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MINUTES

Present:

Chairman: Professor P Farmer	Members: Dr C Allen Dr B Burlinson Dr G Clare Dr J Clements Dr B Elliot Dr D Gatehouse Mrs R Glazebrook 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) Mr K N Mistry (Administrative)
Assessors: Mr A Browning (VMD) Mrs S Daniels (PSD) Dr A Smith (HSE)	In attendance : Dr S Costigan (MHRA item 4) Dr E Damien (MHRA item 4) Mr B Maycock (FSA item 5) Dr D Mason (FSA item 6) Mr K Okona-Mensah (DH Tox unit item 7) Mr S Williams (BP item 6)

CONTENT

Item		Paragraph
1.	Announcements/Apologies for absence	1-3
2.	Minutes of the meeting on 9 February 2006	4
3.	Matters Arising (not covered by later agenda items):	5 - 7
4.	Biological Effects of Wear Debris Generated from Metal on Metal Bearing Surfaces: Evidence for Genotoxicity Discussion with Bristol Implant Research Centre (MUT/06/7)	8
5.	Benzimidazoles: discussion of a common mechanism Group (MUT/06/8).	22
6.	Terephthalic acid : Update on mutagenicity data (MUT/06/9)	35 - 46
7.	Comparative risk assessment: Initial outline discussion paper (MUT/06/10)	47 - 57
8.	Discussion of risk factors for background variance of cytogenicity in peripheral blood lymphocytes (MUT/06/11)	58 - 76
9.	Any Other Business	77 - 80
10.	Papers for information: Do dose response thresholds exist for genotoxic alkylating Agents? Jenkins G et al., Mutagenesis, 20, 389-398, 2005. Using surveys in public participation processes for risk Decision-making: the case of the 2003 British GM Nation? Public debate. Risk Anal, (25)2; 467-79.	81 - 82
11.	Date of next meeting - 12 October 2006 Actions	83

ITEM 1: ANNOUNCEMENTS/APOLOGIES FOR ABSENCE

1. The Chair welcomed the new members Dr Barry Elliot and Dr David Lovell to their first meeting. The Chair also welcomed Dr Elsie Damien (MHRA item 4), Dr Sandra Costigan (MHRA item 4), Mr Barry Maycock (FSA item 5), Dr David Mason (FSA item 6) and Mr Ken Okona-Mensah (DH Tox unit item 7).

2. Apologies for absence were received from Dr N Gooderham (Member), Dr L Hetherington (HPA Secretariat), Ms A Gowers (EA) and Dr H Stemplewski (MHRA).

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

ITEM 2: MINUTES OF MEETING ON 9th February 2006 (MUT/MIN/2006/1)

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

ITEM 3: MATTERS ARISING FROM MUT/MIN/2006/1 (NOT COVERED BY LATER AGENDA ITEMS)

3.1 A comparison of the relative sensitivities of the in vivo rat liver UDS assay and the in vivo COMET assay

5. The secretariat apologised for the delay on a short statement on the comparison the relative sensitivities of the in vivo rat liver UDS assay and the in vivo COMET assay. It was intended that this would be circulated soon after the May meeting.

3.2 Draft OECD guideline for the in vitro micronucleus test

6. The secretariat was aware that UKEMS/IGg were setting up a working group to look at the draft OECD in vitro micronucleus test. This would involve some COM members and HSE would represent regulators. The secretariat would be grateful for any feedback from members from this venture. Members reported that an initial meeting of the group was expected at the end of June 2006. Members also noted that a preliminary JACVAM evaluation of the in-vivo COMET assay might be available in August 2006.

3.3 COT/COC and RCEP report on crop spraying and bystanders

7. Members were informed that the COT/COC discussion paper on the Royal College of Environmental Pollution Report on crop spraying and health of residents and bystanders had been published on the COT and COC internet sites.

ITEM 4: BIOLOGICAL EFFECTS OF WEAR DEBRIS GENERATED FROM METAL ON METAL BEARING SURFACES: EVIDENCE FOR GENOTOXICITY. (MUT/06/7).

8. No interests were declared.

9. The COM had discussed a review paper on the evidence for genotoxicity in patients who had received Metal on Metal hip replacement devices at its previous meeting on the 9th February 2006. The COM was aware of the overriding need for the regulatory authority (MHRA) to balance the risks associated with MoM hip replacements with the benefits e.g. pain relief and improved mobility etc.

10. Members had agreed a number of provisional conclusions (as reported in the minutes of the meeting MUT/MIN/06/1).

11. Members had asked for some additional details regarding the assessment of aneuploidy undertaken by the Bristol Implant Research Centre (BIRC). The COM secretariat and MHRA met with Dr P Case of BIRC on the 31 March 2006. A summary of the meeting was appended at Annex 1 to MUT/06/07. At that meeting a number of additional studies were made available to the secretariat and these were also appended for information.

12. The Committee was asked to consider the additional information provided by Dr Case, including an 'in confidence' prepublication paper, and to comment on an appended first draft working paper.

