&EFA
United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 20460
EPA/600/6-91/002A
February 1991
SAB Review Draft
Response to
Issues and Data
Review
Draft
Submissions on the |P°N°* +>
_ . .. , Cite or Quote)
Carcinogenicity of
Tetrachloroethylene
(Perchloroethylene)
Notice
This document is a preliminary draft. It has not been formally
released by EPA and should not at this stage be construed to
represent Agency policy. It is being circulated for comment on its
technical accuracy and policy implications.
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\ UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
I WASHINGTON. D:C. 20460
?
FEB 22 1991
OFFICE OF
RESEARCH AND DEVELOPMENT
MEMORANDUM
SUBJECT: Charge to Science Advisory Board Regarding Review of
"Response to Issues and Data Submissions on the
Carcinogenicity of Tetrachloroethylene"
FROM: William H. Farland, Ph.D.
Director
Office of Health and Environmental
Assessment (RD-689)
TO: Donald G. Barnes
Executive Director
Science Advisory Board (A-101)
The Office of Health and Environmental Assessment (OHEA)
appreciates the Board's agreement to meet on March 26, 1991 to
review the above-titled document.
By way of background, the subject of a potential cancer
hazard from tetrachloroethylene (perchloroethylene-PCE) is not a
new one in terms of past SAB/EPA dialogue. The most recent
correspondence was a letter of advice dated March 9, 1988 from
the Board to the Administrator regarding the Board's perspectives
on this topic. Since 1988, OHEA has been monitoring research
findings relevant to PCE's carcinogenic potential.
This "response" document has a relatively narrow purpose, as
stated in the introduction, compared to the more typical
comprehensive health assessment document that the committee
usually reviews for OHEA. The objective is to revisit issues and
review data concerning the identification of hazard, i.e., the
weight of the animal evidence bearing on the potential for human
carcinogenicity. Data have been submitted and issues raised in
public comment connected with a variety of recent Agency rule-
making actions. We have also updated our own literature
collection on relevant topics. An earlier version of this
response document is currently in the docket for the recently
promulgated National Primary Drinking Water Standard for
Tetrachloroethylene as published in the Federal Register on
January 30, 1991.
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In general terms, we request that the Board review the
technical adequacy of discussions concerning the animal cancer.
data and related ancillary information, such as mutagenicity and
metabolism, and the relationship of this information base to a
hazard classification of PCE under the Agency's current cancer
guidelines. More specifically, we request that the Board's
focus include the topics or questions listed below.
(1) Technical adequacy of discussions about the three
animal bioassay tumor endpoints, particularly regarding the
relevance of these tumor endpoints to the potential for human
hazard at some dose.
(2) Technical adequacy of discussions about ancillary
information for mutagenicity and metabolism considerations and
the appropriate use of this information in providing a better
understanding of the animal bioassays or the relevance of these
to the potential for human hazard.
(3) Have all important issues been identified and
appropriately considered, recognizing that many more fundamental"
scientific questions may exist but which may not be developed
adequately to meaningfully discuss in a risk assessment context?
(4) The soundness of the rationale used to weigh the
evidence, from each endpoint and in the aggregate for human
hazard potential. This topic relates to the logic of weighing
animal evidence including the relevance of ancillary data to that
process.
It is important to note that the concept of "weight of
evidence" under EPA's Cancer Risk Assessment Guideline identifies
an agent's potential to be a human hazard at some dose.
Questions about the quantitative relationship of dose to response
and mechanisms -of action that affect that relationship are dealt
with as a separate quantitative dose-response assessment which
typically follows the hazard identification part of a
comprehensive assessment. The quantitative dose-response
relationships for tetrachloroethylene needs to be revisited, as
noted in the response document, and we will be doing so in the
future.
We look forward to the Board's advice on this health hazard
identification topic. Should you decide that an overview of
carcinogen risk assessment guideline criteria for weighing the
evidence is useful for background purposes, we will make agenda
arrangements with Sam Rondberg.
cc: Erich Bretthauer
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DO NOT QUOTE OR CITE February 1991
SAB Review Draft
RESPONSE TO ISSUES AND DATA SUBMISSIONS
ON THE CARCINOGENICITY OF
TETRACHLOROETHYLENE
(PERCHLOROETHYLENE)
NOTICE
THIS DOCUMENT IS A PRELIMINARY DRAFT. It has not been formally released by the U.S.
Environmental Protection Agency and should not at this stage be construed to represent
Agency policy. It is being circulated for comment on its technical accuracy and policy
implications.
Human Health Assessment Group
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Washington, D.C. 20460
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DISCLAIMER
This document is a draft for review purposes only and does not constitute Agency
policy. Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
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CONTENTS
Preface °
Authors • '
1. INTRODUCTION 1
2. BACKGROUND 3
2.1. Prior EPA Analyses - • 3
2.2. Animal Studies of Perchloroethylene Carcinogenicity 5
2.3. Purpose of This Paper 6
3. THE METABOLISM OF PERCHLOROETHYLENE 7
4. MUTAGENICITY OF PERCHLOROETHYLENE AND ITS METABOLITES 13
4.1. Data on Mutagenicity of Perchloroethylene per se 13
4.2. Mutagenicity of Perchloroethylene Metabolites 16
5. MOUSE LIVER TUMORS 21
5.1. Carcinogenicity Bioassay Data and EPA's Position 21
5.2. Peroxisome Proliferation and Perchloroethylene 25
6. KIDNEY TUMORS IN MALE RATS 31
6.1. Alpha-2|x-globulin in Renal Carcinogenesis in Male Rats 33
6.2. Sustained Chronic Nephrotoxicity as a Possible Mechanism Independent of
Alpha-2p.-globulin Accumulation 39
6.3. A Mutagenic Mechanism of Perchloroethylene-lnduced Carcinogenesis in Male
Rats • 40
7. MONONUCLEAR CELL LEUKEMIA IN RATS • 44
8. SUMMARY AND CONCLUSIONS 48
58
References
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PREFACE
This document was prepared by the Office of Health and Environmental Assessment
(OHEA) to respond to data and comments submitted to the Agency and to discuss how this
information influences the overall weight-of-evidence classification for a perchloloroethyiene
human cancer hazard. Relevant literature through the fall of 1990 has been critically
evaluated.
iv
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AUTHORS
Members of the Human Health Assessment Group of the Office of Health and
Environmental Assessment (OHEA) prepared this document.
PRIMARY AUTHOR
Jean C. Parker, Ph.D.
Carcinogen Assessment Toxicology Branch
U.S. EPA, Washington, DC
CONTRIBUTING AUTHORS
Vicki Vaughan-Dellarco, Ph.D.
Genetic Toxicology Assessment Branch
U.S. EPA, Washington, DC
David Reese, Ph.D.
Genetic Toxicology Assessment Branch
U.S. EPA, Washington, DC
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1. INTRODUCTION
The scientific debate over the potential carcinogenicity of tetrachloroethylene
(perchloroethylene, perc, PCE) spans several years. The Office of Health and Environmental
Assessment within the U.S. Environmental Protection Agency's Office of Research and
Development has been considering the issues and current thinking pertaining to weight of
evidence for the human cancer hazard from exposure to perchloroethylene. Several issues
were brought up by the EPA's Science Advisory Board (SAB, 1987, 1988) during their review
of an addendum (U.S. EPA, 1986a) to the Health Assessment Document for
Tetrachloroethylene (U.S. EPA, 1985). New information also has become available over the
last two to three years that has bearing on the issues.
Recently generated laboratory data have led to the development of hypotheses about
the mechanisms of perchloroethylene tumorigenesis. Biological arguments have been put
forward suggesting species specificity for some of the proposed tumorigenic mechanisms.
Such arguments imply that certain experimental results are of questionable predictive validity
with respect to human health hazards and risks. While there is some evidence to support
these arguments, several critical experimental elements are missing to determine cause and
effect relationships in the test animals or to answer the human relevancy question with
certainty. Since the data are equivocal regarding mechanisms, a conclusion about the
carcinogenic potential of perchloroethylene in humans must be one of judgment considering
the weight of the pertinent evidence.
The objectives of this paper are: to review the current issues and hypotheses
surrounding perchloroethylene carcinogenesis, to evaluate these hypotheses in light of
recently published studies, and to develop the EPA's response to issues and data submitted
in comments to the Agency on the overall weight of evidence for potential human cancer
hazard. This paper reviews the issues considered by the EPA's Science Advisory Board
during its review of the draft "Addendum to the Health Assessment Document for
Tetrachloroethylene- (U.S. EPA, 1986a) and discusses relevant research data that have been
published since 1986. The topics covered include three tumor end points observed in rodents
exposed to perchloroethylene:
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1. hepatocellular carcinoma in male and female mice,
2. renal tubule neoplasia in male rats, and
3. mononuclear cell leukemia in male and female rats,
and the recent data on metabolism, genotoxicity and mutagenicity, peroxisome proliferation,
and alpha-2ji-globulin.
The EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1986b) provide a
framework for assessing the likelihood of a substance being a human cancer hazard. Under
these guidelines animal studies and human data are first analyzed separately. The evidence
from laboratory animal studies, along with other relevant information, may be classified as
"sufficient," "limited," "inadequate," "no data," or "no evidence" in animals. The human data is
classified as "sufficient," "limited," "inadequate," "no data," or "no evidence" in humans. The
two sets of information are merged with respect to assessing potential carcinogenicity in
humans (see Appendix A: Guidelines for Carcinogen Risk Assessment, 1986). The
classifications refer only to the weight of the experimental evidence that a chemical is
carcinogenic and not to its potency of carcinogenic action. The overall challenge here is not
only to determine how the currently available data influence the Agency's previous
categorization of the experimental animal evidence on the carcinogenicity of perchloroethylene
as "sufficient," but also to determine whether the "sufficient" animal data in the case of
perchloroethylene signify a human hazard potential as would be ordinarily assumed (U.S.
EPA, 1986b; OSTP, 1985; IARC, 1982,1987).
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2. BACKGROUND
2.1. PRIOR EPA ANALYSES
The EPA published a Health Assessment Document (HAD) for Tetrachloroethylene
(Perchloroethylene) in July of 1985 (U.S.EPA, 1985). The Office of Health and Environmental
Assessment (OHEA), in consultation with an Agency workgroup, prepared the HAD to serve
as a source document for the entire EPA (U.S.EPA, 1985, preface). The document underwent
extensive expert peer review and review by the Environmental Health Committee of the
Agency's Science Advisory Board (SAB) prior to publication. Based on the EPA's
interpretation of the overall weight of evidence, the HAD placed perchloroethylene into Group
C (possible human carcinogen). This categorization was in accordance with the Agency's
proposed Guidelines for Carcinogen Risk Assessment (published in final form in September,
1986). The classification was based primarily on the finding that "in a gavage bioassay,
perchloroethylene induced a statistically significant increase of malignant liver tumors in both
male and female B6C3F1 mice." This decision reflected a "limited" number of studies
showing a robust positive response, plus the fact that the response was a commonly observed
tumor type as opposed to a very rare tumor type, rather than "limited" evidence, such as a
borderline response, derived from a number of adequately run studies. In view of the pending
release of National Toxicology Program (NTP) reports on long-term animal inhalation studies
with perchloroethylene, the HAD stated that the carcinogenicity conclusions were interim,
however, and would be updated if necessary when the NTP reports were evaluated (U.S.EPA,
1985, preface).
A draft "Addendum to the HAD for Tetrachloroethylene (Perchloroethylene)" (U.S. EPA,
1986a), prepared by OHEA, analyzed the results of the inhalation bioassays conducted by the
NTP and performed by Battelle Pacific Northwest Laboratories. The results of these studies
revealed perchloroethylene-associated increases in the incidences of hepatocellular
carcinomas-the same tumor type seen in the gavage study-in both sexes of B6C3F1 mice,
mononuclear cell leukemia in both sexes of F 344/N rats, and uncommon renal tubule
neoplasms and some evidence for gliomas of the brain in male rats. The authors of the
addendum concluded that perchloroethylene is a B2 chemical (probable human carcinogen) because:
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• the NTP inhalation bioassay demonstrated that perchloroethylene can induce
carcinogenic effects at multiple sites, in both rats and mice (a replicate finding)
through inhalation exposure (a second route), and
• the earlier NCI bioassay provided positive evidence of hepatocellular carcinomas in
mice administered perchloroethylene by gavage.
On May 15,1986, the draft addendum received peer review by the Halogenated
Organics Subcommittee of the EPA's SAB in a public meeting held in Madison, Wisconsin.
The SAB's initial comments appear in a letter to EPA Administrator Lee Thomas dated
January 27,1987 (SAB, 1987). In this letter the SAB concluded "that perchloroethylene
belongs in the overall weight-of-evidence category C (possible human carcinogen)."
Further, the SAB judged the evidence for carcinogenicity in animals to be "limited"
because, "the National Toxicology Program bioassay does not provide a scientific basis to
associate either lesion (in rats) with inhalational exposure to perchloroethylene,"
thus, "the evidence arises only from a single strain of mouse" and "the kind of tumor
associated with perchloroethylene exposure in this mouse strain makes it difficult to create an
inference regarding human carcinogenicity."
As a result of the SAB's conclusions, Agency scientists and managers reexamined the
assessment of perchloroethylene and again concluded that perchloroethylene should be
classified as a B2 chemical. Administrator Thomas responded to the SAB in an August 3,
1987, letter that clarified the Agency's position on perchloroethylene, and additional
consultative advice was requested of the SAB on specific issues regarding liver tumors in
B6C3F1 mice and kidney tumors in male rats. More detailed comments were presented in
"EPA Staff Comments on Issues Regarding the Carcinogenicity of Perchloroethylene (Perc)
Raised by the SAB," a paper that was enclosed with the Administrator's August 3 letter.
The SAB's response to the second request for advice is contained in a Setter dated
March 9,1988, to Administrator Thomas (SAB, 1988). In this letter the SAB concluded that,
"the overall weight of evidence lies on the continuum between the categories B2 and C of
EPA's risk assessment guidelines for cancer." In an attempt to put this conclusion in
perspective, the SAB also remarked that:
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A substance classified as C (limited evidence in animals) for which human
exposure is high may represent a much greater potential threat to human
health. EPA and other agencies (including those in state governments) may,
therefore wish to take steps to reduce high exposures to substances in the C
category whenever there appears to be a potentially significant threat to human
health (in the sense that the plausible upper bound estimate of potency times
lifetime exposure is above the threshold where regulation may be judged
appropriate).
Since that time, the EPA has received public comments on perchloroethylene issues in
several regulatory actions which include the perchloroethylene weight-of-evidence
classification as a matter considered. These public comments pertain to RCRA listing
(Federal Register, Dec., 1989) and CERCLA reportable quantity rules (Federal Register,
August, 1989), and to MCLG and MCL proposals for drinking water (Federal Register, May,
1989; December, 1990).
2.2. ANIMAL STUDIES OF PERCHLOROETHYLENE CARCINOGENICITY
Perchloroethylene has shown cancer-causing activity in male and female mice and in
male and female rats in the NCI/NTP studies. In both sexes of mice, perchloroethylene
induced dose-related statistically significant increases in hepatocellular carcinomas when
administered by oral gavage or by inhalation. Statistically significant increased incidences of
mononuclear cell leukemia, and the presence of uncommon renal tubule neoplasms and some
evidence of gliomas in the brain were observed in male rats exposed to perchloroethylene by
inhalation. The renal tubule tumors were also detected in male rats exposed by gavage.
Female rats exhibited an increase of mononuclear cell leukemia when exposed to
perchloroethylene by inhalation.
Although perchloroethylene increased the incidence of cancer at three different sites
and in two species, controversy surrounds each of the tumor end points. Considerable
scientific debate has focused on the predictive validity of mouse liver and male rat kidney
tumors as well as mononuclear cell leukemia. In addition to the general controversies
surrounding these tumor end points, chemical-specific data that may be pertinent to the
evaluation of the effect of perchloroethylene on tumor incidence has generated concern.
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2,3, PURPOSE OF THIS PAPER
The intent of this paper is to respond to data and comments submitted to the Agency.
Studies related to perchloroethylene tumorigenesis have been published subsequent to EPA's
1986 draft Addendum, and have been formally submitted to -the EPA. These studies were
designed to elucidate the mechanism of action in animals and provide better understanding of
the relevance of animal data to human hazard. The studies report mechanistic data germane
to a number of issues concerning the etiology of perchloroethylene-induced rodent tumors and
their relevance to humans. The EPA must evaluate information from these studies and report
its assessment of whether the data provide an understanding of underlying tumorigenesis
mechanisms that would lead to a conclusion that modes of perchloroethylene cancer-causing
activity are not operative in humans. The purpose of this paper then, is to provide a response
to the data and comments submitted to the Agency that have bearing on the relevancy issues,
and to discuss how this information influences the overall weight-of-evidence classification for
a perchloroethylene human cancer hazard.
