EPA/635/R-09/001F
www.epa.gov/iris
r/EPA
TOXICOLOGICAL REVIEW
OF
1,1,2,2-TETRACHLOROETHANE
(CAS No. 79-34-5)
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
September 2010
U.S. Environmental Protection Agency
Washington, DC
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DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection
Agency policy and approved for publication. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
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CONTENTS - TOXICOLOGICAL REVIEW OF
1,1,2,2-TETRACHLOROETHANE (CAS No. 79-34-5)
LIST OF TABLES vi
LIST OF FIGURES ix
LIST OF ABBREVIATIONS AND ACRONYMS x
FOREWORD xii
AUTHORS, CONTRIBUTORS, AND REVIEWERS xiii
1. INTRODUCTION 1
2. CHEMICAL AND PHYSICAL INFORMATION 3
3. TOXICOKINETICS 5
3.1. ABSORPTION 5
3.1.1. Oral Exposure 5
3.1.2. Inhalation Exposure 5
3.2. DISTRIBUTION 6
3.3. METABOLISM 7
3.4. ELIMINATION 10
3.5. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELS 11
4. HAZARD IDENTIFICATION 13
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL
CONTROLS 13
4.1.1. Oral Exposure 13
4.1.2. Inhalation Exposure 13
4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
ANIMALS—ORAL AND INHALATION 16
4.2.1. Oral Exposure 16
4.2.1.1. Subchronic Studies 16
4.2.1.2. Chronic Studies 27
4.2.2. Inhalation Exposure 33
4.2.2.1. Subchronic Studies 33
4.2.2.2. Chronic Studies 35
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION.. 35
4.3.1. Oral Exposure 35
4.3.2. Inhalation Exposure 38
4.4. OTHER DURATION- OR ENDPOINT-SPECIFIC STUDIES 39
4.4.1. Acute Studies (Oral and Inhalation) 39
4.4.1.1. Oral Studies 39
4.4.1.2. Inhalation Studies 40
4.4.2. Short-term Studies (Oral and Inhalation) 43
4.4.2.1. Oral Studies 43
4.4.2.2. Short-term Inhalation Studies 48
4.4.3. Acute Injection Studies 49
4.4.4. Immunotoxicological Studies 50
in
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4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF
ACTION 51
4.5.1. Genotoxicity 51
4.5.2. Short-Term Tests of Carcinogen!city 55
4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS 56
4.6.1. Oral 56
4.6.1.1. Human Data 56
4.6.1.2. Animal Data 56
4.6.2. Inhalation 63
4.6.2.1. Human Data 63
4.6.2.2. Animal Data 65
4.6.3. Mode of Action of Noncarcinogenic Effects Information 70
4.7. EVALUATION OF CARCINOGENICITY 71
4.7.1. Summary of Overall Weight of Evidence 71
4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence 72
4.7.3. Mode of Action of Carcinogenicity Information 74
4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 75
4.8.1. Possible Childhood Susceptibility 75
4.8.2. Possible Gender Differences 76
4.8.3. Other Susceptible Populations 76
5. DOSE-RESPONSE ASSESSMENTS 77
5.1. ORAL REFERENCE DOSE (RfD) 77
5.1.1. Subchronic Oral RfD 77
5.1.1.1. Choice of Principal Study and Critical Effect—with Rationale and
Justification 77
5.1.1.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) 80
5.1.1.3. RfD Derivation—Including Application of Uncertainty Factors (UFs) 82
5.1.2. Chronic Oral RfD 83
5.1.2.1. Choice of Principal Study and Critical Effect—with Rationale and
Justification 83
5.1.2.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) 84
5.1.2.3. RfD Derivation—Including Application of UFs 84
5.1.3. RfD Comparison Information 85
5.1.4. Previous RfD Assessment 91
5.2. INHALATION REFERENCE CONCENTRATION (RfC) 91
5.2.1. Choice of Principal Study and Critical Effect—with Rationale and Justification91
5.2.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) 93
5.2.3. Previous RfC Assessment 93
5.3. UNCERTAINTIES IN THE ORAL REFERENCE DOSE AND INHALATION
REFERENCE CONCENTRATION 93
5.4. CANCER ASSESSMENT 95
5.4.1. Choice of Study/Data—with Rationale and Justification 96
5.4.2. Dose-response Data 97
5.4.3. Dose Adjustments and Extrapolation Method(s) 97
5.4.4. Oral Slope Factor and Inhalation Unit Risk 99
5.4.5. Uncertainties in Cancer Risk Values 99
5.4.6. Previous Cancer Assessment 102
IV
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6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE
RESPONSE 103
6.1. HUMAN HAZARD POTENTIAL 103
6.2. DOSE RESPONSE 104
6.2.1. Noncancer/Oral 104
6.2.2. Noncancer/Inhalation 110
6.2.3. Cancer/Oral and Inhalation Ill
7. REFERENCES 114
APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS
AND DISPOSITION A-l
APPENDIX B. BENCHMARK DOSE MODELING RESULTS FOR THE DERIVATION OF
THERfD B-l
APPENDIX C. BENCHMARK DOSE MODELING RESULTS FOR THE DERIVATION OF
THE ORAL SLOPE FACTOR C-l
v
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LIST OF TABLES
2-1. Chemical and physical properties of 1,1,2,2-tetrachloroethane 3
4-1. Final body weights (g) and percent change compared to controls in F344/N rats
exposed to 1,1,2,2-tetrachloroethane in feed for 14 weeks 17
4-2. Absolute liver weights (g) and percent change compared to controls in F344/N rats
exposed to 1,1,2,2-tetrachloroethane in feed for 14 weeks 17
4-3. Relative liver weight (mg organ weight/g body weight) and percent change compared
to controls in F344/N rats exposed to 1,1,2,2-tetrachloroethane in feed for 14 weeks 18
4-4. Serum chemistry and hematology changes in rats exposed to dietary
1,1,2,2-tetrachloroethane for 14 weeks 19
4-5. Incidences of selected histopathological lesions in rats exposed to dietary
1,1,2,2-tetrachlorethanefor 14 weeks 20
4-6. Final body weights (g) and percent change compared to controls in B6C3Fi mice
exposed to 1,1,2,2-tetrachloroethane in feed for 14 weeks 23
4-7. Absolute liver weights (g) and percent change compared to controls in B6C3Fi mice
exposed to 1,1,2,2-tetrachloroethane in feed for 14 weeks 24
4-8. Relative liver weights (mg organ weight/g body weight) and percent change compared
to controls in B6C3Fi mice exposed to 1,1,2,2-tetrachloroethane in feed for 14 weeks 24
4-9. Selected clinical chemistry changes in male mice exposed to dietary
1,1,2,2-tetrachloroethane for 14 weeks 25
4-10. Selected clinical chemistry changes in female mice exposed to dietary
1,1,2,2-tetrachloroethane for 14 weeks 26
4-11. Incidences of selected histopathological lesions in mice exposed to dietary
1,1,2,2-tetrachloroethane for 14 weeks 27
4-12. Incidence of neoplasms in male Osborne-Mendel rats exposed to
1,1,2,2-tetrachloroethane in feed for 78 weeks 29
4-13. Incidence of neoplasms in female Osborne-Mendel rats exposed to
1,1,2,2-tetrachloroethane in feed for 78 weeks 30
4-14. Incidence of nonneoplastic kidney lesions observed in male and female B6C3Fi mice
exposed to 1,1,2,2-tetrachloroethane in feed for 78 weeks 31
4-15. Incidence of hepatocelluar carcinomas in male and female B6C3Fi mice exposed to
1,1,2,2-tetrachloroethane in feed for 78 weeks 32
VI
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4-16. Incidence of additional neoplasms in male and female B6C3Fi mice exposed to
1,1,2,2-tetrachloroethane in feed for 78 weeks 32
4-17. Fetal body weight in CD Sprague-Dawley rats exposed to microencapsulated
1,1,2,2-tetrachloroethane on GDs 4-20 36
4-18. Liver function and other effects observed in Sprague-Dawley rats 60 minutes after
gavage exposure to 1,1,2,2-tetrachloroethane 39
4-19. Results of in vitro and in vivo genotoxicity studies of 1,1,2,2-tetrachloroethane 51
4-20. Pulmonary adenomas in male A/St mice following repeated i.p. injections of
1,1,2,2-tetrachloroethane 55
4-21. Pulmonary adenomas in male and female A/St mice following repeated i.p.
injections of 1,1,2,2-tetrachloroethane 56
4-22. Summary of noncancer results of major studies for oral exposure of animals to
1,1,2,2-tetrachloroethane 58
4-23. Summary of noncancer results of maj or human studies of inhalation exposure to
1,1,2,2-tetrachloroethane 64
4-24. Summary of noncancer results of major studies for inhalation exposure of
animals to 1,1,2,2-tetrachloroethane 66
5-1. Summary of BMD model results for rats exposed to 1,1,2,2-tetrachloroethane 80
5-2. Potential PODs with applied UFs and resulting subchronic RfDs 88
5-3. Incidences of hepatocellular carcinomas in B6C3Fi mice used for dose-response
assessment of 1,1,2,2-tetrachloroethane 97
5-4. HEDs corresponding to duration-adjusted TWA doses in mice 98
5-5. Summary of human equivalent BMDs and BMDLs based on hepatocellular carcinoma
incidence data in female B6C3Fi mice 99
5-6. Summary of uncertainty in the 1,1,2,2-tetrachloroethane cancer risk assessment 100
B-l. Incidences of hepatocellular cytoplasmic vacuolization in rats exposed to dietary
1,1,2,2-tetrachlorethanefor 14 weeks B-l
B-2. Summary of BMD modeling results for the incidence of hepatocellular cytoplasmic
vacuolization in male rats B-2
B-3. Summary of BMD model results for the incidence of hepatocellular cytoplasmic
vacuolization in female rats B-7
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B-4. Selected organ weight and serum chemistry changes in male and female F344 rats
administered 1,1,2,2-tetrachlroethane in the diet for 14 weeks B-15
B-5. Summary of BMD modeling results for absolute liver weight in male rats B-17
B-6. Summary of BMD modeling results for absolute liver weight in female rats B-22
B-7. Summary of BMD modeling results for relative liver weight in male rats B-28
B-8. Summary of BMD modeling results for relative liver weight in female rats B-29
B-9. Summary of BMD modeling results for serum ALT activity in male rats B-3 5
B-10. Summary of BMD modeling results for serum ALT activity in female rats B-40
B-11. Summary of BMD modeling results for serum SDH activity in male rats B-46
B-12. Summary of BMD modeling results for serum SDH activity in female rats B-47
B-13. Summary of BMD results for serum bile acid levels in male rats B-53
B-14. Summary of BMD modeling results for serum bile acid levels in female rats B-58
B-15. Fetal body weight in Sprague-Dawley rats administered 1,1,2,2-tetrachloroethane
in the diet on GDs 4-20 B-64
B-16. Summary of BMD modeling results for fetal body weight following exposure of
pregnant Sprague-Dawley rats on GDs 4-20 B-65
C-l. Incidence of hepatocellular carcinomas in B6C3Fi mice administered
1,1,2,2-tetrachloroethane by gavage for 78 weeks C-l
C-2. Summary of BMD modeling results for the incidence of hepatocellular carcinomas
in male mice C-2
C-3. Summary of BMD modeling results for the incidence of hepatocellular carcinomas in
female mice C-2
Vlll
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LIST OF FIGURES
2-1. Structure of 1,1,2,2-tetrachloroethane 3
3-1. Suggested metabolic pathways of 1,1,2,2-tetrachloroethane 7
5-1. Exposure response array for subchronic and chronic oral exposure to
1,1,2,2-tetrachloroethane 87
5-2. PODs for selected endpoints (with critical effect circled) from Table 5-2 with
corresponding applied UFs and derived sample subchronic oral reference
values (RfVs) 89
5-3. PODs for selected endpoints (with critical effect circled) from Table 5-2 with
corresponding applied UFs and derived sample chronic oral RfVs 90
6-1. PODs for selected endpoints (with critical effect circled) with corresponding applied
UFs and derived sample subchronic oral RfVs 106
6-2. PODs for selected endpoints (with critical effect circled) from Table 5-2 with
corresponding applied UFs and derived sample subchronic oral RfVs 109
IX
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LIST OF ABBREVIATIONS AND ACRONYMS
ACTH adrenocorticotropic hormone
AIC Akaike's Information Criterion
ALP alkaline phosphatase
ALT alanine aminotransferase
AST aspartate aminotransferase
ATP adenosine triphosphate
ATSDR Agency for Toxic Substances and Disease Registry
AUC area under the curve
BMD benchmark dose
BMDL 95% confidence limit (lower bound) on the benchmark dose
BMDS benchmark dose software
BMR benchmark response
CASRN Chemical Abstracts Service Registry Number
CHO Chinese hamster ovary
CNS central nervous system
CYP cytochrome P450
DEN diethylnitrosamine
DF degrees of freedom
DNA deoxyribonucleic acid
FEL frank effect level
FOB functional observational battery
G6Pase glucose-6-phosphatase
GD gestation day
GGT gamma glutamyltranspeptidase
GST glutathione S-transferase
Hb hemoglobin
HED human equivalent dose
i.p. intraperitoneal
IU international units
LCso median lethal concentration
LD50 median lethal dose
LOAEL lowest-observed-adverse-effect level
mA milliampere
NCI National Cancer Institute
NOAEL no-observed-adverse-effect level
NPL National Priorities List
NTP National Toxicology Program
PBPK physiologically based pharmacokinetic
PCNA proliferating cell nuclear antigen
POD point of departure
RBC red blood cell
RfC reference concentration
RfD reference dose
RfV reference value
RNA ribonucleic acid
SCE sister chromatid exchange
x
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SD standard deviation
SDH sorbitol dehydrogenase
TWA time-weighted average
UDS unscheduled DNA synthesis
UF uncertainty factor
U.S. EPA U.S. Environmental Protection Agency
WBC white blood cell
XI
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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 subchronic and chronic
exposure to 1,1,2,2-tetrachloroethane. It is not intended to be a comprehensive treatise on the
chemical or toxicological nature of 1,1,2,2-tetrachloroethane.
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).
xn
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER/AUTHOR
Martin W. Gehlhaus, M.H.S.
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
CONTRIBUTING AUTHORS
AmbujaBale, Ph.D.
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
Geoffrey W. Patton, Ph.D.
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
Susan Rieth, M.S.
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
TedBerner, M.S.
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
Karen Hogan, M.S.
National Center for Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC
CONTRACTING SUPPORT
Mark Osier, Ph.D.
Environmental Science Center
Syracuse Research Corporation
Syracuse, NY
Stephen Bosch, B.S.
Environmental Science Center
Syracuse Research Corporation
Syracuse, NY
xin
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Marc Odin, M.S. DABT
Environmental Science Center
Syracuse Research Corporation
Syracuse, NY
REVIEWERS
This document has been provided for review to EPA scientists, interagency reviewers
from other federal agencies and White House offices, and the public, and peer reviewed by
independent scientists external to EPA. A summary and EPA's disposition of the comments
received from the independent external peer reviewers and from the public is included in
Appendix A.
INTERNAL EPA REVIEWERS
Joyce M. Donohue, Ph.D.
Office of Water
Office of Science and Technology (OST)
Health and Ecological Criteria Division (HECD)
Lynn Flowers, Ph.D., DABT
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Chris Cubbison
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH
EXTERNAL PEER REVIEWERS
Bruce C. Allen, M.S.
Bruce Allen Consulting
James V. Bruckner, Ph.D. (chair)
University of Georgia
Wolfgang Dekant, Dr. Rer. Nat.
University of Wiirzburg
Dale Hattis, Ph.D.
Clark University
Sam Kacew, Ph.D., ATS
University of Ottawa
xiv
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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
1,1,2,2-tetrachloroethane. 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 for
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 a plausible upper bound on
the estimate of risk per mg/kg-day of oral exposure. Similarly, an inhalation unit risk is a
plausible upper bound on the estimate of risk per ug/m3 air breathed.
Development of these hazard identification and dose-response assessments for
1,1,2,2-tetrachloroethane has followed the general guidelines for risk assessment as set forth by
the National Research Council (NRC, 1983). U.S. Environmental Protection Agency (U.S. 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
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Assessment (U.S. EPA, 199 la), Interim Policy for Particle Size and 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, 1996), Guidelines for Neurotoxicity Risk
Assessment (U.S. EPA, 1998), 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 February,
2010.
Portions of this document were developed under a Memorandum of Understanding with
the Agency for Toxic Substances and Disease Registry (ATSDR) as part of a collaborative effort
in the development of human health toxicological assessments.
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2. CHEMICAL AND PHYSICAL INFORMATION
1,1,2,2-Tetrachloroethane (CASRN 79-34-5) is a synthetic, halogenated hydrocarbon that
is a colorless, nonflammable liquid at room temperature. It is highly volatile, somewhat soluble
in water, and miscible with many organic solvents. The structure of 1,1,2,2-tetrachloroethane is
shown below (Figure 2-1), and the chemical and physical properties are presented in Table 2-1.
Cl Cl
H—C-
Cl
-C—H
Cl
Figure 2-1. Structure of 1,1^2,2-tetrachloroethane.
Table 2-1. Chemical and physical properties of l,l?2,2-tetrachloroethane
Characteristic
Chemical name
Synonym(s)
Chemical formula
CASRN
Molecular weight
Color
Freezing point
Boiling point
Density at 20°C
Odor threshold:
Water
Air
Solubility:
Water
Organic solvents
Information
1, 1,2,2-Tetrachloroethane
Acetylene tetrachloride; sym-tetrachloroethane; s-tetrachloro-
ethane; tetrachlorethane; l,l-dichloro-2,2-dichloroethane
C2H2C14
79-34-5
167.85
Colorless
-43.8°C
-36°C
145. 1°C
146.2°C
146.5°C
1.594
1.595
0.50 ppm
1.5 ppm
3-5 ppm
2.87 g/L (20°C)
2.85 g/L (25°C)
Miscible with ethanol, methanol, ether, acetone, benzene,
petroleum, carbon tetrachloride, carbon disulfide, dimethyl
formamide, oils
Reference
NLM, 2009; CAS, 1994
CAS, 1994
CAS, 1994
NLM, 2009; CAS, 1994;
Lide, 1993;Riddicketal.,
1986
Hawley, 1981
Riddicketal., 1986
Lide, 1993
Riddicketal., 1986
Lide, 1993
Merck Index, 1989
Riddicketal., 1986
Lide, 1993
NLM, 2009; Amoore and
Hautala, 1983
Amoore and Hautala, 1983
NLM, 2009
Riddicketal., 1986
Merck Index, 1989
NLM, 2009; Merck Index,
1989; Hawley, 1981
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Table 2-1. Chemical and physical properties of l,l?2,2-tetrachloroethane
Characteristic
Vapor pressure
Partition
coefficients:
log Kow
logKoc
Henry's law constant
Flash point
Conversions:
ppm to mg/m3
mg/m3 to ppm
Information
5.95mmHg(25°C)
9mmHg(30°C)
2.39
1.66
2.78
4.7 x 10'4 atm-m3/mol
4.55 x l(T4 atm-m3/mol
1.80 x 10"3 atm-m3/mol
None - nonflammable
1 ppm =6.87 mg/m3
1 mg/m3 = 0. 146 ppm
Reference
Riddicketal., 1986
NLM, 2009; Flick, 1985
Hansch and Leo, 1985
Chiouetal., 1979
ASTER, 1995
Mackay and Shiu, 1981
NLM, 2009
ASTER, 1995
NLM, 2009; Hawley, 1981
Calculated
Calculated
In the past, the major use for 1,1,2,2-tetrachloroethane was in the production of
trichloroethylene, tetrachloroethylene, and 1,2-dichloroethylene (Archer, 1979).
1,1,2,2-Tetrachloroethane has been identified at numerous National Priorities List (NPL) sites
(ATSDR, 2008). With the development of new processes for manufacturing chlorinated
ethylenes and the availability of less toxic solvents, the production of 1,1,2,2-tetrachloroethane
as a commercial end-product in the United States and Canada has steadily declined since the late
1960s, and production ceased by the early 1990s (NLM, 2009; Environment Canada and Health
Canada, 1993). 1,1,2,2-Tetrachloroethane may still appear as a chemical intermediate in the
production of a variety of other common chemicals. Uses of 1,1,2,2-tetrachloroethane include as
a solvent; in cleaning and degreasing metals; in paint removers, varnishes, and lacquers; in
photographic films; and as an extractant for oils and fats (Hawley, 1981). Although at one time
it was used as an insecticide, fumigant, and weed killer (Hawley, 1981), it presently is not
registered for any of these purposes. It was once used as an ingredient in an insect repellent, but
registration was canceled in the late 1970s.
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3. TOXICOKINETICS
1,1,2,2-Tetrachloroethane is well-absorbed from the respiratory and gastrointestinal tracts
in both humans and laboratory animals, and is extensively metabolized and excreted, chiefly as
metabolites, in the urine and breath. The metabolism of 1,1,2,2-tetrachloroethane in rats and
mice results in the production of trichloroethanol, trichloroacetic acid, and dichloroacetic acid.
The dichloroacetic acid is then broken down to glyoxalic acid, oxalic acid, and carbon dioxide.
When 1,1,2,2-tetrachloroethane undergoes reductive or oxidative metabolism, reactive radical
and acid chloride intermediates, respectively, are produced.
3.1. ABSORPTION
3.1.1. Oral Exposure
There are no known studies that quantify absorption following oral exposure in humans.
However, the health effects resulting from ingestion of large amounts of 1,1,2,2-tetrachloro-
ethane in humans (Section 4.1.1) indicate that 1,1,2,2-tetrachloroethane is absorbed following
oral exposure.
Observations in animals indicate that the oral absorption of 1,1,2,2-tetrachloroethane is
rapid and extensive. Cottalasso et al. (1998) reported hepatic effects only 15-30 minutes
following a single oral exposure in rats, including increases in serum aspartate aminotransferase
(AST) and alanine aminotransferase (ALT), a decrease in microsomal glucose-6-phosphatase
(G6Pase) activity, and an increase in triglyceride levels. Following a single oral exposure of
male Osborne-Mendel rats and B6C3Fi mice to 150 mg/kg of radiolabeled 1,1,2,2-tetrachloro-
ethane in corn oil, only 4-6% of the activity was recovered in the feces 72 hours postexposure
while >90% of the administered activity was found in both species as metabolites, indicating that
the compound was nearly completely absorbed in both rats and mice within 72 hours (Dow
Chemical Company, 1988). Mitoma et al. (1985) exposed groups of male Osborne-Mendel rats
to 25 or 100 mg/kg and B6C3Fi mice to 50 or 200 mg/kg of 1,1,2,2-tetrachloroethane in corn oil
gavage 5 days/week for 4 weeks, followed by a single radiolabeled dose of the compound, and
evaluated the disposition of the radiolabeled 1,1,2,2-tetrachloroethane over the next 48 hours.
While absorption was not quantified, 79% of the dose was metabolized in rats and 68% was
metabolized in mice, suggesting that at least those levels of compound had been absorbed within
48 hours.
3.1.2. Inhalation Exposure
While studies of the systemic toxicity of 1,1,2,2-tetrachloroethane following inhalation in
humans are indicative of some level of systemic absorption, comparatively few studies have
quantitatively addressed this issue. A study in volunteers was carried out in which a bulb
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containing [38Cl]-labeled 1,1,2,2-tetrachloroethane was inserted into their mouths; they
immediately inhaled deeply, held their breaths for 20 seconds, and then exhaled through a trap
containing granulated charcoal. The study showed that approximately 96% of a single breath of
1,1,2,2-tetrachloroethane was absorbed systemically (Morgan et al., 1970). Two subjects were
reported to retain approximately 40-60% of inspired 1,1,2,2-tetrachloroethane after a 30-minute
exposure of up to 2,300 mg/m3 (Lehmann et al., 1936), but additional details were not provided.
The total body burden of 1,1,2,2-tetrachloroethane in male Osborne-Mendel rats and
B6C3Fi mice exposed to a vapor concentration of 10 ppm (68.7 mg/m3) for 6 hours (Dow
Chemical Company, 1988) was 38.7 jimol equivalents/kg in rats (9.50 umol equivalents and
using a body weight of 245 g from the study) and 127 umol equivalents/kg in mice (3.059 umol
equivalents and using a body weight of 24.1 g from the study), indicating that while absorption
occurred in both species, mice absorbed proportionally more 1,1,2,2-tetrachloroethane on a per-
body-weight basis. Ikeda and Ohtsuji (1972) detected metabolites measured as total
trichlorocompounds, trichloroacetic acid, and trichloroethanol, in the urine of rats exposed to
200 ppm (1,370 mg/m3) 1,1,2,2-tetrachloroethane, indicating that absorption had occurred;
however, they did not provide a quantitative estimate of absorption rate or fraction. Similarly,
Gargas and Anderson (1989) followed the elimination of 1,1,2,2-tetrachloroethane as exhaled
breath from the blood after a 6-hour exposure to 350 ppm (2,400 mg/m3), but did not provide
quantitative estimates of absorption.
3.2. DISTRIBUTION
No studies measuring the distribution of 1,1,2,2-tetrachloroethane in humans following
inhalation or oral exposure were located. Following absorption in animals, 1,1,2,2-tetrachloro-
ethane appears to be distributed throughout the body, but may selectively accumulate to a degree
in certain cells and tissues. The human blood-air partition coefficient for 1,1,2,2-tetrachloro-
ethane has been reported to be in the range of 72.6-116 (Meulenberg and Vijverberg, 2000;
Gargas et al., 1989; Morgan et al., 1970). The tissue:air partition coefficients for 1,1,2,2-tetra-
chloroethane in rats have been reported to be 142 (blood), 3,767 (fat), 196 (liver), and
101 (muscle) (Meulenberg and Vijverberg, 2000; Gargas et al., 1989), indicating that
1,1,2,2-tetrachloroethane may partition into fatty tissues, consistent with its low water solubility.
Following a single intravenous injection of radiolabeled 1,1,2,2-tetrachloroethane,
Eriksson and Brittebo (1991) reported selective uptake of nonvolatile radioactivity in the
mucosal tissues of olfactory and tracheobronchial regions of the respiratory tract and in the
mucosae of the oral cavity, tongue, nasopharynx, esophagus, and cardiac region of the
forestomach. High levels of radioactivity were also found in the liver, bile, inner zone of the
adrenal cortices, and interstitium of the testes, although the levels were not quantified.
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3.3. METABOLISM
No studies were located that investigated the metabolism of 1,1,2,2-tetrachloroethane in
humans. Information regarding 1,1,2,2-tetrachloroethane metabolism in animals is summarized
below, and a suggested metabolic scheme based on in vivo and in vitro data is presented in
Figure 3-1.
ci
ci
free radical
CI CI
\ /
reductive /\
dechlorination CI
1,1,2,2-
Ion-enzymatic
dehydrochlorination
CI
loxidation
hydrolytic
cleavage
dichloroacetaldehyde
Trichloroethylene
Cl-
trichloroacetaldehyde
Cf CI
tetrachloroethylene
dichloroacetic acid
P450/ I
\ 7 \
CI OH
CI
CI
CI.
CI
CI
OH
trichloroethanol
Trichloroacetic
oxalic acid
CO2 + C OH
formic acid
Source: Adapted from ATSDR (1996).
Figure 3-1. Suggested metabolic pathways of l,l?2,2-tetrachloroethane.
In vivo and in vitro studies indicate that the metabolism of 1,1,2,2-tetrachloroethane
proceeds via multiple pathways in rodents (Mitoma et al., 1985; Casciola and Ivanetich, 1984;
Halpert, 1982; Koizumi et al., 1982; Halpert andNeal, 1981; Ikeda and Ohtsuji, 1972; Yllner,
1971). The predominant pathway appears to involve production of dichloroacetic acid, formed
as an initial metabolite via stagewise hydrolytic cleavage of 1,1,2,2-tetrachloroethane, yielding
dichloroacetyl chloride and dichloroacetaldehyde as intermediates, or by cytochrome P450
(CYP)-based oxidation of 1,1,2,2-tetrachloroethane (Casciola and Ivanetich, 1984; Halpert and
Neal, 1981; Yllner, 1971). Dichloroacetic acid was identified as the major urinary metabolite in
mice treated with 1,1,2,2-tetrachloroethane by intraperitoneal (i.p.) injection (Yllner, 1971) and
in in vitro systems with rat liver microsomal and nuclear CYP (Casciola and Ivanetich, 1984;
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Halpert, 1982; Halpert and Neal, 1981). Dichloroacetic acid can be further metabolized to
glyoxylic acid, formic acid, and carbon dioxide (Yllner, 1971), with carbon dioxide a potential
major component of the end products (Yllner, 1971). Other pathways may involve the formation
of trichloroethylene via dehydrochlorination or tetrachloroethylene via oxidation as initial
metabolites (Mitoma et al., 1985; Ikeda and Ohtsuji, 1972; Yllner et al., 1971).
Trichloroethylene and tetrachloroethylene are further metabolized to trichloroethanol and
trichloroacetic acid, and oxalic acid and trichloroacetic acid, respectively (Mitoma et al., 1985;
Ikeda and Ohtsuji, 1972; Yllner et al., 1971). 1,1,2,2-Tetrachloroethane may also form free
radicals by undergoing reductive dechlorination (ATSDR, 1996). The formation of free radical
intermediates during 1,1,2,2-tetrachloroethane metabolism has been demonstrated in spin-
trapping experiments (Paolini et al., 1992; Tomasi et al., 1984).
Metabolism of 1,1,2,2-tetrachloroethane is generally extensive, with 68-95% of a total
administered dose found as metabolites (Dow Chemical Company, 1988; Mitoma et al., 1985;
Yllner, 1971). Mice given a single 0.21-0.32 g/kg i.p. dose of [14C]-labeled 1,1,2,2-tetrachloro-
ethane eliminated 45-61% of the administered radioactivity as carbon dioxide in expired air and
23-34% of the radioactivity in urine in the following 3 days (Yllner et al., 1971). Dichloroacetic
acid, trichloroacetic acid, trichloroethanol, oxalic acid, glyoxylic acid, and urea accounted for 27,
4, 10, 7, 0.9, and 2% of the mean urinary radioactivity excreted by the mice in 24 hours,
respectively (Yllner et al., 1971). Yllner et al. (1971) also demonstrated that 20-23% of the
[14C]-tetrachloroethane was converted to glycine following the simultaneous i.p. injection of
[14C]-tetrachloroethane and sodium benzoate and the estimation of [14C]-hippuric acid in the
urine. In rats, trichloroethanol appeared to be present as a urinary metabolite at approximately
fourfold greater levels than trichloroacetic acid following a single 8-hour inhalation exposure
(Ikeda and Ohtsuji, 1972). Several studies have reported that metabolism of 1,1,2,2-tetrachloro-
ethane is greater in mice than in rats, with magnitudes of the reported difference generally in the
range of a 1.1-3.5-fold greater metabolic activity, on a per-kg basis, in mice (Dow Chemical
Company, 1988; Mitoma et al., 1985).
As indicated above, CYP-based metabolism of 1,1,2,2-tetrachloroethane to dichloroacetic
acid has been demonstrated in vitro. Multiple CYP isozymes are likely to be involved, as
demonstrated by studies reporting increased metabolism and covalent binding of metabolites
following pretreatment with phenobarbital (Casciola and Ivanetich, 1984; Halpert, 1982), xylene
(Halpert, 1982), or ethanol (Sato et al., 1980). The isozymes induced by phenobarbital, xylene,
and ethanol include members of the CYP2A, CYP2B, CYP2E, and CYP3A) subfamilies
(Omiecinski et al., 1999; Nebert et al., 1987).
1,1,2,2-Tetrachloroethane has also been reported to produce inactivation of CYP.
1,1,2,2-Tetrachloroethane effectively inactivated the major phenobarbital-inducible CYP
isozyme, but not the major CYP isozyme induced by p-naphthoflavone, in rat liver in vitro
(Halpert et al., 1986). Rat liver nuclear CYP levels were reduced following in vitro incubation
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with 1,1,2,2-tetrachloroethane and a NADPH-generating system (Casciola and Ivanetich, 1984).
In an in vivo study, CYP activity was evaluated in male and female Swiss albino mice 24 hours
after a single 0, 300, or 600 mg/kg i.p. dose of 1,1,2,2-tetrachloroethane (Paolini et al., 1992).
1,1,2,2-Tetrachloroethane treatment statistically significantly (p < 0.01) reduced total CYP
activity 44 and 37% in males and females, respectively, at 300 mg/kg and 85 and 74% in males
and females, respectively, at 600 mg/kg. Treatment with 600 mg/kg statistically significantly
reduced the microsomal activity of CYP isozymes 3A, 2E1, 1A2, 2B1, and 1A1 in both genders,
and 300 mg/kg reduced the activity of CYP3A in both sexes and CYP2B1 in males. Heme
content was reduced 13 and 33% at 300 and 600 mg/kg, respectively, and may have contributed
to the decrease in CYP levels. The 600 mg/kg dose also reduced the activity of glutathione S-
transferase (GST) toward l-chloro-2,4-dinitrobenzene, a general GST substrate, in both genders.
Due to the extensive metabolism of 1,1,2,2 tetrachloroethane to products such as
trichloroethylene and dichloroacetic acid, the relevance of 1,1,2,2-tetrachloroethane interactions
with GST is important. Studies of human GST-zeta polymorphic variants show different
enzymatic activities toward and inhibition by dichloroacetic acid that could reasonably affect the
metabolism of 1,1,2,2-tetrachloroethane (Lantum et al., 2002; Blackburn et al., 2001, 2000;
Tzeng et al., 2000). Dichloroacetic acid may covalently bind to GST-zeta (Anderson et al.,
1999) and inhibit its own metabolism, leading to an increase in the amount of unmetabolized
dichloroacetic acid as the dose and/or duration increases (U.S. EPA, 2003).
Data indicate that 1,1,2,2-tetrachlorethane can be metabolized to dichloroacetic acid
(ATSDR, 1996; Yllner, 1971), suggesting a potential role for this metabolite in some of the
cancer and noncancer effects observed following exposure to 1,1,2,2 tetrachloroethane.
Following an intravenous injection of radiolabeled 1,1,2,2-tetrachloroethane, radioactivity could
not be extracted from epithelium of the respiratory and upper alimentary tracts, or from the liver,
adrenal cortex, or testes (Eriksson and Brittebo, 1991). The presence of tissue-bound metabolites
in the epithelial linings in the upper respiratory tract may demonstrate a first-pass effect by the
respiratory tract (Eriksson and Brittebo, 1991). In addition, the presence of irreversible tissue-
bound metabolites demonstrates the metabolism of 1,1,2,2-tetrachloroethane to reactive
metabolites (Eriksson and Brittebo, 1991). However, the identities of the bound metabolites and
modified proteins or phospholipids were not identified. The presence of radiolabel in the
proteins may have been radiolabeled-incorporated glycine.
Dow Chemical Company (1988) observed radiolabel in hepatic deoxyribonucleic acid
(DNA), although the presence of the radiolabel in the hepatic DNA likely represented the
incorporation of single [14C]-atoms via normal biosynthetic pathways. Mice were found to have
approximately a 1.9-fold greater extent of [14C] activity irreversibly associated with hepatic
macromolecules than rats, which the study authors noted was consistent with the greater
metabolism, on a per-kg basis, in mice compared to rats. After a 4-week oral exposure to
unlabeled 1,1,2,2-tetrachloroethane followed by a single oral dose of labeled
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1,1,2,2-tetrachloroethane, Mitoma et al. (1985) also reported greater levels of hepatic protein-
binding in the tissue of mice compared to rats, and the differences were on the order of twofold
greater binding in mice, which would be consistent both with the Dow Chemical Company
(1988) studies and with the observed differences in metabolism of the two species discussed
above. This may also be related to the 3.2-3.5-fold greater absorption, on a per-kg basis, of mice
compared to rats following inhalation exposure (Dow Chemical Company, 1988).
The kinetic constants of 1,1,2,2-tetrachloroethane metabolism in rats exposed to 350 ppm
of the chemical for 6 hours were determined in gas uptake studies performed by Gargas and
Anderson (1989). The rate of exhalation of 1,1,2,2-tetrachloroethane was measured and,
combined with previously published values for partition coefficients for blood/air, liver/blood,
muscle/blood, and fat/blood, allowed the estimation of the disposition of the chemical in rat
(Gargas et al., 1989). A Km of 4.77 uM and a Vmax of 12 mg/hour (scaled to a 1-kg rat) were
measured.
3.4. ELIMINATION
Morgan et al. (1970) reported that the urinary excretion rate of 1,1,2,2-tetrachloroethane
in humans was 0.015% of the absorbed dose/minute. No other studies measuring the elimination
of 1,1,2,2-tetrachloroethane in humans have been reported.
Available animal data indicate that following absorption into the body, 1,1,2,2-tetra-
chloroethane is eliminated mainly as metabolites in urine, as carbon dioxide, or as unchanged
compound in expired air (Gargas and Anderson, 1989; Dow Chemical Company, 1988; Mitoma
et al., 1985; Ikeda and Ohtsuji, 1972; Yllner et al., 1971). The patterns of elimination in rats and
mice are qualitatively similar (Dow Chemical Company, 1988; Mitoma et al., 1985), although
covalent binding is somewhat greater in mice than rats. Elimination is fairly rapid, with
significant amounts present in the urine and expired air at 48-72 hours postexposure (Dow
Chemical Company, 1988; Mitoma et al., 1985; Ikeda and Ohtsuji, 1972; Yllner et al., 1971).
Only one study quantitatively evaluated the elimination of 1,1,2,2-tetrachloroethane
following inhalation exposure. Dow Chemical Company (1988) followed the excretion of
1,1,2,2-tetrachloroethane for 72 hours following exposure of rats and mice to vapor
concentrations of 10 ppm (68.7 mg/m3) [14C]-l,l,2,2-tetrachloroethane for 6 hours. More than
90% of the absorbed dose was metabolized in both species. The percentage of recovered
radioactivity reported in rats was 33% in breath (25% as CO2 and 8% as unchanged compound),
19% in urine, and 5% in feces. In mice, the percentage of recovered radioactivity was 34% in
breath (32% as CO2 and 2% as unchanged compound), 26% in urine, and 6% in feces.
Radioactivity in urine and feces was nonvolatile (inferred by the researchers to be product(s) of
metabolism), but was not otherwise characterized.