13. The COM was content with the answers provided by Dr Case in response to the committee's questions on the conduct of in vitro cytogenetics and aneugenicity assays. Dr Case had confirmed that in the study by Doherty A et al., 2001 (The Journal of Bone and Joint Surgery vol 83-B, p1075-1081) that the aneuploid index and translocation index had both been derived from the same 300 metaphase spreads/patients. The full data on non-disjunction had not been reported, although it was considered that non-disjunction accounted for most of the aneuploidy observed in this study. It was unlikely that any further information could be retrieved on this study. Members agreed that this study should be interpreted cautiously and to place more reliance upon translocations than aneuploidy. However, the committee was still unclear over the meaning of the aneuploid index and asked whether an explanation could be obtained.

14. The committee considered each of the further papers provided by Dr Case in detail.

Case c et al., 1994. The Journal of Bone and Joint Surgery (Br), vol 76-B, p701-712.

15. Members considered that this study on the distribution of metal ions in various autopsy tissue specimens from patients who had hip replacements had been conducted adequately. The relatively low tissue levels of chromium in patients with Stainless Steel implants was noted.

16. In confidence pre-publication paper on the genotoxic effects of wear debris from worn orthopaedic joint replacements on human cells.

17. The COM considered that this study using the COMET assay had been adequately conducted, but provided insufficient information on interpretation of the COMET assay data. The authors had reported DNA damage in arbitrary units and it was unclear how the DNA damage was distributed among exposed cells. Members considered that in general significant DNA damage would occur in a relatively small number of cells but it was unclear how the data from this study were distributed. The limits of experimental error regarding negative control experiments and information from positive control studies in the cell system used were not available. Members agreed that the results were consistent with a higher extent of DNA damage from Co/Cr wear debris compared to TiVAl wear debris. Members agreed that the influence of cytotoxicity with regard to the biphasic response needed to be considered in addition to the investigation of effects on stimulating DNA repair

Daley B et al., 2004. Journal of Bone and Joint Surgery, vol 86-B, p598-606.

18. This study investigated micronuclei formation in vitro for various metals extracted from wear debris from patients with different types of implant. Titanium, +/- aluminium and vanadium were reported to be correlated with the formation of centromere-positive micronuclei. The concentration of cobalt and chromium +/- nickel and molybdenum were reported to be correlated with the formation of centromere positive and negative micronuclei combined. Members expressed reservations regarding the use of primary amniotic cells for this study, and noted there were no appropriate negative and positive control data for micronuclei induction in this test system. The Committee noted that apparent positive response regarding Stainless Steel implant wear debris but observed this was based on two samples only and the magnitude of the response was small. It was concluded that there was no convincing evidence for a mutagenic response with Stainless steel wear debris. Members also had reservations regarding the reported dose response for micronuclei induction from Co/Cr and TiVAl wear debris. It was agreed that the magnitude of response was relatively small and was suggestive of an effect at the top dose level. However overall this study had not provided convincing evidence of a metal specific effect.

Pilger A et al., 2002. J Tox Env Hlth, part A, vol 65, p655-664.

19. In another study, urinary 8-hydroxyguanosine, a marker for oxidative DNA damage was higher in MoM hip replacement patients (n=46) who had implants for 3-4 years compared to those patients who had implants for 1-2 years. The ratio of smokers in each group was approximately the same. Elevated blood cobalt levels were reported in these patients. There was no correlation between 8-OHdG and SCEs or metal concentrations in the blood. Members noted that 8-OHdG and SCEs were not particularly sensitive endpoints for genotoxicity. Thus 8-OHdG measured the breakdown of damaged nucleotides and the relevance of SCEs to mutagenicity was unclear. It was also noted that is would have been better to have taken urinary samples at 24 hours or to have corrected the data for excretion of creatinine. Overall it was agreed that the reported results were not convincing of a mutagenic effect of metal-on-metal hip replacements.

20. Members considered the draft working paper and its overall conclusions in light of the new data. The COM concluded the following:

[Post meeting note; MHRA asked for the statement to refer to specific metal combinations. Thus metal on metal refers to CoCr on CoCr hip replacements. Metal on polyethylene can refer for CoCr on PE, TiAlV on PE or SS on PE.]

i) The Committee agreed there was good evidence for an association between CoCr- on CoCr and CoCr or TiAlV on polyethylene hip replacements and increased genotoxicity in patients. But, that there was no convincing evidence for increased genotoxicity in patients with stainless steel on polyethylene hip replacements.

ii) The evidence for increased blood levels of chromium and cobalt, in patients with CoCr on CoCr hip replacements or CoCr on polyethylene hip replacements which may be a potential risk of carcinogenicity in humans, gave rise to concern, but the Committee felt that it was not possible to make any definite conclusions which metal ions, or interactions between metal ions or particulate metals might be responsible for the observed genotoxicity.

iii) There was limited evidence available to suggest a possible interaction between chromium and cobalt ions and possible mutagenicity/DNA damage in vitro but not in vivo. There was no convincing evidence for metal specific effects of wear debris with regard to potential for clastogenicity or aneugenicity.

21. The revised draft working paper would be sent to MHRA and circulated to members for comment before publication of the finalised working paper.

