PUBLIC REVIEW
27751
DRAFT CRITERIA DOCUMENT
FOR CARBON TETRACHLORIDE
FEBRUARY 1984
Prepared by
JPB Associates, Inc.
Contract No. 2-813-03-644-09
for
HEALTH EFFECTS BRANCH
CRITERIA AND STANDARDS DIVISION
OFFICE OF DRINKING WATER
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
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27651
DISCLAIMER
This draft document has not been released formally by the
Office of Drinking Water, U.S. Environmental Protection
Agency, and should not at this stage be construed to represent
Agency policy. It is being circulated for comments on its
technical merit and policy implications.
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PREFACE
The objective of this criteria document is to assess the
available data on health effects associated with exposure to
carbon tetrachloride in drinking water and to recommend a
maximum contaminant level. To achieve this objectivet data
on pharmacokinetics, human exposure, acute and chronic health
effects in animals and humans, mechanisms of toxicity were
evaluated!/ Only the reports which were considered pertinent
for the derivation of the maximum contaminant level are cited
in the document. Particular attention .was paid to the utiliza-
tion of primary references for the assessment of carbon tetra—
chloride-induced health effects. Secondary references were
used rarely. For comparison purposes, standards and criteria
developed by other organizations are included and discussed
in Section IX (Quantification of Toxicological Effects of
Carbon Tetrachloride).
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CONTENTS
Page
I. SUMMARY 1-1
II. GENERAL INFORMATION AND PROPERTIES II-l
Analysis of Carbon Tetrachloride in II-4
Drinking Water
Treatment of Carbon Tetrachloride in II-5
Drinking Water
Summary II-6
III. PHARMACOKINETICS III-l
Absorption III-l
Distribution III-5
Metabolism III-8
Excretion 111-13
Summary III-15
IV. HUMAN EXPOSURE* IV-1
V. HEALTH EFFECTS IN ANIMALS V-l
Acute Effects V-l
Chronic Effects V-17
Teratogenicity V-21
Reproductive Effects V-25
Mutagenicity V-28
Carcinogenicity V-34
Summary V-43
VI. HEALTH EFFECTS IN HUMANS VI-1
Case Studies—Acute Effects VI-2
Acute Effects VI~4
*Prepared by the Science and Technology Branch
-2-
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CONTENTS (Contined)
Case Studies — Long-Term Effects
Controlled Studies
Summary
VII. MECHANISMS OF TOXICITY
Formation of Carbonyl Chloride (Phosgene)
Dimerization to Hexachloroethane
Free Radical Binding to Proteinds
Lipid Peroxidation
Summary
VIII. RISK ASSESSMENT
Quantification of Carcinogenic Risk
Sensitive Populations
Interaction of Carbon Tetrachloride with
Other Chemicals
Summary
IX. QUANTIFICATION OF TOXICOLOGICAL EFFECTS OF
CARBON TETRACHLORIDE
Non-Carcinogenic Effects
Quantification of Non-Carcinogenic Effects
Carcinogenic Effects
Quantification of Carcinogenic Effects
Special Considerations
Page
VI- 8
VI- 13
VI-16
VI I- 1
VI I- 1
VI I- 2
VI I- 2
VI I- 8
VII-13
VIII-1
VIII-1
VII I- 3
VIII-5
VII-14
IX- 1
IX- 3
IX-7
IX- 9
IX-12
IX- 15
X. REFERENCES CITED
X-l
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I. SUMMARY
Carbon tetrachloride, also known as tetrachloro-
methane and perchloromethane, is a haloalkane with a wide
range of industrial and chemical applications. In 1980,
322,000 kkg were produced in the United States. Most of the
chemical produced is used in the manufacture of fluorocarbons,
which are used as refrigerants, foam blowing agents, and
solvents. Carbon tetrachloride is also used as a solvent
in metal cleaning and in the manufacture of paints and plas-
tics as well as in fumigants. It is being largely replaced
in grain fumigation by other registered pesticide products.
Inhalation is the most important route of carbon
tetrachloride exposure. There are no major gradients in the
atmospheric distribution of carbon tetrachloride; the
concentrations are similar in the continental and marine air
masses ambient (approximately O.JD0070-0.00084 mg/m3 or
111-133 ppt). The maximum level of carbon tetrachloride
reported was 0.113 mg/m3 (18,000 ppt) in Bayonne, New Jersey.
Carbon tetrachloride has been found in many sampled
waters (including rain, surface^ potable, and sea) at the ppb
level. The National Organics Monitoring Survey (NOMS) sampled
113 public water systems and found carbon tetrachloride at
very low concentations, relative to levels of chloroform
and other organics. Positive results were noted in about 10
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1-2
percent of the samples, with mean values ranging from 2.4-6.4
ppb depending on sampling and analytical procedures. An
accidental discharge of an estimated 70 tons of carbon
tetrachloride into the Kanawha River resulted in levels of
carbon tetrachloride as high as 300 ppb in raw Ohio river
water; the drinking water levels were found to be as high as
100 ppb. Chlorination of raw waters with chlorine gas contami-
nated with carbon tetrachloride may account for the presence
of the compound in some finished drinking waters.
Carbon tetrachloride has been detected in a variety of
foodstuffs other than fish and shellfish in levels ranging from
1-20 ppb. Carbon tetrachloride contaminations of food categories
has been reported as follows: Dairy products (0.2-14.0- ppb);
meat (7.0—9.0 ppb); oils and fats (0.7-18.0 ppb); beverages
(0.2-6.0 ppb); fruits and vegetables (3.0-8.0 ppb); black grapes
(19.9 ppb); and fresh bread (5-1-0 ppb). The amount of carbon
tetrachloride residue in some foodstuffs depends on the
fumigant dosage, storage conditions, length of aeration, and
extent of processing.
No information was found on levels or frequencies of
human dermal exposures. Carbon tetrachloride is no longer on
the market as a component of hair shampoo or cleaning solvents,
and because of .strict OSHA safety regulations, dermal exposure
in the workplace has been minimal.
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1-3
Carbon tetrachloride is absorbed into circulation
through the lungs and the skin. Absorption from the gas-
trointestinal tract also readily occurs but at a slower
rate. Following a single exposure, high concentrations
of carbon tetrachloride were detected in the blood, brain,
kidney, and liver. With repeated exposures, carbon tetra-
chloride is present mostly in the liver and, because of
its lipophilic nature, in the body fat and bone marrow.
In both animals and humans exposed to carbon tetra-
chloride, the liver is the first organ to be affected. Minor
liver damage, as evidenced by increased levels of liver enzymes
in the- serum, progresses through early necrotic changes, in-
creased vacuolization and fat droplets, to necrosis, degen-
eration, and fatty liver. Most acute liver damage is rever-
sible with time. Chronic necrotic and degenerative changes,
however, appear not to be reversed as readily. Exposure to
other hepatotoxins especially ethanol and drugs such as
barbiturates, greatly exacerbates the hepatic toxicity of
carbon tetrachloride. Pulmonary damage occurs, but mostly
at an ultrastructural level and to a much lesser extent
than liver damage. Renal and pancreatic effects also have
been observed, but only following massive doses of carbon
tetrachloride.
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1-4
In "humans, acute toxic effects of carbon tetrachlo-
ride include gastrointestinal disturbances, nausea, and
vomiting. Hepatic injury occurs with all of the signs of
severe cellular damage. Serious renal injury usually does
not become evident until 24-48 hours or several days after
the onset of initial symptoms of poisoning. In severe
cases, death from hepatic coma and uremia occurs within
1-2 weeks. After repeated or prolonged inhalation of less
toxic concentrations of carbon tetrachloride, gastrointesti-
nal and central nervous system symptoms occur such as
nausea, vomiting, visual disturbances, and giddiness.
After repeated exposure, severe hepatorenal damage can
occur. Treatment and cessation of exposure usually result
in disappearance of toxic symptoms and reversal of the
liver and kidney damage.
Carbon tetrachloride has been shown to be carcino-
genic in rats, mice, and hamsters. Although a number of
cases of liver tumors following exposure to carbon tetrachlo-
ride have been reported, the chemical has not been established
to be a human carcinogen. Although it is not mutagenic in
the Salmonella (Ames) assay, carbon tetrachloride has
elicited a mutagenic response in the yeast Saccharomyces
cerevisiae. Carbon tetrachloride has also been shown to
be fetotoxic and to inhibit male fertility in rats.
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1-5
The mechanism of toxicity of carbon tetrachloride has
been extensively studied with special emphasis on the hepatic
effects. Carbon tetrachloride is metabolized by the liver
mixed function oxidase system to trichloromethyl (and other)
free radicals, which can either react with hepatic macromolecules
or be further metabolized via chloroform and phosgene to carbon
(mono/) dioxide. The reaction of the trichloromethyl radical
with cellular lipids initiates a lipid peroxidation resulting
in the progressive and exponential destruction of membranes.
Membrane permeability also is greatly increased as a result
of lipid peroxidation. Although some investigators still
maintain that direct reaction with hepatic macromolecules result
in hepatic damage, the recent ultrastructural evidence tends to
substantiate the lipid peroxidation theory.
By extrapolation from animal studies, the National
Academy of Sciences (NAS) and EPA's Carcinogen Assessment
Group (CAG) have calculated projected incremental excess
cancer risk associated with the consumption of specific
chemicals in drinking water over a 70-year lifetime. Using
the multistage model, NAS estimated that comsumption of 2
liters of water per day over a lifetime at carbon tetrachloride
concentrations of 450, 45, or 4.5/liter would result in the
induction of one excess case of cancer per 10,000, 100,000,
or 1,000,000 people exposed, respectively.
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1-6
Using the "improved" multistage model, CAG esti-
mated that consumption of 2 liters of water per day at
carbon tetrachoride concentrations 42.2 ug/1, 4.2 ug/1, or
0.4 ug/1 would result in the induction of one excess case
of cancer per 10,000, 100,000, or 1/000,000 people exposed,
respectively.
The differences between NAS and CAG risk esti-
mates are partly explained by the fact that the extrapolation
models used by the two groups, although similar, are not
identical. The NAS risk estimate was arrived at using the
multistage model; whereas, the CAG derived their risk esti-
mate using the "improved" version of the multistage model.
In addition, the models also differ in the data selected and
other parameters. The NAS model used rats as the comparison
species; the CAG model used mice.
These modeling methods share the assumption that
there is no threshold level for the action of a carcinogen.
However, no one method can accurately predict the absolute
numbers of excess cancer deaths that will be attributable to
carbon tetrachloride in drinking water. None of the methods
presently used to quantify carcinogenic risk accounts for
increased carcinogenic risk from the interaction of carbon
tetrachloride with other environmental contaminants to
which humans can be exposed.
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Ir 7
It is noteworthy that in assessing CCl4~induced
toxicity, carcinogenicity or any other harmful effect,
compounds that react synergistically or antagonistically
with CC14 must be considered. Identified synergistic
substances include ethanol, kepone, PCB, and PBB. Antago-
nistic effects have been demonstrated with such compounds
as chloramphenicol and catechol. Sensitive populations are
subgroups within the general population which appear at
higher than average risk upon exposure to CC14. Some of
the populations that may be at greater risk include human
fetusesr alcohol consumers, and males of reproductive age.
The quantification of non-carcinogenic effects in
humans could not be undertaken at this juncture due to lack
of acceptable chronic exposure data for this compound. Be-
cause of positive results in animal carcinogenicity studies,
carbon tetrachloride can be considered a suspect human
carcinogen. Thus, the recommended maximum contaminant level
(RMCL) for carbon tetrachloride should be based on its
carcinogenic potential. According to the Safe Drinking Water
Act (42 USC 300F, SDWA, 1974), this level should be zero
(0 mg/L).
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II. GENERAL INFORMATION AND PROPERTIES
Carbon tetrachloride, or tetrachloromethane, is a
colorless liquid with a molecular weight of 154 and a boiling
point of 76.5"C (Weast, 1972). It is a relatively nonpolar
compound that is miscible in alcohol, acetone, and other
organic solvents (Weast, 1972), but is only minimally soluble
in water (0.8 g/liter at 25°C) (Johns, 1976). The octanol/
water partition coefficient of carbon tetrachloride is 2.64
(Johns, 1976).
The properties of carbon tetrachloride favor volati-
lization of the compound from water to air. Carbon tetrachlo-
ride has a high vapor pressure (115.2 mm Hg at 25 *C) (Johns,
1976). The air/water partition coefficient of carbon tetra-
chloride at 20°C is 1.1 by volume, and about 1,000 by weigh t.
(Johns, 1976). The rapid vaporization predicted from these
properties has been confirmed by Billing et al. (1975), who
reported a haIf-life of carbon tetrachloride evaporation of
29 minutes from a dilute aqueous solution at about 25 *C.
The density of carbon tetrachloride is 1.59 g/ml at
20°C (Weast, 1972). Because its density is greater than the
density of water, some carbon tetrachloride from large spills
in water might tend to settle before it is totally dispersed,
emulsified or volatilized.
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II-2
Carbon tetrachloride is produced industrially by the
chlorination of methane, propane, ethane, propylene, or carbon
disulfide (Rams et al., 1979). In 1980, 322,000 kkg were syn-
thesized (USITC, 1981). Carbon tetrachloride is also produced
indirectly during the production of compounds such as vinyl
chloride and'perchloroethylene (Rams et a_l., 1979).
TJhe major use of carbon tetrachloride is in the pro-
duction of chlorofluorocarbons, which are used as refriger-
ants, foam-blowing agents, and solvents. Carbon tetrachlo-
ride is also used in fumigants, and has a variety of minor
uses, including those as a solvent in metal cleaning and in
manufacture of paints and plastics (Rams et_ a_l., 1979). It is
being replaced in grain fumigation by other registered pesti-
cide products (USEPA, 1980a), and its registration for use in
fumigants is presently under review by USEPA (USEPA, 1980b).
Carbon tetrachloride present in the environment
appears to be of anthropogenic origin (Singh et al., 1976).
It can enter natural waters through industrial and agricul-
tural activities. Carbon tetrachloride may be carried to
surface waters through run-off from agricultural, industrial,
and dumping sites, and through industrial effluents. Indus-
trial emissions may also contribute carbon tetrachloride
to the air, from which the compound may reach surface water
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II-3
through rainfall. Carbon tetrachloride may also reach
groundwater through leaching from solid waste sites.
Once in the environment, carbon tetrachloride is
relatively stable. Its half-life for hydrolytic breakdown
in water at pH 1.0-7.0 is estimated to be 70,000 years,
but hydrolysis appears to be accelerated in the presence
of metals such as iron and zinc (Johns, 1976). The high
.stability in water has little practical significance; how-
ever, since carbon tetrachloride vaporizes readily to air.
The atmospheric lifetime of carbon tetrachloride appears
to be on the order of 30-100 years (Singh et al., 1976).
The presence of carbon tetrachloride in the en-
vironment is of concern for two reasons. First, carbon
tetrachloride may contribute to ozone-destroying photochemi-
cal reactions in the stratosphere, which might cause in-
creases in the incidence in human skin cancers and animal
cancers, affect terrestrial and aquatic ecosystems, and
bring about climatic changes (NAS, 1978).
Although levels of carbon tetrachloride in the envi-
ronment are generally in the low ppb range or below (NAS,
1979), this chemical may pose a long-term danger because of
its possible carcinogenic potential. In urban and industrial
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II-4
areas where higher concentrations of carbon tetrachloride
occur, other toxic effects may result (e.g., liver and renal
damage). The following chapters discuss the evidence for
health effects attributed to carbon tetrachloride, with
particular emphasis on ingestion via drinking water.
Analysis of Carbon Tetrachloride in Printing Water
Carbon tetrachloride (and 47 other halogenated organ-
ics) in water can be analyzed by a purge and trap method
{Method 502»1) described by the EPA Environmental Monitoring
and Support Laboratory (USEPA, 1980d). 'This method can be
used to measure purgeable organics at low concentrations.
Purgeable organic compounds are trapped on a Tenax GC-contain-
ing trap at 22*C using a purge gas rate of 40 ml/min for 11
minutes. The trapped material is then heated rapidly to
ISO'C and backflushed with helium at a flow rate of 20-60
ml/min for 4 minutes into the gas chromatographic analytical
column. The programmable gas chromatograph used is capable
of operating at 40"+1*C. The primary analytical column is
stainless steel packed with 1% SP-1000 on Carbopack B (60/80)
mesh (8 ft x 01 in. I.D.) and is run at a. flow rate of 40
ml/min. The temperature program sequence begins at 45 °C for
3 tninutes, increases B*C/min to 22Q°C, and is then held
constant for 15"minutes or until all compounds have eluted.
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11-5
A halogen-specific detector with a sensitivity to 0.10 ug/
liter a relative standard deviation of 10% must be used.
The optional use of GC/MS techniques of comparable accuracy
and precision is acceptable.
Treatment of Carbon Tetrachloride in Drinking Water
The information available on the removal of carbon
tetrachloride from drinking water is limited. However, as
judged from data obtained for industrial waste treatment,
conventional treatment processes are not very effective in
the removal of this compound. An isotherm study of carbon
tetrachloride on Filtration 400 activated carbon (GAG) showed
that at an equilibrium concentration range of 3 x 10~9 to 2.6
x 10~"7 moi/liter-, a maximum surface concentration of 2.6 x
10~5 mol/g was obtained {NAS, 1979). Aeration and adsorption
processes have also been evaluated for removal of this
compound. Powdered activated carbon (PAC) at 2 to 4 mg/liter
was not effective in treating contaminated river water contain-
ing 16.3 mg/liter of carbon tetrachloride. After PAC, coag-
ulation, settling, and filtration, the finished water still
contained 16.0 mg/liter (USEPA, 1980d). Aeration by diffused
air aerator in a laboratory study was found to be more suc-
cessful. At 4:.l air-to-water ratio, a 91 percent removal
efficiency for carbon tetrachloride was achieved (USEPA,
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11-6
1980d). Adsorption by GAG in a pilot scale study revealed
that carbon tetrachloride at an average concentration of 12
rag/liter (Cincinnati tap water) was reduced to less than 0.1
ug/liter for 3 weeks with a 5-minute empty bed contact-time
(EBCT) and for 14-16 weeks with a 10-minute EBCT.
Summary
Carbon tetrachloride is a colorless liquid at am-
bient temperature. Its high vapor pressure favors rapid
volatilization from water to air. Carbon tetrachloride is
produced commercially from the chlorination of methane, pro-
pane, ethane propylene or carbon disulfide and its major use
is in the production of chloroflurocarbons. Carbon tetra-
chloride is present in the enviroment by anthropogenic means
and once in the environment appears relatively stable. Its
presence is of concern because of a possible contribution to
ozone destroying chemical reaction in the atmosphere and
because ingestion via drinking water may present a human health
hazard. Carbon tetrachloride in water may be detected by
the purge and trap method described by the USEPA Enviro-
nmental Monitoring and Support Laboratory. Removal of this
chemical from water may proceed by aeration and filtration
through appropriate media.
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III. PHARMACOKINETICS
Absorption
Part.it.ion Coef ficients. Partition coefficients for
various chlorinated solvents, including carbon tetrachloride,
were determined by several experimenters {Morgan et al. , 1972;
Sato and Nakajima, 1979; Powell, 1945). The partition coeffi-
cient is a measure of the relative solubility of a substance in
two media. The oil/air and oil/water partition coefficients
can be used as indicators of solubility in lipids. The values
of these and other partition coefficients for carbon tetrachlo-
ride, listed in Table III-l, show this chemical to be lipophi-
lic. Because of its lipophilic nature, one would predict that
carbon tetrachloride, could be absorbed by ingestion, inhala-
tion, and skin contact. This prediction is borne out by
results of the experimental studies described below.
TABLE III-l Partition Coefficients for Carbon Tetrachloride
Parameter
20*C
25'C
37°C
Olive oil/air
Olive oil/air
Blood serum/air
Blood/air
Blood/air
Water/air
Olive oil/water
Olive oil/serum
Olive oil/blood
3.6-5.2
142
0.25
1,440
23
361
2.4
150
Adapted from Morgan et al. (1972), Sato and Nakajima (1979), and
Powell (1945).
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III-2
Absorp-tion from the Gas~troint.est.inal Tract.. Absorp-
tion of carbon tetrachloride through the gastrointestinal
tract of dogs was studied by Robbins (1929). In a series of
experiments, the author determined the amount of carbon tetra-
chloride absorbed from the ligated stomach, small intestine,
and colon by measuring the concentration of carbon tetrachlo-
ride found in the exhaled breath. The greatest concentration
of carbon tetrachloride in exhaled air was seen after injec-
tion of the chemical into the small intestine. Direct injec-
tion of carbon tetrachloride into the colon resulted in a
lower concentration of the chemical in exhaled air. After
introduction directly into the stomach by intubation, no
carbon tetrachloride was detected in exhaled air. The method
of detection in these experiments was thermal conductivity,
with stated detection limits of one part in ten. Thus, the
results of the experiment can be viewed as a qualitative indi-
cation of relative absorption from the various components of
the gastrointestinal tract, rather than as quantitatively
accurate results.
Absorption by Inhalation. Von Oettingen et al.
(1950) studied the absorption of carbon tetrachloride by
inhalation in Beagle dogs. The sex was unspecified, but the
authors stated that at least five dogs were used in each
experiment. The dogs inhaled carbon tetrachloride (purity
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II I-3
unspecified) at a concentration of 94,500 mg/m3 (15,000
ppm) for 475 minutes through a two-way valve attached to the
cannulated trachea. Blood samples were taken at unspecified
intervals and analyzed for carbon tetrachloride. Data pre-
sented graphically showed that the concentration of carbon
tetrachloride in blood reached a maximum of 31.2-34.3 mg/100
cc (0.20-0*22 raillimole percent) after approximately 300
minutes of exposure and remained at that level for the dura-
tion of the exposure.
McCollister ejt a_l. (1951) investigated the absorp-
tion of carbon tetrachloride by inhaltion using rhesus mon-
keys. Three female monkeys inhaled 99.9% 14Olabeled
carbon tetrachloride at an average concentration of 290
rag/m3 (46 ppm) for 139, 344, and 300 minutes, respectively.
The authors calculated by difference between inhaled and
exhaled air that the monkeys absorbed an average of 30.4% of
the total amount of carbon tetrachloride inhaled. Analysis
of blood drawn after 270 minutes of exposure showed that the
14C radioactivity was equal to 0.297 mg of carbon tetrachlo-
ride/ 100 g of blood, distributed as follows: 56.2% as carbon
tetrachloride, 16.5% as "acid-volatile" carbonates, and 27.3%
as nonvolatile material. No attempt was made to characterize
metabolites in this study.
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111-4
Absorption Through the Skin. McCollister et al. (1951)
exposed the skins of one male and one female rhesus monkey
to l^c—labeled carbon tetrachloride vapor. To determine
the amount of absorption, blood and exhaled air were analyzed
for l^C radioactivity. After a skin exposure of 240 minutes
to carbon tetrachloride vapor at 3,056 mg/m3 (485 ppm), the
blood of the female monkey contained carbon tetrachloride at
0.012 rag/lOOg and the exhaled air contained 0.0008 mg/liter.
After exposure to 7,345 mg/m3 (1,150 ppm) for 270 minutes,
blood of the male monkey contained carbon tetrachloride at
0.03 mg/lOOg and the exhaled air contained 0.003 mg/liter.
Three human volunteers, sex unspecified, immersed
their thumbs in carbon tetrachloride for 30 minutes in an
experiment to measure skin absorption (Stewart and Dodd, 1964).
Carbon tetrachloride was analyzed by infrared spectroscopy
and was found to contain no detectable impurities- The
concentration of carbon tetrachloride in alveolar air was
used as the indicator of absorption and measured at 10, 20,
and 30 minutes after the start of exposure and at 10, 30,
60, 120, and 300 minutes after cessation of exposure. Carbon
tetrachloride was present in the alveolar air at each time
interval, reached a maximum concentration range of 2.8-5.7
mg/m3 (0.45-0.79 ppra) 30 minutes after exposure, and decreased
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II I-5
exponentially thereafter. The authors concluded that carbon
tetrachloride could be absorbed by the skin in toxic amounts
if the chemical came in contact with arms and hands.
Distribution
Robbins (1929) administered 159 g (100 cc) of carbon
tetrachloride, purity unspecified, to three anesthetized dogs by
stomach tube. The dogs were sacrificed at 6, 23, and 24 hours
after treatment and blood and various tissues were analyzed for
carbon tetrachloride by converting the organic chloride to
inorganic chloride and titrating the inorganic chloride by the
Volhard method, which is accurate to 0.1-0.2%. The results of
the blood and tissue analysis are shown in Table IIT-2.
