OPIS-SECUMICAL ISJTORMAIIOH CS&SMi
EPA-
TOXICITY STUDIES OF SELECTED CHEMICALS
TASK III:
THE TOXICITY OF VINYLIDENE CHLORIDE IN MICE AND RATS AND ITS
ALTERATION BY VARIOUS TREATMENTS
JUNE 1977
DRAFT
FINAL REPORT
ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF TOXIC SUBSTANCES
WASHINGTON, D.C. 20460
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Document is available to the public through the National Technical
Information Service, Springfield, Virginia 22151.
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TOXICITY STUDIES OF SELECTED CHEMICALS
TASK III:
THE TOXICITY OF VINYLIDENE CHLORIDE IN MICE AND RATS AND ITS
ALTERATION BY VARIOUS TREATMENTS
Final Report
Prepared by
Robert D. Short, Jr.
Joseph M. Winston
Jan L. Minor
Chuen-Bin Hong
Brett Ferguson
Timothy Unger
Mary Sawyer
Cheng-Chun Lee
Contract No. 68-01-3242
Joseph Seifter
Project Officer
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, D.C. 20460
June 1977
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PREFACE
This report was prepared at Midwest Research Institute, 425 Volker
Boulevard, Kansas City, Missouri 64110, under Environmental Protection
Agency Contract No. 68-01-3242, MRI Project No. 4128-B, "Toxicity Studies
of Selected Chemicals." The work was supported by the Office of Toxic
Substances of the Environmental Protection Agency. Dr. William Marcus
and Dr. Joseph Seifter are consecutive contract monitors for this project.
the direction of Dr. William B. House between May 1, 1976 and June 1, 1977.
The experimental work was supervised directly by Dr. Cheng-Chun Lee, Assistant
Director, Biological Sciences Division for Pharmacology and Toxicology;
assisted by Dr. Robert D. Short, Jr. (Senior Toxicologist), Dr. Joseph M.
Winston (Associate Toxicologist), Mr. Jan L. Minor (Associate Toxicologist),
Dr. Chuen-Bin Hong (Associate Pathologist), with the technical assistance
of Mr. Brett Ferguson, Mr. Timothy Unger, and Ms. Mary Sawyer.
Approved for:
MIDWEST RESEARCH INSTITUTE
This work was conducted in the Biological Sciences Division under
W. B. House, Director
Biological Sciences Division
ii
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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.
SUMMARY
The toxicity of vinylidene chloride (VDC) was studied in mice and
rats exposed to various concentrations of the vapors for 23 hr a day. In
addition, the ability of various treatments to alter parameters of toxicity
was evaluated. Mice were more sensitive than rats to the acute lethal,
hepatotoxic and renal toxic effects of VDC. Disulfiram treatment reduced
these toxic effects of inhaled VDC and reduced the levels of covalent bound
radioactivity in the liver and kidney after the intraperitoneal administra-
tion of ^C-VDC. Treatment with diethyldithiocarbamate and thiram also
protected mice from the acute lethal effects of VDC.
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I. INTRODUCTION
Vinylidene chloride (1,1-dichloroethyIene; VDC) is an intermediate
used in the production of polymers and the synthesis of other chemicals.
Since environmental contamination and human exposure are inevitable results
of its widespread use, the risks of such exposures should be understood.
1 2/
The toxicity of VDC, which was recently reviewed,—1— has been
studied in several mammalian species.—/ A continuous 90-day inhalation
exposure to 189 mg/m^ (48 ppm) of VDC produced deaths in monkeys and guinea
pigs but did not in dogs and rats. In addition, morphological changes
occurred in livers from monkeys, dogs, and rats and in kidneys from rats.
The hepatic lesions included focal necrosis, hemosiderin deposition, and
fatty metamorphosis. The primary renal lesion was nuclear hypertrophy of
the tubular epithelium.
VDC toxicity was influenced by various parameters. Female rats
were more sensitive than males to the oral toxicity of VDC.— The nutri-
tional status also influenced toxicity. For example, starved rats were
more sensitive to VDC^./ and the diurnal change in toxicity was correlated
with hepatic levels of glutathione (GSH) .6./ Additional studies with diethyl-
maleateZ/ and cysteine^/ pretreatment suggest that hepatic levels of GSH
influence VDC toxicity. Metabolic studies indicate that (a) a major path-
way for detoxification of VDC is by conjugation with GSH and (b) hepatotoxicity
is associated with covalent binding of VDC metabolites in the liver.—'
The purpose of this study was to determine the acute toxicity of
continuously inhaled VDC and evaluate the effect of various treatments on
this parameter. The treatments were selected to alter the metabolic activa-
tion of VDC or promote the detoxification of VDC. In addition, adrenergic
blocking agents were used since VDC exposure sensitized rat hearts to cate-
cholamines
II. METHODS
A. Animals
CD rats and CD-I mice (Charles River Breeding Laboratories, North
Wilmington, Massachusetts) were housed in our animal quarters for at least
7 days prior to use. These quarters were maintained at 24 ± 2°C with a
relative humidity of 50 ± 10% and a 7 AM to 7 FM photoperiod. Rats were
generally housed two per cage and mice six per cage. Animals were given
free access to rodent chow (Wayne Lab-Blox, Allied Mills, Inc., Chicago,
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Illinois) and tap water. During the exposure period, animals were housed
in stainless steel cages with wire mesh bottoms. When feed consumption was
determined, the animals were given powdered rodent chow in stainless steel
feeders (Hoeltge, Cincinnati, Ohio) which were designed to minimize
spillage.
