United States
Environmental Protection
Agency
Environmental Criteria and
Assessment Office
Cincinnati OH 45268
EPA-600/8-82-001 F
September 1984
Final Report
Research and Development
Health Assessment Final
Document for Report
Carbon Tetrachloride
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EPA-600/8-82-001F
September 1984
Final Report
HEALTH ASSESSMENT DOCUMENT FOR
CARBON TETRACHLORIDE
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Research and Development
Office of Health and Environmental Assessment
Environmental Criteria and Assessment Office
Cincinnati, Ohio 45268
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NOTICE
This document has been reviewed In accordance with U.S. Environmental
Protection Agency policy and approved for publication. Mention of trade names
or commercial products does not constitute endorsement or recommendation for
use.
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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 EPA use. Originally the health assessment was developed for use
by the Office of Air Quality Planning and Standards; however, at the request
i
of the Agency Work Group on Solvents, the assessment scope was expanded to
address multimedia aspects.
|
In the development of this assessment document, the scientific literature
t
\
has been Inventoried, key studies have been evaluated, and summaries and con-
clusions have been prepared so that the chemical's toxlclty and related char-
acteristics are qualitatively Identified. Observed effect levels and dose-
response relationships are discussed evaluating the potential toxlclty of
carbon tetrachlorlde. Unit risk estimates for cancer are calculated to
provide a medlaspedf 1c measure of toxlclty. This Information can then be
placed 1n perspective with observed environmental levels.
111
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AUTHORS, CONTRIBUTORS AND REVIEWERS
The EPA Office of Health and Environmental Assessment (OHEA) 1s respon-
sible for the preparation of this health assessment document. The OHEA
Environmental Criteria and Assessment Office (ECAO/C1n) had overall respon-
sibility for preparation and production of the document (Cynthia Sonlch,
Principal Author and Project Manager). Each chapter was originally drafted
by the authors as listed below. The Health Effects Branch, CSD, ODW provid-
ed a draft of the ODW carbon tetrachloMde -document which was used as the
basis for this document.
Authors
Christopher T. DeRosa, Ph.D.
Environmental Criteria and
Assessment Office
C1nc1nnnat1, Ohio
Richard Hertzberg, Ph.D.
Environmental Criteria and
Assessment Office
Cincinnati, Ohio
Sheila Rosenthal, Ph.D.
Reproductive Effects
Assessment Group
Washington, DC
Cynthia Sonlch, M.S.
Environmental Criteria and
Assessment Office
Cincinnati, OH
Health Effects Branch
Criteria and Standards Division
Office of Drinking Water
Washington, DC
Chapters
5, 14, Appendix
10
1, 2, 3, 4, 7,
8, 9, 11, 12,
13, 14, 15
1v
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The OHEA Carcinogen Assessment Group (CAG) was responsible for reviewing
the sections on cardnogenlcHy. Participating members of the CAG are
listed below (principal reviewers for this document are designated by *.).
Roy Albert, M.D. (Chairman)*
Elizabeth L. Anderson, Ph.D.
Larry D. Anderson, Ph.D.*
Steven Bayard, Ph.D.*
David L. Bayllss, M.S.
Chao W. Chen, Ph.D.*
Margaret M.L. Chu, Ph.D.
Herman 0. G1bb, M.S., M.P.H.
Bernard H. Haberman, D.V.M., M.S.
Charallngayya B. Hlremath, Ph.D.
Robert McGaughy, Ph.D.*
Dharm V. Singh, O.V.M., Ph.D.
Todd W. Thorslund, Sc.D.
The OHEA Reproductive Effects Assessment Group (REAG) was responsible
for the authorship and review of sections on mutagenicity. Participating
members of the REAG are listed below (principal author of the mutagenicity
section is designated by *).
Vicki Vaughan-Dellarco, Ph.D.
John R. Fowle III, Ph.D.
K.S. Lavappa, Ph.D.
Sheila Rosenthal, Ph.D.*
Carol Sakai, Ph.D.
Peter Voytek, Ph.D.
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The OHEA Exposure Assessment Group (EAG) was responsible for reviewing
the sections on exposure. Participating members of the EAG are listed below.
James Falco, Ph.D.
Gregory Kew, M.S.
Thomas Mclaughlin, Ph.D.
The OHEA Environmental Criteria and Assessment Office (ECAO/RTP)
provided general peer review of the entire document. Participating members
of the ECAO/RTP are listed below.
Lester Grant, Ph.D.
S1 Duk Lee, Ph.D.
The following Individuals provided peer-review of this draft or earlier
drafts of this document:
U.S. Environmental Protection Agency
Dr. Jerry F. Stara
ECAO-Cin
U.S. Environmental Protection Agency
Dr. Michael Dourson
ECAO-C1n
U.S. Environmental Protection Agency
Mr. Steven D. Lutkenhoff
ECAO-C1n
U.S. Environmental Protection Agency
Dr. Debdas Mukerjee
ECAO-Cin
U.S. Environmental Protection Agency
Dr. Herbert L. Wiser
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
vi
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External Reviewers
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 Plscator
Karollnska Institute
Stockholm, Sweden
Dr. V.M. Sadagopa Ramanujam
IPA Science Advisor to ECAO-C1n
University of Texas Medical Branch
Galveston, Texas
Dr. James Wlthey
Food Directorate
Bureau of Food Chemistry
Tunney's Pasture
Ottawa, Canada
In addition, Initial review of the document was provided by;
Members of the Agency Work Group on Solvents
Elizabeth L. Anderson
Charles H. RIs
Jean Parker
Mark Greenberg
Cynthia Sonlch
Steven D. Lutkenhoff
James A. Stewart
Paul Price
William Lappenbush
Hugh Spitzer
David R. Patrick
Lois Jacob
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
vii
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Arnold Edelman
Josephine Brecher
Hike Rugglero
Jan Jablonski
Charles Delos
Richard Johnson
Prlsdlla Holtzclaw
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
EPA Science Advisory Board
The substance of this document was Independently peer reviewed 1n public
sessions of the Environmental Health Committee of EPA's Science Advisory
Board.
Special Acknowledgement:
Technical Services and Support Staff, ECAO-C1n
U.S. Environmental Protection Agency
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TABLE OF CONTENTS
Page
1. INTRODUCTION 1-1
2. SUMMARY AND CONCLUSIONS 2-1
2.1. SUMMARY 2-1
2.2. CONCLUSIONS 2-6
2.2.1. Major Research Needs 2-8
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 1n Air . . . . . .-., 3-6
3.2.3. Carbon Tetrachlorlde 1n 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 4-1
4!s! ENVIRONMENTAL EXPOSURE"LEVELS ! !!!!!!!'!!! M!! 4-2
4.3.1. Possible Sources and Levels of Carbon
Tetrachloride 1n Water 4-3
4.3.2. Possible Sources and Levels of Carbon
Tetrachloride 1n Air 4-6
4.3.3. Possible Sources and Levels of Carbon
Tetrachloride In Food ..... 4-9
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. A1r . . . 4-18
4.4.3. Food 4-21
4.4.4. Soil 4-21
4.5. SUMMARY 4-21
1x
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Page
5. FATE AND TRANSPORT 5_i
5.1. FATE 5_!
5.1.1. Water 5_1
5.1.2. Air ~. . 5_1
5.1.3. Soil 5-6
5.2. TRANSPORT 5-6
5.2.1. Hater 5-6
5.2.2. A1r 5-6
5.2.3. Soil 5-7
5.3. BIOACCUHULATION/BIOCONCENTRATION. 5-8
5.4. SUMMARY 5-9
6. ECOLOGICAL EFFECTS 6-1
6.1. EFFECTS ON NONTARGET ORGANISMS. 6-1
6.1.1. Aquatic Life Toxicology 6-1
6.1.2. Acute Toxicity ........ 6-2
6.1.3. Chronic Toxicity 6-5
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 6-6
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 7-14
7.5. SUMMARY 7_]4
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Page
8. TOXICOLOGY: ACUTE, SUBCHRONIC AND CHRONIC 8-1
8.1. EXPERIMENTAL ANIMALS 8-1
8.1.1. Acute 8-1
8.1.2. Subchronlc 8-17
8.1.3. Chronic 8-21
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 8-44
8.3.1. Formation of Carbonyl Chloride (Phosgene) 8-44
8.3.2. DlmeMzatlon to Hexachloroethane 8-45
8.3.3. Free Radical Binding to Proteins. 8-45
8.3.4. L1p1d Perox1dat1on 8-46
8.4. SUMMARY 8-49
8.4.1. Experimental Animal Data 8-49
8.4.2. Human Data 8-51
8.4.3. Mechanisms of Toxldty 8-52
9. TERATOGENICITY AND OTHER REPRODUCTIVE EFFECTS 9-1
9.1. TERATOGENICITY 9-1
9.2. OTHER REPRODUCTIVE EFFECTS. ........ 9-3
9.3. SUMMARY 9-8
10. MUTAGENICITY 10-1
10.1. METABOLISM AND COVALENT BINDING TO MACROMOLECULES ..... 10-1
10.1.1. Metabolism 10-1
10.1.2. Covalent Binding to Macromolecules 10-2
10.2. MUTAGENICITY STUDIES IN BACTERIAL TEST SYSTEMS 10-7
10.3. STUDIES IN EUCARYOTIC TEST SYSTEMS 10-12
10.4. OTHER STUDIES INDICATIVE OF DNA DAMAGE. . 10-15
10.5. SUGGESTED ADDITIONAL TESTING. 10-18
10.6. SUMMARY AND CONCLUSIONS . . . : 10-19
11. CARCINOGENICITY 11-1
11.1. EXPERIMENTAL ANIMALS 11-1
11.1.1. Rat Studies 11-1
11.1.2. Mouse Studies 11-9
11.1.3. Hamster Studies 11-28
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Page
11.2. HUMANS 11-31
11.2.1. Case Reports n -11
11.2.2. Studies ;.!!!!!!!! 11-34
11.3. SUMMARY n_37
11.3.1. Experimental Animals 11-37
11.3.2. Humans. 11-37
11.3.3. IARC Evaluation ; . ! ! . 11-37
11.3.4. Conclusion 11-38
12. SYNERGISM AND ANTAGONISM 12-1
12.1. SYNERGISM 12-1
12.2. ANTAGONISM ' 12-7
12.3. SUMMARY .....,....;.......!!! 12-10
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 130
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. Ingestlon 14_8
14.1.2. Inhalation. 14-9
14.1.3. Dermal Exposure 14-10
14.1.4. Mutagenldty 14-10
14.2. SENSITIVE POPULATIONS 14-10
14.3. QUALITATIVE HEALTH HAZARD ASSESSMENT. ."!!.' 14-12
14.3.1. Animal Toxlclty Studies Useful for
Hazard Assessment 14-13
14.3.2. Animal Carc1nogenc1ty Studies .......... .14-15
14.4. FACTORS INFLUENCING HEALTH HAZARD ASSESSMENT 14-16
14.4.1. Exposure 14-16
14.4.2. Estimated Threshold No-effect Levels ! 14-16
14.4.3. Cardnogenldty 14-17
15. REFERENCES 15_1
APPENDIX: UNIT RISK ESTIMATE FOR CANCER A-l
xt1
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No.
LIST OF TABLES
Title
Page
3-1 Physical and Chemical Properties of Carbon Tetrachlorlde, . . 3-2
3-2 Analytical Methods of Carbon TetrachloMde Detection
and Measurement 3-9
4-1 Summary of Atmospheric Concentrations of
Carbon Tetrachlorlde . . . 4-7
4-2 Atmospheric Concentrations of Carbon Tetrachlorlde
Over Seven U.S. Cities 4-8
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 Tetrachlorlde Uptake from Fluids (mg/yr)
Calculated by Assuming 100% Absorption 4-19
4-9 Estimated Human Uptake of Carbon Tetrachlorlde from
the Outdoor Atmosphere. . . . . 4-20
4-10 Carbon Tetrachloride Uptake from Food Supplies (mg/yr)
Calculated by Assuming 10054 Absorption 4-22
4-11 Summary of Carbon Tetrachloride Concentrations
in Food Supplies. 4-23
4-12 Relative Uptake of Carbon Tetrachlorlde from the
Environment by Adult Male 4-24
5-1 Uncertainties 1n Quantifying the Ozone Depletion Caused
by CC14 5-5
6-1 Acute Values for Carbon Tetrachlorlde .... ........ 6-3
7-1 Partition Coefficients for Carbon Tetrachlorlde ....... 7-2
7-2 Carbon Tetrachloride Distribution at Various Times 1n
Anesthetized Dogs after Administration by Stomach Tube. ... 7-7
7-3 Tissue Distribution of [14C]Carbon Tetrachloride
Inhaled by a Female Rhesus Monkey ...... ... 7-9
7-4 Conversion of [14C]Carbon Tetrachlorlde 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 Tetrachlorlde 1n Animals. . 8-2
8-2 SGPT Values of Mice Administered Carbon Tetrachlorlde
Intraperitoneally in "Up and Down" Experiment ... . . . . . 8-4
8-3 Effects of Oral Carbon Tetrachlorlde 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 Tetrachlorlde in "Up and
Down" Experiment 8-10
xiii
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Title
Page
No.
8-6 Exposure Times and Concentrations of Carbon Tetrachlorlde
Vapor In a Controlled Human Study 8-40
8-7 Incorporation of *«C from [14C]Carbon Tetrachlorlde
Into Liplds of Various Rat Tissues 8-50
9-1 Weight Changes 1n Hale Rat Reproductive Organs After
Carbon Tetrachlorlde Treatment. ... . 9.5
10-1 Genetic Effects of Carbon Tetrachlorlde on Strain 07 of
Saccharomvces Cerevlslae following 1-hour
treatment at 37°C 10-14
11-1 Lesions of the Liver 1n Rats Given Subcutaneous Carbon
Tetrachlorlde H_4
11-2 Incidence of the Host Advanced Lesions In Rats
Administered Carbon Tetrachlorlde ... 11-6
11-3 Incidence of Liver Tumors 1n Carbon Tetrachloride-
treated Rats and Colony Controls 11-8
11-4 Occurrence of Hepatomas'in Strain C3H H1ce Following
Administration of Carbon Tetrachlorlde 11-10
11-5 Incidence of Tumors 1n C3H H1ce Ingesting Carbon
Tetrachlorlde 11-12
11-6 Incidence of Tumors in Strain A Mice Ingesting
Carbon Tetrachlorlde 11-13
11-7 Tumors of the Liver in Hale and Female H1ce Receiving
Carbon Tetrachlorlde by Stomach Tube 11-15
11-8 Hepatomas in Hale and Female Strain A Hice Given CCli
via Stomach Tube. . . . 11-17
11-9 Susceptibility of Strain A Hice to Liver Necrosis and
the Incidence of Hepatomas 30 Days After 120 or 30
Doses of Carbon Tetrachlorlde 11-19
11-10 Survival of B6C3F-] H1ce Treated with Carbon Tetrachlorlde . 11-21
11-11 Incidence of Hepatocellular Carcinomas 1n Hice Treated
with Carbon Tetrachlorlde 11-22
11-12 Hepatomas in Hale and Female Hice Given Carbon Tetra-
chloride (0.4 ma 2-3x weekly) by Stomach Tube ........ 11-25
11-13 Hepatomas in Hale Hice Given Olive Oil by Stomach Tube. . . . 11-26
11-14 Studies in Which Liver Necrosis was Induced Using
Carbon Tetrachlorlde 11-32
13-1 Carbon Tetrachloride Inhalation Standards of 11 Countries . . 13-4
14-1 Dose-Related Toxic Effects of Carbon Tetrachlorlde on
Humans and Animals 14_2
14-2 Reproductive Effects of Carbon Tetrachlorlde from
Subchronic Exposure 14_5
14-3 Summary Table for Hutagenicity Studies . . . 14-6
14-4 Carcinogenic!ty Studies Useful for Risk Assessment
of Carbon Tetrachloride 14_7
14-5 Reported No-Effect Levels for Toxicity of Carbon
Tetrachlorlde 14-18
xiv
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LIST OF FISURES
No. Title page
5-1 Estimates of Steady-State Reductions 1n Total Column
Ozone for Continuous Releases of CFCs at Approximately
1975 Rates as Calculated from Different Chemical Models . . . 5-4
7-1 Pathways of Carbon Tetrachlorlde Metabolism .... 7-13
8-1 Free Radical Initiated, Autocatalytlc Peroxldatlon
of Polyenlc Long-Chain Fatty Acids 8-48
xv
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1. INTRODUCTION
Carbon tetrachlorlde (CC14), 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 H is denser than water, thus
under certain environmental conditions, such as groundwater contamination or a
large spill into a cold body of water, the contamination level may remain
relatively constant over time . Its presence in the atmosphere and in water
appears to be of anthropogenic origin. As would be expected from Us parti-
tion coefficients, it is readily absorbed through the lung and gastrointesti-
nal tract and also through the skin.
Toxicological data for nonhuman mammals are extensive and Indicate that
CC1, causes liver and kidney damage primarily but also neurological damage
4
and dermal effects. Case reports on humans document similar effects. The
carcinogenicity of CC1. has been we11-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.
This document 1s an assessment of the literature available up to March
1983 with the exception of the effect of CC14 on stratospheric ozone.
1-1
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2. SUMMARY AND CONCLUSIONS
2.1. SUMMARY
Carbon tetrachlorlde (CC1 ) 1s a relatively nonpolar compound that 1s
slightly soluble 1n water, soluble 1n alcohol and acetone, and misclble 1n
benzene, chloroform and ether. Its density Is 1.59 g/ms, at 4°C which 1s
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.
Carbon tetrachlorlde Is produced commercially from the chlorlnatlon of
several chemicals such as methane, propane, ethane propylene and carbon
dlsulflde. In 1980, 3.22xl08 kg were produced In the United States. A
much smaller amount of CC1. 1s generated during the production of vinyl
chloride and perchloroethylene. The major use of CC1. Is 1n the produc-
tion of chlorofluorocarbons. A reduction 1n the demand for CC1. has
resulted in a 3.5% decrease 1n production over the years 1970-1980. A con-
tinued 1.0% decline 1n production 1s projected each year through 1985.
Carbon tetrachlorlde can be detected in the environment using media-
specific analytical methods. Levels detected In the environment are
generally <0.01 mg/8. 1n water, <0.01 mg/m3 1n air, and <0.01 mg/kg 1n
food although higher levels have been detected 1n urban air and grain
fumigated with CC1.. Food products made from this grain also contain
residues of CC1.. Natural sources of CC1. are unknown so that most, 1f
not all CC1. present 1n the environment can be accounted for by anthropo-
genic activities.
Carbon tetrachlorlde 1s extremely stable 1n the lower atmosphere and
troposphere; however, once In the stratosphere, photod1ssoc1at1on Is
2-1
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relatively rapid. Its presence In the stratosphere Is of concern due to Us
possible contribution to ozone depletion and subsequent modification of UV-B
radiation flux. The extent of this contribution 1s difficult to estimate
for CCl^ due to several uncertainties 1n modeling and data as well as
uncertainties 1n emission projections. These estimates have become much
more stable 1n recent years, particularly because of Improved understanding
of the chemistry. Current estimates of ozone depletion due to CC1. alone
are 0.3-0.5J4 derived from discussions and data presented 1n NAS (1984) and
WHO (1982). The steady-state reduction in total column ozone 1s expected to
be 2-5% as estimated by NAS (1984). Increased UV-B radiation studies 1n the
laboratory have shown adverse effects to a variety of terrestrial plant
species; for example, 1n terms of depressed photosynthetlc activity,
reduced growth rate, Increased somatic mutation rates, and Inhibition of
seed germination. In laboratory studies, some aquatic organisms have also
been shown to be adversely affected. Extension of these laboratory findings
to the natural environment 1s being conducted including the adaptability
potential to small changes 1n UV-B flux. Indirect ecosystem effects associ-
ated with Increases in UV-B radiation flux have been identified to Include
changes 1n genetic material and possible alterations in population composi-
tion. However, the extent to which these findings apply to the natural
environment Is still difficult to determine. An Increased UV-B flux to
human populations 1s likely to lead to an increase 1n skin cancers since
~90J4 of skin cancer (other than melanoma) in the United States is attributed
to sunlight in the UV-B region.
When present in water in dilute concentrations, CC1. is rapidly lost
to the atmosphere by volatilization. However, large quantities of CC1.
spilled into water may settle and persist, due to its high specific gravity,
low water solubility and high chemical stability. In such cases its
2-2
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presence in ambient water or drinking water may present a threat to aquatic
ecosystems or human health.
Humans are potentially exposed to CC1. through various media. For an
adult male, the typical exposure 1s estimated at 13 »»g/day from Inhala-
tion, 9 yg/day from fluid Intake and 4 pg/day from food Intake. Uptake
from air appears to be the major source of exposure. For this reason, the
fate and transport of CC1. has been most extensively studied in air.
Information on CC1. soil contamination 1s limited; consequently, its
contribution to human exposure 1s uncertain.
Ecological impacts have been monitored to a limited extent in freshwater
and saltwater organisms. Only two freshwater fish and one Invertebrate
species have been acutely tested; a 96-hour LC5_ has been determined as
low as 27.3 mg/9. 1n the bluegill, Lepomis macrochirus. Chronic toxiclty
data are not available. However, reported bloconcentration factors are <30
so that tissue residues are insignificant. The only data on saltwater
species deals with toxiclty following acute exposures. Effects have been
identified at levels of 50.0 mg/a. It is noted that the toxic dose for
both freshwater and saltwater species can be lower if more sensitive species
were tested.
In mammals, CC1. is readily absorbed from the lungs and gastrointesti-
nal 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 CC1. is primarily through the
lungs but also occurs in the urine and feces. Quantitative estimates are
available only for inhalation absorption efficiency; these range from 30-65%.
2-3
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Varying degrees of toxlclty have been reported 1n humans and animals
following acute, subchronlc and chronic exposures via Ingestion, Inhalation
or dermal administration. Such effects can occur systemlcally 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 have 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
subchronic and chronic doses have 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 pro-
duces a greater effect upon the liver than exposure to a lower concentration
over a longer period of time, even though the product of time and concentra-
tion 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 CC1. of Impure
or technical grade was not reported; only the use of pure CC14 was
reported.
Adverse effects recorded for humans following CC1. exposure are
similar to those in animals. Damage to the liver, kidney, lungs and central
nervous system has been documented 1n various case reports. Biochemical
alterations have been identified in case reports and one ep1demiolog1cal
study.
Exposure to CC14 did not produce skeletal or functional abnormalities
but did result in signs of fetotoxidty. Rats exposed in utero to CC1.
4
were noted to have fatty Infiltration of the liver from days 1-4 after
birth. Carbon tetrachloride has been shown to be transferred to the neonate
2-4
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through mothers' milk. Other adverse reproductive effects Include changes
in testlcular histology eventually resulting In functional Infertility.
Carbon tetrachloride has been tested for Its mutagenic potential 1n
bacteria, yeast and a mammalian cell line, and for Its DNA damaging poten-
tial In rat hepatocytes when administered In. vivo. Six of the seven point
mutation studies 1n bacteria were negative. The remaining bacterial study
was preliminary and only suggestive of a weak mutagenic response. In none
of the negative studies was it shown that CC1. was activated or metabol-
ized by the exogenous S9 activation system used. Positive mutagenicity and
DNA damage results were found in yeast. In contrast, negative DNA damage
results were reported, using jnn vivo unscheduled DNA synthesis as the assay
endpoint. DNA binding studies by two laboratories indicate that metabol-
Ically activated CC1. may interact with DNA.
Carbon tetrachlorlde has been found to produce carcinogenic or neo-
plastic responses 1) in the liver of six strains of mice (C3H, B6C3F1, A, Y,
C and L); 2) carcinomas 1n the liver of three strains of rat (Osborne-
Mendel, Japanese, Wistar) and hyperplastic nodules 1n a fourth rat strain
(Buffalo) in less than lifetime studies; 3) a small but statistically sig-
nificant (as compared to pooled controls) hepatocellular carcinoma response
1n female Osborne-Mendel rats In lifetime experiments; and 4) hepatocellular
carcinomas in Syrian golden hamsters.
Case reports of human carcinomas developing years after a history of
high - CGI. exposures are suggestive of but not proof of an association
between human carcinogenic risk and exposure to CC1.. Studies on groups
exposed to CC1, Indicate that further research 1s necessary.
Estimates of excess human cancer risk from lifetime exposure to CC1.
have been extrapolated from the cancer studies on animals. Using all models
2-5
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and adjustments as a measure of uncertainty, the upper limit estimates of
unit risk following exposure via ingestion are in the range of 5.8xlO~8 to
3.4xlO~s for exposure to 1 pg/a in drinking water and from 2.3xlO~7
to 1.4xlO~4 for exposure to 1 pg/m3 in air. However, the Agency has
selected the linearized multistage model with the dose per body surface area
conversion across species and with the exponent k=3 in the adjustment for
partial lifetime study. Thus, the upper limit estimates of unit risk are
from S.lxlO"7 to 3.4xlO~s (geometric mean = 3.7xlO~6) for exposure to
1 pg CCl4/s. drinking water and from 1.2xlO~6 to 1.4xlO~4 (geo-
metric mean = 1.5xlO~s) for exposure to 1 pg CCl./m3 air. No single
study was entirely adequate for risk estimation. Thus, the unit risk esti-
mate is based on the geometric mean of the individual unit risk estimates
from the four studies considered. Therefore, the Agency's estimate of unit
risk for water (lifetime risk at a water concentration of 1 pg/ft.) 1s
3.7xlO~6 and for air (lifetime risk at an air concentration of 1
pg/m3) is 1.5xlO~s.
In assessing toxiclty, carcinogenic!ty or any other harmful effect, com-
pounds that react synerglstlcally or antagonistically with CC1. must be
considered. Identified synerglstlc substances Include ethanol, Kepone, PCB
and PBB. Antagonistic effects have been demonstrated with such compounds as
chloramphenlcol and catechol.
2.2. CONCLUSIONS
Carbon tetrachlorlde may contribute to the long term, partial destruc-
tion of stratospheric ozone. However, several uncertainties 1n the emis-
sions and modeling make it difficult to quantitatively estimate the extent
of ozone depletion attributable to CC1..
2-6
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Carbon tetrachloride causes damage to the liver, lungs, kidneys and
central nervous system 1n 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
relationship between the CC1. insult and the toxic response. Furthermore,
the toxicity from CCK 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 CC1. have been demonstrated in mammalian fetuses and
4
neonates exposed directly or indirectly via the placenta or mothers' milk,
respectively. Teratogenic effects have not been noted following CC1,
exposure; however, degenerative changes in the testes and subsequent infer-
tility of the offspring have occurred.
The evidence described in this document is insufficient to allow firm
conclusions to be made concerning the mutagenic potential of CC1.. The
majority of the mutagenicity studies were negative. The borderline positive
results for binding of reactive intermediates to DNA, the positive results
In yeast and the highly preliminary evidence for mutagenicity 1n the one
positive bacterial study are insufficient evidence for mutagenicity, but are
suggestive of weak genotoxic effects and are an indication that further
studies should be done.
2-7
"ffl
-------
Interactions with other chemicals must be considered in assessing the
potential health hazards of exposure to CC1A. Chemicals have been 1dent1-
T" *
fled that potentiate the effects of CC14 as well as those that Inhibit the
effects of CC14.
Cardnogenldty of CC1. has been observed 1n three animal species.
The primary lesions are hepatic neoplasms. Cirrhosis, necrosis and cholan-
gloflbrosls have also been found and have been suggested as Initial lesions
prior to tumorlgenesls 1n liver. Human data on carc1nogen1c1ty are
restricted to case reports and the preliminary results of one epldemlolog-
1cal study. If the International Agency for Research on Cancer (IARC)
criteria were used, this evidence would be classified as sufficient 1n
animals and Inadequate In humans for an overall rating of Group 28. The
IARC description for Group 2B ranking is that carbon tetrachloride is prob-
ably carcinogenic in humans.
2.2.1. Major Research Needs.
•*
Experiments in a number of species designed to derive an
absorption coefficient (inhalation) or an absorption range
(oral) are needed.
• Definitive data on CCl4-1nduced carcinogenic!ty and toxlcity
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
1n the preliminary study on dry cleaners.
• Long-term studies on animals exposed in utero are needed to
assess lifetime effects of such exposures.
Chronic studies on animals exposed to CC14 via drinking
water, air or dermal absorption are needed to establish a
dose-response relationship of CCl^s critical effects.
• The toxicity data on the rat and guinea pig are satisfactory.
Additional data on mice are needed since, at present, dose/
response information is inadequate. Studies on other species
would also be useful.
2-8
-------
Toxic effects of chronic exposure on freshwater and saltwater
organisms need to be documented.
Assessment of the overall ecological Impact 1s sparse. Infor-
mation on soil and air, particularly the stratosphere (levels,
fate and transport processes, relative source contribution),
1s needed, as well as b1oaccumulat1on/b1oconcentrat1on 1n
shellfish.
Additional research to better define the genotoxlc potential
of CC14 1s needed.
2-9
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-------
3. CHEMICAL AND PHYSICAL PROPERTIES/ANALYTICAL METHODOLOGY
3.1. CHEMICAL AND PHYSICAL PROPERTIES
Carbon tetrachlorlde (CC1 ) Is a clear, colorless, nonflammable liquid
with a characteristic odor (Wlndholz, 1976). It 1s a relatively nonpolar
compound that Is slightly soluble 1n water (0.8 g/l at 25°C) (Johns,
1976), soluble 1n alcohol and acetone, and mlsclble 1n benzene, chloroform
and ether (Weast, 1978).
Carbon tetrachlorlde 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 1n water (Johns, 1976). This decomposition
rate 1s considerably accelerated 1n the presence of metals such as Iron
(Pearson and McConnell, 1975). However, hydrolytlc decomposition as a means
of removal from water appears to be Insignificant compared to evaporation,
since the properties (Table 3-1) of CC1. favor volatilization of the
compound from water to air. Carbon tetrachlorlde has a high vapor pressure
of 115.2 mm Hg at 25°C (Oohns, 1976). The air/water partition coefficient
of CC14 at 20°C 1s 1.1 by volume and -1000 by weight (Oohns, 1976). The
rapid vaporization predicted from these properties has been confirmed by
DUlIng et al. (1975), who reported a CC1 evaporation half-life of 29
minutes from a dilute aqueous solution at 25°C.
The density of CC14 Is 1.59 g/ml at 4°C {Weast, 1978). Because Its
density 1s greater than the density of water, CC14 from large spills 1n
water may settle before being totally dispersed, emulsified or volatilized.
Volatilization 1s the major transport process for removal of CC1 from
aquatic systems. Once 1n the troposphere, CC14 remains stable; 1t
exhibits an extremely slow rate of reaction with hydroxyl radicals present
3-1
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TABLE 3-1
Physical and Chemical Properties of Carbon Tetrachlorlde
A. Structure
Cl
Cl-
-Cl
Cl
B. Synonyms
Tetrachloromethane
Methane tetrachlorlde
Perchloromethane
Benzlnoform
Necatorlna
C. Registry Numbers
CAS No. 56-23-5
TSL No. FG 4900000
D. Description
Carbon tetrachlorlde 1s a clear, colorless, nonflammable liquid with a
charcterlstlc odor (Wlndholz, 1976). It Is slightly soluble 1n water, solu-
ble 1n alcohol and acetone, and mlsdble In benzene, ether and chloroform
(Weast, 1978). •
E. Physical Properties
1. Molecular weight
2. Melting point
3. Boiling point at 760 torr
4. Density 1n water at 4°C
at 25°C
5. Vapor pressure at 25°C
6. Solubility 1n water 20°C
7. Log octanol/water parti-
tion coefficient
8. Conversion factors
at 20°C 1 atm.
153.82
-22.9°C
76.54°C
1
1
115
.594 g/ml
.589 g/ml
.2 mm Hg
785 mg/8.
2.64
1 mg/m3 air =
0.156 ppm In air
1 ppm 1n air =
6.402 mg/m3 air
Reference
Weast, 1978
Weast, 1978
Weast, 1978
Weast, 1978
Wlndholz, 1976
Weast, 1978
Pearson and
McConnell, 1975
Neely et al., 1974
Verschuren, 1977
3-2
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in the troposphere. The overall atmospheric lifetime of CC14 1s estimated
at 60-100 years. Carbon tetrachlorlde eventually diffuses Into the strato-
sphere or 1s carried back to the earth during the precipitation process.
Once 1n the stratosphere, CC1. 1s degraded on exposure to shorter wave-
length, higher energy ultraviolet light eventually forming phosgene as the
principal product (Federal Register, 1979). See Chapter 5 for an expanded
discussion of this Issue.
3.2. ANALYTICAL METHODOLOGY
3.2.1. Carbon Tetrachlorlde 1n Water.
3.2.1.1. SAMPLING — Grab samples must be collected 1n glass contain-
ers having a total volume 1n excess of 40 ma.. The sample bottles should
be filled In such a manner that no air bubbles pass through the sample as
the bottle 1s being filled. The bottle should then be sealed so that no
bubbles are entrapped In 1t. The hermetic, nonreactlve seal (such as
Teflon® tape) 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 1s known to contain free or combined
chlorine, sodium thlosulfate preservative (10 mg/40 ma, will suffice for up
to 5 mg/a. Cl ) should be added to the empty sample bottles just before
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 should be analyzed
within 14 days of collection (U.S. EPA, 1982); however, samples have been
shown to be stable up to 21 days. At 27 days, a 90% mean recovery was
measured (Bellar and Llchtenberg, 1981).
3-3
-------
3.2.1.2. ANALYSIS
3.2.1.2.1. Ambient Water ~ Carbon tetrachlorlde (and 47 other
halogenated organlcs) 1n water can be analyzed by a purge and trap method
(Method 502.1) described by the U.S. EPA Environmental Monitoring .and
Support Laboratory (U.S. EPA, 1980b). This method can be used to measure
purgeable organlcs at low concentrations. Purgeable organic compounds are
trapped on a Tenax® GC-conta1n1ng trap at 22°C using a purge gas rate of
40 m!t/m1n for 11 minutes. The trapped material 1s then heated rapidly to
180°C and backflushed with helium at a flow rate of 20-60 mst/m1n for 4
minutes Into the gas chromatographlc analytical column. The programmable
gas chromatograph used 1s capable of operating at 40°il°C. The primary
analytical column 1s stainless steel packed with 1% SP-1000 on Carbopack
B® (60/80) mesh (8 ft x 0.1 1n. l.d.) and 1s run at a flow rate of 40
m&./m1n. The temperature program sequence begins at 45°C for 3 minutes,
Increases 8°C/m1n to 220°C and Is then held constant for 15 minutes or until
all compounds have eluted. A halogen-specific detector with a sensitivity
to 0.10 yg/a and a relative standard deviation of 10% must be used. The
optional use of gas chromatography/mass spectroscopy (GC/MS) techniques of
comparable accuracy and precision 1s acceptable.
Carbon tetrachlorlde can also be detected 1n water using the headspace
gas chromatography method 1n conjunction with electron capture detection
(GC/ECD) described by D1etz and Slngley (1979). A 10 ft. by 4 mm (1.d.)
glass column was used containing 20% SP-2100/0.1% Carbowax 1500® on
100/120 mesh Supelcoport®. With this method, CC1 and other chlorinated
hydrocarbons can be detected from 0.1 pg/s. to the low mg/8. range at
ambient temperatures. The headspace method relies on the fact that when a
water sample containing organic compounds Is sealed 1n a vial, the organlcs
3-4
-------
will equilibrate between the headspace 1n the vial and the water {01etz and
Slngley, 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
Interest. Although 1t Is not clearly stated by the authors, accuracy >98%
can be obtained, and assuming a constant and additive term, precision = 0.1.
3.2.1.2.2. Municipal and Industrial Discharges — The method of
analysis Is designed to meet the requirements of the National Pollutant
Discharge Elimination System (NPDES). In this regard, 1t presupposes a high
expectation of finding the specific compounds of Interest 1n the discharge.
It can be utilized to screen samples; however, the user must develop an
Independent protocol for the verification of Identity.
The method of analysis 1s gas chromatography (GO) and high performance
liquid chromatography (HPLC) {Federal Register, 1979). HPLC has been
developed considerably 1n the past few years and can be used to achieve
separations and measurements that cannot be performed with state-of-the-art
GC. In short, the GC/HPLC method has been developed for the measurement of
solvents and other volatile materials using variations of the Bellar purge
and trap technique. Semlspeclflc detectors are used to minimize background
Interferences. A detailed description of the method Is provided In the
Federal Register (1979).
The sensitivity of this method Is usually dependent upon the level of
Interferences rather than Instrumental limitations. The limit of detection
for CC1. 1s 0.007 vg/8. and represents the sensitivity that can be
achieved In wastewaters under optimum operating conditions.
3-5
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3.2.2. Carbon Tetrachlorlde 1n Air.
3.2.2.1. SAMPLING — The most common method applied to ambient air
sampling of CC1 Involves Its adsorption onto a suitable medium such as
Tenax® GC. Recovery can then be accomplished by thermal desorptlon and
purging with helium Into a liquid nitrogen cooled nickel capillary trap
{PelUzzarl and Bunch, 1979). This method 1s specific to analysis by GC.
Sampling techniques will vary with the analytical methodology.
3.2.2.2. ANALYSIS — Many of the techniques used In analyzing CC1,
4
In water apply to air. There are four practical methods to measure air
concentrations of CC1. [National Academy of Sciences (MAS), 1978]. They
are (1) GC/ECD; (2) GC/MS; (3) long path Infrared absorption spectroscopy,
usually with preconcentratlon of whole air and then separation of the
compounds by gas chromatography (GC/IR); and (4) Infrared solar spectroscopy
(NAS, 1978).
Gas chromatography/electron capture detection 1s by far the most widely
used method (Lovelock et a!., 1973; Lillian et al., 1975; Penkett et al.,
1979). The Instrumentation needed for this method 1s readily obtained and
relatively Inexpensive, costing between $5000 and $10,000 (NAS, 1978). It
1s sturdy and easy to operate and quite sensitive for CC1., which makes It
Ideal for use on aircraft and ships (Sandalls and Hatton, 1977).
The use of GC/MS has been more recent (Grlmsrud and Rasmussen, 1975;
Barkley et al., 1980; Dmltrlev et al., 1980). Although equally as sensitive
as the GC/ECD method, GC/MS has the ability to positively Identify compounds
by their characteristic mass spectra, whereas GC/ECO must rely upon the
somewhat Imprecise method of retention times to Identify compounds {NAS,
1978). Unfortunately, GC/MS Instrumentation 1s quite expensive, costing
$70,000 or more (NAS, 1978). The method employed by Dmltrlev et al. (1980)
3-6
-------
undertakes the chromatographlc separation at room temperature for the first
5 minutes and then at a rising temperature to 150°C at 5°C/m1nute Intervals
for a total chromatography time of -30 minutes, using adsorbants such as
Tenax® or Polysorbamlde®. Each of the chromatographlc fractions can be
analyzed by MS. The authors report a sensitivity level of 1 pg/m3 with
this method.
Long path Infrared absorption spectroscopy with separation by gas
chromatography (GC/IR) has the advantage of real-time continuous measure-
ments; however, the disadvantage of poor sensitivity renders this method
less desirable (NAS, 1978). Furthermore, the method Is expensive, costing
$20,000-1100,000, and cannot be used 1n the field {NAS, 1978). This method
Is used and reported by Hanst et al. (1975) with a detailed description of
sample collection techniques.
Finally, Infrared solar spectroscopy has been described by Rasmussen,
1976 and Murcray et al., 1975 (NAS, 1978). This method uses the solar
spectrum passing through the atmosphere at large zenith angles to obtain the
necessary path length to give sufficient absorption to detect ambient halo-
carbon levels. Although not 1n real-time, this method provides continuous
data on a remote region of the atmosphere (NAS, 1978), I.e., the strato-
sphere. However, 1t 1s limited to that region alone.
3.2.3. Carbon Tetrachlorlde 1n Soil. Little research has been done to
detect CC1. In soil; however, with concern Increasing regarding leachates
from landfills and waste disposal sites, the analysis of soil samples for
organic compounds has become more Important as an Indicator of possible
groundwater contamination. A recent article by DeLeon et al. (1980)
describes a method of analysis for volatile and semlvolatlle organochlorlde
compounds.
3-7
-------
3.2.3.1. SAMPLING — In the method described by DeLeon et al. (1980),
samples were taken from vertical borings 30 feet deep using the split-spoon
method. The samples were then placed 1n jars and sealed with Teflon®-I1ned
screw caps. During shipment, they were maintained at 6-10°C. Upon their
arrival at the analysis site, they were maintained at -20°C until prepared
for analysis.
3.2.3.2. ANALYSIS — The analytical method described by DeLeon et al.
(1980) employs a simple extraction procedure using hexane followed by analy-
sis of the extract using temperature programmed GC on high-resolution glass
capillary columns with ECD, The electron capture gas chromatographlc
results are confirmed by mass spectroscopy much like the method used for
water sample analysis. Reported sensitivity 1s at least 10 vg/g. The
authors demonstrated this technique to be effective In determining the
perimeter and limits of an old chemical waste disposal site and also as an
effective method to assess the extent of leaching of chlorocarbons from the
waste disposal site Into surrounding soils (DeLeon et al., 1980).
3.3. SUMMARY
Carbon tetrachlorlde 1s a clear, colorless, nonflammable liquid with a
characteristic odor and 1s slightly soluble In water. Its high vapor
pressure favors rapid volatilization from water to air. This characteristic
1s utilized 1n most commonly accepted methods of analysis for CC1 . Gas
chromatography, either alone or coupled with mass spectroscopy, Is the most
widely used 'detection method. The compound Is usually separated from other
constHutents 1n the sample by direct volatilization, extraction or heating,
and then analyzed using GC, GC/MS or some other analytical technique. Table
3-2 summarizes the analytical methods discussed 1n this chapter.
3-8
-------
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4. PRODUCTION, USE AND ENVIRONMENTAL EXPOSURE LEVELS
4.1. PRODUCTION
Carbon tetrachloMde is produced Industrially by the chlorlnatlon of
methane, propane, ethane, propylene or carbon dlsulflde (Rams et al., 1979).
In 1980, 322 million kg were manufactured [.U.S. International Trade Commis-
sion (USITC), 1981]. Carbon tetrachloride 1s also produced as a byproduct
during the production of compounds such as vinyl chloride and perchloro-
ethylene (Rams et al., 1979).
Production levels have decreased as a result of the rapid decline 1n Us
major end product area, manufacture of fluorocarbon aerosols. In 1975
fluorocarbon aerosols had a 50% share of the domestic aerosol market;
however, this declined to 20% 1n 1977 due to the non-essential aerosol ban
(Chemical Harketlng Reporter, 1981). Fluorocarbons are expected to show
some strength as solvents, as a refrigerant and In foam-blowing applica-
tions. Growth 1n these latter areas 1s expected to counteract the decrease
1n the manufacture of fluorocarbon aerosols and, therefore, stabilize CC1.
4
production In the 1980s.
4.2. USE
Currently the major use of CC1. 1s 1n the production of chlorofluoro-
carbons, which are used as refrigerants, foam-blowing agents andvsolvents.
These uses accounted for 95% of the total U.S. consumption In. 1980. Carbon
tetrachlorlde 1s also used in fumlgants and has a variety of minor uses (5%
of total 1980 consumption), including those as ^ 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 pesticide products (U.S.
EPA, 1980a), and its registration for use in fumigants was under review by
U.S. EPA (Federal Register, 1980a). Approximately 12% of the total 1980
production was exported.
4-1
-------
4.3. ENVIRONMENTAL EXPOSURE LEVELS
Carbon tetrachloride present In the environment appears to be of anthro-
pogenic origin (Singh et al., 1976). Its presence 1n surface waters occurs
primarily as a result of Industrial and agricultural activities, although
some may reach surface water through rainfall. Groundwater contamination
may be the result of leaching from solid waste sites. This 1s also a source
of soil contamination. A1r 1s the medium wherein the greatest concentra-
tions of CC1. can be found 1n the environment with the major source being
industrial emission. Once in the air, CC1. can be washed Into surface
waters and soil through rainfall.
Once in the environment, CC1. is relatively stable. Its half-life for
hydrolytic breakdown in water at pH 1.0-7.0 is estimated to be 70,000 years,
but hydrolysis appears to be accelerated In the presence of metals such as
Iron and zinc (Johns, 1976). The high stability in water has little prac-
tical significance, however, since CC1. vaporizes readily to'air.' The
atmospheric lifetime of CC1. 1s estimated to be on the order of 30-100
years (Singh et al., 1976).
The presence of CC1. 1n 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 soil.
However, It has also been postulated that CC1. may also contribute to
ozone-destroying photochemical reactions in the stratosphere. If this were
to occur, it might cause increases In the Incidence of human skin cancers
and animal cancers, affect terrestrial and aquatic ecosystems, and bring
about climatic changes (NAS, 1978, 1982).
4-2
-------
Although levels of CC1= 1n the environment are generally 1n the low
ppb range or below (NAS, 1978), CC14 may pose a long-term risk because of
Us possible carcinogenic potential (see Chapter 11). In urban and Indus-
trial areas where higher concentrations of CC1. 1n air may occur, other
toxic effects may result (e.g., liver and renal damage).
No natural sources of CC1. have been reported. The presence of a
natural source was suggested because of the large quantity of CC1 In the
atmosphere and the homogeneity of ambient concentrations In both hemispheres
(Lovelock et al., 1973). However, this suggestion has since been chal-
lenged, since estimates of cumulative worldwide production and emissions of
CC14 appear to account for the CC14 found 1n the environment (Singh et
al., 1976).
4.3.1. Possible Sources and Levels of Carbon Tetrachlorlde in Water.
Carbon tetrachlorlde has been monitored extensively 1n drinking water and,
to a lesser extent, in natural waters. The chemical's concentration in
drinking water has been reported as <0.007 mg/i (Symons et al., 1975).
Samples of ocean, lake and ground water have generally yielded CC1.
concentrations in the ng/a, (ppt) range. There are some indications that
industrial activity may lead to increased CCl. concentrations In surface
4
and groundwater. A discussion of these monitoring studies follows,
In the National Organic* Reconnaissance Survey (NORS), U,S. EPA found
CC14 levels of <0.003 mg/i 1n drinking water 1n 80 cities (Symons et
al., 1975). The more recent National Organlcs Monitoring Survey (NOMS) of
113 public drinking water systems detected CCK 1n the range of
0.0024-0.0064 mg/a 1n 10X of the samples surveyed (U.S. EPA, 1980a).
Carbon tetrachlorlde concentrations in these samples were very low compared
to those of chloroform and other organics.
4-3
-------
Carbon tetrachlorlde has been detected 1n drinking water in Tuscaloosa,
Alabama (Bertsch et al., 1975); the District of Columbia (Schelman et al.,
1974); Durham, North Carolina (McKlnney et al., 1976); and New Orleans,
-N*
Louisiana (Dowty et al., 1975). In the District of Columbia, CC14 1n
drinking water was measured at 0.005 mg/fi. (Schelman et al., 1974). In New
Orleans, higher concentrations of CC1. were found 1n blood plasma than 1n
drinking water, suggesting to the authors the presence of a bloaccumulatlon
mechanism or sources of the compound other than drinking water (Dowty et
al., 1975). The former, however, has not been demonstrated.. Carbon tetra-
chlorlde was also found 1n drinking water 1n Germany In the ng/fi. range
(Sonneborn and Bohn, 1977).
Under unusual conditions, CC1. may be found at high levels 1n raw and
drinking water. After a chemical manufacturer accidentally spilled an
estimated 70 tons of CC1, into the Kanawha River, the U.S. EPA determined
4
that raw Ohio River water contained CC1. levels up to 0.340 mg/s.; drink-
ing water levels were found to be as high as 0.1 mg/st (Landen, 1979). As
Indicated by this incident, levels of CCl. detected in ambient water
should not be construed to be the same levels of CC1, in public drinking
water supplies. Municipal water treatment methods remove some of the
contaminant.
Carbon tetrachlorlde has been found in the ppb range or lower 1n samples
of rain, surface water, potable water and seawater (McConnell et al., 1975).
Trace levels of CC1. have been reported in snow (Su and Goldberg, 1976).
Carbon tetrachlorlde was also detected in the Atlantic Ocean at mean levels
of 60 ppt (£17 ppt). Ocean levels of CC14 were only slightly higher in
the Northern Hemisphere than in the Southern Hemisphere (Lovelock et al.,
1973).
4-4
-------
Lake Zurich and the ground water 1n an Industrial section of Zurich,
Switzerland, were also monitored for CC1.. At various depths of Lake
Zurich, concentrations of this compound of -25 ppt were measured, with no
significant variation. Groundwater levels of CC1. In the Industrial
sector were much larger. The compound was detected in 4 of 18 samples at
levels ranging between 190 and 3600 ppt (61ger et al., 1978).
Carbon tetrachlorlde can be emitted to the environment through the
production and use of the chemical, and through the production and use of
chlorofluorocarbons and other chlorinated compounds that contain CC1.
Impurities. Although small amounts of CC1- may be directly released to
water systems through these processes, most of the CC1. 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 the CC1. eventually
detected 1n water. The chemical may find Its way from air or land to
surface and groundwater systems through rainfall, runoff from agricultural
sites, dumping sites or Industrial sites, and landfill leaching.
High levels of CC1. 1n the ground waters of Zurich, Switzerland have
been attributed to Industrial processes In the Zurich area (Giger et al.,
1978). Levels of CC14 would be expected to be highest In Industrialized
areas because of both Industrial and consumer use of CC1. and Its products
1n these areas.
Carbon tetrachlorlde does not appear to be produced 1n water through
chlorlnatlon reactions, unlike other chlorinated organlcs such as chloroform
(Federal Register, 1979). Recent high levels of CC14 detected in Phila-
delphia drinking water following chlorlnatlon (<0.046 mg/a) were found to
be the result of the use of CC1.-contaminated chlorine. This incident
resulted in a meeting between U.S. EPA and the Chlorine Institute during
4-5
-------
which an Interim maximum level for CC14 In chlorine for drinking water use
was set at 0.1 mg/fi.. The use of chlorine of this purity should result 1n
CC14 levels of <0.001 mg/ii 1n drinking water (U.S. EPA, 1977).
