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MINUTESPresent:
CONTENT
1. The Chairman welcomed Dr Stuart Creton (from the Food Standards Agency) who was to introduce the item on Furan (Item 4) and Dr Karin Fletcher (DH Tox Unit) is attending for items 3.1 and 4 and 7 and Dr Sarah Bull (from the Health Protection Agency) who had provided additional material to MUT/05/19. The Chairman welcomed Mr T Holmes who was replacing Dr D Andrew (PSD) for this meeting as PSD assessor. 2. Apologies for absence were received from Dr B Burlinson (member), Mr A Browning (VMD) and Dr H Stemplewski (MHRA). 3. Members were reminded of the need to declare any interests before discussion of items. ITEM 2: MINUTES OF MEETING ON 26 MAY 2005 (MUT/MIN/2005/2) 4. Members agreed the minutes subject to some minor editorial changes. Regarding the item on Proquinazid, members asked for clarification on the species of fungi controlled by this fungicide. The Chairman noted that Ms Alison Craig from the Pesticides Action Network had submitted a comment on the wording of the minute (para 18, MUT/MIN/2005/2). The Committee agreed that the particular paragraph was an accurate summary of the discussion and there was no need for amendment. Members observed that COM minutes were not intended to be a detailed transcript of discussions. ITEM 3: MATTERS ARISING (NOT COVERED BY LATER AGENDA ITEMS)
5. The Committee was informed that a scientist at the US EPA had queried the COM recommendation for a lower temperature (300C) during bacterial exposure to halonitromethanes than in conventional bacterial mutagenicity tests (ie 370C), the recommendation for triplicate plates and the use of the in-vivo rat liver UDS assay to evaluate in-vivo mutagenicity of halonitromethanes in the liver. Members confirmed their view that exposure at reduced temperature had been validated as a potential method for reducing volatilisation of chemicals and there was experience of using a lower exposure temperature by a number of Japanese investigators. It was noted the plating of bacteria following incubation with the test chemical was undertaken at conventional temperatures. An alternative approach as advocated by UKEMS was to use Teflar bags to prevent evaporative loss. Members acknowledged that availability of test chemical might reduce the extent of testing but considered triplicates to be the optimum approach to use. Although it is possible to offset the loss of power from the use of duplicate treatment plates by increasing the replication of the negative controls this was not done in the study in question. Members felt the in-vivo rat liver UDS assay was appropriate in this instance for the investigation of potential mutagenicity of halonitromethanes in the liver in rodents. It was noted that the finding of DNA adducts in rat liver following in-vivo exposure to dibromonitromethane reported by Dr S Richardson of the EPA supported the view that this compound should be considered as having mutagenic potential in-vivo.
6. Members heard that the COC considered the carcinogenicity data on the cholangiocarcinomas seen rats fed proquinazid for 2 years. COC pathologists were not convinced the lesions seen were neoplastic (eg there was evidence of widespread necrosis but no evidence of increased mitoses, evidence of a widespread inflammatory reaction, fibrosis and goblet cell differentiation were also reported). In this respect the COC agreed with presentation made by Dr Peter Greaves (Leicester) on behalf of the company. The COC agreed to use the terminology suggested by the study pathologist. The COC agreed a non-genotoxic mode of action based on severe acute/chronic response to hepatocellular damage. Subsequent to the last COM, the company submitted a new mouse lymphoma assay. This was circulated by post to relevant COM members. The comments received supported the study as being adequate and providing negative results. The joint COC/COM statement was subsequently agreed and published on the COC/COM internet sites. ITEM 4: BIOMONITORING STUDIES OF GENOTOXICITY IN PESTICIDE APPLICATORS: Draft working paper (MUT/05/19) 7. No interests were declared. 8. Following members comments at the May 2005 meeting, the secretariat undertook a further analysis of the information from biomonitoring studies of genotoxicity in nurses and patients. The information retrieved for cancer patients included high background mutation frequency in lymphocytes, and repopulation of lymphocytes as additional factors to consider when interpreting data on biomonitoring for genotoxicity. It was concluded in the draft working paper that it was difficult to make any use of the data from nurses/cancer patients to help inform on the small increases in mutation frequency reported in a number of biomonitoring studies of pesticide applicators. 9. A further analysis of those pesticide applicator studies which reported exposure data was presented for members information. The evidence supporting any particular active ingredient appeared to be very limited. In addition the evidence supporting exposure to benzimidazoles includes positive data in the COMET and DNA binding assays. It is noted that this class of mutagen is not expected to produce positive results in these assays on theoretical grounds. Members were asked to consider the Bolognesi et al 2004 paper (Mutation Research, 557, 109-117, 2004). A tabulation of all positive studies with exposure data was included in MUT/05/19. It was noted that the Bolognesi study had been listed as positive in the initial tabulation of studies with exposure data and members were asked to comment on this particular conclusion. The Committee was also asked to provide as much advice as possible on any potential biomonitoring study using benzimidazoles. In this respect information on the use of urinary excretion of 5-hydroxy-2-benzimidazole (5HBC) as a biomonitor of absorbed dose of carbendazim had been provided (including a tabled paper Meuling WJA et al. Prediction of Percutaneous Penetration, 38, 598-603, 1993). 10. The Chairman asked members to comment on the topics as set out in MUT/05/19. 