B. Exposure to VDC
1. Duration: Animals were exposed to VDC for 22 to 23 hr a day
for 7 days. Control animals were similarly housed and exposed to room air.
2. Chamber design: Five stainless steel cubical type animal
exposure chambers were used in these studies. Three chambers were of 3.5 m^
volume and two chambers were 4.5 m^ in volume. The contaminants entered the
inlet air stream and were mixed in a plenum at the top of each chamber. Air
was exhausted at the bottom of the chamber.
3. Chamber air supply and flow rate: The air supply to the
chambers was drawn from a stack on the roof of the building. Chamber air
passed through a coarse filter and then over coils for heating, cooling and
dehumidifying. Chamber temperature was maintained at 75°F ± 5° and humidity
at 50% ± 10%.
Air flow rates were measured at the exhaust side of the chambers
with orifice plates connected to magnehelic gauges. Chamber air flows were
calibrated using an Autotronics 100-ssx air flow transducer. Air flow rates
were adjusted to maintain 10 to 15 air changes per hour in each of the
chambers.
4. Chamber safety: All chambers were operated at a slight nega-
tive pressure (0.1-0.2 in. of water) to prevent escape of the contaminant
into the room atmosphere. All air from the chambers traveled under negative
pressure to an incinerator operated at a temperature of 1700° to 1800°F.
5. Generation of VDC vapor: Vinylidene chloride with a purity
of 99% was obtained from the Aldrich Company. VDC vapor was generated by
bubbling nitrogen into VDC contained in a glass flask. A stream of inlet
air was directed through the flask and carried the VDC vapor into the
plenum of the chamber. The rate of VDC generation was determined by the
flay. of nitrogen into the VDC flask. The nitrogen flow was controlled by
th_e use of rotameters.
6. Chamber atmosphere monitoring: VDC was quantified with, a
Varian 2700 gas chromatograph equipped with a flame ionization detector and
a stainless steel column (6 ft x 1/8 in.) packed with 0.4% Carbowax 1500 on
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Carbopak A. The injector, column, and detector temperatures were 135°C,
95°C and 170°C, respectively. A Varian CDS-111 chromatography data system
was used to determine chamber concentrations. The CDS-111 calculates chamber
concentration by integration of peak areas. The CDS-111 was calibrated with
VDC standards at the beginning of each day and provided a direct printout
of chamber concentration in parts per million.
VDC standards were prepared by serial dilution (weight/volume) of
VDC in carbon tetrachloride. The desired final concentrations in a 4 jUl
injection were determined by the following procedure:
mg/liter (fig/ml) = Ppm * ^
24450
The amount of VDC (mol wt = 96.94) in a 1-ml sample from chambers
that contain 10, 50 and 100 ppm VDC is 0.04, 0.20 and 0.40 g, respectively.
For 1 Ml of standard, these same amounts would be obtained from solutions
of 40, 200 and 400 mg/liter; but since 4 fil of standard were injected,
the final dilutions of VDC in carbon tetrachloride were 10, 50 and 100 mg/
liter. Each point on the calibration curve was the mean of three determina-
tions.
Each chamber was equipped with 10 sampling ports on two sides of
the chamber. Preliminary studies were conducted to determine the uniformity
of the concentration of the test material at different points in the chamber.
The concentration at the various sampling points was compared to a reference
point in the center of the chamber. It was found that the average concen-
tration at the various points was ± 3% of the desired chamber concentration.
The concentration at the reference point was 98.4% to 100.4% of the average
chamber concentration. Therefore, the chambers were routinely sampled at
the reference point for determination of chamber concentrations.