With regard to oral exposure, the excretion of 1,1,2,2-tetrachloroethane was followed for
72 hours after oral administration of 150 mg/kg doses to rats and mice (Dow Chemical
10
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Company, 1988). Greater than 90% of the absorbed dose was detected as metabolites in both
species. In rats, 41% was excreted in breath (32% as CO2 and 9% as unchanged compound),
23% in urine, and 4% in feces. In mice, 51% was excreted in breath (50% as CC>2 and 1% as
unchanged compound), 22% in urine, and 6% in feces. Radioactivity in urine and feces was
nonvolatile (inferred by the researchers to be product(s) of metabolism), but was not otherwise
characterized. Mitoma et al. (1985) found that mice given an oral dose of 1,1,2,2-tetrachloro-
ethane excreted about 10% of the dose unchanged in the breath, and the rest was either
metabolized and expired in the breath as carbon dioxide (10%), excreted in the urine and feces
(30%, measured together), or retained in the carcass (27%) after 48 hours. Rats showed similar
patterns of excretion (Mitoma et al., 1985). The most comprehensive study of the metabolism
and excretion of 1,1,2,2-tetrachloroethane was an i.p. study in mice using [14C]-labeled
1,1,2,2-tetrachloroethane. Yllner (1971) showed that after 72 hours, about 4% of the
radioactivity was expired unchanged in the breath, 50% was expired as carbon dioxide, 28% was
excreted in the urine, 1% was excreted in the feces, and 16% remained in the carcass.
Delays in elimination may be the result of covalent binding of 1,1,2,2-tetrachloroethane
metabolites, as reflected in high levels of compound detected in the carcasses of animals.
Mitoma et al. (1985) reported a 30.75% retention in the carcass of rats and a 27.44% retention in
the carcass of mice 48 hours after exposure to a single labeled dose of 25 and 50 mg/kg
1,1,2,2-tetrachloroethane in rats and mice, respectively. Dow Chemical Company (1988)
reported 30% retention in the carcass in rats exposed to 10 ppm by inhalation, 25% in mice
exposed to 10 ppm by inhalation, 23% in rats exposed to 150 mg/kg by gavage, and 17.3% in
mice exposed to 150 mg/kg by gavage. Colacci et al. (1987) reported covalent binding of
radiolabeled 1,1,2,2-tetrachloroethane to DNA, ribonucleic acid (RNA), and protein in the liver,
kidneys, lung, and stomach of rats and mice exposed to a single intravenous dose and analyzed
22 hours postexposure. In vitro binding to calf thymus DNA was found to be greatest when the
microsomal fraction was present, and was inhibited by SKF-525 A, indicating that metabolic
activation was likely required for DNA binding (Colacci et al., 1987). However, Collaci et al.
(1987) did not distinguish between covalent binding and whether the presence of radiolabel in
the DNA, RNA, and protein was the result of incorporated radiolabeled carbon into the
biomolecules through normal biochemical processes.
3.5. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELS
No physiologically based pharmacokinetic (PBPK) models for 1,1,2,2-tetrachloroethane
were located for humans. Meulenberg et al. (2003) used saline:air, rat brain:air, and olive oil:air
partition coefficients to model 28 chemicals from three distinct chemical classes, including
alkylbenzenes, chlorinated hydrocarbons, and ketones. The saline:air, rat brain:air, and olive
oil:air partition coefficients derived for 1,1,2,2-tetrachloroethane were 35.6 ± 6.05, 344 ± 21.0,
and 10,125 ± 547, respectively. The brain partition coefficients for the 28 chemicals were
11
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predicted with accuracy within a factor of 2.5 for 95% of the chemicals. While the study
demonstrates the ability to predict rat brain partition coefficients using a bilinear equation, the
utility of the information for this assessment is limited. Similarly, several PBPK investigations
of 1,1,2,2-tetrachloroethane exposure in fish (McKim et al., 1999; Nichols et al., 1993) provide
little utility for this assessment. In sum, adequate information for PBPK modeling of
1,1,2,2-tetrachloroethane remains a research need.
Chiu and White (2006) presented an analysis of steady-state solutions to a PBPK model
for a generic volatile organic chemical metabolized in the liver. The only parameters used to
determine the system state for a given oral dose rate or inhalation exposure concentration were
the blood-air partition coefficient, metabolic constants, and the rates of blood flow to the liver
and of alveolar ventilation. At exposures where metabolism is close to linear (i.e., unsaturated),
it was demonstrated that only the effective first order metabolic rate constant was needed.
Additionally, it was found that the relationship between cumulative exposure and average
internal dose (e.g., areas under the curve [AUCs]) remains the same for time-varying exposures.
The study authors concluded that steady-state solutions can reproduce or closely approximate the
solutions using a full PBPK model. Section 5.2.2 addresses the applicability of using this model
to conduct a route-to-route extrapolation in this assessment.
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4. HAZARD IDENTIFICATION
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL
CONTROLS
4.1.1. Oral Exposure
A number of case reports provide information on the effects of intentional acute exposure
to lethal oral doses of 1,1,2,2-tetrachloroethane (Mant, 1953; Lilliman, 1949; Forbes, 1943;
Elliot, 1933; Hepple, 1927). Subjects usually lost consciousness within approximately 1 hour
and died 3-20 hours postingestion, depending on the amount of food in the stomach.
Postmortem examinations showed gross congestion in the esophagus, stomach, kidneys, spleen,
and trachea, gross congestion and edema in the lungs, and histological effects of congestion and
cloudy swelling in the lungs, liver, and/or kidneys (Mant, 1953; Hepple, 1927). Amounts of
1,1,2,2-tetrachloroethane recovered from the stomach and intestines of the deceased subjects
included 12 mL (Hepple, 1927), 25 g (Lilliman, 1949), 48.5 mL (Mant, 1953), and 425 mL
(Mant, 1953). Assuming a density of 1.594 g/mL and an average body weight of 70 kg, the
approximate minimum doses consumed in these cases are estimated to be approximately 273,
357, 1,100, and 9,700 mg/kg, respectively. No deaths occurred in eight patients (six men and
two women) who were accidentally given 3 mL of 1,1,2,2-tetrachloroethane (68 mg/kg, using
the above assumptions) or three patients (one young man, one young woman, and one 12-year-
old girl) who were accidentally given 2 or 3 mL (98-117 mg/kg, using the density and reported
body weights) as medicinal treatment for hookworm (Ward, 1955; Sherman, 1953). These
patients experienced loss of consciousness and other clinical signs of narcosis that included
shallow breathing, faint pulse, and pronounced lowering of blood pressure.
4.1.2. Inhalation Exposure
The symptoms of high-dose acute inhalation exposure to 1,1,2,2-tetrachloroethane
commonly include drowsiness, nausea, headache, constipation, decreased red blood cell (RBC)
count, weakness, and at extremely high concentrations, jaundice, unconsciousness, and
respiratory failure (Coyer, 1944; Hamilton, 1917).
An experimental study was conducted in which two volunteers self-inhaled various
concentrations of 1,1,2,2-tetrachloroethane for up to 30 minutes (Lehmann et al., 1936). The
results of this study suggest that 3 ppm (6.9 mg/m3) was the odor detection threshold; 13 ppm
(89 mg/m3) was tolerated without effect for 10 minutes, while 146 ppm (1,003 mg/m3) for
30 minutes or 336 ppm (2,308 mg/m3) for 10 minutes produced irritation of the mucous
membranes, pressure in the head, vertigo, and fatigue. No other relevant information was
reported.
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Minot and Smith (1921) reported that symptoms of industrial 1,1,2,2-tetrachloroethane
poisoning (concentrations not specified) included fatigue, perspiration, drowsiness, loss of
appetite, nausea, vomiting, constipation, headache, and jaundice. Hematological changes
included increased large mononuclear cells, elevated white blood cell (WBC) count, a slight but
progressive anemia, and a slight increase in platelet number. Similar symptoms were reported by
Parmenter (1921) and Willcox et al. (1915). Horiguchi et al. (1964) reported that in 127 coating
workers employed in artificial pearl factories and exposed to 75-225 ppm (500-1,500 mg/m3)
1,1,2,2-tetrachloroethane (along with other solvents), observed effects included decreased
specific gravity of the whole blood, decreased RBC count, relative lymphocytosis, neurological
findings (not specified), and a positive urobilinogen test.
Lobo-Mendonca (1963) observed a number of adverse health effects in a mixed-gender
group of 380 workers at 23 Indian bangle manufacturing facilities (80% of workers employed at
these facilities were examined). In addition to the inhalation exposure, approximately 50% of
the examined workers had a substantial amount of dermal exposure to 1,1,2,2-tetrachloroethane.
Some of the workers were exposed to a mixture of equal parts acetone and 1,1,2,2-tetrachloro-
ethane. Air samples were collected at several work areas in seven facilities. Levels of
1,1,2,2-tetrachloroethane in the air ranged from 9.1 to 98 ppm (62.5-672 mg/m3). High
incidences of a number of effects were reported including anemia (33.7%), loss of appetite
(22.6%), abdominal pain (23.7%), headaches (26.6%), vertigo (30.5%), and tremors (35%). The
significance of these effects cannot be determined because a control group of unexposed workers
was not examined, and coexposure to acetone was possible. The study authors noted that the
incidence of tremors appeared to be directly related to 1,1,2,2-tetrachloroethane exposure
concentrations, as the percentage of workers handling tetrachloroethane and displaying tremors
increased as the air concentration of 1,1,2,2-tetrachloroethane increased.
Over a 3-year period, Jeney et al. (1957) examined 34-75 workers employed at a
penicillin production facility. 1,1,2,2-Tetrachloroethane was used as an emulsifier, and wide
fluctuations in atmospheric levels occurred throughout the day. The investigators noted that the
workers were only in the areas with high 1,1,2,2-tetrachloroethane concentrations for short
periods of time, and gauze masks with organic solvent filters were worn in these areas. During
the first year of the study, 1,1,2,2-tetrachloroethane levels ranged from 0.016 to 1.7 mg/L (16-
1,700 mg/m3; 2-248 ppm). In the second year of the study, ventilation in the work room was
improved and 1,1,2,2-tetrachloroethane levels ranged from 0.01 to 0.85 mg/L (10-850 mg/m3;
1-124 ppm). In the third year of the study, the workers were transferred to a newly built facility
and 1,1,2,2-tetrachloroethane levels in the new facility ranged from 0.01 to 0.25 mg/L (10-
250 mg/m3; 1-36 ppm). At 2-month intervals, the workers received general physical
examinations, and blood was drawn for measurement of hematological parameters, serum
bilirubin levels, and liver function tests; urinary hippuric acid levels were measured every
6 months. It appears that workers with positive signs of liver damage, including palpability of
14
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the liver, rise in bilirubin levels, positive liver function tests, and urobilinogenuria, were
transferred to other areas of the facility and were not examined further.
In the first year of the study, 31% of the examined workers had "general or gastro-
intestinal symptoms." Loss of appetite, bad taste in the mouth, epigastric pain, and a "dull
straining pressure feeling in the area of the liver" were reported by 66% of the workers
experiencing gastrointestinal symptoms. Other symptoms included headaches, general
weakness, and fatigue in 29%, severe weight loss in 4%, and "tormenting itching" in 1%.
Enlargement of the liver was observed in 38% of the screened workers. Urobilinogenuria was
detected in 50% of the workers, most often following more than 6 months of employment, and
31% of the workers with urobilinogenuria also had palpable livers.
In the second year of the study, there was a decline in the number of symptomatic
workers (13% of examined workers) and in workers with positive urobilinogenuria findings
(24%). Liver enlargement was observed in 20% of the examined workers. In the third year, the
number of workers reporting symptoms decreased to 2%, and positive urobilinogen findings
were found in 12%. The investigators noted that the increased urobilinogen levels during the
third year of observation may have been secondary to excessive alcohol consumption or dietary
excess. Enlarged livers were found in 5% of the examined workers.
During the course of the study, no alterations in erythrocyte or hemoglobin (Hb) levels
were found. Leukopenia (defined as leukocyte levels of <5,800 cells/mL) was found in 20% of
the workers, but no relationship between the number of cases and duration of 1,1,2,2-tetrachloro-
ethane exposure was found. A positive relationship between duration of exposure and frequency
of abnormal liver function test results was observed, as statistically significant correlations were
found on the thymol and Takata-Ucko liver function tests, but not the gold sol reaction test. The
thymol liver function test measures the direct precipitation of both lipids and abnormal lipid
protein complexes appearing in liver disease by the addition of a thymol solution (Kunkel and
Hoagland, 1947). The Takata-Ucko (or Takata-Ara) test detects an increase in the amounts of
the globulin components of the serum, signifying liver disease (Kunkel and Hoagland, 1947).
Abnormal hippuric acid levels were only detected in 1% of the examined workers during the first
2 years, and no abnormalities were observed during the third year. Increased serum bilirubin
levels (>1 mg/dL) were observed in 20, 18.7, and 7.6% of the workers during the first, second,
and third years, respectively. The prevalence of hepatitis was assessed using sickness benefit
files. In the 1-year period prior to the study, 21 cases of hepatitis were found (total number of
workers not reported). Three cases of hepatitis were found in the first year of the study, eight
cases in the second year, and four cases in the third year. The lack of a control group and poor
reporting of study design and results precludes using this study for quantitative dose-response
analysis.
Norman et al. (1981) examined the mortality of the employees of 39 chemical processing
plants used by the Army during World War II. Ten plants used 1,1,2,2-tetrachloroethane to help
15
-------
treat clothing, while the others plants used water in the same process. Estimates of exposure
levels were not reported, and coexposure to dry-cleaning chemicals was expected. At the time of
evaluation, 2,414 deaths were reported in the study cohort. No differences in standard mortality
ratios were seen between the tetrachloroethane and water groups for total mortality,
cardiovascular disease, cirrhosis of the liver, or cancer of the digestive and respiratory systems.
The mortality ratio for lymphatic cancers in the tetrachloroethane group was increased relative to
controls or the water group, although the number of deaths was small (4 cases, with an expected
number of 0.85). No other differences were seen between the groups.
4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
ANIMALS—ORAL AND INHALATION
4.2.1. Oral Exposure
4.2.1.1. Subchronic Studies
The National Toxicology Program (NTP, 2004) fed groups of male and female F344 rats
(10/sex/group) diets containing 0, 268, 589, 1,180, 2,300, or 4,600 ppm of microencapsulated
1,1,2,2-tetrachloroethane for 14 weeks. NTP (2004) reported that the microcapsules containing
1,1,2,2-tetrachloroethane were specified to be no greater than 420 jim in diameter, and were not
expected to have any significant effect on the study. The reported average daily doses were 0,
20, 40, 80, 170, or 320 mg/kg-day, and vehicle control (feed with empty microcapsules) and
untreated control groups were used for both genders. Endpoints evaluated throughout the study
included clinical signs, body weight, and feed consumption. Hematology and clinical chemistry
were assessed on days 5 and 21 and at the end of the study; urinalyses were not performed.
Necropsies were performed on all animals, and selected organs (liver, heart, right kidney, lung,
right testis, and thymus) were weighed. Comprehensive histological examinations were
performed on untreated control, vehicle control, and high dose groups. Tissues examined in the
lower dose groups were limited to bone with marrow, clitoral gland, liver, ovary, prostate gland,
spleen, testis with epididymis and seminal vesicle, and uterus. A functional observational battery
(FOB) was performed on rats in the control groups and the 20, 40, and 80 mg/kg-day groups
during weeks 4 and 13. Sperm motility, vaginal cytology, estrous cycle length, and percentage
of time spent in the various estrus stages were evaluated in control groups and the 40, 80, and
170 mg/kg-day groups.
All animals survived to the end of the study, but clinical signs of thinness and pallor were
observed in all animals in the 170 and 320 mg/kg-day groups (NTP, 2004). Final body weights
(Table 4-1) were statistically significantly lower than vehicle controls in males at 80, 170, and
320 mg/kg-day (7, 29, and 65% lower, respectively) and females at 80, 170, and 320 mg/kg-day
(9, 29, and 56% lower, respectively), with both genders at 320 mg/kg-day losing weight over the
course of the study. However, feed consumption by the rats also decreased with increasing dose
level (NTP, 2004).
16
-------
Table 4-1. Final body weights (g) and percent change compared to controls
in F344/N rats exposed to l,l?2,2-tetrachloroethane in feed for 14 weeks
Dose (mg/kg-d)
Vehicle control
20
40
80
170
320
n
10
10
10
10
10
10
Males
366 ± 5a
354 ±9
353 ±6
341 ±6b
259 ± 9b
127 ± 9b
-
-3%
-4
-7
-29
-65
n
10
10
10
10
10
10
Females
195 ±4a
192 ±4
189 ±2
177 ± 2b
139 ±4b
85±3b
-
-2%
-3
-9
-29
-56
aMean ± standard error.
bStatistically significant compared to controls (p < 0.05).
Source: NTP (2004).
Statistically significant increases in absolute liver weights were observed in female rats
exposed to 80 mg/kg-day, and statistically significant decreases in absolute liver weight were
observed at > 170 mg/kg-day in males and at 320 mg/kg-day in females (Table 4-2). Statistically
significant increases in relative liver weights (Table 4-3) were observed at >40 mg/kg-day in
males and females (NTP, 2004). Significant alterations in absolute and/or relative weights were
also observed in the thymus, kidney, heart, lung, and testes primarily at 170 and 320 mg/kg-day.
Table 4-2. Absolute liver weights (g) and percent change compared to
controls in F344/N rats exposed to 1,1^2,2-tetrachloroethane in feed for
14 weeks
Dose (mg/kg-d)
Vehicle control
20
40
80
170
320
n
10
10
10
10
10
10
Males
12.74 ± 0.26a
12.99 ±0.35
14.47 ±0.44
15.54 ±0.39
11.60±0.44b
6.57±0.18b
-
2%
14
22
-9
-48
n
10
10
10
10
10
10
Females
6.84±0.17a
7.03 ±0.12
7.14 ±0.16
7.80±0.08b
6.66 ±0.21
4.94±0.12b
-
3%
4
14
-3
-28
aMean ± standard error.
bStatistically significant compared to controls (p < 0.05).
Source: NTP (2004).
17
-------
Table 4-3. Relative liver weight (mg organ weight/g body weight) and
percent change compared to controls in F344/N rats exposed to l,l?2,2-tetra-
chloroethane in feed for 14 weeks
Dose (mg/kg-d)
Vehicle control
20
40
80
170
320
n
10
10
10
10
10
10
Males
34.79 ±0.42a
36.72 ±0.44
41.03 ±0.85b
45.61 ±0.52b
44.68 ± 0.45b
52.23 ± 1.42b
-
6%
18
31
28
50
n
10
10
10
10
10
10
Females
35.07 ±0.56a
36.69 ±0.36
37.84 ±0.51b
44.20 ± 0.27b
48.03 ±0.89b
58.40 ±1.42b
-
5%
8
26
37
67
"Mean ± standard error.
bStatistically significant compared to controls (p < 0.05).
Source: NTP (2004).
Results of the FOB showed no exposure-related findings of neurotoxicity. The
hematology evaluations indicated that 1,1,2,2-tetrachloroethane affected the circulating erythroid
mass in both genders (Table 4-4). There was evidence of a transient erythrocytosis, as shown by
increases in hematocrit values, Hb concentration, and erythrocyte counts on days 5 and 21 at
>170 mg/kg-day. The erythrocytosis was not considered clinically significant and disappeared
by week 14, at which time minimal to mild, dose-related anemia was evident, as shown by
decreases in hematocrit and Hb at >40 mg/kg-day. For example, although males exposed to
40 mg/kg-day showed a statistically significant decrease in Fib at week 14, the magnitude of the
change was small (3.8%). The anemia was characterized as microcytic based on evidence
suggesting that the circulating erythrocytes were smaller than expected, including decreases in
mean cell volumes, mean cell Fib values, and mean cell Hb concentration in both genders at
>80 mg/kg-day at various time points. At week 14, there were no changes in reticulocyte counts,
suggesting that there was no erythropoietic response to the anemia, which was in turn supported
by the bone marrow atrophy observed microscopically. As discussed by NTP (2004), the
erythrocytosis suggested a physiological response consistent with hemoconcentration due to
dehydration, as well as compromised nutritional status due to the reduced weight gain and food
consumption, both of which may have contributed to the development of the anemia.
18
-------
Table 4-4. Serum chemistry and hematology changes" in rats exposed to
dietary 1,1,2,2-tetrachloroethane for 14 weeks
Oral dose (mg/kg-d)
Vehicle control
20
40
80
170
320
Males (10/group)
Serum total protein (g/dL)
Serum cholesterol (mg/dL)
ALT (IU/L)
ALP (IU/L)
SDH (IU/L)
Bile acids (umol/L)
Hematocrit (%) (automated)
Hb (g/dL)
Mean cell volume (IL)
Mean cell Hb (pg)
Platelets (103/uL)
7.2 ±0.1
73 ±2
48 ±2
256 ±7
23 ±1
29.2 ±2.9
45.2 ±0.5
15.8 ±0.1
50.7 ±0.1
17.7 ±0.1
728.4 ± 12.3
7.3 ±0.1
74 ±3
49 ±2
260 ±5
27±lb
27.5 ±2.7
44.9 ±0.4
15.6 ±0.1
51. 8 ±0.3
18.1±0.1
707.0 ±5.8
7.3 ±0.1
76 ±2
53 ±2
248 ±5
26 ±2
27.2 ±2.7
44.0 ±0.9
15.2±0.3b
52.3 ±0.2
18.0 ±0.1
727.0 ±25.2
7.3 ±0.1
67 ±2
69±3b
245 ±6
31±lb
35.9 ±3.9
43.3 ±0.7
14.9±0.1b
51.3 ±0.2
17.7 ±0.2
716.3 ±9.7
6.7±0.1b
68 ±2
115 ±8b
353 ± 12b
47±2b
92.0±16.6b
43.1±0.6b
14.6±0.1b
49.4 ±0.2
16.8±0.1b
692.8 ± 12.6b
6.0±0.1b
65±2b
292 ± 18b
432 ± 24b
74±4b
332.4 ±47.4b
39.0±l.lb
13.6±0.3b
44.4 ± Q.4b
15.5±0.2b
773.4±23.2b
Females (10/group)
Serum total protein (g/dL)
Serum cholesterol (mg/dL)
ALT (IU/L)
ALP (IU/L)
SDH (IU/L)
Bile acids (umol/L)
Hematocrit (%) (automated)
Hb (g/dL)
Mean cell volume (IL)
Mean cell Hb (pg)
Platelets (103/uL)
7.2 ±0.1
104 ±4
46 ±2
227 ±5
27 ±1
37.0 ±7.1
42.8 ±0.4
15.2 ±0.1
55.4 ±0.1
19.7 ±0.1
742.1 ±20.4
7.3 ±0.0
105 ±3
42 ±1
216 ±4
27 ±1
46.6 ±6.5
43.2 ±0.4
15.3 ±0.1
56.1 ±0.1
19.8 ±0.1
725. 9 ±12.7
7.3 ±0.1
98 ±1
41±2
220 ±3
28 ±2
39.1 ±5.6
42.1 ±0.4
14.9 ±0.1
55.8 ±0.1
19.7 ±0.1
733.9 ±8.8
6.9 ±0.1
81±2b
49 ±2
225 ±11
25 ±1
36.3 ±3.9
40.1±0.5b
14.2±0.2b
53.3±0.2b
18.9±0.1b
727.4 ±14.2
6.4±0.1b
64±3b
112 ±7b
341 ±7b
45±3b
39.3 ±7.9
42.8 ±0.7
14.5±0.2b
49.0 ± 0.2b
16.6±0.2b
639.4 ±9.9b
5.6±0.1b
55±3b
339±18b
468 ± 22b
82±3b
321.5 ±50.6b
34.7±0.7b
12.5±0.2b
44.4 ± 0.4b
16.0±0.2b
662.5 ± 19.4b
"Mean ± standard error.
bStatistically significantly different from control value.
ALP = alkaline phosphatase; IU = international units; SDH = sorbitol dehydrogenase
Source: NTP (2004).
Changes in serum clinical chemistry parameters indicative of liver damage were observed
in both genders, occurring at all time points (day 5, day 21, and week 14) and increasing in
magnitude with increasing dose and time. At week 14 (Table 4-4), these effects included
statistically significant increases in ALT and sorbitol dehydrogenase (SDH) activity in males at
>80 mg/kg-day (41, 134, and 496%, and 15, 74, and 174%, respectively) and females at
>170 mg/kg-day (167 and 707%, and 67 and 204%, respectively), increases in alkaline
phosphatase (ALP) activity in both genders at > 170 mg/kg-day (36 and 66% in males and 58 and
117% in females), increases in bile acid levels in males at >170 mg/kg-day (233 and 1,110%)
and females at 320 mg/kg-day (590%), and decreases in serum cholesterol levels in females at
19
-------
>80 mg/kg-day (23, 39, and 48%, respectively) and males at 320 mg/kg-day (12%). There were
no exposure-related changes in rat serum 5'-nucleotidase activity at week 14, although increases
occurred on day 5 in females at>20 mg/kg-day and on day 21 in males and females at 80, 170,
and/or 320 mg/kg-day.
A summary of histopathological alterations following 1,1,2,2-tetrachloroethane exposure
is presented in Table 4-5. Hepatic cytoplasmic vacuolization was noted in males exposed to
>20 mg/kg-day and in females exposed to >40 mg/kg-day. Although incidence of this alteration
was high in affected groups, severity was only minimal-to-mild and only increased with dose
from 20 to 40 mg/kg-day in males and 40 to 80 mg/kg-day in females. Females exposed to
>80 mg/kg-day showed an increase in the incidence of hepatocyte hypertrophy with an increase
in severity and incidence with increasing exposure level, and males showed similar results at
exposures >170 mg/kg-day. A statistically significant increase in the incidence of hepatocellular
necrosis was observed in male and female rats at 170 and 320 mg/kg-day, accompanied by an
increased severity with an increase in dose. At > 170 mg/kg-day, additional effects in the liver in
both genders were hepatocyte pigmentation and mitotic alteration and mixed cell foci, with bile
duct hyperplasia observed in females only. Pigmentation of the spleen was statistically
significantly increased in male rats exposed to >80 mg/kg-day and in female rats exposed to
>170 mg/kg-day. Other histological effects included statistically significantly increased
incidences of atrophy (red pulp and lymphoid follicle) in the spleen of males at 170 and
320 mg/kg-day and the spleen of females at 320 mg/kg-day. A statistically significant increase
in atrophy of bone (metaphysis) and bone marrow, prostate gland, preputial gland, seminal
vesicles, testes (germinal epithelium), uterus, and clitoral gland, as well as an increase in ovarian
interstitial cell cytoplasmic alterations, was observed in females at>170 mg/kg-day and in males
at 320 mg/kg-day.
Table 4-5. Incidences of selected histopathological lesions in rats exposed to
dietary l,l?2,2-tetrachlorethane for 14 weeks
Dose (mg/kg-d)
Vehicle control
20
40
80
170
320
Males (10/group)
Hepatocyte cytoplasmic vacuolization
Hepatocyte hypertrophy
Hepatocyte necrosis
Hepatocyte pigmentation
Hepatocyte mitotic alteration
Mixed cell foci
Bile duct hyperplasia
Spleen pigmentation
Spleen red pulp atrophy
Spleen lymphoid follicle atrophy
Oa
0
0
0
0
0
0
0
0
0
7b(1.3)
0
0
0
0
0
0
0
0
0
9b (2.0)
0
0
0
0
0
0
1 (1.0)
0
0
10b(1.9)
1 (1.0)
0
0
0
0
0
9b(1.0)
0
0
8b(1.4)
9b(1.3)
8b(1.0)
7b(1.0)
0
o
J
0
9b(1.0)
5b(1.0)
0
0
10b(3.2)
10b(1.6)
10b(1.9)
6b (2.0)
5b
10b(1.7)
9b(1.6)
9b(1.4)
5b(1.0)
20
-------
Table 4-5. Incidences of selected histopathological lesions in rats exposed to
dietary 1,1,2,2-tetrachlorethane for 14 weeks
Dose (mg/kg-d)
Vehicle control
20
40
80
170
320
Females (10/group)
Hepatocyte cytoplasmic vacuolization
Hepatocyte hypertrophy
Hepatocyte necrosis
Hepatocyte pigmentation
Hepatocyte mitotic alteration
Mixed cell foci
Bile duct hyperplasia
Spleen pigmentation
Spleen, red pulp atrophy
Spleen lymphoid follicle atrophy
Oa
0
0
0
0
0
0
1 (1.0)
0
0
0
0
0
0
0
0
0
0
0
0
10b(1.7)
0
0
0
0
0
0
0
0
0
10b (2.2)
4b(1.0)
1 (1.0)
0
0
0
0
4 (1.0)
0
0
4b(1.3)
10b(1.7)
7b(1.0)
10b(1.3)
3 (2.0)
8b
5b(1.0)
8b(l.l)
0
0
0
10b (2.8)
10b(l.l)
10b (2.0)
10b(1.9)
1
10b(1.9)
8b(1.3)
9b(1.6)
3(1.0)
aValues represent number of animals with the lesion, with the severity score in parentheses; severity grades are as
follows: 1 = minimal, 2 = mild, 3 = moderate, 4 = severe.
bSignificantly different from vehicle control group.
Source: NTP (2004).
Epididymal spermatozoal motility was statistically significantly decreased at>40 mg/kg-
day, with statistically significant decreases in epididymis weight at>80 mg/kg-day and cauda
epididymis weight at 320 mg/kg-day. Exposed female rats spent more time in diestrus and less
time in proestrus, estrus, and metestrus than control rats (see Section 4.3.1).
In summary, the NTP (2004) 14-week rat study provides evidence that the liver is a
primary target of 1,1,2,2-tetrachloroethane toxicity. At the lowest dose tested, 20 mg/kg-day,
there was a significant increase in the incidence of hepatic cytoplasmic vacuolization in males.
At 40 mg/kg-day, significant increases in relative liver weights were observed in both males and
females. Hepatocellular hypertrophy and spleen pigmentation were observed at 80 mg/kg-day in
both males and females, although these changes were generally of minimal severity. Increases in
serum ALT and SDH, were observed at 80 mg/kg-day in males and at 170 mg/kg-day in females.
Decreases in serum cholesterol levels were observed in females at 80 mg/kg-day and at
320 mg/kg-day in males. A decrease in body weight (>10%) was observed at 170 mg/kg-day in
both males and females. Increases in serum ALP activity and bile acid levels, hepatocellular
necrosis, bile duct hyperplasia, hepatocellular mitotic alterations, foci of cellular alterations, and
liver pigmentation occurred at 170 and/or 320 mg/kg-day. A no-observed-adverse-effect level
(NOAEL) of 20 mg/kg-day and a lowest-observed-adverse-effect level (LOAEL) of 40 mg/kg-
day was identified by EPA for increased relative liver weight in male and female rats. NTP
(2004) identified a NOAEL of 20 mg/kg-day in rats based on survival and body weight changes
and increased lesion incidences. There were no clinical signs of neurotoxicity at doses as high as
21
-------
320 mg/kg-day or exposure-related findings in the FOB at doses as high as 80 mg/kg-day
(highest tested dose in the FOB), indicating that the nervous system may be less sensitive than
the liver for subchronic dietary exposure.
NTP (2004) also exposed groups of male and female B6C3Fi mice (10/sex/group) to
diets containing 0, 589, 1,120, 2,300, 4,550, or 9,100 ppm of microencapsulated 1,1,2,2-tetra-
chloroethane for 14 weeks, with vehicle and untreated control groups for each gender. The
reported average daily doses were 0, 100, 200, 370, 700, or 1,360 mg/kg-day for males and 0, 80,
160, 300, 600, or 1,400 mg/kg-day for females. Endpoints evaluated throughout the study
included clinical signs, body weight, and feed consumption. Clinical chemistry was assessed at
the end of the study, but hematological evaluations and urinalyses were not performed.
Necropsies were conducted on all animals and selected organs (liver, heart, right kidney, lung,
right testis, and thymus) were weighed. Comprehensive histological examinations were
performed on untreated control, vehicle control, and high dose groups. Tissues examined in the
lower dose groups were limited to the liver, spleen, thymus, preputial gland (in males only), and
lungs (in females only). An FOB (21 parameters) was performed on mice in both control and
160/200, 300/370, and 600/700 mg/kg-day (1,120, 2,300, and 4,550 ppm, respectively) dose
groups during weeks 4 and 13. Sperm motility, vaginal cytology, estrous cycle length, and
percentage of time spent in the various estrus stages were evaluated in both control and 160/200,
600/700, and 1,360/1,400 mg/kg-day (1,120, 2,300, and 4,550 ppm, respectively) dose groups.
All mice survived to the end of the study (NTP, 2004). Thinness was observed clinically
in male mice (3/10, 9/10, 10/10) at 370, 700, and 1,400 mg/kg-day, respectively, and in female
mice (1/10, 2/10, 10/10) at 300, 600, and 1,360 mg/kg-day, respectively. Final body weights
were statistically significantly lower than vehicle controls in male mice at 370, 700, and
1,360 mg/kg-day (12, 16, and 23%, respectively) and female mice at 600 and 1,400 mg/kg-day
(11 and 12%, respectively) (Table 4-6). Feed consumption was less than controls in males at
>700 mg/kg-day, but similar to controls in females.
22
-------
Table 4-6. Final body weights (g) and percent change compared to controls
in B6C3Fi mice exposed to l,l?2,2-tetrachloroethane in feed for 14 weeks
Dose (mg/kg-d)
Vehicle control
100
200
370
700
1,360
n
10
10
10
10
10
10
Vehicle control
80
160
300
600
1,400
10
10
10
10
10
10
Males
30.1±0.6a
30.6 ±0.6
30.0 ±0.3
26.5±0.4b
25.2±0.2b
23.1±0.5b
-
2%
0
-12
-16
-23
Females
24.3±0.5a
24.2 ±0.2
24.3 ±0.6
23. 3 ±0.4
21.7±0.2b
21.5±0.6b
-
0%
0
-4
-11
-12
"Mean ± standard error.
bStatistically significant compared to controls (p < 0.05).
Source: NTP (2004).
Statistically significant increases in absolute liver weights were observed in the male
mice exposed to 200 and 370 mg/kg-day (16 and 10%, respectively), but not at higher doses, and
in female mice exposed to >80 mg/kg-day (11, 29, 27, 22, and 32%, respectively) (Table 4-7).
Statistically significant increases in relative liver weights were observed in male mice at
>200 mg/kg-day (16, 24, 24, and 38%, respectively) and in female mice at>80 mg/kg-day (11,
28, 33, 36, and 49%, respectively) (Table 4-8). Other organ weight changes (increased kidney
weights in males at >370 mg/kg-day and decreased thymus weights in both genders at 1,360/
1,400 mg/kg-day) were considered to be secondary to the body weight changes. Results of the
FOBs showed no exposure-related neurotoxicity.
23
-------
Table 4-7. Absolute liver weights (g) and percent change compared to
controls in B6C3Fi mice exposed to l,l?2,2-tetrachloroethane in feed for
14 weeks
Dose (mg/kg-d)
Vehicle control
100
200
370
700
1,360
n
10
10
10
10
10
10
Vehicle control
80
160
300
600
1,400
10
10
10
10
10
10
Males
1.467 ±0.020
1.557 ±0.039
1.701±0.020b
1.607 ±0.038b
1.531 ±0.052
1.558 ±0.045
-
6%
16
10
4
6
Females
1.048 ±0.028
1.160±0.022b
1.356 ±0.058b
1.336 ±0.037b
1.277 ±0.030b
1.386 ±0.047b
-
11%
29
27
22
32
"Mean ± standard error.
bStatistically significant compared to controls (p < 0.05).
Source: NTP (2004).
Table 4-8. Relative liver weights (mg organ weight/g body weight) and
percent change compared to controls in B6C3Fi mice exposed to
l,l?2,2-tetrachloroethane in feed for 14 weeks
Dose (mg/kg-d)
Vehicle control
100
200
370
700
1,360
n
10
10
10
10
10
10
Vehicle control
80
160
300
600
1,400
10
10
10
10
10
10
Males
48.84 ±1.17
50.94 ±0.93
56.82 ±0.63b
60.63 ± 1.20b
60.71 ± 1.76b
67.43 ± 1.83b
-
4%
16
24
24
38
Females
43.26 ± 1.05
47.90 ±0.85b
55.54 ±1.17b
57.39 ±0.84b
58.73 ±1.23b
64.42 ±1.14b
-
11%
28
33
36
49
"Mean ± standard error.
bStatistically significant compared to controls (p < 0.05).
Source: NTP (2004).
24
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Clinical chemistry findings in the mice are summarized in Tables 4-9 and 4-10 and
included statistically significant decreases in total serum protein levels in males at >200 mg/kg-
day, total serum protein levels in females at >300 mg/kg-day, and serum albumin levels in
females at 1,400 mg/kg-day (NTP, 2004). Decreased serum albumin levels could not fully
account for the decreased total protein levels, suggesting that other factors (e.g., changes in other
protein fractions, hydration status, and/or hepatic function) contributed to the hypoproteinemia
(NTP, 2004). A statistically significant increase of serum SDH activity in females was observed
at >80 mg/kg-day (22, 111, 444, 575, and 1,181%, respectively) and in males at>200 mg/kg-day
(38, 424, 424, and 715%, respectively). A statistically significant decrease in serum cholesterol
levels was observed in females at>160 mg/kg-day (22, 38, 41, and 16%, respectively), and a
statistically significant increase in ALT activity was observed in females at>160 (30, 278, 294,
and 602%, respectively) and in males at>370 mg/kg-day (234, 177, and 377%, respectively).
Total bile acid levels increased statistically significantly in females at>160 mg/kg-day (18, 69,
97, and 290%, respectively) and in males at>370 mg/kg-day (148, 178, and 377%, respectively).
A statistically significant increase in ALP activity was observed in males at >370 mg/kg-day (67,
83, and 136%, respectively) and in females at 300 mg/kg-day (19, 28, 55%, respectively), and a
statistically significant increase in 5'-nucleotidase was observed in males at>370 mg/kg-day (88,
131, and 288%, respectively).
