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MINUTESPresent:
CONTENT
ITEM 1: ANNOUNCEMENTS/APOLOGIES FOR ABSENCE 1. The Chairman welcomed Dr S Bull and Dr K Fletcher from the Department of Health Toxicology Unit. The Chairman also welcomed Ms C Mulholland from the Food Standards Agency and Dr S Dyer, Dr P Edwards and Ms A Grosskurth (afternoon only) who were attending from the Department of Health. 2. Apologies for absence were received from the members Dr B Burlinson, Dr J Clements, Dr D Gatehouse and the assessors Mr R Alexander (NAW), Ms Alison Gowers (EA), Mrs R Kearsley and Dr H Stemplewski (MHRA). 3. The Chairman informed the committee that a letter of resignation from the COM had been received from Ms Langley and added that the committee was grateful for her valuable contribution to the COM over the years. 4. Members were reminded of the need to declare any interests before
discussion of items. ITEM 2: MINUTES OF THE MEETING ON 27 MAY 2004 (MUT/MIN/04/2) 5. The minutes were approved with minor amendments. ITEM 3: MATTERS ARISING NOT COVERED BY LATER AGENDA ITEMS
6. No interest were declared. Finalised reports of two in-vitro mutagenicity studies (chromosomal aberration assay and hgprt gene mutation assay with CHO cells) were considered by the COM. The committee had considered near finalised drafts at the May 2004 meeting in detail and had asked for further information on dosing solution analysis and information on historical control data (particularly on chromosomal and chromatid gaps in the chromosome aberration assay). The dosing analysis indicated reasonable agreement between nominal and actual levels (3% in the chromosome aberration assay and up to 15% in the hgprt assay (4% at the top dose). The contract laboratory had provided further information on historical control data, but this only extended the data already seen by COM by about one year and the specific data on chromosomal and chromatid gaps did not appear to be available. 7. Members were content that the dosing solutions were adequate and agreed
that both studies conformed to OECD testing guidelines and were negative.
The Committee expressed some reservations regarding the conduct of these
studies (possible limitations in sensitivity) and in particular regarding
the repeat in-vitro chromosome aberration study in CHO cells. However,
overall it can be concluded that the balance of the data suggest that
chromium picolinate is not genotoxic in vitro.
9. The committee considered new mutagenicity data on malachite green and leucomalachite green at its meeting in May 2004. The COM confirmed that malachite green had in-vivo genotoxicity in view of DNA adduct formation in the liver of rats and mice and that leucomalachite green should be regarded as an in-vivo mutagen due to the induction of mutations in the liver of DNA in female mice. The COC had considered carcinogenicity data on malachite green and leucomalachite green at its June 2004 meeting. Members were asked to consider a draft joint COM/COC working paper. 10. The COC had concluded that the only convincing evidence for carcinogenicity in NTP bioassays for these two compounds was for leucomalachite green in female mice, based on an increase in hepatocellular adenoma or carcinoma combined. The COC considered that the overall tumour profile for leucomalachite green was not consistent with that of a genotoxic carcinogen, with effects being limited to the liver of female mice. There was no convincing evidence for a non-genotoxic mode of action in the NTP bioassay either. However, there was evidence of cII mutations in the liver of leucomalachite green treated transgenic mice. The in-vivo mutation data provided evidence of in-vivo mutagenicity in the liver, which was a target site for carcinogenicity. Therefore, it was not possible to discount a genotoxic mechanism for the induction of liver tumours in female mice and thus it was considered prudent to regard leucomalachite green as a genotoxic carcinogen. 11. The COM agreed that the results from this dataset were unusual and noted a lack of concordance in the data. Members asked for this to be mentioned in the draft working paper. It was also noted that it was possible that the effect in genomic DNA could be different to that seen in transgenic DNA. 12. The draft joint working paper would be submitted for consideration at the November 2004 COC meeting. It was intended that a revised joint statement would be published after clearance by the Chairs of both the COC and COM.
