MINUTES - 27TH MAY 2004
Present:
| Chairman: Professor P Farmer |
Members: Dr C Allen Dr B Burlinson Dr G Clare Dr J Clements Dr D Gatehouse Mrs R Glazebrook Dr N Gooderham Dr I Mitchell Dr E Parry Professor D Phillips |
Secretariat: Dr R J Fielder (Scientific DH) Dr D Benford (Scientific FSA) Mr J Battershill (Scientific/Minutes DH) Mr K N Mistry (Administrative) |
Assessors: |
In attendance : Dr S Bull (DH Tox Unit) Dr K Fletcher (DH Tox Unit) Ms C Mulholland (FSA Item 4) Mr D Renshaw (FSA Item 5) |
CONTENT
| Item | Paragraph |
|
1. |
Announcements/Apologies for absence | 1 |
2. |
Minutes of the meeting 5 February 2004 (MUT/MIN/04/1) | 4 |
3. |
Matters arising not covered by later agenda items | 5 |
4. |
Mutagenicity of Chromium picolinate (MUT/04/16) | 6 |
5. |
Malachite Green and Leucomalachite Green (MUT/04/12) | 8 |
6. |
2,3-Dichloropropanol; draft statement (MUT/04/16) |
17 |
7. |
Genotoxic carcinogens and DNA repair at low doses Advice requested from COM (MUT/04/14) |
18 |
8. |
SCCNFP recommendations on a strategy for testing |
27 |
9. |
Toxicogenomics: Update on literature (MUT/04/11) |
31 |
10. |
Paper for information (MUT/04/15) |
36 |
11. |
Any other business | 37 |
12. |
Date of next meeting – Thursday, 7 October 2004 |
38 |
ITEM 1: ANNOUNCEMENTS/APOLOGIES FOR ABSENCE
1. The Chairman welcomed Dr Carolyn Allen and Mrs Rosie Glazebrook who were attending COM for the first time as newly appointed lay members. He also welcomed Ms A Gowers (EA) and Mr N Joseph (VMD) who were attending their first COM meeting as Departmental Assessors and Dr K Fletcher and Dr S Bull from the Department of Health Toxicology Unit. The Chairman welcomed Ms C Mulholland and Dr D Renshaw from the Food Standards Agency.
2. Apologies for absence were received from Ms Langley (COM lay member), Mr S Robjohns (Secretariat) and Mr D Andrew (PSD Assessor).
3. Members were reminded of the need to declare any interests before discussion of items.
ITEM 2: MINUTES OF THE MEETING ON 5 FEBRUARY 2004 (MUT/MIN/04/1)
4. The minutes were approved with minor amendments.
ITEM 3: MATTERS ARISING NOT COVERED BY LATER AGENDA ITEMS
5. The Committee was informed that following the discussion of proposals for holding COM meetings in open format at the February 2004 meeting, the secretariat would take steps to introduce arrangements for open meetings from the COM meeting on 7 October 2004. The procedures would be identical to those adopted by the COT. Implementation of the procedures would require the secretariat to liaise with potential attendees and the chair prior to COM meetings and to contact DH Press Office if a request were received from individuals employed by the media.
ITEM 4: MUTAGENICITY OF CHROMIUM PICOLINATE (MUT/04/16)
6. Members heard that the manufacturing company for chromium picolinate was Nutrition 21 based in the U.S.A. Chromium picolinate is a widely available food supplement in the UK. No interests were declared. The minutes of this item are now published - see Paragraphs 39 - 48.
ITEM 5: MALACHITE GREEN AND LEUCOMALACHITE GREEN: CONSIDERATION OF NEW DATA (MUT/04/12)
7. No interests were declared.
8. Malachite green (MG) is a cationic triphenylmethane dyestuff that has been used as a fungicide in freshwater fisheries. Its use in fish for human consumption was banned in the EU in June 2002, but residues continue to be found in fish. The COM considered MG, and its lipophilic metabolite leucomalachite green (LMG) in 1999 and concluded, on the basis of limited data, that it would be prudent to regard both compounds as potential in-vivo mutagens. New data are now available from the NTP programme and the Committee is asked to update its views on the mutagenicity of MG and LMG. The advice of the COC will then be sought on the new carcinogenicity bioassay data, and a joint statement agreed covering both mutagenicity and carcinogenicity.
