xvEPA
United States
Environmental Protection
Agency
Office of Health and
Environmental Assessment
Washington DC 20460
EPA/600/8-82/005FA
April 1986
External Review Draft
Research and Development
Addendum to the Review
Health Assessment Draft
Document for (DO Not
Tetrachloroethylenecite or Quote)
(Perchloroethylene)
Updated
Carcinogenicity
Assessment for
Tetrachloroethylene
(Perchloroethylene,
PERC, PCE)
NOTICE
This document is a preliminary draft. It has not been formally
released by EPA and should not at this stage be construed to
represent Agency policy. It is being circulated for comment on its
technical accuracy and policy implications.
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DRAFT EPA/600/8-82/005FA
DO NOT QUOTE OR CITE April 1986
Review Draft
f
ADDENDUM TO THE HEALTH ASSESSMENT DOCUMENT
FOR TETRACHLOROETHYLENE (PERCHLOROETHYLENE)
Updated Carcinogenicity Assessment
for Tetrachloroethylene (Perch!oroethylene, PERC, PCE)
NOTICE
THIS DOCUMENT IS A PRELIMINARY DRAFT. It has not been formally released by
the U.S. Environmental Protection Agency and should not at this stage be
construed to represent Agency policy. It is being circulated for comment
on its technical accuracy and policy implications.
Office of Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C.
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DISCLAIMER
i
i
This document is an ext&rn.al draft for review purposes .only and .-.doe-s
not constitute Agency .policy. Mention of trade names or commercta-i products
does not constitute endorsement or recommendation for use.
The Health Assessment (Document for Tetraehloroethylene (Perch!oroethylene)
(July 1985; .EPA-600/8-82-005F) i? .available front:
National Technical Information Service
5.285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-465U
Order No.: PB-85-249704
Cost: $22,95 (subject to change)
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CONTENTS
P P£*f f\ f P
Authors! Contributors* and Reviewers !'*'*' ' '''' * ' ' 1'
* '*' ' '''' * ' '
1. SUMMARY AND CONCLUSIONS
2. INTRODUCTION ...... .............
3* ?MriEWi!L!HE NATIONAL TOXICOLOGY PROGRAM INHALATION BIOASSAY
vINIr, 1985; ............... ..... ^ i
3.1. ANIMALS STUDIED. ..... 01
3.2. RESULTS OF THE RAT STUDY ...'*' ' ' ' ......... ,,
3.3. RESULTS OF THE MOUSE STUDY ...*'* ........... ' o"in
3.4. SUMMARY ..... . ..... ...'.'.'. .......... o~Tc
* * * • • * • • • • • -3 ™ *A 0
4. QUANTITATIVE RISK ASSESSMENT .......... ........ 4-r
4.1. DATA AVAILABLE FOR RISK CALCULATION
4.1.1. NTP Rat Inhalation Assay .......... 4 2
4.1.2. NTP Mouse Inhalation Assay ....!*!' 1 !*,*.] 4! 2
4.2,. POSSIBLE MECHANISMS LEADING TO A CARCINOGENIC RESPONSE
• • ft
4.5. CALCULATION OF METABOLIZED DOSE FROM INHALATION 'STUDIES.' .' .' t-8
4.5.1. Direct Estimation from Metabolism Experiments. . . . 4-8
4.5.1.1. Rat. ............ ....... 4-8
4.5.1.2. Mouse ............ *!*.!*,*," 4-11
4.5.2. Metabolized Dose Predicted by a PB-PK Model. . . . . 4-12
' ' ^
....... 4-16
4.7.1. Based on the Metabolized Dose Estimated
Directly from the Empirical Data ..... 415
4.7.2. Based on the Metabolized Dose Calculated
from the PB-PK Model ......... ....... 4_20
4.8. DISCUSSION ....... ...
4.9. SUMMARY OF QUANTITATIVE ASSESSMENT .' .' * ." .* .* ' .* * ' * ' '
5. REFERENCES ............. • ^
***••*•*•••*•••• D~i
APPENDIX A: PHYSIOLOGICALLY-BASED PHARMACOKINETIC MODELS
- AND THEIR APPLICATION IN THE QUANTITATIVE RISK
ASSESSMENT OF PCE ..... ....... ... ..... A-l
iii
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PREFACE
The Office of Health and Environmental Assessment has prepared this adden-
dum to serve as a source document, for EPA use. This addendum updates EPA's
July 1985 Health Assessment Document! for Tetrachloroethylene (perchloroethy-
lene, PERC, PCE) by providing a review of the findings of the draft National
Toxicology Program (NTP) Technical Report on the Toxicology and Carcinogenesi s
Studies of Tetrachloroethylene in F344/N rats and B6C3F1 mice (inhalation
studies). The addendum discusses th|e ways in which the data impact the assess-
I
ment of the weight-of-evidence for the carcinogenicity of PCE, and uses rele-
vant data to develop a revised unit risk estimate for inhalation exposure to
PCE.
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; PREFACE
The Office of Health and Environmental Assessment has prepared this adden-
dum to serve as a source document for EPA use. This addendum updates EPA's
July 1985 Health Assessment Document for Tetrachloroethylene (perch!oroethy-
lene, PERC, PCE) by providing a review of the findings of the draft National
Toxicology Program (NTP) Technical Report on the Toxicology and Carcinogenesis
Studies of Tetrachloroethylene in F344/N rats and B6C3F1 mice (inhalation'
studies). The addendum discusses the ways in which the data impact the assess-
ment of the weight-of-evidence for the carcinogenicity of PCE, and uses rele-
vant data to develop a revised unit risk estimate for inhalation exposure to
PCE.
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1. SUMMARY AND CONCLUSIONS
In the July 1985 Health Assessment Document for Tetrachloroethylene (per-
chloroethylene, PERC, PCE), the U.S. Environmental Protection Agency's Office
of Health and Environmental Assessment (OHEA) summarized the pertinent evidence
for the carcinogenicity of PCE, based primarily on the results of a 2-year bio-
assay conducted by the National Cancer Institute (NCI, 1977). In that study,
PCE induced a statistically significant increase in the incidence of hepatocell-
ular carcinomas in both high- (1072 and 772 mg/kg/day) and low-dose (536 and
386 ng/kg/day) male and female B6C3F1 mice administered PCE by gavage for .a
period of 78 weeks.
In a 1985 inhalation bioassay conducted by the National Toxicology Program
(draft report approved by the Board of Scientific Counselors), groups of 50
male and 50 female F344/N rats and B6C3F1 mice were exposed to PCE (99.9% pure),
6 hours/day, 5 days/week, for 2 years. The exposure concentrations used were
0, 200, and 400 pprn for rats and 0, 100, and 200 ppm for mice. At the 200 and
400 ppm exposure concentrations, PCE induced a marginally statistically signi-
ficant increase of rnononuclear cell leukemia in male rats. PCE also induced an
increased incidence of renal tubular cell adenomas and/or carcinomas (combined)
in male rats. While not statistically significant, there was a dose-related'
trend of these rare tumors. In female rats at both the 200 and 400 ppm expo-
sure concentrations, statistically significant increased incidences of mono-
nuclear cell leukemia were observed. In male and female B6C3F1 mice, PCE
induced a statistically significant increased incidence of hepatocellular
carcinomas at both the 100 and the 200 ppm exposure concentrations. Although
mutagenicity studies of PCE have produced inconclusive or negative results, PCE
epoxide, a reactive metabolite of PCE, has been found to be mutagenic.
1-1
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The NTP (1985, draft) inhalation bioassay demonstrated that PCE -can induce
I
carcinogenic effects in both rats and mice through inhalation exposure, The
earlier NCI (1977) bipassay provided positive evidence of h.epatocellalar car-
cinomas in mice administered PCE bylgavage,. The.se findings, together with tihe
supporting evidence regarding the PCE epoxide (a presumed reactive metabolite
that has been shown to be mutayenic), constitute a "sufficient" level of evi-
dence for the carcinogenicity of P.C.E in animals, using the EPA's Proposed
Guidelines for Carcinogen Risk Assessment. The evidence In rats arid mice, to-
gether with the inconclusive evidence in humans as reported in the 1985 tteailth
Assessment Document for Tetrachloroethylene,, results in the placement of .£CE in
EPA's weight-of-evidence Group B2, meaning that it should be considered a proba-
ble human carcinogen. ;
OHEA is aware of three issues related to the NTP (1985, draft) inhalation
bioassay. These issues are raised in a document presented to EPA by the Halo-
genated Solvent Industry Alliance (hisiA),, and focus on reported carcinogenic
i
responses in male F344 rats, as well as on certain aspects of the conduct of
the bioassay. The validity of a reported marginally statistically significant
I
increased incidence of mononuclear cell leukemia in male F344 rats is in ques-
tion. The finding is dependent on the criteria used for "staging" the leukemia
i
and the approach used in assessing the variable incidence in the untreated
rats. The reasonableness of these approaches, as used in data interpretation,
is at issue. Also at issue is the significance of a reported finding of a low
incidence (not statistically significant) of rare tubular cell adenomas or
carcinomas in male F344 rats. Lastly, alleged deficiencies In animal handling
and identification raise doubts about the reliability of the reported marginal
and low-level incidences of tumors in male rats.
The impact of the issues regarding the findings in male F344 rats relative
I
I
i
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to a weight-of-evidence evaluation is to add to the multiplicity of evidence
if the findings are considered valid; on the other hand, if the findings are
considered uncertain, the remaining evidence in male and female mice and female
i
rats is likely to be judged "sufficient" according to EPA's Proposed Guidelines
for Carcinogen Risk Assessment.
In this addendum, an inhalation unit risk estimate for PCE is developed
using data from the NTP (1985, draft) inhalation bioassay—the first lifetime
inhalation study to show evidence of the carcinogenicity of PCE. Carcinogenic
responses were observed in both rats and mice. These data provide a basis for
directly estimating the risk of PCE by the inhalation route, and an opportunity
to test the reasonableness of the previous inhalation risk estimate, which was
developed using gavage data from the NCI bioassay (1977).
It is generally recognized that the carcinogenic potential of PCE resides
in its biologically reactive metabolites, such as the short-lived, reactive PCE
epoxide intermediate, rather than the parent PCE compound itself. The rate of
metabolism of PCE is dose-dependent--meaning that the effective dose of active
metabolite is not proportional to the administered dose of PCE--and proceeds
according to Michaelis-Menten kinetics. Covalent binding and hepatotoxicity of
PCE are directly proportional to metabolized dose. The tumorigenic response
is also assumed to be directly related to metabolized dose.