13. Members were asked to comment on COM relevant sections of a joint
COT/COC/COM draft working paper on toxicogenomics. Members were informed
that a further section reporting the presentation of Dr David Lovell (held
at COT meeting on 7 September) was being drafted. The COM sections of
the joint draft working paper were agreed with a number of amendments. ITEM 4: BIOMONITORING STUDIES OF GENOTOXICITY IN PESTICIDE APPLICATORS
14. 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 genotoxicity in pesticide applicators and workers exposed to pesticides, particularly focussing on pesticide sprayers/applicators, floriculturists/greenhouse workers, agricultural workers and farmers and forestry workers. It was noted that some degree of overlap between the groups regarding operations undertaken and types of pesticide exposure occurred, and different research groups have applied the terms in different ways. HSE queried the rationale for the current review. The COM secretariat noted the COM advice would be submitted to PSD as the registration authority for agricultural pesticide products and subsequently to the MTP and ACP. 15. Due to the large volume of studies available, a pragmatic decision had been taken by the secretariat and DH Toxicology Unit to limit the initial review paper to studies from the EU, ie not to consider, in the first instance studies from USA, Canada, Asia etc. In addition, studies were compared with the generic guidance available from an IPCS working group, as there was a need to compare the apparently different strategies adopted by the various research groups with an appropriate internationally recognised standard. 16. The Chairman asked for any generic comments before considering each paper in detail. 17. Members agreed that studies reporting data on sister chromatid exchanges could be excluded from further consideration in view of the uncertainty of the biological relevance of this end point to mutagenicity. Members noted the apparent inconsistency between the results of chromosome aberration assay and micronucleus tests in a number of studies and considered this was likely to be due to protocols and the methods of data evaluation used. The protocols used in the micronucleus assays were discussed and members stated that studies using incubation periods of 72 hours should be treated with caution, as many of the cells would have been through three cell divisions post-sampling. The extent of DNA repair and cell loss could complicate the evaluation of such data. Overall, members considered that studies reporting structural chromosome aberrations were likely to be the most informative. 18. Members were concerned about the lack of and limited quality of exposure data presented in the published papers and agreed that this severely limited the evaluation of the available studies. Members also commented on the inadequate matching (sex ratio) and for age in many of the studies. Shipping of blood samples between countries was a source of concern in view of the length of time between blood sampling and processing and also possibly due to the potential damage caused by X-ray scanning if shipped by air. Members also commented that none of the studies reviewed fulfilled IPCS a priori defined criteria for a positive response.
19. Workers were exposed to Mancozeb as a single exposure. Members were satisfied with the protocol used and that cells were not lost during the procedure. Members questioned the fact that all non-smokers were used.
20. Members were satisfied with the protocol used and considered that details of a single exposure to a chemical (diazinon in this case) would be easier to evaluate regarding potential genetic damage than the majority of studies that involved multiple chemical exposures of varying duration. Members questioned whether all subjects were non-smokers as reported by authors.
21. Members suggested the study be eliminated as only sister chromatid exchanges were measured.
22. The cohort consisted of all males and members agreed that counting 10-100 cells/individual for chromosome aberrations was insufficient.
23. Members agreed the approach used was satisfactory but that the preferred indices were the overall number of cells with chromosome aberrations. Members agreed this paper should be considered further.
24. Members considered that the data included in this paper duplicated that in the paper in 1992, hence could did not add to the evaluation.
25. Data were analysed subjectively and very small differences between exposed workers and controls were presented. Members considered the statistical analysis carried out on using group means was inappropriate. Members agreed that data were unconvincing and suggested that little weight should be placed in this study.
26. Members agreed on the use of FISH analysis for centromere positive and negative micronuclei was valuable but incubation times before addition of cytochalasin B were probably too long. Data showed age and sex differences, although no overall significant differences were observed between workers and controls. Members pointed out that a more appropriate protocol for the micronucleus assay was used in some of the earlier studies carried out by Bolognesi and co-workers, as cytochalasin B was added to cultures after 24 hours. Overall, it was felt that the data on centromere positive micronuclei should be considered in more detail.