This paper summarizes OHEA's critical evaluation of three perchloroethylene tumor
end points in rats and mice in light of new scientific findings, and incorporates the recent
information into the weight of evidence for human hazard.
The paper addresses recent literature on perchloroethylene and its biometabolites as
the information relates to: metabolism, mutagenicity, cytotoxicity and proliferative changes in
mouse liver, nephrotoxicity and renal tubule neoplasia in male rats, and mononuclear cell
leukemia in male and female rats.
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3. THE METABOLISM OF PERCHLOROETHYLENE
The cancer-causing activity of halogenated ethylenes is generally considered to reside
primarily in biometabolites rather than in the parent compounds themselves. Studies in both
animals and humans indicate that metabolism of perchloroethylene is relatively limited, as
evidenced by the fact that a high percent of absorbed dose is excreted in the breath as the
parent molecule. In human studies, however, only approximately half of the perchloroethylene
absorbed has been accounted for through the excretion of parent compound or metabolites.
Estimates of the extent of metabolism in humans have been made from balance
studies by accounting for a retained dose after inhalation exposure by measuring trichloro-
compounds excreted in the urine. Metabolites other than those measured may be excreted in
the urine or bile. Thus, the additional perchloroethylene in humans may be metabolized to
compounds that were not measured. Other as yet unrecognized pathways for
perchloroethylene that have not been taken into consideration may exist in humans.
Perchloroethylene is metabolized through at least two distinct pathways. Oxidative
metabolism via the cytochrome P450 system has been extensively reviewed in the HAD (U.S.
EPA, 1985). Recent investigations have revealed a glutathione conjugative pathway which
appears to be a minor but important route that has been shown to generate a mutagenic
constituent. The oxidative and conjugative pathways are summarized in Figures 1, 2, and 3.
Oxidative metabolism of perchloroethylene (dependent on cytochrome P450) probably
occurs mostly in the liver but may occur at other sites. This pathway is operative in humans
as well as rodents and leads to the production of several metabolites (Figure 1). There is no
basis to believe that qualitative differences exist between species with respect to known
pathways of oxidative metabolism of perchloroethylene. However, there are quantitative
differences among the metabolic rates of different species. The major metabolite of the
oxidative pathway is trichloroacetic acid (TCA) which is excreted in the urine of all species
tested. Other identified urinary metabolites are designated in Figure 1. Some of the
intermediates in the oxidative pathway are known to possess cytotoxic/genotoxic activity (e.g.,
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•Identified urinary metabolites.
Rrgure 2. Enzyme-catalyzed metabolism of perchlorothylene to its glutathione conjugate,
followed by removal of the glutamyl and glycine residues to yield its corresponding cysteine
S-conjugate. Conjugation of perchloroethylene with glutathione has been demonstrated in rat
and mouse hepatic cytosolic and microsomal fractions. Further processing to
1,1,2-trichlorovinylcysteine occurs In the kidney. This metabolite gives rise to potent mutagens.
SOURCE: Anders ef.a/., 1988. g
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•Identified urinary metabolites.
Figure 3. Further metabolism in the kidney of the perchloroethytene
intermediate 1,1,2-trichlorovinylcysteine, leading to mutagenic metabolites.
These pathways occur In humans as well as in rodents.
SOURCE: Dekant etal., 1987; Green, 1990; ECETOC, 1990.
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perchloroethylene-epoxide, chloroacetaldehydes; see Section 4) and several have been
shown to cause cancer (e.g., DCA, TCA, and chloroacetaldehydes).
Recent studies in rats (reviewed by Anders et al., 1988 and DeKant et al., 1989) have
demonstrated the formation of a cytotoxic and mutagenic metabolite(s) of perchloroethylene
that arises from hepatic glutathione conjugative pathways (see Figures 2 and 3). This
secondary metabolic pathway is initially catalyzed by hepatic cytosolic and microsomal
glutathione S-transferases to yield S-(1,2,2-trichlorovinyl) glutathione (TCVG). After transport
to the kidney, TCVG is metabolized to S-(1,2,2-trichlorovinyl) cysteine (TCVC) by the
enzymatic removal of glutamyl and glycine residues. TCVC is acetylated to N-Acetyl-S-(1,2,2-
trichlorovinyl)-L-cysteine, which is excreted in the urine. However, TCVC is also a substrate
for renal beta-lyases which cleave TCVC to yield an unstable thiol that may give rise to
cytotoxic and mutagenic intermediates.
In vivo and in vitro experiments in rodents provide evidence to support this conjugative
metabolic scheme. DeKant et al. (1987) and Green et al. (1990) have demonstrated hepatic
conjugation of perchloroethylene with glutathione by rat liver fractions in in vitro experiments.
Vamvakas et al. (1989) found TCVG in the bile excreted by perchloroethylene-perfused rat
livers, and Green et al. (1990) reported the presence of the glutathione conjugate in the bile of
rats administered perchloroethylene by gavage.
There is some limited evidence suggesting that humans may not metabolize
perchloroethylene by the conjugative pathway. Human liver samples have been compared to
rat and mouse liver samples with respect to ability to conjugate perchloroethylene with
glutathione (Green et al., 1990). These investigators found low levels of conjugation by rat
and mouse livers but were unable to demonstrate conjugation by human livers. To confirm
the viability of glutathione S-transferase in the rat and human liver samples studied using a
more powerful protocol, these workers compared the two species with respect to their ability to
conjugate 1-chloro-2,4-dinitrobenzene with glutathione. Both species carried out this
conjugation rapidly and at essentially the same rate. This finding indicates that the reduced
ability of the human liver samples to conjugate perchloroethylene was not attributable to an
inactive glutathione S-transferase. Because of the very low levels of enzyme activity being
measured and the limited numbers of human liver samples tested, it is premature to conclude
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that humans are unlikely to carry out this metabolic step. Additional confirmatory studies are
clearly needed.
The beta-lyase pathway has been shown to produce cytotoxic and mutagenic
metabolites from glutathione and cysteine conjugates of a variety of haloalkenes in a number
of animal models in vivo or in vitro (Anders et al., 1988; Lock, 1988). The same pathway
which leads to the formation of the toxic metabolites in animal models and mutagenic
metabolites in bacterial models is also present in human proximal tubular cells. Human
proximal tubular cells have been shown to be sensitive to the toxicity of glutathione and/or
cysteine conjugates of a variety of chloro- and fluoroalkenes that are activated via the beta-
lyase pathway (Chen et al., 1990).
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4. MUTAGEN1CITY OF PERCHLOROETHYLENE AND ITS METABOLITES
Genetic alterations are critical events in the carcinogenesis process. Thus, evidence
on the ability of an agent to produce heritable genetic lesions (e.g., gene mutations, stable
chromosomal aberrations, aneuploidy) can potentially provide useful mechanistic information
for induced carcinogenesis. Additionally, it is reasonable to assume that mutagenic
mechanisms are universal, and thus evidence of mutagenesis is also regarded as important
information that supports the inference of potential for carcinogenicity in humans. It should be
emphasized, however, that genetic alterations are only one component of carcinogenesis.
Moreover, the most commonly used assays in genetic toxicology are in vitro ones. Thus, the
observations of mutagenic noncarcinogens and nonmutagenic carcinogens as well as
mutagenic carcinogens are to be expected. The use of results from short-term genotoxicity
tests as supporting evidence for or against carcinogenicity of an agent must be undertaken
with caution.
The 1985 EPA Health Assessment Document for Tetrachloroethylene provided a
comprehensive assessment of the genotoxicity of perchloroethylene. The conclusion reached
in the document was that the available data did not clearly support a mutagenic potential for
perchloroethylene. The evidence available at that time indicated that if perchloroethylene is
mutagenic, it is only weakly so. An update of the scientific literature was recently conducted
to determine if the earlier conclusions are still valid.
4.1. DATA ON MUTAGENICITY OF PERCHLOROETHYLENE PER SE
As shown in Table 1, perchloroethylene has not been clearly shown to be an inducer
of gene mutations in routinely used assays. In bacterial assays for reverse mutation
(Sa//77one//a/mammalian microsome test) in the presence or absence of exogenous liver S9
activation, perchloroethylene exposures produced largely negative results (Bartsch et al.,
1979; Margard, 1978; Haworth et al., 1983; Warner et al., 1988). In studies reporting
positive results, the responses were weak and were produced by cytotoxic concentrations.
There was no evidence of clear dose-related effects. These positive findings may have also
been related to the presence of mutagenic contaminants and/or stabilizers in the
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TABLE 1. Summary of Genoloxicity Testing of Tetrachtoroethylene
T
•+ designates positive; - negative; wk weak response; ? inconclusive test. Dose-response relationships
wore not established for the reported + results or wk results.
'Positive results are considered weak because large amounts of material were needed to elicit the
responses. Results may also be explained by mutagente stabilizers or contaminants.
•Questionable evidence for weak or borderline activity in specific data sets.
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perchloroethylene samples tested. When highly purified perchloroethylene was evaluated in
a desiccator using the Sa/mone//a/mammalian microsome test, negative results were obtained
(Shimadaetal., 1985)
Perchloroethylene has also tested negative in yeast for reverse mutations (Callen et
al., 1980; Bronzetti et al., 1983) and Drosophila for sex-linked recessive lethal mutations
(Valencia et al., 1985). Responses after perchloroethylene treatment in the L5178Y TkYTk"
mouse lymphoma cell assay for forward mutations have been either negative or equivocal
(Myhr et al., 1986; McGregor et al., 1988).
Perchloroethylene has not been demonstrated to be a clastogen (chromosome-
breaking activity). Negative results were found for the induction of chromosomal aberrations
in cultured Chinese hamster ovary cells (Galloway et al., 1987) and in the bone marrow assay
for rats and mice (Cema and Kypenova 1977; Rampy et al., 1978; Beliles et al., 1980). A
cytogenetic study of humans exposed to perchloroethylene did not show elevated frequencies
of chromosomal aberrations or sister chromatid exchanges in peripheral lymphocytes (Ikeda et
al., 1980).
Chemical adduct formation is a prerequisite step in certain types of mutagenesis.
Schumann et al. (1980) reported no detectable DNA binding in livers of mice exposed to
inhaled 14C - labeled perchloroethylene. This was not a sensitive test, however, because the
specific activity of the label was too low to preclude the possibility of DNA binding (i.e., this
test could not detect slightly fewer than 10"5 alkylations per nucleotide; recent protocols are
able to detect 10* to 10'12). In a more recent study, Mazzullo et al. (1987) reported low levels
of DNA binding in the liver, kidney, lung, and stomach of the mouse and rat after
intraperitoneal injection of perchloroethylene. These low levels of DNA binding cannot be
distinguished from binding as a result of biosynthetic incorporation of the label into DNA, and
thus, it is questionable whether exposure to perchloroethylene results in the formation of DNA
adducts.
Perchloroethylene exposures have produced negative, questionable, or weak results in
tests that do not measure mutation per se but are indicative of DNA damaging activity. Tests
for DNA repair synthesis in hepatocytes (Shimada et al., 1985; Costa and Ivanetich, 1984;
Goldsworthy et al., 1988), mitotic recombination in yeast (Bronzetti et al., 1983; Koch et al.,
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1988), and sister chromatid exchange formation in culture Chinese hamster cells (Galloway et
al., 1987) have been predominantly negative. Perchloroethylene has been reported to be a
weak inducer of DNA single strand breaks in mouse liver and kidney (Walles, 1986). Although
DNA strand breaks are events that may lead to mutagenteity, agents that can be
demonstrated to Induce only DNA strand breakage should not be viewed to possess the same
genetic hazard potential as agents that have been shown to induce gene mutations or stable
chromosomal aberrations.
4.2. MUTAGENICITY OF PERCHLOROETHYLENE METABOLITES
At the time the 1985 HAD was being prepared, only a limited literature existed on the
mutagenicity of perchloroethylene metabolites. However, several studies are now available
(summarized in Table 2) and their results warrant some consideration.
Oxidative metabolism of perchloroethylene (dependent on cytochrome P4SO) occurs
mostly in the liver. This pathway is operative in both rodents and humans and leads to the
production of several metabolites (Figure 1). Perchloroethylene-epoxide, a hypothesized
intermediate in perchloroethylene oxidative metabolism, has been shown to be rnutagenic in
the Sa/mon0//a/mammalian microsome test (Kline et al., 1982).
Chloral hydrate (trichloroacetaldehyde), a known metabolite of trichloroethylene and
likely a perchloroethylene metabolite, has been produced under both in vitro and in vivo
conditions. Several studies are available on the ability of chloral hydrate to produce
aneuploidy (i.e., loss or gain of whole chromosomes) in both mitotic and meiotic cells,
including yeast (Singh and Sinha, 1976,1979; Kafer, 1985; Gualandi, 1987; Sora and
Carbone, 1987), cultured mammalian somatic cells (Degrassi and Tanzarella, 1988), and
spermatocytes of mice (Russo et al.. 1984; Liang and Pacchierotti, 1988). It should be
pointed out that this type of genetic effect is most likely due to interference of spindle function
rather than a DNA-reactive mechanism. Chloral hydrate has been reported to be weakly
rnutagenic in the Sa/mone/teftnammalian microsome test (Haworth et al., 1983) but negative
for sex-linked recessive lethal mutations in Drosophila (Yoon et al., 1985). It has been
reported to induce single-strand breaks in hepatic DNA of mice and rats (Nelson and Bull,
1988) and mitotic gene conversion in yeast (Bronzetti et al., 1984). Chloral hydrate also has
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TABLE 2. Summary of Genotoxicity Testing of Tetrachtoroethylene Metabolites
1 Metabolite
|
Perchloroethylene-epoxide
Chloral hydrate
-
Trichbroacetic Acid
Dichloroacetaldehyde
Monochloroacetaldehyde
S-(1,2,2-trichloro-
vinyljgluthfone
:
(Result)*/Assay
(+) Salmonella/Ames Assay
(+) Aneupbidy/yeast
(+) Anauploidy/mammalian cells
in vitro
(+) Aneuptoidy/spermatocytes of
mice
(wk) Salmonella/Ames test
(-) Drosophila sex-linked
recessive lethal mutation
(+) DNA strand breaks in mice
and rats
(+) mitotic gene conversion in
yeast
(+) DNA strand break in mice and
rats
(-) DNA strand break in mice and
rats
(?) in vivo cytogenetics
(-) Salmonella/Ames Assay
(+) Salmonella/Ames Assay
(+) DNA strand breaks in human
cells in vitro
(+) DNA strand breaks in human
cells in vitro
(+) Salmonella/Ames Assay
Reference
Kline et al., 1982
Singh and Sinha, 1976, 1979;
Kafer. 1985; Gualandi. 1987;
Sora and Carbane, 1987
Degrassi and Tanzavella, 1988
Russo et al., 1984; Liang and
Pacchierotti, 1988
Haworth et al., 1983
Nelson and Bull, 1988
Bronzetti et al., 1984
Nelson and Bull, 1988
Chang et al., 1989
Bhunya and Behera, 1987
Waskell, 1978
Bignami et al., 1980
Chang et al., 1989
Chang etal., 1989
Vamvakas et at., 1989
•+ designates positive; - negative; wk weak response; ? inconclusive test.
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been observed to cause tumors in mice (Rijhsinghani et al., 1986; Daniel, 1990). Other
chloroacetaldehydes are potentially mutagenic. Dichloroacetaldehyde (DCAA) is mutagenic in
the Sa/mone/fe/mammalian microsome test (Bignami et al., 1980) and both
monochloroacetaldehye and, to a lesser extent DCAA, appear to induce DMA single strand
breaks in cultured human cells (Chang et al., 1989).
Few genotoxicity studies are available on the carcinogenic perchloroethylene
metabolites trichloroacetic acid (TCA) and dichloroacetic acid (DCA). TCA and DCA have
been reported to produce single strand breaks in hepatic DMA of mice and rats. This action is
Independent of peroxisome proliferation and of liver necrosis (Nelson and Bull, 1988; Nelson
etal., 1989). The induction of DNA single strand breaks could not be confirmed by other
laboratories, however, a different methodology was used (Chang et al., 1989). TCA was
reported as positive for the induction of chromosomal aberrations and micronuclei in the bone
marrow of mice (Bhunya and Behera, 1987). This finding is questionable, however, because
of the low background frequencies reported for chromosomal aberrations and the anomalous
dose-response seen for micronuclei formation in normochromatic erythrocytes.
In rats perchloroethylene has been shown to be metabolized to a cytotoxic, mutagenic
fraction through a conjugative-beta-lyase pathway (reviewed by Anders et al., 1988 and
DeKant et al., 1989; see also, Section III [Metabolism] of this document). This secondary
metabolic pathway, which may assume greater importance with saturation of the oxidative
pathway, is initially catalyzed by hepatic cytosolic and microsomal glutathione S -transferases
to yield S-(1,2,2-trichlorovinyl) glutathione (TCVG). After transport to the kidney, TCVG is
metabolized to S-(1,2,2-trichlorovinyl) cysteine (TCVC) by the enzymatic removal of glutamyl
and glycine residues. TCVC is acetylated to N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine, which
is excreted in the urine. However, TCVC is also a substrate for renal beta-lyases which
cleave TCVC to yield an unstable thiol that may give rise to cytotoxic and mutacjenic
intermediates.
