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SERA Health Assessment DRAFT
Document for
Carbon Tetrachloride
COVER 1 SAME SIZE .8% SI 111
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Review Draft
ORA^T
Do not cite or quote
HEALTH ASSESSMENT DOCUMENT
FOR
CARBON TETRACHLORIDE
Notice
This document is a preliminary draft. It has not been
formally released by EPA and should not at this stage be
construed to represent Agency policy. It is being circu-
lated for comment on its technical accuracy and policy im-
plications.
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Environmental Criteria and Assessment Office
Cincinnati, Ohio 45263
Project Manager: Cynthia Sonich
M n.r.tp.-Vion Agency.
\J.$. Hnvironrf.nirM i ._«.- -
*
V
F.£?ie:-> v
y
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DISCLAIMER
This report is an internal draft for review purposes only and does not
constitute Agency policy. Mention of trade names or commercial products does
not constitute endorsement or recommendation for use.
11
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PREFACE
The Office of Health and Environmental Assessment, in consultation with an
Agency work group, has prepared this health assesment to serve as a "source
document" for Agency-wide use. Originally the health assessment was developed
for use by the Office of Air Quality Planning and Standards, however, at the
request of the Agency Work Group on Solvents, the assessment scope was ex-
panded to address multimedia aspects. This assessment will help insure con-
sistency in the Agency's consideration of the relevant scientific health data
associated with carbon tetrachloride
In the development of this assessment document, the scientific literature
has been inventoried, key studies have been evaluated, and summaries and con-
clusions have been prepared so that the chemical's toxicity and related char-
acteristics are qualitatively identified. Observed effect levels and dose-
response relationships are discussed evaluating the potential toxicity of
CCl^. Unit risk estimates for cancer are calculated to provide a media-
specific measure of toxicity. This information can then be placed in perspec-
tive with observed environmental levels.
111
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Members of the Agency Work Group on Solvents
Elizabeth L. Anderson
Charles H. Ris
Jean Parker
Mark Greenberg
Cynthia Sonich
Steven D. Lutkenhoff
James A. Stewart
Paul Price
William Lappenbush
Hugh Spitzer
David R. Patrick
Lois Jacob
Arnold Edelman
Josephine Breeder
Mike Ruggiero
Jan Jablonski
Charles Delos
Richard Johnson
Priscilla Holtzclaw
Office of Health and Environmental Assessment
Office of Health and Environmental Assessment
Office of Health and Environmental Assessment
Office of Health and Environmental Assessment
Office of Health and Environmental Assessment
Office of Health and Environmental Assessment
Office of Toxic Substances
Office of Toxic Substances
Office of Drinking Water
Consumer Product Safety Commission
Office of Air Quality Planning and Standards
Office of General Enforcement
Office of Toxic Integration
Office of Water Regulations and Standards
Office of Water Regulations and Standards
Office of Solid Waste
Office of Water Regulations and Standards
Office of Pesticide Programs
Office of Emergency and Remedial Response
IV
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REVIEWERS (external)
Dr. Roy Albert
New York University Medical Center
Institute of Environmental Medicine
New York, New York
Dr. Tom Clarkson
School of Medicine
University of Rochester
Rochester, New York
Dr. Rolf Hartung
School of Public Health
University of Michigan
Ann Arbor, Michigan
Dr. Magnus Piscator
Karolinska Institute
Stockholm, Sweden
Dr. V.M. Sadagopa Ramanujam
IPA Science Advisor to ECAO-Cin
university of Texas Medical Branch
Galveston, Texas
Dr. James Withey
Food Directorate
Bureau of Food Chemistry
Tunney's Pasture
Ottawa, Canada
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AUTHORS, CONTRIBUTORS AND REVIEWERS
Environmental Criteria and Assessment Office
Office of Health and Environmental Assessment
Cincinnati, Ohio 45268
Health Effects Branch
Criteria and Standards Division
Office of Drinking Water
Washington, D.C. 20460
Lawrence Anderson, Carcinogen Assessment Group
Christopher DeRosa, Environmental Criteria and Assessment Office, Cincinnati
James Falco, Exposure Assessment Group
Les Grant, Environmental Criteria and Assessment Office, RTP
Richard Hertzberg, Environmental Criteria and Assessment Office, Cincinnati
Gregory Kew, Exposure Assessment Group
Si Duk Lee, Environmental Criteria and Assessment Office, RTP
Steven Lutkenhoff, Environmental Criteria and Assessment Office, Cincinnati
Robert McGaughy, Carcinogen Assessment Group
Thomas McLaughlin, Exposure Assessment Group
Sheila Rosenthal, Reproductive Effects Assessment Group
Jerry Stara, Environmental Criteria and Assessment Office, Cincinnati
Peter Voytek, Reproductive Effects Assessment Group
Cynthia Sonich, Document Manager and Principle Author, Environmental
Criteria and Assessment Office, Cincinnati
VI
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TABLE OF CONTENTS
Page
1. INTRODUCTION 1-1
1.1. TH£ COMPOUND 1-1
2. SUMMARY AND CONCLUSIONS 2-1
2.1. SUMMARY 2-1
2.2. CONCLUSIONS 2-5
2.2.1. Major Research Needs 2-7
3. CHEMICAL AND PHYSICAL PROPERTIES/ANALYTICAL METHODOLOGY 3-1
3.1. CHEMICAL AND PHYSICAL PROPERTIES 3-1
3.2. ANALYTICAL METHODOLOGY 3-3
3.2.1 Carbon Tetrachloride in Water 3-3
3.2.2. Carbon Tetrachloride in Air 3-5
3.2.3. Carbon Tetrachloride in Soil 3-7
3.3. SUMMARY 3-8
4. PRODUCTION, USE AND ENVIRONMENTAL EXPOSURE LEVELS 4-1
4.1. PRODUCTION 4-1
4.2. USE 4-1
4.3. ENVIRONMENTAL EXPOSURE LEVELS 4-2
4.3.1. Possible Sources and Levels of Carbon
Tetrachloride in Water 4-3
4.3.2. Possible Sources and Levels of Carbon
Tetrachloride in Air . • • 4-6
4.3.3. Possible Sources and Levels of Carbon
Tetrachloride in Food 4-8
4.3.4. Possible Sources and Levels of Carbon
Tetrachloride in Soil 4-12
4.4. RELATIVE SOURCE CONTRIBUTIONS 4-12
4.4.1. Water 4-18
4.4.2. Air 4-18
4.4.3. ^OOd 4-21
4.4.4. Soil 4-25
4.5. SUMMARY 4-25
Vll
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Page
5. FATE AND TRANSPORT 5_1
5.1. FATE 5-1
5.1.1. Water 5-1
5.1.2. Air 5-1
5.1.3. Soil ' 5-2
5.2. TRANSPORT 5.3
5.2.1. Water 5-3
5.2.2. Air 5_3
5.2.3. Soil 5-4.
5.3. BIOACCUMULATION/BIOCONCENTRATION 5.5
5.4. SUMMARY 5.5
6. ECOLOGICAL EFFECTS 6-1
6.1. EFFECTS ON NON-TARGET ORGANISMS 6-1
6.1.1. Aquatic Life Toxicology 6-1
6.1.2. Acute Toxicity 6-2
6.1.3... Chronic Toxicity 6-4
6.2. TISSUE RESIDUES 6-5
6.3. INDIRECT ECOSYSTEM EFFECTS 6-5
6.3.1. Effect on Stratospheric Ozone 6-5
6.3.2. Effect on UV Flux 6-6
6.4. SUMMARY 6-7
7. COMPOUND DISPOSITION AND RELEVANT PHARMACOKINETICS -. . . 7-1
7.1. ABSORPTION 7-1
7.1.1. Partition Coefficients 7-1
7.1.2. Absorption from the Gastrointestinal Tract 7-3
7.1.3. Absorption by Inhalation 7-4
7.1.4. Absorption Through the Skin 7-5
7.2. DISTRIBUTION 7-6
7.3. METABOLISM 7-10
7.4. ELIMINATION 7-13
7.5. SUMMARY 7-16
8. TOXICOLOGY: ACUTE, SUBCHRONIC AND CHRONIC 8-1
8.1. EXPERIMENTAL ANIMALS 8-1
8.1.1. Acute 8-1
8.1.2. Subchronic 8-17
8.1.3. Chronic 8-21
Vlll
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Page
8.2. HUMANS 8-24
8.2.1. Case Reports 8-25
8.2.2. Controlled/Clinical Studies 8-38
8.3. MECHANISMS OF TOXICITY 8-43
8.3.1. Formation of Carbonyl Chloride (Phosgene) 8-44
8.3.2. Dimerization to Hexachloroethane 8-44
8.3.3. Free Radical Binding to Proteins 8-45
8.3.4. LLpid Peroxidation 8-46
8.4. SUMMARY 8-50
8.4.1. Experimental Animal Data 8-50
8.4.2. Human Data 8-50
8.4.3. Mechanisms of Toxicity 8-51
9. TERATOGENICITY AND OTHER REPRODUCTIVE EFFECTS 9-1
9.1. TERATOGENICTY 9-1
9.2. OTHER REPRODUCTIVE EFFECTS 9-3
9.3. SUMMARY 9-7
10. MUTAGENICITY 10-1
10.1. RELEVANT STUDIES 10-1
10.2. SUMMARY 10-5
11. CARCINOGENICITY 11-1
11.1. RATS 11-1
11.2. MICE 11-7
11.3. HAMSTERS -. . . 11-28
11.4. HUMANS 11-31
11.4.1. Case Reports 11-31
11.4.2. Studies 11-34
11.5. SUMMARY 11-36
11.5.1. Experimental Animals 11-36
11.5.2. Humans 11-36
11.5.3. Conclusion 11-37
12. SYNERGISM AND ANTAGONISM 12-1
12.1. SYNERGISM 12-1
12.2. ANTAGONISM 12-7
12.3. SUMMARY 12-10
IX
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Page
13. REGULATIONS AND STANDARDS 13-1
13.1. WATER 13-1
13.1.1. Ambient Water 13-1
13.1.2. Drinking Water 13-1
13.2. AIR 13-2
13.3. FOOD 13-3
13.4. SUMMARY 13-3
14. EFFECTS OF MAJOR CONCERN AND HEALTH HAZARD ASSESSMENT 14-1
14.1. PRINCIPAL EFFECTS 14-1
14.1.1. Ingestion 14-9
14.1.2. Inhalation 14-10
14.1.3. Dermal Exposure 14-11
14.1.4. Mutagenicity 14-11
14.2. SENSITIVE POPULATIONS 14-11
14.3. QUALITATIVE HEALTH HAZARD ASSESSMENT 14-13
14.3.1. Animal Toxicity Studies Useful for
Hazard Assessment 14-14
14.3.2. Animal Carcinogencity Studies 14-16
14.4. FACTORS INFLUENCING HEALTH HAZARD ASSESSMENT 14-17
14.4.1. Exposure 14-17
14.4.2. Estimated Threshold No-effect Levels 14-18
14.4.3. Carcinogenicity 14-18
15. REFERENCES 15-1
APPENDIX: UNIT RISK ESTIMATE FOR CANCER A-l
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LIST OF TABLES
No. Title Page
3-1 Physical and Chemical Properties of Carbon Tetrachloride. . . 3-2
4-1 Summary of Atmospheric Concentrations of
Carbon Tetrachloride 4-7
4-2 Atmospheric Concentrations of Carbon Tetrachloride
Over Seven U.S. Cities 4-9
4-3 Measured Fluid Intakes 4-13
4-4 Fluid Intake for Reference Individuals 4-14
4-5 Respiratory Volumes for Reference Individual 4-15
4-6 Per Capita Estimates of World Food Consumption by Region. . . 4-16
4-7 Summary of Per Capita Estimates of World Food Consumption . . 4-17
4-8 Carbon Tetrachloride Uptake from Fluids (mg/yr)
Calculated by Assuming 100% Absorption 4-19
4-9 Estimated Human uptake of Carbon Tetrachloride from
the Outdoor Atmosphere 4-20
4-10 Carbon Tetrachloride Uptake from Food Supplies (mg/yr)
Calculated by Assuming 100% Absorption 4-22
4-11 Summary of Carbon Tetrachloride Concentrations
in Food Supplies 4-23
4-12 Relative uptake of Carbon Tetrachloride from the
Environment by Adult Man 4-24
6-1 Acute Values for Carbon Tetrachloride 6-3
7-1 Partition Coefficients for Carbon Tetrachloride 7-2
7-2 Carbon Tetrachloride Distribution at Various Times in
Anesthetized Dogs after Administration by Stomach Tube. . . . 7-7
7-3 Tissue Distribution of C1 C]Carbon Tetrachloride
Inhaled by Rhesus Monkeys 7-9
7-4 Conversion of [1ZtC]Carbon Tetrachloride to [14C]Carbon
Dioxide by Rat Liver Homogenate •.-... 7-12
7-5 Chloroform and Hexachloroethane in Tissues of Rabbits
Given Carbon Tetrachloride Orally 7-15
8-1 Toxic Doses and Effects of Carbon Tetrachloride in Animals. . 8-2
8-2 SGPT Values of Mice Administered Carbon Tetrachloride
Intraperitoneally in "Up and Down" Experiment 8-4
8-3 Effects of Oral Carbon Tetrachloride on Liver Weight
and Liver and Plasma Enzyme Activities in Male Rats 8-6
8-4 Effects of Carbon Tetrachloride on Liver Histopathology
and Serum Enzyme Levels 8-8
8-5 SGPT Activity in Dogs 24 hours After Intraperitoneal
Administration of Carbon Tetrachloride in "Up and
Down" Experiment 8-10
8-6 Exposure Times and Concentrations of Carbon Tetrachloride
Vapor in a Controlled Human Study 8-39
8-7 Incorporation of UC from [1AC] Carbon Tetrachloride
into LJLpids of Various Rat Tissues 8-49
XI
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No. Title =>aqe
9-1 Weight Changes in Male Rat Reproductive Organs After
Carbon Tetrachloride Treatment 9-5
10-1 Mutagenic Effects of Carbon Tetrachloride following
1-hour treatment at 37°C on Strain D7 of
Saccharomyces Cerevisiae 10-3
11-1 Lesions of the Liver in Rats Given Subcutaneous Carbon
Tetrachloride 11-4
11-2 Evidence of the Most Advanced Lesions in Rats
Administered Carbon Tetrachloride 11-5
11-3 Incidence of Liver Tumors in Carbon Tetrachloride-
treated Rats and Colony Controls 11-3
11-4 Incidence of Tumors in C3H Mice Ingesting Carbon
Tetrachloride 11-11
11-5 Incidence of Tumors in Strain A Mice Ingesting
Carbon Tetrachloride 11-12
11-6 Tumors of the Liver in Male and remale Mice Receiving
Carbon Tetrachloride by Stomach Tube 11-14
11-7 Hepatomas in Male and Female Strain A Mice Given CCl^
via Stomach Tube 11-16
11-8 Susceptibility of Strain A Mice to Liver Necrosis and
the Incidence of Heoatomas 30 Days After 120 or 30
Doses of Carbon Tetrachloride 11-18
11-9 Survival of B6C3Fx Mice Treated with Carbon Tetrachloride . 11-20
11-10 Incidence of Heoatocellular Carcinomas in Mice Treated
with Carbon Tetrachloride 11-21
11-11 Hepatomas in Male and Female Mice Given Carbon Tetra-
chloride (0.4 m* 2-3x weekly) by Stomach Tube 11-24
11-12 Hepatomas in Male Mice Given Olive Oil by Stomach Tube. . . . 11-25
11-13 Incidence of Tumors in Rats and Mice Ingesting Carbon
Tetrachloride 11-29
11-14 Studies in Which Liver Necrosis was Induced Using
Carbon Tetrachloride . . . 11-32
13-1 Carbon Tetrachloride Inhalation Standards of 11 Countries . . 13-4
14-1 Dose-Related Toxic Effects of Carbon Tetrachloride on
Humans and Animals 14-2
14-2 Reproductive Effects of Carbon Tetrachloride from
Subchronic Exposure 14-6
14-3 Summary Table for Mutagenicity Studies 14-7
14-4 Carcinogenicity Studies Useful for Risk Assessment
of Carbon Tetrachloride 14-8
14-5 Reported No-Effect Levels for Toxicity of Carbon
Tetrachloride 14-19
xii
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LIST OF FIGURES
No. Title Page
7-1 Pathways of Carbon Tetrachloride Metabolism 7-14
8-1 Free Radical Initiated, Autocatalytic Peroxidation
of Polyenoic Long-Chain Fatty Acids 8-48
xiii
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1. INTRODUCTION
1.1. THE COMPOUND
Carbon tetrachloride (CCl/J, also known as perchloromethane or tetra-
chloromethane, is a haloalkane with a wide range of industrial and chemical
applications. It is a volatile compound yet it is denser than water, thus
rendering it quite stable under certain environmental conditions. Its ores-
ence in the atmosphere and in water appears to be of anthropogenic origin due
to its ubiquity. As would be expected from its partition coefficients, it is
readily absorbed througn the lung and gastrointestinal tract and also through
the skin.
lexicological data for non-human mammals are extensive and indicate that
CC!A causes liver and kidney damage primarily but also neurological damage
and oermal effects. Case reports on humans document similar effects. The
carcinogenicity of CC1, has been well-documented with both the International
Agency for Research on Cancer and the National Cancer Institute identifying it
as an animal carcinogen. It is a suspect human carcinogen.
1-1
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2. SUMMARY AMD CONCLUSIONS
2.1. SUMMARY
Carbon tetrachloride (CCl^) is a relatively non-polar compound that is
slightly soluble in water, soluble in alcohol and acetone, and miscible in
benzene, chloroform and ether. Its density is 1.59 g/mx, at 4°C which is
greater than the density of water. Thus, under favorable conditions, large
amounts spilled into water may settle and not volatilize. However, the high
vapor pressure of CC1. (115.2 mm Hg at 25°C) favors volatilization from
water to air.
CC1. is produced commercially from the chlorination of several chemi-
cals such as methane, propane, ethane propylene and carbon disulfide. In
1980, 3.22 x 10s kg were synthesized in the U.S. This amount of CCl^ is
minimally supplemented indirectly during the production of vinyl chloride
and perchloroethylene. The major use of CCl^ is in the production of
cnlorofluorocarbons. A reduction in the use of CCl^ has resulted in a
3.5?6 decrease in production over the years 1970 to 1980. A continued 1.0%
decline in production is projected each year through 1985.
CC1. can be detected in the environment using media-specific analyt-
ical methods. Levels detected in the environment are generally <.01
mgA in water, <.01 mg/m3 in air and <.01 mg/kg in food although
higher levels have been detected on occasion in urban air and grain fumi-
gated with CC1A. Food products made from this grain also contain residues
of CC1 . Natural sources of CC1, are unknown so that most, if not all
CC1. present in the environment can be accounted for by anthropogenic
activities.
CC1. is extremely stable in the lower atmosphere and troposphere.
However, once in the stratosphere, photodissociation is rapid. Its presence
2-1
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in the stratosphere is of concern due to its possible contribution to the
ozone-destroying chemical reactions and subsequent modification of UV-3
radiation flux. Increased UV-B radiation studies in the laboratory have
shown adverse affects to a variety of terrestrial plant species; for exam-
ple, in terms of depressed photosynthetic activity, reduced growth rate,
increased somatic mutation rates, and inhibition of seed germination. Some
aquatic organisms have also been shown, in laboratory studies, to be
adversely affected. Direct extension of these laboratory findings to the
natural environment should not be made because of adaptability potential to
small changes in UV-B flux. Indirect ecosystem effects associated with
increases in UV-B radiation flux have been identified to include changes in
genetic material and alterations in population composition. However, it is
not known if these laboratory findings apply to the natural environment in
lieu of observed adaptations. The implication to human populations is that
>90% of skin cancer (other than melanoma) in the U.S. is attributed to
sunlight in the UV-B region.
Under favorable conditions, which seldom occur, CC1. settles in water
and, therefore, does not volatilize. In this case, it is extremely stable.
In such capacity, the presence of CCl^ in ambient water and drinking water
may present a threat to aquatic ecosystems and human health.
Despite the fact that humans are potentially exposed to CCl^ through
various media, uptake from air appears to be the major source of exposure
followed by liquids and food. For this reason, the fate and transport of
CC1. has been most extensively studied in air. Information on CCl^ soil
contamination is limited, consequently, its contribution to human exposure
is uncertain.
2-2
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Ecological impacts have been monitored to a limited extent in freshwater
and saltwater organisms. In two freshwater fish and one invertebrate spe-
cies, 96-hour LCcns were determined as low as 27.3 mg/*< in the bluegill
(Lepomis macrochirus). Chronic toxicity data are not available. However,
reported bioconcentration factors are <30 so that tissue residues are
insignificant. The only data on saltwater species deals with toxicity fol-
lowing acute exposures. Effects have been identifed at levels of 50.0
mg/x.. It is noted that the toxic dose for both freshwater and saltwater
species can be lower if a more sensitive species were tested.
In mammals, CC14 is readily absorbed from the lungs and gastrointes-
tinal tract. Absorption also occurs through the skin but at a much slower
rate. Following absorption, CCl^ is distributed to all major organs.
Metabolism of the compound occurs primarily in the liver where it is reduced
to a trichloromethyl radical and thought to be further metabolized and/or
released as a free radical. Excretion of CCl^ is primarily through the
lungs, but also occurs in the urine and feces.
Varying degrees of toxicity have been reported in humans and animals
following acute, subcnronic and chronic exposures via ingestion, inhalation
or dermal administration. Such effects can occur systemically as well as
locally. For example, cirrhosis of the liver has resulted from inhalation
and dermal exposures, whereas lung lesions have resulted from oral inges-
tion. Animals surviving acute doses developed a range of effects such as
damage to the liver, kidney, lung, and central nervous system as well as
dermal effects; biochemical alterations were also noted. Animals receiving
sub-chronic and chronic doses developed kidney and liver damage and, less
frequently, damage to the central nervous system. It has been observed that
exposure to a higher concentration over a shorter period of time produces a
2-3
-------
greater effect upon the liver than exposure to a lower concentration over a
longer period of time even though the product of time and concentration is
equal in both cases.
In an attempt to verify the purity of CC14 used in the testing proto-
cols of the studies in this document, it was observed that CCl^ of imoure
or technical grade was not reported.
Adverse effects seen in humans following CCL exposure are similar to
those seen in animals. Damage to the liver, kidney, lungs and central ner-
vous system has been documented in various case reports. Biochemical alter-
ations have been identified in case reports and one epidemiological study.
Exposure to CCl^ did not produce skeletal or functional abnormalities
but aid result in signs of fetotoxicity. Rats exposed _in utero to CC1,
were noted to have fatty infiltration of the liver from days 1 to 4 after
birth. CC14 is transferred to the neonate through mothers' milk. Other
adverse reproductive effects include changes in testicular histology even-
tually resulting in functional infertility.
Carbon tetrachloride has consistently tested negative in the Salmonella
assay. A positive mutagenic response was seen in an assay using Saccharomy-
ces _cerevisiae, however, there have been problems associated with this study.
Numerous studies report on the carcinogenic effects of CC1, on experi-
mental animals. Both the National Cancer Institute which uses CCl^ as a
positive control in some of its bioassays, and the International Agency for
Research on Cancer have concluded that it is a carcinogenic substance to
experimental animals. The studies on experimental animals indicate that
CCL is carcinogenic to three species: hamsters, mice and rats in order
of decreasing sensitivity.
2-4
-------
Case reports of human carcinomas developing years after a history of
high CCl, exposures offer little conclusive evidence of human carcinogen-
icity. However, they are consistent with the carcinogenic potential of
CCl^ as suggested by animal studies.
In assessing toxicity, carcinogenicity or any other harmful effect, com-
pounds that react synergistically or antagonistically with CC14 must be
consioered. Identified synergistic substances include ethanol, Kepone, PC3
and PBB. Antagonistic effects have been demonstrated with such compounds as
chloramphenicol and catechol.
2.2. CONCLUSIONS
Carbon tetrachloride causes damage to the liver, lungs, kidneys and
central nervous system in humans. These effects are primarily the result of
high oral or inhalation exposures. Less severe effects such as biochemical
alterations, nausea and headache result from lower exposures or are second-
ary to the major health hazards attributed to higher exposures. Similar
responses have been demonstrated in animals. These animal studies provide
useful dose/response data, are well-defined and can identify a causal rela-
tionship between the CC1, insult and the toxic response. Furthermore, the
toxicity from CCL is not only local but also systemic.
Absorption of CC1. varies with species. Based upon both human and
animal data, absorption coefficients of 40% when route of exposure is via
inhalation and 100% when route of exposure is via ingestion are recommended.
The potential exists for embryotoxicity, especially in males. Toxic
effects due to CC14 have been demonstrated in mammalian fetuses and neo-
nates exposed directly or indirectly via the placenta or mothers' milk,
respectively. Teratogenic effects have not been noted following CCl^
exposure, however, degenerative changes in the testes and subsequent infer-
tility of the offspring have occurred.
2-5
-------
Definitive conclusions concerning mutagenicity tests cannot be reached.
CC1, did exhibit a positive mutagenic response in an assay using Saccharo-
myces cerevisiae, however, due to problems associated with the study and the
lack of corroborative studies, the evidence is not adequate to conclude
whether or not CC1. is genotoxic.
Interactions with other chemicals must be considered in assessing the
potential health hazards of exposure to CCl^. Chemicals have been identi-
fied that potentiate the effects of CCl^ as well as those that inhibit the
effects of CC14.
Carcinogenicity of CC1, has been observed in three animal species.
The primary lesions are hepatic neoplasms. Cirrhosis, necrosis and cholan-
giofibrosis have also been found and have been suggested as initial lesions
prior to tumorigenesis in liver. Human data on carcinogenicity are re-
stricted to case reports and one preliminary epidemiological study. The
animal data provide evidence to indicate that carbon tetrachloride is a
potential human carcinogen.
Mechanism of action is a fundamental issue in the assessment of the
potential carcinogenicity of carbon tetrachloride to humans at low doses.
The possibility exists that carbon tetrachloride acts through a nongenotoxic
mechanism to produce a carcinogenic effect; evidence in favor of this possi-
bility includes signs of liver toxicity observed along with liver tumor for-
mation in positive animal carcinogenicity studies and the primarily negative
results from currently available mutagenicity studies. However, Eschen-
brenner and Miller (1946) reported liver tumor development without concomi-
tant necrosis in treated mice in their carcinogenicity study, and in their
carcinogenicity study with five strains of rat, Reuber and Glover (1970)
found greater liver tumor incidence in strains showing lower severity of
2-6
-------
cirrhosis. Furthermore, as indicated in the mutagenicity section herein,
currently available mutagenicity studies do not provide a conclusive judg-
ment on the mutagenic potential of carbon tetrachloride. Further investiga-
tion, particularly in regard to genotoxic potential, is indicated to eluci-
date the carcinogenic mechanism of action for carbon tetrachloride.
2.2.1. Major Research Needs.
Experiments in a numoer of species designed to derive an
absorption coefficient or an absorption range are needed.
Definitive data on 1x14-induced carcinogenicity and toxicity
in humans including the mechanism of action are needed. While
results on short-term exposures are available, they quite
often do not contain adequate dose information. Epidemiology
studies on occupational groups are warranted as demonstrated
in the preliminary study on drycleaners.
Long-term studies on animals exposed jjn utero are needed to
assess lifetime effects of such exposures.
Chronic studies on animals exposed to CCl^ via drinking
water are needed to establish a dose-response relationship.
The toxicity data on the rat and guinea pig are satisfactory.
Additional data on mice are needed since, at present, dose/
response informatipn is inadequate. Studies on other species
would also be useful.
Toxic effects of chronic exposure on freshwater and saltwater
organisms need to be documented.
Assessment of the overall ecological impact is sparse. Infor-
mation on soil and air, particularly the stratosphere (levels,
fate and transport processes, relative source contribution)
are needea, as well as bioaccumulation/bioconcentration in
shellfish.
Additional research to better define the genotoxic potential
and epigenetic potential of CC14.
2-7
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3. CHEMICAL AND PHYSICAL PROPERTIES/ANALYTICAL METHODOLOGY
3.1. CHEMICAL AND PHYSICAL PROPERTIES
Carbon tetrachloride (CCl^) is a clear, colorless, nonflammable liquid
with a characteristic odor (Windholz, 1976). It is a relatively nonpolar
compound that is slightly soluble in water (0.8 g/1 at 25°C) (Johns,
1976), soluble in alcohol and acetone, and miscible in benzene, chloroform
and ether (Weast, 1978).
Carbon tetrachloride may be quite stable under certain environmental
conditions. An estimated 70,000 years are required for half of a given
quantity of CCL to decompose in water (Johns, 1976). This decomposition
rate is considerably accelerated in the presence of metals such as iron
(Pearson and McConnell, 1975). However, hydrolytic decomposition as a means
of removal from water appears to be insignificant as compared to evapora-
tion, since the properties (Table 3-1) of carbon tetrachloriqe favor vola-
tilization of the compound from water to air. Carbon tetrachloride has a
high vapor pressure (115.2 mm Hg at 25°C) (Johns, 1976). The air/water par-
tition coefficient of carbon tetrachloride at 20°C is 1.1 by volume and
about 1000 by weight (Johns, 1976). The rapid vaporization predicted from
these properties has been confirmed by Dilling et al. (1975), who reported a
half-life of carbon tetrachloride evaporation of 29 minutes from a dilute
aqueous solution at 25°C.
The density of carbon tetrachloride in water is 1.59 g/m& at 4°C
(Weast, 1978). 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.
Volatilization is the major transport process for removal of CC1. from
aquatic systems. Once in the troposphere, CC1, remains stable; it
3-1
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TABLE 3-1
Physical and Chemical Properties
of Carbon Tetrachloride
A. Structure
Cl
Cl-
•Cl
Cl
8. Synonyms
Tetrachloromethane
Methane tetrachloride
Perchloromethane
Benzinoform
Necatorina
C. Registry Numbers
CAS No. 56-23-5
TSL No. FG 4900000
0. Description
Carbon tetrachloride is a clear, colorless, nonflammaole liquid with a
charcteristic odor (Windholz, 1976). It is slightly soluble in water, solu-
ble in alcohol and acetone, and misciole in benzene, ether and chloroform
(Weast, 1978).
E. Physical Properties
1. Molecular weight
2. Melting point
3. Boiling point at 760 torr
4. Density in water at 4°C
at 25°C
5. Vapor pressure at 25°C
6. Solubility in water 20°C
7. Log octanol/water partition
coefficient
8. Conversion factors
at 20°C 1 atm.
153.82
-22.9°C
76.54°C
1.594 g/mfc
1.589 g/m£
115.2 mm Hg
785 mg/fc
2.64
0.156 ppm
for 1 mg/m3
6.402 mg/m3
for 1 ppm
Reference
Weast, 1978
Weast, 1978
Weast, 1978
Weast, 1978
Windholz, 1976
Weast, 1978
Pearson and
McConnell, 1975
Neely et al., 1974
Verschuren, 1977
3-2
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exhioits an extremely slow rate of reaction with hydroxyl radicals present
in the troposphere. The atmospheric lifetime of CCl^ is 30 to 100 years.
CC14 eventually diffuses into the stratosphere or is carried back to the
earth during the precipitation process. Once in the stratosphere, CCl^ is
degraded on exposure to shorter wavelength, higher energy ultraviolet light
to eventually form phosgene as the principal initial product (44 FR 68624-
68707).
3.2. ANALYTICAL METHOOGLOGY
3.2.1. Carbon Tetrachloride in Water.
3.2.1.1. SAMPLING — Grab samples must be collected in glass con-
tainers having a total volume in excess of 40 ml. The samole bottles should
be filled in such a manner that no air bubbles pass through the sample as
the bottle is being filled. The bottle should then be sealed so that no
bubbles are entrapped in it. The hermetic seal should be maintained until
the time of analysis.
The samples must be iced or refrigerated from the time of collection
until extraction. If the sample is known to contain free or combined chlor-
ine, sodium thiosulfate preservative (10 mg/40 m*- will suffice for up to 5
mgA C12) should be added to the empty sample bottles just prior to
shipping to the sampling site. In collection of the sample, the bottle
should be filled just to overflowing. After sealing the bottle, the sample
should be shaken vigorously for 1 minute. All samples must be analyzed
within 14 days of collection (44 FR 68624-68707).
3.2.1.2. ANALYSIS
3.2.1.2.1. Ambient Water — Carbon tetrachloride (and 47 other halo-
genated organics) in water can be analyzed by a purge and trap method
(Method 502.1) described by the U.S. EPA Environmental Monitoring and Sup-
port Laboratory (U.S. EPA, 1980b). This method can be used to measure
3-3
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purgeable organics at low concentrations. Purgeable organic compounds are
trapped on a Tenax GC-containing trap at 22°C using a purge gas rate of 40
mVmin for 11 minutes. The trapped material is then heated rapidly to
180°C and backflushed with helium at a flow rate of 20 to 60 mVmin 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 8
(60/80) mesh (8 ft x 0.1 in. I.D.) and is run at a flow rate of 40
mS/min. The temperature program sequence begins at 45°C for 3 minutes,
increases 8°C/min to 220°C, and is then held constant for 15 minutes or
until all compounds have eluted. A halogen-specific detector with a sensi-
tivity to 0.10 vQ/i and a relative standard deviation of 10% must be
used. The optional use of gas chromatography/mass spectometry (GC/MS) tech-
niques of comparable accuracy and precision is acceptable.
Carbon tetrachloride can also be detected in water using the headspace
gas chromatography method in conjunction with electron capture detection
(GC/ECD) described by Dietz and Singley (1979). A 10 *t. by 4 mm (i.d.)
glass column was used containing 20% SP-2100/0.1% Carbowax 15QO on 100/120
mesh Supelcoport. With this method, CCl^ and other chlorinated hydrocar-
bons can be detected from 0.1 vQ/i to the low mg/S, range at ambient
temperatures. The headspace method relies on the fact that when a water
sample containing organic compounds is sealed in a vial, the organics will
equilibrate between the headspace in the vial and the water (Dietz and
Singley, 1979). Distribution of the compounds between the two media depends
on a number of physical and chemical parameters. With these parameters
known, specific measurements can be made on the volatile compounds of inter-
est. Although it is not clearly stated by the authors, accuracy &Q% can
be obtained, and assuming a constant and additive term, precision =0.1.
3-4
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3.2.1.2.2. Municipal and Industrial Discharges — The proposed metnod
is designed to oe used to meet tne National Pollutant Discnarge Elimination
System UNPDES). In this regard, it presupposes a high expectation of find-
ing the specific compounds of interest. It can oe utilized to screen sam-
ples; however, the user must develop an independent protocol for the verifi-
cation of identity.
Tne method of analysis is gas chromatography (GC) and high performance
liquid chromatography (HPLC) for purgeaole halocarbons. HPLC has been
developed considerably in the past few years and can be used to achieve
separations and measurements that cannot De performed with state-of-the-art
GC. In short, the method has been developed for the measurement of solvents
and other volatile materials using variations of the 3ellar purge and trap
technique. Semispecific detectors are used to minimize background interfer-
ences. A detailed description of the method is provided in tne Federal reg-
ister, December 3, 1979 (44 FR 68624-68707)
The sensitivity of this method is usually dependent upon tne level of
interferences rather than instrumental limitations. Tne limit of detection
for carbon tetracnloride is 0.007 ig/1 and represents the" sensitivity
that can be achieved in wastewaters under optimum operating conditions.
3.2.2. Carbon Tetrachloride in Air.
3.2.2.1. SAMPLING — Ambient air sampling of CC1, can involve its
adsorption onto a suitable medium such as Tenax GC. Recovery can then be
accomplished by thermal desorption and purging with helium into a liquid
nitrogen cooled nickel capillary trap (Pellizzari and Bunch, 1979). This
method is the most common and is specific to analysis by GC. Sampling tech-
niques will vary with the analytical methodology.
3-5
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3.2.2.2. ANALYSIS — Many of tne techniques used in analyzing CCi4
in water apply to air. Tnere are four practical methods to measure air con-
centrations of CCi, [National Academy of Sciences (NAS), 1978]. They are
(1) GC/ECD; (2) GC/MS; (3) long path infrared adsorption spectroscopy, usu-
ally with preconcentration of whole air and then separation of the compounds
oy gas cnromatograpny (GC/IR); ano (4) infrared solar spectroscopy (NAS,
1978).
GC/ECO is by far tne most widely used method (LoveiocK et al., 1973;
Lillian et al., 1975; Penkett et al., 1979). The instrumentation needeo. tor
tnis method is readily ootained and relatively inexpensive, costing between
$5000 and $10,000 (NAS, 1978). It is sturdy and easy to operate and quite
sensitive for CCi,, wnicn makes it ideal for use on aircraft (Sdnoaiis and
riattun, 1>77) ano snips.
Tne use of GC/MS has oeen more recent (Grimsrud and Rabmussen, 1975;
Barkley et al., 1980; Dmitriev et al., 1980). Although equally as sensitive
as tne GC/ECD method, GC/MS has the ability to positively identify compounds
oy tneir characteristic mass spectra, whereas GC/ECD must rely upon the
somewhat imprecise method of retention times to identify compounds (NAS,
1978). Unfortunately, GC/MS instrumentation is quite expensive costing
$70,000 or more (NAS, 1978). The method employed by Dmitriev et al. (1980)
undertakes the chromatograpnic separation at room temperature for the first
5 minutes, then at a rising temperature to 150°C at 5°C/minute intervals for
a total chromatograpny time of approximately 30 minutes, using adsoroants
such as Tenax or Polysorbamide. The cnrumatugrapnic fractions are tnen
analyzed oy MS. The authors report a sensitivity level of 1 ug/m3 with
tnis method (Dmitriev et al., 1980).
GC/Irt has tne advantage of real-time continuous measurements; nuwever,
tne disadvantage of poor sensitivity renders tnis method less oesiraole
3-6
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(MAS, 1978). Furthermore, the method is expensive, costing $2Q,000-$100,000
ana cannot be used in the field (NAS, 1978). Tnis metnoo is usea ana
reported oy Hanst et al. (1975) with a detailed description of sample col-
lection techniques.
Finally, infrared soiar spectroscopy has been descriDea by Rasmussen,
i97o ana Murcray et al., 1975 (NAS, 1978). Tnis method uses the solar spec-
trum passing through the atmosphere at large zenith angles tu ootain the
necessary path length to give sufficient absorption to detect amoient halo-
caroon levels. Although not in real-time, this metnod proviaes continuous
data on a remote region of tne atmosphere (NAS, 1978), i.e., the strato-
sphere. However, it is limited to that region alone.
3.2.3. Carbon Tetrachloride in Soil. Little research has been done to
aetect CCl^ in soil; however, with concern increasing regaraing leacnates
from landfills and waste disposal sites, the analysis of soil samples for
organic compounds has oecome more important as an indicator of possible
grouna water contamination. A recent article by DeLeon et al. (1>8G)
descrioes a metnod of analysis for volatile and semivolatile organocnioride
compounas.
3.2.3.1. SAKPLLNG — In tne methoa descrioea by DeLeon et al. (1980),
samples were taken from 30 ft. vertical borings using the splitspoon methoa.
Tne samples were then placea in jars ana seaiea with Teflon-linea screw
caps. During shipment, they were maintained at 6 to 10°C. Upon their arri-
val at the analysis site, they were maintained at -20°C until preparea for
analysis.
3.2.3.2. ANALYSIS — The analytical methoa described (DeLeon et al.,
1980) employs a simple extraction procedure using hexane followed by anal-
ysis of tne extract using temperature programmed GC on high-resolution glass
3-7
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capillary columns with ECO. The electron capture gas chromatographic
results are confirmed by mass spectometry much like the method used for
water sample analysis. Reported sensitivity is at least 10 ug/g. The
authors demonstrate this technique to be effective in determining the peri-
meter and limits of an old chemical waste disposal site and also to be an
effective method to assess the extent of leaching of chlorocarbons from the
waste disposal site into surrounding soils (OeLeon et al., 1980).
