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MINUTES

Present:

Chairman: Professor P Farmer	Members: Dr B Burlinson Dr G Clare Dr J Clements Dr B Elliot Mrs R Glazebrook Dr N Gooderham Dr D Lovell Dr I Mitchell Dr E Parry Professor D Phillips	Secretariat: Mr J Battershill (HPA secretariat) Mr S Robjohns (HPA minutes) Dr D Benford (FSA secretariat) Mrs J Cleverly (Administrative) Mr K N Mistry (DH)
Assessors: Dr D Gray (HSE) Mr S Samuels (PSD)	In attendance : Mr B Maycock (FSA item 3.2) Dr L Hetherington (HPA) Mr K Okona-Mensah (DH Tox Unit item 3.3) A representative (Huntingdon Life Sciences item 4) Dr D Soo Kim (LG Life Sciences item 4)	Observers: Ms Roz Bulleid (ENDS item 5) Mr David Collinge (Sumit Agro items 3.2 & 6)

CONTENT

Item		Paragraph
1.	Announcements/Apologies for absence	1
2.	Minutes of the meeting on 18 May 2006	5
3.	Matters Arising (not covered by later agenda items):	6 - 16
4.	Partial review of Ethaboxam (closed session) (MUT/06/18)	17
5.	Role of methylation status: Transgenerational effects of methylation (MUT/06/15)	18 - 24
6.	Do dose response thresholds exist for genotoxic alkylating agents? GJS Jenkins et al. Mutagenesis 20 (6), 389-398 2005. Evidence for practical thresholds for clastogenic agents (MUT/06/17)	25 - 30
7.	Horizon scanning (MUT/06/19)	31 - 34
8.	Risk factors affecting the formation of chromosomal aberrations and micronuclei in peripheral blood lymphocytes. Draft working paper and additional data (MUT/06/22)	35 - 40
9.	Draft working paper on the use of the in-vivo rat liver UDS assay compared to in vivo rat liver COMET assay (MUT/06/21)	41 - 43
10.	Papers for information: 10.1 Revised procedure for holding committee meetings in open session (MUT/06/20). 10.2 In-vivo MN unclultured T-lymphocytes of male railroad workers and referents. Env Mol Mut, 47, 345-351, 2006) (MUT/06/23)	44 - 45
11.	Any Other Business	46
11.	Date of next meeting - 1 February 2007	47

ITEM 1: ANNOUNCEMENTS/APOLOGIES FOR ABSENCE

1. The Chair welcomed the new administrative secretary Mrs Janice Clevery to the COM for her first meeting. The Chair also welcomed Mr Barry Maycock (FSA), Mr Ken Okona-Mensah (DH Tox unit), the visiting experts Dr W Davies and Dr D Soo (LG Life sciences), and the observers Mr D Collinge (Sumit Agro) and Ms Roz Bulleid (ENDS).

2. The COM chairman and the scientific secretary thanked Mr Khandu Mistry for his greatly valued work and help with the COM over the last 10 years.

3. Apologies for absence were received from the members Dr C Allen and Dr D Gatehouse, the secretariat Ms F Pollit (HPA) and the assessors Dr D Andrew (PSD), Mr Alexander and Dr Sandra Payne (National Assembly for Wales), Dr H Stemplewski (MHRA), Dr B Viegas (DEFRA), Dr S Dyer (DH), Dr A Smith - Douglas Gray attended in his place (HSE) and new member of the DH Tox Unit Dr J Cunningham.

4. Members were reminded of the need to declare any interests before discussion of items.

ITEM 2: MINUTES OF MEETING ON 18 May 2006 (MUT/MIN/2006/2)

5. Members agreed the minutes subject to some minor editorial changes.

ITEM 3: MATTERS ARISING (NOT COVERED BY LATER AGENDA ITEMS)

6. Members were informed that COM statement on metal hip replacements was published at the same time as the MHRA advice (http://www.mhra.gov.uk/home/idcplg?IdcService=SS_GET_PAGE&nodeId=982). MHRA advice included information on risk/benefits of hip replacement. The secretariat apologised that there has been insufficient time to undertake the drafting of a non technical summary of the COM statement, which was intended to be part of the COC/COM risk communication exercise. Members were informed that MHRA were to establish an expert group to assess the clinical significance of the COM findings which would impact on wider risk communication. The COM would be informed of the outcome of these discussions.

3.1 Secretariat letter dated 22 May to HSE regarding RIP 3.3 Phase 2 Endpoint working Group (EWG): Mutagenicity

7. Members heard that following the May 2006 COM meeting, a letter from the COM secretary had been sent to the HSE regarding the mutagenicity testing strategy proposed by an Endpoint Working Group for industrial chemicals for REACH (Regulation Evaluation and Authorisation of Chemicals). The committee were informed that their comments had influenced the proposed testing strategy, and that there would be a further opportunity for members to provide comments within a few weeks time. The Chairman thanked HSE for raising this topic with COM at an early stage of the development of the REACH guidance.

