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EC number: 203-458-1
CAS number: 107-06-2
Negative and positive results have been observed in in vitro and in vivo mutagenicity/genotoxicity tests with 1,2-dichloroethane. Based on a weight-of evidence approach, 1,2-dichloroethane is not considered to be mutagenic in vivo.
Bacterial tests in vitro:
Two reverse mutation assays performed with 1,2-dichloroethane have been reported. In the study by Nestmann et al. (1980), 1,2-dichloroethane (concentrations of 3.6 and 9 mg/plate in a standard plate–incorporation method) did not induce point mutations in the tested strains of S. typhimurium: TA98, 100, 1535, 1537 and 1538, both with and without metabolic activation. A weak (but dose–related) mutagenic response was observed in strains TA1535 and TA100 in the absence and presence of a metabolic activation system when plates were incubated inside desiccators. The response in the TA100 strain was only slightly above background. The maximal response in strain TA 1535 was doubling of mutant colonies. Thus, due to the contrary results obtained in the standard plate–incorporation test compared to results obtained in a modification of this test (incubation of plates in desiccators), the conclusion regarding the mutagenicity potential was ambiguous. In the study by Hemminki et al. (1980), results of the reverse mutation assay (pre–incubation method) conducted in E.coli WP2 uvrA, showed that 1,2-dichloroethane was only weakly mutagenic in the absence of a metabolic activation system at concentrations < 990 μg/ml.
Non–bacterial tests in vitro:
The genotoxicity of 1,2-dichloroethane was studied in a series of in vitro assays using mammalian cells (CHO cells and human-derived AHH-1 and TK6 lymphoblastoid cell lines, primary rat hepatocytes) and investigating end points such as unscheduled DNA-synthesis (UDS) and HGPRT/ K-test. In the HGPRT-assay performed in CHO cells, a dose-related increase in gene mutation was noted both in the absence and presence of metabolic activation at substance concentrations of about 100-5000 μg/mL (1-50 mM) as derived from a loss of the thymidine-kinase activity (Tan and Hsie, 1981). The same result was obtained in HGPRT assays using the human AHH-1 and TK6 lymphoblastoid cell-lines at concentrations of ≥100 and ≥500 μg/mL, respectively, both in a dose-related manner and without metabolic activation (Crespi et al., 1985). In CHL fibroblasts, 1,2-dichloroethane increased the incidence of chromosomal aberrations (chromatid breaks and exchanges, no chromosome breaks), in the presence of metabolic activation at concentrations of 1000 μg/ml after a 6-h exposure with no effect at 0.5 mg/ml. Without metabolic activation no effects were obvious at 200-4000 μg/ml after 24 - or 48 hours, while an ambiguous result was found at 6000 μg/ml (Sofuni et al., 1985). 1,2-dichloroethane was shown to induce micronuclei in human cell lines with a low effective concentration of 1 mM (Doherty et al., 1996). In primary rat hepatocytes, significant induction of unscheduled DNA-synthesis was reported at concentrations of >13 μg/mL in the absence of metabolic activation (Williams et al., 1989). The effects of unscheduled DNA-synthesis, reportedly induced in human lymphocytes, is based on an unsuitable test method using [3H]-TdR incorporation (Perocco and Prodi, 1981): hydroxyurea-induced suppression of replicative DNA synthesis was not a reliable means to discriminate unscheduled from semi-conservative DNA replication, including that of mitochondria. Furthermore, an appropriate positive control substance was not included in the test. The calculation of the so-called DNA-repair value is obscure. In a cell transformation experiment conducted in BALB/C-3T3-cells, no transformation effects were observed at concentrations from 5 to 50 μg/ml without metabolic activation (Tu et al., 1985).