3.3. SUMMARY
Carbon tetrachloride (CCl^) is a clear, colorless, nonflammable liquid
with a characteristic odor and is slightly soluble in water. Its high vapor
pressure favors rapid volatilization from water to air. This characteristic
is utilized in most commonly accepted methods of analysis for CCl^. Gas
chromatography (GO, either alone or coupled with mass spectroscopy (MS), is
the most widely used detection method. The compound is usually separated
from other constitutents in the sample by direct volatilization, extraction
or heating, and then analyzed using GC, GC/MS or some other analytical
technique.
3-8
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4. PRODUCTION, USE AND ENVIRONHENTAL EXPOSURE LEVELS
4.1. PRODUCTION
Carbon tetrachloride is produced industrially by the chlorination of
methane, propane, ethane, propylene or carbon disulfide (Rams et al., 1979).
In 1980, 322 million kg were synthesized [U.S. International Trade Commis-
sion (USITC), 1981]. Carbon tetrachloride is also produced indirectly dur-
ing the production of compounds such as vinyl chloride and perchloroethylene
(Rams et al., 1979).
The demand for carbon tetrachloride in the U.S. is projected at 716 mil-
lion pounds in 1980, 708 million pounds in 1981 and 680 million pounds in
1985 (Chemical Marketing Reporter, 1981). This represents a 1.0% decline
per year through 1985, while the historical trend over the last 10 years
(1970 to 1980) is a decline of 3.5% per year. Carbon tetrachloride produc-
tion in the U.S. has declined at a rate of 7.9% per year since reacning its
peak level of 1974.
Production levels have decreased as a result of the rapid decline in its
major end product area, fluorocarbon aerosols. In 1975 fluorocarbon aero-
sols had a 50% share of the domestic aerosol market; however, this declined
to 20% in 1977 due to the ozone depletion controversy (Chemical Marketing
Reporter, 1981). Fluorocarbon aerosols are expected to show some strength
in refrigerant and foam-blowing applications. Growth in these areas is
expected to stabilize carbon tetrachloride production in the 1980s.
4.2. USE
Currently the major use of carbon tetrachloride is in the production of
chlorofluorocarbons, which are used as refrigerants, foam-olowing agents and
solvents. These uses accounted for 95% of the total U.S. consumption in
1980. Carbon tetrachloride is also used in fumigants and has a variety of
4-1
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minor uses (5% of total 1980 consumption), inciuoing those as a solvent in
metal cleaning and in manufacture of paints and plastics (Rams et al.,
1979). It is being replaced in grain fumigation by other registered pesti-
cide products (U.S. EPA, 1980a), and its registration for use in fumigants
is presently under review by U.S. EPA (45 FR 68534-68584). Approximately
12% of the total 1980 production was exported.
4.3. ENVIRO*€NTAL EXPOSURE LEVELS
Carbon tetrachloride present in the environment appears to be of anthro-
pogenic origin (Singh et al., 1976). Its presence in surface waters occurs
primarily as a result of .industrial and agricultural activities although
some may reach surface water through rainfall. Groundwater contamination
may oe the result of leaching from solid waste sites. This is also a source
of soil contamination. Air is the media wherein the greatest concentrations
of CC1, can be found in the environment. The major source of CC14 in
air is industrial emission. Once in the air, CCl^ can be washeo into sur-
face waters and soil through rainfall.
Once in the environment, carbon tetrachloride is relatively stable. Its
half-life for hydrolytic breakdown in water at pH 1.0 to 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, however, since carbon tetrachloride vaporizes
readily to air. The atmospheric lifetime of carbon tetrachloride appears to
be on the order of 30 to 100 years (Singh et al., 1976).
The presence of carbon tetrachloride in the environment is of concern
for two reasons. As indicated by both animal and human studies, CCl^ may
pose a health problem through direct exposure in the air, water, food and/or
4-2
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soil. However, CCl^ may also contribute to ozone-destroying ohotochemical
reactions in the stratosphere. This might cause increases in the incidence
of human skin cancers and animal cancers, affect terrestrial and aauatic
ecosystems, and bring about climatic changes (NAS, 1978, 1982).
Although levels of carbon tetrachloride in the environment are generally
in the low ppb range or below (NAS, 1978), carbon tetrachloride may pose a
long-term danger because of its possible carcinogenic potential (see Chap-
ter 11). In urban and industrial areas where higher concentrations of car-
bon cetrachloride in air occur, other toxic effects may result (e.g., liver
and renal damage).
No natural sources of carbon tetrachloride have been reported. The
presence of a natural source was suggested because of the large quantity of
carbon tetrachloride in the atmosphere and the homogeneity of ambient con-
centrations in both hemispheres (Lovelock et al., 1973). However, this sug-
gestion has since been challenged, since estimates of cumulative worldwide
production and emissions of carbon tetrachloride appear to account for the
carbon tetrachloride found in the environment (Singh et al., 1976).
4.3.1. Possible Sources and Levels of Carbon Tetrachloride in Water.
Carbon tetrachloride has been monitored extensively in drinking water and,
to a lesser extent, in natural waters. The chemical's concentration in
drinking water has been reported as <.007 mgA (Symons et al., 1975).
Samples of ocean, lake and ground water have generally yieloed carbon tetra-
chloride concentrations in the ppt range. There are some indications that
industrial activity may lead to increased carbon tetrachloride concentra-
tions in surface and ground water. These monitoring studies are discussed
below.
-------
In the National Organics Reconnaissance Survey (NORS), U.S. EPA found
caroon tetracnloride levels of <.003 mg/i in drinking water in 80 cities
(Symons et al., 1975). The more recent National Organics Monitoring Survey
(NOMS) of 113 public drinking water systems detected carbon tetrachloride in
the range of .0024 to .0064 mg/£ in 10% of the samples surveyed (U.S. EPA,
1980a). Carbon tetrachloride concentrations in these samples were very low
compared to those of cnloroform and other organics.
Caroon tetracnloride has been detected in drinKing water in Tuscaloosa,
Alaoama (Sertscn et al., 1975); the District of Columbia (Scheiman et al.,
1974); Durnam, North Carolina (McKinney et al., 1976); ana New Orleans,
Louisiana (Dowty et al., 1975). In the District of Columbia, caroon tetra-
cnloride in drinKing water was measureo at .005 mg/i (Scheiman et aj..,
i>74). In New Orleans, nigher concentrations of caroon tetrachlorioe were
found in olood plasma than in drinking water, suggesting to the authors the
presence of a bioaccumuiation mechanism or sources of tne compound other
than drinking water (Dowty et al., 1975). The former, however, has not been
demonstrated. Caroon tetrachloride was also found in drinking water in
Germany in the ng/& range (Sonneborn and Bohn, 1977).
Under unusual conditions, carbon tetrachloride may be found at high
levels in raw and drinking water. After a chemical manufacturer accidental-
ly spilled an estimated 70 tons of caroon tetrachloride into the Kanawna
River, the U.S. EPA determined that raw Ohio River water contained caroon
tetracniorioe levels up to .340 mg/Jl; drinKing water levels were founo to
oe as nign as .1 mg/il (Landen, 1979). As indicated by tnis incident,
levels of CCl^ detected in ambient water should not be construed to oe the
same levels of CC1 in puoiic drinking water supplies. The treatment pro-
cess ooes remove some of the contaminant.
4-4
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Caroon tetrachloride has been found in the ppo range or lower in samples
of rain, surface water, potable water and seawater (McConnell et al., 1975).
Trace levels of caroon tetrachloride have been reported in snow (Su ana
Goldoerg, 1976). Carbon tetrachloride was also detected in the Atlantic
Ocean at mean levels of 60 ppt (_+17 ppt). Ocean levels of caroon tetra-
cnloride were only slightly higher in tne Northern Hemisphere than in the
Soutnern Hemispnere (Lovelock et al., 1973).
Lake Zurich and the ground water in an industrial section of Zurich,
Switzerland, were also monitored for carbon tetrachloride. At various
depths of Lake Zurich, concentrations of this compound of approximately 25
ppt were measured, with no significant variation. Ground water levels of
carbon tetrachloride in the industrial sector were much larger. The com-
pound was detected in 4 of 18 samples at levels ranging between 190 to 3600
ppt (Giger et al., 1978).
Caroon tetracnioriae can be emitted to the environment througn tne pro-
duction and use of the chemical, and through the production and use of
cmorofluorocaroons and other chlorinated compounds tnat contain caroon
tetracnioride impurities. Although small amounts of caroon _tetracnloride
may oe directly released to water systems through tnese processes, most of
the caroon tetracnioriae emitted to the environment has been estimated to be
released to air or land (Rams et al., 1979). Direct releases to water may
not account for tne caroon tetrachloride eventually detected in water. The
chemical may find its way from air or land to surface and ground water sys-
tems through rainfall, runoff from agricultural sites, dumping sites or
industrial sites, and landfill leaching.
High levels of carbon tetrachloride in the ground waters of Zurich,
Switzerland have been attributed to industrial processes in the Zurich area
(Giger et al., 1978). Levels of carbon tetrachloride would be expected to
4-5
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oe nignest in inuustrializea areas because of bonn inoustrial ana consumer
use or' caroon tetrachloride and its products in these areas.
Caroon tetrachloriae does not appear to be produced in water througn
cnlorination reactions, unlike other chlorinated organics such as chloroform
(44 FR 68624-68707). Recent high levels of carDon tetrachloride detected in
Philadelphia drinking water following chlorination (
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TABLE 4-1
Summary of Atmospheric Concentrations of Carbon Tetrachloride*
Type of Measurement
Carbon Tetrachloride mg/m3
Continental background
Marine background
Urban range
0.00076 ± 0.00008
0.00084 i 0.00006
0.00073 +_ 0.00005
0.00075 +_ 0.00009
0.00081 +_ 0.00003
0.00081 ± 0.00010
0.00070 ± 0.00007
0.00084 +_ 0.00012
0.00075 ± 0.113
0.0088
0.00075 + 0.0094
*Source: NAS, 1978
4-7
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Q.il3 mg/m3 in Bayonne, New Jersey (Lillian et ai., 1^73). A caroon
tetracnioriae concentration of >0.0094 mg/m3 was also detected in tne
air of Grenoole, France (Su and Goldoerg, 1576). In 1980, Singh et al.
measured CCl, concentrations in seven U.S. cities over 2-week periods
(Taole4-2). The highest level detected using GC/ECD was 0.0188 mg/m3
over Houston, Texas.
CarDon tetrachloride in the atmosphere appears to be of anthropogenic
origin, oecause estimates of releases of carbon tetrachloride from indus-
trial processes and uses appear to account for the amount present in tne
atmosphere (Singh et al., 1976; Penkett et al., 1979). In 1978, approxi-
mately 4.5 million pounds of CCl, were emitted from production facil-
ities. The total nationwide emissions of CCl. in 1978 from all sources
were estimated at 65 million pounds. The primary source of tnese emissions
is solvent application.
4.3.3. Possible Sources and Levels of Carbon Tetracnloride in Food. In
British studies, carbon tetracnloride was found in various foods as follows
(McConnell et al., 1975; Pearson and McConnell, 1975):
Concentration
(mg/kg)
Dairy products .0002 - .014
Meat .007 - .009
Oils and fats .0007 - .018
Beverages .0002 - .006
Fruits ano vegetaples .003 - .008
Black grapes (imported) .0197
Fresn bread .005
Fish and seafood .0001 - .006
4-8
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TABLE 4-2
Atmospheric Concentrations of Carbon Tetracnloride
Over Seven U.S. Cities*
CCJ-4 (mg/m3)
City/State
Los Angeles, CA
Phoenix, AZ
OaKlana, CA
Houston, TX
St. Louis, MO
Denver, CO
Riverside, CA
Sampling Dates
Apr. 9
Apr. 23
June 28
May 14
May 29
June 15
July 1
- Apr. 21,
- May 6,
- July 10
- May 25,
- June 6,
- June 28
- July 13,
1979
1979
, 1979
1980
1980
, 1980
1980
Mean
.0014
.0018
.0011
.0026
.0008
.0011
.0011
Max
.0064
.0055
.0063
.0188
.0009
.0018
.0017
Min
.0006
.0008
.0006
.0008
.0007
.0007
.0010
*Source: Singh et al., 1980
4-9
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The oast studied mechanism of caroon tetracnloride contaminatidn in fooo
is its use as a fumigant for grain. Much higher carbdn tetrachloride con-
centrations tnan those reported aoove have been found in fumigated grains,
as nigh as 115 mg/kg in wheat and 21 mg/kg in flour (Lynn and Vorches,
1957). Carbon tetrachloride levels decline dramatically in bread baked from
fumigated wheat with residual levels generally reported as 5 mg/kg car-
Don tetracnioride (wit, 1972). The U.S. EPA Pesticide Laooratory oetected
.005 to 2.61 mg/kg carbon tetrachloride in flour from 11 U.S. cities, with
an average level of .051 mg/kg (45 FR 68534-68584). Carbon _tetracnloride
levels of .0002 to .0003 mg/kg were detected in flour in another study.
However, bread and biscuits made from this flour contained undetectable car-
bon tetracnloride (<5 mg/kg) (Bondi and Alumot, 1972).
In several experiments, researchers have simulated commercial fumigation
conditions to determine residual levels of carbon tetrachloride in foods.
Fumigated wneat, aerated for several weeks, was found to contain 20 to 62
mg/kg caroon tetrachloride. Flour made from this wheat was found to contain
2 to 10 mg/Kg caroon tetracnioride, wnereas white bread maoe from the flour
ccntained <0.007 mg/kg (Wit et al., 1972). In another study, caroon
4-10
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tetrachloride at 200 to 400 mg/kg was detected in wheat and corn after
application of ,a fumigant (Scuaamore and Heuser, 1973). Residual caroon
tetracnloride decreased to 1 to 10 mg/kg 6 months after fumigation. By 12
months after fumigation, the wheat and corn contained a maximum of 4.7 mg/kg
of CCl,. Wneat and oarley were analyzed for carDon tetracnloride in a
study oy Sielorai and Alumot (1966). The initial caroon tetrachloriae con-
centrations of 1.53 mg/kg in wheat and 2.2 mg/kg in oarley decreased to 0.7
ana 0.6 mg/Kg, respectively, oy day i7.
CarDon tetracnloride was detected in levels of 76 to 115 mg/kg in wneat,
iO to 21 mg/kg in flour, 28 to 39 mg/kg in oats and 43 to 88 mg/kg in bran
that had oeen fumigated with recommended fumigant dosages (Lynn and Vorches,
1957). In another study, caroon tetracnloride levels were determined in
fumigated wheat and wheat fractions and in bread prepared from the wheat
(3erck, 1974). Levels in wneat ranged between 3.2 mg/kg (7 weeks of aera-
tion) and 72.6 mg/kg (1 week of aeration). Flour, bran and middlings con-
tained 0.2 to 0.93, 0.43 to 3.53 and 0.2 to 1.65 mg/kg, respectively. Bread
contained £0.13 mg/kg carbon tetrachloride. In a similar study, flour
treated at normal fumigant levels was found to contain caroon^tetrachioride
at levels of 0.6 to 1.6 mg/Kg; levels in bran were 2.9 to 5.3 mg/kg
(JagieisKi et al., 1978). Bread baked from the flour contained <.0i mg/Kg
caroon tetracniorioe. Results of these studies inoicatea that the amount of
caroon tetracnloride remaining as a residue is dependent upon the fumigant
dosage, storage conditions, length of aeration and extent of processing.
In addition to its use as a grain fumigant, carbon tetrachloride may
find its way into other foods through the use of herbicides, insecticides
and fungicides containing carbon tetrachloride as a contaminant (0.18 to
0.4%) in the pesticide formulation. Food may also become contaminated by
carbon tetracnloride in the air (45 FR 68534-68584).
4-11
-------
4.3.4. Possible Sources and Levels of Carbon Tetrachloride in Soil. Car-
bon tetrachloride can occur in soil due to spills, runoff and leaching. As
in ground water contamination, CCl^ may find its way into the soil by run-
off from agricultural, dumping and industrial sites and through landfill
leaching.
Waste water treatment of night soil in Japan also resulted in CCl^
formation. Methanol (MeOH)-water-solution substance was fractionated along
with humic, fulvic and hymatomelanic acids from the effluent of the night
soil treatment plant by the use of Amberlite XAD-2. Each of these four
fractions was chlorinated at pH 7.0. Although all fractions primarily pro-
duced CHC13, CC14 was formed by chlorination of the MeOH-water-solution
substance (Ishikawa et al., 1978).
4.4. RELATIVE SOURCE CONTRIBUTIONS
The widespread distribution of carbon tetrachloride in the environment
can lead to exposure to the chemical through water, food and air. Quanti-
ties of carbon tetrachloride potentially taken into the body as a result of
exposure to carbon tetrachlorioe in air, water and food were estimated by
the National Academy of Sciences (1978). Occupational exposure to carbon
tetrachloride was not considered in these estimates. The NAS computations
were based upon data for fluid consumption and respiratory volume for refer-
ence individuals and worldwide per capita food consumption as compiled by
the International Commission for Radiological Protection (ICRP, 1975).
These data are shown in Tables 4-3 through 4-7.
Data on measured fluid intakes for adults and individuals are presented
in Table 4-3, and calculated intakes of milk, water and other fluids for a
typical (reference) man, woman, 10-year-old child and 1-year-old infant are
given in Table 4-4. Respiratory volumes for these reference individuals are
4-12
-------
TABLE 4-3
Measured Fluid Intakes3
Total Fluidsb Milk
Adults
(normal conditions)
Adults
(high environmental
temperature to 32°C)
Adults
(moderately active)
Children
(5-14 yrs)
(ml/ day)
1000-2400
2840-3410
3256*900
3700
1000-1670
(8/yr) (ml/day) ( «/yr)
365-876 120-450 44-164
1037-1245
1188^329
1351
365-610 330-650 120-237
Tap Water Water-Based Drinks0
(m«/day) ( «/yr) (mi/day) (J/yr)
45-730 16.4-266 320-1450 117-529
- - - -
_
540-790 m«/dayd
197-288 «/yrd
aSource: Adapted from ICRP, 1975
^Numbers in these columns are Independent measurements, not totals from the following six columns. Totals of milk, tap water, and water-
based drinks, therefore, do not correspond to total fluid values.
clncludes tea, coffee, soft drinks, beer, elder, wine, etc.
dCombined tap water and water-based drinks. ,
-------
TABLE 4-4
Fluid Intake for Reference Individuals*
Milk
Tap water
Other
Total
fluid
Adult
(mVday)
300
150
1500
1950
Man
(JZ/yr)
109.5
54.8
547.5
711.8
Adult
(mVday)
200
100
1100
1400
Woman
(X/yr)
73.0
36.5
401.5
511.0
Child,
(mi/ day}
450
200
750
1400
10 yr
(*/yr)
164.3
73.0
273.8
511.0
*Source: Adapted from ICRP, 1975
4-14
-------
TABLE 4-5
Respiratory Volumes for Reference Individual*
(in liters of air breathed)
8 hr working,
light activity
8 hr nonoccupational
8 hr resting
Total per day
Total per year
Adult
Man
9600
9600
3600
2.3 x 10"
8.4 x 10s
Adult
Woman
9100
9100
2900
2.1 x 10"
7.7 x 10s
Child,
10 yr
6240
6240
2300
1.5 x 10*
5.5 x 106
Infant,
1 yr
2500 (10-hr)
1300 (14-hr)
0.38 x 10*
1.4 x 10s
*Source: Adapted from ICRP, 1975
4-15
-------
TABLE 4-6
Per Capita Estimates of World Food Consumption by Region3 (g/day)
Food Group
Cereals'3
Starchy
roots0
Sugar0"
Pulses 4 nuts6
Vegetables
& fruitsf
f MeatQ
1
o\
Eggs1"1
Fish1
MilkJ
Fats & oilsk
Far
East
404
156
22
56
128
24
3
27
51
9
Near
East
446
44
37
47
398
35
5
12
214
20
Africa
330
473
29
37
215
40
4
16
96
19
Latin
America
281
247
85
46
313
102
11
18
240
24
Europe
375
377
79
15
316
111
23
38
494
44
North
America
185
136
113
19
516
248
55
26
850
56
Oceania
243
144
135
11
386
312
31
22
574
45
aSource: Adapted from ICRP, 1975
^Flour and milled rice content.
clncludes sweet potatoes, cassava and other edible roots.
^Includes raw sugar; excludes syrups and honey.
eIncludes cocoa beans.
fFresh equivalent.
9Includes offal, poultry and game expressed as carcass weight, excluding slaughter fats.
^Fresh egg equivalent.
^Landed weight.
JExcludes butter; includes milk products as fresh milk equivalent.
fat content.
-------
TABLE 4-7
Summary of Per Capita Estimates of World Food Consumption3
Worldwide
Food Groups
Cereals0
Starchy roots0
Sugar01
Pulses and nuts6
Vegetables and fruits^
MeatsQ
Eggsn
Fish*
MilkJ
Fats and oilsk
Minimum
(g/day)
185
44
22
11
128
24
!>
12
51
9
Maximum
(g/day)
446
473
135
56
516
312
55
38
850
56
Minimum
(kg/yr)
67.5
16.1
8.0
4.0
46.7
8.8
1.1
4.4
18.6
3.3
Maximum
(kg/yr)
162.8
172.6
49.3
20.4
188.3
113.9
20.1
13.9
310.2
20.4
3Source: Adapted from ICRP, 1975
°Flour and milled
cIncludes sweet potatoes, cassava and other edible roots.
^Includes raw sugar; excludes syrups and honey.
elncludes cocoa beans.
fFresh equivalent.
Olncludes offal, poultry and game expressed as carcass weight, excluding
slaughter fats.
nFresh egg equivalent.
^Landed weight.
JExcludes butter; includes milk products as fresh milk equivalent.
fat content.
4-17
-------
reported in Taole 4-5. Food consumption per capita is reported by geograph-
ical regions in Table 4-6; the worldwide profile is presented in Table 4-7.
The data in these taoies summarize human fluid, respiratory ana food intake.
MAS combined these intake data with information on carbon tetrachloride con-
tamination of water, air and food to estimate human exposure to caroon
tetracnloride. A summary of the MAS computations of carbon tetracnloride
intaKe follows.
4.4.1. Water. Tne potential carbon tetrachlorioe uptake from fluids is
snown in Table 4-8. NAS used in its calculations the levels of carbon
tetracnloriae reported in drinking water in the NORS study (Symons et al.,
1975). These levels were <0.003 mg/i. In a later report (the NOMS)
caroon tetrachloride was detected at levels of 0.0024 to 0.0064 mg/Z in
U.S. drinking water (U.S. EPA, 1980a). Estimation of carbon tetrachloride
consumption derived by using the NOMS maximum value of 0.0064 mg/i were
included in the data in Table 4-8. These data were calculated by multiply-
ing the minimum and maximum concentrations in drinking water by the minimum,
maximum and "reference" consumption of drinKing water. Tnese calculations
were based upon the following assumptions:
1. 100% of the carbon tetrachloriae ingested is aosoroea.
2. Commercially-produced drinks and drinks reconstituted in the
home contain the same level of caroon tetracnloriae as does
drinking water.
3. All fiuias contain the indicated caroon tetracnloriae con-
centration.
Values for total fluia uptake include drinks such as milk, not prepared by
mixing with water.
4.4.2. Air. The potential carbon tetrachloride uptake from air is pre-
sented in Table 4-9. The minimum level for carbon tetrachloride in air used
4-18
-------
TABLE 4-8
Carbon Tetrachloride Uptake from Fluids (rag/yr) Calculated by Assuming 100% Absorption3
E xposure
Minimum
(0.002 mg/t)c
Maximum
(0.003 mg/«.)C
Maximum
(0.0064 mg/«d
Fluid
Tap
water
Other11
Total
fluid
Tap
water
Other13
Total
fluid
Tap
water
Other13
Total
fluid
Adult
Minimum
0.03
0.23
0.73
0.05
0.35
1.10
0.10
0.75
2.34
Man, Fluid
Maximum
0.53
1.06
2.70
0.80
1.59
4.05
1.70
3.39
8.65
Intake
Reference
0.11
1.10
1.42
0.16
1.64
2.14
0.35
3.50
4.56
Adult
Minimum
0.03
0.23
0.73
0.05
0.35
1.10
0.10
0.75
2.34
Woman, Fluid
Maximum
0.53
1.06
2.70
0.80
1.59
4.05
1.70
3.39
8.65
Intake
Reference
0.07
0.80
1.02
0.11
1.20
1.53
0.23
2.57
3.27
Child, Fluid Intake
5-14 yr 10 yr
Minimum Maximum Reference
0.15
0.58
0.55
0.73 1.22 1.02
0.22
0.59 0.86
0.82
i.10 1.83 1.53
U.47
1.26 1.84
1.75
2.34 3.90 3.27
aAdapted from NAS, 1978
^Includes water-based drinks such as tea, coffee, soft drinks, beer, cider, wine.
cCalculated by multiplying the exposure concentration (mg/l) x fluid Intake («/yr) (from Table 4-3 for minimum and maximum intakes and
Table 4-4 for reference intakes). ,
dSimilar calculations based upon NOMS maximum drinking water levels of 6.4 \g/l.
-------
TABLE 4-9
Estimated Human Uptake of Carbon Tetrachloride from the Outdoor Atmosphere3*0
(mg/yr)
i
M
O
Average inhaled0
Minimum absorbed01
Maximum absorbed^
Average inhaled0
Minimum absorbed^
Maximum absorbed^
Adult Man, Exposure
Minimum Typical
6.3 7.9
3.6 4.5
4.1 5.2
Child, 10 yr, Exposure
4.2 5.2
2.4 3.0
2.7 3.4
Adult Woman, Exposure
Maximum
951
542
618
623
355
405
Minimum
5.8
3.3
3.8
Infant,
1.1
0.6
0.7
Typical
7.3
4.1
4.7
1 yr, Exposure
1.3
0.6
0.9
Maximum
872
457
567
159
90.0
103
aSource: Adapted from NAS, 1978
hfhe absorption range used is that reported by Lehmann and Schmidt-Kehl (1936). Based upon human data,
the range given is 57 to 65%, calculated from the concentration of carbon tetrachloride in the inhaled
air and that found in the exhaled breath.
cCalculated by applying respiratory volume per year (see Table 4-5) to the general range of atmospheric
concentrations.
'-'calculated by applying the range of percent absorptions to the total amount inhaled per year.
-------
by NAS was 0.00075 mg/m3, the typical level was 0.00094 mg/m3 and the
maximum level was 0.113 mg/m3, reported by Lillian et al. (1975) for a
monitoring sample for Bayonne, New Jersey. These values were multiplied by
the average quantity of air inhaled per year (from Table 4-5) to arrive at
the average quantity of carbon tetrachloride inhaled per year. In the NAS
report, this quantity was then multiplied by the minimum and maximum values
of the absorption range for carbon tetrachloride in air as reported for
humans by Lehmann and Schmidt-Kehl (1936). These calculations resulted in
values for the minimum and maximum yearly absorption of carbon tetrachloride
from air. The issue of percent absorption of CCl^ has not been clearly
defined. As discussed in Section 7.1, the percent absorbed may vary between
species. The range reported by Lehmann and Schmidt-Kehl and used in the NAS
report, is retained here since it is the only human data available. See
Section 7.1 for a more detailed discussion on absorption of CCl^.
4.4.3. Food. The potential carbon tetrachloride uptake from foods is
presented in Table 4-10. Values of carbon tetrachloride found in foods and
used in these calculations are presented in Table 4-11. To arrive at uptake
values, the minimum and maximum levels of caroon tetrachloride in foods were
multiplied by the minimum and maximum worldwide food intake for individuals
for each food category. NAS calculated a per capita uptake of 0.21 to 7.33
mg carbon tetrachloride from food each year.
The NAS absorption calculations did not include the yearly uptake
through the consumption of bread. The average consumption of carbon tetra-
chloride attributed to bread has been calculated elsewhere as 777 ng/day or
0.3 mg/yr (45 FR 68534-68584). The data on consumption in bread are includ-
ed in the summary of carbon tetrachloride uptake from all sources
(Table 4-12).
4-21
-------
TABLE 4-10
Carbon Tetrachloride Uptake from Food Supplies (mg/yr),
Calculated by Assuming 100% Absorption3>b
Food Group0
Milk products
Eggs
Meats
Fats and oils
Vegetables and
fruits
Fish and seafood
Total, all food
supplies
Worldwide
Food Intaked
Minimum
Maximum
Minimum
Maximum
Minimum
Maximum
Minimum
Maximum
Minimum
Maximum
Minimum
Maximum
Minimum
Average
Maximum
Minimum
0.004
0.06
_ _
0.06
0.80
0.002
0.01
0.14
0.56
<0.001
0.001
0.21
Exposure
Average Maximum
0.26
4.34
0.001
0.01
0.08
1.02
0.06
0.37
0.37
1.51
0.03
0.08
1.12
7.33
aSource: Adapted from NAS, 1978
Calculated by applying worldwide food intake ranges for various food
groups (see Table 4-7) to the range of concentrations found in various
foods (see Table 4-10).
°As used in Tables 4-6 and 4-7.
Table 4-7.
4-22
-------
TABLE 4-11
Summary of Carbon Tetrachloride Concentrations in Food Supplies*
(ppb by weight, ug/kg)
Food Group
Milk products
Eggs
Meats
Fats and oils
Vegetables and fruits
Fish and seafood
Carbon
Minimum
0.2
—
7.0
0.7
3.0
0.1
Tetrachloride
Average Maximum
14.0
0.5
9.0
18.0
8.0
6.0
*Source: Adapted from McConnell et al., 1975; Pearson and
McConnell, 1975
4-23
-------
TABLE 4-12
Relative Uptake of Carbon Tetrachloride from the Environment by Adult Male3
N>
Minimum Exposure*3
Typical Exposure0
Maximum Exposure'-'
Source
Fluid intake
Atmosphere
Food supply
Total
(mg/yr)
0.73
3.60
0.21
4.54
(% of total)
16
79
5
—
(mg/yr)
3.13
4.75
1.42
9.30
(% of total)
34
51
15
—
(mg/yr)
8.65
618
7.63
634.28
(% of total)
1
98
1
aSource: Adapted from NAS, 1978
DFor minimum conditions: assume minimum intake for fluids, minimum absorption from atmosphere, and min-
imum intake for food supplies.
cFor typical conditions: assume reference man intake for fluids, 0.0044 mq/SL as average of concentra-
tions in NOMS study; for intake from atmosphere, assume average of minimum and maximum absorption rates
for typical exposure; for intake from food supplies, assume average exposure and intake from NAS, 1978
plus 0.3 mg/yr from bread consumption (45 FR 68534-68584).
dFor maximum conditions: assume maximum intake for fluids, carbon tetrachloride at 0.0064 mg/Jl as in
NORS study; maximum absorption from atmosphere; and maximum intake from food from NAS, 1978 plus 0.3
mg/yr from bread consumption (45 FR 68534-68584).
-------
4.4.4. Soil. The potential uptake of carbon tetrachloride from soil is
unknown. This includes agricultural runoff as well as uptake from plants.
4.5. SU*WRY
Carbon tetrachloride is produced commercially from the chlorination of
methane, propane, ethane propylene and carbon disulfide. Production has
declined over the last 10 years and a 1.0%/year decline is projected through
1985.
CC1. is ubiquitous in the environment and has been detected in concen-
trations of generally <.01 mg/x, in water, <.01 mg/m3 in air and
<.01 mg/kg in food. Higher levels of carbon tetrachloride have been
detected in urban air, and in grain and food products made from grain.
Increased levels of carbon tetrachloride in urban air are probably due to
the industrial use of carbon tetrachloride and products containing carbon
tetrachloride. The presence of carbon tetrachloride in grains or grain
products is due in part to the use of fumigants containing carbon tetra-
chloride.
Data on the uptake of carbon tetrachloride by the adult male are summa-
rized in Table 4-12. At minimum exposure levels, uptake .from the air
appears to be the major source of exposure (79%), followed by fluids (16%)
and the food supply (5%). At typical exposure levels, uptakes from fluid
(34%) and food (15%) appear to increase with respect to that from air (51%).
At maximum exposure levels, 98% of carbon tetrachloride uptake is estimated
to be from air. Amount of uptake from soil is unknown.
All of the carbon tetrachloride in the environment can be accounted for
by anthropogenic activities. Carbon tetrachloride, unlike chloroform and
other halocarbons, does not appear to be indirectly formed during water
chlorination. No natural sources of carbon tetrachloride are known.
4-25
-------
-------
5. FATE AND TRANSPORT
5.1. FATE
5.1.1. Water. Carbon tetrachloride tends to evaporate from dilute aque-
ous solutions, with a half-life of only 29 minutes (Dilling et al., 1975).
Chemical stability in aquatic environments is hign, witn half-lives of 7 x
10Vc years, where C is concentration in mg/Jl. This formula is derived
from the second order rate constant of 4.8 x lO'Vmoi'Vsec"1 (Maoey
and Mill, 1978). Singn et al. (1976) give an approximate nydrolytic nat-
ure for caroon tetracnioride in oceans as 7 x 101* years. This figure is
lower than that predicted using measured ocean concentrations (Pearson ana
McConnell, 1975) ana the ddove second order formula. For the maximum Atlan-
tic Ocean concentration of 2.4 x 10"6 mg/Jl, the half-life is 2.9 x id6
years. In any case, carbon tetracnioride is extremely staole in water, with
losses primarily due to other factors such as evaporation, sediment adsorp-
tion and organism uptake. Singh et al. (1976) estimate that 2 to 3& of all
Diospneric caroon tetrachloriae is in solution. Although carbon tetrachior-
iae is not easily transported to ground water due to its high volatility,
low solubility ana low mobility in soil, any contamination is likely to per-
sist for several years and accumulate.
5.1.2. Air. Chemical staoility, a long lifetime, and uniform mixing
cnaracterize the fate of carbon tetrachloride in air. Mixing is assumed to
be uniform to approximately 18 Km elevation, witn a pseuao-first oraer re-
moval rate of 1 x 10"3 yr~l, mainly via gas-pnase reactions involving
tne electronic state 0(1D) (Galoally, 197&). Tne principal sinn is
considered to be tne stratosphere (Galbally, 1976; Singn et al., 1976), with
significant loss of caroon tetracnioride limitea by the rate of transport
from the tropospnere. Above 18 km, Galbaliy estimates that stratospneric
5-1
-------
pnotcuysis is dominant, witn a pseudo-first oraer rate constant or
1.7 x 1C"2 yr"1. This pnotodissociation is attributed to UV radiation
mainly in tne 19s to 225 nm region. Gaioaily estimates 50% errors in eacn
of tne aoove rate constants.
UV photolysis was also shown experimentally oy Lillian et al. (1975)
using a 1 I Hanovia photochemical reactor with zero air and a high pres-
sure mercury vapor lamp (0.034 W/cm2 in the 220 to 230 nm region). Simu-
lated tropospheric irradiation produced no discernable dissociation of car-
bon tetrachloride in NO^-air mixtures. The lifetime (time to reach 1/e of
initial concentration) of carbon tetrachloride in the stratosphere is esti-
mated at 53 hours (Lillian et al., 1975). The tropospheric lifetime has
oeen estimated at 330 years (Singh et al., 1976). Tne overall atmuspneric
lifetime of carbon tetracnloride is estimated at 60 to 100 years oy Singh et
al. (1976) and "several decades" by Gaibaliy (1976).
Carbon tetrachioriae in tne stratosphere is considered to De a potential
source of chlorine via photooissociation wnich may, on a moiecule-oy-moie-
cule oasis, have tne potential approximately as strong as CFC-11 and CFC-12
to catalyze tne destruction of the ozone layer (Hanst, 1978; NAS, 1978).
The proposed mechanisms and the potential for destruction of ozone are ten-
tative due to the paucity of data on stratospheric carbon tetrachloride and
to the uncertainties in the structure and evaluation of the transport models
(Sugdon and West, 1980).
5.1.3. Soil. Pertinent information concerning the degradation or trans-
formation of carbon tetrachloride in soil could not be located in the avail-
aole literature. Gaibaliy (1976) calculates that caroon tetracnloride in
ail land Diota is probaoly <0.2% of that in the atmosphere and concludes
that lana biota are an insignificant siriK for caroon tetrachioriae.
5-2
-------
5.2. TRANSPORT
5.2.1. Water. Carbon tetrachionoe introduced into water resdurces will
be transported by movement of surface and grdund water, and by evaporation
into the atmosphere. Studies in N.W. England snow transfer of carbon tetra-
cnloride from tne atmosphere to surface water due to heavy rainfall (McCon-
nell, 1976). In dilute aqueous solutions, the evaporative half-life is 29
minutes (Dilling et al., 1975). Large, concentrated duantities, however,
tend to remain as one mass or "slug". A spill into the Ohio River of ap-
proximately 70 tons of carbon tetrachloride produced a slug that was esti-
mated to be 74 miles long (U.S. Congress, 1977). Degradation of caroon
tetrachloride by hydrolytic reactions is slow; the approximate half-life is
7000 years (Mabey and Mill, 1978). Thus, vdlatilization is considered to be
the main process for removing carbon tetrachloride from aquatic systems.
5.2.2. Air. The high vapor pressure, low water solubility and nign spe-
cific gravity of carbon tetrachloride influence its disposition in air envi-
ronments. Volatilization to the atmosphere from land and water should be
rapid. Actual transport in the air, however, has been infrequently demon-
strated. Penkett et al. (1979) found higher atmospheric values over Great
Britain during easterly winds than during westerly winds, reflecting the
difference in anthropogenic activity under the two trajectories. Other
reports show minor global variation in concentration of carbon tetrachloride
(Singh et al., 1976; Lovelock et al., 1973). Vertical transport from the
lower atmosphere to the stratosphere is estimated at roughly 25%, assuming
an overall atmospheric lifetime of 75 years and a quasi-steady state loading
(Singh et al., 1976). Neely (1977) modeled the transpdrt of carbon tetra-
cnioride vertically from the ocean through the troposphere to tne stiato-
sphere, and horizontally between northern and southern hemispheres. Trans-
5-3
-------
fer between hemispheres was considered substantial, with a first-order rate
constant (in each direction) of 0.9 yr"1. Neely's simulation results
agreed well (within 20%) with observed concentrations.
5.2.3. Soil. There is little quantitative information about adsorption
of carbon tetrachlonae onto seaiments. Following Bnggs (1973), tne sorp-
tion potential can be estimated by the following formula:
log Q = 0.524 log P + 0.618
where P is the octanol/water partition coefficient and Q is the organic
matter/water partition coefficient. For caroon tetracniorioe, log P = 2.64,
and thus Q = 100.3. This value suggests that carbon tetrachloride has "low"
mobility in soil, as defined by Briggs (1973), indicating that it can move
with soil, sediment or oarticulate matter. Morris and Johnson (1976) state
that periods of high agricultural runoff (river turoioity) are directly cor-
related with high chloroform concentration in finished water and imply that
similar associations hold for carbon tetrachloride and naloforms in general.
Statistical analysis of their data, however, shows poor linear correlation
(r = 0.06) between river turbidity and carbon tetracniorioe levels, with
strong peaks occurring during periods of both low and high turbidity. Pear-
son and McConneli (1975) investigated chlorinated hydrocarbons in marine
environments, but could not show any significant relationships between lev-
els in seoiment and levels in overlying seawater, sediment particle size or
geographic features. More research is clearly needed on transport via rain-
fall, directly from the atmosphere and indirectly through agricultural run-
off, in order to assess the importance of soil for transport of carbon
tetrachloride.
5-4
-------
5.3. BIOACCUMULATION/BIOCONCENTRATION
The data of Kopperman et al. (1976) suggest that polar organocnlorine
compounds are easily biodegraded, while non-polar, highly lipophilic com-
pounds accumulate. Bioaccumulation is directly related to the octanol/water
partition coefficient (P) of the compound (Neely et al., 1974). The log
octanol/water partition coefficient (log P) of carbon tetracnloride is 2.64
(Leo et al., 1971; Neely et al., 1974; Chiou et al., 1977), inoicating a
possible tendency for this compound to bioaccumulate unoer conoitions of
constant exposure. However, although carbon tetracnloride ana other organo-
cniorines are lipopnilic and tend to concentrate in fatty tissues, there is
no evidence that they magnify through the food chain (Pearson and McDonnell,
1975). Difficulties in the Pearson and McConnell (1975) study discourage
estimates of bioaccumulation based on their results. Barrows et al. (1980)
reported a carbon tetracnloride bioconcentration factor (BCF = tissue con-
centration divided by water concentration) of 30 for bluegill sunfish
(Lepomis macrochirus) and a tissue half-life of <1 day. Tne authors
stated tnat the short tissue half-life makes it unlikely that carbon tetra-
chloride will biomagnify in fish unless exposure is continuous or prolonged.
