R&S 130214
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R & D REPORT
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DOW CHEMICAL U.S.A.

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Health and Environmental Research

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1976

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COMPARISON OF THE FATE OF VINYL CHLORIDE FOLLOWING SINGLE AND REPEATED

CHI NUMBER

EXPOSURE IN RATS

_______

PACES
IN PULL
REPORT

TOYmAHTH---------------------]------------------------------------------------P. G. Watanabe, J» A. Zempel and P. J. Gehring

autua'iiiii mwiruiirrir
> -

UjttJhudhC--

DATA REFERENCES (bdok and page):

(Refer also to earlier related reports and publications.)
PATENT STATUS:
C3 disclosure submitted CD case filed

Dno patent action required

DESCRIPTIVE SUMMARY WITH CONCLUSIONS:

R&S 130215

Rats were exposed to 5000 ppm nonlabeled vinyl chloride (VC) 6 hours/day, 5 days/week
for 7 weeks. On the last day of exposure 14C-labeled VC was used and the fate of the
14C-VC was followed for 72 hours and compared with the fate in rats subjected to a
single 6-hour exposure to 5000 ppm l4C-VC. The routes and rates of excretion of Re­
activity were the same for the two experimental groups. The activity of microsomal
enzymes, as reflected by aniline hydroxylase and £-nitroanisole 0--demethylase of 9000
x g liver supernatants was essentially the same in rats exposed once, repeatedly or
In nonexposed control rats. Covalent binding to hepatic macromoxecules was greater In
rats repeatedly exposed when compared to those subjected to a single exposure. The
hepatic nonprotein sulfhydryl level of the repeated and single exposed groups Immediately
following exposure was 79% and 39% of control, respectively. These results indicate
that repeated exposure to VC does not induce its biotrasnformation. However, the
increase in hepatic macromolecular binding indicates that repeated exposure augments
the reaction of electrophilic metabolites with macromolecules, and this may be ex­
pected to enhance potential toxicity including carcinogenicity.

j DISTRIBUTION:

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TOXICOLOGY RESEARCH LABORATORY
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TITLE OF REPORT:

.1478-.-

Comparison of the fate of vinyl chloride

following single and repeated exposure in rats.
DATE REPORT ISSUED:
AUTHOR(s):

September 28, 1976

p# G> watanabe, J. A.

Zempel and P. J. Gehring

Notebook(s) - (Number and Page(s):

R&S 130216

1)
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5)

3)

6)

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COMPARISON OP THE FATE OF VINYL CHLORIDE FOLLOWING SINGLE
AND REPEATED EXPOSURE IN RATS

by
P. G. Watanabe, J. A.

Zempel and P. J. Gehring

September 28, 1976

Toxicology Research Laboratory
Health and Environmental Research
Dow Chemical U.S.A.

R&S 130217

COMPARISON OP THE PATE OF VINYL CHLORIDE FOLLOWING
SINGLE AND REPEATED EXPOSURE IN RATS

P. G. Watanabe, J. A. Zempel and P. J. Gehring

ABSTRACT
Rats were exposed to 5000 ppm nonlabeled vinyl chloride
6 hours/day,
exposure

14

5 days/week for 7 weeks.

(VC)

On the last day of

C-labeled VC was used and the fate of the

14

C-VC

was followed for 72 hours and compared with the fate in rats

R&S 130218

subjected to a single 6-hour exposure to 5000 ppm
The routes and rates of excretion of
same for the two experimental groups.

14

14

C-VC.

C-activity were the
The activity of

microsomal enzymes, as reflected by aniline.hydroxylase and
£-nitroanisole O-demethylase of 9000 x g liver supernatants
was essentially the same in rats exposed once, repeatedly
or in nonexposed control rats.

Covalent binding to hepatic

macromolecules was greater in rats repeatedly exposed when
compared to those subjected to a single exposure.

The

hepatic nonprotein sulfhydryl level of the repeated and
single exposed groups immediately following exposure was 79%
and 39% of control, respectively.

These results indicat

that repeated exposure to VC does not induce its biotrans­
formation.

