MINUTES - 10TH FEBRUARY 2005
Present:
| Chairman: Professor P Farmer |
Members: Dr C Allen Dr B Burlinson Dr G Clare Dr J Clements Dr D Gatehouse Dr N Gooderham Dr I Mitchell Dr E Parry Professor D Phillips |
Secretariat: Mr J Battershill (Scientific DH/Minutes) Ms F Pollitt (Scientific DH, item 5) Mr D Renshaw (Scientific FSA) Mr K N Mistry (Administrative) |
Assessors: |
In attendance : Dr S Bull (DH Tox Unit, items 4&9) Mr S Creton (FSA) Dr P Edwards (DH, item 9) Dr K Fletcher (DH Tox Unit, items 5&7) |
Observers: Ms A Craig (PAN-UK item 4) |
CONTENT
| Item | Paragraph |
|
1. |
Announcements/Apologies for absence | 1-4 |
2. |
Minutes of the meeting 7 October 2004 | 5 |
3. |
Matters arising not covered by later agenda items: | 6 |
4. |
Genotoxicity in pesticide applicators: | 7-20 |
| 4.1 Further information and follow-up of October 2004 review | ||
| 4.2 Submitted papers for February 2005 meeting | ||
5. |
Mutagenicity of Halonitromethanes |
21-27 |
6. |
Development of cII transgenic mutation assay. Comparison with lacI and lacZ systems |
28-35 |
7. |
Draft agenda for joint meeting on use of target organ mutagenicity in carcinogen risk assessment |
36 |
8. |
Draft Annual report |
37 |
[CLOSED SESSION: ITEM 9] |
||
| 9. | Additional in-vitro mutagenicity studies on meglumine |
38-41 |
10. |
Any Other Business |
42 |
| 11. | Papers for information: | 43 |
| 12. | Date of next meeting - 26 May 2005 | 44 |
ITEM 1: ANNOUNCEMENTS/APOLOGIES FOR ABSENCE
1. The Chairman welcomed Dr S Bull and Dr K Fletcher from the Department of Health Toxicology Unit and Mr S Creton from the Food Standards Agency. The Chairman noted that Dr Bull would be taking up a new post in the Health Protection Agency (HPA) and conveyed members thanks for her work for COM. The Chairman also noted that Mr S Robjohns had transferred to the Health Protection Agency and conveyed members thanks for his work on the COM minutes over the past four years.
2. Apologies for absence were received from Mrs R Glazebrook (member), and the assessors Ms A Gowers (EA), Mr R Alexander (NAW), (EA), Mrs R Kearsley and Dr H Stemplewski (MHRA).
3. The Chairman informed the committee that the COM had completed eight statements during 2004 and thanked members, secretariat and the DH Toxicology unit for their efforts.
4. Members were reminded of the need to declare any interests before discussion of items.
ITEM 2: MINUTES OF THE MEETING ON 7th OCTOBER 2004 (MUT/MIN/2004/3)
5. The minutes were approved with minor amendments.
ITEM 3: MATTERS ARISING NOT COVERED BY LATER AGENDA ITEMS
3.1 Toxicological reassessment of tobacco products
6. Members were informed that the EU Commission had extended its deadline for submission of comments. An overview of the EU Commission's response to the consultation would be available in due course.
ITEM 4: GENOTOXICITY IN PESTICIDE APPLICATORS (MUT/05/1). SUBMITTED PAPERS FOR FEBRUARY 2005 (MUT/05/6)
7. The Chairman welcomed Ms Alison Craig (PAN-UK) to the meeting. He indicated where members, assessors and secretariat were positioned. Ms Craig told members that PAN-UK was an independent charity with 6 regional centres in the UK. The main interests of the organisation related to health and environmental issues concerning pesticide use.
8. The committee was asked by the Medical and Toxicology Panel (MTP) of the Advisory Committee on Pesticides (ACP) to undertake a review of the available studies of biomonitoring for genotoxicity in pesticide applicators and workers exposed to pesticides, in particularly focussing on pesticide sprayers/applicators, floriculturists/green house workers, agricultural workers and farmers and forestry workers.
9. Selection criteria formerly agreed by the committee were discussed. Members had previously been asked to comment on the exclusion of the study by Munnia et al (Environ Mol Mutagen, 34, 52-6, 1999), and on the use of isolated lymphocytes, and shipping of samples. A report of members' discussion was appended as Annex IV to MUT/05/1. Members agreed that the protocols used in the post labelling studies were adequate and that the use of isolated lymphocytes was acceptable and the shipping of samples was acceptable as long as samples from controls and subjects were shipped at the same time. Members suggested checking to see if control values in such studies were within normal ranges.