11. Members considered that the factors which accounted for the variance in biomonitors of genotoxicity (chromosome aberrations and micronuclei predominantly in circulating blood lymphocytes) in nurses and cancer patients exposed to cytostatic medicines had not been fully evaluated. Members agreed that it was not possible to establish a minimum fold increase in response that was biologically important following exposure to an in-vivo mutagen based on these studies. It was concluded that the data could not be used to set a minimum fold increase for biological importance for use in pesticide applicator biomonitoring studies and that factors affecting variance in biomonitoring indices used in the biomonitoring studies of pesticide applicators which had been reviewed were not understood adequately. In this respect members considered it would be very difficult to infer causality from the small magnitude responses seen in the biomonitoring studies of pesticide applicators. There was a need for further research on factors which were relevant to each biomonitoring indices before interpretation of studies which investigated exposure-response could be interpreted. 12. Members considered the Bolognesi et al 2004 study in detail. Members confirmed that this study should be considered as negative using the criteria agreed by the COM during the review of studies of pesticide applicators. Members agreed this was one of the better studies which had been considered as the investigators had attempted to use a specific index of exposures to tubulin inhibitors such as carbendazim and benomyl, although there were limitations because of the adequacy of the exposure index for measuring dose, the small number of individuals studied and the multiple comparisons used in the analysis. Members considered that the results of the centromere specific investigations were based on limited data and no interpretation of biological significance could be placed on the data. The data provided in this study were consistent with the general conclusions on interpretation which members had reached. 13. The committee reviewed the evidence to support a UK based study of floriculturalists exposed to approved benzimidazole based pesticide products. The committee agreed the evidence for such a study was very weak. Some of the end points listed in the positive studies where exposure had been documented could not be directly associated with exposure to benzimidazoles (eg data from COMET investigations), although it was possible that a positive response could occur at exposures where benzimidazoles induced gross chromosomal damage. The possibility that other potentially mutagenic compounds in the exposures experienced by individuals in these studies could account for the observed responses could not be excluded. Members felt that evidence for an association between benzimidazoles and increases in DNA adducts such as that reported in an investigation of 32P-post labelling was unlikely to be due to benzimidazoles. 14. Members were aware of the guidance available for the conduct on biomonitoring studies of genotoxicity from Albertini et al (Mutation Research, 463, 111-172, 2000) but agreed, that having considered a large data set of studies on pesticide applicators, that more research and guidance on the factors affecting the background variance of biomonitoring indices of genotoxicity was required before such studies could be interpreted particularly with regard to the significance of the small magnitude of response seen in the studies considered. HPA noted this was an important subject for further consideration. Overall the Committee considered that although an association between exposure to pesticides in the available biomonitoring studies and response could not be discounted, there was insufficient evidence to reach definite conclusions concerning which pesticide active ingredients might be responsible for any observed effects on biomonitoring indices of genotoxicity. 15. The Chairman asked members to provide as much guidance as possible with regard to the possible conduct of a study on benzimidazoles approved in the UK and in particular with regard to carbendazim. Members agreed that a longitudinal study where individuals acted as their own controls would be most appropriate. It was agreed that there was supporting evidence that biomonitoring for urinary excretion of 5-hydroxy-2-benzimidazole (5HBC) could be used to assess exposure and uptake of carbendazim. Members commented that the application of Personal Protective Equipment specified for use of carbendazim products (such as in floriculture) meant that exposure would be minimised so that any increase in genotoxicity would be small and unlikely to be detected with any reliability. Any such study would have to be very large and there were doubts as to whether an appropriate exposure group could be identified. 16. PSD suggested that a Margin of Exposure assessment for approved pesticides might help to provide reassurance for the risk assessment of benzimidazoles. It was noted that the ACP would be best placed to advise on the need for such an evaluation. 17. The Committee considered the draft working paper and agreed the wording subject to inclusion of a new section on interpretation of biomonitoring indices of genotoxicity and a clear statement that DNA reactive in-vivo mutagens are not approved for use in pesticide products in the UK. Members agreed the conclusions should be redrafted to reflect the discussion and agreed that a revised draft working paper would be circulated for member's consideration by post. The Chairman would consider members comments and a finalised statement would be published on the COM internet site and forwarded to the ACP for its consideration. Members agreed that conclusion iv) on the potential use of biomonitoring study data to aid in the review of the risk assessment of benzimidazoles should be removed from the draft working paper as the evidence from such studies could not be currently used to assess the adequacy of existing risk assessments.