However, the increase in hepatic macromolecular

2-

-

binding indicates that repeated exposure augments the
reaction of electrophilic metabolites with macromolecules,
and this may be expected to enhance potential toxicity in­
cluding carcinogenicity.

R&S 130219

3-

INTRODUCTION

While many pharmacokinetic and metabolic studies have been
conducted on vinyl chloride

(VC), most of these investi­

gations have concentrated on the fate of VC during and fol­
lowing single exposure.

Since cancer has been induced by

long term repeated exposure of both experimental animals
and man to VC, it is important to consider alterations in
the disposition of VC in the body which may occur after
repeated exposure.

In preliminary studies monochloroacetic acid was tentatively

R&S 130220

identified as a major urinary metabolite following repeated
exposure

(6 hours/day,

nonlabeled VC

5 days/week for 9 weeks)

(Hefner et al., 1975).

to 5000 ppm

However, this meta­

bolite has not been found in rats given a single exposure
to ^C-VC

(Watanabe et al., 1976a) .

This raised the

question whether the biotransformation of VC may be altered
upon repeated exposure.

A change in the biotransformation of VC after repeated ex­
posure is particularly important in assessing the hazard of
exposure to VC since evidence suggests that VC is metabolized
to a reactive metabolite which is ultimately responsible for

-4-

its toxic manifestations.

This concept is supported by

studies showing an enhancement of the mutagenic activity
of VC to bacteria when microsomal and soluble enzymes are
added to the system to provide for metabolic transformation
(Bartsch et al., 1975; Malavielle et al., 1975; Rannug et
al. 1974).

In addition/ recent reports demonstrated that

liver microsomal enzymes, in vitro, mediate the binding of
VC metabolites to the microsomes

(Kappus et al., 1975),

protein sulfhydryl groups, RNA (Bolt et al., 1975), and
adenosine

(Barbin et al., 1975).

That the biotransformation

of VC is involved intimately in its toxic response was
further substantiated when Reynolds et al.

(1975a)

showed

that acute hepatotoxicity could be produced by VC if the
animals were pretreated with phenobarbital or Arochlor 1254,
inducers of microsomal enzyme activity.

Reynolds et al.

(1975b) also reported that a single 6 hour exposure to 5% VC
deactivates cytochrome P-450 and other components of the
mixed function oxidase system.

Since there is evidence suggesting that repeated exposure to

of VC is associated with its toxicity, the objective of the
current study was to determine whether the fate of VC is

R8.S 130221

VC may alter its biotransformation and since the metabolism

-5

indeed altered with repeated exposure.

This was accomplished

by exposing rats to nonlabeled VC for 7 weeks and on the
last exposure to ^c-labeled VC.

The fate of the ^4C-VC

was then followed for 72 hours and compared to animals
receiving a single exposure.

METHOD
Material.

Vinyl chloride gas

(Matheson Gas Products)

99.9% minimum purity was used throughout the study.
labeled VC was synthesized from (lf2

14

C)

R&S 130222

(Wagner and Muelder, 1975).

14

C-

1,2-dichloroethane

(New England Nuclear, Lot #819-292, 4.8 mCi/mmole)
prior to use

of

immediately

The synthesized

14C-VC has been reported to be 95-96% radiochemically pure
(Wagner et aX., 1975).

Nonlabeled VC

ducts) was mixed with the
specific activity.

14

(Matheson Gas Pro-

C-material to obtain the desired

Typically, 40 ml of the

14

C-VC, helium

gas mixture was injected into a 10 liter Saran bag

(Anspec,

Inc.) containing the desired quantity of nonlabeled VC.

Animals.

Male Sprague-Dawley rats

(Spartan Research Labora­

tory) with an initial weight of 160-180 g were used in the
study.

All animals were housed in rooms in which a constant

humidity, temperature and 12 hour light-dark cycle
were maintained.

(8 AM-8 PM)

Pood and water was provided ad libitum

6-

-

except during the exposure.

Exposures were conducted be­

tween 9:00 AM and 4:00 PM (EST).

Exposure.

All rats were exposed under dynamic conditions to

a nominal concentration of 5000 ppm VC
(control)

(treated) or room air

in 30 I glass inhalation chambers.

VC was metered

into the chamber air flow (-6 1/min) with a dual syringe
pump.

