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MINUTESPresent:
CONTENTS
ITEM 1: ANNOUNCEMENTS/APOLOGIES FOR ABSENCE 1. The Chairman welcomed Ms Karen Moizer attending from the Food Standards Agency, Dr Maria Pufulete from Kings College London (previously from DH Toxicology unit at Imperial College) and Dr Claire Cowles (HSE assessor) attending in place of Dr Andrew Smith. The Chairman also welcomed Ms Isabella Lindup (DH Toxicology unit at Imperial College) and Ms Alison Gowers (DH). 2. Apologies for absence were received from Dr J Clements, Dr H Stemplewski, Dr A Smith and Mr A Browning. 3. Members were reminded of the need to declare any interests before discussion of items. ITEM 2: WELCOME TO NEW MEMBERS 4. The Chairman welcomed two new members of the COM, Dr Brian Burlinson (GlaxoSmithKline) and Dr David Gatehouse (Consultant). ITEM 3: MINUTES OF THE MEETING ON 6TH FEBRUARY 2003 (MUT/MIN/03/1) 5. The minutes were approved with minor amendments. ITEM 4: MATTERS ARISING NOT COVERED BY LATER AGENDA ITEMS 6. Members were informed that the COM statement on DEET (Diethyl-m-toluamide) had been sent to the DEET Joint Venture Group who had agreed with the COM conclusions. The advice from the COM would be included in a statement from the COT on the toxicology of DEET. 7. The committee was also informed that the COM statement on flunixin, meglumine and flunixin meglumine had been agreed by chairman’s action. The data holder had agreed to carry out the two in-vitro mutagenicity tests recommended by the COM. 8. New members were informed of COM procedures and working practices, eg indemnity, handling of papers etc. To expand the openness of the working of the COM members heard that certain papers would be placed on the COM website before meetings. ITEM 5: HIGH DOSE BONE MARROW DATA THAT MAY NOT BE INDICATIVE OF IN-VIVO MUTAGENICITY: 3RD DRAFT STATEMENT (MUT/03/10) 9. At the previous two meetings the committee had considered the issue of providing generic guidance on situations where apparently positive in-vivo bone marrow mutagenicity data at excessively high doses (by current guidelines) may not be indicative that the compound should be regarded as an in-vivo mutagen. This was important because a non-threshold approach to risk assessment would normally be adopted for an in-vivo mutagen. A number of potential mechanisms with a threshold of action (eg hypothermia and erythropoiesis) had been considered as well as toxic effects. A guidance document had been drafted and discussed at the last meeting and a revised version circulated for comment. It was noted that a recent UKEMS Industrial Genotoxicity Group meeting had discussed the same topic and had come to similar conclusions as the COM. Members were asked to agree this document and there was an opportunity for new members to comment. The draft guidance document would then be circulated to the COC prior to publication. 10. Members agreed a number of minor editorial changes to the main text of the draft statement and suggested some changes to the conclusion section. Members agreed that conclusions ii) and iii) could be combined. 11. With regard to conclusion (iv) members felt that this should refer to effects being ‘secondary to other non-genotoxic effects’ rather that to ‘toxicity’. Additionally, examples of such mechanisms should be given; these included (but were not limited to) hypothermia, hyperthermia and erythropoiesis. 12. Members asked for clarification of conclusion (V) to state that: 'Only generic advice can be given in this area and it should be emphasised that each compound needs to be considered in a case-by-case basis. However consideration of the above factors, with expert judgement, may provide sufficient evidence to conclude that the positive in-vivo bone marrow data at high dose levels was due to a non-genotoxic effect. A threshold based risk assessment may thus be appropriate’. ITEM 6: 1,3-DICHLOROPROPAN-2-OL (1,3-DCP): NEW IN-VIVO MUTAGENICITY STUDIES (MUT/03/7) 13. Dr Gatehouse declared a personal, non-specific interest. The Chairman noted that he had no involvement in these studies and allowed his participation. 14. The COM considered the mutagenicity of 1,3-DCP in 2001 and noted that the in-vitro data indicated that it had mutagenic potential. In the absence of any in-vivo data in mammals, it was concluded that it would be prudent to assume that 1,3-DCP was potentially genotoxic in-vivo and agreed it should be tested in-vivo using the approach set out in the COM guidelines. Members heard that further reports on 2,3-Dichloropropan-1-ol (also assumed to be genotoxic in-vivo in 2001) would be submitted to the October 2003 COM meeting. 15. Two studies, a bone marrow micronucleus assay and an unscheduled DNA synthesis (UDS) assay had been carried out to investigate the in-vivo genotoxicity of 1,3-DCP. Members discussed the results of the two new studies and considered that both assays had been conducted to relevant guidelines and that the results were clearly negative. It was noted that toxicokinetic samples had been taken in both studies to provide information on plasma levels, but had not been analysed. Members discussed whether it was necessary to measure the levels of 1,3-DCP in the plasma, particularly with regard to the bone marrow micronucleus test. However, doses used in the two in-vivo studies showed clear evidence of systemic toxicity and analysis of these samples would extend beyond the OECD requirements for in-vivo mutagenicity studies. On balance, members felt that analyses of plasma samples were not required in situations where clear systemic toxicity had been demonstrated. 