MINUTES- 25TH MAY 2000
Minutes of the meeting held on Thursday 25 may 2000 at 10.30 am, in Room 102A Skipton House, 80 London Road, Elephant and Castle, London SE1 6LH.
Present:
Chairman:
Professor
Members:
Professor J Ashby
Dr J Clements
Professor P Farmer
Dr N Gooderham
Mrs M Langley
Dr I Mitchell
Professor D Phillips
Professor D Tweats
Secretariat:
Dr R J Fielder (Scientific DH)
Dr J Greig (Scientific FSA]
Mr J Battershill (Minutes)
Mr K Mistry (Administrative)Assessors:
Assessors:
Mr A Browning (VMD)
Dr M Costigan (HSE)
Dr R Shillaker (PSD)
Dr C Steele (MCA)
In attendance:
Professor A Boobis (DH Tox Unit) (Items 1-6)
CONTENTS
1.Apologies for absence/Announcements
2.Minutes of meeting held on 3 February 2000 [Agreed by postal consultation with previous COM]
3.Position regarding COC/COM and Food Standards Agency
4.Update on mutagenicity of Ethanol 6-10.
Thresholds for aneugens 11-16
6.Revised strategy for testing chemicals for mutagenicity
7.Papers For information
8.Any other business .
Date of next meeting: Thursday 12th October 2000
ITEM 1: APOLOGIES FOR ABSENCE
1. Apologies for absence were received from Professor Cooper and Dr A Smith (HSE; Dr Costigan attending).
Announcements
2. The Chairman introduced himself and welcomed all present to the new Committee. He asked Members to outline their scientific interests. He asked Assessors and Secretariat to give a brief statement on their input into the work of the Committee. The Chairman congratulated Professor Tweats upon receiving a chair from the University of Wales, Swansea.
3. Members were reminded of the need to declare any personal and non-personal interests before the discussion of items.
ITEM 2: MINUTES OF THE MEETING HELD ON 3 FEBRUARY 2000 (MUT/MIN/2000/1)
4. Members noted the minutes which had been agreed by postal consultation with the members of the retiring Committee.
ITEM 3: POSITION OF THE COC/COM AND THE FOOD STANDARDS AGENCY
5. Members were informed that the secretariats for the COT, COM, and COC had been reorganised following the establishment of the Food Standards Agency on 3 April 2000. The Committees would report to the Chief Medical Officer (Professor Liam Donaldson) and also to the Chairman of the Board for the Food Standards Agency (Professor Sir John Krebs). The terms of reference for the Committees would be altered in the near future to reflect this change. A joint DH/FSA secretariat had been set up with FSA leading for the COT and DH leading for COC/COM.
ITEM 4: UPDATE ON THE MUTAGENICITY OF ETHANOL (MUT/200/4)
6. In 1995 the COM had provided a statement to an Inter Departmental Working Group as part of a review of the Government's Sensible Drinking message. The Committee concluded that "the consumption of alcoholic beverages does not provide any significant concern with respect to their mutagenic potential." A review of the available mutagenicity data on ethanol and its metabolite acetaldehyde had been prepared by the DH Toxicology Unit. The COM concluded that there was no evidence to suggest that ethanol had mutagenic potential. With regard to acetaldehyde the COM concluded there was evidence that acetaldehyde had "..a direct acting mutagenic potential in-vitro but would only be expected to have the potential of in-vivo activity at sites where it is not rapidly metabolised to acetic acid." MUT2000/4 and the appended review paper had been prepared following a recommendation from the COC in its recently published statement on alcohol and breast cancer that the COM should be asked to update its 1995 opinion on alcohol. The COC had asked for further advice in the context of its review of the association between the consumption of alcoholic beverages and risk of breast cancer and in particular suggestions in the scientific literature that alcohol might induce oxidative damage of DNA in breast tissue which might be important in the induction of breast cancer.
