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MINUTESPresent:
CONTENTS
ITEM 1: ANNOUNCEMENTS/APOLOGIES FOR ABSENCE 1. The Chairman welcomed Dr S Kennedy and Dr R Roberts attending from the COC. The Chairman also welcomed Dr Claire Cowles (HSE), Dr D Andrew (PSD), Mr A Browning (VMD), and Dr J Shavila (FSA). Dr P Wislocki and Dr K Woodward would make a presentation on behalf of Shering-Plough during part of item 4. 2. The chairman informed the committee that it was the last meeting for the members Professor Ashby, Professor Cooper and Professor Tweats who had come to the end of their term of membership. He thanked them for their hard work and valuable contribution to the COM over the years. 3. An apology for absence was received from Professor Ashby. 4. Members were reminded of the need to declare any interests before discussion of items. ITEM 2: MINUTES OF THE MEETING ON 10TH OCTOBER 2002 (MUT/MIN/02/4) 5. The minutes were approved with minor amendments. ITEM 3: MATTERS ARISING FROM THE MEETING OF 10TH OCTOBER 2002
3.1 Malathion 6. The committee was informed that the data holder had responded to the 1st draft statement and there had been an exchange of views between the COM and the data holder particularly with regard to the adequacy of the in-vivo rat liver UDS assay. The Advisory Committee on Pesticides (ACP) had been sent the 1st draft COM/COC statement on malathion and had considered that there was no regulatory need for additional testing. Members were informed that the ACP had given different weighting to the value of carcinogenicity data in reaching their conclusion. A 2nd revised draft COM statement containing additional comments in response to the data holder’s submission had been forwarded to the data holder. The deadline for comment was the 11 February 2003. 7. The data holder sent their comments on the 1st draft statement, which included additional analyses, to both the COM and ACP secretariats, and the latter had tabled them to the ACP meeting on 18 January 2003. The COM secretariat had not been consulted and therefore members had been asked for their initial views over a very short time period (24 hours). A week had been allowed for the full consultation. Members expressed concerns at this process, which clearly allowed inadequate time for COM to comment prior to the ACP consideration of the 1st draft statement. The secretariat agreed to raise the matter with the ACP secretariat and to ensure that COM members were not put in this position again in the future.
3.2 In vitro micronucleus tests) 8. Members heard that the secretariat had written to the Working Group of the International Workshop on Genotoxicity Testing on the In-vitro Micronucleus test. The COM had expressed concern over the IWGT consensus point that the experimental unit was the cell and not the culture as this approach did not conform to that taken in other in-vitro tests, such as the mouse lymphoma assay. Members would be informed when a reply had been received.
ITEM 4: FLUNIXIN, MEGLUMINE AND FLUNIXIN MEGLUMINE (MUT/03/1) 9. Members were reminded that a draft statement was agreed at the last meeting in October 2002 and forwarded to the data holder, Schering-Plough for comment. The data holder had responded with detailed comments both on the mutagenicity and carcinogenicity studies and made alternative proposals for further mutagenicity testing. The Chairman agreed that it would be reasonable to agree to the request for further discussion with the COM and thus the data holder would make a short presentation at this meeting. It had been made clear that in the absence of substantive relevant new data, this would be the last opportunity for consideration of this issue and that a final statement would be drawn up after the meeting. Copies of the correspondence with Schering-Plough were attached to MUT/03/1. In view of the comments made by Schering-Plough about the weight to be attributed to the carcinogenicity bioassay two members of the COC were in attendance. 10. Members considered the papers forwarded by industry for this meeting. The Chairman asked the Committee to identify the key areas for questioning. These were identified as:
Presentation by Data Holder 11. The data holder noted that flunixin-meglumine was a non-steroidal anti-inflammatory veterinary medicine used in food producing animals. It was an ionic compound which dissociated rapidly in aqueous media at physiological pH. The primary purpose of meglumine was to act as a counter ion to keep flunixin soluble. The data holder noted, the Chairman’s request to focus on the approach to mutagenicity testing of meglumine. It was felt important to note that the initial submission for an Maximum Residue Limit (MRL) had been forwarded to the E.U. in 1994. The data holder had responded to queries as appropriate but the process had taken some time, during which the rapporteur had been changed and the European Medicines Evaluation Agency (EMEA) had been established. An MRL had eventually been established for flunixin in 1999. 