Neely et al. (1974) estimated the steady state BCF for rainbow trout (Salmo
gairdneri) to be 17. Data could not be found for bioconcentration of carbon
tetrachloride in shellfish.
5.4. SUMMARY
Transport and fate of carbon tetracnloride have been studieo most exten-
sively in air, with some information on water levels and almost no data on
soil. Carbon tetracnloride is extremely stable in water, the lower atmo-
sphere and the troposphere. Photodissociation in the stratosphere is rapid.
5-5
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Glooal distribution of carbon tetrachloride in air is nearly uniform. Bio-
concentration in fish is low. Research needs are most pronounced for fate
in soil, transport to and fate in the stratosphere, and bioaccumulation/oio-
concentration in shellfish.
5-6
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6. ECOLOGICAL EFFECTS
Data on the ecological effects of caroon tetrachloride are somewhat lim-
ited. The reasons for this can be inferred from the physical and chemical
properties of the compound as discussed in Chapter 3. Carbon tetrachloride
is quite volatile and so does not readily accumulate in either terrestrial
or aquatic environments and is rapidly diluted to low concentrations in the
troposphere. While major spills of carbon tetracnloriae have occurreo, eco-
system effects have not been documented or have not been considered to oe so
significant as to merit further investigation (NAS, 1973). Apparent acute
effects nave oeen minimal and, consequently, cnronic effects on fish and
wilolife associated with long-term exposures at low levels are unlikely.
6.1. EFFECTS ON NON-TARGET ORGANISMS
Because carbon tetrachioride is used as a pesticide, primarily as a
grain and soil fumigant, non-target soil organisms are undoubteoly affected
(NAS, 1978). The interaction of carbon tetrachlorioe with anaerobic organ-
isms has oeen studied by Wood et al. (1968) using extracts of Methanooacil-
lus omelianskii and the N5-methyl tetrahydrofolate-nomocysteine trans-
methylase of Escherichia coli B. The authors founo that low concentrations
of caroon tetrachloride inhibit cobamide-dependent metnyl transfer reactions
in tnese cell extracts. The possibility that caroon tetracnioriae mignt
innioit metnyi transfer systems of microbiai organisms in nature snoulo be
investigated, although there is no imminent hazard (NAS, 1978).
6.1.1. Aquatic Life Toxicology. The majority of the acute toxicity oata
for carbon tetracnlorioe ano aquatic organisms has been determined using
static procedures witn unmeasured test concentrations. Results of these
tests may underestimate the acute toxicity of carbon tetracnlorioe due to
its volatility. No acute or chronic effects were observed at a concentra-
tion lower than 3400
6-1
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6.1.2. Acute Toxicity. Tne 48-hour EC5Q is 35,200 ug/i for Dapnnia
magna (Table 6-1). The bluegill has been tested (Dawson et al., 1977; U.S.
EPA, 1978) and the 96-hdur LC5Q values are 125,000 and 27,300 vq/l,
respectively (see Table 6-1). The reason for this large difference is not
clear, but may have been caused by the volatility of this compound. There
appears to be no great difference in sensitivity between the two tested spe-
cies. A flow-through test result for the fathead minnow is 43,100 v6/£.
However, no comment can be made concerning tne effect of test conditions on
test results.
Carbon tetrachloride (1 rnA/kg, i.p.) produced a 5- to 10-folo increase
in serum GOT, GPT and ICO enzyme activities in rainbow trout (Salmo qairo-
neri), whereas exposure of trout to CCl^ in tank water (1 to 80 mg/i)
produced neither mortality nor changes in serum enzyme activities (Statham
et al., 1978). Tissue residue analysis revealed the highest concentration
of CCl^ in the adipose tissue followed by levels in the liver, brain,
spleen and gills irrespective of tne administration route. Elimination
14
rates of C residues occurred at 2 hours for both i.p. and ambient water
exposure routes and were 4.8 and 0.75 unol/g, respectively.- Histologic
examination of tissues revealed varying degrees of liver and splenic neuro-
sis 6 hours following administration of CC14 (Statham et al., 1978).
A single i.p. injection of CC14 resulted in a significant decrease in
total plasma protein concentration of rainbow trout at 12, 24 ana 36 hours
post-treatment (Pfeifer and Weber, 1981). Plasma albumen was also reduced
significantly at tne 2 m£/kg dose level. The authors suggested that sev-
eral factors, including intestinal inflammation and hemorrhage, may have
contributed to the observed effects on plasma protein concentration. In an
earlier study (Pfeifer and Weber, 1980) using tne same species, it was
6-2
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TABLE 6-1
Acute Values for Carbon Tetrachloride
Species
Cladoceran,
Daphnia magna
Fathead minnow,
Pimephales promelas
Bluegill,
Lepomis macrochirus
Bluegill,
Lepomis macrochirus
Tidewater silversides,
Menidia beryllina
LC5Q/EC5Q Species Acute
Method* Value
(VQ/D (UQ/W
FRESHWATER SPECIES
S, U 35,200 35,200
FT, M 43,100 43,100
S, U 125,000
S, U 27,300 58,000
SALTWATER SPECIES
S, U 150,000 150,000
Reference
U.S. EPA, 1978
Kimbali, manuscript
Dawson et al. , 1977
U.S. EPA, 1978
Dawson et ai., 1977
*S = static, FT = flow-through, U = unmeasured, M = measured
No Final Acute Values are calculable since the 'minimum data base requirements are not met.
-------
determined that a single i.o. dose of CCi. (2.0 mi/kg) oroouceo eitner
oliguria or anuria as early as 1 nour post-treatment. A significant reduc-
tion in urine flow as well as a significant increase in relative wet body
weight were noted. Histological examination of kidney tissue at 24 hours
oost-treatment revealed early localized pathological changes, although
extensive morphological damage was not evident. This, in conjunction with
the early oliguria, suggests that the observed reauction in urine flow rate
was not due to a direct toxic effect on the kidney (Pfeifer and Weber, 1980).
Morphological changes in the liver of two species of carp (Cyprino
carpio and Carassius auratus) were observed following i.p. injection of
CCI. at 0.3 to 5.0 mJl/kg for at least 8 days (Jiang and Zhang, 1979).
The following changes were noted in tne liver tissue of both species:
(1) proliferation in number of vacuoles, (2) nuclei enlargement in some
cells, and (3) contraction and deformation of the nuclear membrane. These
findings are in accord with those of studies discussed previously tnat
reported decreased plasma protein concentrations ana, consequently, reflect
the hepatotoxicity of CCl^.
Only two saltwater fisn and no invertebrate species have been tested.
The 96-nour LC5Q for the tidewater silversides (Menidia beryllina) is
150,000 ug/& (see Table 6-1). Tne other datum is an estimateo 96-nour
LC5Q for the dab (Limanda limanda) of about 50,000 vQ/l.
6.1.3. Chronic Toxicity. No chronic test has been conducted with a
freshwater invertebrate species or any saltwater species. An embryo-larval
test with the fathead minnow (Pimephales promelas) was conducted, and no
adverse effect was observed at carbon tetrachloride concentrations up to
3400 pg/A (U.S. EPA, 1978).
6-4
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6.2. TISSUE RESIDUES
The bluegill (Lepomis macrochirus) bioconcentrated carbon tetrachloride
at equilibrium to a factor of 30 times within 21 days. The biological half-
life in these tissues was less than 1 day. In addition, Neely et al. (1974)
exposed the rainbow trout to carbon tetrachloride and estimated a steady-
state BCF of 17. Similarly, Barrows et al. (1978) determined a BCF for
bluegill sunfish of 30. These results indicate that tissue residues of car-
oon tetrachloride would not pose a potential environmental hazard to aquatic
life.
6.3. INDIRECT ECOSYSTEM EFFECTS
Indirect ecosystem effects of carbon tetrachloride (e.g., those caused
by enhanced UV-B radiation) are similar to those of other halocarbons and
result from photodissociation of the compound and subsequent destruction of
stratospheric ozone (NAS, 1978, 1982). For more discussion of these
effects, see Chapters 5 and 11.
6.3.1. Effect on Stratospheric Ozone. Carbon tetrachloride may contrib-
ute to an overall reduction in stratospheric ozone. Following a relatively
rapid tropospheric mixing with other halogenated methanes, there is a rela-
tively slow entry of CC14 into the stratosphere, followed by a random
ascent to altitudes (in excess of 25 km) where solar ultraviolet (UV) radia-
tion in the range 185 to 225 nm photodissociates CC14 resulting in the
generation of "odd chlorine" (i.e., a variety of chlorine-containing spe-
cies) (NAS, 1978). Dependent upon the rates of a variety of chemical reac-
tions and the rate of odd chlorine-to the troposphere, each odd chlorine is
responsible for the destruction of several thousand ozone molecules. It has
been estimated that CC14, along with hydrochloric acid (HC1) and methyl
chloride (CH3C1), contributes <1% of the total reduction in strato-
6-5
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spheric ozone, comparable to the rate of ozone reduction attributed to
chlorofluoromethanes. The relative importance of HC1 and CH,C1 is
reduced, however, due to atmospheric "rainout" and degradative processes,
respectively (MAS, 1978).
6.3.2. Effect on UV Flux. As indicated above, changes in stratospheric
ozone content result in alterations of UV flux to the earth's surface. This
involves the biologically damaging wavelengths in the 290 to 320 nm range
(UV-8 regions). The molecular basis of the effects of these wavelengths is
tne alteration of protein and nucleic acid structures impacting both genetic
replication and protein synthesis mechanisms (NAS, 1978). While there exist
molecular repair processes that mitigate this damage, they are not totally
effective.
Increased UV-B radiation adversely affects a variety of plant species in
terms of depressed photosynthetic activity and reduced growth rate. Studies
of both agriculturally significant plants and higher non-agricultural spe-
cies (cited by NAS, 1978) demonstrate a diverse response to enriched levels
of UV radiation. These findings include, in addition to depressed photosyn-
thetic activity, inhibition of seed germination and increased somatic muta-
tion rates. However, adaptation of these plant species appears to be suf-
ficient under current ambient levels to maintain food crop yields (NAS,
1982). Although effects on natural ecosystems are difficult to predict, the
ecological impact of such effects may be significant to natural communities
taken collectively, even if only a few constituent species are affected.
The magnitude of such effects cannot be predicted since natural populations
have adapted to current ambient levels to maximum reproduction potential
6-6
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(NAS, 1982). In addition to the direct effect of UV-B wavelengths on these
species, effects on interacting and/or dependent species must be considered
(NAS, 1978) in assessing ecosystem effects.
6.4. SUMMARY
Only two freshwater fish and one invertebrate species have been acutely
tested ana a 96-hour LCco has been determined as low as 27,300 ug/x..
No definitive chronic data are available. Tissue residues of carbon tetra-
chloride do not appear to be a problem since available data suggest a BCF
of <30.
The available data for carbon tetrachloride indicate that acute toxicity
to freshwater aquatic life occurs at concentrations as low as 35,200
MgA and would occur at lower concentrations among species that are more
sensitive than those tested. No data are available concerning the chronic
toxicity of carbon tetrachloride to sensitive freshwater aquatic life.
The available data for carbon tetrachloride indicate that acute toxicity
to saltwater aquatic life occurs at concentrations as low as 50,000 ugA
and would occur at lower concentrations among species that are more sensi-
tive than those tested. No data are available concerning the chronic toxic-
ity of carbon tetrachloride to sensitive saltwater aquatic life.
Indirect effects of CCl^ are associated with reduced levels of atmos-
pneric ozone and concomitant increases of UV-8 radiation flux. There are
known differences in species sensitivities to increases in UV-B radia-
tion. Laboratory studies have identified effects associated with such in-
creases. However, it appears that in the natural ecosystem, some plants and
animals have adapted to current ambient levels. Thus, the magnitude of
effects of enhanced UV-B radiation cannot be predicted either collectively
or for individual species in the natural environment.
6-7
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7. COhFOUND DISPOSITION AMD RELEVANT PHARWCOKINETICS
Tnis chapter is divided into four sections: absorption, distribution,
metaoolism and excretion of caroon tetrachloride. The absorption section
begins witn a discussion of the chemical's partition coefficients. As would
oe expected for a compouno with high partition coefficients for oil/air ana
oil/water, caroon tetrachloride is reported to be aDsorbed readily through
the lungs, ana also through the intestinal tract ana skin. Once aosoroea,
tne chemical and its metaooiites are reported to be distriouted widely
throughout the body, witn hign concentrations in liver, oone marrow, blood,
muscle, fat and brain. Several metabolites of CCl^ have oeen identified,
including chloroform, hexachloroethane and caroon dioxide. Carbonyl cnlor-
iae has been postulated as a meraoolite oy inference based on an _in vitro
stuay of a [ Cjcaroon tetrachloride incubation system. CC1, metabolism
nas oeen proposed to involve a complex with ferrocytochrome heme and forma-
tion of the free radical CC1,. Carbon tetracnloride ana its metabolites
are reported to be excreted principally in exhalea air, but also in urine
and feces.
7.1. ABSORPTION
7.1.1. Partition Coefficients. Partition coefficients for various chlor-
inated solvents, including caroon tetrachlorioe, were determined by several
experiments (Morgan et al.,. 1972; Sato and NaKajima, 1^79; Powell, 1^45).
The partition coefficient is a measure of the relative soluoility of a SUD-
scance in two meaia. The oil/air ana oil/water partition coefficients can
oe used as inaicators of soluoility in lipids. The values of these and
other partition coefficients for carbon tetrachloride, listed in Table 7-1,
snow this chemical to oe lipophilic. Because of its lipopnilic nature, one
7-1
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TABLE 7-1
Partition Coefficients for Carbon Tetrachloride*
Partition Coefficients
Parameter
Olive oil/ air
Blood serum/ air
Blooo/air
rtater/air
Olive oil/ water
Olive oil/ serum
Olive oil/blood
20°C 25°C
142
6
3.6-5.2
0.25
1440
23
— — — —
37°C
361
_••*
2.4
—
—
«
150
*Source: Adapted from Morgan et al., 1972; Sato and
Nakajima, 1979; Powell, 1945
7-2
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would predict that carbon tetrachloride could be absorbed by ingestion,
innaiation and skin contact. This prediction is borne out by results of the
experimental studies described below.
7.1.2. Absorption from the Gastrointestinal Tract. Aosorption of carbon
tetrachloride from the gastrointestinal tract of dogs was studied by Robbins
(1929). In a series of experiments, the author determined the amount of
carbon tetrachloride adsorbed from the ligateo stomach, small intestine and
colon oy measuring the concentration of carbon tetrachloride exnaled. The
greatest concentration of caroon tetrachloride in exnaied air was seen after
injection of the chemical into the small intestine. Direct injection of
caroon 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 1 part in 10 by volume of expired air. Thus, the re-
sults of the experiment can be viewed as a qualitative indication of rela-
tive absorption from the various components of the gastrointestinal tract,
rather than as quantitatively accurate results.
The enhancement in the extent of carbon tetrachloride absorption with
ingestion of fat or alcohol has been reported (Nielsen and Larsen, 1565;
Roooins, 1929; Moon, 1950). It also appears that the absorption of carbon
tetrachioride from tne gastrointestinal tract may vary witn different spe-
cies oecause it occurs more quickly in rabbits than in dogs (Lamson, 1923).
Marcnland et al. (1970) studied the effect of SKF 525A on the distribu-
tion of an oral carbon tetrachlorioe dose on rats. Control animals exposed
only to caroon tetracnloride were found to excrete at least 80% of tne oral-
ly administered dose within 10 hours via the lungs. This indicates that at
least 80% of the carbon tetrachloride dose was absorbed orally.
7-3
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Stokinger and Woodward (1958) report an "accurate" aosorption factor of
50/b via ingestion, nowever, tney do not reference their conclusion.
7.1.3. Absorption by Inhalation. In 1930, von Oettingen et al. studied
the aDsorption of carbon tetrachlorioe by inhalation in beagle dogs. The
sex was unspecified, but the autnors stated that at least five aogs were
usea in each experiment. The dogs inhaled carbon tetrachloride (purity un-
specified) at a concentration of 94,500 mg/m3 for 475 minutes tnrougn 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 tetracnloride in
olood reached a maximum of 31.2 to 34.3 mg/100 cc (0.20 to 0.22 millimole %)
after =300 minutes of exposure and remained at that level for the duration
of the exposure.
McCoilister et al. (1951) investigated the ausorption of caroon tetra-
chioride by inhalation using Rhesus monkeys. Three female monkeys inhaled
9ij.9Vo """ C-labelea carbon tetrachloride vapor at an average concentration
of 29Q mg/m3 for 139, 344 or 300 minutes, respectively. The autnors cal-
culated by difference between inhaled and exhaled air tnat the monkeys
aDsorbeo an average of 30.4% of the total amount of carbon tetrachloride
innaled. Analysis of blood drawn after 270 minutes of exposure showed tnat
the C radioactivity was equal to 0.297 mg caroon tetrachioride/100 g of
blood, distributed as follcws: 56.2% as carbon tetrachloride, 16.5% as
"acid-volatile" caroonates and 27.3% as nonvolatile material. No attempt
was made to characterize metabolites in this study. The radioactivity no
longer associated with the carbon tetrachloride was described by the stage
in the analytical procedure where it was found.
7-4
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Stokinger and Woodward (1958) report an "accurate" absorption factor of
30% via inhalation, nowever, they do not reference their conclusion. Leh-
mann and Schmidt-Kehl (1936) studied humans exposed to CCl^ via inhala-
tion. In separate experiments, individuals (number unknown) were enclosed
in a room of CCL vapors for varying amounts of time. The amount of
CCL available for inhalation was measured. The percent absorbed was
calculated from the concentration of CCL in the inhaled air minus tne
amount found in tne exhaled breath. The reported range of percent apsorp-
tion was 57 to 65%.
7.1.4. Absorption Through the Skin. McCollister et al. (1951) exposed
14
the sKins of one male and one female Rhesus monkey to C-labelea carPon
tetracnloride vapor. To determine the amount of adsorption, blood and
exhaled air were analyzed for C radioactivity. After a skin exposure of
240 minutes to carbon tetrachloride vapor at 3056 mg/m3, the Plood of the
female monkey contained carbon tetrachloride at 0.012 mg/100 g and the
exhaled air contained 0.0008 mg/l. After exposure to 7345 mg/m3 for 270
minutes, blooo of the male monkey contained carbon tetrachloride at 0.03
mg/100 g and the exhaled air contained 0.003 mg/fc.
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). The carbon tetrachlorioe was analyzed by infrared
spectroscopy and was found to contain no detectable impurities. The concen-
tration of carbon tetrachloride in alveolar air was used as the inoicator of
absorption and was measured at 10, 20 and 30 minutes after the start of
exposure and at 10, 30, 60, 120 and 300 minutes after cessation of expo-
sure. Carbon tetrachloride was present in the alveolar air at each time
interval, reached a maximum concentration range of 2.8 to 5.7 mg/m3 30
7-5
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minutes after exposure, ana decreased exponentially thereafter. The autnors
concluded that carbon tetrachloride could be adsorbed by the skin in toxic
amounts if the cnemical came in contact witn arms and hands.
7.2. DISTRIBUTION
Tne distrioution of carbon terrachioride in humans, presumaoly after
cnronic low level exposure through various meoia (food, water ana air), has
oeen reported by McConnell et al. (1975). Measurements were maoe from post-
mortem tissues of several individuals; arithmetic means (in mg/kg) were
aoout: oody fat, .008 (range .002 to .024); liver, .003 (range .001 to
.005); and kidney, .002 (range .001 to .003). Unfortunately these values
represent possible concurrent tricnloroetnane concentrations.
Robbins (1929) administered 159 g (100 cc) carbon tetrachloride, purity
unspecified, to three anesthetized dogs by stomacn tuoe. The dogs were
sacrificed at 6, 23 and 24 hours after treatment. Blood ana various tissues
were analyzea for caroon tetracnioriae by converting the organic chloride to
inorganic cnloriae and titrating tne inorganic chloride oy the Volhard
metnoo, whicn is accurate to 0.1 to Q.2%. 'Tne results of tne blooo ana tis-
sue analysis are shown in Table 7-2. Note tnat the uptaKe_ of CCl^ was
prooaoly reduced oecause of probaole reduced G-I (portal) circulation during
anestnesia (Witney, 1981).
From the experimental data, it appears the limit of detection was in tne
range of 4 to 5 mg caroon tetracnloride/100 g of tissue. In addition, it
appears that the liver, bone marrow, blood and muscle retained the most car-
bon tetrachloride for the longest time.
In 1950, von Oettingen et al. reported the tissue distribution of caroon
tetrachloride in beagle dogs, each weighing =10 kg, exposed to carbon
tetracnloride in air at 94,500 mg/m3 for 475 minutes. The dogs were
7-6
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TABLE 7-2
Caroon Tetracnloride Distrioution at Various Times in Anesthetized
Dogs After Administration Dy Stomach Tube (mg/100 g of tissue)*
Tissue 6 hrs 23 hrs 24 hrs
Brain — 17 9
Biood, portal 26 13 22
Blood, arterial 00 0
Bone marrow — 66
Kidney — 11 13
Liver 15 10 27
Lungs — trace 6
Muscle — trace 20
Pancreas ~ 4.5 14
Spleen — 5
*Source: Roobins, 1929
7-7
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sacrificed at the eno of the exposure. Tissue ana blood samples were taken
ana analyzed for carbon tetrachloride. The concentration of caroon tetra-
chloride (per 100 g of tissue) was 66 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 con-
centration 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 et al. (1951) reported tne tissue distrioution of caroon
tetrachloride in a Rhesus monkey exposed to 290 mg/m3 of [ Cjcarbon
tetracnloride for 300 minutes. The tissue distribution, as calculated from
tne C radioactivity, is shown in Taole 7-3. The concentration of caroon
tetracnloriae was greatest in the fat, followed oy the liver and bone marrow.
Fowler (1969) stuaied the distriuution of caroon tetracnloriae in tne
tissues of rabbits given tne chemical by stomach tube. Five rabbits were
given carbon tetracnloride (1 mil/kg bw) as a 20% (v/v) solution in olive
oil. Analysis of the carbon tetrachloride by GC snowed ji-125 mg/kg hexa-
chloroethane. The rabbits were sacrificed 6, 24 and 48 hours after treat-
ment and the tissues analyzed for carbon tetrachloriae by GC/ECD. Six hours
after carbon tetrachloride was administered, the tissue concentrations (per
kg of tissue) were 787 _+ 289 mg/kg (standard error) in fat, 96 _+ 11 mg/kg in
liver, 21 +_ 12 mg/kg in muscle and 20 _+ 13 mg/kg in kidney. By 48 hours,
tnese 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 kianey and muscles.
It is difficult to compare tnese four distriotuion stuaies, because spe-
cies, sacrifice times, doses and routes of exposure varied. Moreover, not
all important tissues were sampled [e.g., von Oettingen, et al. (1950) did
7-8
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TABLE 7-3
Tissue Distribution of [14C] Carbon Tetrachloride
Inhaled by a Rnesus Monkey*
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
*Source: McCollister et al., 1951
7-9
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not sample the fat]; ana the number of animals in two cases was small [e.g.,
Roobins (1929) reported results on three dogs; McCollister et al. (1951)
reported results on one monkey]. Nonetheless, it appears that the concen-
tration of carbon tetrachloride will be highest in fat, liver, bone marrow,
blood and perhaps kidney or brain after administration by either oral or
inhalation routes. Certainly more work could be done in this area.
7.3. METABOLISM
Chloroform was one of the first caroon tetracnloride metabolites to be
descrioed (Butler, 1961). Eight dogs were exposed to caroon tetrachloride
oy tracneal cannula at the rate of 8000 mg/hr into innaled air for 3 hours.
At tne cessation of exposure, the exhaled air from the dogs was collected
ana analyzed by both gas chromatography and tne Fujiwara reaction, a colori-
metric 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 was estimated at 0.1 to 0.5 mg
oy GC analysis. Tissue homogenates were also shown to metabolize caroon
tetracnloride to cnloroform.
Evidence of metaoolism to a free radical was suggested by studies show-
ing nexacnloroethane to oe a carbon tetrachloride metaoolite (Bini et al.,
1975). Five Wistar rats were administered 160 to 800 mg caroon tetracnior-
iae aissolvea in liquid paraffin by gavage following a 24-hour fast. The
animals were sacrificea 15 minutes to 8 hours after treatment. A graph dis-
playing caroon tetracnloride concentrations in rat liver versus time showed
tne chemical at =0.9 mg/kg of tissue after 15 minutes and at maximal con-
centration (1.7 mg/kg) after 120 minutes. GC analysis showed that cnloro-
form 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 at
7-10
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0.005 mg/kg. The authors explained the formation of botn chloroform ana
nexacnloroethane as carPon tetrachloride metabolites by proposing that the
trichloromethyl free radical was the primary metabolite of carbon tetra-
cnloride.
C-labeled caroon dioxide was detected in the exhaled air of Rhesus
monkeys after a 344-minute exposure to [14C]carbon tetrachloride at 290
mg/m3 oy inhalation (McCollister et al., 1951). The amount of [ C]car-
oon dioxide exnaied during the 7-day period following exposure was reported
to oe 10 to 20% of the total radioactivity expired. The authors fitted
tnese data to a straight line, integrated the resulting equation from 13 to
1800 hours (75 days) and estimated that 4.4 mg or 11% of the total amount of
radioactivity eliminated was excreted as carPon dioxide.
Shah et al. (1979) studied the metabolism of [ Cjcarbon tetrachloride
oy 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 ymole of [ 4C]-
laoeled carbon tetrachloride alone, and with either NADH or NADPH or both.
[ Cjcarbon dioxide was detected by scintillation counting. The results
are shown in Table 7-4. The addition of NADPH or NADH, separately or as a
mixture, appeared to result in substantial conversion of caroon tetracnlor-
iae to carbon dioxide.
Shah et al. (197y) tested for the possioie formation of caroonyi cnior-
ioe in hepatic caroon tetracnloride metabolism oy adding L-cysteine to the
in vitro rat liver system descrioed aoove. Caroonyl chioriue ano L-cryst-
eine are known to react chemically to form a condensation product, 2-oxo-
thiazolioine-4-carooxylic acid. The presence of the condensation product
was confirmed oy thin-layer chromatography (TLC) and mass spectrometry (MS).
The authors inferred from the presence of 2-oxothiazolidine-4-carboxylic
7-11
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TAbLE 7-4
Conversion of [ CjCaroon Tetracnioride to
[l^CjCarbon Dioxide oy Rat Liver Homogenate*
Nucleotide Added [14C]C02 (nmole/g liver, mean +_ standard deviation)
None 27 +_ 5
NADH 373 +_ 17
NADPH 464+33
NADH + NADPH 472 +_ 21
*Source: Snah et al., 1979
7-12
-------
acia that carbonyl chloride was formed in the metaoolism of carbon tetra-
cnloriae Dy rat liver microsomes. The authors postulated a mechanism of
oiotransformation for caroon tetracnioride which involved a sequential oxi-
oation of caroon tetrachloride while oound to a heme (Figure 7-1). Release
of bound intermediates tnen gave rise to different metaoolites at the site
of release.
Fowler (1969) detected hexachloroethane and chloroform in the tissues of
rabbits administered caroon tetrachloride orally. As previously described,
a total of 15 rabbits were given carbon tetrachloride at 1 m&/kg bw and
sacrificed in groups of five at 6, 24 and 48 hours after exposure. Samples
of fat, liver, kidney and muscle tissue were analyzed for chloroform and
hexachloroethane by gas chromatography. The results of the analysis are
shown in Table 7-5. The fat contained the highest amounts of hexachloro-
etnane at each sampling time, but the highest concentrations of cnloroform
appeared in the liver.
For a complete discussion on metaoolic patnways hypotnesized to be mecn-
anisms of toxicity, see Chapter 8, Section 8.3.
7.4. ELIMINATION
14
McColiister et al. (1951) studied the elimination of C-laoeled car-
oon tetrachloride from Rhesus monkeys exposeo by innalation at 290 mg/m3
for 344 minutes. The total C radioactivity in tne blood decreased 12%
during the first 10 minutes after exposure. Graphs of data from the analy-
sis of blood samples ootained periodically for 10 to 12 days following expo-
sure showed that the level of caroon tetrachloride in the blood decreased
exponentially with time. At 10 days, the level of carbon tetracnioride in
blood was =0.009 mg/100 g. The authors estimated that 21% of the total
amount of carbon tetrachloride absorbed was eliminated in expired air during
7-13
-------
Accwtor
' -CClj-Oj"] — Upoperoxidotion_
. Conjugation
+ ZH +
C0 | -HCl
I Acceptor
Acceptor —————- Cl^Cu+t^Q •~~2 HCI •*• C02
FIGURE 7-1
Pathways of Carbon Tetrachloride Metabolism. Products identified as
carbon tetrachloride metabolites are underlined. The electrons utilized in
cytocnrome reductases. Fe + and Fe + denote the respective ferro- and
tne reactions are assumed to come from NADH or NADPH via the flavoprotin
cytocnrome redud
ferricytochromes.
Source: Redrawn from Shan et al., L?79
7-14
-------
TABLE 7-5
Chloroform and Hexacnloroethane in Tissues of Rabbits
Given Caroon Tetrachloriae Orally*
Sample Time
Tissue
CHC13
(ug/g tissue)
C13CCC13
(ug/g tissue)
6 hours
24 hours
48 hours
Fat
Liver
Kidney
Muscle
Fat
Liver
Kidney
Muscle
Fat
Liver
Kidney
Muscle
4.7 + 0.5
4.9 + 1.5
1.4 + 0.6
0.1 i 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 + 0
0.1 + 0.1
4.1 + 1.2
1.6 + 0.5
0.7 + 0.2
0.3 +. 0.2
16.5 + 1.6
4.2 + 1.8
2.2 + 1.1
0.5 +_ 0.2
6.8 + 2.4
1.0 +_ 0.3
Trace
Trace
*Source: Fowler, 196:?
7-15
-------
the first 18 days. By extrapolation of these data, the authors concluded
that after 1800 hours (75 days), approximately 51% of the caroon tetrachlor-
iae initially absoroed would be eliminated in exhaled breath eitner as car-
oon tetrachloride or carbon dioxide. Analysis of urine and feces showed
measuraolfc amounts of radioactivity after 15 ana 12 days, respectively. The
autnors interpreted these findings as indicating that significant quantities
of caroon tetrachloride and/or metabolites may be excreted by these routes.
7.5. SUMMARY
Caroon tetrachloride is readily absorbed from the lungs and the gastro-
intestinal tract, as expected from its partition coefficients. Although few
quantitative data are available on the amount of carbon tetrachloride
absoroed through the lungs, the chemical and its metabolites have been
reported in blood, many tissues, exhaled air, urine and feces after adminis-
tration by this route. Carbon tetrachloride is also absoroed through the
skin.
The oest availaole data concerning CC1, absorption come from two stud-
ies and one report:
•30.4% via inhalation in monkeys (McCollister et al., 1951) .
•57 to 65% via inhalation in humans (Lenmann ana Scnmidt-Kenl,
1936)
•30% via inhalation and 50% via ingestion (Stokinger ana
Woodward, 1958).
The StoKinger and Woodward report has been criticizea for not including
substantiating information or alternatively, citing the specific literature.
There are some considerations associated with extrapolating from animal data
to humans in that animals may alter their breathing patterns during an
experiment or may not be good models insofar as their metabolism is con-
cerned. This is not to say that the animal data or Stokinger and Woodward
7-16
-------
report snouia be overlooked. Tnererore, it is recommenaea tnat a 40/6
aosorption coefficient be used wnen the route of exposure is via inhalation
ana tnat a iOO& adsorption coefficient be used when tne route of exposure is
via ingestion. Tne former being a compromise based upon tne available
information and the latter being tne conservative estimate due to little
information.
In the studies reviewed, caroon tetracnloride appears to be distriouted
to all major organs following absorption. The highest concentrations have
been found in the fat, liver, bone marrow, blood, brain and kidney.
Carbon tetrachloride metabolism has been reported to occur primarily in
tne liver. Carbon tetrachloride has been postulated to be metabolized to a
tricnloromethyl radical bound to an iron atom in the cytochrome heme
moiety. This tricnloromethyl radical is thougnt to be either furtner meta-
oolized or released as a free radical. It is suggestea that the trichloro-
methyl free raoical can unoergo a variety of reactions, inciuoiny macromole-
cular oinoing, nyorogen abstraction to form chloroform, and dimerization to
form nexachloroetnane.
Caroon tetracnloriae and its metabolites have been reported in many
studies to oe excreted primarily in exhaled air, but also in the urine and
feces. However, pharmocokinetic data on these processes are apparently
lacking.
7-17
-------
-------
8. TOXICXOGY: ACUTE, SUBCHRONIC AND CHRONIC
Hepatotoxicity is the major effect reported to be produced Dy acute
exposure of animals to carbon tetrachioride. Hepatic necrosis and fatty
liver degeneration have been documented after both inhalation exposure and
ingestion. Hepatotoxicity has sometimes been accompanied by kianey ana lung
effects. In addition, prenatal toxic effects have been demonstrated after
innalation of tne chemical by pregnant rats. Tnis chapter discusses the
effects from acute exposure (single dose, one day or several days definea in
rodents), suocnronic exposure (2 weeks to somewhat more than 90 days defined
in roaents) and chronic exposure (_>6 months defined in rodents) to carbon
tetracnloride. Emphasis is placea on studies in which dose/response rela-
tionships have oeen developed. Toxic responses occurring prior to carcino-
genicity are reportea in Chapter 11.
8.1. EXPERIMENTAL ANIMALS
8.1.1. Acute. Tne toxicity from acute exposure to carbon tetrachioride
has been documented extensively. Because defining the dose range that pro-
duces minimal health effects is an objective of this report, this section
will concentrate on those studies that (1) describe nonlethal -effects and
(2) provide data on a range of doses from whicn dose-response relationships
can be determined. For this reason a number of studies referring to LD5Qs
will not be discussed. Table 8-1 summarizes some of the lethal dose data
reported for carbon tetrachioride in various species.
Aaministration of carbon tetrachioride results in a numoer of acute sys-
temic effects. Its hepatic effects are tne most pronouncea: carbon tetra-
cnloriae causes necrosis ana fatty liver degeneration. The hepatic effects
causea by carpon tetracnloride have oeen well documentea in many scientific
studies. However, toxicity to other organs has also oeen described. The
following subsections will focus on such organ toxicity.
-------
TABLE 8-1
Toxic Doses and Effects of Carbon Tetrachloride in Animals3
Animal
Rat
Mouse
Dog
Rabbit
Rat
Mouse
Dog
Rabbit
Rat
Mouse
Cat
Guinea pig
Cat
Rabbit
Route of Effect*3
Administration
Oral LD50
LD50
LDLO
LD50
Intraperitoneal 1059
LD5Q
LDLO
LDLO
Inhalation LC5Q
LC5Q
LC(_o
LCLO
Subcutaneous LDi_0
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
624.80 mg/m3
1,487.97 mg/m3
5,952.83 mg/m3
3,124.02 mg/m3
300 mg/kg
3,000 mg/kg
aSource: NIOSH, 1978
, dose lethal for 50% of animals
LC5Q, concentration lethal for 50% of animals
LD|_0, lowest lethal dose
LQ_Q, lowest lethal concentration
8-2
-------
8.1.1.1. LIVER EFFECTS — Functional changes in mouse liver as a re-
sult of carbon tetrachloride exposure were measured by increases in activity
of the enzyme serum glutamic-pyruvic transaminase (SGPT) and in bromsulfo-
phthalein (BSP) retention (Klaassen and Plaa, 1966). Male Swiss-Webster
mice were administered various amounts of analytical grade carbon tetrachlo-
ride i.p. in corn oil in a final volume of 10 mfc/kg bodyweight (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
treatment. 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
DW for BSP retention. The authors did not report the range of carbon tetra-
chloride doses used or the number of animals used at each dose.
In an additional 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 intraperitoneally; the animal's SGPT activity 24
hours after the dose was noted. If the enzyme activity 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 8-2. The authors concluded that 13 mg/kg bw was the median effective
dose (EDcn) of carbon tetrachloride in mice as measured by elevated SGPT
values.
Sein and Chu (1979) studied the effect of carbon tetrachloride on the
level of the liver enzyme glucose-6-phosphatase in mice. Groups of six male
LAC strain mice were treated i.p. with 795, 1590 or 3180 mg/kg bw carbon
8-3
-------
TABLE 8-2
SGPT Values of Mice Administered Carbon Tetracnloride
Intraperitoneally in "Up and Down" Experiment3
Animal
1
2
3
4
5
ED50
Dose
(mg/kg bw)
17.5
13
17.5
13
8
13
Response13
E
N
E
E
N
aSource: Klaassen and Plaa, 1967
°E = Elevated SGPT after 24 hours
N = Normal SGPT after 24 hours
ED5Q = Median effective dose
8-4
-------
tetracnloriae (purity unspecified) in paraffin oil. Tne animals were sacri-
ficed 24 hours after treatment. Control animals (number unspecified) were
given paraffin oil and sacrificed on the same schedule. The livers were re-
moved and analyzed for glucose-6-phosphatase. The results of the analysis
showed that after treatment with carbon tetracnlorioe at 795 or 1590 mg/kg,
the enzyme level fell to 40% of the control value. At a dose of 3180 mg/kg,
the enzyme level had decreased to 20% of the control value.
A series of experiments to determine the effects of single carbon tetra-
cnloriae exposures on rats were performeo by Murphy and Maliey (1969).
Adult male Holtzman rats (250 to 350 g) were orally administered various
ooses of undiluted carbon tetrachloride by gavage. Controi animals were
administered equal volumes of water. At 2 to 20 hours after treatment, ani-
mals were sacrificed and liver enzyme activities and liver weignts were
measured. The results are shown in Table 8-3. The animals receiving carbon
tetrachloride at 1600 mg/kg bw were sacrificed 20 hours after treatment and
the livers examined histopathologically. The examination showed extensive
fatty infiltration, inflammation ana some centrilooular necrosis. Tne
liver-to-body weight ratios were also increased.
Murphy and Maliey (1969) also determined the effect of single exposures
to carbon tetrachloride on tne activities of the corticosterone-inducible
liver enzymes tryptophan pyrrolase and tyrosine-ketoglutarate transaminase.
Groups of rats (4 to 6 in each group, 8 untreated controls) were treated
with carbon tetracnloriae (0, 400, 800 ana 1600 mg/kg bw by gavage) and sac-
rificed 5 hours after treatment. Data graphs snowed that the enzyme levels
were increased roughly in proportion to tne dose.
