vvEPA
United States
Environmental Protection
Agency
Office of Research and
Development
Washington DC 20460
EPA/600/6-91/002F
September 1991
Response to Issues and
Data Submissions on the
Carcinogenicity of
Tetrachloroethylene
(Perchloroethylene)
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EPA/600/6-91/002F
September 1991
RESPONSE TO ISSUES AND DATA SUBMISSIONS
j
ON THE CARCINOGENICITY OF
TETRACHLOROETHYLENE
(PERCHLOROETHYLENE)
Human Health Assessment Group •
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Washington, D.C. 20460 ;
Printed on Recycled Paper
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DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection
Agency policy and approved for publication. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
11
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CONTENTS
Preface vj
Authors ; yii
1. INTRODUCTION l
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 8
4. MUTAGENICITY OF PERCHLOROETHYLENE AND ITS METABOLITES 14
4.1. Data on Mutagenicity of Perchloroethylene per se 14
4.2. Mutagenicity of Perchloroethylene Metabolites 17
5. MOUSE LIVER TUMORS 23
i
5.1. Carcinogenicity Bioassay Data and EPA's Position 23
5.2. Peroxisome Proliferation and Perchloroethylene 27
6. KIDNEY TUMORS IN MALE RATS . 33
6.1. Alpha-2u-Globulin in Renal Carcinogenesis in Male Rats 35
6.2. Sustained Chronic Nephrotoxicity as a Possible Mechanism Independent of
Alpha-2u-Globulin Accumulation 41
6.3. A Mutagenic Mechanism of Perchloroethylene-Induced Carcinogenesis in Male
Rats 42
7. MONONUCLEAR CELL LEUKEMIA IN RATS i 46
m
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CONTENTS (continued)
8. SUMMARY AND CONCLUSIONS 50
References 61
IV
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CONTENTS (continued)
LIST OF TABLES
1. Summary of Genotoxicity Testing of Tetrachloroethylene ..... ......... 15
2. Summary of Genotoxicity Testing of Tetrachloroethylene Metabolites ......... i8
LIST OF FIGURES
1. Oxidative metabolism of perchloroethylene
2. Enzyme-catalyzed metabolism of perchloroethylene to its glutathione conjugate
^^S-co^ °f ** .glUtamyl and ^^ reSidU6S t0 yidd itS ^-Ponding
3. Further metabolism in the kidney of the perchloroethylene intermediate 1 1 2-
trichlorovmylcysteine, leading to mutagenic metabolites ............ '.'... 12
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PREFACE
This document, prepared by the Office of Health.and Environmental Assessment
(OHEA), responds to data and comments submitted to the U.S. Environmental Protection
Agency (EPA) and discusses how this information influences the overall weight-of-evidence
classification for a perchloroethylene human cancer hazard. Relevant literature through early
1991 has been critically evaluated.
The Agency's Science Advisory Board reviewed the February 1991 draft of this
report at a public meeting and found the report to be well written and of high scientific
quality, although the Board did not agree with the Agency's recommended weight-of-
evidence classification for perchloroethylene of B2, probable human carcinogen. The
Science Advisory Board, expressing its views in an August 1991 letter to EPA Administrator
William Reilly, offered advice on the weight-of-evidence classification in a spirit of
flexibility encouraged by the 1986 Risk Assessment Guidelines for Carcinogenicity and
recommended placing perchloroethylene on a continuum between Group B2 and Group C
weight-of-evidence categories. The Board stated that because the major issues arising from
the assessment of perchloroethylene had not changed since 1987, its previous response is
appropriate and cautioned, as in its 1988 response, that "from a scientific point of view, it
seems inappropriate for EPA and other agencies to regulate substances that are classified B2
and not to consider regulations of compounds classified as C, regardless of the level of
human exposure A substance classified as C for which exposure is high may represent a
much greater threat to human health."
VI
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AUTHORS
Members of the Human Health Assessment Group of the Office of Health and
Environmental Assessment (OHEA) prepared this document.
i
i
PRIMARY AUTHOR
Jean C. Parker, Ph.D. i
Carcinogen Assessment Toxicology Branch
U.S. Environmental Protection Agency j
Washington, DC
CONTRIBUTING AUTHORS
Vicki Vaughan-Dellarco, Ph.D.
Genetic Toxicology Assessment Branch
U.S. Environmental Protection Agency i
Washington, DC
David Reese, Ph.D.
Genetic Toxicology Assessment Branch
U.S. Environmental Protection Agency
Washington, DC
Vll
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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 (OHEA) within the U.S. Environmental Protection Agency's
(EPA'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 raised by the EPA's Science Advisory Board (SAB,
personal communication) during its review of an addendum (U.S. EPA, 1986a) to the Health
Assessment Document for Tetrachloroethylene (U.S. EPA, 1985a). New information that
has bearing on the issues also became available over the last two to three years.
Recently generated laboratory data have led to the development of hypotheses about
the mechanisms of perchloroethylene tumorigenesis. Biological arguments suggested 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 some evidence supports 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. Because 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 hi light of
recently published studies, and to develop the EPA's response to issues and data included hi
comments submitted to the Agency on the overall weight of evidence for the 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 published since
1986. The topics include three tumor end points observed hi 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, mutagenicity, peroxisome proliferation, and alpha-2u-
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" hi animals. The human
data are 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 hi humans (see Guidelines for Carcinogen Risk Assessment, U.S. EPA,
1986b). 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
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
hi the case of perchloroethylene signify a potential human hazard, 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 the Health Assessment Document (HAD) for Tetrachloroethylene
(Perchloroethylene) in July 1985 (U.S. EPA, 1985a). The Office of Health and
Environmental Assessment, in consultation with an Agency workgroup, prepared the HAD as
a source document for the entire EPA (U.S. EPA, 1985a, preface). The document
underwent extensive expert peer review and review by the Environmental Health Committee
of the Agency's Science Advisory Board (SAB) before 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 that 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 and
would be updated if necessary when the NTP reports were evaluated (U.S. EPA, 1985a,
preface). !
A draft Addendum to the Health Assessment Document for Tetrachloroethylene
(Perchloroethylene) (U.S. EPA, 1986a), prepared by OHEA, analyzed the results of the
inhalation bioassays performed by Battelle Pacific Northwest Laboratories for the NTP. 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 F344/N rats, and uncommon renal
tubule neoplasms and some evidence for gliomas of the brain in male rats. The authors of
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the addendum concluded that perchloroethylene is a B2 chemical (probable human
carcinogen) because:
• 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 National Cancer Institute (NCI) bioassay provided positive evidence of
hepatocellular carcinomas in mice administered perchloroethylene by gavage.
On May 15, 1986, the draft addendum underwent peer review by the SAB's
Halogenated Organics Subcommittee in a public meeting held in Madison, Wisconsin. The
SAB's initial comments appeared in a letter to EPA Administrator Lee Thomas dated January
27, 1987 (personal communication). 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."
