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Introduction

1. 1,3-dichloropropan-2-ol (1,3-DCP) is a member of a group of chemicals called chloropropanols, which includes 3-monochloropropane-1,2-diol (3-MCPD) and 2,3-dichloropropan-1-ol (2,3-DCP). 1,3-DCP and 3-MCPD can be present as process contaminants in some foodstuffs and as contaminants of polyamine flocculants in drinking water. 1,3-DCP was last considered by the COM and the COC in 2001. COM concluded that it would be prudent to consider 1,3-DCP as potentially genotoxic in vivo but agreed that it should be tested for genotoxicity in vivo using the approach set out in the COM guidelines.

2. 1,3-DCP has been considered by the COC, originally in 1991 and more recently in 2001. The COC noted that in a carcinogenicity study undertaken by Hercules Inc.1, 1,3-DCP was administered in the drinking water of 80 male and 80 female Wistar rats for 104 weeks. Statistically significant positive trends were observed for benign and malignant tumours (intermediate and high dose level) in the liver, kidney, tongue/oral cavity and thyroid. The COC concluded that "it was prudent to assume 1,3-DCP is a genotoxic carcinogen and that exposure to 1,3-DCP should be reduced to as low as technologically feasible".2

COM evaluation: 2001

3. Members agreed that the metabolism of 1,3-DCP was likely to produce a reactive epoxide intermediate that could damage DNA. Members were aware that 1,3-DCP had been found to be mutagenic to Salmonella typhimurium strains TA1535 and or TA 1003-10. Studies with mammalian cells have produced increased frequencies of sister chromatid exchanges and chromosome aberrations11,12. A positive result has been obtained in a mouse lymphoma assay13,14. 1,3-DCP was negative in the wing spot test in Drosophila melanogaster (a somatic mutation and recombination test)15. No in-vivo mammalian studies had been carried out.

4. The Committee concluded that it would be prudent to regard 1,3-DCP as potentially genotoxic in-vivo and agreed that it should be tested for genotoxicity in-vivo using the approach set out in the COM guidelines.

COM evaluation: 2003

5. The Committee considered two new in-vivo genotoxicity studies at its May meeting. These comprised a rat bone-marrow micronucleus test and a rat liver unscheduled DNA synthesis (UDS) assay, both of which are widely used to assess genotoxicity in vivo.

Rat in-vivo bone-marrow micronucleus test16

6. The assay followed the current OECD guideline (No. 474). The highest dose used in the study was selected so that it would produce some signs of toxicity, but not severe effects, based on the results of a range-finding study. In the main study, 1,3-DCP was administered once daily for two consecutive days to groups of six male Han Wistar rats at doses of 25, 50 and 100 mg/kg. Bone marrow was harvested 24 hours after the final dose. A single sex study was considered adequate because no substantial difference in toxicity was observed between males and females in the range-finder.

7. Weight loss (25 mg/kg), piloerection and weight loss (50 mg/kg) and piloerection, weight loss and lethargy (100 mg/kg) were observed in treated animals, indicating that the test was conducted at adequate doses. The Committee noted that the ratios of polychromatic to normochromatic erythrocytes (PCE/NCE) were variable amongst individual animals of the control group. However, the mean value was within the range of historical control group mean data. Rats treated with 1,3-DCP at all doses exhibited group mean PCE/NCE ratios that were similar to those of the control group and within the normal range. Therefore toxicity to the bone marrow was not demonstrated, although 1,3-DCP clearly caused systemic toxicity.

8. There were no statistically significant increases in micronucleus frequency at any dose of 1,3-DCP. The positive control agent, cyclophosphamide, produced a clear increase in micronuclei.

Rat liver in-vivo UDS assay17

9. The UDS assay protocol conformed to the current OECD guideline (No. 486). Based on the results of the range-finder for the micronucleus study, the study was conducted in male rats and the highest dose was 100 mg/kg. Single doses of 40 and 100 mg/kg were administered to groups of four male Han Wistar rats. Hepatocytes were isolated for analysis for UDS by the autoradiographic technique after 12-14 hours in the first study (3 rats per dose group) and at 2-4 hours in the second study (3 rats per dose group).

10. In the 2-4 hour experiment, piloerection was observed in all 1,3-DCP treated rats and lethargy was observed at 100 mg/kg. There were no clinical signs following dosing in the 12-14 hour experiment. There was no evidence for any increase in UDS at either dose level or time point. The positive control compounds 2-AAF and DMN both gave clear positive results.

