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MINUTESPresent:
CONTENT
1. The Chairman informed members that item 4 on proquinazid was a referral from PSD under the Plant Protection Directive 91/414/EC and will be held in closed session. The data holder DuPont was attending to answer questions. The company had requested time to make a presentation. Alison Craig from the Pesticides Action Network was attending as an observer for the review of biomonitoring studies of genotoxicity in pesticide applicators item 5. The Chairman welcomed Dr Nissanka Rajapakse (from the Food Standards Agency) who was to introduce the item on PFOS/PFOA (item 6) and Dr Karin Fletcher (DH Tox Unit is attending to introduce items 3.1 and 5 and Dr Sarah Bull from the Health Protection Agency who had drafted appended papers to MUT/05/12 on item 5. The Chairman welcomed Dr R Shillaker who was replacing Dr D Andrew (PSD) for this meeting as PSD assessor and Dr P Edwards (DH). 2. Apologies for absence were received from Dr B Burlinson, Mrs R Glazebrook, Dr N Gooderham and Professor D Phillips (members), and the assessors Mr R Alexander (NAW), 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 THE MEETING ON 10 FEBRUARY 2005 (MUT/MIN/2005/1) 4. The minutes were approved subject to correction of minor amendments. ITEM 3: MATTERS ARISING NOT COVERED BY LATER AGENDA ITEMS
5. The Committee discussed the need for reference to an in-vitro UDS study as a precursor to undertaking an in-vivo rat liver UDS assay. Members agreed that an in-vitro UDS assay might be a useful pre-screen. Negative results would not obviate the need for a second in-vivo assay. Members asked for an appropriate amendment to be made to the statement. Members also asked for clarification of the term carbocation formation. The committee agreed the suggested strategy for testing HNMs. The finalised statement would be forwarded to the EPA for information.
6. Members agreed the draft working document and the proposal from the secretariat for further consideration of the OECD guidance document at a future meeting. ITEM 4: MUTAGENICITY DATA ON PROQUINAZID (MUT/05/12) 7. Proquinazid is a novel fungicide being considered by the ACP under the plant protection Directive (91/414/EC) The data holder is DuPont Chemicals. The Chairman asked for any declarations of interest. There were no declarations made by members. The Chairman noted that a post-doctorial researcher in his unit at the University of Leicester was the principal investigator for two pieces of research funding, one from the American Chemistry Council and one from CEFIC. Both of these organisations get their money from a large number of organisations world wide including industrial companies. DuPont is a member of both of these organisations. Thus the interest was nonpersonal and nonspecific, and very distant. (The research in Leicester that is funded by ACC and CEFIC is on endogenous alkylation, and on low level ethylene oxide DNA alkylation and mutagenicity respectively. It is not related to any pesticide or proquinazid or DuPont in particular. The Chairman noted that these interests had been declared as non-personal interests in the Committee declarations. Members agreed with the chairman that a deputy should chair this item and that the Chairman could contribute to the discussions http://www.advisorybodies.doh.gov.uk/pdfs/interestsm.pdf). 8. Members heard that proquinazid (6-iodo-2-propoxy-3-propyl-3H-quinazoli-4-one) is a new fungicide intended for use in agriculture and viticulture providing control of powdery mildew in cereals and grapes (Blumeria graminis. Class Ascomycetes) It is a novel class of fungicide acting by inhibiting the development of the appressorial germ tube (which is responsible for penetration of the host) but the mechanism of action was unknown. The ACP deferred making a decision pending advice from the COC and COM with regard to the occurrence of cholangiocarcinoma in the rat carcinogenicity bioassay. The COC/COM have not been asked to review any other tumour reported in rodent carcinogenicity bioassays with proquinazid. 9. Members agreed there were no particular structural alerts with regard to proquinazid but noted that a possibility for generation of metabolites with a quinone type structure which might potentially alert for free radical generation. Members discussed the mutagenicity test data and identified a number of areas where it would be appropriate to seek further information from DuPont.