ITEM 5: BENZIMIDAZOLES: DISCUSSION OF A COMMON MECHANISM GROUP (MUT/06/8)

22. The COT had published a report on the Risk Assessment of mixtures of pesticides and similar substances in September 2002. One of the recommendations was that a scientific and systematic framework should be established to decide when it is appropriate to carry out combined risk assessments of exposures to more than one pesticide and/or veterinary product. The COT recommended the default assumption that the risk assessment of combined exposure should be that chemicals with different toxic actions will act independently, and that those with the same toxic action will act additively. In the latter case, the COT recommended that a toxic equivalency approach might be considered.

23. In response to COT recommendations, government departments and agencies set up a 'Science Group' to consider which pesticides and similar substances could be grouped together based on common mechanisms of mammalian toxicity. Benzimidazoles were selected by the Science Group as substances whose mode of action had not been fully characterised even though they had broad evidence for similar mechanisms of action for cellular effects and would be a suitable test case of the procedure for a common mechanism group (CMG).

24. The discussion paper MUT/06/8 suggested how the US EPA approach to defining CMGs could be modified in relation to benzimidazoles, which have potential aneugenicity via a tubulin depolymerisation mechanism. Potential stages suggested were:

Stage 1: In vitro evidence for mutagenicity and /or aneugenicity and/or pesticidal/veterinary action on tubulin.
Stage 2: Clear evidence of aneugenicity in vivo.
Stage 3: Clear evidence for binding to mammalian tubulin
Stage 4: Rationale for mechanism of aneugenicity
Stage 5: Stages 1-4 considered together

25. The Science Group identified a difficulty in the consistency of the data between compounds. For many of the compounds specific studies investigating the potential for aneugenicity were not available, although some of the regulatory studies may provide an indication of the potential for aneugenicity in vitro or in vivo. Thus, for the majority of compounds there were data gaps. There was not one specific endpoint which has been investigated for all compounds, which raised potential difficulty if compounds assigned to a CMG were to be ranked by comparison of potency for the purposes of a combined risk assessment.

26. The available in vitro and in vivo data on 20 benzimidazoles were briefly summarised at Annex A. Suggested options for defining a CMG included the following:

1. Structure activity relationship + evidence for binding to mammalian tubulin + indication for aneugenicity in vitro + positive in vivo micronucleus assay

2. Structure-activity relationship + any positive micronucleus study either in vivo or in vitro.

3. Structure-activity relationship or inhibition of tubulin polymerisation in any system (e.g. helminth, fungal, plant or mammalian) + positive in vivo micronucleus.

4. A possible fourth option was a mixture of option 2 for benzimidazoles and option 3 for non-benzimidazoles.

27. Members considered that a key step in considering criteria for the CMG should be possession of the benzimidazole ring although it was noted that compounds which also contained a carbamate moiety might have potential clastogenic activity. Members discussed whether the available intelligent Structure Activity Models (e.g DEREK) would be appropriate for structure activity predictions and agreed such models would be valuable with regard to the benzimidazole grouping but possibly of less value with regard to prediction of the influence of ring substituent groups on aneugenicity. In addition to structure, members considered that other key factors which might aid the identification of compounds within a benzimidazole common mechanism group could include evidence suggesting that positive in vitro aneugenicity correlated with in vivo mutagenicity. As an illustrative exercise the LOAELs for relevant aneugenicity effects in vitro and in vivo were reported in MUT/2006/8. As protocols for in vivo studies are designed to identify hazard and not response, there was a difficulty in ranking the potency of these compounds using in vivo data. It was therefore proposed that in vitro data might have some scope for this group of compounds.

28. Members noted that the diversity of the available genotoxicity data and test systems used made evaluation of inclusion into the common mechanism group very difficult. The committee commented that Structure Activity Relationships should also include any metabolites formed from parent pesticides or veterinary medicines. Members were informed by VMD that the data reviewed on omeprazole reflected a possible publication bias towards positive data because that available from comprehensive regulatory studies did not indicate that this compound was an in-vivo aneugen. Members felt that as aneugenicity was an endpoint and did not necessarily reflect a specific mechanism of action by itself, additional information would be required before assigning benzimidazoles to a CMG. The COM agreed that it was essential to have information on molecular interactions with mammalian tubulin as a first step in considering a CMG. It was noted that there were potentially different tubulin binding sites, and that the principle assumption of additivity had not been investigated for benzimidazoles. Members considered an important initial step to considering the development of a CMG would be would be to undertake studies of interaction between a number of compounds likely to be included in the group to demonstrate the nature of interactions and the absence of potential for a synergistic effect for tubulin binding.

29. Members considered the data reviewed on aneugenicity and agreed that the helminth and fungal data were relatively poor predictors of potential in-vivo aneugenicity. However they noted that there was a good correlation for mammalian tubulin binding with in vivo aneugenicity and also that there was also a good correlation between in vitro aneugenicity in mammalian cells and in vivo mutagenicity assays capable of detecting aneugenicity.

