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MINUTES

Present:

Chairman: Professor P Farmer	Members: Dr C Allen Dr G Clare Dr J Clements Dr D Gatehouse Dr D Lovell Dr I Mitchell Dr E Parry Professor D Phillips	Secretariat: Mr J Battershill (HPA secretariat) Mr S Robjohns (HPA minutes) Dr D Benford (FSA secretariat) Dr D Mason (FSA secretariat) Mr K Mistry (DH Acting administrative)
Assessors: Dr S Dyer (DH item 4) Mr M Hosford (EA) Dr A Smith (HSE)	In attendance : Dr K Burnett (DH Tox Unit) Dr P Edwards (HPA) Ms Ehi Idahosa (DH Tox Unit) Dr E Zeiger (Consultant - item 4) Dr D Marroni (PPG - item 4) Dr M Friedman (PPG - item 4) Dr P Ungeheuer (PPG - item 4) (Polyelectrolyte Producers Group)	Observers: Alana Miller (National Institute for Clinical Excellence) Reetan Patel (NICE) Marijke Hermans (University of Sussex item 4)

CONTENT

Item		Paragraph
1.	Announcements/Apologies for absence	1
2.	Minutes of the meeting on 17 May 2007 (MUT/MIN/07/2)	5
3.	Matters Arising (not covered by later agenda items):	6
4.	Review of the Genotoxicity of Acrylamide:	8
	4. 1 Presentation by Dr E Zeiger {Consulting (Chapel Hill NC, USA)}	11
	4.2 Review of submission from PPG (MUT/07/16)	17
	4.3 EU Risk Assessment Summary and Overview of the COM Review strategy (MUT/07/17)	33
5.	Mutagenicity of mixtures. 2nd discussion paper. Evaluation of Interactions using mutagenicity tests (MUT/07/15)	38
6.	Toxicogenomics: Update on 2004 (MUT/07/18)	47
7.	Horizon scanning (MUT/07/19)	55
8.	Aneuploidy: Action both oncogenically and as a tumour Suppressor (MUT/07/20)	63
9.	Papers for information:	69
	9.1 Genotoxicity Test strategies. Pfuhler S et al Tox Sci, 97, 237 - 240, 2007 (MUT/07/21)
	9.2 Revised code of practice for observers (MUT/07/22)
10.	Any other business	70
11.	Date of next meeting: 14 February 2008	73

ITEM 1: ANNOUNCEMENTS/APOLOGIES FOR ABSENCE

1. The committee was informed that Professor Farmer had been re-appointed as the Chair of the COM for a third term from 1 November 2007 to 31 October 2011. He would also serve as an ex-officio member of the COC for the same period.

2. The Chair welcomed Dr K Burnett (DH Toxicology unit), Ms Ehi Idahosa (DH Tox unit), Dr P Edwards (HPA), Dr D Mason (FSA), Dr E Zeiger (Consultant, Chapel Hill NC, USA), Dr D Marroni (Polyelectrolyte Producers Group (PPG)), Dr M Friedman (PPG), Dr P Ungeheur (PPG), Alana Miller (NICE), Reetan Patel (NICE) and Marijke Hermans (University of Sussex item 4).

3. Apologies for absence were received from Dr B Burlinson (COM member), Dr B Elliott (COM member), Mrs R Glazebrook (COM member), Mr S Samuels (PSD), and Dr H Stemplewski (MHRA).

4. Members were reminded of the need to declare any interests before discussion of items.

ITEM 2: MINUTES OF MEETING ON 17th May (MUT/MIN/07/2)

5. Members agreed the minutes subject to some minor editorial changes.

ITEM 3: MATTERS ARISING (NOT COVERED BY LATER AGENDA ITEMS)

3.1 Formaldehyde

6. Members were informed that a COM statement on formaldehyde had previously been agreed (although not yet published on the internet). The COC had considered the COM statement and recent information from the IARC evaluation of formaldehyde. One COC member had a number of comments regarding the potential for a systemic threshold for the mutagenicity of formaldehyde due to a secondary mechanism (eg involving the release of bound formaldehyde and the capacity for rapid metabolism).

7. The chair agreed that the comments from the COC member could be forwarded to COM members for consideration of whether any clarification of their statement was required.

ITEM 4: REVIEW OF GENOTOXICITY OF ACRYLAMIDE (MUT/07/16 and MUT/07/17)

(Tabled papers. Poster Moore et al, Dose response evaluation (MN-GVal) of PPG study undertaken by COM members, revised PPG presentation)

8. The Chairman asked for declarations of interest. Dr Parry reported a declaration of interest. Dr Parry reported her husband been in consultation on one occasion in the past with a member of the PPG team. In answer to questions from the secretariat Dr Parry noted there had been no payment to the Department or to Dr Parry. The Chairman considered Dr Parry could respond to questions raised by the chair on the PPG submission.

