Mutagenesis, Vol. 14, No. 2, 207-215,
March 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Evaluation of 10 aliphatic halogenated hydrocarbons in the mouse bone marrow micronucleus test
1 Istituto Superiore di Sanita', Laboratory of Comparative Toxicology and Ecotoxicology, Viale Regina Elena, 299-00161 Rome, Italy, 2 Istituto di Ricerche Biomediche `Antoine Marxer' RBM S.p.A., 10010 Colleretto Giacosa, Italy and 3 Research Toxicology Centre S.p.A., Via Tito Speri, 12-00040 Pomezia, Italy
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Abstract |
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Ten halogenated aliphatic hydrocarbons (carbon tetrachloride, 1-chlorohexane, 2,3-dichlorobutane, 1,2-dichloroethane, 1,2-dichloroethylene, 1,3-dichloropropane, hexachloroethane, 1,1,2-trichloroethane, 1,2,3-trichloropropane and 1,1,3-trichloropropene), previously assayed in genetic assays in fungi, were evaluated in the mouse bone marrow micronucleus test in order to assess their genotoxicity in vivo. All chemicals were administered once i.p. at 40 and 70–80% of their respective LD50 to male and female CD-1 mice, 24 and 48 h before killing. All treatments produced evident clinical symptoms, but no marked depression of bone marrow proliferation. No statistically significant increases in the incidence of micronucleated polychromatic erythrocytes over the control values were observed at any sampling time with any of the 10 halogenated hydrocarbons assayed. The comparison of the results obtained in this study with the findings provided by in vitro micronucleus assays on the same chemicals, reported by other authors, indicate that mouse bone marrow is weakly sensitive to the genotoxic effects induced by halogenated hydrocarbons in other test systems. This suggests that the role of such an assay in carcinogen screening may be questionable for this chemical class. An examination of mouse bone marrow micronucleus test results with the halogenated aliphatic hydrocarbons classified as carcinogens by IARC supports this conclusion.
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Introduction |
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Since its introduction in the early 1970s (Matter and Schmid, 1971
; Heddle, 1973) the mouse bone marrow micronucleus test has been extensively used in genetic toxicology for the assessment of in vivo mutagenicity of natural and man-made chemicals. At present, the mouse bone marrow micronucleus test plays a central role in many testing strategies (Carere et al., 1995), being regarded as a relatively simple but reliable method, capable of providing evidence for in vivo genotoxicity resulting from different mechanisms. Based on the extensive experience gained world wide, the test protocol has been refined in order to increase its sensitivity and accuracy and such improvements have recently been incorporated in the updated OECD guideline for the conduct of micronucleus test in rodents (OECD, 1998).
Several attempts were made in the past to evaluate, on the basis of literature data, the predictive value of the micronucleus test with respect to chemical carcinogenesis (Mavournin et al., 1990; Shelby et al., 1993; Benigni, 1995). Such estimates showed variable correlation values and were flawed to various extents by uncertainties on the quality of experimental data and/or by the inadequate reporting of the studies. In order to overcome these difficulties, the Mammalian Mutagenicity Study Group of the Environmental Mutagen Society of Japan (JEMS MMS) has recently coordinated a large collaborative study entailing the analysis in the micronucleus test of ~100 chemicals previously classified by IARC as belonging to Group 1 (human carcinogen), 2A (probable human carcinogen) or 2B (possible human carcinogen) (Morita et al., 1997). The generation of such a large database provided the opportunity for a detailed and reliable analysis of the correlation between mouse micronucleus test and rodent carcinogenicity. The analysis highlighted some chemical class specificity, which suggests that the methodology could be poorly sensitive to the genetic effects exerted by some classes of chemical carcinogens, including halogenated hydrocarbons.
Chemical class specificity is an important trait in the operational performance of an assay, which should be taken into account in the selection of tests in new chemical screening, as well as in the evaluation of existing data in chemical safety assessment. This information is most relevant when it concerns chemical classes with wide environmental diffusion and human exposure, such as halogenated hydrocarbons. Therefore, in order to contribute to elucidation of the sensitivity of rodent bone marrow assays to halogenated hydrocarbons, in this paper we present the results obtained in mouse micronucleus assays with 10 chlorinated aliphatics. These chemicals were tested in the framework of a coordinated study on the genetic toxicity of halogenated aliphatics applying an extensive test protocol entailing treatment of animals of both sexes at two doses with two sampling times. Samples of the same test chemicals were also tested in the in vitro micronucleus assay in other laboratories, which reported independently the results obtained (Doherty et al., 1996; Tafazoli and Kirsh-Volders, 1996, Tafazoli et al., 1998). This also provided the opportunity to evaluate the performance of the mouse micronucleus test with respect to in vitro tests addressing the same end-point.