TABLE III-2 Carbon Tetrachloride Distribution at Various Times
After Administration by Stomach Tube (rag/100 g of tissues)
Tissue
Brain
Blood, portal
Blood f arterial
Bone marrow
Kidney
Liver
Lungs
Muscle
Pancreas
Spleen
6 hrs
__
26
0
— _
—
15
—
—
—
—
23 hrs
17
13
0
66
11
10
trace
trace
4.5
5
24 hrs
9
22
0
—
13
27
6
20
14
— —
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nr-6
From the experimental data, it appears that the
limit of detection was in the range of 4-5 mg of carbon tetra-
chloride/ 100 g of tissues. In addition, it appears that the
liver, bone marrow, blood, and muscle retained the most carbon
tetrachloride for the longest time.
Von Oettingen e_t al. (1950) reported the tissue dis-
tribution of carbon tetrachloride in Beagle dogs, each weighing
about 10 kg, exposed to carbon tetrachloride in air at 94,500
mg/m3 (15,000 ppm) for 475 minutes. The dogs were sacrificed
at the end of the exposure. Tissue and blood samples were
taken and analyzed for carbon tetrachloride. The concentration
of carbon tetrachoride (per 100 g of tissue) was 65 mg/100 g
in the brain, 50 mg/100 g in the heart, 36 mg/100 g in the
liver, and 34 mg/100 g in the blood; the concentratiion in
the fat was not determined. The investigators stated that
the accumulation of carbon tetrachloride in the brain was
consistent with its high oil/water partition coefficient and
resulted in its strong narcotic action.
McCollister e_t a_l. (1951) reported the tissue distri-
bution of carbon tetrachloride in rhesus monkeys exposed to
290 mg/m3 (46 ppm) of [14C] carbon tetrachloride for 300
minutes. The tissue distribution, as calculated from the
14C radioactivty, is shown in Table III-3. The concentration
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III-7
of carbon tetrachloride was greatest in the fat, followed by
the liver and bone marrow.
TABLE III-3 Tissues Distribution of [14C] Carbon Tetrachloride
Inhaled by Rhesus Monkeys
Tissue
Fat
Liver
Bone marrow
Blood
Brain
Kidney
Heart
Spleen
Muscle
Lung
Bone
Carbon tetrachloride
(mg/100 g of tissue)
2.46
0.94
0.93
0.31
0.30
0.23
0.14
0.10
0.06
0.04
0.04
Souce: McCollister et al. (1951)
Fowler (1969) studied the distribution of carbon
tetrachoride In the tissues of rabbits given the chemical
by stomach tube. Five rabbits were given carbon tetrachloride
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III-8
(1 ml/kg bw) as a 20% (v/v) solution in olive oil. Analysis
of the carbon tetrachloride by gas chromatography showed
not more than 125 ppb of hexachloroethane. The rabbits were
sacrificed 6, 24, and 48 hours after treatment and the tissues
analyzed for carbon tetrachloride by gas chromatography
equipped with an electron capture detector. Six hours after
carbon tetrachloride was administered, the tissue concentrations
(per kg of tissue) were 787 +_ 289 (Mean ± SEM) mg/kg in fat,
96 +_ 11 mg/kg in liver, 21 +_ 12 mg/kg im muscle, and 20 +_ 13
mg/kg in kidney. By 48 hours, these concentrations had
dropped to 45 +_ 12 mg/kg in fat, 4 + 0.1 mg/kg in liver, and
0.5 ^0.3 mg/kg in kidney and muscles. These data indicate
that most of the dose of carbon tetrachloride is eliminated
from rabbit tissues within 48 hours.
Metabolism
Chloroform was one of the first carbon tetrachloride
metabolites to be described (Butler, 1961). Eight dogs were
exposed to carbon tetrachloride by tracheal cannula at the rate
of 8,000 mg/hr for 3 hours. At the cessation of exposure, the
exhaled air from the dogs was collected and analyzed by both
gas chromatography and the Fujiwara reaction, a colorimetric
procedure for- the identification of chloroform. Chloroform
was detected in the exhaled breath by both of these methods.
The total amount of chloroform exhaled in 2 hours by each dog
-------
III-9
was estimated at 0.10.5 mg by analysis of gas chromatographic
data. Tissue homogenates were also shown to metabolize
carbon tetrachloride to chloroform.
Evidence of metabolism to a free radical was sug-
gested by studies showing hexachloroethane to be a carbon
tetrachloride metabolite (Bini et al., 1975). Five Wistar
rats were administered 160-800 mg of carbon tetrachloride
diluted in liquid paraffin by gavage following a 24-hour
fast. The animals were sacrificed 15 minutes to 8 hours
after treatment. A graph displaying carbon tetrachloride
concentrations in rat liver versus time showed the chemical
at approximately 0-.-9.mg/Xg of tissue after 15 minutes and at
maximal concentration (1.7 mg/kg) after 120 minutes. Analysis
of the gas chromatographic data showed that chloroform, was
maximal at 0.037 mg/kg after 15.minutes; after 4 hours it
had declined to 0.007 mg/kg. Hexachloroethane was also
present after 4 hours 0.005 mg/kg. The authors explained
the formation of both chloroform and hexachloroethane as
carbon tetrachloride metabolites by proposing that the tri-
chloromethyl free radical was the primary metabolite of
carbon tetrachloride.
14C-labeled carbon dioxide was detected in the
exhaled air of rhesus monkeys after a 344-minute exposure to
-------
ux-io
E14C] carbon tetrachloride by inhalation (McCollister at
al. , 1951).- The amount of C14C] carbon dioxide exhaled
during the 7-day period following exposure was reported to
be 10-20% of the total radioactivity expired. The authors
integrated the resulting equation from 18 to 1,800 hours (75
days) and estimated that 4.4 mg or 11% of the total amount of
radioactivity eliminated was excreted as carbon dioxide.
Shah et al. (1979) studied the metabolism of [14C]
carbon tetrachloride by rat liver in vitro^ Samples of liver
homogenate equivalent to 0.167 g of tissue were incubated
for 30 minutes at 37.5*C with 10 umole of 14C-labeled carbon
tetraichloride alone, and with either NADH or NADPH or both.
[i4C3 carbon dioxide was detected by scintillation counting.
The results are shown in Table 1II-4. The addition of NADPH
appeared to result in substantial conversion of carbon tetra-
chloride to carbon dioxide. Addition of NADH and NADPH did
not increase the conversion over that seen with NADPH alone.
Shah et al. (1979) tested for the possible formation
of carbonyl chloride in hepatic carbon tetachloride metabolism
by adding L-cysteine to the in vitro rat liver system described
above. Carbonyl chloride and L-cysteine are known to react
chemically to form a condensation product, 2-oxothiazolidine-
4-carboxylic acid. The presence of the condensation product
-------
I IT- 11
was confirmed by thin-layer chroma tography and mass spectro-
metry. The author inferred from the presence of 2-oxothiazoli-
dine-4-carboxylic acid that carbonyl chloride was formed in
the metabolism of carbon tetrachloride by rat liver microsomes,
The authors postulated a mechanism of biotransformation for
carbon tetrachloride which involves a sequential oxidation of
carbon tetrachloride while bound to a heme (see Figure III-l).
Release of bound intermediates then gives rise to different
unrelated metabolites at the site of release.
TABLE III-4 Conversion of [14C] Carbon Tetrachloride to
Carbon Dioxide by Rat Liver Homogenate
14
Nucleotide added [C}C02 (mole/g liver, mean +_ SEM)
None
NADH
NADPH
NADH + NADPH
27 + 5
373 + 17
464 + 33
472 + 21
Source: Shah e_t al^. (1979)
Fowler (1969) detected hexachloroethane and chloroform
in the tissues of rabbits orally administered carbon tetrachlo-
ride. A total of 15 rabbits were given carbon tetrachloride
-------
111-12
Aceseter
t ft
°2
• CT ^
-CSj-Oj*] —
Meionoieenydt
co
Figure III-l Pathways of Carbon Tetrachloride Metab-
olism. Products identified as carbon tetrachloride
metabolites are underlined. The electrons utilized in
the reactions are assumed to come from NADH or NADPH via
the flavoprotein cytochrome reductases. Fe^4" and Fe^"*"
denote the respective ferro— and f erricytochromes .
Redrawn from Shah et al. .(1979).
at 1 ml/kg bw and sacrificed in groups of five at 6, 24, and
48 hours after exposure. Samples of fat, liver, kidney, and
muscle tissues were analyzed for chloroform and hexachloro-
ethane by gas chroma tography. The results of the analysis are
in Table III-5. The fat contained the highest amounts of hexa
chloroethane at each sampling time, but the highest concentra-
tions of chloroform appeared in the liver.
Excretion
McCollister et al. (1951) studied the elimination of
carbon tetrachloride from rhesus monkeys exposed by
-------
111-13
TABLE ITI-5 Chloroform and Hexachloroethane in Tissues of Rats
Given Carbon Tetrachloride Orally
Sample time
6 hours
24 hours
48 hours
Tissue
Fat
Liver
Kidney
Muscle
Fat.
Liver
Kidney
Muscle
Fat
Liver
Kidney
Muscle
CHCls
(ug/g tissues)
4.7 + 0.5
4.9 + 1.5
1.4 + 0.6
0.1 + 0.1
1.0 + 0.2
1.0 + 0.4
0.4 + 0.2
0»1 + 0.1
0.4 + 0.1
0.8 + 0.2
0.2 -I- 0
0.1 4- 0.1
C13 CCC13
(ug/g tissues)
4.1 + 1.2
1.6 -t- 0.5
0.7 -t- 0.2
0.3 Hr 0.2
16.5 + 1.5
4.2 + 1.8
2.2 + 1.1
0.5 + 0.2
6.8 4- 2.4
1.0 + 0.3
Trace
Trace
.
Sources Fowler (1969)
inhalation for 344 minutes. The total 14C radioactivity in the
blood decreased 12% during the first 10 minutes after exposure.
Graphs of data from the analysis of blood samples obtained
periodically for 10-12 days following exposure showed that the
level of carbon tetrachloride in the blood decreased exponen-
tially with time. At 10 days, the level of carbon tetrachlo-
ride in blood was approximately 0.009 mg/100 g. The authors
estimated that 21% of the total amount of carbon tetrachloride
absorbed was eliminated in expired air during the first 18
-------
nr-14
days. By extrapolation of these data, the authors concluded
that over 1,800 hours (75 days) approximately 51% of the carbon
tetrachloride initially absorbed would be eliminated in exhaled
breath either as carbon tetrachloride or carbon dioxide. Analy-
sis of urine and feces showed measurable amounts of radioacti-
vity after 15 and 12 days, respectively. The authors inter-
preted these findings as indicating that significant quantities
of carbon tetrachloride and/or metabolites may be excreted by
these routes (breath, urine, and feces).
Summary
Carbon tetachloride is readily absorbed from the lungs
and the gastrointestinal tract, as expected from its partition
coefficients. Although few quantitative data are available on
the amount of carbon tetrachloride absorbed through the lungs,
the chemical and its metabolites have been reported in blood,
many tissues, exhaled air, urine, and feces after administration
by inhalation. Carbon tetrachloride has also been absorbed
through the skin, but the reported rate of absorption was
much slower than that of inhalation.
In published studies, carbon tetrachloride has appeared
to be distributed to all major organs following absorption. The
highest concentrations have been found in the liver, fat, blood,
brain, kidney, spleen, and pancreas.
-------
III-15
Carbon tetrachloride metabolism has been reported to
occur primarily in the liver. Carbon tetrachloride has been
postulated to be metabolized to a trichlororaethyl radical
bound to an iron atom in the cytochrome heme moiety. This
trichloromethyl radical was reported to be either further
metabolized or released as a free radical. The trichloro-
methyl free radical was reported to undergo a variety of
reactions; including hydrogen abstraction to form chloroform,
and dimerization to form hexachloroethane. Further metabolism
of the heme-bound trichloromethyl radical was postulated to
result in the eventual formation of carbonyl chloride (phos-
gene) .
Carbon tetrachloride and its metabolites have been
reported in many studies to be excreted primarily in exhaled
air, and also in the urine and-feces.
-------
IV. HUMAN EXPOSURE
Humans may be exposed to carbon tetrachloride in drinking water, food,
and air. Detailed information concerning the occurrence of and exposure to
carbon tetrachloride in the environment is presented in another document
entitled "Occurrence of Carbon Tetrachloride in Drinking Water, Food, and Air"
(Letkiewicz et al. 1983). This section summarizes the pertinent information
presented in that document in order to assess the relative source contribution
from drinking water, food, and air.
Exposure Estimation
This analysis is limited to drinking water, food, and air, since these
media are considered to be general sources common to all individuals. Some
individuals may be exposed to carbon tetrachloride from sources other than the
three considered here, notably in occupational settings and from the use of
consumer products containing - carbon tetrachloride. Even in limiting the
analysis to these three sources, it must be recognized that individual expo-
sure will vary widely based on many personal choices and several factors over
which there is little control. Where one lives, works, and travels, what one
eats, and physiologic characteristics related to age., sex, and health status
can all profoundly affect daily exposure and intake. Individuals living in
the same neighborhood or even in the same household can experience vastly
different exposure patterns.
Unfortunately, data and methods to estimate exposure of identifiable
population subgroups from all sources simultaneously have not yet been
developed. To the extent possible, estimates are provided of the number of
individuals exposed to each medium at various carbon tetrachloride concentra-
tions. The 70-kg adult male is used for estimating dose, which takes into
account the amount of the medium contacted (i.e., water and food ingested; air
breathed) and the amount of carbon tetrachloride actually absorbed into the
body.
a. Water
Cumulative estimates of the U.S. populations exposed to various carbon
tetrachloride levels in drinking water from public drinking water systems are
-------
presented in Table IV-I. The values in the table were obtained using Federal
Reporting Data Systems data (FRDS 1983) on populations served by primary water
supply systems and the estimated number of these water systems that contain a
given level of carbon tetrachloride. An estimated 26,810,000 individuals
(12.5% of the population of 214,419,000 using public water supplies) are
exposed to levels of carbon tetrachloride in drinking water at or above 0.5
ug/1 , while 2,087,000 individuals (1.0%) are exposed to levels above 5 ug/1.
It is estimated that 655,000 individuals are exposed to levels greater than 20
ug/1; none are estimated to be exposed to levels exceeding 30 ug/1.
Table IV-I. Total Estimated Cumulative Population (in Thousands)
Exposed to Carbon Tetrachloride in Drinking Water
Exceeding the Indicated Concentration
Number of Cumulative population (thousands) exposed
people served to concentrations (ug/1) of
in U.S.
System type (thousands)
Groundwater
Surface water
Total
(% of total )
73,473
140,946
214,419
(100%)
_>_ 0.5
2,157
24,653
26,810
(12.5%)
>5
121
1,966
2,087
(1.0%)
>10
43
655
698
(0.3%)
>20
0
655
655
(0.3%)
>30
0
0
0
(0%)
No data were obtained on regional variations in the concentration of
carbon tetrachloride in drinking water. The highest concentrations are
expected to occur near sites of production and use of carbon tetrachloride and
also, in the case of groundwater, near waste disposal sites.
Little information was available on gastrointestinal absorption rates for
carbon tetrachloride. Because of its lipophilic nature, ingested carbon
tetrachloride is expected to be readily absorbed. In one study, rats orally
exposed to carbon tetrachloride were found to excrete at least 80% of the
administered dose within 10 hours via the lungs, indicating that at least 80%
of the dose was absorbed (Marchland et al. 1970 cited in USEPA 1982). Another
study gave an absorption factor of 50% for ingestion, but the report has been
criticized for not including substantiating information or citing the specific
-------
literature (Stokinger and Woodward 1958 cited in USEPA 1982). A conservative
estimate for the rate of gastrointestinal absorption based on the limited data
available is 100% (USEPA 1982).
Daily intake levels of carbon tetrachloride from drinking water were
estimated using various exposure levels and the assumptions presented in Table
IV-II. The data in the table suggest that the majority of the persons using
public drinking water supplies would be exposed to intake levels below 0.014
ug/kg/day.
Table IV-II. Estimated Drinking Water Intake of Carbon Tetrachloride
Persons using supplies
exposed to indicated levels
Exposure level
(ug/1)
>0.5
>5.0
>10
>20
Population
26,810,000
2,087,000
698,000
655,000
% of Total
population
12.5%
1.0%
0.3%
0.3%
Intake (ug/kg/day)
>0.014
>0.14
>0.29
>0.57
Assumptions
70-kg man, 2 liters of water/day, gastrointestinal absorption
rate of 100% (USEPA 1982).
An indication of the overall exposure of the total population to carbon
tetrachloride can be obtained through the calculation of population-concentra-
tion values. These values are a summation of the individual levels of carbon
tetrachloride to which each member of the population is exposed. An explana-
tion of the derivation of these values is presented in Appendix C. Popula-
tion-concentration estimates for carbon tetrachloride in drinking water were
3.3 x 107 ug/1 x persons (best case), 9.7 x 107 ug/1 x persons (mean best
case), 1.1 x 108 ug/1 x persons (mean worst case), and 2.5 x 108 ug/1 x
persons (worst case).
Assuming a consumption rate of 2 liters of water/day and a gastrointes-
tinal absorption rate of 100%, population-dose values of 6.6 x 107 ug/day x
persons (best case), 1.9 x 108 ug/day x persons (mean best case), 2.2 x 10
ug/day x persons (mean worst case),- and 5.0 x 108 ug/day x persons (worst
case) were derived.
-------
b. Diet
The dietary intake of carbon tetrachloride in the United States was
estimated using data on concentrations of carbon tetrachloride in food compo-
sites in the TEAM study (Pellizzari et al. 1982) and data on the average
intake of food by food class (FDA 1980) (Table IV-III). Since levels of
carbon tetrachloride in the composites for the TEAM survey were below the
quantitation limit, daily intakes were calculated by assuming that these
levels were equal to zero (minimum estimate) or to the quantitation limit
{maximum estimate). Dietary intakes of carbon tetrachloride for the food
classes studied, estimated by this method, varied between 0-1.27 ug/day.
Table IV-III. Estimated Adult Dietary Intake of Carbon Tetrachloride
by Food Class3 Using TEAM Survey Data
Average intake
of food class
Average level of
carbon tetrachloride
tug/kg)
Average intake of
carbon tetrachloride
(ug/day)
Food class
I- Dairy
II. Meat, fish,
and poultry
X. Oils and fats
XII. Beverages
TOTAL
(kg/day)b
0.753
0.262
0.073
0.1 28e
Minimum0 Maximum
0 1.0
0 0.9
._0 3.0
Q 0.5
Minimum
0
0
0
o.
0
Maximum
0.75
0.24
0.22
0.064
1.27
aEight food classes not analyzed: grains and cereals (III), potatoes (IV),
leafy legume and root vegetables (V, VI, VII), garden fruits (VIII), fruits
(IX), and sugars and adjuncts (XI).
bFrom FDA 1980.
CA11 nonquantifiable values assumed to be equal to zero.
dA11 nonquantifiable values assumed to be equal to the quantitation limit
(value reported is the average of those composites with known quantitation
limits).
Calculated by subtracting 14-day drinking water consumption from 14-day
beverage consumption (FDA 1980) and dividing by 14.
Several problems arose in the use of the TEAM data:
1) The data are limited, since only five samples from each food group
were analyzed, and they may not be representative of normal carbon
tetrachloride levels in foods.
-------
2) Only four of twelve food classes, those suspected of containing the
highest levels of volatile organics, were analyzed.
3) Composite samples generally contained lower levels of carbon tetra-
chloride than expected from levels in the spiked subcomposite
samples from which they were obtained (i.e., some carbon tetra-
chloride appeared to be lost during compositing).
4) The grains and cereals class, which may contain significant carbon
tetrachloride as a result of grain fumigation, was not analyzed.
Because of these data limitations, the dietary intake values presented
above are considered to be approximations.
Gastrointestinal absorption of carbon tetrachloride was discussed in the
previous section. The estimated absorption rate was 100%. If the average
adult male weighs 70 kg and has a daily intake of 1.27 ug of carbon tetra-
chloride (the maximum estimated in the TEAM study), the estimated adult
dietary intake is 0.018 ug/kg/day.
It is expected that dietary levels of carbon tetrachloride vary somewhat
with geographical location, with higher levels occurring in foods from areas
near sources of carbon tetrachloride exposure. However, no estimates of
variations in intake by geographical region could be made from the available
data. Variances in individual exposure due to differences in diet also could
not be assessed.
c. Air
Exposure to carbon tetrachloride in the atmosphere varies from one loca-
tion to another. The highest level of carbon tetrachloride reported in the
atmosphere was 69,000 ng/m3 (69 ug/m3) (Battelle 1977 cited in Brodzinsky and
Singh 1982). High levels, averaging greater than 10,000 ng/m3 (10 ug/m3),
have been detected in other areas. Normal levels, however, are somewhat
lower. Brodzinsky and Singh (1982) calculated median air levels of carbon
tetrachloride for rural/remote areas, urban/suburban areas, and source domi-
nated areas of 820 ng/m3 (0.82 ug/m3), 1,200 ng/m3 (1.2 ug/m3), and 3,700
ng/m3 (3.7 ug/m3), respectively.
The monitoring data available are not sufficient to determine regional
variations in exposure levels for carbon tetrachloride. However, urban and
industrial areas appear to contain higher levels, as expected.
-------
Pulmonary absorption rates for carbon tetrachloride have been reported in
several studies. In one, rhesus monkeys inhaled ^C-labeled carbon tetra-
chloride vapor at an average concentration of 290 mg/m , and absorption was
measured as the difference between the concentrations of carbon tetrachloride
in inhaled and exhaled air. An average absorption rate of 30.4% was obtained
(McCollister et al. 1951 cited in USEPA 1982). In another study, the absorp-
tion of carbon tetrachloride by humans was studied by the difference in quan-
tity of carbon tetrachloride in inhaled and exhaled air. The reported absorp-
tion range was 57-65% (Lehmann and Schmidt-Kehl 1936 cited in USEPA 1982). A
further study reported an absorption factor for inhalation of 30% (Stokinger
and Woodward 1958 cited in USEPA 1982). The last study, however, has been
criticized for not including substantiating information or citing litera-
ture. From these data, a pulmonary absorption rate of 40% was estimated
(USEPA 1982).
The daily respiratory intake of carbon tetrachloride from air was esti-
mated using the assumptions presented in Table IV-IV and the median and maxi-
mum levels for carbon tetrachloride reported above. The estimates in Table
IV-IV indicate that the daily carbon tetrachloride intake from air for adults
in source dominated areas is approximately 0.5 ug/kg/day. In contrast, the
intake calculated using the maximum carbon tetrachloride level reported is 9.1
ug/kg/day; few if any persons are believed to be exposed at that level. The
values presented do not account for variances in individual exposure or uncer-
tainties in the assumptions used to estimate exposure.
Table IV-IV. Estimated Respiratory Intake of Carbon Tetrachloride
"Exposure (ug/m3) Intake (ug/kg/day)
Rural/remote (0.82) 0.11
Urban/suburban (1.2) °-16
Source dominated (3.7) O*49
Maximum (69) 9**
Assumptions- 70-kg man, 23 m3 of air inhaled/day (ICRP 1975), pulmonary
absorption rate of 40% (USEPA 1982).
-------
In addition to the available monitoring data, Systems Applications (1982)
has provided estimates of atmospheric levels of carbon tetrachloride by apply-
ing air dispersion models to carbon tetrachloride emission sources. The
computed average concentrations of carbon tetrachloride and the number of
individuals estimated to be exposed to these concentrations are presented in
Table IV-V. Specific point sources are individually identified sources with
known locations and modes and rates of emissions. These are generally manu-
facturing plants. General point sources are those which are numerous, small,
or of uncertain location. However, these sources can produce isolated
patterns of significant concentration;. Area sources are numerous and emit
only small concentrations of the chemical (e.g., home chimneys, automo-
biles). These estimates indicate that less than 600,000 persons are exposed
to airborne carbon tetrachloride at concentrations greater than 2,500 ng/m3
(2.5 ug/m3).
Table IV-V also presents a total population-concentration estimate for
carbon tetrachloride of 6.45 x 107 ug/m3 x persons. Assuming an inhalation
rate of 23 m3 of air/day and a 40% pulmonary absorption rate, a population-
dose of 5.93 x 10° ug/day x persons was calculated.
SUMMARY
Table IV-VI presents a general view of the total amount of carbon tetra-
chloride received by an adult male from air, food, and drinking water. Four
separate exposure levels in air, five exposure levels in drinking water, and
one exposure level from foods are shown in the table.
The data presented have been selected from an infinite number of possible
combinations of concentrations for the three sources.. The actual exposures
encountered would represent some finite subset of this infinite series of
combinations. Whether exposure occurs at any specific combination of levels
is not known; nor is it possible to determine the number of persons that would
be exposed to carbon tetrachloride at any of the combined exposure levels.
The data presented represent possible exposures based on the occurrence data
and the estimated intakes.