4.3.2. Possible Sources and Levels of Carbon TetrachloMde in A1r.
Reported concentrations of CCl^ 1n continental and marine air masses 1n
1978 were very similar (0.00070-0.00084 mg/m3) (Table 4-1). As reported
by Lovelock et al. (1973), the levels of. CC14 1n the Southern Hemisphere
were only slightly lower than those in :the Northern Hemisphere. The homo-
geneity of ambient CC14 concentrations throughout the atmosphere has been
attributed to Us slow and continual release to the atmosphere for many
years (Federal Register, 1980a).
In general, CC14 levels over urban areas are only slightly higher than
background levels (see Table 4-1). However, some higher concentrations have
been reported. An average annual concentration of 0.0088 mg/m3 was
reported over Tokyo In 1974-1975 (Ohta et al., 1976). This level was the
highest measured over an extended period of time. Other high ambient
concentrations measured reflected one sample or several covering a short
time period (NAS, 197fr). The maximum concentration ever detected was 0.113
mg/m3 1n Bayonne, New Jersey (Lillian et al., 1975). A CC1. concentra-
tion of >0.0094 mg/m3 was also detected 1n the air of Grenoble, France (Su
and Goldberg, 1976). Singh et al. (1980) measured CC1, concentrations In
4
seven U.S. cities over 2-week periods (Table 4-2). The highest level
detected using GC/ECD was 0.0188 mg/m3 over Houston, Texas.
4-6
-------
TABLE 4-1
Summary of Atmospheric Concentrations of Carbon Tetrachlorlde*
Type of Measurement
Carbon Tetrachlorlde mg/m3
Continental background
Marine background
Urban range
0.00076 i 0.00008
0.00084 ± 0.00006
0.00073 ± 0.00005
0.00075 ± 0.00009
0.00081 i 0.00003
0.00081 ± 0.00010
0.00070 + 0.00007
0.00084
0.00075
0.0088
0.00075
0.00012
0.113
0.0094
*Source: NAS, 1978
4-7
-------
TABLE 4-2
Atmospheric Concentrations of Carbon Tetrachlorlde
Over Seven U.S. Cities*
CC14 (mg/m3)
City/State
Los Angeles, CA
Phoenix,
Oakland,
Houston,
St. Louis
AZ
CA
TX
, HO
Denver, CO
Riverside
, CA
Apr1
Apr.
June
May
May
June
July
Sampling Dates
1 9
23
28
14 -
29 -
15
1 -
- April 21, 1979
- May 6,
- July 10
May 25,
June 6,
- June 28
July 13,
1979
, 1979
1980
1980
, 1980
1980
0
0
0
0
0
0
0
Mean
.0014
.0018
.0011
.0026
.0008
.0011
.0011
Maximum
0
0
0
0
0
0
0
.0064
.0055
.0063
.0188
.0009
.0018
.0017
Minimum
0
0
0
0
0
0
0
.0006
.0008
.0006
.0008
.0007
.0007
.0010
*Source: Singh et al., 1980
4-8
-------
Carbon tetrachlorlde 1n the atmosphere appears to be of anthropogenic
origin, because estimates of releases of CC1 from Industrial processes
and uses appear to account for the amount present 1n the atmosphere (Singh
et al., 1976; Penkett et al., 1979). In 1978, ~2 million kg (-4.5 million
pounds) of CC1 were emitted from production facilities. The total
nationwide emissions of CC1. In 1978 from all sources were estimated at 29
4
million kg (65 million pounds). The primary source of these emissions 1s
solvent application.
4.3.3. Possible Sources and Levels of Carbon Tetrachlorlde 1n Food. In
British studies, CC1 was found 1n various foods as follows (McConnell et
al., 1975; Pearson and McConnell, 1975):
Dairy products
Meat
011s and fats
Beverages
Fruits and vegetables
Black grapes (Imported)
Fresh bread
F1sh and seafood
Concentration
(mq/kq)
0.0002-0.014
0.007-0.009
0.0007-0.018
0.0002-0.006
0.003-0.008
0.0197
0.005
0.0001-0.006
The best studied mechanism of CC1. contamination 1n food 1s Us use as
4
a fumlgant for grain. Much higher CC1. concentrations than those previ-
ously discussed have been found 1n fumigated grains. Residues as high as
115 mg/kg 1n wheat and 21 mg/kg 1n flour were detected by Ramsey (1957)
through work on methods development following fumigation with a normal
dosage of CC1 -CS . The author states that the results "are not highly
accurate" since they were obtained during work on development of methods.
4-9
-------
Higher values (up to 150 mg/kg 1n wheat following 3 hours of aspiration)
were detected by Mapes and Shrader (1957). However, 1t 1s not clear 1f the
doses administered were normal for commercial fumigation. Carbon tetra-
chlorlde levels decline dramatically (dependent on time of aeration) In
bread baked from fumigated wheat with residual levels generally reported as
<10 mg/kg (U.S. EPA, 1980a; Jaglelski et al., 1978). However, Berck (1974)
reported that bread made from wheat aerated for 3 days had residues -0.04
mg/kg 1n the upper and lower crusts and 0.13 mg/kg in the crumbs. In bread
made from wheat aerated for 7 weeks, the upper crust had no residue, the
lower crust had 0.2 mg/kg, and the crumbs had 0.01 mg/kg. The studies of
CCl^ levels 1n fumigated grains and grain products are discussed below.
Wheat, corn and milo fumigated and stored for 1-6 months were shown to
contain CC14 at 2.9-20.4 mg/kg. The levels detected appeared to be depen-
dent upon length of storage and concentration of fumigants (McMahon, 1971).
In a monitoring program for grain imported Into the Netherlands, CC1 was
4
detected 1n half of the cereals sampled. Of these samples, 3% contained
>5 mg/kg CC14 (W1t, 1972). The U.S. EPA Pesticide Laboratory detected
0.005-2.61 mg/kg CC14 1n flour from 11 U.S. cities, with an average level
of 0.051 mg/kg (Federal Register, 1980a). Carbon tetrachlortde levels of
0.0002-0.0003 mg/kg were detected 1n flour 1n another study. However, bread
and biscuits made from this flour contained undetectable CC1 of <5 mg/kg
(Bondl and Alumot, 1972).
In several experiments, researchers have simulated commercial fumigation
conditions to determine residual levels of CC1 1n foods. Fumigated
wheat, aerated for several weeks, was found to contain 20-62 mg/kg CC1..
4-10
-------
Flour made from this wheat was found to contain 2-10 mg/kg CC14, whereas
white bread made from the flour contained <0.007 mg/kg (Wit, 1972), In
another study, CC1 at 200-400 mg/kg was detected 1n wheat and corn after
application of a fumlgant (Scudamore and Heuser, 1973). Residual
decreased to 1-10 mg/kg 6 months after fumigation. By 12 months after
fumigation, the wheat and corn contained a maximum of 4.7 mg/kg CC1 .
Wheat and barley were analyzed for CC14 1n a study by Bleloral and Alumot
(1966). The Initial CC14 concentrations of 1.53 mg/kg 1n wheat and 2.2
mg/kg 1n barley decreased to 0.7 and 0.6 mg/kg, respectively, by day 17.
Carbon tetrachlorlde was detected 1n levels of 76-115 mg/kg In wheat,
10-21 mg/kg 1n flour, 28-39 mg/kg 1n oats and 43-88 mg/kg 1n bran that had
been fumigated with recommended fumlgant dosages (Lynn and Vorches, 1957).
In another study, CC1 levels were determined 1n fumigated wheat and wheat
fractions and 1n bread prepared from the wheat (Berck, 1974). Levels 1n
wheat ranged between 3.2 mg/kg (7 weeks of aeration) and 72.6 mg/kg (1 week
of aeration). Flour, bran and middlings contained 0.2-0.93, 0.43-3.53 and
0.2-1.65 mg/kg, respectively. Bread contained <0.13 mg/kg CC14. In a
similar study, flour treated at normal fumlgant levels was found to contain
CC1 at levels of 0.6-1.6 mg/kg; levels 1n bran were 2.9-5.3 mg/kg
4
(Jag1elsk1 et al., 1978). Bread baked from the flour contained <0.01 mg/kg
CC1 . Results of these studies Indicated that the amount of CC1.
4 •»
remaining as a residue 1s dependent upon the fumlgant dosage, storage condi-
tions, length of aeration and extent of processing.
In addition to Us use as a grain fumlgant, CC14 may find Us way Into
other foods through the use of herbicides, Insecticides and fungicides con-
taining CC1 as a contaminant (0.18-0.4%) 1n the pesticide formulation.
Food may also become contaminated by CC14 In the air (Federal Register,
1980a).
4-11
-------
4.3.4. Possible Sources and Levels of Carbon Tetrachlorlde 1n Soil.
Carbon tetrachloride can occur 1n soil due to spills, runoff and leaching.
As 1n groundwater contamination, CC14 may find Us way Into the soil by
runoff from agricultural, dumping and Industrial sites and through landfill
leaching.
Wastewater treatment of night soil 1n Japan also resulted 1n CC1
4
formation. Methanol (HeOH)-water-solutlon substance was fractionated along
with humlc, fulvlc and hymatomelanlc adds from the effluent of the night
soil treatment plant by the use of Amber lite XAD-2. Each of these four
fractions was chlorinated at pH 7.0. Although all fractions primarily pro-
duced CHC13, CC14 was formed by chloMnatlon of the MeOH-water-solut1on
substance (Ishlkawa et al., 1978).
4.4. RELATIVE SOURCE CONTRIBUTIONS
The widespread distribution of CC14 1n the environment can lead to
exposure to the chemical through water, food and air. Quantities of CC1
potentially taken Into the body as a result of exposure to CC1 1n air,
water and food were estimated by the National Academy of Sciences (1978).
Occupational exposure to CC14 was not considered 1n these estimates. The
NAS computations were based upon data for fluid consumption and respiratory
volume for reference Individuals and worldwide per capita food consumption
as compiled by the International .Commission for Radiological Protection
(ICRP, 1975). These data are shown 1n Tables 4-3 through 4-7,
Data on measured fluid Intakes for adults and Individuals are presented
1n Table 4-3, and calculated Intakes of milk, water and other fluids for a
i
typical (reference) man, woman, 10-year-old child and 1-year-old Infant are
given 1n Table 4-4. Respiratory volumes for these reference Individuals are
reported in Table 4-5. Food consumption per capita Is reported by geograph-
ical regions 1n Table 4-6; the worldwide profile 1s presented 1n Table 4-7.
4-12
-------
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4-13
-------
TABLE 4-4
Fluid Intake for Reference Individuals*
M1lk
Tap water
Other
Total
fluid
Adult
{ ma/day)
300
150
1500
1950
Man
(a/yr)
109.5
54.8
547.5
711.8
Adult
(mil/day)
200
100
1100
1400
Woman
U/yr)
73.0
36.5
401.5
511.0
Child.
(ml/day)
450
200
750
1400
10 yr
U/yr)
164.3
73.0
273.8
511.0
*Source: Adapted from ICRP, 1975
4-14
-------
TABLE 4-5
Respiratory Volumes for Reference Individual*
(1n liters of air breathed)
8 hours working,
light activity
8 hours nonoccupatlonal
8 hours resting
Total per day
Total per year
Adult
Man
9600
9600
3600
2.3 x 10«
8.4 x 106
Adult
Woman
9100
9100
2900
2.1 x 10"
7.7 x 10s
Child,
10 years
6240
6240
2300
1.5 x 104
5.5 x 106
Infant,
1 year
2500 (10-hr)
1300 (14-hr)
0.38 x 104
1.4 x 10«
*Source: Adapted from ICRP, 1975
4-15
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4-16
-------
TABLE 4-7
Summary of Per Capita Estimates of World Food Consumption3
Worldwide
Food Groups
Cereals'3
Starchy roots0
Sugard
Pulses and nuts6
Vegetables and fruits*1
Meatsg
Eggsn
Fish1
M1lk3
Fats and olls^
Minimum
(9/day)
185
44
22
11
128
24
3
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
Source: Adapted from ICRP, 1975
Flour and milled
Includes sweet potatoes, cassava and other edible roots
Includes raw sugar; excludes syrups and honey
e
Includes cocoa beans
Fresh equivalent
gincludes offal, poultry and game expressed as carcass weight, excluding
slaughter fats
Fresh egg equivalent
Landed weight
Excludes butter; Includes milk products as fresh milk equivalent
j.
Pure fat content
4-17
-------
The data 1n these tables summarize human fluid, respiratory and food Intake.
NAS combined these Intake data with Information on CC1 contamination of
the
water, air and food to estimate human exposure to CC1.. A summary of
NAS computations of CC1 Intake follows.
4.4.1. Water. The potential CC14 uptake from fluids is shown in Table
4-8. NAS used in its calculations the levels of CC1. reported 1n drinking
water in the NORS study (Symons et al., 1975). These levels were <0.003
mg/i. In a later report (the NOUS) CC1. was detected at levels of
4
0.0024-0.0064 mg/8, in U.S. drinking water (U.S. EPA, 1980a). Estimation
of CC14 consumption derived by using the NOMS maximum value of 0.0064
mg/s, were included in the data in Table 4-8. These data were calculated
by multiplying the minimum and maximum concentrations In drinking water by
the minimum, maximum and "reference" consumption of drinking water. These
calculations were based upon the following assumptions:
1. 100% of the CC14 ingested is absorbed.
2. Commercially-produced drinks and drinks reconstituted in the
home contain the same level of CC14 as does drinking water.
3. All fluids contain the indicated CC14 concentration.
Values for total fluid uptake include drinks such as milk, not prepared by
mixing with water.
4.4.2. A1r. The potential CC1 uptake from air is presented 1n Table
4-9. The minimum level for CC14 1n air used 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 CC14 inhaled per year. In the NAS report, this quantity was then
4-18
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multiplied by the minimum and maximum values of the absorption range for
CC1 1n air as reported for humans by Lehmann and Schm1dt-Kehl (1936).
These calculations resulted 1n values for the minimum and maximum yearly
absorption of CC1, from air. The Issue of percent absorption of CC1
4 4
has not been clearly defined. As discussed In Section 7.1., the percent
absorbed may vary between species. The range reported by Lehmann and
Schm1dt-Kehl and used In the MAS report, 1s retained here since 1t 1s the
only human data available. See Section 7.1. for a more detailed discussion
on absorption of CC1 .
4.4.3. Food. The potential CC1. uptake from foods 1s presented 1n
Table 4-10. Values of CC1, found 1n foods and used 1n these calculations
4
arc presented 1n Table 4-11. To arrive at uptake values, the minimum and
maximum levels of CC1 1n foods were multiplied by the minimum and maximum
worldwide food Intake for Individuals for each food category. NAS calcu-
lated a per capita uptake of 0.21-7.33 mg CC1. from food each year.
The NAS absorption calculations did not Include the yearly uptake
through the consumption of bread. The average consumption of CC1
attributed to bread has been calculated elsewhere as 777 ng/day or 0.3 mg/yr
(Federal Register, 1980a). The data on consumption 1n bread are Included 1n
the summary of CC1 uptake from all sources (Table 4-12).
4.4.4. Soil. The potential uptake of CC1. from soil 1s unknown. This
Includes agricultural runoff as well as uptake from plants.
4.5. SUMMARY
Carbon tetrachlorlde 1s produced commercially from the chlorlnatlon of
methane, propane, ethane propylene and carbon dlsulflde. Production has
declined over the last 10 years and a 1.0%/year decline Is projected through
1985.
4
4-21
-------
TABLE 4-10
Carbon Tetrachlorlde Uptake from Food Supplies (mg/yr),
Calculated by Assuming 100% Absorpt1ona»b
Food Groupc
H1lk products
Eggs
Heats
Fats and oils
Vegetables and
fruits
F1sh and seafood .
Total, all food
supplies
Worldwide
Food Intake^
Minimum
Maximum
Minimum
Maximum
Minimum
Maximum
Minimum
Maximum
Minimum
Maximum
Minimum
Maximum
Minimum
Average
Maximum
Exposure
Minimum Average
0.004
0.06
0.001
0.01
0.06
0.80
0.002
0.01
0.14
0.56
<0.001
0.001
0.21
1.12
Maximum
0.26
4.34
— —
0.08
1.02
0.06
0.37
0.37
1.51
0.03
0.08
7.33
aSource: Adapted from NAS, 1978
bCalculated 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).
cAs used 1n Tables 4-6 and 4-7.
dFrom Table 4-7.
4-22
-------
TABLE 4-11
Summary of Carbon Tetrachlorlde Concentrations 1n Food Supplies*
{ppb by weight, vg/kg)
Food Group
M1lk products
Eggs
Meats
Fats and oils
Vegetables and fruits
Fish and seafood
Carbon Tetrachlorlde
Minimum Average
0.2
0.5
7.0 __
0.7
3.0
0.1
Maximum
14.0
, —
9.0
18.0
8.0
6.0
*Source: Adapted from McConnell et a!., 1975; Pearson and McConnell, 1975
4-23
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4-24
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Carbon tetrachloride is ubiquitous in the environment and has been
detected 1n concentrations of generally <0.01 mg/a, 1n water, <0.01 mg/m3
1n air and <0.01 mg/kg 1n food. Higher levels of CC1, have been detected
4
1n urban air, and 1n grain and food products made from grain. Increased
levels of CC14 1n urban air are probably due to the Industrial use of
CC14 and products containing CC14. The presence of CC1. 1n grains or
grain products is due in part to the use of fumigants containing CC1..
Data on the uptake of CC14 by the adult male are summarized in Table
4-12. At minimum exposure levels, uptake from the ,a1r 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 CC1 uptake is estimated to be from air. Amount of uptake
from soil is unknown.
All of the CC1 in the environment can be accounted for by anthropo-
genic activities. Carbon tetrachloride, unlike chloroform and other halo-
carbons, does not appear to be, indirectly formed during water chlorination.
No natural sources of CC1. are known.
4
4-25
-------
-------
5. FATE AND TRANSPORT
5.1. FATE
5.1.1. Mater. Carbon tetrachlorlde tends to evaporate from dilute aque-
ous solutions, with a half-life of only 29 minutes (Billing et a!., 1975).
Chemical stability 1n aquatic environments 1s high, with half-lives of
7xl03/C years, where C Is concentration In mg/s,. This formula is
derived from the second order rate constant of 4.8xlO~7/mol~1/sec~1
(Mabey and Mill, 1978). Singh et al. (1976) give an approximate hydrolytlc
half-life for CC1 1n oceans as 7xl04 years. This figure 1s lower than
that predicted using measured ocean concentrations (Pearson and McConnell,
1975) and the above second order formula. For the maximum Atlantic Ocean
concentration of 2.4xlO~6 mg/fi,, the half-life Is 2.9xl06 years. In
any case, CC1. 1s extremely stable In water, with losses primarily due to
other factors such as evaporation, sediment adsorption and organism uptake.
Singh et al. (1976) estimate that 2-3% of all blospherlc CC1. 1s 1n solu-
tion. Although CCl^ 1s not easily transported to groundwater due to Its
high volatility, low solubility and low mobility 1n soil, any contamination
1s likely to persist for many years and accumulate.
5.1.2. A1r. Chemical stability, a long lifetime, and uniform mixing
characterize the fate of CCl^ In air. Mixing 1s assumed to be uniform to
approximately 18 km elevation, with a pseudo-first order removal rate of
IxlO"3 yr"1, mainly via gas-phase reactions Involving the electronic
state Ot'-D) (Galbally, 1976). The principal sink 1s considered to be~ the
stratosphere (Galbally, 1976; Singh et al., 1976), with significant loss of
CC1 limited by the rate of transport from the troposphere. Above 18 km,
stratospheric photolysis 1s dominant. This photodlssodatlon 1s attributed
to UV radiation mainly 1n the 195-225 nm region.
5-1
-------
Simulated tropospheric Irradiation produces no discernible dissociation
of CC1 1n NO -air mixtures. In a laboratory experiment designed to
detect stratospheric reactivity of stable halocarbons, Lilian et al. (1957)
determined the minimum stratospheric half-life of CCla to be 37 hours.;
4
This corresponds to a lifetime (time to reach 1/e of Initial concentration)
of 53 hours. Golombeck (1982) estimated a stratospheric lifetime of 7.5
years using a global three-dimensional model. Using the same model, the
atmospheric lifetime was estimated at -50 years. Estimates of atmospheric
lifetime are also quite variable ranging from 18 years (Krey et al., 1976)
to "several decades" (Galbally, 1976) and 60-100 years (Singh et al., 1976).
Carbon tetrachloride 1n the stratosphere is considered to be a potential
source of chlorine via photodissociation which may, on a molecule-by-mole-
cule basis, have the potential to catalyze the destruction of the ozone
layer approximately as strong as CFC-11 and CFC-12 (Hanst, 1978; MAS, 1978).
Following a relatively rapid tropospheric mixing with other halogenated
methanes, there is a relatively slow entry of CC1 into the stratosphere,
followed by a random ascent to altitudes (>25 km) where solar ultraviolet
(UV) radiation in the range 185-225 nm photodissociates CC1. resulting in
the generation of "odd chlorine" (I.e., a variety of chlorine-containing
species) (NAS, 1978). Dependent upon the rates of a variety of chemical
reactions and the rate of odd chlorine to the troposphere, each odd chlorine
is responsible for the destruction of several thousand ozone molecules. One
projection is that CC1 , along with hydrochloric acid (HC1) and methyl
chloride (CH^Cl), together contribute to a reduction of less than 1% of
o
stratospheric ozone, comparable to but less than 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
O
5-2
-------
processes, respectively (NAS, 1978). Based on the data, assumed release
rates and depletion models discussed 1n NAS (1984) and Hudson et al. (1982)
the steady-state decrease 1n the ozone layer due to CC1. alone can be
estimated at 0.3-0.5%.
The future Impact of CC1. emissions on stratospheric ozone 1s present-
ly difficult to predict, as 1s the collective Impact of all chlorocarbons
(CLC) on stratospheric ozone. Significant advances 1n the chemistry and
modeling aspects (Sugdon and West, 1980) during the past decade concerning
pertinent atmospheric processes, reviewed by NAS (1984), have led to much
Improved modeling estimates of potential decrements 1n total column
atmospheric ozone Illustrated 1n Figure 5-1.
Uncertainties 1n several areas remain to be resolved. Varying assump-
tions (Table 5-1) can be made regarding Important parameters that determine
rates of atmospheric conversions of CC14 and other chlorocarbons, and
regarding their expected furture global emission rates. Anticipated changes
1n atmospheric levels of other compounds (e.g., methane, CO, NO , OH)
could result 1n increases or decreases in the ozone layer. Thus several
different scenarios are possible for future Impacts of CC1. and other
chlorocarbons on stratospheric ozone (see Section 6.3.1. and NAS, 1984). In
particular, the roles of other chemical species such as OL, N?0 and
H20 and their combined effects with chlorinated hydrocarbons are not well
understood (NAS, 1982; Wuebbles and Chang, 1981). It is believed that the
combined effects are not additive, so specific cases would need to be inves-
tigated in order to make a precise prediction of future ozone reduction.
Second, the various chemical cycles Involved seem to be quite dependent on
altitude. Thus, although several of the specific rate constants for the
5-3
-------
251-
20
•S 15
1
u
10
til
I
1974 1975 1976 1977 1978
f
IMPROVED Clx
CHEMISTRY
f
NO + HO2.
DIURNAL
Avg'g
1979
*
1980 1981 1982
i
TEMPERATURE
FEEDBACK
MOLINA + SLOW OH + H0_, HO, + O
ROWLAND CIONO2 HOC(
f
OH + HNO3
FAST HO2. NO2
2 Revised HOX
1983
f
FAST
CIO + NO2
SLOW
CIO + O
REDUCED
O, + kv
FIGURE 5-1
Estimates of steady-state reductions 1n total column ozone for continu-
ous releases of CFCs at approximately 1975 rates as calculated from differ-
ent chemical models. Changes 1n the models and simulation techniques are
Indicated 1n chronological order.
Source: NAS, 1984
5-4
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5-5
-------
chlorine and nitrogen reactions have much Improved estimates, the net result
regarding eventual ozone reduction though understood, has several uncer-
tainties.
5.1.3. Soil. Pertinent Information concerning the degradation or trans-
formation of CC14 1n soil could not be located 1n the available litera-'
ture. Galbally (1976) calculates that CC14 in all land biota 1s probably
-------
in anthropogenic activity under the two trajectories. Other reports show
minor global variation in concentration of CC1. (Singh et al., 1976;
Lovelock et al., 1973). Vertical transport from the lower atmosphere to the
stratosphere accounts for -25% of the atmospheric losses of CC1., assuming
an overall atmospheric lifetime of 75 years and a quasi-steady state loading
(Singh et al., 1976). Neely (1977) modeled the transport of CC14 verti-
cally from the ocean through the troposphere to the stratosphere accounts
for roughly 25% of the atmospheric losses of CC1-, and horizontally
between northern and southern hemispheres. Transfer 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 CC1, onto sediments. Using the approach of Briggs (1973), the sorptlon
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 CC14, log P = 2.64, and thus
Q = 100.3. This value suggests that CC14 has "low" mobility in soil, as
defined by Briggs (1973), indicating that it can move with soil, sediment or
particulate matter. Morris and Johnson (1976) state that periods of high
agricultural runoff (river turbidity) are directly correlated with high
chloroform concentration in finished water and Imply that similar associa-
tions hold for CC1. and haloforms in general. Statistical analysis of
their data, however, demonstrates poor linear correlation (r = 0.06) between
river turbidity and CC1, levels, with strong peaks occurring during
periods of both low and high turbidity. Pearson and HcConnell (1975)
5-7
-------
Investigated chlorinated hydrocarbons in marine environments, but show no
significant relationships between levels in sediment and levels 1n overlying
seawater, sediment particle size or geographic features. More research 1s
clearly needed on transport via rainfall, directly from the atmosphere and
Indirectly through agricultural runoff, in order to assess the importance of
soil for CCK.
5.3. BIOACCUHULATION/BIOCONCENTRATION
The data of Kopperman et al. (1976) suggest that polar organochlorine
compounds are easily biodegraded, while nonpolar, highly llpophilic com-
pounds accumulate. Bioaccumulatlon 1s 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 CC1. Is 2.64 (Leo et al.,
1971; Neely et al., 1974; Chlou et al., 1977), indicating a possible
tendency for this compound to bioaccumulate under conditions of constant
exposure. However, although CC1. and other organochlorlnes are llpophilic
and tend to concentrate In fatty tissues, there is no evidence that they
magnify through the food chain (Pearson and HcConnell, 1975). Difficulties
1n the Pearson and HcConnell (1975) study discourage estimates of bloaccumu-
latlon based on their results. Barrows et al. (1980) report a CC1. bio-
concentration factor (BCF = tissue concentration divided by water concentra-
tion) of 30 for bluegill sunfish, Lepomis macrochirus. and a tissue half-
life of <1 day. The authors state that the short tissue half-life makes H
unlikely for CC14 to biomagnify in fish unless exposure is continuous or
prolonged. Neely et al. (1974) estimate the steady state BCF for rainbow
trout. Salmo gairdnerl. to be 17. Data could not be found for bioconcentra-
tlon of CC14 1n shellfish.
5-8
-------
5.4. SUMMARY
Transport and fate of CCK have been studied most extensively 1n air,
with some Information on water levels and almost no data on soil. Carbon
tetrachloMde 1s extremely stable 1n water, the lower atmosphere and the
troposphere. Photod1ssodat1on In the stratosphere 1s rapid 1n comparison
to the atmospheric lifetime. Global distribution of CC14 in air is nearly
uniform. Bioconcentratlon in fish is low. Research needs are most pro-
nounced for fate in soil, transport to and fate In the stratosphere, and
bioaccumulatlon/bioconcentration in shellfish.
5-9
-------
-------
6. ECOLOGICAL EFFECTS
Data on the ecological effects of CC1, are somewhat limited. The
reasons for this can be Inferred from the physical and chemical properties
of the compound as discussed 1n Chapter 3. Carbon tetrachlorlde 1s quite
volatile and so does not readily accumulate In either terrestrial or aquatic
environments. It 1s also rapidly diluted to low concentrations In the
troposphere. While major spills of CC14 have occurred, ecosystem effects
have not been documented or considered to be significant enough as to merit
further Investigation (MAS, 1978). Apparent acute effects have been minimal
and, consequently, chronic effects on fish and wildlife associated with
long-term exposures at low levels are unlikely.
6.1. EFFECTS ON NONTARGET ORGANISMS
Because CC1, 1s used as a pesticide, primarily as a grain and soil
4
fumlgant, nontarget soil organisms are undoubtedly affected (NAS, 1978).
The Interaction of CC1. with anaerobic organisms was studied by Wood et
al. (1968) using extracts of Hethanobaclllus omellanskir and the Ns-methyl
tetrahydrofolate-homocystelne transmethylase of Escher1ch1a coll B. Low
concentrations of CC1, were found to Inhibit cobamlde-dependent methyl
4
transfer reactions In these cell extracts. The possibility that
might Inhibit methyl transfer systems of microblal organisms In nature
should be Investigated, although there Is no Imminent hazard {NAS, 1978).
6.1.1. Aquatic Life Toxicology. The majority of the acute toxlclty data
for CC1 and aquatic organisms has been determined using static procedures
with unmeasured test concentrations. Results of these tests may under-
estimate the acute toxlclty of CC1. due to Its volatility. No acute or
chronic effects were observed at a concentration lower than 3400
(U.S. EPA, 1978).
6-1
-------
6.1.2. Acute Toxlclty. As shown 1n Table 6-1. the 48-hour LC50/EC50
for Daphnla maqna Is 35,200 Wg/Ji. In two separate studies, Dawson et
al. (1977) determined a 96-hour LC /EC value for the blueglll of
DU DU
125,000 pg/8,, and the U.S. EPA (1978) calculated the value to be 27,300
vg/Sl. The reason for this large difference 1s not clear but may have
been caused by the volatility of this compound. There appears to be no
great difference 1n sensitivity between the two tested species. A flow-
through test result for the fathead minnow Is 43,100 vg/a,. However, no
comment can be made concerning the effect of test conditions on the test
results.
14C-labeled CCl^ (1 ml/kg, 1.p.) produced a 5- to 10-fold Increase
1n serum GOT, GPT, and ICD enzyme activities 1n rainbow trout, Salmo galrd-
nerl., whereas exposure of trout to CC14 1n tank water (1-80 rng/n)
produced neither mortality nor changes In serum enzyme activities. Tissue
residue analysis revealed the highest concentration of CC14 1s found 1n
the adipose tissue followed by levels In the liver, brain, spleen and gills
regardless of the administration route. Elimination rates of **C residues
#
occurred at 2 hours for both 1.p. and ambient water exposure routes totaling
4.8 and 0.75 ymol/g, respectively. H1stolog1c examination of tissues
revealed varying degrees of liver and splenic neurosis 6 hours following
administration of CC1 (Statham et al., 1978).
Pfelfer and Weber (1981) treated rainbow trout with a single 1.p.
Injection of CC14. Initially, fish were treated with 2.0 mil/kg CC1 .
At 3, 6, 12, 18, 24 and 36 hours post-treatment, blood samples were obtained
and analyzed for total protein concentration. Additionally, the fish were
treated with 0.25, 0.50, 1.0 or 2.0 ml/kg CC1 following which total
6-2
-------
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6-3
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plasma protein and albumin concentrations were measured at 24 hours post-
treatment. The single Injection of 2.0 ml/kg CC1 resulted In a signif-
icant decrease In total plasma protein concentration of rainbow trout at 12,
24 and 36 hours post-treatment. Plasma albumin was also reduced signifi-
cantly at the 2.0 mfc/kg dose level 24 hours post-treatment. The authors
suggested that several factors, Including Intestinal Inflammation and
hemorrhage, may have contributed to the observed effects on plasma protein
concentration.
In an earlier study using the same species, Pfelfer and Weber (1980)
determined that a single 1.p. dose of CC1 (2.0 ml/kg) produced either
oUguMa or anurla as early as 1 hour post-treatment. A significant reduc-
tion 1n urine flow as well as a significant Increase 1n relative wet body
weight were noted. H1stolog1cal examination of kidney tissue at 24 hours
post-treatment revealed early localized pathological changes, although
extensive morphological damage was not evident. This, 1n conjunction with
the early ollguria, suggests that the observed reduction 1n urine flow rate
was not due to a direct toxic effect on the kidney.
Morphological changes In the liver of two species of carp, Cyprlno
carplo and Carasslus auratus. were observed following 1.p. Injection of
0.3-5.0 mi/kg CC14 for at least 8 days (Jiang and Zhang, 1979). The
following changes were noted 1n the liver tissue of both species: 1) pro-
liferation In number of vacuoles, 2) nuclei enlargement 1n some cells, and
3) contraction and deformation of the nuclear membrane. These findings are
1n accord with those of studies discussed previously that reported decreased
plasma protein concentrations and, consequently, reflect the hepatotoxldty
of CC1 .
6-4
-------
Only two saltwater fish and no Invertebrate species have been tested
(Dawson et al., 1977), The 96-hour LC50/EC50 for the tidewater silver-
sides, Menidia bervllina. 1s 150,000 ng/8. (see Table 6-1). The other
datum 1s an estimated 96-hour LC ' for the dab, Umanda Umanda. of about
50,000
6.1.3. Chronic Tox1c1ty. No chronic test has been conducted with a
freshwater Invertebrate species or any saltwater species. An embryo-larval
test with the fathead minnow, Plmephales promelas. resulted In no observable
adverse effect at CC14 concentrations up to 3400 yg/Sl (U.S. EPA, 1978).
6.2. TISSUE RESIDUES
The bluegill, Lepoml s macrochlrus, bloconcentrated CCl^ at equilibrium
to a factor of 30 times within 21 days (U.S. EPA, 1978). The biological
half-life In these tissues was <1 day. In addition, Neely et al. (1974)
exposed the rainbow trout to CC1, and estimated a steady-state bioconcen-
tratlon factor (BCF) of 17. Similarly, Barrows et al. (1978) determined a
BCF of 30 for blueglll sunflsh. These results Indicate that tissue residues
of CC1. would not pose a potential environmental hazard to aquatic life.
6.3. INDIRECT ECOSYSTEH EFFECTS
Indirect ecosystem effects of CC14 (e.g., those caused by enhanced
UV-B radiation) are similar to those of other halocarbons and result from
photodissoclation of the compound and subsequent destruction of strato-
spheric ozone (NAS, 1978, 1982). For more discussion of these effects, see
Chapters 5 and 11.
V
6.3.1. Effect on Stratospheric Ozone. Carbon tetrachloride may contrib-
ute to an overall reduction in stratospheric ozone. Estimates of the actual
amount of ozone depletion, even at steady-state, are surrounded by uncer-
tainties. Several chemicals and conditions contribute to overall ozone
6-5
-------
reduction, and the combined influences are thought to be something other
than strictly additive (NAS, 1982), making prediction of the effects
difficult.
6.3.2. Effect on UV Flux. Changes in stratospheric ozone content result
1n alterations of UV flux to the earth's surface. This involves the bio-
logically damaging wavelengths in the 290 to 320 nm range (UV-B regions).
The molecular basis of the effects o,f these wavelengths is the 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 may not be totally effec-
tive at all UV intensities.
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 nonagricultural species
(cited by NAS, 1978) demonstrate a diverse response to enriched levels of UV
radiation. These findings include inhibition of seed germination and
increased somatic mutation rates, in addition to depressed photosynthetic
activity. Although effects on natural ecosystems are difficult to study and
predict, the ecological impact of such effects may be significant to natural
communities taken collectively, even 1f only a few constituent species are
affected. 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) 1n assessing ecosystem effects.
6.4. SUMMARY
Only two freshwater fish and one Invertebrate species have been acutely
tested resulting in a 96-hour LC determined to be as low as 27,300
yg/i. No definitive chronic data are available. Tissue residues of
6-6
-------
CC1. do not appear to be a problem since available data suggest a BCF
of <30.
The cited data for CC14 indicate that acute toxicity to freshwater
aquatic life occurs at concentrations as low as 35,200 pg/l and would
occur at lower concentrations among species that are more sensitive than
those tested. No data are available concerning the chronic toxicity of
CC1, to sensitive freshwater aquatic life.
4
The data also indicate that acute toxicity to saltwater aquatic life
occurs at concentrations as low as 50,000 vg/i and would occur at lower
concentrations among more sensitive species. As for freshwater aquatic
life, no data are available concerning chronic toxicity to sensitive species.
Indirect effects of CC14 are associated with reduced levels of atmo-
spheric ozone and concomitant increases of UV-B radiation flux. There are
known differences in species sensitivities to increases in UV-B radiation.
Laboratory studies have identified effects associated with such increases.
At this time, the determination of the magnitude of effects of enhanced UV-B
radiation collectively or for individual species in the natural environment
is being studied.
6-7
-------
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7. COMPOUND DISPOSITION AND RELEVANT PHARMACOKINETICS
This chapter 1s divided Into four sections: absorption, distribution,
metabolism and excretion of CC1.. The absorption section begins with a
4
discussion of the chemical's partition coefficients. As would be expected
for a compound with high partition coefficients for o1l/a1r and oil/water,
CC1 1s reported to be absorbed readily through the lungs and also through
the Intestinal tract and skin. Once absorbed, the chemical and Us metabo-
lites are reported to be distributed widely throughout the body, with high
concentrations In liver, bone marrow, blood, muscle, fat and brain. Several
metabolites of CC1. have been Identified, Including chloroform, hexa-
chloroethane and carbon dioxide. Carbonyl chloride has been postulated as a
metabolite by Inference based on an in vitro study of a [14C]CC14
Incubation system. Carbon tetrachlorlde metabolism has been proposed to
Involve a complex with ferrocytochrome heme and formation of the free
radical CC1«. Carbon tetrachlorlde and Us metabolites are reported to be
O
excreted principally 1n exhaled air but also In urine and feces.
7.1. ABSORPTION
7.1.1. Partition Coefficients. Partition coefficients for various
chlorinated solvents, Including CC14> were determined by several experi-
ments (Morgan et a!., 1972; Sato and Nakajlma, 1979; Powell, 1945). The
partition coefficient 1s a measure of the relative solubility of a substance
1n two media. The o1l/a1r and oil/water partition coefficients can be used
as Indicators of solubility 1n llplds. The values of these and other parti-
tion coefficients for CC1 , listed 1n Table 7-1, show this chemical to be
UpophlUc. Because of Us UpophlUc nature, one would predict that CCl^
could be absorbed by 1ngest1on, Inhalation and skin contact. This predic-
tion Is borne out by results of the experimental studies described below.
7-1
-------
TABLE 7-1
Partition Coefficients for Carbon Tetrachlorlde*
Parameter
Olive oil/air
Blood serum/air
Blood/air
Water/air
Olive oil/water
Olive oil/serum
Olive oil/blood
Partition Coefficients
20°C 25°C
142
6
3.6-5.2
0.25
1440
23
37°C
361
2.4
—
—
150
*1945°e: Adapted from Horgan et al" 1972' Sato and Nakajlma, 1979; Powell,
7-2
-------
7.1.2. Absorption from the Gastrointestinal Tract. Absorption of
from the gastrointestinal tract of dogs was studied by Robblns (1929). In a
series of experiments, the author determined the amount of CC1. absorbed
from the llgated stomach, small Intestine and colon by measuring the concen-
tration of CC1 exhaled. In all experiments, 3 ms, of CC14 was admin-
istered. The .greatest concentration of CC1 1n exhaled air was seen after
Injection of the chemical Into the small Intestine. Direct Injection of
CC1, Into the colon resulted In a lower concentration of the chemical 1n
4
exhaled air. After Introduction directly Into the stomach by Intubation, no
CC1, was detected 1n exhaled air. The method of detection 1n these
4
experiments was thermal conductivity, with stated detection limits of 1 part
In 10 by volume of expired air. Thus, the results of the experiment can be
viewed as a qualitative Indication of relative absorption from the various
components of the gastrointestinal tract, rather than as quantitatively
accurate results.
The enhancement 1n the extent of CC1. absorption with Ingestlon of fat
or alcohol has been reported (Nielsen and Larsen, 1965; Robblns, 1929; Moon,
1950). It also appears that the absorption of CC14 from the gastrointes-
tinal tract may vary with different species because 1t occurs more quickly
1n rabbits than 1n dogs (Lamson et al., 1923).
Marchland et al. (1970) studied the effect of SKF 525A, the Inhibitor of
drug metabolizing enzymes, on the distribution of CC14 In rats. Prior to
receiving 2 ma./kg [14C]CC1 , male Sprague-Dawley rats were adminis-
tered either the SKF 525A (treated) or saline (control). The control
animals were found to excrete at least 80% of the orally administered dose
within 10 hours via the lungs. This Indicates that at least 80% of the
CC1. dose was absorbed orally.
4
7-3
-------
Stokinger and Woodward (1958) report an "accurate" absorption factor of
50% via Ingestlon; however, they do not reference their conclusion.
7.1.3. Absorption by Inhalation. In 1950~, von Oettlngen et al. studied
the absorption of CC14 by Inhalation in beagle dogs. The sex was unspeci-
fied, but the authors stated that at least five dogs were used In each
experiment. The dogs Inhaled CC14 (purity unspecified) at a concentration
of 94,500 mg/m3 for 475 minutes through a two-way valve attached to the
cannulated trachea. Blood samples were taken at unspecified Intervals and
analyzed for CC14. Data presented graphically showed that the concentra-
tion of CC14 In blood reached a maximum of 31.2-34.3 mg/100 cc (0.20-0.22
mHHmole %) after -300 minutes of exposure and remained at that level for
the duration of the exposure.
McColllster et, al. (1951) Investigated the absorption of CC1 by
Inhalation using Rhesus monkeys. Three female monkeys Inhaled 99.9%
[J-^C]CC14 vapor at an average concentration of 290 mg/m3 for 139, 344
or 300 minutes, respectively. By using the difference between Inhaled and
exhaled air, the authors calculated that the monkeys absorbed an average of
30.4% of the total amount of CC14 Inhaled. Analysis of blood drawn after
270 minutes of exposure showed that the "C radioactivity was equal to
0.297 mg CCyiOO g of blood, distributed as follows: 56.2% as CC1 ,
16.5% as "acid-volatile" carbonates and 27.3% as nonvolatile material. No
attempt was made to characterize metabolites 1n this study. The radio-
activity no longer associated with the CC14 was described by the stage In
the analytical procedure where 1t was found.
Stokinger and Woodward (1958) report an "accurate" absorption factor of
30% via Inhalation; however, they do not reference their conclusion.
7-4
-------
Lehmann and Schmidt-Ken! (1936) studied humans exposed to CC14 via Inhala-
tion. In separate experiments, Individuals (number unknown) were enclosed
1n a room of CCT. vapors for varying amounts of time. The amount of
available for Inhalation was measured. The percent absorbed was
calculated from the concentration of CC1* In the Inhaled air minus the
amount found In the exhaled breath. The reported range of percent absorp-
tion was 57-65%.
7.1.4. Absorption Through the Skin. McColllster et al. (1951) exposed
the skins of one male and one female Rhesus monkey to [14C]CC1 vapor.
Blood and exhaled air were analyzed for 14C radioactivity to determine the
amount of absorption. After a skin exposure of 240 minutes to CC1. vapor
at 3056 mg/m3, the blood of the female monkey contained CC1. at 0.012
mg/100 g and the exhaled air contained 0.0008 mg/a.. After exposure to
7345 mg/m3 for 270 minutes, blood of the male monkey contained CC1 at
0.03 mg/100 g and the exhaled air contained 0.003 mg/ft..
Three human volunteers, sex unspecified, Immersed their thumbs In CC1.
for 30 minutes 1n an experiment to measure skin absorption (Stewart and
Dodd, 1964). The CC1. was analyzed by Infrared spectroscopy and was found
to contain no detectable Impurities. The concentration of CCl^ 1n
alveolar air was used as the Indicator 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 exposure. Carbon tetrachlorlde was present 1n
the alveolar air at each time Interval, reached a maximum concentration
range of 2.8-5.7 mg/m3 30 minutes after exposure and decreased exponen-
tially thereafter. The authors concluded that CC1 could be absorbed by
the skin 1n toxic amounts 1f the chemical came In contact with arms and
hands.
7-5
-------
7.2. DISTRIBUTION
The distribution of CC1 1n humans, presumably after chronic low level
exposure through various media (food, water and air), has been reported by
McDonnell et al. (1975). Measurements were made from postmortem tissues of
several Individuals; arithmetic means (1n mg/kg) were approximately: body
fat, 0.008 (range 0.002-0.024); Hver, 0.003 (range 0.001-0.005); and
kidney, 0.002 (range 0.001-0.003). Unfortunately these values represent
possible concurrent trlchloroethane concentrations.
Robblns (1929) administered 159 g (100 cc) CC1., purity unspecified,
to three anesthetized dogs by stomach tube. The dogs were sacrificed at 6,
23 and 24 hours after treatment. Blood and various tissues were analyzed
s
for CC1. by converting the organic chloride to Inorganic chloride and
4
titrating the Inorganic chloride by the Volhard method, which Is accurate to
0.1-0.2%. The results of the blood and tissue analysis are shown In
Table 7-2. Note that the uptake of CCK was probably reduced because of
probable reduced G-I (portal) circulation during anesthesia.
From the experimental data, 1t appears the limit of detection was 1n the
range of 4-5 mg CC1./100 g of tissue. In addition, H appears that the
Hver, bone marrow, blood and muscle retained the most CC1. for the
longest time.
In 1950, von Oettlngen et al. reported the tissue distribution of CC1.
4
1n beagle dogs, each weighing ~10 kg, exposed to CCl^ 1n air at 94,500
mg/m3 for 475 minutes. The dogs were sacrificed at the end of the
i
exposure. Tissue and blood samples were taken and analyzed for CC1.. The
concentration of CC14 (per 100 g of tissue) was 66 mg/100 g 1n the brain,
50 mg/100 g In the heart, 36 mg/100 g 1n the liver and 34 mg/100 g In the
blood; the concentration In the fat was not determined. The Investigators
7-6
-------
TABLE 7-2
Carbon Tetrachlorlde Distribution at Various Times 1n Anesthetized
Dogs After Administration by Stomach Tube (mg/100 g of tissue)*
Tissue
Brain
Blood, portal
Blood, arterial
Bone marrow
Kidney
Liver
Lungs
Muscle
Pancreas
Spleen
6 hours 23 hours
17
26 13
0 0
66
11
15 , 10
trace
trace
4.5
5
24 hours
9
22
0
—
13
27
6
20
14
—
*Source: Robblns, 1929
7-7
-------
stated that the accumulation of CC14 1n the brain was consistent with Us
high oil-water partition coefficient and resulted 1n Its strong narcotic
action.
HcColllster et al. (1951) reported the tissue distribution of CC1, 1n
4
a Rhesus monkey exposed to 290 mg/m3 of [1«C]CC14 for 300 minutes.
The tissue distribution, as calculated from the 14C radioactivity, 1s
shown 1n Table 7-3. The concentration of CC1 was greatest 1n the fat,
followed by the liver and bone marrow.
Fowler (1969) studied the distribution of CC1, 1n the tissues of
4
rabbits given the chemical by stomach tube. Five rabbits were given CC1.
(1 ma/kg bw) as a 20% (v/v) solution 1n olive oil. Analysis of the CC1.
by GC showed <0.125 mg/kg hexachloroethane. The rabbits were sacrificed 6,
24 and 48 hours after treatment and the tissues analyzed for CC1 by
GC/ECD. Six hours after CC14 was administered, the tissue concentrations
(per kg of tissue) were 787^289 mg/kg (standard error) 1n fat, 96+11 mg/kg
In liver, 2H12 mg/kg 1n muscle and 20+13 mg/kg 1n kidney. By 48 hours,
these concentrations had dropped to 45£12 mg/kg In fat, 4jtp.l mg/kg 1n liver
and 0.5i0.3 mg/kg 1n kidney and muscles.
It 1s difficult to compare these four distribution studies, because
species, sacrifice times, doses and routes of exposure varied. Moreover,
not all Important tissues were sampled [e.g., von Oettlngen, et al. (1950)
did not sample the fat]; and the number of animals In two cases was small
[e.g., Robblns (1929) reported results on three dogs; McColllster et al.
(1951) reported results on one monkey]. Nonetheless, 1t appears that the
concentration of CC14 will be highest In fat, liver, bone marrow, blood
and perhaps kidney or brain after administration by either oral or Inhala-
tion routes. Certainly more work could be done In this area.
7-8
-------
TABLE 7-3
Tissue Distribution of [14C] Carbon Tetrachlorlde
Inhaled by a Female Rhesus Monkey*
Tissue
Fat
Liver
Bone marrow
Blood
Brain
Kidney
Heart
Spleen
Muscle
Lung
Bone
Carbon Tetrachlorlde
(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: McColllster et al., 1951
7-9
-------
7.3. METABOLISM
Chloroform was one of the first CC1. metabolites to be described
4
(Butler, 1961). Eight dogs were exposed to CC1 by trachea! cannula at
the rate of 8000 mg/hr Into Inhaled air for 3 hours. At the, cessation of
exposure, the exhaled air from the dogs was collected and analyzed by both
gas chromatography and the Fujiwara reaction, a colorlmetrlc 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 0.1-0.5 mg by GC analysis. Tissue
homogenates were also shown to metabolize CC1. to chloroform.
Evidence of metabolism to a free radical was suggested by studies show-
Ing hexachloroethane to be a CC14 metabolite (B1n1 et a!., 1975). Five
Wlstar rats were administered 160-800 mg CC14 dissolved 1n liquid paraffin
by gavage following a 24-hour fast. The animals were sacrificed 15 minutes
to 8 hours after treatment. A graph displaying CC1 concentrations 1n rat
liver versus time showed the chemical at -0.9 mg/kg of tissue after 15
minutes and at maximal concentration (1.7 mg/kg) after 120 minutes. Gas
chromatography analysis showed that chloroform was maximal at 0.037 mg/kg
after 15 minutes; after 4 hours It had declined to 0.007 mg/kg. Hexachloro-
ethane was also present after 4 hours at 0.005 mg/kg. The authors explained
the formation of both chloroform and hexachloroethane as CC1 metabolites
by proposing that the trichloromethyl free radical was the primary metabo-
lite of CC1,.
4
14C-labeled carbon dioxide (E14C]C02) was detected in the exhaled
air of Rhesus monkeys after a 344-mlnute exposure to [14C]CC14 at 290
mg/ma by Inhalation (McCollister et a!., 1951). The amount of
[14C]CO exhaled during the 7-day period following exposure was reported
7-10
-------
to be 10-20% of the total radioactivity expired. The authors fitted these
data to a straight line, Integrated the resulting equation from 18-1800
hours {75 days) and estimated that 4.4 mg or 11% of the total amount of
radioactivity eliminated was excreted as C0?.