Because 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 requested additional
consultative advice from the SAB on specific issues relating to 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 responded to the second request for advice in a letter dated March 9, 1988,
to Administrator Thomas. In this letter the SAB concluded that "the overall weight of
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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:
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 then, the EPA has received public comments on perchloroetitrylene in several
regulatory actions that include consideration of the perchloroethylene weight-of-evidence
classification. These public comments pertain to Resource Conservation and Recovery Act
listing (U.S. EPA, 1989a) and Comprehensive Environmental Response Compensation and
Liability Act reportable quantity rules (U.S. EPA, 1989b), and to maximum contaminant
level goal and maximum contaminant level proposals for drinking water (U.S. EPA, 1989c).
After its review of a 1991 draft of this report on perchloroethylene, the Agency's
Science Advisory Board concluded that its previous advice remained appropriate and that the
weight of evidence indicates that perchloroethylene lies on a continuum between categories
B2 and C. The Board also maintained its stance that, from a scientific perspective, exposure
should be considered more important than classification category in determining potential
threat to human health and whether or not a chemical substance should be regulated.
2.2. ANIMAL STUDIES OF PERCHLOROETHYLENE CARCINOGrENICITY
Perchloroethylene has shown cancer-causing activity in male and female mice and in
male and female rats in the NCI and NTP studies. In both sexes of mice, perchloroethylene
administered by oral gavage or by inhalation induced dose-related statistically significant
increases in hepatocellular carcinomas. Statistically significant increased incidences of
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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. 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 rat mononuclear cell leukemia. In addition to the general controversies
surrounding these tumor end points, chemical-specific data that may be pertinent to
evaluating the effect of perchloroethylene on tumor incidence have generated concern.
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
assess whether the data reflect 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-related
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.
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The paper addresses recent literature on perchloroethylene and its biometabolites as
they relate 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
animals and humans indicate that metabolism of perchloroethylene is relatively limited, as
evidenced by the high percentage of absorbed dose excreted hi the breath as the parent
molecule (U.S. EPA, 1985a, 1986a; Ohtsuki et al., 1983; Ikeda et al., 1980; Henschler,
1977a; Yllner, 1961). In human studies, however, only about half of the perchloroethylene
absorbed has been accounted for through excretion of parent compound or metabolites (U.S.
EPA, 1985a, 1986a).
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 hi 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 considered may exist hi 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, 1985a). Recent investigations have revealed a glutathione conjugative pathway that
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 P45o) probably
occurs mostly hi the liver but may occur at other sites. This pathway is operative in humans
as well as in 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. There are, however, quantitative
differences among the metabolic rates of different species. The major metabolite of the
oxidative pathway is trichloroacetic acid (TCA), which is excreted hi the urine of all species
tested. Other identified urinary metabolites are designated in figure 1. Some of the
intermediates hi the oxidative pathway are known to possess cytotoxic or genotoxic activity
8
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(e.g., 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 cytotoxic and mutagenic metabolites of perchloroethylene that
arise 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-(l,2,2-trichlorovinyl) glutathione (TCVG). After transport to the
kidney, TCVG is metabolized to S-(l,2,2-trichlorovinyl) cysteine (TCVC) by the enzymatic
removal of glutamyl and glycine residues. TCVC is acetylated via a reversible reaction to
N-acetyl-S-(l,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 (Vamvakas et al., 1987, 1989a,
1989b, 1989c).
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. (1989b) 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.
Some limited evidence suggests that humans may not metabolize perchloroethylene by
the conjugative pathway. Human liver samples have been compared with 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 by 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
10
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Cl
Cl
X - _ « X
.A* -""^v
ci' sci
Perchloroethylene
GSH (glutatMone)
C'
GSH-S-transferas*
NH-CH2-COOH
.C=<
or
i-CH9-CH
AH
1,1,2-TrichIorovlnylgIutathlone
O COOH
II I
C-CH2-CH2-CH
NH,
a-glutamyltranspeptldase
civ
cix
.Cl
gtutamate
NH-CH9-COOH
"C
NS-CH2-CH
NH,
cystelnylglydnedlpeptldase
gtydne
Ov OH
V
-------
Cl
Cl'
Cl
C N-acetyltransterase^
!-CH9-CH « -
NH,
1 ,1 ^-Trichloro vlnylcystelne*
Cl
Ci
rearrangement
Cl SH
Trlchlorovlnylthlol
Cl
Cl
Cl
xs. I
S-CH2-CH O
2 I II
NH-C-CH3
1,1,2-Trichlorovinyl-N-acetylcysteIne*
Cl Cl
V=<
Cl' S
Dichlorothioacetyl Chloride
+ Pyruvate
+ Ammonia
Cl
vc=c=s
Cl'
Dichlorothloketene
H2o
OH
Cl
Cl
DIchloroacetIc Acid*
•Identified urinary metabolites.
Figure 3. Further metabolism in the kidney of the perchloroethylene intermediate
1,1,2-trichIorovinylcysteine, leading to mutagenic metabolites. These pathways occur
in humans as well as in rodents.
Source: Dekant et al., 1987; Green et ah, 1990; ECETOC, 1990.
12
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of human liver sampies tested, it „ premature ^ d
c step » untteiy to occur in humans. Additional confirmatory studies ^clearly
needed. Human liver docs conjugate glutathione with hexach,oro-l 3-LdienlT
cMoroalkene rdated to percUoroethylene (Oesch and Wo,f 19m 'l^™' *
Lvorry ystetae metaboute- ta reiaaveiy ^ «™ «-» *- «-.
s have been measured in the urine of occupation^ exposed subjects These data
a h,gn ^iihood of perciUoroethyiene being n^ed via this athway ^N
.cys.in, rhe precursors of these urtaa, metaLKs
icagen^hcnbioacUvatedbybeta-.yase.
activdy
He bewyase paftway has been shown to produce cytotoxic and mutagcnic
.-. to .e fo^on of .e toxic metaboltes in anim d «
~,es in bacteria, mode, is aiso present in human proxima, tubuiar cei^h et a,
1990; Green et al. , 1990). Human proximal tubular cells have been shown r
* ~» of g-utathione andVor cysteine conjugates of a ^££.
fluoroalkenes that are activated via the beta-lyase pathway (Chen et a,., 1990).
13
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4. MUTAGENICITY OF PERCHLOROETHYLENE AND ITS METABOLITES
Genetic alterations are critica, events in the carcinogenesis process. Thus, evidence
of an agent to produce heritable genetic lesions (e.g., gene mutauons, stable
-r: ir. :===
observations of mutagenic noncarcinogens and nonmutagenic carcmogens as well as
985 EPA Heal* Assessment Document for TetracMoroethylene provded a
to determine if the earlier conclusions are still valid.
