United States Environmental Protection Agency Office of Health and Environmental Assessment Washington DC 20460 Research and Development EPA/600/S6-85/001 Sept. 1985 SERA Project Summary Assessment of the Mutagenic Potential of Carbon Disulfide, Carbon Tetrachloride, Dichloromethane, Ethylene Dichloride, and Methyl Bromide: A Comparative Analysis in Relation to Ethylene Dibromide Vicki Vaughan-Dellarco, John R. Fowle III, and Sheila Rosenthal The Reproductive Effects Assessment Group was requested by the Haiard Evaluation Division of the Office of Pesticide Programs (OPP) to prepare a mutagenicity assessment of proposed pesticide alternatives to the fumigant, ethylene dibromide. These alternatives included carbon disulfide, carbon tetra- chloride, dichloromethane, ethylene dichloride, and methyl bromide. This mutagenicity assessment is to serve as a "source document" for OPP's use. In the development of this document, the scientific literature has been inven- toried, and key studies have been crit- ically evaluated. The Environmental Mutagen, Carcinogen, and Teratogen Information Department at the Oak Ridge relational Laboratory identified the published literature. Three sections of Chapter 4 in the full report were taken from the health assessment documents prepared by the Office of Health and Environmental Assessment (OHEA) for the Office of Air Quality Planning and Standards. These sections include data evaluations of carbon tetrachloride, dichlorometh- ane, and ethylene dichloride. The Health Assessment Document for Carbon Tet- rachloride has received full administra- tive and peer review. The Health As- sessment Documents for Dichlorometh- ane and for Ethylene Dichloride are undergoing public review and comment and EPA Science Advisory Board re- view. The reader is referred to the health assessment documents (U.S. EPA, 1983a. 1984b, 1984c), if additional information is needed regarding health effects other than mutagenicity or back- ground information such as physical- chemical properties. This Project Summary was developed by EPA's Office of Health and Environ- mental Assessment, Washington, DC, to announce key findings of the research project that is fully documented in a separate report of the same title (see Project Report ordering information at back). Introduction This Summary provides a brief evalua- tion of the mutagenic potential of five proposed alternatives to the use of the fumigant, ethylene dibromide. The alter- native compounds are carbon disulfide, carbon tetrachloride, dichloromethane, ethylene dichloride, and methyl bromide, Figure 1. The evaluation involved a survey ------- Chemical Empirical Formula Structure Carbon Disulfide Carbon Tetrachloride Ethylene Dibromide (1,2 -Dibromoethanei Ethylene Dichloride (1,2 -Dichloroethane) Methyl Bromide (Bromomethane) Methylene Chloride (Dichloromethane) CSZ ecu CH3Br Cl I CI — C — CI I Cl H H Br — C — C—Br H H H H I I CI — C — C—Cl I I H H H I fir — C —H I H H I CI — C — CI \ H figure 1. Chemical structures of ethylene dibromide and proposed alternatives. and critical analysis of relevant studies. A separate analysis of the mutagenicity of each proposed alternative is found in the individual sections of Chapter 4 in the full report. The evaluation of the five proposed alternative fumigants included a deter- mination of the intrinsic mutagenic po- tential of each agent and its ability to reach germinal tissue in intact mammals. Ethylene dibromide is not included as a separate section in the full report because it has been evaluated previously by OPP. A comparative analysis of mutagenicity between each of the proposed altenatives and C2H4Br2 is presented. The spectrum of genetic damage induced by each agent is discussed and mutagenic potencies are compared whenever appropriate. Be- cause judgments cannot be reached due to gaps in current knowledge, recom- mendations are made for additional stud- ies that could be conducted to determine if a potential mutagenic risk exists. Comparative Analysis of Ethylene Dibromide and Proposed Alternatives This comparison considers the genetic damage that is induced by each agent and mutagenic potencies in selected tests. Of the five proposed alternative fumi- gants, there is sufficient evidence on ethylene dichloride, dichloromethane, and methyl bromide, in addition to ethyl- ene dibromide itself, to classify them as mutagens. These chemicals have been reported as positive in two or more gene mutation tests in phylogenetically differ- ent organisms. Table 1. There is also ancillary information regarding their DNA-damaging potential (e.g., SCE, DNA repair, DNA alkylation). The evidence that ethylene dichloride is a presumed mam- malian mutagen is stronger than that for dichloromethane or methyl bromide be- cause of (1) the larger number of positive tests conducted in different laboratories, (2) the suggestive evidence that ethylene dichloride causes somatic gene mutations in whole mammals, and (3) a study demonstrating the alkylation of DNA in somatic tissues of whole mammals. Al- though the data on the ability of these agents to cause chromosomal aberrations are limited, none of the chemicals appear to be strong clastogens. It is uncertain whether these agents produce a similar( array of other types of genetic damage (e.g., nondisjunction, SCEs, mitotic re- combination), because they have not all been sufficiently evaluated for the induc- tion of other types of genetic alterations. Furthermore, it is uncertain