27. Members pointed out that cytochalasin B was added after 44 hours of incubation, allowing the cells to undergo several cell divisions and pointed out that the negative results observed might be due to loss of damaged cells or failure to convert chromatid damage into acentric fragments that are then lost as micronuclei during exposure and harvest. Members were also concerned about the lack of matching of controls to exposed subjects as the percentage of males in the groups differed (85% in exposed group; 68% in controls). Overall, members suggested the study should not be considered further.
28. Members were satisfied with the protocol used in this study and thought that it should be considered further.
29. Members were satisfied with the protocol used in this study as cells were incubated for a shorter period, and thought that it should be considered further.
30. Members were satisfied with the protocol used in this study as cells were incubated for a shorter period, although data were replicated from previous papers.
31. Members noted that lymphocytes were isolated from peripheral blood and post sampling manipulation could have led to chromosome damage, although it was unclear when the blood samples were collected. Longer incubation times (72 hours) were needed in order to attain cells in second division in order to assess numerical aberrations. Members commented on the different groups used in the study ie healthy workers with no exposure, healthy workers exposed to pesticides and workers exposed to pesticides hospitalized for bladder cancer and agreed this study should not be considered further.
32. Members stated that the 72 hour incubation period used in the micronucleus assay was inappropriate.
33. Members noted that samples were shipped to another laboratory for processing and queried the influence of this on results. Small increases in MN frequency were observed. Members queried whether the data reflected a treatment-related mutagenic effect.
34. Members noted that samples were collected from subjects in Denmark and shipped to Finland for processing. Members noted that increases potentially associated with exposure were predominantly owing to chromatid gaps.
35. Members were concerned about the low level of adducts (<1 x 108), and it was unclear whether a direct interaction with DNA had been observed. No correlation was seen with exposure. Members suggested that the study should not be considered further.
36. Members expressed concerns regarding the protocol used for the 32P-post-labelling assay, as butanol extraction was not performed, and suggested the study be eliminated.
37. Members were satisfied with the protocol used for the comet assay. However, concerns were expressed over the low number of subjects used in the study, as only 30 males were used and members questioned the power of the study to detect an effect.
38. Members noted that samples were shipped to a central laboratory for analysis, and expressed concerns over the protocol used as cells were incubated for 72 hours. Members agreed that buccal cells could be used for micronuclei analysis. Although the study was large, members thought it was difficult to make any conclusions from the data.
39. Members expressed concerns about the high number of females in the controls group and the long incubation time used and agreed the study should not be considered further.
40. Members were concerned about the long incubation time used in the micronucleus assay and agreed the study should not be considered further.
41. Members were concerned about the long incubation time used in the micronucleus assay and agreed the study should not be considered further.
42. Members were concerned about the long incubation time used in the micronucleus assay and agreed the study should not be considered further. Lucero et al, 2000 43. Members were concerned about the long incubation time used in the micronucleus assay and agreed the study should not be considered further.
44. Members questioned results in annex II, paragraph 400, which stated that the Wilcoxon test revealed a significant decrease (p<0.001) in the individual percentage of CA (chromatid-type aberrations, total aberrations and aberrant cells) following periods of lower exposure. Members agreed the results should be considered further but noted damage was confined to chromatid breaks.
45. Members agreed the data from this study repeated similar findings to Carbonell 1995. Members noted that isolated lymphocytes were used, which may have lead to the increase in damage.
46. Members suggested the study should not be considered further as only sister chromatid exchange was measured.
47. Members expressed concerns regarding the low cell number scored for chromosome aberrations and that the chromosome identity of the karyotyping was not reported. It was agreed the study should not be considered further.
48. Members expressed concerns over the protocol used for the micronucleus assay and noted high background values. The sister chromatid exchange assay was not considered further.
49. Members expressed concerns about small numbers of individuals studied and inconsistencies in the data reported. It was agreed the study should not be considered further.