In vivo tests
Several in vivo mutagenicity studies with 1,2-dichloroethane are available in which endpoints such as induction of micronuclei (MN), sister chromatid exchanges (SCE), germ cell mutations, DNA-breakage and sex-linked-recessive-lethal mutations, somatic mutations and recombinations in Drosophila melanogaster were investigated. In a well-conducted micronucleus test in male and female NMRI mice, no increases in the number of micronucleated PCEs were noted in bone marrow cells 6 hr after the last dose when the maximum possible dose of 396 mg/kg bw of the material was given twice in an interval of 24 hr by i.p. injection for a total dose of 798 mg/kg bw; the dose was selected from previous toxicity testing ranging from non-toxic to approximately lethal doses (King et al., 1979). Likewise, a second well-conducted micronucleus test on lymphoma-prone transgenic mice (Eμ-PIM-1) using repeated oral dosing of 1,2-dichloroethane (200 mg/kg bw in males and 300 mg/kg bw in females, due to toxicity reduced to 100 and 150 mg/kg bw, respectively) in corn oil by gavage failed to demonstrate any deleterious effect on peripheral polychromatic and normochromatic erythrocytes after exposure for 14 and 41 weeks (Armstrong and Galloway, 1993). A third micronucleus test was negative after a single i.p. injection of 100 mg/kg bw into male CBA mice. The results of this study may be of limited value because of the use of a relatively low single dose level and the late sampling time of 30 hrs (Jenssen and Ramel, 1980). In an SCE study on bone marrow cells of male Swiss mice, animals were administered 1,2-dichloroethane in peanut oil by i.p. injection at doses of 0, 0.5, 1.0, 2.0, 4.0, 8.0 and 16 mg/kg bw. A dose-dependent increase in the number of SCEs, which are considered as non-mutation endpoints and provide evidence of exposure, at 1.0 mg/kg bw and above (p <0.01 at 2 mg/kg bw and higher) was observed. A dose of 4 mg/kg bw caused a doubling of the spontaneous SCE rate (background: approx. 3 events per cell). At 0.5 mg/kg bw, no increase in the number of SCEs was observed. No positive control was included (Giri and Que-Hee, 1988). Within the scope of a two-generation reproduction study, a dominant lethal test was undertaken twice in male Swiss mice of the first (F1) and second (F2) descendants’ generations which had been delivered from pre-treated parents (F0 and F1). After administration of 1,2-dichloroethane in drinking water (0, 0.03, 0.09 or 0.29 mg/ml; designed to yield daily doses of 0, 5, 15, or 50 mg/kg bw), those repeatedly treated F1 and F2 males were mated to virgin untreated females. There was no evidence of increases in pre- or post-implantation losses, no significant effects on the number of foetal implants or viable foetuses. Therefore, the ability of 1,2-dichloroethane to produce genotoxic effects on germ cells of male mice was considered unlikely (Lane et al., 1982). However, the incomplete documentation of the dosage regimen and the selection of relatively low doses – although up to 50 mg/kg bw repeated dosing for 2 generations - did not allow a firm conclusion. Studies were conducted in male B6C3F1 mice to investigate the DNA-breaking potential of 1,2-dichloroethane (in vivo/in vitro DNA unwinding assay): single doses were administered orally, by i.p. injection and by inhalation, and the presence of single strand breaks and alkali labile sites in isolated hepatic double-strand DNA was examined by using the in vitro alkaline DNA-elution technique (Storer et al., 1984). At 4-hour following oral or i.p. application of sub-lethal and sub-toxic doses of 100-200 mg/kg bw (oral) and 150-300 mg/kg bw (i.p.) DNA damage was induced, as demonstrated by a distinct decrease in the double-strand fractions as compared to the vehicle controls. After a 4-hour inhalation exposure, no such effect was found at sub-toxic concentrations up to 500 ppm (approx. 2000 mg/m³), whereas clear DNA damage occurred at hepatotoxic and lethal exposures to 1000 and 2000 ppm. The differences were explained by the completely different absorption, distribution, and elimination kinetics for the various exposure routes (see toxicokinetics). 