5
Genotoxicity

Whether tetrachloroethylene and its metabolites are genotoxic (and if so at what doses) is an important consideration in evaluating potential modes of action for carcinogenic effects in the Environmental Protection Agency (EPA) draft Integrated Risk Information System (IRIS) assessment. The evidence on the genotoxicity of tetrachloroethylene is summarized in Section 4.3 of the draft assessment (EPA 2008). The committee found that the publications cited and discussed by EPA are relevant but that the summary does not reflect the entire knowledge base available on the topic and does not provide transparent means for assessing the genotoxicity of tetrachloroethylene itself or its metabolites. The draft IRIS assessment predominantly reports positive studies, whereas good studies that had negative results are not mentioned or in some cases are incorrectly described as having had positive results. The committee therefore recommends that a more balanced, transparent, and inclusive approach be used to consider the evidence. The sections below offer some specific guidance.

ORGANIZATION AND EVALUATION OF DATA

The draft IRIS assessment’s consideration of genotoxicity lacks cohesive structure, and the organization of the data presentation should be revised. Specifically, the section should be subdivided into sections on tetrachloroethylene itself, its metabolites, and evidence of indirect genotoxicity. Each section should include a table that lists all primary publications, the results related to tetrachloroethylene in the assays that it was tested in, and comments regarding strengths or weaknesses of each dataset. How the studies were selected should be articulated. It would be helpful if the studies were organized according to the general test systems used; for example, data on nonmammalian systems, in vitro mammalian cells, intact animals, and humans should be delineated separately. A good example of such table may be found in recent monographs of the International Agency for Research on Cancer (IARC). The text that accompanies each table should provide an assessment of the quality of each study cited. At the end



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5 Genotoxicity Whether tetrachloroethylene and its metabolites are genotoxic (and if so at what doses) is an important consideration in evaluating potential modes of ac- tion for carcinogenic effects in the Environmental Protection Agency (EPA) draft Integrated Risk Information System (IRIS) assessment. The evidence on the genotoxicity of tetrachloroethylene is summarized in Section 4.3 of the draft assessment (EPA 2008). The committee found that the publications cited and discussed by EPA are relevant but that the summary does not reflect the entire knowledge base available on the topic and does not provide transparent means for assessing the genotoxicity of tetrachloroethylene itself or its metabolites. The draft IRIS assessment predominantly reports positive studies, whereas good studies that had negative results are not mentioned or in some cases are incor- rectly described as having had positive results. The committee therefore recom- mends that a more balanced, transparent, and inclusive approach be used to con- sider the evidence. The sections below offer some specific guidance. ORGANIZATION AND EVALUATION OF DATA The draft IRIS assessment’s consideration of genotoxicity lacks cohesive structure, and the organization of the data presentation should be revised. Spe- cifically, the section should be subdivided into sections on tetrachloroethylene itself, its metabolites, and evidence of indirect genotoxicity. Each section should include a table that lists all primary publications, the results related to tetra- chloroethylene in the assays that it was tested in, and comments regarding strengths or weaknesses of each dataset. How the studies were selected should be articulated. It would be helpful if the studies were organized according to the general test systems used; for example, data on nonmammalian systems, in vitro mammalian cells, intact animals, and humans should be delineated separately. A good example of such table may be found in recent monographs of the Interna- tional Agency for Research on Cancer (IARC). The text that accompanies each table should provide an assessment of the quality of each study cited. At the end 50