The nominal concentration of VC was determined from

the ratio of the rate at which the VC gas was dispensed and
the total chamber air flow.

The analytical concentration of

VC was monitored continuously by recirculating a fraction of
the chamber atmosphere through an infrared spectrophotometer
(Wilks)

set at 10.6 y.

The rats repeatedly exposed to VC

were exposed 6 hours/day, 5 days/week for 7 weeks
posures in 44 days).

(32 ex­

The mean analytical concentration of

VC over the 7 week exposure period was 4775+908

(SD) ppm.

On the final day of exposure the animals were subjected to
a 6 hour exposure to

14

C-labeled VC generated xn the same
On this final day of exposure,

the chamber atmosphere was also analyzed at hourly intervals by gas chromatography, and at the same times the

14

C-

activity was determined by bubbling 1 ml aliquots of the
chamber atmosphere into a scintillation solution containing
Concifluor

(Mallinckrodt Chemical), 2-methoxyethanol, toluene

R&S 130223

manner as described above.

-7

(6:11:83)

(Watanabe et al.,

1976a).

The radioactivity was

determined by counting in a liquid scintillation spectro­
meter.

The mean analytical concentration determined on the

final exposure day when the animals were exposed to
labeled VC was 4600 ppm ± 311

(SD).

14

c-

The specific activity

was 50 dpm per microgram VC.

The inhalation chamber was operated in a laboratory fume
hood to prevent contamination of the working environment.
After transit through the inhalation chamber the

R&S 130224

absorbed on activated charcoal.

14

C-VC was

These traps were disposed

of as radioactive waste according to standard regulations.

Procedure.

Eight rats were exposed repeatedly to VC as

described previously.

On the last day, 5 additional•un­

exposed rats and the 8 rats exposed repeatedly were exposed
to 5000 ppm
posure to

14

C-VC for 6 hours.

Following this final ex-

C-VC, 3 of the 8 exposed repeatedly and 2 of the

5 exposed once were placed in glass Roth-type metabolism
cages for the collection of urine, feces and expired air.

Room air was drawn through the cages at 400-500 ml/min.

The

exiting air was passed through a series of traps to collect
the expired

14

14
C-VC and . CC^.

The air leaving the chamber

8-

-

was passed first through a glass tube containing about 40 g
of Drierite

(W. A. Hammond Drierite Co.) to remove moisture.

Subsequent transit through a series of two cold finger traps
containing 50 ml of toluene, 2-methoxyethanol

(80:20), and a

single trap containing 120 ml of 5 M ethanolamine in 2methoxyethanol enabled the collection of
respectively.

14

C-VC and

14

CO2,

The cold finger traps were immersed in 2-

methoxyethanol, dry ice baths throughout the collection
periods.

The trap for COj was maintained at room temperature.

Samples of excreta were collected for 72 hr after termination of exposure and analyzed for

14

C activity.

Expired

VC was collected at 0.5 hr intervals for 3 hr? the COg trap
(immersed in dry ice bath) were changed

at 12 hr intervals for 72 hr; and feces were collected every
24 hr.

At the termination of the study

(72 hr) the animals

were killed by a blow to the head, and samples of tissues
(fat, kidney, liver) were collected for analysis of

14

C

activity.

The remaining carcass was skinned and homogenized

(50% w/v)

in distilled water and analyzed for

14

. .
C activity.

The samples of excreta and tissue were prepared for scintil­
lation counting as described previously
1976a).

(Watanabe et al.,

R&S 130225

and urine receptacle

9-

Carbon 14 activity was determined by counting in a Mark II
or Mark III liquid scintillation spectrometer.

External

standard channel ratios were used to determine the counting
efficiency.

The counts per minute were converted to dis­

integrations per minute using a standard quench curve.
t

The remaining rats in the groups exposed repeatedly and
singly to VC,

5 and 3 respectively along with a group of

4 controls exposed to room air for 6 hours were killed by a
blow to the head immediately following exposure.

A piece

of liver was sampled and used for determining hepatic non­
protein sulfhydryl content by a modification of the method

R&S 130226

of Sedlak and Lindsay

(1968).