3.2 Benzimidazoles (MUT/0614)

8. Members were reminded of their consideration of the potential for the use of a common mechanism group for the risk assessment of benzimidazoles and other tubulin binding substances at the May 2006 meeting. Members had agreed that there was a good correlation between aneugenicity in mammalian cells in vitro and aneugenicity in vivo, but that non-mammalian data, such as data for fungi and helminths were less predictive. A good correlation between mammalian tubulin binding in vitro and in vivo aneugenicity was also noted. Members had felt at the May 2006 COM meeting that it would be necessary to demonstrate that binding occurred to mammalian tubulin at the same site and that chemicals acted in a similar manner (eg tubulin depolymerisation or stabilization) and additively before they could be included in a common mechanism group. Members had also agreed that a possible scoring system could be developed to determine potential inclusion in a common mechanism group ie for the tubulin active benzimidazoles.

9. The secretariat and the Food Standards Agency had considered this subject further, and had drafted a paper that suggested the use of a decision tree for including a substance in a common mechanism group (MUT/2006/14) might be more appropriate than a weighting of data. The paper also summarised the current state of knowledge in this area, suggested that other tubulin binding substances could subsequently be added to a common mechanism group (CMG), provided that they were aneugenic in vivo and demonstrated additivity with at least one compound in the benzimidazoles CMG.

10. From the data summarised in MUT/2006/14, members agreed that the example chemicals demonstrated binding at different tubulin binding sites eg there was evidence for oxibendazole and fenbenadozole binding to the colchicine binding site, but evidence that benomyl binds to a novel site. Members noted that the effects produced were difficult to predict without conducting case by case studies. There was evidence that some chemicals such as dilantin and vinblastine interacted with tubulin by different mechanisms and different binding sites, but that their combined effects on microtubule inhibition was additive. It was also noted that different chemicals binding to the same site could induce different mechanistic effects ie stabilisation or depolymerisation of tubulin, and that the specific effect produced could vary depending on concentration. It was noted that tubulin binding studies for anti-cancer chemotherapeutics had been specifically investigated for potential additivity and synergism. It was possible that more published data might be available which might be valuable to COM consideration. Thus, members suggested based on the data reviewed in MUT/06/14 that it would be more useful and important to demonstrate a common biological endpoint, such as inhibition of tubulin polymerisation or stimulation of depolymerisation, rather than consideration of the specific binding site or additivity.

11. Regarding the proposed decision tree for allocation to a benzimidazole CMG, members agreed that a combination of in-vitro and in-vivo aneugenicity data (along with data on target tissue exposure) was required and queried the order of the steps set out in figure 2 to MUT/06/14. The FSA secretariat noted that the sequence had been derived for decisions regarding inclusion in the CMG rather than following a traditional mutagenicity testing strategy.

12. The key area of risk assessment related to co-exposure to more than one benzimidazole. The general approach as recommended by the COT working group on risk assessment of pesticides and similar chemicals (WIGRAMP) was that for chemicals with a similar mechanism the default was to assume additivity of effect. This approach would be relevant for benzimidazoles which acted through a threshold-related mechanism. The committee discussed the extent of data required to reach a conclusion on additivity for benzimidazoles and considered whether data were required on a compound-by- compound basis. Members considered that there were too few data to predict combined effects for chemicals that bind to the same and different tubulin sites at present. The COM agreed that if the mechanism of action was known, there was scope for allocation to a common mechanism group for aneugens other than benzimidazoles. Members raised a number of observations which the secretariat would consider when revising the proposed approach. One member commented on potential for redundancy of target molecules in describing thresholds for tubulin binding effects. Another member emphasised the need to base assignment into the CMG on the basis of functional effects on tubulin rather than detailed information on binding sites. The chair thanked members for their comments and asked for any further comments to be sent to the secretariat. A further paper would be drafted for the February 2007 meeting.

3.3 Comparative risk assessment update of COC discussion (MUT/06/16)

13. The committee was provided with a brief update on the comparative risk assessment project and the discussions held at the July 2006 COC regarding environmental carcinogens and communication of associated cancer risk to the public. An initial discussion paper had been considered at the previous COM meeting in May, and this had been taken to the COC in July. Three initial approaches had been outlined in the paper ie the Calman Risk scale, the Paling perspective and Dr Frank Duckwork's Riskometer. The DH Toxicology unit and the secretariat had subsequently identified a Comparative Risk Assessment (CRA) approach developed by the National Radiological Protection board (now part of the HPA) for radiation exposure.

14. The COC Chairman had noted that this was a useful exercise and that the COC had previously considered that a comparison of risks from environmental carcinogens with smoking tobacco was not helpful, for a numbers of reasons eg inappropriate comparison of an involuntary risk with the voluntary risk of smoking. The COC agreed with the COM proposal that current approaches to drafting statements should be improved by including non technical summaries. The COC considered that COM/COC documents should provide more information on the reasons for undertaking reviews. COC members felt that the 'Risk it' game, developed by the NRPB mixed actuarial and estimated risk scenarios and that it did not consider benefits in the comparisons made. It was suggested that it would be more useful to compare more closely connected risks eg those of natural toxins with synthetic pesticide residues that are both present in the same food.