COM discussion

11. Members agreed that the two new studies met the previously stated requirement that 1,3-DCP should be tested for genotoxicity in-vivo using the approach set out in the COM guidelines. The studies were adequately conducted and gave clear negative results, and therefore Members considered that these studies provided evidence that 1,3-DCP was not an in-vivo mutagen. Members then gave consideration as to possible mechanisms whereby mutagenic activity observed in vitro was not expressed in vivo.

12. The role of metabolism in the in-vitro mutagenicity is unclear. Most bacterial studies have shown mutagenicity both in the presence and absence of metabolic activation. Two studies indicated that metabolism increased the mutagenicity in TA100 and/or TA1535 7,9, whereas one reported a decrease in TA100 revertants in the presence of metabolic activation8. Activation was found to result in reduced frequency of sister chromatid exchanges in V79 cells11.

13. It has been postulated that metabolism to epichlorhydrin could be responsible for the mutagenicity of 1,3-DCP18. There is some evidence that bacteria can convert 1,3-DCP to epichlorhydrin19, which could account for the direct activity seen in the bacterial mutagenicity tests, although no data are available with respect to Salmonella typhimurium. It has been suggested that epichlorhydrin may be formed non-enzymically during the pre-incubation stage of the SOS chromotest assay with Escherichia coli stain GC47986.

14. An alternative active metabolite is 1,3-dichloroacetone. It has been postulated that this may be formed from 1,3-DCP by action of alcohol dehydrogenase20 or CYP2E121. Glutathione conjugation is believed to be a detoxification pathway since 1,3-DCP depletes glutathione both in vitro22 and in vivo23, and glutathione depletion has been shown to potentiate the toxicity of 1,3-DCP to rat hepatocytes22.

15. One known route of 1,3-DCP metabolism in rats involves hydroxylation to 3-monochloropropane-1,2-diol (3-MCPD), accounting for 1 to 14% of a 10 mg subcutaneous dose in rats24. The COM has concluded that 3-MCPD can be regarded as having no significant genotoxic potential in vivo25.

16. The COM considered that the metabolism of 1,3-DCP had not been fully elucidated. Metabolic activation in vivo to two active metabolites had been postulated. In both cases the compound formed would be expected to be rapidly de-activated in vivo by glutathione. In one case deactivation would also occur by the action of epoxide hydrolase. Thus once formed, the active metabolite is rapidly detoxified and hence 1,3-DCP would be unlikely to have significant activity in vivo. This is supported by the negative results obtained in the two new in-vivo mutagenicity assays.

Conclusions

17. The Committee concluded that both the rat bone-marrow micronucleus test and the rat liver UDS test had been carried out to an acceptable standard and were negative. Thus the additional information recommended by the COM as being necessary to provide adequate reassurance that the mutagenic activity seen in vitro was not expressed in vivo had now been provided.

18. The Committee noted the uncertainties with regard to routes of metabolic activation of 1,3-DCP and agreed that the two new mutagenicity studies supported the view that reactive metabolites, if formed, did not produce genotoxicity in vivo in the tissues assessed.

19. The Committee concluded that 1,3-DCP can be regarded as having no significant genotoxic potential in vivo.

October 2003

References

1. Hercules Inc. (1986). 104-Week Chronic Toxicity and Oncogenicity Study with 1,3-Dichlor-propanol-2-ol in the Rat. Unpublished Report No. 017820 from Research and Consulting Company AG, ltingen, Switzerland.

2. Carcinogenicity of 1,3-dichloropropan-2-ol (1,3 DCP) and 2,3 -dichloropropan-1-ol (2,3 DCP). COC Statement - May 2001 (COC/01/S1) http://www.doh.gov.uk/coc.htm

3. Hahn H. Eder E and Deininger C. (1991). Genotoxicity of 1,3-dichloro-2-propanol in the SOS chromotests and in the Ames tests. Elucidation of the genotoxic mechanism. Chem. Biol. Interactions, 80: 73-88.

4. Silhankova L. Smid F, Cerna M, Davidek J, and Velisek J. (1982). Mutagenicity of glycerol chlorohydrins and of their esters with higher fatty acids present in protein hydrolysates. Mutation Research, 103: 77-81.

5. Stolzenberg S.J. & Hine C.H. (1980). Mutagenicity of 2- and 3-carbon halogenated compounds in the Salmonella/mammalian-microsome test. Environmental Mutagenesis, 2: 59-66.

6. Zeiger E, Anderson B, Haworth S, Lawlor T, Mortelmans K. (1988) Salmonella mutagenicity tests: IV. Results from the testing of 300 chemicals. Environ Molec Mutagen 1 (S12): l - 158.

7. Nakamura A. Tateno N, Kogima s, Kaniwa M-A and Kawamura T (1979). The mutagenicity of halogenated alkanols and their phosphoric acid esters for Salmonella typhimurium. Mutation Research, 66: 373-380.