30. The committee agreed that it was possible to use a weight of evidence approach to derive conclusions on a CMG, where a scoring system using points for a number of parameters might be used. Members felt that the greatest weight should be allocated to in vivo aneugenicity detected from in vivo micronuclei formation with a sliding scale of grades used for other data. Supplementary data would include evaluation of toxicokinetic and metabolism data to ascertain whether an aneugenic metabolite was formed and in the case of compounds with no evidence of in-vivo aneugenicity to ascertain exposure of target tissues to the compound and metabolites.

31. Members considered that the following option for defining a common mechanism group for benzimidazoles could be used when combined with a weight of evidence scoring system. The secretariat were asked to consider options for the possible development of such a system:

Structure activity relationship + evidence for binding to mammalian tubulin and indication for aneugenicity in vitro + positive in vivo micronucleus assay

32. The COM discussed the relationship of aneugenicity to other target organ toxicological effects. It was noted that for the benzimidazole and other tubulin binding compounds reviewed, there was no common target organ affected in the available studies. Members queried how the data on common mechanism group for aneugenicity could be integrated into risk assessment for the compounds under review. The secretariat noted that there was a considerable amount of evaluation required to reach the stage of a risk assessment for a CMG and this might also involve seeking advice from COT. However one possible proposal based on approaches used for dioxins would be to select a potent aneugenic compound with a full toxicological database and relate all other compounds to this through a Toxicological Equivalency Factor (TEF) approach. The NOAEL for the reference compound would be used for risk assessment and the contribution of other compounds in the CMG towards any end effect incorporated into the risk assessment by calculating Toxic Equivalents compared to the referent compound (i.e. TEF x concentration in exposure source). A critical assumption in this approach was additivity of effect, and it was noted that the Committee had clearly identified this as a key data requirement before a benzimidazole common mechanism group could be derived. In addition, although not all compounds in the polychlorinated dibenzodioxin and polychlorinated dibenzofuran group had been tested for a complete range of target organ toxicological effects, there was a presumption that these compounds all produced a similar range of toxicological effects in experimental animals. The alternative option was to relate the CMG to aneugenicity alone and to develop a risk assessment model for this end effect in isolation.

33. The Committee discussed the available data on potential ranking of benzimidazole ranking using in vitro and/or in vivo data. Members noted the variance in reported potency depended on the experimental approach used, end point, selection of doses, and the relative potency of the parent compound and/or metabolites. In addition there would be additional uncertainty in extrapolating from in vitro potency to in vivo potency. A complete set of data using in vivo studies with a protocol designed to evaluate dose-response supported by toxicokinetic data for exposure of target organs would represent the ideal data set but this was unlikely to be achieved. Members agreed that a pragmatic ranking for selection for further studies could be obtained from in vitro studies but the in vitro data couldn't be used for in vivo potency ranking.

34. The secretariat agreed to report back to the Committee regarding the development of a weight of evidence approach to benzimidazole common mechanism grouping.

ITEM 6: MUTAGENICITY OF TEREPHTHALIC ACID (MUT/06/9)

35. Terephthalic acid (TPA) is a monomer used in the manufacture of polyethylene terephthalate (PET), a common food contact material. Low levels of TPA (0.8 mg/kg of food) have been detected following leaching from can coatings. Whilst terephthalic acid was found at 0.84 to 29 mg/kg in 33 samples of plastic packaging, no migration into food simulants was detected. Commission Directive 2002/72/EC set a specific migration limit for TPA of 7.5 mg/kg food.

36. In 2000, the COT concluded that based on the available data, the reported exposure to TPA was not of concern. However, in light of the urinary tumours in rats after long-term administration of TPA, the COM view was sought on the potential in vivo genotoxicity of this compound. The COM considered the limited genotoxicity data in 2001, including an intraperitoneal mouse bone marrow micronucleus test. All assays were negative; however additional assays were requested to support the whole data package including a metabolism (toxicokinetics) study to support the micronucleus test.

37. Subsequently, a multi-generation reproductive toxicity study was evaluated by the COT in 2005, which concluded that dietary administration of 20 mg/kg diet TPA for two successive generations did not result in any alterations in reproductive performance. However, changes (transitional epithelial hyperplasia, cystitis, inflammatory or mononuclear cell infiltration and haemorrhage) were observed in the urinary bladder of animals of both sexes receiving 20 mg/kg diet TPA, which instigated extensive histological examination. No changes were observed in the bladder of animals receiving 1 and 5 mg/kg diet TPA or in controls, therefore the COT determined a NOAEL of 5 mg/kg bw/day. This did not indicate a need to reduce the temporary TDI of 0.125 mg/kg bw/day proposed by the SCF. However, the COT decided that a final statement should not be issued until the COM had evaluated the additional mutagenicity data on TPA.

38. New studies were submitted to the COM. Two in vitro cytogenetics assays, a mouse metabolism study to support the earlier intraperitoneal micronucleus test and an in vivo UDS assay.