10. Members agreed that this study had been adequately conducted and no further information was required.
11. Members agreed that this study had been adequately conducted and no further information was required.
12. Members noted the information provided by Du Pont where it had been suggested that two sampling times helped to offset the need for a continuous exposure trial. Members noted the further comment that marked cytotoxicity of the test material would make it difficult to undertake a continuous exposure trial but agreed that it was not impossible. Members considered that additional estimates of cytotoxicity other than mitotic index would be desirable. However, it was common practice to use mitotic index in cytogenetic studies with human lymphocytes as anything else presented practical or interpretation difficulties. It was agreed that further information on these aspects should be sought from DuPont.
13. Members recalled their previous evaluation of hprt mutagenicity assays undertaken in CHO cells where it had been agreed that exposure of 107cells was required for an adequate assay. It was noted that less cells had been used in the hprt mutagenicity assay undertaken with proquinazid in CHO cells. Members noted that there was considerable inter-trial variation in the recorded mutation frequency and the use of acetone as the solvent is an added complication. This might reflect some of the difficulties in undertaking and evaluating this particular assay. The laboratory criteria for a positive response of a mutation frequency of >40 x 106 was noted but members agreed that the data presented could not be interpreted and no conclusion could be reached. Members confirmed their view that the mouse lymphoma assay was preferable since higher cell numbers could be treated and the higher background mutation frequency at the tk locus than compared to the hprt locus made it easier to undertake reproducible adequate studies. It was agreed that further information on these aspects should be sought from DuPont.
14. Members noted that cytotoxicity had been determined using release of lactate dehydrogenase and microscopic examination, although exclusion of trypan blue uptake and microscopical examination were more usual. Members agreed to seek further information from DuPont.
15. Members agreed that the data provided were indicative of negative findings in these assays. Members agreed the evidence for small increases in percentage miconucleated polychromatic erythrocytes seen at some doses most probably represented fluctuation in the background incidence of micronuclei. Members agreed that further information should be sought from DuPont with regard to exposure of the bone marrow in these studies. 16. The Committee held a short preliminary discussion on what further mutagenicity studies should be undertaken. Members were of the view that a mouse lymphoma assay undertaken to internationally accepted standards and a continuous exposure part of a mammalian cell cytogenetics assay were required. These two assays would extend the existing data set to a satisfactory level given that evidence for cholangiocarcinoma had been documented in the rat carcinogenicity study. Members heard an overview of the mutagenicity test requirements under 91/414/EEC which were broadly similar to the COM guidance.
17. The representatives from Du Pont (Dr Karen Bentley, Mr G Stuart) were welcomed by the chairman for this item. The secretariat indicated where COM members, the secretariat and assessors from Government Departments were sitting in the room. The DuPont representative indicated that they wished to make a presentation and then take questions from COM members. (Overheads were made available to members. One additional overhead was included in the presentation but was not available for distribution to COM members.) 18. Dr Bentley (Global Regulatory Toxicology, Du Pont Crop Protection) told members that the purpose of the presentation was to provide DuPont's rationale on the adequacy of the genetic toxicology data base for proquinazid and to specifically discuss the results of the mammalian cell in-vitro chromosome aberration assay in human lymphocytes and the in-vitro mammalian cell gene hprt mutation assay in CHO cells. DuPont wanted to seek the committee's agreement that proquinazid is not genotoxic and further testing is unnecessary. 19. Dr Bentley provided a short overview of the structure and known information on fungicidal activity of proquinazid. DuPont was seeking to market formulations containing proquinazid for application to cereals (UK) and cereals and grapes (EU). The UK Pesticide Safety Directorate was acting as the EU rapporteur for consideration of proquinazid. A dossier of information had been provided to PSD in November 2004. DuPont was aware of minor limitations in the in-vitro chromosomal assay and in-vitro gene mutation assay in mammalian cells. Despite these limitations and in the absence of structural alerts for DNA reactivity, PSD had concluded that proquinazid does not cause genetic damage. However the ACP had recommended at its 13 January 2005 meeting that a further mouse lymphoma assay might be warranted. 