Mutagenesis vol. 18 no. 5 pp. 401-404,
September 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
In vivo genotoxicity studies with 3-monochloropropan-1,2-diol
Public Health Group, Department of Health, Skipton House, 80 London Road, London SE1 6LH, 1Covance Laboratories, Harrogate HG3 1PY and 2Directorate of Health and Social Care – North, Sunley Tower, Manchester M1 4BE, UK 3Present address: Genetic Toxicology, AstraZeneca, Mereside, Alderley Park, Macclesfield SK10 4TG, UK
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
3-Monochloropropan-1,2-diol (3-MCPD) is a contaminant of polyamine flocculants used in the production of drinking water, but more significantly for human exposure it can arise also in certain foodstuffs containing acid-hydrolysed vegetable protein. It is carcinogenic in the rat, producing tumours in males in the testes, mammary gland and the preputial gland and also kidney tumours in both sexes. It has given positive results in in vitro mutagenicity studies, but there have been no satisfactorily conducted in vivo studies in somatic cells published in the peer reviewed literature. As a result, and because of the absence of appropriate in vivo evidence, several international regulatory agencies had previously judged it prudent to assume that 3-MCPD possessed mutagenic activity in vivo and considered 3-MCPD to be a genotoxic carcinogen. We present in this paper results from two in vivo mutagenicity studies with 3-MCPD, namely a bone marrow micronucleus test in the rat and unscheduled DNA synthesis in the rat liver, both conducted in accordance with relevant OECD protocols. These studies show that 3-MCPD does not possess genotoxic activity in vivo in the tissues examined. On the basis of these findings, and along with evidence that tumours may be induced by mechanisms involving either hormonal disturbances or sustained cytotoxicity, we believe that 3-MCPD may now be considered to be carcinogenic to rodents via a non-genotoxic mechanism.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
3-Monochloropropan-1,2-diol (3-MCPD) is a member of a group of compounds known collectively as chloropropanols, a group that includes the genotoxic animal carcinogen 1,3-dichloropropan-2-ol (Olsen, 1993
; Committee on Carcinogenicity of Chemicals in Food, Consumer Products and the Environment, 2001). 3-MCPD itself is an animal carcinogen producing tumours at various sites in male F344 rats (mammary tissue, testes and preputial gland) and renal tubular adenomas and carcinoma in both sexes of F344 rats (Sunahara et al., 1993; Lynch, 1998). It has given positive results in in vitro mutagenicity studies (Silhankova et al., 1982; Zeiger et al., 1988; Olsen, 1993; Lynch, 1998) but there have been no satisfactorily conducted in vivo studies in somatic cells published in peer reviewed journals.
As a result, and because of the absence of appropriate in vivo evidence, several international regulatory agencies had previously judged it prudent to assume that 3-MCPD possessed mutagenic activity in vivo and considered 3-MCPD to be a genotoxic carcinogen. Due to its mutagenic potential, practice in the UK had previously been to reduce exposure to 3-MCPD to as low a level as practicable. 3-MCPD has been detected as a contaminant of several foods and food ingredients, including acid-hydrolysed vegetable protein, malts and soy sauces (Food Standards Agency, 2001). The current approach in the UK is to ensure that levels of 3-MCPD in foods and food ingredients are <0.02 mg/kg based on 40% dry matter, the EU limit (European Commission, 2001). In respect of exposure through drinking water, the UK Drinking Water Inspectorate (1999) has set controls on impurity levels of 3-MCPD and on maximum usage rates of flocculant addition to water to achieve a maximum theoretical level in drinking water of 0.1 µg/l [note that this is identical to the level set in the EC Drinking Water Quality Directive (European Commission, 1998) for potentially genotoxic polymer-derived contaminants of drinking water such as acrylamide and epichlorohydrin].
The key missing evidence required to enable an adequate assessment of the health risks of 3-MCPD has been in vivo mutagenicity studies conducted to current standards. In this paper we present data on two new studies with 3-MCPD, namely a rat bone marrow erythrocyte micronucleus test and an assay of unscheduled DNA synthesis (UDS) in rat liver cells.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The methods used for the micronucleus and UDS assays were based on current regulatory guidelines for these tests (OECD, 1997
).
Reagents
3-MCPD (98.5% pure) was obtained from Sigma-Aldrich Chemical Co. (Gillingham, UK) and stored at room temperature in the dark. It was diluted in purified water prior to dosing. Negative control animals received purified water alone. 2-Acetamidofluorene (2-AAF) (Sigma Chemical Co., Poole, UK), suspended in corn oil, and dimethylnitrosamine (DMN) (Sigma Chemical Co.), dissolved in purified water, were used as positive controls for the UDS assay. Cyclophosphamide (CPA) (Aldrich Chemical Co., Gillingham, UK) was dissolved in saline to serve as the positive control for the micronucleus test.