12. The data holder accepted that the mutagenicity data on meglumine were difficult to evaluate and there were equivocal results. The main rationale for suggesting further in-vivo testing, as opposed to the COM suggestion of additional in-vitro testing was that the prime concern was to evaluate meglumine and potential metabolites which might occur as residues in food producing animals and hence in-vivo testing in the rat was considered more appropriate than further in-vitro testing. The approach was also consistent with the VICH recommendations. In addition the data holder had some concerns regarding "over-sensitivity" (ie false positives) with the mouse lymphoma assay and had also considered that further testing in mice was likely to repeat the inconsistent and equivocal results already reported to the COM. Discussion between Data holder and COM/COC members 13. In answer to questions from Members, the data holder confirmed their view that the dissociation of flunixin-meglumine was rapid. There were no data on the ADME of meglumine available either from the data holder or in the literature. It was proposed by the data holder that metabolism would be rapid on the assumption that the chemical rapidly degraded to amino acids present in intermediary metabolism. Members felt that appropriate reassurance from well conducted ADME studies might help to allay any concerns that residues of meglumine might be present in feed producing animals. The data holder noted this was unlikely in view of the withdrawal time for animals treated with flunixin-meglumine of 3-7 days. In addition, in answer to member’s question, the data holder stated that milk from the first three milkings after treatment with flunixin-meglumine was discarded. It was noted that milk was usually bulked and that the VMD had not detected flunixin-meglumine in surveys for residues in milk. This information provided reassurance to members with regard to potential for residues in food. 14. Members noted that the mouse lymphoma assay was used routinely in regulatory submissions and considered the false positive rate to be no worse than other in-vitro assays in mammalian cells. Members felt it important to establish whether meglumine had any mutagenic potential in-vitro first before considering the need for further in-vivo testing. Members thought that provision of three adequately conducted in-vitro tests (http://www.doh.gov.uk/com/guidance.pdf) as outlined in COM guidance with negative results would obviate the need for any further in-vivo testing. The purpose of these assays would be initial screening of meglumine and its metabolites in-vitro. The data holder accepted this proposal. 15. With regard to members enquiries on the carcinogenicity studies, the data holder presented additional data regarding the carcinogenicity studies. With regard to the study of flunixin-meglumine in rats, which had been initiated in 1976, there had been concerns that the poor survival due to toxicological effects associated with flunixin had limited its value. The data holder had established that two additional separate histopathology evaluations had been undertaken for this study in addition to the original study histopathology including recutting of tissues in the third assessment. The additional data ensured that full histopathology had been undertaken for all animals in this study. There was no evidence for tumourigenicity. This additional information had not been submitted as part of the request for an MRL. With regard to the mouse carcinogenicity study, additional information presented noted that the dose levels were lower that in the rat study. The top dose level of 6 mg/kg bw/day expressed as flunixin-meglumine (hence actual dose of flunixin was lower than stated, ca 3.2 mg/kg bw/day). However an MTD had been achieved as suppression of body weight gain and evidence of bone marrow hyperplasia had been documented. 16. The data holder asked how the statement would be taken forward. The chairman replied that the draft statement would be revised and provided to the data holder for comment. The finalised statement and minutes would be made publicly available via the COM internet site and the Annual report. The statement would also be forwarded to the Food Standards Agency who had asked the Committee to review flunixin-meglumine, flunixin and meglumine. 