7. The Committee noted that there were no new in-vitro mutagenicity studies using ethanol. No conclusions regarding mutagenicity could be drawn from the investigation of the effects of ethanol on sperm function. Studies using human lymphoma cells had confirmed earlier studies that acetaldehyde induced protein-DNA cross links, but only at concentrations which resulted in cell death (Costa et al (1997) J Toxicol Environ Health, 50, 433-449). In addition acetaldehyde induced HPRT mutations in human T cells (Lambert et al (1994) Environ Health Perspect, 102, 135-138.). Members agreed that no conclusions could be drawn from the finding of acetaldehyde DNA adducts in peripheral white blood cells of alcoholics in view of lack of control for the effects of smoking by alcoholics in the study group and the well known abnormalities in metabolism in alcoholics (Fang and Vaca (1997). Carcinogenesis, 18, 627-632). Members also agreed that these reservations also applied to the investigations of clastogenicity in alcoholics and print workers (i.e lack of control for effects of smoking). Members agreed that no conclusions could be drawn from the studies in Drosophila.
8. The Committee reaffirmed the opinion of the COM with respect to ethanol and acetaldehyde reached in 1995. Members agreed that conclusions with regard to acetaldehyde should take into consideration the rapid metabolism of acetaldehyde to acetic acid.
9. Members reviewed the hypothesis published by Wright et al (1999 Free Radical Biology and Medicine, 26, 348-354.) and agreed that the suggestion put forward by Wright RM et al should be examined in detail. Wright and colleagues had noted the finding that alcohol metabolism is known to produce reactive oxygen species (ROS) and mammary tissue contains the necessary metabolising enzymes to produce ROS from alcohol. In addition, Wright and colleagues noted two further observations which supported their hypothesis, namely that breast cancer is associated with high levels of hydroxyl modified DNA and iron which has been proposed to catalyse the formation of ROS accumulated with time in breast tissue. The Committee was aware that the COC had not reached a conclusion regarding whether the association between alcohol and breast cancer was causal. Members agreed that there was evidence that ethanol and its metabolites induced the formation of free radicals in-vitro, but the evidence in-vivo was conflicting. Members commented that the observations reported by Wright and colleagues might be a result of tumour progression rather than an initiator of cancer. In addition it was noted that co-administration of iron and alcohol to rats in the initiation phase of a two stage model for hepatocarcinogenesis did not result in any genotoxic effects (Stahl P et al (1997) J Hepatol, 27, 562-571). Overall, the COM concluded that that there was insufficient evidence to support the Wright et al hypothesis regarding breast cancer. However further assessment of the available mechanistic data pertaining to the induction of cancers which were causally related to alcohol would be helpful.
10. The Committee agreed that conclusions regarding ethanol and acetaldehyde could be reached. Members agreed that further work on the available information regarding alcoholic beverages was needed before final conclusions could be reached and a statement prepared covering alcoholic beverages as well as ethanol and acetaldehyde.
ITEM 5: THRESHOLDS FOR ANEUGENS (MUT/2000/5)
11. Dr Clements declared a non-personal specific interest.
12. Benomyl, carbendazim and thiophanate-methyl belong to the methyl benzimadazole carbamate (MBCs) class of chemicals. The MBC class of chemicals are widely used in approved pesticide products as fungicides and also in veterinary medicines in particular as antihelmintics in both food producing and companion animals. These chemicals act by interfering with microtubule formation during cell division. The COM provided advice to the U.K Regulatory Authorities, namely the Pesticides Safety Directorate (PSD) and the Veterinary Medicines Directorate (VMD) of the Ministry of Agriculture, Fisheries and Food on the most appropriate approach for the risk assessment of MBCs in 1993, 1995 and in 1996. The COM have agreed that it was reasonable to assume that aneuploidy inducing chemicals (particularly those that function by interfering with the spindle apparatus of cell division) have a dose-dependent threshold of action. Studies to demonstrate a threshold have been reported for these three MBCs.