15. The committee had agreed that a 'Margin of exposure' (MOE) approach, such as that currently being developed by the EFSA and ILSI could be useful. Such an approach would be consistent with the 'Minimal Risk Level' approach outlined in the current COC guidance. A similar process had been used by JECFA for risk ranking purposes, and therefore could be extended for a CRA. The COC had discussed a number of examples where the risks of exposure to environmental carcinogens had been reported by the media eg Sudan 1. The FSA were keen to develop a MOE approach. COC members had felt that comparisons with lifetime risk eg loss of life expectancy were more useful than annual rates and that some comparisons were inappropriate for chemical carcinogens such as slipping in the bath.

16. Overall, the COM agreed with the COC that further consideration of the MOE approach was warranted and that a presentation by a risk communication expert would be helpful.

ITEM 4: PARTIAL REVIEW OF ETHABOXAM (MUT/06/18)

17. The discussion of this in-confidence item was conducted in a closed session. The minutes will be published when the COM statement has been finalised and published. Now available see paragraphs 48 - 70.

ITEM 5: ROLE OF METHYLATION STATUS: TRANSGENERATIONAL EFFECTS OF METHYLATION (MUT/06/15)

18. The COM had agreed to undertake an initial evaluation of the role of methylation status and transgenerational effects of methylation at its horizon scanning exercise in 2005. This was in response to the Medical and Toxicology panel of the Advisory Committee on Pesticides (ACP), which had also requested consideration of this topic.

19. The Medical and Toxicology Panel (MTP) of the ACP had reviewed a recent paper, which reported on investigations in to the potential for vinclozolin or methoxychlor to induce transgenerational effects via the male line, following a short duration of exposure of pregnant females to relatively high doses (Anway et al Science 308, 1466-69, 2005). The DH Toxicology Unit had provided a summary of the Anway et al 2005 paper, appended to MUT/06/15. Decreased spermatogenic capacity and reduced fertility were reported over four generations. The authors suggested that the effects on reproduction correlated with altered DNA methylation. The MTP had also noted that there was literature on other chemicals regarding transgenerational effects in experimental animals (eg with diethylstilbestrol by Newbold R 2004, Toxicol Appl Pharm, 199, 142-150) and thus it was important to consider the scope of any review work, potential epigenetic mechanisms, and end points. Members were informed that the draft discussion paper was based on a limited number of chemicals in order to help consideration on possible future areas of work, the possibility of testing for DNA methylation changes, and consideration of the significance of transgenerational DNA methylation changes in risk assessment.

20. The DH Toxicology Unit provided a review of the mechanisms by which chemicals may induce epigenetic alterations and consequent potential to cause effects in offspring (appended to MUT/06/15). The phenomenon of an increase in tumourigenic and teratogenic effects in transplacentally exposed F1 offspring of treated mothers, also observed in subsequent F2 and F3 generations, had been documented approximately 30 years ago. Paternal transmission of heritable effects have also been recognised and studied both for carcinogenic and teratogenic effects and behavioural and neurochemical effects. Members noted the observation that the high frequency of effects, not adhering to Mendelian inheritance, had been cited as possible evidence that the mechanisms did not involve mutation. Members observed that loss of genomic imprinting possibly induced by DNA methylation could result in gene silencing or activation and might be important with regard to transgenerational effects. There was a tendency for decreased expression of the examined imprinted genes associated with higher methylation levels. There was evidence that the observed effects were predominantly due to DNA methylation pattern changes occurring at specific cytosine-guanosine dinucleotides (CpG sites) resulting in subsequent alterations in gene expression.

21. Members discussed the data presented on the three examples and agreed that there was evidence for transgenerational effects. Anway et al 2005, showed that maternal exposure (F0 only) to relatively high doses of vinclozlin (an antiandorogenic endocrine disrupter) significantly reduced sperm apoptosis, sperm counts and motility through four generations after subsequent breeding. The high incidence of changes (>90%) were considered unlikely to be explained by a 'normal' mutational DNA sequence mechanism, and evidence for an alteration in methylation patterns was also found. In addition members noted that Anway and colleagues had recently published additional studies confirming these effects in two strains of rat (Anway MD et al J of Andrology, e-publication 11 July 2006) There was evidence that exposure to diethylstilbestrol (DES) during particular periods of development in utero resulted in malignancies of reproductive organs/tissues in the offspring of both experimental animals and humans (reviewed in Newbold et al 2004). There was also evidence that these effects were transmitted to a second generation in the female line of mice (Walker and Haven 1997). Wu et al 2004, found evidence that TCDD affected fetal development via methylation and imprinted genes. However, members felt that the reported evidence for transgenerational carcinogenic effects induced by chromium III was very limited and no definite conclusions could be reached. It was noted that the information provided on vinclozolin, methoxychlor and DES showed evidence for effects through more than one generation.