9. Members agreed that the structure of both MG and LMG contained groupings that provide structural alerts for potential mutagenicity and thus it was plausible, therefore, to consider both compounds as being potentially mutagenic.
10. The Committee then considered the new in-vitro data. It was agreed that the new data on MG using the Comet assay was clearly positive in the presence of S-9. Other data were consistent with earlier studies. LMG was negative in the Comet assay, but the available in-vitro mutagenicity data on this compound were still inadequate to draw any definite conclusions.
11. Full details were now available of the 32P post-labelling studies to investigate liver DNA adduct formation in F344 rats and B6C3F1 mice treated with MG and LMG for 28 days in the rat, and with MG in the mouse only. Although the data clearly indicated adduct formation (except with LMG in mice), with evidence of a dose response, it was noted that the extent of binding was very low, around 1 - 6 adducts per 108 nucleotides. Similar results were obtained in 32P post-labelling studies in Big Blue rats using LMG and up to 32 weeks exposure. Again the extent of binding was very low (1 in 108 nucleotides).
12. The induction of lac I mutations in the liver was also investigated in these studies in Big Blue rats. Evidence for an increase in mutations was seen only at one mid-time point (16 weeks) at the top dose level. The significance of this transient increase was felt to be questionable. The authors argued that the effects were due to disproportionate expansion of spontaneous lac I mutations. Members noted that the effects were only marginally different from the controls, and believed that the results should be considered as equivocal.
13. In addition to the liver mutations and adducts, the ability of LMG to induce micronuclei in bone marrow and hprt mutations in spleenic lymphocytes was investigated in these studies in Big Blue rats. Although negative results were obtained, members felt that it should be noted that the experimental design (subchronic dietary administration for 4-32 weeks) did not optimise the chances of obtaining a positive response and no definite conclusions could be drawn.
14. Members felt that the detection of adducts in the liver but not mutations may be a reflection of the different sensitivities in the assays. The extent of adduct formation was low (of the order of 1 adduct per 108 nucleotides), which was probably too low to be produce detectable effects in the mutation assay. Other factors are also important with regard to the association between DNA adducts and mutations, for example the extent of error-prone or error-free repair.
15. The studies with MG and LMG in Big Blue mice were then considered. These used a 16 week dietary exposure period with up to about 400ppm LMG and 450ppm MG. A range of end points was investigated, namely liver DNA adducts, micronuclei in peripheral lymphocytes, hprt mutations in spleenic lymphocytes, and CII mutations in the liver. As in previous studies, MG gave evidence of low level DNA binding to liver DNA but no other evidence of a mutagenic response. In contrast LMG produced no evidence for formation of DNA adducts in the liver but did produce a mutation spectra that differed from that seen in the control animals with a clear increase in CII mutations. It was noted that the CII mutations in liver appeared to mirror the tumour data in the bioassays, with LMG producing a clear increase in hepatic tumours in female mice, but with equivocal evidence in the rat.
16. The Committee agreed that in view of the demonstration of DNA adduct formation in samples from both rats and mice, MG should be regarded as an in-vivo mutagen. Additionally, in view of the demonstration of the induction of mutations in liver DNA of female B6C3F1 mice, LMG should be regarded as an in-vivo mutagen.
ITEM 6: 2,3- DICHLOROPROPAN-1-OL: DRAFT STATEMENT (MUT/04/10)
17. No interests were declared. The COM agreed the draft statement subject to a number of minor editorial amendments.
ITEM 7: GENOTOXIC CARCINOGENS AND DNA REPAIR AT LOW DOSES (MUT/04/14)
18. No interests were declared.
19. An overview paper on the significance of DNA repair induction at low doses with respect to thresholds had been discussed at the February 2004 COM meeting. Members had asked that information be provided on a few direct acting chemical mutagens as examples to facilitate consideration of this area. This had been done in MUT/04/14 using MMNG, EMS and MMS. In view of the amount of data on these 3 compounds, ethylene oxide had not been used as an example.
20. Most information was available on the O6-methyltransferase (O6-MT) repair system, mainly in bacterial systems but some information was also available in mammalian cells. Members agreed that these data provided some evidence for thresholds in the adaptive state that could be attributed to the induction of the O6-MT.