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51 Genotoxicity of each section, an evaluation of the strength of the evidence of genotoxicity of a particular compound should be included by way of summarizing the totality of data available. Finally, there should be an integrative assessment, including spe- cies-specific kinetics and metabolism of tetrachloroethylene and of genotoxicity and mutagenicity in intact animals and humans. STUDIES OF TETRACHLOROETHYLENE Nonmammalian Systems A considerable number of mutagenicity studies of pure tetrachloroethylene that used Salmonella strains, Escherichia coli, and Saccharomyces have been per- formed with and without exogenous metabolic activation by liver S9 fractions from rats, mice, and hamsters (including animals pretreated with Aroclor or phenobarbi- tal). The results have been essentially negative. The studies should be documented in a table (see above for specific format suggestions). However, when tetrachloroethyl- ene was incubated with purified glutathione S-transferase (GST), glutathione, and rat kidney fractions, formation of S-(1,2,2-trichlorovinyl) glutathione (TCVG) was found, and mutagenic activity in Salmonella was clearly demonstrated as correctly described in the EPA draft. The committee recommends that EPA also consider the negative results in the National Toxicology Program study (NTP 1986) of sex-linked lethal muta- tions in Drosophila. Mammalian Cells in Vitro EPA should describe the mutation study with mouse lymphoma L5178Y cells (NTP 1986), which appears to be the only available mammalian mutation test performed with tetrachloroethylene. This well-done study revealed that tet- rachloroethylene at a variety of concentrations, with and without S9 for meta- bolic activation (but not with GST and rat kidney fractions), did not enhance the frequency of mutations at the thymidine kinase locus. Likewise, investigations of chromosomal aberrations and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells (NTP 1986; Galloway et al. 1987) showed no evi- dence of tetrachloroethylene-induced genetic activity, although for technical reasons the weight of these studies was somewhat limited. In addition, the nega- tive studies of chromosome aberrations in Chinese hamster lung cells by Sofuni et al. (1985) should be reported. The work of Hartmann and Speit (1995) is addressed in the draft IRIS as- sessment, but it is incorrectly quoted in a statement that tetrachloroethylene in- duced genetic damage, which was not shown. Hartmann and Speit investigated SCEs and DNA integrity (by using the single-cell gel electrophoresis or comet assay) in human blood cells exposed to tetrachloroethylene in vitro. The study was well performed, with negative and positive controls, without and with

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52 Review of the EPA’s Draft IRIS Assessment of Tetrachloroethylene metabolic activation, and with assay repeats. Although the highest concentration of tetrachloroethylene used in the comet assay was cytotoxic, there clearly was no evidence that tetrachloroethylene at any dose caused increases in SCEs or comet. EPA’s review of the study should be corrected. Concerning the study of Doherty et al. (1996), the EPA draft correctly re- ports that tetrachloroethylene induced micronuclei in two novel cell lines of hu- man lymphoblastoma origin (h2E1 and MCL-5) through either clastogenic or aneugenic mechanisms. Cells were genetically engineered to express human enzymes (CYP2E1 or CYP1A2, 2A6, 3A4, 2E1) and epoxide hydrolase stably. The committee recommends that EPA acknowledge that those cell lines were not validated as test systems and that other compounds tested in the study, such as hexane and toluene, that are generally regarded as nongenotoxic also led to formation of micronuclei—an indication that the new cell lines may be oversen- sitive and may provide false-positive results. Micronucleus formation in MCL-5 cells by tetrachloroethylene was confirmed by White et al. (2001), and Wang et al. (2001) found increases in micronuclei in CHO-K1 cells, as mentioned in the draft IRIS assessment. Tetrachloroethylene’s effects on unscheduled DNA synthesis were studied in human fibroblasts (WI-38) (Beliles et al. 1980), in primary hepatocytes from rats and mice (Shimada et al. 1985; Costa and Ivanetich 1984; Milman et al. 1988), and in human lymphocytes (Perocco et al. 1983); the results were mostly negative. Although those studies are limited in performance or reporting, EPA should discuss them to provide a full account of the existing database. In Vivo Studies in Animals EPA correctly reports that the study of Walles (1986) showed occurrence of DNA single-strand breaks in liver and kidneys but not lungs of mice 1 hour after intraperitoneal injection of tetrachloroethylene at 650-1,300 mg/kg dis- solved in 0.05 mL of Tween 80. EPA fails to mention the full reversibility of that effect at 24 hours. Furthermore, the relevance of the unphysiologic mode of application (intraperitoneal injection in Tween) should be discussed. Tetra- chloroethylene is a known irritant of skin and mucosa, and intraperitoneal injec- tion may trigger the release of inflammatory mediators that will stimulate secre- tion of reactive oxygen species and cytokines in liver and kidney. In addition, the high toxic dose of tetrachloroethylene may produce cell death associated with endonucleolytic DNA fragmentation (Storer et al. 1996). No increase in renal single-strand breaks in DNA was seen 24 hours after oral administration of tetrachloroethylene in rats, but single-strand breaks were enhanced after applica- tion of the genotoxins dimethylnitrosamine and diethylnitrosamine (Potter et al 1996). The EPA draft quotes the paper by Mazullo et al. (1987), which reports low levels of DNA binding 22 hours after intraperitoneal injection of radioac- tively labeled tetrachloroethylene in mice or rats. Binding was calculated at 2.9