Shah et al. (1979) studied the metabolism of [14C]CC1. by rat liver
i *
1_n vitro. Samples of Hver homogenate equivalent to 0.167 g of tissue were
Incubated for 30 minutes at 37.5°C with 10 vmole of [14C]CC1. alone,
and with either NADH or NADPH or both. [14C]carbon 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 1n
substantial conversion of CC1 to CO .
Shah et al. (1979) tested for the possible formation of carbonyl
chloride 1n hepatic CC1 metabolism by adding L-cyste1ne to the j_n vitro
rat liver system described above. Carbonyl chloride and L-cryste1ne are
known to react chemically to form a condensation product, 2-oxoth1azol1d1ne-
4-carboxyHc acid. The presence of the condensation product was confirmed
by thin-layer chromatography (TLC) and mass spectrometry (MS). The authors
Inferred from the presence of 2-oxoth1azol1d1ne~4-carboxyl1c acid that
carbonyl chloride was formed 1n the metabolism of CC1 by rat liver micro-
somes. The authors postulated a mechanism of blotransformatlon for CC1
4
which Involved a sequential oxidation of CC1, while bound to a heme
(Figure 7-1). Release of bound Intermediates then gave rise to different
metabolites at the site of release.
Fowler (1969) detected hexachloroethane and chloroform In the tissues of
rabbits administered CC1 orally. As previously described, a total of 15
rabbits were given CC1. at 1 mfc/kg bw and sacrificed 1n groups of five
at 6, 24 and 48 hours after exposure. Samples of fat, liver, kidney and
7-11
-------
TABLE 7-4
Conversion of [14C]Carbon TetrachloMde to
[14C]Carbon Dioxide by Rat Liver Homogenate*
Nucleotlde Added
[X*C]C02 (nmole/g liver, mean i standard deviation)
None
NADH
NADPH
NADH * NADPH
27 ± 5
373 i 17
464 ± 33
472 + 21
*Source: Shah et al., 1979
7-12
-------
Acceptor
I ^Acceptor
WIS ^ P.
60
I
a3cca3
"|+e CD+HCOO*
s 2* -003-02"] — Upoperoxidotion
1, Conjugotion
*2H Moionaidehyde
\3*-CI3CQH]"
-HC!
Acceptor — CljCO+HjO 2 HCJ * CC2
FIGURE 7-1
Pathways of Carbon Tetrachlorlde Metabolism. Products Identified as
carbon tetrachlorlde metabolites are underlined. The electrons utilized 1n
the reactions are assumed to come from NADH or NADPH via the flavoproteln
cytochrome reductases. Fe * and Fe * denote the respective ferro- and
ferrlcytochromes.
Source: Redrawn from Shah et al., 1979
7-13
-------
muscle tissue were analyzed for chloroform and hexachloroethane by gas
chromatography. The results of the analysis are shown 1n Table 7-5. The'
fat contained the highest amounts of hexachloroethane at each sampling time,
but the highest concentrations of chloroform appeared 1n the liver.
For a complete discussion on metabolic pathways hypothesized to be
mechanisms of toxlclty, see Chapter 8, Section 8.3.
7.4. ELIMINATION
McColllster et al. (1951) studied the elimination of ["ClCCl from
4
Rhesus monkeys exposed by Inhalation at 290 mg/m3 for 344 minutes. The
total 14C radioactivity in the blood decreased 12% during the first 10
minutes after exposure. Graphs of data from the analysis of blood samples
obtained periodically for 10-12 days following exposure showed that the
level of CCl^ in the blood decreased exponentially with time. - At 10 days,
the level of CC14 in blood was -0.009 mg/100 g. The authors estimated
that 21% of the total amount of CC14 absorbed was eliminated in expired
air during the first 18 days. By extrapolation of these data, the authors
concluded that after 1800 hours (75 days), -51% of the CC1. initially
4
absorbed would be eliminated in exhaled breath either as CC1 or CO .
4 2
Analysis of urine and feces showed measurable amounts of radioactivity after
15 and 12 days, respectively. The authors Interpreted these findings as an
indication that significant quantities of CC14 and/or metabolites may be
excreted by these routes.
7.5. SUMMARY
Carbon tetrachloride 1s 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 CC1. absorbed through the
7-14
-------
TABLE 7-5
Chloroform and Hexachloroethane 1n Tissues of Rabbits
Given Carbon Tetrachlorlde Orally*
CHC13
C13CCC13
Sample Time
6 hours
24 hours
48 hours
Tissue
fat
liver
kidney
muscle
fat
Hver
kidney
muscle
fat
liver
kidney
muscle
(ug/g tissue)
4.7 + 0.5
4.9 * 1.5
1.4 4- 0.6
0.1 ± 0.1
1.0 4- 0.2
1.0 4- 0.4
0.4 4- 0.2
0.1 i 0.1
0.4 4- 0.1
0.8 4- 0.2
0.2 4- 0
0.1 4- 0.1
(yg/g tissue)
4.1 * 1.2
1.6 4- 0.5
0.7 «• 0.2
0.3 * 0.2
16.5 * 1.6
4.2 4- 1.8
2.2 4- 1.1
0.5 t 0.2
6.8 4- 2.4
( 1.0 ± 0.3
trace
trace
*Source: Fowler, 1969
7-15
-------
:lungs, the chemical and Its metabolites have been reported 1n blood, many
tissues, exhaled air, urine and feces after administration by this route.
Carbon tetrachloMde 1s also absorbed through the skin.
The best available data concerning CC1 absorption come from two
studies and one report:
30.4% via Inhalation 1n monkeys (McColllster et al., 1951)
57-65% via Inhalation 1n humans (Lehmann and Schm1dt-Kehl, 1936)
30% via Inhalation and 50% via 1ngest1on (Stoklnger and Wood-
ward, 1958).
The Stoklnger and Woodward report has been criticized for not Including
substantiating Information or alternatively, citing the specific literature.
There are some considerations associated with extrapolating from animal data
to humans 1n that animals may alter their breathing patterns during an
experiment or may not be good models Insofar as their metabolism 1s con-
cerned. This is not to say that the animal data or Stoklnger and Woodward
report should be overlooked. Therefore, 1t Is recommended that a 40%
absorption coefficient be used when the route of exposure 1s via Inhalation
and that a 100% absorption coefficient be used when the route of exposure Is
via Ingestlon. The former 1s a compromise based upon the available Informa-
tion and the latter 1s the conservative estimate due to little Information.
In the studies reviewed, CC1 appears to be distributed to all major
organs following absorption. The highest concentrations have been found 1n
the fat, Hver, bone marrow, blood, brain and kidney.
Carbon tetrachlorlde metabolism has been reported to occur primarily 1n
the liver. Carbon tetrachlorlde has been postulated to be metabolized to a
trlchloromethyl radical bound to an Iron atom In the cytochrome heme moiety.
7-16
-------
This tMchloromethyl radical 1s thought to be either further metabolized or
released as a free radical. It 1s suggested that the trlchloromethyl free
radical can undergo a variety of reactions, Including macromolecular
binding, hydrogen abstraction to form chloroform, and d1mer1zat1on to form
hexachloroethane.
Carbon tetrachlorlde and Us metabolites have been reported In many
studies to be excreted primarily 1n exhaled air, but also 1n the urine and
feces. However, pharmocok1net1c data on these processes are apparently
lacking.
7-17
-------
-------
8. TOXICOLOGY: ACUTE, SUBCHRONIC AND CHRONIC
Hepatotoxldty 1s the major effect reported to be produced by acute
exposure of animals to CC1.. Hepatic necrosis and fatty liver degenera-
tion have been documented after both Inhalation exposure and 1ngest1on.
Hepatotoxldty has sometimes been accompanied by toxic effects to the kidney
and lungs. In addition, prenatal toxic effects have been demonstrated after
Inhalation of the chemical by pregnant rats. This chapter discusses the
effects from acute exposure (single dose, 1 day or several days defined 1n
rodents), subchronlc exposure (2 weeks to somewhat more than 90 days defined
1n rodents) and chronic exposure (>6 months defined 1n rodents) to CC1 .
Emphasis Is placed on studies 1n which dose/response relationships have been
developed. Toxic responses occurring prior to carc1nogen1c1ty are reported
1n Chapter 11.
8.1. EXPERIMENTAL ANIMALS
8.1.1. Acute. The toxlclty from acute exposure to CC1. has been docu-
mented extensively. Because defining the dose range that produces minimal
health effects 1s 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 which dose-response relationships can be determined.
For this reason a number of studies referring to LDc/.s will not be
bu
discussed. Table 8-1 summarizes some of the lethal dose data reported for
CC1 1n various species.
Administration of CC1 results 1n a number of acute systemic effects.
Its hepatic effects are the most pronounced; CC1. causes necrosis and
fatty liver degeneration. These effects have been well documented 1n many
scientific studies. However, toxlclty to other organs has also been
described. The following subsections will focus on such organ toxlclty.
8-1
-------
TABLE 8-1
Toxic Doses and Effects of Carbon Tetrachlorlde \n Animals3
Animal
Route of
Administration
^Source: U.S. EPA 1981
Effect^
dose lethal for 50% of animals
concentration lethal for 50% of animals
lowest lethal dose
LC|_0, lowest lethal concentration
Dose
Rat oral
House
Dog
Rabbit
Rat Intraperltoneal
House
Dog
Rabbit
Rat Inhalation
House
Cat
Guinea pig
Cat subcutaneous
Rabbit
L°50
LD50
LDLo
LD50
LD50
LD50
LD.
Lo
LDLo
LC50
LC50
LCLO
LCLO
LDLo '
LDLo
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
8-2
-------
8.1.1.1. LIVER EFFECTS — Functional changes In mouse liver as a
result of CC1 exposure were measured by increases 1n activity of the
enzyme serum glutamic-pyruvlc transamlnase (SGPT) and 1n sulfobromophthaleln
(BSP) retention (Klaassen and Plaa, 1966). Male Swiss-Webster mice were
administered various amounts of analytical grade CC1, i.p. 1n corn oil in
a final volume of 10 ma/kg 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 CC1 as 15.9 mg/kg bw for elevation of SGPT
activity and 94 mg/kg bw for BSP retention. The authors did not report the
range of CC1. 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 CC1. exposure and elevated SGPT levels in
mice. They used the "up and down" method in which one dose (given 1n Table
8-2) of the compound was given Intraperltoneally; 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 (ED,-_) of CC1 in mice as measured by elevated SGPT values.
Sein and Chu (1979) studied the effect of CC1. on the level of the
liver enzyme glucose-6-phosphatase 1n mice. Groups of six male LAC strain
mice were treated i.p. with 795, 1590 or 3180 mg/kg bw CC1. (purity
unspecified) in paraffin oil. The animals were sacrificed 24 hours after
treatment. Control animals (number unspecified) were given paraffin oil and
8-3
-------
TABLE 8-2
SGPT Values of Mice Administered Carbon Tetrachlorlde
IntrapeMtoneally 1n "Up and Down" Experiment3
Animal
1
2
3
4
5
ED50
Dose
(mg/kg bw)
17.5
13
17.5
13
8
13
Response**
E
N
E
E
N
aSource: Klaassen and Plaa, 1967
N
Elevated SGPT after 24 hours
Normal SGPT after 24 hours
= Median effective dose
8-4
-------
sacrificed on the same schedule. The livers were removed and analyzed for
glucose-6-phosphatase. The results of the analysis showed that after treat-
ment with CC1 at 795 or 1590 mg/kg, the enzyme level fell to 40% of the
4
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 CCl^
exposures on rats were performed by Murphy and Halley (1969). Adult male
Holtzman rats (250-350 g) were administered various doses of undiluted
CC1 by gavage. Control animals were administered equal volumes of water.
At 2-20 hours after treatment, animals were sacrificed and Hver enzyme
activities and Hver weights were measured. The results are shown 1n Table
8-3. The animals receiving CC1 at 1600 mg/kg bw were sacrificed 20 hours
after treatment and the Hvers examined hlstopathologlcally. The examina-
tion showed extensive fatty Infiltration, Inflammation and some centrllobu-
lar necrosis. The Hver-to-body weight ratios were also Increased.
The effect of single exposures on the activities of the cortlcosterone-
Indudble liver enzymes tryptophan pyrrolase and tyroslne-ketoglutarate
transamlnase was studied. Groups of rats (4-6 In each group, 8 untreated
controls) were treated with CC1, (0, 400, 800 and 1600 mg/kg bw by gavage)
and sacrificed 5 hours after treatment. Data graphs showed that the enzyme
levels were Increased roughly 1n proportion to the dose.
Similar studies on the effect of CC14 administration on serum activity
of Hver enzymes 1n rats were performed by Drotman and Lawhorn (1978).
Groups of four male Cox rats were administered CC1 l.p. at 60, 120, 240
or 480 mg/kg bw 1n a total volume of 1 ms, In corn oil and exsanguinated at
specified time Intervals. Serum activities were determined for the enzymes
8-5
-------
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in
0)
U
1
c
«r-
UJ
CO
+1
in
c
re
u
8-6
-------
sorbltol dehydrogenase (SSDH), ornlthlne carbamyl transferase (SOCT), aspar-
tate amlnotransferase (SAST) and 1soc1tr1c dehydrogenase (SICDH). Liver
specimens were taken from each animal and scored for h1stopatholog1cal
changes. The results of the enzyme analyses and hlstopathology are tabu-
lated 1n Table 8-4 by dose and hours after dose. The SOCT activities showed
the best correlation with liver hlstopathology 1n time of appearance as well
as extent of damage. The authors concluded that SOCT levels were a sensi-
tive Indicator of liver damage.
Effects of acute exposure to low levels of CC1. were also reported by
4
Korsrud et al. (1972). Male Wlstar rats (260-400 g; 8-10 animals per treat-
ment group) were administered single oral doses of CC1 (0-4000 mg/kg bw)
In corn oil (5 ml/kg bw). The rats were fasted for 6 hours before treat-
ment and for 18 hours afterward, and then sacrificed. Assays Included liver
weight and fat content, serum urea and arglnlne levels, and levels of nine
serum enzymes produced mainly In the liver. At 20 mg/kg bw there was hlsto-
pathologlc evidence of toxic effects on the liver. These changes Included a
loss of basophlllc stippling, a few swollen cells and minimal cytoplasmlc
vacuolatlon. At 40 mg/kg bw, Hver fat, Hver weight, serum urea and levels
of five of the nine liver enzymes were Increased while serum arglnlne
decreased. At higher doses the remaining four enzyme levels were also
elevated.
In addition to the study on hepatic effects of CC1. In mice described
earlier 1n this section, Klaassen and Plaa (1967) also Investigated the
hepatic effects of CC1 exposure on dogs. Male and female mongrel dogs
were treated 1.p. with CCl^ at 22-38 mg/kg bw 1n an "up and down" experi-
mental design. Blood samples were taken for measurement of SGPT 24 hours
after administration. Control dogs had serum SGPT activity of 36£7 units.
8-7
-------
TABLE 8-4
Effects of Carbon Tetrachlorlde on Liver Hlstopathology
and Serum Enzyme Levels3
Dose
(rag/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
H1stologyb
0
2
1
1
0
0
3
2
1
0
0
3
4
1
0
0
3
4
1
0
Serum Enzyme Concentrations
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
^Source: Adapted from Drotman and Lawhorn, 1978
N) = No observable changes.
1 = Minimal changes. Large central vein, swelling of hepatocyte, etc.
2 = H1ld degenerative change. Loss of cord arrangement.
3 = Moderate degenerative change. Pale cytoplasm, spindle cell.
4 = Marked degenerative change. CentMlobular fatty degeneration.
*S1gn1f1cantly different from zero time as determined by one-way analysis of
variance of the log-transformed data (p<0.01).
8-8
-------
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 1n Table
8-5. The SGPT returned to normal In 17-18 days. Animals were then sacri-
ficed and the livers were examined hlstopathologlcally. They showed moder-
ate vacuolatlon of the centrllobular and mldzonal hepatocytes as well as
traces of brown material In the cytoplasm of centrllobular Kupffer cells.
Gardner et al. (1924) also studied the acute toxic effects from Inges-
tlon of CC1. 1n dogs. Effects ranged from no apparent effect at 0.01
mS,/kg {15.89 mg/kg) to centrllobular necrosis at 0.05 ml/kg (79.45
mg/kg). Rabbits similarly treated experienced Hver necrosis at 0.1 mil/kg
(158.9 mg/kg).
Kronevl et al. (1979) administered CC1 eplcutaneously 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 necrotlc changes.
Altered Hver morphology was seen after 16 hours when hepatocytes 1n the
central two-thirds of each lobule showed marked hydropic changes which were
characterized by large, clear cytoplasmlc spaces. It was noted thefe was
also a tendency toward necrotlc lesions characterized by homogeneous,
slightly eos1noph1Hc and slightly PAS-pos1t1ve structures within the cyto-
plasm. Glycogen was absent and the nuclei showed a tendency to degeneration.
In studying the renal effects of CC1 1n cats (see Section 8.1.1.2.),
Wong and DIStefano (1966) noted a delayed liver reaction. Liver weights
were significantly Increased 24 hours following Inhalation exposure.
8.1.1.2. KIDNEY EFFECTS — In experiments conducted by Plaa and
Larson (1965) using CCl high doses did not Induce renal failure as
measured by phenolsulfonphthaleln (PSP) excretion In mice although patho-
logical kidney alterations were present. Male Swiss mice (18-30 g) were
8-9
-------
TABLE 8-5
SGPT Activity 1n Dogs 24 Hours After Intraperltoneal Administration
of Carbon TetrachloMde 1n "Up and Down" Experiment3
Animal
1
2
3
4
5
"50
Dose
(mg/kg)
22.2
30.2
22.2
30.2
38
32
Response**
N
E
N
N
E
aSource: Adapted from Klaassen and Plaa, 1967
bN » normal SGPT after 24 hours
E » elevated SGPT after 24 hours
Median effective dose
8-10
-------
given 1.p. Injections of CC14 (1600-6400 mg/kg bw) dissolved 1n 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 CC1. doses lethal 1n some animals (>6400 mg/kg bw) failed to
cause renal dysfunction 1n the majority of survivors, as measured by excre-
tion of PSP, urinary protein and glucose. At a high nonlethal dose (3260
mg/kg bw), minimal renal dysfunction was observed after 96 hours. Hlsto-
loglc examination of kidney sections from five mice administered this dose
Indicated that one of the mice had necrosis of proximal convoluted tubules,
and four of five mice had swelling of the tubules.
Carbon tetrachlorlde did, however, decrease activity of glucose-6-phos-
phatase 1n the kidney (Seln and Chu, 1979). Male mice (40-50 days old,
weighing 24-28 g) were Injected l.p. with CC14 at 795, 1590 or 3180 mg/kg
bw In paraffin oil. Twenty-four hours after Injection of 795 or 1590 mg/kg
bw, 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
lower doses and decreased further to 20% of the control value at the 3180
mg/kg bw dose. The authors attributed these differences to the limited
metabolic capacity of the kidneys.
Klaassen and Plaa (1967) studied the effect of CC1. exposure on kidney
*T
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 l.p. with CCl^ at 22-38 mg/kg bw, and the
24-hour excretion rate for PSP was determined. Control dogs were used to
8-11
-------
determine a normal range for PSP excretion. None of the dogs treated with
CCl^ exhibited decreased PSP excretion. However, on hlstopathlc examina-
tion of the kidneys from the treated dogs, the Bowman's capsules appeared
dilated with some contraction of glomerular tufts and calcification of a
small number of tubules 1n the medulla.
Striker et al. (1968) examined the structural and functional changes 1n
the rat kidney during CC14 Intoxication. Exposure was 2.5 ma/kg bw 1n
an equal volume of mineral oil by gastric Intubation. A progressive In-
crease 1n 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-
v
1ble. The earliest morphological change was 1n the mitochondria, followed
by cellular swelling manifested by loss of basllar 1nterd1g1tat1ons and
swollen mlcrovllH. Occurring later was the appearance of large aggregates
of smooth-surfaced membranous profiles. By 5 days after exposure, all
alterations were reversed.
Also apparent were differences 1n serum parameters reflecting kidney
function. Serum creatlnlne and blood urea nitrogen peaked at 12 hours and
24 hours, respectively. Total serum blllrubln was elevated 12-48 hours. By
5 days all values returned to normal (Striker et al., 1968).
Wong and DIStefano (1966) examined llpld accumulation and hlstologlc
changes 1n the kidney of the cat following Inhalation of 10,000 ppm CC1.
for periods of 15, 30, 60 and 240 minutes. Kidney welghttbody weight ratios
Increased significantly following 60- and 240-m1nute exposures. The renal
cortical llpld content Increased significantly within 15 minutes and an
accumulation of fat droplets was seen hlstologlcally In 30-60 minutes. One
8-12
-------
hour after the termination of CCl^ Inhalation, the kidneys were enlarged
and the weight continued to Increase past the 24-hour observation period.
8.1.1.3. LUNG EFFECTS — Boyd et al. (1980) Investigated the effect
of 1ngest1on and Inhalation of CC14 on pulmonary Clara cells 1n Swiss
mice. For the 1ngest1on study, the mice were treated with CCl^ (4000
mg/kg bw) In a 50% sesame oil solution and sacrificed 16 hours after treat-
ment. The lungs were removed and examined by electron microscopy. The
examination showed the Clara cells to have massive dilation of vesicles of
smooth endoplasmlc retlculum, Increased mitochondria! staining density,
rlbosomal dlsaggregatlon, nuclear condensation and occasional cellular
•\
necrosis. Additional experiments with oral CC14 doses of <1600 mg/kg bw
did not produce any pulmonary lesions visible by light microscopy. Doses of
2400-4800 mg/kg bw produced Clara cell lesions similar on electron micro-
scopic examination to those previously described. The extent of damage was
proportional to the dose administered. In addition, the time course of the
Clara cell damage caused by 1ngest1on of CC1. (4000 mg/kg bw) was studied.
Pulmonary tissue 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 toxldty of Inhaled CC1 was also studied by Boyd et al.
(1980). Swiss mice were exposed to CC14 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 reported to be more frequent after
Inhalation than after oral exposure, but no effort to quantify this finding
was reported.
8-13
-------
Gould and Smuckler (1971) detailed the structural alterations 1n rat
lungs following CC14 1ngest1on. Male Sprague-Daw!ey rats (200-250 g) were
fasted 16 hours prior to administration of CC1 (4000 mg/kg bw) by gavage.
The animals exhibited pHoerectlon and lassitude 3-4 hours after treatment.
Necropsies were performed on all animals. Microscopic examination of the
lungs of treated rats revealed perlvascular edema and mononuclear Infiltra-
tion 1n the first 4 hours after treatment. These areas were local but were
estimated to Involve 10% of the parenchyma. Areas of atelectasls and 1ntra-
alveolar hemorrhage Involving 15-20% of the parenchyma were observed 8-12
hours after treatment.
Electron micrographs of rat lungs after CC1 Ingestlon showed granular
pneumocytes containing swollen Inclusions with decreased osmlophlUa and
attenuated lamellae 1 hour after treatment. These changes were more severe
4 hours after treatment. By 4-8 hours after treatment, cytoplasmlc edema,
dislocation of dense rlbosomal aggregates and mltochondrlal disruption were
apparent. Multlveslcular bodies were "conspicuously decreased" within the
granular pneumocytes. Necrosis was evident 12-24 hours after treatment.
One hour after administration, endothellal cells displayed markedly In-
creased plnocytotlc vesicles. Severe disruption of endothellal cells was
evident from 8 hours onward. Ultrastructural damage was seen 1n all compo-
nents of the alveolar wall, and fibrin was observed within alveoli. The
authors Interpreted these findings as showing significant alterations 1n
vascular permeability.
Lesions of the Clara cells In the lungs of male Sprague-Dawley rats
orally treated with CC14 were observed by Boyd et al. (1980). The CC1
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
8-14
-------
cell lesions occurred at the two highest doses. The authors stated that the
lesions were less pronounced than those 1n mice exposed to comparable
amounts of CC1,.
4
Chen et al. (1977) examined the lung effects of rats given a single
CC1. exposure by gastric Intubation or by Inhalation. Exposures were: 2.5
mi/kg bw by gastric Intubation, and 30 minutes 1n air containing 4.38%
CClfl (280,400 mg/m3) by Inhalation. Both the orally administered and
4
Inhaled CC1 markedly modified the lung and liver cytochrome P-450 con-
tent, but there was a greater response 1n the pulmonary tissue. Inhalation
resulted 1n a less significant depression of activity In both organs (Chen
et al., 1977). Morphologic analyses of the lung revealed focal changes by
1-7 days 1n pulmonary architecture consisting of areas of alveolar collapse,
septal thickening, transudatlon and modification of type II pneumonocytes.
8.1.1.4. DERMAL EFFECTS — Kronevl et al. (1979) examined the effects
on guinea pigs following eplcutaneous administration o,f 1 ma. CCl^.
Slight karyolysls was seen. After 15 minutes, marked sponglosls developed.
Fifteen minutes and onward, karyopyknosls was evident. The authors saw
progressing nuclear pyknosls and functional separation between the basement
membrane and the basal cells along with Induced sponglosls appearing before
the functional separation attributed to the CC14 exposure.
8.1.1.5. BIOCHEMICAL AND OTHER EFFECTS — Merkur'eva et al. (1979)
studied the effects of continuous Inhalation of CC14 on the rat enzyme
system. Three stages of pathological changes 1n the liver were defined as
characterized by morphological and biochemical parallels In the development
of effects. Random-bred, sexually mature male albino rats (strain not
given), totaling 55, were exposed to 300 mg/m3 CC14- The exposure
resulted 1n a 45% reduction 1n the activity of N-acetyl-B-d-glucosamlnldase
8-15
-------
24 hours following Initiation, and an overall reduction 1n the activity of
the same enzyme by the 14th day of the experiment. The activity of serum
hyaluronldase Increased to a maximum 3 days Into the experiment with the
average Increase being 96%. After 3 days, a gradual decrease was observed.
However, at 14 days, levels were stm Increased over controls by 68%.
The peak 1n activity of serum hyaluronldase was accompanied by an
Increase 1n the activity of add phosphatase (by 38%). In addition, the
activities of Blglucosldase and 8-galactos1dase decreased to 62% and 73%,
respectively, at 8 days. Fourteen days following Initiation, a 3-fold
Increase 1n the activity of 6-glucos1dase was observed along with a decrease
(by 30%) In the activity of add phosphatase. Finally, a 39% Inhibition of
N-acetyl-neuram1n1c acid aldolase was observed.
Following Injection of CC14, Wyrebowska and Jerzykowskl (1980) saw a
change 1n enzyme activity 1n rat serum. Mature male and young Wlstar rats
were given a single 2 mil/kg bw dose of CC1 l.p. dissolved 1n sterile
vegetable oil (1:1) or 1 mil/kg CC14. Following this dose, both the
alkaline and add forms of amlnopropanol dehydrogenase appeared 1n the blood
serum. Maximum activity occurred 24 hours after administration 1n the young
rats and 12 hours after administration 1n the mature male rats.
Mikhail et al. (1978) Injected male and female adult rats 1ntraper1to-
neally with 0.5 ms, of a 1:1 mixture of CC1 In mineral oil per 100 g bw
(0.005 mi of the mixture per g bw). This resulted 1n an Increase 1n serum
Iron, copper, zinc, calcium, potassium and sodium 24 hours after administra-
tion. There was no change observed In serum magnesium. The authors attrib-
ute the rise 1n some serum chemistries to the known hepatotoxlc effect of
CCl^. They conclude that the disturbance 1n minerals metabolism Is one of
the earliest lesions In CC14 poisoning (Mikhail et al., 1978).
8-16
-------
David et'al. (1981) examined the effect of different exposure schemes
upon biochemical and morphological changes 1n the rat liver. Groups of six
male Wlstar rats, 7 months old, were exposed to CC1 vapor under different
exposure schedules. Schedules were designed In such a way to expose each
group to a constant 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 shorter 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.
The authors conclude that sensitivity of the liver 1s more Influenced by the
concentration of CC1, 1n the Inhaled air than 1n the total amount Inhaled,
4
the theory being that the former allows more CC14 Into the blood entering
the Hver. As the authors explain, this Information 1s Important In putting
time weighted averages (TWAs) 1n the workplace.
8.1.2. Subchronlc. The toxldty followng subchronlc exposure to CC14
has not been as extensively described as the toxldty following acute expo-
sure. Paquet and Kamphausen (1975) examined biochemical changes 1n female
Wlstar rats following subchronlc exposure to CC1 . Administration of
CC1 was by subcutaneous Injection of 1 ml/kg bw In an equal amount of
peanut oil at 7-day Intervals for 8 weeks. The authors described the
changes In stages. In stage 1 there was a'decrease 1n pseudochollnesterasls
Indicative of the stage of liver necrosis. In stage 2, the trlglycerldes
reached a high plateau; there was an Increase In SGOT, an Increase In BSP
retention and a continued decrease In pseudochollnesterasls. The second
8-17
-------
stage was characterized by massive fatty Infiltration of the liver and In-
creased necrosis with liver flbrosls at the end. In stage 3, SCOT continued
to Increase along with hydroxyprollne, trlglycerldes and BSP retention.
This final stage was characterized by a reduced synthetical ability and
atrophy of the liver.
B1z1n et al. (1977) performed a comparative study of the effect on rats
of continuous and Intermittent exposure to CC1 . Continuous Inhalation of
500 mg CCl^/m3 for 10 days Induced toxldty symptoms 4- to 5-fold as
rapidly as did Inhalation for 6 hours dally for 40 days (B1z1n et al., 1977).
Additionally, Alumot et al. (1976) reported the effects upon groups of
six weanling rats (strain not given) 4 weeks old fed a diet containing
CC14 at 150, 275 or 520 mg/kg of feed for 5 weeks (females) or 6 weeks
(males). The fumigated feed was stored 1n airtight containers; CC1. loss
during the storage period of 7-10 days was determined to be 5%. The animals
were allowed access to the feed only at set time Intervals to minimize loss
of CC14 by volatilization. The authors calculated that the amount of
remaining 1n the consumed feed was 60-70% of the amount Initially
present; the total decrease reflected amounts lost during storage and after
removal from storage to feeding troughs. From these data and the weights of
the animals, the authors calculated that 275 mg/kg" of feed represented a
dally dose of 40 mg/kg bw. By assuming that all parameters were the same
and that the delivered dose was proportional to the concentration 1n feed,
diets of 150 and 520 mg/kg of feed can be calculated to represent dally
doses of 22 and 76 mg/kg bw, respectively (U.S. EPA, 1981). At the end of
the experiment the animals were weighed and killed. Of the three doses,
only the highest, 76 mg/kg bw (520 mg/kg of feed), caused significantly
depressed weight gain 1n males. Weight gain 1n females appeared to be
8-18
-------
unaffected by all doses. Total I1p1d and trlglycerlde levels 1n the liver
were significantly higher 1n animals fed CC1. at 40 and 76 mg/kg bw than
1n controls or animals fed 22 mg/kg bw. Levels of liver phosphollplds
(measured 1n females) were not affected at any dose. Of the three doses
used 1n 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 Hartley guinea pigs to CCT 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 Hvers examined hlstopathologlcally. The
examination revealed fatty Infiltration, flbrosls, bile duct proliferation,
hepatic cell degeneration and regeneration, focal Inflammatory cell Infil-
tration, alteration of lobular structure and early portal cirrhosis. The
I1p1d content of the guinea pig liver was reported to be 35.4±10.7%, much
higher than the control value of ll.Ojh3.6H.
In addition, the Investigators continuously exposed 15 Hartley guinea
pigs to CC1 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 "high Incidence" of enlarged and discolored livers was
reported on gross pathological examination. Hlstopathologlc examination of
the Hvers revealed fatty changes, flbroblastlc proliferation, collagen
deposition, hepatic cell degeneration and regeneration, and structure alter-
ation of the liver lobule. Enzymatic studies showed that only the sucdnlc
dehydrogenase (SDH) activity was moderately reduced as compared to that In
controls.
8-19
-------
Prendergast et al. (1967) also exposed Hartley guinea pigs, number
unspecified, to CC1 vapor continuously at 6.1 mg/m3 for 90 days. The
authors reported that "no visible signs of tox1c1ty" were seen during this
study. L1p1d content of the liver and serum urea nitrogen were within
normal range. The authors concluded that no pathologic changes could be
attributed to CC1. exposure.
In addition to their studies on guinea pigs, Prendergast et al. (1967)
*
studied the effects of both repeated and continuous exposure to CC1. on
three squirrel monkeys, three New Zealand albino rabbits, and two beagle
dogs for each exposure regime. The experimental designs were the same as
those described for the guinea pigs. All the animals showed a weight loss
during repeated exposure to 515 mg/m3. Fatty changes were noted 1n the
Hver of all species being most severe 1n rabbits, followed by dogs and
monkeys. In the continuous exposure to 61 mg/m3 for 90 days, all species
exhibited a depressed weight gain, as did guinea pigs. Liver changes were
also noted, but enzyme activities (as measured by NADH, NADPH, SDH, LDH and
66PD) were within the normal range. At a continuous exposure of 6.1
mg/m3, no toxic signs were noted.
Finally, the same Investigators {Prendergast et al., 1967) studied the
effects of repeated (515 mg/m3) and continuous (61 mg/ma or 6.1 mg/m3)
exposure on 15 Long-Evans or Sprague-Dawley rats following the same method-
ology described above. With repeated exposure, there was a high percentage
of mottled livers. H1stopatholog1c examination revealed morphologic changes
1n lungs and Hvers but no changes 1n the heart, spleen or kidney. Fatty
changes also developed 1n the liver. Following continuous exposure to 61
mg/m3, depressed growth curves resulted as compared to control animals.
8-20
-------
Examination upon autopsy revealed a high Incidence of enlarged and/or dis-
colored livers. H1stopatholog1c liver changes Included fatty Infiltration,
flbroblastlc proliferation, collagen deposition, hepatic cell degeneration
and regeneration, and alteration 1n the structure of the liver lobule.
There were no adverse effects following 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 were
mentioned) and vague Information such that only the general conclusion that
liver damage follows CC1. Inhalation can be made (EnvlroControl, 1981).
8.1.3. Chronic. Smyth et al. (1936) studied the toxlclty of CC1. on
rats after chronic Inhalation exposure. Groups of 24 Wlstar rats were
exposed to CC14 concentrations of 315, 630, 1260 or 2520 mg/m3 for 8
hours/day, 5 days/week for 10.5 months. The CC14 was found to contain
<0.003% carbon dlsulflde. Control rats were used, but the number was
unspecified. Growth retardation was observed with the 2520 mg/m3 dose.
At the 630 and 1260 mg/m3 dose, growth was the same as In controls, and at
the 315 mg/m3 dose the growth was stimulated. Cirrhosis developed 1n the
630, 1260 and 2520 mg/m3 groups after 173, 115 and 54 exposures, respec-
tively, but not 1n the 315 mg/m3 group. When exposure was stopped fatty
liver degeneration resolved within 50 days. Surface alterations (hobnail
liver) did not resolve until 156 days after cessation of exposure. Unspeci-
fied renal damage was observed after 52 exposures to the 315 mg/m3 concen-
tration and after 18-20 exposures at the higher concentrations, but was
termed "not extreme."
In this same study, groups of 24 guinea pigs (strain not given) were
exposed to CC14 vapor at 315, 635, 1260 or 2520 mg/m3. The frequency of
8-21
-------
exposure was 8 hours/day, 5 days/week for <10.5 months. Marked mortality
occurred 1n exposed animals: 9/24 at the 315 mg/m3 dose after a median of
44 exposures (exposure terminated at 135 days), 16/24 at the 630 mg/m3
dose after a median of 10 exposures, 13/24 at the 1260 mg/m3 dose after a
median of three exposures, and 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 cirrhosis and
hobnail surface alterations of the liver in 105 exposures. The authors
concluded that survival of guinea pigs at higher doses was of insufficient
duration to allow development of cirrhosis. In addition, granular swelling
was observed 1n adrenal glands of guinea pigs exposed to CC1 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 the sciatic nerves. Dense
clumps of black granules (osmic acid stain) were observed paralleling the
large majority of fibers.
Rhesus monkeys were also examined. They inhaled CC1. 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 postexposure. Two of four monkeys exposed
to 1280 mg/m3 for 8.7 and 10.5 months showed definite damage to the
sciatic nerve.
In summary, guinea pigs were found to be more sensitive to the toxic
effects of CC1. than were rats or monkeys. This may be due, 1n part, to
the diet provided to the guinea pigs (low in calcium and lacking in animal
8-22
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fat and protein). All concentrations provided, produced some effect 1n the
rats, and very little harm was observed 1n the monkeys. The liver and
kidney Injury produced 1n the monkeys reversed completely 28 days following
exposure.
In a chronic oral exposure study (Alumot et al., 1976), groups of 36
rats (18 male and 18 female Uttermates, strain not given) were fed mash
containing CC14 at 0, 80 or 200 mg/kg of feed. The feed was stored 1n
airtight containers, assayed for CC1. content, and consumed soon after
removal to feeding troughs. The authors calculated that the 200 mg/kg of
feed represented a dally dose of 10-18 mg/kg bw. During this 2-year study,
several parameters were measured. No effects were noticed on body weight
gain for up to 13 weeks. Throughout the study measurements of male and
female fecundity remained essentially normal. After 2 years, the surviving
animals were killed. In these animals, serum values for glucose, protein,
albumin, urea, uric add, cholesterol, SGOT and SGPT 1n the treated animals
did not differ from those 1n controls. No fatty livers were detected 1n the
treated animals. Thus, the authors found no biochemical, hlstopathologlc,
reproductive or other abnormalities attributable to CC1. exposure.
However, Interpretation of the results was complicated by the widespread
Incidence of chronic respiratory disease 1n the animals which started at
about 14 months Into the experiment. More than half the animals were dead
at 21 months, although at 18 months the survival ranged from 61-89%.
Although the authors Indicated that 10-18 mg/kg bw (200 mg/kg of feed) 1s a
no-observed-adverse-effect level (NOAEL) of CC1. over 2 years, this con-
clusion may be questioned because of the chronic respiratory Infection and
hence poor survival of the animals 1n the latter part of the experiment.
8-23
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Yet, 1t may be Inferred from these results that a level of 10-18 mg/kg
bw/day over a 1-year period caused no observable adverse effects.
A chronic Inhalation study by Adams et al. (1952) also presented Incom-
plete data. No numbers of animals tested, surviving, or affected were
given; therefore, 1t 1s not possible to determine what measurements were
made at different exposures. The only conclusion possible 1s that at some
exposures ranging from 32.5-2600 mg/m3, 7 hours/day, 5 days/week for 258
days, some Hver damage occurred In albino Wlstar rats, albino rabbits
(strain not given) and guinea pigs (strain not given). A few rhesus monkeys
were tested, but only limited Information 1s presented. At least one monkey
exposed to 25 ppm CC1 , 7 hours/day, 5 days/week, survived for 212 days
with no apparent adverse effects. The limited Information and confusing
description precludes a more detailed appraisal of the study.
In a study previously discussed (Merkur'eva et al., 1979), the authors
also examined the effect of chronic CC1. Inhalation. Long-term exposure
to 300 mg CCl^/m3 caused a considerable Increase In the DMA-synthesizing
connective tissue cells (Merkur'eva et al., 1979).
Rotenberg (1978) examined the dynamics of liver bloenergetlc system
responses following the chronic exposure of 150 rats (strain not given) to
small concentrations of CC1 1n air (14 mg CClVm3, 5 hours/day, 5
4 4
days/week .for 5 months). As stated by the authors, the exposure caused
phasic changes 1n hepatic energy-producing processes as evidenced by altera-
tions 1n respiratory rate, phosphorylatlon and sensitivity of respiratory
enzymes to respiration Inhibitors.
8.2. HUMANS
Many poisonings have resulted from the accidental or suicidal 1ngest1on
of CC1, or from Its medicinal use as an anthelmlntlc. For Us medicinal
4
8-24
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use, the therapeutic dose recommended for adults was 2-3 ma. 1n capsule
form and 0.13 mfc/year for Infants and children up to 15 years of age (von
Oettlngen, 1964). As emphasized by von Oettlngen (1964), such doses, which
are followed by doses of Epsom salts, have caused toxic effects only excep-
tionally. Horrocks (1934) reported one fatality from Us medicinal use.
The vast majority of poisonings, however, have resulted from the Inhalation
of Its vapors when used as a solvent or dry cleaning agent {von Oettlngen,
1964). Stm other poisonings have been the result of dermal exposures
through the use of CC14 1n shampoos (NIOSH, 1975). Finally, some have
resulted from Us use 1n fire extinguishers (Dudley, 1935).
Norwood et al. (1950) reported the occurrence of 2 fatalities, 1 near
fatality, 4 poisonings requiring hospHallzatlon, and 51 mild Industrial
poisonings In two communities over a period of 1 year. In 1935, Smyth
(1935) noted 28 fatalities, 14 of which resulted from the 1ngest1on of
CCl^; 120 acute and subchronlc poisonings; and 7 cases of chronic poison-
Ing. By 1964, an additional 28 poisonings resulting from CCK 1ngest1on
(Including 10 fatalities) and 202 cases from Inhalation (Including 29 fatal-
ities) were reported. The actual Incidence of such poisonings was doubtless
much greater, since many poisonings were not attributed to CC1. and others
were not published In the medical literature (von Oettlngen, 1964). Since
1964, there have been additional poisonings and case reports (Bagnasco et
al., 1978; Bonltenko and Bruk, 1979; Shlmanko 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 CC1 have
4
occurred to a great extent, as reported by a number of authors (Docherty and
8-25
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Burgess, 1922; Seattle, et al. 1944; NIOSH, 1975; K1rkpatr1ck and Suther-
land, 1956; Oawborn et al., 1961). A summary of the symptoms of such oral
poisoning 1s given below (von Oettlngen, 1964). Following Ingestlon of
CC1., the patient experiences a burning sensation 1n the mouth, esophagus,
and stomach. Depending upon the dose, this 1s sooner or later complicated
by abdominal pain, nausea, and vomiting. Some patients develop hiccoughs.
The tongue 1s coated. These symptoms are soon followed by diarrhea, which
later may be followed by constipation and occasionally by gastric and
Intestinal hemorrhages which, 1n rare cases, may also be seen In the mouth
and pharynx. Again, depending upon the dose along with other factors, the
patient becomes jaundiced, the liver becomes enlarged and tender; this may
be associated with asdtes and generalized edema. Soon after the Ingestlon,
the patient feels dizzy, may suffer from headache and become confused,
semiconscious and delirious. The patient may become restless and develop
choreatlc movements. Finally, consciousness 1s lost and the patient passes
Into coma. Some patients complain of visual disturbances and edema of the
eyelids and develop hemorrhages of the sclerae. In severe cases, circula-
tory disturbances may develop, characterized by lowered or Increased blood
pressure, thin and rapid pulse, and signs of congestive heart failure with
cyanosis.
A fatality attributed to Ingestlon of CC1 was reported by Smetana
(1939). The victim, a photographer described as having "a history of
chronic alcoholism," died 10 days after consuming an unknown amount of "some
fluid containing carbon tetrachlorlde." He presented symptoms Including
nausea, vomiting, jaundice, anurla and semlstupor. In the final clinical
diagnosis, death was attributed to CC1. poisoning.
8-26
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A case of attempted suicide by Ingestlon of CC1, was reported by
Stewart et al. (1963). The victim, a 29-year-old female who Ingested one
pint of a CC1 :methanol solution (2:1), experienced ringing 1n the ears
Immediately after Ingestlon and lost consciousness. She was hospitalized
for 3 weeks. Three hours after Ingestlon, CC1. In the exhaled breath and
blood was confirmed by Infrared analysis. The exhaled breath was then
monitored throughout the hospltallzatlon; CC1. was reported to decrease
exponentially. Because of the toxldty of the methanol and the possibility
of synerglstlc reactions with the CC1., hemodlalysis was performed soon
after admission. Mannltol solution was given by continuous Intravenous
Infusion. Clinical laboratory analyses during hospltallzatlon showed some
elevation of SGOT, which reached a maximum of 75 units on day 6, and an ele-
vation of urinary uroblUnogen to a maximum of 7.8 Ehrllch units on day 10.
Other laboratory findings Included elevation of serum Iron and depression of
serum protein concentration and albumin fractions. The retention time of
bromosulfophthaleln was Increased. These findings were Interpreted as
evidence of minimal hepatocellular Injury. Acute renal dysfunction was not
observed; the authors credited the mannltol treatment with preventing renal
damage.
Lamson and M1not (1928) studied the lethal effects of CC1 on patients
receiving CC1. and magnesium sulfate orally as a treatment for hookworms.
The authors reported the treatment of thousands of patients with a single
dose of 2.5-15 ms, CO! without 111 effects. One man was reported to
have safely Ingested 40 mfi, of CC1 . However, an "extremely small" popu-
lation of adults died after receiving 1.5 ma of CC1.; doses of 0.18-0.92
ma, were reported to be fatal to children. Susceptibility 1n adults was
8-27
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correlated with alcoholic Intake (chronic alcoholism or exposure to alcohol
shortly after treatment), the presence of ascarld worms, and the Intake of
foods, particularly of high fatty content.
There are few postmortem reports on pathological changes 1n patients
after the Ingestlon of CCi.. McMahon and Weiss (1929) examined a 34-year-
4
old male alcoholic who died 5 days after drinking one ounce of CCl.. They
discovered some reddish-brown fluid 1n the abdominal cavity, early athero-
matous lesions In the heart, congested and edematous lungs with scattered
petechlal hemorrhages, enlarged and congested kidneys, marked erosion of the
esophagus, and a congested and enlarged fatty liver.
Cases of acute toxldty associated with CCl. Inhalation by humans have
been more numerous. Bilateral peripheral constriction of the ocular color
fields, resulting 1n symptoms of toxic amblyopla 1n three males, was attrib-
uted to the Inhalation of CCl vapors (Wlrtschafter, 1933). Five male
employees of dry cleaning establishments who had been exposed to CCl. (of
unknown concentration) from 8-10 hours dally for 1-6 months were examined.
Two men also had signs of conjunctivitis. Three of the men complained of
visual disturbances characterized by blurred vision or spots before the
eyes. Wlrtschafter concluded that toxic amblyopla may result from exposure
to CCl. vapor.
One fatality occurred 1n two cases of CCl. poisoning reported by
Smetana (1939). In the fatality, a dry cleaner and Interior decorator
described as being "a steady and heavy drinker" was exposed for several
hours to CCl vapors (concentration unknown) during work. Upon returning
from work, he noted dyspnea. Several hours after the exposure, headache,
dizziness and malaise developed, accompanied by nausea and repeated vomiting
that persisted for several days. The patient also suffered labored breath-
ing and cough with bloody sputum before he died 9 days after exposure.
8-28
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The second Inhalation case reported by Smetana was a housemaid also
described as having a history of chronic alcoholism. Three days before
hosp1ta!1zat1on, the patient cleaned dresses with CC1 for 3 hours In a
poorly ventilated room. Soon after exposure, she began to vomit. She
suffered symptoms similar to those described for the other case. After ~1.5
months of hospltallzatlon, this patient was released; her condition several
weeks later was described as "much Improved."
Seven of the cases of CC1. poisoning reported by Norwood et al. (1950)
resulted from both occupational and nonoccupatlonal Inhalation exposures.
In the three cases described as "severe" poisonings, there was a history of
chronic alcoholism; two fatalities occurred 1n this group. In one fatal
case, the victim had been exposed for about 25 minutes to an atmosphere
containing CC1 at an estimated 1575 mg/m3 (this estimate was made by
duplicating the conditions). H1stopatholog1cal examination of liver and
kidney tissue from the fatalities revealed liver necrosis and degeneration
of the renal tubules. The four remaining cases were characterized as "mild
Industrial" exposures. After CC1. exposure, all subjects suffered varied
i
symptoms Including nausea, vomiting, diarrhea, headache, muscular ache, pain
or numbness, labored breathing and dizziness.
In another case, a 31-year-old janitor suffered malaise, back and lower
abdominal pain, nausea and vomiting the morning after working for 5 hours In
a closed room with an unknown concentration of CC1, (Klttleson and Borden,
1956). He reportedly consumed two bottles of beer during the exposure
period. The patient required 2 months of hospltallzatlon for treatment of
acute renal Insufficiency as a result of CC14 Intoxication.
Elevated SGOT activities with concomitant liver changes were reported 1n
two men occupatlonally exposed to unreported concentrations of CC1
8-29
-------
(Lachnlt and Pletschmann, 1960). One became 111 after exposure to CC1,
for 3 hours 1n a relatively well-ventilated room. He was hospitalized 3
days after exposure. His liver was slightly enlarged, with the SGOT value
elevated by 6000 units. This value rapidly decreased and by the 10th day
had returned to normal. A biopsy of the Hver taken on the 8th day showed
necrosis In the centers of the lobull, but the surrounding tissue was
undamaged. An additional needle biopsy of the liver taken on the 28th day
showed that the cells had almost returned to normal. In the second case a
male similarly exposed to CC1 entered the hospital 12 days after expo-
sure. The SGOT had Increased to 80 units. A liver needle biopsy on the
22nd day showed only moderate changes, some of a degenerative nature.
In a chemical packing plant, use of CC1 by two workers for equipment
cleaning as a substitute for the customarily used acetone, resulted 1n the
hospUallzatlon of 4 of 43 workers at the plant (Folland et a!., 1976). Ten
additional workers became 111. Eight of- the 43 workers fell 111 within 12
hours following the start of the 2-hour exposure; six others followed within
the next 36 hours. The four hospitalized workers showed evidence of severe
disruption of liver function: one case had an SGOT level of 13,390 units.
All patients recovered within 90 days. All hospitalized workers, as well as
most of the others taken 111, had worked near a bottle-filling operation for
Isopropyl alcohol at the northern end of the plant, adjacent to the CCK
4
cleaning area.
Carbon tetrachlorlde concentrations at the time of exposure were not
ascertained; acetone was normally used for cleaning. Isopropyl alcohol
concentrations at the northern end of the plant averaged 2624 mg/m3.
Acetone 1n alveolar air samples of workers 1n the northern area averaged
121.6 mg/ma. The authors ascribed the toxic episode to CC1 toxldty
8-30
-------
potentiated by Isopropyl alcohol. Because CCl^ concentrations were
unknown and Isopropyl alcohol (and possibly other chemicals) were present,
the health effects reported 1n this study cannot be attributed to CC1.