Another piece of liver was

used to prepare a 9000 x g supernatent in 1.15% KC1 in order
to determine aniline hydrolyxlase
nitroanisole O-demethylase
vity.

(LaDu et al., 1971)

(Kinoshita et al., 1966)

and £-

acti­

Macromolecular binding of radioactivity to hepatic

tissue was determined by the method of Jollow et al.

(1974).

The carcass was analyzed for total radioactivity as des­
cribed above.

RESULTS

Excretion of

14

C-activity within 72 hours after a single or

repeated exposure to 5000 ppm

4

14

C-VC is shown in Table 1.

10-

-

The percentage of

14

C-activity excreted by each route as

well as the total mg equivalents VC recovered were essen­
tially identical for the singly and repeatedly exposed
groups.

The majority of

14

C-activity eliminated was expired

as VC per se.

The time course for expiration of
and urinary excretion of

14

14

C-VC per se

C-activity

(Figure 1)

(Figure 2) were also

essentially identical for the singly and repeatedly exposed
rats.

The curves were fit by linear regression analysis of

the logarithmically transformed data.

The estimate of the

apparent first order rate constant for expiration of VC was
0.023 min-^ i 0.01
30 minutes.

The elimination of urinary

14

C-activity was

An estimate of the apparent first order rate

constant for the initial portion of the urinary excretion
curve from 12-36 hours was 0.155 hr”^ ± 0.002
corresponds to a half-life of 4.47 hours.

(SD) which

The data for the

slow phase of urinary excretion were extremely variable;
therefore no attempt was made to estimate the excretion
rate.

Less than 1 percent of the radioactivity excreted in

the urine occurred during the slow phase.

Urinary

14

C-activity was separated by thin layer chromato­

graphy in rj-butanol, acetone, H20

(50:20:30) on cellulose

R&S 130227

biphasic.

(SD) which corresponds to a half-life of

11-

-

and n-butanol, acetic acid, 1^0

(80:20:20) on silica gel.

The profile of radioactivity for rats exposed repeatedly
or singly were qualitatively similar and no significant
radioactivity was associated with the R^ value of a
standard of monochloroacetic acid.

The concentration of radioactivity detected in tissue 72
hours after exposure revealed no statistically significant
difference between rats exposed once or repeatedly to VC
(Table 2).

It does appear that in those exposed repeatedly

more radioactivity may have been retained in the liver and
skin; however, the number of animals used does not provide
for an adequate statistical evaluation.

The effect of VC on xenobiotic drug metabolism by liver 9000
x g supernatents as reflected by aniline hydroxylase and £nitroanisole-O-demethylase activity is presented in Table 3.
Neither single or repeated exposures to 5000 ppm VC altered
discernibly the enzyme activity in either system when com­
pared to air exposed controls.

R&S 130228

i

The total amount of VC biotransformed, the hepatic macromolecular binding of

14

C-activity and the hepatic nonprotem

sulfhydryl content following single and repeated exposure

12-

-

are shown in Table 4.

The total amount of VC biotransformed

was not significantly different between the two groups.
However the hepatic macromolecular binding appears to be
increased in the rats exposed repeatedly.

When the protein

binding was corrected for the amount of VC biotransformed
(B/A x 100) a statistically significant increase was found.
*

This parameter indicates that a larger fraction of the
biotransformed VC reacts covalently with hepatic macro­
molecules in rats exposed repeatedly.

This result strengthens

the significance of the previous observation that 72 hours
after the last exposure more radioactivity was found in the
liver of rats exposed repeatedly than those exposed once.

In contrast to the results of macromolecular binding,
hepatic nonprotein sulfhdyryl content was depressed to a
greater extent in rats receiving a single exposure than
in those exposed repeatedly.

DISCUSSION
14
Repeated exposure of rats to 5000 ppm . C-VC did not alter

activity or qualitatively the excretory products formed
from VC or VC per se.

These results negate the previous

preliminary observation that monochloroacetic may be a major
biotransformation producer of VC

(Hefner et al^., 1975).

R&S 130229

discernibly the routes or rates of excretion of radio­

13-

-

No differences were found in the activity of the enzymes,
aniline hydrolylase and g-nitroanisole O-demethylase, in the
liver of rats exposed repeatedly to 5000 ppm VC, in rats
exposed once and in nonexposed rats.