Similar studies on the effect of carbon tetrachloriae administration on
serum activity of liver enzymes in rats were performea by Drotman ana
8-5
-------
lAldLt U-3
£.1 Tects Ot Oral Carbon Itttruchloride on Liver Weiyltt anu
Liver anil Plasma Liuyme Activities in Male Ratsd
oo
Ch
Dose Tune
(my /kg bw) (hr)
0
3200 2
3200 5
32UJ 20
2400 20
1600 20
600 20
Number of Plasma
Animals AKT"
7 2.6 ± 0.3C
4 2.1 ± 0.2
5 13.2 *_ 3.4
5 35.2 ± 4.2
4 35.2 j- 3.4
5 lb.1 ± 4.3
4 9.3^2.3
AKfb Tk
2360 ;t ib2 *3
170
1U13 ^ 331 330
1174 t 55y 3ol
15U5 +_ 14b 305
15^6 * 1V4 2V4
2120 +_ 1U2 130
Liverc
U° AH"
+ ii 14. y ^ i.o
_»_ 12 14.5 _* 1.0
4_ 36 14.2 +.1.3
^ 42 34.6 t i.b
+_ 21 37.1 t 1.6
f 61 33.3 t 3.6
_f 5 ^y.l ^ 6.5
WeiyiiL ly/iUDy Dw)
2.75
2.V4
3.26
4.36
3.V5
3.VO
3.4/
«^ O.Oo
^ 0.04
<_ O.iO
± 0.06
i_ O.O/
*. 0.05
*_ O.O/
aSource: Adapted from Murphy and Malley,
bAKT = alanine-o-ketoglutarate Lransaininasc
TKl = tyroiirte-o-ketoalutarate transaminase
«P = alkaline phospnatase
^ St in niicroittaleb of product forineU per graiw of fresh liver or miiHliter of piasuia per houi
-------
Lawnorn (1978). Groups of four male Cox rats were administered carbon
tetracnloride i.D. at 60, 120, 240 or 480 mg/kg bw in a total volume of
1 ml in corn oil ana exsanguinated at specified time intervals. Serum
activities were determined for tne enzymes sorbitol denydrogenase (SSDH),
ornitnine carbamyl transferase (SOCT), aspartate aminotransferase (SAST) and
isocitric denydrogenase (SICDH). Liver specimens were taken from each ani-
mal and scored for nistopatnological changes. The results of the enzyme
analyses and nistopathology are tabulated in Table 8-4 by dose ana hours
after dose. The SOCT activities snowed the best correlation with liver
histopatnology 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 tetracnloride were
also reported by Korsrud et al. (1972). Male Wistar rats (260 to 400 g; 8
to 10 animals per treatment group) were administered single oral doses of
carbon tetrachloride (0 to 4000 mg/kg bw) in corn oil (5 mi/kg bw). The
rats were fasted for 6 hours before treatment 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 tne liver. At 20 mg/kg bw there was histopathologic evidence of toxic
effects on the liver. These changes included a loss of basophilic stip-
pling, a few swollen cells and minimal cytoplasmic vacuolation. At 40 mg/kg
bw, liver fat, liver weight, serum urea and levels of 5 of tne 9 liver
enzymes were increased while serum arginine decreased. At higher doses the
remaining four enzyme levels were also elevated.
In addition to the study on hepatic effects of carbon tetracnloride in
mice described earlier in this section, Klaassen ana Plaa (1967) also inves-
tigated the hepatic effects of carbon tetrachioride exposure on dogs. Male
3-7
-------
TABLE 8-4
Effects of Carbon Tetrachloride on Liver Histopathology
and Serum Enzyme Levels3
Serum Enzyme Concentrations
Dose
(mg/kg)
60
120
240
480
Hours
After
Dose
0
6
12
24
36
0
24
48
96
168
0
24
48
96
168
0
24
48
96
168
Histologyb
0
2
1
1
0
0
3
2
1
0
0
3
4
1
0
0
3
4
1
0
Relative to Pretreatment Levels
SOCT
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
SSDH
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
SAST
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
SICDH
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
aSource: Adaoted from Drotman and Lawhorn, 1978
bO = No observable changes.
1 = Minimal changes. Large central vein, swelling of heoatocyte, 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<0.01).
8-8
-------
and female mongrel dogs were treated intraperitoneally with carbon tetra-
chloride at 22 to 38 mg/kg bw in an "up and down" experimental design.
Blood samples were taken for measurement of SGPT 24 hours after administra-
tion of carbon tetrachloride. Control dogs had serum SGPT activity of 36 +_
7 units. Therefore, 36+2 standard deviations or 50 units was chosen as
the upper limit of the normal value. The results of the analysis are shown
in Table 8-5. The SGPT returned to normal in 17 to 18 days. Animals were
then sacrificed and the livers were examined histopathologically. They
showed moderate vacuolation of the centrilobular and midzonal hepatocytes as
well as traces of brown material in the cytoplasm of centrilobular Kupffer
cells.
Gardner et al. (1924) also studied the acute toxic effects from inges-
tion of CC1. in dogs. Effects ranged from no apparent effect at 0.01
mJi/kg (15.89 mg/kg) to centrilobular necrosis at 0.05 mi/kg (79.45
mg/kg). Rabbits similarly treated experienced liver necrosis at 0.1 m£/kg
(158.5 mg/kg).
Kronevi et al. (1979) administered CC1 epicutaneously to guinea pigs.
Dermal effects were seen (see Section 8.1.1.4.) along with effects on the
liver. Liver morphology was characterized by hydropic and necrotic changes.
Altered liver morphology was seen after 16 hours when hepatocytes in the
central two-thirds of each lobule showed marked hydropic changes which were
characterized by large, clear cytoplasmic spaces. It was noted that there
was also a tendency to necrotic lesions characterized by homogeneous,
slightly eosinophilic and slightly PAS-positive- structures within the cyto-
plasm. Glycogen was absent and the nuclei showed a tendency to degeneration
(Kronevi et al., 1979).
8-9
-------
TABLE 8-3
SGPT Activity in Dogs 24 Hours After Intraperitoneai Administration
of CarDon Tetrachioride in "Up and Down" Experiment3
Animal
1
2
3
4
5
ED50
Dose
(mg/kg)
22.2
30.2
22.2
30.2
38
32
Response0
N
E
N
N
E
aSource: Adapted from Kiaassen ana
Piaa, 1967
ON = normal SGPT after 24 hours
E = elevated SGPT after 24 hours
ED5Q = Meaian effective dose
8-10
-------
In studying the renal effects of CCl^ in cats (see Section 8.1.1.2.),
Wong and DiStefano (1966) noted a delayed liver reaction. Liver weights
were significantly increased 24 hours following exposure by inhalation.
8.1.1.2. KIDNEY EFFECTS — In experiments conducted by Plaa and
Larson (1965) using CC1., high doses failed to induce renal failure as
measured by phenolsulfonphthalein (PSP) excretion in mice although patho-
logical kidney alterations were present. Male Swiss mice (18 to 30 g) were
given i.p. injections of carbon tetrachloride (1600 to 6400 mg/kg bw) dis-
solved in corn oil at a final amount of 10 mg/kg bw. The animals were then
hydrated with tap water (50 mg/kg bw) by gavage and placed on a urinary
collection unit for 2 hours. Even carbon tetrachloride doses lethal in some
animals (>6400 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 (3260 mg/kg bw), minimal renal dysfunction was ob-
served after 96 hours. Histologic examination of kidney sections from five
mice that had been administered this dose showed that one of the mice showed
necrosis of proximal convoluted tubules, and 4 of 5 mice showed swelling of
the tubules.
Carbon tetrachloride did, however, decrease activity of glucose-6-phos-
phatase in the kidney (Sein and Chu, 1979). Male mice (40 to 50 days old,
weigning 24 to 28 g) were injected i.p. with carbon tetracnloride at 795,
1590 or 3180 mg/kg bw in paraffin oil. Twenty-four hours after injection of
795 or 1590 mg/kg ow, the kidney glucose-6-phosphatase activity decreased to
77 or 65% of the control value, respectively. Increasing the dose to 3180
mg/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
8-11
-------
lower doses and decreased further to 20% of the control value at the 3130
rag/kg bw dose. The authors attributed these differences to the limited
metabolic capacity of the kidneys.
Klaassen and Plaa (1967) studied the effect of carbon tetrachloride
exposure on kidney function in dogs. PSP excretion of <39% of control
value was considered indicative of renal dysfunction. An unspecified number
of male and female mongrel dogs were treated i.p. with carbon tetrachloride
at 22 to 38 mg/kg bw, and the 24-hour excretion rate for PSP was deter-
mined. Control dogs were used to determine a normal range for PSP excre-
tion. None of the dogs treated with carbon tetrachloride exhibited
decreased PSP excretion. However, on histopathic examination of the kidneys
from the treated dogs, the Bowman's capsules appeared dilated with some con-
traction of glomerular tufts and calcification of a small number of tubules
in the medulla.
Striker et al. (1968) examined the structural and functional changes in
the rat kidney during CCl^ intoxication. Exposure was 2.5 mil/kg bw in
an equal volume of mineral oil by gastric intubation. A progressive in-
crease in the size and paleness of the kidney was apparent during the first
2 days after exposure, with maximum effects seen at 48 hours. Alterations
were limited to the proximal tubule and appeared to involve primarily the
middle and lower segments. The alterations seen were sequential and revers-
ible. The earliest morphological change was in the mitochondria, followed
by cellular swelling manifested by loss of basilar interdigitations and
swollen microvilli. Occurring later was the appearance of large aggregates
of smooth-surfaced membranous profiles. By 5 days after exposure, all
alterations were reversed (Striker et al., 1968).
8-12
-------
Also apparent were differences in serum parameters reflecting kidney
function. Serum creatinine and blooa urea nitrogen peaked at 12 hours and
24 hours, respectively. Total serum bilirubin was elevated 12 to 48 nours.
By 5 days all values returned to normal (Striker et al., 1968).
Wong and DiStefano (1966) examined lipid accumulation and histologic
cnanges in the kidney of tne cat following CCi. innalation. Kidney
weignt:body weight ratids increased significantly following 60- and 240-min-
ute exposures. The renal cortical lipid content increased significantly
within 15 "minutes and an accumulation of fat droplets was seen nistological-
ly in 30 to 60 minutes. One hour after the termination of CCI. innala-
tion, the kidneys were enlarged and the weight continued to increase past
the 24-hour observation period (Wong and DiStefano, 1966).
8.1.1.3. LUNG EFFECTS — Boyd et al. (1980) investigated the effect
of ingestion and inhalation of carbon tetracnlorioe on pulmonary Clara cells
in Swiss mice. For the ingestion study, the mice were treated with carbon
tetracnlorioe (4000 mg/kg ow) in a 50% sesame oil solution and sacrificed 16
hours after treatment. The lungs were removed and examined by electron
microscopy. The examination showed the Clara cells to have massive dilation
of vesicles of smooth endoplasmic reticulum, increased mitochondria! stain-
ing density, ribosomal disaggregation, nuclear condensation ana occasional
cellular necrosis. Additional experiments with oral carbon tetracnloride
doses of <1600 mg/kg bw did not produce any pulmonary lesions visible by
light microscopy. Doses of 2400 to 4800 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
8-13
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caused by ingestion of carbon tetrachloride (4000 mg/kg bw). Pulmonary tis-
sue was evaluated by light microscopy 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.
The pulmonary toxicity of inhaled carbon tetrachloride 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, respectively. The animals were sacrificed 24 hours after exposure,
and the lungs were examined. Marked Clara cell lesions similar to those
seen after oral exposure were seen at all exposure levels; necrosis was re-
ported to be more -frequent after inhalation than after oral exposure, but no
effort to quantify this finding was reported.
Gould and Smuckler (1971) detailed the structural alterations in rat
lungs following carbon tetrachloride ingestion. Male Sprague-Dawley rats
(200 to 250 g) were fasted 16 hours prior to administration of carbon tetra-
chloride (4000 mg/kg bw) by gavage. The animals exhibited piloerection and
lassitude 3 to 4 hours after treatment. Necropsies were performed on all
animals. Microscopic examination of the lungs of treated rats revealed
perivascular edema and mononuclear 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 hemorrhage involving 15
to 20% of the parenchyma were observed 8 to 12 hours after treatment.
Electron micrographs of rat lungs after carbon tetrachloride ingestion
(Gould and Smuckler, 1971) showed granular pneumocytes containing swollen
inclusions with decreased osmiophilia and attenuated lamellae 1 hour after
treatment. These changes were more severe 4 hours after treatment. By 4 to
8-14
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8 hours after treatment, cytoplasmic edema, dislocation of dense ribosomal
aggregates and mitochondria! disruption were apparent. Multivesicular
bodies were "conspicuously decreased" within the granular pneumocytes.
Necrosis was evident 12 to 24 hours after treatment. One hour after admin-
istration, endothelial cells displayed markedly increased pinocytotic vesi-
cles. Severe disruption of endothelial cells was evident from 8 hours on-
ward. Ultrastructural damage was seen in all components of the alveolar
wall, and fibrin was observed within alveoli. The authors interpreted these
finoings as showing significant alterations in vascular permeability.
Lesions of the Clara cells in the lungs of male Sprague-Oawley rats
orally treated with carbon tetrachloride were observed by Boyd et al.
(1980). The carbon tetrachloride was administered by gavage at doses of
3816, 5088 and 7155 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 were less pronounced than those
in mice exposed to comparable amounts of carbon tetrachloride.
Chen et al. (1977) examined the effects upon the lungs of rats given a
single CCl^ exposure by gastric intubation or by inhalation-. Exposures
were: 2.5 mil/kg bw by gastric intubation, and 30 minutes in air containing
4.38% CCl^ (280,400 mg/m3) by inhalation. Both the orally administered
and innaled CC1 markedly modified the lung and liver cytochrome P-450
content, but there was a greater response in the pulmonary tissue. Inhala-
tion resulted in a less significant depression of activity in both organs
(Chen et al., 1977). Morphologic analyses of the lung revealed focal
changes by 1 to 7 days in pulmonary architecture consisting of areas of
alveolar collapse, septal thickening, transudation and modification of type
II pneumonocytes.
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8.1.1.4. DERMAL EFFECTS — Kronevi et ai. (197*) examined tne effects
on guinea pigs following epicutaneous administration of 1 mSL CCi,.
Slignt Karyolysis was seen. After 15 minutes, marked spongiosis developed.
Fifteen minutes and onward, karyopyknosis was evident. The authors saw pro-
gressing nuclear pyknosis and junctional separation between the basement
membrane and the basal cells along with induced spongiosis appearing before
the junctional separation attributed to the CC1, exposure.
8.1.1.5. BIXHEMICAL AND OTHER EFFECTS — Merkureva et al. (1979)
stuoieo the effects of continuous inhalation of CCL on the rat enzyme
system. Exposure of 300 mg/m3 CC14 in air resulted in 55% reduction in
the activity of N-acetyl-ki-Q-glucosaminidase 24 hours foiiowiny initiation.
The activity of nyaluronioase of blood serum was increased oy 96% 3 days
after exposure. The activities of 3-giucosioase, acio pnospnatase ana
id-gaiactosiaase were also affected. Finally, a 39% inhioition of N-acetyl-
neuraminic acid aiaolase was seen.
Following injection of CC1., Wyrebowska ana JerzyKowsKi (i960) saw a
cnange in enzyme activity in rat serum. Mature male ana young Wistar rats
were given a single 2 mi/kg bw dose of CCl^ i.p. dissolved- in sterile
vegetable oil (1:1). Following this dose, both the alkaline and acid forms
of aminopropanol denydrogenase appeared in the blood serum. Maximum activ-
ity occurreo 24 hours after administration in the young rats and 12 hours
after administration in the mature male rats.
Mikhail et al. (1978) injected male ana female adult rats intraperitone-
ally with 0.5 mil of a 1:1 mixture of CC1 in mineral oil per 100 g bw
(0.005 mS, of tne mixture per g ow). This resulted in an increase in serum
iron, copper, zinc, caicuim, potassium ana soaium 24 hours after aaministra-
tion. There was no cnange observed in serum magnesium. The authors attrib-
8-16
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ute tne rise in some serum chemistries to tne known hepatotoxic effect of
CC1. . They conclude that the disturoance in minerals mitaoolism is one of
the earliest lesions in CC1 poisoning (Miknail et al., 1978).
David et al. (1981) examined the effect of different exposure schemes
upon Diocnemical and morphological changes in the rat liver. Groups of six
male Wistar rats, 7 months oia, were exposeo to CC1, vapors with differing
exposure scneoules. Schedules were designed in sucn a way to give a con-
stant product of concentration and time (CT = 1950 mg/m3) for 4 successive
days a week.
It was found that a higher dose given over a snorter period of time had
a greater effect than a lower dose given over a longer period of time (1625
mg/m3 for 72 minutes vs. 325 mg/m3 for 6 hours). Also, continuous expo-
sure (18 minutes) to 6500 mg/m3 produced a greater effect than intermit-
tent exposure (3 minutes, 6 times with 1 hour intervals) to 6500 mg/m3.
Tne authors conclude that sensitivity of the liver is more influenced by the
concentration of CCl^ in the inhaled air than in the total amount inhaled,
the tneory being that the former allows more CC1, into the blood entering
tne liver. As tne authors explain, this information is important in putting
time weigntea averages (TWAs) in the workplace.
8.1.2. Suochronic. The toxicity followng subchronic exposure to CC14
nas not oeen as extensively described as the toxicity following acute expo-
sure. Paquet and Kamphausen (1975) examined biocnemical changes in rats
following suocnronic exposure to CCl^. Administration of CC1, was by
subcutaneous injection of 1 m£/kg bw in an equal amount of peanut oil at
7-day intervals for 8 weeks. The authors describe tne changes in stages.
In stage 1 there is a decrease in pseudocholinesterasis indicative of the
stage of liver necrosis. In stage 2, the triglycerides reach a high pla-
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teau, there is an increase in SCOT, an increase in BSP retention and a con-
tinued decrease in pseudocnolinesterasis. The second stage is characterized
oy massive fatty infiltration of the liver and increased necrosis witn liver
fibrosis at the end. In stage 3, SCOT continues to increase along witn
nydroxyproiine, trigiycerides and BSP retention. This finai stage is char-
acterized by a reouceo synthetical ability and atrophy of the liver.
Bizin et al. (1977) performeo a comparative stuoy of the effect on rats
of continuous and intermittent exposure to CC1,. Continuous inhalation of
500 mg CCl^/m3 for 10 days inducea toxicity symptoms 4- to 5-fold as
rapidly as did innalation for 6 hours daily for 40 days (Bizin et al., 1577).
Additionally, Alumot et al. (1976) reported the effects upon groups of
six weanling rats 4 weeks old 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 tetracnloride loss
during the storage period of 7 to 10 days was determined to be 5%. The ani-
mals were allowed access to the feed only at set time intervals to minimize
loss of carbon tetrachloride by volatilization. The autnors calculated that
the amount of caroon tetracnloride remaining in the consumed feed was 60 to
70% of the amount initially present; the total decrease reflected amounts
lost during storage ana after removal from storage to feeoing trougns. From
these aata and tne weights of the animals, the authors calculated that 275
mg/Kg of feeo representeo a daily dose of 40 mg/kg ow. By assuming tnat all
parameters were the same and that tne delivered dose was proportional to the
concentration in feed, diets of 150 ana 520 mg/kg of feea can be calculated
to represent daily doses of 22 and 76 mg/kg bw, respectively (U.S. EPA,
1981). At the ena of the experiment the animals were weighed and killed.
Of the three doses, only the highest, 76 mg/kg bw (520 mg/kg of feea),
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caused significantly Depressed weignt gain in males. Weight gain in females
appeared to oe unaffected oy all doses. Total lipid and triglycerioe levels
in tne liver were significantly higher in animals fed caroon tetrachloride
at 40 and 76 mg/kg bw tnan 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.
Prendergast et al. (1967) repeatedly (8 hours/day, 5 days/week) exposed
15 guinea pigs to caroon tetrachloride (purity unspecified) at 515 mg/m3
over a period of 6 weeks and observed hepatic changes. Three guinea pigs
died on days 20, 22 and 30. All the animals showed a body weight loss. The
surviving animals were sacrificed at 6 weeks and the livers examined histo-
pathologically. The examination revealed fatty infiltration, fibrosis, bile
duct proliferation, nepatic ceil degeneration and regeneration, fdcal in-
flammatory cell infiltration, alteration of loPular structure and early por-
tdl cirrhosis. The lipio content of tne guinea pig liver was reported to be
35.4 _+ 10.7%, much higner than the control value of 11.0 +_ 3.6%.
In addition, Prendergast et al. (1967) also continuously exposed guinea
pigs td caroon tetrachloride vapor at 61 mg/m3 for 90 days. Three of the
15 guinea pigs died on days 47, 63 and 74. All the exposed animals showed a
depressed weight gain. A "hign incidence" of enlarged and discolored livers
was reported on gross pathological examination. Histopathologic examination
of tne livers revealed fatty changes, fibroblastic proliferation, collagen
deposition, hepatic cell degeneration and regeneration, and structure alter-
ation of the liver lobule. Enzymatic studies showed that only the succinic
dehyorogenase (SDH) activity was moderately reouced as compared to that in
controls.
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Prendergast et al. (1967) also exposed guinea pigs, number unspecified,
to carbon tetrachloride vapor continuously at 6.1 mg/m3 for 90 days. Tne
authors reported that "no visible signs of toxicity" were seen during this
study. Lipid content of the liver and serum urea nitrogen were within nor-
mal range. Tne authors concluded that no pathologic changes could be at-
tributed to carbon tetrachloride exposure.
In addition to their studies on guinea pigs, Prenoergast et al. (1967)
studied tne effects of ooth repeated and continuous exposure to caroon
tetracnlorioe on tnree squirrel monKeys, three New Zealana raooits, ana two
oeagie dogs for eacn exposure regime. The experimental designs were the
same as tnose oescrioed for the guinea pigs. Ail tne animals snowed a
weight loss during repeated exposure to 515 mg/m3. Fatty changes were
noted in tne liver of all species; they were most severe in raobits, fol-
lowed by dogs and monkeys. In the continuous exposure to 61 mg/m3 for 90
days, all species exnibited a depressed weignt gain, as did guinea pigs.
Liver changes were also noted, but enzyme activities (as measured by NADH,
NADPH, SDH, LDH anc G6PD) were within the normal range. At a continuous
exposure of 6.1 mg/m3, no toxic signs were noted.
Finally, the same investigators (Prenoergast et al., 1967) studied the
effects of repeated (515 mg/m3) and continuous (61 mg/m3 or 6.1 mg/m3)
exposure of rats following tne same metnodology descrioed above. Witn re-
peated exposure, there was a nigh percentage of mottled livers. Histopatno-
logic examination revealeo morphologic cnanges in lungs and livers but no
changes in the neart, spleen or Kidney. Fatty cnanges also developed in tne
liver. Following continuous exposure to 61 mg/m3, depressed growth curves
resulted as compared to control animals. Examination upon autopsy revealed
a high incidence of enlarged and/or discolored livers. Histopathologic
8-20
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liver cnanges included fatty infiltration, fiDroDiastic proliferation, col-
lagen deposition, hepatic cell degeneration and regeneration, and alteration
in the structure of the liver lobule. Tnere were no adverse effects follow-
ing continuous exposure to 6.1 mg/m3.
The studies done by Prendergast et al. (1967) have been criticized due
to small sample size (only rats and guinea pigs had numbers >10), incon-
sistent reporting (over 10 different descriptions of liver damage are men-
tioned) and vague information such that only the general conclusion that
liver carnage follows CCL inhalation can be made (EnviroControl, 1981;.
8.1.3. Chronic. Smyth et ai. (1936) studied the toxicity of caroon
tetracnioride on rats after chronic inhalation exposure. Groups of 24
Wistar rats were exposed to carpon tetrachiorioe concentrations of 315, 630,
1260 or 2520 mg/m3 for 8 nours/day, 5 days/weeK for 10.5 months. The
CC1 was found to contain <0.003& carbon disulfide. Control rats were
4
used, but the number was unspecified. Growth retardation was observed with
the 2520 mg/m3 oose. At the 630 and 1260 mg/m3 dose, growth was tne
same as in controls, and at the 315 mg/m3 dose the growth was stimulated.
Cirrhosis developed in the 630, 1260 and 2520 mg/m3 groups after 173, 115
and 54 exposures, respectively, but not in the 315 mg/m3 group. When
exposure was stopped fatty liver degeneration resolved within 50 days. Sur-
face alterations (hoonail liver) did not resolve until 156 days after cessa-
tion of exposure. Unspecified renal damage was observed after 52 exposures
to tne 315 mg/m3 concentration ano after 18 to 20 exposures at the hiyner
concentrations, out was termed "not extreme."
In this same study (Smytn et al., 1936), groups of 24 guinea pigs were
exposed to caroon tetracnioride vapor at 315, 635, 1260 or 2520 mg/m3.
Tne frequency of exposure was 8 hours/day, 5 days/weeK for <_1U.5 montns.
8-21
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Marked mortality occurred in exposed animals: 9/24 at tne 315 mg/m3 dose
after a median of 44 exposures (exposure terminated at 155 days), 16/24 at
tne 630 mg/m3 dose after a median of 10 exposures, 13/24 at the 1260
mg/m3 dose after a median of three exposures, ana 19/24 at the 2520
mg/m3 dose also after a median of three exposures.
The guinea pigs exposed to the 315 mg/m3 dose developed cirrnosis and
nobnail surface alterations of tne liver in 105 exposures. The authors con-
cluded that survival of guinea pigs at higner doses was of insufficient
duration to allow development of cirrhosis (Smyth et al., 1936). In addi-
tion, granular swelling was ooserveo in adrenal glanas of guinea pigs ex-
posed to caroon tetracnloride at 315, 630 and 1260 mg/m3 for 8, 7 and 17
exposures, respectively. Exposure to higher concentrations (1260 or 2520
mg/m3) or continued exposure to lower concentrations resulted in marked
damage to tne sciatic nerves. Dense clumps of black granules (osmic acid
stain) were observed paralleling the large majority of fibers.
Rhesus monkeys were also examined. They innaled CCl, at concentra-
tions of 320 and 1280 mg/m3 for 8 hours/day, 5 days/week for 10.5 months
(Smyth et al., 1936). Monkeys exposed to 1280 mg/m3 showed an 8% less
weight gain than the controls. At both 320 and 1280 mg/m3, monkeys had
livers with slight fatty degeneration following 8.7 months of exposure. The
livers returned to normal 28 days post exposure. Two of four monkeys ex-
posed to 1280 mg/m3 for 8.7 and 10.5 months showed definite damage to the
sciatic nerve.
In a cnronic oral exposure study (Alumot et al., 1976), groups of 36
rats (16 male and 18 female littermates) were fed mash containing caroon
tetracnloride at 0, 80 or 200 mg/kg of feed. The feed was stored jpn air-
tight containers, assayed for carbon tetracnloride content, and consumed
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soon after removal to feeaing trougns. The autnors calculated that the 200
mg/kg of feea represented a daily dose of 10 to 18 mg/kg bw. During this
2-year stuay, several parameters were measured. Up to 13 weeks no effects
were noticed on Dody weight gain. Throughout the study measurements of male
and female fecundity remained essentially normal. After 2 years, the sur-
viving animals were killed. In these animals, 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 detect-
ed in the treated animals. Thus, the authors found no biochemical, histo-
pathologic, reproductive or other abnormalities attributable to carbon
tetracnloride exposure. However, interpretation of the results was compli-
cated by the widespread incidence of chronic respiratory disease in the
animais which started at about 14 months into the experiment. More tnan
half the animals were dead at 21 months, although at 18 months the survival
ranged from 61 to 89%. Although the authors indicated that 10 to 18 mg/kg
ow (200 mg/kg of feed) is a no-ooservaole-adverse-effect level (NOAEL) of
caroon tetracnioride over 2 years, this conclusion may be questioned because
of the chronic respiratory infection and hence poor survival of-the animals
in the latter part of the experiment. Yet, it may be inferred from these
results that a level of 10 to 18 mg/kg bw/oay over a 1-year period caused no
ooservable adverse effects.
A chronic inhalation study by Adams et al. (1952) also presented incom-
plete data. No numbers of animals tested, surviving, or affected are given
and it is not possible to determine what measurements were maae at different
exposures. The only conclusion possible is that at some exposures ranging
from 32.5 to 2600 mg/m3, 7 hours/day, 5 days/week for 256 days, some liver
damage occurred in rats and rabbits.
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In a study previously discussed (Merkureva et al., 1979), tne authors
also examined the effect of chronic CCl^ inhalation. Long-term exposure
to 300 mg CCl^/m3 caused a considerable increase in the DNA-synthesizing
connective tissue cells (Merkureva et al., 1979).
Rotenoerg (1978) examined the dynamics of liver bioenergetic system re-
sponses following the chronic exposure of rats to small concentrations of
CC14 in air (14 mg CCl^/m3, 5 hours/oay, 5 days/week for 5 months).
As stated oy the authors, the exposure caused phasic changes in hepatic
energy-producing processes as evidenceo by alterations in respiratory rate,
phosphorylation ana sensitivity of respiratory enzymes to respiration in-
hibitors (Rotenoerg, 1978).
8.2. HUMANS
Many poisonings have resulted from the accidental or suiciaal ingestion
of CCl^ or from its medicinal use as an anthelmintic. For its medicinal
use, the tnerapeutic dose recommended for adults was 2 to 3 mil in capsule
form ana 0.13 m£/year for infants and children up to 15 years of age (von
Oettingen, 1964). As emphasized by von Oettingen (1964), such doses, which
are followed by doses of Epsom salts, have caused toxic effects-only excep-
tionally. Horrocks (1934) reported one fatality from its medicinal use.
Tne vast majority of poisonings, however, have resulted frum the innaiation
of its vapors when used as a solvent or dry cleaning agent (von Oettingen,
1964). Still other poisonings have oeen the result of dermal exposures
through the use of CC14 in shampoos (NIOSH, 1975). Finally, some have
resulted from its use in fire extinguishers (Dudley, 1935).
Norwood et ai. (1950) reported the occurrence of 2 fatalities, 1 near
fatality, 4 poisonings requiring hospitalization, and 51 mild industrial
poisonings in two communities over a period of 1 year. In 1935, Smyth
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(1935) noted 28 fatalities, 14 of which resulted from the ingestion of
CCi ; 120 acute and suochronic poisonings; and 7 cases of chronic poison-
ing. By 1964, an additional 28 poisonings resulting from CCi^ ingestion
(including 10 fatalities) ano 202 cases from innaiation (including 29 fatal-
ities) were reported. The actual incidence of such poisonings is douotless
much greater, since many poisonings are not attributed to CCl^ and others
are not published in the medical literature (von Qettingen, 1964). Since
1964, there nave been additional poisonings ano case reports (Bagnasco et
al., 1978; Bonitenko and Bruk, 1979; Shimanko et al., 1979; Campbell et al.,
1980); however, the total number has not been compiled.
8.2.1. Case Reports.
8.2.1.1. ACUTE — Oral poisonings from acute exposure to CC14 have
occurred to a great extent, as reported by a numoer of authors (Docherty and
Burgess, 1922; Seattle, et al. 1944; NIOSH, 1975; KirkpatriCK and Suther-
land, 1956; Dawoorn et al., 1961). A summary of the symptoms of sucn oral
poisoning is given below (von Oettingen, 1964). Following ingestion of
CCi,, tne patient experiences a burning sensation in the mouth, esophagus,
ana stomach. Depending upon the dose, this is sooner or later- complicated
oy aodominal pain, nausea, and vomiting. Some patients develop hiccoughs.
The tongue is coated. These symptoms are soon followed by diarrhea, which
later may oe followed by constipation and occasionally by gastric and in-
testinal hemorrhages which, in rare cases, may also be seen in the mouth and
pharynx. Again, depending upon the dose along with other factors, the pat-
ient becomes jaundiced, the liver becomes enlarged and tender; this may be
associated with ascites and generalized edema. Soon after the ingestion,
the patient feels dizzy, may suffer from headache and become confused, semi-
conscious and delirious. The patient ray become restless and develop
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cnoreatic movements. Finally, consciousness is lost ana the patient paoses
into coma. Sume patients complain of visual disturoances ana eaema of the
eyelids ana develop nemorrnages of the scierae. In severe cases, circula-
tory disturbances may develop, cnaracterizea Dy lowered or increased olood
pressure, tnin and rapia pulse, ana signs of congestive heart failure witn
cyanosis.
A fatality attriDuted to ingestion of caroon tetracnioriae was reportea
oy Smetana (1939). The victim, a photographer descrioed as having "a his-
tory of chronic alcoholism," died 10 oays after consuming an unKnown amount
of "some fluid containing caroon tetrachloride." He presentee symptoms in-
cluding nausea, vomiting, jaundice, anuria and semistupor. In the final
clinical diagnosis, death was attributed to caroon tetrachloride poisoning.
A case of attempted suicide by ingestion of caroon tetracnioride was
reported oy Stewart et al. (1963). The victim, a 29-year-old female who
ingested one pint of a carbon tetrachloride:metnanoi solution (2:1), experi-
enced ringing in the ears immediately after ingestion and lost conscious-
ness. Sne was nospitalized for 3 weeKs. Tnree hours after inyestion, car-
Don tetracnioride in the exhaiea breath ana blood was confirmed oy infrared
analysis. The exhaled breath was tnen monitored throughout the hospitaliza-
tion; CC1. was reported to aecrease exponentially. Because of the toxic-
ity of the methanol ana tne possibility of synergistic reactions with the
caroon tetrachloribe, hemoaialysis was performed soon after admission. Man-
nitol solution was given by continuous intravenous infusion. Clinical lao-
oratory analyses during hospitalization showed some elevation of SCOT, which
reached a maximum of 75 units on day 6, and an elevation of urinary urooiii-
nogen to a maximum of 7.8 Ehrlich units on day 10. Other laooratory find-
ings included elevation of serum iron ana depression of serum protein con-
8-26
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centration and aloumin fractions. The retention time of bromosulfopnthaiein
was increased. These findings were interpreted as evidence of minimal hepa-
toceiiular injury. Acute renal dysfunction was not observed; tne autnors
credited the mannitol treatment with preventing renal damage.
Lamson ana Minot (1928) studied the lethal effects of carbon tetrachio-
riae on patients receiving carbon tetrachloride and magnesium sulfate orally
as a treatment for hookworms. The authors reported the treatment of thou-
sands of patients with a single dose of 2.5 to 15 m£ carbon tetrachloride
without ill effects. One man was reported to have safely ingested 40 mH
of carbon tetrachloride. However, an "extremely small" population of adults
died after receiving 1.5 ml of carbon tetrachloride; doses of 0.18 to 0.92
mt were reported to be fatal to children. Susceptibility in adults was
correlated with alcoholic intake (chronic alcoholism or exposure to alcohol
shortly after treatment), the presence of ascarid worms, and the intake of
foods, particularly of nign fatty content.
Tnere are few postmortem reports on pathological changes in patients
after tne ingestion of CCl^. McMahon ana Weiss (1929) examined a 34-year-
oia male alcoholic who died five days after drinking one ounce of CCl*.
They discovered some reddish-brown fluid in the abdominal cavity, early
atheromatous lesions in the heart, congested and edematous lungs with scat-
tered petecnial hemorrhages, enlarged and congested kidneys, marked erosion
of the esophagus, and a congested and enlarged fatty liver.
Cases of acute toxicity associated with CCl^ inhalation by humans have
been more numerous. Bilateral peripheral constriction of the ocular color
fields, resulting in symptoms of toxic amblyopia in three males, was attrib-
uted to the inhalation of carbon tetrachloride vapors (Wirtschafter, 1933).
3-27
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Five male employees of dry cleaning establishments who had been exposed to
caroon tetrachloriae (of unknown concentration) from 3 to 10 hours daily for
1 to 6 months were examined. Two men also had signs of conjunctivitis.
Tnree of the men complained of visual disturbances characterized by blurred
vision or spots before the eyes. Wirtschafter concluded that toxic amblyo-
pia may result from exposure to caroon tetrachloride vapor.
One fatality occurred in two cases of carbon tetrachloride poisoning
reported by Smetana (1939). In the fatality, a dry cleaner ana interior
decorator described as being "a steady and heavy drinker" was exposed for
several hours to carbon tetrachloride vapors (concentration unknown) during
work. Upon returning from work, he noted dyspnea. Several hours after tne
exposure, heaoacne, dizziness and malaise developed, accompanied by nausea
ana repeated vomiting tnat persisted for several days. The patient also
suffered labored breathing and cough with blooay sputum before he diea 9
days after exposure.
The secono innalation case reportea by Smetana was a housemaid also de-
scribed as having a history of chronic alcoholism. Three days before hospi-
talization, 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 describeo for the otner case. After
approximately 1.5 months of hospitalization, this patient was released; her
condition several wee«s later was described as "much improved."
Seven of the cases of carbon tetrachloride poisoning reported by Norwooa
et al. (1950) resultea from both occupational and nonoccupational innalation
exposures. In the three cases described as "severe" poisonings, there was a
nistory of cnronic alconolism; two fatalities occurred in this group. In
one fatal case, the victim had oeen exposed for about 25 minutes to an
8-28
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atmosphere containing carbon tetrachloride at an estimated 1575 mg/m3
(tnis estimate was maae by duplicating the conditions). Histopathological
examination of liver and kidney tissue from the fatalities revealed liver
necrosis ana Degeneration of the renal tuoules. The four remaining cases
were characterized as "mild industrial" exposures. After exposure to caroon
tetracnloride, all subjects suffered varied symptoms including nausea,
vomiting, diarrhea, headache, muscular ache, pain or numbness, laoored
oreathing ana aizziness.
In another case, a 31-year-old janitor suffered malaise, bacK and lower
aboominal pain, nausea and vomiting the morning after working for 5 hours in
a closed room with carbon tetrachloride (concentration unknown) (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 SCOT activities with concomitant liver changes were reported in
two men occupationally exposed to unreported concentrations of carbon tetra-
cnloriae (Lachnit ano Pietschmann, 1960). One became ill after .exposure to
carbon tetracnloride for 3 hours in a relatively well-ventilated room. He
was hospitalized 3 days after exposure. His liver was sligntly enlarged,
with the SCOT value elevated by 6000 units. This value rapidly decreased
ano by the 10th day had returned to normal. A biopsy of the liver taken on
the 8th day showed necrosis in the centers of the lobuli, but the surround-
ing tissue was undamaged. An additional needle biopsy of the liver taken on
the 28tn day showed that the cells had almost returned to normal. In the
second case a male similarly exposed to carbon tetrachloride entered the
8-29
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hospital 12 cays after exposure. The SCOT nac increased to 80 units. A
liver needle Diopsy on the 22nd day showed only moderate changes, some of a
degenerative nature.
In a chemical packing plant, use of caroon tetrachloride by two workers
for equipment cleaning as a substitute for the customarily used acetone,
resulted in the hospitalization of 4 of 43 workers at the plant (Folland et
si., 1976). Ten additional workers became ill. Eight of the 43 workers
fell ill within 12 hours following the start of the 2-nour 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 D,390 units. All patients recovered within 90 days. All
hospitalized workers, as well as most of the others taken ill, ha a wor«ed
near a bottle-filling operation for isopropyl alcohol at the northern end of
tne plant, adjacent to the carpon tetracnloriae cleaning area.
Carbon tetrachloride concentrations at the time of exposure were not
ascertained; acetone was normally used for cleaning. Isopropyl alcohol con-
centrations at the northern end of the plant averagec 2624 mg/m3. Acetone
in alveolar air samples of workers in the northern area averaged 121.6
mg/m3. The authors ascribed the toxic episode to carbon tetrachloride
toxicity potentiated by isopropyl alcohol. Because carbon tetrachloride
concentrations were unknown and isopropyl alcohol (and possibly otner chemi-
cals) were present, the nealth effects reported in this stuay cannot be
attributed tc carbon tetrachloride exposure alone.
Some of these reports and others (Davis, 1934; Stewart et al., 1961;
Smith, 1950; NIOSH, 1975; von Oettingen, 1964) indicate that with single
exposure to low concentrations, there is consideraole variation in symptoms
among different persons and that tne acute toxicity is relatively low in
3-30
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contrast to that with repeated exposure. Cases in which exposure is light
may be restricted to such symptoms as moderate irritation of the eyes, mod-
erate dizziness and neadache, which disappear promptly upon discontinuation
of the exposure.
The immediate effects from acute inhalation exposure to higher concen-
trations of CCl^ consist of the same symptoms as descrioed above, but in
aooition the patient may become nauseated ana suffer from loss of appetite,
mental confusion, agitation and the feeling of suffocation. In severe
cases, the patient may lose consciousness and develop fever and chills. The
tongue may be furred and the patient may suffer from vomiting with bloody or
oile-stained vomitus which may last for days, colicky pain and diarrhea with
liquid brown-black or bloody stools (von Oettingen, 1964). This ten- dency
for hemorrhages may also result in bleeding from the gums and nose,
hemorrhages under the skin and macular papular rashes. The colicky pain may
be associatea with a marked abdominal resistance simulating the "acute
abdomen" and thus has been mistaken for appendicitis and peptic ulcer. Fol-
lowing such an acute episode, the patient feels tirea ana weak and frequent-
ly suffers from headache. The patient may develop muscular twitchings and
epileptic convulsions. In a few instances, paralysis (hemiplegia) ana poly-
neuritis have been reported (von Oettingen, 1964).