16. In the micronucleus assay, Members noted that the PCE:NCE ratio was very variable amongst controls and that the top and bottom ends of the range was outside the range for historical controls. These findings were not considered to invalidate the study but Members requested that the contract laboratory be asked to comment on the reason for the variability. 17. Members considered that it would be appropriate to consider these studies provided evidence that 1,3-DCP was not an in-vivo mutagen and thus not a genotoxic carcinogen. However, Members considered that it would help if an explanation of why the in-vitro results with 1,3-DCP were not expressed in-vivo could be provided in the statement. 18. A draft statement will be prepared for the next meeting in October 2003. ITEM 7: 2-PHENYLPHENOL: UPDATE ON MUTAGENICITY (MUT/03/9) 19. Members were informed that 2-Phenylphenol is a broad spectrum fungicide that is approved in the UK for use as a non-agricultural pesticide (wood preservative); it is also used as a biocide. The COM had previously advised on the mutagenicity of this compound, most recently in 1997, in the context of its pesticide use. Further data are now available and the Committee was requested to update the previous advice to inform the UK position regarding consideration of its use in biocidal products [HSE lead on the implementation of the Biocidal Products Directive in the UK], particularly with regard to whether a threshold approach to risk assessment was appropriate. 20. At high dose levels 2-phenylphenol induces bladder tumours in the rat and it is particularly important to be able to exclude a genotoxic mechanism being involved. In 1992 the COM noted that although negative results were obtained in bone marrow assays and germ cell assays for mutagenicity in-vivo, some conflicting results were obtained in in-vivo assays for effects on the DNA in bladder epithelium. The COM recommended that data from an in-vivo study to investigate DNA adduct formation in bladder epithelium should be carried out using more sensitive methods, to provide definitive information regarding the absence of a genotoxic mechanism. At that time the Committee felt that there was insufficient concern to recommend a departure from a risk assessment approach based on the use of uncertainty factors to estimate safe levels of exposure (ie a threshold approach was adopted). 21. In 1997 the Committee considered data from an in-vivo 32P post-labelling study on DNA adducts in the bladder but they had concerns at the limitations of the method used and recommended that this be further investigated. This recommendation was not made into a regulatory request because of other data on carcinogenicity provided to the ACP who felt that the post review regulatory requirements had been provided, and that a threshold based risk assessment was appropriate. Considerable additional data has now been published relating to the metabolism and mutagenicity of 2-phenylphenol, including further in-vivo DNA adduct work; this was considered by the Committee. 22. Members agreed that the additional data on the metabolism of 2-phenylphenol indicated that it had a similar profile in the rat and the mouse, and that it was difficult to explain the specific effect in the rat bladder on this basis. Quinone derivatives phenylbenzoquinone (PBQ) and phenylhydroquinone (PHQ), which gave rise to some concern because of their potential for oxidative DNA damage, were formed at higher dose levels in both species, essentially only in conjugated form. 23. Members noted that the in-vivo 32P post labelling study using rat bladder epithelium DNA that the committee had seen earlier had now been published. They did not agree with the explanation given as to why appropriate extraction and enrichment techniques were not used, and maintained their view that the method was insensitive, and that no definite conclusions could be drawn. However, they agreed that the new AMS study provided good evidence for the lack of covalent DNA binding in the male rat bladder. The method was a particularly sensitive technique and the study had been well performed. Protein but not DNA binding had been clearly shown. Members agreed that the weight of evidence with regard to the DNA binding studies in the rat bladder was now sufficient to conclude that no significant DNA binding occurred. However, it was noted that most of the available data were from short term studies, and one, limited, subchronic study had provided some evidence for adduct formation in bladder DNA. The committee felt that the possibility of prolonged high level exposure producing DNA adducts could not be entirely discounted. 