The ingestion and inhalation unit risk estimates reported in the 1985
Health Assessment Document for Tetrachloroethylene were extrapolated from a .
gavage cancer bioassay study in mice. Metabolism and kinetic information were
incorporated into these estimations, expressing dose in terms of the amount
metabolized, or a "metabolized dose." Thus, the inhalation unit risk estimate
for PCE reported in the 1985 document was based on tumor incidence data from a
gavage study and the assimilated dose metabolized, accomplished by incorporating
1-3
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metabolism and kinetics information fpr both animals and humans. On this
I
basis, the inhalation unit risk is 4.|8 x 10-7/jig/m3. The unit risk for
ingestion of 1 ug PCE/L in drinking water is 1.5 x 1CH>.
t
The new unit risk estimate for i'nhalation also utilizes the metabolized
dose-tumor incidence relationships derived from pertinent exposure concentra-
tion dose-metabolism relationship data in both animals and humans. The line-
I
arized multistage model is used for t'he low-dose extrapolation. The details
\
of this procedure are presented in the 1985 Health Assessment Document for
Tetrachloroethylene. The typical uncertainty associated with extrapolating
i
dose from animals to humans is reduced by the consideration of actual human
exposure concentration dose-metabolism relationship data, where urinary tri-
chloroacetic acid is taken as an index of the amount of PCE metabolized, in
addition to the use of comparable ani,mal data. The inhalation unit risk esti-
mates calculated on the basis of animal inhalation carcinogenicity studies
(rats and mice) are comparable to that previously derived from the gavage
j
carcinogenicity data in mice. The upper-bound estimates of the incremental
o
cancer risk due to lifetime exposure 'to I pg/m^ of PCE in air are calculated
for six sets of dose-tumor incidence data by three methods. The ranges of the
i
six estimates under each of the three methods are as follows: (a) 2.9 x 10"7
to 9.5 x 10-7/yg/m3; (b) 2.9 x ID-6 to 1.1 x 10-5/yg/m3; (c) 9.6 x 10"7 to
3.6 x 10-6/ug/m3. j
The first range of estimates isjcalculated on the basis of metabolized
i
doses that are derived directly from;a single-exposure balance study (assuming
that the total amount metabolized over a 72-hour period from a one-time expo-
sure is comparable to the steady-state metabolized dose under the NTP multiple-
exposure cancer bioassay study). The second and third range of estimates are
I
calculated on the basis of metabolized doses that are derived from a physiolog-
: 1-4
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ically-based pharmacokinetic (PB-PK) model. Such models, which piece together
the relevant physiological and biochemical information, can be useful tools for
predicting absorption, distribution, biotransformation, and elimination of .-
chemicals across species when sufficient data are available. However, until
more data become available, EPA's Carcinogen Assessment Group (CA6) recommends
that the first range of estimates be used to represent the inhalation risk for
PCE, since there are uncertainties with parameters used in the PB-PK model
that were estimated from the same empirical data set used in the direct estima-
tion, and the model cannot be considered to give a better estimate. That is,
the revised upper-bound estimate of the incremental cancer risk due to lifetime
exposure of 1 pg/m3 of PCE in air ranges from 2.9 x 10~7 to 9.5 x 1Q-7. This
unit risk estimate is applicable only for low-level exposures where the rela-
tionship between the ambient air concentration and metabolized dose is linear.
The upper-bound nature of these estimates is such that the true risk for humans
is not likely to exceed these values and may be lower. Expressed in terms of
relative potency, PCE ranks in the lowest quartile among 55 suspect or known
carcinogens evaluated by the CAG.
1-5
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. ; 2. INTRODUCTION
The July 1985 Health Assessment Document for Tetrachloroethylene (per-
ch! oroethylene, PERC, PCE) (U.S. EPA, 1985) summarized the evidence that PCE
administered by gavage induced malignant tumors pf the liver in both male and
female B6C3F1 mice (NCI, 1977). The Health Assessment Document also cited the
results of several epidemiologic studies, which were characterized as providing
inconclusive findings. According to the Agency's Proposed Guidelines for
Carcinogen Risk Assessment (U.S. EPA, 1984), the carcinogenic evidence avail-
able as of July 1985 for PCE in animals was judged to be limited, and the
epidemiologic data inconclusive. The overall weight-of-evidence for the car-
cinogenicity of PCE was classified as belonging in EPA Group C, meaning that
PCE should be considered a "possible" human carcinogen. Quantitative human
unit risk estimations for ingestion and inhalation were developed using the NCI
mouse gavage study, relevant metabolism and kinetics data, and the linearized,
multistage model for low-dose extrapolation.
Since publication of the July 1985 Health Assessment Document for Tetra-
chloroethylene, the National Toxicology Program inhalation bioassay draft
report (NTP, 1985) on PCE carcinogenicity in rats and mice has become available.
The study findings have been audited and validated for technical accuracy, and
the report has been peer-reviewed by the NTP Board of Scientific Counselors.
Male F344/N rats in the study showed a statistically significant increased
incidence of mononuclear cell leukemia and a dose-related trend for rare renal
tubular neoplasms. Female F344/N rats showed statistically significant inci-
dences of mononuclear cell leukemia. In addition, PCE induced a statistically
significant increased incidence of hepatocellular carcinomas in both male and
female B6C3F1 mice.
2-1
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This addendum reviews the new NTPj (1985, draft) inhalation bioassay of PCE
in detail, and discusses the ways in which the data impact the assessment of
the weight of evidence for the carcinogenicity of PCE. This addendum also in-
cludes a section on unit risk estimation which incorporates relevant •inhalation
i
metabolism and kinetics data. '
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.3. REVIEW OF THE NATIONAL TOXICOLOGY PROGRAM INHALATION
CARCINOGENICITY BIOASSAY (NTP, 1985)
A 2-year inhalation cancer bioassay of^PCE in F344/N rats and B6C3F1 mice
has been performed by Battelle Pacific Northwest Laboratories, under the spon-
sorship of the National Toxicology Program {NTP, ;1.985). The study provides
positive evidence for the carcinogenicity .of PCE in bothiof the, above species.
A description of the various aspects of the study'follows.; - ';
The study utilized high-purity PCE (99.9$) obtained from the Dow Chemical
Company. The purity and identity analyses of the PCE were performed by the
Midwest Research Institute. The PCE was stabilized with n-methylmorpholine
(53 ppm) to prevent decomposition. The PCE was ,then vaporized 'at 100°C to
110°C, diluted with air, and introduced into the inhalation chambers. The
concentrations in the chambers were monitored 8 to 12 times per exposure period
by means of a Hewlett-Packard 5840A gas chromatograph equipped with a flame
ionization detector.
3.1. ANIMALS STUDIED
The 8- to 9-week-old male and female F344/N rats and B6C3F1 mice used in
this study were obtained from the Charles River Breeding Laboratories. Groups
of 50 rats:'of each ;sex were exposed to air containing PCE at concentrations of i
0 (chamber^controlE 200, or 400 ppm, 6 hours/day, 5,days/week for 103 weeks. V:
Groups of 50 mice of each sex were also exposed to PCE at 0, 100, and 200 ppm. ; :
These doses /were selected based on the results of an earlier 13-week study in :
which incidence ;of death at 1,600 ppm and incidence of liver lesions at lower
concentrations had been observed for rats. Incidence of death at 1600. ppm, an.d
of hepatic and renal lesions at lower concentrations, were observed for mice.
Rats and mice were housed individually. The animals were given a diet (NIH07
3-1
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Open Formula) and water freely, except during the exposure period, when water
was available.
All animals were observed two tijmes per day. Clinical signs were recorded
once per month. Body weights were recorded once each week for the first 13
weeks of the study and once per month! thereafter. Mean body weights were
calculated for each group. Moribund janimals, as well as animals that survived
I
to the end of the study, were killed.; A necropsy was performed on all animals.
Vital organs were examined for any grjossly visible lesions. Tissues were
preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned,
and stained with hematoxylin and eosih, with other stains being used as needed.
The tissue slides were examined microscopically to determine pathology.
3.2. RESULTS OF THE RAT STUDY ;
Figure 3-1 presents the growth curves for male and female rats exposed to
PCE by inhalation for 2 years. The mean body weights of PCE-exposed male and
female rats were comparable with those of controls.
i
Survival data for rats in the 2-j^ear inhalation study are given Table 3-1
and Figure 3-2. The survival of malejrats exposed to 400 ppm was significantly
I
reduced (control, 23/50; 200 ppm, 20/50; 400 ppm, 12/50) but not that of females
i
(control, 23/50; 200 ppm, 21/50; 400 ppm, 24/50). Most of the unscheduled deaths
(87%) in the 400 ppm dose group occurred late in the study (week 82 or later)
and may have been related to the high incidence of mononuclear cell leukemia.
Table 3-2 presents the incidence [of mononuclear cell leukemia in male and
i
female rats exposed to 200 and 400 ppm PCE. Incidences of mononuclear cell
i
leukemia in male rats were marginally'statistically significant: control,
28/50; 200 ppm, 37/50 (p = 0.046); 400 ppm, 37/50 (p = 0.046). In female rats,
the incidences of mononuclear cell leukemia were also statistically signifi-
cant: control, 18/50; 200 ppm, 30/50j(p =0.014); 400 ppm, 29/50 (p = 0.022).
i
3-2
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6OO.O
«, 450.0-
3E
100.0
>
500.0
«0 460.0-
O
400.0-
j_ 360.0-
X
o
uj 300.0-
100.0
i
MALE RATS
D = UNTREATED
O = 2OO PPM
AS 400 PPM
—T~
30
45 90 76
WEEKS ON STUDY
00
106
3
g
Q@ 88
§8
FEMALE RATS
D = UNTREATED
O = 200 PPM
A = 400 PPM
15 30 45 6O 75
WEEKS ON STUDY
90
105
Figure 3-1. Growth curves for rats exposed to PCE by
inhalation for 2 years.
SOURCE: NTP, 1985.
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TABLE 3-1. 'SURVIVAL OF RATS
IN THE NTP 2-YEAR INHALATION BIOASSAY OF PCE
Group C
Males
Animals initially
in the study
Nonacci dental deaths
Died during termination
period
Killed at termination
Females
Animals initially
in the study
Nonacci dental deaths
Killed at termination13
ontrol 200 ppma
50 50
27 (54%) 3.0 (60%)
0 1 (2%)
23 (46%) 19 (38%)
50 50
27 (54%) 29 (58%)
23 (46%) 21 (42%)
4QQ ppm
50
38 (76%)
1 (2%)
11 (22,%)
50
26 (52%)
24 (48%)
£1 ppm is equivalent to 6908 yg/m3.
bTerminal kill period: week 104.
SOURCE: NTP, 1985.
3-4
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1.0
0.9-
> 0.8-
«C
u.
O
0.6-
t-
^0.5H
CD
ffi
O0.4-
E
a.
0.3-
0.2
0.9
> 0.8-
oc
= 0.7-1
o.e-
0.5-
rt
a.
0.3-
O.2
MALE RATS
O = UNTREATED
0= *00 PPM
A = 400 PPM
15 SO 45 «0 76
WEEKS ON STUDY
FEMALE RATS
D s UNTREATED
O = 200 PPM
A = 400 PPM
90 105
80 45 60 7'6
WEEKS ON STUDY
105
Tl9nor ?"2' KaP1an-Meier survival curves for rats exposed
to PCE by inhalation for 2 years.
SOURCE: NTP, 1985.
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TABLE; 3-2. MQNONUCLpAR, CE.LL LEUKEMIA IN RATS
EXPOSED TO pCE BY INHALATION
Group
Control
200 ppma 400: p;pma
Hales
Mononuclear cell
leukemia
28/50 ($6%-).
37/50 (74%)
p = 0.046
37/50: (74%)
p = 0..046
Females
Mononuclear cell
leukemia
18/50 (36%)
30/50 (60%)
p. =• 0.014;
29/50 ('58%)
p = 0.022
ap-values were calculated using the Flisher Exact Test.,
• 3-6
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The diagnoses of mononuclear cell leukemia were classified as stage 1.(early),
stage 2 (intermediate), or stage 3 (advanced and probably fatal).
The following criteria were used for assigning these stages:
Stajej, - Spleen not enlarged or only moderately enlarged, with few '
suspicious cells surrounding Malpighian corpuscles of the spleen, and
only very few neoplastic cells in the blood vessels of the lung.
Stage^ - More severe than in the first category; spleen and/or liver
only moderately enlarged, with neoplastic cells in the liver sinusoids
and in the blood vessels of other organs.
StajLl " Enlarged spleen and liver, with many neoplastic cells in -
the liver sinusoids and in the blood vessels of other organs.
Table 3-3 summarizes the distribution of these three stages of mononuclear cell
leukemia in the male and female rats studied.
The strength of the evidence for carcinogenicity presented here for F344
rats hinges on the resolution of issues raised regarding the uncertainty in '
the assignment of incidence within stages of mononuclear cell leukemia in the
treated F344 rats and the high and variable incidence of mononuclear cell leu-
kemia in.F344 untreated rats, particularly males.
Table 3-4 presents an analysis of adenomas and carcinomas in the rats ex-
posed to PCE. Tubular cell adenomas or carcinomas were observed only in male
rats, but with a dose-related incidence that is not statistically significant.