The relative source contribution data for carbon tetrachloride account
for differential absorption rates for the chemical by the respiratory and
gastrointestinal routes. Thus, relative doses of the chemical directly enter-
-------
Table IV-V. Exposure and Dosage Summary for Airborne Carbon Tetrachloride
Population
Concentration
level
(ug/m3)
829
500
25GL
100
50
25
10
5
2.5
1
0.5
0.25
0.1
3.8 x 10"5
Specific General
point point
source source
15
133
625
1,652
4,318
9,146
23/759
36,719
62,189
121,852
213,423
--
—
7,979,115
0
0
0
0
0
0
0
0
0
0
0
0
0
0
exposed (persons)
Area source
0
0
0
0
0
0
0
0
505,140
9,149,730
33,072,205
83,219,704
142,928,535
158,679,135
U.S. total
15
133
625
1,652
4,318
9,146
23,759
36,719
567,329
9,271,582
33,285,628
--
--
--
Dosage (ug/m x persons)
Specific General
point point
source source
12,600
88,200
257,000
404,000
576,000
729,000
950,000
1,040,000
1,130,000
1,230,000
1,290,000
--
--
1,530,000
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Area source
0
0
0
0
0
0
0
0
2,327,400
17,913,784
35,194,859
51,528,284
61,879,083
62,926,300
U.S. total
12,600
88,200
257,000
404,000
576,000
729,000
950,000
1,040,000
3,457,400
19,143,784
--
--
__
64,456,300
Note: The use of "--" as an entry indicates that the incremental
not significant (relative to the last entry in that column
that the exposure of the same population may be counted in
Source: Systems Applications 1982
increase In the dosage or
or to an entry in another
another column.
the population exposed Is
column at the same row) or
-------
ing the body are compared. However, it should be noted that the relative
effects of the chemical on the body may vary by different routes of exposure.
Brodzinsky and Singh (1982) calculated a median urban/suburban air level
of carbon tetrachloride of 1.2 ug/m3 based on air monitoring data. Assuming
an air level of 1.2 ug/m3 and the estimated carbon tetrachloride intake of
0.018 ug/kg/day in foods, drinking water would be the predominant source of
carbon tetrachloride exposure in the adult male at drinking water levels above
6.3 ug/1. An accurate assessment of the number of individuals for which
drinking water is the predominant source of exposure cannot be determined from
the data since specific locations containing high concentrations of carbon
tetrachloride in drinking water and low concentrations of carbon tetrachloride
in ambient air and food are unknown.
Population-dose estimates for carbon tetrachloride in drinking water and
air were presented previously. Estimates for drinking water ranged from 0.66-
5.0 x 108 ug/day x persons; the estimate for ambient air was 5.9 x 109 ug/day
x persons. These estimates suggest that ambient air may be a greater source
of exposure to carbon tetrachloride than drinking water on a general popula-
tion basis. Comparison of these estimates, however, may be deceiving since
the same population-dose level can occur if: 1) a whole population is exposed
to moderate levels of a chemical or 2X some segments of the same population
are exposed to high levels and others to low levels. The population-dose
values presented give no indication of the relative predominance of drinking
water and air as specific sources of carbon tetrachloride on a site-by-site or
subpopulation basis.
-------
Table IV-VI. Estimated Dose of Carbon Tetrachloride Absorbed
from the Environment by Adult Males in ug/kg/day
(X from Drinking Water)
Concentration in Concentration in air
drinking water Rural /remote Urban/suburban
(ug/1) (0.82 ug/m3) (1.2 ug/m3}
0 0.13 (0%) 0.18 (0%)
0.5a 0.14 (9.9%) 0.19 (7.3%)
5.0b 0.28 (50%) 0.32 (44%)
10C 0.42 (69%) 0.47 (62%)
20d 0.71 (80%) 0.75 (76%)
Intake from each source (see Sections 5.1-5.3):
Water: 0.5 ug/1: 0.014 ug/kg/day
5.0 ug/1 : 0.14 ug/kg/day
10 ug/1 : 0.29 ug/kg/day
20 ug/1: 0.57 ug/kg/day
Air: 0.82 ug/m;*: 0.11 ug/kg/day
1.2 ug/m;*: 0.16 ug/kg/day
3.7 ug/m;*: 0.49 ug/kg/day
69 ug/m3: 9.1 ug/kg/day
Source dominated
(3.7 ug/m3)
0.51 (0%)
0.52 (2.7%)
0.65 (22%)
0.80 (36%)
1.1 (52%)
Maximum
(69 ug/m3)
9.1 (0%)
9.1 (0.2%)
9.3 (1.5%)
9.4 (3.1%)
9.7 (5.9%)
Food: 0.018 ug/kg/day
a26,810,000 individuals using public drinking water systems are estimated to
be exposed to levels >_ 0.5 ug/1 (12.5% of population using public water
supplies).
b2,087,000 individuals using public drinking water systems are estimated to be
exposed to levels > 5.0 ug/1 (1.0% of population using public water supplies).
C698,000 individuals using public drinking water systems are estimated to be
exposed to levels > 10 ug/1 (0.3% of population using public water supplies).
d655,000 individuals using public drinking water systems are estimated to be
exposed to levels > 20 ug/1 (0.3% of population using public water supplies).
10
-------
REFERENCES
Battalia. 1977. Environmental monitoring near industrial sites: Methyl -
chloroform. Prepared by Battelle Columbus Laboratories, Columbus, OH, for
U/S. Environmental Protection Agency. EPA-560/6-77-025. Cited in Brodzinsky
and Singh 1982.
Brodzinsky R, Singh HB. 1982. Volatile organic chemicals in the atmosphere:
An assessment of available data. Prepared by SRI International, Menlo Park,
CA, for Environmental Sciences Research Laboratory, Office of Research and
-Development, U.S. Environmental Protection Agency, Research Triangle Park,
NC. Contract No. 68-02-3452.
FDA. 1980. Food and Drug Administration. Compliance program report of
findings: FY 77 total diet studies — adult (7320.73). Washington, DC:
Industry Programs Branch, Food and Drug Administration.
FRDS. 1983. Federal Reporting Data System. Facilities and population served
by primary water supply source (FRDS07), April 19, 1983. U.S. Environmental
Protection Agency, Washington, DC,
ICRP. 1975. International Commission on Radiological Protection. Report of
the task group on reference man. New York: Pergamon Press. ICRP Publication
23.
Lehmann KB, Schmidt-Kehl L. 1936. The thirteen most important chlorinated
aliphatic hydrocarbons from the standpoint of industrial hygiene. Arch.
Hyg. 116:132-200. Cited in USEPA 1982.
Letkiewicz F, Johnston P, Macaluso C, Elder R, Yu U, Bason C. 1983.
Occurrence of carbon tetrachloride in drinking water, food, and air. Prepared
by JRB Associates, McLean, VA, for Office of Drinking Water, U.S.
Environmental Protection Agency, Washington, DC. EPA Contract No. 68-01-6388.
Marchland C, McLean S, Plaa GL. 1970. The effect of SKF 525A on the
distribution of carbon tetrachloride in rats. J. Phar. Exper. Ther. 174:232.
McCollister OD, Beamer WH, Atchison GJ, Spencer HC. 1951. The absorption,
distribution, and elimination of radioactive carbon tetrachloride by monkeys
upon exposure to low vapor concentrations. J. Pharmacol. Exp. Ther. 102:112-
124. Cited in USEPA 1982.
Pellizzari ED, Hartwell T, Zelon H, Leninger C, Erickson M, Sparacino C.
1982. Total exposure assessment methodology (TEAM): Prepilot study -
Northern New Jersey. Prepared by Research Triangle Institute for Office of
Research and Development, U.S. Environmental Protection Agency, Washington,
DC. EPA Contract 68-01-3849.
11
-------
Stokinger HE, Woodward RL. 1958. Toxicological methods for establishing
drinking water standards. J. Am. Water Works Assoc. 52:515-529. Cited in
USEPA 1982.
Systems Applications. 1982. Human exposure to atmospheric concentrations of
selected chemicals. Prepared by Systems Applications, Inc. for Office of Air
Quality Planning and Standards, U.S. Environmental Protection Agency, Research
Triangle Park, NC. Contract No. 68-02-3066.
USEPA. 1982. U.S. Environmental Protection Agency. Health assessment
document for carbon tetrachloride. Washington, DC: Office of Research and
Development, U.S. Environmental Protection Agency. EPA-600/8-82-001.
12
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V. HEALTH EFFECTS IN ANIMALS
This section discusses the acute and chronic effects of
carbon tetrachloride exposure* with emphasis on studies in which
dose-response relationships for minimal adverse effects have been
developed. The subsection on teratogenicity, rautagenicity, and
carinogenicity outline the various study designs in more detail
and elaborate on their deficiencies where appropriate.
Acute Effects
The acute toxicity of carbon tetrachloride has been
extensively documented. This subsection will concentrate on
those studies that (i) describe nonlethal effects and (ii)
provide data on a range of doses from which dose-response
relationships can be determined. For this reason a number of
studies referring to LDso's will not be discussed. However,
Table V-l summarizes some of the lethal dose data reported for
carbon tetrachloride in various species.
Liver Effects. Functional changes in mouse liver as
a result of carbon tetrachloride exposure were measured by
increases in the activity of the enzyme serum glutamic-pyruvic
transaminase (SGPT) and in bromsulfophthalein (BSP) retention
(Klaassen and Plaa, 1966). Male Swiss-Webster mice were admin-
istered various amounts of analytical grade carbon tetrachloride
intraperitoneally in corn oil at a final volume of 10 ml/kg
-------
V-2
TABLE v-1 Toxic Doses and Effects of Carbon Tetrachloride
in Animals
Route of
Animal administration Effect*
Rat Oral LD50
Mouse LD50
Dog LDLO
Rabbit LD50
Rat Intraperitoneal ^-^50
Mouse ^^50
Dog ^DLQ
Rabbit LDLO
Rat Inhalation ^50
Mouse LC5Q
Cat LCLO
Guinea pig L^LO
Cat Subcutaneous LDLO
Rabbit LDLO
Dose
2,800 mg/kg
12,800 mg/kg
1,000 mg/kg
6,380 mg/kg
1,500 mg/kg
4,675 mg/kg
1,500 mg/kg
478 mg/kg
4,000 ppm/4 hrs
9,526 ppm/8 hrs
38,110 ppm/2 hrs
20,000 ppm/2 hrs
300 mg/kg
3,000 mg/kg
aLD50, dose lethal for 50% of animals
LC50' concentration lethal for 50% of animals
LDi,O' lowest lethal dose
Source: NIOSH1 (1978)
-------
V-3
of body weight (bw). Mice treated only with corn oil were
used to establish the normal range of values for BSP retention
and SGPT activity, which were determined 24 hours after treat-
ment. The authors reported the median effective doses .of
carbon tetrachloride as 15.9 mg/kg bw for elevation of SGPT
activity and 94 mg/kg bw for BSP retention. The authors did
not specify the range of carbon tetrachloride doses used or the
number of animals used at each dose.
In another study, Klaassen and Plaa (1967) further
defined a dose-response relationship for carbon tetrachloride
exposure and elevated SGPT levels in mice. They used the "up
and down" method in which one dose of the compound was given
to an animal and the animal's SGPT activity 24 hours after
the dose was noted. If the enzyme was elevated, the dose
was decreased 40% and the experiment repeated in another
animal. If no effect was noted, the dose was increased 40%
and the experiment repeated in another animal. This series
was repeated three times after one positive and one negative
response had been obtained. The results for mice are shown
in Table V-2. The authors concluded that 13 mg/kg bw was
the median effective dose of carbon tetrachloride in mice as
measured by elevated SGPT values.
-------
V-4
Table V-2 SGPT Values of Mice Administered Carbon Tetrachloride
Intraperitoneally in "Up and Down" Experiment
Animal
1
2
3
4
5
ED50
Dose
(rag/kg faw)
17.5
13
17.5
13
8
13
Response3
E
N
E
E
N
aE, = Elevated SGPT after 24 hours
N = Normal SGPT after 24 hours
Source: Klaassen and Plaa (1967)
Sein and Chu (1979) studied the effect of carbon
tetrachloride on the level of the liver enzyme glucose-6-phos-
phatase in mice. Groups of six male LAC strain mice were
treated intraperitoneally with 795, 1,590, or 3,180 mg/kg bw
of carbon tetrachloride (purity unspecified) in paraffin
oil. The animals were sacrificed 24 hours after treatment.
Control animals, number unspecified, were given paraffin oil
and sacrificed on the same schedule. The livers were removed
and analyzed for glucose-6-phosphatase. The results of the
analysis showed that after treatment with carbon tetrachloride
at 795 or 1,590 mg/kg, the enzyme level fell to 40% of the
-------
V-5
control value. At a dose of 3,18'0 rag/kg, the enzyme level
had decreased to 20% of the control value.
A series of experiments to determine the effects of
single carbon tetrachloride exposures on rats were performed
by Murphy and Malley (1969). Adult male Holtzman rats (250-
350 g) were orally administred various doses of undiluted
carbon tetrachloride by gavage. Control animals' were admin-
istered equal volumes of water. At 2-20 hours after treatment,
animals were sacrificed and liver enzyme activities and
liver weights were measured. The results are shown in Table
V-3. The animals receiving carbon tetrachloride at 1,600
mg/kg bw were sacrificed 20 hours after treatment and the
livers examined histopathologically. The examination showed
extensive fatty infiltration, inflammation, and some centrolo—
bular necrosis. The liver-to-body weight ratios were also
increased.
Murphy and Malley (1969) also determined the effect
of single exposures to carbon tetrachloride on the activities
of the corticosterone-inducible liver enzymes tryptophan pyr-
rolase and tyrosine-«*«-ketoglutarate transaminase. Groups of
rats (4-6 in each group, 8 untreated controls) were treated
with carbon tetrachloride (0, 400, 800, and 1,600 mg/kg bw)
-------
V-6
and sacrificed 5 hours after treatment. Data showed that
the enzyme levels were increased roughly in proportion to
the dose.
Similar studies on the effect of carbon tetrachloride
administration on serum activity of liver enzymes in rats were
performed by Drotman and Lawhorn (1978). Groups of four male
Cox rats were admministered carbon tetrachloride intraperito—
neally at 60, 120, 240, or 480 mg/kg bw in a total volume of
1 ml in corn oil and exsanguinated at specified time intervals.
Serum activities were determined for the enzymes sorbitol
dehydrogenase (SSDH), ornithine carbamyl transferase (SOCT),
aspartate aminotransferase (SAST), and isocitric dehydrogenase
(SICDH). Liver specimens were taken from each animal and scored
for histopathologial changes. The results of the enzyme analyses
and histopathology are tabulated., in Table V-4 by dose and
hours after dose. The SOCT activities showed the best corre-
lation with liver histopathology in time of appearance as
well as extent of damage. The authors concluded that SOCT
levels are a sensitive indicator of liver damage.
Effects of acute exposure to low levels of carbon
tetrachloride were also reported by Korsrud et al. (1972).
Male Wistar rats (260-400 g; 8-10 animals per treatment group)
were administered single oral doses of carbon tetrachloride
-------
V-7
Table V-3 Effects of Oral Carbon Tetrachloride on Liver Weight and
Liver and Plasma Enzyme Activities in Male Rats
Dose Time
(rag. kg bw) (hr)
0
3200
3200
3200
2400
1600
800
-
2
5
20
20
20
20
Number of Plasma
animals AKTa
7 2.6 +
4 2.1 +
5 13.2 +
5 35.2 +
4 35.2 +
5 18.1 +
4 9.3 +
0.3
0.2
3.4
4.2
3.4
4.3
2.3
AKTa
b 2360 + 182
-
1813 ± 331
; 1174 + 559
1585 + 148
1596 + 194
2120 + 182
Liverb
TKTa APa Weight (g/lOOg bw)
93 + 11 14.9 + 1.0 2.75 + 0.06
170 + 12 14.5 + 1.0 2.94 + 0.04
330 +36 14.2+1.3 3.26+0.10
361 + 42 34.6 + 5.8 4.36 + 0.06
305 + 21 37.1 + 1.6 3.95 + 0.07
294 + 61 33.3 + 3.6 3.90 + 0.05
138 + 5 29.1 + 6.5 3.47 + 0.07
aAKT =
TKT =
AP =
b =
alanine-«x*-ketoglutarate
tyros ine
alkaline
means +
-*e-ketoglutarate
phosphatase
SE in micromoles
transaminase
transaminase
of
product formed
per gram of fresh liver or
milliliter of plasma per hour
Adapted from Murphy and Malley (1969)
-------
V-8
(0-4,000 mg/Tcg bw) in corn oil (5 ml/kg bw). The rats were
fasted for 6 hours before treatment and for 18 hours after-
ward, and then sacrificed. Assays included liver weight and
fat content, serum urea and arginine levels, and levels of
nine serum enzymes produced mainly in the liver. At 20
mg/kg bw there was histopathologic evidence of toxic effects
on the liver. These changes included a loss of basophilic
stippling, a few swollen cells, and minimal cytoplasmic
vacuolation. At 40 mg/kg bw, liver fat, liver weight, serum
urea, and levels of five of the nine liver enzymes were
increased while serum arginine decreased. At higher doses
the remaining four enzyme levels were also elevated.
Alumot et £l. (1976) reported the effects of sub-
chronic exposure to feed that had been fumigated with carbon
tetrachloride. Groups of six weaning rats 4 weeks old were
fed a diet containing carbon tetrachloride at 150, 275, or
520 mg/kg of feed for 5 weeks (females) or 6 weeks (males).
The fumigated feed was stored in airtight containers; carbon
tetrachloride loss during the storage period of 7-10 days
was determined to be 5%. The animals were allowed access to
the feed only at set time intervals to minimize loss of
carbon tetrachloride by volatilization. The authors calcu-
lated that the amount of carbon tetrachloride remaining in
the consumed feed was 60-70% of the amount initially present;
-------
V-9
Table V-4
Effects of Carbon Tetrachloride on Liver
Histopathology and Serum Enzyme Levels
Hours
Dose after
(mg/kg) dose Histologya
Serum enzyme concentrations
relative to pretreatment levels
SOCT SSDH SAST SICDH
60
120
240
480
0
6
12
24
36
0
24
48
96
168
0
24
43
96
168
0
24
48
96
168
0
2
1
1
0
0
3
2
1
0
0
3
4
1
0
0
3
4
1
0
1.0
9.6*
8.2*
5.7*
1.0
1.0
14.0*
7.4*
1.8
1.0
1.0
31.0*
180.0*
6.6*
1.1
1.0
28.4*
465.5*
1.0
1.0
1.0
2.5
4.4*
1.7
1.0
1.0
7.2*
1.0
1.7
1.0
1.0
17.4*
43.4*
4.7*
1.0
1.0
90.0*
163.5*
8.4*
1.4
1.0
2.0
2.5*
2.0
1.0
1.0
2.1*
1.0
1.3
1.0
1.0
5.8*
17.0*
3.6*
1.9
1.0
6.1*
18.4*
1.8*
1.0
1.0
1.4
1.1
1.3
1.0
1.0
1.1
1.0
1.3
1.0
1.0
7.2*
7.4
2.0
2.0
1.0
5.4*
50.4*
2.0
2.1
aO = No observable changes.
1 = Minimal changes. Large central vein, swelling of hepato-
cyte, etc.
2 = Mild degenerative change. Loss of cord arrangement.
3 = Moderate degenerative change. Pale cytoplasm, spindle cell.
4- = Marked degenerative change. Centrilobular fatty degeneration,
* Significantly different from zero time as determined by one-way
analysis of variance of the log-transformed data (P £.01).
Adapted from Drotman and Lawhorn (1978)
-------
V-10
the total decrease reflected amounts lost during storage and
after removal from storage to feeding troughs. From these
data and the weights of the animals, the authors calculated
that 275 rag/kg of feed represented a daily dose of 40 rag/kg
bw. (By assuming that all parameters were the same and that
the delivered dose was proportional to the concentration in
feed, diets of 150 and 520 rag/kg of feed were calculated by
JRB to represent daily doses of 22 and 76 mg/kg bw, respec-
tively. ) At the end of the experiment the animals were
weighed and sacrificed. Of the three doses, only the highest,
76 mg/kg bw (520 mg/kg of feed), caused significantly depressed
weight gain in males. Weight gain in females appeared to be
unaffected by all doses. Total lipid and triglyceride levels
in the liver were significantly higher in animals fed carbon
tetrachloride at 40 and 76 mg/kg bw than in controls or animals
fed 22 mg/kg bw. Levels of liver phospholipids (measured in
females) were not affected at any dose. Of the three doses
used in this experiment, the lowest, 22 mg/kg bw, failed to
produce effects on the measured parameters.
In addition to the study of hepatic effects of carbon
tetrachloride in mice, described earlier in this section,
Klaassen and Plaa (1967) also investigated the hepatic effects
of carbon tetrachloride exposure in dogs. Male and female mon-
grel dogs were treated intraperitoneally with carbon tetrachlo-
ride at 22-38 mg/kg bw in an "up and down" experimental design.
-------
V-ll
Blood samples were taken for measurement of SGPT 24 hours after
administration of carbon tetrachloride. Control dogs had serum
SGPT activity of 36+7 units. Therefore, 36+2 standard devia-
tions or 50 units were chosen as the upper limit of the normal
value. The results of the analysis are shown in Table V-5.
The SGPT values returned to normal in 17-18 days.
Animals were then sacrificed and the livers were examined his-
topathologically. They showed moderate vacuolation of the
centrolobular and midzonal hepatocytes.as well as traces of
brown material in the cytoplasm of centrolobular Kupffer cells.
Table V-5 SGPT Activity in Dogs 24 Hours After Intraperitoneal
Administration of Carbon Tetrachloride in "Up and
Down" Experiment
Animal
1
2
3
4
5
ED50
Dose
(mg/Tcg)
22.2
30.2
22.2
30.2
38
32
Responsea
N
E
N
N
E
aN = normal SGPT after 24 hours
E = elevated SGPT after 24 hours
Adapted from Klaassen and Plaa (1967)
-------
V-12
Kidney Effects* Carbon tetrachloride, even at
high doses, failed to induce renal failure as measured by
phenolsulfonphtalein (PSP) excretion in mice although patho-
logical kidney alterations were present (Plaa and Larson,
1965) . Male Swiss mice (18-30 g) were given intraperitoneal
injjections of carbon tetrachloride (1,6006,400 mg/kg bw)
dissolved in corn oil at a final volume of 0.1 ml/10 g bw.
The animals were then hydrated with tap water (50 ml/kg bw)
by gavage and placed on a urinary collection unit for 2
hours. Even carbon tetrachloride doses lethal in some
animals (>^ 6,400 mg/kg bw) failed to cause renal dysfunction,
measured as excretion of PSP, urinary protein, and glucose,
in the majority of survivors. At a high nonlethal dose
(3,260 mg/kg bw) minimal renal dysfunction was observed
after 96 hours. Histologic examination of kidney sections
from five mice that had been administered this dose showed
necrosis of proximal convoluted tubules (n=l) and swelling
of the tubules (n=4).
Carbon tetrachloride decreased the activity of
glucose-6-phosphatase in the kidney (Sein and Chu, 1979).
Male mice (40-50 days old, weighing 24-28 g) were injected
intraperitoneally with carbon tetrachloride at 795, 1,590, or
3,180 mg/kg bw in paraffin oil. Twenty-four hours after injec-
tion of 795 or 1,590 mg/kg bw, the renal glucose-6-phosphatase
-------
activity decreased to 77% or 65% of the control value, respec-
tively. Increasing the dose to 3,180 rag/kg bw had no further
effect on the kidney enzyme level.
These results were in contrast to the liver glucose-
6-phosphatase level discussed earlier, which decreased to 40%
of the control value at the two lower doses and decreased
further to 20% of the control value at 3,180 rag/kg bw. The
authors attributed these differences to the limited meta-
bolic capacity of the kidneys.
Klaassen and Plaa (1967) studied the effect of carbon
tetrachloride exposure on kidney function in dogs. PSP excre-
tion of less than 39% of control values was considered indica-
tive of renal dysfunction. An unspecified number of male
and female mongrel dogs were treated intraperitoneally with
carbon tetrachloride at 22-38 mg/kg bw and the 24 hour excre-r
tion rate for PSP was determined. Control dogs were used to
determine a normal range for PSP excretion. None of the
dogs treated with carbon tetrachloride exhibited decreased
PSP excretion. However, on histological examination of the
kidneys from the treated dogs, the Bowman's capsules appeared
dilated with some contraction of glomerular tufts and calcifi-
cation of a small number of tubules in the medulla.