10. One member commented on the subjective measurement of comet tails in the comet assay, and thought studies that were previously excluded on these grounds (Piperakis et al (Environ Mol Mutagen, 41,104-110, 2003), Lebailley et al (Cancer Epidemiology Biomarkers Prev, 7, 917-927, and 929-940, 1998 and Occupational Environmental Medicine, 60, 910-7, 2003) could be included in the review. Members agreed.
11. Members considered the exclusion of the MN assay when using a 72 hour incubation period and agreed that such studies could be included if data were presented regarding the proliferation of cells. Members agreed that all studies should be evaluated using the same criteria.
12. One member suggested that the criteria for inclusion of studies measuring CA be amended, to include only those where data on the % aberrant cells excluding gaps was either presented, or could be determined from the information available, in order to compare the magnitude of any response with a common denominator. Studies that presented data as CA per cell or % aberrant cells with gaps should be excluded.
13. Members thought that studies that did not use matched controls should be excluded. The COM secretariat stated that advice had been sought from an epidemiologist who advised not to exclude such studies. Members asked for additional epidemiological advice.
14. Members discussed that the variation in controls within each study should be presented and compared to historical controls, although the lack of historical control data in such variable populations could limit the usefulness of any evaluation of these data. Members requested that mean or median ± SD and 95% CI be calculated.
15. The HSE assessor queried the measurement of exposure to pesticides in the different studies, and raised the argument that the use of pesticides considered to have mutagenic potential (eg ethylene bromide and methyl bromide and several others classified for mutagenicity relatively recently) by workers in the selected studies could make judgements about relevance to the UK situation difficult. Such pesticides may no longer be approved for use in the UK. The COM secretariat noted that advice had been sought from PSD regarding the comparison of pesticide use in the selected studies and to practices in the UK. In addition studies that had investigated exposure to single chemicals that were due to be phased out or had now been revoked (such as methyl bromide) had not been included. Members agreed this approach.
16. One member noted that the potential for synergistic or additive effects of pesticides could not be evaluated from the available studies. This limitation would need to noted in the COM advice.
17. None of the studies had an a priori statement regarding the fold increase that would be needed for the data to be 'clearly' positive and have a biological significance, although several authors had claimed a positive response from what appeared to be relatively modest increases.
18. Members considered that the evidence presented did not agree with the conclusion made by Bolognesi (Mutation Research, 543, 251-272, 2003), regarding cumulative exposure and agreed the data were insufficient to conclude that cumulative exposure led to cytogenetic damage, although cumulative exposure should be evident in resting lymphocytes. Members noted that both the papers by Garry et al (Cancer Epidemiol Biomarkers Prevention, 5, 11-6 1996 and, Environ Health Perspect, 109, 495, 2001) were well conducted and could potentially provide the best approach to evaluating cumulative exposure by examining stable aberrations such as translocations and inversions.
19. Members asked the secretariat to circulate a revised set of criteria and to amend the list of selected studies appropriately. It was agreed that one member would act as liaison with the DH Toxicology Unit and the secretariat. The information from studies from the rest of the world was not individually assessed at this meeting. It was agreed the secretariat could agree inclusion/exclusion of these studies following consultation with a nominated member outside the meeting. Members were informed that an epidemiological overview would be presented to the next COM meeting on 26 May 2005.
20. The Chairman asked if the observer if she had any comments to make. Ms Craig noted that exposure assessment was often the weakest part of pesticide assessments. The secretariat told members that advice on this aspect was currently being sought from PSD. Ms Craig asked if the exposure assessment advice from PSD would be published, and was told that it would be published in the COM minutes as part of the overall COM consideration. Ms Craig expressed anxiety over the exclusion criteria being developed by the COM and questioned the rationale used for cytogenetics studies. Members explained the rationale behind excluding data for chromosome/chromatid gaps.
ITEM 5: HALONITROMETHANES (HNMs) (MUT/05/3)
21. Halonitromethanes (HNM's) are a class of compound characterised by the presence of one or more halogen atoms together with a nitro moiety on a single central carbon. They differ from the Halomethanes (HM's) by the presence of the nitro moiety. The HNM's have recently been identified as disinfection by-products (DBP) in drinking water in the US (Richardson et al 1999) and had received a high priority ranking for investigation by the Environmental Protection Agency (Weinberg et al 2002 http://www.epa.gov/athens/publications/EPA_600_R02_068.pdf).
22. Members were told that the only available published data related to the in-vitro studies in the three publications appended to MUT/05/3. Some preliminary findings had been reported by the EPA to the DH Toxicology Unit regarding in-vivo testing in transgenic medaka fish. Members noted that a negative in-vivo bone marrow mouse micronuclei test with trichloronitromethane (TCNM; chloropicrin) but that there were no appropriate in-vivo mutagenicity data available for other HNMs.