1,2-Dichloroethane induced DNA adducts in rats and mice. The extent and pattern of DNA binding was noted to vary with differences in factors such as species, strain, sex, target organ and route and dose of exposure. An early study by Reitz et al. (1982) found low levels of DNA alkylation in the liver, spleen, kidney and stomach 4 hr following administration of 14C-DCE to Osborne-Mendel rats. Compared with inhalation exposure, DCE via gavage was associated with a 2- to 5-fold greater extent of DNA alkylation but 1.5- to 2-fold lower levels of macromolecular binding. In a study by Watanabe et al. (2007), the level of S-[2-(N7-guanyl)ethyl]GSH, the primary adduct, in liver or kidney of rats treated with 1,2-dibromoethane was∼1 adduct/105DNA bases; in male or female mice, the level was approximately one-half of this. The levels of 1,2-dichloroethane adducts were 10–50-fold lower than for 1,2-dibromoethane. None of four known in vitro GSH–DNA adducts was detected at a level of >2/108DNA bases from dibromomethane or dichloromethane. DNA binding occurs with 1,2-dichloroethane but is considerably less than from 1,2-dibromoethanein vivo, and low exposure to dihalomethanes does not produce appreciable DNA adduct levels in rat or mouse liver and kidney at the doses used.
A study conducted by Gocke et al. (1983) evaluated the mutagenic potential of 1,2-dichloroethane exposurein vivo using the mammalian spot test to detect somatic gene mutations in mouse embryos. In the progeny of female C57/BL mice exposed to 300 mg/kg on gestational day 10, somatic gene mutations were evident and significantly increased compared to negative controls (p = 0.03) but not compared to the vehicle control (p = 0.18). Only one dose was examined for each chemical assayed. Therefore, although a significant increase was observed at this dose, it would not be considered positive for mutagenicity based on the current OECD guidelines. In Drosophila melanogaster, 1,2-dichloroethane produced significant increases in somatic mutations (SMART-test) and germ-cell mutations (SLRL-test) after both feed and gas-phase exposure (Kramers et al., 1991; King et al., 1979; Ramel et al., 1990; Romert et al., 1990). Furthermore, after feed administration mutagenic effects were enhanced after pre-treatment with the cytochrome P 450 inducer phenobarbital, and reduced after pre-treatment with the glutathione-S-transferase inhibitor buthionine sulfoximine (Ramel et al., 1990; Romert et al., 1990). In a study conducted to address mode-of-action (MOA), a Comet assay was used to assess the DNA damage (single- and double-strand breaks) of inhaled vapours of 1,2-dichloroethane in the mammary gland (target tissue) of rats repeatedly exposed to a high concentration (200 ppm) of DCE (6h/day, 7days/week for at least 28 exposures), compared to air-exposed (0 ppm DCE) control rats. Mammary epithelial cells (MEC) are presumed to be the target cell population for DCE-induced effects leading to the formation of mammary tumours; therefore, a multistep isolation procedure was used to prepare a mono-disperse population of mammary gland cells highly enriched in MEC for Comet assay analysis to increase the specificity and sensitivity of the Comet assay for this tissue. MEC isolated from a positive control group of rats treated with MNU (a potent genotoxic agent) were analysed under the same conditions as MEC isolated from control and DCE-exposed rats. The significant increase in tail intensity in MNU-treated control tissue demonstrated that the assay could detect DNA damage in isolated MEC. Under the conditions of this study, no exposure-related genotoxic effects in isolated MEC were seen in rats repeatedly exposed to a high concentration of DCE for 28-31 days, compared to air-exposed (0 ppm DCE) control rats. Under these repeated exposure conditions, inhaled DCE did not damage DNA in MEC isolated from female F344/DuCrl rats.
The human genotoxicity data for DCE are limited to a study of occupational DCE exposure in vinyl chloride (VCM) manufacturing plants examining chromosomal aberrations. The authors conclude that smoking and exposure to air concentration of 1 ppm (4.05 mg/m3) DCE increased SCE frequency, whereas higher levels (5 ppm; 20.24 mg/m3) are probably necessary to observe the same effect from VCM exposure. The overall results of the study are not conclusive.