17. The data holder thanked members for the opportunity to make a presentation and withdrew. COM/COC consideration of presentation 18. Members agreed that the discussion had identified all appropriate areas and were content that the data holder had indicated willingness to undertake appropriate in-vitro mutagenicity studies. COC members considered that the additional information on the carcinogenicity studies had provided reassurance with regard to the assessment of flunixin-meglumine. They indicated that the carcinogenicity studies were not informative with regard to meglumine. Provision of appropriate ADME studies on meglumine might help to resolve any concerns with regard to exposure to meglumine from treated animals. MCA raised concerns regarding the adequacy of the carcinogenicity studies and absence of concomitant kinetic investigations. However COM/COC members felt the studies were adequate for the time when undertaken and the most appropriate way forward would be to undertake the two in-vitro mutagenicity studies requested by COM. 19. Members considered two additional papers provided by FSA reporting evidence for mutagenic effects on ionic contrast media in human peripheral lymphocytes. (Parvesz Z et al, Mutation Research vol 188, 233-239, 1987 and Mozdarani H and Fadaei S, Toxicology Letters, vol 98, 25-30, 1998.) These reported evidence for increased micronuclei and clastogenicity attributable to ionic contrast media containing meglumine. However the quality of the evidence was not convincing with regard to meglumine. It was noted that there were other papers in the literature regarding ionic contrast media. Abstracts of these latter papers would be circulated to members, but no specific reference was required for the statement on flunixin-meglumine, flunixin and meglumine. 20. Members considered the draft statement and made a number of revisions. The revised draft statement, additional data provided by the data holder would be circulated to members for comment and finalised by chairman’s action. ITEM 5: GUIDANCE ON CONSIDERATION OF HIGH DOSE POSITIVE IN-VIVO MUTAGENICITY DATA IN THE IN VIVO BONE MARROW ASSAY (MUT/03/2) 21. At the previous meeting the COM had given some initial consideration to when apparently positive in-vivo mutagenicity data in bone marrow assays at excessively high doses (by current guidelines) associated with severe toxicity and lethality could be discounted with regard to considering a compound as an in-vivo mutagen. This was important because positive results in an in-vivo mutagenicity assay normally led to the adoption of a non-threshold approach to risk assessment. A number of potential mechanisms with a threshold of action (eg hypothermia and erythropoiesis) had been considered as well as severe toxic effects. The secretariat had prepared a first draft guidance document that the COM could publish in this area. This placed emphasis on considering all the available data on a case-by-case basis, including results from adequately conducted carcinogenicity bioassays. The COM and COC consider it prudent to regard in-vivo mutagens as potential genotoxic carcinogens. Due to the close association between mutagenicity and carcinogenicity, two members of the COC attended for this item to discuss the weight of evidence to be given to carcinogenicity studies in this regard. 22. Members agreed that consideration needed to be given to the whole data package, including chemical reactivity, toxicokinetics and whether the carcinogenicity data of a compound represented the profile of a genotoxic carcinogen. Chemicals needed to be considered on a case-by-case basis. A negative carcinogenicity bioassay on its own could not discount a ‘high dose’ positive in-vivo mutagenicity result. Conversely a positive result in a bioassay may not necessarily mean that a chemical was a mutagen, as there were ‘non-genotoxic’ mechanisms that could induce cancer. 23. The committee had previously agreed that in-vivo mutagenicity data from dose levels associated with lethality (ie clearly above the top dose recommended by OECD guidelines) could not be interpreted with any certainty due to the confounding effects of toxicity. If adequate information were not available from lower doses then further testing would be required with more appropriate lower doses. Members considered that mechanistic data were valuable in discounting positive mutagenicity results seen at toxic doses i.e. to demonstrate the response was secondary to toxicity. 24. It was agreed that adequately conducted carcinogenicity bioassays provided important information to help evaluate the significance of positive results from mutagenicity studies at ‘high’ doses. Adequate negative carcinogenicity bioassays, preferably in two species, could be used to help discount evidence of mutagenicity at lethal doses, and where there was some concern at the relevance of the in-vivo mutagenicity data. Members discussed the need to take into account the differences in the maximum dose levels in these studies. It was noted that consideration of the differences in maximum dose levels would be of limited value in view of the differences in duration of dosing (usually 1-2 days compared to 2 years). However consideration should always be given to available toxicokinetic data. 25. Overall the COM considered that a weight of evidence approach using the whole data package on a case by case basis had to be used when considering whether a ‘high’ dose positive in-vivo result could be discounted in somatic cells due to a biologically plausible mechanism. 