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www.epa.gov/iris
vvEPA
TOXICOLOGICAL REVIEW
OF
CARBON TETRACHLORIDE
(CAS No. 56-23-5)
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
May 2008
NOTICE
This document is an External Peer Review draft. This information is distributed solely for the
purpose of pre-dissemination peer review under applicable information quality guidelines. It has
not been formally disseminated by EPA. It does not represent and should not be construed to
represent any Agency determination or policy. It is being circulated for review of its technical
accuracy and science policy implications.
U.S. Environmental Protection Agency
Washington, DC
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DISCLAIMER
This document is a preliminary review draft for review purposes only. This information
is distributed solely for the purpose of pre-dissemination peer review under applicable
information quality guidelines. It has not been formally disseminated by EPA. It does not
represent and should not be construed to represent any Agency determination or policy. Mention
of trade names or commercial products does not constitute endorsement or recommendation for
use.
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CONTENTS —TOXICOLOGICAL REVIEW OF CARBON TETRACHLORIDE
(CAS No. 56-23-5)
DISCLAIMER ii
LIST OF TABLES vi
LIST OF TABLES vi
FOREWORD xiii
AUTHORS, CONTRIBUTORS, AND REVIEWERS xiv
1. INTRODUCTION 1
2. CHEMICAL AND PHYSICAL INFORMATION 3
3. TOXICOKINETICS 6
3.1. ABSORPTION 6
3.1.1. Oral Exposure 6
3.1.2. Inhalation Exposure 7
3.1.3. Dermal Exposure 7
3.2. DISTRIBUTION 8
3.2.1. Oral Exposure 8
3.2.2. Inhalation Exposure 8
3.2.3. Dermal Exposure 10
3.2.4. Lactational Transfer 10
3.3. METABOLISM 11
3.4. ELIMINATION 14
3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELS 17
4. HAZARD IDENTIFICATION 28
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL
CONTROLS 28
4.1.1. Oral Exposure 28
4.1.2. Inhalation Exposure 30
4.1.3. Dermal Exposure 38
4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
ANIMALS—ORAL AND INHALATION 38
4.2.1. Oral Exposure 39
4.2.2. Inhalation Exposure 47
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION. 64
4.3.1. Oral Exposure 64
4.3.2. Inhalation Exposure 66
4.4. OTHER DURATION- OR ENDPOINT-SPECIFIC STUDIES 68
4.4.1. Acute and Short-term Toxicity Data 68
4.4.2. Genotoxicity Studies 72
4.4.3. Neurotoxicity Studies 114
4.4.4. Immunotoxicity Studies 114
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4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF
ACTION 118
4.5.1. Metabolism Is Required for Toxicity 118
4.5.2. Role of Free Radicals 119
4.5.3. LipidPeroxidation 120
4.5.4. Depletion of Glutathione 124
4.5.5. Disruption of Calcium Homeostasis 126
4.5.6. Immunological and Inflammatory Effects 129
4.5.7. Changes in Gene Expression 132
4.5.8. Mechanisms of Kidney Toxicity 133
4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS 134
4.6.1. Oral Exposure 134
4.6.2. Inhalation Exposure 139
4.6.3. Mode of Action Information 144
4.7. EVALUATION OF CARCINOGENICITY 146
4.7.1. Summary of Overall Weight-of-Evidence 146
4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence 147
4.7.3. Mode of Action Information for Liver Tumors 152
4.7.4. Mode of Action Information for Pheochromocytomas 166
4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 166
4.8.1. Possible Childhood Susceptibility 167
4.8.2. Possible Effects of Aging 169
4.8.3. Possible Gender Differences 172
4.8.4. Nutritional Status 172
4.8.5. Disease Status 173
4.8.6. Exposure to Other Chemicals 174
5. DOSE-RESPONSE ASSESSMENTS 176
5.1. ORAL REFERENCE DOSE(RfD) 176
5.1.1. Choice of Principal Study and Critical Effect—with Rationale and
Justification 176
5.1.2. Methods of Analysis—Including Models 176
5.1.3. RfD Derivation—Including Application of Uncertainty Factors 182
5.1.4. RfD Comparison Information 185
5.1.5. Previous RfD Assessment 190
5.2. INHALATION REFERENCE CONCENTRATION (RfC) 190
5.2.1. Choice of Principal Study and Critical Effect—with Rationale and
Justification 190
5.2.2. Methods of Analysis—Including Models 193
5.2.3. RfC Derivation—Including Application of Uncertainty Factors 202
5.2.4. RfC Comparison Information 204
5.2.5. Previous RfC Assessment 210
5.3. UNCERTAINTIES IN THE ORAL REFERENCE DOSE AND INHALATION
REFERENCE CONCENTRATION 210
5.4. CANCER ASSESSMENT 214
5.4.1. Nonlinear Extrapolation Approach 215
5.4.2. Linear Extrapolation Approach 216
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5.4.3. Choosing an Extrapolation Approach for Assessing Cancer Risk 236
5.4.4. Uncertainties in Cancer Risk Values 239
5.4.5. Previous Cancer Assessment 245
6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF_HAZARD AND DOSE
RESPONSE 247
6.1. HUMAN HAZARD POTENTIAL 247
6.2. DOSE RESPONSE 249
6.2.1. Noncancer- Oral Exposure 249
6.2.2. Noncancer- Inhalation Exposure 251
6.2.3. Cancer 252
7. REFERENCES 260
APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS
AND DISPOSITION A-l
APPENDIX B. DOSE-RESPONSE MODELING FOR DERIVING THE RfD B-l
APPENDIX C. PBPK MODELING C-l
APPENDIX D. BENCHMARK DOSE MODELING FOR DERIVING THE RfC D-1
APPENDIX E. CANCER ASSESSMENT: BMD MODELING OUTPUTS FOR LOW-DOSE
LINEAR EXTRAPOLATION APPROACH E-l
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LIST OF TABLES
Table 3-1. AUC, Cmax, and Tmax in rat tissues following administration of 179 mg/kg carbon
tetrachloride by inhalation (1000 ppm for 2 hours), oral bolus dosing, or gastric
infusion over 2 hours 10
Table 3-2. Metabolic rate constants for hepatic microsomes in vitro 14
Table 3-3. Elimination half-life (ti/2) and apparent clearance of carbon tetrachloride from rat
tissues following administration of 179 mg/kg (1000 ppm, 2 hours) by inhalation,
oral bolus dosing, or gastric infusion over 2 hours 16
Table 3-4. Physiological parameters for the rat, monkey, and human PBPK models for
carbon tetrachloride 19
Table 3-5. Comparison of metabolism from in vitro and in vivo studies 22
Table 4-1. Mean of selected serum chemistry and hematology variables in relation to carbon
tetrachloride exposure in British chemical workers 33
Table 4-2. Urinalysis results in rats after 2-year exposure to carbon tetrachloride 55
Table 4-3. Incidence of selected nonneoplastic lesions in F344 rats exposed to carbon
tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week) 57
Table 4-4. Incidence of liver tumors in F344 rats exposed to carbon tetrachloride vapor
for 104 weeks (6 hours/day, 5 days/week) 58
Table 4-5. Incidence of selected nonneoplastic lesions in BDF1 mice exposed to carbon
tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week) 62
Table 4-6. Incidence of liver and adrenal tumors in BDF1 mice exposed to carbon
tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week) 63
Table 4-7. Hepatic toxicity in rats exposed to carbon tetrachloride by inhalation or by
equivalent oral dosing as bolus or 2-hour gastric infusion 72
Table 4-8. Genotoxicity studies of carbon tetrachloride in prokaryotic organisms 73
Table 4-9. Genotoxicity studies of carbon tetrachloride in non-mammalian eukaryotic
organisms 76
Table 4-10. Genotoxicity studies of carbon tetrachloride in mammalian cells in vitro 78
Table 4-11. Genotoxicity studies of carbon tetrachloride in mammalian cells in vivo 82
Table 4-12. Challenges in evaluating carbon tetrachloride genotoxicity 93
Table 4-13. Oral toxicity studies for carbon tetrachloride 135
Table 4-14. Inhalation toxicity studies for carbon tetrachloride 140
Table 4-15. Exposure levels for necrosis/degeneration and hyperplasia/regeneration in liver
following subchronic or chronic exposure to carbon tetrachloride by gavage or
inhalation 150
Table 4-16. Dose considerations of mechanistic studies of carbon tetrachloride 159
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Table 4-17. Temporal sequence and dose-response relationship for key events and liver
tumors in male and female F344 rats exposed to carbon tetrachloride vapor for
13 and 104 weeks (6 hours/day, 5 days/week) 163
Table 5-1. Serum enzyme data in male rats after 10- or 12-week exposure to carbon
tetrachloride 177
Table 5-2. Severity of liver lesions in male rats after 12-week exposure to carbon
tetrachloride 179
Table 5-3. Incidence of selected liver lesions in mice treated with carbon tetrachloride
for 90 days 179
Table 5-4. Nonneoplastic lesions (fatty change) in F344 rats exposed to carbon tetrachloride
vapor for 104 weeks (6 hours/day, 5 days/week) 194
Table 5-5. Comparisons of internal dose metrics predicted from PBPK rat models (Paustenbach
etal., 1988; Thrall etal., 2000) 197
Table 5-6. HEC values corresponding to BMDL values for incidence data for fatty changes of
the liver in male F344 rats 200
Table 5-7. HEC values corresponding to BMDL values for incidence data for fatty changes of
the liver in female F344 rats (high dose dropped) 200
Table 5-8. Incidence of liver tumors in F344 rats and BDF1 mice exposed to carbon
tetrachloride vapor for 104 weeks (6 hours/day, 5 day/week) 219
Table 5-9. Incidence of adrenal tumors (pheochromocytomas) in BDF1 mice exposed to carbon
tetrachloride vapor for 104 weeks (6 hours/day, 5 day/week) 219
Table 5-10. Internal dose metrics predicted from Fisher et al. (2004) and Thrall et al. (2000)
PBPK mouse models 223
Table 5-11. BMDL values for incidence data for liver tumors (adenoma plus carcinoma) in
female F344 rats and corresponding HEC andHED values 226
Table 5-12. BMDL values for incidence data for liver tumors (adenoma plus carcinoma) in
female F344 rats (high dose dropped) and corresponding HEC and HED values ...227
Table 5-13. BMDL values for incidence data for liver tumors (adenoma plus carcinoma) in
female BDF1 mice (high dose dropped) and corresponding HEC and HED
values 228
Table 5-14. BMDL values for incidence data for liver tumors (adenoma plus carcinoma) in
female BDF1 mice (2 highest doses dropped) and corresponding HEC and HED
values 229
Table 5-15. BMDL values for incidence data for liver tumors (adenoma plus carcinoma) in male
BDF1 mice (high dose dropped) and corresponding HEC and HED values 230
Table 5-16. BMDL values for incidence data for pheochromocytomas in female BDF1 mice and
corresponding HEC and HED values 231
Table 5-17. BMDL values for incidence data for pheochromocytomas in male BDF1 mice and
corresponding HEC and HED values 231
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Table 5-18. Summary of inhalation unit risk estimates using linear low-dose extrapolation
approach 234
Table 5-19. Summary of oral slope factor estimates using linear low-dose extrapolation approach
and route-to-route extrapolation 236
Table 5-20. Summary of uncertainty in the carbon tetrachloride cancer risk assessment 244
Table B-l. Serum enzyme data in male rats after 10- or 12-week exposure to carbon
tetrachloride B-l
Table B-2. Model predictions for changes in serum SDH levels (ITJ/mL) in male rats
exposed to carbon tetrachoride for 10 weeks B-2
Table B-3. Model predictions for changes in serum OCT levels (nmol CCVmL) in male rats
exposed to carbon tetrachloride for 10 weeks B-6
Table B-4. Model predictions for changes in serum ALT levels (ITJ/mL) in male rats
exposed to carbon tetrachloride for 10 weeks B-7
Table C-l. Comparison of predicted and observed values for selected parameters from
toxicokinetic data collected from rats and mice 48 hours post exposure to a 4-hour
nose-only inhalation exposure (20 ppm carbon tetrachloride) C-3
Table C-2. Parameter values for rat and human models C-5
Table C-3. Parameter values for mouse models C-6
Table C-4. Interspecies conversion factors based on MCA dose metric (VMAXC=0.04) C-9
Table C-5. Interspecies conversion factors based on MCA dose metric (VMAXC=0.65) C-10
Table C-6. Interspecies conversion factors based on MCA dose metric (VMAXC=1.49) C-l 1
Table C-7. Interspecies conversion factors based on MCA dose metric (VMAXC=1.70) C-12
Table C-8. Interspecies conversion factors based on MRAMKL dose metric
(VMAXC=0.04) C-13
Table C-9. Interspecies conversion factors based on MRAMKL dose metric
(VMAXC=0.65) C-15
Table C-10. Interspecies conversion factors based on MRAMKL dose metric
(VMAXC=1.49) C-17
Table C-l 1. Interspecies conversion factors based on MRAMKL dose metric
(VMAXC=1.70) C-19
Table C-12. Sensitive parameters (indicated with +) in the human model C-31
LIST OF FIGURES
Figure 2-1. Carbon tetrachloride 3
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Figure 3-1. Metabolic scheme for carbon tetrachloride 12
Figure 3-2. Two-compartment model for simulating gastrointestinal absorption of carbon
tetrachloride administered to mice as a single gavage dose in Emulphor
(Fisher et al., 2004) 26
Figure 4-1. Survival curves for male and female rats 54
Figure 4-2. Survival curves for male and female mice 60
Figure 4-3. Lipid peroxidation 121
Figure 4-4. Hypothesized carcinogenic mode of action 153
Figure 5-1. Liver toxicity: oral 187
Figure 5-2. Developmental toxicity: oral 188
Figure 5-3. Organ-specific oral RfDs 189
Figure 5-4. Process for analyzing animal bioassay data for deriving noncancer toxicity values
and cancer unit risks and slope factors using PBPK modeling 195
Figure 5-5. Internal dose metrics predicted by the PBPK rat model (Paustenbach et al., 1988;
Thrall et al., 2000) 198
Figure 5-6. Liver toxicity: inhalation 207
Figure 5-7. Kidney toxicity: inhalation 208
Figure 5-8. Organ-specific inhalation RfCs 209
Figure 5-9. Internal dose metrics predicted from the Fisher et al. (2004) and Thrall et al. (2000)
PBPK mouse models 224
Figure B-l. Power model fit to the SDH data of Bruckner etal. (1986) B-3
Figure C-l. Comparison of observed and predicted chamber carbon tetrachloride concentrations
in closed chamber studies conducted in rats C-l
Figure C-2. Comparison of observed and predicted chamber carbon tetrachloride concentrations
in closed chamber studies conducted in mice C-2
Figure C-3. Comparison of the actual versus predicted concentration of carbon tetrachloride in
the expired breath of humans exposed to 10 ppm of carbon tetrachloride for 180
minutes (data from Stewart et al., 1961) C-4
Figure C-4. Atmospheric clearance of carbon tetrachloride from gas uptake chambers containing
mice (initial concentrations about 50, 130, 450, or 1250 ppm) C-7
Figure C-5. Relationship between internal dose metric MCA (time-averaged arterial blood
concentration of carbon tetrachloride) and equivalent exposure concentration (EC,
left panel) and values for % delta for trend lines (right panel). VMAX=0.40
mg/hr/kg BW°-70 C-21
Figure C-6. Relationship between internal dose metric MCA (time-averaged arterial blood
concentration of carbon tetrachloride) and equivalent exposure concentration (EC,
left panel) and values for % delta for trend lines (right panel). VMAX=0.65
mg/hr/kg BW°-70 C-22
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Figure C-7. Relationship between internal dose metric MCA (time-averaged arterial blood
concentration of carbon tetrachloride) and equivalent exposure concentration (EC,
left panel) and values for % delta for trend lines (right panel). VMAX=1.49
mg/hr/kg BW°-70 C-23
Figure C-8. Relationship between internal dose metric MCA (time-averaged arterial blood
concentration of carbon tetrachloride) and equivalent exposure concentration (EC,
left panel) and values for % delta for trend lines (right panel). VMAX=1.70
mg/hr/kg BW°-70 C-24
Figure C-9. Relationship between internal dose metric MCA (time-averaged arterial blood
concentration of carbon tetrachloride) and equivalent rate of uptake from the GI tract
to liver (RGIL, left panel) and values for % delta for trend lines (right panel).
VMAX=0.40 mg/hr/kg BW°-70 C-25
Figure C-10. Relationship between internal dose metric MCA (time-averaged arterial blood
concentration of carbon tetrachloride) and equivalent rate of uptake from the GI tract
to liver (RGIL, left panel) and values for % delta for trend lines (right panel).
VMAX=0.65 mg/hr/kg BW°-70 C-26
Figure C-l 1. Relationship between internal dose metric MCA (time-averaged arterial blood
concentration of carbon tetrachloride) and equivalent rate of uptake from the GI tract
to liver (RGIL, left panel) and values for % delta for trend lines (right panel).
VMAX= 1.49 mg/hr/kg BW°-70 C-27
Figure C-12. Relationship between internal dose metric MCA (time-averaged arterial blood
concentration of carbon tetrachloride) and equivalent rate of uptake from the GI tract
to liver (RGIL, left panel) and values for % delta for trend lines (right panel).
VMAX=1.70 mg/hr/kg BW°-70 C-28
Figure C-13. Relationship between internal dose metric MRAMKL (mean rate of carbon
tetrachloride metabolism in the liver) and equivalent concentration (EC) and values
for % delta for trend lines C-29
Figure C-14. Relationship between internal dose metric MRAMKL (mean rate of carbon
tetrachloride metabolism in the liver) and equivalent rate of uptake from the GI tract
to liver (RGIL) and values for % delta for trend lines C-30
Figure C-l5. Standardized sensitivity coefficients for the MCA dose metric (average
concentration of carbon tetrachloride in blood, umol/L) simulated with the human
carbon tetrachloride PBPK model C-32
Figure C-l6. Standardized sensitivity coefficients for the MRAMKB dose metric (average rate of
metabolism of carbon tetrachloride umol/hr/kg body weight) simulated with the
human carbon tetrachloride PBPK mode C-33
Figure E-l. Histogram of the shape parameter E-36
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LIST OF ABBREVIATIONS AND ACRONYMS
ACGIH American Conference of Governmental Industrial Hygienists
ACSL Advanced Continuous Simulation Language
AIC Akaike's Information Criterion
ALP Alkaline phosphatase
ALT Alanine aminotransferase
AST Aspartate aminotransferase
ATSDR Agency for Toxic Substances and Disease Registry
AUC Area under the curve
BCF Bioconcentration factor
BMD Benchmark dose
BMDL Benchmark dose, 95% lower bound
BMDS Benchmark dose software
BMR Benchmark response
BrdU 5-Bromo-2'-deoxyuridine
BUN Blood urea nitrogen
BW Body weight
CASRN Chemical Abstracts Service Registry Number
CBZ N-benzyloxycarbonyl-valine-phenylalanine methyl ester
CC14 Carbon tetrachloride
CHO Chinese hamster ovary
CI Confidence interval
CITI Chemicals Inspection and Testing Institute
Cmax Maximum tissue concentration
CPN Chronic progressive nephropathy
CPK Creatine phosphokinase
CYP450 Cytochrome P450
DMSO Dimethyl sulfoxide
EPA Environmental Protection Agency
FEL Frank effect level
G6Pase Glucose-6-phosphatase
GCL y -Glutamylcysteine ligase
GD Gestational day
GDH Glutamate dehydrogenase
GGT y -Glutamyl transferase
GI Gastrointestinal
GSH Glutathione (reduced)
HA Hemagglutinin
HEC Human equivalent concentration
4-HNE 4-Hydroxynonenal
IFN- Y Interferon- y
Ig Immunoglobulin
i.p. Intraperitoneal
IRIS Integrated Risk Information System
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JBRC Japan Bioassay Research Center
Km Michaelis-Menten constant
LAP Leucine aminopeptidase
LDH Lactate dehydrogenase
LOAEL Lowest-observed-adverse-effect level
MCA mean arterial concentration
MCL mean liver concentration
MDA Malondialdehyde
MRAMKL Mean rate of metabolism in the liver
MN Micronucleus
MW Molecular weight
NCI National Cancer Institute
NHL Non-Hodgkin's lymphoma
NK Natural killer
NLM National Library of Medicine
NOAEL No-observed-adverse-effect level
NRC National Research Council
OCT Ornithine carbamoyl transferase
8-OHdG 8-hydroxy-2'-deoxyguanosine
OR Odds ratio
PBPD Physiologically based pharmacodynamic
PBPK Physiologically based pharmacokinetic
PFC Plaque-forming cell
PNMT Phenylethanolamine-N-methyltransferase
PND Postnatal day
POD Point of departure
RfC Reference concentration
RfD Reference dose
SAM S-adenosylmethionine
SCE Sister chromatid exchange
SD Standard deviation
SDH Sorbitol dehydrogenase
SMR Standardized mortality ratio
SOS Inducible DNA repair system
SRC Syracuse Research Corporation
ti/2 Half-life
TEA Total bile acids
TEARS Thiobarbituric acid-reactive substances
TGF Tumor growth factor
Tmax Time at which the maximum occurred
TNF- a Tumor necrosis factor a
UF Uncertainty factor
USD Unscheduled DNA synthesis
Vmax Maximum velocity of enzyme reaction
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FOREWORD
The purpose of this Toxicological Review is to provide scientific support and rationale
for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to carbon
tetrachloride. It is not intended to be a comprehensive treatise on the chemical or toxicological
nature of carbon tetrachloride.
The intent of Section 6, Major Conclusions in the Characterization of Hazard and Dose
Response, is to present the major conclusions reached in the derivation of the reference dose,
reference concentration, and cancer assessment, where applicable, and to characterize the overall
confidence in the quantitative and qualitative aspects of hazard and dose response by addressing
the quality of the data and related uncertainties. The discussion is intended to convey the
limitations of the assessment and to aid and guide the risk assessor in the ensuing steps of the
risk assessment process.
For other general information about this assessment or other questions relating to IRIS,
the reader is referred to EPA's IRIS Hotline at (202) 566-1676 (phone), (202) 566-1749 (fax), or
hotline.iris@epa.gov (email address).
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER/AUTHOR
Susan Rieth
National Center for Environmental Assessment
Office of Research and Development
Reeder Sams
National Center for Environmental Assessment
Office of Research and Development
AUTHORS
Mary Manibusan
Health Effects Division
Office of Pesticide Programs
Jennifer Jinot
National Center for Environmental Assessment
Office of Research and Development
Leonid Kopylev
National Center for Environmental Assessment
Office of Research and Development
Paul White
National Center for Environmental Assessment
Office of Research and Development
Paul Schlosser
National Center for Environmental Assessment
Office of Research and Development
Marc Odin
Environmental Science Center
Syracuse Research Corporation
Syracuse, NY
Gary Diamond
Environmental Science Center
Syracuse Research Corporation
Syracuse, NY
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Margaret Fransen
Environmental Science Center
Syracuse Research Corporation
Syracuse, NY
Julie Klotzbach
Environmental Science Center
Syracuse Research Corporation
Syracuse, NY
David Eastmond
Environmental Toxicology Graduate Program
University of California, Riverside
Riverside, CA
REVIEWERS
INTERNAL EPA REVIEWERS
Joyce Donohue, Office of Water/OST
Anthony DeAngelo, Office of Research and Development/NHEERL
Karen Hammerstrom, Office of Research and Development/NCEA
Genetic Toxicology
Larry Valcovic, Office of Research and Development/NCEA
YinTak Woo, Office of Prevention, Pesticides and Toxic Substances
Channa Keshava, Office of Research and Development/NCEA
Immunotoxicology
Andrew Rooney, Office of Research and Development/NCEA
PBPK
Rob Dewoskin, Office of Research and Development/NCEA
Marina Evans, Office of Research and Development/NHEERL
Mode of Action
YinTak Woo, Office of Prevention, Pesticides and Toxic Substances
Vicki Dellarco, Office of Pollution Prevention
Rita Schoeny, Office of Water/OST
Julie Du, Office of Water/OST
John Whalan, Office of Research and Development/NCEA
Danielle Devoney, Office of Research and Development/NCEA
Jean Zodrow, U.S. EPA Region 10
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EXTERNAL PEER REVIEWERS
Summaries of the external peer reviewers' comments and the disposition of their
recommendations are provided in Appendix A.
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1. INTRODUCTION
This document presents background information and justification for the Integrated Risk
Information System (IRIS) Summary of the hazard and dose-response assessment of carbon
tetrachloride. IRIS Summaries may include oral reference dose (RfD) and inhalation reference
concentration (RfC) values for chronic and other exposure durations, and a carcinogenicity
assessment.
The RfD and RfC, if derived, provide quantitative information for use in risk assessments
for health effects known or assumed to be produced through a nonlinear (presumed threshold)
mode of action. The RfD (expressed in units of mg/kg-day) is defined as an estimate (with
uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human
population (including sensitive subgroups) that is likely to be without an appreciable risk of
deleterious effects during a lifetime. The inhalation RfC (expressed in units of mg/m3) is
analogous to the oral RfD but provides a continuous inhalation exposure estimate. The inhalation
RfC considers toxic effects for both the respiratory system (portal of entry) and effects peripheral
to the respiratory system (extrarespiratory or systemic effects). Reference values are generally
derived for chronic exposures (up to a lifetime), but may also be derived for acute (<24 hours),
short-term (>24 hours up to 30 days), and subchronic (>30 days up to 10% of lifetime) exposure
durations, all of which are derived based on an assumption of continuous exposure throughout
the duration specified. Unless specified otherwise, the RfD and RfC are derived for chronic
exposure duration.
The carcinogenicity assessment provides information on the carcinogenic hazard
potential of the substance in question and quantitative estimates of risk from oral and inhalation
exposure may be derived. The information includes a weight-of-evidence judgment of the
likelihood that the agent is a human carcinogen and the conditions under which the carcinogenic
effects may be expressed. Quantitative risk estimates may be derived from the application of a
low-dose extrapolation procedure. If derived, the oral slope factor is an upper bound on the
estimate of risk per mg/kg-day of oral exposure. Similarly, a unit risk is an upper bound on the
estimate of risk per |ig/m3 air breathed.
Development of these hazard identification and dose-response assessments for carbon
tetrachloride has followed the general guidelines for risk assessment as set forth by the National
Research Council (NRC, 1983). EPA Guidelines and Risk Assessment Forum Technical Panel
Reports that may have been used in the development of this assessment include the following:
Guidelines for the Health Risk Assessment of Chemical Mixtures (U.S. EPA, 1986a), Guidelines
for Mutagenicity Risk Assessment (U.S. EPA, 1986b), Recommendations for and Documentation
of Biological Values for Use in Risk Assessment (U.S. EPA, 1988), Guidelines for
Developmental Toxicity Risk Assessment (U.S. EPA, 1991), Interim Policy for Particle Size and
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Limit Concentration Issues in Inhalation Toxicity (U.S. EPA, 1994a), Methods for Derivation of
Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA,
1994b), Use of the Benchmark Dose Approach in Health Risk Assessment (U.S. EPA, 1995),
Guidelines for Reproductive Toxicity Risk Assessment (U.S. EPA, 1996a), Guidelines for
Neurotoxicity Risk Assessment (U.S. EPA, 1998a), Science Policy Council Handbook: Risk
Characterization (U.S. EPA, 2000a), Benchmark Dose Technical Guidance Document (U.S.
EPA, 2000b), Supplementary Guidance for Conducting Health Risk Assessment of Chemical
Mixtures (U.S. EPA, 2000c), A Review of the Reference Dose and Reference Concentration
Processes (U.S. EPA, 2002), Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a),
Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens
(U.S. EPA, 2005b), Science Policy Council Handbook: Peer Review (U.S. EPA, 2006a), and A
Framework for Assessing Health Risks of Environmental Exposures to Children (U.S. EPA,
2006b).
The literature search strategy employed for this compound was based on the Chemical
Abstracts Service Registry Number (CASRN) and at least one common name. Any pertinent
scientific information submitted by the public to the IRIS Submission Desk was also considered
in the development of this document. The relevant literature was reviewed through December
2007.
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2. CHEMICAL AND PHYSICAL INFORMATION
Carbon tetrachloride is a colorless liquid with a sweetish odor (NLM, 2003; Lewis,
1997). Synonyms include tetrachloromethane and perchloromethane (NLM, 2003; O'Neil and
Smith, 2001). The chemical structure of carbon tetrachloride is shown in Figure 2-1. Selected
chemical and physical properties of carbon tetrachloride are listed below.
Cl
Cl —C —Cl
Cl
Figure 2-1. Carbon tetrachloride.
CAS number:
Molecular weight (MW):
Chemical formula:
Boiling point:
Melting point:
Vapor pressure:
Density:
Vapor density (air=l):
Water solubility:
Other solubility:
Partition coefficient:
Flash point:
Autoignition temperature:
Latent heat of vaporization:
Heat of fusion:
Critical temperature:
Critical pressure:
Viscosity:
Surface tension:
Henry's law constant:
OH reaction rate constant:
56-23-5 (Lide, 2000)
153.82 (O'Neil and Smith, 2001)
CC14 (O'Neil and Smith, 2001)
76.8 °C (NLM, 2003; Lide, 2000)
-23 °C (NLM, 2003; Lide, 2000)
1.15 x 102mmHgat25°C(NLM, 2003)
1.5940 g/mL at 20 °C (NLM, 2003; Lide, 2000)
5.32 (NLM, 2003; U.S. Coast Guard, 1999);
5.41 (O'Neil and Smith, 2001)
7.93 x 102 mg/L at 25 °C (NLM, 2003; Horvath, 1982)
Miscible with alcohol, benzene, chloroform, ether,
carbon disulfide, petroleum ether, oils (NLM, 2003;
O'Neil and Smith, 2001)
log Kow = 2.83 (NLM, 2003; Hansch et al., 1995)
Not flammable (NLM, 2003; U.S. Coast Guard, 1999)
>1000 °C (Holbrook, 1993)
1.959 x 105 J/kg (U.S. Coast Guard, 1999)
5.09 cal/g (NLM, 2003; U.S. Coast Guard, 1999)
556.35 °C (Daubert and Danner, 1995)
4.56 x 106 Pa (Daubert and Danner, 1995)
0.922 cp at 24 °C (U.S. Coast Guard, 1999)
0.027 N/m at 20 °C (U.S. Coast Guard, 1999)
2.76 x 1Q~2 atm m3/mol at 25 °C (NLM, 2003; Leighton
andCalo, 1981)
1.20 x 10~16 cm3/molecule second at 25 °C (NLM,
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2003; Atkinson, 1989)
Koc: 71 (NLM, 2003)
Bioconcentration factor (BCF): 3.2-7.4 (NLM, 2003; CITI, 1992)
Conversion factors: 1 mg/m3 = 0.16 ppm (25 °C)
1 ppm = 6.29 mg/m3 (25 °C)
In the United States, carbon tetrachloride is most commonly prepared by chlorinating
methane or by a chlorinating cleavage reaction with less than or equal to €3 hydrocarbons or
chlorinated hydrocarbons (Rossberg, 2002). Prior to the late 1950s, carbon tetrachloride was
produced primarily by carbon disulfide chlorination (NLM, 2003; Rossberg, 2002).
Carbon tetrachloride has been used as a dry-cleaning agent, fabric-spotting fluid, solvent,
reagent in chemical synthesis, fire extinguisher fluid, and grain fumigant (NLM, 2003; Holbrook,
1993), but its primary use was in chlorofluorocarbon (CFC) production (NLM, 2003; Rossberg,
2002). Since the mid-1970s, annual use and production has generally declined. The Consumer
Product Safety Commission banned the use of carbon tetrachloride in consumer products in the
1970s. Decline in the use of carbon tetrachloride also accompanied EPA's increased regulation
of the use of CFCs in propellants (a ban on CFCs in aerosol products went into effect in 1978),
and the adoption of the Montreal Protocol, an international agreement to reduce environmental
concentrations of ozone-depleting chemicals, which was implemented in the U.S. via Title VI of
the Clean Air Act Amendments of 1990 (ATSDR, 2003; Doherty, 2000; Holbrook, 1993). The
ban on production and import of carbon tetrachloride in developed countries, including the U.S.,
took effect on January 1, 1996. Excluded from the production and import ban is the manufacture
of a controlled substance that is subsequently transformed or destroyed and small amounts
exempted for essential laboratory and analytical uses (40 CFR Part 82; 72 Fed Reg 52332, Sept
13, 2007).
Production figures for carbon tetrachloride since the 1970s reflect the regulatory history
of the chemical. Carbon tetrachloride production peaked in the early 1970s, with annual U.S.
production exceeding one billion pounds. Production in the early 1990s had declined to
approximately 300 million pounds (Doherty, 2000). According to ATSDR (2005), manufacture
of carbon tetrachloride in the U.S. in the early 2000s was limited to one company (Vulcan
Materials Company) at two plants with a combined 130 million pound capacity; however, these
capacities were considered flexible because other chlorinated solvents are made using the same
equipment.
Historically, carbon tetrachloride was released into the environment predominantly
through direct emissions to air, with lower amounts discharged to soil and water (ATSDR,
2003). Carbon tetrachloride released to soil or water is expected to volatilize to air based on its
vapor pressure and Henry's Law constant (NLM, 2003). In air, carbon tetrachloride will exist as
a vapor, as indicated by its vapor pressure (NLM, 2003). The behavior of carbon tetrachloride in
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the atmosphere is the most important aspect of this chemical's environmental fate. Carbon
tetrachloride does not undergo photodegradation (Holbrook, 1993) or absorb light at wavelengths
found in the troposphere and hence does not undergo direct photolysis in that region of the
atmosphere (NLM, 2003). Carbon tetrachloride that remains in the troposphere eventually rises
into the stratosphere, where it is photolyzed by the shorter wavelength light (Molina and
Rowland, 1974). When carbon tetrachloride photolyzes in the stratosphere, the chlorine radicals
responsible for the destruction of atmospheric ozone are released.
In soil, carbon tetrachloride is expected to be highly mobile based on its Koc and is
expected to leach to lower soil horizons and groundwater (NLM, 2003). BCF values indicate
that carbon tetrachloride will not bioconcentrate appreciably in aquatic or marine organisms
(NLM, 2003). Carbon tetrachloride may biodegrade in soil or water under anaerobic conditions;
however, biodegradation of carbon tetrachloride under aerobic conditions does not occur readily
(NLM, 2003; U.S. EPA, 1996b; Semprini, 1995).
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3. TOXICOKINETICS
Carbon tetrachloride is rapidly absorbed by any route of exposure in humans and animals.
Once absorbed, it is widely distributed among tissues, especially those with high lipid content,
reaching peak concentrations in less than 1-6 hours, depending on exposure concentration or
dose. It is metabolized by the liver, lung, and other tissues. Carbon tetrachloride is rapidly
excreted, primarily in exhaled breath.
3.1. ABSORPTION
3.1.1. Oral Exposure
Carbon tetrachloride is readily absorbed through the gastrointestinal tract in humans and
animals. There is evidence of gastrointestinal absorption in humans based on reports of toxicity
following poisoning incidents (Ruprah et al., 1985; Gosselin et al., 1976; von Oettingen, 1964;
Stewart et al., 1963; Umiker and Pearce, 1953). In male Sprague-Dawley rats receiving gavage
bolus doses of approximately 18 or 180 mg/kg, peak concentrations of carbon tetrachloride were
detected in the liver within 1 minute and in the blood within 10 minutes (Sanzgiri et al., 1995;
Bruckner et al., 1990). Total absorption was reduced by 37-56% when the same doses were
administered by infusion over a 2-hour period. An oral dose of about 3200 mg/kg attained a
peak blood concentration in about 2 hours in rats (Marchand et al., 1970). After radiolabeled
carbon tetrachloride was injected into the duodenum of rats, at least 82% was absorbed based on
recoveries of label in exhaled air (Paul and Rubinstein, 1963).
Administration of carbon tetrachloride in a vehicle changes the rate and percentage of
gastrointestinal absorption. Peak blood concentrations were achieved within 3.5-6.0 minutes
after oral exposure in male Sprague-Dawley rats dosed with 25 mg/kg of neat (i.e., undiluted)
carbon tetrachloride (Kim et al., 1990a, b; Gillespie et al., 1990). Relative to the neat compound,
the initial rate of gastrointestinal absorption of 25 mg/kg of carbon tetrachloride was faster with
administration as a saturated solution in water or 0.25% aqueous Emulphora emulsion but slower
when administered in corn oil. Although the initial rate of absorption in the presence of corn oil
was relatively slow, the total percentage absorbed over 9 hours when administered in corn oil
(83.1%) exceeded the percent absorption for the neat compound (62.8%) and was comparable to
that for the 0.25% aqueous emulsion (85.4%). The highest percent absorption was obtained from
a water vehicle (91.9%). Pharmacokinetic data suggested that corn oil vehicle resulted in slower
absorption from the gastrointestinal tract and subsequently lower peak blood concentrations and
a Emulphor is a polyethoxylated vegetable oil used to incorporate volatile organic compounds
(VOCs) and other lipophilic compounds into aqueous solutions.
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delayed removal from the blood stream (Kim et al., 1990a).
3.1.2. Inhalation Exposure
Data from humans and animals suggest that carbon tetrachloride is rapidly absorbed
through the lungs, which is inferred from the rapid onset of symptoms of toxicity or detection of
carbon tetrachloride in blood or in exhaled air. In volunteers exposed to 10 ppm for 180
minutes, carbon tetrachloride was detectable in exhaled air within 15 minutes (Stewart et al.,
1961). Human subjects exposed to 60 mg/L (9600 ppm) or higher reported symptoms of toxicity
within the first minute of exposure; symptoms appeared after 3 minutes in subjects exposed to 30
mg/L (4800 ppm) (Lehmann and Schmidt-Kehl, 1936). After male Sprague-Dawley rats were
exposed at 100 or 1000 ppm, carbon tetrachloride was detected in arterial blood in the initial 5-
minute samples (Sanzgiri et al., 1995; Bruckner et al., 1990); blood levels rose during the 2-hour
exposure period to a near steady-state level. In dogs exposed to 5000 ppm of carbon
tetrachloride, blood levels reached a near steady-state level within 2 hours (von Oettingen et al.,
1950).
Lehmann and Schmidt-Kehl (1936) estimated that approximately 63% of inhaled carbon
tetrachloride vapor was absorbed by the lungs in human subjects exposed to "a few mg per liter."
In monkeys exposed to carbon tetrachloride at 46 ppm for periods between 2 and 5 hours, an
average of 30% of the total amount inhaled was absorbed, and the rate of absorption averaged
0.022 mg/kg-minute (McCollister et al., 1951). Rats that were exposed at 4000 ppm for 6 hours
had initial body burdens of approximately 14 mg of carbon tetrachloride and 257 ug of its
metabolite chloroform (Dambrauskas and Cornish, 1970). Initial body burdens in rats, mice, and
hamsters that were exposed to 20 ppm of carbon tetrachloride vapor for 4 hours were 7.7, 10.6,
and 4.0 mg/kg, respectively (Benson and Springer, 1999). In vitro experiments of carbon
tetrachloride indicated blood/air partition coefficients of 2.73-4.20 for human blood (Fisher et
al., 1997; Gargas et al., 1989) and 4.52 for rat blood (Gargas et al., 1986).
3.1.3. Dermal Exposure
Carbon tetrachloride is absorbed rapidly through the skin. The chemical was detected in
alveolar air within 10 minutes in human subjects who immersed their thumbs in neat liquid
(Stewart and Dodd, 1964). Animal studies have found similar results. Carbon tetrachloride was
detected in blood within 5 minutes of dermal application of neat liquid in guinea pigs (Jakobson
et al., 1982). The percutaneous absorption rate for carbon tetrachloride applied neat to the
abdominal skin of male ICR mice was estimated as 53.6 ± 9.3 nmoles/minute/cm2 (Tsuruta,
1975). Morgan et al. (1991) compared dermal absorption of carbon tetrachloride in rats when
applied neat or in aqueous solution. With neat application, maximum blood levels were reached
within 30 minutes, and approximately one quarter of the applied volume (0.54 mL) was absorbed
in a 24-hour period. With application in saturated aqueous solution, absorption was slower (peak
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blood levels were not attained until 10 hours after exposure), and a somewhat lower amount
(0.39 mL) was absorbed in 24 hours.
Dermal absorption of radiolabeled carbon tetrachloride vapor was low in monkeys
exposed to 485 or 1150 ppm for about 4 hours (McCollister et al., 1951). Blood concentrations
at the end of exposure were approximately equivalent to 0.012-0.03 mg carbon tetrachloride/100
g blood but were undetectable after 48 hours; concentrations in exhaled air were equivalent to
0.0008-0.003 mg carbon tetrachloride/L but were undetectable 120 hours later. The authors
concluded that, for whole-body exposures to carbon tetrachloride vapor, the dermally absorbed
fraction would be negligible.
3.2. DISTRIBUTION
3.2.1. Oral Exposure
No data are available for the distribution of carbon tetrachloride in humans. Animal
studies indicate that the largest fraction of an absorbed oral dose of carbon tetrachloride is
initially distributed to fat. After administration of about 3200 mg/kg to rats, peak levels of
radiolabeled carbon tetrachloride were observed after about 2 hours in blood, muscle, liver, and
brain and after 5.5 hours in fat (Marchand et al., 1970). Peak tissue levels of carbon tetrachloride
were similar in blood and muscle but were twice as high in the brain, 5 times higher in liver, and
50 times higher in fat. Similar results were obtained in rabbits treated with a low dose of carbon
tetrachloride (Fowler, 1969). Six hours after an oral dose of 1.6 mg/kg, recoveries of parent
compound totaled 787 ug/g in fat, 96 ug/g in liver, 20 ug/g in kidney, and 21 ug/g in muscle;
distributions of the carbon tetrachloride metabolites chloroform and hexachloroethane were
highest in fat and liver but were below 5 ug/g. Forty-eight hours after dosing, tissue
concentrations of the parent compound were 45 ug/g in fat, 3.8 ug/g in liver, and <1 ug/g in the
other tissues; chloroform was present at <1 ug/g in the four tissues, whereas hexachloroethane
was present at 6.8 ug/g in fat, 1 ug/g in liver, and <1 ug/g in other tissues.
3.2.2. Inhalation Exposure
A similar pattern of distribution has been found in animals exposed to carbon
tetrachloride by inhalation. Rats exposed to 4000 ppm for 6 hours showed the largest
concentrations of carbon tetrachloride in the fat (1674 ug/g), followed by the brain (407 ug/g),
kidney (233 ug/g), liver (136 ug/g), and blood (64 ug/g) (Dambrauskas and Cornish, 1970). The
liver also contained 10 ug/g of chloroform (as a carbon tetrachloride metabolite). Monkeys
exposed to 46 ppm of radiolabeled carbon tetrachloride vapor for 5 hours had the highest
concentration of label in fat, with decreasing amounts in the liver, bone marrow, blood, brain,
kidney, heart, spleen, muscle, lung, and bone (McCollister et al., 1951). The concentrations in
fat and liver were eightfold and threefold higher, respectively, than concentrations in blood.
Bergman (1983) followed the distribution of radiolabeled carbon tetrachloride by whole-
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body autoradiography in mice exposed by inhalation for 10 minutes and sacrificed at time points
up to 24 hours; sections were either processed at low temperatures to retain volatile radioactivity
(primarily parent compound), evaporated to retain only nonvolatile radioactivity (metabolites), or
evaporated and then extracted to retain only protein- and nucleic acid-bound radioactivity
(metabolites covalently bound to protein and nucleic acids). Immediately after exposure by
inhalation, high levels of volatile radioactivity were detectable in fat, bone marrow, and nervous
tissues (spinal cord and white matter of the brain). Nonvolatile and partly nonextractable
radioactivity was detected in the liver, kidney cortex, lung, bronchi, gastrointestinal mucosa
(especially in the glandular stomach, colon, and rectum), nasal mucosa, salivary glands, vaginal
and uterine mucosa, and, interstitially, in the testis; nonvolatile radioactivity was also detected in
urine and bile. The distribution pattern of volatile carbon tetrachloride and its nonvolatile
metabolites was similar 30 minutes after exposure. Volatile radioactivity was detectable at
relatively high levels in the nervous system at 4 hours and in fat at 8 hours but not at 24 hours.
The pattern of labeling in the liver demonstrated a centrilobular concentration. Bergman (1983)
reported a good correlation between nonextractable radioactivity and published tissue
concentrations of cytochrome (CYP) P450.
Sanzgiri et al. (1997) compared the tissue distribution of carbon tetrachloride
administered by inhalation (1000 ppm for 2 hours) and the equivalent oral dose (179 mg/kg)
given as a single bolus dose or gastric infusion over 2 hours. Table 3-1 shows area under the
curve (AUC) for the 24-hour monitoring period, the maximum tissue concentrations (Cmax), and
the times (Tmax) at which the maxima occurred. Maximal tissue concentrations were reached
quickest by gavage dosing, followed by inhalation and then gastric infusion. By all routes,
attainment of maximal levels was slower in fat than in other tissues. Maximal levels in fat were
considerably in excess of the maximal levels in other tissues, regardless of route of exposure.
Among tissues other than fat, distribution kinetics of carbon tetrachloride were generally similar
for the different tissues, except that maximal levels were higher and attained more quickly in the
liver than in other tissues following bolus oral administration.
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Table 3-1. AUC, Cmax, and Tmax in rat tissues following administration of
179 mg/kg carbon tetrachloride by inhalation (1000 ppm for 2 hours),
oral bolus dosing, or gastric infusion over 2 hours
Tissue
Liver
Kidney
Lung
Brain
Fat
Heart
Muscle
Spleen
Inhalation
AUC
(Hgxminute/
mL)
2823
3064
2952
3255
230,699
2571
3248
2035
^max
G»g/g)
20
25
24
28
1506
18
18
13
T
A max
(min)
30
30
30
30
240
30
30
30
Oral bolus
AUC
(ugxminute/
mL)
1023
3029
2908
4223
235,471
2747
4117
4096
^max
G»g/g)
58
14
10
15
246
10
7
12
T
A max
(min)
1
5
15
15
120
5
60
5
Gastric infusion
AUC
(Hgxminute/
mL)
149
800
2842
2683
165,983
1900
2164
1660
^max
G»g/g)
0.5
4
6
10
179
8
10
6
T
Amax
(min)
120
120
180
150
360
120
150
150
Source: Sanzgirietal., 1997.
Benson et al. (2001) compared the initial and delayed tissue distribution of inhaled
carbon tetrachloride in rats, mice, and hamsters exposed to 20 ppm of radiolabeled carbon
tetrachloride for 4 hours. Immediately after exposure, the percentage of the initial body burden
present in major tissues was 30% in rats and hamsters and 40% in mice; the highest proportion at
that time was in the liver of mice and hamsters and in the fat in rats. Two days later, the liver
contained the highest amount in all three species. The results in rats reflect the initial lipophilic
distribution of carbon tetrachloride and the subsequent accumulation in the liver.
3.2.3. Dermal Exposure
Few data are available regarding tissue concentrations of carbon tetrachloride following
dermal exposure. One study of guinea pigs given topical application of carbon tetrachloride
found that blood concentrations of the chemical increased during the first half hour of exposure
but then declined to about 25% of peak levels despite continued exposure over a 6-hour period
(Jakob son et al., 1982).
3.2.4. Lactational Transfer
Fisher et al. (1997) experimentally derived a human milk/blood partition coefficient of
3.26 for carbon tetrachloride, which would suggest a potential sensitive subpopulation of nursing
infants based on the possibility of lactational transfer.
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3.3. METABOLISM
Carbon tetrachloride is metabolized in the body, primarily by the liver, but also in the
kidney, lung, and other tissues containing CYP450. The percent of a given dose that is
metabolized varies with dose, as discussed in Section 3.4.
The metabolism of carbon tetrachloride has been extensively studied in in vivo and in
vitro mammalian systems. Based on available data, a proposed metabolic scheme for carbon
tetrachloride is illustrated in Figure 3-1. There is considerable evidence that the initial step in
biotransformation of carbon tetrachloride is reductive dehalogenation: reductive cleavage of one
carbon-chlorine bond to yield chloride ion and the trichloromethyl radical (Reinke and Janzen,
1991; Tomasi et al., 1987; McCay et al., 1984; Mico and Pohl, 1983; Slater, 1982; Poyer et al.,
1980, 1978; Lai etal., 1979).
The initial reaction step is catalyzed by an NADPH-dependent CYP450 that is inducible
by phenobarbital or ethanol (Castillo et al., 1992; Noguchi et al., 1982a; Sipes et al., 1977). In
humans and animals, CYP2E1 is the primary enzyme involved with carbon tetrachloride
bioactivation, while CYP3A may be involved under high exposure conditions (Zangar et al.,
2000; Raucy et al., 1993). As demonstrated in studies with CYP2E1 genetic knockout mice, this
enzyme is required for the development of hepatotoxicity (as measured by elevated liver
enzymes and liver histopathology) in mice exposed to carbon tetrachloride (Wong et al., 1998).
The fate of the trichloromethyl radical is dependent on the availability of oxygen and
includes several alternative pathways for anaerobic or aerobic conditions. Anaerobically, the
trichloromethyl radical may dimerize to form hexachloroethane, which has been detected in
animal tissues (Uehleke et al., 1973; Fowler, 1969). Addition of a proton and an electron to the
radical results in the formation of chloroform (CHCb), which has been detected in exposed rats
and rabbits (Reynolds et al., 1984; Ahr et al., 1980; Glende et al., 1976; Uehleke et al., 1973;
Dambrauskas and Cornish, 1970; Fowler, 1969). The trichloromethyl radical can undergo
further reductive dehalogenation catalyzed by CYP450 to form dichlorocarbene (:CC\2), which
can bind irreversibly to tissue components or react with water to form formyl chloride (HCOC1),
which decomposes to carbon monoxide (Galelli and Castro, 1998; Pohl et al., 1984; Ahr et al.,
1980; Wolf et al., 1977). The trichloromethyl radical can bind directly to microsomal lipids and
proteins (Fanelli and Castro, 1995; Ansari et al., 1982; Villarruel et al., 1977), as well as the
heme portion of CYP450.
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Carbon Tetrachloride
CCL
R-CCI
adduct
•O-O-CCI
Trichloromethyl
peroxy radical
Lipid Peroxidation
R-CO+
2HCI
Cysteine
Oxothiazolidine
carboxylic acid
-e- ( CYP450
•ecu
Phosgene
(carbonyl chloride)
2HCI
GSCSG
Diglutathionyl
dithiocarbonate
Dimerization CI3CCCI3
Hexachloroethane
Carbon
monoxide
H20
2HCI
CO2
Carbon
dioxide
Figure 3-1. Metabolic scheme for carbon tetrachloride.
CYP450, usually CYP2E1, but also CYP3A; R = acceptor molecule, such as protein or lipid.
Source: ACGIH, 2001.
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Aerobically, the trichloromethyl radical can be trapped by oxygen to form the
trichloromethyl peroxy radical, which can bind to tissue proteins (Galelli and Castro, 1998;
Packer et al., 1978) or decompose to form phosgene (COC12) (Pohl et al., 1984) and an
electrophilic form of chlorine (Pohl et al., 1984). The trichloromethyl peroxy radical is the
primary initiator of lipid peroxidation that occurs from exposure to carbon tetrachloride (Boll et
al., 2001a; McCay et al., 1984; Rao and Recknagel, 1969). Carbon dioxide is generated by the
hydrolytic cleavage of phosgene (Shah et al., 1979). Phosgene may also be conjugated to
reduced glutathione (GSH) to form diglutathionyl dithiocarbonate or to cysteine to form
oxothiazolidine carboxylic acid (U.S. EPA, 200la).
Continued exposure to carbon tetrachloride has been shown to temporarily reduce its
initial toxicity in rat studies (Glende, 1972). This phenomenon is related to the loss of CYP450
content (suicide inactivation), which has also been observed in treated rats (de Toranzo et al.,
1978), resulting from the formation of reactive intermediates, such as the trichloromethyl radical
(Fernandez et al., 1982; Noguchi et al., 1982b; de Groot and Haas, 1981; Glende, 1972). Under
anaerobic conditions, heme tetrapyrrolic structures of the human or rat CYP450 enzymes are
destroyed in a process that follows pseudo first-order kinetics (Manno et al., 1992, 1988).
Although the fast and slow half-lives for the two species are similar (3.2 and 28.9 minutes for the
rat and 4.0 and 29.8 minutes for the human), inactivation is more severe in the rat, with 1
molecule of rat CYP450 enzyme lost for every 26 molecules of substrate metabolized, compared
with a loss of 1 molecule of human enzyme for every 196 molecules of substrate processed
(Manno et al., 1992, 1988). Based on the studies by Manno et al. (1992, 1988), deactivation of
the CYP450 enzyme is reduced more in rats than in humans. Accordingly, enzyme deactivation
is less significant in humans than in rats.
As demonstrated qualitatively by the distribution of nonvolatile radioactivity
(metabolites) in the autoradiography study by Bergman (1983) and quantitatively in other in vivo
assays (see Section 3.2), carbon tetrachloride is metabolized in many tissues throughout the body
but most significantly in the liver. The amount of carbon tetrachloride metabolized in a given
tissue is related to the CYP450 content of the tissue (Bergman, 1983; Villarruel et al., 1977). In
the liver, the greatest accumulation of carbon tetrachloride metabolites occurs in the centrilobular
region, which has high CYP450 levels (Bergman, 1983).
Zangar et al. (2000) measured carbon tetrachloride metabolic rate constants for human
and animal hepatic microsomal preparations in vitro (Table 3-2). Results suggest that the
metabolic rate in humans is more similar to the rate in rats than in other rodent species.
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Table 3-2. Metabolic rate constants for hepatic microsomes in vitro
Species
Human
Rat
Mouse
Hamster
K.,'
OiM)
56.8
59.1
29.3
30.2
V b
~ max
(nmol/minute/mg protein)
2.26
3.1
2.86
4.1
aKm= Michaelis-Menten constant.
b Vmax = Maximum velocity of enzyme reaction.
Source: Zangar et al., 2000.
Metabolism of carbon tetrachloride can be induced by chemicals that increase the
expression of CYP2E1 or CYP3A (see Section 4.8.6. for further discussion).
3.4. ELIMINATION
In humans and animals exposed to carbon tetrachloride by any route, the unmetabolized
parent compound is excreted in exhaled air. Additionally, animal studies show that volatile
metabolites are released in exhaled air, whereas nonvolatile metabolites are excreted in feces and
to a lesser degree in urine.
Six hours after an attempted suicide by ingestion of an unknown amount of carbon
tetrachloride in a mixture with methanol, the concentration of carbon tetrachloride in expired air
was -2500 ug/L and declined to -120 ug/L after 1 day and to -1 ug/L after 20 days (Stewart et
al., 1963). In a worker acutely exposed to mixed solvent vapors, the concentration of carbon
tetrachloride in alveolar air declined from an initial value of-4000 ppm to -0.003 ppm after 15
days (Stewart et al., 1965). Human subjects (n=6) who inhaled carbon tetrachloride vapor at 10
ppm for 3 hours had concentrations in expired air of 1 ppm 15 minutes postexposure and about
0.28 ppm 5 hours postexposure (Stewart et al., 1961). Approximately 33% of the absorbed dose
was excreted in exhaled air within 1 hour in human subjects who inhaled radiochlorine-labeled
carbon tetrachloride in a single breath (Morgan et al., 1970). Following dermal exposure to neat
carbon tetrachloride, excretion into alveolar air was detectable within 10 minutes in three human
subjects (Stewart and Dodd, 1964). Concentrations in alveolar air ranged from 0.11-0.83 ppm
by the end of a 30-minute exposure, peaking 30 minutes postexposure and beginning to decline 1
hour postexposure; after 5 hours, the concentrations were 0.12-0.14 ppm. Using a physiological
four compartment model, Sato and Nakajima (1987) calculated that 93% of inhaled carbon
tetrachloride vapor was removed unchanged via the lungs (assuming an alveolar ventilation rate
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of 336 L/hour), while 7% was cleared metabolically in humans.
Animal studies evaluated elimination of carbon tetrachloride following oral or inhalation
exposures. In rats receiving equivalent doses by inhalation or bolus gavage, terminal elimination
half-lives (ti/2) were about 4 hours (Bruckner et al., 1990).
Reynolds et al. (1984) evaluated elimination parameters during a 24-hour period in rats
exposed by gavage to [14C]-carbon tetrachloride at doses ranging from 15 to 4000 mg/kg. At the
low dose of 15 mg/kg, 19% of the administered dose was eliminated in exhaled air as the parent
compound, 28% as CC>2 (accounting for 83% of metabolites), and 0.11% as chloroform (0.3% of
metabolites); 2.9% of metabolites remained bound in the liver, while 2.7% were excreted in
urine and 11% in feces. At doses >600 mg/kg, >76% of the administered dose was exhaled as
parent compound, <2% was exhaled as CC>2 (accounting for 50-60% of metabolites), and
<0.40% as chloroform (11-19% of metabolites); 2-4% of metabolites remained bound in the
liver, while 3-9% of metabolites were excreted in urine and 7-30% in feces. At 15 mg/kg, peak
exhalation rates were 11, 2.6, and 0.02 umoles/hour per kg for CC>2, parent compound, and
chloroform, respectively; the timing of the peak rates occurred in 15-45 minutes, within 2 hours,
and slightly after 2 hours for CC>2, parent compound, and chloroform, respectively. At 4000
mg/kg, peak exhalation rates were 88, 1550, and 3.40 umoles/hour per kg for CC>2, parent
compound, and chloroform, respectively; compared with the lower doses, peak rates were
achieved more quickly for CO2 than for parent compound and chloroform.
In monkeys exposed by inhalation to radiolabeled carbon tetrachloride at 46 ppm for 5.75
hours, 21% of the total absorbed dose was eliminated during the initial 18 hours as carbon
dioxide and parent compound or volatile metabolite (McCollister et al., 1951). Within 75 days
following the end of exposure, 11% was eliminated as carbon dioxide and 40% as parent
compound or volatile metabolite in exhaled breath. The majority of urinary and fecal excretion
occurred in the 5 days following exposure; a small amount of label was detectable in feces after
12 days and in urine after 15 days.
In rats exposed to radiolabeled carbon tetrachloride vapor by inhalation at 100 or 1000
ppm for 8 hours for 1-5 days, no fecal elimination was detected (Page and Carlson, 1994); in
comparison, intravenous administration resulted in biliary and nonbiliary fecal elimination that
was less than 1% of the administered dose.
Sanzgiri et al. (1997) measured the elimination of carbon tetrachloride from tissues in rats
exposed to 1000 ppm via inhalation for 2 hours or the equivalent oral dose of 179 mg/kg
administered as a single bolus dose or by intragastric infusion over 2 hours. The half-lives of
elimination from various tissues are given in Table 3-3. Elimination half-lives were slowest for
fat, which is poorly perfused, but similar for the other tissues.
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Table 3-3. Elimination half-life (ti/i) and apparent clearance of carbon
tetrachloride from rat tissues following administration of 179 mg/kg
(1000 ppm, 2 hours) by inhalation, oral bolus dosing, or gastric infusion
over 2 hours
Tissue
Liver
Kidney
Lung
Brain
Fat
Heart
Muscle
Spleen
Inhalation
tl/2
(minutes)
249
204
226
248
665
274
218
273
Clearance
(mL/minute/kg)
63
58
61
55
0.8
70
55
88
Oral bolus
tl/2
(minutes)
323
278
442
313
780
490
649
472
Clearance
(mL/minute/kg)
175
59
62
42
0.8
65
43
44
Gastric infusion
tl/2
(minutes)
269
190
249
250
358
216
262
208
Clearance
(mL/minute/kg)
1198
224
72
67
1
94
83
108
Source: Sanzgirietal., 1997.
Benson et al. (2001) compared elimination parameters in rats, mice, and hamsters
exposed to 20 ppm of [14C]-labeled carbon tetrachloride for 4 hours. In the 48 hours following
exposure, approximately 65-83% of the initial body burdens were eliminated as volatile organic
compounds or CC>2 in exhaled air. Elimination half-times were 7.4, 8.8, and 5.3 hours for CC>2
and 4.3, 0.8, and 3.6 hours for the volatile organic compounds for rats, mice, and hamsters,
respectively. Elimination in the urine and feces combined constituted less than 10% of the initial
body burden in rats and less than 20% in mice and hamsters.
Paustenbach et al. (1986a, b) and Veng-Pedersen et al. (1987) compared the
pharmacokinetics of carbon tetrachloride in rats exposed to 100 ppm of carbon tetrachloride
vapor in scenarios that mirror human work schedules: 8 hours/day for 5 days or 11.5 hours/day
for 4 days. Additional groups were exposed on a 2-week schedule for 5 or 3 additional days,
respectively. Following 2 weeks of exposure at 8 hours/day, 45% of the label was eliminated in
exhaled air (-97.5% as parent compound) and 48% in feces. Exposure at 11.5 hours/day for 2
weeks resulted in elimination of 32% in exhaled air and 62% in feces. On either schedule, less
than 8% was excreted in urine and less than 2% was exhaled as CC>2. The elimination profiles
for exhaled air were biphasic. For the 2-week 8 hours/day and 11.5 hours/day schedules,
elimination of the parent compound in breath had half-lives for the fast and slow phases of 96
and 455 minutes and 89 and 568 minutes, respectively. Similarly, half-lives for the fast and slow
phases of elimination of CO2 were 305 and 829 minutes on the 8-hour schedule and 455 and
1824 minutes on the 11.5-hour schedule. The authors concluded that the longer daily exposure
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placed more of the absorbed dose into the poorly-perfused fat compartment. The half-lives of
elimination in urine and feces for the 2-week exposures were 1066 and 3700 minutes for the 8-
hour schedule and 944 and 6700 minutes for the 11.5-hour schedule.
Rats or gerbils intraperitoneally injected with carbon tetrachloride at a dose of 128-159
mg/kg eliminated 80-90% in exhaled air as carbon tetrachloride and less than 1% as CC>2
(Young and Mehendale, 1989).
3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELS
Physiologically based pharmacokinetic (PBPK) models are available for carbon
tetrachloride for exposures by the inhalation route (Yoon et al., 2007; Fisher et al., 2004; Thrall
et al., 2000; Benson and Springer, 1999; Evans et al., 1994; Paustenbach et al., 1988, 1987;
Gargas et al., 1986) and the oral route (Fisher et al., 2004; Semino et al., 1997; Gallo et al.,
1993). The models are based primarily on experimental data from rodents. However, Thrall et
al. (2000) derived in vivo metabolic rate constants for humans based on human in vitro metabolic
constants and in vivo/in vitro ratios for metabolic rate constants derived from animals (also
reported in Benson and Springer, 1999).
Gargas et al. (1986)
Gargas et al. (1986) used the PBPK model framework developed by Ramsey and
Andersen (1984) for styrene, together with experimentally derived tissue partition coefficients
and gas uptake data for carbon tetrachloride, to estimate in vivo metabolic rate constants for
carbon tetrachloride in rats. The model comprises a series of differential equations describing
the rate of carbon tetrachloride entry into and exit from a series of body compartments, including
liver, fat, muscle, and viscera (richly perfused organs), as well as arterial and venous blood.
Gas-uptake data were obtained in a closed recirculated exposure system. Partition coefficients
were experimentally derived in a series of in vitro studies using the tissues of interest. The
researchers found that the uptake kinetics of carbon tetrachloride were adequately described by
modeling metabolism of the compound as a single saturable process with Vmax of 0.92 jimol/hour
(0.14 mg/hour) and Km of 1.62 |imol/L (0.25 mg/L).
Paustenbach et al. (1988, 1987)
Paustenbach et al. (1988, 1987) developed a four-compartment PBPK model (similar in
structure to Gargas et al., 1986) to describe the disposition of carbon tetrachloride absorbed
during inhalation, based on the framework developed by Ramsey and Andersen (1984) and the
parameter values reported by Gargas et al. (1986). Metabolism, assumed to occur only in the
liver compartment, was modeled as a single, saturable pathway. Metabolites were apportioned
into three separate storage compartments, leading to elimination in the exhaled breath, urine, and
feces, respectively. In order to accommodate the observed biphasic elimination of CC>2,
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equations were included to allow for the interconversion from the urinary or fecal pools to
production of CO2. The model also included a time delay of 23.5 hours for fecal excretion to
account for the observed delay in appearance of radioactivity in the feces. Parameter values
needed to run the model included partition coefficients (determined experimentally by vial
equilibration), biochemical constants for carbon tetrachloride metabolism (determined
experimentally by gas uptake studies), and physiological parameters (estimated from the
literature, from previous pharmacokinetic studies, and from the process of fitting the carbon
tetrachloride data during model development). Selection of the optimal parameters for fat
compartment volume, blood flow, Vmax, and Km were determined by the quality of the visual fit
of the model predictions with laboratory data; sensitivity analysis indicated that changes to other
parameters had little effect on the simulation and were thus not subject to optimization. Model
parameters are presented in Table 3-4. Calibration of the rat model was done using data for
Sprague-Dawley rats exposed to 100 ppm of carbon tetrachloride for 4, 5, 7, or 10 exposures as
reported in Paustenbach et al. (1986a, b). The model reliably predicted values for the following
experimental parameters: concentration of [14C] activity in adipose tissue, concentration of [14C]-
carbon tetrachloride in the expired breath, concentration of 14CC>2 in the expired breath, activity
of 14C in the urine, and activity of [14C] in the feces.
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Table 3-4. Physiological parameters for the rat, monkey, and human
PBPK models for carbon tetrachloride
Parameter
Cardiac output (liters blood/hour)
Alveolar ventilation (liters air/hour)
Rat (0.42 kg)
8.15
7.91
Monkey (4.6 kg)
41.2a
43. 9a
Human (70 kg)
256a
254a
Tissue volumes (percent of total)
Liver
Fat
Muscle
Richly perfused organs
4
8
74
5
4
10
72
5
4
20C
62
5
Blood flow (percent of total)
Liver
Fat
Muscle
Richly perfused organs
Metabolism
Vmax (mg/hour)
Km (mg/liter)
25
4
20
51
25
4
20
51
25
6
18
51
0.35
0.25
1.9T
0.25b
12.72a
0.25b
a Allometrically scaled from the rat data using (body weight)0 75.
b Assumed to be the same as in rats.
0 Tissue volume for fat in humans is shown in Table 2 of Paustenbach et al. (1998) as 10%; however, the
text of this paper states that the rat model was scaled up to humans using a fat compartment of 20% of
body weight. The 20% value was determined to be correct.
Source: Paustenbach et al., 1988.
In order to extend the model to monkeys and humans, the rat model was scaled up,
resulting in models for monkeys and humans that were used to predict the concentration of
carbon tetrachloride in expired air. For both the monkey model and the human model, cardiac
output, alveolar ventilation, and Vmax were estimated using (body weight)0'75, and the Km was
assumed to be the same as for the rat. The rat model was scaled to monkeys, using a body
weight (BW) of 4.6 kg, a body fat estimate of 10%, and fat perfusion of 4% of cardiac output;
other parameters were assumed to be the same as in the rat. The monkey model was calibrated
by using the data of McCollister et al. (1951), which measured the concentration of expired
carbon tetrachloride after a 370-minute exposure to 50 ppm. The time course was accurately
predicted, except for long periods (>240 hours) after exposure in which the model predicted
lower concentrations than were demonstrated experimentally. The study authors suggested that
small amounts (0.4%) of carbon tetrachloride may have been converted into C2C16, which has a
much longer half-life in adipose tissue and would account for the slow elimination of small
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amounts of radiolabel. The rat model was scaled up to humans by using an experimentally
measured human blood:air partition coefficient, a body weight of 70 kg, and a fat compartment
of 20% BW. Model simulations of concentration of carbon tetrachloride in expired air over time
were compared with the data of Stewart et al. (1961), who exposed human volunteers to 49 ppm
of carbon tetrachloride for 70 minutes or to 10 ppm of carbon tetrachloride for 180 minutes;
there was good agreement between the model simulation and the measured results. The model
predicted that at concentrations up to 100 ppm, the rat, monkey, and human metabolize carbon
tetrachloride in a similar manner. Because of physiological differences, the models predicted
species differences in carbon tetrachloride accumulation in fat. The rat PBPK model accurately
described carbon tetrachloride concentrations in adipose tissue where no significant day-to-day
accumulation in fat or blood was observed following repeated exposure to 100 ppm for 8 or
11.5 hours/day, whereas the human model predicted day-to-day increases in carbon tetrachloride
in fat following inhalation exposure to 5 ppm for 8 hours/day.
Thrall et al. (2000); Benson and Springer (1999)
Thrall et al. (2000) and Benson and Springer (1999) expanded the rat PBPK model of
Paustenbach et al. (1988) to include parameters for the mouse and the hamster. The mouse and
hamster models consist of five compartments identical to the rat model (lung, liver, fat, muscle,
and richly perfused tissues). Metabolism is still assumed to occur only in the liver and is
modeled by a single, saturable pathway that results in products that may be eliminated in the
expired air, urine, or feces. For the mouse, tissue:air partition coefficients were assumed to be
equal to those for the rat, with the exception of the blood:air coefficient, which was measured
with the vial equilibration technique. Tissue:blood partition coefficients were then calculated by
dividing the tissue:air coefficients by the blood:air coefficients. Metabolic rate constants (i.e.,
Vmax and Km) were measured in whole animals by using gas uptake studies with a closed
recirculating chamber; in comparison to the rat, the mouse has a slightly higher capacity (higher
in vivo Vmax) and lower affinity (higher in vivo Km) for metabolizing carbon tetrachloride.
Physiological parameters for the mouse model were based on published values in the literature
(Andersen et al., 1987). Model predictions for initial body burden, exhaled carbon tetrachloride,
and exhaled CC>2 were compared with data collected over a 48-hour period following a 4-hour
inhalation exposure to 20 ppm of [14 C]-carbon tetrachloride (data from a personal
communication and not presented in the manuscript); ratios of predicted/observed concentrations
ranged from 1.1 to 1.4, indicating very good agreement among observed and predicted values.
For the hamster, coefficients for blood:air, muscle:air, liverair, and fatair were determined by
the vial equilibration technique. Hamster tissue:air partition coefficients did not differ
significantly from those of the rat. Tissue:blood partition coefficients were then calculated by
dividing the tissue:air coefficients by the blood:air coefficients. Metabolic rate constants (i.e.,
Vmax and Km) were measured in whole animals by using gas uptake studies with a closed
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recirculating chamber; in comparison to the rat, the hamster has a higher capacity (higher in vivo
Vmax) and lower affinity (higher in vivo Km) for metabolizing carbon tetrachloride. Physiological
parameters for the hamster model were those used in the rat model. The hamster model tended
to overpredict uptake from exposure at low concentrations and underpredict the uptake from
exposure at high concentrations (1800 ppm exposure). Model predictions for initial body
burden, exhaled carbon tetrachloride, and exhaled CC>2 were compared with data collected over a
48-hour period following a 4-hour inhalation exposure to 20 ppm of [14 C]-carbon tetrachloride
(data from a personal communication and not presented in the manuscript); ratios of
predicted/observed concentrations ranged from 0.6 to 2.1 for all three species, and from 0.6 to
1.4 for rats and mice (see Appendix C for a comparison of model predictions and
experimentally-derived data).
Thrall et al. (2000) and Benson and Springer (1999) used in vitro data on metabolism of
carbon tetrachloride by human liver microsomes (Zangar et al., 2000), together with in vitro and
in vivo rodent data, to estimate the in vivo human metabolic rate constants. The calculation is
presented in Table 3-5. Briefly, in vivo Vmax/Km ratios were obtained for the rodent species after
Vmax was normalized for milligrams of liver protein. The corresponding in vitro Vmax/Km ratios
were calculated in the same manner, and the in vivo/in vitro ratios were calculated, giving values
of 1.40, 1.01, and 1.70 for the rat, mouse, and hamster, respectively. As these values were very
similar, a human in vivo Vmax/Km ratio of 1.37 was estimated as the mean of the rat, mouse, and
hamster ratios. Because the human Km in vitro is similar to that of the rat, the in vivo human Km
was assumed to be the same as that of the rat, allowing for the calculation of a human in vivo
Vmax of 29.15 mg/hour. The researchers used the new value for Vmax in the human PBPK model
of Paustenbach et al. (1988), with other parameters remaining as previously described, and
compared it with the human data of Stewart et al. (1961). The model simulation of expired
carbon tetrachloride levels provided good agreement with the experimental data, particularly at
longer periods postexposure (see Appendix C for a comparison of model predictions and
experimentally-derived data).
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Table 3-5. Comparison of metabolism from in vitro and in vivo studies
BW (kg)
Liver weight (g)a
mg protein/g liverb
In vivo Vmax (mg/hour/kg BW)C
In vivo Vmax (mg/hour)d
In vivo Vmax (mg/hour/mg protein)
In vivo Km (mg/L)°
In vivo Vmax/Km
In vitro Vmax (umol/hour/mg protein)6
In vitro Km (umol/L)e
In vitro Vmax/Km (L/hour/mg protein)
Ratio (in vivo/in vitro)
Rat
0.25
10
13.8
0.4
0.15
l.lxKT3
0.25
4.4 xKT3
0.186
59.1
3.15xKT3
1.4
Mouse
0.025
1
21.9
0.79
5.97xlO~2
2.7 xKT3
0.46
5.9xKT3
0.1712
29.3
5.86xlO"3
1.01
Hamster
0.15
6
17.8
6.39
1.69
0.016f
1.14
0.014s
0.246
30.2
8.14xlO"3
1.7
Human
70
2800
12.8
1.49
29.15
S.lxKT4
0.25h
3.2xKT3
0.135
56.8
2.38xlO"3
1.37
a Calculated as 4% of body weight.
b From Reitz et al. (1996), except hamster, which was estimated as the mean of mouse and rat.
0 Rodents: experimentally measured; humans: calculated (see text).
dRodents: calculated from in vivo Vmax (mg/hour/kg BW) using BW°7 (personal communication; email
dated 9/5/2006, from Dr. Karla Thrall, Pacific Northwest National Laboratory, to Susan Rieth, U.S.
EPA); humans: calculated (see text).
e Data from Zangar et al. (2000).
f Corrected from value of 0.16 in Table 5 of Thrall et al. (2000) (personal communication; email dated
9/5/2006, from Dr. Karla Thrall, Pacific Northwest National Laboratory, to Susan Rieth, U.S. EPA).
8 Corrected from value of 0.14 in Table 5 of Thrall et al. (2000) (personal communication; email dated
9/5/2006, from Dr. Karla Thrall, Pacific Northwest National Laboratory, to Susan Rieth, U.S. EPA).
h Assumed to be equal to the rat based on in vitro Km comparisons.
'Calculated as the average of the rat, mouse, and hamster in vivo/in vitro ratios.
Source: Thrall etal., 2000.
Other Extensions of the Paustenbach et al. (1988) Model
Several other models have been developed as extensions of the Paustenbach et al. (1988)
model. Semino et al. (1997) added a gastrointestinal compartment to the inhalation model of
Paustenbach et al. (1988) to describe uptake of carbon tetrachloride administered by a single
gavage dose at levels of 25 or 50 mg/kg in corn oil or at a dose of 17.25 mg/kg in 0.25% aqueous
Emulphor to male F344 rats. The gastrointestinal compartment was divided into a series of
sequential absorption subcompartments, each characterized by three parameters: emptying time,
absorption rate constant (describing input to the portal circulation), and bioavailability. These
parameters were optimized against the experimental results for concentrations of parent carbon
tetrachloride in arterial blood or exhaled air. The number of subcompartments was also varied;
nine subcompartments were needed to obtain a good fit of this data set for delivery by corn oil
gavage, whereas only six or seven subcompartments were needed for aqueous Emulphor. The
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model simulated the higher rapid initial uptake with the aqueous vehicle and the more pulsatile
absorption profile observed from corn oil delivery following a single exposure. The
subcompartments were not intended to correspond to actual anatomic segments of the
gastrointestinal tract, and the values generated for oral uptake parameters were not intended to
represent true physiological measurements.
Thrall and Kenny (1996) adapted the PBPK model of Paustenbach et al. (1988) to
simulate an intravenous route of exposure in the male F344 rat. The model added equations to
simulate the introduction of carbon tetrachloride into the mixed venous blood pool.
Physiological parameters were adjusted to account for the smaller body size of F344 rats
compared with Sprague-Dawley rats, using data from Arms and Travis (1988). The model was
used to predict the concentration of carbon tetrachloride in the expired air after a single
intravenous exposure and was compared with real-time monitoring data from rats given a single
injection of carbon tetrachloride at 0.6 or 1.5 mg/kg BW. With the exception of underestimation
of the initial peak in exhalation, the model predictions were in good agreement with the
measured data.
El-Masri et al. (1996) modified the PBPK rat model of Paustenbach et al. (1988) to
include a linked physiologically based pharmacodynamic (PBPD) model for hepatocellular
injury and animal death. First-order rate constants governed simulated cell mitosis and birth,
injury (due to carbon tetrachloride-induced vacuolation and incidental injury), repair, delay of
mitosis and repair, cell death, and phagocytosis by macrophages. Animal death was simulated to
occur when >50% of hepatocytes died. The data of Lockard et al. (1983) was used to visually
optimize the PBPD model rate constants.
Other models of carbon tetrachloride disposition were developed independent of Thrall et
al. (2000) or Paustenbach et al. (1988) and are discussed further below.
Galloetal. (1993)
Gallo et al. (1993) developed a physiological and systems analysis hybrid
pharmacokinetic model for blood concentration-time data obtained during intravenous or oral
administration. The systems analysis procedure was based on a disposition-decomposition
method for deriving an absorption input function for each regimen. Equations were derived,
representing input into the blood, distribution to and from the blood to the peripheral tissues, and
elimination from the blood, allowing for the estimation of arterial and venous blood
concentrations but not concentrations in target tissues. Experimental data were collected for
male Sprague-Dawley rats given a single oral dose of 25 mg/kg in one of four ways (undiluted,
in corn oil, as an emulsion in 0.25% Emulphor, or in water) and from other rats receiving the
same dose in aqueous polyethylene glycol 400 as an intravenous bolus injection. A hybrid
model that combined model parameters available in the literature with the absorption input
functions obtained by systems analysis adequately described the observed blood concentration-
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time data. The same model using conventional first-order absorption inputs provided less
accurate fits to the data. Both the standard model and the hybrid model overestimated the initial
concentration in blood for the oral or intravenous routes.
Evans et al. (1994)
Evans et al. (1994) developed a PBPK model for carbon tetrachloride in rats based on the
Ramsey and Andersen (1984) model for styrene. Flow-limited compartments for liver, fat, and
rapidly and slowly perfused tissues were connected by arterial and venous blood. The
investigators derived partition coefficients from blood, liver, fat, and muscle samples of naive
male Fischer-344 rats. Physiological parameter values were taken from the literature.
Metabolism of carbon tetrachloride was constrained to the liver and described by Michaelis-
Menten kinetics. Vmax and Km were estimated by optimizing the model to closed-chamber gas
uptake data, generated by the study authors, for adult male Fischer-344 rats exposed to 25, 100,
250, or 1000 ppm carbon tetrachloride for 6 hours. The resulting Vmaxc and Km values were
0.37 mg/hr/kg and 1.3 mg/L, respectively. The predicted decreases in chamber carbon
tetrachloride concentrations were very similar to observations for all exposure levels and time
points. A sensitivity analysis was performed on all of the model parameters. For the low
exposure (25 ppm), the blood:air partition coefficient (5.49), followed by the fatblood partition
coefficient (51.3) and fat tissue volume (8%), had the greatest effects on simulated chamber
concentration. However, the fatblood partition coefficient and fat tissue volume dominated the
decrease in chamber concentration in the 1000-ppm exposure.
The model of Evans et al. (1994) was applied to examine the effect of methanol
pretreatment of rats (10,000 ppm for 6 hours) at 24 and 48 hours prior to 6-hour closed-chamber
carbon tetrachloride exposures of 25, 100, 250, or 1000 ppm (Evans and Simmons, 1996). Vmaxc
was optimized against the gas uptake data from all exposure levels. A Vmaxc value of
0.48 mg/hr/kg for the 24-hour methanol pretreatment group resulted in very good agreement of
the predicted and observed chamber concentrations at all exposure levels, indicating that
induction of carbon tetrachloride metabolism could be adequately simulated. Good agreement
was also achieved between predicted and observed chamber concentrations at all exposure levels
for the 48-hour methanol pretreatment group. The estimated Vmaxc value of 0.18 mg/hr/kg,
which was very close to the carbon tetrachloride-only value of 0.11 mg/hr/kg (from Evans et al.,
1994), indicated that the effect of methanol induction of carbon tetrachloride metabolism had
practically ceased by this time.
Yoshida et al. (1999)
Yoshida et al. (1999) used a classical compartment pharmacokinetic model to derive rates
of absorption of carbon tetrachloride in rats exposed at low concentrations in a closed chamber
system. Experimentally, rats were exposed at initial concentrations between 10 and 1000 ppb,
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and the changes in chamber concentrations were measured over 6 hours. The model, like the
experimental system, had three compartments: a tank containing barium chloride to capture the
compound, the exposure chamber into which the compound was injected, and the rat. The model
consisted of three differential equations describing the apparent volumes of distribution for the
three compartments. The model included single rate constants for inhalation, exhalation, and
metabolic elimination processes in the rat. The rate constant for exhalation was determined to be
higher than that for elimination. Metabolic elimination of carbon tetrachloride was estimated as
0.53 |imol/hour/kg at 10 ppm.
Andersen et al. (1996) developed a model to describe the anaerobic in vitro metabolism
of carbon tetrachloride in a two-phase, closed-chamber headspace vial. Data were generated
from hepatic microsomal preparations from fed or fasting adult male F344 rats. Partition
coefficients were experimentally derived for phosphate buffer to air and microsomal suspension
to air. In addition to the Michaelis-Menten kinetic constants, a first-order loss-rate constant was
required for accurate fitting of the model. The model described the kinetics of anaerobic
transformation of carbon tetrachloride to chloroform.
Fisher et al. (2004)
Fisher et al. (2004) developed a PBPK model for simultaneous exposures to carbon
tetrachloride and tetrachloroethylene in mice. The model contained a 4-compartment structure
(liver, fat, and richly and slowly perfused tissues) for carbon tetrachloride based on the Ramsey
and Andersen (1984) model and tetrachloroethylene based on a modified form of the Gearhart et
al. (1993) model. Absorption from the gastrointestinal tract was simulated as a 2-compartment,
3-parameter model (Figure 3-2). Rate coefficients were estimated by visually fitting these
parameters to blood data following single oral gavage doses of carbon tetrachloride (20, 50, or
100 mg/kg carbon tetrachloride alone, 10 or 100 mg/kg tetrachloroethylene alone, and 1, 5, 20,
50, or 100 mg/kg carbon tetrachloride followed 1 hour later by 10 or 100 mg/kg
tetrachloroethylene; all oral bolus doses were administered in aqueous emulsion vehicle).
Metabolism for both chemicals was represented as a saturable Michaelis-Menten pathway in the
liver only. Carbon tetrachloride-induced suicide inhibition was modeled with a second-order
inhibition constant, KD, which was used to calculate the loss of metabolic capacity (Vmaxc) for
both carbon tetrachloride and tetrachloroethylene. A submodel for trichloroacetic acid, the sole
metabolite of tetrachloroethylene oxidation, was included in which the rate of trichloroacetic
acid production in the liver was equal to the rate of tetrachloroethylene metabolism. Four
compartments for trichloroacetic acid were included: liver, kidney, and rapidly and slowly
perfused tissues.
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Liver
K1
K3
Oral dose
C1
K2
C1
Figure 3-2. Two-compartment model for simulating gastrointestinal absorption of
carbon tetrachloride administered to mice as a single gavage dose in Emulphor
(Fisher et al., 2004).
Values for rate coefficients were derived by visual fit of model predictions to observed blood carbon
tetrachloride kinetics in mice. The value for Kl was dose dependent (0.4 hr"1 for 20 mg/kg dose and 10 hr"1
for 50 and 100 mg/kg doses). Values for K2 and K3 were 2 and 0.05 hr"1, respectively.
Carbon tetrachloride partition coefficients for blood, liver, fat, and muscle (representing
slowly perfused tissue) were determined by the study authors (Fisher et al., 2004) using the vial
equilibration method of Gargas et al. (1989). Partition coefficients for tetrachloroethylene and
trichloroacetic acid were taken from Gearhart et al. (1993) and Abbas and Fisher (1997),
respectively. Physiological constants for mice were taken from the compendium of Brown et al.
(1997). Data for carbon tetrachloride gas uptake exposures of 130 ppm (Thrall et al., 2000) and
50, 450, or 1250 ppm (Fisher et al., 2004) in male B6C3F1 mice were used to optimize Vmaxc
and Km, resulting in values of 1 mg/hr/kg0'75 and 0.3 mg/L, respectively. For tetrachloroethylene,
gas uptake-derived Vmaxc and Km values of 6 mg/hr/kg0'75 and 3 mg/L, respectively, were taken
from Gearhart et al. (1993). Oral absorption rate constants for carbon tetrachloride and
tetrachloroethylene were visually fitted from the blood concentration data for each chemical.
The value for KD was estimated by optimization of the model to blood trichloroacetic acid
concentrations following co-exposures of tetrachloroethylene and carbon tetrachloride via oral
bolus dosing. See Appendix C for a summary of parameter values used in the Fisher et al.
(2004) model.
Yoonetal. (2007)
Yoon et al. (2007) explored the effect of extrahepatic carbon tetrachloride metabolism in
rats and humans on estimates of hepatic Vmax and Km. The investigators developed an 8-
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compartment, flow-limited PBPK model, including compartments for lung, liver, brain, kidney,
fat, rapidly and slowly perfused tissues, and the gastrointestinal tract. Physiological parameter
values were taken from the literature (Delp et al., 1991; U.S. EPA, 2000e; Brown et al., 1997).
Tissue partition coefficients for the rat were taken from Evans et al. (1994). Gas uptake data
from closed-chamber experiments (Evans et al., 1994) were used to estimate values of Vmax (0.13
mg/kr/kg0'75) and Km (1.10 mg/L) in the liver. Data for estimation of extrahepatic metabolism
were generated from in vitro CYP2E1-mediated microsomal metabolism of carbon tetrachloride
in liver, brain, skin, kidney, lung, and fat. No metabolic activity was detected in the fat, brain, or
skin. Estimates of extrahepatic in vivo metabolism in the lung and kidney were modeled as the
liver Vmax adjusted by the tissue volume-normalized ratio of VmaX: ;n vitro tissue / VmaX; invitro iiver.
Simulations of open-chamber inhalation exposures (ATSDR, 2003) were used to compare the
effect of the presence or absence of extrahepatic metabolism on the following dose metrics:
carbon tetrachloride blood Cmax, AUC for carbon tetrachloride in blood over a 24-hour period,
total carbon tetrachloride metabolized in the body, and carbon tetrachloride metabolized in the
liver (normalized for liver volume). The presence or absence of extrahepatic metabolism did not
affect either the estimation of hepatic Vmax and Km or the predicted dose metrics. The proportion
of liver metabolism estimated for the lung and kidney was quite small, 0.79 and 0.93%,
respectively, based on the microsomal studies. This resulted in identical values for Vmax and all
of the examined dose metrics, and similar values for Km (1.10 and 1.14 mg/L without and with
extrahepatic metabolism, respectively).
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4. HAZARD IDENTIFICATION
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL
CONTROLS
4.1.1. Oral Exposure
4.1.1.1. Human Poisoning Incidents
Case reports reveal that individuals acutely poisoned with carbon tetrachloride can
exhibit gastrointestinal toxicity (nausea, vomiting, diarrhea, and abdominal pain) and
neurotoxicity (drowsiness, coma, or seizures) (Ruprah et al., 1985; Stewart et al., 1963; New et
al., 1962). Hepatic involvement has been demonstrated by liver enlargement and significant
elevations in serum enzyme (>100-fold increases in alanine aminotransferase [ALT] or aspartate
aminotransferase [AST]) and bilirubin levels (Ruprah et al., 1985; Stewart et al., 1963). One of
two individuals who received one 5 mL dose of carbon tetrachloride as an antihelmintic
exhibited microscopic pathology in the liver (granular degeneration); a third person who received
a second dose 2 weeks later had fatty degeneration of the liver, as well as swelling of the
proximal tubules of the kidney (Docherty and Nicholls, 1923; Docherty and Burgess, 1922).
Renal effects (oliguria and increases in blood urea nitrogen [BUN]) may occur within 1-8 days
of acute exposure (New et al., 1962). Umiker and Pearce (1953) noted that, after ingestion of
fatal doses of carbon tetrachloride, the primary cause of death during the first week was hepatic
injury and afterwards was renal insufficiency. Pulmonary lesions (lung congestion, edema,
bronchopneumonia, fibrinous exudate, alveolar epithelial proliferation) appear about 8 days after
exposure and have been considered to be secondary effects of renal failure (Umiker and Pearce,
1953). Human fatalities from ingestion of carbon tetrachloride may occur with ingestion of
amounts as low as 2-3 mL (45-68 mg/kg, based on the reference adult BW of 70 kg) (Ruprah et
al., 1985; Gosselin et al., 1976).
4.1.1.2. Epidemiology Studies
Epidemiological studies have investigated possible associations between oral exposure to
carbon tetrachloride and a variety of adverse birth outcomes (Croen et al., 1997; Bove et al.,
1995, 1992a, b); however, because of multiple chemical exposures and insufficient power, these
studies are considered limited and insufficient to determine whether there is an association
between carbon tetrachloride exposure and adverse birth outcomes.
Bove etal. (1995, 1992a,b)
Bove et al. (1995, 1992a,b) evaluated the relationship between contamination of public
drinking water with organic compounds (including carbon tetrachloride) and adverse birth
outcomes in a cross-sectional study of births in four counties in northern New Jersey. The study
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population consisted of registered live births and fetal deaths occurring from January 1, 1985, to
December 31, 1988, in 75 towns (selected from a total of 146 in the four counties), where most
residents were served by public water systems and most births occurred in the state. After
exclusion of plural births and fetal deaths from therapeutic abortions or chromosomal anomalies,
the subjects totaled 80,938 live births and 594 fetal deaths. Fetal death certificates available for
all fetal deaths with gestational age greater than 20 weeks and the New Jersey Birth Defects
Registry were used to gather data on a selection of adverse birth outcomes. A comparison group
of 52,334 births that had no adverse outcomes was included in the study to evaluate categorical
outcomes. Exposure to organic compounds was estimated from the monthly records of the 49
water companies serving the study population (water samples were collected at the tap). In
addition to carbon tetrachloride, other contaminants in the drinking water included
trihalomethanes (primarily chloroform), 1,2-dichloroethane, dichloroethylenes, 1,1,1-
trichloroethane, trichloroethylene, tetrachloroethylene, and benzene. Levels of all of these
compounds, other than benzene, were higher than carbon tetrachloride; levels of trihalomethanes
were 20- to 40-fold higher. For carbon tetrachloride, the exposed population was defined in one
of two ways: those with exposure to >1 ppb in the drinking water or those with any detectable
amount in the drinking water. In either case, the size of the comparison group with exposure to
carbon tetrachloride was small: 357 births where levels >1 ppb were detected and 1993 births
where any carbon tetrachloride was detected.
Carbon tetrachloride and the other contaminants were evaluated for effects on 13 selected
birth outcomes (birth weight among term births, term low birth weight, small for gestational age,
preterm birth, very low birth weight, fetal death, central nervous system defects, neural tube
defects, oral clefts, major cardiac defects, ventricular septal defects, all cardiac defects, and all
surveillance defects). Odds ratios (ORs) for an association between each outcome and carbon
tetrachloride were calculated as the ratio of the risk of the outcome in the population with the
specified exposure (either > nd or >1 ppb) to the risk in the population without the specified
exposure. ORs were adjusted for maternal age, race, education, parity, adequacy of prenatal
care, and sex of the child. Positive associations were found between exposure to carbon
tetrachloride in drinking water at concentrations above 1 ppb and certain adverse outcomes: low
birth weight (<2.5 kg) among term births (OR = 2.26, 95% confidence interval [CI]: 1.41-3.60)
and small (at or below their race-, sex- and gestation week-specific tenth percentile weight) for
gestational age (OR = 1.34, 95% CI: 1.02-1.80). These same effects, however, were also
significantly associated with exposure to trihalomethanes, which were present in much higher
levels and were much more prevalent in the drinking water supply (i.e., had a much larger
exposed population and number of cases). While there was a statistically positive association
between exposure to >1 ppb carbon tetrachloride and occurrence of neural tube defects (OR =
5.39, 95% CI: 1.31-22.2), it was based on only two cases in the exposed population. Using a
criterion of OR >1.5 without consideration of CIs, the authors also reported positive relationships
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between carbon tetrachloride and several of the other adverse outcomes tested. However, the
reliability of these purported relationships is suspect without statistical support. Maternal
interviews were conducted for a sample of the study population to collect more detailed
information about potential confounders, such as maternal occupational exposures, smoking,
medical histories, height, and gestational weight gain. Adjustment for these additional risk
factors had no appreciable effect on the results for carbon tetrachloride. Interpretation of the
study results is hindered by simultaneous exposure to multiple chemicals in the drinking water,
the relatively small number of people exposed to carbon tetrachloride and the low levels to
which they were exposed, and the limited characterization of exposure to carbon tetrachloride
(and the other chemicals tested).
Croenetal, 1997
Croen et al. (1997) used data from two population-based case-control studies to
determine whether maternal residential proximity to hazardous waste sites increased the risk for
certain birth defects in California. Residential histories were obtained by interviews with
mothers of infants with specific birth defects (neural tube defects [507 cases] in one study; heart
defects [201 cases] and oral cleft defects [439 cases] in the other) and mothers of controls in the
two studies (517 for the neural tube study and 455 for the other two defects). Information was
collected on 764 inactive waste sites as well as 105 National Priority List sites. Multivariate
analysis was used to control for potential confounding effects, such as maternal race/ethnicity,
income, and education. The study found no increased risk of heart defects or oral cleft defects
among offspring of mothers living near a waste site containing carbon tetrachloride, but this
study had little power to detect effects. Odds ratios for neural tube defects associated with
carbon tetrachloride were not provided.
4.1.2. Inhalation Exposure
4.1.2.1. Acute Exposure Incidents
The initial acute effects of carbon tetrachloride in humans exposed by inhalation are
similar to effects reported from humans exposed orally (Stewart et al., 1965; New et al., 1962;
Norwood et al., 1950); these effects include gastrointestinal symptoms (nausea and vomiting,
diarrhea, abdominal pain), hepatic effects (elevated serum AST, mild jaundice, and, in fatal
cases, necrosis of the liver), and neurological effects (headache, dizziness, weakness). As with
acute oral exposure, inhalation exposure causes renal effects (oliguria, elevated BUN) that
appear 1-8 days after exposure, with an average delay of 4 days (New et al., 1962). Renal
histopathological effects in fatal cases include nephrosis, degeneration, and interstitial
inflammation of the kidney (Norwood et al., 1950). Pulmonary edema is a secondary
consequence of renal insufficiency (Umiker and Pearce, 1953; Norwood et al., 1950). Some case
reports noted that a high intake of alcohol, which can enhance carbon tetrachloride toxicity, was
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common among the patients intoxicated by inhaled carbon tetrachloride (New et al., 1962;
Norwood et al., 1950).
Lehmann and Schmidt-Kehl (1936) described the neurological symptoms in humans
exposed briefly to carbon tetrachloride vapor at concentrations of 20 mg/L (3200 ppm) and
above. No effect was observed following exposure at 20 mg/L for 5 minutes. Exposure at
30 mg/L (4800 ppm) for 2.5 minutes resulted in slight drowsiness after 5 minutes. Exposures at
40 mg/L (6400 ppm) for 3 minutes resulted in tremor and drowsiness, followed by staggering.
The highest tested exposure, 89 mg/L (14,100 ppm) for 0.8 minutes, resulted in loss of
consciousness. Stewart et al. (1961) reported no adverse effects (such as nausea or dizziness) in
male volunteers exposed to carbon tetrachloride vapor at 49 ppm for 70 minutes or 10-11 ppm
for 180 minutes.
4.1.2.2. Epidemiology Studies
Occupational exposure to unknown concentrations of carbon tetrachloride vapor for
periods between 6 weeks and 3 months resulted in gastrointestinal effects (nausea, vomiting,
abdominal pain, anorexia), hepatic effects (jaundice), and neurological effects (headache,
dizziness) (Norwood et al., 1950). Kazantzis and Bomford (1960) described symptoms in 17
workers exposed to carbon tetrachloride vapor at concentrations between 45 and 97 ppm without
adequate ventilation. Symptoms in 15/17 workers included anorexia and nausea and, in more
than half of the workers, vomiting, epigastric discomfort or distension, depression, irritability,
headache, or giddiness. Symptoms typically developed in the latter half of the workweek and
cleared over the weekend. One of the workers, who reported having symptoms for 2 years,
previously had an increased serum AST level, but levels were normal for this individual and
seven others examined by the authors for this study. Similarly, Elkins (1942) reported results of
industrial hygiene evaluations in 11 plants in which workers were exposed to carbon
tetrachloride vapor. At concentrations between 5 and <85 ppm, nausea was the most common
symptom, but vomiting, headache, and body weight loss were also observed.
Tomemon et al., 1995
Tomenson et al. (1995) conducted a cross-sectional study of hepatic function in 135
carbon tetrachloride-exposed workers in three chemical plants in northwest England and in a
control group of 276 unexposed workers. The latter came from two sites, including one of the
plants that provided workers for the exposed group and a plant nearby where carbon tetrachloride
was not used. Controls had not held jobs with potential exposure to carbon tetrachloride or other
known hepatotoxins during the previous 5 years. Subjects were administered a questionnaire
that collected information on medical history, alcohol consumption, and length of service in a job
exposed to carbon tetrachloride. Blood samples were obtained from subjects after a 12-hour fast
that included abstinence from alcohol; samples were collected for about 60 subjects over 2 weeks
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in November 1986 and for the remaining subjects over 8 weeks starting in February 1987. Blood
samples were analyzed for ALT, AST, alkaline phosphatase (ALP), y -glutamyl transferase
(GOT), glutamate dehydrogenase (GDH), 5'-nucleotidase, total bile acids, cholesterol,
triglycerides, and hematological variables.