20. A battery of seven genotoxicity studies had been conducted with technical grade material during the period 1996-1998 (in-life dates of studies). The company stated that the studies complied with the EU directive 91/414/EEC, the COM guidance of 1989, and OECD test guidelines (current at the time of testing). Two batches of technical material were evaluated (which had been produced by different manufacturing methods, the earlier batch DPX-KQ926-45 was 97% pure and the latter batch DPX-KQ926-75 was 97.9% pure). Mutagenicity tests undertaken with the 45 batch included the Ames test, the in-vitro chromosomal mutation assay, the in-vitro gene mutation assay in CHO cells, the in-vitro UDS assay using primary rat hepatocytes, an in-vivo bone-marrow MN assay. Mutagenicity tests undertaken with the 75 batch included an Ames test and an in-vivo bone marrow MN assay. 21. Dr Bentley reviewed the design of and results obtained in the in-vitro chromosomal mutation assay in human lymphocytes. She noted the negative results and adequate performance of the positive control trials. Some additional historical positive control data was presented (additional slide not in information tabled for members). COM members asked for further information on these additional positive control data The information was intended to show the lower limit of positive control responses in the test facility both before and after the in-life phase of the in-vitro chromosomal aberration assay. The data were consistent with those already provided for members data and were generated from a number of tests with cyclophosphamide and MMC. PSD had commented to DuPont that the study complied with the 1983 OECD test guideline (experimental phase was in 1996) but had noted the lack of continuous exposure and the marked cytotoxicity in the repeat assay. PSD had noted that the limitation was offset by marked toxicity seen at all dose levels at the 24 h harvest in the repeat assay. DuPont had noted that the study complied an earlier proposed revision of the OECD guideline and included a delayed harvest and that marked cytotoxicity was seen at the 24 h and 48 h harvests. In addition the consistent negative findings in this assay were supported by negative findings in two in-vivo bone marrow MN assays in mice where dose levels up to 2g/kg po had been tested with the occurrence of clinical signs of toxicity/mortality in mice and evidence of bone marrow exposure in the rat metabolism study. 22. Dr Bentley reviewed the design of and results obtained in the in-vitro mammalian cell gene mutation assay (CHO/hprt). She noted that two independent assays had been conducted for each treatment and that a third trial had been performed with activation due to a lack of toxicity in the second trial. The study was a qualitative assessment of mutagenic potential. A positive response was obtained if the mutation frequency was > 40 x 106 cells at two or more consecutive concentrations. Plating of 106 cells was sufficient to detect a positive response. Dr Bentley reported that there was no evidence for a positive response in the assay. Mutation frequencies obtained with the test material were within the negative historical control range and there was a lack of reproducible dose-dependent increases. Positive controls produced marked increases in mutation frequency in all trials and it was noted that there was comparatively little variance in the positive control data between trials using the same treatment condition. DuPont had noted a comment from PSD that the low mutation frequencies in the negative control cultures of some assays suggested uncertainty regarding the sensitivity of the study. PSD had also commented that the clear positive control responses provided some reassurance. DuPont had responded that all negative control mutation frequencies were within the range of historical controls for the test laboratory, the positive controls exhibited consistent responses despite the fluctuation in negative control frequencies and the negative findings were supported by a lack of genotoxicity in the battery of other in-vitro and in-vivo genotoxicity studies. 23. Dr Bentley concluded that proquinazid does not pose a mutagenic concern. There were negative findings in a battery of seven in-vitro and in-vivo studies. There was no result to suggest a positive or weak positive response. Neither proquinazid, nor metabolites formed in rats, contain structural alerts for DNA reactivity. The studies were conducted to the prevailing guidelines at the time. The limitations noted by PSD with regard to the in-vitro chromosomal aberration assay and the mammalian cell gene mutation assay (CHO/hprt) do not impact on the overall genotoxic assessment of proquinazid. Thus further testing was unnecessary to conclude that proquinazid is not genotoxic.