4 ! DATA ON MUTAGENICTTY OF PERCHLOROETHYIJSNE PER SE
Til in table 1, perchloroethvlene has not been clearly shown to be an mducer
o, gene mutations in routinery used assays. In bacteria, assays for reverse mutation
1— /mammalian microsome test) in the presence or — «^T
activation, perchloroemylene exposures produced largely negative results (Bartsch e, a.
r979 Majard, 1978; Haworth e, a,., 1983; Warner et a,., 1988). In srud.es reportmg
!Tpi tested. Jen highly purified perch.oroethy.ene was evaluated in a des,ccator with
14
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Table 1. Summary of Genotoxicity Testing of Tetrachloroethylene
A. Gene Mutation Tests
Salmonella/Ames assay
Escherichia coll K12/343/113
Multipurpose test
Yeast reverse mutation test
Drosophila sex-linked recessive lethal test
L5178YTK+/TK- mouse lymphoma cell assay
B. Chromosomal Aberration Tests
Chinese hamster ovary cells (CHO)
Rat bone marrow assay
Peripheral lymphocytes from exposed humans
C. Other Tests Indicative of DNA-Damaging
Activity
Unscheduled DNA synthesis in WI-38
Hepatocyte primary cuIture/DNA repair test
Mitotic recombination tests in yeast
Sister chromatid exchange formation in CHO cells
DNA strand breaks (alkaline elution test assay) in
mouse kidney and liver cells
D. DNA Binding Studies
Mice
Mice and rats
Results3
mostly —
+6
-
-
-
-, ?c
-
_c
-
_c
+b,-
+,-
-
wk
-
9
References
Shimada et al., 1985; Margard, 1978;
Bartsch et al., 1979; Haworth et al., 1983;
Warner et al., 1988
Henschler, 1977b; Greim et al., 1975
Callen et al., 1980; Bronzetti et al.; 1983
Beliles et al., 1980; Valencia et al., 1985
Myhr et al., 1986; Galloway et al., 1987;
McGregor et al., 1988
I
Galloway et al., 1987
Rampy et al., 1978); Beliles et al., 1980
Dceda et al., 1980
Beliles et al., 1980
Shimada et al., 19S5; Costa and Ivanetich,
1984; Goldsworthy et al., 1988
Callen et al., 1980; Bronzetti et al., 1983
Koch et al., 1988; Galloway et al., 1987
Walles, 1986
Schumann et al., 1980
Mazzullo et al., 1987
a+ designates positive; - negative; wk weak response; ? inconclusive test. Dose-response relationships were 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 mutagenic stabilizers or contaminants. :
Questionable evidence for weak or borderline activity in specific data sets.
15
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the Salmonella/mammalian microsome test, negative results were obtained (Shimada et al.,
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
Tk+/Tk~ 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 (Cerna 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
etal., 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. It is important to note that 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 10'9 to 1042). 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., 1988), and sister chromatid exchange formation in culture Chinese hamster cells
16
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(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 may lead ,o mutagenieity, agents tot can be
demonstrated to induee only DNA strand breakage shou!d not be viewed as possessing the
same genetie hazard potential as agents mat have been shown to induce gene mutations or
stable chromosomal aberrations.
4.2. MUTAGENICITY OF PERCHLOROETHYLENE METABOLITES
When the 1985 HAD was being prepared, literature on the mutagenicity of
Perchloroethylene metabolites was limited. However, several studies are now available
(summarized in table 2), and their results warrant some consideration. \
Oxidative metabolism of perchloroethylene (dependent on cytochrome P450) occurs
mostly in the liver. This pathway is operative in both rodents and humans and leads to the
production of several metabolites (U.S. EPA, 1985a; Yllner, 1961) (figure 1).
Perchloroethylene-epoxide, a hypothesized intermediate in perchloroethylene oxidative
metabolism (Henschler et al., 1977a,b), has been shown to be mutagenic in the
Salmonella/mammalian microsome test (Kline et al., 1982)
Chloral hydrate (trichloroacetaldehyde), a known metabolite of trichloroethylene and
likely a perchloroethylene metabolite, has been produced both in vitro and in vivo 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 Agostini-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 also been shown to block spindle elongation in insect
spermatocytes (Ris, 1949). Chloral hydrate has been reported to be weakly mutagenic in the
Salmonella/mammon 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
17
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Table 2. Summary of Genotoxicity Testing of Tetrachloroethylene Metabolites
Metabolite
_... —
Perchloroethylene-epoxide
—i •— ""
Chloral hydrate
Trichloroacetic acid
Dichloroacetaldehyde
Monochloroacetaldehyde
S-<1,2,2-trichloro-vinyl)
glutathione
(Result)3/Assay
(+) SalmonellatAiass assay
(+) Aneuploidy/yeast
•
(+) Aneuploidy/mammalian cells in vitro
(+) Aneuploidy/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
—
(+) Salmonellal'Ames assay
(+) DNA strand breaks in human cells
in vitro
(+) DNA strand breaks in human cells
in vitro
(+) Salmonellal'Ames assay
(+) unscheduled DNA synthesis in
LLC-PK, cells
Reference
_
Kline et al., 1982
_.
Singh and Sinha, 1976, 1979; Kafer,
1985; Gualandi, 1987; Sora and
Aeostini-Carbone, 1987
Degrassi and Tanzarella, 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
"
—. —
Bignamiet al., 1980
Chang etal., 1989
~
•
Chang et al., 1989
Green and Odum, 1985; Dekant et
al., 1986
_«^_i^—^—^—•
Vamvakas et al., 1989b
Vamvakas et al., 1989a
»+ designates positive; - negative; wk weak response; ? inconclusive test.
18
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gene conversion in yeas. (Bronzetti e, al., 1984). Chloral hydrate also has been observed ,o
cause tumors in mice (Rijhsinghani e, a,., 1986). Other chtoroacetaMehydes are potently
mutagenic. Dichloroacetaldehyde (DCAA) is mutagenic in «he &«a/mamma,ian
m.crosome test (Bignami et a,., 1980), and monochloroacetaMehye and, to a lesser extent
DCAA appear to induce DNA single-strand breaks in cultured human ceils (Chang et al.,'
Few genotoxicity studies are available on the carcinogenic perchloroethylene
metabolites trichloroacetic acid (TCA) and dichloroaeetic acid (DCA). TCA and DCA have
been reported to produce stogie-strand breaks to hepatic DNA of mice and rats. This action
» mdependen. of peroxisome proliferation and of liver necrosis (Nelson Snd Bull 1988-
Nelson e, al., 1989). The induction of DNA stogfe-strand breaks could not be confirmed by
other laboratories, but a different methodology was used (Chang et al., 1989) TCA was
reported as positive for the induction of chromosomal aberrations and micronuclei to the
bone marrow of mice (Bhunya and Behera, 1987). This finding is questionaWe, however
because of the low background frequencies report for chromosomal aberrations and the'
anomalous dose response seen for mieronuclei formation in nonnochromatic erythrocyfcs
In rats, perchloroethytene has been shown to be metabolized ,o a cytotoxic, mutagenic
fractton through a conjugate beta-lyase pathway (reviewed by Anders e, al. 1988 and
Dekant e, al., 1989; see also section 3 of this document). This secondary metabolic
pathway, although quantitatively minor, may be of major toxicologic importance The
pathway is toitiaily catalyzed by hepatic cy.oso.ic and microsoma. glutathione S-^ansferases
to y,e.d S-a.^-trichlorovinyl) glutathkme ^^ After ^^ ^ ^ ^
me.abo.ized to S-(l,2,2-tricblorovmyl) cysteme (TCVC) by the enzymatic remova! of
glutamy, and giyeme r^idues. TCVC is ace«yla.ed by a reversible reaction to N-acetyl-S-
(l,2,2-tnchlorovtayl)-L-cysteine, which is excreted in the urine. However, TCVC is also a
substrate for renal beta-lyases that cleave TCVC to yie.d an unstab.e thio, that may give rise
to cytotoxic and mutagenic 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
19
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fractions in in vitro experiments. Vamvakas et al. (1989b) 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.