Animals
Outbred Crl:HanWist (Glx:BRL) BR rats were obtained from Charles River UK Ltd (Margate, UK) or Harlan (UK) Ltd (Bicester, UK). The animals were housed in solid floored polypropylene cages (45 x 28 x 20 cm), with wood shavings for bedding. They were acclimatised for at least 6 days after arrival and were approximately 8 weeks of age (190–239 g) at the start of dosing. Animals were provided with a special-quality control (SQC) rat mouse maintenance diet [RM1 (E) SQC, Special Diets Services Limited, Witham, UK] ad libitum. All procedures carried out as part of this study were subject to the provisions of the UK Animals (Scientific Procedures) Act, 1986.
Dose selection
The doses of 3-MCPD selected for each assay were based on preliminary dose range finding experiments in which rats were given a single administration (UDS assay) or were dosed once daily for two consecutive days (micronucleus test) by oral gavage at a dose volume of 10 ml/kg. Signs of toxicity were then recorded. Maximum doses of 100 mg/kg and 60 mg/kg/day were selected for the UDS and micronucleus assays as being close to the maximum tolerated doses for single and double administration, respectively. Although signs of toxicity at these doses in the subsequent genotoxicity assays were limited, the results of the range finder demonstrated that it would not have been reasonable to administer appreciably higher doses. The UDS and micronucleus tests were performed using males only, as no substantial difference in toxicity was apparent between males and females.
Micronucleus test
Groups of six rats were dosed once daily by gavage for two consecutive days with the vehicle (water) or 3-MCPD at 15, 30 or 60 mg/kg/day. CPA was given as a single administration at 40 mg/kg, on the second day of dosing. All animals were killed and bone marrow sampled 24 h after the second 3-MCPD administration. Bone marrow smears were fixed in methanol and stained for 4 min in 12.5 µg/ml acridine orange made up in 0.1 M phosphate buffer, pH 7.4. Slides were rinsed in phosphate buffer and allowed to dry. Slides were randomised and 2000 polychromatic erythrocytes (PCE) scored for micronuclei per animal. The ratio of PCE to normochromatic erythrocytes (NCE) was determined based on a total of at least 1000 PCE + NCE.
UDS assay
Animals were dosed in groups of four with vehicle or 3-MCPD at 40 or 100 mg/kg via oral gavage. The assay was conducted as two experiments. In the first experiment, animals were sampled 2–4 h after administration using DMN as the concurrent positive control. In the second, rats were sampled 12–14 h after treatment using 2-AAF as the positive control. At the appropriate sampling time, each animal was maintained under deep anaesthesia with halothane to prevent recovery and the liver perfused with collagenase solution via the hepatic portal vein. The liver was cut free and the hepatocytes teased out into phosphate buffer. The separated hepatocytes were then resuspended in Williams E Medium–Complete and a sample of the suspension counted after staining with 0.4% (w/v) trypan blue. Each suspension was diluted to provide 
1.5 x 105 viable cells/ml and plated into 6-well multi-well plates (3 ml/well), each well containing a 25 mm round plastic Thermanox coverslip. Cultures were incubated at 37°C in an atmosphere of 5% CO2 in air for at least 90 min to allow the cells to attach. The medium was removed from the cells, the monolayers washed and medium containing 10 µCi/ml [3H]thymidine added. After 4 h incubation the medium was removed, the cells washed with three changes of Williams E medium-Incomplete (WE-I) containing 0.25 mM cold thymidine and the cultures incubated overnight in the same medium.
To prepare the coverslips for autoradiography they were washed with phosphate-buffered saline and the cells fixed with three changes of acetic acid:ethanol (1:3 v/v). They were then washed with purified water, allowed to dry and mounted onto microscope slides, cells side up. The slides were dipped in Ilford K2 liquid emulsion, which was then allowed to gel. The slides were packed in light-tight boxes containing desiccant and refrigerated for 14 days. At the end of this time, the emulsion was developed in Kodak D19 developer and fixed using Ilford Hypam fixer. The cell nuclei and cytoplasm were stained with Meyers haemalum/eosin Y, dehydrated in ethanol, cleared in xylene and mounted with coverslips. Grain counting was performed using a microscope with a video camera connected to an image analysis system (Perceptive Instruments, Haverhill, UK).