The exposure assessment was based on historical personal monitoring data for various
jobs at the three plants. Subjects were placed into one of three exposure categories (low,
medium, or high), according to their current jobs. When objective monitoring data were not
available for a particular combination of job and location (as was the case for 23 of 40 in the low
exposure group, 35 of 54 in the medium exposure group, and 2 of the 61 in the high exposure
group), an industrial hygienist classified the exposure qualitatively based on comparison with
similar groups. The quantitative exposure levels nominally associated with each of these
categories were: <1 ppm for "low," 1.1-3.9 ppm for "medium," and 4 ppm-11.9 ppm for "high."
Exposed workers were also categorized according to length of time in job (<1 year, 1-5 years,
and >5 years).
Study and control groups were found to be well matched for age, height, weight, work
patterns, and, generally, alcohol consumption. Almost all (97-98%) control and exposed
workers were current drinkers, and the proportions of low, medium, and high alcohol drinkers
were roughly similar in the two groups (p = 0.30 for Chi-square comparison of 4 levels of
alcohol use between exposed and non-exposed). However, there was a slightly higher proportion
of very high drinkers (5-7 units every day or > 8 units at least 3-4 times per week) in the exposed
group (27%) than in controls (20%) (p = 0.20 for Chi-square comparison of high alcohol use
between exposed and non-exposed). Serum levels of GOT, bile acids, and triglycerides were
significantly increased in the high and/or very high alcohol consumption groups. In addition,
serum levels of GGT, cholesterol, triglycerides, AST, and 5'-nucleotidase were found to be
significantly related to age. Ages of workers in both control and exposed groups were
approximately normally distributed, with similar means and ranges.
Analysis of variance was used to investigate the relationship between carbon
tetrachloride exposure and serum chemistry and hematology variables, while controlling for age,
sampling time, and alcohol consumption. Initial analyses also included an interaction term
between carbon tetrachloride and alcohol consumption, but no evidence for any interaction was
found and the term was dropped from subsequent analyses. No analyses based on length of time
on job (i.e., duration of exposure) are presented in the published paper.
Multivariate analysis, based on simultaneous consideration of ALT, AST, ALP, and GGT
as dependent variables, revealed a statistically significant (p<0.05) difference between exposed
and unexposed workers. There was no evidence, however, of a dose-response across the levels
of exposure. In univariate analyses, in which each dependent variable was assessed separately,
there were no significant differences between the carbon tetrachloride-exposed group and the
control group for any of the serum chemistry variables. However, there was evidence of
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increased levels of ALP and GGT in the medium and high exposure groups, with the differences
between the medium exposure group and controls being statistically significant (p < 0.05) (see
Table 4-1). GDH was significantly increased in the medium-exposure group but declined in the
high-exposure group to the level seen in controls (see Table 4-1). There was little difference in
the mean adjusted serum ALT, AST, bile acids, and 5'-nucleotidase levels across exposure
categories.
Table 4-1. Mean of selected serum chemistry and hematology variables in
relation to carbon tetrachloride exposure in British chemical workers
Variable"
ALT (mU/mL)b
AST (mU/mL)b
ALP (mU/mL)b
GGT (mU/mL)b
GDH (mU/mL)b
Total bile acids (umol/L)b
5'-Nucleotidase (mU/mL)
Hemoglobin (g/dL)
Packed cell volume (%)
Red blood cell count (x 1012/L)
Control
20.54 (1.03)
16.48 (1.02)
125.79 (1.02)
26.89 (1.05)
3 (1.05)
1.06(1.06)
5.89(1.03)
15.97 (0.08)
48.54 (0.23)
5.61 (0.03)
Exposure group
Low
20.35 (1.08)
15.25 (1.05)
122.2 (1.05)
26.89(1.11)
3.26(1.10)
1 (1.00)
6.54(1.08)
15.6(0.19)
47.32C (0.54)
5.5 (0.08)
Medium
20.82 (1.05)
15.88(1.04)
137.10C(1.04)
33.17C(1.08)
3.57C(1.07)
1.25 (1.25)
6.25 (1.06)
15.39C(0.14)
47.32C (0.39)
5.47C (0.06)
High
19.39(1.06)
15.62 (1.04)
135.1 (1.04)
31.5(1.08)
2.98 (1.07)
1.28(1.28)
5.75 (1.06)
15.71(0.14)
48.05 (0,41)
5.5 (0.06)
aResults are presented as least square means, adjusted for age, sampling time, and alcohol consumption.
bAnalyzed after logarithmic transformation; values are geometric means with standard error of the mean
(SEM).
°p<0.05 (pairwise comparison).
Source: Tomensonetal., 1995.
Statistically significant changes were found for some of the hematological variables
(decreased red blood cell count, hemoglobin, and packed cell volume) in the univariate analyses
but without a dose response. Compared with the unexposed controls, there were very slight
(2.5-3.5%) statistically significant decreases in all three of these variables in the medium
exposure group and in packed cell volume in the low-exposure group (Table 4-1). Values for all
three hematological variables were similar to controls in the high-exposure group.
In an alternative analysis, a normal range was determined for each serum chemistry and
hematology variable based on the 2.5 and 97.5% quantiles in the control group. The proportion
of exposed workers exceeding the normal range was significantly elevated for ALT (8%) and
GGT (11%) but not for the other serum chemistry or hematology variables. This analysis did not
include any adjustment for alcohol intake or other potential confounders. The researchers noted
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that, for the serum chemistry variables, the upper normal limits defined based on the control
group were notably higher than the upper limits of the reference ranges for these tests supplied
by the manufacturers, indicating a difference between the control group and the population used
to derive the reference values, which are often hospital or university employees. This may have
been related to high alcohol consumption in the study controls, whose alcohol intake was similar
to the exposed group.
Individuals with one or more test results in excess of three standard deviations (SDs)
outside the control group mean were examined by a gastroenterologist. One exposed worker had
clinically detectable liver disease, but this could not be related to exposure to carbon
tetrachloride. The only other clinical findings were non-Hodgkin's lymphoma (NHL) in an
exposed worker and hemochromatosis in a control worker.
The observed decreases in hemoglobin, packed cell volume, and red blood cell count
were not considered to indicate a biologically significant effect of carbon tetrachloride, as the
observed changes were minimal and not clearly related to level of carbon tetrachloride exposure.
The results were generally suggestive of an effect on the liver, but were not consistent across the
liver variables or exposure levels. The overall difference seen in the multivariate analyses of the
four enzymes (ALT, AST, ALP, GGT) seemed to be driven by the increase in GGT, and to a
lesser extent in ALP, in the medium and high exposure groups. For GGT, the levels in the
medium and high carbon tetrachloride exposure groups were similar to the levels seen in the high
and very high alcohol use categories (geometric mean 30.04 and 32.32 mU/mL, respectively, in
these two alcohol use groups compared with 24.6 mU/mL in the low alcohol use groups). There
was little difference between the low carbon tetrachloride exposure group (<1 ppm estimated
exposure levels) and the no exposure group on any of the liver enzymes.
It is unclear to what extent the observed changes in serum enzyme levels reflect clinically
significant changes. The researchers suggest that their results show some enzyme leakage from
cells but without a measurable deficit in liver function (as assessed by total bile acid levels), and
they note that no effects of clinical significance were observed. Increased serum levels of ALT,
AST, ALP and GGT are indicators of liver damage (with ALP and GGT increased in exposed
workers), but none are specific for liver disease. Elevated ALP is used in the diagnosis of
hepatobiliary disease and bone disease, and elevated GGT in the diagnosis of liver disease. The
measurement of serum GTT levels can be used to ascertain whether observed elevations of ALP
are due to skeletal disease of reflect the presence of a hepatobiliary condition (Tietz, 1976).
One limitation of the study is the lack of information pertaining to the reliability (e.g.,
coefficient of variation, comparison with known standards) of the enzyme measures. The
investigators noted that a follow-up study conducted at one site 3 years later revealed clear
evidence of differences in laboratory procedures between the laboratories that had performed the
testing of blood samples in the cross sectional and follow-up studies. In addition, it was noted
that differences in the hematological variables (i.e., hemoglobin, packed cell volume, and red
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blood count) were observed between the samples collected in November 1986 and those
collected in February and March of 1987.
Overall, this study provides suggestive evidence of an effect from occupational carbon
tetrachloride exposure on hepatic serum enzymes, indicative of effects on the human liver.
Specifically, serum enzyme results suggested an exposure-related effect in the medium and high
exposure categories (>l-3.9 ppm [>6.3-24.5 mg/m3] and 4-11.9 ppm [25.2-75 mg/m3]). ALP
and GOT were elevated to a similar degree in both medium and high exposure categories
(although the difference was statistically significant only in the medium exposure category), and
enzyme levels in these exposure groups were comparable to the levels of ALP and GGT seen in
very high alcohol consumers. Confidence in the exposure monitoring for the medium exposure
group is relatively low, where exposures were estimated for over half (35/54) of the workers.
Confidence in the exposure monitoring for the high exposure group, where exposures were
measured for 59/61 workers, is higher. Because enzyme levels in these two groups were
comparable, an average concentration of the medium and high exposure groups (weighted by
number of subjects within specific exposure ranges) of 5.5 ppm (35 mg/m3) was considered to be
an estimate of the lowest-observed-adverse-effect level (LOAEL).b No effects on serum enzyme
levels were seen in the low exposure category (i.e., <1 ppm [<6.3 mg/m3]). Because exposures
were estimated for more than half (23/40) of the workers in this exposure category and because
this category covers exposures less than 1 ppm, a NOAEL could not be determined.
Seidler et al, 1999
Seidler et al. (1999) evaluated the association between maternal occupational exposure to
b An average exposure concentration for medium and high exposure categories (weighted by number of subjects
within specific exposure ranges) was calculated as follows using data in the appendix to Tomensen et al. (1995):
Exposure category
Medium
High
Sum
Average cone, for medium and
high exposure categories (ppm)
Exposure cone, (ppm)
[mid-point of range]
1.5
2.5
3.5
2.5 (estimated)*
5
7
9
11
8 (estimated)*
Number of
subjects
4
10
5
35
14
14
16
15
2
115
Product of cone, x number of subjects
(ppm-subject)
6
25
17.5
87.5
70
98
144
165
16
629
5 5**
* Estimated exposures were assumed to be the mid-point of the exposure category.
** Average calculated as the sum of the product of exposure concentration x number subjects for the individual
exposure ranges in the medium and high exposure categories divided by the total number of subjects, or 629 ppm-
subject^- 115 subjects = 5.5 ppm.
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chemicals and the risk of infants small for gestational age in singleton births in a prospective
cohort study of 3946 pregnant women in West Germany from 1987 to 1988. The final group of
1865 women included those who completed a questionnaire on sociodemographic, psychosocial,
nutritional, environmental, and occupational factors, for whom pregnancy outcomes were known
and who were working at the time of the interview. Women with stillbirths, multiple births, and
incompletely recorded outcomes were excluded. A semiquantitative job-exposure matrix,
incorporating consideration of likelihood of exposure, intensity of exposure, and proportion of
time at work, was used to classify occupational exposure to eight chemicals or chemical groups,
including carbon tetrachloride. ORs were calculated, adjusting for age, smoking status, alcohol
consumption, body mass index, number of former births, and income as potential confounders.
The study found no association between occupational exposure to carbon tetrachloride and the
risk of infants small for gestational age. The power of this study was limited. Of the 1865
births, only 64 mothers had potential exposures to carbon tetrachloride characterized as "low" or
"moderate."
Cancer studies
Several epidemiological studies have investigated potential associations between cancers
of various types and exposure to carbon tetrachloride. The subjects of all of these studies
experienced multiple chemical exposures, and the exposures were estimated qualitatively based
on historical information. These studies, therefore, can provide only suggestive evidence for
such associations.
Exposure to carbon tetrachloride was not found to be associated with cancer risk in case-
control studies for astrocytic brain cancer in white males (300 cases and 320 controls) from three
areas of the U.S. where a high proportion of the workforce is employed in petroleum refining and
chemical manufacture (after adjustment for several potential confounders) (Heineman et al.,
1994), for lung cancer in male employees (308 cases and 588 controls) of a Texas chemical plant
(Bond et al., 1986), for pancreatic cancer in residents (63,097 cases and 252,386 controls) from
24 U.S. states (Kernan et al., 1999), for renal cell carcinoma in Minnesota residents (438 cases
and 687 controls) (Dosemeci et al., 1999), for rectal cancer in Montreal residents (257 cases and
533 controls) (Dumas et al., 2000), or for lymphoma in a population (age 18-80 years) recruited
from six study regions in Germany. In the general population-based case-control studies (Seidler
et al., 2007; Kernan et al., 1999: Dosemeci et al., 1999; Dumas et al., 2000), occupation/industry
information obtained from questionnaires, interviews or death certificates in combination with a
job exposure matrix was used to characterize chemical exposures. There was evidence for a
weak association between exposure to carbon tetrachloride and excess risk for breast cancer
among white female residents of 24 U.S. states; the OR was 1.21 (95% CI: 1.1-1.3) for those
thought to have had the highest intensity of exposure to carbon tetrachloride [based on
occupation listed on death certificates] (Cantor et al., 1995). Among white male workers at a
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rubber manufacturing plant in Akron, Ohio, there was a significant age-adjusted association
between exposure to carbon tetrachloride and death from lymphosarcoma (6 exposed out of 9
cases, OR = 4.2,/?<0.5) and lymphocytic leukemia (8 exposed out of 10 cases, OR = 15.3,
/K0.001) (Wilcosky et al., 1984; Checkoway et al., 1984). Kubale et al. (2005) reported that
exposure to solvents (including carbon tetrachloride and benzene) was significantly associated
with leukemia mortality in civilian workers at the Portsmouth Naval Shipyard in Kittery, Maine
(OR = 1.03, 95% CI: 1.01-1.06). The findings with respect to carbon tetrachloride are uncertain,
however, because solvent exposures cannot be separated, exposure misclassification was
considered likely, and the phase out of carbon tetrachloride began in 1948, whereas the cohort
considered deaths between 1952 and 1996. No case-control studies were identified that looked
for an association between carbon tetrachloride and liver tumors or adrenal gland tumors (the
tumor types found in laboratory bioassays with carbon tetrachloride).
Spirtas et al. (1991) conducted a retrospective cohort study of 14,457 aircraft
maintenance workers at Hill Air Force Base in Utah to evaluate mortality associated with
workplace exposures, particularly trichloroethylene. Carbon tetrachloride was one of more than
20 chemicals include in the study. Spirtas et al. found increased mortality for NHL in white
female workers who had been exposed to carbon tetrachloride, in comparison with the Utah
population (Spirtas et al., 1991). However, in a follow-up study of the same cohort (Blair et al.,
1998) that extended the follow-up of worker mortality from 1982 to 1990, the relative risk
(calculated as the ratio of the rate of NHL mortality in the exposed and unexposed portions of the
cohort, adjusted for date of birth, calendar year of death, and sex) of NHL mortality was not
significantly increased in the female cohort (relative risk = 3.3, 95% CI: 0.9-12.7). A cohort of
dry cleaners in St. Louis, Missouri, showed slight significant excesses for deaths from all cancers
(standardized mortality ratio [SMR] = 1.2, 95% CI: 1.0-1.3), esophageal cancer (SMR = 2.1,
95% CI: 1.1-3.6), and cervical cancer (SMR = 1.7, 95% CI: 1.0-2.0) (Blair et al., 1990, 1979).
Risk of esophageal cancer was increased specifically in workers with the highest cumulative
exposure (SMR = 0.9, 0.3, and 2.8 in the low, medium, and high cumulative exposure
categories). There also appeared to be an increase in the risk of lymphatic and hematopoietic
cancers in the high-exposure group (SMR = 4.0), although this apparent increase was based on
only five cases. While some of these workers are likely to have been exposed to carbon
tetrachloride, no separate analysis was conducted for those exposed to carbon tetrachloride or
any other individual chemical. A cohort of Finnish laboratory workers exposed to carbon
tetrachloride and other chemicals showed no increased risk of cancer of any type, although the
average follow-up time of 15.7 years for the cohort may have been too short to reveal risks for
rare cancers with longer latency periods (Kauppinen et al., 2003).
An association between inhalation of carbon tetrachloride and liver cancer in humans was
suggested by two case reports (Tracey and Sherlock, 1968; Johnstone, 1948). Johnstone (1948)
reported the death of a 30-year-old female from liver cancer after 2-3 years of occupational
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exposure (assistant to a metallurgist) to carbon tetrachloride at levels that produced signs of
central nervous system toxicity, fatigue, and jaundice. Carbon tetrachloride exposure levels were
not assessed. Prior to carbon tetrachloride exposure, the woman had a history of "biliary colic"
and jaundice and had been studied for "gall bladder disease." A 66-year-old man died of
hepatocellular carcinoma 7 years after acute inhalation exposure from carpets that had been
cleaned with carbon tetrachloride (Tracey and Sherlock, 1968). The man was asymptomatic for
5 days after exposure but then developed vomiting, diarrhea, anuria, and jaundice. Although the
patient had no prior history of liver disease, he reported daily consumption of "several alcoholic
drinks"; the duration of alcohol consumption was not given. At the time of death, the liver tumor
was extensive, with very little normal tissue remaining. The potential contribution of alcohol
consumption to liver disease in this patient could not be ruled out. Because of complicating
factors (e.g., alcohol consumption, previous history of liver disease), small number of individuals
involved, single exposure in one case, and relatively short time spans between exposure and
tumor appearance, a causal relationship between carbon tetrachloride and liver tumors cannot be
established from these case reports.
4.1.3. Dermal Exposure
There is evidence from one case report of health effects from exposure to carbon
tetrachloride that can at least partially be attributed to absorption across the skin (Farrell and
Senseman, 1944). The worker was exposed 8 hours/day by using a fine spray of carbon
tetrachloride to saturate a cloth wrapped around the fingers. Although some exposure is likely to
have occurred by inhalation, the authors considered absorption through the skin of the hands to
be the primary route of exposure. After an unspecified period of time at this job, the worker
developed polyneuritis. Symptoms included weakness, pain in the limbs, and loss or reduction
of certain reflexes. The patient, whose body weight was not reported, lost 8 pounds in the month
between onset of illness and hospitalization. The signs and symptoms of neurotoxicity reversed
after several months without exposure.
4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
ANIMALS—ORAL AND INHALATION
Consistent with human data, toxicity assays in animals exposed orally or by inhalation
identify the liver to be the major target organ, with oral NOAELs between 0.71 and 0.86 mg/kg
and oral LOAELs between 7.1 and 17.8 mg/kg. Hepatic carcinogenicity has also been reported
in rats and mice exposed orally or by inhalation to carbon tetrachloride. While the liver appears
to be the primary target organ for both oral and inhalation studies, the kidney is also a sensitive
target organ for carbon tetrachloride exposure. Nephritis and nephrosis are very common effects
following inhalation exposure to carbon tetrachloride.
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4.2.1. Oral Exposure
4.2.1.1. Subchronic Toxicity
Litchfield and Gartland, 1974
Litchfield and Gartland (1974) conducted a series of assays evaluating hepatic effects in
beagle dogs treated with carbon tetrachloride in gelatin capsules prior to their daily food intake.
In one experiment, groups of six male and six female young adult dogs were dosed with 797
mg/kg-day for up to 28 days. Blood samples taken before treatment and at 7-day intervals were
evaluated for serum ALT, AST, ALP, ornithine carbamoyl transferase (OCT), and creatine
kinase. At termination, livers were examined for histopathology. In a second experiment, three
female dogs were given 32 mg/kg-day for 8 weeks. Blood was sampled before treatment and at
2, 3, 5, 6, 7, and 8 weeks. Livers were examined for histopathology after sacrifice. Control
values were obtained from untreated dogs. No clinical signs of toxicity were observed. In dogs
treated at 797 mg/kg-day, increases in serum ALT levels (2- to 34-fold in 4/6 males and 6/6
females) and OCT (2- to 20-fold in 3/6 males and 6/6 females) were observed after 14-28 days.
All dogs exhibited hepatic histopathology (minimal to moderately severe centrilobular fatty
vacuolization, sometimes accompanied by single cell necrosis), the severity of which correlated
with the level of serum ALT and OCT in individual dogs. Dogs that showed no enzyme level
effect or a twofold increase only in ALT had minimal vacuolization with very occasional
necrosis. Dogs that had two- to eightfold increases in ALT and two- to threefold increases in
OCT had minimal to moderate vacuolization with occasional necrosis. Dogs with 8- to 11-fold
increases in ALT and 4- to 7-fold increases in OCT had moderate vacuolation with single cell
necrosis, and those with 18- to 34-fold increases in ALT and 20-fold increases in OCT had
moderately severe vacuolation with single cell necrosis. The female dogs given 32 mg/kg-day
for 8 weeks showed no change in serum enzyme levels and no histopathology of the liver. In
this study, 797 mg/kg-day was a LOAEL based on reported hepatic effects in six male and six
female dogs, and 32 mg/kg-day was a NOAEL based on no hepatic effects reported in three
female dogs. Given the wide dose spacing in this study, there is considerable uncertainty about
the assigned value of the NOAEL and LOAEL.
Bruckner et al, 1986
Groups of 15-16 adult male Sprague-Dawley rats were given doses of 0, 1, 10, or 33
mg/kg of analytical-grade carbon tetrachloride by gavage in corn oil 5 days/week for 12 weeks
(time-weighted average doses of 0, 0.71, 7.1, or 23.6 mg/kg-day). Body weight was measured
twice weekly. Blood samples were taken from five rats from each group at 2-week intervals
(each individual animal served as a blood donor twice, at 6-week intervals). After 12 weeks, 7-9
animals from each group were sacrificed. The remaining animals were maintained without
carbon tetrachloride treatment for an additional 2 weeks and then sacrificed. Following sacrifice,
a terminal blood sample was taken by cardiac puncture. The liver and kidneys were removed,
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weighed, and processed for histopathological examination. Blood samples were used for
determination of serum ALT, OCT, and sorbitol dehydrogenase (SDH), all of which are
indicators of liver injury, and BUN, an indicator of kidney damage. At the end of the exposure
period, substantial toxicity was evident in rats exposed to 23.6 mg/kg-day. Body weight gain in
this group was significantly reduced by about 6% after 30 days and 17% after 90 days. Liver
toxicity in this group was manifested by significantly elevated ALT (up to 34 times control
levels), SDH (up to 50 times control levels), and OCT (up to 8 times control levels) from week 2
through the end of exposure, significantly increased liverbody weight ratio, and extensive
occurrence of degenerative lesions. Observed liver lesions included lipid vacuolization, nuclear
and cellular polymorphism, bile duct hyperplasia, and periportal fibrosis. Severe degenerative
changes, such as Councilman-like bodies (single-cell necrosis), deeply eosinophilic cytoplasm,
and pyknotic nuclei, were occasionally noted as well. No evidence of nephrotoxicity was
observed. Only moderate effects were seen in animals exposed to 7.1 mg/kg-day. Body weight
gain was similar to controls, and liver toxicity was shown only by a significant (two- to
threefold) elevation of SDH during the second half of the exposure period and the presence of
mild centrilobular vacuolization in the liver. During the 2-week recovery period, serum ALT
and SDH levels returned towards control levels in both mid- and high-dose rats. Hepatic lesions
were still present in both groups, but severity was reduced for lesions other than fibrosis and bile
duct hyperplasia, the severity of which did not change. No effects were observed in rats exposed
to 0.71 mg/kg-day. This study identified a NOAEL of 0.71 mg/kg-day and a LOAEL of 7.1
mg/kg-day for carbon tetrachloride-induced liver toxicity.
Allisetal, 1990
Allis et al. (1990) conducted a study to investigate the ability of rats to recover from
toxicity induced by subchronic exposure to carbon tetrachloride. Groups of 48 60-day-old male
F344 rats were given 0, 20, or 40 mg/kg of carbon tetrachloride 5 days/week for 12 weeks
(average daily doses of 0, 14.3, or 28.6 mg/kg-day) by gavage in corn oil. Food consumption by
cage was measured throughout the study. Rats were weighed several times during the first week
and once a week thereafter. After 12 weeks, treatment with carbon tetrachloride was stopped.
Six animals from each group were sacrificed 1, 3, 8, and 15 days after exposure termination.
Upon sacrifice, a terminal blood sample was taken for determination of total bilirubin,
triglycerides, cholesterol, ALT, AST, ALP, and lactate dehydrogenase (LDH). The liver was
weighed, and samples were taken for light microscopic examination and determination of protein
and CYP450. The remaining 24 animals were used to determine liver uptake relative to the
spleen for a sulfur colloid labeled with technetium-99m and for tritiated 2-deoxyglucose0. Rats
0 Relative efficiency of liver uptake of the labeled sulfur colloid is a diagnostic test for human cirrhosis and
considered by investigators to be an indirect measure of hepatocyte function. Hepatic uptake of 2-deoxy glucose is
an indicator of hepatic glucose utilization.
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used for this purpose were maintained as long as 22 days postexposure. The only toxicity
endpoint measured in these "remaining" animals was liver weight. Both doses of carbon
tetrachloride were hepatotoxic, although the high dose produced significantly greater toxicity
than the low dose. One day after the end of exposure, significant dose-related changes were
found for liverbody weight ratio and serum ALT, AST, and LDH (all increased) and liver
CYP450 (decreased) in both dose groups. In addition, serum ALP and cholesterol were
increased in the high-dose group. Histopathological examination of the liver revealed, among
low-dose rats, cirrhosis in 2/6 and vacuolar degeneration and hepatocellular necrosis in 6/6 and,
among high dose rats, cirrhosis (as well as degeneration and necrosis) in 6/6. Serum enzyme
levels and CYP450 returned to control levels within 8 days of the end of exposure. Severity of
microscopic lesions declined during the postexposure period, but cirrhosis persisted in the high-
dose group through the end of the experiment. Relative liver weight decreased during the
postexposure period but did not reach control levels in the high-dose group even after 22 days.
Neither of the radiolabeled tracer techniques detected a decreased functional capacity in cirrhotic
livers, a finding that could not be explained by the investigators. The low dose of 14.3 mg/kg-
day was a LOAEL for hepatic toxicity in this study.
Koporec etal, 1995
Koporec et al. (1995) evaluated the effect of different dosing vehicles on the subchronic
oral toxicity of carbon tetrachloride in the rat. Groups of 11 male Sprague-Dawley rats were
treated with carbon tetrachloride by gavage at doses of 0, 25, or 100 mg/kg, 5 days/week for 13
weeks (average daily doses of 0, 17.8, or 71.4 mg/kg-day). The compound was administered in
corn oil or as an aqueous emulsion in 1% Emulphor. An untreated control group was followed in
addition to vehicle controls. Blood samples were taken from 4-5 rats/group after weeks 4 and 8
for analysis of SDH and ALT. All surviving rats were sacrificed at the end of exposure at which
time additional blood samples were collected and the liver was weighed and sampled for
histopathology and biochemical studies (triglyceride, microsomal protein, CYP450, and glucose-
6-phosphatase [G6Pase]).
Mortality was found in all treated groups. The number of deaths was higher for rats
treated with the Emulphor vehicle than with corn oil and increased with dose for both vehicles.
Mortality was about 75% and 25% in the high- and low-dose Emulphor groups and about 45%
and 10% in the high- and low-dose corn oil groups. No deaths occurred in any of the control
groups. Body weight decreased in a dose-related fashion throughout the study to a comparable
extent in rats treated with either vehicle. Terminal body weights were reduced about 25%
(statistically significant) in the high-dose groups (both vehicles) and about 6% in the low-dose
groups (both vehicles). Serum chemistry analyses showed statistically significant dose-related
increases in SDH and ALT at both dose levels after 4-13 weeks of treatment with either vehicle.
Increases in SDH were as high as 10-fold in the low-dose groups and 100-fold in the high-dose
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groups, while increases in ALT were about twofold in the low-dose groups and 25-fold in the
high-dose groups. The results were similar for rats treated in either vehicle. Liver microsomal
enzyme activities (CYP450 and G6Pase) were significantly reduced only in the high-dose
groups, and, again, the magnitudes of the effects were similar for rats treated in either vehicle.
Absolute and relative liver weights were slightly but significantly increased in the high-dose rats
treated in Emulphor but in no other groups. The researchers noted that the livers were perfused
with saline to facilitate collection of biochemical data and suggested that this procedure may
have influenced the liver weight results. Liver histopathology findings were similar in rats
treated in either vehicle. In the low-dose groups, lesions, seen in almost all animals, consisted
primarily of minimal-to-slight vacuolation and minimal fibrosis. In the high-dose groups,
vacuolation and fibrosis were moderate-to-moderately severe (all animals), and other lesions
were also seen in all animals, including minimal-to-slight necrosis and moderate-to-moderately
severe cytomegaly, nodular hyperplasia, oval-cell hyperplasia, and bile-duct hyperplasia. The
low dose of 17.8 mg/kg-day, which produced hepatic effects in rats with either the corn oil or the
Emulphor vehicle, was considered a frank effect level (FEL) by the U.S. EPA because of the
increased mortality at this dose level. Vehicle did not influence hepatotoxicity in this study, but
lethality appeared to be enhanced by dosing in Emulphor.
Condie et al, 1986
A study comparing the effects of two different gavage vehicles on subchronic toxicity of
carbon tetrachloride was also performed in mice. CD-I mice (12/sex/group) were treated with 0,
1.2, 12, or 120 mg/kg of carbon tetrachloride (98.2% pure) by gavage in either corn oil or 1%
Tween-60 aqueous emulsion 5 days/week for 12 weeks (average daily doses of 0, 0.86, 8.6, or 86
mg/kg-day) (Condie et al., 1986). The mice were caged in groups of six and provided with food
and water ad libitum. Food and water consumption and body weights were measured twice
weekly. At terminal sacrifice, blood samples were drawn for determination of serum ALT, AST,
and LDH. The livers were examined grossly, weighed, and processed for histopathological
examination. Fifteen deaths occurred during the study, half of which were attributed to gavage
error; the others were not dose related. These early deaths were scattered over dose groups and
did not appear to influence the study outcome. Body weight was not affected by treatment in any
exposure group. Hepatotoxicity was indicated in the high-dose group (86 mg/kg-day) by
significantly elevated liver weight and liverbody weight ratio; significantly elevated ALT (77-
89 times control levels in corn oil and 10-19 times control levels in Tween-60), AST (14-15
times control levels in corn oil and 3-4 times control levels in Tween-60), and LDH (12-15
times control levels in corn oil and 2-3 times control levels in Tween-60); and increased
incidence and severity of hepatic lesions, such as hepatocellular vacuolization, inflammation,
hepatocytomegaly, necrosis, and portal bridging fibrosis. At this dose, the only difference
between gavage vehicles was a greater incidence and severity of necrosis in mice given carbon
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tetrachloride in corn oil. The difference between vehicles was more apparent at the middle dose
of 8.6 mg/kg-day. This dose produced significantly elevated ALT and mild-to-moderate liver
lesions in mice gavaged with corn oil but was identified as a NOAEL for mice gavaged with
Tween-60. The low dose of 0.86 mg/kg-day was identified as the NOAEL for mice gavaged
with corn oil. In general, both sexes responded similarly, with severity of histopathologic
changes in males slightly greater than females.
Hayes etal, 1986
Another study in mice was conducted at higher doses. CD-I mice (20/sex/group) were
gavaged daily with 0, 12, 120, 540, or 1200 mg/kg-day of carbon tetrachloride (high
performance liquid chromatography grade, purity >99%) in corn oil for 90 days (Hayes et al.,
1986). An untreated control group of 20 male and 20 female mice was maintained as well. The
mice were observed for clinical signs of toxicity twice daily and weighed weekly. At
termination of exposure, the mice were sacrificed, blood was collected by cardiac puncture, and
gross necropsy was performed. Organ weights were determined for brain, liver, spleen, lungs,
thymus, kidneys, and testes, and samples were taken from the liver and kidney for
histopathological examination. The blood samples were used for comprehensive hematological
and clinical chemistry analyses. Urinalysis was also performed, although collection of urine was
not described. Determination of effect was made by comparing test groups to the vehicle
controls. Untreated controls were also compared with the vehicle controls. Observed effects
were reported in mice of both sexes at all dose levels and generally appeared to be dose-related.
These effects included increases in serum LDH, ALT, AST, ALP, and 5'-nucleotidase and a
decrease in serum glucose. Absolute and relative liver, spleen, and thymus weights were
increased. A variety of treatment-related lesions were observed in the liver, including fatty
change, hepatocytomegaly, karyomegaly, bile duct hyperplasia, necrosis, and chronic hepatitis.
No treatment-related lesions were observed in the kidney. No changes were found in urinalysis
or hematology parameters. It should be noted that, compared with untreated controls, vehicle
controls themselves had significantly elevated serum LDH and ALT, altered organ weights, and
increased incidence of liver lesions (e.g., necrosis in 5/19 versus 0/20 in untreated controls and
20/20 in 12 mg/kg-day group). This study failed to identify a NOAEL; the low dose of 12
mg/kg-day was a LOAEL for hepatic effects.
4.2.1.2. Chronic Toxicity and Carcinogenicity
4.2.1.2.1. Early National Cancer Institute studies
Edwards, 1941
Researchers at the National Cancer Institute (NCI) performed a series of early
experiments on the tumorigenicity of orally ingested carbon tetrachloride in mice. In the first of
these experiments, groups of 143 male strain C3H mice (2-3.5 months old) were treated with 0.1
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mL of a 40% solution of carbon tetrachloride in olive oil (0.04 mL or 64 mg of carbon
tetrachloride) by gavage two or three times/week for a total of 23-58 doses per mouse over a
period of 8-16 weeks (Edwards, 1941). [Because body weights were not provided, doses in
mg/kg-day could not be estimated.] This dose produced parenchymal necrosis of the liver but no
renal damage and was not lethal with repeated administration. Necropsies performed 2-147 days
after the last feeding, when the animals were between 6 and 10 months of age, found hepatomas
in 126/143 mice (88%). Tumors were typically multiple and were similar in appearance to
spontaneous hepatoma. No metastases were found. As in spontaneous hepatoma, the tumor
cells were morphologically similar to hepatic parenchymal cells. An olive oil control group
consisted of 23 male C3H mice given 39-50 gavage doses of 0.1 mL of olive oil (two or three
per week) and autopsied between 9 and 11 months of age. Only 1 of the 23 mice in this group
(4%) had a hepatoma. In untreated male C3H mice from the same stock, autopsies performed on
17 animals at 8.5-9 months of age found no hepatic tumors, while the incidence was 10% in
animals autopsied at 11 months of age and 26% in 341 animals autopsied at 11-19 months of
age.
Edwards andDalton, 1942; Edwards et al, 1942; Edwards, 1941
Similar experiments performed by the same researchers in other strains of mice with
lower spontaneous incidence of hepatoma than C3H mice (strains A, C, Y, and L) produced
similar results (Edwards and Dalton, 1942; Edwards et al., 1942; Edwards, 1941). A lower, but
still hepatotoxic (based on histopathologically observed cirrhosis), dose was administered in one
experiment. A group of 58 strain A female mice 2.5 months of age were treated with 0.1 mL of
5% carbon tetrachloride in olive oil (0.005 mL or 8 mg of carbon tetrachloride) three times
weekly for 25-29 doses over a 2-month period (Edwards and Dalton, 1942). [Because body
weights were not provided, doses in mg/kg-day could not be estimated.] The mice were
autopsied from 2 days to 4.5 months after the last dosing. The incidence of hepatoma was 71%.
The tumors were morphologically similar to those seen in mice treated with the higher dose. In a
related experiment by the same investigators, doses ranging from 0.005 mL (8 mg) to 0.04 mL
(64 mg) did not produce any hepatomas in 2-month-old mice treated only one to three times and
autopsied 2-12 months later. The livers of mice in this latter experiment showed complete
regeneration, with only limited evidence of the earlier damage caused by dosing. These studies,
and a subsequent one designed specifically to investigate the possibility of a sex-related
difference in susceptibility to carbon tetrachloride tumorigenicity in C3H mice (Andervont,
1958), found no evidence of any such difference between the sexes.
Eschenbrenner and Miller, 1946
A study with multiple dose levels was conducted by Eschenbrenner and Miller (1946) in
order to investigate the relationship between necrotic damage and regenerative processes in the
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liver and induction of hepatoma. Strain A mice (five/sex/group) were treated by gavage with 0,
0.125, 0.25, 0.5, or 1% of carbon tetrachloride in olive oil, receiving either 30 doses of 0.02
mL/g BW at 4-day intervals or 120 doses of 0.005 mL/g BW daily. Doses of carbon
tetrachloride, then, were 0, 10, 20, 40, or 80 mg/kg-day daily or 0, 40, 80, or 160 mg/kg-day
every 4 days for 120 days. The mice were 3 months old at the start of treatment and 7 months
old at the end of treatment. Mice were maintained for one month without treatment. One
additional dose was given 24 hours before sacrifice (at 8 months of age). Mice were examined
for presence of hepatomas and necrotic lesions in the liver. No necrosis or hepatoma was found
in control animals. No necrosis was observed in mice treated with either 0.005 or 0.02 mL/g of
0.125% solution (i.e., 120 doses of 10 mg/kg-day or 30 doses of 40 mg/kg-day). Although no
hepatomas were found by gross examination, two mice in the group that received 30 intermittent
40 mg/kg-day doses were found to have very small tumors (hepatomas) by microscopic
examination. Necrosis was produced only with 30 intermittent doses of 80 and 160 mg/kg-day.
Hepatomas were produced with 30 intermittent doses of 80 and 160 mg/kg-day as well as 120
continuous doses of 20, 40, or 80 mg/kg-day. The investigators observed, based on results of
separate experiments involving 1 or 2 doses, that all dose levels under both dosing regimens
(except 120 daily doses of 10 mg/kg-day) were expected to have produced initial liver necrosis,
although it was not observed at terminal sacrifice.
Delia Porta et al, 1961
An oral cancer bioassay for carbon tetrachloride in hamsters was also conducted. Delia
Porta et al. (1961) treated Syrian golden hamsters (10/sex) with carbon tetrachloride by gavage
weekly for 30 weeks. For the first 7 weeks, 0.25 mL of 5% carbon tetrachloride in corn oil (12.5
uL or 20 mg of carbon tetrachloride) was administered; this dose was halved for the remainder of
the exposure period. [Because body weight was not provided, doses in mg/kg-day could not be
estimated.] Animals were observed for an additional 25 weeks prior to sacrifice. Four females
and five males died during the treatment period, and three more females died during the
observation period. The remaining three females and five males were sacrificed at the end of the
55th week. Cirrhotic changes in the liver were seen in the animals that died during treatment and
to a lesser extent in the other animals as well. Of the 10 hamsters (five males and five females)
that died or were killed between weeks 43 and 55, all had liver-cell carcinomas, typically
multiple, and one had metastasized to the mesenteric and cervical lymph nodes. No liver-cell
tumors were observed in an untreated group of 109 male and 145 female hamsters from the same
breeder or in another group of 50 males and 30 females given 0.5 mL of corn oil by gavage twice
weekly for 45 weeks.
4.2.1.2.2. NCI bioassay. NCI (1977, 1976a, b; Weisburger, 1977) used carbon tetrachloride as a
positive control in cancer assays for chloroform, trichloroethylene, and 1,1,1-trichloroethane in
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rats and mice, and findings are reported in appendices to the bioassay reports for these other
chlorinated solvents. Neoplastic and nonneoplastic incidence data were also available through
the National Toxicology Program database search application (NTP, 2007).d Groups of
Osborne-Mendel rats (50/sex/group) were administered carbon tetrachloride by corn oil gavage
at time-weighted average doses of 47 or 94 mg/kg for males and 80 or 159 mg/kg for females, 5
days/week for 78 weeks. Rats were maintained without treatment for an additional 32 weeks.
Only 7/50 (14%) males and 14/50 (28%) females in the high-dose group and 14/50 (28%) males
and 26/50 (52%) females in the low-dose group survived to 110 weeks. In the pooled negative
control group, 26/100 (26%) males and 51/100 (51%) females survived to the end of the study.
Both doses of carbon tetrachloride resulted in marked heptotoxicity (including fatty changes),
with resultant fibrosis, cirrhosis, bile duct proliferation, and regeneration. Based on the NTP
database of neoplastic and nonneoplastic incidences (NTP, 2007), all other major organ systems
were examined for histopathological changes; however, no treatment-related effects other than
those in the liver were reported. The incidence of liver tumors was low in all groups.
Hepatocellular carcinoma was recorded in 1/99 pooled control, 2/49 low-dose, and 2/50 high-
dose males and in 0/98 pooled control, 4/49 low-dose, and 2/49 high-dose females. Neoplastic
nodules in the liver were seen in 0/99 pooled controls and 2/50 low-dose and 1/50 high-dose
males, and in 2/98 pooled controls and 2/49 low-dose and 3/49 high-dose females. The increase
in carcinomas was statistically significant in low-dose females in relation to pooled controls.
High early mortality, particularly in the high-dose group, may have affected the power of this
study to detect a carcinogenic effect.
In the same study, groups of male and female B6C3F1 mice received gavage doses of
1250 or 2500 mg/kg, 5 days/week for 78 weeks, and were maintained without treatment for 32
additional weeks. Mortality was markedly increased in treated mice. Survival was about 20% in
low-dose groups and <10% in high-dose groups at 78 weeks (versus 70% in control males and
90% in control females), and only one treated mouse survived to study termination at 92 weeks
(versus 50% in control males and 80% in control females). Liver toxicity (cirrhosis, bile duct
proliferation, toxic hepatitis, and fatty liver) was reported in only a few treated mice. According
to the NTP database of neoplastic and nonneoplastic incidences (NTP, 2007), the only other
nonneoplastic lesions in mice that appeared to be increased in a dose-related fashion was chronic
murine pneumonia in the lungs. Almost all treated mice, even those that died early, had
hepatocellular carcinomas (49/49 low-dose males, 47/48 high-dose males, 40/41 low-dose
females, and 43/45 high-dose females). In pooled controls, incidence was only 5/77 (6%) in
males and 1/80 (1%) in females. The incidence of adrenal adenoma and pheochromocytoma was
also increased in male mice (concurrent control: 0/18, low-dose: 28/49, high-dose: 27/48) and
d In a few instances, the tumor incidence values differed slightly between the NCI bioassay reports where carbon
tetrachloride was included as a positive control, the Weisburger (1977) review, and the NTP database. In those
instances, the incidence value included in the Toxicological Review was taken from the NTP database.
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female mice (concurrent control: 0/18, low-dose: 15/41, high-dose: 10/45) (NTP, 2007;
Weisburger, 1977).
4.2.2. Inhalation Exposure
4.2.2.1. Subchronic Toxicity
Smyth etal, 1936
Smyth et al. (1936) exposed groups of 24 guinea pigs (strain not specified) and 24
Wistar-derived rats (mixed sexes of both species) to 50, 100, 200, or 400 ppm (315, 630, 1260,
or 2520 mg/m3) of carbon tetrachloride vapor (>99% pure), 8 hours/day, 5 days/week for up to
10.5 months. The guinea pigs in this study received a purely vegetarian diet, but, because the
authors felt that low calcium in this diet may have affected the toxicity results, additional groups
of 16 guinea pigs fed diets supplemented with calcium were tested at concentrations of 25 ppm
(157 mg/m3), as well as 50, 100, and 200 ppm. In addition to the rats and guinea pigs, groups of
four monkeys (species and sex not specified) were exposed to 50 or 200 ppm using the same
protocol. Use of controls was not described, although controls apparently were included in the
study. All animals were weighed weekly. Blood counts (all species) and urinalysis (guinea pigs
and monkeys) were performed monthly. The fertility of rats and guinea pigs, which were housed
in mixed-sex groups and produced litters during the study, was monitored. All animals that
survived to scheduled sacrifice (including some animals that were sacrificed only after recovery
periods of varying durations) and most of those dying during the study were examined for gross
pathology. Tissue samples for histopathological examination were taken from the liver, kidney,
adrenal gland, spleen, heart, sciatic and optic nerves, and ocular muscle. Serum chemistry
analyses were performed on some animals as well. No statistical tests were conducted.
Guinea pigs of all exposure groups, including those that received diets supplemented with
calcium, suffered substantial mortality (>25-80% among "uninfected" guinea pigs). Mortality in
controls was not reported. In contrast, mortality among "uninfected" rats was limited to two
animals exposed to 400 ppm. No monkeys died during the study. Body weight gain was
reported to be markedly reduced among survivors in all groups of guinea pigs, compared with
that in controls. Body weight gain was also reduced by about 30% among rats exposed to 400
ppm. Too few litters were born to guinea pigs during the study to determine if exposure had any
effect, but, in rats, fertility was reduced in the 200 and 400 ppm groups. In guinea pigs, fatty
changes in the liver were seen at all dose levels, and cirrhosis developed at >50 ppm. In rats,
fatty changes were seen at >50 ppm and cirrhosis at > 100 ppm. In monkeys, mild fatty
degeneration of the liver was found at both 50 and 200 ppm. Other pathological changes in
animals exposed to these concentrations included renal tubular degeneration, degeneration of the
adrenal glands (with necrosis in guinea pigs), and damage to the sciatic nerve. This study did not
include concentrations low enough to identify a NOAEL for any of the three species tested. For
guinea pigs, the low concentration of 25 ppm was a frank effect level that produced substantial
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mortality. For rats and monkeys, the low concentration of 50 ppm was a LOAEL that produced
fatty changes in the liver. This study provides evidence of the progression of toxic liver effects
from fatty changes in the liver at lower doses to liver cirrhosis at higher doses. Because of the
age of the study, knowledge that bacterial and viral infections were a common problem at that
time, and the confounding that pregnancy (or lack of pregnancy) could have had on body
weights, the findings from this study much be interpreted with caution.
Adams etaL, 1952
Adams et al. (1952) conducted studies in which Wistar-derived rats (15-25/sex), outbred
guinea pigs (5-9/sex), outbred rabbits (1-2/sex), and rhesus monkeys (1-2 of either sex) were
exposed to carbon tetrachloride vapor (>99% pure), 7 hours/day, 5 days/week for 6 months at
concentrations of 5, 10,25,50, 100, 200, or 400 ppm (31, 63, 157,315,630, 1260, or 2520
mg/m3). Matched control groups, both unexposed and air exposed, were included in these
experiments. Animals were observed frequently for appearance and general behavior and
weighed twice weekly. Selected animals were used for hematological analyses periodically
throughout the study. Moribund animals and those surviving to scheduled sacrifice were
necropsied. The lungs, heart, liver, kidneys, spleen, and testes were weighed, and sections from
these and 10 other tissues were prepared for histopathological examination. In many cases,
terminal blood samples were collected and used for serum chemistry analyses, and part of the
liver was frozen and used for lipid analyses.
In this study, the primary target of carbon tetrachloride in all species was the liver. In
guinea pigs, liver effects progressed from a slight, statistical increase in relative liver weight in
females, but not males, at 5 ppm (not considered adverse by itself) to include slight-to-moderate
fatty degeneration and increases in liver total lipid, neutral fat, and esterified cholesterol at 10
ppm, and cirrhosis at 25 ppm. Liver effects became progressively more severe at higher
concentrations. Growth retardation was first observed at 25 ppm and progressed to rapid loss of
weight at 200 ppm. In the kidney, slight tubular degeneration was first observed at 200 ppm and
increased kidney weight at 400 ppm. Mortality was increased at >100 ppm. A similar
progression of effects was seen in rats, with no effects at 5 ppm, mild liver changes at 10 ppm,
cirrhosis at 50 ppm, and liver necrosis, kidney effects, testicular atrophy, growth depression, and
mortality at >200 ppm. In rabbits, 10 ppm was without effect, 25 ppm produced mild liver
changes, 50 ppm produced moderate liver changes, and 100 ppm produced growth depression.
Monkeys were the most resistant species tested, with evidence of adverse effects (mild liver
lesions and increased liver lipid) only at 100 ppm, the highest concentration tested. This study
identified NOAEL and LOAEL values, respectively, of 5 and 10 ppm in rats and guinea pigs, 10
and 25 ppm in rabbits, and 50 and 100 ppm in monkeys, all based on hepatotoxic effects.
Prendergast et al., 1967
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Prendergast et al. (1967) exposed groups of 15 Sprague-Dawley or Long-Evans rats, 15
Hartley guinea pigs, three New Zealand rabbits, two beagle dogs, and three squirrel monkeys
(sex not specified) to carbon tetrachloride vapor ("highest purity available") either by continuous
exposure to 1 or 10 ppm (6.1 or 61 mg/m3) for 90 days or intermittent exposure (8 hours/day, 5
days/week) to 82 ppm (515 mg/m3) for 6 weeks. The control group consisted of 304 rats, 314
guinea pigs, 48 rabbits, 34 dogs, and 57 monkeys. In order to generate the 1 ppm concentration,
the researchers found it necessary to dilute the carbon tetrachloride in 10 ppm of n-octane.
Therefore, a vehicle control group exposed to 10 ppm of n-octane was included in this study.
Animals were observed routinely for signs of toxicity and weighed monthly. Blood samples for
hematological analysis were taken at the end of the exposure period. Following sacrifice,
animals were necropsied and sections of the heart, lung, liver, spleen, and kidney were taken for
histopathological examination. Serum chemistry and liver lipid analyses were performed on
some animals. No statistical tests were conducted.
Intermittent exposure to 82 ppm resulted in the death of 3/15 guinea pigs and 1/3
monkeys. [This compares to mortality in the control groups of 7/304 (2.3%) rats, 2/314 (0.64%)
guinea pigs, 2/48 (4.2%) rabbits, 0/34 dogs, and 1/57 (1.7%) monkeys.] Body weight gain was
reduced in all species relative to the controls, and all species except rats actually lost weight
during the study. Mottled livers were seen in all species except dogs. Histopathological
examination of the liver revealed fatty changes that decreased in severity from guinea pigs to rats
to rabbits to dogs to monkeys. Liver lipid content of guinea pigs was increased about threefold
compared with controls. The only other effect noted was interstitial inflammation in the lungs of
all species. Continuous exposure to 10 ppm resulted in the deaths of 3/15 guinea pigs. Body
weight gain was depressed in all species relative to the controls, and monkeys appeared visibly
emaciated. Gross examination showed the presence of enlarged/discolored livers in all species
except dogs. Microscopic examination revealed fatty changes in the liver that were most
prominent in rats and guinea pigs but were present in the other species as well. Lung effects
were not reported in this group. Continuous exposure to 1 ppm produced no mortality or clinical
signs of toxicity. Weight gain relative to the controls was reduced in guinea pigs, rabbits, dogs,
and monkeys but not in rats. The only histopathological findings were nonspecific inflammatory
changes in the liver, kidney, heart, and lungs. No effects were noted in the n-octane control
group. The results of this study suggest a NOAEL of 1 ppm (6.1 mg/m3) and a LOAEL of 10
ppm (61 mg/m3) for rats, guinea pigs, rabbits, dogs, and monkeys based on hepatotoxicity.
Effects on growth were reported at both exposure levels, but the data are difficult to interpret, as
only starting body weights and percent change are reported, the changes did not occur in a dose-
related manner in all species, and no statistical comparisons were performed. It is unclear
whether inflammatory changes observed in the lungs of some exposed animals occurred in
controls as well.
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Nagano et al, 2007a [Japan Bioassay Research Center (JBRC), 1998]
Groups of F344/DuCrj rats (10/sex/group) were exposed (whole body) to 0, 10, 30, 90,
270, or 810 ppm (0, 63, 189, 566, 1700, or 5094 mg/m3) of carbon tetrachloride (99.8% pure)
vapor for 6 hours/day, 5 days/week for 13 weeks (Nagano et al., 2007a). [This study was
previously available as an unpublished study by the Japan Bioassay Research Center (JBRC,
1998).] Rats were observed once a day for clinical signs, behavioral changes, and mortality and
were weighed weekly. Urinalysis (pH, protein, occult blood, glucose, ketone body, bilirubin,
and urobilinogen) was performed at the end of the dosing period. Blood for hematological
(erythrocytes, hemoglobin, hematocrit, platelets, and leukocyte differential) and serum chemistry
analyses (AST, ALT, LDH, ALP, total bilirubin, creatine phosphokinase, urea nitrogen,
creatinine, total protein, albumin, albumin/globulin ratio, glucose, total cholesterol, phospholipid,
sodium, potassium, chloride, calcium, and inorganic phosphorus) was taken during euthanization
at the scheduled sacrifice after overnight fasting. All organs and tissues were examined for gross
lesions, and organ weights were recorded for the thymus, adrenal gland, ovary, testis, heart, lung,
kidney, spleen, liver and brain. Tissues (not specified) were fixed for histopathological analysis;
lesions were presented for selected tissues (liver and kidney). Additionally, livers of control and
810-ppm male rats were sectioned for examination of hepatic altered cell foci, a preneoplastic
lesion, by immunohistochemical staining with anti-GST-P using an avidin-biotin-peroxidase
complex method.
No deaths occurred in any group. Body weight in the 810 ppm males was lower than in
controls throughout the study. At termination, the decrease was about 20% (p<0.01). Body
weight was consistently lower than controls in the 810 ppm females as well, but the difference at
termination was slight (4%) and not statistically significant. Statistically significant, dose-related
decreases in hemoglobin and hematocrit were observed at. 90 ppm in both males and females. At
810 ppm, red blood cell count was also significantly decreased in both sexes. Serum chemistry
changes included large, statistically significant and dose-related increases in ALT, AST, LDH,
ALP, and LAP (leucine aminopeptidase) in males at 270 ppm and females at 90 ppm. Total
bilirubin was significantly increased in male rats at 810 ppm and female rats at >270 ppm.
Serum levels of creatine phosphokinase (CPK) were statistically increased in females at 30 ppm
and above, but there was little change as exposure level increased from 90 to 810 ppm. CPK
levels in males were not statistically different from those in controls. In the urine, protein levels
were increased in males at 270 ppm and in females at 90 ppm. Urinary pH was decreased and
the presence of occult blood was noted in males and females at 810 ppm. Relative liver weights
were significantly increased in a dose-related fashion in male rats (>10 ppm) and female rats
(>30 ppm). Significant, dose-related increases in absolute and relative weights were also
recorded for the kidneys, spleen, heart, and lungs in both males and females, primarily at 90 ppm
and above. Females at 810 ppm also had significant reductions in absolute and relative ovary
weights. Males at 270 or 810 ppm had significantly reduced absolute testes weights, but relative
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weights were similar to those in controls. Dose-related increases in the incidence and severity of
histopathological lesions of the liver were observed at 10 ppm in both sexes. At the low level of
10 ppm, treatment-related lesions included slight fatty change, cytological alteration, and
granulation. Additional lesions at higher levels included ceroid deposits, fibrosis, pleomorphism,
proliferation of bile ducts, and cirrhosis. Altered cell foci were observed in male rats at >270
ppm and in female rats at >90 ppm (based on H&E-stained sections). The altered cell foci in
810-ppm male rats also stained positively with the anti-GST-P antibody. Renal lesions
(localized glomerulosclerosis) were seen in the 810 ppm males and females. The low
concentration of 10 ppm was a LOAEL for hepatic effects in rats (increased liver weight and
histopathology). A NOAEL was not identified.
These researchers conducted a similar study in mice. Groups of Crj:BDFl mice
(10/sex/group) were exposed (whole body) to 0, 10, 30, 90, 270, or 810 ppm (0, 63, 189, 566,
1700, or 5094 mg/m3) of carbon tetrachloride (99.8% pure) vapor for 6 hours/day, 5 days/week
for 13 weeks. Endpoints monitored were the same as described above for the 13-week rat study.
No treatment-related deaths occurred. Body weights were lower than in controls for most of the
study in males at 30 ppm; at termination, the decreases in these groups ranged from 8% to 15%
and were statistically significant. Body weights in treated females were similar to those in
controls throughout the study. Hematology findings included slight, significant decreases in red
blood cell count and hemoglobin at. 270 ppm and hematocrit at 810 ppm in females and in
hemoglobin at 810 ppm in males. Serum chemistry changes of note included significant
increases in ALT and LAP in males and females at 90 ppm (and ALP in males at >30 ppm),
slight significant increases in total protein and/or albumin in males and females at. 270 ppm, and
a significant increase in AST in males at 810 ppm. Urinalysis revealed no treatment-related
changes in males but a significant decrease in the pH of urine in females at 810 ppm. Organ
weight changes in treated mice included significant increases in absolute and/or relative weights
of the liver, kidney, and spleen in males and females, primarily at 90 ppm and above. Organ
weight changes in males were confounded by body weight decreases in most treated male
groups. Histopathological changes in mice were found only in the liver. In both sexes, the
hepatic lesions exhibited dose-related increases in incidence and severity. The only effect at the
low level of 10 ppm was an increase in incidence of slight cytoplasmic globular and fatty change
(large droplets) in males. Additional liver lesions noted in the higher exposure groups were:
nuclear enlargement with atypia and altered cell foci (>270 ppm) and collapse (presumably
resulting from the necrotic loss of hepatocytes) (>30 ppm). Altered cell foci included
acidophilic, basophilic, clear cell and mixed cell foci. The lowest exposure level of 10 ppm is a
minimal LOAEL for hepatic effects (slight cytological alterations) in male mice.
Benson and Springer, 1999
Groups of F344 rats, B6C3F1 mice, and Syrian hamsters (10 males/species) were
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exposed by inhalation to carbon tetrachloride vapor at concentrations of 0, 5, 20, or 100 ppm
(31.5, 126, or 630 mg/m3) for 6 hours/day, 5 days/week for 12 weeks (Benson and Springer,
1999; Nikula et al., 1998). An indicator of DNA replication, 5-bromo-2'-deoxyuridine (BrdU),
was administered to animals of all species several days prior to sacrifice. Additional satellite
groups of 5-6 animals/species were sacrificed after 1 and 4 weeks. At sacrifice, blood was
collected for ALT and SDH determinations, and liver sections were collected for
histopathological examination (quantitative evaluation of necrosis in the hepatic parenchyma)
and BrdU detection. Serum levels of ALT and SDH were significantly increased in mice at 320
ppm and in rats and hamsters at 100 ppm. The increases in mice and hamsters were larger than
those in rats. The actual magnitude of the changes could not be assessed from the graphical
presentation of the data. The volume percent of the hepatic parenchyma that was necrotic also
was significantly increased in mice at >20 ppm and in rats and hamsters at 100 ppm. No
necrosis was seen in controls or 5 ppm animals of any species. After 12 weeks, the volume
percent of necrosis in the liver of the groups showing statistically significant increases ranged
from approximately 5-10% in all species. More precise measures of necrosis could not be
determined from the graphical presentation of the data. BrdU labeling indices were also
significantly increased in mice at >20 ppm and hamsters at 100 ppm but were not increased in
rats at any concentration tested (except for a small nonsignificant increase at 100 ppm). In mice,
the percent of BrdU positive hepatocytes at 12 weeks was about 20% at 20 ppm and 60% at 100
ppm. In hamsters at 100 ppm, the percent of BrdU positive hepatocytes at 12 weeks was about
40%. In controls, the percent of BrdU positive hepatocytes at 12 weeks was approximately 2%.
These results show the occurrence of hepatocellular proliferation only at doses that also
produced necrotic damage. The study identified 5 ppm as a NOAEL and 20 ppm as a LOAEL
for hepatotoxicity in mice. Hamsters and rats were less sensitive than mice, with NOAEL values
of 20 ppm and LOAEL values of 100 ppm in these species.
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4.2.2.2. Chronic Toxicity and Carcinogenicity
Nagano et al, 2007b [Japan Bioassay Research Center (JBRC), 1998]
Groups of F344/DuCrj rats (50/sex/group) were exposed (whole body) to 0, 5, 25, or 125
ppm (0, 31.5, 157, or 786 mg/m3) of carbon tetrachloride (99.8% pure) vapor for 6 hours/day, 5
days/week for 104 weeks (Nagano et al., 2007b). [This study was previously available as an
unpublished study by the Japan Bioassay Research Center (JBRC, 1998).] Animals were
observed daily for clinical signs, behavioral changes, and mortality. Body weights were
measured once a week for the first 14 weeks and every 2 weeks thereafter. Urinalysis,
hematology, and clinical chemistry tests were conducted at study termination as described above
for the 13-week rat study, except that GGT was added to the list of serum enzymes monitored.
All organs and tissues were examined for gross lesions and organ weights were recorded for the
adrenal gland, testis, ovary, heart, lung, kidney, spleen, liver and brain. All major tissues were
examined for histopathologic changes.
Survival curves are for the male and female rat are shown in Figure 4-1. Survival was
high in all groups through week 64. After week 64, survival declined precipitously in the
125-ppm males and females. Only three males and one female from this group survived to 104
weeks. Liver tumors and chronic progressive nephropathy were the main causes of death.
Survival in the other treated groups (19-28/50 in males and 39-43/50 in females) was similar to
controls and adequate for evaluation of late developing tumors. Body weights were reduced
throughout most of the study in 125 ppm males (reduced 22% at termination) and after week 84
in 25 ppm males (reduced approximately 10% at termination). In females, body weight was
reduced during the second year of the study in both the 125 ppm (reduced 45% at termination)
and 25 ppm (reduced approximately 10% at termination) groups. The body weight decreases in
the 25 ppm males and females at termination were statistically significant. Low survival of rats
in the 125 ppm group limited statistical comparison of this group with controls.
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Male rat
Female rat
OTOI «0 : oou
IH1JUL 1 HI
rerarr im i n
AKIKAL fflMBESS
Figure 4-1. Survival curves for male and female rats
Source: JBRC (1998)
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Hematology analyses showed trends for decreased red blood cell count, hemoglobin, and
hematocrit in males and females at 25 and 125 ppm, although only the decreases for hemoglobin
and hematocrit in 25 ppm females were statistically significant (there was no statistical
evaluation for the 125 ppm group). Serum chemistry changes included statistically significant
increases in AST (males), ALT (males and females), LDH (females), and GPT (females) at 25
ppm; the increases over control in individual serum chemistry parameters at 25 ppm ranged from
1.2- to twofold. There were also significant increases in BUN in both males and females at 25
ppm (25 to 63% over controls). At 125 ppm, BUN, creatinine, and inorganic phosphate were
increased by two- to threefold over the control (but were untestable statistically because of the
small number the surviving animals at 125 ppm). Consistent with the subchronic rat study, there
was a significant increase in CPK in 25 ppm females but not males. An increase was reported in
the number of male and female rats with high levels of proteinuria in the 5 and 25 ppm groups
(too few data to test in the 125 ppm group) (Table 4-2).
Table 4-2. Urinalysis results in rats after 2-year exposure to carbon
tetrachloride
Concentration
(ppm)b
Protein content of urine"
+
2+
3+
4+
Male
0
5C
25C
125
0/22 (0%)
0/31 (0%)
0/19 (0%)
0/3 (0%)
2/22 (9%)
2/31 (6%)
1/19 (5%)
0/3 (0%)
20/22 (91%)
5/31 (16%)
3/19 (16%)
3/3 (100%)
0/22 (0%)
24/31 (77%)
15/19 (79%)
0/3 (0%)
Female
0
5C
25C
125
1/39 (3%)
0/43 (0%)
0/40 (0%)
0/1 (0%)
2/39 (5%)
2/43 (5%)
0/40 (0%)
0/1 (0%)
35/39 (90%)
15/43 (35%)
3/40 (8%)
1/1 (100%)
1/39 (3%)
26/43 (60%)
37/40 (92%)
0/1 (0%)
a Urine protein concentrations were measured with a semi-quantitative dipstick test. Equivalent
concentrations are: +: 30 mg/dl; 2+: 100 mg/dl; 3+: 300 mg/dl; 4+: 1000 mg/dl (letter dated March 8,
2004, from Kasuke Nagano, JBRC, to Mary Manibusan, U.S. EPA).
bThe exposure concentrations adjusted to continuous exposure (i.e., multiplied by 5/7 x 6/24) = 0.9, 4.5,
and 22.3 ppm.
°The study report indicated that urine protein results in male and female rats in the 5- and 25-ppm groups
were statistically elevated (p<0.01) based on a %2 test. Whether the statistical test represented a trend test
or pairwise comparison of the graded responses was unclear from the study report.
Source: JBRC, 1998.
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Organ weight changes were generally unremarkable and limited to the 25 and 125 ppm
groups, where they were confounded by body weight decreases in both males and females. Clear
increases in the incidence and severity of nonneoplastic liver lesions (fatty change, fibrosis,
cirrhosis) were seen at 25 and 125 ppm in both males and females (Table 4-3). Liver lesions
(e.g., fatty liver, granulation) in the 5 ppm group were of similar type, incidence, and severity as
controls. In the kidney, there was a dose-related increase in the severity of chronic nephropathy
(progressive glomerulonephrosis6) at 25 and 125 ppm in both males and females (Table 4-3).
Nephropathy was characterized as severe in most members of the 125 ppm group. Other dose-
related histopathological changes were increased severity of eosinophilic change (eosinophilic
globules in cytoplasm) in the nasal cavity at >25 ppm in males and >5 ppm in females and
increased incidence and severity of granulation in the lymph nodes at 125 ppm in both sexes
(Table 4-3).
e Chronic nephropathy (progressive glomerulonephrosis) is another term for the progressive renal disease in aging
rats more recently referred to as chronic progressive nephropathy (CPN) (Peter et al., 1986).
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Table 4-3. Incidence of selected nonneoplastic lesions in F344 rats exposed
to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week)3
Lesion
Male
0 ppm
5 ppm
25 ppm
125 ppm
Female
0 ppm
5 ppm
25 ppm
125 ppm
Liver
Fatty change
+
2+
3+
3/50
1/50
7/50
30/50
9/50
27/50
22/50
5/50
1/50
3/50
4/50
18/50
27/50
4/50
17/50
29/50
Fibrosis
+
2+
43/50
2/50
34/50
11/50
Cirrhosis
+
2+
1/50
14/50
26/50
1/50
1/50
23/50
27/50
Kidney
Chronic nephropathy
+
2+
3+
16/50
26/50
7/50
8/50
32/50
9/50
9/50
23/50
18/50
8/50b
9/50b
33/50b
31/50
13/50
37/50
7/50
1/50
19/50
25/50
5/50
5/50
7/50
38/50
Nasal cavity
Eosinophilic change
+
2+
43/50
47/50
25/50
25/50
13/50
34/50
39/50
33/50
16/50
25/50
25/50
4/50
46/50
Lymph nodes
Granulation
+
2+
4/50
9/50
1/50
11/50
1/50
6/50
27/50
3/50
5/50
11/50
2/50
12/50
28/50
a A blank cell indicates that the incidence of the histopathologic finding at that severity level was zero.
The exposure concentrations adjusted to continuous exposure (i.e., multiplied by 5/7 x 6/24) = 0.9, 4.5,
and 22.3 ppm.
b The published paper of the JBRC bioassay shows an incidence (all scores combined) of 49/50 125-ppm
male rats. The study report shows a total incidence of 50/50.
Source: Nagano et al., 2007b; JBRC, 1998.
The low exposure level of 5 ppm was associated with an increase in the severity of
proteinuria in male and female rats at this concentration; however, there was no effect on the
incidence of proteinuria at any exposure level. Histopathological examination revealed clear
evidence of treatment-related glomerular damage (increased severity of glomerulonephrosis) in
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male and female rats exposed to 25 or 125 ppm. Increases in BUN (at >25 ppm) and serum
creatinine and inorganic phosphorus (primarily at 125 ppm) show impairment of glomerular
function (i.e., decrease in glomerular filtration rate) at the same concentrations as the observed
lesions. The increased proteinuria at 5 and 25 ppm could be related to the glomerular changes
indicated by histopathology and serum chemistry results at 25 and 125 ppm. For reasons
discussed more fully in Section 4.6.2., interpretation of the observed proteinuria in the F344 rat,
a strain with a high spontaneous incidence of renal lesions, is problematic. Therefore, 5 ppm
was considered a NOAEL and 25 ppm a LOAEL for effects on the liver and kidney.
Tumor incidence data for rats are presented in Table 4-4. The incidence of hepatocellular
adenomas and carcinomas was statistically significantly increased in male and female rats at 125
ppm. The incidence of hepatocellular carcinomas in female 25-ppm rats (6%) was not
statistically elevated compared with the concurrent control, but did exceed the historical control
range for female rats from JBRC (0-2%). The increase in liver carcinoma over historical control
(2/1797) was statistically significant (based on Fisher's exact test; two-tailed p-value = 0.0002).
No other tumors occurred with an increased incidence in treated rats. Incidences of hepatic
altered cell foci (preneoplastic lesions of the liver), including clear, acidophilic, basophilic, and
mixed cell foci, were significantly increased in the 25-ppm female rats; in males, only the
incidence of basophilic cell foci was increased at 125 ppm.
Table 4-4. Incidence of liver tumors in F344 rats exposed to carbon
tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week)a
Tumor
Hepatocellular
adenoma
Hepatocellular
carcinoma
Hepatocellular
adenoma or
carcinoma
Male
0 ppm
0/50b
l/50b
l/50b
5 ppm
1/50
0/50
1/50
25 ppm
1/50
0/50
1/50
125 ppm
21/50C
32/50c
40/50C
Female
0 ppm
0/50b
0/50b
0/50b
5 ppm
0/50
0/50
0/50
25 ppm
0/50
3/50d
3/50d
125 ppm
40/50C
15/50C
44/50c
a The exposure concentrations adjusted to continuous exposure (i.e., multiplied by 5/7 x 6/24) = 0.9, 4.5,
and 22.3 ppm.
b Statistically significant trend for increased tumor incidence by Peto's test (p<0.0l).
c Tumor incidence significantly elevated compared with that in controls by Fisher Exact test (p<0.0l).
d Statistically significant (p < 0.001 by Fisher Exact test) in comparison to the historical control incidence
(2/1797).
Note: The historical control incidence of liver tumors in F344/DuCrj rats in JBRC studies was 1.7% (0-
8%) in males and 1.2% (0-6%) in females for hepatocellular adenoma and 0.3% (0-2%) in males and
0.1% (0-2%) in females for hepatocellular carcinoma (based on data from 36-39 carcinogenicity studies
carried out by JBRC; email dated April 5, 2007, from Kasuke Nagano, JBRC, to Susan Rieth, U.S. EPA).
Source: Nagano et al., 2007b; JBRC, 1998.
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These researchers also conducted a 2-year study using Crj :BDF1 mice. Groups of
Crj:BDFl mice (50/sex/group) were whole-body exposed to 0, 5, 25, or 125 ppm (0, 31.5, 157,
or 786 mg/m3) of carbon tetrachloride (99% pure) vapor for 6 hours/day, 5 days/week for 104
weeks. Endpoints monitored were the same as described above for the 2-year rat study. Survival
was high until week 64 of the study in all groups (see survival curves in Figure 4-2). Survival
decreased rapidly in 125 ppm males and females, starting at week 64, and in 25 ppm males and
females, starting at week 84. The decreases in survival were statistically significant in both
sexes at both concentrations. At 104 weeks, only one male and one female survived in the 125
ppm group and 25 males and 10 females in the 25 ppm group (versus 35 males and 26 females in
the control group). Investigators reported that liver tumors were the main cause of death at 125
ppm. At 25 ppm, deaths prior to study termination were also largely attributable to the presence
of tumors (with liver adenomas or carcinomas present in 33/39 female mice and 22/23 male mice
that died or were sacrificed prior to study termination). Body weights were markedly depressed
throughout the study in 25 and 125 ppm males and females (22 to 39% reduction at termination).
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Male
mouse
Female
mouse «-
sma no •- am
mm. -, fx'r met
IBWT IITE i »
SDHVITAL 0IMI. fflJHBERS
Figure 4-2. Survival curves for male and female mice
Source: JBRC(1998)
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The survival of only one mouse of each sex at 125 ppm prevented statistical comparisons
involving this group. Statistically significant increases in red blood cell count, hemoglobin, and
hematocrit were found in 25 ppm females. Values for these variables were also higher than in
controls (but not statistically increased) in the 25 ppm males and in the 125 ppm male and
female. This is in contrast with the significant decreases in these variables seen in the
subchronic mouse study and the rat studies.
Serum chemistry changes of interest were large, statistically significant increases in ALT,
AST, LDH, ALP, protein, total bilirubin, and BUN in males and females at 25 ppm (increases
over control ranged from 1.3- to 18-fold) and, for most of these variables, still larger increases in
the 125 ppm male and female (based on one surviving mouse/sex at terminal sacrifice).
Statistically significant decreases in ALT, AST, LDH, and CPK in 5 ppm males were not
considered to be biologically significant by the researchers (letter dated March 8, 2004, from
Kasuke Nagano, JBRC, to Mary Manibusan, U.S. EPA). The decreases were inconsistent with
the large increases seen at higher doses in males or the results in females and appeared to reflect
unusually high serum levels of these enzymes in male controls rather than reduced levels in the 5
ppm males. Levels of these enzymes in control males exceeded historical control values for
male Crj:BDFl mice in 2-year studies from the same laboratory by 1.5- to 2.5-fold; this is in
contrast to the results in females, where control values for all of these variables were within 10%
of historical control values (historical control data provided in a letter dated March 9, 2004, from
Kasuke Nagano, JBRC, to Mary Manibusan, U.S. EPA). Urinary pH was significantly decreased
in males and females at 25 ppm. The only organ weight changes of note were large significant
increases in absolute (~2.5-fold) and relative (-three- to fourfold) liver weight in 25 ppm males
and females. Liver weight data in the surviving 125 ppm male and female were consistent with
these results as well. Treatment-related nonneoplastic lesions occurred in the 25 and 125 ppm
males and females; these included increased incidence and/or severity of degeneration, cyst
formation, and deposit of ceroid in the liver, protein casts in the kidney, and extra medullary
hematopoiesis in the spleen (Table 4-5). The 25 ppm concentration was a LOAEL in this study
for effects on the liver (increased weight, serum chemistry changes indicative of damage, and
lesions), kidney (serum chemistry changes and lesions), and spleen (lesions); decreased growth;
and reduced survival. The 5 ppm level was a NOAEL.
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Table 4-5. Incidence of selected nonneoplastic lesions in BDF1 mice
exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5
days/week)3
Lesion
Male
0 ppm
5 ppm
25 ppm
125 ppm
Female
0 ppm
5 ppm
25 ppm
125 ppm
Liver
Degeneration
+
2+
3+
1/50
4/50
3/50
1/50
7/50
2/50
1/50
4/50
9/50
6/50
6/50
Cyst formation
+
2+
1/50
3/50
10/50
1/50
5/50
3/50
3/50
1/50
2/49
10/50
2/50
3/50
3/50
Deposition of ceroid
+
2+
Bile duct
proliferation
Centrilobular
hydropic
change
2/50
0/50
1/50
1/50
0/50
0/50
28/50
8/50
19/50
8/50
22/50
14/50
22/50
9/50
0/50
1/50
0/49
0/49
22/50
6/50
5/50
13/50
22/50
13/50
9/50
12/50
Kidney
Protein casts
+
2+
1/50
1/50
5/50
6/50
1/50
2/50
9/50
3/50
Spleen
Extramedullary hematopoiesis
+
2+
3+
15/50
12/50
1/50
15/50
8/50
2/50
14/50
25/50
5/50
5/50
26/50
12/50
8/50
7/50
3/50
11/49
4/49
5/49
11/50
18/50
7/50
4/50
30/50
9/50
a A blank cell indicates that the incidence of the histopathologic finding at that severity level was zero.
The exposure concentrations adjusted to continuous exposure (i.e., multiplied by 5/7 x 6/24) = 0.9, 4.5,
and 22.3 ppm.
Source: Nagano et al., 2007b; JBRC, 1998.
Tumor incidence data in mice are presented in Table 4-6. The incidences of liver tumors
in control mice (18% in males and 4% in females for hepatocellular adenomas and 34% in males
and 4% in females for hepatocellular carcinomas) were similar to historical control data for liver
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tumors in Crj:BDFl mice in 20 studies at JBRC (see Table 4-6 for historical control liver tumor
incidence). The gender differences in unexposed mice are thought to be related to inhibition of
liver tumor formation by female estrogen levels. The incidences of hepatocellular adenomas and
carcinomas were significantly elevated in both sexes at >25 ppm. At 5 ppm, the incidence of
liver adenomas in female mice (8/49 or 16%) was not statistically significantly elevated
compared to the concurrent control, but did exceed the historical control range (2-10%).
Table 4-6. Incidence of liver and adrenal tumors in BDF1 mice exposed to
carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week)3
Tumor
Hepatocellular
adenoma
Hepatocellular
carcinoma
Hepatocellular
adenoma or carcinoma
Adrenal
pheochromocytoma6
Male
0 ppm
9/50b
17/50b
24/50b
0/50b
5 ppm
10/50
12/50
20/50
0/50
25 ppm
27/50c
44/50c
49/50c
16/50C
125 ppm
16/50
47/50c
49/50c
32/50c
Female
0 ppm
2/50b
2/50b
4/50b
0/50b
5 ppm
8/49d
1/49
9/49
0/49
25 ppm
17/50C
33/50c
44/50c
0/50
125 ppm
5/49
48/49c
48/49c
22/49c
"The exposure concentrations adjusted to continuous exposure (i.e., multiplied by 5/7 x 6/24) = 0.9, 4.5,
and 22.3 ppm.
b Statistically significant trend for increased tumor incidence by Peto's test (£><0.01).
0 Tumor incidence significantly elevated compared with controls by Fisher Exact test (/?<0.01).
d Tumor incidence significantly elevated compared with controls by Fisher Exact test (p<0.05).
e All pheochromocytomas in the mouse were benign with the exception of one malignant
pheochromocytoma in the 125-ppm male mouse group.
Note: Liver historical control data in Crj:BDFl mice in 20 studies at JBRC: 17.1% (4-34%) in males and
5.2% (2-10%) in females for hepatocellular adenoma and 20.1% (2-42%) in males and 2.4% (0-8%) in
females for hepatocellular carcinoma (letter dated March 8, 2004 and email dated March 9, 2004, from
Kasuke Nagano, JBRC, to Mary Manibusan, U.S. EPA).
Pheochromocytoma historical control data in Crj:BDFl mice in 32 studies at JBRC: 0.3% (range: 0 to
2%) in both males and females (email dated October 15, 2005, from Kasuke Nagano, JBRC, to Mary
Manibusan, U.S. EPA).
Source: Nagano et al., 2007b; JBRC, 1998.
The incidence of adrenal pheochromocytoma was significantly increased in males at >25
ppm and in females at 125 ppm. This incidence exceeded the historical control incidence of
pheochromocytomas in Crj:BDFl mice in JBRC studies of 0.3% (range: 0 to 2%) in both males
and females (email dated October 15, 2005, from Kasuke Nagano, JBRC, to Mary Manibusan,
U.S. EPA).
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4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION
4.3.1. Oral Exposure
No adequate reproductive toxicity studies have been conducted in animals exposed by the
oral route. Teratogenicity has not been observed in the offspring of rats orally exposed to carbon
tetrachloride. However, total litter loss has been described at maternally toxic doses that are
much higher than those associated with liver and kidney toxicity.
Alumotetal, 1976
Reproductive performance was monitored in an oral study in which rats of an unspecified
strain (18/sex/group) were fed for up to 2 years on experimental diets that had been fumigated
with carbon tetrachloride for 48 hours (Alumot et al., 1976). Doses could not reliably be
estimated. Serial matings were performed throughout the study. Rats fed fumigated food
showed no effects on reproduction (male and female fertility, litter size, and pup mortality and
body weight at birth and weaning). There was widespread occurrence of chronic respiratory
disease in animals from all groups after 14 months, but this probably did not affect the
reproductive outcomes because most reproductive activity took place during the first year of the
study (only seven successful matings occurred during the second year). Treatment-related
parental toxicity was not reported, but only parental body weight was monitored concurrently
with the reproductive part of the study. No evidence of liver toxicity was found by serum
analyses or biochemical tests at the end of the study. This study found no evidence of
reproductive or maternal effects, but doses received by the experimental animals are unknown.
Wilson, 1954
Wilson (1954) administered daily doses of 478 mg of carbon tetrachloride by gavage in
corn oil to 29 pregnant rats (strain not specified) on 1 or 2 successive days of gestation beginning
between gestational days (GDs) 7 and 11. The experiment was terminated on GD 20 at which
time surviving dams were sacrificed, uteri were examined for resorptions, and litters were
examined for external malformations. Fifty-nine percent of the dams failed to produce offspring;
this included 6 of 29 dams (21%) that died (a rate less than the 50% mortality for nonpregnant
rats given the same dose) and 11 of 29 dams (38%) that had total litter loss from early resorption.
For the 12 of 29 dams (41%) that produced offspring, the resorption rate was within normal
limits (9.1%), no fetuses were malformed, and only one litter contained fetuses with retarded
growth. Because the single dose level of carbon tetrachloride used in this study caused 21%
mortality in the dams, it is difficult to determine whether the observation of total litter loss was a
direct effect of carbon tetrachloride or was secondary to maternal toxicity.
Narotsky andKavlock, 1995; Narotsky et al., 1997a, b, 1995
Narotsky and Kavlock (1995) reported the results of a developmental toxicity screening
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study in rats. Groups of 16-21 timed pregnant F344 rats were treated with 0, 112.5, or 150
mg/kg-day of carbon tetrachloride by gavage in corn oil on days 6-19 of gestation. Maternal
body weight was monitored periodically throughout gestation. The dams were allowed to litter.
Pups were examined on postnatal days (PNDs) 1, 3, and 6 and weighed on PNDs 1 and 6. Pups
found dead without gross external malformations were dissected and examined for visceral
malformations. After the final examination of their litters, dams were sacrificed and their uteri
examined for implantation sites. Dams that did not litter by presumed day 24 of gestation were
sacrificed for uterine examination. Ammonium sulfide stain was used as needed to detect full-
litter resorption. No dams died during the study. The number of females actually pregnant in
each group was 13, 9, and 14 in the control-, low-, and high-dose groups, respectively. Both
doses of carbon tetrachloride caused maternal weight loss (4-8%) early in the treatment period
and reduced extrauterine weight gain (35-45% lower than controls) over the treatment period as
a whole. The incidence of full-litter resorption was markedly increased in both dose groups: 4/9
(44%) and 10/14 (71%) in the 112.5 and 150 mg/kg-day groups, respectively (versus 0/13 in
controls). As a result, prenatal loss (reported as percent loss per litter) was significantly
increased in both dose groups. Implantation sites of the resorbed litters were not grossly visible
in most cases, requiring ammonium sulfide stain to find them. This suggested to the researchers
that the resorptions occurred early in pregnancy. Among dams that maintained their
pregnancies, resorptions were not increased nor were postnatal losses. Pup body weight was not
markedly affected by treatment. No malformations were associated with carbon tetrachloride
exposure. Reduced maternal weight gain and full-litter resorption were found at the low dose of
112.5 mg/kg-day in this study. In follow-up investigations, the researchers suggested that the
all-or-none nature of the observed resorptions points to a maternally mediated response and
produced evidence that the response is associated with reduced levels of progesterone and
luteinizing hormone in the dams (Narotsky et al., 1997a, 1995).
Narotsky et al. (1997b) compared the developmental toxicity of carbon tetrachloride
administered to rats by gavage in corn oil or an aqueous emulsion (10% Emulphor). Groups of
12-14 timed pregnant F344 rats received carbon tetrachloride at doses of 0, 25, 50, or 75 mg/kg-
day in either vehicle on GDs 6-15. Maternal body weights were determined on GDs 5, 6, 8, 10,
13, 16, and 20. All dams were examined for clinical signs of toxicity and the day of parturition
was recorded. Pups were examined for viability and body weight on PND 1 and 6. Pups that
died without gross malformations were examined macroscopically for soft tissue alterations.
Dams were sacrificed on PND 6 and uterine implantation sites were counted. The uteri of
females that did not deliver were stained with 10% ammonium sulfide to detect sites of early
resorption. There was no maternal mortality. Dose-related piloerection was observed in dams at
>50 mg/kg-day for both vehicles but was seen in more animals and for longer periods in the corn
oil groups. Dams exposed to 75 mg/kg-day in corn oil also exhibited kyphosis (rounded upper
back) and marked weight loss. Dams exposed to 50 and 75 mg/kg-day in water showed only
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significantly reduced body weight gain. Full-litter resorption occurred with an incidence of 0/13,
0/13, 5/12 (42%), and 8/12 (67%) in the control through high-dose corn oil groups and 0/12,
0/12, 2/14 (14%), and 1/12 (8%) in the respective aqueous groups. The difference between
vehicles was statistically significant at the high dose. Among the surviving litters, there were no
effects on gestation length, prenatal or postnatal survival, or pup weight or morphology. The 25
mg/kg-day dose was a NOAEL and the 50 mg/kg-day dose a LOAEL for full-litter resorption
and maternal toxicity (piloerection) with either corn oil or aqueous vehicle, although these
effects were more pronounced with the corn oil vehicle.
Hamlinetal, 1993
Hamlin et al. (1993) treated pregnant female B6D2F1 mice with 0, 82.6, or 826 mg/kg of
carbon tetrachloride by gavage in corn oil on days 1-5 of gestation. In this strain, days 1-5 of
gestation are characterized by sequential cleavage of the fertilized oocyte to generate a hatched
blastocyte, with implantation occurring on day 5 and organogenesis occurring subsequently.
Therefore, dosing in this study was limited to the preimplantation period. A total of 31 pregnant
females were included in the experiment, with a minimum of 8 in each dose group (actual group
sizes were not reported). Dams were allowed to give birth; litter size was recorded; and neonates
were weighed, measured for crown-rump length, and checked for obvious birth defects. During
lactation, the pups were weighed and measured for crown-rump length weekly. Lower incisor
eruption and eye opening were assessed in all pups on postpartum days 11 and 15, respectively.
Pups were weaned on postpartum day 22 and sacrificed. Dams were weighed weekly during
pregnancy and on postpartum day 22 just prior to sacrifice. The liver and kidneys from the dams
were removed and weighed. Liver and kidney tissue samples were collected for possible
histopathological examination at a later date but were not examined for this report. Treatment
with carbon tetrachloride had no effect on dam body weight during pregnancy or on absolute or
relative liver or kidney weight at sacrifice. Treatment also had no effect on litter size, pup size at
birth, the timing of developmental milestones (incisor eruption and eye opening), or pup growth
through weaning (a statistically significant difference in body weight between high-dose pups
and controls on day 15 postpartum was not considered to be biologically significant by the
researchers because crown-rump length was not affected and no other body weight differences
were found). No stillbirths or malformations were observed. The study report included only a
limited presentation of the results and no data were shown.
4.3.2. Inhalation Exposure
The potential for reproductive toxicity of carbon tetrachloride in animals is suggested by
Bergman's (1983) finding of partly nonextractable radiolabel in the interstitial testis of mice
exposed by inhalation to [14C]-carbon tetrachloride vapor. In the subchronic inhalation study by
Adams et al. (1952), testicular atrophy was observed in rats exposed to 200 or 400 ppm (1260 or
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2520 mg/m3) of carbon tetrachloride vapor 7 hours/day, 5 days/week for 6 months. Testicular
degeneration has also been reported in rats following repeated intraperitoneal (i.p.) doses of 1.5
mL/kg (Kalla and Bansal, 1975; Chatterjee, 1966). Smyth et al. (1936) found that fertility was
reduced in rats exposed to 200 or 400 ppm (1260 or 2520 mg/m3) of carbon tetrachloride vapor 8
hours/day, 5 days/week for up to 10.5 months.
The most detailed inhalation exposure study (Schwetz et al., 1974) suggests that
developmental effects of carbon tetrachloride occur at concentrations toxic to the mother and at
exposure concentrations higher than those associated with liver and kidney toxicity.
Oilman, 1971
As described in an abstract of an unpublished doctoral dissertation, Oilman (1971)
exposed groups of pregnant albino Sprague-Dawley rats to ambient air or 250 ppm (1575 mg/m3)
of carbon tetrachloride vapor for 8 hours/day on GDs 10-15. There were no adverse effects on
maternal body weight, litter size, the ratio of live to still births, or the incidence of skeletal
abnormalities.
Schwetz etal, 1974
Groups of 22-23 pregnant female Sprague-Dawley rats were exposed by inhalation to
carbon tetrachloride vapor at concentrations of 0, 334, or 1004 ppm (0, 2101, or 6316 mg/m3) for
7 hours/day on GDs 6-15 (Schwetz et al., 1974). Exposures to the two different dose levels were
not performed concurrently, so two separate control groups were used. Data from the two
control groups were combined except where they differed significantly (e.g., incidence of
delayed ossification of sternebrae). The rats were observed daily throughout pregnancy. Food
intake was monitored every other day during the experiment, and body weight was determined
on days 6, 13, and 21 of gestation. Following sacrifice on GD 21, the number and uterine
position of live, dead, and resorbed fetuses were recorded. The fetuses were weighed, measured,
and examined for external anomalies. Half of the fetuses in each litter were prepared so as to
enable detection of soft tissue anomalies upon subsequent examination, and the remainder were
prepared and examined for skeletal abnormalities. The litter was considered the unit of treatment
and observation when comparing the results from the different exposure groups. Nonpregnant
female rats were exposed simultaneously with the pregnant rats in order to monitor effects on the
liver. Serum ALT was determined in these rats throughout exposure, and some were sacrificed
for gross examination of the liver at the end of the exposure period. The remainder were
sacrificed 6 days later (corresponding to the end of gestation in the pregnant rats) for ALT
analysis, gross examination of the liver, and determination of liver weight. In the 334- and 1004-
ppm groups, significant reductions in fetal body weight (7% and 14%, respectively) and crown-
rump length (3.5% and 4.5%, respectively) were found. The incidence of delayed ossification of
the sternebrae was significantly elevated in the high-dose group (13%) compared with the
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concurrent control (2%) but not compared with the low-dose group or its concurrent control. No
other effects attributable to carbon tetrachloride exposure were found. No anomalies were seen
upon gross examination. A significant increase in subcutaneous edema was observed at 334 ppm
but not at 1004 ppm. No other increases in individual soft tissue or skeletal anomalies were
reported. Maternal toxicity was also observed in both dose groups. Food consumption and body
weight were significantly reduced compared with controls, and hepatotoxicity was indicated by
significantly elevated serum ALT (fourfold increase over control), gross changes in liver
appearance, and significantly increased liver weight (26% at 334 ppm and 44% at 1004 ppm).
This study, therefore, detected both maternal and developmental toxicity at a LOAEL of 334
ppm.
4.4. OTHER DURATION- OR ENDPOINT-SPECIFIC STUDIES
4.4.1. Acute and Short-term Toxicity Data
4.4.1.1. Oral Exposure
In animals acutely exposed to carbon tetrachloride by gavage, the liver appears to be the
primary target organ; damage to the kidney appears to occur at slightly higher doses (Blair et al.,
1991; Kim et al., 1990a, b; Bruckner et al., 1986; Hayes et al., 1986; Nakata et al., 1975;
Litchfield and Gartland, 1974; Korsrud et al., 1972; Gardner et al., 1925). Lung effects have also
been noted (Boyd et al., 1980; Gould and Smuckler, 1971). Hepatic toxicity is frequently
measured by significant increases in serum enzyme activities that peak between 24 and 48 hours
after dosing: ALT, AST, SDH, and OCT. The serum enzyme changes represent leakage from
damaged hepatocytes. Korsrud et al. (1972) indicated that overt hepatic necrosis was
unnecessary for detectable increases in serum enzymes. Reductions in the levels of microsomal
protein, microsomal enzymes (G6Pase), and CYP450 levels also occur after carbon tetrachloride
dosing (Kim et al., 1990a, b). Histopathological effects in the liver include centrilobular fatty
vacuolization, degeneration, necrosis, and inflammation.
Wangetal, 1997
Wang et al. (1997) monitored the time course of hepatic injury in Wistar rats treated with
3188 mg/kg of carbon tetrachloride by gavage in corn oil. There were immediate steep declines
in the hepatic microsomal protein and CYP450 content, so that metabolic rates declined by 50%
or more, as measured in microsomal CYP content. Plasma levels of AST and ALT increased
100-fold by 24 hours. Immediate histopathological lesions of the liver included hepatocellular
degeneration, necrosis, and hydropic swelling. Inflammatory cell infiltration was detectable
within 3 hours, and proliferation of mesenchymal cells began after 24 hours.
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Leeetal, 1998
Lee et al. (1998) examined the time course and distribution of toxicity and repair in the
livers of male Sprague-Dawley rats 24, 36, and 48 hours after receiving 40 or 400 mg/kg carbon
tetrachloride by gavage in corn oil. Cell proliferation was monitored by pulse-labeling with
BrdU 1 hour before sacrifice. The high dose caused extensive damage in the perivenous-to-
midlobular zones. Administration of 40 mg/kg induced regenerative hepatocyte proliferation, as
indicated by a significant elevation in BrdU-positive cells in the periportal zone (the site of
necrosis) at 24 hours, increasing at 36 hours and plateauing at 48 hours. BrdU-positive cells
were close to the portal tract at 24 hours and then increasingly in the outer periportal and
midlobular zones at later times. A few hepatocytes in the perivenous zone adjacent to the area of
cell damage were labeled at all time points.
Steupetal, 1993
Steup et al. (1993) also found significantly elevated serum ALT and SDH levels in male
F344 rats 3-72 hours after they received a single dose of 80 mg/kg carbon tetrachloride by
gavage in 10% Emulphor; peak enzyme levels were at 24 hours. Hepatic GSH concentrations
were significantly elevated in treated rats at 48 hours after dosing. Six hours after treatment,
hepatocytes near terminal venules (zone 3) showed some depletion of glycogen and ballooning.
Small collections of lymphocytes were adjacent to focal necrosis of single hepatocytes. More
extensive injury involved confluent areas of necrotic cells. Hepatocellular lysis was evident by
48 hours and a mononuclear cell infiltrate concentrated around terminal hepatic venules. Mitotic
figures predominated in the cells of the surrounding tissue. By 72 hours, recovery was evident
with only a mild infiltrate of mononuclear cells at the site of injury.
Evidence of regeneration of livers in animals treated with carbon tetrachloride appears
within 48 hours of dosing. In strain A mice dosed with 2550 mg/kg of carbon tetrachloride in
olive oil, necrosis was detectable in half the hepatocytes at 24 hours, and mitotic activity
appeared 48 hours after dosing (Eschenbrenner and Miller, 1946). Wistar rats treated with 7970
mg/kg had peak ALT levels at 24 hours, peak AST levels at 48 hours, and significantly elevated
levels for activities of DNA-synthesizing enzymes thymidine kinase and thymidylate synthetase
at 48 and 72 hours (Nakata et al., 1975); activity levels for DNA-synthesizing enzymes were
reduced at 96 hours. Doolittle et al. (1987) found that, in male CD-I mice administered a single
oral gavage dose or multiple (1, 7, or 14) daily doses of carbon tetrachloride in corn oil (up to
100 mg/kg-day), dose levels high enough to elicit significant increases in serum ALT and AST
also significantly increased the number of hepatocytes in S-phase, beginning 24 hours after
dosing. Multiple doses tended to lower the concentration required to induce hepatotoxicity and
increased the number of hepatocytes in S-phase (DNA-synthesizing phase of the cell-replication
cycle).
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The effect of dosing vehicle on carbon tetrachloride-induced hepatic toxicity has been
investigated in several studies. Kim et al. (1990a, b) reported that administration in a corn oil
vehicle resulted in lower acute hepatotoxicity (as measured by serum SDH and ALT levels over
a 72-hour period) compared with administration in an aqueous emulsion or as undiluted carbon
tetrachloride. Raymond and Plaa (1997) reported no consistent difference in serum ALT levels
measured 48 hours after dosing in male Sprague-Dawley rats given carbon tetrachloride (5.2 to
25.8 mmol/kg) in corn oil, 5% aqueous Emulphor emulsion, or Tween-85 (undiluted carbon
tetrachloride was not tested).
Damage to the lung has been noted in rodents exposed to carbon tetrachloride by gavage.
After male Sprague-Dawley rats received a single dose of 4000 mg/kg in mineral oil, pulmonary
histopathological effects included perivascular edema and mononuclear infiltration after 4 hours
and atelectasis (collapsed lung) and intraalveolar hemorrhages after 8 hours (Gould and
Smuckler, 1971). In male Swiss mice or Sprague-Dawley rats, there were significant reductions
in pulmonary CYP450 levels and the activity of the microsomal enzyme benzphetamine
demethylase 16 hours after receiving a single dose of 4000 mg/kg of carbon tetrachloride in 50%
sesame oil (Boyd et al., 1980). Clara cells showed histopathological changes (swelling and
necrosis with pyknotic nuclei), whereas the adjacent ciliated bronchiolar cells had normal
histology.
4.4.1.2. Inhalation Exposure
The central nervous system and the liver are the primary targets in acute toxicity studies
in animals exposed by inhalation. Suppression of the central nervous system occurs at relatively
high concentrations and is an immediate effect. In Wistar rats exposed for 7 hours, stupor was
observed at 4600 ppm, incoordination at 7300 ppm, and unconsciousness at 12,000 ppm (Adams
et al., 1952); 16-24 hours after exposure, these rats exhibited increased liver weights and
centrilobular fatty degeneration of the liver. Significant elevations in serum enzymes (ALT,
AST, SDH, and GDH) have been observed within 24 hours of acute inhalation exposures
(Paustenbach et al., 1986a, b; Siegers et al. 1985; Brondeau et al., 1983; Jaeger et al., 1975). In
addition, hepatic histopathology within 24 hours of a 4-hour exposure showed centrilobular
hydropic or necrotic parenchymal cell damage (Magos et al., 1982).
Hepatotoxicity, and to a lesser extent nephrotoxicity, appear to be the primary effects of
short-term duration inhalation exposures. Exposures of male Sprague-Dawley rats at 100 ppm, 8
or 11.5 hours/day for 5 or more days resulted in fatty changes in the liver (Paustenbach et al.,
1986a, b); nephrosis (degenerative changes in the kidney) was characterized as minor in rats
exposed for 8 hours/day but was more significant in rats exposed for 11.5 hours/day.