In more severe innalation poisoning blood pressure may be lowered, but
as renal complications develop, the blood pressure is usually elevated and
the cardiac output decreases because of increased peripheral resistance.
The pulse may be accelerated. In the case of severe inhalation poisoning,
the patient may collapse. Electrocardiograms have shown changes character-
istic of myocardial injury characterized by sinus bradycardia and followed
by auriculoventricular arrhythmia, auricular fiorillation and sinus
arrhythmias (von Oettingen, 1964).
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Depending upon the condition of the patient, respiration may oe normal,
rapid and shallow, or slow and labored. The latter is evident especially if
circulatory failure is imminent and pulmonary edema develops. Thompson
(1946) found that early roentgenograms of the lungs may show pulmonary in-
volvement.
In most instances after the severe inhalation exposure, the oatient
develops signs of liver injury within a few days. The patient becomes jaun-
diced and the liver becomes enlarged and tender. This is toxic hepatitis,
which may pass into yellow atrophy and, in more protracted cases, eventually
into cirrhosis of the liver. In the early stages of liver injury, even be-
fore a marked enlargement occurs and while liver function tests such as the
cephalin-flocculation test are still normal, the SCOT level may be marKedly
elevated (von Oettingen, 1964).
As signs of liver injury develop, ano sometimes in their absence, injury
of tne kidneys may dominate the clinical picture and oe responsible for
early death (von Oettingen, 1964). Kittleson and Borden (1956) character-
ized renal failure by three phases. The first phase is characterized by
poiyuria and nocturia, which may result in severe dehydration, -followed by
oliguria and finally by diuresis. The renal injury may result in acute
nephritis with albumin, red and white cells, and casts in the urine. In
some patients, the presence of acetone and sugar in the urine has been re-
ported. The oliguria may be associated with increased blood levels of po-
tassium, indican, phenol, cresol, creatinine and urea; the latter may re-
sult in uremia. In other instances, the injury may consist of necrotizing
nepnrosis with comparatively little change in the urinary composition. The
renal blood flow and glomerular filtration rate are decreased; and the form-
er seems to oe mainly responsiole for the maintenance of oliguria, being the
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sequela rather than the cause of renal failure (von Oettingen, 1964).
During the early stage of oliguria, abnormal tuDular back diffusion of the
filtrate may play an important role. Oliguria may develop as early as 24
hours or 3 to 4 days after onset of the poisoning and may persist for 12 to
14 days and even longer (von Oettingen, 1964).
In the early stages after severe inhalation poisoning and during the
period of polyuria, the blood may show some polycythemia, but later this may
oe followed by anemia ana lowering of the hematocrit levels because of hemo-
»
dilution. The most important changes in the blood are, however, related to
the biochemical composition of the blood wnich reflects the renal ana hepa-
tic injury. As soon as the renal injury develops, the nonprotein nitrogen
and urea nitrogen levels in the blood are increased and may reach extremely
high values. The creatinine, indican, phenol, and cresol levels may also be
increased. In the case of liver injury, as related to the blood, the icter-
ic index is usually increased, and the levels of sugar and phospolipids,
along with the ratio of cnloesterol esters over chloestrol, are reduced.
The protnrombin time and the fibrinogen content may be reduced, resulting in
an increased clotting time. The chloride level is frequently, lowered by
nemodilution or severe vomiting, and the potassium level may be elevated.
Tnis increase in potassium may contribute to ventricular fibrillation or
cardiac arrest (von Oettingen, 1964).
In addition to constriction of the visual field and toxic amblyopia
(wirtscnafter, 1933), carbon tetrachloride poisoning can also result in
blurred and double vision. Conjunctival hemorrhages are common. Retinal
hemorrhages and exceptional cases of the degeneration of the optic nerve
have been reported (von Qettingen, 1964).
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Conway ana Haven (1946) report tnat after ingestion of CC1,, certain
electrocardiograpnic cnanges may be observeo indicating degenerative pro-
cesses in the heart muscle, such as sinus braaycaraia, followea oy auricula-
ventricular rhythm, auricular fiorillation, and sinus arrhytnmias. The res-
piration varies with the condition of the patient. If the patient is in
collapse, it will be rapid and shallow; if the patient is comatose, it may
be labored and dyspneic, and pulmonary edema ana hemorrnages may develop.
Eventually, disturbances develop characterized by polyuria and followed by
aliguria which may pass into anuria. The urine of such patients is ricn in
albumin and may contain blood and casts. If the liver is damaged, the urine
will contain urobilinogen, urobilin, ana bile pigments. The nonprotein
nitrogen level in the blood will be increased and the patient (ray suffer
from hypoprothromoinemia, nypochloremia, ana signs of aciaosis. Death may
ensue after 3 nours, or 3, 5 or 10 days, and sometimes later.
Two casts involving the pancreas following inhalation or an acute expo-
sure of CCl^ were reported by Jahnke (1953). Both patients oecame list-
less and aevelopea hepatic and circulatory disturoances ana sensitivity of
tne pancreas to pressure. Such disturbances were long term ana had not com-
pletely suosiaed after 10 months.
Guild et al. (1958) report on 20 cases of CC1A intoxication which re-
sulted in renal damage manifested by anuria. Four of these were the result
of ingestion. Exposures varied: one drank 15 cc; one drank 4 ounces; 2
drank small amounts, but were also consuming alcohol (see Chapter 12). The
remaining 16 cases inhaled CCl^. Exposure times ranged from 30 minutes to
11 hours. Two of the four that ingested the CCl. died whereas three died
of the 16 exposed through inhalation.
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Severe liver involvement was seen in all four of those ingesting CCl,.
Eleven of the 16 who inhaled CCl^ had "moderate to severe" liver involve-
ment whereas the remaining 5 had no liver involvement (Guild et al., 1958).
These discrepancies can be attributed in part to the variaoility in exposure
amounts and duration; however, this cannot be resolved from the report. The
role of alcohol consumption also cannot be defined; however> this report
does ieno some support to the research discussed in Chapter 12.
Stevens and Forster (1953) report on the neurological effects of CCl, .
Fifteen cases of CC1. intoxication with varying amounts and routes of
exposure are discussed. Thirteen occurred in the home and two occurred in
commercial cleaning plants. Of the D cases occurring at home, 2 were chil-
dren (the youngest being 9 months old) who drank the fluia. Two were adults
wno accidentally orank it, and the rest were exposed via inhalation. There
were five deaths. Eleven of the 13 were alchoholic. Four of the deaths
occurred in alcoholics; for the fifth, no history was availaole (Stevens and
Forster, 1953). Seven of the fifteen experienced neurological symptoms. Of
these, two children who ingested the chemical displayed stupor, drowsiness
or unconsciousness. The adults displayed headache, vertigo-, weakness,
Dlurred vision, lethargy and coma.
8.2.1.2. SUBCHRONIC/CHRONIC — Subchronic/chronic human exposures to
carbon tetrachloride are nearly always by inhalation. Heacache, fatigue,
dizziness, nausea, and vomiting occur at suostantially lower concentrations
in suochronic and chronic exposures than in acute exposure (Elkins, 1942;
Kazantzis and Bomford, 1960). As in acute exposures fatty and necrotic liv-
ers are the common pathological findings (Gray, 1947; Dellian and Wittgens,
1962; Barnes and Jones, 1967). In some cases, renal injury is the major
3-35
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finding. Renal tubular necrosis has been reportec ooth with ana without
concomitant liver disease (Hamourger et al., 1958; Richet et al., 1959; von
Oettingen, 1964).
CC1, poisoning as a result of chronic innalation of low dose exposures
nave oeen reported by Butsch (1932), Wirtscnafter (1933), Straus (1954), von
Oettingen (1964) and others. The clinical picture after chronic CC14
exposure is mucn less characteristic than that after acute exposure, von
Oettingen (1964) has reviewed the symptoms. With chronic exposure, patients
may complain of fatigue, lassitude, giddiness, anxiety, and headache. They
suffer from paresthesias and muscular twitchings and show increased reflex
excitability. They may be moderately jaundiced, have a tendency to hypogly-
cemia, and biopsy specimens of the liver may show fatty infiltration. Pa-
tients may complain of lack of appetite, nausea, and occasionally diarrhea.
In some instances, the blood pressure is lowered whicn is accompanied by
pain in the cardiac region and mild anemia. Other patients aevelop pain in
tne Kidney region, dysuria and slight nocturia ana have urine containing
small amounts of albumin and a few reo blood cells. Burning of the eyes
and, in a few instances, blurreo vision are frequent complaints of those
exposed. If these symptoms are not pronounced or of long standing, recovery
usually takes place upon discontinuation of the exposure if the proper
treatment is received (von Oettingen, 1964).
Straus (1954) suggested a possible causal relationship between carbon
tetrachloride exposure and aplastic anemia. Three males had been exposed
via inhalation and dermal absorption to carbon tetracnloride at unknown con-
centrations for 2 months to 3 years. Autopsy findings included hypoplasia
of the bone marrow. However, a causal relationship between caroon tetra-
cnloride and aplastic anemia suspected oy the author in these cases is not
3-36
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supported adequately. One of the men nad also been exposed to kerosene for
3 years. Anotner was an auto mechanic who worxed in a garage. Tne occupa-
tion of the third was not specified although his exposure to carbon tetra-
chloride 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 expected in carbon
tetrachloride poisoning. In one case the liver was reported to have peri-
portal fibrosis and fatty infiltration. These findings were attributed to
toxic hepatitis wnich was considered to be the result of carbon tetrachlo-
riae poisoning. The information reported in these case studies tends not to
substantiate the author's suggestion that the patients' illnesses may have
been caused oy caroon tetrachloride.
Postmortem reports on pathological changes in patients after inhalation
of CCl^ are generally limited to findings in the liver and kidneys. The
liver may snow nutmeg appearance and fatty degeneration even in the aosence
of clinical signs and symptoms of liver injury. In other instances, centri-
iobular necrosis and hemorrnages with infiltration of leukocytes and histio-
cytes and collapse of the lobules with condensation of the reticular frame-
work within these areas are seen. After chronic exposure, there may be evi-
dence of regeneration of the liver cells (von Oettingen, 1964).
Postmortem changes in the kidney are characterized by nepnrosis, by a
distention of Bowman's capsule with albuminous precipitates, and py swelling
of the lining cells. The cells of the convoluted tuoules may be swollen ano
vacuolated; later, degenerative changes may be seen in Henle's loops, asso-
ciated with granular, hyaline, and cellular casts in the tubules. After
chronic exposure, regenerative changes may oe visible in these regions. In
other cases, the kidneys may offer the picture of acute hemorrnagic nephri-
tis (von Oettingen, 1964).
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Other postmortem organ changes are less characteristic for CC1, poi-
soning anci vary consideraoly with the clinical picture. Some changes may
occur that are a direct result of tne changes occurring in the primary tar-
get organs of CC1,. Stasis of various organs is the most outstanding fea-
ture of caraiac failure. The brain and lungs may be eaematous. The intes-
tines may be hyperemic and covered with numerous petecnial nemorrhages, and
tne spleen may oe enlarged and hyperemic. Occasionally the adrenal glanas
may show Degenerative changes of the cortex, and the heart may undergo toxic
myocarditis (von Oettingen, 1964).
8.2.2. Controlled/Clinical Studies. Human volunteers were exposed to
known concentrations of carbon tetrachloride vapor in an effort to correlate
physiological and/or biochemical changes to the magnitude of exposure
(Stewart et al., 1961). Eight healthy male volunteers were exposed to car-
bon tetracnloride vapors in a series of three separate experiments performed
1 month apart. Prior to exposure, data on blood pressure, SCOT and urinary
uropiiinogen were optainea for each suoject. Samples of pre-exposure ex-
naleo oreath, 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 curing the experiment but an
air supply grill was left open. A fan circulated air across the dish. Car-
oon tetrachloride amoient concentrations were monitored with 3 Davis halioe
meter and an infrared spectrometer. The carbon tetrachloride concentration
ranges and exposure times are given in Taole 8-6.
Carpon tetrachloride was detected in exhaled breath in all three experi-
ments. Graphs showed an exponential decrease in concentration of carbon
tetrachloride versus time. The exact values were not qiven.
8-38
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TABLE 8-6
Exposure Times ana Concentrations of Caroon Tetrachloride Vapor
In a Controlled Human Study*
Experiment
1
2
3
Average Concentration,
Time-weighted (mg/m3)
309
69
63
Concentration
Range (mg/m3)
192-548
63-88
57-88
Exposure
(minutes)
70
180
180
*Source: Stewart et al., 1961
8-39
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The serum iron showed an initial decrease in 3 of 6 suojects at the 309
mg/m3 exposure level but had returned to normal in two of these suojects
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 suojects
exposed to carbon tetracnloride at 309 mg/m3, the serum transaminase level
was sligntly elevated in some and depressed in otners, but remained within
the normal range. Carbon tetrachloride was not detected in the blood or
urine at any exposure time or dose, but tne analytical technique used
(infrared metnod) was not a sensitive one. The authors concluded that no
ill effects were observed from exposure to carbon tetracnloride at 63
mg/m3 for 180 minutes, although the small changes in serum iron at the 309
mg/m3 aose might have been an indication of liver insult.
A survey conducted by Kazantis and Bomford (1960) was the result of the
complaints of a worker in a factory processing raw quartz. Intermittently
for 2 years, he experienced anorexia, nausea and occasional vomiting with
aodominal discomfort. He noticed that his symptoms or dyspepsia worsened
during the work week but got better on the weekends. The investigators
interviewed 17 of the 18 employees working in the same area taKing meoical
and occupational histories. Their ages ranged from 16 to 54. Fifteen of
tne 17 complained of symptoms similar to the initial case, primarily nausea,
anorexia, vomiting, flatulence, epigastric discomfort and depression. These
symptoms had been occurring among the workers from 1 week to 2 years. Envi-
ronmental monitoring measured CC14 atmospheric concentrations of 292.5 to
650 mg/m3. Once the cause of the hign concentration was found, changes
were made in the processing procedure. Within 1 week following these
changes, the symptoms disappeared in all workers. Follow-up for 6 months
revealed no recurrences.
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Direct application of CC1. to human sKin causes a burning ana stinging
sensation within 5 minutes. The maximum pain is reached 6 minutes later and
is associated with erythema, hyperemia, and wheal formation, later followed
by vesication (Oettel, 1936).
The aosorption 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 carbon tetra-
chloride was analyzed by infrared spectroscopy, and no impurities were de-
tected. Sequential sensations of burning and cooling were experienced by
all volunteers during the immersion. 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
tetrachioride ,vas detected in the alveolar air of each suoject within 10
minutes of immersion of tneir thumbs. The concentration in the expired
oreath rose continuously to a maximum of 4.0 mg/m3 10 to 30 minutes after
the exposure period ended, and then decreased exponentially. The mean con-
centration of carbon tetrachloride 2 hours after the end of exposure was 2.0
mg/m3; at 5 hours after exposure, the alveolar air concentration was still
>0.6 mg/m3. The authors concluded tnat carbon tetrachloride could be
absorbed through the skin in toxic quantities.
Hall (1921a,b) demonstrated the effectiveness of CCl^ as a vermicioal
agent in treatment of hookworm infestations. The usage of CC1. in this
capacity stimulated considerable research efforts to investigate the pharma-
cologic and physiologic effects of CCl^ on humans (NIOSH, 1975). The ef-
fects of oral ooses of CC1. as a human anthelmintic, administered to con-
aemned prisoners in Ceylon has been reported (Docnerty and Burgess, 1922;
8-41
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Docherty and Nicholls, 1923). Three of the prisoners receiveo 4 mil
CCL, two received 5 mil CCl^, and one received 5 mSL plus an addi-
tional 3 mil two weeks after the first dose. Execution of the prisoners
occurred 3 to 15 days after the CC1 administration. Autopsies were per-
formed and the finoings varied. The livers of some showed no major micro-
scopic or macroscopic changes wnereas the livers of other showed marked
ratty degeneration. From sucn data, a dose-response relationsnip would be
difficult to determine (NIOSH, 1975).
A cross-sectional epidemiologic study (Sonich et al., 1981) examineo the
effect upon persons exposed to CC14 through their drinking water. Seventy
tons of CC1, were spilled in the Kanawha and Ohio Rivers in 1977. Mea-
surements of raw water revealed maximum concentrations of 0.340 mg/H.
Twenty-one cities situated along tne river were involved in the study.
These cities represent areas that draw their drinking water directly from
tne river and/or areas that draw their drinking water from sources not in-
fluenced Dy the quality of the river water. Measurements at each of the
faur major cities along the river showed a decrease in the CCl, concentra-
tion in the river with the number of river miles from the spill: Hunting-
ton, West Virginia - 0.210 mg/Z; Cincinnati, Ohio - 0.180 mg/2,; Louis-
ville, Kentucky - 0.110 mg/2,; Evansville, Indiana - 0.060 mg/Jl. Fin-
ished drinking water in Cincinnati contained a peak level of 0.087 mg/2..
A control period was identified in 1976 when there were non-detectable to
trace amounts of CCl, in the river as indicated by monitoring efforts at
Cincinnati. By using river volumes and flow rates, periods of high exposure
(1977) ano low exposure (1976) to CCl^ were estimated for each city along
the river. A total of 35 nospitals in these cities provided medical data
on patients admitteo during these estimated time periods. Tne results of
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routine tests measuring serum chemistries reflecting liver ana kianey func-
tion along with basic epidemiologic information were aostracted from approx-
imately 6000 medical records. It is hypothesized tnat CCl^ would cause a
rise in some or all of these serum chemistries. The data were categorically
analyzed to test for a oose/response relationship. In this capacity, tne
ratio of tne odds ratios (ROR) for each serum chemistry for each city group
were computed as ROR = OR77/OR7 where
the odds of naving an elevated test result
wnile drinking Ohio River water in 1976
oods rati076 = tne odds of naving an elevated test result
(ORy^) wnile drinking water in 1976 not affected
oy the quality of the Ohio River
and
the odus of having an elevatea test result
while drinking Ohio River water in 1977
odds ratioyy = the odds of having an elevated test result
(ORyy) while drinking water in 1977 not affected
Oy the quality of the Ohio River.
The results obtained for creatinine show a positive and statistically sig-
nificant (pc 0.05) dose/response relationship between the CC1, exposure
ano the ROR or frequency of elevated levels of serum creatinine in exposed
patients in relation to the controls as determined by a test for linearity
of trend. Otner parameters analyzed were alkaline phosphatase, -total oili-
rubin, olood urea nitrogen, lactic dehydrogenise, SGUT and y-giutamyl
transpeptioase. Nu similar results were found for these parameters.
8.3. MECHANISMS OF TOXICITY
The toxicity of caroon tetrachloride to an organism depends upon the
aoility of the organism to metaoolize the compound; unmetaoolized caroon
tetracnioride does not appear to be significantly toxic (Recknagel and
Glende, 1973). In mammals, as discussed in Chapter 7, carbon tetrachloride
is thought to be metabolized in the endoplasmic reticulum of the liver by
the mixed-function oxidase system of enzymes. A reaction sequence proposed
8-43
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in the literature for carbon tetrachloride metabolism is outlined in Fig-
ure 7-1. Two free radicals have been postulated as metabolic intermed-
iates: 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 (pnosgene), dimerization to hexachloroethane, free radical binding
to protein, and lipio peroxidation. In this chapter each of these proposed
patnways will pe presented in conjunction with the toxic effects attrioutea
to it.
8.3.1. Formation of Carbonyl Chloride (Phosgene). From the results of an
in vitro study of carbon tetrachloride metabolism, Shan et al. (1979) postu-
lated tne formation of carbonyl cnlorioe from the trichloromethyl radical.
As previously discussed, the authors incubated L-cysteine and [ Cjcarbon
tetrachlorioe with rat liver nomogenate and looked for tne formation of
2-oxothiozolidine-A-carboxylic acid. This compound is formed from the reac-
tion of L-cysteine and carbonyl chloride. Analysis of tne metabolic prod-
ucts by mass spectroscopy showed a fragmentation pattern consistent with
2-oxothiozolidine-4-carboxylic acid. The authors inferred from- these ana-
lytical data that carbonyl chloride was formed as a metabolic product of
carbon tetrachloride. Although carbonyl chloride (pnosgene) is not reported
to be a carcinogen, the authors pointed out that the compound is hignly
toxic and that the reactive chlorines could react with macromolecules in
ways similar to alkylating agents.
8.3.2. Dimerization to Hexachloroetnane. Hexachloroetnane has been iaen-
tifiea as a metabolite of carnon tetrachloride by Fowler (1969). The forma-
tion of this compound is believed to take place by the dimerization of the
trichloromethyl raoical. Although hexachloroethane is a hepatotoxin, its
8-44
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toxicity is less than tnat seen in carbon tetrachloride poisoning. There-
fore, other mechanisms probably account for the severe toxicity of carbon
tetracnloriae.
8.3.3. Free Radical Binding to Proteins. Free radical binding to pro-
teins nas oeen postulated as one cause of toxicity associated with carbon
tetrachloride (Recknagel and Glende, 1973). The binding was reported to
involve reactions with cellular proteins, particularly those with sulfnydryl
groups. Oelvillarruel et al. (1977) found a good qualitative correlation
between intensity of CCl^ activation and P-450 content of various organs
in male, virgin female, or pregnant female rats. No correlation was found
between CCl. activation and cytochrome c reductase activity. The authors
state that their results suggest that P-450 is involved in CCl. activation
ana that irreversible binding of CCl. metabolites to cellular components,
rather than lipid peroxidation, is responsible for some biochemical and/or
ultrastructurai lesions reported in different tissues. In another stuoy,
L 4C]caroon tetracnloriae has oeen observeo to bind irreversioly to raboit
microsomal proteins at a rate of approximately 20 nmole/mg protein/hour
(Uehleke and Werner, 1975). Binding of caroon tetracnloriae (or-its metaoo-
lites) to hepatic macromolecules was enhanced in the absence of oxygen, con-
sistent with the proposal that the trichloromethyl radical is the reactive
metabolite. Furthermore, the oroer of species susceptibility to liver
necrosis from caroon tetrachloride more closely parallels the species order
for'C Cjcarbon tetrachloride binding to cellular components than the spe-
cies order for lipid peroxidation (Diaz Gomez et al., 1975):
Effect + Most susceptible Least susceptible ->•
Liver necrosis mouse > guinea pig = hamster > rat > chicken
f
[I^CJCCI^ binding (rouse = hamster > guinea pig > rat > cnicken
Lipid peroxidation rat > hamster = guinea pig > cnicken = mouse
3-45
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These authors also report that in mice liver necrosis proceeded 24 hours in
the; aosence of lipid peroxidation.
Although it has oeen shown that [ C] from caroon tetrachloride binds
to proteins, the question of caroon tetrachloride binding to poiynucleotides
remains. The issue is important because of its implications for the mechan-
ism of caroon tetrachloride carcinogenicity and mutagenicity. In the only
experiment addressing this question, Uehleke and Werner (1975) incubated
[ Cjcaroon tetracnloride with either isolated liver microsomes (rat or
mouse, species not identified) or with added soluble RNA. They reported no
[ C] binding to ribosomal RNA or exogenous RNA. Experimental details
were not presented, but it appears possible to tentatively conclude that
proteins — rather than nucleic acids — are trie main sites of macromolecu-
lar caroon tetracnloride oinding.
8.3.4. Lipid Peroxidation. A numoer of the hepatic effects resulting
from caroon tetracnloride exposure, including the fatty liver syndrome, are
oeiieved to arise as a result of iipid perioxidation (RecKnagel and Glende,
1^73). Tne mechanism proposed for the peroxidation is presented oelow, fol-
lowed by a discussion of the evidence that this biochemical sequence results
in the hepatic lesions associated with carbon tetrachloride poisoning.
The first step in the reaction sequence proposed for lipid peroxidation
is production of free radicals, especially the tricnloromethyl free radical.
The radical initiates a chain reaction by reacting with the hydrogen atom of
a -OU-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 disintegrates in
trto fashions: (1) intramolecular cyclization to form malonic dialoenyde and
two new free radicals, or (2) simple homolytic fission that also yields two
3-46
-------
free radicals. This whole process occurs autocatalytically: each free rad-
ical gives rise to two new free radicals. Figure 8-1 summarizes this hypo-
thesis (Recknagel and Glende, 1973).
A number of indices have been used in _in vivo ana _in vitro assays of
lipid peroxidation: pentane and ethane levels in exhaled air (arising from
fatty acid decomposition) and malonic dialdenyae concentrations in hepato-
cytes (arising from intramolecular 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 i.p. administration of caroon tetra-
chloride doses of 160, 480 and 1440 me/kg bw, respectively (Sagai and
Tappel, 1979).
A mechanism for the pathogenesis of carbon tetrachloride-induced hepatic
lesions based on lipid peroxidation has been proposed recently (Pasquali-
Ronchetti et al., 1980). According to this hypothesis, lipid peroxidation
is suggested to affect primarily unsaturated acyl chains of membrane pnos-
pnolipids, resulting in breakage of the hydrocarbon and loss of phospholip-
ids from the membrane. Lipid peroxidation would therefore produce progres-
sive degenerative changes in the assembly of memoranous struc-tures sucn as
(rat) liver endoplasmic reticulum, or its in vitro counterpart, microsomes.
This hypothesis is supported by studies showing that treatment with car-
bon tetracnloride produced lipid peroxidation in rat liver enaoplasmic reti-
cuium at a concentration of 0.5 mi/100 g bw (Pasquali-Ronchette et al.,
1980), caused disintegration of enduplasmic reticulum _in vitro within 10
minutes at a concentration of 636 mg/i (Pasquali-Roncnetti et al., 1980),
ana was incorporated predominantly into liver phospholipids in rats
(Table 8-7) (Ciccoli and Casini, 1978).
8-47
-------
H H H
-c=c-c-c=c-c-c=c-c-c=c-
H H H
HCC13 • CCJ/jTrichlormethyl
Free Radical
-oc-c-c=c-c-c=c-c-oc-
RESONANCE (All
Possible Forms Not
Shown.)
Organic Free Radical
Y Q a a Q y
OC-C-C- C=C-C=C-C-C=C-
Peroxide •
formation 02 diene conjugation, \nax=233mu
Y
8
a
•c-c-c=c-c=c-c-c=
o
o
H
c-
Organic Peroxide (Unstable)
Intramolecular cyclization
and decomposition to yield
malonic dialdehyde and two
new organic free radicals.
Decomposition to yield two free
radicals. Eventual stable decom-
position products highly organo-
leptic.
FIGURE -3-1
Free Radical Initiated, Autocatalytic Peroxidation of
Polyenoic Long-Chain Fatty Acids
Source: Adapted from Recknagel and Glende, 1973
8-48
-------
TABLE 8-7
Incorporation of 14C from [14CJCaroon Tetracniorioe
into Lipias of Various Rat Tissues3»D
Tissues
Total Lipids
(opm/mg)
Acetone Precipitate
(pnospnolipios)
(dpm/mg)
Acetone Supernatant
Lipids
(dpm/mg)
Liver (6) 112.6 +_ 7.4
Intestinal mucosa 61.8 +.7.5
Kidney (6)
Adrenals0
Lung (5)
Spleen (6)
Testas (6)
brain (6;
Heart (6)
23.4 ±2.6
8.0
11.3 +_ 1.8
8.7 +_ 1.2
6.3 _+ 1.5
3.3 +_ 0.3
2.2 +_ 0.8
Skeletal muscle (6) 0.7 +_ 0.1
Piasmad 5.1
135.7 +_ 14.6
69.5 +_ 7.7
25.6 +_ 3.9
22.0
15.2 +_ 1.4
7.4 ± 2.3
5.2 +_ 1.4
3.7^ 0.3
2.6 -v 1.4
2.9± 0.7
trace
53.3 +_ 4.3
48.3 ^ 8.4
11.4 +_ 2.0
2.4
6.5 +_ 0.8
4.0 ^ 1.0
2.3 ^ 1.0
0.7 +_ 0.2
0.8 ^0.5
0.6 ^ 0.2
3.7
aSource: Adapted from Ciccoli and Casini, 1978
D[14C]CC14 dose: 4000 mg/kg bw (58.6 x 10s dpm). Values are expressed
as means j-_ S.E.M. The number of rats is reported in parentheses.
cEight poolea adrenals.
dPiasma of two animals.
dpm = disintegrations per minute
8-49
-------
8.4. SU>WARY
Carbon tetrachloride is toxic to humans and animals following inhala-
tion, ingestion or dermal administration. Acute, sub-chronic and chronic
exposures primarily affect the central nervous system, liver and kidneys.
Sporadic cases of ocular toxicity also occur following subchronic and
chronic exposure to carbon tetrachloride vapor. However, these ocular signs
do not correlate with exposure levels or other organ toxicities. Ingestion
of alconol appears to increase susceptioility to carbon tetrachloride toxic-
ity, but the mechanisms are unknown.
8.4.1. Experimental Animal Data. The toxicity of carbon tetrachloride
following acute inhalation, ingestion and dermal exposures has been reported
for various species. Animals surviving acute dcses of carbon tetrachloride
developed liver damage ana, in some cases, kidney damage. These injuries
were dose related.
Subchronic/chronic studies of carbon tetrachloride exposure in rats,
monkeys, rabbits, dogs and guinea pigs demonstrated liver, kidney, sciatic
nerve, optic nerve and ocular muscle damage.
It has been observed that exposure to a higher concentration over a
shorter period of time produces a greater effect upon the liver than expo-
sure to a lower concentration over a longer period of time even though the
product of time and concentration is equal in both cases.
8.4.2. Human Data. Considerable human exposure to carbon tetrachloride
through inhalation has occurred through its use as an industrial solvent and
dry cleaning fluid. Ingestion of carbon tetrachloride or of mixtures con-
taining carbon tetrachloride has also been documented in various case
reports. Ingestion has occurred under different circumstances by persons of
diverse occupations and ages. These acute exposures have been followed by
hepatoxic effects accompanied by acute nephrosis.
8-50
-------
Hepatic necrosis ana renal pathology appear to be cnaracteristic effects
of acute human exposure to caroon tetrachloride. If exposure is terminated,
the liver snows regeneration in most cases. In cases of acute renal dys-
function, kidney function returns to normal after exposure to carbon tetra-
chloride is terminated and medical treatment is given.
In many of the case reports and older studies, the investigators present
tne data in narrative form. Although interesting, these type of data are
not suitaole to quantitative analysis since numbers are not adequately pre-
sented. Furthermore, there usually are a numoer of uncontrolled variaoles
(alcohol intake, age, simultaneous exposures) or unknown variaoles (exposure
amount) making it difficult to attrioute the outcome solely to the CC1.
exposure. The experimental human studies are not numerous, yet they are
important since they can either support or challenge the experimental animal
stuoies ana can aia qualitatively in the extrapolations from animals to
humans.
8.4.3. Mechanisms of Toxicity. The chemical pathology of CC1. liver
injury is generally viewed as an example of lethal cleavage, where the
CCLj-Cl bond is split in the mixed function oxidase syste'm of hepato-
cytes. Two major sequences of this cleavage have been suggested; both views
presume tne formation of free radicals from the homolytic cleavage of the
CClj-Cl bond (i.e., CCly and Cl-). One sequence entails the direct
attack (via alxylation) by free radicals on cellular constituents, notably
protein sulfnyoryl groups. The second sequence involves the abstraction of
a nyorogen atom oy the free trichloromethyl raaicai from a long-chain fatty
acid to form chloroform and a fatty acid free radical. Molecular oxygen,
because of its triplet ground state, binds witn the unpaired election on tne
fatty acid radical to form an organic peroxide. The peroxide is unstaole
8-51
-------
and decomposes to form more organic free radicals, wnich in turn form more
organic peroxides (Recknagel and Glenae, 1973). Tnis process appears to
lead to fatty acid chain decomposition, with the resulting breakdown of mem-
orane structure (Recknagel ana Glende, 1973). This breakdown may lead to a
nalt in lipid excretion via the Golgi apparatus, with fatty liver occurring
as a consequence. Cell necrosis would also follow directly from lipid
destruction. The mecnanism oy which lipid peroxidation could lead to cell
transformation is not explained at present, and tne molecular events leading
to CC14 carcinogenicity remain unknown.
In addition to these proposed mechanisms of toxicity, two minor metabol-
ic pathways have been postulated: dimerization of two trichloromethyl free
radicals to form hexachloroethane (Fowler et al., 1969) and the formation of
a trichloromethyl peroxy radical which may result in production of phosgene
and carbon dioxide (Shah et al., 1979). Both hexacnloroethane and phosgene
are toxic, but the extent of their contribution to observed hepatotoxicity
is un«nown. Carbon tetrachloride has not been found to bind to cellular
polynucleotides, but presently only one investigation studying the Dinding
of CC1, to such nucleotides has been reported.
3-52
-------
9. TERATOGENICITY AND OTHER REPRODUCTIVE EFFECTS
9.1. TERATOGENICITY
In two inhalation studies on the teratogenic ana prenatal toxicologic
effects of caroon tetracnloride, the chemical was reported to produce pre-
natal toxicity out not teratogenicity. In the first to be discussed,
Schwetz et ai. (1974) exposed pregnant Sprague-Oawley rats to caroon tetra-
cnioride at 1800 or 6300 mg/m3 for 7 hours/aay on days 6 to 15 of gesta-
tion. Statistically significant decreases in fetal oody weight and crown-
rump length were observed. Other parameters examined such as sex ratio,
live fetuses/litter and resorptions were not significantly different from
those of controls. Two other statistically significant fetal effects were
noted: an increased incidence of litters with sterneoral anomalies in the
6300 mg/m3 group and an increased incidence of litters with subcutaneous
edemas in tne 1800 mg/m3 group. The incidence of litters witn edema in
the 6300 mg/m3 group (50%), although apparently increased, was not signif-
icantly different from the control incidence (33%). The dams exposed to
both concentrations of carbon tetracnloride snowed a decreased food consump-
tion compared to control animals and a statistically significant-decrease in
weight gain. 6otn of these effects were greater at the higher dose level.
Hepatotoxicity, as measured oy significantly increased SGPT activity, was
also seen in tne dams following daily exposure to 1800 or 6300 mg/m3
CCl^ out the increase was greater at the lower dose level. Tne authors
did not establish any consistent pattern between fetal toxicity and maternal
toxicity at tne subanesthetic levels of carbon tetrachloride used in this
experiment. They concluded that carbon tetrachloride was not highly emoryo-
toxic at the concentrations used in this experiment. The evidence of ma-
ternal toxicity precluded any statement aoout the teratogenic potential of
CC14.
-------
Anotner study reported no teratogenic effects following exposure of
pregnant rats to caroon tetrachioride at 1575 mg/m3 8 hours/day for five
consecutive days Detween days 10 to 15 of pregnancy (Oilman, 1971). Concom-
itant exposure to 15% ethanol in drinking water also did not result in tera-
togenic effects. Caroon tetracnioride exposure, however, did decrease the
viaoility index to 83% as compared to 99% for controls (p<0.1), resulting
in a decrease in the number of pups per litter, 9.2 as compared to 10.3 for
controls. The lactation index was also decreased to 83% as compared to 98%
for controls. Concomitant ethancl exposure exacerbated tne former effect:
8.48 pups per litter as compared to 10.3 for controls. Although the numoer
of animals used was small, 10 control ano 25 experimental, thus lessening
tne sensitivity, the results of this study tend to support those of Schwetz
et al. (1974), indicating prenatal toxicity.
Suocutaneous exposure of carPon tetrachlorioe to pregnant rats has been
reported to result in liver damage in fetuses ano neonates (Bnattacharyya,
1965). Administration of 1600 mg/kg CCl^ subcutaneously on days 20 or 19
of gestation resulted in small areas of focal hepatic necrosis in neonates
corn 48 or 72 hours later, respectively. Histologic findings generally
included a sharply demarcated area of centrilobular necrosis and prolifera-
tion changes in nonnecrotic lobes.
These authors also treated fetuses directly 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 wail
(Bhattacharyya, 1965). Liver changes following injection of 6 mg carbon
tetrachloride were variable; cells generally became extremely pale in cen-
trilooular ano miozonal areas, indicating fatty infiltration. Livers
remained aDnormal until at least 4 days after birth. Nu necrosis, hemor-
rhage or regeneration was observed.
9-2
-------
Sensitivity or' tne liver of neonate racs to caroon tecracnioriae
reported to oe low 1 nour dfter oirth, then to rise aDove the aoult level at
19 nours ana to aecline to adult levels ay 3 to 7 days after birtn. Thus,
oruy 2 of 10 1-nour-ola neunates receiving caroon tetracnloride (1600 mg/Kg)
suocutaneousiy showed centriloduiar necrosis after 24 hours. In aoaition,
nepatic portai areas contained numerous neutrophils, out in contrast to
findings in adult animals, no bile duct proliferation could be ooserved.
Nineteen-nour neonates showed more pronounced hepatic damage than 1-hour
neonates. Damage declined in 3- and 4-day-old neonates; that in 5-, 6- and
7-day-olds was similar in appearance to that of adults.
Neonates can apparently be exposed to CCl tnrougn mothers' milk
(Bhattacnaryya, 1965). Suocutaneous administration of caroon tetracnloride
at 1600 or 3200 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 offspring. Levels in the miiK were not
reported.
Tne pre- ana postnatal toxicoiogic studies descrioed above do not meet
current aesign criteria for nazard assessment purposes in that fewer than
tnree dose levels were used and a second, nonrodent species was not studied
(U.S. EPA, 1981). Furthermore, positive controls were not used. No study
of specific postnatal functional lesions was available.
9.2. OTHER REPRODUCTIVE EFFECTS
Testicular degeneration was ooserved in rats receiving caroon tetra-
chioride at 4800 mg/kg bw i.p. (Chatterjee, 1966). One group of six male
rats received carbon tetrachloride as a 1:1 mixture in coconut oil. Tne
vehicle control group received only an equal volume of coconut oil. On day
15 all animals were sacrificed. Body weights were similar for treated and
9-3
-------
control animals. However, the relative tastes weignt decreased from 15.5
(+0.4) g/kg bw in controls to 9.8 (+1.2) g/kg bw in exposed animals. A
decrease in testis size and weight is a good indicator of a decline in male
spermatogenic process. 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,
however, increased: 50.0 (+1.4) mg/kg bw in treated as compareo to 32.4
(+0.9) mg/kg bw in control animals. This increase would be expected if
feedback mechanism from the testis is obliterated and pituitary concentra-
tions of gonadotrophin were increased. However, in this study, actual
pituitary content of gonadotrophins was not measured. Therefore, no conclu-
sions can be made on the effect of the chemical on the feedback mechanism.
Thus, tne significance of the increase in pituitary weignts is difficult to
interpret.
Histological examination of testes in CCl.-treateo animals showeo tes-
ticular atrophy and "some abnormality" in spermatogenesis. The authors
proposed a mecnanism for carbon tetrachlorioe-inducea testicular atrophy in
which blockage of pituitary hormone release results in atrophy of Leydig
cells within the seminal vesicles, followed by an abnormal spermatogenesis.
In another study (Kalla and Bansal, 1975), intraperitoneal administra-
tion of carbon tetrachloride (4800 mg/kg bw as a 1:1 mixture of coconut oil)
to male rats for 10, 15 or 20 days (Group I, II or III, respectively) led to
impairments in spermatogenesis as indicated by histological examination.