9. The Chairman asked the representatives from PPG to introduce themselves. Dr Marroni (chair of PPG) noted the Polyelectrolyte Producers Group was a European association of Polyacrylamide manufacturers who were the main users of acrylamide. Dr Zeiger reported that he was acting as a consultant to PPG. Dr Friedman informed members that he was acting as an independent consultant to PPG but had considerable experience of acrylamide toxicology including the conduct of two long term carcinogenicity bioassays. Dr Ungeheuer reported that he acted as secretary to the PPG.

10. The secretariat reported that the COM had been asked by HSE for an assessment of germ cell mutagenicity data on acrylamide in January 2007. There had been insufficient time to bring the evaluation of germ cell mutagenicity data to a full meeting of the COM and a postal consultation had been undertaken with the agreed COM advice posted on the COM internet site (http://www.advisorybodies.doh.gov.uk/com/acryla.htm). The PPG had written to the chair on 8 May 2007 outlining a number of queries regarding the COM view on germ cell mutagenicity. The COM had considered this at their meeting of the 17 May and had agreed to a request from HSE to consider a submission of data from PPG. FSA had requested a full review of the genotoxicity data on acrylamide.

Item 4.1 Presentation: An analysis of Acrylamide Genotoxicity. (Dr E Zeiger, consultant, Chapel Hill, NC, USA)

11. The Chairman asked Dr Zeiger to make the presentation on behalf of PPG and asked members to ask questions following the presentation.

PPG presentation

12. Dr Zeiger informed the COM that the presentation augmented the submission forwarded in August 2007 to the secretariat and focussed on effects of acrylamide and glycidamide on protein function, the evaluation of a big Blue transgenic mutation assay in mice conducted with acrylamide and glycidamide, (Manjanatha MG et al, Environmental and Molecular Mutagenesis, 47, 6-17, 2006) presentation of new information from the dose response study of micronuclei formation (MN), haemoglobin (Hb) and DNA adduct formation in mice given repeated oral doses of acrylamide via the drinking water (and comparison with other studies) and additional information on mechanisms of aneuploidy of acrylamide. Dr Zeiger noted that acrylamide and glycidamide had multiple actions at the cellular level and were highly efficient inhibitors of kinesin activity and inducers of oxidative stress. With regard to acrylamide, Dr Zeiger noted metabolism to glycidamide and the relatively high protein alkylation compared to DNA alkylation of acrylamide and glycidamide. Acrylamide and glycidamide were highly effective at inhibiting microtubule binding to KIFC5A (resulting in misalignment of microtubules) and inhibition of microtubule disassembly by krp2. Acrylamide inhibited kinesin motility in vivo. The structurally related methylene bis acrylamide and propionamide had no effect on kinesin motility. He commented that acrylamide did not produce point mutations in vitro but was clastogenic in vitro, and was a relatively weak in-vivo clastogen. Acrylamide was carcinogenic to rodents. Dr Zeiger considered these molecular actions of acrylamide and glycidamide could explain the clastogenic and aneugenic effects documented in vivo.

13. Dr Zeiger outlined the Big Blue transgenic mouse study undertaken with acrylamide and glycidamide where the test material had been administered via the drinking water. He noted that the highest dose level of acrylamide had been terminated after 3 weeks as overt neurotoxicity had been induced. Statistically significant increases in micronucleated reticulocytes (MN-RET) in the high dose males (low dose males and females not scored), HGPRT mutations in males and females at all doses and in cII mutations at the high dose level in males and females were reported. Further analysis of the cII mutations by the PPG study group found the majority occurred at a GGGG- frameshift hotspot and were more likely to be caused by frameshifts or small deletions than base-pair changes. Dr Zeiger considered the HGPRT mutations were most likely to represent deletions or chromosome rearrangements. He noted the smaller size of the cII gene was not amenable to the detection of small deletions.