8. Ohkubo T, Hayashi T, Watanabe E, Endo H, Goto S, Mizoguchi T, Mori Y (1995) Mutagenicity of chlorohydrins. Nippon Suisan Gakkaishi 61: 596-601 (in Japanese).

9. Gold MD, Blum A and Ames B (1978). Another flame retardant, Tris-(1,3-dichloro-2-propyl)-phosphate, and its expected metabolites are mutagens. Science 200: 785-787.

10. Lynn RK, Wong K Garvie-Gould C and Kennish JM. (1981). Disposition of the flame retardant, Tris (1,3-dichloro-2-propyl) phosphate in the rat. Drug metabolism and Disposition, 9: 434-451.

11. von der Hude W. Scheutwinkel M, Gramlich U, Fissler B and Basler A (1987). Genotoxicity of three-carbon compounds evaluated in the SCE test In-Vitro. Environmental Mutagenesis, 9: 401-410.

12. Putman D. & Morris M. (1990). Sister chromatid exchange and chromosome aberration assay in Chinese hamster ovary cells, 1,3-dichloro-2-propanol. Microbiological Associates, Inc. Laboratory study No. T9250.337 NTP.

13. San R. & Blanchard M. (1990). L5178Y TK+/- mouse lymphoma mutagenesis assay, 1,3-dichloro-2-propanol. Microbiological Associates, Inc. Laboratory study No. T9250.702.

14. Henderson LM, Bosworth HJ, Ransome SJ, Banks SJ, Brabbs CE and Tinner AJ . (1987) An assessment of the mutagenic potential of 1,3-dichloro-2-propanol, 3-chloro-1,2-propanediol and a cocktail of chloropropanols using the mouse lymohoma TK locus assay. Unpublished report No ULR 130 ABC/861423 from Huntingdon Research Centre, Huntingdon, Cambridgeshire England.

15. Frei H. & Wurgler F (1997) The vicinal chloroalcohols 1,3-dichloro-2-propanol (DC2P), 3-chloro-1,2-propanediol (3CPD) and 2-chloro-1,3-propanediol (2CPD) are not genotoxic in-vivo in the wing spot test of Drosophila melanogaster. Mut Res 394: 59-68.

16. Howe, J. (2002) 1,3-Dichloropropan-2-ol (1,3-DCP): Induction of micronuclei in the bone marrow of treated rats. Report no 2150/1-D6172 from Covance Laboratories Ltd, Harrogate, North Yorkshire, England. Available from the Food Standards Agency.

17. Beevers, C. (2003) 1,3-Dichloropropan-2-ol (1,3-DCP): Induction of unscheduled DNA synthesis in rat liver using an in vivo/in vitro procedure. Report no 2150/3-D6173 from Covance Laboratories Ltd, Harrogate, North Yorkshire, England. Available from the Food Standards Agency.

18. Jones AR & Fakhouri G (1979). Epoxides as obligatory intermediates in the metabolism of halohydrins. Xenobiotica 9: 595-599.

19. Nakamura T, Nagasawa T, Yu F, Watanabe I & Yamada (1992). Resolution and some properties of enzymes involved in enantioselective transformation of 1,3-dichloro-2-propanol to (R)-3-chloro-1,2-propanediol by Corynebacterium sp. strain N-1074. J. Bacteriol 174: 7613-7619.

20. Eder E & Weinfutner (1994). Mutagenic and carcinogenic risk of oxygen containing chlorinated C-3 hydrocarbons: Putative secondary products of C-3 chlorohydrocarbons and chlorination of water. Chemosphere 29: 2455-2466.

21. Hammond AH & Fry (1997). Involvement of cytochrome P4502E1 in the toxicity of dichloropropanol to rat hepatocyte cultures. Toxicol 118: 171-179.

22. Hammond AH, Garle MJ & Fry (1996). Toxicity of dichloropropanols in rat hepatocyte cultures. Environ Toxicol Pharmacol 1: 39-43.

23. Fry JR, Sinclair D, Holly Piper C, Townsend S-L & Thomas NW (1999). Depression of glutathione content, elevation of CYP2E1-dependent activation, and the principal determinant of fasting-mediated enhancement of 1,3-dichloro-2-propanol hepatotoxicity in the rat. Food Chem Toxicol 37: 351-355.

24. Koga M, Inoue N, Imazu K, Yamada N & Shinoki Y (1992). Identification and quantitative analysis of urinary metabolites of dichloropropanols in rats. J Univ Occup Environ Health Japan 14 13-22.

25. Mutagenicity of 3-monochloropropane-1,2-diol (3-MCPD). COM Statement - October 2000 (COM/00/S4) http://www.doh.gov.uk/com.htm

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