24. Consideration was then given to the data on the mutagenicity of the quinone metabolites PBQ and PHQ: There was some evidence that the metabolites could produce DNA damage in-vitro and also some limited evidence for induction of aneuploidy. It was noted however that much of the data involved HL60 cells. These cells were highly sensitive due to their highly oxidant environment, with little capacity for detoxification by, for example, sulphotransferase or sulphation enzymes. This was also true, but to a lesser extent, with the other system used, V-79 cells. Both were ‘biased’ toward an oxidative environment. Since the effects were seen with high concentrations of metabolites rather than the parent compound the significance of the results to the in-vivo situation was questionable. Members agreed that it was not possible to exclude oxidative DNA damage at high doses contributing to the induction of bladder tumours in the male rat, but such an effect would not be expected to occur at low dose levels. Therefore, this did not preclude the assumption of a threshold in the risk assessment process. 25. Members felt that no conclusions could be drawn from the studies using a novel approach to identify micronuclei in cells taken from the bladder of rats exposed to 2-phenylphenol. This was mainly due to problems in the methodology such as failure to distinguish micronuclei from cell debris. It was noted that the FISH studies using a DNA probe for rat chromosome 4 did not provide any clear evidence for the induction of aneuploidy by 2-phenylphenol. However, because of the overall limitations of this study no conclusions could be drawn. 26. Data from the in-vivo COMET assay were then considered. Although there were some concerns as to whether the method used at that time would have adequately distinguished between genotoxicity and cytotoxicity, it was felt that the results were suggestive of high dose effects in some tissues (stomach, liver, lung, kidney but not bladder). 27. The Committee agreed that overall it would be reasonable to adopt a threshold based risk assessment for 2-phenylphenol and its sodium salt (rather than assuming that it was a genotoxic carcinogen with no threshold). 28. A statement on the mutagenicity of 2-phenylphenol will be drawn up in the light of these discussions for circulation to the committee (and will be cleared by chairman’s action). ITEM 8: POLYCYCLIC AROMATIC HYDROCARBONS: CONSIDERATION OF SOME ASPECTS IN DEVELOPMENT OF INHALATION POTENCY EQUIVALENCY FACTORS (PEFS). (MUT/03/6) 29. The Committee was informed that the COC had recently evaluated published carcinogenicity data on dibenzo(a,l)pyrene (DB(a,l)P) and had agreed that this compound was between 10-100 times more potent than benzo(a)pyrene depending on the test system used. Thus a paper had been drafted which considered the potential impact of DB(a,l)P and other high carcinogenic potency PAHs on the existing approaches to risk assessment of PAHs in air. Three approaches to carcinogenic risk assessment have been advocated: the use of Potency Equivalency Factors (PEFs) (equivalent to Toxicity Equivalent Factors, TEFs, for general toxicity); the complete mixture method; and use of B(a)P as a surrogate carcinogen for all PAHs. It was possible that the B(a)P surrogate approach which is currently used to monitor for compliance with UK air pollution standard, might not be appropriate if high potency PAHs were present in air pollution and if the concentrations of these compounds varied significantly when compared to B(a)P. A review of the possible approaches to risk assessment has been undertaken, taking into account available data on carcinogenicity, including a discussion of kinetics of PAH uptake via the lung and self-induction of PAH metabolism. COM advice was particularly sought on the proposals for use of DNA adducts or mutations as surrogate end-points for carcinogenic potency. 30. The Chairman asked members to comment on the metabolism of PAHs before considering the proposed approaches to ranking carcinogenic potency. He noted that it had been proposed that only a broad ranking of these compounds was possible. 31. Members agreed that the majority of these compounds were activated in-vivo to diol-epoxides. It was possible to make informed predictions regarding the pathways of metabolism and reaction of diol-epoxides with DNA from the structure of PAHs. Members agreed that PAHs could be considered as members of a single group of genotoxic carcinogens whose mechanism of activation, formation of DNA adducts and metabolism were all broadly similar. There were some exceptions where PAHs were also metabolised by another minor route also leading to activation and DNA binding, for example the bis-diol-epoxide formed by dibenz(a,h)anthracene (DB(a,h)A). It was agreed that PAHs activated by routes other than via a diol-epoxide would need to be considered carefully. Members noted the carcinogenic process leading to PAH induced tumourigenicity was complex and included factors such as tumour promotion and cell proliferation which would also affect carcinogenic potency. PAHs also had other molecular targets (e.g. oestrogen receptors) which might potentially impact on the carcinogenic process in target tissues. 