24. The Chairman thanked Dr Bentley for her presentation and invited members to ask questions or make comments on the presentation. 25. Members agreed that some aspects of the in-vitro chromosome aberration assay in human lymphocytes had been conducted in excess of requirements but felt that a prolonged exposure assay was required so that cells were exposed over the whole cell cycle. Members did not consider that the test had adequately covered this aspect. Such a test would address in-vitro potential for aneugenicity as an end-point. Members observed that the dose selection had resulted in high levels of cytotoxicity and that the positive control response reported (towards the lower limit of a very wide range, ca 6-80% cells with aberrations) limited the value of this assay. The Committee agreed that it would be advisable to request a continuous 20 h exposure in a mammalian cell chromosomal aberration assay (in human lymphocytes) in the absence of exogenous metabolic activation. This would ensure that all stages of the cell cycle were exposed. It would be appropriate to include estimation of polyploidy as an indicator of potential for aneugenicity. The study should be conducted to internationally accepted standards. The Committee was aware of the need to adequately explain the mode of action of the cholangiocarcinomas reported in the long-term bioassay of proquinazid in rats and agreed that this information would help to complete the in-vitro mutagenicity evaluation of proquinazid which was an important part of the carcinogenicity risk assessment. 26. Dr Bentley felt that the limitations noted were offset by the negative results in two in-vivo bone marrow micronuclei tests with high dose levels. Members asked about the evidence for exposure of the bone-marrow in these latter two studies. Dr Bentley referred to the evidence of rapid and extensive oral absorption in the rat kinetic studies and the finding of similar acute toxicity signs in the rat and the mouse for proquinazid. 27. Members commented that the data from the in-vitro mammalian cell hprt gene mutation assay in CHO cells was consistent with the conclusions COM had previously published on practical difficulties in undertaking this assay.( http://www.advisorybodies.doh.gov.uk/com/mut033.htm) There was considerable inter-trial variance in mutation frequency in the proquinazid assay and the finding of very low mutation frequencies in some trials (<0.6 x 106 cells) limited the conclusions that could be drawn. Thus it was noted that the mutation frequency in the third trial in the acetone control and test material experiments was low at all dose levels. Members concluded that the data were not interpretable. The Committee agreed that a mouse lymphoma assay conducted to internationally accepted standards should be requested. The Committee was aware of the need to adequately explain the mode of action of the cholangiocarcinomas reported in the long-term bioassay of proquinazid in rats and agreed that this information was essential to complete the in-vitro mutagenicity evaluation of proquinazid which was an important part of the carcinogenicity risk assessment. 28. Dr Bentley considered that all the data were within the background range for this test laboratory and the results for the positive controls indicated a consistent response in each trial. 29. Members noted the potential for quinone formation in the metabolism studies with proquinazid in rats and asked if DuPont had considered this aspect. Dr Bentley indicated DuPont had undertaken an evaluation of potential toxicity of rat metabolites which included use of the TOPKAT and DEREK data bases which had only indicated potential for thyroid toxicity. She noted thyroid tumours had been reported in the rat long-term carcinogenicity bioassay. (It was noted that the ACP referral related to cholangiocarcinoma formation.) 30. Members queried the evaluation of cytotoxicity in the in-vitro UDS assay using rat hepatocytes. Dr Bentley considered the microscopic evaluation of slides for cell morphology and in particular cell degeneration was an important determinant of the dose levels selected for UDS evaluation. 31. There was a general discussion of the quality of mutagenicity data on proquinazid. The Chairman noted that as the committees had been asked to evaluate the potential mechanism of cholangiocarcinomas seen in the rat long-term carcinogenicity bioassay with proquinazid, there was a need to have a high level of confidence in the mutagenicity data on proquinazid. Whilst members agreed there was no convincing evidence for positive results in any of the tests, it was not possible to conclude that the in-vitro cytogenetic assay in human lymphocytes and the in-vitro hprt mammalian gene mutation assay in CHO cells were negative in view of the limitations. Members accepted that some reassurance was gained from the in-vivo bone marrow micronuclei assays in mice (although further information regarding acute toxicity of proquinazid in rats and mice was required to determine whether or not it was valid to extrapolate exposure data from rats to mice as evidence of exposure of mouse bone marrow to proquinazid and metabolites). Dr Bentley restated DuPont's view that the data base was sufficiently robust to draw an overall conclusion that proquinazid was non genotoxic. 32. The Chairman noted that COM advice would be passed to the COC. A joint COM/COC statement would be prepared. The statement would be made available for DuPont to comment on (20 working days) before forwarding to the ACP. Dr Bentley noted that DuPont were to make a presentation to the COC where they believed it was possible to explain the mechanism of the cholangiocarcinomas seen in rats. 33. The representatives from DuPont withdrew.