14. Dr Zeiger outlined the experimental protocol used for the PPG study (GLP compliant) where groups of male mice were administered acrylamide orally for 28 days (11 dose groups and control). Hb adducts in peripheral blood erythrocytes and DNA adducts in peripheral blood lymphocytes were measured. MN formation was assessed using flow cytometry of 1.3 million NCES and 200,000 PCEs per mouse. Dr Zeiger outlined the contributions from scientists who were responsible for the PPG study and noted that additional data were currently being evaluated for a peer review publication. A dose related increase in MN-RET and MN-NCE had been documented. The PPG study group had noted that an accumulation of MN-NCEs with dose had not been documented (Dr Zeiger noted this had been reported in similar dose-response studies using benzene). The NOEL in the PPG study was reported to be 6.0 mg/kg/day for MN-RET and 4.0 mg/kg bw/day for MN-NCE. The evaluation of dose-response had used both linear and non linear models. The non linear model had provided a slightly better fit. Overall a threshold was reported by Dr Zeiger of approximately 1-2 mg/kg/day. The PPG study group had compared their results with that the Abramsson-Zetterberg study (Mutation Research, 535, 215-222, 2003) and a further study which had been recently accepted for publication (Witt et al [reference to be supplied by PPG]). Dr Zeiger noted there was a remarkable agreement regarding the dose-response relationships for MN-NCE and MN-RET between the three studies with similar magnitude responses. Dr Zeiger noted that haemoglobin adducts (AAVal- acrylamide valine, and GAVal- glycidamide-valine adduct) could be used as internal estimate of dose. Plotting MN-NCE and MN-RET against log of GAVal produced a curvilinear response indicative of a threshold response. The N-7-(2-carbamoyl-2-hydroxyethyl) guanine and N-3(2-carbamoyl-2-hydroxyethyl) adenine (N-7GAGua and N-3GAAde) had been measured to report on DNA adduction. It was noted that these adducts were spontaneously converted to apurinic sites. The data presented in graphical form (for DNA alkylation of mouse liver compared to dose and GAVal, and MN-NCE and MN-RET plotted against N-7GAGua) had not yet been subject to statistical analysis. Dr Zeiger noted that aneuploidy had not been reported in the Abramsson-Zetterberg study. Dr Zeiger noted published studies supporting the in vivo aneugenic action of acrylamide. He commented on Shriver-Schwemmer G (Mutagenesis, 12, 201-207, 1997) which had shown MN with whole chromosomes were more likely to extruded from erythrocytes than MN with acentric fragments. A table of relative potency of acrylamide for acute toxicity, neurotoxicity, mutagenicity and carcinogenicity was presented. He referred to the COM statement on in vivo risk assessment of mutagens (COM/01/S3) and noted that COM had previously agreed that a threshold for mutagenicity existed for aneugenicity induction by tubulin inhibitors and the interaction with non-DNA targets. In this respect he proposed that non-DNA components could include kinesin and proteins that drive the mitotic and meiotic processes.

15. Dr Zeiger outlined a number of conclusions reached by PPG and noted in particular the need to consider that genetic toxicity of acrylamide is mediated through kinesin effects which may occur at doses below those that induce genotoxic responses. Dr Zeiger noted that inhibition of kinesin was a possible mode of genotoxicity and that this could have led to the observed chromosomal effects of acrylamide based on the dose-response characteristics reported. Acrylamide and glycidamide induce oxidative stress in-vivo which may be related to chromosome breakage and mutation. Acrylamide induces non disjunction (aneuploidy in male germ cells and cells in culture). Acrylamide and glycidamide are clastogenic to rodent sperm cells. Acrylamide is a weak and inconsistent clastogen in mouse somatic cells and is negative in rat somatic cells. Glycidamide is a very weak DNA binder. Glycidamide DNA adducts have a low mutagenic efficiency. Germ cell (dominant lethal) effects correlate with protamine but not DNA adducts. The non-DNA reactive effects of acrylamide at doses below those needed to produce a noticeable increase in chromosome damage, support an indirect mode of action. Such a mode of action would predict a threshold to the genotoxic response, which is consistent with the response seen in bone marrow MN test.

16. The Chairman thanked Dr Zeiger for his presentation and asked members for questions on the presentation first and then for questions on the submission.

Item 4.2 Review of submission from The Polyelectrolyte Producers Group (PPG) (MUT/07/16)

COM questions on PPG presentation

17. One member asked for PPG's evaluation of heritable translocations. Dr Zeiger considered these resulted from chromosomal breakage. Members asked several questions regarding the evaluation of dose-response in PPG MN study in mice. Members considered the dose-response data for the PPG MN study and noted the data showed little variability in the lower doses and could be interpreted as evidence for small increases below the 1-2 mg/kg bw/day. Members considered that there was little difference between the application of linear and non linear models to dose response fitting and queried whether the statistical approach used had been derived post hoc. Members queried the plotting of doses on a logarithmic scale. Dr Zeiger considered the PPG study had been fully GLP compliant and the statistical aspects had been considered in the design. The use of log plots had been necessitated by the emphasis of the design on the use of multiple low dose levels. Members asked for historical control data for MN from the laboratory to be submitted. PPG noted these data had become available just before leaving for the UK and presented some data (the mean for MN NCE was 1.46±1.1 and for MN-RET 2.58±0.48 %o) and would be forwarded in full to the secretariat. Members queried the nature of MN identified in the PPG and other dose-response studies. It was possible that these included acentric fragments. Members noted that appropriate data to investigate the nature of MN formed was not available. PPG considered the weight of evidence from the available data regarding protein interactions of acrylamide and the germ cell mutagenicity data supported an aneugenic mechanism for MN formation in the PPG study.