Plummer et al. (1990) conducted a 4-week inhalation toxicity study in male Wistar rats
exposed to carbon tetrachloride vapor continuously at 16 ppm (100 mg/m3) for 24 hours/day, 7
days/week except for 1.5-hour periods on Mondays and Fridays, or discontinuously at 87 ppm
70 DRAFT - DO NOT CITE OR QUOTE
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(50 mg/m3) for 6 hours/day, 5 days/week. The total time-weighted average exposures
(concentration H time) were the same: 10,507 ppm-hours for the continuous regimen and 10,458
ppm-hours for the discontinuous regimen. Liver histopathology (fibrosis and cirrhosis) was
indistinguishable between the two groups, suggesting that inhalation toxicity from carbon
tetrachloride is proportional to the product of concentration x time. In another 4-week study,
Bogers et al. (1987) exposed groups of Wistar rats to 6-hour daily exposures of carbon
tetrachloride vapor at 63 or 80 ppm, either uninterrupted or in 2-hour sessions with an
interruption of 1.5 hours; peak loads were added for some groups. At 80 ppm, serum enzyme
levels were slightly but significantly increased in the interrupted-exposure groups compared with
the uninterrupted-exposure groups (the 63 ppm groups were not compared).
4.4.1.3. Acute Studies Comparing Oral and Inhalation Exposures
The effect of route of administration on the hepatic toxicity of carbon tetrachloride has
been evaluated in rats (Sanzgiri et al., 1997; Bruckner et al., 1990). In both studies, male
Sprague-Dawley rats were exposed (nose only) to carbon tetrachloride vapor at 100 or 1000 ppm
(630 or 6300 mg/m3) for 2 hours. The systemically absorbed doses were calculated from
measurements of minute volume and differences between concentrations in inhaled and exhaled
air over time; the doses were calculated as 18.9 and 186 mg/kg by Bruckner et al. (1990) and as
17.5 and 179 mg/kg by Sanzgiri et al. (1997). Subsequently, groups of four to nine rats were
exposed by inhalation for 2 hours or given the same doses by gavage as a bolus delivery or as a
gastric infusion over 2 hours. Hepatotoxicity was measured by activities of SDH and ALT in
serum samples taken 24 hours after dosing, and the concentration of CYP450 and activity of
G6Pase per mg of hepatic microsomal protein. The results of the two studies are similar; those
for Sanzgiri et al. (1997) are presented in Table 4-7. SDH and ALT values were not significantly
affected by inhalation exposure at 100 ppm or gastric infusion at 17.5 mg/kg but were
significantly elevated at 1000 ppm or 179 mg/kg. In comparison, oral bolus dosing caused more
severe elevations at both dose levels. CYP450 levels were significantly reduced in all treated
groups, with more severe effects for the gastric routes at 17.5 mg/kg and the oral bolus route at
179 mg/kg. Suppression of microsomal G6Pase activity was most severe for gastric infusion at
both doses, followed by bolus delivery at both doses. Inhalation exposure at 100 ppm slightly
decreased G6Pase activity, but exposure at 1000 ppm was not significantly different from the
control. Overall, the results indicate more severe hepatic toxicity when carbon tetrachloride is
administered as a single bolus, compared with the same dose administered by inhalation or
gastric infusion over a longer period of time.
71 DRAFT - DO NOT CITE OR QUOTE
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Table 4-7. Hepatic toxicity in rats exposed to carbon tetrachloride by
inhalation or by equivalent oral dosing as bolus or 2-hour gastric
infusion
Exposure
Control3
Inhalation13
Gastric infusion
Oral bolus
Inhalation13
Gastric infusion
Oral bolus
Dose
(mg/kg)
0
17.5
17.5
17.5
179
179
179
SDH
(mU/mL)
5.2±1.0C
11.3±3.7C
6.0 ± 1.6C
64.6±12.5d
87.6±25.7d
96.9±18.0d
269.0 ± 44.7e
ALT
(mU/mL)
24.4±2.2C
19.3±1.7C
15.9±2.3C
55.5±9.9d
53.3±14.7d
81.0±8.2d
176.5 ±17.4e
P450
(nmol/mg protein)
0.81±0.02C
0.65±0.05d
0.46 ± 0.04e
0.49 ± 0.06e
0.61±0.04d
0.63±0.05d
0.47 ± 0.04e
G6Pase
(umol/hour/mg protein)
14.5±0.7C
10.9±0.5d
7.3±0.7e
12.5±0.1d
14.3±0.9C
7.8±0.7e
8.9 ± 0.3d
""Controls were treated with corn oil by gavage.
b!00 or 1000 ppm for 2 hours.
°"eMeans of each parameter that are statistically equivalent share the same superscript.
Source: Sanzgirietal., 1997.
Magos et al. (1982) compared the isotoxic oral and 4-hour inhalation concentrations of
carbon tetrachloride in Porton-Wistar or Fischer rats. For exposures by either route, Fischer rats
were twice as sensitive to hepatotoxic effects (based on SGPT and extent of liver centrilobular
damage) of carbon tetrachloride as the Porton-Wistar rats. Fischer rats required an inhalation
concentration 1.5 times lower and an oral dose 3.3 times lower than Porton-Wistar rats to
produce a 10-fold increase in serum ALT levels, measured 20 hours after exposure.
4.4.2. Genotoxicity Studies
The results of genotoxicity studies of carbon tetrachloride are summarized in Tables 4-8
to 4-11. These tables are not intended to provide an exhaustive list of genotoxicity studies for
carbon tetrachloride, but rather represent a reasonably comprehensive summary of the available
genotoxicity literature.
72
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Table 4-8. Genotoxicity studies of carbon tetrachloride in prokaryotic organisms
Test system
Salmonella typhimurium
TA100, TA1535
Salmonella typhimurium his
G46, TA1950
Salmonella typhimurium his
G46, TA1950
S. typhimurium TA98, TA100,
TA1535, TA1537, TA1538
S. typhimurium TA97, TA98,
TA100
S. typhimurium TA98, TA100,
TA1535, TA1537
S. typhimurium TA1535,
TA1538
S. typhimurium TA97, TA98,
TA100, TA1535, TA1537
S. typhimurium TA97, TA98,
TA100, TA1535
S. typhimurium TA98, TA100,
TA1535, TA1537,TA1538
S. typhimurium TA100,
TA1535
S. typhimurium TA98, TA100,
TA1535
S. typhimurium TA100,
TA1535, TA1537
S. typhimurium TA98
Endpoint
Reverse mutation
Reverse mutation
Reverse mutation
Reverse mutation
Reverse mutation
Reverse mutation
Reverse mutation
Reverse mutation
Reverse mutation
Reverse mutation
Reverse mutation
Reverse mutation
Reverse mutation
Reverse mutation
Test conditions
Plate incorporation assay
Spot test
Host-mediated assay in male
NMRImice
Plate incorporation assay
Plate incorporation assay
Plate incorporation assay
Preincubation assay using
capped tubes
Preincubation assay using
capped tubes
Preincubation assay using
capped tubes
Gas phase exposure in
dessicator for 7-10 hours
Gas phase exposure in
dessicator for 7-8 hours
Gas phase exposure in closed
incubation system for 48 hours
Gas phase exposure in a gas
sampling bag for 24 hours
Gas phase exposure in a gas
sampling bag for 24 hours
Results3
Without
activation
-
-
NA
-(T)
-
+d
-
-
-
-
-
-
-(T)
±
With
activation1"
-
-
-
-(T)
-
d
-
-
-
-
-
-
-(T)
-
Dosec
10,000 ug/plate
4000 ug/plate
6400 mg/kg
10,000 ug/plate
in DMSOd
1000 ug/plate in
DMSOd
2460 ug/plate in
methanol
1230 ug/mL
3333 ug/plate in
DMSO
3333 ug/plate in
DMSO
ND
ND
2830 ug/plate
50,000 ppm
10,000 ppm
Reference
McCann et al., 1975
Braun and
Schoneich, 1975
Braun and
Schoneich, 1975
De Flora, 1981
Bramsetal., 1987
Varmaetal., 1988
Uehlekeetal., 1977
Zeigeretal., 1988
Zeigeretal., 1988
Simmon etal., 1977
Simmon and Tardiff,
1978
Barber etal., 1981
Araki etal., 2004
Araki etal., 2004
73
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Table 4-8. Genotoxicity studies of carbon tetrachloride in prokaryotic organisms
Test system
Escherichia coli
WP2ttvrA/pKM101
E. co//WP2/pKM101
E. coli WP2wvrA
S. typhimurium BA13 and
BAL13
S. typhimurium BA13 and
BAL13
S. typhimurium
TA1535/pSK1002
E. coli PQ37
E. coli WP2, WP67, CM871
E. coli WP2, WP67, CM871
E. coli WP2, WP67, CM871
E. coli K-12 343/636, K-12
343/591
Endpoint
Reverse mutation
Reverse mutation
Reverse mutation
Forward mutation
Forward mutation
DNA repair
DNA repair
Differential DNA
repair
Differential DNA
repair
Differential DNA
repair
Differential DNA
repair
Test conditions
Gas phase exposure in a gas
sampling bag for 24 hours
Gas phase exposure in a gas
sampling bag for 24 hours
Gas phase exposure in a
desiccator
Preincubation assay for L-
arabinose resistance (AraR test)
Preincubation assay for L-
arabinose resistance (AraR test)
SOS response indicated by umu
gene expression
SOS chromotest
Liquid micromethod using
sealed plates
Preincubation assay in sealed
tubes
Spot test
Preincubation assay
Results"
Without
activation
±
+
ND
-
±
-
-
+
+
-
-
With
activation1"
±
+e
±
-
-
-
-
+
ND
ND
-
Dosec
10,000 ppm
5000 ppm
25,000 ppm
1230 ug/plate in
DMSOd
384 ug/plate in
DMSOd
5300 ug/mL
1540 ug/mL in
DMSO
12.5 ug
ND
ND
15,400 ug/mL
Reference
Arakietal.,2004
Arakietal.,2004
Norpoth et al., 1980
Roldan-Arjona et al.,
1991
Roldan-Arjona and
Pueyo, 1993
Nakamura et al.,
1987
Bramsetal., 1987
De Flora etal., 1984
De Flora etal., 1984
De Flora etal., 1984
Hellmer and
Bolcsfoldi, 1992
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Table 4-8. Genotoxicity studies of carbon tetrachloride in prokaryotic organisms
Test system
Endpoint
Test conditions
Results"
Without
activation
With
activation1"
Dosec
Reference
a + = positive, ± = equivocal or weakly positive, - = negative, (T) = toxicity, ND = no data.
b Exogenous metabolic activation used, typically induced rat liver S9.
0 Lowest effective dose for positive results, highest dose tested for negative results, ND = no data, NA = not applicable.
d Increase in revertants not dose-related and cytotoxicity not discussed.
e Results similar with or without glutathione added to the S9 mix. Positive response is based on the magnitude of response as statistical analyses were
not performed.
75
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Table 4-9. Genotoxicity studies of carbon tetrachloride in non-mammalian eukaryotic organisms
Test system
Saccharomyces cerevisiae
D7
S. cerevisiae D7
S. cerevisiae D7
S. cerevisiae RSI 12
S. cerevisiae RSI 12
S. cerevisiae RSI 12
S. cerevisiae RS112
(arrested in S phase)
S. cerevisiae RSI 12
(arrested in S phase)
S. cerevisiae RSI 12
(arrested in Gl phase)
S. cerevisiae RSI 12
(arrested in Gl phase)
S. cerevisiae AGY3
(arrested in G2 phase or
growing normally)
S. cerevisiae D6 1 .M
Endpoint
Gene conversion
Mitotic recombination
Reverse mutation
Intrachromosomal
recombination
Intrachromosomal
recombination
Interchromosomal
recombination
Intrachromosomal
recombination
Interchromosomal
recombination
Intrachromosomal
recombination
Interchromosomal
recombination
Intrachromosomal
recombination
Aneuploidy
Test conditions
Preincubation assay in capped
tubes
Preincubation assay in capped
tubes
Preincubation assay in capped
tubes
Preincubation assay
Preincubation assay
Preincubation assay
Preincubation assay
Preincubation assay
Preincubation assay
Preincubation assay
Preincubation assay
Standard 16-hour incubation or
cold-interruption regimen
Results3
Without
activation
+ (T)
+ (T)
+ (T)
+ (T)
+ (T)
+ (T)
-
-
+ (T)
+ (T)
+ (T)
-
With
activation1"
ND
ND
ND
ND
+ (T)
+ (T)
ND
ND
ND
ND
ND
ND
Dosec
5230 ug/mL
5230 ug/mL
5230 ug/mL
2000 ug/mL
4000 ug/mL
4000 ug/mL
8000 ug/mL
8000 ug/mL
5000 ug/mL
5000 ug/mL
8000 ug/mL
6400 ug/mL
Reference
Callenetal., 1980
Callenetal., 1980
Callenetal., 1980
Brennan and
Schiestl, 1998
Schiestletal., 1989;
Galli and Schiestl,
1998
Galli and Schiestl,
1998
Galli and Schiestl,
1998
Galli and Schiestl,
1998
Galli and Schiestl,
1996; Galli and
Schiestl, 1998
Galli and Schiestl,
1996; Galli and
Schiestl, 1998
Galli and Schiestl,
1995
Whittaker et al.,
1989
76
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Table 4-9. Genotoxicity studies of carbon tetrachloride in non-mammalian eukaryotic organisms
Test system
Aspergillus nidulans PI
A. nidulans 35
A. nidulans PI
A. nidulans PI
Drosophila melanogaster
Endpoint
Somatic segregation
due to cross over and
aneuploidy
Forward mutation
Somatic segregation
(positive for
aneuploidy; negative
for cross over)
Somatic segregation
(positive for
aneuploidy; negative
for cross over)
Mutation
Test conditions
Plate incorporation assay
Plate incorporation and
growth-mediated assays
Mitotic segregation assay
Mitotic segregation assay
Sex-linked recessive lethal
assay
Results"
Without
activation
+ (T)
±(T)
+(T)
+ (T)
With
activation1"
ND
ND
ND
ND
NA
Dosec
0.5%
0.5%
0.04%
0.0275%
25,000 ppm in
feed or 2000
ppm injection
Reference
Gualandi, 1984
Gualandi, 1984
Crebelli et al., 1988
Benignietal., 1993
Fouremanetal.,
1994
a + = positive, ± = equivocal or weakly positive, - = negative, (T) = toxicity, ND = no data.
b Exogenous metabolic activation not used for most tests because fungi have metabolic capabilities.
0 Lowest effective dose for positive results, highest dose tested for negative results, ND = no data, NA = not applicable.
77
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Table 4-10. Genotoxicity studies of carbon tetrachloride in mammalian cells in vitro
Test system
Human peripheral lymphocytes
Go
Human peripheral lymphocytes
Go
Human lymphocytes from 2
donors
Human lymphocytes
Human lymphocytes
Lamb peripheral lymphocytes
Lamb peripheral lymphocytes
Lamb peripheral lymphocytes
h2El cell line (cDNA for
CYP2E1)
MCL-5 cell line (cDNA for
CYPs 1A2, 2A6, 3A4, and
2E1, and epoxide hydrolase)
AHH-1 cell line (expresses
CYP1A1)
Chinese hamster ovary cells
Endpoint
Chromosomal
aberrations
Sister chromatid
exchange
Micronucleus
formation
DNA breaks
Unscheduled DNA
synthesis
Chromosomal
aberrations
Micronucleus
formation
Sister chromatid
exchange
Micronucleus
formation
Micronucleus
formation
Micronucleus
formation
Chromosomal
aberrations
Test conditions
30 Minute incubation in sealed
tubes
30 Minute incubation in sealed
tubes
Test conducted in capped tubes
Comet assay
4-Hour culture, autoradiography
48-Hour incubation
48-Hour incubation
48-Hour incubation
Immunofluorescent labeling of
kinetochore proteins
Immunofluorescent labeling of
kinetochore proteins
Immunofluorescent labeling of
kinetochore proteins
Assay conducted in sealed flasks
Results3
Without
activation
-(T)
-(T)
(2-)d
-
-
-
+
+
+ e(T)
+ e(T)
-
-
With
activation1"
-(T)
-(T)
±
(l-)d
-
-
ND
+
±
ND
ND
ND
-
Dosec
76 ug/mL
48 ug/mL
1540 ug/mL
3080 ug/mL
16,000 ug/mL
16 ug/mL
8 ug/mL (w/out
activation)
16 ug/mL
(w/activation)
4 ug/mL
308 ug/mL
308 ug/mL
1540 ug/mL
3000 ug/mL in
DMSOf
Reference
Garry etal., 1990
Garry etal., 1990
Tafazolietal., 1998
Tafazolietal., 1998
Perocco and Prodi,
1981
Sivikova etal., 2001
Sivikova etal., 2001
Sivikova etal., 2001
Doherty et al., 1996
Doherty et al., 1996
Doherty et al., 1996
Loveday et al., 1990
78
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Table 4-10. Genotoxicity studies of carbon tetrachloride in mammalian cells in vitro
Test system
Chinese hamster ovary cells
Chinese hamster ovary cells
V79 Chinese hamster lung cell
line
V79 Chinese hamster lung cell
line
Syrian hamster embryo cells
Mouse lymphoma L5 178Y
cells
Mouse lymphoma L5 178Y
cells
RLj cultured cell line derived
from rat liver
RLj cultured cell line derived
from rat liver
Hepatocytes- primary cultures
from 4 human donors
Hepatocytes isolated from male
Sprague-Dawley rats
Hepatocytes isolated from rats
Endpoint
Sister chromatid
exchange
Lagging
chromosomes and
multipolar spindles
Aneuploidy
c-Mitosis (spindle
disturbance)
Morphological
transformation
Mutation at tk locus
DNA strand breaks
Chromosomal
aberrations
Sister chromatid
exchange
Unscheduled DNA
synthesis
Unscheduled DNA
synthesis
DNA single strand
breaks
Test conditions
Assay conducted in sealed flasks
Anaphase analysis
3 -Hour incubation
30-Minute incubation
Clonal assay
4-Hour incubation
Alkaline elution
Assay conducted in sealed flasks
Assay conducted in sealed flasks
21.5 to 24 hr incubation periods
Autoradiography and flow
cytometric assays
Alkaline elution
Results"
Without
activation
-(T)
+
+
±(T)
±g
ND
ND
-
-
ND
-
±(T)
With
activation1"
ND
ND
ND
ND
-(T)
+(T)
ND
ND
(4-)d
ND
ND
Dosec
1490 ug/mL
(w/out
activation) 2930
ug/mL(w/
activation) note:
both inDMSOf
8000 ug/mL
246 ug/mL
492 ug/ml
3 ug/mL
635 ug/mL
1007 ug/mL
0.02 ug/mL in
DMSOd
0.02 ug/mL in
DMSOd
154 ug/mL
154 ug/mL
461 ug/mL
Reference
Loveday et al., 1990
Coutino, 1979
Onfelt, 1987
Onfelt, 1987
Amacher and
Zelljadt, 1983
Wangenheim and
Bolcsfoldi, 1988
Garberg et al., 1988
Dean and Hodson-
Walker, 1979
Dean and Hodson-
Walker, 1979
Butterworthetal.,
1989
Seldenetal., 1994
Sinaetal., 1983
79
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Table 4-10. Genotoxicity studies of carbon tetrachloride in mammalian cells in vitro
Test system
Hepatocytes isolated from
female Wistar rats
Hepatocytes isolated from
female Wistar rats
Hepatocytes isolated from
female Wistar rats
Calf thymus DNA
CalfthymusDNA
Mouse liver chromatin
Hepatocytes isolated from
Sprague-Dawley rats
Hepatocytes isolated from C3H
mice
Hepatocytes isolated from
Syrian golden hamsters
Endpoint
DNA single strand
breaks
DNA adduct
formation
DNA adduct
formation
DNA binding of
radiolabeled
chemical
DNA binding of
radiolabeled
chemical
DNA binding
DNA binding
DNA binding
DNA binding
Test conditions
Comet assay
MidG adducts formed secondary
to lipid peroxidation
SoxodG adducts formed
secondary to lipid peroxidation
30 min incubation with rat and
mouse microsomes
60 min incubation under a N2
atmosphere
2 and 4 hr incubation with
binding measured in DNase I-
sensitive and -resistant
chromatin DNA
Measured as radioactivity bound
to DNA after a 1 hr incubation
with microsomes
Measured as radioactivity bound
to DNA after a 1 hr incubation
with microsomes
Measured as radioactivity bound
to DNA after a 1 hr incubation
with microsomes
Results"
Without
activation
±
±
±(T)
+
ND
ND
±
±
±
With
activation1"
ND
ND
ND
+
+
+
±
±
±
Dosec
154 ug/mL
154 ug/mL
615 ug/mL
5.6 ug/mL
154 ug/ml
192 ug/mL
31 ug/mL
31 ug/mL
31 ug/mL
Reference
Beddowes et al.,
2003
Beddowes et al.,
2003
Beddowes et al.,
2003
Rocchietal., 1973
DiRenzoetal., 1982
Oruambo and Van
Duuren, 1987
Castro etal., 1989
Castro etal., 1989
Castro etal., 1989
80
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Table 4-10. Genotoxicity studies of carbon tetrachloride in mammalian cells in vitro
Test system
Endpoint
Test conditions
Results"
Without
activation
With
activation1"
Dosec
Reference
a + = positive, ± = equivocal or weakly positive, - = negative, (T) = toxicity, ND = no data.
b Exogenous metabolic activation used, typically induced rat liver S9.
0 Lowest effective dose for positive results, highest dose tested for negative results, ND = no data, NA = not applicable.
d Results for the individual donors are presented.
e Increase mostly in kinetochore-positive (aneugenic) micronuclei which occurred at the lower (308 ug/ml) concentration, and some increase in
kinetochore-negative (clastogenic) micronuclei which was significantly increased at the highest (1538 ug/ml) test concentration.
f DMSO = dimethyl sulfoxide
g Although declared positive by the authors, the induced frequency is well within the currently accepted control range.
81
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Table 4-11. Genotoxicity studies of carbon tetrachloride in mammalian systems in vivo
Test system
Mouse (101/H, male)
Rat (Sprague-Dawley, male)
Mouse (BDF1, male)
Mouse (BDF1, male)
Mouse (BDF1, male)
Mouse (CD-I, male)
Mouse (CD-I, male and
female)
Mouse (CD-I, male)
Rat (F344, male)
Endpoint
Chromosomal
aberrations in bone
marrow
Chromosomal
aberrations in bone
marrow
Micronucleus
formation in bone
marrow
Micronucleus
formation in bone
marrow
Micronucleus
formation in
peripheral blood
Micronucleus
formation in
peripheral blood
Micronucleus
formation in bone
marrow
DNA damage in
stomach, kidney,
bladder, lung, brain,
and bone marrow
DNA breakage
Test conditions
Metaphase analysis of samples
collected 6 to 48 hr after
dosing
Metaphase analyses from
animals sacrificed 24 hr after
dosing
Analyzed polychromatic
erythrocytes from specimens
prepared 24 hours after dosing
Analyzed polychromatic
erythrocytes from specimens
prepared 24 hours after dosing
Analyzed reticulocytes from
specimens prepared 24-72
hours after dosing
Analyzed reticulocytes from
specimens prepared 24-72
hours after dosing
Analyzed polychromatic
erythrocytes from femur bone
marrow of mice killed 24 or
48 hours after dosing
Comet assay on stomach,
kidney, bladder, lung, brain,
and bone marrow obtained 0,
3, or 24 hours after dosing
Comet assay on peripheral
blood cells
Results3
Without
activation
- (T)
"
-(T)
-(T)
"
d
-(T)
±(T)
With
activation1"
NA
NA
NA
NA
NA
NA
NA
NA
NA
Dosec
8000 mg/kg
injected i.m.
1600 mg/ml by
gavage
2000 mg/kg by
gavage (2x)
2000 mg/kg by
gavage
3000 mg/kg by
i.p. injection
2000 mg/kg by
gavage in olive
oil
3000 mg/kg
i.p. in olive oil
2000 mg/kg by
gavage
120 mg/kg by
i.p. injection
Reference
Lil'p, 1982
Rossi etal., 1988
Moritaetal., 1997;
Suzuki etal., 1997
Moritaetal., 1997;
Suzuki etal., 1997
Suzuki etal., 1997
Moritaetal., 1997
Crebelli et al., 1999
Sasaki et al., 1998
Kadiiska et al., 2005
82
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Table 4-11. Genotoxicity studies of carbon tetrachloride in mammalian systems in vivo
Test system
Mouse (NMRI, male and
female)
Rat (Wistar, female, partially
hepatectomized)
Rat (F-344, male)
Rat (strain and sex not
specified)
Rat (BD-VI, male)
Rat (Sprague-Dawley, male)
Mouse (CD-I, male)
Rat (Sprague-Dawley CD stain,
female)
Rat (Sprague-Dawley, male)
Rat (Wistar, male)
Endpoint
DNA strand breaks
in liver
DNA damage in
liver
DNA strand breaks
in liver
DNA breaks in liver
DNA strand breaks
in liver
DNA damage in
liver
DNA strand breaks
in liver
DNA strand breaks
in liver
DNA strand breaks
in liver
DNA strand breaks
in liver
Test conditions
Alkaline elution of sample
collected 4 hr after dosing
Caffeine elution 4 or 24 hours
after dosing
Alkaline elution on primary
hepatocytes isolated from rats
sacrificed 2-48 hours after
dosing
Alkaline elution on liver
nuclei obtained 1 hr after
dosing
Alkaline elution on primary
hepatocytes isolated from rats
sacrificed 4 hours after dosing
Viscometric assay on rats
sacrificed 2 hours after dosing
Alkaline elution
Alkaline elution on primary
hepatocytes isolated from rats
dosed 21 and 4 hr before
sacrifice
DNA strand breaks in
hepatocytes were measured by
a fluorometric assay for DNA
unwinding 1 hr after dosing
Breaks in DNA of non-
parenchymal cells identified
by in situ nick translation 12
to 96 hr after dosing.
Results"
Without
activation
-
"
-(T)
-
+ (T)
±(T)e
With
activation1"
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Dosec
4000 mg/kg by
gavage
800 mg/kg by
gavage in corn
oil
400 mg/kg by
corn oil gavage
4 mg/kg by i.p.
injection
4000 mg/kg by
i.p. injection
200 mg/kg by
i.p. injection
80 mg/kg by
corn oil gavage
1050 mg/kg by
oral gavage in
corn oil (2x)
160 mg/kg in
corn oil by i.p.
1600 mg/kg
i.p. in olive oil
Reference
Schwarzetal., 1979
Stewart, 1981
Bermudez et al.,
1982
Kittaetal., 1982
Barbinetal., 1983
Brambillaetal.,
1983
Cans and Korson,
1984
Kitchin and Brown,
1989
Ikegwuonu and
Mehendale, 1991
Nakamura and
Hotchi, 1992
83
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Table 4-11. Genotoxicity studies of carbon tetrachloride in mammalian systems in vivo
Test system
Rat (Wistar, male)
Mouse (CD-I, male)
Rat (Wistar, male)
Rat (Wistar, male)
Rat (Wistar, female)
Rat (Wistar, female)
Rat (F-344, male)
Endpoint
DNA strand breaks
in liver
DNA damage in
liver
DNA fragmentation
in liver
DNA fragmentation
in liver
Unscheduled DNA
synthesis in liver
Unscheduled DNA
synthesis in liver
Unscheduled DNA
synthesis in liver
Test conditions
Breaks in DNA of non-
parenchymal cells identified
by in situ nick translation after
dosing twice a week until
week 12 with sacrifices at 3,
6, 9, 12, 15, and 18 weeks.
Comet assay on liver obtained
0, 3, or 24 hours after dosing
TUNEL f assay on rats
sacrificed 1 day after the
second dose
TUNEL f assay on rats
sacrificed at 10, 15, 20, 25 and
30 hr after dosing
Animals injected with
hydroxyurea (to stop de novo
DNA synthesis) and then
[3H]-thymidine 2 hours after
dosing
Animals injected with
hydroxyurea (to stop de novo
DNA synthesis) and then
[3H]-thymidine 17 hours after
dosing
Rats sacrificed 2 hr after
dosing; primary hepatocytes
isolated by liver perfusion and
cultured with [3H]-thymidine
Results"
Without
activation
±(T)e
+ (T)
+ (T)
+ (T)
+ (T)
With
activation1"
NA
NA
NA
NA
NA
NA
NA
Dosec
2000 mg/kg
(24x)
1000 mg/kg by
gavage
800 mg/kg by
ip; (2x)
240 mg/kg in
corn oil by ip
4000 mg/kg by
gavage in
liquid paraffin
4000 mg/kg by
gavage in
liquid paraffin
100 mg/kg by
corn oil gavage
Reference
Nakamura and
Hotchi, 1992
Sasaki et al., 1998
Cabreetal., 1999
Yasuda et al., 2000
Craddock and
Henderson, 1978
Craddock and
Henderson, 1978
Mirsalis and
Butterworth, 1980
84
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Table 4-11. Genotoxicity studies of carbon tetrachloride in mammalian systems in vivo
Test system
Rat (F-344, male)
Mouse (B6C3F1, male)
Mouse (B6C3F1, female)
Mouse (CD-I, male)
Rat (Sprague-Dawley, male)
Mouse (DC-1, male)
Rat (F-344, male)
Rat (F-344, male)
Endpoint
Unscheduled DNA
synthesis in liver
Unscheduled DNA
synthesis in liver
Unscheduled DNA
synthesis in liver
Unscheduled DNA
synthesis in liver
Unscheduled DNA
synthesis
Chromosomal
fragments and
bridges in liver
Chromosomal
aberrations in liver
Sister chromatid
exchange in liver
Test conditions
Rats sacrificed 2-48 hours
after dosing; primary
hepatocytes isolated by liver
perfusion and cultured with
[3H]-thymidine
Rats sacrificed 12 hr after
dosing; primary hepatocytes
isolated by liver perfusion and
cultured with [3H]-thymidine
Rats sacrificed 12 hr after
dosing; primary hepatocytes
isolated by liver perfusion and
cultured with [3H]-thymidine
Mice sacrificed 3-48 hours
after dosing; liver cells
isolated and analyzed by
autoradiography
Unscheduled DNA synthesis
by labeling of DNA in
hydroxyurea-treated animals 1
hr after dosing
Anaphase analysis of squash
preparations prepared 72 hr
after dosing
Analyzed primary hepatocytes
cultured for 48 hr from rats
sacrificed 0-72 hours after
dosing
Analyzed primary hepatocytes
cultured for 48 hr from rats
sacrificed 0-72 hours after
dosing
Results"
Without
activation
-(T)
-(T)
-(T)
-(T)
±
With
activation1"
NA
NA
NA
NA
NA
NA
NA
NA
Dosec
400mg/kgby
corn oil gavage
lOOmg/kgby
oral gavage
lOOmg/kgby
oral gavage
100 mg/kg by
corn oil gavage
160 mg/kg in
corn oil by i.p.
8000 mg/kg
1600 mg/kg by
corn oil gavage
1600 mg/kg by
corn oil gavage
Reference
Mirsalisetal., 1982
Mirsalis, 1987;
Madleetal., 1994
Mirsalis, 1987;
Madleetal., 1994
Doolittle et al., 1987
Ikegwuonu and
Mehendale, 1991
Curtis and Tilley,
1968
Sawadaetal., 1991
Sawadaetal., 1991
85
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Table 4-11. Genotoxicity studies of carbon tetrachloride in mammalian systems in vivo
Test system
Rat (F-344, male)
Rat (Wistar, male)
Rat (Wistar, male)
Mouse (CBAxC575BL/6,
male)
Mouse (B6C3F1, lad
transgenic; Big Blue™, male)
Mouse (CD2F1 lacz transgenic,
Mutamouse™, male)
Mouse (CD2F1 lacz transgenic,
Mutamouse™, male)
Endpoint
Micronucleus
formation in liver
Micronucleus
formation in liver
Micronucleus
formation in liver
Micronucleus
formation and
ploidy levels in
liver
Mutations in lad
transgene in liver
Mutations in the
lacz transgene in
liver
Mutations in the
lacz transgene in
liver
Test conditions
Analyzed primary hepatocytes
cultured for 48 hr from rats
sacrificed 0-72 hours after
dosing
Analyzed primary hepatocytes
harvested 72 hr after dosing,
an optimal time to detect
micronuclei.
Analyzed primary hepatocytes
harvested 72 hr after dosing,
an optimal time to detect
micronuclei.
Analyzed primary hepatocytes
from rats sacrificed 5 days
after dosing and compared
with a partially
hepatectomized control.
The target lad gene is
recovered from genomic DNA
after 5 daily doses and the
animals sacrificed 7 days after
the first dose
The target lacz gene is
recovered from genomic DNA
after a single dose with the
animals being sacrificed 14
days later
The target lacz gene is
recovered from genomic DNA
after dosing with the animals
being sacrificed 7, 14 or 28
days later
Results"
Without
activation
±(T)
+ (T)8
-(T)
- (T)
-(T)1
With
activation1"
NA
NA
NA
NA
NA
NA
NA
Dosec
1600 mg/kg by
corn oil gavage
3 200 mg/kg by
gavage in corn
oil
3 200 mg/kg by
gavage in corn
oil
15 -Minute
inhalation at
0.05-0.1
mL/5L
35 mg/kg-day
(5x)
80 mg/kg by
gavage in corn
oil
1400 mg/kg by
gavage
Reference
Sawadaetal., 1991
Van Goethem et al.,
1993
Van Goethem et al.,
1995
Uryvaeva and
Delone, 1995
Mirsalisetal., 1994
Tombolan et al.,
1999; Lambert etal.,
2005
Hachiya and
Motohashi, 2000;
Lambert et al., 2005
86
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Table 4-11. Genotoxicity studies of carbon tetrachloride in mammalian systems in vivo
Test system
Rat (Wistar, male)
Mouse (Swiss, male)
Rat (Sprague-Dawley, male)
Mouse (A/J, male)
Mouse (A/J, male)
Rat (Sprague Dawley, male)
Rat (Sprague-Dawley, male)
Mouse (C3H, male)
Hamster (Syrian golden, male)
Endpoint
DNA binding in
liver
DNA binding in
liver
DNA binding in
liver
DNA binding in
liver
DNA binding in
liver
DNA binding to
mitochondria and
nucleus
DNA binding in
liver
DNA binding in
liver
DNA binding in
liver
Test conditions
DNA extracted from liver of
rats (with or without
methylcholanthrene
pretreatment) sacrificed 12
hours after dosing
DNA extracted from liver of
mice (some pretreated with
methylcholanthrene)
sacrificed 12 hours after
dosing
DNA isolated from liver slices
of rats sacrificed 6 hours after
dosing
DNA isolated from liver slices
of mice sacrificed 6 hours
after dosing
DNA isolated from liver slices
of mice sacrificed 6 hours
after dosing
Mitochondrial DNA isolated
from the livers at 5 and 24 hr
after dosing
DNA isolated from liver slices
of rats sacrificed 6 hours after
dosing
DNA isolated from liver slices
of mice sacrificed 6 hours
after dosing
DNA isolated from liver slices
of hamsters sacrificed 6 hours
after dosing
Results"
Without
activation
+h
±
±
+ (T)
+ (T)
±
±
±
With
activation1"
NA
NA
NA
NA
NA
NA
NA
NA
NA
Dosec
56 mg/kg i.p.
56 mg/kg i.p.
1.4 mg/kg i.p.
in olive oil
1.4 mg/kg i.p.
in olive oil
3200 mg/kg
i.p. in olive oil
3.2 mg/kg in
corn oil
1200 mg/kg
i.p. in olive oil
1200 mg/kg
i.p. in olive oil
1200 mg/kg
i.p. in olive oil
Reference
Rocchietal., 1973
Rocchietal., 1973
Diaz Gomez and
Castro, 1980a
Diaz Gomez and
Castro, 1980a
Diaz Gomez and
Castro, 1980a
Levy and Brabec,
1984
Castro et al., 1989
Castro et al., 1989
Castro et al., 1989
87
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Table 4-11. Genotoxicity studies of carbon tetrachloride in mammalian systems in vivo
Test system
Rat (strain and sex not
specified)
Rat (Sprague-Dawley, sex not
specified)
Rat (strain and sex not
specified)
Hamster (Syrian golden,
female)
Rat (F-344, male)
Rat (F-344, female)
Rat (Fischer, male)
Rat (F-344, male)
Endpoint
DNA adducts in
liver
DNA adducts in
liver
DNA adducts in
liver
DNA adducts in
liver and kidney
DNA adducts in
liver
DNA adducts in
liver, kidney, lung,
colon, and
forestomach
DNA adducts in
liver
DNA adducts in
liver
Test conditions
Deoxyguanosine-
malondialdehyde adducts
measured 48 hr after dosing
MidG adducts formed
secondary to lipid
peroxidation measured 4 days
after dosing
Deoxyguanosine-
malondialdehyde adducts
measured 48 hr after dosing
13-HPO and
malondialdehyde-derived
adducts formed secondary to
lipid peroxidation detected by
32P-postlabelling analysis 4 hr
after treatment
HNE-dG adducts formed
secondary to lipid
peroxidation
HNE-dG adducts formed
secondary to lipid
peroxidation. Samples
collected 4, 8, 16 or 24 hr after
final dose.
8-OHdG adducts were
measured by
immunohistochemistry and
electrochemical detection at
times from 6 hr to 7 days
8-OHdG adducts measured at
the end of week 1 after dosage
on days 1 and 4
Results"
Without
activation
+ (T)
+ (T)
±(T)
+ (T)
+ (T)
+ (T)
±(T)
With
activation1"
NA
NA
NA
NA
NA
NA
NA
NA
Dosec
1600mg/kgby
gavage
0.1 mg/kgby
corn oil gavage
160 mg/kg by
oral gavage
160 mg/kg by
corn oil gavage
3200 mg/kg
i.p. in olive oil
500 mg/kg i.p.
(Ior4x)
3 200 mg/kg by
gavage in olive
oil
400 mg/kg by
s.c. injection
(2x)
Reference
Hadley and Draper,
1990
Chaudhary et al.,
1994
Draper etal., 1995
Wang and Liehr,
1995
Chung et al., 2000
Wacker etal., 2001
Takahashi et al.,
1998
Iwai et al., 2002
DRAFT - DO NOT CITE OR QUOTE
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Table 4-11. Genotoxicity studies of carbon tetrachloride in mammalian systems in vivo
Test system
Rat (F344, male)
Mouse (CD-I, female)
Mouse (ICR, male)
Mouse (ICR, male)
Rat (F-344, male)
Rat (Wistar, male)
Endpoint
DNA adducts in
urine
DNA binding in
liver
DNA binding in
liver
DNA binding in
liver
DNA methylation in
liver
DNA hypo-
methylation in liver
Test conditions
8-OHdG adducts measured in
the urine 7 and 16 hr after a
single dose
8-oxodG measured in the
livers of 2 month and 14
month animals dosed for 3
days and sacrificed on day 4.
32P -Postlabeling was used to
identify indigenous adducts
present 24 hr after a single
injection
32P -Postlabeling was used to
identify indigenous and
exogenous adducts present 1,
4, and 8 weeks after two
injections given a week part.
Hydrolyzed DNA was
analyzed for aberrant
methylation as increases in 7-
methylguanine and O6-
methylguanine, 12 hr after
dosing
The in vitro incorporation of
3H-methyl groups into isolated
hepatic DNA was increased
indicating that the DNA was
hypomethylated.
Results"
Without
activation
+ (T)
+
+ (T)
-(T)
+ (T)
+
With
activation1"
NA
NA
NA
NA
NA
NA
Dosec
120 mg/kg by
i.p. injection
43 mg/kg i.p.
in mineral oil
1200 mg/kg by
i.p. in corn oil
1200 mg/kg by
i.p. in corn oil
1000 mg/kg in
corn oil
800 mg/kg by
i.p. injection
2X per week
for 3 weeks
Reference
Kadiiska et al., 2005
Lopez-Diazguerrero
etal.,2005
Nathetal., 1990
Nathetal., 1990
Barrows and Shank,
1981
Varela-Moreiras et
al., 1995
89
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Table 4-11. Genotoxicity studies of carbon tetrachloride in mammalian systems in vivo
Test system
Endpoint
Test conditions
Results"
Without
activation
With
activation1"
Dosec
Reference
a + = positive, ± = equivocal or weakly positive, - = negative, (T) = toxicity, ND = no data.
b Exogenous metabolic activation not applicable (NA) for these in vivo studies.
0 Lowest effective dose for positive results, highest dose tested for negative results, ND = no data, NA = not applicable.
d The small statistically significant increase detected was considered biologically insignificant by the authors (and other reviewers).
e At this dose a roughly 3 fold increase in micronucleus formation was seen along with a decrease in binucleated cells (about 35-50%) indicating a
cytostatic and cytotoxic effect.
f TUNEL - terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling
8 Increase was in both centromere-lacking (5.5-fold) and centromere-containing (3.6-fold) micronuclei.
h With methylcholanthrene pretreatment only.
Note 1: The data in the paper by Sarkar and associates (Sarkar et al., 1999) was judged to be insufficiently reliable to be included in the table.
90
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4.4.2.1. Genotoxicity Studies: Prokaryotic Organisms
Studies of bacterial reverse mutation assays (see Table 4-8) indicate that, in most cases,
carbon tetrachloride is not directly genotoxic at concentrations below cytotoxic levels. Carbon
tetrachloride was negative in most standard plate incorporation assays for reverse mutation in
Salmonella typhimurium, with or without addition of a mammalian metabolic activation system
(Brams et al., 1987; de Flora, 1981; McCann et al., 1975). Increases in reversion frequency were
reported by Varma et al. (1988), but the changes were not dose-related. The positive responses
were reported at lower doses and negative responses at higher doses. Varma et al. (1988) did not
present data for the positive controls nor discuss cytotoxicity, making it unclear how to interpret
these data. Some S. typhimurium reversion studies used modified testing techniques in order to
account for the volatile nature of carbon tetrachloride. Preincubation assays conducted in capped
tubes were performed by Uehleke et al. (1977) and Zeiger et al. (1988). Both of these research
groups obtained negative results. Gas-phase exposure studies have been conducted in various
closed systems (Araki et al., 2004; Barber et al., 1981; Simmon and Tardiff, 1978; Simmon et
al., 1977). Results were negative in most of these studies, although Araki et al. (2004) found a
small increase in reversion frequency in TA98 at concentrations of 1% (10,000 ppm) and above,
when tested without activation. It should be noted that the average control frequency of 13
revertants per plate in this study is unusually low, and even the elevated response of 31
revertants per plate seen at the 50,000 ppm concentration is well within the range of spontaneous
revertants typically seen in TA98 controls (30-50 revertants per plate) (Maron and Ames, 1983).
In other studies using S. typhimurium, negative or equivocal results were reported for
carbon tetrachloride in a preincubation forward mutation assay using strains BA13 and BAL13
with and without metabolic activation (Roldan-Arjona and Pueyo, 1993; Roldan-Arjona et al.,
1991), and in an SOS induction assay using strain TA1535/pSK1002 (Nakamura et al., 1987).
More varied results were seen in experiments using E. coli. Carbon tetrachloride was negative in
a SOS chromotest assay (Brams et al., 1987), in a spot test (De Flora et al., 1984), and a
preincubation assay when evaluated for differential DNA repair (Hellmer and Bolcsfoldi, 1992).
In contrast, using strains that are more sensitive to oxidative mutagens, increases in DNA repair
were reported by De Flora et al. (1984) and increases in reverse mutation were reported by Araki
et al. (2004) and Norpoth et al. (1980). In the DeFlora et al. (1984) study, carbon tetrachloride
was more toxic to the E. coli strain CM871 (uvrA- recA- lexA-) than it was to the isogenic
repair-proficient WP2 strain or WP67 (uvrA-polA-). Although a similar pattern was seen in the
presence of metabolic activation, carbon tetrachloride was more active in the absence of
activation. The differential toxicity was seen initially using the liquid micromethod, and then
confirmed using a 2-hour pre-incubation assay. In the report of Araki et al. (2004), carbon
tetrachloride produced a modest 2.5-fold increase in mutations in the WP2uvrA/pKM101 strain
of E. coli both in the presence and absence of metabolic activation. The peak response was seen
after 24 hours of exposure at a high (20,000) ppm concentration. The control frequencies appear
91 DRAFT - DO NOT CITE OR QUOTE
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to be unusually low and the induced response was within the control values reported by others
(Martinez et al., 2000; Damment et al., 2005). Additionally, a weak positive response
(statistically significant but well less than a twofold increase) for E. coli WP2wvrA was reported
by Norpoth et al. (1980) at high levels (about 25,000 ppm) in another gas phase exposure study.
A stronger mutagenic response was seen when carbon tetrachloride was tested in the
repair-proficient WP2/pKM101 strain of E. coli. A doubling in mutant frequency was observed
at the 5000 ppm carbon tetrachloride concentration and reached a fivefold increase compared to
pooled controls at the 20,000 ppm concentration. The increase was seen in experiments with and
without metabolic activation as well as with S9 plus reduced glutathione. Because the WP2
strains of E. coli have an AT base pair at the critical mutation site within the trpE gene, they
have been recommended for screening oxidizing mutagens (Gatehouse et al., 1994; Martinez et
al., 2000). This increased sensitivity to oxidative damage may help explain both the Araki et al.
(2004) and the DeFlores et al. (1984) isolated positive results, although some aspects of the
studies are still unusual. The greater response in the repair-proficient strain seen in the Araki et
al. (2004) study as compared to the repair-deficient strain was unexpected, and led the authors to
postulate that a cross-linking metabolite might be responsible. If true, this could also be related
to oxidative damage as lipid peroxidation-derived products have been shown to form DNA and
DNA-protein cross-links (Kurtz and Lloyd, 2003; Niedernhofer et al., 2003). Again, the control
frequencies reported by Araki (2004) are lower than those reported by others (Watanabe et al.,
1998), but in this case, the induced mutant frequencies substantially exceed the control range of
either group. Araki et al. (2004) reported a tenfold increase in mutants in the WP2/pKM101
experiments without S9. However, approximately half of the observed increase appeared to be
due to an unusually low mutant frequency. Also, it should be noted that the results were not
statistically analyzed as the experiments were not performed in triplicate.
Some caution should be exercised in the interpretation of these and other in vitro studies
as a number of the factors listed in Table 4-12 could potentially influence the outcome of the
assays and contribute to both positive and negative results. For example, the bioactivation of
carbon tetrachloride to a mutagenic species can be affected in a variety of ways. The initial step
in the bioactivation of carbon tetrachloride is a cytochrome P450 monooxygenase-mediated
formation of the trichloromethyl radical (Halliwell and Gutteridge, 1999; Weber et al., 2003).
This radical is highly reactive, and as a result, may not be able to cross the bacterial cell wall or
membranes to access the bacterial DNA. The trichloromethyl radical or a derived species can
also react with and inactivate the monooxygenase activation system (Weber et al., 2003), which
could also affect the outcome of the in vitro assays. In addition, many of the commonly used
vehicle solvents used for in vitro testing such as methanol, DMSO, and ethanol are also
metabolized by the cytochrome P450 2E1 isoform CYP2E1 (Hyland et al., 1992), the isoform
primarily involved in carbon tetrachloride metabolism, and may have interfered with the
92 DRAFT - DO NOT CITE OR QUOTE
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bioactivation of carbon tetrachloride in these test systems. In addition, DMSO can act as a free
radical scavenger (Halliwell and Gutteridge, 1999).
Similarly, when standard inducing procedures (Arochlor 1254 or the combination of
phenobarbitone and 6eta-naphthoflavone) have been used, the levels of CYP2E1 in the rat liver
are markedly suppressed (Burke et al., 1994). This would lead to a decrease in CYP2E1 in the
S9 used for the test and could potentially contribute to the observed negative results.
Furthermore, although carbon tetrachloride has been evaluated many times in the standard
Salmonella test strains, it has not been tested in either TA102 or TA104 and only a few times in
the E. coli WP2 strains, the strains that would be the most sensitive to the oxidative DNA
damage likely to be generated during carbon tetrachloride toxicity. Because of the many
possible confounding factors, the in vitro carbon tetrachloride results should be interpreted
cautiously.
Table 4-12. Challenges in evaluating carbon tetrachloride genotoxicity
Large number of genotoxicity studies
Elevated error rates related to multiple statistical tests and comparisons
Requirement to test to high levels of toxicity to ensure a true negative response
Non-specific effects that can occur at very high chemical concentrations
Potential volatility from culture media
Requirement for metabolic activation
Downregulation of CYP2E1 synthesis shortly after carbon tetrachloride administration
Inhibition of cytochrome P450 monoxygenases by primary carbon tetrachloride
metabolite(s)
Competitive inhibition of CYP2E1 by common solvents used as vehicles (ethanol, methanol,
DMSO)
Free radical-scavenging properties of common vehicles such as DMSO
Possible inability of reactive trichloromethyl radical generated extracellularly by rat
postmitochondrial supernatant to cross the bacterial cell wall or eukaryotic cell membrane
and damage the DNA of the cell being tested
Commonly used enzyme inducers suppress CYP2E1 levels in the rat liver S9
Possible influence of dosing vehicle (corn oil, olive oil) in vivo
Concurrence of cytotoxicity and genotoxicity
Occurrence of DNA breakage during apoptotic and necrotic cell death
Occurrence of multiple reactive species and potential mechanisms of genotoxicity
Difficulties in distinguishing direct and indirect genotoxic effects
Generation of genotoxic products secondary to lipid peroxidation
Genotoxic responses occurring secondary to inflammatory responses
93
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4.4.2.2. Genotoxicity Studies: Non-Mammalian Eukaryotic Organisms
Carbon tetrachloride has also been tested in the yeast Saccharomyces cerevisiae and the
mold Aspergillus nidulans (Table 4-9). In contrast to the bacterial results, the majority of the
studies conducted in these species have yielded positive results. However, the results obtained
from the two fungal species differ significantly, most likely due to the test strains selected and
the endpoints chosen for examination. In initial studies by Callen et al. (1980), carbon
tetrachloride induced strong (>20-fold) increases in gene conversion and mitotic conversion and
a weak (2.5-fold) increase in reverse mutations when tested at high concentrations in the yeast
D7 strain in a preincubation assay employing capped tubes. The increases were only seen at the
highest test concentration of 34 mM, one that caused extensive toxicity (90%). These initial
results were followed by a series of studies by Schiestl and co-workers using yeast strains that
were designed to detect intrachromosomal recombination (DEL assay) that results from double
stranded DNA breakage. Interchromosomal recombination can also be measured in these strains.
In the initial study using the DEL assay (Schiestl et al., 1989), carbon tetrachloride at a
concentration of 8000 |ig/mL induced a strong (25-fold) increase in intrachromosomal
recombinants with no increase in interchromosomal recombination. Toxicity was greater than
99% at the highest test concentration where the increase in recombinants was seen. Follow-up
studies showed that the induced recombinants occurred during the Gl and G2, but not S phase of
the cell cycle, and in some cases an increase in interchromosomal recombination was also seen.
The dose-response curves tended to be steep and occurred concurrently with significant toxicity
(Galli and Schiestl, 1996; Galli and Schiestl, 1995). Since carbon tetrachloride did not induce
recombination during S phase even though it was toxic, the authors suggested that carbon
tetrachloride acted by prematurely pushing Gl cells into S phase and G2 cells into cell division
(Galli and Schiestl, 1998). The inability to completely repair damaged DNA prior to replication
or cell division might result in DNA strand breakage and subsequent recombination. Brennan
and Schiestl (1998) showed that yeast cells treated with carbon tetrachloride showed an increase
in oxidative radical species as measured by the intracellular oxidation of 2,7-dichlorofluorescein
diacetate. N-acetylcysteine did not exhibit a protective effect on carbon tetrachloride-induced
DEL recombination, although the results are difficult to interpret as increased toxicity was seen
in cells jointly treated with carbon tetrachloride and this sulfhydryl-containing agent.
In contrast to the recombinogenic effects seen with Saccharomyces cerevisiae, the assays
using Aspergillus nidulans primarily detected an abnormal segregation of chromosomes.
Following treatment with high concentrations (0.5%) of carbon tetrachloride, Gualandi (1984)
observed a significant (>20-fold) increase in abnormal chromosome segregation but only a weak
(~2.5-fold) increase in forward mutations. Toxicity at the test concentration was approximately
70%. Additional studies showed a strong correlation between toxicity and altered segregation
leading to aneuploid cells. Cysteamine (a free-radical scavenger) was also co-administered with
carbon tetrachloride and showed some protection against the induced alterations in chromosome
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segregation. In a series of related studies, carbon tetrachloride was consistently shown to
interfere with chromosome segregation leading to aneuploidy. Crebelli et al. (1988)
demonstrated that carbon tetrachloride induced a ten-fold increase in chromosome segregation at
the highest (0.08%) concentration tested. Toxicity at this concentration was 72%. More modest
effects (~3-fold) were seen beginning at lower concentrations (0.04%) that were less toxic
(18%). Notably, no increase in crossing over was seen in these experiments. Similar results
both on chromosome segregation and crossing over were observed in a follow-up study using a
narrower and somewhat lower dose range (0.01 to 0.03%; Benigni et al., 1993). In a related
quantitative structure-activity-relationship study of carbon tetrachloride and 23 other chlorinated
aliphatic hydrocarbons, the ease at which the compounds were able to accept electrons, as
characterized by the energy of lowest unoccupied molecular orbital, was the best predictor of
their aneuploidy-inducing properties (Crebelli et al., 1992).
As indicated in Table 4-9, the genotoxic effects were seen in both Saccharomyces and
Aspergillus experiments without the use of exogenous metabolic activation. This is consistent
with studies that have shown actively growing cells of both species contain cytochrome P450
monooxygenase enzymes capable of bioactivating promutagens to mutagens (Bignami et al.,
1981; Callen et al., 1980). As indicated above, the studies in Saccharomyces detected primarily
recombination whereas those in Aspergillus detected primarily alterations in chromosome
segregation. This difference in outcome appears to be due primarily to the nature of the specific
strains used and the endpoints selected for evaluation by the investigators. There was a close
association seen between cytotoxicity and the recombinogenic and aneugenic effects measured in
the two systems.
Additionally, carbon tetrachloride did not produce sex-linked recessive lethal mutations
in Drosophila melanogaster (Foureman et al., 1994).
4.4.2.3. Genotoxicity Studies: Mammalian Cells In Vitro
Numerous studies have been performed to evaluate the ability of carbon tetrachloride to
cause genotoxic effects or precursor lesions in mammalian cells in vitro (Table 4-10). These
studies have been performed using both model cell systems frequently with exogenous metabolic
activation and hepatocytes that retain their xenobiotic-metabolizing capabilities.
Studies in non-target mammalian cells. In studies using peripheral blood lymphocytes
or lymphoblastoid cells, carbon tetrachloride yielded mixed results. As part of a study of
fumigants, Garry et al. (1990) exposed G0 lymphocytes to carbon tetrachloride for 30 minutes,
then cultured the lymphocytes and measured the frequencies of chromosome aberrations and
sister chromatid exchanges (SCEs). No increases in structural aberrations or SCEs were seen.
Tafazoli et al. (1998) used the micronucleus assay to measure chromosome loss or breakage in
the peripheral lymphocytes obtained from two donors. Exposure to different concentrations of
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carbon tetrachloride ranging from 1 to 40 mM did not induce a statistically significant increase in
micronucleated cells at any concentrations except at 10 mM in one donor with S9 mix and at
5 mM in the second donor without S9 mix. Cell division was not affected at these mutagenic
concentrations; however, the authors identified a cytotoxic concentration of 40 mM both with
and without S9 mix in one donor. To measure the amount of DNA strand breaks, Tafazoli et al.
used the in vitro Comet assay with isolated lymphocytes from the donors. No statistically
significant response was found for either tail length or tail moment at concentrations tested (5 to
20 mM) either with or without S9 mix. Carbon tetrachloride was also reported to be negative
when assayed for unscheduled DNA synthesis (UDS) in lymphocytes (Perocco and Prodi, 1981).
Each of these studies either used high carbon tetrachloride concentrations (>1500 |ig/mL) or
tested to toxic concentrations.
In contrast, when tested at relatively low concentrations, Sivikova et al. (2001) reported
that cultured ovine peripheral lymphocytes exposed to carbon tetrachloride exhibited modest
twofold increases in micronuclei in both the absence and presence of S9, and an approximately
25% increase in SCEs in the absence of S9. Under similar conditions, no increase in structural
chromosome aberrations was seen although a decrease in the mitotic index was detected.
Interestingly for both the MN and SCE experiments, the addition of vitamin E and selenium to
the cultures protected against the increases in MN and SCE, implicating a role for free radicals in
the observed genotoxic effects. In spite of the protective effects of the antioxidants, these studies
would still appear to be anomalous given the observations of effects at fairly low concentrations
and the greater activity in the absence of S9.
Doherty et al. (1996) reported that carbon tetrachloride induced micronuclei in two
human lymphoblastoid cell lines - one expressing CYP2E1 (h2El) and the other expressing
CYP1A2, 2A6, 3A4, 2E1 and microsomal epoxide hydrolase (MCL-5) - but not the CYP1 Al-
expressing AHH-1 cell line. Treatment of the cells with 10 mM carbon tetrachloride resulted in
a five- and a ninefold increase in micronucleated cells in the h2El and the MCL-5 cell lines,
respectively. The increases occurred mostly in kinetochore-positive micronuclei, indicating an
origin from chromosome loss. Smaller increases (-two to fourfold) in micronuclei originating
from chromosomal breakage (kinetochore-negative) were also seen. At the 10 mM
concentration, the percentage of binucleated cells, an indicator of cell proliferation and an
indirect indicator of cytotoxicity, was 6 - 7% of the control values indicating that the increase in
micronuclei occurred primarily under conditions producing potent cytotoxic or cytostatic effects.
In other studies involving non-target cell culture systems, carbon tetrachloride was
negative for inducing structural chromosome aberrations and SCEs in Chinese hamster ovary
(CHO) cells (Loveday et al., 1990). However, in a number of other assays using CHO and V79
cells, carbon tetrachloride in the absence of exogenous activation was reported to produce
modest increases in c-mitoses, generate multipolar spindles and lagging chromosomes during
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anaphase, and interfere with chromosome segregation resulting in aneuploidy (Onfelt, 1987;
Coutino, 1979).
Carbon tetrachloride was also tested for its ability to induce morphological
transformation in Syrian hamster embryo cells (Amacher and Zelljadt, 1983). In the
transformation assay, carbon tetrachloride was tested in both RPMI 1640 media with horse
serum and DMEM with fetal bovine serum. It was negative in the RPMI medium with 0
transformants among 2665 colonies. In DMEM, one transformed colony was seen in 2003
colonies scored. Although this was considered a positive result by the authors, the increase is not
statistically significant, does not meet current criteria for a positive result (Kerckaert et al.,
1996), and falls within the normal control frequencies of 0 to 0.8% reported for this type of
transformation assay (LeBoeuf et al., 1996).
In studies using mouse lymphoma (L5178Y) cells with exogenous activation, carbon
tetrachloride was inactive in inducing mutations at the tk locus when tested up to toxic
concentrations (Wangenheim and Bolcsfoldi, 1988). In a follow-up study employing similar
cells and conditions, DNA strand breaks were induced as measured by the alkaline elution assay.
The increases in strand breaks were accompanied by increases in cytotoxicity (Garberg et al.,
1988).
Studies in liver cells. Carbon tetrachloride has also exhibited mixed results when tested
in vitro using isolated hepatocytes or cell lines derived from the rat liver. In early studies by
Dean and Hodgson-Walker, carbon tetrachloride was negative for inducing structural
chromosome aberrations or SCEs when tested at a low concentration in a metabolically
competent rat liver cell line (Dean and Hodson-Walker, 1979). Similarly, no increase in UDS
was seen by Selden et al. (1994) in their studies using rat hepatocytes or by Butterworth et al.
(1989) in their UDS studies employing primary hepatocyte cultures from four human donors. In
contrast, using an alkaline elution assay on isolated rat hepatocytes, Sina and colleagues reported
a 3.1- to 5.0-fold increase in strand breaks at the highest concentration tested (3 mM), a dose that
also resulted in approximately 50-60% toxicity (Sina et al., 1983). A modest dose-related
increase in DNA strand breaks was also seen in the single cell gel electrophoresis (Comet) assay
by Beddowes et al. (2003). The increase in breaks reported by Beddowes was accompanied by
similar increases in the formation of the oxidative DNA adducts, 8-oxodeoxyguanosine and a
malondialdehyde deoxyguanosine adduct.
The ability of bioactivated carbon tetrachloride to react directly with DNA has been
investigated by a number of investigators using isolated DNA and nuclear preparations obtained
from hepatocytes. Initial studies by Rocchi and colleagues demonstrated that when radiolabeled
carbon tetrachloride was incubated with microsomes from uninduced and 3-methylcholanthrene-
induced mice and rats, modest increases in radiolabel were recovered following extensive
washing and extraction of the DNA with several solvents (Rocchi et al., 1973). This binding was
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greater in the incubations containing the 3-methylcholanthrene-induced microsomes. Similarly
DiRenzo et al. (1982) reported that significant binding of carbon tetrachloride to DNA (0.39
nmol/mg DNA) occurred following the incubation of radiolabeled carbon tetrachloride with
pronase-pretreated calf thymus DNA and microsomes from phenobarbital-induced rats. The
incubation was performed under a N2 atmosphere using conditions that in previous studies had
resulted in maximal binding to proteins and lipids. Oruambo and Van Duuren (1987)
investigated the binding of radiolabeled carbon tetrachloride to various regions of mouse
chromatin. Following a two-hour incubation with mouse hepatic microsomes, hepatic
chromatin, and radiolabeled carbon tetrachloride, the authors concluded that the carbon
tetrachloride metabolite(s) bound equally to both DNase I-sensitive and -resistant regions. After
4 hours of incubation, more radiolabel was recovered associated with DNase I-resistant DNA
than with DNase I-sensitive DNA. This preferential binding to transcriptionally inactive (DNase
I-resistant) sites in chromatin was seen as unique among carcinogens, and could be attributable
to changes in chromatin conformation or differential DNA repair. In addition, Castro et al.
(1989) investigated the ability of radiolabeled carbon tetrachloride to bind to the DNA of
purified nuclear preparations obtained from the livers of Sprague-Dawley rats, a strain resistant
to carbon tetrachloride carcinogenicity, and C3H mice and Syrian golden hamsters, two strains
that are sensitive to carbon tetrachloride hepatocarcinogenesis. Low levels of binding were
observed, which were increased in the mouse and hamster incubations when NADPH was
included in the microsomal incubation. The authors noted that there was no correlation between
sensitivity to carbon tetrachloride carcinogenesis (hamster > mouse » rat) and the binding of
carbon tetrachloride metabolites to DNA, either in vitro or in vivo (in vivo: hamster = mouse =
rat; in vitro with NADPH: hamster = mouse = rat; in vitro without NADPH: rat > mouse =
hamster).
Overall, these data indicate that under certain conditions carbon tetrachloride can induce
genotoxic effects in mammalian cells exposed in vitro. Although numerous negative studies
were seen, there are indications from multiple studies that at high doses, bioactivated carbon
tetrachloride is able to cause DNA breaks leading in some cases to chromosome breakage. There
are also multiple studies indicating that carbon tetrachloride is able to interfere with chromosome
segregation resulting in modest levels of chromosome loss and aneuploidy. However, since
exogenous bioactivation was required in some studies and not others, the observed effects may
result from both specific and non-specific mechanisms, some of which may not be operable in
vivo. The binding studies using radiolabeled carbon tetrachloride have significant weaknesses
(for discussion, see the following sections), but provide limited evidence that bioactivated carbon
tetrachloride can bind directly to DNA. The overall magnitude of the covalent binding appears
to be low. As seen in non-mammalian assay systems, in most cases where genotoxic effects
were observed, they occurred concurrently with significant cytotoxicity.
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4.4.2.4. Genotoxicity Studies: Mammalian Cells In Vivo
Carbon tetrachloride has been extensively tested for genotoxicity in mammalian systems
in vivo (Table 4-11). A number of these studies have been conducted using standard protocols
and examined genotoxicity in highly proliferating non-target organs such as the bone marrow. In
addition, a large number of studies have examined genotoxic effects or precursor lesions such as
DNA adducts occurring in the rodent liver. A summary of the important studies by target organ
and endpoint is presented below.
Chromosomal alterations and DNA breakage in non-target organs. In studies of
chromosomal alterations occurring in the bone marrow, carbon tetrachloride has shown negative
results for the induction of structural chromosome aberrations in the bone marrow of male
Sprague-Dawley rats and 101/H mice (Rossi et al., 1988; Lil'p, 1982), as well as for the
formation of micronuclei in the bone marrow and peripheral blood erythrocytes of male BDF1
mice (Suzuki et al., 1997; Morita et al., 1997). Negative results were also seen for the induction
of micrenucleated erythrocytes in the bone marrow and peripheral blood of both male and female
CD-I mice (Crebelli et al., 1999). In the Comet assay, no evidence of DNA breakage was seen
in the nucleated cells of the stomach, kidney, bladder, lung, brain or bone marrow of male CD-I
mice administered 2000 mg/kg carbon tetrachloride with sampling at 0, 3 and 24 hours after
dosing (Sasaki et al., 1998). In these same animals, significant increases in DNA breakage were
seen in the liver, although this was considered by the authors to be a false positive result because
it was accompanied by evidence of necrosis in the liver. In a biomarker study, carbon
tetrachloride was also reported to induce an isolated significant increase in DNA breakage in the
Comet assay in nucleated peripheral blood cells of male F344 rats (Kadiiska et al., 2005). The
increase is of questionable relevance as it was only seen at one of the three time points tested and
only at the lower of the two doses tested.
DNA breakage in rodent liver cells. Within the rodent liver, carbon tetrachloride has
been evaluated for a range of genotoxic effects across a considerable dose range. Fourteen
studies employed the alkaline elution or similar method to determine if carbon tetrachloride is
able to induce DNA breaks in liver cells in vivo. Negative results were seen in eight of the
studies, equivocal or weak responses were seen in two, and positive results were seen in four
studies. When positive or equivocal responses were seen, they consistently occurred at doses
where extensive toxicity or regenerative proliferation was manifest. For most of the studies that
showed a positive response, the responses appear to be more related to a general cytotoxic effect
rather than a specific genotoxic effect. A brief overview of each of the positive studies is
provided below.
Nakamura and Hotchi (1992) observed a modest increase in DNA breakage in their
studies of DNA breakage in non-parenchymal cells. The DNA breaks were identified using an in
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situ nick translation approach at time points ranging from 12 hours to 18 weeks after dosing.
Although breaks were seen, the authors argued that the breaks were most likely physiological in
nature, reflecting changes in proliferation and gene expression rather than direct carbon
tetrachloride-mediated DNA damage. In another series of experiments involving the adaptation
of the liver to long-term continuous carbon tetrachloride administration to mice, Gans and
Korson (1984) noted changes in the DNA synthesis of the liver nuclear DNA. As one aspect of
the study, the authors used an alkaline elution approach to study DNA damage in the liver of
CD-I mice. A maximal increase in DNA damage was seen 18 hours after administration. The
normal pattern of sedimentation was restored by 24 to 36 hours. The authors stated that "these
changes were observed only following doses of carbon tetrachloride which resulted in liver
necrosis. Doses of carbon tetrachloride which did not produce necrosis did not result in a shift in
the sedimentation of DNA."
Similarly, Cabre and associates detected DNA breaks in rats treated with two high doses
of carbon tetrachloride using the terminal deoxynucleotidyl transferase-mediated deoxyuridine
triphosphate nick end labeling (TUNEL) technique (Cabre et al., 1999). The TUNEL assay is
commonly used to measure DNA strand breaks occurring in apoptotic cells but also detects
breaks occurring in necrotic cells (Higami et al., 2004). Similarly, Yasuda and colleagues used
the TUNEL assay to study necrotic cell death induced by carbon tetrachloride and
dimethylnitrosamine (Yasuda et al., 2000). In the Yasuda studies of carbon tetrachloride-treated
livers, TUNEL staining was closely associated with the release of lysosomal enzymes into the
cytoplasm, and an intranuclear localization of lysosomal enzymes occurred at an early stage of
subcellular damage. This pattern was notably different from that seen with the alkylating agent,
dimethylnitrosamine. Given the high doses administered and the known hepatotoxicity of
carbon tetrachloride, the observed detection of DNA strand breaks in these and the other studies
is not surprising. As mentioned earlier and for the same reason, Sasaki et al. (1998) considered
the DNA strand breaks that they observed using the Comet assay to be false positives and not
relevant to assessing genotoxic potential since evidence of necrosis was present.
Unscheduled DNA synthesis in the rodent liver. A number of studies have been
performed to investigate the ability of carbon tetrachloride to induce UDS in the liver of rats and
mice treated in vivo. In an initial study of de novo and repair replication of DNA in the livers of
treated rats, Craddock and Henderson (1978) reported that oral administration of 4000 mg/kg
carbon tetrachloride increased the synthesis of DNA in non-replicating hydroxyurea-treated
hepatocytes 17 hours, but not 2 hours, after treatment. In the absence of the hydroxyurea
treatment, extensive DNA synthesis was seen at the 17-hour time point. Diethylnitrosamine,
ethyl ethanesulfonate, aflatoxin, and retrosine induced DNA repair replication at the earlier two-
hour sampling. The delay seen with carbon tetrachloride was suggested by the authors as
indicating that the repair was associated with damage caused by an indirect mechanism such as
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deoxyribonuclease activity resulting from lysosomal damage; however, the extensive DNA
synthesis occurring at the 17-hour time point is almost certainly due to proliferation following
extensive cell death induced by carbon tetrachloride. Under these conditions, it is not clear how
efficient the hydroxyurea inhibition of DNA synthesis would be. In a more recent study using
the hydroxyurea approach, Ikegwuonu and Mehendale (1991) saw similar results, although they
saw no increase in DNA breakage using an alkaline elution technique in a parallel study. The
observations of DNA repair in the absence of detectable DNA breaks are inconsistent and the
authors concluded that the hydroxyurea repair results were attributable to induced de novo
synthesis (post replication repair) rather than true DNA repair. It should also be noted that the
use of the hydroxyurea method to measure UDS is generally not recommended because of the
complex effects of hydroxyurea in the cell and its ability to directly induce UDS (for additional
details, see Madle et al., 1994).
Six other studies have been conducted using the currently recommended and more
reliable autographic method of detecting UDS. No increase in UDS induced by carbon
tetrachloride was seen even at doses exhibiting significant toxicity. With the autographic
method, DNA uptake is measured in individual cells allowing UDS to be clearly distinguished
from de novo synthesis.
To summarize the UDS results, eight in vivo studies have been performed investigating
UDS in the rodent liver following carbon tetrachloride administration. Two major methods for
measuring UDS were employed, the autographic method that allows UDS in individual cells to
be measured and that is considered to be more reliable, and a less reliable method that measures
DNA synthesis in the presence of hydroxyurea, an inhibitor of global de novo DNA synthesis.
The six studies that used the autoradiographic method yielded negative results whereas the two
that used the hydroxyurea method produced results most appropriately characterized as false
positives.
Chromosome aberrations and micronuclei in rodent liver cells. In cytogenetic assays of
hepatocytes isolated from treated rodents, carbon tetrachloride produced mixed, largely negative
results. In an early study by Curtis and Tiley (1968), no increase in chromosomal fragments or
bridges occurring in anaphase cells was seen in liver squash preparations of mice treated with a
high (8000 mg/kg) dose of carbon tetrachloride. Similar negative results for structural
chromosome aberrations, SCEs and micronuclei were reported at all time points in time course
studies conducted by Sawada et al. (1991). Negative results were also reported for micronucleus
formation and altered ploidy by Uryvaeva and Delone (1995).
In two studies conducted by Van Goethem and colleagues, however, an increase in
micronuclei was reported. In their initial study investigating the early stages of hepatic
carcinogenesis (Van Goethem et al., 1993), carbon tetrachloride was administered to male Wistar
rats at 3200 mg/kg and the frequency of micronuclei was measured in hepatocytes harvested 72
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hours later. Initial studies of the mitotic index and the percent binucleated cells indicated that 72
hours was the optimal time to harvest hepatocytes for the detection of micronuclei. High intra-
animal variability was seen, but the results suggested that the hepatocytes of the carbon
tetrachloride- (and CT+NaCl-) treated mice exhibited an increase in micronuclei (1.7-7.2%) as
compared to those of control (and NaCl-treated) mice (0.2-1%). In a follow-up study, Van
Goethem and associates repeated portions of their earlier experiment (Van Goethem et al., 1995).
Three animals received carbon tetrachloride and three served as controls. The frequency of
micronucleated hepatocytes increased from 1.5% in the controls to 7.6% in the carbon
tetrachloride-treated rats, a significant fivefold difference. Using fluorescence in situ
hybridization with a multi-centromeric rat probe, the authors attributed the increase in MN
primarily to chromosomal breakage. Based on the frequencies given in the paper, chromosome
breakage can be calculated to be 5.5-fold over the control whereas chromosome loss can be
calculated as a 3.5-fold increase. It should be noted that the observed difference in the
proportion of centromere-containing and -lacking micronuclei in the study is attributable to a low
frequency of centromere-containing micronuclei in only one rat and is unlikely to be either
statistically or biologically significant. Based on their work and that of others (Craddock and
Henderson, 1978), the authors attributed the results to chemically-induced oxidative cellular
damage, and suggested that free radicals produced from carbon tetrachloride may disrupt
cytoplasmic organelles releasing DNase and tissue-destructive hydrolases within the cell leading
to DNA strand breaks and tissue damage. Although the sample sizes of the studies are quite
small, the two studies indicate that the micronucleus results are reproducible and that under
regenerative conditions following toxicity, an increase in chromosome breakage and possibly
chromosome loss can be detected in the regenerating cells of carbon tetrachloride-treated rats.
In should also be noted that Sarkar et al. (1999) reported that the administration of carbon
tetrachloride to mice over a five-week period resulted in increases in structural chromosome
aberrations in liver cells. However, there appear to be numerous and significant methodological
issues with these experiments. For example, the methods section does not adequately explain
how metaphases were obtained from either the treated or control mice that would allow structural
chromosome aberrations to be scored. Given the low number of mitotic cells in the untreated
mouse liver, it would be very difficult if not impossible without mitotic stimulation to obtain 50
well spread metaphases without the use of colchicine or other spindle-disrupting agent. In
addition, the reported frequencies of structural aberrations including some classes of aberration,
such as ring chromosomes, are unusually high (32-48% including gaps) when compared to other
studies. Because of these concerns, this paper has not been included in Table 4-11.
Mutations in transgenic mice. The ability of carbon tetrachloride to induce mutations in
hepatocytes in vivo has been investigated in three studies using transgenic mice. Negative
results were seen in each of the three studies. As reported by Mirsalis and coworkers, transgenic
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B6C3F1 lacl mice were treated with 5 daily doses of carbon tetrachloride at 35 mg/kg-day and
the animals were sacrificed 7 days after the first dose (Mirsalis et al., 1994; Mirsalis, 1995).
Mice were implanted with an osmotic pump that released [3H]thymidine at the beginning of the
study to measure the percent of hepatocytes in S phase (labeling index). Controls had a labeling
index of 0.07% and a mutant frequency of <6xlO"5. Carbon tetrachloride produced a nearly
1000-fold increase in the labeling index with no increase in the mutant frequency. The authors
concluded that short bursts of cell proliferation induced by carbon tetrachloride do not result in
mutations in the liver.
As part of another study to investigate the impact of cell proliferation on liver
mutagenesis, carbon tetrachloride at 80 mg/kg was administered by i.p. injection to lacz
transgenic CD2F1 mice (Muta™Mice) and the animals were sacrificed 14 days later (Tombolan
et al., 1999; Lambert et al., 2005). The mutant frequency in the carbon tetrachloride-treated
animals (8.6 x 10"5) was not significantly increased over that seen in the controls (5.4 x 10"5). In
non-transgenic CD2F1 mice receiving an intragastric dose of carbon tetrachloride, significant
increases in absolute and relative liver weights were seen beginning two days after treatment.
The percent of hepatocytes labeling with BrdU during the last two hours before sacrifice peaked
at 59 times that of the controls at 3 days after treatment and returned to control levels by day 7.
In the third study reported by Hachiya and Motohashi (2000), the frequency of mutations
the lacZ transgene in liver of male CD2F1 lacZ transgenic mice (Muta™Mice) was determined
14 days after administration of 700 mg/kg carbon tetrachloride (by oral gavage) or 7, 14, or 28
days after administration of 1400 mg/kg. A small increase in mutant frequency, considered
biologically insignificant by the authors, was seen. The mutant frequencies for six of the nine
carbon tetrachloride-treated animals were within the control range (53 x 10"6 to 100.4 x 10"6).
The mutant frequencies for the other three mice exceeded the upper end of the control range by 3
to 49%. The results as analyzed by Fishers exact test were statistically significant in part
because of the large number of plaques evaluated and the fact that the Fisher's exact test does not
account for animal-to-animal variability. The authors concluded that no biologically significant
increase in the mutant frequency was seen in the carbon tetrachloride-treated mice. Other
reviewers have concurred with this conclusion (Lambert et al., 2005).
As indicated in Heddle et al. (2000), a commonly used cut-off value for a positive
response in this type of transgenic assay is at least a twofold increase over the historical negative
control mutant frequency. Although a historical control range for the Hayashi and Motohashi lab
was not presented, the range for the concurrent controls was 5.3 x 10"5 to 10 x 10"5 with a mean
of 8.2 x 10"5. For comparison, a general control range suggested by Heddle et al. (2000) used for
sample size calculations is 4 x 10"5 to 7 x 10"5. Using this as a historical control, no treatment
group exceeded twofold that of the control and only one treated animal in the study was outside
of this range. As a caveat, the numbers of animals used in the three studies were small, and the
dosing and sampling protocols did not follow those currently recommended (Heddle et al., 2000;
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Lambert et al., 2005). However, the results of these three in vivo studies are consistent and
provide no evidence for the formation of carbon tetrachloride-induced mutations in the liver
following acutely toxic doses.
DNA binding by carbon tetrachloride-derived metabolites. A number of studies have
investigated the potential of carbon tetrachloride to bind covalently to DNA. Additional studies
have investigated whether DNA adducts derived from reactive oxygen species or from lipid
peroxidation-derived products are elevated following carbon tetrachloride administration. DNA
adducts from both pathways have been reported in carbon tetrachloride-treated mice, rats and
hamsters.
In initial studies, Rocchi et al. (1973) investigated the ability of 14C-labeled carbon
tetrachloride to bind to the DNA, RNA and proteins in the liver of male Wistar rats and male
Swiss mice. Carbon tetrachloride was injected i.p. at 56 mg/kg and the animals were sacrificed
12 hours later and the livers from the treatment groups were pooled. Half of the animals had
been previously treated with 3-methylcholanthrene to induce hepatic metabolism.
Radiochemical binding to nuclear and cytoplasmic proteins but not DNA was seen in the 3-
methylcholanthrene-pretreated and non-pretreated rats. Binding to rRNA was also seen in the 3-
methylcholanthrene-pretreated rats. In the mouse studies, DNA binding was seen in the livers of
mice pretreated with 3-methylcholanthrene but not in mice not previously pretreated. Protein
binding was seen in both groups of mice. Since the livers of the treatment groups were pooled
for analysis, no measure of variability or statistical significance could be established. In
addition, although the article mentions that the counts per minute (cpm) of the samples was at
least twice that of the background, there is no mention of controls nor information on how the
samples were corrected for radioactivity in the control samples.
Diaz Gomez and Castro (1980a) also studied the ability of 14C-labeled carbon
tetrachloride to bind to DNA, nuclear proteins and nuclear lipids in the liver of male Sprague
Dawley rats and male Strain A/J mice. Carbon tetrachloride was injected i.p. at 1.4 mg/kg, and
the animals were sacrificed 16 hours later. Three samples, each comprised of one rat liver or the
pooled livers from 10 mice, were measured per experimental group. A small but significant
increase in radiocarbon binding was seen in both the mouse and rat samples in this experiment.
Binding to nuclear proteins and lipids was also seen in parallel experiments. In another series of
experiments, mice previously treated with phenobarbital or 3-methylcholanthrene to induce
hepatic metabolism were administered carbon tetrachloride at 1.4 mg/kg. Another group was
administered a higher (3200 mg/kg) toxic carbon tetrachloride dose. Radiochemical binding to
mouse liver DNA was reported for the phenobarbital and 3-methylcholanthrene-pretreated mice
as well as for the mice treated with the toxic carbon tetrachloride dose. DNA binding was
slightly increased in the 3-methylcholanthrene-pretreated mice (0.84 pmol/mg) and the high-dose
mice (2.803 pmol/mg) as compared to the low-dose carbon tetrachloride-treated mice (0.72
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pmol/mg). The levels of low-dose carbon tetrachloride binding to DNA were considered to be
quite low in both species with the binding in the mouse liver slightly higher than that in the rat
liver. Negative control information was not presented. In place of a true negative control, the
background radioactivity counted in the presence of DNA of 78 dpm (disintegrations per
minute). This was approximately double the background of 38 detected in the absence of DNA
and was deducted from each experimental determination.
In a follow-up study, Castro et al. (1989) investigated the relationship between the
intensities of covalent binding to liver DNA and nuclear proteins in vivo in samples obtained
from C3H mice, Syrian golden hamsters, and Sprague-Dawley rats - three species with different
susceptibilities to carbon tetrachloride-induced liver cancer - administered 1200 mg/kg
radiolabeled carbon tetrachloride ([14C]CCU). The authors reported that there was no correlation
between the intensity of the carcinogenic effects in these species and DNA binding, either in
vitro or in vivo. However, a good correlation was found between carcinogenicity and covalent
binding to total nuclear proteins both in vitro and in vivo. Covalent binding to liver DNA in all
three species was similar [(2.2-2.3 pmol carbon tetrachloride/mg DNA or 1.4-1.5 mol
nucleotides/mol carbon tetrachloride metabolites (x 106)]. Higher levels of covalent binding to
nuclear proteins, particularly the acidic nuclear protein fractions, were seen when expressed on a
pmol per mg basis. The authors discussed that the acidic nuclear proteins often have regulatory
functions in gene expression and that this may be important in carbon tetrachloride-induced
carcinogenesis. Again, the authors indicated that they subtracted for background radioactivity
(35 dpm), but presented no data on control binding or how they corrected for control
radioactivity - a serious limitation for the use of this and other studies in assessing genotoxic
potential.
Levy and Brabec (1984) also investigated the ability of radiolabeled carbon tetrachloride
to bind to different types of DNA. After the administration of a single dose of 14C- carbon
tetrachloride to male Sprague-Dawley rats, elevated levels of radioactivity were recovered bound
to purified mitochondrial and nuclear DNA. At both a low non-necrotizing and a high dose, 20-
to 50-fold more radioactivity was recovered bound to mitochondrial DNA than to nuclear DNA.
Binding to mitochondrial DNA also occurred when radiolabeled carbon tetrachloride was
incubated anaerobically with isolated mitochondria. Carbon tetrachloride is known to be
bioactivated in the mitochondria (Weber et al., 2003), so this report of elevated binding close to
the site of activation seems plausible. Again, there is no mention of a negative control or how
the samples were corrected for control radioactivity or counts. There is also no indication of
variability, the number of samples analyzed, or statistical significance of the results.
As described above, four studies have reported that following administration of
radiolabeled carbon tetrachloride, detectable amounts of radioactivity were recovered bound to
the extracted nuclear DNA. Significant methodological problems with each of the studies create
difficulties in interpreting the results. For one or two of the studies, basic information on sample
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size, variability and statistical significance is not provided. In addition, all studies failed to
provide data for untreated controls or indicate that the treatment samples were corrected for
control radioactivity (or dpm). For agents that bind weakly to DNA such as carbon tetrachloride,
even small increases in dpm in the controls can substantially alter the amount of binding
attributed to the chemical treatment.
Following the administration of a radiolabeled compound to an animal, the recovery of
radioactivity strongly associated with the isolated and extracted DNA is assumed to represent
covalent binding of the chemical or its metabolite to DNA. However, binding to proteins or
lipids can occur and may be recovered as contaminants within the DNA preparation (Kitta et al.,
1982). In addition, metabolic incorporation of the radiocarbon into DNA can also occur through
entry into the carbon pool of the cell with subsequent incorporation into DNA (Phillips et al.,
2000). This is a concern with carbon tetrachloride as metabolic studies have shown that
complete dechlorination of carbon tetrachloride can occur during cellular metabolism (Weber et
al., 2003; Halliwell and Gutteridge, 1999). It is therefore possible that part of the radiolabel
recovered in the in vivo 14C studies represents carbon tetrachloride-derived carbon that was
incorporated into DNA. For both of these reasons, it is important to identify the carbon
tetrachloride-derived DNA adducts to confirm that they occur in vivo. Unfortunately, this has
not yet occurred. Studies in nonaqueous model systems have shown that the trichloromethyl
radical can adduct nucleotides (Castro et al., 1994; Diaz Gomez and Castro, 1981), but it is not
clear to what extent this would occur in aqueous systems or in vivo. Assuming that all of the
radiocarbon recovered represents adducts and that the levels of radioactivity in the controls are
equivalent to background, the magnitude of the DNA binding even at high toxic concentrations
is relatively low (Castro et al., 1989; Lutz, 1986; Levy and Brabec, 1984; Diaz Gomez and
Castro, 1980a; Lutz, 1979; Rocchi et al., 1973). Overall, there is limited evidence for the ability
of carbon tetrachloride metabolites to bind covalently to DNA in vivo.
Oxidative- andlipidper•oxidation-derived'DNA adducts. Since reactive oxygen species
as well as lipid peroxidation-derived degradation products are also known to bind covalently to
DNA, numerous investigators have investigated whether oxidative adducts can be detected
following the administration of carbon tetrachloride to animals. Adducts derived from both
reactive oxygen and lipid peroxidation have been detected. Four studies employing a wide range
of doses attempted to detect DNA adducts derived from the lipid peroxidation product
malondialdehyde (MDA) or similar reactive species, in the hepatic DNA of rats or hamsters. Of
the four studies, two were positive, one was equivocal, and one produced negative results. In
addition, two studies detected DNA adducts formed in the liver (as well as other tissues) from
rram--4-hydroxy-2-nonenal (4-HNE), another reactive species formed during lipid peroxidation.
A brief description of the individual studies follows.
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In the initial study, Hadley and Draper (1990) briefly mention that the excretion of a
newly identified guanine-malondialdehyde adduct in the urine was increased 2.5-fold after the
oral administration of carbon tetrachloride to rats. No data were provided. In a later study using
a sensitive mass spectrometric method, Chaudhary et al. (1994) demonstrated that four days after
the administration of a 0.1 mg/kg oral dose of carbon tetrachloride to Sprague Dawley rats, the
liver levels of the major endogenous malondialdehyde deoxyguanosine adduct increased 1.8-fold
from 2.1 per 107 bases in the controls to 3.8 per 107 bases. The level of isoprostane, another
product of lipid peroxidation, was increased 16-fold in the treated animals.
In the report by Draper et al. (1995), the concentration of a deoxyguanosine-
malondialdehyde adducts in the liver was determined 48 hours after oral administration of 160
mg/kg carbon tetrachloride to a group of five rats. A significant decrease in the level of this
adduct was seen in the carbon tetrachloride-treated rats as compared to controls. The authors
suggested that in some undetermined fashion the liver DNA was protected from the increasing
amounts of malondialdehyde formed. They noted that under the same conditions, previous
studies have shown that large concentrations of malondialdehyde adducts with lysine, but not
deoxyguanosine-malondialdehyde, are excreted in the urine.
As part of another study to identify DNA adducts contributing to lipid hydroperoxide-
mediated carcinogenesis, Wang and Liehr (1995) performed 32P-postlabeling to measure and
quantify the influence of carbon tetrachloride on the presence of endogenous adducts in Syrian
golden hamsters four hours after treatment with 160 mg/kg and 1600 mg/kg carbon tetrachloride.
Treatment of the hamsters with the 160 mg/kg dose resulted in a doubling of renal and liver lipid
hydroperoxide levels. At the higher dose, renal lipid hydroperoxide levels were raised by 30%
but those in the liver were lowered by 50%, presumably due to lipid hydroperoxide-mediated
inactivation of metabolic enzymes required for the activation of carbon tetrachloride. The levels
of lipid hydroperoxide-derived DNA adducts in the kidney and liver varied in a comparable
manner; the measured endogenous adducts in the liver increased from ~9 in the controls to -14
(expressed as relative adduct level x 108 adducts) at the low dose and decreased to ~8 at the high
carbon tetrachloride dose. Adduct levels in the kidney increased from ~11 in the controls to -25
at the low dose and -16 at the high dose. A very good correlation between measured lipid
hydroperoxide levels and endogenous adducts was seen. The authors noted that the decreased
levels that were seen at the high dose were consistent with decreases in polar adducts observed
by Nath et al. (1990) in the livers of mice treated with carbon tetrachloride at 1200 mg/kg. The
observed decrease is also similar to the decrease in the deoxyguanosine-malondialdehyde adduct
seen by Draper et al. (1995). It would appear that at times there can be an unusual relationship
between carbon tetrachloride dose and lipid peroxide-derived DNA adducts.
Using 32P postlabeling combined with high-performance liquid chromatography, the
formation of trans-4-hydroxy-2-nonenal-derived cyclic adducts with deoxyguanosine was seen in
untreated rat and human tissues indicating that they are endogenous in origin (Chung et al.,
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2000). Significant increases in the formation of the HNE-dG adduct were seen in the livers of
F344 rats treated with a single 3200 mg/kg dose of carbon tetrachloride. Twenty-four hours after
treatment the levels of the HNE-dG adducts were increased 37-fold as compared to those of
control animals (104 nmol/mol guanine vs. 2.8 nmol/mol guanine). The adducts appeared to be
persistent as significant levels of the HNE-dG adducts (88 nmol/mod guanine) were present 72
hours after dosing.
The formation of l,N2-propanodeoxyguanosine adducts of trans-4-hydroxy-2-nonenal
(HNE-dGp-adducts) were measured in tissues of rats treated with carbon tetrachloride and
compared to those in control rats (Wacker et al., 2001). Carbon tetrachloride at a dosage of 500
mg/kg was administered by a single i.p. injection with sacrifices at 4, 16 and 24 hours post
injection, or by 4 injections at 24-hour intervals with the sacrifice occurring 8 hours after the
final dose. In the single injection studies, increases in HNE-dGp adducts were seen in the lung
and colon at various times and in the forestomach at all three time points. HNE-dGp adduct
levels also showed a nonsignificant increase in the liver and no change in the kidney. The
maximum increases seen were approximately 1.5 to 2-fold. In the multi-dose studies, significant
increases were seen in the liver (2.2-fold) and the forestomach (1.7-fold). The levels of HNE-
dGp adducts detected in the liver (2.8 per 107 normal nucleotides) in this study were of the same
order of magnitude as the adduct levels formed from malondialdehyde (MDA) in the liver after
treatment with carbon tetrachloride (3.8 per 107 normal nucleotides; Chaudhary et al., 1994) and
HNE adducts found in the liver (22 per 107 normal nucleotides; Chung et al., 2000).
The formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) is one of many adducts
formed between reactive oxygen species and DNA. Because of its prevalence and ease of
measurement, it is frequently used as a measure of oxidative DNA damage. Four studies have
attempted to measure 8-OHdG following the administration of carbon tetrachloride to rats or
mice. All four of the studies were positive although the response in one was relatively weak.
In the initial study by Takahashi et al. (1998), the suitability of an antibody to detect 8-
OHdG for immunohistochemistry was determined by measuring adduct levels in hepatocyte
nuclei in a time-course study following the treatment of rats with carbon tetrachloride. Rats were
administered carbon tetrachloride at 3200 mg/kg by gavage and sacrificed at 6 hours, 12 hours,
1, 2, 3, and 7 days. Severe centrilobular necrosis was present by day 1. By days 2 and 3, anti-8-
OHdG antibody staining was present in the mononuclear cells infiltrating the necrotic
centrilobular regions as well as in the hepatocytes in the midzonal and periportal regions, and
sinusoidal endothelial cells. At the day 2 time point, the formation of 8-OHdG in DNA and 8-
oxo-dGTPase mRNA expression were also increased by 5.1- and 1.7-fold, respectively. MDA
plus 4-NHE showed peaks at 6 hours and 3 days. The findings suggested that increased lipid
peroxidation, rather than an excessive formation of 8-OHdG, was the main contributing factor in
the massive hepatic necrosis observed. The observed increase in 8-OHdG was attributed to the
infiltrating mononuclear cells.
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In the studies reported by Iwai et al. (2002), carbon tetrachloride was administered by
subcutaneous injection to rats twice a week at a dose of 200 mg/kg for the first 10 weeks, then at
400 mg/kg for the next 10 weeks. The rats were sacrificed at the end of week 22. At week 1, an
approximately twofold increase in 8-OHdG was seen in liver DNA of the treated rats when
compared with untreated controls. Consistent with this, the treated rats also exhibited higher
levels of 8-oxo-guanine DNA glycosylase 1 (OGG1) mRNA when measured using reverse-
transcriptase PCR.
Recently as part of an investigation into the susceptibility of young and old mice to
oxidative stressors, Lopez-Diazguerrero et al. (2005) administered carbon tetrachloride at a dose
of 43 mg/kg by i.p. injection on 3 consecutive days to young (2 month old) and older (14 month
old) female CD-I mice. Twenty-four hours post-treatment, liver DNA in carbon tetrachlori de-
treated young and old mice exhibited significant increases in 8-oxo-7,8-dihydro-2'-
deoxyguanosine (8-oxodG). The 8-oxodG levels increased from 0.5 residues/106 dG in the
young controls to 7.4 residues/106 dG in the carbon tetrachloride-treated young animals. In the
older animals, the 8-oxo-dG levels increased from 2.6 residues/106 dG in the controls to 10.1
residues/106 dG in the treated animals. The 8-oxodG levels between the treated young and old
animals did not differ significantly.
Similarly as part of a larger study of oxidative biomarkers, Kadiiska et al. (2005)
measured the levels of 8-OHdG in the urine of male Fischer 344 rats previously administered
carbon tetrachloride at 120 mg/kg and 1200 mg/kg by i.p. injection (urine collected 2-7 hours
and 7-16 hours after carbon tetrachloride injection). Significant increases in 8-OHdG compared
to the control were seen for the low dose at 16 hours and the high dose at both sample times.
The high dose resulted in a seven- and threefold increase in the excreted adducts at the two
successive time points.
Available studies provide considerable evidence of DNA adducts derived from reactive
oxygen species or lipid peroxidation following in vivo administration. In some cases, the
relationship between dose and adduct levels appeared to be complex, without a monotonic
relationship between dose and response. In comparing the results from the various binding
studies, it should be remembered that the binding measured in radiocarbon binding studies
reflects all DNA adducts that contain the 14C label. In contrast, 8-OHdG and MDA and 4-HNE
adducts represent only a few of the many types of oxidative adducts (De Bont and van Larebeke,
2004; Halliwell and Gutteridge, 1999). When increases in these marker adducts are seen, the
total number of oxidative DNA adducts is undoubtedly much larger. The overall consistency
and magnitude of the results from the oxidative adduct studies indicate that they likely represent
the major class of DNA lesion occurring in the rodent liver following carbon tetrachloride
administration.
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Endogenous adducts. Using the 32P-post-labeling assay, Nath et al. (1990) investigated
the effects of carbon tetrachloride on presence of hepatic "I" spots (DNA adducts believed to be
formed from endogenous compounds) in both acute and long-term studies using 10-12 month-
old ICR mice. For the acute study, carbon tetrachloride was injected i.p. at a dose of 1200
mg/kg. Twenty-four hours after the injection, the intensity of non-polar I-spots in the liver DNA
was increased as compared to those in corn oil-treated controls while the intensity of one polar I
spot was reduced. In contrast, in a long-term study of carbon tetrachloride, mice given two
consecutive injections of carbon tetrachloride (1200 mg/kg) and sacrificed at 1, 4, 8, 12, and 22
weeks after the final injection, the total liver I compound levels were reduced to 17-49% of the
corresponding controls. Although there was a trend in recovery between weeks 8 and 22, the I-
compound levels remained significantly lower at week 22. The authors suggested that the
persistent reduction in I-compound levels may point towards a nongenotoxic effect of carbon
tetrachloride contribution to hepatocarcinogenesis. As mentioned by the authors, previous
studies have shown a significant reduction in I-compound levels following treatment with a
number of nongenotoxic carcinogens and by other treatments/conditions associated with rat liver
carcinogenesis. Of particular note, the authors reported that "neither the acute nor the chronic
experiments with carbon tetrachloride produced extra spots indicative of DNA adducts"
indicating that exogenous adducts were not seen in the carbon tetrachloride-treated mice.
Altered DNA methylation. Following carbon tetrachloride administration, a number of
studies have reported alterations in liver DNA methylation. In early studies performed by
Barrows and Shank (1982), increases in 7-methylguanine and O6-methylguanine were seen in
liver DNA 12 hours after rats were administered a single 1000 mg/kg dose of carbon
tetrachloride. This increase was also seen in hydrazine- and ethanol-treated rats, and there was
some evidence in the hydrazine-treated rats that S-adenosylmethionine (SAM) was the methyl
donor. Based on the observed results, the authors suggested that aberrant DNA methylation may
be a non-specific response to chemical injury to the liver.