1,2-Dichloroethane was weakly mutagenic in bacterial test systems. It was positive in in vitro gene mutation and cytogenetic assays in mammalian cells. Metabolic activation which involves both the cytochrome-P450 and the glutathione-dependent pathways is required to cause these effects. In general, an increased level of DNA damage is observed related to the GSH-dependent bioactivation of DCE. Increased chromosomal aberrations with increased CYP450 expression were suggestive of a role for the oxidative metabolites of DCE in inducing chromosomal damage.
There is evidence that 1,2-dichloroethane interacts with DNA in exposed animals in the absence of overt toxicity. The in vivo effects include DNA strand breaks and observations of DNA binding at the macromolecular level and are dose-dependent and systemic, with multiple organs involved. However, many of these assays are limited for assessing the genotoxic potential of 1,2-dichloroethane due to the use of high doses and/or an inadequate study design. Limited DNA damage and/or binding was demonstrated following inhalation exposure compared to oral and intraperitoneal exposures.
The results of the available in vivo studies failed to show a mutagenic potential of 1,2-dichloroethane as the substance was clearly negative in 3 micronucleus assays (2 by i.p. and one repeated dosing by the oral route) and in a dominant lethal test with repeated exposure on 2 generations. Some evidence of DNA interaction is presented by positive results in an SCE assay (a non-mutagenic endpoint), in DNA strand-break assays, and in a test with Drosophila. Moreover, the ability of 1,2-dichloroethane to produce genotoxic effects in germ cells of male mice was considered unlikely. In particular, a recently conducted Comet assay in mammary gland epithelial cells (the target organ for carcinogenicity) after in vivo inhalation exposure of female rats to 200 ppm 1,2-dichloroethane for 4 weeks was clearly negative.
Based on a weight-of-evidence approach it is concluded that 1,2-dichloroethane is not mutagenic in vivo. The results of the available mutagenicity and genotoxicity assays do not provide convincing support for a genotoxic mode of action for the carcinogenicity of 1,2-dichloroethane.
Justification for selection of genetic toxicity endpoint
A number of in vitro and in vivo studies area available for 1,2-dichloroethane. Based on a weight-of-evidence approach, it has been concluded that 1,2-dichloroethane is not mutagenic in vivo.
Short description of key information:
1,2-dichloroethane was weakly mutagenic in bacterial test systems, but showed more pronounced effects in mammalian cytogenetic and gene mutation assays. Metabolic activation was primarily required to cause these effects, which was in line with the known metabolism of the material involving the cytochrome-P450- and the glutathione-dependent pathways, where both pathways were considered as possible steps in the bioactivation cascade leading to reactive metabolites. Multiple studies demonstrating DNA and protein adducts in bacterial and mammalian cells. The results of available in vivo studies failed to show a mutagenic potential of 1,2-dichloroethane in several micronucleus assays. Some evidence of DNA damaging in-vivo activity/genotoxicity was presented by positive results in SCE assay, in DNA strand break assays (Comet), and in a SLRL test in Drosophila melanogaster analysis. Based on a weight-of-evidence approach it is concluded that 1,2-dichloroethane is not mutagenic in vivo. The results of on the available mutagenicity and genotoxicity assays do not provide convincing support for a genotoxic mode of action for the carcinogenicity of 1,2-dichloroethane.
Endpoint Conclusion: No adverse effect observed (negative)
Negative and positive results have been observed in in
vitro and in vivo mutagenicity/genotoxicity tests with
1,2-dichloroethane. Based on a weight-of evidence approach, the
applicant does not consider 1,2-dichloroethane as mutagenic in vivo.
Therefore, there is no need to classify the substance as mutagenic
according to Regulation 1272/2008/EC (CPL) and Directive 67/548/EEC
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