TV.

REPORT
e.T"• .V.
DOW CHEMICAL U S A.
RESTRICTED: l«r w>« within i S* Dow CK#ihi£«I Compos?

UIDIltoiiv
on t coos
HITI K-l 7 1 1-CM)
CiA^t illuio
Sept. 3H , 1 97li
Vi r*lo, ftnoiuu no
1 ; .7 0,0 ,0 ,7,1 ,

Health and Environmental Re*1.ear. n
t • t 1C
COMPARISON OP THE FATE Of VINYL (.III.OR I Df POI.I.o'.; i ;.i; NiNOl.E AN!) REPEATED

PACES
IN FULL
REPORT

EXPOSURE IN RATS
ft u f ►■*£>• • FT
l1.

G*

WiitanaiH1 ,

J.

A*

I

.mil I',

.1.

C-Tiriii:;

lUiMa^'ir i^NTfufcrm---------------------------------------------------------

(Refer also to earlier related reports and nublteatlone.)
PATENT STATUS:

□dlaclonuro submitted

CZ3 case filed

Duo patent action required

DESCRIPTIVE SUMMARY WITH CONCLUSIONS:
Rats were exposed to 5000 ppm -.ion I a lie 1 ed vinyl chloride (VC) h hours/day, 3 days/week
for 7 week-;.
On Lite InsL dav oi exposure 1 ’C-1 .i h*-1 ed V( was used nod the late of Lite
1 'C-VC was toi lowed tor 71! hour-: .nn! ■ ompa rv'd wiiii tin- tale in rats subjected Lo a
single (j-liOti)’ exposure Lo 3000 ppm ‘ ‘C--V0.
1 lie mutes and r.iLe'. oi excretion of
activity were Lite sane for the two experimental -.roups.
The activity of microsomal
enzymes, as reflected bv aniline hydroxylase and n-n i t roan i so 1 <• O-drmethy ] ase of 9000
x
liver supernatants was t ■■ tent i a 1 1 y Lie.- sare m rats exposed oun , repeatedly or
in nonexposod conLroi rats.
Covaient binding t’ hepatic macromu i ecu] us was greater In
rats repeatedly exposed when (umpai'ed to those subjected to a single exposure,
i he
hepatic nonproteiti sull’iiydryl level of the repeated and single exposed groups Immediately
following exposure was 79/i and 397'. m control, respectively.
The.se results Indicate
that repeated exposure to VC does not induce its hjntr.isniormation.
However, the
increase in hepatic tnacromoloculur binding, indicates that repeated exposure augments
l lie reaction of electrophilic me La bo 1 i Los with macromo 1 ecu 1 es , and tills may be expicted to enhance potential Loxirity ;m lading carcinogenicity.

DISTRIBUTION:

l

** * ■
DI-6560

See Back Page

»»*#♦«•♦
9-8-7S

R&S 022442

R & D

AND REPEATED EXPOSURE IN RATS

by
P.

G*

Watanabe,

J.

A.

Zempel and P.

September 23,

J-

Gehring

1976

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

■ w

a. -a

R&S 022443

COMPARISON OF THE FATE OF VINYL CHLORIDE FOLLOWING SINGLE

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

P.

G.

Watanabe,

J.

A.

Zcmpel and P.

J.

Gehrinq

ABSTRACT
Rats wore exposed to 5000 ppm nonlabclcd viny]
6 hours/day,

5 days/weck for 7 weeks.

exposure ^c-labeled VC was used and
was

followed

chloride

On the last day of
the fate of

for 72 hours and compared with the

the

same for the two experimental groups.
microsomal enzymes,

14

^C-VC

fate in rats

subjected to a single 6-hour exposure to 5000 ppm
The routes and rates cf excretion of

(VC)

14

C-VC.

C-activity were the
The activity of

as reflected by aniline hydroxylase and

p-nitroanisole O-demethylasc of

9000 x g liver supernatants

was essentially the same in rats exposed once,
or in nonexposed control rats.

repeatedly

Covalent binding to hepatic

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

The

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 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 022445

r""

-r^y-Y

-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 monochlorcacetic acid was
identified as a major urinary metabolite
exposure

(6 hours/day,

nonlabeled VC

following repeated

5 days/week for 9 weeks)

(Hefner et al.,

1975).

tentatively

However,

to 5000 ppm
this meta­

bolite has not been found in rats given a single exposure
(Watanabe et, a_l. ,

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

-r ■ '**♦—*—

R&S 022446

to 14C-VC

-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_. ,
al.

1974).

1975;

Malavielle ct. a_l. ,

In addition,

in. vitro,

VC metabolites to the microsomes
protein sulfhydryl groups,
(Barbin et al_ ,

Rannug ct

recent reports demonstrated that

liver microsomal enzymes,

adenosine

1975;

RNA

mediate the binding of

(Kappus et^ al;*/

(Bolt et al.,

1975).