-------
V-14
Lung Effects. Boyd et al. (1980) investigated the
effect of ingestion and inhalation of carbon tetrachloride
on pulmonary Clara cells in Swiss mice. For the ingestion
study, the mice were treated with carbon tetrachloride (4,000
m9/k9 fcw) in a 50% sesame oil solution and sacrificed 16
hours after treatment. The lungs were removed and examined
by electron microscopy. Clara cells exhibited massive
dilation of vesicles of smooth endoplasmic reticulum, increased
mitochondrial staining density, ribosomal disaggregation, nuclear
condensation, and occasional cellular necrosis. Additional
experiments with oral carbon tetrachloride doses of less
than 1,600 ing/Kg bw did not produce any pulmonary lesions
visible by light microscopy. Doses of 2,400-4,800 mg/kg bw
produced Clara cell lesions similar on electron microscopic
examination to those previously.-described. The extent of
damage was proportional to the dose administered. Boyd et
al. (1980) also studied the time course of the Clara cell
damage caused by ingestion of carbon tetrachloride (4,000
mg/Tcg bw). Pulmonary tissue was evaluated by light micros-
copy at 12, 24, 36, 48, 96, and 168 hours. The lesions were
present at 12 hours, maximal at 24 hours, and less intense
at 36 hours. By 48 hours, the lesions were seen infrequently
and at 96 and 168 hours the pulmonary bronchioles appeared
normal.
-------
V-15
The pulmonary toxicity of inhaled carbon tetrachlo-
ride was also studied by Boyd et al. (1980). Swiss mice were
exposed to carbon tetrachloride vapor at 71,800, 144,000,
287,000 or 574,000 mg/m3 for 60, 60, 12, or 2 minutes, respec-
tively. The animals were sacrificed 24 hours after exposure,
and the lungs examined. Marked Clara cell lesions similar to
those seen after oral exposure were seen at all exposure levels
and necrosis was reported to be more frequent after inhalation
than after oral exposure, but no effort to quantify this find-
ing was reported.
Gould and Smuckler (1971) investigated the structural
alterations in rat lungs following carbon tetrachloride inges-
tion. Male Sprague-Dawley rats (200-250 g) were fasted 16
hours prior to administration of carbon tetrachloride (4,000
mg/kg bw) by gavage. The animals exhibited piloerection
and lassitude 3-4 hours after treatment. They were sacrificed
1, 4, 8, 12, or 24 hours following treatment. Necropsies were
performed on all animals. Microscopic examination of the
lungs of treated rats revealed perivascular edema and mono-
nuclear infiltration in the first 4 hours after treatment.
These areas were local but were estimated to involve 10% of
the parenchyma. Areas of atelectasis and intraalveolar hemor-
rhage involving 15-20% of the parenchyma were observed 8-12
hours after treatment.
-------
V-16
Electron micrographs of rat lungs after carbon tetra-
chloride ingestion showed granular pneumocytes containing
swollen inclusions with decreased osraiophilia and attenuated
lamellae 1 hour after treatment (Gould and Smuckler, 1971).
These changes were more severe 4 hours following treatment.
By 4—8 hours after treatment, cytoplasmic edema, dislocation
of dense ribosomal aggregates, and mitochondrial disruption
were apparent. Multivesicular bodies were "conspicuously
decreased" within the granular pneumocytes. Necrosis was
evident 12-24 hours after treatment. One hour after administra-
tion, endothelial cells displayed markedly increased pinocytotic
vesicles. Severe disruption of endothelial cells was evident
from 8 hours onward. Ultrastructural damage was seen in all
components of the alveolar wall, and fibrin was observed
within alveoli. The authors interpreted these findings as
indicative of significant alterations in vascular permeability.
Lesions of the Clara cells in the lungs of male
Sprague-Dawley rats orally treated with carbon tetrachloride
were observed by Boyd et al. (1980). The carbon tetrachloride
was administered by gavage at doses of 3,816, 5,088, and
7,155 mg/kg as a 50% solution in sesame oil. Control animals
received sesame oil only. Clara cell lesions occurred at
the two highest doses. The authors stated that the lesions
-------
V-17
were less pronounced than those, in mice exposed to comparable
amounts of carbon te^rachloride.
Chronic Effects
Smyth et al. (1936) studied the chronic effects of
carbon tetrachloride inhalation exposure in rats. Groups of
24 Wistar rats were exposed to carbon tetrachloride concentra-
tions of 315, 630, 1,260, or 2,520 mg/m3 (50, 100, 200, or
400 ppm) for 8 hours a day, 5 days a week for 10.5 months.
The carbon tetrachloride was found to contain less than
0.003% carbon disulfide. Control rats were used, but the
number was unspecified. Growth retardation was observed
at 2,520 mg/m3. At 630 and 1,260 mg/m3, growth was the
same as in controls, and at 315 mg/m3 growth was stimulated.
Cirrhosis developed in rats exposed to 630, 1,260, and 2,520
mg/m3 after 173, 115, and 54 exposures, respectively. When
exposure was stopped, fatty liver degeneration resolved within
50 days. Surface alterations- (hobnail liver) did not resolve
until 156 days after cessation of exposure. Unspecified
renal damage was observed after 52^ exposures to 315 mg/m3 and
after 12-20 exposures at the higher concentrations.
In a chronic oral exposure study (Alumot et aj..,
1976), groups erf 36 rats (18 male and 18 female littermates)
were fed mash containing carbon tetrachloride at 0,'80, or
200 mg/kg of feed. The feed was stored in airtight containers,
-------
V-1S
assayed for carbon tetrachloride content, and consumed soon
after removal to feeding troughs. The authors calculated
that 200 mg/kg of feed represented a daily dose of 10-18
m9/fcg bw. After 2 years, the surviving animals were sacri-»
ficed. In these animals, serum values for glucose, protein^
albumin, urea, uric acid, cholesterol, SCOT, and SGPT in jfche
treated animals did not differ from those £n controls. No
fatty livers were detected in the treated animals. Thus,
the authors found no biochemical abnormalities attributable
to carbon tetrachloride exposure. However, interpretation
of the results was complicated by the widespread incidence
»
of chronic respiratory disease in the animals. More than
half the animals were dead at 21 months, although at 18
months the survival ranged from"¥l-89%. The authors
indicated that 10-18 mg/kg bw (200 mg/kg of feed) is a no-
adverse-effect level of carbon tetrachloride over 2 years.
However, this conclusion may be questioned because of the
poor survival rate and widespread respiratory infection of
experimental animals.
Groups of 24 guinea pigs were exposed to carbon tetra-
chloride vapor at 315, 635, 1,260, 2,500 mg/m3 in a study by
Smyth et al. (1936). The frequency of exposure was 8 hours per
day, 5 days per week for up to 10.5 months. Marked mortality
occurred in exposed animals: 9/24 at 315 mg/m3 after a median
-------
V-19
of 44 exposures (exposure terminated at 135 days), 16/24 at
630 mg/m3 after a median of ten exposures, 13/24 at 1,260
mg/m3 after a median of three exposures, and 19/24 at
2,520 mg/m3 after a median of three exposures.
Guinea pigs exposed to the 315 mg/m3 dose in the Sroyth
et a_l., (1936) study developed cirrhosis and hobnail surface
alterations of the liver in 105 exposures. The authors
concluded that survival of guinea pigs at higher doses was
of insufficient duration to allow development of cirrhosis.
In addition, granular swelling was observed in adrenal glands
of guinea pigs exposed to carbon tetrachloride at 315, 630,
and 260 mg/m3 for 8, 7, and 17 exposures, respectively.
Exposure to higher concentrations (1,260 or 2,520 mg/m3) or
continued exposure to lower concentrations resulted in marked
damage to the sciatic nerves. Dense clumps of black granules
(osmic acid stain) were observed paralleling the large majority
of fibers.
Prendergast et al. (1967) repeatedly exposed 15 guinea
pigs to carbon tetrachloride (purity unspecified) at 515 mg/m3
(82 ppm) over a period of 6 weeks and observed hepatic changes.
Three guinea pigs died on days 20, 22, and 30, respectively.
All the animals showed a body weight loss. The surviving
animals were sacrificed at 6 weeks and the livers examinined
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V-20
histopathologically. The examiniation revealed fatty infiltra-
tion, fibrosisr bile duct proliferation, hepatic cell degenera-
tion and regeneration, focal inflanunatory cell infiltration,
alteration of lobular structure, and early portal cirrhosis.
The hepatic lipid content of carbon tetrachloride-treated
animals (35.4 _+ 10.7%) was higher than that of the controls
(11.0 + 3.6%) .
In addition, Prendergast e_t a_l. (1967) exposed
continuously guinea pigs to carbon tetrachloride vapor at 61
mg/m3 (10 ppm) for 90 days. Three of the 15 guinea pigs
died: on days 47, 63, and 74 respectively. All the exposed
animals showed a depressed weight gain. A "high incidence"
of enlarged and discolored livers was reported on gross
pathological examination. Histopathologic examination of
the livers revealed fatty changes, fibroblastic proliferation,
collagen deposition, hepatic cell degenertion and regeneration,
and structure alteration of the liver lobule. Enzymatic
studies showed that only the succinic dehydrogenase (SDH)
activity was moderately reduced as compared to that in controls
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V-21
In addition to their studies on guinea pigs, Pren-
dergast et al. (1967) studied the effects of both repeated
and continuous exposure to carbon tetrachloride on three
squirrel monkeys, three New Zealand rabbits, and two beagle
dogs for each exposure regimen. The experimental designs
were the same as those described for the guinea pigs. All
the animals showed a weight loss during repeated exposure to
515 mg/ra3 (82 ppm). Fatty changes were noted in the liver
of all species; they were most severe in rabbits, followed
by dogs and monkeys. In the continuous exposure to 61 mg/m3
(10 ppm) for 90 days, all species exhibited a depressed
weight gain, as did guinea pigs. Liver changes were also
noted, but enzyme activities (as measured by NADH, NADPH,
SDH, LDH G6PI) were within the normal range. At a continuous
exposure of 6.1 mg/m3 (1 ppm)-r no toxic signs were noted.
Teratogenicity
In two studies on the teratogenic and prenatal toxi-
cologic effects of carbon tetrachloride, the chemical was
reported to produce prenatal toxicity following inhalation
exposure.
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V-22
Schwetz et al. (1974) exposed pregnant rats to car-
bon tetrachloride at 1,800 or 6,300 rag/m3 (300 or 1,000
ppm) for 7 hours per day on days 6-15 of gestation. Statisti-
cally significant decreases in fetal body weight and crown-
rump length were observed. Other parameters examined were
not significantly different from those of controls. The
authors concluded that carbon tetrachloride was not terato—
genie at the concentrations used in this experiment. Two
other statistically significant fetal effects were noted:
an increased incidence of sternebral anomalies in the 6,300
mg/m3 group and an increased incidence of subcutaneous edema
in the 1,800 mg/m^ group. Edema was not seen in the 6.300
mg/m3 group. The dams exposed to both concentrations of
carbon tetrachloride showed a statistically significant
decrease in weight gain and also decreased food consumption
compared to control animals. Hepatotoxicity as measured by
significantly increased SGPT activity was also seen in the
dams. However, the authors did not establish any consistent
pattern between fetal toxicity and maternal toxicity at the
subanesthetic levels of carbon tetrachloride used in this
experiment.
Another study reported no teratogenic effects fol-
lowing exposure of pregnant rats to carbon tetrachloride at
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V-23
1,575 mg/m3 (250 ppm) 8 hours per day for 5 consecutive
days between days 10-15 of pregnancy (Oilman, 1971). Concomit-
ant exposure to 15% ethanol in drinking water also did not
result in teratogenic effects. Carbon tetrachloride exposure,
however, did decrease the viability index to 83% as compared
to 99% for controls. The lactation index was also decreased
to 83% as compared to 98% for controls. A decrease in the
number of pups per litter also occurred: 9.2 as compared
to 10.3 for controls. Concomitant ethanol exposure exacerbated
this effect: 8.48 pups per litter as compared to 10.3 for con-
trols.
No teratogenic effects were noted in rats fed diets
containing carbon tetrachloride at either 80 or 200 mg/kg of
feed for up to 2 years (Alumot et al., 1976). Groups of 18
female rats were mated, at 3 months of age (6 weeks of exposure)
with untreated males. Thereafter, they were mated every 2
months with groups of treated males.
Exposure to carbon tetrachloride in utero has been
reported to result in liver damage in rat fetuses and neonates
(Bhattacharyya, 1965). In one case, subcutaneous administra-
tion of carbon tetrachloride at 1,600 mg/kg bw to a pregnant
rat on day 20 of gestation resulted in small areas of focal
hepatic necrosis in a fetal liver 24 hours later. Similar
treatment resulted in focal hepatic necrosis in neonates born
48 or 72 hours after treatment of dams on day 19 or 20 of
-------
V-24
gestation. Histologic findings generally included a sharply
demarcated area of centrolobular necrosis and proliferative
changes in nonnecrotic lobes.
In addition, fetuses were directly treated with
carbon tetrachloride by subjecting the mother to a laparotomy
and either injecting the chemical directly into the fetus or
into the amniotic sac through the uterine wall (Bhattacharyya,
1965). Liver changes following injection of 6 mg of carbon
tetrachloride were variable: cells generally became extremely
pale in centrolobular and midzonal areas, indicating fatty
infiltration. Livers remained abnormal until at least 4
days after birth. No necrosis, hemorrhage, or regeneration
was observed.
Sensitivity of neonate rats to carbon tetrachloride
was reported to be low 1 hour after birth, then to rise
above the adult level at 19 hours and to decline by 3-7 days
after birth. Thus, only two of ten 1-hour-old neonates
receiving carbon tetrachloride (1,600 mg/kg) subcutaneously
showed centrolobular necrosis after 24 hours. In addition,
hepatic portal areas contained numerous neutrophils, but no
bile duct proliferation could be observed, in contrast to
findings in adult animals. Following the same treatment,
19-hour neonates showed more pronounced hepatic damage than
1-hour neonates. Damage declined in 3-and 4-day old neonates;
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V-25
that in 5-,6-, and 7-day-olds was similar in appearance to
that of adults.
Carbon tetrachloride can apparently be transferred
to the neonates through mother's milk (Bhattacharyya, 1965).
Subcutaneous -administration of carbon tetrachloride at 1,600
or 3,200 mg/kg bw to four- nursing rats resulted in hepatic
damage in the neonates 24 or 48 hours later. A dose of 800
mg/kg bw to dams did not produce any hepatic damage to off-
spring.
Reproductive Effects
Testicular degeneration was observed in rats receiv-
ing carbon tetrachloride at 4,800 mg/kg bw intraperitoneally
(frequently unstated) (Chatterjee, 1966). One group of six
male rats received carbon tetrachloride as a 1:1 mixture in
coconut oil; a control group received only an equal volume
of coconut oil. On day 15 all animals were sacrificed.
Body weights were similar for treated and control animals.
However, the relative testes weight decreased from 15.5 (+
0.4) g/kg bw in controls to 9.8 (+ 1.2) g/kg bw in exposed
animals. Relative weight of seminal vesicles showed an even
more pronounced decrease: 1.27 (+0.171) g/kg bw in treated
as compared to 3.10 ( + 0.059) g/kg bw in control animals.
Relative pituitary weight was also increased: 50 ( +_ 1.4)
-------
V-26
mg/kg bw in treated as compared to 32.4 (j^ 0.9) mg/kg bw in
control animals.
Histological examination of testes showed testicular
atrophy and "some abnormality" in spermatogenesis in carbon
tetrachloride-treated animals. The authors proposed a mechan-
ism for carbon tetrachloride-induced testicular atrophy in
which blockage of pituitary hormone release results in atrophy
of Leydig cells within the seminal vesicles, followed by an
abnormal spermatogenesis.
Intraperitoneal administration to male rats of carbon
tetrachloride (4,800 mg/kg bw as a 1:1 mixture of coconut oil)
for 10, 15, or 20 days (Group I, II, or III) led to impairments
in spermatogenesis as indicated by histological examination
(Kalla and Bansal, 1975). Controls were administered equal
volumes of coconut oil. Weights of testes, seminal vesicles,
epididyrais and prostates were decreased in exposed animals,
whereas the weight of adrenal glands increased (see Table V-6).
The gonadosomatic index (GSI) (equal to body weight x testes
weight/100) was also decreased in treated animals. A slight
decrease in pituitary weight was observed following a 10-day
treatment but not after either the 15- or 20-day treatment. As
Table V-5 shows, the ratio of germinal to nongerminal area
steadily decreased from Group I to Group III and was always
higher in treated than control animals. Significant differences
-------
V-27
in total germinal area between treated and control animals,
however, were observed only at 20 days. Histological examination
did not reveal any abnormalities in testes from Group I.
Clusters of mature sperm were present in the lumen. In Group
II, slight testicular damage was observed; a decrease in
spermatogenic cells and increased lumen size. In Group III,
shrinkage of the tubules and increased area of the lumen were
observed. Arrangement of the germ cells was disrupted; early
gonadal cells were present in the lumen of many of the tubules.
No spermatids were observed. Intersititial material was "dam-
aged" and in many places the basement membrane was detached
from the epithelium.
Thus, carbon tetrachloride, at a total dose of 48
g/kg bw over 10 days, had a distinct but minor effect on male
rat reproductive physiology, whereas a total dose of 96 g/kg
bw over 20 days resulted in severe disturbances of spermato
genesis.
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V-28
Table V-6 Weight Changes in Male Reproductive Organs After Carbon Tetrachloride Treatment
Treatment Body weight (g)
and Before After
Period treatment treatment
Group I
10 days
Control 257+5* 269+4.5
Treated 257+13.71 247+10.76
Group II
15 days
Control 230+15.49 235+5.0
Treated 230U5.49 173+13.42
Group III
20 days
Control 234+5.5 235+6.5
Treated 230+3 .5 2 10+0 . 5
Test is
(gAg bw)
12.36+0.94
8 .84+1 .04;
10.47+0.59
9.46+0.33
11.37+0.06
9.90+0.65
Seminal
vesicles
(gAg bw)
4.25+0.55
—i
2.41+0.14
3.9+0.52
1.17+0.26
3.24+0.31
1.53+0.28
Epididymis
(gAg/bw)
4.45+0.45
2.88+0.38
4.12+0.18
2.63+0.18
3.72+0.09
2.52+^0.22
Prostate
(gAg/bw)
2.1+0.7
1.26+0.24
2.19+0.31
1.68+0.13
1.78+0.04
1. 29+J). 01
Adrenal
(gAg/bw)
0.1B+.02
0.24+0.03
0.22+0.01
0.32+0.05
0.12+0.01
0.25+0.02
GSI
7.94+0.45
5.68+0.31
6.02+0.21
5.44+0.28
6.64+0.23
5.69+0.38
aMean +_ standard deviation.
Adapted from Kalla and Bansal (1975).
-------
V-29
Mutagenicity
There have been no reports of rautagenic activity
associated with carbon tetrachloride in any of the various
Salmonella (Ames) assays. However, mutagenic activity asso-
ciated with carbon tetrachloride has been reported in a eukary-
otic test system using the yeast Saccharomyces cerevisiae.
This recent report has not been confirmed and should not be
accepted as conclusive evidence of carbon tetrachloride rauta-
genicity. Nevertheless, these eukaryotic test systems can be
used to screen for mutagenic chromosomal effects that cannot
be detected in prokaryotic systems. Gallon et al. (1980)
have suggested that the yeast system is more sensitive than
the Salmonella assay system because active metabolites are
produced in much closer priximity to the nucleus than they
are with liver S-9 in vitro activation systems used in bac-
terial assays* Proximity to the nucleus would be advantageous
if the active metabolites were"particularly unstable or reac-
tive, as is likely to be the case for those of halogenated
hydrocarbons such as carbon tetrachloride.
Gallon et_ a_l. (1980) reported that carbon tetrachlo-
ride, in addition to six other hydrocarbons, induced a mutagenic
response in S_. cerevisiae strain D7. Unlike the Salmonella
system, in which promutagens must be treated with exogenous
liver S-9 activation systems, the D7 strain of S_. cerevisiae
contains an endogenous cytochrome P-450 dependent mono-oxy-
genase activation systems. In addition, strain D7 can be
used to detect both gene crossover and mitotic recombination.
Three concentrations of carbon tetrachloride (21, 28, and 34
mM) were incubated at 37°C for either 1 or 4 hours in 3-ml
-------
V-30
aliquots of the yeast cell suspension (3 x 10^ eelIs/ml).
Treatment was terminated by the addition of 40 ml of ice-cold
buffer followed by centrifugation to remove the cells from
the media. The resuspended cells were then plated on appro-
priate media to allow for estimation of mutagenic activity.
A spectral analysis of the cell suspension demonstrated that
carbon tetrachloride was gradually altered during the incuba-
tion periods; the authors concluded that it was absorbed and
metabolized by the cells.
The 1-hour treatment of cells with carbon tetrachlo-
ride resulted in significant increases in gene crossover and
mitotic recombination. Results for the different loci are
presented in Table V-7. When cells were treated with increasing
carbon tetrachloride concentrations there was decreased cell
survival and increased incidence of gene conversion and mitotic
recombination. There was a nonlinear correlation between cell
survival and the concentration of carbon tetrachloride in the
incubation mixture. Only marginal mutagenic activity was noted
when incubation was continued for 4 hours (data not reported
by authors). This observation could have resulted from toxicity
masking the genetic effects, the continued metabolism of active
metabolites to inactive products, or the destruction of the
metabolic system. Gallon et al. (1980) noted that carbon
tetrachloride had been reported to be inactive in test systems
which used an exogenous mammalian activation system and suggested
that their yeast system was more sensitive than some of the
other in vitro test systems.
-------
V-31
TABLE V-7 Mutagenic Effects of Carbon Tetrachloride
on Strain D7 of Saccharomyces Cerevisiae3-
Concentration (ing/liter)
0 3234 4312 5128
Survival
Total colonies 1454 1252 1120 152
% of control 100 86 77 10
trp 5 locus (gene conversion)
Total convertants 285 331 350 506
Convertants/I05 survivors 2.0 2.6 3.1 61.7
ade 2 locus (mitotic recombination)
Total twin spots 1 3 3 10
Mitotic recombinants/104 survivors 1.6 5.3 5.8 40.1
Total genetically altered
colonies 11 19 16 65
Total genetically altered
colonies/10^ survivors 1.7 3.4 3.1 33.3
ilv 1 locus (gene reversion)
Total revertants
Revertants/106 survivors
38 41 57 11
2.6 3.3 5.1 7.2
a The total number of colonies in the different classes represent total
counts of colonies from five plates in the case of survival, conversion,
and revertant-frequency estimations. Mitotic recombination was estimated
from counts of colonies growing on a total of 30 plates, 20 plates con-
taining medium of which all surviving cells grew and 10 plates containing
medium on which only trp 5 convertants grew.
Adapted from Callen et al. (1980)
-------
V-32
Studies to determine the rautagenic activity of carbon
tetrachloride in the Salmonella typhimurium system have been
uniformly negative (see reviews: McCann et_ a^., 1975; Fishbein,
1976; and Rinkus and Legator, 1979). Carbon tetrachloride has
also been void of mutagenic activity when tested in Escherichia
coli (Uehleke et al., 1976).
Carbon tetrachloride did not induce chromosome damage
(i.e., chromatid gaps, deletions, or exchanges) during an in
vitro chromosomal assay using cultures rat-liver cells (Dean
and Walker, 1979). Mirsalis and Butterworth (1980) found that
treatment of adult male Fischer-344 rats (200-250 g) with car-
bon tetrachloride (10 or 100 mg/kg, po) produced no increase in
unscheduled DNA synthesis in cultures of primary rat hepato-
cytes. According to the authors these results indicate that
carbon tetrachloride does not act through a genotoxic mechanism
and confirm the report of Craddock and Henderson (1978) that
this chemical does not induce DNA repair in hepatocytes immedi-
ately following treatment.
Uehleke et al. (1977) studied the interaction of car-
bon tetrachloride in a liver microsome system used in conjunc-
tion with assays in S_. typhimuriuro strains TA 1535 (test for
base pair substitutions) and TA 1538 (tests for frame shift
mutations). The authors incubated 3-4C-labeled carbon tetrachlo-
ride (1 mM) with rabbit liver microsomes pretreated with pheno-
barbital (5 mg of protein/ml) and a NADPH regenerating system
at 37°C for 60 minutes. A total of 10% of the radioactivity
-------
V-33
from carbon tetrachloride was irreversibly (covalently) bound
to endoplasraic portein and more than 30% was bound to microsomal
lipid. No mutagenic activity was observed in S3. typhimurium
strains TA 1535 or TA 1538 which were incubated with 8 mM carbon
tetrachloride and microsomal suspensions. The authors concluded
that a reactive species generated in the biological system may
not distribute into the incubation medium and thus may be
inaccessible to the test bacteria. They also speculated that
the active products of carbon tetrachloride may have very short
half-lives.
Carcinogenicity
The carcinogenic effects of carbon tetrachloride have
been well documented (IARC, 1979). IARC has judged the evidence
from animal studies demonstrating that carbon tetrachloride in-
duced hepatic neoplasms as conclusive for experimental animal
carcinogenesis (IARC, 1979).