Renal tubular adenomas were: in control, 1/49; 200 ppm, 3/49; 400 ppm, 2/50.,
Carcinomas were: in control, 0/49; 200 ppm, 0/49; 400 ppm, 2/50. The his-
torical incidences of renal tubular adenomas in the NTP studies were 4/1720, or
less than 0.2% ± 0.7%; no malignant tubular cell carcinomas have been observed
historically. In addition, dose-related increased incidences of renal.tubular
karyomegaly were observed in both male and female rats. Tubular cell hyper-
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TABLE 3-3. CLASSIFICATION OF!NONONUCLEAR CELL LEUKEMIA IN RATS
EXPOSED TOJPCE BY INHALATION
Group
Males
Control
200 ppm
400 ppm
Females
Control
200 ppm
400 ppm
Number j
of rats [
with <
mononuclear \
cell leukemia
28
37
37
18
30
29
1
1
1
5
6
4
3
6
2
Stage
2
3
7
6
5
6
6
3
20
24
27
10
18
21
SOURCE: NTP, 1985.
3-8
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TABLE 3-4. TUBULAR CELL ADENOMAS AND CARCINOMAS
OF THE KIDNEY IN RATS EXPOSED TO PCE BY INHALATIONS
GrouP Control 200 ppma
400 ppma
Males
Tubular cell
adenomas
Tubular. cell
adenocarcinomas
Tubular cell
adenomas or
adenocarcinomas
1/49 (2%)
0/49 (0%)
1/49 (2%)
3/49 (6%)
0/49 (0%)
3/49 (6%)
p = 0.259&
2/50 (4%)
2/50 (4%)
4/50 (8%)
p = 0.070&
Females
Adenomas611 °/5° (°%) °/49 (°%) 0/5°
Tabular cell 0/50 (0%) 0/50 (0%) 0/50 (0%)
adenocarcinomas v '
"adenomas"^ °/5° (°%) °/49 (°%) 0/5° ^
adenocarcinomas
amstorical incidence at the Battelle Pacific Northwest Laboratories: 0/100-
historical incidence in NTP studies: 4/1,720, 0.2% ± 0.7%; no malignant '
tubular cell tumors were observed.
bp-values were calculated for tubular cell adenomas or carcinomas using the
Msher Exact Test.
SOURCE: NTP, 1985.
3-9
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plasias were also observed;'these may be preneoplastic lesions.
In the rats, there were no statistically significant increased incidences
I
of tumors In other organs examined.; In particular, no neoplastic changes were
observed in the respiratory tracts of either male or female rats. However,
there was an increased incidence ofjsquamous cell metaplasia in the nasal cavi-
ties of the exposed male rats (0/50), control; 5/50, 200 ppm; 5/50, 400 ppm).
3.3. RESULTS OF THE MOUSE STUDY
i
I
Figure 3-3 presents the growth!curves for male and female mice exposed to
i
PCE by inhalation for 2 years. As indicated in the figure, there was no signi-
ficant change in the mean body weights of dosed animals as compared with con-
trols. The numbers' of mice surviving the 2-year inhalation study of PCE are
[
shown in Table 3-5 and Figure 3-4. [The survival of male mice was significantly
reduced at both 100 and 200 ppm PCE exposures, as compared with controls: low
dose, 25/50 (50%); high dose, 32/50j(64%); control, 46/50 (96%). In female
I
mice, survival was reduced at 200 ppm as compared with controls: low dose,
31/50 (62%); high dose, 17/50 (34%); control, 36/50 (72%). Survival of male
control mice was quite high. The high early mortality observed in female mice
[31/50 (60%)] may be attributed to line high incidence of hepatocellular car-
cinomas. ;
The incidences of liver tumors 'in the male and female B6C3F1 mice exposed
i
to PCE are summarized in Table 3-6. In the male mice, PCE induced statisti-
i
cally significant increased incidences of hepatocellular carcinomas at both the
100 and 200 ppm exposure levels, as [compared with controls. These tumor inci-
I
dences were: low dose, 25/49 (51%) (p < 0.001); high dose, 26/50 (52%) (p <
r
0.001); control, 7/49 (14%). Hepatocellular adenomas alone were not statistic-
I
ally significant at either the 100 ppm or the 200 ppm exposure concentration.
i
The combined incidences of hepatocetlular adenomas and carcinomas were sta-
i . •
3-10
-------
z
< 35.0
ac
o
X
o
IU
30.0-
>- 25.0
O
o
< 20.0-
IU
15.0
D
40.0
CO
< 35.0
OC
O
30.0
> 26.0
Q
O
CD
,< 20.0
ui
15.0
8°
o
MALE MICE
n s UNTREATED
O « 1OO PPM
A B 200 PPM
15 30 45 «0 76
WEEKS ON STUDY
90
105
S
a
A O _
O D O O °
O A
FEMALE MICE
D•UNTREATED
OB 100 PPM
A K 200 PPM
0 16 30 45 «0 76
WEEKS ON STUDY
90
105
ear' Gr°Wth curves for mice exposed to PCE by inhalation
SOURCE: NTP, 1985.
3-11
-------
TABLE 3-5. SURVIVAL OF MICE
IN THE NTP Z-YEAR IINHALATION BIOASSAY OF PCE
Group
Control
100 ppm
200 ppm
Males ,
i
Animals initially in study 50 i
i
Nonaccidental deaths 3 [(6%)
Animals missexed 1 |(2%)
i
Killed at termination 46 (92%)
i
Females
Animals initially in study 50
I
Nonaccidental deaths 11 !(22%)
Accidentally killed 2 |(4%)
Animals missexed 1 j(2%)
i
Killed at termination 36 |(72%)
i
aTwo mice died during termination period.
50
25 (50%)
0 (0%)
25 (50%)
50
17 (34%)
2 (4%)
0 (0%)
31 (62%)
50
18 (36%)
0 (0%)
32 (64%)
50
30 (60%)
1 (2%)
0 (0%)
17 (34%)a
3-12
-------
•.9
O.7-
M.
O
I- O.C +
0.6-
O
c
0.4-
O.3
0.8-
-0.8-
0.7-
t o.«H
O.6-
0.4-
0.34
0
*1
^
MALE MICE
O =* UNTREATED
O=100 PPM
A = 200 PPM
00
16 SO 46 CO 76
WEEK* ON STUDY
00 105
n -----
FEMALE MICE
D = UNTMEATEO
0=10O PPM
A = 200 PPM
*
-------
TABLE 3-6. LIVER TUMORS IN[MICE EXPOSED TO PCE BY INHALATION
Group
Control3
100
200 ppma»b
Males
Hepatocellular adenoma
Hepatocellular carcinoma
Hepatocellular adenoma
or carcinoma
Females
11/49j(22%)
7/49 j(14%)
16/49 l(33%)c
8/49 (16%)
N.S.
25/49 (51%)
p<0.001
31/49 (63%)d
p=0.002
18/50 (36%)
N.S.
26/50 (5.2%)
p<0.001
40/50 (80%)e
p<0.001
Hepatocel
Hepatocel
Hepatocel
or carci
lular
lular
lular
noma
adenoma
carcinoma
adenoma
3/48
(6%)
1/48 (2%)
4/48
™ 1 rt G ft O in rtrtm rt a 4* /*\ t^^ r*^\»-i*%/^f»j-***4" 4- W « mi i.-«l*. »»
(8%)
6/50
N.S.
13/50
p<0.
(12%)
(26%)
001
17/50 (34%)f
, p=0.001
2/50
N.S.
36/50
p<0.
38/50
p<0.
(4%)
(72%)
001
(76%)
001
— —,^..,.,.v.~w. o i v.^1 v^jtm, one nuiiiuci 3 of animals examined histologically.
Dp-values were calculated using the Fiisher Exact Test.
^Controls, male - 2 mice had both adehoma and carcinoma.
°Low dose, male - 2 mice had both adenoma and carcinoma.
=High dose, male - 4 mice had both adenoma and carcinoma.
TLow dose, female - 2 mice had both adenoma and carcinoma.
N.S. - not significant.
SOURCE: NTP, 1985. \
3-14
-------
tistically significant as compared with the controls. The incidences were:
low dose, 31/49 (63%) (p = 0.002); high dose, 40/50 (80%) (p < 0.001); control,
16/49 (33%).
In female mice, PCE induced a statistically significant increased inci-
dence of hepatocellular carcinomas at the 100 and 200 ppm exposure concentra-
tions, as compared with the controls. These incidences were: low dose, 13/50
(26%) (p =< 0.001); high dose, 36/50 (72%) (p < 0.001); control, 1/48 (8%).
Incidences of adenomas were not statistically significant. However, the com-
bined incidences of adenomas and carcinomas were statistically significant as
compared with controls. The incidences were: low dose, 17/50 (34%) (p =
0.001);;high dose, 38/50 (76%) (p < 0.001); control, 4/48 (8%). Carcinogenic
effects were not seen in any of the other organ sites, including the respira-
tory system. In addition, dose-related increases in liver degeneration,
hepatocellular necrosis, and hepatocellular nuclear inclusion were observed
in both male and female mice exposed to PCE. Dose-related renal tubular cell
karyomegaly was observed in the kidneys of both male and female mice, but no
dose-related tubular cell carcinomas were found in the mice.
The NTP mouse inhalation bioassay further confirms the earlier National
Cancer Institute study in mice (NCI, 1977) in which PCE induced a statistically.
significant increased incidence of hepatocellular carcinomas by gavage.
The NTP (1985, draft) study was audited by Dynamac Corporation for complete-
ness, consistency, accuracy, and for procedures consistent with good laboratory
practice requirements. The audit revealed no major problem in the conduct of
the study or in the collection and documentation of the experimental data.
3.4. SUMMARY
In the NTP (1985, draft) inhalation bioassay, groups of 50 male and 50
female F344/N rats and B6C3F1 mice were exposed to PCE (99.9% pure), 6 hours/
3-15
-------
day, 5 days/week, for 2 years. The exposure concentrations used were 0, 200,
and 400 ppm for rats and 0, 100, and 200 ppm for mice. At the 200 and 400 ppm
exposure concentrations, PCE induced |a marginally statistically significant
j
increase of mononuclear cell leukemia in male rats. PCE also induced an in-
t
creased incidence of renal tubular cell adenomas or carcinomas (combined) in
male rats. This incidence shows a do[se-related trend, although it is not
statistically significant.
In female rats at the 200 and 400 ppm exposure concentrations, a statis-
tically significant increased incidence of mononuclear cell leukemia was ob-
served. In male and female B6C3F1 mice, PCE induced a statistically signifi-
cant increased incidence of hepatocelflular carcinomas and/or adenomas at both
the 100 and the 200 ppm exposure concentrations.
3-16
-------
• 4. QUANTITATIVE RISK ASSESSMENT
This section updates the inhalation unit risk estimate for PCE reported in
the July 1985 Health Assessment Document for Tetrachloroethylene by using data
from the NTP (1985) inhalation bioassay, which is the first lifetime inhalation
study to show evidence of the carcinogenicity of PCE in mice and rats. These
data provide a basis for directly estimating the risk of PCE by the inhalation
route, and an opportunity to test the reasonableness of the July 1985 risk
estimate, which was developed using the gavage data from the NCI bioassay
(1977).' The inhalation unit risk estimate for PCE based on tumor incidence
data from a gavage study in mice and the assimilated dose metabolized, was
accomplished by utilizing metabolism information for both animals and humans,
expressing dose as the amount of PCE metabolized. On this basis the reported
inhalation unit risk is 4.8 x 10-7/yg/m3. This estimate was found to be
comparable to three other estimates using different data bases and/or different
methods of calculation. The details of these calculations are presented in the
Health Assessment Document (U.S. EPA, 1985, pp. 9-53 .to 9-61).
The new unit risk estimate for inhalation also utilizes metabolized dose-
tumor incidence relationships derived from exposure dose-metabolism relationship
data in both animal and human experiments. The uncertainty associated with ex-
trapolating from animals to humans is reduced by the use of actual human expo-
sure dose-metabolism relationship estimates, using urinary trichloroacetic acid
as a measure of metabolism, in addition to the use of comparable animal data.
For comparison, we have also used a physiologically-based pharmacokinetic
model to predict the amount of PCE metabolized in the NTP bioassay animals.
4.1. DATA AVAILABLE FOR RISK CALCULATION
The NTP (1985) inhalation bioassay shows evidence of the carcinogenicity
4-1
-------
of PCE in rats (mononuclear cell leukemia) and mice (hepatocellular adenomas
and carcinomas). The assays for botlh species have been selected for estimation
of carcinogenic risk to humans for inhalation exposure.