Table 4-9. Selected clinical chemistry changes in male mice exposed to
dietary 1,1,2,2-tetrachloroethane for 14 weeks
Dose (mg/kg-d)
Serum total protein (g/dL)
Serum cholesterol (mg/dL)
ALT (IU/L)
ALP (IU/L)
SDH (IU/L)
5'-Nucleotidase (IU/L)
Bile acids (umol/L)
Vehicle control
5.4±0.r
131±7
66 ±8
85 ±2
55 ±3
18 ±1
25.3 ± 1.2
100
5.2 ±0.1
125 ±4
62 ±19
78 ±2
53 ±2
16 ±1
22.8 ±1.5
200
5.1±0.1b
94±3b
74 ±8
89 ±2
76±3b
18 ±0
24.8 ±0.6
370
5.1±0.1b
110±5
207 ± 18b
130 ±3b
288 ± 20b
30±2b
56.5 ± 5. lb
700
5.1±0.1b
112±4
172 ± 18b
143 ± 7b
288 ± 29b
37±3b
63.3±7.5b
1,360
5.1±0.1b
126 ±5
296 ± 24b
184±llb
448 ± 25b
62±7b
108.7 ± 8. lb
aMean ± standard error.
bStatistically significantly different from control value.
Source: NTP (2004).
25
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Table 4-10. Selected clinical chemistry changes in female mice exposed to
dietary 1,1,2,2-tetrachloroethane for 14 weeks
Dose (mg/kg-d)
Serum total protein (g/dL)
Serum cholesterol (mg/dL)
ALT (IU/L)
ALP (IU/L)
SDH (IU/L)
5'-Nucleotidase (IU/L)
Bile acids (umol/L)
Vehicle control
5.6±0.r
109 ±2
34 ±5
131±5
36 ±1
59 ±3
27.2 ±1.2
80
5.6 ±0.1
109 ±3
50 ±15
126 ±2
44±3b
71±2
26.1 ±1.9
160
5. 5 ±0.0
85±3b
65±5b
139 ±5
76±4b
84±5b
30.9±l.lb
300
5.4±0.1b
68±2b
189±33b
150 ± 3b
197±15b
62 ±2
44.2±3.9b
600
5.4±0.0b
64±3b
197±21b
161 ±7b
243 ± 23b
62 ±3
51.5±3.6b
1,400
5.1±0.1b
92±4b
351±35b
195 ±6b
461±59b
83±4b
101.7 ±12.0b
"Mean ± standard error.
bStatistically significantly different from control value.
Source: NTP (2004).
The histopathological results in the B6C3Fi mice are summarized in Table 4-11. A
statistically significant increased incidence of minimal to moderate hepatocyte hypertrophy was
observed at > 160 mg/kg-day in females and >200 mg/kg-day in males. The incidence of
hepatocellular necrosis was statistically significantly increased in male mice at>370 mg/kg-day
and in female mice at >300 mg/kg-day. A statistically significant increased incidence of
pigmentation and bile duct hyperplasia occurred at>300 mg/kg-day in females and >370 mg/kg-
day in males. Additionally, the histological findings included an increased incidence of preputial
gland atrophy in males in the 100, 700, and 1,360 mg/kg-day dose groups (Table 4-11), but this
effect did not appear dose-related. Based on the increase in serum SDH activity and increased
absolute and relative liver weights at 80 mg/kg-day in female mice, as well as serum chemistry
changes at > 160 mg/kg-day and clear evidence of histopathology at higher doses, a LOAEL of
80 mg/kg-day was identified based on liver toxicity.
26
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Table 4-11. Incidences of selected histopathological lesions in mice exposed
to dietary 1,1,2,2-tetrachloroethane for 14 weeks
Males (10/group)
Oral dose (mg/kg-d)
Hepatocyte hypertrophy
Hepatocyte necrosis
Liver focal pigmentation
Bile duct hyperplasia
Preputial gland atrophy
Vehicle control
Oa
0
0
0
0
100
0
0
0
0
4b (2.0)
200
7b(1.0)
1 (2.0)
0
0
2 (1.0)
370
10b (2.2)
8b(l.l)
10b(1.2)
7b(1.4)
0
700
10b (2.8)
8b(1.0)
10b(1.4)
9b(1.3)
4b (2.5)
1,360
10b(3.1)
9b(1.0)
8b(1.3)
10b (2.0)
5b (2.2)
Females (10/group)
Oral dose (mg/kg-d)
Hepatocyte hypertrophy
Hepatocyte necrosis
Liver focal pigmentation
Bile duct hyperplasia
Vehicle control
Oa
0
0
0
80
2(1.5)
0
0
0
160
9b(1.0)
0
2 (1.0)
0
300
10b(1.9)
3 (1.0)
9b(1.0)
8b(1.0)
600
10b (2.5)
7b(1.0)
8b(1.0)
10b(1.4)
1,400
10b(3.0)
4b(1.0)
7b(l.l)
10b (2.0)
aValues represent number of animals with the lesion, with the severity score in parentheses; severity grades are as
follows: 1 = minimal, 2 = mild, 3 = moderate, 4 = severe.
bSignificantly different from vehicle control group.
Source: NTP (2004).
4.2.1.2. Chronic Studies
Information on the chronic oral toxicity of 1,1,2,2-tetrachloroethane is available from a
bioassay in rats and mice. The National Cancer Institute (NCI, 1978) exposed groups of 50 male
and 50 female Osborne-Mendel rats to 1,1,2,2-tetrachloroethane in corn oil via gavage
5 days/week for 78 weeks. Vehicle and untreated control groups (20 animals/sex/species) were
also used. The initial low and high doses used for rats of both genders were 50 and 100 mg/kg-
day. At week 15, the doses were raised to 65 mg/kg-day for low-dose males and 130 mg/kg-day
for high dose males. At week 26, the doses were decreased to 40 mg/kg-day for the low-dose
females and 80 mg/kg-day for the high-dose females. Beginning at week 33, intubation of all
high-dose rats was suspended for 1 week followed by 4 weeks of dosing, and this cyclic pattern
of dosing was maintained for the remainder of the treatment period. Low-dose rats were not
subject to this regimen. The reported time-weighted average (TWA) doses were 62 and
108 mg/kg-day for male rats and 43 and 76 mg/kg-day for female rats. The exposure period was
followed by a 32-week observation period in which the rats were not exposed to 1,1,2,2-tetra-
chloroethane. Clinical signs, survival, body weight, food consumption, gross pathology, and
histology (32 major organs and tissues as well as gross lesions) were evaluated.
There were no clear effects on survival in the male rats. In females, survival in the
vehicle control, low-dose, and high-dose groups at the end of the study was 70, 58, and 40%,
respectively. Although there was a statistically significant association between increased
mortality and dose in the females, the increased mortality was affected by the deaths of 10 high-
27
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dose females, 8 with pneumonia and 2 with no reported lesions, during the first 5 weeks of the
study. The study authors also stated that there was no evidence that the early deaths were tumor-
related. The male and female rats also demonstrated an increased incidence of endemic chronic
murine pneumonia. Respective incidences of chronic murine pneumonia in the vehicle control,
low-, and high-dose groups were 40, 68, and 76% in females and 55, 50, and 65% in males.
Clinical observations included squinted or reddened eyes in all control and treated groups of both
genders, but these effects occurred with greater frequency in the exposed rats. There was a low
or moderate incidence of labored breathing, wheezing, and/or nasal discharge in all control and
treated groups during the first year of the study; near the end of the study these signs were
observed more frequently in the exposed animals.
Dose-related decreases in body weight gain were observed. However, as the study
approached termination (weeks 100-110), the differences in body weight across the dose groups
decreased.
Histopathological effects included a dose-related increased incidence of hepatic fatty
metamorphosis in high-dose males (2/20, 0/20, 2/50, and 9/49 in the untreated control, vehicle
control, low-dose, and high-dose groups, respectively). In addition, inflammation, focal cellular
changes, and angiectasis were observed in male and female rats but were not statistically
significant or biologically relevant. NCI (1978) stated that the inflammatory, degenerative, and
proliferative lesions observed in the control and dosed animals were similar in incidence and
type to those occurring in naturally-aged rats.
A statistically significant increase in tumor incidence was not observed in the rats;
however, two hepatocellular carcinomas, which are rare tumors in male Osborne-Mendel rats
(NCI, 1978), as well as one neoplastic nodule, were observed in the high-dose males
(Table 4-12). A hepatocellular carcinoma was also observed in an untreated female control.
Although interpretation of this study is complicated by the chronic murine pneumonia, it is
unlikely to have contributed to the fatty metamorphosis observed in the liver of male rats.
28
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Table 4-12. Incidence of neoplasms in male Osborne-Mendel rats exposed to
l,l?2,2-tetrachloroethane in feed for 78 weeks
Neoplasm
Papilloma, stomach
Squamous cell carcinoma, stomach
Neoplastic nodule/carcinoma, liver
Follicular-cell carcinoma, thyroid
Hemangiosarcoma, all sites
Adenocarcinoma, mammary gland
Fibroadenoma, mammary gland
Chromophode adenomas, pituitary
Islet-cell adenomas, pancreatic islets
Fibroma, subcutaneous tissue
Dose (mg/kg-d)
Control
0/20
0/20
0/20
1/19
0/20
1/20
1/20
2/20
0/20
0/20
Vehicle control
0/20
0/20
0/20
3/20
0/20
2/20
1/70
5/14
2/20
1/20
62
0/50
0/50
0/50
0/49
2/50
2/50
1/50
5/48
2/49
2/50
108
1/48
1/48
3/49
2/48
3/49
0/49
0/49
5/48
2/49
2/49
Source: NCI (1978).
In addition, one papilloma of the stomach, one squamous-cell carcinoma of the stomach,
two follicular-cell carcinomas of the thyroid, and three hemangiosarcomas were each observed in
high-dose males (Table 4-12). In the low-dose males, two mammary gland adenocarcinomas
(2/20 in vehicle controls) and two hemangiosarcomas (0/20 in vehicle control) were observed.
Adenomas were observed as follows: pituitary chromophobe adenomas in the vehicle control
(5/14) and low- and high-dose males (5/48 and 5/48, respectively); pancreatic islet-cell
adenomas in the vehicle control (2/20) and low- and high-dose males (2/49 and 2/49,
respectively); mammary gland fibroadenomas in the vehicle control (1/20) and low-dose males
(1/50); and subcutaneous tissue fibromas in the vehicle control (1/20) and low- and high-dose
females (2/50 and 2/49, respectively). In male rats, the incidence of chromophobe adenomas,
islet-cell adenomas, and follicular-cell carcinomas in the vehicle controls was significantly
increased over the incidence in historical controls (NCI, 1978).
In the female rats (Table 4-13), one follicular-cell carcinoma was observed in both the
low- and high-dose groups. One mammary gland adenocarcinoma was observed in a low-dose
female, and two were observed in the high-dose group. One hemangiosarcoma was observed in
a low-dose female. Adenomas were observed as follows: pituitary chromophobe adenomas in
the vehicle control (3/20) and low- and high-dose females (11/49 and 6/48, respectively); one
pancreatic islet-cell adenoma in a low-dose female; mammary gland fibroadenomas in the
vehicle control (9/20) and low- and high-dose females (13/50 and 11/50, respectively); and
subcutaneous tissue fibromas in the vehicle control (1/20) and low- and high-dose females
(2/50 and 1/50, respectively). The incidence of fibroadenomas of the mammary gland in the
vehicle control group was statistically significantly increased over the incidence in historical
controls (NCI, 1978).
29
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Table 4-13. Incidence of neoplasms in female Osborne-Mendel rats exposed
to l,l?2,2-tetrachloroethane in feed for 78 weeks
Neoplasm
Adenocarcinoma, mammary gland
Fibroadenoma, mammary gland
Hemangiosarcomas, uterus
Chromophode adenomas, pituitary
Islet-cell adenomas, pancreatic islets
Follicular-cell carcinoma, thyroid
Fibroma, subcutaneous tissue
Dose (mg/kg-d)
Control
2/20
2/20
0/20
6/19
1/20
0/20
0/20
Vehicle control
0/20
9/20
0/20
3/20
0/20
0/20
1/20
43
1/50
13/50
1/50
11/49
1/50
1/49
2/50
76
2/50
11/50
0/50
6/48
0/50
1/50
1/50
Source: NCI (1978).
NCI (1978) also exposed groups of 50 male and 50 female B6C3Fi mice to 1,1,2,2-tetra-
chloroethane in corn oil via gavage 5 days/week for 78 weeks. Initial dose levels were 100 and
200 mg/kg-day in both genders. In week 19, the doses were increased to 150 and 300 mg/kg-
day, respectively. Three weeks later, the doses were increased to 200 and 400 mg/kg-day,
respectively. In week 27, the doses were decreased to 150 and 300 mg/kg-day, respectively.
The reported TWA doses were 142 and 284 mg/kg-day for male and female mice, respectively.
The exposure period was followed by a 12-week observation period in which the mice were not
exposed to 1,1,2,2-tetrachloroethane. Vehicle and untreated control groups (20 animals/sex) and
a pooled vehicle control were also used. The pooled vehicle control group comprised the vehicle
controls from the studies of 1,1,2,2-tetrachloroethane and chloropicrin. Clinical signs, survival,
body weight, food consumption, gross pathology, and histology (32 major organs and tissues as
well as gross lesions) were evaluated.
A statistically significant association between mortality and dose was observed, as
survival was markedly decreased in the high-dose male and female mice. Terminal survival data
were not reported for the males, although acute toxic tubular nephrosis was determined to be the
apparent cause of death in 33 high-dose males dying between weeks 69 and 70. Survival in the
vehicle control, low-dose, and high-dose females at the end of the study was 75, 74, and 34%,
respectively, but the cause of death in the high-dose females was not reported. The male and
female mice also demonstrated an increased incidence of endemic chronic murine pneumonia.
Respective incidences of chronic murine pneumonia in the vehicle control, low-, and high-dose
groups were 11, 0, and 2% in males and 5, 13, and 18% in females.
A high incidence (approximately 95%) of pronounced abdominal distension, possibly
resulting from liver tumors, was observed in the high-dose females beginning in week 60 and
continuing throughout the recovery period. Nodular hyperplasia and organized thrombus were
30
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observed in male and female mice, but the incidences were not statistically significant.
Nonneoplastic lesions observed included hydronephrosis (16/46) and chronic inflammation in
the kidneys (5/46) in high-dose females and chronic inflammation in the kidneys in the low-
(13/39) and high-dose (10/47) males (Table 4-14). In addition, acute toxic tubular nephrosis was
observed and was the apparent cause of death, as identified by the study authors, in high-dose
male mice that died during weeks 69 and 70.
Table 4-14. Incidence of nonneoplastic kidney lesions observed in male and
female B6C3Fi mice exposed to l,l?2,2-tetrachloroethane in feed for
78 weeks
Lesion
Dose (mg/kg-d)
Control
Vehicle control
142
284
Males
Chronic inflammation - kidney
7/19
5/18
13/39
10/47
Females
Hydronephrosis
Chronic inflammation
0/19
0/19
0/20
0/20
0/46
0/46
16/46
5/46
Source: NCI (1978).
Statistically significant increases in the incidences of hepatocellular carcinomas occurred
in both sexes and at both dose levels (Table 4-15). The incidences in the vehicle control, pooled
vehicle control, 142, and 284 mg/kg-day groups were 1/18, 3/36, 13/50, and 44/49, respectively,
in males and 0/20, 1/40, 30/48, and 43/47, respectively, in females. Information on the
progression from preneoplastic pathology to hepatocellular carcinoma is not available due to the
lack of interim sacrifices. The hepatocellular carcinomas varied in microscopic appearance, with
some tumors composed of well-differentiated cells and a relatively uniform rearrangement of
cords, and other tumors composed of anaplastic cells with large hyperchromatic nuclei with
eosinophilic inclusion bodies and/or vacuolated pale cytoplasm. In addition, a decrease in the
time to tumor for the hepatocellular carcinomas was also evident in both genders of mice. The
spontaneous tumor rate for hepatocellular carcinoma in the historical vehicle controls at the
testing laboratory was 74/612 (12%) for male B6C3Fi mice and 8/560 for female B6C3Fi mice.
31
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Table 4-15. Incidence of hepatocelluar carcinomas in male and female
B6C3Fi mice exposed to l,l?2,2-tetrachloroethane in feed for 78 weeks
Hepatocellular carcinoma
Dose (mg/kg-d)
Vehicle control
Pooled vehicle control
142
284
Males
Incidence
Time to first tumor (wks)
1/18
72
3/36
NA
13/503
84
44/49*
52
Females
Incidence
Time to first tumor (wks)
0/20
NA
1/40
NA
30/483
58
43/47a
53
"Significantly different from control groups.
Source: NCI (1978).
In addition to the liver tumors, alveolar/bronchiolar adenomas in the lung were observed
in the male matched vehicle controls (1/18), male and female pooled-vehicle controls (1/36 and
1/40, respectively), low-dose males and females (2/39 and 1/46, respectively), and high-dose
males and females (2/47 and 1/44, respectively) (Table 4-16). Lymphomas were observed in
low- and high-dose males (4/50 and 3/49, respectively), in female pooled vehicle controls (2/40),
and in low- and high-dose females (7/48 and 3/47, respectively).
Table 4-16. Incidence of additional neoplasms in male and female B6C3Fi
mice exposed to l,l?2,2-tetrachloroethane in feed for 78 weeks
Neoplasm
Dose (mg/kg-d)
Matched control
Pooled vehicle control
142
284
Males
Alveolar/bronchiolar adenomas, lung
Lymphomas, multiple organ
1/18
0/18
1/36
0/36
2/39
4/50
2/47
3/49
Females
Alveolar/bronchiolar adenomas, lung
Lymphomas, multiple organ
0/20
0/20
1/40
2/40
1/46
7/48
1/44
3/47
Source: NCI (1978).
For chronic inflammation in the kidneys of male mice, a LOAEL of 142 mg/kg-day was
selected. A NOAEL was not identified. For hydronephrosis and chronic inflammation in the
kidneys in females, a NOAEL of 142 mg/kg-day and a LOAEL of 284 mg/kg-day were selected.
32
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4.2.2. Inhalation Exposure
4.2.2.1. Subchronic Studies
Truffert et al. (1977) exposed groups of female Sprague-Dawley rats (55/dose) to
1,1,2,2-tetrachloroethane vapor at reported calculated atmospheric concentrations of 0 or
560 mL/m3 5 days/week for 15 weeks (78 exposures). The daily exposure duration was 6 hours
for the first 8 exposures and 5 hours for the remaining 70 exposures. There is uncertainty
regarding the actual concentration employed due to the unusual unit of exposure (i.e., mL/m3). It
is assumed that mL/m3 is a volume/volume vapor concentration, so the reported concentration is
equivalent to 560 ppm (3,909 mg/m3). Interim sacrifices were conducted after 2, 4, 9, 19, 39,
and 63 exposures, although the number of animals killed at each time period was not reported.
This study is limited by poor reporting quality and minimal quantitative data.
Pronounced prostration was observed "after the first exposures to 1,1,2,2-tetrachloroethane,
followed by recovery". Body weight gain was decreased at the end of the study, but the
magnitude of the change was not reported. Increases in relative liver weights were observed
beginning 15 days after exposure initiation, but were not quantified. Hematological alterations
consisting of a decrease in hematocrit "confirmed by the joint RBC and WBC counts" were
observed at the end of the study, but were not quantified. A marked increase (313%) in
thymidine uptake in hepatic DNA was observed after four exposures, but by the ninth exposure
the thymidine uptake had decreased to levels similar to controls. Histological alterations were
observed in the liver after nine exposures and included granular appearance, cytoplasmic
vacuolization, and evidence of hyperplasia (increase in the number of binucleated cells and the
appearance of mitosis), but the alterations regressed after 19 exposures and were no longer
observed after 39 exposures. Incidences and severity of the liver lesions were not reported.
Considering the lack of incidence and severity data and other inadequately reported results, lack
of information on dose-response due to the use of a single exposure level, and uncertainty
regarding the exposure concentration, a NOAEL or LOAEL cannot be identified from this study.
Horiuchi et al. (1962) exposed one adult male monkey (Macaco, cynomolga Linne) to
1,1,2,2-tetrachloroethane for 2 hours/day, 6 days/week for a total of 190 exposures in 9 months.
The exposure level was 2,000-4,000 ppm (13,700-27,500 mg/m3) for the first 20 exposures,
1,000-2,000 ppm (6,870-13,700 mg/m3) for the next 140 exposures, and 3,000-4,000 ppm
(20,600-27,500 mg/m3) for the last 30 exposures. The TWA concentration was 1,974 ppm
(13,560 mg/m3). The authors noted that the monkey was weak after approximately seven
exposures and had diarrhea and anorexia between the 12th and 15th exposures. Beginning at the
15th exposure, the monkey was "almost completely unconscious falling upon his side" for 20-
60 minutes after each exposure. The authors noted a gradual increase in body weight during
months 3-5 followed by a gradual decrease until the study was terminated. Hematological
parameters demonstrated sporadic changes in hematocrit and RBC and WBC counts, but the
significance of these findings cannot be determined because there were no clear trends, only one
33
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monkey was tested, and there was no control group. Histological alterations consisted of fatty
degeneration in the liver and splenic congestion, and no effects were observed in the heart, lung,
kidneys, pancreas, or testes. This study cannot be used to identify a NOAEL or LOAEL for
subchronic exposure due to the use of a single animal without a control.
A 6-month inhalation study in rats was performed by the Mellon Institute of Industrial
Research (1947). Groups of 12 male and 12 female albino rats were exposed to 0 or 167 ppm
(1,150 mg/m3) of 1,1,2,2-tetrachloroethane for 7 hours/day on alternate days for the 6-month
study period. A statistically significant increase (15%) in kidney weight was observed in the
1,1,2,2-tetrachloroethane-exposed rats. The rats also appeared to develop lung lesions following
exposure to tetrachloroethane; however, the study authors stated that the pathology reported for
tetrachloroethane must be discounted due to approximately 50% of the control animals
demonstrating major pathology of the kidneys, liver, or lung. Meaningful interpretation of these
results is precluded by the observed endemic lung infection, which resulted in significant early
mortality in all of the rats (57 and 69% mortality in the control and tetrachloroethane-exposed
groups, respectively). This study also included one mongrel dog that followed the same study
design and evaluation as the rats. Serum phosphatase activity levels, mean of 33 units/100 mL,
and blood urea nitrogen levels, mean of 20.66%, were increased in the treated dog compared to
control values of 5.72/100 mL and 14.94%, respectively. The dog survived the 6-month
exposure with effects that included cloudy swelling of the liver and of the convoluted tubules of
the kidneys, and light congestion of the lungs. Identification of a LOAEL or NOAEL is
precluded by poor study reporting, high mortality in the rats, and the use of a single treated
animal in the dog study.
Kulinskaya and Verlinskaya (1972) examined effects of 1,1,2,2-tetrachloroethane on the
blood acetylcholine system in Chinchilla rabbits exposed to 0 or 10 mg/m3 (0 or 1.5 ppm)
3 hours/day, 6 days/week for 7-8.5 months. The animals were immunized twice, at 1.5-2 and
4 months, subcutaneously with a 1.2 and 1.5 billion microbe dose of typhoid vaccine in an
attempt to reveal changes in the immunological reactivity following 1,1,2,2-tetrachloroethane
exposures. The exposed group contained six animals, and the size of the control group was not
specified. In comparison with both initial and control levels, serum acetylcholine levels were
decreased after 1.5 months, significantly increased after 4.5 months, and significantly decreased
at the end of the study. The concentration of acetylcholine in the blood was increased following
the first immunization. No changes in serum acetylcholinesterase activity were reported,
although serum butyrylcholinesterase activity was reduced after 5-6 months of exposure. This is
a poorly reported study that did not examine any other relevant endpoints. A NOAEL or
LOAEL could not be identified because the changes in acetylcholine levels were inconsistent
across time and incompletely quantified, and the biological significance of the change is unclear.
34
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4.2.2.2. Chronic Studies
In a chronic inhalation study by Schmidt et al. (1972), groups of 105 male rats were
exposed to 0 or 0.0133 mg/L (13.3 mg/m3) 1,1,2,2-tetrachloroethane for 4 hours daily for up to
265 days. Subgroups of seven treated and seven control rats were killed after 110 or 265 days of
exposure and 60 days after exposure termination, with the remaining animals observed until
natural death. There were no significant alterations in survival. Weight gain in exposed rats was
2.1, 11.6, and 12.2% less than controls on study days 110, 260, and 324, respectively, although
the only statistically significant decreases in body weight gain occurred between days 90 and
170. Other statistically significant changes compared to controls included increased leukocyte
(89%) and Pi-globulin (12%) levels after 110 days, and an increased percentage of segmented
nucleated neutrophils (36%), decreased percentage of lymphocytes (17%), and increased
percentage of liver total fat content (34%) after 265 days. There was a statistically significant
decrease in y-globulin levels (32%) at 60 days postexposure and a decrease in adrenal ascorbic
acid content (a measure of pituitary adrenocorticotropic hormone [ACTH] activity) at all three
time periods (64, 21, and 13%, respectively). This study is insufficient for identification of a
NOAEL or LOAEL for systemic toxicity because the experimental design and results were
poorly reported, and histological examinations were not conducted.
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION
4.3.1. Oral Exposure
Gulati et al. (1991a) exposed timed-pregnant CD Sprague-Dawley rats (8-9 animals/
group) to diets containing 0, 0.045, 0.135, 0.27, 0.405, or 0.54% microencapsulated
1,1,2,2-tetrachloroethane from gestation days (GDs) 4 through 20. Based on body weight and
food consumption data, the reported estimated doses of 1,1,2,2-tetrachloroethane were 0, 34, 98,
180, 278, or 330 mg/kg-day. Dams were sacrificed and litters were evaluated on GD 20.
Evaluations included maternal body weight, feed consumption and clinical signs, uterine weight,
and numbers of implantations, early and late resorptions, live fetuses, and dead fetuses.
Necropsies were performed on the maternal animals, but fetuses were not examined for
malformations.
All dams survived to study termination on GD 20. Compared to controls, maternal body
weight was statistically significantly decreased 9, 11, 14, and 24% at 98, 108, 278, and
330 mg/kg-day, respectively, and demonstrated a dose-dependent and time-dependent decrease
in all dose groups. However, an increase in maternal body weight on day 20, compared to body
weight on day 4, was apparent for all dose groups. Daily food consumption was significantly
decreased in all dose groups, and this may have contributed to the decreased body weights
observed in the study. Four out of nine rats in the 278 mg/kg-day dose group had slightly rough
fur beginning on GD 10, while rough fur was present in all animals in the 330 mg/kg-day dose
group. No statistically significant changes were observed in the numbers of live fetuses/litter,
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dead fetuses/litter, resorptions/litter, or implants/litter. One dam in the 98 mg/kg-day group and
four of nine dams in the 330 mg/kg-day group completely resorbed their litters. At scheduled
sacrifice, average fetal weights were statistically significantly decreased 3.9, 12.7, 10.5, and
20.6% in the 98, 180, 278, and 330 mg/kg-day dose groups, respectively (Table 4-17). Gravid
uterine weight was statistically significantly reduced only in the 330 mg/kg-day animals. Small
but statistically significant decreases were seen in maternal body weight and average fetal weight
at >98 mg/kg-day. Using statistical significance and a 10% change as the criterion for an
adverse change in maternal body weight, aNOAEL of 34 mg/kg-day and LOAEL of 98 mg/kg-
day were selected for changes in maternal body weight. A NOAEL of 34 mg/kg-day and
LOAEL of 98 mg/kg-day were selected for developmental toxicity based on the lowest dose that
produced a statistically significant decrease in fetal body weight.
Table 4-17. Fetal body weight in CD Sprague-Dawley rats exposed to
microencapsulated l,l?2,2-tetrachloroethane on GDs 4-20
Dose (mg/kg-d)
0
34
98
180
278
330
n
9
8
8
9
9
5
Mean
2.28
2.17
2.19
1.99
2.04
1.81
SD
0.12
0.11
0.08
0.15
0.42
0.26
% change
-
4.8
3.9
12.7
10.5
20.6
Source: Gulatietal. (1991a).
Gulati et al. (1991b) exposed timed-pregnant Swiss CD-I mice (n = 5-11) to diets
containing 0, 0.5, 1, 1.5, 2, or 3% microencapsulated 1,1,2,2-tetrachloroethane from GDs 4
through 17. Based on body weight and food consumption data, the reported estimated doses of
1,1,2,2-tetrachloroethane were 0, 987, 2,120, 2,216, or 4,575 mg/kg-day; an average dose could
not be calculated for the 3% group due to early mortality. Dams were sacrificed, and litters were
evaluated on GD 17. Evaluations included maternal body weight, feed consumption and clinical
signs, uterine weight, and numbers of implantations, early and late resorptions, live fetuses, and
dead fetuses. Necropsies were performed on the maternal animals, but fetuses were not
examined for malformations.
All animals (9/9) in the 3% group died prior to the end of the study. Mortality was 0/11,
0/9, 2/10, 4/5, and 5/7 in the 0, 987, 2,120, 2,216, or 4,575 mg/kg-day groups, respectively, and
the mortality in the higher dose groups affected the statistical power of the study for those
groups. Maternal body weights were statistically significantly decreased compared to controls at
>2,120 mg/kg-day beginning on study day 9, although the day 17 data were not statistically
significantly different from controls for any treatment group. Average daily feed consumption
36
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was statistically significantly decreased in all treated groups except in the 987 mg/kg-day
animals. Gross hepatic effects were reported in dams from all groups except the 987 mg/kg-day
group and included pale or grey and/or enlarged livers and a prominent lobulated pattern.
Complete litter resorption occurred in 1/11, 0/9, 2/8, 1/1, and 1/2 dams in the 0, 987, 2,120,
2,216, and 4,575 mg/kg-day groups, respectively. No changes in developmental endpoints were
noted in the 987 or 2,120 mg/kg-day groups. The 2,120 and 4,575 mg/kg-day groups had too
few litters, due to maternal toxicity, to permit statistical analysis of the findings. The high
mortality in the exposed mice precluded the identification of a NOAEL or LOAEL for this study.
NTP (2004) conducted a 14-week study in which groups of 10 male and 10 female
F344 rats were fed diets containing microencapsulated 1,1,2,2-tetrachloroethane at reported
average daily doses of 0, 20, 40, 80, 170, or 320 mg/kg-day. The main part of this study is
summarized in Section 4.2.1.1. Reproductive function (fertility) was not evaluated. Endpoints
relevant to reproductive toxicity included histology (testis with epididymis and seminal vesicle,
preputial gland, prostate gland, clitoral gland, ovary, and uterus) and weights (left cauda
epididymis, left epididymis, and left testis) of selected reproductive tissues in all control and
treated groups. Sperm evaluations and vaginal cytology evaluations were performed in animals
in the 0, 40, 80, and 170 mg/kg-day dose groups. The sperm evaluations consisted of spermatid
heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and
concentration. The vaginal cytology evaluations consisted of measures of estrous cycle length.
Sperm motility was 17.1, 14.9, and 24.0% lower than in vehicle controls at 40, 80, and
170 mg/kg-day, respectively. Other statistically significant effects in the males included
reductions in absolute epididymis weight at>80 mg/kg-day and absolute left cauda epididymis
weight at 170 mg/kg-day, and statistically significant increases in the incidences (90-100%) of
minimal to moderate atrophy of the preputial and prostate gland, seminal vesicle, and testicular
germinal epithelium at 320 mg/kg-day. Effects in the females included statistically significant
increases in incidences of minimal to mild uterine atrophy (70-90%) at > 170 mg/kg-day and
clitoral gland atrophy (70%) and ovarian interstitial cell cytoplasmic alterations (100%) at
320 mg/kg-day. The vaginal cytology evaluations indicated that the females in the 170 mg/kg-
day group spent more time in diestrus and less time in proestrus, estrus, and metestrus than did
the vehicle controls. Body weight loss and reduced body weight gain at the lower dose levels
may have contributed to the atrophy and other effects observed in both genders (NTP, 2004).
NTP (2004) also tested groups of 10 male and 10 female B6C3Fi mice that were
similarly exposed to 1,1,2,2-tetrachloroethane for 14 weeks at reported average daily dietary
doses of 0, 100, 200, 370, 700, or 1,360 mg/kg-day (males) or 0, 80, 160, 300, 600, or
1,400 mg/kg-day (females). The main part of this study is summarized in Section 4.2.1.1.
Reproductive function (fertility) was not evaluated, and toxicity endpoints in reproductive organs
are the same as those evaluated in the rat part of the study summarized above. The sperm and
37
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vaginal cytology evaluations were performed in the 200-, 700- and 1,360-mg/kg-day male
groups and the 160-, 600- and 1,400-mg/kg-day female groups.
Effects observed in the male mice included statistically significant increases in the
incidence of preputial gland atrophy at 100, 700, and 1,360 mg/kg-day (incidences in the control
to high dose groups were 0/10, 4/10, 2/10, 0/10, 4/10, and 5/10, respectively), decreased absolute
testis weight at >700 mg/kg-day and absolute epididymis and cauda epididymis weights at
1,360 mg/kg-day, and decreased epididymal spermatozoal motility at 1,360 mg/kg-day (3.1%
less than vehicle controls). In female mice, the length of the estrous cycle was significantly
increased at 1,400 mg/kg-day (8.7% longer than vehicle controls). The pronounced decreases in
body weight gain or body weight loss were similar to those observed in rats.
4.3.2. Inhalation Exposure
Male rats were exposed to 0 or 15 mg/m3 (2.2 ppm) 1,1,2,2-tetrachloroethane 4 hours/day
for up to 8 days in a 10-day period (Gohlke and Schmidt, 1972; Schmidt et al., 1972).
Reproductive function was not tested, but evaluations included histological examinations of the
testes in groups of seven control and seven treated males following the second, fourth, and eighth
exposures, as detailed in Schmidt et al. (1972) in Section 4.2.2.2. This study is limited by
imprecise and incomplete reporting of results. It was noted that testicular histopathology,
described as atrophy of the seminal tubules with strongly restricted or absent spermatogenesis,
was observed in five exposed animals following the fourth exposure; data for the other time
periods and the control group were not reported.
The Schmidt et al. (1972) chronic inhalation study, summarized in Section 4.2.2.2,
included a limited reproductive function/developmental toxicity assessment. Male rats were
exposed to 0 or 13.3 mg/m3 (1.9 ppm) 1,1,2,2-tetrachloroethane 4 hours/day for 265 days, as well
as during the mating period. One week before the end of the exposure period, seven control and
seven exposed males were each mated with five unexposed virgin females. Dams were
permitted to deliver and the offspring were observed for 84 days and were examined
macroscopically for malformations. The percentage of mated females having offspring, littering
interval, time to 50% littered, total number of pups, pups/litter, average birth weight, postnatal
survival on days 1, 2, 7, 14, 21, and 84, sex ratio, and average body weight on postnatal day 84
were also measured. No macroscopic malformations or significant group differences in the other
indices were found, indicating that 13.3 mg/m3 was aNOAEL for male reproductive toxicity.
No effects attributable to 1,1,2,2-tetrachloroethane were reported in rats exposed to 5 or
50 ppm (34.3 or 343 mg/m3, respectively) 7 hours/day for 5 days in a dominant lethal test
(McGregor, 1980). A viral infection may have resulted in increased numbers of early deaths in
all groups, including the control group, possibly affecting study sensitivity. The frequency of
sperm with hook abnormalities was statistically significantly increased in the 343 mg/m3 group,
but not at 34.3 mg/m3.
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4.4. OTHER DURATION- OR ENDPOINT-SPECIFIC STUDIES
4.4.1. Acute Studies (Oral and Inhalation)
4.4.1.1. Oral Studies
Oral (single-dose gavage) median lethal dose (LDso) values of 250-800 mg/kg have been
reported in rats (NTP, 2004; Schmidt et al., 1980b; Gohlke et al., 1977; Smyth et al., 1969).
Cottalasso et al. (1998) described a series of experiments evaluating the effect of a single gavage
dose of 1,1,2,2-tetrachloroethane on the liver of exposed rats. In the first experiment, male
Sprague-Dawley rats (5/group) were given a single gavage dose of 0, 143.5, 287, 574, or
1,148 mg/kg in mineral oil and five animals from each group were sacrificed 5, 15, 30, or
60 minutes later. Sixty minutes after treatment, statistically significant, dose-related increases in
serum activity levels of AST (66, 129, and 201%, respectively) and ALT (54, 88, and 146%,
respectively) were observed at>287 mg/kg. The increase in rat serum activities of AST and
ALT were also increased in a time-dependent manner. Serum AST increased 13-130% from
5 to 60 minutes in rats at 574 mg/kg-day and serum ALT increased 8-88% from 5 to 60 minutes.
A statistically significant decrease in hepatic microsomal G6Pase activity (19, 36, and 47%,
respectively) was observed at>287 mg/kg. A statistically significant decrease in levels of
dolichol, a polyisoprenoid compound believed to be important in protein glycosylation reactions,
in the liver (41 and 56%, respectively) and a statistically significant increase in triglyceride
levels in liver homogenate (60 and 83%, respectively) were observed at >574 mg/kg. A
statistically significant increase in the trigylceride levels in liver microsomes (46, 65, and 97%,
respectively) was observed at>287 mg/kg. See Table 4-18 for a summary of these acute liver
toxicity results. A time-dependent effect was observed in the decrease in G6Pase, in the increase
in triglyceride levels, and in the decrease in levels of dolichol in the liver at 574 mg/kg from 5 to
60 minutes.
Table 4-18. Liver function and other effects observed in Sprague-Dawley
rats 60 minutes after gavage exposure to 1,1^2,2-tetrachloroethane
Dose
(mg/kg)
0
143.5
287
574
1,148
Serum AST
(IU/L)
62 ±9
80 ±10
103±21a
143 ± 13a
187 ± 24a
Serum
ALT
(IU/L)
26 ±4
32 ±6
40±7a
49±6a
64±9a
Microsomal G6Pase
(nmol/min/mg
protein)
361 ±29
342 ± 43
291±39a
230 ± 18a
191±31a
Homogenate
triglycerides
(mg/g liver)
14.5 ±2.0
15.9 ±2.3
19.7 ±3.2
23.2±2.8a
26.5±3.4a
Microsomal
triglycerides
(mg/g liver)
1.61 ±0.12
1.95 ±0.21
2.35±0.30a
2.65±0.35a
3.17±0.42a
Homogenate total
dolichol levels
(ng/mg protein)
335 ±0.28
302 ±53
268 ± 45
197±25a
147±21a
"Significantly different from control.
Source: Cottalasso et al. (1998).
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Schmidt et al. (1980b) administered 0 or 100 mg/kg doses of 1,1,2,2-tetrachloroethane in
corn oil by gavage to groups of 10 male Wistar rats, followed immediately by increased
environmental temperatures, and evaluated hepatic effects 20-22 hours post administration.
