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Introduction

1. The COM had requested a discussion paper on the comparison of the in-vivo rat liver UDS assay and the in-vivo Comet assay during the horizon scanning discussion in October 2005. (http://www.advisorybodies.doh.gov.uk/pdfs/mut0521.pdf)

This request had originated from the discussion at the joint COM/COC meeting on the use of target organ mutagenicity in the risk assessment of genotoxic carcinogens held in June 2005 (http://www.advisorybodies.doh.gov.uk/com/tom.htm) The DH Toxicology Unit and Secretariat had drafted a discussion paper based on available published literature which provided comparative data for 16 compounds ^1-16 (http://www.advisorybodies.doh.gov.uk/pdfs/mut063.pdf) The majority of the data were obtained from a limited number of papers which had been expressly aimed at examining the general applicability of the two assays under consideration.^{9,10,11,12,13} It was difficult to make direct comparisons between the two assays as for several compounds UDS data were only available from rats and Comet data from mice and there were differences in dose levels used, routes of administration. Some of the available Comet assays had investigated multiple organs in rats and mice.¹³ The Committee was asked to evaluate the data presented and to draw generic conclusions as far as was possible and to identify individual compounds which might require additional evaluation. (In respect of the latter request it is noted that that a full evaluation of the mutagenicity data of the chemicals under consideration was not part of the remit of the current review.)

COM consideration of data presented on rat liver UDS and Comet (liver) data.

Overall comments

2. The Committee agreed that a broad interpretation of the data presented could be derived for results obtained for rat liver using both the UDS and Comet assays. In this respect members considered that a significant reservation in reaching conclusions related to the quality of the available Comet assays and in particular the use of isolated nuclei in the Comet assay.^11,13 Members noted that procedures were still being developed for different organs in the Comet assay and hence it was difficult to draw any conclusions on the utility of the assay at the present time. Members also commented that in general intra peritoneal dosing for the rat liver UDS and Comet assays could complicate the interpretation of data.

3. A broad interpretation of the current review paper, accepting the results as presented based only on response in rat liver was that there was a good degree of concordance in positive results with six chemicals (aflatoxin^9,10,13, benzidine^1,10,13, 2,4 diaminotoluene^10,11,13, 1,2 dimethylhydrazine^3,7,12, diethylnitrosamine^9,13, methylmethane sulphonate^11,13), negative results in three chemicals acrylamide^4,8, benzidine^8,13, and o-anisidine^1,13), with discordant results in chorodibromomethane^13,14 (positive in Comet¹³ and negative in rat liver UDS¹⁴).

Comments on data on specific chemicals reviewed

4. The Committee briefly discussed the data presented on acrylamide and chlorobromodiemthane in more detail.

5. With regard to acrylamide (an established genotoxic carcinogen in rodents), although the data suggested a negative result for both rat liver UDS and for rat liver Comet (using oral administration), it was considered based on the relatively poor results with concurrent positive control (MMS) that the Comet assay had underperformed in this instance. In additional acrylamide had produced borderline positive or equivocal results in other organs including brain and testes.⁸ Members commented that positive Comet data were available for acrylamide in a range of mouse tissues following intraperitoneal dosing and negative data were reported for CYP2E1 null mice which indicated that the metabolite of acrylamide glycinamide, mediated the genotoxicity of acrylamide in rodents.¹⁷ It was agreed that there was no need for further consideration arising from the current comparative review of results obtained from rat liver UDS and Comet assays.

6. With regard to chlorodibromomethane (a water disinfection by product), members recalled that the COM and COC had considered this compound, which induced malignant liver tumours in rats, in detail in 1994/5, and had concluded that it was not a genotoxic carcinogen on the basis of adequate negative bone marrow MN assays and rat liver UDS assays.¹⁸ The available Comet data indicated a clear a positive result in both rats and mice in the liver.¹³ However members expressed reservations regarding the conduct of these assays which used isolated nuclei and considered that a repeat test for rat liver Comet would be appropriate supported, if possible, by a repeat rat liver UDS assay conducted concurrently.

Use of Comet assay to identify potential cancer target organs in rodents.

7. The use of the Comet assay to identify cancer target organs in rodents was not the primary focus of the current review. However members noted the positive results in Comet assays of the bladder mice and rats dosed with o-anisidine and the finding of a positive results in the stomach in rats and mice dosed with benzyl acetate.¹³

COM conclusions

8. Members concluded that the approach used in the review was relevant to empirical comparisons between in-vivo mutagenicity assays but that any discussion on the role of the UDS assay and the Comet assay in overall testing strategy also needed to include consideration of using in-vivo assays in the context of the data provided by the in-vitro assessment of mutagenicity.