In vivo and in vitro experiments provide evidence to support this metabolic and
cytotoxic/mutagenic scheme. DeKant et al. (1987) and Green et al. (1990) have
demonstrated hepatic conjugation of perchloroethylene with glutathione by rodent liver
fractions in in vitro experiments. Vamvakas et al. (1989) found TCVG in the bile excreted by
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perchloroethylene-perfused rat livers and Green et al. (1990) reported the presence of the
glutathione conjugate In the bile of rats administered perchloroethylene by gavage.
Human liver samples have been compared to rat and mouse liver samples with respect
to ability to conjugate perchloroethylene with glutathione (Green et al., 1990). These
investigators found low levels of conjugation by rat and mouse livers but were unable to
demonstrate conjugation by the human livers. To confirm the viability of glutathione S-
transferase in the rat and human liver samples studied, these workers compared the two
species with respect to their ability to conjugate 1-chloro-2,4-dinitrobenzene with glutathione.
Both species carried out this conjugation rapidly and at essentially the same rate. This finding
indicates that failure of human liver samples to conjugate perchloroethylene was not
attributable to an inactive glutathione S-transferase.
An inability of human liver to conjugate perchloroethylene with glutathione would
indicate that toxicologic effects attributable to conjugative metabolites in animals would have
little, if any, relevance to human health hazard. However, because of the limited numbers of
human livers tested, it is impossible to conclude that humans are unable to carry out this
metabolic step. Additional confirmatory studies are clearly needed.
Vamvakas et al. (1989) studied the mutagenicity of chemically synthesized TCVG in a
modified Ames protocol employing Salmonella typhimurium TA100. In the absence of an
exogenous activating system the conjugate produced a weak mutagenic response. In the
presence of rat kidney microsomes, mitochondria or cytosolic fractions (sources of gamma
glutamyl transferase [GGT] and dipeptidase), TCVG caused marked, dose-related mutagenic
responses. These responses were reduced when the protein fraction was pretreated with
either a beta-lyase or a GGT inhibitor. A mutagenic response was not observed when hepatic
enzymes were used in place of kidney fractions.
The results of these experiments show that TCVG requires metabolic activation to
express its marked mutagenic activity. Further, the enzymes required to carry out this
activation are found in the rat kidney, not in the liver. This distribution of enzymes is
consistent with the production of renal tubule neoplasia in perchloroethylene-treated rats.
Bile, collected from rat livers perfused with perchloroethylene, was found to contain
TCVG and, when tested in the Ames protocol using kidney paniculate fractions as the
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activating system, was clearly mutagenic. As in the experiment with synthetic TCVG,
inhibition of renai beta-lyase or GGT reduced the mutagenicity of the bile samples (Vamvakas
etal., 1990).
Green et al. (1990) reported the presence of N-acetyl-S^1,2,2-trichlorovir»yl)-L-cysteine
in the urine of rats dosed with perchloroethylene by gavage and rats and mice dosed by
inhalation. These investigators have also shown that renal cytosolic beta-lyase from rats,
mice, and humans are capable of metabolizing TCVC. Others also have shown that the beta-
lyase pathway is present in human proximal tubular cells, and is responsible for activating
glutathione and /or cysteine conjugates of a variety of chloro- and fluoroalkenes to reactive
metabolites capable of binding to cellular macromolecules (Chen et al., 1990).
In additional studies, TCVG has been found to induce unscheduled DNA synthesis in a
porcine kidney cell line and TCVG and N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine have both
been found to be mutagenic in the Ames test (DeKant et al., 1989).
The available data indicate that metabolism would be a prerequisite for
perchloroethylene mutagenicity. The data do not support classifying the parent compound per
se as a mutagen. Although certain metabolites of oxidative metabolism may be mutagenic
(e.g., the chloroacetaldehydes including chloral hydrate), these positive data are
predominantly limited to in vitro studies. Moreover, perchloroethylene was assayed in the
presence of several types of metabolic activation systems (e.g., liver homogenates and intact
hepatocytes) that would favor oxidative metabolism and, under these conditions,
predominantly negative results were found.
Perchloroethylene may also be activated by a minor pathway involving conjugation with
giutathione followed by renal processing of the S-conjugate. This S-conjugate is a beta-lyase
dependent mutagen in the Sa/mone/te/mammalian microsome assay. Mutagenic metabolites
formed in the kidney could conceivably contribute to the tumors observed in male rat kidneys.
However, these mutagenicity studies of perchloroethylene metabolites formed by the kidney
are in vitro and have been conducted in only one laboratory.
The mutagenicity studies on metabolites of perchloroethylene emphasize the need for
further studies concerning a mutagenic role for them in perchloroethylene carcinogenesis.
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5. MOUSE LIVER TUMORS
5.1. CARCINOGENICITY BIOASSAY DATA AND EPA'S POSITION
In carcinogenicity bioassays, perchloroethyiene has been shown to cause a statistically
significant increase in the incidence of hepatocellular carcinoma in both sexes of B6C3F1
mice, following either oral gavage administration or inhalation exposure (NCI, 1967; NTP,
1986).
In a study conducted by NCI (NCI.1976), groups of 50 male mice received time-
weighted average doses of 536 or 1,072 mg/kg of perchloroethyiene in com oil by intragastric
gavage for 78 weeks (450 or 900 mg/kg for 11 weeks, then 550 or 1,100 mg/kg for 67 weeks).
Groups of 50 female mice received time-weighted average doses of 386 or 772 mg/kg of
perchloroethyiene in com oil by gavage (300 or 600 mg/kg for 11 weeks, then 400 or 800
mg/kg for 67 weeks). Mice were dosed 5 days per week. The perchloroethyiene used in the
study was greater than 99% pure, but identification of impurities was not made (NCI, 1976;
U.S. EPA, 1985). However, the test sample was estimated to contain epichlorohydrin
concentrations of less than 500 ppm (U.S. EPA, 1985). It was considered unlikely, however,
that the tumor response resulted from this low concentration of epichlorohydrin.
Perchloroethylene caused statistically significant (p < 0.001) increases in the incidences of
hepatocellular carcinoma in both sexes of mice in both treatment groups when compared to
untreated controls or to vehicle controls. The Time to tumor was decreased in treated mice.
Additional studies reported by the NTP confirmed the finding of hepatocellular
carcinoma in B6C3F1 mice exposed to perchloroethyiene. Groups of 50 mice of each sex
were exposed to perchloroethyiene concentrations of 0,100, or 200 ppm by inhalation
exposure, 6 hours a day, 5 days per week, for 103 weeks. Perchloroethyiene caused dose-
related statistically significant increases in the incidences of hepatocellular carcinoma in both
sexes.
The biologic significance of chemically-induced mouse-liver tumors, with respect to
human hazard identification and the use of such tumor data in assessing cancer risk to
humans, is a subject of extensive debate. The controversy surrounding the liver tumor
response in the B6C3F1 mouse specifically is well recognized and has been going on for
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some time. Several meetings and symposia on the subject have been held and numerous
publications have appeared dealing with different aspects of the subject (e.g., Popp, 1984;
Stevenson et al.. I990). EPA is aware of the divergent scientific views regarding the predictive
validity of mouse liver tumors in the assessment of carcinogenic risk in general, and in the
case of perchloroethylene in particular. The EPA undertook an extensive review of the issues
concerning mouse liver tumors while it was developing its guidelines for carcinogen risk
assessment, and has kept abreast of the issues since that time.
The relevance of mouse liver tumors to the assessment of carcinogenicity in humans
has been questioned because of:
' - the high, and sometimes variable, background incidence of sj.ontaneously
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(McConnell, 1990). Of the chemicals evaluated as carcinogens by EPA, fewer than 10% have
been found to cause only mouse liver cancer (Seal, 1990).
At this time, the Agency's position is that increased incidences of mouse liver tumors
are considered evidence for human carcinogenic potential, although the evidence may be
downgraded on a case-by-case basis according to chemical-specific data. The current EPA
policy for evaluating mouse liver tumor data is described in the Guidelines for Carcinogen Risk
Assessment, published in 1986 (U.S. EPA, 1986):
An increased incidence of neoplasms that occur with high spontaneous background
incidences (e.g., mouse liver tumors and rat pituitary tumors in certain strains)
generally constitutes "sufficient" evidence of carcinogenicity but may be changed to
"limited" when warranted by the specific information available on the agent (p 1-7)."
"For a number of reasons, there are widely diverging scientific views about the
validity of mouse liver tumors as an indication of potential carcinogenicity in
humans when such tumors occur in strains with high spontaneous background
incidence and when they constitute the only tumor response to an agent.
These Guidelines take the position that when the only tumor response is in the
mouse liver and when other conditions for a classification of "sufficient"
evidence in animal studies are met (e.g., replicate studies, malignancy; see
section IV), the data should be considered as "sufficient" evidence of
carcinogenicity. It is understood that this classification could be changed on a
case-by-case basis to "limited," if warranted, when factors such as the following
are observed: an increased incidence of tumors only in the highest dose group
and/or only at the end of the study; no substantial dose-related increase in the
proportion of tumors that are malignant; the occurrence of tumors that are
predominantly benign; no dose-related shortening of the time to the appearance
of tumors; negative or inconclusive results from a spectrum of short-term tests
for mutagenic activity; the occurrence of excess tumors only in a single sex
(pp.1-5, 1-6).
Thus, in the absence of convincing evidence to the contrary, the Agency considers
increased incidences of mouse liver tumors in replicate studies to be "sufficient" evidence of
carcinogenicity.
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The 1987 EPA paper on the weight-of-evidence classification for perchloroethylene1
was in keeping with the Agency's Guidelines for Carcinogen Risk Assessment. The EPA
paper stated that:
a strong carcinogenic response has been demonstrated in two separate
experiments, in different laboratories, using different routes of exposure,
producing similar dose-related responses, increases the weight of the evidence
that the response is indicative of a carcinogenic response in animals. While
this interpretation can be debated because the response is seen in mousie liver
and is accompanied by some non-neoplastic pathology, the confirmatory finding
as well as the nature of the response is viewed by many in the science
community as "sufficient evidence" of an animal carcinogenic response, ias is
stated in the Agency's Carcinogen Risk Assessment Guidelines. Additional
support for this view comes from the recent deliberations on the classification of
perchloroethylene by IARC /(see footnote 5 in the conclusion section)/.... It is
the position of the Agency, therefore, that the new inhalation liver tumor data
from the NTP study should add to the weight-of-evidence determination tor
perchloroethylene.
The EPA paper and the most recent letter from the SAB concerning perchloroethylene
(SAB, 1988) are consistent regarding statements about the mouse liver tumors. The SAB
letter in fact stated that the Board's consensus on the significance of mouse liver tumors was,
"that mechanistic explanations are not sufficiently well developed and validated at this time to
change EPA's present approach expressed in its risk assessment guidelines for
carcinogenicity." The SAB concluded that,
the generation of mouse liver tumors by chemicals is an important predictor of potential
risks to humans. Of the several mechanistic models under consideration (including
regenerative hyperplasia, oncogene activation and trihalomethyl radical formation) the
one most promising for immediate application to risk assessment is characterized by
proliferation of peroxisomes, an intracellular organelle, in the liver."
1The staff paper sent to the SAB as an attachment to the Augus 3,1987 letter from EPA s
AdminSrator written in response to the formal comments submitted to the Agency by the
Salogenatedpanics Subcommittee regarding the public SAB review of the draft addendum
to the HAD on perchloroethylene.
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5.2. PEROXISOME PROLIFERATION AND PERCHLOROETHYLENE
Beginning in 1986, additional information regarding the possible link between
peroxisome proliferation and liver cancer in B6C3F1 mice exposed to perchloroethylene, has
been published (Odum et a!., 1988, Green et al., 1986; DeAngelo et al., 1989; Goldsworthy
and Popp, 1987). These newer data warrant an evaluation with respect to the interpretation of
perchloroethylene mouse liver tumor data as it relates to human health hazard.
A chemically-induced increase in numbers of hepatic peroxisomes, generally referred
to as peroxisome proliferation, has been suggested as the underlying mechanism through
which perchloroethylene induces hepatocellular carcinomas in B6C3F1 mice (Odum et al.,
1988). Carcinomas are proposed to arise as a result of oxidative damage to the cell, possibly
at the level of DMA, caused by elevated concentrations of hydrogen peroxide, a peroxisome
degradation product. Hydrogen peroxide is normally degraded by a peroxisomal catalase,
however the activity of this enzyme does not increase in a parallel fashion with peroxisomes
and other peroxisomal enzymes following perchloroethylene exposure. This enzymic
imbalance may result in the accumulation of cytotoxic concentrations of hydrogen peroxide.
Although DNA is identified as a potential ultimate target of oxidative damage, the mechanism
is still described as "epigenetic" or "nongenotoxic." These terms are used in this context to
contrast DNA damaging events that are secondary to other effects caused by perchloro-
ethylene from DNA damage produced by a direct, primary interaction of perchloroethylene or
its metabolites with DNA.
Assuming that the observed perchloroethylene-induced peroxisomal proliferation and
liver hepatocellular carcinomas in the B6C3F1 mouse are related, one course of reasoning
supports the hypothesis that the liver tumors could be unique to that species if this is the only
mechanism, therefore, decreasing their predictive validity relative to human health hazard.
The central points of the supporting rationale are:
• a major metabolite of perchloroethylene, trichloroacetic acid (TCA), is the
peroxisome-inducing agent and, therefore, the cancer-causing agent in mouse liver,
• because of a lower rate of metabolism, and metabolic enzyme saturation at
relatively low concentrations of perchloroethylene, rats are not as efficient as mice
in metabolizing perchloroethylene to TCA, which explains the lack of an
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hepatocarcinogenic effect in rats. This also implies that a threshold level of TCA
must be exceeded before peroxisomal proliferation and liver cancer can occur,
humans are even less efficient metabolizers of perchloroethylene than rats,
human liver cells would probably not respond to the peroxisome-proliferating activity
of TCA even if sufficient levels of the chemical could be produced in humans
mecauslwnen human liver cells iare challenged in vitro with TCA at concentrations
known to cause peroxisome proliferation in cultured mouse and rat hepatocytes, no
evidence of increased peroxisomal activity can be detected), and
• human hepatocytes are also not as responsive as rodents to other known
peroxisome proliferators such as the hypolipidemic drugs and phthalate esters.
The conclusion from the above rationale is that humans, because of an inability to
generate sufficient TCA levels from perchloroethylene metabolism, and a general
unresponsiveness to peroxisome-proliferating agents, are unlikely to show a hepatocellular
carcinogenic response to perchloroethylene, assuming this is the mechanism of action in
mice.
Many aspects of the above statements are supported to some extent by experimental
data.
• TCA has been shown to be a major metabolite of perchloroethylene (Odum et al
1988)- TCA has been shown to cause peroxisome proliferation (as measured by an
increase in peroxisomal enzyme activity) in hepatocytes of mice and rats both in
vivo and in vitro after short-term exposure (Elcombe, 1985). Further, TCA has been
shown to be a hepatocellular carcinogen in the B6C3F1 mouse (Herren-Freund et
al.. 1987).
• Perchloroethylene oxidative metabolism approaches saturation at lower levels in
rats than in mice. Saturation in rats occurs at atmosphenc concentrat,on'-n excess
of 100 ppm (Ikeda et al., 1972). Consequently, at high atmosphenc ^ncentrat,ons
of perchloroethylene (i.e., > 100 ppm) mice generate relafveiy more TCA.than do
rate (Odum et al., 1988). Following 6 hours of inhalat.on exposure to 400 ppm, the
cumulative blood concentrations of TCA in mice were 6 to 7 times greater than
concentrations in rats; peak blood levels of TCA were found to be 13-fold h,gher m
mice than in rats.
These results show that there is a quantitative difference between rats and mice
with respect to their abilities to metabolize perchloroethylene to TCA Such a
difference* consistent with the known species variability in responsiveness to the
hepatocarcinogenic effects of perchloroethylene. In view of the peroxisome-
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inducing activity of TCA in the rat, which for equivalent doses may be even more
responsive than the mouse (Elcombe, 1985), and the belief that peroxisome
proliferation is a necessary prerequisite to perchloroethylene-induced hepatocellular
carcinogenesis, these results also suggest that TCA per se should be
hepatocarcinogenic in rats if sufficient blood levels are achieved (Elcombe, 1985).
Although a two-year bioassay was stated to be in progress (Elcombe, 1985), EPA is
unaware of published studies on the hepatocellular carcinogenicity of TCA in rats.