More recently, Varela-Moreiras et al. (1995) investigated the effect of short-term
administration of carbon tetrachloride on hepatic DNA methylation and on SAM and S-
adenosylhomocysteine (SAH) in male Wistar rats administered 800 mg/kg carbon tetrachloride
by i.p. injection 2 times/week, for 3 weeks. Rats treated with carbon tetrachloride exhibited
hypomethylation of their hepatic DNA as measured by the extent to which the liver DNA from
the treated animals could be methylated in vitro using [3H-methyl]-SAM as a methyl donor. In
addition, decreased levels of SAM, methionine and folate as well as increased levels of SAH and
homocysteine were seen. No changes were observed in the levels of cystathionine, reduced
glutathione, or in the activity of SAM-synthetase. The magnitude of the observed changes was
substantially reduced in animals co-administered SAM with carbon tetrachloride. The authors
proposed that "carbon tetrachloride disrupts the distribution of homocysteine between
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remethylation and its degradation via the transsulphuration pathway, and that SAM, by resetting
the methylation ratio, restores this equilibrium." In eukaryotic and mammalian cells, gene
expression is influenced by the extent and patterns of DNA methylation, so the observed changes
in hepatic DNA methylation could represent an epigenetic alteration that could contribute to
carbon tetrachloride carcinogenesis.
4.4.2.5. Genotoxicity Studies: Summary of the Evidence for Genotoxic andMutagenic Effects
EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) identify a number
of criteria that should be considered in judging the adequacy of mechanistic data. These include
mechanistic relevance, number of studies of each endpoint, consistency of results in different test
systems and species, conduct of the tests according to generally accepted protocols, and degree
of consensus and general acceptance among scientists regarding the interpretation of the results.
In addition to these general considerations, evaluation of the genotoxicity data on carbon
tetrachloride poses some unique challenges. First, the genotoxicity data for carbon tetrachloride
are derived from a large number of experiments performed over a period spanning almost 40
years. Some assays were at early stages of development when performed, whereas others were
conducted under well-established protocols. As a result, the quality of the data varies widely. In
spite of this, most studies provide worthwhile information that can provide insights into the
potential of carbon tetrachloride to cause genotoxic effects. In addition, because of the large
numbers of tests performed, one would expect a number of studies to be positive due to random
chance or elevated error rates resulting from multiple comparisons. Some of the unique
challenges associated with evaluated carbon tetrachloride genotoxicity are outlined in
Table 4-12.
In accordance with the EPA mutagenicity risk assessment guidelines (U.S. EPA, 1986b),
when evaluating genotoxicity results, more weight has been given to tests performed in vivo in
mammalian systems than to those performed in vitro using mammalian cells or in sub-
mammalian systems such as yeast and bacteria. Preference has also been given to results seen in
the rodent liver over those seen in other non-target tissues. This prioritization scheme is also
consistent with the current EPA carcinogen risk assessment guidelines (U.S. EPA, 2005a), which
state "Although important information can be gained from in vitro test systems, a higher level of
confidence is generally given to data that are derived from in vivo systems, particularly those
results that show a site concordance with the tumor data."
As indicated in Tables 4-8 to 4-11, well over 100 studies have been performed to assess
the genotoxic and mutagenic effects of carbon tetrachloride. A few experiments have been
conducted using human cells but none were located describing genotoxic effects in humans. A
summary evaluation by major type of genetic alteration is presented below.
Gene mutations. Intragenic or point mutations have been found in many cancer-related
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genes and have been shown to play a determining role in chemical carcinogenesis (Stanley,
1995; Anderson et al., 1992; Harris, 1991). The ability of a chemical to form mutations in model
systems is an important consideration in establishing whether an agent acts through a mutagenic
mode of action. There is little direct evidence that carbon tetrachloride induces intragenic or
point mutations in mammalian systems. The mutation studies that have been performed using
transgenic mice have yielded negative results, as have the vast majority of the mutagenesis
studies that have been conducted in bacterial systems. Since oxidative DNA adducts can be
converted into mutations, the inability to detect mutations in the transgenic mouse assays may be
an indication of efficient repair of oxidative lesions, a preferential formation of large
chromosomal mutations that are inefficiently detected in the transgenic models, or a reflection of
the limitations and sensitivity of the specific assays that were performed with carbon
tetrachloride. The two positive mutation/DNA damage studies conducted in E. coli were seen in
strains that are particularly sensitive to oxidative damage. Moreover, the intrachromosomal
recombination induced by carbon tetrachloride in S. cerevisiae is believed to result from double
stranded DNA breaks leading to deletion mutations. These results are consistent with DNA
breakage originating from oxidative or peroxidative stress that occurs concurrently with
cytotoxicity.
DNA strand breakage. DNA strand breakage is not a measure of mutation per se, but
can be a useful indicator of DNA damage and can contribute to an evaluation of an agent's
mutagenic potential. However, DNA breaks can also be formed during apoptotic and necrotic
cell death even by noncarcinogenic agents (Higami et al., 2004; Bergman et al., 1996; Grasl-
Kraupp et al., 1995; Elia et al., 1994), so the potential contribution of cytotoxicity to the
observed results needs to be carefully evaluated in studies reporting DNA damage. There is
some evidence that carbon tetrachloride administration results in DNA breakage and
fragmentation in the liver of treated mice and rats; however, extensive hepatotoxicity was seen in
each of the studies where DNA damage has been reported. While some of the damage may be
due to reactive species formed during carbon tetrachloride metabolism and lipid peroxidation,
much of observed damage appears to be more related to a cytotoxic response associated with cell
death than a genotoxic response leading to mutation. Indeed, the TUNEL assay used in two of
the positive carbon tetrachloride studies is commonly used as an early indicator of apoptotic and
necrotic cell death (Higami et al., 2004; Grasl-Kraupp et al., 1995).
Structural and numerical chromosome aberrations. Non-random structural and
numerical chromosomal aberrations are commonly seen in cancer cells and are believed to play
an important role in carcinogenesis (Pedersen-Bjergaard et al., 2002; Solomon et al., 1991;
Hansen and Cavenee, 1987; Oshimura and Barrett, 1986; Yunis, 1983). Furthermore, elevated
frequencies of chromosomal aberrations have been observed in humans exposed to
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environmental chemicals, and recent investigations have indicated that individuals with elevated
levels of these alterations have increased risks of developing cancer (Hagmar et al., 2004;
Hagmar et al., 1998; Sorsa et al., 1992). Chromosomal alterations, measured in cell culture
systems or in animals treated in vivo, are commonly induced by carcinogenic agents, and the
evaluation of chromosomal aberrations or micronuclei is an important component of commonly
accepted genotoxicity testing schemes (Muller et al., 1999). Although less prone to problems of
cytotoxicity than the DNA breakage assays, under conditions of severe toxicity or stress,
increases in structural chromosome aberrations and micronuclei have been shown to occur
through indirect mechanisms (Galloway, 2000; Galloway et al., 1987). While aberrations
formed by noncarcinogenic agents under extreme conditions are not believed to be relevant to
mutagenic risks (Galloway, 2000), the significance of aberrations formed by carcinogens under
such conditions is less clear. For screening new chemicals, protocols have been established, at
least in vitro, to limit genotoxicity testing to concentrations that do not exhibit high toxicity
(Muller and Sofuni, 2000).
In the genotoxicity studies conducted on carbon tetrachloride, there is no evidence for
chromosomal damage when carbon tetrachloride has been tested in conventional assays for
chromosomal damage in the rat or mouse bone marrow. There is some evidence that following
high cytotoxic doses of carbon tetrachloride, increases in chromosome breakage and loss can
occur in the rat liver. It has not been established, however, whether these represent independent
genotoxic events due to the formation of reactive metabolites or the result of chromosomal
damage occurring at an early stage of necrosis or apoptosis. Regardless of their origin, the
increases that have been observed have occurred exclusively at hepatotoxic doses and have been
limited in magnitude.
DNA adducts. The formation of DNA adducts within the liver following carbon
tetrachloride exposure is indicative of DNA damage occurring in the target organ. Because
adducts may be converted into mutations or DNA strand breaks, but can also be efficiently
repaired or remain unchanged in less critical non-coding sequences of DNA, these DNA adducts
represent precursor lesions rather than specific mutagenic or genotoxic effects. It is generally
recognized that the types of DNA adducts formed after exposure can also provide valuable
insights into the mechanisms underlying an agent's genotoxic and mutagenic effects. There is
strong evidence of increases in DNA adducts formed from reactive oxygen species (i.e., 8-
OHdG) and lipid peroxidation products such as MDA and 4-HNE in the liver of rodents
following administration of carbon tetrachloride. Based on both in vivo and in vitro studies,
there is some (limited) evidence for the formation of DNA adducts derived directly from carbon
tetrachloride; however, this has not been adequately established and serious methodological
problems limit the interpretation and usage of the results from the existing studies.
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Unscheduled DNA synthesis. The unscheduled synthesis of DNA is a measure of DNA
repair and is commonly used to assess DNA damage produced by mutagenic chemicals in the
livers of treated animals. Based on the reliable studies conducted to date, there is no evidence of
UDS in the livers of carbon tetrachloride-treated rats or mice even when tested under conditions
producing significant hepatotoxicity.
4.4.3. Neurotoxicity Studies
High-dose, acute toxicity studies in humans and animals reported neurotoxic effects of
carbon tetrachloride. Human case reports mention headache, drowsiness, comas, or seizures
occurring after exposure by ingestion or inhalation (Stewart et al., 1965; New et al., 1962;
Norwood et al., 1950). Lehmann and Schmidt-Kehl (1936) reported neurological symptoms
occurring after exposures of 30 mg/L (-4800 ppm) or higher. In an acute inhalation study in
rats, signs of central nervous system depression occurred at >4600 ppm (Adams et al., 1952).
Frantik et al. (1994) quantified the air concentrations of carbon tetrachloride and other
solvents that would produce an acute neurotoxic effect in rats and mice. Whole-body exposures
at various concentrations were undertaken for groups of four male albino Wistar rats for 4 hours
or female H mice for 2 hours; animals were then tested for the inhibition of propagation and
maintenance of an electrically evoked seizure discharge. Testing was conducted by application
of a short electrical impulse (0.2 seconds, 50 Hz, 180 volts in rats and 90 volts in mice) through
ear electrodes. The most consistent sensitive measure was the duration of tonic extension
through the hind limbs in rats and the velocity of toxic extension (reciprocal of latency) in mice.
The authors reported the "isoeffective concentration" of carbon tetrachloride in air by
interpolating to the level that would produce one-third of the maximum effect. The isoeffective
concentrations were 611 ppm (one-tailed 90% CI: 98 ppm) for rats and 1370 ppm (one-tailed
90% CI: 465 ppm) for mice.
4.4.4. Immunotoxicity Studies
Immunological effects of carbon tetrachloride have been evaluated in mice and rats
exposed by the parenteral (Kaminski et al., 1990, 1989), oral (Guo et al., 2000; Ladies et al.,
1998; Ahn and Kim, 1993; Smialowicz et al., 1991; Kaminski et al., 1989), and inhalation (Ban
et al., 2003) routes. Results of available studies indicate that carbon tetrachloride produces
adverse effects on T-cell-dependent immunity at doses that are hepatotoxic. However, it is
important to note that immunological effects appear to be, at least in part, secondary to
hepatotoxicity and the process of hepatic repair. Information regarding the mechanism of
immune system effects and the relationship of immunotoxicity to hepatotoxicity, inflammation,
and repair, including activation of Kupffer and stellate cells, is reviewed in Section 4.5.6.
Effects of parenteral exposure of mice to carbon tetrachloride on immune function was
studied by Kaminski et al. (1990, 1989). Carbon tetrachloride was injected intraperitoneally to
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female B6C3F1 mice at doses of 0, 500, 1000, or 1500 mg/kg-day in corn oil for 7 consecutive
days. Systemic toxicity endpoints included body weight, selected organ weights (liver, spleen,
lung, kidney, and thymus), and serum chemistry. Humoral antibody responses (the number of
antibody-forming cells) to T-cell-dependent antigen (sheep erythrocytes) and T-cell-independent
antigen (DNP-ficoll) were evaluated in vivo and in vitro. Treatment with carbon tetrachloride
had no significant effect on survival, clinical signs, body weight gain, or organ weights, except
for a decrease in thymus weight at >500 mg/kg-day. There were significant increases in serum
ALT and bilirubin at >500 mg/kg-day, albumin at >1000 mg/kg-day, and total protein at 1500
mg/kg-day. In vivo response to T-cell-dependent antigen was suppressed in a dose-related
manner: by 36% at 500 mg/kg-day to 53% at 1500 mg/kg-day. The in vivo response to T-cell-
independent antigen was suppressed by 16% at the highest dose. T-cell-dependent responses
were more vulnerable to carbon tetrachloride than were T-cell-independent responses.
Kaminski et al. (1990) conducted a series of immunotoxicity experiments in female
B6C3F1 mice given carbon tetrachloride by i.p. injection or gavage in corn oil. Oral or i.p.
administration of 500-5000 mg/kg-day for 7 consecutive days significantly reduced in vivo T-
dependent antibody response to sheep erythrocytes; the route of administration had no significant
effect. Intraperitoneal injection of 25 mg/kg-day for 30 consecutive days also significantly
reduced the in vivo T-dependent antibody response. Intraperitoneal injection at 500 or 1000
mg/kg-day on 8 consecutive days significantly increased serum ALT (by five- and sevenfold,
respectively), but treatment at 250 mg/kg-day had no effect; no effects on body or organ weights
(spleen, liver, or thymus) were observed. Intraperitoneal injection with 5-1000 mg/kg-day on 7
consecutive days significantly reduced the total microsomal protein content per gram of liver.
Whereas treatment at 25-100 mg/kg-day for 3 days had no effect on the T-cell-dependent
antibody response, pretreatment with 4 g/kg ethanol caused significant immunosuppression at 50
or 100 mg/kg-day. The authors concluded that immunosuppression following treatment with
carbon tetrachloride is related to its bioactivation by microsomal enzymes.
The effects of oral exposure to carbon tetrachloride have been studied in mice (Guo et al.,
2000; Ahn and Kim, 1993) and rats (Ladies et al., 1998; Smialowicz et al., 1991). Guo et al.
(2000) administered carbon tetrachloride at doses of 0, 50, 100, 500, or 1000 mg/kg-day by
gavage in corn oil to B6C3F1 mice on 14 consecutive days. Mice were examined for gross
pathology at which time organ weights were recorded for thymus, lungs, liver, spleen, and
kidneys with adrenals. Blood was collected for hematology and serum chemistry analyses.
Immunological endpoints included quantification of T- and B-cells in the spleen and spleen
immunoglobulin (IgM) antibody-forming cell response and antibody liters to a T-dependent
antigen, sheep red blood cells; in addition, cellular-mediated immunity was evaluated in host
responses to infection by two bacterial strains. Treatment had no effect on mortality, the
incidence of clinical signs, body weight gain, or the weights of brain, spleen, lung, thymus, and
kidneys and no biologically significant effect on hematology parameters. Absolute liver weight
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was significantly increased by 23% at 500 mg/kg-day compared with that in vehicle controls.
Significant, dose-related increases in relative liver weights were observed at 350 mg/kg-day.
Treated groups showed histopathology in the liver (cloudy swelling of hepatocytes and
centrilobular necrosis) but not in other organs. Significant dose-related changes in serum
parameters included increases in ALT (19-fold at 50 mg/kg-day), total protein (9% at 100
mg/kg-day), BUN (34% at 500 mg/kg-day), and globulin (20% at 1000 mg/kg-day) and a
decrease in glucose (by 20% at 1000 mg/kg-day). Exposure to carbon tetrachloride had no effect
on the mixed leukocyte response, cytotoxic T-lymphocyte activity, or natural killer (NK) cell
activity. Exposure to carbon tetrachloride reduced the humoral immune response; the IgM
antibody-forming cell response to sheep erythrocytes was suppressed at >50 mg/kg-day,
maximally by 43% at 1000 mg/kg-day. IgM serum liters to sheep erythrocytes were
significantly reduced at >100 mg/kg-day. Absolute numbers of CD4+CD8+ T-cells were reduced
by 40% in all dosed groups compared with vehicle controls; absolute numbers and percentages
of CD4+CD8 T-cells were reduced in the 500 mg/kg-day group. Treatment with carbon
tetrachloride reduced host resistance to both Streptococcuspneumoniae and Listeria
monocytogenes at 500 and >50 mg/kg-day, respectively. In mice, the low dose of 50 mg/kg-day
was a LOAEL for immunotoxic effects of carbon tetrachloride by oral exposure, affecting
primarily T-cell-dependent responses.
The immunotoxicity of carbon tetrachloride was investigated in male ICR mice
administered 1 mL/kg (1590 mg/kg) carbon tetrachloride in olive oil twice weekly by gavage
(Ahn and Kim, 1993) for 4 weeks. Systemic endpoints included relative weights of liver, spleen,
and thymus. Immune response to sheep erythrocytes was assessed using hemagglutinin (HA)
liters, assays of plaque-forming cells (PFCs) and delayed-type hypersensitivity reaction, and
measurement of NK cell and phagocytic activity. Compared with control (olive oil) mice,
relative liver weights were significantly increased by 12% in mice treated with carbon
tetrachloride. Relative weight of thymus and spleen were significantly decreased by 6 and 25%,
respectively, compared with that in controls. The HA titer against sheep erythrocytes and the
PFC response, both measures of T-cell-dependent antibody response, were significantly inhibited
by 56 and 40%, respectively, in mice treated with carbon tetrachloride. The delayed-type
hypersensitivity response, a measure of in vivo cell-mediated immunity, was significantly
increased by carbon tetrachloride treatment, indicating that carbon tetrachloride alters T-helper
cell function. In carbon tetrachloride-treated mice, the number of rosette-forming cells (1.90%)
was significantly decreased compared with controls (4.18%). Natural killer cell activity, activity
of phagocytic cells, and the number of circulating leukocytes were significantly decreased by 61,
40, and 34%, respectively, in carbon tetrachloride-treated mice compared with controls. These
results demonstrate that treatment with carbon tetrachloride alters humoral and cell-mediated
immune functions.
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The effect of carbon tetrachloride on humoral immunity was assessed by the IgM
response to intravenously injected sheep erythrocytes in male CD rats administered 0, 12.5, or 25
mg/kg carbon tetrachloride (eight rats per group) in corn oil by gavage 5 days/week for 30 or 90
days (Ladies et al., 1998). Carbon tetrachloride-induced hepatotoxicity was assessed by
examination of the liver by light microscopy and measurement of serum SDH activity in rats
injected with sheep erythrocytes or control vehicle. In rats treated for 30 days, administration of
12.5 and 25 mg/kg carbon tetrachloride decreased sheep erythrocyte-specific serum IgM levels
by 42 and 45%, respectively. In contrast, sheep erythrocyte-specific serum IgM levels were
unchanged compared with controls in the 12.5 mg/kg group and increased by 50% in the 25
mg/kg group in rats treated for 90 days. The authors proposed that time-dependent decreases in
metabolism of carbon tetrachloride contributed to the increased IgM response observed after 90
days of treatment with 25 mg/kg. Exposure to carbon tetrachloride did not alter the population
of splenic lymphocyte subsets (numbers of T-helper cells, T-cyt/sup cells, total T-cells, total B-
cells) or weights or morphology of lymphoid organs (spleen and thymus). Exposure to 25 mg/kg
carbon tetrachloride for 30 or 90 days and to 12.5 mg/kg for 90 days produced hepatotoxicity, as
indicated by increased relative liver weight, histopathological alterations (centrilobular fatty
changes), and increases in serum SDH activity. Results of hepatotoxicity assessments in rats
treated with sheep erythrocytes were similar to controls, indicating that exposure to sheep
erythrocytes did not interfere with the histopathological examination or measurement of serum
SDH activity.
Smialowicz et al. (1991) evaluated immunotoxicity in male F344 rats given carbon
tetrachloride by gavage at doses of 0, 5, 10, 20, or 40 mg/kg-day on 10 consecutive days.
Endpoints included body weight gain, organ weights (liver, kidney, spleen, and thymus), hepatic
microsomal protein levels, serum chemistry, and the histopathology of liver and kidney.
Immunological endpoints included NK cell activity of splenocytes, cytotoxic T-lymphocyte
responses, and proliferative responses of splenic lymphocytes to T-cell mitogens
(phytohemagglutinin and concanavalin A), a B-cell mitogen (S. typhimurium), and a T- and B-
cell mitogen (pokeweed mitogen). Primary antibody responses to a T-cell-dependent antigen
(sheep erythrocytes) were also tested following treatment with carbon tetrachloride at 0, 40, 80,
or 160 mg/kg-day for 10 days. Treatment at >80 mg/kg-day significantly reduced body weight
gain; separate analysis by two-way analysis of variance of 40 mg/kg-day groups and their
respective controls in three experiments indicated a significant decrease in body weight gain.
Treatment had no significant effect on the absolute or relative weights of the spleen, thymus, or
kidney or on absolute liver weight; relative liver weight was significantly increased at 40 mg/kg-
day. There were dose-related increases in AST and ALT: 47% and twofold, respectively, at 20
mg/kg-day. Whereas no hepatic histopathology was detected in control rats, there were dose-
related increases in the incidence and severity of vacuolar degeneration (minimal at 5 mg/kg-day
to mild/moderate at 40 mg/kg-day) and hepatic necrosis (none-to-minimal at 10 mg/kg-day to
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minimal/mild at 40 mg/kg-day). Treatment had no significant effect on kidney histopathology or
renal serum parameters. Treatment had no effect on immunological parameters in rats at doses
that caused hepatic toxicity.
The effects of inhaled carbon tetrachloride on systemic and local immune response were
investigated in female BALB/c mice exposed to 0, 100, 200, or 300 ppm (630, 1260, or 1890
mg/m3) of carbon tetrachloride vapor (Ban et al., 2003). Exposure duration was not reported;
however, the maximum exposure period was most likely less than 24 hours. Immune function
was assessed for systemic (spleen) and local (lung-associated lymph nodes) effects using the
IgM response to sheep erythrocytes and interferon-y (IFN-y) production by spleen and lung-
associated lymph node cells isolated from exposed mice. Assessments of other systemic effects
of carbon tetrachloride (e.g., hepatotoxicity) were not conducted. The IgM response of spleen
cells to sheep erythrocytes, as measured by the number of PFCs, was unaffected by carbon
tetrachloride treatment. In lung-associated lymph nodes, the PFC number was significantly
increased (1.7-fold increase) in mice exposed to 300 ppm carbon tetrachloride compared with
controls, but no differences were observed in the 100 or 200 ppm carbon tetrachloride groups. In
spleen cells, carbon tetrachloride exposure had no effect on IFN-y release, whereas IFN-y release
from lung-associated lymph node cells was significantly increased by 150 to >600% of controls
in all carbon tetrachloride groups. Results of this study indicate that inhaled carbon tetrachloride
exerts immunotoxicity at the point of entry.
4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF
ACTION
There is considerable in vivo and in vitro evidence that may contribute to an
understanding of the mode of action by which carbon tetrachloride produces toxic effects in
animals (Weber et al., 2003; Jaeschke et al., 2002; Plaa, 2000; Omura et al., 1999; Mehendale,
1990; Recknagel et al., 1989; DiRenzo et al., 1982; Slater, 1982; Gillette, 1973; Recknagel and
Glende, 1973; Castro etal., 1973, 1972; Castro and Diaz Gomez, 1972). Representative studies
that provide information on the roles of metabolism, lipid peroxidation, and disruption of
calcium homeostasis are summarized below.
4.5.1. Metabolism Is Required for Toxicity
Numerous studies show that metabolism of carbon tetrachloride is required for toxicity.
As discussed in Section 3.3, the initial step of carbon tetrachloride metabolism is reductive
dehalogenation by CYP450, primarily CYP2E1. Studies using CYP450 inhibitors (e.g., SKF-
525A, colchicine, silymarin, and allylisopropylacetamide) have shown that these compounds,
which inhibit activity of CYP450 enzymes and consequently prevent metabolism of carbon
tetrachloride, prevent carbon tetrachloride-induced liver damage (Martinez et al., 1995; Letteron
et al., 1990; Mourelle et al., 1988; Bechtold et al., 1982; Weddle et al., 1976).
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Carbon tetrachloride itself has been shown to temporarily protect against carbon
tetrachloride toxicity by inhibiting activity of CYP450 and reducing its own metabolism. Glende
(1972) found that rats pretreated with a small, nonlethal dose of carbon tetrachloride were
protected against toxicity from a subsequent large and ordinarily lethal challenge dose of carbon
tetrachloride. Protection was not yet evident when the challenge occurred only 6 hours after the
initial dose but was complete for challenge doses administered 1-3 days after pretreatment and
was gradually less effective for subsequent challenge doses. CYP450 activity measured in this
study showed a sharp decline after the initial dose that reached a minimum at 1 day after
treatment. Gradual increases in CYP450 activity were observed at 4 days and later. The close
parallel between time course of effects on CYP450 activity and toxicity in this study is further
evidence that metabolism of carbon tetrachloride by CYP450 is required for toxicity.
Wong et al. (1998) demonstrated the specific significance of CYP2E1 to carbon
tetrachloride-induced hepatotoxicity in mice using CYP2E1 knockout mice (cyp2eT~). Twenty-
four hours after i.p. injection of 1 mL/kg (1.59 g/kg) of carbon tetrachloride to wild type mice
(cyp2el+ +), there were no significant effects on survival or liver/body weight ratios, but there
was a 422-fold increase in serum ALT, a 125-fold increase in serum AST, and significant
necrosis in the centrilobular hepatocytes. In cyp2el+ + mice, serum ALT was found to be
significantly increased at 12 hours and peaked 24 hours after carbon tetrachloride dosing
(Avasarala et al., 2006). Administration of the same dose to knockout mice (cyp2eJ~/~) resulted
in no increase in AST, only a slight elevation in serum ALT (within normal range), and absence
of liver histopathology. Additionally, Badger et al. (1997) demonstrated that treatment of
Sprague-Dawley rats with gadolinium chloride (GdCb) decreased CYP450 levels in liver
preparations from these animals, which may explain the protective role of GdCls in carbon
tetrachloride-treated animals (See Section 4.5.6).
Conversely, it has been demonstrated that chemical inducers of CYP450 that increase the
activity of CYP450, and particularly those that induce the activity of CYP2E1 specifically,
potentiate carbon tetrachloride hepatotoxicity. See Section 4.8.6 for a list of chemical CYP450
inducers, and associated references, shown to potentiate carbon tetrachloride hepatotoxicity. In
vitro, it has been shown that hepatocyte cell lines that over-express CYP450 have increased
levels of carbon tetrachloride-induced cytotoxicity (Jaeschke et al., 2002; Takahashi et al., 2002;
Dai and Cederbaum, 1995).
4.5.2. Role of Free Radicals
The products of carbon tetrachloride metabolism by CYP2E1 include trichloromethyl and
trichloromethyl peroxy radicals (see Section 3.3). Studies with radical scavengers, such as N-
acetylcysteine, and spin-trapping agents, such as 7V-fert-butyl-a-(4-nitrophenyl)nitrone, have
shown that these agents confer a protective effect against carbon tetrachloride-induced liver
toxicity (Brennan and Schiestl, 1998; Stoyanovsky and Cederbaum, 1996; Slater, 1982),
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indicating that free radicals released via metabolism of carbon tetrachloride may contribute to
carbon tetrachloride toxicity.
The trichloromethyl and trichloromethyl peroxy radicals are highly reactive species that
may produce cellular damage by covalently binding to cellular macromolecules to form nucleic
acid, protein, and lipid adducts (Recknagel and Glende, 1973). Studies using radiolabeled
carbon tetrachloride have shown irreversible binding to cellular DNA, proteins, nuclear proteins,
and lipids, following bioactivation in various in vitro and in vivo systems (Boll et al., 2001b;
Azri et al., 1991; Castro et al., 1989; DiRenzo et al., 1982; Diaz Gomez and Castro, 1980a;
Castro and Diaz Gomez, 1972; Gordis, 1969). Pulse radiolysis experiments showed that the
trichloromethyl peroxy radical is far more reactive towards cellular macromolecules than the
trichloromethyl radical (Slater, 1981; Packer et al., 1978). The trichloromethyl radical binds to
macromolecules strongly but more slowly than the more reactive trichloromethyl peroxy radical.
However, Slater (1981) concluded that most covalent binding involved the trichloromethyl
radical, because binding with the trichloromethyl peroxy radical, although faster, produces a less
stable product. This process involving the binding of the trichloromethyl radical to
macromolecules is known as haloalkylation (Dianzani, 1984).
4.5.3. Lipid Peroxidation
Under oxygen rich conditions, the trichloromethyl radical is converted to the more
reactive trichloromethyl peroxy radical. The trichloromethyl peroxy radical can attack polyenoic
(polyunsaturated) fatty acids in the cellular membrane, forming fatty acid free radicals that
initiate subsequent autocatalytic lipid peroxidation through a chain reaction (see Figure 4-3).
Although the trichloromethyl radical can also initiate lipid peroxidation, it does so at a
very slow rate compared to the more reactive trichloromethyl peroxy radical (Slater, 1981). In
this process, the trichloromethyl peroxy radical abstracts a hydrogen from the methylene carbon
between two double bonds in the polyunsaturated fatty acid, generating a lipid free radical.
Rearrangement of the double bonds into a conjugated pattern shifts the location of the free
radical electron to an adjacent tetrahedral carbon, and reaction of the free radical carbon with
molecular oxygen produces a peroxylipid free radical. The peroxylipid radical can abstract a
hydrogen from a donor molecule, forming a lipid hydroperoxide, a first step in the oxidation of
the fatty acid. If the hydrogen donor is another polyunsaturated fatty acid, the process begins
again, perpetuating the lipid peroxidation (Klaassen, 1996). If the donor is a small hydrocarbon
free radical, an alkane can form.
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Any hydrogen can be removed;
hydrogens on CH2 between
two cis double bonds are
particularly accessible.
CH3-CH2-CH=CH-CH2-(CHm)n-COORi
•CH, or 'OO-CH,
r
\4 or H-OO-CH3
CH3-CH2-CH=CH-CH-(CHm)n-COOR1
m = 1 or 2. Unsaturated fatty
acids will have one or more
CH=CH-CH2 combinations.
In all other cases m = 2.
R! = remainder of a membrane
phospholipid, sphingolipid, or
glycolipid.
CH3-CH2-CH-CH=CH-(CHm)n-COOR1
^02
Alkanes
or
alkenes
CH3-CH2-CH-CH=CH-(CHm)n-COOR1
.
CH3-CH2-CH-CH=CH-(CHm)n-COORi
Propagation: when R2 is another fatty
acid moiety.
Termination: when R2 is an antioxidant
or GSH.
When the fatty acid is polyunsaturated,
r^Tj !,^TT!,^TT_,^TT /^TT x ^r»r»u it is likely to undergo further
3-CH2yCH|CH-CH-(CHm)n-COOR1 peroxidation.
:O : \ ,R2-H
Lipid .
remnant
Aldehydes
i
i
t
Acids
•R
Hydroxylated
fatty acids
t
Malondialdehyde,
4-hydroxynonenal
Figure 4-3. Lipid peroxidation.
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Numerous studies have demonstrated the occurrence of lipid peroxidation following
carbon tetrachloride exposure, either by detection of conjugated dienes (a characteristic marker
of lipid peroxidation) in liver lipids (Tribble et al., 1987; Lee et al., 1982; Recknagel and Glende,
1973; Rao and Recknagel, 1969), increased exhalation of ethane or pentane (end degradation
products of peroxidized T-3 and T-6 polyunsaturated fatty acids, respectively) in treated rats
(Younes and Siegers, 1985; Gee et al., 1981), or occurrence of reactive aldehydes, such as
malonaldehyde and 4-hydroxyalkenals, frequently measured as thiobarbituric acid-reactive
substances (TEARS) (de Zwart et al., 1997; Gasso et al., 1996; Ichinose et al., 1994; Fraga et al.,
1987; Comporti, 1985; Comporti et al., 1984). TEARS form when the oxidation of the fatty acid
progresses from the hydroperoxide, facilitated by the oxidation of Fe2+to Fe3+ in a Fenton
reaction, leading to breaks in the fatty acid chain and the formation of aldehydes from the fatty
acid fragments (Klaassen, 1996). Among the many different aldehydes formed from lipid
peroxidation are 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA).
In vitro studies have shown 4-HNE at high concentrations (> 10 uM) is a cytotoxic
product of liver microsomal lipid peroxidation because of degradation of T-6 unsaturated fatty
acids (Esterbauer et al., 1991; Van Kuijk et al., 1990). The formation of HNE-dGp-adducts may
be relevant to the formation of cancer when these promutagenic lesions are insufficiently
repaired (Wacker et al., 2001). Wacker et al. (2001) developed a sensitive detection method for
l,N2-propanodeoxyguanosine adducts of FINE (promutagenic adducts), a specific marker for
genotoxic interaction of reactive oxygen species and lipid peroxidation products. Background
levels of adducts in various tissues in F344 rats were found in the range of 18-158 adducts/109
nucleotides. Levels of endogenous DNA adducts were higher in the liver, and lower levels were
found in kidney, lung, and colon. After induction of lipid peroxidation by a single i.p.
application of 50 uL carbon tetrachloride at a dosage of 500 mg/kg body weight, levels of HNE-
dG-adducts in the liver were elevated 1.5- to twofold compared with those in controls. The
authors concluded that these promutagenic adducts are evidence of radical-initiated lipid
peroxidation, which if not repaired effectively can lead to cancer. Other studies have also
indicated that lipid peroxidation by-products could inhibit certain DNA repair systems and thus
indirectly increase the rate of spontaneous mutations (Curren et al., 1988; Krokan et al., 1985).
Chung et al. (2000) identified lipid peroxidation as the cause of the 37-fold increase of
UNE-dG adducts in liver tissue DNA of F344 rats after treatment with 3.2 g/kg carbon
tetrachloride via i.p. administration. Wang and Liehr (1995) found that MDA induced DNA
adducts in hamsters treated with an oral administration of 0.1 mL/kg carbon tetrachloride, and
the levels of adducts formed were directly correlated with lipid hydroperoxide concentrations.
These reactive aldehydes can form DNA adducts causing frameshift or base mispairing (G to T
and G to A mutations).
Similar to 4-HNE, MDA is a result of oxidative degradation of polyunsaturated fatty
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acids with more than two methylene-interrupted double bonds. In mammalian tissues, precursors
for MDA are arachidonic acid and docosahexenoic acid.
Ichinose et al. (1994) compared the in vitro production of MDA per mg microsomal
protein from hepatic microsomes in several species. The rat generated the highest amount of
MDA over 2 hours, followed by monkey, mouse, pig, cow, rabbit, sheep, horse, and dog. Using
tissue slices from male Sprague-Dawley rats incubated in 1 mM carbon tetrachloride for 2 hours,
Fraga et al. (1987) found significant increases over control values in TEARS (nmol/g tissue)
released from treated liver (-fourfold), kidney (-threefold), spleen (-twofold), and testis
(-fivefold). Abraham et al. (1999) reported significantly elevated lipid peroxide levels in the
lung (65%), testis (200%), kidney (85%), and liver (200%) of Wistar rats exposed to carbon
tetrachloride vapor over a 12-week period. The results of Fraga et al. (1987) and Abraham et al.
(1999) show that lipid peroxidation can occur in other tissues besides the liver, specifically in the
kidney, testis, spleen, and lung.
Lipid peroxidation has been proposed to disrupt cellular membranes, resulting in loss of
membrane integrity (Recknagel and Glende, 1989) and the production of reactive aldehydes that
can attack tissues and form protein and DNA adducts (Comporti, 1985; Comporti et al., 1984).
These aldehydes may diffuse from the membranes and traverse intracellularly or extracellularly
away from the point of origin to attack distant targets, acting as secondary toxicants.
Immunohistochemical procedures using antibodies directed against MDA- and 4-HNE protein
adducts have been used to detect adducts in rat liver sections treated with carbon tetrachloride
(Bedossa et al., 1994). Abraham et al. (1999) reported significantly elevated protein carbonyl
content, a measure of protein adduct formation, in the liver (238%), lungs (51%), and testis
(21%) of carbon tetrachloride vapor-treated rats compared with controls.
Hartley et al. (1999) studied the temporal relationship between carbon tetrachloride-
initiated lipid peroxidation, hepatocellular damage, and formation of 4-HNE and MDA-hepatic
protein adducts, using immunohistochemical detection of aldehyde-adducted proteins in liver
sections and immunoprecipitation and immunoblotting procedures to detect and characterize
4-HNE and MDA-adducted proteins in liver homogenates from male highly alcohol-sensitive
rats treated with 1 mL/kg (1.59 g/kg) of carbon tetrachloride in mineral oil by gavage. Mineral
oil alone elicited subtle centrilobular steatosis, a slight increase in necrosis at 12 hours, and a
slight elevation of serum ALT at 24 hours. The livers of rats treated with carbon tetrachloride in
mineral oil exhibited a significant number of ballooned hepatocytes and inflammatory cells at
12 hours and progressive, massive centrilobular steatosis, inflammation, and necrosis at 18-48
hours. There was a fivefold increase in serum ALT at 6 hours after treatment, peaking at
36 hours with a 32-fold increase in ALT over control. Between 18 and 36 hours posttreatment,
TEARS values in liver homogenates of treated rats were maximal at a 2.5-fold increase over
controls. MDA-amine and 4-hydroxynonenal-sulfhydryl protein adducts were detectable at
6 hours in the midzonal region and in the centrilobular region at 12-36 hours. The
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correspondence in time course and location for lipid peroxidation, production of protein adducts,
and liver damage suggests that protein adducts resulting from lipid peroxidation contribute to
hepatocellular injury in carbon tetrachloride-treated rats.
Evidence of the relationship between hepatotoxicity and lipid peroxidation was also
reported by Younes and Siegers (1985). These researchers found that administration of an iron-
chelating agent, deferoxamine, suppressed both lipid peroxidation (ethane exhalation) and
hepatotoxicity (serum ALT and SDH levels) in GSH-depleted mice treated with carbon
tetrachloride. This result suggests that the observed hepatotoxic effect was secondary to lipid
peroxidation. Administration of the antioxidant vitamin E (a-tocopherol) was shown to reduce
lipid peroxidation (pentane exhalation) and metabolism (chloroform generation) in another rat
study (Gee et al., 1981).
Lipid peroxidation by-products can also form promutagenic DNA adducts and modify
double-stranded DNA by formation of amino-imino propene crosslinks between the NH2 group
of the guanosine base and complementary cytosine base. In rat hepatocytes cultured with 0.25, 1
or 4 mM carbon tetrachloride, Beddowes et al. (2003) showed that carbon tetrachloride caused a
dose-dependent increase in the formation of DNA strand breaks, 8-oxodG and MDA-DNA
adducts. The increased formation of DNA strand breaks and MDA-DNA adducts was
statistically significant at 1 and 4 mM. The level of 8-oxodG was statistically elevated only at 4
mM, a concentration that caused a decrease in cellular viability. Carbon tetrachloride induced
lipid peroxidation carbonyl product formation (>2-fold) at 4 mM; lower concentrations were not
studied. The formation of MDA-DNA adducts appeared to correlate with the ability of carbon
tetrachloride to induce lipid peroxidation, although failure to measure lipid peroxidation at the
two lower concentrations (0.25 and 1 mM) somewhat limits the ability to establish this
correlation.
4.5.4. Depletion of Glutathione
Reduced glutathione is capable of donating a hydrogen to quench a free-radical chain
reaction and can play a key role in limiting the damage to cellular membranes caused by lipid
peroxidation. The efficacy of reduced glutathione in quenching a free radical reaction is
dependent on the activity of GSH peroxidase, the enzyme that facilitates the transfer of hydrogen
to hydrogen peroxide with the formation of glutathione disulfide (GSSG) and water. Cellular
levels of reduced glutathione are restored through the activity of GSH reductase using NADPH +
H+ as the hydrogen donor (Klaassen, 1996a).
Cabre et al. (2000) assessed the temporal relationships between hepatic lipid
peroxidation, GSH metabolism, and development of cirrhosis in groups of 10 male Wistar rats
exposed to carbon tetrachloride. Rats were injected intraperitoneally with 0.5 mL of carbon
tetrachloride in olive oil twice weekly for 9 weeks to induce hepatic cirrhosis. By the second
week, 10/10 livers were fibrotic. Cirrhosis appeared in all 10 animals by week 9. Hepatic GSH
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levels were significantly reduced, beginning at week 5, and GSH peroxidase activity was
significantly decreased at week 7 in carbon tetrachloride-treated rats; the activity of GSH
peroxidase is dependent on a sufficient level of GSH. Cytosolic GSH S-transferase activity was
also significantly inhibited in rats receiving carbon tetrachloride at week 1. TEARS (lipid
peroxides) began to be elevated by week 7. The findings of this study show that induction of
cirrhosis in rats by carbon tetrachloride produces a decrease in several components of the hepatic
GSH antioxidant system. Impairment of this hepatoprotective system was related to an increased
generation of lipid peroxides.
Gorla et al. (1983) confirmed that oral pretreatment of male Sprague-Dawley rats with 2
g/kg of GSH 30 minutes before an i.p. injection of carbon tetrachloride (1.59 mg/kg) partially
prevented the hepatic necrosis that normally occurs 24 hours after carbon tetrachloride dosing.
Treatment with cysteine, which is a precursor of GSH and, like GSH, is able to conjugate
phosgene (from chloroform) produced from carbon tetrachloride, also protected against carbon
tetrachloride hepatotoxicity when given orally 30 minutes before or 1 hour after i.p. injection of
carbon tetrachloride (de Ferreyra et al., 1974).
Gasso et al. (1996) investigated the effects of S-adenosylmethionine (SAM) availability
on lipid peroxidation and liver fibrogenesis in male Wistar rats with carbon tetrachloride-induced
cirrhosis. SAM is essential for the production of the GSH precursor homocysteine, which
provides the sulfur for the endogenous synthesis of cysteine (the source of the reactive -SH
functional group in glutathione). A SAM deficiency can also limit transmethylation reactions
that function in DNA and RNA methylation and the production of thymine for DNA repair.
Gasso et al. (1996) found that depletion of GSH triggers a feedback mechanism, leading to
inactivation of SAM synthetase, which in turn causes a further decrease in GSH. SAM
synthetase is responsible for the endogenous production of SAM from the essential amino acid
methionine. The deficit of SAM could be corrected by exogenous administration of SAM but
not methionine. Accordingly, the deficit appeared to be the result of enzyme inhibition rather
than methionine availability.
Carbon tetrachloride-treated rats receiving SAM for 6 weeks had significantly higher
SAM synthetase activity (156 ± 5.6 pmol/minute/mg protein) than rats treated with carbon
tetrachloride alone (89.4 ±3.4 pmol/minute/mg protein) (Gasso et al., 1996). The hepatic GSH
was significantly decreased in carbon tetrachloride-treated rats (2.7 ±13 nmol/g tissue) and
returned to normal in rats receiving SAM for 3 or 6 weeks (3.7 ± 0.13 and 3.9 ± 0.11 nmol/g
tissue). Carbon tetrachloride-treated rats receiving SAM for 6 weeks had significantly lower
liver toxicity (collagen and propyl hydroxylase activity, reduced lipid peroxidation, and less
advanced liver fibrosis). The hepatic TEARS, markers of lipid peroxidation, were also
significantly lower in rats treated with carbon tetrachloride and SAM for 6 weeks (98 ± 5 nmol/g
tissue) than rats treated with only carbon tetrachloride (134 ± 12 nmol/g tissue). In rats treated
with carbon tetrachloride and SAM for 6 weeks, serum AST (76 ± 6 U/L) and ALT (57 ± 4 U/L)
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were lower than rats treated with only carbon tetrachloride (321 ± 33 U/L and 185 ± 21 U/L,
respectively). These data provide evidence that hepatic lipid peroxidation is increased during
hepatic fibrogenesis and that exogenous SAM may lead to an increase of GSH levels, which
could prevent SAM synthetase inactivation, inhibit lipid peroxidation, and, consequently,
attenuate the development of liver fibrosis and cirrhosis.
Will et al. (1999) demonstrated in 11 untreated mammalian cell lines that the intrinsic
levels of GSH expression were inversely correlated with the background level of oxidative DNA
modifications, such as 8-hydroxyguanine. Depletion of GSH with buthionine sulphoximine, an
inhibitor of y -glutamyl-cysteine, the precursor to GSH (Edgren and Revesz, 1987), increased the
basal levels of oxidative DNA base modifications. Schisandrin B, a compound that enhances the
GSH antioxidant status in hepatic mitochondria, was hepatoprotective against carbon
tetrachloride exposure in Balb/c mice (Chiu et al., 2003).
4.5.5. Disruption of Calcium Homeostasis
Calcium plays an essential role in cellular physiology. Levels of calcium in the cell are
maintained far below extracellular levels by resistance of the plasma membrane to passive
diffusion of calcium across the membrane and by active transport of calcium across the cell
membrane and into the extracellular space (Klaassen, 1996). Calcium within the cell is actively
transported across the microsomal membrane into the endoplasmic reticulum and across the
mitochondrial membrane into the mitochondria. Maintenance of calcium homeostasis is vital to
cellular function, and interference with calcium homeostasis is suspected to cause cell death
(Farber, 1981).
Calcium ATPase helps maintain calcium-level homeostasis within the cell. When
cytosolic calcium levels are highly elevated, the calcium ATPase, located in the plasma
membrane, is activated. Activation of calcium ATPase triggers the transport of two calcium ions
from the cytosol to the endoplasmic reticulum hydrolyzing one ATP in this process. This
process also requires Mg2+to be tightly complexed to ATP. A rise in cytosolic calcium also
induces the binding of calcium ions to regulatory calcium-binding proteins, like calmodulin (a
148-residue protein found in many cells and an essential subunit of the plasma membrane
calcium ATPase). Binding of cytosolic calcium to calmodulin triggers an allosteric activation of
calcium ATPase that accelerates the uptake of calcium ions from the cytosol by the endoplasmic
reticulum to maintain a low cytosolic concentration of less than 1 jiM calcium. While calmodulin
complements calcium ATPase, it also modulates the activities of a large number of calcium-
dependent proteins (Garrett and Grisham, 1999).
Studies conducted with carbon tetrachloride have reported 100-fold or more increases in
the cytosolic concentration of calcium following exposure (Agarwal and Mehendale, 1986, 1984;
Long and Moore, 1986; Kroner, 1982). In a study in which hepatocytes were incubated in a
medium containing EGTA, a calcium-specific chelator, but no added calcium, treatment with
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carbon tetrachloride elicited an increased calcium-dependent conversion of glycogen
phosphorylase "b" to phosphorylase "a" by phosphorylase kinase, which is stimulated by
increased intracellular calcium levels (Long and Moore, 1986). The lack of extracellular calcium
in this experimental system indicates that the carbon tetrachloride exposure released sequestered
calcium, probably from microsomes. The authors suggested that calcium could contribute to cell
death by the overstimulation of calcium-responsive cellular enzymes that initiate a cascade of
events, resulting in irreversible cell injury.
Hepatocytes treated with carbon tetrachloride had an impaired ability to maintain proper
calcium levels that was associated with inactivation of the calcium ATPase of the endoplasmic
reticulum (Lowrey et al., 1981; Moore, 1980). Administration of carbon tetrachloride caused an
85% reduction of ATP-dependent calcium uptake and calcium-sequestering capacity of the
hepatocyte endoplasmic reticulum (Moore et al., 1976). Hemmings et al. (2002) showed that
carbon tetrachloride decreased active calcium transport across the plasma and mitochondrial
membranes, as well as the endoplasmic reticulum, in rat liver. In vitro experiments confirmed
that inhibition of the plasma membrane calcium transport system by carbon tetrachloride was
rapid (within a minute) and strong (>90%) (Hemmings et al., 2002).
Carbon tetrachloride can also increase cytoplasmic calcium levels by opening certain
calcium transport channels in membranes. Liver endoplasmic reticulum contains ryanodine-
sensitive calcium-binding sites (Feng et al., 1992). Ryanodine is an alkaloid, usually found in
the skeletal and cardiac sarcoplasmic reticulum, that induces calcium release from liver
microsomes by binding to certain calcium release channels. Stoyanovsky and Cederbaum (1996)
showed that hepatic ryanodine-sensitive calcium channels may be involved in the elevation of
cytosolic calcium levels in the liver following carbon tetrachloride dosing. These researchers
observed elevated cytosolic calcium levels after treatment of hepatic microsomes with 50 uM of
carbon tetrachloride. Ruthenium red, a specific inhibitor of the ryanodine receptor calcium
release channel, has been shown to block the carbon tetrachloride-induced release of calcium.
Activation of calcium-dependent cysteine proteases andphospholipases
The increase in cytosolic calcium and inhibition of the calcium pump can activate a
number of calcium-dependent cysteine proteases (e.g., calpains, known for their involvement in
proteolysis of proteins during mitosis, apoptosis, and necrosis) and phospholipases (particularly
phospholipase A2) that preferentially hydrolyze membrane lipids. Activation of these enzymes
can contribute to toxicity of carbon tetrachloride in the liver.
When calcium homeostasis has been disrupted because of the loss of microsomal
membrane integrity, increased levels of calcium leakage activate a number of cytosolic and
lysosomal degradative enzymes that are also leaked out into the extracellular space from dying
cells; these degradative enzymes can subsequently attack neighboring cells. Limaye et al. (2003)
demonstrated the involvement of calpain, a calcium-dependent cytosolic neutral cysteine
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protease that leaks out from injured hepatocytes, in degrading cytoskeletal and membrane
proteins (e.g., a-fodrin, talin, filamin) and other macromolecules crucial to maintaining cellular
integrity, culminating in cell lysis and hepatocyte cell death. Calpain causes cell death by
attacking the plasma membrane, and, once the integrity of the membrane is lost, cells are
rendered highly vulnerable to destruction. Limaye et al. (2003) showed how calpain inhibition
with calpain-specific inhibitor N-benzyloxycarbonyl-valine-phenylalanine methyl ester (CBZ)
after carbon tetrachloride treatment substantially reduced the progression of injury and improved
animal survival. After 48 hours, the elevation in calpain activity was substantially in the carbon
tetrachloride + CBZ-treated rats than the carbon tetrachloride + DMSO-treated rats. [DMSO
was the vehicle used for CBZ administration.] More significantly, in rats challenged with a
normally lethal dose of carbon tetrachloride (3 mL/kg, i.p.), 75% of the male Sprague-Dawley
rats that received CBZ (60 mg/kg) 1 hour after carbon tetrachloride administration survived,
while rats treated with carbon tetrachloride alone or carbon tetrachloride and DMSO experienced
75% mortality. All control rats survived.
This study also evaluated the degradative effect of calpain on a-fodrin, a membrane
protein (Limaye et al., 2003). Calpain is known to degrade the 240-kDa fodrin to produce a 150-
kDa fragment. In rats receiving CBZ after carbon tetrachloride, the breakdown of a-fodrin was
similar to that in controls, indicating that inhibition of calpain released from dying hepatocytes
resulted in lower cellular damage. To confirm that cell death was caused by calpain, fresh
hepatocytes were incubated with calpain and 2.5 mM calcium. By the end of 240 minutes, cell
viability was decreased to 75%. Dying cells were found to develop plasma membrane blebs,
indicating cytotoxicity, which is typical of cytoskeletal damage induced by calpain. In the
presence of CBZ, hepatocytes were completely protected from calpain-mediated cell death.
Additional experiments with E64, a cell-impermeable inhibitor of calpain, also significantly
reduced plasma ALT levels, suggesting that the presence of calpain in the extracellular space is
responsible for the damage to some hepatocytes.
While these results suggest that calpain is a major contributor in the progression of liver
injury, other degradative enzymes are also released into the extracellular space, such as
nucleases, acid phosphatases, and phospholipases. Loss of calcium sequestration capacity
caused by in vitro metabolism of carbon tetrachloride by isolated rat liver microsomes (e.g.,
Lowrey et al., 1981) correlates with carbon tetrachloride-dependent activation of phospholipase
A2, measured by lysophosphatide formation or release of arachidonic acid from the hydrolysis of
esterified arachidonic acid from the sn-2 position of hepatocyte phospholipids (Glende and
Pushpendran, 1986). Studies with rat hepatic microsomes demonstrated a progressive loss of
phospholipid after incubation in 5 mM CaCb, with time-dependent losses of microsomal protein
activity (G6Pase and CYP450) that reached 80% by 3 hours (Chien et al., 1980). Quinacrine, a
phospholipase A2 inhibitor at 150 mg/kg i.p., has been shown to prevent carbon tetrachloride-
induced liver necrosis at 24 hours when administered 30 minutes before or 6 or 10 hours after
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carbon tetrachloride exposure (2.5 mL/kg orally) (Gonzalez Padron et al., 1993). The authors of
this study concluded that phospholipase A2 plays a major role in carbon tetrachloride-induced
liver necrosis.
Glende and Pushpendran (1986) prelabeled hepatocytes with 3H-arachidonic acid or
[14C]-ethanolamine and subsequently incubated the cells with carbon tetrachloride. Calcium-
activated phospholipase A2 activity was determined by measuring the release of 3H-arachidonic
acid from cellular phospholipids labeled with arachidonate or the formation of [14C]-
lysophospholipids from cellular phospholipids labeled with ethanolamine. Treatment with 0.23-
1.3 mM of carbon tetrachloride increased the endogenous phospholipase A2 activity 1.4- to 5.3-
fold beginning within 30-60 minutes. A similar study in isolated hepatocytes revealed that
carbon tetrachloride stimulated phospholipase A2 activity (monitored by production of
lysophosphatidylethanolamine) within 15 minutes, succeeded within 15 minutes by
hepatotoxicity, as measured by the release of LDH from the cells into the medium (Glende and
Recknagel, 1992). This same study demonstrated that related compounds (chloroform,
bromotrichloromethane, and 1,1-dichloroethylene) similarly activate phospholipase A2 activity in
hepatocytes. The authors suggested that phospholipase A2 could contribute to hepatocyte
pathology by two different means: by increasing the hydrolysis of membrane lipids at rates
exceeding the rate of repair and/or by the phospholipase A2-dependent generation of toxic
prostanoids via initiation of the arachidonic acid cascade.
4.5.6. Immunological and Inflammatory Effects
Immunological effects of carbon tetrachloride appear to be, at least in part, secondary to
hepatotoxicity and the process of hepatic repair. Carbon tetrachloride induces a regenerative
response in the liver similar to that observed following administration of other hepatotoxic
chemicals (e.g., acetaminophen) or partial hepatectomy (Jeon et al., 1997; Delaney et al., 1994).
The regenerative process involves complex interactions among several cell types and cell
mediators, including the hepatic synthesis and release of serum-borne growth factors
(hepatotrophic factors) that act directly on liver cells to induce mitosis (Luster et al., 2000).
Hepatotrophic factors also appear to act on peripheral organs, most notably the spleen (Delaney
and Kaminski, 1994; Delaney et al., 1994). Results of studies on the effects of hepatotrophic
factors indicate that immune effects of carbon tetrachloride, and other hepatotoxic chemicals,
may be mediated by tumor growth factor (TGF)- pi released from the liver during the
regenerative process (Jeon et al., 1997; Delaney et al., 1994; Delaney and Kaminski, 1993).
A series of experiments conducted by Delaney and coworkers suggest that carbon
tetrachloride-induced suppression of T-cell function is mediated through serum-borne factors
(Delaney et al., 1994; Delaney and Kaminski, 1993). Serum from B6C3F1 mice treated with
250 or 500 mg/kg carbon tetrachloride in corn oil by gavage for 7 days, a dose regimen that
produced hepatotoxicity, suppressed the sheep erythrocyte-induced antibody response of carbon
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tetrachloride-naive spleen cells in vitro (Delaney and Kaminski, 1993). In a subsequent study,
Delaney et al. (1994) demonstrated that carbon tetrachloride-induced suppression of the T-cell-
dependent humoral response is at least partially mediated by TGF- pi. Suppression of the sheep
erythrocyte antibody response of naive spleen cells in vitro by serum of mice exposed to carbon
tetrachloride (single oral dose of 500 or 1000 mg/kg carbon tetrachloride in corn oil) was
abolished upon addition of TGF- pi-specific antibodies to the assay. Jeon et al. (1997) reported
elevations of TGF- pi mRNA in the liver of B6C3F1 mice treated with a single hepatotoxic dose
(500 mg/kg) of carbon tetrachloride within 24 hours of exposure. Although direct effects of
carbon tetrachloride on the immune system by carbon tetrachloride have not been ruled out,
results of in vitro and in vivo studies suggest that immunotoxicity is, in part, mediated by TGF-
Pl secreted by the liver during tissue repair.
Inflammation contributes to the development of chemical-induced hepatotoxicity and
possibly to immunotoxic effects. Kupffer cells are hepatic macrophages that respond to signals
from injured hepatocytes by releasing biologically active mediators, such as prostaglandins,
reactive oxygen species, and cytokines (Luckey and Petersen, 2001). Factors released by
Kupffer cells after activation by carbon tetrachloride include nitric oxide, tumor necrosis factor-
ex, TGF- p, and interleukins-6, -8, and -10. The mediators produced by Kupffer cells are
involved in the regulation of the inflammatory response and fibrotic response following hepatic
injury. As discussed earlier, TGF- pi released from the liver plays an important role in the
immunotoxic effects of carbon tetrachloride, providing a possible link between hepatic
inflammation and Kupffer cell activation by immunotoxic events.
Stellate cells are hepatic fat-storing cells that respond to liver injury by proliferating,
migrating towards damaged areas, releasing nitric oxide and extracellular signal-regulated
kinases that perform various functions in different tissues, and increasing production of
extracellular matrix, thereby promoting fibrosis (Weber et al., 2003; Marra et al., 1999). Stellate
cells are activated by TGF-a. Acute treatment with carbon tetrachloride increases the activity of
extracellular signal-regulated kinases from stellate cells (Marra et al., 1999).
Carbon tetrachloride has been shown to stimulate increases in the numbers of
immunodetectable Kupffer cells in the livers of treated rats, as well as increases in releases of
various cytokines and reactive oxidative species, corresponding to different stages of liver
histopathology (Luckey and Petersen, 2001; Alric et al., 2000). Towner et al. (1994) reported
that i.p. administration of 1275 mg/kg of carbon tetrachloride to male Wistar rats was
characterized by hepatic edema from the accumulation of vacuoles and lipid droplets in
parenchymal cells and accumulation of phagosomes (large secondary lysosomes) and extrusion
of pseudopods in enlarged Kupffer cells. With a 1-hour intravenous pretreatment with 10 mg/kg
gadolinium trichloride (GdCb), an inhibitor of Kupffer cell activation, the parenchymal cells
were normal and Kupffer cells contained only a few secondary lysosomes. The protective effect
of GdCb was not associated with a change in detectability of carbon tetrachloride-generated
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trichloromethyl radical by electron spin resonance spectroscopy.
The effects of GdCl3 on carbon tetrachloride-induced hepatic toxicity were evaluated in
other studies. Muriel et al. (2001) treated male Wistar rats with 4000 mg/kg of carbon
tetrachloride by gavage in corn oil, with or without i.p. injection of 2000 mg/kg GdCb. Twenty-
four hours later, rats treated with carbon tetrachloride showed typical hepatotoxicity (increased
serum enzymes and bilirubin, 2.5-fold increase in hepatic lipid peroxidation, and liver
histopathology: ballooning necrotic hepatocytes). Treatment with GdCls eliminated the
increases in serum biomarkers of membrane damage and hepatic lipid peroxidation and
significantly reduced the severity of hepatic necrosis. In a follow-up study of similar design,
male Wistar rats were treated with carbon tetrachloride (400 mg/kg by i.p. injection in mineral
oil 3 times/week), GdCls (20 mg/kg i.p. in saline daily), or both for 8 weeks (Muriel and
Escobar, 2003). Cotreatment with GdCb resulted in partial or complete protection against the
effects of carbon tetrachloride on serum ALT, GGT, ALP, and bilirubin; liver MD A content
(index of lipid peroxidation); liver hydroxyproline content (index of collagen content and
fibrosis); and histopathology (both necrosis and fibrosis). Depletion of liver glycogen by carbon
tetrachloride was not affected by GdCb, and GdCb itself produced a significant depletion of
glycogen.
Although multiple studies have indicated that GdCl3 treatment reduces or inhibits carbon
tetrachloride-induced hepatotoxicity through inactivation of Kupffer cells, GdCl3 may also
reduce carbon tetrachloride toxicity through other cellular mechanisms. Rose et al. (2001)
demonstrated both in vivo and in vitro that GdCb stimulated hepatocyte proliferation through a
mitogenic mechanism involving TNF-a, and promoted recovery from liver damage. GdCb has
also been shown to inhibit free radical-induced hepatocyte damage by nonselective blockage of
Na+ channels that induce necrosis in an in vitro model (Barros et al., 2001). Critical to carbon
tetrachloride-induced toxicity is the generation of reactive metabolites by CYP2E1 for which
GdCls downregulates the gene expression in vivo (Okamoto, 2000; Badger et al., 1997).
Overall, multiple cellular mechanisms have been demonstrated by which GdCb reduces carbon
tetrachloride-induced toxicity and indicates that toxicity is not mediated exclusively through
inactivation of Kupffer cells.
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4.5.7. Changes in Gene Expression
Changes in gene expression in response to exposure to carbon tetrachloride have been
investigated in the liver of rats and mice and in the human hepatoma cell line (lessen et al., 2003;
Fountoulakis et al., 2002; Bartosiewicz et al., 2001; Holden et al., 2000; Columbano et al., 1997;
Menegazzi et al., 1997). Many of the known upregulated genes are related to stress, DNA
damage and repair, and signal transduction, but for the most part their specific contributions to
hepatotoxicity are not known. Fountoulakis et al. (2002) reported a fivefold increase in
expression of some genes related to stress and DNA damage repair in the livers of male Wistar
rats 6 hours after they received 400 mg/kg carbon tetrachloride. Rats receiving 3190 mg/kg
showed 10-fold increases in expression in some genes. Some of the stress- and DNA-damage-
related genes upregulated by both doses at 24 hours included GADD45, GADD153, heat-shock
proteins, heme oxygenase, p53, c-myc, and c-jun. There were some qualitative differences in
altered gene expression at 6 and 24 hours between the two doses administered in this study,
which possibly provides a basis for the different hepatocellular responses to carbon tetrachloride-
induced injury. The hepatic expression of the Cdk inhibitor p21 in mice treated with carbon
tetrachloride occurs just prior to necrosis at 6 hours, and mice deficient in that gene do not
exhibit necrosis in response to carbon tetrachloride (Kwon et al., 2003); p21 also contributes to
the cessation of cellular proliferation that occurs later.
Intraperitoneal injection of Sprague-Dawley rats with 160 mg/kg of carbon tetrachloride
in corn oil activated c-fos and c-jun gene expression in the liver within 30 minutes (Gruebele et
al., 1996). Pretreatment of rats with diallyl sulfide, an inhibitor of CYP2E1, 3 hours before
dosing with carbon tetrachloride reduced c-jun mRNA levels by 76%. Treatment with carbon
tetrachloride also increased hepatic nuclear levels of the NF-6B transcription factor, which
regulates genes involved in responses to inflammation, apoptosis, hepatocyte proliferation, and
liver regeneration.
Columbano et al. (1997) investigated the relationship between immediate early genes and
hepatocyte proliferation through comparison of the hepatic levels of c-fos, c-jun and LRF-1
transcripts during mouse liver cell proliferation under two conditions: (1) direct hyperplasia
induced by the primary mitogen (and hepatocarcinogen) l,4-bis[2-(3,5-
dichloropyridyloxy)]benzene (TCPOBOP), and (2) compensatory regeneration caused by a
necrogenic dose of carbon tetrachloride (single intragastric dose of 2 mg/kg in oil) or by
performing a 2/3 partial hepatectomy. A striking difference in the activation of early genes was
observed. In spite of a rapid stimulation of S phase by the mitogen TCPOBOP, there were no
changes in the expression of c-fos, c-jun and LRF-1 or in steady state mRNA hepatic levels of
IGFBP-1 (a gene highly expressed in rat liver following partial hepatectomy), and only a slight
increase in c-myc and PRL-1. In contrast, a rapid, massive and transient increase in the hepatic
mRNA levels of all these genes was observed during carbon tetrachloride-induced regeneration
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that was comparable to those seen following 2/3 partial hepatectomy. In similar research from
the same laboratory, the pattern of immediate early gene and growth factor gene expression in
the rat liver induced by primary mitogens (including lead nitrate (LN), cyproterone acetate, or
nafenopin) was shown to differ from that observed following compensatory liver regeneration
occurring after cell loss/death and direct hyperplasia resulting from a partial 2/3 hepatectomy or
a necrogenic dose (2 mL/kg) of carbon tetrachloride (Menegazzi et al., 1997). In this study, the
following indicators of gene expression were examined: modifications in the activation of two
transcription factors, NF-kappaB and AP-1; steady-state levels of tumor necrosis factor alpha
(TNF-alpha) messenger RNA (mRNA); and induction of the inducible nitric oxide synthase
(iNOS). Liver regeneration after treatment with carbon tetrachloride was associated with an
increase in steady-state levels of TNF-alpha mRNA, activation of NF-kappaB and AP-1, and
induction of iNOS. LN induced NF-kappaB, TNF-alpha and iNOS mRNA but not AP-1,
whereas direct hyperplasia induced by the other two primary mitogens occurred in the complete
absence of modifications in the hepatic levels of TNF-alpha mRNA, activation of NF-kappaB
and AP-1, or induction of iNOS, although the number of hepatocytes entering S phase 18 to 24
hours after NAF was similar to that seen after PH. The findings from these two studies indicate
that regenerative proliferation alone does not explain the tumorigenic response associated with
carbon tetrachloride in chronic bioassays, but these data do not preclude regenerative
proliferation as a biologically based marker of such causal events.
4.5.8. Mechanisms of Kidney Toxicity
Limited data suggest that some of the same mechanisms by which carbon tetrachloride
produces damage to the liver can also operate in the kidney. Dogukan et al. (2003) observed
moderate renal histopathology (tubular necrosis, dilatation, atrophy, glomerular hypercellularity,
capillary obliteration, and interstitial fibrosis) in male Wistar rats subcutaneously injected three
times/week with 240 mg/kg of carbon tetrachloride in olive oil for 7 weeks. The tissue damage
was associated with a significant increase in renal MDA (+34%), indicating lipid peroxidation,
and the researchers attributed the effects to oxidative stress. The tissue damage was also
accompanied by a significant decrease in renal GSH peroxidase, indicating a depletion of renal
GSH as contributing to the observed tissue damage. Studies by Fraga et al. (1987) using rat
tissue slices in vitro and Abraham et al. (1999) in rats in vivo also showed lipid peroxidation in
the kidney resulting from carbon tetrachloride exposure.
Ozturk et al. (2003) evaluated the levels of antioxidants in the kidney of Sprague-Dawley
rats subcutaneously injected with 1594 mg/kg-day of carbon tetrachloride on 4 consecutive days.
Compared with control kidneys, treated kidneys had significantly elevated activity levels for
superoxide dismutase (+30%) and catalase (+46%) but reduced activity for GSH peroxidase
(-44%) 24 hours after the last injection. The authors attributed the reduced activity of GSH
peroxidase to decreased availability of renal GSH in its reduced form. Treated kidneys showed
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severe and extensive cortical histopathology: focal glomerular necrosis, tubular dilation,
epithelial vacuolization or necrosis (with detachment from the basement membrane), and protein
casts. A parallel group treated with carbon tetrachloride and betaine (a methyl group donor)
showed no differences from the control group for superoxide dismutase or GSH peroxidase,
whereas catalase was significantly elevated (+34%). Kidneys of rats treated with carbon
tetrachloride plus betaine had normal glomerular histology and only sparse tubular dilatation,
epithelial vacuolization, and few cell detachments. The authors suggested that the beneficial
effect of betaine on renal histology and GSH peroxidase activity was related to its promotion of
SAM levels, as has been demonstrated in the liver by other investigators. This study suggests
that similar toxicological mechanisms may occur in the liver and kidney of rats treated with
carbon tetrachloride.
Cytosolic phospholipase A2 levels were significantly elevated in the renal cortex and
medulla of rats with carbon tetrachloride-induced cirrhosis and ascites (Niederberger et al.,
1998). The authors attributed the increase in phospholipase A2 to the increased renal production
of prostaglandins in cirrhosis.
4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS
Hepatic and renal effects are the most sensitive noncancer effects of oral or inhalation
exposure to carbon tetrachloride in humans and animals.
4.6.1. Oral Exposure
No long-term toxicity data are available for humans with quantified oral exposures to
carbon tetrachloride, but case reports identify the liver and kidney as the primary target organs
following acute exposures. Evidence of acute oral hepatotoxicity in humans comes from
observations of liver enlargement, elevated serum enzyme (AST and/or ALT), bilirubin levels, or
histopathology (hepatocyte degeneration) (Ruprah et al., 1985; Stewart et al., 1963; Docherty
and Nicholls, 1923; Docherty and Burgess, 1922). Other acute oral effects in humans include
renal toxicity, usually delayed relative to hepatic toxicity (New et al., 1962) and lung effects
secondary to renal failure (Umiker and Pearce, 1953). The prominence of hepatic injury in
acutely exposed humans suggests that hepatic toxicity observed in subchronic animal studies is
an important and relevant consideration for human health risk assessment of carbon
tetrachloride.
Studies in laboratory animals indicate that hepatic toxicity is the predominant noncancer
effect of subchronic or chronic oral exposure to carbon tetrachloride (Table 4-13). In these
studies, evidence of hepatic damage included liver histopathology (fatty degeneration, necrosis,
fibrosis, cirrhosis, inflammation, and regenerative activity), along with increases in liver weight
and serum markers for hepatotoxicity (ALT, AST, OCT, SDH, and bilirubin) (Koporec et al.,
1995; Allis et al., 1990; Bruckner et al., 1986; Condie et al., 1986; Hayes et al., 1986; NCI, 1977,
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1976a, b; Weisburger, 1977; Litchfield and Gartland, 1974; Delia Porta et al., 1961;
Eschenbrenner and Miller, 1946; Edwards and Dalton, 1942; Edwards et al., 1942; Edwards,
1941). Liver damage was produced at doses as low as 7-9 mg/kg-day in rats and mice in 90-day
corn oil gavage studies (Table 4-13). The corresponding NOAEL values were 0.7-0.9 mg/kg-
day (Bruckner et al., 1986; Condie et al., 1986). The lowest dose to produce hepatotoxicity in
90-day aqueous gavage studies was 18 mg/kg-day (Koporec et al., 1995).
Table 4-13. Oral toxicity studies for carbon tetrachloride
Species
Dose/duration
NOAEL
(mg/kg-day)
LOAEL
(mg/kg-day)
Effects at the LOAEL
Reference
Subchronic studies
Dog
(6/sex)
Dog
(3F)
Rat
(15-16 M/
group)
Rat
(6M7
group and
sacrifice
time)
Rat
(11 M/
group)
Rat
(11 M/
group)
Mouse
(12/sex/
group)
Mouse
(12/sex/
group)
28 days in gelatin
capsule: 797 mg/kg-
day
8 weeks in gelatin
capsule: 32 mg/kg-
day
5 days/week for 12
weeks by corn oil
gavage: 0, 1, 10, or
33 mg/kg-day
5 days/week for 12
weeks by corn oil
gavage: 0, 20, or 40
mg/kg-day;
sacrificed at
intervals from 1-15
days post-exposure
5 days/week for 13
week by corn oil
gavage: 0, 25, or
100 mg/kg-day
5 days/week for 13
weeks by gavage in
1% Emulphor: 0,
25, or 100 mg/kg-
day
5 days/week for 12
weeks by corn oil
gavage: 0, 1.2, 12,
or 120 mg/kg-day
5 days/week for 12
weeks by gavage in
l%Tween-60:0,
1.2, 12, or 120
mg/kg-day
Not
determined
32
1 [0.71]a
Not
determined
Not
determined
Not
determined
1.2 [0.86] a
12 [8.6] a
797
Not
determined
10 [7.1]a
20 [14.3] a
25 [17.8[a
(PEL)
25 [17.8] a
(PEL)
12 [8.6] a
120 [86] a
Increased ALT, OCT; fatty
vacuolization with single
cell necrosis in liver
No increases in serum
enzymes; no liver
histopathology
Two- to threefold increase
in SDH; mild centrilobular
vacuolization in liver
Increased liver weight,
ALT, AST, LDH; reduced
liver CYP450; cirrhosis,
necrosis, and degeneration
in liver
10% Mortality; increased
ALT, SDH; slight hepato-
cellular vacuolization and
minimal fibrosis in liver
25% Mortality; increased
ALT, SDH; slight hepato-
cellular vacuolization and
minimal fibrosis in liver
Increased ALT; mild to
moderate hepatic lesions
(hepatocytomegaly,
necrosis, inflammation)
Increased liver weight,
ALT, AST, LDH; hepato-
cytomegaly, vacuolation,
inflammation, necrosis,
and fibrosis in liver
Litchfield and
Gartland, 1974
Litchfield and
Gartland, 1974
Bruckner etal.,
1986
Allis etal.,
1990
Koporec et al.,
1995
Koporec et al.,
1995
Condie et al.,
1986
Condie et al.,
1986
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Table 4-13. Oral toxicity studies for carbon tetrachloride
Species
Mouse
(20/sex/
group)
Mouse
(5/sex/
group)
Dose/duration
7 days/week for 13
weeks by corn oil
gavage: 0, 12, 120,
540, or 1200 mg/kg-
day
30 times in 120
days by olive oil
gavage:
0, 40, 80, or 160
mg/kg-day
NOAEL
(mg/kg-day)
Not
determined
40
LOAEL
(mg/kg-day)
12
80
Effects at the LOAEL
Increased liver weight,
ALT, AST, ALP, LDH, 5'-
nucleotidase; fatty change,
hepatocytomegaly,
necrosis, and hepatitis
Necrosis in liver
Reference
Hayes etal.,
1986
Eschenbrenner
and Miller,
1946
Chronic studies
Rat
(50/sex/
group)
Mouse
(50/sex/
group)
5 days/week for 78
weeks by corn oil
gavage: 0, 47, or 80
mg/kg-day for
males; 0, 94, or 159
mg/kg-day for
females
5 days/week for 78
weeks by corn oil
gavage: 0, 1250, or
2500 mg/kg-day
Not
determined
Not
determined
47
1250
Increased mortality;
cirrhosis in liver
Markedly increased
mortality; cirrhosis and
other toxic lesions in liver;
adrenal
pheochromocytoma
NCI, 1977,
1976a, b
NCI, 1977,
1976a, b
Gestational exposure studies
Rat
(29 gravid
F)
Rat
(9-14
gravid F/
group)
Rat
(12-14
gravid F/
group)
Rat
(12-14
gravid F/
group)
Mouse
(>8 gravid
F/ group)
2 days on CDs 7-11
by corn oil gavage:
478 mg/kg-day
CDs 6-19 by corn
oil gavage: 0, 112.5,
or 150 mg/kg-day
GDs 6-15 by corn
oil gavage: 0, 25,
50, or 75 mg/kg-day
GDs 6-15 by
gavage in 10%
Emulphor: 0, 25,
50, or 75 mg/kg-day
GDs 1-5 by gavage
in corn oil: 0, 83, or
826 mg/kg-day
Not
determined
Not
determined
25
25
826
478
112.5
50
50
Not
determined
21% Maternal mortality;
59% of dams had no
offspring, 3 8% because of
full-litter resorption
Reduced maternal weight
gain; markedly increased
full-litter resorption
Piloerection; markedly
increased full-litter
resorption
Piloerection; slightly
increased full-litter
resorption
No effect on dams or pups
Wilson, 1954
Narotsky and
Kavlock, 1995
Narotsky et al.,
1997b
Narotsky et al.,
1997b
Hamlinetal.,
1993
""Duration adjusted dose provided in brackets (e.g., 1 mg/kg-day x (5 days per week/7 days per week) =
0.71 mg/kg-day.
136
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Subchronic oral studies that also examined nonhepatic endpoints (Bruckner et al., 1986;
Hayes et al., 1986) did not observe effects in the kidneys or other organs. There was some
evidence for impairment of T-cell-dependent immunity in mice treated with 40 mg/kg-day for 14
days but not in rats at hepatotoxic doses (160 mg/kg-day for 10 days) (Guo et al., 2000;
Smialowicz et al., 1991; Kaminski et al., 1990).
There is no direct evidence for effects on reproduction or development in humans
exposed orally to carbon tetrachloride. One epidemiological study (Bove et al., 1995, 1992a, b)
suggested associations between maternal exposure to carbon tetrachloride in drinking water and
adverse birth outcomes (the strongest relationship was for low term birth weight), but subjects
were exposed to multiple chemicals and the study included only a limited characterization of
exposure. Studies in animals have found that relatively high oral doses of carbon tetrachloride
(50 mg/kg-day and above) given on days 6-15 of gestation produce significant prenatal loss by
increasing the incidence of full-litter resorptions (Narotsky et al.,1997a, b, 1995; Narotsky and
Kavlock, 1995; Wilson, 1954); some evidence exists that reproductive effects are a consequence
of a maternally mediated response to alterations in hormonal levels (Narotsky et al., 1995,
1997a). The doses producing litter resorption also produced overt toxic effects in dams
(piloerection, kyphosis (or rounded upper back), and marked weight loss) and are well above the
LOAELs for liver toxicity with longer-term exposure. Although the NOAELs and LOAELs
were the same, both the clinical signs and litter resorptions were more pronounced when carbon
tetrachloride was administered in corn oil versus aqueous emulsion. Mice treated with carbon
tetrachloride early in gestation apparently did not show these effects (Hamlin et al., 1993).
Adrenal adenoma and pheochromocytomas were observed in mice exposed to carbon
tetrachloride by gavage in an NCI bioassay in which carbon tetrachloride was used as a positive
control for liver tumors (Weisburger, 1977); none of these tumors were specifically identified as
malignant. These tumors may indicate a potential noncancer health risk, as well as a cancer risk.
Benign pheochromocytomas are tumors that originate in chromaffin cells of the adrenal gland
medulla and secrete excessive amounts of catecholamines, usually epinephrine and
norepinephrine. Because pheochromocytomas are not innervated, catecholamine secretion is
unregulated, producing sustained sympathetic nervous system hyperactivity leading to
hypertension, tachycardia, and cardiac arrhythmias (Hansen, 1998). Health effects related to
pheochromocytoma formation in mice were not assessed in the NCI (1977) cancer bioassay.
Therefore, the potential for secondary effects of pheochromocytoma on the cardiovascular
system can only be inferred. The lowest exposure level associated with benign
pheochromocytomas in mice (LOAEL of 1250 mg/kg-day, 5 days/week [approximately
900 mg/kg-day]) is approximately 2 orders of magnitude higher than levels at which liver effects
become apparent in experimental animals. Therefore, the available data do not identify the
137 DRAFT - DO NOT CITE OR QUOTE
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adrenal gland as a sensitive target organ for carbon tetrachloride by oral administration.
Effect of Dosing Vehicle on Carbon Tetrachloride Toxicity
A number of investigators have demonstrated that the vehicle used in gavage studies to
administer carbon tetrachloride and other chlorinated solvents may affect the test chemical's
toxicity. Several investigators reported that carbon tetrachloride toxicity was enhanced if
administered in corn oil compared to an aqueous solution (Narotsky et al., 1997; Condie et al.,
1986), whereas Kaporec et al. (1995) found that corn oil as a vehicle (compared to an aqueous
vehicle) did not significantly alter carbon tetrachloride hepatotoxicity following subchronic
exposure, and Kim et al. (1990b) observed that administration in an aqueous solution enhanced
carbon tetrachloride toxicity as compared to corn oil. Raymond and Plaa (1997) and Narotsky et
al. (1997) found that the influence of vehicle could be dose-dependent. In their study of
developmental toxicity, Narotsky et al. (1997) reported that maternal toxicity was slightly more
pronounced when carbon tetrachloride was administered in aqueous vehicle, but at higher doses
was more pronounced when administered in corn oil vehicle. Sanzgiri and Bruckner (1997)
found that Emulphor, a polyethoxylated vegetable oil used as an emulsifier for VOCs and other
lipophilic compounds, had no significant effect on carbon tetrachloride acute hepatotoxicity in
Sprague-Dawley rats (as measured by elevation of serum enzyme activities of SDH and ALT)
when carbon tetrachloride was administered as a single oral doses at two dose levels (10 and 180
mg/kg) and at four concentrations of Emulphor (1, 2.5, 5 and 10%). Blood carbon tetrachloride
concentrations in these rats (measured at intervals up to 12 hours postdosing) revealed no
significant differences as a function of Emulphor concentration, suggesting that Emulphor did
not significantly affect carbon tetrachloride absorption or distribution.
A number of explanations of the influence of vehicle on the oral toxicity of carbon
tetrachloride have been offered. Kim et al. (1990b) reported that corn oil delays carbon
tetrachloride absorption from the digestive track and thereby decreases its arterial blood
concentration. Such alterations in carbon tetrachloride pharmacokinetics could influence the
resulting toxicity. It is possible that the preservation state of corn oil might influence toxicity;
cell membranes could be altered by older oil stored under improper conditions and contaminated
with peroxides or by heated and oxygenated corn oil that could lead to the formation of reactive
oxygen radicals (Raymond and Plaa, 1997). It has been proposed that corn oil might induce
CYP450 metabolizing enzymes that could enhance metabolism of carbon tetrachloride to
reactive, cytotoxic forms (Raymond and Plaa, 1997; Kaporec et al., 1995). High lipid intake
could possibly increase lipid levels in the liver, thereby enhancing target organ deposition of
lipophilic carbon tetrachloride. Corn oil could also directly affect the lipid composition of cell
membranes; the effects of carbon tetrachloride-derived trichloromethyl free radicals on hepatic
microsomal proteins and lipids might then be enhanced (Kim et al., 1995).
13 8 DRAFT - DO NOT CITE OR QUOTE
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Kaporec et al. (1995) proposed that a possible explanation for the observation of less
pronounced hepatotoxicity in mice dosed with halocarbons in aqueous media involves method
preparation. Even using methods to minimize carbon tetrachloride loss, Kaporec et al. (1995)
found that there was typically about a 20% loss of carbon tetrachloride from an aqueous
emulsion (Emulphor), but none from corn oil dosing solutions. Thus, findings of less severe
toxicity with an aqueous vehicle than corn oil vehicle may have been the result of animals
receiving a lower daily dose.
Thus, it is possible that the vehicle used in oral gavage studies to administer carbon
tetrachloride could be a potential confounding factor in toxicity assays; however, the magnitude
of the confounding and the nature of the interaction of corn oil remain uncertain.
4.6.2. Inhalation Exposure
Case reports of acute high-level exposure to carbon tetrachloride vapor or long-term
occupational exposure provide evidence of hepatotoxic and nephrotoxic effects of carbon
tetrachloride in humans. Observations indicative of an effect on the liver in these cases include
jaundice, increased serum enzyme levels, and, in fatal cases, necrosis of the liver (Stewart et al.,
1965; New et al., 1962; Kazantzis and Bomford, 1960; Norwood et al., 1950). Delayed effects
on the kidney have also been reported in acute overexposure cases. Other effects associated with
carbon tetrachloride exposure in humans are gastrointestinal symptoms (nausea and vomiting,
diarrhea, and abdominal pain) and neurological effects indicative of central nervous system
depression (headache, dizziness, and weakness). Tomenson et al. (1995) conducted a cross-
sectional epidemiology study of hepatic function in workers exposed to carbon tetrachloride.
They found suggestive evidence of an effect of occupational carbon tetrachloride exposure on
serum enzymes indicative of hepatic effects at workplace concentrations in the range of 1 to
4 ppm.
The liver and kidney are the most prominent targets of carbon tetrachloride in subchronic
and chronic inhalation studies of laboratory animals. Hepatic toxicity in these studies was
demonstrated by histopathology (centrilobular fatty degeneration, necrosis, fibrosis, cirrhosis,
hepatitis, and regenerative activity) as well as increases in liver weight and serum markers for
liver damage (Nagano et al., 2007a,b; Benson and Springer, 1999; JBRC, 1998; Prendergast et
al., 1967; Adams et al., 1952; Smyth et al., 1936). Hepatic effects were observed in animals
exposed to carbon tetrachloride concentrations as low as 2 ppm (adjusted to continuous
exposure, see Table 4-14). Renal damage was reported less frequently in these animal studies
and generally at higher concentrations than liver damage. The JBRC chronic bioassay (Nagano
et al., 2007b; JBRC, 1998) found renal damage, as evidenced by histopathology (increased
severity of chronic nephropathy in the rat and protein casts in the mouse) and changes in serum
chemistry and urinalysis variables at a concentration of 4 ppm (adjusted to continuous exposure,
see Table 4-14). There is evidence that liver effects produced by carbon tetrachloride are
139 DRAFT - DO NOT CITE OR QUOTE
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proportional to the product of concentration and time (C x T) (Plummer et al., 1990) and,
therefore, that the duration adjusted exposure concentration is the most appropriate dose metric
to use as the basis for comparison among studies. This is assumed to be the case for other
systemic targets as well.
Table 4-14. Inhalation toxicity studies for carbon tetrachloride
Species
Duration/
concentration
NOAEL
(ppm)
LOAEL
(ppm)
Effects at the LOAEL
Reference
Subchronic studies
Rat
(24 mixed
sex/group)
Guinea pig
(24 mixed
sex/group)
Monkey
(4/group)
Rat
(15-25/sex/
group)
Guinea pig
(5-9/sex/
group)
Rabbit
(1-2/sex/
group)
Monkey
(1-2/group)
Rat
(15/group)
8 hours/day, 5
days/week for 10.5
months: 0, 50, 100,
200, or 400 ppm
8 hours/day, 5
days/week for 10.5
months: 0, 25, 50,
100, 200, or 400 ppm
8 hours/day, 5
days/week for 10.5
months: 0, 50, or 200
ppm
7 hours/day, 5
days/week for 6
months: 0, 5, 10, 25,
50, 100, 200, or 400
ppm
7 hours/day, 5
days/week for 6
months: 0, 5, 10, 25,
50, 100,200, or 400
ppm
7 hours/day, 5
days/week for 6
months: 0, 5, 10, 25,
50, or 100 ppm
7 hours/day, 5
days/week for 6
months: 0, 5, 10, 25,
50, or 100 ppm
24 hours/day, 7
days/week for 13
weeks: 0, 1 (in n-
octane), or 10 ppm
Not
determined
Not
determined
Not
determined
5 [If
5 [If
10 [2]a
50 [10]a
1
50 [12]a
25 [6]a
50 [12]a
10 [2]a
10 [2]a
25 [5]a
100 [21]a
10
Fatty change in liver;
effects in other organs
were reported, but the
LOAEL for these effects
was unclear
Increased mortality;
reduced body weight
gain; fatty change in
liver; effects in other
organs were reported, but
LOAEL for these effects
was unclear
Mild fatty change and
degeneration in liver;
effects in other organs
were reported, but
LOAEL for these effects
was unclear
Increased liver weight;
fatty degeneration in liver
Increased liver weight;
fatty degeneration in liver
Increased liver weight;
fatty degeneration and
slight cirrhosis in liver
Slight fatty degeneration
and increased lipid
content in liver
Reduced body weight
gain; enlarged liver with
fatty change
Smyth et al.,
1936
Smyth et al.,
1936
Smyth et al.,
1936
Adams et al.,
1952
Adams et al.,
1952
Adams et al.,
1952
Adams et al.,
1952
Prendergast
etal., 1967
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Table 4-14. Inhalation toxicity studies for carbon tetrachloride
Species
Guinea pig
(15/group)
Rabbit
(3/group)
Dog
(2/group)
Monkey
(3/group)
Rat (107
sex/group)
Mouse (107
sex/group)
Rat
(10 M/
group)
Mouse
(10 M/
group)
Hamster
(10 M/
group)
Duration/
concentration
24 hours/day, 7
days/week for 13
weeks: 0, 1 (in n-
octane), or 10 ppm
24 hours/day, 7
days/week for 13
weeks: 0, 1 (in n-
octane), or 10 ppm
24 hours/day, 7
days/week for 13
weeks: 0, 1 (in n-
octane), or 10 ppm
24 hours/day, 7
days/week for 13
weeks: 0, 1 (in n-
octane), or 10 ppm
6 hours/day, 5
days/week for 13
weeks: 0, 10, 30, 90,
270, or 8 10 ppm
6 hours/day, 5
days/week for 13
weeks: 0, 10, 30, 90,
270, or 8 10 ppm
6 hours/day, 5
days/week for 12
weeks: 0, 5, 20, or 100
ppm
6 hours/day, 5
days/week for 12
weeks: 0, 5, 20, or 100
ppm
6 hours/day, 5
days/week for 12
weeks: 0, 5, 20, or 100
ppm
NOAEL
(ppm)
1
1
1
1
Not
determined
Not
determined
20 [4]a
5 [0.9]a
20 [4]a
LOAEL
(ppm)
10
10
10
10
10 [2]a
10 [2]a
100 [18]a
20 [4]a
100 [18]a
Effects at the LOAEL
Three died; reduced body
weight gain; enlarged
liver with fatty change
Reduced body weight
gain; enlarged liver with
fatty change
Reduced body weight
gain; fatty change in liver
Visibly emaciated;
enlarged liver with fatty
change
Increased liver weight;
fatty change in liver
Slight cytological
alterations in the liver
Increased ALT, SDH;
necrosis in liver
Increased ALT, SDH;
necrosis and cell
proliferation in liver
Increased ALT, SDH;
necrosis and cell
proliferation in liver
Reference
Prendergast
etal., 1967
Prendergast
etal., 1967
Prendergast
etal., 1967
Prendergast
etal., 1967
Nagano etal.,
2007a;
JBRC, 1998
Nagano etal.,
2007a;
JBRC, 1998
Benson and
Springer,
1999
Benson and
Springer,
1999
Benson and
Springer,
1999
Chronic studies
Rat
(50/sex/
group)
6 hours/day, 5
days/week for 104
weeks: 0, 5, 25, or 125
ppm
5 [0.9]a
25 [4]a
Reduced body weight
gain; increased AST,
ALT, LDH, GPT, BUN,
CPK; lesions in the liver
(fatty changes, fibrosis,
cirrhosis) and kidney
(progressive
glomerulonephrosis)
Nagano et al,
2007b;
JBRC, 1998
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Table 4-14. Inhalation toxicity studies for carbon tetrachloride
Species
Mouse
(50/sex/
Group)
Duration/
concentration
6 hours/day, 5
days/week for 104
weeks: 0, 5, 25, or 125
ppm
NOAEL
(ppm)
5 [0.9]a
LOAEL
(ppm)
25 [4]a
Effects at the LOAEL
Reduced survival late in
study (because of liver
tumors); reduced body
weight gain; increased
ALT, AST, LDH, ALP,
protein, total bilirubin,
and BUN; decreased
urinary pH; increased
liver weight; lesions in
the liver (degeneration),
spleen (extra medullary
hematopoiesis), and
kidney (protein casts);
benign
pheochromocytoma
(males)
Reference
Nagano etal.,
2007b;
JBRC, 1998
Gestational exposure study
Rat
(22-23
gravid
F/group)
7 hours/day on GDs
6-15: 0, 334, or 1004
ppm
Not
determined
334 [97]a
Dam: reduced body
weight; increased liver
weight and ALT; altered
gross appearance of liver
Fetus: reduced body
weight and crown-rump
length
Schwetz et
al., 1974
""Duration adjusted concentration is provided in brackets (e.g., 10 ppm x (6 hours per day724 hours per
day x 5 days per week/7 days per week) = 2 ppm).
In the subchronic studies, effects on the kidneys were generally observed at
concentrations above the LOAEL for liver effects and thus are not listed in Table 4-14. With
chronic exposure, the sensitivity of the kidney and liver as target organs appears to be
comparable in the rodent. The JBRC chronic rat study (Nagano et al., 2007b, JBRC, 1998)
reported liver toxicity (serum enzyme changes, fatty liver, fibrosis, cirrhosis) and kidney toxicity
(increases in BUN, creatinine, inorganic phosphorus, and severity of chronic progressive
nephropathy [CPN]) at exposure concentrations of 25 ppm (>4 ppm, duration adjusted) (Table
4-14). An increase in the severity of proteinuria was reported in male and female rats at the
lowest tested concentration of 5 ppm (0.9 ppm, duration adjusted). While the increased severity
of proteinuria could be related to the nephropathy observed at >25 ppm, the biological
significance of the finding of proteinuria at 5 ppm is unclear. Proteinuria (or protein in the urine)
was found in essentially 100% of the rats - both control and carbon tetrachloride-exposed, and
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90% or more of all rats - again control and carbon tetrachloride-exposed - had protein content in
the urine graded as either 3+ or 4+ (see Table 4-2). In the carbon tetrachloride-exposed animals,
however, rats showed an increase in the severity of proteinuria relative to controls (i.e., relatively
more carbon tetrachloride-exposed animals had protein content in urine graded 4+ than 3+).
After two years of exposure to carbon tetrachloride, proteinuria in 5-ppm rats did not appear to
progress, i.e., rats at this concentration did not show treatment-related increases in incidence or
severity of renal changes recognized as clearly adverse (e.g., progressive glomerulonephrosis [or
CPN] or measures of impaired glomerular function, including increased levels of BUN,
creatinine, and inorganic phosphorus) that were observed at higher exposure concentrations.
Complicating interpretation of kidney effects in this study is the fact that the F344 rat is
known for its high incidence of spontaneous, age-related CPN (Hard and Seely, 2005; Chandra
and Frith, 1993/94). Chandra and Firth (1993/94) reported a background incidence of CPN of
88.8% in male and 74.5% in female F344 rats based on an examination of 491 controls from
several 2-year carcinogenicity/chronic toxicity bioassays. CPN can be seen as early as 3 months
and severity of the lesion increases with age. The presence of CPN can confound kidney lesion
diagnosis (Hard and Seely, 2005). Kidney lesions in the JBRC 13-week study of carbon
tetrachloride (Nagano et al., 2007a; JBRC, 1998) were examined with the thought that the
confounding encountered in older (2-year old) rats would be minimized and treatment-related
lesions could be more easily distinguished from spontaneous old-age renal lesions. In the 13-
week study, the severity of proteinuria was statistically significantly increased at a concentration
of >90 ppm in females and >270 ppm in males; histopathological changes in the kidney occurred
in both sexes at >810 ppm. These effect levels are approximately 20- to 50-fold higher than the
5-ppm concentration in the chronic study at which an increase in severity of proteinuria was
observed. It is unexpected that the effect level for kidney effects would decrease by such a large
margin between subchronic and chronic exposure durations. Thus, the findings from the
subchronic study by JBRC (Nagano et al., 2007a; JBRC, 1998) are not clearly consistent with a
LOAEL for renal toxicity following chronic exposure of 5 ppm. Finally, the body of literature
for carbon tetrachloride suggests that the rat liver is a more sensitive target organ that the kidney
following exposures of subchronic duration (e.g., Nagano et al., 2007a; JBRC, 1998; Bruckner et
al., 1986; Adams et al., 1952); there are no adequate chronic studies of carbon tetrachloride
(beyond JBRC, 1998) to confirm whether the kidney may be a more sensitive target organ than
the liver following chronic exposure. The above uncertainties raise questions as to the relevance
of the finding of proteinuria in 5-ppm rats to human health assessment.
In addition to adverse effects on the liver and kidney, the observation of benign
pheochromocytomas in mice exposed to carbon tetrachloride by inhalation in the JBRC chronic
study (Nagano et al., 2007b; JBRC, 1998) may indicate a potential noncancer health risk. As
noted in Section 4.6.1, benign pheochromocytomas are tumors that originate in chromaffm cells
of the adrenal gland medulla and secrete excessive amounts of catecholamines, usually
143 DRAFT - DO NOT CITE OR QUOTE
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epinephrine and norepinephrine. Because pheochromocytomas are not innervated,
catecholamine secretion is unregulated, producing sustained sympathetic nervous system
hyperactivity leading to hypertension, tachycardia, and cardiac arrhythmias (Hansen, 1998).
Health effects related to pheochromocytoma formation in mice were not assessed in the JBRC
chronic inhalation exposure study. Therefore, the potential for secondary effects of
pheochromocytoma on the cardiovascular system can only be inferred. Exposure levels
associated with benign pheochromocytomas in mice (LOAELs of 4 and 22 ppm, duration
adjusted, in male and female mice, respectively) were equal to or greater than levels associated
with hepatic and renal toxicity; thus, the adrenal gland does not appear to be the most sensitive
target organ for carbon tetrachloride following inhalation exposure.
There is no evidence for reproductive or developmental toxicity in humans exposed by
inhalation to carbon tetrachloride. One epidemiological study found no association between
maternal occupational exposure to carbon tetrachloride and infants born small for gestational age
(Seidler et al., 1999). Carbon tetrachloride has been found to produce effects in mouse testis
(Bergman, 1983), testicular atrophy, and reduced fertility in rats exposed intermittently to high
concentrations (>200 ppm) for 6 or more months (Adams et al., 1952; Smyth et al., 1936).
Testicular degeneration has also been reported in rats following repeated i.p. doses of 1.5 mL/kg
(Kalla and Bansal, 1975; Chatterjee, 1966). A definitive reproductive toxicity study has not been
performed, however. In a developmental toxicity study, Schwetz et al. (1974) found significant
reductions in fetal body weight and crown-rump length in rats exposed to carbon tetrachloride
vapor in the air during gestation but at a high concentration (334 ppm, 7 hours/day) that also
produced hepatotoxicity and reduced growth in the dams.
4.6.3. Mode of Action Information
The mode of action of carbon tetrachloride-induced hepatotoxicity has been the subject of
extensive research. Mechanistic studies (described in Section 4.5) provide evidence that
metabolism of carbon tetrachloride via CYP2E1 to highly reactive free radical metabolites plays
a role in its mode of action (Wong et al., 1998; Martinez et al., 1995; Letteron et al., 1990;
Mourelle et al., 1988; Bechtold et al., 1982; Weddle et al., 1976). The primary metabolites,
trichloromethyl and trichloromethyl peroxy free radicals, are highly reactive and are capable of
covalently binding to cellular macromolecules (Boll et al., 2001b; Azri et al., 1991; DiRenzo et
al., 1982; Diaz Gomez and Castro, 1980a; Castro and Diaz Gomez, 1972; Gordis, 1969).