18. No interests were declared. 19 The COM had been asked for advice on the mutagenicity of furan by
the COT who were considering research that would be helpful regarding
the risk assessment of this contaminant. Members agreed there were no
clear structural alerts but metabolism mediated by CYP2E1 could lead to
epoxide formation which was consistent with the finding of cis-2-butene-1,4-dial.
It was 20. The Committee considered that the available mutagenicity tests of furan in bacteria had been adequately conducted. A weak positive result had been documented with furan in one assay using Salmonella typhimurium TA 100 in the presence of exogenous metabolic activation. It was noted that CYP2E1 would not be significantly induced in Arochlor 1254-induced rat liver S-9 mix. The positive result in Salmonella typhimurium TA 104 with cis-2-butene-1,4-dial was consistent with this compound being a reactive aldehyde. Members noted the positive results reported in chromosomal aberrations tests in CHO cells in both the presence and absence of exogenous metabolic activation but commented that the concentrations where positive results had been identified (ca 125 mM and 250 mM) were considerably greater than the OECD upper limit of 10 mM for non cytotoxic test materials. There was no information on cytotoxicity in the NTP CHO test report. 21. Members considered that the L5178Y mouse lymphoma assay reported
by the NTP had been conducted to standards acceptable at the time using
plating in soft agar but that a trial had not been conducted in the presence
of an exogenous metabolising fraction. It was considered the cells might
have expressed some CYP2E1 activity. A negative response had been reported
in trial 1 but a dose related increase in mutation frequency was seen
in trials 2 and 3 which included dose levels where there was only a small
decrease in relative total growth. It was noted that dose levels used
in 22. The Committee considered the available in-vivo mutagenicity studies. The key in-vivo bone marrow chromosomal aberration assay was undertaken as part of the NTP programme on furan. The investigators had used several dose levels and multiple sampling times. The two trials which had used an extended harvest time of 36 hours reported a statistically significant increase in chromosome aberrations (12 and 11 fold respectively) at 250 mg/kg bw administered by gavage. No information on toxicity was available. However it was noted that other published data suggested that a single oral gavage dose of furan at 50 mg/kg bw resulted in moderate histopathological signs of liver toxicity whilst a dose level of 250 mg/kg bw resulted in extensive hepatocellular necrosis and was the highest possible based on lethality. Members agreed that the data from this study could not be interpreted with certainty because of the confounding effects of toxicity at the dose level used. 23. The Committee agreed that negative results had been obtained in in-vivo liver UDS studies in rats and mice. Covalent binding studies using oral or intraperitoneal doses of radiolabelled 2,5-14C-furan in rats were available. There was evidence for dose related binding of furan to protein. Members considered the studies were limited by use of low specific activity radiolabelled furan, low sensitivity of radiolabel analysis and the incorporation of carbon dioxide formed from furan into intermediary metabolism. The results of in-vitro studies of the reaction of cis-2-butene-1,4-dial with deoxynucleosides indicated reactions with 2'-deoxyadenosine, 2'-deoxyguanosine and 2'-deoxycytidine but not thymidine. The authors had proposed that cis-2-butene-1,4-dial might form DNA cross links. 24. Members agreed that no definite conclusions could be drawn from the studies of proto-oncogene mutation spectra in hepatocellular adenomas and carcinomas from control and furan treated mice. 25. The Committee concluded that furan should be regarded as an in-vitro mutagen but there was insufficient evidence to reach a conclusion on the available in-vivo mutagenicity data. 26. The Chairman noted that the Food Standards Agency had asked for advice on what research could be used to determine whether a threshold approach to risk assessment would be appropriate. Members felt that adequate in-vivo mutagenicity testing would be useful. It was suggested this might include a bone marrow micronucleus test or DNA binding studies in cancer target organ tissues. It was noted that the critical carcinogenic effect was cholangiocarcinoma and there would be difficulties in isolating bile duct cells during DNA binding studies. There was some discussion regarding whether a study using Accelerator Mass Spectrometry (AMS) would be appropriate. This would require the formation of stable DNA adducts. The potential for DNA-protein cross links would need to be considered. Members considered that further consideration of the mechanisms of in-vitro mutagenicity and the possible use of scavenging agents (eg N-acetylcysteine) to investigate whether the primary mode of action was cytotoxicity would also be useful. It was noted that any further in-vitro studies would need to consider the selection of solvents carefully as many commonly used solvents (eg DMSO, acetone, ethanol) would compete with furan for metabolism via CYP2E1. 27. It was agreed that the COM advice would be passed to COC for consideration during its discussions on furan. The COM and COC advice would be reported back to COT as conclusions for inclusion in a COT statement.