22. The committee considered that carcinogenesis and reproductive effects appeared to be the main endpoints for transgenerational DNA methylation changes. It was suggested that such gene expression could be examined by using a micro array approach. Members noted that the chemicals looked at so far, for their ability to affect DNA methylation changes and produce epigenetic effects, were structurally very diverse, and thus difficult to predict or to devise a testing strategy or to integrate this with mutagenicity testing. One member recalled studies with 5-azacytidine which reduced overall cellular methylation and considered it was possible this effect was, in addition to mutational effects, related to the carcinogenicity of this chemical. The COM noted that certain important genes in the carcinogenic process, such as Kras could be affected by methylation.

23 Regarding future research, members suggested that vinclozolin could be used as a model compound to further investigate gene changes in relation to toxicological outcome. More generally, a micro array approach to analysis could be used to examine the effects of chemicals and methylation on specific gene expression eg whether up regulated or down regulated. It was noted that DNA methylation and subsequent histone changes could also be important, but that this would be difficult to distinguish between the relative importance of these changes. The committee felt that it would be very useful to review other compounds, and when more was known about DNA methylation and its effects on heritable risks, there may be a need to for incorporation in the COM strategy.

24. The COM felt that DNA methylation effects would be a very important area for future research for a potentially wide range of toxicological effects, particularly for carcinogenesis, and considered that this topic was something that the COC and COT would need to be involved. It was suggested that a joint workshop and an invitation to key researchers in this area to attend would be useful.

ITEM 6: DO DOSE RESPONSE THRESHOLDS EXIST FOR GENOTOXIC ALKYLATING AGENTS? GLS JENKINS et al., 20, (6), 389-398, 2005. EVIDENCE FOR PRACTICAL THRESHOLDS FOR CLASTOGENIC AGENTS (MUT/06/17)

25. A copy of the paper by Jenkins and colleagues was provided for information at the May 2006 meeting. Members had agreed to have a detailed consideration of the paper at the October meeting. An overview of the paper by Jenkins and colleagues was provided in MUT/06/17 and the Jenkins et al 2005 paper was appended.

26. The concepts of absolute threshold, non-linear dose-response and NOEL were outlined. The key area of discussion concerned the concept of a practical threshold, where the threshold for DNA adducts is lower than the threshold for subsequent mutation. The concept of 'not biologically significant' and 'biological significant' effects representing doses in the LOEL range was outlined. The practical threshold was said to be determined by chemical specific mechanisms ie redundant targets such as microtubules, membranes, cytoplasmic elements, DNA repair, and differences in the conversion of different adducts to mutations. The main sections of the paper concerned the evidence for thresholds for DNA reactive alkylating agents. These included ENU, MNU, EMS and MMS as they formed two different groups of alkylating agents that had been comparatively well characterized and information on these chemicals could help to understand the concepts of thresholds in general.

27. Some of the evidence reported for alkylating agents reviewed by Jenkins et al 2005, had been considered in an earlier COM paper on DNA repair mechanisms at low doses of mutagens. The COM had concluded that there was evidence to support a threshold mechanism in vitro for mutagenicity in bacteria with proficient O⁶-methyl transferase activity and suggested that in in-vivo threshold was likely, but not proven. Jenkins and colleagues concluded that more information was needed to determine mutation thresholds experimentally and the mechanisms of repair pathways. This included more evidence for thresholds for repair of O⁶G and N7G adducts.

28. Members agreed with Jenkins et al that the current evidence only referred to acute exposures to single agents, whilst most environmental chemical exposures occurred to mixtures over extended and often chronic durations. Thus, the available data were limited in their usefulness in demonstrating a practical threshold for mutation ie due to uncertainties in extrapolating to longer and combined exposure scenarios. Members also noted the problem posed by the much higher sensitivity for DNA adduct detection compared with the detection of any subsequent mutation. The biological significance of low levels of DNA adducts and of individual DNA adducts had not yet been fully established and this presented a difficulty in identifying a threshold for mutation. Members observed that the DNA repair mechanisms considered (such as DNA alkyltransferases) would follow Michaelis-Menton kinetics and thus would presumably be suboptimal at concentrations below the K_m. Members noted that there would be different approaches to consider regarding mechanisms for potential thresholds for direct and indirect mutagens relating to metabolic activation and detoxication.