Because the toxicity of carbon tetrachloride is secondary to its metabolism, the liver is
expected to be an important target organ on the basis of its high CYP2E1 content. Subchronic
gavage studies report that the liver is the sole target organ (see Section 4.6.1), probably related to
a first-pass effect. The literature for carbon tetrachloride also suggests that the rat liver is a more
sensitive target organ compared to the kidney following exposures of subchronic duration (e.g.,
Nagano et al., 2007a; JBRC, 1998; Bruckner et al., 1986; Adams et al., 1952). Additionally,
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there are no adequate chronic studies of carbon tetrachloride (other than Nagano et al., 2007b;
JBRC, 1998) to confirm whether the kidney may be a more sensitive target organ than the liver
following chronic exposure (see Section 4.6.2).
The trichloromethyl peroxy and trichloromethyl radical may induce multiple cellular
effects including lipid peroxidation (de Zwart et al., 1997; Gasso et al., 1996; Ichinose et al.,
1994; Tribble et al., 1987; Lee et al., 1982; Recknagel and Glende, 1973; Rao and Recknagel,
1969) decreases in antioxidant levels (Cabre et al., 2000; Gasso et al., 1996 Gorla et al., 1983),
alterations in calcium homeostasis, and activation of calcium dependent phospholipases as
discussed in Section 4.5 (Limaye et al., 2003; Hemmings et al., 2002; Gonzalez Padron et al.,
1993; Agarwal and Mehendale, 1986, 1984; Long and Moore, 1986; Kroner, 1982; Moore et al.,
1976). Additionally, products of lipid peroxidation include reactive aldehydes that can form
protein adducts that may contribute to hepatotoxicity (Beddowes et al., 2003; Abraham et al.,
1999; Hartley et al., 1999; Bedossa et al., 1994; Comporti, 1985; Comporti et al., 1984). At this
time it is uncertain the exact sequence or contribution of cellular mechanisms leading from the
key event of metabolism to carbon tetrachloride-induced hepatotoxicity (cell death). A
description of how the noncancer mode of action coincides with the carcinogenic mode of action
can be found in Figure 4-4.
Although most mechanistic studies for carbon tetrachloride have concentrated on hepatic
effects, some studies provide evidence for a similar mode of action for noncancer effects in the
kidney. The distribution study of Bergman (1983) provided evidence that nonvolatile
metabolites of carbon tetrachloride accumulate in the kidney as well as the liver of mice
immediately following a 10-minute inhalation exposure (see Section 3.2). Like the liver, the
kidney contains both CYP2E1 and CYP3A, which are able to metabolize carbon tetrachloride to
the trichloromethyl radical (Warrington et al., 2004; Koch et al., 2002; Haehner et al., 1996).
Histopathological examination in multiple studies revealed clear evidence of treatment-related
glomerular damage (increased in severity of glomerulonephrosis, BUN, proteinuria, tubular
degeneration, organ weight, and protein casts) in male and female rats exposed to carbon
tetrachloride (Nagano et al., 2007a,b; Benson and Springer, 1999; JBRC, 1998; Prendergast et
al., 1967; Adams et al., 1952; Smyth et al., 1936). Mechanistic similarities also exist between
the liver and kidney regarding increases in lipid peroxidation products (Natarajan et al., 2006;
Dogukan et al., 2003; Abraham et al., 1999; Fraga et al., 1987), reductions in GSH peroxidase
activity, attributable to depleted stores of GSH (Natarajan et al., 2006; Dogukan et al., 2003;
Ozturk et al., 2003) and increased levels of cytosolic phospholipase A2 (Niederberger et al.,
1998). Based on the available data, the kidney and liver effects associated with carbon
tetrachloride appear to operate via a similar mode-of-action pathway.
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4.7. EVALUATION OF CARCINOGENICITY
4.7.1. Summary of Overall Weight-of-Evidence
Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), carbon
tetrachloride can be classified as likely to be carcinogenic to humans by all routes of exposure.
This cancer weight of evidence determination is based on (1) inadequate evidence of
carcinogenicity in humans and (2) sufficient evidence in animals (i.e., concordant expression of
hepatic tumors in several species and by several routes of exposure in response to carbon
tetrachloride and evidence of pheochromocytomas in mice by two routes of exposure).
Carbon tetrachloride has been shown to be a liver carcinogen in rats, mice, and hamsters
in eight bioassays of various experimental design by oral and inhalation exposure. A general
correspondence has been observed between hepatocellular cytotoxicity and regenerative
hyperplasia and the induction of liver tumors. At lower exposure levels, this correspondence is
less consistent. In particular, in the JBRC 2-year inhalation cancer bioassay in the mouse
(Nagano et al., 2007b, JBRC, 1998), the lowest exposure concentration tested (5 ppm or 0.9 ppm
adjusted; see Tables 4-5 and 4-6) was not hepatotoxic, whereas the incidence of liver adenomas
in female mice at this exposure concentration displayed a statistically significant increase
compared to concurrent and historical controls.
A hypothesized carcinogenic mode of action for carbon tetrachloride has been proposed
and includes the following key events: (1) metabolism to the trichloromethyl radical by CYP2E1
and subsequent formation of the trichloromethyl peroxy radical, (2) radical-induced mechanisms
leading to hepatocellular cytotoxicity, and (3) sustained regenerative and proliferative changes in
the liver in response to hepatotoxicity. A substantial amount of data exists that supports these
key events in the cancer mode of action for carbon tetrachloride. A weight of evidence analysis
of the genotoxicity literature suggests that carbon tetrachloride is more likely an indirect than
direct mutagenic agent. Results of extensive testing for genotoxic and mutagenic potential are
largely negative. There is little direct evidence that carbon tetrachloride induces intragenic or
point mutations in mammalian systems. The mutagenicity studies that have been performed
using transgenic mice have yielded negative results, as have the vast majority of the mutagenesis
studies that have been conducted in bacterial systems. Under highly cytotoxic conditions,
bioactivated carbon tetrachloride can exert genotoxic effects. These tend to be modest in
magnitude and are manifested primarily as DNA breakage and related sequelae. Chromosome
loss leading to aneuploidy may also occur to a limited extent. The fact that carbon tetrachloride
overall has not been found to be a potent mutagen and that positive genotoxic results are found
only at high exposure levels and generally in concert with cytotoxic effects (see Tables 4-8 to
4-11) indicates that carbon tetrachloride does not likely induce genotoxic effects through direct
binding or damage to DNA. The majority of genotoxicity studies, however, have been
conducted at relatively high exposure levels, which does not provide information regarding low
doses of carbon tetrachloride (e.g., as potentially elicited through biological mechanisms such as
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the formation of DNA adducts through carbon tetrachloride-induced oxidative stress, lipid
peroxidation, and direct modification of DNA). Because carbon tetrachloride is metabolized to
reactive species (trichloromethyl and trichloromethyl peroxy radical), the potential exists for
these biologically active metabolites of carbon tetrachloride to react with macromolecules at low
exposures (i.e., exposure levels below doses that are cytotoxic). Data to characterize this low-
exposure activity are limited. Therefore, the mode of action of carbon tetrachloride at low
exposure levels can be hypothesized, but is unknown at this time.
4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence
Studies in humans are inadequate to show an association between exposure to carbon
tetrachloride and carcinogenicity. There is some evidence for certain types of cancer in
occupational populations thought to have had some exposure to carbon tetrachloride, including
NHL (Blair et al., 1998; Spirtas et al., 1991), lymphosarcoma and lymphatic leukemia
(Checkoway et al., 1984; Wilcosky et al., 1984), esophageal and cervical cancer (Blair et al.,
1990, 1979), breast cancer (Cantor et al., 1995), astrocytic brain cancer (Heineman et al., 1994),
and rectal cancer (Dumas et al., 2000). In these cases exposure to carbon tetrachloride was
poorly characterized and confounded by simultaneous exposures to other chemicals.
Additionally, these studies were designed to evaluate tetrachloroethylene and trichloroethylene
and had only limited ability to examine other chemical exposures such as carbon tetrachloride.
None of the human epidemiology studies reported associations to cancer of the liver, which is the
main site of carcinogenicity in animal studies, but this may be because of a lack of power to
detect a relatively rare human tumor.
Carbon tetrachloride has been shown to induce hepatocellular carcinomas in rodents by
oral, inhalation, and parenteral exposure. Researchers at the NCI conducted a series of gavage
studies in mice of various strains and found large increases in the incidence of liver tumors in
treated mice (Andervont, 1958; Edwards and Dalton, 1942; Edwards et al., 1942; Edwards,
1941). A similar result was obtained in hamsters (Delia Porta et al., 1961). These animal studies
were generally conducted using a single high dose of carbon tetrachloride, but one early study
was conducted with multiple dose levels in order to investigate dose-response relationships for
induction of liver tumors (Eschenbrenner and Miller, 1946). This study was conducted using
small groups of five mice of each sex per group and two dosing regimens (gavage administration
in olive oil daily or every 4 days for 4 months) that gave the same total exposure. Liver tumors
(hepatomas) were found in all strain A male and female mice that received average daily doses
as low as 20 mg/kg-day. No gross or microscopic tumors were found in mice receiving only 10
mg/kg-day. Interestingly, the incidence of hepatomas was somewhat higher in mice dosed daily,
whereas liver necrosis appeared to be somewhat more prevalent in mice treated intermittently
(every 4 days). Liver necrosis and hepatomas were not clearly concordant as would be expected.
The fact that mice were sacrificed one month after dosing ended complicates interpretation of the
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necrosis findings.
Oral bioassays of carbon tetrachloride using groups of 50 animals/sex were conducted in
mice and rats by NCI (1977, 1976a, b) as a positive control for bioassays of chloroform,
trichloroethylene, and 1,1,1-trichloroethane. The bioassay in mice employed very high doses
(1250 or 2500 mg/kg, 5 day/week for 78 weeks) that produced close to 100% incidence of
hepatocellular carcinoma. The incidence of adrenal adenoma and pheochromocytoma was also
significantly increased in both dose groups in male and female mice. The bioassay in rats (47 or
94 mg/kg for males and 80 or 159 mg/kg for females, 5 days/week for 78 weeks) produced only
a low incidence of liver tumors, but high early mortality, particularly in the high-dose group,
may have affected the power of this study to detect a carcinogenic effect. Even so, the increase
in carcinomas was statistically significant in low-dose females (4/49) in relation to pooled
controls (1/99).
Carbon tetrachloride produced clear evidence of carcinogenicity in inhalation bioassays
in rats and mice (Nagano et al., 2007b; JBRC, 1998). In rats, intermittent exposure (6 hours/day,
5 days/week) to 125 ppm for 2 years produced marked significant increases in the incidence of
hepatocellular carcinomas and adenomas in both males and females. The incidence of tumors
was not increased in rats exposed to 5 or 25 ppm by the same protocol although the incidence of
liver carcinoma (3/50) in 25-ppm females exceeded the range of historical control incidence
from JBRC bioassays. In mice, marked significant increases in hepatocellular carcinomas and
(to a lesser extent) adenomas occurred at both 25 and 125 ppm in both sexes. Also, a statistically
significant increase in the incidence of liver adenomas in female mice at 5 ppm (0.9 ppm
adjusted) was observed compared to the concurrent control and exceeded the historical control
range for hepatocellular adenomas from JBRC 2-year bioassays. The assays in mice also found
significant increases in the incidence of benign adrenal pheochromocytomas in males at 25 or
125 ppm and females at 125 ppm, exposure levels at or above those associated with liver
hepatocellular carcinoma and adenoma. Specifically, pheochromocytomas were identified in
32/50 high-dose male mice, only one of which was classified as malignant (the remaining 31
pheochromocytomas were benign) (JBRC, 1998). Benign pheochromocytomas were identified
in 22/49 high-dose female mice; none were malignant. In addition to the potential cancer risk
suggested by these tumors, benign pheochromocytomas may represent a noncancer health risk
because of the excessive secretion of catecholamines, leading to sustained and unregulated
sympathetic nervous system hyperactivity (see Section 4.6.2).
Some data from parenteral studies are also available. Subcutaneous injections of carbon
tetrachloride at an average dose of 0.29 mg/kg-day for 33-47 weeks induced hepatocellular
carcinomas in Osborne-Mendel, Japanese, and Wistar rats but not in Sprague-Dawley or black
rats (Reuber and Glover, 1970, 1967a, b). Intraperitoneal injections at an average of 86 mg/kg-
day induced hepatomas in C3H mice (Kiplinger and Kensler, 1963).
Overall, carbon tetrachloride has been extensively studied for its genotoxic and
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mutagenic effects with largely negative results. There is little direct evidence that carbon
tetrachloride induces intragenic or point mutations in mammalian systems (Section 4.4.2). The
mutagenicity studies that have been performed using transgenic mice have yielded negative
results, as have the vast majority of the mutagenesis studies that have been conducted in bacterial
systems. However, since oxidative DNA adducts can be converted into mutations, the inability
to detect mutations in the transgenic mouse assays may be an indication of efficient repair of
oxidative lesions, a preferential formation of large chromosomal mutations that are inefficiently
detected in the transgenic models, or a reflection of the limitations and sensitivity of the specific
assays that were performed with carbon tetrachloride (see Table 4-12). The two positive
mutation / DNA damage studies conducted in E. coli were seen in strains that are particularly
sensitive to oxidative damage. Moreover, the intrachromosomal recombination induced by
carbon tetrachloride in S. cerevisiae is believed to result from double stranded DNA breaks
leading to deletion mutations. These results are consistent with DNA breakage originating from
oxidative stress or lipid peroxidation products that occur concurrently with cytotoxicity.
An evaluation based on the weight of evidence suggests that carbon tetrachloride is more
likely an indirect than a direct mutagenic agent. In general, genotoxic effects have been
observed in a consistent and close relationship with cytotoxicity, lipid peroxidation, and/or
oxidative DNA damage. Mutagenic effects, if they occur, are likely to be generated through
indirect mechanisms resulting from oxidative stress or lipid peroxidation products. Under highly
cytotoxic conditions, bioactivated carbon tetrachloride can exert genotoxic effects. These tend to
be modest in magnitude and are manifested primarily as DNA breakage and related sequelae.
Chromosome loss leading to aneuploidy may also occur to a limited extent.
Challenges in evaluating the carbon tetrachloride genotoxicity database must be
acknowledged (e.g., see Table 4-12). Although the cellular effects of carbon tetrachloride are
described adequately at doses at or above those that induce cytotoxicity, there is a paucity of data
describing DNA damaging events at doses below those that are cytotoxic. Additionally, there
exists some level of uncertainty as to whether assays used to assess the genotoxicity of carbon
tetrachloride were of sufficient quality to assess genotoxicity at doses that do not induce
cytotoxicity.
The database for carbon tetrachloride provides evidence that hepatic regeneration is
related to hepatic carcinogenicity. Acute toxicity studies on rodents treated orally with carbon
tetrachloride report hepatic necrosis within 6-24 hours of dosing and evidence of compensatory
hepatocellular proliferation (mitosis, BrdU-positive labeling, or increases in DNA-synthesizing
enzymes and increases in cells in S-phase) at the same time or within 48 hours (Lee et al., 1998;
Wang et al., 1997; Steup et al., 1993; Doolittle et al., 1987; Nakata et al., 1975; Eschenbrenner
and Miller, 1946). Table 4-15 shows the necrotic and regenerative lesions observed in
subchronic and chronic oral and inhalation studies of carbon tetrachloride (only studies explicitly
reporting necrotic or regenerative lesions are included). In these studies, hepatic necrosis or
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degeneration was usually found in conjunction with some type of proliferative lesion, either
regenerative hepatocellular changes (Nagano et al., 2007a,b; Benson and Springer, 1999; JBRC,
1998; Prendergast et al., 1967) or proliferation or hyperplasia of the bile duct (Nagano et al.,
2007a; JBRC, 1998; Koporec et al., 1995; Bruckner et al., 1986; Hayes et al., 1986; NCI, 1977,
1976a, b; Prendergast et al., 1967). The only detailed study of both chronic toxicity and
carcinogenicity of carbon tetrachloride was the JBRC inhalation study in rats and mice (Nagano
et al., 2007b; JBRC, 1998). The occurrence of liver adenomas in female mice exposed to carbon
tetrachloride at 5 ppm (0.9 ppm adjusted) makes the relationship of the hypothesized key events
cytotoxicity and regenerative proliferation with tumor formation less clear since there are no
available data that supports these key events at this dose level (see Table 4-15). Eschenbrenner
and Miller (1946) reported development of tumors in mice at doses that did not evidently
produce necrosis, but the design of this study may have influenced this result, as animals were
sacrificed and examined one month after the end of the main treatment period (animals were,
however, given one last dose 24 hours prior to sacrifice). Currently, there are no data to
characterize the liver changes that may have occurred and what effect this would have on
eliciting or abating cellular cytotoxicity 24 hours prior to terminal sacrifice. The investigators
noted that all doses that induced hepatomas were likely to have caused initial necrosis based on
separate studies using one or two doses. Regenerative changes were not investigated in this part
of the study.
Table 4-15. Exposure levels for necrosis/degeneration and hyperplasia/
regeneration in liver following subchronic or chronic exposure to carbon
tetrachloride by gavage or inhalation
Strain, species
Sprague-Dawley
rat (male)
F344 rat (male)
Sprague-Dawley
rat (male)
CD-I mouse
CD-I mouse
Strain A mouse
B6C3F1 mouse
Exposure
Oral, 12 weeks
24 mg/kg-day (adjusted)
Oral, 12 weeks
14 mg/kg-day (adjusted)
Oral, 13 weeks
71 mg/kg-day (adjusted)
Oral, 13 weeks
12 mg/kg-day
Oral, 12 weeks
8.6 mg/kg-day (adjusted)
Oral, 120 days (30 doses)
80 mg/kg-dayb
Oral, 78 weeks,
892 mg/kg-dayb (adjusted)
Hepatic necrosis/
degeneration
Necrosis
6/6 Necrosis
Necrosis
Necrosis
Necrosis
Necrosis
Hyperplasia/
regeneration
Bile duct hyperplasia
Nodular hepatic, bile
duct, and oval cell
hyperplasia
Bile duct hyperplasia
Bile duct proliferation
Reference
Bruckner et al.,
1986
Allisetal., 1990
Koporec et al.,
1995
Hayes et al.,
1986
Condie et al.,
1986
Eschenbrenner
and Miller, 1946
NCI, 1977,
1976a, b
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Table 4-15. Exposure levels for necrosis/degeneration and hyperplasia/
regeneration in liver following subchronic or chronic exposure to carbon
tetrachloride by gavage or inhalation
Strain, species
F344 rat (male)
B6C3F1 mouse
(male)
Syrian hamster
(male)
Wistar rat
Hartley guinea
Pig
Hartley guinea
pig; Sprague-
Dawley or Long-
Evans rat
F344 rat
BDF1 mouse
F344 rat
BDF1 mouse
Exposure
Inhalation, 12 weeks
1 8 ppm (adjusted)3
Inhalation, 12 weeks
4 ppm (adjusted)3
Inhalation, 12 weeks
18 ppm (adjusted)3
Inhalation, 6 months
42 ppm
Inhalation, 13 weeks
10 ppm (continuous)
Inhalation, 6 weeks
20 ppm (adjusted)3
Inhalation, 13 weeks
2 ppm (adjusted)3
Inhalation, 13 weeks
5-48 ppm (adjusted)3
Inhalation, 104 weeks
5-22 ppm (adjusted)3
Inhalation, 104 weeks
5 ppmb (adjusted)3
Hepatic necrosis/
degeneration
Necrosis
Necrosis
Necrosis
Necrosis
Hepatocellular
degeneration
Necrosis,
hepatocellular
degeneration
Degeneration in
males; necrosis in
females
Hyperplasia/
regeneration
BrdU-negative
hepatocytes
BrdU-positive
hepatocytes
BrdU-positive
hepatocytes
Hepatocellular
regeneration
Hepatocellular
regeneration, bile duct
proliferation
Mitosis, bile duct
proliferation, foci
Bile duct proliferation: 5
ppm, F; 16 ppm, M;
mitosis: 16 ppm, M; 48
ppm, F; foci: 48 ppm
both sexes
Foci: 5 ppm, F;
22 ppm, M3
Reference
Benson and
Springer, 1999
Benson and
Springer, 1999
Benson and
Springer, 1999
Adams et al.,
1952
Prendergast et
al., 1967
Prendergast et
al., 1967
Nagano etal.,
2007a; JBRC,
1998
Nagano etal.,
2007a; JBRC,
1998
Nagano etal.,
2007b; JBRC,
1998
Nagano etal.,
2007b; JBRC,
1998
3 This concentration was adjusted to continuous exposure, e.g., a factor of 6/24 x 5/7 applied used for an
inhalation exposure administered 6 hours/day, 5 days/week.
b Hepatic tumors detected at this level.
In summary, repeated oral or inhalation exposure to carbon tetrachloride causes
degeneration or necrosis of the liver, and there is evidence for hepatocellular regeneration in
repeated inhalation exposure studies.
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4.7.3. Mode of Action Information for Liver Tumors
4.7.3.1. Hypothesized Mode of Action and Identification of Key Events
The hypothesized mode of action for carbon tetrachloride-induced liver tumors is
described graphically in Figure 4-4. Key events in the carcinogenicity of carbon tetrachloride
include: (1) metabolism to the trichloromethyl radical by CYP2E1 and subsequent formation of
the trichloromethyl peroxy radical, (2) radical-induced mechanisms leading to hepatocellular
toxicity, and (3) sustained regenerative and proliferative changes in the liver in response to
hepatotoxicity. In rodent models (Nagano et al., 2007b; JBRC, 1998; NCI, 1977, 1976a, b; Delia
Porta et al., 1961; Andervont, 1958; Edwards and Dalton, 1942; Edwards et al., 1942; Edwards,
1941), tumorigenicity occurs consistently in the liver. Metabolism of carbon tetrachloride is
identified as a key event based on the following: (1) reactive metabolites are present in the liver
(Stoyanovsky et al., 1999; Conner et al., 1986), (2) CYP450 inhibitors prevent carbon
tetrachloride-induced liver damage (Martinez et al., 1995; Letteron et al., 1990; Mourelle et al.,
1988; Bechtold et al., 1982; Weddle et al., 1976), (3) treatment of knockout mice specific for
CYP2E1 (cyp2el"") with carbon tetrachloride does not result in hepatocellular cytotoxicity as
compared to wild type (cyp2el +/+) mice, and (4) treatment with compounds that induce
CYP450's result in potentiating effects to carbon tetrachloride-induced toxicity (Section 4.8.6).
The resulting hepatocellular cytotoxicity has been demonstrated in numerous studies (Table 4-
15) as measured by increases in liver enzymes (i.e., ALT, AST, SDH, and LDH) in plasma or by
histopathological examination. As a result of cytotoxicity in the liver of carbon tetrachloride-
treated animals, significant regenerative cellular proliferation occurs to compensate for the
necrotic or damaged tissue. As discussed in Section 4.7.2, there is a general correlation
(particularly at higher doses) between occurrence of hepatotoxicity and/or
regenerative/proliferative lesions and development of tumors. Findings from the study by JBRC
(Nagano et al., 2007b; JBRC, 1998), the only detailed study of both chronic toxicity and
carcinogenicity of carbon tetrachloride available, are generally consistent with the hypothesis
that liver tumors occur at exposure levels that produced hepatotoxicity in both rats and mice.
Liver adenomas did occur, however, in female mice at exposures below which hepatotoxicity
was observed. Tumorigenesis through this hypothesized mode of action resulting from carbon
tetrachloride-induced toxicity is believed to require persistent hepatocellular cytotoxicity and
regenerative cellular proliferation for tumor formation. Other biological mechanisms (e.g.,
mutagenicity) may contribute to the tumorigenic response and may represent additional key
events or other operable modes of action; however, the contribution of these key events or modes
of action has not been fully established.
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Key Events
Chronic exposure
to CCL
T
Metabolism of
CCI4byCYP2E1
i
Hepatocellular
regenerative
proliferation
Liver tumors
Hepatocellular
cytotoxicity
4
"»
Mechanistic Events /
Areas of Uncertainty
Lipid
peroxidation
Disruption of
Ca" homeostasis
Genetic damage
or alterations
A.
B.
Demonstrated at high
doses compared to chronic
bioassays (g/kg vs mg/kg)
Level of contribution to
cytotoxicity unknown?
A.
B.
Demonstrated at lower
levels in vitro (0.5 mtvl)
Unknown cause or effect of
cytotoxicity?
Background mutational
events
Reactive aldehyde-induced
mutations (HNE, MDA)
Cytotoxicity-induced
damage
Oxidative stress (8-OH-dG)
Figure 4-4. Hypothesized carcinogenic mode of action
As mentioned above there exist several important mechanistic events, including lipid
peroxidation, disturbances in calcium homeostasis, and genetic damage, that are possibly
involved in this biological process; however, the available data at doses below those that induce
cytotoxicity are not adequate to evaluate their relative significance. Therefore, whether these
mechanistic events represent key events remain areas of uncertainty in carbon tetrachloride's
carcinogenic mode of action.
In general, mechanistic studies that have evaluated the induction of lipid peroxidation
have been conducted at doses that induce significant levels of cytotoxicity (see Table 4-15).
Representative studies evaluating the occurrence of lipid peroxidation are provided in
Table 4-16, Section 4.7.3.2.2. These studies are not definitive regarding the cellular responses
that occur below those that have been found to induce tumors in chronic bioassays.
Additionally, it is not clear at what dose lipid peroxidation or generation of reactive aldehydes
would begin to contribute to the toxicity of carbon tetrachloride. Although carbon tetrachloride
is not considered likely to be directly genotoxic, it is possible that lipid peroxidation products
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generate compounds (reactive aldehydes) that may covalently bind to DNA. The low molecular
weight aldehydes generated by lipid peroxidation have sufficiently long biological half-lives to
diffuse from their site of formation to other parts of the cell (Slater, 1982, 1981). Nuclear DNA
adducts to these aldehydes in hepatocytes have been demonstrated in a number of studies
(Beddowes et al., 2003; Wacker et al., 2001; Chung et al., 2000; Wang and Liehr, 1995;
Chaudhary et al., 1994). One of these compounds, malonaldehyde, has been shown to be
tumorigenic in Swiss mice when applied repeatedly to the skin (Shamberger et al., 1974). In
cultured rat hepatocytes, however, the lowest concentration producing a statistically significant
increase in DNA breaks and DNA adducts generated by lipid peroxidation approached the
concentration that induced cytotoxicity (LDH leakage) (Beddowes et al., 2003). The possibility
exists that reactive aldehydes generated at low levels of carbon tetrachloride could result in
increased levels of endogenous MDA and 4-HNE DNA adducts that may contribute to the
genotoxicity of carbon tetrachloride. Additionally, based on current data sets that characterize
the generation of lipid peroxidation induced by carbon tetrachloride (Table 4-16, Section
4.7.3.2.2), the doses at which this effect have been demonstrated do not allow a clear
determination as to whether lipid peroxidation induces cytotoxicity or whether cytotoxicity
induces lipid peroxidation.
Disruption of calcium homeostasis as a process by which carbon tetrachloride may
induce toxicity is an area of extensive research (Hemmings et al., 2002; Long and Moore, 1986;
Kroner, 1982; Moore et al., 1976). Similar to research conducted on carbon tetrachloride-
induced lipid peroxidation, it is not established if disruption of calcium homeostasis is a cause or
an effect of cellular cytotoxicity. Some studies present evidence that disturbances in calcium
homeostasis may not be a necessary event for cell death to result from carbon tetrachloride
(Albano et al., 1989; Clawson, 1989). Likewise, evaluation of the carbon tetrachloride dose
required to induce disturbances in calcium homeostasis does not confirm this as a key event
(Hemmings et al., 2002; Long and Moore, 1986; Kroner, 1982; Moore et al., 1976).
Genetic damage or alteration to the DNA represents an additional area of uncertainty for
the mode(s) of action for carbon tetrachloride that is at the present time not adequately
characterized. Several cellular processes have been proposed that may account for how genetic
damage may occur, ultimately leading to genotoxic events. The trichloromethyl and
trichloromethyl peroxy free radicals are capable of covalently binding to nucleic acids.
However, the reactivity of these radicals is such that they are not expected to diffuse very far
from their site of formation (Slater, 1982, 1981). As a result, the amount reaching the cell
nucleus from microsomes would be negligible. Studies have indicated small increases in
covalent binding of trichloromethyl radical to nuclear DNA, as well as nuclear proteins and
lipids, apparently as a result of bioactivation of carbon tetrachloride by CYP450 in the nuclear
membrane (Fanelli and Castro, 1995; Castro et al., 1989; Levy and Brabec, 1984; Diaz Gomez
and Castro, 1980a, b; Rocchi et al., 1973). However, there are significant methodological
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problems with each of these studies that create difficulties in interpreting the results (see Section
4.4.2.4). Additionally, the fact that carbon tetrachloride overall has not been found to be a potent
mutagen and that the few positive genotoxic results are found only at high exposure levels and
generally in concert with cytotoxic effects (Tables 4-8 to 4-11) indicates that carbon tetrachloride
does not likely induce genotoxic effects through direct binding or damage to DNA. As for lipid
peroxidation-induced DNA damage, development of mutations by this mechanism could occur
and would likely result from the production of radicals exceeding the cell's capacity to quench
and/or repair these alterations.
Genetic damage could also result from background or spontaneous mutations. In vivo
studies have estimated that background mutation frequencies may increase many fold over the
lifetime of an organism (Morley and Turner, 1999). It is generally accepted that sustained cell
proliferation in response to cell death from toxicity or other causes is a significant risk factor for
cancer (Holsapple et al., 2005). Hepatic regeneration following injury from carbon tetrachloride
has the potential to result in carcinogenesis as a result of replication errors becoming fixed
mutations before DNA repair can be completed.
Many studies have characterized the formation of endogenously produced DNA adducts
(Beddowes et al., 2003; Wacker et al., 2001; Chung et al., 2000; Wang and Liehr, 1995;
Chaudhary et al., 1994), DNA strand breaks (Kadiiska et al., 2005; Yasuda et al., 2000; Gans and
Korson, 1984), chromosomal aberrations (Sawada et al., 1991), and micronucleus formation
(Uryvaeva and Delone, 1995; Van Goethem et al., 1995). However, to date no measure of
genetic damage to DNA has been well characterized at or below doses at which tumors are
observed (Nagano et al., 2007b; JBRC, 1998; NCI, 1977, 1976a, b; Eschenbrenner and Miller,
1946). Adequate dose-response studies for assays that measure genetic damaging events at or
below dose levels for which carbon tetrachloride induces tumors in chronic bioassays would help
clarify whether or not carbon tetrachloride is carcinogenic at dose levels that do not cause
cytotoxicity and cell regeneration. In the absence of adequate characterization of genetic
damaging events that may lead to or contribute to background mutational levels, EPA is
choosing to characterize the full range of carcinogenic potential for human exposure to carbon
tetrachloride (see Section 5.3).
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4.7.3.2. Experimental Support for the Hypothesized Mode of Action
4.7.3.2.1. Strength, consistency, specificity of association. Carcinogenicity studies of carbon
tetrachloride have consistently reported carcinogenicity in the liver, independent of species,
gender, or route of administration. Hepatic toxicity (cytotoxicity), necrosis, and regenerative
proliferation have generally been reported in animals exposed to carbon tetrachloride orally or by
inhalation and are correlated with the CYP450 content. However, it remains to be established if
genotoxic events could occur at dose levels below those that induce tumors or are induced by
cytotoxicity and clonal expansion due to regenerative proliferative events and could represent a
primary mode of action for carbon tetrachloride. In the 2-year inhalation studies in rats and
mice, which are the best documented of chronic studies, livers with adenomas or carcinomas also
expressed nonneoplastic lesions (Nagano et al., 2007b; JBRC, 1998) with the exception of liver
adenomas at the lowest dose (5-ppm or 0.9-ppm adjusted) in female mice. Additionally,
multiple reports confirm the occurrence of adenomas and carcinomas in carbon tetrachloride-
treated animals (NCI 1977, 1976a, b; Delia Porta et al., 1961; Andervont, 1958; Eschenbrenner
and Miller, 1946; Edwards and Dalton, 1942; Edwards et al., 1942; Edwards, 1941) (see also
Table 4-15).
4.7.3.2.2. Dose-response concordance. Carbon tetrachloride-induced liver tumors were seen in
rats, mice, and hamsters after oral bolus dosing in oil and in rats and mice exposed by inhalation.
Several oral studies were conducted using only a single-dose level (i.e., studies in the mouse by
Edwards, 1941; Edwards and Dalton, 1942; Edwards et al., 1942; and a study in the hamster by
Delia Porta et al., 1961) and, therefore, did not provide information on the relationship between
dose and tumor induction. The NCI (1977, 1976a, b) bioassay included two dose levels, but high
early mortality in the rat study, particularly at the high dose, limited interpretation of the results.
In the mouse study, liver carcinomas were produced at .100% incidence in male and female mice
of both dose groups (179/182 mice). Eschenbrenner and Miller (1946) observed liver tumors in
all mice treated daily with 20 mg/kg-day or more (n = 29), but none in the 10 mice treated with
10 mg/kg-day. In the inhalation studies (Nagano et al., 2007b; JBRC, 1998), liver tumors were
markedly increased at 125 ppm in male and female rats and 25 and 125 ppm in male and female
mice. In both species at the next lower exposure concentration (i.e., 25 ppm in rats or 5 ppm in
mice) the liver tumor incidence was elevated only in female animals. In the 25-ppm female rat,
the incidence of hepatocellular carcinomas did not differ statistically from the concurrent
controls, but exceeded the historical control range from the research center that conducted the
bioassay. In the 5-ppm female mouse, the incidence of hepatocellular adenomas was statistically
elevated over the concurrent control and historical controls for that research center. In male
mice, the incidence of carcinomas was very high in the 25 ppm group (88%), and there was only
a modest increase as the concentration was increased to 125 ppm (94%). In the female mice, the
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incidence of carcinomas was lower in the 25-ppm group (66%), and there was a larger increase
as the concentration was increased to 125-ppm (98%).
The dose-response relationship between hepatic cytotoxicity and tumor formation is best
demonstrated by the JBRC cancer bioassay in rats and mice, which examined histopathological
changes to the liver after 13 and 104 weeks and tumor formation after 104 weeks of exposure to
carbon tetrachloride by inhalation (Nagano et al., 2007a,b; JBRC, 1998). Carbon tetrachloride
concentrations evaluated were 0, 10, 30, 90, 270, and 810 ppm in the 13-week study and 0, 5, 25,
and 125 ppm in the 104-week study. In rats exposed for 13 weeks, histopathological changes
indicative of cellular damage ("fatty change") and inflammation (granulation) were observed in
all carbon tetrachloride treatment groups. At concentrations >30 ppm, proliferative
(pleomorphism and increased mitosis) and regenerative (fibrosis, proliferative ducts, cirrhosis)
responses occurred. At concentrations >270 ppm, eosinophilic and basophilic foci, which are
associated with hyperplastic or preneoplastic changes, were observed. Similar nonneoplastic
hepatic lesions (fatty changes, granulation, cirrhosis) were observed in livers of rats exposed to
>25 ppm for 104 weeks; the incidence of nonneoplastic lesions in rats exposed to 5 ppm for 104
weeks appeared similar to that in controls. The incidence of liver tumors in rats was
significantly increased only in the 125-ppm group compared with that in concurrent controls,
although an increase in hepatocellular carcinomas in 25-ppm female rats exceeded the historical
control range. Thus, liver tumors were observed at an exposure level associated with
hepatotoxicity following subchronic and chronic exposure, but tumors were not observed at an
exposure level below the level that induced cytotoxicity (<10 ppm for 13-week exposure and 5
ppm for 104-week exposure). A similar dose-response relationship for cytotoxicity and tumor
formation was observed for mice (Nagano et al., 2007b; JBRC, 1998), although the relationship
was less consistent at the lowest exposure concentration. In mice exposed for 13 weeks, dose-
dependent histopathological findings indicative of cytotoxicity, damage, proliferation, and
preneoplastic changes (e.g., fatty liver, pleomorphism, increased mitosis, proliferative ducts, and
eosinophilic and basophilic foci) were observed; "cytological alterations" occurred at the lowest
concentration tested (10 ppm). Similar histopathological findings were observed in mice
exposed to >25 ppm for 104 weeks. The incidence of liver adenomas and carcinomas in mice
was increased compared to concurrent controls at >25 ppm; thus, an increase in hepatic tumors in
mice was observed at an exposure level that produced cytotoxicity. Additionally, at 5 ppm (0.9-
ppm adjusted), where hepatocellular damage was not observed in the 104-week study, the
incidence of hepatocellular adenomas was statistically elevated in female mice compared to
concurrent controls.
Thus, results of this dose-response analysis suggest that liver tumors generally occur at
the same or higher exposure levels as cytotoxicity. Although cytotoxicity was not observed at
the 5-ppm (0.9-ppm adjusted) exposure level, evidence for cytotoxicity does exist at the 25-ppm
level (5-ppm adjusted) (Nagano et al., 2007b) and regenerative proliferation at the 30-ppm level
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(7-ppm adjusted) (Nagano et al., 2007a). The dose-response relationships between cytotoxicity
and liver tumors demonstrated by the JBRC bioassay in rats and mice generally support
cytotoxicity, regeneration, and proliferation as the predominant mode of action for carbon
tetrachloride-induced carcinogenesis at higher exposure levels but may not adequately
characterize critical events that would support other plausible modes of action, particularly at
lower exposure levels.
As summarized in Table 4-15, several subchronic inhalation and oral studies have
demonstrated that carbon tetrachloride produces hepatic toxicity and regeneration. In rodents
exposed to carbon tetrachloride vapor for 12 weeks to 6 months, LOAELs for tissue damage
were reported at concentrations ranging from 4 to 42 ppm (adjusted) and for hyperplasia/
regeneration at concentrations ranging from 4 to 20 ppm (adjusted). Thus, results of subchronic
exposure studies are consistent with results of the JBRC study in rats, showing cytotoxicity at
>10 ppm (>2 ppm adjusted) and hyperplasia/proliferation at >30 ppm (>5.4 ppm adjusted) after
13 weeks of exposure (Nagano et al., 2007a; JBRC, 1998) and cytotoxicity and
hyperplasia/regeneration at>25 ppm (>4.5 ppm adjusted) after 104 weeks of exposure (Nagano
et al., 2007b; JBRC, 1998). In rats and mice exposed orally to carbon tetrachloride for 12-17
weeks, LOAELs for tissue necrosis ranged from 8.6 to 80 mg/kg-day and for
hyperplasia/regeneration ranged from 12 to 71 mg/kg-day. Durations of the subchronic studies
were too short to evaluate tumor formation; thus, data from subchronic studies do not allow for
further definition of the dose-response relationship and time course for cytotoxicity and tumor
formation.
Significant research has been conducted on the mechanistic events that precede carbon
tetrachloride-induced hepatocellular cytotoxicity (see Section 4.5). Much of this research has
focused on lipid peroxidation (de Zwart et al., 1997; Gasso et al., 1996; Ichinose et al., 1994;
Tribble et al., 1987; Lee et al., 1982; Recknagel and Glende, 1973; Rao and Recknagel, 1969),
decreases in antioxidant levels (Cabre et al., 2000; Gasso et al., 1996 Gorla et al., 1983),
alterations in calcium homeostasis, and activation of calcium-dependent phospholipases (Limaye
et al., 2003; Hemmings et al., 2002; Gonzalez Padron et al., 1993; Agarwal and Mehendale,
1986, 1984; Long and Moore, 1986; Kroner, 1982; Moore et al., 1976). Compared to doses that
result in tumor formation in chronic bioassays (5-125 ppm: Nagano et al., 2007b; JBRC, 1998;
20 mg/kg-day: Eschenbrenner and Miller, 1946), these mechanistic studies were conducted at
relatively high exposure levels (see Table 4-16). In most, if not all, mechanistic studies,
exposure levels greatly exceeded those used in chronic bioassays (e.g., on the order of grams per
kilogram (in vivo) or millimolar concentrations (> 1 mM) of carbon tetrachloride). The
relevance of the mechanistic findings at these high exposure levels to lexicologically relevant
exposures is uncertain (Weber et al., 2003; Clawson, 1989; Recknagel and Glende, 1989; Dolak
et al., 1988). The degree to which lipid peroxidation, depletion of cellular antioxidants,
alterations in calcium homeostasis, and activation of calcium-dependent phospholipases
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contribute to the process of cytotoxicity, regenerative proliferation, and tumorigenesis, and the
possible reversibility of these effects, constitutes an area of uncertainty (Weber et al., 2003;
Rikans et al., 1994; Kefalas and Stacey, 1989; Dolak et al., 1988; Sandy et al., 1988; Stacey and
Klaassen, 1981).
Table 4-16. Dose considerations of mechanistic studies of carbon
tetrachloride
End point
Lipid peroxidation
Lipid peroxidation
Lipid peroxidation
Lipid peroxidation
Lipid peroxidation
Lipid peroxidation
Lipid peroxidation
Lipid peroxidation
Lipid peroxidation
Lipid peroxidation
Lipid peroxidation
Dose of Carbon
Tetrachloride
1 ml/kg
(1590 mg/kg)a
0.5 ml/kg
(800 mg/kg)a
0.5 ml (2.38 ml/kg)
injected i.p.
(800 mg/kg)a
ImM
(154 mg/L)
ImM
(154 mg/L)
500 mg/kg
3200 mg/kg
0.1 ml/kg
(160 mg/kg)a
1590 mg/kg
2.5 ml/kg p.o. or 1
ml/kg (5 ml/kg as a
20% solution.)
injected i.p.
(3980 or 1590
mg/kg)a
1590 mg/kg
Test System
Sprague-Dawley
rats; three strains
of mice ( A/J,
BALB/cJ, and
C57B1/6J
Rats and mice
Male Wistar rats
In vitro, liver
microsomes
(multiple species)
Liver slices from
Sprague-Dawley
Rats
Female F344 rats
Female F344 rats
Hamsters
Rat
Male Sprague-
Dawley rats
Rat
Result
Increased conjugated
dienes in carbon
tetrachloride (CC14)
treated animals
compared to controls
Ethane production
increased in CC14-
treated animals; iron
binding eliminated
lipid peroxidation
(ethane) in CC14-
treated animals
TEARS significantly
lower in animals
receiving SAM
Increased MDA
DNA adducts
Significant increase
in TEARS
2-fold induction of
HNE-dG adducts
37-fold induction of
HNE-dG adducts
MDA DNA adducts
Significant increase
in 4-HNE and MDA
adducts in liver
Conjugated dienes or
incorporation of C14
labeled CC14 was not
significantly
prevented by several
antioxidants
2. 5 -fold increase
TEARS over
Reference
Leeetal., 1982
Younes and
Siegers, 1985
Gassoetal., 1996
Ichinoseetal., 1994
Fragaetal., 1987
Wackeretal. 2001
Chung et al. 2000
Wang and Liehr,
1995
Hartley et al., 1999
de Ferreyra et al.,
1975
Hartley et al., 1999
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Table 4-16. Dose considerations of mechanistic studies of carbon
tetrachloride
End point
Lipid peroxidation
Protein carbonyl
(protein adducts)
GSH modulation
GSH modulation
GSH modulation
GSH modulation
Altered Ca++
homeostasis
Altered Ca++
homeostasis
Altered Ca++
homeostasis
Dose of Carbon
Tetrachloride
1 and 4 mM
(154 and 615 mg/L)
1 and 4 mM
(154 and 615 mg/L)
0.5 ml (2.38 ml/kg)
injected i.p.
(800 mg/kg)a
Pretreated with 2
g/kg GSH 30
minutes prior to
1590 mg/kgi.p.
carbon tetrachloride
1600 mg/kg, twice
weekly for 6 weeks,
i.p.
0.1 ml/kg
(160 mg/kg)a
0.3-10mM
(46-1540 mg/L)
Iml/kg injected i.p.
(1590 mg/kg)a
2.5 ml/kg oral dose
by feeding tube
(3980 mg/kg)a
Test System
In vitro rat
hepatocytes
In vitro rat
hepatocytes
Wistar rats
Male Sprague-
Dawley rats
Rat
Female Balb/c
mice
In vitro
hepatocytes
Female Wistar rats
Male Sprague-
Dawley rats
Result
controls
Significant increase
in MDA adducts
2. 5 -fold increase at 4
mM
GSH decreased at 5
weeks
GSH pretreatment
partially prevented
hepatic necrosis
Significant decrease
in GSH; SAM
partially prevented
liver toxicity
SchisandrinB-
partially prevented
hepatotoxicity and
GSH depletion
Increased activity of
phosphorylase a and
decreased activity of
endoplasmic
reticulum Ca++
pump; effects only
observed at
concentrations >
ImM
Significant decrease
in microsomal Ca++
concentration;
significant increase
in mitochondrial
Ca++ concentration
85% reduction in
ATP-dependent
Ca++ uptake and
endoplasmic
reticulum capacity
Reference
Beddowes et al.,
2003
Beddowes et al.,
2003
Cabreetal, 2000
Gorlaetal, 1983
Gassoetal., 1996
Chiu et al., 2003
Long and Moore,
1986
Kroner, 1982
Moore etal, 1976
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Table 4-16. Dose considerations of mechanistic studies of carbon
tetrachloride
End point
Altered Ca++
homeostasis
Altered Ca++
homeostasis
Phospholipase
activity
Phospholipase
activity
Phospholipase
activity
Phospholipase
activity
Dose of Carbon
Tetrachloride
0.03 ml/100 g to
0.125 ml 7100 g
body weight (1.25
ml /kg by feeding
tube)
(0.48 - 1990
mg/kg)a
50 uM
(7.7 mg/L)
3 ml/kg i.p.
(4770 mg/kg)a
1 ml/kg injected i.p.
(1590 mg/kg)a
0.23 - 1.3 mM
(35-200 mg/L)
1.2 mM
(185 mg/L)
Test System
Male F344 rats
In vitro
hepatocytes
Male Sprague-
Dawley rats
Male Sprague-
Dawley rats
In vitro
hepatocytes
In vitro
hepatocytes
Result
Decreased Ca++
transport across
plasma membrane
and mitochondria
Elevated cytosolic
Ca++ levels
Co-treated with CBZ
(calpain inhibitor),
decreased mortality
50% from lethal dose
ofCC14
Pretreated with
quinacrine
(phospholipase A2
inhibitor)
Increased
phospholipase A2
activity 1.4- to 5.3-
fold
Increased
phospholipase A2
activity and
hepatocyte
degeneration (LDH
release)
Reference
Hemmings et al.,
2002
Stoyanovsky and
Cederbaum, 1996
Limaye etal., 2003
Gonzalez Padron et
al., 1993
Glende and
Pushpendaran, 1986
Glende and
Recknagel, 1992
a Dose in mg/kg estimated using a density for carbon tetrachloride of 1.5940 g/ml at 20 °C.
An additional area of significant uncertainty for dose-response concordance is the
possibility of genetically damaging events occurring at or below doses that induce tumors in
laboratory rodents. Because genotoxicity and mechanistic data in this portion of the dose-
response curve are limited, the possible contribution of genetic damage to the formation of liver
tumors due to carbon tetrachloride exposure cannot be established.
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4.7.3.2.3. Temporal relationship. Carbon tetrachloride is metabolized to trichloromethyl and
peroxy free radicals, which may result in radical-induced mechanisms including lipid
peroxidation and disruption of calcium homeostasis leading to hepatocellular cytotoxicity. Initial
metabolism of carbon tetrachloride to reactive radicals and subsequent events leading to
cytotoxicity are ongoing processes that occur throughout exposure.
The temporal progression of nonneoplastic liver lesions following acute and subchronic
exposure is consistent with the hypothesized cytotoxic-proliferative mode of action. Initial
histopathological changes in the liver following acute exposure to carbon tetrachloride include
fatty degeneration, inflammatory cell infiltration, and necrosis (Lee et al., 1998; Wang et al.,
1997; Steup et al., 1993). Hepatocyte regeneration has been observed within 24 hours of
exposure (Lee et al., 1998). As reviewed in Sections 4.2.1.1 and 4.2.2, numerous subchronic
exposure studies report histopathological findings consistent with an ongoing cycle of hepatic
damage, repair, and proliferation (e.g., fatty vacuolization and degeneration, necrosis, nuclear
pleomorphism, hyperplasia, fibrosis, and cirrhosis) (Nagano et al., 2007a; JBRC, 1998; Allis et
al., 1990; Bruckner et al., 1986; Condie et al., 1986; Litchfield and Gartland, 1974). Smyth et al.
(1936), Adams et al. (1952), and Benson and Springer (1999) clearly show a progression of liver
toxicity from fatty degeneration of the liver to liver cirrhosis and hepatocellular proliferation
only at doses that produce necrotic damage.
A temporal and dose-related progression of key events (hepatotoxicity, repair,
proliferation, and tumor development) is supported by the results of the JBRC inhalation cancer
bioassay in rats (Nagano et al., 2007b; JBRC, 1998), in which the development of hyperplastic or
preneoplastic lesions (eosinophilic and basophilic foci) following subchronic exposure to
cytotoxic levels, with subsequent development of liver tumors, is demonstrated (see Table 4-17).
Thus, in the rat, the temporal relationship of the key events is consistent with the mode of action
for carbon tetrachloride carcinogenesis. This relationship, however, is not as clearly defined for
the increased incidence of liver adenomas in female mice (Nagano et al., 2007a,b).
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Table 4-17. Temporal sequence and dose-response relationship for key events and liver
tumors in male and female F344 rats exposed to carbon tetrachloride vapor for 13 and 104
weeks (6 hours/day, 5 days/week)
Key event (time •>)
Exposure
level3
(ppm)
5 (0.9)
10(1.8)
25 (4.5)
30 (5.4)
90(16.1)
125 (22.3)
270 (48.2)
810(145)
Metabolism &
formation of
•0-0-CCb
(immediate and
ongoing)
+ b
+ b
+ b
+ b
+ b
+ b
+ b
+ b
13 weeks
Hepato-
toxicity c
+/— e
+
+
+
+
Regeneration
and
proliferation d
—
+
+
+
+
104 weeks
Hepato-
toxicity0
—
+
+
Regeneration
and
proliferation d
—
—
+
Liver tumors
(104 weeks)
—
+/-f
+
a The exposure concentration in parentheses is the concentration adjusted to continuous exposure (i.e.,
multiplied by 5/7 x 6/24)
+ b = Studies demonstrating key event were not conducted as part of the JBRC 13- and 104-week bioassays.
Based on data from acute exposure and in vitro studies (Avasarala et al., 2006; Zangar et al., 2000; Raucy
et al., 1993), metabolism of carbon tetrachloride to reactive metabolites has been demonstrated and is
assumed to occur immediately and continue throughout the duration of exposure to carbon tetrachloride at
all exposure levels. Although metabolism to reactive metabolites has been specifically demonstrated at
relatively high doses, it can reasonably be assumed that such metabolism would occur at lower exposures.
0 As indicated based on histopathological findings, including fatty change, fibrosis, cirrhosis, and/or
necrosis.
d As indicated based on histopathological findings, including proliferation and hyperplasia (and in the 13-
week study, mitosis).
e An increased incidence of fatty change was observed that was not statistically significant.
f The incidence of hepatocellular carcinomas in female 25-ppm rats was not statistically elevated compared
to concurrent controls, but did exceed the historical control range for female rats from JBRC (0-2%), and
increase that was statistically significant compared to the historical control.
Note: Different exposure concentrations were used in the 13-week and 104-week JBRC bioassays. Blank
cells indicate exposure concentrations not tested in either the 13-week or 104-week study.
+ = Evidence demonstrating key event.
— = No evidence demonstrating key event.
+/— = equivocal
Source: Nagano et al., 2007a,b; JBRC, 1998.
4.7.3.2.4. Biological plausibility and coherence. The theory that sustained cell proliferation to
replace cells killed by toxicity or viral or other insults, such as physical abrasion of tissues, can
be a significant risk factor for cancer is plausible and generally accepted (Correa, 1996). It is
logical to deduce that sustained cytotoxicity and regenerative cell proliferation may result in a
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greater likelihood of mutations (whether spontaneous, or directly or indirectly induced by the
chemical) being perpetuated, with the possibility of one or more of these resulting in loss of cell
cycle control and tumor development. It may also be that continuous stimulus of proliferation by
growth factors involved in inflammatory responses (e.g., TGF-a in the hepatic response to
carbon tetrachloride) increases the probability that damaged cells may slip through cell cycle
checkpoints carrying DNA alterations that would otherwise be repaired. Current views of cancer
processes support both possibilities. A high proliferation rate alone is not assumed to cause
cancer; tissues with naturally high rates of turnover do not necessarily have high rates of cancer,
and tissue toxicity in animal studies does not invariably lead to cancer. Nevertheless,
regenerative proliferation associated with persistent cytotoxicity appears to be a risk factor of
consequence.
4.7.3.3. Other Possible Modes of Action
Section 4.4.2 provides a critical review of the genotoxicity literature for carbon
tetrachloride. Various confounding factors and other challenges in evaluating genotoxicity
studies are highlighted in Table 4-12; these general features of the carbon tetrachloride literature
as well as various methodological and reporting issues in individual studies were taken into
account in the current review of the genotoxicity literature. Many of the positive genotoxicity
findings, including the following, are consistent with compounds that induce oxidative events
leading to genetic damage: (1) two positive mutation/DNA damage studies in E. coli strains
particularly sensitive to oxidative damage; (2) intrachromosomal recombination induced by
carbon tetrachloride in S. cerevisiae consistent with DNA breakage originating from oxidative
stress that occurs concurrent with cytotoxicity; (3) evidence from in vitro and in vivo assays of
DNA breakage and fragmentation in association with extensive hepatotoxicity; and (4) DNA
adducts formed from reactive oxygen species and lipid peroxidation products (e.g., MDA and 4-
HNE) in the liver of rodents following carbon tetrachloride administration. A limited number of
positive genotoxicity findings in the absence of cytotoxicity have been reported (see Section
4.4.2 and Table 4-8 to 4-11); methodological or reporting issues with many of these studies have
been identified.
As a whole, the literature suggests that carbon tetrachloride is more likely an indirect than
direct mutagenic agent and that mutagenic effects, if they occur, are likely to be generated
through indirect mechanisms resulting from oxidative damage stress or lipid peroxidation by-
products, which have been observed with cytotoxicity at high doses of carbon tetrachloride
(Table 4-16). Nevertheless, uncertainties in this complex database must be acknowledged. To
that end, if carbon tetrachloride-associated DNA damage occurs above background levels and
contributes to low-dose mutagenic activity, some nonzero risk of carcinogenicity at doses below
those associated with cytotoxicity would be predicted. Under this scenario, a quantitative
approach that accounts for possible carbon tetrachloride-associated DNA damage in a mode of
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action would apply and would dictate an approach consistent with a nonthreshold response (see
Section 5.3.2).
Thus, the possible contribution of a low-dose mutagenic effect in the mode of action or
alternative modes of action cannot be excluded.
4.7.3.4. Conclusions About the Hypothesized Mode of Action
The weight of evidence supports reductive dehalogenation of carbon tetrachloride by
CYP2E1, sustained cytotoxicity, and regenerative cell proliferation as key events in the mode of
action for carbon tetrachloride-induced tumors of the liver. A wide range of evidence across
different species, sexes, and routes of exposure implicates reductive dehalogenation by CYP2E1
as the initial step leading to hepatic toxicity. Hepatocellular cytotoxicity leading to regenerative
proliferation and subsequent tumorigenesis as key events have experimental support at high
exposure levels in terms of strength, consistency and specificity of association; dose-response
concordance; temporal relationship; and biological plausibility and coherence. Considerable
empirical evidence provides support for a hypothesis that liver carcinogenicity is presumed to
occur at exposures that also induce hepatocellular toxicity and a sustained regenerative and
proliferative response, and that exposures that do not cause hepatotoxicity are not expected to
result in liver cancer. This hypothesized mode of action for carbon tetrachloride liver
carcinogenicity is consistent with a nonlinear approach to low-dose extrapolation (see Section
5.3.1).
However, the temporal relationship for cytotoxicity, regenerative hyperplasia, and liver
tumors in the female mouse (Nagano et al., 2007b) is not consistent with the above mode of
action (see Section 4.7.3.2.3 for additional discussion). Furthermore, relatively little mode of
action information is available at lower exposure levels (i.e., exposures that are not cytotoxic).
Whether carbon tetrachloride-induced biological events occur at low exposures that could lead to
increased cancer risk is uncertain. Possible mechanistic events that could be operating at low
doses include mutagenicity as a key event or a mode of action in the formation of tumors. These
events could be operating at all exposure levels. Currently, information on such events is
unavailable at low exposure levels. At higher exposures there are some studies indicating
equivocal, or possibly positive, genetic toxicity. Additionally, at high exposures both the
cytotoxicity-based mode of action and the mutagenicity-based mode of action may be operative,
but it is not possible to delineate the contribution of these possible mode(s) of action to carbon
tetrachloride tumor response. It is important to establish the presence of mutational events
following carbon tetrachloride treatment, and the mechanistic role such events may play in tumor
response, especially at low exposure levels. Thus, alternative nonthreshold approaches (i.e., low-
dose linear extrapolation procedures) to carbon tetrachloride carcinogenicity have also been
considered (see Section 5.3.2).
In summary, biological support exists for a hypothetical mode of action involving
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metabolism of carbon tetrachloride by CYP2E1, sustained cytotoxicity, and regenerative cell
proliferation as a major mode of action driving the steep nonlinear increase in liver tumor dose-
response at relatively high carbon tetrachloride exposures. Inconsistencies and uncertainties at
the low end of the experimental exposure range suggest that other (or another) mode(s) of action
that are independent of cytotoxicity and regenerative cell proliferation may be operative in this
range.
4.7.3.5. Relevance of the Hypothesized Mode of Action to Humans
Although there is no evidence for hepatic cancer resulting from exposure to carbon
tetrachloride in humans, the potential modes of action are considered relevant to humans.
Humans express ethanol-inducible CYP2E1 and phenobarbital-inducible CYP3A in the liver,
both of which are associated with the generation of trichloromethyl radical in animals exposed to
carbon tetrachloride. The antioxidant systems in animals and humans are similar. Therefore,
both the mode of action and the endogenous protective mechanisms likely have related processes
in animals and humans. Furthermore, humans exhibit the same signs of liver toxicity that have
been observed in animal studies (cirrhosis, fibrosis, steatosis, necrosis, and liver enzyme
changes). Finally, the types of tumors, hepatocellular adenoma and carcinoma, expressed
consistently in several animal species exposed to carbon tetrachloride are also found in humans.
4.7.4. Mode of Action Information for Pheochromocytomas
An increased incidence of pheochromocytomas (a neuroendocrine tumor of adrenal
chromaffm cells) associated with carbon tetrachloride administration has been observed in male
and female mice by oral (NCI, 1977, 1976a,b; Weisburger, 1977; NTP, 2007) and inhalation
exposure (Nagano et al., 2007b; JBRC, 1998), but not in rats by either route of exposure. The
mode of action by which carbon tetrachloride induces pheochromocytomas in mice is not known.
Unlike liver tumors, it is not known whether the parent compound or an active metabolite is
responsible for tumor induction, and none of the key events in the development of carbon
tetrachloride-induced pheochromocytomas has been elucidated. In general, few chemicals have
been reported to cause pheochromocytomas in mice. Of 514 technical reports published by the
National Toxicology Program (NTP), only seven chemicals have been associated with
pheochromocytomas in mice with no apparent common denominator (Tischler et al., 2004; Hill
et al., 2003).
In the absence of any information on mode of action for carbon tetrachloride induction of
pheochromocytomas, the framework for evaluating a hypothesized mode(s) of action as
described in U.S. EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) has not
been applied.
4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES
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Age (e.g., childhood, senescence), gender, nutritional status, disease status, and exposure
to other chemicals are all factors that might influence susceptibility to carbon tetrachloride. Each
of these factors is described below in more detail.
4.8.1. Possible Childhood Susceptibility
Key events leading to carbon tetrachloride-induced liver toxicity and carcinogenicity
(e.g., metabolism to trichloromethyl radical by CYP2E1 and subsequent formation of
trichloromethyl peroxy radical, cytotoxicity, and sustained regenerative and proliferative changes
in the liver in response to hepatotoxicity; or possibly, DNA damage and fixation leading to
mutagenic activity) involve metabolic and cellular processes common to cells at all life stages,
and are therefore considered plausible in the developing organism. For these key events, limited
data are available to evaluate the relative susceptibility of children to carbon tetrachloride.
As observed in adult animals, the initiating event for liver toxicity and carcinogenicity is
metabolism of carbon tetrachloride by CYP2E1 to reactive metabolites. Assuming that this is
the initiating key event in the mode of action for all age groups, susceptibility to carbon
tetrachloride at all life stages is related to the presence of functional microsomal enzymes
(particularly CYP2E1 but also CYP3A). Hepatic concentrations of CYP2E1 do not achieve
adult levels until sometime between 1 and 10 years, although large increases in hepatic CYP2E1
protein occur postnatally between 1 and 3 months in humans (Vieira et al., 1996). Thus, age-
related differences in CYP450, as described below, could potentially affect susceptibility. To the
extent that hepatic CYP2E1 levels are lower, infants and children would be less susceptible to
free radical-induced liver injury from carbon tetrachloride than adults. There is some evidence
from the therapeutic drug literature that CYP3 A levels also change with age, but in a pattern
different from CYP2E1. Based on half-life results for several therapeutic drugs metabolized by
the CYP3 A family (Ginsberg et al., 2002), enzyme levels appeared to be lower than the adult up
to 2 months of age, but from 6 months to 2 years of age were significantly higher than the adult.
To the extent that CYP3 A levels are relatively higher than the adult and CYP3 A plays a
significant role in carbon tetrachloride metabolism, infants and young children could be
relatively more susceptible to liver injury from carbon tetrachloride. Work conducted by Zangar
et al. (2000), however, suggests that CYP2E1 is the major human enzyme in the adult
responsible for carbon tetrachloride bioactivation at lower, environmentally relevant levels (i.e.,
levels that are not hepatotoxic). Only at higher carbon tetrachloride levels, CYP3 A and possibly
other CYP450 forms may contribute to carbon tetrachloride metabolism. Therefore, assuming
CYP2E1 is the more effective metabolizing enzyme in children as it is in adults at
environmentally-relevant exposure levels, infants and children would likely be less susceptible to
liver injury from carbon tetrachloride than adults to the extent that hepatic CYP2E1 levels are
lower. Carbon tetrachloride-specific enzyme data for younger populations are not available,
however, to confirm these assumptions.
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There is no information to suggest that key events subsequent to metabolism of carbon
tetrachloride would exhibit age-dependence.
Low levels of CYP2E1 mRNA begin to be elevated in human fetal brains after week 7
and increase thereafter to week 16 (the oldest stage examined) (Brzezinski et al., 1999).
CYP2E1 function was analyzed in prenatal human brain and liver tissues (7 to 17 weeks of
gestation) using three assays (Boutelet-Bochan et al., 1997). Low levels of CYP2E1 expression
were detected in fetal brain tissue, with some evidence for increasing expression at later stages of
gestation; weaker levels were identified in fetal liver. In fetal brain, CYP2E1 was not detected
with the less sensitive assay (Northern blot), and expression measured with the two more
sensitive assays (RT-PCR and RNase protection assays) were considerably weaker than those
measured in adult human or rat liver samples. The results suggested that, during gestation weeks
8-17, the fetal brain might be more vulnerable than the liver to toxic effects from exposure to
carbon tetrachloride. Carpenter et al. (1996) detected functional CYP2E1 in human fetal livers at
19 weeks of gestation. However, when related to weight unit of microsomal protein, the
CYP2E1 content of fetal livers was considerably lower than in adults. In an in vitro experiment,
exposure to ethanol or clofibrate induced expression of CYP2E1 in hepatocytes from a 20-week
fetus, which suggests that maternal alcohol intake might enhance CYP2E1 in the human fetus.
Given that the maternal liver mass and hepatocellular CYP2E1 content are so much higher than
the fetal values, it would seem that fetuses would have only a slight vulnerability from maternal
exposure to carbon tetrachloride at low levels. Because for inhalation exposures the arterial
blood flow does not perfuse the liver before reaching the fetus, this observation may apply more
to oral exposures than to inhalation exposures.
An unknown factor in fetal vulnerability is the expression of CYP450 in the placenta.
Two different laboratories have detected CYP2E1 in human placentas. Hakkola et al. (1996)
detected several different enzymes in human placentas, including CYP2D6, CYP4B1, and
several forms of CYP3A and CYP2E1; there was considerable variation in expression among the
different individuals. Rasheed et al. (1997) compared the levels of CYP2E1 protein in western
immunoblots of microsomes taken at delivery from placentas of 12 African-American women.
None of the women who abstained from ethanol had detectable levels of placental CYP2E1,
whereas the protein was detectable in blots for 6/8 drinkers. The median head circumference at
birth was significantly smaller (33.2 cm) in children with detectable CYP2E1 compared with
those without detectable enzyme (37 cm,/> = 0.04). The study provides suggestive evidence that
placental CYP2E1 is inducible by alcohol consumption, although there are individual variations.
Theoretically, fetuses of mothers who drink ethanol would be potentially more susceptible to
injury from carbon tetrachloride exposure.
Carpenter et al. (1996) measured the amount and activity of CYP2E1 in fetal (GD 20)
and maternal rat liver and brain, following maternal exposure to a 5% ethanol diet. Rates of
metabolism for chlorzoxazone and N-nitrosodimethylamine were used to evaluate functional
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activity of CYP2E1. In untreated or pair-fed rats, the amount of CYP2E1 in maternal or fetal
brain was several hundred-fold lower than in the respective livers. Ethanol exposure increased
the level of CYP2E1 protein by 1.4-fold in the maternal liver and 2.4-fold in the fetal liver
compared with the untreated or pair-fed groups but had no effect on CYP2E1 levels in maternal
or fetal brain. Hepatic CYP2E1 function, as exemplified by chlorzoxazone 6-hydroxylation, was
elevated 2.1-fold in ethanol-exposed maternal liver but not significantly in fetal liver.
Demethylation of N-nitrosodimethylamine was elevated about 1.5-fold in maternal and fetal
livers after ethanol exposure. Cambon-Gros et al. (1986) demonstrated the formation of
trichloromethyl radicals in maternal and fetal rat liver exposed to carbon tetrachloride on GD 20.
The results of these studies suggest that maternal ethanol ingestion might increase the
susceptibility of fetuses to hepatotoxicity from exposure to carbon tetrachloride.
Developmental studies in rats demonstrated that total litter loss was the primary effect of
maternal exposure between GDs 6 and 15 (Narotsky et al., 1997b; Narotsky and Kavlock, 1995).
The mode of action for developmental effects has not been explored, so it is unknown whether
placental expression of CYP2E1 may contribute to the litter loss, as CYP2E1 contributes to liver
cytotoxicity.
While some information is available on the activity of enzymes involved in the
metabolism of carbon tetrachloride in children, little lifestage-specific information on the levels
of antioxidants (e.g., GSH) was identified.
In summary, there is no direct evidence for increased or decreased susceptibility to
carbon tetrachloride in children. The relatively lower activity of CYP2E1 (the major human
enzyme responsible for carbon tetrachloride bioactivation at environmentally-relevant exposure
levels) in infants and children compared to adults suggests the possibility of lower susceptibility
to carbon tetrachloride-induced liver injury for younger life stages. Too little is known,
however, about changes in activity of other enzyme levels with age to support a conclusion that
children are at decreased risk. CYP3 A levels are higher in children 6 months to 2 years than in
adults (although CYP3 A is less likely to contribute to carbon tetrachloride metabolism at
environmentally-relevant exposure levels than CYP2E1). Further, little lifestage-specific
information on the levels of antioxidants (e.g., GSH), another factor likely to contribute to
susceptibility to carbon tetrachloride toxicity, is available. No information is available to support
an evaluation of differences in childhood susceptibility to possible effects of carbon tetrachloride
on the adrenal medulla (as suggested by the increased incidence of pheochromocytomas in
mice).
4.8.2. Possible Effects of Aging
The overall vulnerability to carbon tetrachloride is affected directly by the rate of
generation of reactive intermediates, a function of microsomal CYP activity, and inversely by the
antioxidant content. Compared with young/mature adults, older organisms exhibit changes,
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usually decreases, in these parameters that vary independently in different tissues.
Studies evaluating the capacity for drug metabolism in the human liver during different
life stages reported a reduction in activity for CYP3A3/4 and CYP2E1 in the elderly (i.e.,
individuals older than 65 years) (reviewed in Tanaka, 1998). Total immunoreactive CYP3A
protein (the sum of CYP3A4 and CYP3A5) per mg hepatic microsomal protein was significantly
reduced by 90% in samples from men aged 61-72 (n = 5) compared with those from men aged
21-40 years (n = 5) (Patki et al., 2004). McLean and Le Couteur (2004) suggested that the
reduction in phase I enzyme activity may be related not to deficits intrinsic to the liver
microsomal monooxygenase systems, but rather to structural changes in the liver with age (e.g.,
thickening and defenestration of the sinusoidal endothelium of the liver) that may reduce oxygen
availability for phase I enzymes that are directly dependent on oxygen supply as a substrate.
Studies in experimental animals also provide evidence of age-related changes in CYP
activity. Although no significant age-related variations in hepatic CYP2E1 mRNA content were
noted in adult (18-month-old) male Wistar rats compared with 8-month-old rats, CYP2E1
microsomal protein levels were 20% reduced (not statistically significant), and CYP2E1 activity
(assayed as chlorzoxazone oxidation) was significantly reduced by 46% in the older group
(Wauthier et al., 2004); this study found no age-related changes for hepatic CYP3A1, 3A2, 3A9,
or 3A23 mRNA or protein levels in rats. Wauthier et al. (2004) attributed age-related reductions
in hepatic CYP2E1 activity to posttranslational modifications, possibly from the reactive oxygen
species commonly generated by this CYP. A photoperiodicity study reported that increases in
hepatic CYP3 A-dependent erythromycin N-demethylase activity, which is elevated after Wistar
rats are exposed to a dark cycle, were twofold lower in the livers of 22-month-old rats compared
with 10-week-old rats (Martin et al., 2003). Total immunoreactive CYP3A content was reduced
in the hepatic microsomes of 2-year-old compared with 1-year-old male CD-I mice and was
associated with a reduced clearance of the substrate midazolam (Warrington et al., 2000). These
results suggest that the metabolism of carbon tetrachloride would be slower in the liver of old
compared with younger organisms.
Warrington et al. (2004) compared age-related changes in microsomal CYP3A and
NADPH-reductase in the liver and kidney in male F344 rats at 2-4 months (young), 13-14
months (intermediate), and 25-26 months (old). Expression of CYP3A protein in the kidney was
only 1% of that in the liver. The net CYP3A content of the liver was significantly reduced in old
rats compared with young or intermediate rats and involved both immunodetectable bands in
western blots. Conversely, a 50% increase in one isoform of CYP3 A was detected in the kidneys
of old rats compared with the intermediate group; an 11% net increase in renal CYP3A was not
statistically significant. Age-related decreases (by 23-36%) in the expression of NADPH-
reductase occurred in the liver and kidney of male F344 rats, but compared with that in young
rats the decline was statistically significant only in the liver of old rats (Warrington et al., 2004).
The results of this study suggest that the capacity to initiate the metabolism of carbon
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tetrachloride is reduced in the liver but possibly increased in the kidney of older organisms
compared with younger animals.
Antioxidant content is also reduced in aging animals compared with younger life stages.
Hepatic GSH content was 35% lower in 24-28-month-old male F344 rats compared with 2-5-
month-old rats (Suh et al., 2004); the decline was related to significant decreases in the level and
activity of y -glutamylcysteine ligase (GCL), the rate-controlling enzyme in the synthesis of
GSH. The ultimate reduction in enzyme activity in old rats was related to an age-related
decrease in a transcription factor, nuclear factor erythroid-related factor 2, that governs the
expression of GCL (Suh et al., 2004). In the liver of 18-month-old male Wistar rats, the GSH
content was significantly reduced by 34% compared with 8-month-old rats, and the level of
TEARS was increased by 287% compared with that in 3-month-old rats (Wauthier et al., 2004).
One study reported a significant age-related reduction in GSH peroxidase activity in the kidney,
but not the liver, of 24-month-old male F344 rats compared with 6-month-old rats (Tian et al.,
1998). Significant decreases in GSH (-20% and -15%), GSH peroxidase activity (-59% and
-37%), and increases in TEARS (+54% and +23%) were noted, respectively, in the liver and
kidney of 22-month-old Wistar rats compared with those of 10-week-old animals (Martin et al.,
2003). These studies suggest that older animals are at greater risk than younger animals of
oxidative damage following exposure to carbon tetrachloride. Studies vary as to whether the
age-related changes are more significant in the kidney or liver, possibly because of strain
differences.
In general, aging is associated with constriction of the kidney arterioles and reduced renal
blood flow as well as with reductions in kidney mass and the number of functioning nephrons
(U.S. EPA, 2001b). The result of these changes is a decrease in glomerular filtration rate.
Because of their reduced glomerular function, aged adults are likely to be more sensitive than
younger adults to a chemical, such as carbon tetrachloride, that targets the glomerulus. The
manifestations of renal disease in 2-year-old rats that had been exposed to high concentrations of
carbon tetrachloride in air for most of their lifetimes were increased severity of glomerular
lesions associated with aging (progressive glomerulonephrosis) and impaired glomerular
function (decreased glomerular filtration rate, as indicated by increases in serum levels of BUN,
creatinine, and inorganic phosphorous) in comparison with untreated concurrent controls.
Whether older populations would likely be more susceptible to carbon tetrachloride
toxicity is difficult to determine. Evidence for a reduction in CYP3A and CYP2E1 activity in
the liver with age would suggest an age-related reduction in the generation of reactive
metabolites from carbon tetrachloride and possibly a corresponding reduction in susceptibility;
however, evidence for reduction in antioxidant content in aging animals would result in a relative
increased risk of oxidative damage in older animals. Functional changes in the kidney with age
and increases in kidney CYP3 A activity (as suggested by experimental animal studies) indicate
that older populations may be at greater risk of carbon tetrachloride-associated kidney damage.
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4.8.3. Possible Gender Differences
The extent to which men and women differ in susceptibility to carbon tetrachloride
toxicity is not known. No human data are available to suggest there are gender differences in the
toxicity or carcinogen!city of carbon tetrachloride.
Animal subchronic and chronic toxicity studies by the oral or inhalation route did not
report any significant gender differences in susceptibility to cancer or noncancer effects from
carbon tetrachloride. One study in rats exposed by i.p. injection measured a 2.5-fold increase in
the serum level of hepatic enzymes, a longer period of hepatic injury, and more evidence of
hepatic regeneration in females compared with males (Moghaddam et al., 1998); male livers had
20% more CYP2E1 activity than female livers. The significance of this observation is uncertain,
given the modest difference and the absence of other corroborating data. There appears to be no
basis for assuming gender differences in susceptibility.
4.8.4. Nutritional Status
Fasting or food deprivation has been shown to increase the toxicity of carbon
tetrachloride, as demonstrated by histopathology of the liver, increased serum enzyme levels, or
increased generation of chloroform (Qin et al., 2007; Seki et al., 2000; Shertzer et al., 1988; Sato
and Nakajima, 1985; Pentz and Strubelt, 1983; Yoshimine and Takagi, 1982). Decreasing levels
of GSH have been detected in food-restricted animals (Gonzalez-Reimers et al., 2003; Harris and
Anders, 1980; Nakajima and Sato, 1979). The basis for the increased toxicity caused by fasting
is the increase in lipolysis, which generates acetone, an inducer of CYP2E1 (Bruckner et al.,
2002). Bruckner et al. (2002) established that a circadian rhythmicity of vulnerability to carbon
tetrachloride in rats was based on the increased levels of acetone that occur during overnight
fasting. Peak levels of serum SDH, ALT, and isocitrate dehydrogenase were significantly higher
in fasted rats than in fed rats (for example, peak SDH levels were seven times higher with
fasting). Fasted rats also showed significantly more covalent binding of radiolabeled carbon
tetrachloride to microsomal protein and significantly higher CYP2E1 activities.
Carbon tetrachloride toxicity is also affected by the level of antioxidants in the diet. Rats
fed a diet low in vitamin E, methionine, and selenium (a cofactor for GSH reductase) showed an
increase in lipid peroxidation and liver damage that was reversed by supplementing the diet with
one or more of the antioxidants (Parola et al., 1992; Sagai and Tappel, 1978; Hafeman and
Hoekstra, 1977; Taylor and Tappel, 1976). Addition of vitamin A (retinoic acid or retinol) to
basal diet reduced the hepatic effects of carbon tetrachloride in mice (Rosengren et al., 1995;
Kohno et al., 1992), although it had the opposite effect in rats (Badger et al., 1996; El Sisi et al.,
1993a, b).
Dietary mineral content can also be important. Rats fed a diet deficient in zinc showed
an increase in hepatotoxicity from carbon tetrachloride (DiSilvestro and Carlson, 1994). Cabre
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et al. (2000) assessed the time course of hepatic lipid peroxidation and GSH metabolism in
Wistar rats injected with 0.5 mL of carbon tetrachloride to induce hepatic cirrhosis. Inclusion of
zinc in the diet delayed the appearance of cirrhosis and prevented the rise in lipid peroxides. The
protective effect of zinc was independent of GSH levels, which were reduced by carbon
tetrachloride.
4.8.5. Disease Status
Based on experimental findings from rodent studies, there is some reason to suspect that
people with diabetes may have altered susceptibility to hepatotoxic effects from carbon
tetrachloride. Studies in rats have found that rats made diabetic by pretreatment with the
diabetogenic agents alloxan or streptozotocin display markedly enhanced hepatotoxicity in
comparison with nondiabetic rats (Sawant et al., 2007, 2004; Watkins et al., 1988; Hanasono et
al., 1975). The relevance of this finding to humans is uncertain, although it has been reported
that diabetics have nearly twofold higher risk of acute liver failure due to drug-induced toxicities
and chronic liver disease (Sawant et al., 2007). Streptozotocin-induced diabetes not only failed
to enhance the hepatotoxicity of carbon tetrachloride but actually protected against lethality of
the compound in mice (Shankar et al., 2003; Gaynes and Watkins, 1989).
There has been some investigation of the mechanism by which diabetes potentiates
carbon tetrachloride hepatotoxicity in rats. Diabetic rats do not gain weight as normal rats do,
raising the possibility that the enhanced toxicity in diabetic rats is a result of associated
starvation (see Section 4.8.4). However, data for a pair-fed control group in the Hanasono et al.
(1975) study showed that the restriction in food intake could account for only a small portion of
the observed hepatotoxicity in diabetic Sprague-Dawley rats. [Diabetes was induced by
treatment with alloxam monohydrate or streptozotocin.] Treatment of diabetic rats with insulin
controlled the diabetic state and prevented any enhancement of carbon tetrachloride
hepatotoxicity in these rats (Watkins et al., 1988; Hanasono et al., 1975), suggesting the diabetic
state and not the presence of inducer chemicals potentiates carbon tetrachloride hepatotoxicity.
Serum glucose levels in the diabetic rats were not sensitive predictors of the extent of
hepatotoxicity in the Hanasono et al. (1975) study (e.g., 40 mg alloxan and 65 mg streptozotocin
produced similar plasma glucose levels, but the increase in serum ALT associated with carbon
tetrachloride treatment was twofold higher in the latter experiment), suggesting that other
metabolic effects of diabetes are more important to the effect on carbon tetrachloride toxicity.
Because ketones and compounds metabolized to ketones have been found to potentiate
the toxicity of carbon tetrachloride and other haloalkanes (see Section 4.8.6), presumably by
enhancing expression of CYP2E1 leading to increased activation of the hepatotoxicant, it has
been suggested that ketosis associated with diabetes might be responsible for the observed effect
(Hewitt et al., 1980). However, there are several lines of evidence suggesting that ketonemia and
increased bioactivation may not be the critical features of diabetes leading to enhanced toxicity
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of carbon tetrachloride. In the study by Hanasono et al. (1975), alloxan and streptozotocin both
potentiated carbon tetrachloride-induced hepatotoxicity, even though alloxan-induced diabetes in
rats is characterized by a marked persistent increase in ketone bodies and streptozotocin-induced
diabetes is not. Both alloxan and streptozotocin have been reported to decrease CYP450 activity
(Watkins et al., 1988; Hanasono et al., 1975). Sawant et al. (2004) found no effect on hepatic
microsomal CYP2E1 levels or activity, lipid peroxidation, GSH, or covalent binding of carbon
tetrachloride in the liver in rats with streptozotocin-induced diabetes. Time course studies
performed by Sawant et al. (2004) found that the initial liver injury produced by carbon
tetrachloride in diabetic rats was similar to that in nondiabetic rats but that the effect progressed
only in the diabetic rats. Sawant et al. (2007) reported that liver injury initiated by non-lethal
doses of carbon tetrachloride progressed to hepatic failure and death of diabetic Sprague-Dawley
rats because liver cells failed to advance from Go/Gi to S-phase, thereby unabling S-phase DNA
synthesis (a critical step in cell division) and inhibiting tissue repair. A more detailed
understanding of the mechanism would be needed to predict how diabetes might affect carbon
tetrachloride toxicity in humans.
4.8.6. Exposure to Other Chemicals
Factors that increase the expression of CYP2E1 or CYP3A are likely to increase
susceptibility to carbon tetrachloride exposure (all other things being the same) because the
relatively higher rate of metabolism on a per cell basis would significantly increase the rate of
generation of trichloromethyl radicals in the liver and kidney. Heavy consumers of ethanol,
which induces CYP2E1, are therefore more vulnerable to carbon tetrachloride (Manno et al.,
1996). Manno et al. (1996) described case reports of two workers who consumed 120 or 250
grams of ethanol per day and were the only individuals to develop severe hepatonephrotoxicity
following a 2-hour exposure to carbon tetrachloride vapors used in a fire extinguisher (Manno et
al., 1996); their nonsymptomatic colleagues, who also were exposed, consumed less than 50
grams of ethanol per day. Cases of acute carbon tetrachloride poisoning often involved
individuals who were alcohol consumers (New et al., 1962). Enhanced toxicity from
concomitant or preceding ethanol consumption and exposure to carbon tetrachloride has been
verified in animal studies (Wang et al., 1997; Plummer et al., 1994; Hall et al., 1991; Ikatsu et
al., 1991; Kniepert et al., 1990; Reinke et al., 1988; Sato and Nakajima, 1985; Strubelt, 1984;
Teschke et al., 1984; Harris and Anders, 1980; Sato et al., 1980).
Potentiation of carbon tetrachloride hepatotoxicity has also been observed following
exposure to other chemical inducers of CYP450, including isopropanol which converts to
acetone (Rao et al., 1996; Folland et al., 1976; Traiger and Plaa, 1971), methanol (Allis et al.,
1996; Harris and Anders, 1980), 2-butanol (Traiger and Bruckner, 1976), tert-butanol (Ray and
Mehendale, 1990; Harris and Anders, 1980), and other aliphatic alcohols (Ray and Mehendale,
1990); acetone, methyl ethyl ketone, methyl isobutyl ketone, 2-butanone, and other ketones
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(Raymond and Plaa, 1995; Charbonneau et al., 1986; Pilon et al., 1986; Plaa and Traiger, 1972);
phenobarbital (Abraham et al., 1999; Sundari et al., 1997; Hocher et al., 1996; Cornish et al.,
1973; Garner and McLean, 1969); DDT (McLean and McLean, 1966); poly chlorinated and
polybrominated biphenyls (Kluwe et al., 1979); and mirex and chlordecone (Soni and
Mehendale, 1993; Kodavanti etal., 1992; Mehendale, 1992, 1991, 1990; Bell and Mehendale,
1987, 1985; Curtis et al., 1979). Coexposure to nicotine in drinking water also increased hepatic
effects of carbon tetrachloride, although this was thought to be because of a synergistic effect on
lipid peroxidation produced by both chemicals rather than induction of CYP450 (Yuen et al.,
1995).
There is also limited evidence for a reduction in carbon tetrachloride hepatotoxicity
associated with reduced bioactivation of the chemical. Coexposure to carbon tetrachloride and
carbon disulfide both in rats and human workers resulted in hepatic and neurological effects
associated with carbon disulfide but no effects characteristic of carbon tetrachloride (Peters et al.,
1987; Seawright et al., 1980). The researchers attributed this result to destruction of CYP450 by
carbon disulfide and reduced bioactivation of carbon tetrachloride. Pretreatment with lead
nitrate reduced the hepatotoxicity of carbon tetrachloride, apparently because of the ability of
lead to inhibit CYP450 (Calabrese et al., 1995).
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5. DOSE-RESPONSE ASSESSMENTS
5.1. ORAL REFERENCE DOSE (RfD)
5.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification
Epidemiological studies of long-term exposure to carbon tetrachloride are inadequate to
establish whether an association exists between oral exposure and adverse birth outcomes (the
only health outcome evaluated in these studies). Case reports of human poisoning identify the
liver and kidney as primary target organs of acute carbon tetrachloride exposure, but do not
provide data useful for dose-response analysis.
Several subchronic oral toxicity studies, including Bruckner et al. (1986), Condie et al.
(1986), Hayes et al. (1986) and Allis et al. (1990), provide liver toxicity data that was considered
for dose-response analysis. Hayes et al. (1986) and Allis et al. (1990) reported liver toxicity at
the lowest dose tested (i.e., a NOAEL was not identified) and thus are less suitable for defining a
point of departure (POD) for the RfD. Further, in the Hayes et al. (1986) study, which included
both a vehicle (corn oil) and untreated control group, the vehicle controls themselves had
significantly elevated serum enzyme levels, altered organ weights, and increased incidence of
liver necrosis. This type of corn oil vehicle response was not seen in other studies. The Allis et
al. (1990) protocol also provided data less amenable to dose-response analysis. Male rats were
sacrificed in groups of six at various time points after exposure was terminated (1, 3, 8, and 15
days), and results at these various time points could not be combined.
Subchronic gavage studies by Bruckner et al. (1986) in male rats and Condie et al. (1986)
in male and female mice provided the best available characterizations of the dose response for
ingested carbon tetrachloride at low doses. Bruckner et al. (1986) identified a NOAEL of
1 mg/kg and a LOAEL of 10 mg/kg in rats treated by gavage in corn oil, while Condie et al.
(1986) identified a NOAEL of 1.2 mg/kg and a LOAEL of 12 mg/kg in similarly treated mice.
In both studies, the LOAEL of 10-12 mg/kg (average daily dose of 7-9 mg/kg-day) produced
hepatotoxicity, indicated by increased serum activity of enzyme markers of liver damage and
direct histopathological determination of liver lesions. More marked effects on the liver were
found at higher doses in both studies. Liver effects were also observed in numerous other studies
in animals. The LOAELs from Bruckner et al. (1986) and Condie et al. (1986) are consistent
with the LOAELs from Hayes et al. (1986) [12 mg/kg-day] and Allis et al. (1990) [14.3 mg/kg-
day].
5.1.2. Methods of Analysis—Including Models
The most sensitive endpoints identified for effects of carbon tetrachloride by oral
exposure relate to liver toxicity in the subchronic corn oil gavage studies of Bruckner et al.
(1986) in male rats and Condie et al. (1986) in male and female mice. Sensitive endpoints in
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both studies were evaluated for suitability for benchmark dose (BMD) modeling. For suitable
data sets, BMD modeling methodology (U.S. EPA, 2000c, 1995) was used to analyze the data.
The Bruckner et al. (1986) study identified serum enzyme changes and liver
histopathology as the most sensitive endpoints for carbon tetrachloride. Serum chemistry data
from Bruckner et al. (1986) are presented in Table 5-1. Of the enzymes monitored, only SDH
shows a clear statistically and biologically significant increase in the 10 mg/kg dose group. The
data for the 10- and 12-week blood draws are similar. The 10-week data were used for BMD
modeling because the precise group sizes were not known for the 12-week data (a range of 7-9
rats per group was reported).
Table 5-1. Serum enzyme data in male rats after 10- or 12-week exposure
to carbon tetrachloride
Daily dose
(mg/kg-day)
0
1
10
33
SDH (IU/mL)a
10 weeks
3.5 ±0.4
2.3 ±0.6
7.6±2.5b
134.8 ±15.0b
12 weeks
3.2 ±0.4
1.9±0.1
8.7±2.0b
145.7 ±57.9b
OCT (nmol CO2/mL)a
10 weeks
28 ±8
23 ±3
55 ±10
148 ± 48b
12 weeks
45 ±4
61 ±12
69 ±16
247±31b
ALT (IU/mL)a
10 weeks
18 ±1
20 ±1
23 ±1
617 ±334
12 weeks
20 ±0.3
19 ± 1
27±2b
502±135b
aValues presented are mean standard error for groups of five rats at 10 weeks and seven to nine rats at 12
weeks.
V<0.05.
Source: Bruckner et al., 1986.
All of the models for continuous data in U.S. EPA's BMD software (BMDS) (version
1.4.1) (U.S. EPA, 2007) were fit to the 10-week SDH data. An increase in SDH activity two
times the control mean, representing an increase in serum enzyme level considered to be
biologically significant, was used as the benchmark response (BMR). Several expert
organizations, particularly those concerned with early signs of drug-induced hepatotoxicity, have
identified an increase in liver enzymes compared with concurrent controls of two to fivefold as
an indicator of concern for hepatic injury (EMEA, 2006; Boone et al., 2005; FDA Working
Group, 2000). Dr. James Bruckner, University of Georgia and principal investigator of the study
used to derive the RfD, considered a twofold increase in SDH to be an indication of a
lexicologically significant response (personal communication, November 7, 2006, with Susan
Rieth, U.S. EPA). Because ALT is the liver enzyme that is generally measured clinically, most
expert organizations similarly focus on ALT as an indicator of liver injury in preclinical (animal)
studies. Because SDH, like ALT, is one of the more specific indicators of hepatocellular damage
in most animal species and generally parallels changes in ALT in toxicity studies where liver
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injury occurs, a similar twofold increase in SDH is considered indicative of liver injury in
experimental animals.
BMD modeling results are summarized in Appendix B. The 3rd degree polynomial and
power models provided adequate fits of the 10-week SDH data (based on a goodness-of-fit p-
value of >0.1). The power model provided the better fit of the data (based on the lower AIC
value) and therefore was selected as the basis for a candidate POD; this model estimated a
BMD2X of 7.32 mg/kg-day and BMDL2X of 5.46 mg/kg-day.
BMD modeling was also performed using the 10-week OCT and ALT data from
Bruckner et al. (1986). OCT data could not adequately be fit by the models available in BMDS.
The power model provided an adequate fit of the 10-week ALT data; however, as shown in
Table 5-1, the standard error of the mean ALT for the high-dose (33 mg/kg-day) male rats was
extremely large (617 ± 334). Bruckner et al. (1986) noted: "There was a pronounced rise in GPT
[ALT] at 10 and 12 weeks. Scrutiny of values of individual animals revealed that dramatic
increases in two rats at each time point were largely responsible for the late increase in GPT
[ALT] activity." In light of the large variation in response at 33 mg/kg-day, using the ALT data
set for quantitative analysis was not considered appropriate.
Condie et al. (1986) also reported liver enzyme changes in carbon tetrachloride-exposed
mice; however, the median of 8 to 12 determinations was reported without a standard error (SE)
or standard deviation (SD) (only the minimum and maximum of the range were reported).
Without a mean and SE or SD, BMD analysis cannot be performed.
Liver lesion incidence data from the Bruckner et al. (1986) study in male rats and the
Condie et al. (1986) study in male and female mice support a nonlinear induction of hepatic
lesions due to carbon tetrachloride somewhere below 10-12 mg/kg. Table 5-2 presents liver
pathology data from the Bruckner et al. (1986) study. Data were displayed as mean severity
scores. Incidence data were not presented directly, although it can be inferred that incidence was
0% where severity is 0. In addition, a statement in the text implied that incidence was 100% for
lipid vacuolation in the 10 mg/kg dose group.
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Table 5-2. Severity of liver lesions in male rats after 12-week exposure to
carbon tetrachloride
Daily dose
(mg/kg-day)
0
1
10
33
Lipid vacuolation"
Ob
0
3.7C
4
Nuclear and cellular
pleomorphism"
0
0
0
5.7
Bile duct
hyperplasia"
0
0
0
4
Periportal fibrosis"
0
0
0
3.7
""Severity graded from 0 (absent) to 8 (severe); values presented are means for groups of 6-7 rats.
bSeverity score of 0 implies incidence of 0%.
°Text reports that "each animal" in this group showed the lesion, implying incidence of 100%.
Source: Bruckner et al., 1986.
It can be seen that lipid vacuolation was the only lesion to occur in the 10 mg/kg group,
making this the most sensitive pathology endpoint in the study, and that the incidence (not
reported but assumed from the text of the paper) of this lesion increased from 0% at 1 mg/kg to
100% at 10 mg/kg.
In the Condie et al. (1986) study, exposure to carbon tetrachloride by gavage in corn oil
or Tween-60 aqueous emulsion produced a variety of liver lesions (hepatocellular vacuolization,
inflammation, hepatocytomegaly, necrosis, portal bridging fibrosis) in male and female mice at
the high dose of 120 mg/kg. However, only necrosis (minimal to mild) in males and
hepatocytomegaly (severity unranked) in males and females treated using corn oil vehicle
occurred with statistically elevated incidence in the 12 mg/kg dose group. Incidence data for
these lesions, which represent the most sensitive effects of carbon tetrachloride in mice, are
shown in Table 5-3.
Table 5-3. Incidence of selected liver lesions in mice treated with carbon
tetrachloride for 90 days
Sex
M
M
F
Vehicle
Corn oil
Corn oil
Corn oil
Lesion
Necrosis
Hepatocytomegaly
Hepatocytomegaly
Incidence at daily dose
0 mg/kg-day
0/10
0/10
0/10
1.2 mg/kg-day
0/9
0/9
0/9
12 mg/kg-day
9/10a
8/10a
6/10a
120 mg/kg-day
9/10a
10/10a
9/9a
a/><0.05 by Fishers Exact test conducted for EPA.
Source: Condie etal., 1986.
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For all three of these data sets, incidence increased from 0% in the 1.2 mg/kg group to
60-90% in the 12 mg/kg group.
The histopathology data from Bruckner et al. (1986) and Condie et al. (1986) are,
therefore, consistent with a POD between 1 and 10 mg/kg in male rats and 1.2 and 12 mg/kg in
mice but do not provide sufficient information on response in the vicinity of the BMR (typically
10% for quantal data) (U.S. EPA, 2000c) to objectively inform the shape of the dose-response
curve in the region of interest. At the LOAEL in these studies, the response rate was 60 to
100%, whereas the response at the dose below the LOAEL was 0%. The incident data do,
however, support the BMD2X of 7.32 mg/kg and BMDL2X of 5.46 mg/kg estimated from the
increase in 10-week serum SDH observed in the Bruckner et al. (1986) study.
Serum activity of SDH is widely used in toxicity studies as an indicator of hepatocellular
injury. It is a specific and sensitive biomarker of liver damage. SDH is located in the cytosol
and mitochondria of liver cells. It is found at low levels in normal serum and erythrocytes.
Presence of increased activity in serum indicates leakage from hepatocytes secondary to cell
damage. In acute studies with carbon tetrachloride, serum SDH activity was a particularly
sensitive indicator of liver toxicity, with increases found at doses similar to, or even lower than,
those producing cellular damage visible by light microscopy (Paustenbach et al., 1986b; Korsrud
et al., 1972). In the Bruckner et al. (1986) study, the lowest administered dose at which serum
SDH activity was increased was also the lowest dose at which liver lesions were observed.
Use of elevated serum SDH activity as the critical effect for derivation of the RfD is
supported by results of a study examining the use of serum liver enzymes as predictors of
hepatotoxicity (Travlos et al., 1996). The relationship between the activity of serum liver
enzymes (ALT, SDH, ALP, and TEA [total bile acids]) and liver histopathology was examined
for 50 chemicals and three chemical mixtures using 1-, 2-, 3-, and 13-week clinical chemistry
measurements and 13-week histopathology assessments in male and female F344 rats, although
carbon tetrachloride was not tested. Treatment-related changes in serum liver enzymes were
determined using the Jonksheere-Terpstra trend test at the 0.05 level or Dunn's test at the 0.01
level; serum liver enzyme activities were not reported. An association was observed between
treatment-related increases in SDH and ALT activities and the development of histopathological
changes to the liver. SDH appeared to be a more sensitive predictor of histopathological changes
than ALT, with SDH activity predicting 13-week histopathological changes in rats of both sexes
with 76-92% accuracy, compared with 56-83% accuracy for ALT. If both SDH and ALT were
elevated, positive terminal histopathological changes were predicted with 100% accuracy from
the 2-, 3-, and 13-week clinical chemistry measurements. TEA and ALP were predictive of
histopathology results with 20-85% accuracy and 29-82% accuracy, respectively. Based on
these findings, statistically significant elevations in serum SDH and ALT activity appear to be
sensitive markers for liver toxicity, with SDH predicting histopathological changes to the liver
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with higher accuracy than ALT. As shown in Table 5-1, serum liver enzyme activity for SDH in
the Bruckner et al. (1986) study was significantly elevated after 10 and 12 weeks of exposure in
the mid- and high-dose groups and ALT was significantly elevated in the mid- and high-dose
groups after 12-weeks of exposure. In addition, treatment-related histopathologic findings were
observed in the mid-dose group (lipid vacuolization), with more extensive findings in the high-
dose group (lipid vacuolization, nuclear and cellular pleomorphism, bile duct hyperplasia, and
periportal fibrosis) after 12-weeks of exposure. Thus, carbon tetrachloride-induced elevations in
SDH and ALT appear to be valid markers of histopathological changes to the liver in the
Bruckner et al. (1986) study.
The BMDL2X of 5.46 mg/kg-day estimated from the increase in serum SDH activity in
male rats in the Bruckner et al. (1986) study was used as the POD for derivation of the RfD. Use
of the modeled BMDL provides an inherent advantage over use of a NOAEL or LOAEL by
making greater use of all of the data. The BMDS was able to achieve adequate fit to the SDH
data, providing a better estimate of the dose-response relationship for this endpoint than for other
endpoints monitored, which were less sensitive and/or had data less suited to dose-response
analysis. The BMD results based on SDH are supported by the histopathology data both in rats
and mice. Serum SDH activity is a specific and sensitive biomarker of hepatic injury by carbon
tetrachloride, comparable to histopathology in terms of sensitivity.