4.1.1. NTP Rat Inhalation Assay !
Long-tern (103 weeks) carcinogenicity studies were conducted by inhalation
exposure (0, 200, and 400 ppm, 6 hours/day, 5 days/week) on groups of 50 male
and 50 female Fischer 344/N rats. The animals were placed on study at 8 to 9
weeks of age and killed at 112 to lib weeks of age.
Table 3-2 shows the overall incidence rates for mononuclear cell leukemia.
Although the survival of male rats in the hiyh-dose group is significantly
reduced when compared to the control group, the difference appears to be signi-
ficant only at the late stage (after! 80 weeks) of the experiment. Because the
survivalship differs only at the Iat4 stage of the experiment and a large pro-
[
portion of leukemias were found at the termination of the bioassay, it is more
appropriate to use the incidence rat6 data, rather than the time-to-death data,
to calculate the lifetime cancer risk.
4.1.2. NTP House Inhalation Assay '
- _ . .
Lifetime (103 weeks) carcinogenicity studies were conducted by inhalation
exposure (0, 100, and 200 ppm, 6 hourfs/day, 5 days/week) on groups of 50 male
t
and 50 feniale B6C3F1 mice. The animals were placed on study at 8 to 9 weeks
I
and killed at 112 to 113 weeks. Exposure to PCE at 100 or 200 ppm reduced
survival of male mice, and at 200 ppm reduced survival of female mice. Most
of the deaths in treated animals occurred after week 82, and there is evidence
that the unscheduled deaths may have been influenced by the high incidence of
hepatocellular carcinomas. However, !for the same reasons as discussed for
rats, the incidence data are considered more appropriate for risk calculation
than the time-to-death data. Table 3|-4 shows the overall incidence rates for
4-2
-------
hepatocellular adenomas and carcinomas/which, combined, show a dose-related
incidence of hepatocellular neoplasms in both male and female mice.
4.2. POSSIBLE MECHANISMS LEADING TO A CARCINOGENIC RESPONSE FOR PCE
Possible mechanisms proposed for carcinogenesis include direct interaction
of a chemical or its metabolites with DNA, long-term tissue injury, stimulation
of cell, proliferation, immunosuppression, hormonal imbalances, or release of
altered cells from growth control (Weisburger and Williams, 1980, 1981). The
mechanism(s) by which PCE causes cancer is not known and therefore a matter of
some speculation. The current knowledge of PCE metabolism and the acute cell-
ular toxicity directly related to reactive metabolites suggest different cell-
ular processes that may possibly lead to carcinogenic activity. One process
may involve cell death induced by the cellular toxicity of PCE followed by the
consequent stimulation of DNA replication associated with cell multiplication,
resulting in an indirect or promoting effect. The continuing process of DNA
replication results in increased proportions of single-strand DNA, which is
more susceptible to irreversible binding by reactive intermediates than is
double-strand DNA. Alternatively, it is possible that PCE acts by stressing
the fidelity of replication, thus increasing the possibility of introducing DNA
transcript errors. A general process proposing that necrosis constututes a
necessary precursor for PCE liver carcinogenicity is consistent with a thresh-
old effect at the necrotizing doses at which all death and turnover occur. It
is also consistent with the negative or inconclusive results of the majority of
mutagenicity assays.
Peroxisome proliferation has been linked with cancer in rodents (Moody and
Reddy, 1978; Reddy et al., 1980; Prout et al., 1985; Green and Prout, 1985). •
Trichloroacetic acid (TCA) resulting from the biotransformation of PCE has been
shown to induce liver peroxisome proliferation in mice but not in rats (Green,
4-3
-------
1986). It has been hypothesized that the species difference in the hepatocar-
cinogenicity of PCE seen between rats and mice is possibly due to a species
difference in peroxisome proliferation resulting from higher TCA blood levels
I ,
in the mice (Green, 1986). |
Increased reactive oxygen species formed by the peroxisomes may result in
DNA damage, or the peroxisome proliferation may result in formation of the low
amounts of DNA alkylation that have been observed following exposure to PCE.
However, a cause-and-effect relationship between liver peroxisome proliferation
and PCE hepatocarcinogenicity in mice! has not been established. This mechanism
does not account for the mononuclear cell leukemias and renal tubular neoplasms
seen in rats.
Another general process that may[ lead to carcinogenicity involves the
f
metabolic production of active PCE metabolites (tetrachloroethylene oxide) and
I
their direct interaction with DNA. This genotoxic process could be expected
1
to be modulated by various' homeostatic mechanisms, such as DNA repair and im-
munological surveillance, but it is generally regarded as lacking a threshold.
This mechanism is supported by the mujbagenicity of the PCE-epoxide and the fact
that DNA binding of PCE metabolites has been observed, even though only at low
doses.
In the absence of definitive evidence solely supporting any specific one
of the likely processes operative in the carcinogenic activity of PCE, the risk
assessment performed by the Carcinogenic Assessment Group considers the process
which is generally accepted—the genotoxic mechanism—to be associated with the
greatest risk. The risk of PCE carcinogenicity by this process, with its lack
of threshold, is appropriately estimated by a mathematical model predicting
zero incremental risk only at zero exposure. In addition, at the present time,
the lack of sufficient understanding about the mechanisms of action involved
4-4
-------
in carcinogenesis does not allow for a distinction between chemicals acting
directly with DNA and those which do not, nor can those which have not been
shown to be genotoxic be considered to have identifiable population thresh-
olds or to be safer than those which are considered to be genotoxic (Pereira,
1984). More research is required on the mechanism(s) of action of PCE before
a threshold model would be appropriate for carcinogen risk assessment (Pereira,,
1984).
4.3. RELEVANCE OF METABOLISM TO QUANTITATIVE RISK ASSESSMENT
The general considerations involved in the scaling of toxicologic effects,
including carcinogenicity, among species (including man) have been outlined for
PCE in the Health Assessment Document for Tetrachloroethylene (U.S. EPA, 1985),
which also presented information on the kinetics, metabolism, and covalent
binding properties of PCE in rat and mouse species and metabolism and kinetics
in humans. This information may be summarized as follows:
(1) .There is evidence that the liver and kidney toxicity and the carci-
nogenic potential of PCE and other halogenated ethylenes are dependent on the
metabolic conversion of these compounds to reactive epoxides and other bio-
logically: reactive intermediates of metabolism (Figure 4-1) (Bolt et a!., 1982;
U.S. EPA, 1985, Chap. 5).
(2) The reactive metabolites .of PCE bind irreversibly to cellular macro-
molecules jm vitro and jm \nvo_ (Costa and Ivanetich, 1980; Pegg et a!., 1979;
Schumann et al., 1980). Significant binding to hepatic DNA has not been
demonstrated to occur in. vjrvo (Schumann et al., 1980). Irreversible binding
to other cellular macromolecules (proteins and lipids) in vivo occurs indepen-
dently of route of administration (gavage or inhalation) but proportionally
to the amount metabolized (Pegg et al., 1979; Schumann et al., 1980). Indices
of hepatocellular toxicity and cellular damage have been shown to correlate '
4-5
-------
CHLORIDE
MIGRATION
DECARBOXYLATION
+H Cl
TCA
CO
Figure 4-1. Postulated scheme for the metabolism of PCE.
SOURCE: U.S. EPA, 1985.
4-6
-------
linearly with metabolism (Buben and O'Flaherty, 1985).
(3) The metabolism and kinetics of PCE have been studied in three species:
mice, rats, and humans. In each of these species there is evidence that metabo-
lism of PCE after oral (mice, rats) or inhalation exposure (mice, rats, humans)
is rate-limited and proceeds according to Michaelis-Menten kinetics (Filser and
Bolt, 1979; Pegg et al., 1979; Schumann et al., 1980; Buben and O'Flaherty, '
1985; Ohtsuji et al., 1983; U.S. EPA, 1985, Chap. 5). There is no comparative
experimental evidence that the metabolic pathway for PCE qualitatively differs
for the above-mentioned species. The metabolic capacity for the metabolism of
PCE by mice and rats has been found to be proportional to body surface area for
these two species (U.S. EPA, 1985, Chap. 9).
(4) Elimination of PCE following inhalation exposure in humans is tri-
phasic, with a terminal first-order elimination half-time of 55 to 65 hours;
for the rat, the dominant half-time of elimination is 7 hours after inhalation
or oral exposure. These long half-times indicate that with chronic daily
exposures in humans or animals (conditions of carcinogenicity bioassays) com-
plete elimination (by metabolic and pulmonary routes) would not occur within
the 24-hour dosing interval and that body accumulation would occur until a
plateau of equilibrium is reached between body concentration and inhalation or
oral exposure dose.
4.4. INHALATION EXPOSURE IN HUMANS
PCE is readily absorbed through the lungs into the blood by first-order
passive diffusion processes. Two major processes account for the known eli-
mination of PCE from the body: 1) pulmonary excretion of unchanged PCE, and
2) metabolism of PCE to urinary metabolites. The metabolism of PCE appears
to be very limited in humans, as it is in experimental animals. Only a small
percentage of the estimated amounts absorbed by inhalation are metabolized to
4-7
-------
trichloroacetic acid (TCA) and other chlorinated metabolites found in the urine
|
(U.S. EPA, 1985, Chap. 5). Saturation of metabolism has been estimated to
occur in humans at relatively low exposure concentrations (between 100 and 400
ppm PCE in inspired air). Estimates of the extent of PCE metabolism in humans
have been made from balance studies by measuring urinary metabolites after
accounting for a retained inhalation exposure dose (U.S. EPA, 1985, Chap,. 5).
I
However, there are several difficulties associated with balance studies in
humans. For example, problems are encountered'in obtaining an accurate measure-
i
ment of the retained dose of PCE from!inhalation exposure. In addition, the
available balance studies in humans are incomplete. One shortcoming in some of
the older PCE studies has been the imprecision of the analytical methodologies
!
used; for example, the Fujiwara reaction for measuring metabolites. Also,
urinary sampling may not have been sufficient considering the long half-life of
PCE metabolites. In addition, individual variations among subjects can be
i
relatively high. Only urinary TCA was used in these studies as a measure of
metabolism, and the possibility exists that significant amounts of metabolites
other than TCA are excreted in the urine or by other routes. It has been
proposed that a much higher percentage of the dose is actually metabolized, and
that a so-far unrecognized pathway may exist for PCE and for other metabolites
i
which have not yet been identified, However, measuring the amount of urinary
I
TCA is one approach that may be used to compare the metabolized dose of PCE
among species. The metabolized dose may be considered to be the effective dose
in organ toxicity and carcinogenicity.
4.5. CALCULATION OF METABOLIZED DOSE SFROM INHALATION STUDIES
4*5.1. Direct Estimation from Metabolism Experiments
4.5.1.1. RaJ>-Pegg et al. (1979) and Schumann et al. (1980) determined body
burdens of Sprague-Dawley rats and B6q3Fl mice after 6-hour inhalation expo-
I
4-8
-------
sures. The animals were exposed to 14C-PCE and the amount metabolized as a
fraction of the body burden was estimated from radioactivity in urine, feces,
expired air, etc., and unchanged 14C-PCE in exhaled air. Their results for
exposed rats are given in Table 4-1.
Total recovery of 14C-radioactivity from 6-hour inhalation of 10 ppm an,d
600 ppm l^C-PCE 1n rats was nearly directly proportional to exposure concen- -
tration; However, the absolute amount metabolized (10 ppm, 0.47 mg-equiva-
lents; 600 ppm, 9.11 mg-equivalents PCE) was not proportional to exposure con-
centration and decreased both in absolute proportions and as a percentage of the
total assimilated dose (10 ppm, 32%; 600 ppm, 12%). Thus, these data from rats
confirm in this species limited and saturable metabolism of PCE consistent with
dose-dependent Michael1s-Menten kinetics, as has been indicated by kinetic in-
halation studies of Filser and Bolt (1979). These data are also in accord with
the demonstration by Buben and O'Flaherty (1985) of dose-dependent Michaelis-
Menten kinetics for PCE in mice.