Each slide was examined to ensure that the culture was viable. For each cell analysed, the number of grains over the nucleus was first counted. The field of view was moved and counts obtained for three separate adjacent areas of cytoplasm of equal size. Nuclear and mean cytoplasmic grain counts were then recorded and the net grains/nucleus (NNG) determined. One hundred cells were analysed per animal.
Data analysis
Micronucleus test
For each group, inter-individual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity 
2 test (Lovell et al., 1989). The numbers of micronucleated PCE in each treated group were then compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine 2 (Lovell et al., 1989) and also compared with the laboratory historical negative control range.
UDS assay The population average NNG, the percentage of cells responding or in repair (i.e. % NNG) and the population average cytoplasmic and nuclear grain counts were calculated for each slide, animal and dose point (cells with 5 NNG or more were considered to be ‘in repair’). Results for 3-MCPD-treated animals were compared with concurrent negative controls.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Micronucleus test
The results of the micronucleus test are shown in Table I. Rats treated with 3-MCPD had group mean ratios of PCE to NCE which were lower than that seen in the vehicle control group. This is considered to be a sign of toxicity and a clear demonstration of exposure of the target cells. Group mean frequencies of micronucleated PCE were similar and not significantly different from the value for the concurrent vehicle control group and fell within the laboratory historical negative control range.
|
UDS assay
Summary data for the UDS assay are shown in Table II. Treatment with 3-MCPD at all doses yielded NNG values <0, producing group mean NNG values at the two sampling times in the range –3.3 to –2.4, well below the threshold value of 0 NNG required for a positive response (Kennelly et al., 1993
; OECD, 1997). No more than 0.3% cells were seen in repair at any dose of 3-MCPD.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
3-MCPD has a chemical structure which suggests that it may be metabolised to genotoxic intermediates, particularly glycidol. A number of studies have shown 3-MCPD to be mutagenic in the Ames assay both in the presence and absence of S9 metabolic activation (Stolzenberg and Hine, 1979, 1980
; Silhankova et al., 1982; Majeska and Matheson, 1983; Zeiger et al., 1988; Ohkubo et al., 1995). 3-MCPD has been reported to be mutagenic in mammalian cells (Olsen, 1993; Lynch, 1998) and to yeast (Rossi et al., 1983). Malignant transformation of mouse M2 fibroblasts in culture has also been demonstrated (Piasecki et al., 1990). In vivo mutagenicity studies have shown 3-MCPD to be negative in the dominant lethal test in the mouse and rat (Jones et al., 1969; Epstein et al., 1972; Jones and Jackson, 1976). However, there are no data in the published literature relating to in vivo studies in somatic cells.
As 3-MCPD is clearly mutagenic in vitro in the Salmonella assay and in the mouse lymphoma assay in the absence of metabolic activation, it can be assumed that non-microsomal pathways can generate DNA-reactive species in vitro. The new in vivo data indicate that activity is not expressed in bone marrow or liver. Consideration of the metabolic profile of 3-MCPD indicates that, in rats, the predominant urinary metabolite following oral or i.p. doses of 3-MCPD was ß-chlorolactic acid (Jones et al., 1978), a metabolite which is produced by an oxidative pathway not generating glycidol. Furthermore, a degradation product of ß-chlorolactic acid, namely oxalic acid, has been documented to induce the nephrotoxic effect which is a key feature of 3-MCPD toxicity (Jones et al., 1979; Olsen, 1993). One study (Jones, 1975) has shown that 3-MCPD may also be metabolised to glycidol by a minor pathway, but this then undergoes conjugation with glutathione and ultimately forms a mercapturic acid [N-acetyl-S-(2,3-dihydroxypropyl)cysteine] in urine of rats. This suggests that glycidol may arise at low levels, but that it is subsequently inactivated. While it is appreciated that the metabolism of 3-MCPD in rats has not been fully examined, the available evidence supports the findings from the two new in vivo mutagenicity studies, that significant DNA-reactive metabolites are not produced in the whole animal.
The negative findings in the in vivo studies are also consistent with postulated non-genotoxic mechanisms for tumour formation in rats (Lynch et al., 1998). Tumours were reported in both sexes in the kidney and in males only at hormonally responsive sites (i.e. the testes, mammary gland and preputial gland) at dose levels which exceeded the maximum tolerated dose (Sunahara et al., 1993). In the kidney, tumours in both sexes were benign (renal tubular adenoma) and these were accompanied by chronic progressive nephropathy. As discussed above, metabolism to ß-chlorolactic acid, and subsequently oxalate, is a major pathway in the rat. Oxalate is known to induce severe renal cytoxicity (Jones et al., 1979; Olsen, 1993) and this and other evidence, such as a study by Jones et al. (1978) in which crystals of oxalate were detected in the urine of rats treated with 3-MCPD (single dose of 100 mg/kg i.p.), supports a role for sustained cytotoxicity as a possible mechanism for the induction of kidney tumours in rats.