29. The COM considered that the concept of a threshold for biological significance could be a useful way forward, but felt that this needed to be considered in the context of the possible DNA repair mechanisms involved and the available dose-response data available (including the sensitivity of the method to detect a NOEL). The COM considered the Jenkins et al review with regard to the COM conclusions reached in 2001 on thresholds for in-vivo mutagens and genotoxic carcinogens (http://www.advisorybodies.doh.gov.uk/com/comivm.htm) and agreed that there was no need to change its current view that for in-vivo mutagens and genotoxic carcinogens it is prudent to assume that there is no threshold for mutagenicity. It may be possible to identify a possible threshold when appropriate data on DNA adduction, mutation mechanisms, DNA repair were available. However such data needed to be generated on a chemical-by-chemical basis.

30. Regarding future work, members agreed with Jenkins et al that further studies with paired alkylating agents with similar/dissimilar adduct types with repair deficient cell lines could be informative. It was agreed that it would be important to monitor future literature in the area of thresholds for in vivo mutagens. Members noted that there was currently a lot of interest particularly within the USA in using flow cytometry for the analysis of micronuclei in relation to potential thresholds. However, it was felt that this method may improve precision of a NOEL by allowing measurements of a greater number of cells from each animal, but it might not necessarily improve sensitivity due to the natural variance between animals and possible experimental variation resulting from the flow cytometric procedure.

ITEM 7: HORIZON SCANNING 2006 (MUT/06/19)

31. The 2006 horizon scanning paper had been prepared by a literature search strategy using PUBMED, which indicated several thousand publications in 2005/6 which might be relevant. About 2,000 references were identified by using terms such as "potent mutagen", "mutagenicity", and "mutagenicity testing". Additionally, the contents lists of Environmental and Molecular Mutagenesis and Mutagenesis were scanned. The literature search was briefly scanned to highlight chemicals, exposures and generic areas of mutagenicity evaluation that could be of interest to the COM. A brief overview was provided in MUT/06/19, as an initial starting point for members view on future work. The horizon scanning exercise provided an opportunity for members and advisers from Government Departments/Regulatory agencies to discuss topics for further work. Members were asked for their views on what areas should be considered for further work.

32. One main area which emerged was the evaluation of mixtures (eg water/air contamination) by testing components or combustion products (in the case of tobacco), and the approaches used to evaluate the interactions between components. A number of questions regarding mixtures were included, which were:

i. developing advice on possible strategies for assessing the mutagenicity of chemical mixtures in order to help define the potential marker compounds or mixtures specific approaches to regulation.

ii. developing advice on possible strategies to assess the adequacy of regulatory standards for mixtures, which are based on single marker compounds (It is noted that COC/COM have agreed that mutagenic potency and DNA binding at the site of contact can act as a surrogate for site of contact carcinogenic potency for PAHs).

33. Other areas identified included the use of mutational finger prints, testing strategies, the use of flow cytometry in the in-vitro MN assay, the possible use of Expanded Simple Tandem Repeats for detection of germ cell line mutagens, the development of an in-vivo skin MN assay, and the development of in-silico approaches. The main potential areas identified at the end of the paper were:

A. Evaluation of chemical mixtures for mutagenicity including specific evaluation of exposure scenarios and evaluation of approaches to risk assessment.

B. Evaluation of generic areas of testing relevant to future consideration of testing strategy.

C. Use of mutational fingerprints in molecular epidemiology.

D. Further consideration of approach to risk assessment of in-vivo mutagens (links with the Jenkins paper item 6).

34. Members felt that a comprehensive selection of potential areas of interest had been identified and noted that it would not be possible to consider all of these suggested topics in detail. The committee agreed that considering approaches to the risk assessment of mixtures of chemical mutagens should be a priority. Members also agreed that mutation "fingerprints" would be a useful area to monitor, for example the measurement of mutation "hotspots" in the analysis of the carcinogenic process. It was possible that both of these projects could be undertaken jointly with COC. It would also be necessary to keep a watching brief on the literature regarding the potential for thresholds for in-vivo mutagens (as discussed in item 6). Consideration of the relative potency of various in-vivo mutagens regarding risk communication was felt to be important, although members believed that it would be difficult to rank the potency of individual in-vivo mutagens.

ITEM 8: RISK FACTORS AFFECTING THE FORMATION OF CHROMOSOMAL ABERRATIONS AND MICRONUCLEI IN PERIPHERAL BLOOD LYMPHOCYTES. DRAFT WORKING PAPER AND ADDITIONAL DATA (MUT/06/22)

35. The COM had considered a review of the available data on the background variance of micronuclei (MN) formation and chromosomal aberrations (CAs) in peripheral blood lymphocytes (PBLs) at its February and May 2006 meetings. The objective was to prepare a statement which would assist Government Departments and Agencies in evaluating the data and in the design of biomonitoring studies for genotoxicity in populations and occupational groups with potential exposure to genotoxic chemicals. The covering paper MUT/06/22 summarised additional information required to complete the review, which related to information on the influence of drinking alcohol on background levels of MN formation and CAs in peripheral blood lymphocytes. Further data on the effects of infection, disease state, and conditions of stress, such as physical exercise were also provided. A review of these new data was appended at Annex 1.