In a NCI bioassay program, carbon tetrachloride was
used as a positive control in the bioassays of chloroform and
1,1,1-trichloroethane (NCI 1976, 1977). The positive control
groups described in both bioassays were of the same strain and
source as the treated animals and were housed identically.
Groups of 50 Osborne-Mendel rats of each sex were administered
carbon tetrachloride in corn oil by gavage five times weekly
for 78 weeks at two dose levels: 47 and 94 rag/kg bw for males,
80 and 160 mg/kg bw for females. This treatment resulted in
toxicity: at 110 weeks at the highest dose, only 7 of 50 males
-------
V-34
and 14 of 50 females survived as compared to 26 of 100 males and
51 of 100 females for controls. The incidence of hepatocellular
carcinomas was increased in animals exposed .to carbon tetrachlo-
ride as compared to controls (see Table V-8). Absolute incidence
of hepatic neoplasms was low. The apparent decrease in the
incidence of hepatocellular carcinomas in female rats at the
high dose was attributed to increased lethality, i.e., females
died before tumors could be expressed. The incidence of other
neoplasms in these rats was acknowledged but not quantified.
Table V-8. Incidence of Liver Tumors in Carbon
Tetrachloride-Treated Rats and Colony Controls
Animal group Hepatocellular carcinoma Neoplastic nodule
Males
Females
Controls
Low dose
High dose
Controls
Low dose
High dose
1/99.
2/50
2/50
0/98
4/49
1/49
0/99
2/50
1/50
2/98
2/49
3/49
Sources NCI (1976)
Groups of 50 B6C3F1 mice of each sex were administered
carbon tetrachloride in corn oil by gavage five times weekly for
78 weeks at two doses, 1,250 and 2,500 mg/kg bw, for both males
and females. High exposure-related lethality occurred in all
groups. At 78 weeks at the high dose, only 2 of 50 males and 4
of 50 females survived as compared to 53 of 77 V-3& males and 71
of 80 females for controls. At 91-92 weeks at the high dose,
-------
V-35
none of 50 males and 1 of 50 females survived as compared
to 38 of 77 male and 65 of 80 female controls. An exposure-
related increase in the incidence of hepatocellular carcinomas
was observed (see Table v-9). The average latency for appearance
of liver tumors was also significantly decreased in carbon
tetrachloride exposed animals. In high-dose males, the latency
was 26 weeks, as compared to 48 weeks in low-dose males and 72
weeks in control males. In high-dose females, the latency was
19 weeks, as compared to 16 weeks in low-dose and 90 weeks in
control female mice.
TABLE V-9 Comparison of Hepatocellular Carcinoma Incidence
in Carbon Tetrachloride-Treated Mice Vehicle-Treated Controls
Animal
Males
Females
group Hepatocellular carcinoma
Controls
Low dose
High dose
Controls
Low dose
High dose
5/77
49/49
47/48
1/80
40/40
43/45
Source: NCI (1976)
The comparative carcinogenicity of carbon tetrachlo-
ride has been studied in five rat species: Japanese, Osborne-
Medel, Wistar, Black, and Sprague-Dawley (Reuber and Glover
1970). Groups of 12-17 male rats of each strain were given
-------
twice weekly subcutaneous injections of carbon tetrachloride
(2,080 rag/kg bw as a 50% solution in corn oil). Treated animals
were sacrificed when moribund; controls for each strain were
sacrificed at the time as the last experimental animal. Incidence
of hepatic lesions is given in Table V-10. The data indicate
that: (i) sensitivity to carbon tetrachloride-induced neoplasms
varies widely among strains; and (ii) the trends in incidence of
neoplasms and cirrhosis run exactly opposite. Varying amounts
of toxicity occurred, all experimental animals of the Black rat
strain were dead at 18 weeks, and those of Sprague-Dawley Strain
at 16 weeks; the failure to find carcinomas in those strains may
have been caused in part by an insufficient latency time. in all
three other strains, significant toxicity (i.e., lethality)
occurred. Toxicity decreased in the same order as carcinogenicity
increased. Thus, it appears that there is no causal connection
between the degree of toxicity and carcinogenicity.
Other neoplasms were also observed, all in the Osborne—
Mendel and Japanese strains. (It is unclear from the text whether
these were in experimental or control animals.) Heman-giomas of
the spleen were seen in three rats: two Japanese and one Osborne-
Mendel. Six carcinomas of the thyroid gland were observed: Three
in Japanese and three in Osborne-Mendel rats. Multicystic kidneys
were observed in two Osborne-Mendel and three Japanese rats. One
rat of the Japanese strain had a subcutaneous leiomyosarcoma.
Relative organ weights were decreased for testes and
increased for liver, spleen, and kidneys of all experimental
-------
V-37
animals' as compared to strain controls. The extent of atrophy
of the testes, prostate, and seminal vesicles was correlated
with the degree of cirrhosis.
The carcinogenicity of carbon tetrachloride in hams-
ters has also been described (Delia Porta et al., 1961). Groups
of 10 Syrian golden hamsters of each sex were administered weekly
by gavage 20 mg of carbon tetrachloride (5% solution in corn
TABLE V-10. Evidence of the Most Advanced Lesions
In Rats Administered Carbon Tetrachloride
Japanese Osborne- Wistar Black Sprague-
Mendel Dawley
No hyperplasia
Hyperplasia
Hyperplastic nodule
Small carcinoma
Large carcinoma
Total 'Carcinoma
No cirrhosis
Mild cirrhosis
Moderate cirrhosis"
Severe cirrhosis
0/15
0/15
3/15
4/15
8/15
12/15
0/15
9/15
5/15
1/15
0/13
1/13
4/13
4/13
4/13
8/13
0/13
2/13
7/13
4/13
0/12
1/12
7/12
3/12
1/12
4/12
0/12
0/12
6/12
6/12
4/17
6/17
7/17
0/17
0/17
0/17
0/17
0/17
4/17
13/17
8/16
6/16
2/16
0/16
0/16
0/16
0/16
0/16
0/16
16/16
Adapted from Reuber and Glover (1970)
-------
V-38
oil) for 7 weeks, followed by 10 mg for 23 weeks (equivalent
to 200 and 100 mg/kg bw). Survivors were sacrificed at 55
weeks. Postnecrotic cirrhosis was observed in all animals that
died in week 41. Postnecrotic cirrhosis was described by the
authors to involve "regenerative hypexplastic nodules." In
current experiments, these probably would have been described
as neoplastic nodules in view of the following micropathological
findings: nodules had obliteration of normal lobular architec-
ture and were surrounded by fibrpus tissues; cells were irregular
in shape and size? nuclei and cytoplasm stained abnormally with
uneven distribution of glycoge^i. Each of the other 10 animals
had one or more hepatic carcinomas. A total of 22 neo-plasms
was observed: 12 in five females and 10'in five males. Some
were sizeable, measuring 5-30 mnu
Thus, Syrian golden hamsters appear sensitive to the
carcinogenic effects of carbon tetrachloride. Although, the
number of animals in this study was small, the authors consider-
ed the results to be significant because the reported control
rate of hepatic tumors in hamsters were 0/254.
Carbon tetrachloride was also reported to be carcino-
genic in C3H mice (Andervont, 1958). Groups of 30-77 female or
male C3H mice were administered by gavage 6.46 mg of carbon
tetrachloride once weekly for 2 weeks, followed by administra-
tion of 9.6 mg once weekly for 17 weeks {equivalent of 213 and
320 mg/kg bw). Pathogen-free or normal C3H mice were used. For
no difference in the incidence of hepatomas was observed between
-------
pathogen-free and normal mice: 79% as compared to 49% in con-
trols^ The average number of hepatoraas per animal was 1.8 in
treated animals and 1.3 in controls. In females, a difference
between the incidence and hepatomas in pathogen-free and normal
rats was observed: 46% and 29%, respectively as compared to 3%
in controls. The average number of hepatomas per mouse was 1.5,
1.2, and 1.0, respectively, indicating that both the incidence
as well as the average number of tumors per animal increased in
the order: controls
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V-4O
Table V-ll Susceptibility of Strain A Mice to Liver Necrosis and the Incidence of Hepatones 30 Days
After 120 or 30 Doses of Carbon Tetrachloride3
Carbon tetrachloride dose15
0
9600 mg/kg 4800 mg/kg 2400 mgAg 1200 mg/kg (olive oil)
OJ W
23 > 0) H
•H H
r-j r-H C "j
10 fl) fll
2.31.13 1 2.
ri 50 -H to q q
•c-l p M-l O •" •"
|I|I j i
Sex Dose Conditions "o
-------
V-41
were detected by microscopic examination in two males. The
authors concluded that repeated liver necrosis and its associated
chronic regenerative state are probably not necessary for the
induction of tumors with carbon tetrachloride.
A third study also reported the induction of hepato-
mas in mice by exposure to carbon tetrachloride (Edwards, 1941).
Of 143 female C3H mice administered 64 mg of carbon tetrachloride
two or three times weekly for 36-55 doses, 126 or 88.1% developed
hepatomas. In similar experiments, a 100% incidence of hepatomas
was observed in 54 male and female strain A mice having received
23—58 doses. The first hepatoma was observed following 23 doses.
Summary
Carbon tetrachloride-induced hepatic effects have been
reported after both acute and chronic exposure. The degrees of
toxicity and of hepatic damage have appeared to be dose-related,
as measured by liver enzyme activity in at least four species:
mouse, rat, guinea pig, and dog. In parallel acute studies in
mice and dogs in which dose-response relationships for minimal
toxic effects were developed, mice have appeared more sensitive
to the toxic effects of carbon tetrachloride than have dogs.
Liver damage from acute exposure to carbon tetrachloride has
been reported to be reversible. At 36 hours after acute
exposure to various doses of carbon tetrachloride, histopatho-
logical examination has shown no abnormalities in liver speci-
mens. In chronic exposure, liver damage has been reported to
-------
V-42
be reversible if the damage did not advance to the necrosis
stage.
The degenerative liver changes observed in both
acute and chronic exposure have been increased serum/plasma
levels of hepatic enzymes, progressing through more pronounced
cellular degenerative changes such as fatty liver and pro-
liferation of rough endoplasmic reticulum, to fatty liver
degeneration and necrosis. Lung damage has included similar
cellular effects, with the majority of changes occurring
within the Clara cells. Degenerative kidney effects have
also been observed, but appeared significant only after high
doses of carbon tetrachloride.
Carbon tetrachloride has produced prenatal toxic
effects, which could not be well correlated with extent of
maternal exposure. Rats exposed to carbon tetrachloride in
utero have shown hepatic abnormalities at birth, but the
lifetime effects of these changes were not reported.
Carbon tetrachloride has produced distinct degenera-
tive changes in testicular histology, eventually resulting
in aspermatogenesis and functional male infertility. These
effects occurred at medium to high doses.
Carbon tetrachloride has elicited a mutagenic response
in a Saccharomyces cervisiae test system, but has consistently
tested nagative in the Salmonella (Ames) assay. Investigators
have attributed the failure of carbon tetrachloride to induce
-------
V-43
a mutagenic response in Salmonella to the absence of an in
vivo activating system in the prokaryote. No other mutagenic
responses to carbon tetrachloride have been reported in the
literature.
Carbon tetrachloride has been reported to be carcino-
genic in numerous animal studies. Hepatocellular carcinomas
have been the neoplasm induced in all species. Hamsters have
been the most sensitive species studied, followed by mice and
then rats. A significant strain difference has been observed
in rats. Females have appeared less sensitive to the chronic
toxic effects and more sensitive to the carcinogenic effects
of carbon tetrachloride in both rats and mice.
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VI. HEALTH EFFECTS IN HUMANS
Considerable human exposure to carbon tetrachlo-
ride through inhalation has come through its use as an
industrial solvent and dry cleaning fluid. Ingestion of
carbon tetrachloride or mixtures containing carbon
tetrachloride has also been documented in various case
reports. Ingestion has occurred under different circum-
stances (e.g., suicide attempts, medical use) by persons
of diverse occupations and ages. These acute exposures
have been followed by hepatoxic effects accompanied by
acute nephrosis.
In the following section the effects of carbon tetra-
chloride exposure are presented as reported in case studies and
in controlled studies for humans. The case studies are divided
into reports of acute and long-term effects. The human case
studies are often anecdotal, with missing or imcomplete medical
descriptions of clinical signs of poisonings. In the controlled
studies using human volunteers, changes .in serum and urine
chemistry were measured after exposure to carbon tetrachloride,
but no histologic specimens were taken. Because of the
limitations in these studies of humans, they are presented
as supporting evidence for the harmful effects of carbon
tetrachloride' in humans.
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VI-2
Case Studies-—-Acute Effects
Ingestion. Lamson et al. (1928) studied the lethal
effects of carbon tetrachloride in patients receiving carbon
tetrachloride and magnesium sulfate orally as a treatment for
hookworms. The authors reported the treatment of thousands
of patients with a single dose of 2.5-15 ml of carbon tetra-
chloride without ill effects. One man was reported to have
safely ingested 40 ml of carbon tetrachloride. However, an
"extremely small" population of adults died after receiving
1.5 ml of carbon tetrachloride; doses of 0.18-0.92 ml were
reported to be fatal to children. Susceptibility in adults
was cprrelated with alcoholic intake (chronic alcoholism or
exposure to alcohol shortly after treatment), the presence of
ascarid worms, and the intake of foods, particularly of high
fatty content.
A fatality attributed to ingestion of carbon tetra-
chloride was reported by Smetana (1939). The victim, a photo-
grapher described as having "a history of chronic alcoholism,"
died 10 days after consuming an unknown amount of "some fluid
containing carbon tetrachloride." He presented symptoms includ-
ing nausea, vomiting, jaundice, anuria, and semistupor. In the
final clinical diagnosis, death was attributed to carbon tetra-
chloride poisoning.
A case of attempted suicide by ingestion of carbon
tetrachloride was reported by Stewart et al. (1963). The
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VI-3
victim, a 29-year-old female who ingested 1 pint of a carbon
tetrachloride: methanol solution (2:1), experienced ringing
in the ears immediately after ingestion and lost consciousness.
She was hospitalized for 3 weeks. Three hours after ingestion,
carbon tetrachloride in the exhaled breath and blood was con-
firmed by infrared analysis. The exhaled breath was then
monitored throughout the hospitalization, and was reported to
decrease exponentially. Because of the toxicity of the methanol
and the possibility of synergistic reactions with the carbon
tetrachloride, hemodialysis was performed soon after admission.
Mannitol solution was given by continuous intravenous infusion.
Clinical laboratory analyses during hospitalization showed some
elevation of SCOT, which reached a maximum of 75 units at day 6,
and an elevation of urinary urobilinogen to a maximum of 7.8
Ehrlich units at day 10. Other laboratory findings included
elevation of serum iron and depression of serum protein concen-
tration and albumin fractions. The retention time of bromo-
sulfophthalein was increased. These finding were interpreted
as evidence of minimal hepatocellular injury. Acute renal
dysfunction was not observed; the authors credited the mannitol
treatment with preventing renal damage.
Acute Effects
Inhalation. Bilateral peripheral constriction of the
ocular color fields, resulting in symptoms of toxic amblyopia
in three males, was attributed to the inhalation of carbon
tetrachloride vapors (Wirtschafter, 1933). Five male employees
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VI-4
of dry cleaning establishments who had been exposed to carbon
tetrachloride (of unknown concentration) from 8-10 hours daily
for 1-6 months were examined. Two men also had signs' of con-
junctivitis. Three of the men complained of visual disturbances
characterized by blurred vision or spots before the eyes. The
author concluded that toxic amblyopia may result from exposure
to carbon tetrachloride vapor.
One fatality occurred in two cases of carbon tetra-
chloride poisoning reported by Smetana (1939). In the fatality,
a dry cleaner and interior decorator described as being "a steady
and heavy drinker" was exposed for several hours to carbon tetra-
chloride vapors during work. Upon returning from work, he noted
dyspnea. Several hours after the exposure, headache, dizziness,
and malaise developed, accompanied by nausea and repeated vomit-
ing that persisted for several days. The patient also suffered
labored breathing and cough with bloody sputum before he died 9
9 days following exposure.
The second inhaltion case reported by Smetana was a
housemaid also described as having a history of chronic alcoho-
lism. Three days before hospitalization, the patient cleaned
dresses with carbon tetrachloride for 3 hours in a poorly
ventilated room. Soon after exposure, she began to vomit. She
suffered symptoms similar to those described for the other case.
After approximately 1-1/2 months of hospitalization, this
patient was released from the hospital her condition several
weeks later was described as "much improved."
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VI-5
Seven cases of carbon tetrachloride poisoning reported
by Norwood et al. (1950) resulted from both occupational and non-
occupational inhaltion exposures. In the three cases described
as "severe" poisonings* there was a history of chronic alcoholism;
two fatalities occurred in this group. In one case, the victim
had been exposed for about 15 minutes to an atmosphere containing
carbon tetrachloride at an estimated 1,575 mg/m^ (this estimate
was made by duplicating the conditions). Histopathologic exami-
nation of liver and kidney tissue from the fatalities revealed
liver necrosis and degenaration of the renal tubules. The four
remaining cases were characterized as "mild industrial"
exposures. After exposure to carbon tetrachloride, all subjects
suffered varied symptoms including nauseas, vomiting, diarrhea,
headache, muscular ache, pain, or numbness, labored breathing,
and dizziness.
In another case, a 31-year-old janitor suffered ma-
laise, back and lower abdominal pain, nausea, and vomiting the
morning after working for 5 hours in a closed room with carbon
tetrachloride (Kittleson and Borden, 1956). He reportedly
consumed two bottles of beer during the exposure period. The
patient required 2 months of hospitalization for treatment of
acute renal insufficiency as a result of carbon tetrachloride
intoxication.
Elevated serum glutamic oxaloacetic transaminase (SCOT)
activities with concomitant liver changes were reported in two
men occupationally exposed to unreported concentrations of
-------
VI-6
carbon tetrachloride (Lachnit and Pietschmann, 1960). One
became ill after exposure to carbon tetrachloride for 3 hours
in a relatively well-ventilated room. He was hospitalized 3
days after exposure. His liver was slightly enlarged, with
the (SGOT) value elevated by 6,000 units. This value rapidly
decreased and returned to normal by the 10th day. A biopsy of
the liver taken on the 8th day showed necrosis in the centers
of the lobuli, but the surrounding tissue was undamaged. An
additional needle biopsy of the liver taken at the 28th day
showed that the cells had almost returned to normal. In the
second case a male similarly exposed to carbon tetrachloride
entered the hospital 12 days after exposure. The SGOT had
increased to 80 units. A liver needle biopsy on the 22nd day
showed only moderate changes, some of a degenerative nature.
In a chemical packing plant, use of carbon tetra-
chloride by two workers for equipment cleaning, as a substitue
for the customarily used acetone, resulted in the hospitaliza-
tion of 4 of 43 workers at the plant (Folland e_t al., 1976).
Ten additional workers also became ill. Eight of the 43 work-
ers fell ill within 12 hours following the start of the 2-hour
exposure? six others followed within the next 36 hours. The
four hospitalized workers showed evidence of severe disruption
of liver function: one case had an SCOT level of 13,390 units.
All patients recovered within 90 days. All hospitalized work-
ers as well as most of the others taken ill had worked near a
bottle-filling operation for isopropyl alcohol at the northern
end of the plant, adjacent to the carbodn tetrachloride cleaning
area.
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VI-7
Carbon tetrachloride concentrations at the time of expo-
sure were not ascertained; acetone was normally used for cleaning.
Isoporopyl alcohol concentrations at the northern end of the plant
average 410 ppm. Acetone in alveolar air samples of workers in the
northern area averaged 19 ppm. The authors described the toxic
episode to carbon tetachloride toxicity potentiated by isopropyl
alcohol. Because carbon tetrchloride concentrations were unknown
and isopropyl alcohol (and possibly other chemicals) were present,
the health effects reported in this study cannot be attributed
to carbon tetrachloride exposure alone.
Case Studies Long-Term Effects
Straus (1954) suggested a possible causal relationship
between carbon tetrachloride exposure and aplastic anemia. Three
males had been exposed to carbon tetrachloride at unknown concen-
trations for 2 months to 3 years. Autopsy findings included hypo-
plasia of the bone marrow. However, a causal relationship between
carbon tetrachloride and aplastic anemia suspected by the author
in these cases is not supported adequately. One of the men had
also been exposed to kerosene for 3 years. Another was an auto
mechanic who worked in a garage. The occupation of the third
was not specified although his exposure to carbon tetrachloride
was occupationally related. Thus the effects of other chemicals
cannot be discounted. The autopsy findings of two of the patients
included no liver or kidney damage of the type that would be ex-
pected in carbon tetrachloride poisoning. In one case the liver
was reported to have toxic hepatitis.which was considered to be
the result of carbon tetrachloride poisoning. The information
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VI-8
reported in these case studies tends not to substantiate the
author's suggestion that the patients' illnesses may have been
caused by carbon tetrachloride.
Carcinogenicity The possibility of carcinogenic
effects of carbon tetrachloride in humans has been raised in a
number of case reports. These reports do not establish a causal
link between carbon tetrachloride exposure'xand the incidence of
neoplasms (heptomas)^ Thus, the suggestion that carbon tetra-
chloride is carcinogenic in humans remains purely speculative.
A 59-year-old man with a history of moderate alcohol
consumption returned from a cocktail party and noticed the vapor
of carbon tetrachloride used to clean a rug in his apartment
earlier that evening. Five days later he developed nausea, vomit-
ing, and diarrhea and within 10 days of exposure he developed
jaundice (Tracey and Sherlock, 1968K The patient recovered
following'a long and complex hospitalization and was discharged
after 9 weeks. Four years after hospitalization for jaundice,
he was found to have a smooth, enlarged, nontender liver. He
denied alcohol consumption within the intervening period. Three
years after this checkup, the patient was readmitted with a
history of nausea, vomiting, and diarrhea. A liver biopsy was
diagnosed as hepatocellular carcinoma. No treatment was admin-
istered until readmission 5 months later when he received X-ray
radiation dose of 3000 R. He died 2 weeks after discharge.
Postmortem examination revealed the liver to be extensively
involved with the tumor. Little normal liver tissue remained.
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VI-9
The connection between carbon tetrachloride exposure/
potentiated by prior alcohol use, and the induction of jaundice
appears well established. In contrast, as the authors state, no
causal relationship between the carbon tetrachloride exposure and
hepatocellular carcinoma can be drawn from this report. Aside.
from the acute exposure to carbon tetrachloride 7 years before
diagnosis of cancer, the patient's possible additional exposure
to this and other toxic chemicals -was not reported. Ho medical
history was given for the 3 years before the final diagnosis.
A. study of residents of an area surrounding a solvent
recovery plant in rural Maryland found a great increase in the
incidence of lymphatic cancer (Capurro, 1979). The mortality
experience of residents of a 1.5 Icm^ area around the plant—which
emitted at least 31 chemicals (identified by gas chromatography),
including carbon tetrachloride—-was followed from October 1968 to
October 1974. Six deaths, one due to cancer, were expected over
this period. Fourteen deaths were observed, including seven
from cancer. Four of seven malignancies were lymphomas, more
than 60 times the expected incidence. These deaths were not
attributed to any particular chemical.
Other case reports of human neoplasms developing after
exposure to carbon tetrachloride have appeared. In one case a
woman developed modular cirrhosis of the liver followed by cancer
of the liver after exposure to carbon tetrachloride, and died 3
years after the first exposure (Johnstone, 1948). However, she
had suffered from periodic jaundice for 5 years prior to exposure
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VI-10
to carbon tetrachloride. In a second case, a fireman developed
cirrhosis and an "epithelioma" of the liver 4 years after acute
carbon tetrachloride intoxication (Sintler et al., 1964). In
none of the cases could a causal link between carbon tetrachlo-
ride exposure and development of neoplasms be established.
Because of concomitant exposure to other chemicals
this study does establish a causative association between carbon
tetrachloride exposure and increased mortality.
Epidemiology. A retrospective study of laundry and dry
cleaning workers was conducted by Blair, ejt a!U (1979) to determine
if occupational exposure to carbon tetrachloride, trichloroethy-
lene, tetrachloroethylene, and petroleum solvents resulted in
increased morbidity or mortality. Data for cases were obtained
from union records benefits lists. Sex, race, age at death, and
and underlying and contributing cause of death were abstracted
from death certificates. The age, race, sex, and cause distribu-
tion for all deaths in the United States from 1957-1970 served
as the control standard. Causes of death were analyzed using
the proportionate mortality method. The results of analysis
demonstrated an excess of lung and cervical cancer, and slight
excesses of leukemia and liver cancer.