9. The Committee concluded that the current comparative review of the rat liver UDS and Comet assays should be considered in the context of the available published data reviewed, the limitations of the experiments considered, the ongoing development of the Comet assay for rodent tissues and the possibility of relevant data held by industry but not available in the public domain. Overall it was agreed that;

i) the available data was consistent with the view that rat liver UDS assay and the rat liver COMET assay had broadly similar response with a limited number of known rodent carcinogens.

ii) a further repeat rat liver Comet assay was desirable for chlorodibromomethane.

iii) no further evaluation of the mutagenicity acrylamide was required at the present time for the comparative review of results obtained in the rat liver UDS and Comet assays. .

December 2006

References

1. Ashby J, Lefevre P.A, et al (1991) The non-genotoxicity to rodents of the potent rodent bladder carcinogens o-anisidine and p-cresidine. Mut. Res, 250 115-133

2. Ashby J, Beije B. (1985) concomitant observations of UDS in the liver and micronuclei in the bone marrow of rats exposed to cyclophosphamide or 2-acetylaminofluorene Mut. Res. 150 383-392

3. Brendler-Schwab, SY, Volkner W, Fautz R, Herbold BA. (2002) Dimethylhydrazine: a reliable positive control for the short sampling time in the UDS assay in vivo. Mut. Res, 520 57-62

4. Butterworth BE, Eldridge SR, et al (1992) Tissue-specific genotoxic effects of acrylamide and acrylonitrile. Environ. Mol. Nut. 20 148-155

5. Franke, S.I, Pra D, et al (2005) Influence of orange juice over the genotoxicity induced by alkylating agents. Mutagenesis 230 279-283

6. Hartmann A, Schmacher M, et al (2004) Use of the alkaline in vivo Comet assay for mechanistic genotoxicity investigations. Mutagenesis 19(1) 51-59

7. Kennelly, JC (1995) Design and interpretation of rat liver UDS assays. Mutagenesis 10(3) 215-221

8. Maniere I, Godard T, et al (2005) DNA damage and DNA adduct formation in rat tissues following oral administration of acrylamide. Mut. Res. 580 110-129

9. Mirsalis JC, Tyson KC and Butterworth BE (1982). Detection of Genotoxic carcinogens in the in-vivo-in-vitro hepatocytes DNA repair assay. Environmental Mutagenesis, 4, 553-562.

10. Mirsalis JC, Tyson CK, Steinmetz KL, Loh EK, Hamilton CM, Bakke JP, Spalding JW. (1989). Measurement of unscheduled DNA synthesis and S-phase synthesis in rodent hepatocytes following in-vivo treatment: testing of 24 compounds. Envionmental Molecular Mutagenesis, 14, 155-164.

11. Sasaki YF, Nishidate E, et al (1997) Simple detection of chemical mutagens by the alkaline single cell electrophoresis assay in multiple mouse organs. Mut.Res. 391 215-231.

12. Sasaki YF, Saga A, et al (1998) Organ-specific genotoxicity of the potent rodent colon carcinogen 1,2-dimethylhydrazine and three hydrazine derivatives. Mut.Res. 415 1-12.

13. Sekihashi K, Yamamoto A, et al (2002) Comparative investigation of multiple organs of mice and rats in the comet assay. Mut. Res. 517 53-74.

14. Stocker KJ, Statham J et al (1997) Assessment of the potential in vivo genotoxicity of three trihalomethanes: chlorodibromomethane, bromodichloromethane and bromoform. Mutagenesis 12 169-173.

15. Tinwell H., Lefevre P.A., Ashby J. (1998) Relative activities of Methyl Methane sulphonate (MMS) as a genotoxin, clastogen and gene mutagen to the liver and bone marrow of MutaMouse mice. Environ. Molec. Mutagenesis. 32 163-172.

16. Vrzoc M, Petras M.L. (1997) Comparison of alkaline single cell gel (COMET) and peripheral blood micronucleus assays in detecting DNA damage caused by direct and indirect acting mutagens. Mut. Res. 381 31-40.

17. Ghanayem BI, Witt KL, Kissling GE, Tice RR, Reico L.(2005) Absence of acrylamide-induced genotoxicity in CYP2E1-null mice: evidence consistent with a glycinamide-mediated effect. Mutation Research, 578, 284-97.

18. Committees on Toxicity, Mutagenicity and Carcinogenicity of chemicals in Food, Consumer Products and the Environment (COT, COM, COC) (1994). Annual report, publ HMSO.

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