21. The difficulty in establishing unequivocal evidence for a threshold was discussed, as opposed to a no-observable-effect-level depending on the sensitivity of the assay. The importance of some knowledge of the underlying mechanism to support the biological plausibility of a threshold (as emphasised in the current COM guidance) was acknowledged.
22. Only limited data were available on systems other than the O6-MT repair, and members considered that these did not provide any significant evidence regarding thresholds.
23. In the general discussion it was noted that evidence regarding DNA adducts induced by bulky compound such as aromatic amines was consistent with a linear dose response and no threshold. It may thus have been useful to have considered data on such compounds as well as simple alkylating agents. However, it was recognised that this would have introduced the added complication of the need for metabolic activation. It was also pertinent that the key issue was what happened to the DNA adduct with regard to error-prone or error-free repair, the latter being a potentially threshold related pathway.
24. Overall, the Committee agreed that there was no clear evidence for a J shaped dose response in any of the data considered. Data regarding the O6-MT induction suggested that an in-vivo threshold was likely, but not proven. No conclusions could be drawn from the limited data on other DNA repair mechanisms.
25. When considering whether any further work would be appropriate it was important to consider if this would be cost effective. The difficulty in designing experimental studies with adequate sensitivity to provide definite answers needed to be recognised.
26. The Committee agreed that these data considered in this paper do not warrant any reconsideration of the Committee's current advice that it is prudent to assume that in-vivo mutagens do not have a threshold, unless there is good evidence to the contrary.
ITEM 8: SCCNFP RECOMMENDATIONS ON A STRATEGY FOR TESTING COSMETIC INGREDIENTS FOR MUTAGENICITY (MUT/04/13)
27. Dr Gooderham declared a personal interest. The Chairman asked him not to contribute to this item.
28. The Committee were asked to comment on the strategy recently proposed by the EC's Scientific Committee on Cosmetics and Non-Food Consumer Products (SCCNFP) for permitted cosmetic ingredients (Opinion SCCNFP/0755/03 adopted April 2004). In particular the strategy proposed for hair dyes, which is based on their opinion of June 2003, is of particular concern as this differs from the COM recommended strategy both in the number of in-vitro tests being proposed, and the importance placed in the SHE cell transformation assay. The Committee were asked whether they were aware of any data that would warrant re-consideration of their current views that the SHE cell transformation assay should not be used for regulatory screening of chemicals for potential carcinogenicity, as outlined in their statement of April 2002.
29. The Committee agreed that the SCCNFP's general recommendations for in-vitro screening of cosmetic ingredients, based on an in-vitro gene mutation test in mammalian cells and an in-vitro micronucleus test were reasonable. However, the proposals for testing hair dye ingredients based on the SCCNFP's June 2003 recommendations, gave rise to concern. The requirement for both a metaphase analysis for clastogenicity and an in-vitro micronucleus test introduced unnecessary redundancy; the Committee has always felt that, as regards the clastogenicity end-point, these 2 assays should give equivalent data. Furthermore the Committee felt that there was little to be gained by the additional in-vitro assay for DNA damage.
30. Regarding the SHE Cell Transformation Assay, members were aware that considerable work was being carried out on the development of this assay, much of which would be discussed at a meeting of the EMS later in the year. It was agreed that the COM should consider these data when they are published, with a view to revising their statement on this assay if necessary.
ITEM 9: TOXICOGENOMICS: UPDATE ON LITERATURE (MUT/04/11)
31. The Committee had reviewed the available published literature and initial results from the ILSI/HESI trial on the utility of using toxicogenomic methods to screen for mutagens at the 5 February 2004 meeting. The majority of in-vitro data reviewed at the 5 February 2004 meeting concerned studies using mouse lymphoma L5178Y tk+/- cells. Members had not been able to derive any definite conclusions regarding these studies. There were concerns regarding reproducibility of the cDNA microarray experiments undertaken in these trials and the interpretation of the gene expression data.
32. The current paper presented to COM (MUT/04/11 and addendum) concerned more recent published in-vitro work with data presented on the use of Hep2G cells in a screening evaluation (Van Delft et al, accepted for publication in Carcinogenesis). This study had used 20 animal carcinogens (11 genotoxic carcinogens, 9 non-genotoxic carcinogens) in a series of 16 learning set and 6 validation experiments. Members expressed concern regarding the apparent variation in toxicity reported for dibenz(a,h)anthracene in the data from two trials which had been reported. The Committee agreed that the authors had been able to distinguish between genotoxic and non-genotoxic carcinogens but only when a number of genotoxic compounds (predominantly methylating agents) were excluded. Overall these experiments provided useful preliminary data but there was a need for considerable additional research before a satisfactory approach to screening for mutagenicity could be developed. This would involve consideration of multiple dose levels and sampling time points as well as additional work on methods, statistics and information from collaborative trials.