36. Since the May 2006 meeting, three additional papers had been retrieved that could be useful to the COM in coming to its final conclusions. These were provided at Annex 2. Abramsson-Zetterberg et al 2006 (Mutation Research, 603, 33-40), reported on the effect of folate within the normal range of serum folate. A study by Patino-Garcia and colleagues (Mutagenesis, 21, 191-197, 2006) investigated the scoring variability of MN formation in PBLs in a case-control study, where MN determinations were undertaken over a period of 18 months. This extended the earlier work that the COM had seen regarding the variance due to scoring, which had been investigated following irradiation of PBLs at a single time point. A review article by Bonassi S et al (Environmental and Molecular Mutagenesis, 45, 258-270, 2005) reviewed a large number of studies and provided a highly valuable commentary on the application of MN and CA in PBLs to the biomonitoring of genotoxicity.

37. Regarding alcohol, members agreed that the evidence for alcohol drinking induced increases micronuclei was inconclusive. However, an increase in MN formation had been documented in drinkers of alcoholic beverages who also had the ALDH2* polymorphism (associated with a slower metabolism of acetaldehyde). An increase in MN formation had been documented in alcoholics still consuming alcohol, but not in those who had abstained from drinking for one year. This latter effect was also observed for CAs.

38. It was noted that certain disease conditions (bacterial/viral infections) and intense physical exercise may increase chromosome damage. Thus, such information should also be gathered during biomonitoring studies analysing for MN and CA formation in PBLs in relation to environmental chemical exposures.

39. Members noted the results reported by Abramsson-Zetterberg L et al, Mutation Research 603, 33-40, 2006. A statistically significant correlation between reduced serum folate concentration in the normal range and increased MN formation in PBLs had been documented when the results of three cross-sectional studies had been considered together. Members considered that these data were heavily influenced by one outlier and thus only limited conclusions should be drawn. One member asked for further consideration of the available data on vegetarian diet and MN formation in PBLs before the statement was finalised. Members suggested that a peer review publication might be based on this work.

40. Due to limited time there was a lack of opportunity for members to provide detailed comments on the draft working paper MUT/06/22 at the meeting. Members were asked to provide more detailed comments by postal circulation and it was intended that the working paper would be finalised by Chairman's action.

ITEM 9: DRAFT WORKING PAPER ON THE USE OF IN-VIVO RAT LIVER UDS ASSAY COMPARED TO RAT LIVER COMET ASSAY DATA (MUT/06/21)

41. The committee had requested a discussion paper on the comparison of the in-vivo rat liver UDS assay and the in-vivo comet assay during the horizon scanning process in October 2005. A draft working paper had been prepared based on the COM discussion in February 2006. Members had concluded that the approach used in the review was relevant to empirical comparisons between in-vivo mutagenicity assays but that any discussion on the role of the UDS assay and the comet assay in overall testing strategy also needed to include consideration of using in-vivo assays in the context of the data provided by the in-vitro assessment of mutagenicity.

42. The Committee had also concluded that the current comparative review of the rat liver UDS and comet assays should be considered in the context of the available published data reviewed, the limitations of the experiments considered, the ongoing development of the comet assay for rodent tissues and the possibility of relevant data held by industry but not available in the public domain. Members commented that the limitation of the isolated nuclei method for the comet assay in the data reviewed related predominantly to the approach used by the investigators but overall the isolated nuclei method should not be discounted. It had been agreed that:

i. the available data was consistent with the view that rat liver UDS assay and the rat liver comet assay had broadly similar response with a limited number of known rodent carcinogens.

ii. a further repeat rat liver comet assay was desirable for chlorodibromomethane.

iii. no further evaluation of the mutagenicity acrylamide was required at the present time for the comparative review of results obtained in the rat liver UDS and comet assays.

43. Members were asked to send any comments to the secretariat and the draft working paper would be finalised by Chairman's action.

ITEM 10: PAPERS FOR INFORMATION

44. 10.1 - Revised procedure for holding committee meetings in open session (MUT/06/20)

45. 10.2 - In-vivo MN in uncultured T-lymphocytes of male railroad transit workers and referents. (Env Mol Mut, 47, 345-351,2006) (MUT/06/23)

ITEM 11: ANY OTHER BUSINESS

46. Members were informed that, following the transition of duties from DH to the HPA , the process for the reimbursement of expenses via the HPA Payroll Systems had now been implemented and payments would commence in October 2006.

ITEM 12: DATE OF NEXT MEETING

47. 1 February 2007

ITEM 4: PARTIAL REVIEW OF ETHABOXAM (MUT/06/18)

48. The Chairman told the meeting that this item would be held in closed session and asked all observers and attendees to leave the room. He asked for declarations of interest. Dr Burlinson reported a direct interest in the compound and left the room. Dr Clare reported a lapsed indirect interest in that she had been study director at the contract laboratory where some of the mutagenicity studies had been undertaken with ethaboxam. The Chairman considered that Dr Clare could be present for the item and could answer members queries on the conduct of the studies whilst members were deliberating the questions to ask the data holder and their representative but should not take part in deriving COM conclusions.