Statistically significant increases in serum leucine aminopeptidase activity, hepatic ascorbic acid,
and hepatic triglyceride levels (10.5, 22.3, and 125% greater than control levels, respectively)
were observed, but changes in body weight, liver weight, hepatic N-demethylation of
aminopyrine, and serum ALT activity were not observed. The report includes a general
statement that all chemicals tested in this study led to necrosis and fatty degeneration, which
suggests that 100 mg/kg was a hepatotoxic dose of 1,1,2,2-tetrachloroethane. However, the
significance of the histology results cannot be assessed due to a lack of incidence and severity
measures. No other 1,1,2,2-tetrachloroethane-related histological data were reported in this
study.
Wolff (1978) exposed 8-10-week-old female Wistar rats in groups of 8-10, to a single
gavage dose of 0, 25, or 50 mg/kg 1,1,2,2-tetrachloroethane 30 minutes prior to testing for
passive avoidance (shock level of 0.4 milliamperes [mA]). Passive avoidance was measured by
allowing the test rats to explore the test apparatus, which consisted of a larger, lit box and a
smaller, dark box. After 180 seconds, the darkened box received an electrical shock through the
grid floor. During the 180 seconds, the rats remained in the darkened box approximately 80% of
the time. The test was repeated 24 hours later. No differences in avoidance were observed
between the control and 25 mg/kg groups, but decreased passive avoidance behavior was
reported following exposure to 50 mg/kg. In the second test series, the shock level was increased
to 0.8 mA and the 1,1,2,2-tetrachloroethane dose was increased to 50 mg/kg. The 1,1,2,2-tetra-
chloroethane doses were then increased to 80 mg/kg and then to 100 mg/kg. Increasing the
shock level to 0.8 mA resulted in no significant differences in avoidance between the controls
and the 50 mg/kg dose group (n = 10). Passive avoidance was altered at 80 mg/kg (n = 10), and
at 100 mg/kg, the animals (n = 10) were ataxic and did not learn to avoid the shock. The authors
stated that the treatment with 1,1,2,2-tetrachloroethane may have affected the threshold of
perception of the shock, rather than memory (Wolff, 1978). This conclusion would be consistent
with the high-dose anesthetic effects characteristic of volatile organic compounds in general.
4.4.1.2. Inhalation Studies
Schmidt et al. (1980a) established a 24-hour median lethal concentration (LCso) of
8,600 mg/m3 (1,256 ppm) for 1,1,2,2-tetrachloroethane in rats for a single 4-hour exposure.
Carpenter et al. (1949) found that a 4-hour exposure to 1,000 ppm 1,1,2,2-tetrachloroethane
(6,870 mg/m3) was lethal in Sherman rats, with mortality in "2/6, 3/6, or 4/6" animals.
Price et al. (1978) exposed rats and guinea pigs to 576, 5,050, and 6,310 ppm
1,1,2,2-tetrachloroethane for 30 minutes. Rats exposed to 576 ppm (3,950 mg/m3) for
30 minutes showed a slight reduction in activity and alertness, while increasing the concentration
40
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to 5,050 or 6,310 ppm (34,700 or 43,350 mg/m3) caused lacrimation, ataxia, narcosis, labored
respiration, and 30-50% mortality (Price et al., 1978). Eye closure, squinting, lacrimation, and
decreased activity were observed in guinea pigs exposed to 576 ppm for 30 minutes; exposure to
5,050 ppm resulted in tremors, narcosis, and labored breathing, and exposure to 6,310 ppm
produced 30% mortality (Price et al., 1978). Organ weight measurements and gross pathology
and histology evaluations performed 14 days following the 30-minute exposures did not result in
chemical-related effects in the lungs, liver, kidneys, heart, brain, adrenals, testes, epididymides,
ovaries, or uterus in either species.
Pantelitsch (1933) exposed groups of three mice to 1,1,2,2-tetrachloroethane
concentrations of 7,000, 8,000-10,000, 17,000, 29,000, or 34,000 mg/m3 (1,022, 1,168-1,460,
3,060, 5,220, or 6,120 ppm, respectively) for approximately 1.5-2 hours and examined changes
in clinical status of the animals. All concentrations resulted in disturbed equilibrium, prostration,
and loss of reflexes, with deaths occurring at >8,000-10,000 mg/m3; increasing the concentration
resulted in a more rapid onset of symptoms.
Horvath and Frantik (1973) determined that effective concentrations of 1,1,2,2-tetra-
chloroethane following a single 6-hour exposure in rats were 360 ppm (2,470 mg/m3) for a 50%
decrease in spontaneous motor activity and 200 ppm (1,370 mg/m3) for a 50% increase in
pentobarbital sleep time. No additional relevant information was reported.
Schmidt et al. (1980a) exposed groups of 10 male Wistar rats to 0, 410, 700, 1,030,
2,100, or 4,200 mg/m3 (0, 60, 102, 150, 307, or 613 ppm, respectively) 1,1,2,2-tetrachloroethane
(mean concentrations) for 4 hours and evaluated the animals immediately (within 15-
100 minutes), at 24 hours, or at 120 hours following exposure. The purpose of this study was to
determine a threshold concentration for effects on the liver following inhalation exposure.
Evaluation of this study is complicated by imprecise and incomplete reporting of results,
exposure levels, and observation durations. For example, results for endpoints other than liver
histology, ascorbic acid content, and histochemistry were not reported for the lowest
concentration (410 mg/m3), and liver ascorbic acid content and serum and liver triglyceride
levels were the only results reported quantitatively. Histological effects included diffuse fine
droplet fatty degeneration in the liver at 410 and 700 mg/m3 (24 hours postexposure),
nonspecific inflammation and Councilman bodies (eosinophilic globules derived from necrosis
of single hepatocytes) in the liver at 4,200 mg/m3 (24 hours postexposure), and interstitial
nephritis in the kidneys at 700 mg/m3 (120 hours postexposure). Additional information on these
findings, including incidences and results for other exposure concentrations, was not reported.
Hepatic ascorbic acid levels were statistically significantly increased in groups exposed
to >700 mg/m3 immediately after exposure (2, 64, 29, 167, and 182% higher than controls at
410, 700, 1,030, 2,100, and 4,200 mg/m3, respectively), but returned to control levels within
24 hours. Serum triglyceride concentrations were statistically significantly decreased at
>700 mg/m3 after 24 hours (35, 23, 29, and 56% at 700, 1,030, 2,100, and 4,200 mg/m3,
41
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respectively) and at 2,100 and 4,200 mg/m3 (39 and 42%, respectively) after 120 hours. Hepatic
triglyceride levels were significantly increased at 2,100 and 4,200 mg/m3 (92 and 76%,
respectively) at 24 hours postexposure. Hexobarbital sleep time was increased at 2,100 and
4,200 mg/m3 (not quantified). Assessing the biological significance and adversity of the effects
in this study is complicated by factors that include the lack of liver lesion incidence data, the
paucity of other quantitative data, and other reporting insufficiencies. The authors concluded
that the threshold for effects on the liver was between 410 and 700 mg/m3 because the fine
droplet fatty degeneration was not considered to be biologically significant in the absence of
accompanying serum and liver biochemical changes.
Hepatic effects were also reported by Tomokuni (1969), who administered a single
3-hour exposure of 600 ppm (4,120 mg/m3) 1,1,2,2-tetrachloroethane to female Cb mice. Total
hepatic lipids and triglycerides were statistically significantly increased following exposure and
continued to increase for 8 hours postexposure. Hepatic triglyceride levels increased more than
total lipid levels for 8 hours postexposure. Total hepatic adenosine triphosphate (ATP) levels
were decreased immediately following exposure and continued to decrease over the next 8 hours.
A later study by the same investigator (Tomokuni, 1970) evaluated female Cb mice (5-8/group)
exposed to 800 ppm (5,490 mg/m3) 1,1,2,2-tetrachloroethane for 3 hours and then followed the
time-course of the changes in hepatic lipids and phospholipids over the next 90 hours. Increased
triglyceride and decreased phospholipid levels were seen for the first 30-45 hours postexposure,
but the effects generally resolved by 90 hours postexposure, demonstrating that hepatic effects
resolved after exposure was terminated.
Horiuchi et al. (1962) exposed 10 male mice for a single 3-hour period to an atmosphere
containing 5,900 ppm (-40,500 mg/m3) or 6,600 ppm (-45,300 mg/m3) 1,1,2,2-tetrachloroethane
and then observed the animals for 1 week following exposure. Tissues were obtained for
histologic evaluation from animals at sacrifice or when discovered dead. Three mice exposed to
5,900 ppm and four mice exposed to 6,600 ppm died prior to those sacrificed at the end of the
study. The histological results reported by Horiuchi et al. (1962) are similar to the repeated
vapor exposure study in mice, described in Section 4.4.2.2, where slight to moderate congestion
and fatty degeneration of the liver and congestion of the other male tissues were observed.
Deguchi (1972) administered a single 6-hour exposure of 0, 10, 100, or 1,000 ppm (0, 69,
690, or 6,900 mg/m3, respectively) 1,1,2,2-tetrachloroethane to male rats and evaluated serum
AST and ALT activities up to 72 hours postexposure. This study was reported in Japanese and
included an English translation of the abstract. Based on information in the English abstract and
data graphs in this Japanese study, there was a minimal increase in serum AST at all exposure
concentrations 72 hours postexposure.
42
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4.4.2. Short-term Studies (Oral and Inhalation)
4.4.2.1. Oral Studies
Dow Chemical Company (1988) exposed groups of male Osborne-Mendel rats (n = 5) to
daily gavage doses of 0, 25, 75, 150, or 300 mg/kg-day 1,1,2,2-tetrachloroethane every 24 hours
for 4 days, followed by an injection of [3H]-thymidine for DNA incorporation studies 24 hours
following the last 1,1,2,2-tetrachloroethane dose. The fourth dose was not administered to the
300 mg/kg-day group due to signs of central nervous system (CNS) depression and debilitation,
and one animal in this group died before [3H]-thymidine injection. Terminal body weights of the
300 mg/kg-day animals were statistically significantly decreased, 17% compared to controls.
Absolute liver weights at the highest dose were decreased and relative liver weights were
statistically significantly increased 14% in the 150 mg/kg-day dose group.
Histological examinations of the livers showed increased numbers of hepatocytes in
mitosis in the 75, 150, and 300 mg/kg-day groups, although this response was variable in high-
dose rats, possibly due to the increased toxicity observed in this group (Dow Chemical
Company, 1988). Increased numbers of reticuloendothelial cells were seen at 300 mg/kg-day.
Increased hepatic glycogen content was found in hepatocytes of 75 and 150 mg/kg-day animals,
although this could be an outcome of altered feeding patterns resulting from sedative effects of
dosing (Dow Chemical Company, 1988).
Hepatic DNA synthesis ([3H]-thymidine incorporation) was increased 2.8-, 4.8-, and
2.5-fold at 75, 150, and 300 mg/kg-day, respectively; the decline at 300 mg/kg-day may have
been due to the poor clinical status of the rats in this group (Dow Chemical Company, 1988).
Total hepatic DNA content was not increased. Other endpoints were not evaluated. The
300 mg/kg-day dose is a frank effect level (FEL) based on the CNS depression and mortality.
The 75 mg/kg-day dose may represent a NOAEL for increased relative liver weight in rats.
However, the increase in DNA synthesis and mitosis are not necessarily indicative of
hepatotoxicity, and the histological examinations showed no accompanying degeneration or
other adverse liver lesions.
Dow Chemical Company (1988) similarly exposed groups of male B6C3Fi mice (n = 5)
to daily gavage doses of 0, 25, 75, 150, or 300 mg/kg-day 1,1,2,2-tetrachloroethane for 4 days,
followed by [3H]-thymidine injection for the DNA incorporation studies. All animals survived
treatment, and changes in body weight were not observed at any dose level. Absolute and
relative liver weights were increased 13 and 11%, respectively, at 150 mg/kg-day and 19 and
72%, respectively, at 300 mg/kg-day, although only the increase in relative liver weight at
300 mg/kg-day was statistically significant.
Histopathologic examination of the liver revealed centrilobular swelling with a
corresponding decrease in hepatocyte size in the periportal region due to decreased glycogen
content in mice at >75 mg/kg-day. Increased hepatocyte mitosis was also observed in mice at
300 mg/kg-day. Hepatic DNA synthesis was increased 1.7-fold at 150 mg/kg-day and 4.4-fold at
43
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300 mg/kg-day, although total hepatic DNA content was not increased. Other endpoints were
not evaluated.
TSI Mason Laboratories (1993a, unpublished) administered 1,1,2,2-tetrachloroethane in
corn oil to groups of male and female (n = 5) F344/N rats at 0, 135, 270, or 540 mg/kg-day for
12 days over a 16-day period. Rats were weighed prior to dosing, after 7 days, and prior to
euthanasia, and all surviving rats were euthanized and subject to necropsy. Study endpoints
included clinical observations, body weight, necropsy, selected organ weights (liver, kidneys,
thymus, lung, heart, and testes), and histology of gross lesions. All of the high-dose rats died by
day 5 of the study. Male rats exposed to 270 mg/kg-day displayed an increase in body weight of
37% from day 1 through day 17, compared to an increase of 64% in controls. Female rats
exposed to 270 mg/kg-day displayed a decrease in body weight of 3% from day 1 through day
17, compared to an increase of 30% in controls. The automatic watering system for the low- and
high-dose males failed prior to the administration of 1,1,2,2-tetrachloroethane, and the low and
high doses of the study were repeated in a subsequent study by TSI Mason Laboratories (1993b,
unpublished).
Clinical signs were absent in the 135 mg/kg-day animals, but animals exposed to 270 or
540 mg/kg-day were lethargic following treatment. Absolute liver weights were statistically
significantly increased (19%) in the 135 mg/kg-day female rats, while relative liver weights were
statistically significantly increased at both 135 and 270 mg/kg-day (16 and 34%, respectively).
No changes in absolute or relative liver weights were seen in exposed male rats. Absolute right
kidney weight was significantly increased 9 and 37% in females at 135 and 270 mg/kg-day,
respectively. Absolute thymus weight was statistically significantly decreased in the mid-dose
group of male rats (33% at 270 mg/kg-day), while absolute (45%) and relative (32%) thymus
weights were statistically significantly decreased in only the mid-dose females. Relative right
testis weight was statistically significantly increased (10% at 270 mg/kg-day) in male rats.
Absolute but not relative lung weights were statistically significantly decreased in 270 mg/kg-
day females (17%), while relative heart weights were statistically significantly increased (14%)
in females.
Gross and microscopic lesions were observed in the liver (i.e., hepatodiaphragmatic
nodules) of one control, one mid-dose, and one high-dose rat, but these were common
spontaneous lesions.
In another study, TSI Mason Laboratories (1993b, unpublished) exposed groups of male
F344/N rats (n = 5) to 0, 135, 270, or 540 mg/kg-day 1,1,2,2-tetrachloroethane by gavage in corn
oil for 12 days in a 16-day period. Study endpoints included clinical observations, body weight,
necropsy, selected organ weights (liver, kidneys, thymus, lung, heart, and testes), and histology
of gross lesions. All animals exposed to 540 mg/kg-day died by day 3 of the study. Rats in the
270 and 540 mg/kg-day groups were extremely lethargic following administration of the test
article, with recovery observed only in the 270 mg/kg-day rats.
44
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The weight gain observed in the low- and mid-dose rats was 55.2 and 28%, respectively.
At 135 mg/kg, statistically significant increases of 17 and 13% in absolute and relative liver
weights, respectively, were observed compared to controls. In the mid-dose group, statistically
significant decreases in absolute testes weight (7%), absolute kidney weight (9%), absolute and
relative heart weight (10 and 6%, respectively), and absolute and relative thymus weight (33 and
21%, respectively) were observed. Statistically significant increases in relative thymus (10%),
liver (16%), and kidney weights (7%) were observed at 270 mg/kg-day compared to controls.
Gross and microscopic lesions were observed in the liver of one 270 mg/kg-day male and
in the glandular stomach of one 540 mg/kg-day male, but these were diagnosed as spontaneous
lesions commonly observed in F344/N rats. The lesion observed in the liver was a dark nodule
on the median lobe and corresponded histomorphologically to a hepatodiaphragmatic nodule,
and the lesion observed in the glandular stomach was a pale foci.
TSI Mason Laboratories (1993c, unpublished) exposed groups of five male and five
female B6C3Fi mice to 0, 337.5, 675, or 1,350 mg/kg-day 1,1,2,2-tetrachloroethane by gavage in
corn oil for 12 days during a 16-day period. Study endpoints included clinical observations,
body weight, necropsy, selected organ weights (liver, kidneys, thymus, lung, heart, and testes),
and histology of gross lesions. All mice of both genders in the 1,350 mg/kg-day groups were
found dead or euthanized by day 3 of the study. Additionally, one 675 mg/kg-day female died
and one 337.5 mg/kg-day female was euthanized prior to the end of the study.
No significant changes in body weight were reported in treated groups. Animals in the
675 and 1,350 mg/kg-day groups appeared lethargic within 15 minutes of dosing, and the
1,350 mg/kg-day mice failed to recover after the third treatment. Lethargy also occurred in the
337.5 mg/kg-day female that was sacrificed, but not in other animals in the same exposure group.
In male mice, relative liver weight was statistically significantly increased 9% at 337.5 mg/kg,
and absolute and relative liver weights were statistically significantly increased 28 and 37%,
respectively, at 675 mg/kg-day. In female mice, absolute and relative liver weights were
statistically significantly increased by 50 and 42%, respectively, at 675 mg/kg-day.
Gross hepatic changes, described as pale livers, were noted in one male and three females
at 337.5 mg/kg-day and in four males and three females at 675 mg/kg-day. Histological
examination of the gross lesions showed that they correlated with centrilobular hepatocellular
degeneration characterized by hepatocellular swelling, cytoplasmic rarefaction, and
hepatocellular necrosis in the 675 and 1,350 mg/kg-day males and the 337.5, 675, and
1,350 mg/kg-day females. Hepatocellular necrosis was the most common lesion observed at
675 mg/kg-day.
In a study examining the potential renal toxicity of orally administered halogenated
ethanes, groups of five male F344/N rats received 0, 0.62, or 1.24 mmol/kg-day 1,1,2,2-tetra-
chloroethane by gavage in corn oil (0, 104, or 208 mg/kg-day, respectively) for 21 consecutive
days (NTP, 1996). All rats in the high-dose group died or were killed moribund on days 13-14
45
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and were not evaluated further. Evaluations of the 0 and 104 mg/kg-day animals included
weekly body weights, end-of-study urinalysis (volume, specific gravity, creatinine, glucose, total
protein, AST, y-glutamyl transpeptidase, and N-acetyl-p-D-glucosaminidase), gross necropsy,
selected organ weights (right kidney, liver, and right testis), selected histopathology (right
kidney, left liver lobe, and gross lesions), and kidney cell proliferation analysis (proliferating cell
nuclear antigen [PCNA] labeling index for proximal and distal tubule epithelial cells in S phase).
Clinical signs in the high-dose animals included thinness and lethargy (5/5 rats), diarrhea,
abnormal breathing, and ruffled fur (3/5 rats). In the low-dose group, no effects on survival,
body weight gain, urinalysis parameters, absolute or relative kidney weights, renal or testicular
histopathology, or kidney cell PCNA labeling index were observed.
Hepatic effects in the low-dose group included increased absolute and relative liver
weights (24 and 29% greater than controls, respectively) and cytoplasmic vacuolization of
hepatocytes. The vacuolation occurred in hepatocytes of all low-dose rats and consisted of
multifocal areas with clear droplets within the cytoplasm. Changes in the kidneys of the male
rats were not observed.
In a range-finding study, the NTP (NTP, 2004; TSI Mason Laboratories, 1993d) exposed
male and female F344/N rats (5/sex/group) to 0, 3,325, 6,650, 13,300, 26,600, or 53,200 ppm
1,1,2,2-tetrachloroethane in the diet (microcapsules) for 15 days. Unexposed and vehicle control
groups were also evaluated, with the latter being given feed with empty microcapsules. Study
endpoints included clinical observations, body weight, food consumption, necropsy, selected
organ weights (liver, kidneys, thymus, lung, heart, and testes), and histology of gross lesions;
histology was not evaluated in animals without gross lesions. The study authors reported that
average daily doses for the three lowest concentrations were 300, 400, or 500 mg/kg-day for both
genders. All rats exposed to 26,600 or 53,200 ppm were killed moribund on day 11. The
average daily doses for these groups were not reported.
Female rats exposed to 400 mg/kg-day and both genders exposed to 500 mg/kg-day were
thin and displayed ruffled fur. Body weight at study termination was statistically significantly
lower than controls in both genders of all treated groups. Male rats exposed to 300 mg/kg-day
showed decreased weight gain compared to controls and those exposed to higher doses lost
weight, with final body weights in male rats 28, 46, and 53% less than vehicle controls at 300,
400, and 500 mg/kg-day, respectively. Females lost weight at doses of >300 mg/kg-day, with
final body weights in female rats 25, 38, and 47% less than vehicle controls at 300, 400, and
500 mg/kg-day, respectively. Decreased feed consumption likely contributed to the decreased
weight gains because consumption was reduced in a dose-related manner in both genders of all
treated groups (NTP, 1996).
Absolute thymus weights were decreased 24, 69, and 84% in male rats and 37, 61, and
81% in female rats at doses of >300 mg/kg-day, and relative thymus weights were decreased
42 and 65% in male rats and 38 and 65% in female rats at >400 mg/kg-day (NTP, 2004; TSI
46
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Mason Laboratories, 1993d). In male rats, absolute liver weights were decreased 22, 49, and
60% compared to controls at 300, 400, and 500 mg/kg-day, respectively. Relative liver weight
was increased 7% compared to controls at 300 mg/kg-day and decreased 14% compared to
controls at 500 mg/kg-day. In female rats, absolute liver weight was decreased 25 and 34%
compared to controls at 400 and 500 mg/kg-day, respectively, and relative liver weight was
increased 34 and 23% compared to controls at 300 and 500 mg/kg-day, respectively. Relative
kidney weights were increased 14, 26, and 18% in male rats at 300, 400, and 500 mg/kg-day,
respectively, and 17 and 36% in female rats at 400 and 500 mg/kg-day, respectively. Absolute
kidney weights were decreased 17, 32, and 45% in males and 16, 27, and 27% in females at 300,
400, and 500 mg/kg-day, respectively. Other organ weight decreases were considered a
reflection of the decreased body weights.
Focal areas of alopecia occurred on the skin of four female rats in the 500 mg/kg-day
group, and these lesions correlated with minimal to moderate acanthosis, which is an abnormal,
benign increase in the thickness of the stratum spinosum of the epidermis, a layer of cells that is
capable of undergoing mitotic cell division. In the liver, mild or moderate centrilobular
degeneration was observed microscopically in the exposed male and female rats.
Groups of five male and five female B6C3Fi mice were exposed to 0, 3,325, 6,650,
13,300, 26,600, or 53,200 ppm of encapsulated 1,1,2,2-tetrachloroethane in the diet for 15 days
(NTP, 2004; TSI Mason Laboratories, 1993d). Organ weights, gross necropsy, and histology of
gross lesions were evaluated in surviving mice at the termination of the study. Average daily
doses were not determined by the study authors because feed consumption could not be
measured accurately due to excessive scattering of feed. All male and female mice exposed to
53,200 ppm, all males exposed to 26,600 ppm, and two males exposed to 13,300 ppm were
sacrificed in extremis before the end of the study. Final body weights were decreased 16, 24,
and 22%, in comparison to vehicle controls, in males at 3,325, 6,650, and 13,300 ppm,
respectively. In females, final body weights were decreased 9, 20, 31, and 34% at 3,325, 6,650,
13,300, and 26,600 ppm, respectively.
Clinical findings included hyperactivity in males and females exposed to 3,325, 6,650, or
13,300 ppm and in females in the 26,600 ppm group. Males in the 26,600 and 53,200 ppm
groups were lethargic. Males exposed to >6,650 ppm and females exposed to 26,600 and
53,200 ppm were thin and had ruffled fur. A statistically significant decrease in absolute (31, 47,
82, and 81%, respectively) and relative (22, 33, 74, and 72%, respectively) thymus weights
compared to controls was observed in all exposed female mice. Relative liver weights were
statistically significantly increased 22, 31, and 34% in male mice at 3,325, 6,650, and
13,300 ppm, respectively. Absolute liver weights were statistically significantly decreased 11, 9,
and 5% in female mice at 6,650, 13,300, and 26,600 ppm, respectively, and relative liver weight
increased 30 and 44% at 13,300 and 26,600 ppm, respectively. Other organ weight changes
were associated with changes in body weight. Pale or mottled livers were noted in all exposed
47
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groups of male and female mice and correlated microscopically with hepatocellular
degeneration, which was characterized by hepatocellular swelling, cytoplasmic rarefaction,
single paranuclear vacuoles, hepatocellular necrosis, and infrequent mononuclear infiltrates. The
severity of the hepatic changes increased with increasing exposure concentration.
The histological examinations in the surviving mice showed hepatocellular degeneration
in 3/3, 4/4, 4/4, 1/1, and 1/1 males, and 4/4, 4/4, 3/3, 3/3, and 3/3 females, at 3,325, 6,650,
13,300, 26,600, and 53,200 ppm, respectively (TSI Mason Laboratories, 1993d). For both
genders, the lesions tended to be minimal to mild at 3,325 and 6,650 ppm, with more moderate to
marked severity observed at the higher doses.
NCI (1978) conducted a range-finding study in rats and mice in order to estimate the
maximum tolerated dose for administration in the chronic bioassay. In this study, Osborne-
Mendel rats (5/sex/group) received gavage doses of 0 (vehicle control group), 56, 100, 178, 316,
or 562 mg/kg-day 1,1,2,2-tetrachloroethane in corn oil 5 days/week for 6 weeks, followed by a
2-week observation period. B6C3Fi mice (5/sex/group) were similarly exposed to 0, 32, 56,
100, 178, or 316 mg/kg-day 1,1,2,2-tetrachloroethane. It appears that mortality and body weight
gain were the only endpoints used to assess toxicity and determine the high-dose levels for the
NCI (1978) chronic bioassays in rats and mice. In the rats, one male exposed to 100 mg/kg-day
and all five females exposed to 316 mg/kg-day died (mortality rates in the 562 mg/kg-day groups
were not reported). Body weight gain was reduced 3, 9, and 38% in male rats and 9, 24, and
41% in female rats at 56, 100, and 178 mg/kg-day, respectively. No deaths or significant
alterations in body weight gain were observed in the mice. In male rats, 100 and 178 mg/kg-day,
were selected as the NOAEL and LOAEL, respectively, for the observed decrease in body
weight, while in female rats the NOAEL and LOAEL were 56 and 100 mg/kg-day, respectively,
for the same endpoint. The highest dose in mice, 316 mg/kg-day, was selected as the NOAEL
for body weight changes and mortality.
4.4.2.2. Short-term Inhalation Studies
Rats (n = 84) were exposed to 0 or 15 mg/m3 (2.2 ppm) 1,1,2,2-tetrachloroethane
4 hours/day for up to 8 days in a 10-day period (Gohlke and Schmidt, 1972; Schmidt et al.,
1972). Following the first, third, and seventh exposures, seven control and exposed rats were
given an unknown amount of ethanol. Evaluations were performed on seven males from the
control and treated groups, with and without ethanol, following the second, fourth, and eighth
exposures.
Statistically significant changes included increased serum total protein and decreased
serum on- and (X2-globulin fractions compared to controls after the eighth exposure (day 10),
although the difference was not quantified (Schmidt et al., 1972). Histological effects included a
fine to medium droplet fatty degeneration of the liver that involved increasing numbers of
animals with increasing duration of exposure, although the incidences and severity were not
48
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reported (Gohlke and Schmidt, 1972). The results of the serum and histochemical evaluations
were illegible in the best copy of the translated reference available. Testicular atrophy in the
seminal tubules was observed in five treated animals following the fourth exposure (Gohlke and
Schmidt, 1972). This study is limited by imprecise and incomplete reporting of results.
Assessment of the adversity of liver and other effects in this study is complicated by the
reporting insufficiencies, particularly the paucity of incidence and other quantitative data, as well
as effects that were not consistently observed in the three time periods and a lack of information
on dose-response due to the use of a single exposure level.
Horiuchi et al. (1962) exposed nine male mice to an average concentration of
approximately 7,000 ppm (48,000 mg/m3) 1,1,2,2-tetrachloroethane for 2 hours once/week for a
total of five exposures over 29 days. All animals died during the study with none of the deaths
occurring during exposure, and most (5/9) of the mice died within 5 days of the first exposure.
The only other reported findings in the exposed animals were slight to moderate congestion and
fatty degeneration of the liver and congestion of "other main tissues."
Horiuchi et al. (1962) exposed six male rats to an average concentration of 9,000 ppm
(62,000 mg/m3) 1,1,2,2-tetrachloroethane 2 hours/day, 2-3 times a week for 11 exposures in
29 days. All rats died during the study. No changes in body weight were reported. Exposed
animals generally showed hypermotility within the first few minutes of exposure, followed by
atactic gait within approximately 20 minutes and eventual near-complete loss of consciousness
1-1.5 hours after the onset of exposure. Hematology was assessed in three rats that survived
beyond 2 weeks, and two of these animals showed a decrease in RBC count and Hb content.
Exposed animals generally showed moderate congestion and fatty degeneration of the liver and
congestion of "other main tissues."
As discussed in Section 4.2.2.1, one monkey was exposed to varying concentrations
(2,000-4,000 ppm for the first 20 exposures, 1,000-2,000 ppm for the 20th-160th exposure, and
3,000-4,000 ppm for the remaining exposures) of 1,1,2,2-tetrachloroethane for 2 hours/day,
6 days/week for 9 months (Horiuchi et al., 1962). Effects of short-term exposure included
weakness after seven exposures, diarrhea and anorexia between the 12th and 15th exposures, and
beginning at the 15th exposure, near-complete unconsciousness for 20-60 minutes after each
exposure.
4.4.3. Acute Injection Studies
Paolini et al. (1992) exposed groups of male and female Swiss Albino mice to a single
i.p. dose of 0, 300, or 600 mg/kg 1,1,2,2-tetrachloroethane and sacrificed the animals 24 hours
after dosing to assess hepatotoxicity. An LDso of 1,476 mg/kg for 1,1,2,2-tetrachloroethane was
calculated using six animals/dose and eight dose groups. At 600 mg/kg, absolute and relative
liver weights were statistically significantly decreased 16 and 37%, respectively, in female mice.
No changes in total microsomal protein were noted. Statistically significant decreases (37-74%)
49
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in hepatic CYP enzymes of numerous classes were reported at both dose levels in male and
female mice (see Section 3.3). Other hepatic enzymes with statistically significantly decreased
activity included NADPH-cytochrome c-reductase, 5-aminolevulinic acid-synthetase,
ethoxyresorufm-O-deethylase, pentoxyresorufm O-depentylase, GST (600 mg/kg-day only), and
epoxide hydrolase. Total hepatic heme was reduced at both doses, and heme oxygenase activity
was increased in a dose-related manner, but was statistically significant only in high-dose males
and females.
Wolff (1978) exposed groups of female Wistar rats to a single i.p. dose of 0, 20, or
50 mg/kg 30 minutes prior to testing for passive avoidance of a 0.4 mA electric shock. No
differences between the control and 25 mg/kg groups were reported, but doses of 50 mg/kg
resulted in decreased passive avoidance behavior. Similarly, no differences were seen in the
open-field test at any dose level. In male ICR-mice, a single i.p. dose of 20 mg/kg resulted in a
significant reduction in spontaneous locomotor activity, and 50-60 mg/kg resulted in a 50%
reduction (Wolff, 1978).
In an abstract, Andrews et al. (2002) described the exposure of a rat whole embryo
culture system to 1,1,2,2-tetrachloroethane. GD 9 embryos were exposed to concentrations
between 0.5 and 2.9 mM 1,1,2,2-tetrachloroethane for 48 hours and then evaluated for
morphological changes. At concentrations >1.4 mM, 1,1,2,2-tetrachloroethane resulted in
rotational defects and anomalies of the heart and eye. Embryo lethality was observed at
>2.4 mM.
4.4.4. Immunotoxicological Studies
Shmuter (1977) exposed groups of 12 Chinchilla rabbits to 0, 2, 10, or 100 mg/m3 (0, 0.3,
1.5, or 14.6 ppm, respectively) 1,1,2,2-tetrachloroethane 3 hours/day, 6 days/week for 8-
10 months. Animals were vaccinated with 1 mL of a 1.5 x 109 suspension of heated typhoid
vaccine 1.5, 4.5-5, and 7.5-8 months after the initiation of 1,1,2,2-tetrachloroethane exposure.
Significant increases and decreases in total antibody levels were observed in the 2 and
100 mg/m3 groups, respectively. No significant changes in 7S-typhoid antibody levels were
observed. Significant alterations in the levels of "normal" hemolysins to the Forsman's antigen
of sheep erythrocytes were observed in the 10 and 100 mg/m3 groups, as levels were increased in
the 10 mg/m3 group after 1.5, 2, and 2.5 months of exposure, decreased after 4 months, and
absent at 5 months of exposure. Levels of these hemolysins were decreased in the 100 mg/m3
group during the first 6 months of exposure. Increases in the electrophoretic mobility of specific
antibodies following 1,1,2,2-tetrachloroethane were also reported. Exposure to 100 mg/m3
1,1,2,2-tetrachloroethane resulted in a decrease in the relative content of antibodies in the
y-globulin fraction and an increase in the T and PD fractions. This is a poorly reported study that
provides inadequate quantitative data. The inconsistent dose-response patterns preclude
assessing biological significance and identification of a NOAEL or LOAEL.
50
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4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF
ACTION
4.5.1. Genotoxicity
As discussed in Section 3.4, radiolabeled 1,1,2,2-tetrachloroethane may covalently bind
to DNA and RNA (Colacci et al., 1987), suggesting the potential for mutagenicity. A summary
of the results of genotoxicity studies of 1,1,2,2-tetrachloroethane is presented in Table 4-19.
Table 4-19. Results of in vitro and in vivo genotoxicity studies of
l,l?2,2-tetrachloroethane
In vitro gene mutation assays
Test system
Endpoint
Cells/strain
Concentrations
Results
-S9
+S9
Reference
(a) Bacterial assays
Salmonella
typhimurium
(Ames test)
Escherichia coli
Saccharomyces
cerevisisae
Aspergillus
nidulans
Reverse
mutation
Forward
mutation
DNA damage
Gene
conversion
Gene
reversion
Gene
recombina-
tion
Mitotic
crossover
TA100, 1535,
1537, 1538, 98
TA1530, 1535,
1538
TA1535, 1537,
98
TA1535
TA97, 98, 100,
1535, 1537
TA98, 100,
1535, 1537
TA98, 100,
1535, 1537
TA100
BA13
pol A+/pol Af
WP2S(I)
D7
D7
D7
PI
NA
10 uL/plate
10 uL/plate
NA
10-3,333 uL/plate
NA
5-1,000 uL/plate
NA
0.06-2,979 nmol/
10 uL/plate
15-236 mM
3.1-7.3 mM
NA
3.1-7.3 mM
NA
3.1-7.3 mM
0.01-0.04%v:v
-
NP
-
-
-
-
-
-
-
NP
+
NP
NP
NP
NP
NP
NP
-
+
-
-
-
-
-
-
-
+
-
+
-
+
-
+
+
Nestmann et al., 1980
Rosenkranz, 1977; Brem
etal., 1974
Mitomaetal., 1984
Onoetal., 1996
NTP, 2004
Milmanetal., 1988
Haworth et al., 1983
Warner etal., 1988
Roldan-Arjona et al.,
1991
Rosenkranz, 1977; Brem
etal., 1974
DeMarini and Brooks,
1992
Callenetal., 1980
Nestmann and Lee, 1983
Callenetal., 1980
Nestmann and Lee, 1983
Callenetal., 1980
Crebellietal., 1988
51
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(b) Mammalian cell assays
Mouse lymphoma Gene T „ , _ov
L-i J 1 / O 1
mutation
Hepatocytes DNA repair Osborne
(primary) Mendel rats
B6C3FJ mice
25-500 nL/mL
NA
NA
—
NP
NP
—
—
-
NTP, 2004
Milmanetal., 1988;
Williams, 1983
In vitro chromosomal damage assays
Test system
Cells/organs
Concentrations
Results
Reference
Mammalian cells
Chromosomal
aberrations
SCEs
UDS
CHO cells
CHO cells
BALB/c-3T3 cells
Human embryonic
intestinal fibroblasts
453-804 ug/mL
16.8-558 ug/mL
500-1,000 ug/mL
<15,869 ug/mL
—
+
+
—
—
+
+
NP
NTP, 2004; Galloway et
al., 1987
NTP, 2004; Galloway et
al., 1987
Colaccietal., 1992
McGregor, 1980
Other in vitro assays
Cell transformation
(initiation)
Cell transformation
(promotion)
BALB/c-3T3 cells
1-250 ug/mL
1-250 ug/mL
125-1,000 ug/mL
NA
0. 1-1,000 ng/mL
NP
NP
+
-
NP
-
-
+
-
—
Arthur Little, Inc., 1983
Tuetal., 1985
Colaccietal., 1990
Milmanetal., 1988
Colaccietal., 1996
In vivo bioassays
Test system
Cells/organs
Doses
Results
Reference
Chromosomal damage: mammalian
Chromosomal
aberrations
Micronucleus
UDS
DNA alkylation
Rat bone marrow cells,
male
Rat bone marrow cells,
female
Mouse peripheral blood
erythrocytes
Mouse hepatocytes
Mouse hepatocytes, male
Mouse hepatocytes, female
Mouse hepatocytes
50ppm
50 ppm
589-9, 100 ppm
200 mg/kg
50-1,000 (mg/kg)
50-1,000 mg/kg
150 mg/kg
—
+
+
+
-
-
+
McGregor, 1980
NTP, 2004
Miyagawa et al., 1995
Mirsalisetal., 1989
Dow Chemical Company,
1988
Other in vivo assays
S-phase DNA
synthesis
Mitotic recombination
Recessive lethal
mutation
Mouse hepatocytes, male
Mouse hepatocytes, female
Drosophila melanogaster
D. melanogaster
200-700 mg/kg
200-700 mg/kg
500-1,000 ppm
800 ppm (injected)
1,500 (feed)
-
+/-
-
—
Mirsalisetal., 1989
Vogel and Nivard, 1993
Woodruff etal., 1985
+ = positive; - = negative/no change; CHO = Chinese hamster ovary; NA = not available; NP = assay not
performed; SCE = sister chromatid exchange; UDS = unscheduled DNA synthesis
52
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1,1,2,2-Tetrachloroethane has been shown to be predominantly inactive in reverse
mutation assays in Salmonella typhimurium (strains TA97, TA98, TA100, TA1530, TA1535,
TA1537, and TA1538), either with or without the addition of S9 metabolic activating mixture,
even at concentrations that lead to cytotoxicity (NTP, 2004; Ono et al., 1996; Milman et al.,
1988; Warner et al., 1988; Mitoma et al., 1984; Haworth et al., 1983; Nestmann et al., 1980).
