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Mutagenicity of 3-monochloro propane 1,2-diol (3-MCPD)
COM statement COM/00/S4 - October 2000

Introduction

1. 3-Monochloro propane 1,2,-diol (3-MCPD) can be present as a contaminant in epichlorhydrin/amine copolymers used as flocculants or coagulent aids in water treatment. These polyamine flocculants have been available for many years as approved products for use in water treatment and thus 3-MCPD may be present in drinking water from their use. 3-MCPD is a member of a group of contaminants known as chloropropanols. This group includes some known genotoxic carcinogens in animals such as 1,3 dichloropropan-2-ol. The COM was asked in 1999 to evaluate the available mutagenicity data on 3-MCPD and to provide conclusions for the Committee on Carcinogenicity (COC) who had been asked to consider the carcinogenicity data on 3-MCPD. The COM was aware that 3-MCPD had been detected as a contaminant of several foods and food ingredients, including acid hydrolysed vegetable protein (acid-HVP) and that the EU Scientific Committee for Food had published an opinion in 1994 where it was agreed that 3-MCPD should be regarded as a genotoxic carcinogen.(1)

2. In 1999 the COC noted that in a carcinogenicity study undertaken by Sunhara et al (1993) 1,3-MCPD was administered via drinking water to groups of 50 male and 50 female (aged 6 weeks at start) F344 rats for a period of 104 weeks. Statistically significant increases in leydig cell adenomas (intermediate and high dose level) and mammary gland fibroadenomas (high dose level) had been noted in males and a statistically significant increase in kidney tumours had been noted in females at the high dose level. The COC had noted in 1999 that the high dose level had exceeded the Maximum Tolerated Dose. The COC therefore asked the COM for an assessment of the mutagenicity data on 3-MCPD as part of its evaluation of the mechanism for the carcinogenic effects seen in rats.

COM evaluation: 1999

3. The Committee was aware that 3-MCPD had been detected as a contaminant of savoury food ingredients, including acid hydrolysed vegetable protein (acid-HVP) and that the EU Scientific Committee for Food had published an opinion in 1994 where it was agreed that 3-MCPD should be regarded as a genotoxic carcinogen.(1) The Committee also had access to published mutagenicity data on 3-MCPD, a safety evaluation prepared by CanTox.Inc (Ontario, Canada) for the International Hydrolysed Protein Council,(3) a review document published by the Institute of Toxicology, National Food Agency of Denmark,(4) and one in-vivo mutagenicity submitted in an in-confidence basis.(5) In reviewing these documents, members commented that the available metabolism data on 3-MCPD were relatively old and focused on metabolic pathways following intraperitoneal administration. There was no oral mass balance investigation available. The Committee considered the proposal by CanTox Inc regarding the formation of bacterial-specific mutagens and agreed that there was no evidence to support this speculation.

4. The Committee reached the following conclusions on the mutagenicity data available in 1999.

i) 3-MCPD was mutagenic in Salmonella typhimurium in the absence of exogenous metabolic activation.(6-9) The addition of S-9 mix did not increase the mutagenic response observed.

ii) Positive results have also been reported in the mouse lymphoma assay in the presence of metabolic activation,(4) but the full report of the study was not available to the Committee. Positive results were also reported in tests in yeast (Schizosaccharomyces pombe)(10) and in tests for Sister Chromatid Exchange in mammalian cells.(4)

iii) The Committee concluded that 3-MCPD had mutagenic activity in-vitro.

iv) Negative results have been reported from a bone marrow micronucleus assay in mice using a single oral dose of up to120 mg/kg bw and sampling of bone marrow at 24, 48 or 72 hours post administration. The authors stated that higher doses would result in significant weight loss and mortality. The Committee noted that no evidence for a reduction in the ratio of polchromatic to normochromatic erythrocytes (i.e. ratio of PCE/NCE) and thus there was no evidence to show exposure of the bone marrow to the test material and its metabolites had occurred.(5)

v) The Committee agreed that no conclusions could be drawn from the investigation of colonic micronuclei in mice (5) in view of the limited database available for this assay or from the inadequately reported dominant lethal assays.(11,12)

vi) The Committee agreed that further negative results in an in-vivo mutagenicity test in a second tissue namely rat liver UDS were required in order to provide adequate reassurance that the activity seen in-vitro is not expressed in-vivo.

COM evaluation: 2000

5. The Committee considered two new in-vivo mutagenicity studies commissioned by the U.K Drinking Water Inspectorate at its October 2000 meeting. These comprise a rat bone-marrow micronucleus test and a rat liver UDS assay, both of which are widely used to assess genotoxicity in-vivo.

Rat in-vivo bone-marrow micronucleus test (13)

6. The assay protocol conformed to OECD 474. A single sex (male (Crl: HanWist BR): group size 6) was used as there was no substantial sex differences in toxicity. The top dose was selected from a range-finding study in which single oral doses of between 20-100 mg/kg bw were administered once daily for two consecutive days to groups of male and female rats. Dose levels of 60 mg/kg bw resulted in severe toxicity and some deaths. In the main study, doses of 15, 30 and 60 mg/kg bw were given for two consecutive days. Signs of toxicity were seen at the top dose level (piloerection) which was also associated with a clear reduction in polychromatic erythrocytes to normochromatic erythrocytes, indicating that bone marrow cytotoxicity (and hence that 3-MCPD and/ or its metabolites reached the bone-marrow).

7. There was no increase in the number of micronucleated PCEs at any dose level in 3-MCPD treated animals (2000 polychromatic erythrocytes scored/animal). The positive control, cyclophosphamide produced a clear increase in micronuclei.