26. The secretariat would use members’ comments to amend the draft guidance document on consideration of ‘high’ dose positive in-vivo mutagenicity data. 27. A revised paper would be circulated to members for their comments and also forwarded to COC for endorsement. The finalised paper would also be forwarded to the ACP. ITEM 6: PHENOL: RECONSIDERATION OF MUTAGENICITY DATA IN THE LIGHT OF NEW DATA RELATING TO THE HIGH DOSE BONE MARROW MICRONUCLEUS ASSAY (MUT/03/3) 28. The COM had previously considered phenol on a number of occasions (1994, 1995 & 2000). The in-vitro data were poor. The committee had decided that phenol should be regarded as an in-vivo somatic cell mutagen based on positive results at high doses in the bone marrow assays for clastogenicity. Negative results were obtained in carcinogenicity bioassays in rats and mice. The committee agreed that a threshold for mutagenicity could be assumed for the oral route because there was evidence to show that any phenol active metabolites formed in-vivo were rapidly detoxified by multiple pathways following ingestion. But a threshold for mutagenicity for exposure by other routes, such as inhalation or dermal, could not be assumed. 29. Since then, data have become available to provide a plausible mechanism to support the view that positive results in the bone marrow assays were not due to a direct mutagenic effect of phenol, but were due to a secondary threshold toxic effect, namely hypothermia occurring at dose levels associated with positive results in the micronucleus assays. 30. The committee was asked to consider whether these new unpublished data together with the absence of other in-vivo mutagenicity data and the negative results in the carcinogenicity bioassays indicated that phenol did not produce clastogenic effects at doses below those that produced hypothermia. These new data indicate that phenol can produce hypothermia in mice at doses that result in micronuclei formation. An intraperitoneal dose of 300 mg/kg produced significant and prolonged hypothermia with a drop in body temperature of up to 7 degrees centigrade. No effects on body temperature were seen at doses below 300 mg/kg, and single doses of 400 and 500 mg/kg reduced body temperature and cause marked lethality. To investigate micronucleus induction single intraperitoneal doses of phenol were administered at 0, 30, 100 or 300 mg/kg and bone marrow harvested at 24 and 48 hours post dose (cyclophosphamide was used as a positive control and produced micronuclei). An increase in micronuclei was seen only at 300 mg/kg phenol, which was associated with significant and prolonged hypothermia. The investigators argued that the induction of micronuclei by phenol at the maximum tolerated dose is threshold related and may be causally related to hypothemia. 31. The COM considered that the new data provided a plausible mechanism. Members agreed the function of spindle fibres could be inhibited at low body temperatures, which could result in adverse chromosome effects, such as aneuploidy. The committee was also aware of other data that indicated that hyperthermia can also induce chromosome damage both in-vitro and in-vivo, and that high body temperature induces micronuclei in mouse bone marrow. Members agreed that inhibition of the spindle function and disturbance of the mitotic apparatus was also a possible mechanism for this effect. 32. Members agreed that before definite conclusions could be drawn on the significance of these new data they would like to see a peer reviewed published report of this study. Members requested further data on the dose-response of hypothermia induced by phenol. It was also agreed that strong evidence to support this hypothesis would be provided if micronuclei were not induced by phenol in a separate group of animals maintained at normal body temperature (eg by the use of heated plates and warm beds). If such information could be provided members agreed that phenol could be regarded as having no significant in-vivo mutagenic potential at dose levels that do not produce any significant toxic effects (hypothemia). ITEM 7: MALACHITE GREEN: NEW DATA (MUT/03/4) 33. The COM last considered the mutagenicity of malachite green and leucomalachite green in 1999. A statement had been forwarded to the Committee on Toxicity. It had been concluded that on the basis of the limited data available, and specifically a 32P post-labelling study, both compounds were considered as potential in-vivo mutagens. Further results were now available from the NTP studies on the mutagenicity and carcinogenicity of leucomalachite green. These new data included results of lacI mutations in the livers of Big Blue rats fed leucomalachite green at levels of up to 543 ppm in the diet for 4, 16 and 32 weeks and results of DNA adducts measurements in the livers of rats fed leucomalachite green for 4 weeks using 32P-postlabelling assays. (Culp SJ et al, Mutation Research vol 506-507, 55-63, 2002). The Food Standards Agency had asked the COM for advice on these new data. The mutagenicity data were from studies on leucomalachite green fed to BigBlue® rats. An approximate 3-fold increase in lacI mutations was reported in the liver only at the top dose level at the 16-week time interval. An apparent dose-related increase in DNA adducts using the 32P post-labelling assay was reported. 