18. Members considered the Big Blue transgenic mutagenicity data and queried whether the available data suggested that a predominantly aneugenic mechanism. Members considered that the HPGRT mutation data supported the involvement of glycidamide and the occurrence of point mutations. Members discussed the investigation of acrylamide mutagenicity in CYP2E1 proficient bacteria. Dr Zeiger considered the negative results could potentially be due to lack of activation to glycidamide in sufficient amounts combined with rapid metabolism of glycidamide by epoxide hydrolase. He noted that positive data in this test system had also been reported with glycidamide. Members noted that these data for glycidamide were not reported in the published paper (Emmert B et al Toxicology, 228, 66-76, 2006) and might be important with regard to the evaluation of gene mutagenicity of acrylamide.

[Post meeting amendment for paragraph 18 of MUT/MIN/2007/3 arising from COM discussion at 14 February 2008 meeting, (paragraph 23 of MUT/MIN/2008/1). PPG are currently seeking a laboratory to test glycidamide in CYP2E1 proficient bacteria.]

19. FSA asked for clarification of the potency data reported in the presentation and queried why a mouse carcinogenicity TD50 value had been compared to LOEL and BMD₁₀ data for other end points in rodent species. It was noted the rat carcinogenicity studies had indicated evidence for higher carcinogenic potency in rats compared to mice. A BMDL₁₀ in rats of 0.3 mg/kg bw/day had been reported (Joint FAO/WHO Expert Committee on Food Additives, WHO Food Additives Series 55, Nutrition Paper 82, WHO Geneva, 2006). Dr Zeiger noted that a further mouse long term carcinogenicity bioassay was currently being undertaken by the US National Centre for Toxicological Research.

20. The Chairman asked for questions on the written submission and suggested that members consider the ten sections in the order presented in Annex 4 to MUT/07/16.

Acrylamide adducts to haemoglobin and the 7-position in guanine in mammalian cells

21. The Chairman asked for further consideration of DNA adducts other than N7-GAGua which might be formed from acrylamide exposure. Dr Zeiger noted that N3-GAAde was formed at substantially lower levels than N7-GAGua and that other DNA adducts could not be detected in the available studies.

DNA breaks are unrelated to sites of alkylation and rapidly repaired

22. Members noted the evidence for DNA repair in human lymphocytes and considered the data were similar for other compounds and did not exclude the possibility of low levels of persistent DNA adducts. The Chairman noted the recent abstract of a study in LY5178Y cells (Moore et al tabled abstract). These mammalian cell mutagenicity data were consistent with different modes of action of acrylamide (loss of heterozygosity) and glycidamide (DNA adducts). The authors had not identified any glycidamide formation from acrylamide in this test system. Members queried whether CYP2E1 was the only mode of acrylamide metabolic activation. Members were aware of the data from CYP2E1 knockout mice. PPG noted that other data showed the toxicity of glycidamide formed from acrylamide was different to that of exogenously dosed glycidamide.

Prokaryotic cells are not mutated by acrylamide

23. Members noted the strain of Salmonella typhimurium used by Emmert et al (2006) YG71083ERb₅ was derived from TA1535 which responded to N7 alkylation. Members emphasised the need for submission of the glycidamide investigations cited by PPG group for this strain.

Induction only of chromosome aberrations in mammalian cells in culture

24. Members noted the need for additional evaluation of the mutation response in vitro in mammalian cells for the identification of point mutations and in particular the response in HPRT assays.

Acrylamide inhibits kinesin and causes oxidative stress in vivo

25. Members had no additional questions on this topic.

The Big Blue Mouse data confirm frameshifts, rather than base-pair substitutions are the major component of the increased mutagenic response in vivo.

26. The secretariat noted that Manjanatha et al (2006) had reported A-->G transitions as well as A-->C and A-->T transversions, all in the range of 5-10% total and considered that it was conceivable that the N1 and N3 dA adducts produced by glycidamide may at least be partially responsible for these mutations. Dr Zeiger considered that the response in this Big Blue mouse study was a shift in mutagenic pattern rather than a specific increase in mutants such as base-pair transitions. He noted that PPG was aware of other transgenic mutation assays which had not been published and were attempting to obtain these for further evaluation. The secretariat indicated they would also attempt to identify these studies. Members agreed this would be relevant information.

The data reveal a threshold in the micronucleus test in vivo

27. Members had no further comments to raise on the PPG study.

Acrylamide binds protamines in vivo

28. Members recognised contamination had been previously noted as a problem with regard to AMS studies. Members noted that the possibility of alkylation with regard to male germ cell effects could not be discounted on the basis of these data.

The induction of heritable translocations is non-linear

29. Members considered that the arguments proposed were generic for compounds which induced heritable translocations. A biphasic dose -response might not necessarily infer a threshold.

Acrylamide is only a weak clastogen in vivo

30. Members asked how the estimates of Bench Mark Doses related to plasma levels of acrylamide and glycidamide. Dr Zeiger indicated that the preferred dosimeter would be GAVal Hb adducts. Members noted that the shape of dose-response curves from the information contained in the presentation suggested little difference between intraperitoneal and oral exposure. The possibility of localised metabolism in the gastro intestinal tract was noted.

