United States
Environmental Protection
1=1 m m Agency
EPA/690/R-07/027F
Final
9-28-2007
Provisional Peer Reviewed Toxicity Values for
Pentachloroethane
(CASRN 76-01-7)
Superfund Health Risk Technical Support Center
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
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Acronyms and Abbreviations
AIC Akaike Information Criterion
BMD benchmark dose
BMDL 95% lower bound on the benchmark dose
cc cubic centimeters
CD Caesarean Delivered
CERCLA Comprehensive Environmental Response, Compensation and Liability Act
of 1980
CNS central nervous system
cu.m cubic meter
DWEL Drinking Water Equivalent Level
FEL frank-effect level
FIFRA Federal Insecticide, Fungicide, and Rodenticide Act
g grams
GI gastrointestinal
HEC human equivalent concentration
Hgb hemoglobin
i.m. intramuscular
i.p. intraperitoneal
IRIS Integrated Risk Information System
IUR inhalation unit risk
i.v. intravenous
kg kilogram
L liter
LEL lowest-effect level
LOAEL lowest-observed-adverse-effect level
LOAEL(ADJ) LOAEL adjusted to continuous exposure duration
LOAEL(HEC) LOAEL adjusted for dosimetric differences across species to a human
m meter
MCL maximum contaminant level
MCLG maximum contaminant level goal
MF modifying factor
mg milligram
mg/kg milligrams per kilogram
mg/L milligrams per liter
MRL minimal risk level
MTD maximum tolerated dose
MTL median threshold limit
NAAQS National Ambient Air Quality Standards
NOAEL no-ob served-adverse-effect level
NOAEL(ADJ) NOAEL adjusted to continuous exposure duration
NOAEL(HEC) NOAEL adjusted for dosimetric differences across species to a human
NOEL no-ob served-effect level
OSF oral slope factor
p-IUR provisional inhalation unit risk
p-OSF provisional oral slope factor
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p-RfC
provisional inhalation reference concentration
p-RfD
provisional oral reference dose
PBPK
physiologically based pharmacokinetic
POD
point of departure
ppb
parts per billion
ppm
parts per million
PPRTV
Provisional Peer Reviewed Toxicity Value
RBC
red blood cell(s)
RCRA
Resource Conservation and Recovery Act
RDDR
Regional deposited dose ratio (for the indicated lung region)
REL
relative exposure level
RfC
inhalation reference concentration
RfD
oral reference dose
RGDR
Regional gas dose ratio (for the indicated lung region)
s.c.
subcutaneous
SCE
sister chromatid exchange
SDWA
Safe Drinking Water Act
sq.cm.
square centimeters
TSCA
Toxic Substances Control Act
UF
uncertainty factor
Hg
microgram
|j,mol
micromoles
voc
volatile organic compound
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PROVISIONAL PEER REVIEWED TOXICITY VALUES FOR
PENTACHLOROETHANE (CASRN 76-01-7)
Background
On December 5, 2003, the U.S. Environmental Protection Agency's (EPA's) Office of
Superfund Remediation and Technology Innovation (OSRTI) revised its hierarchy of human
health toxicity values for Superfund risk assessments, establishing the following three tiers as the
new hierarchy:
1. EPA's Integrated Risk Information System (IRIS).
2. Provisional Peer-Reviewed Toxicity Values (PPRTV) used in EPA's Superfund
Program.
3. Other (peer-reviewed) toxicity values, including:
~ Minimal Risk Levels produced by the Agency for Toxic Substances and Disease
Registry (ATSDR),
~ California Environmental Protection Agency (CalEPA) values, and
~ EPA Health Effects Assessment Summary Table (HEAST) values.
A PPRTV is defined as a toxicity value derived for use in the Superfund Program when
such a value is not available in EPA's Integrated Risk Information System (IRIS). PPRTVs are
developed according to a Standard Operating Procedure (SOP) and are derived after a review of
the relevant scientific literature using the same methods, sources of data, and Agency guidance
for value derivation generally used by the EPA IRIS Program. All provisional toxicity values
receive internal review by two EPA scientists and external peer review by three independently
selected scientific experts. PPRTVs differ from IRIS values in that PPRTVs do not receive the
multi-program consensus review provided for IRIS values. This is because IRIS values are
generally intended to be used in all EPA programs, while PPRTVs are developed specifically for
the Superfund Program.
Because new information becomes available and scientific methods improve over time,
PPRTVs are reviewed on a five-year basis and updated into the active database. Once an IRIS
value for a specific chemical becomes available for Agency review, the analogous PPRTV for
that same chemical is retired. It should also be noted that some PPRTV manuscripts conclude
that a PPRTV cannot be derived based on inadequate data.
Disclaimers
Users of this document should first check to see if any IRIS values exist for the chemical
of concern before proceeding to use a PPRTV. If no IRIS value is available, staff in the regional
Superfund and RCRA program offices are advised to carefully review the information provided
in this document to ensure that the PPRTVs used are appropriate for the types of exposures and
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circumstances at the Superfund site or RCRA facility in question. PPRTVs are periodically
updated; therefore, users should ensure that the values contained in the PPRTV are current at the
time of use.
It is important to remember that a provisional value alone tells very little about the
adverse effects of a chemical or the quality of evidence on which the value is based. Therefore,
users are strongly encouraged to read the entire PPRTV manuscript and understand the strengths
and limitations of the derived provisional values. PPRTVs are developed by the EPA Office of
Research and Development's National Center for Environmental Assessment, Superfund Health
Risk Technical Support Center for OSRTI. Other EPA programs or external parties who may
choose of their own initiative to use these PPRTVs are advised that Superfund resources will not
generally be used to respond to challenges of PPRTVs used in a context outside of the Superfund
Program.
