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Introduction

1. 2,3-dichloropropan-1-ol (2,3-DCP) is a member of a group of chemicals called chloropropanols, which includes 3-monochloropropane-1,2-diol (3-MCPD) and 1,3-dichloropropan-2-ol (1,3-DCP). 2,3-DCP and 3-MCPD can be present as process contaminants in some polyamine flocculants used in water treatment and may therefore potentially be present in drinking water. 1,3-DCP is found as a process contaminant in food stuffs where acid-hydrolysed vegetable protein has been used as an ingredient in soy sauce or similar oriental sauces. It is not currently known if 2,3-DCP is present in food. 2,3-DCP was last considered by the COM in 2001. COM concluded that it would be prudent to consider 2,3-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.

COM evaluation 2001

2. Members were aware that there was very little data on the absorption, distribution, and excretion of 2,3-DCP. Theoretically, 2,3-DCP could be metabolised to produce epichlorohydrin (and subsequently glycidol) and therefore there were structural alerts for genotoxicity and carcinogenicity.

3. The Committee noted 2,3-DCP was mutagenic in Salmonella typhimurium strains TA 100 and TA 1535 in a study with and without metabolic activation¹, and mutagenic in another Ames test.² Positive results were also obtained for sister chromatid exchange with Chinese Hamster V79 cells both with and without metabolic activation.³ No in-vivo studies in mammals have been carried out.

4. The Committee concluded that in the limited studies conducted, 2,3-DCP was genotoxic in-vitro with and without metabolic activation in bacterial and mammalian cells.

COM evaluation 2004

5. The Committee considered two new in-vivo genotoxicity studies at its February 2004 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 test⁴

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 70, 140 and 280 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. Clinical signs of toxicity, including lethargy, eye closure and piloerection were observed at 140 and 280 mg/kg bw. A dose related decrease in the mean ratios of polychromatic to normochromatic erythrocytes (PCE/NCE) compared to vehicle control was documented indicating that the test material was toxic to the bone marrow.

8. There were no statistically significant increases in micronucleus frequency at any dose of 2,3-DCP. The positive control agent, cyclophosphamide, produced a clear increase in micronuclei.

Rat liver in-vivo UDS assay⁵

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 280 mg/kg. Single doses of 110 and 280 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. The investigators reported notable weight loss in animals dosed with 280 mg/kg bw at 12-14 hours post-dose. It was noted that for a one animal, at 110 mg/kg bw in the 12-14 hour trial, the number of cells scored was below the recommended number. However there were sufficient cells scored from all other animals in the study. There was clear evidence of toxicity at the top dose level used. There was no evidence of an increase in Net Nuclear Grain counts in treated animals at either 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 2,3-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 2,3-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. Members agreed that 2,3-DCP was metabolised to 2,3-dichloroacetaldehyde and from this to the corresponding acid. One research group had provided some in vitro data to suggest that induction of CYP2E1 resulted in 2,3-DCP mediated hepatotoxicity and glutathione depletion.⁶ Members noted the findings of Koga et al⁶ which suggested dechlorination/ hydroxylation occurred but agreed these authors had not provided evidence for the formation of an epoxide. Members concluded that there were insufficient data to draw conclusions on the metabolic activation of 2,3-DCP but overall the evidence suggested metabolic activation of 2,3-DCP differed from 1,3-DCP.

13. The COM considered that the metabolism of 2,3-DCP had not been fully elucidated. Metabolic activation in vivo to active metabolites had been postulated but had not been proven. The Committee agreed that 2,3-DCP was not mutagenic in the two tissues assessed which provided some assurance that active metabolites were not formed in-vivo.

Conclusions

14. 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.

15. The Committee noted the uncertainties with regard to routes of metabolic activation of 2,3-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.

16. The Committee concluded that 2,3-DCP can be regarded as having no significant genotoxic potential in vivo.

May 2004

References

1. 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.

2. 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.

3. 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.

4. Beevers C. (2003) 2,3-Dichloropropan-2-ol (1,3-DCP): Induction of micronuclei in the bone marrow of treated rats. Report no 2150/2-D6172 from Covance Laboratories Ltd, Harrogate, North Yorkshire, England. Available from the Food Standards Agency.

5. Beevers, C. (2003) 2,3-Dichloropropan-2-ol (1,3-DCP): Induction of unscheduled DNA synthesis in rat liver using an in vivo/in vitro procedure. Report no 2150/4-D6172 from Covance Laboratories Ltd, Harrogate, North Yorkshire, England. Available from the Food Standards Agency.

6. Hammond AH and Fry JR . (1999). Effect of cyanamide on toxicity and glutathione depletion in rat hepatocyte cultures: differences between two dichloropropanol isomer. Chemical Biological Interactions, 122, 107-115.

7. Koga M, Inoue N, Imazu K, Yamada N and Shinoki T (1992). Identification and quantitative analysis of urinary metabolites of dichloropropanols in rats. J UOEH (University Occupational and Environmental Health, 14, 13-22.

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