C. Treatments to Alter VDC Toxicity
Groups of mice were treated with one of several compounds in order
to alter the toxicity of VDC. The chemical structure of some of these com-
pounds is shown in Figure 1. The compounds used in this study were: disulfiram
(0.10% in feed 2 to 3 days before and during exposure), diethyldithiocarbamate
(0.12% in feed 3 days before and during exposure), thiram (0.10% in feed 3
days before and during exposure), cysteine CO.10% or 0.50% in feed 3 days
before and during exposure), methionine (0.10% or 0.50% in feed 3 days before
and during exposure), N-acetylcysteine (1,200 mg/kg orally every day during
exposure), SKF 525-A (50 mg/kg ip every day during exposure), cobaltous chlor-
ide* 6 H2O (60 mg/kg ip daily for 2 days before exposure), BAL (50 mg/kg sc
daily during exposure), phenoxybenzamine (10 mg/kg ip daily for first 2 days
of exposure), propranolol (10 mg/kg ip daily during exposure), Vitamin C
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(100 mg/kg ip daily during exposure), DL-a-tocopherol acetate (Vitamin E,
750 mg/kg orally once 2 days before exposure and on first day of exposure),
ethanol (15% in drinking water 2 days before and during exposure), sodium
phenobarbital (0.1% in drinking water 7 days before and during exposure),
or selenium (1 ppm in feed 10 days before and during exposure).
D. Observations
1. General: Mortality, general well being, and in some cases,
feed consumption were determined for animals exposed to VDC.
2. Histopathology: Livers, kidneys, and hearts from male mice
and rats exposed to VDC were examined for microscopic lesions. The tissues
were fixed in neutral buffered 10% formalin, processed in an Autotechnicon
and stained with H and E stain.
3. Serum enzymes: Serum glutamic-oxaloacetic transaminase (SGOT)—^
and serum glutamic-pyruvic transaminase (SGPT)i?/ were determined in cardiac
blood from mice and aortic blood from rats.
4. Hepatic non-protein sulfhydryl concentrations: A colorimetric
method—^ was used to measure this parameter in: (a) male and female rats
exposed to 0 and 75 ppm VDC for 1, 2, 4, 8, and 16 days. As the result of
logistic problems, these assays were conducted 3 to 4 hr after the animals
were removed from the chamber; and (b) male mice that received various treat-
ments for a total of 10 days (i.e., 3 days before exposure and 7 days during
exposure to room air).
5. Glucose 6-phosphate dehydrogenase activity: Male rats were
exposed to 0 and 75 ppm VDC for 16 days. The rats were sacrificed by de-
capitation and the liver was removed, weighed, and homogenized in 16 volumes
of Buffer A (55 mM Tris buffer that contained 3.3 mM MgC^ with a pH of
7.8). The homogenate was centrifuged at 20,000 g for 15 min. The enzyme
assays contained 2.7 ml Buffer A, 0.1 ml of 6 mM NADP, 0.1 ml of 100 nM
glucose 6-phosphate, and 0.1 ml of the liver supernatant. The assay blank
contained everything except glucose 6-phosphate. The rate of reaction was
measured by the production of NADPH which was followed spectrophotometrically
at 340 nm. The protein concentration-^/ was determined on the supernatant.
6. Covalently bound radioactivity: Covalent bound radioactivity
was measured in male mice after the intraperitoneal administration of 3 mg/kg
¦^C-VDC (20 fiCi/kg) which was obtained from New England Nuclear (Boston,
Massachusetts) with a specific activity of 0.652 |iCi/nimole. The ^C-VDC was
slowly bubbled into the peanut oil vehicle with nitrogen. The tissues were
homogenized in cold water and macromolecules were precipitated with an equal
volume of 1 N perchloric acid (PCA). The precipitate was washed with 5 ml
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of 0.2 N PCA, 0.2 N PCA, 95% ethanol saturated with sodium acetate, absolute
ethanol, ethanol:ether (3:1), heated 1 hr at 37°C in 4 ml of 0.5 N sodium
hydroxide, and afterwards 3 ml of 30% thichloroacetic acid (TCA) was added.
The precipitate was washed with 5 ml of 5% TCA and heated in 5% TCA for 20
min on a boiling water bath. The pellet was washed with 5 ml of 5% TCA and
dissolved in 10 ml of 0.3 N sodium hydroxide. Radioactivity and proteinic/
were determined on this fraction and the results were expressed as DPM/mg
protein.
E. Statistics
Mortality data were evaluated in terms of the concentration of VDC
required to kill 50% of the animals (LC^q)—^ and the time required to kill
50% of the animals at 20 ppm of VDC (LT^q)^ Other data were analyzed
by the two sample rank testiZ/ or Fisher exact probability test—^ with a
level of significance selected as p 0.05. Data are reported as the mean +
S.E.
III. RESULTS
A. Toxicity of VDC in Mice and Rats
1. General: VDC was more toxic, as measured in terms of lethality
(dead/exposed) and hepatotoxicity (SG0T and SGPT), in mice than rats (Table 1).
Since the cause of death is not known, the toxicity of VDC, as discussed in
this report, is subdivided into lethality and organ toxicity. Organ toxicity
is measured in terms of serum enzymes (SG0T and SGPT) for the liver and histo-
pathology for the liver, kidney and heart.
2. Lethality: The lethality of VDC inhaled by mice and rats for
various days is presented in Table 2. Mice had a reduced feed consumption
(data presented below in part B), weight loss, rough coats, and were lethargic.
In addition, the body temperature of debilitated mice was reduced by 5 to 7°C.