28. No interests were declared. 29. The risk assessment of nanomaterials has been identified by COT/COC/COM as an area of interest during horizon scanning discussions for 2004. The Committee's interest in this area was prompted by the publication of the Royal Society review of nanotechnology. (http://www.nanotec.org.uk/) The appended documents have been drafted to provide baseline toxicology information for all three committees. These comprise; The objective is to collect initial views from the COC (21 April), COM (26 May, deferred to 13 October 2005) and COT (12 July) and draft a short statement. The COT overview paper has been submitted to COM for information (MUT/05/18). 30. There was in-vitro evidence that nanoparticulate titanium dioxide was mutagenic and induced clastogenicity and MN. However a number of published studies were available which showed that commercial samples of titanium dioxide which presumably included µmetre sized particles also induced mutagenic effects in-vitro in mammalian cells. In some instances there were no data on particle size reported (eg Lu PJ et al Mutation Research, 414, 15-20, 1998). It had been suggested that a free radical mechanism is appropriate. Members had reservations regarding the description of titanium dioxide test materials used. It was considered that titanium dioxide particles had not been solubilised in any of the studies and that it was most appropriate to describe the test materials as suspensions. There was no evidence to clarify what size of particles had been tested or whether particle agglomeration or disagglomeration had taken place. Members discussed the potential mechanisms of particle uptake and noted that there was no information in this regard with respect to the bacterial and mammalian cell systems commonly used in mutagenicity tests. It was agreed that no definite conclusions could be reached with regard to the studies with nanometre titanium dioxide reported by Rahman Q et al, Environmental Health Perspectives, 110, 797-800. A positive in-vivo BM MN has been documented some years ago with commercial grade titanium dioxide. (Shelby D et al Env Mol Mutagenesis, 21, 160-179, 1993). 31. The data on zinc oxide could not be interpreted as there was insufficient information regarding particulate size tested and the properties of the test material in the vehicles chosen. Members considered that zinc oxide formed a solution in dilute hydrochloric acid and hence the evidence for clastogenicity reported by Hikiba et al (J Pharmacol Sci, 97, 146-152, 2005) related to soluble zinc ions. Members also concluded that there was insufficient information regarding the presentation of fullerene molecules in the available mutagenicity studies undertaken in various strains of Salmonella typhimurium. Members agreed with the conclusion reached by Sera et al (Carcinogenesis, 17, 2163-2169, 1996) that the photomutagenicity of C60 fullerene (>99.9% pure dissolved in polyvinyl-pyrrolidone) tested in plate incorporation assays in S.typhimurium TA102, TA 104 and YG3003 (repair deficient strain derived from TA 102) in the presence of rat liver microsomes occurred most probably via a free radical generation mechanism involving rat liver microsomal phospholipids. 32. The COM considered that specific information on particle size was required to assess mutagenicity studies undertaken with nanomaterials. Thus the available information on titanium dioxide did not allow an assessment of the agglomeration/disagglomeration of particles in the vehicles used and it was not possible to conclude what particles had been tested. The COM agreed that it might be appropriate to provide imaging data on particle sizes in order to evaluate in-vitro mutagenicity tests nanomaterials. ITEM 7: HORIZON SCANNING PAPER FOR 2005 33. The secretariat and DH Toxicology unit had drafted a horizon scanning paper. Members were asked to consider the proposals and to add any additional items and to suggest priorities. The following areas of work were agreed which have been grouped into three areas of priority. 34. Areas with high priority were:
ITEM 8: DRAFT WORKING PAPER ON JOINT COM/COC MEETING JUNE 9 ON USE OF TARGET ORGAN MUTAGENICITY ASSAYS IN CARCINOGEN RISK ASSESSMENT (MUT/05/23) 37. The Chairman asked members to forward any comments to the secretariat. It was noted that the two working groups had reached apparently different conclusions regarding the comet assay. The secretariat was asked to liaise with COM and COC and consider if any amendment to the draft statement was necessary. ITEM 9: ANY OTHER BUSINESS 38. The Committee was informed that the DH part of the secretariat for COM and COC would transfer to the HPA from the beginning of 2006. This would not affect the operation of the committees. ITEM 10: PAPERS FOR INFORMATION 39. COT review of nanomaterials (MUT/05/18). ITEM 11: DATE OF NEXT MEETING 40. Thursday 10 February 2006.
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