MINUTES - 11TH OCTOBER 2001
Present:
| Chairman: Professor J M Parry (up to item 6) Professor P Farmer (from item 6) |
Members: Professor J Ashby Dr J Clements Professor C Cooper Dr N Gooderham Ms M Langley Dr I Mitchell Professor D Phillips Professor D Tweats |
Secretariat: Dr R J Fielder (Scientific DH) Dr D Benford (Scientific FSA) Mr J Battershill (Scientific DH) Mr S Robjohns (Minutes) Mr K N Mistry (Administrative) |
| Assessors: Mr A Browning (VMD) Dr D Shillaker (PSD) Dr C Cowles (HSE) |
In attendance : Mr B Groves (FSA, items 7 & 8) Dr J Shavila (FSA, item 6) Dr D Renshaw (FSA, items 7 & 8) |
CONTENTS
| Item | Paragraph |
|
1. |
Announcements | 1 |
2. |
Apologies for absence | 4 |
3. |
Minutes of the extraordinary meeting of 23 July 2001 | 5 |
| 4. | Matters arising: |
6-8 |
4.1. Dichlorvos |
||
5. |
SHE Cell Transformation Assay: Results from ILSI/HESI Research Programme (MUT/01/26) | 9 |
6. |
Terephthalic acid (MUT/01/25) | 10-14 |
7. |
Dimetridazole (MUT/01/27) | 15 |
8. |
Flunixin meglumine (MUT/01/28) | 16 |
9. |
Any other business | 17 |
10. |
Date of next meeting | 18 |
ITEM 1: ANNOUNCEMENTS
Announcements regarding new Chair
1. Members were informed that this was the last COM meeting to be chaired by Professor Parry. The Committee thanked Professor Parry for his contribution and leadership of the COM over many years.
2. The COM welcomed its new Chair Professor Farmer who would take over the role as Chair at item 6.
3. Members were reminded of the need to declare any interests before discussion of items.
ITEM 2: APOLOGIES FOR ABSENCE
4. An apology for absence was received from Dr D Jones (MCA).
ITEM 3: MINUTES OF THE EXTRAORDINARY MEETING HELD ON 23 JULY 2001 (MUT/MIN/01/3)
5. The minutes were approved with no amendments.
ITEM 4: MATTERS ARISING
4.1 Dichlorvos
6. Members were informed that the COM statement had not been published shortly after the 23 July 2001 meeting. One pesticide approval holder had obtained an injunction preventing publication. A preliminary hearing regarding whether a judicial review would take place was to be heard on the 25 October 2001.
4.2 Nitrosation of nicotine
7. This item was not discussed, as MCA were unable to attend this meeting.
4.3 P-53 mutations in tobacco induced carcinogenesis
8. Members were informed that a review of the paper by Rodin and Rodin (Proc Natl Acad Sci, US, 97 12244-12249, 2000) on the importance of p53 mutations in tobacco induced carcinogenesis (MUT/01/3) had been drafted. This review would be circulated to members before the next meeting.
ITEM 5: SHE CELL TRANSFORMATION ASSAY: RESULTS FROM ILSI/HESI RESEARCH PROGRAMME (MUT/01/26)
9. The minutes of this item now published as paragraphs 42 to 51.
ITEM 6. TEREPHTHALIC ACID (MUT/01/25)
10. No interests were declared.
11 The COT considered TPA last year and requested advice from the COM on the potential in vivo genotoxicity of this compound, which produced bladder tumours in a carcinogenicity bioassay in the rat. It was important to exclude a genotoxic mechanism.