4
exposure alone.
Some of these reports and others (Davis, 1934; Stewart et al., 1961;
Smith, 1950; NIOSH, 1975; von Oettlngen, 1964) Indicate that with single
exposure to low concentrations, there 1s considerable variation 1n symptoms
among different persons and that the acute toxldty 1s relatively low 1n
contrast to that with repeated exposure. Cases 1n which exposure 1s light
may be restricted to such symptoms as moderate Irritation of the eyes,
moderate dizziness and headache, which disappear promptly upon discontinua-
tion of the exposure.
The Immediate effects from acute Inhalation exposure to higher concen-
trations of CC1 consist of the same symptoms as described above; however,
the patient may also become nauseated and 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 eject bloody or b1le-sta1ned
vomltus which may last for days, and experience colicky pain and diarrhea
with liquid brown-black or bloody stools (von Oettlngen, 1964). This
tendency for hemorrhages may also result 1n bleeding from the gums and nose,
hemorrhages under the skin and macular papular rashes. The colicky pain may
be associated with a marked abdominal resistance simulating the "acute
abdomen" and thus has been mistaken for appendicitis and peptic ulcer.
Following such an acute episode, the patient feels tired and weak and fre-
quently suffers from headache. The patient may develop muscular twltchlngs
and epileptic convulsions. In a few Instances, paralysis (hemlplegla) and
polyneuHtls have been reported (von Oettlngen, 1964).
8-31
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In more severe Inhalation poisoning blood pressure may be lowered, but
as renal complications develop, the blood pressure 1s usually elevated and
the cardiac output 1s decreased 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 myocardlal Injury characterized by sinus bradycardla and followed
by aurlculoventrlcular arrhythmia, auricular fibrillation and sinus arrhyth-
mias (von Oettlngen, 1964).
Depending upon the condition of the patient, respiration may be normal,
rapid and shallow, or slow and labored. The latter 1s evident especially 1f
circulatory failure 1s Imminent and pulmonary edema develops. Thompson
(1946) found that early roentgenograms of the lungs may show pulmonary
Involvement.
In most Instances after severe Inhalation exposure, the patient develops
signs of liver Injury within a few days. The patient becomes jaundiced and
the liver becomes enlarged and tender. This Is toxic hepatitis, which may
pass Into yellow atrophy and, 1n more protracted cases, eventually Into
cirrhosis of the Hver. In the early stages of liver Injury, even before a
marked enlargement occurs and while Hver function tests such as the
cephaHn-flocculatlon test are still normal, the SGOT level may be markedly
elevated (von Oettlngen, 1964).
As signs of liver Injury develop, and sometimes 1n their absence, Injury
of the kidneys may dominate the clinical picture and be responsible for
early death (von Oettlngen, 1964). KHtleson and Borden (1956) character-
ized renal failure by three phases. The first phase 1s characterized by
polyurla and nocturla, which may result 1n severe dehydration, followed by
ollgurla and finally by diuresis. The renal Injury may result 1n acute
8-32
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nephritis with albumin, red and white cells, and casts In the urine. In
some patients, the presence of acetone and sugar 1n the urine has been
reported. The ollguria may be associated with Increased blood levels of
potassium, Indlcan, phenol, cresol, creatlnlne and urea; the latter may
result 1n uremia. In other Instances, the Injury may consist of necrotlzlng
nephrosls with comparatively IHtle change 1n the urinary composition. The
renal blood flow and glomerular filtration rate are decreased; and the
former seems to be mainly responsible for the maintenance of ollguria, being
the sequela rather than the cause of renal failure (von Oettlngen, 1964).
During the early stage of ollguria, abnormal tubular back diffusion of the
filtrate may play an Important role. Ollguria may develop as early as 24
hours or 3-4 days after onset of the poisoning and may persist for 12-14
days and even longer (von Oettlngen, 1964).
In the early stages after severe Inhalation poisoning and during the
period of polyurla, the blood may show some polycythemla, but later this may
be followed by anemia and lowering of the hematocrlt levels because of hemo-
dllutlon. However, the most Important changes 1n the blood are related to
the biochemical composition of the blood which reflects the renal and hepat-
ic Injury. As soon as the renal Injury develops, the nonproteln nitrogen
and urea nitrogen levels In the blood are Increased and may reach extremely
high values. The creatlnlne, Indlcan, phenol and cresol levels may also be
Increased. In the case of Hver Injury, as related to the blood, the Icter-
ic Index Is usually Increased, and the levels of sugar and phospollplds,
along with the ratio of cholesterol esters over cholestrol, are reduced.
The prothrombln time and the flbrlnogen content may be reduced, resulting In
an Increased clotting time. The chloride level 1s frequently lowered by
8-33
-------
hemod1lut1on or severe vomiting, and the potassium level may be elevated.
This Increase 1n potassium may contribute to ventricular fibrillation or
cardiac arrest (von Oettlngen, 1964).
In addition to constriction of the visual field and toxic amblyopla
(Wlrtschafter, 1933), CC14 poisoning can also result 1n blurred and double
vision. Conjunctiva! hemorrhages are common. Retinal hemorrhages and
exceptional cases of the degeneration of the optic nerve have been reported
(von Oettlngen, 1964).
Conway and Hoven (1946) report that after 1ngest1on of CC1., certain
electrocardiograph^ changes may be observed Indicating degenerative pro-
cesses 1n the heart,.muscle, such as sinus bradycardla, followed by aurlculo-
ventrlcular rhythm, auricular fibrillation, and sinus arrhythmias. The
respiration varies with the condition of the patient. If the patient 1s 1n
collapse, It will be rapid and shallow; 1f the patient 1s comatose, It may
be labored and dyspnelc, and pulmonary edema and hemorrhages may develop.
Eventually, disturbances develop characterized by polyurla and followed by
ollguria which may pass Into anurla. The urine of such patients 1s rich 1n
albumin and may contain blood and casts. If the liver 1s damaged, the urine
will contain uroblllnogen, urobllln, and bile pigments. The nonproteln
nitrogen level 1n the blood will be Increased and the patient may suffer
from hypoprothromblnemla, hypochloremla, and signs of addosls. Death may
ensue after 8 hours, or 3, 5 or 10 days, and sometimes later.
Two cases Involving the pancreas following Inhalation of an acute
exposure of CC14 were reported by Jahnke (1953). Both patients became
listless and developed hepatic and circulatory disturbances and sensitivity
of the pancreas to pressure. Such disturbances were long-term and had not
completely subsided after 10 months.
8-34
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Guild et al. (1958) reported on 20 cases of CCl^ Intoxication which
resulted In renal damage manifested by anurla. Four of these were the
result of Ingestlon. Exposures varied: one drank 15 cc; one drank 4
ounces; two drank small amounts but were also consuming alcohol (see Chapter
12). The remaining 16 cases Inhaled CCK with exposure times ranging from
30 minutes to 11 hours. Two of the four that Ingested the CC1. died
whereas three died of the 16 exposed through Inhalation.
Severe liver Involvement was seen 1n all four of those Ingesting CC1 .
Eleven of the 16 who Inhaled CC1, had "moderate to severe" liver Involve-
4
ment whereas the remaining 5 had no liver Involvement (Guild et al., 1958).
These discrepancies can be attributed 1n part to the variability In exposure
amounts and duration; however, this cannot be resolved from the report. The
role of alcohol consumption also cannot be defined, although this report
does lend some support to the research discussed 1n Chapter 12.
Stevens and Forster (1953) reported on the neurological effects of
CC14> Fifteen cases of CC14 Intoxication with varying amounts and
i
routes of exposure are discussed. Thirteen occurred In the home and two
occurred 1n commercial cleaning plants. Of the 13 cases occurring at home,
2 were children (the youngest being 9 months old) who drank the fluid. Two
were adults who accidentally drank 1t, and the others were exposed via
Inhalation. There were five deaths. Eleven of the 13 were alchohollc.
Four of the deaths occurred In alcoholics; for the fifth, no history was
available. Seven of the 15 experienced neurological symptoms. Of these,
two children who Ingested the chemical displayed stupor, drowsiness or
unconsciousness. The adults displayed headache, vertigo, weakness, blurred
vision, lethargy and coma.
8-35
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8.2.1.2. SUBCHRONIC/CHRONIC — Subchronlc/chronlc human exposures to
CC1. are nearly always by Inhalation. Headache, fatigue, dizziness,
nausea, and vomiting occur at substantially lower concentrations 1n these
exposures than In acute exposure (Elklns, 1942; Kazantzls and Bomford,
1960). As 1n acute exposures fatty and necrotlc Hvers are the common
pathological findings (Gray, 1947; Delllan and Wlttgens, 1962; Barnes and
Jones, 1967). In some cases, renal Injury 1s the major finding. Renal
tubular necrosis has been reported both with and without concomitant liver
disease (Hamburger et a!., 1958; Rlchet et a!., 1959; von Oettlngen, 1964).
Carbon tetrachlorlde poisoning as a result of chronic Inhalation of low
dose exposures has been reported by Butsch (1932), Wlrtschafter (1933),
Straus (1954), von Oettlngen (1964) and others. The clinical picture after
chronic CC1. exposure 1s much less characteristic than that after acute
exposure. With chronic exposure, patients may complain of fatigue, lassi-
tude, giddiness, anxiety, and headache. They suffer from parestheslas and
muscular twltchlngs and show Increased reflex excitability. They may be
moderately jaundiced, have a tendency to hypoglycemla, and biopsy specimens
of the liver may show fatty Infiltration. Patients may complain of lack of
appetite, nausea, and occasionally diarrhea. In some Instances, the blood
pressure 1s lowered accompanied by pain 1n the cardiac region and mild
anemia. Other patients develop pain In the kidney region, dysurla and
slight nocturla and have urine containing small amounts of albumin and a few
red blood cells. Burning of the eyes and, 1n a few Instances, blurred
vision are frequent complaints of those exposed. If these symptoms are not
pronounced or of long standing, recovery usually takes place upon discontin-
uation of the exposure If the proper treatment 1s received (von Oettlngen,
1964).
8-36
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Straus (1954) suggested a possible causal relationship between
exposure and aplastlc anemia. Three males had been exposed via Inhalation
and dermal absorption to CC1, at unknown concentrations for 2 months to 3
4
years. Autopsy findings Included hypoplasla of the bone marrow. However, a
causal relationship between CC1, and aplastlc anemia suspected by the
author 1n these cases 1s not supported adequately. One of the men had also
been exposed to kerosene for 3 years. Another was an auto mechanic who
worked 1n a garage. The occupation of the third was not specified although
his exposure to CC1 was occupatlonally-related. Thus the effects of
t
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 1n CC1 poisoning. In the third case the liver was reported to
have perlportal flbrosls and fatty Infiltration. These findings were
attributed to toxic hepatitis which was considered to be the result of
CC1. poisoning. The Information reported 1n these case studies tends not
to substantiate the author's suggestion that the patients' Illnesses may
have been caused by CC1 .
Postmortem reports on pathological changes In patients after Inhalation
of CC1. are generally limited to findings 1n the liver and kidneys. The
liver may show nutmeg appearance and fatty degeneration even In the absence
of clinical signs and symptoms of liver Injury. In other Instances, centrl-
lobular necrosis and hemorrhages with Infiltration of leukocytes and hlstlo-
cytes and collapse of the lobules with condensation of the retlcular frame-
work within these areas are seen. After chronic exposure, there may be
evidence of regeneration of the liver cells (von Oettlngen, 1964).
Postmortem changes 1n the kidney are characterized by nephrosls, by a
dlstentlon of Bowman's capsule with albuminous precipitates, and by swelling
8-37
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of the lining cells. The cells of the convoluted tubules may be swollen and
vacuolated; later, degenerative changes may be seen 1n Henle's loops,
associated with granular, hyaline, and cellular casts 1n the tubules. After
chronic exposure, regenerative changes may be visible 1n these regions. In
other cases, the kidneys may offer the picture of acute hemorrhaglc
nephritis (von Oettlngen, 1964).
Other postmortem organ changes are less characteristic for CC1.
poisoning and vary considerably with the clinical picture. Some changes may
occur that are a direct result of the changes occurring 1n the primary
target organs of CC1.. Stasis of various organs 1s the most outstanding
feature of cardiac failure. The brain and lungs may be edematous. The
Intestines may be hyperemlc and covered with numerous petechlal hemorrhages,
and the spleen may be enlarged and hyperemlc. Occasionally the adrenal
glands may show degenerative changes of the cortex, and the heart may
undergo toxic myocarditis (von Oettlngen, 1964).
8.2.2. Controlled/Clinical Studies. Human volunteers were exposed to
known concentrations of CC1 vapor 1n 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 CC1. vapors In a
series of three separate experiments performed 1 month apart. Prior to
exposure, data on blood pressure, SGOT and urinary urobHlnogen were ob-
tained for each subject. Prior to exposure, samples of exhaled breath,
urine and blood showed no detectable Cd.. The volunteers were seated 1n
4
a closed room (11 x 12 x 7.5 feet) where 99% pure CC1. was poured Into a
dish and covered with a towel. An exhaust system grill and door were closed
during the experiment but an air supply grill was left open. A fan circu-
lated air across the dish. Ambient concentrations of CC1. were monitored
4
8-38
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with a Davis hallde meter and an Infrared spectrometer. The CCI^ concen-
tration ranges and exposure times are given In Table 8-6.
Carbon tetrachlorlde was detected 1n exhaled breath 1n all three experi-
ments. Graphs showed an exponential decrease 1n concentration of CCK
versus time. The exact values were not given.
The serum Iron showed an Initial decrease 1n 3 of 6 subjects at the 309
mg/ma exposure level but had returned to normal 1n two of these subjects
68 hours after exposure. The remaining subject showed a 31% depression 1n
serum Iron at 68 hours, but the value was within the normal range. Serum
Iron was not analyzed 1n the other two experiments. Of the six subjects
exposed to 309 mg/m3 CC1 „ the serum transamlnase level was slightly
elevated 1n some and depressed 1n others, but remained within the normal
range. Carbon tetrachlorlde was not detected In the blood or urine at any
exposure time or dose, but the analytical technique used (Infrared method)
was not a sensitive one. The authors concluded that no 111 effects were
observed from exposure to CCl^ at 63 mg/m3 for 180 minutes, although the
small changes In serum Iron at the 309 mg/m3 dose might have been an
Indication of liver Insult.
A survey conducted by Kazantls and Bomford (1960) was the result of the
complaints of a worker 1n a factory processing raw quartz. Intermittently
for 2 years, he experienced anorexia, nausea and occasional vomiting with
abdominal discomfort. He noticed that his symptoms or dyspepsia worsened
during the workweek but got better on the weekends. The Investigators
Interviewed 17 of the 18 employees working In the same area taking medical
and occupational histories; their ages ranged from 16-54. Fifteen of the 17
complained of symptoms similar to the Initial case, primarily nausea,
anorexia, vomiting, flatulence, epigastric discomfort and depression. These
8-39
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TABLE 8-6
Exposure Times and Concentrations of Carbon Tetrachlorlde Vapor
In a Controlled Human Study*
Experiment Average Concentration,
Time-weighted (mg/m3)
Concentration
Range (mg/m3)
Exposure
(minutes)
1
2
3
309
69
63
192-548
63-88
57-88
70
180
180
*Source: Stewart et al., 1961
8-40
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symptoms had been occurring among the workers from 1 week to 2 years.
Environmental monitoring measured CC1 atmospheric concentrations of
292.5-650 mg/m3. Once the cause of the high concentration was found,
changes were made 1n the processing procedure. Within 1 week following
these changes, the symptoms disappeared 1n all workers. Follow-up for 6
months revealed no recurrences.
Direct application of CC1, to human skin causes a burning and stinging
rr 4
sensation within 5 minutes. The maximum pain 1s reached 6 minutes later and
Is associated with erythema, hyperemla, and wheal formation, later followed
by veslcatlon (Oettel, 1936).
The absorption of CC1. through human skin was measured by Immersion of
the thumbs of three male and female volunteers 1n a sample of this compound
for 30 minutes (Stewart and Oodd, 1964). The CC1 was analyzed by Infra-
red spectroscopy, and no Impurities were detected. 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 tetrachlorlde was detected 1n the
alveolar air of each subject within 10 minutes of Immersion of their thumbs.
The concentration In the expired breath rose continuously to a maximum of
4.0 mg/m3 10-30 minutes after the exposure period ended, and then
decreased exponentially. The mean concentration of CC1 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 that CC1
could be absorbed through the skin In toxic quantities.
Hall (1921a,b) demonstrated the effectiveness of CC14 as a vermlcldal
agent In treatment of hookworm Infestations. The usage of CC1. 1n this
8-41
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capacity stimulated considerable research efforts to Investigate the pharma-
cologlc and physiologic effects of CC14 on humans (NIOSH, 1975)., The
effects of oral doses of CC1 as a human anthelmlntlc, administered to
condemned prisoners 1n Ceylon has been reported (Docherty and Burgess, 1922;
Docherty and Nlcholls, 1923). Three of the prisoners received 4 ma
CC14, two received 5 mil CCl^, and one received 5 ma plus an addi-
tional 3 ma 2 weeks after the first dose. Execution of the prisoners
occurred 3-15 days after the CC14 administration. Autopsies were per-
formed and the findings varied. The Hvers of some showed no major micro-
scopic or macroscopic changes whereas the Hvers of others showed marked
fatty degeneration. From such data, a dose-response relationship would be
difficult to determine (NIOSH, 1975).
A cross-sectional ep1dem1olog1c study (Sonlch et al., 1981) examined the
effect upon persons exposed to CC14 through their drinking water. Seventy
tons of CC14 were spilled 1n the Kanawha and Ohio Rivers In 1977.
Measurements of raw water revealed maximum concentrations of 0.340 mg/a.
Twenty-one cities situated along the river were Involved 1n the study.
These cities represent areas that draw their drinking water directly from
the river and/or areas that draw their drinking water from sources not
Influenced by the quality of the river water. Measurements at each of the
four major cities along the river showed a decrease 1n the CCK concen-
4
tratlon In the river with the number of river miles from the spill: Hunt-
Ington, West Virginia - 0.210 mg/a; Cincinnati, Ohio - 0.180 mg/a;
Louisville, Kentucky - 0.110 mg/a; Evansvllle, Indiana - 0.060 mg/a.
Finished drinking water 1n Cincinnati contained a peak level of 0.087
mg/a. A control period was Identified 1n 1976 when there were nondetect-
able to trace amounts of CC1 1n the river as Indicated by monitoring
8-42
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efforts at Cincinnati. By using river volumes and flow rates, periods of
high exposure (1977) and low exposure (1976) to CC1 were estimated for
each city along the river. A total of 35 hospitals 1n these cities pro-
vided medical data on patients admitted during these estimated time periods.
The results of routine tests measuring serum chemistries reflecting liver
and kidney function along with basic ep1dem1olog1c Information were
abstracted from -6000 medical records. It Is hypothesized that CC14 would
cause a rise In some or all of these serum chemistries. The data were
categorically analyzed to test for a dose/response relationship. In this
capacity, the ratio of the odds ratios (ROR) for each serum chemistry for
each city group were computed as ROR = OR77/OR?& where
odds rati075
(OR76)
and
odds
(OR77)
the odds of having an elevated test result
while drinking Ohio River water In 1976
the odds of having an elevated test result
while drinking water In 1976 not affected
by the quality of the Ohio River
the odds of having an elevated test result
while drinking Ohio River water In 1977
the odds of having an elevated test result
while drinking water 1n 1977 not affected
by the quality of the Ohio River.
The results obtained for creatln'me show a positive and statistically sig-
nificant (p<0.05) dose/response relationship between the CC14 exposure and
the ROR or frequency of elevated levels of serum creatlnlne 1n exposed
patients 1n relation to the controls as determined by a test for linearity
of trend. Other parameters analyzed were alkaline phosphatase, total
blUrubln, blood urea nitrogen, lactic dehydrogenase, SGOT and Y
transpeptldase. No similar results were found for these parameters.
8-43
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8.3. MECHANISMS OF TOXICITY
The toxldty of CC14 to an organism depends upon the ability of the
organism to metabolize the compound; unmetabollzed CC1 does not appear to
be significantly toxic (Recknagel and Glende, 1973). In mammals, as dis-
cussed 1n Chapter 1, CC1 1s thought to be metabolized 1n the endoplasmlc
retlculum of the Hver by the mixed-function oxldase system of enzymes. A
reaction sequence proposed 1n the literature for CC14 metabolism 1s out-
lined 1n Figure 7-1. Two free radicals have been postulated as metabolic
Intermediates: the trlchloromethyl radical and the chlorine radical. The
toxldty of CC1 has been attributed to subsequent reactions of the
trlchloromethyl radical. These reactions Include formation of carbonyl
chloride (phosgene), d1mer1zat1on to hexachloroethane, free radical binding
to protein, and I1p1d peroxldatlon. In this chapter each of these proposed
pathways will be presented 1n conjunction with the toxic effects attributed
to 1t.
8,3.1. Formation of Carbonyl Chloride (Phosgene). From the results of an
In vitro study of CCl^ metabolism, Shah et al. (1979) postulated the for-
mation of carbonyl chloride from the trlchloromethyl radical. As previously
discussed, the authors Incubated L-cyste1ne and [14C]CC1. with rat liver
homogenate and looked for the formation of 2-oxQth1ozol1d1ne-4-carboxyl1c
add. This compound 1s formed from the reaction of L-cystelne and carbonyl
chloride. Analysis of the metabolic products by mass spectroscopy showed a
fragmentation pattern consistent with 2-oxoth1ozol1d1ne-4-carboxyl1c add.
The authors Inferred from these analytical data that carbonyl chloride was
formed as a metabolic product of CC1.. Although carbonyl chloride
(phosgene) Is not reported to be a carcinogen, the authors pointed out that
the compound 1s highly toxic and that the reactive chlorines could react
with macromolecules 1n ways similar to alkylatlng agents.
8-44
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8.3.2. Dlmerlzatlon to Hexachloroethane. Hexachloroethane has been Iden-
tified as a metabolite of CC1 by Fowler (1969). The formation of this
4
compound Is believed to take place by the d1mer1zat1on of the trlchloro-
methyl radical. Although hexachloroethane 1s a hepatotoxln, Us toxlclty Is
less than that seen 1n CC14 poisoning. Therefore, other mechanisms
probably account for the severe toxlclty of CC14.
8.3.3. Free Radical Binding to Proteins. Free radical binding to
proteins has been postulated as one cause of toxlclty associated with CC14
(Recknagel and Glende, 1973). The binding was reported to Involve reactions
with cellular proteins, particularly those with sulfhydryl groups.
DelvUlarruel et al. (1977) found a good qualitative correlation between
Intensity of CC1, activation and P-450 content of various organs 1n male,
virgin female, or pregnant female rats. No correlation was found between
CC1 activation and cytochrome c reductase activity. The authors state
4
that their results suggest that P-450 1s Involved 1n CC14 activation and
that Irreversible binding of CC14 metabolites to cellular components,
rather than I1p1d peroxldatlon, 1s responsible for some biochemical and/or
ultrastructural lesions reported In different tissues. In another study,
[14C]CC14 has been observed to bind Irreversibly to rabbit mlcrosomal
proteins at a rate of ~20 nmole/mg protein/hour (Uehleke and Werner, 1975).
Binding of CC14 (or Us metabolites) to hepatic macromolecules was
enhanced 1n the absence of oxygen, consistent with the proposal that the
trlchloromethyl radical 1s the reactive metabolite. Furthermore, the order
of species susceptibility to liver necrosis from CC14 more closely
8-45
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parallels the species order for ["CJCC14 binding to cellular components
than the species order for I1p1d peroxldatlon (D1az Gomez et al., 1975):
Effect
Liver necrosis
["C]CC14 binding
Llpld peroxldatlon
«• Most susceptible Least susceptible ->
mouse > guinea pig = hamster > rat > chicken
mouse = hamster > guinea pig > rat > chicken
rat > hamster = guinea pig > chicken = mouse
These authors also report that 1n mice liver necrosis proceeded 24 hours In
the absence of I1p1d peroxldatlon.
Although 1t has been shown that [^C] from CC1 binds to proteins,
the question of CC14 binding to polynucleotldes remains. The Issue 1s
Important because of Its Implications for the mechanism of CC1 carclno-
genlclty and mutagenldty. In the only experiment addressing this question,
Uehleke and Werner (1975) Incubated [«C]CC1 "with either Isolated liver
mlcrosomes (rat or mouse, species not Identified) or with added soluble RNA.
They reported no [^C] binding to Mbosomal RNA or exogenous RNA. Experi-
mental details were not presented, but 1t appears possible to tentatively
conclude that proteins — rather than nucleic adds — are the main sites of
macromolecular CC14 binding.
8.3.4. Llpld Peroxldatlon. A number of the hepatic effects resulting
from CC14 exposure, Including the fatty liver syndrome, are believed to
arise as a result of llpld peroxldatlon (Recknagel and Glende, 1973). The
mechanism proposed for the peroxldatlon 1s presented below, followed by a
discussion of the evidence that this biochemical sequence results 1n the
hepatic lesions associated with CC1 poisoning.
8-46
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The first step In the reaction sequence proposed for I1p1d peroxldatton
Is production of free radicals, especially the trlchloromethyl free radical.
The radical Initiates a chain reaction by reacting with the hydrogen atom of
a -CH9-group 1n an unsaturated fatty add, generating a fatty add free
radical. On reaction with molecular oxygen, the fatty add free radical 1s
converted Into an unstable organic peroxide. The peroxide disintegrates 1n
two fashions: (1) Intramolecular cycllzatlon to form malonlc dlaldehyde and
two new free radicals, or (2) simple homolytlc fission that also yields two
free radicals. This whole process occurs autocatalytlcally: each free
radical gives rise to two new free radicals. Figure 8-1 summarizes this
hypothesis (Recknagel and Glende, 1973).
A number of Indices have been used In in vivo and In. vitro assays of
Upld peroxldatlon: pentane and ethane levels 1n exhaled air {arising from
fatty add decomposition) and malonlc dlaldehyde concentrations 1n hepato-
cytes {arising from Intramolecular cycllzatlon). Pentane production 1n male
Sprague-Daw!ey rats Increased by factors of 4.6, 13.2 and 26.4 over that 1n
mineral oil controls within 30 minutes following 1.p. administration of
CC1. doses of 160, 480 and 1440 mg/kg bw, respectively (Sagal and Tappel,
4
1979).
A mechanism for the pathogenesis of CC1 -Induced hepatic lesions based
on llpld peroxldatlon has been proposed recently (PasquaH-Ronchettl et al.,
1980). According to this hypothesis, llpld peroxldatlon Is suggested to
affect primarily unsaturated acyl chains of membrane phosphoHplds, result-
Ing In breakage of the hydrocarbon and loss of phosphollplds from the
membrane. Llpld peroxldatlon would therefore produce progressive degenera-
tive changes 1n the assembly of membranous structures such as {rat) liver
endoplasmlc retlculum, or Us 1n vitro counterpart, mlcrosomes.
8-47
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H H H
-c=c-c-c=c-c-c=c-c-c=c-
H /HK H
HCC13
CCljTrichlormethyl
Free Radical
-C-C-C-C-CTC-C-C>C-C=C
RESONANCE (All
Possible Forms Not
Shown.)
Organic Free Radical
Y Q a J a B y
oc-c-c- oc-c=c-c-c<-
Peroxide •
formation 02 diene conjugation, Amax=233mu
C-C-C=C-C=C-C-C=G
Organic Peroxide (Unstable)
Intramolecular cyclization
and decomoosition to yield
malonic dialdehyde and two
new organic free radicals.
Decomoosition to yield two free
radicals. Eventual stable decom-
position products highly organo-
leotic.
FIGURE 8-1
Free Radical Initiated, Autocatalytlc Peroxidatlon of
Polyenlc Long-Chain Fatty Adds
Source: Adapted from Recknagel and Glende, 1973
8-48
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This hypothesis Is supported by studies showing that treatment with
produced llpld peroxldatlon 1n male Wlstar rat liver endoplasmlc
retlculum at a concentration of 0.5 mil/100 g bw (Pasquall-Ronchette et
al., 1980), caused disintegration of endoplasmlc retlculum Ijn vitro within
10 minutes at a concentration of 636 mg/!l {Pasqual1-Ronchett1 et al.,
1980), and was Incorporated predominantly Into Uver phosphol1p1ds In rats
(Table 8-7) (C1ccol1 and Cas1n1, 1978).
8.4. SUMMARY
Carbon tetrachlorlde 1s toxic to humans and animals following Inhala-
tion, Ingestlon or dermal administration. Acute, subchronlc and chronic
exposures primarily affect the central nervous system, liver and kidneys.
Sporadic cases of ocular toxlcity also occur following subchronlc and chron-
ic exposure to CC1. vapor. However, these ocular signs do not correlate
with exposure levels or other organ tox1c1t1es. Ingestlon of alcohol
appears to Increase susceptibility to CC1. toxldty, but the mechanisms
are unknown.
8.4.1. Experimental Animal Data. The toxlcity of CC1. following acute
Inhalation, Ingestlon and dermal exposures has been reported for various
species. Animals surviving acute doses of CC1 developed Uver damage
and, 1n some cases, kidney damage. These Injuries were dose related.
• Subchron1c/chron1c studies of CC1 exposure 1n rats, monkeys, rabbits,
dogs and guinea pigs demonstrated Uver, 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 Uver than expo-
sure to a lower concentration over a longer period of time even though the
product of time and concentration 1s equal 1n both cases.
8-49
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TABLE 8-7
Incorporation of 14C from [14C]Carbon Tetrachlorlde
Into L1p1ds of Various Rat T1ssuesa»b
Tissues Total Llplds
( dpm/mg )
Liver (6) 112.6 ± 7.4
Intestinal mucosa
Kidney (6)
Adrenals0
Lung (5)
Spleen (6)
Testls (6)
Brain (6)
Heart (6)
Skeletal muscle (6)
Plasmad
61.8 + 7.5
23.4 i 2.6
8.0
11.3 + 1.8
8.7 ± 1.2
6.3 ± 1.5
3.3 i 0.3
2.2 ± 0.8
0.7 ± 0.1
5.1
Acetone Precipitate
(phosphollplds)
(dpm/mg)
135.7 ± 14.6
69.5 i
25.6 i
22.0
15.2 ±
7.4 i
5.2 i
3.7 +
2.6 +
2.9 ±
trace
7.7
3.9
1.4
2.3
1.4
0.3
1.4
0.7
Acetone Supernatant
L1p1ds
(dpm/mg)
53.3 ±
48.3 +
11.4 i
2.4
6.5 +
4.0 i
2.3 ±
0.7 ±
0.8 ±
0.6 ±
3.7
4.8
8.4
2.0
0.8
1.0
1.0
0.2
0.5
0.2
aSource: Adapted from C1ccol1 and Caslnl, 1978
b[14C]CCl4 dose: 4000 mg/kg bw (58.6 x 106 dpm). Values are expressed as
means £ S.E.H. The number of rats 1s reported 1n parentheses.
cE1ght pooled adrenals
dPlasma of two animals
dpm = disintegrations per minute
8-50
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It Is recognized that Interspecles differences exist particularly with
regard to the metabolism of CC1. and other pharmacoklnetlc parameters as
well as sensitivity to CC14- However, data quantifying such differences
have not been found 1n the available literature. Methods of dose conversion
from animal species to humans are discussed 1n the Appendix.
8.4.2. Human Data. Considerable human exposure to CC1 through Inhala-
tion has occurred through Its use as an Industrial solvent and dry cleaning
fluid. Ingestlon of CC1 or of mixtures containing CC1 has also been
documented In various case reports. Ingestlon has occurred under different
circumstances by persons of diverse occupations and ages. These acute
exposures have been followed by hepatoxlc effects accompanied by acute
nephrosls.
Hepatic necrosis and renal pathology appear to be characteristic effects
of acute human exposure to CC1 . If exposure 1s terminated, the liver
shows regeneration 1n most cases. In cases of acute renal dysfunction,
kidney function returns to normal after exposure 1s terminated and medical
treatment 1s given.
In many of the case reports and older studies, the Investigators present
the data 1n narrative form. Although Interesting, these type of data are
not suitable to quantitative analysis since numbers are not adequately pre-
sented. Furthermore, there usually are a number of uncontrolled variables
(alcohol Intake, age, simultaneous exposures) or unknown variables (exposure
amount) making It difficult to attribute the outcome solely to the CC14
exposure. The experimental human studies are not numerous, yet they are
Important since they can either support or challenge the experimental animal
studies and can aid qualitatively 1n the extrapolations from animals to
humans.
8-51
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8.4.3. Mechanisms of Tox1c1ty. The chemical pathology of CC14 liver
Injury 1s generally viewed as an example of lethal cleavage, .where the
CC13-C1 bond 1s split 1n the mixed function oxldase system of hepatocytes.
Two major sequences of this cleavage have been suggested; both views presume
the formation of free radicals from the homolytlc cleavage of the CC1 -Cl
O
bond (I.e., *CC13 and •€!). One sequence entails the direct attack
(via alkylatlon) by free radicals on cellular constituents, notably protein
sulfhydryl groups. The second sequence Involves the extraction of a hydro-
gen atom by the trlchloromethyl free radical from a long-chain fatty add to
form chloroform and a fatty acid free radical. Molecular oxygen, because of
Us triplet ground state, binds with the unpaired electron on the fatty add
radical to form an organic peroxide. The peroxide 1s unstable and decom-
poses to form more organic free radicals, which In turn form more organic
peroxides (Recknagel and Glende, 1973). This process appears to lead to
fatty acid chain decomposition, with the resulting breakdown of membrane
structure (Recknagel and Glende, 1973). This breakdown may lead to a halt
1n Upld excretion via the Golgl apparatus, with fatty liver occurring as a
consequence. Cell necrosis would also follow directly from I1p1d destruc-
tion. The mechanism by which I1p1d peroxldatlon could lead to cell trans-
formation 1s not explained at present, and the molecular events leading to
CCl^ cardnogenldty remain unknown,
In addition to these proposed mechanisms of toxlclty, two minor metabol-
ic pathways have been postulated: d1mer1zat1on of two trlchloromethyl free
radicals to form hexachloroethane (Fowler, 1969) and the formation of a
trlchloromethyl peroxy radical which may result In production of phosgene
and carbon dioxide (Shah et a!., 1979). Both hexachloroethane and phosgene
8-52
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are toxic, but the extent of their contribution to observed hepatotoxlclty
1s unknown. Carbon tetrachlorlde has not been found to bind to cellular
polynucleotldes, but presently only one Investigation studying the binding
of CC1. to such nucleotldes has been reported (Uehleke and Werner, 1975).
4
8-53
-------
-------
9. TERATOGENICITY AND OTHER REPRODUCTIVE EFFECTS
9.1. TERATOGENICITY
In two Inhalation studies on the teratogenlc and prenatal toxlcologlc
effects of CCl^, the chemical was reported to produce prenatal toxlclty
but not teratogenlclty. Schwetz et al. (1974) exposed pregnant Sprague-
Dawley rats to CC14 at 1800 or 6300 mg/m3 for 7 hrs/day on days 6-15 of
gestation. Statistically significant decreases 1n fetal body weight and
crown-rump length were observed. Other parameters examined such as sex
ratio, live fetuses/Utter and resorptlons were not significantly different
from those of controls. Two other statistically significant fetal effects
were noted: an Increased Incidence of Utters with sternebral anomalies 1n
the 6300 mg/m3 group and an Increased Incidence of Utters with subcuta-
neous edemas 1n the 1800 mg/ma group. The Incidence of litters with edema
1n the 6300 mg/m3 group (50%), although apparently Increased, was not
significantly different from the control Incidence (33%). The dams exposed
to both concentrations of CC1 showed a decreased food consumption com-
pared to control animals and a statistically significant decrease 1n weight
gain. Both of these effects were greater at the higher dose level. Hepato-
toxldty, as measured by significantly Increased SGPT activity, was also
seen 1n the dams following dally exposure to 1800 or 6300 mg/m3 CC1, but
the Increase was greater at the lower dose level. No consistent pattern was
established between fetal toxlclty and maternal toxlcity at the subanesthet-
1c levels of CCl^ used 1n this experiment. The authors concluded that
CC14 was not highly embryotoxlc at the concentrations used. The evidence
of maternal toxlclty precluded any statement about the teratogenlc potential
of CC14.
9-1
-------
The other Inhalation study resulted In no teratogenlc effects following
exposure of pregnant rats to CC1 at 1575 mg/m3 8 hrs/day for 5 consecu-
tive days between days 10-15 of pregnancy (Gllman, 1971). Concomitant
exposure to 15% ethanol 1n drinking water also did not result 1n teratogenlc
effects. Carbon tetrachlorlde exposure, however, did decrease the viability
Index to 83% as compared to 99% for controls (p<0.1), resulting 1n a
decrease 1n the number of pups per Utter, 9.2 as compared to 10.3 for con-
trols. The lactation Index was also decreased to 83% compared to 98% for
controls. Concomitant ethanol exposure exacerbated the former effect: 8.48
pups per Utter compared to 10.3 for controls. Although only 10 control and
25 experimental animals were used, thus lessening the sensitivity, the
results of this study Indicate prenatal toxlclty and tend to support those
of Schwetz et al. (1974).
Subcutaneous exposure of CC1 to pregnant rats has resulted In liver
damage 1n fetuses and neonates (Bhattacharyya, 1965). Administration of
1600 mg/kg CC14 s.c. on days 20 or 19 of gestation resulted 1n small areas
of focal hepatic necrosis 1n neonates born 48 or 72 hours later, respec-
f
tlvely. H1stolog1c findings generally Included a sharply demarcated area of
centrllobular necrosis and proliferation changes 1n nonnecrotic lobes.
The Investigator also treated fetuses directly with CC1 by subjecting
the dam to a laparotomy and either Injecting the chemical directly Into the
fetus or Into the amnlotlc sac through the uterine wall. Liver changes
following Injection of 6 mg CC1. were variable; cells generally became
extremely pale In centrllobular and mldzonal areas, Indicating fatty
Infiltration. Livers remained abnormal until at least 4 days after birth.
No necrosis, hemorrhage or regeneration was observed.
9-2
-------
Liver sensitivity of neonate rats to CC14 was reported to be low 1
hour after birth, then to rise above the adult level at 19 hours and to
decline to adult levels by 3-7 days after birth. Thus, only 2 of 10
1-hour-old neonates receiving 1600 mg/kg CC1. s.c. showed centMlobular
necrosis after 24 hours. In addition, hepatic portal areas contained
numerous neutrophlls, but In contrast to findings 1n adult animals, no bile
duct proliferation could be observed. More pronounced hepatic damage was
found In 19-hour neonates than 1-hour neonates. Damage declined 1n 3- and
4-day-old neonates; that 1n 5-, 6- and 7-day-olds was similar 1n appearance
to that of adults.
Neonates can apparently be exposed to CC1 through mothers' milk
(Bhattacharyya, 1965). Subcutaneous administration of CC1. at 1600 or
3200 mg/kg bw to four nursing rats resulted 1n hepatic damage 1n the neo-
nates 24 or 48 hours later. A dose of 800 mg/kg bw to dams did not produce
hepatic damage to offspring. Levels 1n the milk were not reported.
The pre- and postnatal toxlcologlc studies described above do not meet
current design criteria for hazard assessment purposes 1n that fewer than
three 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
Testlcular degeneration was observed 1n rats receiving CC1. at 4800
mg/kg bw 1.p. (Chatterjee, 1966). One group of six male rats received
CC1 as a 1:1 mixture In coconut oil. The vehicle control group received
only an equal volume of coconut oil. All animals were sacrificed on day 15.
Body weights were similar for treated and control animals. However, the
relative testes weight decreased from 15.5 (±0.4) g/kg bw 1n controls to 9.8
9-3
-------
(£1.2) g/kg bw In exposed animals. A decrease In testls size and weight 1s
a good Indicator of a decline 1n male spermatogenlc process. Relative
weight of seminal vesicles showed an even more pronounced decrease: 1.27
(±0.171) g/kg bw 1n treated as compared to 3.10 (±0.059) g/kg bw 1n control
animals. Relative pituitary weight, however, Increased: 50.0 (±1.4) mg/kg
bw 1n treated as compared to 32.4 (±0.9) mg/kg bw 1n control animals. This
Increase would be expected 1f the feedback mechanism from the testls was
obliterated and pituitary concentrations of gonadotrophln were Increased.
However, 1n this study actual pituitary content of gonadotrophlns was not
measured. Therefore, no conclusions can be made on the effect of the
chemical on the feedback mechanism, making the significance of the Increase
1n pituitary weights difficult to Interpret.
Further, hlstologlcal examination of testes In CC1 -treated animals
showed testlcular atrophy and "some abnormality" 1n spermatogenesls. The
author proposed a mechanism for CC1.-Induced testlcular atrophy 1n which
blockage of pituitary hormone release results In atrophy of Leydlg cells
within the seminal vesicles, followed by an abnormal spermatogenesls.
In another study (Kalla and Bansal, 1975), Intraperltoneal administra-
tion of CC1. (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
1n spermatogenesls as Indicated by hlstologlcal examination. Vehicle
controls were administered equal volumes of coconut oil. Weights of testes,
seminal vesicles, ep1d1dym1s and prostates were decreased In exposed
animals, whereas the weight of adrenals Increased (Table 9-1). The gonado-
somatlc Index (GSI: equal to body weight x testes weight/100) was also
decreased 1n treated animals. A slight decrease 1n pituitary weight was
observed following a 10-day treatment but not after either the 15- or 20-day
9-4
-------
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pituitary content of various hormones, measurements of the synthesized,
stored and released hormones should be made. In addition to these criti-
cisms, the two studies described In this section had shortcomings with
regard to current experimental design criteria for hazard assessment: less
than three dose levels and no data for a second, nonrodent, species.
The relationship between maternal toxldty and reproductive effects was
Investigated by WHson (1954). The author compared fetal and maternal
tox1c1t1es of rats (strain not given) across several treatments and conclud-
ed that not all physiological disturbances of the mother will alter fetal
development. In fact, little correlation existed across treatments between
the severity of maternal toxldty and the extent of effect to the surviving
offspring. In the treatment consisting of CC1 poisoning, surviving
Utters were virtually normal, even though several dams died and others had
premature termination of pregnancy (I.e., resorptlon). The exposure
schedule was 0.3 cc CCl^ by stomach tube or 0.8 cc subcutaneously In corn
oil given 2 or 3 times on as many successive days during gestation.
Teratogenlc effects 1n humans caused by CC1. exposure have, not been
reported. However, human fetuses 1n one study appeared to have selectively
accumulated CCl^ from the mothers' circulation (Dowty and Laseter, 1976).
Maternal blood samples were taken from 11 women either before or directly
after vaginal delivery (prior exposure of the women to toxic chemicals was
not reported). Paired cord blood samples were obtained Immediately after
delivery. All volatHes were analyzed by gas chromatography and mass
spectrometry. Carbon tetrachlorlde, benzene and chloroform were present 1n
higher concentrations In cord blood as compared to maternal blood.
9-7
-------
9.3. SUMMARY
Carbon tetrachlorlde has produced prenatal toxic effects, some of which
(I.e., subcutaneous edema) could not be associated with extent of maternal
exposure. Rats exposed to CC1. in utero have shown hepatic abnormalities
at birth, but the fetal rat liver appears to be less sensitive than the
adult liver to the hepatotoxlc effects of CC1 .
Carbon tetrachlorlde has produced distinct degenerative changes 1n
testlcular histology 1n rats, eventually resulting 1n aspermatogenesls and
functional male Infertility. These effects occurred following 1ntraper1-
toneal Injection at relatively high doses. Unfortunately, low doses were
not tested.
Due to the limited number and scope of the studies reported In this
chapter, 1t Is difficult to evaluate the potential of CC1. to cause
adverse teratogenlc, embryotoxlc 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 teratogenlc or reproductive effects according to testing
criteria such as those currently used for U.S. EPA Office of Pesticides
Programs or Office of Toxic Substances.
9-8
-------
10. MUTAGENICITY
The mutagenlc potential of CC1 has been assessed by evaluating the
results of seven bacterial studies, one yeast study, one in vitro mammalian
chromosome damage study and three Iji vivo DNA damage studies 1n rodents.
The majority of these studies were negative. Information relating to the
metabolism of CC1 and to covalent binding of the metabolites to cellular
macromolecules (Including DNA) 1s presented before the sections assessing
the genotoxlclty of CC1.. This was done to set the stage for the discus-
sion of the largely negative results obtained 1n the mutagenldty studies
described below and for the suggestion that CC1 may be a weak mutagen.
In addition, suggestions for additional testing are presented.
10.1. METABOLISM AND COVALENT BINDING TO MACROMOLECULES
The evidence described In this section suggests that CC1. 1s metabo-
lized In the Hver to highly reactive Intermediates (the trlchloromethyl
free radical and phosgene). The evidence also Indicates that metabollcally
activated CC1. covalently binds to protein, llpld and DNA, suggesting that
CC1. may have genotoxlc potential.
10.1.1. Metabolism. Carbon tetrachlorlde Is metabolized 1n the liver
endoplasmlc retlculum by the cytochrome P-450 component of the mixed
function oxldase system (Reynolds and Moslen, 1980). The available evidence
Indicates that metabolism of CC1 results In the generation of the
trlchloromethyl free radical -CClg (Reynolds and Moslen, 1980; Trudell
et a!., 1982) and phosgene (Shah et al . , 1979; Kublc and Anders, 1980; Pohl
et al., 1981). These two substances are the most likely of the metabolites
to exhibit reaction with tissue macromolecules because of their high
reactivity. By using human cytochrome P-450 reconstituted 1n phospho!1p1d
10-1
-------
vesicles, Trudell et al. (1982) have demonstrated that »CC13 1s the
major product of the reductive metabolism of CC1 as determined by mass
spectral Identification of the adducts formed between »CC1~ and the
phosphollpld dloleoyl phosphatldylchoHne.
Phosgene results from further metabolism of »CC1_ (Shah et al.,
1979) (see Figure 7-1).
CC1,
•CC13 -»-»•» C1-C-C1
Pohl et al. (1981) measured the amount of phosgene (as dlglutathlonyl
dHhlocarbonate) produced from the aerobic metabolism of CC1., CHC1« and
*\ o
CBrCl- by Hver mlcrosomes (from phenobarbltal-treated rats) plus
O
cofactors. The results Indicate that phosgene production from CC1. Is
only 4% of that produced from CHClg. Thus, the level of phosgene produc-
tion from aerobic metabolism of CC1. Is small. The carclnogenlclty and
DNA binding of phosgene are currently under Investigation In Dr. B.L. Van
Duuren's laboratory at New York University Medical Center. Preliminary
evidence Indicates that phosgene binds to DNA (Dr. S1pra Banerjee, personal
communication). Metabolites of CC1. are so reactive that they bind to and
$
Inactivate the cytochrome P-450 enzymes that were responsible for their
generation (suicide mechanism) (Valnlo et al., 1976; S1pes et al., 1977;
De Groot and Haas, 1980; Cooper and WUmer, 1982).
10.1.2. Covalent Binding to Macromolecules. Metabollcally activated
CC1 has been found to bind covalently* to Upld and protein both jm vivo
(Rocchl et al., 1973; D1az Gomez and Castro, 1980a,b; and in vitro (Rocchl
et al., 1973; Uehleke et al., 1977). Uehleke et al. (1977) measured
*Amount of label bound was determined after washing and extraction, Indicat-
ing that the binding was covalent.
10-2
-------
covalent binding of 1 mM [14C]CC14 (0.25 Cl/mol) to mlcrosomal protein
and Upld In liver mlcrosome suspensions (5 mg protein/ma, plus cofactors)
from phenobarbltal-treated rabbits. About 10% of the 14C label was
covalently bound to endoplasmlc retlculum protein and >30% was bound to
mlcrosomal I1p1d. Extramlcrosomal binding was evaluated by the addition of
5 mg of bovine serum albumin per ma. to the CC1 /mlcrosome mixture,. The
binding of metabollcally activated [14C]CC14 to added bovine serum albu-
min (1.4 nmol/mg 1n 60 minutes) was about 1.5% of that bound to mlcrosomal
protein (20.0 nmol/mg) plus I1p1d (76.0 nmol/mg). Thus, 1t appears that
binding of metabollcally activated [14C]CC1. to extramlcrosomal macro-
molecules Is negligible compared to binding to mlcrosomal constituents.
(Mutagenlclty studies were also described 1n this paper and are discussed 1n
the next section.)
Evidence that the CC1 metabolite, phosgene, reacts with proteins was
obtained by Cessl et al. (1966) when they measured the In vivo binding of
CC1. to rat Hver proteins and compared It to the jn_ vitro acylatlon of
poly-L-lys1ne and serum albumin by phosgene. Similar reaction products were
obtained 1n both systems, suggesting that phosgene reacts with the lyslne
e-am1no groups In proteins, leading to cross-linked carbonyl derivatives:
0 NH
C1-C-C1 + 2 lyslne
phosgene proteins cross-linked proteins
H 0 H
-HI
-*- lys1ne-N-C-N-lys1ne
or
C1-C-C1
phosgene
NH,
NH,
• f\ •»• • rj
...ly'slne lyslne.
protein
NH-C-NH
-*-lys1ne .lyslne
cross-linked protein
10-3
-------
Such cross-linked proteins would undoubtedly exhibit Impaired biological
activity. It 1s also possible that similar cross-Unking reactions of
phosgene can occur with amlno groups 1n DNA, resulting 1n alterations 1n DNA
structure and function.
Binding of metabollcally activated CC1. to DNA was found by two
groups. Rocchl et al. (1973) studied the binding of CC1. to nucleic adds
and protein. "C-labeled CCl^ (367 ymol/kg) was Injected Into rats
and mice and the amount of metabollte(s) of CC1. that covalently bound to
liver DNA, RNA, nuclear proteins, and cytoplasmlc proteins was measured.
Significant amounts of labeled material were found associated with rRNA,
nuclear proteins and cytoplasmlc proteins In rats. When the rats were
pretreated with 3-methylcholanthrene (5 mg, 24 hours before treatment with
CC1.), the amount of label associated with the macromolecules Increased.
No label was associated with DNA In the rat studies. Similar studies In
mice Indicated that DNA binding occurred (108 ymol CC1 /mol DNA), but
only after pretreatment with 3-methylcholanthrene (1 mg, 24 hours before
CC1 dosing).