50. Members expressed similar concerns about the study compared to Lebailly 1998a and agreed it should not be considered further.
51. Members agreed that the data on urinary mutagenesis and predicted exposure using the UK POEM (Predicted Operator Exposure Model) should be considered further.
52. Members noted that in-vivo and in-vitro experiments carried out with 2,4-D appeared adequately undertaken. The data from the biomonitoring component should be considered further.
53. Members suggested the study should not be considered further as only sister chromatid exchange was measured.
54. Members considered the general conclusions suggested in the DH Toxicology Unit paper and proposed a number of revisions. It was agreed that the lack of appropriate exposure would severely limit any conclusions from the review. It was agreed that no weight should be placed on the results of sister chromatid exchange investigations. Members agreed that the secretariat would proceed to identify studies for further evaluation by an independent epidemiologist. A further paper would be submitted to the February 2005 COM meeting.
55. Members confirmed that no weight could be placed on the studies appended
to MUT/04/20. It was agreed that the number of discrepancies between the
different reports severely limited the value of the research. ITEM 5: RE-ASSESMENT OF TOXICOLOGICAL TESTING FOR TOBACCO PRODUCTS (MUT/04/21) 56. The Chairman welcomed observers representing a number of tobacco manufacturers. He asked for declarations of interest. Dr Mitchell declared a personal interest and left the room. Dr Parry and Dr Clare declared non-personal, non-specific interests. The chair agreed that they could take part in the discussion. 57. The Department of Health has asked for advice on the strategies which have been used to assess tobacco products and in particular Potentially Reduced Exposure Tobacco Products (PREPS). The advice is needed in the context of the request from the European Commission (EU). To submit, no later than 31 December 2004, a report on the application of Directive 2001/37/EC and in particular in respect of:
58. Paper MUT/04/21 discusses generic aspects of toxicology testing to tobacco products. The approaches to toxicology testing that have been used in practice are briefly summarised in paragraphs 14-17 of the paper. These essentially consist of in-vitro assays for mutagenicity and cytotoxicity in a range of tests, supported by short-term exposure studies in experimental animals. In some instances biological monitoring for exposure to tobacco constituents has been undertaken in volunteer studies using regular smokers. 59. The Chair asked members to consider the information presented on the in-vitro mutagenicity tests with reference to assessment of tobacco products. Members agreed that the approaches used were consistent with COM guidance. Such studies could provide information on the mixtures tested and could provide information on mutagenic potency in the assay used but could not easily be extrapolated to the in-vivo situation. No conclusions regarding risk of cancer associated with "PREPS" could be derived from such studies. The HSE assessor noted that the COM test strategy was designed for screening individual chemicals and had not been designed for screening complex mixtures or changes in complex mixtures. 60. Members noted that the limitations of toxicogenomic methods had been recently discussed by COM. The same comments applied to the application of these techniques to assessment of tobacco products. Some useful information on mechanisms might potentially be derived from such studies but the data would be complex and difficult to evaluate. 61. Members reviewed the available biomonitoring studies. With regard to Breland et al (Tobacco Control 2003, vol 12, 317-321), members agreed the study had been adequately designed and performed. 4-(Methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolites were considered to be valid biomarkers of tobacco usage. A reduction in urinary total 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanol (NNAL) was documented for Advance™ compared to own brand. Members noted the adaptation of smoking activity might, in part, explain these results. The major limitation to this study and other similar biomonitoring studies was the absence of data regarding other recognised carcinogens present in tobacco smoke. It was not possible to reach conclusions regarding risk of cancer from such studies. Members agreed that no additional data on mutagens/carcinogens could be derived from Breland et al Tobacco Control, 2002, vol 11, 376-378. 62. Members agreed the study by Hughes et al (Tobacco Control, 2004, vol 13, 175-179) had been adequately conducted. Overall members agreed that no statistically significant effects on urinary total NNAL levels had been documented for Omni compared to own brand. 