The relationship between the rate of PCE metabolism in adult male Sprague-
Dawley rats (or the amount of PCE metabolized per unit body weight per 6-hour
exposure, M) and exposure concentration dose, d (ppm), can be expressed by a
Michaelis-Menten-like equation
M = V x d/(K + d)
where V and K are constants to be estimated from the data. In order to utilize
the human data (metabolites in urine) for interspecies extrapolation, the amount
of urinary metabolites in animals is used to establish the dose-metabolism rela-
tionship. Using the data of urinary metabolites for rats and assuming Fischer
344 rats metabolize PCE qualitatively similar to Sprague-Dawley rats with inha-
4-9
-------
TABLE 4-1. RECOVERY 0F 14C-PCE RADIOACTIVITY
AFTER INHALATION EXPOSURE FOR 6 HOURS
TO SPRAGUE-DAWLEY RATS AND B6C3F1 MICE*
Rats (avl of 3)a
10 ppm | 600 ppm
(mg-equivalents PCEb)
Mice (av. of 3)
10 ppm
Expired unchanged
Metabolized
14co2
Urine
Feces
Carcass
Total
1.008 (68%)C
1
0.053
0.275 ;
0.076
0.063
0.467 (32%)c i
1.475
68.39 (88%)c
0.54
4.54
2.36
1.67
9.11 (12%)C
77.50
0.048 (12%)c
0.032
0.285
0.027
0.012
0.356 (88%)c
0.404
aAverage experimental animal weights (drams): 250, rat; 24.5, mouse. The
animals were exposed to 10 or 600 ppm ^C-PCE for 6 hours and maintained in
metabolism cages for 72 hours after exposure.
"Each value represents the mean of data from three animals.
cNumbers in parenthesis indicate the fraction of total administered dose.
i
SOURCES: Adapted from Pegg et al., 197;9 and Schumann et al., 1980.
4U10
-------
latloh exposure (Table 4-1), the dose-metabolism relationship is determined to
be
M (mg/kg) = V x d (ppm)/(K + d)
with V and K estimated to be 24.64 mg/kg and 213.96 ppm, respectively.
This relationship is used to estimate the amount of PCE metabolized for a 6-
hour exposure at 200 or 400 ppm in the NTP (1985) rat bloassay. The amounts
metabolized (urinary excretion) are 11.90 mg/kg/day and 16.05 mg/kg/day for
6-hour exposures, corresponding, respectively, to 200 and 400 ppm ambient air
concentrations. It may be noted that doubling the daily inhalation exposure
concentration from 200 to 400 ppm results in only a 30% increase in the amount
of PCE metabolized. The two doses represent approximately 48% and 65% of
total metabolic capacity. These observations may, in part, explain the fail-
ure to observe an exposure dose-leukemia response relationship for this rat
bioassay.
4.5.1,2. Mouse-The dose-dependent Michaelis-Menten kinetics for the metabol-
ism of PCE by mice after oral administration have been amply demonstrated by
Buben and O'Flaherty (1985). For inhalation exposure, however, few data are
available pertinent to the conditions of the NTP (1985) bioassay, from which to
derive a comparative relationship between exposure concentration and metabol-
ism, as was done in the Buben and O'Flaherty (1985) oral study.
Watanabe and associates (Pegg et al., 1979; Schumann et al., 1980) assess-
ed the amount of PCE metabolism accrued from a 10 ppm, 6-hour exposure to
B6C3F1 mice (Table 4-1). The metabolism of PCE in mice was not determined at
other exposure concentrations. The exposure levels, of the NTP inhalation bio-
assay in mice were 10- and 20-fold higher. At the 10 ppm exposure level, the
absolute amount of metabolites in urinary excretion is comparable between the
4-11
-------
two species; however, expressed in mg/^kg, the amount of PCE metabolites is
about 10 times greater for mice than for rats (11.6 mg/kg vs. 1.1 mg/kg). This
ratio should not be expected to hold at higher concentrations because at higher
concentrations the metabolic rate is cjominated by Vmax in the Michaelis-Menten
equation. Theoretically, the ratio of Vmax between two species equals unity if
i
Vmax is expressed in terms of metabolic rate as represented by mg/W2' , or, if
Vmax is expressed in terms of mg/kg, is equal to (W1/W2)1/3 where W's are body
i
weights for animals of each species. Therefore, at high doses one might expect
to see that mice metabolize about twice the amount of PCE, in terms pf mg/kg,
i
as do rats [i.e., (0.25/0.0245)1/3 = 2.2]. This ratio is supported by the
observation from oral studies (Table 4-2) that at 500 mg/kg, the ratio of the
i
absolute amount of urinary PCE metabolites between mice and rats is 0.267,
or 2.7 on a mg/kg basis [i.e., (1.53/0.0245)7(5.72/0.25) = 2.7]. The amount
metabolized of a 500 mg/kg oral dose in rats is approximately equal to the
amount metabolized of a 600 ppm inhalation exposure in this species. To calcu-
i
late the metabolized dose for the NTPjmice, it is assumed that mice metabolize
approximately 5 times (in terms of mg/kg) more than rats at the cancer bioassay
doses of 100 and 200 ppm on the basis[that mice metabolize greater amounts (in
I
terms of mg/kg) than rats, ranging from 10 times greater at 10 ppm to 2 to 3
i
times greater at higher doses. There-Tore, the metabolized doses of PCE for
mice, based on urinary metabolites, corresponding to 100 and 200 ppm 6-hour
exposures are, respectively, 39.24 mg/kg/day and 59.50 mg/kg/day—or approxi-
I
mately five times the values calculated from the dose-metabolism relationship
for rats, when expressed in terms of mg/kg.
j
i
4.5.2. Metabolized Dose Predicted by a PB-PK Model
In addition to the (urinary) metabolized doses that are calculated direct-
I
ly from the empirical data and are prjesented in the previous sections, we have
: 4-12
-------
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also calculated the amount metabolized by using the physiologically-based phar-
macokinetic (PB-PK) model. An explanation of the construction of the model is
given in Appendix A. The PB-PK model I predicts only the total amount metabo-
lized, not the urinary metabolites as j previously calculated^. The metabolized
I
doses predicted by the PB-PK model are comparable to those calculated directly
i
from the empirical data (Table 4-3), considering the fact that urinary metabo-
lites account for about 50% to 80% of the total metabolites and the fact that
the metabolized doses for mice are slightly underestimated (approximately three
times), by the PB-PK model, as discussed in section A.3.2.2. of Appendix A.
_
4."6. HUMAN METABOLIZED DOSE AT LOW-LtVEL AMBIENT EXPOSURE
i
Estimation of the extent of metabolism in humans have been made from sev-
eral balance studies by accounting for a retained dose after inhalation expo-
sure through measuring metabolites excreted in the urine. The difficulties
associated with these balance studies Jin humans have already been mentioned in
section 4.3. However, urinary TCA measurements can reasonably be used to make
species comparisons of metabolized dose. The data in humans are represented
here by studies selected on the basis of the lower exposure levels more com-
mensurate with anticipated environmental concentrations.
A study by Bolanowska and Golacka (1972) provides some information on the
relationship between the air concentration of PCE and the amount metabolized in
urinary excretion. In this study, five subjects were exposed to 390,000 pg/m3
of PCE (approximately 50 ppm) for 6 hours. The excretion rate of metabolites
in urine (mg/hr) is presented graphically over' the 20-hour period during and
after exposure. The total amount of ryietabolites in urine (area under curve)
is estimated to be about 13 mg. The area under the curve from 20 hours to
i
infinity is calculated by C x ti/2/0.693, where C is the concentration at the
t
last sampling time (i.e., 20 hours) and ti/2, the half-life, is assumed to be
4-14
-------
TABLE 4-3. COMPARATIVE ESTIMATES OF METABOLIZED DOSES
IN THE NTP (1985) INHALATION BIOASSAY
Total
metabolites
Exposure Direct estimate of estimated by
concentration urinary metabolites PB-PK model
SPecies (PPm) (mg/kg) (mg/kg)*
Rats
Mi ce
200
400
100
200
11.90
16.05
39.24
59.50
19.39
28.57
19.77b
30.00b
The PB-PK model can predict only the total amount of metabolites. The values
are total metabolized dose over 72 hours after a single 6-hour exposure.
These values are presented here for the purpose of comparison only: they are
not used for risk calculation. J
DThe metabolized doses for mice are slightly underestimated (approximately 3
times) due to the use of under-valued physiological parameters, in particular
the alveolar ventilation rate (see section A.3.2.2. in Appendix A).
4-15
-------
100 hours. Under the assumption that the amount metabolized is linearly pro-
portional to the air concentration below this concentration, the amount meta-
bolized (in urine) associated with 1 pg/m3 of PCE in air is 13 mg/390,000 =
3.33 x 10~5 mg for a 6-hour exposures.
Another study that supports the above estimate is that of Fernandez et al,
(1976) in which humans were exposed! to PCE vapor at 150 ppm for 8 hours. The
i
quantity of PCE metabolized and eliminated in urine over 72 hours was about
25 mg. This is clearly less than the total amount metabolized from the 8-hour
I
exposure because of the long half-ljife in the final phase of urine elimination.
As an approximation, the (urinary) metabolized dose associated with 1 pg/m3
of PCE in air is calculated to be
i
1 ug/m3 x (25 mg/150 ppm) x (6 hr/8 hr)/6908/ug/m3/ppm = 1.8 x 10-5
mg
for a 6-hour exposure, given the fatt that 1 ppm = 6908 yg/m3. This value is
less than the value of 3.33 x 10~5 mg calculated on the basis of Bolanowska and
Golacka (1972). The value 3.33 x lO'5 mg/6 hours will be used for risk calcu-
lation because it is obtained from a lower concentration (50 ppm), where the
assumption that the dose-metabolism:relationship is linear is not unreasonable.
4.7. CALCULATION OF UNIT RISK
i
4:7.1. Based on the Metabolized Dose Estimated Directly from the Empirical
Data
In this section, the incremental lifetime cancer risk over background due
to 1 yg/m3 of PCE in ambient air (iie., unit risk) is calculated. The calcu-
i
lation procedure, the interpretation, and the appropriate use of the concept of
unit risk were presented in the Health Assessment Document for PCE (U.S. EPA,
1985). The data used for the risk calculation are given in Tables 4-4 and 4-5.
4-16
-------
TABLE-4-4. NTP METABOLIZED DOSE
AND INCIDENCE OF MONONUCLEAR CELL LEUKEMIA
IN FISCHER 344 RATS
Group
Males
Exposure
concentration
(ppm)
0
200
400
Human equivalent
metabolized dose
(mg/W2/3/day)a
0
6.26
8.45
Incidence
rate
28/50
37/50
37/50
Females 0 0 18/50
200 5.81 30/50
400 7.84 29/50
aMetabolized dose = M(mg/kg/day) x (5/7) x wl/3, where M(mg/kg/day) is the
direct.estimate of the dose metabolized taken from Table 4-3- the factor 5/7
reflects that animals were exposed only 5 days/week; and W = 0.40 kg fon male
rats and 0.32 kg for female rats. For convenience, the notation mg/W2/3 is
used to indicate the amount metabolized in terms of mg per two-thirds of body
weight, which is assumed to be proportional to the body surface area.
4-17
-------
TABLE 4-5. NTP METABOLIZED DOSE
AND INCIDENCE OF'LIVER TUMORS IN MICE
Group
Exposure
concentration
(ppm)
Males
0
100
200
Human i
equivalent I
metabolized
dose
(mg/W2/3/day)a
0
9.37
14.21
Incidence rate
Carcinomas Adenomas/carcinomas
7/49b
25/47
26/50
16/49
31/47
40/50
Females
0
100
200
0 ;
8.92
13.52
1/46
13/42
36/47
4/46
17/42
38/47
Metabolized dose = M(mg/kg/day) x (5/7) x W1/3, where M(mg/kg/day) is the
direct estimate of metabolized dose taken from Table 4-3; 5/7 is a factor
to adjust for 5 days exposure in a we'ek; and W. = 0.0374 kg for male mice
and 0.0322 kg for female mice. \
bOnly animals surviving beyond 60 week's are included in the denominator. The
first death from liver tumor occurred at 60 weeks.