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53 Genotoxicity pmol/mg for mouse liver DNA and 0.2-0.5 pmol/mg for rat liver and rat and mouse kidney, lung, and stomach DNA. Thus, there was no evidence of in- creased binding to rat kidney DNA as misleadingly reported by EPA. Moreover, EPA fails to mention that RNA and protein were labeled much more highly than DNA (up to 420 pmol/mg in the case of RNA). That seriously limits the weight of the study because DNA may have been contaminated by RNA or protein (ap- parently, DNA was not purified to constant specific activity) and 14C may have been incorporated into DNA via the intermediary metabolism. Overall, those limitations should be taken into account by EPA in the evaluation of the study. The in vivo micronucleus study in mice by Murakami and Horikawa (1995) is potentially of key importance in the evaluation of tetrachloroethylene’s effects on intact organisms. The authors investigated the appearance of micro- nucleated cells in peripheral blood and liver. However, the draft IRIS assess- ment is partially incorrect: it reports increased frequencies of micronuclei in peripheral blood reticulocytes after intraperitoneal injection of tetrachloroethyl- ene, but the paper says the opposite (that is, there was no increase in micronuclei in reticulocytes). EPA correctly quotes from the paper in saying that hepatocytes showed small increases in micronuclei when mice received intraperitoneal injec- tions of tetrachloroethylene at high doses 24 hours after partial hepatectomy but not when tetrachloroethylene was injected before partial hepatectomy. The fre- quency of micronuclei increased less than two-fold but was statistically signifi- cant; the positive control diethylnitrosamine produced a 10-fold increase. Sev- eral restrictions should be considered by EPA in interpreting the study. The effects were observed at high doses (1,000 and 2,000 mg/kg were effective, but not 500 mg/kg). Given that hepatic toxicity in mice increases from a lowest ob- served-adverse-effect level of 100 mg/kg (EPA 2008, Section 4.4.2.1), the high doses necessary to enhance micronucleus formation must have been severely toxic to the residual hepatocytes and to the whole organism. The toxic load on the residual liver would have been aggravated by the intraperitoneal tetrachloro- ethylene application and by the likely release of cytokines and reactive oxygen species. Overall, the small observed increase in micronuclei in mouse hepato- cytes might have been due to nonspecific toxic effects. In conclusion, this in vivo study clearly found no increase in reticulocyte micronuclei, and the data suggesting formation of micronuclei in hepatocytes are not convincing. EPA should mention the in vivo unscheduled DNA synthesis test per- formed on kidney. Tetrachloroethylene was administered to rats orally (1 g/kg at 0 and 12 hours); at 24 hours, no evidence of unscheduled DNA synthesis in iso- lated renal cells was observed (Goldsworthy et al. 1988, abstract). A recent paper by Cederberg et al. (2009) describes the results of an in vivo study in which the alkaline Comet assay was performed on the liver and kidney of CD1 mice treated orally with tetrachloroethylene at 1,000 or 2,000 mg/kg dissolved in corn oil. A slight increase in DNA damage was reported; the effect was significant for one of two end points (tail intensity, but not tail mo- ment) in the liver. No increases were found in the kidney. The study had been performed by a contract laboratory, and the study director had concluded from