With regard to the sex-specific tumours in male rats (in the testes, mammary gland and preputial gland), it has been argued (Lynch et al., 1998) that the testicular tumours need to be viewed against the high spontaneous incidence of Leydig cell tumours common in the ageing F344 rat. Also, 3-MCPD has been shown to induce a prolonged increase in circulating hormone levels, a single i.p. dose of 80 mg/kg body wt causing increased serum levels of follicle stimulating hormone, luteinising hormone and prolactin (Morris and Jackson, 1978). It is possible that increases in the spontaneous rate of Leydig cell tumours may have been promoted by hormonal imbalance caused by 3-MCPD. Subsequently, the increase in tumours at other hormone-responsive sites (i.e. in the male mammary gland and the preputial gland) may be secondary to further hormonal disturbances, which are known to be induced by proliferating Leydig cells.
It is concluded that a non-genotoxic mechanism for the carcinogenicity of 3-MCPD in rodents is plausible and that these new negative in vivo mutagenicity studies suggest that reactive metabolites, if formed, do not produce genotoxicity in the bone marrow and liver.
The UK Department of Health referred these two in vivo studies to the Committtee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment and the Committee on Carcinogenicity of Chemicals in Food, Consumer Products and the Environment and they also concluded that 3-MCPD could be regarded as having no significant genotoxic potential in vivo (Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment, 2000; Committee on Carcinogenicity of Chemicals in Food, Consumer Products and the Environment, 2000).
|
Acknowledgements |
|---|
The financial assistance of the Drinking Water Inspectorate (an agency of the Department of the Environment, Food and Rural Affairs) in funding this work is gratefully appreciated. The opinions presented in this paper are those of the authors and in no way commit the Department of Health or other Departments.
|
Notes |
|---|
4To whom correspondence should be addressed. Tel: +44 20 7972 1765; Fax: +44 20 7972 5156; Email: stephen.robjohns{at}doh.gsi.gov.uk
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
Committee on Carcinogenicity of Chemicals in Food, Consumer Products and the Environment (2000) Statement on Carcinogencity of 3-Monochloro-propane-1,2-diol (3-MCPD), COC/00/S5. Available at www.doh.gov.uk/mcpd1.htm
Committee on Carcinogenicity of Chemicals in Food, Consumer Products and the Environment (2001) Statement on Carcinogenicity of 1,3-Dichloropropan-1-ol (1,3-DCP) and 2,3-Dichloropropan-1ol (2,3-DCP), COC/01/S1. Available at www.doh.gov.uk/cocdcp.htm
Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment (2000) Statement on Carcinogencity of 3-Monochloro-propane-1,2-diol (3-MCPD), COM/00/S4. Available at www.doh.gov.uk/mcpd2.htm
Drinking Water Inspectorate (1999) Polyamine Flocculants—Review of Conditions of Approval, Regulation 25, Letter 4/99 dated 12 November 1999. Committee on Chemicals and Materials of Construction for use in Public Water Supply and Swimming Pools. DWI, London, UK.
European Commission (1998) Directive on the Quality of Water Intended for Human Consumption, Council Directive 98/83/EC, 3 November 1998. European Commission, Brussels, Belgium.
European Commission (2001) Setting Maximum Levels for Certain Contaminants in Foodstuffs, Commission Regulation no. 466/2001, 8 March 2001. European Commission, Brussels, Belgium.