The feed consumption and body weights of rats was also reduced; however, there
was no effect of 60 ppm VDC on body temperature. Mice were more sensitive to
the acute lethal effects of VDC than rats and most of the deaths occurred within
the first 3 days of exposure. Male mice were more sensitive to 19 and 41 ppm
of VDC than females; however, this effect was not significant at 80 ppm.
3. Organ toxicity:
a. Serum enzymes: The serum enzymes of male mice exposed to
0, 15, 30 and 60 ppm of VDC as a function of time are presented in Table 3.
After 1 exposure day, there was a dose-related increase in both SGOT and SGPT.
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Towards the end of the 5 days exposure period, these values tended to
decline. No mice survived a 3-day exposure to 60 ppm of VDC.
The serum enzymes in male rats were elevated after exposure to
60 ppm of VDC (Table 4). This change was not as dramatic as in mice simi-
larly exposed and required a longer exposure period before it was evident.
b. Histopathology: Microscopic lesions observed in male
mice exposed to 15, 30 and 60 ppm of VDC as a function of time are pre-
sented in Table 5. Although there was no hepatic midzonal necrosis at 15 ppm
of VDC, the severity of this lesion increased as the VDC concentration was
raised from 30 to 60 ppm. However, hepatocellular degeneration, which con-
sisted of centrilobular cloudy swelling and/or cytoplasmic vacuolization, was
evident at all of the VDC concentrations. An increased number of mitotic
figures in hepatocytes, was observed at both 15 and 30 ppm of VDC. A very
high incidence of tubular nephrosis was observed in the kidneys of mice ex-
posed to 15, 30 and 60 ppm of VDC. In addition, there was evidence of tubu-
lar regeneration in mice exposed to 15 ppm of VDC for 5 days. A mild focal
myocardial degeneration was observed in a few mice exposed to 15 and 30 ppm
of VDC. None of these lesions were observed in control mice at any of the
observation periods.
The microscopic lesions observed in rats exposed to 60 ppm
of VDC for various days are presented in Table 6. Centrilobular degenera-
tion and/or necrosis and mild hyperplasia of the bile duct occurred in the
rats exposed to VDC. In addition, mild focal myocardial degeneration
occurred in one rat exposed to 60 ppm of VDC for 3 days. None of these
lesions were present in the control group at any of the observation periods.
B. Effect of Various Treatments on the Lethality of VDC in Mice
Groups of mice were given one of several compounds during initial
studies to identify compounds that altered the toxicity of VDC. Ethanol
and BAL increased the lethality of 20 ppm VDC in male mice (Table 7). in
contrast, phenobarbital treatment reduced the mortality of 74 ppm VDC in
female mice. Selenium did not alter the toxicity of VDC.
Additional studies were conducted to identify compounds that modi-
fied the lethal effects of VDC. Some of these compounds were incorporated
into the feed. The feed was given prior to VDC exposure, since mice ex-
posed to VDC had a reduced feed consumption (Table 8). Although, mice that
received cysteine (0.1% in diet), methionine (0.1% in diet) and disulfiram
had a reduced feed consumption during VDC exposure, they did consume the diets
containing these compounds in the absence of VDC. Disulfiram was the only one
of these compounds that altered the LC^q value of VDC, which was calculated
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after the first exposure day (Table 9). In addition, cobaltous chloride
did not alter this measure of toxicity.
The LCjq of VDC, which was calculated after the second day of ex-
posure, was increased in mice pretreated with disulfiram, diethyldithio-
carbamate (DDC), and thiram (Table 10). However, treatment with N-acetyl-
cysteine, methionine (0.5% in diet), cysteine (0.5% in diet), propranolol,
Vitamin C, and Vitamin £ did not alter this parameter.
If lethality was expressed in terms of the time required to kill
50% of the mice exposed to 20 ppm of VDC (LT^g), then disulfiram diethyl-
dithiocarbamate (DDC), and thiram provided protection (Table 11). In addi-
tion, this presentation of the data suggested that methionine (0.5% in diet)
and cysteine (0.5% in diet) also provided a degree of protection. None of
the other treatments presented in Table 11 protected mice against the lethal
effects of VDC.
C. Effect of Disulfiram Treatment on the Organ Toxicity of VDC in Mice
1. Serum enzymes: The hepatotoxicity of VDC, as measured by ele-
vated SGOT and SGPT, was reduced in male mice treated with disulfiram
(Table 12). This protective effect was evident after the first, but not the
second or third, exposure days. No control mice survived 3 days in an atmo-
sphere containing 60 ppm VDC. In contrast, disulfiram treated mice survived
a similar exposure to VDC. Serum enzymes in control mice exposed to room
air are presented in Table 3. Treatment with disulfiram did not alter these
values.