13. The Committee was asked by the ACP to consider the conclusions reached by the European Commission's Group of Specialised Experts who were considering the classification and labelling of benomyl, carbendazim and thiophanate-methyl under the Dangerous Substances Directive 67/548/EEC. The Committee considered a draft summary record of the meeting of the Specialised Experts held on the 1-2 September 1999. The Committee was asked by the ACP to advise on the applicability of extrapolating to germ cells, evidence for thresholds for induced aneuploidy obtained in studies on somatic cells, and the relevance of those conclusions for the approach used by PSD to evaluate aneuploidy data in the risk assessment of agricultural pesticides. The Committee has not been asked to comment on the proposals for classification and labelling of these MBCs.
14. The Specialised Experts had concluded that "..current knowledge does not allow extrapolation to meiotic cells of the in-vitro finding of a threshold [for induced aneuploidy in somatic cells]." The COM considered that the critical piece of evidence used to reach this conclusion came from the publication by de Stoppelaar et al (Mutagenesis, 14, 621-631,1999) which reported diploidy (i.e but not aneuploidy) in sperm of rats exposed to carbendazim. de Stoppelaar et al concluded that their findings suggested that diploidy in sperm is induced at a lower dose level than micronuclei in peripheral blood erythrocytes (no micronuclei were seen in this study). The main finding in the first was a small but dose related increase in the absolute frequency of diploid sperm (0.03% to 0.22%) following oral dosing and analysis of sperm at 31 days after treatment. The Committee noted that a smaller increased frequency of diploid sperm was reported in the second experiment in animals given an oral dose of 800 mg/kg bw carbendazim which may have resulted from sub-optimal exposure conditions in the experiment. No micronuclei were induced in peripheral blood erythrocytes following oral treatment of up to 800 mg/kg bw after sampling peripheral blood at 48 or 72 hours post treatment. The Committee noted that the mechanism of action of carbendazim involved interference with the formation of polar microtubules. This effect combined with differences in the type of nuclear organising centres (NOC's) for germ cells in first meiotic division in males (a single NOC) and in females (multiple NOCs) would result in carbendazim inducing polyploidy in sperm and aneuploidy in oocytes.
15. The Committee considered that the results obtained by de Stoppelaar et al did not provide evidence for a lower threshold for aneuploidy in germ cells compared to somatic cells. The Committee felt that the analysis of peripheral blood samples for micronuclei in rats undertaken by de Stoppelaar et al was suboptimal in that a 24 hour sampling time point should have been used as micronucleated erythrocytes may have been efficiently removed by the spleen in rats and exposure conditions used may not have been optimal for the production of micronuclei. The Committee agreed that a more appropriate study in somatic cells for comparison with germ cells in the rat would be an investigation of the dose-response for the formation of micronuclei containing aneuploid chromosomes in polychromatic erythrocytes obtained in bone marrow smears from rats treated using a similar protocol to that used by de Stoppelaar et al. The Committee noted that such data would be informative with regard to any differences between aneuploidy in somatic and germ cells and agreed to review the subject when appropriate results were available. Even if it could be established that the effects on sperm occur at lower doses than for somatic cells, this would not invalidate the concept of a threshold effect.
16. The Chairman asked Members to consider a draft statement which would be circulated by post for comment. The statement was needed by the ACP secretariat by 15 June 2000. The Committee would reconsider the subject when more information on the effects of carbendazim on micronuclei formation in the bone-marrow of rats was available.