12. Members noted that TPA contained no structural alerts for mutagenicity and was practically insoluble in water. In discussion, the Committee agreed that the non-genotoxic mechanism (formation of insoluble crystals in the bladder and subsequent chronic tissue damage) suggested that the induction of bladder tumours with this compound was plausible. The Committee commented on the poor quality of the in-vitro mutagenicity test data but accepted that the evidence from the studies using bacteria suggested that TPA was not mutagenic in a limited number of Salmonella typhimurium strains. The cytogenetics test in lung fibroblasts had not been conducted to internationally accepted standards, i.e. no short duration exposure had been used and no trials using exogenous metabolic activation had been undertaken. Members were aware that TPA could be used in a potentially large number of food contact applications and thus intakes could be higher than quoted in the COT statement (2000/08). The Committee agreed that an adequately conducted in-vitro cytogenetics test in mammalian cells was required to bring the in-vitro mutagenicity data on this compound up to an acceptable level.
13. Members reviewed the in-vivo micronucleus assay conducted with TPA in ICR mice. The test material had been given as a suspension in corn oil by the intraperitoneal route and there was no direct measurement of exposure to the bone marrow and no toxicokinetics data were available to assist. However, signs of toxicity reported suggested that the test material had been absorbed into the blood circulation and thus dose selection had been adequate. Members noted the relatively low spontaneous background incidence of micronuclei in ICR complicated the interpretation of the data. The Committee agreed that this study had given negative results but a decision on further in-vivo mutagenicity testing should await the outcome of the in-vitro cytogenetics testing requested by the Committee.
14. The Committee noted that TPA is used widely in food contact applications and agreed that the available in vitro mutagenicity data in bacterial test systems suggested that TPA is not mutagenic in a limited number of Salmonella typhimurium strains. It was also agreed that the available in-vivo micronucleus assay conducted with TPA had given negative results. The Committee recommended that an adequately conducted in-vitro cytogenetics test in mammalian cells is needed before any definite conclusions were reached that this compound did not have any significant mutagenic potential, and thus that the bladder tumours in the rat carcinogenicity bioassay arose from a non-genotoxic mechanism.
ITEM 7: DIMETRIDAZOLE (DMZ) (MUT/01/27)
15. The minutes now published as paragraphs 24 to 32 which were allocated in the earlier version of the minutes.
ITEM 8. FLUNIXIN MEGLUMINE MUT/01/28
16. The minutes of this item now published as paragraphs 33 to 41. Please also see statement COM/03/S2.
ITEM 9: ANY OTHER BUSINESS
17. Members were informed of a paper in Mutagenesis on the susceptibility of children's lymphocytes, which would be made available at the next meeting.
ITEM 10: DATE OF NEXT MEETING
18. Date of next meeting 7th February 2002.
Secretariat
February 2002
ACTIONS
| Item | Action | Responsibility |
| Item 5 SHE Assay | Draft statement and circulate to Members | Secretariat |
| Item 6 Terephthalic acid | Draft statement | Secretariat |
| Item 7 Dimetridazole | Draft statement and circulate to members | Secretariat |
| Item 8 Flunixin meglumine | Draft interim statement | Secretariat |
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The paragraph numbers 24 to 32 relate to the earlier version of the minutes and were added in October 2002.
ITEM 7: DIMETRIDAZOLE (DMZ) (MUT/01/27)
24. No interests were declared.
25. Dimetridazole (DMZ) is used in the UK both as a feed additive, for turkeys and guinea fowl, and as in veterinary medicine for game birds. There is controversy about its mutagenicity. The EU Committee on Veterinary Products (CVMP) had concerns regarding the mutagenicity, and felt that further data was needed. This was not forthcoming, and they have recommended that it should not be used in food producing animals. The UK has a derogation allowing use in pheasant and partridges. With regard to food additive use the EU's Scientific Committee on Animal Nutrition (SCAN) have concluded that there was no genotoxic hazard to consumers.
26. The Food Standards Agency had therefore sought advice from the COM. Members were told that it had not been possible to obtain full reports of all the relevant mutagenicity studies.