2. Hlstopathology: The microscopic lesions observed in control
and disulfiram treated male mice exposed to 60 ppm VDC for various days is
presented in Table 13. None of these lesions were observed in either control
or disulfiram treated mice that were exposed to room air. As a result of the
high mortality in control mice exposed to 60 ppm VDC, it is valuable to com-
pare the disulfiram treated group (Table 13) with the various concentrations
of VDC previously presented (Table 5). No midzonal necrosis was observed in
mice treated with disulfiram and exposed to VDC for 1 day. However, midzonal
necrosis was observed in this group after the second exposure day. Disulfiram
also reduced the severity of renal tubular nephrosis early in the exposure
period; however, as the duration of exposure increased, the severity of this
lesion also increased.
D. Biochemical Observations on the Toxicity of VDC and the Effect
of Various Treatments
1. Covalent binding: Radioactivity, covalently bound to protein,
was measured in the liver and kidney at 4 and 24 hr after the intraperitoneal
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administration of ^C-VDC (Table 14). The level of bound radioactivity was
reduced at both times in mice pretreated with disulfiram.
2. Hepatic non-protein sulfhydryl concentration in rats exposed
to VDC: The non-protein sulfhydryl concentration in the livers from male
and female rats exposed to 75 ppm of VDC for various days is presented in
Table 15. These values increased in a biphasic fashion over the 16-day
observation period. Since these observations were made 3 to 4 hr after ex-
posures were terminated, these values do not represent the non-protein
sulfhydryl concentration during exposure.
3. Hepatic non-protein sulfhydryl concentration in the male
mice after various treatments: This value was measured to determine if
various treatments elevated the hepatic non-protein sulfhydryl concentra-
tion. Male mice exposed to room air and fed diets that contained disul-
firam, diethyldithiocarbamate, thiram, cysteine (0.5% in feed), or methionine
(0.5% in feed) for 10 days did not have elevated concentrations of hepatic non-
protein sulfhydryl groups (Table 16).
4. Hepatic glucose 6-phosphate dehydrogenase activity in male rats
exposed to VDC: The activity of this enzyme was increased about four-fold
in the liver of male rats exposed to 75 ppm of VDC for 16 days (Table 17).
IV. DISCUSSION
The results of this study demonstrate that (1) mice are more sensi-
tive than rats to the lethal, hepatotoxic and renal toxic effects of VDC, (2)
disulfiram reduces the acute lethal and organotoxic effects of inhaled VDC
and reduces the levels of covalent bound radioactivity in the liver and kidney
after ^C-VDC, and (3) diethyldithiocarbamate, thiram and, to a lesser extent,
methionine and cysteine also protect mice from the lethal effects of VDC.
Microscopic examination of the liver and kidney revealed that renal
tubular nephrosis was the lesion that occurred most frequently at the concen-
tration of VDC studied. In addition, there was evidence that this lesion was
reversible, as demonstrated by the presence of tubular regeneration, after a
5-day exposure to 15 ppm. However, there was no similar evidence of reversi-
bility in mice exposed to 15 ppm for 1, 2 and 3 days or 30 and 60 ppm for any
of the times studied. Renal lesions are of interest in view of the occurrence
of kidney tumors in mice exposed to VDC.—^ However, no kidney tumors were
observed in CD-I mice, the strain used in the present study, during a 12-month
exposure to 55 ppm of VDC.—^
The cause of death is a topic for speculation. The data presented
in this report suggest that death is due to hepato-renal failure. However,
other mechanisms involving, for example, the cardiovascular or respiratory
system may also be involved.
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Disulfiram, diethyldithiocarbamate, and thiram are structurally
related dithiocarbamates. Disulfiram is used clinically in alcohol therapy
programs and thiram is used both in the agricultural and rubber industry.
Disulfiram is metabolized to diethyldithiocarbamateZl/ and both compounds
alter the metabolism of xenobioticsi-l/ and protect against several types
of drug-induced toxicities.^3,24/ in addition, members of the dithiocarba-
25/
mate class have radioprotective properties.—'
Although the mechanisms by which the dithiocarbamates tested pro-
tected against VDC toxicity is uncertain, speculations may be offered con-
cerning such mechanisms. For these speculations, it is assumed that VDC is
metabolized by the hepatic mixed function oxidase system to a compound that
produces organotoxicity which results in death. If disulfiram reduces the
metabolic activation of VDC, then SKF 525-A—^ and cobaltous chloride^!/
should also have provided protection. If treatments protected by providing
additional sulfhydryl groups for the detoxification of VDC epoxides, then
doses of N-acetylcysteine that protect mice from acetaminophen toxicity^/
should also have protected against VDC toxicity. Although cysteine and
methionine, which are also sulfhydryl containing compounds, provide a degree
of protection, the effect is not as dramatic as with the dithiocarbamates.
The failure of these compounds to alter the hepatic nonprotein sulfhydryl
concentration may be due to (1) pharmacokinetic properties of the compounds
and/or (2) adaptation of the liver to an increased supply of sulfhydryl
containing compounds.