1975),

1975),

and

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.
(1975b)

Reynolds et al.

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
VC may alter its biotransformation and since the metabolism
of VC is associated with its toxicity,

the objective of the

current study was to determine whether the fate of VC is

ft*.

indeed altered with repeated exposure.

This was accomplished

by exposing rats to nonlabcled VC for 7 weeks and on the
last exposure to "^C-labeled VC.

The fate of the ^ C-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
(New England Nuclear,
prior to use

Dot

(1,2

#819-292,

(Wagner and Muelder,

14

C)

14

C-

1,2-dichloroethane

4.8 mCi/mmole)

1975).

of

immediately

The synthesized

^C-VC has been reported to be 95-96% radiochemically pure
(Wagner et al.,
ducts)

1975).

Nonlabeled VC

(Matheson Gas Pro­

was mixed with the ^C-material to obtain the desired

specific activity.

helium

gas mixture was injected into a 10 liter Saran bag

(Anspec,

containing the desired quantity of nonlabeled VC.

Animals.
tory)
study.

40 ml of the

14

C-VC,

Inc.)

Typically,

Male Sprague-Dawley rats

(Spartan Research Labora­

with an initial weight of 160-180 g were used in the
All animals were housed in rooms in which a constant

humidity,

temperature and 12 hour light-dark cycle

were maintained.

(8 AM-8 PM)

Food and water was provided ad libitum

R&S 022448

-5-

-G-

except during the exposure.
tween 9:00 AM and 4:00 PM

(EST).

All rats were exposed under dynamic conditions to

a nominal concentration of 5000 ppm VC
(control)

in 30

f, glass inhalation chambers.

into the chamber air flow
pump.

(treated)

(-6

1/min)

or room air

VC was metered

with a dual syringe

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
the chamber atmosphere through an
(Wilks)

set at 10.6 u.

The

were exposed 6 hours/day,
posures in 44 days).

fraction of.

infrared spectrophotometer

cats repeatedly exposed to VC

5 days/week

for 7 weeks

(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 ^C-labeled VC generated
manner as described above.

in the same

On this final day of exposure,

the chamber atmosphere was also ana)yzed 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 022449

Exposure.

Exposures were conducted be­

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

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

311

(SD).

14

Ine

C-

The specific activity,

was 50 dpm per microgram VC.

The inhalation chamber was operated

in a

laboratory fume

After transit through the inhalation chamber the
absorbed on activated charcoal.

C-VC was

These traps were disposed

of as radioactive waste according

Procedure.

14

to standard regulations.

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 14C-VC for
posure to ^4C-VC,

6 hours.

Following this

final ex­

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 ^4C-VC and ^4CC>2.

The air leaving the chamber

R&S 022450

hood to prevent contamination of the working environment.

was passed first through a
of Drierite

(W.

A.

glass tube containing about 40 g

Hammond Drieritc Co.)

Subsequent transit through a series of
containing 50 ml of toluene,
single trap containing

to remove moisture.

two cold

2-:nethoxyethanol

120 ml of

The cold

methoxyethanol,
periods.

finger

(30:20),

5 M cthnnolamine in
14

methoxyethanol enabled the collection of
respectively.

finger traps

traps were

C-VC and

immersed

and a

214

C02.

m 2-

dry ice baths throughout the collection

The trap for CC>2 was maintained at room temperature.

Samples of excreta were collected

for 72 hr after terrtu14
C activity.

VC was collected at 0.5

for 3 hr;

and urine receptacle
at 12 hr
24

hr.

hi

intervals

(immersed

intervals for 72 hr;

in dry
and

ice bath)

At the termination of the study

kidney,

liver)

the CC>2 trap
were changed

feces were collected every

were killed by a blow to the head,
(fat,

Expired

wore collected

(72 hr)'the animals

and samples of tissues
for analysis of

14

C

activity.

The remaining carcass was skinned and homogenized

(50% w/v)

in distilled water and analyzed for

The samples of excreta and

. ■
C art;vj‘.y.

tissue were prepared for nciutil-

lation counting as described previously
1976a).

14

(Watanabe et a_l. ,

R&s 022451

nation of exposure and analyzed for

*v #

-9~

Carbon 14 activity was determined by counting
or Mark III

in a Mark

liquid scintillation spectrometer.

standard channel
efficiency.

ratios were used

External

to determine the counting

The counts per minute were converted to dis­

integrations per minute using a

The remaining rats
singly to VC,

in

a and

4 controls exposed

3

standard quench curve.

she groups exposed repeated]’/ and
respectively

to room air for

d.(.na with a group of
G hours were killed by

blow to the head immediately fallowing exposure.
of

IT

liver was sampled and used

A piece

for d-. tormining hepatic non­

protein sulfhydryl content by a modification of

the method

of Sedlak and Lindsay

liver was

used to prepare a

(19GB).