49. The secretariat noted that ethaboxam had been referred to the COM by the Pesticides Safety Directorate. The referral statement was; "PSD have asked for advice from the COM on the most appropriate strategy to be taken with regard to the testing for aneugenicity of ethaboxam in-vivo and the potential for risk of site of contact in-vivo anegenicity effects and their likely significance for risk assessment. This is not a full referral of the mutagenicity data to the COM." Ethaboxam is a new fungicide and is formulated as a 100 g/L suspension concentrate for control of grapevine downy mildew caused by Plasmopara viticola. The proposed pattern of use is for up to 5 applications per season, with an interval of 7 to 10 days depending on weather conditions and disease pressure. Thus the key sites of contact for evaluation are the upper respiratory tract (in particular the nasal passages) and possible skin contact to formulated concentrate and in-use dilution.

COM consideration of areas for discussion

50. The Committee considered the submitted data which included the draft PSD evaluation which presented information on structure, use, ADME studies, general toxicology , mutagenicity, carcinogenicity and reproduction, and copies of the mutagenicity studies which had been submitted to PSD. Members considered the areas of mutagenicity evaluation to raise with the data holder and their representative.

51. There was potential for epoxidation and metabolism at a secondary amine in ethaboxam. There was no evidence for metabolism via these routes in the available studies and overall, members considered there were no clear indications from structure and metabolism with regard to mutagenicity and in particular aneugenicity.

52. There were no questions to raise on the in-vitro bacterial mutagenicity test using Salmonella typhimurium.

53. With regard to the in-vitro cytogenetics assay using human peripheral blood lymphocytes (PBLs), members queried whether sufficient investigation of the potential for polyploidy (a possible indicator for aneugenicity) had been undertaken. This had included a quantitative analysis for polyploidy at the highest dose level used. It was noted that the effects on cell division with the high frequency of metaphase arrest masked the potential for identifying polyploidy cells. However it was agreed to ask whether lower dose levels could have been studied.

54. With regard to the in-vitro mouse lymphoma assay (MLA), it was noted that an apparent dose related increase in mutant frequency was observed in 24 hour trials in the absence of exogenous metabolic activation at three dose levels of 4µg/ml and above where survival was below 10%. Members noted that an appropriate level of toxicity (ca 10-20%) was not achieved when using percentage relative survival. However, these data would be consistent with that reported for other chemicals with aneugenic potential such as colchicine. Members agreed that the data holder and representative should be asked for their interpretation of both toxicity and mutagenicity data from this study.

55. With regard to the in-vitro assay with PBLs designed to investigate effects on mitosis, members noted that a cytochalasin B block method had been used to asses the proportion of binucleate to mononucleate cells and queried why an investigation of micronuclei had not been undertaken at this stage, given the available information on the fungicidal mode of action for ethaboxam.

56. With regard to the in-vitro MN assay in PBLs subsequently undertaken at the request of the ACP, members queried the interpretation of the dose response and noted the relatively stringent statistical criteria for a positive response which had been applied (ie use of multiple comparison test and a P value of 0.01). Members considered that the NOEL was most likely below the reported 5µg/ml in the absence of exogenous metabolic activation. Members noted a higher NOEL of 50 µg/ml had been reported in the presence of exogenous metabolic activation. However members queried on the basis of the available ADME data whether all or part of the increase in the NOEL reported could be attributed to metabolism or whether some other process such as sequestration in microsomes might have occurred. It was noted that in vitro treatment in the presence of S9 is reduced to 3 hours (compared to 20 hours in the absence of S9) so that there would be reduced exposure. Members also noted that for other aneugenic substances such as benomyl the NOEL for non disjunction was below that for chromosome loss/gain and hence the NOEL for non disjunction of ethaboxam could be below that reported for MN induction in PBLs. Member considered that the in-vitro MN assay using PBLs needed additional evaluation for whole chromosomes at each dose level to investigate the relative proportion of aneugenicity to clastogenicity.

57. With regard to the first in-vivo bone marrow MN assay in rats using oral administration, members noted that the vehicle chosen was 0.5% carboxymethyl cellulose (CMC). However 1% CMC with the addition of Tween 80 had been used in the oral absorption study in rats and this had resulted in a relatively poor absorption of approximately 60%. Overall, it was felt that the oral absorption in the oral bone marrow MN assay in rats would have been limited. Members agreed to ask for comments on the selection of vehicle in the oral bone marrow MN assay in rats.