In an in vitro experiment, Rocchl et al. (1973) used rat or mouse liver
mlcrosomes to activate labeled CC1, 1n the presence of calf thymus DNA.
Pretreatment of animals with 3-methylcholanthrene enhanced the amount of
label associated with DNA. Furthermore, pH 5 enzyme preparations, contain-
ing activating enzymes (Keller and Zamecnlk, 1956), were also found to
Increase the amount of label bound to DNA. Therefore, from these results,
1t appears that metabolites of CC1, can Interact with DNA; however, for
4
optimal binding conditions, mlcrosomal enzymes had to be activated with
3-methylcholanthrene and the binding assay carried out 1n the presence of
pH 5 enzymes.
10-4
-------
Diaz Gomez and Castro (1980a) have also measured binding of metabolic-
ally activated CC1 to cellular macromolecules. The results show that
14C from [14C]CC14 (specific activity, 27 Cl/mol) Irreversibly bound
\n vivo to liver nuclear DNA, protein and llplds 1n strain A/3 mice and
Sprague-Dawley male rats. Mouse and rat liver nuclear DNA Isolated from
animals given [^CJCCT, 16 hours before they were killed exhibited a
4
small but significant labeling (mice, 0.72 pmol/mg; rats, 0.52 pmol/mg).
The count from the assay done 1n the presence of unlabeled DNA was sub-
tracted from the experimental counts before binding was calculated. In
contrast to the results of Rocchl et al. (1973), Induction of liver enzymes
by 3-methylcholanthrene was not required for binding of 14C from
[14C]CC1. to DNA. It should be mentioned that the purified DNA samples
contained 0.2% protein. However, contamination by protein at this low level
could not account for all the covalent binding measured in the DNA sample.
Significant Vn vitro binding of metabolically activated CC1. to iso-
lated mouse liver DNA (1.81 pmol/mg) was observed by Diaz Gomez and Castro
(1980a) in anaerobic incubation mixtures containing microsomes and NADPH.
It was also found that -CC13 chemically produced by reaction of CCl^
with benzoyl peroxide interacted with DNA (826 pmol/mg). This result
suggests that -CC1 may be the species involved in the binding to DNA.
O
In addition to the above observations that metabolites of CC1. bind to
4
DNA, Diaz Gomez and Castro (1980a) also observed significant binding in vivo
of metabolically activated CC1 to rat liver nuclear protein and lipld.
The label bound to nuclear protein was 47.7 pmol/mg and that bound to
nuclear lipld was 113.5 pmol/mg. The authors suggested that the binding of
metabolically activated CC1. to nuclear lipids is significant in terms of
the carcinogenicity of CCU, because nuclear lipld is derived from the
10-5
\
-------
nuclear membrane which contains the P-450 necessary for activation of CC14
to the probable reactive metabolites •CCla and phosgene. Since these
O
metabolites are highly unstable and therefore are not likely to exist long
enough to travel from the endoplasmlc retlculum P-450 enzymes to nuclear
components, activation by nuclear membrane P-450 enzymes Is more Hkely to
yield Interactions • of the metabolites with nuclear components (e.g., ONA)
than will activation 1n the endoplasmlc retlculum.
D1az Gomez and Castro (1980b) have assessed the CC1 activation poten-
tial of purified rat Hver nuclei by measuring covalent binding of nuclear-
activated CC1 to nuclear protein and llpld. Binding to DNA was not
measured 1n this study. The results were compared to results obtained from
similar Incubation mixtures containing mlcrosomes Instead of purified
nuclei. The Incubation mixtures containing either nuclei (1.3 mg pro-
tefn/ma.) or mlcrosomes (1.56 mg protein/mi) were Incubated for 30
minutes In 37.6 nM [1*C]CC14 (6.94 Cl/mol) and an NADPH generating
system 1n an 02-free N2 atmosphere.
The authors observed that the extent of binding to proteins In the
nuclear preparations was 43.5% of that observed for mlcrosomes (nuclear
suspensions, 21.9 pmol/mg; mlcrosomes, 50.3 pmol/mg). Binding to nuclear
Uplds was 77.3% of that observed for mlcrosomes (nuclear suspension, 147
pmol/mg; mlcrosomes, 190 pmol/mg). Thus, Isolated nuclei were less effi-
cient than mlcrosomes 1n metabolizing CCU but the results were within the,
same order of magnitude.
This study suggests that, metabolism of CC1 to reactive Intermediates
can occur In nuclear membranes and Indicates that the in vivo binding
observed 1n the previous study (D1az Gomez and Castro, 1980a) may have been
due to nuclear rather than mlcrosomal activation of CC1 . It should be
4
mentioned, however, that the nuclear preparations were contaminated with
10-6
-------
trace amounts of endoplasmlc retlctrtum, which may have been sufficient to
result 1n at least part of the nuclear activation observed.
D1az Gomez and Castro (1981) have published preliminary evidence that
•CC1«, chemically generated from the benzoyl peroxide-catalyzed decompo-
O
s1t1on of CC14, reacts primarily with guanlne and adenlne and to a lesser
extent with cytoslne and thymlne. This result suggests that *CC13 may
bind to DMA in vivo by Interaction with the deoxyrlbonuclelc add bases.
In summary, 1t has been shown that CC1 1s metabolized to reactive
Intermediates {•CC1« and phosgene). It has also been shown that meta-
o
bollcally activated CC1. binds to DNA, protein and I1p1d. These results
suggest that CC1. has genotoxlc potential. The negative results In six of
the seven bacterial mutagenlclty studies described In the next section may
be due to Inadequate metabolic activation 1n the test systems used or may
result from the scavenging by protein or I1p1d of any very reactive meta-
bolic Intermediates formed {e.g., 'cc^3 and phosgene) under conditions
of exogenous activation, thus limiting their availability for reaction with
DMA. For these reasons, test systems used to assess the genotoxlclty of
CC1. should Incorporate adequate metabolic activation and an Indication
that any highly reactive metabolites formed were not scavenged by mlcrosomal
protein or llpld before reaching the DNA of the test organism.
10.2. MUTASENICITY STUDIES IN BACTERIAL TEST SYSTEMS
Studies to determine the mutagenlc activity of CC1A 1n the Salmonella
«J.
tvphlmuMum revertant system have been primarily negative. A review article
written by McCann et al. (1975) stated that an assay using Aroclor-lnduced
S9 activation and strains TA100 and TA1535 was negative. This article con-
tained no details of the procedure used to generate this negative result.
10-7
-------
Another review article (F1shbe1n, 1976) 1n which no data were presented
contained a statement that CCl^ was not mutagenlc when assayed 1n a spot
test with the TA1950 strain.
Uehleke et al. (1977) tested the mutagenldty of CC1 1n suspension
assays with S. typh1mur1um strains TA1535 and TA1538. No mutagenlc activity
was observed. About 6-9xl08 bacteria were Incubated for 60 minutes under
N2 1n tightly closed test tubes with 8 mM CC14 and mlcrosomes (5 mg
protein) plus cofactors. The mutation frequencies (his* colony forming
units/108 his" colony forming units) were <10 for both strains and the
spontaneous mutation frequencies were 3.9^3.7 for strain TA1535 and 4.4*3.5
for strain TA1538. At this concentration of CC1 , survival of the
bacteria was at least 90%. Thus, the negative results for CC1, cannot be
4
Interpreted since concentrations of CCl^ resulting In <90% survival should
also have been tested. D1methyln1trosam1ne, cyclophosphamlde, 3-methylcho-
lanthrene and benzo(a)pyrene were the positive controls used 1n this study.
Although these chemicals were mutagenlc In the presence of the S9 activation
system, they may not be appropriate as controls for CC1. because they are
not halogenated alkanes and are, therefore, not metabolized like them.
Because metabollcally activated CC1 did not significantly bind to
exogenously added bovine serum albumin (see binding studies above), Uehleke
et al. (1977) concluded that any reactive species generated by the micro-
somes may not have distributed Into the Incubation medium and, thus, may
have been Inaccessible to the test bacteria, resulting 1n the negative
mutagenldty response observed. However, 1t 1s not clear from the descrip-
tion provided In this report whether rat, mouse or rabbit mlcrosomes were
used 1n the mutagenldty studies. It 1s clearly stated that rabbit mlcro-
somes were used for the binding studies described above. If mouse or rat
10-8
-------
mlcrosomes rather than rabbit mlcrosomes were used for the mutagenldty
experiments, 1t cannot be assumed that CC1 was sufficiently activated,
since activation sufficient for binding of [14C]CC1 to macromolecules
was shown 1n this paper only with rabbit mlcrosomes. Another deficiency 1n
this study 1s that the Ames strains TA98 and TA100 were not used. These
strains contain an R factor plasmld that Increases the sensitivity of the
tester strains to certain mutagens. Because of these deficiencies, the
negative mutagenldty results 1n this paper are judged to be Inconclusive.
The mutagenldty of CC1 was also tested 1n a study designed to
evaluate the mutagenlc potential of chemicals Identified 1n drinking water
(Simmon et a!., 1977). No mutagenlc activity was detected with CC1,. The
4
authors tested 71 of the 300 chemicals that had been Identified In public
water supplies. Carbon tetrachlorlde was tested In this study 1n a desic-
cator to assess mutagenldty due to vapor exposure and to avoid excessive
loss of CCl^ to the atmosphere. The desiccator contained a magnetic
stlrrer which acted as a fan to aid 1n evaporation of the measured amount of
CC1 and to maintain an even distribution of the vapors. Plates were
exposed to the vapors for 7-10 hours and then removed from the desiccators,
covered, and Incubated -40 hours before scoring. Mutagenlc activity was not
observed and no Information on toxldty was provided.
This study by Simmon et al. (1977), although lacking some specific
details of the CC14 assay, clearly Identifies certain trlhalomethanes
(CHBr3, CHBr2Cl, CHBrCl2) as mutagens 1n the vapor assay In desic-
cators. Methyl bromide, methyl chloride, methyl Iodide and methylene
chloride were also found to be mutagenlc .In the desiccator assay. However,
these seven halogenated compounds did not require metabolic activation to
exhibit mutagenlc activity. It may be that CC1 Itself Is not mutagenlc
10-9
-------
and the rat liver S9 does not effectively metabolize CCl^ to a potential
mutagenlc reactive Intermediate {? •CClg), even though the demonstration
of mutagenldty of three of the chemicals tested [b1s(2-chloro1sopropyl)~
ether, vinyl chloride and vlnylldene chloride] required or was enhanced by
this S9 mix. It may also be that a reactive Intermediate was formed, but 1t
was too reactive or short-lived to be detected 1n a test system that uses
exogenous metabolic activation.
Another negative result for the mutagenldty of CC1. was obtained 1n a
recent study using the Salmonella/mlcrosome assay In which escape of the
volatile compounds was prevented by the use of a specially designed, closed,
Inert Incubation system (Barber et a!., 1981). Seven of the 10 halogenated
alkane solvents tested gave positive mutagenldty results when the Ames
assays were carried out In the closed Incubation system. Under standard
conditions (1n which volatilization was not prevented), only 2 of the 10
solvents tested gave a positive result. Thus, the closed Incubation system
allowed for the detection of five more mutagens than could be detected under
standard conditions. Carbon tetrachlorlde was one of the three solvents
tested that gave a negative result 1n both the standard and closed Incuba-
tion systems. The Investigators Indicated that CC1. was tested at concen-
trations high enough to produce observable toxlclty (determined by absence
of background lawn). The Salmonella strains used were TA1535, TA1537,
TA1538, TA98 and TA100. Levels of CC1 tested were 4.7, 5.7, 10.2, 12.3
and 18.4 ymol per plate. No dose-related response was observed. The
seven solvents that tested positive 1n this closed system did not require
\
metabolic activation by S9 mix prepared from Aroclor-lnduced rats (the S9
mix did activate the positive control 2-am1noanthracene). It 1s possible
that the S9 mix used, although adequate for activation of 2-amlnoanthracene,
was not adequate to metabolize the other three solvents (Including CC1.).
10-10
-------
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LPUIOSOJD^UI Aq paBuaApas uaaq aApq Apiu (uia^sAs
a^pnbapp UP Buiuinssp) pa^pjauaB auaBsoqd jo
A"UP 'sa^pn^s asaq^ ^o Aupui u^ pasn ajaw suia^sAs UO^P
snouaBoxa asnpoag 'aAH^eaJ ^[qB^q pup atqpjsun ajp '1?L33
aq^ 'auaBsoqd pup Ci33«
a^pnbappu^ uaaq aApq APUJ pasn sma^sAs
p 0} anp pau^p^qo uaaq aApq ptnoo
ajp sa^pn^s LBU^^seq aAoqp aq^ iuoa^
aqi *i
jo jaquinu
asnpoaq
aq^ Ajpuauns
-------
11-01
*(6S aaAH. HQqpa qsaa^ 0,0 asn pus 4V1Q3 40 aouasaad aq} ui ucnsuadsns
\i\ eia^^PEQ a.o aansodxa 'uoisua) uaBA'xo MOL) suoniPUOD A"BSSB
aapun A"i.uo inq 'tesss sauiy aq} UL otuaBBinui aq ABUU L33
A^JBUtuiiL9Jd sa^n^usuoo ^[nsaa aAUtsod >|caM stm -uis
e oBaapun ^BH^ spunoduiOD aLn^LOA Bu^sa^ JQ^ tua^sAs atqe^tns e aq
uj. Acsse uo^suadsns aq^ ^eq^ papruouoo sjoq^ne aqi
0}
paseaaoap uonepixoaad
yuu
%Q8 pasneo ijui/Louirt g !/IBSSB uo^suadsns
uoqaeo
paseqoand
sew
-ao)
[Buosaad 'aauiHM
apsafi oi.a}auio}oqdoa}Dads SBM *L33 9M1 'A'H
DtuaBB}nui ou pa^q^qxa 53 Hqqsa q}LM S^BSSB a}B|.d asnBDaq paAaasqo
aq} aoj a|.q|.suodsaa SBM auo|.B 5$ aaAt|_ uqqsa aq}
ia}sAs uoi}BAV}OB aq} SB pasn SBM 6S aaAit
uaqM ALUO PUB s}uauitaadxa a}BaBdas OM} ut paAaasqo SBM }tnsaa
>|BaM aqi *}oea}sqB s^q} u^ pa}aodaa }ou SBM diqsuoj.}BLaa asuodsaa-asop B
aoj aouaptA3 *(s}UB}aaAaa OS^LI^L 133 Lol)urt S Is}UB}aaAaa &+ZL 'punoaB
->|DBq) punoaB>|OBq aAoqB s}UB}aaAaa u^ asBaaou^ p[o^~2 6 u^ pa}[nsaa sa}B|_d
^o uoueaBdaad aao^aq (saqn} aau^B}noBA uui 9) uo^sua} uaBA'xo MOJ. }B uo^s
h
-uadsns pinbu TSUI [ ^ u^ sa}nutui QZ aoj L33 o^ OOLV1
j.o aansodxa }Bq} pa}aodaa (286L) JaulHM Pue aadooo '}OBa}sqB }U33aj B
'VNQ L^Laa^^Pq 9M1-
aao^aq saLnoaLouioaoBiu auBaquiaiu [B^aa}OBq ao (M0[aq aas) s}uauoduio3 [BUIOS
pa}DBaa 'pauiao^ ^ 'sa}^oqB}aiu aA^}DB }Bq} aj.qissod OSJ^B s^ }j
-------
1s essentially constant, amounts In suspension will vary. Extracellular or
membrane effects may have resulted In the high toxldty observed at
5.13 g/a..
Results of the Callen et al. study are presented 1n Table 10-1. A
1-hour treatment of cells with CC1 a.t the highest amount tested
(5.13 g/8,) resulted 1n significant Increases In gene conversion (31-fold)
and mltotlc crossing over (25-fold). Gene reversion was also Increased, but
to a lesser extent (3-fold Increase). Survival was only 10% at this concen-
tration of CO!.. At the Intermediate level (4.31 g/8.) of CC14, much
smaller effects In the three genetic systems were observed (2- to 3-fold
Increases). Survival at this level was 77%. Thus, the data 1n the Callen
et al. (1980) study are suggestive of a weak positive response, but addi-
tional studies are needed before 1t can be conclusively stated that CC1.
causes genetic effects In yeast.
Negative results have been obtained 1n a recently developed Vn vitro
chromosome assay that utilized an epithelial-type cell line derived from rat
liver (Dean and Hodson-Walker, 1979). This cell line has sufficient metabo-
lizing activity to activate various chemical mutagens and carcinogens with-
out the need for an exogenous activating system. Sealed-flask cultures were
treated for 22 hours with CC1 dissolved 1n growth medium at 0.005, 0.010
and 0.020 mg/8,. Carbon tetrachlorlde at these low concentrations did not
Induce any chromatld or chromosomal aberrations, whereas a number of
direct-acting mutagens and several requiring metabolic activation produced
chromatld deletions, gaps and exchanges. However, none of the other sub-
stances tested was a heavily chlorinated compound. In addition, one of
these substances was assayed at levels comparable to that used for CCl^
and the remaining 10 compounds were assayed at doses several orders of
10-13
-------
TABLE 10-1
Genetic Effects of Carbon TetrachloMde on Strain 07 of
Saccharomvces cerevlslae following 1-hour treatment at 37°Ca»b
Concentration (g/£)
3.23
4.31 5.13
Survival
Total colonies
% of control
trpS locus (gene conversion)
Total convertants
Convertants/105 survivors
ade2 locus (mltotlc crossing over)
Total twin spots
Mltotlc cross-overs/104 survivors
Total genetically altered colonies
1454
100
285
2.0
1
1.6
11
1252
86
331
2.6
3
5.3
19
1120
77
350
3.1
3
5.8
16
152
10
506
61.7
10
40.1
65
Total genetically altered colonies/
10s survivors
1.7
3.4
3.1
33.3
llh/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
bThe total number of colonies 1n the different classes represent total
counts of colonies from five plates 1n the case of survival, conversion and
revertant-frequency estimations. MHotlc crossing over was estimated from
counts of colonies growing on a total of 30 plates, 20 plates containing
medium on which all surviving cells grew and 10 plates containing medium on
which only trpS convertants grew.
10-14
-------
magnitude larger. The doses chosen for each substance assayed were deter-
mined from cytotoxldty assays. Apparently, CC14 was very toxic to rat
liver cells. EDTA (0.1 mM) has been found to decrease the cytotoxldty of
CC1 without affecting mutagenldty 1n bacteria (Cooper and WUmer, 1982),
4
apparently by decreasing membrane I1p1d peroxldatlon (Masuda and Murano,
1977). Perhaps addition of EDTA to a mammalian In. vitro chromosome assay,
such as 1n the study carried out by Dean and Hodson-Walker (1979), would
allow for the use of larger concentrations of CCl^.
10.4. OTHER STUDIES INDICATIVE OF DNA DAMAGE
M1rsal1s and Butterworth (1980) measured unscheduled DNA synthesis (UDS)
In primary rat hepatocyte cultures following la vivo treatment of adult male
Fischer-344 rats (200-250 g) with CC14 (certified ACS grade, Fischer
Scientific Co.) by oral gavage. Control rats received corn oil by gavage.
Acetylamlnofluorlne and d1methyln1trosam1ne were also tested. At 2 hours
after treatment, the livers were perfused la situ and hepatocytes were
Isolated. Approximately 6xl05 viable cells were seeded In culture dishes
containing coversllps and allowed to attach to the coversllps for 90
minutes. After the coversllp cultures were washed, they were Incubated 1n a
medium containing 10 yd [3H]thym1d1ne (42 d/mmol) per ma for 4
hours. The cultures were washed again and Incubated In medium containing
0.5 mM cold thymldlne for 14-16 hours. The extent of UDS was assessed by
autoradlography. Net grains/nucleus were calculated 'as the silver grains
over the nucleus minus the highest grain count of three adjacent
nuclear-sized areas over the cytoplasm. The area of the silver grains was
counted rather than the grain number, so that densely labeled cells where
silver grains were touching could be accurately measured.
10-15
-------
Cells from control animals ranged from -2 to -6 net grains/nucleus.
Treatment of rats with d1methyln1trosam1ne (1.p.) at 10, 1 or 0.1 mg/kg
yielded 36.6, 6.4 and -0.9 net grains/nucleus, respectively; dlmethylnltros-
amlne at 10 mg/kg (p.o.) produced 22.2 net grains/nucleus. Oral doses of
acetylamlnofluorlne at 50 or 5 mg/kg yielded 14.0 and 6.4 net grains/nucle-
us, respectively. Carbon tetrachlorlde at 100 or 10 mg/kg (p.o.) yielded
-3.2 and -5.1 net grains/nucleus, respectively. Thus, dose-related
Increases In UDS were observed for d1methyln,1trosam1ne and acetylamlno-
fluorlne, whereas CC14 produced no response.
In this study two doses of CC14 were tested: 10 mg/kg and 100 mg/kg.
The oral LDgo for CC14 in rats Is 2800 mg/kg. It was not determined In
this study at what dose hepatic cell tox1c1ty would occur under the condi-
tions used. Therefore, 1t 1s not clear that adequate doses of CC1. were
tested. Also, 1t 1s not clear whether the 2-hour time period between expo-
sure of the rats to CC14 and Isolation of the hepatocytes was sufficient
for observation of UDS. In a study by Popp et al. (1978), In which CC14-
Induced hepatocellular changes were noted, the shortest exposure period
studied was 6 hours. If M1rsal1s and Butterworth had shown that the 2-hour
exposure period was sufficient for activation of the CC1, to a reactive
4
Intermediate, perhaps by demonstrating alkylatlon of protein by ^.CCl
J
the negative results they obtained would be more convincing. Thus, this
negative result could truly reflect the. Inability of CC14 to cause UDS in
the system used or it could be a false negative result due to factors such
as an Inadequate dose or an Inadequate exposure time period.
Craddock and Henderson (1978) carried out an in vivo UDS study In which
hepatocyte nuclei were Isolated and then assayed for radioactivity by
scintillation counting rather than by grain counting. This method may be
10-16
-------
superior to the whole-cell grain counting procedure used by M1rsal1s and
Butterworth (1980) as described above, particularly for chemicals, that may
cause low UDS activity, because a small effect could be obscured when back-
ground cytoplasmlc grain counts are subtracted from the nuclear grain
counts. In this study, Craddock and Henderson used a CC14 dose of 4000
mg/kg, which 1s significantly larger than the oral LD5Q (2800 mg/kg).
Negative results were obtained after a 2-hour exposure. A positive effect
was observed after a 17-hour exposure. The authors suggested that this
result may have been due to secondary effects such as lysosomal damage,
which may result 1n release of DNA degradatlve enzymes.
In their latest study, M1rsal1s et al. (1982) used combinations of doses
(up to 400 mg/kg) and exposure times (up to 48 hours) that resulted 1n Hver
toxldty, as measured by an Increase 1n Hver cell mitosis. Therefore, the
above mentioned criticisms of the previous studies relating to Inadequate
dose and exposure appear to have been obviated by this study. Carbon tetra-
chlorlde tested negative In this study also. However, H Is still uncertain
whether the method of assaying for UDS (subtraction of cytoplasmlc grain
counts from nuclear grain counts) will allow for detection of a weak
response (see above). In addition, benzo(a)pyrene, 7,12-d1methylbenz(a)-
anthracene and N-methyl-N'-n1tro-N-n1trosoguan1d1ne were negative 1n this In.
vivo UDS assay, whereas these chemicals tested positive In the In. vitro rat
hepatocyte UDS assay (Williams, 1981). This discrepancy suggests that the
In vitro test may be more sensitive than the In. vivo assay. Carbon tetra-
\
chloride has not been tested 1n the in vitro rat hepatocyte UDS assay.
In summary, the above in vivo UDS results suggest that CC14 may not
damage DNA. However, in vitro UDS studies are needed before a decision on
the DNA damaging potential of CC14 can be made. Also, use of a procedure
10-17
-------
for the UDS assay 1n which the radioactivity of Isolated nuclei 1s assayed,
rather than one 1n which grain counts of nuclei are corrected for grain
counts 1n the cytoplasm, may better allow for detection of low levels of UOS.
10.5. SUGGESTED ADDITIONAL TESTING
Suggested additional testing falls Into six categories:
1. The DNA damage studies reported by Craddock and Henderson (1978),
H1rsal1s and Butterworth (1980) and M1rsal1s et al. (1982), suggesting
that CC14 does not damage DNA following in vivo treatment of rats,
should be corroborated by the In. vitro rat hepatocyte UDS assay of
Mil Hams (1981). The \jn vitro test may be more sensitive than the In
vivo test for potential weak genotoxlc effects. Also, use of a proce-
dure. 1n which nuclei are Isolated and assayed for radlolabel 1s recom-
mended rather than use of the standard procedure 1n which cytoplasmlc
grain counts are subtracted from nuclear grain counts. The former
procedure may better allow for detection of a weak response.
2. Studies using experimental procedures that are sensitive enough to
detect the formation of liver DNA-chem1cal adducts after exposure of
animals to CCl^ are needed to confirm the low level of DNA binding
observed by D1az Gomez and Castro (1980a) and by Rocchl et al. (1973).
3. Additional data are needed to corroborate the Callen et al. (1980) study
1n the yeast system, which utilizes an endogenous activation system and
1s capable of assaying for point mutations, mltotlc crossing over, and
gene conversion. The Callen et al. study 1s the only yeast study
reported In the literature.
10-18
-------
4. Additional cytogenetlc testing for chromosome effects 1n mammalian
systems Is needed before CC1 can be considered to be adequately
tested for chromosome damage. Because EDTA has been reported to
decrease cytotoxlclty due to CC14 1n bacteria (Cooper and Wltmer,
1982), perhaps In. vitro mammalian liver cell cytogenetlc assays should
be carried out In the presence of EDTA so that higher levels of CC14
could be assayed than were used 1n the Dean and Hodson-Walker {1979}
study. In addition, la vivo cytogenetlc studies such as the bone marrow
mlcronucleus test, are needed.
5. Corroboratlon of the preliminary evidence for the weakly mutagenlc
response 1n the Ames test reported by Cooper and Wltmer (1982) 1s
needed. The same experimental conditions (fresh rabbit liver S9 and
exposure of the bacteria 1n suspension to CC14 under reduced oxygen
tension 1n the presence of EDTA) should be used and several concentra-
tions of CC1. should be tested to establish whether or not a
dose-response relationship exists. In addition, In. vitro mammalian
mutagenlclty studies should be carried out using these same experimental
conditions.
6. Studies on the ability of CC1. to reach reproductive organs and cause
germ cell mutation were not available. If results from the tests
suggested above for somatic cell mutation are positive, then studies
assessing heritable risk are needed. (See Federal Register, 1980c for
guidance on such tests.)
10.6. SUMMARY AND CONCLUSIONS
Carbon tetrachlorlde has been tested for Its mutagenlc potential 1n
bacteria, yeast and a mammalian cell line and for Its DNA damaging potential
1n rat hepatocytes when administered Vn vivo.
10-19
-------
Six of the seven point mutation studies 1n bacteria were negative. The
remaining bacterial study (Cooper and Wltmer, 1982) was preliminary and only
suggestive of a weak mutagenlc response. In none of the negative studies
was 1t shown that CC14 was activated or metabolized by the exogenous S9
activation system used. Metabolism of 2-am1noanthracene or vinyl compounds
(used as positive controls) 1s probably an Inadequate Indication that the
"activation system can metabolize halogenated alkanes such as CC1 . These
4
substances are very different from CC14. A better Indication that the
activation system 1s sufficient for metabolism of CC14 may be to show that
It metabolizes ["C]CC14 to Intermediates that bind to macromolecules.
It 1s also conceivable that potentially mutagenlc reactive Intermediates of
CC14 (such as the free radical -CC13 and phosgene) are generated 1n
the presence of an S9 activation system but that they are too short-lived to
Interact with DNA In in vitro test systems.
The Callen et al. (1980) study was designed to overcome this problem by
the use of an jin vivo activation system 1n yeast. Positive mutagenlcHy and
DNA damage results were reported. In contrast, negative DNA damage results
were reported, using in vivo UDS as the assay endpolnt. However, these
negative results need confirmation by the potentially more sensitive In.
vitro hepatocyte UDS assay of Williams (1981). Binding studies by Rocchl et
al. (1973) and by D1az Gomez and Castro (1980a,b, 1981) Indicate that
metabollcally activated CC14 may Interact with DNA.
Therefore, the negative genotoxldty test results that have been report-
ed may be due to any of four (or more) factors: 1) CC14 1s not mutagenlc;
2) excessive volatilization and escape of CC14 if appropriate precautions
are not taken; 3) Inadequate activation of CC14 by the S9 system used to a
metabolite capable of causing mutations (e.g.. .-CClg or phosgene), or
10-20
-------
4) Inability of any reactive Intermediates formed to reach DNA before being
scavenged by I1p1d and protein (particularly under conditions of exogenous
activation, such as 1n the Ames test).
Additional tests should be conducted In which appropriate measures are
taken to ensure that: 1) volatilization and escape of CC14 does not
decrease exposure of the test organism or cell to levels of CC1 that are
too low to be effective, 2) metabolic activation Is occurring, and 3) DNA 1s
exposed to the activated chemical species.
In summary, evidence described In this report Is Insufficient to allow
firm conclusions to be made concerning the genotoxldty of CC1 . The
borderline results for binding of reactive Intermediates to DMA {Rocchl et
al., 1973; 01az Gomez and Castro, 1980a, 1981), the study of Callen et al.
(1980) and the highly preliminary evidence for mutagenlclty In the Ames
assay (Cooper and Wltmer, 1982) are Insufficient evidence for genotoxldty,
but are suggestive of weak genotoxlc effects and are an Indication that
further studies should be done.
10-21
-------
-------
11. CARCINOGENICITY
Carcinogenic effects of CC1. have been studied 1n rats, mice and
hamsters. These studies are reviewed In Section 11.1. Human case reports
and one occupational study are reviewed 1n Section 11.2.
11.1. EXPERIMENTAL ANIMALS
11.1.1. Rat Studies. Studies performed on rats have primarily used subcu-
taneous Injection as the route of exposure. Hepatomas were found as well as
toxic effects such as cirrhosis, hyperplasla and cholanglof1bros1s. Neo-
plasms also developed following exposure by oral 1ngest1on.
Cameron and Karunaratne (1936) looked at CC1. cirrhosis 1n relation to
liver regeneration In the rat. Albino rats (strain not given) weighing
~150 g each were administered subcutaneous Injections of 0.1-0.25 ma,
CC1. twice a week. Changes developed In the liver after 6-10 doses and
4
disappeared within 7-10 days after cessation of treatment. With longer per-
iods of exposure, the liver showed less and less tendency to return to a
normal appearance when administration of the chemical was discontinued.
Cirrhosis of the Hver developed after several doses and was severe and
Irreversible after 40 doses.
Upon examination the Hver was found to be pale, tough and finely granu-
lar. Extensive flbrosls radiated from the portal areas, thereby dividing
the Hver Into small Irregular masses. Hyperplastlc nodules were seen 1n
different parts of the liver.
In summary, rats given subcutaneous Injections of CC1 readily devel-
oped cirrhosis of the Hver. Hyperplastlc nodules of the liver were also
.noted.
11-1
-------
Reuber and Glover (1967a) administered subcutaneous Injections of CC1.
to Inbred Buffalo male and female rats 4, 12, 24 and 52 weeks old. Addi-
tionally, newborn rats were obtained and given Injections of CC1. at 4
days of age. Each age group contained 10-14 rats of each sex. Control
groups consisted of six animals per each age and sex. All experimental ani-
mals were given 1.3 mi/kg bw of a 50% solution of CCl 1n corn oil twice
4
a week for 12 weeks. Control animals were Injected with the same amount of
corn oil.
The 4-day-old animals died 1n an average of 8 days with hepatic and
renal necrosis. The other age-groups survived for the duration of the
study. During this period, the 52-week-old rats maintained their weight,
and the -12-week-old rats each gained from 20-30 g. The 4-week-old females
weighed 3 times their starting weights and males weighed 4 times their
starting weights.
At sacrifice complete necropsies were done. All organs were examined
histologically, including such tissues as diaphragm, tongue and skeletal
muscle. Special staining was done for glycogen, mucin, connective tissue,
cerold, canaliculi, hemosiderin and lipld.
The males given subcutaneous injections at 52 weeks of age had more
hyperplastic lesions than the other males. Hyperplastic nodules were found
in 6 of 14 (43%) rats, with one having a small hepatic carcinoma. The only
other males with nodules were the 24-week-old rats, 2/11 (18%). The remain-
ing 52-week-old and 24-week-old male rats had hyperplasia of the liver.
Hyperplasia developed in less than half of the 12-week-old male rats.
Hyperplastic lesions and hyperplasia were not observed in control male rats.
11-2
-------
The 24- and 52-week-old females had more hyperplastlc nodules than did
the younger females. The most striking lesions were In the 24-week-old
female rats. In this group, 8/10 rats (80%) had hyperplastlc nodules and
one rat had a small carcinoma of the liver. There were more hyperplastlc
nodules per liver and larger lesions 1n the females than 1n the males.
Lesions were not present 1n control female rats.
There were two kinds of hyperplastlc lesions In the Hver, one located
1n the perlportal region and the other around central veins. Cirrhosis var-
ied from mild to severe, but was unrelated to the hyperplastlc lesions 1n
Individual rats. The severity and the hlstologlc pattern of the cirrhosis
were related to age and sex. The hyperplastlc nodules seen were similar to
those known to be preneoplastlc {Reuber and Glover, 1967a). If the study
had been continued for a longer period of time, It 1s possible that the
hyperplastlc nodules could have become overt tumors. Results of this study
are given In Table 11-1.
In summary, 24- and 52-week-old Inbred Buffalo rats of both sexes given
subcutaneous CC1 developed more hyperplastlc hepatic nodules, as well as
an occasional early carcinoma of the Hver, than did rats of other ages.
The number of hyperplastlc lesions per liver and the size of the lesions
were larger 1n females than In males. Four-day-old rats died with necrosis
of the liver and kidney.
Reuber and Glover (1967b) also studied cholanglof1bros1s of the liver 1n
male and female Buffalo strain rats of varying ages. Rats were given
subcutaneous Injections of 1.3 ml/kg bw of a 50% solution of CC1 and
4
corn oil twice weekly for 12 weeks. Cholanglof1bros1s may be a precursor of
cholanglocardnomas of the liver {Reuber and Glover, 1967b); H Is a lesion
composed of ducts lined by Irregular epithelial cells and surrounded by
11-3
-------
TABLE 11-1
Lesions of the Liver In Rats Given Subcutaneous Carbon Tetrachlorlde*
(1.3 mil/kg bw In 50% solution with corn oil)
Age
(weeks)
HALE
4
12
24
52
FEMALE
4
12
24
52
Hyperplasla
6/14 (43%)
4/11 (36%)
8/11 (73%)
7/14 (50%)
4/11 .(36%)
5/11 (45%)
1/10 (10%)
4/11 (36%)
Hyperplastlc
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, 1967a
11-4
-------
connective tissue. Cholangiof1bros1s of the Hver developed 1n male and
female rats receiving Injections of CC14. The lesion was present 1n male
rats of all ages, except those 4 weeks of age. Some rats were fed 3-methyl-
cholanthrene (MCA) 1n the diet 1n addition to receiving the subcutaneous
CC1 Injections. The lesion was Increased In male rats 5 weeks of age
given both CC14 and MCA, whereas 1t was decreased 1n rats of all other
ages. Most female rats given both chemicals also had cholanglof1bros1s.
The comparative carc1nogen1c1ty of CC14 has been studied In five rat
strains: Japanese, Osborne-Mendel, Wlstar, Black and Sprague-Daw!ey (Reuber
and Glover, 1970). Groups of 12-17 male rats of each strain were given
twice weekly subcutaneous Injections of CC1 (2080 mg/kg bw as a 50%
solution 1n corn oil) for varying durations due to the different survival
patterns of these strains. Treated animals were killed when moribund;
controls for each strain were killed at the same time as the last experi-
mental animal. Incidence of hepatic lesions 1s given 1n Table 11-2.
Lesions other than hepatic also occurred. Hemanglomas of the spleen were
present 1n two Japanese rats and 1n one of the Osborne-Mendel strain. There
were carcinomas of the thyroid gland In three Osborne-Mendel and three
Japanese rats. One Japanese rat had a subcutaneous lelomyosarcoma; two
Osborne-Mendel and three Japanese rats had chronic renal disease.
The data Indicate that: (1) sensitivity to CC1 -induced neoplasms
varies widely among these five rat strains; and (2) the trends in incidence
of neoplasms and severe cirrhosis appear to be Inversely related. Varying
amounts of toxicity occurred: all experimental animals of the Black rat
strain were dead at 18 weeks, and those of the Sprague-Dawley strain at 16
weeks; the failure to find carcinomas in those strains may have been caused
11-5
-------
TABLE 11-2
Incidence of the Most Advanced Hepatic Lesions 1n Rats
Administered Carbon Tetrachlorlde3
{2080 mg/kg bw 1n 50% solution with corn oil)
Lesion
No hyperplasla
Hyperplasla
Hyperplastlc nodule
Small carcinoma
Large carcinoma
Total carc1nomab
No cirrhosis
Mild cirrhosis
Moderate cirrhosis
Severe cirrhosis
Japanese
0/15
0/15
3/15
4/15
8/15
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
Wlstar
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-
Dawiey
8/16
6/16
2/16
0/16
0/16
0/16
0/16
0/16
0/16
16/16
aSource: Adapted from Reuber and Glover, 1970
bThese Incidence rates may not be comparable due to large differences 1n
average survival for the different strains.
11-6
-------
in part by an Insufficient study duration. In all three other strains,
toxldty (e.g., cirrhosis, hepatic vein thrombosis, cholangloflbrosls)
occurred. Toxldty was Inversely related to carclnogenlclty. Development
of Hver carcinomas was related to the severity of cirrhosis and survival
time. Sprague-Dawley, Black and to a lesser extent Wlstar males died from
moderate or severe cirrhosis before they could develop carcinomas. Japanese
and Osborne-Mendel rats were less susceptible to cirrhosis; thus, they
survived and developed carcinomas.
In the NCI bioassays for chloroform (NCI, 1976a), 1,1,1-trichloroethane
(NCI, 1977) and trichloroethylene (NCI, 1976b), CC14 was used as the posi-
tive control. The positive control groups of 50 Osborne-Mendel rats of each
sex were administered CC1 in corn oil by gavage 5 times weekly for 78
weeks at two dose levels: 47 and 94 mg/kg bw for males, 80 and 159 mg/kg bw
for females. This treatment resulted In some toxiclty (cirrhosis, fatty
liver) and death. At 110 weeks using the high dose, only 7/50 males and
14/50 females survived; at the low dose, 14/50 males and 20/50 females sur-
vived as compared to 26/100 males and 51/100 females for controls. The med-
ian survival times were 92 and 102.5 weeks for males given the low and high
doses, respectively, and 67.5 and 102.5 for females given the low and high
doses, respectively. The incidence of hepatocellular carcinomas was In-
creased in animals exposed to CC1 as compared to pooled colony controls
(Table 11-3). However, this was statistically significant only for low dose
females as compared to the colony controls and not the matched controls.
Absolute Incidence of hepatic neoplasms was low (-5% 1n the animals exposed
to CC1 ). The apparent decrease in the incidence of hepatocellular carci-
nomas 1n female rats at the high dose was attributed to increased lethality
(i.e., females died before tumors could be expressed). The Incidence of
11-7
-------
TABLE 11-3
Incidence of Liver Tumors In Carbon
Tetrachlorlde-Treated Rats and Pooled Colony Controls*
Animal Group
Hepatocellular
Carcinoma
NeoplastU
Nodule
Hales Controls (pooled)
Low dose (47 mg/kg bw)
High dose (94 mg/kg bw)
Females Controls (pooled)
Low dose (80 mg/kg bw)
High dose (159 mg/kg bw)
1/99
2/50
2/50
0/98
4/49
1/49
0/99
2/50
1/50
2/98
2/49
3/49
*Source: NCI, 1976a,b, 1977
11-8
-------
,
4
,
4
other neoplasms was acknowledged but not quantified. The National Research
Council used these data in estimating carcinogenic risks for CC1. (NAS,
1978).
11.1.2. Mouse Studies. Several studies have been reported which indicate
Induction of liver tumors in various strains of mice treated with CC1
either by oral ingestion (which has been the primary route of exposure),
subcutaneous injection or rectal administration. Signs of liver toxlcity,
such as necrosis and cirrhosis, have also been a frequent result of CC1
treatment In the carcinogenlcity studies 1n mice.
In a study by Andervont (1958), groups of 30-77 female or male C3H mice
were administered 6.46 mg CC1. by gavage once weekly for 2 weeks, followed
by administration of 9.6 mg CC14 once weekly for 17 weeks (equivalent to
213 and 320 mg/kg bw). Pathogen-free and pathogen-carrying C3H mice were
used. For males, no difference in the incidence of hepatomas was observed
between the pathogen-free and pathogen-carrying groups; thus, they were
combined. The average number of hepatomas per animal was 1.8 in treated
animals (pathogen-free and pathogen-carrying combined) and 1.3 1n controls.
In females, a difference between the average number of hepatomas in
pathogen-free mice (1.5) and pathogen-carrying mice (1.2) was observed. In
control females, the average number was 1.0. In males, the tumor incidence.
was 79% in treated animals (pathogen-free and pathogen-carrying combined)
and 49% in controls. In females, the tumor incidence in pathogen-free mice
was 46% and in pathogen-carrying mice was 29%. In control females, the
incidence was 3% (Table 11-4). 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 pathogen-carrying mice < treated pathogen-free mice.
11-9
-------
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A study by Edwards (1941) also reported the Induction of hepatomas 1n
mice by exposure to CC1 . The animals used were 207 male C3H mice, aged
3-6 months, and 133 male and female strain A mice, aged 2-3.5 months. They
were given 0.1 mil of a 40% olive oil solution of CCl^ (0.04 cc CCl^)
by stomach tube two or three times weekly for 8-16 weeks. Autopsy was per-
formed up to 21 weeks after the last treatment.
Olive oil was administered by stomach tube 1n doses of 0.1 mil 2 or 3
times weekly to control male C3H and A mice from the same stock as those
used 1n treated groups. In total, 23 strain C3H mice were given CC1 from
39-50 times and were killed and examined from 9-11 months of age. A high
percentage of the treated animals developed hepatomas.
Of 143 C3H mice, varying from 6-10 months of age at autopsy, 126 (88.1%)
showed hepatomas (Table 11-5). Similar tumors were present 1n all of the 54
strain A mice whose ages varied from 4.5-12 months (Table 11-6). It should
be noted that the Incidence of spontaneous hepatomas In both the C3H and A
strains Is markedly below that of the Induced tumors 1n the treated mice.
Autopsies performed on 17 C3H male mice 8.5-9 months of age and of the same
stock as that used 1n the study failed to show any hepatic tumors.
Edwards et al. (1942) performed another study on mice. The mice used
were Inbred strain L (their Incidence of spontaneous hepatomas Is extremely
low), 2.5-3.5 months or 3.5-7.5 months of age at the start of the experi-
ment. The number of mice varied from 8-39 per group. Carbon tetrachlorlde
of a high degree of purity was administered 1n olive oil by stomach tube
usually three, but occasionally two, times weekly. Each treatment consisted
of 0.1 cc of a 40% solution or 0.04 ml of CC14. Mice were given 46
administrations of CC1. over a 4-month period and were killed and necrop-
sled 3-3.5 months after the last treatment. The mice varied from 8.5-14
months of age at necropsy. The liver was examined hlstologlcally.
11-11
-------
TABLE 11-5
Incidence of Tumors 1n C3H Mice Ingesting
Carbon Tetrachlorlde*
Group
Controls
Controls w/OUve oil
Treated Animals
(Olive oil and
0.04 mfi. CC14)
Number of
Mice
Au tops led
17
23
143
Number of
Nice. with
Hepatomas
0
1
126
Incidence
of Hepatomas
(%)
0
4.3
88.1
*Source: Edwards, 1941
11-12
-------
TABLE 11-6
Incidence of Tumors In Strain A Mice Ingesting
Carbon Tetrachlorlde*
Group
Controls
Controls w/0l1ve oil
{0.1 mi 2 or 3x
weekly)
Number of
Mice
Autopsled
200
22
Number of
Mice with
Hepatomas
1
0
Incidence
of Hepatomas
(X)
0.5
0
Treated Animals
(0.01 ma, of a 40% soln.
of CC14 In Olive oil
2 or 3x weekly)
54
54
100.0
*Source: Edwards, 1941
11-13
-------
Hepatomas developed 1n 34/73 mice (47%) given CC14. Hepatomas were
observed 1n 7/15 younger male mice (47%), 21/39 older male mice (54%), 3/8
younger females (38%), and 3/11 older females (27%). Cirrhosis of the liver
was not mentioned. The results are summarized 1n Table 11-7.
Historically, the Incidence of spontaneous hepatomas 1n strain L mice 1s
extremely low: 2/152 (1%) 1n untreated mice. One of 23 untreated virgin
male mice (4%) and 0 of 28 females (0%), necropsled at 15 months of age, had
tumors of the liver. Tumors were not present 1n 22 males and 28 females 18
months of age or 1n 27 female breeders 12-23 months of age. One of 24 male
breeders (4%) had a tumor.
In summary, strain L male and female mice were highly susceptible to the
Induction of hepatomas by CC1., and male mice were slightly more suscept-
ible than female mice.
Eschenbrenner and Miller (1943) studied the effects of size and spacing
of multiple CC1 doses 1n the Induction of hepatomas. Strain A mice were
used because of their normal low Incidence of hepatomas In untreated mice
{
-------
TABLE 11-7
Tumors of the Liver 1n Male and Female Strain L Mice Receiving
Carbon TetrachloMde by Stomach Tube3
(0.04 ma. CC14 2 or 3x weekly)
Age (months)
2.5 - 3.5
3.5 - 7.5
2.5 - 7.5b
Males
7/15 (47%)
21/39 (54%)
28/54 (52%)c
Females
3/8 (38%)
3/11 (27%)
6/19 (32%)c
^Source: Edwards et al., 1942
bThese values represent total number of tumors observed 1n mice In both
age groups.
C01d control mice of this strain exhibit a very low Incidence, as compared
to CCl4-treated mice. Hepatomas were present 1n 2/71 untreated males
(3%) and 0/81 untreated females (0%).
11-15
-------
The experimental and control groups were divided Into five subgroups
according to the Interval between successive doses (1, 2, 3, 4 or 5 days)
and the total period of treatment (29, 58, 87, 116 or 145 days). Equal num-
bers of male and female mice were used In each of the experimental and the
five control groups. All mice were examined for the presence of hepatoma
150 days after the first dose. Some of them were killed at that time;
others were subjected to laparotomy. If hepatomas were not present, laparo-
tomles were performed at monthly Intervals thereafter to determine If hepa-
tomas eventually did appear. The gross diagnoses of hepatoma were confirmed
by hlstologlcal examinations.
In the lower dosage and shorter Interval groups, hepatomas were few 1n
number and small In size. With Increases 1n dose and 1n Intervals between
successive doses, there was progressive Increase 1n the number of small
hepatomas and the size of hepatomas for a given mouse. There was no differ-
ence 1n the Incidence of hepatomas between males and females.
The authors present the data 1n a matrix showing dose by Interval. An
adaptation of their matrix Is given 1n Table 11-8. Excluding the 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 roughly Increased nonlinearly with the time span
between onset and termination of CC1, exposure. All animals were examined
4
for the presence of hepatomas 150 days after the first dose.
11-16
-------
TABLE 11-8
Hepatomas 1n Male and Female Strain A Mice
Given CC14 via Stomach Tube3
Interval Between Doses (days)
ml of CCl4/g bw
30 doses
16 x 10~«
Male
Female
8 x 10"*
Male
Female
4 x NT*
Male
Female
2 x TO"4
Male
Female
1 x 10~«
Male
Female
0
Male
Female
1
0/6^
0/6
0/6
0/6
0/6
0/6
1/6
0/6
0/6
0/6
0/3
0/3
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
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
bNumber hepatomas/number mice
11-17
-------
In another study, liver necrosis and hepatomas were noted after NCI
strain A mice were treated with CC1 for 4 months (Eschenbrenner and
HUler, 1946). Treatment with CC1. was started when 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 CC1. by gavage at 0, 1200,
2400, 4800 or 9600 mg/kg bw. Dose schedules were either 120 doses 1n 119
days (dally) or 30 doses 1n 116 days (4-day Intervals). Animals were
necropsled at 8 months (30 days after cessation of administration). All
mice were given one additional dose of the solution 24 hours prior to
necropsy. Note that mice 1n the two groups at each dose level were admin-
istered the same total amount of CC1 over the same period of time, but
with a variation In the number of doses Into which the total amount was
divided and, therefore, 1n the size of each dose. The doses for mice 1n
group one (120 doses 1n 119 days) were previously determined as being
"necrotlzlng" and "non-necrotlzlng" and 1n group two (30 doses 1n 116 days)
as "only necrotlzlng." Two control groups of mice received 0.02 or 0.005
mi olive o1l/g bw/dose. The Incidence of hepatomas and necrotlc lesions
1s noted 1n Table 11-9.
At the three high group one doses on the (120 doses 1n 119 days) dose
schedule, a TOOK Incidence of hepatomas was observed. At the highest dose,
the mice had hepatomas of up to 1 cm In diameter. At the two Intermediate
doses the neoplasms were smaller 1n size. No hepatomas were observed 1n the
1200 mg/kg group. The Incidence of hepatomas was decreased 1n the animals
on the group two (30 doses 1n 116 days) dose schedule as compared to those
on the group one (120 doses 1n 119 days) dose schedule. At the 1200 mg/kg
dose, however, very small hepatomas were detected by microscopic examination
In two males. Mice given olive oil did not develop tumors.
11-18
-------
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-------
The presence or absence of hepatomas and of hepatic necrosis was deter-
mined. The localization of necrosis after chronic administration of CC1
4
did not appear to follow a definite pattern, 1n contrast to the regular
pattern of centrHobular necrosis seen after a single dose was administered
to strain A mice.
In summary, mice receiving non-necrotlzlng doses of CC1 developed as
4
many, If not more, tumors of the liver than mice given necrotlzlng doses
+
despite the fact that equal amounts of CC14 were administered. Mice given
1200 mg/kg did not develop gross tumors; most mice receiving either 2400,
4800 or 9600 mg/kg bw did develop tumors.
