mice. Members agreed it was difficult to explain why such a strong positive response had been documented following dermal application compared to intraperitoneal dosing. The test material (a technical grade material manufactured in Argentina) had been dissolved in corn oil which would have led to residual vehicle on the skin. Members noted that dermal absorption in human volunteers ranged form 5% to 15% depending on formulation and dilution with water. It was not possible to quantify the extent of dermal absorption in this study. There would have also been some oral ingestion arising by grooming of the animals, but his could not be quantified. Members noted that there was no evidence for a dose response following dermal administration in this study, but felt that the results could not be refuted and thus there was a need for a repeat study. 48. The COM reviewed the published data provided in respect of the studies undertaken by Pluth and colleagues (Mutation Research 397, 137-148, 1998) on mutational spectra in human T-lymphocytes exposed to malathion and agreed the data supported a conclusion that malathion was mutagenic in-vitro but there were doubts as to whether a mutational spectra for malathion had been identified. 49. Members considered it was important to balance the evidence for mutagenicity in-vivo where the evidence suggested a complex pattern of results with the evidence from the dietary carcinogenicity bioassays in rats and mice that malathion was not a genotoxic carcinogen. In this respect Members concurred that there was no evidence for mutagenicity in a well conducted oral bone-marrow cytogenetics assay in rats undertaken by the pesticide data holder. No definite conclusions could be drawn from the oral liver UDS assay in rats undertaken by the pesticide data holder in view of the isolated positive findings in two animals and the concerns raised by members regarding the adequacy of this study. There was some limited evidence for a clastogenic effect in mice following oral dosing of a technical material manufactured in India by Giri and colleagues (Mutation Research vol 514, 223-231, 2002). The Committee agreed that in view of the lack evidence for carcinogenicity in the long-term feeding carcinogenicity bioassay in mice, that there was no need for a further oral study in mice at this juncture. Overall the Committee concluded that malathion was mutagenic in-vivo in mice dosed by the intraperitoneal route, and possibly also by the oral and dermal routes. Conversely, there was no evidence of in vivo mutagenicity in rats dosed orally. These data might indicate a species-specific in-vivo mutagenicity in mice. 50. Members agreed there was evidence for a mutagenic effect in-vivo in mice by the intraperitoneal route and also in limited evidence in mice following oral and dermal application using technical grade materials. It was noted that some of these studies were relatively old and the test materials used did not correspond to that supplied in the UK. However members felt, there was no clear explanation for these data but the limited evidence did suggest provisionally, that there was a mutagenic hazard in-vivo in mice following these routes of exposure. In view of the potential for dermal exposure following the use of malathion as a pesticide or in the treatment of head lice, the Committee considered it was important that the study reported by Dulout et al (1982) in mice was repeated in order that definite conclusions could be drawn with regard to mutagenic hazard following dermal exposure to malathion.
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