Thus, exposure to VC

at this concentration does not appear to influence micro­
somal metabolism.

In contrast to this conclusion, core the

findings of Reynolds et al.

(1975b) that the cytochrome P-

450 content and the oxidative N-demethylation of aminoantipyrine and ethylmorphine were markedly depressed in rats
following exposure to 50,000 ppm VC for 6 hrs.

However, the

extremely high exposure concentration renders suspect the
relevance of any conclusion based on the Reynolds study.

R&S 130230

A most significant finding in the study reported herein was
a significantly increased amount of radioactivity bound co­
valently to macromolecules of rats exposed repeatedly to VC
versus those exposed once.

An associated observation was

the retention of an apparently greater level of radio­
activity in the liver of repeatedly exposed rats 72 hours
after exposure than those exposed once.

These results

indicate that toxic manifestations, including carcinogenicity,

-14-

associated with the reaction of reactive metabolites of VC
with macromolecules may be enhanced by repeated exposure to
VC.

In rats exposed repeatedly, the enhanced covalent binding
of VC with macromolecules occurred in spite of a less
significant depression of the nonprotein sulfhydryl content
of the liver when compared to those rats exposed once.
Previous studies have demonstrated that detoxification of
the reactive metabolites of VC occurs via their enzymatic
conjugation with glutathione

(Watanabe et al., 1976b;

Watanabe et al., 1976c) and subsequent excretion.

Glu­

tathione is reportedly the major constituent of the non­
protein sulfhydryl content of the liver.

The foregoing results are somewhat perplexing because an
increased covalent binding of radioactivity is normally
thought to be associated with either an enhanced biotrans­
formation to reactive metabolites or a decreased detoxi­
fication of the reactive metabolites.

The former pos­

R&S 130231

sibility is negated by the absence of a qualitative or

15-

-

quantitative change in the biotransformation of VC in rats
exposed repeatedly as well as the lack of evidence that
microsomal enzymes are induced by repeated exposure to VC.
The finding that the hepatic nonprotein sulfhydryl content
was reduced less in rats exposed repeatedly to VC suggests
that detoxification of reactive metabolites formed from VC
by conjugation with GSH should not be depressed.

Therefore,

both logical reasons for the increased covalent binding of
reactive metabolites of VC to macromolecules appear non­
operative.

However, not to be overlooked is the fact that

the nonprotein sulfhydryl content of the liver rather than
GSH content was measured.

It is conceivable that the GSH

level of the liver was actually decreased.

If this were

true, the binding of reactive metabolites to macromolecules
may be enhanced inspite of an apparently normal hepatic

R&S 130232

nonprotein sulfhydryl content.

It may be expected that

nonprotein sulfhydryl compounds other than GSH will be less
effective in detoxifying reactive metabolites of VC.

In conclusion, the results of these studies show that re­
peated exposure to high levels of VC cause a persistent but
partially compensated depression of the nonprotein sulfhdyryl

16-

-

content of the liver.

More significantly, the binding of

reactive metabolites of VC with hepatic macromolecules
appears to be enhanced by repeated exposure to high levels
of VC.

Associated with this may be expected an enhanced

toxicity, including carcinogenicity.

The reason for the

enhanced covalent binding with repeated exposure is‘under
investigation.

WRITTEN BY:

P. G. Watanabe, Ph.D.
Toxicology Research Laboratory
1803 Building

Toxicology Research Laboratory
1803 Building

Director, ToxicologyResearch Laboratory
Health and Environmental Research
1803 Building

REVIEWED BY:

L. W. Rampy, Ph.D.//
Toxicology Research Laboratory
Health and Environmental Resaerch
1803 Building

JO
&
CO

13 0 2 3 3

17-

-

REFERENCES

Barbin. A., Bresil, H., Croisy, A., Jacguignon, P.,
Malavielle, C., Montesano, R. and Bartsch, H.
(1975). . Liver microsome mediated formation of
alkylating agents from vinyl bromide and vinyl
chloride.
Biochem. Biophys. Res. Comm., 67,
596-603.
Bartsch, H., Malavielle, C., and Montesano, R. (1975).
Human rat, and mouse liver mediated mutagenicity
of vinyl chloride in Salmonella typhimurium strains.
Int. J. Cancer, 15, 429-437.
Bolt, H. M., Kappus, H., Buchter, A., and Bolt, W.
Metabolism of vinyl chloride.
Lancet, 1425.