58. With regard to the intraperitoneal bone marrow MN assay in mice which had been undertaken in 1996 but had only recently been submitted to PSD, it was noted that the test material was relatively impure (ca 90.4%), In the first analysis at 48 h post dose, a significant increase in MN was noted at 488 mg/kg (which was associated with a high level of toxicity and mortality). The trend test at the sampling time (48h) was significant. The investigators had rescored at this sampling time using a higher number of 2000 polychromatic erythrocytes and had not reported a statistically significant effect. Members considered that the treatment related increase and apparent trend in MN formation in the reanalysis were still evident and noted that P values of the statistical analysis had not been reported. However, they felt these P values may have been relevant information because a stringent P,0.01 value had been used for determining statistical significance. Members agreed to ask the data holder and their representative for an interpretation of this study. Overall, the COM considered that the study could not be clearly considered as negative and the usual approach COM would recommend would be a retest including lower doses.

Data holder presentation

59. The data holder (LG Life Sciences) and representative (Huntingdon Life Sciences) were asked to make a short presentation to the COM and to answer members queries. All comments were made by the HLS representative.

60. HLS noted that some of the fungicidal activity of ethaboxam was due to interference with the fungal cytoskeleton possibly by inhibition of tubulin subunits. The negative findings in the Ames and MLA tests indicated no potential for gene mutagenicity. The in-vitro cytogenetics and MN tests in PBLs were considered to be positive in the absence of exogenous metabolic activation. However two negative bone marrow MN assays were available in the rat (oral dosing) and in the mouse (intraperitoneal dosing). In addition following a request from the ACP, a re-analysis of sections from the gastrointestinal tract from the rat 2 year chronic toxicity/carcinogenicity assay had not indicated any effects on mitosis. HLS also noted that no adverse toxicity had been reported in a 28 day dermal toxicity study in the rat. It was also considered that potential site of contact concentrations (skin and via oral ingestion) would be low.

COM questions for data holder and HLS

61. Members raised one generic comment that the estimated site of contact concentrations reported by HLS should also include some estimate of respiratory exposure.

62. The Chairman asked members to introduce their questions (which have been outlined in paragraphs 3-13 above. A summary of the responses is given below.

63. HLS noted that the initial strategy had placed a weight of evidence on the mouse lymphoma assay and the available in-vivo MN assays and thus the initial in-vitro study in PBLs had not specifically incorporated an investigation of MN formation. HLS noted the comments made by the COM and evaluation of the in-vitro chromosomal aberration assay and the mouse lymphoma assay. HLS noted the evaluation of the COM with regard to the in-vitro MN assay in PBLs regarding the assessment of dose response for aneugenicity and clastogenicity and the possible effects of adding exogenous metabolic activation systems. HLS considered that the use of 0.5% carboxymethyl cellulose was a relatively normal approach to oral dosing in in-vivo bone marrow MN assays. The COM considered that oral absorption would have been limited in comparison to that seen in the oral rat kinetics study (up to 60%) using radiolabelled ethaboxam which had used 1% CMC and Tween 80 as a surfactant to aid solubility in the dosing vehicle. HLS noted the interpretation of the in-vivo bone marrow MN assays in rats and mice reached by the COM.

64. The secretariat informed the data holder and HLS that a draft working document (statement) should be forwarded for comments in around 10 working days. Comments received from the data holder and their representative would be forwarded to the COM chair for his consideration but there was no obligation to alter the statement. The final agreed statement would be forwarded to the ACP who advise ministers on the approval of pesticides. The statement and minutes would be published in consultation with PSD.

65. The data holder and HLS withdrew from the meeting.

COM consideration and conclusions

66. The COM considered that an appropriate evaluation of polyploidy as an indicator for potential aneugenicity had been undertaken given the predominant effects of ethaboxam on cell division. The COM agreed that more evaluation of the mouse lymphoma data to predict potential for aneugenicity might have been possible.

67. The COM agreed that a further evaluation for aneugenicity and clastogenicity should be undertaken in-vitro to describe appropriate dose-response data and to determine the NOELs. The COM noted that the potential NOEL for non disjunction might be lower than for chromosome loss or gain and this end point should also be investigated.

68. The COM agreed the NOEL for MN induction in PBLs in-vitro was likely to be lower than the value of 5 µg/ml reported in the existing study in PBLs, but probably above the test concentration of 1.0 µg/ml.

69. The COM considered that it would be difficult to undertake an evaluation of site of contact aneugenicity in-vivo for ethaboxam given the current knowledge of potential approaches. A pragmatic in-vivo testing strategy should include a re-test using intraperitoneal dosing in mice with evaluation of MN in the bone marrow to include evaluation of whole chromosomes. Members agreed that consideration should also be given to investigating MN formation in other tissues in this study such as the liver, germ cells and splenocytes. Members considered it was important to measure plasma and tissue concentrations of ethaboxam.

70. The Chairman thanked members for their comments. He agreed to write to the chairman of the ACP outlining a possible interpretation of the above studies and to note some of COM members comments on other aspects of the evaluation of ethaboxam (ie the thyroid accumulation of ethaboxam derived material that was radiolabel site specific, the possible mechanism of leydig cell tumours and effects on sperm at high dietary doses in the multigeneration study.)

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