Human liver samples have been compared with rat and mouse liver samples with
respect to their 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 l-cWoro-2,4-dirntrobenzene 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 indicates
that toxicologic effects attributable to conjugative metabolites in animals would have little, if
any, relevance to human health hazard. However, because of the limited number 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. Human liver does conjugate other
chloroalkenes, including trichloroethylene, with glutathione (Oesch and Wolf, 1989).
Vamvakas et al. (1989b) 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 jesponse 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.
20
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Bile collected from rat livers perfused with perchloroethylene was found to contain
TCVG and, when tested in the Ames protocol using kidney particulate fractions as the
activating system, was clearly mutagenic. As in the experiment with synthetic TCVG,
inhibition of renal beta-lyase or GOT reduced the mutagenicity of the bile samples
(Vamvakas et al., 1989b).
Green et al. (1990) reported the presence of N-acetyl-S-(l,2,2-trichlorovinyl)-L-
cysteine in the urine of rats dosed with perchloroethylene by gavage and in rats and mice
dosed by inhalation. These investigators have also shown that renal cytosolic beta-lyase from
rats, mice, and humans is 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 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 (Vamvakas et al., 1989a), 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). I
The available data indicate that metabolism is 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 glutathione followed by renal processing of the S-conjugate. This S-conjugate is a beta-
lyase-dependent mutagen in the Salmonella/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 only.
21
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The mutagenicity studies on metabolites of perchloroethylene emphasize the need for
further studies concerning a mutagenic role for them hi perchloroethylene carcinogenesis.
22
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5. MOUSE LIVER TUMORS
5.1. CARCINOGENICITY BIOASSAY DATA AND EPA'S POSITION
In carcinogenicity bioassays, perchloroethylene 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, 1977; NTP,
1986a).
In a study conducted by NCI (NCI, 1977), groups of 50 male mice received time-
weighted average doses of 536 or 1,072 mg/kg of perchloroethylene in corn 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 perchloroethylene in corn 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 perchloroethylene
used in the study was greater than 99 percent pure, but impurities were not identified (NCI,
1977; U.S. EPA, 1985a). The test sample, however, was estimated to contain
epichlorohydrin concentrations of less than 500 ppm (U.S. EPA, 1985a). 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 hi both sexes of mice in both treatment groups when
compared with untreated controls or vehicle controls. The tune to tumor was decreased in
treated mice.
I
Additional studies reported by the NTP confirmed the finding of hepatocellular
carcinoma in B6C3F1 mice exposed to perchloroethylene. Groups of 50 mice of each sex
were exposed to perchloroethylene concentrations of 0, 100, or 200 ppm by inhalation
exposure, 6 hours a day, 5 days per week, for 103 weeks. Perchloroethylene caused dose-
related statistically significant increases hi the incidences of hepatocellular carcinoma in both
sexes.
The biologic significance of chemically induced mouse liver tumors, with respect to
identifying human hazard and using such tumor data in assessing cancer risk to humans, is a
subject of extensive debate. The controversy surrounding the liver tumor response in the
23
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B6C3F1 mouse specifically is well recognized and has been ongoing for some time. Several
meetings and symposia on the subject have been held, and numerous publications deal with
different aspects of the subject (e.g., Popp, 1984; Stevenson et al., 1990). The 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 extensively reviewed 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 tune.
The relevance of mouse liver tumors to the assessment of carcinogenicity hi humans
has been questioned because of:
• The high, and sometimes variable, background incidence of spontaneously
occurring tumors in certain strains of mice, particularly the male B6C3F1 mouse
used in the perchloroethylene studies conducted by NCI and NTP;
• The observation that liver cancer is a relatively uncommon cause of death in the
United States (although not worldwide) (Pickle et al., 1987; Page and Asire,
1985); and
• Some of the hypothesized mechanisms for mouse liver tumorigenesis that many
scientists believe would be unlikely to occur in humans.
On the other hand, many scientists believe that mouse liver tumors are as relevant as
any other tumor type observed in laboratory test animals. This point of view concurs with
the philosophy of using a sensitive model to detect a response in small numbers of test
animals. Also, certain proposed mechanisms, such as oncogene activation, involve steps that
are comparable to those observed hi the development of other tumor types both in animals
and in humans (McConnell, 1990). At least 8 of the fewer than 30 known human
carcinogens cause liver tumors hi mice, and most of these chemicals also cause other types
of tumors hi rodents (IARC, 1987).
Hepatocellular tumors are common end points hi rodent carcinogenicity studies. Of
the chemicals tested hi the NTP's bioassay program, 50 percent of those testing positive
caused increased incidences of liver tumors hi mice. Most, however, also caused other
24
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tumors in mice or rats as well (Maronpot et al., 1987; Haseman et al., 1984b). Only about
5 to 6 percent of the compounds studied in the NTP carcinogenesis bioassays induced only
mouse liver tumors (McConnell, 1990). Of the chemicals evaluated as carcinogens by the
EPA, fewer than 10 percent have been found to cause only mouse liver cancer (Deal, 1990).
At this tune, 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, 1986b): !
An increased incidence of neoplasms that occur with high spontaneous
background incidences (e.g., mouse liver tumors and rat pituitary tumors hi
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). I
i
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 hi 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 hi a single sex (pp. 1-5, 1-6).
Thus, hi 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.
25
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The 1987 EPA paper on the weight-of-evidence classification for perchloroethylene1
was hi 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, hi 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 mouse 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, as is
stated hi 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
for perchloroethylene.
The EPA paper and the most recent letter from the SAB concerning perchloroethylene
(personal communication) 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, hi the liver.
staff paper sent to the SAB as an attachment to the August 3, 1987, letter from EPA's
Administrator, written hi response to the formal comments submitted to the Agency by the
Halogenated Organics Subcommittee regarding the public SAB review of the draft addendum
to the HAD on perchloroethylene.