Vehicle controls were administered equal volumes of coconut oil. Weights of
testes, seminal vesicles, epididymis and prostates were decreased in exposeo
animals, whereas the weight of adrenals increased (Table 9-1). The gonado-
somatic index (GSI) (equal to booy weight x testes weight/100) was also
9-4
-------
TABLE 9-1
Weight Changes in Male Rat Reproductive Organs After Carbun Tetrauhloride Treatment"1 >u
(4800 mg/kg bw i.p. as a 1:1 mixture ot coconut oil)
U1
Treatment and Period
Group I
10 dayb
Control
Treated
Group II
15 days
Control
Treated
Group III
20 days
Control
Treated
Body
Before
Treatment
257_f 5.0
257+13.71
230+15.49
230+15.49
234^ 5.5
230^ 3.5
Weight (q)
After
Treatment
269+- 4.5
247_»_10.76
235+ 5.0
173+^13.42
235_t 6.5 '
219±0.5
Testis
(g/kg bw)
12.36+0.94
8.84+1.04
10.47+0.59
9.46+0.33
11.37_+p.06
9.9 +0.65
Semina 1
Vesicles
(g/kg bw)
4.25+0.55
2.41+0.14
3.9+0.52
1.17+0.26
3.24+U.31
1.53+0.20
Epidldymis
(g/kg bw)
4.45+0.45
2.88t0.38
4.12+O.ia
2.63+p.ia
3.72+0.09
2.52+0.22
Prostate
(g/kg bw)
2.1 _+0.7
1.26+0.24
2.19_.0.31
1.68^11.13
1.78U1.04
1.29+0.01
AUrei a 1
(g/kg bw)
O.ld+0.02
0.24+0.03
0.22+O.Oi
0.32+0.05
0.12+0.01
0.25+0.02
o,.
7.MJ1.W
5.6U+0.31
6. 02+0. /I
5.44U1.28
6.04+0.23
5.0^+0. 3d
abixirce: Auapted froin Kulia and Bansal, 197i>
uHudii _+ btanoard deviation
cGunadosoiiutic index = budy weight x testis weight/100
-------
Decreased in treated animais. A slignt decrease in pituitary weignt was
ooservea following a iO-oay treatment but not after eitner the 15- or 20-oay
treatment. As reported, the ratio of germinal to nongerminal area steadily
decreased from Group I to Group III and was always higher in treated tnan
control animals. Significant differences in total germinal area between
treated and control animals, however, were observed only at 20 days. Histo-
logical examination did not reveal any abnormalities in testes from Group I.
Clusters of mature sperm were present in the lumen. In Group II, slignt
testicular damage was observed: a decrease in spermatogenic cells and
increased lumen size, in Group III, shrin«age of the tuoules 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 ooserved. Interstitial material was "damaged"
and in many places the oasement memurane was detacneo from the epithelium.
Proolems associated with this study include that the authors do not
report numoers of animals studied at each dose level, frequency of treat-
ments, or whether the dose of 3 mA/kg bw was the total dose or the dose
given at each treatment, although the former is assumed. Thus,- noting the
amoiguity in the reporting of dose levels, CCl^ at an assumed total dose
of 3 mil/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 spermatogenesis. In view of the
increased pituitary weignt and the decreased adrenal weignt, a possioie
mechanism of action of carbon tetrachloride's male reproductive effect could
be tne suppression of pituitary gonadal axes. However, as pointed out
earlier in this section, pituitary weight by itself is almost a meaningless
parameter. Many hormones are located in the total pituitary, and reproouc-
9-6
-------
tive hormones, such as gonadotrophins, are located only in the anterior
pituitary. In addition, before a conclusion can be made about pituitary
content of various hormones, measurements of the synthesized, stored and
released hormones should be made. Despite these criticisms, the two studies
describee in this section ha a snortcomings with regard to current experi-
mental aesign criteria for hazard assessment: less than three oose levels
and no oata for a second, nonrooent species.
Teratogenic effects in humans causeo by carbon tetrachionde exposure
nave not been reported. However, human fetuses in one stuoy appeareo to
have selectively accumulated caroon tetracnloride from the mothers' circula-
tion (Dowty ano Laseter, 1976). Maternal blood samples were taKen from 11
women eitner oefore or directly after (vaginal) delivery (prior exposure of
the women to toxic chemicals was not reported). Paired cord blood samples
were ootained immediately after delivery. All volatiles were analyzed by
gas chromatography and mass spectrometry. Carbon tetracnloride, benzene and
cnloroform were present in higher concentrations in cord blood as compared
to maternal olood.
9.3. SUMMARY
Carbon tetrachlorioe has produced prenatal toxic effects, some of wnicn
(i.e., suocutaneous edema) could not be associated with extent of maternal
exposure. Rats exposed to caroon tetracniorioe _in utero have shown hepatic
aonormalities at birth, out the fetal rat liver appears to be less sensitive
tnan the aouit liver to the hepatotoxic effects of CCiA.
Caroon tetrachlorioe has produced distinct degenerative changes in tes-
ticular histology, eventually resulting in aspermatogenesis and functional
male infertility. These effects occurred following intraperitoneal injec-
tion at relatively high doses. Unfortunately, low doses were not tested.
9-7
-------
Due to the limited number and scope of the studies reported in this
chapter, it is difficult to evaluate the potential of CC1, to cause
4
adverse teratogenic, embryotoxic or reproductive effects. Some of the
specific limitations are provided within the discussion of each study. In
general, the studies do not provide adequate dose groups for concluding the
existence of teratogenic or reproductive effects (according to testing
criteria such as those currently used for U.S. EPA Office of Pesticides
Programs or Office of Toxic Suostances).
9-8
-------
10. MUTAGENICITY
10.1. RELEVANT STUDIES
Studies to determine the mutagenic activity of caroon tetracnloride in
the Salmonella typhimurium revertant system have been uniformly negative. A
review article written by McCann et al. (1575) stated that an assay using
Arocnlor-induced S9 activation and strains TA100 and TA1535 was negative.
Tne McCann et al. article contained no details of the procedure usea to gen-
erate tnis negative result. Another review article (Fisnoein, 1976) in
wnicn no data .were presented, contained a statement that carbon tetrachlor-
iae was not mutagenic wnen assayed in a spot test with the TA1950 strain.
Simmon and Tardiff (1978) assayed the mutagenicity of caroon tetrachloride
as a gas in a desiccator to avoid loss of the substance to the atmosphere.
It was previously found that many volatile alkyl halides are mutagenic only
when tested in a desiccator apparatus. Negative results were obtained in
tnis study with strains TA1535 and TA100 with and without Arocnior-inducea
rat liver S9 activation.
In an aostract, Uehleke et al. (1976) reportea that carbon tetracruoriae
was not mutagenic in Salmonella typnimurium (strains TA1535 and TA1538) and
Escnericnia coli K12 incuoatea with raboit liver microsomes. However, tnese
results cannot be evaluated because no data were presented. Uehleke et al.
(1977) studied the interaction of carbon tetracnloride witn liver microsomes
from phenooarpital-treated rabbits and used this system for suspension
assays witn Salmonella typhimurium strains TA1535 and TA1538. APout 10% of
the C-labeled carbon tetracnloride (ImM) was covalently bound to endo-
plasmic prptein and greater than 30% was bound to microsomal lipid. (The
metabolism of carbon tetrachloride with subsequent lipid peroxidation is
presumaoly one mechanism for the liver toxicity observed in animals and
humans exposed to carbon tetrachloride. See Section 8.3.) Interaction of
10-1
-------
caroon tetracnoriae witn nucleic acia was not tested. No mutagenic activity
was Deserved in tne bacteria, wnicn were incuoated under nitrogen gas in
tigntiy closed test tuoes with 8 mM (1.23 g/&) carbon tetrachioriae arid
rrdcrosomes. Since tne solubility of carbon tetracnloride in water is 0.6
g/i at 25°C, 1.23 g/i may be just above the solubility level at 37°C.
Tne authors concluded that a reactive species (such as free radicals) gen-
erated in tne biological system may not distribute into the incubation med-
ium and, thus, may be inaccessiole to the test bacteria. They also specu-
lated that any potential reactive metabolites of carbon tetrachloride may be
very short lived.
Callen et al. (1980) carried out a study in yeast that was designed to
overcome tne proolem of a snort-lived intermediate's having to react with
inacessioie DNA. Tne 07 strain of Saccnaromyces cerevisiae contains an
endogenous cytocnrome P-450 depenoent mono-oxygenase activation system.
Three different genetic effects can oe examined under tnis system: gene con-
version, mitotic recomoination and gene reversion. These effects were mea-
sured oy using cells exposed in suspension at 3.23, 4.31 and 5.13 g of car-
bon tetracnlorioe per liter of buffer, well above the solubility level of
caroon tetracnloride in water (0.8 g/4 at 25°C). Therefore, a dose-
response relationship could not be obtained because the dose was essential-
ly tne same in all cases - the solubility level of carbon tetrachloride in
water at 37°C. Volatilization of the caroon tetrachloride is not expected
to nave occurred to any significant extent, because the incubations were
carried out in screw-capped glass tubes. Although the dose is essentially
constant, amounts in suspension will vary. Extracellular or membrane
effects may result in tne nigh toxicity ooservea at 5.13 g/i. Results of
the Calien et al. stuoy are presented in Taoie 10-1. A 1-nr treatment of
10-2
-------
TABLE 10-1
Mutagenic Effects of Caroon Tetrachloride following
1-hour treatment at 373C on Strain 07 of
Saccharomyces cereyisiaea'°
Concentration (mg/£)
3234
4312
Total genetically altered
colonies
Total genetically altered
colonies/103 survivors
11
1.7
3.4
16
3.1
5128
Survival
Total colonies
% of control
nrp-5 locus (gene conversion)
Total convertants
Convertants/105 survivors
aae-2 locus (mitotic recombination)
Total twin spots
Mitotic recombinants/101* survivors
1454
100
285
2.0
1
1.6
1252
86
331
2.6
3
5.3
1120
77
350
3.1
3
5.8
152
10
506
61.7
10
40.1
65
33.3
ilv-1 locus (gene reversion)
Total revertants
Revertants/106 survivors
38
2.6
41
3.3
57
5.1
11
7.2
aSource: Adapted from Callen et al., 1980
'-'The total numoer 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 recomoination was estimated from
counts of colonies growing on a total of 30 plates, 20 plates containing
mealum of which all surviving cells grew and 10 plates containing medium on
which only trp-5 convertants grew.
10-3
-------
cells with carbon tetracnloride at the highest amount treatea resulted in
significant increases in gene conversion and mitotic recomoination. How-
ever, survival was only 10% at tnis dose. Therefore, increases in gene con-
version and mitotic recombination may be influenced Dy the high toxic levels
used. Alternatively, since concentrations in these studies were very high
(above the solubility), the increases in these frequencies may be caused by
an impurity in the carbon tetrachloride sample used in this study. Informa-
tion on the identification and concentration of impurities is forthcoming
from Mallinckrodt.
There was questionable increase in gene reversion. The greatest value
of total revertants counted with carbon tetrachloride-treated cells (57 at
4.31 g/i) was less than the control value (61) in the chloroform experi-
ment reported in the same study. The authors aid not demonstrate that the
increase in revertant frequency observed represented true mutation induction
and not simply selective killing.
Negative results have also oeen obtained in a recently developed _in
vitro chromosome assay that utilized an epithelial-type cell line derived
from rat liver (Dean and Hudson-Walker, 1979). This cell line_ has suffi-
cient metabolizing activity to activate various chemical mutagens and car-
cinogens without the need for an extrinsic activating system. Sealed-flask
cultures were treated with carbon tetrachloride dissolved in growth medium
at 0.005, 0.010 and 0.020 mg/4. Carbon tetrachloride did not induce any
chromosomal aberrations, whereas a number of direct-acting mutagens and sev-
eral requiring metabolic activation produced chromatid breaks, gaps and
exchanges.
Rocchi et al. (1973) studied the binding of carbon tetrachloride with
nucleic acids and protein. C-Labeled carbon tetracnloride (367
ynol/kg) was injected into rats and mice following which the amount of
10-4
-------
metaooiite(s) of caroon tetrachlqriae that covaientiy bind to liver DNA,
RNA, nuclear proteins and cytopiamic proteins was measured. The autnors
reported chat a significant amount of laoeieo. material was found to be asso-
ciated with RNA, nuclear proteins and cytopiasmic proteins in rats. Rats
pretreated witn 3-metnylcnoloanthrene (5 mg, 24 hr before treatment with
caroon tetracnlorioe) increased the amount of label associated with the
macromoiecules. No laoel was associated with DNA in the rat stuoies. Sim-
ilar studies in mice indicated that DNA was labeled but only after pretreat-
ment with 3-methylcnolanthrene (1 mg, 24 hr before caroon tetrachloride
dosing).
In an in vitro experiment, Roccni et al. (1973) used rat or mouse liver
microsomes to activate laoeled carbon tetrachloride in the presence of calf
thymus DNA. Tney found that pretreatment of animals witn 3-fiiethylcnol-
anthrene ennanced the amount of label associated witn DNA. Furthermore, pH
5 enzyme preparations were also found to increase the amount of laoel bound
to uNA. Tnerefore, from these results, it appears that caroon tetrachlorioe
(metaooiites) can interact with DNA, but that for optimal binoing cunoi-
tions, microsomal enzymes nao to be activated with 3-methylcholanthrene in
tne presence of pH 5 enzyme preparations.
10.2. SUMMARY
Carbon tetrachloride has oeen tested for its mutagenic potential in bac-
teria, yeast and a mammalian cell line. All point mutation studies were
negative. It is conceivable that potentially mutagenic reactive intermedi-
ates of carbon tetracnloride (such as the free radical 'CC1,) are gen-
erated in an S9 system but that they are too short-lived to interact with
DNA in _in vitro test systems. The Callen et al. stuay (I960) was designed
to overcome this proolem oy the use of an _in_ vivo activation system in
10-5
-------
yeast. However, because of otner proolems witn this stuay ana the laCK of
corroborative stuuies, the evidence is inadequate to conclude that carbon
tetracnloride is not genotoxic.
Binding studies by Rocchi et al. (1973) indicate that caroon tetracnlo-
riae can interact with DNA dut in order to achieve optimal conditions, cer-
tain precautions must be ta«en. Therefore, some of the negative point muta-
tion test results that nave oeen reporteo may be oue to inaaequate activa-
tion of caroun tetracniorioe to a metaooiite capaoie of causing mutations.
Aoaitionai tests snould oe conducted at levels for wnicn the toxicity of
carbon tetracniorioe i^ not a factor ana appropriate measures snouia oe
to assure that activation is occurring.
10-6
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li. CARCINOGENICITY
The carcinogenic effects of caroon tetracnloride nave been well docu-
mented. Tne International Agency for Research on Cancer (IARC) concludes
tnat tne evidence from animal studies demonstrating CCl^-induced hepatic
neoplasms is sufficient to indicate experimental animal carcinogenesis
(IARC, 1979). The National Cancer Institute (NCI) also identifies CCl^ as
an animal carcinogen and has used it as the positive control in three of its
oioassays.
Tnis section will focus on tne carcinogenicity of CCi. demonstrated in
various species. Some of the studies showing carcinogenesis have also shown
severe toxic effects in tne same animals. A discussion of these toxic
ertects is inciuoeo here to ofrer a clearer picture of the induced malady.
11.1. RATS
Studies performed on rats have primarily used suocutaneous injection as
tne route of exposure. Hepatomas were found as well as toxic effects such
as cirrnosis, hyperplasia ano choiangiofiorosis. Neoplasms also developed
following exposure oy oral ingestion.
Cameron and Karunaratne (1936) looked at CCI, cirrnosis in relation to
liver regeneration in the rat. Albino rats weighing about 150 g each were
administered subcutaneous injections of O.i to 0.25 mH caroon tetracnic-
ride twice a week. After 6 to 10 doses, cnanges whicn developed in the
liver disappeared within 7 to 10 days after cessation of treatment. With
lunger periods of exposure, the liver snowed less ana less tenaency to
return to a normal appearance wnen the cnemical was aiscontinued. Cirrndsis
or tne liver developed after several doses ano was severe ana irreversiuie
dfter 40 doses.
Tne liver was pale, tougn ana finely granular. There was extensive
fiorosis radiacing from the portal areas, thereoy dividing tne liver into
li-l
-------
small irregular masses. Hyperplastic nodules were seen in different parts
or' tne liver.
In this study, rats given suocutaneous injections of caroon tetracnio-
ride readily developed cirrhosis of the liver. Also, there were hyper-
piastic nooules of the liver.
Reuoer ano Glover (1^67) administered suocutaneuus injections of CC-u
t«ace a weet< for 12 weeKS to inored Buffalo male ana female rats 4, 12, 24
ana 52 weeKS ala. Tnere were 10 to 14 rats of eacn sex anu age. Aoaition-
aiiy, newoorn rats were ootained and given CCl^ at 4 days of age. All
rats were given 1.3 mil/kg ow of a 50% solution of CCl^ and corn oil.
Control rats, six per group, were inj'ected with the same amount of corn oil.
Tne 4-oay-old animals oied in an average of 8 days with hepatic ano
renal necrosis. The otner rats survived for the 12 weeks of the study.
During this period, the 52-week-old rats maintained their weignt, and the
12-week-old rats eacn gained from 20 to 30 g. The 4-week-old females
weigned three times their starting weights ana males weighed four times
tneir starting weights.
At sacrifice complete necropsies were done. Ail organs were examined
nistologicaliy, including sucn tissues as diapnragm, tongue and SKeletai
muscle. Special staining was oone for glycogen, mucin, connective tissue,
ceroid, canaliculi, nemosiderin ana lipid.
Tne males given suocutaneous injections at 52 weeKS of age hao more
hyperpiastic lesions than the other males. Six of 14 rats (43%) nao hyper-
plastic nooules, with one having a small hepatic carcinoma. Tne only otner
males with nodules were the 24-week-ola rats, 2/11 (18%). The remaining
52-week-old rats, and all but one of the 24-week-old rats, hao hyperplasia
of the liver. Hyperplasia developed in less than half of the 12-weeK-old
rats. Hyperplastic lesions and hyperplasia were not observed in control
male rats.
11-2
-------
The 24- ana 52-week-ola females had more hyperplastic nooules than aid
the younger females. The most striking lesions were in the 24-week-old
rats. In this group, 8/10 rats (80%) had hyperplastic nooules and one rat
had a small carcinoma of the liver. There were more hyperplastic nodules
per liver and larger lesions in the females than in the males. Lesions were
not present in control female rats.
There were two kinds of hyperplastic lesions in the liver, one located
in the periportal region and the other around central veins. Cirrhosis var-
ied from mild to severe, out was unrelated to the hyperplastic lesions in
individual rats. The severity and the histologic pattern of the cirrhosis
were related to age and sex. The hyperplastic nodules seen were similar to
those known to be preneoplastic (Reuber and Glover, 1967). If the study had
oeen continued for a longer period of time, it is possible tnac the hyper-
plastic nocules could nave become overt tumors. Results of this study are
given in Table ll-l.
In summary, 24- and 52-week-old rats of both sexes given subcutaneous
carcon tetrachloride developed more hyperplastic hepatic nodules, as well as
an occasional early carcinoma of the liver, than did rats of ,other ages.
The number of hyperplastic lesions per liver and the size of lesions were
larger in females than in males, f7our-day-old rats died with necrosis of
the liver and kidney.
Reuber and Glover (1967) also studied cholangiofiorosis of the liver in
male and female Buffalo strain rats of varying ages. Cholangiofibrosis, may
oe a precursor of cholangiocarcinomas of the liver; it is a lesion composed
of ducts lined oy irregular epithelial cells and surrounded by connective
tissue (Reuoer and Glover, 1967). ChoiangiofiDrosis of the liver developed
in rale and female rats receiving injections of carbon tetrachlorioe. The
lesion was present in male rats of all ages, except those 4 weeks of age.
11-3
-------
TABLE 11-1
Lesions of the Liver in Rats Given Subcutaneous Caroon Tetrachloride*
(1.3 mil/kg Dw in 50% solution with corn oil)
Age
( weeKs )
MALE
4
12
24
52
FEMALE
4
12
24
52
Hyperplasia
6/14 (43%)
4/11 (36%)
8/11 (73%)
7/14 (50%)
4/11 (36%)
5/11 (45%)
1/10 (10%)
4/11 (36%)
Hyperplastic
Nodules
0/14 (0%)
0/11 (0%)
2/11 (18%)
6/14 (43%)
0/11 (0%)
3/11 (27%)
8/10 (80%)
6/11 (54%)
Carcinoma
0/14 (0%)
0/11 (0%)
0/11 (0%)
1/14 (7%)
0/11 (0%)
0/11 (0%)
1/10 (10%)
1/11 (9%)
Total
Nodules Plus
Carcinoma
0/14 (0%)
0/11 (0%)
2/11 (18%)
7/14 (50%)
0/11 (0%)
3/11 (27%)
9/10 (90%)
7/11 (64%)
*Source: Reuber and Glover, 1967
11-4
-------
Tne lesion was increased in male rats 5 weeKs of age given both CCi, ana
3-methylcholantnrene (MCA), wnereas it was decreased in rats of all other
ages. Most female rats'given both chemicals also naa cholangiofiDrosis.
The comparative carcinogenicity of carbon tetrachloride has been studied
in five rat species: Japanese, Osoorne-Menaei, Wistar, BidCk ana Sprague-
Dawiey (Reuber and Glover, 1970). Groups of 12 to 17 male rats of each
strain were given twice weexly suocutaneous injections of caroon tetracnlo-
riae (2080 mg/Kg ow as a 50% solution in corn oil). Treated animals were
Killed wnen monouna; controls for eacn strain were kiiieo. at the same time
as tne last experimental animal. Incidence of nepatic lesions is given in
Taole ii-/. Lesions other than nepatic also occurrea. Hemangiomas of tne
spleen were present in two Japanese rats and in one of the Osborne-Mendel
strain. Tnere were carcinomas of tne thyroia gland in three Osoorne-Mendei
and three Japanese rats. One Japanese rat nad a subcutaneous leiomyosar-
coma; two OsDorne-Mendel ana three Japanese rats had chronic renal disease.
The oata indicate that: (1) sensitivity to carbon tetrachloride-induced
neoplasms varies widely among strains; ana (2) the trenus in inciaence of
neoplasms ana cirrhosis appear to be inversely related. Varying amounts of
toxicity occurrea: all experimental animals of the BlacK rat strain were
aeaa at 18 weeks, and tnose of tne Sprague-Oawiey strain at 16 weeks; tne
failure to fina carcinomas in tnose strains may have oeen caused in part by
an insufficient latency time. In all three other strains, toxicity (e.g.,
cirrnusis, hepatic vein tnrombosis, cnolangiotiorusis) occurrea. Tuxicity
(ana survival time) were inversely related to carcinogenicity. It thus
appears that there is no causal connection between tne degree of toxicity
ana carcinogenicity.
11-5
-------
TABLE 11-2
Evidence of trie Most Advanced Lesions in Rats
Administered Carbon Tetrachloriae*
(2080 mg/kg bw in 50% solution with corn oil)
Lesion
No hyperplasia
Hyperplasia
Hyperplastic nodule
Small carcinoma
Large carcinoma
Total carcinoma
No cirrnosis
Mild cirrnosis
Moderate cirrhosis
Severe cirrhosis
Japanese
0/15
0/15
3/15
4/15
3/15
12/15
0/15
9/15
5/15
1/15
Osborne-
Mendel
0/13
1/13
4/13
4/13
4/13
8/13
0/13
2/13
7/13
4/13
Wistar
0/12
1/12
7/12
3/12
1/12
4/12
0/12
0/12
6/12
6/12
Black
4/17
6/17
7/17
0/17
0/17
0/17
0/17
0/17
4/17
13/17
Sprague-
Dawley
8/16
6/16
2/16
0/16
0/16
0/16
0/16
0/16
0/16
16/16
*Source: Adapted from Reuber and Glover, 1970
11-6
-------
In an NDI (1976) oioassay for trichioroetnyiene, carbon tetrachioride
was used as tne positive control. The positive control groups of 50
Gsborne-Menael rats of each sex were acministerea CCl^ in corn oil by gav-
age five times weekly for 78 weeks at two aose levels: 47 and 94 mg/kg bw
for males, 80 and 159 mg/kg bw for females. This treatment resulted in some
toxicity (cirrhosis, fatty liver) and death: at 110 weeks at the nigh dose,
only 7 of 50 males and 14 of 50 females survived and at the low dose, 14 of
50 males ana 26 of 50 females survived as compared to 26 of 100 males ana 51
of 100 females for controls. Tne median survival times were 92 ana 102.5
weeks for males given the low and high doses, respectively ana 67.5 and
102.5 for females given the low and high doses, respectively. Tne incidence
of hepatocellular carcinomas was increased in animals exposed to CC1A as
compared to pooled colony controls (Taole 11-3). However, this was statis-
tically significant only for low dose females as compared to the colony con-
trols ana not the matched controls. Absolute incidence of nepatic neoplasms
was low (=5% in the animals exposed to CC1,). This may be attriouteo to
tnt resistance oy this rat strain to such chlorinated hydrocarbons. The
apparent decrease in the incidence of hepatocellular carcinomas in female
rats at the high dose was attributed to increased lethality (i.e., females
oied before tumors could be expressed). The incidence of other neoplasms
was acknowledged but not quantified. This study is used Dy the National
Researcn Council in determining the carcinogenic risk estimate for CC1.
cue to the oose levels used ano the appropriate length of the study (NAS,
1978).
11.2. MICE
Several studies nave been reported which indicate induction of liver
tumors in various strains of mice treated with CCl^ either by oral inges-
11-7
-------
TABLE 11-3
Incidence of Liver Tumors in Carbon
Tetrachloride-Treated Rats and Colony Controls*
Hepatocellular Neoplastic
Animal Group Carcinoma Nodule
Males Controls 1/99 0/99
Low oose (47 mg/kg bw) 2/50 2/50
High dose (90 mg/kg bw) 2/50 1/50
Females Controls 0/98 2/98
Low dose (80 mg/kg bw) 4/49 2/49
High dose (159 mg/kg bw) 1/49 3/49
*Source: NCI, 1976
11-8
-------
tion wnich has been the primary route of exposure, subcutaneous injection or
rectal administration. Signs of liver toxicity such as necrosis and cirrho-
sis have also oeen a frequent result of CCl, treatment in the carcino-
genicity studies in mice.
In a study by Andervont (1958), groups of 30 to 77 female or maie C3H
mice were administered 6.46 mg CCl. by gavage once weexly for 2 weeks,
followed by administration of 9.6 mg CCl once wee«ly for 17 weeKS (equi-
valent of 213 and 320 mg/kg DW). Pathogen-free C3H mice were used. For
males, no difference in the incidence of hepatomas was observed between the
pathogen-free and pathogen-carrying «groups combined, and control mice: 79%
as compared to 49%. The average number of hepatomas per animal was 1.8 in
treated animals (pathogen-free and pathogen-carrying combined) and 1.3 in
controls. In females, a difference between the incidence of hepatomas in
patnogen-free and pathogen-carrying mice was observed: 46 and 29%, respec-
tively, as compared to 3% in controls. The average number of hepatomas per
female mouse in the pathogen-free, pathogen-carrying and control groups 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: control
mice < treated normal mice < treated pathogen-carrying mice.
A study by Edwards (1941) also reported on the induction of hepatomas in
mice oy exposure to carbon tetrachloride. Two hundred and seven male C3H
mice, aged 3 to 6 months, and 133 maie and female strain A mice, aged 2 to
3.5 months, were used. They were given 0.1 m& of a 40% olive oil solution
of carbon tetrachloride (0.04 cc CCi^) by stomach tube two or three times
weekly for 8 to 16 weeks. Autopsy was performed up to 21 weeKs after the
last treatment.
11-9
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Olive oil was administered by stomach tube in doses of 0.1 m£ 2 or 3
times wee«ly to control male C3H ana A strain mice from the same stocK as
those mice useo in treated groups. Twenty-three strain C3H mice were given
CCi, from 39 to 50 times and were killed and examined from 9 to 11 months
4
of age. A nigh percentage of the treated animals developed hepatomas.
Of 143 C3H mice, which varied from 6 to 10 months of age at autopsy, 126
(38.1%) showed hepatomas (Table 11-4). Similar tumors were present in all
of the 54 strain A mice whose ages varied from 4.5 to 12 months (Table
11-5). It should be notea that the incidence of spontaneous hepatomas in
both the C3H and A strains is markedly below that of the induced tumors in
the treated mice. Autopsies performed on 17 C3H male mice 8.5 to 9 months
of age and tne same stock as that used in the study failed to show any hepa-
tic tumors.
In 1942, Eowaros et al. performed another study on mice. The mice useo
in this study were inbred strain L (their incidence of spontaneous nepatomas
is extremely low), 2.5 to 3.5 montns or 3.5 to 7.5 months of age at the
start. The number of mice varied from B to 39 per group. Carbon tetra-
chloride of a high degree of purity was administered in olive oil by stumach
tube usually three, but occasionally two, times weekly. Each treatment con-
sisted of 0.1 cc of a 40% solution or 0.04 mil of CCI, . Mice were given
46 administrations of CCl^ over a 4-month period and were killed and
necropsied 3 to 3.5 months after the last treatment. The mice varied from
8.5 to 14 months of age at necropsy. The liver was examined histologically.
Thirty-four of 73 mice (47&) given CCI. developed hepatomas. The
incidence of tumors in tne younger mice was essentially similar to that for
tne older mice, with the exception of the older females where the incidence
was considerably lower. Tumors of the liver were observed in 7/15 younger
11-10
-------
TABLE 11-4
Incidence of Tumors in C3H Mice Ingesting
Carbon Tetracnloride*
Group
Controls
Controls «/01ive oil
Treated Animals
(Olive oil and
0.04 nA CC14)
Number of
Mice
Autopsied
17
23
143
Number of
Mice with
Hepatomas
0
1
126
Incidence
of Hepatomas
(in percent)
0
4.3
88.1
*Source: Edwards, 1941
11-11
-------
TABLE 11-5
Incidence of Tumors in Strain A Mice Ingesting
Caroon Tetracniorioe*
Number of
Group Mice
Autopsied
Numoer of
Mice witn
Hepa comas
Incioence
of Hepatomas
(in percent)
Controls 200 i J.5
Controls w/Olive oil
(0.1 mil 2 or 3x 22 0 Q
weekly)
Treated Animals
(0.01 mH of a 40% soln. 54 54 100.0
of CCl^ in Olive oil
2 or 3x weekly)
*Source: Edwaras, 1941
11-12
-------
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.
Hepatomas were observed in 2/152 (1%) untreated strain L mice. One of
23 untreated virgin male mice (4%) and 0 of 28 females (0%), necropsied at
15 months of age, haa tumors of the liver. Tumors were not oresent in 22
males and 23 females 18 months of age or in 27 female breeders 12 to 23
inonths of age. One of 24 male breeders (4%) haa a tumor. The results are
summarizea in Table 11-6.
In summary, strain L male and female mice were highly susceptible to the
inouction of nepatomas by carbon tetrachloride, ana male mice were slightly
more susceptiDle than female mice.
Eschenbrenner ana Miller (1943) studied the effects of size ana spacing
of multiple CC1, aoses in the induction of hepatomas. Strain A mice were
used because of their normal low incidence of tumors of the liver in un-
treated mice (<_!%). Male and female mice were 2.5 to 3 months old at the
beginning of the study.
Solutions of CCL in olive oil were administerea by stomach tube. All
mice received 30 doses of the solution or olive oil alone. Five dilutions
of CC17 were used: 32, 16, 8, 4 and 2% solutions. Mice received 0.005
m2- of solutions per gram bw containing 16 x 10"1*, 8 x 10"1*, 4 x
10"*, 2 x 10""* or 1 x 10"1* m2,, respectively, of CCL. Central
necrosis of the liver was producea by each of these doses. Control mice
received 0.005 mi olive oil/g bw.
The experimental and control groups were diviaed into five subgroups
according to the interval between successive doses (1, 2, 3, 4 ana 5 days)
ana the total period of treatment (29, 58, 87, 116 or 145 days). Equal num-
oers of male and female mice were used in each of the experimental and the
11-13
-------
TABLE 11-6
Tumors of the Liver in Male and Female Mice Receiving
Carbon Tetracnloride oy Stomach Tube3
(0.04 mZ, CC14 2 or 3x weekly)
Age (months)
2.5
3.5
2.5
- 3.5
- 7.5
- 7.5°
Males
7/15 (47%)
21/39 (54%)
28/54 (52%)C
Females
3/8 (38%)
3/11 (27%)
6/19 (32%)c
aSource: Edwards et ai., 1942
°Tnese values represent total numoer of tumors observed in mice in both
age groups.
C01o control mice of this strain exhibit a very low incidence,, as compared
to CCl^-treated mice. Hepatomas were present in 2/71 untreated males
(3%) and 0/81 untreated females (0%).
11-14
-------
five control groups. All mice were examined for the presence of hepatoma
130 oays after the first oose. Some of tnem were killed at that time;
otners were subjected to laparotomy. If nepatomas were not present, laparo-
tomies were performed at montnly intervals thereafter to determine if hepa-
tomas eventually did appear. The gross diagnoses of hepatoma were confirmed
oy nistological examinations.
In tne lower dosage and snorter interval groups, hepatomas were few in
numper ano small in size. With increases in dose and in intervals between
successive doses, there was progressive increase in the number of small
nepatomas and the size of hepatomas for a given mouse. There was no differ-
ence in the incidence of tumors of the liver between males and females.
The authors present the data in a matrix showing dose by interval. An
adaptation of their matrix is given in Table 11-7. Excluding tne values for
the 1-day interval, the data were summed across intervals and sexes. A x2
test for trend among proportions was significant at p <0.01.
In this study, the incidence of hepatomas rcugnly increased non-linearly
witn tne total duration of CCi^ exposure. A given incidence of nepatomdS
was obtained with progressively less total amount of caroon tetracruorioe as
tne duration of administration was increased.
In another study, liver necrosis and hepatomas were noted after NCI
strain A mice were treated with carbon tetrachloride for 4 months (Eschen-
orenner and Miller, 1946). Treatment with CC1, was started wnen the mice
were 3 months of age and was terminated when they were 7 months old. Groups
of five male or female mice were administered caroon tetrachloride by gavage
at 0, 1200, 2400, 4800 or 9600 mg/kg bw. Dose schedules were either 120
doses in 119 days (120/119) i.e., daily, or 30 doses in 116 days (30/116)
i.e., 4-day inter- vals. Animals were necropsied at 8 months (30 days after
11-15
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TABLE 11-7
Hepatomas in Male and Female Strain A Mice
Given CC1/, via Stomach Tube3
ON
mR.
16
8 x
4 x
2 x
1 x
0
of CCl4/g bw
30 doses
x 10""
Male
Female
10""
Male
Female
10" "
Male
Female
10-
Male
Female
10-
Maie
Female
Male
Female
1
0/6b
0/6
0/6
0/6
0/6
0/6
1/6
0/6
0/6
0/6
0/3
0/3 '
Interval
2
5/7
3/5
3/6
2/6
0/6
3/6
0/6
1/6
1/6
0/6
0/3
0/3
Between Doses (days)
3
6/6
2/6
5/6
3/6
4/6
4/6
4/6
2/6
4/6
3/6
0/3
0/3
4
5/8
6/7
5/8
6/7
6/8
3/7
5/5
7/10
7/8
5/7
0/2
0/3
Total Excluding
5 Interval 1
2/4
4/5 33/48
3/4
5/5 32/48
2/4
3/4 25/47
2/4
1/5 22/48
1/4
2/5 23/48
0/2
0/3 0/22
aSource: Adapted from Eschenbrenner and Miller, 1943
. hepatorras/no. mice
-------
cessation of administration). All mice were given one additional dose of
the solution 24 hours prior to necropsy. Note that mice in the two grouos
at each'dose level were administered the same total amount of CCl^ over
the same period of time, but with a variation in the number of doses into
whicn the total amount was divided, and therefore in the size of each dose.
The doses for mice in group one (120/119) were previously determined as
being "necrotizing" and "non-necrotizing" and in group two (30/116) as "only
necrotizing". Two control groups of mice received 0.02 or 0.005 mx. olive
oil/g bw/dose. The incidence of hepatomas and necrotic lesions is noted in
Table 11-3.
At the three highest doses on the 120/119 dose schedule, a 100% inci-
dence of hepatomas was observed. At the highest dose, the mice had numerous
hepatomas of up to 1 cm in diameter. At the two intermediate doses the neo-
plasms were less frequent and smaller in size. No hepatomas were observed
in the 1200 mg/kg group. The incidence of hepatomas was decreased in the
animals on the 30/116 schedule as compared to those on the 120/119 sched-
ule. At the 1200 mg/kg dose, however, very small hepatomas were detected by
microscopic examination in two males. Mice given olive oil did not have
tumors.
The presence or absence of hepatomas and of hepatic necrosis was deter-
mined. When necrosis of the liver was found in mice with tumors, necrosis
was not observed in the hepatomas. The localization of necrosis after
chronic administration of CC14 did not appear to follow a definite pat-
tern, in contrast to the regular pattern of centrilobular necrosis seen
after a single dose was administered to strain A mice.
In summary, mice receiving "non-necrotizing" doses of CCl^ developed
as many, if not more, tumors of the liver than mice given "necrotizing"
11-17
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TABLE 11-8
Susceptibility of Strain A Mice to Liver Necrosis and the Incidence of
Hepatomas 30 Days After 120 or 30 Doses of Carbon Tetrachloride3»b
OD
Sex
F
M
F
M
Carbon Tetrachloride Dose
9600 mg/kg 4800 mg/kg 2400 mg/kg 1200 mg/kg
Dose Conditions ABC ABC ABC ABC
(doses/ days)
120/119 + 0/5 5/5 + 0/5 5/5 ± 0/5 5/5 - 0/5 0/5
120/119 + 0/4 4/4 -i- 0/5 5/5 ± 0/5 5/5 - 0/5 0/5
30/116 + 2/4 2/4 + 2/5 3/5 + 0/5 0/5
30/116 + 4/4 3/4 + 3/5 4/5 + 0/5 0/5C
0
(olive oil)
ABC
- 0/5 0/5
- 0/5 0/5
- 0/5 0/5
- 0/5 0/5
aSource: Adapted from Eschenbrenner and Miller, 1946
DBased on gross and microscopic examination
cTwo animals showing no tumor on gross examination were found each to have a very small hepatoma on
microscopic examination of a random section of the livers.
A = Probable initial liver necrosis (+ = presence, - = absence)
B = Observed final liver necrosis
C = Hepatoma incidence
-------
doses despite the fact that equal amounts of CCl^ were administered. Mice
given 1200 mg/kg did not have gross tumors; most mice receiving either 2400,
4800 or 9600 mg/kg bw did have tumors.
NCI (1976) performed a bioassay on trichloroethylene in which CCl^ was
used as the positive control. Tests were done using B6C3F, male and
female mice (35 days of age, 50 per group). Treatment by oral gavage 5
times per week occurred for 78 weeks. Surviving mice were sacrificed at 92
weeks from the start of the study. The doses of CC1, were 1250 or 2500
mg/kg bw for mice of both sexes. There were 20 control mice of each sex
that were given corn oil only. A necropsy was performed on all mice along
with complete histological examinations.
Most male and female mice treated with CC1, were dead by 78 weeks
(Table 11-9). Median survival times were 63 and 72 weeks for the males
given the low and high doses, respectively and 58.5 and 68.5 weeks for the
females given the low and high doses, respectively. Hepatocellular carcino-
mas were found in practically all mice receiving CC1., including those
dying before termination of the test (Table 11-10). 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. Some liver toxicity occurred, identified as cirrhosis, bile duct
proliferation, toxic hepatitis and fatty liver; however, these cases were
few in number. In summary, this study found carbon tetrachloride to be
highly carcinogenic for liver in mice. It is used by the Carcinogen Assess-
ment Group (CAG) of the U.S. EPA in determining the carcinogenic risk esti-
11-19
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TABLE 11-9
Survival of B6C3F]_ Mice Treated with Carbon Tetrachloride3
administered by oral gavage)
Dose
MALE
Control
Matcned
Pooled
Low Dose0
High Dosec
FEMALE
Control
Matcned
Pooled
Low Dose0
High Dose0
Initial
20
77
50
50
20
80
50
50
78 Weeks
13 (65%)
53 (69%)
11 (22%)
2 (4%)
18 (90%)
71 (89%)
10 (20%)
4 (8%)
91-92 Weeks
7 (35%)
38 (49%)
0 (0%)
0 (0%)
17 (85%)
65 (81%)
0 (0%)
1 (2%)
aSource: NCI, 1976
D1250 mg/kg bw
C2500 mg/kg bw
11-20
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TABLE 11-10
Incidence of Hepatocellular Carcinomas in Mice
Treated with Carbon Tetrachloride3
(CC14 administered by oral gavage)
Dose
Hepatocellular
Carcinomas
MALE
Pooled Controls
Low Dose*3
Hign Dosec
FEMALE
Pooled Controls
Low Dose13
High Dosec
2/19
49/49 (100%)
47/48 (98%)
1/20 (5%)
40/40 (100%)
43/45 (96%)
^Source: NCI, 1976
b!250 mg/kg bw
C2500 mg/kg DW
11-21
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mate for CC14, in spite of the high doses but justified by the marked
effects.