Epstein,S.S., Arnold,E., Andrea,J., Bass,W. and Bishop,Y. (1972) Detection of chemical mutagens by the dominant lethal assay in the mouse. Toxicol. Appl. Pharmacol., 23, 288–325.[CrossRef][Web of Science][Medline]
Food Standards Agency (2001) Survey of 3-Monochloropropane-1,2-Diol (3-MCPD) in Soy Sauce and Related Products, no. 14/01. Food Standards Agency. Available at www.foodstandards.gov.uk/science/surveillance/ fsis-2001/3
Jones,A.R., Davies,P., Edwards,K. and Jackson,H. (1969) Antifertility effects and metabolism of 
- and epi-chlorohydrins in the rat. Nature, 224, 83.[Medline]
Jones,A.R. (1975) The metabolism of 3-chloro, 3-bromo and 3-iodopropan-1,2-diol in rats and mice. Xenobiotica, 5, 155–165.[Web of Science][Medline]
Jones,A.R., Milton,D.H. and Murcott,C. (1978) The oxidative metabolism of alpha-chlorohydrin in the male rat and the formation of spermatoceles. Xenobiotica, 8, 573–582.[Medline]
Jones,A.R., Gadiel,P. and Murcott,C. (1979) The renal oxidative toxicity of the rodenticide alpha-chlorohydrin in the rat. Naturwissenschaften, 66, 425.[Medline]
Jones,P. and Jackson,H. (1976) Antifertility and dominant lethal mutation studies in male rats with dl-alpha-chlorohydrin and an amino-analogue. Contraception, 13, 639–646.[Medline]
Lovell,D.P., Anderson,D., Albanese,R., Amphlett,G.E., Clare,G., Ferguson,R., Richold,M., Papworth,D.G. and Savage,J.R.K. (1989) Statistical analysis of in vivo cytogenetic assays. In Kirkland,D.J. (ed.), Statistical Evaluation of Mutagenicity Test Data, UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III. Cambridge University Press, Cambridge, UK, pp. 184–232.
Kennelly,J.C., Waters,R., Ashby,J., Lefevre,P.A., Burlinson,B., Benford,D.J., Dean,S.W. and Mitchell,I.deG. (1993) In vivo rat liver UDS assay. In Kirkland,D.J. and Fox,M. (eds), Supplementary Mutagenicity Tests UKEMS Recommended Procedures. Cambridge University Press, Cambridge, UK, pp. 52–77.
Lynch,B.S., Bryant,D.W., Hook,G.J., Nestmann,E.R. and Munro,I.C. (1998) Carcinogenicity of monochloro-1,2-propanediol (alpha-chlorohydrin, 3-MCPD). Int. J. Toxicol., 17, 47–76.
Majeska,J.B. and Matheson,D.W. (1983) Quantitative estimate of mutagenicity of tris-[1,3-dichloro-2-propyl]-phosphate (TCPP) and its possible metabolites in Salmonella. Environ. Mutagen., 5, 478. (Abstract)
Morris,I.D. and Jackson,C.M. (1978) Gonadotrophin changes in male rats following a sterilising dose of alpha-chlorohydrin. Int. J. Androl., 1, 85–95.
OECD (1997) Genetic toxicology: micronucleus test. In OECD Guidelines for the Testing of Chemicals, Test Guideline 474. OECD, Paris, France.
OECD (1997) Genetic toxicology: unscheduled DNA synthesis (UDS) test with mammalian liver cells in vivo. In OECD Guidelines for the Testing of Chemicals, Test Guideline 486. OECD, Paris, France.
Ohkubo,T., Hayashi,T., Watanabe,E., Endo,H., Goto,S., Endo,O., Mizoguchi,T. and Mori,Y. (1995) Mutagenicity of chlorohydrins. Nippon Suisan Gakkai Shi, 61, 596–601. (In Japanese)
Olsen,P. (1993) Chloropropanols. In Joint FAO/WHO Expert Committee on Food Additives. Toxicological Evaluation of Certain Food Additives and Contaminants, WHO Food Additives Series no. 32. World Health Organization, Geneva, Switzerland, pp. 267–285.
Piasecki,A., Ruge,A. and Marquardt,H. (1990) Malignant transformation of mouse M2-fibroblasts by glycerol chlorohydrins contained in protein hydrolysates and commercial food. Arzneim.-Forsch/Drug Res., 40, 1054–1055.
Rossi,A.M., Migliore,L., Lascialfari,D., Sbrana,I., Loprieno,N., Tortoreto,M., Bidoli,F. and Pantarotto,C. (1983) Genotoxicity, metabolism and blood kinetics of epichlorohydrin in mice. Mutat. Res., 118, 213–226.[Medline]
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. Mutat. Res., 103, 77–81.[Medline]
Stolzenberg,S.J. and Hine,C.H. (1979) Mutagenicity of halogenated and oxygenated three-carbon compounds. J. Toxicol. Environ. Health, 5, 1149–1158.[Medline]
Stolzenberg,S.J. and Hine,C.H. (1980) Mutagenicity of 2- and 3-carbon halogenated compounds in the Salmonella/mammalian-microsome test. Environ. Mutagen., 2, 5946.
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, Vevey, Switzerland.
Zeiger,E., Anderson,B., Haworth,S., Lawlor,T. and Mortelmans,K. (1988) Salmonella mutagenicity tests: IV. Results from the testing of 300 chemicals. Environ. Mol. Mutagen., 11 (suppl. 12), 1–158.[Web of Science][Medline]
Received on September 26, 2001; revised on May 15, 2003; accepted on May 22, 2003.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||