26
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5.2. PEROXISOME PROLIFERATION AND PERCHLOROETHYLENE
Beginning in 1986, additional information about the possible link between peroxisome
proliferation and liver cancer in B6C3F1 mice exposed to perchloroethylene has been
published (Odum et al., 1988; 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 they relate 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 DNA, caused by elevated concentrations of hydrogen peroxide, a peroxisome
degradation product. Hydrogen peroxide is normally degraded by a peroxisomal catalase,
but the activity of this enzyme does not increase in a parallel fashion with peroxisomes and
other peroxisomal enzymes following perchloroethylene exposure. This enzymic unbalance
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 perchloroethylene 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, 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
27
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in metabolizing perchloroethylene to TCA, which explains the lack of a
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 (because when human liver cells are 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).
• 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 that 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 hi peroxisomal enzyme activity) in hepatocytes of mice and rats both in
vivo and hi vitro after short-term exposure (Elcombe, 1985). Further, TCA has
been shown to be a hepatocellular carcinogen in the B6C3F1 mouse (Herren-
Freundetal., 1987).
Perchloroethylene oxidative metabolism approaches saturation at lower levels in
rats than hi mice. Saturation in rats occurs at atmospheric concentrations in
excess of 100 ppm (Ikeda et al., 1972). Consequently, at high atmospheric
concentrations of perchloroethylene (i.e., > 100 ppm) mice generate relatively
more TCA than do rats (Odum et al., 1988). Following 6 hours of inhalation
exposure to 400 ppm, the cumulative blood concentrations of TCA in mice were
six to seven times greater than concentrations in rats; peak blood levels of TCA
were found to be 13-fold higher in mice than in rats.
28
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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 is consistent with the known species variability in responsiveness to the
hepatocarcinogenic effects of perchloroethylene. In view of the peroxisome-
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), the
EPA is unaware of published studies on the hepatocellular carcinogemcity of TCA
hi rats.
Saturation of human perchloroethylene metabolic processes has been reported to
occur at perchloroethylene concentrations of approximately 100 ppm to 400• ppm
(US EPA, 1985a; Ikeda et al., 1972; Ohtsuki et al, 1983). Odum e 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
to be 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 that 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 (Canot
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 also have provided suggestive evidence that
29
-------
human hepatocytes are relatively unresponsive to hypolipidemic drugs and
ter plasticizers (Elc°mbe and Mitchell, 1986; Butterworto et al
e T? IT"™*' ** Sampl£ Size is lhnited to a few human "v'ers.
tion of the data is subject, therefore, to the same uncertainty as the in
vitro TCA studies on human hepatocytes.
Although evidence exists to support the hypothesis that mouse liver cancer associated
with exposure to perchloroethylene 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
1C A. 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
DeAngelo et al. (1989), however, reported that TCA was more potent as a '
peroxisome proliferator than was DCA in male B6C3F1 mice. Nelson et al
DPA I Sr?p°rd ^ TC/Produced Sreater 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., 1988) 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 percent incidence). DEHP produced no liver
lesions 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
tachJoroethylene, has been studied in the Swiss mouse (Henschler et al 1984)
Tnchloroethylene, which is metabolized to TCA, induces peroxisome proliferation
30
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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 administered by
Henschler et al. were from three to five 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
perchloroethy lene.
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 hi DNA was detected with an
alkaline unwinding assay (Nelson et al., 1989). No increase in peroxisomal
palmitoyl-CoA oxidase activity was detected for periods up to 24 hours 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 hi demonstrating single-strand breaks hi DNA with TCA and DCA
(Chang etal., 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 oncogenes 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 hi 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
31
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different pattern of oncogene activation in chemically induced mouse liver tumors
compared with 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; Oshimura et al., 1988).
In summary, some evidence supports the hypothesis that perchloroethylene-induced
hepatic carcinogenesis may be related to peroxisome proliferation, but 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-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.
32
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6. KIDNEY TUMORS IN MALE RATS
The inhalational administration of perchloroethylene to male and female F344/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 proliferate lesions of the renal tubular cells (tubular cell hyperplasia, adenoma,
and adenocarcinoma) in male rats (NTP, 1986a). 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 F344/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 incidence 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: ,
^
. percem]). Likewise, the overall historic* control mcidence of renal
tubule tumors in male F344/N vehicle controls in gavage studies is 1/1,943 (0. 05
nercent) The incidence is even lower in female controls. This is supported by
s«ou?«ml lule tumor incidence rates recorded for other rat strams £.«..
Osbcme-Mendel, males 0.3 percent; females 0 percent; Goodman et al., 1980).
Tne ^oearance of tubule neoplasms in 8% of perchloroethylene-dosed animals
JowloSTd h$,lse groups combined) is convincing evidence of a treatment-
related effect. ,
• No malignant renal tubule neoplasms have been observed in any control rats
elaSnefby me NTP. This includes the chamber controls from the performing
Satory an?me untreated controls and the vehicle controls from gavage studies.
33
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Two of the tumors observed in high-dose animals in the NTP study were
carcinoma^ The probability of two rare carcinomas appearing by^chTnS in a
group of 50 animals has been calculated to be less than 0.00 "(US Epl 1987)
In addition to the NTP study findings of renal tubule tumors in male F344/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 rats dosed
with other chlorinated ethanes and ethylenes (NTP, 1983, 1988; and unpublished results cited
in NTP, 1986b). There is some evidence that nontumor pathology is not unique to
perchloroethylene; 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 perchloroethylene
produces nephrotoxicity in both sexes of mice and rats and an increased incidence of
proliferate 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 evidence exists to suggest that renal effects
induced in male rats by chemicals causing alpha-2u-globulin accumulation are unlikely to
occur in any species not producing alpha-2u-g,obulin or a protein with a structurally similar
binding domain, in the large quantities typically seen in the male rat. Thus, if a chemical
34
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induces alpha-2u-globulin accumulation in hyaline droplets and a carcinogenic response in the
male rat kidney, the tumor response may not constimte evidence of a carcinogenic hazard to
humans.
The EPA is presently developing criteria that will define a weight-of-ev,dence
approach for evaluating, on a case-by-casc basis, the role of a,pha-2u-g.obulin in rat kidney
tumor formation (U.S. EPA, 1991). A report (U.S. EPA, 199!) currently being developed
by a technical panel of EPA's Risk Assessment Forum provides guidance on determnung
when it is reasonable to presume that a renal tumor in male rats results from alpha-2u-
globulin accumulation and on selecting appropriate procedures to use in extrapohmon 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-2u-globulin. Data on renal
tumors in fl>e male ra, will fall into several categories depending on whether the tumors are
attributable solely to alpha-2u-globuUn accumulation, whether anofher mechanism apphes,
whether several mechanisms are feasible, one of which involves alpha-2u-globuhn, or
whether the available information is inadequate to determine the role of alpha-2u-globulm.
