Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment
Minutes of the meeting held on 7th October 1999
MUT/MIN/99/3
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Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment Minutes of the meeting held on 7th October 1999
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Minutes of the meeting held on Thursday 7 October 1999 at 10.30 am, in the Cathedral room, Richmond House, Whitehall, London Present: Chairman: Professor J M Parry Members: Dr P Bryant Professor C Cooper Professor D Davies Professor MHL Green Ms M Langley Dr D Tweats Dr S Venitt Secretariat: Dr R J Fielder (Scientific) Mr J Battershill (Scientific) Mr M Al-Derzi (Minutes) Mr K N Mistry (Administrative) Assessors: Mr A Browning (VMD) Dr C Fisher (MAFF-JFSSG items ) Dr A Smith (HSE) Dr RO Shillaker (PSD) Dr H Stemplewski (MCA) In attendance Miss A Achampong (HSE) Miss J Andrews (DH) Dr T Barlow (FSP (D)) Dr P Ridgeway (HSE) Mrs J Walden (DH) (Item7) Dr A Sewart (FSP(D)) CONTENTS
ITEM 1: APOLOGIES FOR ABSENCE 1. Apologies for absence were received from Professors Ashby, Davies, and Newbold, and from Dr P Tromans (National Assembly of Wales), Announcements 2. Members were reminded of the need to declare any interests before discussion of items. 3. The Chairman welcomed Ms Margaret Langley as new lay-member on the COM (and COC). Members introduced themselves. ITEM 2: MINUTES OF THE MEETING HELD ON 20TH MAY 1999 (MUT/MIN/99/2) 4. The minutes of the meeting held on 20th May 1999 were agreed subject to one minor typographical amendment. ITEMS 3 & 4: DRAFT CONCLUSIONS FOR MEETING HELD ON 20TH MAY 1999 AND MATTERS ARISING. 5. 3-MCPD: The conclusions on 3-MCPD (including the COC conclusions) were finalised following consultation with Nestle and placed on the COM Internet site. 6. Di-isopropylnaphthalene (DIPNs): Members reservations regarding the conduct of the mouse lymphoma assay with DIPNs were forwarded to the contract laboratory who had now agreed to repeat the assay. Conclusions on DIPNs would be finalised following receipt and evaluation of the new study ITEMS 5 & 6 : HYDROQUINONE AND PHENOL (MUT/99/36, 37 and 41) 7. Members were told that in-confidence data were submitted to the Committee in 1994/5 by Kodak (Eastman) Ltd. Data provided for the current review came from published papers and from research sponsored jointly by HSE and a contract laboratory. No interests were declared. 8. The COM considered the mutagenicity data on hydroquinone and phenol in 1994/5. It was concluded that hydroquinone was an in-vivo mutagen in somatic cells but not germ cells. Similarly there was evidence that phenol was an in-vivo mutagen in somatic cells but that it had been inadequately investigated in germ cells. The potential for a threshold via the oral route existed with both hydroquinone and phenol since there were two protective mechanisms, namely rapid conjunction and detoxification via the glutathione pathway, which could substantially reduce systemic exposure. There was no evidence to support a threshold for exposure via the respiratory tract or skin. Further data submitted by Kodak in 1995 provided additional toxicokinetic/metabolism data on hydroquinone and phenol derivatives in the lung and skin. The Committee considered this information but agreed that the data supplied did not adequately address the question of presystemic metabolism. They asked for further studies to be conducted including studies in rats or dogs following administration of hydroquinone or phenol via a bronchoscope with early sampling for free/conjugated test substance in the blood and additional studies of percutaneous absorption in volunteers. 9. The Committee first considered the published mutagenicity data on phenol not previously considered. Members agreed that no significance could be attached to the negative results reported in the studies published by Tsutsui et al (Mutation Research, 373, 113-123, 1997). These investigators had examined a number of end-points (i.e gene mutations, chromosomal aberrations and aneuploidy and ployploidy) in in-vitro tests in SHE cells but the highest dose selected was inadequate as cell survival was reported to be 78%. A small but statistically significant increase in micronucleated polychromatic erythrocytes was reported in a bone marrow micronucleus assay (Marrazini A et al. Mutation Research, 341, 29-446, 1994) at a dose of 120 mg/kg (give by intraperitoneal injection) not previously considered by the COM. Members agreed that the increase (3/1000 PCEs, cf 2/1000 PCEs in