4-18
-------
The NTP bioassay doses for rats and mice have been converted into metabolized
doses using the dose-metabolism relationship previously established. The
metabolized dose, expressed in mg/W2/3/day, where W is the body weight in kg,
is assumed to be "equivalent" (equally potent) among species. The carcinogenic
potencies (slopes) of PCE are calculated using the multistage model on the
basis of "equivalent" doses and their corresponding tumor incidence rates. The
slope Is defined as the 95% upper confidence limit of the linear parameter in a
multistage model. These slope estimates are presented in Table 4-6, and have
been used for calculating the unit risk of PCE. It should be noted that these
slope values are expressed in terms of "equivalent" doses. These values are
only used in the intermediate steps of the derivation of the risk to humans due
to 1 ug/m3 of PCE in air. The slope estimates calculated on the basis of six
different data sets are comparable, ranging from 3.64 x 10-2/(mg/w2/3/day) to
1.21 x 10-1/(mg/W2/3/day). It is possible to express slopes in terms of ppm
ambient air concentrations by using the dose-metabolism relationship previously
determined. Since such a conversion does not affect our derivation of risk to
humans, it suffices to point out that slope values per ppm for mice are greater
than those for rats because the rate of metabolism is greater in mice than in
rats.
To calculate the human risk due to 1 yg/m3 of PCE in air, it is neces-
sary to estimate the corresponding amount metabolized in humans. As previously
estimated, the amount metabolized is 3.33 x 10'5 mg when an individual is ex-
posed to 1 yg/m3 of PCE in air for 6 hours. As an approximation, the amount
metabolized corresponding to a continuous (24 hours) exposure to 1 yg/m3 of
PCE in air is 3.33 x 10-5 mg x (24 hours/6 hours) = 1.33 x 1(H.ing/day, or 7.83
x lO-6 mg/W2/3/day for a 70-kg person.
An upper-bound estimate of the incremental risk due to 1 yg/m3 of PCE .
4-19
-------
(i.e., unit risk) is calculated as 7.83 x 10~6 x slope. The risk estimates
calculated on the basis of six different data sets (Table 4-6) are comparable,
ranging from 2.85 x 10"? to 9.47 x 10["7, with a geometric mean equal to 5.78
x 1Q~7. All of these values are comparable to our previous unit risk estimate
l
of 4.8 x 10~7, which is based on hepajtocellular carcinomas in mice in the NCI
(1977) gavage study.
4.7.2. Based on the Metabolized Dosej Calculated from the PB-PK Model
i
For comparison, the inhalation unit risk for PCE is also calculated on the
basis of metabolized dose derived frorn the PB-PK model, which has gained in-
i
creasingly broad acceptance over the [last few years as an effective tool for the
study of the absorption, distribution, biotransformation, and elimination of
chemicals in the body. However, due jto the lack of data, some of the parameters
that are used in the modeling (including the Michaelis-Menten kinetic parameters
Vm and Km) must be estimated from thej same data that were used in the approach
presented in the previous section. For this reason, the model so derived is
not necessarily more reliable than th'at approach to predict the amount metabo-
lized in the NTP bioassay. The metabblized dose (mg/W2/3/day) in the NTP study,
and the corresponding tumor incidence! rate used in the risk calculation, are
i
presented in Tables 4-7 and 4-8. Tab^le 4-9 presents the resultant unit risk
estimates calculated from the six difjferent data sets. The risk calculated by
I
this approach is also comparable to those calculated previously (Table 4-6),
considering the fact that the risk calculated on the basis of mouse data is
I
overestimated due to the underestimation of the metabolized dose by the PB-PK
model (see section A.3.2.2. in Appendix A), in particular, when the risk
estimates (in terms of mg/W2/3) in the second columns of Tables 4-6 and 4-9
are compared. The risk estimates in the second columns are calculated directly
from animal data, and thus do not reflect the uncertainty associated with the
4-20
-------
TABLE 4-6. UNIT RISK ESTIMATES
CALCULATED ON THE BASIS OF DIFFERENT DATA SETS
Data set
Risk due to
one unit (mg/W2/3)a
of urinary
metabolized dose
Risk due to
1 pg/m3
of PCE in air
Rat (leukemia):
Males
Females
Mouse (liver carcinomas):
Males
Females
Mouse (liver adenomas/
carcinomas):
Males
Females
Geometric mean:
1.21 x 10-1
1.01 x 10-1
6.67 x 10-2
3.64 x ID'2
1.08 x 10-1
5.05 x ID"2
7.38 x 10-2
9.47 x 10-7
7.91 x 10-7
5.22 x 10-7
2.85 x 10-7
8.46 x 10-7
3.95 x 10-7
5.78 x 10'-7
aThe metabolized dose expressed in mg/w2/3 (wnere w is body weignt in kq) is
assumed to be equivalent, i.e., equally potent, among species.
4-21
-------
TABLE 4-7. NTP RAT METABOLIZED DOSE PREDICTED BY PB-PK MODEL
AND THE CORRESPONDING MONONUCLEAR CELL LEUKEMIA INCIDENCE RATE
Human
Exposure j equivalent
concentration metabolized dose
Sex (ppm) !(mg/W2/3/day)a Incidence rate
Males i
0 | 0 28/50
200 i 10.95 . 37/50
i
400 13.68 37/50
Females
0 0 18/50
I
200 10.16 30/50
400 1 12.70 29/50
aMetabo!ized dose (mg/W^/^/day) = d(mg/kg/day) x W^/3, where d - 14.86 mg/kg/
day and 18.57 mg/kg/day, respectively, for the 200 ppm and 400 ppm groups.
The body weight W is 0.4 kg for male [rats and 0.32 kg for female rats. For
convenience, d is derived on the basis of a 0.35 kg, rat, rather than calcula-
ted separately for 0..4-kg and 0.32-kg rats.
,4-22
-------
TABLE 41L xTP M°USE METABOLIZED DOSE PREDICTED BY PB-PK MODEL
AND THE CORRESPONDING LIVER TUMOR INCIDENCE RATE
SeX
Males
Females
Exposure
concentration
(ppm)
100
200
0
100
200
Human
equivalent
metabolized
Incidence rate
~
ion dose
(mg/W2/3/day)a
0
4.49
6.55
0
4.27
6.23
Carcinomas
7/49
25/47
26/50
1/46
13/42
36/47
Carcinomas/adenomas
16/49
31/47
40/50
4/46
17/42
38/47
4-23
-------
TABLE 4-9. UNIT RISK ESTIMATES BASED ON PB-PK PREDICTED METABOLIZED DOSE
Data base
Risk due to
one unit (mg/W2/3)
of total
metabolites
Risk to humans
due to 1 yg/rn3
of PCE in air
Method Ia
Method IIb
Rat (leukemia):
Males 7.32 x 10--2
Females 6.13 x 10~2
Mouse (liver carcinomas)0:
Males 1.42 x 10-1
Females 7.74 x 10~2
Mouse (liver adenomas/
carcinomas):
Males 2.30 ^ 10'1
j
Females 1.05 % 10"1
. i
Geometric mean: 1.03 x 10-1
3.40 x 10-6
2.85 x 10-6
6.60 x 10-6
3.60 x 10-6
1.06 x ID'5
4.88 x 10-6
4.77 x ID"6
1.15 x 10-6
9.62 x 10-7
2.23 x 10-6
1.21 x 10-6
3.60 x lO-6
1.65 x 10-6
1.61 x 10-6
aMethod I uses the human metabolized^dose actually predicted by the PB-PK
model. For a 70-kg person, the metabolized dose (at steady-state) corre-
sponding to 1 ug/m3 of PCE in air is 7.89 x 10~4 mg or 4.65 x .10-5 mg/W2/3.
The risk is calculated by multiplying 4.65 x lO'5 to the values in the second
column. i
bMethod II uses the metabolized dose [calculated from the direct observation of
humans by Bolanowska and Golacka (1972). As discussed in section 4.6.1., the
metabolites in the urinary excretion were estimated to be 7.83 x 10~6 mg/w2/3
for a 70-kg person exposed to 1 ug/m3 of PCE in air. If the urinary excre-
tion is assumed to account for 50% of the total metabolites, the metabolized
dose corresponding to 1 ug/m3 of PCE in air would be 1.57 x 10-5 mg/w2/3.
This value is multiplied in the second column to obtain the risk. The assump-
tion about the percentage of urinary! metabolites in the total metabolites is
based on the observation in rat and mouse studies where urinary metabolites
account for more than 50% of total metabolites.
cThe unit risks calculated on the basis of mouse data are somewhat over esti-
mated because the metabolized dose was underestimated (see section A.3.2.2.
in Appendix A for explanation).
4-24
-------
dose-metabolism relationship for humans.
4.8. DISCUSSION
In calculating the dose-response relationship for PCE, the amount metabo-
lized is considered to be an effective dose. The use of this surrogate effec-
tive dose may not eliminate the uncertainty associated with the low-dose ex- :
trapolation because the dose actually reaching the receptor sites may not be
linearly proportional to the total amount metabolized, and the shape of the
dose-response curve is still unknown. However, it seems reasonable to expect
that the uncertainty with regard to the low-dose extrapolation would be some-
what reduced by the use of the metabolized dose because the metabolized dose
better reflects the dose-response relationship, particularly in the high-dose
region. To extrapolate from animals to humans, the metabolized dose per body
surface area (which is proportional to W2/3) is assumed to be equivalent (i.e.,
equally potent) among species. This assumption appears to be reasonable, since
it is partially supported by the fact that the carcinogenic potency (i.e.,
slope) values are comparable between rats and mice when these values are ex-
pressed in terms of mg/W2/3. it" is interesting to note that the use of meta-
bolic and kinetic information, when available, improves the accuracy of risk
estimation, as evidenced by the fact that the PCE inhalation unit risk, calcu-
/
ulated previously on the basis of gavage data, is shown to be a reasonable
estimate when the newly released inhalation bioassay data are used.
The CAG has also employed the PB-PK model to calculate the amount metabol-
ized in the NTP bioassay. This modeling technique provides insight into vari-
ous assumptions used in the risk assessment. The most serious limitation for
the PB-PK modeling is that the metabolism data reported in the literature do
not represent periodic sampling over an interval of time. A detailed discussion
dealing with PB-PK modeling is presented in Appendix A. The risk calculated by
4-25
-------
this approach is also comparable to the previous risk estimates, considering
the fact that the risk calculated on the basis of mouse data is overestimated
f
due to the underestimation of the metabolized dose by the PB-PK model (see
section A.3.2.2. in Appendix A); in particular, when one compares the risk
i
estimates (expressed in terms of mg/W2/3) in the second columns of Tables 4-6
i
and 4-9, which are calculated from the animal data.
4.9. SUMMARY OF QUANTITATIVE ASSESSMENT
L
This section updates the inhalation unit risk estimate for PCE, using data
i
from the NTP (1985, draft) bioassay, which is the first lifetime inhalation
study to show clear evidence of the carcinogenicity of PCE in both rats and
mice. These data provide a basis foridirectly estimating the risk of PCE by
i
the inhalation route, and an opportunity to test the reasonableness of the
[
earlier risk estimate which was calculated using tumor incidence data from the
NCI gavage cancer bioassay (1977) and the assimilated dose metabolized. The
inhalation unit risk estimate reported in the 1985 Health Assessment Document
for Tetrachloroethylene was 4.8 x 10~7/ug/m3.
The new unit risk estimate for inhalation also utilizes the metabolized
i
dose-tumor incidence relationships derived from pertinent exposure concentra-
I
tion dose-metabolism relationship data in both animal and human experiments.
t
t
The uncertainty associated with extrapolating from animals to humans is reduced
by the consideration of actual human exposure concentration dose-metabolism re-
lationship data where urinary TCA is taken as an index of the amount of PCE
metabolized, in addition to the use of comparable animal data. It is of con-
siderable interest to note that the air unit risk estimates calculated on the
basis of animal inhalation carcinogenicity studies (rats and mice) are compara-
ble to estimates derived previously from the oral gavage cancer data in mice,
when a metabolized dose is used for the dose-tumor incidence relationships.