For instance, if the perchloroethylene alpha-Zu-globuUn data are subsequently judged to be
me only definitive explanation for the occurrence of male rat kidney tumors, mis tumor end
point may not have relevance for human health hazard assessment. TOs can be further
evaluated as the EPA's criteria for identifying chemicals inducing alpha-2u-globuhn
accumulation become available to apply to the perchloroethylene-specific data.
6 1 ALPHA-2U-GLOBULIN IN RENAL CARCINOGENESIS 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 nephropamy (NTP, 1983, 1986b, 1987, 1990; Alden et al., 1985;
MacNaughton and Uddin, 1984; Alden et a,., 1984; PhiUips ct al., 1987). The accumulate
of the protein alpha-2u-globu.in is believed to be the reason for an excessive number of
hyaline droplets (Stonard et a!., 1986; Olson et al., 1987). A normal urinary protein tn the
j male rat, alpha-2u-globulin is synthesized in me liver under hormonal control, bu, „ has no,
35 :
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been detected in the liver of fema,e rats or in other species, including humans. Among the
chemicals tested so far in chronic animal bioassays, those tha, invoked mis specific type of
protem droplet nephropathv in male rats also produced renal tubule tumors in male rats but
dtd 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 epitheHal cells of
proximal tubules (UAREP, 1983; Alden e. al., 1984; Haider et a., 1984; Swenberg et al
1989):
• Excessive accumulation of hyaline droplets in proximal tubules
'^ '° ^ Cdl "'«''• ">'
reDresentins,
of tubules within the renal medulla. Y'
• The chronic progressive nephropathy characteristically found in aging rats is
exacerbated as a consequence of the induced nephrotoxicity. g
• Renal tubule hyperplasia and neoplasia develop subsequently.
A number of investigators hypothesize that the increased proliferate 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 me
acute and chronic renal effects induced in male rats by these chemicals will be unlikely to
occur in any species not producing alpha-2u-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; 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 alpha-2u-globulin
accumulation and tumorigenesis in male rats must be established on a chemical-by-chemical
36
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basis before it is reasonable to discount the significance of the tumor induced by a particular
chemical. Guidance for evaluating data concerning alpha-2u-globulin and kidney tumors m
male rats is being developed by
-------
Alpha-2u-Globulin Response for Perchloroethylene Is R(
Although the a,pha-2u-globulin response occurs in male rats exposed to
per^oroethylene, it has been observed following only Ugh dose, ^ ^
tester inhaled doses of perch.oroetby.ene (up ,„ 400 ppm 6 hours per day or
wun arnmaU sacnficed within 18 hours of termination of the fina, exposure) in rats but thTre
was no evince of hyaline dropiet formation although .here may have been tin, for
recovery efore sacrifice. ,t is noteworthy that the 400 ppm concentration was the same
, but fte age of the rats as weH as the ,ength of time Oaat ed
between fina, exposure and sacrifice may expiain some of the differences. Mineraiization in
tne mner meduHa and papiiia of ^e Hdney, a characteristic trait of a-pha-^obuHn
nephropathy, was not seen, however (NTP, 1986a).
It is possible to longer term exposure to the 400 ppm concentration of
r* for "" hyaltoe ^ accumulation to «- ^ of -
, 1990). Alpha-2u-globu,in accumu,a«iOn can be demons.a.ed, however, after
only short-term exposures (even a single administration) to severa, agents such as d-
TssHlTt' ^^ gaS°ltae' "* "«*»*-»- (Charbonneau et a,., 1987; NTP
1988). lack of hyaline drop,et formation or increase in alpha-2u-globulin or signs of the
charactenstic rena, nephropamy at the high-dose level of the NTP inhalation study may
-ndtcate a threshold effect and thus diminish the like,ihood tha, the rena, tumors associated
w th exposure to perchloroethylene are induced through this mechanism (Green e, a, ,990)
Phannaco^eUc 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 percMoroethylene for up to 2 year, These doses "
38
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associated with the production of renal tubule neoplasms in male rats. The fact that the NTP
did not report the presence of hyaline droplets in either the 14-day, 90-day, or 2-year studies
is not definitive, however, because the NTP protocol at that time was not designed to detect
hyaline droplets or alpha-2u-globulin accumulation in the kidney (NTP, 1990). Thus, the
procedures followed at the time of the study were not necessarily conducive to detecting
hyaline droplets. For example, in the chronic study of perchloroethylene, at least 1 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-2u-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-2u-globulin accumulation
(NTP, 1986a; letter from Scot Eustis to William Farland, 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-2u-globulin accumulation (e.g., for
pentachloroethane, the incidence of renal papillar mineralization is 8 percent in controls; 59
percent, low dose; 58 percent, high dose). In addition, some aspects of toxic tubular
nephropathy were also observed in female rats and male mice exposed to perchloroethylene.
Perchloroethylene induces alpha-2u-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 the mechanism 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 data supporting other mechanisms, including possible genotoxicity
discussed below, makes it difficult to conclude that perchloroethylene-induced renal tumors
can be attributed solely to this hypothesized species- or sex-specific mechanism.
39
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Chronically Induced Perchloroethylene Nonneoplastic Kidney Lesions Exhibit Neither
Species Nor Sex Specificity.
In contrast to most other chemicals inducing alpha-2u-globulin accumulation that have
been tested by the NTP in chronic carcinogenicity bioassays, renal lesions occurring in
animals exposed to perchloroethylene were not limited 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; 1 of 50 female rats exposed at the high
dose developed tubule 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 (NTP, 1989). On the other hand,
trichloroethylene, which was also tested by the 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-2u-globulin (Goldsworthy et al., 1988). Consequently, kidney tumors induced
by this compound are not considered to be associated with alpha-2u-globulin accumulation.
Perchloroethylene is structurally closely related to trichloroethylene, and both chemicals have
been shown to be metabolized in the kidney to mutagenic compounds.
40
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6.2. SUSTAINED CHRONIC NEPHROTOXICITY AS A POSSIBLE MECHANISM
INDEPENDENT OF ALPHA-2u-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 chemicals and
may include tubular cell cytomegaly, karyomegaly and pleomorphism, tubular cell dilation,
or the formation of granular casts. Certain 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-2u-globulin 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-2u-globulin accumulation. If this is the case, renal tubule neoplasia in these
experiments would not be expected to be a species- or 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. Because both trichloroethylene and perchloroethylene produce renal tubule tumors,
but no enhanced cell replication was seen with trichloroethylene, it is difficult to conclude
41
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that perchloroethylene induces the renal tumors solely by a nephrotoxic mechanism apart
from nephropathy associated with alpha-2u-globulin 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.
6.3. 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, 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 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 3 and 4
on metabolism and mutagenicity; also see Dekant et al., 1989; Vamvakas et al., 1989b).