controls) was of marginal biological significance. Similar small increases in micronucleated PCEs were also documented in a separate bone marrow micronucleus assay where phenol was administered intraperitoneally on each of three consecutive days at 100 or 160 mg/kg bw (Chen H and Estmond DA. Carcinogenesis, 16, 1963-1969, 1995). Thus the available data on phenol suggested that a small but consistent mutagenic effect could be detected in in-vivo mutagenicity tests conducted to OECD guideline or similar standards at intraperitoneal doses of 100-160 mg/kg bw. 10. The Committee considered the new transgenic mutagenicity test with phenol using the LacZ transgene in MutaTM mice. Animals were given either dermal doses of 100 mg/kg bw or exposed for a period of 2 hours to a vapour containing 100 ppm phenol on five consecutive days. Samples of tissues (liver, bone marrow, and blood, and also for inhalation exposure nasal epithelia and lung) were taken at a number of time points after dosing and the DNA extracted and packaged for analysis of LacZ mutants. Members noted that positive control (benzo (a)pyrene) had been used for the dermal studies but no positive control had been used for the inhalation studies presumably in view of the potential hazards involved in handling and controlling exposures to test animals. Members acknowledged that there would be an observable degree of inter-animal variation in results for in-vivo mutation assays such as LacZ, which complicates the assessment of data but agreed that the results reported for the study concerned could not be assessed in view of the failure to obtain acceptable levels of DNA packaging in many of the trials. The Committee considered that inhalation exposure to phenol followed with assessment of mutation frequency in nasal tissue was the critical data for identification of site of contact mutagenicity and felt that a further study with acceptable levels of DNA packaging was needed before any conclusions regarding site of contact mutagenicity could be reached. 11. Members considered that the relatively old studies to investigate the potential for germ cell clastogenicity with phenol reported in a Polish paper had been inadequately performed and reported and thus no interpretation of the results was possible (Bulsiewicz H. Folia Morphol, 36, 13-22, 1977). 12. Thus the new mutagenicity data on phenol supported the overall conclusion reached in 1994/5 namely, that phenol was an in-vivo mutagen but effects on germ cells could not be assessed from the available data. Members agreed that the conclusions on phenol should be amended to reflect the new data. 13. The Committee considered the new data on hydroquinone and agreed that the positive results obtained in a bone marrow micronucleus assay published by Chen H and Estmond DA (Carcinogenesis, 16, 1963-1969, 1995) were consistent with previous studies. Members then considered the new toxicokinetic data. They noted the written comments provided by Professor Davies and agreed that the new toxicokinetic study in which rats were given a single intratracheal dose of 14C-hydroquinone showed detectable free hydroquinone in arterial blood within 5-10 seconds after dosing (Deisinger PJ and English JC. Drug Metabolism and Disposition, 27, 442-448, 1999) This new information suggested a potential risk of site of contact and systemic mutagenic effects with hydroquinone following inhalation exposure. The Committee agreed that the conclusions on hydroquinone should be altered to reflect these new data. Members also agreed that the further paper on in-vitro percutaneous absorption of hydroquinone through rat and human skin did not significantly alter the Committees overall conclusions on hydroquinone with respect to risk following dermal exposure. 14. The Committee agreed that the conclusions on phenol and hydroquinone should be redrafted to reflect the new data but that overall there was insufficient evidence to support a threshold approach to risk assessment with respect to inhalation or dermal exposure to these two chemicals. ITEM 7: GUIDANCE ON THE STRATEGY FOR TESTING AND EVALUATION OF MUTAGENICITY OF CHEMICALS (MUT/99/38) 15. The Committee considered draft sections revising the 1989 guidelines at the May 1999 meeting where it was agreed that the next draft document should concentrate on revising the strategy for testing. The second draft guideline (Annex 1 to MUT/99/38) had been based on the first draft presented at the February meeting (MUT/99/22) and took the discussion at the May 1999 meeting into account including the issues raised in an HSE discussion paper (MUT/99/25), and comments received from PSD and HSE on an initial revised draft document prepared by the secretariat. The draft guidelines submitted for members' consideration were intended to provide a clear strategy for testing and evaluation. It was recognised that there were a number of key issues still to be resolved, for example the requirement for aneugenicity testing and guidance on the selection of the most appropriate second tissue in-vivo assay. 