Because of the multiple historical exposures experi-
enced by this population, it is difficult to establish a causal
association between specific substances and increased mortality
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VI-J.1
trends. This study is presented as evidence of a possible link
between carbon tetrachloride exposure and increased mortality
rather than as a study in which quantitative extrapolations of
the chemical's effects on human health are possible.
A cross-sectional epidemiologic study (Sonich et al.,
unpublished) examined health effects of CC14 ingestion in humans.
Seventy tons of carbon tetrachloride were spilled in the Kanawha
. and Ohio River in 1977. Measurements of raw water revealed maxi-
mum concentration of 0.340 mg/1. Twenty-one cities situated along
the river were involved in the study. These cities represent
areas that obtained their drinking water directly from the river
and/or area that obtained their drinking water from sources not
influenced by the quality of the river water. By using river
volumes and flow rates/ periods of high exposure (1977) and low
exposure (1976) to carbon tetrachloride were estimated for each
city along the river. The results' of routine tests measuring
serura chemistries reflecting liver and kidney function along with
basic epideraiologic information were abstracted from approximated
6,000 medical records. The results obtained for creatinine show
a positive and statistically significant (p<.05) relationship
between the carbon tetrachloride exposure and the frequencly of
elevated levels of serum creatinine in exposed patients. No
similar results were found for the other parameters analyzed.
Controlled Studies
Inhalation. Human volunteers were exposed to known
concentrations of carbon tetrachloride vapor in an effort to
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VI-12
correlate physiological and/or biochemical changes to the mag-
nitude of exposure (Stewart et a_l., 1961). Eight healthy male
volunteers were exposed to carbon tetrachloride vapors in a
series of three separate experiments performed 1 month apart.
Prior to exposure, data on blood pressure, SCOT, and urinary
urobilinogen were obtained for each subject. Samples of pre-
exposure exhaled breath, urine, and blood showed no detectable
carbon tetrachloride. The volunteers were seated in a closed
room (11 x 12 x 7.5 feet) where 99% pure carbon tetrachloride
was poured into a dish and covered with a towel. An exhaust
system grill and door were closed during the experiment but an
air supply grill was left open. A fan circulated air across
the dish. Carbon tetrachloride ambient concentrations were
monitored with a Davis halide meter and an infrared spectra-
meter. The carbon tetrachloride concentration ranges and
exposure times are given below in Table VI-1.
TABLE VI-1 Exposure Times and Concentrations of Carbon Tetra-
chloride Vapor in a Study by Stewart et. al. (1961)
Experiment
Average concentration,
time-weighted (mg/m^)
Concentration
range
Exposure
(minutes )
1
2
3
309
69
63
192-548
63-38
57-88
70
180
180
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VI-13
Carbon tetrachloride was detected in exhaled breath in
all three experiments. Graphs showed an exponential decrease in
concentration of carbon tetrachloride versus time. The exact
values were not given.
The serum iron showed an initial decrease in three of
six subjects at the 309 mg/m3 exposure level but had returned
to normal in two of these subjects 68 hours after exposure. The
remaining subject showed a 31% depression in serum iron at 68
hours, but the value was within the normal range. Serum iron was
not analyzed in the other two experiments. Of the six subjects
exposed to carbon tetrachloride at 309 mg/m3, the serum trans-
minase level was slightly elevated in some and depressed in
others, but remained within the normal range. Carbon tetrachlo-
ride was not detected in the blood or urine at any exposure time
or dose, but the analytical technique used was not a sensitive
one. The authors concluded that no ill effects were observed
from exposure to carbon tetrachloride at 63 mg/m3 for 180 minutes,
although the small changes in serum iron at the 309 mg/m3 dose
might have been an indication of liver insult.
Absorption through Skin. The absorption of carbon
tetrachloride through human skin was measured by immersion of
the thumbs of three male and female volunteers in a sample of
this compound for 30 minutes (Stewart and Dodd, 1964). The car-
bon tetrachloride was analyzed by infrared spectroscopy, and no
impurities were detected. Sequential sensations of burning and
cooling were experienced by all volunteers during the immersion.
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VI-14
Burning ceased about 10 minutes after removal from the solvent.
The thumbs of all volunteers appeared scaly and red, a condition
that improved within several hours after exposure. Carbon tetra-
chloride was detected in the alveolar air of each subject within
10 minutes of immersion of their thumbs. The concentration in
the expired breath rose continuously to a maximum of 4.0 mg/rn-*
10 to 30 minutes after the exposure period ended, and then
decreased exponentially. The mean concentration of carbon tetra-
chloride was 2.0 mg/m3, 2 hours after the end of exposure; at 5
hours after exposure, the alveolar air concentration was still
greater than 0.6 mg/m^. The authors concluded that carbon tetra—
chloride could be absorbed through the skin in toxic quantities.
Reproductive Effects. No teratogenic effects in humans
caused by carbon tetrachloride exposure have been reported. How-
ever, human fetuses in one study appeared to have selectively
accumulated carbon tetrachloride from the mother's circulation
(Dowty et al., 1976). Maternal blood samples were taken from 11
women either before or directly after (vaginal) delivery. (Prior
exposure of the women to toxic chemicals was not reported.)
Paired cord blood samples were obtained immediately after de-
livery. All volatiles were analyzed by gas chromatography and
mass spectrometry. Carbon tetrachloride, benzene, and chloroform
were present in higher concentratation in cord blood as compared
to maternal blood.
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VI-15
Summary
Hepatic necrosis and renal pathology appear to be
characteristic effects of acute human exposure to carbon
tetrachloride. If exposure is terminated, the liver shows
regeneration in most cases. In cases of acute renal dysfunc-
tion, Sidney function returns to normal after exposure to
carbon tetrachloride is terminated and medical treatment is
given.
The possibility of an association between carbon
tetrachloride exposure and cancer or aplastic anemia is not
substantiated in epidemiological and case studies. These
studies are limited in number, and all suffer from the con-
founding factor of exposure to multiple chemicals.
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VII. MECHANISMS OF TOXICITY
The toxicity of carbon tetrachloride to an organism
depends upon the ability of the organism to metabolize the com-
pound. Thus, unmetabolized carbon terachloride does not appear
to be significantly toxic (Rechnagel and Glende, 1973). In mam-
mals, carbon tetrachloride is thought to be metabolized in the
endoplasmic reticulum of the liver by the mixed-function oxidase
system of enzymes. The reaction sequence proposed in the litera-
ture for carbon tetrachloride metabolism was outlined in Section
III, Pharmacokinetics. Two free radicals have been postulated as
metabolic intermediates: the trichloromethyl radical and the
chlorine radical. The toxicity of carbon tetrachloride has been
attributed to subsequent reactions of the trichloromethyl radical,
These reactions include formation of carbonyl chloride (phogene),
dimerizatiion to hexachloroethane, free radical binding protein,
and lipid peroxidation. In this section each of these proposed
pathways will be presented in conjuction with the toxic effects
attributed to it.
Formation of Cabonyl Chloride (phosgene)
From the results of an ir± vitro study of carbon tetra-
chloride metabolism, Shah e_t al^. (1979) postulated the formation
of carbonyl chloride from the trichloromethyl radical. The
authors incubated L-cysteine and f^C] carbon tetrachloride with
rat liver homogenate and looked for the formation of 2-oxothio-
zolidine-4-carboxylic acid. This compound is formed from the
reaction of L-cysteine and carbonyl chloride. Analysis of the
metabolic products by mass spectroscopy showed a fragmentation
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VI I-2
pattern consistent with 2-oxothiozolidine-4-carboxylic acid.
The authors inferred from these analytical data that carbonyl
chloride was formed as a metabolic product of carbon tetrachlo-
ride. Although carbonyl chloride (phosgene) is not reported
to be a carcinogen, the authors pointed out that the compound
is highly toxic and that the reactive chlorines could react
with macromolecules in ways similar to alkylating agents.
Dimerization to Hexachloroethane
Hexachloroethane has been identified as a metabolite
of carbon tetrachloride by Fowler (1969). The formation of this
compound is believed to take place by the dimerization of the
trichloromethyl radical. Although hexachloroethane is a hepato-
toxin, its toxicity is less than that seen in carbon tetrahloride
poisoning. Therefore, other mechanisms probably account for the
severity of the toxicity associated with carbon tetrachloride.
Free Radical Binding to Proteins
Free radical binding to proteins had been postulated as
one cause of toxicity associated with carbon tetrachloride (Rech-
nagle and Glended, 1973). The binding was reported to involve
reactions with cellular proteins, particularly those with sulf-
hydryl groups. Experimental results have not confirmed this
theory of carbon tetrachloride toxicity, nor is it supported
by the pathological changes seen in the liver resulting from
carbon tetrachloride exposure. In one study, [14C] carbon
tetrachloride has been observed to bind irreversibly to rabbit
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VII-3
microsomal proteins at a rate of approximately 20 mole per mg
of protein per hour (Uehleke and Werner, 1975). Binding of
carbon tetrachloride (or its metabolites) to hepatic macromole-
cules was enhanced in the absence of oxygen, consistent with
the proposal that the trichloromethyl radical is the reactive
metabolite.
Although it has been shown that 14C from carbon tetra-
chloride binds to proteins, the question of carbon tetrachloride
binding to polynucleotides is still open. The question is impor-
tant because of its implications for the mechanism of carbon
tetrachloride carcinogenicity and mutagenicity. Rocchi et al.
(1973) examined the possible binding of carbon tetrachloride with
nucleic acids in Wistar rats and Swiss mice in vivo, and with
DNA and polynucleotides rn vitro. !4C-labelled carbon tetra-
chloride (367 umol/kg) binds jjn vivo to DNA of mouse liver and
to ribosomal RNA of rat liver if the animals have been pretreated
with 3-methylchloanthrene (MCA). The pretreatment with MCA
increases the amount of the binding due to a higher activity of
the microsomal system activating carbon tetrachloride. In_ vitro
carbon tetrachloride (0.218 umol) is activated by microsomes and
pH 5 enzymes of MBA-treated animals to a metabolite which can
react with DNA and polynucleotides. No binding of carbon tetra-
chloride metabolites to hepatic DNA from control mice or rats was
detected. On the other hand, Uehleke and Werner (1975) incubated
[14C] carbon tetrachloride with either isolated liver microsomes
(rat or mouse, species not identified) or with soluble RNA, they
-------
VII-4
observed no [14C] binding to ribosomal RNA or exogenous RNA.
Experimental details were not presented.
Diaz Gomez and Castro (1980) reported that 1*C from
carbon tetrachloride irreversibly binds in vivo to hepatic
nuclear DNA from strain A/J mice and Sprague-Dawley rats (Table
VII-1). Also binding of 14C from carbon tetrachloride to DNA
was observed in vitro in incubation mixtures containing micro—
somes and a NADPH generating system as well as in tissue slices
(Table VII-2) . Liver nuclear proteins (Table VII-3) and lipids
(Table VII-4) irreversibly bind carbon tetrachloride metabolites.
The authors concluded that: (a) the differences between the
results of Rocchi et al. (1973) (pretreatment with MCA required)
and theirs are possibly related to the use of different strains
of mice (Swiss vs A/J) and rats (Wistar vs Sprague-Dawley);
(b) the interaction of carbon tetrachloride metabolites with DNA
and nuclear proteins could be relevant to carbon tetrachloride
induced liver tumors and hepatoxic effects; and (c) the
epigenetic mechanisms for chemical induction of cancer, not
involving carbon tetrachloride-DNA interactions as shown in
their study, could also be relevant.
Lipid Peroxidation
A number of the hepatic effects resulting from carbon
tetrachloride exposure, including the fatty liver syndrome, are
believed to arise as a result of lipid peroxidation (Rechnagel
and Glende, 1973). The mechanism proposed for the peroxidation
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VII-5
TABLE VII-1
Covalent Binding of 14C from 14CC14
to Rat and Mouse Liver DNA In Vivo**
Species
pmol/mg + SD
14C from 14CC11 in DNAb
mol nucleotide/mol of CC14
metabolite (x
A/J mice
0.72 + 0.05
4.54
Sprague-Dawley
0.52 0.05
6.25
a 14CC14(27 mCi/mmol) was administered ip as an olive oil solu-
tion <2§ uCi/ml) at a dose of 10 ml of solution/kg bw. Animals
were sacrificed 6 hr after 14CC14 and DNA was isolated and coun
ted. For calculations it was 'considered that 1 mg of DNA con-
tained 3,237 umol of nucleotides.
b Results are the mean of 3 samples. Each sample was a pool
of 10 livers in the case of mice and one liver for rats.
Values for rats were significantly lower than those of mice
(p<.05).
Adapted from Diaz Gomez and Castro (1980)
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VI1-6
TABLE VIII-2 Covalent Binding of 14C from 14CC14 to
DNA In Vitro
14« From 14CCl/lb
DNA
DNA mol nucleotide/mol Of
Experimental pmol/mg +_ SD CC14 metabolite
Microsomal activation 1.81 ± 0.13 1.75 X 106
Microsomal activation 4302 £ 300 7.5 X 102
(33 mM CC14)
Chemical activation 826 + 250 3.84 X 103
aAnaerobic mixtures containing microsomes, NADPH, and mouse liver
DNA were incubated for 30 min at 370 °C with 10 ul of an ethanol
solution of I4CC14 (27 mCi/mmol) at a concentration of 13.63
uCi/ml. DNA was isolated and counted. In the second experiment
on the microsomal activation, the incubation system was as before
except that cold CC14 was added (final concentration, 33 mM)
and specific activity was 0.0137 mCi/mmol. When chemical activa-
tion was studied DNA/acetyltrimethyl ammonium bromide salts (3 mg)
were heated for 16 hr at 80 °C in a sealed vial under N? atmosphere
with 3 ml of an alcoholic solution of 14CC14 (1.82 uCi/ml) in the
presence of benzoyl peroxide. DNA was isolated and counted.
results are the mean of triplicate simultaneous experiments
Adapted from Diaz Gomez and Castro (1980).
-------
VII-7
TABLE VI1-3
Covalent Binding of 14C from 14CC14 to Nuclear
Protein Fractions From Rat Livera
Nuclear Protein Fraction
14C from 1
in protein10
(pmol/mg)
Percentage total
of label in each
fraction
I
II
III
IV
V
Histones
Residual
4.9
17.9
8.8
68.1
39.3
43.6
45.7
- 7.5
- 10.1
- 5.3
-52.6
- 28.5
- 56.8
- 63.5
18.25 -
29.73 -
7.76 -
12.14 -
6.55 -
7.13 -
18.44 -
19.95
23.61
5.33
8.23
4.85
14.07
23.96
a 14CC14 administration and time of treatment as in Table VII-1.
Isolated nuclear proteins were dissolved in formic acid and counted.
Fraction I and II correspond to nuclear sap proteins. Fraction III
and IV are deoxuribonucleoproteins, ° -'— TT n'~ *"** i-sv.™™r-i«»rw.
protein.
Fraction V is acid ribonucleo-
b Pooled livers from 4 rats were used in each experiment. These
results are 2 separate experiments .
Adapted from Diaz Gomez and Castro (1980).
-------
VI1-8
TABLE VI1-4 Covalent Binding of 14C from 14CC14 to
Different. Lipid Fraction From Rat Livera
Percentage of total label
Lipid Fraction in each fraction*3
Phospholipids 75.69-65.11
Diglycerides 16.73-24.07
Cholesterol esters 0.36 - 0.24
Triglycerides 0.22 - 0.37
Free fatty acids 1.19 - 0.64
Cholesterol 5.70 - 9.50
Undetermined or loss 0.11 - 0.07
a 14CC14 administration as in Table VII-1. Animals were sacrificed
3 hr after 14CC14 administration. Total lipid fractions were used
for counting. Covalent binding to total nuclear lipids was 113.5
pmol/mg.
k Pooled livers from 4 rats were used in each experiment. These
results are two separate experiments.
Adapted from Diaz Gomez and Castro (1980).
-------
VI I-9
is presented below, followed by a discussion of the evidence
that it's biochemical sequence results in the hepatic lesions
associated with carbon tetrachloride poisoning.
The first step in the reaction sequence proposed for
lipid peroxidation is the production of free radicals, especially
the trichloromethyl free radical. The radical initiates a chain
reaction by reacting with the hydrogen atom of a -CH2~group in an
unsaturated fatty acid, generating a fatty acid free radical. On
reaction with molecular oxygen, the fatty acid-free radical is
converted into an unstable organic peroxide. The peroxide disin-
tegrates in two fashions: (i) intramolecular cyclization to form
malonic dialdehyde and two new free radicals, or (ii) simple
homolytic fission that also yields two free radicals. This whole
process occurs autocatalytically: each free radical gives rise to
two new free radicals. Figure "VII-1 summarizes this hypothesis
(Rechnagel and Glende, 1973).
A number of indicators have been used in in vivo and in
vitro assays of lipid peroxidation: pentane and ethane levels in
exhaled air (arising from fatty acid decomposition) and- malonic
dialdehyde concentrations in hepatocytes (arising from intramolec-
ular cyclization). Pentane production in male rats increased by
factors of 4.6,. 13.2, and 26.4 over that in mineral oil controls
within 30 minutes following intraperitoneal administration of
carbon tetrachloride doses of 160, 430, and 1,440 mg/kg bw,
respectively (Sagai and Tappel, 1979).
-------
VII-10
A mechanism for the pathogenesis of^carbon tetrachlo—
rideinduced hepatic lesions based on lipid peroxidation has been
proposed (Pasquali-Ronchetti e_t ad., 1980). According to this
hypothesis, lipid peroxidation is suggested to affect primarily
unsaturated acyl chains of membrane phospholipids, resulting in
breakage of the hydrocarbon and loss of phospholipids from the
membrane. Lipid peroxidation would therefore produce progressive
degenerative changes in the assembly of membranous structures
such as microsomes and of (rat) liver endoplasmic reticulum.
This hypothesis is supported by studies showing that
treatment with carbon tetrachloride produced lipid peroxidation
in rat liver endoplasmic reticulum at a concentration of 0.5
ml/100 g bw (Pasquali-Ronchetti et a_l., 1980); caused disinte-
gration of endoplasmic reticulum in vitro within 10 minutes at
a concentration of 636 mg/liter (Pasquali-Ronchetti et al.,
1980); and was incorporated predominantly into liver phospho-
lipids in rats (Table VII-5) (Ciccoli e_t al_., 1978).
Two pieces of contrary evidence have been presented by
Diaz Gomez, e_t al. (1975). One was that the order of species
susceptibility to liver necrosis from carbon tetrachloride more
closely parallels the species order for [14C] carbon tetrachlo-
ride binding to cellular components than the species order for
lipid peroxidation:
-------
VII-11
H H H
c=c-c- c=c-c- c=o-oc=c-
H H H
HCC1- »CCi-Trichisraeehyl Fres
J Sadical
-c=c-c-c=c-c-c=c-c-oc-
R2S03ANCZ (All / * Organic Frse Radical
Possible Foras Not
Shown.)
J ft * * a fi .
- c=c-c-c-oc-c=c-c-c=c-
Peroxide v *N*
formation OjT diene coajuge.ian) ^_ »233cu
SS^C
-C=C-C-C-C-C-C=C-C-C=C-
V Organic Psroxide (Uasrabla)
H
\
Inrraaolectilar cyclizaclon Decanpasitioa co yield cvo free
and deconposirion to yield radicals. Evericuai stable decan-
nalonic dialdehyde and cvo posicion products highly organo-
new organic free radicals. leptic.
Figure VII-1. Free Radical Initiated, Autocatalytic
Peroxidation of Polyenoic Long-Chain
Fatty Acids
(Adapted from Recknagel and Glende 1973).
-------
VI1-12
Liver necrosis mouse> guinea pig.= hamster> rat> chicken
[ C]CC1^ binding mouse = hamster> guinea pig> chicken = mouse
Lipid peroxidation rat> hamster = guinea pig> chicken = mouse
A second result of their experiments was that for mice,
necrosis proceeded for 24 hours in the absence of lipid peroxida-
tion. Together, these pieces of evidence imply that caution is
required in accepting the lipid peroxidation mechanism for carbon
tetrachloride toxicity.
Summary
The metabolism of carbon tetrachloride is thought to
proceed through the formation of the trichloromethyl free radi-
cal. The hepatotoxicity of carbon tetrachloride has been attri-
buted to subsequent lipid peroxidation initiated by this radical
in the following manner: a hydrogen atom is abstracted by the
free trichloromethly radical from a long-chain fatty acid to form
chloroform and a. fatty acid-free radical. Molecular oxygen,
because of its triplet ground state, binds with the unparied
electron on the fatty acid radical to form an organic peroxide.
The peroxide is unstable and decomposes to form more organic-
free radicals, which in turn form more organic peroxides. This
process appears to lead to fatty acid chain decomposition, with
the resulting breakdown of membrane structure. This breakdown
may lead to a halt in lipid excretion via the Golgi apparatus,
with fatty liver occurring as a consequence. Cell necrosis would
also follow directly from lipid destruction. The mechanism by
-------
VII-13
which lipid peroxidation could lead to cell transformation is
not explained yet, and the molecular events leading to carbon
tetrachloride careinogenicity remain unknown.
In addition to this proposed lipid peroxidation
mechanism, which produces chloroform, two minor metabolic path-
ways have been; postulated: dimerization to two trichloromethyl
free radicals to form hexachloroethane and the formation of a
trichloromethyl peroxy radical which may result in production
of phosgene 'and carbon dioxide. Both hexachloroethane and
phosgene are toxic, but the extent of their contribution to
observed hepatotoxicity is unknown. A fourth possible
mechanism of hepatotoxicity is the binding of trichloromethyl
radical to cellular proteins.
-------
VIII. RISK ASSESSMENT
The possibility that carbon tetrachloride is carcino-
genic in humans has been a subject of concern, and has resulted
in the development of several human risk assessments. This
section of the report summarizes carcinogenic risk assessments
prepared by the National Academy of Sciences (NAS) and the U.S.
Environmental Protection Agency (EPA).
Current methods used to estimate carcinogenic risk
have in common the assumption that there is no threshold level
for the action of a carcinogen. The state-of-the-art and data
now available are such that no one method can accurarely predict
and/or model the absolute numbers of excess cancer deaths attri-
butable to carbon tetrachloride in drinking water. Because of
biological variability and the assumptions used, each of the
methods used to quantify carcinogenic risk leads to a different
value. The estimates and their interpretation may vary widely.
In addition, none of the method's now used to quantify carcino-
genic risk can account for the increased risk of carbon tetra-
chloride exposure to sensitive populations.
Quantification of Carcinogenic Risk
Because of positive results in animal carcinogenicity
studies, carbon tetrachloride can be considered a suspect human
carcinogen. Data from these animal studies have been used by
the NAS and EPA's Carcinogen Assessment Group (CAG) to calcu-
late the number of additional cancer cases that may occur when
-------
VIII-2
carbon tetrachloride is consumed in drinking water over a 70-
year lifetime. The results of these calculations are shown in
Table VIII-1.
The criteria for the CAG (USEPA 1980c, 1983) and NAS
(1977) risk calculations differ in several respects: (1) NAS
used the multistage model, while CAG used an "improved" multi-
stage model. (2) NAS used the data set from the National Cancer
Institute (NCI) negative study in male rats while CAG used the
data set from NCI's positive study in male mice. Because of
these differences, the carbon tetrachloride levels reported by
CAG for cancer risk levels are approximately 1/10th those
reported by NAS.
The U.S. Environmental Protection Agency has also
developed Ambient Water Quality Criteria based on estimates
of the increased lifetime cancer risk resulting from a life-
time consumption of both drinking water (2 liters per day) and
aquatic life (6.5 g of fish and shellfish per day). These risk
estimates differ from those in the previous risk assessments,
which are based on consumption of drinking water alone. These
criteria for lifetime cancer risks of 10~5, and 10~6, and 10~7
are 4.0, 0.40, and 0.04 ug/liter, respectively (USEPA 1980a).
Sensitive Populations
Sensitive populations are subgroups within the general
population which appear at higher than average risk upon exposure
to carbon tetrachloride. Some of the populations that may be
-------
VIII-3
Table VIII-1
Estimat.es of Additional Carcinogenic Risk
Following Exposure of Humans to Carbon Tetra-
chloride in Drinking Watera
Carbon tetrachloride concentrations
(ug/liter)
Excess cancer
risk/lifetime
GAG
(upper 95%
confidence
limit)
NAS
(upper 95%
confidence
limit)
NAS
(point estimate)
(95% confidence
range)
10-4
10-5
icr6
42.2
4.2
0.4
450
45
4.5
1100-900
110-90
11-9
aAn average daily drinking water consumption of 2 liters per day
was assumed.
at greater risk include human fetuses, alcohol consumers, and
males of reproductive age. This section will deal with the
possible effects of age, sex, and nutritional status on toxicity
of carbon tetrachloride.