Questions Regarding PPRTVs
Questions regarding the contents of the PPRTVs and their appropriate use (e.g., on
chemicals not covered, or whether chemicals have pending IRIS toxicity values) may be directed
to the EPA Office of Research and Development's National Center for Environmental
Assessment, Superfund Health Risk Technical Support Center (513-569-7300), or OSRTI.
INTRODUCTION
No assessment for pentachloroethane is available on IRIS (U.S. EPA, 2007a), the
HEAST (U.S. EPA, 1997), or the Drinking Water Standards and Health Advisories list (U.S.
EPA, 2004). No relevant documents were located in the Chemical Assessment and Related
Activities (CARA) list (U.S. EPA, 1991b, 1994). AT SDR (2007), and WHO (2007) have not
produced documents regarding pentachloroethane. IARC (1999a) concluded that
pentachloroethane is not classifiable as to its carcinogenicity in humans (Group 3).
Comprehensive literature searches were conducted in 2006 of the following databases:
TOXLINE (1965-2006), CANCERLINE (1970-2006), MEDLINE (1966-2006), GENETOX,
DART, CCRIS, CHEMID, RTECS, EMIC, ETICBACK and TSCATS for toxicity studies of
pentachl oroethane.
REVIEW OF PERTINENT LITERATURE
Human Studies
There are no epidemiologic data relevant to the carcinogenicity of pentachloroethane in
humans (IARC, 1999a).
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Animal Studies
In conjunction with a carcinogenesis bioassay, the National Toxicology Program (NTP,
1983) performed short term (14-day) and subchronic (13-weeks) toxicity studies for
pentachloroethane in F344/N rats and B6C3F1 mice. In the 14-day study, groups of 5 F344/N
rats/sex/dose and 5 B6C3F1 mice/sex/dose were administered by gavage 10, 50, 100, 500 or
1000 mg/kg pentachloroethane in corn oil for 14 days. Vehicle controls received only corn oil.
All animals at the 1000 mg/kg-day dose died. Sixty percent of the animals at the 500 mg/kg-day
dose died. The only clinical sign observed was lethargy among the rats at the two highest doses.
No gross or microscopic lesions were found in the animals that died. Body weights of the
animals that survived at the 500 mg/kg-day dose were slightly reduced compared to controls
(10% and 15% in the males and females, respectively). No compound-related gross or
microscopic (examined liver, lungs and spleen) changes were noted in any of the treated animals.
The dose level of 500 mg/kg-day is identified as an FEL for mortality. Given the lack of
histopathological effects and the minimal number of animals, a NOAEL or LOAEL cannot be
determined with any confidence.
In the NTP (1983) subchronic study, groups of 10 F344/N rats/sex/dose were
administered by gavage 5, 10, 50, 125 or 250 mg/kg pentachloroethane in corn oil for 13 weeks
(5 times/week). Groups of 10 B6C3F1 mice/sex/dose received 5, 10, 50, 100 or 500 mg/kg by
the same protocol. Vehicle controls received only corn oil. The composition of the material was
89.5%) pentachloroethane, 10.4%> hexachloroethane and 0.55%> tetrachloroethylene. All rats
survived the 13 week exposure period, and no compound-related gross or histopathologic effects
were observed. All male mice survived to the end of the 13 week study period, and their body
weight gains were comparable to controls. One female mouse in the 500 mg/kg dose group died.
No compound-related gross or histopathologic effects were observed in either rats or mice. Final
body weights were depressed by 5%> for male and 9%> for female rats, compared to control
groups. No statistical analysis was reported by NTP (1983). An analysis of the mean body
weights of rats administered pentachloroethane (presented in Table 4 in the NTP, 1983 report)
shows reductions in body weight gain of 10%> for male rats and 17%> for female rats at the 250
mg/kg-day dose level compared to controls. No statistical analyses can be performed on these
data, as neither the individual animal data nor standard errors are presented. The final body
weight of high dose females was reduced 8%> compared with controls. An analysis of mean body
weights of mice administered pentachloroethane (presented in Table 8 in the NTP, 1983 report)
shows a 29% reduction in body weight gain for female mice at 500 mg/kg-day compared to
controls. A 14%> reduction in body weight gain was calculated for female mice at both 5 and 100
mg/kg-day dose groups, with the two intermediate dose groups (10 and 50 mg/kg-day) showing
no effects on body weight gain (i.e., 100%> of controls). Thus, the significance of the 19%>
reduction at 5 mg/kg-day is questionable. Based on the body weight gain data, a LOAEL of 250
mg/kg-day (LOAELadj =180 mg/kg-day after adjusting for gavage schedule) is suggested for
rats and 500 mg/kg-day (LOAELadj = 357 mg/kg-day) for mice. The respective NOAELs are
125 mg/kg-day (NOAELadj = 89 mg/kg-day) for rats and 100 mg/kg-day (NOAELadj = 71
mg/kg-day) for mice.