16. Members noted that a number of additional papers had been submitted (MUT/99/28, 30-35, 40,42) and agreed to consider the information in these papers at the appropriate times during the discussion of the draft guideline document (Annex 1 to MUT/99/38). 17. The Chairman asked the Committee to make as much progress at this meeting as was possible but commented that a further meeting of the committee to discuss the guidelines would be needed in December so that an agreed document could be finalised at the February 2000 meeting. The document would then be forwarded to UK professional groups, industry, academia and Government Departments for consultation. The Committee considered the comments provided by the Industry Genotoxicity Group (IGG, MUT/99/42) and agree that although helpful, the comments were largely anecdotal points raised by individuals rather than a concerted view of IGG on the proposed COM strategy for testing. 18. Members made a number of general comments on the strategy before making detailed comments on the draft document. It was agreed that the issues to consider when devising a strategy and rationale for the COM strategy should be outlined in the introduction section, but with less detail regarding individual Structure Activity models, since there was no evidence that such models were any better than use of expert judgement and the Committee had no preference for any one particular model. Members agreed that the revised document should clearly separate screening of chemicals for potential mutagenic activity (i.e. stage 1 in-vitro tests) from hazard assessment (stage 2 in-vivo tests) and risk assessment (i.e stage 3 tests). It was agreed that the detection of aneugenic activity should be incorporated into the strategy. 19. Members considered some of the issues raised in the supporting papers appended to MUT/99/28. Thus members agreed with Kirkland (Mutation Research, 404, 173-185, 1998) that the in-vitro Micronucleus assay was likely to replace conventional metaphase analysis as the screening tests of choice for clastogenicity. Members noted that the proposed strategy for stage 1 testing had more emphasis upon the use of in vitro cytogenetics than that of the ICH guidelines for mutagenicity testing of pharmaceuticals. Members disagreed with the views proposed by Marshall R and Obe G (Environmental and Molecular Mutagenesis, 32, 212-222, 1998) and considered that chromosomal painting techniques such as FISH would be of considerable value in mutagenicity testing, for example in the identification of potential aneugens. Members agreed that evidence available (MUT/99/40) supported the routine use of the COMET assay in stage 2 as a potential assay in tissues other than the bone-marrow. Members were aware that a large collaborative study was underway in Japan which would provide more supporting data. In addition a draft guideline for the conduct of in-vitro micronucleus assay was to be submitted for consideration by OECD in the near future (a copy was circulated for any comments from members). 20. The following comments were made on the text of the second draft guideline document. Introduction 21. Omit reference to cell transformation. Redraft section to define "genotoxic" as a pragmatic term describing direct or indirect interaction with DNA. Clearly state COM position on evidence for end-point specificity (i.e gene mutagens will also induce clastogenicity under appropriate conditions, although there are some clastogens which may not be detected in gene mutation assays). General principles of testing strategy 22. Reduce discussion of SAR models. Emphasise that strategy relates to mutagenicity testing of individual substances and not mixtures where two or more substances are present. Stage 1 Screening 23. Ensure diagram representing strategy is near to introductory paragraph. Clarify introduction particularly regarding the evidence needed to conclude that a substance has no mutagenic activity. Incorporate screening for aneugenicity (with reference to discussion in MUT/99/30 and MUT/99/40) into