whether the proposed alternatives reach and interact with mammalian germ-cell DNA, but ethylene dibromide is known to do so and this is presumed to be a human germ-cell mutagen. Ethylene dichloride, methyl bromide, and dichloromethane are posi- tive in the Drosophila sex-linked recessive lethal test. This test organism has germ- cell stages analogous to those in mam- mals and provides some information regarding germ-cell risk in intact animals. Although the data bases are not equally complete for each of the compounds, none of the proposed alternatives appear to be as mutagenic as ethylene dibromide when results from similar tests are compared. The mutagenic potential of the other two alternatives, carbon disulfide and carbon tetrachloride, could not be judgec because of insufficient information. The available studies suggest, however, thai if they are mutagenic, they are weakly so This conclusion does not necessarily appl^ to chromosome non-disjunction because carbon tetrachloride has not been evalu ated for its ability to disrupt spindle structures or function, and inadequate evidence is available for the induction o numerical chromosomal aberrations bi carbon disulfide. The reader is referredt< Chapter 4 of the full report for a critica analysis of the data pertaining to th< mutagenicity of these five propose! alternative fumigants and for a detailet summary for each chemical. Mutagenic Potencies The mutagenic potencies of each of th> five proposed alternative fumigants wer compared with those of ethylene dibro mide using results from the Salmonell assay. This test was the only one in whic all chemicals have been evaluatec Several criteria were imposed to selec appropriate experiments for this analysis Only experiments using the desiccate procedure were included because all c the chemicals are volatile, and testing i sealed containers is more appropriat than in the standard plate assay in whic the volatile test material evaporates an escapes. In addition, results were consic ered only if there were at least tw nonzero dose points; spontaneous coum were reported, and revertant data wet given in the report. Results on tests ------- Table 1. Qualitative Comparison of the Mutagenicity of Ethylene Dibromide and Proposed Alternatives Chemical Bacteria Fungi Gene Mutation Clastogenicity Numerical Other Chromo- Indicators of Higher Mammalian Whole some DNA DNA Plants Drosophila Cells Mammals In Vitro In Vivo Mutation Damage Binding Carbon disulfide -d) K3) K2) 1(2) -12) 1(1), UDS Carbon tetra- 1(4) chloride -(2) Dichloro- methane Ethylene dibromide Ethylene dichloride Methyl bromide +(12) 1(2)' -(4) W(7f 1(3) +(4) W(J) -d) +(3) +(3) +(1) KD +(3) KD -d) KD +(2) Wft) KD -(2) -12) K2) W(1), YMR -(3), UDS (I). YMR /+). YMR w(2), see -(2), UDS +(1). YMR +(1). UDS -d). UDS +d). Pol +(1). SCE 1(1). UDS Id). Pol 1(2). UDS +(2) Information on ethylene dibromide is based on OPP documents (U.S. EPA, 1983a; Mauer, 1979; Lee. 1980). "+" designates a positive result; "-" a negative result; "I" an inconclusive study; "W" a weak, borderline, or suggestive result. Numbers of studies are indicated in parentheses. UDS = unscheduled DNA synthesis; YMR = yeast mitotic recombination; SCE = sister chromatid exchange; Pol = bacteria. "Plants only. tStronger response with an S9 metabolic activation system. ^Drosophila only. strain TA100 in the presence or absence of metabolic activation were used be- cause this was the only strain for which data were available on all compounds. A simple linear regression analysis was used on the linear portion of the dose- responses; linear regression calculations with correlation coefficients less than 0.90 were not accepted. It was clear from these tests that eth- ylene dibromide is a much more potent mutagen than the corresponding chlori- nated compounds and more mutagenic than methyl bromide based on structural- activity relationships. Ethylene dibromide is a bi-functional agent, and thus more biologically reactive. Carbon tetrachloride and carbon disulfide are predominantly negative in bacterial tests. Potencies were also examined in the Drosophila sex-linked recessive lethal test. Data were available for ethylene dibromide, ethylene dichloride, dichloro- methane, and methyl bromide. Although certain germ-cell stages appear to be more sensitive than others, the total lethal frequencies were compared and it was found that ethylene dibromide is a more potent mutagen than the alterna- tives, whether data from inhalation or feeding experiments are compared. It is uncertain if ethylene dichloride is more active than methyl bromide. There is some overlap of the lethal frequencies per unit of exposure for these alternatives. Different strains of Drosophila were used, which could account for differences in lethal frequencies. Although carbon di- sulfide has been reported as negative, the possibility of weak effects cannot be excluded. In feeding experiments, ethyl- ene dichloride seems to be more active than dichloromethane. However, these experiments were conducted in different laboratories, and factors such as stocks of Drosophila and solvents differed; these variations could contribute observed dif- ferences among the alternatives in lethal frequencies. Nevertheless, in their total- ity, the data show that ethylene dibromide is more mutagenic than the proposed alternatives in the Drosophila sex-linked recessive lethal test. The Drosophila results are therefore consistent with those in Salmonella. The mutagenicity