The pentachloroethane carcinogenesis/general toxicity bioassay part of the NTP (1983)
study was also performed in F344/N rats and B6C3F1 mice. This was the only available study
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that evaluated the chronic (noncancer) toxicity and carcinogenicity of pentachloroethane in
animals. Male and female F344/N rats (50/sex/dose) were administered doses of 75 or 150
mg/kg pentachloroethane for 103 weeks (5 times/week) by gavage. The composition of the
material was 95.5% pentachloroethane, 4.2% hexachloroethane and 0.125% trichloroethylene,
which differed slightly from the material used in the shorter-term studies (10.4%
hexachloroethane and 0.55% tetrachloroethylene impurities). Male rats maintained normal body
weights through the first 76 weeks of the study, after which body weights were slightly less than
control animals. Female rats maintained normal body weights through the first 42 weeks of the
study, after which body weights were decreased compared to control animals. Final mean body
weights were decreased 4-5% for treated males compared to controls, and 8-12% for treated
females compared to controls. No dose-response relationship was observed for decreased body
weight gain, although there was a trend for reduced body weight for all treated animals
beginning at week 40 for females and week 70 for males (Figure 1; NTP, 1983). Survival among
male rats at the end of the study was 82% for controls, 66% for the low dose group, and 52% for
the high dose group. Early mortality was evident for all treated males compared to controls. A
sharp downward trend in survival was apparent starting at week 15, with survival down to 88%
by week 18 for both treatment groups (Figure 2; NTP, 1983). Male control animals did not
experience reduced survival until after week 80. Survival among female rats at the end of the
study was 76% for controls, 72% for the low dose group, and 50% for the high dose group. A
reduced survival trend was evident for high-dose females, only. In regards to nonneoplastic
lesions, chronic inflammation of the kidney (nephropathy) and interstitial inflammation of the
lung were observed in male rats with a positive dose-response relationship (Table 1).
TABLE 1. Incidences of Chronic Noncancer Effects in Male F344/N Rats Exposed
for 103 Weeks to Pentachloroethane
Endpoint
Control
75 mg/kg (low dose)
150 mg/kg (high dose)
Nephropathy
4/50 (8%)
14/49 (29%)
33/50 (66%)
Renal papilla
mineralization
4/50 (8%)
29/49 (59%)
29/50 (58%)
Lung interstitial
inflammation
5/50 (10%)
10/49 (20%)
15/50 (30%)
Acute/chronic lung
inflammation
27/50 (54%)
31/49 (63%)
19/50 (38%)
Nephropathy in the male rat was characterized by interstitial fibrosis, interstitial
accumulation of mononuclear inflammatory cells, and severe tubular dilation in the inner cortex
with some evidence of giant cells and casts. This toxic endpoint is distinct from "aging"
nephropathy where interstitial fibrosis and tubular dilation are not as severe; giant cells within
tubules are not characteristic of aging nephropathy. Glomerular hyalinization was also observed.
Treated rats displayed increased incidences of mineralization of the renal papilla. In the lung,
male rats displayed interstitial inflammation. However, an association between lung
inflammation and pentachloroethane exposure could not be conclusively established because of
the higher incidence of acute/chronic lung inflammation in the control group compared to the
high dose group. Female rats did not exhibit any exposure-related nonneoplastic lesions.
However, because of the decreased survival of males in both treatment groups, an FEL of 54
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mg/kg-day (75 mg/kg-day dose after adjustment for dosing schedule) is established in this study
(NTP, 1983).
Table 2 summarizes the noncancer effects observed in the NTP (1983) report.
TABLE 2. Table of Noncancer Effects (NTP, 1983)
Species
Duration
Dose/Exposure
Endpoint
NOAEL
(mg/kg-day)a
LOAEL
(mg/kg-day)a
Male F344/N rats
13 weeks
5, 10, 50, 125 or
250 mg/kg
Decreased body weight
gain
89
180
Female F344/N rats
13 weeks
5, 10, 50, 125 or
250 mg/kg
Decreased body weight
gain
89
180
Male B6C3F1 mice
13 weeks
5, 10, 50, 100 or
500 mg/kg
No effects observed
500
None
Female B6C3F1
mice
13 weeks
5, 10, 50, 100 or
500 mg/kg
One mouse in 500
mg/kg group died, body
weight decreased
71
357
Male F344/N rats
103 weeks
75 or 150 mg/kg
Nephropathy, reduced
survival
None
54b
Female F344/N rats
103 weeks
75 or 150 mg/kg
No effects observed
110
None
Male B6C3F1 mice
103 weeks
250 or 500
mg/kg
Decreased body weight
gain, high mortality
both dose groups
None
180
(FEL)
Female B6C3F1
mice
103 weeks
250 or 500
mg/kg
Decreased body weight
gain, high mortality
both dose groups
None
180
(FEL)
a adjusted for exposure regimen (from experimental gavage dosing for 5 days per week to 7 days per week)
b also an FEL for reduced survival
The incidences of primary tumors exhibited negative dose-response trends when
statistically analyzed. Male rats exhibited negative trends for incidences of subcutaneous tissue
fibromas and pituitary adenomas (primarily chromophobe). Female rats exhibited a negative
trend for the incidence of pituitary adenomas. The peer-review panel (for NTP, 1983)
commented that these negative trends could be explained by the decreased survival by the end of
the study. Additionally, after re-examination of the slides, NTP (1983) determined that the
incidence of renal tubular-cell adenomas was increased in treated male rats with a dose-related
trend (p < 0.05). The incidence of this (historically rare) tumor was 0/50, 1/49 and 4/50 for the
control, low-dose and high-dose groups, respectively. In addition, kidney adenocarcinomas were
observed in one control and one low-dose male rats, with an additional carcinoma of the kidney
reported for another low-dose male.