33. Members considered the accepted manuscript from Professor Parry and colleagues from the Centre for Molecular Genetics and Toxicology, University of Swansea. The approach adopted compared gene expression (using cDNA microarray analysis of a targeted set of 96 human genes) of RNA extracted from human lymphoblastoid line AHH-1 cells treated with clastogenic agents which were active either at low cytotoxicity (4-nitroquinoline-N-oxide) or high cytotoxicity (8-hydroxyquinoline). Members thought the approach provided useful preliminary information on an approach, which could be used to aid in the evaluation of cytogenetic assays. The evidence suggested that clastogenic effects reported under conditions where there was high cytotoxicity showed an increased expression of heat shock proteins. Further results to substantiate these findings would be valuable with regard to developing an aid for the interpretation of results of cytogenetics assays at levels associated with appreciable cytotoxicity.
34. Members reviewed some recent in-vitro studies of mutagens using proteomic analysis, but agreed that the data were too preliminary to draw any conclusions regarding patterns of protein expression.
35. Members were informed that Dr David Lovell had agreed to make a presentation to the 7 September 2004 COT meeting on statistics and bioinformatics in toxicogenomics. It was hoped that following this presentation, a joint statement would be prepared for COT/COC/COM to update the statement of 2001. This would be provided in draft form for the October COM meeting.
36. Members noted the in-confidence paper on in-silico p53 mutation hotspots in lung cancer which had been provided for information.
ITEM 11: ANY OTHER BUSINESS
37. There were no other items of business.
ITEM 12: DATE OF NEXT MEETING38. Thursday, 7 October 2004.
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ITEM 4: MUTAGENICITY OF CHROMIUM PICOLINATE (MUT/04/16)
39. Members heard that the manufacturing company for chromium picolinate was Nutrition 21 based in the USA. Chromium picolinate is a widely available food supplement in the UK. No interests were declared.
40. The adverse effects of chromium were recently reviewed by the Expert Group on Vitamins and Minerals (EVM). The reports of genotoxicity associated with chromium picolinate were noted by EVM and chromium picolinate was excluded from their recommendations for a safe upper level for this dietary supplement. The Food Standards Agency had asked the COM to review the available information on chromium picolinate in order to provide advice on mutagenicity and if appropriate to recommend what further mutagenicity studies, if any, would be required to draw definite conclusions on mutagenicity.
41. The COM had reached conclusions on the available published mutagenicity data at the October 2003 meeting. The Committee considered that the in vitro studies had given some indication of mutagenic activity and recommended that the critical tests should be repeated using commercial grade material (the previously tested material had been synthesised by the testing laboratory) before any definite conclusions could be drawn. Thus a further in vitro hprt assay and an in vitro chromosome aberration study were recommended with chromium picolinate (both tests using CHO cells and to international standards) to determine whether the published results could be confirmed with a commercial grade sample. It was also recommended that the hprt assay should include a 48-hour incubation experiment in the absence of S-9 in addition to the standard time points of analysis. The Committee had commented on the draft text of a statement at the February 2004 COM meeting where it had been agreed to await the results of the further in vitro tests before finalising the statement.
42. The Chairman asked members to comment on the test material used in the tests and then on the individual reports. He noted that members had previously been concerned that the material used in the in-vitro tests giving positive results had been synthesised by the testing laboratory and there were uncertainties regarding the level and nature of impurities in the test materials used. Members confirmed that they were content with the technical specification information provided to the contract laboratory.