COM discussion

31. The Chairman thanked Members for their comments and questions. He noted that several COM members had been unable to attend at short notice and suggested that following circulation of the minutes of this meting, that any further queries from COM members would be forwarded to PPG. He asked members to consider the overall questions in paragraph 11 of the covering paper. The secretariat noted additional data on the PPG study, information on the Witt study and data outlined during the discussion with PPG were required. Members asked that the secretariat overview paper currently under preparation required consideration of the source of exogenous metabolising fraction in the in vitro tests. (Additional comments on topics to be included in the review would be discussed during the consideration of item 4.3) Members agreed a full COM statement on the genotoxicity of acrylamide should be produced. The Chairman thanked PPG for their presentation of the data.

32. HSE asked if a provisional conclusion could be reached regarding whether acrylamide should be considered in accordance with COM approach to evaluation of in vivo mutagens as a non threshold compound (cf COM Statement 03/01 http://www.advisorybodies.doh.gov.uk/com/comivm.htm). The COM chairman supported by the secretariat considered that there were additional data to consider which were relevant to this topic and therefore it would be premature to reach an interim conclusion. Overall, the COM could provide interim advice only if the balance of evidence was such that there was little prospect that additional data could change a conclusion from a default position of assuming no threshold.

Item 4.3 EU risk assessment summary and overview of the COM review strategy (MUT/07/17)

33. The secretariat asked members to consider the two topics included in MUT/07/17 separately. Members were asked if the EU risk assessment report drafted by HSE and agreed by an EU Technical Group could be used as a basis for the secretariat overview. Questions for the committee to consider were set out in paragraph 8 of the draft discussion paper. Members agreed the overall conclusion reached in the EU risk Assessment report. Members agreed with previous comments under item 4.1 that there was uncertainty regarding whether there was metabolic activation in mammalian cells and the pathways of metabolism involved. Members agreed that the transgenic mutation assays considered in the EU risk assessment report were relatively early studies in the development of these assays but the data were consistent with positive results. Members were content to use the EU risk assessment report as the basis for the COM review. Members asked that the COM review of acrylamide and glycidamide genotoxicity data post 1995 should present information on end point, possible mechanism(s) for each end point and role of metabolism.

34. Members considered the literature searching strategy and the exclusion and inclusion criteria used and agreed these were acceptable. It was noted that post 1995 a total of 67 published references had been identified for review of which a small number had been exclusively identified either by PPG or by the COM secretariat. Members queried whether the search strategy would identify all studies where DNA damage/oxidative stress had been studied. It was agreed some additional searches should be undertaken on this aspect. Members noted that a small number of references published in 1995 or earlier had been identified by the secretariat and agreed these should be included in the review. The secretariat reported that the list of references to be included in the COM review would be made available to PPG and interested Government Departments.

35. Members agreed with the decision reached under items 4.1 and 4.2 that a full COM statement on the genotoxicity of acrylamide and glycidamide should be drafted.

36. Members commented that taking all the available information presented during this meeting into account, there was some provisional evidence to support the contention proposed by the PPG group for an indirect mechanism for at least a proportion of acrylamide-induced aneugenicity and clastogenicity. However members considered that further evaluation of the data on gene mutation and the positive responses reported in the literature in a range of tests systems was required in order to reach a definite conclusion on the possibility of a threshold and no decision could be taken at this meeting.

37. The Chairman thanked members for their comments and asked that members not present be asked for any additional comments on item 4.3. The secretariat noted that they would need to seek a further meeting with PPG to obtain the further information requested by COM. The Secretariat requested that PPG should direct all correspondence through the secretariat.

ITEM 5: MUTAGENICITY OF MIXTURES 2ND DISCUSSION PAPER: EVALUATION OF MIXTURES USING MUTAGENICITY STUDIES (MUT/07/15)

38. There were no declarations of interest.

Members were reminded that the first discussion paper considered at the February 2007 meeting presented a review of integrative and dissective approaches to genotoxicity evaluation of mixtures. An overview strategy for monitoring complex mixtures was presented. A revised scheme should be brought forward when a draft working paper is considered. MUT/07/15 presented a systematic approach that had been undertaken to identify papers. A quality screen was used to rank papers for review (Borgert et al, Hum Ecol Risk Assess, 7 (2), 259-306, 2001). Only 15 out of the 91 articles retrieved met all the quality screen criteria and the review had focused predominantly on these papers. Members' attention was brought to a commentary on the review provided by one member (Annex B to MUT/07/15).