23. The COM discussed the in-vitro COMET studies which had all been conducted using CHO cells. Members noted that HNMs tested were cytotoxic in CHO cells and that apoptosis induction had not been recorded in these studies. In addition none of the trials had included the addition of an exogenous metabolising fraction. However members agreed that HNMs were probably genotoxic in this test assay and that the rank order reported by the study authors was a reasonable guide to relative potency. It was noted that the data precluded a determination of absolute potency of HNMs compared to the positive controls used in the study but the data were consistent with HNMs being more potent in the test system than ethyl methanesulphonate.
24. The COM reviewed the available bacterial mutagenicity data obtained from plate incorporation trials using Salmonella typhimurium strains both in the presence and absence of an exogenous metabolising fraction (Kundu B et al, Mutation research, 562, 39-45, 2004). The strains tested included TA 98, TA 100, TA 104 and a strain RSJ100 which expresses rat glutathione transferase theta (GSTT1-1) known to activate halomethanes. Members agreed that a standard plate incorporation approach had been undertaken in this study using two plates per dose point (instead of the normal three) but that reproducible small increases in the number of revertant colonies had been reported. It was noted that HNMs might volatilise and therefore procedures to prevent evaporation might have been more appropriate. In another study (Kundu B et al, Mutation research, 554, 335-350, 2004) trials had been undertaken using pre-incubation in screw-capped glass vials using Salmonella typhimurium strain TA 100 at 370C for 30 minutes followed by plate incorporation assessment of revertant colonies. Members felt that some residual loss of HNMs could still have occurred and that it would have been appropriate to undertake pre-incubation at a lower temperature of 300C. Overall members considered that the estimates of relative potency should be interpreted with caution.
25. Members considered the potential mechanisms of HNM induced mutagenicity in Salmonella typhimurium strains and agreed this included both direct acting and metabolically activated mutagenic responses. This might include an oxidative pathway but it was evident that that there were possible differences between the nine chemicals in the HNM group. Whether direct activity was due to HNM carbocation formation and DNA alkylation in an SN1reaction or alternatively SN2 substitution, was not clear. Members felt that unlike the halomethane group of compounds there was no convincing evidence for a glutathione mediated pathway with HNMs. Members observed that it was difficult to derive clear conclusions regarding mechanism and potency in the bacterial mutagenicity tests but overall brominated HNMs appeared from the limited data to be more potent than chlorinated HNMs.
26. The COM discussed the potential testing strategy that might be applied to HNMs and agreed that each individual HNM needed to be tested. It was agreed that the COM strategy was appropriate in this instance but could be modified so that the in-vivo rat liver UDS assay was the first in-vivo test followed by a site of contact COMET assay (possibly in the stomach). Members felt that an in-vitro liver UDS assay would be a useful pre-screen.
27. The Chairman agreed that a short statement should be prepared for circulation to members. This could be shared with the EPA at an early stage.
ITEM 6: DEVELOPMENT OF cII TRANSGENIC MUTATION ASSAY (COMPARISON WITH lacl AND lacz) (MUT/05/4)
28. The Committee recalled that one important piece of evidence regarding leucomalachite green came from the finding of an increased mutagen frequency in the liver in an in-vivo mutagenicity assay using the cII transgene in Big Blue mice fed a diet containing leucomalachite green. The objective of MUT/05/4 was to provide members with an overview of the development of the cII transgenic mutation assay and to pose some questions regarding sensitivity, the potential uses in target organ mutagenesis, and uses of sequencing data from the cII transgene.
29. One member noted that the OECD were in the process of drafting a guidance document on in-vivo mutagenicity tests in transgenic animals and a draft would be issued for comment soon. (Post meeting note; a link to the OECD guidance document has been forwarded to members).
30. Members commented that the cII transgene was smaller than the lacI and lacZ transgenes. The Chairman noted the finding in control studies that approximately 18% of lytic cycle plaques had no detectable mutations. Members agreed this could occur with selection assays dependent on differentiating between lysogenic and lytic cell cycles of bacteriophage transgenes. It was therefore important to undertake sequencing of mutant colonies to confirm that false positive results had not been obtained.
31. The Committee agreed that an acute dosing approach to the cII assay could be potentially insensitive to the identification of mutagens. There was limited published information on the influence of repeated dosing on mutation induction in tissues which supported the conclusion that the duration of exposure necessary to obtain a plateau in the increase in mutation frequency was related predominantly to the cell turnover of target cells in tissues and organs. The current approaches used were pragmatic involving repeated administration for 5-28 days followed by multiple sampling times dependent on tissue or organ being sampled.
32. Members observed the study using PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), which had examined the induction of cII and lacI mutations in the colon of Big Blue® F344, showed that the cII transgene was not responsive to frameshift or deletion mutations. (Stuart GR et al, 452, 101-121, 2000). In addition members noted the study from Monroe et al (Mutation Research, 421, 121-136, 1998) provided evidence that the cII transgene did not respond to MNU (N-nitroso-N-methylurea) mutagenicity in splenic T cells whereas a positive result could be demonstrated with lacI.