Two studies reported reverse mutation activity in S. typhimurium (Rosenkranz, 1977; Brem et
al., 1974). Results of studies employing methods to prevent volatilization were not notably
different from those that did not use those methods. 1,1,2,2-Tetrachloroethane also did not
induce forward mutations (L-arabinose resistance) in S. typhimurium strain BA13 (Roldan-
Arjona et al., 1991). Assays with Escherichia coli indicated that 1,1,2,2-tetrachloroethane
induced DNA damage, as shown by growth inhibition in DNA polymerase deficient E. coli
(Rosenkranz, 1977; Brem et al., 1974) and induction of prophage lambda (DeMarini and Brooks,
1992). In Saccharomyces cerevisiae, 1,1,2,2-tetrachloroethane induced gene conversion,
reversion, and recombination in one study (Callen et al., 1980), whereas another study found no
conversion or reversion (Nestmann and Lee, 1983). In Aspergillus nidulans, 1,1,2,2-tetrachloro-
ethane induced aneuploidy, but no crossing over (Crebelli et al., 1988).
1,1,2,2-Tetrachloroethane did not induce trifluorothymidine resistance in L5178Y mouse
lymphoma cells, with or without S9, at concentrations up to those producing lethality (NTP,
2004). Primary hepatocytes from rats and mice exposed in vitro to 1,1,2,2-tetrachloroethane did
not show altered DNA repair at concentrations that were not cytotoxic (Milman et al., 1988;
Williams, 1983). McGregor (1980) reported no increase in unscheduled DNA synthesis (UDS)
in human embryonic intestinal fibroblasts exposed to 1,1,2,2-tetrachloroethane. Treatment of
Chinese hamster ovary (CHO) cells with up to 653 ug/mL (which was cytotoxic) did not result in
increased induction of chromosomal aberrations (NTP, 2004; Galloway et al., 1987) but did
produce an increased frequency of sister chromatid exchanges (SCEs) at concentrations of
>55.8 ug/mL (NTP, 2004; Galloway et al., 1987). SCEs were also induced in BALB/c-3T3 cells
treated in vitro with high concentrations (>500 ug/mL) of 1,1,2,2-tetrachloroethane, either with
or without S9 activating mixture (Colacci et al., 1992).
In BALB/C-3T3 cells, 1,1,2,2-tetrachloroethane exposure of up to 250 ug/mL in the
absence of exogenous metabolic activation did not result in increased numbers of transformed
cells (Colacci et al., 1992; Milman et al., 1988; Tu et al., 1985; Arthur Little, Inc., 1983);
survival was generally >70%. Higher concentrations (>500 ug/mL) were capable of
transforming the cells but also showed higher levels of cytotoxicity (Colacci et al., 1990).
However, even relatively low levels (31.25 ug/mL) of 1,1,2,2-tetrachloroethane used as an
initiating agent, followed by promotion with 12-O-tetradecanoylphorbol-13-acetate, resulted in
increased numbers of transformed cells (Colacci et al., 1992). 1,1,2,2-Tetrachloroethane did not
act as a promoter in BALB/C-3T3 cells in vitro without metabolic activation (Colacci et al.,
1996).
53
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1,1,2,2-Tetrachloroethane tested negative for sex-linked recessive lethal mutations and
mitotic recombination mDrosophila melanogaster (NTP, 2004; Vogel and Nivard, 1993;
Woodruff et al., 1985; McGregor, 1980). Replicative DNA synthesis was increased in
hepatocytes isolated from male B6C3Fi mice exposed to a single gavage dose of 200 mg/kg (24
and 48 hours postexposure) or 400 mg/kg (24, 39, and 48 hours postexposure) relative to
hepatocytes from unexposed mice (Miyagawa et al., 1995). Hepatocytes isolated from mice
following a single gavage dose of up to 1,000 mg/kg did not show an increase in UDS or S-phase
DNA synthesis (Mirsalis et al., 1989). Hepatocytes isolated from B6C3Fi mice 6 hours after a
single gavage dose of 150 mg/kg in corn oil demonstrated irreversible alkylation of hepatic DNA
(Dow Chemical Company, 1988). Inhalation exposure to 5 or 50 ppm (34.3 or 343 mg/m3) for
7 hours/day, 5 days/week did not result in increased frequency of chromosomal aberrations in
bone marrow cells isolated from male rats (McGregor, 1980); female rats exposed to 50 ppm
(343 mg/m3), but not to 5 ppm (34.3 mg/m3), showed an increase in bone marrow cell
aberrations other than gaps (McGregor, 1980).
In summary, genotoxicity studies provide limited evidence of a mutagenic mode of
action. 1,1,2,2-Tetrachloroethane has some genotoxic activity, but in vitro genotoxicity tests
generally reported negative results. Similarly, in vivo studies had mostly negative results with
the exception of chromosomal aberrations in female rat bone marrow cells and micronucleus
formation in mouse bone marrow peripheral erythrocytes. The results of rat liver preneoplastic
foci and mouse BALB/C-3T3 cell neoplastic transformation assays suggest that 1,1,2,2-tetra-
chloroethane may have initiating and promoting activity. Overall, results of genotoxicity studies
for 1,1,2,2-tetrachloroethane are mixed and insufficient for establishing a mutagenic mode of
action.
54
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4.5.2. Short-Term Tests of Carcinogenicity
Treatment of partially hepatectomized male Osborne-Mendel rats with a single
100 mg/kg gavage dose of 1,1,2,2-tetrachloroethane, followed by 7 weeks of promotion with
phenobarbital in the diet, did not result in increased numbers of preneoplastic (gamma-glutamyl
transpeptidase [GGT]-positive) foci in the liver (Milman et al., 1988; Story et al., 1986).
Exposure of partially hepatectomized male Osborne-Mendel rats to a single i.p. dose of
diethylnitrosamine (DEN) as an initiating agent followed by promotion with 100 mg/kg-day of
1,1,2,2-tetrachloroethane by gavage 5 days/week for 7 weeks produced a significantly increased
number of GGT-positive foci in the liver (Milman et al., 1988; Story et al., 1986). 1,1,2,2-Tetra-
chloroethane also significantly increased the number of GGT-positive foci in rats administered
the promotion protocol in the absence of the DEN initiator. The study authors concluded that
1,1,2,2-tetrachloroethane induces hepatocarcinogenesis primarily through a promoting
mechanism (Story et al., 1986).
Using a mouse strain that had been shown to be susceptible to pulmonary adenomas
when exposed to organic chemicals, Theiss et al. (1977) administered i.p. injections of 80, 200,
or 400 mg/kg 1,1,2,2-tetrachloroethane in Tricaprylin 5-18 times to groups of 20 male A/St mice
for 8 weeks. There was a dose-related increase in number of lung tumors/mouse (Table 4-20),
and the dose-response was nearly statistically significant (Theiss et al., 1977).
Table 4-20. Pulmonary adenomas in male A/St mice following repeated i.p.
injections of l,l?2,2-tetrachloroethane
Dose/injection (mg/kg)
Number of i.p. injections
Total dose (mg/kg)
Number of surviving animals
Number of lung tumors/mouse
0
24
0
15/20
0.27 ±0.15
80
5
400
10/20
0.30 ±0.21
200
18
3,600
15/20
0.50 ±0.14
400
16
6,400
5/20
1.00 ±0.45
Source: Thiess etal. (1977).
Maronpot et al. (1986) tested 65 chemicals at three doses in 6- to 8-week-old male and
female strain A/St or A/J mice housed 10/cage. Doses were set based on the highest dose
exhibiting a lack of overt toxicity from a preliminary dose-setting study, with the mid and low
dose as half the higher dose. Mice were injected i.p. 3 times/week for 8 weeks. Lungs were
examined histologically. The data for 1,1,2,2-tetrachloroethane-exposed male and female strain
A/St are presented in Table 4-21.
55
-------
Table 4-21. Pulmonary adenomas in male and female A/St mice following
repeated i.p. injections of l,l?2,2-tetrachloroethane
Compound
Dose/injection (mg/kg)
Vehicle
Untreated
control
-
-
Saline
vehicle
control
-
-
Tricaprylin
vehicle
control
-
-
Urethan
positive
control
1,000
-
1,1,2,2-Tetrachloroethane
62.5
Tricaprylin
99
Tricaprylin
187.5
Tricaprylin
Male A/St mice
Number of surviving
animals3
Percent survivors with
tumors
Tumors per mouseb
119/120
2
0.017
45/50
9
0.089
54/60
13
0.167
47/50
96
11.9
10/10
10
0.1
8/10
0
0
5/10
0
0
Female A/St mice
Number of surviving
animals3
Percent survivors with
tumors
Tumors per mouseb
79/80
8
0.076
44/50
14
0.186
54/60
11
0.11
47/50
96
10.3
9/10
0
0
5/10
20
0.2
3/10
0
0
"Numerator is number of mice alive at study termination; denominator is number of mice started on study.
bBased on all surviving mice at study termination.
Source: Maronpotetal. (1986).
4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS
4.6.1. Oral
4.6.1.1. Human Data
Information on the acute oral toxicity of 1,1,2,2-tetrachloroethane in humans is available
from several case reports. Based on amounts of 1,1,2,2-tetrachloroethane recovered from the
gastrointestinal tract of deceased subjects following intentional ingestion (Mant, 1953; Sherman,
1953; Lilliman, 1949; Forbes, 1943; Elliot, 1933; Hepple, 1927), estimated lethal doses ranged
from 273 to 9,700 mg/kg. Patients who accidentally consumed a known volume of 1,1,2,2-tetra-
chloroethane, corresponding to single doses ranging from 68 to 117 mg/kg, as medicinal
treatment for hookworm experienced loss of consciousness and other clinical signs of narcosis
(Ward, 1955; Sherman, 1953). Chronic oral effects of 1,1,2,2-tetrachloroethane in humans have
not been reported in the literature.
4.6.1.2. Animal Data
Few studies have evaluated acute oral toxicity in animals, and the endpoints assessed
consist of data on lethality and neurological and liver effects (Table 4-22). Oral LD50 values
ranged from 250 to 800 mg/kg in rats (NTP, 2004; Schmidt et al., 1980a; Gohlke et al., 1977;
Smyth et al., 1969). Neurological effects of acute, oral 1,1,2,2-tetrachloroethane administration
56
-------
revealed ataxic effects and decreased passive avoidance behavior (Wolff, 1978). Hepatic
changes were noted in two separate acute oral toxicity studies. Male Sprague-Dawley rats
administered between 287 and 1,148 mg/kg 1,1,2,2-tetrachloroethane had dose-dependent
increases in the serum activity levels of AST and ALT as well as a decrease in hepatic
microsomal G6Pase activity (Cottalasso et al., 1998). Male Wistar rats were administered
100 mg/kg 1,1,2,2-tetrachloroethane and had increases in hepatic ascorbic acid levels and serum
leucine aminopeptidase activity, but no changes in serum ALT activity (Schmidt et al., 1980a, b).
Both studies noted increases in triglyceride levels in the liver.
57
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Table 4-22. Summary of noncancer results of major studies for oral exposure of animals to l,l?2,2-tetrachloroethane
Species
Sex
Average daily
dose
(mg/kg-d)
Exposure
duration
NOAEL
(mg/kg-d)
LOAEL
(mg/kg-d)
Response
Comments
Reference
Acute exposure
Rat
(Wistar)
Rat
(Sprague-
Dawley)
Rat (Wistar)
F
M
M
0, 25, 50, 80, or
100
(gavage)
0, 143.5, 287,
574, or 1,148
(gavage)
0 or 100
Single dose
Single dose
Single dose
25
143.5
100
50
287
ND
Increased electric shock
perception threshold.
Increased serum AST
activity and ALT activity,
increased liver triglyceride
levels; decreased liver
dolichol levels.
Increased hepatic ascorbic
acid levels and serum
leucine aminopeptidase
activity.
Results suggestive of a subtle
anesthetic effect. Ataxia observed
at 100 mg/kg.
Evaluations performed 1 hr
postexposure. Approximately
twofold increases in AST and ALT
at >574 mg/kg. Liver histology and
neurotoxicity not assessed.
No changes in serum ALT.
Wolff, 1978
Cottalasso et al.,
1998
Schmidt etal.,
1980a,b
Short-term exposure
Rat
(Osborne-
Mendel)
Mouse
(B6C3FO
Rat (F344/N)
Rat (F344/N)
Mouse
(B6C3FO
Rat (F344/N)
M
M
M,F
M
M,F
M
0, 25, 75, 150, or
300
(gavage)
0, 25, 75, 150, or
300
(gavage)
0, 135, 270, or
540
(gavage)
0, 135, 270, or
540
(gavage)
0, 337.5, 675, or
1,350
(gavage)
0, 104, or 208
(gavage)
3-4 d
4d
12 doses in
16 d
12 doses in
16 d
12 doses in
16 d
13-21 d
150
300
135
135
ND
ND
300 (PEL)
ND
270
270
337.5
104
CNS depression and
mortality. No
histopathological changes
in liver.
Decreased body weight in
females, plus lethargy and
increased organ weights.
Lethargy, decreased body
weight gain.
Hepatocellular
degeneration (females).
Hepatic cytoplasmic
vacuolization at low dose,
mortality at high dose.
Increased hepatocellular DNA
synthesis and mitosis at>75 mg/kg-
d; increased liver weight at
>150 mg/kg-d. No nonhepatic
endpoints evaluated.
Centrilobular swelling at
>75 mg/kg-d and increased
hepatocellular DNA synthesis and
mitosis at > 150 mg/kg-d. No
nonhepatic endpoints evaluated.
The highest dose caused 100%
mortality. Limited histology3.
Mortality at 540 mg/kg-d. Limited
histology3.
Lethargy, increased liver weight,
and mortality at higher doses.
Limited histology3.
No changes in body weight, kidney
weights, kidney histology, or
urinalysis.
Dow Chemical
Company, 1988
Dow Chemical
Company, 1988
TSI Mason
Laboratories,
1993a, unpubl.
TSI Mason
Laboratories,
1993b, unpubl.
TSI Mason
Laboratories,
1993c, unpubl.
NTP, 1996;
58
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Table 4-22. Summary of noncancer results of major studies for oral exposure of animals to l,l?2,2-tetrachloroethane
Species
Rat (F344/N)
Mouse
(B6C3FO
Rat
(Osborne-
Mendel)
Mouse
(B6C3FO
Sex
M, F
M, F
M, F
M, F
Average daily
dose
(mg/kg-d)
0, 300, 400, or
500
(diet)
3,325, 6,650,
13,300, 26,600, or
53,200 ppm
0, 56, 100, 178,
316, or 562
0, 32, 56, 100,
178, or 3 16
Exposure
duration
15 d
15 d
5 d/wk for
6 wks
5 d/wk for
6 wks
NOAEL
(mg/kg-d)
ND
ND
100 (male)
56 (female)
316
LOAEL
(mg/kg-d)
300
ND
178 (male)
100 (female)
ND
Response
Decreased body weight
gain.
Decreased body weight,
hyperactivity, decreased
absolute and relative
thymus weight, increased
relative liver weight, pale
or mottled livers,
hepatocellular
degeneration.
Decreased body weight
gain.
Body weight changes and
mortality.
Comments
Changes in liver and kidney
weights and clinical signs at higher
doses. Limited histology3.
Feed consumption could not be
measured accurately due to feed
scattering; thus average daily doses
(mg/kg-d) were not estimated.
Mortality and body weight gain
were the only endpoints used to
assess toxicity.
Mortality and body weight gain
were the only endpoints used to
assess toxicity.
Reference
NTP, 2004
NTP, 2004; TSI
Mason
Laboratories,
1993d
NCI, 1978
NCI, 1978
Subchronic exposure
Rat (F344)
Mouse
(B6C3FO
M,F
M, F
0, 20, 40, 80, 170,
or 320
(diet)
0, 100, 200, 370,
700, or 1,360
(male); 0, 80,
160, 300, 600, or
1,400 (female)
(diet)
14 wks
14 wks
20
40
80
40
80
160
Increased liver weight, as
well as decreased sperm
motility.
Increased serum ALT
activity, SDH activity, and
cholesterol levels, reduced
epididymis weight.
Increased liver weight,
increased ALT activity,
ALP activity, SDH
activity, and bile acid
levels.
Comprehensive study. More
serious hepatic effects, including
hepatocyte necrosis and bile duct
hyperplasia, as well as effects on
other organs, at >170 mg/kg-d.
Comprehensive study. Wide array
of endpoints evaluated, including
histopathology. More serious
hepatic effects, including
hepatocyte necrosis and bile duct
hyperplasia, as well as effects on
other organs, at >300 mg/kg-d.
NTP, 2004
NTP, 2004
Chronic exposure
59
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Table 4-22. Summary of noncancer results of major studies for oral exposure of animals to l,l?2,2-tetrachloroethane
Species
Rat
(Osborne-
Mendel)
Mouse
(B6C3FO
Sex
M, F
M, F
Average daily
dose
(mg/kg-d)
0, 62, or
108 (male)
0, 43, or
76 (female)
(gavage)
0, 142, or 284
(gavage)
Exposure
duration
78wks
78wks
NOAEL
(mg/kg-d)
62 (M)
76 (F)?
ND
142
LOAEL
(mg/kg-d)
108 (M)
ND(F)
142 (M)
284 (F)
Response
Fatty changes in liver.
Reduced survival. Acute
toxic tubular nephrosis,
hydronephrosis, and
chronic inflammation in
the kidneys.
Comments
Study is confounded by endemic
chronic murine pneumonia, but this
is unlikely to have contributed to
the liver pathology.
Reference
NCI, 1978
NCI, 1978
Developmental exposure
Rat
(Sprague-
Dawley)
Mouse
(CD-I)
F
F
0, 34, 98, 180,
278, or 330
(diet)
0, 987, 2,120,
2,216, or 4,575
(diet)
CDs 4-20
CDs 4-17
34
ND
98
ND
Decreased maternal and
fetal body weights.
Maternal mortality and
litter resorptions.
Effects were more pronounced at
higher doses.
High mortality in the exposed mice
precluded the identification of a
NOAEL or LOAEL.
Gulati et al.,
1991a
Gulati et al.,
1991b
aHistology only evaluated in animals with gross lesions.
F = female; M = male; ND = Not determined
60
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Short-term oral exposure (Table 4-22) to 1,1,2,2-tetrachloroethane produced clinical
signs of neurotoxicity and mortality at doses as low as 208-300 mg/kg-day by gavage in rats
(NTP, 1996; TSI Mason Laboratories, 1993a, b; Dow Chemical Company, 1988). Body weight
gain was decreased at similar dose levels in rats exposed by gavage or diet (NTP, 2004; TSI
Mason Laboratories, 1993a, b; Dow Chemical Company, 1988; NCI, 1978). Hepatic effects
consisted of increased DNA synthesis and centrilobular swelling in mice exposed to 75 mg/kg-
day in the diet (Dow Chemical Company, 1988) and hepatocellular cytoplasmic vacuolation in
rats exposed to 104 mg/kg-day (NTP, 1996). At higher doses (337.5 mg/kg-day), hepatocellular
degeneration was observed in mice (TSI Mason Laboratories, 1993c).
Subchronic and chronic oral administration studies (Table 4-22) with 1,1,2,2-tetrachloro-
ethane in animals indicated that the liver is the most sensitive organ for toxicity. Oral toxicity
studies in F344 and Osborne-Mendel rats and B6C3Fi mice were evaluated (NTP, 2004, NCI,
1978). The 14-week subchronic study by NTP (2004) in both F344 rats and B6C3Fi mice was
the most comprehensive evaluation of 1,1,2,2-tetrachloroethane-mediated toxicity through an
orally administered route. NCI (1978) conducted a chronic study on Osborne Mendel rats and
B6C3Fi mice in which dosing regimens were modified during the course of the study.
In F344 rats, an increased incidence of hepatocellular cytoplasmic vacuolization was
observed at 20 mg/kg-day in males and 40 mg/kg-day in females, increased relative liver weights
were observed at 40 mg/kg-day, and hepatocellular hypertrophy was observed at 80 mg/kg-day
in the subchronic NTP (2004) study. Additional hepatic effects included increases in serum ALT
and SDH activity at 80 mg/kg-day, decreases in serum cholesterol levels at 80 mg/kg-day, and
increases in serum ALP activity and bile acid levels, hepatocellular necrosis, bile duct
hyperplasia, hepatocellular mitotic alterations, foci of cellular alterations, and hepatocyte
pigmentation at 170 and 320 mg/kg-day. A NOAEL of 20 mg/kg-day and a LOAEL of
40 mg/kg-day was selected based on the increase in relative liver weight; however, it should be
noted that an increased incidence of hepatocellular cytoplasmic vacuolization was observed at
20 and 40 mg/kg-day in male and female rats, respectively. In the Osborne-Mendel rats,
significant increases in hepatic fatty metamorphosis were observed in male rats following a
chronic exposure to 108 mg/kg-day (TWA, based on changes in dosing regimen) (NCI, 1978).
Mortality was significantly increased in female rats dosed at a TWA dose of 43 and 76 mg/kg-
day; however, the increased mortality was affected by the deaths of 10 high-dose females, 8 with
pneumonia and 2 with no reported lesions, during the first 5 weeks of the study. A NOAEL of
62 mg/kg-day and a LOAEL of 108 mg/kg-day were identified in male rats based on an
increased incidence of hepatic fatty metamorphosis (NCI, 1978).
Mice appear to be less sensitive than rats to noncancer effects mediated by orally
administered 1,1,2,2-tetrachloroethane. Relative liver weight was statistically significantly
increased in female and male B6C3Fi mice at 80 and 200 mg/kg-day, respectively. Effects in the
mice also included minimal hepatocellular hypertrophy, increased serum SDH activity, ALT
61
-------
activity, and bile acid levels, and decreased serum cholesterol levels at 160-200 mg/kg-day, as
well as increased serum ALP and 5'-nucleotidase activities, necrosis, pigmentation, and bile duct
hyperplasia at 300-370 mg/kg-day. Based on the increase in relative liver weight observed in
the NTP (2004) study, a NOAEL of 100 mg/kg-day and a LOAEL of 200 mg/kg-day in male
mice and a LOAEL of 80 mg/kg-day in female mice were identified. In addition, male and
female B6C3Fi mice were evaluated for chronic oral toxicity by NCI (1978). For this study, a
LOAEL of 142 mg/kg-day was selected for chronic inflammation in the kidneys of male mice,
while a NOAEL of 142 mg/kg-day and a LOAEL of 284 mg/kg-day were selected for
hydronephrosis and chronic inflammation in the kidneys of female mice.
Comprehensive neurobehavioral testing showed no evidence of neurotoxicity in either
species at doses equal to or higher than the LOAELs based on liver effects (NTP, 2004),
indicating that the liver is more sensitive than the nervous system to subchronic dietary exposure
to 1,1,2,2-tetrachloroethane.
Developmental parameters were significantly affected by oral administration of
1,1,2,2-tetrachloroethane in rats and mice. Significant decreases in rat maternal and fetal body
weights were noted at doses of >98 mg/kg-day (Gulati et al., 1991a). Using statistical
significance and a 10% change as the criteria for establishing an adverse effect in maternal body
weight, a NOAEL of 34 mg/kg-day and LOAEL of 98 mg/kg-day were selected for
developmental toxicity based on the lowest dose that produced a statistically significant decrease
in fetal body weight. In mice, the PEL based on maternal toxicity and resorption of litters is
2,120 mg/kg-day (Gulati et al., 1991b). The high mortality in the exposed mice precluded the
identification of a NOAEL or LOAEL from this study.
Toxicity to reproductive tissues following 1,1,2,2-tetrachloroethane exposure to adult rats
and mice was observed at dose levels as low as 40 mg/kg-day (NTP, 2004). In male rats, sperm
motility was decreased at >40 mg/kg-day. Higher doses resulted in decreased epididymal
absolute weight and increased atrophy of the preputial and prostate gland, seminal vesicle, and
testicular germinal epithelium. In female rats, minimal to mild uterine atrophy was increased at
>170 mg/kg-day, and clitoral gland atrophy and ovarian interstitial cell cytoplasmic alterations
were increased at 320 mg/kg-day. Female F344 rats in the 170 mg/kg-day group spent more
time in diestrus than did the vehicle controls.
Male B6C3Fi mice had increased incidences of preputial gland atrophy at > 100 mg/kg-
day. Less sensitive effects included decreases in absolute testis weight (>700 mg/kg-day) and
absolute epididymis and cauda epididymis weights (1,360 mg/kg-day) and a decrease in
epididymal spermatozoal motility (1,360 mg/kg-day). The only noted reproductive toxicity
parameter in female mice affected was a significant increase in the length of the estrous cycle at
a dose of 1,400 mg/kg-day (NTP, 2004).
62
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4.6.2. Inhalation
4.6.2.1. Human Data
Limited information is available on the acute inhalation toxicity of 1,1,2,2-tetrachloro-
ethane in humans (Table 4-23). The results of an early, poorly reported experimental study with
two volunteers suggest that 3 ppm (6.9 mg/m3) was the odor detection threshold. Irritation of the
mucous membranes, pressure in the head, vertigo, and fatigue were observed at 146 ppm
(1,003 mg/m3) for 30 minutes or 336 ppm (2,308 mg/m3) for 10 minutes. Common reported
symptoms of high-level acute inhalation exposure to 1,1,2,2-tetrachloroethane in humans include
drowsiness, nausea, headache, and weakness, and at extremely high concentrations, jaundice,
unconsciousness, and respiratory failure (Coyer, 1944; Hamilton, 1917).
63
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Table 4-23. Summary of noncancer results of major human studies of inhalation exposure to l,l?2,2-tetrachloroethane
Study
population
Sex
Exposure
level (mg/m3)
Exposure NOAEL
duration (mg/m3)
LOAEL
(mg/m3)
Response
Comments
Reference
Acute exposure
Two volunteers
NS
6.9-2,308
30 min
ND
ND
Irritation, vertigo, head
pressure, fatigue.
Effect levels could not be
determined due to limited
analysis.
Lehmannet al., 1936
Occupational exposure
127 coating
workers
Workers from
39 chemical
processing
plants
380 workers
from
23 factories
34-75 workers
in penicillin
production
NS
NS
M,F
NS
500-1,500
NS
62.5-672
10-1,700
NS
NS
Generally <1 yr
Up to 3 yrs
ND
ND
ND
ND
ND
ND
ND
ND
Decreased whole blood
specific gravity,
decreased RBC count,
lymphocyte sis,
unspecified neurological
findings.
Increased mortality for
lymphatic cancers.
Anemia, loss of appetite,
abdominal pain,
headache, vertigo, and
tremors.
Loss of appetite,
epigastric pain, hepatic
enlargement,
urobilinogenuria,
weakness, fatigue, weight
loss, and itching.
Effect levels could not be
determined due to limited
analysis.
Mortality from cardiovascular
disease, cirrhosis of the liver, and
digestive or respiratory cancers
was not elevated.
Effect levels could not be
determined due to a lack of a
control population and possible
coexposure.
Effect levels could not be
determined due to a lack of a
control population, limited
reporting, and possible
coexposure.
Horiguchi et al., 1964
Norman etal., 1981
Lobo-Mendonca, 1963
Jeney etal., 1957
F = female; M = male; ND = not determined; NS = not stated
64
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Chronic toxicity of inhaled 1,1,2,2-tetrachloroethane in humans (Table 4-23) resulted in
neurological symptoms including headache, weakness, fatigue, and hematological changes such
as anemia and elevated WBC count (Norman et al., 1981; Lobo-Mendonca, 1963; Jeney et al.,
1957; Minot and Smith, 1921). Most occupational exposure studies failed to evaluate hepatic
endpoints, other than an urobilinogen test. Jeney et al. (1957) reported a positive relationship
between duration of exposure and frequency of abnormal liver function test results, loss of
appetite, bad taste in the mouth, epigastric pain, and a "dull straining pressure feeling in the area
of the liver".
4.6.2.2. Animal Data
Acute inhalation exposures in animals (Table 4-24) resulted in near-lethal or lethal effects
at levels >1,000 ppm (Schmidt et al., 1980a; Price et al., 1978; Horiuchi et al., 1962; Carpenter et
al., 1949; Pantelitsch, 1933). Death was typically preceded by signs of CNS toxicity (e.g.,
incoordination, loss of reflexes, labored respiration, prostration, and loss of consciousness) and
was often accompanied by congestion and fatty degeneration of the liver. Nonlethal exposures
increased lipid and triglyceride levels in the liver in mice following exposure to 600-800 ppm
(4,120-5,490 mg/m3) for 3 hours (Tomokuni, 1970, 1969). Nonlethal exposures also reduced
motor activity in rats following exposure to 576 ppm (3,950 mg/m3) for 30 minutes (Price et al.,
1978) and 360 ppm (2,470 mg/m3) for 6 hours (Horvath and Frantik, 1973) and in guinea pigs
following exposure to 576 ppm (3,950 mg/m3) (Price et al., 1978).
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Table 4-24. Summary of noncancer results of major studies for inhalation exposure of animals to
l,l?2,2-tetrachloroethane.
Species
Sex
Exposure
level (mg/m3)
Exposure
duration
NOAEL
(mg/m3)
LOAEL
(mg/m3)
Response
Comments
Reference
Acute exposure
Rat
Rat (Wistar)
Rat (Sherman)
Rat
Guinea pig
Rat
(NR)
Mouse (Cb)
Mouse (Cb)
Mouse
Mouse
Rat
NR
M
NR
NR
NR
NR
F
F
NS
M
M
NR
0, 410, 700,
1,030, 2,100,
or 4,200
6,870
3,950,
34,700, or
43,350
3,950,
34,700, or
43,350
1,370 or
2,470
4,120
5,490
7,000, 8,000-
10,000,
17,000,
29,000, or
34,000
40,500 or
45,300
0, 69, 690, or
6,900
4hrs
4Hrs
4hrs
30 mins
30 mins
6 hrs
3hrs
3hrs
1.5-2 hrs
3hrs
6 hrs
NR
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
8,600
ND
ND
3,950
3,950
2,470
4,120
ND
7,000
ND
69
LC50
24-Hr observation.
Hepatic effects included histological alterations and
increases in serum enzymes and liver triglycerides.
Identification of a NOAEL or LOAEL precluded by
reporting inadequacies.
Mortality
Slight reduction in activity and alertness; lacrimation,
ataxia, narcosis, labored respiration, and 30-50%
mortality when concentration increased.
Eye closure, squinting, lacrimation, and decreased
activity; tremors, narcosis, labored breathing, and
mortality when concentration increased
Effective concentration
for a 50% decrease in
spontaneous motor
activity.
Increased hepatic lipid
and triglyceride levels,
decreased hepatic
ATP.
Increased triglyceride
and decreased
phospholipid levels.
Disturbed equilibrium,
prostration, and loss of
reflexes.
Effective concentration for a
50% increase in pentobarbital
sleep time was 1,370 mg/m3.
A limited number of
endpoints were evaluated.
Effects generally resolved by
90 hours postexposure.
Limited number of endpoints
and poor reporting. Mortality
at >8,000 mg/m3.
Mortality: 3/10 and 4/10, respectively
Minimal increase in serum AST at all exposure
concentrations 72 hrs postexposure.
Schmidt etal., 1980a
Schmidt etal., 1980a
Carpenter etal., 1949
Price etal., 1978
Price etal., 1978
Horvath and Frantik, 1973
Tomokuni, 1969
Tomokuni, 1970
Pantelitsch, 1933
Horiuchi et al., 1962
Deguchi, 1970
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Table 4-24. Summary of noncancer results of major studies for inhalation exposure of animals to
l,l?2,2-tetrachloroethane.
Species
Sex
Exposure
level (mg/m3)
Exposure
duration
NOAEL
(mg/m3)
LOAEL
(mg/m3)
Response
Short-term exposure
Rat
Rat
Mouse
M
M
M
Oorl5
62,000
48,000
4 hrs/d for up to
8 exposures in
10 d
2 hrs/d, 2-
3 times/wk for
1 1 exposures in
29 d
2 hrs/d for
5 exposures in
29 d
ND
ND
ND
ND
ND
ND
Comments
Reference
Increases in serum proteins and histological
alterations in the liver. Identification of a NOAEL or
LOAEL precluded by reporting inadequacies.
All rats died during the study. No changes in body
weight were reported. Exposed animals generally
showed moderate congestion and fatty degeneration
of the liver.
Moderate congestion
and fatty degeneration
of the liver.
Subchronic exposure
Rat
(Sprague-
Dawley)
Monkey
(Macaca sp.)
Rat
Mongrel dog
Rabbit
F
M
M,F
M
NS
0 or 3,909
13,560
0 or 1,150
0 or 1,150
OorlO
5-6 hrs/d,
5d/wkfor
15 wks
2 hrs/d, 6 d/wk
for total of
190 exposures in
9 mo
7 hrs/d for 6 mo
7 hrs/d for 6 mo
3 hrs/d, 6 d/wk
for 7-8.5 mo
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Most (5/9) of the mice died
within 5 days of the first
exposure.
Gohlke and Schmidt,
1972; Schmidt etal., 1972
Horiuchi et al., 1962
Horiuchi et al., 1962
Increased liver weight, transient liver cytoplasmic
vacuolization. Identification of a NOAEL or LOAEL
precluded by reporting inadequacies.
Fatty degeneration and splenic congestion.
Identification of a LOAEL or NOAEL is precluded
by the use of a single animal and lack of control.
Pathological effects in the liver, kidney, and lung,
precluded by an endemic lung infection.
Increased serum phosphatase and blood urea nitrogen
levels, cloudy swelling of the liver and convoluted
tubule of the kidney, and light congestion of the
lungs. A NOAEL or LOAEL was not identified due
to single treated dog.
Altered serum acetylcholine levels. A NOAEL or
LOAEL cannot be identified due to incomplete
quantitation.
Truffertetal., 1977
Horiuchi et al., 1962
Mellon Institute of
Industrial Research, 1947
Mellon Institute of
Industrial Research, 1947
Kulinskaya and
Verlinskaya, 1972
67
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Table 4-24. Summary of noncancer results of major studies for inhalation exposure of animals to
l,l?2,2-tetrachloroethane.
Species
Rabbit
Sex
NS
Exposure
level (mg/m3)
0, 2, 10, or
100
Exposure
duration
3 hrs/d, 6 d/wk
for 8-10 mo
NOAEL
(mg/m3)
ND
LOAEL
(mg/m3)
ND
Response
Comments
Increase and decrease in total antibody levels,
increase in the mobility
of specific antibodies,
decrease in the relative content of y-globulin
antibodies and an increase in the T and (3D fractions.
Poorly reported study that provides inadequate
quantitative data.
Reference
Shmuter, 1977
Chronic exposure
Rat
M
0 or 13. 3
4 hrs/d, 110 or
265 d
ND
ND
Increased leukocyte and Pi -globulin levels, increased
percentage of segmented nucleated neutrophils,
decreased percentage of lymphocytes, increased liver
total fat content. Experimental design and results
were poorly reported and histological examinations
do not appear to have been conducted.
Schmidt etal., 1972
F = female; M = male; ND = not determined; NS = not specified
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Acute and short-term inhalation exposure (Table 4-24) to high concentrations
(>7,000 ppm) of 1,1,2,2-tetrachloroethane produced mortality and neurological and liver effects
in animals. Mortality occurred in mice exposed to 7,000 ppm (48,000 mg/m3) for 2 hours,
once/week for a total of 4 exposures in 29 days and in rats exposed to 9,000 ppm (62,000 mg/m3)
for 2 hours/day, 2-3 times/week for a total of 11 exposures in 29 days. Congestion and fatty
degeneration in the liver (mice and rats), as well as a biphasic change in neurological motor
activity (hyperactivity followed by ataxia, rats only), were also reported (Horiuchi et al., 1962).
At the lowest inhalation exposure of 2.2 ppm (15 mg/m3) for 4 hours/day (8-10 days), rats had
fine droplet fatty degeneration in the liver and changes in levels of serum proteins, but no
neurological changes were reported (Gohlke and Schmidt, 1972; Schmidt et al., 1972).
There are a few subchronic inhalation exposure studies and one chronic exposure study
with 1,1,2,2-tetrachloroethane (Table 4-24). Overall these studies either had poor study designs,
one exposure concentration, low numbers of animals, or a combination of the above. The
available subchronic and chronic inhalation studies indicate that the liver was the most sensitive
organ to 1,1,2,2-tetrachloroethane exposure. Increased relative liver weights were reported at
exposures of 560 ppm (3,909 mg/m3) for 15 weeks (Truffert et al., 1977). Other transient hepatic
changes (e.g., histological alterations and cytoplasmic vacuolation) were observed, but these
effects did not persist (Truffert et al., 1977). In the chronic exposure study, rats exposed to
13.3 mg/m3 (1.9 ppm) 1,1,2,2-tetrachloroethane 4 hours/day for 265 days exhibited increased
liver fat content (Schmidt et al., 1972). In the third rat study (Mellon Institute of Industrial
Research, 1947), none of the effects noted from 1,1,2,2-tetrachloroethane exposure could be
evaluated since the control animals experienced a high degree of pathological effects in the
kidneys, liver, and lung. Hepatic effects from long-term exposure to 1,1,2,2-tetrachloroethane
were also reported in a study with one mongrel dog with cloudy swelling of the liver at 167 ppm
(1,150 mg/m3) for 6 months (Mellon Institute of Industrial Research, 1947) and one male
monkey with fatty degeneration of the liver at 1,974 ppm (13,560 mg/m3) for 9 months (Horiuchi
etal., 1962).