Saturation of human perchloroethylene metabolic processes has been reported to
occur at perchloroethylene concentrations of approximately 100 ppm to 400 ppm
(U.S. EPA, 1985; Ikeda et al.,1972; Ohtsuki et al., 1983). Odum et al. (1988)
summarized evidence suggesting that humans exposed to perchloroethylene would
be "exposed to lower concentrations of TCA than mice or rats."
In in vitro experiments conducted to compare rat, mouse and human hepatocytes
with respect to susceptibility to TCA-induced peroxisome proliferation, mouse
hepatocytes are more responsive than rat, and human hepatocytes have been
found relatively unresponsive (Elcombe, 1985).
If peroxisome proliferation is required for perchloroethylene-induced liver
carcinogenesis, reduced human hepatocyte responsiveness to TCA combined with
reduced ability to form TCA supports the hypothesis that perchloroethylene is
unlikely to be carcinogenic in humans. Inasmuch as this study was based on only
two human livers, the evidence cannot be considered persuasive at this point,
however. Considerable individual variation in function may be expected to exist
between human livers, particularly in specimens from donors who may have been
treated with a variety of drugs. More human liver samples need to be examined
before a convincing argument can be made.
There is some evidence that hepatocytes from humans and other primates are
relatively unresponsive to a variety of agents which cause peroxisomal proliferation
in rodents, although this evidence is limited in quantity and scope. Microscopic
studies on liver biopsies from humans chronically dosed with hypolipidemic drugs
suggest that these agents do not cause peroxisome proliferation in humans (Cariot
et al., 1983; De La Inglesia et al., 1982; Hanefeld et al., 1983). Biochemical data,
such as peroxisomal enzyme measurements, have not been reported, however.
Nafenopin, a peroxisome inducer in rodents, was inactive in cultured marmoset
hepatocytes (Bieri et al., 1988), but the drug did cause increased cell proliferation.
Several in vitro studies have also provided suggestive evidence that human
hepatocytes are relatively unresponsive to hypolipidemic drugs and phthalate-ester
plasticizers ( Elcombe and Mitchell, 1986; Butterworth et al., 1989). Here again,
however, the sample size is limited to a few human livers. Interpretation of the
data is subject, therefore, to the same uncertainty as the in vitro TCA studies on
human hepatocytes.
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Although evidence does exist to afford support for the hypothesis that mouse liver
cancer associated with exposure to perchlofoethylene may be secondary to peroxisome
proliferation, there are points that run counter to the hypothesis and thus its validity remains
questionable. For example:
• If peroxisome proliferation is causally related to the induction of liver cancer, one
would expect to detect a quantitative relationship between the two events. That is,
potent peroxisome proliferators should also be potent hepatocarcinogens. This
does not appear to be the case.
In a chronic 65-week study of DCA and TCA in male B6C3F1 mice, Herren-Freund
et al.(1987) found that DCA was a more potent hepatocarcinogen than TCA.
Equidoses (5 g/L in drinking water) resulted in a nearly threefold higher incidence of
liver cancer in DCA-dosed animals than in TCA-dosed animals. DeArigelo et al.
(1989) however, reported that TCA was more potent as a peroxisome proliferator
than was DCA in male B6C3F1 mice. Nelson et al. (1989) also reported that TCA
produced greater peroxisome proliferation than did DCA in B6C3F1 mice dosed for
only 10 days.
An even more pronounced lack of correlation was reported in a study of the
peroxisome proliferators, DEHP and Wy-14643 (Marsman et al., 19S8). At doses
producing equivalent increases in peroxisome volume density and peroxisomal
enzyme activity for the two compounds, the liver lesions and tumors were produced
only by Wy-14643 (100% incidence). DEHP produced no liver lesionis in dosed
rats.
• Studies on the Swiss mouse also raise questions about the connection between
peroxisome proliferation and cancer. If mouse liver peroxisome proliferation in
response to perchloroethylene is correlated to the induction of liver cancer, one
would expect strains of mice that exhibit peroxisome proliferation to also exhibit
hepatocellular carcinoma induction. While the EPA is unaware of perchloroethylene
bioassays being conducted in mouse strains other than B6C3F1 and Strain A, the
closely related hepatocarcinogen in B6C3F1 mice, trichloroethylene, has been
studied in the Swiss mouse (Henschler et al., 1984). Trichloroethylene, which is
metabolized to TCA, induces peroxisome proliferation in Swiss mice (Elcombe,
1985). Hepatic peroxisome proliferation was induced as measured by both an
increase in peroxisomal enzymes and peroxisome density volume. Induction of the
peroxisome marker enzyme, cyanide insensitive palmitoyl CoA oxidase activity,
increased linearly with increasing trichloroethylene doses of 0.05-0.5 g/kg/day
following 10-day exposure periods (Elcombe, 1985). TCA, a major metabolite of
trichloroethylene, also induced peroxisome proliferation in Swiss mice. The daily
oral administration of trichloroethylene to Swiss mice for 18 months did not produce
liver cancer in either sex, however (Henschler et al., 1984). The doses
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administered by Henschier et al. were from 3 to 5 times higher than doses found by
Elcombe to induce peroxisome proliferation in these mice.
• The results of recent studies have raised the possibility that genotoxicity may occur
independently of peroxisomal proliferation following exposure to perchloroethylene.
Recent studies have shown that the TCA and DCA metabolites of perchloroethylene
have DNA-damaging activity without increasing the levels of peroxisomal enzymes.
Within 1 hour after a single dose of TCA or DCA at 0.5 g/kg, a significant increase
in single-strand breaks in DNA was detected using an alkaline unwinding assay
(Nelson et al., 1989). No increase in peroxisomai palmitoyl-CoA oxidase activity
was detected for periods up to 24 hr after dosing. This study raises the possibility
that genotoxicity, and thus potential mutagenicity, may occur independently of
peroxisome proliferation following perchloroethylene exposure. As discussed
earlier, other investigators have been unsuccessful in demonstrating single-strand
breaks in DNA with TCA and DCA (Chang et al., abstr., 1989).
Other metabolites, such as chloroacetaldehydes like chloral hydrate, might also
contribute to liver tumorigenesis. A preliminary study in mice indicates that chloral
hydrate is a hepatocarcinogen in mice (Rijhsinghani et al., 1986; ). Chloral
hydrate has been shown to produce aneuploidy and may be mutagenic (see
Section 4.2.).
• More recently, Nelson et al. (1990) reported that TCA administration significantly
increased expression of the C-H-ras and c-myc oncogene in hepatocellular
carcinomas in B6C3F1 mice. The data indicate that elevated expression of these
oncogenes may play an important role in the development of liver tumors in these
mice. As discussed by these authors, the consequence of increased c-myc
expression in most cells is loss of cellular differentiation. The oncogene does not
induce cell division, but appears to play a permissive function in relation to cell
division. These workers suggest that TCA increased c-myc expression may prevent
initiated cells from differentiating, thereby increasing their probability of progressing
to hepatocellular carcinoma. Several investigators have reported a different pattern
of oncogene activation in chemically induced mouse liver tumors compared to that
observed in the tumors of untreated animals. This indicates that the chemicals do
not simply promote spontaneous background tumors (Fox et al., 1990; Reynolds et
al., 1987).
In summary, there is some evidence to support the hypothesis that perchloroethylene-
induced hepatic carcinogenesis may be related to peroxisome proliferation. However, critical
review of the scientific literature reveals significant data gaps regarding the relationship
between the proliferative effect and neoplasia. The recent demonstration of a peroxisome-
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proliferator-activated receptor (Issemann and Green, 1990) should lead to increased
understanding of the mechanism of action for chemicals causing this phenomenon. Also, the
recent demonstration, that the major metabolite of perchloroethylene, TCA, causes the
expression of the c-myc oncogene in B6C3F1 mice, requires experimental exploration.
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6. KIDNEY TUMORS IN MALE RATS
The inhalational administration of perchloroethylene to male and female F 344/N rats
and B6C3F1 mice produced dose-related increases in the incidences of nontumor
nephrotoxicity in both sexes of both species and a nonstatistically significant increase in the
incidence of proliferative lesions of the renal tubular cells (tubular cell hyperplasia, adenoma,
and adenocarcinoma) in male rats (NTP, 1986). A slight increase in renal tumors was also
observed in male Sprague-Dawley rats receiving perchloroethylene by gavage or by inhalation
in other studies (Maltoni and Cotti, 1986; Rampy et al., 1978).
In the NTP studies groups of 50 male and 50 female F 344/N rats were exposed by
inhalation to atmospheres containing 0, 200, or 400 ppm perchloroethylene for 6 hours a day,
5 days per week for 103 weeks. Tubular cell hyperplasia, was observed in male rats (control
0/49, low dose 3/49, and high dose 5/50) and in one high dose female. Renal tubule
neoplasms were observed in male rats (control 1/49, low dose 3/49, and high dose 4/50).
Although the incidences of renal tubule neoplasms in perchloroethylene exposed male
rats was not statistically significant (p > 0.05) relative to concurrent controls, the production of
the lesions is considered to be evidence of a carcinogenic effect in rats. This is supported by
the following facts:
• Renal tubule tumors occur only rarely in F 344/N rats. Historically, the NTP has
found renal tubule neoplasms in only 0.2% of male F 344/N rats (chamber controls
from the performing laboratory, 1/249 (0.4%) and untreated controls from non-
inhalation studies, 4/1968 (0.2%)). Likewise, the overall historical control 'ncidence
of renal tubule tumors in male F 344/N vehicle controls in gavage studies is 1/1943
(0 05%) The incidence is even lower in female controls. This is supported by
spontaneous renal tubule tumor incidence rates recorded for other rat strains (e.g.,
Osborne-Mendel, males 0.3%; females 0%; Goodman et al., 1980). The
appearance of tubule neoplasms in 7% of perchloroethylene-dosed animate (low-
and high-dose groups combined) is convincing evidence of a treatment-related
effect.
No malignant renal tubule neoplasms have been observed in any control rats
examined by the NTP. This includes the chamber controls from the performing
laboratory, and the untreated controls and the vehicle controls from gavage studies.
Two of the tumors observed in high-dose animals in the NTP study were
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carcinomas. The probability of two rare carcinomas appearing by chance in a
group of 50 animals has been calculated to be less than 0.001 (U.S. EPA, 1987).
• When statistically compared to historical control incidences of renal tubule tumors,
there is a significant dose-related positive trend, and tumor incidences in both low-
and high-dose groups are significantly elevated. Although standard statistical
analyses of tumor incidence data did not reveal a significanlI increase n kidney
tumors, when the incidences of tubular cell hyperplas.a (0/49, 3/49, 5/bO) and
neoplasms (1/49, 3/49, 4/50) and tumor severity (two adenocarcmoma® in the high-
dose group) are all considered, a dose-response relationship is apparent. Thus,
although the tumor incidence is not statistically significant when compared with
concurrent controls, there is a positive trend.
In addition to the NTP study findings of renal tubule tumors in male F 344/N rats,
Rampy et al. (1978) and Maltoni and Cotti (1986) reported slight increases in renal tumors in
Sprague-Dawley rats dosed with perchloroethylene by inhalation and gavage, respectively.
There is good evidence that the tubule tumors are not unique to the administration of
perchloroethylene. The NTP has found low incidences of tubule neoplasms in tats dosed with
other chlorinated ethanes and ethylenes (NTP, 1983,1988; and unpublished results cited in
NTP, 1986). There is some evidence that nontumor pathology is not unique to
percnloroethylene; however, there is also evidence that the nephrotoxicity observed with
certain chemicals of this group, such as pentachloroethane, may be different from that seen
with others of these compounds, such as trichloroethylene.
The data support the conclusion that the chronic administration of perchiloroethylene
produces nephrotoxicity in both sexes of mice and rats and an increased incidence of
proliferative lesions of the kidney tubules in male rats. The use of these data to infer risk of
carcinogenesis to humans, however, is a focus of scientific debate. Of particular
consequence in this debate is the possibility that the induction of renal tubule tumors by
perchloroethylene may be unique to male rats, and therefore, is inappropriate for deducing
potential human health hazard. This is because reasonable evidence exists to suggest that
renal effects induced in male rats by chemicals causing alpha-2n-globulin accumulation are
unlikely to occur in any species not producing alphas-globulin or a protein with a structurally
similar binding domain, in the large quantities typically seen in the male rat. Thus, if a
chemical induces alphas-globulin accumulation in hyaline droplets, and a carcinogenic
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response in the male rat kidney, the tumor response may not constitute evidence of a
carcinogenic hazard to humans.
The EPA is presently developing criteria which will define a weight-of-evidence
approach for evaluating, on a case by case basis, the role of alphas-globulin in rat kidney
tumor formation (U.S. EPA, 1991). A report (U.S. EPA, 1991) currently being developed by a
technical panel of EPA's Risk Assessment Forum, provides guidance on determining when it
is reasonable to presume that a renal tumor in male rats results from alpha-2|i-globulin
accumulation, and on selecting appropriate procedures to use in extrapolation to humans
under such circumstances. The report also defines other situations that suggest a different
approach and calls for research to clarify questions raised because of the existence of human
proteins that may be structurally similar to alpha-2ji-globulin. Data on renal tumors in the
male rat will fall into several categories depending on whether the tumors are attributable
solely to alpha-2n.-globulin accumulation, whether another mechanism applies, whether
several mechanisms are feasible, one of which involves alpha-2n-globulin, or whether the
available information is inadequate to determine the role of alpha-2|x-globulin. For instance, if
the perchloroethylene alpha-2p. data are subsequently judged to be the only definitive
explanation for the occurrence of male rat kidney tumors, this tumor end point may not have
relevance for human health hazard assessment. This can be further evaluated as the EPA's
criteria for identifying chemicals inducing alphas-globulin accumulation become available to
apply to the perchloroethylene-specific data.
6.1. ALPHA-2U-GLOBULIN IN RENAL CARCINOGENE3IS IN MALE RATS
A variety of organic compounds studied by the NTP and others have been shown to
produce sex- and species-specific lesions in the renal tubules of male rats in the form of
hyaline droplet nephropathy (NTP, 1987,1986, 1988, 1983; Alden et al., 1985; MacNoughton
and Uddin, 1984; Alden et al., 1984; Phillips et al., 1987). The accumulation of the protein,
alpha-2|i-globulin, is believed to be the reason for an excessive number of hyaline droplets
(Stonard et al., 1986; Olson et al., 1987). A normal urinary protein in the male rat, alpha-2|i-
globulin is synthesized in the liver under hormonal control but it has not been detected in the
liver of female rats nor in other species, including humans (HEI, 1988). Among the chemicals
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tested so far in chronic animal bioassays, those that invoked this specific type of protein
droplet nephropathy in male rats also produced renal tubule tumors in male rats, but did not
produce renal tubule tumors in other species tested. The renal tubule tumors appear to be
the end product in the following sequence of functional changes in the epithelial cells of
proximal tubules (UAREP, 1983; Alden et al., 1984 Haider et al., 1984; HEI, 1988; Swenberg
eta!., 1989): .;
• Excessive accumulation of hyaline droplets in proximal tubules, representing
lysosomal overload, leads to tubule cell degeneration, cell loss, and regenerative
cellular proliferation.
• Cell debris in the form of granular casts accumulates at the corticomedullary
junction with associated dilation of the affected tubule segment and more distally,
mineralization of tubules within the renal medulla.
• The chronic progressive nephropathy characteristically found in aging rats is
exacerbated as a consequence of the induced nephrotoxicity.
• Renal tubule hyperplasia and neoplasia develop subsequently.
A number of investigators hypothesize that the increased proliferative response caused
by the chemically-induced cytotoxicity results in clonal expansion of spontaneously initiated
renal tubule cells and increased incidence of renal tumor formation (Trump et al., 1984; Alden,
1989; Swenberg et al., 1989). This line of reasoning leads to a conclusion that the acute and
chronic renal effects induced in male rats by these chemicals will be unlikely to occur in any
species not producing alphas-globulin, or a very closely related protein, in the large
quantities typically seen in the male rat (Alden, 1989; Borghoff et al.. 1990; Green et al., 1990;
Olson et al., 1990; Flamm and Lehman-McKeeman, 1991).
This proposed mechanism of tumorigenesis seems plausible and may provide an
adequate explanation of the specific susceptibility of the male rat to the induction of renal
tubule tumors by certain chemicals. However, definitive links between alphas-globulin
accumulation and tumorigenesis in male rats must be established on a chemical-by-chemical
basis before it is reasonable to discount the significance of the tumor induced by a particular
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chemical. Guidance for evaluation of data concerning alphas-globulin and kidney tumors in
male rats is being developed by EPA (U.S. EPA, 1991).
Goldsworthy and his coworkers (1988) observed increases in alpha-2p, hyaline droplets
and crystalloid accumulation in the cytoplasm oHhe P2 segment of proximal tubules of male,
but not female, F 344/N rats, following 10 days of gavage with 1000 mg/kg perchloroethylene.