^4-26
-------
Furthermore, the risk per unit of metabolite is found to be comparable between
rats and mice even though the risk for mice corresponding to a unit of ambient
air concentration is higher than that for rats. The implication is that the
cancer risk might be overestimated if it were assumed that PCE in the ambient
air is equally potent among species. The upper-bound estimates of the incre-
mental lifetime cancer risk due to lifetime exposure to 1 yg/m3 of PCE in air
are calculated by three methods for six sets of dose-tumor incidence data. The
range of the six risk estimates under each of the three methods are as follows:
(a) 2.9 x ID'7 to 9.5 x 10-7/yg/m3; (b) 2.9 x IQ~6 to 1.1 x 10-5/pg/m3; and
(c) 9.6 x ID'7 to 3.6 x 10-6/yg/m3.
The first range of estimates is calculated on the basis of metabolized
doses that are derived directly from the single-exposure metabolism study,
assuming that the total amount metabolized over the 72-hour period from the
single exposure is comparable to the steady-state metabolized dose under the
NTP multiple-exposure pattern. The second and third ranges of estimates are'
calculated on the basis of metabolized doses that are derived from the physi-
ologically-based pharmacokinetic (PB-PK) model. Such models, which piece
together the relevant physiological and biochemical information, can be useful
tools for predicting absorption, distribution, biotransfonnation, and elimina-
tion of chemicals across species when sufficient data are available. However,
until more data become available, the CAG recommends that the first range of
of estimates be used to represent the inhalation risk for PCE, since there are
uncertainties with parameters used in the PB-PK model that were estimated from
the same empirical data set used in the direct estimation, and the model cannot
be considered to give a better estimate. That is, the upper-bound estimate of
the incremental cancer risk due to lifetime exposure of 1 yg/m3 of PCE in air
ranges from 2.9 x 10'7 to 9.5 x lO"7. This range includes the previous estimate
4-27
-------
of 4.8 x 10~7/yg/m3 derived from the NCI (1977) gavage data. Thus, the
inhalation risk estimated from gavagejcarcinogenicity data is supported by the
inhalation risk developed from the use of inhalation carcinogenicity data.
4-2,8
-------
5, REFERENCES
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?ne 'r hUmaPS Under exP^imental conditions. Medycyna Pracy
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Bolt, H.M.; laib, R.J.; Filser, J.G. (1982) Reactive metabolites and car-
cinogemcity of halogenated ethylenes. Pharmacology 31(l):l-4.
Buben, JR.; O'FJaherty, E.J. (1985) Delineation of the role of metabolism
in the hepatotoxicity of trichloroethylene and perch! oroethylene: a
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hepatic microsomal cytochrome P-450 system. Biochem. Pharmacol. 20:2863
Fernandez T.; Guberman E.; Caperos, J. (1976) Experimental human exposures
Am.^^yg0:^1.6^ (K^lsT '^ '" ^ ^ ^Mm"
of halogenated ethylenes 1n
Green T; Prout M.S. (1985) Species differences in response to trichloro-
Green, T. (1986) To be published.
Moody, D.C.; Reddy O.K. (1978) Hepatic peroxisome (microbody) proliferation
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National Toxicology Program (NTP). (1985, Aug.) NTP technical report on the
toxicology and carcinogenesis studies of tetrachloroethylene in F344/N
rats and B6C3F1 mice (inhalation studies). Draft.
Ohtsuji, T.; Sato, K.; Koijumi, A.; Kumai, M.; Ikeda, M. (1983) Limited
r>^nar>i't-\//-\-Fl-»f«m-»*>^.4-«. j__i_~i_? j_j_ 11 ...
ze tetrachloroethylene. Int. Arch. Occup.
ttrhiMiif-H-; Watanabe' P-G- (1979) Disposition of
tetrachloro(iltC)ethylene following oral and inhalation exposure in rats.
Toxicol. Appl. Pharmacol. 51:465-474.
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Pereira, M. (1984) The genotoxic/epijgenetic distinction: relevance to cancer
policy. Env. Res. 34:175-191. !
Prout, M.S.; Provan, W.M.; Green, T. i(1985) Species differences in response
to trichloroethylene. I. Pharmacokinetics in rats and mice. Toxicol.
Appl. Pharmacol. 79:389-400.
Ramsey, J.C.; Anderson, M.E. (1984) JA physiologically based description of
the inhalation pharmacokinetics of styrene in rats and humans. Toxicol.
Appl. Pharmacol. 73:159-175. !
Reddy, O.K.; Azarnoff, D.L.; Higuita,
C.E. (1980) Hypolipidemic hepatic
peroxisome proliferation form a novel class of chemical carcinogens.
Nature 283:397-398.
Schumann, A.M.; Quast, T.F.; Watanabe,1 P.G. (1980) The pharmacokinetics of
perch!oroethylene in mice and rats as related to oncogenicity. Toxicol.
Appl. Pharmacol. 55:207-219. !
U.S. Environmental Protection Agency, j (1984, Nov. 23) Proposed guidelines for
carcinogen risk assessment. Federal Register 49(227):46294-46301.
I
U.S. Environmental Protection Agency. ; (1985, July) Health assessment document
for tetrachloroethylene. Final report. EPA-600/8-82-005F.
Weisburger, J.H.; Williams, G.M. (1980) Chemical carcinogens. In: Toxicol-
ogy: the basic science of poisons (Doull, J.; Klaassen, C.D.; Amdur, M.D.,
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i
Weisburger, O.H.; Williams, G.M. (1981) Carcinogen testing. Current problems
and new approaches. Science 214:'401-407.
-------
APPENDIX A
PHYSIOLOGICALLY-BASED PHARMACOKINETIC MODELS
AND THEIR APPLICATION IN THE QUANTITATIVE RISK ASSESSMENT OF PCE*
Physiologically-based pharmacokinetic (PB-PK) models have gained increas-
ingly broad acceptance in recent years as effective tools for studying the
absorption, distribution, biotransformation, and elimination of chemicals in
the body. The most desirable characteristic of these models is their ability
to piece together all of the relevant physiological and biochemical information
to formulate a tool for predicting absorption, distribution, biotransformation,
and elimination across species. In calculating the unit risk estimate for PCE,
the PB-PK approach is used to supplement the risk calculations that are done on
the basis of more directly estimated metabolized dose. The PB-PK model to be
developed here is only applicable to inhalation exposures. The PB-PK approach
is not considered to be superior to the approach that was used previously in
Section 4.6.1. for the following reasons:
(1) Some of the parameters used in the PB-PK model must be estimated from
the same set of data that is used in the crude approach. There are
uncertainties with the parameters used in the model. Therefore, the
amount of metabolites predicted by the PB-PK model is not necessarily
more reliable.
'Dr. Jerry Blancato of the Exposure Assessment Group provided valuable advice
during the preparation of this Appendix, and Dr. Curtis Travis of the Oak Ridge
National Laboratory provided valuable information regarding his work on PCE.
Some of the data used in this Appendix are taken from a report entitled "Inter-
pretation of Metabolic Data in Exposure Analysis," conducted by Oak Ridge
National Laboratory and sponsored by the Exposure Assessment Group of the U.S.
Environmental Protection Agency.
A-l
-------
(2) Available data on the dose-metabolism relationship are primarily for
i
rats, with limited data avjailable for mice and humans. Therefore,
I
the uncertainty associated with species conversion is somewhat re-
duced by the direct use ofj these data.
The use of the PB-PK approach in calculating the unit risk estimate for
PCE has provided insight into the problems of using metabolism/kinetic data in
quantitative assessment, and has pointed out a need for further research.
A.I. DESCRIPTION OF THE PB-PK MODEL' FOR INHALED PCE
PCE in vapor form in air is readily absorbed through the lungs into blood
by first-order diffusion processes. Pulmonary uptake of PCE is largely deter-
mined by the ventilation rate, duration and concentration of exposure, solu-
bility in blood and body tissues, and metabolism. PCE is eliminated by pulmo-
nary excretion and by metabolism (primarily in the liver). Figure A-l depicts
the PB-PK model used to simulate PCE; distribution and metabolism over time.
This model was used by Ramsey and Aniderson (1984) to study the behavior of
i
inhaled styrene. Abbreviations usedi in the figure are given in Table A-l.
i
The model in Figure A-l is described by a system of differential equations
which quantify the rate of change of < PCE concentration in each tissue group
over time. Pulmonary uptake and elimination 'are described by the following
equation of mass balance of PCE entering and leaving the lungs:
- Ca)|= Qt(Cart - Cven)
This equation assumes a concentration equilibrium between arterial blood and
alveolar air. By substituting the relationship N = Cart/Ca into the equation
above, we have
A-2
-------
'van
"vf
'vr
'vp
'vl
Alveolar Space
Lung Blood
Fat Tissue
Group
Richly Perfused
Tissue Group
Poorly Perfused
Tissue Group
Liver
(Metabolizing)
Tissue Group
m
"H;
'•rt
'art
'art
Q,
'art
'art
K
Metabolites
m
Figure A-l. Diagram of the physiologically-based pharmacokinetic
model. Abbreviations are defined in Table A-l.
A-3
-------
TABLE A-l. ABBREVIATIONS USED IN FIGURE A-l
Qa: Alveolar ventilation rate (L/min)
Qt: Cardiac blood output (L/min) !
|
Q^: Blood flow rate to liver (metabolizing) tissue group (L/min)
Qf: Blood flow rate to fat tissue group (L/min)
Qr: Blood flow rate to richly-perfuse|d tissue group (L/min)
Qp: Blood flow rate to poorly-perfused tissue group (L/min)
Y£, Vf, Vp, and Vp: Volumes of tissue^ groups (L) corresponding, respectively,
to liver, fat, richly-perfused, and poorly-perfused tissue groups
N: Bloodrair partition coefficient
P£, Pf, Pr, and Pp-. Tissuerblood partition coefficient corresponding,
respectively, to liver, fat, richly-perfused, and poorly-perfused tissue
groups
m
Maximum velocity of metabolism (m'g/min)
Michael is constant (mg/L blood)
Amount metabolized (mg)
: Concentration in arterial blood (mg/L)
Cven: Concentration in venous blood (mg/L)
I
Cjj., Cf, Cp, and Cp: Concentrations in tissue groups corresponding, respec-
tively, to liver, fat, richly-perfused, and poorly-perfused tissue groups
Cj: Concentration in inhaled air (mg/L)
Ca: Concentration in alveolar air (mg!/L)
A-4
-------
' cart = (Qacl + Qtcven) / (Qt
where venous blood concentration is given by
Cven = (Q^^/P^ + QfCf/Pf + QrCr/Pr + QpCp/Pp) / Qt
The metabolism of PCE occurs mainly in the liver, with a rate of metabo-
lism characterized by the Michaelis-Menten type equation
dA
The PCE concentrations in each of the four tissue groups are described by
the following equations:
Metabolism tissue group:
= o (c - c
g ( C
dAm
dt~ * art
Non-metabolism tissue groups:
dC,-
where subscript i represents fat, richly-perfused, and poorly-perfused tissue
groups.
A.2. PARAMETERS USED IN THE MODEL
Table A-2 presents the physiological and biochemical parameters used in
the model. How these parameters were obtained is described below.
A-5
-------
TABLE A-2. PHYSIOLOGICAL AND BIOCHEMICAL PARAMETERS USED IN THE MODEL
Parameters
Body weight (kg)
Alveolar ventilation rate
Qa
Blood flow rate
Qt
QA
Qf
Qr
Qp
Tissue volume
VA
Vf
vr
VP
Partition coefficient
N
PA
Pf
Pr
PP
vm
Km
Rats
I
0.35
0.083
0.104
0.0389
I
0.00920
' 0.0434
' 0.0126
i 0.0140
i 0.0315
0.0150
0.2550
18.9
3.719
108.994 -
3.719
•• 1.058
0.00586
2.9378
Mice
0.04
0.018
0.0225
0.00836
0.00198
0.00940
0.00272
0.00160
0.00359
0.00199
0.02.920
16.9
4.159
121.893
4.159
1.183
0.00138
2.9378
Humans
70
3.421
4.31
1.607
0.387
1.800
0.506
0.8000
6.2970
3.4980
51.085
10.3
3.719
108.994
3.719
1.058
0.2004
2.9378
•
A-6
1
-------
A.2.1. Physiological Parameters
These parameters are taken from Ramsey and Anderson (1984), with adjust-
ment for the difference in body weight. This adjustment is performed by assum-
ing that within a given species the ventilation and blood flow rates are pro-
portional to the two-thirds power of body weight, and the volume of each tissue
group is directly proportional to the body weight. For example, the alveolar
ventilation rate for a 0.35-kg rat is calculated by
Qa = 0.075 x (0.35/0.3)2/3 = 0.083 L/min
where 0.075 L/min is the ventilation rate for a 0.30-kg rat that is given in
Ramsey and Anderson (1984).