Other endpoints that were observed following subchronic and chronic inhalation
exposure are described below. Hematological alterations including increased leukocyte and
Pi-globulin levels, increased percentage of segmented nucleated neutrophils and decreased
percentage of lymphocytes, decreased y-globulin, and decreased adrenal ascorbic acid levels
were observed in rats exposed to 1.9 ppm (13.3 mg/m3) for 265 days (Schmidt et al., 1972), and
splenic congestion was noted in a study of a single monkey (Horiuchi et al., 1962). In the
mongrel dog study noted above, cloudy swelling of the convoluted tubules of the kidneys and
light congestion of the lungs were observed (Mellon Institute of Industrial Research, 1947).
Kulinskaya and Verlinskaya (1972) observed alterations in serum acetylcholine levels in rabbits
exposed to 10 mg/m3 (1.5 ppm) 3 hours/day, 6 days/week for 7-8.5 months. Shmuter (1977)
69
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observed immunological alterations (changes in antibody levels) in rabbits exposed to 2-
100 mg/m3 (0.3-14.6 ppm) 3 hours/day, 6 days/week for 8-10 months.
A reproductive toxicity assessment was conducted on seven male rats exposed to
13.3 mg/m3 1,1,2,2-tetrachloroethane for 258 days. No significant changes in reproductive
parameters were observed, indicating that 13.3 mg/m3 (1.9 ppm) was aNOAEL for male
reproductive effects in the rat (Schmidt et al., 1972).
4.6.3. Mode of Action of Noncarcinogenic Effects Information
1,1,2,2-Tetrachloroethane is rapidly and extensively absorbed following both oral and
inhalation exposures, with absorption of 70-100% following oral exposure in animals (Dow
Chemical Company, 1988; Mitoma et al., 1985) and 40-97% following inhalation exposures in
humans (Morgan et al., 1970; Lehmann et al., 1936). Following absorption, the chemical is
distributed throughout the body, although the high tissue:air partition coefficient for fat (Gargas
et al., 1989) suggests that it may accumulate more in lipid-rich tissues. Metabolism is extensive,
with >68% of a total administered dose generally found as metabolites (Dow Chemical
Company, 1988; Mitoma et al., 1985; Yllner, 1971), and is believed to occur mostly in the liver.
Urinary elimination occurs mainly as metabolites, including dichloroacetic acid, glyoxalic acid,
formic acid, trichloroethanol, and trichloroacetic acid, while a fraction of an absorbed dose may
be eliminated in expired air as parent compound or carbon dioxide.
Metabolism of 1,1,2,2-tetrachloroethane to reactive products is likely to play a key role in
the observed noncancer effects. Both nuclear and microsomal CYP enzymes have been
implicated in the metabolism of the compound, possibly forming a number of biologically active
compounds including aldehydes, alkenes, acids, and free radicals (see Figure 3-1 in Section 3.3),
which may react with biological tissues. Evidence for metabolism to reactive compounds comes
from studies of radiolabel incorporation following single doses of radiolabeled 1,1,2,2-tetra-
chloroethane in which incorporated radiolabel was enhanced by pretreatment with phenobarbital,
xylene, or ethanol, and the variety of inducers capable of influencing this effect suggest that
multiple CYP isozymes may be involved (Casciola and Ivanetich, 1984; Halpert, 1982; Sato et
al., 1980), including members of the CYP2A, CYP2B, CYP2E, and CYP3A subfamilies
(Omiecinski et al., 1999; Nebert et al., 1987). Additionally, mice are known to metabolize
1,1,2,2-tetrachloroethylene at a 1.1-3.5-fold greater rate than rats and have been demonstrated to
have approximately a twofold greater binding of radiolabel to tissues, further implicating
metabolic activation as a possible step in the mode of action of noncarcinogenic effects.
However, there is uncertainty as to whether the presence of radiolabel in proteins, DNA, and
RNA may be radiolabeled carbon that has been incorporated into biomolecules through normal
biochemical processes. Studies providing additional mode of action information for the
1,1,2,2-tetrachloroethane-induced noncancer toxicological effects are not available.
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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) 1,1,2,2-tetra-
chloroethane is "likely to be carcinogenic to humans" based on data from an oral cancer bioassay
in male and female Osborne-Mendel rats and B6C3Fi mice (NCI, 1978). In B6C3Fi mice, a
statistically significant increase in the incidence of hepatocellular carcinomas in both genders
was observed at doses of 142 and 284 mg/kg-day. A decrease in the time to tumor in both
genders of mice was also observed. In this same bioassay, male Osborne-Mendel rats exhibited
an increased incidence of hepatocellular carcinomas, a rare tumor in this strain (NCI, 1978), at
the high dose only, although this increased incidence was not statistically significant. An
untreated female control rat also developed a hepatocellular carcinoma. In the high-dose male
mice, acute toxic tubular nephrosis was characterized as the cause of death in the mice that died
prior to study termination, although hepatocellular carcinomas were observed in most of these
mice.
The predominant proposed metabolic pathway for 1,1,2,2-tetrachloroethane involves
production of dichloroacetic acid (Casciola and Ivanetich, 1984; Halpert and Neal, 1981; Yllner,
1971). Dichloroacetic acid was identified as the major urinary metabolite in mice treated with
1,1,2,2-tetrachloroethane by i.p. injection (Yllner et al., 1971) and in in vitro systems with rat
liver microsomal and nuclear CYP (Casciola and Ivanetich, 1984; Halpert, 1982; Halpert and
Neal, 1981). Other pathways may involve the formation of trichloroethylene via
dehydrochlorination or tetrachloroethylene via oxidation as initial metabolites (Mitoma et al.,
1985; Ikeda and Ohtsuji, 1972; Yllner et al., 1971). 1,1,2,2-Tetrachloroethane may also form
free radicals by undergoing reductive dechlorination (ATSDR, 1996).
Dichloroacetic acid induces hepatocellular carcinomas in both genders of F344 rats and
B6C3Fi mice (DeAngelo et al., 1999; DeAngelo et al., 1996; Pereira, 1996; Pereira and Phelps,
1996; Ferreira-Gonzalez et al., 1995; Richmond et al., 1995; Daniel et al., 1992; DeAngelo et al.,
1991; U.S. EPA, 1991b; Bull et al., 1990; Herren-Freund et al., 1987). Trichloroethylene, also a
metabolite of 1,1,2,2-tetrachloroethane, has been shown to produce hepatocellular carcinomas
and hepatocellular adenomas in male and female B6C3Fi mice, respectively, but did not
demonstrate carcinogenicity in Osborne-Mendel or Sprague-Dawley rats (NTP, 1990; NCI,
1976). Tetrachloroethylene, another metabolite of 1,1,2,2-tetrachloroethane, was characterized
by NCI (1977) as a liver carcinogen in B6C3Fi mice, but an evaluation of carcinogenicity in
Osborne-Mendel rats was inadequate due to early mortality. In a study by NTP (1986),
tetrachloroethylene demonstrated evidence of carcinogenicity in F344 rats, as shown by
increased incidences of mononuclear cell leukemia, and in B6C3Fi mice, as shown by increased
incidences of hepatocellular adenomas and carcinomas in males and carcinomas in females.
Additional information on the carcinogenic potential comes from studies on the tumor
initiating and promoting activity in mammalian cells (Colacci et al., 1996, 1992). The results of
71
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the in vivo and in vitro genotoxicity studies for 1,1,2,2-tetrachloroethane, which were generally
negative, provide limited evidence of a mutagenic mode of action.
No animal cancer bioassay data following inhalation exposure to 1,1,2,2-tetrachloro-
ethane are available. However, U.S. EPA's Guidelines for Carcinogen Risk Assessment (2005a)
indicates that, for tumors occurring at a site other than the initial point of contact, the cancer
descriptor generally applies to all routes of exposure that have not been adequately studied unless
there is convincing information to indicate otherwise. No additional information is available for
1,1,2,2-tetrachloroethane (e.g., toxicokinetic data that absorption does not occur by other routes).
Thus, based on the observance of systemic tumors following oral exposure, and in the absence of
information to indicate otherwise, 1,1,2,2-tetrachloroethane is considered "likely to be
carcinogenic to humans" by any route of exposure.
The weight of evidence for the carcinogenicity of 1,1,2,2-tetrachloroethane could be
strengthened by additional cancer bioassays demonstrating tumor development. Currently, the
NCI (1978) bioassay is the only study available demonstrating 1,1,2,2-tetrachloroethane
tumorgenicity. The NCI (1978) study was a 78-week study, compared to a 104-week bioassay,
and the limitations of the study included increased mortality in male and female mice, the
variable doses given to the mice over the course of the 78-week exposure period, and the acute
toxic tubular nephrosis, characterized as the cause of death, in the high-dose male mice that died
prior to study termination (although hepatocellular carcinomas were observed in most of these
mice).
4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence
Only one study in humans evaluated the possible carcinogenic effects of 1,1,2,2-tetra-
chloroethane. Norman et al. (1981) evaluated groups of clothing-treatment workers employed
during World War II in which some workers used 1,1,2,2-tetrachloroethane and some used
water. Inhalation exposure concentrations and durations were not reported and dermal exposures
were likely. In addition, coexposures to dry-cleaning chemicals occurred. No differences in
standard mortality ratios were seen between the 1,1,2,2-tetrachloroethane and water groups for
total mortality, cardiovascular disease, cirrhosis of the liver, or cancer of the digestive and
respiratory systems. The mortality ratio for lymphatic cancers in the 1,1,2,2-tetrachloroethane
group was increased relative to controls and the water group, although the number of deaths was
small (4 cases observed compared to 0.85 cases expected). No other information was located
regarding the carcinogenicity of 1,1,2,2-tetrachloroethane in humans.
The only comprehensive animal study that evaluated the carcinogenicity of 1,1,2,2-tetra-
chloroethane was performed by the NCI (1978). Male and female Osborne-Mendel rats were
exposed to TWA doses of 0, 62, or 108 mg/kg-day (males) or 0, 43, or 76 mg/kg-day (females)
5 days/week for 78 weeks, followed by a 32-week observation period during which the rats were
not exposed. No statistically significant increases in tumor incidences were observed in rats.
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However, two hepatocellular carcinomas, which were characterized by NCI (1978) as rare in
Osborne-Mendel rats, and one neoplastic nodule were observed in the high-dose male rats. A
hepatocellular carcinoma was also observed in a female rat in the control group. NCI (1978)
characterized the carcinogenic results in male rats as "equivocal." Male and female B6C3Fi
mice were exposed to TWA doses of 0, 142, or 284 mg/kg-day 5 days/week for 78 weeks,
followed by a 12-week observation period during which the mice were not exposed. Statistically
significant, dose-related increases in the incidence of hepatocellular carcinoma were observed in
males (3/36, 13/50, and 44/49 in the control, low-, and high-dose groups, respectively) and
females (1/40, 30/48, and 43/47, respectively). In addition, a decrease in the time to tumor for
the hepatocellular carcinomas was also evident in both genders of mice. Lymphomas were also
seen in the male and female mice, but the incidences were not found to be statistically
significant. The only other available study observed pulmonary adenomas in female Strain A/St
mice given 99 mg/kg-day injections i.p. 3 times/week for 8 weeks (Maronpot et al., 1986).
In vitro studies of the genotoxicity of 1,1,2,2-tetrachloroethane have yielded mixed,
though mainly negative, results. Mutagenicity studies in S. typhimurium were predominantly
negative, with only 2 of 10 available studies reporting activity (NTP, 2004; Ono et al., 1996;
Roldan-Arjona et al., 1991; Milman et al., 1988; Warner et al., 1988; Mitoma et al., 1984;
Haworth et al., 1983; Nestmann et al., 1980; Rosenkranz, 1977; Brem et al., 1974). Mixed
results were reported for gene conversion, reversion, and recombination in S. cerevisiae
(Nestmann and Lee, 1983; Callen et al., 1980), and aneuploidy but not mitotic cross over was
induced in A nidulans (Crebelli et al., 1988). Tests for DNA damage in E. coli were positive
(DeMarini and Brooks, 1992; Rosenkranz, 1977; Brem et al., 1974). 1,1,2,2-Tetrachloroethane
was not mutagenic in mouse L5178Y lymphoma cells (NTP, 2004) and was negative in tests for
DNA damage in other mammalian cells, including induction of DNA repair in primary rat or
mouse hepatocytes (Milman et al., 1988; Williams, 1983), induction of chromosomal aberrations
in CHO cells (NTP, 2004; Galloway et al., 1987), and induction of cell transformation in
BALB/C-3T3 cells (Colacci et al., 1992; Milman et al., 1988; Tu et al., 1985; Arthur Little, Inc.,
1983). 1,1,2,2-Tetrachloroethane was positive for induction of SCEs in both BALB/C-3T3
(Colacci et al., 1992) and CHO cells (NTP, 2004; Galloway et al., 1987) and for induction of cell
transformation in BALB/C-3T3 cells at high (cytotoxic) doses (Colacci et al., 1990).
1,1,2,2-Tetrachloroethane also had mixed results for genotoxicity following in vivo
exposure. Tests for sex-linked recessive lethal mutations and mitotic recombination in
Drosophila were negative (NTP, 2004; Vogel and Nivard, 1993; Woodruff et al., 1985;
McGregor, 1980). Both positive (Miyagawa et al., 1995) and negative results (Mirsalis et al.,
1989) have been reported in mouse hepatocytes tested for UDS, and tests for S-phase DNA
induction in hepatocytes were negative in male mice and equivocal in female mice (Mirsalis et
al., 1989). Rat bone marrow cells were negative for chromosomal aberrations in male rats, but
positive in female rats (McGregor, 1980).
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1,1,2,2-Tetrachloroethane showed promoting activity but limited initiating activity in rat
liver preneoplastic (GGT-positive) foci assays (Milman et al., 1988; Story et al., 1986).
1,1,2,2-Tetrachloroethane initiated but did not promote neoplastic transformation in mouse
BALB/c-3t3 cells (Colacci etal., 1996, 1992).
4.7.3. Mode of Action of Carcinogenicity Information
The mode of action of the carcinogenic effects of 1,1,2,2-tetrachloroethane is unknown.
Colacci et al. (1987) reported possible covalent binding of radiolabeled 1,1,2,2-tetrachloroethane
to DNA, RNA, and protein in the liver, kidneys, lung, and stomach of rats and mice exposed to a
single intravenous dose and analyzed 22 hours postexposure. However, the conclusion of
covalent binding may be influenced by the presence of radiolabel in the DNA, RNA, and protein
that was the result of incorporated radiolabeled carbon into the biomolecules through normal
biochemical processes.
The mutagenicity data for 1,1,2,2-tetrachloroethane are inconclusive, with in vitro
genotoxicity tests generally reporting negative results, except for assays of SCE and cell
transformation, and in vivo tests of genotoxicity showing a similar pattern. Several studies have
reported increases in the number of hepatocytes in mitosis, but the possible role these effects
may have on the carcinogenicity of 1,1,2,2-tetrachloroethane has not been evaluated. The results
of rat liver preneoplastic foci and mouse BALB/C-3T3 cell neoplastic transformation assays
suggest that 1,1,2,2-tetrachloroethane may have initiating and promoting activity (Colacci, 1996,
1992; Milman et al., 1988; Story et al., 1986), but tumor initiation and promotion studies have
not been conducted.
Tumor formation by 1,1,2,2-tetrachloroethane may involve metabolism to one or more
active compounds with the predominant pathway leading to the production of dichloroacetic acid
(Casciola and Ivanetich, 1984; Halpert andNeal, 1981; Yllner, 1971). 1,1,2,2-Tetrachloroethane
is metabolized extensively following absorption, at least in part, by CYP enzymes from the
members of the CYP2A, CYP2B, CYP2E, and CYP3A subfamilies (see Section 3.3). Mice are
known to metabolize 1,1,2,2-tetrachloroethane to a greater extent than rats, which may in part
account for the fact that liver tumors occurred in mice at statistically significant levels but not in
rats following chronic oral exposure.
Dichloroacetic acid, which appears to be the main metabolite of 1,1,2,2-tetrachloro-
ethane, induces hepatocellular carcinomas in both genders of F344 rats and B6C3Fi mice
(DeAngelo et al., 1999; DeAngelo et al., 1996; Pereira, 1996; Pereira and Phelps, 1996; Ferreira-
Gonzalez et al., 1995; Richmond et al., 1995; Daniel et al., 1992; DeAngelo et al., 1991; U.S.
EPA, 1991b; Bull et al., 1990; Herren-Freund et al., 1987). Dichloroacetic acid is recognized as
hepatocarcinogenic in both genders of two rodent species.
1,1,2,2-Tetrachloroethane may be metabolized to form free radicals, which may, in turn,
covalently bind to macromolecules including DNA. Formation of free radicals during
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1,1,2,2-tetrachloroethane metabolism has been demonstrated in spin-trapping experiments
(Tomasi et al., 1984). Both nuclear and microsomal forms of CYP enzymes have been
implicated in this process, as increased metabolism and covalent binding of metabolites
following pretreatment with phenobarbital (Casciola and Ivanetich, 1984; Halpert, 1982), xylene
(Halpert, 1982), or ethanol (Sato et al., 1980) have been reported. The presence of covalently
bound label has been reported following inhalation (Dow Chemical Company, 1988), oral
(Mitoma et al., 1985), and intravenous (Eriksson and Brittebo, 1991) administration of
radiolabeled 1,1,2,2-tetrachloroethane.
In summary, only limited data are available regarding the possible mode(s) of action of
1,1,2,2-tetrachloroethane carcinogenicity. Metabolism to one or more active compounds may
play a role in tumor development. Results of genotoxicity studies of 1,1,2,2-tetrachloroethane
are mixed and provide inconclusive evidence for establishing a mutagenic mode of action.
There is some evidence to indicate that the mode of carcinogenic action may involve
tumor promotion. Milman et al. (1988) and Story et al. (1986) concluded that 1,1,2,2-tetra-
chloroethane induces hepatocarcinogenesis primarily through a promoting mechanism following
treatment of partially hepatectomized male Osborne-Mendel rats with a single 100 mg/kg gavage
dose of 1,1,2,2-tetrachloroethane, followed by 7 weeks of promotion with phenobarbital in the
diet. This regimen failed to result in increased numbers of preneoplastic (GGT-positive) foci in
the liver. However, an exposure of partially hepatectomized male Osborne-Mendel rats to a
single i.p. dose of DEN as an initiating agent followed by promotion with 100 mg/kg-day of
1,1,2,2-tetrachloroethane by gavage 5 days/week for 7 weeks produced a significantly increased
number of GGT-positive foci in the liver.
4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES
4.8.1. Possible Childhood Susceptibility
Studies in humans and laboratory animals are not available to determine whether early
life stages are particularly susceptible to 1,1,2,2,-tetrachloroethane exposures. However, the
Gulati rat study (Gulati et al., 1991b) demonstrated that fetuses exposed in utero can be
adversely affected. At scheduled sacrifice, average fetal weights were statistically significantly
decreased in all dose groups except the 34 mg/kg-day group. In the Gulati mouse study (Gulati
et al., 1991a), complete litter resorption occurred in mice in 1/11, 0/9, 2/8, 1/1, and 1/2 dams in
the 0, 987, 2,120, 2,216, and 4,575 mg/kg-day dose groups, respectively. The limited data
evaluating the effect of 1,1,2,2-tetrachloroethane on the developing organism have not indicated
effects on the offspring at levels that did not also produce maternal effects.
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4.8.2. Possible Gender Differences
Studies evaluating the differences in potency of 1,1,2,2-tetrachloroethane in male and
female rodents are not available. Some toxicity studies which evaluated both genders in the
same study showed close concordance between genders with often no more than one dose
distinguishing between response levels for a given effect. Men normally have a smaller volume
of body fat than women, even accounting for average size differences, contributing to differential
disposition of organic solvents between genders (Sato and Nakajima, 1987). Rats have
pronounced sex-specific differences in CYPs, primarily involving the CYP2C family which is
not found in humans, but humans have not demonstrated sex-specific isoforms of CYP (Mugford
and Kedderis, 1998). Humans have differences in CYP 3A4 activity related to estrogen and
progesterone, but these differences are regulated by hormones at the level of gene expression
(Harris et al., 1995). Other differences may occur at the Phase 2 level attributable to
conjugation. Overall, no consistent differences have been reported between women and men in
the handling of xenobiotics such as 1,1,2,2-tetrachloroethane by CYP isoforms (Shimada et al.,
1994). These distinctions make it difficult to predict from the animal data gender-relevant
differences for human exposure to 1,1,2,2-tetrachloroethane.
4.8.3. Other Susceptible Populations
As metabolism is believed to play an important role in the toxicity of 1,1,2,2-tetrachloro-
ethane, particularly in the liver, individuals with elevated levels of CYP enzymes may have an
increased susceptibility to the compound. Halpert (1982) reported an increase in in vitro
metabolite formation and in covalently bound metabolites following pretreatment with xylene or
phenobarbital, both of which increased CYP activity. Sato et al. (1980) similarly reported an
increased metabolism of 1,1,2,2-tetrachloroethane in rats following ethanol pretreatment. Since
1,1,2,2-tetrachloroethane has been demonstrated to inhibit CYP enzymes (Paolini et al., 1992;
Halpert, 1982), presumably through a suicide inhibition mechanism, it is also possible that
people coexposed to chemicals that are inactivated by CYP enzymes will be more susceptible to
those compounds.
In addition, studies of human GST-zeta polymorphic variants show different enzymatic
activities toward and inhibition by dichloroacetic acid that could affect the metabolism of
1,1,2,2-tetrachloroethane (Lantum et al., 2002; Blackburn et al., 2001, 2000; Tzeng et al., 2000).
Dichloroacetic acid may covalently bind to GST-zeta (Anderson et al., 1999), irreversibly
inhibiting one of two stereochemically different conjugates, thus inhibiting its own metabolism
and leading to an increase in unmetabolized dichloroacetic acid as the dose and duration of
exposure increases (U.S. EPA, 2003). GST zeta is a hepatic enzyme that also functions in the
pathway for tyrosine catabolism. Populations or single individuals may be more sensitive to
1,1,2,2-tetrachloroethane toxicity depending on which GST-zeta variant they possess.
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5. DOSE-RESPONSE ASSESSMENTS
5.1. ORAL REFERENCE DOSE (RfD)
5.1.1. Subchronic Oral RfD
5.1.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification
The data available on subchronic oral exposure to 1,1,2,2-tetrachloroethane are limited to
experimental studies in animals. Although a number of case reports provide information on
effects of intentional acute oral exposure to lethal oral doses of 1,1,2,2-tetrachloroethane (Mant,
1953; Lilliman, 1949; Forbes, 1943; Elliot, 1933; Hepple, 1927), no subchronic studies of oral
exposure to 1,1,2,2-tetrachloroethane in humans exist. A single, well-designed 14-week
subchronic study in rats and mice that tested multiple dose levels and examined an array of
endpoints and tissues in rats is available (NTP, 2004). Furthermore, a developmental toxicity
study in rats and mice exists (Gulati et al., 1991a, b). These studies in laboratory animals
provide evidence suggesting that the liver and the developing fetus may be targets of toxicity
following subchronic oral exposure to 1,1,2,2-tetrachloroethane.
NTP reported multiple effects on the livers of both male and female rats and mice
following subchronic oral exposure to 1,1,2,2-tetrachloroethane. Specifically, NTP (2004)
exposed F344 rats (10/sex/group) to 0, 20, 40, 80, 170, or 320 mg/kg-day (both males and
females) and B6C3Fi mice (10/sex/group) to 0, 100, 200, 370, 700, or 1,360 mg/kg-day (males)
and 0, 80, 160, 300, 600, or 1,400 mg/kg-day (females) in the diet for 14 weeks. A statistically
significant decrease in body weight gain (<10%) in both male and female rats at >80 mg/kg-day
was observed. Low dose effects observed in the liver included statistically significantly
increased relative liver weights in both male and female rats at>40 mg/kg-day. In addition,
hepatocellular vacuolization was observed at >20 mg/kg-day in male rats and >40 mg/kg-day in
female rats. The severity of vacuolization was reported to be minimal to mild. Serum enzyme
activity levels of both male and female rats were also affected. For example, increases in serum
ALT and SDH activity were observed at >80 mg/kg-day in male rats and >170 mg/kg-day in
female rats. In addition, increased cholesterol levels and ALP activity were observed in female
rats at >80 and 170 mg/kg-day, respectively. Additional histopathology observed in the liver
included a statistically significantly increased incidence of minimal to moderate hepatocellular
hypertrophy at>170 mg/kg-day in females and>200 mg/kg-day in males. Also, increased
incidence of necrosis and pigmentation were observed at>80 mg/kg-day and hepatocellular
mitotic alterations and foci of cellular alterations were observed at>80 and >170 mg/kg-day,
respectively, in male rats. In females, increased incidence of hepatocellular hypertrophy was
observed at >80 mg/kg-day, and necrosis, pigmentation, and foci of cellular alterations were
reported at > 170 mg/kg-day. Bile duct hyperplasia, increased bile acids, spleen pigmentation,
and spleen atrophy were also observed in both male and female rats at the two highest doses.
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Evidence of liver effects was also observed in mice by NTP (2004). A statistically
significant increase in relative liver weights was observed in both male and female mice at
>200 and 80 mg/kg-day, respectively. Increases in serum ALT and ALP activity, bile acid
levels, and hepatic 5'-nucleotidase activity (males only) were observed in males and females at
>370 and 160 mg/kg-day, respectively. The study authors also reported an increase in SDH
activity at >200 and 80 mg/kg-day in male and female mice, respectively. Serum cholesterol
levels were statistically significantly increased in female mice at>160 mg/kg-day. The
incidence of hepatocellular necrosis was statistically significantly increased in male mice at
>370 mg/kg-day and in female mice at >700 mg/kg-day. Hepatocellular hypertrophy was also
reported in both genders at > 160-200 mg/kg-day. A statistically significant increase in incidence
of liver pigmentation and bile duct hyperplasia occurred at>300 mg/kg-day in females and
>370 mg/kg-day in males.
In addition to effects on the liver, NTP (2004) also observed effects associated with
reproduction in adult rats and mice following subchronic exposure to 1,1,2,2-tetrachloroethane at
dose levels as low as 40 mg/kg-day. In male rats, sperm motility was decreased at>40 mg/kg-
day, and higher doses resulted in decreased epididymis weight and increased atrophy of the
preputial and prostate gland, seminal vesicle, and testicular germinal epithelium. In female rats,
minimal to mild uterine atrophy was increased at > 170 mg/kg-day and clitoral gland atrophy and
ovarian interstitial cell cytoplasmic alterations were increased at 320 mg/kg-day. Female F344
rats in the 170 mg/kg-day group also spent more time in diestrus compared to controls. Male
mice had increased incidences of preputial gland atrophy at > 100 mg/kg-day. Less sensitive
effects included decreases in absolute testes weight (>700 mg/kg-day), absolute epididymis, and
cauda epididymis weights (1,360 mg/kg-day), and a decrease in epididymal spermatozoal
motility (1,360 mg/kg-day). The only noted reproductive toxicity parameter in female mice
affected was a significant increase in the length of the estrous cycle at a dose of 1,400 mg/kg-
day.
A developmental toxicity study by Gulati et al. (1991a) demonstrated that the developing
fetus may be sensitive to 1,1,2,2-tetrachloroethane exposure. Gulati et al. (1991a) exposed
pregnant CD Sprague-Dawley rats to 0, 34, 98, 180, 278, or 330 mg/kg-day 1,1,2,2-tetrachloro-
ethane from GDs 4 through 20. Small but statistically significant decreases were observed in
maternal body weight and average fetal weight at>98 mg/kg-day. No other maternal or fetal
effects were reported by the study authors. In a second study, Gulati et al. (1991b) exposed
pregnant Swiss CD-I mice to 0, 987, 2,120, 2,216, or 4,575 mg/kg-day 1,1,2,2-tetrachloroethane
from GDs 4 through 17. All animals (9/9) in the high-dose group died prior to the end of the
study, precluding calculation of the average dose in this exposure group. Maternal body weights
were statistically significantly decreased compared to controls at>2,120 mg/kg-day beginning on
study day 9. Gross hepatic effects such as pale or grey and/or enlarged livers and a prominent
lobulated pattern were also reported in dams from all groups except at the low dose. Complete
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litter resorption occurred in 1/11, 0/9, 2/8, 1/1, and 1/2 dams in the 0, 987, 2,120, 2,216, and
4,575 mg/kg-day groups, respectively. No other developmental effects were reported. Gulati et
al. (1991a, b) suggested that the developing fetus may be a target of 1,1,2,2-tetrachloroethane-
induced toxicity. However, these developmental studies were conducted at doses higher than the
subchronic NTP (2004) study, which demonstrated liver effects at lower doses. Therefore,
Gulati et al. (1991a, b) was not selected as the principal study, and the observed reproductive
effects were not selected as the critical effect following subchronic exposure to 1,1,2,2-tetra-
chloroethane. Nevertheless, potential PODs based on the observed developmental effects from
Gulati et al. (1991a) were provided for comparison (see Section 5.1.2 and Appendix B).
In consideration of the available studies reporting effects of subchronic oral exposure to
1,1,2,2-tetrachloroethane in animals, NTP (2004) was chosen as the principal study for the
derivation of the subchronic RfD. This study was conducted in both genders of two species,
used five dose levels and a concurrent control group, measured a wide-range of endpoints and
tissues, and provides data that were transparently and completely reported. NTP (2004)
identified the liver as the most sensitive target organ of 1,1,2,2-tetrachloroethane-induced
toxicity. Specifically, NTP (2004) identified effects on the liver including increased liver weight
and increased incidence of hepatocellular vacuolization at low dose levels. Other liver effects
observed in rats and mice at higher doses included increased liver weight, increased ALT, ALP,
and SDH serum activity levels, increased bile acid levels, and an increased incidence of
hepatocellular vacuolization and necrosis.
Based on the available data from the NTP (2004) study, the liver appears to be the most
sensitive target organ for 1,1,2,2-tetrachloroethane-induced toxicity. Thus, the observed effects
in the liver were considered in the selection of the critical effect for the derivation of the
subchronic RfD. Specifically, liver effects including increased liver weight, increased ALT,
ALP, and SDH serum levels, increased bile acid levels, and an increased incidence of
hepatocellular vacuolization were modeled and considered for the determination of the critical
effect and POD (Section 5.1.1.2 and Appendix B). EPA selected increased liver weight as the
critical effect because this effect may represent a sensitive endpoint that occurs early in the
process leading to hepatocellular necrosis associated with subchronic oral exposure to
1,1,2,2-tetrachloroethane; however, chemical-specific data demonstrating the relationship
between increased liver weight and hepatocellular necrosis are not available. The increase in
relative liver weight, as opposed to an increase in absolute liver weight, was selected because
the calculation of relative liver weight takes into account the substantive, dose-dependent
decreases in body weight that were observed in both genders of rats. Rats were selected as the
representative species because they appeared to be more sensitive than mice to the hepatotoxic
effects of 1,1,2,2-tetrachloroethane.
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5.1.1.2. Methods of Analysis—Including Models (PBPK, BMD, etc.)
BMD modeling was conducted using the EPA's benchmark dose software (BMDS,
version 2.1.1.) to analyze the hepatotoxic effects associated with subchronic exposure to
1,1,2,2-tetrachloroethane (see Appendix B for modeling details). The software was used to
calculate potential PODs for deriving the subchronic RfD by estimating the effective dose at a
specified level of response (BMDX) and its 95% lower bound (BMDLX). For all continuous
endpoints, a benchmark response (BMR) of 1SD of the control mean was considered appropriate
for derivation of the RfD under the assumption that it represents a minimally biologically
significant response level. A BMR of 1 standard deviation (SD) of the control mean was also
included for comparative purposes. For the dichotomous data (i.e., the incidence of
hepatocellular cytoplasmic vacuolization), a BMR of 10% extra risk was considered appropriate
for derivation of the RfD under the assumption that it represents a minimally biologically
significant response level. The effects modeled include liver weight changes, serum ALT and
SDH, bile acids, hepatocellular cytoplasmic vacuolization, and rat fetal body weights. Table 5-1
summarizes the BMD modeling results for the selected toxicological endpoints.
Table 5-1. Summary of BMD model results for rats exposed to l,l?2,2-tetra-
chloroethane
Endpoint
Model
BMR
BMD (mg/kg-d)
BMDL (mg/kg-d)
Males
Cytoplasmic vacuolization
Relative liver weight
Absolute live weight
ALT
SDH
Bile acids
Polynomial
None
Polynomial
Polynomial
None
Power
10% extra risk
NA
1 SD
1 SD
NA
1 SD
1.7
NA
30
41
NA
72
1.1
NA
23
26
NA
57
Females
Cytoplasmic vacuolization
Relative liver weight
Absolute liver weight
ALT
SDH
Bile acids
Weibull
Polynomial
Polynomial
Hill
Power
Polynomial
10% extra risk
1 SD
1 SD
1 SD
1 SD
1 SD
31
22
36
82
157
188
19
15
26
69
113
170
Developmental
Rat fetal weight
Linear
1 SD
83
60
Changes in hepatocellular cytoplasmic vacuolization, ALT, SDH, ALP, and bile acid
serum levels from NTP (2004), as well as mean rat fetal weights from Gulati et al. (1991a), were
modeled for comparison in identifying a POD. For serum ALT levels in female rats, a BMD of
82 mg/kg-day and a BMDL of 69 mg/kg-day was derived from the Hill model. For serum SDH
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in female rats, a BMD of 157 mg/kg-day and a BMDL of 113 mg/kg-day was derived from the
power model. The serum ALP data were not amenable to BMD modeling; a LOAEL of 160
mg/kg-day was identified. For bile acid levels in female rats, a BMD of 188 mg/kg-day and a
BMDL of 170 mg/kg-day were derived from the polynomial model. BMD modeling derived a
BMD of 83 mg/kg-day and a BMDL of 60 mg/kg-day from a linear model for decreased rat fetal
weight.
A BMD of 31 mg/kg-day and BMDL of 19 mg/kg-day were derived from the multistage
model for the increased incidence of hepatocellular cytoplasmic vacuolization in female rats.
The POD for the increased incidence of hepatocellular vacuolization is approximately an order
of magnitude lower than the POD for increased relative liver weight, and would result in a lower
RfD than that derived for increased relative liver weight (See Sections 5.1.1.2 and 5.1.3 for more
information). However, the biological significance of this effect following 1,1,2,2-tetrachloro-
ethane exposure is unclear based on the following considerations. Vacuoles are defined as
cavities bound by a single membrane that serve several functions, usually providing storage areas
for fat, glycogen, secretion precursors, liquid, or debris (Osol, 1972). Vacuolization is defined as
the process of accumulating vacuoles in a cell or the state of accumulated vacuoles (Grasso,
2002). This process can be classified as either a normal physiological response or may reflect an
early toxicological process. As a normal physiological response, vacuolization is associated with
the sequestration of materials and fluids taken up by cells, and also with secretion and digestion
of cellular products (Henics and Wheatley, 1999). In addition, Robbins et al. (1976)
characterized vacuolization (i.e., intracellular autophagy) as a normal cellular functional,
homeostatic, and adaptive response.
Vacuolization is not only a normal physiological response. Vacuolization has been
identified as one of four principal types of chemical-induced injury (the other three being cloudy
swelling, hydropic change, and fatty change) (Grasso, 2002). It is one of the most common
responses of the liver following a chemical exposure, typically in the accumulation of fat in
parenchymal cells, most often in the periportal zone (Plaa and Hewitt, 1998). The ability to
detect subtle ultrastructural defects, such as vacuolization, early in the course of toxicity often
permits identification of the initial site of the lesion and thus can provide clues to possible
biochemical mechanisms involved in the pathogenesis of liver injury (Hayes, 2001).
The hepatocellular vacuolization reported by NTP (2004) was not observed consistently
across species (i.e., reported only in male and female rats); whereas the other observed liver
effects were reported in both sexes of both species. In addition, NTP (2004) did not characterize
the vacuole content following exposure to 1,1,2,2-tetrachloroethane. The study authors indicated
that the severity of the hepatocellular vacuolization was minimal to mild and was concentration
independent, but NTP (2004) did not report the localization of the vacuolization in the liver. The
observed vacuolization in the liver at low doses appeared to diminish as dose increased.
Specifically, hepatocellular vacuolization increased in a dose dependant manner from 20 to
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80 mg/kg-day in male rats. At 80 mg/kg-day, 100% of male rats were affected, and at doses of
>80 mg/kg-day, the incidence of vacuolization began to decrease. Concurrent with this decrease
in incidence of vacuolization, an increased incidence of hepatocellular hypertrophy, necrosis, and
pigmentation were observed. In female rats, the incidence of vacuolization was 100% at 40 and
80 mg/kg-day, followed by a diminished response at the two highest doses. Necrosis and
pigmentation were observed in the females at the two high doses. Thus, the qualitative and
quantitative biological relationship between the observed hepatocellular toxicity (i.e., hepato-
cellular necrosis) and the increased incidence of hepatocellular cytoplasmic vacuolization in
NTP (2004) is unknown.
The BMDiso of 22 mg/kg-day and BMDLiso of 15 mg/kg-day based on increased
relative liver weight in the female rat was selected as the POD for the subchronic RfD. The
observed changes in liver weights, serum liver enzyme levels, and hepatocellular necrosis
combine to support hepatotoxicity as the major toxic effect following 1,1,2,2-tetrachloroethane
exposure.
5.1.1.3. RfD Derivation—Including Application of Uncertainty Factors (UFs)
To derive the subchronic RfD, the BMDLiso of 15 mg/kg-day for increased relative
liver weight in female rats is divided by a total UF of 300. The UF of 300 comprises component
factors of 10 for interspecies extrapolation, 10 for interhuman variability, and 3 for database
deficiencies.
A default UF of 10 was selected to account for the interspecies variability in
extrapolating from laboratory animals (rats) to humans because information was not available to
quantitatively assess toxicokinetic or toxicodynamic differences between animals and humans
for 1,1,2,2-tetrachloroethane.
A default UF of 10 was selected to account for intraspecies variability (UFH) in
susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of 1,1,2,2-tetrachloroethane in humans. However, studies of human GST-zeta
polymorphic variants demonstrate different enzymatic activities toward and inhibition by
dichloroacetic acid that could affect the metabolism of 1,1,2,2-tetrachloroethane (Lantum et al.,
2002; Blackburn et al., 2001, 2000; Tzeng et al., 2000). Populations or single individuals may be
more sensitive to 1,1,2,2-tetrachloroethane toxicity depending on which GST-zeta variant they
possess. Animal toxicity studies did not show consistent sex-related differences.