27. Members agreed that the nitro grouping is a structural alert for mutagenicity. DMZ was mutagenic in bacteria. Studies using nitroreductase proficient and deficient strains of Salmonella typhimurium TA100 showed that this pathway accounted for a proportion but not necessarily all of the mutagenicity seen in bacteria. Positive results were reported in Saccharomyces cerevisiae D4 demonstrating that DMZ was mutagenic in eukaryotic cells. Members noted that there was inconsistency in the interpretation of the in-vitro data for the mutation assays in mouse lymphoma assay. For the cytogenetics assay in CHO-K1 cells the data were reported as negative but were not traceable. In addition there were very limited details available regarding the in-vitro UDS assay in V79 Chinese hamster lung fibroblasts and no conclusions could be reached on the results of this test.
28. However, sufficient details were available to assess the genotoxicity of DMZ in human lymphocytes using the comet assay. It was established that DMZ was genotoxic under aerobic conditions in the absence of exogenous metabolic activation. The addition of Aroclor 1254 induced rat liver S-9 fraction abolished the genotoxicity of TPA in this test system. Using the same treatment concentration of DMS, the magnitude of the response (mean tail moment derived from 200 cells) was reduced under anaerobic conditions and abolished by the addition of anti-oxidants (8-hydroxyquinoline, vitamin C, catalase or superoxide dismutase). Thus, DMZ was genotoxic in the in-vitro comet assay in human lymphocytes and that the activity observed in this assay was consistent with oxidative DNA damage. The Committee agreed that DMZ was mutagenic in-vitro.
29. Negative results were reported in in-vivo micronucleus assays in the mouse using intraperitoneal and oral routes of administration and in in-vivo rat liver UDS assay but no conclusions could be reached by the Committee in view of the limited details of these tests that were available. Members noted that evidence for an increase in dominant lethal mutations had been reported in male CD-1 mice dosed with 2-hydroxymethyl-1-methyl-5-nitroimidazole (DMZ-OH, a metabolite of DMZ) three weeks after receiving oral doses of DMZ-OH for up to 5 days. (It was noted that DMZ-OH had also given positive results in Salmonella typhimurium.) The Committee agreed that definite conclusions could not be reached in the absence of full details of this study, but the available data were consistent with a positive result and thus it was important to follow-up the information provided in MUT/01/27 and to obtain further data from this study.
30. Members agreed that no weight could be given to the negative results reported in the sex-linked recessive lethal mutation assay in Drosophila melanogaster. The Committee agreed that the available long-term carcinogenicity bioassays in rats had not been conducted to contemporary standards. Benign mammary tumours had been documented in two of these studies and some evidence was available to support a hormonal mechanism for the induction of these tumours. However, the blood concentration of progesterone was raised in female rats but not in males, whereas benign mammary tumours had been produced in both sexes. Overall, no reassurance regarding absence of genotoxicity could be derived from the available carcinogenicity bioassays. The Committee therefore agreed that, in view of the absence of appropriate information from two adequately conducted in-vivo mutagenicity studies, it would be prudent to assume that DMZ had in-vivo mutagenic activity.
31. Members noted that veterinary use was limited to game birds and agreed that consumer exposure would most likely occur as a result of DMZ use as a feed additive for turkeys and guinea-fowl. Members confirmed that it was important to ensure that mutagenicity testing for feed additives was compliant with the COM guidance and asked the Food Standards Agency to raise this subject with the appropriate colleagues in Europe.
32. The Committee agreed that a statement should be drafted and agreed by postal circulation.
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ADDED PARAGRAPHS 33 TO 41 on 28 March 2003
ITEM 8. FLUNIXIN MEGLUMINE MUT/01/28
33. No interests were declared.
34. Flunixin Meglumine is a non-steroidal anti-inflammatory agent that is used in veterinary medicine including food-producing animals. The relevant EU Committee, CVMP, has concluded that flunixin is not genotoxic in vivo and they have not considered meglumine as it is regarded as an excipient. The Food Standards Agency had concerns at the possible genotoxicity of meglumine and flunixin meglumine and therefore requested advice from COM.