The studies of Reuber and Glover (1968) with rats
suggest that sensitivity to the effects of carbon tetrachloride
may vary with age and sex. In these studies, inbred Buffalo
rats 4, 12, 24, or 52 weeks of age were injected subcutaneously
twice weekly for 12 weeks with carbon tetrachloride (1000 mg/kg
bw in corn oil). Cirrhosis of the liver following exposure
increased with age in male rats, whereas female sensitivity
-------
VIII-4
increased up to 24 weeks and declined thereafter. Female
sensitivity exceeded male sensitivity in the 4- and 12-week
groups; this pattern reversed after 24 weeks.
Nutritional status may also affect the degree of
toxicity following exposure to carbon tetrachloride in rats.
Gyorgy et al. (1946) exposed young rats on various diets to
approximately 300 ppm of carbon tetrachloride in a gas chamber
7 hours per day, 5 days per week for 5 months. Animals were
then sacrificed and histopathological effects on the liver and
kidneys were determined. Compared to these signs of toxicity
in animals fed standard chow, these effects were more severe
in animals fed a diet high in lipid and low in carbohydrate,
or a diet low in protein. Methionine appeared to protect
against the increased toxicity (particularly kidney damage)
caused by low-protein diets.
Interaction of Carbon Tetrachloride with Other Chemicals
The interaction of carbon tetrachloride with certain
chemicals has resulted in an enhancement of the toxic effects
produced in animals by either chemical alone. Exposure of
animals to certain environmental carcinogens in combination with
carbon tetrachloride has resulted in an increase in carcinogenic
efficacy. In. addition, certain chemicals appear to increase the
toxic effects of carbon tetrachloride on the liver and other
organs of experimental animals. There is also clinical evidence
that two of these chemicals, isopropanol and ethanol, may poten-
tiate carbon tetcachloride toxicity in humans (see Section VII).
-------
VIII-5
Carcinogenic Effects. The interaction of carbon tetra-
chloride with several carcinogens has been studied. The effects
of carbon tetrachloride on dimethylnitrosamine-induced carcino-
genicity were studied in male Sprague-Dawley rats treated with a
single dose of carbon tetrachloride (4,000 mg/kg bw) by gavage
42 or 60 hours prior to a single intraperitoneal dose of dimethyl-
nitrosamine (20 or 40 mg/kg bw). This treatment resulted in a
greater than additive increase in incidence of tumors or tumor-
like lesions of the liver and kidney at 12 months as compared to
rats treated with carbon tetrachloride or dimethylnitrosaraine
along (Pound e_t al.r 1973). If the pretreatment took place more
than 60 hours before injection of dimethylnitrosamine, the
incidence of kidney neoplasms decreased, while that of liver
neoplasms increased further.
Similar results were- obtained with N-butylnitrosurea
(Takizawa et al., 1975). Experimental male ICR/JCL mice were
treated with 80 mg of carbon tetrachloride subcutaneously 1 day
before administration of 10 or 20 mg of N-butylnitrosurea in 50%
ethanol by gavage. This treatment resulted in the induction of
hepatomas after 15 months in 12 of 23^mTce"(dose groups combined)
as compared to 1 of 18 mice given N-butylnitrosurea but not car-
bon tetrachloride. However, since the ethanol vehicle was not
given to all'controls receiving carbon tetrachloride alone, the
possible ethanol potentiation of carbon tetrachloride effects
could not be fully evaluated (see the following discussion of
ethanol effects on carbon tetrachloride toxicity).
-------
VIII-6
The effects of carbon tetrachloride on the carcinogeni-
city of 2,7-bis-(acetoamido)fluorene (2,7-AAF) were also studied.
Carbon tetrachloride (1,4000 mg in corn oil) was given by gavage
once weekly for 8 weeks to male SMA/Ms mice fed chow containing
0.025% of 2,7-AAF. A greater than additive effect on the incidence
of hepatomas was observed in these animals as compared to controls,
which received carbon tetrachloride or 2,7-AAF for 8 weeks, or
carbon tetrachloride for one 8-week period and 2,7-AAF for another
8-233k period (Kozuka and Sassa, 1976).
Toxicologic Effects. Many chemicals have been reported
to potentiate noncarcinogenic effects of carbon tetrachloride.
Hewitt et al. (1980"t, in their review of studies on potentiation
of hepatotoxic effects of carbon tetrachloride by various agents,
have postulated that ketones (e.g., acetone, methyl ethyl ketone)
or chemicals that can be metabolized to ketones (e.g., isopropa-
nol, 2-butanol, 1,3-butanediol) potentiate the effects of carbon
tetrachloride and other haloalkanes.
Traiger and Plaa (1971) administered isopropanol (2,000
mg/kg bw by gavage) to male Sprague-Dawley rats 18 hours before
intraperitoneal injection with carbon tetrachloride (160 mg/kg
bw in corn oil). The SGPT activity of these animals was 22 times
higher than that in controls receiving carbon tetrachloride alone.
In addition, SGPT activities in animals receiving isopropanol and
carbon tetrachloride at 160 mg/kg bw were higher than those in
animals receiving carbon tetrachloride at 1,600 mg/kg bw without
isopropanol.
-------
VIII-7
Later studies on isopropanol potentiation (reviewed by
Hewitt et al., 1980) using an inhibitor of isopropanol metabolism
demonstrated that potentiation probably depended on the metabolism
of isopropanol to acetone. When the blood concentration of iso-
propanol was kept high and that of acetone was low, the potenti-
ating capacity of isopropanol was significantly reduced. Acetone
administered directly (2,000 mg/kg bw) potentiated the hepato-
toxic effects of carbon tetrachloride (160 mg/kg bw) given 18
hours later. Therefore, acetone rather than isopropanol appeared
to be responsible for the potentiation observed.
Isopropanol was also implicated as the potentiating
factor in carbon tetrachloride toxicity (renal and hepatic)
in humans after accidental exposure to carbon tetrachloride
in an isopropanol packaging plan (Folland et al., 1976). Ele-
vated levels of acetone were found in samples of the expired
air of these workers.
Traiger and Bruckner (1976) studied the effects of 2-
butanol and its ketone metabolite 2-butone (methyl ethyl ketone)
on the potentiation of carbon tetrachloride hepatotoxicity.
2-Butanol (1,800 mg/kg bw) or 2-butanone (1,500 mg/kg bw) were
administered orally to male Sprague-Dawley rats 16 hours prior
to intraperitoneal injection of carbon tetrachloride (160 mg/kg
bw in corn oil) . SGPT activity 24 hours after carbon tetrachlo-
ride exposure was 30-fold higher in both groups than in controls
given carbon tetrachloride alone. Glucose-6-phosphatase acti-
vity decreased and triglyceride levels increasd in experimental
-------
VIII-8
animals as compared to controls. 2-Butanol had an apparent
oral half-life of 2.5 hours; by the time carbon tetrachloride
was administered, most of the 2-butanol present had been
metabolized to 2-butanone or eliminated. The dose of 2-butanone
used approximated the level of 2-butanone in the blood after
administration of 2-butanol. Because the effect of both treat-
ments were similar, the authors proposed that 2-butanone was
responsible for carbon tetrachloride potentiation in both
treatments. Therefore, a similar mechanism was implicated
for 2-butanol as for isopropanoli metabolism to the ketone.
Administration of 1,3-butanediol to rats has been
associated with a rapid rise in the blood concentration of ketone
bodies. This compound was therefore used to test the hypothesis
that matabolic ketosis results in an increase in carbon tetra-
chloride hepatotoxicity (Hewitt and Plaa, 1979). Male Sprague-
Dawley rats received 1,3-butariediol (5 g/kg bw) three times daily
for 3 days. On day 3, the rats received an intraperiotoneal dose
of carbon tetrachloride (160 mg/kg bw in corn oil), and were
sacrificed 24 hours later. SGPT activity in experimental animals
incrased 18-fold over that in controls receiving carbon tetra-
chloride alone, while hepatic glucose-6-phosphatase activity
in experimental animals decreased compared to controls. These
results, therefore, also implicated ketone's in the potenti-
ation of carbon tetrachloride toxicity.
-------
VIII-9
Several other chemicals that potentiate carbon tetra-
chloride toxicity, although complex in structure, also contain
ketone groups (i.e., phenobarbital, pentobarbital, triamcinolone,
progesterone, and Kepone). Phenobarbital has been demonstrated
to markedly enhance carbon tetrachloride liver damage in mice and
rats. Cans et al. (1976) detected an increase in liver weight,
protein synthesis, and DMA synthesis in male Swiss mice given both
phenobarbital and carbon tetrachloride as compared to those given
either compound alone. Tuchweber and Kovacs (1971) found that
pretreatment of female ARS/Sprague-Dawley rats with phenobarbital
resulted in increased mortality, liver damage, and triglyceride
accumulation after carbon tetrachloride exposure as compared to
animals given carbon tetrachloride alone. Pani et al. (1973)
also demonstrated an increase in mortality of male Wistar rats
given phenobarbital in conjunction with carbon tetrachloride as
compared to those given carbon tetrachloride alone. In addition,
serum enzyme activities were increased in animals given both
phenobarbital and carbon tetrachloride compared to those given
only carbon tetrachloride. Lindstrom and Anders (1978) detected
an increase in diene conjugation in hepatic microsomal lipids in
male Sprague-Dawley rats after administration of phenobarbital
and carbon tetrachloride compared to that in controls receiving
carbon tetrachloride or phenobarbital alone. In further studies,
isolated rat hepatocytes were prepared from male Sprague-Dawley
rats that had been treated with phenobarbital; control hepatocytes
were prepared from untreated rats (Lindstrom et al., 1978). In
cultures containing carbon tetrachloride, release of lactic
-------
VIII-10
dehydrogenase was potentiated in hepatocytes from treated animals
compared to controls. Chang-Tsui and Ho (1980), in an experiment
with pentobarbital, demonstrated potentiation of carbon tetrachlo-
ride toxicity in male ICR mice with increases in SGPT and SGOT
following carbon tetrachloride exposure.
Triamcinolone and progesterone have also been shown to
potentiate carbon tetrachloride hepatotoxicity (Tuchweber and
Kovacs 1971)» Female ARS/Sprague-Dawley rats were treated orally
twice daily with 5 mg of progesterone or 2 mg of triamcinolone.
After 4 days, the animals received carbon tetrachloride at either
16 or 4,000 ing/kg bw. These treatments resulted in increases in
mortality, liver damage, and triglyceride accumulation in compar-
ison to controls given only carbon tetrachloride.
Another Tcetone, Kepone, also appears to potentiate the
hepatotoxic effects of carbon tetrachloride (Curtis et a_l., 1979).
After being fed chow containing 0-10 ppm of Kepone for 15 days,
male Sprague-Dawley rats received a single intraperitoneal dose
of carbon tetrachloride (0-320 mg/kg bw in corn oil). Compared
to controls given carbon tetrachloride but no Kepone, Kepone-fed
animals showed increased impairment of biliary excretion at carbon
tetrachloride doses of 80 mg/kg bw or above, elevated SGPT and
SGOT (carbon tetrachloride doses of 160 mg/kg bw and above), and
morphological changes in the (carbon tetrachloride doses of SO
mg/kg bw and higher). Some animals in these studies were also
exposed to pentobarbital during anesthesia and possibly to acetone
residue in the food, since acetone was used as a vehicle for
-------
VIII-11
Kepone. The possible effects of these exposures on the results
could not be determined. Hewitt et_ _al. (1980) reported that
Kepone potentiated the toxic effects of chloroform, a compound
similar to carbon tetrachloride (i.e., 1 a haloalkane3.
In addition to the alcohols previously discussed,
ethanol and methanol have been shown to potentiate the effects
of carbon tetrachloride. In one study, hepatotoxic effects of
carbon tetrachloride were potentiated by ethanol and methanol in
male Swiss-Webster mice and by ethanol in male Sprague-Dawley
rats (Traiger and Plaa, 1971). SGPT activity was increased in
mice exposed to both ethanol and carbon tetrachloride compared
to controls receiving carbon tetrachloride alone. Rats treated
with both ethanol and carbon tetrachloride also showed increased
levels of hepatic triglycerides and decreased activity of hepatic
glucose-6-phosphatase. Cornish and Adefuin (1966) found that a
single oral dose of ethanol followed by exposure to carbon tetra-
chloride vapors resulted in increased levels of SGPT, SCOT, and
serum isocitric dehydrogenase in male Sprague-Dawley rats. Con-
trol animals receiving carbon tetrachloride or ethanol alone had
lower levels of these enzymes, although some effect occurred with
carbon tetrachloride alone. Maling, et a_l. (1975) also found an
increase in SGPT activity in male Sprague-Dawley rats treated both
with ethanol and carbon tetrachloride compared to rats treated
with carbon tetrachloride alone. Increases were also noted in
liver triglycerides, hepatic necrosis, and the covalent binding
of carbon tetrachloride to liver protein and lipid.
-------
VIII-12
Strubelt et al. (1978) also reported increases in
serum enzyme activities and changes in liver morphology in male
Wistar rats exposed to ethanol plus carbon tetrachloride compared
to controls given either chemical alone. Cantilena et al. (1979)
demonstrated the methanol potentiation of carbon tetrachloride
hepatoxicity in male Sprague—Darley rats. Plasma alanine amino-
transferase activity and hepatic triglyceride levels were in-
creased and hepatic glucose-6-phosphotase activity was decreased
compared to controls given carbon tetrachloride alone.
Ethanol and methanol have been judged to be less effec-
tive than isopropanol in potentiating the toxic effects of carbon
tetrachloride in mice and rats (Traiger and Plaa, 1971) . In
humans, the effects of potentiation of ethanol and methanol on
carbon tetrachloride toxicity have not been quantified. Never-
theless, case reports appear to indicate ethanol potentiating
effects in humans qualitatively similar to those in animals.
Another class of chemicals which have been reported to
interact with carbon tetrachloride to produce toxic effects is
polychlorinated biphenyls. Carlson (1975) reported the potenti-
ation of carbon tetrachloride hepatotoxicity in male albino rats
by the polychlorinated biphenyls Arochlor 1254, 1221, and 1260.
The animals received intraperitoneal injections of one of these
compounds in corn oil for 6 days. Twenty-four hours after the
last injection, the rats were exposed to carbon tetrachloride
vapors (3,700-26,400 mg/m3; 590-4,200 ppm) for 2 hours. Changes
-------
VI11-13
were demonstrated in various serum and liver enzyme activities
after these treatments as compared to activities after carbon
tetrachloride treatment or Arochlor treatment alone.
Summary
Carbon tetrachloride is a known animal carcinogen and
a suspect human carcinogen. The risk of cancer from human expo-
sure to carbon tetrachloride in drinking water has been estimated
by the NAS and the CAG. These quantification efforts differed
in methodology and in animal data. used. An excess cancer risk
of 10~5 over a 70-year lifetime would result from exposure to
drinking water containing tetrachloride at 4.2 ug/liter in the
CAG estimate or 45 ug/liter in the NAS estimate.
In addition to the quantitative estimation of risk, two
factors that can be considered in evaluating risk of exposure to
a chemical are the variation in sensitivity among populations and
the interaction of the chemical with others. In the case of
carbon tetrachloride, studies in rats have suggested that young
females (less than 12 weeks old) are more sensitive to the
chemical's cirrhotic effects, with the sensitivity pattern revers-
ing at older ages. Furthermore, dietary changes (high-lipid/
low-carbohydrate diets, or low protein diets) were reported to
render rats more sensitive to liver and kidney damage following
carbon tetrachloride exposure. These conclusions can be only
tentative.
Greater than additive toxicity resulting from exposure
to carbon tetrachloride concomitantly with other chemicals has
-------
VIII-14
been reported in many animal studies. The interactions have
been of two forms.
1) Enhanced carcinogencity when carbon tetrachloride
was given before a carcinogen. Greater than additive numbers
of liver and kidney tumors were seen when dimethylnitrosamine,
n-butylurea, or 2-AAF was given after carbon tetrachloride.
2) Enhanced hepatotoxic effects when carbon tetra-
chloride was given along with a second chemical/ usually a
ketone or an alcohol that is metabolized to a ketone. Poten-
tiating effects on carbon tetrachloride toxicity have been
best documented for isopropanol and butanol. Kinetic and
metabolic inhibition studies have been consistent with the
hypothesis that these alcohols must be metabolized to ketones
(acetone and methyl ethyl ketone, respectively) before they
exert their potentiating effects. Among the other alcohols
reported to potentiate carbon tetrachloride toxicity are 1,3-
butanediol (which is associated with a rapid rise in ketone
bodies), ethanol, and methanol. In addition, potentiating
effects have been reported for several chemicals with ketone
groups (phenobarbital, pentobarbital, triamcinolone,
progesterone, and Kepone), and for several polychlorinated
biphenyls.
-------
IX. QUANTIFICATION OF TOXICOLOGICAL EFFECTS OF CARBON TETRACHLORIDE
The quantification of toxicological effects of a chemical
consists of an assessment of the noncarcinogenic and carcinogenic
effects. In the quantification of noncarcinogenic effects,
an Acceptable Daily Intake (ADI) is calculated. From this an
Adjusted Acceptable Daily Intake (AADI) and Health Advisory
(HA) values for the chemical are calculated to define the
appropriate drinking water concentrations to limit human
exposure. For ingestion data, this approach is illustrated
as follows:
ADI = (NOAEL or LOAEL in mg/kg/day) (Body weight in kg) = mg/day
Uncertainty/Safety factor
AADI » ADI = mg/L
Drinking water volume in L/day
where:
NOAEL = no-observed-adverse-effeet level.
LOAEL = lowest-obvserved-adverse-effeet level.
Body weight = 70 kg for adult or 10 kg for child.
Drinking Water volume = 2 L per day for adults or 1 L
per day for children.
Uncertainty/Safety factor = 10, 100 or 1,000.
Utilizing these equations, the following drinking water
concentrations are developed for noncarcinogenic effects:
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IX-2
1. A one-day HA for 10-kg child.
2. A one-day HA for 70-kg adult.
3. A ten-day HA for 10-kg child.
4. A ten-day HA for 70-kg adult.
5. A lifetime AADI for a 70-kg adult.
The distinctions made between the HA calculations (items
1 through 4) are associated with the duration of anticipated
exposure. Items 1 and 2 assume a single acute exposure to
the chemical. Items 3 and 4 assume a limited period of
exposure (possibly 1 to 2 weeks). The HA values will not be
used in establishing a drinking water standard for the
chemical. Rather, they will be used as informal scientific
guidance to municipalities and other organizations when
emergency spills or contamination situations occur. The AADI
value (item 5} is intended to provide the scientific basis
for establishing a drinking water standard based upon
noncarcinogenic effects.
A NOAEL or LOAEL is determined from animal toxicity data
or human effects data. For animal data, this level is divided
by an uncertainty factor because there is no universally
acceptable quantitative method to extrapolate from animals to
humans. The possibility must be- considered that humans are
more sensitive to the toxic effects of chemicals than are
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IX-3
animals. For human data, an uncertainty factor is also used
to account for the heterogeneity of the human population in
which persons exhibit differing sensitivity to toxic chemicals.
A modification of the guidelines set forth by the National
Academy of Sciences (NAS 1977, 1980) are used in establishing
uncertainty factors as follows:
o An uncertainty factor of 10 is used when good acute- or
chronic human exposure data are available and supported by
acute or chronic toxicity data in other species.
o An uncertainty factor of 100 is used when good acute or
chronic toxicity data identifying NOEL/NOAEL are available
for one or more species, but human data are not available.
o An uncertainty factor of 1,000 is used when limited or
incomplete acute or chronic toxicity data in all species are
available or when the acute or chronic toxicity data identify
a LOAEL (but not NOEL/NOAEL) for one or more species, but
human data are not available.
If toxicological evidence requires the chemical to be
classified as a potential carcinogen (i.e., carbon tetrachloride),
mathematical models are used to calculate the estimated excess
cancer risks associated with the ingestion of the chemical
via drinking water. The bioassay data used in these estimates
are from animal experiments. In order to predict the risk
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IX-4
for humans from these data, it must be converted to an
equivalent human dose. This conversion includes correction
for non-continuous animal feeding, non-lifetime studies and
for the difference in size. The factor that compensates for
the size difference is the cube root of the ratio of the
animal and human body weights. It is assumed that the average
human body weight is 70 kg and that the average human consumes
2 liters of water per day. The multistage model is then fit
to the equivalent human data to estimate the risk at low
doses. The upper 95% confidence limit of this estimate is
used. Excess cancer risks can also be estimated using other
models such as the one-hit model, the Weibull model, the
logit model and the probit model. There is no basis in the
current understanding of the biological mechanisms involved
in cancer to choose among these models. The esitmates of
low doses for these models can differ by several orders of
magnitude. The multistage model does not necessarily give
the highest or lowest risk estimates at low doses. Whether-'
it is the most conservative, least conservative or predicts a
risk in the middle of the range of risks predicted by other
'models is chemical specific. The multistage model is used
because CAG and NAS use it.
The scientific data base used to calculate and support
the setting of risk rate levels has an inherent uncertainty.
This is because the tools of scientific measurement, by their
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IX-5
very nature,- involve both systematic and random error. In
most cases, only studies using experimental animals have been
performed. There is thus uncertainty when the data are
extrapolated to humans. When developing risk rate levels,
several other areas of uncertainty exist, such as (1) incomplete
knowledge concerning the health effects of contaminants
in drinking water, (2) the impact of test animal age, sex and
species and the nature of target organ systems examined on
the toxicity study results and (3) the actual rate of exposure
of internal targets in test animals or humans. Dose-response
data are usually only available for high levels of exposure,
not for the lower levels of exposure for which a standard is
being set. When there is exposure to more than one contaminant,
additional uncertainty results from a lack of information
about possible synergistic or antagonistic effects.
A. Noncarcinogenic Effects
Varying degrees of carbon tetrachloride (CC14)-induced
toxicity have been reported in humans and animals following acute
and chronic exposures via ingestion, inhalation or dermal admini-
stration. The following paragraphs discuss the results of perti-
nent studies to be considered for the derivation of the RMCL at
a later date.
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IX-6
Effects of acute exposure to low levels of CCl$ in rats
were reported by Korsrud ejt a_l. (1972). Male rats (260-400 g)
were administreed single oral doses of CC14 (0 to 4,000 mg/kg bw)
in corn oil. The rats were fasted for 6 hours before dosing and
for 18 hours afterward, and then sacrificed. Assays included
liver weight and fat content, serum urea and arginine levels, and
levels of nine serum enzymes, produced mainly in the liver. At
20 mg/kg bw, there was histopathologic evidence of toxic effects
on the liver. At 40 mg/kg bw, liver fat, liver weight, serum
urea, serum arginine, and levels of six of the nine liver enzymes
were increased. At higher doses the remaining three enzyme levels
were also elevated. The histologic changes seen at the minimum
effect level, 20 mg/kg bw, included a loss of basophilic stippling,
a few swollen cells, and minimal cytoplasmic vacuolation. This
study was used by EPA's Office -of Drinking Water to derive the
existing one-day and ten-day health advisories for CC14 (USEPA,
1981d).
Murphy and Malley (1969) investigated the effects of
single oral doses of CC14 on the corticosterone-inducible liver
enzymes, tyrosine-«c-ketoglutarate transaminase, alkaline phos-
phatase, and tryptophan pyrrolase in rats. Specifically, groups
of 4-7 male rats were administered by gavage 400, 800, 1600, 2400,
or 3200 mg/kg undiluted CC14. Single doses of 400 mg/kg or greater
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IX-7
of CC14 increased liver tyrosine-^c-ketoglutarate transaminase
acid and alkaline phosphatase, but not tryptophan pyrrolase
activity within 5 hours. The National Academy of Sciences
(NAS) used this experiment to calculate one-day and seven-day
suggested no-adverse-response levels (SNARLs) for CC14
(NAS, 1980).
In a chronic oral exposure study (Alumot et al., 1976),
groups of 36 rats (18 males and 18 females) were fed
mash containing CC14 at 0, 80, or 200 mg/kg of feed. The
authors calculated that the 200 mg/kg of feed represented a
daily dose of 10-18 mg/kg bw. After 2 years, the surviving
animals were sacrificed. Serum values for glucose, protein,
albumin, urea, uric acid, cholesterol, SCOT, and SGPT in the
treated animals did not differ from those in controls. No
fatty livers were detected in the treated animals. The
authors found no biochemical changes attributable to CC14
exposure. However, interpretation of the results was compli-
cated by the widespread incidence of chronic respiratory
disease in the animals. At 18 months, the survival ranged
from 61-89%, and more than half the animals were dead at 21
months. Although the authors indicated that 10-18 mg/kg bw
(200 mg/kg of feed) is a no-adverse-effect level of CC14
over 2 years, this conclusion may be questioned because of
the poor survival and chronic respiratory infection of
experimental animals.