NCI performed bloassays on chloroform (NCI, 1976a), 1,1,l-tr1chloro-
ethane {NCI, 1977) and trlchloroethylene (NCI, 1976b) 1n which CC1 was
4
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. A necropsy was performed on all mice along
with complete hlstologlcal examinations.
Host male and female mice treated with CC1, were dead by 78 weeks
4
(Table 11-10). 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
/f
females given the low and high doses, respectively. Hepatocellular carcino-
mas were found 1n practically all mice receiving CC1 , Including those
dying before termination of the test (Table 11-11). The first carcinomas
were observed In low dose female mice at 16 weeks, In high dose female mice
at 19 weeks, 1n low dose males at 48 weeks, and 1n high dose males at 26
weeks, compared to 90 weeks for vehicle control females and 72 weeks for
11-20
-------
TABLE 11-10
Survival of B6C3Fi Mice Treated with Carbon TetrachloMde3
(CC14 administered by oral gavage)
Dose
MALE
Control
Matched
Pooled
Low Dose*3
High Dosec
FEMALE
Control
Matched
Pooled
Low Dose^
High Dosee
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%)
^Source: NCI, 1976a,b, 1977
b!250 mg/kg bw
C2500 mg/kg bw
11-21
-------
TABLE 11-11
Incidence of Hepatocellular Carcinomas
1n Mice Treated with Carbon TetrachloMde3
(CC14 administered by oral gavage)
Dose
Hepatocellular
Carcinomas
HALE
Pooled Controls
Low Doseb
High Dose0
FEMALE
Pooled Controls
Low Dose'3
High Dosec
5/77 (6%)
49/49 (100%)
47/48 (98%)
1/80 (1%)
40/40 (100%)
43/45 (96%)
aSource: NCI, 1976a,b, 1977
b!250 mg/kg bw
C2500 mg/kg bw
11-22
-------
vehicle control males. Some liver toxldty occurred, Identified as cir-
rhosis, bile duct proliferation, toxic hepatitis and fatty liver; however,
these cases were few In number. In summary, this study found CCl^ to be
highly carcinogenic for liver 1n mice.
Confer and Stenger (1966) studied nodules 1n the livers of C3H mice
after long-term CC1 administration. Twenty-five male mice, 5 weeks of
age, received rectal Instillation of 0.1 ma. of a 40% solution of
dissolved 1n olive oil 2 times a week for 20-26 weeks. Only olive oil was
given to 10 control mice. Fourteen mice were killed 9 days after the last
treatment, and the remaining mice were killed at periods of 3-37 weeks. The
livers were examined by light and electron microscopy and revealed that 5/14
mice (36%) killed after 9 days and 8/11 mice (73%) killed later developed
hyperplastlc hepatic nodules. Cirrhosis was not observed 1n the liver.
In summary, mice given CC14 by rectal Instillation had hyperplastlc
nodules that persisted after the discontinuation of the chemical, but did
not develop cirrhosis of the liver. Confer and Stenger (1966) proposed that
hyperplastlc nodules, as observed 1n their study, are precursors of liver
carcinomas.
Edwards and Dalton (1942) studied the CC14 Induction of cirrhosis of
the liver and hepatomas 1n 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. All mice were started 1n the study at
1-5 months-of age.
In one experiment, a dose of 0.1 ma of a 40% solution of CCl^ (with
no Impurities) In olive oil was administered by stomach tube 2 or 3 times
(
per week. The total number of treatments varied from 23-58. In order to
11-23
-------
study any early pathologic changes, a number of mice were killed after
receiving 1-23 doses. In another experiment, male mice were given 0.1 ma
of olive oil 2 or 3 times a week for 39-62 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 hlstological techniques
were used to examine a number of primary and transplanted tumors. These
Included techniques for the presence of fat, glycogen or alkaline phos-
phatase and those for studying the mitochondria and Golgi apparatus.
Hepatomas were observed in 88% of C3H male mice treated with CC1 ,
whereas they occurred in 4% of untreated mice of the same age and strain.
Liver tumors developed in 60% of male and female strain Y mice treated with
CC14 and occurred in only 2% of 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 strain C 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 control groups are given in Tables 11-12 and 11-13. Tumors did
not appear to have been Induced 1n any of the other organs.
Low dose administration (0.1 ma. of 5% CC L in olive oil or 0.005
4
mjt) occurred 3 times weekly by stomach tube to 58 strain A female mice,
2.5 months of age, for 2 months. Mice were necropsUnl ? clays to 4.5 months
after the last treatment. Hepatomas were present In 41 mice (71%), and some
had cirrhosis of the Hver.
11-24
-------
TABLE 11-12
Hepatomas 1n Male and Female Mice Given Carbon Tetrachlorlde
(0.04 mfi. 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 1n Untreated Male and Female Nice
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 (OH)
8/400 (2%)
*Source: Edwards and Dalton, 1942
11-25
-------
TABLE 11-13
Hepatomas 1n Hale Mice Given Olive 011 by Storoach Tube*
Strain
C3H
C
A
Age (months)
10-11
12
5-12
Incidence
4*
0%
0%
*Source: Edwards and Dalton, 1942
11-26
-------
The total dose (0.125-0.145 ml CCl^) 1s comparable to the total dose
of 0.120 ml CC14 In the experiment In which mice were given treatments
of 0.04 ma each. The tumors of the Hver were morphologically similar 1n
\
both studies.
Limited treatment Involved strain A female mice, 2 months of age. There
were 21-62 mice 1n three treatment groups. The CC14 used was dissolved 1n
olive o1l9 the volume of the mixture administered amounting to 0.1 ml.
The mice were given 1-3 treatments. The hepatotoxlc doses were 0.04, 0.01
or 0.005 ml CC1,. Eleven mice received olive oil only. The mice were
4
necropsled 2-12 months after the start of the experiment.
Tumors of the liver were not found 1n these mice. There was pigment 1n
Kupffer cells, occasional foci of basophHlc debris, and an Increase 1n con-
nective tissue and retlculum.
In summary, CC1. Induced significant numbers of tumors of the Hver,
as well as cirrhosis, 1n four strains of mice.
Since successful transplantation Is frequently considered to be a cri-
terion of neoplasla, Leduc and Wilson (1959) attempted to transplant CC14~
Induced tumors of the liver 1n mice. At first
"numerous failures to establish a transplantable CCl4-1nduced
hepatoma supported the Idea that, 1f transplantabllHy Is a cri-
terion, the nodules might be hyperplastlc but not neoplastlc. Sub-
sequently, however, several such nodules were successfully 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
been found 1n this strain, up to Us 40th generation. Carbon tetrachlorlde
was administered by stomach tube In doses of 0.1 ml of a 40% solution In
olive oil (0.04 ma CC1 ) per treatment. Carbon tetrachlorlde was given
3 times a week for a total of 45-66 doses. About one-third of the mice were
given three dally Intravenous (l.v.) Injections of 0.2 ml of thorotrast
11-2?
-------
before CC14 administration was started. As discussed by Leduc and Wilson
(1959), thorotrast 1s useful 1n the detection of hepatomas but has been
Involved (as Indicated by other Investigators), 1n tumor production.
The first-generation tumor transplants were made subcutaneously. Subse-
quently, both subcutaneous and 1ntrasplen1c transplants were made. Under
light ether anesthesia, Implants of tumor Into the spleen were made by an
Incision through the dorsal body wall. The spleens were examined periodi-
cally by laparotomy.
Hepatomas did not develop 1n 20 control mice given thorotrast only.
Hepatomas did occur 1n CCl4-treated mice that were free of thorotrast.
The CC14 hepatomas (5 of 7) that were successfully transplanted dif-
fered from those that did not grow 1n new hosts because a longer time period
elapsed between CC14 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 after the last treatment. The authors concluded that chronic
CC14 Injury to the liver Induces the development of both hyperplastlc
nodules and hepatomas. They found the livers of CC1 -treated mice to be
drrhotlc with numerous hyperplastlc nodules.
11.1.3. Hamster Studies Hamsters have not been studied as extensively as
rats and mice. There has been only one report of the Induction of tumors 1n
hamsters by CCK.
Delia Porta et al. (1961) orally administered CC14 to SyMan golden
hamsters as a part of a larger Investigation of the response of this species
to carcinogens that Induced liver neoplasms 1n other species. Ten female
and 10 male Syrian golden hamsters, 12 weeks old, were used. At the beginn-
ing of the experiment, males weighed an average of 99 g and females weighed
11-28
-------
an average of 109 g. At the end of the experiment, the average weight was
104 g for both sexes. The treatment consisted of weekly administration by
stomach tube of a 5% solution of CC1. 1n corn oil for 30 weeks. Controls
4
cited were historical controls kept by the Investigators 1n the same labora-
tory for a Hfespan. A total of 145 female and 109 male hamsters of the
same strain, fed the same diet, did not develop liver-cell tumors. The
authors also cited controls for a different study they conducted. In this
latter study, 30 female and 50 male hamsters fed the same diet but given
0.5 ml corn oil via stomach tube twice weekly for 45 weeks also did not
develop liver-cell tumors. During the first 7 weeks of the former experi-
ment, 0.25 ma of the solution containing 12.5 ySL CC14 was given each
week. This dose was then reduced to 0.125 ml and contained 6.25 yt of
CCT.. After this treatment, the survivors were kept under observation for
4
25 additional weeks and then killed. Detailed hlstopathologlcal examina-
tions of all hamsters were conducted, except for one female lost through
cannabaHsm at the 28th week.
Weights of the hamsters varied Irregularly during'the period following
treatment. In general, the weights Increased. Females weighed an average
of 114 g and males 118 g. One female died at the 10th week of treatment;
three females and five males died or were killed between the 17th and the
28th week. Three females died at weeks 41, 43 and 54. The surviving three
females and five males were killed at the end of the 55th week.
Hamsters dying during the treatment and at the 41st week had cirrhosis,
as well as hyperplastlc nodules that were two to several layers thick. The
cells showed Irregularities 1n the shape, size and staining qualities of
their cytoplasm and nucleus, with an uneven distribution of glycogen.
11-29
-------
All of the animals, five males and five females, dying or killed 13-25
weeks after the end of the treatment, had one or more liver-cell carcinomas
(a total of 22 tumors: 12 1n the females and 10 1n the males). No mention
was made of toxldty 1n these animals. Liver cell carcinomas were not found
1n the other animals dying before week 43.
Transplantation efforts were not successful. The authors note 1n their
discussion that this negative result deserves further Investigation since
"many other tumors of hamsters have been successfully transplanted to non-
Inbred hamsters 1n this and other laboratories."
In summary, Syrian golden hamsters appear sensitive to the carcinogenic
effects of CCl^. Although the number of animals 1n this study was small,
the authors considered the results to be significant because the reported
historical control Incidence of hepatic tumors 1n hamsters was 0/254.
Hyperplastlc nodules appeared during treatment, and carcinomas appeared
after CC14 administration had been discontinued, which suggests that the
nodules or benign tumors were precursor lesions for carcinomas. It should
be noted that this study 1s the only report found 1n the available litera-
ture of CC14 Induction of tumors 1n hamsters.
It 1s recognized that differences exist among species regarding their
sensitivities. However, data quantifying such differences have not been
found 1n the available literature. Nevertheless, such differences should be
noted 1n light of the predominance of research/data on the rodent species.
Methods of dose conversion from animals to humans are discussed 1n the
Appendix.
In concluding this section on animals, 1t should be noted that some of
the research reported suggests that hepatomas occur only after liver necro-
sis and flbrosls have occurred (Edwards, 1941; Edwards and Dalton, 1942;
11-30
-------
Delia Porta et al., 1961; Reuber and Glover, 1967, 1970). The results have
been Interpreted to mean that "as far as the liver 1s concerned, hepatoma 1s
an occasional consequence of the Induction of postnecrotlc cirrhosis and
that CC14 1s not a direct liver carcinogen" (Lourla, 1977). The results
reported by Eschenbrenner and Miller (1946), however, refute Lourla's state-
ment. These authors concluded that If CC1. 1s, In fact, a carcinogenic
4
agent, tumors should be obtained with non-necrotlzlng doses. As discussed
earlier 1n this chapter, their series of experiments examining the Issue,
revealed:
"While 1t was found that a correlation exists between the degree of
liver necrosis and the Incidence of hepatomas 1n relation to dose,
the use of a graded series of necrotlzlng and non-necrotlzlng doses
Indicated that repeated liver necrosis and Its associated chronic
regenerative state are probably not necessary for the Induction of
tumors with carbon tetrachlorlde" {Eschenbrenner and MUler, 1946).
The small number of animals used 1n this study must be noted.
A list of authors addressing the Issue of liver necrosis Induced by
CC14 1s provided 1n Table 11-14.
11.2. HUMANS
11.2.1. Case Reports. As mentioned In the section dealing with human tox-
Iclty, 1n many of the case reports the Investigators present the data 1n
narrative form. Although Interesting, these type of data are not suitable
to quantitative analysis since numbers are not adequately presented. Fur-
thermore, there usually are a number of uncontrolled variables (alcohol
Intake, age, simultaneous exposures) or unknown variables (exposure amount)
making 1t difficult to attribute the outcome solely to the CC1. exposure.
Despite these limitations, they are Important since they can lend support or
challenge the experimental animal studies and c®n aid qualitatively Ira the
extrapolations from animals to humans.
11-31
-------
TABLE 11-14
Studies 1n Which Liver Necrosis was Induced
Using Carbon Tetrachlorlde
Species
Mice
Mice
Mice
Hamsters
Rats
Human
Rats
Route
gavage
1ngest1on
1ngest1on
1ngest1on
subcutaneous
Injection
Ingestlon
subcutaneous
Injection
Reference
Edwards, 1941
Edwards and Dalton, 1942
Eschenbrenner and Miller, 1946
Delia Porta et al., 1961
Reuber and Glover, 1967a
Hashimoto et al., 1968
Reuber and Glover, 1970
11-32
-------
The carcinogenic effect of exposure to CC14 In humans has been sug-
gested 1n 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
CC1 used to clean a rug 1n his apartment earlier that evening. Five days
4
later he developed nausea, vomiting and diarrhea and within 10 days of expo-
sure he developed jaundice. The patient recovered following a long and com-
plex hosp1ta!1zat1on and was discharged after 9 weeks. Four years after
hosp1tal1zat1on for jaundice, he was found to have a smooth, enlarged, non-
tender 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 Ingestlon of alcohol. A
liver biopsy was diagnosed as hepatocellular carcinoma. Postmortem examina-
tion revealed the liver to be extensively Involved with tumor. Little nor-
mal liver tissue remained.
Aside from the acute exposure to CC14 7 years before diagnosis of can-
cer, the patient's possible additional exposure to this and other toxic
chemicals was not reported. No medical history was given for the 3 years
before final diagnosis.
Other case reports of human neoplasms developing after exposure to
CC1, have appeared. In one, a woman developed modular cirrhosis of the
4
liver followed by cancer of the liver after exposure to CC14, and died 3
years after the first exposure (3ohnstone, 1948). However, she had suffered
from periodic jaundice for 5 years prior to the CCl^ exposure. In a
second, a fireman developed cirrhosis and an eplthelloma of the liver 4
years after acute CC1 Intoxication (Slmler et al., 1964). In none of the
cases could a causal link between CCl^ exposure and development of neo-
plasms be established.
11-33
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11.2.2. Studies. A study by Capurro (1979) reports a series of cancer
cases 1n a rural valley polluted by vapors from a solvent recovery plant,
Odor and pollution problems existed 1n the valley from 1961-1971. A variety
of solvents Including CC14 were Identified 1n the air. Blood tests were
done on 24 residents; solvents were detected 1n the blood of all those
tested. A 11st of these solvents detected 1s not given but those "most
easily detected" (as cited by the author) were: benzene, chloroform, methyl
Isobutyl ketone and tMchloroethylene. Levels were not reported.
A study population was defined consisting of 117 Caucasians who lived
within 1.5 km2 of the 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
that were not residents. The author's analysis focused on four cases of
lymphoma (2 lymphosarcoma, ICD 200.0 and 2 retlculum cell sarcoma ICO
200.1). All four of these cases had a history of having worked 1n the paper
mill which preceded the solvent recovery plant at the same location. The
author reported that a study of paper mill workers {Hllham, 1976) found that
the proportionate mortality ratio [PMR = (observed deaths/expected deaths) x
100] Increased 1.8 times for lymphosarcoma. No significant Increase was
found for retlculum cell sarcoma. However, as Capurro (1979) reported, "the
excess lymphomas observed by us 1s far above the one described by Mllham."
A mortality ratio Indicating a 160-fold Increase for these two cancers (ICO
200.0 and 200.1) was calculated. However, 1t should not be reported due to
errors 1n Us calculation. Three observed deaths were used, rather than
four, with no explanation and the study population was rounded off to 120
Instead of using the defined 117. The ratio was not standardized by age or
sex, nor was 1t described as race-specific (all Caucasians). The assumption
11-34
-------
was made that since the age distribution of the study population 1s compar-
able to that of the nation, age standardization was not necessary. However,
the study population was then compared to the population of the State of
Maryland rather than the nation. The discussion of lymphomas diagnosed 1n
the county and 1n the area surrounding the plant lacks Important Information
on population distribution, time period and comparison group as well as
methods used. Due to Us lack of specificity and Its questionable statis-
tical methods, this study 1s of limited value. Thus, 1n light of these
deficiencies, the conclusion of the author that the Incidence of malignant
lymphoma 1s abnormal 1n the area exposed to solvent vapors 1s not supported.
In a preliminary study designed to determine 1f occupational exposure to
CC1 , trlchloroethylene and tetrachloroethylene resulted 1n Increased mor-
tality, Blair et al. (1979) studied causes of death 1n 330 laundry and dry
cleaning workers. Sex, race and age along with underlying and contributing
causes of death were abstracted from death certificates. The underlying
cause was classified according to the International Classification of Dis-
eases. Seventh Revision, by a trained nosologlst. 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 comparison group) and statistically signifi-
cant (x2 = 6.423, 1 df, significant at p<0.05). The excess deaths con-
sisted of lung, cervical and Hver cancers, and leukemia. The authors note
that the lung and cervical cancer excess may reflect the lower sodoeconomlc
status of these workers. The slight excess of liver cancer 1s consistent
with bloassay studies showing liver abnormalities. The authors conclude
that ep1dem1olog1c studies of this occupational group are warranted due to
the Increased risk observed.
11-35
-------
The paper has numerous faults. First, the authors place too much confi-
dence 1n the PMR. The PMR does not estimate risk but Instead Indicates the
possibility of an Increased risk. Any conclusions made by the authors con-
cerning an Increased risk associated with the exposure 1s unfounded. The
exposed population is compared to the United States population to determine
the expected number of deaths. Ideally, the exposed population should have
been compared to another working population to control for the "healthy
worker effect." However, more serious reservations arise when examining the
data and conclusions. Based on the United States population, 126 deaths due
to circulatory diseases were expected. One hundred deaths due to circula-
tory diseases were observed in the exposed population. This lower than
expected number of deaths 1s, as noted by the authors, necessarily asso-
ciated with the elevated PHR for cancer deaths (68 expected, 87 observed).
Thus, it 1s difficult to conclude that the elevated PMR for cancer deaths Is
1n excess of expectations but rather that it may merely be the result of
lower than expected relative frequencies of other causes of death. There-
fore, one can only conclude from this study that there is a need for addi-
tional work on this occupational group to clarify the issues raised. An
excess risk of carcinogeniclty following exposure to CC1. 1s not indicated.
Finally, increases 1n certain types of skin cancers can be attributed to
Increased atmospheric CC1 . As explained 1n Chapters 5 and 6, CC1 may
result in an overall reduction of stratospheric ozone, eventually leading to
Increased UV-B radiation. It is estimated that a 2 to 5% increase In basal
cell skin cancer will occur per 1% decrease 1n 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
unclear, thus rendering quantitative estimates uncertain. However, sunlight
may be a causal factor 1n the development of malignant melanoma.
11-36
-------
11.3. SUMMARY
11.3.1. Experimental Animals. Carbon tetrachlorlde 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 1n adrenal tumors In male and female mice was seen 1n
one study. Hamsters, although only used 1n one study, have been the most
sensitive species studied, followed by mice and then rats. A difference 1n
sensitivity to CCl.-1nduced neoplasms has been observed among five rat
strains. Female rats have appeared less sensitive to the chronic toxic
effects but more sensitive to the carcinogenic effects of CC1..
11.3.2. Humans. A number of cases of hepatomas appearing 1n humans years
following exposure to CC1 have been reported. A study examining the
effect of solvent vapors (one of which was CC1.) on a group of environ-
mentally exposed people concluded the existence of an abnormal Incidence of
malignant lymphoma. However, 1t should not be used as evidence of the car-
clnogenldty of CC1. due to the concomitant exposures and poor study tech-
niques. A preliminary epldemlologlcal study of a group occupatlonally
exposed to CC1. revealed a slight excess of liver cancer. Although an
excess risk of carc1nogen1c1ty following exposure to CCl^ 1s not Indi-
cated, this study does Indicate the need for additional research.
11.3.3. IARC Evaluation. The International Agency for Research on Cancer
(IARC, 1979) has devised a rating scheme for carcinogenic potential based on
animal and human studies. The IARC evaluations for CC1. are:
"sufficient" evidence for carclnogenlclty 1n animals, "Inadequate"
evidence for carclnogenlclty In humans, an overall evaluation that
CC14 1s "probably carcinogenic to humans" (group 2B).
11-37
-------
The animal category of "sufficient evidence" Indicates that Increased
Incidence of malignant tumors was shown 1n multiple species or strains, or
1n multiple studies, or to an unusual degree regarding Incidence, site,
tumor type, or age of onset.
The human category of "Inadequate evidence" Indicates that few data
exist, the available positive evidence did not exclude chance, bias or
confounding, and/or studies exist which did not show evidence of carcino-
genldty.
The overall evaluation of "probably carcinogenic to humans", group 28,
reflects the Important role played by the results of animal studies, with
limited or no data on humans. The combination of "sufficient" evidence for
animals and "Inadequate" evidence for humans usually results 1n the overall
classification of 2B.
11.3.4. Conclusion. In conclusion, there 1s evidence that CC1. may be a
human carcinogen based upon the following studies: (1) positive findings on
mice 1n the NCI bloassay for trlchloroethylene (1976) 1n which CC1. was
used as the positive control; (2) the hamster study by Delia Porta et al.,
(1961); (3) the rat studies by Reuber and Glover (1967, 1970) and NCI
(1976); and (4) the mice studies by Edwards (1941), Edwards et al. (1942),
Edwards and Dalton (1942) and Welsburger (1977). Human data as reported by
Blair et al. (1979) Indicate the need for additional ep1dem1olog1cal
research. The combined evidence of animal and human studies has been
evaluated by the IARC as Indicating that this compound 1s probably carcino-
genic for humans.
11-38
-------
12. SYNERGISN AND ANTAGOMISM
12.1. SYNER6ISM
A description of the entire clinical picture of CC1. toxlclty should
consider the role played by alcohol 1n the genesis of severe CCl^ poison-
Ing (yon Oettlngen, 1964). A number of researchers have reported on this
phenomenon (Stevens and Forster, 1953; K1rkpatr1ck and Sutherland, 1956;
Joron et al., 1957; New et al., 1962; Markham, 1967). It has been estab-
lished that habitual 1ngest1on of alcoholic beverages and also their
occasional use may Increase the dangers from comparatively moderate exposure
(Markham, 1967; Tracey and Sherlock, 1968). This fact 1s Illustrated by
reports on simultaneous exposure of abstinent persons and consumers of
alcohol to the same CC1. concentration with only the latter becoming
seriously 111 (von Oettlngen, 1964).
An example of this was given by Smetana (1939) who reported on three
cases of CC1. 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 CC14- Several hours later, he developed the
typical acute symptoms of dyspnea, cough and bloody sputum associated with
exposure to CC1,. His condition worsened and he subsequently died. A
coworker of his, a teetotaler, had been working 1n the same room for the
same amount of time receiving the same exposure to the CC1 . The author
stated that "although he felt the effect of the exposure and suffered from
headache and gastrointestinal distress, he recovered quickly after breathing
fresh air" (Smetana, 1939).
Hypotheses have been advanced to rationalize this apparent synerglstlc
reaction between alcohol and CCl.. Lamson et al. (1928) postulated that
4
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 1n the hepatic cells of alcoholics
may have some role 1n the greater susceptibility to CC1 poisoning. This
latter theory 1s supported by the findings that neonates are protected
against CCl^ poisoning and that neonates have an Increased concentration
of liver glycogen (Bhattacharyya, 1965).
Tralger and Plaa (1971) Investigated the potentiating capacity of
aliphatic alcohols on CC14 toxldty. Hale Swiss-Webster mice were given a
single dose by gavage of methanol, ethanol or Isopropanol equivalent to 50%
of the LD50- After 20 hours, an 1.p. Injection of 0.0075 mfi,/kg CC1.
1n corn oil was administered. Controls received only the corn oil. Blood
samples were analyzed for SGPT activity. All alcohols produced Increased
activity but the effect was most marked among the mice pretreated with
Isopropanol. The administration of the alcohols or CC1, alone did not
4
change the SGPT levels.
In an effort to ascertain the differences of the alcohols 1n the poten-
tlatlon 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 3-48 hours prior to the 1.p.
Injection of 0.1 ml/kg CC14 1n corn 611. Controls received only the
corn oil. After 24 hours, the animals were sacrificed and blood and Hver
allquots were taken for SGPT, glucose-6-phosphatase, blUrubln and hepatic
trlglycerlde analysis. Isopropanol produced a more marked change 1n the
activities of SGPT, glucose-6-phosphatase and trlglycerldes. Isopropanol
pretreatment produced hyperbH1rub1nem1a whereas ethanol pretreatment or
alone at a 10-fold Increase 1n dosage did not. Also, a 10-fold
12-2
-------
dosage Increase In CCl^ alone produced an Increased level of S6PT activ-
ity; however, It was only -25% of the SGPT activity found among rats pre-
treated with Isopropanol.
We1 et al. (1971) Investigated the potentlatlon of CC14 hepatotoxlclty
by ethanol and cold. Sprague-Dawley rats were exposed to either ethanol
(50% v/v 1n water Intubated per os), cold (2-4°C for 18 hours) or warmth
(31-33°C for 18 hours). Indices of hepatotoxlclty were SGPT levels and
Hver trlglycerlde levels. In both male and female rats, the SGPT levels
Increased after both ethanol and cold exposures 1n response to the CC1 .
The authors postulate that the ethanol as well as cold, stimulates release
of noreplnephrlne, which 1n turn Increases the susceptibility of the Hver
to CC1 . Warmth was found td reduce the potentiating effects of ethanol
on serum enzyme release.
According to Davis (1934), very obese or undernourished persons suffer-
ing from pulmonary diseases or gastric ulcers or having a tendency to vomit-
Ing, Hver or kidney diseases, diabetes or glandular disturbances are
especially sensitive to the toxic effect of CC1 (von Oettlngen, 1964).
Strubelt et al. (1978) found that CCl,-1nduced Hver damage was sig-
nificantly greater In rats concomltantly exposed to ethanol than In control
rats not exposed to ethanol. ftale Wlstar 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, CGI was administered Intraperltoneally at a
dose of 0.1 mg/kg. Hepatotoxlc effects were evaluated by measuring the
serum activities of SGOT, SGPT arid sorbltal dehydrogenase (SDH) as well as
hlstologlcal Investigations.
12-3
-------
The potentlatlon noted was fully developed following 1 week of exposure
and was greater 1n those rats provided a 15% ethanol solution than 1n the
rats provided a 5% ethanol solution, thus appearing to be dose-dependent.
Hasumura et al. (1978) also examined the potentlatlon of CCK hepato-
4
toxlclty by ethanol. They designed their study to determine 1f hepatic
mlcrosomal changes, which are secondary to chronic ethanol consumption, play
a role 1n the mechanism of CC1 hepatotoxldty. Rats were pair-fed a
liquid diet containing ethanol (36% of calories) or 1socalor1c carbohydrate
for a period of 4-5 weeks. Carbon tetrachlorlde, at a dose of 0.5 mil/kg,
was administered 1ntragastr1cally 15 hours after ethanol withdrawal. IsJHhln
24 hours there was an Increase 1n liver I1p1ds and serum ornlthlne carbamyl
transferase activity and a decrease 1n the activities of hepatic amlnopyrene
N-demethylase and glucose-6-phosphatase. These changes were determined to
be significantly greater 1n the ethanol-fed rats versus the control rats
indicating in vivo potentlatlon of CC1. hepatotoxldty 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, Hver micro-
somes were Incubated with [14C]CC1 and a NADPH-generatlng system. The
authors found an enhancement of: 1) the covalent binding of i4C to mlcro-
somal protein 1n vitro, and 2) the blotransformatlon of [14C1CC1 to
4
X4C02 in vitro. This suggests that ethanol pretreatment stimulates the
mlcrosomal formation of an active metabolite of CC1.. Thus, mlcrosomal
changes are responsible, at least partially for the Increased CCK hepato-
toxldty (Hasumura et al., 1978).
12-4
-------
In addition to ethanol, other compounds have been Investigated as to
their effect on CC1 hepatotoxldty. Curtis et al. (1979a) and Davis and
Mehendale (1980) have studied the potentlatlon of CC14 hepatotoxldty by
exposure to chlordecone (Kepone®).
Curtis'et al. (1979a) found greatly potentiated hepatotoxldty reflected
1n the form of elevated SPGT and SGOT activities between groups of rats
which had undergone a 15-day feeding of 0 or 10 mg/kg chlordecone, and a
single 1ntraper1toneal challenge of CC1 . The CC1 challenge was either
0, 0.025, 0.05, 0.1 or 0.2 mi/kg. The SPGT and SGOT activities Increased
1n excess of 30-fold and 10-fold respectively 1n chlordecone-fed rats chal-
lenged with CC1 of 0.1 and 0.2 ml/kg. The authors concluded that the
data Indicate a great potential for the production of severe liver damage
resulting from Interactions of CC1. and chlordecone exposure at levels
which may be Independently nontoxlc.
Davis and Mehendale (1980) conducted a similar study using a single per
os administration of chlordecone (5 mg/kg) followed 48 hours later by 1ntra-
perltoneal administration of CC1 {0.2 ml/kg). Twenty-four hours later
the hepatic excretory function of the animals treated with chlordecone and
CC14 had decreased (20% of controls), while plasma transaminase activities
and biHrubln were elevated. Parameters measured or assessed which were not
affected by chlordecone pretreatment were: hepatic mixed function oxidase
activity, irreversible binding of label from [14C]CC14 to hepatic
protein or lipid, hepatic and renal glutathione concentrations and CC1 -
Induced I1p1d peroxldation of liver tissue measured in vitro and In vivo.
The authors conclude that the mechanism for the enhanced toxidty is still
unknown; however, the results suggest the interaction between chlordecone
12-5
-------
and CCl^ 1s a subtle one, not causally Involving Increased covalent bind-
ing of the toxin, Increased susceptibility of tissue I1p1ds to peroxldatlve
damage or decreased hepatic glutathlone.
Curtis et al. (1979b) Investigated the effect of pre-exposure to 50
mg/kg photomlrex on CC1 hepatotoxlclty 1n rats. Photomlrex 1s a photo-
degradation product of the Insecticide mlrex, and 1s also a structural
analog of chlordecone. The photom1rex/CCl Interaction resulted 1n Hver
hypertrophy, 7- and 8-fold elevations 1n SGOT and SGPT over the control rats
and rats treated with photomlrex alone, and considerable centrHobular
necrosis. Liver weight, SGOT and SGPT were unaffected by the CCK
challenge alone; therefore, pre-exposure to photomlrex does potentiate
CCl^ hepatotoxlclty as does chlordecone, a structural analog.
In a review article, Falk (1976) reported on the effects of different
chemicals on CC1 toxldty. A summary of this review 1s presented here.
Benzo(a)pyrene (BAP) 1n conjunction with CC14 has been shown to enhance
tumor production 1n laboratory animals (Kotln et al., 1962; Proetzel et al.,
1964) while CC1. alone was found to produce no sarcomas. Welsburger et
al. (1965) found that treating rats with acetylamlnofluorene (AAF) produced
an Increase 1n hepatomas In female rats, from 0% without to 81% with CC1,.
4
In males, the Increase was 73-100%. A change 1n metabolism of AAF toward
greater N-hydroxylat1on on CC1 treatment was found to be the reason for
4
the enhanced tumor Incidence (Welsburger and Welsburger, 1963).
Kluwe et al. (1979) found that CC1 produced liver damage 1n male rats
fed 100 mg/kg polybromlnated blphenyls (PBB) or 200 mg/kg polychloMnated
blphenyls (PCB) 28 days before Injection of CC14, as represented by
Increased levels of SGOT. The liver damage was greater 1n the rats fed PBB
than 1n those fed PCB which 1n turn showed greater damage than the control
12-6
-------
rats. Functional renal damage was produced by CCl^ and several other sol-
vents such as TCE and 1,1,2-trichloroethane. Carbon tetrachloride-lnduced
renal dysfunction was found to be potentiated by PBB but not PCB.
01etz and Tralger (1979) found that pretreatment of rats with either
2-butanone or 2,3-butanedlol markedly enhanced the hepatotoxic response to
CC1, as measured by SGPT and hepatic trlglycerldes. The administration of
4
CC1. and qulnalphos (an Insecticide) was found to cause morphological
changes in the liver, kidney and testes of male rats (D1ksh1th et a!.,
1980). The authors suggest that pretreatment of CC1 rendered the animals
susceptible to the toxic effects of qulnalphos. x
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 methlonine against I1p1d peroxidatlon Induced by CC1-. In the
first of three experiments, 21-day-old male weanling rats were fed a diet
deficient in vitamin E and Se and low in methlonine. Dietary supplements of
these three variables were administered either alone or 1n combination. To
assess the effect of CC1 , lipid peroxidatlon was monitored by the evolu-
tion of ethane, an auto-oxidation product of w-3-unsaturated fatty acids.
To study the effect of Increasing dietary -3-unsaturated fat, was substituted for lard. A dose of 2 ma CCl./kg
bw was administered via 1.p. injection. The investigators found that 1n all
dietary groups, CC1 stimulated ethane evolution. However, among mice
given dietary supplements of vitamin E, Se and methlonine, ethane evolution
was reduced 17, 26 and 39%, respectively. The substitution of OLD for lard
in the diets resulted in a 6-fold increase in ethane evolution. If only
12-7
-------
CCl4-1nduced ethane evolution 1s considered, only dietary supplements of
vitamin E and Se resulted 1n a statistically significant (p<0.05) reduction
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 toxldty of CC1. was decreased
1n correlation with ethane evolution. Thus, vitamin E, Se and methlonlne
protected against CCl4-1nduced I1p1d peroxldatlon, probably by maintaining
Intracellular glutathlone and glutathlone .peroxldase.
In two other experiments, the authors sought to determine the effective-
ness of each protective factor alone or In combination and to estimate
peroxldatlon rates 1n rats 1n which CC1. caused early mortality. In these
experiments, a dose of 1 mil CCl4/kg bw was administered via 1.p. Injec-
tion. Results Indicated that vitamin E, Se and methlonlne supplements 1n
all combinations protected against CC1 toxldty and that supplements of
either vitamin E or Se, or both with methlonlne, completely prevented
CCl.-1nduced mortality.
Reserplne, carbon dlsulflde and d1ethyld1th1ocarbamate reportedly
diminish the toxic effects of CC14 on liver 1n experimental animals. The
mechanisms are unknown, but some of the Investigators have speculated that
metabolism via the mlcrosomal enzyme system may be Involved (Douglas and
Clower, 1968; Seawrlght et a!., 1980; Siegers et a!., 1978). Chlorpromazlne
prevented liver necrosis from CC1. 1n short-term rat experiments without
affecting I1p1d peroxldatlon or binding of CCl4-react1ve metabolites (not
Identified), but 1t also lowered body temperatures. The Investigators were
of the opinion that chlorpromazlne only delayed the onset of liver necrosis
(Marzl et a!., 1980). Cagen and Klaassen (1980) reported that the release
of alanlne amlnotransferase and aspartate amlnotransferase Into plasma 24
12-8
-------
hours following various doses of CC1. was markedly lower 1n rats pretreat-
ed for 3 days with zinc chloride (150 ymol/kg/day) than 1n control rats.
Administration of zinc chloride solution was found to Increase hepatic con-
centrations of metallothloneln. The authors suggest that metalloth1one1n
may protect against CC1 -Induced liver damage by sequestering reactive
metabolites of CC1,. .
4
Seawrlght and McLean (1967) conducted a study to Investigate the de-
crease 1n CC1. toxldty brought about by a protein-free diet. Hale Wlstar
rats of a Porton strain were orally administered 2.5 ml/kg [14C]CC1.
diluted 1n an equal volume of paraffin oil. Those rats maintained on
protein-free diets >7 days exhibited a decrease In the metabolism of CC1.
to CtL 1_n vivo as well as 1n mlcrosomal preparations, compared to rats
maintained on a stock diet. However, rats Injected subcutaneously with 100
mg/kg DDT 1 week before death regained their sensitivity to the toxic
effects of CCK as measured by their ability to metabolize CC1, and
Pyramldon demethylatlon.
Falk (1976) also reported on the effects of diet on CC1. toxldty. A
low protein diet fed to rats reduced the hepatic mlcrosomal hydroxylase
activity and dramatically reduced the toxldty of CC1 . The L0__ of
CC1. was 14.7 ma./kg on the protein-free diet compared to 6.4 ma on a
4
regular diet. When the rats were pretreated with phenobarbltal the LD_
was 0.5 mil/kg.
Mikhail et al. (1978) examined the protective effect of adenoslne mono-
phosphate (AMP) on OCT. toxldty. Three groups of male and female adult
4
rats were studied: a control group, a group treated with 0.5 ma of a 1:1
\
mixture of CC1. 1n mineral oH/100 g bw via 1ntraper1toneal Injection and
12-9
-------
a group treated with 30 mg AMP via Intraperltoneal Injection 1/2 hour prior
to treatment with CC1 . The CC1 resulted In elevated serum Iron,
copper, 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 there were no changes In serum calcium, magne-
sium, potassium and sodium levels as compared to the CCl.-treated group.
The normalization of zinc may be due to the action of AMP on hormone secre-
tion. Thus, the authors conclude that the normalization of Iron and copper
may be due to some protective effect of AMP on the liver (Mikhail et al.,
1978).
Additional work Is reported In the literature dealing with antagonism
but will only briefly be mentioned here. Kleczka et al. (1981) determined
that an oxidized catechol metabolite rather than the catechol molecule
Itself may be responsible for the enzymatic Inhibition of the activation
•CC1_ radicals
O .
step of CC14 or may Interfere with reactions of the
formed. The administration of chloramphenlcol early 1n CC1. Intoxication
prevents I1p1d peroxldatlon of endoplasmlc retlculum membranes 1n rats
(Dold and Brabec, 1978). Dzhlolev and Balanskl (1974) found that CC1
4
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 synerglstically affect CC1.
toxlclty. Ethanol has been shown to potentiate CC1. toxlclty even when
the ethanol consumption had taken place prior to exposure. This effect has
been documented In case studies and more recently quantified In animal
studies. Several other environmental pollutants such as Kepone®, PBB and
12-10
-------
PCB have been shown to potentiate CC1. toxlcity at doses where both sub-
stances are not considered toxic. Carbon tetrachloride toxicity has been
shown to be Inhibited by several compounds, such as chloramphenicol and
catechol.
While this Information Is Interesting, It can only be used qualita-
tively. The probabilities of such synergistic or antagonistic reactions
occurring 1n the natural environment are not available. Once such Informa-
tion is quantified, methods of altering "safe" exposures can be investigated.
12-11
-------
-------
13. REGULATIONS AND STANDARDS
13.1. WATER
13.1.1. Ambient Water. In 1980 the U.S. EPA announced availability of 64
ambient water quality criteria documents and associated criteria {Federal
Register, 1980b). The criteria associated with the CC14 document were
4.0, 0.4 or 0.04 yg/8, based on estimated human lifetime cancer risks of
10~5, 10~6 or 10~7, respectively. These criteria were derived based
on the assumption of a daily contaminated water intake of 2 a, and contami-
nated fish Intake of 6.5 g per person. Criteria associated with only the
consumption of 6.5 g of contaminated fish were 69, 6.9 or 0.69 yg/8,,
respectively.
13.1.2. Drinking Water. The NAS recommended "Suggested No-Adverse-
Response Levels" (SNARLs) for CC14 In drinking water (NAS, 1980). The
recommended 24-hour value was 14 mg/8. based on data which indicated a
toxic effect to the liver of rats 5 hours after one exposure to CCl^ at
400 mg/kg bw, a 1000-fold safety factor, and an assumed daily water consump-
tion of 2 8. for a 70 kg adult. NAS recommended a 7-day SNARL of 2 mg/8,
based on the assumed cummulative effects of CC1, after repeated daily
4
exposure (i.e., 14 mg/8.Yr 7 = 2 mg/8.). NAS did not recommend a "chronic
exposure" SNARL because CC1. is a carcinogen in some animal species.
The Office of Drinking Water (ODW) of the U.S. EPA has recommended draft
SNARLs for CC1 . The 1-day value is 0.2 mg/ft. based on data which
indicate adverse effects in rats after an acute exposure of CCl^ at 20
mg/kg bw, a 1000-fold safety factor, and an assumed daily water consumption
of 1 a, for a 10 kg child (Ohanian, 1981). ODW recommended a draft 10-day
SNARL at 0.02 mg/8.. A longer-term EPA-SNARL was not developed due to the
lack of acceptable data on chronic exposure to this compound.
13-1
-------
The differences In the NAS and ODW SNARLs are 3-fold: different data
bases were used, the NAS-SNARL was calculated for a 70 kg adult rather than
a 10 kg child, and the adult was assumed to Ingest 2 8. of water/day as
compared to 1 8,/day for a child. Both methods assume 100% absorption of
Ingested CGI,.
4
13.2. AIR
The American Conference of Industrial Governmental Hygienists (ACGIH,
1980) has recommended threshold-llmlt-values (TLVs) for CC1 both as a
time-weighted average (TWA) and a short-term-exposure limit (STEL) of 30 and
125 mg/m3, respectively. Previously, ACGIH (1979) recommended values of
65 and 130 mg/m3, respectively. A concise history of other relevant ACGIH
recommendations and accompanying logic can be found in the Ambient Water
Quality Criteria Document for Carbon Tetrachloride (U.S. EPA, 1980a).
The Occupational Safety and Health Administration (OSHA), U.S. Depart-
ment of Labor, adopted the American National Standards Institute (ANSI)
standard Z37.17-1967 (ANSI, 1967) as the Federal standard for CC1 (29 CFR
4
1910.1000). This standard is 65 mg/m3 for an 8-hour TWA exposure, with an
acceptable ceiling exposure concentration of 162.5 mg/m3, and an accept-
able maximum peak above the acceptable celling concentration for an 8-hour
shift of 1300 mg/m3 for 5 minutes in any 4 hours. This adopted ANSI
standard was based on human experience and extensive studies on animals.
References cited to support 1t were Adams et al. (1952), Stewart et'al.
(1961, 1965), Stewart and Dodd (1964), von Oettingen (1964) and Irish (1963).
The National Institute for Occupational Safety and Health (NIOSH, 1975)
recommended a CCl^ 1WA 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 exposed to CC1 . In a
4
13-2
-------
1976 revision, NIOSH recommended that the concentration of CC14 not be
>12.6 mg/m3 of breathing zone air 1n a 45 8, air sample taken over a
period not to exceed 1 hour 1n duration (NIOSH, 1976)
Inhalation standards for various toxic substances 1n the working
environment of several other countries have been published (International
Labor Office, 1970): Carbon tetrachlorlde Inhalation standards are shown 1n
Table 13-1. the USSR values are maximum allowable concentrations (HACs)
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 CC1, permitted 1n cooked cereal as 50 yg/kg. This value was
4
derived from a FAO/WHO expert committee 1n 1972. No other Information was
found concerning guidelines, criteria or standards for CC14 1n food.
13.4. SUMMARY
Protective levels for CCK In air 1n 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 toxlclty
data 1n this area. Protective levels for CC14 1n 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 1s needed 1n this latter area.
13-3
-------
TABLE 13-1
Carbon Tetrachlorlde Inhalation Standards of 11 Countries3
Country
United States
ACGIH
OSHA
NIOSH
Czechoslovakia
Finland
Hungary
Japan
Poland
Rumania
UAR and SAR
USSR
Yugoslavia
Standard
(mg/m3)
30b
125b
65
162.5
1300
12. 6°
12. 6b
50
250
160
20
100
10
20
50
625
20
65
Qualifications
TWA
STEL
TWA
Celling exposure concentration
Maximum peak of celling
Concentration not to be
exceeded for 5 minutes 1n any
4 hour period.
TWA
45 8, a1r/60 minutes
MAC
Single short exposure
8 hours continuous exposure
8-hour average
30 minutes
MAC
aSource: Adapted from NIOSH, 1975
bRecommended standards
13-4
-------
14. EFFECTS OF MA30R CONCERN AND HEALTH HAZARD ASSESSMENT
The assessment of health hazards from CC1. requires Information which
relates specific adverse health effects to dose-exposure conditions.
Studies which report the route of exposure, the dose, the duration of
exposure, the animal species and the nature of adverse effects are therefore
most useful 1n hazard assessment, Involving the prediction of effects from
given environmental or occupational exposure situations. Since the route of
Intake 1s often Important, experimental studies should use the typical
routes of human exposure (I.e., Ingestlon, Inhalation or dermal contact).
Therefore, studies based on exposure by Intraperltoneal or subcutaneous
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, subchronlc and chronic effects, followed by summaries on reproduc-
tive, mutagenlc and carcinogenic effects. Specific Information on dose,
exposure, test animal, effects and references are presented In Table 14-1
for acute, subchronlc and chronic studies; In Table 14-2 for reproduction
studies; In Table 14-3 for mutagenidty studies; and 1n Table 14-4 for car-
dnogenldty studies. Note that these tables summarize the toxic!ty data.
Further analysis leading to unit risk estimates for humans 1s presented 1n
the Appendix.
14.1. PRINCIPAL EFFECTS
Carbon tetrachlorlde 1s 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 1n malignant tumors of the liver
1n three animal species. Several studies have produced satisfactory dose-
response Information, Including estimated "no-effect" levels for humans and
14-1
-------
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TABLE 14-3
Summary Table for Mutagen1c1ty Studies
Assay
Response
Reference
Ames test
£. coll reversion test
In. vitro chromosome assay
Yeast cells
negative
negative
negative
positive
HcCann et a!., 1975
Uehleke et a!., 1976
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14-6
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four animal species. These studies are classified as satisfactory In that
they not only provide all of the necessary quantitative Information (e.g..
number of animals 1n control and treated groups), but also Include suffi-
cient detail to demonstrate the high quality and defens1b1!1ty of the
research.
14.1.1. Ingestlon. The only dose-related human data with complete quanti-
tative Information for oral 1ngest1on (see Section 8.8.2.) are from an
ep1dem1olog1cal study which found elevated serum creatlnlne levels following
an estimated 1-day exposure to CC14 1n drinking water. Several animal
"t
studies showed dose-related effects from CC14 Ingested via water, gavage
or diet. Host of these studies Involved a single dose (e.g., Korsrud et
a!., 1972). Acute effects (with Increasing dose) Included greater Hver
weight than normal, greater amount of liver fat, changes 1n enzyme levels or
activity, some liver necrosis, reversible lung and kidney structural
changes, lung lesions and behavioral abnormalities. One short-term (6-week)
study 1n rats showed dose-related Increases 1n total serum Uplds and
trlglycerldes and depressed weight gain (Alumot et al., 1976).
Several authors reported that CC14 does not appear to be a teralogen
but that 1t can affect reproduction following subchronlc exposure. Oral
administration of CC14 to rats for either 2 or 3 days during days 7-14 of
gestation was reported to cause a reduction 1n Utter sizes and an Increase
1n the number of fetal resorptlons (Wilson, 1954).
Carbon tetrachloMde has produced Hver tumors In hamsters, mice and
rats. A number of experiments have been conducted using mice of various
strains (B6C3FV C3H, A and L) and different dosage regimens of CC1
(Edwards et al., 1942; NCI, 1976). The types of tumors observed have
Included hepatomas, hepatocellular carcinomas and hyperplastlc hepatic
14-8
-------
nodules. Hyperplastlc hepatic nodules were also Induced 1n Syrian golden
hamsters by oral doses of CC1 (Delia Porta et al., 1961). Effects
Induced by CC1. 1n several strains of rats also Include cholang1of1bros1s,
hepatic hyperplasla, hyperplastlc hepatic nodules and hepatic carcinomas
(Reuber and Glover, 1967, 1970).
14.1.2. Inhalation. The majority of the toxldty studies encountered 1n
the available literature Involved Inhalation of CGI. and Included a wide
4
range of exposure levels. One 70-mlnute exposure study found decreased
serum Iron and altered serum transamlnase levels, Implicating liver damage
(Stewart et al., 1961). A 2-year exposure 1n humans showed reversible
nausea, anorexia, vomiting and epigastric discomfort (Kazantls and Bomford,
1960).
Effects from short-term exposure on animals appear to be dose-dependent
and Include: Increased kidney and Hver weights, reduction 1n 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
subchronlc exposures are highly species-specific. For example, reversible
Hver damage was seen 1n the monkey at the same dose and duration which
caused liver cirrhosis and Increased mortality 1n the guinea pig. Other
effects due to subchronlc exposures Include minor Hver damage, weight loss
and damage to the sciatic nerve.
Only one chronic Inhalation animal study (of 10 months duration) was
found 1n the literature. Liver damage was reported. The authors did not
report Hver tumors (Smyth et al., 1936).
When pregnant rats were exposed to CC1 by Inhalation on days 6-15 of
gestation, the exposure resulted 1n decreased fetal body weights and lengths
(Schwetz et al., 1974). In a three-generation Inhalation study Involving
14-9
-------
rats, a dose of 238 mg/kg/day did not affect reproductive functions, while a
dose of 475 mg/kg/day caused reduced Utter sizes (Smyth et al., 1936).
Single subcutaneous Injection of pregnant animals with CCla has also been
4
reported to produce hlstologlcal and biochemical changes 1n the livers of
the offspring; thus, Bhattacharyya (1965) concludes that CCK may be
4
fetotoxlc and can cross the placenta! barrier.