9000 x g

Another piece of

supurnstent

to determine aniline.* hydroiyxiasc
nitroanisole o-demetr.yl ase
vity.

i

in 1.151 KC1

(LaDu ct

(Kmoshita et al . ,

■

in order

^971)

I960)

and p~

acti­

Macromolecular binding of radioactivity to hepatic

The carcass war. analyzed for total

(1974 ) ,

radioactivity as des­

cribed above.

RESULTS

Excretion of

^C-activicy within 72 hours after a single or

repeated exposure to 5000 ppm

i). M? .pr

-VC is shown

m Table

1.

R&s 022452

tissue was determined by the method of Jollow c_t a^L.

The percentage of

14C-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

^C-activity eliminated was expired

as VC per se.

The time course for expiration of

^C-VC per se

and urinary excretion of ^C-activity

(Figure 2)

(Figure 1)
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-1

+

30 minutes.
biphasic.

0.01

(SD)

which corresponds to a half-life of

The elimination of urinary

14

• ■
C-activity was

An estimate of the apparent fir'.t order rate

constant for the initial portion of the urinary excretion
curve from 12-36 hours was 0.155 hr ^
corresponds

±

0.002

to a half-life of 4.47 nours.

(SD)

which

The data for the

slow phase of urinary excretion were extremely variable;
therefore no attempt was made to estimate the excretion
Less than 1 percent of the radioactivity excreted in

the urine occurred during the slow phase.

Urinary ^C-activity was separated by thin layer chromato­
graphy in n-butanol,

acetone,

H^O

(50:20:30)

on cellulose

R&S 022453

rate.

[g I Mill*11 'i*~^

n, •)

11-

-

and n-butanol,

acetic acid,

H^O

(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

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 :n 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.

The total amount of VC biotransformed,
molecular binding of

14

the hepatic macro-

c-activity and the hepatic nonprotein

sulfhydryl content following single and repeated exposure

R&S 022454

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
Repeated exposure of rats to 5000 ppm ^ C-VC did not alter
discernibly the routes or rates of excretion of radio­
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 022455

12-

-

^ i i ilH —Jttsutta,

-13-

No differences were found in the activity of the enzymes,
aniline hydrolylase and p-nitroanisole O-demethylase,

exposed once and in nonexposed rats.

Thus,

in rats

exposure to VC

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

In contrast to this conclusion,

findings of Reynolds e_t al..

(1975b)

are the

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.

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.
indicate that toxic manifestations,

These results

including carcinogenicity,

R&S 022456

liver of rats exposed repeatedly to 5000 ppm VC,

in the

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 loss
significant depression of the nonprotein sulfhydryl content
of the liver when compared to those rats exposed once.
Previous studies have demonstrated that detoxification of

conjugation with glutathione
Watanabe et a3L.,

1976c)

(Watanabe et. a^. ,

1976b;

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

sibility is negated by the absence of a qualitative or

R&S 022457

the reactive metabolites of VC occurs via their enzymatic

-15-

quant itative 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 nonoperative.

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

true,

If this were

the binding of reactive metabolites to macromolecules

may be enhanced inspite of an apparently normal hepatic
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

’1--------

R&S 022458

level of the liver was actually decreased.

-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- &

_______

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

J . A. 21jmp£
Toxicology Research Laboratory
1803 Building

P. J./Gehring,
Ph.D.
Director, Toxicology atesearch Laboratory
Health and Environmental Research
1803 Building

J: REVIEWED BY:

R&S 022459

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

R&S 022460

1

-17-

REFERENCES

Barbin, A., Bresil, H., Croisy, A., Jacquignon, 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.
Jollow, D. J., Thorgeirsson, S. S., Potter, W. 2.,
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
1‘‘C-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.) fi971).
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., 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.
J.

Sedlak, J. And Lindsay, R. M. (1968).
Estimation of total
protein-bound, and nonprotein sulfhydryl groups in
tissue with Ellman's Reaqent, Analyt. Biochem., 25,
192-205.
Wagner, E. R. and Muelder, W. W. (1975).
A procedure for
preparing ^C-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
1‘*C-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 *1‘‘C-vinyl chloride following
inhalation exposure in rats.
Toxicol. Appl. Pharmacol.,
in press.
Watanabe, P. G., McGowan, G. R. and Gehring, P. J. (1976b).
Fate of 1‘*C-vinyl chloride after single oral adminis­
tration in rats, Toxicol. Appl. Pharmacol., in press.
Watanabe, P. G., Hefner, "... 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.