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54 Review of the EPA’s Draft IRIS Assessment of Tetrachloroethylene the same data that tetrachloroethylene did not increase DNA damage because of the inconsistent effects on the two end points, the low magnitude of increases, the high inter-animal variation, and lack of statistically significant increases in a statistical test (Dunnet). Overall, the paper by Cederberg et al. does not present convincing evidence for a genotoxic activity of tetrachloroethylene. It would also be useful to add the results of studies of hepatic-tumor initia- tion by tetrachloroethylene although this end point does not necessarily reflect mutagenic activity. When 10 male Osborne Mendel rats were given tetrachloro- ethylene at 1,000 mg/kg and then phenobarbital as a promoting treatment for 7 weeks (an initiation protocol), the tetrachloroethylene did not induce an increase in the number of gamma-glutamyl transpeptidase-positive cell foci in the liver (Milman et al 1988). Likewise, tetrachloroethylene did not produce liver foci in neonatal female Wistar rats exposed at 2,000 ppm 8 hours/day 5 days/week for 10 weeks (Bolt et al. 1982). Thus, two independent studies did not indicate an initiation potential of tetrachloroethylene in rat liver. Studies in Humans Toraason et al. (2003) studied oxidative damage (measured as 8- hydroxydeoxyguanosine [8-OHdG]) in leukocyte DNA of 18 female dry clean- ers exposed to tetrachloroethylene and compared it with oxidative damage in 20 female laundry workers who were not exposed to tetrachloroethylene. Blood concentrations in the exposed workers were greater than in unexposed workers by two orders of magnitude. There was a statistically significant reduction in 8- OHdG in the exposed workers and no difference in urinary 8-OHdG or in a uri- nary lipid peroxidation biomarkers between the two groups. The data from this small sample provide no evidence of oxidative DNA damage under the condi- tions of the study. EPA should report the studies by Ikeda et al. (1980a,b), who investigated chromosomal aberrations, SCEs, and modified cell-cycle kinetics in human lymphocytes after 3 days in culture with phytohemagglutinin. Lymphocytes were obtained from 10 workers who had been exposed to tetrachloroethylene and from 11 control subjects. Although no significant effects were found in the exposed group with respect to any of the end points, the limitations of the stud- ies, such as small samples, will need to be considered in evaluating the results. STUDIES OF METABOLITES OF TETRACHLOROETHYLENE EPA briefly describes studies that identify TCVG, S-(1,2,2-trichloro- vinyl)-L-cysteine (TCVC), and N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine (N- Ac-TCVC) as bacterial mutagens that act either directly or after activation by rat renal microsomes. It also mentions the induction of unscheduled DNA synthesis by TCVC in a porcine renal-cell line and the key role of renal ß-lyase in the final activation step as demonstrated in these studies.