Cell replication was enhanced in the male rats, specifically in damaged P2 segments,
suggesting a link between the alpha-2|o.-globulin accumulation and kidney tumors. These
investigators reported similar findings for pentachloroethane, but at a dose of 150 mg/kg for
10 days. Trichloroethylene, structurally very closely related to perchloroethylene, was not
found to cause an increase in protein droplets or cell replication in either male or female rats
administered 1000 mg/kg for 10 days.
In short-term, high-dose studies, Green et al. (1990) found that the oral administration
of from 1000 to 1500 mg/kg of perchloroethylene daily for up to 42 days caused an
accumulation of alpha-2ji-globulin in the proximal tubules of male rats. The animals were
killed within 24 hours of the last dose of perchloroethylene. The effect was accompanied by
evidence of nephrotoxicity, with the formation of granular tubular casts and evidence of tubular
cell regeneration. The effects were not observed in female rats or in mice. Inhalation
exposure to 1000 ppm of perchloroethylene for 10 days resulted in the formation of hyaline
droplets in the kidneys of male rats, but granular casts and tubule cell regeneration were not
observed, although the time period may have been too short. These results show that very
high doses of perchloroethylene are capable of precipitating hyaline droplet nephropathy in
male rats and that male rats are far more sensitive to the effect than are female rats or either
sex of mice. Therefore, alpha-2n,-globulin accumulation may play a role in the tumorigenesis
observed in male rats exposed to perchloroethylene.
The following points, however, show that factors other than the specific protein droplet
nephropathy may have as much or more of a significant role in explaining renal tumor
formation resulting from perchloroethylene exposure, although some contribution of alpha-2^.-
globulin accumulation cannot be ruled out.
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Ths AlDha-2u-ResDonse for Perchloroethvlene is Relatively Mild, and Renal Tumors Have
Been Observed at Doses Lower than the Ones Shown to Causa the Alpha-2u.-Response.
Although the alphas-response does occur in male rats exposed to perchloroethylene,
It has been observed following only high doses. While Green et al. (1990) tested lower
inhaled doses of perchloroethylene (up to 400 ppm 6 hours per day for 28 days with animals
being sacrificed within 18 hours of termination of the final exposure) in rats, there was no
evidence of hyaline droplet formation although there may have been time for recovery before
sacrifice. It is noteworthy that the 400 ppm concentration was the same exposure level used
for the high-dose rats in the NTP inhalation carcinogenicity bioassay. In the NTP study the
400 ppm concentration caused a high incidence of non-tumor nephropathy and resulted in the
formation of kidney tubule adenomas and adenocarcinomas. The renal pathology of rats in
the NTP study was reported to be different from the specific alpha-2^i-nephropa1:hy, but the
age of the rats, as well as the length of time that elapsed between final exposure and
sacrifice, may explain some of the differences. Mineralization in the inner medulla and papilla
of the kidney, a characteristic trait of alpha-2|i-nephropathy, was not seen, however (NTP,
1986).
It is possible that longer-term exposure to the 400 ppm concentration of
perchloroethylene is required for the production of hyaline droplet accumulation in the kidney
of rats (ECETOC, 1990). Alpha-2p.-globulin accumulation can be demonstrated, however,
after only short-term exposures (even a single administration) to several agents such as d-
limonene, decalin, unleaded gasoline, and trimethylpentane (Charbonneau et al., 1987; NTP,
1988). Lack of hyaline droplet formation or increase in alpha-2n-globulin or signs of the
characteristic renal nephropathy at the high-dose level of the NTP inhalation study may
indicate a threshold effect and thus diminish the likelihood that the renal tumors; associated
with exposure to perchloroethylene are induced through this mechanism (Green, 1990).
Pharmacokinetic differences between oral and inhalation exposure may contribute to the
observed discrepancies in some of the results.
The NTP did not report the presence of hyaline droplets in rats that had been exposed
to either 200 or 400 ppm of perchloroethylene for up to 2 years. These doses; were
associated with the production of renal tubule neoplasms in male rats. The fact that the NTP
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did not report the presence of hyaline droplets in either the 14-day, 90-day, or 2-year studies
is not definitive, however, since the NTP protocol at that time was not designed to detect
hyaline droplets or alpha-2n-globulin accumulation in the kidney (NTP, 1990). Thus, the
procedures followed at the time of the study were not necessarily conducive to detection of
hyaline droplets. For example, in the chronic study of perchloroethylene, at least one week
elapsed between the final perchloroethylene exposure and the scheduled sacrifice of the
surviving animals. It is possible that had the hyaline droplets been present they could have
regressed. Also, the nephropathy observed at the end of a 2-year bioassay could be difficult
to distinguish from the old-age nephropathy that occurs in these rats. Other investigators
(Goldsworthy et al., 1988; Green et al., 1990) have observed hyaline droplets containing
alpha-2n-globulin following high doses of perchloroethylene administered to male rats.
In the NTP bioassay, however, the renal pathology reported is not entirely consistent
with the results generally found for chemicals where there is alpha-2n- accumulation (NTP,
1986; letter from Scot Eustis to William Fariand, 1988). For example, as mentioned above,
there was no mineralization in the inner medulla and papilla of the kidney, a frequent finding in
bioassays of chemicals inducing alpha-2ji-globulin accumulation (e.g., for pentachloroethane,
the incidence of renal papillar mineralization is 8% in controls; 59%, low dose; 58%, high
dose). In addition, some aspects of toxic tubular nephropathy were also observed in female
rats and male mice exposed to perchloroethylene.
Perchloroethylene does induce alpha-2n--globulin accumulation and some of its
associated nephropathy in male rats, although the evidence for this exists only at high doses.
Nevertheless, the hypothesis of hyaline droplet formation leading to renal tubule tumors in
male rats is a valid proposal for mechanisms of tumorigenesis. The absence of evidence that
chronic inhalational exposure to 200 or 400 ppm of perchloroethylene causes the
accumulation of hyaline droplets and its associated nephropathy in the kidneys of male rats,
considered along with the data supporting other mechanisms, including possible genotoxicity
discussed below, makes it difficult to conclude, however, that perchloroethyiene-induced renal
tumors can be attributed solely to this hypothesized species/sex specific mechanism.
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Chronically-Induced Perchloroethvlene Nonneoplastic Kidnev Lesions Exhibit Neither Species
Nor Sex Specificity.
In contrast to most other chemicals inducing alpha-2ji-globulin accumulation that have
been tested by NTP in chronic carcinogenicity bioassays, renal lesions occurring in animals
exposed to perchloroethylene were not confined to the male rat. Although the female rat did
not develop any renal tubule tumors, the incidence of karyomegaly was significantly elevated
in the female rat as well as in the male rat; one of 50 female rats exposed at the high dose
developed tubular cell hyperplasia.
In the mouse, "nephrosis" was observed at increased incidences in dosed females,
casts were observed at increased incidences in dosed males and high-dose females, and
karyomegaly of the tubular cells was observed at increased incidences in both sexes of
treated mice. The severity of the renal lesions was dose related and one low-dose male had
a renal tubular cell adenocarcinoma.
In the NCI gavage study of perchloroethylene, toxic nephropathy, not detected in the
control animals, occurred in both male and female Osborne-Mendel rats administered
perchloroethylene. Unfortunately, the animal survival in this study was not adequate to
support any conclusions about perchloroethylene carcinogenicity.
Other chlorinated ethanes and ethylenes produce nephrotoxicity and renal tubule
tumors in laboratory animals as well. Hexachloroethane causes accumulation of hyaline
droplets and renal tubule tumors in male rats. On the other hand, trichloroethylene, for
example, which was also tested by NTP, induces kidney tumors in male rats only (NTP,
1988b) but does not cause an accumulation of hyaline droplets or an increase in levels of
alpha-2p,-globulin (Goldsworthy et al., 1988). Consequently, kidney tumors induced by this
compound are not considered to be associated with alpha-ap. accumulation.
Perchloroethyiene is closely related structurally to trichloroethylene, and both of these
chemicals have been shown to be metabolized in the kidney to mutagenic compounds.
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6.2. SUSTAINED CHRONIC NEPHROTOXICITY AS A POSSIBLE MECHANISM
INDEPENDENT OF ALPHAS-GLOBULIN ACCUMULATION
Numerous compounds such as perchloroethylene, trichloroethylene, and
pentachloroethane have been reported to produce nephrotoxicity in male and female rats and
mice. This toxicity, although appearing to be characteristic of chronic administration of
chlorinated ethanes and ethylenes, manifests itself differently with specific ones of these
chemicals and may include tubular cell cytomegaly, karyomegaly and pleomorphism, tubular
cell dilation, or the formation of granular casts. Certain of these compounds cause kidney
tumors in male mice only (vinylidene chloride), male rats only (trichloroethylene), and both
male rats and male mice (chloroform).
As previously discussed for the alpha-2|i-nephropathy, sustained kidney damage may
be a risk factor for tumorigenesis. Thus, there may be a link between renal toxicity and
tumorigenesis, and it is reasonable to suspect that renal tubule neoplasia in male rats may be
influenced by perchloroethylene-induced cytotoxicity and subsequent cellular regeneration. It
also has been suggested that renal neoplasms induced by perchloroethylene may be
secondary to renal cytotoxicity and subsequent cellular proliferation without regard to alpha-
2|i-globulin accumulation. If this is the case, renal tubule neoplasia in these experiments
would not be expected to be a species/sex-specific response to chronic administration of
perchloroethylene because the nontumor lesions appeared in both sexes of both species.
Perchloroethylene-induced cytomegaly and karyomegaly appeared in both rats and mice
during the early phases of the NTP inhalation study indicating that animals of both species
surviving to the scheduled termination of the study had long-standing nephrotoxicity. If renal
tubule neoplasia were directly consequent to this pathology, tumors would likely have been
found in dosed female rats or male and female mice. Goldsworthy et al. (1988) determined
that cell-replication rates increased specifically in the histologically damaged tubule segments
of male rats, but not in female rats, after perchloroethylene exposure. Cell replication did not
differ from controls in trichloroethylene-treated male or female rats, however. Since both
trichloroethylene and perchloroethylene produce renal tubule tumors, but no enhanced cell
replication was seen with trichloroethylene, It is difficult to conclude that perchloroethylene
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Induces the renal tumors by a nephrotoxic mechanism apart from nephropathy associated with
a!pha-2ji-g!obulin accumulation.
The fact that there is little doubt that the kidney is a target organ for perchloroethylene
and other chlorinated ethanes and ethylenes in mammalian species contributes to the overall
concern regarding the kidney tumor end point. Although nephrotoxicity may play a role, more
supportive evidence is needed to define such a role.
63. A MUTAGENIC MECHANISM OF PERCHLOROETHYLENE-INDUCED
CARCINOGENESIS IN MALE RATS
The possible explanations of perchloroethylene-induced renal carcinogenesis
discussed earlier have centered on nonmutagenic (epigenetic) mechanisms. This is because
mutagenicity studies of perchloroethylene have produced largely negative or only weakly
positive results. The early studies of genetic toxicology of perchloroethylene have centered on
the effects of perchloroethylene per se and later on certain of its products of oxidative
metabolism. A secondary metabolic pathway for perchloroethylene (hepatic conjugation with
glutathione and subsequent degradation by renal beta lyases) has been discovered in rats
(see sections III and IV on metabolism and mutagenicity; also see Dekant et al., 1989;
Vamvakas et al., 1989). Perchloroethylene is conjugated with hepatic glutathione to form S-
(1,2,2-trichlorovinyl) glutathione (TCVG). The conjugative pathway in the liver appears to be
the minor of two competitive pathways; its activity is thought to increase as the oxidative
pathway approaches saturation. The glutathione S-conjugate metabolite thus formed in the
liver is either excreted into the bile or transported to the kidney where it is acted upon by
gamma glutamyl transferase and dipeptidase to form its corresponding cysteine S-conjugate,
S-(1,2,2-trichlorovinyl)-l-cysteine (TCVC) (DeKant et al.. 1987). TCVC may undergo N-
acetylation and be excreted in the urine, or it may become a substrate for renal beta-lyases
which cleave TCVC to form a mutagenic fragment that probably includes electrophilic
acylating and alkylating agents. The mutagenic activities of TCVG, in the presence of hepatic-
and renal-activating systems, and TCVC, in the presence of a renal-activating system, have
been demonstrated in an Ames test protocol. The conjugative pathways producing mutagenic
metabolites in the kidney are operative for trichloroethylene, a close structural ainalog of
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perchloroethylene (Anders et al., 1988; DeKant et al., 1989). Trichloroethylene also induces
renal tumors in male rats, but is not a chemical that induces alpha-2|i-globulin accumulation
(NTP, 1988b; Goldsworthy et al., 1988; U.S. EPA, 1991). The trichloroethylene conjugates
that lead to mutagenic constituents are dichlorovinylglutathione and dichlorovinylcysteine
(Anders et al., 1988; DeKant et al., 1989).
Although the beta-lyase enzymes necessary for the metabolism to the mutagenic
metabolite are found in the kidneys of rats, mice, and humans (Green et al., 1990; Chen et
al., 1990), in vitro conjugation of perchloroethylene with glutathione by human liver was not
detected ( Green et al., 1990). If the human is incapable of conjugating perchloroethylene
with glutathione, this potential mechanism of carcinogenesis may not be relevant to projecting
human health hazards associated with perchloroethylene.
Green et al., (1990) have reported species differences with respect to
perchloroethylene-glutathione conjugation. In vivo conjugation by rat liver occurred at a
relatively low rate but this rate was five times greater than that observed for mouse liver.
Using a limited number of human liver samples, Green et al. were unable to demonstrate
conjugation of perchloroethylene. Few liver samples were studied, however, and a
conjugation rate tenfold lower than that observed for rats would fall below the limits of
detection of the method employed. Tenfold differences in enzyme activities within the human
population are not uncommon. Consequently, it remains a distinct possibility that humans
may conjugate perchloroethylene, although probably at a very low rate as indicated by the low
rate measured in rodent tissue.
The roles of this metabolic pathway in the production of renal tumors in male rats and
in the carcinogenic potential of perchloroethylene in humans remain to be established. It has
been pointed out that the conjugative pathway is minor and may be noticeably more active
only when the oxidative pathway approaches saturation. Human (and rat) oxidative
metabolism of perchloroethylene is recognized as being saturable. The quantitative
relationships between various degrees of saturation of the oxidative pathway and concomitant
importance of the conjugative pathway requires close scrutiny before it can be concluded that
conjugation is unimportant in perchloroethylene metabolism by humans, particularly since
products include potential mutagens.
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Also, if the glutathione/beta-lyase pathway provides a mechanism for the induction of
renal tumors it is difficult to explain why female rats and both sexes of mice did mot exhibit
renal tumors. The metabolic processes required for the generation of the mutagenic
Intermediate are operative in both sexes of both species, albeit to a lesser extent: in female
rats and both sexes of mice. The male rat tumor rate was relatively low, but the incidences in
female rats or male and female mice might be expected to be still lower. The rates in female
rats and both sexes of mice might be too low to be detected with the small numbers of
animals subjected to testing.
In summary, since the NTP discovery that chronic administration of perchloroethylene
i
induces a low level of renal tubule tumors in male rats, significant research has been
conducted to explain the mechanism of the carcinogenic effect. This research has resulted in
at least three possible explanations:
1. The tumors may be secondary to the renal accumulation of the low molecular
weight protein, alpha-2p.-globulin. Since only male rats produce the protein, the
tumors would have little or no predictive validity with respect to human health
hazard on a site- or mechanism-specific basis. Perchloroethylene induces kidney
tumors at lower doses than those required to cause alpha-2ji-globulin accumulation,
however. The EPA is presently developing criteria which will define a weight-of-
evidence approach for evaluating, on a case by case basis, the role of alpha-2ji-
globulin in rat kidney tumor formation (U.S. EPA, 1991).
2. The chronic administration of perchloroethylene produces nephrotoxicity and it has
been suggested that tumor production is secondary to sustained cytotoxicity and
cellular regeneration. Although certain "nephrotoxicity" occurs in both sexes of rats
and mice, implying that kidney tumors would occur in rats and mice of both sexes in
the carcinogenicity bioassays, ceil replication occurs in male but not ini female rats
suggesting that any nephrotoxic mechanism would likely be associated with alpha-
2|i-globulin accumulation. On the other hand, the mechanism of perchloroethylene
tumorigenesis may be similar to that of its structural analog, trichloroethylene.
Trichloroethylene induces kidney tumors in male rats, but does not enhance cell
replication and does not cause alpha-2p,-globulin accumulation.