Consideration of PBPK Models for Interspecies Extrapolation
Three PBPK models of oral exposures have been reported; two rat models (Semino et al.,
1997; Gallo et al., 1993) and a mouse model (Fisher et al., 2004). These models implement
different approaches to simulate the complex kinetics of absorption of carbon tetrachloride that
follows an oral gavage dose of carbon tetrachloride in corn oil or emulsifiers (e.g., Emulphor).
Oral absorption of carbon tetrachloride in corn oil (and Emulphor) exhibits a pulsatile behavior,
evident from multiple peaks of carbon tetrachloride concentrations in blood that occur during the
first 12-20 hours following an oral gavage dose (Fisher et al., 2004; Semino et al., 1997; Gallo et
al., 1993). Semino et al. (1997) successfully modeled this pulsatile behavior in the rat with a
multi-compartment model in which first-order absorption from 6-9 compartments was scheduled
at different times following the dose (i.e., absorption was zero until the scheduled activation of
each compartment). The scheduling was accomplished using the SCHEDULE commend in
ACSL, which cannot be implemented repeatedly; therefore, the implementation is not directly
amenable to continuous simulation of multiple exposures. The approach also required
calibration of the model against blood concentration kinetics for a specific dose of carbon
tetrachloride (e.g., 25 mg/kg). The dose-dependence of the resulting parameter values was not
evaluated and, therefore, extrapolation to other dose levels would be highly uncertain. Gallo et
al. (1993) successfully simulated the oral absorption of carbon tetrachloride in corn oil with
multiple zero-order absorption rates (e.g., |ig/hr) that were estimated by fitting to observed blood
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carbon tetrachloride kinetics. Although this approach successfully reproduced the blood carbon
tetrachloride absorption kinetics following a 25 mg/kg dose to the rat, implementation of this
approach would require calibration of the zero-order absorption rates to each data set (i.e., blood
kinetics following the dose levels of interest). Fisher et al. (2004) simulated oral absorption of
carbon tetrachloride in an aqueous emulsion vehicle (similar to Emulphor) in the mouse with a
2-compartment, 3-parameter model (see Figure 3-2). Rate coefficients were estimated by
visually fitting these parameters to blood kinetics following single oral gavage doses of carbon
tetrachloride. One of the parameters in the absorption model was varied with dose in order to
simulate dose-dependent absorption kinetics; as a result, similar to the Gallo et al. (1993)
approach, implementation of the 2-compartment, 3-parameter model would require calibration to
blood kinetics for the dose levels of interest.
The above approaches to simulating oral absorption kinetics of carbon tetrachloride were
not implemented in the dosimetry analysis of oral bioassay data for two major reasons:
(1) predictions of oral absorption kinetics of carbon tetrachloride would be highly uncertain for
doses other than those to which the above models had been specifically calibrated; and
(2) extrapolation of these absorption models to humans also would be highly uncertain. An
alternative approach that simulates a time-averaged daily absorption rate and bioavailability
might suffice for simulating long-term average blood (arterial) concentrations of carbon
tetrachloride that would result from repeated oral exposures to carbon tetrachloride. Estimates of
liver metabolism rates would be less certain, however, since carbon tetrachloride is simulated in
the PBPK models as a non-linear function of carbon tetrachloride delivery to the liver (i.e., from
absorption and from arterial blood). (See additional discussion of PBPK modeling in Section
5.4.2.3.4). As a result, large fluctuations in absorption rate could result in similarly large
fluctuations in metabolism rates that may not be accurately represented by simulations of time-
averaged rates of absorption.
The BMDL2X of 5.46 mg/kg-day was derived from a study (Bruckner et al., 1986) with
an intermittent dosing schedule. In the absence of a suitable PBPK model, the BMDL is adjusted
to an average daily dose according to the following equation:
BMDL 2X-ADJ = BMDL2X x 5 days/7 days
= 5.46 mg/kg-day x 5 days/7 days
3.9 mg/kg-day (5-1)
5.1.3. RfD Derivation—Including Application of Uncertainty Factors
An RfD of 0.0039 mg/kg-day for carbon tetrachloride is derived by applying a composite
uncertainty factor (UF) of 1000 to the BMDL2X-ADJ of 3.9 mg/kg-day, as follows:
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RfD = BMDL2X-ADJ/UF
3.9mg/kg-day/1000
0.004 mg/kg-day (5-2)
The composite UF of 1000 includes a factor of 3 (10°5) to extrapolate from a subchronic
to chronic duration of exposure, a factor of 10 to protect susceptible individuals, a factor of 10 to
extrapolate from rats to humans, and a factor of 3 to account for an incomplete database, lacking
an adequate multigeneration study of reproductive function.
• A default 10-fold UF for intraspecies differences was selected to account for variability
in susceptibility among members of the human population in the absence of quantitative
information on the variability of human response to carbon tetrachloride. Factors that
could contribute to a range of human response to carbon tetrachloride were discussed in
Section 4.8. Variations in CYP450 levels because of age-related differences or other
factors (e.g., exposure to other chemicals that induce or inhibit microsomal enzymes)
could alter susceptibility to carbon tetrachloride toxicity. Individual variability in
nutritional status, alcohol consumption, or the presence of underlying disease could also
alter metabolism of carbon tetrachloride or antioxidant protection systems. To account
for these uncertainties, a factor of 10 was included for individual variability.
• A default 10-fold UF for interspecies extrapolation was selected to account for potential
pharmacokinetic and pharmacodynamic differences between rats and humans.
Metabolism of carbon tetrachloride to reactive species is the initial key event in the
development of carbon tetrachloride toxicity. Also critical to carbon tetrachloride
toxicity are cellular antioxidant systems that function to quench the lipid peroxidation
reaction, thereby preventing damage to cellular membranes. PBPK models available for
carbon tetrachloride were found unsuitable for repeat-dose oral scenarios, and could not
be used for interspecies extrapolation. In the absence of data to quantify specific
interspecies differences for key events of the mode of action and a suitable PBPK model,
a UF of 10 is included.
• A UF of 3 (10°5) for subchronic to chronic extrapolation was selected based on the
following: (1) Qualitative information demonstrating that the target of toxicity following
chronic oral exposure is the liver. The NCI oral cancer bioassay in rats and mice (NTP,
2007; NCI, 1977, 1976a,b; Weisburger, 1977) did not include an adequate evaluation of
low-dose exposures; in rats, there was marked hepatotoxicity at the lowest dose tested,
and in mice survival was low in dosed animals because of the high incidence of liver
tumors. For these reasons, the bioassay was not suitable for dose-response analysis.
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Nevertheless, complete nonneoplastic incidence data available through an NTP (2007)
database of neoplastic and nonneoplastic data did not identify carbon tetrachloride-related
histopathological changes in any organ systems or tissues other than the liver. Therefore,
the NCI bioassay clearly identified the liver as a target organ following chronic
exposures, consistent with the findings from subchronic oral studies and subchronic and
chronic inhalation studies.
(2) Knowledge of the relationship between effect levels in subchronic and chronic
inhalation studies. The JBRC inhalation bioassay, which included 13-week and 2-year
inhalation studies in rats and mice (Nagano et al., 2007a,b; JBRC, 1998), provides
information on the relationship between NOAELs and LOAELs from subchronic and
chronic exposure durations. In the 13-week study, liver toxicity (increased liver weight
and fatty liver) was observed in rats and mice at the lowest exposure concentration tested
(LOAEL = 2 ppm, duration adjusted). Following chronic exposure, the LOAEL based on
liver and kidney effects was 4 ppm (duration adjusted) and the NOAEL was 0.9 ppm
(duration adjusted); the LOAEL concentration in the chronic study was, in fact, twofold
higher than the LOAEL from the subchronic study. Other subchronic inhalation studies
in rats and mice support a NOAEL in the range of 0.9 to 4 ppm (see Table 4-14), which is
similar to or within fourfold of the NOAEL from the JBRC chronic inhalation bioassay.
Thus, the inhalation data do not support a full default UF of 10.
(3) Early onset of liver toxicity. Cytotoxicity occurs early in the sequence of events. For
example, Bruckner et al. (1986) observed increases in liver enzymes and liver cell
vacuolization after four days of exposure in an 11-day oral toxicity study, and increases
in liver enzymes at week two in a 12-week oral toxicity study. Thus, early appearance of
liver toxicity in carbon tetrachloride-exposed animals similarly does not support a full 10-
fold UF for subchronic to chronic extrapolation.
• An UF to account for extrapolation from a LOAEL to a NOAEL was not used because
the current approach is to address this extrapolation as one of the considerations in
selecting a BMR for BMD modeling. In this case, a BMR represented by an increase in
SDH activity two times the control mean was selected under an assumption that it
represents a minimal biologically significant change.
• A database UF of 3 (10°5) was selected. The oral database for this chemical includes
extensive testing for subchronic toxicity in animals, a number of tests of immunotoxic
potential, limited chronic oral bioassays in both rats and mice, and limited human data.
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Developmental toxicity testing by the oral route has been conducted. Testing for
developmental toxicity by two groups of investigators (Narotsky and Kavlock, 1995;
Wilson, 1954) found full-litter resorption at doses accompanied by some degree of
maternal toxicity, ranging from piloerection to mortality. Because both studies used
relatively high doses, neither study identified a NOAEL. The low dose of carbon
tetrachloride (25 mg/kg-day) used in Narotsky et al. (1997b) caused neither maternal nor
developmental effects when administered in either aqueous or corn oil vehicles, albeit the
group sizes (12-14 dams/dose level) were smaller than the group size used in the typical
developmental toxicity study. Nevertheless, the NOAEL in this developmental study (25
mg/kg-day) exceeds the POD for the RfD based on liver effects by over 6-fold and the
LOAEL (50 mg/kg-day) by 13-fold, and is consistent with developmental toxicity
endpoints as less sensitive than measures of hepatotoxicity. Also, as noted in Section
4.8.1 (Possible Childhood Susceptibility), the available life stage information on
microsomal enzyme activity, and in particular CYP2E1, suggests that the developing
organism would be no more susceptible to free radical-induced liver injury from carbon
tetrachloride than adults. The carbon tetrachloride database lacks an adequate
multigeneration study of reproductive function by any route of exposure. A database UF
of 3 was applied to account for the lack of a multigeneration reproductive toxicity study.
5.1.4. RfD Comparison Information
PODs and oral RfDs based on selected studies included in Table 4-13 are arrayed in
Figures 5-1 to 5-3, and provide perspective on the RfD supported by Bruckner et al. (1986).
These figures should be interpreted with caution because the PODs across studies are not
necessarily comparable, nor is the confidence in the data sets from which the PODs were derived
the same. PODs in these figures may be based on a NOAEL, LOAEL, or BMDL (in the case of
the principal study), and the nature, severity, and incidence of effects occurring at a LOAEL are
likely to vary. To some extent, the confidence associated with the resulting RfD is reflected in
the magnitude of the total UF applied to the POD (i.e., the size of the bar); however, the text of
Sections 5.1.1 and 5.1.2 should be consulted for a more complete understanding of the issues
associated with each data set and the rationale for the selection of the critical effect and principal
study used to derive the RfD.
The predominant noncancer effect of subchronic and chronic oral exposure to carbon
tetrachloride is hepatic toxicity. Figure 5-1 provides a graphical display of dose-response
information from five studies that reported liver toxicity in experimental animals following
subchronic oral exposure to carbon tetrachloride, including the PODs that could be considered in
deriving the oral RfD. As discussed in Sections 5.1.1 and 5.1.2, among those studies that
demonstrated liver toxicity, the study by Bruckner et al. (1986) provided the data set most
appropriate for deriving the RfD. Possible RfDs that might be derived from each of these studies
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are also presented. Although the RfD based on Bruckner et al. (1986) is not the lowest among
candidate studies, it is considered the most scientifically rigorous. The POD is based on BMD
methods, which has an inherent advantage over use of a NOAEL or LOAEL by making greater
use of all the data from the study. Because the studies by Hayes et al. (1986) and Allis et al.
(1990) identified only a LOAEL for liver effects, the RfD associated with these studies is driven
lower by use of a larger composite UF.
Studies in experimental animals have also found that relatively high doses of carbon
tetrachloride during gestation can produce prenatal loss; these doses also produced overt toxic
effects in the dams. A graphical display of dose-response information from three developmental
studies is provided in Figure 5-2.
Figure 5-3 displays PODs for the major targets of toxicity associated with oral exposure
to carbon tetrachloride. For the reasons discussed in Section 5.1.2, liver effects in the rat
observed in the study by Bruckner et al. (1986) are considered the most appropriate basis for the
carbon tetrachloride RfD. The POD is lower than that for developmental toxicity, and the
resulting RfD should adequately protect against developmental effects of carbon tetrachloride.
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Figure 5-1. Liver toxicity: oral
100
10
O)
o> 0.1
0)
i/)
0.01
0.001
0.0001
•
The Rf D in the dashed
circle w as selected as
the final RfDfor carbon
tetrachloride.
• POD
UjAnimal-to-human
Q Human variation
g| LOAEL to NOAEL
n Subchr to Chronic
| Database deficiencies
oRfD
Condieetal, 1986; 12-
wk mouse study (corn
oil gavage); liver
enzyme activity,
histopathology; NOAEL
Hayes etal, 1986; 13- Allis etal, 1990; 12-wk
wk mouse study; liver rat study; liver wt,
wt, enzyme activity, enzyme activity,
histopathology; LOAEL histopathology; LOAEL
(1) (2)
Bruckner etal, 1986;
12-wk rat study; SDH
activity; BMDL-2x
Condieetal, 1986; 12-
wk mouse study
(Tween-60 gavage);
liver wt, enzyme activity,
histopathology; NOAEL
(1) Magnitude of effect at the LOAEL: liver weight (f 15-19%); enzyme activity (f <6-fold); 100% necrosis.
(2) Magnitude of effect at the LOAEL: liver weight (f 30%); enzyme activity (f 3-5X); 100% necrosis.
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Figure 5-2. Developmental toxicity: oral
1000.00
100.00
•>;
TO
7
O)
Ja:
"3)
£.
0)
i/)
10.00
1.00
0.10
0.01
1
• POD
dm Animal-to-human
| | Human variation
^ LOAEL to NOAEL
I I Subchr to Chronic
H Database deficiencies
O RfD
Narotskyand Kavlock, 1995; rat;
GD 6-19; resorptions; LOAEL (1)
Narotskyetal, 1997b; rat; GD6-15
(corn oil gavage); resorptions;
NOAEL
Narotsky etal, 1997b; rat; GD6-15
(Emulphor gavage); resorptions;
NOAEL
(1 ) Magnitude of effect at the LOAEL: 44% resorptions
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Figure 5-3. Organ-specific oral RfDs
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y
fhe RfD in the dashed
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he final RfD for carbon
etrachloride.
• POD
[f[T|Animal-to-human
Q Human variation
^LOAELtoNOAEL
| | Subchr to Chronic
| Database deficiencies
ORfD
POD based on:
Liver: BMDLfor
elevated serum SDH
activity in the rat; 12-
week oral (gavage)
study (Bruckner etal.,
1986)
Developmental:
NOAELfor
resorptions in the rat;
exposure on GD 6-15
(Narotskyetal,
1997b)
Li\«r
De\«lopmental
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5.1.5. Previous RfD Assessment
The previous oral RfD for carbon tetrachloride (verified on 05/20/85 and posted on the
IRIS database in 1987) was 0.0007 mg/kg-day, based on the NOAEL of 1 mg/kg (daily dose of
0.7 mg/kg-day) and the LOAEL of 10 mg/kg (daily dose of 7 mg/kg-day) for liver lesions
(evidenced by mild centrilobular vacuolation and significantly increased serum SDH activity) in
rats treated for 12 weeks (5 days/week) with carbon tetrachloride by gavage in corn oil by
Bruckner et al. (1986). [A 1983 draft of the Bruckner et al. (1986) study was used as the basis
for the RfD by the RfD Work Group. The published version of the study did not necessitate a
change to the RfD.] The RfD of 0.0007 mg/kg-day was calculated by applying a UF of 1000
(three factors of 10 to account for interspecies and interhuman variability and extrapolation from
subchronic to chronic exposure) to the NOAEL of 0.7 mg/kg-day.
The current RfD relies on the same principal study as the previous RfD, but applies
benchmark dose analysis to derive the POD (3.9 mg/kg-day), whereas the previous RfD used the
NOAEL (0.7 mg/kg-day) as the POD. Both RfDs were derived using a total UF of 1000,
although some of the individual UFs differed. The previous RfD incorporated a UF of 10 to
account for extrapolation from subchronic to chronic exposure, whereas the current RfD includes
a subchronic to chronic UF of 3 based from a more thorough analysis of the available oral and
inhalation literature. The current RfD also includes a database UF of 3; the previous RfD
(posted in 1987) predated the institution of the database UF.
5.2. INHALATION REFERENCE CONCENTRATION (RfC)
5.2.1. Choice of Principal Study and Critical Effect—with Rationale and Justification
As noted in Section 4.6.2., the predominant targets of toxicity of carbon tetrachloride in
humans (based on case reports of acute, high-level exposure or long-term occupational exposure)
and experimental animals following inhalation exposure are the liver and kidney. Only one
cross-sectional epidemiological study of hepatic function in workers (Tomenson et al., 1995)
provides data that can be considered for use in dose-response analysis.
Tomenson et al. (1995) conducted a cross-sectional study of hepatic function in 135
carbon tetrachloride-exposed workers in three chemical plants in northwest England and in a
control group of 276 unexposed workers. The exposure assessment was based on historical
personal monitoring data for various jobs at the three plants. Subjects were placed into one of
three exposure categories—low (<1 ppm), medium (1.1-3.9 ppm), or high (>4 ppm)—according
to their current jobs. Multivariate analysis, based on simultaneous consideration of ALT, AST,
ALP, and GOT as dependent variables, revealed a statistically significant (p<0.05) difference
between exposed and unexposed workers. Univariate analyses (in which each dependent
variable was assessed separately) showed evidence of increased levels of ALP and GOT in the
medium and high exposure groups, with the differences between the medium exposure group and
controls being statistically significant (p < 0.05). In an alternative analysis, the proportion of
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exposed workers exceeding the normal range (i.e., the 2.5 and 97.5% quantiles of the control
group) was significantly elevated for ALT (8%) and GOT (11%) but not for the other serum
chemistry variables. There was little difference between the low carbon tetrachloride exposure
group (<1 ppm estimated exposure levels) and the control group on any of the liver enzymes.
Overall, this study provides suggestive evidence of an effect of occupational carbon tetrachloride
exposure on the liver at exposures in the range of >1 to 3.9 ppm (6.3 to 24.5 mg/m3); this
exposure range is considered to be a LOAEL. The low exposure category in this study (<1 ppm
or <6.3 mg/m3) appears to be a NOAEL. Because of study uncertainties described in Section
4.1.2.2, these values of the NOAEL and LOAEL must be considered similarly uncertain.
A number of experimental animal studies that identified liver and kidney as targets of
carbon tetrachloride toxicity were considered as the basis for RfC derivation. The most robust
study was the 2-year inhalation bioassay by JBRC (Nagano et al., 2007b; JBRC, 1998), which
used 50 animals/sex/group and examined an extensive set of endpoints of toxicity. The exposure
concentration of 25 ppm, 6 hours/day, 5 days/week in this study (corresponding to a continuous
.p
exposure level of 4.5 ppm) produced evidence of liver and renal toxicity in both male and
female F344/DuCrj rats. The lowest exposure concentration in this study, 5 ppm (0.9 ppm,
adjusted to continuous exposure), was considered to be a NOAEL. As described in Section
4.2.2.2., carbon tetrachloride-induced liver toxicity at >25 ppm was evidenced by serum
chemistry changes (including significant increases in ALT, AST, LDH, LAP and GOT) and
histopathologic changes (fatty change, fibrosis, and cirrhosis) (see Table 4-3). In the kidney,
there was a dose-related increase in the severity of chronic nephropathy (progressive
glomerulonephrosis or CPN) (see Table 4-3) and a significant increase in BUN in rats exposed to
>25 ppm. Because of the high spontaneous rate of chronic nephropathy in F344 rats, the
incidence of chronic nephropathy was close to 100% in all dose groups, including the control,
and a dose-related increase in incidence could not be demonstrated. As discussed in Section
4.6.2, the severity (but not incidence) of proteinuria was increased in all carbon tetrachloride-
exposed rats. Because this observation was difficult to interpret and its biological significance
was uncertain, it was not used to define the NOAEL and LOAEL for kidney effects. For these
reasons, hepatic effects in this study were considered the more appropriate and sensitive measure
of carbon tetrachloride-related toxicity.
Hepatic effects observed in the chronic rat inhalation study are consistent with the overall
carbon tetrachloride database. Subchronic studies in a number of experimental species (Adams
et al., 1952; Prendergast et al., 1967; Benson and Springer, 1999) identified a NOAEL for liver
effects in the range of 0.9 to 4 ppm (adjusted to continuous exposure). These subchronic studies
used exposure durations of 12 to 26 weeks (versus 104 weeks in the JBRC bioassay) and
f The exposure of 25 ppm for 6 hours/day, 5 days/week was adjusted to continuous exposure as follows: 25 ppm x 6
hours/24 hours x 5 days/7 days = 4.5 ppm
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experimental protocols that were less rigorous than the JBRC bioassay. Therefore, these studies
were considered less appropriate as the basis for the RfC. In the chronic mouse study by JBRC
(Nagano et al., 2007b; JBRC, 1998), the NOAEL for liver toxicity was 0.9 ppm (adjusted to
continuous exposure). This NOAEL is the same as that for rats in the JBRC bioassay; however,
the incidences of specific liver lesions in the mouse were lower than those in the rat. Hepatic
toxicity as the critical effect is also consistent with the epidemiological literature, in particular a
cross-sectional study of hepatic function in carbon tetrachloride-exposed workers (Tomenson et
al., 1995). Tomenson et al. (1995) reported suggestive evidence of carbon tetrachloride-
associated effects on hepatic serum enzymes.
Renal effects were observed in the JBRC chronic mouse study (Nagano et al., 2007b;
JBRC, 1998) and in subchronic animal studies, but generally at concentrations higher than those
that produced liver effects or occurred at a lower incidence than liver effects.
At the lowest tested concentration of 5 ppm in the JBRC study (corresponding to a
continuous exposure level of 0.9 ppm), an increase in severity of proteinuria in male and female
rats was reported. As discussed in Section 4.6.2., the adversity of the proteinuria findings at this
exposure concentration is uncertain, and the evidence as a whole does not support the finding of
a LOAEL at 5 ppm based on proteinuria data.
In addition to proteinuria, the only other effect reported at 5 ppm in the chronic rat study
was an increase in severity of eosinophilic change in the nasal cavity of the female rats (Nagano
et al., 2007b; JBRC, 1998). A similar effect in males was seen only at 25 ppm and above. This
change, by itself, is not considered to represent an adverse effect. Even in the high-exposure
group that experienced severe renal and hepatic effects, the nasal lesion was graded at only
moderate severity and was not accompanied by any other, more clearly adverse effects in the
nasal cavity. Nonvolatile and partly nonextractable radioactivity was detected in the nasal
mucosa after inhalation of radiolabeled carbon tetrachloride in mice (Bergman, 1983),
suggesting that some inhaled carbon tetrachloride is metabolized in the nasal cavity. However,
there are no other reports of lesions or irritant effects produced by carbon tetrachloride vapor in
either humans or animals.
By inhalation, benign pheochromocytomas were reported in mice in the JBRC inhalation
bioassay (Nagano et al., 2007b; JBRC, 1998). This benign tumor was observed only in mice
(i.e., no increase in pheochromocytomas was observed in rats in either NCI (1977) or Nagano et
al. (2007b)] and thus may represent a strain-specific finding. No data are available, however, to
establish whether this response is species specific. Developmental toxicity (reduced fetal body
weight and crown-rump length) was reported in a single inhalation study (Schwetz et al., 1974)
at a concentration that also produced toxicity in the dam. Because neither benign
pheochromocytomas nor developmental toxicity occurred at a concentration below those
associated with liver toxicity and because level of response was less robust than for endpoints of
liver toxicity, these endpoints were not considered most appropriate as the basis for the RfC.
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The hepatic effects observed in the JBRC chronic inhalation bioassay (Nagano et al.,
2007b; JBRC, 1998) were considered the most appropriate basis for RfC derivation. Fatty
change in the liver of rats was selected as the specific endpoint for dose-response analysis
because this histopathologic lesion is indicative of cellular damage and appears to be a more
sensitive endpoint than other histopathologic changes that were also present in 25-ppm rats in the
JBRC study. Liver serum enzyme activities were also increased in male and female rats and
mice exposed to 25 ppm; however, serum enzyme levels were considered a less consistent and
reliable indicator of liver damage in this study than histopathologic changes. In the mouse, the
overall increase in liver enzyme levels was not monotonic (i.e., levels at 5 ppm were lower than
control levels). In the rat, liver enzyme level increases at 25 ppm were considered modest (i.e.,
increases over control of only 40 to 90%). Further, reliable liver enzyme data were not available
for 125-ppm rats or mice because of the high mortality at this exposure concentration (1 to 3
surviving animals/group at study termination) and because blood biochemistry was not
performed on animals that died before study termination. Therefore, liver enzyme data were
considered a less appropriate endpoint for dose-response analysis.
The occupational study by Tomensen et al. (1995) was also considered as the basis for
RfC derivation, using the estimated LOAEL of 5.5 ppm (35 mg/m3) as the POD. As discussed
more fully in Section 4.1.2.2, exposures for almost two-thirds of the workers were estimated, so
that there is some uncertainly in the value of the LOAEL. Although the data from the Tomensen
et al. (1995) study was not used to derive the RfC, the study was considered in an examination of
RfC values that would be obtained using alternative PODs (see Section 5.2.4).
5.2.2. Methods of Analysis—Including Models
Candidate RfCs for carbon tetrachloride were derived from data on fatty changes to the
liver in male and female rats; incidence data are summarized in Table 5-4
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Table 5-4. Nonneoplastic lesions (fatty change) in F344 rats exposed to carbon
tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week)
Species
Rat
Rat
Sex
Mb
Fc
Lesion type
fatty change
fatty change
Lesion severity"
1+ and 2+
1+, 2+, and 3+
Number of rats with lesions
Dose
0 ppm
4
6
5 ppm
7
7
25 ppm
39
49
125 ppm
49
46
""Severity rating: +, slight; 2+, moderate; 3+ marked.
bNumber of male rats examined: 50/group; number of male rats surviving to study termination: 0 ppm,
22/50; 5 ppm, 29/50; 25 ppm, 19/50; 125 ppm, 3/50.
°Number of female rats examined: 50/group; number of female rats surviving to study termination: 0
ppm, 39/50; 5 ppm, 43/50; 25 ppm, 39/50; 125 ppm, 1/50.
Source: Nagano et al., 2007b; JBRC, 1998.
The general procedure for analysis of the animal bioassay data is depicted in Figure 5-4.
Exposure levels studied in the 2-year rat bioassay (Nagano et al., 2007b; JBRC, 1998) were
converted to estimates of internal doses by application of a PBPK model. BMD modeling
methodology (U.S. EPA, 2000c, 1995) was used to analyze the relationship between the
estimated internal doses and response (i.e., fatty change of the liver). The resulting BMDL
values were converted to estimates of equivalent human exposure concentrations (HECs) by
applying a human PBPK model.
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Animal External Doses or
Exposure Concentrations
T
Animal PBPK
r
Animal Internal Doses
(MCA, MRAMKL)
T
Dose-respons
r
Benchmark Doses (BMDLs)
Expressed in Units of
Internal Dose (MCA,
MRAMKL)
T
Human PBPK
r
Human External Doses or
Exposure Concentrations
Corresponding to BMDLs
T
r
Point of Departure
Figure 5-4. Process for analyzing animal bioassay data for deriving noncancer
toxicity values and cancer unit risks and slope factors using PBPK modeling.
BMDL, lower confidence limit on benchmark dose; BMDS, Benchmark Dose Software; MCA, time-
averaged arterial blood concentration of carbon tetrachloride (umol/L); MRAMKL, time-average rate of
metabolism of carbon tetrachloride (junol/hr/kg liver); PBPK, physiologically-based pharmacokinetics
model
5.2.2.1. PBPK Modeling for Internal Dose Metrics
Estimation of internal doses corresponding to the exposure concentrations studied in the
2-year rat bioassay (Nagano et al., 2007b; JBRC, 1998) was accomplished using a PBPK model
for the rat (Thrall et al., 2000; Benson and Springer, 1999; Paustenbach et al., 1988) (see
Sections 3.5 for description of the model). The review, selection and application of the chosen
PBPK models was informed by an EPA report (U.S. EPA, 2006), which addresses the
application and evaluation of PBPK models. The PBPK model was used to simulate internal
dose metrics corresponding to exposure concentrations studied in the 2-year bioassay: 5, 25, and
125 ppm, 6 hours/day, 5 days/week (Nagano et al., 2007b; JBRC, 1998). Internal dose metrics
were selected that were considered to be most relevant to the toxicity endpoints of interest (e.g.,
liver toxicity), based on consideration of evidence for mode of action of carbon tetrachloride.
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Two dose metrics were selected based on available information on the mechanisms of carbon
tetrachloride liver toxicity: (1) time-averaged arterial blood concentration of carbon tetrachloride
(MCA, jimol/L); and (2) time-averaged rate of metabolism of carbon tetrachloride (MRAMKL,
|imol/hr/kg liver). Liver metabolism rate was selected as the primary dose metric for liver
effects, based on evidence that metabolism of carbon tetrachloride via CYP2E1 to highly
reactive free radical metabolites plays a crucial role in its mode of action in producing liver
toxicity (described in Section 4.5). Uncertainty regarding the accuracy of available PBPK
models to simulate carbon tetrachloride is recognized. These uncertainties include the following:
(1) estimates of the Km and Vmax for the CYP2E1 pathway in the rat and human and potential
dose-dependence of these parameters (e.g., suicide inhibition); (2) relative contributions of extra-
hepatic tissues to carbon tetrachloride metabolism (all of which is assigned to the liver in PBPK
models used in this analysis); and (3) magnitude of direct contribution of carbon tetrachloride
(i.e., parent compound) to liver toxicity. Given the above uncertainties, arterial blood
concentration of carbon tetrachloride was also included in the analysis as a more proximal dose
metric to liver metabolism.
The two dose metrics, MCA and MRAMKL, were simulated in the rat PBPK model as
time-averaged values, with the averaging time being the chronic exposure period (e.g., 2 years).
The time-averaged dose metrics were calculated as follows (Equations 5-1 and 5-2):
Eq. (5-1)
MRAMKL =~~~RAMKL = ^L
t t Eq. (5-2)
where:
MCA = time-averaged arterial blood concentration of carbon tetrachloride (|imol/L)
AUCcA = area under the arterial concentration (CA) - time profile (|imol-hr/L)
MRAMKL = time-averaged rate of metabolism of carbon tetrachloride (|imol/hr/kg liver
weight)
AUCRAMKL = area under the rate of metabolism (RAMKL) - time profile (|imol/kg liver
weight)
AMKL = cumulative amount of carbon tetrachloride metabolized (|imol/kg liver)
t = time (hours)
Internal dose metrics corresponding to the exposure concentrations studied in the 2-year
rat inhalation bioassay (Nagano et al., 2007b; JBRC, 1998) are presented in Table 5-5. Two
values for Vmaxc (maximum rate of hepatic metabolism of carbon tetrachloride) have been
reported for the rat; both estimates are represented in the data presented in Table 5-5. Gargas et
al. (1986) derived a value for Vmaxc of 0.4 mg/hr/kg BW°'70, based on the results of gas uptake
studies in rats. Paustenbach et al. (1988) derived a value of 0.65 mg/hr/kg BW°'70, based on a
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reanalysis of data for a subset of the rats used in the Gargas et al. (1986) study. Increasing Vmaxc
from 0.4 to 0.65 mg/hr/kg BW°'70 resulted in lower values for the MCA dose metric and higher
values for the MRAMKL dose metric (Table 5-5). Comparisons of internal doses predicted for
various exposure concentrations are shown in Figure 5-5. The effect of varying Vmaxc on
MRAMKL becomes more pronounced as exposure concentration increases. This pattern reflects
the increasing influence of Vmax on rate of metabolism at higher exposures concentrations that
result in liver carbon tetrachloride concentrations that exceed the Km.
Table 5-5. Comparisons of internal dose metrics predicted from PBPK rat models
(Paustenbach et al., 1988; Thrall et al., 2000)
Exposure
(ppm)
5
25
125
MCA
(umol/L)
VmaxC=0.40
0.128
0.708
3.892
Vmax=0.65
0.116
0.653
3.775
MRAMKL
(umol/hr/kg liver)
VmaxC=0.40
3.813
12.092
24.320
Vmax=0.65
4.991
17.626
36.266
Values are for 0.452 kg rat.
MCA, time-averaged arterial concentration of carbon tetrachloride; MRAMKL, time-averaged rate of
metabolism of carbon tetrachloride per kg liver, VmaxC, maximum rate of metabolism of carbon
tetrachloride (mg/hr/kg BW070)
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7.0
6.0
5.0
VmaxC=0.40
- - - •VmaxC=0.65
0
50 100 150
AIR(ppm)
200
250
50
45
40
35
* 30
< 25
I 20
15
10
5
0
•VmaxC=0.40
•VmaxC=0.65
0
50 100 150
AIR(ppm)
200
250
Figure 5-5. Internal dose metrics predicted by the PBPK rat model (Paustenbach et
al., 1988; Thrall et al., 2000).
Dose metrics shown are time-averaged arterial concentration of carbon tetrachloride (MCA, [j,mol/L, upper
panel), and time-averaged rate of metabolism of carbon tetrachloride (MRAMKL, [j,mol/hr/kg liver, lower
panel). The dose metrics are plotted against exposure concentration (6 hours/day, 5 days/week, 2 years) for
a 0.452 kg rat.
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5.2.2.2. Benchmark Dose Modeling
BMD modeling methodology (U.S. EPA, 2000c, 1995) was used to analyze data on
estimated internal doses (i.e., MCA, MRAMKL) and incidence data (e.g., fatty changes of the
liver) from the 2-year rat bioassay (Nagano et al., 2007b; JBRC, 1998). All of the models for
dichotomous data in U.S. EPA's HMDS (version 1.4.1) (U.S. EPA, 2007) were fit to the
incidence data for rats.
Internal doses associated with a benchmark response (BMR) of 10% extra risk were
calculated. A BMR of 10% extra risk of fatty changes in the liver was selected because the POD
associated with this BMR fell near the low end of the range of experimental data points (see
plots in Appendix D). As noted U.S. EPA (2000), "[t]he major aim of benchmark dose modeling
is to model the dose-response data for an adverse effect in the observable range (i.e., across the
range of doses for which toxicity studies have reasonable power to detect effects) and then select
a 'benchmark dose' at the low end of the observable range to use as a 'point of departure'."
In the male rat, the best fit of the data was provided by the log-logistic model using MCA
as the dose metric and the logistic model using MRAMKL as the dose metric (based on %2/>>0.1
and lowest AIC value). For female rats, no models provided an adequate fit to the data when all
dose groups were included, as assessed by the %2 goodness-of-fit test (i.e., application of the
models in BMDS yielded j^ p values in all cases <0.1). After dropping the highest dose, the
multistage model provided the best fit of the female incidence data (based on %2 p>0.\ and
lowest AIC value) using either dose metric. Summaries of the resulting BMDio and BMDLio
values for male and female rats are shown in Tables 5-6 and 5-7 (columns 3 and 4). Details of
the BMD modeling are provided in Appendix D.
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Table 5-6. HEC values corresponding to BMDL values for incidence data for fatty
changes of the liver in male F344 rats
BMR
(l)b
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
Metric
(2)
MCA
MRAMKL
BMD modeling"
VMAXCR=0.4
(3)
BMD10:0.14
BMDL10: 0.079
BMD10: 3.26
BMDL10: 2.59
VMAXCR=0.65
(4)
BMD10:0.12
BMDL10: 0.071
BMD10: 4.60
BMDL10: 3.65
VMAXCH
(5)
0.40
0.65
1.49
1.70
0.40
0.65
1.49
1.70
HEC
VMAXCR=0.4
(6)
5.396
5.712
6.338
6.436
23.793
17.160
11.826
11.343
VMAXCR=0.65
(7)
4.830
5.113
5.671
5.760
35.243
24.773
16.794
16.093
Rats were exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week). Doses
modeled correspond to exposure concentrations: 0, 5, 25, 125 ppm.
BMR, benchmark response; HEC, human equivalent concentration, mg/m3; MCA, time-averaged arterial
blood concentration, umol/L; MRAMKL, time-averaged rate of metabolism per kg liver, umol/hr/kg liver;
VMAXC, maximum rate of metabolism in humans (H) or rat (R), mg/hr/kg BW°70
aMCA, log-logistic model provided the best fit; MRAMKL, logistic model provided the best fit.
b Number in parentheses indicates the column number.
Table 5-7. HEC values corresponding to BMDL values for incidence data for fatty
changes of the liver in female F344 rats (high dose dropped)
BMR
(l)b
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
Metric
(2)
MCA
MRAMKL
BMD modeling"
VMAXCR=0.4
(3)
BMD10:0.12
BMDL10: 0.085
BMD10: 3.77
BMDL10: 2.82
VMAXCR=0.65
(4)
BMD10:0.11
BMDL10: 0.078
BMD10: 5.42
BMDL10: 3.75
VMAXCH
(5)
0.40
0.65
1.49
1.70
0.40
0.65
1.49
1.70
HEC
VMAXCR=0.4
(6)
5.815
6.156
6.831
6.937
26.259
18.838
12.935
12.405
VMAXCR=0.65
(7)
5.298
5.608
6.222
6.319
36.337
25.478
17.246
16.524
Rats were exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week). Doses modeled
correspond to exposure concentrations: 0, 5, 25 ppm (125 ppm dose dropped).
BMR, benchmark response; HEC, human equivalent concentration, mg/m3; MCA, time-averaged arterial
blood concentration, umol/L; MRAMKL, time-averaged rate of metabolism per kg liver, umol/hr/kg liver;
VMAXC, maximum rate of metabolism in humans (H) or rat (R), mg/hr/kg BW°70
a MCA, multistage (2); MRAMKL, multistage (3)
b Number in parentheses indicates the column number.
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5.2.2.3. PBPK Modeling of Human Equivalent Exposure Concentrations
Interspecies extrapolation (i.e., rat-to-human) of carbon tetrachloride inhalation
dosimetry was accomplished using a human PBPK model described in Paustenbach et al. (1988),
Thrall et al. (2000), and Benson and Springer (1999). The human PBPK model was used to
estimate continuous human equivalent concentrations (HECs, in mg/m3) that would result in
values for the internal dose metrics, MCA or MRAMKL, equal to the BMDLio values for fatty
changes of the liver.
The approach used to derive the HECs for each dose metric was as follows:
(1) The human PBPK model was used to calculate internal doses corresponding to a series of
exposure concentrations (EC, continuous exposure, mg/m3). For the dose metric MCA, the
human PBPK model was run at intervals over the range from 0.1 to 100 ppm (0.63 to
629 mg/m3); for MRAMKL, the human PBPK model was run at intervals from 1 to 300 ppm
(6.3 to 1887 mg/m3).
(2) For each internal dose, conversion factors were calculated as the following corresponding
ratios:
• EC/MCA (to relate a continuous chronic human inhalation exposure in mg/m3 [EC] to
an internal dose using MCA as the dose metric);
• EC/MRAMKL (to relate a continuous chronic human inhalation exposure in mg/m3
[EC] to an internal dose using MRAMKL as the dose metric); and
(3) Conversion factors were calculated for each of four assumed values of Vmaxc in the human
PBPK model: 0.40, 0.65, 1.49, or 1.70 mg/hr/kg BW0'70. These conversion factors are provided
in Appendix C. Trend equations were also developed to permit the calculation of EC for any
value of MCA or MRAMKL (see Appendix C).
Estimates of the dose metrics, MCA and MRAMKL, were sensitive to the value assigned
to the VmaxC parameter (see Figure 5-5). Several values for VmaxC in animals and humans have
been reported (Thrall et al., 2000, Benson and Springer, 1999; Paustenbach et al., 1988; Gargas
et al., 1986); therefore, evaluation of uncertainty in this parameter was introduced into the
analysis by assuming various reported values for Vmaxc in the estimation of HECs. Thrall et al.
(2000) and Benson and Springer (1999) derived a value of 1.49 mg/hr/kg BW°'70 for humans,
based on an analysis of data on in vivo (gas uptake) studies in rodents and in vitro studies of
metabolism of carbon tetrachloride in rodent and human liver samples. Thrall et al. (2000) also
derived a value of 1.7 mg/hr/kg BW0'70 for hamsters, based on the results of closed chamber gas
uptake studies. The value of 1.49 mg/hr/kg BW0'70 for humans (Thrall et al., 2000; Benson and
Springer, 1999), the value of 1.70 mg/hr/kg BW0'70 for the hamster (Thrall et al., 2000), and the
two values estimated for the rat (0.4, 0.65 mg/hr/kg BW0'70; Paustenbach et al., 1988; Gargas et
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al., 1986) were used in the estimation of HECs. Estimated values for HECs corresponding to
BMDLio values for fatty changes of the liver as reported in the 2-year rat inhalation bioassay
(Nagano et al., 2007b; JBRC, 1998) for alternative values of VmaxC in the rat and human are
presented in Tables 5-6 and 5-7 (columns 6 and 7).
A human Vmaxc estimated from in vitro human data can reasonably be presumed to be
more relevant than a human Vmaxc based entirely on rodent data. In addition, because the mode
of action for carbon tetrachloride-induced hepatotoxicity involves metabolism to reactive
metabolites in the liver, HECs based on the MRAMKL dose metric is the most proximate to the
critical effect. Therefore, the human Vmaxc estimated from in vitro human data (1.49 mg/hr/kg
BW°70) and the dose metric MRAMKL are considered to yield the most appropriate estimate of
the HEC. No information is available to establish a rat Vmaxc of either 0.4 or 0.65 mg/hr/kg
BW°70 as the more scientifically defensible value for this parameter. Therefore, HECs derived
using these two rat Vmaxc values were averaged to derive the POD for the carbon tetrachloride
RfC. Accordingly, the POD based on male rat data was calculated as (11.826 + 16.794) - 2 =
14.3 mg/m3. In the female rat, the HEC was similarly calculated as (12.935 + 17.246) -2 = 15.1
mg/m3. The HEC based on data for the male rat (14.3 mg/m3) is the lower of the two values, and
is selected as the POD for RfC derivation.
5.2.3. RfC Derivation—Including Application of Uncertainty Factors
An RfC of 0.1 mg/m3 for carbon tetrachloride is derived by applying a composite UF of
100 to the HEC of 14.3 mg/m3, as follows:
RfC = HEC/UF (5-3)
14.3 mg/m3/100
0.143 mg/m3 or 0.1 mg/m3
The composite UF of 100 includes a factor of 10 to protect susceptible individuals, a
factor of 3 (10°5) to adjust for pharmacodynamic differences in the extrapolation from rats to
humans, and a factor of 3 (1005) to account for an incomplete database lacking an adequate
multigeneration study of reproductive function.
• A default 10-fold UF for intraspecies differences was selected to account for variability
in susceptibility among members of the human population in the absence of quantitative
information on the variability of human response to carbon tetrachloride. Factors that
could contribute to a range of human response to carbon tetrachloride were discussed in
Section 4.8. Variations in CYP450 levels because of age-related differences or other
factors (e.g., exposure to other chemicals that induce or inhibit microsomal enzymes)
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could alter susceptibility to carbon tetrachloride toxicity. Individual variability in
nutritional status, alcohol consumption, or the presence of underlying disease could also
alter metabolism of carbon tetrachloride or antioxidant protection systems. To account
for these uncertainties, a factor of 10 was included for individual variability.
A UF of 3 (10°5) was selected for interspecies extrapolation to account for potential
pharmacodynamic differences between rats and humans. As pharmacokinetic and
pharmacodynamic components are assumed to contribute equally to the uncertainty in
interspecies extrapolation and the product of the two components is assumed by default
to be 10, a numeric value of 10°5 (3.2, expressed as the numeral 3 after rounding) is
assigned to each component. Cellular antioxidant systems function to quench the lipid
peroxidation reaction and prevent damage to cellular membranes. In the absence of data
to quantify specific interspecies differences for cellular protective mechanisms, a UF of 3
is included to account for species differences in pharmacodynamics. A pharmacokinetic
model was used to adjust for pharmacokinetic differences across species; therefore, an
additional UF was not included for pharmacokinetic differences between species.
An UF to account for extrapolation from a LOAEL to a NOAEL was not used because
the current approach is to address this extrapolation as one of the considerations in
selecting a BMR for BMD modeling. In this case, a BMR of a 10% change in fatty
changes of the liver was selected under an assumption that it represents a minimal
biologically significant change.
An UF to extrapolate from a subchronic to a chronic exposure duration was not necessary
because the RfC was derived from a study using a chronic exposure protocol.
A database UF of 3 (10°5) was selected. The inhalation database for this chemical
includes extensive testing for subchronic toxicity in animals, 2-year chronic inhalation
bioassays in both rats and mice, one study of immunotoxic potential, and human
epidemiology data. Testing for developmental toxicity was limited to one inhalation
study in the rat that found effects only at high, maternally toxic exposure concentrations.
This study did not use an exposure concentration low enough to identify a NOAEL for
either maternal or fetal toxicity. Nevertheless, the developmental effects at the LOAEL
were modest, and were limited to decreased fetal body weight (7%) and decreased crown-
rump length (3.5%). The LOAEL for developmental effects (in the presence of maternal
toxicity) in this study (334 ppm) was 66-fold higher than the NOAEL from the principal
study (5 ppm). Developmental toxicity has been tested more extensively by the oral
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route, although all adequate studies were conducted in the same species (rat); the oral
NOAEL for developmental toxicity exceeded both the oral NOAEL and LOAEL for liver
toxicity. As noted in Section 4.8.1. (Possible Childhood Susceptibility), microsomal
enzymes that are responsible for metabolizing carbon tetrachloride, particularly CYP2E1,
are lower in the developing organism than the adult, and in humans do not achieve adult
levels until sometime between one and 10 years. Thus, life stage information on
microsomal enzyme activity suggests that the developing organism would be no more
susceptible to free radical-induced liver injury from carbon tetrachloride than adults. On
balance, the available information suggests that further developmental toxicity testing
would not likely result in a POD smaller than that based on liver toxicity. The database
lacks an adequate multigeneration study of reproductive function by any route of
exposure.
5.2.4. RfC Comparison Information
PODs and inhalation RfCs based on selected studies included in Table 4-14 are arrayed in
Figures 5-6 to 5-8, and provide perspective on the RfC supported by Nagano et al. (2007b;
JBRC, 1998). These figures should be interpreted with caution because the PODs across studies
are not necessarily comparable, nor is the confidence in the data sets from which the PODs were
derived the same. PODs in these figures may be based on a NOAEL, LOAEL, or BMDL (in the
case of the principal study), and the nature, severity, and incidence of effects occurring at a
LOAEL are likely to vary. In addition, PBPK modeling for animal to human extrapolation was
applied to data from the principal study, whereas the default approach (i.e., application of an UF
of 10) was used for other animal data sets. To some extent, the confidence associated with the
resulting RfC is reflected in the magnitude of the total UF applied to the POD (i.e., the size of the
bar); however, the text of Sections 5.2.1 and 5.2.2 should be consulted for a more complete
understanding of the issues associated with each data set and the rationale for the selection of the
critical effect and principal study used to derive the RfC.
As discussed in Section 4.6.2, the liver and kidney are the predominant targets of carbon
tetrachloride toxicity in subchronic and chronic inhalation studies in laboratory animals (Nagano
et al., 2007a,b; Benson and Springer, 1999; JBRC, 1998; Prendergast et al., 1967; Adams et al.,
1952; Smyth et al., 1936) and in humans based on case reports and studies in exposed workers.
Benign pheochromocytomas from the adrenal gland medulla, that could represent a potential
noncancer health hazard, were observed by inhalation only in mice in the JBRC chronic bioassay
(Nagano et al., 2007b; JBRC, 1998). A single study of developmental toxicity (Schwetz et al.,
1974) found significant reductions in fetal body weight and crown-rump length in rats at a
carbon tetrachloride concentration that also produced hepatotoxicity and reduced growth in the
dams. This set of literature was evaluated in selecting the most appropriate study and endpoint
to use as the basis for the RfC, with particular consideration given to the overall strength of the
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evidence for a given measure of toxicity, consistency of the finding across studies, relevance to
humans, sensitivity of the endpoint, and rigor of a given study.
Figure 5-6 provides a graphical display of dose-response information from one
occupational cross sectional study and five experimental animal data sets that reported liver
toxicity; all animal studies identified a NOAEL for liver toxicity of approximately 6 mg/m3 or
0.9 ppm (adjusted to continuous exposure) and the study of exposed workers (Tomensen et al.,
1995) identified a LOAEL of approximately to 12.5 mg/m3 or 2 ppm (also adjusted to continuous
exposure).8 As discussed in Section 5.2.1, the JBRC study in the rat (Nagano et al., 2007b;
JBRC, 1998), which identified a NOAEL for liver toxicity of 5.7 mg/m3 or 0.9 ppm (adjusted to
continuous exposure), was determined to be a sensitive and well-conducted study of carbon
tetrachloride toxicity, and was selected as the basis for the RfC. Dose-response analysis of the
data from this study, which included BMD and PBPK modeling, yielded a POD of 14.3 mg/m3.
Possible RfCs that might be derived from other studies demonstrating liver toxicity are also
presented in Figure 5-6. Although the RfC based on the JBRC rat data is not the lowest among
candidate studies, it is considered to be the most scientifically rigorous and associated with a
lower degree of uncertainty than other experimental animal studies. The POD is based on a
study of chronic toxicity data (rather than the subchronic exposures used in Benson and Springer,
1999, and Adams et al., 1952), the application of BMD methods, which has an inherent
advantage over the use of a NOAEL or LOAEL by making greater use of all the data from the
study, and the use of PBPK modeling for interspecies extrapolation. As shown in Figure 5-6, the
use of PBPK modeling also resulted in the application of a smaller composite uncertainty factor
to the POD, i.e., smaller degree of uncertainty than with other data sets to which the default
uncertainty factor of 10 for interspecies extrapolation was applied. The RfC derived using data
from the JBRC rat study is consistent with the RfC derived from Tomensen et al. (1995).
Tomensen et al. reported a statistically significant increase in two of four serum enzymes
indicative of liver function in workers exposed to approximately 35 mg/m3 (5.5 ppm) carbon
tetrachloride (adjusted to continuous exposure: 12.5 mg/m3). Using 12.5 mg/m3 as the POD and
applying a composite UF of 300 (10 for variation in sensitivity in the human population, 10 for
extrapolation from a LOAEL to a NOAEL, and 3 for database deficiencies), the RfC is estimate
to be 0.04 mg/m3. Because the Tomensen et al. (1995) noted that "there was no evidence of
effects of clear clinical significance on the liver function of workers exposed to carbon
tetrachloride at the levels indicated," it could be argued that a UF for LOAEL to NOAEL
extrapolation of 3 (rather than a full UF of 10) might be appropriate. In this case, the RfC
estimated from Tomensen et al. (1995) serum enzyme data would be 0.1 mg/m3. Thus, the RfC
estimated from Tomensen et al. (1995) of 0.04 to 0.1 mg/m3 is consistent with the RfC of
8 The workplace exposure concentration of 35 mg/m3 was adjusted to continous exposure by multiplying by
(10 m3/day -^ 20 m3/day) x (5 days/week + 7 days/week), where 10 m3/day is an estimate of an 8-hour time-weighted
average occupational respiratory rate and 20 nrVday an estimate of an average daily respiratory rate.
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0.1 mg/m3 derived from the JBRC rat bioassay (Nagano et al., 2007b; JBRC, 1998), and supports
the RfC for liver effects derived from animal data.
The most sensitive study of kidney toxicity was the JBRC bioassay in the rat and mouse
(Figure 5-7) (Nagano et al., 2007b; JBRC, 1998). As discussed in Section 5.2.1., kidney effects
occurred at a concentration similar to liver effects, but at lower incidence.
Figure 5-8 displays PODs for all major targets of carbon tetrachloride toxicity by the
inhalation route, including liver, kidney, adrenal gland, and developmental toxicity. For the
reasons discussed in Section 5.2.1., liver effects in the rat observed in the JBRC study are
considered the most appropriate basis for the carbon tetrachloride RfC. The POD based on liver
effects is similar to the PODs associated with kidney effects and effects on the adrenal gland
(benign pheochromocytomas); however, a smaller composite UF was applied to the POD for
liver effects because PBPK modeling was used for interspecies extrapolation. The greatest
degree of uncertainty is associated with the RfC for developmental toxicity. While this relatively
large UF drives down the value of the RfC for developmental toxicity, the RfC based on liver
effects should be adequately protective.
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Figure 5-6. Liver toxicity: inhalation
100
10
O)
o
§
Ǥ
0.1
0.01
0.001
A
/ • \
ftf/ttfj
/%%
i
H|
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I*
.
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(
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\
\
\
I
* /
/
^^1 ^^H^^ The RfC in the
H^H ^ dashed circle was
^ ^ selected as the
final RfC for carbon
tetrachloride.
• POD
[]]J| Animal-to-human
Q Human variation
^LOAELtoNOAEL
Q Subchr to Chronic
• Database deficiencies
ORfC
Note: The RfC based on
Nagano et al. (2007b)
used BMD methods and
PBPK modeling, and
therefore is not directly
comparable to the other
RfCs for liver endpoints.
Tomensen et al, 1 995; Benson & Springer JBRC 1 998; 2-y r Adams et al, 1 952; Adams et al, 1 952; Nagano et al., 2007b;
occupational epid 1999; 12-wk mouse mouse study; liver 6-month rat study; 6-month guinea pig 2-yr rat study; liver
study; liver enzymes; study; liver enzymes, weight, enzyme liver weight, fatty study; liver weight, enzymes, histopath,
LOAEL(1) histopath; NOAEL activity, histopath; liver; NOAEL fatty liver; NOAEL including fatty liver;
NOAEL BMDL10[HEC]
(1) Magnitude of effect at the LOAEL: liver enzyme levels (f <23%)
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Figure 5-7. Kidney toxicity: inhalation
10.00
1.00
O)
o
I
<§
0.10
0.01
• POD
fTflAnimal-to-human
~| Human variation
^LOAELto NOAEL
~~] Subchr to Chronic
^ Database deficiencies
oRfC
Nagano etal., 2007b; 2-yr rat study; serum chemistry,
histopathology; NOAEL
Nagano etal., 2007b; 2-yr mouse study; serum chemisty,
histopathology; NOAEL
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Figure 5-8. Organ-specific inhalation RfCs
1000
100
• POD
ITU Animal-to-human
n Human variation
0LOAELto NOAEL
CD Subchr to Chronic
| Database deficiencies
ORfC
10
O)
o
0)
u
Ǥ
0.1
0.01
Kidney
Adrenal gland (benign
pheochromocytoma)
Liver
TlieRfCinthe
dashed circle was
selected as the final
Rf C for carbon
tetrachloride.
Developmental
POD based on:
Kidney: NOAEL for serum
chemistry and histopathology
changes in the rat and mouse;
2-year inhalation study
(Nagano et al., 2007b)
Benign pheochromocvtoma:
NOAEL in the mouse; 2-year
inhalation study (JBRC, 1998)
Liver: BMDL[HEq for fatty liver
in the rat; 2-year inhalation
study (JBRC, 1998)
Developmental: LOAEL for
decreased fetal body weight
and crown-rump length;
exposure on GD 6-15 (Schwetz
etal., 1974)
Note: The RfC based on liver
toxicity used BMD methods
and PBPK modeling, and
therefore is not directly
comparable to the other organ-
specific RfCs.
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5.2.5. Previous RfC Assessment
An inhalation assessment for carbon tetrachloride was not previously available on IRIS.
5.3. UNCERTAINTIES IN THE ORAL REFERENCE DOSE AND INHALATION
REFERENCE CONCENTRATION
Risk assessments need to describe associated uncertainty. The following discussion
identifies uncertainties associated with the RfD and RfC for carbon tetrachloride. As presented
earlier in this section (5.1.2 and 5.1.3 for the RfD; 5.2.2 and 5.2.3 for the RfC), the uncertainty
factor approach (U.S. EPA, 2002, 1994b) was used to derive the RfD and RfC for carbon
tetrachloride. Using this approach, the POD was divided by a set of factors to account for
uncertainties associated with a number of steps in the analysis, including extrapolation from
responses observed in animal bioassays to humans and from data from subchronic exposure to
chronic exposure, a diverse population of varying susceptibilities, and to account for database
deficiencies. Because information specific to carbon tetrachloride was unavailable to fully
inform many of these extrapolations, default factors were generally applied.
A broad range of animal toxicity data and more limited range of human study data are
available to assess carbon tetrachloride hazard (see Section 4). Human studies include case
reports of acute human exposure (both oral and inhalation) and occupational epidemiology
studies. The animal toxicology literature includes subchronic and chronic animal studies by the
oral and inhalation routes, developmental toxicity studies by the oral and inhalation routes,
studies of immunotoxic potential, extensive literature on genotoxicity, and numerous mechanistic
toxicity studies. In addition, carbon tetrachloride has been used in hundreds of studies as a
classic inducer of liver toxicity. Nevertheless, gaps in the carbon tetrachloride database have
been identified; uncertainties associated with these data deficiencies are discussed more fully
below.
Selection of the critical effect for reference value determination. Liver toxicity was
selected as the critical effect for both the RfD and RfC (specifically, elevated liver enzymes
[Bruckner et al., 1986] in the case of the RfD and fatty change of the liver [Nagano et al., 2007b;
JBRC, 1998] in the case of the RfC. The liver has been established as a sensitive target of
toxicity across animal species and routes of exposure. Case reports of human poisonings identify
the liver as a target organ of acute carbon tetrachloride exposure, and an occupational
epidemiology study of workers exposed to carbon tetrachloride (Tomenson et al., 1995) provides
evidence of impaired liver function in humans following prolonged exposure. Thus, there is
little uncertainty related to the relevance of the critical effect to human health assessment.
Kidney toxicity associated with carbon tetrachloride inhalation exposure has been seen
less consistently in experimental animal studies. Nagano et al. (2007b; also reported as JBRC,
1998) reported an increase in the severity of proteinuria in rats at the lowest concentration tested
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in a two-year bioassay. This kidney finding occurred at an exposure level lower than the
concentration associated with fatty changes of the liver; however, given the uncertainties in this
endpoint discussed in Section 4.6.2, proteinuria was not used as the critical effect for the RfC.
Use of proteinuria data as the basis for the RfC would have yielded a lower POD than liver data.
Dose-response modeling. BMD modeling was used to estimate the POD for both the
RfD and RfC. BMD modeling has advantages over a POD based on a NOAEL or LOAEL
because, in part, the latter are a reflection of the particular exposure concentration or dose at
which a study was conducted. A NOAEL or LOAEL lacks characterization of the dose-response
curve and for this reason is less informative than a POD obtained from BMD modeling. The
selected models—the power model in the case of the RfD and the logistic model in the case of
the RfC—provided the best mathematical fits to the experimental data sets (as determined by the
lowest AIC), but do not necessarily have greater biological support over the various models
included in BMDS. Other models in BMDS yield estimates of the POD both higher and lower
than the PODs used to derive the RfD and RfC in the current assessment.
Animal to human extrapolation. Extrapolating dose-response data from animals to
humans is another source of uncertainty. The effect and the magnitude of the effect at the POD
in rodents are extrapolated to human response. Uncertainty in interspecies extrapolation can be
separated into two general areas—toxicokinetic and toxicodynamic. A UF of 3 was used to
account for toxicodynamic difference between animals and humans. A PBPK model was
available for the inhalation pathway and was used in deriving the RfC to address the
toxicokinetic portion of interspecies extrapolation. Availability of an inhalation PBPK model
generally reduces the toxicokinetic component of uncertainty associated with animal to human
extrapolation by moving away from default assumptions about kinetic differences between
animals and humans. Any PBPK model, however, has its own associated uncertainties. In the
carbon tetrachloride RfC analysis, uncertainty was examined by using two dose metrics and
alternative values of Vmaxc. MRAMKL was considered the more scientifically appropriate dose
metric for liver toxicity; MCA was included given uncertainties in modeling carbon tetrachloride
metabolism and uncertainties regarding the magnitude of direct contribution of carbon
tetrachloride (as parent compound) to liver toxicity. MRAMKL provided HEC (and thus RfC)
values that were 2- to 7-fold higher than those derived using MCA (depending on the value of
VmaxC USed).
Estimates of the dose metrics, MCA and MRAMKL, were sensitive to the value assigned
to the Vmaxc parameter (see Figure 5-5 and Tables 5-6 and 5-7). Several values for Vmaxc in
animals and humans have been reported (Thrall et al., 2000, Benson and Springer, 1999;
Paustenbach et al., 1988; Gargas et al., 1986); therefore, evaluation of uncertainty in this
parameter was introduced into the analysis by assuming various reported values for Vmaxc in the
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estimation of HECs. Thrall et al. (2000) and Benson and Springer (1999) derived a value of
1.49 mg/hr/kg BW°'70 for humans, based on an analysis of data on in vivo (gas uptake) studies in
rodents and in vitro studies of metabolism of carbon tetrachloride in rodent and human liver
samples. Thrall et al. (2000) also derived a value of 1.7 mg/hr/kg BW°'70 for hamsters, based on
the results of closed chamber gas uptake studies. The value of 1.49 mg/hr/kg BW°'70 for humans
(Thrall et al., 2000; Benson and Springer, 1999), the value of 1.70 mg/hr/kg BW0'70 for the
hamster (Thrall et al., 2000), and the two values estimated for the rat (0.4, 0.65 mg/hr/kg BW°'70;
Paustenbach et al., 1988; Gargas et al., 1986) were used in the estimation of HECs. In general,
increasing Vmaxc from 0.4 to 1.7 mg/hr/kg BW0'70 resulted in higher values for HECs based on
the MCA dose metric and lower values for HECs based on the MRAMKL dose metric. This
pattern reflects the effect of higher rates of metabolism and blood clearance at any given
exposure concentration that result from higher values for Vmax. Higher rates of metabolism
decrease the corresponding exposure concentration required to achieve a given value of
MRAMKL and increase the corresponding exposure concentration required to achieve a given
value of MCA. The effect of increasing Vmaxc was more pronounced on HECs based on the
MRAMKL dose metric. This pattern reflects the increasing influence of Vmax on metabolism
rate at higher exposure concentrations that result in liver carbon tetrachloride concentrations that
exceed the Km. The VmaxC upon which the RfC was based, i.e., a VmaxC based on in vitro human
data, was considered most scientifically defensible; other values of VmaxC yielded HEC (and thus
RfC) values that ranged from 4% smaller to 2-fold higher.
A sensitivity analysis was also performed for the human PBPK model (see Section C.4 in
Appendix C). Other sensitive chemical-specific parameters included the blood:air partition
coefficient and Michaelis-Menten coefficient for metabolism (Kmx) using MCA as the internal
dose metric, and liverblood, slowly-perfused:blood, and readily-perfused:blood partition
coefficients for MRAMKL as the dose metric.
An adequate PBPK model for the oral pathway was not available and thus PBPK
modeling could not be used for interspecies extrapolation in developing the RfD. In the absence
of information to quantitatively assess oral toxicokinetic or toxicodynamic differences between
animals and humans, a 10-fold UF was used to account for uncertainty in extrapolating from
laboratory animals to humans in the derivation of the RfD associated with this 10-fold UF.
The magnitude of possible over- or underestimation of interspecies differences
introduced by the use of default factors cannot be determined.
Intrahuman variability. Heterogeneity among humans is another source of uncertainty.
Carbon tetrachloride-specific data on human variation is not available. In addition, there is an
absence of quantitative information on variation in hepatic levels of CYP2E1 or other
metabolizing enzymes that can influence carbon tetrachloride toxicity, as well as an absence of
quantitative information on levels of metabolizing enzymes in other tissues (e.g., brain) during
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various stages of development. Accordingly, a default UF of 10 was used to account for
uncertainty associated with human variation in the derivation of the RfD and RfC. Human
variation may be larger or smaller; however, carbon tetrachloride-specific data to examine the
potential magnitude of over- or underestimation is unavailable.
Subchronic to chronic exposure extrapolation. Because the available chronic oral
toxicity studies for carbon tetrachloride were not considered adequate for derivation of the oral
RfD, subchronic toxicity studies were used, and a UF of 3 was applied to extrapolate those data
obtained from a study of subchronic exposure to chronic exposure. This UF is based on the
assumption that an effect seen at a shorter duration will also be seen after a lifetime of exposure,
but at a lower exposure level or with greater severity. In the absence of information to inform
this extrapolation, a subchronic to chronic UF of 10 is typically applied. Inhalation data for
carbon tetrachloride and other chemical-specific information (see Section 5.1.3) indicate that a
full default UF of 10 would overestimate the difference in response following subchronic and
chronic oral exposures. The availability of carbon tetrachloride-specific information reduces the
uncertainty in extrapolating from subchronic to chronic exposure data.
Data gaps. To the extent that the database for carbon tetrachloride is incomplete, it is
possible that certain endpoints of toxicity or certain sensitive lifestages have not been evaluated
that could result in PODs lower than those for which study data are available. The carbon
tetrachloride database lacks an adequate multigeneration study of reproductive toxicity by any
route of exposure. The absence of these types of studies introduces uncertainty in the RfD and
RfC. The magnitude of this uncertainty cannot be quantified.
Vehicle effects. The vehicle used in oral gavage studies to administer carbon
tetrachloride could be a potential confounding factor in toxicity assays. Investigators have
variably reported that (compared to an aqueous vehicle) corn oil either enhanced carbon
tetrachloride toxicity (Narotsky et al., 1997; Condie et al., 1986), did not significantly affect
toxicity (Kaporec et al., 1995), or reduced toxicity (Kim et al., 1990b), or that influences of
vehicle could be dose-dependent (Raymond and Plaa, 1997; Narotsky et al., 1997). The
polyethoxylated vegetable oil Emulphor has been shown not to influence carbon tetrachloride
acute hepatotoxicity, absorption, or distribution (Sanzgiri and Bruckner, 1997). Thus, it is
possible that the vehicle used in oral gavage studies to administer carbon tetrachloride could
influence the observed toxicity; however, given the variable effects of corn oil (versus an
aqueous vehicle), the magnitude of the confounding and the nature of the interaction of corn oil
remain uncertain.
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5.4. CANCER ASSESSMENT
Several epidemiological studies (including several case-control studies and one
retrospective cohort study) have investigated potential associations between cancers of various
types and exposure to carbon tetrachloride. In all the available studies, subjects experienced
multiple chemical exposures and exposures were estimated qualitatively based on historical
information. These studies, therefore, can provide only suggestive evidence for an association
between carbon tetrachloride exposure and cancer, and are not useful for dose-response analysis.
Studies in experimental animals suggest that the primary cancer risk associated with
exposure to carbon tetrachloride is development of liver cancer. Carbon tetrachloride produced
hepatocellular carcinomas in rats, mice, and hamsters in oral studies and in rats and mice by
inhalation exposure. In addition to liver tumors, adrenal pheochromocytomas were observed in
male and female mice by oral and inhalation exposure (Nagano et al., 2007b; JBRC, 1998;
Weisburger, 1977). No increase in pheochromocytomas was observed in rats.
Examination of rodent liver tumors reveals a general correspondence between
hepatocellular cytotoxicity and regenerative hyperplasia and the induction of liver tumors,
although at lower exposure levels this correspondence is somewhat less consistent. A weight of
evidence analysis of the genotoxicity literature suggests that carbon tetrachloride is more likely
an indirect than direct mutagenic agent; however, the nature of the genotoxicity database poses
distinct challenges to the evaluation of carbon tetrachloride genotoxicity. Positive genotoxicity
findings have generally been observed at exposures that induce cytotoxicity and regenerative cell
proliferation. Due to the difficulties in detecting genotoxic effects following treatment with
carbon tetrachloride, many studies were conducted at relatively high doses that lack information
regarding dose-response. This has resulted in a database that does not characterize the role of
genotoxicity at low doses of carbon tetrachloride.
The extensive mechanistic literature related to carbon tetrachloride-induced liver tumors
informs the mode of action for liver tumors. As noted in Section 4.7.3 and in the following
section, the empirical evidence for carbon tetrachloride, particularly data from relatively high-
exposure studies, provides support for the hypothesis that liver carcinogenicity is presumed to
occur at exposures that also induce hepatocellular toxicity and a sustained regenerative and
proliferative response, and that exposures that do not cause hepatotoxicity are not expected to
result in liver cancer. This mode of action for carbon tetrachloride liver carcinogenicity is
consistent with a nonlinear approach to low-dose extrapolation. A nonlinear low-dose
extrapolation approach is presented in Section 5.4.1.
Although much of the empirical data is consistent with hepatocellular toxicity and a
sustained regenerative and proliferative response as key events in the mode of action for rodent
liver tumors, liver findings from the JBRC bioassay (Nagano et al., 2007b; JBRC, 1998) suggest
that mouse hepatocarcinogenicity cannot simply be explained in terms of this mode of action.
An increased incidence of hepatocellular adenomas occurred in the low-dose (5-ppm or 0.9-ppm
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adjusted) female mouse in the absence of nonneoplastic liver toxicity, raising the possibility of
another mode of action operating in addition to or in conjunction with the cytotoxic-proliferative
mode of action. As discussed in Section 4.7, considerable evidence points to the involvement of
highly reactive metabolites in the induction of liver toxicity and carcinogenicity by carbon
tetrachloride. In addition, subsequent chemical reactions of carbon tetrachloride metabolites
with cellular constituents lead to formation of reactive oxygen species that also can damage
DNA and other macromolecules. Although the extensive genotoxicity database for carbon
tetrachloride suggests that carbon tetrachloride is not likely a direct acting mutagen, the database
is complex and raises various issues (see Table 4-12) that make it difficult to reach a firm
judgement about the potential genotoxicity of carbon tetrachloride at exposures below which
there is overt toxicity. In light of the mouse liver findings from the JBRC bioassay, the
fundamental reactivity of both direct and indirect products of carbon tetrachloride metabolism,
and limited information about carbon tetrachloride's biological activity at low exposures, an
argument can be made for application of a nonthreshold approach to carbon tetrachloride
carcinogenicity. Section 5.4.2 presents low-dose linear extrapolation approaches to carbon
tetrachloride carcinogenicity.
5.4.1. Nonlinear Extrapolation Approach
As noted above, much of the empirical evidence for carbon tetrachloride, particularly
from studies using relatively high exposure levels, supports a mode of action for liver tumors that
includes the following key events: (1) metabolism to the trichloromethyl radical by CYP2E1 and
subsequent formation of the trichloromethyl peroxy radical, (2) radical-induced mechanisms
leading to hepatocellular toxicity, and (3) sustained regenerative and proliferative changes in the
liver in response to hepatotoxicity. These key events are consistent with a hypothesis that liver
carcinogenicity occurs at exposures that also induce hepatocellular toxicity and a sustained
regenerative and proliferative response, and that exposures that do not cause hepatotoxicity are
not expected to result in liver cancer. For this hypothesized mode of action for carbon
tetrachloride liver carcinogenicity, a nonlinear approach to low-dose extrapolation is considered
appropriate.
The RfD of 0.004 mg/kg-day and RfC of 0.1 mg/m3 derived in Sections 5.1 and 5.2
represent the outcome of nonlinear assessments based on hepatotoxicity associated with oral
exposures (RfD) and inhalation exposures (RfC) to carbon tetrachloride. Consistent with the
hypothesized mode of action for liver tumors consisting of metabolism, cytotoxicity, and
sustained regeneration and proliferation, doses (or concentrations) of carbon tetrachloride below
the RfD (or RfC) would not be expected to produce liver tissue damage and therefore would not
be expected to produce an increase in liver cancer risk. This harmonized approach between
noncancer and cancer endpoints transparently utilizes a key event (cytotoxicity or hepatotoxicity)
in the hypothesized nonlinear mode of action to derive the RfD and RfC. Based on the mode of
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action consistent with nonlinearity, the RfD of 0.004 mg/kg-day and RfC of 0.1 mg/m3 can be
used to assess the potential risk of liver cancer from carbon tetrachloride exposure.
The application of a nonlinear approach for liver tumors is based on mode of action
information specific to that tumor type and does not apply to the occurrence of
pheochromocytomas. As noted above, the pheochromocytomas in mice reported in the JBRC
104-week bioassay (Nagano et al., 2007b; JBRC, 1998) were, with one exception, characterized
as benign rather than malignant. Unlike liver tumors associated with carbon tetrachloride
exposure, which have been observed in numerous bioassays in multiple species and by multiple
routes of exposure, pheochromocytomas have been observed only in the mouse. Thus, the
finding of pheochromocytomas in the mouse may be a species-specific finding and, as such, may
present a less certain human cancer risk than does the finding of liver tumors in experimental
animals. Nevertheless, the RfD and RfC based on liver toxicity cannot be assumed to be
protective for the potential cancer risk associated with carbon tetrachloride-induced
pheochromocytomas in the mouse.
5.4.2. Linear Extrapolation Approach
This section develops estimates of carbon tetrachloride cancer risk using approaches
incorporating low-dose linearity. Available data are not sufficient to support a biologically-
based dose-response model for the relationship of carbon tetrachloride and cancer. Judgements
regarding the potential dose-response relationships for carbon tetrachloride cancer risks at low
dose are informed by bioassay and mechanistic data for carbon tetrachloride as well as broader
scientific considerations.
The application of linear extrapolation is consistent with several pieces of evidence
suggesting that carbon tetrachloride carcinogenicity may not be attributable to a nonlinear mode
of action only. As noted above, the JBRC bioassay (Nagano et al., 2007b; JBRC, 1998) revealed
an increased incidence of hepatocellular adenomas in the low-dose (5-ppm or 0.9-ppm adjusted)
female mouse in the absence of cytotoxicity, suggesting that mouse hepatocarcinogenicity cannot
be explained in terms of a cytotoxic-proliferative mode of action. In addition, carbon
tetrachloride induced pheochromocytomas in male and female mice by oral (NTP, 2007;
Weisburger, 1977) and inhalation (Nagano et al., 2007b; JBRC, 1998) exposure. Because the
mode of action for pheochromocytomas in the mouse is unknown, linear low-dose extrapolation
as a default approach is applied to data for this tumor type.
As discussed in Section 4.7, considerable evidence points to the involvement of highly
reactive metabolites (with the capacity to chemically interact with DNA and other cellular
macromolecules) in the processes of toxicity and carcinogenicity of carbon tetrachloride. In
addition, subsequent chemical reactions of carbon tetrachloride metabolites with cellular
constituents lead to formation of reactive oxygen species that also can damage DNA and other
macromolecules. As evaluated in this assessment, the data available at this time do not
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demonstrate that carbon tetrachloride or its metabolites are direct acting mutagens. However, the
genotoxicity database, while large, is complex and there are various interpretive issues (see
Table 4-12) regarding the potential for genotoxicity of carbon tetrachloride at doses below those
associated with overt toxicity. In this situation, the fundamental reactivity of both direct and
indirect products of carbon tetrachloride metabolism provides a cogent argument in favour of
some degree of nonthreshold response to carbon tetrachloride carcinogenicity. Further research
may inform both the dosimetry for DNA (or other macromolecules) exposure to direct and
indirect reactive products resulting from carbon tetrachloride exposure and the dose-response
relationships for subsequent events resulting from damage of DNA or other macromolecules.
Thus, in the case of liver tumors, bioassay evidence inconsistent with a nonlinear mode of
action in the range of experimental observations and uncertainties in carbon tetrachloride's
biological activity at low exposures suggest that other (or another) modes of action may be
operative. Given a lack of understanding for the mode(s) of action for liver tumors, a default
linear low-dose extrapolation approach to carbon tetrachloride cancer data (see Section 5.4.2) is
presented in addition to the nonlinear approach (see Section 5.4.1) for carbon tetrachloride-
induced liver tumors. Given a lack of understanding of the mode of action for
pheochromocytomas, a default linear low-dose extrapolation approach to carbon tetrachloride
cancer data is applied consistent with the 2005 Guidelines for Carcinogen Risk Assessment (U.S.
EPA, 2005a) (see Section 5.4.2).
Broader science considerations based on scientific literature not specific to carbon
tetrachloride also support inferences about potential risks of carbon tetrachloride at lower doses.
EPA guidance and reports from expert advisory bodies have provided broad and long-standing
scientific arguments in favor of low-dose linear approaches to cancer risk assessment. This
perspective is based on the following principles:
• A chemical's carcinogenic effects may act additively to ongoing biological processes,
given that diverse human populations already have substantial background incidence of
various tumors (e.g., Crump et al, 1976);
• A broadening of the dose-response curve in the human population (i.e., less rapid fall-off
with dose) and, accordingly, a greater potential for risks from low-dose exposures (see
Zeise et al., 1987; Lutz et al., 2005) would result for two reasons. First, even if there is a
threshold concentration at the cellular level, that threshold is likely to be different among
different individuals. Secondly, greater variability in response to exposures in the
heterogeneous human population would be anticipated than in controlled laboratory
species and conditions (due to, e.g., genetic variability, disease states, nutrition, age).
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• The general use of linear extrapolation provides plausible upper-bound risk estimates and
also provides consistency across assessments.
5.4.2.1. Choice of Study Data—with Rationale and Justification
5.4.2.1.1. Inhalation data. As noted previously, epidemiological studies of populations exposed
to carbon tetrachloride provide only suggestive evidence for an association between carbon
tetrachloride exposure and human cancer and are not adequate for dose-response analysis.
The only chronic bioassay of carbon tetrachloride by the inhalation route is the 104-week
inhalation bioassay in rats and mice conducted by JBRC (Nagano et al., 2007b; JBRC, 1998), a
bioassay that provides data adequate for dose-response modeling. In this bioassay, F344 rats and
BDF1 mice were exposed to 0, 5, 25, or 125 ppm carbon tetrachloride, 6 hours/day, 5 days/week,
for 2 years. Carbon tetrachloride produced a statistically significant increase in hepatocellular
adenomas and carcinomas in rats and mice of both sexes, and adrenal pheochromocytomas in
mice of both sexes.
5.4.2.1.2. Oral data. Studies of carbon tetrachloride carcinogenicity by the oral exposure route
are not sufficient to derive a quantitative estimate of cancer risk using low-dose linear
approaches. No epidemiological investigations of the possible carcinogenicity of carbon
tetrachloride associated with oral exposure have been performed. The cancer studies by Edwards
et al. (1942) in the mouse and Delia Porta et al. (1961) in the hamster included a control and only
one dose group, and animals were dosed for less than a lifetime (2 months and 30 weeks,
respectively). Neither study provided body weight information, so that doses could not be
estimated with certainty. Despite the relatively short dosing periods and the fact that animals
were kept on study for less than a lifetime (approximately 6.5 months in the case of Edwards et
al., 1942, and approximately 1 year in the case of Delia Porta et al., 1961), liver tumor incidence
was very high (71% in the case of Edwards et al., 1942, and 100% of the hamsters that died or
were sacrificed between weeks 43 and 55 in the case of Delia Porta et al., 1961). In the NCI
bioassays (1977, 1976a,b), liver tumor incidence in the mouse was virtually 100% in both dose
groups. In the rat, liver tumor incidence was low and failed to show a dose-response relationship
(in the female rat, tumor incidence was higher in the low-dose group [4/46] than in the high-dose
group [1/30], presumably because early mortality in the high-dose group precluded tumor
formation). Thus, none of the available oral studies of carbon tetrachloride carcinogenicity
provided data sets amenable to dose-response modeling.
5.4.2.2. Dose-Response Data
5.4.2.2.1. Inhalation data. Dose-response modeling was performed for five tumor responses
from the JBRC bioassay: adenoma and carcinoma of the liver in female rats, adenoma and
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carcinoma of the liver in male and female mice, and pheochromocytomas in male and female
mice. Incidence data for liver tumors and pheochromocytomas are summarized in Tables 5-8
and 5-9 below.
Table 5-8. Incidence of liver tumors in F344 rats and BDF1 mice exposed
to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week)
Tumor
Male
0 ppm
5 ppm
25 ppm
125 ppm
Female
0 ppm
5 ppm
25 ppm
125 ppm
RAT
Hepatocellular
adenoma or
carcinoma
l/50a
1/50
1/50
40/50b
0/50a
0/50
3/50
44/50b
MOUSE
Hepatocellular
adenoma or
carcinoma
24/50a
20/50
49/50b
49/50b
4/50a
9/49
44/50b
48/49b
a Statistically significant trend for increased tumor incidence by Peto's test (£><0.01).
bTumor incidence significantly elevated compared with that in controls by Fisher Exact test (p<0.0l).
Source: Nagano et al, 2007b; JBRC, 1998
Table 5-9. Incidence of adrenal tumors (pheochromocytomas) in BDF1
mice exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day,
5 days/week)
Tumor
Adrenal
pheochromocytoma0
Male
0 ppm
0/50a
5 ppm
0/50
25 ppm
16/50b
125 ppm
32/50b
Female
0 ppm
0/50a
5 ppm
0/49
25 ppm
0/50
125 ppm
22/49b
a Statistically significant trend for increased tumor incidence by Peto's test (p
-------
female rat compared with the male rat, such that the female rat data would provide the higher
estimate of risk of the two data sets.
For the female mouse, the bioassay data set contained two exposure concentrations (mid-
and high-exposure concentrations) at which close to maximal responses were seen. Preliminary
fitting of a multistage model revealed that: (1) a fit with an adequate chi square based p-value
was not obtained, and (2) the fit and parameter estimates were highly sensitive to the precise
finding of 48/49 tumors at the highest concentration. (A hypothetical shift of the data to 49/49
tumors led to a good model fit with different powers of the multistage model involved in the fit.)
As these distinctions were not judged biologically based, multistage model fits below were
conducted without use of the highest exposure concentration data, an approach commonly used
in BMD modeling when very high dose data are not compatible with model fits.)
Dose-response modeling was also conducted for pheochromocytomas observed in the
JBRC mouse bioassay. These tumors, with one exception, were characterized as benign rather
than malignant. Unlike liver tumors associated with carbon tetrachloride exposure, which have
been observed in numerous bioassays in multiple species, pheochromocytomas have been
observed in only one species (mouse). Thus, the finding of pheochromocytomas in the mouse
may present a less certain human health hazard than does the finding of liver tumors in
experimental animals. The decision to develop dose-response models for pheochromocytomas
was based on guidance provided in the 2005 Guidelines for Carcinogen Risk Assessment (U.S.
EPA, 2005a), which states that "benign tumors that are not observed to progress to malignancy
are assessed on a case-by-case basis." The benign tumor type seen in the mouse has a human
equivalent that is damaging to human health and can lead to fatal sequelae. In humans,
pheochromocytomas are rare and usually benign neuroendocrine tumors, but may also present as
or develop into a malignancy (Eisenhofer et al., 2004). Salmenkivi et al. (2004) noted that
approximately 10% of pheochromocytomas in humans metastasize. The presence of one
observed malignant tumor in the mouse study also suggests potential for these benign tumors to
progress to malignancy. The oral NCI bioassay characterized adrenal gland tumors simply as
"pheochromocytoma" (incidence data are provided in Weisburger, 1977, and NTP, 2007). This
characterization suggests that the tumors were not malignant, although the status as benign or
malignant was not clearly established. Finally, Salmenkivi et al. (2004) observed that while
most pheochromocytomas are benign, differentiating a benign from a malignant tumor only by
histological criteria is very difficult. Thus, it was considered appropriate to conduct dose-
response modeling for pheochromocytomas and to address the potential cancer risk using linear
extrapolation as a default approach.
In the analyses of the mouse and rat carbon tetrachloride inhalation data that follow,
incidence reflects that of benign or malignant tumors combined. Data are not available to
indicate whether malignant tumors developed specifically from progression of the benign
tumors; however, etiologically similar tumor types, i.e., benign and malignant tumors of the
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same cell type, were combined for these analyses because of the possibility that the benign
tumors could progress to the malignant form (U.S. EPA, 2005a).
5.4.2.2.2. Oral data. As noted above, oral cancer bioassay data for carbon tetrachloride are not
adequate for dose-response analysis. Therefore, PBPK modeling was applied to extrapolate
inhalation tumor data to the oral route. Because liver tumors and pheochromocytomas have been
observed in experimental animals following both inhalation and oral exposures, the data sets
evaluated as the basis for the inhalation unit risk were considered appropriate for estimation of
an oral slope factor. The route-to-route extrapolation method is described further below.
5.4.2.3. Dose Adjustments and Extrapolation Methods
5.4.2.3.1. General approach to modeling and extrapolation of animal data to humans. Cancer
risk estimates were obtained by straight line extrapolation from the POD to zero as described in
the EPA's Guidelines for Carcinogenic Risk Assessment (U.S. EPA, 2005a). As stated in the
guidelines, "The linear approach is to draw a straight line between a point of departure from
observed data, generally as a default, an LED [lower bound of effective dose] chosen to be
representative of the lower end of the observed range, and the origin (zero incremental dose, zero
incremental response)." Linear extrapolation is used as the approach in the absence of data
supporting a biologically based model for extrapolation outside of the observed range (U.S. EPA,
2005a).
The general procedure for deriving the POD from animal bioassay data is the same as
that used to derive the POD for RfC derivation and is depicted in Figure 5-4. Exposure levels
studied in the 2-year rat and mouse bioassays (Nagano et al., 2007b; JBRC, 1998) were
converted to estimates of internal doses by application of the rat and mouse PBPK models.
BMD modeling methodology (U.S. EPA, 2000c, 1995) was used to analyze the relationship
between the estimated internal doses and response (i.e., liver tumors and pheochromocytomas).
The resulting BMDL values were converted to estimates of equivalent human exposure
concentrations and doses (HECs and HEDs) by applying the human PBPK model.
5.4.2.3.2. PBPK modeling for internal dose metrics. Estimation of internal doses
corresponding to the exposure concentrations studied in the 2-year rat and mouse bioassays
(Nagano et al., 2007b; JBRC, 1998) was accomplished using PBPK models of the rat (Thrall et
al., 2000; Benson and Springer, 1999; Paustenbach et al., 1988) and mouse (Fisher et al., 2004;
Thrall et al., 2000), respectively (see Sections 3.5 and Appendix C for description of the models).
The review, selection and application of the chosen PBPK models was informed by an EPA
report (U.S. EPA, 2006), which addresses the application and evaluation of PBPK models. The
PBPK models were used to simulate internal dose metrics corresponding to exposure
concentrations studied in the 2-year bioassays: 5, 25, and 125 ppm, 6 hours/day, 5 days/week
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(Nagano et al., 2007b; JBRC, 1998). Internal dose metrics were selected that were considered to
be most relevant to the toxicity endpoints of interest (i.e., liver tumors and pheochromocytomas),
based on consideration of evidence for mode of action of carbon tetrachloride. Two dose metrics
were selected based on available information on the mechanisms of carbon tetrachloride liver
toxicity: (1) time-averaged arterial blood concentration of carbon tetrachloride (MCA, jimol/L);
and (2) time-averaged rate of metabolism of carbon tetrachloride (MRAMKL, |imol/hr/kg liver).
Liver metabolism rate was selected as the primary dose metric for liver effects based on evidence
that metabolism of carbon tetrachloride via CYP2E1 to highly reactive free radical metabolites
plays a crucial role in its mode of action in producing liver toxicity (described in Section 4.5).
Because of acknowledged uncertainties regarding the accuracy of available PBPK models to
simulate carbon tetrachloride (see Section 5.2.2.1), arterial blood concentration of carbon
tetrachloride was also included in the analysis of liver tumor data as a more proximal dose metric
to liver metabolism.
Data on incidence of adrenal pheochromocytomas in mice were also analyzed. The
MRAMKL dose metric was excluded from consideration in the analysis of pheochromocytomas
on the basis that reactive metabolites of carbon tetrachloride formed in the liver are unlikely to
be sufficiently stable to contribute to toxicity or transformations of cells in the adrenal gland.
Although it is possible that local generation of reactive metabolites may contribute to the
production of pheochromocytomas, PBPK models available for this analysis do not simulate
uptake and metabolism of carbon tetrachloride in the adrenal gland. [The model of Yoon et al.
(2007) is the only one available that includes extra-hepatic metabolism, specifically in lung and
kidney. Metabolism in each of these tissues was estimated to be less than 1% of that in the liver,
and they had a negligible effect on MCA and MRAMKL.] It would be expected, however, that
rates of metabolism in all tissues, including the adrenal gland, would be dependent on delivery of
carbon tetrachloride to these tissues and, thereby, would be correlated with blood concentrations
of carbon tetrachloride. Therefore, the MCA dose metric was used to represent the internal dose
in BMD modeling of pheochromocytoma incidence in mice.
The two dose metrics, MCA and MRAMKL, were simulated in the rat and mouse PBPK
models as time-averaged values, with the averaging time being the chronic exposure period (e.g.,
2 years). See Equations 5-1 and 5-2 (Section 5.2.2.1) for the calculation of the time-averaged
dose metrics.
Internal dose metrics corresponding to the exposure concentrations studied in the 2-year
rat inhalation bioassay (Nagano et al., 2007b; JBRC, 1998) for two values of VmaxC were
provided previously in Table 5-5 (see Section 5.2.2.1). Internal dose metrics corresponding to
the exposure concentrations studied in the 2-year mouse inhalation bioassay (Nagano et al.,
2007b; JBRC, 1998) as derived from the Fisher et al. (2004) and Thrall et al. (2000) PBPK
models are presented in Table 5-10. The Fisher et al. (2004) model predicts lower values for
MCA than the Thrall et al. (2000) model. This is at least partly explained by the higher values
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for tissue:blood partition coefficients in the Fisher et al. (2004) model, which results in a larger
fraction of the body burden outside of the vascular compartment. The Fisher et al. (2004) model
predicts higher values for MRAMKL at exposure concentrations above approximately 40 ppm.
At least two factors contribute to this pattern: (1) the higher liverblood partition coefficient in
the Fisher et al. (2004) model results in higher concentrations of carbon tetrachloride in the liver;
and (2) the higher Vmaxc in the Fisher et al. (2004) model results in increases in liver metabolism
rate at any given liver concentration of carbon tetrachloride, with the more pronounced
enhancement of metabolism at liver concentrations above the Km. The exposure concentration-
dependence of the dose metrics estimated from both models is shown in Figure 5-9.
Table 5-10. Internal dose metrics predicted from Fisher et al. (2004) and Thrall et
al. (2000) PBPK mouse models
Exposure
(ppm)
5
25
125
MCA
(umol/L)
Fisher
0.111
0.603
3.315
Thrall
0.213
1.226
6.856
MRAMKL
(umol/hr/kg liver)
Fisher
12.666
41.675
71.589
Thrall
15.456
43.599
63.596
Values are for 0.036 kg mouse.
MCA, time-averaged arterial concentration of carbon tetrachloride; MRAMKL, time-averaged rate of
metabolism of carbon tetrachloride per kg liver.
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12.0
10.0
8.0
<
O 6.0
4.0
2.0
0.0
0
50 100 150
AIR(ppm)
200
250
0
0
50 100 150
AIR(ppm)
200
250
Figure 5-9. Internal dose metrics predicted from the Fisher et al. (2004) and Thrall
et al. (2000) PBPK mouse models.
Dose metrics shown are time-averaged arterial concentration of carbon tetrachloride (MCA, :mol/L, upper
panel), and time-averaged rate of metabolism of carbon tetrachloride (MRAMKL, umol/hr/kg liver, lower
panel). The dose metrics are plotted against exposure concentration (6 hours/day, 5 days/week, 2 years) for
a 0.036 kg mouse.
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5.4.2.3.3. Benchmark dose modeling of response data from animal bioassays. BMD modeling
methodology (U.S. EPA, 2000c, 1995) was used to analyze data on estimated internal doses (i.e.,
MCA, MRAMKL) and incidence data (i.e., liver tumors in rats, and liver tumors and adrenal
pheochromocytomas in mice) from the 2-year rat and mouse inhalation bioassays (Nagano et al.,
2007b; JBRC, 1998). The multistage model in U.S. EPA's BMDS (version 1.4.1) (U.S. EPA,
2007) was fit to the tumor incidence data for rats and mice. When adequate fit could not be
achieved with the multistage model, other models from the BMDS suite of models were fit. The
results of the BMD modeling are summarized below; detailed model outputs are provided in
Appendix E.
Female F344 rat — hepatocellular adenomas + carcinomas
Internal doses associated with a BMR of 5% extra risk of liver tumors were calculated. A
BMR of 5% excess risk was in the low range of experimental data for the rat (see Appendix E).
In addition, a BMR of 5% excess risk was preferred over a BMR of 10% in the interest of
moving the POD further from the range where hepatocellular toxicity and a
proliferative/regenerative response was observed and where tumor induction is more likely
influenced by a cytotoxic-proliferative mode of action.
BMD modeling using the multistage model in BMDS was performed using the female rat
liver tumor incidence data shown in Table 5-8 and internal doses shown in Table 5-5. A
summary of the resulting BMDs and BMDLs values is presented in Table 5-11 (columns 2
and 3).
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Table 5-11. BMD values for incidence data for liver tumors (adenoma plus
carcinoma) in female F344 rats and corresponding HEC and HED values
Metric
(l)b
MCA
MRAMKL
BMD modeling"
VMAXCR=
0.4
(2)
BMD5: 0.61
BMDL5: 0.39
BMD5: 9.82
BMDL5: 8.40
VMAXCR
= 0.65
(3)
BMD5: 0.59
BMDL5: 0.35
BMD5: 14.6
BMDL5: 12.3
VMAXCH
(4)
0.40
0.65
1.49
1.70
0.40
0.65
1.49
1.70
HEC
VMAXCR
= 0.4
(5)
26.083
27.605
27.605
31.273
107.759
63.915
39.635
37.771
VMAXCR
= 0.65
(6)
23.922
25.318
28.203
28.667
236.171
105.882
59.326
56.236
HED
VMAXCR
= 0.4
(7)
3.65
4.27
6.35
6.87
5.10
3.03
1.88
1.79
VMAXCR
= 0.65
(8)
3.37
3.96
5.95
6.44
11.19
5.01
2.81
2.66
Rats were exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week). Internal doses
modeled correspond to exposure concentrations: 0, 5, 25, or 125 ppm.
HEC, human equivalent concentration, mg/m3; HED, human equivalent dose, mg/kg-day; MCA, time-
averaged arterial blood concentration, umol/L; MRAMKL, time-averaged rate of metabolism per kg liver,
umol/hr/kg liver; VMAXC, maximum rate of metabolism in humans (H) or rat (R), mg/hr/kg B W°70
a MCA, multistage (2-stage); MRAMKL, multistage (4-stage)
BMR (benchmark response) = 5%
b Number in parentheses indicates the column number.
A second analysis was performed to examine the effect on the cancer risk estimate of
using only carbon tetrachloride cancer response data at exposure levels below those associated
with evidence of cell replication. In the female F344 rat, the 3/50 hepatocellular carcinoma
response at 25 ppm (an exposure concentration at which cytotoxicity occurred but below which
regenerative proliferation was reported; see Table 4-17) is statistically significant (two-tailed p-
value of 0.0002) when compared to the historical control incidence of'211191 for female rats for
the same strain and research center (email data April 5, 2007, from Kasuke Nagano, JBRC, to
Susan Rieth, U.S. EPA). A comparison to concurrent controls in the JBRC study did not yield a
statistically significant difference in response; however, because the observed carcinomas in
female rats at 25 ppm are part of a trend of increasing carcinoma incidences with increasing
exposure, it is reasonable to consider the tumors to be biologically significant.
As noted above, cytotoxicity was reported in female rats at 25 ppm in the 104-week
study, but regeneration and proliferation were not reported at this exposure level; additionally,
regeneration and proliferation were not observed in 13-week studies at 30 ppm and below
(Table 4-17). Thus, the tumor response at 25 ppm can be considered as potentially independent
of, or at most minimally influenced by, regenerative proliferation.
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A multistage POD model of the control, 5 ppm, and 25 ppm exposure groups
(Table 5-12, columns 2 and 3) is provided for comparison with the results above for the full data
set (see Table 5-11, columns 2 and 3).
Table 5-12. BMD values for incidence data for liver tumors (adenoma plus
carcinoma) in female F344 rats (high dose dropped) and corresponding HEC and
HED values
Metric
(l)b
MCA
MRAMKL
BMD modeling"
VMAXCR=
0.4
(2)
BMD5: 0.65
BMDL5: 0.35
BMD5: 11.6
BMDL5: 6.92
VMAXCR
= 0.65
(3)
BMD5: 0.60
BMDL5: 0.32
BMD5: 16.7
BMDL5: 9.76
VMAXCH
(4)
0.40
0.65
1.49
1.70
0.40
0.65
1.49
1.70
HEC
VMAXCR
= 0.4
(5)
23.339
24.701
27.512
27.965
79.943
50.626
32.384
30.918
VMAXCR
= 0.65
(6)
21.459
22.713
25.288
25.701
140.519
77.275
46.414
44.157
HED
VMAXCR
= 0.4
(7)
3.29
3.88
5.84
6.33
3.79
3.05
1.53
1.46
VMAXCR
= 0.65
(8)
2.40
3.61
5.48
5.95
6.66
3.66
2.20
2.09
Rats were exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week). Internal doses
modeled correspond to exposure concentrations: 0, 5, 25, or 125 ppm.
HEC, human equivalent concentration, mg/m3; HED, human equivalent dose, mg/kg-day; MCA, time-
averaged arterial blood concentration, umol/L; MRAMKL, time-averaged rate of metabolism per kg liver,
umol/hr/kg liver; VMAXC, maximum rate of metabolism in humans (H) or rat (R), mg/hr/kg B W°70
a MCA, multistage (2-stage); MRAMKL, multistage (2-stage)
BMR (benchmark response) = 5%
b Number in parentheses indicates the column number.