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rx-a
Recently, Bruckner et al. (manuscript in preparation)
investigated the oral toxicity of CC14 in male Sprague
Dawley rats. In Study I, rats weighing 300-350 g were randomly
divided into groups of 5 animals each. The animals were
administered by gavage 0, 20, 40, or 80 mg CCl4/kg bw (in
corn oil) daily for 5 days, allowed 2 day without dosing,
and then dosed once daily for 4 additional days. One group
of animals at each dosage level was sacrificed at 1, 4, and
11 days following the initiation of the dosing. The following
observations were made: (1) One-day treatment: At 20 and
40 mg/kg, there were no significant changes in blood urea
nitrogen (BUN), glutamic-pyruvic transaminase (GPT) activity,
sorbitol dehydrogenase (SDH) activity, ornithine-carbamy1
tranferase (OCT) activity or histopathological changes in
the liver or kidneys. At 80 mg/kg, increased GPT (p<0.05)
activity was observed. Furthermore, there was vacuolization
of centrilobular cells adjacent to the central vein in each
liver. (2) Four-day treatment: Animals exposed to 20 mg/kg
did not exhibit any significant alterations in enzymatic
activities. At 40 and 80 mg/kg, there was an increase (p<0.05)
in SDH activity. GPT activity was elevated (p<0.05) in rats
given 80 mg/kg. Centrilobular vacuolization was noted in
groups receiving 20 and 40 mg/kg. Midzonal vacuolization was
observed in rats exposed to 80 mg/kg. (3) Eleven-day
treatment: The lowest dose (20 mg/kg) produced an increase
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IX-9
(p<0.01) in SDH activity whereas 40 and 80 mg/kg increased
(p<0.05 and <0.001) serum levels of all three enzymes. The
extent of morphological changes in the liver was dose-related.
In Study II, rats weighing 200-250 g were randomly
divided into groups of 15 to 16 animals each. The animals were
given by gavage 0, 1, 10, or 33 mg CCl4/kg bw (in corn oil). The
animals were dosed on a daily basis, 5 times weekly, for a total
period of 12 weeks. Blood samples were obtained from alternate
animals at the following intervals: 2, 4, 6, 8, 10, and 12 weeks
post-treatment. The serum was analyzed for BUN, GPT, SDH and
OCT. At 1 mg/kg, there were no significant biochemical/histo-
pathological changes at any time during the study. SDH, the
most sensitive index of hepatotoxicity, was elevated (p<0.05)
in rats receiving 10 mg/kg throughout the study. Also,
these rats exhibited mild hepatic centrilobular vacuoli-
zation. At 33 mg/kg, levels of GPT, SDH and OCT were markedly
increased (p<0.01) and severe hepatic lesions were apparent.
Prominent fibrosis, bile duct hyperplasia, and hepatocellular
vacuolization were seen in the portal and periportal regions
of hepatic lobules. Nuclear pleomorphic and severe cytoplasmic
degenerative changes were commonly present in mid- and centrilobular
hepatocytes. There was no evidence that CC14 was nephrotoxic.
Studies I and II will be used for the derivation of one-day
and ten-day Health Advisories; and lifetime AADI.
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IX-10
A cross-sectional epidemiclogic study (Sonich et al.,
unpublished) examined health effects of CC14 ingestion in humans.
Seventy tons of CC14 were spilled in the Kanawha and Ohio Rivers
in 1977. Measurements of raw water revealed maximum concentra-
tions of 0.340 mg/1. Twenty-one cities situated along the river
were involved in the study. These cities represented areas that
obtained their drinking water directly from the river and/or
areas that obtained their drinking water from sources not in-
fluenced by the quality of the river water. By using river
volumes and flow rates, periods of high exposure (1977) and low
exposure (1976) to CC14 were estimated -for each city along the
river. The results of routine tests measuring serum chemistries
reflecting liver and kidney function along with basic epidemiologic
information were abstracted from approximated 6,000 medical records,
The results obtained for serum creatinine show a positive and
statistically significant (p<0.65) relationship between the CC14
exposure and the frequency of elevated levels of serum creatinine
in exposed patients. No similar results were found for the other
parameters analyzed.
Stewart ejt al. (1961) reported the toxic effects of
experimental exposure of human volunteers to CC14 vapor. Healthy
males, 30-59 years of age, were exposed to concentrations of 63,
69, and 309 mg/m3 of CC14 (99% pure) in an exposure chamber for
180 minutes at the two lower doses or 70 minutes at the highest
dose. All subjects had undergone periodic physical examinations;
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IX-11
some participated in more than one of the exposure experiments,
which were conducted more than 4 weeks apart. Six subjects
exposed to the highest concentration experienced no nausea or
light-headedness, and CC14 was not detected in blood and urine
during or after exposure. One of these six subjects had an
increased level of urinary urobilinogen 7 days after exposure.
In addition, two of four subjects exposed to the highest concen-
tration and monitored for serum iron showed a decrease within 48
hours after exposure. CC14 was also not detected in the blood
or urine of volunteers exposed at 63 or 69 rng/m-^, and the
volunteers reported no physiologic effects. No changes in blood
pressure, serum transaminase levels, or urinary urobilinogen
levels were noted.
B. Quantification of Noncarcinogenic Effects
Korsrud et_ al. (1972) reported that the lowest acute
oral dose of CC14 inducing an adverse effect in rats was 20
mg/kg bw (lowest-observed-adverse-effect level) (Korsrud et
al., 1972). The length of this study was only 18 hours. Alumot
et al. (1976) proposed that 10 mg CCl4/kg is the acceptable
daily intake or no-adverse-effect-level for rats of both sexes
fed CC14 for a period of 2 years. However, as discussed earlier
this study could not be considered at this time due to high
morbidity/mortality rate among experimental rats and their
respective controls. It is noteworthy that the results for the
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IX-12
first year of this study was used by ECAO-Cin. (USEPA, 1982a)
to derive the ambient water level for CC14 of 3.3 mg/1 for pro-
tection against CCl4~induced noncarcinogenic effects. It should
be noted that values for consumption of contaminated water/fish
(2 I/day and 0.0065 kg/day) and bioconcentration factor for CC14
(18.75 I/kg) were taken into consideration during the calculation
of this protective level. The corresponding "drinking water
only" value is 3.5 mg/1.
EPA's existing health advisories andi NAS' SNARLs for
CC14 could be summarized as follows:
EPA-health advisory ' - NAS-SNARL
One-day 0.2 mg/1 14 mg/1
Seven-day - 2 ^g/1
Ten-day 0.02 mg/1
Longer-term None3 None0
a EPA did not calculate a longer-term health advisory for
CC14 due to lack of acceptable chronic exposure data.
.b NAS did not determine a longer-term SNARL for CC14
because this chemical is a carcinogen in animals.
The assessment of human health risks, that is, the
likelihood of certain adverse effects from given exposure
scenarios, is hampered by the paucity of good dose-response
data in humans. A human no-observed-adverse-effect level
(NOAEL) for oral ingestion was reported as 0.2 mg/day ( ss 0.1
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IX-13
ppm) for L-day exposure (Sonich et al. , 198-1, unpublished). The
observed effect was a dose-related increase in the frequency of
elevated serum creatinine levels in the exposed population.
However, these findings have not been published at this time. A
human no-observed-effect level (NOEL) for inhalation was reported
as 63 mg/m^ ( fs 10 ppm) for 3-hour exposure (Stewart et al.,
1961). The monitored effects were changes in serum enzyme and
iron levels.
At this juncture, the two studies of Bruckner et al.
manuscript in preparation provide us with acceptable data
(dose-response relationship, length of exposure, etc.) to revise
the existing health advisories (USEPA, 1981d) and to derive a
lifetime AADI as follows:
Study I (Bruckner £t_ al_. , manuscript in preparation)
showed that one-day exposure to 20 or 40 mg CCl4/kg did
not produce any significant changes in BUN, GPT, SDH, OCT,
or histopathological changes in the liver and kidneys. At
80 mg/kg, increased GPT activity and vacuolization of
centrilobular cells adjacent to the central vein in each
liver were observed. Therefore, the largest dose with no
significant biochemical or histopathological effects is 40 mg
CCl4/kg (NOAEL).
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IX-14
One-Day Health Advisory (HA)
a. One-Day Health Advisory for Child
(40 mg/kg/day) (10 kg) = 4 mg/1
(100) (1 I/day)
where: 40 mg/kg/day = NOAEL following one-day exposure
10 kg = weight of child
100 = uncertainty factor based upon a good
animal study revealing NOAEL
1 I/day = assumed water consumption by a
10-kg child
b. One-day Health Advisory for Adult
(40 mg/kg/day) (70 kg) = 14 mg/1
(100) (2 I/day)
where: 40 mg/kg/day = NOAEL following one-day exposure
70 kg = weight of adult human
100 = uncertainty factor based upon a good animal
study revealing NOAEL
1 I/day = assumed water consumption by a
70-kg adult human
Study I (Bruckner et_ al_. , manuscript in preparation)
found that eleven-day exposure to the lowest dose (20 mg
CCl4/kg) induced an increase in SDH whereas 40 and 80 mg
CCl4/kg increased serum levels of GPT, SDH, and OCT. The
extent of morphological changes in the liver was dose-related
Thus, the lowest dose of 20 mg/kg should be considered as
the NOAEL.
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IX-15
Ten-Day Health Advisory
a. Ten-day Health Advisory for Child
(20 mg/kg/day) (5 days) (10 kg) = 0.142 mg/1 or 142 ug/1
(1000) {7 days) (1 I/day)
where: 20 mg/kg/day - NOAEL following 11-day exposure
5/7 days = fraction converting from 5 to 7-day
oral exposure
10 kg = weight of child
1000 = uncertainty factor based upon a good
animal study revealing NOAEL
1 I/day = assumed water consumption by a
10-kg child
b. Ten-day Health Advisory for Adult
(20 mg/kg/day) (5 days) (70 kg) = 0.5 mg/1 or 500 ug/1
(1000) (7 days) (2 I/day)
where: 20 mg/kg/day = NOAEL following 11-day exposure
5/7 days = fraction converting from 5 to 7-day
oral exposure
70 kg = weight of adult human
1000 = uncertainty factor based upon a good
animal study revealing NOAEL
2 I/day = assumed water consumption by a
70-kg adult human
Study II (Bruckner et_ aA., manuscript in preparation)
showed that, following 90-day exposure to 1 mg CCl4/kg,
there were no significant biochemical/histopathological
changes in rats. At 10 mg/kg/ SDH, the most sensitive index
of hepatotoxicity, was elevated, and mild hepatic centrilobular
vacuolization was detected.
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IX-16
Lifetime Adjusted Acceptable Daily Intake (AADI) For Adult
ADI = (1 mg/kg/day) (5 days) (70 kg) = 0.050 mg/day or 50 ug/day
(100) (10) (7 days)
where: 1 mg/kg/day = NOAEL following 90-day exposure
5/7 days = fraction converting from 5 to 7 day
oral exposure
70 kg = weight of adult human
100 = uncertainty factor based upon a good
animal study revealing NOAEL
10 = uncertainty factor to take into account
the length of exposure (i.e., convert
90-day to lifetime exposure)
AADI = 50 ug/day = 25 ug/day
2 I/day
where: 50 ug/day = ADI
2 I/day = assumed water consumption by a 70-kg
adult human
C. Carcinogenic Effects
The carcinogenic effects of CC14 have been well
documented. Oral administration of CC14 have been shown to
be carcinogenic in rats, mice, and hamsters. In all three
species, liver neoplasms developed although hamsters appeared
to be the most sensitive.
The International Agency for Research on Cancer (IARC)
concluded that the evidence from animal studies demonstrating
CCl4-induced hepatic neoplasms was sufficient to indicate
experimental animal carcinogenesis (IARC, 1979). The National
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IX-17
Cancer Institute (MCI) also identifies CC14 as an animal
carcinogen and has used it as the positive control in three' of
its bioassays. The following paragraphs will focus on pertinent
studies demonstrating the carcinogenicity of CC14.
In an NCI (1976) bioassay for trichloroethylene, CC14
was used as the positive control. Rats; The positive control
groups of 50 Osborne-Mendel rats of each sex were administered
CC14 in corn oil by gavage five times weekly for 78 weeks at
two dose levels: 47 and 94 mg/kg bw for males, 80 and 159
mg/kg bw for females. The incidence of hepatocellular carci-
nomas was increased in animals exposed to CC14 as compared
with pooled colony controls. However, this was statistically
significant only for females given the low dose as compared
with the colony controls and not the matched controls. Absolute
incidence of hepatic neoplasms was low (5% in the animals exposed
to CC14). This may be attributed to the resistance by this
rat strain to CC14. The incidence of other neoplasms was
acknowledged but not quantified. This study was used by NAS
(1978) in determining the carcinogenic risk estimate for CC14
due to the dose levels used and the appropriate length of the
study. Mice; B6C3F1 male and female mice (35 days of age, 50
per group) were given CC14 (1,250 or 2,500 mg/kg bw) in corn
oil by gavage five times weekly for 78 weeks. Surviving mice
were sacrificed at 92 weeks from the start of the study. There
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IX-18
were 20 control mice af each sex that were given corn oil only.
A necropsy was performed on all mice along with complete histo-
logical examinations.
Most male and female mice treated with CC14 were dead
by 78 weeks. Hepatocellular carcinomas were found in practically
all mice receiving CCl^, including those dying before termination
of the test. The first carcinomas were observed in low dose
female mice at 16 weeks, in high dose female mice at 19 weeks,
in high dose males at 26 weeks and in low dose males at 48 weeks,
compared to 72 weeks for pooled control males and 90 weeks for
pooled control females. Cystic endometrial hyperplasia occurred
in both control and treated female mice. Thrombosis of the
atrium of the heart was seen in 9 of 41 high dose female mice
(22%), all of which died with carcinomas of the liver. In summary,
this study found CC14 to be high-ly carcinogenic for liver in
mice and is used by the World Health Organization (WHO, 1981) in
ascertaining the carcinogenic risk estimates for CC14.
Edwards et_ a.1. (1942) investigated the carcinogenic potential
of CC14 in mice. The mice used were inbred strain L with
extremely low incidence of spontaneous hepatomas, 2.5-3.5 months
or 3.5-7.5 months of age at the onset of the experiment. The
number of mice varied from 8-39 per group. Carbon tetrachloride
was administered in olive oil by stomach tube usually three, but
occasionally two, times weekly. Each treatment consisted of
0.1 cc of a 40% solution or 0.04 ml of CC14. Mice were given
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IX-19
46 administrations of CC14 over a 4-month period and were
sacrificed and necropsied 3-3.5 months after the last treatment.
The mice varied from 8.5-14 months of age at necropsy. The liver
.was examined histologically.
Hepatomas developed in 34/73 mice (47%) given CC14.
Ftepatomas were observed in 7/15 younger male mice (47%), 21/39
older male mice (54%), 3/8 younger females (38%), and 3/11 older
females (27%). Cirrhosis of the liver was not mentioned. Histori-
cally, the incidence of spontaneous hepatomas in strain L mice is
extremely low: 2/152 (1%) in untreated mice. One of 23 untreated
virgin male mice (4%) and 0 of 28 females (0%),. necropsied at 15
months of age, had tumors of the liver. Tumors were not present
in 22 males and 28 females 18 months of age or in 27 female
breeders 12-23 months of age. One of 24 male breeders (4%) had a
tumor. In summary, strain L male and female mice were highly
susceptible to the induction of hepatomas by CC14, and male mice
were slightly more susceptible than female mice.
Delia Porta e_t_ al. (1961) exposed Syrian golden hamsters
to CC14 in order to investigate the response of this species to
carcinogens that induced liver neoplasms in other species. Ten
female and 10 male Syrian golden hamsters, 12 weeks old, were
used. At the onset of the experiment, males weighed an average
of 99 g and females weighed an average of 109 g. At the end of
the experiment, the average weight was 104 g for both sexes.
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IX-20
'The treatment consisted of weekly administration by stomach
tube of a 5% solution of CC14 in corn oil for 30 weeks. Controls
cited were historical controls kept by the investigators in the
same laboratory for a lifespan. A total of 145 female and 109
male hamsters of the same strain, fed the same diet, did not
develop hepatic tumors. The authors also cited controls for a
different study they conducted. In this latter study, 30 female
and 50 male hamsters fed the same diet but given 0.5 ml corn oil
via stomach tube twice weekly for 45 weeks also did not develop
hepatic tumors. During the first 7 weeks of the former experiment,
0.25 ml of the solution containing 12.5 ul CC14 was given each
week. This dose was then reduced to 0.125 ml and contained 6.25
ul of CC14. After this treatment, the survivors were kept
under observation for 25 additional weeks and then sacrificed.
Detailed histopathological examinations of all hamsters were
conducted, except for one female lost through cannabalism at
the 28th week.
Weights of the hamsters varied irregularly during the
period following treatment. In general, the weights increased.
Females weighed an average of 114 g and males 113 g. One female
died at the 10th week of treatment; three females and five males
died or were sacrificed between the 17th and the 28th week. Three
females died at weeks 41, 43 and 54. The surviving three females
land five males were sacrificed at the end of the 55th week.
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IX-21
Hamsters dying during the treatment and at the 41st week
manifested cirrhosis, as well as hyperplastic nodules that were .two
to several layers thick. The cells showed irregularities in the
shape, size and staining qualities of their cytoplasm and nucleus,
with an uneven distribution of glycogen.
All of the animals, five males and five females, dying or
sacrificed 13-25 weeks after the end of the treatment, had one or
more hepatic carcinomas (a total of 22 tumors: 12 in the females
and 10 in the males). No mention was made of toxicity in these
animals. Hepatic carcinomas were not found in the other animals
dying before week 43.
In summary, Syrian golden hamsters appear sensitive to
the carcinogenic effects of 0014. Although the number of animals
in this study was small, the authors considered the results to be
significant because the reported historical control incidence of
hepatic tumors in hamsters was 0/254. Hyperplastic nodules ap-
peared during treatment, and carcinomas appeared after CC14 admin-
istration had been discontinued, which suggests that the nodules
or benign tumors were precursor lesions for carcinomas. It should
be noted that this study is. the only report found in the available
literature of CC14 induction of tumors in hamsters.
The above studies by NCI (1976), Edwards e_t al_. (1942),
and Delia Porta et al. (1961) were used by EPA's Office of Health
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IX-22
and Environmental Assessment (OHEA) to calculate the unit risk
estimates for CCl^ (USEPA, 1983).
Although several investigators noted that toxic effects
of CC14 are concurrent with liver tumors (hepatomas), it has
not been established that tissue damage (i.e., necrotic cirrhosis)
is a necessary precursor to CCl4-induced carcinogenesis.
Despite a wealth of data on its toxic effects, there is
little definitive information on its metabolism or its mode of
carcinogenic action. Among reported metabolic reactions in liver
are conversion to carbon dioxide, chloroform, hexachloroethane,
carbonyl chloride (phosgene), and binding to lipids and proteins.
Diaz Gomez and Castro (1980) reported that L4C from 4CC14
irreversibly binds in vivo to hepatic nuclear DNA from mice and
rats. Also, binding of 14C from 14CC14 to DNA was detected
in vitro in incubation mixtures containing microsomes and a
NADPH-generating system as well as in tissue slices. Liver
nuclear proteins and lipids irreversibly bind CC14 metabolites.
The authors concluded that (a) the interaction of CC14 metabolites
with DNA and nuclear proteins could be relevant to CCl4-induced
liver tumors and hepatoxic effects; and (b) the epigenetic mechan-
isms for chemical induction of cancer, not involving CC14-DNA
interactions could also be relevant. There have been no reports
of mutagenic activity associated with CC14 in any of the various
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IX-23
Salmonella (Ames) assays. However, mutagenic activity associated
with CC14 has been observed in a eukaryotic test system using
the yeast Saccharomyces cerevisiae (Gallon el: al., 1980). This
report has not been confirmed and should not be accepted as
conclusive evidence of CC14 mutagenicity. Carbon tetrachloride
did not cause chromosome damage (i.e., chromatid gaps, deletions,
or exchanges) during an in vitro chromosome assay using cultured
rat-liver cells (Dean and Walker, 1979). Mirsalis and Butterworth
(1980) found that treatment of male rats with CC14 (10 or 100
ing/kg administered by gavage) produced no increase in unscheduled
DNA synthesis in cultures of primary rat hepatocytes. According
to the authors, this observation indicates that CC14 does not
act through a genotoxic mechanism. Thus, the genotoxic potential
of CC14 obviously needs further investigation.
D. Quantification of Carcinogenic'Risk
Because of positive results in animal carcinogenicity
studies, CC14 can be considered a suspect human carcinogen.
Data from the animal studies have been used by NAS (1977) and
OHEA (USEPA, 1980a; 1983) to calculate the upper bound on the
number of additional cancer cases that may occur when CC14 is
consumed in drinking water over a 70-year lifetime. As shown in
Table IX-I, using the OHEA and NAS data, estimates of additional
carcinogenic risk following the exposure of humans to CC14 may
be derived.
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IX-24
Table IX-I
Estimates of Additional Carcinogenic Risk Following Exposure to
CC14 in Drinking Water3
CC14 Concentrations (ug/1)
Excess Cancer
Risk
(Lifetime )
10-4
10-5
10~6
OHEA
(USEPA, 1980a)J
42.2
4.2
0.4
(USEPA, 1983)e
27.0
2.7
0.3
NAS ( 1 9 7 7 ) <-"
450
45
4.5
a assuming an average daily drinking water consumption of 2 liters
b lower 95% confidence limit
c based on NCI (1976) (rats)
d based on NCI (1976) (mice)
e based on NCI (1976) (rats and mice), Edwards e_t al_. (1942) (mice),
and Delia Porta et al. (1961) (hamsters)
-------
IX-25
The criteria for the OHEA and NAS risk calculations
differ in several respects: (1) NAS used the multistage model,
while OHEA used an "improved" multistage model, (2) NAS used the
data set from the National Cancer Institute (NCI) study in male
rats while OHEA used the data set from NCI's study in male mice
(USEPA, 1980a), and used a geometric mean of four studies (NCI,/
1976 - mice; NCI, 1976 - rats; Edwards e_t a_l. , 1942 - mice; and-
Delia Porta et_ al_. , 1961 - hamsters) (USEPA, 1983).
EPA's Ambient Water Quality Criteria for CC14 (USEPA,
1980a) were based on increased lifetime cancer risk estimates
of 10-5 (4.0 ug/1), 10~6 (0.40 ug/1), and 10~7 (0.04 ug/1)
calculated by--OHEA. It is noteworthy that these estimates were
derived by assuming a lifetime consumption of both drinking
water (2 I/day) and aquatic life (6.5 g fish and shellfish/day)
grown in waters containing the corresponding CC14 levels.
Specifically, OHEA's daily CC14 exposure assumptions were as
follows: 94% from ingesting drinking water and 6% from consuming
seafood "fish factor." The corresponding "drink water only" con-
centrations are 4.4, 0.44, and 0.04 ug/1, respectively.
Using the same data set as OHEA and a linear multistage
model, WHO (1983) derived a recommended tentative limit for CC14
of 3 ug/1. This level should give rise to less than 1 additional
cancer per 100,000 population for a lifetime of exposure assuming
a 2-liter daily consumption of drinking water.
-------
IX-26
In addition to MAS', OHEA's and WHO'S estimates of addi-
tional carcinogenic risk following exposure of humans to CC14 in
drinking water, OHEA calculated a unit risk estimate for humans
from exposure to CC14 in water as follows: 0.37 x 10~5 for a
person continuously exposed to 1 ug CC14 per liter of water,
(USEPA, 1983). Since no single study was entirely adequate for
risk assessment, this estimate is based upon the geometric
mean of four studies discussed above and correspond to drinking
water concentrations presented in Table IX-I. It should be noted
that EPA's Science Advisory Board approved OHEA's approach for
calculating unit risk estimates for 0014.
E. Special Considerations
It is noteworthy that in assessing CCl4~induced toxicity,
carcinogenicity or any other harmful effect, compounds that
react synergistically or antagonistically with CC14 must be
considered. Identified synergistic substances include ethanol,
kepone, PCB, and PBB. Antagonistic effects have been demonstrated
with such compounds as chloramphenicol and catechol.
Sensitive populations are subgroups within the general
population which appear at higher than average risk upon exposure
to CC14. Some of the populations that may be at greater risk
include human fetuses, alcohol consumers, and males of repro-
ductive age.
-------
IX-27
F. Summary
The recommended values for one-day, ten-day for both
children and adult humans, the lifetime AADI for adult humans, and
the estimated lifetime cancer risks are summarized in Table IX-II.
Table IX-II
Summary of Quantification of Toxicological Effects of CC14
Drinking Water
Concentration
10-kg Child 70-kg Adult
One-Day Health Advisory 4 mg/1 14 mg/1
Ten-Day Health Advisory 142 ug/1 500 ug/1
Lifetime AADI 25 ug/1
Excess Cancer Risk
10-4 27 ug/1
10-5 2.7 ug/1
10-6 0.3 ug/1
-------
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