39. When TPA was tested in an in vitro cytogenetic assay in human lymphocytes it was found to be positive following 20h incubation in the absence of S9 metabolic activation. However, the possibility that clastogenicity was the result of reduced pH of the culture medium was considered (since pH had limited the maximum concentration tested in this assay). To address this, the assay was repeated using sodium terephthalate. This test material did not reduce the pH up to 10 mM (the maximum concentration for this assay). Small, but statistically significant increases in the percentage of aberrant cells were observed following 3 h incubation in the presence and absence of S9 metabolic activation. The study author suggested that these were not biologically relevant. The UDS assay was reported as negative.

40. The metabolism study reported that the test compound administered by an intraperitoneal dose was extensively absorbed into the systemic circulation and was widely distributed to all tissues, including the bone marrow, and was rapidly excreted via the urine as the sulphate conjugate. Members noted that a vehicle different to the one that had been used in the in vivo micronucleus study and considered that interpretation was difficult due to the large variability in tissue levels of TPA seen between animals in the same dose group. The committee felt that this metabolism study was not helpful in demonstrating target tissue exposure had been achieved in the micronucleus test. However, members noted there was evidence of systemic toxicity in the preliminary toxicity study and in the in vivo micronucleus test. Also, that there had been a reduction in the Polychromatic/Normochromatic erythrocyte ratio (P/N ratio) in the micronucleus test, which suggested that target tissue exposure to TPA, had occurred.

41. Regarding the in vivo UDS assay, a single 2000 mg/kg bw oral dose of TPA had been given to male rats. Groups of three rats were sampled at 2 and 16 hours post administration. Members agreed that this study had been adequately conducted to GLP and OECD Guideline 486 and was negative.

42. Members had reservations that the positive clastogenic result seen with TPA, in the in vitro cytogenetics assay with human lymphocytes, could be entirely explained by the associated reduction in pH. Members noted that the degree of damage was high and that there had not been a large shift in pH ( approximately 1 pH unit). Further, human lymphocytes were not considered to be particularly sensitive to pH induced chromosome damage.

43. The COM also had concerns over the negative result reported in the follow up human lymphocyte cytogenetics assay. Members considered that a larger number of cells should have been scored and that the effects seen in the controls were low. It was felt that the criteria for a positive response had not been fulfilled, but members could not be certain that sodium TPA produced no effect.

44. The COM requested further in vitro cytogenetic data to provide reassurance that the positive result seen with TPA was due to a lowering in pH and that the repeat test with sodium TPA was negative. This could be taken forward by the FSA.

45. Overall the committee concluded that:

i) That the metabolism study provided inconclusive evidence on whether the bone marrow was exposed to TPA, but noted that there was a presumption of systemic exposure regarding the in vivo micronucleus test where systemic toxicity and some evidence of bone marrow exposure had been seen.

ii) The in vitro cytogenetics test with sodium terephthalic acid was inconclusive and requested a further evaluation to include scoring more cells.

iii) The in vitro cytogenetics evidence was inconclusive on whether the terephthalate ion itself was an in vitro clastogen.

iv) Overall the evidence was consistent with a non-genotoxic mechanism for the bladder tumours seen in the rat carcinogenicity bioassay. This conclusion would be reinforced if further investigation of the in vitro, cytogenetics data confirmed a negative result.

46. The further presentation of results from the in vitro cytogenetics assay tabled at the COM meeting in human lymphocytes did not materially alter the COM's conclusions. The Chair asked the observer from BP for any comments. BP responded that they would undertake the appropriate work and forward the results to FSA.

ITEM 7: COMPARATIVE RISK ASSESSMENT PROJECT INITIAL OUTLINE DISCUSSION PAPER (MUT/06/10)

47. At the COC 2005 Horizon Scanning meeting, the secretariat proposed that a review on comparative risk assessment (CRA) should be conducted that compared the carcinogenic risk of selected environmental carcinogens with risks associated with familiar hazards which would be understood by a lay audience. COC Members ranked this as high priority but did not support the proposal to use smoking cigarettes as a comparator. COC Members suggested that the former Chief Medical Officer's 'risk scale' could be used as a possible alternative approach. COC Members also agreed with the COM's recommendation that both Committees should collaborate together to improve how advice on the potential carcinogenicity and mutagenicity of chemicals is presented to the general public.

48. The DH Toxicology unit and secretariat had drafted an initial outline discussion paper which provided an introduction to the current approaches used by the COC/COM to report information on risks, the possible approaches to undertaking comparative risk assessment, and how the outcomes of such approaches could be used to provide better communication of risks associated with environmental mutagens/carcinogens. The second part of the review, reported on publications which dealt with strategies for better risk communication.

Comparative risk Assessment.

49. One key objective which arose from the COC discussions was to attempt to compare risks of environmental carcinogenesis with other involuntary risks. Three approaches were identified, namely the Calman risk scale, the Paling Perspective scale, and the Duckworth 'riskometer'. Members' views on these approaches were sought. Members' views were also requested on the proposal to select potent genotoxic carcinogens and compare their evaluation using these different scales.

Risk communication

50. The usual outcome of COM/COC work is published statements designed to be used by Government Departments and Regulatory Agencies in formulating policy or risk management strategies. In a relatively small number of cases, a decision has been made to produce a non-technical summary which would be aimed at a wider audience.