The results suggest that disulfiram protects against toxicity by
a mechanism that involves more than an inhibition of VDC activation or an
increase in VDC detoxification. Possibly, disulfiram is superior to the
other nondithiocarbamate compounds because both mechanisms are operating
simultaneously. In other words, disulfiram and its metabolisms may not only
reduce the activation of VDC but also increase the extent of detoxification.
In this regard, dithiocarbamates may serve as more effective molecules for
detoxifying VDC metabolites than some of the sulfhydryl containing compounds
that were tested.
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against carbon tetrachloride-induced lipid peroxidation in vitro.
Biochem. Pharmacol. 22:1729 (1973).
Wattenberg, L. W. Inhibition of carcinogenic and toxic effects of poly-
cyclic hydrocarbons by several sulfur-containing compounds. J. Nat.
Cane. Inst. 52:1583 (1974).
12
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25. Barnes, J. H., et al. Synthese et effects radioprotecteurs d'alkanebis-
dithiocarbamates disodiques, d'acides u-aminoalkyldithiocarbamiques
et de leurs derives N,N'-dimethyles. Eur. J. Med. Chem-Chimica
Therap. 10:619 (1975).
26. Conney, A. H., et al. Enzyme induction and inhibition in studies on the
pharmacological actions of acetophenetidin. J. Pharmacol. Exp. Ther.
151:133 (1966).
27. Tephly, T. R., and Hibbeln, P. The effect of cobalt chloride administra-
tion on the synthesis of hepatic microsomal cytochrome P-450. Biochem.
Biophys. Res. Comm. 42:589 (1971).
28. Piperno, E., and Berssenbruegge, D. A. Reversal of experimental para-
cetamol toxicosis with N-acetylcysteine. Lancet, October 2:738 (1976).
13
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CHEMICAL STRUCTURES
CI.
CI'
^C = CH2 VINYLIDENE CHLORIDE
c2H5v ^2^5
N-C-S-S-C-N
C2H5' I I "c2h5
NH2-CH-CH2-SH
1
COOH
DISULFIRAM
CYSTEINE
C2H5.
C2H5'
N-C-S" Na
NH2-CH-CH2-CH2-SCH3
COOH
DIETHYLDITHIOCARBAMIC ACID,Na
METHIONINE
CH3
CH3
/CH3
^N-C-S-S-C-N^
ch3
Ac-NH-CH-CH2-SH
COOH
THIRAM
N-ACETYLCYSTEINE
ch2-ch2-ch2oh
t I
SH SH
BAL
Figure 1 - Chemical Structures of Some Compounds Used in the
Study to Alter VDC Toxicity
14
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TABLE 1
TOXICITY OF 60 PPM VDC IN MICE AND RATS
Mice
Rats
Days
Exposed
1
2
1
2
Dead /
Exposed
2/10
8/10
0/10
0/10
SGOT
(IU/L)
1,946 + 211—
751 + 150
74+6
264 + 33
SGPT
(IU/L)
3,043 + 209
1,112 + 226
44 + 7
198 + 29
a/ Mean + S.E. for 2 to 5 determinations.
TABLE 2
LETHALITY OF VDC INHALED BY MICE AND RATS
Species Sex
Mice Male
Rats
Days Exposed
VDC fppm)
19
41
oa/
20
5(&/
30 ,
50^
4C&{
70^
80
20
100
100
100
Female
19
0
0
0
0
41
0
0
0
0
80
10
70
80
80
Male
313
0
0
0
0
at Percent dead (10 animals/group).
b/ Lethality in males significantly different from females (Fisher
exact probability test).
15
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TABLE 3
EFFECT OF VDC EXPOSURE ON SERUM ENZYMES IN MALE MICE
Days Serum
Exposed Enzyme
VDC Concentration (ppm)
15
30
60
\*U
SGOT 56+8 (5)2/ 249 + 28 (4) 466 + 50 (5) 1,946 + 271 (4)
SGPT 32 + 6 (5) 200 + 19 (4) 474 + 66 (5) 3,043 + 209 (4)
SGOT 82 + 30 (4) 91+5 (5) 705 + 120(6) 751 + 150 (2)
SGPT 38+4 (4) 189 + 26 (5) 675 + 81 (6) 1,112 + 226 (2)
SGOT
SGPT
64 + 14 (5)
29+2 (5)
138 + 16
166 + 33
(4) 448 + 179(4)
(4) 514 + 200(4)
No
Survivors
SGOT
SGPT
60+5 (5)
32+2 (5)
118 + 18
47+5
(6) 294 + 50 (2)
(6) 109 + 8 (2)
No
Survivors
a/ Mean + S.E. (number of mice).