of the alternative compounds in cultured mammalian cells cannot readily be compared to that of eth- ylene dibromide because test results are based on different cell lines and different loci. However, in one study using human lymphoblasts and another study using Chinese hamster ovary cells, ethylene dibromide was a more potent mutagen than ethylene dichloride. Ethylene dibromide, ethylene dichlo- ride, and methyl bromide have been examined for DNA adduct formation in whole mammals after inhalation expo- sure. Although there is DNA binding data with carbon tetrachloride, it is derived from intraperitoneal injection experi- ments and therefore were not used in comparison with the other compounds. Liver was the only organ in which DNA ------- alkylations could be compared for the three chemicals. It was found in these experiments that methyl bromide binds DNA to a much lesser extent than does ethylene dibromide; approximately five orders of magnitude difference were observed. Ethylene dichloride appears to bind to DNA to a greater extent than methyl bromide but to a lesser extent than eth- ylene dibromide. It should be cautioned, however, that the measurements for ethylene dichloride were derived from a different rodent species (rat) than those for ethylene dibromide and methyl bro- mide (mouse). Although there were two orders of magnitude difference in the alkylations, a species difference could conceivably account for the amount of binding. Given the available data, few conclu- sions can be drawn about DNA alkylation by methyl bromide and ethylene dichlo- ride, except that both agents interact with DNA. Methyl bromide appears to do so to a lesser extent than does ethylene di- bromide. Information from experiments that involve measurements at different time intervals to determine the stability of various DNA adducts formed by these compounds would be a valuable addition to current knowledge. If stable adducts are formed in testicular DNA, which is a target tissue for heritable risk, then the induced genetic damage could accumu- late during the cellular life cycle of the gonial cells. The gonia are an important cell type relevant for human genetic risk assessment. In the case of ethylene dibromide, it is known that adducts are formed in testicular DNA. Data regarding the degree of alkylation is more useful if it includes information on the type of ad- ducts formed and the stability of these adducts. Such information is needed for ethylene dichloride, methyl bromide, and dichloromethane in germinal tissue of intact mammals. Conclusions and Recommendations The five proposed alternatives do not appear to be as mutagenic as ethylene dibromide. Two alternatives, carbon tet- rachloride and carbon disulfide, have been primarily negative in mutagenicity testing, however it cannot be stated that they do not pose a mutagenic risk because the available information is limited and sometimes inadequate. Additional testing would be necessary for them to be classified. It should be noted that even if these agents do not pose a mutagenic hazard, they do pose other health hazards; for example, carbon disulfide is extremely toxic and carbon tetrachloride is extreme- ly toxic and carcinogenic in mice and rats. The alternative compounds that are mutagenic in several short-term gene mutation assays are ethylene dichloride, dichloromethane, and methyl bromide. It cannot be concluded that one of these agents is more mutagenic than the other because of limited data. It does appear, however, that these agents are not strong mutagens, because rather large, and often toxic, doses are required to elicit mutagenic responses. Delineation of dif- ferences in mutagenic activity among these agents will require dose-response data that are generated in the same laboratory so as to minimize technical and biological variation. The proposed alternatives are all volatile chemicals and precautions are therefore essential to prevent excessive evaporation of test material. Several different assay systems, including mammalian systems, should be used to determine a rank order for mutagenic potency. If these experiments were coupled with molecular dosimetry, the relationship of mutation frequency could be compared to target dose rather than to exposure. If a similar rank order of potency is observed in different species, it might be reasonable to assume that a similar ranking may exist in humans. After these determinations, whole mam- mal germ-cell studies would have to be conducted to estimate heritable risk. The EPA authors. Vicki L. Vaughan-Dellarcofalso the EPA Project Officer, see below), John R. f ovule III, and Sheila Ftosenthal are with Office of Health and Environmental Assessment, Washington, DC 20460. The complete report, entitled "Assessment of the Mutagenic Potential of Carbon Disulfide.'Carbon Tetrachloride, Dichloromethane, Ethylene Dichloride, and Methyl Bromide: A Comparative Analysis in Relation to Ethylene Dibromide," (Order No. PB 85-241 800/AS; Cost: $16.00, subject to change) will be available only from: National Technical Information Service 5285 Port Royal Road Springfield, VA22161 Telephone: 703-487-4650 The EPA Project Officer can be contacted at: Office of Health and Environmental Assessment U.S. Environmental Protection Agency Washington, DC 20460 United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 BULK RATE POSTAGE & FEES P EPA PERMIT No. G-3f Official Business Penalty for Private Use $300 EPA/600/S6-85/001 ------- |