Confer and Stenger (1966) studied nodules in the livers of C3H mice
after long-term CCl^ administration. Twenty-five male mice, 5 weeks of
age, received rectal instillation of 0.1 m*. of a 40% solution of carbon
tetrachloride dissolved in olive oil two times a week for 20 to 26 weeks.
Ten control mice were given only olive oil. Fourteen mice were killed 9
days after the last treatment, and the remaining mice were killed at periods
of 3 to 37 weeks. The livers were examined by light and electron micro-
scopy. Five of the 14 mice (36%) killed after 9 days, and 8 of 11 mice
(73%) killed later, developed hyperplastic hepatic nodules. Cirrhosis was
not observed in the liver.
In summary, mice given carbon tetrachloride by rectal instillation had
hyperplastic nodules that persisted after the discontinuation of the chemi-
cal, but did not develop cirrhosis of the liver. Confer and Stenger (1966)
proposed hyperplastic nodules, as observed in their study, as precursors of
liver carcinomas.
In 1942, using CCl^, Edwards and Dalton studied the induction of cir-
rhosis of the liver and hepatomas in mice. They investigated the outcome of
high, dose, low dose and limited treatment. For high dose administration,
strain C3H male mice, male and female strain A mice, male and female strain
Y mice and strain C female mice were used. They were started on the study
at 1 to 5 months of age.
In one experiment, a dose of 0.1 m*- of a 40% solution of CC1A (with
no impurities) in olive oil was administered by stomach tube two or three
times per week. The total number of treatments varied from 23 to 58. In
order to study any early pathologic changes, a number of mice were killed
11-22
-------
after receiving 1 to 23 doses. In another experiment, male mice were given
<»
0.1 mx. of olive oil 2 or 3 times a week for 39 to 62 doses.
Animals were killed at 1 year of age or younger by cervical disloca-
tion. Subcutaneous transplants of tumor tissue were made by the trocar
technique into mice of homologous strains. Special histological techniques
were used to examine a number of primary and transplanted tumors. These
include techniques for the presence of fat, glycogen or alkaline phosphatase
and those for studying the mitochondria and Golgi apparatus.
Hepatomas were observed in 88% of C3H male mice treated with CCl^;
whereas they occurred in 4% of untreated mice of the same age and strain.
Tumors of the liver developed in 60% of male and female Y strain mice,
whereas only 2% were seen in untreated mice of that strain. Liver tumors
were seen in 98% of strain A mice of both sexes, whereas only 2% of these
mice developed the tumor spontaneously. Hepatic tumors were found in 83% of
C strain females, compared with 0% of untreated mice of the same age and
strain. The hepatic tumors observed in this study were usually multiple —
as many as 10 occurring in one liver. Results of both the treated and con-
trol groups are given in Tables 11-11 and 11-12. Tumors did _not aopear to
have been induced in any of the other organs.
Low dose administration (0.1 mx. of 5% CC14 in olive oil - 0.005
m*-) occurred three times weekly by stomach tube to 58 strain A female
mice, 2.5 months of age, for 2 months. Mice were necropsied 2 days to 4.5
months after the last treatment. Hepatomas were present in 41 mice (71%),
and some mice had cirrhosis of the liver.
The total dose (0.125 to 0.145 mx. CCl^) is comparable to the total
dose of 0.120 m* CC1. in the experiment in which mice were given treat-
ments of 0.04 iw. each. The tumors of the liver were morphologically sim-
ilar in both studies.
11-23
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TABLE 11-11
Hepatomas in Male and Female Mice Given Carbon Tetrachloride
(0.04 ml 2-3x weekly) by Stomach Tube*
Strain
C3H
Y
C
A
Age (months) Males Females
6-10 126/143 (88%)
4-12
6-7 - 34/41 (83%)
4-12
Both
-
9/15 (60%)
-
161/164 (98%)
Hepatomas in Untreated Male and Female Mice
Strain
C3H
C3H
Y
C
A
A
Age (months) Males Females
8-11 ' 2/50 (4%)
12-19 86/320 (27%)
10-16
13-24 - 0/150 (0%)
4-8
12-16
Both
-
-
3/129 (2%)
-
0/400 (0%)
8/400 (2%)
*Source: Edwards and Dalton, 1942
11-24
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TABLE 11-12
Hepatomas in Male Mice Given Olive Oil
by Stomach Tube*
Strain
C3H
C
A
Age (months)
10-11
12
5-12
Incidence
4%
0%
0%
*Source: Edwards and Dalton, 1942
11-25
-------
Limited treatment involved strain A femaie mice, 2 montns of age. Tnere
were 2i to 62 mice in three treatment groups. The CCl^ used was dissolved
in olive oil, the volume of the mixture administered amounting to 0.1 mi.
The mice were given 1 to 3 treatments. The doses, which were hepatotoxic,
were 0.04, 0.01 or 0.005 mil CC1.. Eleven mice received olive oil only.
The mice were necropsied 2 to 12 months after the start of the study.
Tumors of the liver were not found in these mice. There was pigment in
Kupffer ceils, occasional foci of oasopnilic oebris, ana an increase in con-
nective tissue ana reticuium.
In summary, carbon tetrachioriae inaucea significant numoers of tumors
of the liver, as weil as cirrhosis, in three strains of mice (Eowaras and
Dai tun, 1242).
Since successful transplantation is frequently considered to oe a cri-
terion of neopiasia, Leouc and Wilson (1959) attempted to transplant CCl^-
inoucea tumors of the liver in mice. At first
"numerous failures to estaolish a transplantable CC^-inoucea
hepatoma supported the idea that, if transplantabiiity is a crite-
rion, the nodules might be nyperplastic but not neoplastic. Suose-
quently, however, several such nodules were successfuily trans-
planted from a host that was allowed to live for a long period
after the CC14 administration ceased" (Leduc and Wilson, 1959)".
Male mice of the BUB strain were used. Spontaneous hepatomas have not
oeen found in this strain, up to its 40th generation. Carbon tetrachloride
was administered by stomach tube in doses of 0.1 mH of a 40* solution in
olive oil (0.04 m& CCi^) per treatment. Caroon tetracnioriae was given
3 times a weex for a total of 45 to 66 doses. About one-tnira of the mice
were given tnree Gaily intravenous i.v. injections of 0.2 mi of thorotrast
before CCi administration was started. As discussed by Leouc ana Wilson
11-26
-------
(1959), thorotrast is useful in the detection of hepatomas but has been
involved (as indicated by other investigators), in tumor production.
Tne first-generation tumor transplants were made subcutaneously. Sub-
sequently, both subcutaneous and intrasplenic transplants were made. Unaer
lignt ether anesthesia, implants of tumor into the spleen were made oy an
incision through the dorsal body wall. The spleens were examined periodic-
ally by laparotomy.
Hepatomas did not develop in 20 control mice given thorotrast only.
Hepatomas did occur in CCl^-treated mice that were free of thoratrast.
The CC1 hepatomas (5 of 7) that were successfully transplanted dif-
fered from those that did not grow in new hosts because a longer time period
elapsed between CCl^ administration and tumor transplantation. The five
successful transplants were obtained from a single host killed 8 months
after the last treatment, whereas those that did not grow were transplanted
11 weeks or so after the last treatment. . The authors concluded that chronic
CC1. injury to the liver induces the development of both hyperplastic nod-
ules and hepatomas based on their results. They found the livers of CC1.-
treated mice to be cirrnotic with numerous hyperplastic nodules.
Weisburger (1977) did a series of carcinogenicity studies on halogenated
hydrocarbons using both mice and rats. Carbon tetrachloride was used as the
positive control. The compounds were tested by oral intubation in 200
Osborne-Mendel rats and 200 B6C3F-, mice of both sexes. Maximum dose
levels were 2500 mg/kg for both male and female mice and 100 mg/kg and 150
mg/kg for male and female rats, respectively. A high yield of both hepato-
cellular carcinomas and adrenal tumors was seen in male and female mice.
11-27
-------
Neopiastic nooules and a few carcinomas of the liver were observed in the
rats, which, as the author points out, was lower than anticipated (presum-
aoly based upon earlier findings). Some of the results are proviaed in
Table 11-13.
This study can be criticized in that the report is poorly written by
omitting some important information. Only the maximum tolerated dose is
reported for each species and each sex. The low dose is not given. The
percent survival is also not reported, nor are final numbers in each group.
Finally, tumor type is not specific. The study, however, is included here
since it is a part of the literature dealing with the carcinogenicity of
CCL. However, it is recommended that the data on CCL given in the NCI
(1976) oioassay for trichloroethylene be used instead.
11.3. HAMSTERS
Hamsters have not been studied as extensively as have rats ana mice.
There has oeen only one report of the induction of tumors in hamsters by
cci4.
Delia Porta et al. (1961) orally administered caroon tetrachloride to
Syrian golden hamsters as a part of a larger investigation of the response
of this species to carcinogens that induced neoplasms of the liver in other
species. Ten female and 10 male Syrian golden hamsters, 12 weeks old, were
used. Males weighed an average of 109 g and females 99 g. The treatment
consisted of weekly administration by stomach tube of a 5% solution of
CCL in corn oil for 30 weeks. No information was given on control ani-
mals. During the first 7 weeks, 0.25 md of the solution containing 12.5
U& CC1 was given each week. This dose was then reoucea to 0.125 mil
ana contained 6.25 yi of CCL. After this treatment, the survivors
were kept under observation for 25 additional weeKs and tnen killed.
11-28
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TABLE 11-13
Incidence of Tumors in Rats and Mice Ingesting Carbon Tetrachloride*
V£>
Species
Rat
Mouse
Tumor Type
Adrenal tumors
Thyroid-adenoma
and carcinoma
Liver-neoplastic
nodule
Liver hepatocellular
carcinoma
Total tumors
# animals examined
# animals with tumors
Liver hepatocellular
carcinoma
Adrenal adenoma and
pheochromocytoma
Total tumors
# animals examined
# animals with tumors
Control
1
1
0
0
12
20
7
3
0
4
18
4
Male
Low
1
1
9
2
29
49
21
49
28
81
49
49
Female
High
3
1
3
2
26
50
24
47
28
75
48
47
Control
0
2
1
1
17
20
10
I
0
3
18
3
Low
1
2
11
4
46
50
34
40
15
56
42
40
High
0
4
9
2
24
49
20
43
10
54
45
43
*Source: Weisburger, 1977
-------
Detailed histopatnological examinations of all hamsters were conouctea,
except for one female lost through cannabalism at the 28th week.
Weights of the hamsters variea irregularly during the period following
treatment. In general, the weights increased. Females weighed an average
of 114 g and males 118 g. One female died at the 10th week of treatment;
three females ana five males died or were killed between the 17th and the
28tn week. Three females died at weeks 41, 43 and 54. The surviving three
females and five males were killed at the ena of the 55th week.
Hamsters dying during the treatment and at the 41st week had cirrhosis,
as well as hyperplastic nodules that were two to several layers thick. The
cells showed irregularities in the shape, size ana staining qualities of
their cytoplasm and nucleus, with an uneven distribution of glycogen.
All of tne animals, five males and five females, dying or killed 13 to
25 weeks after the end of the treatment, had one or more liver-cell carcin-
omas (a total of 22 tumors: 12 in the 5 females and 10 in the 5 males). No
mention was made of toxicity in these animals. Liver cell carcinomas were
not found in the other animals which died before week 43.
Transplantation efforts were not successful. The authors note in tneir
discussion that this negative result deserves further investigation since
"many other tumors of hamsters have been successfully transplanted to non-
inbred hamsters in this and other laooratories" (Delia Porta et al., 1961).
In summary, Syrian golden hamsters appear sensitive to the carcinogenic
effects of carbon tetrachloride. Although the numper 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 appeared during treatment, and
carcinomas appeared after CC1. administration had been discontinued, whicn
11-30
-------
suggests that the nodules or benign tumors were precursor lesions for car-
cinomas. It should be noted that this study is the only report found in the
available literature, of the induction of tumors in hamsters by CCl^.
In concluding this section, it should be noted that some of the research
reported suggests that hepatomas occur only after liver necrosis and fibre-
sis have occurred (Edwards, 1941; Edwards and Dalton, 1942; Delia Porta et
al., 1961; Reuber ana Glover, 1967, 1970). The results have been interpret-
ed to mean that "as far as the liver is concerned, hepatoma is an occasional
consequence of the induction of postnecrotic cirrhosis and that CC1. is
not a direct liver carcinogen" (Louria, 1977). The results reported by
Escnenbrenner and Miller (1946), however, refute Louria's statement. These
authors concluded that if carbon tetrachloride is, in fact, a carcinogenic
agent, tumors should be obtained with non-necrotizing doses. As discussed
earlier in this chapter, their series of experiments examining the issue,
revealed:
While it was found that a correlation exists between the degree of
liver necrosis and the incidence of hepatomas in relation to dose,
the use of a graded series of necrotizing and non-necrotizing doses
indicated that repeated liver necrosis and its associated chronic
regenerative state are probably not necessary for the inouction of
tumors with carbon tetrachloride (Eschenbrenner and Miller, 1946).
The small number of animals used in this study must be noted.
A list of authors addressing the issue of liver necrosis induced by car-
bon tetracnloride is provided in Table 11-14.
11.4. HUMANS
11.4.1. Case Reports. As mentioned in the section dealing with human
toxicity, in many of the case reports the investigators present the data in
narrative form. Although interesting, these type of data are not suitable
to quantitative analysis since numbers are not adequately presented.
11-31
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TABLE il-14
Studies in Which Liver Necrosis was Induced
Using Carbon Tetrachloride
Species—Route
Reference
Mice — gavage
Mice — ingestion
Mice — ingestion
Hamsters — ingestion
Rats — subcutaneous
injection
Human — ingestion
Rats — subcutaneous
injection
Edwards, 1941
Edwards and Dalton, 1942
Eschenbrenner and Miller, 1946
Delia Porta et al., 1961
Reuber and Glover, 1967
Hashimoto et al., 1968
Reuber and Glover, 1970
11-32
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Furthermore, there usually are a number of uncontrolled variaoles (alcohol
intake, age, simultaneous exposures) or unknown variables (exposure amount)
making it difficult to attribute the outcome solely to the CC14 exposure.
Despite these limitations, they are important since they can either support
or challenge the experimental animal studies ana can aid qualitatively in
the extrapolations from animals to numans.
The carcinogenic effect of exposure to carbon tetracnloride in humans
has been suggested in a number of case reports by physicians. One of these
was reported by Tracey and Sherlock (1968). A 59-year-old man with a
history of moderate alcohol consumption returned from a cocktail party and
noticed the vapor of carbon tetrachloriae used to clean a rug in his
apartment earlier that evening. Five days later he developed nausea,
vomiting ana diarrhea and within 10 days of exposure he developed jaundice.
The patient recovered following a long and complex hospitalization ana 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.
He again denied ingestion of alcohol. A liver biopsy was diagnoseo as
hepatocellular carcinoma. Postmortem examination revealed the liver to be
extensively involved with tumor. Little normal liver tissue remained.
Aside from the acute exposure to carbon tetracnloride 7 years before
diagnosis of cancer, the patient's possible additional exposure to this and
other toxic chemicals was not reported. No medical history was given for
tne 3 years before final diagnosis.
11-33
-------
Other case reports of human neoplasms developing after exposure to car-
oon tetrachloride have appeared. In one, a woman developed modular cir-
rnosis of tne liver followed by cancer of tne liver after exposure to caroon
tetrachioride, and died 3 years after the first exposure (Johnstone, 1948).
However, sne had suffered from periodic jaundice for 5 years prior to tne
CC14 exposure. In a second, a fireman developed cirrhosis and an "epithe-
a" or the liver 4 years after acute caroon tetracniorice intoxication
(Simiar et al. , 1964). In none of the cases could a causal link oetween
caroon tetracnioride exposure and development of neoplasms be estaolisned.
11.4.2. Studies. A study by Capurro (1379) reports a series of cancer
cases in a rural valley polluted py vapors from a solvent recovery plant.
Due to its lack of specificity and questionable statistical methods, the
study is of limited value. However, in an effort to be complete, it is
described ano critiqued. Odor and pollution problems existed in this valley
from 1961 to 1971. A variety of solvents including caroon tetracnioride
were identified in the air. Blood tests were done on 24 residents; solvents
were detected in the olood of ail those tested. A list of solvents detected
in tne oioud is not given out those "most easily detected" (as cited by the
author) were: oenzene, cnloroform, metnyi isooutyi ketone ana trichioro-
etnylene. Levels were not reported.
A study population was defined consisting of 117 Caucasians wno lived
witnin 1.5 Km2 of tne plant during the appropriate time for greatest expo-
sure. These persons were followed for a 6-year period.
Ten cancer cases occurred during this time period including two cases
tnat were not residents. The author's analysis focuses on four cases of
lympnoma (2 lymphosarcoma , ICO 200.0 and 2 reticulum cell sarcoma ICD
200.1). All four of these cases had a history of work in the paper mill
11-34
-------
wnich preceded tne solvent recovery plant at the same location. Tne autnor
reports that a study of paper mill workers (Milham, 1976) snowed a propor-
tionate mortality ratio [PMR = (ooserved deaths/expected deaths) x j.00] of
l.b for lympnosarcoma, out no significant increase of reticulum cell
sarcoma. However, as Capurro (1979) reports, "the excess lympnomas ooserved
oy us is far aoove the one described by Milham". A mortality ratio indicat-
ing a 160-fold increase for these two cancers (ICD 200.0 and 200.1) was cal-
culated. However, it should not be reported due to the errors in its calcu-
lation. Three observed deaths are used, rather than four, with no explana-
tion and the study population is rounded off to 120 instead of using the
defined 117. The ratio is not standardized by age or sex, nor is it
descrioed as race-specific (all Caucasians). The assumption is made that
since the age distribution of tne study population is comparable to that of
the nation, age stanciaraization is not necessary. However, the study popu-
lation is then compared to tne population of the State of Maryland ratner
than the nation. The discussion of lymphomas diagnosed in the county and in
tne area surrounding the plant lacks important information on population
distrioution, time period and comparison group as well as methods used.
Thus, in lieu of these deficiencies, the conclusion that tne incidence of
malignant lymphoma is abnormal in the area exposed to solvent vapors is not
supported.
In a preliminary study designed to determine if occupational exposure to
carbon tetrachloride, trichloroethylene and tetracnloroethylene resulted in
increased mortality, Blair et al. (1979) studied causes of death in 330
laundry and dry cleaning workers. Sex, race and age along with underlying
and contributing causes of death were abstracted from death certificates.
11-35
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The underlying cause was classified according to the International Classifi-
cation of Diseases, Seventh Revision, by a trained nosologist. The control
standard was the age, race, sex and cause specific distribution of U.S.
deaths from the same time period. The PMR for all malignant neoplasms was
128 (100 would signify no difference from the control) and statistically
significant (xz test, p<0.05). The excess deaths consisted of lung,
cervical and liver cancers, and leukemia. The authors note that the lung
and cervical cancer excess may reflect the lower socioeconomic status of
these workers. The slight excess of liver cancer is consistent with bio-
assay studies showing liver abnormalities. The authors conclude that epi-
demiologic studies of this occupational group are warranted due tP the
increased risk observed.
Finally, increases in certain types of skin cancers can be attributed to
increased atmospheric CCl^. As explained in Chapters 5 and 6, CCl^ may
result in an overall reduction of stratospheric ozone, eventually leading to
increased UV-8 radiation. It is estimated that a 2 to 5% increase in basal
cell skin cancer will occur per 1% decrease in ozone and that a 4 to 10%
increase in squamous cell skin cancer will occur per 1% decrease in ozone
(MAS, 1982). The relationship between sunlight and malignant melanoma is
not clear at present.
11.5. SUMMARY
11.5.1. Experimental Animals. Carbon tetrachloride has been reported to
be carcinogenic in numerous animal studies. Hepatocellular carcinomas have
been the neoplasm induced in all species evaluated (rats, mice and ham-;
sters). An increase in adrenal tumors in male and female mice was seen in
one study. Hamsters, although only used in one study, have been the most
sensitive species studied, followed by mice and then rats. A significant
11-36
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strain difference has been observed in rats. Females have appeared less
sensitive to the chronic toxic effects but more sensitive to the carcinogen-
ic effects of carbon tetrachloride in rats.
11.5.2. Humans. A number of cases of hepatomas appearing in humans years
following exposure to CCl^ have been reported. A study examining the
effect of solvent vapors (one of which was CC1J on a group of environ-
mentally exposed people concluded the existence of an abnormal incidence of
malignant lymphoma. However, it should not be used as evidence of the car-
cinogenicity of CCl^ due to the concomitant exposures and poor study tech-
niques. A preliminary epidemiological study of a group occupationally
exposed to CCl^ revealed a slight excess of liver cancer in this group
which offers better evidence of human carcinogenesis associated with CCl^
exposure, and points the way to further research needs.
11.5.3. Conclusion. In conclusion, there is evidence that CCl^ is a
potential human carcinogen based upon the positive findings on mice in the
NCI bioassay for trichloroethylene (1976) in which CC14 was used as the
positive control. Other studies on animals support this conclusion such as
the hamster study by Delia Porta et al., (1961), the rat studies by Reuber
and Glover (1967, 1970) and NCI (1976), and the mice studies by Edwards
(1941), Edwards et al. (1942), Edwards and Dalton (1942) and Weisburger
(1977). Human data as reported by Blair et al. (1979) also are consistent
with this conclusion.
11-37
-------
-------
12. SYNERGISM AND ANTAGONISM
12.1. SYNERGISM
A description of the entire clinical picture of the toxicity of
should consider the role played by alcohol in the genesis of severe CC14
poisoning (von Oettingen, 1964). A number of researchers have reported on
this phenomenon (Stevens and Forster, 1953; Kirkpatrick and Sutherland,
1956; Joron et al., 1957; New et al., 1962; Markham, 1967). It has oeen
established that habitual ingestion of alcoholic beverages and also tneir
occasional use may increase the Gangers from comparatively moderate exposure
(MarKham, 1967; Tracey ana Sherlock, 1968). This fact is illustrated by
reports on simultaneous exposure of abstinent persons and consumers of alco-
hol to the same concentration with only the latter becoming seriously ill
(von Oettingen, 1964).
An example of this was given by Smetana (1939) wno reporteo on three
cases of CC14 poisoning. In one case, a 35-year-old male dry cleaner/
interior decorator (a "steady and heavy drinker") had been cleaning furni-
ture and draperies with CCl^. Several hours later, he developed the typi-
cal acute symptoms of dyspnea, cough and bloody sputum associated with expo-
sure to CCL. His condition worsened and he subsequently died. A co-
worker of his, a teetotaler, had been working in the same room for the same
amount of time receiving the same exposure to the CC1.. The author stated
that "altnough he felt the effect of the exposure and suffered from headacne
and gastrointestinal distress, he recovered quickly after breathing frtsh
air" (Smetana, 1939).
Hypotheses have been advanced to rationalize this apparent synergistic
reaction Detween alcohol and CCl^. Lamson et al. (1928) postulated that
the alcohol either caused a greater absorption from the gastrointestinal
12-1
-------
tract or a greater penetration into the liver. Smetana (1939) theorized
that the reduction of the glycogen store in the hepatic cells of alcoholics
may have some role in the greater susceptibility to CC1. poisoning. [This
latter tneory is supported by the findings that neonates are protected
against CC1, poisoning and that neonates have an increased concentration
of liver glycogen (Bhattacharyya, 1S65)].
Traiger and Plaa (1971) investigated the potentiating capacity of ali-
phatic alcohols on CCl^ toxicity. Male Swiss-Webster mice were given a
single dose by gavage of methanol, ethanol or isopropanol equivalent to 50%
of the LD5Q. After 20 hours, an i.p. injection of 0.0075 mi/kg CCl^
in corn oil was administered. Controls received only the corn oil. Blood
samples were analyzed for SGPT activity. All alconols produced increased
activity out the effect was most marked among the mice pretreated with iso-
propanol. The administration of the alcohols or CC1, alone did not change
the SGPT levels.
In an effort to ascertain the differences of the alcohols in the poten-
tiation of CCl^, the authors performed more detailed biochemical studies
on male Sprague-Dawley rats. In these experiments, a single dose of ethanol
or isopropanol was administered by gavage 3 to 48 hours prior to the i.p.
injection of 0.1 mi/kg CCL in corn oil. Controls received only the
corn oil. After 24 hours, the animals were sacrificed and blood and liver
aliquots were taken for SGPT, glucose-6-phosphatase (G6PO), biliruoin and
hepatic triglyceride analysis. Isopropanol produced a more marked change in
the activities of SGPT, G6PO and triglycerides. Isopropanol pretreatment
produced hyperbilirubinemia whereas ethanol pretreatment or CCl^ alone at
a 10-fold increase in dosage did not. Also, a 10-fold dosage increase in
12-2
-------
l^ alone producea an increasea level of SGPT activity; however, it was
only approximately 25% of the SGPT activity found among rats pretreated with
isopropanol.
Wei et al. (1971) investigated the potentiation of CCl^ hepatotoxicity
by ethanol and cold. This was accomplished by pretreating rats witn ethanol
and exposing rats to a cold temperature (18 hours at 4°C). Indices of hepa-
totoxicity were SGPT levels and liver triglyceride levels. In both male and
female rats, the SGPT levels increased after both ethanol and cold exposures
in response to the CCl,. The authors postulate that the ethanol releases
norepinepnrine, which increases the susceptibility of the liver to CCi^.
According to Davis (1934), very obese or undernourished persons suffering
from pulmonary diseases or gastric ulcers or having a tendency to vomiting,
liver or kidney diseases, diaoetes or glandular disturbances are especially
sensitive to the toxic effect of CC1. (von Oettingen, 1964).
Struoelt et al. (1978) found that carbon tetrachloriae-induced liver
aamage was significantly greater in rats concomitantly exposed to ethanol
than in control rats not exposed to ethanol. Male Wistar rats were given
either a 5% or 15% ethanol solution as their sole source of fluid (11.4 or
24.9% of total colonies, respectively). Controls were provided with tap
water. Following 1, 2 and 3 weeks of exposure, CCl^ was administered
intraperitonea11y at a dose of 0.1 mg/kg. Hepatotoxic effects were evaluat-
ed by measuring the serum activities of glutamic oxaloacetic transaminase
(SCOT), glutamic pyruvic transaminase (SGPT) and sorbital dehyarogenase
(SDH) as well as histological investigations.
The potentiation noted was fully developed following 1 week of expo-
sure and was greater in those rats provided a 15% ethanol solution than in
the rats provided a 5% ethanol solution, thus appearing to be dose-dependent.
12-3
-------
Hasumura et al. (1978) also examined the potentiation of carbon tetra-
chloride hepatotoxicity by ethanol. They designed their study to determine
if hepatic microsomal changes which are secondary to chronic ethanol con-
sumption, play a role in the mechanism of CC1. hepatotoxicity. Rats were
pair-fed a liquid diet containing ethanol (36% of calories) or isocaloric
caroonyarate for a period of 4 to 5 weeks. Carbon tetrachloride, at a dose
of 0.5 mil/kg, was administered intragastrically 15 hours after ethanol
withdrawal. Within 24 hours there was an increase in liver lipids and serum
ornithine caroamyl transferase activity and a decrease in the activities of
hepatic aminopyrene N-demethylase and glucose-6-phosphatase. These changes
were determined to be significantly greater in the ethanol-fed rats versus
the control rats indicating _in vivo potentiation of carbon tetrachloride
hepatotoxicity by chronic ethanol consumption, even in the absence of
ethanol at the time of exposure (Hasumura et al., 1978).
In an attempt to determine the mechanism of this effect, liver micro-
somes were incubated with CCl. and a NADPH-generating system. The
14
authors found an enhancement of: (1) the covalent binding of C to
14
microsomal protein in vitro and (2) the biotransformation of CCl^ to
CCL _in vitro. This suggests that etnanol pretreatment stimulates the
microsomal formation of an active metabolite of CCl thus, microsomal
changes are responsible, at least partially for the increased CC14 hepato-
toxicity (Hasumura et al., 1978).
In addition to ethanol other compounds have been investigated as to
their effect on carbon tetrachloride hepatotoxicity. Curtis et al. (1979a)
and Davis and Mehendale (1980) have studied the potentiation of carbon
tetrachloride hepatotoxicity by exposure to chlordecone (Kepone).
12-4
-------
Curtis et al. (1979a) founo greatly potentiated hepatotoxicity reflected
in tne form of elevated SPGT and SCOT activities between groups of rats
wnicn had undergone a 15-day feeding of 0 or 10 mg/kg chlordecone, and a
single intraperitoneal challenge of CCl^. The CCL challenge was either
0, ,025, .05, .1 or .2 mi/kg. The SPGT and SCOT activities increased in
excess of 30-fold and 10-fold respectively in chlordecone-fed rats chal-
lenged with CCl^ of .1 and .2 mi/kg. The authors concluded that the
cata indicate a great potential for the production of severe liver damage
resulting from interactions of carbon tetrachloride and chlordecone exposure
at levels wnich may be independently non-toxic.
Davis and Mehendale (1980) conducted a similar study using a single per
os administration of chlordecone (5 mg/kg) followed 48 hours later by intra-
peritoneal administration of CCl^ (.2 mil/kg). Twenty-four hours la*er
the hepatic excretory function of the animals treated with chioroecone and
CC14 had Decreased (20% of controls), while plasma transaminase activities
and oiliruoin were elevated. Parameters measured or assessed which were 'not
affected by chlordecone pretreatrrent were: hepatic mixed function oxidase
activity, irreversible binding of label from CC14 to hepatic protein
or lipid, hepatic and renal glutathione concentrations and CC1,-induced
lipid peroxidation of liver tissue measured _in_ vitro and _in_ vivo. The
authors conclude that the mechanism for the enhanced toxicity is still un-
known; however, the results suggest the interaction between chlordecone and
CCl^ is a subtle one, not causally involving increased covalent binding of
the toxin, increased susceptibility of tissue lipids to peroxidative damage
or decreased hepatic glutathione.
Curtis et al. (1979b) investigated the effect ot pre-exposure to 50
mg/kg pnotomirex on carbon tetracnloride hepatotoxicity in rats. Photomirex
12-5
-------
is a photoaegradation proouct of the insecticide mirex ana is also a struc-
tural analog of cnloraecone. The photomirex/CCl, interaction resulted in
liver nypertrophy, 7- and 8- fold elevations in SCOT and SGPT over tne con-
trol rats and rats treated with photomirex alone, and considerable centri-
iddular necrosis. Liver weight, SCOT and SGPT were unaffected by the CC14
cnailenge alone, therefore pre-exposure to photomirex does potentiate CCi^
hepatotoxicity as does cnloroecone, a structural analog.
In a review article, Falk (1976) reported on the effects of different
chemicals on carbon tetracnloride toxicity. A summary of this review is
presented here. A low protein diet fed to rats reduced the hepatic micro-
somal hydroxylase activity and dramatically reduced the toxicity of carbon
tetrachloride. The LD5Q of CC14 was 14.7 mil/kg on the protein free
diet compared to 6.4 mil on a regular diet. When the rats were pretreated
with pnenobarbitaol the LD5Q was 0.5 mil/kg.
6enzo(a)pyrene (BAP) in conjunction with caroon tetracniuriae has been
.^
snown to enhance tumor production in laboratory animals (Kotin et al., 1262;
Proetzel et ai., 1964) while CCL alone was founa to produce no sarcomas
at all. weisourger et ai. (1965) found that treating rats witn acetylamino-
fluorene (.AAF) produced an increase in hepatomas in female rats, from Q%
without to 81% with CC1.. In males, the increase was 73* to 100%. A
cnange in metabolism of AAF toward greater N-hydroxylation on CCl^ treat-
ment was found to be the reason for the enhanced tumor incidence (Weisburger
and Weisburger, 1963).
Kluwe et al. (1980) found that CCi^ produced liver damage, as repre-
sented oy increased levels of SCOT in rale rats fed with 100 mg/kg poly-
brominated biphenyls (PBB) or 200 mg/kg poiycnlorinated biphenyls (PCB) 28
days before injection of CCl^. The liver damage was greater in tne rats
12-6
-------
fed PBB than in those fed PCS which in turn showed greater damage than the
control rats. Functional renal damage was produced by CC14 and several
other solvents such as TCE and 1,1,2-trichloroethane. CCl^-inouced renal
dysfunction was found to be potentiated by PBB but not PCS.
Dietz and Traiger (1979) found that pretreatment of rats with either
2-butanone or 2,3-butanediol markedly enhanced the hepatotoxic response to
CC1, as measured Dy SGPT and hepatic triglycerides. The administration of
CC1, and quinalphos (an insecticide) was founo to cause morphological
changes in the liver, kidney and testes of male rats (Dikshith et al.,
1980). The authors suggest that pretreatment of CC1, made the animals
susceptible to quinalphos.
12.2. ANTAGONISM
As to the antagonistic compounds associated with CC1,, Hafeman and
Hoekstra (1977) report a protective effect of dietary vitamin E, selenium
(Se) and methionine against lipid peroxication induced by CC1.. In the
first of three experiments, 21-oay-old male weanling rats were fed a diet
deficient in vitamin E and Se and low in methionine. Dietary supplements of
these three variables were administered either alone or in combination. To
assess the effect of CC1., they monitored lipid peroxidation by the
evolution of ethane, an auto-oxidation product of u-3-unsaturated fatty
acids. To study the effect of increasing dietary oi-3-unsaturated fat on
CClA-inouced etnane evolution, coa liver oil (CLO), which is rich in
oj-3-unsaturated fat, was substituted for lard. A dose of 2 mil CC1 /kg
bw was administered via i.p. injection. The investigators found that in all
dietary groups, CCL stimulated ethane evolution. However, among mice
given dietary supplements of vitamin E, Se and methionine, ethane evolution
was reduced 17, 26 and 39%, respectively. The substitution of CLO for lard
12-7
-------
in the diets resulted in a 6-fold increase in ethane evolution. If only
CC1 -induced ethane evolution is considered, only dietary supplements of
vitamin E and Se resulted in a statistically significant (pcQ.05) reduc-
tion of ethane evolution. It was also noted that among rats given CC1.
with no supplements there was pronounced mortality which correlated well
with ethane evolution. The authors conclude that the toxicity of CCl^ was
decreased in correlation with ethane evolution. Thus, vitamin E, Se and
metnionine protected against CCL-induced lipid peroxidation, prooaoly by
maintaining intracellular glutathione and glutathione peroxioase.
In two other experiments, the authors sought to determine tne effective-
ness of eacn protective factor alone or in comoination and to estimate
peroxioation rates in rats in which CC1. caused early mortality. In- these
experiments, a dose of 1 mi CCl^/kg bw was administered via i.p. injec-
tion. Results indicate that vitamin E, Se and methionine supplements in all
combinations protect against CCl^ toxicity and that supplements of either
vitamin E or Se, or both with methionine, completely prevented CC1.-
induced mortality.
Reserpine, carbon disulfide and diethyldithiocarbamate reportedly dimin-
ish the toxic effects of carbon tetrachloride on liver in experimental ani-
mals. Tne mechanisms are unknown, but some of the investigators have specu-
lated that metabolism via the microsomal enzyme system may be involved
(Douglas and Glower, 1968; Seawright et al., 1980; Siegers et al., 1978).
Chlorpromazine prevented liver necrosis from carbon tetrachloride in short-
term rat experiments without affecting lipid peroxidation or binaing of car-
bon tetrachloride-reactive metaoolites (not identified), but it also lowered
oody temperatures. The investigators were of the opinion that cnlorpro-
mazine only delayed the onset of liver necrosis (Marzi et al., 1980). Cagen
12-8
-------
ana Klaassen (1980) reported that the release of alanine aminotransferase
ana aspartate aminotransferase into plasma 24 hours following various doses
of CC1A was marKediy lower irr rats pretreatea for 3 days with zinc chlo-
ride (150 umol/KO/day) than in control rats. Administration of zinc chiu-
ride solution was found to increase hepatic concentrations of metdiiotniu-
neiri. Tne autnors suggest that metallothionein ray protect against CC14-
inouced liver damage oy sequestering reactive metaoolites of CCl^.
MiKhaii et ai. (1973) examined the protective effect of adcnosine mono-
pnosphate (AMP) on CCi, toxicity. Three groups of male and female aouit
rats were studied: a control group, a group treated with 0.5 mi of a 1:1
mixture of CCI in mineral oil/100 g bw via intraperitoneal injection and
a group treated with 30 mg AMP via incraperitoneal injection 1/2 hour prior
to treatment with CCl^. The CCl^ resulted in elevated serum iron, cop-
per, zinc, potassium, sodium and calcium 24 hours after administration.
However, pretreatment with AMP led to a normalization of the levels of serum
iron, copper and zinc while tnere were no cnanges in serum calcium, magne-
sium, potassium and sodium levels as compared to the CCi^-treated group.
The normalization of zinc may De due to the action of AMP on hormone secre-
tion. Thus, tne autnors conclude tnat tne normalization of iron and copper
may oe due to some protective effect of AMP on tne liver (Mikhail et al.,
1978).
Additional worK is reported in tne literature dealing witn an'cagorusm
out win only oriefly be mentioned nere. Kieczka et al. (1981) determined
tnat an oxioized catechol metaoolite rather tnan tne catecnol molecule
itself may be responsible for the enzymatic inhipition of the activation
step of CCI. or may interfere witn reactions of tne *CC1_ radicals
formed. The administration of chloramphenicol early in CCI, intoxication
12-9
-------
prevents lipid peroxidation of endoplasmic reticulum memoranes in rats
(Dolci and Brabec, 1978). Dzhioiev and Balanski (1574) found tnat CC14
injected prior to the administration of urethane reduced the incidence of
adenomas by 32 and 47%.
12.3. SUMMARY
Numerous substances have been shown to synergistically affect carbon
tetracnloride toxicity. Ethanol has been shown to potentiate CC1. toxic-
ity even when the ethanol consumption had taken place prior to exposure.
This effect has been documented in case studies ana more recently quantified
in animal studies. Several other environmental pollutants such as Kepone,
PBB ana PCB have been shown to potentiate CCi. toxicity at doses where
ooth substances are not considered toxic. CCI, toxicity nas oeen shown to
be innibitea oy several compounds such as cnloramphenicol and catechol.
12-10
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13. REGULATIONS AND STANDARDS
13.1. WATER
13.1.1. Ambient Water. The U.S. EPA recently announced avaiiaoility of 64
ambient water quality criteria documents and associated criteria (45 FR
7*318). Tne criteria associated with tne carbon tetracnionae document were
4.0, 0.4 or 0.04 yg/i based on estimated human lifetime cancer risks of
10"s, 10"6 or 10"7, respectively. These criteria weie derived based
on tne assumption of a daily contaminated water intake of 2 I and con-
tamiiiateo fish intake of 6.5 g per person. Criteria associated witn oruy
tne consumption of 6.5 g of contaminated fisn were 69, 6.9 or 0.69 ug/i,
respectively.