TABLE 4
EFFECT OF VDC EXPOSURE ON SERUM ENZYMES IN MALE RATS
Days Serum VDC Concentration (ppm)
Exposed Enzymes 0_ 60
1 SGOT 66 + 10 (5)—^ 74+6 (5)
SGPT 28 + 2 (5) 44 + 7 (5)
2 SGOT 63+4 (5) 264 + 33 (5)
SGPT 34 + 4 (5) 198 + 29 (5)
3 SGOT 81 + 4 (5) 238 + 47 (5)
SGPT 34+6 (5) 122 + 29 (5)
a/ Mean + S.E. (number of rats).
16
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TABLE 5
MICROSCOPIC LESIONS IN ORGANS OF MICE EXPOSED TO VDC
VDC (ppm)
15
30
Mitotic figures of hepatoctes
Slight increase
Moderate increase
Hepatocellular degeneration
60
Exposure Days:
1
2
3
5
1
2
3
5
1
2
Number of Mice Examined
5
5
5
7
5
5
4
2
4
2
Liver lesions
Midzonal necrosis
Qa/
Mild
0
0
0
20
40
50
0
0
50
Moderate
0
0
0
0
0
0
0
0
50
50
Marked
0
0
0
0
0
0
0
0
50
0
0
60
40
0
0
0
0
0
0
0
0
0
0
0
0
0
25
0
0
0
20
40
20
86^/
20
40
0
50
25
0
Kidney lesions
Tubular nephrosis
Moderate
0
0
0
57
0
0
0
0
0
0
Marked
0
0
0
43
0
0
0
0
0
0
Severe
10(£/
100^/
100^/
0
10(£/
100^/
10(£/
ioo^./
50
100^
Tubular regeneration
0
0
0
86^/
0
0
0
0
0
0
Cardiac lesions
Mild focal 0 0 0 28 0 0 0 50 00
myocardial degeneration
a/ Percent of mice examined with the indicated lesion. None of these lesions were observed in the control
group (five mice/day) exposed to room air.
b/ Significantly different from control group (Fisher exact probability test).
-------
TABLE 6
MICROSCOPIC LESIONS IN
ORGANS OF RATS EXPOSED TO
60 PPM VDC
Exposure Days 1
2 3
Number of Rats Examined
5
5 5
Liver lesions
Centrilobular degeneration
and/or necrosis
Mild
40
o
00
Moderate
0
0 60
Mild hyperplasia of
60
40 0
bile duct
Cardial lesions
Mild focal 0 0 20
myocardial degeneration
a/ Percent of rats examined with the indicated lesion. None of
these lesions were observed in the control group (five mice/
day) exposed to room air.
bJ Significantly different from control group (Fisher exact pro-
bability test).
TABLE 7
MORTALITY IN MICE EXPOSED TO VDC FOR 7 DAYS
Sex Treatment VDC (ppm) % Dead
Male None 20 40
Ethanol 20 100
BAL 20 100
Female None 74 90
a/
Phenobarbital 74 0—
a/ Significantly different from non-pretreated group
(Fisher exact probability test).
18
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TABLE 8
FEED CONSUMPTION MALE MICE EXPOSED TO VDC
VDC Concentration (ppm)
Treatment 0 41 80
None 4.4 + 0.1—^ 1.5 + 0.3 1.7 + 0.1
Cysteine (0.1%) 4.6 + 0.2 1.8 + 0.6 1.8 + 0.3
Methionine (0.1%) 4.8 + 0.1 1.5 + 0.1 1.8 + 0.1
Disulfiram (0.1%) 4.2 + 0.1 3.3 + 0.3 2.8 + 0.1
a/ Gqi/mouse/day determined on a cage basis for a total of 10 mice
housed 5 mice/cage.
TABLE 9
ONE DAY LC?n (PPM) OF VDC IN MICE
Treatment Male Female
Control 98 (82-118)-' 105 (92-121)
Disulfiram (0.10%) >320 >320
Cysteine (0.10%) 98 (76-127) 92 (74-113)
Methionine (0.10%) 113 (93-138) 113 (93-138)
CoCl, 113 (81-157) 123 (85-179)
a/ LC50 in ppm (95% confidence limits) or approximation
°f LCj0.
19
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TABLE 10
TWO DAY LCcQ (PPM) OF VDC IN MICE
Treatment Male
Control
35
(25-47)-'
Disulfiram (0.10%)
>160
DDC (0.12%)
>160
Thiram (0.10%)
>160
N-Acetylcysteine
20
(7-25)
Methionine (0.50%)
38
(28-51)
Cysteine (0.50%)
43
(31-58)
SKF 525-A
26
(17-35)
Phenoxybenzamine
35
(25-47)
Propranolol
28
(19-37)
Vitamin C
35
(25-46)
Vitamin E
35
(25-47)
a/ LC50 *n PP111 (957. confidence limits) or
approximation of LC
TABLE 11
LTgn (DAYS) AT 20 PPM OF VDC
Treatment
Male
Control
4.0
(3.6-4.4)—7
Disulfiram (0.10%)
>7
DDC (0.12%)
>7
Thiram (0.10%)
>7
Cysteine (0.50%)
>7
Methionine (0.50%)
7
N-Acetylcysteine
1-2
SKF 525-A
2.4
(1.7-3.4)
Phenoxybenzamine
3-4
Propranolol
2.7
(2.0-3.7)
Vitamin C
2.6
(1.9-3.5)
Vitamin E
3-4
a/ ^50 days (95% confidence limits) or
approximation of LT^Q.