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55 Genotoxicity The EPA draft mentions the positive test for bacterial mutagenicity of tet- rachloroethylene epoxide. A discussion of existing studies of the genotoxicity of trichloroacetyl chloride should be added. As to trichloroacetic acid (TCA), the draft states (EPA 2008, p. 4-5) that “as reviewed by Moore and Harrington- Brock (2000), the oxidative metabolite TCA, the major urinary excretion prod- uct, exhibits little, if any, genotoxic activity.” That statement is followed by brief descriptions of numerous studies of single-strand breaks, which had incon- sistent results. Increases in single-strand breaks might have been caused by cyto- toxic effects and necrosis at high doses of TCA because of endonucleolytic deg- radation of DNA (Storer et al. [1996], as reported by EPA). The purpose of the description of studies devoted exclusively to DNA single-strand breaks after exposure to TCA is not clear. The committee recommends integration of the data on single-strand breaks into a balanced review of all available genotoxicity studies of TCA (including a table and a discussion of the studies’ strengths and weaknesses) to support the conclusion that TCA exhibits little if any evidence of genotoxicity by an evaluation of the weight of evidence. Clarity regarding the genotoxicity studies of chloral hydrate and di- chloroacetic acid (DCA) is also needed. As recommended earlier, this would be facilitated by an overview of all published data displayed in tables, and there should be a weight-of-evidence evaluation to support EPA’s conclusion that chloral hydrate and DCA are genotoxic. That conclusion generally agrees with a recent IARC assessment, but according to IARC (2004), genotoxicity of DCA was limited to high doses that probably are not relevant to tetrachloroethylene carcinogenicity; EPA should consider this argument. TCVC sulfoxide, another reactive metabolite of tetrachloroethylene, which is nephrotoxic (Elfarra and Krause 2007), does not appear to have been studied for genotoxicity. EVIDENCE OF INDIRECT GENOTOXICITY Two studies by Toraason et al. (1999, 2003) are briefly described in the draft IRIS assessment. They revealed no evidence of oxidative DNA damage in rats after a single intraperitoneal dose of tetrachloroethylene at up to 1,000 mg/kg in rats or in humans after occupational exposure to tetrachloroethylene. EPA should add the important information from the animal study by Toraason et al. (1999) that the similar chemical trichloroethylene applied at the same doses as tetrachloroethylene increased oxidative DNA damage in rat liver, whereas tetrachloroethylene did not. As reported in the IARC (2004) monograph on TCA, the frequency of 8- hydroxydeoxyguanosine-DNA adducts in the liver of B6C3F1 mice was not modified after application of TCA via drinking water (Parrish et al. 1996), was slightly increased after administration through gavage (Austin et al. 1996), and was clearly increased after intraperitoneal injection (Von Tungeln et al. 2002). That comparison of study results again suggests that the route of application

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56 Review of the EPA’s Draft IRIS Assessment of Tetrachloroethylene (oral vs intraperitoneal) should be considered in evaluating genotoxic effects of tetrachloroethylene and its metabolites. FORMATION OF REACTIVE METABOLITES IN ANIMALS AND HUMANS As described in Section 3 of the draft IRIS assessment, the metabolic flux of tetrachloroethylene through glutathione conjugation and β-lyase cleavage is much lower in humans than in rats. TCVG formation in liver, β-lyase activity in kidney, and N-Ac-TCVC excretion in urine are all much lower in humans than in rats (Dekant et al. 1986b; Green et al. 1990; Volkel et al 1998). Furthermore, Pahler et al. (1998, 1999) generated monospecific antibodies to the protein adducts of the reac- tive intermediates either of the glutathione (GSH) conjugation or the oxidative path- way, namely to N-dichloroacetyl-L-lysine and N-trichloroacetyl-L-lysine. The anti- bodies allow determination of the amounts of reactive metabolites formed in the two main pathways. Comparing binding in rat kidney and rat liver subfractions, the di- chloro adduct (indicating the GSH conjugation pathway) predominates in the kidney with only faint bands in liver; the trichloro adduct (indicating the oxidative pathway) predominates in the liver. Pahler et al. (1999) also compared protein adducts in rat plasma and human plasma obtained from six volunteers. Both adducts were present in rat plasma; in human plasma, the dichloro adducts were below the detection limit, and the trichloro adduct was much lower than in rat plasma. It can be calculated from the data that after exposure to tetrachloroethylene at the same concentration (40 ppm) and duration (6 hours), dichloro adducts were at least 40-fold lower in human plasma than in rat plasma. Trichloro adducts were not quantifiable with gas chroma- tography for technical reasons (Pahler et al. 1999). Overall, those results show that humans produce smaller amounts of the reactive metabolites; this is consistent with the overall greater metabolism of tetrachloroethylene in rats. A possible risk of mutagenic effects posed by tetra- chloroethylene metabolites with known genotoxic activity should therefore be substantially lower in humans than in rats. However, not all possible metabolites have been assessed for mutagenic activity, and techniques for identifying some metabolites in human samples are not readily available. Generally, the committee recommends that EPA integrate the qualitative and quantitative data from toxicokinetic, metabolic, and toxicodynamic studies in its assessment of the current knowledge of the toxic potential of tetrachloro- ethylene and specifically in its mode-of-action considerations. CELL-TRANSFORMATION ASSAYS The committee recommends that EPA include at least the more recent cell-transformation studies of tetrachloroethylene (Tu et al 1985, Milman et al. 1988).