I

R&S 022461

Reynolds, E. S., Maslen, M. T., Szabo, S. and Jaeger, R.
(1975b).
Vinyl chloride induced deactivation of
cytochrome P-450 and other components of the liver
mixed function oxidase system:
on in vivo study.
Res. Comm. Chem. Path. Pharmacol., IT(4) , 685-693.

LEGENDS

Figure 1

Expired vinyl chloride

(mg)

versus time

following a 6 hour exposure to 5000 ppm
14

C-VC.

Repeatedly exposed animals

singly exposed animals

Figure 2

14

(x)

and

(*)-

C-activity excreted in the urine expressed

as percentage of the recovered radioactivity
versus time following a 6 hour exposure to
5000 ppm
(x)

14

C-VC-

Repeatedly exposed animals

and singly exposed aninal

(•)-

R&S 022462
\

i

TABLE 1

Percentage ^4C-Activity Eliminated During 72 Hours Following
Inhalation Exposure to 5000 ppm Vinyl Chloride3

Percent

14

. .
b
C-Activity

Single

Repeated

Expired:
as VC

54.5±3.5C (14.0)d

53.712.1° (12.94)'

8.011.4

'2.05)

9.611.0

(2.27)

Urine

27.112.1

(6.93)

25.711.4

(6.21)

Feces

3.212.5

(0.80)

1.410.4

(0.32)

Carcass and
Tissues

7.312.5

(1.89)

9.711.6

(2.32)

as C02

Total mg Equivalents
VC Recovered

(25.67)

(24.07)

aRGLs exposed to 5000 ppm VC for 6 hours/day, 5 days/weck
for 7 weeks (repeated*) or one 6 hour exposure (single) .
^Expressed as percentage of the total
2 rats.

^Milligram equivale
eMean + SD,

3 rats.

R&S 022463

cMean ± SD,

^4C-actwitv recovered,

TABLE 2

Percentage ^C-Activity per Gram Tissue 72 Hour Following
Inhalation Exposure to 5000 ppm Vinyl Chloride3

Percentage

14

. .
b
C-Activity

Single

Repeated

Liver

0.119+0.022c

d
0.157 + 0.028'

Kidney

0.062+0.026

0.070+0.006

Tissue

N.D.e

Fat

N.D.

Skin

0.046+0.015

0.080+0.019

Carcass

0.030+0.014

0.039+0.011

aRats exposed to 5000 ppm VC for 6 hours/day, 5 days/week
for 7 weeks (repeated)* or one 6 hour exposure (single).
h
14
Expressed as percentage of the total
C-activity metabolized
cMean + SD,

3 rats.

dMean ± SD,

2 rats.

eNot detectable, detection limit of 3 ug VC eguiv./g fat
or 0.03 percent 14C-activity metabolized per g tissue.

?i,*'

- 1

TABLE 3

Effect of Vinyl Chloride on Drug Metabolism By
A 9,000 x g Supernatent Fraction of Liver3

pq product/q liver/hour_
_
_
_
_
_
_
_
_

Control

Aniline
Hydroxylase

£-Nitroanisole
Q-Demethylase

65±16C

226+22

71+7

254+45

83±10

217+31

(4)'

Single VC
Exposure

(3)

Repeated VC
Exposure (5)

aRats exposed to 5000 ppm VC *or 6 hours/day, 5 days/week
for 7 weeks (repeated) or one 6 hour exposure (single).
Animals were killed immediately following the last ex­
posure and enzyme activity assayed.
^Number rats/group

cMean + SD

TABLE 4

Total Metabolism,

Hepatic Macromolecular Binding and Hepatic Nonprotein

Sulfhydryl Levels Following Single Or Repeated Exposure to
5000 ppm Vinyl Chloride2

B

A

Single Exposure
Repeated Exposure

pg VC Equivalents
Metabolized

ug VC Equivalents
Bound per g Protein

B/A x 10{ft

Hepatic GSH
(% of Control)

9265+1467°

113.5+10.4

1.12+0.13c

3 9e

8718 + 895d

123.8+10.3

1.43+0.16f

r*
1ST

aRats exposed to 5000 ppm VC for 6 hours/day, 5 days/week for 7 weeks (repeated*
one 6 hour exposure (single) .
^The ratio of B/A x 100 was calculated from the individual animal data.
c.Mean

+ SD,

3 rats

drtean + SD,

5 rats

c Statistically significant from air exposed controls,

Student t-test

or

(P<0.05) .

^Statistically significant from the single exposure.

99frZZ0 S*U

R&S 022467

mg V C Expired

FIGURE 1

^

022468

Percent o f Recovered 14C -A ctivity

Si

v r

FIGURE 2

U