35. The Chairman asked Members to consider the available data on flunixin, meglumine and flunixin meglumins separately. As a general comment Members queried the relatively high level of micronuclei seen in all of the in-vivo bone-marrow micronuclei studies submitted on these chemicals which had all been undertaken in the early 1980s by one contract laboratory.
36. The available mutagenicity data on flunixin were relatively old and of limited value. Negative results were reported in Salmonella typhimurium and in an in-vivo micronucleus assay. The DNA damage assay in Escherichia coli p3478 repair deficient strain showed an increase in DNA damage at one interim concentration, but this finding was not seen in a repeat test. Overall, Members agreed that there was no evidence to suggest that flunixin had mutagenic potential.
37. Members agreed that the structure for meglumine in the papers presented to the Committee was incorrect and that this chemical contained a secondary amine. Negative results were reported in old plate incorporation assays in a limited number of Salmonella typhimurium strains. A positive result had been seen in a bone-marrow micronucleus assay in BSI mice using intraperitoneal administration of 500 or 1000 mg/kg bw meglumine suspended in 0.25% methylcellulose. A repeat test using 1000 mg/kg bw was also positive. Members queried how the repeat test could have been undertaken on the same day as the initial test. It was noted that negative results were obtained in a separate in-vivo micronucleus assay using intraperitoneal administration of up to 600 mg/kg bw of meglumine to CD1 mice. Members noted that a clear positive control response had only been seen in males and not females. Overall the data suggested that meglumine had in-vivo mutagenic potential but it would be necessary to see full reports from two adequately conducted in-vivo assays before any definite conclusions could be reached.
38. Members agreed that the mutagenicity data on flunixin meglumine (ie the mixture used in veterinary medicines) was difficult to assess. Negative results were reported in old plate incorporation assay in a limited number of Salmonella typhimurium strains. Negative results were also reported in an in-vitro UDS assay using rat hepatocytes. Positive results were documented in a mitotic gene conversion assay in Saccharomyces cerevisiae. There was also evidence for DNA damage in two separate tests in Escherichia coli p3478 repair deficient strain but not in a third test. However there was limited evidence for a mutagenic effect in three separate mouse lymphoma assays and evidence for an equivocal response in a cytogenetics test. Members noted that negative results had been documented in an in-vivo micronucleus assay in BRS1mice using intraperitoneal administration but there were concerns with regard to the adequacy of the selection of dose levels.
39. Members agreed that further consideration of the full reports of these assays was required before conclusions could be reached.
40. Members noted that although negative results were obtained in carcinogenicity bioassays in the rat and the mouse these were not done to an adequate standard and no conclusions can be drawn regarding the carcinogenicity of flunixin meglumine.
41. The Chairman asked the secretariat to draft an interim draft statement and to circulate the statement for consideration. He asked a number of members to consider the full reports of some of the mutagenicity studies and to provide evaluations for the February 2002 meeting. The Committee would consider conclusions at the next meeting.
ADDED PARAGRAPHS 42 TO 51 on 28 March 2003
ITEM 5: SHE CELL TRANSFORMATION ASSAY: RESULTS FROM ILSI/HESI RESEARCH PROGRAMME (MUT/01/26)
42. Dr Clements declared a non-personal interest and was permitted by the Chair to take part in the discussion.
43. At its last meeting the COC considered the results from the International Life Science Institute (ILSI) and the Health and Environmental Science Institute (HESI) programme on a number of alternative models to the chronic animal bioassay. The COC has asked the COM for its opinion on the use of the in-vitro Syrian Hamster Embryo (SHE) cell transformation assay for regulatory assessments.
44. The COM has previously considered this assay in 1994 and 1996. On both occasions the Committee felt that the assay could not be used for regulatory purposes and that further work was required on both validation and understanding the mechanism of action. Members were asked to consider suggested draft text for the COM contribution to the statement regarding the data from the ILSI/HESI programme on alternative cancer models.