A.2.2. Partition Coefficients
The parameters for rats and mice were provided by Dr. Curtis Travis of the
Oak Ridge National Laboratory, which is the contractor for the EPA project,
"Interpretation of Metabolic Data in Exposure Analysis," sponsored by the
Exposure Assessment Group, Office of Health and Environmental Assessment.
It is our understanding that these parameters were obtained by Dr. Melvin E.
Anderson of the Air Force Medical Research Laboratory at Wright-Patterson Air
Force Base, using the equilibrium vial technique. The tissue:blot)d concentra-
tion ratio was calculated by taking the ratio of two concentration ratios,
air/blood and air/homogenate tissue. The tissuetblood coefficients for humans
are assumed to be identical to those of rats.
A.2.3. Metabolism Rate Constants, Vm and Km
Rats: The constants Vm and Km were estimated by least-square optimization
using the system of differential equations and data from a metabolism study by
Pegg et al. (1979). As presented in Table 4-1, the amounts metabolized over 72
A-7
-------
hours for a 0.25-kg rat exposed to PCE at 10 ppm and 600 ppm for 6 hours were
t
recorded to be, respectively, 0.467 mg and 9.11 mg. These two data points
(72 hours, 0.467 mg) and (72 hours, 9.11 mg), were used to fit the system of
differential equations, described previously, with Vm and Km as unknown para-
i
meters. The resultant estimates are[Vm = 0.004685 mg/min and Km = 2.9378 mg/L.
For the NTP bioassay rats, which had (approximately 0.35 kg body weight, the
estimates are Vm = 0.004685 x (0.35/0.25)2/3 = 0.00586 mg/min, with Km the same,
independent of body weight.
Mice: An approach similar to trie one used for rats cannot be used for
mice because there is only one data point for mice; the amount metabolized over
72 hours was 0.356 mg when mice were [exposed to 10 ppm of PCE for 6 hours (see
I
Table 4-1). To derive Vm and Km for Imice, we assume that the ratio of Vm
between two species is equal to the two-thirds power of the ratio of their body
weights, and that Km is the same acro|ss species. For a 0.04-kg mouse,
i
i
Vm = 0.004685 x (0.04;/0.25)2/3 = 0.0013S mg/min
and
Km = 2.9378 mg/L :
These results are presented in Table A-2.
Humans: The metabolism constants for humans were calculated in a similar
r
way as the constants for mice. The rjesults are presented in Table A-2.
A.3. RESULTS AND APPLICATIONS TO RISJC ASSESSMENT
A.3.1. Results Under a Single Exposure Scenario
Figure A-2 describes the amount metabolized over an 80-hour (4,680-,min)
i
period for a 0.35-kg rat exposed to 200 ppm and 400 ppm of PCE for 6 hours.
! A-8
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A-9
-------
Fron this figure, it is seen that the|curve for the 200-ppm group reaches
asymptotic value at about 72 hours, whereas the curve for the 400-ppm group
approaches its asymptotic value at on beyond 80 hours. Thus, the assumption
I
that the metabolites collected over the 72-hour period represent the total
amount metabolized is justified only for the 200-ppm group and represents,
at most, a very crude approximation for the higher dosed group. Figures A-3
through A-7 depict blood and tissue cbncentration curves over an 80-hour
(4,680-min) period. These curves indicate that PCE is rapidly removed in all
of the tissue groups except the fat tissue group.
A.3.2. Results Under the NTP Multiple Exposure Scenario
I
Animals in the NTP bioassay were1 exposed to PCE 6 hours/day, 5 days/week.
i
It would be of interest to investigate whether the amount metabolized reaches
steady state during the weekday exposure period and whether the metabolites are
eliminated over the weekend. i
i
A.3.2.1. Results for Rats—From Table A-3, it is seen that the amount meta-
i
i
bolized per day reaches steady state [quickly at the second day of exposure.
The steady-state values are about 5.2; mg/day and 6.5 mg/day, respectively,
I
for the 200-ppm and 400-ppm dosed grolips. Although the amounts metabolized
I
in the first and the last two days of} the week are less than the. steady-state
i
values, these values increased from t|he first week to the second week, and thus
i
are expected to increase to larger values than the values presented in Table
i
A-3. As an approximation, the steady-state values are taken as the effective
daily dose rate for the risk calculation (see Table 4-7).
A.3.2.2. Results for Mice—The results for mice, are more uncertain than for
rats because the metabolism constants: Vm and Km must be converted from those
for rats. Using the converted Vm and Km for mice, the amount metabolized for a
0.025-kg mouse that is exposed to 10 |ppm of PCE for 6-hours is predicted to be
i
A-10
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A-15
-------
TABLE A-3. DAILY METABOLITES (mg)
I-UK t
EXPOSED TO PCE AT THE
Week Day
1 Mon.
Tues.
Wed.
Thur.
Fri.
Sat.
Sun.
2 Mon.
Tues.
Wed.
Thur.
Fri..
Sat.
Sun.
VU.-50-Kg KAI
IJJTP BIOASSAY
| Exposure
' 200 ppm
4.575
5,017
5.134
5.166
5.175
2.260
1 0.670
4.6.14
i 5,032
\
5.137
5.167
I 5,175
2.260
I
0,670
EXPOSURE PATTERN
conGentration (mg)
400 ppm
5.94,6
6.346
6,439
6.467
6.474
3.79.1
1,409
:5.987
6,35.2
6.441
6.467
6,475
3.792
1.413
! . -
A-1.6
-------
0.10 mg, which is only one-third of that observed in the metabolism study by
Pegg et al. (1979). The reason for the underestimation becomes obvious when
one examines the following calculation. For a 0.025-kg mouse, the alveolar
ventilation rate (given by Ramsey and Anderson, 1984) is 0.0132 L/min. At 10
ppm (i.e., 0.06908 mg/L), 6-hour exposure, the total uptake (including the
amount metabolized) is no more than
' 0.0132 L/min x 0.06908 mg/L x 6 hr x 60 min/hr = 0.328 mg
which is even less than the metabolized value of 0.356 mg observed by Pegg et
al. (1979). Thus, the underestimation is probably due to use of undervalued
physiological parameters; in particular, the ventilation rate. Although it is
possible to adjust the ventilation rate and other parameters in the model, we
choose not to do so because the difference between the observed and predicted
metabolized dose is not large. However, one should keep in mind that the risks
calculated on the basis of mouse data (using the PB-PK model) may be overesti-
mated due to the underestimation of the metabolized dose. Table A-4 presents
the amount metabolized for mice.
The NTP mice reached steady state quickly, with steady-state values at
0.74 mg/day and 1.07 mg/day, respectively, for the 100-ppm and 200-ppm dosed
groups. However, the mice appeared to metabolize faster than the rats, as
evidenced by the fact that the metabolized dose almost Reached steady state on
the first day of exposure. Furthermore, the mice also eliminated faster than
the rats, as evidenced by the fact that the metabolites were almost totally
removed over the nonexposed weekend. Therefore, it is more appropriate to use
as the daily dose, the averaged amount metabolized over 7 days. That is, the
effective doses for the 100-ppm and 200-ppm dosed groups are respectively 0.54
A-17
-------
EXPOSED
Day
Mon.,
Tues .
Wed.,
Thur.
Frt-
Sat.
Sun.
j
i
TABLE A-4»» DAILY METAMILHES (ing:)1
FOR A. 0.04-kg HOUSE
TO* PCE AT THE Np BEOASSA^ EXPOSURE" PATTERN-
i
1
Exposure
i
100 ppm
0.710
0.740
O.J42:;
O.J43-
0.743
0.084!
0.006'
; concenlrra'tfonr (mg)
ZOO; ppm
1.027
1.064
1..067'
1.067
1..067'
0.,17:S
0:313
A-18-
-------
mg/day and 0.78 mg/day. These effective daily doses have been used in the risk
calculation (see Table 4-8).
A.3.3. Results for Human Exposure
In the previous risk calculation (sections 4.5 and 4.6.1), the study by
Bolanowska and Golacka (1972) was used to estimate the human dose-metabolism
relationship. In that study, five subjects were exposed to 390,000 ug/m3
of PCE for 6 hours. The metabolites in the urinary excretion were estimated
to be 13 mg. Assuming that the urinary excretion accounts for 50% of the total
metabolites, one would expect that the PB-PK model predicts 26 mg of the total
metabolites. This assumption is based on the observation from rat and mouse
studies that urinary metabolites accounted for at least 50% of total metabo-
lites. The amount of metabolites actually predicted by the PB-PK model under
the above exposure scenario is 64.34 mg, which is about 2.5 times greater
than the expected value of 26 mg. Therefore, if the Bolanowska and Golacka
study is considered adequate, the PB-PK model may somewhat overpredict the mag-
nitude of human metabolites and thus lead to an overestimation of human risk by
a factor of about 2 to 3. However, it is remarkable that the PB-PK model could
predict with such accuracy despite the fact that the metabolism for humans must
be estimated from that of rats.
To calculate the unit risk for humans, it is necessary to calculate the
amount metabolized associated with a continuous (24-hour) exposure to 1 ug/m3
of PCE in air. Table A-5 presents the consequences of the PCE exposure over a
2-week (14-day) period during which the steady state is reached. The steady-
state value of the amount metabolized is 7.9 mg/day. This value was used to
calculate the unit risk of PCE in Table 4-9.
Table A-6 presents the consequences of continuous (24 hours/day) exposure
to PCE at higher concentrations. The values presented in Table A-6 represent
A-19
-------
TABLE A-5. AMOUNT METABOLIZED, PER DAY FOR A 70-kg PERSON
Day
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Amount of metabolite (m.g:) at end of each day
4.36
x 10-4
6.108 x 10-4
6.43
6.92
7.25
7.47
7.62
7.71
7.78
7.82
7.85
7.87
7.88
7.88
x 10-4
x 10-4
x 10-4
x 10-4
x 10-4
x 10-4
x 10-4
x 10-4
x 10-4
x 10-4
x 10-4
x 10-4 ;
A-2.0
-------
TABLE A-6. STEADY-STATE METABOLIZED DOSE (rug/day)
FOR A 70-kg PERSON
CONTINUOUSLY EXPOSED TO PCE IN AIR
Ambient air Amount
concentration metabolized
(ppro) (mg)
150 221.96
125 211.71
100 197.82
75 178.02
50 147.76
25 96.89
1 5.37
A-21
-------
the amount metabolized per day 14 days after first exposure; these values are
considered to be steady-state values.
A.4. COMMENTS ;
As demonstrated in this Appendix, the PB-PK model can be a useful to.ol
I
for quantitative risk assessment. It: provides not only the estimation of the
metabolized dose in the NTP bioassay,* but also an insight into, the validity of
various assumptions that must be made in risk assessment, such as the validity
of using data from a single-exposure experiment in place of multiple-exposure
bioassay data. For the PCE PB-PK modeling, we find the following data to be
desirable:
i
(1) The tissue concentrations and the amount metabolized over time are
i
useful. For PCE, only metabolism! data at 72 hours are available. The
measurements at several time points and at different exposure concentra-
i .
tions are necessary for estimating the parameters and verifying the ade-
quacy of the model constructed. ;
(2) The physiological and biochemical parameters for the animals used in
a metabolism/kinetic study are useful. Although the physiological para-
meters for most species are known to be within certain ranges, the use of
t
exact parameter values for the animals could greatly improve the accuracy
of the prediction by the model. We feel that work should be initiated to
develop a methodology that better quantifies key metabolic parameters in
j
humans. !
A-22
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