43. The COM reviewed the new in-vitro chromosomal aberration study using CHO cells. Members noted that the highest dose level used (770 µg/ml) was determined by the solubility of chromium picolinate using DMSO as a solvent in the treatment medium. Evidence of a visible precipitate had been reported in the treatment medium at this dose level. Dose levels 231 µg/ml were reported to be soluble in the test medium. The two other dose levels chosen for the cytogenetics analysis were 192.5 µg/ml and 385 µg/ml. No evidence of a precipitate was reported at 385 µg/ml. Members were aware that evidence for cytotoxicity (based on relative cloning efficiency) had been documented at concentrations above 150 µg/ml in the hprt assay in CHO cells using a 5 hour exposure period but no evidence of cytotoxicity was documented in the chromosomal aberration assay in CHO cells at either exposure period based on cell growth inhibition. The available data did not allow members to evaluate whether the dose levels in the chromosomal aberration study might have given rise to cytotoxicity as well as growth inhibition. Members were aware that chromium picolinate was poorly soluble in aqueous media and that the report of the dosing solution analysis was not available. Members asked to see this report to evaluate whether there was a satisfactory agreement between nominal and actual test concentrations.
44. Members observed that the number of cultures reported to show no evidence of aberrations was higher than they would have expected. It was also noted that the positive control response was towards the lower end of the historical control range. Members also commented that the historical positive control range both in the absence and presence of exogenous metabolic activation was unexpectedly wide (based on 2000/2 data for structural aberrations was 7.5-87.0% in non activated and 8.0-84.0% in S-9 activated tests). The Committee considered that these data were of limited value in assessing the adequacy of the test and requested to see data pertaining to 2003/4. Members were particularly interested to see historical positive control data for chromosomal/chromatid gaps (not shown in the reported historical data) as there was some evidence for an increase in the frequency of gaps in chromium picolinate treated CHO cells, particularly after the 20 hour continuous exposure.
45. Overall members considered that although the conduct of the study conformed to international guidelines and had given negative results, the quality of the data gave some cause for concern. The Committee considered that these data indicated a relatively insensitive assay for which the power of the statistical analysis would be low. Provision of the additional information requested would assist the Committee in reaching a definite conclusion.
46. The COM discussed the new hprt assay in CHO cells. Two reports had been provided. The first test reported on exposure of CHO cells for 5 hours both in the absence and presence of exogenous metabolic activation and the conduct conformed to internationally accepted guidelines. Dosing solution analyses were not provided. The second test report concerned the additional 48 hour exposure study requested by COM, where the elongated exposure time was not specifically covered by international test guidelines. Members noted that the highest dose level tested (500 µg/ml in DMSO) had been shown to give rise to evidence of cytotoxicity in a preliminary study – after 5 hours to this dose level, the relative cloning efficiency (RCE) in the absence or presence of exogenous metabolic activation was 50% and 60%, respectively. There was less evidence for cytotoxicity in the main experiment using 5 hour exposure (eg in the independent repeat trial, the RCE was 89 % in absence and 74% in the presence of exogenous metabolic activation). Members queried the setting of the positive response level at 40 mutants/ clonable 106 cells and felt that this might miss a number of mutagenic test materials. Members considered it more appropriate to use the general criteria established in the relevant OCED guideline (476) for evaluating the results of such tests.
47. Members felt there was evidence for excessive variance between the plates. Collectively, the data at the highest dose level (500 µg/ml) in the 5 hour exposure studies indicated the possibility of a weak positive response . However, there was no evidence for a positive trend in dose response. The data from the 48 hour exposure study were negative. The overall conclusion was that there was no clear evidence for a mutagenic effect of the test material in this assay.
48. Members compared the data from these studies with the results reported by Stearns DM et al (Mutation research, volume 513, 135-142, 2002). Members agreed the data from the new studies were markedly different and suggested that chromium picolinate was not mutagenic in-vitro. The Committee discussed the need for any further tests and were aware that the NTP carcinogenicity bioassay was ongoing. Members were informed that the in-life phase would be completed during July 2004 and thus a report would not be expected until some time thereafter. Members felt that the submitted studies were negative, although a number of queries were raised. The answers to these questions should allow the Committee to reach a definite conclusion on the submitted tests.
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ACTIONS
| Item | Action | Responsibility |
| 3.1 Openness | Publish proforma on COM internet site |
Secretariat |
| 4. Chromium picolinate | Seek further information on in-vitro tests | FSA |
| 5. Malachite Green Leucomalachite green | Forward advice to COC | Secretariat |
| 7. DNA repair at low doses of carcinogens | Forward advice to COC | Secretariat |
| 8. SCCNFP testing strategy for hair dyes |
Forward advice to DTI | Secretariat |
| 9. Toxicogenomics |
Advise COM members on attendance of COT presentation. Begin to draft statement |
Secretariat |