14.1.3. Dermal Exposure. Guinea pigs exposed to pure CC1. In a skin
painting experiment showed epidermal sponglosls and karyopyknosls, altered
liver morphology and some liver necrosis (Kronevl et al., 1979).
14.1.4. Hutagen1c1ty. Carbon tetrachlorlde has produced negative results
1n the Ames Salmonella test, 1n both the presence and the absence of mlcro-
somal activation (HcCann et al., 1975) and 1n the Escher1ch1a coll K12 test
(Uehleke et al., 1976). Carbon tetrachlorlde produced negative results and
was nonclastogenlc 1n a chromosome assay using an epithelial-type cell line
derived from rat Hver which possessed Intrinsic metabolizing activity (Dean
and Hudson-Walker, 1979).
However, Callen et al. (1980) found CC1 to be genetically active and
cytotoxlc 1n strain D yeast cells of Saccharomvces cerevlslae. In these
cells, which contain a cytochrome P-450-dependent mono-oxygenase system,
CC14 caused Increased frequencies of "gene conversion and mltotlc recombi-
nation" and decreased cell survival.
14.2. SENSITIVE POPULATIONS
Studies on human sensitivity are limited. There Is clinical evidence,
discussed 1n Chapter 12, that Isopropanol and ethanol may potentiate CC1
4
tox1c1ty 1n humans. As described by Moon (1950), a repeated history of
alcoholism 1n cases of fatal CC14 poisoning may Indicate a synerglstlc
effect between alcohol and CC1 . In other case reports, very obese and
14-10
-------
undernourished persons suffering from pulmonary diseases, gastric ulcers,
liver or kidney diseases, diabetes or glandular disturbances seem especially
sensitive to the toxic effects of CC1,.
4
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). Chaturvedl
(1969) examined age and sex as factors 1n CC1 toxldty. The findings
revealed that female rats are less susceptible to the adverse effects of
different hepatotoxlc agents. Chaturvedl (1969) postulated that this was
due to different hormonal and enzyme patterns and the lack of certain
proteins 1n the female liver. The sex difference noticed 1n adult rats was
not as apparent 1n young rats.
Nutritional status may also affect the degree of toxldty following
exposure to CC1 1n rats. Gyorgy et al. (1946) exposed young rats on
various diets to -300 mg/kg CC1. 1n a gas chamber 7 hours/day, 5 days/week
for 45 months. Animals were then sacrificed, and hlstopathologlcal effects
1n the liver and kidneys were determined. When animals fed standard chow
were compared to other groups, these effects were more severe In animals fed
/
a diet high 1n I1p1d and low 1n carbohydrate, or a diet low 1n protein.
Meth1on1ne appeared to protect against Increased toxldty, particularly
kidney damage, caused by low-protein diets (Gyorgy et al., 1946).
The Interaction of CC1. with other chemicals has resulted 1n an
4
enhancement of the toxic effects produced 1n animals by either chemical
alone. Exposure of animals to selected environmental carcinogens 1n combi-
nation with CC1 has resulted 1n an Increase 1n carc1nogen1c1ty. In
addition, certain chemicals appear to Increase the toxic effects of CC1
on the liver and other organs of experimental animals.
14-11
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14.3. QUALITATIVE HEALTH HAZARD ASSESSMENT
The assessment of human health risks, that 1s, the likelihood of certain
adverse effects from given exposure scenarios, 1s hampered by the paucity of
good dose-response data In humans. Of the three human studies discussed
above, the acute ep1dem1olog1cal study involving oral 1ngest1on (Sonlch et
a!., 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 (Kazantls 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 1n humans and the determination of no-effect levels for subsequent
use 1n a quantitative hazard or risk assessment, are primarily estimated
from the many animal studies showing dose-related effects.
The major reported sublethal health hazards to humans from exposure to
CC1. are damage to the liver, lungs, kidneys and central nervous system.
Less severe adverse effects Include al.tered enzyme activities following
Ingestlon and Inhalation, gastrointestinal disturbances following Inhala-
tion, and epidermal damage following dermal contact. In animal studies,
some effects are seen 1n areas distant from the contact Interface. These
Include lung damage from oral Ingestlon, liver damage from inhalation and
from dermal contact and, to a lesser degree, enzyme disturbances from
Inhalation and Ingestlon.
Teratogenlc, mutagenlc and carcinogenic effects have not been demon-
strated 1n humans, and only cardnogenldty has been shown 1n experimental
animals. In addition, fetotoxlcity and neonatal toxldty have been shown 1n
rats {Schwetz et al., 1974; Bhattacharyya, 1965). Reproductive efficiency
has also been affected In rats evidenced by reduced Utter sizes (Smyth et
14-12
-------
al., 1936). Negative mutagenicity has been demonstrated 1n four studies
Including Salmonella typhimurium (McCann et al., 1975; Simmon and Tardiff,
1977), E_. coll (Uehleke et al., 1976), and 1n a recently developed epithe-
lial-type cell chromosome assay (Dean and Hudson-Walker, 1979). The single
positive mutagenlcHy test 1n yeast cells, Saccharomvces cerevisiae. there-
fore requires further confirmation (Callen et al., 1980).
Carc1nogen1c1ty has been demonstrated in three experimental animal
species, but predominantly several strains of rats and mice (NCI, 1976a,b;
Edwards et al., 1942; Reuber and Glover, 1970). Thus, CC1. should be
considered a potential human carcinogen, even though no definitive cause-
and-effect human data exist.
14.3.1. Animal Toxldty 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
phenomena. The threshold region of toxiclty 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 1n
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.
14-13
-------
PEL Frank-Effect Level: That exposure level which produces
unmistakable adverse effects, ranging from reversible hlsto-
pathologlcal damage to Irreversible functional Impairment or
mortality, at a statistically significant Increase 1n 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 1n dose-response studies on
CC]., with 12 satisfactory studies (as defined earlier) representing 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 results may be
consistent. The Infrequent, minor liver cell changes reported by Korsrud et
al. may have been present 1n 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 endpolnt Investigated. Extensive single-
dose Information on lung effects from oral 1ngest1on was reported by Boyd et
al. (1980), who studied two dose levels 1n both rats and mice, the lower 1n
each case resulting 1n a "lung NOEL." Other studies which provide dose-
related effect data for single oral exposures are listed 1n Table 14-1. The
only subchronlc 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.
Intercomparlson of four studies 1n rats showed a dose-dependent progression
1n severity of effects, with doses ranging from 300-285,000 mg/m3
(Herkur'eva et al., 1979; Wong and DIStefano, 1966; Boyd et al., 1980; Chen
et al., 1977). Useful subchronlc Inhalation data were provided by Prender-
gast et al. (1967), who used three dose levels (6.1-515 mg/m3) Including a
14-14
-------
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 1n rats were predominantly on the liver, whereas 1n the
guinea pigs the endpolnt 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
exposures at a distant site.
14.3.2. Animal Carcinogenldty Studies. Liver tumors have been demon-
strated in several species following oral exposure to CCK. Only a few
studies, however, were designed to demonstrate the relation between tumor
incidence and dose or exposure duration. No suitable inhalation studies
with dose-response cancer data were located in the available literature.
Three oral studies provided adequate information on dose and incidence 1n
controls and treated animals (see Table 14-4). Edwards et al. (1942)
reported dose-related Incidence of tumors for inbred strain L mice of two
age groups, NCI (1976a,b, 1977) demonstrated significant tumor incidence for
Osborne-Hendel rats and treated B6C3F1 mice, and Delia Porta et al. (1961)
showed significant tumor incidence in Syrian golden hamsters. It should be
noted that 1n most of the animal studies showing carclnogenlcity either the
length of the study was too short, the incidence rate was too high, or
survival was too poor for a dose-response estimation for lifetime exposure
14-15
-------
(NAS, 1978). The National Research Council recognizes this problem and uses
the rat NCI (1976a,b) bloassay for trichloroethylene (with CC14 as a
positive control) for determining a carcinogenic risk estimate for
14.4. FACTORS INFLUENCING HEALTH HAZARD ASSESSMENT
14.4.1. Exposure. Carbon tetrachlorlde is persistent 1n air and ground
water. Contamination of surface water and soil by CC14 Is not likely to
present long-term hazards due to Us rapid volatilization. However, large
quantities of CC14 1n bodies of cold water, such as lakes, are likely to
remain submerged and be relatively stable, contaminating the body of water
for several years. Carbon tetrachlorlde 1s readily absorbed through the
lungs, but 1t 1s also rapidly exhaled; 1t 1s excreted through all routes,
predominantly as the parent compound. The relative contributions of these
factors to the bloaval lability and body burden of CC14 1n humans are not
well defined. Also, experimental animals may differ substantially from
humans 1n terms of oral and dermal efficiencies of absorption. Thus,
application of human or animal pharmacokinetic information to quantitative
human health hazard assessment does not seem feasible at this time.
Consequently, the usable exposure information is from monitoring data of
ambient CC1, levels in air, water and other liquids, and food.
4
14.4.2. Estimated Threshold No-effect Levels.* The dose-response data for
CC14 are quite limited, especially regarding effects on humans (Table
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.
*Animal studies discussed herein indicate that CC14 is a potential human
carcinogen, and hence a no-effect level may not exist. These no-effect
levels are supplied for comparison purposes.
14-16
-------
A human NOEL for Inhalation was reported as 63 mg/m3 (~IO 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 equlva- lent) for
Inhalation ranging from 6.1-315 mg/m3, depending on length of exposure.
Insufficient Information exists to allow estimation of NOAELs based upon
long-terra exposures.
In light of the uncertainties and Inadequacies associated with the data
base for CC14> 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 1s the main obstacle to accurate
conversion of the animal exposure data to human equivalent exposures.
14.4.3. Carc1nogen1c1ty. Liver tumors have been shown to result from oral
exposure to CC14 by three species of animals. Although several authors
note that toxic effects are concurrent with liver tumors, 1t has not been
established that tissue damage 1s a necessary precursor to CC1, carcino-
genesis. The estimation of carcinogenic risk (Appendix) Is based on the
assumption of low-dose linearity, which Is supported by studies on muta-
genesis. The Inconclusive nature of the presently available mutagenlclty
evidence for CC14 then adds considerable uncertainty to any risk estima-
tion based on low-dose extrapolation.
14-17
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15. REFERENCES
ACGIH (American Conference of Government Industrial Hyg1en1sts). 1979.
Threshold limit values for chemical substances 1n workroom air. Cincinnati,
OH. p. 12.
ACGIH (American Conference of Government Industrial Hyg1en1sts). 1980.
Threshold limit values for chemical substances 1n workroom air. Cincinnati,
OH. p. 12.
Adams, E.M., et al. 1952. Vapor toxldty of carbon tetrachlorlde deter-
mined by experiments on laboratory animals. Arch. Ind. Hyg. Occup. Med.
6: 50.
Albert, R. 1983. Report on the January 13, 1983 CAG Extrapolation Model
Conference. U.S. EPA Memorandum.
Alumot, E., E. Nachtoml, E. Mandel and P. Holsteln. 1976. Tolerance and
acceptable dally Intake of chlorinated fumlgants 1n the rat diet. Food
Cosmet. Toxlcol. 14: 105-110.
American National Standards Institute. 1967. American standard maximum
acceptable concentration of carbon tetrachlorlde. Z37.17. New York.
Andervont, H.B. 1958. Induction of hepatomas In strain C3H mice with
4-o-tolylazo-o-tolu1d1ne and carbon tetrachlorlde. J. Natl. Can. Inst.
20: 431-438.
15-1
-------
Anonymous. 1983. NCAB Committee close to definition of quantitative risk
assessment. Cancer Lett. 9: 6-7.
Bagnasco, P.M., B. Stringer and A.M. Muslim. 1978. Carbon tetrachlorlde
poisoning: Rad1ograph1c findings. N.Y. State 0. Med. 78(4): 646-647.
Barber, E.D., W.H. Donlsh and K.R. Mueller. 1981. A procedure for the
quantitative measurement of the mutagenldty of volatile liquids 1n the Ames
Salmonella/mlcrosome assay. Mutat. Res. 90: 31-48.
Barkley, 3., 3. Bunch, 3.T. Bursey, et al. 1980. Gas chromatography-mass
spectrometry computer analysis of volatile halogenated hydrocarbons 1n man
and his environment -- A multimedia environmental study. Blomed. Mass.
Spectrom. 7(4): 139-147.
Barnes, R. and R.C. Jones. 1967. Carbon tetrachlorlde poisoning. Am. Ind.
Hyg. Assoc. 3. 28: 557.
Barrows, M.E., S.R. PetrocelH, K.3. Macek and 3.3. Carrol. 1980. Blocon-
centratlon and elimination of selected water pollutants by blueglll sunflsh
(Lepomls macrochlrus). In.: Dynamics, Exposure and Hazard Assessment of
Toxic Chemicals, R. Haque, Ed. Ann Arbor Science Pub!., Inc., Ann Arbor, HI.
Seattle, 3., et al. 1944. In.: W.F. von Oettlngen. 1964. Br. Med. 3.
1: 209.
15-2
-------
Rellar, T. and 3.3. Llchtenberg. 1981. The Detection of Halogenated
Chemicals In Water by the Purge and Trap Method. U.S. EPA Environmental
Monitoring and Support Laboratory, Cincinnati, OH. EPA 600/4-81-059.
Berck, B. 1974. Fumlgant residues of carbon tetrachloMde, ethylene
dlchlorlde, and ethylene dlbromlde In wheat, flour, bran, middlings, and
bread. 3. Agrlc. Food Chem. 22: 977-985.
Bertsch, W., E. Anderson and G. Holzer. 1975. Trace analysis of organic
volatlles 1n water by gas chromatography-mass spectometry with glass capil-
lary columns. J. Chromatog. 112: 701-718.
Bhattacharyya* K. 1965. Foetal and neonatal responses to hepatotoxlc
agents. J. Pathol. Bact. 90: 151-161.
;
B1elora1, B. and E. Alumot. 1966. Determination of residues of a fumlgant
mixture 1n cereal grain by electron capture gas chromatography. J. Agrlc.
Food Chem. 4: 622.
B1n1, A., G. Vecchl, G. V1vol1, V. Vann1n1 and C. Cessl. 1975. Detection
of early mataboHtes 1n rat liver after administration of CC1 and
4
. Pharmacol. Res. Comm. 7: 143-149.
Blzln, Y.P., G.M. Gorban and Z.I. PlUpynk. 1977. Comparative study of the
effect on animals of continuous and Intermittent carbon tetrachlorlde
[Inhalation], G1g. Sanlt. 3: 27-29.
15-3
-------
Blair, A., P. Decoufle and D. Grauman. 1979. Causes of death among laundry
and dry cleaning workers. Am. 3. Public Health. 69: 508-511.
Bondl, A. and E. Alumot. 1972. Evaluations of some pesticide residues 1n
food. WHO Pesticide Residues Series, No. 1. As reported by E.E. Kenaga,
1971, IUPAC Meeting. The Monographs, World Health Organization, Geneva.
Bon1tenk1, Y.Y. and A.A. Bruk. 1979. Cl1n1cal-enzymolog1cal diagnosis of
acute Inhalation. G1g. Tr. Prof. Zabol. 1: 18.
Boyd, M.R., C.N. Statham and N.S. Longo. 1980. The pulmonary Clara cell as
a target for toxic chemicals requiring metabolic activation; studies with
carbon tetrachlorlde. J. Pharmacol. Exp. Ther. 212: 109-114.
Brlggs, G.G. 1973. A simple relationship between soil adsorption of
organic chemicals and their octanol/water partition coefficients. Proc. 7th
Br. Insect. Fung. Conf.
Butler, T.C. 1961. Reduction of carbon tetrachlorlde in vivo and reduction
of carbon tetrachlorlde and chloroform In. vitro by tissue and tissue con-
stituents. 3. Pharmacol. Exp. Ther. 134: 311-319.
Butsch, W.L. 1932. In.: W.F. von Oettlngen. 1964. 3. Am. Med. Assoc.
99: 728.
Cagen, S.Z. and C.D. Klaassen. 1980. Protection of carbon tetrachlorlde
Induced hepatotoxldty by zinc: Role of metallothloneln. Toxlcol. Appl.
Pharmacol. 51(1): 107-116.
15-4
-------
Calabrese, E.JL 1982. Principles of Animal Extrapolation. WHey-Inter-
sclence, NY. 603 p.
Callen, D.F., C.R. Wolf and R.M. Phllpot. 1980. Cytochrome P-450 mediated
genetic activity and cytotoxldty of seven halogenated aliphatic hydro-
carbons 1n Saccharomyces cerevlslae. Mutat. Res. 77: 55-63.
Cameron, G.R. and W.A.E. Karunaratne. 1936. Carbon tetrachloMde cirrhosis
1n relation to Hver regeneration. 3. Pathol. Bacterlol. 42: 1.
Campbell, C.B., O.M. Collins and A. Van Tongeren. 1980. Serum bile..adds
and other liver function tests 1n hepatocellular damage from carbon tetra-
chlorlde 1ngest1on. N.Z. Med. 3. 91(660): 381-384.
Capurro, P.U. 1979. Cancer In a community subject to air pollution by
solvent vapors. Cl1n. Toxlcol. 14: 285-294.
Cessl, C., C. Colomblnl and L. Mamell. 1966. The reaction of liver pro-
teins with a metabolite of carbon tetrachlorlde. Blochem. 3. 101: 46c-47c.
Chatterjee, A. 1966. Testlcular degeneration 1n rats by carbon tetra-
chlorlde Intoxication. Exper1ent1a. 22: 395-396.
Chaturvedl, U.C. 1969. Influence of sex on hepatic Injury In albino rats.
Ind. 3. Med. Scl. 23: 374.
Chemical Marketing Reporter. 1981. A.R. Kavaler, Ed. May 4, 1981
219(18): 9.
15-5
-------
Chen, W.J., E.Y. Chi and E.A. Smuckler. 1977. Carbon tetrachlor1de-1nduced
changes 1n mixed function oxldases and mlcrosomal cytochromes 1n the rat
lung. Lab. Invest. 36(4): 388.
Chlou, C.T., V.H. Freed, D.W. Schmeddlng and R.L. Kohnut. 1977. Partition
coefficient and b1oaccumulat1on of selected organic chemicals. Environ.
Sc1. Techno!. 11(5): 475-478.
C1ccol1, L. and A.F. Cas1n1. 1978. Free radical damage, produced by carbon
tetrachlorlde 1n the I1p1ds of various rat tissues. Agents Actions. 8(3):
303-310.
Confer, D.B. and R.J. Stenger. 1966. Nodules In the livers of C3H mice
after long-term carbon tetrachlorlde administration. A light and electron
microscope study. Cancer Res. 26: 834.
Conway, H.B. and F. Hoven. 1946. In.: W.F. von Oettlngen. 1964. U.S. Nav.
Hed. Bull. 46: 593.
Cooper, K. and C. WUmer. 1982. Low oxygen tension and Us effects on
CC1. toxldty and mutagenldty 1n the Ames test (Abstract Bh-6). Environ-
mental Mutagen Society 13th Annual Meeting, February 26-March 1. 13: 99.
Cox, C.R. 1972. Regression model and life tables. 3. Roy. Stat. Soc. B.
34: 187.
Craddock, V.H. and A.R. Henderson. 1978. De novo and repair replication of
DMA In Hver of carcinogen-treated animals. Cancer Res. 38: 2135-2143.
15-6
-------
Crump, K.S. 1982. Implications of the multistage model to risks from
partial lifetime exposure. Prepared for U.S. EPA, ECAO-C1nc1nnat1, P.O.
C217NAST.
Curtis, L.R., W.L. Williams and H.M. Mehendale. 1979a. Potentlatlon of the
hepatotoxldty of carbon tetrachlorlde following preexposure to chlorodecone
(Kepone) 1n the male rat. Toxlcol. Appl. Pharmacol. 51: 283-293.
Curtis, L.R., A.K. Thureson-Kleln and H.M. Mehendale. 1979b. Specificity
of kepone-lnduced potentlatlon of carbon tetrachlorlde hepatotoxldty:
Comparison with mlrex and photomlrex. Fed. Proc. (3236) 38(3): 845.
David, A., E. Frantlk, R. Holusa and 0. Novakova. 1981. Role of time and
concentration on carbon tetrachlorlde toxldty 1n rats. Int. Arch. Occup.
Environ. Health. 48: 49-60.
Davis, M.E. and H.H. Mehendale. 1980. Functional and biochemical corre-
lates of chlordecone exposure and Us enhancement of CC1 hepatotoxldty.
Toxicology. 15: 91-103.
Davis, P.A. 1934. Carbon tetrachlorlde as an Industrial hazard. J. Am.
Med. Assoc. 103: 962.
Dawborn, 3.K., et al. 1961. In.: W.F. von Oettlngen. 1964. Br. Med. J.
11: 493.
15-7
-------
Daws on, G.W., et al. 1977. The acute toxldty of 47 Industrial chemicals
to fresh and saltwater fishes. J. Hazard. Mater. 1: 303.
Dean, B.J. and 6. Hudson-Walker. 1979. An In. vitro chromosome assay using
cultured rat-liver cells. Mutat. Res. 64: 329-337.
De Groot, H. and W. Haas. 1980. Op-Independent damage of cytochrome
P-450 by CC1. metabolites 1n hepatic mlcrosomes. FEBS Lett. 115: 253-256.
DeLeon, I.R., H.A. Maberry, E.B. Overton, et al. 1980. Rapid gas chromato-
graphlc method for the determination of volatile and sem1volat1le organo-
chlorlne compounds 1n soil and chemical waste disposal site samples. J.
Chrom. Sc1. 18: 85-88.
Delia Porta, G., B. Terrac1n1 and P. Shublk. 1961. Induction with carbon
tetrachlorlde of Hver cell carcinomas 1n hamsters. J. Natl. Can. Inst.
26: 855-863.
DelHan, V.L. and H. WHtgens. 1962. Labor hygiene experience with carbon
tetrachlorlde 1n railroad workshops. Zentralbl Arbeltsmed. 12: 216-223.
(Ger.)
Delvlllarruel, H., E.G.D. DeToranzo and 3.A. Castro. 1977. Carbon tetra-
chlorlde activation, Upld peroxldatlon and the mixed function oxygenase
activity of various rat tissues. Toxlcol. Appl. Pharmacol. 41(2): 337-344.
15-8
-------
Diaz Gomez, M.I. and 3.A. Castro. 1980a. Covalent binding of carbon tetra-
chlorlde metabolites to liver nuclear DMA, proteins, and I1p1ds. Toxlcol.
Appl. Pharmacol. 56: 199-206.
Diaz Gomez, M.I. and 3.A. Castro. 1980b. Nuclear activation of carbon
tetrachlorlde and chloroform. Res. Commun. Chem. Pathol. Pharmacol.
27: 191-194.
Diaz Gomez, M.I. and 3.A. Castro. 1981. Reaction of trlchloromethyl free
radicals with deoxyrlbonuclelc add bases. Res. Commun. Chem. Pathol.
Pharmacol. 32: 147-153.
D1az Gomez, M.I., C.R. De Castro, N. C'Acosta, O.M. De Fenos, E.C. De
Ferreya and 3.A. Castro. 1975. Species differences 1n carbon tetrachlo-
r1de-1nduced hepatotoxlclty: The role of CC1. activation and of I1p1d
peroxldatlon. Toxlcol. Appl. Pharmacol. 34: 102-114.
D1etz, E.A. and K.F. Slngley. 1979. Determination of chlorinated hydro-
carbons 1n water by headspace gas chromatography. Anal. Chem. 51(11):
1809-1814.
Dletz, F.K. and G.3. Tralger. 1979. Potent1at1on of CC1. of hepatotoxlc-
Hy In rats by a metabolite of 2-butanone: 2,3-Butaned1ol. Toxicology.
14: 209-215.
Dlkshlth, T.S.S., K.K. Datta and R.B. Ralzada. 1980. Response of carbon
tetrachlorlde-treated male rats to benzene hexachlorlde and qulnalphos.
Ind. J. Exp. Blol. 18: 1267-1272.
15-9
-------
Dllllng, W.L., N.B. Tefertlller and G.J. Kallos. 1975. Evaporation rates
and reactivities of methylene chloride, chloroform, 1,1,1-tMchloroethane,
trlchloroethylene, tetrachloroethylene and other chlorinated compounds 1n
diluted aqueous solutions. Environ. Sc1. Technol. 9: 833.
Dm1tr1ev, M.T., E.G. Rast1ann1kov, S.A. Volkov, A.G. Malysheva and B.H.
Samokhodk. 1980. Chromatographlc, mass-spectrometrlc determination of
toxic substances 1n the air. G1g. Sanlt. 5: 42-44.
Docherty, S.F. and E. Burgess. 1922. The action of carbon tetrachlorlde on
the liver. Br. Hed. 3. 907.
Docherty, J.F. and L. Nlcholls. 1923. Report of three autopsies following
carbon tetrachlorlde treatment. Br. Med. 3. 753.
Dolcl, E.D. and M.3. Brabec. 1978. Antagonism by chloramphenlcol of carbon
tetrachlorlde hepatotoxldty: Examination of mlcrosomal cytochrome P-450 and
Upld peroxldatlon. Exp. Mol. Pathol. 28(1): 96-106.
Doll, R. 1971. Welbull distribution of cancer: Implications for models of
carclnogenesls. 3. Roy. Stat. Soc. A. 13: 133.
Douglas, B.H. and B.R. Glower. 1968. Hepatotoxlc effect of carbon tetra-
chlorlde during pregnancy. Am. 3. Obstet. Gynecol. 102: 236-239.
Dowty, B.3. and 3.L. Laseter. 1976. The transplacental migration and
accumulation 1n blood of volatile organic constituents. Pedlat. Res.
10: 696-701.
15-10
-------
Dowty, B.3., D. Carlisle and 3.L. Laseter, 1975. Halogenated hydrocarbons
In New Orleans drinking water and blood plasma. Science. 187(4171): 75-77.
Drotman, R.B. and G.T. Lawhorn. 1978. Serum enzymes as Indicators of
chemically Induced liver damage. Drug Chem. Toxlcol. 1: 163-171.
Druckrey, H. 1967. Quantitative aspects 1n chemical carclnogenesls. In:
Potential Carcinogenic Hazards from Drugs. Sprlnger-Verlag, Berlin.
p. 60-78.
Dudley, S.F. 1935. Ijr. W.F. von Oettlngen. 3. Ind. Hyg. 1964. 17: 19.
Dzh1o1ev, F.K. and R.H. Balanskl. 1974. Inhibition of urethane carclno-
genesls by antloxldants and CC1.. Vopr. Onkol. 20(9): 98-102.
Edwards, 3.E. 1941. Hepatomas 1n mice Induced with carbon tetrachlorlde.
3. Natl. Can. Inst. 2(1): 197-199.
Edwards, 3. and A. Dalton. 1942. Induction of cirrhosis of the liver and
hepatomas In mice with carbon tetrachlorlde. 3. Natl. Cancer Inst. 3: 19.
Edwards, 3., et al. 1942. Induction of the carbon tetrachlorlde hepatoma
1n strain L. mice. 3. Natl. Cancer Inst. 3: 297.
Elklns, H.B. 1942. Maximal allowable concentrations. II. Carbon tetra-
chlorlde. 3. Ind. Hyg. Toxlcol. 24: 233.
15-11
-------
EnvlroControl. 1981. Atlas Document for Carbon TetrachloMde. March 3,
1981. Prepared for Environmental Criteria and Assessment Office, U.S.
Environmental Protection Agency, Cincinnati, Ohio. Contract No. 68-03-3048.
Eschenbrenner, A.B. and E. Miller. 1943. Studies on hepatomas — Size and
spacing of multiple doses 1n the Induction of carbon tetrachlorlde hepa-
tomas. 3. Natl. Cancer Inst. 4: 385.
Eschenbrenner, A.B. and E. Miller. 1946. Liver necrosis and the Induction
of carbon tetrachlorlde hepatomas 1n strain A mice. 3. Natl. Can. Inst.
6: 325-341.
Falk, H.L. 1976. Possible mechanisms of combination effects 1n chemical
cardnogenesls. Oncology. 33(2): 77-85.
Federal Register. 1979. National Interim Primary Drinking Water Regula-
tions; Control of Trlhalomethanes 1n Drinking Water; Final Rule. November
29, 1979. p. 68624-68707.
Federal Register. 1980a. Carbon Tetrachlorlde, Pesticides Programs;
Rebuttable Presumption Against Registration and Continued Registration of
Certain Pesticide Products. October 15, 1980. p. 68534-68584.
Federal Register. 1980b. Water Quality Criteria Documents; Availability.
November 28, 1980. p. 79318-79379.
15-12
-------
Federal Register. 1980c. Mutagen1c1ty Risk Assessment: Proposed Guide-
lines. November 13, 1980. p. 74984-74988.
F1shbe1n, L. 1976. Industrial mutagens and potential mutagens. I. Halo-
genated aliphatic derivatives. Mutat. Res. 32: 267-308.
Folland, D.S., W. Schaffner, H.E. Glnn, O.B. Crofford and D.R. McMurray.
1976. Carbon tetrachlorlde toxlclty potentiated by Isopropyl alcohol.
Investigation of an Industrial outbreak. 0. Am. Med. Assoc. 236(16):
1853-1856.
Fowler, J.S.L. 1969. Carbon tetrachlorlde metabolism 1n the rabbit. Br.
3. Pharmacol. 37: 733-737.
Galbally, I.E. 1976. Man-made carbon tetrachlorlde In the atmosphere.
Science. 193: 573-576.
Gardner, G.H., et al. 1924. Studies on the pathological histology of
experimental carbon tetrachlorlde poisoning. Bull. Johns Hopkins Hosp.
36: 107-133.
Glger, W., E. Molnar-Kublca and S. Wakeham. 1978. Volatile chlorinated
hydrocarbons In ground and lake waters. Aquatic Pollut: Transform. B1ol.
Eff. 1977. Pergamon Ser. Environ. Sc1. pp. 101-123.
Gllman, M.R. 1971. A preliminary study of the teratogenlc effects of
Inhaled carbon tetrachlorlde and ethyl alcohol consumption 1n the rat.
Dissertation, Drexel University.
15-13
-------
Golombeck, A. 1982. A global three-dimensional model of the circulation
and chemistry of long-lived atmospheric species. Ph.D. Thesis, Mass. Inst.
of Tech., Cambridge, HA. (Cited 1n Slmmonds et al., 1983)
Gould, V.E. and E.A. Smuckler. 1971. Alveolar Injury 1n acute carbon
tetrachlorlde Intoxication. Arch. Intern. Hed. 128: 109-117.
Gray, I. 1947. Carbon tetrachlorlde poisoning — Report of seven cases
with two deaths. NY State J. Med. 47: 2311-2315.
Grlmsrud, E.P. and R.A. Rasmussen. 1975. Survey and analysis of halo-
carbons 1n the atmosphere by gas chromatograhy-mass spectrometry. Atmos.
Environ. 9: 1014-1017.
%
Guild, W.R., et al. 1958. Anurla due to carbon tetrachlorlde Intoxication.
Ann. Int. Med. 48: 1221-1227.
Gyorgy, P., J. Selfter, R.M. Tomarelll and H. Goldblatt. 1946. Influence
of dietary factors and sex on the toxldty of carbon tetrachlorlde In rats.
J. Exp. Med. 83: 449-462.
Hafeman, D.G. and W.G. Hoekstra. 1977. Protection against carbon tetra-
chlor1de-1nduced Hpid petroxldatlon 1n the rat by dietary vitamin E, sele-
nium and methlonlne as measured by ehtane evolution. J. Nutr. 107: 656.
Hall, M.C. 1921a. The use of carbon tetrachlorlde for the removal of hook-
worms. J. Am. Med. Assoc. 77: 1671.
15-14
-------
Hall, M.C. 1921b. Carbon tetrachlorlde for the removal of parasitic worms,
especially hookworms. J. Agrlc. Res. 21: 157.
Hamburger, J., et al. 1958. Anurla caused by Inhalation of carbon tetra-
chlorlde. Acquls. Med. Recent, p. 15-34. (Fre.)
Hanst, P.L. 1978. Part II: Halogenated pollutants. Noxious trace gases 1n
the air. Chemistry. 51(2): 6-12.
Hanst, P.L., L.L. Splller, D.M. Watts, J.W. Spence and M.F. Miller. 1975.
Infrared measurement of fluorocarbons, carbon tetrachlorlde, carbonyl
sulflde and other atmospheric trace gases. J. A1r Pollut. Con. Assoc.
25(12): 1220-1226.
Hashimoto, S., E.A. Glende and R.O. Rechnagel. 1968. Hepatic I1p1d peroxl-
datlon 1n acute fatal human carbon tetrachlorlde poisoning. New Engl. 3.
Med. 279: 1082.
Hasumura, Y., R. Teschke and C.S. Lleber. 1978. Mechanism of increased
CC1 hepatotoxldty after chronic ethanol consumption. Vol. 65. (Abstr.)
Horrocks, G.E. 1934. In.: W.F. von Oettingen. 1964. M1ll1t. Surgeon.
74: 246.
IARC (International Agency for Research on Cancer). 1979. IARC Monographs
on the Evaluation of Carcinogenic Risk of Chemicals to Man. 20: 371-399.
15-15
-------
ICRP (International Commission for Radiological Protection). 1975. Report
of the Task Group on Reference Manual, ed. no. 23. Pergamon Press, New York.
International Labor Office. 1970. In: National Institute for Occupational
Safety and Health. 1975. Criteria for a Recommended Standard. Occupation-
al exposure to carbon tetrachlorlde. U.S. Dept. Health Edu. Welfare, p. 87.
Irish, D.D. 1963. Halogenated hydrocarbons. I. Aliphatic. In.: Industrial
Hygiene and Toxicology, 2nd ed, F.A. Patty, Ed. Toxicology Intersdence
Publishers, New York.
Ishlkawa, T., Y. Ose, T. Sato and B. Tomlta. 1978. Waste water treatment
of night soil. IX. Formation of organohalldes 1n the chlorlnatlon process
of a night soil treatment plant. E1se1 Kagaku. 24(5): 235-240. (Abstr;)
Jag1elsk1, J., K.S. Scudamore and S.G. Heuser. 1978. Residues of carbon
tetrachlorlde and 1,2-d1bromoethane 1n cereals and processed foods after
liquid fumlgant grain treatment for pest control. Pesticide Sc1. 9: 117.
Jahnke, K.H. 1953. In.: W.F. von Oettlngen. 1964. Aerztl. Wschr. 8: 1051.
Jiang, L. and R. Zhang. 1979. H1stolog1cal studies on the toxic effects of
phenol and CCl^ on the Hver and kidney of common carp and Crucian carp.
Acta Zool. S1n. 25(4): 311-314.
Johns, R. 1976. A1r pollution assessment of carbon tetrachlorlde. Pre-
pared by Mitre Corporation for U.S. Environmental Protection Agency. Mitre
Corp., Washington, DC. Contract No. 68-02-1495. MHre No. MTR-7144.
15-16
-------
3ohnstone, R.T. 1948. Occupational Medicine and Industrial Hygiene. C.V.
Mosby Co., St. Louis, MO. p. 157.
3oron, G.E., et al. 1957. Carbon tetrachlorlde — An underrated hazard.
Can. Med. Assoc. 3. 76: 173.
Kalla, M.R. and M.P. Bansal. 1975. Effect of carbon tetrachlorlde on
gonadal physiology 1n male rats. Act. Anat. 91: 380-385.
Kazantzls, G. and R.R. Bomford. 1960. Dyspepsia due to Inhalation of
carbon tetrachlorlde vapor. Lancet. 1: 360.
Keller, E.B. and P.C. Zamecnlk. 1956. The effect of guanoslne dlphosphate
and trlphosphate on the Incorporation of labeled amlno adds Into proteins.
3. B1ol. Chem. 221: 45-59.
Kleczka, H., H. Remmer and H. Kappus. 1981. Influence of oxygen on the
Inhibition of liver mlcrosomal activation of carbon tetrachlorlde by the
catechol 2-hydroxyestrad1ol-l7B. Blochem. Pharmacol. 30: 319-324.
Klroball, G. The effects of lesser known metals and one organic to fathead
minnows (Plmephales dromelas) and Daohnla magna. (Manuscript).
Klrkpatrlck, H.3.R. and 3.M. Sutherland. 1956. In: M.F. von Oettlngen.
1964. 3. CUn. Pathol. 9: 242.
15-17
-------
KUtleson, K.D. and C.W. Borden. 1956. Acute renal failure due to carbon
tetrachlorlde poisoning. Northwestern Univ. Med. School Mag. 30: 117-123
Klaassen, C.D. and G.L. Plaa. 1966. Relative effects of various chlori-
nated hydrocarbons on Hver and kidney function In mice. Toxlcol. Appl.
Pharmacol. 9: 139-151.
Klaassen. C.D. and G.L. Plaa. 1967. Relative effects of various chlori-
nated hydrocarbons on liver and kidney function 1n dogs. Toxlcol. Appl.
Pharmacol. 10: 119-131.
Kluwe. W.M., C.L. Herrman and J.B. Hook. 1979. Effects of dietary poly-
chlorinated blphenyls and polybromlnated blphenyls on the renal and hepatic
toxlcltles of several chlorinated hydrocarbon solvents 1n mice. J. Toxlcol.
Environ. Health. 5: 605-615.
Kopperman. J.L., D.W. Kuehl and G.E. Glass. 1976. Chlorinated compounds
found m waste treatment effluents and their capacity to bloaccumulate.
Proceedings of the Conference on the Environmental Impact of Water Chlorlna-
tlon, Oak Ridge, TN. October 22-24, 1975. p. 327-345.
Korsrud, G.O., H.C. GMce and J.M. McLaughlan. 1972. Sensitivity of
several serum enzymes 1n detecting carbon tetrachlor1de-1nduced Hver damage
1n rats. Toxlcol. Appl. Pharmacol. 22: 474-483.
Kotln, P., et al. 1962. Effect of carbon tetrachloMde Intoxication on
metabolism of benzo(a)pyrene In rats and mice. 3. Natl. Cancer Inst.
28: 725-745.
15-18
-------
Krey, P.M., R. Lagomarslno, M. Schonberg and I.E. Toonkel. 1976. CCl^ ,1n
ground level air and stratosphere. Health Safety Lab. Environ. Quart.
p. 1-21 through 1-49. (Cited 1n Simmonds et al.t 1983)
Kronevl, T., J. Wahlberg and 8. Holmberg. 1979. Hlstppathology of skin,
Hver and kidney after eplcutaneous administration of five Industrial
solvents to guinea pigs. Environ. Res. 19: 56-59.
Kubic, V.L. and M.W. Anders. 1980. Metabolism of carbon tetrachloride to
phosgene. Life Sc1. 26: 2151-2155.
Lachnlt, V. and H. Pietschmann. 1960. Activity of serum glutamic-oxalo-
acetic transamlnase and aldolase in workers exposed to halogenated hydro-
carbons. Ind. Med. Surg. 29: 523-526.
Lamson, P.O., A.S. M1not and B.H. Robbins. 1928. The prevention and treat-
ment of carbon tetrachloride intoxication. J. Am. Med. Assoc. 90: 345-346.
Lamson, P.O., et al. 1923. In.: W.F. von Oettingen. 1964. J. Pharmacol.
Exp. Ther. 22: 215.
Landen, W.O. 1979. Micro assay of carbon tetrachloride in beverages and in
pharmaceutical preparations. Bull. Environ. Contam. Toxlcol. 22: 431-438.
Leduc, E.H. and J.W. Wilson. 1959. Transplantation of carbon tetrachlo-
ride-1nduced hepatomas in mice. J. Natl. Cancer Inst. 22: 581.
15-19
-------
Lehmann, K.B. and L. Schm1dt-Kehl. 1936. The thirteen most Important chlo-
rinated aliphatic hydrocarbons from the standpoint of Industrial hygiene.
Arch. Hyg. 116: 132-200.
Leo, A., C. Hansch and D. Elklns. 1971. Partition coefficients and their
uses. Chem. Rev. 71(6): 525-612.
Lillian, D., H.B. Singh, A. Appleby, et al. 1975. Atmospheric fates of
halogenated compounds. Environ. Sc1. Technol. 9(12): 1042-1048.
Lourla, O.B. 1977. The dangers from limited exposure to carbon tetrachlo-
rlde. Prepared for Allied Chemical Corp.
Lovelock, J.E., R.J. Maggs and R.J. Wade. 1973. Halogenated hydrocarbons
1n and over the Atlantic. Nature. 241: 194-196.
Mabey, W. and T. Mill. 1978. Critical review of hydrolysis of organic com-
pounds 1n water under environmental conditions. J. Phys. Chem. Ref. Data.
7: 103.
Mantel, N. and M.A. Schnelderman. 1975. Estimating "safe levels", a
hazardous undertaking. Cancer Res. 35: 1379.
15-20
-------
Mapes, D.A. and S.A. Shrader. 1957. Determination of total and Inorganic
bromide residues 1n fumigated products. In.: Residues In Foods and Feeds
Resulting from Fumigation of Grains with the Commoner Liquid Formulations of
Carbon D1sulf1de, Carbon Tetrachlorlde, Ethylene D1chlor1de and Ethylene
D1brom1de, G.E. Lynn and F.A. Vorhes, Jr., Ed. J. Assoc. Off. Agrlc. Chem.
40(1): 189-196.
Marchland, C., S. McLean and G.L. Plaa. 1970. The effect of SKF 525A on
the distribution of carbon tetrachlorlde 1n rats. J. Pharmacol. Exp. Ther.
174: 232.
Markham, T.N. 1967. Ann Arbor Case Reports: Renal failure due to carbon
tetrachlorlde. Ann Arbor Reports. 9(1): 16-17.
Marzl, A., E.G.O. OeToranzo and J.A. Castro. 1980. Mechanism of chlorpro-
mazlne prevention of carbon tetrachlor1de-1nduced liver necrosis. Toxlcol.
App.l. Pharmacol. 52: 82-88.
Masuda, Y. and T. Murano. 1977. Carbon tetrachlorlde-lnduced I1p1d peroxl-
datlon of rat liver mlcrosomes 1n vitro. Blochem. Pharmacol. 26: 2275-2282.
/
McCann, J., E. Choi, E. Yamasakl and B.N. Ames. 1975. Detection of carcin-
ogens as mutagens 1n the Salmonella/mlcrosome test: Assay of 300 chemicals.
Proc. Natl. Acad. Sc1. 72: 5135-5139.
15-21
-------
McCollister, D.D., W.H. Beamer, G.J. Atchison and H.C. Spencer. 1951. The
absorption, distribution and elimination of radioactive carbon tetrachloMde
by monkeys upon exposure to low vapor concentrations. J. Pharmacol. Exp.
Ther. 102: 112-124.
HcConnell, G. 1976. Halo organlcs 1n water supplies. J. Inst. Water.
30(8): 431-445.
HcConnell, G., O.M. Ferguson and C.R. Pearson. 1975. Chlorinated hydro-
carbons and the environment. Endeavour. 34: 13-18.
McKlnney, J.D., R.R. Haurer, J.R. Mass and R.O. Thomas. 1976. Possible
factors 1n the drinking water of laboratory animals causing reproductive
failure. In.: Identification and Analysis of Organic Pollutants 1n Water,
L.H. Keith, Ed. Ann Arbor Science Publishers, Inc., Ann Arbor, HI.
p. 417-432.
McHahon, B. 1971. Analyses of commercially fumigated grains for residues
of organic fumlgants. J. Assoc. Off. Anal. Chem. 54: 964-965.
HcHahon, H.E. and S. Weiss. 1929. In: W.F. von Oettingen. 1964. Am. J.
Pathol. 5: 623.
Herkur'eva, R.V., T.I. Bonashevskaya, I.S. Shatemikova, et al. 1979.
Comparative biochemical and morphological Investigation of the Hver of
experimental animals 1n the process of hepatotropic effect of atmospheric
pollution (on the model of carbon tetrachloride). J. Hyg. Epidemic!. Micro.
Immunol. 23(4): 368-377.
15-22
-------
Mikhail, T.H., R. Awadallah, E.A. El-Dessoukey. 1978. Effect of AMP on
serum minerals 1n CC1 hepatotoxlclty. Ze1tschr1ft fur Ernahrungswissen-
schaft. 17(1): 47-51.
Milham, S. 1976. In.: P.U. Capurro. 1979. Cl1n. Toxlcol. 14: 285-294.
M1rsal1s, J.C. and B.E. Butterworth. 1980. Detection of unscheduled DNA
synthesis 1n hepatocytes isolated from rats treated with genotoxic agents:
an in vivo-jjn vitro assay for potential carcinogens and mutagens. Carcino-
genesis. 1: 621-625.
Mirsalis, J.C., C.K. Tyson and B.E. Butterworth. 1982. Detection of geno-
toxic carcinogens in the iji vivo-in vitro hepatocyte DNA repair assay.
Environ. Mutagen. 4: 553-562.
Moon, H.D. 1950. Pathology of fatal carbon tetrachloride poisoning with
special reference to histogenesis of the hepatic and renal lesions. Am. J.
Pathol. 26: 1041.
Morgan, A., A. Black and D.R. Belcher. 1972. Studies on the absorption of
halogenated hydrocarbons and their excretion 1n breath using 38C1 tracer
techniques. Ann. Occup. Hyg. 15: 273-282.
Morris, R.L. and L.6. Johnson. 1976. Agricultural runoff as a source of
halomethanes in drinking water. J. Am. Water Works Assoc. p. 492-494.
15-23
-------
Hurphy, S.D. and S. Hal ley. 1969. Effect of carbon tetrachloMde on Induc-
tion of liver enzymes by acute stress or cortlcosterone. Toxlcol. Appl.
Pharmacol. 15: 117-130.
NAS (National Academy of Sciences). 1978. Panel on Low Molecular Weight
Halogenated Hydrocarbons of the Coordinating Committee for Scientific and
Technical Assessments of Environmental Pollutants. Chloroform, Carbon
TetrachloMde, and other Halomethanes: An Environmental Assessment.
Washington, DC.
NAS (National Academy of Sciences). 1980. Drinking Water and Health.
3: 96-98. National Academy Press, Washington, DC.
NAS (National Academy of Sciences). 1982. Causes and Effects of Strato-
spheric Ozone Reduction: An Update. Washington, DC.
NAS (National Academy of Sciences). 1984. Causes and Effects of Changes 1n
Stratospheric Ozone: Update 1983. Washington, DC.
NCI (National Cancer Institute). 1976a. Report on the Cardnogenesls Bio-
assay of Chloroform. Cardnogenesls Program, Division of Cancer Cause and
Prevention. Harch 1, 1976.
NCI (National Cancer Institute). 1976b. Cardnogenesls Bloassay of Tr1-
chloroethylene. National Cancer Institute Cardnogenesls Technical Report
Series, No. 2. NCI-C6-TR-2. February, 1976.
15-24
-------
NCI (National Cancer Institute). 1977. Bloassay of 1,1,1-TMchloroethane
for Possible Carclnogenldty. National Cancer Institute Cardnogenesls
Technical Report Series, No. 3. NCI-CG-TR-3. January, 1977.
Neely, W.B. 1977. Material balance analysis of trlchlorofluoromethene and
carbon tetrachlorlde 1n the atmosphere. Sc1. Total Environ. 8: 267-274.
Neely, W.B., et al. 1974. Partition coefficient to measure bloconcentra-
tlon potential of organic chemicals 1n fish. Environ. Sc1. Techno!.
8: 1113.
New, P.S., et al. 1962. Acute renal failure associated with carbon tetra-
chlorlde Intoxication. J. Am. Hed. Assoc. 181: 903.
Nielsen, V.K. and J. Larsen. 1965. Acute renal failure due to carbon
tetrachlorlde poisoning. Acta. Hedlca. Scand1nav1ca. 178: 363.
NIOSH (National Institute for Occupational Safety and Health). 1975.
Criteria for a recommended standard. Occupational exposure to carbon tetra-
chlorlde. U.S. DHEW Pub. No. 76-133.
NIOSH (National Institute for Occupational Safety and Health). 1976.
Revised Recommended Carbon Tetrachlorlde Standard. U.S. DHEW Pub. No.
76-133.
Norwood, W.O., P.A. Fuqua and B.C. Scudder. 1950. Carbon tetrachlorlde
poisoning. Ind. Hyg. Occup. Med. 90-100.
15-25
-------
Oettel, H. 1936. In: W.F. von Oettlngen. 1964. Arch. Exp. Pathol.
Pharmacol. 183: 641.
Ohanlan, E. 1981. Telephone conversation with Or. M. Dourson. Office of
Drinking Hater, U.S. EPA.
Ohta, T., H. Horlta and I. H1zogucch1. 1976. Local distribution of
chlorinated hydrocarbons 1n the ambient air 1n Tokyo. Atmos. Environ.
10: 557-560.
Paquet, K.J. and V. Kamphausen. 1975. The carbon tetrachlorlde hepato-
toxlclty as a model of liver damage. First report: Long-time biochemical
changes. Acta. Hepato-Gastro. 22: 84-88.
Pasquall-Ronchettl, I., A. B1n1, B. Bottl, G. De Alojslo, C. Fornlerl and V.
Vann1n1. 1980. Ultrastructural and biochemical changes Induced by progres-
sive Upld peroxidation on Isolated mlcrosomes and rat Hver endoplasmlc
retlculum. Lab. Invest. 42: 457-468.
Pearson, C.R. and 6. McConnell. 1975. Chlorinated C] and C» hydro-
carbons 1n the marine environment. Proc. Roy. Soc. London. Series B.
189(1096): 305-322.
PelUzzarl, E.D. and J.E. Bunch. 1979. Ambient air carcinogenic vapors.
Improved sampling and analytical techniques and field studies. EPA
600/2-79-081.
15-26
-------
Penkett, S.A., K.A. Brlce, R.G. Derwent and A.E.J. Eggleton. T979.
Measurement of CCl
November 1977. Atmos. Environ. 13: 1011-1019.
and CC14 at Harwell over the period January 1975-
Pfelfer, K.F. and L.J. Weber. 1980. The effect of carbon tetrachlorlde
treatment on the urine flow rate of rainbow trout. Toxlcol. Appl.
Pharmacol. 52(2): 347-350.
Pfelfer, K.F. and L.J. Weber. 1981. The effect of carbon tetrachlorlde on
the total plasma protein concentration of rainbow trout, Salmo qalrdnerl.
Comp. Blochem. Physio!. 64C(1): 37-42.