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Materials and methods |
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Chemicals
Chemical names, structures, CAS numbers, purity and sources of the halogenated aliphatic hydrocarbons evaluated in this study are presented in Table I
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Animals
Crl: CD-1 (ICR) BR mice, weighing ~25–35 g at the time of killing, were purchased from Charles River Italia S.p.A. (Calco, Lecco, Italy). Shortly after arrival, animals were randomly allocated to treatment groups. Mice were housed in polycarbonate cages, 5 animals/cage, by sex, under the following ambient conditions: temperature 22 ± 2°C, relative humidity 55 ± 10%, dark/light cycle 12 h. Filtered fresh water and a pelleted balanced rodent chow were supplied ad libitum. Animal care, treatments and killing were conducted in strict accordance with Directive 86/609/EEC on the protection of laboratory animals.
Treatments
Treatments were carried out either at the Rome Toxicology Centre (RTC, Pomezia, Rome, Italy) or at the Istituto di Ricerche Biomediche `Antoine Marxer' (RBM S.p.A., Colleretto Giacosa, Italy) using fairly comparable experimental protocols. The compounds 1,1,3-trichloropropene, 2,3-dichlorobutane, 1,2-dichloroethane, hexachloroethane and carbon tetrachloride were assayed at RTC; 1,1,2-trichloroethane, 1,2-dichloroethylene, 1,3-dichloropropane, 1,2,3-trichloropropane and 1-chlorohexane were assayed at RBM. Vehicle and positive control substances were tested at both laboratories. Slides of substances tested at RTC were scored by two experienced readers. Slides of substances assayed at RBM were scored by a single reader. In order to minimize possible bias due to variability between scorers, for each substance the whole slide set, including vehicle and positive controls, was scored by a single reader. The distribution of tasks is shown in Table II.
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Test substances were administered by single i.p. injection to groups of five male and five female mice at ~40 and 70–80% of their LD50. Acute toxicity was determined in preliminary toxicity trials following the EEC Guideline B.1 and the OECD Guideline 401. For 1,2-dichloroethane and 1,1,2-trichloroethane, dose levels were selected on the basis of published LD50 values. When the i.p. LD50 exceeded 4.0 g/kg body wt, the maximum dose level used was 4.0 g/kg and the lower dose level was 2.0 g/kg body wt. Olive oil was used as vehicle at 10 ml/kg body wt. Animals were killed by cervical dislocation under pentobarbital anaesthesia (1 ml/kg body wt 5% v/v solution, i.p.), 24 and 48 h after treatment. Doses, sampling times and toxicity findings are summarized in Table II
. The positive control substances colchicine (COL) and mitomycin C (MMC), both from Sigma Chemical Co., were dissolved in water and given to groups of three (at RBM) or five (at RTC) male and female mice. At RTC, colchicine (1.0 mg/kg body wt) and mitomycin C (2.0 mg/kg body wt) were administered 24 h before sacrifice. At RBM colchicine (1.5 mg/kg body wt) and mitomycin C (8.0 mg/kg body wt) were administered 24 and 48 h before killing, respectively.
Slide preparation
After death femurs were rapidly removed, cut at the proximal end and flushed with fetal calf serum in order to extract bone marrow cells. Cell suspensions were centrifuged for 10 min at 1000 g, the supernatant carefully taken off and the pellet smeared on clean microscope slides. Slides were air dried and stained with May–Gruenwald and Giemsa solutions in phosphate buffer (pH 6.8).
Scoring
1000–2000 polychromatic erythrocytes (PCE) per animal were scored for the presence of micronuclei using coded slides. The ratio polychromatic/normochromatic erythrocytes (PCE/NCE) was calculated by counting 1000 PCE/animal in each microscope field.
Statistics
The incidences of micronuclei in control and treated groups were compared by the 
2 test, controlling for within-group heterogeneity by analysis of variance. The PCE/NCE ratio in control and treated groups were compared by the t-test. Variability between readers was tested by ANOVA.
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Results |
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All test chemicals produced, at the dose levels assayed, more or less severe clinical signs (Table II
). In some cases (1-chlorohexane, 1,2-dichloroethylene, 1,3-dichloropropane, 1,2,3-trichloropropane) the top dose, originally set at 80% of LD50, had to be lowered to 70% of LD50 because of the severity of symptoms. Sporadic deaths occurred in some treated groups (i.e. 24 and 48 h after high dose treatment with 1,1,3-trichloropropene); dead mice were replaced by reserve animals, taken from satellite groups included in each experiment.