(1975).

Hefner, R. E. Jr., Watanabe, P. G., and Gehring, P. J.
(1975).
Preliminary studies of the fate of inhaled
vinyl chloride monomer (VCM) in rats, Ann. N.Y.
Acad. Sci., 246, 135-148.

R&S 130234

Jollow, D. J., Thorgeirsson, S. S., Potter, W. Z.,
Hashimoto, M. and Mitchell, J. R. (1974).
Ace­
taminophen-induced hepatic necrosis VI., Pharma­
cology, 12, 251-271.
Kappus, H., Bolt, H. M., Buchter, A. and Bolt, W. (1975).
Rat liver microsomes catalyze covalent binding of
14C-vinyl chloride to macromolecules. Nature, 257,
134-135.
Kinoshita, F. K., Frawley, J. P. and DuBois, K. P. (1966).
Quantitative measurement of induction of hepatic
microsomal enzymes by various dietary levels of DDT
and toxaphene in rats.
Toxicol. Appl. Pharmacol.,
9, 505-513.
LaDu, B. N., Mandel, H. G. and Way, E. L. (ed.) (1971).
Fundamentals of Drug Metabolism and Drug Disposition,
pp. 566-569, The Williams and Wilkins Co., Baltimore,
Maryland.
Malavielle, C., Bartsch, H., Barbin, A., Camus, A. M.
and Montesano, R. (1975).
Mutagenicity of vinyl
chloride, chloroethyleneoxide, chloroacetaldehyde
and chloroethanol.
Biochem. Biophys. Res. Comm.,
63, 363-370.

18-

-

Rannug, U.f Johansson, A., Ramel, C. and Wachtmeister,
C. A. (1974).
The mutagenicity of vinyl chloride
after metabolic activation, Ambio, 3, 194-197.
Reynolds, E. S., Moslen, M. T., Szabo, S., Jaeger, R. J.
and Murphy, S. D. (1975a).. Hepatotoxicity of vinyl
chloride and 1,1-dichloroethylene.
Am. J. Path.,
81(1), 219-231.
Reynolds, E. S., Maslen, M. T., Szabo, S. and Jaeger, R. J.
(1975b).
Vinyl chloride induced deactivation of
cytochrome P-450 and other components of the livfer
mixed function oxidase system:
on in vivo study.
Res. Comm. Chem. Path. Pharmacol., T?(4), 685-693.
Sedlak, J. And Lindsay, R. M. (1968).
Estimation of total
protein-bound, and nonprotein sulfhydryl groups in
tissue with Ellman's Reagent, Analyt. Biochem., 25,
192-205.
Wagner, E. R. and Muelder, W. W. (1975).
A procedure for
preparing 14C-labeled vinyl chloride.
Ann. N.Y.
Acad. Sci., 246, 152-153.
Wagner, E. R., Muelder, W. W., Watanabe, P. G., Hefner,
R. E. Jr., Braun, W. H., and Gehring, P. J. (1975).
Gas chromatographic method for the preparation of
14C-labeled vinyl chloride, J-. Labeled Compounds,
11, 535-542.
Watanabe, P. G., McGowan, G. R., Madrid, E. 0. and Gehring,
P. J. (1976a).
Fate of 14C-vinyl chloride following
inhalation exposure in rats.
Toxicol. Appl. Pharmacol.,
in press.

Watanabe, P. G., Hefner, R. E. Jr., and Gehring, P. J.
(1976c).
Vinyl chloride induced depression of
hepatic nonprotein sulfhydryl content and effects
on bromosulphthalein (BSP) clearance in rats.
Toxicology, in press.

R&S 130235

Watanabe, P. G., McGowan, G. R. and Gehring, P. J. (1976b).
Fate of 14C-vinyl chloride after single oral adminis­
tration in rats, Toxicol. Appl. Pharmacol., in press.