Plaa, 6.L. and R.E. Larson. 1965. Relative nephrotoxlc properties of
chlorinated methane, ethane, and ethylene derivatives 1n mice. Toxlcol.
Appl. Pharmacol. 7: 37-44.
Pohl, L.R., R.V. Branchflower, R.J. Hlghet, et al. 1981. The formation of
dlglutathlonyl dlthlocarbonate as a metabolite of chloroform, bromotrl-
chloromethane, and carbon tetrachlorlde. Drug Metab. D1spos1t. 9: 334-339.
Popp, J.A., S. H1sash1 and E. Farber. 1978. The protective effects of
diethyldlthlocarbamate and cyclohexlmide on the multiple hepatic lesions
Induced by carbon tetrachlorlde 1n the rat. Toxlcol. Appl. Pharmacol.
45: 549-564.
Powell, J.F. 1945. The solubility or distribution on coefficient of carbon
tetrachlorlde In water, whole blood, and plasma. Br. J. Ind. Hed.
2: 212-216.
15-27
-------
Prendergast, J.A., R.A. Jones, L.J. Jenkins and J. Selgel. 1967. Effects
on experimental animals of long-term Inhalation of trlchlorethylene, carbon
tetrachlorlde, 1,1,1-trichlorethane, dlchlorodlfluoromethane, and
I,l-d1chloroethylene. Toxlcol. Appl. Pharmacol. 10: 270-289.
Proetzel, H., et al. 1964. The effect of liver Imbalance on the develop-
ment of oral tumors 1n mice following the application of benzpyrene or
tobacco tar. Oral Surg. 18: 622-635.
Rams, J.H., H. Pilgrim, S. Rauth, G. Hunt, T. Shannon and K. SUmak. 1979.
Draft report. Level I materials balance: Carbon tetrachlorlde. Prepared by
JRB Associates for Office of Pesticides and Toxic Substances, U.S. Environ.
Prot. Agency, Washington, DC. Contract No. 68-01-5793.
Ramsey, L.L. 1957. Color1metr1c determination of carbon tetrachlorlde 1n
fumigated cereal products. In.: Residues 1n Foods and Feeds Resulting from
Fumigation of Grains with the Commoner Liquid Formulations of Carbon
D1sulf1de, Carbon Tetrachlorlde, Ethylene D1chlor1de and Ethylene D1brom1de,
G.E. Lynn and F.A. Vorhes, Jr., Ed. J. Assoc. Off. Agrlc. Chem. 40(1):
175-180.
Recknagel, R.O. and E.A. Glende. 1973. Carbon tetrachlorlde hepatotox1c->
Uy: An example of lethal cleavage. In.: Critical Reviews 1n Toxicology, L,
Golberg, Ed. CRC Press, p. 263-297.
Reuber, H.D. and E.L. Glover. 1967a. Hyperplastlc and early neoplastlc
lesions of the liver in buffalo strain rats of various ages given subcutane-
ous carbon tetrachlorlde. J. Natl. Cancer Inst. 38: 891.
15-28
-------
Reuber, M.O. and E.L. Glover. 1967b. Cholanglof1bros1s In the liver of
Buffalo strain rats Injected with carbon tetrachlorlde. Br. J. Exp.
Pathol. 48(3): 319-322.
Reuber, M.O. and E.L. Glover. 1970. Cirrhosis and carcinoma of the liver
1n male rats given subcutaneous carbon tetrachlorlde. J. Natl. Cancer Inst.
44: 419-427.
Reynolds, E.S. and M.T. Moslen. 1980. Free-radical damage 1n liver. In.:
Free Radicals 1n Biology, Vol. IV, W.A. Pryor, Ed. Academic Press, New
York. p. 49-94.
Rlchet, G., et al. 1959. Anurla caused by carbon tetrachlorlde poisoning;
25 case reports. Rev. Prat. Paris. 9: 519-610. (Fre.)
Robblns, B.H. 1929. The absorption, distribution and excretion of carbon
tetrachlorlde 1n dogs under various conditions. J. Pharmacol. Exp. Ther.
37: 203-216.
Rocchl, P., G. Pordl, S. Gr1ll1 and A.M. FerreM. 1973. In vivo and In.
vitro binding of CC1 with nucleic adds and proteins 1n rat and mouse
liver. Int. J. Cancer. 11: 419-425.
Rotenberg, Y.S. 1978. Dynamics of liver bloenergetlc system responses fol-
lowing long-term exposure to small concentrations of carbon tetrachlorlde.
G1g. Tr. Prof. Zabol. 11: 55-56. (Abstr.)
15-29
-------
Sagal, H. and A.E. Tappel. 1979. L1p1d peroxldatlon Induced by some halo-
methanes as measured by In. vivo pentane production 1n the rat. Toxlcol.
Appl. Pharmacol. 49: 283-291. ,,s
Sandalls, F.J. and D.B. Hatton. 1977. Measurements of atmospheric concen-
trations of trlchlorofluoromethane, d1chlorod1fluorpmethane and carbon
tetrachlorlde by aircraft sampling over the British Isles. Atmos. Environ.
11: 321-327.
Sato, A. and T. Nakajlma. 1979. A structure-activity relationship of some
chlorinated hydrocarbons. Arch. Environ. Health. 34: 69-75.
Schelman, M.A., R.A. Saunders and F.E. Saalfeld. 1974. Organic contami-
nants 1n the District of Columbia water supply. J. Blomed. Mass. Spectrom.
1: 209-211.
Schwetz, B.A., B.K.J. Leong and P.J. Gehrlng. 1974. Embryo- and fetotoxlc-
1ty of Inhaled carbon tetrachlorlde, I,l-d1chloroethane and methyl ethyl
ketone 1n rats. Toxlcol. Appl. Pharmacol. 28: 452-464.
Scudamore, K.A. and S.G. Heuser. 1973. Determination of carbon tetrachlo-
rlde 1n fumigated cereal grains. Pestle. Sc1. 4: 1-12.
Seawrlght, A.A. and A.E. McLean. 1967. The effect of diet on carbon tetra-
chlorlde metabolism. Blochem. J. 105: 1055.
15-30
-------
Seawrlght, A.A., I.M. WHkie, P. Costlgan, J. Hrdlicka and D.P. Steele.
1980. The effect of an equimolar mixture of carbon, tetrachlorlde and carbon
dlsulphlde on the liver of the rats. Biochem. Pharmacolv 29: 1007.
Sein, K.T. and N. Chu. 1979. Liver and kidney glucose-6-phosphatase levels
1n carbon tetrachlorlde- and ODT-adm1n1stered mice. Enzyme. 24: 72-74.
Shah, H., S.P. Hartman and S. Welnhouse, 1979. Formation of carbonyl
chloride in carbon tetrachlorlde metabolism by rat liver iin vitro. Cancer
Res. 39: 3942-3947.
Shlmanko, 1.1., L.A. ITchenko and B.T. Rlmalis. 1979. Pulmonary hyper-
hydratlon 1n carbon tetrachlorlde poisoning. Klin. Med. 57(7): 85-88.
Siegers, C.P., J.6. Fllser and H.H. Bolt. 1978. Effect of dlthlocarbon
metabolism and covalent bonding of carbon tetrachlorlde. Toxlcol. Appl.
Pharmacol. 46: 709.
Slmler, M,, M, Maurer and J.C. Mandard. 1964, Liver cancer after cirrhosis
due to carbon tetrachloride. Strasbourg Med. 15: 910-918. .
Simmon, V.F. and R.G. Tardlff. 1978. The mutagenic activity of halogenated
compounds found 1n chlorinated drinking water. Water Chlorf 2: 417-431.
Simmon, V.F., K. Kauhanen and R.G. Tardlff. 1977. Mutagenic activity of
chemicals identified 1n drinking water. In.: Progress in Genetic Toxicology,
D. Scott, B.A. Bridges and F.H. Sobels, Ed. Elsevler/Northr-Holland Blomedi-
cal Press, Mew Yprk. p. 249-258.
15-31
-------
Slmmonds, P.6., F.N. Alyea, C.A. Cardellno, et al. 1983. The atmospheric
lifetime experiment. 6. Results for carbon tetrachlorlde based on 3 years
data. J. Geophys. Res. 88{C13): 8427-8441.
Singh, H.B., D.P. Fowler and T.O. Peyton. 1976. Atmospheric carbon tetra-
chlorlde: Another man-made pollutant. Science. 192(4234): 1231-1234.
Singh, A.B., L.J. Salas, A. Smith, R. Stiles and H. Sh1ge1sh1. 1980. Atmo-
spheric measurements of selected hazardous organic chemicals. Prepared for
Environmental Sciences Research Laboratory EPA/RTP by Atmospheric Science
Center, SRI International.
S1pes, 6.I., G. Krishna and J.R. Gillette. 1977. B1oact1vat1on of carbon
tetrachlorlde, chloroform and bromotrlchloromethane: Role of cytochrome
P-450. Life Sc1. 20: 1541-1548.
Smetana, H. 1939. Nephrosls due to carbon tetrachlorlde. Arch. Intern.
Hed. 63: 760-777.
Smith, A.R. 1950. Optic atrophy following Inhalation of carbon tetra-
chlorlde. Arch. Ind. Hyg. Occup. Med. 1: 348.
Smyth, H.F. 1935. In.: W.F. von Oettlngen. 1964. Ind. Med. 4: 12.
Smyth, H.F., H.F. Smyth, Jr. and C.P. Carpenter. 1936. The chronic toxlc-
1ty of carbon tetrachlorlde; animal exposure and field studies. J. Ind.
Hyg. Toxlcol. 18: 277-298.
15-32
-------
Sonlch, C., D.F. Kraemer, S.J. Samuels and J.B. Lucas. 1981. An epldemlo-
loglcal study of acute effects of a low level exposure to carbon tetrachlo-
rlde. Am. J. Ep1dem1ol. (Unpubl.)
Sonneborn, M. and 8. Bonn. 1977. Formation and occurrence of haloforms 1n
drinking water. In.: Water Chlorination. Environmental Impact and Health
Effects, Vol. 2, R.L. Jolley, H. Gorchev and D.H. Hamilton, Jr., Ed. Ann
Arbor Science Publishers, Inc., Ann Arbor, MI.
Stara, J.F. and D. Kello. 1979. Relationship of long-term animal studies
to human disease. ITK Assessing Toxic Effects of Environmental Pollutants,
S Lee and J.B. Mudd, Ed. Ann Arbor Science, Ann Arbor, MI. p. 43-76.
Statham, C.N., W.A. Croft and J.J. Lech. 1978. Uptake, distribution and
effects of carbon tetrachlorlde 1n rainbow trout (Salmo galrdnerl).
Toxlcol. Appl. Pharmacol. 45(1): 131-140.
Stevens, H. and P.M. Forster. 1953. Effect of carbon tetrachloride on the
nervous system. Arch. Neurol. Psychlat. 70: 635-649.
Stewart, R.D. and H.C. Oodd. 1964. Absorption of carbon tetrachlorlde
trlchloroethylene, tetrachloroethylene, methylene chloride, and T,l,1-tr1-
chloroethane through the human skin. Am. Ind. Hyg. Assoc. J. 25: 439-446.
Stewart, R.O., H.H. Gay, D.S. Erley, C.L. Hake and J.E. Peterson. 1961.
Human exposure to carbon tetrachlorlde vapor — Relationship of expired air
concentration to exposure and toxlcity. J. Occup. Med. 3: 586-590.
15-33
-------
Stewart, R.D., E.A. Boettner, R.R. Southworth and J.C. Cerny. 1963. Acute
carbon tetrachlorlde Intoxication. J. Am. Med. Assoc. 183: 994-997.
Stewart, R.D., et al. 1965. Diagnosis of solvent poisoning. J. Am. Med.
Assoc. 93: 1097.
Stoklnger, H.E. and R.L. Woodward. 1958. Tox1colog1c methods for estab-
lishing drinking water standards. J. Am. Water Works Assoc* 52t 515-529.
Straus, B. 1954. Aplastlc anemia following exposure to carbon tetrachlo-
rlde. J. Am. Med. Assoc. 155(8): 737-739.
Striker, G.E., E.A. Smuckler, P.W. Kohneh and R.B. Nagle. 1968. Structural
and functional changes
Pathol. 53(5): 769-789.
and functional changes 1n rat kidney during CC1. Intoxication. Am. J.
Strubelt, 0., F. Obermeler, C.P. Siegers and M. Volpel. 1978. Increased
carbon tetrachlorlde hepatotoxldty after low-level ethanol consumption.
Toxlcol. 10: 261-270.
Su, C. and E.D. Goldberg. 1976. Environmental concentrations and fluxes of
some halocarbons. In,; Marine Pollutant Transfer^ H.L. Wlndoms and R.A.
Duce, Ed. Lexington Books, D.C. Heath and Co., Lexington, MA. p. 353-374.
Sugdon, T.M. and T.F. West. 1980. Chlorofluorocarbohs 1n the Environment:
The Aerosol Controversy. John Wiley and Sons, New York.
15-^34
-------
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U.S. EPA. 1981. Final draft for the drinking water criteria document on
carbon tetrachlorlde. Office of Drinking Water, Washington, DC. Contract
No. 2-813-03-644-09. (Draft)
U.S. EPA. 1982. Handbook for Sampling and Sample Preservation of Water and
Wastewater. U.S. EPA Environmental Monitoring and Support Laboratory,
Cincinnati, OH. EPA 600/4-82-029.
USITC (U.S. International Trade Commission). 1981. Preliminary report on
U.S. production of selected synthetic organic chemicals (Including synthetic
plastics and resin materials).
Va1n1o, H., H.G. Parkki and J. Harnlemi. 1976. Effects of aliphatic
chlorohydrocarbons on drug-metabolizing enzymes in rat liver Jm vivo.
Xenoblotica. 6: 599-604.
Verschuren, K. 1977. Handbook of Environmental Data on Organic Chemicals.
Van Nostrand Relnhold Company, New York.
von Oettingen, W.F. 1964. The halogenated hydrocarbons of industrial and
toxlcologlcal importance. In.: Elsevier Monographs on Toxic Agents, E.
Browning, Ed. Elsevier Publishing Co., New York.
von Oettingen, W.F., C.C. Powell, N.E. Sharpless, W.C. Alford and L.J.
Pecora. 1950. Comparative studies of the toxiclty and pharmacodynamic
action of chlorinated methanes with special reference to their physical and
chemical characteristics. Arch. Int. Pharmacodyn. 81: 17-34.
15-37
-------
Weast, R.C., Ed. 1978. Physical constants of organic compounds: Chloro-
form. In.: CRC Handbook of Chemistry and Physics, 59th ed. CRC Press, Inc.,
West Palm Beach, FL. p. C-244.
We1, E., L.C.K. Wong and C.H. H1ne. 1971. Potent1at1on of carbon tetra-
chlorlde hepatoxldty by ethanol and cold. Toxlcol. Appl. Pharmcol.
18: 329.
Welsburger, E.K. 1977. Carc1nogen1city studies on halogenated hydro-
carbons. Environ. Health Perspect. 21: 7-16.
Welsburger, J.H. and E.K. Welsburger. 1963. Endogenous and exogenous
factors 1n chemical cardnogenesls by N-2-fluorenylacetamlde. Acta Un. Int.
Cancer. 19: 513-518.
Welsburger, J.H., et al. 1965. Carc1nogenes1s by simultaneous action of
several agents. Toxlcol. Appl. Pharmacol. 7: 502.
Williams, 6.H. 1981. Liver culture Indicators for the detection of chemi-
cal carcinogens. In.: Short-Term Tests for Chemical Carcinogens, H.F. Stlch
and R.H.C. San, Ed. Sprlnger-Verlag, New York. p. 275-289.
Wilson, J.G. 1954. Influence of the offspring of altered physiologic
states during pregnancy 1n the rat. Ann. N.Y. Acad. Sc1. 57: 517-525.
Wlndholz, H., Ed. 1976. Carbon Tetrachlorlde. In.: The Merck Index, 9th
ed. Merck &Co., Inc., Rahway, N.J. p. 232.
15-38
-------
Wlrtschafter, Z.T. 1933. Toxic amblyopla and accompanying physiological
disturbances 1n carbon tetrachlorlde Intoxication. Am. J. Public Health.
22: 1035-1038.
W1t, S.L. 1972. Residues of Insecticides 1n Cereals and Related Products
Incorporated 1n the Netherlands 1964/1966. 1971 Evaluation of Some Pesti-
cide Residues 1n Food Monography, WHO Pestle. Res. Sev. Vol.
WHO (World Meteorological Organization). 1982. The Stratosphere 1981:
Theory and Measurements. WHO Global Ozone Research and Monitoring Project.
Report No. 11. Geneva, Switzerland.
Wong, L.C.K. and V. DiStefano. 1966. Rapid accumulation of renal fat in
cats after single Inhalations of carbon tetrachlorlde. Toxicol. Appl.
Pharmacol. 9: 485.
Wood, J.M., F.S. Kennedy and R.S^ Wolfe. 1968. The reaction of multihalo-
genated hydrocarbons with free and bound reduced vitamin 8-12. Biochem.
7: 1707-1713.
Wyrebowska, J. and T. Jerzykowski. 1980. Some properties of amlnopropanol
dehydrogenase in rat serum studied 1n normal conditions and 1n acute carbon
tetrachloride poisoning. J. Toxicol. Environ. Health. 6: 613-620.
15-39
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APPENDIX
Unit Risk Estimates for Cancer
DEFINITION
Unit risk 1s one Index of the relative carcinogenic potential of a chem-
ical. Unit risk 1s defined here as the lifetime risk to humans of contract-
Ing cancer from a dally exposure to a concentration of 1 yg/si In water
via 1ngest1on or a dally exposure to 1 yg/m3 in air via Inhalation. The
main assumptions for such risk estimates are 70 kg bw, 2 a/day consumption
of water and 20 mVday Inhalation rate (ICRP, 1975). The unit risk
represents only the estimated risk at the stated exposure concentrations.
It should not be Interpreted as the slope at any exposure level since the
shape of the curve 1n the low-dose region 1s not known.
The unit risk estimate for CC1 represents an extrapolation below the
4
dose-risk range of experimental data. There 1s currently no solid scientif-
ic basis for any mathematical extrapolation model that relates exposure to
cancer risk at the extremely low concentrations, Including the unit concen-
tration given above, that must be dealt with 1n evaluating environmental
hazards. For practical reasons the correspondingly low levels of risk
cannot be measured directly either by animal experiments or by ep1dem1olog1c
studies. Low-dose extrapolation must, therefore, be based on current under-
standing of the mechanisms of cardnogenesls. At the present time the domi-
nant view of the carcinogenic process Involves the concept that most agents
that cause cancer also cause Irreversible damage to DNA. This position 1s
' based 1n part on the fact that a very large proportion of agents that cause
cancer are also mutagenlc. There 1s reason to expect that the quanta!
response that 1s characteristic of mutagenesls 1s associated with a linear
non-threshold dose-response relationship. Indeed, there 1s substantial
evidence from mutagenlclty studies with both Ionizing radiation and a wide
A-l
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variety of chemicals that this type of dose-response model 1s the appro-
priate one to use. This 1s particularly true at the lower end of the dose-
response curve; at higher doses, there can be an upward curvature probably
reflecting the effects of multistage processes on the mutagenlc response.
The linear non-threshold dose-response relationship 1s also consistent with
the relatively few ep1dem1olog1c studies of cancer responses to specific
agents that contain enough Information to make the evaluation possible
(e.g., radiation-Induced leukemia, breast and thyroid cancer, skin cancer
Induced by arsenic 1n drinking water, liver cancer Induced by aflatoxln 1n
the diet). Some supporting evidence also exists from animal experiments
(e.g., liver tumors Induced 1n mice by 2-acetylam1nofluorene In the large
scale EDni study at the National Center for Tox1colog1cal Research and the
Initiation stage of the two-stage :carc1nogenes1s model In rat Hver and
mouse skin).
Because 1t has the best, albeit limited, scientific basis of any of the
current mathematical extrapolation models, the non-threshold model which 1s
linear at low doses has been adopted as the primary basis for risk extrapo-
lation to low levels of the dose-response relationship. The risk estimates
made with such a model should be regarded as conservative, representing the
most plausible upper-limit for the risk, I.e., the true, risk Is not likely
to be higher than the estimate, but 1t could be lower.
The mathematical formulation chosen to describe the dose-response
relationship at low doses Is the linearized multistage model. This model
employs enough arbitrary constants to be able to fit almost any monotonlc-
ally Increasing dose-response data. It 1s constrained to ensure linearity
1n the low dose region at least for the upper confidence limit by requiring
non-negative values for the fitted coefficients. Furthermore, there exists
A-2
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a procedure for estimating an upper confidence limit on the slope at low
extrapolated doses that 1s based on fitting the data at all experimental
dose levels. Other dose-response models have been proposed which are also
linear 1n the low dose region. The procedure recommended by the Carcinogen
Assessment Group of EPA, however, Involves estimating a most plausible upper
limit of the slope at low doses. The other models (discussed later) can be
shown to give lower slopes for the same data set than does the linearized
multistage model, when extrapolated to the low dose region. Thus, the
extrapolation model preferred by the Carcinogen Assessment Group 1s the
multistage model.
EXPERIMENTAL STUDIES USED IN UNIT RISK ESTIMATES
Three oral studies on animals have sufficient Information to allow
estimation of unit risk. The oral studies are the positive control data for
mice and rats used 1n three of Us bloassays (NCI, 1976a,b, 1977), the
Edwards et al. (1942) mice data and the Delia Porta et al. (1961) hamster
data. The Incidence data and other pertinent quantitative Information on
these studies are presented 1n Table 1. For all studies, male and female
data were combined. This was done because of the small sample sizes 1n the
groups segregated by sex. No appropriate Inhalation studies or human oral
studies were found 1n the available literature.
Each of these oral studies has one or several characteristics which make
1t less than Ideal for risk estimation for continuous dally exposure over a
lifetime. Delia Porta et al. (1961) did not report results for a control
group, although they did report the Incidence rate for vehicle controls 1n a
different study. Moreover, the dose was administered only once per week and
was reduced by half after 7 weeks, forcing the use of a time-weighted
average approximation to a dally dose. The sample size (19) was also small.
Edwards et al. (1942) exposed the mice for only 4 months and observed them
A-3
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for ~8 months, much less than the desired lifetime experiment (1.5-2 yrs).
The ages varied; the animals Initially ranged from 2.5-7.5 months of age.
In the NCI (1976a,b, 1977) study on mice, both low and high dose groups
showed virtually 100% response (89/89 and 90/93, respectively), so that no
Information was available on the slope of the dose-response curve. As a
consequence, risk projections for doses lower than those used 1n the study
will be underestimated by an unknown amount. In the NCI (1976a,b, 1977)
study on rats, survival to the end of exposure was poor, the dose was
changed forcing use of a TWA dose estimate, and when segregated by sex, the
sample sizes were small so that only the low-dose females were statistically
significantly different from controls. The combining of the male and female
rats when different, though similar, doses were used (see Table 1) may add
further uncertainty.
Insufficient metabolic and pharmacoklnetlc data precluded the selection
of the most appropriate species for use 1n estimating human risk. Because
no study could be selected as "best" and "most appropriate", all four data
sets are usedv1n separate estimates of unit risk. In addition, an average
unit risk estimate 1s also calculated which 1s the geometric mean of the
four separate estimates.
DESCRIPTION OF THE PREFERRED LOW-DOSE ANIMAL EXTRAPOLATION MODEL
Let P(d) represent the lifetime risk (probability) of cancer at dose d.
The multistage model has the form
P(d) = 1 - exp [-(q0 + q]d * q2d2 + ... + qkdk)]
where all coefficients (qQ, q] ...qk) are non-negative. The unit risk
estimates are based on excess or extra risk over the background rate at
dose d, I.e. the effect of treatment:
P(d) - P{o)
Pt(d) =
1 - P(o)
A-5
-------
qkdk)]
It follows that
Pt(d) = 1 - exp [-{q
The point estimate of the coefficients q., 1 = 0, 1, 2, ..., k, and
consequently the extra risk function Pt(d) at any given dose d, 1s calcu-
lated by maxlmzlng the likelihood function of the data. The point estimate
and the 95% upper confidence limit of the extra risk ?t(d) are calculated
by using the computer program GLOBAL 79 developed by Crump and Watson
(1979). The upper confidence limit for the extra risk calculated at low
doses 1s always linear with dose. This 1s conceptually consistent with the
linear non-threshold concept discussed earlier. The slope parameter q *
Is taken as an upper bound (at low doses) of the potency of the chemical 1n
Inducing cancer.
In fitting the dose-response model, the number of terms In the polyno-
mial 1s chosen equal to (h-1), where h 1s the number of dose groups 1n the
experiment Including the control group.
Whenever the multistage model does not fit the data sufficiently well,
the data point at the highest dose 1s deleted and the model 1s refitted to
the rest of the data. This Is continued until an acceptable fit to the data
1s obtained. To determine whether or not a fit 1s acceptable, the ch1-
square statistic
h
X2 = E
1=1
d-Pl)
Is calculated where
th
Is the number of animals 1n the 1"" dose group,
X* Is the number of animals In the 1 dose group with a tumor response,
P^ 1s the probability of a response In the 1 dose group estimated by
fitting the multistage model to the data, and h Is the number of dose
2
groups. The fit 1s determined to be unacceptable whenever X 1s larger
A-6
-------
than the cumulative 99% point of the ch1-square distribution with f degrees
of freedom, where f equals the number of dose groups minus the number of
non-zero multistage coefficients.
CONSIDERATIONS IN SELECTING INCIDENCE DATA
The tumor Incidence data are separated according to organ sites or tumor
types. The set of data (I.e., dose and tumor Incidence) used 1n the model
1s the set where the Incidence Is statistically significantly higher than
the control for at least one test dose level and/or where the tumor
Incidence rate shows a statistically significant trend with respect to dose
level. Usually, the conservative approach adopted by the Carcinogen
Assessment Group selects the data set which gives the highest estimate of
the unit risk for humans. Because of the difficulties with each CC1
4
study described earlier, no selection was deemed appropriate. Instead, the
separate estimates and their geometric mean are presented.
If two or more significant tumor sites are observed in the same study,
and if the data are available, the number of animals with at least one of
the specific tumor sites under consideration 1s used as Incidence data In
the model.
DESCRIPTION OF THE PREFERRED METHOD FOR CALCULATING HUMAN EQUIVALENT UNIT
RISK
The method adopted by the Carcinogen Assessment Group for calculating a
human equivalent estimate of unit risk from animal data employs two adjust-
ments (Federal Register, 1980b) reflecting species differences and the
influence of exposure duration on lifetime cancer risk. First, the animal
doses are expressed as the time-weighted-average (TWA) daily dose over the
duration of the experiment, and the low-dose extrapolation model is fitted
to the resulting dose-incidence data. The risk is then estimated for a
daily dose of 2 yg/day, which is the oral dally exposure corresponding to
A-7
-------
a water concentration of 1 vg/8. and human Intake of 2 a/day. This
risk 1s then multiplied by each of the above adjustment factors to obtain
the human equivalent oral unit risk.
The models used to develop the species and duration adjustment factors
represent the best scientific judgment based on available data. Other
approaches have been suggested and are discussed in a later section.
The preferred model for equltoxic dose across species, or equlvalently
for risk at a constant dose across species, 1s based on an adjustment for
metabolic differences. Metabolic rate has been suggested to be roughly
proportional to body surface area (Mantel and Schnelderman, 1975; Calabrese,
1982), thus the equltoxic model 1s dose/surface area = constant. Equating
the animal and human ratios and solving for the human dose gives:
"h • "a
where d 1s daily dose (mg/day), S is surface area, and a and h refer to
animal and human, respectively. The surface area is roughly proportional to
the 2/3 power of body weight, and the proportionality constant is approxi-
mately the same (-10) for a variety of species. The human dose is then
approximated by
. . ... ... ,2/3
d, = d (W./W )
h aha
The unit risk estimate represents the lifetime risk for lifetime
exposure to the carcinogen. When the animal experiment is partial lifetime,
an adjustment is necessary to allow for positive responses that would have
occurred had sufficient time been allowed for the tumors to develop. The
risk 1s then adjusted upward (or equlvalently the dose downward) to reflect
the missing responders in the short experiment. The adjustment coefficient
is (L/L )3, where L is the animal lifespan and Lfi 1s the duration of
the experiment. The exponent 3 is supported in part by Doll (1971), who
A-8
-------
showed that age-specific rates for humans Increase by at least the second
power of age, thus the cumulative tumor rate should Increase by at least the
third power of age. The choice of 3 for the exponent 1s also supported by
Druckrey (1967) who showed that for a constant Incidence rate, the dose-
duration relationship was represented by dtn = constant with n ranging
from 2-4 1n his experiments. With n=3, Druckrey's results suggest that the
dose used for half-lifetime exposure (and observation) can be reduced to
one-eighth Its value for lifetime exposure and the Incidence rate will be
the same. Druckrey's results then reflect the Influence of both exposure
duration and observation period on the resulting Incidence rate.
This adjustment factor 1s consistent with the proportional hazard model
proposed by Cox (1972) and the t1me-to-tumor model considered by Crump
(1979, 1982) where the probability of cancer by age t at dose d 1s
P(d,t) = 1 - exp [-f(t)*g(d)].
For comparison, Crump's (1982) t1me-to-tumor model 1s also used to estimate
unit risk for the NCI rat and mice studies (NCI, 1976a,b, 1977) which had
t1me-to-tumor data. In the above model, g(d) 1s the multistage polynomial,
i,
and f(t) 1s (t-tQ) , where tQ may be Interpreted as minimum Induction
time.
Interpretation of Quantitative Estimates
For several reasons, the unit risk estimate based on animal bloassays Is
only an approximate Indication of the absolute risk 1n populations exposed
to known carcinogen concentrations. First, there are Important species
differences 1n uptake, metabolism, and organ distribution of carcinogens, as
well as species differences 1n target site susceptibility, 1mmunolog1cal
responses, hormone function, dietary factors and disease. Second, the
concept of equivalent doses for humans compared to animals on a mg/surface
A-9
-------
area basis is virtually without experimental verification regarding carcino-
genic response. Finally, human populations are variable with respect to
genetic constitution and diet, living environment, activity patterns, and
other cultural factors. The exposure levels used 1n the unit risk calcula-
tions (1 vg/fc, 1 yg/m3) correspond to estimated Intake of 2 yg/day
via 1ngest1on and 20 yg/day via Inhalation. The expected human Intake
rates for CC1, are 1n the same range: 4 yg/day from food, 9 yg/day
4
from fluids and 13 yg/day from air (see Chapter 4). The assumed dose-
response curve 1s quite linear 1n this dose range so that the risk 1s
proportional to exposure level, I.e., the upper limit estimate of risk 1s
the unit risk multiplied by the exposure concentration.
The unit risk estimate' can give a rough Indication of the relative
potency of a given agent compared with other carcinogens. The comparative
potency of different agents 1s more reliable when the comparison 1s based on
studies 1n the same test species, strain and sex, and by the same route of
exposure. For unit risk estimates for air, the preferable studies would use
exposure by Inhalation.
The quantitative aspect of the carcinogen risk assessment 1s Included
here because 1t may be of use 1n the regulatory decision-making process
(e.g., setting regulatory priorities, evaluating the adequacy of technology-
based controls, etc.). However, 1t should be recognized that the estimation
of cancer risks to humans at low levels of exposure 1s uncertain. At best,
the low-dose linear extrapolation model used here provides a rough, but
plausible estimate of the upper-limit of risk, I.e., 1t 1s not likely that
the true risk would be much more than the estimated risk, but 1t could very
well be considerably lower. The risk estimates presented 1n subsequent
sections should not be regarded as accurate representations of the expected
A-10
-------
cancer risks even when the exposures are accurately defined. The estimates
presented may be factored Into regulatory decisions to the extent that the
concept of upper risk limits 1s found to be useful.
Unit Risk Estimates for Ingestlon Exposure
The unit risk estimates based on human equivalent doses as discussed
above are given 1n Table 2. Since the NCI studies (1976a,b, 1977) on rats
and mice were the only ones to present t1me-to-tumor data, they are the only
data sets evaluated using the t1me-to-tumor model. Both the maximum likeli-
hood estimates (MLE) and upper 95% confidence limits are presented, as well
as their geometric means. For the NCI (1976a,b) mouse data, the goodness-
of-f1t criterion was not satisfied (x2 = 14.4) for the multistage (and
one-hit) model. The risk estimates are presented anyway since the model
cannot be fitted to the data 1f the high dose group 1s deleted, due to the
100% response at the low dose. Because of the protective approach discussed
earlier which led (1n part) to the adoption of the multistage model, and
because the MLE does not account for estimation errors due to small sample
sizes, the 95% upper limit on risk 1s preferred. Furthermore, since no
study was entirely adequate for risk assessment purposes, the geometric mean
of the upper confidence limits 1s preferred as the most plausible upper
limit estimate of unit risk. For lifetime Ingestlon of 2 a/day of water,
the recommended (based upon present Information and current understanding of
cardnogenesls) estimate of unit risk (concentration of 1 pg/a.) 1s
3.7x10-®.
ALTERNATIVE METHODOLOGICAL APPROACHES
The methods described above, which have been adopted by the Carcinogen
Assessment Group, are consistently conservative, I.e., tending toward high
estimates of cancer risk. The aspect which contributes most to this
conservatism 1s the choice of the linearized multistage model for low-dose
A-ll
-------
TABLE 2
Human Equivalent Unit Risk Estimates for Ingestlon Exposure
with Specific Adjustments3
Data Set
Delia Porta et al. (1961)
Edwards et al. (1942)
NCI (1976) mouse
NCI (1976) rat
All (geometric mean)
Mul
HLE
2.1E-5
7.1E-6
1.4E-6
1.9E-7
2.5E-6
Extrapolation
t1staqeb
l)L
3.4E-5
9.4E-6
1.8E-6
3.1E-7
3.7E-6
Model
T1me-to-Tumorb
MLE
1.8E-6
3.1E-7
7.5E-7
UL
£/
£./
-2.2E-6
5.3E-7
1.1E-6
aFor Ingestlon of 1 u9 CC14 per a. water dally for life. Species
conversion uses dose/body surface area. Duration adjustment for partial
lifetime experiment used for Edwards et al. (1942) study 1s (fraction life-
span)"3.
&MLE » Maximum likelihood estimate; UL = upper 95% confidence limit.
°No t1me-to-tumor data were available for these studies.
A-12
-------
extrapolation. Other extrapolation models have been suggested, and are
Included below for comparison. These other models generally give lower
estimates of risk than does the multistage model.
The various adjustment factors can also be calculated 1n different ways.
The uncertainties related to the several models and adjustment factors and
their Influences on the risk estimates are discussed below. Generally, most
of the uncertainty 1n estimating cancer risk from animal data 1s due to the
limited data available 1n the bloassays, especially due to the high dose
levels used, so that almost nothing Is known about the shape of the dose-
response curve at low doses or about the differences In low-dose Incidence
rates across species.
Low-Dose Extrapolation Models
Four models are used to extrapolate from the region of the experimental
1ngest1on data to the levels corresponding to 1 vq/l (Albert, 1983).
All of these models relate exposure level to the Incidence of tumor-bearing
animals (Table 3). The "linearized" multistage model 1s constrained to have
non-negative parameter values, and has the same number of parameters as the
number of dose groups (Including the control group). The one-hit model has
two parameters. These two models are linear at sufficiently low doses. The
Welbull model has three parameters, When only two dose groups exist, the
Welbull exponent 1s set at 1, and then the Welbull model 1s also linear at
low doses. The log problt model 1s used to represent a class of models
which are not linear at low doses. The multistage reduces to the one-hit 1f
only two dose groups exist. With only two dose groups, the Welbull param-
eter k 1s set to 1 and the Welbull model also reduces to the one-hit model.
Currently, as discussed previously, there 1s Insufficient evidence to
provide strong support for any low-dose extrapolation model, although there
1s some justification for low-dose linearity.
A-13
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A-14
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In addition, since the NCI (1976a,b, 197*7) data provide information
pertaining to early mortality and t1me-to-tumor estimates, a multistage
Welbull model with five parameters (Crump, 1982) 1s also used (see Table 3).
The t1me-to-tumor estimates are based largely on t1me-to-mortal1ty with
subsequent discovery of tumors. Since the NCI (1976a,b, 1977) studies are
the only ones Involving more than two dose groups, they are also the only
data sets to which all four previous models can be applied.
Unit Risk Calculation Approach
\
Each low-dose extrapolation model 1s applied to the original unadjusted
animal data. The resulting risk estimate 1s then converted Into equivalent
human unit risk by multiplication by several factors to adjust for experi-
ment duration (If partial lifetime), species differences and, 1f necessary,
route conversion. Uncertainties 1n each adjustment factor are Investigated
by changing the choice of each adjustment model and/or the assumed parameter
values 1n the model. There 1s Insufficient Information to allow the alter-
natives to be characterized by likelihood or error distributions; hence, all
decisions are based on scientific judgment. The adjustment categories and
their decision alternatives are given 1n Table 4.
Each animal risk estimate obtained by fitting a low-dose extrapolation
model to the animal data 1s presented as the maximum likelihood estimate
(MLE) and,, when possible, as the upper 95% confidence limit. The confidence
limit 1s statistically more stable; the MLE may show substantial sensitivity
to small changes 1n the original data. Some data errors are expected due to
diagnostic uncertainties generally leading to underdetectlon, I.e., missed
tumors. The confidence limit on risk, however, reflects sample size and
random variability, and may be much higher than the MLE. Both estimates aYe
Investigated for their sensitivities to such data errors.
A-15
-------
TABLE 4
Alternatives for Judgmental Decisions 1n Cancer Unit Risk Estimation
Decision Category
Alternatives
Low-dose extrapolation
Equltoxlc dose across species
Adjustment for partial lifetime
experiment
Model: Multistage, one-hit, Welbull,
log problt
Estimator: Maximum likelihood estimate,
upper 95% confidence limit
Model: Dose/bw, dose/body surface area
Model: (L/Le)k, k = 1-4
A-16
-------
The risk estimate from the low-dose extrapolation 1s based on average
dally Intake level. Gavage studies add uncertainty since gastrointestinal
effects may be due to the repeated high local concentrations and may not
occur 1f the same dally dose were given continuously, hence at a lower local
concentration. Since Insufficient pharmacoklnetlc Information exists to
adjust for Intermittent exposure, the TWA dally Intake rate 1s used.
The extrapolation from risk based on partial lifetime experiments to
if
risk from lifetime exposure uses the factor: (L/L ) , where L 1s life-
span and L 1s the duration of the experiment. As discussed previously,
there 1s some evidence from human and animal studies to support an exponent
of 3. Druckrey (1967) found that, at constant risk, fitting the model
dtn = constant to a series of data on nltrosamlnes gave values of n of
2-4. Studies of age-specific cancer rates 1n humans give estimates of k at
3 or higher. The concept of total dose (dose rate x duration) or similarly
time-weighted-average dose for the experiment has been used as an Indication
of toxic severity, but has not been verified for cancer risk. It Is In-
cluded here for completeness and 1s represented by the case k=l 1n the
adjustment factor. There 1s Insufficient Information at present to allow
precise determination of k for most chemicals. In the absence of such
Information, the sensitivity of the risk estimate to k 1s demonstrated by
varying k between 1 and 4, and displaying the resulting range of risk
estimates.
The conversion factor for species differences 1s presently based on
models for equltoxlc dose. The two general models currently used are based
on body burden, dose/bw = constant (Stara and Kello, 1974); and metabolic
rate, dose/body surface area = constant (Mantel and Schnelderman, 1975;
Calabrese, 1982). In the absence of pharmacoklnetlc data related to
A-17
-------
toxlclty on the test chemical for the experimental species and for humans,
both models are used.
The conversion of administered dose from one route to another {e.g.,
1ngest1on to Inhalation) 1s not well understood. The approach by Stocklnger
and Woodward (1958) uses approximate net absorption fractions and dally
Intake rate to convert from one route to an equltoxic exposure level via
another route. This approach, discussed below, 1s used to give approximate
estimates of exposure levels when Insufficient data exist for the desired
route, although the estimates are acknowledged as being highly uncertain and
probably Inaccurate.
Unit Risk Estimates for Ingestlon Exposure
The unit risk estimates based on human equivalent doses consistent with
previous guidelines (Federal Register, 1980b) are given In Table 5 for the
four data sets and the applicable models. Note that for the NCI mouse and
rat data, the unconstrained Welbull and unconstrained log problt models
could not be used to estimate risk. The failure of the computer algorithm
to yield meaningful results 1s attributed to the virtually flat slope at the
low and high dose data. This caused the extrapolated risk to be essentially
dose-Independent, I.e., the same as the risk 1n the dosed groups regardless
of the level of dose. The successfully applied models were fitted to the
original animal data using average dally Intake (mg/day); their parameter
estimates are given 1n Table 6. The human risk was then calculated by mul-
tiplying the animal unit risk by the adjustment coefficients reflecting par-
tial lifetime exposure and species conversion. The low-dose extrapolation
of the NCI rat data 1s shown 1n Figure 1 as an example of the difference 1n
unit risk estimation (risk at 1 vg/9.) due only to selection of extrapo-
lation model. The risk projections have not been adjusted for species
A-18
-------
TABLE 5
Unit Risk Estimates for Ingestlon3
Model (type of estimate)*3
Study
Multistage0
(HLE)
(UL)
T1me-to-Tumor
(HLE) (UL)
Welbull Log ProbH
Delia Porta
et al. (1961)
Edwards et al .
(1942)
NCI (1976) mouse
MCI (1976) rat
2.1E-5
7.1E-6
1.4E-6
1.9E-7
3.4E-5 d/ e/
9.4E-6 d/ e/
1.8E-6 1.8E-6 2.2E-6 £/
3.1E-7 3.1E-7 5.3E-7 f/
§./
e/
I/
I/
aFor Ingestlon of 1 yg CC14 per a. water dally for life. Species
conversion uses dose/body surface area.
bMLE = Maximum likelihood estimate; UL = upper 95% confidence limit.
cThe one-hit model results agreed with the multistage model results to
three significant figures.
t1me-to-tumor data were available for these studies.
eThese models have three parameters which could not be fit by the two dose
groups In these studies.
unconstrained Welbull and unconstrained log problt. models could not
be used to estimate unit risk for these studies. See text.
A-19
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A-21
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differences so that the actual rat response rates (the 4 data points) can be
plotted. The logarithmic plot precluded the display of the control group
response (OJ4).
The possibility of errors 1n the Incidence data Increase the uncertainty
1n the unit risk estimates. Such errors are usually under-detectlon of
tumors (Anonymous, 1983), causing positively responding animals to be class-
ified as nonresponders, and leading to underestimates of the unit risk.
Furthermore, previous studies have suggested that the multistage maximum
likelihood estimate, q^, 1s more sensitive to such data errors than 1s the
upper confidence limit, q^.' The sensitivities of q and q * to data
changes are Investigated for each of the three oral studies under consider-
ation.
\
Discussion of possible detection errors 1n Incidence data at the
"ED01" conference* suggested the following: that mlsclasslfIcatlon 1s
more Hkely to underestimate the Incidence of tumor-bearing animals than
overestimate the incidence, and that having one animal mlsclasslfled in a
study of 50 animals 1s not rare. The sensitivity of the parameter estimates
to data errors was determined here by Increasing the number of responders by
1 for every 50 animals tested (e.g., 1 for up to 50 animals, 2 for 51-100
animals, etc.), and then recalculating the parameters. The results are
presented in Table 7. For each of these studies, the HLE (q ) seems to be
\ I
approximately as sensitive as the upper bound (q *) to the data changes.
Note that the parameter estimates are obtained from the raw data, are
unadjusted and, thus, are not comparable across studies.
*"Workshop on Biological and Statistical Implications of the EDQ1 Study
and Related Data Bases," Deer Creek State Park, Ohio, September 13-16, 1981.
A-22
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In Table 5, 1n line with previous guidelines (Federal Register, 1980b),
the species conversion model was dose per body surface area and partial
lifetime risk was adjusted to lifetime risk by the ratio (L/L )3. The
effects of using dose per body weight Instead for species conversion, as
well as exponents of 1 (total dose), 2 or 4 for the partial lifetime to
lifetime adjustment are shown by the coefficients 1n Table 8 and the range
of unit risk estimates 1n Table 9. Note that the adjustment coefficients 1n
Table 8 are comparable only within a study, not across studies. The unit
risk estimates 1n Table 9 are comparable across studies.
UNIT RISK ESTIMATES FOR INHALATION EXPOSURE
The unit risk for Inhalation exposure 1s the excess cancer risk for
lifetime exposure to 1 yg CCl4/m3 air. No Inhalation cancer studies
have been located which contain adequate dose-response information.
However, the unit risk can be estimated from 1ngest1on studies by assuming
that the same daily intake rate results in the same lifetime risk. This
assumption has not been thoroughly tested with other chemicals. In addi-
tion, each of the studies used for estimating oral unit risk has deficien-
cies. Therefore, the unit risk estimate for inhalation exposure should be
considered approximate based on assumptions that have yet to be proven.
To estimate the risk corresponding to the concentration of 1 \*q
air, the effective dose in terms of mg/kg/day corresponding to
must first be estimated. Assuming an air Intake of 20 mVday
(ICRP, 1975) and a 40% absorption rate by inhalation for humans (as recom-
mended in this document), this effective dose can be estimated for a 70 kg
human:
air
ms/dav x °-40 x
x 10~3
= 1.14 x 10~4 mg/kg/day = 7.98 x 10~3 mg/day
A-24
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which 1s 4.0 times the unit Intake for oral exposure (1 yg/a. = 2xlO~s
mg/day). The unit risk for Inhalation 1s then estimated from the oral
studies by multiplying the oral unit risk by 4.0. Based on the range of
recommended risks (based upon present Information and current understanding
of cardnogenesls) derived from the four oral data sets, and using the
linearized multistage model with the dose per body surface area conversion
across species, and with the exponent k=3 1n the adjustment for partial
lifetime study, the upper limit estimate of unit risk for Inhalation
exposure ranges from 1.2xlO~6 to 1.4xlO~4 with a geometric mean of
1.5xlO~s. As a measure of uncertainty, all models and adjustments were
considered (see Table 9). The resulting upper limit unit risk estimate for
Inhalation exposure ranged from 2.3xlO~7 to 1.4xlO~«.
Because of the uncertainties 1n both the qualitative and quantitative
aspects of risk assessment, the actual cancer risks may be lower than the
best unit risks presented above, which should be regarded only as plausible
upper-limits. The unit risk estimates are calculated using a dose-response
extrapolation model which 1s linear at low doses. This low-dose linearity
1s based on mutageniclty studies and on some similarities between mutagene-
s1s and cardnogenesls. Since the results on the mutageniclty of CCK are
Inconclusive, the selected extrapolation model may be Inappropriate, and
hence the unit risk estimates are uncertain.
MULTIPLE EXPOSURE SITUATIONS
The above Information provides recommended route-specific cancer risk
estimates associated with exposure to given units of CC1 . These
estimates may be conservative due to the choice of the multistage model for
dose extrapolation and the various adjustment factors. Nevertheless, unit
A-27
-------
risks for cancer presented above are defined for Independent water and air
exposures 1n that their computation assumes 100% of the Insult is via the
stated route.
When exposure 1s by both oral and Inhalation routes, an add1t1v1ty
assumption can be used to calculate the risk associated with the concurrent
exposures. It 1s a general recommendation to use the add1t1v1ty assumption
which 1s made since the available data on CC1. are limited and do not
allow the presentation of a defensible alternative. As new Information
becomes available, other alternatives should be considered. Here the
add1t1v1ty assumption 1s that the risk associated with exposure to a given
chemical via two routes concurrently 1s roughly the sum of the risks asso-
ciated with each Independent route-specific exposure. Since Interactions
between the concurrent routes of Intake cannot be quantified, uncertainty
surrounds the resulting risk estimate that 1s derived from the concurrent
risks.
In applying the assumption of add1t1v1ty, the risks rather than the
doses associated with each route are added, but the mere summation of these
risks 1s presently justifiable only when doses are low enough that no Inter-
action occurs between the two routes. Furthermore, the amounts of
1 pg/a. and 1 yg/m3 are concentrations 1n water and air, respec-
tively, not doses. The dose can be estimated by assuming consumption of
2 a water/day over the lifetime. Thus, the dally dose corresponding to a
concentration of 1 yg/a water would be 2 I/day x 1 pg/ft =
2 pg/day.
COMPARISON OF RISK ESTIMATES FOR VARIOUS CARCINOGENS
The carcinogenic risk from exposure to CC1 1s compared to the risk
from exposure to other potential carcinogens 1n Table 10. For comparison
A-28
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purposes, the risks are based on lifetime esposure to 1 mg/kg/day. These
estimates are derived from various studies on humans and animals, for oral
and Inhalation exposure. These estimates are derived with the linearized
multistage model with species adjustment using dose/body surface area and
adjustment for partial lifetime study by using the exponent k=3. Carbon
tetrachlorlde has a relatively low potency compared to the others 1n the
group.
A relative potency index, proposed by the Carcinogen Assessment Group,
is also presented in Table 10 for each chemical. This index represents the
risk posed by dally exposure to 1 mMol of carcinogen per kg body weight, and
thus allows comparison of risks from exposure to the same number of
molecules. The frequency distribution of the relative potency Indices,
rounded to the nearest order of magnitude, 1s shown in Figure 2.
SUMMARY/CONCLUSIONS
No single study was entirely adequate for risk estimation. Thus, the
unit risk estimate is based on the geometric mean of the individual unit
risk estimates from the four studies considered. The studies contained data
on three animal species: rats, mice and hamsters. From these data, the
recommended upper limit unit risk estimates (based upon present information
and current understanding of carcinogenesls) for ingestlon are in the range
of 3.1xlO~7 to 3.4xlO~s with a geometric mean of 3.7xlO~6. Using
these same oral data, unit risk estimates for Inhalation are in the range of
1.2xlO~6 to 1.4xlO~4 with a geometric mean of 1.5xlO~s. Since no
study was entirely adequate for risk assessment purposes, the geometric mean
of the upper confidence limits is preferred as the most plausible upper
limit estimate of unit risk.
A-33
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FIGURE 2
Histogram Representing the Frequency Distribution of the Potency Indices
of 54 Suspect Carcinogens Evaluated by the Carcinogen Assessment Group
*U.S. GOVERNMENT PRINTING OFFICE:! 988 -Sit8-158/ 87032
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