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3. A glutathione-beta lyase conjugation pathway of perchloroethylene metabolism has
been discovered in rats and also shown in mice. This minor pathway leads to the
formation of a cytotoxic/mutagenic metabolite product. It is interesting to note that
the structural analog of perchloroethylene, trichloroethylene, forms a
cytotoxic/mutagenic metabolite via this pathway and also causes kidney tumors in
male rats; but trichloroethylene does not cause alpha-2n-globulin accumulation.
Humans may or may not have the capacity to carry out the initial conjugation step.
If the human cannot form the perchloroethylene-glutathione conjugate, this pathway
is irrelevant with respect to human risk projection.
While there is some evidence to support each of the proposed mechanisms, there are
also significant quantitative and qualitative gaps in the supportive data. The mode of
perchloroethylene-induced renal tumorigenesis in male rats is not yet understood.
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7. MONONUCLEAR CELL LEUKEMIA IN RATS
The NTP (1986) reported that the chronic inhalationai administration of
perchloroethylene to male and female F 344/N rats caused positive trends in the incidence of
mononuclear cell leukemia (MCL) in both sexes. Pairwise comparisons of tumoir incidences in
dosed and control groups of males (life table analysis) revealed statistically significant
Increases in both the low and high dose groups (controls, 28/50; low dose, 37/50; p = 0.046:
high dose, 37/50, p = 0.004; trend test p = 0.004). Analysis of the data for female rats
revealed a marginally significant trend (p = 0.053) and a significant increase in the low-dose
group and a marginally significant increase in the high-dose group (control, 18/50; low dose,
30/50, p « 0.023; high dose, 29/50, p = 0.053).
Interpretation of these data is somewhat clouded by the fact that overall incidences of
MCL in the concurrent chamber control groups were high relative to historical chamber control
groups at the performing laboratory (males 28/50, 56% versus 117/250, 47%; females 18/50,
36% versus 73/249, 29%). The concurrent control group rates were also higher than the NTP
Program historical rate for untreated control groups (males 583/1,977, 29% and females
375/2,021,18%).
Because of these factors the NTP conducted supplemental analyses of the progression
of the disease, the effect of perchloroethylene on the time of onset of advanced MCL, and the
contribution of MCL to early deaths in control and dosed animals. The results of these
supplemental analyses showed that:
• In both males and females, perchloroethylene produced a dose-related increase in
the severity of MCL.
• Perchloroethylene exposure significantly shortened the time to onset of MCL in
female rats.
• Although there was no remarkable effect of perchloroethylene exposure on survival
of female rats, there was an increased incidence of advanced MCL in female rats
that died prior to the scheduled termination of the study. Thus, a more appropriate
statistical analysis was conducted in which only the incidences of advanced MCL in
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rats were considered. Significantly positive trends and significant increases in the
incidences of advanced MCL in both male and female rats in the high-dose groups
were observed.
In 1987 the EPA Science Advisory Board took exception to the use of these special
analyses since they did not represent generally accepted approaches to the evaluation of
increased incidences of MCL According to the NTP report, however, the interpretation of
MCL incidences in the perchloroethylene study was based on standard methods of data
evaluation (NTP, 1986). The special analyses were conducted to support, rather than
establish, the interpretation.
Under the conditions of the NTP study there was evidence of a carcinogenic effect of
perchloroethylene in male and female rats as evidenced by significant increases in the
incidences of MCL in both sexes. However, the usefulness of increased incidences of MCL in
the prediction of human carcinogenic risk associated with exposure to perchloroethylene has
been questioned on several grounds:
MCL is a Common and Variable Tumor that Occurs Spontaneously in F 344/N Rats. Marginal
Increases in Incidences are of Questionable Biological Significance.
MCL is recognized as a common neoplasm in rats and its rate of appearance in
historical control groups is highly variable. In the five contemporary inhalation studies
conducted at the performing laboratory the incidences of MCL in chamber control male rats
ranged from 32 to 68% In female chamber control rats the incidences ranged from 22 to
36%. A similarly high variability has been observed among NTP Program-wide untreated
control groups (males, 10 to 60%; females, 6 to 38%).
Concurrent controls represent the most appropriate groups to use for the purpose of
determining the statistical significance of observed differences between experimental groups.
However, it is recognized that in the case of spontaneous and highly variable tumors,
comparisons of treatment groups to historical control groups may be helpful in the
interpretation of experimental results. When the overall rates of MCL in male rats in the
perchloroethylene studies are compared to the range of tumor incidences in historical controls,
the perchloroethylene-treated animals were essentially identical to those in the historical
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control group with the highest Incidence (74% in perchloroethylene-dosed animals versus 68%
In the historical control group). However, the incidences of MCL in perchloroethylene-dosed
female rats (60 and 58%) were elevated relative to the highest incidence in historical control
groups (38%).
The Pathobioloov of MCL is Too Poorly Understood to Allow the Tumors to be Used to
Determine Human Health Risk.
MCL is a relatively well defined and understood rodent neoplasm. The disease per se,
which is splenic in origin, is readily and unequivocally diagnosed by the use of standard
histopathological techniques. MCL is known to be a rapidly progressing and fatal neoplasm
whose Incidence is age related. The tumor is transplantable; and its etiological factor is
unknown. It has been suggested that a cellular oncogene may be responsible for the
induction of MCL
Although the specific mechanism of leukemogenesis in rats is not understood, it is
Interesting to note eariy reports of toxicity of cysteine S-conjugates where S-(1,2,-
dichlorovinyl)-L-cysteine was implicated in induction of aplastic anemia and marked
biochemical alteration of DMA in bone marrow, lymph nodes, and thymus in calves (McKinney
et al., 1957; Schultz et al., 1959; Bhattacharya and Schultz, 1971,1972). As discussed
earlier, the giutathione conjugate of perchloroethylene is hydrolyzed in the kidney to the
cysteine S-conjugate, a compound that can be cleaved to form a mutagenic fragment.
Humans as well as rodents activate the conjugate via the b-lyase pathway. Thus, the
possibility exists that the perchloroethylene S-conjugate, S-(1,2,2-trichlorovinyl)-L-cysteine,
may be involved in the leukemia induction in rats and may have the potential to produce blood
dyscrasias in humans as well.
MCL is a Rodent Specific Tumor with no Human Correlate.
MCL is a neoplasm whose incidence and progression can be influenced by chemical
agents. While human leukemias originate in bone marrow, MCL is splenic in origin.
Discounting a rodent neoplasm simply because it has no exact human counterpart is not a
scientifically defensible reason, however. Site concordance is not a requirement for relevancy
46
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in extrapolation of hazard potential, although its actual presence can strengthen belief in a
particular hazard e.g., many aromatic amines are probable bladder carcinogens in humans but
are likely to produce Zymbal gland tumors rather than bladder tumors in rats. The human
does not develop Zymbal gland tumors.
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8. SUMMARY AND CONCLUSIONS
The Office of Health and Environmental Assessment has reviewed the daita currently
available regarding perchloroethylene carcinogenicity. Because the human epidemiology data
are inadequate for determining the carcinogenicity of perchloroethylene, the focus of this
paper is on the animal data and other information as they are relevant to potential cancer-
causing activity in humans. Two questions now must be considered: first, whether the data
are judged as "sufficient" evidence of carcinogenicity in animals, and second, whether the
general assumption that "sufficient1 animal data leads to an overall weight-of-evidence
classification of B2, probable human carcinogen, holds up in the case of perchloroethylene.
js There Sufficient Evidence of Carcinonenicitv in Animals?
EPA's Guidelines for Carcinogen Risk Assessment provide criteria to follow in weighing
the scientific evidence. The Guidelines also permit the exercise of professional judgment
throughout the process, with explanations for the judgment calls. The positive animal
evidence for perchloroethylene2 clearly meets the criteria for "sufficient" animal data as
depicted in the Guidelines. Sufficient evidence of carcinogenicity indicates that there is an
increased incidence of malignant tumors or combined malignant and benign tumors in (1)
multiple species or strains; in (2) multiple experiments (e.g., with different routes of
administration or using different dose levels); or (3) to an unusual degree in a single
experiment with regard to high incidence, unusual site or type of tumor, or early age at onset.
2Perch!oroethvlene has been shown to cause multiple tumor end polnte-hepatocellular
carcSas rbo^sexef of mice, kidney tumors and some indication of gliomas ,n male rats,
SeukemSB in both male and female rats. The incidences of liver tumors m rruce_and the
leukerntes in rats are statistically significantly elevated when compared to controls. The
SS^S^WM and gliomas in male rats is significantly elevated when compared to
his SSs, and these tumors are biologically significant because they are rare tumors.
Ca cSe^esis has been demonstrated by both inhalation and oral routes of exposure.
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The perchloroethylene data meets, at least to some degree, each of the three
independent criteria for attaining "sufficient" animal evidence3-4. More robust responses in
the rat would add strength to the concern for the response seen in that species, however, the
perchloroethylene mouse liver tumor data alone are believed by some to meet the Guidelines
criteria for "sufficient" animal evidence since there are multiple experiments having different
exposure routes and no appreciable downgrading of the evidence according to the Guideline
provisions for mouse liver tumor responses. The leukemia response in both male and female
rats, while not momentous, is nevertheless a second and valid end point. The kidney and
perhaps glioma responses in male rats are also suggestive and add to having a response in a
second animal species which in turn is supportive of the sufficient evidence call. Mutagenicity
data are primarily negative for the parent compound, however, metabolism seems to produce
mutagenic metabolites.
Does Sufficient Animal Data Indicate Human Hazard?
Ordinarily, animal data labeled as "sufficient," as are the data that exist for
perchloroethylene, would be highly indicative of potential human hazard and would lead to an
overall weight-of-evidence classification of B2, probable human carcinogen, when considered
with the "inadequate" epidemiologic data (EPA, 1985; Brown and Kaplan, 1987; Blair et al.,
NTP (1986) study of perchloroethylene received peer review by the NTP Board of
Scientific Counselors. The panel of experts agreed (nine affirmative votes) that there was
clear evidence of carcinogenicity of perchloroethylene for B6C3F1 mice as shown by
increased incidences of both hepatocellular adenomas and carcinomas in males and of
hepatocelluiar carcinomas in females. The panel approved (eight affirmative votes, one
negative vote) conclusions of some evidence in female F 344/N rats of carcinogenicity of
perchloroethylene as shown by increased incidences of mononuclear cell leukemia. The
panel concluded (five affirmative votes, four negative votes) that there was clear evidence of
carcinogenicity of perchloroethylene in male F344/N rats as shown by an increased incidence
of mononuclear cell leukemia and uncommon renal tubular cell neoplasms. (Two members of
the expert panel abstained from voting due to employment conflict of interest.)
4There is agreement between IARC and EPA that the animal evidence is closer to "sufficient"
than to "limited." IARC has classified perchloroethylene in the IARC category that is more
similar to EPA's sufficient animal evidence, B2. The IARC phraseology, however, is "possible-
human carcinogen, whereas EPA uses the term "probable" human carcinogen.
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1990). Laboratory data published subsequent to the perchloroethylene carcinogenicity
bloassays have led to the development of new hypotheses about the mechanisms of
perchloroethylene tumorigenesis, however, and reasoning, discussed in this paper, has been
put forward suggesting species specificity for certain metabolic pathways as well as the
proposed tumorigenesis mechanisms involving peroxisomes in the livers of mice and hyaline
droplets in male rat kidneys. Such hypotheses imply that certain experimental results from the
animal carcinogenicity bioassays are of questionable predictive validity with respect to human
health hazards, and so the question now must be answered whether the general assumption
of "sufficient" animal data indicating a human hazard potential remains a reasonable
assumption for perchloroethylene.
Implications of Perchloroethvlene Metabolism
It is generally considered that the toxicity, mutagenicity, and carcinogenicity of
perchloroethylene resides in reactive metabolites. The data do not lend support to classifying
the parent compound per se as a mutagen, although certain perchloroethylene metabolites
may be mutagenic (e.g., chloroacetaldehydes including chloral hydrate). Several
perchloroethylene metabolites have been shown to be cytotoxic, and certain metabolites (e.g.,
TCA, DCA, and trichloroacetaldehyde), cause liver tumors in mice.
Studies in both animals and humans indicate that metabolism of perchloroethylene is
relatively limited as evidenced by the fact that a high percent of absorbed dose is excreted in
the breath as the parent molecule. In human studies, however, only approximately half of the
perchloroethylene absorbed has been accounted for through the excretion of parent
compound or its metabolites.
There is no reason to believe that qualitative differences exist between species with
respect to oxidative metabolism of perchloroethylene, although there are quantitative
differences. For example, metabolic rates differ among species, saturation of metabolism
pathways occurs at different levels in different species, and other parameters such as peak
blood levels and half times of the parent compound and its metabolites may vay among
species. Thus, higher blood levels of TCA, for example, may be reached in mice, but TCA
production and half life is much longer in humans. Metabolites from oxidative pathways
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probably contribute to the development of liver tumors in mice. Several perchloroethylene
oxidative metabolites may be potentially involved in different mechanisms hypothesized for
mouse hepatocarcinogenicity.
Although certain metabolites of oxidative metabolism may be mutagenic (e.g., the
chloroacetaldehydes), these positive data are predominantly limited to in vitro studies.
Moreover, perchloroethylene was assayed in the presence of several types of metabolic
activation systems (e.g., liver homogenates and intact hepatocytes) that would favor oxidative
metabolism and under these conditions predominantly negative results were found.
Perchloroethylene may also be activated by a minor, but possibly important, secondary
pathway involving hepatic conjugation with glutathione followed by renal processing of the S-
conjugate. This S-conjugate is a potent beta-lyase dependent mutagen in the
Sa/mone//a/mammalian microsome assay. Mutagenic metabolites formed in the kidney would
be consistent with the tumors observed in male rat kidneys. However, these mutagenicity
studies are in vitro and have been conducted in only one laboratory. The beta-lyase pathway
activation of the S-cysteine conjugate may lead to metabolites that are also nephrotoxic.
Glutathione conjugation of perchloroethylene by human liver has not been shown,
although only a very few human liver samples have been examined, and even the enzyme
activity observed in rodents was low. The quantitative relationships between various degrees
of saturation of the oxidative pathway and concomitant enhancement of the conjugative
pathway requires close scrutiny before it can be concluded that conjugation is not important in
perchloroethylene metabolism by humans.
The available data indicate that metabolism would be a prerequisite for
perchloroethylene cytotoxicity and mutagenicity. Several studies are now available on the
mutagenicity of certain perchloroethylene metabolites, and their results warrant further
consideration. The mutagenicity studies on metabolites of perchloroethylene emphasize the
need for additional studies concerning the mutagenic role of intermediates in
perchloroethylene carcinogenesis. Other pathways such as the further metabolism of TCA, or
other routes to CO2 initially proposed in the 1985 HAD, or as yet undiscovered metabolic
pathways may be important.
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Mouse Liver Tumors
At this time, the Agency's position is that mouse liver tumors are considered evidence
for human carcinogenic potential, although the evidence may be downgraded on a case-by-
case basis according to chemical-specific data. The current EPA policy for evaluating mouse
liver tumor data is described in the guidelines for carcinogen risk assessment, published in
1986 (U.S. EPA, 1986).
Many different mechanisms have been proposed for hepatocarcinogenicily in B6C3F1
mice. One of the mechanisms hypothesized for perchloroethylene liver tumor induction in
these mice is postulated to result from TCA-induced peroxisome proliferation, and is said to
be, species-specific. There is some evidence to support the hypothesis that
perchloroethylene-induced hepatic carcinogenesis may be related to peroxisome proliferation;
critical review of the scientific literature, however, reveals significant data gaps regarding the
relationship between the proliferative effect and neoplasia.
For example, if peroxisome proliferation is causally related to the induction of liver
cancer, one would expect to detect a quantitative relationship between the two events. That
is, potent peroxisome proliferators should also be potent hepatocarcinogens. This does not
appear to be the case. Also, if mouse liver peroxisome proliferation in response to
perchloroethylene is correlated to the induction of liver cancer, strains of mice that exhibit
peroxisome proliferation should also develop liver tumors. Trichloroethylene, a structurally-
related hepatocarcinogen in B6C3F1 mice, and its metabolite TCA, induce peroxisome
proliferation in Swiss mice, but trichloroethylene does not cause liver tumors in this strain.
The results of recent studies have raised the possibility that genotoxicity may occur
independently of peroxisomal proliferation following exposure to perchloroethylene. Other
evidence indicates mutagenicity of metabolites should be further considered.
The role of the c-myc and C-H-ras oncogenes in the development of liver tumors in
B6C3F1 mice needs further evaluation. Nelson et al. (1990) reported that TCA administration
significantly increased expression of the c-myc oncogene in hepatocellular carcinomas in
B6C3F1 mice. These workers suggest that TCA increased c-myc expression may prevent
initiated cells from differentiating, thereby increasing their probability of progressing to
hepatocellular carcinoma.