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57 Genotoxicity FINDINGS AND RECOMMENDATIONS In vitro studies did not provide evidence of mutagenic activity of tetra- chloroethylene in mouse lymphoma cells or in bacterial and yeast mutation as- says except in the few tests in which metabolites of the GSH pathway were gen- erated, and no increases in chromosomal aberrations and SCEs were found in CHO cells. Tetrachloroethylene did not increase SCE and comet formation in human blood cells (this was incorrectly reported in the EPA draft); increases in the frequency of micronuclei were found in genetically altered human lymphoid cell lines and in a CHO cell line. In vitro studies of unscheduled DNA synthesis were mostly negative. The key question is whether the reactive metabolites of tetrachloroethyl- ene are formed and become available to sensitive cells in vivo and have genotoxic effects in intact organisms. Tetrachloroethylene did not induce un- scheduled DNA synthesis in rat kidney. It induced single-strand breaks in mouse liver and kidney at 1 hour but not at 24 hours after intraperitoneal injection and not in rat kidney 1 day after oral administration. The increase at 1 hour may be nonspecific because of intraperitoneal application and high doses. Tetrachloro- ethylene did not increase micronucleated reticulocytes in peripheral blood of mice (this was incorrectly reported in the EPA draft) and did not increase mi- cronucleated hepatocytes when administered before partial hepatectomy. When injected after partial hepatectomy, tetrachloroethylene slightly increased micro- nucleus formation, but this effect may be nonspecific because of severe liver toxicity caused by the high doses of tetrachloroethylene and the intraperitoneal application of this irritant substance. A study with 14C-labeled tetrachloroethyl- ene suggested a low level of binding to mouse liver DNA and even less to rat liver DNA and mouse and rat kidney, lung, and stomach DNA. These effects are considered nonspecific because DNA was not purified to constant radioactivity and because labeling via the intermediary metabolism appeared likely. In hu- mans exposed to tetrachloroethylene, no evidence of genetic alterations was noted, although the studies are of limited weight. Two studies in rats found no evidence of tumor-initiating activity of tetrachloroethylene (when liver foci were used as the end point). In conclusion, there is no convincing evidence that tetrachloroethylene has important genotoxic or mutagenic activity in intact organisms. The committee agrees with EPA’s conclusion that several metabolites of tetrachloroethylene are clearly genotoxic: TCVG, TCVC, N-Ac-TCVC, tetrachloroethylene oxide, DCA, and chlo- ral hydrate. However, it is still questionable whether the metabolites of tetrachloro- ethylene play an important role in the mode of action of tetrachloroethylene carcino- genesis (see Chapters 6-8) in view of the absence of convincing evidence of mutagenic and tumor-initiating activity of tetrachloroethylene in vivo. Additional studies of genotoxicity in vivo with state-of-the-art methods would be valuable. As noted above, the committee recommends that EPA provide an ex- panded and more integrated discussion of the genotoxicity data. The presenta- tion could be improved by the use of tables detailing the primary evidence, by

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58 Review of the EPA’s Draft IRIS Assessment of Tetrachloroethylene separate discussion of the genotoxic evidence on tetrachloroethylene and its metabolites, and by a more critical analysis of the studies.