The results of mouse bone marrow micronucleus tests with the halogenated aliphatic hydrocarbons selected for this study are summarized in Tables III–XII. For each chemical, the average incidence of micronucleated PCEs in each treatment group, as well as the number of micronucleated PCEs/scored PCEs and the PCE/NCE ratio (mean of the ratio values for the individual animals) are shown. Five of the test chemicals (1,2-dichloroethane, hexachloroethane, carbon tetrachloride, 2,3-dichlorobutane and 1,1,3-trichloropropene) were assayed at the same time, thus they share vehicle and positive controls groups. Different set of slides from these animals were scored by two microscopists (B and C, see Table II). Two-way ANOVA showed no significant difference between the two scorers (F = 1.104, P = 0.346).
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Despite the evident signs of acute toxicity, most of the halogenated compounds tested did not exhibit significant toxicity on bone marrow cells. No marked decrease in the PCE/NCE ratio was observed at any sampling time in females; in males, significant decreases in the PCE/NCE ratio were only observed in the low dose groups 48 h after administration of carbon tetrachloride and 1-chlorohexane. On the other hand, these findings were not confirmed by the high dose groups. Following treatments with colchicine and mitomycin C, a slight decrease in the PCE/NCE ratio was observed in almost all experiments, suggesting that positive controls exerted a mild toxic effect on bone marrow cells. This effect did not achieve statistical significance in all cases, because of the wide dispersion of individual animal scores.
The incidence of micronucleated PCEs in vehicle-treated mice (males 0.74
, range 2.5–0; females 0.68, range 2.0–0) was within the accepted spontaneous range for this strain of mouse (Salamone and Mavournin, 1994), with no significant sex difference.
Examining the results from treated male and female animals, either combined or separately, no statistically significant increases in the incidence of micronucleated PCEs over the control values were observed at any sampling time with any of the 10 halogenated hydrocarbons assayed.
Statistically significant increases in the incidence of micro-nucleated PCEs over the control values were observed following treatments with the positive control substance mitomycin C for both sexes. The comparison of results obtained at 24 h (Tables III, V, VI, IX and XII) with those obtained at 48 h (Tables IV, VII, VIII, X and XI) shows that mitomycin C was relatively more effective as an inducer of micronuclei in PCEs at the shorter harvest time, according to the time course of micronuclei induction reported in the literature (Tice et al., 1990). Also, colchicine, employed as an additional positive control substance, produced significant increases in micronucleated PCEs in both sexes, confirming the sensitivity of the experimental system to the detection of both clastogenic and aneugenic agents.
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Discussion |
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The results presented in the previous section indicate that all 10 halogenated hydrocarbons tested are unable to induce significant increases in micronuclei in bone marrow cells of CD-1 mice.
The compounds tested in this study are a subset of a larger group of halogenated aliphatic hydrocarbons previously assayed in fungi (Crebelli et al., 1988, 1992) and used to derive quantitative structure–activity relationship models for aneuploidy induction (Benigni et al., 1993; Crebelli et al., 1995). This subset of congeneric molecules was selected on the basis of structural and electronic parameters in order to be representative of the whole group and used for the further assessment of the genotoxicity of haloalkanes in vitro and in vivo.
The results of in vitro micronucleus assays were reported independently (Doherty et al., 1996; Tafazoli and Kirsch-Volders, 1996; Tafazoli et al., 1998) and are summarized herein in Table XIII. These studies showed that all chemicals induced significant increases in micronuclei in at least one cell type. The characterization of micronuclei for kinetochore content by immunofluorescent labelling and fluorescence in situ hybridization (Doherty et al., 1996) demonstrated the induction of both clastogenic and aneugenic effects for six compounds (carbon tetrachloride, 1,2-dichloroethane, 1,2-dichloroethylene, 1,3-dichloropropane, 1,2,3-trichloropropane and 1,1,3-trichloropropene). Moreover, evidence of DNA damaging properties was obtained for all chemicals, excluding carbon tetrachloride and hexachloroethane, by means of single cell gel electrophoresis (Tafazoli and Kirsch-Volders, 1996; Tafazoli et al., 1998), basically confirming the findings of other in vitro mutagenicity short-term assays performed on this class of chemicals (data reviewed in Doherty et al., 1996).
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Several lines of evidence indicate that short-chain halogenated hydrocarbons require biotransformation to reactive metabolites in order to exert genotoxic or toxic effects (Ahmed et al., 1980
; Anders and Jakobson, 1985; Macdonald, 1982; Raucy et al., 1993). Also, this group of compounds proved to be in general more active in in vitro genotoxicity assays in the presence of exogenous metabolic activation (Tafazoli and Kirsch-Volders, 1996; Tafazoli et al., 1998) or in metabolically competent cell lines (Doherty et al., 1996). Yet, as shown in this study, the administration of the same compounds to mice in vivo failed to exert detectable clastogenic/aneugenic effects in bone marrow cells.