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Thus, although evidence does exist that supports the hypothesis of mouse liver cancer
induction being secondary to peroxisome proliferation, the validity of the hypothesis remains
questionable. It is clear that perchloroethylene, probably through its metabolites, causes
peroxisome proliferation and also causes hepatocellular carcinoma in B6C3F1 mice, but a
cause-and-effect relationship between the two effects has not been shown. It is not clear
what role, if any, peroxisome proliferation actually plays in perchloroethylene carcinogenesis.
Additional studies of the mouse peroxisome- proliferator-activated receptor, a member of the
steroid hormone receptor superfamily, may contribute to understanding any role the
peroxisome proliferation phenomenon might have in tumorigenesis.
Also, several other proposed mechanisms need to be further investigated. For
example, the recent demonstration that the major metabolite of perchloroethylene, TCA,
causes the expression of the c-myc oncogene in B6C3F1 mice requires experimental
exploration. Possible roles of other toxic metabolites, including mutagenicity, need further
consideration as well.
Kidnev Tumors in Male Rats
Since the NTP discovery that chronic administration of perchloroethylene induces a low
level of renal tubule cell tumors in male rats, significant research has been conducted to
explain the mechanism of the carcinogenic effect. This research has resulted in at least three
possible explanations.
The tumors may be secondary to the renal accumulation of the low molecular weight
protein, alphas-globulin. Since only male rats produce the protein, if this is the mode of
tumor production, the tumors may have no predictive validity with respect to human health
hazard on a site- or mechanism-specific basis.
Hyaline droplet accumulation in rat kidneys has not been demonstrated to occur,
however, at the perchloroethylene inhalation doses that caused renal tumors in male rats.
The alpha-2|i-globulin response for perchloroethylene is relatively mild, and the pathology
reported is not entirely consistent with the results generally observed for chemicals that cause
alpha-2n,-globulin accumulation. Renal hyperplasia was observed in a female rat and kidney
adenocarcinoma was identified in a male mouse. In the case of perchloroethylene it is
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feasible, however, that the renal tumors could result in part from the accumulation of the
alpha-2p. protein. Enhanced cell replication, associated with hyaline droplet formation, and
mutagenic metabolites from the beta-lyase pathway may together lead to the observed renal
tumors. The role of alphas-globulin in perchloroethylene-induced rat kidney tumorigenesis is
not understood; thus, the tumors cannot be discounted. The EPA is presently developing
criteria which will define a weight-of-evidence approach for evaluating, on a .case by case
basis, the role of alphas-globulin in rat kidney tumor formation.
The chronic administration of perchloroethylene produces nephrotoxicity and it has
been suggested that tumor production is secondary to sustained cytotoxicity and cellular
regeneration. If so, it is probably related to alphas-globulin nephropathy, although sustained
chronic nephrotoxicity independent of alphaS-globulin accumulation has been implicated as
a possible mechanism of perchloroethylene-induced renal carcinogenesis in male rats.
Perchloroethylene-induced renal tubule neoplasms have been detected only in male rats, but
certain nontumor pathology associated with the chronic administration of perchloroethylene
has been detected in female rats and in both sexes of mice, as well as in male rats. These
chronically-induced perchloroethylene nonneoplastic kidney lesions exhibit neither species- nor
sex-specificity. Such a mechanism implies that kidney tumors would not occur solely in male
rats. If tubule cell neoplasia were directly consequent to this pathology, tumors would likely
have been found in dosed female rats or male or female mice. Although nephrotoxicity may
play a role, more supportive evidence is needed to define what that role may be. The fact
that there is little doubt that the kidney is a target organ for perchloroethylene and other
chlorinated ethanes and ethylenes in mammalian species contributes to the overall concern
regarding the kidney tumor end point.
A glutathione-beta lyase conjugation pathway of perchloroethylene metabolism has
been discovered in rodents. This minor pathway leads to the formation of a
cytotoxic/mutagenic metabolite(s). Humans may or may not have the capacity to carry out the
initial conjugation reaction. If the human cannot form the perchloroethylene-glutathione
conjugate, this pathway is irrelevant with respect to hazard projection.
OHEA is unable at this time to conclude that results with human liver samples are
indicative of a general inability of humans to conjugate perchloroethylene. This is because
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such few liver samples were studied, and a conjugation rate tenfold lower than that observed
for rats would fall below the limits of detection of the method employed. Tenfold differences in
enzyme activities within the human population are not uncommon.
While there is some evidence to support each of the proposed mechanisms, there are
also significant quantitative and qualitative gaps in the supportive data. The mode of
perchloroethylene-tumorigenesis in male rats is not yet understood.
Leukemia in Rats
Under the conditions of the NTP study there was evidence of a carcinogenic effect of
perchloroethylene in male and female rats as evidenced by significant increases in the
incidences of MCL in both sexes. However, the usefulness of increased incidences of MCL in
the prediction of human carcinogenic risk associated with exposure to perchloroethylene has
been questioned on several grounds: high spontaneous background incidences, use of special
supplemental analyses to aid in data interpretation, and the relevance of MCL in F344/N rats
because this type of leukemia does not occur in humans.
The leukemia incidences were statistically significantly increased in both male and
female rats. In both sexes, perchloroethylene caused a dose-related increase in severity of
MCL and shortened the time to tumor in female rats. There was an increased incidence of
advanced MCL in female rats that died before the scheduled termination of the study. The
supplemental analyses were based on standard methods of data evaluation and lent support
to the data interpretation.
While human leukemias originate in bone marrow, MCL is splenic in origin. Despite
the fact that the disease occurs only in rats, it is a neoplasm whose incidence and progression
can be influenced by chemical agents. Discounting a rodent neoplasm simply because it has
no exact human counterpart is not reasonable. Many aromatic amines are probable bladder
carcinogens in humans but are likely to produce Zymbal gland tumors rather than bladder
tumors in rats. The human does not have a Zymbal gland.
There is no one specific mechanism that explains all of the tumor end points. Of
several modes of action that have been proposed for perchloroethylene tumorigenesis,
different mechanisms have been hypothesized to cause the three distinct tumor types
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observed in rats and mice. Data exist to support certain of these mechanisms. Scientific
evidence also exist to support species specificity for particular mechanisms, but the
incompleteness of the data limits the scope of the conclusions that can be deduced although
the mechanisms are certainly plausible. The modes of action for perchloroethylene
carcinogenesis are not yet well understood. With regard to the kidney tumors, evidence
exists for more than a single mechanism, all of which may play some role in the tumor
development independently or in concert. A cause and effect relationship cannot be
effectively demonstrated between the peroxisome proliferation and development of liver
tumors in mice. Several other mechanisms may contribute to hepatocarcinogenicity in mice.
The possibility that there may well be a mutagenic component in the development of the
tumors, especially the kidney tumors, cannot be entirely ruled out. The evidence that
metabolic pathways in rodents may not occur in humans is not convincing.
Because mechanisms of perchloroethylene carcinogenesis are not well enough
understood, each individual tumor type is viewed as contributing not just to the "sufficient-
evidence in animals, but to the overall weight-of-evidence determination that perchloroethylene
is a probable human carcinogen. In each case, reasonable doubt exists that the mode of
tumorigenesis is only through mechanisms species-specific to rodent strains. Other
mechanisms are feasible that would not be specific to rodents. All three tumor types can
therefore be considered valid as indicators relevant to potential carcinogenicity in humans,
although some uncertainty does exist concerning relevance to humans.
EPA's Guidelines for Carcinogen Risk Assessment (EPA, 1986) suggest that the
weight of evidence increases with the increase in number of animal species, strains, sexes,
and number of experiments and doses showing a carcinogenic response, with the increase in
number of tissue sites affected , with the occurrence of dose-response relationships as well as
statistical significance of the increased tumor incidence in treated compared to control groups,
when there is decreased time-to-tumor occurrence or death with tumor, and when there is a
dose-related increase of malignant tumors. All of these criteria are met in some way.
Perchloroethylene causes at least three types of tumors in rodents, each of which can be
considered as contributing in some way to the concern for cancer-causing potential in
humans. Indications of cancer-causing activity were seen in two species, in two sexes, by
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inhalation and oral exposure, and is called "sufficient" animal evidence; and, although there is
some scientific uncertainty concerning relevance to humans for some of the data, the totality
of the animal data for perchloroethylene is not only closer to the "sufficient" evidence
category, but also can be considered relevant for extrapolation of hazard potential to humans.
Therefore, although the relevance of some of the data is less than certain, the
inclusive animal data for perchloroethylene, taken as a whole, along with the considerations of
inadequate human data, information on metabolism, and mutagenicity data on metabolites are
most logically categorized in Group B2, thus classifying perchloroethylene as a probable
human carcinogen. It must be remembered that classifications refer only to the weight of the
experimental evidence that a chemical is carcinogenic and not to its potency of carcinogenic
action. This paper has not addressed the quantitative estimation of risk. Mechanistic
considerations may justify special interpretation of the dose-response data with respect to
projecting human carcinogenesis risk.
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REFERENCES
Alden, C.L (1989)
Alden, C.L; Kanerve, R.L; Ridder, Q.; Stone, LC. (1984) The pathogenesis of the
nephrotoxicity of volatile hydrocarbons in the male rat. In: Mehlman, M.A.; Hemstreet.
G P • Thorpe, J.J.; Weaver, N.K., eds. Advances in modem environmental toxicology.
Vol."?, Renal effects of petroleum hydrocarbons. Princeton, NJ: Princeton Scientific
Publishers, Inc., pp.1 07-1 20.
Alden, C.L; Ridder, G.; Stone, L; Kanerva, R.L (1 985) Pathology of petrochemical fuels in male
rats: acute toxicity. In: Bach, P.H.; Lock, E.A., eds. Renal heterogeneity and target cell
toxidty. New York, NY: John Wiley and Sons, pp. 461-472.
Anders, M.W.; L Lash; Dekant, W. (1988) Biosynthesis and biotransformation of glutathione S-
conjugates to toxic metabolites. CRC Grit. Rev. Toxicol. 1 8:31 1 -341 .
Bartsch, H.; Malaveille, C.; Barbin, A.; Planche, G. (1979) Mutagenic and alkylaling metabolites
of halo-ethylenes, chlorobutadienes and dichlorobutenes produced by rodent or human
liver tissues. Arch. Toxicol. 41 249-277.
Seal D (1990) Use of mouse liver tumor data in risk assessment performed by the US
' Environmental Protection Agency. In: Mouse liver carcinogenesis: mechanisms and
species comparisons. Alan R. Liss, Inc. p. 5-18.
Beliles, R.P.; Brusisk, D.J.; Mecler, F.J. (1980) Teratogenic mutagenlc
contaminants: trichloroethylene, perchloroethylene, and carbon disulfide. Prepared by/for
_ under Contract No. 210-77-0047. Litton Bionetics, Inc., Kensington,
Maryland.
Bhattacharya, R.K.; Schultz, M.O. (1971) Arch. Biochem. Biophys. 145:565.
Bhattacharya, R.K.; Schultz, M.O. (1972) Arch. Biochem. Biophys. 153:105.
Bhunya S.P.; Behera, B.C. (1 987) Relative genotoxicity of trichloroacetic acid (TCA) as revealed
by different cytogenetic assays: bone marrow chromosome aberration micronucleus ana
sperm-head abnormality in the mouse. Mutat. Res. 188215-221.
Bieri,F.; Stubli, W.; Waechter, F.; Muakkassah-Kelly, S.; Bentley, P. (1988) Stimulation of DNA
synthesis but not peroxisome B-oxidation by nafenopin in pnmary cultures of marmoset
hepatocytes. Cell Biol.lnt.Rep.1 2,1 077-1 087.
58
-------
Bignami, M.; Conti, G.; Conti, L; Crebelli, R.; Misuraca, F.; Puglia, A.M.,; Randazzo, R.;
Sciandrello, G.; Carere, A. (1980) Mutagenicity of halogenated alphatic hydrocarbons in
Salmonella typhimurium Streptomyces coelicx)lor and Aspergillus nidulans. Chem. Biol.
Interact. 30:9-23.
Blair, .; et al. (1990) Cancer and other causes of death among a cohort of dry cleaners.
Br. J. Ind.Med. 47:162-168.
Borghoff et al. (1990)
Bronzetti, G.; Bauer, C.; Corsi, C.; Del Carratore, R.; Galli, A.; Nieri, R.; Paolini, M. (1983)
Genetic and biochemical studies on perchloroethylene in vitro and in vivo. Mutat. Res.
116:323-331.
Bronzetti etal. (1984)
Brown, _.; Kaplan, _. (1987) Retrospective Cohort Mortality Study of Dry Cleaner Workers
Using Perchloroethylene. J. Occup. Med. 29:13536-841.
Butterworth, B.E.; et al. (1989) Use of primary cultures of human hepatocytes in toxicology
studies. Cancer Res.49,1075-1084.
Callen, D.F.; Wolf, C.R.; Philpot, R.M. (1980) Cytochrome P-450 mediated genetic activity and
cytotoxicity of seven halogenated aliphatic hydrocarbons in Saccharomyces cerevisiae.
Mutat. Res. 77:55-63.
Cariot, P.; Barrat, E.; Mejean, L.; Pointel, J.P.; Drouin, P.; Debbry, G. (1983) Fenofibrate and
human liver. Lack of proliferation of peroxisomes. Arch.Toxicol.53,151-163.
Cema, N.; Kypenova, H. (1977) Mutagenic activity of chloroethylenes analyzed by screening
system test. Mutat. Res. 46:36 (Abstract).
Chang, L.W.; DeAngelo, A.B.; Daniel, F.B. (1989) Evaluation of DMA strand breaks (SB) by
chloroacetic acids (CAs) and chloroacetaldehydes (CADs) in vivo and in vitro.
Carcinogenesis 30 (Abstract 578).
Charbonneau, M.; Short, B.G.; Lock, E.A.; Swenberg, J.A. (1987) Mechanism of petroleum-
induced sex-specific protein droplet nephropathy and renal cell proliferation in Fischer-344
rats: Relevance to humans. Hemphill, D.D., ed. Trace Substances In Environmental
Health, Vol. 21. Columbia, MO: University of Missouri, pp.263-273.
Chen, .; et al. (1990) Renal Cysteine Conjugate Beta-Lyase Mediated Toxicity Studied with
Primary Cultures of Human Proximal Tubular Cells. Toxicol Appl. Pharmacol. 103:463-473.
Costa, A.K. (1984) Chlorinated ethylenes: their metabolism and the effect on DNA repair in rat
' hepatocytes. Carcinogenesis (London) 5:1629-1636.
Costa and Ivanetich (1984)
59
-------
Daniel (1990)
De La Iglesfa, FA; Lewis, J.E.; Buchanan, R.A.; Marcus, E.L; McMahon, G. (1982) Light and
electron microscopy of liver in hyperlipoproteineimic patients under long-term gemfibrozil
treatment. Atherosclerosis 43,1 9-37.
DeAngelo, A.B.; Daniel, F.B.; McMillan, L; Wernsing, P.; Savage, R.E., Jr. (1989) Species and
strain sensitivity to the induction of peroxisome proliferation by chloroacetic acids.
Toxicol.Appl.Pharmacol.1 01 ,285-298.
Degrassi, F.; Tanzarella, C. (1988) Immunofluorescent staining of kinetochores in micronuclei:
A new assay for the detection of aneuploidy. Mutat Res. 203: 339-345.
DeKant, W.; Vamvakas, S. (1989) Bioactivation of nephrotoxic haloalkenes by glutathione
conjugation: Formation of toxic and mutagenic intermediates by cysteine conjugate B-
lyase. Drug Metab. Rev. 20:(1) 48-83.
DeKant etal. (1980)
Dekant, W.; Martens, G.; Vamvakas, S. (1987) Role of glutathione s-transfeirase-catalyzed
conjugation versus cytochrome P-450 dependent phospholipid alkylation. Drug Metab.
Dispos. 15:(5) 702-709.
E C E T O C . (1990) Tetrachloroethylene: Assessment of Human Carcinogenic Hazard.
Brussels.
Elcombe, C.R. (1985) Species differences in carcinogenicity and peroxisome proliferation due
to trichloroethylene: A biochemical human hazard assessment. Arch.Toxicol.(8),6-7.
Elcombe, C.R.; Mitchell, A.M. (1986) Peroxisome proliferation due to di(2-ethylhexyl)phthalate
(DEHP): Species differences and possible mechanisms. Environ.Health Perspect.70,21 1-
219.
Flam and Lehman-McKeeman (1991)
Fox, T.R.; Schumann, A.M.; Watanabe, P.G.; Yano, B.L.; Maher, V.M.; McCormick, J.J. (1990)
Mutational analysis of the H-ras oncogene in spontaneous C57BL/6 x C3H/He mouse liver
tumors and tumors induced with genotoxic and nongenotoxic hepatocarcinogens. Cancer
Res. 50:4014-4019.