34. Members considered that the company had provided some relevant information and comments but had not been convinced that the in-vitro mutagenicity test package was adequate. Members felt that a key element of any proposal regarding mechanism of proquinazid induced cholangiocarcinoma in the rat would require a full evaluation of mutagenicity and hence it was important to complete the mutagenicity test package. Members asked for further information on acute toxicity in the rat and mouse with regard to the evaluation of tissue exposure in the mouse bone marrow micronucleus tests. 35. The Chairman summarised that the COM requested a mouse lymphoma assay
and agreed it would be advisable to undertake a continuous 20 h exposure
in a mammalian cell chromosomal aberration assay (in human lymphocytes)
in the absence of exogenous metabolic activation. Both studies should
be conducted to internationally accepted standards. ITEM 5: BIOMONITORING STUDIES OF GENOTOXICITY IN PESTICIDE APPLICATORS (MUT/05/11 AND MUT 05/12. MUT 05/10 PREVIOUSLY DISTRIBUTED TO MEMBERS) 36. The Chairman welcomed Ms Alison Craig (PAN-UK) to the meeting as an observer. He asked members to consider the epidemiology overview paper first and then to consider the further information provided in response to the discussion held at the February 2005 COM meeting. 37. The secretariat informed members that the epidemiology overview of the studies selected by COM following the February 2005 meeting had been drafted by Dr Lesley Rushton (from the MRC Institute for Environment and Health and COT member specialising in epidemiology ). The secretariat had asked Dr Rushton whether the available biomonitoring studies could be ranked for quality. Dr Rushton had reported that all of the studies were limited in design, particularly with regard to study size, the assessment of selection and recruitment biases. Many of the studies provided information on demographics, medical history, lifestyle factors, potential occupational exposures to materials other than pesticides (eg solvents, radiation), and also information on type of pesticides used, duration and frequency of exposure and use of protective measures. However these data had generally not been used in the analyses reported and the majority of papers did not provide a specific analysis of individual pesticides. It was noted that the majority of studies were not sufficiently large enough to allow an evaluation of all the variables for which data might be available. Study designs were generally cross sectional, although a few had taken multiple samples (eg at different time points in a growing season). The time interval between exposure and sampling thus varied considerably between studies and this might affect the conclusions which could be drawn. There were limitations in the statistical approaches used in many of the studies. Thus for example many did not consider whether or not population distributions were normal. The reporting of modelling was variable and in most cases was not adequate. Dr Rushton noted that the papers tended to focus on statistical significance even when the absolute difference between groups was tiny. Overall it was not possible to identify any particular study that was clearly better in design and reporting than the other papers in the 24 studies identified by COM. 38. Members commented that the review helped to clarify the limitations in the data set under review and the need to draft conclusions carefully based on the weight of evidence available. 39. COM members noted the relatively small increase in indices of genotoxicity or mutagenicity in many of the studies considered at the October 2004 and February 2005 meetings and asked for an evaluation of control data and consideration of criteria for a positive response. An initial evaluation was presented in MUT/05/10 which was circulated to members. A more detailed evaluation was presented in MUT/05/12. Modelling of the data from the 24 selected studies suggested that MN data were normally distributed whilst there was evidence for a skewed distribution of CA as would be expected. Members felt the available data suggested that the distribution of micronuclei and chromosomal aberrations in human peripheral blood lymphocytes might be either bimodal or possibly trimodal. It was considered that more data would help to resolve the actual distribution of these indices in peripheral blood lymphocytes. The large overall variation seen (approximately 16 fold for micronuclei and chromosomal aberrations) suggested that it was not possible to define a historical control range. Members agreed that statistical significance from adequately conducted studies in combination with magnitude of response represented the most appropriate approach to evaluating the results of studies. Members agreed that the distribution of data should be assessed prior to consideration of the most appropriate statistical approach to analysis and that the effect of confounding factors should be clearly evaluated. 