63. Members agreed that the study by Hatsukami et al (Journal National Cancer Institute, 2004, vol 96, 844-852) had been adequately designed and conducted. Members noted that more information had been published in this paper compared to the other biomonitoring studies presented to COM. Urinary levels of total NNAL were reduced in smokers who switched from own to the Omni brand, but no statistically significant reduction in 1-hydroxypyrene (a marker for Polycyclic Aromatic Hydrocarbon exposure) was documented. Members considered that future biomonitoring studies of tobacco products should examine a wider range of carcinogens present in tobacco smoke. 64. The Chair noted that Omni had a palladium catalyst and asked observers from tobacco companies whether any mutagenicity data were available for the catalyst. One observer commented that no data were available. 65. The Chair asked members to consider the two questions posed in para 25 of the covering paper MUT/04/21. 66. The Committee considered the two questions posed on the covering
paper. It was agreed that in-vitro screening mutagenicity tests
had the potential to be informative on changes in levels of mutagens in
tobacco products, although protocols would need to be adapted. However,
the results could not be extrapolated to predict mutagenic risk of tobacco
products in-vivo. 68. The observers were asked for any comments. One observer noted that
additional data might be available. The secretariat replied that such
data could be forwarded, but in order to meet the deadline for providing
an input to the EU review, the Committees would finalise their statement
based on the information provided in MUT/04/21. ITEM 6: HORIZON SCANNING PAPER 2004 (MUT/04/22) 69. The COM undertakes 'Horizon scanning' exercises at regular intervals
to identify new and emerging issues which have the potential to impact
on public health and which might require COM advice. Members considered
paper MUT/04/22 which focussed on target organ mutagenesis and carcinogenicity
risk assessment, low dose DNA adducts, mutagenicity of micronised chemicals
and effects of micronutrients on mutagenicity. 71. Members agreed that the link between target organ mutation and cancer had not been investigated in detail at present. The committee considered that factors that determined target organs for carcinogenicity were poorly understood and the process was complex. The COM agreed that it would be valuable to have a joint COM/COC meeting on the topic. A number of independent experts would be invited to attend. 72. Techniques to assess DNA adducts had improved in recent years and highly sensitive methods such as 32P-postlabelling were available. The committee noted that ILSI/HESI had looked at the use of low dose DNA adducts in risk assessment at its meeting in April 2004. The COM agreed to await a review on this topic by a subgroup of ILSI/HESI before considering the matter further. 73. The COM agreed that genotoxicity testing of nanotechnology products was potentially important. There were a few examples where micronised particles of traditionally accepted non-mutagenic chemicals had given positive results in in-vitro tests (eg zinc oxide and titanium dioxide). Aspects to consider included whether nanoparticles could be more readily absorbed, whether conventional mutagenicity tests were suitable for particles and whether any genotoxicity could be secondary to toxic effects such as the generation of reactive oxygen species. Members agreed that a periodic review of this subject would be useful. 74. The committee was aware that there was considerable attention given
to the role of micronutrients in the prevention of cancer. There were
a number of topics of interest. It had been postulated that the variation
in cancer incidences within populations with similar diets may be explained
by genetic factors eg polymorphisms. Diets lacking in certain micronutrients
(known to contribute methyl groups) could result in altered DNA methylation
status, which could in turn affect the incidence of mutation and possibly
cancer. Antioxidants such as vitamins C and E, selenium and lycopene were
considered to affect in-vivo mutagenesis. It was agreed to raise
these issues with the Scientific Advisory Committee on Nutrition. ITEM 7: PAPERS FOR INFORMATION (MUT/04/23 & MUT/04/24) 75. Members noted three papers on test methods (MUT/04/23) and a paper reporting a NOEL for in vivo mutagenicity in Big Blue transgenic rats given MeIQx (MUT/04/24), which had been provided for information. ITEM 8: ANY OTHER BUSINESS 76. There were no other items of business. ITEM 9: DATE OF NEXT MEETING 77. To be announced.
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