ITEM 6: REVISED STRATEGY FOR TESTING CHEMICALS FOR MUTAGENICITY (MUT/2000/6) AND COMMENTS (MUT/2000/7, MUT/2000/11)
17. Members were informed that the draft consultation document "Guidelines on the Strategy for testing of Chemicals for Mutagenicity " (MUT/2000/6) had been circulated to relevant professional societies in the UK and had also been placed on the Internet for wider consultation. Detailed comments had been received from the Industrial Genotoxicity Group (IGG) which had been tabled for consideration (MUT/2000/11). In addition extensive comments had been received from laboratories and research groups from across Europe, the U.S.A and Japan. A summary of these comments had been presented for discussion (MUT/2000/7). In addition members were informed that there had been considerable critical debate of the COM guidelines at the Environmental Mutagen Society meeting in the U.S.A. a few days earlier. The Chairman commented that he was aware that there had also been criticism of individual COM members. He asked for a note to be made in the minutes that members had provided advice during the drafting of the document as independent scientists and did not represent any organisation.
18. Members noted that there had been much confusion regarding the role of the COM and the relationship of the draft guidelines with respect to the available regulatory guidelines (and in particular the ICH guidelines for pharmaceuticals). It was acknowledged that considerable efforts had been made to gain acceptance of the ICH guidelines within U.S. Regulatory Agencies. The Committee agreed that the COM document should be entitled "Guidance on the strategy for testing of chemicals for mutagenicity" and should contain a preface section to give the background to the role of the COM in providing up to date scientific advice on the testing of chemicals for mutagenicity. Members agreed that unnecessary repetition should be removed, the referencing of the document improved and examples quoted where possible.
19. Members considered that further clarification of the rationale for end-point specificity in Stage 1 in-vitro testing, the strategy for investigating aneuploidy (e.g why polyploidy in-vitro may not give an adequate screen for aneugenicity), the value of the in-vitro micronucleus assay and use of FISH in cytogenetics tests. Members noted that some results of an international collaborative study being co-ordinated in France would contribute to the validation of the in-vitro micronucleus assay. A draft protocol had recently been submitted to OECD for assessment but being pragmatic it would take 2 years for acceptance. The Committee agreed that additional guidance on the likely exposure scenarios which would attract 3 in-vitro tests in stage 1 was needed.
20. Members also suggested that some more recent information on the use of DNA binding assays was now available and could be incorporated. It was agreed that a reference to DNA strand breakage assays other than the COMET assay should be made although these were little used. Members reaffirmed that the document should be limited to consideration of the most critical and valuable assays with the objective of providing scientific advice.
21. Members noted the comments from IGG which also proposed resolution to a number of problems with the revised COM strategy (MUT/2000/11). These were to be discussed in detail at a special UKEMS/IGG workshop to be held on 26 May 2000. Members commented that an appropriate preface to the COM document would be particularly helpful in resolving many of the topics raised by IGG. The aim was to revise the document in the light of comments received and the discussion at the meeting of 26 May 2000 and to finalise the document at the October meeting. It was recognised that there would be a need to consult Members of the COM who made substantial contributions to the document but had retired from the Committee.
ITEM 7: PAPERS FOR INFORMATION
7.1 Membership list (MUT/2000/8)
7.2 For information: Recommendations for categorisation of germ cell mutagens: MAK document (MUT/00/9)
7.3 For information: Report from the In Vitro Micronucleus Working Group (MUT/00/10)
ITEM 9: ANY OTHER BUSINESS
23. Members were informed that the Chairman had been invited by Sir Robert May (CSO) on 5 June 2000 to discuss approaches used by Advisory Committees to undertake risk assessment of chemicals in food. This meeting was part of a consultation process being undertaken by the Office of Science and Technology (OST) on this subject at the request of the Chief Medical Officer (Professor Liam Donaldson) and the Chairman of the Board of the Foods Standards Agency (Professor Sir John Krebs). An OST report of the review would be available in due course
ITEM 10: DATE OF NEXT MEETING
24. Thursday 12th October 2000
ACTIONS
| Items | Action | Responsibility |
| 5. Alcohol | Undertake further literature search for information on alcoholic beverages Draft Statement |
Secretariat/DH Tox Unit |
| 6. Thresholds | Draft statement, circulate and finalise for submission to ACP July meeting | Secretariat |
| 7.Strategy | Revise in light of members comments and from consultation exerciseSecretariat |