In order to reconcile the apparent discrepancy between the results of in vitro and in vivo assays, a few points need to be taken into account. Firstly, despite the high doses applied, which sometimes exceeded the currently recommended limit dose of 2 g/kg (OECD, 1998) and the evident clinical symptoms produced by treatments, no toxicity to bone marrow was revealed by the concurrent determination of PCE/NCE ratios. Even in the absence of relevant toxicokinetic data, this result suggests that tissue concentrations of putative active metabolites of the parent compounds were too low to exert detectable genotoxicity in the bone marrow. In this respect it should be noted that, even though the evidence of systemic toxicity demonstrates the presence of biologically effective doses of the administered compounds in blood, and hence in bone marrow, the concentration of active metabolites may be considerably lower in the target tissue. In the case of haloalkanes in particular, symptoms of acute toxicity are mainly related to the depressant action of the parent compounds on the central nervous system, caused by their incorporation into the nerve cell membrane with impairment of ionic transfer (Savoilanen, 1977; McFarland, 1986), whereas genotoxic effects involve reactive derivatives mainly produced by liver metabolism (Ahmed et al., 1980; McDonald, 1982; Anders and Jakobson, 1985; Raucy et al., 1993). For this reason, the evidence of acute toxicity does not imply that effective concentrations of active metabolites were achieved in bone marrow. Consequently, the negative results obtained in this study should not be taken alone as proof of absence of genotoxicity in vivo, because of the possible activity of the chemicals at other sites more accessible to short-lived metabolites. On the other hand, the possibility that a chemical inactive in bone marrow assays may elicit a genotoxic effect in other tissues is widely acknowledged and accepted by regulatory bodies which recommend the consideration of at least another tissue, in addition to bone marrow, for the assessment of in vivo genotoxicity.
The apparent lack of sensitivity of the bone marrow micronucleus test to haloalkanes raises some concern in view of the role of this assay in carcinogen prescreening. In this respect, it can be observed that five of the chemicals tested with negative results in this study provided at least some evidence of carcinogenic activity in rodents (Table XIII). A compilation of results in mouse bone marrow micronucleus tests with the halogenated aliphatic hydrocarbons classified in IARC Groups 1–2B is given in Table XIV. The data, largely based on the results reported by Morita et al. (1997), show that within this chemical class only 1/10 of carcinogens elicited an unambiguous positive response in the mouse bone marrow micronucleus assay. Interestingly, most of the carcinogens listed in Table XIV did not target the haematopoietic system, suggesting that target specificity may be a relevant feature affecting the outcome of in vivo genotoxicity assays with chemical carcinogens. No established non-carcinogens were available for the analysis, consequently no further evaluation of specificity and predictive values can be done. The data, however, clearly show that the mouse bone marrow micronucleus test displays low sensitivity in the screening of carcinogenic halogenated hydrocarbons. A poor correlation between carcinogenicity and genotoxicity test results is conceivable in the case of epigenetic, non-genotoxic carcinogens. Among the haloalkanes listed in Table XIV, a non-genotoxic mechanism of carcinogenicity was proposed for chloroform (World Health Organization, 1994) and possibly for carbon tetrachloride (McGregor and Lang, 1996). For the majority of the remaining chemicals, a genotoxic mechanism can be hypothesized on the basis of structural alerts and positive findings in short-term mutagenicity assays (IARC, 1987a; Morita et al., 1997). Literature data on DNA damage in rodent liver confirm, at least for 1,2-dichloroethane (Storer and Conolly, 1983; Storer et al., 1984) and ethylene dibromide (IARC, 1987a), the induction of genotoxic effects in vivo, whereas negative results were reported for the supposedly epigenetic carcinogens carbon tetrachloride and chloroform (IARC, 1987a).
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In conclusion, the results presented in this paper show that 10 halogenated hydrocarbons, positive to various extents in in vitro micronucleus assays, are unable to significantly raise the incidence of micronuclei in mouse PCEs when given by the i.p. route of aministration at doses close to the LD50. These findings raise some concern as to the sensitivity of the mouse bone marrow micronucleus test to halogenated hydrocarbons and support the need to consider data from additional tissues for the assessment of the in vivo genotoxicity and potential carcinogenicity of this class of compounds.
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Acknowledgments |
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The authors are grateful to Dr R.Benigni for his advice in the selection of tested compounds. The technical assistance of Barbara Crochi is gratefully acknowledged. The helpful criticism of referees is also appreciated.
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Notes |
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4 To whom correspondence should be addressed. Tel: +39 6 4990 2840; Fax: +39 6 4938 7139; Email: crebelli{at}net.iss.it
5 Present address: Synthélabo Recherche, Z.I. Limay/Porcheville, 78440 Gargenville, France
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References |
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Received on June 29, 1998; accepted on October 7, 1998.
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