An UF of 3 was selected to account for deficiencies in the database. The NTP (2004)
14-week study provides comprehensive evaluations of systemic toxicity and neurotoxicity in two
species. The NTP (2004) study provides information on effects on sperm, estrous cycle, and
male and female reproductive tissues in rats and mice, but the database lacks a two-generation
reproductive toxicity study. Available developmental toxicity studies provide information on
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embryo or fetotoxicity in orally exposed rats and mice (Gulati et al., 199 la, b), but the studies
did not include skeletal and visceral examinations.
An UF for LOAEL-to-NOAEL extrapolation was not used because the current approach
is to address this factor as one of the considerations in selecting a BMR for BMD modeling. In
this case, a BMR associated with a change of 1 SD from the control mean was selected under an
assumption that it represents a minimally biologically significant change.
The subchronic RfD for 1,1,2,2-tetrachloroethane is calculated as follows:
Subchronic RfD = BMDLiSD ^
15 mg/kg-day -3 00
= 0.05 mg/kg-day (or 5 x 10"2 mg/kg-day)
5.1.2. Chronic Oral RfD
5.1.2.1. Choice of Principal Study and Critical Effect — with Rationale and Justification
Information on the chronic oral toxicity of 1,1,2,2-tetrachloroethane is limited to a
78-week cancer bioassay in rats and mice that were exposed by gavage (NCI, 1978).
Interpretation of the rat study may be confounded by high incidences of endemic chronic murine
pneumonia, although it is unlikely that this contributed to effects observed in the liver. Based on
an increased incidence of hepatic fatty changes, the NOAEL and LOAEL for liver effects were
62 and 108 mg/kg-day, respectively. In the mouse study, a LOAEL of 142 mg/kg-day was
selected for chronic inflammation in the kidneys of males, and a NOAEL of 142 mg/kg-day and
a LOAEL of 284 mg/kg-day were selected for hydronephrosis and chronic inflammation in the
kidneys of females, respectively.
The 14-week dietary study in rats and mice (NTP, 2004) used to derive the subchronic
RfD was also considered for the derivation of the chronic RfD. The subchronic NTP (2004)
study appears to be a more sensitive assay than the chronic NCI (1978) bioassay. The NTP
(2004) study also uses lower dose levels and a wider dose range than the NCI (1978) study, and
thereby provides a better characterization of the dose-response curve in the low-dose region.
Additionally, dietary exposure is a more relevant route of exposure for the general population
exposed to 1,1,2,2-tetrachloroethane in the environment than is gavage exposure. For these
reasons, the NTP (2004) subchronic study was selected as the principal study.
EPA selected increased liver weight as the critical effect because this effect may
represent a potential sensitive endpoint that may occur early in the process leading to
hepatocellular necrosis associated with subchronic oral exposure to 1,1,2,2-tetrachloroethane.
The increase in relative liver weight, as opposed to an increase in absolute liver weight, was
selected because the calculation of relative liver weight takes into account the substantive, dose-
dependent decreases in body weight that were observed in both sexes of rats. Additional liver
effects observed included increased liver weight, increased ALT, ALP, and SDH serum levels,
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increased serum bile acid levels, and increased incidence of hepatocellular vacuolization and
necrosis.
5.1.2.2. Methods of Analysis—Including Models (PBPK, BMD, etc.)
The subchronic BMDLiso of 15 mg/kg-day based on the increased relative liver weight
in female rats was used as the POD for the chronic RfD. The observed increases in liver
weights, serum liver enzyme levels, and incidence of hepatocellular necrosis combine to support
hepatotoxicity as the critical effect of toxicity of 1,1,2,2-tetrachloroethane.
5.1.2.3. RfD Derivation—Including Application ofVFs
To derive the chronic RfD, the subchronic BMDLiso of 15 mg/kg-day, based on
increased relative liver weights in female rats, was divided by a UF of 1,000. The UF of 1,000
comprises component factors of 10 for interspecies extrapolation, 10 for interhuman variability,
3 for subchronic to chronic duration extrapolation, and 3 for database deficiencies, as explained
below.
A default UF of 10 was selected to account for the interspecies variability in
extrapolating from laboratory animals (rats) to humans because information was not available to
quantitatively assess toxicokinetic or toxicodynamic differences between animals and humans
for 1,1,2,2-tetrachloroethane.
A default UF of 10 was selected to account for interindividual variability (UFH) in
susceptibility in the absence of quantitative information to assess the toxicokinetics and
toxicodynamics of 1,1,2,2-tetrachloroethane in humans. However, studies of human GST-zeta
polymorphic variants demonstrate different enzymatic activities toward and inhibition by
dichloroacetic acid that could affect the metabolism of 1,1,2,2-tetrachloroethane (Lantum et al.,
2002; Blackburn et al., 2001, 2000; Tzeng et al., 2000). Populations or single individuals may be
more sensitive to 1,1,2,2-tetrachloroethane toxicity depending on which GST-zeta variant they
possess. Animal toxicity studies which evaluated both sexes in the same study did not show
consistent sex-related differences. Developmental toxicity studies in animals are limited in
scope, but have not indicated effects on the offspring at levels that did not also cause maternal
effects.
An UF of 3 was selected to account for extrapolation from a subchronic exposure
duration study to a chronic RfD. The study selected as the principal study was a 14-week study
by NTP (2004), a study duration that is minimally past the standard subchronic (90-day) study
and falls well short of a standard lifetime study. In addition, some data are available to inform
the nature and extent of effects that would be observed with a longer duration of exposure to
1,1,2,2-tetrachloroethane. Specifically, the available chronic cancer bioassay data (NCI, 1978)
suggest that liver damage observed in F344 rats following subchronic exposure to 1,1,2,2-tetra-
chloroethane (NTP, 2004) (e.g., increased liver weight and incidence of necrosis, and altered
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serum enzyme and bile levels) may not progress to more severe effects following chronic
exposures. The chronic cancer bioassay was conducted in Osborne-Mendel rats and did not
measure liver enzyme levels. However, NCI (1978) observed minimal alterations in liver
pathology including inflammation, fatty metamorphosis, focal cellular change, and angiectasis in
rats, and organized thrombus and nodular hyperplasia in mice. NCI (1978) reported that the
study authors performed complete histological analysis on the liver, but specific endpoints
assessed were not included. The available database does not abrogate all concern associated
with using a subchronic study as the basis of the RfD. For these reasons, a threefold UF was
used to account for the extrapolation from subchronic to chronic exposure duration for the
derivation of the chronic RfD.
An UF of 3 was selected to account for deficiencies in the database. The NTP (2004)
14-week study provides comprehensive evaluations of systemic toxicity and neurotoxicity in
both rats and mice. However, the database is limited by the lack of a two-generation
reproductive toxicity study. The NTP (2004) study provides information on effects on sperm,
estrous cycle, and male and female reproductive tissues in rats and mice, but the database lacks a
two-generation reproductive toxicity study. Available developmental toxicity studies provide
information on embryo or fetotoxicity in orally exposed rats and mice (Gulati et al., 1991a, b),
but the studies did not include skeletal and visceral examinations.
An UF for LOAEL-to-NOAEL extrapolation was not used because the current approach
is to address this factor as one of the considerations in selecting a BMR for BMD modeling. In
this case, a BMR associated with a change of 1 SD from the control mean was selected under an
assumption that it represents a minimally biologically significant change.
The chronic RfD for 1,1,2,2-tetrachloroethane is calculated as follows:
Chronic RfD = BMDLiso - UF
15 mg/kg-day-1,000
0.02 mg/kg-day (or 2 x 10"2 mg/kg-day)
5.1.3. RfD Comparison Information
Figure 5-1 is an exposure-response array that presents NOAELs, LOAELs, and the dose
range tested corresponding to selected health effects. The effects observed in the subchronic and
chronic studies were considered candidates for the derivation of the sample subchronic and
chronic RfDs.
In addition to the increase in relative liver weight and the increased incidence of
hepatocellular cytoplasmic vacuolization, changes in absolute liver weight and serum levels of
ALT and SDH, bile acid levels, and serum cholesterol levels were considered for comparison.
Mean rat fetal weights observed following subchronic or chronic exposure to 1,1,2,2-tetrachloro-
ethane were also considered for comparison. Table 5-2 provides a tabular summary of sample
85
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PODs and resulting subchronic sample RfDs for these endpoints in female rats. Additionally,
Figure 5-2 provides a graphical representation of this information. This figure should be
interpreted with caution since the PODs across studies are not necessarily comparable, nor is the
confidence the same in the data sets from which the PODs were derived. Figure 5-3 provides a
graphical representation of the derivation of sample chronic RfDs for sample PODs from the
subchronic data.
86
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the range of
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a given study.
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-------
Table 5-2. Potential PODs with applied UFs and resulting subchronic RfDs
Effect
Hepatocellular cytoplasmic vacuolization
Relative liver weight
Absolute liver weight
ALT
SDH
Bile acids
Fetal body weight
POD
(mg/kg-d)
l.lb
15C
23C
26C
113C
57C
60d
Gender and
species
Male Rat
Female Rat
Male Rat
Male Rat
Female Rat
Male Rat
Rat
UFsa
A
10
10
10
10
10
10
10
H
10
10
10
10
10
10
10
L
-
-
-
-
-
-
-
s
-
-
-
-
-
-
-
D
3
3
3
3
3
3
3
Total
300
300
300
300
300
300
300
Subchronic
RfD
4 x 10"3
5 x 10'2
8 x 10"2
9 x 10'2
0.38
0.20
0.20
aUFs: A = animal to human (interspecies); H = interindividual (intraspecies); L = LOAEL to NOAEL;
S = subchronic-to-chronic duration; D = database deficiency.
bPOD based on BMDL determined through BMD modeling of a 10% response; source: NTP (2004).
CPOD based on BMDL determined through BMD modeling of a 1 SD response; source: NTP (2004).
dPOD based on BMDL determined through BMD modeling of a 5% response; source: Gulatietal. (1991a).
-------
1000
100
10
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0)
0.1
0.01
1001
/ \
/ \
POD 4
UFA
UFD
RfD
hepatocellular relative liver weight- absolute liver weight increased ALT -$ increased SDH-? increased bile acids - decreased fetal body
cytoplasrric ? rats (NTP, 2004) - $ rats (NTP, 2004) rats (NTR 2004) rats (NTP, 2004) $ rats (NTP, 2004) weight-rats (Gulati
vacuolization - $ rats et al, 1991 a)
(NTP, 2004)
Figure 5-2. PODs for selected endpoints (with critical effect circled) from Table 5-2 with corresponding applied
UFs and derived sample subchronic oral reference values (RfVs).
89
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100
10
ti
TJ
6)
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0.1
0.01
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POD
UFA
UFH
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RfD
hepatocellular relative liver weight- absolute liver weight increased ALT -$ increased SDH-? increased bile acids-decreased fetal body
cytoplasmic ? rats (NTP, 2004) - $ rats (NTP, 2004) rats (NTP, 2004) rats (NTP, 2004) $ rats (NTP, 2004) weight-rats (Gulati
vacuolization - $ rats etal, 1991 a)
(NTP, 2004)
Figure 5-3. PODs for selected endpoints (with critical effect circled) from Table 5-2 with corresponding applied
UFs and derived sample chronic oral reference values (RfVs).
90
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5.1.4. Previous RfD Assessment
An oral assessment for 1,1,2,2-tetrachloroethane was not previously available on IRIS.
5.2. INHALATION REFERENCE CONCENTRATION (RfC)
5.2.1. Choice of Principal Study and Critical Effect—with Rationale and Justification
Information on the inhalation toxicity of 1,1,2,2-tetrachloroethane is limited. In the
Truffert et al. (1977) study, rats were exposed to a presumed concentration of 560 ppm
(3,909 mg/m3) for a TWA duration of 5.1 hours/day, 5 days/week for 15 weeks. Findings
included transient histological alterations in the liver including granular appearance and
cytoplasmic vacuolation, which were observed after 9 exposures and were no longer evident
after 39 exposures. Because of the uncertainty regarding the actual exposure concentration for
the single dose, and a lack of incidence and severity data, this report cannot be used to identify a
NOAEL or LOAEL or for possible derivation of an RfC.
Horiuchi et al. (1962) observed fatty degeneration of the liver and splenic congestion in a
single monkey exposed to a TWA of 1,974 ppm (15,560 mg/m3) 1,1,2,2-tetrachloroethane
2 hours/day, 6 days/week for 9 months. The monkey was weak after approximately seven
exposures and had diarrhea and anorexia between the 12th and 15th exposures. Beginning at the
15th exposure, the monkey was "almost completely unconscious falling upon his side" for 20-
60 minutes after each exposure. Also, hematological parameters demonstrated sporadic changes
in hematocrit and RBC and WBC counts, but the significance of these findings cannot be
determined. This study cannot be utilized to identify a NOAEL or LOAEL due to the use of a
single test animal with no control group.
Mellon Institute of Industrial Research (1947) observed an increased incidence of lung
lesions and an increase in kidney weight in rats following a 6-month exposure to 200 ppm
1,1,2,2-tetrachloroethane, but these results were not evaluated because the control animals
experienced a high degree of pathological effects in the kidney, liver, and lung, and because of
the presence of an endemic lung infection in both controls and treated groups. Mellon Institute
of Industrial Research (1947) also observed increased serum phosphatase levels and blood urea
nitrogen levels in a dog exposed to 200 ppm 1,1,2,2-tetrachloroethane, compared to control
values, along with cloudy swelling of the liver and the convoluted tubules of the kidney, and
light congestion of the lungs. However, identification of a LOAEL or NOAEL is precluded by
poor study reporting, high mortality and lung infection in the rats, and the use of a single treated
animal in the dog study.
Kulinskaya and Verlinskaya (1972) observed inconsistent changes in acetylcholine levels
in Chinchilla rabbits exposed to 10 mg/m3 (1.5 ppm) 1,1,2,2-tetrachloroethane 3 hours/day,
6 days/week for 7-8.5 months. A NOAEL or LOAEL was not identified because the changes in
acetylcholine were not consistent across time and incompletely quantified, and the biological
significance of the change is unclear.
91
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Shmuter (1977) observed increases in antibody levels in Chinchilla rabbits at 2 mg/m3
1,1,2,2-tetrachloroethane and decreases in antibody levels at 100 mg/m3. Exposure to
100 mg/m3 1,1,2,2-tetrachloroethane also resulted in a decrease in the relative content of
antibodies in the y-globulin fraction and an increase in the T and P fractions. This is a poorly
reported study that provides inadequate data, including reporting limitations, toxicological
uncertainty in the endpoints, and inconsistent patterns of response, which preclude the
identification of a NOAEL or LOAEL.
Effects following the chronic inhalation toxicity of 1,1,2,2-tetrachloroethane included
hematological alterations and increased liver fat content in rats exposed to 1.9 ppm (13.3 mg/m3)
4 hours/day for 265 days (Schmidt et al., 1972). Statistically significant changes included
increased leukocyte (89%) and Pi-globulin (12%) levels compared to controls after 110 days,
and an increased percentage of segmented nucleated neutrophils (36%), decreased percentage of
lymphocytes (17%), and increased liver total fat content (34%) after 265 days. A statistically
significant decrease in y-globulin levels (32%) at 60 days postexposure and a decrease in adrenal
ascorbic acid content (a measure of pituitary ACTH activity) were observed at all three time
periods (64, 21, and 13%, respectively). This study is insufficient for identification of a NOAEL
or LOAEL for systemic toxicity because most of the observed effects occurred at a single dose or
time point, or there was a reversal of the effect at the next dose or time point. A reproductive
assessment in the Schmidt et al. (1972) study was sufficient for identification of a NOAEL for
the single dose tested, 1.9 ppm (13.3 mg/m3), for reproductive effects in male rats, including
percentage of mated females having offspring, littering interval, time to 50% littered, total
number of pups, pups per litter, average birth weight, postnatal survival on days 1, 2, 7, 14, 21,
and 84, sex ratio, and average body weight on postnatal day 84. However, macroscopic
malformations or significant group differences in the other indices were not observed at
13.3 mg/m3. The lack of information on the reproductive toxicity precludes utilizing the selected
NOAEL in the derivation of the RfC.
In addition, effects of chronic exposure to 1,1,2,2-tetrachloroethane included alterations
in serum acetylcholinesterase activity in rabbits exposed to 1.5 ppm (10 mg/m3) 1,1,2,2-tetra-
chloroethane 3 hours/day, 6 days/week for 7-8.5 months (Kulinskaya and Verlinskaya, 1972)
and immunological alterations in rabbits exposed to 0.3-14.6 ppm (2-100 mg/m3) 3 hours/day,
6 days/week, for 8-10 months (Shmuter, 1977). These studies are inadequate for identification
of NOAELs or LOAELs for systemic toxicity due to inadequate study reporting.
The inhalation toxicity database lacks a well-conducted study that demonstrates a dose-
related toxicological effect following subchronic and/or chronic exposure to 1,1,2,2-tetrachloro-
ethane. Therefore, an inhalation RfC was not derived.
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5.2.2. Methods of Analysis—Including Models (PBPK, BMD, etc.)
A route-to-route extrapolation using the computational technique of Chiu and White
(2006), as described in Section 3.5, was considered. However, U.S. EPA (1994b) recommends
not conducting a route-to-route extrapolation from oral data when a first-pass effect by the liver
or respiratory tract is expected, or a potential for a portal-of-entry effect in the respiratory tract is
indicated following an analysis of the available short-term inhalation, dermal irritation, and in
vitro studies, or after evaluation of the physical/chemical properties. In the case of 1,1,2,2-tetra-
chloroethane, a first-pass effect by the liver is expected. In addition, the presence of tissue-
bound metabolites in the epithelial linings in the upper respiratory tract may demonstrate a first-
pass effect by the respiratory tract (Eriksson and Brittebo, 1991). Lehmann et al. (1936)
observed irritation of the mucous membranes of two humans following inhalation of 146 ppm
(1,003 mg/m3) for 30 minutes or 336 ppm (2,308 mg/m3) for 10 minutes, indicating the potential
for portal-of-entry effects in the respiratory system.
5.2.3. Previous RfC Assessment
An inhalation assessment for 1,1,2,2-tetrachloroethane was not previously available on
IRIS.
5.3. UNCERTAINTIES IN THE ORAL REFERENCE DOSE AND INHALATION
REFERENCE CONCENTRATION
The following discussion identifies some uncertainties associated with the RfD for
1,1,2,2-tetrachloroethane. As presented earlier (Sections 5.1.2 and 5.1.3; 5.2.2 and 5.2.3), EPA
standard practices and RfC and RfD guidance (U.S. EPA, 1994b) were followed in applying a
UF approach to a POD, a BMDLiso for the subchronic and chronic RfDs. Factors accounting
for uncertainties associated with a number of steps in the analyses were adopted to account for
extrapolating from an animal bioassay to human exposure, a diverse human population of
varying susceptibilities, and database deficiencies. These extrapolations are carried out with
standard approaches given the lack of extensive experimental and human data on 1,1,2,2-tetra-
chloroethane to inform individual steps.
An adequate range of animal toxicology data is available for the hazard assessment of
1,1,2,2-tetrachloroethane, as described in Section 4. Included in these studies are short-term and
long-term bioassays and a developmental toxicity bioassay in rats and mice, as well as numerous
supporting genotoxicity and metabolism studies. Toxicity associated with oral exposure to
1,1,2,2-tetrachloroethane is observed in the liver, kidney, and developing organism, including
decreased fetal body weight and increased number of litter resorptions.
Consideration of the available dose-response data to determine an estimate of oral
exposure that is likely to be without an appreciable risk of adverse health effects over a lifetime
led to the selection of the 14-week oral dietary study in rats (NTP, 2004) and increased relative
93
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liver weight in females as the principal study and critical effect, respectively, for deriving the
subchronic and chronic RfDs for 1,1,2,2-tetrachloroethane. The NTP (2004) data demonstrate
hepatocellular damage including increased liver weight, increased serum liver enzyme levels,
and increased incidence of hepatic necrosis. Increased liver weight was chosen as the critical
effect because it may represent a sensitive indicator of 1,1,2,2,-tetrachloroethane-induced
hepatoxicity and occurs at a dose lower than the observed overt liver necrosis. However,
chemical-specific data demonstrating a relationship between increased liver weight and
hepatocellular necrosis is not available. The increase in relative liver weight was selected as the
basis for the selection of the POD because this analysis takes into account the substantive, dose-
dependent decreases in body weight that were observed in both sexes of rats. The dose-response
relationships between oral exposure to 1,1,2,2-tetrachloroethane and fetal body weight in rats
and mice are also suitable for deriving an RfD, but are associated with BMDLs that are less
sensitive than the selected critical effect and corresponding BMDL.
For comparison purposes, Figure 5-2 presents potential PODs, applied UFs, and derived
potential RfDs for the additional endpoints that were modeled using the EPA's BMDS, version
2.1.1. The additional endpoints included increased absolute liver weight, changes in serum ALT
and SDH, increased bile acids, and increased incidence of hepatocellular necrosis, all of which
support the liver as the target of 1,1,2,2-tetrachloroethane-induced toxicity following oral
exposure. A decrease in rat fetal weight was also modeled. The change in serum ALP was
modeled, but a model with adequate fit was not available.
The selection of the BMD model for the quantitation of the RfD does not lead to
significant uncertainty in estimating the POD, since benchmark effect levels were within the
range of experimental data. However, the selected model, the polynomial model, does not
represent all possible models one might fit, and other models could be selected to yield more
extreme results, both higher and lower than those included in this assessment.
Extrapolating from animals to humans embodies further issues and uncertainties. An
effect and its magnitude associated with the concentration at the POD in rodents are extrapolated
to human response. Pharmacokinetic models are useful in examining species differences in
pharmacokinetic processing; however, dosimetric adjustment using pharmacokinetic modeling
was not possible for the toxicity observed following oral and inhalation exposure to 1,1,2,2-tetra-
chloroethane. Additional interspecies uncertainty may arise from the rate of metabolism across
species, as it has been demonstrated that mice have greater metabolic capacity following
exposure to tetrachloroethylene than rats and humans (Reitz et al., 1996). Reitz et al. (1996)
demonstrated that mice possessed a greater relative ability to metabolize tetrachloroethylene than
rats and humans, and, although data are not available, a similar situation may exist for
1,1,2,2-tetrachloroethane.
Heterogeneity among humans is another uncertainty associated with extrapolating from
animals to humans. Uncertainty related to human variation needs to be considered; also,
94
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uncertainties in extrapolating from a subpopulation, say of one sex or a narrow range of life
stages typical of occupational epidemiologic studies, to a larger, more diverse population need to
be addressed. In the absence of 1,1,2,2-tetrachloroethane-specific data on human variation, a
factor of 10 was used to account for uncertainty associated with human variation in the
derivation of the RfD. Human variation may be larger or smaller; however, 1,1,2,2-tetrachloro-
ethane-specific data to examine the potential magnitude of over- or underestimation are
unavailable.
Extrapolating from subchronic PODs to derive chronic reference values (RfVs) is also an
uncertainty encountered in this assessment. A threefold UF was selected to account for
extrapolation from a subchronic exposure duration study to a chronic RfD. Based on the
available data for 1,1,2,2-tetrachloroethane, the toxicity observed in the liver does not appear to
increase over time. The use of data from a subchronic study to derive a chronic RfD becomes a
concern when the damage, in this case hepatoxicity, has the potential to accumulate; however, if
the progression of the effect is not apparent, a reduced UF may be considered (U.S. EPA,
1994b). Specifically, liver damage observed in F344 rats following subchronic exposure to
1,1,2,2-tetrachloroethane (NTP, 2004) (e.g., increased incidence of necrosis or altered serum
enzyme and bile levels) did not progress to more severe effects such as cirrhosis or major liver
disease following chronic exposures (NCI, 1978). NCI (1978) observed minimal alterations in
liver pathology including inflammation, fatty metamorphosis, focal cellular change, and
angiectasis in rats, and organized thrombus and nodular hyperplasia in mice. Therefore, the
available database does not abrogate all concern associated with using a subchronic study as the
basis of the RfD, but supports the utilization of a database UF of 3.
Data gaps have been identified that are associated with uncertainties in database
deficiencies specific to the developmental and reproductive toxicity of 1,1,2,2-tetrachloroethane
following oral exposure. The developing fetus may be a target of toxicity, and the absence of a
study specifically evaluating the full range of developmental toxicity endpoints represents an
area of uncertainty or gap in the database. The database of inhalation studies is of particular
concern due to the paucity of studies, especially subchronic and chronic studies, a multi-
generational reproductive study, and a developmental toxicity study.
5.4. CANCER ASSESSMENT
As discussed in Section 4.7, under U.S. EPA's Guidelines for Carcinogen Risk
Assessment (U.S. EPA, 2005a), 1,1,2,2-tetrachloroethane is "likely to be carcinogenic to
humans" based on data from an oral cancer bioassay in male and female Osborne-Mendel rats
and B6C3Fi mice (NCI, 1978) demonstrating an increase in the incidence of hepatocellular
carcinomas in both sexes of mice. In this study, the incidence of hepatocellular carcinomas was
statistically significantly increased in both sexes of B6C3Fi mice at 142 (13/50 males;
30/48 females) and 284 mg/kg-day (44/49 males; 43/47 females), with incidences in the male
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and female controls of 3/36 and 1/40, respectively. NCI (1978) also demonstrated a decrease in
the time to tumor in both sexes of mice. Male rats exhibited an increased incidence in
hepatocellular carcinomas, characterized as rare tumors, but the increased incidence was not
statistically significantly different from controls. NCI (1978) has characterized the carcinogenic
results in male rats as "equivocal."
The epidemiological human data available are inadequate for evaluation for cancer risk
(IARC, 1999). There are a limited number of positive results from genotoxicity studies which
suggest that 1,1,2,2-tetrachloroethane treatment in animals can result in UDS (Miyagawa et al.,
1995), chromosomal aberrations (McGregor, 1980), SCE (NTP, 2004; Colacci et al., 1992), and
micronucleus formation (NTP, 2004). The ability of 1,1,2,2,-tetrachloroethane to alkylate
enzymatically purified hepatic DNA was observed following a single oral dose of 150 mg/kg of
1,1,2,2-tetrachloroethane in B6C3Fi mice (Dow Chemical Company, 1988). 1,1,2,2-Tetra-
chloroethane may have tumor initiating and promoting activity in mammalian cells (Colacci et
al., 1996, 1992; Milman et al., 1988; Story et al., 1986).
5.4.1. Choice of Study/Data—with Rationale and Justification
The only carcinogenicity bioassay for 1,1,2,2-tetrachloroethane is a chronic gavage study
in Osborne-Mendel rats and B6C3Fi mice performed by NCI (1978). This study was conducted
in both sexes in two species with an adequate number of animals per dose group, with
examination of appropriate toxicological endpoints in both sexes of rats and mice. Selection of
doses was aided by range-finding toxicity tests. The rat study did not identify statistically
significant increases in tumor incidences in males or females. Three rare liver tumors in high-
dose male rats were noted. Limitations in the study included increased mortality in male and
female mice, the variable doses given to the mice over the course of the 78-week exposure
period, and the exposure duration of the study (78 weeks) was less than the standard 104 week
chronic exposure duration. In the high-dose male mice, acute toxic tubular nephrosis was
characterized as the cause of death in the mice that died prior to study termination, although
hepatocellular carcinomas were observed in most of these mice.
The mouse study identified statistically significant, dose-related increases in the
incidences of hepatocellular carcinomas in both sexes. Based on these increases in
hepatocellular carcinomas, NCI (1978) concluded that orally administered 1,1,2,2-tetrachloro-
ethane is a liver carcinogen in male and female B6C3Fi mice. NCI (1978) stated that there was
no evidence for carcinogenicity of 1,1,2,2-tetrachloroethane in Osborne-Mendel rats (NCI,
1978). The tumor data in mice from the NCI study was used for dose-response analysis for oral
exposure.
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5.4.2. Dose-response Data
Data on the incidences of hepatocellular carcinomas in male and female mice from the
NCI (1978) study were used for cancer dose-response assessment. These data are shown in
Table 5-3. The control data were pooled from vehicle control groups. The cancer bioassay for
1,1,2,2-tetrachloroethane demonstrated evidence of increased incidence of tumors in both sexes
of one species.
Table 5-3. Incidences of hepatocellular carcinomas in B6C3Fi mice used for
dose-response assessment of l,l?2,2-tetrachloroethane
Sex
Male
Female
Dose (mg/kg-d)a
0
3/36b
l/40b
142
13/50
30/48
284
44/49
43/47
aTWA dose administered by gavage 5 d/wk for 78 wks.
bPooled vehicle (corn oil) control groups from this and another concurrent bioassay. Pooling based on identical
housing and care, similar spontaneous tumor rates, placed on test at about the same time, and examined by the same
pathologists.
Source: NCI (1978).
5.4.3. Dose Adjustments and Extrapolation Method(s)
Conversion of the doses in the NCI (1978) mouse study to human equivalent doses
(HEDs) to be used for dose-response modeling was accomplished in three steps. The mice were
treated with 1,1,2,2-tetrachloroethane by gavage 5 days/week for 78 weeks and then observed
untreated for 12 weeks for a total study duration of 90 weeks. Because the reported TWA doses
were for a 5 day/week, 78 week exposure, they were duration-adjusted to account for the partial
week exposure (by multiplying by 5 days/7 days) and untreated observation period (by
multiplying by 78 weeks/90 weeks). These duration-adjusted animal doses were then converted
to HEDs by adjusting for differences in body weight and lifespan between humans and mice. In
accordance with the U.S. EPA (2005a) Guidelines for Carcinogen Risk Assessment., a factor of
BW3 4 was used for cross-species scaling. Because the study duration (90 weeks) was less than
the animal lifespan (104 weeks), the scaled dose was then multiplied by the cubed ratio of
experimental duration to animal lifespan to complete the extrapolation to a lifetime exposure in
humans. The equation and data used to calculate the HEDs are presented below, and the
calculated HEDs are presented in Table 5-4.
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HED = Dose* x (W/70 kg)1/4 x (Le/L)3
Where:
Dose = average daily animal dose (* TWA converted for 5/7 days, 78/90 weeks)
W = average animal body weight (0.030 kg for male and female B6C3Fi mice [U.S.
EPA, 1988]).
70 kg = reference human body weight (U.S. EPA, 1988)
Le = duration of experiment (90 weeks)
L = reference mouse lifespan (104 weeks) (U.S. EPA, 1988)
Table 5-4. HEDs corresponding to duration-adjusted TWA doses in mice
Duration-adjusted dose in male and female mice (mg/kg-d)
HED for use with both male and female mouse incidence data (mg/kg-d)
Dose (mg/kg-d)
0
0
87.9
8.22
175.8
16.5
The mode of action of 1,1,2,2-tetrachloroethane carcinogenicity is unknown. It appears
that metabolism to one or more active compounds is likely to play a role in the development of
the observed liver tumors, but insufficient data preclude proposing a specific mode of action.
Dichloroacetic acid, a metabolite of 1,1,2,2-tetrachloroethane, induces hepatocellular carcinomas
in male and female B6C3Fi mice and F344 rats. Trichloroethylene (NTP, 1990; NCI, 1976) and
tetrachloroethylene (NTP, 1996; NCI, 1977), also metabolites of 1,1,2,2-tetrachloroethane, have
also been shown to be hepatocarcinogens in rodents.
Results of genotoxicity and mutagenicity studies of 1,1,2,2-tetrachloroethane are mixed
and insufficient for informing whether 1,1,2,2-tetrachloroethane carcinogenicity is associated
with a mutagenic mode of action. Given that the mechanistic and other information available on
cancer risk from exposure to 1,1,2,2-tetrachloroethane is sparse and that the existing data do not
inform the mode of action of carcinogenicity, a linear low-dose extrapolation was conducted as a
default option for the derivation of the oral slope factor.
Dose-response modeling was performed to obtain a POD for quantitative assessment of
cancer risk. The data sets for hepatocellular carcinoma in both sexes of mice were modeled for
determination of the POD. In accordance with the U.S. EPA (2005a) cancer guidelines, the
BMDLio (lower bound on dose estimated to produce a 10% increase in tumor incidence over
background) was estimated by applying the multistage cancer model in the EPA's BMDS
(version 2.1.1.) for the dichotomous incidence data and selecting the results of the model that
best characterized the cancer incidences. The BMD modeling of the male mouse data did not
achieve adequate model fit for any of the dichotomous models; thus, a cancer slope factor was
not derived from the male data. The 1° multistage model was selected for the derivation of the
cancer slope factor from the female data because this model provided adequate model fit and the
lowest Akaike's Information Criterion (AIC) when compared to the results of the 2° multistage
model. In addition, the 2° multistage model had insufficient degrees of freedom (DF) to test the
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goodness-of-fit. The BMDL of 0.65 mg/kg-day from the modeling of the tumor incidence data
in female mice was selected as the POD for use in calculation of an oral slope factor (Table 5-5).
Details of the BMD modeling are presented in Appendix C.
Table 5-5. Summary of human equivalent BMDs and BMDLs based on
hepatocellular carcinoma incidence data in female B6C3Fi mice
Female mice
BMR (% extra risk)
10
BMD (mg/kg-d)a
0.81
BMDL10(mg/kg-d)a
0.65
aHED.
5.4.4. Oral Slope Factor and Inhalation Unit Risk
The oral slope factor was derived from the BMDLio (the lower bound on the exposure
associated with a 10% extra cancer risk) by dividing the BMR by the BMDLio, and represents an
upper bound on cancer risk associated with a continuous lifetime exposure to 1,1,2,2-tetrachloro-
ethane. In accordance with the U.S. EPA (2005a) guidelines, an oral slope factor (mg/kg-day)"1
was calculated by dividing the human equivalent BMDLio into 0.1 (10%) (Appendix C). The
BMDLio is 0.65 mg/kg-day, and the cancer slope factor (the slope of the linear extrapolation
from the BMDLio to 0) is 0.10/0.65 = 0.2 per mg/kg-day. The slope of the linear extrapolation
from the central estimate (i.e., BMD) is 0.1/0.81 mg/kg-day or 0.1 per mg/kg-day.
In the absence of any suitable data on the carcinogenicity of 1,1,2,2-tetrachloroethane via
the inhalation route, an inhalation unit risk has not been derived.
5.4.5. Uncertainties in Cancer Risk Values
Extrapolation of data from animals to estimate potential cancer risks to human
populations from exposure to 1,1,2,2-tetrachloroethane yields uncertainty. Several types of
uncertainties may be considered quantitatively, but other important uncertainties cannot be
considered quantitatively. Thus, an overall integrated quantitative uncertainty analysis is not
presented. This section and Table 5-6 summarize the principal uncertainties.
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Table 5-6. Summary of uncertainty in the l,l?2,2-tetrachloroethane cancer
risk assessment
Consideration/
approach
Low-dose
extrapolation
procedure
Dose metric
Cross-species
scaling
Statistical
uncertainty at POD
Bioassay
Species/gender
combination
Human relevance of
mouse tumor data
Human population
variability in
metabolism and
response/sensitive
subpopulations
Impact on oral slope
factor
Departure from U.S.
EPA's Guidelines for
Carcinogen Risk
Assessment POD
paradigm, if justified,
could | or t slope
factor an unknown
extent
Alternatives could t
or J, slope factor by an
unknown extent
Alternatives could J,
or t slope factor (e.g.,
3. 5 -fold J, [scaling by
BW] or twofoldf
[scaling by BW2/3])
J, slope factor if MLE
of the POD is used
rather than lower
bound on POD (i.e.,
LEC)
Alternatives could t
or J, slope factor by an
unknown extent
Human risk could J, or
t, depending on
relative sensitivity
Human relevance of
mouse tumor data
could J, slope factor
Low-dose risk f or J,
to an unknown extent
Decision
Multistage cancer
model to determine
POD, linear low-
dose extrapolation
from POD
Used administered
exposure
BW3/4
LEC (method for
calculating
reasonable upper
bound slope factor)
NCI study
Female mice liver
cancer
Liver tumors in
mice are relevant
to human exposure
Considered
qualitatively
Justification
Available mode of action data do not inform
selection of dose-response model; linear approach
used in absence of an alternative as per U.S.
EPA's Guidelines for Carcinogen Risk
Assessment.
Experimental evidence supports a role for
metabolism in toxicity, but actual responsible
metabolites are not clearly identified.
There are no data to support alternatives. Because
the dose metric was not an AUC, BW3/4 scaling
was used to calculate equivalent cumulative
exposures for estimating equivalent human risks.
Limited size of bioassay results in sampling
variability; lower bound is 95% confidence
interval on administered exposure.
Alternative bioassays were unavailable.
There are no mode of action data to guide
extrapolation approach for any choice. It was
assumed that humans are as sensitive as the most
sensitive rodent gender/species tested; true
correspondence is unknown. The carcinogenic
response occurs across species. Generally, direct
site concordance is not assumed; consistent with
this view, some human tumor types are not found
in rodents, and rat and mouse tumor types also
differ.
1,1,2,2-Tetrachloroethane is carcinogenic through
an unknown mode of action.
No data to support range of human
variability/sensitivity, including whether children
are more sensitive. Metabolic activation mode of
action (if fully established) could indicate t or J,
early-life susceptibility.
LEC = lower confidence limit on a concentration producing a given effect; MLE = maximum likelihood estimate
Choice of low-dose extrapolation approach. The mode of action is a key consideration in
clarifying how risks at low-dose exposures should be estimated. A linear low-dose extrapolation
approach was used to estimate human carcinogenic risk associated with 1,1,2,2-tetrachloroethane
100
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exposure due to the unavailability of data that supports any specific mode of carcinogenic action
for 1,1,2,2-tetrachloroethane.
The extent to which the overall uncertainty in low-dose risk estimation could be reduced
if the mode of action for 1,1,2,2-tetrachloroethane were known is of interest, but data on the
mode of action of 1,1,2,2-tetrachloroethane are not available.
Dose metric. 1,1,2,2-Tetrachloroethane is metabolized to intermediates with
carcinogenic potential. Dichloroacetic acid is recognized as hepatocarcinogenic in male B6C3Fi
mice and F344 rats (U.S. EPA, 2003). However, it is unknown whether a metabolite or some
combination of parent compound and metabolites is responsible for the observed toxicity. If the
actual carcinogenic moiety is proportional to administered exposure, then use of administered
exposure as the dose metric is the least biased choice. On the other hand, if this is not the correct
dose metric, then the impact on the slope factor is unknown.
Cross-species scaling. An adjustment for cross-species scaling (BW3 4) was applied to
address toxicological equivalence of internal doses between the rodent species and humans,
consistent with the 2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a). It is
assumed that equal risks result from equivalent constant lifetime exposures.