39. The Committee agreed that the FSA secretariat had undertaken a good approach to data collection and quality screening of the papers and that the studies had been summarised to a good standard. Members noted that a particular difficulty in assessing genotoxicity studies of interaction between chemicals related to the terminology used by the authors (eg the terminology of non-interaction based on dose or response additivity and the definition of synergy) and the different design of studies and statistical approach used to evaluate results. The concept of the envelope of additivity was considered to be a helpful approach to graphically presenting the results of studies and to help identify non-interaction (additive responses) and interaction responses (synergy and antagonism) (Lutz et al Tox Sci, 86 (2), 318-323, 2005). Members were alerted to the unifying approach for application of statistical methods in chemical mixture research based on the shape of the dose response curve and changes in the slope of the dose-response in studies using two or more chemicals (Gennings C et al Tox Sci, 88 (2), 287-297,2007. This paper linked the traditional statistical models of interaction (as found in the general linear model / factorial ANOVA models) to the different concepts of joint toxic action. The unification of the approaches is achieved by showing that there is no interaction if the dose-response relationship of one chemical is not changed by the presence of other chemicals. An interaction exists if there is a change in the slope of the response. This concept of interaction related to underlying statistical models of additivity. Members agreed that the approach suggested by Gennings et al 2005 could be potentially helpful when assessing genotoxicity studies of interaction between chemicals.

40. Members considered that an important part of the assessment of genotoxicity studies of interaction between chemicals would be reproducing results seen in one test system with other appropriate genotoxicity tests (eg confirming results seen in bacterial gene mutation assays in mammalian cell gene mutation assays). This could be used in a weight of evidence assessment of interactions and would be particularly important for interactive effects such as synergy or antagonism.

41. The strategy would also need to include in vivo tests with consideration of toxicokinetics and exposure of sampled tissues. Members commented that the available published literature presented a number of examples where interaction had been reported, but there was essentially no appropriate independent confirmation of the results in separate tests, or within an appropriate genotoxicity testing strategy for the identification of interactions.

The Chairman asked members to review the four studies highlighted by the secretariat which reported the best evidence for interaction

42. Homme M et al (Mutation Research, 461, 211-219, 2000) had documented synergistic DNA damage using UDS assays in human fibroblasts between 4-nitroquinoline-1-oxide (4-NQO) and non-effective methyl methanesulfonate (MMS). The authors had proposed that the ultimate DNA reactive metabolites formed from 4-NQO resulted in unwinding of super helical DNA so that more molecules of MMS could reach the bases of DNA resulting increased methylation and mutation. Members considered that a viable hypothesis had been proposed. It would be necessary to undertake independent confirmation of the results and to include additional combinations of mutagens with and without 4-NQO to provide further data to investigate the proposed mechanism. At present no definite conclusions could be reached on this specific example of an interaction.

43. Kojima H et al (Mutation Research, 266, 171-180, 1992) had investigated the potential for interaction between MMS and EMS in Chinese hamster V79 cells using cell killing, induction of 6-thioguanine mutants (6TG resistant mutants) and chromosome aberrations. These authors had reported evidence for synergistic interactions for both cell killing and 6TG mutation and evidence for additivity with regard to chromosome aberrations. The authors had suggested that the DNA damage produced by one alkylating agent could be increased in the presence of a small amount of another alkylating agent. Members noted the predominant S_N2 mechanism of MMS and the S_N1 mechanism of EMs and considered that these differences could form the basis for a hypothesis of interactive effects with regard to genotoxicity. However members considered there was a need for independent confirmation of these results and further investigations of other alkylating agents before any definite conclusions could be reached.

44. Lutz et al (Toxicol Sciences, 86 (2), 318-323, 2005) had reported evidence for antagonism using a combination of N-methyl-N-nitrosourea (MNU) and the topoisomerase-II inhibitor genistein (GEN) in the mouse lymphoma assay in LY5178Y cells. In separate tests when MMS was combined with GEN an additive response (reported to be within the envelope of additivity) was reported. The authors hypothesised that the profile of DNA methylation and/or epigenetic effects were responsible for the different responses reported for the binary combinations tested. Members agreed these data raised interesting hypotheses for further testing but no definite conclusions could be reached on these data.

45. Marrazzini A et al (Mutation Research, 341, 29-46, 1994) had undertaken in vivo mouse bone marrow MN tests in mice using intraperitoneal administration of binary combinations of hydroquinone, catechol and phenol. Mixtures of hydroquinone and phenol and catechol and phenol were reported to result in synergistic induction of micronuclei. Members noted that it was not possible to discern a potential mechanism of interaction from these studies which could be used to support hypotheses for further testing.

46. The COM was asked to comment on the overall questions outlined in para 22 of the covering paper. Members commented that the different interpretations of synergy used by the research groups and limitations in the available data made it difficult to reach any definite conclusions. Overall there was insufficient evidence to conclude that the studies reviewed provided conclusive evidence for interaction (either synergy or antagonism). However a number of the studies provided evidence to suggest hypotheses for interaction which could be further examined in appropriately designed testing strategies. Members agreed the potential for interactions with regard to genotoxicity needed to be studied on a case-by-case basis. The Chairman thanked members for their comments and noted that a draft working paper summarising the committee's considerations of mixtures would be prepared for the February 2008 meeting.