Perchloroethylene is conjugated with hepatic glutathione to form S-(l,2,2-trichlorovinyl)
glutathione. 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 on by gamma glutamyl transferee and
dipeptidase to form its corresponding cysteine S-conjugate, S-(l,2,2-trichlorovinyl)-L-
cysteine (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 that 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 analog of perchloroethylene (Anders et al.,
42
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1988; Dekant et al., 1989). Trichloroethylene also induces renal tumors in male rats but is
not a'chemical that induces alpha-2u-globulin accumulation (NTP, 1988; 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 hi rodent tissue.
The roles of this metabolic pathway in producing 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 (Green et al., 1990). 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 require close scrutiny before it can be concluded that
conjugation is unimportant in perchloroethylene metabolism by humans, particularly since
products include potential mutagens. Moreover, human liver is known to conjugate other
43
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chloroalkenes, related closely to perchloroethylene, with gutathione (see sections on
metabolism and mutagenicity). For example, N-acetyl-S.(dichlorovmyl)-L-cysteine has been
measured in urine of humans exposed to trichloroethylene in the occupational setting.
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 not 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
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-2u-globulin. Because only male rats product £±SSn
*e tum0rs would have little or no predictive validity with respect to 1C health
hazard on a site- or mechanism-specific basis. Perchloroethylene indu™dnev
tumors at lower doses than those required to cause alpto-^Sto *
acamnilation, however. The EPA is developing criteria thatwill define a weight-
of-ev dence approach for evaluating, on a case-by-case basis, the role of alia 2u-
globulrn 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 susfcdned cyS«Sy
and cellular regeneration. Although certain "nephrotoxicity" occurs HoSLs
of rats and mice, implying that kidney tumors would occur in rats and mfce of
SSSTiS CarT°gtCity bi°aSSayS' Cdl rePlication occurs * nTbut not
in female rats suggesting that any nephrotbxic mechanism would likely be
mSech^mW1f alp?r2ut°bUlin accu^tion. On the other hand me
mechanism of perchloroethylene tumorigenesis may be similar to that of its
structural analog, trichloroethylene. Trichloroethylene induces kidney
in
44
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3
> .
m
pathway is irrelevant with respect to human risk projection.
Although 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.
45
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7. MONONUCLEAR CELL LEUKEMIA IN RATS
The NTP (1986a) reported that the chronic inhalational administration of
perchloroethylene to male and female F344/N rats caused positive trends in the incidence of
mononuclear cell leukemia (MCL) in both sexes. Pairwise comparisons of tumor incidences
m 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 m the concurrent chamber control groups were high relative to historical chamber
control groups at the performing laboratory (males 28/50, 56 percent versus 117/250 47
percent; females 18/50, 36 percent versus 73/249, 29 percent). The concurrent control
group rates were also higher than the NTP historical rate for untreated control groups (males
583/1,977, 29 percent and females 375/2,021, 18 percent).
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:
' PercMor0eth^e Produ-d a dose-related
increase in
S°ratf ^^ eXP°SUre Significantly shorten^ the time to onset of MCL in
Although there was no remarkable effect of perchloroethylene exposure on
survival of female rats, there was an increased incidence of Tdvanced MCL in
female rats that died before the scheduled termination of the smdy Thus a more
appropriate statistical analysis was conducted in which only the ScidenTes' of
advanced MCL m rats were considered. Significantly posftive trends and
46
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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 because they did not represent generally accepted approaches to evaluating increased
i
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, 1986a). The special analyses were conducted to support, rather than establish, the
interpretation.
Under the conditions of the NTP study a carcinogenic effect of percfaloroethylene in
male and female rats was evidenced by significant increases in the incidences of MCL in
both sexes. However, the usefulness of increased incidences of MCL in predicting 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 F344/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 percent. In female chamber control rats, the incidences ranged from
22 to 36 percent. A similarly high variability has been observed among the NTP untreated
control groups (males, 10 to 60 percent; females, 6 to 38 percent).
Concurrent controls represent the most appropriate groups to use for determining the
statistical significance of observed differences between experimental groups. It is
recognized, however, that in the case of spontaneous and highly variable tumors,
comparisons of treatment groups to historical control groups may be helpful in interpreting
experimental results (Haseman et al., 1984a). When the overall rates of MCL hi male rats
hi the perchloroethylene studies are compared with the range of tumor incidences hi
historical controls, the perchloroethylene-treated animals were essentially identical to those in
47
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the historical control group with the highest incidence (74 percent in perchloroethylene-dosed
animals versus 68 percent in the historical control group). However, the incidences of MCL
in perchloroethylene-dosed female rats (60 percent and 58 percent) were elevated relative to
the highest incidence in historical control groups (38 percent).
The Pathobiology 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 characterized by
infiltration of pleomorphic blastlike mononuclear cells in numerous organs. The disease per
se, which is splenic hi origin but later infiltrates the liver, lung, bone marrow, lymph nodes,
and other organs, is readily and unequivocally diagnosed by standard histopathological
techniques. MCL has also been described as large, granular, lymphocytic leukemia and is
known to be a rapidly progressing and fatal neoplasm whose incidence is age related. The
tumor is transplantable; 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 early reports of toxicity of cysteine S-conjugates where S-(l,2,-
dichlorovinyl)-L-cysteine was implicated in induction of aplastic anemia and marked
biochemical alteration of DNA hi bone marrow, lymph nodes, and thymus in calves
(McKinney et al., 1957; Schultze et al., 1959; Bhattacharya and Schultze, 1971, 1972). As
discussed earlier, the glutathione 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 beta-lyase pathway. Thus, the
possibility exists that the perchloroethylene S-conjugate, S-(l,2,2-trichlorovinyl)-L-cysteine,
may be involved hi inducing leukemia hi rats and may have the potential to produce blood
dyscrasias hi 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 hi bone marrow, MCL is splenic in origin.
48
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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 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.
49
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8. SUMMARY AND CONCLUSIONS
The Office of Health and Environmental Assessment has reviewed the data 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 "sufficient" animal data lead to an overall weight-of-evidence
classification of B2, probable human carcinogen, holds up in the case of perchloroethylene.
Is There Sufficient Evidence of Carcinogenicity 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 (1) in
multiple species or strains; (2) in 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.