Male and female B6C3F1 mice (50/sex/dose) were administered doses of 250 or 500
mg/kg pentachloroethane for 103 weeks (5 times/week) by gavage. The composition of the
material was 95.5% pentachloroethane, 4.2% hexachloroethane and 0.125% trichloroethylene,
which differed slightly from the material used in the shorter-term studies (10.4%
hexachloroethane and 0.55% tetrachloroethylene impurities). Pentachloroethane exposure
resulted in a significant and dose-related adverse effect on survival. For the high dose males,
42/50 (84%>) had died by week 41 of the study. The 8 remaining high dose males were killed
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during week 41 for histopathologic evaluation. To provide control samples, 25 male control
mice were killed in week 44. Of the remaining male control mice, 19/25 (76%) survived to the
end of the study. For the low dose males, 22/50 (44%) survived to the end of the study. Of the
female control mice, 38/50 {16%) survived to the end of the study. Survival in the low dose
female group was 9/50 (18%) at study termination. The high dose females were all dead by
week 74. Both exposure levels resulted in significantly decreased body weight gain in both
sexes. The high dose males stopped growing appreciably beginning in week 12. By week 52,
the low dose males had significantly lower body weights than controls. While male control mice
body weights remained constant, low dose males exhibited a mean body weight decrease of 30%
between weeks 42 and 104. High dose and low dose females exhibited lower body weights than
controls beginning at weeks 26 and 72, respectively. The body weights for high dose females
remained constant between weeks 26 and 74, although the last high dose female died at week 74.
Body weights of the low dose females were decreased by more thanl0% after weeks 75-80.
Due to the high mortality in the male high dose group and the killing of 25 male control
mice in week 45, tumor incidences were compared in male mice at 0-52 weeks, 53-103 weeks,
and at study termination. Individual time interval comparisons were combined statistically to
obtain an overall result. Neoplastic lesions were observed primarily in the liver of mice of both
sexes. Incidences of hepatocellular carcinomas and hepatocellular adenomas were significantly
increased in exposed female mice compared to control (Table 3). Male mice also exhibited
statistically increased incidences of hepatocellular carcinomas, although early mortality of high
dose males precluded an evaluation of their lifetime incidence of hepatocellular carcinomas. The
combined incidence of hepatocellular adenomas and hepatocarcinomas in female mice, with
poly-3 adjustment (Bailer and C.J. Portier, 1988), was selected for oral cancer slope factor
derivation.
NTP concluded that: "Under the conditions of this bioassay, technical grade
pentachloroethane containing 4.2% hexachloroethane (a known carcinogen in mice) was not
carcinogenic in F344/N rats. The decreased survival of dosed rats might have reduced the
sensitivity for a carcinogenic response in this species. Pentachloroethane was nephrotoxic to
male rats. Technical grade pentachloroethane was carcinogenic for B6C3F1 mice, causing
hepatocellular carcinomas in males and females, and adenomas in females."
This study was reported in the scientific literature by Mennear et al. (1982). This NTP
(1983) study was cited by IARC (1986, 1999) as limited evidence for the carcinogenicity of
pentachloroethane in experimental animals. Combined with the lack of epidemiological data for
exposure to pentachloroethane, IARC (1999) stated that pentachloroethane was not classifiable
as to its carcinogenicity in humans.
Other Studies
NTP (1983) stated in their discussion that NTP mutagenicity tests were negative in
Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 either in the presence or
absence of an exogenous metabolic activation system (S9) from the livers of Aroclor-induced
rats or hamsters (NTP unpublished results, as cited in IARC, 1986).
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TABLE 3. Incidences of Neoplastic Lesions in Mice
Endpoint
Control
250 mg/kg (low dose)
500 mg/kg (high dose)
Female Mice
Hepatocellular
1/46 (2%)
28/42 (67%)
13/45 (29%)
carcinomas
Hepatocellular
2/46 (4%)
8/42 (19%)
19/45 (42%)
adenomas
Combined tumors
3/44 (7%)
36/40 (90%)
32/34 (93%)
(poly-3 adjusted)
Male mice
Hepatocellular
carcinomas
Overall
4/48 (8%)
26/44 (59%)
7/45 (16%)
0-52 weeks
0/25 (0%)
1/2 (50%)
7/45 (16%)
53-103 weeks
0/4 (0%)
9/18 (50%)
0/0
Terminal kill
4/19 (21%)
16/24 (67%)
0/0
Hepatocellular
adenomas
Overall
10/48 (21%)
4/44 (9%)
7/45 (16%)
0-52 weeks
5/25 (20%)
0/2 (0%)
7/45 (16%)
53-103 weeks
0/4 (0%)
2/18(11%)
0/0
Terminal kill
5/19 (26%)
2/24 (8%)
0/0
Galloway et al. (1987) reviewed and summarized the data on sister chromatid exchanges
(SCE) and chromosome aberrations in Chinese hamster ovary (CHO) cells for 108 chemicals.
Pentachloroethane was positive for SCE when no exogenous metabolic system was present, but
negative when rat liver S9 was added. Chromosome aberrations were negative both with and
without exogenous rat liver S9.