40. Members were surprised at the small magnitude of response in the biomonitoring studies of nurses or patients exposed to cytostatic medicines. The mean fold increase in nurses (1.8, range 1.5-2.2) and in patients (mean 2.1, range 1.5-2.7) derived from studies for either micronuclei or chromosomal aberrations was similar to that reported for pesticide applicators in the studies reviewed by COM (1.7, range 0.8-5). Members noted that the higher maximum fold increase in pesticide applicators compared to nurses or patients exposed to cytostatic medicines most likely reflected differences in the extent of control for confounding factors between studies. The Committee agreed that this analysis provided valuable information on the outcome of biomonitoring studies for genotoxicity but that it was not possible to draw a definite conclusion on a minimum fold increase in genotoxicity indices associated with exposure to genotoxic compounds. 41. The committee noted the limited information on exposure. The only direct exposure measurements were reported in the study by Garry et al (Environmental Health Perspectives, vol 109, 495-500, 2001) for exposure to 2,4-dichlorophenoxyacetic acid. Information had been provided on the identity of pesticides which applicators had used in 14 (11 reporting positive results and 3 reporting negative results) out of the 24 studies reviewed. This information had been reviewed in the context of information from the Pesticide Usage Survey regarding information on use over the period 1993-2002 and also with regard to the available information on classification status under Directive EC/67/548 with regard to mutagenicity provided by HSE. [cf post meeting note diuron is not classified as a category 3 mutagen.] Members noted that the magnitude of exposure in these studies had not been recorded in these studies and application of personal protective clothing had not been reported in most reports. It was also uncertain to what extent the application practices cited in the published reports were relevant to UK agricultural practice. 42. The reported use of pyrethroids, thiocarbamates and benzimidazoles was higher in the positive studies compared to the negatives studies. At least one pyrethroid and/or a benzimidazole was cited in 73% of the positive studies compared to 33% of the negative studies and at least one thiocarbamate was cited in 36% of the positive studies whereas none were used in the negative studies. Using information from the Pesticide Usage Survey, the amount of metam sodium and carbendazim increased over part of the period 1993-2001 in floricultural and green house applications. The area of outdoor bulbs and flowers sprayed with carbendazim was reported to increase. Increases in the use of bifenthrin, metam sodium and thiram were reported in agricultural practices. The Committee noted that with regard to pesticide active ingredients that were currently approved for use in the UK and which were also classified with regard to mutagenicity under EC/67/548 , carbendazim and thiophanate-methyl were used in 5/11, and 1/11 positive studies respectively but not in the negative studies. This represented a very small and incomplete amount of information and there were a number of other classified mutagens listed in the positive studies not approved for use in the UK. 43. The Committee were informed of written comments from a member who had been unable to attend. It was suggested that the evidence supported the conclusions that a UK based study should be undertaken and that it was important that the design of the study took into account the limitations noted in the currently available published studies. Members discussed whether any proposed study should focus on an occupational group (such as floriculture) or on specific pesticides. Members acknowledged that the evidence was limited particularly with regard to the design, conduct, reporting and analysis of the available studies both with respect to identifying either an occupational category or specific pesticides. One member noted that a limitation in the available published literature concerned relevant information on the mutagenicity of mixtures of pesticides. The secretariat referred members to the COT report on mixtures of pesticides (the WIGRAMP report http://www.food.gov.uk/science/ouradvisors/toxicity/COTwg/wigramp/). The COT working group had identified benzimidazoles as a possible common mechanism group of compounds for further evaluation. A draft report on benzimidazoles was currently under consideration. HSE made the observation that occupational exposure to chemicals that were genotoxic (ie classified as mutagens) needed to be well controlled. 