32. The Committee reviewed the available data regarding use of DNA adducts as a surrogate marker for PAH carcinogenic potency. There was evidence that DNA adducts may be an adequate endpoint for this group of genotoxic carcinogens from two research groups using different experimental approaches. Dermal application of PAHs to the skin of mice showed a good correlation for DNA adducts in skin and lung with applied skin dose. Studies using intraperitoneal administration of PAHs to A/J mice showed a good correlation between total DNA adducts (Time Integrated DNA Adduct Levels; TIDAL) in lung and administered PAH dose. Both research groups had documented a good correlation between DNA adducts and tumourigenicity. It was suggested that DNA adducts could serve as a pragmatic marker (it is argued for reactive diol-epoxide DNA adduct formation) that correlates with tumourigenicity. It was noted that there was also data from studies in A/J mice using administration of mixtures of PAHs that suggested that any departure from additivity was limited. 33. Members noted the evidence for a lack of correlation between PAH induced mutation frequency in transgenic mice given oral doses of PAHs and target organ for carcinogenicity. Members felt that it would be important to evaluate dose-response for PAH induced mutation frequency in cancer target tissues in a similar way to the DNA adduct data before conclusions regarding the utility of mutations as a surrogate marker for PAH induced carcinogenicity could be drawn. 34. The Committee considered the proposals for experiments to rank carcinogenic potency of PAHs. It was agreed that intratracheal instillation of small doses in rats, with measurement of total DNA adducts in lung, would be appropriate for inhalation of PAHs. Members also considered that measurement of DNA adducts in skin following topical treatment would also yield a similar ranking of PAHs. It was suggested that it might be possible to undertake DNA adduct measurements and determination of PAH induced mutation frequency in the same animals, although it was acknowledged this would represent a very large research project and that it was not a practical approach for ranking PAHs. Members briefly considered whether in-vitro approaches could yield relevant data but agreed that differences in metabolism between in-vitro cultures and whole animals would limit the value of in-vitro studies. 35. Members asked for information on how the results of such research could be used. The secretariat noted that information on air levels of high potency PAHs were being evaluated and this would help decide whether data from relative ranking was used within a risk assessment for inhaled PAHs based on either a B(a)P surrogate or a PEF approach. Members suggested that it would be worthwhile commenting on the potential for interaction between PAHs and other air pollutants with regard to risk of lung cancer. Members were informed that the paper would also be forwarded to COC for comment before submission to a peer review journal. Members asked for information regarding air levels of highly potent carcinogenic PAHs when available. ITEM 9: HORIZON SCANNING (MUT/03/8) 36. The committee was informed that the Office for Science and Technology (OST) Guidance for Scientific Advisory Committees outlines a number of areas where horizon scanning should be considered. Specifically these related to:
37. The COM considered a number of topics for potential future consideration. Members discussed whether a future review of bisphenol A was needed but heard that there was considerable on going work on this compound. It was for example, a priority chemical under the EUs Existing Substances Regulations and a comprehensive risk assessment had led to further data being requested from industry. Members discussed some generic issues which arose from the recently published work on the formation of acrylamide in potato based food products during processing/cooking (particularly frying). One possible approach would be to evaluate the in-vitro mutagenicity of such processed foods followed by detailed chemical analysis. It was also suggested that the significance of mitochondrial mutation and the possible genotoxicity of phytoestrogens could be possible topics for future COM consideration. 38. Regarding new methods of evaluation, members agreed there could be a review of changes in gene expression induced by chemical mutagens. Also further consideration could be given to the appropriate weight of evidence provided by negative in-vivo results in two tissues (as suggested by the COM guidance) when discounting positive results in-vitro. 39. It was agreed that members could raise new issues or developments in chemical induced mutagenicity at any time by contacting the secretariat or chair or by raising the matter as AOB at a meeting. However, there would need to be an initial discussion at COM meeting before a formal review of a topic could be considered. This might involve liaison with other Governmental Departments/Regulatory Agencies who may have legislative/policy responsibility for specific areas of chemical exposure (eg Pesticide Safety Directorate of DEFRA for agrochemical pesticides). 40. It was also agreed that the committee should formally discuss issues relating to horizon scanning at least once per year. ITEM 10: ANY OTHER BUSINESS 41. There was no other business. ITEM 11: DATE OF NEXT MEETING 42. 2 October 2003.
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