Perchloroethylene has been shown to cause multiple tumor end points-hepatocellular
carcinomas m both sexes of mice, kidney tumors and some indication of gliomas in male
H th v and female rats' The incidences of liver tumors in mice
and the leukemias in rats are statistically significantly elevated when compared with
controls. The incidence of renal tumors and gliomas in male rats is significantly elevated
when compared with historical controls, and these tumors are biologically sigScant
has been demo^ted byU inhalation and
50
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The perchloroethylene data meet, at least to some degree each of the three
======
OuideL criteria for 'sufficient- animal evidence because there are mufcp.e
having different exposure routes and no appreciable d°™^°f ^
would * ative of potent, human hazard and wou.d .ead ;
Ividence dassificaUon of B2, prooaWe human carcinogen, When cons.dered w* the
increased incidences of bom hepato^llular adenomas a affirmative votes, one
hepatocellular carcinomas in females. The p anel wr o ^ s carcinogenicity of
negative vote) conclusions of some evince inl ^^^.^ MU leukemia. The
pe?chloroethylene, as shown by mcr^™*n^o™; tot there was clear evidence of
panel concluded (five affirmative votes, four ne|M ve vo ^ ^ ^^^
carcinogenicity of P-te"*^^.^! mbular cell neoplasms. (Two
o
voting due to employment conflict of interest,
*There is agreement between the It-temationa, Agency for
EPA that flie animal '"
more similar to EPA's sufficient
"Possible" human carcinogen,
51
-------
"inadequate" epidemiologic data (U S EPA tos* D
be answered is whether fte genera, ~ ^ ">*
reasonable
Implications of Perchloroethylene Metabolism
I. is generally considered tot the toxicity, mutagenicjtyi and
e reside in reac,ive mua^ The ^ do „ d
^ as; mutagen' although — '-*-*• —
c (e.g., TCVC, ohloroacetaldehydes including cUoral hydrate) Severa,
Perchloroethylene metabolites have been shown to be cytotoxic . „
TCA DPA anH , • t, cytotoxic, and certain metabolites (e g
1CA, DCA, and trichloroacetaldehyde) cause liver tumors in mice
52
-------
— '
e oon
53
-------
Mouse Liver Tumors
case basis according ,o uc
tun™ data is
(U.S. EPA,
liver
boiwsed for
—•
are
°" a
.ouse
publislled in
54
-------
S although evidence exists that supports the hypothesis of mouse iiver cancer
second to peroxisome proliferation, the vaUdity ot the nypotnests remams
*. . , - .—— - — « :^ rrr a
peroxisome proliferation and also causes hepatocellular carcmoma » B6OF1 nuce bu
clL-eL relationship between the two effects has not been shown. It „ not dear
T, , if any peroxisome proliferation actually plays in perchloroethylcne
role the peroxisome proliferation phenomenon may have in tumongenes,
1, severa, other proposed mechanisms need to be further mvest.ga.d. For
the recent demons^atiou that the major metabolite of perchioroethylene TCA,
consideration as well.
was identified in a male mouse. !n .e case of percMoroe.y.ene, U .
55
-------
rrricrr.r? •
-A «.. tat cenam nonto.or pathology associated with the chronic administn,^ of
n
genera, .
o
of humans ,„ conjugate perchtoroethyiene. Ms i
56
is because
-------
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.
Although some evidence supports each of the proposed mechanisms, there also are
significant quantitative and qualitative gaps hi the supportive data. The mode of
perchloroethylene tumorigenesis hi male rats is not yet understood.
Leukemia in Rats
Under the conditions of the NTP study, a carcinogenic effect of perchloroethylene hi
male and female rats was evidenced by significant increases hi the incidences of MCL in
both sexes. However, the usefulness of increased incidences of MCL hi predicting 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 hi 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 hi severity of
MCL and shortened the tune 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.
Supplemental analyses were based on standard methods of data evaluation and supported 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 hi rats. The human does not have a Zymbal gland.
57
-------
Conclusion
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 observed
in rats and mice. Data support certain of these mechanisms. Scientific evidence also
supports 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 hi 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 rnutagenic 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 understood well
enough, 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 exists concerning relevance to humans.
The EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 1986b) 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 with
control groups, when there is decreased tune to tumor occurrence or death with tumor, and
58
-------
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
inhalation and oral exposure, and are called "sufficient" animal evidence. 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
can most logically be categorized as a Group B2 probable human carcinogen, although the
EPA's SAB does not fully agree. 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 directly the quantitative estimation of risk.
Mechanistic considerations may justify special interpretation of the dose-response data with
respect to projecting human carcinogenesis risk. j
The Agency's quantitative estimates of carcinogenic risk from exposure to
perchloroethylene have not been recently updated. Existing estimates are relatively simplistic
and are calculated using a nonthreshold model that is linear at low doses. The risk estimates
made using such a model are regarded as conservative and represent a plausible upper limit
for the risk such that the true risk is not likely to be higher than the estimate, but it is likely
to be lower and could even be zero. Relative to other potential carcinogens evaluated by the
Agency, perchloroethylene carcinogenic potency index ranks in the lowest quartile, so
perchloroethylene is not viewed as a very potent carcinogen.
Because it is generally accepted that the carcinogenic potential of perchloroethylene
resides in its metabolites, the amount of the compound metabolized was considered as being
directly proportional to the dose to the target tissue and was thus factored into the calculation
of the risk estimates as the dose rather than using the administered dose. Since amount of
perchloroethylene metabolized was used as the dose and since metabolism is nonlinear, it is
59
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important to realize that the metabolism curve should be factored in when back calculating
from the unit risk estimate to the risk to humans at high exposures.
The upper-bound estimate of the incremental unit risk for inhalation of 1 /ig/m3 of
perchloroethylene hi air is 4.8 X 10'7, and the upper-bound estimate of the incremental
cancer risk due to 1 /zg/L of perchloroethylene in drinking water is 1.5 x 10"6 (U.S. EPA,
1985a), based on hepatocellular carcinomas observed in oral studies in mice. Calculations
based on inhalation studies hi which hepatocellular carcinomas were detected in the same
mouse strain resulted hi similar risk estimates (U.S. EPA, 1986a). It is of interest to note
that the air unit risk estimates calculated on the basis of leukemias in rats exposed to
perchloroethylene via inhalation are also comparable to the estimates derived from the mouse
studies. Thus, risk estimates calculated on the basis of six different inhalation data sets are
comparable, ranging from 2.9 X 10 "7 to 9.5 X 10'7. In addition, all of these values are
comparable to the unit risk estimate of 4.8 X 10'7, derived from the oral gavage study in
mice, a risk estimate that falls approximately hi the middle of the range of estimates.
An ED10 value (effective dose in 10 percent) has been calculated for
perchloroethylene based on the the geometric mean of tumor incidences from the six
inhalation data sets. The ED10 is the dose level expected to cause cancer in 10 percent of
the population exposed. The ED10 for oral exposure is 83 mg/kg/day. The ED10 for
inhalation exposure is 2.9E-5 jug/m3. The ED10 can be divided by the environmental level,
or the exposure level of concern, to give a margin of exposure.
The Agency's Science Advisory Board reviewed a draft of this report and deemed it
to be well written and of high scientific quality. In an August 1991 letter to the EPA
Administrator, however, the Board maintained as still being appropriate its previous advice
regarding the weight-of-evidence classification for perchloroethylene—that it lies on a
continuum between categories B2 and C. This differs somewhat from the EPA position of a
B2 classification for perchloroethylene. The Board also adhered to its earlier opinion that,
from a scientific perspective, exposure should be considered more important than
classification category hi determining potential threat to human health and whether or not a
chemical substance should be regulated.
60
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