20
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TABLE 12
TOXICITY OF 60 PPM VDC IN CONTROL AND DISULFIRAM TREATED MICE
Diet
Control
Disulfiram
Days
Exposed
to VDC
1
2
3
1
2
3
SGOT
(IU/L)
1,946+271 (4)-'
751 + 150 (2)
No Survivors
140 + 38 (5)
784 + 332 (4)
2,292 + 965 (3)
a/
SGPT
(IU/L)
3,043 + 209 (4)
1,112 + 226 (2)
No Survivors
66 + 11 (5)
1,236 + 668 (4)
3,182 + 1,488 (3)
a/ Mean + S.E. (number of observations).
21
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TABLE 13
MICROSCOPIC LESIONS IN ORGANS OF MICE EXPOSED
TO 60 PPM VDC AND TREATED WITH DISULFIRAM
Treatment None Disulfiram^/
Exposure Days: 1 2 12 3
Number of Mice Examined 4 2 5 5 3
Liver lesions
Midzonal necrosis
Mild
Ob/
50
0
0
0
Moderate
50
50
0
0
0
Marked
50
0
0
60
0
Severe
0
0
0
0
67
Hepatocellular degeneration
25
0
0
20
33
Kidney lesions
Tubular nephrosis
Mild
0
0
20
40
0
Moderate
0
0
20
20
0
Marked
50
0
0
40
100
Severe
50
100
0
0
0
a/ Disulfiram (0.1%) in feed 3 days before and during exposure
to VDC.
b/ Percent of mice examined with the indicated lesion. None of these
lesions were observed in groups (five mice/day) fed either control
or disulfiram diets and exposed to room air.
22
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TABLE 14
COVALENTLY BOUND RADIOACTIVITY AFTER 14C-VPC IN CONTROL
AND DISULFIRAM TREATED MICE
Tissue
Liver
Hr after
14C-VPC
4
24
DPM/mg Protein
Control
88 + lW
58 + 2
Disulfiram
31 + 7
23 + 5
Kidney
4
24
134 + 9
88 + 2
32+7
27 + 4
a/ Mean + S.E. for four determinations.
TABLE 15
HEPATIC NON PROTEIN SULFHYDRYL
CONCENTRATION IN RATS EXPOSED
TO 75 PPM OF VDC
Concentration—^
Days Exposed (7o of Control)
To VDC Males Females
1 145 + 5^ 149 + 4
2 100+8 118 + 8
4 95+4 138 + 11
8 184 + 22 158 + 6
16 201 +9 170+8
a/ Determined 3 to 4 hours after rats
removed from chamber,
b/ Mean + S.E. for three to four de-
terminations.
23
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TABLE 16
HEPATIC NON-PROTEIN SULFHYDRYL
CONCENTRATION IN MALE MICE
AFTER VARIOUS TREATMENTS
Concentration
Treatment—^ (% of Control)
None 100 + .
Disulfiram (0.10%) 102 + 2
DDC (0.127.) 100 + 3
Thiram (0.10%) 95+2
Cysteine (0.50%) 84+2
Methionine (0.50%) 87+1
a/ Mean + S.E. for four observations,
TABLE 17
HEPATIC GLUCOSE 6 PHOSPHATE
DEHYDROGENASE ACTIVITY
IN MALE RATS EXPOSED
TO VDC FOR 16 DAYS
VDC (ppm) Activity—^
0 20.2 + 1.6^
75 80.7 + 4.1
a/ nMoles NADPH formed/min/mg
protein.
b/ Mean + S.E. for four deter-
minations .
24
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TECHNICAL REPORT DATA
(Please read fnurnctioiis on the reverse before completing)
. REPORT NO.
EPA-
3. RECIPIENT'S ACCESSIOI*NO.
4. TITLE AND SUBTITLE
Toxicity Studies of Selected Chemicals Task III:The
Toxicity of Vinylidene Chloride in Mice and Rats and
its Alteration by Various Treatments
5. REPORT DATE
June 1977
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
R. D. Short, J. M. Winston, J. L. Minor, C. B. Hong,
B. Ferguson, T. Unger, M. Sawyer, and C. C. Lee
8. PERFORMING ORGANIZATION REPORT NO.
9. Pfc R FORMING ORGANIZATION NAME AND ADDRESS
Midwest Research Institute
425 Volker Boulevard
Kansas City, MO 64110
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
68-01-3242
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Protection Agency
Office of Toxic Substances
Washington, DC 20460
13. TYPE OF REPORT AND PERIOD COVERED
Draft Final
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES . , . - T7. 1 JJ r
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