See Appendix E for the BMDS model outputs and graphs of the modeled data.
Female BDF1 mouse — hepatocellular adenomas + carcinomas
Internal doses associated with a BMR of 10% extra risk of liver tumors were calculated.
As with the female rat liver tumor data, EPA considered a BMR of 5% excess risk in the interest
of moving the POD further from the range where hepatocellular toxicity and a
proliferative/regenerative response was observed and where tumor induction may more likely be
influenced by a cytotoxic-proliferative mode of action. In the case of the female mouse liver
tumor data, however, a BMR of 5% fell well below the experimental range; therefore, a BMR of
10% was used in the BMD modeling of female mouse liver tumor data.
BMD modeling using the multistage model in BMDS was performed using the female
mouse liver tumor incidence data shown in Table 5-8 and internal doses shown in Table 5-10.
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As noted in Section 5.4.2.2.1, the multistage model fits below were conducted without use of the
highest exposure concentration data, an approach commonly used in BMD modeling when very
high dose data are not compatible with model fits. A summary of the resulting BMDio and
BMDLio values is presented in Table 5-13 (columns 2 and 3).
Table 5-13. BMD values for incidence data for liver tumors (adenoma plus
carcinoma) in female BDF1 mice (high dose dropped) and corresponding HEC and
HED values
Metric
(l)b
MCA
MRAMKL
BMD modeling"
Fisher
(2)
BMD10:0.10
BMDL10: 0.047
BMD10: 9.71
BMDL10: 6.32
Thrall
(3)
BMD10:0.19
BMDL10: 0.088
BMD10: 10.4
BMDL10: 7.59
VMAXCH
(4)
0.40
0.65
1.49
1.70
0.40
0.65
1.49
1.70
HEC
Fisher
(5)
3.197
3.385
3.753
3.811
70.278
45.526
29.466
28.152
Thrall
(6)
6.042
6.396
7.097
7.208
91.709
56.492
35.646
34.005
HED
Fisher
(7)
0.50
0.61
0.99
1.08
3.33
2.16
1.40
1.33
Thrall
(8)
0.94
1.14
1.82
2.00
4.34
2.68
1.69
1.61
Mice were exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week). Doses
modeled correspond to exposure concentrations: 0, 5, or 25 ppm (125 ppm exposure dropped)
HEC, human equivalent concentration, mg/m3; HED, human equivalent dose, mg/kg-day; Fisher, Fisher et
al. (2004) model; MCA, time-averaged arterial blood concentration, umol/L; MRAMKL, time-averaged
rate of metabolism per kg liver, umol/hr/kg liver; Thrall, Thrall et al. (2000) model; VMAXCn, maximum
rate of metabolism in humans, mg/hr/kg BW°70
a MCA, multistage (2-stage); MRAMKL, multistage (2-stage)
BMR (benchmark response) = 10%
b Number in parentheses indicates the column number.
As with the rat, a second analysis was performed with female mouse liver tumor data to
examine the effect on the cancer risk estimate of using only carbon tetrachloride cancer response
data at exposure levels below those associated with evidence of cell replication. A multistage
model POD calculation using only the control and 5-ppm exposure group (Table 5-14, columns 2
and 3) is provided for comparison with the results above for the full data set (Table 5-13,
columns 2 and 3).
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Table 5-14. BMD values for incidence data for liver tumors (adenoma plus
carcinoma) in female BDF1 mice (2 highest doses dropped) and corresponding HEC
and HED values
Metric
(l)b
MCA
MRAMKL
BMD modeling"
Fisher
(2)
BMD10:0.10
BMDL10: 0.044
BMD10: 11.6
BMDL10: 5.05
Thrall
(3)
BMD10: 0.20
BMDL10: 0.085
BMD10: 14.2
BMDL10:6.16
VMAXCH
(4)
0.40
0.65
1.49
1.70
0.40
0.65
1.49
1.70
HEC
Fisher
(5)
3.025
3.202
3.550
3.605
52.187
35.277
23.367
22.358
Thrall
(6)
5.792
6.132
6.804
6.910
67.796
44.180
28.683
27.410
HED
Fisher
(7)
0.48
0.58
0.94
1.03
2.47
1.67
1.11
1.06
Thrall
(8)
0.91
1.09
1.75
1.92
3.21
2.09
1.36
1.30
Mice were exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week). Doses
modeled correspond to exposure concentrations: 0, 5, or 25 ppm (125 ppm exposure dropped)
HEC, human equivalent concentration, mg/m3; HED, human equivalent dose, mg/kg-day; Fisher, Fisher et
al. (2004) model; MCA, time-averaged arterial blood concentration, umol/L; MRAMKL, time-averaged
rate of metabolism per kg liver, umol/hr/kg liver; Thrall, Thrall et al. (2000) model; VMAXCH, maximum
rate of metabolism in humans, mg/hr/kg BW°70
a MCA, multistage (2-stage); MRAMKL, multistage (2-stage)
BMR (benchmark response) = 10%
b Number in parentheses indicates the column number.
See Appendix E for the BMDS model outputs and graphs of the modeled data.
Male BDF1 mouse — hepatocellular adenomas + carcinomas
Internal doses associated with a BMR of 10% extra risk of liver tumors were calculated
for the male mouse. As with the female mouse liver tumor data, a BMR of 10% was used in the
BMD modeling.
Similar to the male rat data for liver adenomas and carcinomas, the male mouse data
provided poor resolution of the dose-response relationship for liver tumors. Tumor incidence in
5-ppm male mice was below the control level, and was close to maximal response (49/50) at the
mid- and high-exposure groups, without any intervening dose levels having submaximal
responses. BMD modeling of this data set (shown in Table 5-8) and internal doses (shown in
Table 5-10) revealed that none of the dichotomous models in BMDS provided an adequate fit of
the liver tumor data. Therefore, multistage model fits were conducted without use of the highest
exposure group (125-ppm) data. A marginal fit of the data was obtained when the multistage
model was applied to this reduced data set. A summary of the resulting BMDio and BMDLio
values is presented in Table 5-15 (columns 2 and 3).
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Table 5-15. BMD values for incidence data for liver tumors (adenoma plus
carcinoma) in male BDF1 mice (high dose dropped) and corresponding HEC and
HED values
Metric
(l)b
MCA
MRAMKL
BMD modeling"
Fisher
(2)
BMD10:0.19
BMDL10: 0.064
BMD10: 13.4
BMDL10:7.31
Thrall
(3)
BMD10: 0.39
BMDL10:0.12
BMD10: 14.2
BMDL10: 8.82
VMAXCH
(4)
0.40
0.65
1.49
1.70
0.40
0.65
1.49
1.70
HEC
Fisher
(5)
4.33
4.59
5.09
5.17
86.55
53.95
34.25
32.68
Thrall
(6)
8.26
8.74
9.72
9.88
116.95
67.89
41.70
39.72
HED
Fisher
(7)
0.68
0.83
1.33
1.46
4.10
2.56
1.62
1.55
Thrall
(8)
1.28
1.56
2.48
2.71
5.54
3.22
1.98
1.88
Mice were exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week). Doses
modeled correspond to exposure concentrations: 0, 5, or 25 ppm (125 ppm exposure dropped)
HEC, human equivalent concentration, mg/m3; HED, human equivalent dose, mg/kg-day; Fisher, Fisher et
al. (2004) model; MCA, time-averaged arterial blood concentration, umol/L; MRAMKL, time-averaged
rate of metabolism per kg liver, umol/hr/kg liver; Thrall, Thrall et al. (2000) model; VMAXCH, maximum
rate of metabolism in humans, mg/hr/kg BW°70
a MCA, multistage (3-stage); MRAMKL, multistage (3-stage)
BMR (benchmark response) = 10%
b Number in parentheses indicates the column number.
See Appendix E for the BMDS model outputs and graphs of the modeled data.
Female and male BDF1 mouse - pheochromocytomas
Internal doses associated with a BMR of 10% extra risk of pheochromocytomas were
calculated. BMD modeling in BMDS was performed using the female and male mouse
pheochromocytoma incidence data shown in Table 5-9 and internal doses shown in Table 5-10.
The multistage model was used to fit female mouse pheochromocytoma data. The multistage
model did not provide an adequate fit of the male mouse data for this tumor type; therefore, for
this data set, other models for dichotomous data in BMDS were run. The log-probit model
without restriction on the slope parameter provided the best fit of the male mouse
pheochromocytoma data (based on %2p>0.1 and lowest AIC value). Bayesian analysis (see
Appendix E) confirmed BMDS results and provided an explanation as to why the slope
parameter of the log-probit model should not be constrained. Summaries of the resulting BMDio
and BMDLio values for the female and male mouse are presented in Table 5-16 (columns 2
and 3) and Table 5-17 (columns 2 and 3), respectively.
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Table 5-16. BMD values for incidence data for pheochromocytomas in female
BDF1 mice and corresponding HEC and HED values
Metric
(l)b
MCA
BMD modeling"
Fisher
(2)
BMD10: 1.43
BMDL10: 1.14
Thrall
(3)
BMD10: 2.95
BMDL10: 2.34
VMAXCH
(4)
0.4
0.65
1.49
1.7
HEC
Fisher
(5)
74.551
78.636
88.173
89.826
Thrall
(6)
149.096
156.027
174.686
178.325
HED
Fisher
(7)
9.66
10.73
14.20
15.05
Thrall
(8)
18.54
19.90
24.34
25.44
Mice were exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week). Doses
modeled correspond to exposure concentrations: 0, 5, or 25 ppm (125 ppm exposure dropped)
HEC, human equivalent concentration, mg/m3; HED, human equivalent dose, mg/kg-day; Fisher, Fisher et
al. (2004) model; MCA, time-averaged arterial blood concentration, umol/L; MRAMKL, time-averaged
rate of metabolism per kg liver, umol/hr/kg liver; Thrall, Thrall et al. (2000) model; VMAXCn, maximum
rate of metabolism in humans, mg/hr/kg BW°70
a Multistage (2-stage) model
BMR (benchmark response) = 10%
b Number in parentheses indicates the column number.
Table 5-17. BMD values for incidence data for pheochromocytomas in male BDF1
mice and corresponding HEC and HED values
Metric
(l)b
MCA
BMD modeling3
Fisher
(2)
BMD10: 0.26
BMDL10:0.15
Thrall
(3)
BMD10: 0.53
BMDL10: 0.30
VMAXCH
(4)
0.40
0.65
1.49
1.70
HEC
Fisher
(5)
10.19
10.79
12.00
12.20
Thrall
(6)
19.96
21.13
23.56
23.95
HED
Fisher
(7)
1.56
1.91
3.04
3.33
Thrall
(8)
2.87
3.41
5.21
5.67
Mice were exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week). Doses
modeled correspond to exposure concentrations: 0, 5, or 25 ppm (125 ppm exposure dropped)
HEC, human equivalent concentration, mg/m3; HED, human equivalent dose, mg/kg-day; Fisher, Fisher et
al. (2004) model; MCA, time-averaged arterial blood concentration, umol/L; MRAMKL, time-averaged
rate of metabolism per kg liver, umol/hr/kg liver; Thrall, Thrall et al. (2000) model; VMAXCH, maximum
rate of metabolism in humans, mg/hr/kg BW°70
a log-probit model
BMR (benchmark response) = 10%
b Number in parentheses indicates the column number.
5.4.2.3.4. PBPK modeling of human equivalent exposure concentrations and doses.
Interspecies extrapolation (i.e., rat-to-human, mouse-to-human) and route-to-route extrapolation
of carbon tetrachloride inhalation dosimetry was accomplished using the human PBPK model
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described in Paustenbach et al. (1988), Thrall et al. (2000), and Benson and Springer (1999).
The human PBPK model was used to estimate human equivalent concentrations (HECs, in
mg/m3) or human equivalent doses (HEDs, i.e., daily ingested doses, in mg/kg-day) that would
result in values for the internal dose metrics, MCA or MRAMKL, equal to the respective
BMDLs for each toxicity endpoint (i.e., liver tumors in rats, liver tumors and adrenal
pheochromocytomas in mice).
The approach used to derive the HECs and HEDs for each dose metric was as follows:
(1) The human PBPK model was used to calculate internal doses corresponding to a series of
exposure concentrations (EC, continuous exposure, mg/m3). For the dose metric MCA, the
human PBPK model was run at intervals over the range from 0.1 to 100 ppm (0.63 to
629 mg/m3); for MRAMKL, the human PBPK model was run at intervals from 1 to 300 ppm
(6.3 to 1887 mg/m3).
(2) For each of these internal doses, the human PBPK model was also used to calculate
equivalent rates of uptake of carbon tetrachloride from the gastrointestinal tract to liver (RGIL)
that yielded the same internal doses. Uptake was expressed in units of mg/kg-day. This simple
approximation method assumed continuous infusion of carbon tetrachloride from the human
gastrointestinal tract to the liver. It should be noted that doses extrapolated from inhalation to
oral exposures in this analysis are approximations because they do not account for oral
bioavailability or absorption kinetics, information that is not available for carbon tetrachloride.
(3) For each internal dose, conversion factors were calculated as the following corresponding
ratios:
• EC/MCA (to relate a continuous chronic human inhalation exposure in mg/m3 [EC] to
an internal dose using MCA as the dose metric);
• RGIL/MCA (to relate the rate of uptake of carbon tetrachloride from the
gastrointestinal tract to the liver (i.e., chronic daily ingested dose in mg/kg-day
[RGIL] to an internal dose using MCA as the dose metric);
• EC/MRAMKL (to relate a continuous chronic human inhalation exposure in mg/m3
[EC] to an internal dose using MRAMKL as the dose metric); and
• RGIL/MRAMKL (to relate the rate of uptake of carbon tetrachloride from the
gastrointestinal tract to the liver in mg/kg-day [RGIL] to an internal dose using
MRAMKL as the dose metric).
(4) Conversion factors were calculated for each of four assumed values of Vmaxc in the human
PBPK model: 0.40, 0.65, 1.49, or 1.70 mg/hr/kg BW°'70. These conversion factors are provided
in Appendix C. Trend equations were also developed to permit the calculation of EC or RGIL
for any value of MCA or MRAMKL (see Appendix C).
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Estimated values for inhalation HECs corresponding to BMDLs for the 2-year rat and
mouse inhalation bioassays (Nagano et al., 2007b; JBRC, 1998) for different tumor types and
alternative values of VmaxC are presented in Tables 5-11 to 5-17, columns 5 and 6. Estimated
values for oral HEDs are presented in Tables 5-11 to 5-17, columns 7 and 8. As noted in the
discussion of the RfC derivation, estimates of the dose metrics, MCA and MRAMKL, were
sensitive to the value assigned to the Vmaxc parameter (see Figures 5-5 and 5-9), and the
inclusion of these alternative Vmaxc values provides some indication of the uncertainty in the
modeling. As in the derivation of the RfC, the human Vmaxc estimated from in vitro human data
(1.49 mg/hr/kg BW0'70) was considered to yield the most appropriate estimate of the HEC and
HED, and was used as the basis for cancer risk estimates. As discussed in Section 5.4.2.3.2, the
dose metric MRAMKL was considered to be the most appropriate dose metric to represent
internal doses in modeling liver tumors in rats and mice, and MCA was considered to be the
appropriate dose metric to represent internal doses in modeling pheochromocytoma incidence in
mice; these dose metrics were used as the basis for cancer risk estimates.
For the rat model, no information is available to establish whether a rat Vmaxc of 0.4 or
0.65 mg/hr/kg BW0'70 is the more scientifically defensible value for this parameter. Therefore,
the cancer risk values derived using these two rat Vmaxc values were averaged to derive the final
cancer risk values for carbon tetrachloride. Similarly, for the mouse, it cannot be established
whether the Fisher et al. (2004) or Thrall et al. (2000) model provides the more accurate
prediction of the internal dose for the mouse. Therefore, the cancer risk values derived using
these two mouse models were averaged to derive the final cancer risk values for carbon
tetrachloride (see Section 5.4.2.4 below).
5.4.2.4. Inhalation Unit Risk and Oral Slope Factor
5.4.2.4.1. Inhalation unit risk. Inhalation unit risk (IUR) estimates based on the five tumor data
sets analyzed in Section 5.4.2.3.3 are provided in Table 5-18. The highest IUR was associated
with pheochromocytomas in the male mouse [6 x 10"6 (jig/m3)"1]. Incidence of liver tumors was
also increased in male mice. Because different internal dose metrics were used in the dose-
response analysis of liver tumors (MRAMKL) and pheochromocytomas (MCA), the addition of
individual tumor risks to obtain a composite risk for the male mouse could not be performed.
Uncertainty in the estimate of the IUR associated with male mouse liver tumors also argues
against risk addition. As noted in Section 5.4.2.3.3, data from the male mouse provided a poor
resolution of the dose-response relationship for liver tumors. A marginal fit of this data set with
the multistage model in BMDS was obtained only when the highest dose group was dropped.
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Table 5-18. Summary of inhalation unit risk estimates using linear low-dose
extrapolation approach
Tumor
Female rat
hepatocellular
adenoma + carcinoma
Female mouse
hepatocellular
adenoma + carcinoma
Male mouse
hepatocellular
adenoma + carcinoma
Female mouse
pheochromocytoma
Male mouse
pheochromocytoma
Dose Groups
Modeled
0, 5, 25, 125 ppm
0, 5, 25 ppm
0, 5, 25 ppm
0, 5 ppm
0, 5, 25 ppm
0, 5, 25, 125 ppm
0, 5, 25, 125 ppm
Model
Parameters
MRAMKL; VmaxR = 0.4
BMR = 5%
MRAMKL; VmaxR = 0.65
BMR = 5%
MRAMKL; VmaxR = 0.4
BMR = 5%
MRAMKL; VmaxR = 0.65
BMR = 5%
MRAMKL; Fisher model
BMR = 10%
MRAMKL; Thrall model
BMR = 10%
MRAMKL; Fisher model
BMR = 10%
MRAMKL; Thrall model
BMR = 10%
MRAMKL; Fisher model
BMR = 10%
MRAMKL; Thrall model
BMR = 10%
MCA; Fisher model
BMR = 10%
MCA; Thrall model
BMR = 10%
MCA; Fisher model
BMR = 10%
MCA; Thrall model
BMR = 10%
HEC
(mg/m3)
39.63
59.32
32.33
46.41
29.46
35.64
23.37
28.68
34.25
41.70
88.54
173.77
12.00
23.56
Inhalation Unit Risk
Estimate (jig/m3) '
1.3 x 10"6
8.4 x 10"7
1.5 x 10"6
l.lxlO"6
3.4 xlO"6
2.8 x 10"6
4.3 x 10"6
3.5 x 10"6
2.9 x 10"6
2.4 x 10"6
l.lxlO"6
5.8xlO"7
8.3 x 10"6
4.2 x 10"6
Average
rounded to one
signif. figure =
1 x 10"6
Average
rounded to one
signif. figure =
1 x 10"6
Average
rounded to one
signif. figure =
3 x 10"6
Average
rounded to one
signif. figure =
4 x 10"6
Average
rounded to one
signif. figure =
3 x 10"6
Average
rounded to one
signif. figure =
8 x 10"7
Average
rounded to one
signif. figure =
6 x 10"6
Carbon tetrachloride also induced both liver tumors and pheochromocytomas in the
female mouse. For the same reason as the male mouse (i.e., different internal dose metrics were
used in the dose-response analysis), the risks associated with female liver tumors and
pheochromocytomas could not be summed. To ensure that the composite tumor risk in female
mouse did not exceed that associated with pheochromocytomas in the male mouse, a bounding
exercise was performed by summing the lURs for female mouse liver tumors and
pheochromocytomas [i.e., 3 x 10"6 + 8 x 10"7 (jig/m3)"1 = 4 x 10"6 (jig/m3)"1], a procedure that
results in an overestimation of composite risk. This bounding exercise confirms that the highest
value of the IUR is derived from male mouse pheochromocytoma data.
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Therefore, in consideration of the goal of providing an upper bound on riskh, the IUR for
carbon tetrachloride via the inhalation pathway is estimated as 6 x 10"6 (ug/m3)"1 based on
pheochromocytomas in the male mouse. This data set was judged to be applicable, scientifically
sound, and yielded the highest estimate of risk.
5.4.2.4.2. Oral slope factor. Oral slope factor (SF) estimates based on the five inhalation tumor
data sets analyzed in Section 5.4.2.3.3 and use of the human PBPK model of Paustenbach et al.
(1988) and Thrall et al. (2000) to perform route-to-route extrapolation are provided in Table 5-
19. The highest oral SF [7 x 10"2 (mg/kg-day)"1] was associated with female mouse
hepatocellular adenomas or carcinomas (using tumor data from the 0, 5, and 25-ppm exposure
groups). An analysis of liver tumor data using only the 0 and 5 ppm groups yielded a higher SF,
but because it is based on only two data points and thus provides a less informative
characterization of the dose-response curve for female mouse liver tumors, the SF based on
analysis of data from the 0, 5, and 25-ppm groups is considered more reliable. The analysis
based on tumor response data using only the 0 and 5-ppm groups was performed to examine the
effect on the liver cancer risk estimate of using only carbon tetrachloride response data at
exposure levels below those associated with evidence of cell replication. This analysis reveals
that dropping the 25-ppm group data had a relatively small impact on the SF [i.e., 7 x 10"2 vs
8 x 10"2 (mg/kg-day)"1]. A similar analysis of female rat liver tumor data revealed a similarly
negligible impact of performing a dose-response analysis on data points below those associated
with evidence of cell replication (i.e., 2 x 10"2 vs 3 x 10"2 (mg/kg-day)"1; see Table 5-19).
Carbon tetrachloride also induced pheochromocytomas in the female mouse. For the
same reason provided for the male mouse tumor data used to derive the IUR, the estimated risks
from the individual tumors could not be summed because different internal dose metrics were
used in the dose-response/PBPK analysis. Because the SF associated with pheochromocytomas
is an order of magnitude smaller than the SF associated with liver tumors in the female mouse,
the pheochromocytoma data would be expected to contribute negligibly to the total cancer risk
estimate.
Therefore, in consideration of the goal of providing an upper bound on risk1, the oral
slope factor for carbon tetrachloride is estimated as 7 x 10"2 (mg/kg-day)"1 based on female
mouse liver tumors. This data set was judged to be applicable, scientifically sound, and yielded
the highest estimate of risk.
h According to EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA,2005A), "[t]he use of upper bounds
generally is considered to be a health-protective approach for covering the risk to susceptible individuals, although
the calculation of upper bounds is not based on susceptibility data." Upper bound is defined in the IPJS glossary as
a plausible upper limit to the true value of a quantity, and is usually not a true statistical confidence limit
().
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Table 5-19. Summary of oral slope factor estimates using linear low-dose
extrapolation approach and route-to-route extrapolation
Tumor
Female rat
hepatocellular
adenoma + carcinoma
Female mouse
hepatocellular
adenoma + carcinoma
Male mouse
hepatocellular
adenoma + carcinoma
Female mouse
pheochromocytoma
Male mouse
pheochromocytoma
Dose Groups
Modeled
0, 5, 25,
125 ppm
0, 5, 25 ppm
0, 5, 25 ppm
0, 5 ppm
0, 5, 25 ppm
0, 5, 25,
125 ppm
0, 5, 25,
125 ppm
Model
Parameters
MRAMKL; VmaxR = 0.4
BMR = 5%
MRAMKL; VmaxR = 0.65
BMR = 5%
MRAMKL; VmaxR = 0.4
BMR = 5%
MRAMKL; VmaxR = 0.65
BMR = 5%
MRAMKL; Fisher model
BMR = 10%
MRAMKL; Thrall model
BMR = 10%
MRAMKL; Fisher model
BMR = 10%
MRAMKL; Thrall model
BMR = 10%
MRAMKL; Fisher model
BMR = 10%
MRAMKL; Thrall model
BMR = 10%
MCA; Fisher model
BMR = 10%
MCA; Thrall model
BMR = 10%
MCA; Fisher model
BMR = 10%
MCA; Thrall model
BMR = 10%
RED
(mg/kg-d)
1.88
2.81
1.53
2.20
1.40
1.69
1.11
1.36
1.62
1.98
14.2
24.34
3.03
5.21
Oral Slope Factor Estimate
(mg/kg-day)1
2.7 x 10"2
1.8 xlO"2
3.3 x 10"2
2.3 x 10"2
7.2 x 10"2
5.9 x 10"2
9.0 x 10"2
7.4 x 10"2
6.2 x 10"2
5.1xlO"2
7.0 x 10"3
4.1xlO"3
3.3 x 10"2
1.9 xlO"2
Average
rounded to one
signif. figure =
2 x 10"2
Average
rounded to one
signif. figure =
3 x 10"2
Average
rounded to one
signif. figure =
7 x 10"2
Average
rounded to one
signif. figure =
8 x 1Q-2
Average
rounded to one
signif. figure =
6 x 1Q-2
Average
rounded to one
signif. figure =
6 x 1Q-3
Average
rounded to one
signif. figure =
3 x 1Q-2
5.4.3. Choosing an Extrapolation Approach for Assessing Cancer Risk
According to EPA's (2005a) Guidelines for Carcinogen Risk Assessment, a nonlinear
extrapolation approach should be selected for assessing cancer risk:
"when there are sufficient data to ascertain the mode of action and conclude that it is not
linear at low doses and the agent does not demonstrate mutagenic or other activity
consistent with linearity at low doses. Special attention is important when the data
support a nonlinear mode of action but there is also a suggestion of mutagenicity.
Ibid.
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Depending on the strength of the suggestion of mutagenicity, the assessment may justify
a conclusion that mutagenicity is not operative at low doses and focus on a nonlinear
approach, or alternatively, the assessment may use both linear and nonlinear approaches."
A linear extrapolation approach is used as the default approach:
"[w]hen the weight of evidence evaluation of all available data are insufficient to
establish the mode of action for a tumor site and when scientifically plausible based on
the available data,... because linear extrapolation generally is considered to be a health-
protective approach."
Both linear and nonlinear approaches may be presented:
"[w]here alternative approaches with significant biological support are available for the
same tumor response and no scientific consensus favors a single approach"
or
"when there are multiple modes of action."
The Guidelines for Carcinogen Risk Assessment also suggest that:
"[i]f there are multiple modes of action at a single tumor site, one linear and another
nonlinear, that both approaches are used to decouple and consider the respective
contributions of each mode of action in different dose ranges."
Alternative cancer assessment approaches are presented for carbon tetrachloride liver
tumors in Sections 5.4.1 and 5.4.2. At high exposure levels, rodent bioassay data reveal a
general correspondence between hepatocellular cytotoxicity and regenerative hyperplasia and the
induction of liver tumors. Extensive mechanistic data support a hypothesized mode of action for
carbon tetrachloride-induced liver tumors at relatively high exposure levels that includes the
following key events: (1) metabolism to the trichloromethyl radical by CYP2E1 and subsequent
formation of the trichloromethyl peroxy radical, (2) radical-induced mechanisms leading to
hepatocellular cytotoxicity, and (3) sustained regenerative and proliferative changes in the liver
in response to hepatotoxicity. A weight of evidence analysis of the genotoxicity literature
suggests that carbon tetrachloride is more likely an indirect than direct mutagenic agent. As a
whole, this empirical evidence provides significant biological support for the hypothesis that
liver carcinogenicity by this mode of action occurs at carbon tetrachloride exposures that also
induce hepatocellular toxicity and a sustained regenerative and proliferative response, and that
exposures that do not cause hepatotoxicity are not expected to result in liver cancer. This
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hypothesis is consistent with a nonlinear approach to cancer assessment for liver tumors.
Several pieces of evidence suggest that carbon tetrachloride carcinogenicity may not be
explained by a cytotoxic-proliferative mode of action alone. These pieces of evidence, described
further in the paragraphs that follow, include: an increased incidence of liver tumors in the low-
dose female mouse (Nagano et al., 2007b) in the absence of nonneoplastic liver toxicity;
induction of pheochromocytomas in mice, a tumor for which the mode of action is unknown;
fundamental reactivity of the chemical; and absence of data on low-dose genotoxicity.
At lower exposure levels the correspondence between hepatocellular cytotoxicity and
regenerative hyperplasia and the induction of liver tumors is reveals inconsistencies. In
particular, liver findings from the JBRC bioassay (Nagano et al., 2007b; JBRC, 1998) suggest
that mouse hepatocarcinogenicity cannot be explained in terms of the cytotoxic-proliferative
mode of action. An increased incidence of hepatocellular adenomas occurred in the low-dose
(0.9-ppm adjusted) female mouse in the absence of nonneoplastic liver toxicity, raising the
possibility of another mode of action operating in addition to or in conjunction with the
cytotoxic-proliferative mode of action. It should be noted, however, that cytotoxicity and
cellular regeneration are observed at comparable doses (5 ppm adjusted; see Table 4-15) even
though they may not be directly observed at the dose level (0.9 ppm adjusted) inducing a
significant increase in liver adenomas in the mouse model. These data add to the complexity of
evaluating the weight of evidence for the hypothesized mode of action.
Carbon tetrachloride also induced pheochromocytomas in male and female mice by oral
(NTP, 2007; Weisburger, 1977) and inhalation (Nagano et al., 2007b; JBRC, 1998) exposure.
The mode of action for the induction of pheochromocytomas in the mouse is unknown. Where
the mode of action for a tumor site is unknown, linear extrapolation is used as the default.
Other considerations suggest that the carbon tetrachloride database is insufficient for
ruling out other modes of action at low exposure levels, in particular considerations related to the
compound's genotoxicity and general reactivity. Carbon tetrachloride is metabolized to reactive
species (trichloromethyl and trichloromethyl peroxy radical), and subsequent chemical reactions
of carbon tetrachloride metabolites with cellular constituents lead to formation of reactive
oxygen species that also can damage DNA and other macromolecules. A concern exists
regarding the potential biological activity of carbon tetrachloride with macromolecules at low
exposures (i.e., exposure levels below doses that are cytotoxic). Data to characterize this low-
exposure activity is limited.
Results of extensive testing for genotoxic and mutagenic potential are largely negative,
and a weight of evidence analysis of the genotoxicity literature suggests that carbon tetrachloride
is more likely an indirect than direct mutagenic agent; however, the nature of the database does
not characterize the role of genotoxicity at low doses of carbon tetrachloride There is little
direct evidence that carbon tetrachloride induces intragenic or point mutations in mammalian
systems. The mutagenicity studies that have been performed using transgenic mice have yielded
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negative results, as have the vast majority of the mutagenesis studies that have been conducted in
bacterial systems. Under highly cytotoxic conditions, bioactivated carbon tetrachloride can exert
genotoxic effects. These tend to be modest in magnitude and are manifested primarily as DNA
breakage and related sequelae. Chromosome loss leading to aneuploidy may also occur to a
limited extent. The fact that carbon tetrachloride overall has not been found to be a potent
mutagen and that positive genotoxic results are found only at high exposure levels and generally
in concert with cytotoxic effects (see Tables 4-8 to 4-11) indicates that carbon tetrachloride does
not likely induce genotoxic effects through direct binding or damage to DNA. The majority of
genotoxicity studies, however, have been conducted at relatively high exposure levels such that
the potential for genotoxic activity at low doses cannot be determined.
Thus, as summarized above and in Section 4.7.3.4, biological support exists for a
hypothesized cytotoxicity-regenerative mode of action as a major mode of action driving the
steep nonlinear increase in liver tumor dose-response at relatively high carbon tetrachloride
exposures. Inconsistencies and uncertainties at the low end of the experimental exposure range
(including bioassay evidence from the JBRC bioassay that indicates that female mouse liver
tumors cannot simply be explained in terms of the cytotoxic-proliferative mode of action, the
findings of pheochromocytomas in mouse by oral [NCI bioassay] and inhalation [JBRC
bioassay] exposure for which the mode of action is unknown, and insufficient data at low doses
to rule out the possibility of low-dose genotoxicity or other biological responses to a reactive
chemical), suggest that other (or another) modes of action independent of cytotoxicity and
regenerative cell proliferation may be operative in this range. It is the low end of the
experimental range that best informs the choice of the low-dose extrapolation approach. Given
an incomplete understanding of the cancer mode of action for carbon tetrachloride, linear low-
dose extrapolation as a default approach is therefore recommended for assessing carbon
tetrachloride cancer risk. The IUR provided in Section 5.4.2.4.1 of 6 x 10"6 (jig/m3)"1 and the
oral SF provided in Section 5.4.2.4.2 of 7 x 10"2 (mg/kg-day)"1 should be used for assessing
cancer risk under a linear low-dose extrapolation approach.
5.4.4. Uncertainties in Cancer Risk Values
As in most risk assessments, extrapolation of the available experimental data for carbon
tetrachloride to estimate potential cancer risk in human populations introduces uncertainty in the
risk estimation. Several types of uncertainty may be considered quantitatively, whereas others
can only be addressed qualitatively. Thus, an overall integrated quantitative uncertainty analysis
cannot be developed. Major sources of uncertainty in the cancer assessment for carbon
tetrachloride are summarized in this section and in Table 5-20 at the end of this section.
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Relevance to humans. The relevance of the mode of action of liver tumor induction to
humans was considered in Section 4.7.3.5. There is no evidence in humans for hepatic cancer
associated with carbon tetrachloride exposure. The experimental animal literature, however,
shows carbon tetrachloride to consistently induce liver tumors across species and routes of
exposure. Further, there are similarities between experimental animals and humans in terms of
carbon tetrachloride metabolism, antioxidant systems, and evidence for the liver as a sensitive
target organ. Together, this evidence supports a conclusion that experimental evidence for liver
cancer is relevant to humans.
Pheochromocytomas, on the other hand, were observed in only one species (the mouse).
In humans, pheochromocytomas are rare catecholamine-producing neuroendocrine tumors that
are usually benign, but may also present as or develop into a malignancy (Eisenhofer et al., 2004;
Salmenkivi et al., 2004; Tischler et al., 1996). In humans, hereditary factors have been identified
as important in the development of pheochromocytomas (Eisenhofer et al., 2004). In the mouse,
few chemicals have been reported to cause mouse adrenal medullary tumors (Hill et al., 2003),
and the mode of action for this tumor in mice is unknown. The relevance of mouse
pheochromocytomas to humans is similarly unknown, although parallels between this tumor in
the mouse and human led investigators to concluded that the mouse might be an appropriate
model for human adrenal medullary tumors (Tischler et al., 1996). Like the human,
pheochromocytomas in the mouse are relatively rare, as are metastases. Both the morphological
variability of the mouse pheochromocytomas and the morphology of the predominant cells are
comparable to those of human pheochromocytomas. An important characteristic of mouse
pheochromocytomas is expression of immunoreactive phenylethanolamine-N-methyltransferase
(PNMT); human pheochromocytomas are also usually PNMT-positive (Tischler et al., 1996).
Overall, this evidence supports a conclusion that experimental evidence for pheochromocytomas
is potentially relevant to humans.
Choice of low-dose extrapolation approach. The mode of action is a key determinant of
which approach to apply for estimating low-dose cancer risk. For liver tumors, two approaches
to low-dose cancer risk estimation were developed reflecting inconsistencies and uncertainly in
the mode of action at low carbon tetrachloride exposures.
The mode of action of carbon tetrachloride liver carcinogenicity has been investigated
extensively. Much of this research was conducted at relatively high exposure levels. The
mode(s) of action at low exposure levels is not known. Presentation of a linear and nonlinear
approach to cancer assessment is likely to bracket the risk of liver cancer associated with carbon
tetrachloride exposure. Additional mode of action information in the low-dose region to
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establish whether a linear or nonlinear approach applies to carbon tetrachloride liver tumors
would significantly reduce the uncertainty associated with magnitude of liver tumor risk.
The nonlinear extrapolation approach for liver tumors assumes that the RfD and RfC can
be used to assess the potential risk of liver cancer from carbon tetrachloride. This assumption is
based upon the RfD and RfC which were both quantitatively derived from hepatotoxicity
(cytotoxicity) as a noncancer endpoint. Hepatotoxicity is identified as a key event in the
hypothesized nonlinear mode of action for liver tumors (see Section 4.7.1 and 5.4.1).
Uncertainties in the derivation of the RfD and RfC are discussed in Section 5.3.
The effect on risk estimates derived using a linear extrapolation approach of using only
data on carbon tetrachloride liver tumor response at levels below those associated with increased
cell replication was examined. The risk calculations did not prove particularly sensitive to the
limitation of data points to those below which increased cell replication was reported (see Tables
5-18 and 5-19). This consistency in cancer risk estimates provided some confidence that the IUR
and SF estimates based on liver tumor data are not driven by high doses associated with
significant hepatotoxicity.
In data sets where early mortality is observed, methods that can reflect the influence of
competing risks and intercurrent mortality on site-specific tumor incidence rates are preferred.
Survival curves for female rats and mice from the JBRC bioassay (see Figures 4-1 and 4-2) show
early mortality in some treated groups. Because liver tumors were the primary cause of early
deaths in these groups, failure to apply a time to tumor analysis is not likely to significantly
influence the inhalation unit risk for liver tumors. The impact on the unit risk from
pheochromocytomas is unknown.
Cancer risk estimates were calculated by straight line extrapolation from the POD to zero,
with the multistage model used to derive the POD. (The one exception is the male mouse
pheochromocytoma data set, where the log-probit model was used.) It is unknown how well this
extrapolation procedure predicts low-dose risks for carbon tetrachloride. The multistage model
does not represent all possible models one might fit, and other models could conceivably be
selected to yield different results consistent with the observed data, both higher and lower than
those included in this assessment.
For pheochromocytomas, only a linear low-dose extrapolation approach was used to
estimate human carcinogenic risk in the absence of any information on the mode of action for
this tumor. Mode of action information to establish whether a linear or nonlinear approach
applies to carbon tetrachloride-induced pheochromocytomas would significantly reduce the
uncertainty associated with the magnitude of risk from exposure to this tumor type.
Cancer risk estimates for liver tumors and pheochromocytomas developed using a linear
low-dose extrapolation approach were not combined because different dose metrics were used in
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the dose-response/PBPK analysis of these two tumor types. Deriving the IUR or oral SF for data
on one tumor site, however, may underestimate the carcinogenic potential of carbon
tetrachloride. For the IUR based on male mouse pheochromocytomas, because of the poor
resolution of the dose-response relationship for male rodent liver tumors, the magnitude of the
potential risk underestimation cannot be characterized. Because the SF based on female mouse
liver tumors was an order of magnitude greater that that for female mouse pheochromocytomas,
any underestimation of the SF is expected to be small.
Interspecies extrapolation. Extrapolating dose-response data from animals to humans
was accomplished using PBPK models in the rat, mouse, and human. Availability of a PBPK
model generally reduces the pharmacokinetic component of uncertainty associated with animal
to human extrapolation; however, any PBPK model has its own associated uncertainties.
Specific uncertainties in the PBPK modeling for carbon tetrachloride were discussed previously
in Section 5.3.
Route-to-route extrapolation for the oral SF. Studies of carbon tetrachloride
carcinogenicity by the oral route were determined to be insufficient to derive a quantitative
estimate of cancer risk. Therefore, a human PBPK model was used to extrapolate inhalation data
to the oral route. A simple approximation method was used that assumed continuous infusion of
carbon tetrachloride from the human gastrointestinal tract to the liver. Doses extrapolated from
inhalation to oral exposures in this analysis were approximations because they did not account
for oral bioavailability or absorption kinetics, information that is not available for carbon
tetrachloride. The magnitude of uncertainty introduced by these assumptions cannot be
quantified.
Statistical uncertainty at the point of departure. Parameter uncertainty can be assessed
through confidence intervals. Each description of parameter uncertainty assumes that the
underlying model and associated assumptions are valid. For the log-probit model applied to the
male mouse pheochromocytoma data, there is a reasonably small degree of uncertainty at the
10% excess incidence level (the point of departure for linear low-dose extrapolation); the lower
bound on the BMD (i.e., the BMDLio) is 1.8-fold lower than the BMD. For the multistage
model applied to the female mouse liver tumor data, there is similarly a reasonably small degree
of uncertainty at the 10% excess incidence level; the lower bound on the BMD (i.e., the
BMDLio) is approximately 1.5-fold lower than the BMD.
Bioassay selection. The study by Nagano et al. (2007b; also reported as JBRC, 1998)
was used for development of the inhalation unit risk. A full report of the bioassay findings was
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published in 2007, although the study itself was conducted in the mid-1980s. Although not a
recently conducted study, this bioassay was well-designed, and included both sexes in two
species, an adequate number of animals per dose group, and an appropriate untreated control
group. Examination of toxicological endpoints in both sexes of rats and mice was appropriate.
No issues were identified with this bioassay that might have contributed to uncertainty in the
cancer assessment. Alternative bioassays for developing an inhalation unit risk were
unavailable.
Choice of species/gender. For liver tumors, modeling was performed using JBRC
inhalation bioassay data for the female mouse and female rat. The male rat liver tumor data were
not modeled because these data sets lacked the resolution desired for dose-response modeling;
the male mouse liver data were modeled, but provided similarly poor dose-response curve
resolution. Tumor frequencies jumped from control levels to close to maximal responses without
any intervening dose levels having submaximal responses. In the female mice and rats, lower
but biologically significant levels of response were seen at intermediate dose levels. Also,
notably, increased levels of hepatocellular proliferation were not reported for rodents at these
intermediate levels, increasing the likelihood that dose-response modeling may be relevant to
lower (noncytotoxic) dose conditions. There is no indication that male rodents are more
sensitive to carbon tetrachloride liver tumor induction and that use of female data only
underestimated potential risk. For pheochromocytomas, JBRC inhalation data sets for both male
and female mice were amenable to modeling, and the data set yielding the highest estimate of
cancer risk could be selected.
Human population variability. Neither the extent of interindividual variability in carbon
tetrachloride metabolism nor human variability in response to carbon tetrachloride has been fully
characterized. Factors that could contribute to a range of human response to carbon tetrachloride
include variations in CYP450 levels because of age-related differences or other factors (e.g.,
exposure to other chemicals that induce or inhibit microsomal enzymes), nutritional status,
alcohol consumption, or the presence of underlying disease that could alter metabolism of carbon
tetrachloride or antioxidant protection systems. Incomplete understanding of the potential
differences in metabolism and susceptibility across exposed human populations represents a
source of uncertainty.
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Table 5-20. Summary of uncertainty in the carbon tetrachloride cancer risk
assessment
Consideration/
Approach
Impact on cancer
risk estimate
Decision
Justification
Human relevance
of rodent tumor
data
If rodent tumors
proved not to be
relevant to humans,
unit risk would not
apply, i.e., human
risk would J,
Liver tumors in rats
and mice and
pheochromocytomas
in mice are relevant
to human exposure
Liver: There is no evidence in humans for hepatic
cancer associated with carbon tetrachloride
exposure. The experimental animal literature,
however, shows carbon tetrachloride to consistently
induce liver tumors across species and routes of
exposure. Further, there are similarities between
experimental animals and humans in terms of
carbon tetrachloride metabolism, antioxidant
systems, and evidence for the liver as a sensitive
target organ. Together, this evidence supports a
conclusion that experimental evidence for liver
cancer is relevant to humans.
Pheochromocvtomas: Pheochromocytomas were
observed in the mouse only. In humans,
pheochromocytomas are rare catecholamine-
producing neuroendocrine tumors that are usually
benign, but may also present as or develop into a
malignancy. Hereditary factors have been
identified as important in pheochromocytoma
development. The mouse has been characterized as
possibly an appropriate model for human adrenal
medullary tumors.
Low-dose
extrapolation
approach
Departure from
EPA's Guidelines
for Carcinogen Risk
Assessment POD
paradigm, if
justified, could J, or
t unit risk an
unknown extent
Liver: Nonlinear
approach and linear
approach presented.
Under the linear
extrapolation
approach, a POD-
based straight-line
extrapolation was
applied
Pheochromocvtoma:
Linear approach,
using a POD-based
straight-line
extrapolation
Liver: Biological support is available for a
cytotoxic-proliferative mode of action (MOA) that
is consistent with a nonlinear extrapolation
approach; however, other evidence suggests that
hepatocarcinogenicity may not be explained only in
terms of this MOA. Where data are not strong
enough to ascertain the MOA, EPA's 2005
Guidelines for Carcinogen Risk Assessment
recommend application of a linear low-dose
extrapolation approach in addition to a nonlinear
approach.
Pheochromocvtoma: Application of a linear
approach where the MOA has not been established
is consistent with EPA's 2005 Guidelines for
Carcinogen Risk Assessment.
Interspecies
extrapolation
using PBPK
model
I run
PBPK modeling used
to extrapolate rodent
tumor data to humans
PBPK modeling is considered to reduce the
uncertainty in extrapolating rodent tumor data to
humans.
Route-to-route
extrapolation
using PBPK
model
The magnitude of
uncertainty cannot
be quantified.
A human PBPK
model was used to
extrapolate inhalation
data to the oral route
Studies of carbon tetrachloride carcinogenicity by
the oral route were determined insufficient to derive
a quantitative estimate of cancer risk. A simple
approximation method was used that assumed
continuous infusion of carbon tetrachloride from
the human gastrointestinal tract to the liver.
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Table 5-20. Summary of uncertainty in the carbon tetrachloride cancer risk
assessment
Consideration/
Approach
Statistical
uncertainty at
POD
Bioassay
Species/gender
combination
Human
population
variability in
metabolism and
response/
sensitive
subpopulations
Impact on cancer
risk estimate
ilURandSFby 1.5
to 1.8-fold if BMD
used as the POD
rather than lower
bound on POD
Alternative
bioassay, if
available, could t or
I slope factor by an
unknown extent
Human risk could t
or I, depending on
relative sensitivity
Low-dose risk could
t or I to an
unknown extent
Decision
BMDL (preferred
approach for
calculating
reasonable upper
bound slope factor)
JBRC bioassay
Female mouse and
rat liver tumors
Male and female
mouse pheo-
chromocytomas
Considered
qualitatively
Justification
Limited size of bioassay results in sampling
variability; lower bound is 95% confidence interval
on administered exposure.
Alternative bioassays were unavailable.
It was assumed that humans are as sensitive as the
most sensitive rodent gender/species tested; true
correspondence is unknown.
For liver tumors, female mouse and female rat data
from the JBRC bioassay were considered more
amenable for modeling and demonstrating a
response that may be more relevant to lower dose
conditions than males. For pheochromocytomas,
JBRC inhalation data sets for both male and female
mice were amenable to modeling, and the data set
yielding the highest estimate of cancer risk could be
selected.
No data to support range of human
variability/sensitivity. Factors that could contribute
to a range of human response to carbon
tetrachloride include variations in CYP450 levels,
nutritional status, alcohol consumption, or the
presence of underlying disease that could alter
metabolism of carbon tetrachloride or antioxidant
protection systems. On balance, available data do
not indicate that children would necessarily be
more sensitive.
5.4.5. Previous Cancer Assessment
The previous cancer assessment for carbon tetrachloride was posted on the IRIS database
in 1987. At that time, carbon tetrachloride was classified as a B2 carcinogen (probable human
carcinogen), based on the finding of treatment-related hepatocellular carcinomas in rats, mice
and hamsters. In the previous assessment, an oral slope factor of 1.3 x 10"1 (mg/kg-day)"1 was
derived using linear extrapolation procedures and liver tumor data sets from the hamster (Delia
Porta et al., 1961), mouse (Edwards et al., 1942; NCI, 1977, 1976a, b), and rat (NCI, 1977,
1976a, b). In the current assessment, the available oral bioassay data were not considered
adequate for dose-response analysis, and a SF was derived instead by application of a PBPK
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model to extrapolate inhalation bioassay data to the oral route. The resulting SF
[7 x 10"2 (mg/kg-day)"1] is approximately 2-fold smaller than the previous SF.
An inhalation unit risk of 1.5 x 10"5 (jig/m3)"1 was derived previously from the oral slope
factor by route-to-route extrapolation (assuming an air intake of 20 m3/day, body weight of
70 kg, and 40% absorption rate by humans). The current IUR [6 x 10"6 (jig/m3)"1] was derived
using a chronic inhalation bioassay (Nagano et al., 2007) that was not available at the time of the
previous assessment and PBPK modeling for interspecies extrapolation.
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6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF
HAZARD AND DOSE RESPONSE
6.1. HUMAN HAZARD POTENTIAL
Carbon tetrachloride is rapidly absorbed by any route of exposure. Once absorbed, it is
widely distributed among tissues, especially those with high lipid content, reaching peak
concentrations in less than 1-6 hours, depending on dose. It is efficiently metabolized by the
liver, lung, and other tissues. The initial step in metabolism is reductive dehalogenation to
trichloromethyl radical by CYP450. The fate of the trichloromethyl radical is dependent on the
availability of oxygen and includes several alternative pathways for anaerobic or aerobic
conditions. Unmetabolized parent compound is excreted in exhaled air. Volatile metabolites are
also released in exhaled air, whereas nonvolatile metabolites are excreted in feces and, to a lesser
degree, in urine.
Hepatic and renal toxicities are the primary noncancer effects of oral or inhalation
exposure to carbon tetrachloride. In humans, damage to both the liver and kidney was observed
in acute poisoning cases. Suggestive evidence of hepatotoxicity was also seen in workers
exposed to carbon tetrachloride for an extended period of time in the workplace. Numerous
animal studies confirmed the toxic effect of carbon tetrachloride to the liver by oral exposure and
to both the liver and kidney by inhalation exposure. Exposure to high levels of carbon
tetrachloride by the oral or inhalation routes can also produce effects on reproduction and
development. Animal studies reported degeneration of the testes, reduced male fertility, delayed
fetal growth, and whole litter resorption following high-level carbon tetrachloride exposure.
Carbon tetrachloride was also carcinogenic in animal studies, inducing hepatocellular
carcinomas in rats, mice, and hamsters in oral studies and in rats and mice by inhalation
exposure. Pheochromocytomas were reported in mice in one oral and one inhalation bioassay.
The toxic effects of carbon tetrachloride are generally attributed to reactive products of
metabolism. The first step of carbon tetrachloride metabolism results in the production of the
trichloromethyl radical. In the presence of molecular oxygen, the trichloromethyl radical forms a
transient, but more potent, trichloromethyl peroxy radical that can induce lipid peroxidation. The
two reactive intermediates can also covalently bind to cellular components, causing disruption of
the cellular membrane. Increased permeability of cellular membranes interferes with cellular
processes dependent on calcium sequestration and also results in the release of hydrolytic
enzymes that may attack adjacent cells.
Examination of rodent liver tumors reveals a general correspondence between
hepatocellular cytotoxicity and regenerative hyperplasia and the induction of liver tumors,
although at lower exposure levels this correspondence is less consistent. Studies of genotoxic
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and mutagenic potential are largely negative. There is little direct evidence that carbon
tetrachloride induces intragenic or point mutations in mammalian systems. Mutagenicity studies
performed using transgenic mice have yielded negative results, as have the vast majority of the
mutagenesis studies that have been conducted in bacterial systems. Under highly cytotoxic
conditions, bioactivated carbon tetrachloride can exert genotoxic effects. These tend to be
modest in magnitude and are manifested primarily as DNA breakage and related sequelae.
Chromosome loss leading to aneuploidy may also occur to a limited extent. The fact that carbon
tetrachloride overall has not been found to be a potent mutagen and that positive genotoxic
results are found only at high exposure levels and generally in concert with cytotoxic effects
indicates that carbon tetrachloride does not likely induce genotoxic effects through direct binding
or damage to DNA. The nature of the genotoxicity database, however, poses distinct challenges
to the evaluation of carbon tetrachloride genotoxicity, particularly at low exposure levels.
Extensive mechanistic study data informs the mode(s) of action for carbon tetrachloride-induced
liver tumors. The empirical evidence for carbon tetrachloride, particularly data from relatively
high-exposure studies, provides support for the hypothesis that liver carcinogenicity is presumed
to occur at exposures that also induce hepatocellular toxicity and a sustained regenerative and
proliferative response, and that exposures that do not cause hepatotoxicity are not expected to
result in liver cancer. In the JBRC 2-year mouse bioassay (Nagano et al., 2007b; JBRC, 1998),
however, an increased incidence of hepatocellular adenomas in the low-dose (5-ppm or 0.9-ppm
adjusted) female mouse occurred in the absence of cytotoxicity, suggesting that mouse
hepatocarcinogenicity cannot simply be explained in terms of a cytotoxic-proliferative mode of
action. Information on the biological activity of carbon tetrachloride at low exposures is far less
complete than at higher (cytotoxic) exposure levels. Considerable evidence points to the
involvement of reactive metabolites and reaction products of carbon tetrachloride with cellular
constituents in the induction of liver toxicity and carcinogenicity by carbon tetrachloride. In
light of the fundamental reactivity of both direct and indirect products of carbon tetrachloride
metabolism and uncertainties about genotoxic activity at low exposures, the mode(s) of action
for carbon tetrachloride-induced liver tumors at low exposure levels cannot be characterized.
The mode of action for pheochromocytomas induced by carbon tetrachloride is unknown.
Carbon tetrachloride can be classified as likely to be carcinogenic to humans by all routes of
exposure.
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6.2. DOSE RESPONSE
6.2.1. Noncancer - Oral Exposure
The most sensitive endpoints identified for effects of carbon tetrachloride by oral
exposure relate to liver toxicity in the subchronic corn oil gavage studies of Bruckner et al.
(1986) in rats and Condie et al. (1986) in mice. The Bruckner et al. (1986) study identified
serum enzyme changes and liver histopathology as the most sensitive endpoints for carbon
tetrachloride. Serum SDH was the most sensitive serum chemistry endpoint and was considered
a marker of histopathologic changes. Another target of carbon tetrachloride toxicity following
oral exposure considered in the selection of the critical effect was the developing organism.
Studies in experimental animals found that relatively high doses of carbon tetrachloride during
gestation can produce prenatal loss; these doses also produced overt toxic effects in the dams.
Carbon tetrachloride doses associated with liver toxicity were much lower than those associated
with developmental toxicity.
BMD modeling methods were used to calculate the POD for deriving the RfD by
estimating the effective dose at a specified level of response (BMDX) and its 95% lower bound
(BMDLX) for liver enzyme changes. An increase in SDH activity two times the control mean
was used as the BMR. All of the models for continuous data in U.S. EPA's BMDS (version
1.4.1) (U.S. EPA, 2007) were fit to the 10-week SDH data. The power model, which provided
the best fit to the data, estimated a BMD2x of 7.32 mg/kg-day and a BMDL2x of 5.46 mg/kg-day.
Liver lesion incidence data from the Bruckner et al. (1986) study in rats and the Condie et
al. (1986) study in mice do not provide adequate information in the response region of concern
(i.e., 10% increase in extra risk over controls) to warrant BMD modeling of these endpoints
(U.S. EPA, 2000c). The NOAEL of 1 and LOAEL of 10-12 mg/kg-day in these studies do,
however, support the BMD2X of 7.32 mg/kg-day and the BMDL2x of 5.46 mg/kg-day estimated
from the increase in serum SDH observed in the Bruckner et al. (1986) study.
The BMDL2X of 5.46 mg/kg estimated from the increase in serum SDH activity in rats in
the Bruckner et al. (1986) subchronic toxicity study was used as the POD for derivation of the
RfD. Use of the modeled BMDL provides an inherent advantage over use of a NOAEL or
LOAEL by making greater use of the available data. Because of the absence of a suitable PBPK
model for oral exposure to carbon tetrachloride, one was not used for this assessment. Because
the BMDL2x of 5.46 mg/kg was derived from a study (Bruckner et al., 1986) with an intermittent
dosing schedule, it was adjusted to an average daily dose prior to application of UFs (BMDL2x-
ADJ = 3.9 mg/kg-day). Applying a composite UF of 1000 to the BMDLAoj of 3.9 mg/kg-day
yields an RfD of 0.004 mg/kg-day for carbon tetrachloride. The composite UF of 1000 includes
a factor of 10 to protect susceptible individuals, a factor of 10 to extrapolate from rats to humans,
a factor of 3 (1005) to extrapolate from a subchronic to a chronic duration of exposure, and a
factor of 3 (10°5) to account for an incomplete database. Information was unavailable to
quantitatively assess toxicokinetic or toxicodynamic differences between animals and humans
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and the potential variability in human susceptibility (factors that could contribute to a range of
human response include variations in CYP450 levels, nutritional status, alcohol consumption, or
the presence of underlying disease); thus, the UF selected for uncertainties related to both
interspecies and intraspecies was the default of 10. A UF of 3 for subchronic to chronic
extrapolation was selected based on: (1) qualitative information demonstrating that the target of
toxicity following chronic oral exposure as the liver; (2) knowledge of the relationship between
effect levels in subchronic and chronic inhalation studies; and (3) early onset of liver toxicity. A
database UF of 3 was selected to account for an incomplete database lacking an adequate
multigeneration study of reproductive function.
To provide perspective on the RfD supported by Bruckner et al. (1986), PODs and oral
RfDs based on other selected studies of carbon tetrachloride oral toxicity are arrayed in Figures
5-1 to 5-3 presented in Section 5. The predominant noncancer effect of subchronic and chronic
oral exposure to carbon tetrachloride is hepatic toxicity. Figure 5-1 provides a graphical display
of five studies that reported liver toxicity in experimental animals following subchronic oral
exposure, including the PODs, applied uncertainly factors, and RfDs for comparison to that
derived from the Bruckner et al. study. Studies in experimental animals have also reported
developmental toxicity (prenatal loss) at relatively high doses of carbon tetrachloride during
gestation. A graphical display of information from three developmental studies is provided in
Figure 5-2. Figure 5-3 displays PODs for the major targets of toxicity associated with oral
exposure to carbon tetrachloride. For the reasons discussed in Section 5.1.2, liver effects in the
rat observed in the study by Bruckner et al. (1986) are considered the most appropriate basis for
the carbon tetrachloride RfD. The text of Sections 5.1.1 and 5.1.2 should be consulted for a
more complete understanding of the issues associated with each data set and the rationale for the
selection of the critical effect and principal study used to derive the RfD.
Confidence in the principal study, Bruckner et al. (1986), is medium. The 12-week
gavage study is a well-conducted, peer-reviewed study that used three dose groups plus a control
and collected interim data at two-week intervals. The study is limited by relatively small group
sizes (5 to 9 rats/group) and investigation of only two target organs (liver and kidney).
Confidence in the oral database is medium. Two chronic oral animal studies were designed as
cancer bioassays, and one of the two included only limited investigation of noncancer endpoints.
The second chronic bioassay by NCI provided complete nonneoplastic incidence data; however,
because of the marked hepatotoxicity in dosed rats at the lowest dose tested and the low survival
in dosed mice as a result of the high incidence of liver tumors, the bioassay was not suitable for
dose-response analysis. The toxicity of carbon tetrachloride has been more thoroughly
investigated in a number of oral toxicity studies of subchronic duration, and a number of tests of
immunotoxic potential are available. The oral database lacks an adequate multigeneration study
of reproductive function. Testing for developmental toxicity has been performed in only one
species. Overall confidence in the RfD is medium.
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6.2.2. Noncancer - Inhalation Exposure
The most sensitive endpoint identified for effects of carbon tetrachloride by inhalation
exposure was liver toxicity in the chronic rat study by JBRC (Nagano et al., 2007b; JBRC,
1998), manifested at an exposure concentration of 25 ppm by elevated serum enzymes, fatty
change, fibrosis and cirrhosis. Other targets of carbon tetrachloride toxicity considered in the
selection of the critical effect included the kidney, the adrenal gland, and the developing
organism.
PBPK and BMD modeling methods were used to calculate the POD for deriving the RfC.
Exposure levels studied in the 2-year JBRC rat bioassay were converted to estimates of internal
dose metrics by application of PBPK models (Paustenbach et al., 1988; Thrall et al., 2000;
Benson and Springer, 1999); rate of carbon tetrachloride metabolism in the liver was considered
the most appropriate dose metric for liver toxicity. BMD modeling methodology (U.S. EPA,
2000c, 1995) was used to analyze the relationship between the estimated internal doses and
response (i.e., fatty change of the liver) by estimating the effective dose at a specified level of
response (BMDX) and its 95% lower bound (BMDLX). A 10% extra risk of fatty changes of the
liver was used as the BMR. All of the models for dichotomous data in U.S. EPA's BMDS
(version 1.4.1) (U.S. EPA, 2007) were fit to the incidence data for fatty liver in male and female
rats. In the male rat, the logistic model provided the best fit of the data. For female rats, no
models provided an adequate fit to the data when all dose groups were included, as assessed by
the i2 goodness-of-fit test. After dropping the highest dose, the multistage model provided the
best fit of the data. The resulting BMDLio values (expressed as internal doses) were converted
to estimates of equivalent human exposure concentrations (HECs) by applying a human PBPK
model and assuming a value for the human Vmaxc estimated from in vitro human data. An HEC
of 14.3 mg/m3 is used as the POD for RfC derivation. An RfC of 0.1 mg/m3 for carbon
tetrachloride is derived by applying a composite UF of 100 to the FIEC of 14.3 mg/m3. The
composite UF of 100 includes a factor of 10 to protect susceptible individuals, a factor of 3
(10°5) to extrapolate from rats to humans, and a factor of 3 (10°5) to account for an incomplete
database. Information was unavailable to quantitatively assess the potential variability in human
susceptibility (factors that could contribute to a range of human response include variations in
CYP450 levels, nutritional status, alcohol consumption, or the presence of underlying disease);
thus, a default UF of 10 was selected to account for the uncertainty in intraspecies variability. A
pharmacokinetic model was used to adjust for pharmacokinetic differences across species. A UF
of 3 was selected for interspecies extrapolation to account for potential pharmacodynamic
differences between rats and humans. A database UF of 3 was selected to account for an
incomplete database lacking a multigeneration reproductive toxicity.
To provide perspective on the RfC derived using data from the JBRC inhalation bioassay
in the rat, PODs and inhalation RfCs based on other selected studies of carbon tetrachloride
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inhalation toxicity are arrayed in Figures 5-6 to 5-8 presented in Section 5. The liver and kidney
are the predominant targets of carbon tetrachloride toxicity in subchronic and chronic inhalation
studies in laboratory animals and in humans based on case reports and studies in exposed
workers. Figures 5-6 and 5-7 provide graphical displays of information from studies that
reported liver or kidney toxicity in experimental animals following subchronic oral exposure,
including the PODs, applied uncertainty factors, and RfDs for comparison to that derived from
JBRC liver data. Benign pheochromocytomas from the adrenal gland medulla, that could
represent a potential noncancer health hazard, were observed following inhalation exposure only
in mice in the JBRC chronic bioassay. A single study of developmental toxicity found
significant reductions in fetal body weight and crown-rump length in rats at a carbon
tetrachloride concentration that was also toxic to the dams. Figure 5-8 displays PODs for all
major targets of carbon tetrachloride toxicity by the inhalation route. For the reasons discussed
in Section 5.2.2, liver effects in the rat observed in the study by JBRC are considered the most
appropriate basis for the carbon tetrachloride RfC. The text of Sections 5.2.1 and 5.2.2 should be
consulted for a more complete understanding of the issues associated with each data set and the
rationale for the selection of the critical effect and principal study used to derive the RfC.
Confidence in the principal study, the JBRC bioassay, is high. This chronic study was
well conducted, using two species and adequate numbers of animals. The JBRC chronic study
was preceded by a 13-week subchronic study, and an extensive set of endpoints was examined in
both studies. Confidence in the database, which includes the JBRC two-year chronic inhalation
bioassays in rats and mice, subchronic toxicity studies, and one study of immunotoxic potential,
is medium. Testing for developmental toxicity by inhalation exposure found effects only at high,
maternally toxic exposure concentrations but was limited to a single inhalation study in a single
species that did not test an exposure concentration low enough to identify a NOAEL for maternal
or fetal toxicity. The database lacks an adequate inhalation multigeneration study of
reproductive function. Overall confidence in the RfC is medium.
6.2.3. Cancer
Two approaches to low-dose extrapolation were applied in the dose-response assessment
for carbon tetrachloride carcinogenicity.
Nonlinear Approach. At high exposure levels, rodent bioassay data reveal a general
correspondence between hepatocellular cytotoxicity and regenerative hyperplasia and the
induction of liver tumors. Extensive mechanistic data support a hypothesized mode of action for
carbon tetrachloride-induced liver tumors that includes the following key events: (1) metabolism
to the trichloromethyl radical by CYP2E1 and subsequent formation of the trichloromethyl
peroxy radical, (2) radical-induced mechanisms leading to hepatocellular cytotoxicity, and
(3) sustained regenerative and proliferative changes in the liver in response to hepatotoxicity. A
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weight of evidence analysis of the genotoxicity literature suggests that carbon tetrachloride is
more likely an indirect than direct mutagenic agent. As a whole, this empirical evidence
provides significant biological support for the hypothesis that liver carcinogenicity by this mode
of action occurs at carbon tetrachloride exposures that also induce hepatocellular toxicity and a
sustained regenerative and proliferative response, and that exposures that do not cause
hepatotoxicity are not expected to result in liver cancer. This hypothesis is consistent with a
nonlinear approach to cancer assessment for liver tumors. The RfD and RfC were quantitatively
derived based upon hepatotoxicity (cytotoxicity). Hepatotoxicity is a key event for the
hypothesized nonlinear mode of action. Under an assumption of nonlinearity, the RfD of 0.004
mg/kg-day and RfC of 0.1 mg/m3 can be used to assess the potential risk of liver cancer from
carbon tetrachloride exposure for oral and inhalation exposures, respectively.
Linear Approach. Some bioassay data also reveal that at lower exposure levels the
correspondence between hepatocellular cytotoxicity and regenerative hyperplasia and the
induction of liver tumors is less consistent. In particular, liver findings from the JBRC bioassay
(Nagano et al., 2007b; JBRC, 1998) suggest that mouse hepatocarcinogenicity cannot simply be
explained in terms of the cytotoxic-proliferative mode of action. An increased incidence of
hepatocellular adenomas occurred in the low-dose (5-ppm) female mouse in the absence of
nonneoplastic liver toxicity, raising the possibility of another mode of action operating in
addition to the cytotoxic-proliferative mode of action.
Carbon tetrachloride also induced pheochromocytomas in male and female mice by oral
(NTP, 2007; Weisburger, 1977) and inhalation (Nagano et al., 2007b; JBRC, 1998) exposure.
The mode of action for pheochromocytomas in the mouse is unknown. Where the mode of
action for a tumor site is unknown, linear extrapolation is used as the default.
Other considerations suggest that the carbon tetrachloride database is insufficient for
ruling out other modes of action at low exposure levels, in particular considerations related to the
compound's genotoxicity and general reactivity. Carbon tetrachloride is metabolized to reactive
species (trichloromethyl and trichloromethyl peroxy radical), and subsequent chemical reactions
of carbon tetrachloride metabolites with cellular constituents lead to formation of reactive
oxygen species that also can damage DNA and other macromolecules. A concern exists
regarding the potential biological activity of carbon tetrachloride with macromolecules at low
exposures (i.e., exposure levels below doses that are cytotoxic). Data to characterize this low-
exposure activity is limited.
A weight of evidence analysis of the genotoxicity literature suggests that carbon
tetrachloride is more likely an indirect than direct mutagenic agent; however, the nature of the
genotoxicity database poses distinct challenges to the evaluation of carbon tetrachloride
genotoxicity. Positive genotoxicity findings have generally been observed at exposures that
induce cytotoxicity and regenerative cell proliferation. The majority of genotoxicity studies,
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however, have been conducted at relatively high exposure levels such that the potential for
genotoxic activity at low doses cannot be determined.
The above considerations provide support for the application of a low-dose linear
extrapolation approach to carbon tetrachloride carcinogenicity.
The 104-week inhalation bioassay in rats and mice conducted by JBRC (Nagano et al.,
2007b; JBRC, 1998) provided data adequate for dose-response modeling of the inhalation
pathway and was used as the basis for the IUR. Exposure levels studied in the 2-year JBRC rat
and mouse bioassay were converted to estimates of internal dose metrics by application of a
PBPK model. BMD modeling methodology (U.S. EPA, 2000c, 1995) was used to analyze the
relationship between the estimated internal doses and response (i.e., liver tumors in rats and mice
and pheochromocytomas in mice). The resulting BMDL values were converted to estimates of
equivalent human exposure concentrations (HECs) by applying a human PBPK model. Data for
male mouse pheochromocytomas yielded the highest estimate of the IUR of those data sets
modeled [i.e., 6 x 10'6 (ng/m3)'1].
Studies of carbon tetrachloride carcinogenicity in humans and experimental animals by
the oral exposure route are not sufficient to derive a quantitative estimate of cancer risk using
low-dose linear approaches. Therefore, PBPK modeling was applied to extrapolate inhalation
tumor data to the oral route. Because liver tumors and pheochromocytomas have been observed
in experimental animals following both inhalation and oral exposures, the data sets evaluated as
the basis for the IUR were considered appropriate for estimation of an oral SF. Data for female
mouse liver tumors yielded the highest estimate of the SF of those data sets modeled [i.e.,
7x 10"2 (mg/kg-day)"1].
Choosing an Extrapolation Approach. A linear low-dose extrapolation approach is
recommended for assessing carbon tetrachloride cancer risk for both liver tumors and
pheochromocytomas. For liver tumors, this recommendation was reached in light of (1)
evidence from the JBRC bioassay suggesting that mouse hepatocarcinogenicity cannot simply be
explained in terms of a cytotoxic-proliferative mode of action alone; (2) considerable evidence
that points to the involvement of highly reactive metabolites (with the capacity to chemically
interact with DNA and other cellular macromolecules) in the processes of toxicity and
carcinogenicity of carbon tetrachloride, and subsequent chemical reactions of carbon
tetrachloride metabolites with cellular constituents that can lead to formation of reactive oxygen
species that also can damage DNA and other macromolecules; and (3) a genotoxicity database
that, while large, is complex and has various issues that make it difficult to reach a firm
judgement about the potential for genotoxicity of carbon tetrachloride at doses below which
there is overt toxicity. Linear extrapolation is supported for pheochromocytomas (observed in
the male and female mouse by oral [NCI bioassay] and inhalation [JBRC bioassay] exposure) in
the absence of any understanding of the cancer mode of action for this tumor. The IUR of
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6 x 10"6 (jig/m3)"1 and oral SF of 7 x 10"2 (mg/kg-day)"1 should be used for assessing cancer risk
under a linear low-dose extrapolation approach.
Uncertainties in the Cancer Dose-Response Assessment. Major uncertainties in the
cancer assessment are described below:
Relevance to humans. The relevance of the mode of action of liver tumor induction to
humans was considered in Section 4.7.3.5. There is no evidence in humans for hepatic
cancer associated with carbon tetrachloride exposure. The experimental animal literature,
however, shows carbon tetrachloride to consistently induce liver tumors across species
and routes of exposure. Further, there are similarities between experimental animals and
humans in terms of carbon tetrachloride metabolism, antioxidant systems, and evidence
for the liver as a sensitive target organ. Together, this evidence supports a conclusion
that experimental evidence for liver cancer is relevant to humans.
Pheochromocytomas, on the other hand, were observed in only one species (the
mouse). In humans, pheochromocytomas are rare catecholamine-producing
neuroendocrine tumors that are usually benign, but may also present as or develop into a
malignancy (Eisenhofer et al., 2004; Salmenkivi et al., 2004; Tischler et al., 1996). In
humans, hereditary factors have been identified as important in the development of
pheochromocytomas (Eisenhofer et al., 2004). In the mouse, few chemicals have been
reported to cause mouse adrenal medullary tumors (Hill et al., 2003), and the mode of
action for this tumor in mice is unknown. The relevance of mouse pheochromocytomas
to humans is similarly unknown, although parallels between this tumor in the mouse and
human led investigators to concluded that the mouse might be an appropriate model for
human adrenal medullary tumors (Tischler et al., 1996). Like the human,
pheochromocytomas in the mouse are relatively rare, as are metastases. Both the
morphological variability of the mouse pheochromocytomas and the morphology of the
predominant cells are comparable to those of human pheochromocytomas. An important
characteristic of mouse pheochromocytomas is expression of immunoreactive
phenylethanolamine-N-methyltransferase (PNMT); human pheochromocytomas are also
usually PNMT-positive (Tischler et al., 1996). Overall, this evidence supports a
conclusion that experimental evidence for pheochromocytomas is potentially relevant to
humans.
Choice of low-dose extrapolation approach. The mode of action is a key determinant of
the approach to apply for estimating low-dose cancer risk. For liver tumors, two
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approaches to low-dose cancer risk were developed reflecting inconsistencies and
uncertainty in the mode of action at low carbon tetrachloride exposures.
The mode of action of carbon tetrachloride liver carcinogenicity has been
investigated extensively. Much of this research was conducted at relatively high
exposure levels, such that the mode(s) of action at low exposure levels cannot be
determined. Additional mode of action information in the low-dose region to establish
whether a linear or nonlinear approach applies to carbon tetrachloride liver tumors would
significantly reduce the uncertainty associated with magnitude of liver tumor risk.
The nonlinear extrapolation approach for liver tumors assumes that the RfD and
RfC can be used to assess the potential risk of liver cancer from carbon tetrachloride.
Uncertainties in the derivation of the RfD and RfC are discussed in Section 5.3. This
assumption is based upon the RfD and RfC which were both quantitatively derived from
hepatotoxicity (cytotoxicity) as a noncancer endpoint. Hepatotoxicity is identified as a
key event in the hypothesized nonlinear mode of action for liver tumors (see Section
4.7.1 and 5.4.1).
The effect on risk estimates derived using a linear extrapolation approach of using
only data on carbon tetrachloride liver cancer response at levels below those associated
with increased cell replication was examined. The risk calculations did not prove
particularly sensitive to the limitation of data points to below which increased cell
replication was reported. This consistency in cancer risk estimates provided some
confidence that the IUR and SF estimates based on liver tumor data are not driven by
high doses associated with significant hepatotoxicity.
In data sets where early mortality is observed, methods that can reflect the
influence of competing risks and intercurrent mortality on site-specific tumor incidence
rates are preferred. Survival curves for female rats and mice from the JBRC bioassay
(see Figures 4-1 and 4-2) show early mortality in some treated groups. Because liver
tumors were the primary cause of early deaths in these groups, failure to apply a time to
tumor analysis is not likely to significantly influence the inhalation unit risk for liver
tumors. The impact on the unit risk from pheochromocytomas is unknown.
Cancer risk estimates were calculated by straight line extrapolation from the POD
to zero, with the multistage model used to derive the POD. (The one exception is the
male mouse pheochromocytoma data set, where the log-probit model was used.) It is
unknown how well this extrapolation procedure predicts low-dose risks for carbon
tetrachloride. The multistage model does not represent all possible models one might fit,
and other models could conceivably be selected to yield more extreme results consistent
with the observed data, both higher and lower than those included in this assessment.
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For pheochromocytomas, only a linear low-dose extrapolation approach was used
to estimate human carcinogenic risk in the absence of any information on the mode of
action for this tumor. Mode of action information to establish whether a linear or
nonlinear approach applies to carbon tetrachloride-induced pheochromocytomas would
significantly reduce the uncertainty associated with the magnitude of risk from exposure
to this tumor type.
Cancer risk estimates for liver tumors and pheochromocytomas developed using a
linear low-dose extrapolation approach were not combined because different internal
dose metrics were used in the dose-response/PBPK analysis of these two tumor types.
Deriving the IUR or SF for data on one tumor site, however, may underestimate the
carcinogenic potential of carbon tetrachloride. For the IUR based on male mouse
pheochromocytomas, because of the poor resolution of the dose-response relationship for
male mouse liver tumors, the magnitude of the potential risk underestimation cannot be
characterized. Because the SF based on female mouse liver tumors was an order of
magnitude greater that that for female mouse pheochromocytomas, any underestimation
of the SF is expected to be small.
Interspecies extrapolation. Extrapolating dose-response data from animals to humans
was accomplished using PBPK models in the rat, mouse, and human. Availability of a
PBPK model generally reduces the pharmacokinetic component of uncertainty associated
with animal to human extrapolation; however, any PBPK model has its own associated
uncertainties. Specific uncertainties in the PBPK modeling for carbon tetrachloride are
discussed in Section 5.3.
Route-to-route extrapolation for the oral SF. Studies of carbon tetrachloride
carcinogenicity by the oral route were determined to be insufficient to derive a
quantitative estimate of cancer risk. Therefore, a human PBPK model was used to
extrapolate inhalation data to the oral route. A simple approximation method was used
that assumed continuous infusion of carbon tetrachloride from the human gastrointestinal
tract to the liver. Doses extrapolated from inhalation to oral exposures in this analysis
were approximations because they did not account for oral bioavailability or absorption
kinetics, information that is not available for carbon tetrachloride. The magnitude of
uncertainty introduced by these assumptions cannot be quantified.
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Statistical uncertainty at the point of departure. Parameter uncertainty can be assessed
through confidence intervals. Each description of parameter uncertainty assumes that the
underlying model and associated assumptions are valid. For the log-probit model applied
to the male mouse pheochromocytoma data, there is a reasonably small degree of
uncertainty at the 10% excess incidence level (the point of departure for linear low-dose
extrapolation); the lower bound on the BMD (i.e., the BMDLio) is 1.8-fold lower than the
BMD. For the multistage model applied to the female mouse liver tumor data, there is
similarly a reasonably small degree of uncertainty at the 10% excess incidence level; the
lower bound on the BMD (i.e., the BMDLio) is approximately 1.5-fold lower than the
BMD.
Bioassay selection. The study by Nagano et al. (2007b; also reported as JBRC, 1998)
was used for development of the inhalation unit risk. A full report of the bioassay
findings was published in 2007, although the study itself was conducted in the mid-
1980s. Although not a recently conducted study, this bioassay was well-designed, and
included both sexes in two species, an adequate number of animals per dose group, and
an appropriate untreated control group. Examination of toxicological endpoints in both
sexes of rats and mice was appropriate. No issues were identified with this bioassay that
might have contributed to uncertainty in the cancer assessment. Alternative bioassays for
developing an inhalation unit risk were unavailable.
Choice of species/gender. For liver tumors, modeling was performed using JBRC
inhalation bioassay from the female mouse and female rat. The male rat liver tumor data
were not modeled because these data sets lacked the resolution desired for dose-response
modeling; The male mouse liver data were modeled, but provided similarly poor dose-
response curve resolution. Tumor frequencies jumped from control levels to close to
maximal responses without any intervening dose levels having submaximal responses. In
the female mice and rats, lower but biologically significant levels of response were seen
at intermediate dose levels. Also, notably, increased levels of hepatocellular proliferation
were not reported for rodents at these intermediate levels, increasing the likelihood that
dose-response modeling may be relevant to lower dose conditions. There is no indication
that male rodents are more sensitive to carbon tetrachloride liver tumor induction and that
use of female data only underestimated potential risk. For pheochromocytomas, JBRC
inhalation data sets for both male and female mice were amenable to modeling, and the
data set yielding the highest estimate of cancer risk could be selected.
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Human population variability. Neither the extent of interindividual variability in carbon
tetrachloride metabolism nor human variability in response to carbon tetrachloride has
been fully characterized. Factors that could contribute to a range of human response to
carbon tetrachloride include variations in CYP450 levels because of age-related
differences or other factors (e.g., exposure to other chemicals that induce or inhibit
microsomal enzymes), nutritional status, alcohol consumption, or the presence of
underlying disease that could alter metabolism of carbon tetrachloride or antioxidant
protection systems. Incomplete understanding of the potential differences in metabolism
and susceptibility across exposed human populations represents a source of uncertainty.
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7. REFERENCES
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APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC
COMMENTS AND DISPOSITION
[to be added after external peer review and public comment period]
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APPENDIX B. DOSE-RESPONSE MODELING FOR DERIVING THE RfD
Serum enzyme data (indicators of liver toxicity) from Bruckner et al. (1986) are
summarized in Table B-l.
Table B-l. Serum enzyme data in male rats after 10- or 12-week
exposure to carbon tetrachloride
Daily dose
(mg/kg-day)
0
1
10
33
SDH (IU/mL)a
10 weeks
3. 5 ±0.4
2.3 ±0.6
7.6±2.5b
134.8 ±15.0b
12 weeks
3.2 ±0.4
1.9±0.1
8.7±2.0b
145.7 ± 57.9b
OCT (nmol CO2/mL)a
10 weeks
28 ±8
23 ±3
55 ±10
148 ± 48b
12 weeks
45 ±4
61 ±12
69 ±16
247±31b
ALT (IU/mL)a
10 weeks
18 ±1
20 ±1
23 ±1
617 ±334
12 weeks
20 ±0.3
19 ±1
27±2b
502±135b
aValues presented are mean standard error for groups of five rats at 10 weeks and seven to nine rats at 12
weeks.
V<0.05
Source: Bruckner et al., 1986.
B.I. BMD Modeling of SDH
SDH data for the 10-week time point were used for BMD analysis. Although serum
enzyme data for the 10- and 12-week time points are similar, the 10-week data were modeled
because the precise group sizes were not known for the 12-week data (a range of 7-9 rats per
group was reported), and these data are needed to run the BMD model.
All of the models for continuous data in U.S. EPA's BMDS (version 1.4.1) (U.S. EPA,
2007) were fit to the 10-week serum SDH data from Bruckner et al. (1986), which are shown in
Table B-l, column 2. Because of the nonhomogeneous variances in the SDH data, a
nonhomogeneous variance model was used in running each of the models in BMDS. A twofold
increase in mean control SDH was used as the BMR (see Section 5.1.2. for the rationale for
using this BMR), with "relative deviation" selected as the BMR type. As stated in U.S. EPA's
benchmark dose technical guidance (U.S. EPA, 2000c), relative deviation means the BMR will
be the background estimate (PO) plus (or minus) the product of the background estimate times
the BMR Factor (BRMF) entered by the user, or
BMR = PO ± (BMRF*PO)
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To achieve a doubling of the control mean, a BMRF of one was used. Thus, the BMR was
calculated as PO + (1 x PO) or 2 x PO. It should be noted that BMDS uses the fitted, or estimated,
value for the mean and standard deviation to calculate the BMR and BMD. The value estimated
by BMDS for the control SDH mean is 2.71 lU/mL (see detailed model run; a box appears
around the estimated mean). Thus the BMR using relative deviation (as the BMR type) and a
BMRF of 1 was calculated as BMR = 2.71 + (1 x 2.71) = 5.42.
Modeling results are summarized in Table B-2. The 3rd degree polynomial and power
models provided adequate fits of the 10-week SDH data (based on a goodness-of-fit p-value
>0.1); with both models, the modeling of the variance (test 3 in BMDS output) was marginally
adequate (p-value = 0.07515). The power model provided the better fit of the data (based on the
lowest AIC value) and therefore was selected as the basis for deriving the RfD; this model
estimated a BMD2X of 7.32 mg/kg-day and BMDL2X of 5.46 mg/kg-day. Figure B-l shows the
power model fit to the SDH data and the associated BMD2X and BMDL2x; the detailed model
run is provided at the end of this section.
Table B-2. Model predictions for changes in serum SDH levels (lU/mL) in
male rats exposed to carbon tetrachloride for 10 weeks
Model
Linearb
Polynomial (3rd degree)b'c
Powerd
Hilld
/rvalue"
O.0001
0.253
0.264
NAe
AICf for fitted
model
138.26
85.95
85.88
87.84
BMD2X
(mg/kg-day)
5 x 10"8
7.15
7.32
8.88
BMDL2X
(mg/kg-day)
4.5 x 10"8
4.29
5.46
5.49
ap-value for Test 4: Does the model fit? Values <0.10 fail to meet conventional goodness-of-fit criteria.
b Betas restricted to. 0.
0 Insufficient degrees of freedom to fit higher degree polynomials.
d Power restricted to 1.
e Insufficient degrees of freedom.
f AIC = Akaike's Information Criterion.
For purposes of comparison across chemicals, the BMD and BMDL corresponding to a
change in the mean response equal to one control standard deviation (SD) from the control mean
were also calculated, consistent with BMD guidance (U.S. EPA, 2000c):
: 5.5 mg/mg-day
o: 3.8 mg/kg-day
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Power Model with 0.95 Confidence Level
I
o
ro
CD
150
100
50
0
Power
BMDL, , , , BMP
0 5
13:2712/282006
10
15 20
dose
25 30
Figure B-l. Power model fit to the SDH data of Bruckner et al. (1986)
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BMDS MODEL RUN - Power Model
The form of the response function is:
Y[dose] = control + slope * dose^power
Dependent variable = MEAN
Independent variable = Dose(mg/kg-d)
The power is restricted to be greater than or egual to 1
The variance is to be modeled as Var(i) = alpha*mean(i)^rho
Total number of dose groups = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
alpha = 289.698
rho = 0
control = 2.3
slope = 0.0106715
power = 2.69605
Asymptotic Correlation Matrix of Parameter Estimates
alpha rho control slope
alpha
rho
control
slope
power
Parameter Estimates
power
Wald Confidence Interval
Lower Conf. Limit
-0.163949
1.13449
1.85783
-0.00952409
2.09436
0
1
10
33
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Likelihoods of Interest
Model Log(likelihood) # Param's
Al -64.456951 5
A2 -34.731110 8
A3 -37.319331 6
fitted -37.942951 5
R -91.888765 2
Test 1: Do responses and/or variances differ among Dose levels? (A2 vs. R)
Test 2: Are Variances Homogeneous? (Al vs A2)
Test 3: Are variances adequately modeled? (A2 vs. A3)
Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)
(Note: When rho=0 the results of Test 3 and Test 2 will be the same.)
Tests of Interest
Test -2*log(Likelihood Ratio) Test df p-value
Test 1 114.315 6
Test 2 59.4517 3
Test 3 5.17644 2
Test 4 1.24724 1
The p-value for Test 3 is less than .1. You may want to consider a different variance model.
The p-value for Test 4 is greater than .1. The model chosen seems to adequately describe the
data.
Benchmark Dose Computation
Specified effect = 1
Risk Type = Relative risk
Confidence level = 0.95
BMD = 7.32096
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B.2. BMD Modeling of OCT and ALT
BMD modeling was also conducted for OCT and ALT. Available continuous-variable
models in the EPA BMDS (linear, polynomial, power, and Hill models; BMDS version 1.4.1;
U.S. EPA, 2007) were fit to the data shown in Table B-l for changes in serum OCT and ALT in
male rats exposed to carbon tetrachloride for 10 weeks (Bruckner et al., 1986). For each of these
endpoints, a twofold increase in mean enzyme level was used as the BMR (see Section 5.1.2.),
with relative deviation as the BMR type and a BMRF of one (see Section B.I). A
nonhomogeneous variance model was used in running each of the models in BMDS.
Modeling results are summarized in Tables B-3 and B-4. None of the models for
continuous data provided an adequate fit to the 10-week OCT data (based on a goodness-of-fit p-
value >0.1). The power model provided an adequate fit of the 10-week ALT data; however, as
shown in Table B-l, the standard error of the mean ALT for the high-dose (33 mg/kg-day) rats
was extremely large (617 ± 334). Bruckner et al. (1986) noted: "There was a pronounced rise in
GPT [ALT] at 10 and 12 weeks. Scrutiny of values of individual animals revealed that dramatic
increases in two rats at each time point were largely responsible for the late increase in GPT
[ALT] activity." In light of the large variation in response at 33 mg/kg-day, using this data set
for quantitative analysis was not considered appropriate.
Table B-3. Model predictions for changes in serum OCT levels (nmol COi/mL) in
male rats exposed to carbon tetrachloride for 10 weeks
Model
Linearb
Polynomial (2nd degree)b'c
Powerd
Hilld
p value"
0.0449
0.0427
0.0553
NAf
AICe for fitted
model
157.57
157.47
157.04
158.60
BMD2X
(mg/kg-day)
8.04
11.4
11.04
10.12
BMDL2X
(mg/kg-day)
4.44
5.86
6.19
6.52
a Values <0.10 fail to meet conventional goodness-of-fit criteria.
b Betas restricted to >0.
0 Insufficient degrees of freedom to fit higher degree polynomials.
d Power restricted to>l.
e AIC = Akaike's Information Criterion.
fNA = not available; insufficient degrees of freedom.
Source: Bruckner et al., 1986.
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Table B-4. Model predictions for changes in serum ALT levels (lU/mL) in male
rats exposed to carbon tetrachloride for 10 weeks
Model
Linearb
Polynomial (3rd degree)b'c
Powerd
Hilld
p value"
O.0001
0.01022
0.1145
NAf
AICf for fitted
model
291.27
123.31
118.70
120.70
BMD2X
(mg/kg-day)
33.05
13.66
14.66
NAf
BMDL2X
(mg/kg-day)
0.0071
12.71
13.21
NAf
a Values <0.10 fail to meet conventional goodness-of-fit criteria.
b Betas restricted to >0.
0 Insufficient degrees of freedom to fit higher degree polynomials.
d Power restricted to >1.
e AIC = Akaike's Information Criterion.
f NA = not available; insufficient degrees of freedom (BMD software could not generate a model output).
Source: Bruckner et al, 1986.
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APPENDIX C. PBPK MODELING
C.I. Paustenbach et al. (1988) and Thrall et al. (2000) PBPK Models (rat, mouse, human)
Detailed summaries of the Paustenbach et al. (1988) and Thrall et al. (2000) PBPK
models appear in Section 3.5. Source code for the rat, mouse, and hamster models (reported in
Thrall et al., 2000) in Advanced Continuous Simulation Language (ACSL) was graciously
provided to Syracuse Research Corporation (SRC) by Dr. Karla Thrall. Included with the code
were data collected from gas uptake studies conducted in these species (also reported in Thrall et
al., 2000). Accuracy of the implementation of the rat and mouse models in ACSL (version
11.8.4) was verified by comparing model predictions to observations from the closed chamber
studies. These simulations are shown in Figures C-l and C-2. The comparisons of observed and
predicted closed chamber CCU concentrations as a function of exposure times match those
reported in Figure 2 of Thrall et al. (2000).
10000
1000
Q.
Q.
ra
'c
c
3
(5
.Q
6
100,
Gas Chamber Simulations - Rat
Figure C-l. Comparison of observed and predicted chamber carbon tetrachloride
concentrations in closed chamber studies conducted in rats.
Data points are observations (provided by Thrall) for exposures for 3 rats per chamber
(body weight, 0.24 kg); lines are simulations. The non-specific loss rate of carbon
tetrachloride from the chamber was assumed to be 0.05 hr"1 (from Thrall). Partition
coefficients were from Thrall source code.
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10000
E
Q.
CL
c
g
|
I
o
O
.
E
ra
.c
O
1000 ,
100 :
Gas Chamber Simulations - Mouse
Figure C-2. Comparison of observed and predicted chamber carbon
tetrachloride concentrations in closed chamber studies conducted in mice.
Data points are observations (provided by Thrall) for exposures for 7 mice per
chamber (body weight, 0.024 kg); lines are simulations. The non-specific loss
rate of CCU from the chamber was assumed to be 0.05 hr"1 (from Thrall source
code). Partition coefficients were from Thrall source code.
As noted above, Thrall et al. (2000) compared model predictions for the rat and mouse
with experimental data collected over a 48-hour period following a 4-hour nose-only inhalation
exposure to 20 ppm of [14C]-carbon tetrachloride (data from a personal communication and not
presented in Thrall et al. (2000)). This comparison of PBPK model-predicted and
experimentally-observed values for selected parameters is provided in Table C-l. Thrall et al.
(2000) also compared the model simulation for humans with human data of Stewart et al. (1961)
(see Figure C-3). As this figure shows, the model simulation of expired carbon tetrachloride
levels provided good agreement with the experimental data, particularly at longer periods
postexposure.
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Table C-l. Comparison of predicted and observed values for selected parameters
from toxicokinetic data collected from rats and mice 48 hours post exposure to a 4-
hour nose-only inhalation exposure (20 ppm carbon tetrachloride)
Species
Rat
Mouse
Parameter
Initial body burden
Total amount trapped by KOHb
Total amount trapped on charcoal0
Total amount metabolized"1
Initial body burden
Total amount trapped by KOHb
Total amount trapped on charcoal0
Total amount metabolized"1
Model
(umol)
7.8
2.8
4.1
3.7
2.2
0.95
0.94
1.3
Data
(umol equivalents of
CO4±SD)a
11.7 ±0.54
2.7 ±0.25
7.4 ±0.44
3.7 ±0.22
2.0 ±0.48
0.69 ±0.11
0.76 ±0.37
1.2±0.11
Ratio
(predicted/observed)
0.7
1.0
0.6
1.0
1.1
1.4
1.2
1.1
an= 3-4 animals.
b 14CO2 measured using a KOH trap.
0 Parent compound (14CC14) measured using a charcoal trap.
d Represents the sum of radioactivity (in umol equivalents) in urine, feces, and trapped on KOH (CO2).
Source: Thrall et al. (2000); Benson and Springer (1999).
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100
Time, hr
Figure C-3. Comparison of the actual versus predicted concentration of carbon
tetrachloride in the expired breath of humans exposed to 10 ppm of carbon
tetrachloride for 180 minutes (data from Stewart et al., 1961).
Source: Thrall et al. (2000); Benson and Springer (1999)
Parameter values for the rat and human models used in the Paustenbach et al. (1988) and
Thrall et al. (2000) models are summarized in Table C-2. Parameter values for the mouse are
shown in Table C-3.
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Table C-2. Parameter values for rat and human models3
Parameter
BW
VLC
VFC
VSC
VRC
QCC
QPC
QLC
QFC
QSC
QRC
PB
PL
PF
PS
PR
Vmaxc
Kmx
Definition
Body weight (kg)
Liver volume (fraction of body)
Fat volume (fraction of body)
Slowly-perfused tissue volume (fraction of body)
Rapidly -perfused tissue volume (fraction of body)
Cardiac output (L/hour-kg BW)
Alveolar ventilation rate (L/hour-kg B W)
Liver blood flow (fraction of cardiac output)
Fat blood flow (fraction of cardiac output)
Slowly-perfused blood flow (fraction of cardiac output)
Rapidly -perfused blood flow (fraction of cardiac output)
Blood:air partition coefficient
Liverblood partition coefficient
Fatblood partition coefficient
Slowly -perfused partition coefficient
Readily -perfused partition coefficient
Maximum rate of metabolism (mg/hour-kg B W)
Michaelis-Menten coefficient for metabolism (mg/L)
Rat model
0.452b
0.04c'd
0.08c'd
0.74c'd
0.05c'd
15c'd
15c'd
0.25c'd
0.04c'd
0.2c'd
0.51c'd
4.52e
3.14e
79.42e
1"
3.14f
0.4e,0.65c
0.25c'd
Human model
70
0.04C
0.2g
0.62C
0.05C
15C
15C
0.25C
0.06C
0.18C
0.51C
2.64C
3.14e
79.42e
1"
3.14f
0.4e, 0.65C, 1.49d,
1.7d
0.25c'd
aSee summary of the Paustenbach et al. (1988) and Thrall et al. (2000) models in Section 3.5 for
discussion the source of parameter values.
bTime-weighted mean body weight for the exposure group of interest (0.452 kg, male rats) and an
exposure of 3 ppm, 6 hours/day, 5 days/week (based on Nagano et al., 2007b; JBRC, 1998).
'Paustenbach et al., 1988.
dThrall et al., 2000.
"Gargasetal, 1986.
Partition coefficient for readily-perfused is assumed to be equal to that of liver.
8Adjusted from reported value of 0.1 in Paustenbach et al., 1988.
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Table C-3. Parameter values for mouse models3
Parameter
BW
VLC
VFC
VSC
VRC
QCC
QPC
QLC
QFC
QSC
QRC
PB
PL
PF
PS
PR
Vmaxc
Kmx
Kl
K2
K2
Definition
Body weight (kg)
Liver volume (fraction of body)
Fat volume (fraction of body)
Slowly-perfused tissue volume (fraction of body)
Richly-perfused tissue volume (fraction of body)
Cardiac output (L/hour-kg BW^)411
Alveolar ventilation rate (L/hour-kg BWSF)d'h
Liver blood flow (fraction of cardiac output)
Fat blood flow (fraction of cardiac output)
Slowly-perfused blood flow (fraction of cardiac output)
Richly-perfused blood flow (fraction of cardiac output)
Blood:air partition coefficient
Liverblood partition coefficient
Fatblood partition coefficient
Slowly -perfused partition coefficient
Richly-perfused partition coefficient
Maximum rate of metabolism (mg/hour-kg B WSF)fj
Michaelis-Menten coefficient for metabolism (mg/L)
GI absorption rate coefficient Cl-liver (hour"1)
GI absorption rate coefficient C1-C2 (hour"1)
GI absorption rate coefficient C2 -liver (hour"1)
Thrall et al.
(2000)
0.036b
0.04C
0.04C
0.78C
0.05C
28c'd
28c'd
0.24C
0.05C
0.19C
0.52C
7.83C
2.08e
23.0e
0.61e
2.08e
0.79e'f
0.46e
~
~
~
Fisher et al.
(2004)
~
0.04s
0.04g
0.69g
0.14s
30g"h
30g'h
0.24s
0.05g
0.17g
0.54s
3.8h
4.8h
91.4h
2.5h
4.8h
rj
0.31
0.4, 10k
2k
0.051
aSee Paustenbach et al. (1988) and Thrall etal. (2000) for discussion the source of parameter values.
bReference value for mouse body weight in a chronic study (0.036 kg; U.S. EPA, 1988)
"Andersen etal., 1987
d SF, scaling factor; QC (L/hour)=QCC*B W°74; QP (L/hour)=QPC*B W°74
"Thrall source code (CARBON TETRACHLORIDE PBPK MODEL KD THRALL 3/98
ITRICCL4.ACSL). Thrall et al. (2000) reported the tissue:blood partition coefficients for the mouse were
based on values for blood:air for the mouse (7.83) from Thrall et al. (2000) and tissue:air values
(liver:air=14.2; muscle:air=4.54; fat:air=359) from Gargas et al. (1986). The corresponding tissue:blood
values would be: PL=1.81; PF=45.85; PS=0.58; PR=1.81.
f SF, scaling factor; VMAX=VBMAXC*BW°70
gBrownetal., 1997
h SF, scaling factor; QC (L/hour)=QCC*BW075; QP (L/hour)=QPC*B W°75
'Fisher et al. (2004) vial equilibrium measurements
J VMAX=VBMAXC*BW075
kFisher et al. (2004) fit to closed chamber data.