ITEM 6: UPDATE ON TOXICOGENOMICS (MUT/07/18)

47. The COT/COC/COM held a joint symposium on the issue of genomics and proteomics in October 2001 and published a joint statement in December 2004 on the use of Toxicogenomics in toxicology. This was based on literature review of 50 studies and included information from the International Life Sciences Institute/Health and Environmental Sciences Institute (ILSI/HESI) collaborative programme of research.

48. This topic was identified during the 2006 horizon scanning exercise for an updated review. The DH Toxicology unit drafted a short overview (Annex 2 of MUT/07/18) of a number of new relevant in-vitro studies, which included data on gene expression changes in studies on DNA adducts and mutagenicity. A large number of papers had been retrieved, but those selected for review were specifically chosen with the aim of identifying any advancement in the field, which may affect the conclusions drawn in the last statement.

49. The studies evaluated only included information from transcriptomic approaches using a number of mammalian cell lines which were either p53 proficient (TK) cells or p53 deficient (LY5178Y). Test compounds used were predominantly genotoxins acting by a variety of mechanisms in the absence of metabolic activation. The use of statistical methods and bioinformatics in toxicogenomics had not been addressed, but a more detailed review on these aspects would be included in the proposed review for the COT. There were also a number of approaches including some in-vivo studies using proteomic/metabonomic approaches that would need to be evaluated before the COM could review its 2004 conclusions. However, it was felt that the papers reviewed so far, would be useful in initiating the COM review.

50. The committee was informed that the COC had also considered a short overview paper at its July 2007 meeting and the minutes were provided for information. The COT review was not expected until later in the year. It was also noted that there were a number of ongoing international investigations of toxicogenomic approaches to chemical risk assessment including an investigation by ILSI/HESI. A presentation of some of the initial ILSI/HESI results was made available, and the COM was informed that a HESI Genomics Committee symposium in November 2007 would feature the first public release of results from its 2005 - 2007 research programmes.

51. The Committee noted that considerable progress in the field of toxicogenomics had been made since the last COM review and considered this to be a very interesting area. Members agreed that although many of the papers identified appeared to be robust and well conducted the methods used in the studies varied (eg in microarray platforms used, approaches to normalisation and replication, statistical methods used and approaches to gene mapping), which made comparison difficult. . Thus it was difficult to derive conclusions on the gene responses to alkylating agents (eg MMS) from the available in vitro transcriptomic assays reviewed.

52. Members agreed that there was some evidence for using transcriptomic assays to aid in the discrimination between cytotoxicity and genotoxicity and between genotoxic compounds that reacted directly with DNA and genotoxic compounds that did not interact directly with DNA. Members felt that interpretation of gene pathways or networks might possibly, in the future, provide more useful approaches than consideration of individual gene patterns from high density microarrays, as it was more likely that such information could be extrapolated from system to system. The COM felt that in the future, it may be possible to only look at a relatively small number of specific pathways that had been identified as the most important but further information was needed to reach conclusions on this aspect.

53. Regarding individual gene responses, members thought that a small change in expression (eg a 2 fold upregulation) for normally inactive genes could be more important than a relative large change in gene expression for a normally active gene and hence it was preferable to undertake unsupervised analysis without specified thresholds for inclusion in analyses. Members agreed the value of obtaining supportive data for the results of transcriptomic investigations with information from proteomic and metabonomic analyses. Thus it was possible that increased gene expression at the mRNA level did not lead to increased gene activity within the cell.

54. The COM noted that the available evidence in the ILSI/HESI presentation suggested that DNA adduct formation was more sensitive than gene expression changes. Overall, the COM agreed that it would be able to update its 2004 views on toxicogenomics, but they would first need to see the additional review of new information and wait for the outcome of the ILSI/HESI trial and November 2007 symposium, before drawing any new conclusions. The committee agreed that at present, toxicogenomic studies were not as sensitive as standard genotoxicty assays and could not be used as primary assays. However, it was felt that there was potential for toxicogenomics studies to provide additional useful information on the mode of action. This topic would come back to the committee at the February 2008 meeting.

ITEM 7: HORIZON SCANNING (MUT/07/19)

55. The annual horizon scanning exercise was intended to provide an opportunity for members and advisers from Government Department/Agencies to discuss and suggest topics for further work. The COM was informed that a more limited literature search had been undertaken this year compared to previous years due to time constraints.

56. Considerable progress on the items identified in the 2006 horizon scanning exercise had been made, although it was noted that review on mutational spectra had not been initiated.

57. The 2007 literature search used PUBMED and initially indicated several thousand publications in 2006/7, which could be potentially relevant. The search strategy subsequently focused on a number of areas such as, genotoxicity test strategy, novel mutagens, mutagens in the environment, genotoxicity biomonitoring, high potency mutagens, potency of genotoxins, mutagen spectra/spectrum and chemical mutagens, and was summarised in MUT/07/19.