44. The Chairman thanked members for their contributions and asked for brief comments on the concluding paragraphs of MUT/05/12. Members agreed that consideration of statistical significance and magnitude of effects from adequately conducted studies was the most appropriate approach to evaluating the available data. Members confirmed that the available information was severely limited and hence no definite conclusions could be drawn with regard to UK agricultural applications of pesticides or to individual pesticide active ingredients. Members agreed that there was limited evidence to suggest that an appropriate study of floriculturalists using carbendazim might represent a reasonable first hypothesis to examine whether genotoxicity could be identified in a biomonitoring study of pesticide applicators in the UK. The Committee was aware that a threshold approach to the risk assessment of carbendazim mutagenicity had been agreed by the COM in 1996. Such a study would also aid in the evaluation of the adequacy of personal protective clothing and the overall risk assessment for carbendazim. 45. The Chairman asked the secretariat to prepare a draft working paper for consideration by COM at the October 2005 meeting. A short interim update paper would be produced for the Medical and Toxicology Panel of the ACP for its July 2005 meeting. 46. The Chairman asked the observer if she had any comments to make.
Ms Craig was concerned that undertaking a study in the UK would delay
any appropriate regulatory activity and agreed with the comment from the
HSE assessor. She queried declarations of interest in respect of involvement
in the pesticides industry in particular and having a general "vested"
interest in further scientific investigations. Members stated that declarations
of interest were closely monitored. Appropriate information is on the
COM internet site. (http://www.advisorybodies.doh.gov.uk/pdfs/interestsm.pdf)
ITEM 6: MUTAGENICITY OF PERFLUOROOCTANE SULPHONATE (PFOS) AND PERFLUOROOCTANOIC ACID (PFOA) (MUT/05/14) 47. Dr Clare and Dr Clements declared a non-specific, non-personal interests.
The Chairman agreed that they could take part in the discussion. These
chemicals were formerly manufactured and distributed by 3M who ceased
production in 2002. A number of other companies (the Fluoropolymers Manufacturers
Group and other unspecified manufacturers) are continuing to supply these
materials. They are used in a widespread number of industrial applications
(such as in electronics, plastics, textiles and consumer materials) 49. The Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment (COT) recently concluded that the available toxicology data indicate that PFOS and PFOA do not demonstrate sufficient similarities to be assessed jointly. COT Members also recommended that the COM and COC be consulted for advice on the mutagenicity and carcinogenicity of PFOS and PFOA. 50. These compounds are multifluorinated chemicals based on an eight carbon linear molecule (some branched chain and lower alkane fluorinated carbon impurities may be present at low levels). PFOS and PFOA had unusual physico-chemical properties forming microdispersion micelles in aqueous systems. There were no apparent structural alerts for mutagenicity and the evidence from animal studies is that absorbed material is not metabolised. However there is evidence for target organ toxicity in repeat dose studies with both PFOS and PFOA in rats and monkeys and evidence for adverse effects on reproduction and the induction of endocrine target organ tumours with PFOS and PFOA and liver tumours in rats with PFOS. The mechanisms for these toxicological effects had yet to be evaluated by COC and COT. 51. Members discussed the mutagenicity data on PFOS and agreed that the physico-chemical properties of PFOS considerably complicated the formulation of appropriate test solutions for mutagenicity studies. The use of DMSO as a solvent using dose levels up to those which induced cytotoxicity or precipitation was agreed as acceptable. 