Matsuoka et al. (1996) used a Chinese hamster lung fibroblast cell line (CHO/IU) to
evaluate the ability of pentachloroethane to induce chromosomal aberrations. This study used a
different cell line and protocol than the negative NTP study. Three different exposure times (6,
24 or 48 hours) were paired with two recovery times (0 or 18 hours). Marginal results for
structural chromosomal aberrations were observed for the 24 hour exposure/0 hour recovery
protocol and the 6 hour exposure/18 hour recovery; the addition of rat liver S9 resulted in
negative results. Pentachloroethane induced polyploidy in 24 hour exposure treatments with a
dose-response relationship. However, polyploidy induction was marginal in 48 hour exposure
treatments, most likely due to spindle poisons. The overall assessment by the study authors of
pentachloroethane's ability to induce chromosomal aberrations was positive.
Sofuni et al. (1996) summarized the results from various laboratories using the mouse
lymphoma assay (L5178Y (TK+/TK-)) for the detection of in vitro clastogens and spindle
poisons. Pentachloroethane was positive in the absence of a rat liver S9 fraction, but negative
when S9 was present.
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Nastainczyk et al. (1982) evaluated the metabolism of pentachloroethane using rat liver
microsomes from male Sprague-Dawley rats pretreated with sodium phenobarbitone (PB) or 3-
methylcholanthrene (3-MC). PB and 3-MC are cytochrome P450- specific inducers. The rates
of formation of trichloroethylene and tetrachloroethane are presented in Table 4. Liver
microsomes from PB-treated rats metabolized pentachloroethane to trichloroethylene (96%) and
tetrachloroethane (4%). PB pretreatment resulted in a large induction of trichloroethylene and
tetrachloroethane formation, whereas 3-MC pretreatment had no significant effect. Carbon
monoxide, a high affinity cytochrome P450 inhibitor, effectively inhibited the metabolism (99%
inhibition) of pentachloroethane, confirming that cytochrome P450 enzymes are the primary
enzymes involved in pentachloroethane metabolism. Metyrapone (10"4 M) inhibited formation
of trichloroethylene and tetrachloroethane by 47 ± 10%, 27 ± 6% and 22 ± 3% for PB-treated
rats, 3-MC-treated rats, and untreated controls, respectively. This study clearly indicates that the
major metabolites of pentachloroethane are trichloroethylene and tetrachloroethane, and that the
metabolism is catalyzed by PB-inducible cytochrome P450 isoforms.
TABLE 4. Rates of Formation of Pentachloroethane Metabolites by Rat Liver
Microsomes Induced by PB or 3-MC
Pretreatment
Cytochrome P450
(nmol/mg protein)
Pentachloroethane (0.5 mM)
T richloroethylene
(nmol/mg protein/min)
Tetrachloroethane
(nmol/mg protein/min)
PB
2.2
28.5 + 1.7
1.20 + 0.04
3-MC
1.1
4.2 + 0.5
0.54 + 0.08
Control
0.75
6.4 + 0.5
0.64 + 0.07
Results are mean + SD from 3 experiments
Yllner (1971) evaluated the metabolism and elimination of pentachloroethane in female
NMRI mice. Pentachloroethane was administered subcutaneously at doses of 1.1-1.8 g/kg, and
excretion was monitored for 3 days. Over the course of 3 days, approximately 32% of the
administered dose was excreted unchanged via urine; 23.5% of the dose was excreted as
trichloroethanol; 16.1% of the dose was excreted as trichloroacetic acid. In expired air, 7.4% of
the dose was excreted as tetrachloroethane and 8.5% as trichloroethylene. Trichloroethanol and
trichloroacetic acid are urinary metabolites produced during hepatic oxidation of
trichloroethylene and tetrachloroethane.
Goldsworthy et al. (1988) evaluated the role of protein droplet accumulation in the renal
carcinogenicity of male rats exposed to pentachloroethane. Male and female F344 rats were
gavaged with 150 mg/kg pentachloroethane for 10 days. On day 10, the rats were euthanized
and histopathology of kidney was carried out. Pentachloroethane caused significant increases in
kidney protein droplet accumulation and immunohistochemical detection of alpha2U-globulin
protein in male rats, but not female rats. Proximal tubule sections were assessed for cell
proliferation using a labeling index (percentage of labeled cells). The labeling index was not
affected by pentachloroethane treatment in female rats. Male rats exhibited statistically
significant increases in labeling index compared to male control rats. The authors hypothesized
that increased incidences of renal tumors in male rats following pentachloroethane exposure are
caused by nephrotoxicant-induced increases in cell replication. In summary, the nephrotoxic
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effects of the pentachloroethane can be concluded to arise from an accumulation of alpha2u-
globulin, a mode of action that is not relevant to humans (U.S. EPA, 1991a).
NTP (1983) reported increases in protein hyaline droplet accumulation in the renal
tubules of male rats exposed to pentachloroethane. The male rats exhibited nephrotoxic effects
including severe tubular dilation in the pars recta (inner cortex), giant cells and casts in the
dilated tubules, significantly increased incidence of mineralization of the renal papilla, and
prominent interstitial fibrosis and interstitial accumulation of mononuclear inflammatory cells.
These nephrotoxic effects have been associated with alpha2u-globulin-characteristic nephropathy
that is typically only observed in male rats. These renal effects are also frequently associated
with a spontaneous age-related nephropathy syndrome (i.e., not related to alpha2u-globulin)
commonly referred to as chronic progressive nephropathy. However, NTP (1983) suggested that
the nephrotoxic effects observed with exposure to pentachloroethane were distinct from chronic
progressive nephropathy based on the severity of effects and the presence of giant cells within
the tubules which are not characteristic of aging nephropathy. Goldsworthy et al. (1988) also
reported significant increases in protein droplet accumulation. The alpha2U-globulin protein was
identified within the hyaline droplets via immunohistochemical staining. Evidence of cell
proliferation in the proximal tubules as shown by significant increases in labeling index was
observed in male rats administered pentachloroethane.