51. A paper by Leiss et al was summarised and a copy of the recent Royal Society/FSA workshop on risk communications was appended. Also, a short overview of the COT discussions was provided for information.

52. The COM was asked whether there were any potential approaches outlined, which would be valuable for COC/COM, and whether the committee had any other ideas/areas for discussion on comparative risk assessment or risk communication of committee advice.

COM discussion

53. It was recognised that the committee's main role was communication of specialist scientific advice to government departments and regulatory agencies and that risk communication was mainly a matter for these groups. Members felt that the COM generally advised on hazard i.e. whether a particular chemical was mutagenic or not. It was difficult to convey the concept that a small increase in risk of mutagenicity equated to a small increase in risk of other hazards (such as cancer or effects on reproduction) and even harder to quantify the relationships between chemical induced mutagenicity and other potential end effects. The default assumption of no-threshold (in the absence of chemical specific evidence to the contrary) was supported by scientific data and had proved valuable for Regulatory Agencies and Government Departments. It was agreed that more work on the use of relative potency and development of Margins of Exposure approaches would be helpful.

54. The committee considered that non-technical summaries of technical statements should be published when possible, but agreed that this was not always appropriate. However, this should be acknowledged in published statements where non-technical statements were not published. Members considered that the discussion of the agenda item on metal hip replacements was an appropriate topic to consider drafting a non-technical summary. The secretariat would liaise with MHRA in this respect. Members felt that the OECD guidance on risk communication could provide a good template for considering non-technical summaries in the future.

55. With regard to a paper by Williams et al., 2004, members considered that of the suggested risk comparison options A and C were the most relevant to the COM. Option A referred to intrachemical comparisons, to show that a particular source of the same exposure was significant/negligible e.g. comparing exposure to mercury fillings with exposure to mercury from eating fish. Option C referred to comparison to background levels of risk e.g. comparing exposure to arsenic from treated wood to exposures from natural environmental sources. There would need to be consideration of such approaches on a case-by case basis.

56. Members considered the comparative risk scales reviewed were probably more relevant to COC discussions. Some members felt that a logarithmic scale of comparison similar to the Richter scale for earthquakes might be a useful approach if applied to carcinogenic risk. Generally the committee had concerns over the types of risk comparisons identified in the published literature. It was felt that it was not helpful to express mutagen/carcinogen risks estimates on an annual basis as this might be difficult for a wider audience to understand. Members considered that comparisons of carcinogenic risk estimates with actuarial risks (where the data existed to determine the actual risk) were not appropriate. Thus it would be difficult to convey the information on the distribution and uncertainty associated with risk estimates associated with carcinogen exposures and then compare to the actuarial risk estimates used in the scales (e.g the risk of death resulting from falling over in the bath). Members also agreed that comparisons of involuntary risks (e.g. to environmental contaminants) with voluntary risks should not be made and hence agreed with COC that smoking was not an appropriate comparator. It was also noted individual perception of the magnitude and acceptability of particular risks varied greatly.

57. Members noted that any adaptations to risk communication procedures used by the COM would need to be judged acceptable by an wider audience than the one for which scientific statements from COM were primarily used. Evaluating the success of any approach would be difficult. This discussion paper would be taken to the COC for further consideration. Members agreed that it would be helpful to have a presentation on comparative risk assessment and improving risk communication. The secretariat agreed to identify appropriate individuals but commented that it was unlikely that there would be sufficient time to arrange a presentation for the July COC meeting.

ITEM 8: BACKGROUND VARIATION IN CHROMOSOMAL ABERRATIONS IN PERIPHERAL BLOOD LYMPHOCYTES; DISCUSSION PAPER ON RISK FACTORS (MUT/06/11)

58. The COM had previously identified the need for further evaluation of the factors affecting the formation of biomarkers of genotoxicity in peripheral blood lymphocytes (PBLs) and for key guidance regarding the evaluation of results from biomonitoring studies of exposure to chemicals (see statement on pesticide applicators
http://www.advisorybodies.doh.gov.uk/pdfs/pesapp.pdf)

59. The discussion paper MUT/06/11 overviewed the published literature specifically in relation to studies which provided information on the background variation of the formation of chromosomal aberrations in PBLs. The review included information on a variety of assay procedures undertaken with PBLs including classical metaphase analysis using staining techniques such as Giemsa, the use of banding techniques such as G-banding to identify specific aberrations in individual or groups of chromosomes at metaphase, and the use of Fluorescence In Situ Hybridisation (FISH) techniques for individual and groups of chromosomes at metaphase and interphase. These different approaches varied in their suitability to detect different types of cytogenetic damage.

60. The data were reviewed with respect to the impact of age, sex, smoking, diet, micronutrient level, and polymorphisms on the level of chromosomal aberrations in control populations (a review of alcohol consumption and cytogenetic changes in PBLs would be brought to a future COM meeting). For some aspects, such as age and smoking, there was as much data available as had been previously identified for the COM review of micronuclei formation. For other potential factors such as diet and micronutrients, comparatively few data were retrieved. There were a number of papers that presented evaluation of combined data from several laboratories, although none were as comprehensive as the HUMN project data for micronuclei formation (see MUT/06/01). The review also considered the impact of different methods used on the background variance of chromosomal aberrations in PBLs in control populations.