33. The Committee considered that studies using AAF (2-acetylaminofluorene), a compound that induced frameshift mutations, would provide relevant information on the potential uses of the cII transgene. It was noted that no studies with AAF had been retrieved in the literature search for the COM paper.
34. Members agreed that no conclusion could be reached on the limited published information available regarding the response of the cII transgene in cancer target organs in experimental animals. The Committee agreed that sequencing of the cII transgene in mutant plagues was essential for determining a positive result and also for evaluating equivocal mutagenic response based on fold increase in mutation frequency.
35. A short statement would be drafted by the secretariat to reflect the views of the committee.
ITEM 7: DRAFT AGENDA FOR JOINT MEETING ON THE USE OF TARGET ORGAN MUTAGENICITY IN CARCINOGEN RISK ASSESMENT (MUT/05/8)
36. Members agreed the overall plan for the joint meeting but asked that the number of presentations in the morning be reduced to a manageable level. Members suggested that the correlation between rodent species regarding target organs and sex/species response and the correlation between DNA adducts in target and non target organs be covered during the meeting.
ITEM 8: DRAFT ANNUAL REPORT (2004) (MUT/05/5)
37. Members were asked to forward any comments to the secretariat over the next two weeks. The secretariat were asked to check whether an entry for the evaluation of high dose positive in-vivo tests was required. The HSE assessor advised that a more appropriate title for the current evaluation of pesticides being undertaken by the COM at the request of the ACP would be "Biomonitoring studies for genotoxicity in pesticide applicators". The Committee agreed with this request.
[CLOSED SESSION]
ITEM 9: REVIEW OF ADDITIONAL IN-VITRO MUTAGENICITY STUDIES ON MEGLUMINE(MUT/05/2)
38. Dr Clements and Dr Clare declared non-personal, non-specific declarations of interest. The Chair considered that they could take part in the discussion.
39. The COM provided advice on the mutagenicity of Flunixin, meglumine and Flunixin (a non-streoidal anti-inflammatory veterinary medicine)-meglumine (an excipient, exempt from EU regulations) in a statement COM/03/S2 in response to a request for advice from the FSA. The available data on these compounds in 2003 were relatively poor and the COM agreed the best approach was to consider the data on each entity separately. The evaluation of meglumine posed the greatest difficulty in that there were inadequate in-vitro data on this compound but some clear positive results had been documented in relatively old in-vivo bone-marrow assays using intraperitoneal administration of 2 doses of meglumine followed by harvest 6 hours after the last dose. A number of confirmatory in-vivo studies undertaken for the Committee had yielded negative/inconclusive findings. The Committee had concluded that there was a need for two adequately undertaken in-vitro studies (namely a mouse lymphoma assay and an in-vitro chromosome aberration assay in human peripheral blood lymphocytes) before conclusions could be reached.
40. There were a number of inaccuracies in the summary submitted which conflicted with the reports but members agreed that the two studies submitted did not provide any evidence for a mutagenic effect and the assays had been adequately conducted. Members noted evidence for variance in relative growth in mouse lymphoma cells.
41. The Chairman asked members to consider the draft working paper provided as Annex 5 to MUT/05/2. The text of this document was agreed subject to a number of minor editorial changes. The company would be consulted for any comments and these forwarded to the Chairman. A statement would be agreed through Chairman's action. Members had no additional comments regarding Flunixin or Flunixin meglumine.
ITEM 10: ANY OTHER BUSINESS
42. The secretariat informed members that a paper on Proquinazid (a pesticide currently under regulatory review by the Advisory Committee on Pesticides) would be submitted to the May 2005 COM meeting. The discussion of this item would be held in closed session. The COC is also to be asked for advice. The applicant for pesticide approval was DuPont. Members were asked if there were any declarations of interest to make before the papers were distributed.
ITEM 11: PAPERS FOR INFORMATION
43. A number of papers were submitted for information.
ITEM 12: DATE OF NEXT MEETING
44. 26 May 2005.
ACTIONS
| Item | Action | Responsibility |
| 4.1 Biomonitoring from EU of genotoxicity of pesticides | Circulate criteria to members, send papers to epidemiologists |
Secretariat |
| 5 Halonitromethanes | Draft short working paper | Secretariat |
| 6 cII Transgenic mutation assay | Draft short working paper | Secretariat |
| 7 Joint meeting | Amend draft agenda, set up meeting | Secretariat/DH toxicology unit |
| 8 Annual report | Undertake minor amendments | Secretariat |
| 9 Meglumine | Amend draft working paper seek industry comments | Secretariat |