The EPA (U.S. EPA, 1991a) established criteria for determining if alpha2u-globulin
nephropathy is acting as a factor in the development of renal tumors. Positive evidence in three
categories is required: increased number and size of hyaline droplets in renal proximal tubule
cells; accumulating protein in the hyaline droplets is identified as alpha2U-globulin; and
additional aspects of the pathological sequence of lesions associated with alpha2U-globulin
nephropathy are present. The evidence in male rats suggests that the nephrotoxic effects of
pentachloroethane may arise from an accumulation of alpha2U-globulin and may not be relevant
to humans. However, considering that the pathological sequence of lesions has not been fully
established, the latter conclusion cannot be stated with certainty.
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC RfDs
FOR PENTACHLOROETHANE
Chronic and subchronic (NTP, 1983) studies following oral exposure are available.
ANOAEL and LOAEL (adjusted for exposure regimen) for pentachloroethane were determined
to be 89 and 180 mg/kg-day, respectively, for a 15% reduction in body-weight gain in both male
and female rats in the 13-week NTP (1983) study. A similar reduction of 14% in female mice at
71 mg/kg-day is judged to be non-significant given the apparently random fluctuations in body
weight gain for animals dosed at lower levels. The 29% body weight gain reduction for female
mice at 357 mg/kg-day is considered to be biologically significant. In addition, no effect on
body weights for either mice or rats were observed in the chronic study at doses up to 107
mg/kg-day (NTP, 1983). The subchronic mouse study suggests an equivocal NOAEL of 71
mg/kg-day, with a LOAEL of 357 mg-kg-day for reduction in body weight gain. The latter
could also be considered to be a FEL, given the death of one animal at that dose. In addition,
given the FEL of 180 mg/kg-day for mice in the 2-year NTP (1983) study, an overall NOAEL
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and LOAEL for the pentachloroethane for mice cannot be determined. The FEL of 54 mg/kg-day
for mortality in rats and mice in the chronic study (evident for rats as early as 18 weeks)
confounds the derivation of subchronic and chronic p-RfDs, given the close proximity to the
subchronic NOAELs (71 and 89 mg/kg-day for mice and rats, respectively). Thus, the data are
considered to be inadequate for the quantitative derivation of subchronic and chronic p-RfDs.
DERIVATION OF PROVISIONAL SUBCHRONIC AND CHRONIC RfCs FOR
PENTACHLOROETHANE
Subchronic and chronic provisional RfCs cannot be derived for pentachloroethane
because no toxicology information from the inhalation route of exposure is available. A route-
to-route extrapolation could not be performed because of the lack of information on the
absorption, metabolism and distribution of pentachloroethane following inhalation exposure.
PROVISIONAL CARCINOGENICITY ASSESSMENT FOR
PENTACHLOROETHANE
The derivation of oral cancer slope factors was based on the female mouse hepatocellular
adenomas observed in the chronic bioassay conducted by NTP (1983). Tumor incidences from
this study are reported in Table 2. Data from male mice could not be used due to the high
mortality in the high dose group, which precluded an evaluation of their lifetime incidences of
hepatocellular carcinoma and adenoma. Hepatocellular carcinomas in female mice were
statistically significantly increased in both dose groups compared to controlHepatocellular
adenomas in female mice exhibited a statistically significant dose-related increase in incidence.
The combined data set will be used for derivation of oral cancer slope factors.
NTP (1983) concluded that: "Under the conditions of this bioassay, technical grade
pentachloroethane containing 4.2% hexachloroethane (a known carcinogen in mice) was not
carcinogenic in F344/N rats. The decreased survival of dosed rats might have reduced the
sensitivity for a carcinogenic response in this species. Pentachloroethane was nephrotoxic to
male rats. Technical grade pentachloroethane was carcinogenic for B6C3F1 mice, causing
hepatocellular carcinomas in males and females, and adenomas in females."
Pentachloroethane was not mutagenic in Ames Salmonella typhimurium mutagenicity
tests, both in the presence and absence of an exogenous metabolic activation system (NTP,
1983). It was also reported to be negative in CHO cells for chromosome aberrations (Galloway
et al., 1987). Pentachloroethane has been observed to cause other forms of genotoxicity such as
sister chromatid exchanges (Galloway et al., 1987), polyploidy (Matsuoka et al., 1996), and
clastogenicity (Sofuni et al., 1996). Based on this evidence, it is unclear if pentachloroethane is a
mutagenic carcinogen.
The tumorigenicity observed in male and female mice along with some evidence of
genotoxicity suggests that pentachloroethane is "Likely to Be Carcinogenic to Humans'"
according to U.S. EPA Guidelines for Carcinogen Risk Assessment (2005a). Since the mode of
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action for pentachloroethane carcinogenicity cannot be determined due to the limited information
available, the default linear extrapolation approach was used to calculated the oral slope factor
(U.S. EPA, 2005a). The application of age-dependent adjustment factors for early-life exposure
when a mutagenic mode of carcinogenic action has been determined is not recommended. The
mode of action of pentachloroethane is unknown and evidence for a mutagenic mode of action is
not available.