Galloway, S.M.; Armstrong, J.J.; Reuben, C.; Colman, S.; Brown, B.; Cannon, C.; Bloom, A.D.;
Nakamura, F.; Ahmed, M.; Duk, S.; Rimpo, J.; Margolin, B.H.; Resnick, M.A.; Anderson,
B.; Zeiger, E. (1987) Chromosome aberrations and sister-chromatid exchanges in
Chinese hamster ovary cells: Evaluations of 108 chemicals. Environ. Molec. Mutagen.
Goldsworthy and Popp (1987)
60
-------
Goldsworthy, T.L.; Smith-Oliver, T.; Loury, D.J.; Popp, J.A. (1988) Assessment of chlorinated
hydrocarbon-induced genotoxicity and cell replication in rat kidney cells. Environ. Molec.
Mutagen. 11:(11)39.
Goodman etal. (1980)
Green etal. (1986)
Green, T.; Odum, J.; Foster, J.K. (1990) Perchloroethylene-induced rat kidney tumors: An
investigation of the mechanisms involved and their relevance to humans. Toxicol. Apl.
Pharmacol. 103:77-89.
Greimetal. (1975)
Gualandi, G. (1987) Use of alpha-and beta-tublin mutants for the study of spontaneous and
induced chromosomal mis-distribution in Aspergillus nidulans. Mutat. Res. 178:33-41.
HEI (1988)
Haider etal. (1984)
Hanefeld, M.; Kemmer, C.; Kadner, E. (1983) Relationship between morphological changes and
lipid-lowering action of p-chlorphenoxyisobutyric acid (CPIB) on hepatic mitochondria and
peroxisomes in man. Atherosclerosis 46,239-246.
Haseman, J.K.; Huff, J.; Boorman, G.A.; (1984) Use of historical control data in carcinogenicity
studies in rodents. Toxicol. Pathol. 12:126-135.
Haseman, J.K.; Crawford, D.D.; Huff, J.E.; Boorman, G.A.; McDonnell, E.E. (1984) Results from
86 two-year carcinogenicity studies conducted by the NTP. J. Toxicol. Environ. Health
14:621-639.
Haworth, S.; Lawlor, T.; Mortelmans, K.; Speck, W.; Zeiger, E. (1983) Salmonella mutagenicity
test results for 250 chemicals. Environ. Mutagen. 5:(1)1-142.
Henschler (1977)
Henschler, D.; Elsasser, H.; Romen, W.; Eder, E. (1984) Carcinogenicity study of
trichloroethylene, with and without epoxide stabilizers, in mice. Cancer Res.Clin. Oncol.
107:149-156.
Herren-Freund, S.L.; Pereira, M.A.; Knoury, M.D.; Olson, G. (1987) The carcinogenicity of
trichloroethyiene and its metabolites, trichloroacetic acid and dichloroacetic acid, in mouse
liver. Toxicol. Appl. Pharmacol. 90:183-189.
International Agency for Research on Cancer (IARC). (1982) Evaluation of the carcinogenic risk
to humans: IARC Monogr. (Suppl) 4.
61
-------
International Agency for Research on Cancer (IARC). (1987) Overall evaluations of
carcinogenicity: An updating of IARC monographs Volumes 1 to 42. IARC Monogr. Eval.
Care. RIskChem. Hnr. 7 (Suppl): 1-440.
Ikeda, M.; Ohtsujl, H.; Imamura, T.; Komoike, Y. (1972) Urinary excretion of total trichloro-
compounds, trichloroethanol and trichloroacetic acid as a measure of exposure to
trichloroethylene and tetrachloroethylene. Br. J. Ind. Med. 29:328-333.
Ikeda etal. (1980)
Issemann, .; Green, . (1990) Activation of a member of the steroid hormone receptor
superfamily by peroxisome proliferators. Nature 347:645.
Kafer, E. (1985) Tests which distinguish induced aneuploidy and crossing-over from secondary
segregation in Aspergillus treated with cloral hydrate or x-rays. Mutat. Res. 167:9-34.
Kline etal. (1982)
Koch etal. (1988)
Koch, R.; Wolf, H.U. (1988) Genetic effects of chlorinated ethylenes in the yeiast saccaryces
cervisiar. Mutat. Res. 206:209-216.
Liang, J.C.; Paccheirotti, F. (1988) Cytogenetic investigation of chemically-induced aneuploidy
in mouse spermatocytes. Mutat. Res. 201:325-335.
Lock, E.A. (1988) Studies on the mechanism of nephrotoxicity and nephrocarcinogenicity of
halogenated alkenes. CRC Grit Rev. Toxicol. 19, 23-42.
MacNaughton, M.G.; Uddin, D.E. (1984) Toxicology of mixed distillate and high-energy synthetic
fuels. In: Mehlmen.M.A. Hemstreet, G.P., Thorpe, J.J., Weaver, N.K. (eds.), Advances
in Modem Envoronmental Toxicology. Vol. 7, Renal Effects of Petroleum Hydrocarbons.
Princeton Scientific Publishers, Inc., Princeton, New Jersey, pp. 121-132.
MaltonI, C.; Cotti, G. (1986) Results of long-term carcinogenicity bioassays of
tetrachloroethylene on Sprague-Dawley rats administered by ingestion. Acta Oncologica
1,11 -26. As cited in Green, 1990. Chloroethylenes: A mechanistic approach to human risk
evaluation. Ann. Rev. Pharmacol. Toxicol.
Margard, W. (I978) In vitro bioassay of chlorinated hydrocarbon solvents. Battelle Laboratories.
Unpublished document for Detrex Chemical Industries.
Maronpot, R.R.; Haseman, J.K.; Boorman, G.A.; Eustis, S.E.; Rao, G.N.; Huff, J.fiE. (1987) Liver
lesions B6C3F1 mice: The National Toxicology Program, experience and position. Arch.
Toxicol. (Suppl) 10:10-26.
62
-------
Marsman, D.S.; CattJey, B.C.; Conway, J.G.; Popp, J.A. (1988) Relationship of hepatic
peroxisome proliferation and replicative DNA synthesis to hepatocarcinogenicity of the
peroxisome proliferators di(2-ethylhexyl)phthalate and [4-chloro-6-(2,3-xyIidino)-2-
pyrimidinylthiojacetic acid (Wy-14,643) in rats. Cancer Res.48,6739-6744.
Mazzullo, M.; Grilli, S.; Lattanzi, G. (1987) Evidence of DNA binding cavity of perchloroethylene.
Res. Commun. Chem. Pathol. Pharmacol. 58:(2)215-235.
McDonnell, E.E. (1990) Mouse liver tumor: the problem in mouse liver carcinogenesis:
Mechanisms and Species Comparisons p. 1-3. Alan R. LJss, Inc.
McGregor, D.B.; Brown, A.; Cattanach, P.; Edwards, I. (1988) Responses of the L5178Y
TK+/TK- mouse lymphoma cell forward mutation assay. 3.72 coded chemicals. Environ.
Molec. Mutagen. 12:85-154.
McKinney, L.L; Weakley, F.B.; Eldridge, A.C.; Campbell, R.E.; Cowan, J.C.; Picken, J.C; Blester,
H.E. (1957) S(dichlorovinyl) L-cysteine: an agent causing fatal aplastic anemia in calves.
J. Am. Chem. Soc. 79:3932.
MOUSE LIVER CARCINOGENESIS: Mechanisms and Species Comparisons. Proceedings of
the Symposium Held in Austin, Texas, November 30-December 3,1988. Stevenson, D.E.,
McClain, R.M., Popp, J.A., Slaga, T.J., Ward, J.M., Pitot, H.C. Progress in Clinical And
Biological Research Volume 331. Wiley-Uss, New York. 444 p.
Myhr, B.C.; Bowers, L.R.; Caspary, W.J. (1986) Results from the testing of coded chemicals in
the L5178Y TK +/-mouse lymphoma mutagenesis assay. Environ. Mutagen. 8:(6)58.
Nebert, D.W. (1990) Growth Signal Pathways. Nature 347:709.
Nelson, M.A.; Bull, R.J. (1988) Induction of strand breaks in DNA by trichloroethylene and
metabolites in rat and mouse liver in vivo. Toxicol. Appl. Pharmacol. 94:45-54.
Nelson, M.A.; Lansing, A.J.; Sanchez, I.M.; Bull, R.J.; Springer, D.L (1989) Dichloroacetic acid
and trichloroacetic acid-induced DNA strand breaks are independent of peroxisome
proliferation. Toxicology 58:239-248.
Nelson, .; et al. (1990) Increased expression of c-myc and cH-ras in dichloroacetate- and
trichloroacetate-induced liver tumors in B6C3F1 mice. Toxicology 64, 47-5
NTP. (1983) Carcinogenesis bioassay of pentachloroethane (CAS No. 76-01-7) in F344/N rats
and B6C3F1 mice (gavage study). NTP-81-34. NIH Publication No. 83-1788. NTP TR
232. April, 1983.
NTP. (1986) Toxicology and carcinogenesis of tetrachloroethylene (perchloroethylene) (CAS No.
127-18-4) in F344/N rats and B6C3F., mice (inhalation studies) NIH publication No. 86-
2567. NTP TR 311.
63
-------
NTP. (1986) Toxicology and carcinogenesis studies of isophorone (CAS No. 78-59-1) in F344/N
rats and B6C3F1 mice (gavage studies). NIH publication No. 86-2547. NTP TR 291.
Jan, 1986.
NTP. (1987) Toxicology and carcinogenesis studies of dimethyl methylphosphonate (CAS No.
756-79-6) in F344/N rats and B6C3F1 mice (gavage studies). NIH Publication No. 87-
2579. NTP TR 323, Nov., 1987.
NTP. (1988) Toxicology and carcinogenesis studies of d-limonene (CAS No. 5989-27-5) in
F344/N rats and BeCgF, mice (gavage studies). NIH publication No. 88-2902. NTP TR
347.
NTP. (1988b)
NTP. (1989) Toxicology and carcinogenesis studies of hexachloroethane (CAS No. 67-72-1) in
F344/N rats (gavage studies. NIH publication No. 89-2816. NTP TR 361 dated August,
1989.
NTP. (1990) 347 Toxicology and carcinogenesis studies of d-limonene.
Odum, J.; Green, T.; Foster, J.R.; Hext, P.M. (1988) The role of trichloroacetic and peroxisome
proliferation in the differences in carcinogenicity of perchloroethylene in the mouse and
rat. Toxicol.Appl. PharmacoI.92,103-112.
Office of Science and Technology Policy (OSTP) (1985) Chemical carcinogens: review of the
science and its associated principles. Federal Register 50:10372-10422.
Ohtsukietal. (1983)
Olson etal. (1987)
Olson etal. (1990)
Oshimura, M.; Koi, M.; Ozawa, N. (1988) Role of chromosome loss in ras/myc-induced Syrian
hamster tumors. Cancer Res. 48:1623-1632.
Page and Asire (1985)
Phillips, R.D.; Moran, E.J.; Dodd, D.E.; Fowler, E.H.; Dary, C.D.; O'Donoghue, J. (1987) A 14-
week vapor inhalation toxicity study of methyl isobutyl ketone. Fundam. Appl. Toxicol. 9:
380- 388.
Pickle etal. (1987)
Rampy, L.W.; Quast, J.F.; Balmer, M.F.; Leong, B.K.J.; Gehring, P.J. (1978) Results of a long-
term inhalation toxicity study: perchloroethylene in rats. Toxicology Research Laboratory.
Health and Environmental Research. The Dow Chemical Company. Midland, Ml.
Unpublished.
64
-------
Reynolds, S.H.; Stowers, S.J.; Patterson, R.M.; Maranpot, R.R.; Aaronson, S.A.; Anderson, M.W.
(1987) Activated oncogenes in B6C3F1 mouse liver tumors: implications for risk
assessment. Science 237:1309.
Rijhsinghani, .; et al., (1986) (muta)
Russo, A.; Pacchierotti, F.; Metalli, P. (1984) Nondisjunction induced in mouse spermatogenesis
by chloral hydrate, a metabolite of trichloroethylene. Environ. Mutagen. 6:695-703.
Science Advisory Board (1985). Meeting of the Carcinogen Risk Assessment Guidelines Panel
of the Science Advisory Board. April 22-23, 1985.
SRI International (1983)
Schultz, M.O.; Klubes, P.; Perman, V.; Mizuno, W.S.; Bates, F.W.; Sautter, J.H. (1959) Blood
dyscrasia in calves induced by S(dichlorovinyl) L-cysteine. Blood 14:1015.
Schumann, A.M.; Quasi, J.F.; Watanabe, P.G. (1980) The pharmacokinetics and
macromolecular interactions of perchloroethylene in mice and rats as related to
oncogenicity. Toxicol. Appl. Phamnacol. 55:207-219.
Science Advisory Board (SAB). (1987) Letter from the SAB to EPA Administrator Lee Thomas
dated January 27,1987.
Science Advisory Board (SAB). (1988) Letter from the SAB to EPA Administrator Lee Thomas
dated March 9,1988.
Shimada, T.; Swanson, A.F.; Leber, P.; Williams, G. (1985) Activities of chlorinated ethane and
ethylene compounds in the Salmonella/rat microsome mutagenesis and rat
hepatocyte/DNA repair assays under vapor phase exaposure conditions. Cell Biol.
Toxicol. 1:159-179.
Singh, M.; Sinha, U. (1976) Chloral hydrate induced haploidization in Aspergillus nidulans.
Experientia. 32:114-1145.
Singh, M.; Sinha, U. (1979) Mitotic haploidization and growth of Aspergillus nidulans on media
containing chloral hydrate. J. Cytol. Genet. 14:1-4.
Solleveld, H.A.; Haseman, J.K.; McConnell, E.E. (1984) National history of body weight gain,
survival, and neoplasia in the F344 rat. J. Natl. Cancer Inst. 72:929-940.
Sora, S. (1987) Chloral hydrate, methylmercury hydroxide and ethidium bromide affect
chromosomal segregation during meiosis of saccharomyces cervisiae. Mutat. Res.
190:13-17.
Sora and Carbone (1987)
Stevenson et al. (1990)
65
-------
Stonnard, .; et al., (1986) d-2u-globulin: measurement in rat kidney following administration
of 2,3,4-trimethylpentane. Toxicology 41:161-168.
Swenberg et al. (1989)
Trump etal. (1989)
U.S. Environmental Protection Agency (U.S. EPA). .{1985) Health Assessment Document for
Tetrachloroethylene (Perchloroethylene). Prepared by the Office of Health and
Environmental Assessment, Washington, DC. EPA/600/8-82/005F. Available from:
National Technical Information Service, Springfield, VA; PB-85-249704/AS.
U.S. Environmental Protection Agency (U.S. EPA). (1985b) Health Assessment Document for
Trichloroethylene. Prepared by the Office of Health and Environmental Assessment,
Washington, DC. EPA/600/8-82/006F. Available from: National Technical Information
Service, Springfield, VA; PB-85-249696/AS.
U.S. Environmental Protection Agency (U.S. EPA). (1986a) Addendum to the Health
Assessment Document for Tetrachloroethylene (Perchloroethylene). Prepared by the
Office of Health and Environmental Assessment, Washington, DC. EPA/600/8-82/005FA.
Available from: National Technical Information Service, Springfield, VA; PB-86-
174489/AS.
U.S. Environmental Protection Agency (U.S. EPA). (1986b) Guidelines for carcinogen risk
assessment. Federal Register 51:33992-34003.
U.S. Environmental Protection Agency (U.S. EPA). (1987) Evaluation of the caircinogenicity of
unleaded gasoline. Prepared by the Office of Health and Environmental Assessment,
Washington, DC. EPA/600/6-87/001. Available from the National Technical Information
Service, Springfield, VA; PB .
U.S. Environmental Protection Agency (U.S. EPA). (1991) Alpha-2^-globulin: Association with
chemically induced renal toxicity and neoplasia in the male rat. Risk Asessment Technical
Panel document draft.
Valencia, R.; Mason, J.M.; Woodruff, R.C.; Zimmerming, S. (1985) Chemical mutagenesis
testing on Drosophila. II. Results of 48 coded compounds tested for the National
Toxicology Program. Environ. Mutagen. 7:325-348.
Vamvakas, S.; Herkenhoff, M.; Dekant, W.; Henschler, D. (1989) Mutagenicity of
tetrachloroethane in the Ames test: metabolic activation by conjugation with glutathione.
J. Biochem. Toxicol. 4:(1 )21 -27.
Walles, S.A.S. (1986) Induction of single-strand breaks in DNA of mice by trichforethylene and
tetrachloroethane. Toxicol. Lett. 31:31-35.
66
-------
Warner, J.R.; Hughes, T.J.; Claxton, LD. (1988) Mutagenicity of 16 volatile organic chemicals
in a evaporation technique with Salmonella typhimurium TA100. Environ. Molec.
Mutagen. 11(11 ):111-112.
Waskell (1978)
Yoon, .; et al., (1985) (muta)
67
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