45. The Committee considered a draft paper by Mauthe RJ and colleagues, which had been submitted to Toxicologic Pathology. The Committee had considerable reservations on several aspects of the ILSI study relating to the assay system, the study design, the data obtained and the data analyses. There is still no mechanistic basis to underpin the assay and the endpoint (transformed foci) remains subjective as there is a lack of a clear, objective definition of transformed foci. There were reservations about the selection and categorisation of test chemicals, concentration ranges were considered to be too narrow for reasonable analysis of dose-response and, crucially, there were no repeat tests. Control values were unsatisfactorily low (0 to 6) and in some tests there was a lack of consistency of result between closely spaced doses. Finally, the definition of positive and negative results relied on statistical analyses that seemed inappropriate to the data. Most importantly, there was no correction for multiple comparisons. Also methods had been used (Fisher exact and binomial-based) that assume uniformity of sampling circumstance (i.e. that all cells have an equal chance of transformation under fixed experimental conditions). This is unlikely to be true for the mixture of cell types derived from embryos. No tables of historical negative and positive control ranges (and/or confidence limits) were given to enable general variability, test validity and biological importance to be assessed. Some members of the committee considered that two-sided (-tailed) statistical analyses would be more appropriate as it is probable that compounds may have negative as well as positive effects on transformation . The Committee considered that the results of some of the tests reported as positive were most likely to be either equivocal or negative if the requirement in the criteria for an indication of a statistically positive dose response trend was used. Members also thought that the difficulties in objectively identifying transformed cells combined with the limited number of repeat trials suggested that interpretation of the ILSI/HESI data was particularly problematic.
46. The Committee noted that phenacetin was a genotoxic carcinogen in rodents and considered that a positive response should have been obtained in the SHE cell transformation assay, but noted that negative responses had been consistently obtained in the ILSI/HESI programme. Members considered that ampicillin had given a positive response at the highest dose tested (3000 g/ml, cf 13 transformed cells) whereas a dose level of 2450 g/ml had not yielded any transformed cells. Overall using the ILSI/HESI criteria, it was agreed that ampicillin had given an equivocal or negative response in the SHE cell transformation assay. There was a need for further testing with ampicillin before a definite conclusion could be reached.
47. Members agreed that difficulties in the morphological identification of transformed foci and obtaining appropriate interlaboratory agreement as result was a particular problem in performing the SHE assay between laboratories. The Committee agreed that there was little prospect of obtaining reproducible results with the SHE transformation assay as a screen for the identification of chemical carcinogens, without an objective molecular marker to identify cell transformanants. Members considered that there was a need to explain on a mechanistic basis why chemical carcinogens with disparate mechanisms of action should be expected to produce cell transformation in an embryonic cell line.
48. Members considered that whilst positive results could be obtained for benzo(a)pyrene indicating that SHE cells (and or its feeder layer) could activate carcinogens, there was very little information available to define the metabolic competency of SHE cells and the feeder layer used.
49. Members noted that there was a tendency to report positive results for the SHE cell transformation assay, is illustrated by the data set from the ILSI/HESI trial. However Members felt that no particular value could currently be ascribed to either positive or negative results obtained in the SHE cell transformation assay in the absence of an objective endpoint for the assay and an understanding of the mechanism underlying the process of cell transformation. Many of the chemical selected for this trial are rodent carcinogens that are not human carcinogens. There are considerations that this assay has poor specificity for detection of human carcinogens.
50. The COM concluded by reaffirming its previous opinion, that the SHE assay should not be used for the regulatory screening of chemicals for potential carcinogenicity. There were insufficient data on validation of the SHE cell transformation assay to justify the development of an OECD test guideline for this assay. The Committee had considerable reservations regarding the mechanistic basis underpinning the rationale for using the SHE cell transformation assay to screen for chemical carcinogenesis. The Committee had considerable reservations regarding the validity of using morphological assessment to define transformed foci in the SHE cell transformation assay . The development of an objective molecular marker is essential before validation work can proceed.
51. The Committee agreed that a statement should be drafted and agreed by postal circulation.