The oral slope factor was derived using the combined incidence of hepatocellular
adenomas and hepatocarcinomas in female B6C3F1 mice (NTP, 1983), assuming that the
adenomas are direct precursors to the carcinomas. The doses used in this study (0, 250 mg/kg,
and 500 mg/kg; 5 times/week) were first adjusted for continuous exposure by adjusting for
exposure regimen (from experimental gavage dosing for 5 days per week to 7 days per week),
resulting in continuous doses of 0, 178.5 and 357.1 mg/kg-day. These continuous doses were
scaled to human doses by multiplying the continuous animal dose with the animal:human body
weight ratio to the Vi power: (animal weight/human weight)174, according to U.S. EPA (2005a).
The human equivalent doses (HEDs) are 0, 25.7 and 51.4 mg/kg-day, respectively, assuming
(default) body weights of 0.03 kg for mice and 70 kg for humans. The combined tumor
incidences were adjusted for survival (early death of tumor-free animals) by the poly-3 method
(Bailer and C.J. Portier, 1988). The poly-3 adjusted tumor incidences for these doses were 3/44,
36/40 and 32/34, respectively.
U. S. EPA Benchmark Dose Software version 1.4.1 was used for determination of the
point of departure (U.S. EPA, 2007b). The dichotomous 1st order cancer multi-stage model was
applied to the data (U.S. EPA, 2000). The model fit was adequate, with a Chi-square p-value of
0.117. The BMDLhed/io was 1.13 mg/kg-day, with a cancer slope factor of 0.0883 (mg/kg-day)"1
(see Appendix A).
The oral cancer slope factor (p-OSF) for pentachloroethane is 0.09 per mg/kg-day.
The oral cancer slope factor for pentachloroethane should not be used with exposures
exceeding the point of departure (1 mg/kg-day; BMDLHed/io), because above this point the slope
factor may not approximate the observed dose-response relationship adequately.
The presence of hexachloroethane in the dosing preparation is a confounding factor in
this assessment. Hexachloroethane produces liver tumors in mice but not in rats. Therefore, at
least some of the tumor response observed for the pentachloroethane-treated mice could be a
result of hexachloroethane exposure. However, it is unlikely that hexachloroethane is a major
factor in the observed tumorigenic response in this study. Hexachloroethane comprises only
about 4% of the technical pentachloroethane material and the OSF for hexachloroethane is 0.014
(mg/kg-day) (U.S. EPA, 1987) which is much less than the OSF estimated for
pentachloroethane. Therefore, the OSF for pure pentachloroethane is unlikely to be much less
than the estimate of 0.09 per mg/kg-day.
Provisional inhalation unit risk estimates (p-IUR) could not be derived for
pentachloroethane because of the lack of inhalation toxicology data, and the lack of information
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on the absorption, metabolism, and distribution of pentachloroethane after inhalation exposure.
A route-to-route extrapolation cannot be performed without this information.
REFERENCES
ATSDR (Agency for Toxic Substances and Disease Registry). 1999. Toxicological profile for
Chlorophenols. Review Draft. U.S. Public Health Service. Atlanta, GA. TP-107. Available at
http://www.atsdr.cdc.gov/toxprofiles/tpl07.html
Bailer, AJ and C.J. Portier. 1988. Effects of treatment-induced mortality and tumor-induced
mortality on tests for carcinogenicity in small samples. Biometrics 44:417-431.
Galloway, S.M., M.J. Armstrong, C. Reuben et al. 1987. Chromosome aberrations and sister
chromatid exchanges in Chinese hamster ovary cells: evaluations of 108 chemicals. Environ.
Mol. Mutagen. 10(SUPPL 10): 1-175.
Goldsworthy, T.L., O. Lyght, V.L. Burnett et al. 1988. Potential role of alpha-2 mu-globulin,
protein droplet accumulation, and cell replication in the renal carcinogenicity of rats exposed to
trichloroethylene, perchloroethylene, and pentachloroethane. Toxicol. Appl. Pharmacol.
96(2):367-379.
IARC (International Agency for Research on Cancer). 1986. Some halogenated hydrocarbons
and pesticide exposures. IARC monographs on the evaluation of carcinogenic risks to humans.
Vol. 41. Lyon, France: International Agency for Research on Cancer, p. 99-130.
IARC (International Agency for Research on Cancer). 1999a. Re-evaluation of some organic
chemicals, hydrazine and hydrogen peroxide. IARC monographs on the evaluation of
carcinogenic risks to humans. Vol. 71. Lyon, France: International Agency for Research on
Cancer, p. 1519-1524.
IARC (International Agency for Research on Cancer). 1999b. Re-evaluation of some organic
chemicals, hydrazine and hydrogen peroxide. IARC monographs on the evaluation of
carcinogenic risks to humans. Vol. 71. Lyon, France: International Agency for Research on
Cancer, p. 817-828.
IARC (International Agency for Research on Cancer). 1999c. Re-evaluation of some organic
chemicals, hydrazine and hydrogen peroxide. IARC monographs on the evaluation of
carcinogenic risks to humans. Vol. 71. Lyon, France: International Agency for Research on
Cancer, p. 1133-1142.
Matsuoka, A., K. Yamakage, H. Kusakabe et al. 1996. Re-evaluation of chromosomal
aberration induction on nine mouse lymphoma assay "unique positive' NTP carcinogens. Mutat.
Res. 369(3-4):243-252.
Mennear, J.H., J.K. Haseman, D.J. Sullivan et al. 1982. Studies on the carcinogenicity of
pentachloroethane in rats and mice. Fundam. Appl. Toxicol. 2(2):82-87.