'Fisher et al. (2004) fit to oral gavage blood data. Kl values are 0.4 hr"1 for 20 mg/kg dose and 10 hr"1 for
50 and 100 mg/kg dose.
C-6
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C.2. Fisher PBPK Model (mouse)
A detailed summary of the mouse PBPK model developed by Fisher et al. (2004) is
provided in Section 3.5. This model was reconstructed from the information provided in their
paper.
Fisher et al. (2004) performed gas uptake experiments with mice at four concentrations of
carbon tetrachloride to estimate metabolic constants. As shown in Figure C-3, metabolic
constants provided a good fit between model predictions and observations for the gas uptake
study.
Parameter values for the mouse used in the Fisher et al. (2004) model are summarized in
Table C-3 and are compared with the mouse parameter values from the Thrall et al. (2000)
model. Values for Km and Vmaxc used in the two models are similar: 0.3 mg/L, 1 mg/hr/kg0'75
(Fisher et al., 2004) compared to 0.46 mg/L, 0.79 mg/hr/kg0'70 (Thrall et al., 2000); although
different allometric scaling factors were used to scale Vmax to body weight. The corresponding
Vmax values for a 0.036-kg mouse are 0.077 mg/hr (Thrall et al., 2000) and 0.082 mg/hr (Fisher
et al., 2004). Tissue partition coefficients used in the Fisher et al. (2004) model were 2-4 times
higher than in the Thrall et al. (2000) model.
14000
I ii
1 imt (hrs)
Figure C-4. Atmospheric clearance of carbon tetrachloride from gas uptake
chambers containing mice (initial concentrations about 50,130, 450, or
1250 ppm). At initial concentrations of 50, 450 and 1250 ppm: 3 mice in a 2-liter
chamber; at 130 ppm: 7 mice in a 9-liter chamber. Metabolic constants for carbon
tetrachloride (Vmaxc (mg/h/kg) and Km (mg/1)) were estimated by fitting the gas
uptake data with the carbon tetrachloride PBPK model.
Source: Fisher et al. (2004)
C-7
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C.3. PBPK Modeling of Human Equivalent Concentrations and Doses
Interspecies extrapolation (i.e., rat-to-human, mouse-to-human) and route-to-route
extrapolation of carbon tetrachloride inhalation dosimetry was accomplished using a human
PBPK model described in Paustenbach et al. (1988), Thrall et al. (2000), and Benson and
Springer (1999). The human PBPK model was used to estimate the continuous chronic human
inhalation exposure in mg/m3 (abbreviated as EC in the following tables) or the rate of uptake of
carbon tetrachloride from the GI tract to the liver (i.e., chronic daily ingested dose) in mg/kg-day
(abbreviated RGIL in the following tables) that would result in values for the internal dose
metrics, MCA or MRAMKL, equal to the respective BMDLs for each toxicity endpoint (i.e.,
RfC: fatty liver degeneration; cancer: liver tumors in rats, liver tumors and adrenal
pheochromocytomas in mice). This procedure is described in Section 5.4.2.3.4.
Conversion factors that relate EC or RGIL to the two dose metrics (MCA and
MRAMKL) for each of the assumed values of human Vmaxc (0.40, 0.65, 1.49, or 1.70 mg/hr/kg
BW°-70) are provided in Tables C-4 to C-l 1. Figures C-5 to C-12 display plots of MCA and
corresponding values of EC or RGIL predicted from the human PBPK model, with trend
equations developed to permit the calculation of EC or RGIL for any value of MCA. Trend
equations shown on the plots are power functions fit to each data set using the method of least
squares (Microsoft Excel). The corresponding fit to the PBPK model predictions were evaluated
by R2 (shown on the trend plots) and the magnitude of the difference between PBPK model
predictions and the trend function predictions (i.e., shown in the plots of % delta, where % delta
= 100*[Trend-PBPK]/PBPK). If values for % delta uniformly <5% could not be achieved with
single trend functions applied to the full ranges of internal dose metric values presented in Tables
C-4 to C-l 1, trend functions were developed for subsets of the full MCA range that yielded
achieved % delta values <5%. Similar plots were developed for the dose metric MRAMKL (see
Figures C-13 and C-14).
C-8 DRAFT - DO NOT CITE OR QUOTE
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Table C-4. Interspecies conversion factors based on MCA dose metric
(VMAXC=0.40)
EC
(ppm)
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
2
3
4
5
6
7
8
9
10
20
30
40
50
60
70
80
90
100
EC
(mg/m3)
0.6290
1.258
1.887
2.516
3.145
3.774
4.403
5.032
5.661
6.290
12.58
18.87
25.16
31.45
37.74
44.03
50.32
56.61
62.90
125.8
188.7
251.6
314.5
377.4
440.3
503.2
566.1
629.0
MCA
(jimol/L)
0.009182
0.01837
0.02757
0.03678
0.04599
0.05522
0.06445
0.07369
0.08293
0.09219
0.1852
0.2790
0.3735
0.4687
0.5646
0.6611
0.7583
0.8560
0.9543
1.961
2.995
4.045
5.103
6.167
7.234
8.304
9.375
10.447
RGIL
(mg/kg/day)
0.1016
0.2021
0.3019
0.4007
0.4987
0.5959
0.6923
0.7880
0.8829
0.9772
1.887
2.752
3.584
4.392
5.183
5.961
6.729
7.490
8.245
15.67
23.06
30.47
37.91
45.36
52.82
60.29
67.77
75.25
RGIL/EC
(mg/kg/day/
mg/m3)
0.1614
0.1607
0.1600
0.1592
0.1586
0.1579
0.1572
0.1566
0.1560
0.1554
0.1500
0.1458
0.1424
0.1396
0.1373
0.1354
0.1337
0.1323
0.1311
0.1245
0.1222
0.1211
0.1205
0.1202
0.1200
0.1198
0.1197
0.1196
EC/MCA
(mg/m3/
jimol/L)
68.51
68.48
68.45
68.42
68.38
68.35
68.32
68.29
68.26
68.23
67.94
67.65
67.37
67.11
66.85
66.60
66.36
66.14
65.92
64.17
63.01
62.21
61.63
61.20
60.87
60.60
60.39
60.21
RGIL/MCA
(mg/kg/day/
jimol/L)
11.06
11.00
10.95
10.89
10.84
10.79
10.74
10.69
10.65
10.60
10.19
9.864
9.595
9.370
9.180
9.016
8.874
8.749
8.640
7.992
7.699
7.534
7.428
7.355
7.302
7.261
7.229
7.203
EC, air exposure concentration; MCA, time-averaged arterial concentration of carbon tetrachloride; RGIL,
rate of uptake of carbon tetrachloride from Gl-tract to liver; VMAXC, maximum rate of metabolism of
carbon tetrachloride (mg/hr/kg B W°70).
C-9
DRAFT - DO NOT CITE OR QUOTE
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Table C-5. Interspecies conversion factors based on MCA dose metric
(VMAXC=0.65)
EC
(ppm)
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
2
3
4
5
6
7
8
9
10
20
30
40
50
60
70
80
90
100
EC
(mg/m3)
0.6290
1.258
1.887
2.516
3.145
3.774
4.403
5.032
5.661
6.290
12.58
18.87
25.16
31.45
37.74
44.03
50.32
56.61
62.90
125.8
188.7
251.6
314.5
377.4
440.3
503.2
566.1
629.0
MCA
(jimol/L)
0.008674
0.01735
0.02604
0.03474
0.04344
0.05215
0.06087
0.06959
0.07832
0.08706
0.1748
0.2633
0.3525
0.4424
0.5330
0.6243
0.7162
0.8087
0.9019
1.864
2.866
3.893
4.936
5.988
7.047
8.110
9.176
10.244
RGIL
(mg/kg/day)
0.1182
0.2350
0.3504
0.4645
0.5774
0.6890
0.7995
0.9088
1.0171
1.1243
2.147
3.097
3.994
4.853
5.683
6.489
7.279
8.055
8.821
16.21
23.51
30.85
38.22
45.63
53.05
60.50
67.95
75.41
RGIL/EC
(mg/kg/day/
mg/m3)
0.1879
0.1868
0.1857
0.1846
0.1836
0.1826
0.1816
0.1806
0.1797
0.1787
0.1706
0.1641
0.1588
0.1543
0.1506
0.1474
0.1447
0.1423
0.1402
0.1289
0.1246
0.1226
0.1215
0.1209
0.1205
0.1202
0.1200
0.1199
EC/MCA
(mg/m3/
jimol/L)
72.52
72.49
72.46
72.43
72.40
72.37
72.34
72.31
72.28
72.25
71.96
71.66
71.37
71.09
70.81
70.54
70.27
70.00
69.75
67.51
65.85
64.63
63.72
63.03
62.48
62.05
61.70
61.41
RGIL/MCA
(mg/kg/day/
jimol/L)
13.63
13.54
13.45
13.37
13.29
13.21
13.14
13.06
12.99
12.91
12.28
11.760
11.331
10.969
10.661
10.395
10.164
9.961
9.780
8.699
8.203
7.923
7.743
7.619
7.529
7.460
7.406
7.362
EC, air exposure concentration; MCA, time-averaged arterial concentration of carbon tetrachloride; RGIL,
rate of uptake of carbon tetrachloride from Gl-tract to liver; VMAXC, maximum rate of metabolism of
carbon tetrachloride (mg/hr/kg B W°70).
C-10
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-6. Interspecies conversion factors based on MCA dose metric
(VMAXC=1.49)
EC
(ppm)
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
2
3
4
5
6
7
8
9
10
20
30
40
50
60
70
80
90
100
EC
(mg/m3)
0.6290
1.258
1.887
2.516
3.145
3.774
4.403
5.032
5.661
6.290
12.58
18.87
25.16
31.45
37.74
44.03
50.32
56.61
62.90
125.8
188.7
251.6
314.5
377.4
440.3
503.2
566.1
629.0
MCA
(jimol/L)
0.007827
0.01566
0.02349
0.03133
0.03917
0.04702
0.05487
0.06272
0.07058
0.07844
0.1573
0.2365
0.3161
0.3962
0.4766
0.5575
0.6388
0.7205
0.8027
1.650
2.545
3.482
4.454
5.453
6.470
7.501
8.542
9.590
RGIL
(mg/kg/day)
0.1742
0.3457
0.5146
0.6808
0.8447
1.0060
1.1651
1.3219
1.4766
1.6291
3.053
4.326
5.487
6.559
7.564
8.514
9.419
10.288
11.130
18.67
25.69
32.67
39.74
46.90
54.13
61.42
68.76
76.13
RGIL/EC
(mg/kg/day/
mg/m3)
0.2770
0.2748
0.2727
0.2706
0.2686
0.2665
0.2646
0.2627
0.2608
0.2590
0.2427
0.2293
0.2181
0.2085
0.2004
0.1934
0.1872
0.1817
0.1769
0.1484
0.1361
0.1299
0.1263
0.1243
0.1229
0.1221
0.1215
0.1210
EC/MCA
(mg/m3/
jimol/L)
80.37
80.35
80.33
80.31
80.29
80.27
80.25
80.23
80.21
80.19
79.99
79.80
79.60
79.39
79.19
78.98
78.78
78.57
78.36
76.24
74.16
72.26
70.61
69.22
68.06
67.09
66.28
65.59
RGIL/MCA
(mg/kg/day/
jimol/L)
22.26
22.08
21.90
21.73
21.56
21.40
21.23
21.07
20.92
20.77
19.41
18.294
17.358
16.557
15.871
15.272
14.744
14.278
13.864
11.316
10.095
9.384
8.922
8.601
8.367
8.188
8.049
7.938
EC, air exposure concentration; MCA, time-averaged arterial concentration of carbon tetrachloride; RGIL,
rate of uptake of carbon tetrachloride from Gl-tract to liver; VMAXC, maximum rate of metabolism of
carbon tetrachloride (mg/hr/kg B W°70).
C-ll
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-7. Interspecies conversion factors based on MCA dose metric
(VMAXC=1.70)
EC
(ppm)
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
2
3
4
5
6
7
8
9
10
20
30
40
50
60
70
80
90
100
EC
(mg/m3)
0.6290
1.258
1.887
2.516
3.145
3.774
4.403
5.032
5.661
6.290
12.58
18.87
25.16
31.45
37.74
44.03
50.32
56.61
62.90
125.8
188.7
251.6
314.5
377.4
440.3
503.2
566.1
629.0
MCA
(jimol/L)
0.007709
0.01542
0.02314
0.03086
0.03858
0.04630
0.05403
0.06177
0.06950
0.07724
0.1548
0.2327
0.3110
0.3896
0.4686
0.5480
0.6277
0.7078
0.7883
1.616
2.488
3.403
4.355
5.336
6.340
7.361
8.394
9.435
RGIL
(mg/kg/day)
0.1882
0.3735
0.5557
0.7351
0.9118
1.0857
1.2571
1.4259
1.5924
1.7565
3.284
4.642
5.873
7.005
8.060
9.051
9.993
10.893
11.758
19.40
26.38
33.28
40.25
47.32
54.49
61.73
69.03
76.36
RGIL/EC
(mg/kg/day/
mg/m3)
0.2993
0.2969
0.2945
0.2922
0.2899
0.2877
0.2855
0.2834
0.2813
0.2792
0.2610
0.2460
0.2334
0.2227
0.2135
0.2055
0.1986
0.1924
0.1869
0.1542
0.1398
0.1323
0.1280
0.1254
0.1238
0.1227
0.1219
0.1214
EC/MCA
(mg/m3/
jimol/L)
81.60
81.58
81.56
81.54
81.53
81.51
81.49
81.47
81.46
81.44
81.26
81.09
80.91
80.73
80.54
80.36
80.17
79.98
79.79
77.83
75.84
73.94
72.22
70.73
69.45
68.36
67.45
66.67
RGIL/MCA
(mg/kg/day/
jimol/L)
24.42
24.22
24.02
23.82
23.63
23.45
23.26
23.09
22.91
22.74
21.21
19.948
18.885
17.978
17.200
16.517
15.920
15.390
14.915
12.003
10.602
9.780
9.242
8.868
8.595
8.386
8.224
8.093
EC, air exposure concentration; MCA, time-averaged arterial concentration of carbon tetrachloride; RGIL,
rate of uptake of carbon tetrachloride from Gl-tract to liver; VMAXC, maximum rate of metabolism of
carbon tetrachloride (mg/hr/kg B W°70).
C-12
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-8. Interspecies conversion factors based on MRAMKL dose metric
(VMAXC=0.40)
EC
(ppm)
1
2
3
4
5
6
7
8
9
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
EC
(mg/m3)
6.290
12.58
18.87
25.16
31.45
37.74
44.03
50.32
56.61
62.90
125.8
188.7
251.6
314.5
377.4
440.3
503.2
566.1
629.0
691.9
754.8
817.8
880.7
943.6
1006
1069
1132
1195
1258
1321
1384
1447
1510
1573
1636
1698
MRAMKL
(nmol/hr/kg
liver)
0.7352
1.433
2.093
2.719
3.311
3.872
4.402
4.903
5.377
5.826
9.196
11.24
12.57
13.48
14.15
14.65
15.05
15.36
15.62
15.84
16.02
16.18
16.31
16.43
16.53
16.63
16.71
16.78
16.85
16.91
16.97
17.02
17.06
17.11
17.15
17.19
RGIL
(mg/kg/day)
0.2980
0.5960
0.8940
1.192
1.490
1.788
2.086
2.384
2.682
2.980
5.959
8.938
11.92
14.89
17.87
20.85
23.83
26.80
29.78
32.75
35.73
38.70
41.68
44.65
47.63
50.60
53.57
56.54
59.52
62.49
65.46
68.43
71.40
74.37
77.34
80.31
RGIL/EC
(mg/kg/day/
mg/m3)
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04736
0.04736
0.04735
0.04735
0.04735
0.04734
0.04734
0.04734
0.04733
0.04733
0.04733
0.04732
0.04732
0.04732
0.04732
0.04731
0.04731
0.04731
0.04730
0.04730
0.04730
0.04730
0.04729
0.04729
0.04728
EC/MRAMKL
(mg/m3/
jimol/hr/kg liver)
8.556
8.782
9.015
9.254
9.498
9.749
10.004
10.264
10.529
10.798
13.681
16.792
20.025
23.329
26.675
30.049
33.442
36.849
40.265
43.689
47.119
50.553
53.990
57.430
60.873
64.318
67.765
71.213
74.662
78.112
81.563
85.015
88.468
91.921
95.375
98.830
RGIL/MRAMKL
(mg/kg/day/
jimol/hr/kg liver)
0.4053
0.4161
0.4271
0.4384
0.4500
0.4618
0.4739
0.4862
0.4987
0.5115
0.6480
0.7953
0.9483
1.105
1.263
1.423
1.583
1.744
1.906
2.068
2.230
2.393
2.555
2.718
2.880
3.043
3.206
3.369
3.532
3.695
3.858
4.021
4.184
4.347
4.510
4.673
C-13
DRAFT - DO NOT CITE OR QUOTE
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Table C-8. Interspecies conversion factors based on MRAMKL dose metric
(VMAXC=0.40)
280
290
300
1761
1824
1887
17.22
17.25
17.28
83.28
86.24
89.21
0.04728
0.04728
0.04728
102.284
105.740
109.195
4.836
4.999
5.162
EC, air exposure concentration; MRAMKL, time-averaged rate of metabolism of carbon tetrachloride;
RGIL, rate of uptake of carbon tetrachloride from Gl-tract to liver; VMAXC, maximum rate of metabolism
of carbon tetrachloride (mg/hr/kg B W°70).
C-14
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. Interspecies conversion factors based on MRAMKL dose metric
(VMAXC=0.65)
EC
(ppm)
1
2
3
4
5
6
7
8
9
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
EC
(mg/m3)
6.290
12.58
18.87
25.16
31.45
37.74
44.03
50.32
56.61
62.90
125.8
188.7
251.6
314.5
377.4
440.3
503.2
566.1
629.0
691.9
754.8
817.8
880.7
943.6
1006
1069
1132
1195
1258
1321
1384
1447
1510
1573
1636
1698
MRAMKL
(nmol/hr/kg
liver)
0.9770
1.920
2.830
3.706
4.550
5.361
6.140
6.888
7.607
8.296
13.772
17.33
19.71
21.36
22.57
23.47
24.18
24.74
25.19
25.57
25.89
26.16
26.40
26.60
26.78
26.94
27.08
27.21
27.33
27.43
27.52
27.61
27.69
27.76
27.83
27.89
RGIL
(mg/kg/day)
0.2980
0.5960
0.8940
1.192
1.490
1.788
2.086
2.384
2.682
2.980
5.959
8.938
11.92
14.89
17.87
20.85
23.83
26.80
29.78
32.75
35.73
38.71
41.68
44.66
47.63
50.60
53.57
56.55
59.52
62.49
65.46
68.44
71.40
74.38
77.34
80.32
RGIL/EC
(mg/kg/day/
mg/m3)
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04736
0.04736
0.04736
0.04735
0.04735
0.04735
0.04734
0.04734
0.04734
0.04733
0.04733
0.04733
0.04733
0.04732
0.04732
0.04731
0.04731
0.04731
0.04730
0.04730
0.04730
0.04730
0.04730
0.04729
0.04729
EC/MRAMKL
(mg/m3/
jimol/hr/kg liver)
6.438
6.552
6.669
6.789
6.913
7.041
7.171
7.305
7.443
7.583
9.135
10.889
12.768
14.723
16.726
18.760
20.815
22.886
24.967
27.057
29.153
31.254
33.359
35.467
37.578
39.691
41.805
43.922
46.039
48.158
50.278
52.398
54.519
56.641
58.764
60.887
RGIL/MRAMKL
(mg/kg/day/
jimol/hr/kg liver)
0.3050
0.3104
0.3159
0.3216
0.3275
0.3335
0.3397
0.3461
0.3525
0.3592
0.4327
0.5157
0.6047
0.697
0.792
0.888
0.986
1.084
1.182
1.281
1.380
1.479
1.579
1.679
1.778
1.878
1.978
2.078
2.178
2.278
2.378
2.479
2.579
2.679
2.779
2.879
C-15
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-9. Interspecies conversion factors based on MRAMKL dose metric
(VMAXC=0.65)
280
290
300
1761
1824
1887
27.95
28.01
28.06
83.28
86.25
89.22
0.04728
0.04728
0.04728
63.010
65.134
67.259
2.979
3.080
3.180
EC, air exposure concentration; MRAMKL, time-averaged rate of metabolism of carbon tetrachloride;
RGIL, rate of uptake of carbon tetrachloride from Gl-tract to liver; VMAXC, maximum rate of metabolism
of carbon tetrachloride (mg/hr/kg BW070).
C-16
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-10. Interspecies conversion factors based on MRAMKL dose metric
(VMAXC=1.49)
EC
(ppm)
1
2
3
4
5
6
7
8
9
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
EC
(mg/m3)
6.290
12.58
18.87
25.16
31.45
37.74
44.03
50.32
56.61
62.90
125.8
188.7
251.6
314.5
377.4
440.3
503.2
566.1
629.0
691.9
754.8
817.8
880.7
943.6
1006
1069
1132
1195
1258
1321
1384
1447
1510
1573
1636
1698
MRAMKL
(nmol/hr/kg
liver)
1.3834
2.749
4.095
5.423
6.731
8.020
9.289
10.537
11.764
12.971
23.832
32.48
39.11
44.09
47.83
50.68
52.88
54.62
56.01
57.15
58.10
58.90
59.57
60.16
60.67
61.11
61.50
61.85
62.17
62.45
62.70
62.93
63.14
63.34
63.52
63.68
RGIL
(mg/kg/day)
0.2980
0.5960
0.8940
1.192
1.490
1.788
2.086
2.384
2.682
2.980
5.960
8.940
11.92
14.90
17.87
20.85
23.83
26.81
29.79
32.76
35.74
38.71
41.69
44.66
47.64
50.61
53.59
56.56
59.53
62.51
65.47
68.45
71.42
74.39
77.36
80.33
RGIL/EC
(mg/kg/day/
mg/m3)
0.04737
0.04738
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04736
0.04736
0.04736
0.04736
0.04735
0.04735
0.04735
0.04734
0.04734
0.04734
0.04733
0.04733
0.04733
0.04733
0.04732
0.04732
0.04732
0.04731
0.04731
0.04730
0.04730
0.04730
0.04730
EC/MRAMKL
(mg/m3/
jimol/hr/kg liver)
4.547
4.577
4.608
4.640
4.672
4.706
4.740
4.776
4.812
4.850
5.279
5.810
6.434
7.134
7.891
8.689
9.516
10.365
11.230
12.107
12.992
13.885
14.783
15.685
16.590
17.499
18.410
19.323
20.238
21.154
22.071
22.989
23.909
24.829
25.750
26.671
RGIL/MRAMKL
(mg/kg/day/
jimol/hr/kg liver)
0.2154
0.2168
0.2183
0.2198
0.2213
0.2229
0.2246
0.2263
0.2280
0.2297
0.2501
0.2752
0.3048
0.338
0.374
0.411
0.451
0.491
0.532
0.573
0.615
0.657
0.700
0.742
0.785
0.828
0.871
0.914
0.958
1.001
1.044
1.088
1.131
1.175
1.218
1.261
C-17
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-10. Interspecies conversion factors based on MRAMKL dose metric
(VMAXC=1.49)
280
290
300
1761
1824
1887
63.83
63.97
64.10
83.30
86.27
89.24
0.04729
0.04729
0.04729
27.593
28.516
29.439
1.305
1.349
1.392
EC, air exposure concentration; MRAMKL, time-averaged rate of metabolism of carbon tetrachloride;
RGIL, rate of uptake of carbon tetrachloride from Gl-tract to liver; VMAXC, maximum rate of metabolism
of carbon tetrachloride (mg/hr/kg BW070).
C-18
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-ll. Interspecies conversion factors based on MRAMKL dose metric
(VMAXC=1.70)
EC
(ppm)
1
2
3
4
5
6
7
8
9
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
210
220
230
240
250
260
270
EC
(mg/m3)
6.290
12.58
18.87
25.16
31.45
37.74
44.03
50.32
56.61
62.90
125.8
188.7
251.6
314.5
377.4
440.3
503.2
566.1
629.0
691.9
754.8
817.8
880.7
943.6
1006
1069
1132
1195
1258
1321
1384
1447
1510
1573
1636
1698
MRAMKL
(nmol/hr/kg
liver)
1.4401
2.865
4.273
5.665
7.040
8.398
9.738
11.060
12.365
13.650
25.429
35.14
42.84
48.77
53.31
56.79
59.49
61.62
63.33
64.72
65.88
66.84
67.66
68.37
68.98
69.51
69.99
70.40
70.78
71.11
71.42
71.69
71.94
72.17
72.38
72.57
RGIL
(mg/kg/day)
0.2980
0.5960
0.8940
1.192
1.490
1.788
2.086
2.384
2.682
2.980
5.960
8.939
11.92
14.90
17.88
20.85
23.83
26.81
29.79
32.76
35.74
38.71
41.69
44.66
47.64
50.61
53.59
56.56
59.53
62.51
65.48
68.45
71.42
74.39
77.36
80.34
RGIL/EC
(mg/kg/day/
mg/m3)
0.04737
0.04738
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04737
0.04736
0.04736
0.04736
0.04735
0.04735
0.04735
0.04735
0.04734
0.04734
0.04734
0.04733
0.04733
0.04733
0.04733
0.04732
0.04732
0.04731
0.04731
0.04731
0.04731
0.04730
0.04730
EC/MRAMKL
(mg/m3/
jimol/hr/kg liver)
4.368
4.392
4.417
4.442
4.468
4.494
4.522
4.550
4.579
4.608
4.947
5.370
5.874
6.448
7.080
7.754
8.459
9.187
9.933
10.691
11.459
12.234
13.015
13.801
14.591
15.383
16.179
16.976
17.775
18.576
19.378
20.181
20.985
21.790
22.596
23.403
RGIL/MRAMKL
(mg/kg/day/
jimol/hr/kg liver)
0.2069
0.2081
0.2092
0.2104
0.2117
0.2129
0.2142
0.2155
0.2169
0.2183
0.2344
0.2544
0.2782
0.305
0.335
0.367
0.401
0.435
0.470
0.506
0.543
0.579
0.616
0.653
0.691
0.728
0.766
0.803
0.841
0.879
0.917
0.955
0.993
1.031
1.069
1.107
C-19
DRAFT - DO NOT CITE OR QUOTE
-------
Table C-ll. Interspecies conversion factors based on MRAMKL dose metric
(VMAXC=1.70)
280
290
300
1761
1824
1887
72.75
72.92
73.07
83.30
86.28
89.24
0.04730
0.04730
0.04729
24.210
25.017
25.825
1.145
1.183
1.221
EC, air exposure concentration; MRAMKL, time-averaged rate of metabolism of carbon tetrachloride;
RGIL, rate of uptake of carbon tetrachloride from Gl-tract to liver; VMAXC, maximum rate of metabolism
of carbon tetrachloride (mg/hr/kg BW070).
C-20
DRAFT - DO NOT CITE OR QUOTE
-------
400
234
MCA (umol/L)
400
234
MCA (umol/L)
80
70
60
§50
I 40
O 30
LU
20
10
MCA (0.01 - 1), VMAXC=0.40
0.0
0.2
0.4 0.6
MCA (umol/L)
0.8
1.0
o -1
LU
-3 -
-4 --
-5
MCA (0.01 - 5), VMAXC=0.40
tt- -4-^*^-2-- --3-- --A-- --5-- --(>
MCA (umol/L)
'S
0
-o
#
o
LU
0
-1 <
MCA (1 -5), VMAXC=0.40
}- -A-- --2-- -3- -A-- --S-- -4>
MCA (umol/L)
J3 1 .
0 n
"° n
# °
0 -1 <
m n
MCA (0.01 - 1), VMAXC=0.40
B
;_____ _T ,
^ ' ' . ' •
}- --•&--•-- 0-- -4- --1-- --1-- --
MCA (umol/L)
Figure C-5. Relationship between internal dose metric MCA (time-averaged arterial blood
concentration of carbon tetrachloride) and equivalent exposure concentration (EC, left
panel) and values for % delta for trend lines (right panel). VMAXC=0.40 mg/hr/kg BW°70.
C-21
DRAFT - DO NOT CITE OR QUOTE
-------
234
MCA (umol/L)
400
350
300
|250
I 200
O 150
LU
100
50
0
MCA (1 -5), VMAXC=0.65
23456
MCA (umol/L)
80
70
_ 60
E 50
| 40
O 30
^20
10
0
0.0
0.2
0.4 0.6
MCA (umol/L)
0.8
1.0
S*
0
£
O
III
10 -,
I
o
2\
10 -
MCA (0.01 - 5), VMAXC=0.65
i .»•••••""""""*"""""
• ' ' ' 'A '„.•••••-?*""*
} «**+**~^ 2 3- 4 5 (
MCA (umol/L)
?
0
-D
#
O
LU
n
-1 <
c _
MCA (1 - 5), VMAXC=0.65
***•* .w»ml- •?•?•?•••
}- -^-- --2-- --3- -4-- --5-- --(>
MCA (umol/L)
5
4 -
3
2
^ 1 -
- 01
<£ °
0-10
m -2-
-3
-4 -
c
MCA (0.01 - 1), VMAXC=0.65
t
_ ; __ f .
^ ' ' . ' •
0 Or2- ? - -0-.4- 0.6 -05 H>- 1-2
MCA (umol/L)
Figure C-6. Relationship between internal dose metric MCA (time-averaged arterial blood
concentration of carbon tetrachloride) and equivalent exposure concentration (EC, left
panel) and values for % delta for trend lines (right panel). VMAXC=0.65 mg/hr/kg BW° 70.
C-22
DRAFT - DO NOT CITE OR QUOTE
-------
2 3
MCA (umol/L)
400
O 150 - - - -.X^ - y = 78,4969^ _
234
MCA (umol/L)
80
70
60
fso
40 --
O 30
LLI
20
10 --
0.0 0.2 0.4 0.6 0.8 1.0 1.2
MCA (umol/L)
MCA (umol/L)
£*
0
~O
#
O
LU
n
-1 <
c _
MCA (1 -5), VMAXC=1.49
•.
x
•*••..:.....»•*•••
>- -^-- --2-- --3-- --4-- -','>
MCA (umol/L)
EC % delta)
4 -
3
2
1 -
n -
-10
-2
-3
-4 -
c
MCA (0.01 - 1), VMAXC=1.49
V * T • '
V '.".'.' . ' « ' * '
Q Or2 0.4 6.6 -03 1-,6- 42
MCA (umol/L)
Figure C-7. Relationship between internal dose metric MCA (time-averaged arterial blood
concentration of carbon tetrachloride) and equivalent exposure concentration (EC, left
panel) and values for % delta for trend lines (right panel). VMAXC=1.49 mg/hr/kg BW
0.70
C-23 DRAFT - DO NOT CITE OR QUOTE
-------
2 3
MCA (umol/L)
400
234
MCA (umol/L)
80
70
60
fso
40 --
O 30
LLI
20
10 --
0.0 0.2 0.4 0.6 0.8 1.0
MCA (umol/L)
1.2
MCA (umol/L)
£*
0
~O
^
O
LU
-1 <
c _
MCA (1 -5), VMAXC=1.70
•
S. ;.;--•••"- —
•••••,'„•••••••*••
>- -^-- --2-- --3-- --4-- --','>
MCA (umol/L)
EC % delta)
4 -
3
2
1 -
n -
-10
-2
-3
-4 -
c
MCA (0.01 - 1), VMAXC=1.70
^ '" '
^* . . '.' '•' '* '*
Q Or2 0.4 6.6 -0^ -h& 42
MCA (umol/L)
Figure C-8. Relationship between internal dose metric MCA (time-averaged arterial blood
concentration of carbon tetrachloride) and equivalent exposure concentration (EC, left
panel) and values for % delta for trend lines (right panel). VMAXC=1.70 mg/hr/kg BW
0.70
C-24 DRAFT - DO NOT CITE OR QUOTE
-------
234
MCA (umol/L)
50
40
30
20 --
10 --
MCA (0.1 -5), VMAXC=0.40
y=8.5959x_
R2 = 0.9999
234
MCA (umol/L)
0.00
0.02
0.04 0.06
MCA (umol/L)
0.08
0.10
12
0
•Q
£ 0
* 4'
o: -4
i
» MCA (0.01 - 5), VMAXC=0.40
•
., , , ^M*-: , , "•" •••:•:• , ,
I ,.**1 2 3 4 5 fi
«•*
MCA (umol/L)
s
{£
c _,
n
-1 <
5 _
MCA (0.1 -5), VMAXC=0.40
..•••••••'""••••••...^
)_•_ _y_ 4 _ _ _2_ _ _ -3. _ . _ 4*"'"-«& - - -(>
MCA (umol/L)
c
4 -
3
I 2-
0
-o 1 -
e o-
CD -10:
K -2-
-3
-4 -
5
MCA (0.01 - 0.1), VMAXC=0.40
. . • '
' ' '*' ' i ' . ' '•' ' * ' *
30- - - - 6.62 - - -6:64 0:66 &.08 0.10
MCA (umol/L)
Figure C-9. Relationship between internal dose metric MCA (time-averaged arterial blood
concentration of carbon tetrachloride) and equivalent rate of uptake from GI tract to liver
(RGIL, left panel) and values for % delta for trend lines (right panel). VMAXC=0.40
mg/hr/kg BW° 70.
C-25
DRAFT - DO NOT CITE OR QUOTE
-------
50
;40 -
'30 --
)
'20 --
10 --
MCA (0.01 - 5), VMAXC=0.65
234
MCA (umol/L)
234
MCA (umol/L)
0.00
0.05
0.10
MCA (umol/L)
0.15
0.20
1
•Q
u9
o
a:
on -,
0
/i $
-4
Q
°
12
MCA (0.01 - 5), VMAXC=0.65
,
. ^^•••••'•••••M' ••••••••••••••
[' ' _.;•*•
_>*T 2 _3 4_ 5 (>
N.'*
MCA (umol/L)
MCA (umol/L)
c
4 -
3
I 2-
« 1 -
# n
^^
a: -2
-3
-4 -
c;
MCA (0.01 - 0.2), VMAXC=0.65
•
* '
•
30-- " --TW35--*- --Or1f)-- -€H5-- - - 6.
MCA (umol/L)
20
Figure C-10. Relationship between internal dose metric MCA (time-averaged arterial
blood concentration of carbon tetrachloride) and equivalent rate of uptake from GI tract to
liver (RGIL, left panel) and values for % delta for trend lines (right panel). VMAXC=0.65
mg/hr/kg BW
0.70
C-26
DRAFT - DO NOT CITE OR QUOTE
-------
234
MCA (umol/L)
234
MCA (umol/L)
0.00 0.05 0.10 0.15
MCA (umol/L)
0.20
40
30
"0
S, 10
CD
-10 --
-20
MCA (0.01 -5), VMAXC=0.1.49
MCA (umol/L)
ra on
0
-o
^ -in
_i
CD n
K °
(
MCA (0.2 -5), VMAXC=1.49
) 1 2 3 4 !i
MCA (umol/L)
jg 2 -
^ n -
MCA (0.01 -0.2), VMAXC=1.49 •
.
.
30- *- r ft.05-"- - - -Or K)- - - -OrlS - - - -0:
MCA (umol/L)
20
Figure C-ll. Relationship between internal dose metric MCA (time-averaged arterial
blood concentration of carbon tetrachloride) and equivalent rate of uptake from GI tract to
liver (RGIL, left panel) and values for % delta for trend lines (right panel). VMAXC=1.49
mg/hr/kg BW
0.70
C-27
DRAFT - DO NOT CITE OR QUOTE
-------
1234
MCA (umol/L)
2345
MCA (umol/L)
0.00 0.05 0.10 0.15 0.20
MCA (umol/L)
8
0
T3
_l
CD
15 -
1
_ _MCA (0.01_- 5)LVJV1AXC=0^170 _
i . ...•••••••••
, ..,••••**"***
} 4 >a^»*" - 3- - 4 ^i
1 X
MCA (umol/L)
f s
J
0
-o
n -
-1 •<
MCA (0.2-5), VMAXC=1.70
..•••••***"""*••••....
• .••* '••••'.. '
}- -V --2-- -3- --4-- --!i
• t.»
•**
MCA (umol/L)
RGIL(% delta)
4 -
3
2
1 -
n -
-10:
-2
-3
-4 -
MCA (0.01 -0.2), VMAXC=1.70
•
•
30- - -*-«-.-».0&-*-"- --OT«)-- --07-I5-- -6r!0
MCA (umol/L)
Figure C-12. Relationship between internal dose metric MCA (time-averaged arterial
blood concentration of carbon tetrachloride) and equivalent rate of uptake from GI tract to
liver (RGIL, left panel) and values for % delta for trend lines (right panel). VMAXC=1.70
mg/hr/kg BW
0.70
C-28
DRAFT - DO NOT CITE OR QUOTE
-------
60
50 H
40
1 30
O
m
20 -
10 -
Vmax = 0.40
fit = 0.49541 *MRAMKL +
15.082*MRAMKL/(18.176 - MRAMKL).
0.03
+ 0.02
70
0.01 8
E
c
£U
+ 0 ^
3
- -0.01 2
- -0.02
-0.03
6 9
MRAMKL
12
15
12
10 -
8 -
I 6
O
7 0.03
Vmax =1.49
fit = 0.49547*MRAMKL +
15.078*MRAMKL/(67.700 - MRf
-0.03
6 9
MRAMKL
12
15
a. 19
Q. 1^
O
LJJ
t
Vmax = 0.65 ^ /
fit = 0.49544*MRAMKL + /
15.081*MRAMKL/(29.535 - MRAMKL) /
~ >/
/
/
^/
D 3 6 9 12 1
MRAMKL
73
0.01
£
c
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(I)
3
o"-
n fY3
5
-
o
o
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0
c
Vmax = 1.70 ^ y
fit = 0.49543*MRAMKL + /
15.082*MRAMKL/(77.246 - MRAMKLJX
_/
/
/
/
) 3 6 9 12 1
MRAMKL
0.01
-------
mg/kg/d)
O
S
12-
9 -
t
Vmax = 0.40 /
fit =0.1 4773* MRAMKL + *~/*
4. 4926* MRAMKL/(18.176 - MRAMKL)?*.
• •'••••/
Z
D 3 6 9 12 1
MRAMKL
7J
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J a>
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n m "*"3
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5
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^)
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D)
_l
O o
a:
S
i
Vmax = 0.65 >
fit =0.14770*MRAMKL+ - — -f
4.4925*MRAMKL/(29.535 - MRAMKL) /
' /• • •
/. • ". ' •/..•
• '/' ' ' '
//
^Z
D 3 6 9 12 1
MRAMKL
n n^
-0.02
-0.01 »
- 0.00 |
- -0.01 "p
- -0.02
n (Y3
5
3.6
3
S 1.8 -
s
a: ,
0.6 -
Vmax = 1.49
fit = 0.14767*MRAMKL +
4.4929*MRAMKL/(67.71 1 - MRAMj
Z
6 9
MRAMKL
12
0.03
-- 0.02
-- 0.01
a.
0.00 ^
3
-0.01 "3
-- -0.02
-0.03
15
5
O)
^
P"
_i
CO
a:
2.4 -
1.2
Vmax = 1.70 *
fit = 0.14767*MRAMKL + *~/~
4.4929*MRAMKL/(77.252 - MRAMX1)
>^
^ > m •/_
"**^ • • / . . • •
z
/
/
73
0.01 g
a.
c
G)
(D
5
-0.01 "p
0 3 6 9 12 15
MRAMKL
Figure C-14. Relationship between internal dose metric MRAMKL (mean rate of
carbon tetrachloride metabolism in the liver) and equivalent rate of uptake from GI
tract to liver (RGIL) and values for % delta for trend lines.
C-30
DRAFT - DO NOT CITE OR QUOTE
-------
C.4. Sensitivity Analysis
Univariate sensitivity analysis consisted of running the model after perturbing values for
single parameters by a factor of 0.01, in the up and down directions. Parameter sensitivities were
assessed from comparison of standardized sensitivity coefficients:
e^_ r./^_/(* + A*)-/(*-A) x
/(*)
Eq. (1)
where SC is the standardized sensitivity coefficient, f(x) is the output variable at parameter value
x, and ) is the perturbation of x (i.e., O.Olx). Figures C-15 and C-16 show sensitivity coefficients
for each internal dose metric (i.e., MCA, MRAMKB) for the human model. Absolute values of
sensitivity coefficients that were >0.01 are shown in these figures. Conversion to absolute value
removes information on the direction of the change in the output variable, allowing the
magnitudes of the influence of each parameter on the output variable to be directly compared.
Parameters having sensitivity coefficients >0.1 can be considered to be highly influential
parameters. Chemical parameters in this category (i.e., sensitivity coefficient >0.1) are shown in
Table C-12 (indicated with +). The mouse and rat models yielded the same rank order of
sensitivity coefficients as the human model.
Table C-12. Sensitive parameters (indicated with +) in the human model
Parameter
PB
PL
PF
PS
PR
Vmaxc
Km
Definition
Blood:air partition coefficient
Liverblood partition coefficient
Fat:blood partition coefficient
Slowly -perfused:blood partition coefficient
Readily-perfused:blood partition coefficient
Maximum rate of metabolism (mg/hour-kg B W)
Michaelis-Menten coefficient for metabolism
(mg/L)
Internal Dose Metric
MCA
+
+
+
MRAMKB
+
+
+
+
C-31
DRAFT - DO NOT CITE OR QUOTE
-------
SC - MCA 960 hr
1.0E-02 1.0E-01 1.0E+00
Parameter
QRC
PBLD
QSC
QIC
QPC
QCC
VMAXC
KMX
QFC
VFC
PF
PS
VSC
PR
VRC
VLC
PL
I
I
I
I
I
I
I
I
Figure C-15. Standardized sensitivity coefficients for the MCA dose metric
(average concentration of carbon tetrachloride in blood, umol/L) simulated
with the human carbon tetrachloride PBPK model.
Absolute values of coefficients >0.01 are shown. The simulation was of a
continuous exposure to 2.5 ppm for 980 hours (rank order of sensitivity
coefficients was not dependent on exposure time).
C-32
DRAFT - DO NOT CITE OR QUOTE
-------
SC - MRAMKB 960 hr
1.0E-02 1.0E-01 1.0E+00
Parameter
VLC
PR
QRC
PS
PL
VMAXC
QCC
VSC
VRC
QSC
QFC
PF
QPC
VFC
QIC
PBLD
KMX
I
I
|
I
I
I
I
I
I
Figure C-16. Standardized sensitivity coefficients for the MRAMKB dose
metric (average rate of metabolism of carbon tetrachloride umol/hr/kg body
weight) simulated with the human carbon tetrachloride PBPK model.
Absolute values of coefficients >0.01 are shown. The simulation was of a
continuous exposure to 2.5 ppm for 980 hours (rank order of sensitivity
coefficients was not dependent on exposure time).
C-33
DRAFT - DO NOT CITE OR QUOTE
-------
APPENDIX D. BENCHMARK DOSE MODELING FOR DERIVING THE RfC
MALE RAT:
Incidence data for fatty changes of the liver
Male F344 rats exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week)
Doses modeled: 0, 5, 25, 125 ppm
BMR=10%
Model
Vmax = 0.4
AIC
X2/> value3
BMC10
BMCL10
Vmax = 0.65
AIC
X2/> value"
BMC10
BMCL10
MCA (umol/L)
Gammab
Logistic0
Log-Logistic'
Multistage 1-
degree4'
Probitc
Log-probitc
Quantal-linear
Weibullb
144.336
155.104
137.403
142.388
169.521
138.408
142.388
142.388
0.0007
0.0000
0.4355
0.0074
0.0000
0.1761
0.0074
0.0074
0.0793248
0.170834
0.136715
0.0714015
0.22329
0.124953
0.0714017
0.0714016
0.0551873
0.137191
0.0790319
0.0550523
0.17626
0.0755939
0.0550523
0.0550523
144.772
156.51
137.463
142.778
171.234
138.529
142.778
142.778
0.0005
0.0000
0.4087
0.0031
0.0000
0.1581
0.0031
0.0031
0.0689847
0.157857
0.123076
0.0665234
0.21463
0.112257
0.0665234
0.0665235
0.051179
0.126743
0.0707077
0.0511645
0.168317
0.0803264
0.0511645
0.0511645
MRAMKL (umol/hr-kg liver)
Gammab
Logistic'
Log-Logistic0
Multistage 2-
degreee- f
Probitc
Log-probitc
Quantal-linear
Weibullb
137.468
136.747
136.933
137.073
138.891
136.871
151.674
138.997
0.4177
0.3444
0.8012
0.2702
0.0826
0.9538
0.0008
0.1316
3.98707
3.25675
4.56744
3.55184
2.97807
4.27176
1.01942
3.34831
2.6343
2.58557
3.08461
2.02617
2.41619
3.06539
0.831472
2.18252
137.338
136.513
136.996
138.991
138.712
136.872
148.898
138.601
0.4760
0.3671
0.7246
0.0944
0.0728
0.9470
0.0025
0.1751
5.31098
4.60057
6.20422
4.99656
4.23817
5.73628
1.45532
4.4781
3.35649
3.65284
4.00273
2.5022
3.44383
3.97844
1.18412
2.81908
aValues <0.1 fail to meet conventional goodness-of-fit criteria; p value from the i2 test.
bPower restricted to >1.
°Slope restricted to >1.
dUsed smallest degree polynomial available with an adequate fit; the 2- and 3-degree polynomials provided the same
fit as the 1-degree.
dBetas restricted to >0.
fUsed smallest degree polynomial available with an adequate fit; the 3-degree polynomial provided the same fit as
the 2-degree.
D-l
DRAFT - DO NOT CITE OR QUOTE
-------
FEMALE RAT:
Incidence data for fatty changes of the liver
Female F344 rats exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week)
Doses modeled: 0, 5, 25,125ppm
BMR=10%
None of the models in BMDS provided an adequate fit of the female rat data.
Incidence data for fatty changes of the liver
Female F344 rats exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week)
Doses modeled: 0, 5, 25 ppm [high dose dropped]
BMR=10%
Model
Vmax = 0.4
AIC
X2/> value"
BMC10
BMCL10
Vmax = 0.65
AIC
X2/> value"
BMC10
BMCL10
MCA (umol/L)
Gammab
Logistic0
Log-Logistic0
Multistage* e
Probit0
Log-probit°
Quantal-linear
Weibullb
92.9928
93.4185
92.9928
2ncl degree
92.4089
3rd degree
94.9928
93.6833
92.9928
111.424
92.9928
NA
0.1121
NA
0.2442
NA
0.0968
NA
0.0000
NA
0.187771
0.106984
0.182663
0.123631
0.213915
0.100288
0.174053
0.0363563
0.213201
0.107455
0.0803379
0.111838
0.0851972
0.090506
0.0779817
0.112578
0.0277405
0.102923
92.9928
93.3172
92.9928
2nd degree
92.3049
3ri degree
92.9928
93.5689
92.9928
111.025
92.9928
NA
0.1201
NA
0.2617
NA
0.1043
NA
0.0001
NA
0.170979
0.0979754
0.166144
0.113721
0.195194
0.0919928
0.158234
0.0332712
0.194228
0.0971536
0.0734707
0.101213
0.0775873
0.08177
0.0714911
0.101889
0.0253689
0.0930656
MRAMKL (umol/hr-kg liver)
Gammab
Logistic0
Log-Logistic0
Multistage* e
Probit0
Log-probit°
Quantal-linear
Weibullb
92.9928
99.7262
92.9928
2ntl degree
100.7
3rcl degree
92.2866
100.988
92.9928
127.034
92.9928
NA
0.0020
NA
0.0039
0.2650
0.0013
NA
0.0000
NA
4.85516
2.45785
4.84705
2.43344
3.76974
2.16088
4.69168
0.817323
5.3798
3.42634
1.90371
3.48106
1.99357
2.82488
1.70134
3.49658
0.634088
3.29131
92.9928
97.8675
92.9928
2nd degree
98.1134
3rd degree
91.5964
98.8142
92.9928
123.548
92.9928
NA
0.0064
NA
0.0124
0.4421
0.0044
NA
0.0000
NA
6.52318
3.34536
6.48806
3.42266
5.42354
2.98448
6.26103
1.12472
7.27174
4.43018
2.58247
4.51798
2.75565
3.74923
2.34695
4.54001
0.870515
4.24944
aValues <0.1 fail to meet conventional goodness-of-fit criteria; p value from the %2 test.
bPower restricted to >1.
°Slope restricted to >1.
dUsed smallest degree polynomial available with an adequate fit.
eBetas restricted to >0.
D-2
DRAFT - DO NOT CITE OR QUOTE
-------
Male Rat
Dose metric: MCA
Vmax = 0.4 mg/hr/kg BW
0.07
Log-Logistic Model with 0.95 Confidence Level
0.8
0.6
c
I 0-4
0.2
Log-Logistic
E8MQL
BMD
0.5
1.5
2
dose
2.5
3.5
10:5910/122007
Logistic Model. (Version: 2.9; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\RFC RAT LIVER\MALE RAT\MCA-
VMAX=0.4\RAT-FATTYLIVER-MCA-4.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\RFC RAT LIVER\MALE
RAT\MCA-VMAX=0.4\RAT-FATTYLIVER-MCA-4.pit
Fri Oct 12 10:59:34 2007
The form of the probability function is:
P[response] = background+(1-background)/[1 + EXP(-intercept-slope*Log(dose) ) ;
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-00<:
Parameter Convergence has been set to: le-008
D-3
DRAFT - DO NOT CITE OR QUOTE
-------
background
intercept
slope
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
background
intercept
slope
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -65.434 4
Fitted model -65.7017 3 0.535433 1 0.4643
Reduced model -138.619 1 146.371 3 <.0001
AIC:
d.f. = 1 P-value = 0.435
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 0.136715
BMDL = 0.0790319
D-4 DRAFT - DO NOT CITE OR QUOTE
-------
Male Rat
Dose metric: MCA
Vmax = 0.65 mg/hr/kg BW
0.07
Log-Logistic Model with 0.95 Confidence Level
T3
0.8
0-6
1 0-4
0.2
Log-Logistic
0 0.5
1.5 2
dose
2.5
3.5
11:1210/122007
Logistic Model. (Version: 2.9; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\RFC RAT LIVER\MALE RAT\MCA-
VMAX=0.65\RAT-FATTYLIVER-MCA-65.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\RFC RAT LIVER\MALE
RAT\MCA-VMAX=0.65\RAT-FATTYLIVER-MCA-65.pit
Fri Oct 12 11:12:25 2007
The form of the probability function is:
P[response] = background+(1-background)/[1 + EXP(-intercept-slope*Log(dose) ) ;
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-00<:
Parameter Convergence has been set to: le-008
D-5
DRAFT - DO NOT CITE OR QUOTE
-------
Variable Estimate Std. Err.
background
intercept
slope
Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
Log(likelihood) # Param's Deviance Test d.f.
-65.434 4
-65.7316 3 0.595159 1
-138.619 1 146.371 3
P-value
Dose
d.f. = 1
P-value = 0.4087
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 0.123076
BMDL = 0.0707077
D-6
DRAFT - DO NOT CITE OR QUOTE
-------
Male Rat
Dose metric: MRAMKL
Vmax = 0.4 mg/hr/kg BW
0.07
Logistic Model with 0.95 Confidence Level
0.8
I
0.2
Logistic
BMDL
BMD
10 15
dose
20
25
11:1710/122007
Logistic Model. (Version: 2.9; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\RFC RAT LIVER\MALE RAT\MRAMKL-
VMAX=0.4\FATTY_LIVER_MRAMKL-4.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\RFC RAT LIVER\MALE
RAT\MRAMKL-VMAX=0.4\FATTY_LIVER_MRAMKL-4.pit
Fri Oct 12 11:17:49 2007
The form of the probability function is:
P[response] = I/[1+EXP(-intercept-slope*dose)]
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le
Parameter Convergence has been set to: le-008
D-7
DRAFT - DO NOT CITE OR QUOTE
-------
*** The model parameter(s) -background
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
intercept slope
1 -0.82
-0.82 1
Variable
intercept
slope
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
-3.43685 -1.93488
0.228273 0.390994
Model
Full model
Fitted model
Reduced model
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-65.434 4
-66.3737 2 1.87944 2
-138.619 1 146.371 3
Est. Prob.
Goodness of Fit
Expected Observed Size
4 50
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 3.25675
BMDL = 2.58557
D-8
DRAFT - DO NOT CITE OR QUOTE
-------
Male Rat
Dose metric: MRAMKL
Vmax = 0.65 mg/hr/kg BW
0.07
0.8
I 0-4
0.2
0
Logistic Model with 0.95 Confidence Level
Logistic
BMDL
0 5
11:2310/122007
10
15 20
dose
25
30
35
Logistic Model. (Version: 2.9; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\RFC RAT LIVER\MALE RAT\MRAMKL-
VMAX=0.65\MRAT_FATTY_LIVER_MRAMKL-65.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\RFC RAT LIVER\MALE
RAT\MRAMKL-VMAX=0.65\MRAT_FATTY_LIVER_MRAMKL-65.pit
Fri Oct 12 11:23:29 2007
The form of the probability function is:
P[response] = I/[1+EXP(-intercept-slope*dose)]
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le
Parameter Convergence has been set to: le-008
D-9
DRAFT - DO NOT CITE OR QUOTE
-------
intercept slope
intercept
slope
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
intercept -2.59592 0.370821 -3.32272 -1.86913
slope 0.207777 0.0278282 0.153235 0.26232
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -65.434 4
Fitted model -66.2567 2 1.64536 2
Reduced model -138.619 1 146.371 3
AIC:
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 4.60057
BMDL = 3.65284
D-10 DRAFT - DO NOT CITE OR QUOTE
-------
Female Rat
Dose metric: MCA
Vmax = 0.4 mg/hr/kg BW
0.07
o
0.8
0.4
0.2
0 :
Multistage Model with 0.95 Confidence Level
Multistage
BMDL , BMD
0 0.1
11:4210/122007
0.2
0.3 0.4
dose
0.5
0.6
0.7
Multistage Model. (Version: 2.8; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\RFC RAT LIVER\FEMALE RAT\MCA-
VMAX=0.4\FRAT-FATTYLIVER-MCA-4.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\RFC RAT LIVER\FEMALE
RAT\MCA-VMAX=0.4\FRAT-FATTYLIVER-MCA-4.pit
Fri Oct 12 11:42:22 2007
BMDS MODEL RUN
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose^l-beta2*dose^2) ]
The parameter betas are restricted to be positive
Dependent variable = FattyLiver
Independent variable = umol/L
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0746099
Beta(l) = 0
Beta(2) = 7.64624
D-ll
DRAFT - DO NOT CITE OR QUOTE
-------
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta(l)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Background
Background
Beta(2)
1
-0.21
Beta(2)
-0.21
1
Parameter Estimates
Variable
Background
Beta(l)
Beta(2)
Estimate
0.0951491
0
6.89319
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-43.4964
-44.2044
-101.707
92.4089
# Param's
3
2
1
Deviance Test d.f.
1.41613
116.422
P-value
0.234
<.0001
Goodness of Fit
Dose
0.0000
0.1280
0.7080
Est. Prob.
0.0951
0.1918
0.9714
Expected
4 .757
9.589
48 .571
Observed
6
7
49
Size
50
50
50
Scaled
Residual
0.599
-0.930
0.364
=1.36
d.f. = 1
P-value = 0.2442
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
0.123631
0.0851972
0.148857
Taken together, (0.0851972, 0.148857) is a 90
interval for the BMD
% two-sided confidence
D-12
DRAFT - DO NOT CITE OR QUOTE
-------
Female Rat
Dose metric: MCA
Vmax = 0.65 mg/hr/kg BW
0.07
Multistage Model with 0.95 Confidence Level
0.8
0.6
0.4
0.2
Multistage
0 :
BMDL , BMD
0 0.1
11:4710/122007
0.2
0.3
dose
0.4
0.5
0.6
Multistage Model. (Version: 2.8; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\RFC RAT LIVER\FEMALE RAT\MCA-
VMAX=0.65\FRAT-FATTYLIVER-MCA-65.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\RFC RAT LIVER\FEMALE
RAT\MCA-VMAX=0.65\FRAT-FATTYLIVER-MCA-65.plt
Fri Oct 12 11:47:23 2007
BMDS MODEL RUN
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose^l-beta2*dose^2) ]
The parameter betas are restricted to be positive
Dependent variable = FattyLiver
Independent variable = umol/L
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0765787
Beta(l) = 0
Beta(2) = 8.98383
D-13
DRAFT - DO NOT CITE OR QUOTE
-------
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta(l)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Background
Background
Beta(2)
1
-0.21
Beta(2)
-0.21
1
Parameter Estimates
Variable
Background
Beta(l)
Beta(2)
Estimate
0.095736
0
8.14699
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-43.4964
-44.1525
-101.707
92.3049
# Param's
3
2
1
Deviance Test d.f.
1.31215
116.422
P-value
0.252
<.0001
Goodness of Fit
Dose
0.0000
0.1160
0.6530
Est. Prob.
0.0957
0.1896
0.9720
Expected
4 .787
9.481
48 .599
Observed
6
7
49
Size
50
50
50
Scaled
Residual
0.583
-0.895
0.344
= 1.26
d.f. = 1
P-value = 0.2617
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
0.113721
0.0775873
0.137047
Taken together, (0.0775873, 0.137047) is a 90
interval for the BMD
% two-sided confidence
D-14
DRAFT - DO NOT CITE OR QUOTE
-------
Female Rat
Dose metric: MRAMKL
Vmax = 0.4 mg/hr/kg BW
0.07
Multistage Model with 0.95 Confidence Level
0.8
0.6
0.4
0.2
0 :
Multistage
BMD
6
dose
10
12
11:5210/122007
Multistage Model. (Version: 2.8; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\RFC RAT LIVER\FEMALE
RAT\MRAMKL-VMAX=0.4\FRAT_FATTY_LIVER_MRAMKL-4.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\RFC RAT LIVER\FEMALE
RAT\MRAMKL-VMAX=0.4\FRAT_FATTY_LIVER_MRAMKL-4.pit
Fri Oct 12 11:52:42 2007
BMDS MODEL RUN
Observation # < parameter # for Multistage model.
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose^l-beta2*dose^2-beta3*dose^3)]
The parameter betas are restricted to be positive
Dependent variable = FattyLiver
Independent variable = umol/hr-kgL
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 4
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0769299
Beta(l) = 0
D-15
DRAFT - DO NOT CITE OR QUOTE
-------
Beta(2) =
Beta(3) =
0.00216647
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta(l) -Beta(2)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Background
Background
Beta(3)
1
-0.21
Beta(3)
-0.21
1
Parameter Estimates
Variable
Background
Beta(l)
Beta(2)
Beta(3)
Estimate
0.0958436
0
0
0.00196673
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-43.4964
-44.1433
-101.707
92.2866
# Param's
3
2
1
Deviance Test d.f.
1.29386
116.422
P-value
0.2553
<.0001
Goodness of Fit
Dose
0.0000
3 .8130
12 .0920
Est. Prob.
0.0958
0.1892
0.9721
Expected
4 .792
9.462
48 .603
Observed
6
7
49
Size
50
50
50
Scaled
Residual
0.580
-0.889
0.340
= 1.24
d.f. = 1
P-value = 0.2650
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
3.76974
2.82488
4.26949
Taken together, (2.82488, 4.26949) is a 90
interval for the BMD
two-sided confidence
D-16
DRAFT - DO NOT CITE OR QUOTE
-------
Female Rat
Dose metric: MRAMKL
Vmax = 0.65 mg/hr/kg BW
0.07
Multistage Model with 0.95 Confidence Level
0.8
0.6
0.4
0.2
0 :
Multistage
• BMDL BiyiD ^
0 2
11:5710/122007
10 12 14 16 18
dose
Multistage Model. (Version: 2.8; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\RFC RAT LIVER\FEMALE
RAT\MRAMKL-VMAX=0.65\FRAT_FATTY_LIVER_MRAMKL-65.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\RFC RAT LIVER\FEMALE
RAT\MRAMKL-VMAX=0.65\FRAT_FATTY_LIVER_MRAMKL-65.pit
Fri Oct 12 11:57:06 2007
BMDS MODEL RUN
Observation # < parameter # for Multistage model.
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose^l-beta2*dose^2-beta3*dose^3)]
The parameter betas are restricted to be positive
Dependent variable = FattyLiver
Independent variable = umol/hr-kgL
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 4
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0
Beta(l) = 0
Beta(2) = 0
Beta(3) = 0.000714264
D-17
DRAFT - DO NOT CITE OR QUOTE
-------
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta(l) -Beta(2)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Background
Background
Beta(3)
1
-0.19
Beta(3)
-0.19
1
Parameter Estimates
Variable
Background
Beta(l)
Beta(2)
Beta(3)
Estimate
0.101433
0
0
0.000660435
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-43.4964
-43.7982
-101.707
91.5964
# Param's
3
2
1
Deviance Test d.f.
0.603632
116.422
P-value
0.4372
<.0001
Dose
Est. Prob.
Goodness of Fit
Expected Observed Size
Scaled
Residual
0.0000
4 .9910
17.6260
0.1014
0.1723
0.9759
= 0.59
d.f. = 1
5.072 6 50
8.613 7 50
48.793 49 50
P-value = 0.4421
0.435
-0.604
0.191
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
5.42354
3.74923
6.17189
Taken together, (3.74923, 6.17189) is a 90
interval for the BMD
two-sided confidence
D-18
DRAFT - DO NOT CITE OR QUOTE
-------
APPENDIX E. CANCER ASSESSMENT: BMD MODELING OUTPUTS FOR LOW-
DOSE LINEAR EXTRAPOLATION APPROACH
E.I. Benchmark Dose Analysis
Liver tumors (adenoma + carcinoma)
Female F344 rats exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week)
Doses modeled: 0, 5, 25, 125 ppm
Multistage; MCA: 2-stage model MRAMKL: 4-stage model
BMR
(extra risk)
Vmax = 0.4
AIC
0.05
61.6602
0.05
63.3399
value"
0.9842
0.6503
BMC
0.609955
9.8151
BMCL
BMR/
BMCL
Vmax = 0.65
AIC
MCA
(umol/L)
0.387377
0.129
61.5904
MRAMKL
(umol/hr-kg liver)
8.40334
0.00595
62.8343
7*P
value"
0.9916
0.7440
BMC BM
0.588686 0.35
14.582 12.2
CL BMR/
BMCL
4766 0.141
867 0.00407
"Values <0.1 fail to meet conventional goodness-of-fit criteria; p value from the II test.
Liver tumors (adenoma + carcinoma)
Female F344 rats exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week)
Doses modeled: 0, 5, 25 ppm
Multistage; 2-stage model
BMR
(extra risk)
Vmax = 0.4
AIC
0.05
24.8957
value"
0.9507
BMC
0.655398
BMCL
0.345984
BMR/
BMCL
Vmax = 0.65
.,„ x2/; BMC BM
A_H_, _ a
value
MCA
;umol/L)
0.144
24.8889 0.9523 0.604144 0.31
CL BMR/
BMCL
7726 0.157
MRAMKL
(umol/hr-kg liver)
0.05
25.2825
0.8571
11.5604
6.92352
0.00722
25.1734 0.8831 16.6986 9.76
339 0.00512
aValues <0.1 fail to meet conventional goodness-of-fit criteria; p value from the %2 test.
Note: 3-stage model did not provide a sufficiently improved model fit.
E-l
DRAFT - DO NOT CITE OR QUOTE
-------
Liver tumors (adenoma + carcinoma)
Female BDF1 mouse exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week)
Doses modeled: 0, 5, 25 ppm
Multistage; MCA: 2-stage model MRAMKL: 2-stage model
BMR
(extra risk)
Fisher
AIC
0.1
117.307
1?P
value"
NA
BMC
| 0.10186
BMCL
BMR/
BMCL
MCA
(umol/L)
0.0467576
2.14
Thrall
AIC X,
vah
| 117.307 N;
p BMC BM
iea
V 0.194624 0.08S
CL BMR/
BMCL
S5305 1.13
MRAMKL
(umol/hr-kg liver)
0.1
115.912
0.4437
| 9.70893
6.3204
0.0158
| 117.341 | O.lf
554 10.4557 7.5S
>255 0.0132
aValues <0.1 fail to meet conventional goodness-of-fit criteria; p value from the II2 test.
Note: 3-stage model did not provide a sufficiently improved model fit.
Liver tumors (adenoma + carcinoma)
Female BDF1 mouse exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week)
Doses modeled: 0, 5 ppm
Multistage; 2-stage model
BMR
(extra risk)
Fisher
AIC
0.1
80.6149
0.1
80.6149
f?P
value"
NA
NA |
BMC BM<
0.101967 | 0.04'
11.6352 5.046
CL BMR/
BMCL
Thrall
7*p BMC BM
A1C value3
MCA
(umol/L)
1224 2.26
80.6149 NA 0.195666 0.084
MRAMKL
(umol/hr-kg liver)
31 0.0198
80.6149 NA 14.1982 6.15
CL BMR/
BMCL
8621 1.18
788 0.0162
aValues <0.1 fail to meet conventional goodness-of-fit criteria; p value from the %2 test.
E-2
DRAFT - DO NOT CITE OR QUOTE
-------
Liver tumors (adenoma + carcinoma)
Male BDF1 mouse exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week)
Doses modeled: 0, 5, 25 ppm
Note: models could not fit data with all 4 dose groups; highest dose group dropped
BMR = 0.1
Multistage; 3 -stage model
BMR
(extra risk)
Fisher
AIC
1?P
value"
BMC BMC
L BMR/
BMCL
Thrall
AIC X,
val
•p BMC BM
uea
CL BMR/
BMCL
MCA
(umol/L)
0.1
151.192
0.3562
0.191106 | 0.0636
50 1.57
151.158 0.3
560 0.388392 0.12
2027 0.819
MRAMKL
(jimol/hr-kg liver)
0.1
152.089
0.1864
13.3804 | 7.307
35 0.0137
152.924 0.1
386 14.185 8.82
145 0.0113
"Values <0.1 fail to meet conventional goodness-of-fit criteria; p value from the i2 test.
E-3
DRAFT - DO NOT CITE OR QUOTE
-------
Pheochromocytomas
Female BDF1 mouse exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week)
Doses modeled: 0, 5, 25, 125 ppm
Multistage; 2-stage model
BMR=10%
BMR
(extra risk)
Fisher
AIC
0.1
71.4077
1?P
value"
0.7947
BMC BM
1.42662 1.13
CL BMR/
BMCL
Thrall
AIC f^p BMC BM
value"
MCA
(umol/L)
753 0.0879
71.3358 0.8039 2.94801 2.34
CL BMR/
BMCL
113 0.0427
aValues <0.1 fail to meet conventional goodness-of-fit criteria; p value from the %2 test.
Note: 3-stage model did not provide a sufficiently improved model fit.
E-4
DRAFT - DO NOT CITE OR QUOTE
-------
Pheochromocytomas
Male BDF1 mouse exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week)
Doses modeled: 0, 5, 25,125ppm
Cancer Multistage
BMR=10%
Cancer Multistage (restricted mode) model did not provide an adequate Jit of the male
pheochromocytoma data (1, 2, and 3 stage models provided the same outputs); therefore other models in
BMDS were used (see table below).
BMR
(extra risk)
Fisher
AIC
fp
value"
BMC
BMCL
BMR/
BMCL
Thrall
AIC
fp
value"
BMC
BMCL
BMR/
BMCL
MCA
(umol/L)
1st, 2"" & 3rd
0.1
139.129
0.0513
0.292123
0.230102
0.435
139.077
0.0488
0.600117
0.472644
0.212
aValues <0.1 fail to meet conventional goodness-of-fit criteria; p value from the %2 test.
Pheochromocytomas
Male BDF1 mouse exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week)
Doses modeled: 0, 5, 25, 125 ppm
Models other than Multistage
BMR = 0.1
Model
Fisher
AIC
1?P
value"
BMC
BMCL
BMR/
BMCL
Thrall
AIC
1?P
value"
BMC
BMCL
BMR/
BMCL
MCA (umol/L)
Gammab
Gamma —
unrestricted
Logistic0
Logistic —
unrestricted
Log-logistic0
Log-logistic -
unrestricted
Probit0
Probit -
unrestricted
Log-probit°
Log-probit —
unrestricted
Quantal-linear
Weibullb
Weibull -
unrestricted
139.129
140.755
161.228
161.228
138.661
138.661
159.808
159.808
141.637
137.136
139.129
139.129
140.513
0.0513
0.0401
0.0000
0.0000
0.0978
0.0978
0.0000
0.0000
0.0044
0.1533
0.0513
0.0513
0.0497
0.292124
0.238028
0.929566
0.929566
0.24731
0.247311
0.851235
0.851235
0.423924
0.264859
0.292124
0.292124
0.226525
0.230102
0.10463
0.75614
0.75614
0.147398
0.130943
0.702221
0.702221
0.340228
0.150882
0.230102
0.230102
0.10562
0.435
0.956
0.132
0.132
0.678
0.764
0.142
0.142
0.294
0.663
0.435
0.435
0.947
139.077
140.587
161.353
161.353
138.467
138.467
159.949
159.949
141.988
136.945
139.077
139.077
140.316
0.0488
0.0428
0.0000
0.0000
0.1050
0.1050
0.0000
0.0000
0.0035
0.1648
0.0488
0.0488
0.0535
0.600118
0.473653
1.9184
1.9184
0.492945
0.492945
1.75643
1.75643
0.867906
0.527758
0.60012
0.60012
0.45102
0.472644
0.204957
1.56019
1.56019
0.297393
0.257935
1.44878
1.44878
0.696011
0.297349
0.472644
0.472644
0.207636
0.212
0.488
0.064
0.064
0.336
0.388
0.069
0.069
0.144
0.336
0.212
0.212
0.482
E-5
DRAFT - DO NOT CITE OR QUOTE
-------
Pheochromocytomas
Male BDF1 mouse exposed to carbon tetrachloride vapor for 104 weeks (6 hours/day, 5 days/week)
Doses modeled: 0, 5,
25, 125 ppm
Models other than Multistage
BMR = 0.1
Model
Fisher
??P
value"
BMC BM
CL BMR/
BMCL
Thrall
. „ Y2 p
value
BMC BM
CL BMR/
BMCL
"Values <0.1 fail to meet conventional goodness-of-fit criteria; p value from the % test.
bPower restricted to >1.
°Slope restricted to >1.
E-6
DRAFT - DO NOT CITE OR QUOTE
-------
Female F344 rat — hepatocellular adenomas + carcinomas (0, 5, 25, 125 ppm dose groups)
Dose metric: MRAMKL
Vmax = 0.4 mg/hr/kg BW
° °7
Multistage Cancer Model with 0.95 Confidence Level
0.8
0.6
°-4
ro
0.2
Multistage Cancer
Linear extrapolation
10 15
dose
20
25
10:0010/162007
Multistage Cancer Model. (Version: 1.5; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE RAT LIVER\MRAMKL-
VMAX=0.4\FRAT_LIVER_ADCAR_MRAMKL-4.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE RAT
LIVER\MRAMKL-VMAX=0.4\FRAT_LIVER_ADCAR_MRAMKL-4.pit
Tue Oct 16 10:00:27 2007
BMDS MODEL RUN
Observation # < parameter # for Multistage Cancer model.
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose^l-beta2*dose^2-beta3*dose^3-beta4*dose^4)]
The parameter betas are restricted to be positive
Dependent variable = IncLiverTumor
Independent variable = umol/hr-kgL
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 5
Total number of specified parameters = 0
Degree of polynomial = 4
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0
Beta(l) = 0
Beta(2) = 0
Beta(3) = 0
E-7
DRAFT - DO NOT CITE OR QUOTE
-------
Beta(4) = 6.11699e-006
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Beta(l) -Beta(2) -Beta(3)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Beta(4)
Beta(4) 1
Parameter Estimates
Variable
Background
Beta(l)
Beta(2)
Beta(3)
Beta(4)
Estimate
0
0
0
0
5.52689e-006
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-29.6946
-30.67
-109.05
63.3399
# Param's
4
1
1
Deviance Test d.f.
1.95065
158.71
P-value
0.5827
<.0001
Goodness of Fit
Dose
0.0000
3 .8130
12 .0920
24 .3200
Est. Prob.
0.0000
0.0012
0 .1114
0.8554
Expected
0.000
0.058
5.572
42 .768
Observed
0
0
3
44
Size
50
50
50
50
Scaled
Residual
0.000
-0.242
-1.156
0.495
=1.64
d.f. = 3
P-value = 0.6503
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.05
Extra risk
0.95
9.8151
8.40334
10.5331
Taken together, (8.40334, 10.5331) is a 90
interval for the BMD
% two-sided confidence
Multistage Cancer Slope Factor =
0.00595002
Eo
-O
DRAFT - DO NOT CITE OR QUOTE
-------
Dose metric: MRAMKL
Vmax = 0.65 mg/hr/kg BW
0.07
I
'•8
ro
0.8
0.6
0.4
0.2
0
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
BMDL
0 5
13:0912/142007
10
15 20
dose
25
30
35
Multistage Cancer Model. (Version: 1.5; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE RAT LIVER\MRAMKL-
VMAX=0.65\FRAT_LIVER_ADCAR_MRAMKL-65.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE RAT
LIVER\MRAMKL-VMAX=0.65\FRAT_LIVER_ADCAR_MRAMKL-65.pit
Tue Oct 16 10:05:09 2007
Observation # < parameter # for Multistage Cancer model.
The form of the probability function is:
Dependent variable = IncLiverTumor
Independent variable = umol/hr-kgL
E-9
DRAFT - DO NOT CITE OR QUOTE
-------
Asymptotic Correlation Matrix of Parameter Estimates
Parameter Estimates
Std. Err.
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-29.6946 4
-30.4171 1 1.44504 3
-109.05 1 158.71 3
P-value
0
0
3
44
d.f. = 3
Benchmark Dose Computation
Specified effect = 0.05
Risk Type = Extra risk
Confidence level =
BMD =
BMDL =
BMDU =
E-10
DRAFT - DO NOT CITE OR QUOTE
-------
Female F344 rat — hepatocellular adenomas + carcinomas (0, 5, 25 ppm dose groups)
Dose metric: MRAMKL
,0.07
Vmax = 0.4 mg/hr/kg BW
Multistage Cancer Model with 0.95 Confidence Level
o
t3
ro
0.15
0.1
0.05
Multistage Cancer
Linear extrapolation
BMDL
BMD
6
dose
8
10
12
08:2310/122007
Multistage Cancer Model. (Version: 1.5; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE RAT LIVER\MRAMKL-
VMAX=0.4\FRAT_LIVER_ADCAR_MRAMKL-4.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE RAT
LIVER\MRAMKL-VMAX=0.4\FRAT_LIVER_ADCAR_MRAMKL-4.pit
Fri Oct 12 08:23:17 2007
BMDS MODEL RUN
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose^l-beta2*dose^2)]
The parameter betas are restricted to be positive
Dependent variable = IncLiverTumor
Independent variable = umol/hr-kgL
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0
Beta(l) = 0
Beta(2) = 0.00044169
E-ll
DRAFT - DO NOT CITE OR QUOTE
-------
Asymptotic Correlation Matrix of Parameter Estimates
Beta(2)
*** The model parameter(s) -Background -Beta(l)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Beta(2)
1
Parameter Estimates
Variable
Background
Beta(l)
Beta(2)
Estimate
0
0
0.000383811
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-11.3484
-11.6412
-14.7059
25.2825
# Param's Deviance
3
Test d.f.
0.585705
6.71498
P-value
0.7461
0.03482
Dose
Est. Prob.
Goodness of Fit
Expected Observed Size
Scaled
Residual
0.0000
3 .8130
12 .0920
0.0000
0.0056
0.0546
= 0.31
d.f. = 2
0.000 0 50
0.278 0 50
2.729 3 50
P-value = 0.8571
0.000
-0.529
0.169
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.05
Extra risk
0.95
11.5604
6.92352
30.5183
Taken together, (6.92352, 30.5183) is a 90
interval for the BMD
% two-sided confidence
Multistage Cancer Slope Factor =
0.00722176
E-12
DRAFT - DO NOT CITE OR QUOTE
-------
Dose metric: MRAMKL
Vmax = 0.65 mg/hr/kg BW
0.07
Multistage Cancer Model with 0.95 Confidence Level
0.15
£ o.i
<
o
0.05
Multistage Cancer
Linear extrapolation
BMDL
BMD
10
12
14
16
18
dose
08:3510/122007
Multistage Cancer Model. (Version: 1.5; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE RAT LIVER\MRAMKL-
VMAX=0.65\FRAT_LIVER_ADCAR_MRAMKL-65.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE RAT
LIVER\MRAMKL-VMAX=0.65\FRAT_LIVER_ADCAR_MRAMKL-65.pit
Fri Oct 12 08:35:44 2007
BMDS MODEL RUN
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose^l-beta2*dose^2)]
The parameter betas are restricted to be positive
Dependent variable = IncLiverTumor
Independent variable = umol/hr-kgL
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0
Beta(l) = 0
Beta(2) = 0.000206402
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Beta(l)
E-13
DRAFT - DO NOT CITE OR QUOTE
-------
Beta(2)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Beta(2)
1
Parameter Estimates
Variable
Background
Beta(l)
Beta(2)
Estimate
0
0
0.000183949
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-11.3484
-11.5867
-14.7059
25.1734
# Param's Deviance
3
Test d.f.
0.476667
6.71498
P-value
0.7879
0.03482
Dose
Est. Prob.
Goodness of Fit
Expected Observed Size
Scaled
Residual
0.0000
4.9910
17.6260
0.0000
0.0046
0.0555
ChiA
0.25
d.f. = 2
0.000 0 50
0.229 0 50
2.777 3 50
P-value = 0.8831
0.000
-0.479
0.137
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.05
Extra risk
0.95
16 .6986
9.76339
43 .9237
Taken together, (9.76339, 43.9237) is a 90
interval for the BMD
% two-sided confidence
Multistage Cancer Slope Factor =
0.00512117
E-14
DRAFT - DO NOT CITE OR QUOTE
-------
Female BDF1 mouse — hepatocellular adenomas + carcinomas (0, 5, 25 ppm dose groups)
Dose metric: MRAMKL
Fisher model
Multistage Cancer Model with 0.95 Confidence Level
0.8
0.6
1 0.4
ro
0.2
0 :
Multistage Cancer
Linear extrapolation
BMPL
BMD,
10
15
20 25
dose
30
35
40
12:0410/152007
Multistage Cancer Model. (Version: 1.5; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE MOUSE
LIVER\MRAMKL-FISHER\FMOUSE_LIVER_ADCAR_MRAMKL-FISHER.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE MOUSE
LIVER\MRAMKL-FISHER\FMOUSE_LIVER_ADCAR_MRAMKL-FISHER.pit
Fri Oct 12 08:54:44 2007
BMDS MODEL RUN
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose^l-beta2*dose^2)]
The parameter betas are restricted to be positive
Dependent variable = IncLiverTumor
Independent variable = umol/hr-kgL
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0482072
Beta(l) = 0
Beta(2) = 0.00119035
E-15
DRAFT - DO NOT CITE OR QUOTE
-------
Asymptotic Correlation Matrix of Parameter Estimates
Background
Beta(2)
*** The model parameter(s) -Beta(l)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Background Beta(2)
1 -0.38
-0.38 1
Parameter Estimates
Variable
Background
Beta(l)
Beta(2)
Estimate
0.0693295
0
0.00111772
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-55.6537
-55.9559
-99.1295
115.912
# Param's
3
2
1
Deviance Test d.f.
0.604318
86.9516
P-value
0.4369
<.0001
Dose
Est. Prob.
Goodness of Fit
Expected Observed Size
Scaled
Residual
0.0000
12 .6660
41.6750
= 0.59
0.0693
0.2221
0.8664
d.f. = 1
3.466 4 50
10.883 9 49
43.321 44 50
P-value = 0.4437
0.297
-0.647
0.282
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
Taken together, (6.3204
interval for the BMD
0.1
Extra risk
0.95
9.70893
6.3204
11.2942
, 11.2942) is a 90
two-sided confidence
Multistage Cancer Slope Factor =
0.0158218
E-16
DRAFT - DO NOT CITE OR QUOTE
-------
Dose metric: MRAMKL
Thrall model
Multistage Model with 0.95 Confidence Level
0.8
I °'6
0.4
0.2
0 :
Multistage
BMPL
BMD
10
15
20 25
dose
30
35
40
45
12:1010/152007
Multistage Model. (Version: 2.8; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE
MOUSE LIVER\MRAMKL-THRALL\FMOUSE_LIVER_ADCAR_MRAMKL-THRALL.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS
FEMALE MOUSE LIVER\MRAMKL-THRALL\FMOUSE_LIVER_ADCAR_MRAMKL-THRALL.pit
Fri Oct 12 09:01:03 2007
BMDS MODEL RUN
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose^l-beta2*dose^2)]
The parameter betas are restricted to be positive
Dependent variable = IncLiverTumor
Independent variable = umol/hr-kgL
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0162478
Beta(l) = 0
Beta(2) = 0.00110173
Asymptotic Correlation Matrix of Parameter Estimates
E-17
DRAFT - DO NOT CITE OR QUOTE
-------
user,
Background
Beta(2)
( *** The model parameter(s) -Beta(l)
have been estimated at a boundary point, or have been specified by the
and do not appear in the correlation matrix )
Background Beta(2)
1 -0.4
-0.4 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Background 0.0643165 * * *
Beta(l) 0 * * *
Beta(2) 0.000963757 * * *
* - Indicates that this value is not calculated.
Limit
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f.
-55.6537 3
-56.6705 2 2.03362 1
-99.1295 1 86.9516 2
117.341
P-value
0.1539
<.0001
Goodness of Fit
Dose
0.0000
15.4560
43 .5990
Est. Prob.
0.0643
0.2567
0.8502
Expected
3 .216
12 .580
42 .510
Observed
4
9
44
Size
50
49
50
Scaled
Residual
0.452
-1.171
0.590
Chi 2 =1.92
d.f. = 1
P-value = 0.1654
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
10.4557
7.59255
12.107
Taken together, (7.59255, 12.107 ) is a 90
interval for the BMD
% two-sided confidence
E-18
DRAFT - DO NOT CITE OR QUOTE
-------
Female BDF1 mouse — hepatocellular adenomas + carcinomas (0, 5 ppm dose groups)
Dose metric: MRAMKL
Fisher model
Multistage Cancer Model with 0.95 Confidence Level
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
Multistage Cancer
Linear extrapolation
BMDL
6
dose
10
12
12:4910/152007
Multistage Cancer Model. (Version: 1.5; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE MOUSE
LIVER\MRAMKL-FISHER\FMOUSE_LIVER_ADCAR_MRAMKL-FISHER.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE MOUSE
LIVER\MRAMKL-FISHER\FMOUSE_LIVER_ADCAR_MRAMKL-FISHER.pit
Fri Oct 12 09:15:17 2007
The parameter betas are restricted to be positive
Total number of observations = 2
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Default Initial Parameter Values
Background = 0.24898
E-19
DRAFT - DO NOT CITE OR QUOTE
-------
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l) Beta(2)
Background 1 -2.2e-008 8.3e-009
Beta(l) -6e-009 1 -1
Beta(2) -3.2e-009 -1 1
Parameter Estimates
Std. Err.
* - Indicates that this value is not calculated.
Error in computing chi-sguare; returning 2
Analysis of Deviance Table
Model
Full model
Fitted model
Log(likelihood) # Param's Deviance Test d.f. P-value
-37.3075 2
-37.3075 3 2.84217e-014 -1 NA
1 2.38238 1 0.1227
Dose
Goodness of Fit
Est._Prob. Expected Observed
4.000 4
9.000 9
d.f. = -1
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 11.6352
BMDL = 5.04631
E-20
DRAFT - DO NOT CITE OR QUOTE
-------
Dose metric: MRAMKL
Thrall model
Multistage Model with 0.95 Confidence Level
0.35
0.3
0.25
T3
| 0.2
<
c
•| 0.15
ro
LL
0.1
0.05
Multistage
, ...BMDL
BMD
10
12
14
16
dose
12:5010/152007
Multistage Model. (Version: 2.8; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE MOUSE
LIVER\MRAMKL-THRALL\FMOUSE_LIVER_ADCAR_MRAMKL-THRALL.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE MOUSE
LIVER\MRAMKL-THRALL\FMOUSE_LIVER_ADCAR_MRAMKL-THRALL.pit
Fri Oct 12 09:17:46 2007
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose^l-beta2*dose^2 ) ]
The parameter betas are restricted to be positive
Total number of observations = 2
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
E-21
DRAFT - DO NOT CITE OR QUOTE
-------
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l) Beta(2)
Background 1 NA NA
Beta(l) NA NA NA
Beta (2) NA NA NA
NA - This parameter's variance has been estimated as zero or less.
THE MODEL HAS PROBABLY NOT CONVERGED!!!
Parameter Estimates
Std. Err.
* - Indicates that this value is not calculated.
At least some variance estimates are negative.
THIS USUALLY MEANS THE MODEL HAS NOT CONVERGED!
Try again from another starting point.
Error in computing chi-sguare; returning 2
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
-1
1
-38.4987
80.6149
Goodness of Fit
Est._Prob. Expected Observed Size
d.f. = -1
Benchmark Dose Computatior
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
P-value =
Taken together, (6.15788, 2.64632e+014) is a 90
interval for the BMD
% two-sided confidence
E-22
DRAFT - DO NOT CITE OR QUOTE
-------
Male BDF1 mouse — hepatocellular adenomas + carcinomas (0, 5, 25 ppm)
Dose metric: MRAMKL
Fisher model
Multistage Cancer Model with 0.95 Confidence Level
1
0.9
0.8
§ 0.6
••6
,r 0.5
0.4
0.3
0.2
Multistage Cancer
Linear extrapolation
BMDL
BMD
10
15
20
dose
25
30
35
40
12:0312/042007
Multistage Cancer Model. (Version: 1.5; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS MALE MOUSE LIVER\MRAMKL-
FISHER\MMOUSE_LIVER_ADCAR_MRAMKL-FISHER.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS MALE MOUSE
LIVER\MRAMKL-FISHER\MMOUSE_LIVER_ADCAR_MRAMKL-FISHER.pit
Tue Dec 04 12:03:25 2007
BMDS MODEL RUN
Observation # < parameter # for Multistage Cancer model.
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose^l-beta2*dose^2-beta3*dose^3)]
The parameter betas are restricted to be positive
Dependent variable = IncLiverTumor
Independent variable = umol/hr-kgL
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 4
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.352068
Beta(l) = 0
Beta(2) = 0
Beta(3) = 4.77425e-005
E-23
DRAFT - DO NOT CITE OR QUOTE
-------
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta(l) -Beta(2)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Background
Background
Beta(3)
1
-0.22
Beta(3)
-0.22
1
Parameter Estimates
Variable
Background
Beta(l)
Beta(2)
Beta(3)
Estimate
0.41973
0
0
4.398186-005
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-73.1699
-74.0443
-99.6096
152.089
# Param's
3
2
1
Deviance Test d.f.
1.74874
52.8795
P-value
0.186
<.0001
Dose
Est. Prob.
Goodness of Fit
Expected Observed Size
Scaled
Residual
0.0000
12 .6660
41.6750
0.4197
0.4693
0.9760
=1.75
d.f. = 1
20.987 24 50
23.467 20 50
48.798 49 50
P-value = 0.1864
0.864
-0.982
0.187
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
13.3804
7.30705
15.6428
Taken together, (7.30705, 15.6428) is a 90
interval for the BMD
two-sided confidence
Multistage Cancer Slope Factor =
0.0136854
E-24
DRAFT - DO NOT CITE OR QUOTE
-------
Dose metric: MRAMKL
Thrall model
Multistage Cancer Model with 0.95 Confidence Level
-o
1
:
H
z BMDL B,MD :
0 5 10 15 20 25 30 35 40 45
dose
13:1212/142007
Multistage Cancer Model. (Version: 1.5; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS MALE MOUSE LIVER\MRAMKL-
THRALL\MMOUSE_LIVER_ADCAR_MRAMKL-THRALL.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS MALE MOUSE
LIVER\MRAMKL-THRALL\MMOUSE_LIVER_ADCAR_MRAMKL-THRALL.plt
Tue Dec 04 12:19:57 2007
BMDS MODEL RUN
Observation # < parameter # for Multistage Cancer model.
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose^l-beta2*dose^2-beta3*dose^3)]
The parameter betas are restricted to be positive
Dependent variable = IncLiverTumor
Independent variable = umol/hr-kgL
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 4
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.317881
Beta(l) = 0
Beta(2) = 0
Beta(3) = 4.21166e-005
E-25
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Asymptotic Correlation Matrix of Parameter Estimates
Background
Beta(3)
*** The model parameter(s) -Beta(l) -Beta(2)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Background Beta(3)
1 -0.26
-0.26 1
Parameter Estimates
Variable
Background
Beta(l)
Beta(2)
Beta(3)
Estimate
0.410703
0
0
3.691436-005
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-73.1699
-74.462
-99.6096
152.924
# Param's
3
2
1
Deviance Test d.f.
2.58426
52.8795
P-value
0.1079
<.0001
Goodness of Fit
Dose
0.0000
15.4560
43 .5990
Est. Prob.
0.4107
0.4858
0.9724
Expected
20.535
24 .289
48 .618
Observed
24
20
49
Size
50
50
50
Scaled
Residual
0.996
-1.214
0.330
Chi 2 =2.57
d.f. = 1
P-value = 0.1086
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
14.185
8.82145
16.5171
Taken together, (8.82145, 16.5171) is a 90
interval for the BMD
two-sided confidence
Multistage Cancer Slope Factor =
0.011336
E-26
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BDF1 mouse (female) — pheochromocytomas
Dose metric: MCA
Fisher model
Multistage Cancer Model with 0.95 Confidence Level
0.6
0.5
0 04
t>
< 0.3
g
| 0.2
0.1
Multistage Cancer
Linear extrapolation
BMDL| BMP
0.5
1
1.5
2.5
dose
09:49 10/122007
Multistage Cancer Model. (Version: 1.5; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE
PHEOCHROMOCYTOMAS\FISHER\FMOUSE_PHEOCHROMOCYTOMA_MCA-FISHER.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE
PHEOCHROMOCYTOMAS\FISHER\FMOUSE_PHEOCHROMOCYTOMA_MCA-FISHER.pit
Fri Oct 12 09:49:11 2007
The form of the probability function is:
Dependent variable = Pheochrom
Independent variable = umol/L
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
E-27
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0
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Beta(l)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Parameter Estimates
Std. Err.
Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
Log(likelihood) # Param's Deviance Test d.f.
-33.7087 4
-34.7039 1 1.99041 3
-69.0688 1 70.7202 3
Dose
Chi'
d.f.
P-value =
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0. 95
1. 42662
1.13753
1.72224
Taken together, (1.13753, 1.72224) is a 90
interval for the BMD
two-sided confidence
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Dose metric: MCA
Thrall model
Multistage Cancer Model with 0.95 Confidence Level
•g
1
o
TO
LL
0.6
0.5
0.4
0.3
0.2
0.1
0
: ' ' ' '. ' ' ' ' ' '' :
L / \
/^
^^
\ ]\^=^f^^
! BMDL PMD :
01234567
dose
09:5310/122007
Multistage Cancer Model. (Version: 1.5; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE
PHEOCHROMOCYTOMAS\THRALL\FMOUSE_PHEOCHROMOCYTOMA-MCA-THRALL.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS FEMALE
PHEOCHROMOCYTOMAS\THRALL\FMOUSE_PHEOCHROMOCYTOMA-MCA-THRALL.plt
Fri Oct 12 09:53:23 2007
The form of the probability function is
Dependent variable = Pheochrom
Independent variable = umol/L
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Default Initial Parameter Values
Background = 0
Beta(l) = 0
Beta(2) = 0.0128084
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*** The model parameter(s) -Background -Beta(l)
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Parameter Estimates
95.0% Wald Confidence Interval
Std. Err. Lower Conf. Limit Upper Conf. Limit
Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f.
Full model -33.7087 4
Fitted model -34.6679 1 1.91847 3
Reduced model -69.0688 1 70.7202 3
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 2. 94801
BMDL = 2.34113
BMDU = 3.55893
E-30 DRAFT - DO NOT CITE OR QUOTE
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BDF1 mouse (male) — pheochromocytomas
Dose metric: MCA
Fisher model
0.8
0.7
0.6
t3 u-°
it
< 0.4
c
g
t3 0.3
2
LL
0.2
0.1
0
Probit
BMDL
Probit Model with 0.95 Confidence Level
BMD
0 0.5
12:5511/302007
1.5
2.5
dose
Probit Model. (Version: 2.8; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS MALE
PHEOCHROMOCYTOMAS\FISHER\MMOUSE_PHEOCHROMOCYTOMA_MCA-FISHER.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS MALE
PHEOCHROMOCYTOMAS\FISHER\MMOUSE_PHEOCHROMOCYTOMA_MCA-FISHER.pit
Fri Nov 30 12:55:04 2007
The form of the probability function is:
P[response] = Background
+ (1-Background) * CumNorm(Intercept + Slope*Log(Dose) ) ,
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
User has chosen the log transformed model
E-31
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intercept
slope
Parameter Estimates
NA - Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -64.0144 4
2
1
AIC: 137.136
Goodness of Fit
Dose Est. Prob. Expected Observed
11.
d.f.
P-value =
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 0.264859
BMDL = 0.150882
E-32
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Dose metric: MCA
Thrall model
Probit Model with 0.95 Confidence Level
Affected
o
••6
ro
LL
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
^^~-~~~^ \
^-^ \
> // H
^
-II ;
BMDL .BMD :
01234567
dose
13:1511/302007
Probit Model. (Version: 2.8; Date: 02/20/2007)
Input Data File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS MALE
PHEOCHROMOCYTOMAS\THRALL\MMOUSE_PHEOCHROMOCYTOMA_MCA-THRALL.(d)
Gnuplot Plotting File: G:\CARBON TET\BMD\BMD MODELING 10-2007\TUMORS MALE
PHEOCHROMOCYTOMAS\THRALL\MMOUSE_PHEOCHROMOCYTOMA_MCA-THRALL.plt
Fri Nov 30 13:15:12 2007
The form of the probability function is:
where CumNorm(.) is the cumulative normal distribution function
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-00<:
Parameter Convergence has been set to: le-008
Default Initial (and Specified) Parameter Values
background = 0
intercept = -0.965049
slope = 0.776315
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intercept
slope
*** The model parameter(s) -background
have been estimated at a boundary point, or have been specified
and do not appear in the correlation matrix )
intercept
1
Parameter Estimates
Estimate
Variable
background
intercept
slope
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood) # Param's Deviance Test d.f.
-64.0144 4
-66.4723 2 4.91585 2
-110.216 1 92.4032 3
Est. Prob.
Scaled
Residual
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 0.527758
BMDL = 0.297349
E-34
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E.2. A Bayesian Approach to Modeling Pheochromocytoma Incidence in Male Mice
A Bayesian analysis was conducted utilizing the log-probit model in order to: 1) provide
an alternative to modeling the pheochromocytoma incidence data in male mice using the profile
likelihood method implemented in BMDS; and 2) investigate the distribution of the slope
parameter in the log-probit model.
This Bayesian approach was used to generate a probability distribution of risk estimates.
This formal application of Bayesian methods to the evaluation of uncertainty in dose-response
modeling, although conceptually simple, relies on recent computational advances that allow use
of Markov Chain Monte Carlo (MCMC) methods. The analysis here takes advantage of the
computational power of WinBugs 1.4.1, free software (Spiegelhalter et al., 2003) for the
Bayesian analysis of statistical models using MCMC methods (e.g., Brooks, 1998; Gilks et al.,
1998; Chib and Greenberg, 1995; Casella and George, 1992; Smith and Gelfand, 1992).
More specifically, the use of MCMC methods (via WinBugs) to derive a distribution of
BMDs for the multistage model in BMDS has been recently described by Kopylev et al. (2007).
This same methodology can be straightforwardly generalized to derive a distribution of BMDs
for the log-probit model. For this analysis, diffuse (high variance) Gaussian prior distributions
for both the intercept and slope parameters were used, truncated at zero to exclude negative
parameter values. A uniform (0,1) prior was used for the background parameter. The posterior
distributions of parameters and BMDs are based on three Markov chains of 550,000 simulations
each with a burn-in of 50,000 and thinning rate 10 so that 150,000 total simulations were used
for deriving the posterior distributions of the parameters and the BMDs. Standard practices of
MCMC analysis were followed for verifying convergence using multiple chains and for checking
sensitivity to initial values. The mean and 5th percentile of the posterior distribution provide
estimates of the BMD and the BMDL ("lower bound"), respectively.
Using outputs from the Thrall model and MCA as the dose metric, the BMDio and
BMDLio calculated by this analysis were 0.57568 |imol/L and 0.3177 |imol/L, respectively;
these values are very close to the modeling results generated in BMDS for the log-probit model
(BMDio = 0.5278 |imol/L and BMDLio = 0.2973 |imol/L), thus confirming the results of the
BMDS analysis. Additionally, Figure E-l below shows the posterior distribution of the slope or
shape parameter for the log-probit model generated by the Bayesian analysis. This graph shows
that more than 99% of the posterior distribution for the shape parameter is below 1; whereas in
BMDS the slope parameter for the log-probit model is typically constrained to be greater than 1.
Clearly, constraining the slope parameter in this situation leads to misspecifying the statistical
model and should be avoided.
E-3 5 DRAFT - DO NOT CITE OR QUOTE
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g
5!
o
S
S J
8 -
i
0.4
0.6 0.8
shape parameter
1.0
'.2
Figure E-l. Histogram of the shape parameter
E-36 DRAFT - DO NOT CITE OR QUOTE
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