52. Members agreed that the in-vitro plate incorporation test using five strains of Salmonella typhimurium and the D4 strain of Saccharomyces cerevisiae gave negative results. Members agreed that the reverse mutation assay using Escherichia coli gave negative results. The difficulty in formulating an adequate suspension of the test material was noted for the in-vitro chromosomal aberration assay in human lymphocytes. Members agreed that this study had yielded negative results. Members agreed that the in-vitro UDS assay in rat liver primary hepatocytes gave negative results. Members commented on the difficulty in adequately formulating PFOS for oral dosing of mice in the bone-marrow micronucleus test. Members noted that only 1000 micronuclei had been evaluated at each dose level. However overall the study was considered to be acceptable and provided negative results. 53. The Committee agreed that the studies undertaken with PFOS were acceptable and that PFOS should be regarded as not mutagenic. 54. The Committee discussed the data on PFOA. It was noted that mutagenicity assays had been conducted using aqueous solutions of sodium or ammonium perfluorooctanoate. 55. Members agreed that the plate incorporation bacterial mutagenicity tests using strains of Salmonella typhimurium and Escherichia coli using sodium or ammonium perfluorooctanoate were adequately undertaken and gave negative results. The Committee noted that the in-vitro hprt assay in CHO cells using ammonium perfluorooctanoate had reported negative results. 56. Members considered that a mutagenic response had been documented in the in-vitro chromosomal aberration assay of sodium perfluorooctanoate in CHO cells in the presence of exogenous metabolic activation at the two highest concentrations. This finding had been reproduced in a confirmatory assay in the presence of exogenous metabolic activation at the highest concentration tested. No evidence for increased chromosome aberrations had been documented in the absence of exogenous metabolic activation. However it was unclear as to what extent the results reported from this in vitro study were due to cytotoxicity. An increase in chromosome aberrations was also documented in an assay using ammonium perfluorooctanoate at the highest dose tested in the presence of exogenous metabolic activation. There was clear evidence of cytotoxicity at this dose level in this particular assay. It was agreed that sodium perfluorooctanoate, in the absence and presence of metabolic activation, had not induced chromosomal aberrations in cultured human whole blood lymphocytes when tested up to doses that were cytotoxic. Members commented that the available information indicated that absorbed PFOA was not metabolised by mammals. 57. No evidence for a mutagenic effect was found in a mouse bone marrow micronucleus assay where mice had been given a single oral gavage dose of up to 5000 mg/kg bw sodium perfluorooctanoate or 1990 mg/kg bw ammonium perfluorooctanoate. The test materials had been solubilised in deionised water. There was clear evidence of toxicity (mortality of both males and females) and reduced PCE/NCE ratios in animals dosed with 5000 mg/kg bw sodium perfluorooctanoate. A dose level of 1990 mg/kg bw ammonium perfluorooctanoate did result in one male death but did not have an effect on the PCE/NCE ratio. Members commented that the in-vivo bone marrow mouse micronucleus studies had been adequately conducted although there was no direct measure of exposure of the bone marrow to the test materials and also noted that absorbed PFOA was highly bound to proteins. 58. The Committee discussed what further testing might be appropriate
for PFOA salts and considered whether an investigation of in-vivo DNA
binding or further evaluation of the mechanism of in-vitro chromosomal
aberration assays were appropriate. Overall, the Committee concluded that
the results from the in-vitro chromosomal aberration assays with PFOA
salts were likely to represent a cytotoxic response. It was agreed that
a plausible in-vitro mechanism for the positive response in CHO cells
was required to reassure the Committee of this conclusion. ITEM 7: MUTAGENICITY DATA ON NANOMATERIALS (MUT/05/15) 59. This item was deferred to the October 2005 meeting. ITEM 8: ANY OTHER BUSINESS 60. There were no other items of business. ITEM 9: PAPERS FOR INFORMATION 61. MUT/05/18: COT/COC/COM overview of nanomaterial toxicology (deferred to the October 2005 meeting). ITEM 10: DATE OF NEXT MEETING 62. 13 October 2005
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