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Nastainczyk, W., H.J. Ahr and V. Ullrich. 1982. The reductive metabolism of halogenated
alkanes by liver microsomal cytochrome P450. Biochem. Pharmacol. 31(3):391-396.
NTP (National Toxicology Program). 1983. Carcinogenesis bioassay of pentachloroethane in
F344/N rats and B6C3F1 mice (gavage studies). National Institute of Environmental Health
Sciences, Public Health Service, U.S. Department of Health and Human Services, Research
Triangle Park, NC; NTP TR 232. Available at: http://ntp-server.niehs.nih.gov
Sofuni, T., M. Honma, M. Hayashi et al. 1996. Detection of in vitro clastogens and spindle
poisons by the mouse lymphoma assay using the microwell method: interim report of an
international collaborative study. Mutagenesis. ll(4):349-355.
U.S. EPA. 1987. Reference Dose for Chronic Oral Exposure for Hexachloroethane. Integrated
Risk Information System (IRIS). Online. Office of Research and Development, National Center
for Environmental Assessment, Washington, DC. Accessed 2007.
www.epa.gov/iris/subst/0167.htm
U.S. EPA. 1991a. Alpha2U-globulin: association with chemically induced renal toxicity and
neoplasia in the male rat. Risk Assessment Forum, Washington, DC; EPA/625/3-91/019F.
Available at: http://cfpub.epa.gov/ncea/cfm/recordisplav.cfm?deid=54883
U.S. EPA. 1991b. Chemical Assessments and Related Activities (CARA). Office of Health and
Environmental Assessment, Washington, DC. April.
U.S. EPA. 1994. Chemical Assessments and Related Activities (CARA). Office of Health and
Environmental Assessment, Washington, DC. December.
U.S. EPA. 1997. Health Effects Assessment Summary Tables. Annual Update. FY-1997.
Office of Research and Development, Office of Emergency and Remedial Response,
Washington, DC. July 1997. EPA/540/R-97/036. NTIS PB97-921199.
U.S. EPA. 2000. Benchmark dose technical guidance document (external review draft). Risk
Assessment Forum, Washington, DC; EPA/63O/R-OO/OOl. Available at:
http: //cfpub. epa. gov/ncea/cfm/recordi spl ay. cfm? dei d=20871
U.S. EPA. 2004. 2004 Edition of the Drinking Water Standards and Health Advisories. Office
of Water, Washington, DC. EPA/822/R-02/038. Available at
http://www.epa.gov/waterscience/drinking/standards/dwstandards.pdf
U.S. EPA. 2005a. Guidelines for carcinogen risk assessment. Risk Assessment Forum,
Washington, DC; EPA/630/P-03/001F. Federal Register 70(66): 17765-17817. Available online
at http://www.epa.gov/raf
U.S. EPA. 2005b. Supplemental guidance for assessing susceptibility from early-life exposure
to carcinogens. Risk Assessment Forum, Washington, DC; EPA/630/R-03/003F. Available at:
http://www.epa.gov/iris/backgr-d.htm
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U.S. EPA. 2007a. Integrated Risk Information System (IRIS). Online. Office of Research and
Development, National Center for Environmental Assessment, Washington, DC.
http://www.epa.gov/iris
U.S. EPA. 2007b. Benchmark dose software (BMDS) version 1.3.2 (last modified 2000).
Available at: http://cfpub.epa.gov/ncea/cfm/recordisplav.cfm?deid=20167
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29(5-6):481-489.
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Appendix A: Benchmark Dose Derivations
Appendix A contains the output from the Benchmark Dose Software version 1.4.1 for the
combined hepatocellular adenoma and hepatocarcinoma tumor incidence in female mice (NTP,
1983; U.S. EPA, 2007b).
Multi-stage 1° Polynomial
Multistage Cancer Model. (Version: 1.5; Date: 02/20/2007)
Input Data File: C:\BMDS\PENTACHLOROETHANE.(d)
Gnuplot Plotting File: C:\BMDS\PENTACHLOROETHANE.plt
Wed Sep 26 21:07:41 2007
BMDS MODEL RUN
The form of the probability function is:
P[response] = background + (l-background)*[l-EXP(
-betal*doseAl)]
The parameter betas are restricted to be positive
Dependent variable = rcomb
Independent variable = HED
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.296723
Beta(l) = 0.0538105
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Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.38
Beta(l) -0.38 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0.0700696 * * *
Beta(l) 0.0706968 * * *
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log(likelihood) #Param's Deviance Testd.f. P-value
Full model -31.5618 3
Fitted model -32.6066 2 2.08954 1 0.1483
Reduced model -79.3336 1 95.5436 2 <.0001
AIC: 69.2131
Goodness of Fit
Scaled
Dose Est.Prob. Expected Observed Size Residual
0.0000 0.0701 3.083 3 44 -0.049
25.7600 0.8495 33.980 36 40 0.893
51.3700 0.9754 33.163 32 34 -1.287
ChiA2 = 2.46 d.f. = 1 P-value = 0.1170
Benchmark Dose Computation
Specified effect = 0.1
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Risk Type = Extra risk
Confidence level = 0.95
BMD = 1.49031
BMDL = 1.13237
BMDU = 1.97227
Taken together, (1.13237, 1.97227) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.0883104
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
"o 0.8
0
o
it= 0.6
<
° 0.4
-4—'
o
(0
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0.2
BMD
0
10
20
30
40
50
dose
07:41 09/27 2007
A-3
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