61. Members asked for revisions to the classification of cytogenetic damage to be incorporated into the COM statement (thus asymmetrical damage was generally unstable whereas symmetrical damage was generally stable). Members agreed that a formal systematic review (meta-analysis) of cytogenetics studies would be very difficult given the heterogeneity of the methods used and end points analysed. It was suggested that a FUNEL plot could be used to evaluate for publication bias towards reporting of positive results. Overall members agreed that the secretariat had evaluated the possible causes of variance in the formation of chromosomal aberrations in PBLs and agreed that without a very large controlled study it would not be possible to quantify the impact of all the risk factors for variance in background chromosomal aberrations in PBLs. Members noted that some laboratories employed a finder/selector of acceptable metaphases for analysis and a different technician scored the slides. This would be a different situation to the approach used in the studies reviewed in the COM discussion paper.

62. Members asked for a number of minor areas of the paper to be further considered. This included the evidence, if any, for linearity in age related increases clastogenic changes in PBLs, a consideration of the findings reported by Galloway et al (Cytogenet Cell Genet, 20, 78-95, 1978) with regard to difference between males and females in hypoploidy after 48 and 72 hour incubation. The Committee agreed a number of conclusions with regard to the factors affecting background variance in chromosomal aberrations in PBLs which had been considered. No specific conclusion was reached on impact of diet in view of limited data available.

Conclusion; age

63. There was good evidence for an age related increase in chromosomal aberrations (excluding gaps). This included breaks, exchanges and aneuploidy. There was good evidence from studies using FISH that stable translocations also increased with age. The evidence regarding unstable chromosomal changes such as dicentrics was unclear, with both positive and negative findings reported, which may have been affected by the method used to score dicentrics (see assay variables below). It was also noted that smoking may be a risk factor for dicentric formation.

Conclusion smoking

64. The results of metaphase analysis studies were consistent with an effect for smoking on chromosomal aberrations, although it was difficult to assess the level of smoking required for an effect on chromosomes in view of the limitations of the smoking consumption data. Overall, the increase in unstable aberrations (e.g. dicentrics) was evident in heavy smokers (>20/d). G-banding studies supported this conclusion. There was less evidence for a cytogenetic effect of smoking from Fluorescence In Situ Hybridisation (FISH) studies. The retrospective evaluation of data from a number of laboratories concluded that there was not a statistically significant association between smoking and translocations (some evidence was presented for certain age groups). The differences between the data from metaphase analysis, G-banding and FISH may relate to the adequacy of the methods for evaluating unstable chromosomal changes, the size of FISH studies, and the limited number of heavy smokers included in the FISH studies. It was noteworthy that the limited data on multi vitamin intervention did not report an effect for vitamin C, E and Se intervention (12 weeks) with metaphase analysis for chromosomal aberrations (see below).

Conclusion; Gender

65. There was good evidence for sex chromosome non-disjunction and X-chromosome loss or gain in females, which was age related. There was evidence for sex- chromosome non-disjunction and Y-chromosome loss in males, but insufficient evidence regarding Y-chromosome gain in males. It was difficult to conclude whether the overall rate of aneuploidy differed between females and males based on the available metaphase analyses and G-banding studies. There was insufficient evidence to conclude whether there was any gender related differences in cytogenetic changes (e.g. the frequency of unstable chromosomal changes). There was no evidence from FISH studies for any gender related cytogenetic effects with the possible exception of malsegregation of the X-chromosome in females, an observation supporting the finding of aneuploidy in metaphase analyses and G-banding studies.

66. No specific conclusion was reached on impact of diet in view of limited data.

Conclusion; Micronutrients

67. There was no evidence from the available limited trials that vitamin supplementation independently affected cytogenetic damage in PBLs. However, the studies retrieved did not include a specific investigation of folate or vitamin B12 supplementation and thus the data could not be compared to the available data for MN formation in PBLs.

Conclusion; Effects of micronutrients on sensitivity to clastogens

68. There was some limited evidence that vitamin supplementation may affect sensitivity of PBLs to chemically induced cytogenetic damage, but the data were inadequate to draw any firm conclusions particularly with regard to specific vitamins that might be relevant with regard to reduction of chemically induced cytogenetic damage.

Conclusion; Individual variance

69. No definite conclusions can be reached regarding the effect of genotype on background frequency of chromosomal damage in PBLs. The available evidence regarding slow NAT2 acetylation may reflect exposure to tobacco smoke.

Conclusion; Assay variables

70. Interlaboratory trials using experimental studies and photomicrograph data from metaphase analyses reported considerable variation in results due to individual scorer selection of metaphases and scoring of aberrations with a low frequency (in particular unstable aberrations). A variation in the amount of cytogenetic damage assessed in metaphase analysis after radiation exposure was reported, which was a similar finding to that reported for MN formation in PBLs. It was noted that the variation in the reporting of dicentrics in metaphase analysis may be confounded by heavy smoking. There were relatively few data on variation in G-banding studies, but the available information for hypoploidy was consistent with that reported for standard metaphase analysis. The available studies on FISH analysis in PBLs suggested that this approach may be an appropriate method for identifying dicentrics. Variation in FISH studies due to selection of cells and scoring for other aberrations, in particular translocations has been reported. There was also the possibility of variation due to the hybridization techniques adopted. There was a quantitative assessment of the overall impact of assay variation in the assessment of cytogenetic damage in PBLs (as reported in HUMN project for MN formation.)