13.1.2. Drinking Water. The NAS recommended "Suggested No-Adverse-
Response Levels" (SNARLs) for caroon tetrachloride in drinking water (NAS,
I960). The recommended 24-hour value was 14 mg/i oased on data wnich
indicated a toxic effect to the liver of rats 5 hours after one exposure to
carbon tetracnloride at 400 mg/kg bw, a 1000-fold safety factor, and an
assumed daily water ccnsumptidn of 2 & for a 70 kg aouit. NAS recom-
mended a 7-oay SNARL of 2 mg/i based on tne assumed cumnuiative. effects of
carbon tetracnloride after repeated daily exposure (i.e., 14 mg/2, T 7 =
2 mg/i). NAS did not recommend a "chronic exposure" SNARL because care-on
tetracniorioe is a carcinogen in some animal species.
Tne Office of DrinKing Water (ODW) of the U.S. EPA has recommended draft
SNARLs for caroon tetracnloride. Tne 1-day value is 0.2 mg/S, based on
oata wnicn indicate adverse effects in rats after an acute exposure of car-
oon tetracniorioe at 20 mg/kg bw, a 1000-fold safety factor, and an assumed
daily water consumption of 1 I for a 10 kg child (Ohanian, 1>81). ODW
13-1
-------
recommended a draft iO-oay SNARL at 0.02 mg/A. A longer-term EPA-SNARL
-------
The National Institute for Occupational Safety and Health (NIOSH, 1975)
recommended a carbon tetrachloride TWA value of 12.6 mg/m3 for a 10-hour
work day, 40-hour week over a working lifetime. This recommendation was
based on liver and eye changes found in workers chronically exoosed to
carbon tetrachloride. In a 1976 revision, NIOSH recommended that the con-'
centration of CCl^ not be >12.6 mg/m3 of breathing zone air in a
45 * air sample taken over a period not to exceed 1 hour in duration
(NIOSH, 1976)
Inhalation standards for various toxic substances in the working envi-
ronment of several other countries have been published (International Labor
Office, 1970). Carbon tetrachloride inhalation standards are shown in
Table 13-1. The USSR values are maximum allowable concentrations (MACs)
never to be exceeded. Several countries follow USSR standards; others
follow the recommendations of ACGIH.
13.3. FOOD
The National Academy of Sciences (NAS, 1978) reported maximum concentra-
tions of carbon tetrachloride permitted in cooked cereal as 50 ug/kg.
This value was derived from a FAQ/WHO expert committee in 19J2. No other
information was found concerning guidelines, criteria or standards for car-
oon tetrachloride in food.
13.4. SUMWRY
Protective levels for carbon tetrachloride in air in the workplace have
been suggested by several countries and by several groups within the United
States. The number of suggested protective levels demonstrates the wealth
of toxicity data in this area. Protective levels for carbon tetrachloride
in water (both drinking and ambient) have recently been suggested by the
U.S. EPA and NAS. Only one protective level has been suggested for food:
that of cooked cereal. Obviously, more work is needed in this latter area.
13-3
-------
TABLE 13-1
Carbon Tetrachloride Inhalation Standards of 11 Countries3
Country
United States
ACGIH
OSHA
NIOSH
Czechoslovakia
Finland
Hungary
Japan
Polano
Rumania
UAR and SAR
USSR
Yugoslavia
Standard
mg/m3
30°
125°
65
162.5
1300
12.6°
12.6°
50
250
160
20
• 100
10
20
50
625
20
65
Qualifications
TWA
STEL
TWA
Ceiling exposure concentration
Maximum peak of ceiling
Concentration not to be
exceeded for 5 minutes in any
4 hour period.
TWA
45£ air/60 minutes
MAC
Single short exposure
8 hours continuous exposure
8-hour average
30 minutes
-
MAC
aSource: Adapted from NIOSH, 1975
DRecommended standaros
13-4
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14. EFFECTS OF MAXR CONCERN AMD HEALTH HAZARD ASSESSKENT
The assessment of health hazards from carbon tetrachloride requires
information whicn relates specific adverse health effects to dose-exposure
conditions. Studies which report the route of exposure, the dose, the dura-
tion of exposure, the animal species and the nature of adverse effects are
therefore most useful in hazard assessment, which involves the prediction of
effects from given environmental or occupational exposure situations. Since
the route of intake is often important, experimental studies should use the
typical routes of human exposure (i.e., ingestion, inhalation or dermal con-
tact). Therefore, studies based on exposure by intraperitoneal or subcuta-
neous injection are not included.
The discussion of health effects is organized according to the route of
exposure. Within each subsection, the discussion first lists the pertinent
acute, sub-chronic and chronic effects, followed by summaries on reproduc-
tive, mutagenic and carcinogenic effects. Specific information on dose,
exposure, test animal, effects and references are presented in Table 14-1
for acute, sub-chronic and chronic studies, in Table 14-2 for reproduction
studies, in Table 14-3 for mutagenicity studies, and in Table,14-4 for car-
cinogenicity studies. Note that these tables summarize the toxicity data
and require further analysis before risk estimates can be made for humans.
14.1. PRINCIPAL EFFECTS
Carbon tetrachloride is toxic to humans and animals. Sublethal exposure
affects several organs; however, the primary target organs are the liver and
the lung. Long-term exposure has resulted in malignant tumors of the liver
in three animal species. Several studies have produced satisfactory dose-
response information, including estimated "no-effect" levels for humans and
four animal species. These studies are classified as satisfactory in that
14-1
-------
TABLE 14-1
Dose-Related Toxic Effects of Carbon Tetrachloride
an Mumans and Animals
Route3 Animal
Ingest Ion Human
Human
Human
Human
Rat
Rat
Dog
Rabbit
Dog
Mouse
Rat
Rat
Mouse
Rat
Doseb
0.06
-------
TABLE 14-1 (cont.)
Ji-
I
Route3 Animal
Rat
Rat
Rat
Rat
Rat
Inhalation Human
Human
Human
Rat
Rat
Mice
Rat
Doset>
4000 ing/kg
5088 mg/kg
22 mg/kg
40 mg/kg
76 mg/kg
63 rog/m'
309 ntg/m'
290-650 mg/m'
300 mg/m'
65 g/m1
71.8 g/m1
285 g/m1
Exposure
Single dose
Single dose
6 wk
6 wk
6 wk
3 hr
1 hr
2 yr
24 hr
0.3-4.0 hr
1 hr
0.5 hr
Reported Effects0
Behavior changes, diverse lung cell
changes, altered liver C-450 content
Clara cell lesions In lung
NOEL
Higher levels of total llpids and
triglycerides
Depressed weight gain, higher levels
of total llpids and trlglycerldes
NOEL
Decreased serum Iron, altered serum
transamlnase levels
Reversible nausea, anorexia, vomiting,
discomfort
Reduction in enzyme activity
Increased liver and kidney weights
Clara cell lesions in lung
Morphological and cellular changes in
Reference
Gould and Smuckler,
Boyd et al., 1980
Alumot et al., 1976
Alumot et al., 1976
Alumot et al., 1976
Stewart et al., 1961
Stewart et al., 1961
Kazantis and Bomford
1971
, 1960
Merkur'eva et al., 1979
Wong and 01 Stefano,
Boyd et al., I960
Chen et al., 1977
1966
lung
Rat
6.1 mg/m'
6 wk6
NOEL
Prenriergast et al., 1967
-------
TABLE 14-1 (cont.)
Route3 Animal
Guinea
pig
Rat
Guinea
pig
Rat
£. Guinea
pig
Rat
Rat
Guinea
pig
Monkey
Rat
Guinea
pig
Dose0
6.1 mg/m'
61 mg/m1
61 mg/m'
515 mg/m'
515 mg/m'
14 mg/m'
315 mg/m1
315 mg/m'
315 mg/m'
630 mg/m'
630 mg/ra'
Exposure
90 days6
6 wke
90 days6
6 wk6
6 wk6
5 hr/day, 5 day/
wk, 5 mo
10 mo6
10 moe
10 mcf-
10 moe
10 moe
Reported Effects0
NOEL
Fatty infiltration and some degenera-
tion of liver
3 deaths (of 15 animals), depressed
weight gain, liver damage
Fatty degeneration of liver, morpho-
logical changes In liver and lung
3 deaths (of 15 animals), weight loss,
liver damage, morphological changes
in liver and lung
Changes in hepatic energy producing
processes
Growth stimulation, NOAEL
Mortality (9/24) with median 44 expo-
sures, liver cirrhosis
Reversible liver degeneration
Liver cirrhosis after 173 exposures
Mortality (16/24) with median 10 expo-
sures
Reference
Prendergast
Prendergast
Prendergast
Prendergast
Prendergast
et al.,
et al.,
et al.,
et al.,
et al. ,
1967
1967
1967
1967
1967
Rotenberg, 1978
Smyth et al.
Smyth et al.
Smyth et al.
Smyth et al.
Smyth et al.
, 1936
, 1936
, 1936
, 1936
, 1936
Rat
1260 rag/in' 10 mo6
Liver cirrhosis after 115 exposures
Smyth et al., 1936
-------
TABLE 14-1 (cont.)
Route3 Animal
Guinea
pig
Monkey
Rat
Guinea
pig
Dermal Guinea
pig
Guinea
pig
Dose0 Exposure
1260 mo/in1 10 mo6
1260 mg/m1 10 mo6
2520 mg/nt1 10 mo6
2520 mg/ra* 10 mo6
1 m*/ 0.3 hr
3. 1 cm2
1 n*-/ 16 hr
3. 1 cm1
Reported effects0
Mortality (13/24) with median 3 expo-
sures
Reversible liver degeneration, de-
pressed weight gain, sciatic nerve
damage
Growth retardation, liver cirrhosis
after 54 exposures
Mortality (19/24) with median 3 expo-
sures
Epidermal sponqinsis and karyopyknosis
Pronounced epidermal spongiosls, karyo-
pyknosis and functional separation,
marked hydropic changes in liver,
liver necrosis
Reference
Smyth et al., 1936
Smyth et al., 1936
Smyth et al., 19?6
Smyth et al., 1936
Kronevi et al., 1979
Kronevi et al., 1979
exposure includes gavage, drinking water and diet.
tflral
Oral exposure is in mg CCl^, or mg of CCU/kg body weight. Inhalation exposure is in weight of CCl^/cubic
When ppm is supplied by the referenced article, conversion is 6.5 mg/m = 1 ppm. Dermal exposure is volume of C
meter of air.
per area of
skin. These are the reported animal exposure levels and are not human equivalent exposure levels. Therefore, doses are not compar-
able and may not be directly applicable to the human exposure situation.
cThe type of effect level (NOEL, etc.) was determined for this document and was not necessarily reported by the original authors.
is the ratio of odds ratios (see text, Section 8.2.2). Corresponding dose Is estimated for drinking water by assuming that
the Cincinnati ratio of drinking water concentration to river water concentration (1:2) is constant for all cities in the study. A
test for linear trend of ROR on dose is significant (pfiO.05).
Exposure: 8 hr/day, 5 days/wk.
-------
TABLE 14-2
Reproductive Effects of Carbon Tetrachloride
from Subchronic Exposure
Route
Inhalation
Inhalation
Oral
Dose Level
and Species
622 mg/kg
Rat
475 mg/kq
Rat
2370 mg/kg
Rat
Exposure
Duration
Daily; days 6-15
of gestation
Daily; for three
generations
2 or 3 days; during
gestation
Biological
Endpoint
Retarded fetal
development
Reduced fertility
Reduced fertility
Reference
Schwetz et al
Smyth et al. ,
Wilson, 1954
. , 1974
1936
-------
TABLE 14-3
Summary Table for Mutagenicity Studies
Assay Response Reference
Ames test Negative McCann et al., i975
§• co-*-J- reversion test Negative Uehleke et al., 1976
Ln vitro chromosome
assay Negative Dean and Huason-Walker, 1979
Yeast cells Positive Callen et al., 1980
14-7
-------
TABLE 14-4
Carclnogenicity Studies Useful for Risk Assessment of Carbon Tetrachloride8
t— •
.p-
1
CO
Species/
Strain
Mice/
Inoi ed L
Mice/
Njinber0
73 Treated
152 Control
200 Treated
40 Control
Dose0
Regimen
2-3x/wk
5x/wk
in corn
oil
Duration
Dose Exposure Observation
0.04 ml 4 mo 7.0-7.5 mo
(=•2100 rag/kg)
1250 mg/kg 78 wk 92 wk
2500 mg/kg
Animals with Tumors
Control Treated
1% 47%
8* 100*
97X
Tumor
Type Reference
Liver tumor Edwarus et
al., W42
Hepato- NCI, li/7b
cellular
carclnocna
abtudles piovlding both a NOAEL and a LOAEL
bl(iciudes males and females
cBy guvage or gastric intubation
-------
they not only provide all of the necessary quantitative information (e.g.,
number of animals in control and treated groups) but also include sufficient
detail to demonstrate the high quality and defensibility of the research.
14.1.1. Ingestion. The only dose-related human data with complete quanti-
tative information for oral ingestion (see Section 8.8.2.) are from an epi-
demiological study which found elevated serum creatinine levels following
approximately 1-day exposure to carbon tetrachloride in drinking water.
Several animal studies showed dose-related effects from carbon tetrachloride
ingested via water, gavage or diet. Most of these studies involved a single
aose (e.g., Korsrud et al., 1972). Acute effects (with increasing dose)
included greater liver weight than normal, greater amount of liver fat,
cnanges in enzyme levels or activity, some liver necrosis, reversible lung
and kidney structural changes, lung lesions and behavioral abnormalities.
One short-term (6-week) study in rats showed dose-related increases in total
serum lipids and triglycerides and depressed weight gain (Alumot et al.,
1976).
Several authors reported that carbon tetrachloride does not appear to be
a teratogen but that it can affect reproduction following subchronic expo-
sure. Oral administration of carbon tetrachloride to rats for either 2 or 3
days during days 7 to 14 of gestation was reported to cause a reduction in
litter sizes and an increase in the number of fetal resorptions (Wilson,
1954).
Carbon tetrachloride has produced liver tumors in hamsters, mice and
rats. A number of experiments have been conducted using mice of various
strains (86C3Fp C3H, A and L) and different dosage regimens of carbon
tetrachloride (Edwards et al., 1942; NCI, 1976). The types of tumors
observed have included hepatomas, hepatocellular carcinomas and hyperplastic
14-9
-------
hepatic nodules. Hyperplastic hepatic nodules were also induced in Syrian
golden hamsters by oral doses of carbon tetrachloride (Delia Porta et al.,
1961). Effects induced by carbon tetrachloride in several strains of rats
also include cholangiofibrosis, hepatic hyperplasia, hyperplastic hepatic
nodules and hepatic carcinomas (Reuber and Glover, 1967, 1970).
14.1.2. Inhalation. The majority of the toxicity studies encountered in
the available literature involved inhalation of carbon tetrachloride and
included a wide range of exposure levels. One 70 minutes exposure study
found decreased serum iron and altered serum transaminase levels, implicat-
ing liver damage (Stewart et al., 1961). A 2-year exposure in humans showed
reversible nausea, anorexia, vomiting and epigastric discomfort (Kazantis
and Bomford, 1960).
Effects from short-term exposure on animals appear to be dose-dependent
and include: increased kidney and liver weights, reduction in activities of
various enzymes, morphological and cellular changes in the liver and the
lung, and Clara cell lesions in the lung. Dose-related effects from sub-
chronic exposures are highly species-specific. For example, reversible
liver damage was seen in the monkey at the same dose and duration which
caused liver cirrhosis and increased mortality in the guinea pig. Other
effects due to subchronic exposures include minor liver damage, weight loss
and damage to the sciatic nerve.
Only one chronic inhalation animal study (of 10 months duration) was
found in the literature. Liver damage was reported. The authors did not
report liver tumors (Smyth et al., 1936).
When pregnant rats were exposed to carbon tetrachloride by inhalation on
days 6 to 15 of gestation, the exposure resulted in decreased fetal body
weights and lengths (Schwetz et al., 1974). In a three-generation inhala-
14-10
-------
tion study involving rats, a dose of 238 mg/kg/day did not affect reproduc-
tive functions, while a dose of 475 mg/kg/day caused reduced litter sizes
(Smyth et al., 1936). Single subcutaneous injection of pregnant animals
with carbon tetrachloride has also been reported to produce histological and
biochemical changes in the livers of the offspring; thus, Bhattacharyya
(1965) concludes that carbon tetrachloride may be fetotoxic and can cross
the placental barrier.
14.1.3. Dermal Exposure. Guinea pigs exposed to pure carbon tetrachloride
in a skin painting experiment showed epidermal spongiosis and karyopyknosis,
altered liver morphology and some liver necrosis (Kronevi et al., 1979).
14.1.4. Mutagenicity. Carbon tetrachloride has produced negative results
in the Ames Salmonella test, in both the presence and the absence of micrc-
somal activation (McCann et al., 1975) and in the Escherichia coli K12 test
(Uehleke et al., 1976). Carbon tetrachloride produced negative results and
was non-clastogenic in a chromosome assay using an epithelial-type cell line
derived from rat liver which possessed intrinsic metabolizing activity (Dean
and Hudson-Walker, 1979).
However, Callen et al. (1980) found carbon tetrachloride .to be geneti-
cally active and cytotoxic in strain 0 yeast cells of Saccharomyces cerevi-
siae. In these cells, which contain a cytochrome P-450-dependent mono-oxy-
genase system, carbon tetrachloride caused increased frequencies of "gene
conversion and mitotic recombination" and decreased cell survival.
14.2. SENSITIVE POPULATIONS
Studies on human sensitivity are limited. There is clinical evidence,
discussed in Chapter 12, that two chemicals, isopropanol and ethanol, may
potentiate carbon tetrachloride toxicity in humans. As described by Moon
(1950), a repeated history of alcoholism in cases of fatal CC1A poisoning
14-11
-------
may indicate a synergistic effect between alcohol and CC1,. In other case
reports, very obese and undernourished persons suffering from pulmonary
diseases, gastric ulcers, liver or kidney diseases, diabetes or glandular
disturbances seem especially sensitive to the toxic effects of CCl^.
Supportive studies on rats suggest that older animals are more suscepti-
ble to the toxic effects of CC1, than are younger animals, and that males
are more susceptible than females (Rueber and Glover, 1967). Chaturvedi
(1969) examined age and sex as factors in CCl^ toxicity. The findings
revealed that female rats are less susceptible to the adverse effects of
different hepatotoxic agents. Chaturvedi (1969) postulated that this was
due to different hormonal and enzyme patterns and the lack of certain pro-
teins in the female liver. The sex difference noticed in adult rats was not
as apparent in young rats.
Nutritipnal 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 -300 mg/kg carbon tetrachloride in a gas
chamber 7 hours/day, 5 days/week for 45 months. Animals were then sacri-
ficed, and histopathological effects in the liver and kidneys were deter-
mined. When animals fed standard chow were compared to other groups, these
effects were more severe in animals fed a diet high in lipid and low in car-
bohydrate, or a diet low in protein. Methionine appeared to protect against
increased toxicity, particularly kidney damage, caused by low-protein diets
(Gyorgy et al., 1946).
The interaction of carbon tetrachloride with other chemicals has result-
ed in an enhancement of the toxic effects produced in animals by either
chemical alone. Exposure of animals to selected environmental carcinogens
in combination with carbon tetrachloride has resulted in an increase in car-
14-12
-------
cinogenicity. In addition, certain chemicals appear to increase the toxic
effects of carbon tetrachloride on the liver and other organs of experiment-
al animals.
14.3. QUALITATIVE HEALTH HAZARD ASSESSMENT
The assessment of human health risks, that is, the likelihood of certain
adverse effects from given exposure scenarios, is hampered by the paucity of
gooo oose-response data in humans. Of tne three human studies discussed
above, the acute epidemiological study involving oral ingestion (Sonich et
al., 1981) and the acute inhalation study (Stewart et al., 1961) show serum
alterations. The short-term inhalation study shows reversible, minor CNS
and gastrointestinal effects (Kazantis and Bomford, 1960). In other
instances, although several case reports document human effects, concentra-
tions and exposures are not reported. Therefore, the prediction of toxic
effects in humans and the determination of no-effect levels for subsequent
use in a quantitative hazard or risk assessment, are primarily estimated
from the many animal studies showing dose-related effects.
The major reported suolethal health hazards to humans from exposure to
caroon tetrachloride are damage to the liver, lungs, kidneys -ana central
nervous system. Less severe adverse effects include altered enzyme activi-
ties following ingestion and inhalation, gastrointestinal disujroances fol-
lowing inhalation, and epidermal damage following dermal contact. In animal
studies, some effects are seen in areas distant from the contact interface.
These include lung damage from oral ingestion, liver damage from inhalation
and from dermal contact and, to a lesser degree, enzyme disturbances from
inhalation and ingestion.
Teratogenic, mutagenic and carcinogenic effects have not been demon-
strated in humans, and only carcinogenicity has been shown in experimental
14-13
-------
animals. In addition, fetotoxicity and neonatal toxicity have been shown in
rats (Schwetz et al., 1974; Bhattacharyya, 1965). Reproductive efficiency
has also been affected in rats evidenced by reduced litter sizes (Smyth et
al., 1936). Negative mutagenicity has been demonstrated in four studies
including Salmonella typhimurium (McCann et al., 1975; Simmon and Tardiff,
1977), E. coli (Uehleke et al., 1976), and in a recently developed epithe-
lial-type cell chromosome assay (Dean and Hudson-Walker, 1979). The single
positive mutagenicity test in yeast cells, Saccharomyces cerevisiae, there-
fore requires further confirmation (Callen et al., 1980).
Carcinogenicity has been demonstrated in three experimental animal spe-
cies, but predominantly several strains of rats and mice (NCI, 1976; Edwards
et al., 1942; Reuber and Glover, 1970). Thus, carbon tetrachloride should
be considered a potential human carcinogen, even though no definitive cause-
and-effect human data exist.
Adverse effects on the biosphere from partial depletion of the ozone
layer by carbon tetrachloride and the subsequent increase in UV flux are of
concern for future research. The lack of stratospheric CC1, data and the
uncertainties relative to the currently used, simplified transport models
prevent reliable hazard assessment.
14.3.1. Animal Toxicity Studies Useful for Hazard Assessment. The pre-
ferred studies for hazard assessment are those which provide definite effect
levels. Adverse effects are defined here as functional impairment and/or
pathological lesions which may affect the performance of the whole organism,
or which reduce an organism's ability to respond to an additional challenge.
Adverse effects which are not carcinogenic are assumed to be threshold
14-14
-------
phenomena. The threshold region of toxicity is estimated by evaluating four
types of effect levels:
NOEL No-Observed-Effect Level: That exposure level at which
there are no statistically significant increases in fre-
quency or severity of effects between the exposed population
and its appropriate control.
NOAEL No-Observed-Adverse-Effect Level: That exposure level at
which there are no statistically significant increases in
frequency or severity of adverse effects between the exposed
population and its appropriate control. Effects are pro-
duced at this level, but they are not considered to be
adverse.
LOAEL Lowest-Observed-Adverse-Effect Level: The lowest exposure
level in a study or group of studies which produces statis-
tically significant increases in frequency or severity of
adverse effects between the exposed population and its
appropriate control.
FEL Frank-Effect Level: That exposure level which produces
unmistakable adverse effects, ranging from reversible histo-
pathological damage to irreversible functional impairment or
mortality, at a statistically significant increase in fre-
quency or severity between an exposed population and its
appropriate control.
Studies providing both a NOAEL and a LOAEL, therefore, are the most useful
studies for hazard assessment.
The rat was the most commonly used animal in dose-response studies on
carbon tetrachloride, with 12 satisfactory studies (as defined earlier) rep-
resenting 22 dose-exposure groups (see Table 14-1). Korsrud et al. (1972)
reported a single-dose LOAEL for rats of 20 mg/kg, virtually identical to
the 6-week rat NOEL of 22 mg/kg given by Alumot et al. (1976). These re-
sults may be consistent. The infrequent, minor liver cell changes reported
by Korsrud et al. may have been present in the Alumot et al. animals without
affecting the serum levels monitored in the latter study. This illustrates
the dependence of effect-level category on the endpoint investigated.
14-15
-------
Extensive single-dose information on lung effects from oral ingestion was
reported by Boyd et al. (1980), who studied two dose levels in both rats and
mice, the lower in each case resulting in a "lung NOEL." Other studies
whicn provide dose-related effect data for single oral exposures are listed
in Taole 14-1. The only subchronic oral study (6 weeks) provided a NOEL and
two higher adverse effect levels for rats (Alumot et al., 1976).
Acute inhalation studies did not provide dose-related effect data.
Intercomparison of four studies in rats showed a dose-dependent progression
in severity of effects, with doses ranging from 300 to 285,000 mg/m3
(Merkureva et al., 1979; Wong and OiStefano, 1966; Boyd et al., 1980; Chen
et al., 1977). Useful subchronic inhalation data were provided by Prender-
gast et al. (1967), who used three dose levels (6.1 to 515 mg/m3) includ-
ing a NOEL, resulting in progressive effects on the liver of rats. The
authors also applied identical dose levels to guinea pigs, the lowest also
being a NOEL, with more severe effects resulting from the two higher
levels. The only chronic inhalation study involved rats and guinea pigs and
demonstrated effects from four dose levels administered for 10 months (Smyth
et al., 1936). The effects in rats were predominantly on the liver, whereas
in the guinea pigs the endpoint was mortality, dependent on dose and length
of exposure.
The only dermal study providing dose-response information involved a
single dose for short durations (0.3 and 16 hours) using guinea pigs
(Kronevi et al., 1979). This study is most important for the duration-
related effects on the liver, demonstrating definite effects of dermal expo-
sures at a distant site.
14.3,2. Animal Carcinogenicity Studies. Liver tumors have been demon-
strated in several species following oral exposure to carbon tetrachloride.
14-16
-------
uruy a few studies, nowever, were designed to demonstrate tne relation
oetween tumor incidence and dose or exposure duration. Two studies provided
definitive information on dose ano incidence in controls ana treated animals
(.see Taoie 14-4). Edwards et ai. (1942) reported dose-related incidence of
tumors for inored strain L mice of two age groups, and NCI (1976) demon-
strated significant tumor incidence for treated B6C3F1 mice. It should be
noted that in most of tne animal studies showing carcinogenicity either the
lengtn of the study was too short or the dose level was too high for a dose-
response estimation of lifetime exposure (NAS, 1978). The National Research
Council recognizes this proolem and for this reason uses the NCI (1976) bio-
assay for tricnloroetnyiene (witn CC1, as a positive control) for deter-
mining a carcinogenic risK estimate for caroon tetrachloride.
14.4. FACTORS INFLUENCING HEALTH HAZARD ASSESSMENT
14.4.1. Exposure. Caroon tetracniorioe is persistent in air and ground
water. Contamination of surface water and soil oy carbon tetracnloride is
not liKeiy to present long-term nazaros due to its rapio volatilization.
However, large quantities of CC1, in bodies of cold water, sucn as lakes,
are liKeiy to remain submerged and oe relatively staole, contaminating the
oooy of water for several years. Carbon tetrachloride is readily aosorbed
througn the lungs, but it is also rapidly exhaled; it is excreted through
all routes, predominantly as the parent compound. The relative contribu-
tions of tnese factors to tne bioavailaoility and body burden of CC1, in
numans are not well defined. Also, experimental animals may differ suostan-
tiaily from humans in terms of oral and dermal efficiencies of absorption.
Tnus, application of human or animal pnarmacoKinetic information to quanti-
tative human neaitn nazard assessment does not seem feasiole at this time.
Consequently, tne usaole exposure information is from monitoring data of
amoiant CCi. levels in air, water ana otner liquids, and fdod.
14-17
-------
14.4.2. Estimated Threshold No-effect Levels.* The dose-response data for
carbon tetrachloride are quite limited, especially regarding effects on
humans (Taole 14-5). A human NOAEL for oral ingestion was reported as 0.2
mg/day (- 0.1 ppm) for 1-day exposure. The observed effect was a dose-
related increase in the frequency of elevated creatinine levels in the study
population. A human NOEL for inhalation was reoorted as 63 mg/m3
(-10 ppm) for 3-hour exposure. The monitored effects were serum enzyme
and iron levels. Animal data are slightly more complete, with rat NOELs
(not human equivalent) for inhalation ranging from 6.1 to 315 mg/m3,
depending on length of exposure. Insufficient information exists to allow
estimation of NOAELs based upon long-term exposures.
In light of the uncertainties and inadequacies associated with the data
base for CCl^, particularly with the human NOEL and NOAEL given above,
calculations of acceptable chronic exposure levels must be approached with
caution. The lack of good absorption data is the main obstacle to accurate
conversion of the animal exposure data to human equivalent exposures.
14.4.3. Carcinogenicity. Liver tumors have been shown to result from oral
exposure to carbon tetrachloride by three species of animals. Although
several authors note that toxic effects are concurrent with liver tumors, it
has not been established that tissue damage is a necessary precursor to
CCl^ carcinogenesis. However, in view of the inconclusive nature of the
presently available evidence for the muzagenicity of CC14, the upper bound
of risk is presently regarded as having only limited plausibility.
*Animal studies discussed herein indicate that carbon tetrachloride is a
potential human carcinogen, and hence a no-effect level may not exist.
These no-effect levels are supplied for comparison purposes.
14-18
-------
TABLE 14-5
Reported No-Effect Levels for Toxicity of Carbon Tetrachlorlde3
Route
Oral
Exposure
Single dose
Acute
Subchronic
Animal
Rat
Mouse
Dog
Hi nan6
Rat
No-Effect
Levelb
£/
1600 mg/kg
116 mg/kg
0.2 mg/day
22 mg/kg
Lowest-Observable-
Adverse-Effect
Levelc
20 mg/kg
4000 mg/kg
Til mg/kg
—
40 mg/kg
Observed
Effects
Liver cell changes
Widespread lung cell changes
Liver necrosis
Elevated scrum cieatinint!
Elevated serum liplds and
Reference
Korsrud et «u., 1972
Boyd et al., 19bO
Gardner et «.!., 1924
Sunich et ai., 1981
Alumot et al., ±976
Chronic
Rat
mg/kg
trlglycerides
Reproductive perfornance and
body weight
Alumut et ai., U76t
-------
TABLE 14-5 (cont.)
Route
Inhalation
Exposure
Acute
Subchronic
Animal
Human
Rat (6 wk)
No-Effect
Levelb
63 mg/ro'
6.1 mg/m'
Lowest-Observahle-
Adverse-Effect
Level0
309 mg/m'
61 mg/m'
Observed
Effects
Decreased serum iron and
altered transaminase levels
Liver degeneration and fatty
Reference
Stewart et al. ,
Prendergast et ;
1961
il. ,
Chronic
Rat (5 mo) 14 mg/m1
Guinea pig 6.1 mg/m' 61 mg/m'e
Rat 315 mg/m' 630 mg/m'
infiltration
Altered respiratory rates and
sensitivity of respiratory
enzymes to inhibitors
Increased mortality, liver
damage
Liver cirrhosis
1967
Rotenberg, J97H
Prendergast et al. ,
1967
Smyth et al., 1
K)
Q
^Extracted from Table 14-1.
^Includes no-observable-effect levels and no-ohservable-adverse-effect levels (see Table 14-1). These are the reported animal exposure
levels and are not human equivalent exposure levels. Therefore, doses are not comparable and may not be directly applicable to the human
exposure situation.
Lowest or only adverse-effect level reported.
*lNo NOEL was reported for the rat in this study. Boyd et al. (1980) gives a rat NOEL of 3816 mg/kg; however, this is for lunq effects only.
eThe elevated serum creatinine levels are not considered to be an adverse effect. No human data exist showing dose-related, adverse effects
from oral ingestion of carbon tetrachlorirte.
One-year results inferred from a 2-year study; see discussion in Section 0.1.3.
-------
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Appendix: Unit Risk Estimate for Cancer
Unit riSK is defined as the lifetime cancer risk associated witn contin-
uous lifetime exposure to water or air containing a unit concentration of a
pollutant in eacn respective media. Using the units of 1 \iQ/i and 1
ug/m3 for water ana air, respectively, route-specific unit risk esti-
mates are calculated for carbon tetrachloride (CCi4). Upper bounds of
risk nave been estimated for individuals undergoing either of two hypothet-
ical lifetime exposure situations: 1) continuous exposure to 1 yg
CCl4/m3 of air, or 2) continuous exposure to 1 ug CCi4/& in drink-
ing water. These estimates are based upon the results of the National
Cancer Institute (NCI, 1976) bioassay for trichloroethylene, in which CC14
of unspecified purity, was used as the positive control. This bioassay
examineo the incioence of hepatocellular carcinoma in male mice. Tne
effects of CC14 have oeen studied extensively in a number of other studies
examining the effects of ingestion and innaiatian exposures upon mice as
well as rats, hamsters and dogs. However, upon evaluation of these studies,
eitner the length uf the stuoy was too snort or the dose level was too niyn
to ootain a oose-response estimation of lifetime exposure (NAS, 1978).
In this NCI study, male mice received CC14 oy gavage at 1250 mg/kg and
2500 mg/kg 5 cays/week for 78 weeks (546 days). Liver tumors were observed
at 92 weeks (644 days) when the experiment was terminated. Using the re-
ported data, the parameters of the extrapolation model are:
Dose Liver Cancer Incidence
(mg/kg/day) (no. affected/no, examined)
0 (pooled venicle control) 5/77 (6%)
1250 49/49 (100%)
2500 47/48 (98%)
le = 78 weeks = 546 cays
Le = 92 weeks = 644 days
L = 92 weeks = 644 days
w = 0.028 kg
A-l
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As given in the Guidelines for determining the impact of pollutants upon
numan healtn (45 PR 79351), tne following procedure is used to calculate the
carcinogenic potency (q^*) of CC1, for humans. Lifetime time-weighted
average daily doses (d) for animals are computed:
d = dose (mg/kg/day) x 5/7 x le/Le
Thus,
dlow = 1250 mg/kg/day x 5/7 x 546/644 = 754 mg/kg/day
d high = 2500 mg/kg/day x 5/7 x 546/644 = 1508 mg/kg/day
Using these values, the carcinogenic potency (a,*) for animals is computed
via the linearized multistage model. The linearized form of the multistage
model can be expressed:
P = 1 - exp[-q * x d -i- higher order terms in dose]
Tnis is derived and discussed in the Guidelines as citeo aoove. For low
doses, the higher order terms are negligible compared to the q-,*d term.
The animal q,* is then converted to a q,* for humans using the body
weights of 0.028 kg for mice and 70 kg for humans with the assumption that
doses producing equivalent effects are calculated in terms of mg chemical/
surface area of animal.
Thus, the CCl^ carcinogenic potency factor for humans is estimated:
q1* = 8.275 x 10" 2 (mg/kg/day)^
From this factor the carcinogenic potency of CC1, can be expressed in
terms of exposure via water or air as follows.
Unit Risk Estimate for Humans from Exposure to Carbon Tetrachloride in Water.
To estimate the risk corresponding to the concentration of 1 ug
CC1 /i water, the effective dose in terms of mg/Kg/day corresponding to
A-2
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1 ug/Jl must first be estimated. Assuming a water intake of 2 £/day
and a 100% absorption rate by ingestion for a 70 kg human:
awater = t2 A/day x 1'00 x 1/(^70kQ^ x 10"3 m9/Jl
= 2.86 x 10"5 mg/kg/day
Using the multistage model, the upper bound of the risk corresponding to
awater is e^imated:
P(dwater) = 1 - exp[-8.275 x 10'2 x 2.86 x 10"5]
= 0.236 x 10"5
Therefore, tne unit cancer risk estimate for humans from exposure to
CC1. in water, that is, the risk corresponding to 1 yg/i, is
0.236 x lO'5.
Unit Risk Estimate for Humans from Exposure to Carbon Tetrachloride in Air.
To estimate the risk corresponding to the concentration of 1 yg
air, the effective dose in terms of mg/kg/day corresponding to
3 must first be estimated. Assuming an air intake of 20 mVday
and a 40% absorption rate by inhalation for humans (as recommended in this
document), this effective dose can be estimated for a 70 kg humane
cL,_ = [20 mVday x 0.40 x I/(70kg)] x 10"3 mg/m3
d -Li
= 1.14 x 10"1* mg/kg/day
Using the multistage model, the upper oound of the risk corresponding to
CL ._ is estimated:
d .11.
P(d ) = 1 - expC-8.275 x 10'2 x 1.14 x 10'1*]
3 XI.
= 0.945 x 10"5
Therefore, the unit cancer risk estimate for humans from exposure to
^ in air is directly obtained from the potency such that the risk
orresponding to 1 yg/m3 is 0.945 x 10"s .
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In this calculation, the linearized multistage model is used to give the
following estimates of a plausible upper bound of lifetime cancer risk:
0.236 x 10"5 for a person continuously exposed to 1 ug
per liter of water, or
0.945 x 10"s for a person continuously exposed to 1 ug
per cubic meter of air.
Because of the uncertainties in both the qualitative and quantitative
aspects of risk assessment, the actual cancer risks may be lower than those
indicated above, whicn should be regarded as plausible upper-limits and may
approach zero. However, in view of the inconclusive nature of the presently
available evidence for the mutagenicity of CCl,, the upper bound of risk
is presently regarded as having limited plausibility.
The above information provides route-specific cancer risk estimates
associated with exposure to given units of CCl^. These estimates may be
conservative due to the mathematical model employed. Nevertheless, unit
risks for cancer are defined for independent water and air exposures in that
their computation assumes 100% of the insult is via the stated route.
When information is available to indicate the occurrence of concurrent
exposures, two steps should be considered in estimating one risk. First,
the risks associated with independent exposures can be adjusted for concen-
tration, if known to be different from that defined by the unit risk esti-
mate. This adjustment can be done by simple multiplication or division and
is justified in that exposure is in the low dose region where risk is di-
rectly proportional to dose. For example, if it is known that the concen-
tration in air is 1.5 yg/m3 then the upper bound of the risk associated
with 1 ug/m3, 0.95 x 10"s, can be multiplied by 1.5 to obtain:
(0.945 x 10'5) (1.5) = 1.42 x 10'5
A-4
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Thus, it can be inferred that the upper bound of the risk associated with
tne concentration of 1.5 ug/m3 CC1. in air is 1.42 x 1CTS .
Once such an adjustment is made, if necessary, the additivity assumption
can be used to calculate the risk associated with concurrent exposures via
two routes. It is a general recommendation to use the additivity assumption
which is made since the available data on CCl^ are limited and do not
allow the presentation of a defensible alternative. As new information
oecomes availaole, other alternatives should be consiaered. Here, tne addi-
tivity assumption is that the risk associated with exposure to a given chem-
ical via two routes concurrently is roughly the sum of the risks associated
with each independent route-specific exposure. Since interactions between
the concurrent routes of intake cannot be quantified, uncertainty surrounds
the resulting risk estimate that is derived from the concurrent risks.
In applying the assumption of additivity, the risks rather than the
doses associated with each route are added, but the mere summation of these
risks is presently justifiable only at low doses. Furthermore, the amounts
of 1 ug/i and 1 ug/m3 are concentrations in water and air, respec-
tively, not doses. To state an example, the risk of 0.236 xlO-5 is asso-
ciated with a lifetime exposure to 1 ug CCl^/l water. The dose can be
estimated by assuming consumption of 2 I water/day over the lifetime.
Tnus, the daily dose corresponding to a concentration 1 yg/2- water would
oe 2 £/day x 1 ug/& = 2 ug/day.
A-5
-------
In conclusion, based upon the data for CC1. given in the NCI oioassay
for trichloroetnylene, in which CCl^ was used as a positive control, the
upper bounds of route-specific unit risk estimates for cancer are computed
for exposures via water and air:
The unit cancer risk estimate for humans from exposure to a concen-
tration of 1 ug CC14/4 water is 0.236 x 1CTS.
The unit cancer risk estimate for humans from exposure to a concen-
tration of 1 yg CCl^/m3 air is 0.945 x 10"s.
However, each upper bound of risk is presently regarded as having limit-
ed plausibility due to the inconclusive nature of the available evidence for
the mutagenicity of CCL. Furthermore, because of tne uncertainties in
Doth the qualitative and quantitative aspects of risk assessment, the actual
cancer risks may be lower than those indicated above and may approach zero.
U S. Er-vi-onmontal Protection Agency
r -on V, Library
?r:} soul'' Dc;:rbo'n Street
C;iica~go, Illinois 60604
A-6
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