34. Members agreed that the results for lacI mutations were unusual in that the claimed positive response seen at 16 weeks feeding had disappeared after 32 weeks of feeding leucomalachite green. It was noted that the inter-animal variation in mutation frequency was high and the apparent positive result might relate to one out of the five animals fed leucomalachite green for 16 weeks. The committee was not convinced that a clear positive response had been obtained in this mutagenicity study. A reported increase in DNA adducts in the liver of female BigBlue® rats did correlate with the increasing dose of leucomalachite green. However, it was noted that adduct levels produced were very low; the highest level of DNA adducts found equated to approximately 1 adduct per 10-8 nucleotides (this was considered to be a difficult adduct formation frequency to detect). The author’s explanation was that the reported increase in mutation frequency at 16 weeks may have been due to a proliferative response of background mutations rather than induced by leucomalachite green. The committee did not find this particular argument convincing and agreed that overall no conclusions could be reached from the investigations reported in this published paper. 35. The preliminary results of the new NTP two-year carcinogenicity bioassay in rats with leucomalachite green were summarised in the published paper submitted to COM and suggested a dose-related increase in the incidence of alveolar/bronchiolar adenomas in the lungs of male rats but not females. There was no increased incidence of liver tumours in rats. Members noted that reference to preliminary findings in a mouse carcinogenicity study and also to further planned mutagenicity studies in BigBlue mice were reported. Members agreed that it was likely that additional data would be published by these authors on leucomalachite green. 36. In answer to the questions posed by the FSA, members agreed that no conclusions could be reached from the new mutagenicity data on leucomalachite green summarised in the paper by Culp SJ et al (Mutation Research vol 506-507, 55-63, 2002). No conclusions could be reached with regard to the provisional information on carcinogenicity of leucomalachite green reported in this paper. The Committee agreed there was no need to alter its previous advice on malachite green and leucomalachite green, but that the situation should be reviewed when full reports of the carcinogenicity bioassay became available. ITEM 8: DEET (MUT/03/4) 37. The COM considered the available mutagenicity data on DEET in April 2002 and concluded that whilst there were apparently no concerns regarding mutagenicity, there was a need to consider the full reports of the two unpublished studies (carried out for the DEET Joint Venture Group), namely a metaphase assay for clastogenicity in CHO cells and an assay for UDS in rat hepatocytes. The secretariat had now received copies of the full reports of these studies, together with a Salmonella assay. The advice from COM would be included in a statement from the COT on the toxicology of DEET. 38. Members noted that DEET appeared to be highly toxic in-vitro to mammalian cells in both CHO cells and rat hepatocytes in the UDS assay and hence adequate results were only available from a relatively narrow concentration test range. Members also noted that that the exogenous metabolising fraction used in the CHO chromosome aberration assay had performed poorly as indicated by the positive control data with cyclophosphamide. There was some discussion on whether a repeat cytogenetics test was required. 39. The COM concluded that there were limitations in all of the submitted studies. In particular, the COM considered that the exogenous metabolising fraction used in the in-vitro chromosome aberration assay had not produced satisfactory results with cylophosphamide. However, overall the results of the Ames test, an in-vitro cytogenetics assay in CHO cells and an in-vitro UDS assay in hepatocytes were negative. This information together with the information previously reviewed by the Committee (lack of structural alerts with DEET, negative Ames tests and negative carcinogenicity studies) suggest there is no concern with regard to the mutagenicity of DEET. ITEM 9: ANY OTHER BUSINESS
40. Members were requested to provide any comments they might have on the draft COT/COC/COM annual report. ITEM 10: DATE OF NEXT MEETING 41. 29th May 2003. ACTIONS
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