Overall impact of risk factors

71. There were comparatively fewer data for direct observation of chromosomal aberrations than were available for the evaluation of MN formation. Overall, it was suggested that the differences between assay methods, variables within methods and the endogenous factors of age and sex were relevant for the design of biomonitoring studies. Smoking had less impact (similar conclusion to that reported for MN formation). However there were insufficient data to draw conclusions regarding micronutrient such as folate and vitamin B12 with regard to cytogenetics.

72. Members were asked to consider a number of questions in the review, which were designed to help draft a statement on guidance for consideration in evaluating biomonitoring studies of chromosomal aberrations in PBLs.

73. Members agreed that a number of factors influenced the background frequency of chromosomal aberrations (including aneuploidy) in PBLs. These included age, heavy smoking (>20/d), gender (e.g. X chromosome loss or gain in females and Y chromosome loss in males) and individual variance. Members noted that it was unclear whether there was an effect for lower levels of smoking. Regarding age, it was felt that it was more helpful if possible to estimate the size of the effect on an annual basis.

74. Overall the COM agreed that the impact of risk factors affecting background variance of clastogenicity in PBLS was very similar to those affecting the formation on micronuclei with the exception of the gender effect (MN formation females> males) seen with regard to MN formation.

75. The Committee discussed the selection of most appropriate method for biomonitoring studies of chemical exposure using cytogenetics in PBLs. Regarding the different methods for measuring cytogenetic damage (e.g. metaphase analysis, G-banding and FISH) it was felt that the most appropriate method would depend on the endpoint, the sensitivity for detection of a clastogenic effect, the target population to be monitored and relevant exposures to mutagenic chemicals, and the availability of funds for the study. FISH was considered to be appropriate for the determination of long-term cumulative damage whereas classical metaphase analysis was appropriate for the determination of recent DNA damage. G-banding was considered to be generally to be technically difficult and costly to apply to routine studies of biomonitoring for exposure to mutagenic chemicals. Members agreed that if possible more information should be included on a number of factors that could affect background levels of cytogenetic damage. These included alcohol (which was to be reviewed), ionising radiation such as X-rays, viral infections, exercise, stress, health status, and occupations where exposure was predominantly in open situations where differences in the weather might potentially impact on chromosomal aberrations. The Committee noted the lack of appropriate studies investigating the effect of folate status on formation of chromosomal damage in PBLs.

76. Members were asked to pass any further comments to the secretariat on any issues raised in the review. It was hoped that a draft working paper would be brought to the October 2006 meeting.

ITEM 9: ANY OTHER BUSINESS

9.1 Proposed mutagenicity testing for chemicals under REACH

77. The HSE alerted the COM to a proposed mutagenicity strategy suggested by an Endpoint Working Group for industrial chemicals to be considered under REACH (Regulation Evaluation and Authorisation of Chemicals).

78. The committee was informed that the European REACH risk assessment process would have to consider a large number of industrial chemicals e.g. 10,000 chemicals. In order to limit the number of animal tests required and to devise a pragmatic approach the Endpoint Working Group had made a number of suggestions and approaches that could be taken to assess mutagenicity. This included no in vivo mutagenicity tests for a chemical with an Ames positive result where there were two negative mammalian cell tests; incorporation of an in vivo mutagenicity test into 28-day toxicity tests; no default second tissue in vivo test where a positive in vitro test had been obtained. HSE asked for advice on a number of questions which could be fed back to the EWG.

79. The Chairman expressed concern that the suggested approach would not be in accordance with the COM guidance and lacked a scientific rationale. It was suggested that the COM secretariat would need to provide a formal response to HSE regarding the testing issues raised by the EWG and that COM members should have an opportunity to discuss the REACH mutagenicity testing strategy with the EWG.

9.2 Procedure for holding committee meetings in open session

80. The committee was informed that a code of practice for open sessions of committee meetings had been drafted. Members were asked to forward any comments to the secretariat. Members noted that any proposal to allow observers up to 5 minutes to comment on each item would result in considerable disruption to Committee procedures.

ITEM 10: PAPERS FOR INFORMATION/WRITTEN COMMENTS

81. Do dose response thresholds exist for genotoxic alkylating agents? Jenkins G et al., 2005. Mutagenesis, 20, 389-398 (MUT/06/12). The Committee agreed that some discussion of this paper at the next meeting would be valuable.

82. Pidgeon N et al., 2005. Using surveys in public participation process for risk decision making: the case of the 2003 British GM Nation? Public debate. Risk Anal, (25)2:467-79 (MUT/06/13).

ITEM 11: DATE OF NEXT MEETING

83. 12 October 2006

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