document text (particularly with reference to the in-vitro micronucleus assay) and also into the flow diagram. Clarify need to complete stage 1 testing even if a positive result is identified in one of the screening tests. Stage 2 Hazard 24. Redraft introduction section to remove ambiguities regarding number of tests and to clearly state the COM proposal to in-vivo testing. Revise strategy to show COMET assay on equal standing with the UDS assay. Consider DNA binding and 32P-postlabelling assays as appropriate in specialised circumstances. Undertake review of recent literature on utility of mutagenicity assays in transgenic mice. Significantly reduce reference to methods not recommended in stage 2. Clarify drafting of summary section. Stage 3 Risk 25. Undertake further consideration of studies in transgenic animal models for the assessment of germ cell mutations. Investigation of Aneuploidy and Polyploidy 26. Incorporate text into Stages 1 and 2. Amend figures 1 and 2 to reflect evaluation of aneugenicity. Role of mutation signatures 27. Undertake editorial changes. Evaluation /Risk Assessment/Summary 28. Redraft to reflect the amendments to the strategy. 29. The Committee agreed that a revised draft of the guidelines should be submitted to an additional meeting of the Committee to be held in early December. Members asked for some of the supporting documents to be resubmitted. ITEM 8 MEASUREMENT OF CARCINOGEN DNA AND PROTEIN ADDUCTS USING AMS (MUT/99/39) 30. The Committee was asked to comment on some recent research using Accelerator Mass Spectrometry as a sensitive technique for measuring levels of adducts at environmentally relevant levels of exposure which has been submitted to MAFF. The project report summarises DNA/protein adduct data from rodents and volunteers exposed to a number of carcinogens found in food (aflatoxin(AFB1), heterocyclic amines (MeIQx and PhIP) and poylcyclic aromatic hydrocarbons (BaP). 31. Members will recall that the COM and COC held a joint meeting on the issue of the significance of low levels of exposure to DNA adduct inducing chemicals in 1996 at which Professor Garner from the Jack Birch Unit for Environmental Carcinogenesis, University of York, gave a presentation of the preliminary work using Accelerator Mass Spectrometry to detect levels of DNA adducts after exposure to low doses of carcinogens . Overall, it was concluded that DNA adducts were a useful indicator of exposure and uptake, (i.e. biomarkers, but that currently adduct formation alone could not be used to undertake risk assessments. 32. The COM and COC also provided the Department of Health with advice on research priorities in 1996 in respect of mutagenicity and carcinogenicity assessment of chemicals. Both committees agreed on the following suggested research area in respect of the potential use of adduct data for risk assessment: "The establishment of a database of biological samples from individuals with documented exposure to known chemical carcinogens to identify biomarkers such as adducts and mutagens, combined with long-term epidemiological surveillance" 33. Members agreed that AMS was a very sensitive method for measuring the formation of DNA adducts following dosing of small amounts of radioactive carcinogens. The method can in some cases be ethically used in certain groups of humans e.g. cancer patients. The COM pointed out a number of potential limitations of the methodology: i) The sensitivity of detection is predominately related to the quality and effectiveness of the AMS (hplc) separation used in sample preparation ii) Limited conclusions can be reached regarding interspecies comparisons of DNA adducts from the available data or in respect of the rank order of adduct formation reported for the rat. iii) The biological significance of DNA adducts detected at these very low levels of carcinogen exposure is currently difficult to interpret. iv) The health status of the humans studied may also influence the outcome of adduct formation. 34. Members agreed that a more comprehensive literature review of AMS was required and that the Committees preliminary comments be incorporated, along with those of the COC, into a statement for publication on the Internet site. ITEM 9: PAPERS FOR INFORMATION 35. No additional papers have been forwarded to the Committee for this meeting ITEM 10 ANY OTHER BUSINESS 36. There were no other items of business. ITEM 11. DATES FOR MEETINGS IN 2000 37. These have been arranged for 3 February, 18 May, and 5 October.
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