Rat Liver UDS assay (14)

8. The UDS assay protocol conformed to OECD protocol 486. The top dose, a single oral dose of 100 mg/kg bw was chosen on the basis of a sighting toxicity study which had shown severe toxicity at oral doses of 150 mg/kg bw. In the main study single oral doses of 40 mg/kg bw or 100 mg/kg bw were administered to male rats (Han Wistar) and hepatocytes recovered for UDS analysis using autoradiography after 12-24 hours (4 animals/dose level) and 2-4 hours (5 animals/dose level). No signs of toxicity were seen at either dose level. There was no evidence for any increase in UDS at either dose level or time point. The two positive control substances (2-AAF and DMN) both gave clear positive results.

COM Discussion

9. Members agreed that 3-MCPD has a chemical structure which suggests that it may be metabolised to genotoxic intermediates (particularly glycidol). 3-MCPD was clearly mutagenic in-vitro in the salmonella assay and in the mouse lymphoma assay in the presence of metabolic activation.

10. The committee noted that the predominant urinary metabolite in rats fed or given intraperitoneal doses of 3-MCPD was b -chlorolactic acid (15), i.e. by a pathway not producing glycidol or other genotoxic intermediates. A degradation product of b-chlorolactic acid, namely oxalic acid, has been documented to induce the nephrotoxic effects seen with 3-MCPD. (5,16) One study has also shown that 3-MCPD may be metabolised by a minor pathway and undergo conjugation with glutathione to ultimately form a mercapturic acid in urine of rats (N-acetyl-S-(2,3-dihydroxypropyl) cysteine (17), suggesting the formation of a reactive metabolite glycidol at low levels that are subsequently inactivated. The COM considered that the metabolism of the 3-MCPD in rats had not been fully examined, but agreed that evidence from the two new in-vivo mutagenicity studies supported the view that reactive metabolites were not produced in the tissues where genotoxicity was assessed. Thus the Committee reached the following conclusions.

COM Conclusion

11. 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.

12. The Committee agreed that the major urinary metabolite b-chlorolactic acid in rats was formed by oxidation of 3-MCPD and that the two new mutagenicity studies supported the view that reactive metabolites if formed did not produce genotoxicity in-vivo in the tissues assessed.

13. The Committee concluded that 3-MCPD can be regarded as having no significant genotoxic potential in-vivo.

References

1. SCF (1994). Opinion on 3-Monochloro-propane, 1,2-diol (3-MCPD). Expressed 16 December 1994. Reports of the Scientific Committee for Food (thirty-sixth series), 1994.

2. Sunahara G, Perrin I, and Marchessini M (1993). Carcinogenicity study on 3- monochloro propane 1,2,-diol (3-MCPD) administered in drinking water to Fischer 344 rats. Report No RE-SR93003 Nestec Ltd, Research and Development Switzerland.

3. Lynch BS, Bryant DW, Hook GJ, Nestmann ER, and Munro IC (1998). Carcinogenicity of monochloro-1,2-propanediol (a-chlorohydrin, 3-MCPD). International Journal of Toxicology, 17, 47-76.

4. Olsen P (1993). Chloropropanols In JECFA. Toxicological Evaluation of Certain Food Additives and Contaminants. 41st Meeting of WHO Food Additives Series, 32, 267-285. World Health Organisation, Geneva, Switzerland.

5. Jaccaud E and Aeschbacher HU (1989). Evaluation of 3-chloro-1,2-propanediol (3-MCPD) in the bone marrow and colonic micronucleus test in mice. Unpublished report No 1265, pp1-57, Nestec Ltd, Research Centre.

6. Zeiger E, Anderson B, Haworth S, Lawlor T and Mortlemans K (1988). Salmonella mutagenicity tests: IV. Results from the testing of 300 chemicals. Environmental and Molecular Mutagenesis, 11, (S12), 1-158.

7. 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.

8. Stolzenberg SJ and Hine CH (1979). Mutagenicity of halogenated and oxygenated three-carbon compounds. Journal of Toxicology and Environmental Health, 5, (6), 1149-1158.

9. Stolzenberg SJ and Hine CH (1980). Mutagenicity o 2- and 3- carbon halogenated compounds in the Salmonella/mammalian microsome test. Environmental Mutagenesis, 2, 5946.

10. Rossi AM, Miglore L, Lasialfari D, Sbrana I, Loprieno N, Tororeto M, Bidoli F and Pantarotto C (1983). Genotoxicity, metabolism and blood kinetics of epichlorhydrin in mice. Mutation Research, 118, 213-226.

11. Jones AR and Murcott C (1976). Antifertility and dominant lethal mutation studies in male rats with dl-alpha-chlorohydrin and an amino analogue. Contraception, 13 (5), 639-646.

12. Epstein SS, Arnold E, Andrea J, Bass W, and Bishop Y (1972). Detection of chemical mutagens by the dominant lethal assay in the mouse. Toxicology and Applied Pharmacology, 23, 288-325.

13. Marshall R M (2000). 3-MCPD: induction of micronuclei in the bone-marrow of treated rats. Covance Laboratories Report Number 1863/2-D5140.

14. Fellows M (2000). 3-MCPD: Measurement of Unscheduled DNA synsthesis in rat liver using an in-vitro/in-vivo procedure. Convance Laboratories Report Number 1863/1-D5140.

15. Jones, A R, Milton, D H, and Murcott, C (1978). The oxidative metabolism of a-chlorohydrin in the male rat and the formation of spermatoceles. Xenobiotica, 8, 573-582.

16. Jones, A R, Gadiel, P and Murcott C (1979). The renal toxicity of the rodenticide a-chlorohydrin in the rat. Naturwissenschaften, 66, 425.

17. Jones A R (1975). The metabolism of 3-chloro, 3-bromo, and 3-idopropan 1,2-diol in rats and mice. Xenobiotica,,5 (3), 155-165