58. Members were asked for their views on the identified areas and for additional suggestions for further work.

59. Furan had been identified as a possible chemical for review by the literature search. A COC member had recently identified a paper reporting negative findings for furan in a peer reviewed publication of an in-vitro micronucleus assay and also for an in-vivo micronucleus assay. The COM had previously advised the COC during 2005 that furan should be regarded as an in-vitro mutagen, but that a conclusion on in-vivo mutagenicity could not be reached and had suggested further work. However, members agreed that there was no need to consider furan at present, due to substantial research on this compound taking place in the EU.

60. Phenol was another chemical that had been identified for further work. The COM had reviewed unpublished data in 2003, which suggested a plausible mechanism for secondary indirect effect for positive mutagenic results seen in the bone marrow ie hypothermia occurring at dose levels associated with positive micronucleus assays. Members had agreed that before definite conclusions could be drawn on the significance of such data they would need to see a peer reviewed published report of the study. A publication on this data was now available. The committee agreed that it could review this hypothesis ie the potential for false positive effects due to hypothermia or hyperthermia using phenol as one example compound.

61. The committee agreed that the review of the approaches to the genotoxicity testing of mixtures should be completed. Members agreed to initiate an update of the current COM guidance on mutagenicity testing, but noted that this would be a long process which should first involve an overview of how the field of mutagenicity testing had progressed since the last COM Guidance published in 2000. The overview would have to consider new draft Guidance on mutagenicity that had recently been published or was about to be published by other authoritative organisations eg WHO and US EPA.

62. Members' suggestions for other areas for future work included the significance of aneuploidy, its causes and possible approaches to risk assessment, also mitochondrial mutation and its potential involvement in various diseases, and epigenetics. Members agreed a focused review of the utility of the Ames test for evaluation of low levels of mutagenic impurities in test materials would be valuable.

ITEM 8: ANUEPLOIDY: ACTION BOTH ONCOGENICALLY AND AS A TUMOUR SUPPRESSOR (MUT/07/20)

63. One member of the Advisory Committee on Pesticides had recently noted a paper on aneuploidy (Weaver BAA et al, Cancer Cell, 11, p25 - 36, 2007) that put forward the hypothesis that aneuploidy may also be associated with tumour suppression if aneuploid cells are directed towards apoptosis (aneuploidy is normally considered to be a common feature of human cancer cells and believed to play a role in tumourigeneisis).

64. The authors reported that cells and animals that are heterozygous for centromere-linked protein (CENP-E^+/-) become aneuploid due to random malsegregation of one or a few chromosomes at high rates in the absence of DNA damage. CENP-E is an essential, mitosis specific, cell-cycle regulated protein that accumulates primarily in late G2, used in mitosis and then degraded at the end of mitosis. It is thought that CENP-E maintains chromosome interactions with microtubules and might also have a dual function in mitotic checkpoint signalling.

65. The researchers reported that reduced CENP-E in heterozygotes can promote tumourigenesis in mice, but can also lead to inhibition of spontaneous liver formation in the same mice. A group of 25 homozygous CENP-E mice and 13 heterozygous CENP-E mice were given a single dose of 7,12-DMBA on Post Natal day (PND) 3-5. It was reported that 40% of the homozygous mice developed lung tumours compared with 31% of the heterozygous mice. The lung tumours in the heterozygous mice were also of a smaller size and this was considered to provide some limited evidence that anueploidy may inhibit tumourigenesis.

66. The default risk assessment approach has been to consider chemically induced in-vivo aneugenicity to be hazardous even if there are no particular biological effects reported in standard animal toxicology tests (eg impaired fertility or tumourigenesis).

67. Members noted that this was an interesting, although small study, and were surprised at the abnormally high level of background aneuploidy in the mouse strain used which probably affected the results of the studies reported (ie substantial background DNA damage resulting from reduced CENP-E activity might predispose cells towards apoptosis). It was felt that the results of tumour suppression would have been expected due to the high level of DNA damage, which would tend to cause cell death rather than tumour progression. No details of a statistical test were provided to show whether a significant difference in tumuorigenesis had occurred or not.

68. Overall, the COM considered this to be a valuable paper but that it provided unconvincing evidence for a change to the default assumption of regarding aneuploidy as a hazardous effect.

ITEM 9: PAPERS FOR INFORMATION

69. The following papers were provided for information:

9.1 Genotoxicity test strategies: Article from FORUM series from Toxicological Sciences published in 2007. Pfuhler S et al, Toxicological Science, 97, 237-40, 2007. (MUT/07/21)

9.2 Revised code of practice for observers (MUT/07/22)

ITEM 10: ANY OTHER BUSINESS

70. Members were informed that the FSA would be drafting a review paper on sodium and potassium sorbates and benzoates which would come to the COM.

71. The committee was also informed that members would be forwarded WHO and US EPA Guidance on genotoxicity testing for comment.

72. A typographical error in the COM revised code of conduct for observers was pointed out to the secretariat.

ITEM 11: DATE OF NEXT MEETING

73. 14th February 2008

ACTIONS

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