Statistical uncertainty at the POD. Parameter, or probabilistic, uncertainty can be
assessed through confidence intervals. Each description of parameter uncertainty assumes that
the underlying model and associated assumptions are valid. For the multistage cancer model
applied to the female mice data, there is a reasonably small degree of uncertainty at a 10%
increase in tumor incidence (the POD for linear low-dose extrapolation).
Bioassay selection. The study by NCI (1978) was used for development of an oral slope
factor. This study was conducted in both sexes in two species with an adequate number of
animals per dose group, with examination of appropriate toxicological endpoints in both sexes of
rats and mice. Alternative bioassays were unavailable. Both genders of mice exhibited liver
tumors. Uncertainties associated with the use of this study in the derivation of the oral slope
factor arise, primarily, from the study design. The dose levels used in the study were poorly
selected and were modified over the exposure duration, and the exposure duration of the study
(78 weeks) was less than the standard 104-week chronic exposure duration. In addition, the
bolus nature of the 1,1,2,2-tetrachloroethane gavage exposures in NCI (1978), as well as the use
of corn oil as the gavage vehicle, may lead to more pronounced irritation, inflammation, cell
death, and an eventual increase in tumor incidence at portals of entry; however, chemical-
specific data demonstrating this progression are not available. There was also an increased
incidence of endemic chronic murine pneumonia in male and female rats and mice, and while
interpretation of this study is complicated by the chronic murine pneumonia, it is unlikely to
have contributed to the carcinogenicity results observed in male and female rats.
Choice of species/gender. The oral slope factor for 1,1,2,2-tetrachloroethane was
quantified using the tumor incidence data for female mice. The hepatocelluar carcinoma data in
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male mice demonstrated tumorigenicity, but the data in male mice did not achieve adequate
model fit for any of the dichotomous models when BMD modeled. The male and female rat
tumor incidence data were not suitable for deriving low-dose quantitative risk estimates, and NCI
described the rat strain as relatively insensitive to the carcinogenic effects of chlorinated organic
compounds.
Relevance to humans. The oral slope factor is derived from the incidence of
hepatocellular carcinomas in female B6C3Fi mice. Using liver tumors in B6C3Fi mice as the
model for human carcinogenesis is a concern because of the prevalence of and susceptibility to
developing liver tumors in this strain of mice, which may result in the derivation of an oral slope
factor that is overly health protective in relation to human risk assessment. Hasemen et al.
(1998) reported an increased liver carcinoma rate of 17.9 and 8.4% for male and female B6C3Fi
mice, respectively, from NTP carcinogenicity feeding bioassays, and a combined adenoma and
carcinoma rate of 42 and 24% for male and female B6C3Fi mice, respectively. However, the
incidence in the control B6C3Fi mice in NCI (1978) was 1/18 in the male vehicle controls and
0/20 in the female vehicle controls, and 3/36 and 1/40 in male and female pooled-vehicle
controls, respectively, and comparison of an experimental group with its concurrent controls has
been considered to be the most appropriate comparison (Haseman et al., 1992; Tarone et al.,
1981; Gait et al., 1979 as cited in Haseman et al., 1998; Goodman et al., 1980).
Additional interspecies uncertainty may arise from the rate of metabolism across species.
Reitz et al. (1996) demonstrated that mice possessed a greater relative ability to metabolize
tetrachloroethylene than rats and humans, and, although data are not available, a similar situation
may exist for 1,1,2,2-tetrachloroethane.
In addition, the genotoxicity and mutagenicity studies provide limited evidence of a
mutagenic mode of action, with 1,1,2,2-tetrachloroethane displaying equivocal results of
mutagenic activity. There are inadequate data to support any mode of action hypothesis.
Human population variability. The extent of interindividual variability in animals for
1,1,2,2-tetrachloroethane metabolism has not been characterized. A separate issue is that the
human variability in response to 1,1,2,2-tetrachloroethane is also unknown. This lack of
understanding about potential differences in metabolism and susceptibility across exposed
animal and human populations, thus, represents a source of uncertainty.
5.4.6. Previous Cancer Assessment
In the previous IRIS assessment, posted to the IRIS database in 1987, 1,1,2,2-tetrachloro-
ethane was characterized as "Classification — C; possible human carcinogen" based on the
increased incidence of hepatocellular carcinomas in mice observed in the NCI (1978) bioassay.
An oral slope factor of 0.2 (mg/kg-day)"1 was derived using the increased incidence of
hepatocellular carcinomas in female mice (NCI, 1978) and a linearized multistage approach.
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6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE
RESPONSE
6.1. HUMAN HAZARD POTENTIAL
1,1,2,2-Tetrachloroethane (CAS No. 79-34-5) has been used as an insecticide, fumigant,
and weed killer (Hawley, 1981), although it presently is not registered for any of these purposes.
It was once used as an ingredient in an insect repellent, but registration was canceled in the late
1970s. In the past, the major use for 1,1,2,2-tetrachloroethane was in the production of
trichloroethylene, tetrachloroethylene, and 1,2-dichloroethylene (Archer, 1979). It was also used
as a solvent, in cleaning and degreasing metals, in paint removers, varnishes, and lacquers, in
photographic films, and as an extractant for oils and fats (Hawley, 1981). With the development
of new processes for manufacturing chlorinated ethylenes, the production of 1,1,2,2-tetrachloro-
ethane as a commercial end-product in the United States and Canada steadily declined since the
late 1960s and had ceased by the early 1990s (NLM, 2009; Environment Canada and Health
Canada, 1993). 1,1,2,2-Tetrachloroethane may still appear as a chemical intermediate in the
production of a variety of other common chemicals.
1,1,2,2-Tetrachloroethane is well absorbed from the respiratory and gastrointestinal
tracts, is rapidly and extensively metabolized, and is eliminated mainly as metabolites in the
urine and breath. Both reductive and oxidative metabolisms occur, producing reactive radical
and organochlorine intermediates, respectively. Trichloroethanol, trichloroacetic acid, and
dichloroacetic acid are initial metabolites that subsequently yield glyoxalic acid, oxalic acid, and
carbon dioxide.
A limited amount of information is available addressing the toxicity of 1,1,2,2-tetra-
chloroethane in humans. CNS depression was the predominant effect of high-dose acute oral
and inhalation exposures, although acute inhalation also caused irritation of the mucous
membranes. Occupational studies suggest that repeated exposure to 1,1,2,2-tetrachloroethane
can affect the liver and the nervous system.
Animal studies have established that the CNS and liver are the main targets of toxicity at
high levels of oral and inhalation exposures. Death in laboratory animals typically was preceded
by signs of CNS depression (e.g., lethargy, incoordination, loss of reflexes, depressed
respiration, prostration, and loss of consciousness), and postmortem examinations mainly
showed fatty degeneration in the liver. The most sensitive target of sublethal ingestion and
inhalation appears to be the liver, and short-term and subchronic exposures caused hepatic
effects that included serum chemistry changes, hepatocellular degeneration, and other
histopathological alterations. Comprehensive neurobehavioral testing in 14-week feeding studies
showed no effects in rats or mice, indicating that the liver was more sensitive than the nervous
system for subchronic oral exposure (Chan, 2004). A limited amount of information is available
103
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on other effects of 1,1,2,2-tetrachloroethane. Reduced body weight gain and weight loss were
effects of repeated oral exposures in rats and mice that generally occurred at high doses and may
have contributed to mild anemia and atrophy in the spleen, bone, bone marrow, and reproductive
tissues in these animals. Kidney lesions (acute toxic tubular necrosis and chronic inflammation)
occurred in mice that were chronically exposed to oral doses that also caused reduced survival.
Adequate immunological testing of 1,1,2,2-tetrachloroethane has not been performed.
The reproductive and developmental toxicity of 1,1,2,2-tetrachloroethane has not been
adequately evaluated. Significant decreases in maternal and fetal body weights were observed in
rats. In mice, litter resorption was observed along with high maternal mortality. Toxicity to
reproductive tissues following 1,1,2,2-tetrachloroethane exposure to adult rats and mice was
observed in F344 rats and B6C3Fi mice. Effects observed in rats and/or mice include:
decreased sperm and spermatozoal motility; decreased testis and epididymal weight; increased
atrophy of the preputial and prostate gland, seminal vesicle, testicular germinal epithelium,
uterus, and clitoral gland; ovarian interstitial cell cytoplasmic alterations; and lengthened estrus
cycle. Chronic low-level inhalation caused no effects on reproductive function in male mice, but
multigeneration or other tests of reproductive function in females have not been conducted for
any route of exposure. Developmental toxicity was assessed in rats and mice that were
gestationally exposed to 1,1,2,2-tetrachloroethane in the diet. These studies did not include
examinations for skeletal or visceral abnormalities, although effects that included reduced fetal
body weight gain in rats and litter resorptions in mice occurred at doses that were maternally
toxic.
The carcinogenicity of 1,1,2,2-tetrachloroethane was evaluated in a chronic gavage study
in rats and mice conducted by NCI (1978). Hepatocellular carcinomas were induced in male and
female mice, but there were no statistically significant increases in tumor incidences in the rats.
Three rare tumors in high dose male rats were noted. Thus, 1,1,2,2-tetrachloroethane is "likely
to be carcinogenic to humans" by any route of exposure, according to the Guidelines for
Carcinogen Risk Assessment (U.S. EPA, 2005a).
6.2. DOSE RESPONSE
6.2.1. Noncancer/Oral
The NTP (2004) study was selected as the principal study because it was a well-designed,
subchronic dietary study, conducted in both sexes in two rodent species with a sufficient number
of animals per dose group. The number of test animals allocated among three dose levels and an
untreated control group was acceptable, with examination of appropriate toxicological endpoints
in both sexes of rats and mice. The liver was the most sensitive target in both species, and the
rats were more sensitive than the mice. In addition to the observed liver weight increases, there
is evidence of hepatocellular effects including increased serum liver enzyme levels and an
increased incidence of both hepatocellular cytoplasmic vacuolization and necrosis from the
104
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subchronic NTP (2004) study. EPA selected increased liver weight as the critical effect because
this effect may represent an indicator of liver toxicity that occurs early in the process leading to
hepatocellular necrosis associated with subchronic oral exposure to 1,1,2,2-tetrachloroethane;
however, chemical-specific data demonstrating the relationship between increased liver weight
and hepatocellular necrosis is not available.
Potential PODs for a subchronic RfD were derived by BMD modeling of dose-response
data for increases in liver weight, increases in serum levels of ALT, SDH, and ALP, increased
levels of bile acids, and increased incidence of hepatocellular cytoplasmic vacuolization in rats.
All available dichotomous models in the EPA's BMDS (version 2.1.1) were fit to the incidence
data for hepatocellular cytoplasmic vacuolization, and all available continuous models in the
software were applied to the data for liver weight and serum enzyme levels, as well as the data
for rat fetal body weight. A BMR of 10% (10% extra risk above control) was selected for
derivation of the BMDL for hepatocellular cytoplasmic vacuolization in female rats, and a BMR
of 1 SD (a change in the mean equal to 1 SD from the control mean) was selected for the
derivation of the BMDL for the continuous female rat liver weight and rat fetal body weight
data.
The BMDiso of 22 mg/kg-day and BMDLiso of 15 mg/kg-day based on the relative liver
weight effects seen in the female rat was selected as the POD for the derivation of the RfD. To
derive the subchronic RfD, the 15 mg/kg-day BMDLiso based on female rat relative liver weight
was divided by a total UF of 300, yielding a subchronic RfD of 0.05 mg/kg-day. The UF of
300 comprises component factors of 10 for interspecies extrapolation, 10 for interhuman
variability, and 3 for database deficiencies.
The choice of BMD model is not expected to introduce a considerable amount of
uncertainty in the risk assessment since the chosen response rate of 1 SD is within the observable
range of the data. Additional BMD modeling for other amenable data sets, including serum liver
enzyme levels, liver lesions, and fetal body weight, were also conducted to provide other PODs
for comparison purposes (see Appendix B). A graphical representation of these potential PODs
and resulting subchronic RfVs is shown below in Figure 6-1.
105
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1000
100
10
w
TJ
6)
0)
0.1
0.01
/ \
/ \
POD 4
UFA
UFD
RfD
1001
hepatocellular relative liver weight- absolute liver weight increased ALT -$ increased SDH-? increased bile acids - decreased fetal body
cytoplasrric ? rats (NTP, 2004) - $ rats (NTP, 2004) rats (NTP, 2004) rats (NTP, 2004) $ rats (NTP, 2004) weight-rats (Gulati
vacuolization - $ rats et al, 1991a)
(NTP, 2004)
Figure 6-1. PODs for selected endpoints (with critical effect circled) with corresponding applied UFs and
derived sample subchronic oral RfVs.
106
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The default UF of 10 for the extrapolation from animals to humans is a composite of
uncertainty to account for toxicokinetic differences and toxicodynamic differences between the
animal species in which the POD was derived and humans.
PBPK models can be useful for the evaluation of interspecies toxicokinetics; however,
information was unavailable to quantitatively assess toxicokinetic or toxicodynamic differences
between animals and humans and the potential variability in human susceptibility; thus, the
interspecies and intraspecies UFs of 10 were applied for a total UF of 100. Human variation may
be larger or smaller; however, 1,1,2,2-tetrachloroethane-specific data to examine the potential
magnitude of human variability of response are unknown.
In addition, a threefold database UF was applied due to the lack of information
addressing the potential reproductive toxicity associated with 1,1,2,2-tetrachloroethane.
Uncertainties associated with data gaps in the 1,1,2,2-tetrachloroethane database have been
identified, specifically, uncertainties associated with database deficiencies characterizing
reproductive toxicity associated with oral exposure to 1,1,2,2-tetrachloroethane. The developing
fetus may be a target of toxicity (Gulati et al., 1991a), and the absence of a study specifically
evaluating the full range of developmental toxicity represents an additional area of uncertainty or
gap in the database.
The overall confidence in this subchronic RfD assessment is medium. Confidence in the
principal study (NTP, 2004) is high. Confidence in the database is medium. Reflecting high
confidence in the principal study and medium confidence in the database, confidence in the
subchronic RfD is medium.
Information on the chronic oral toxicity of 1,1,2,2-tetrachloroethane consists of a limited
78-week gavage study in rats and mice (NCI, 1978). The high incidences of hepatocellular
tumors in all treated groups of mice precluded evaluation of noncancer effects in the liver and
identification of a NOAEL or LOAEL. Additionally, the NCI (1978) study performed
histological examinations on the animals when they died or at the termination of the study, which
was beyond the point at which more sensitive hepatotoxic effects, including nonneoplastic
effects, would be expected. The 14-week dietary study (NTP, 2004) was used to derive the
subchronic oral RfD. The NTP (2004) study also utilized a more relevant type of exposure (i.e.,
oral feeding) for the general population exposed to 1,1,2,2-tetrachloroethane in the environment.
The chronic RfD of 0.02 mg/kg-day was calculated by dividing the subchronic BMDLiso
of 15 mg/kg-day for increased relative liver weight by a total UF of 1,000: 10 for interspecies
extrapolation, 10 for interhuman variability, 3 for subchronic to chronic duration extrapolation,
and 3 for database deficiencies.
The choice of BMD model is not expected to introduce a considerable amount of
uncertainty in the risk assessment since the chosen BMR of 1 SD from the control mean is within
the observable range of the data. Additional BMD modeling for other amenable data sets,
including serum liver enzyme levels, liver lesions, and fetal body weight, were also conducted to
107
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provide other PODs for comparison purposes (see Appendix B). A graphical representation of
these potential PODs and resulting chronic RfVs is shown below in Figure 6-2.
108
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IUUU
inn
IUU
m
•o
0) 1 i
^ I "
o
n 1 -
nni
U.U I
nnm -
^
8
\ \ \
\\\
/
i
'
i
\
»
^.^
/ N
>
WsS;
88w
\ \ \
\ \ \
\ /
\
\
\
\
1
I
1
1
^
^i^
twffl
*
^
^^
^^v
^
Kvvvv
AVVW
\\\
\ \ \
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\\\
\ \ \
^
A
VjVji\\
B
\\\
\ \ \
*
POD 4
UFA ;i;i
UFH "~
UFo B
1 1C ^
Ursc ^ ^ *"
\ \ \
RfD •
hepatocellular
cytoplasmic
vacuolization - $ rats
(NTP, 2004)
relative liver weight- absolute liver weight increased A LT-c? increased SDH-? increased bile acids - decreased fetal body
? rats (NTP, 2004) - $ rats (NTP, 2004) rats (NTP, 2004) rats (NTP, 2004) $ rats (NTP, 2004) weight-rats (Gulati
etal.,1991a)
Figure 6-2. PODs for selected endpoints (with critical effect circled) from Table 5-2 with corresponding applied UFs
and derived sample subchronic oral RfVs.
109
-------
The default UF of 10 for the extrapolation from animals to humans is a composite of
uncertainty to account for toxicokinetic differences and toxicodynamic differences between the
animal species in which the POD was derived and humans.
PBPK models can be useful for the evaluation of interspecies toxicokinetics; however,
information was unavailable to quantitatively assess toxicokinetic or toxicodynamic differences
between animals and humans and the potential variability in human susceptibility; thus, the
interspecies and intraspecies UFs of 10 were applied for a total UF of 100. Human variation may
be larger or smaller; however, 1,1,2,2-tetrachloroethane-specific data to examine the potential
magnitude of human variability of response are unknown.
A threefold UF was applied for extrapolation from a subchronic exposure duration study
to a chronic RfD. Based on the available data for 1,1,2,2-tetrachloroethane, the toxicity observed
in the liver does not appear to increase over time. Specifically, liver damage observed in
F344 rats following subchronic exposure to 1,1,2,2-tetrachloroethane (NTP, 2004) (e.g.,
increased incidence of necrosis or altered serum enzyme and bile levels) did not progress to more
severe effects such as cirrhosis or major liver disease following chronic exposures (NCI, 1978).
Therefore, the available database does not abrogate all concern associated with using a
subchronic study as the basis of the RfD but supports the utilization of a database UF of 3.
In addition, a threefold database UF was applied due to the lack of information
addressing the potential reproductive toxicity associated with 1,1,2,2-tetrachloroethane.
Uncertainties associated with data gaps in the 1,1,2,2-tetrachloroethane database have been
identified, specifically, uncertainties associated with database deficiencies characterizing
reproductive toxicity associated with oral exposure to 1,1,2,2-tetrachloroethane. The developing
fetus may be a target of toxicity (Gulati et al., 1991a), and the absence of a study specifically
evaluating the full range of developmental toxicity represents an additional area of uncertainty or
gap in the database.
The overall confidence in this chronic RfD assessment is medium. Confidence in the
principal study (NTP, 2004) is high. Confidence in the database is medium. Reflecting high
confidence in the principal study and medium confidence in the database, confidence in the
chronic RfD is medium.
6.2.2. Noncancer/Inhalation
An RfC was not calculated due to insufficient data. Information on the subchronic and
chronic inhalation toxicity of 1,1,2,2-tetrachloroethane is limited to the results of one study in
rats that found transient liver effects (Truffert et al., 1977). Reporting inadequacies preclude
identification of a NOAEL or LOAEL and derivation of an RfC in the usual manner.
A route-to-route extrapolation using the computational technique of Chiu and White
(2006), as described in Section 3.5, was considered. However, U.S. EPA (1994b) recommends
not conducting a route-to-route extrapolation from oral data when a first-pass effect by the liver
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or respiratory tract is expected, or a potential for portal-of-entry effects in the respiratory tract is
indicated following analysis of short-term inhalation, dermal irritation, in vitro studies, or
evaluation of the physical properties of the chemical. In the case of 1,1,2,2-tetrachloroethane, a
first-pass effect by the liver is expected. In addition, the presence of tissue-bound metabolites in
the epithelial linings in the upper respiratory tract may demonstrate a first-pass effect by the
respiratory tract (Eriksson and Brittebo, 1991). Lehmann et al. (1936) observed irritation of the
mucous membranes of two humans following exposure to 1,1,2,2-tetrachloroethane air
concentrations of 146 ppm (1,003 mg/m3) for 30 minutes or 336 ppm (2,308 mg/m3) for
10 minutes, indicating the potential for portal-of-entry effects in the respiratory system.
Information regarding the chronic inhalation toxicity of 1,1,2,2-tetrachloroethane is
available from four animal studies that include limited data on liver effects and serum
acetylcholinesterase, or hematological and immunological alterations (Shmuter, 1977;
Kulinskaya and Verlinskaya, 1972; Schmidt et al., 1972; Mellon Institute of Industrial Research,
1947). However, the reporting of results from these chronic bioassays is inadequate for
identification of NOAELs or LOAELs for systemic toxicity. A chronic NOAEL was identified
for reproductive effects in male rats (Schmidt et al., 1972); however, macroscopic malformations
or significant group differences in the other indices were not observed at 13.3 mg/m3. This lack
of information on reproductive toxicity precludes utilizing this selected NOAEL in the derivation
ofanRfC.
6.2.3. Cancer/Oral and Inhalation
Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), 1,1,2,2-tetra-
chloroethane is characterized as "likely to be carcinogenic to humans", based on the existence of
evidence of the compound's turn origeni city in a single study in two animal species (NCI, 1978).
The epidemiological human data available are inadequate for evaluation of cancer risk (IARC,
1999). The NCI (1978) provided evidence that 1,1,2,2-tetrachloroethane causes hepatocellular
tumors in male and female mice, and rare tumors (not statistically significant) were seen in high-
dose male rats.
The only carcinogenicity bioassay for 1,1,2,2-tetrachloroethane was a chronic gavage
study in Osborne-Mendel rats and B6C3Fi mice performed by NCI (1978). This was a well-
designed study, conducted in both sexes in two rodent species with an adequate number of
animals per dose group and with examination of appropriate toxicological endpoints in both
sexes of rats and mice. Although limitations in the study included increased mortality in male
and female mice, the variable doses given to the mice over the course of the 78-week exposure
period, and the exposure duration of the study (78 weeks) was less than the standard 104 week
chronic exposure duration.
The rat study found no statistically significant increases in tumor incidences in males or
females. Three rare hepatocellular tumors in high-dose male rats were noted, and NCI (1978)
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characterized the carcinogenic results in male rats as "equivocal." The mouse study found
significant, dose-related increases in the incidences of hepatocellular carcinomas in both sexes.
Based on the increased incidences of hepatocellular carcinomas, NCI (1978) concluded that
orally administered 1,1,2,2-tetrachloroethane is a liver carcinogen in male and female B6C3Fi
mice. This NCI study was used for dose-response analysis for oral exposure.
Data on the incidences of hepatocellular carcinomas in male and female mice from the
NCI (1978) study were used for cancer dose-response assessment. Conversion of the doses in
the NCI (1978) mouse study to HEDs to be used for dose-response modeling was accomplished
in two steps. The mice were treated with 1,1,2,2-tetrachloroethane by gavage 5 days/week for
78 weeks, and then observed untreated for 12 weeks for a total study duration of 90 weeks.
Because the reported TWA doses were doses for 5 days/week for 78 weeks, they were duration-
adjusted to account for the partial week exposure (by multiplying by 5 days/7 days) and
untreated observation period (by multiplying by 78 weeks/90 weeks). The duration-adjusted
animal doses were converted to HEDs by adjusting for differences in body weight and lifespan
between humans and mice. In accordance with U.S. EPA (2005a) Guidelines for Carcinogen
Risk Assessment, a factor of BW3 4 was used for cross-species scaling. Because the study
duration (90 weeks) was less than the animal lifespan (104 weeks), the scaled dose was then
multiplied by the cubed ratio of experimental duration to animal lifespan to complete the
extrapolation to a lifetime exposure in humans.
The mode of action of 1,1,2,2-tetrachloroethane carcinogenicity is unknown. It appears
that metabolism to one or more active compounds is likely to play a role in the development of
the observed liver tumors, but insufficient data preclude proposing this as a mode of action.
Results of genotoxicity and mutagenicity studies of 1,1,2,2-tetrachloroethane are mixed and
insufficient for informing the mode of action. Given that the mechanistic and other information
available on cancer risk from exposure to 1,1,2,2-tetrachloroethane is sparse and that the data
that do exist are equivocal, there is inadequate information to inform the low dose extrapolation.
Dose-response modeling was performed to obtain a POD for quantitative assessment of
cancer risk. The incidences of hepatocellular carcinomas in both sexes of mice were modeled for
determination of the POD. In accordance with the U.S. EPA (2005a) cancer guidelines, the
BMDLio (lower bound on dose estimated to produce a 10% increase in tumor incidence over
background) was estimated by applying the multistage cancer model in the EPA's BMDS
(version 2.1.1) for the dichotomous incidence data and selecting the results for the model that
best fit the data. The BMD modeling of the male mouse data did not achieve adequate fit for any
of the dichotomous models; thus, a cancer slope factor was not derived from the male data. The
1° multistage model was selected for the derivation of the cancer slope factor from the female
data because this model provided adequate model fit and the lowest AIC when compared to the
results of the 2° multistage model. In addition, the 2° multistage model had insufficient DF to
test the goodness-of-fit. The BMDLio of 0.65 mg/kg-day from the modeling of the tumor
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incidence data in female mice is selected as the POD for use in calculation of an oral slope
factor. In accordance with the U.S. EPA (2005a) guidelines, an oral slope factor of 0.2 (mg/kg-
day)"1 is calculated by dividing the human equivalent BMDLio of 0.65 mg/kg-day into 0.1 (10%)
(Appendix C).
In the absence of any data on the carcinogenicity of 1,1,2,2-tetrachloroethane via the
inhalation route, an inhalation unit risk has not been derived in this evaluation.
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APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC
COMMENTS AND DISPOSITION
The Toxicological Review of 1,1,2,2-tetrachloroethane (dated August, 2009) has
undergone a formal external peer review performed by scientists in accordance with EPA
guidance on peer review (U.S. EPA, 2006a, 2000a). An external peer-review workshop was held
January 27, 2010. The external peer reviewers were tasked with providing written answers to
general questions on the overall assessment and on chemical-specific questions in areas of
scientific controversy or uncertainty. A summary of significant comments made by the external
reviewers and EPA's responses to these comments arranged by charge question follow. In many
cases, the comments of the individual reviewers have been synthesized and paraphrased in
development of Appendix A. EPA did not receive any scientific comments from the public.
EXTERNAL PEER REVIEW PANEL COMMENTS
The reviewers made several editorial suggestions to clarify specific portions of the text.
These changes were incorporated in the document as appropriate and are not discussed further.
In addition, the reviewers provided comments specific to particular decisions and
analyses presented in the Toxicological Review under multiple charge questions. These
comments were organized and responded to under the most appropriate charge question.
A. General Comments
1. Is the Toxicological Review logical, clear and concise? Has EPA clearly synthesized the
scientific evidence for noncancer and cancer hazard?
Comments: The reviewers generally commented that the Toxicological Review was
logically written. One reviewer recommended an improvement to the clarity of the document
by reducing the text describing the available studies and presenting the individual study data
in a bulleted format, and this was echoed by another reviewer who recommended condensing
the study summaries and discussions.
Response: The content of the Toxicological Review is consistent with the current outline for
IRIS Toxicological Reviews, although an effort has been made to streamline the document
and reduce the redundancy. The general structure of a Toxicological Review is to present a
factual summary of toxicity studies and a qualitative synthesis of these studies in Section 4
and a quantitative critical interpretation in Section 5.
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2. Please identify any additional studies that should be considered in the assessment of the
noncancer and cancer health effects of 1,1^2,2-tetrachloroethane.
Comments: Several reviewers did not provide additional studies. One reviewer identified
the following studies:
Ashley, DL; Bonin, MA; Cardinal!, FL; et al. (1994) Blood concentrations of volatile
organic compounds in a nonoccupationally exposed US population and in groups with
suspected exposure. Clin Chem 40(7 Pt 2): 1401-4.
Matsuoka, A; Yamakage, K; Kusakabe, H; et al. (1996) Re-evaluation of chromosomal
aberration induction on nine mouse lymphoma assay "unique positive' NTP carcinogens.
MutatRes 12;369(3-4):243-52.
Sofuni, T; Honma, M; Hayashi, M; et al. (1996) Detection of in vitro clastogens and
spindle poisons by the mouse lymphoma assay using the microwell method: interim
report of an international collaborative study. Mutagenesis ll(4):349-55.
Response: The references (Matsuoka et al. [1996]; Sofuni et al. [1996]; Ashley et al. [1994])
were examined but have not been added to the Toxicological Review, as these references do
not contribute significant information to the discussion and analysis in the document.
B. Oral Reference Dose (RfD) for 1,1,2,2-tetrachloroethane
1. Subchronic and chronic RfDs for l,l?2,2-tetrachloroethane have been derived from a
13-week oral gavage study (NTP, 2004) in rats and mice. Please comment on whether
the selection of this study as the principal study has been scientifically justified. Please
identify and provide the rationale for any other studies that should be selected as the
principal study.
Comment: The reviewers generally agreed that the selection of the NTP (2004) report as the
principal study was scientifically justified.
Response: Comment acknowledged.
Comment: One reviewer commented that the Gulati et al. (1991a, b) study is the only other
study that could be a candidate principal study and provides what may be a more significant
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endpoint for human health protection; but also states that EPA has made a reasonable
selection in the NTP study.
Response: The Gulati et al. (199la, b) developmental studies were conducted at doses higher
than the subchronic NTP (2004) study, which demonstrated liver effects at lower doses.
Therefore, the Gulati et al. (1991a, b) studies were not selected as the principal studies.
However, potential PODs based on the observed developmental effects from Gulati et al.
(1991a) were provided in the document for comparison purposes.
Comment: One reviewer requested additional explanation regarding the statement that high
incidences of hepatocellular tumors in all mouse groups of the NCI (1978) study precluded
evaluation of noncancer effects in the liver.
Response: The statement in Section 5.1.2.1., Choice of Principal Study and Critical Effect -
with Rationale and Justification, regarding the high incidence of hepatocellular tumors in all
mouse dose groups precluding the evaluation of noncancer effects in the liver was deleted.
The effects observed in the NCI (1978) study were considered in the identification of the
principal study and critical effect, and a LOAEL of 142 mg/kg-day was identified for chronic
inflammation in the kidneys of male mice, while a NOAEL of 142 mg/kg-day and a LOAEL
of 284 mg/kg-day were identified for hydronephrosis and chronic inflammation in the
kidneys of female mice. This information is included in Section 5.1.2.1.
2. Increased relative liver weight was selected as the critical effect for the derivation of the
subchronic and chronic RfDs. Please comment on whether the rationale for the
selection of this critical effect has been scientifically justified. Please provide a detailed
explanation. Please identify and provide the rationale for any other endpoints that
should be considered in the selection of the critical effect.
Comment: The reviewers generally agreed that the selection of increased relative liver
weight as the critical effect for the derivation of the subchronic and chronic RfDs was
justified. However, one reviewer did not concur with the selection of the critical effect and
stated that there is no scientific evidence to support the conclusion that the increase in liver
weight represents a sensitive endpoint early in the process leading to hepatocellular necrosis.
A second reviewer questioned whether increases in liver weight reflect other, earlier changes
that have been going on long enough to cause the cell proliferation, inflammation, or other
effects responsible for the observed weight gain.
One reviewer commented that increased relative liver weight is a less lexicologically
significant index of liver change than increased absolute liver weight, due to the treatment-
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induced loss of body weight; whereas another reviewer believed the change in relative liver
weight is more appropriate than absolute liver weight where body weights in general are
being affected. Another reviewer commented that increased serum enzyme activity is an
alternative critical effect and a true measure of hepatocellular damage, and the most
toxicologically-significant endpoint should be selected as the critical effect. A reviewer
commented that the only other endpoint that is a candidate critical effect is reduced fetal
body weight in the Gulati et al. (1991a, b) studies, but also states that EPA's selection of the
relative liver weight as the critical effect is reasonable.
Response: EPA considered that, given the available data, increased liver weight represents
the most sensitive effect observed in the liver. In addition to increased liver weight following
subchronic exposure, the evidence of hepatocellular damage includes increased serum
concentrations of hepatocellular enzymes (ALT and SDH), decreased serum cholesterol, and
increased incidences of hepatocellular necrosis, bile duct hyperplasia, hepatocellular mitotic
alterations, and hepatic pigmentation. Evidence of the 'earlier changes' reflected by the
increase in liver weight as suggested by one reviewer is unavailable. Thus, EPA retained
increased liver weight as a critical effect for the subchronic and chronic RfDs. Clarification
text has been added to Section 5.1.1.1, Choice of Principal Study and Critical Effect - with
Rationale and Justification addressing the lack of chemical-specific data demonstrating the
relationship between increased liver weight and hepatocellular necrosis.
The increase in relative liver weight was selected as the basis for the POD because
the relative liver weight analysis takes into account the substantive, dose-dependent
decreases in body weight that were observed in both sexes of rats.
The increase in serum enzyme activity was included as a comparison to the increased
liver weight in Section 5.1.3., RfD Comparison Information, however, it was determined that
the observed increase in liver weight may represent the most sensitive effect that occurs early
in the process of 1,1,2,2-tetrachloroethane-induced hepatotoxicity.
The reduction in fetal body weight was observed at doses higher than the
demonstrated liver effects from the subchronic NTP (2004) study. Therefore, the decrease in
fetal body weight was not selected as the critical effect. However, PODs based on the
observed developmental effects from Gulati et al. (1991a) were provided in the document for
comparison purposes.
3. Hepatocellular vacuolization was observed at the lowest dose in the principal study
(NTP, 2004). This effect was not selected as the critical effect for the determination of
the POD for derivation of the subchronic and chronic RfDs. Please comment on the
rationale and justification for not selecting this endpoint as the critical effect.
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Comment: The reviewers generally considered the rationale and justification for not
selecting hepatocellular vacuolization as the critical effect as reasonable, justified, logical,
and comprehensive. One reviewer recommended slight refinements to the justification, and
questioned whether the comments that vacuolization was not observed across species and the
severity was not dose-dependent supported the conclusion. Another reviewer asked if NTP
(2004) specified the lobular distribution of the vacuoles.
Response: The decision to not select hepatocellular vacuolization as the critical effect
involved more than a consideration of cross species observations and severity (see Section
5.1.1.1., Choice of Principal Study and Critical Effect - with Rationale and Justification).
The biological significance of the hepatocellular vacuolization observed following
1,1,2,2-tetrachloroethane exposure was unclear based on the paucity of available information.
NTP did not specify the lobular distribution of the observed vacuoles.
4. The subchronic and chronic RfDs have been derived utilizing benchmark dose (BMD)
modeling to define the point of departure (POD). All available models were fit to the
data in both rats and mice for increased absolute and relative liver weight, increased
incidence of hepatocellular cytoplasmic vacuolization (rats only), increased levels of
ALT, SDH, and bile acids, and decreased fetal body weight. Has the BMD modeling
been appropriately conducted? Is the benchmark response (BMR) selected for use in
deriving the POD (i.e., one standard deviation from the control mean) scientifically
justified? Please identify and provide the rationale for any alternative approaches
(including the selection of the BMR, model, etc.) for the determination of the POD and
discuss whether such approaches are preferred to EPA's approach.
Comment: Three reviewers stated that the BMD modeling was appropriate. One reviewer
disagreed with the reasoning provided in the document for eliminating the two highest dose
groups from the BMD modeling analysis for all of the endpoints, and stated that dropping
doses is typically only done when the issues of model fit are encountered. A second reviewer
commented that EPA should at least show earlier BMD modeling results with the highest
doses included and show the lack of model fit that led to the elimination of the two highest
doses.
Response: In agreement with the reviewers' comments, the reasoning, provided in Section
5.1.1.2 of the document, Methods of Analysis—Including Models (PBPK, BMD, etc.), for
dropping the two highest dose groups (exceeding the MTD) was removed. In its place, a
rationale for dropping dose groups based on adequacy of model fit was employed. In
addition, the endpoints in Table 5-1 were remodeled using all of the dose groups within the
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dataset. The BMD modeling results (generated using version 2.1.1 of BMDS) with the
highest dose groups included are presented in Appendix B. This analysis demonstrated that
lack of model fit led to the elimination of one or more of these dose groups in order to obtain
adequate fit. As a result of this remodeling, the critical effect, increased relative liver weight,
was based on the data in female rats, where before, relative liver weight in male rats had been
selected as the most sensitive species/sex.
5. Please comment on the selection of the uncertainty factors applied to the POD for the
derivation of the RfDs. For instance, are they scientifically justified? If changes to the
selected uncertainty factors are proposed, please identify and provide a rationale(s).
Please comment specifically on the following uncertainty factor:
• A database uncertainty factor of 3 was used to account for the lack of oral
reproductive and developmental toxicity data for 1,1,2,2-tetrachloroethane.
Please comment on whether the application of this uncertainty factor has been
scientifically justified.
Comment: The reviewers generally considered the applications of the UFs to be adequate,
acceptable, reasonable, and appropriate.
Response: Comment acknowledged.
Comment: One reviewer requested a comparison between the RfD derived from the
subchronic NTP study and an approximate RfD derived from the chronic NCI study.
Response: The RfD from the subchronic NTP study was based on a study that used lower
dose levels and a wider dose range than the NCI (1978) study, and thereby provided a better
characterization of the dose-response curve in the low-dose region. Additionally, the route of
exposure used in the NTP study (dietary exposure) is a more relevant route of exposure for
the general population exposed to 1,1,2,2-tetrachloroethane in the environment than the
gavage exposure used in the NCI study. However, if one were to use the observance of
chronic inflammation in the kidneys of male mice in the NCI study as a LOAEL, for
purposes of comparison, the POD of 142 mg/kg-day could be divided by a total UF of 300 to
yield a potential RfD of 0.5 mg/kg-day. This information is shown in Figure 5-1.
Comment: A reviewer recommended the addition of text addressing the major metabolites of
1,1,2,2-tetrachloroethane (dichloroacetic acid, trichloroethylene, perchloroethylene) and how
the results of these assessments compare to those derived for 1,1,2,2-tetrachloroethane.
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Response: The RfDs derived for the major metabolites are not an appropriate comparison
because the principal studies, critical effects, PODs, and UFs are chemical-specific, and are
not directly comparable, nor is the confidence the same in the data sets from which the PODs
were derived. While the datasets may have similarities, the overall assessment development
will be quite different.
Comment: One reviewer commented that there is a considerable amount of information
about the toxicokinetics of related halocarbons (e.g., trichloroethylene, perchloroethylene,
chloroform, 1,1,1-trichloroethane) in rodents and humans, and that the rank of metabolic
activation of the compounds is: mice » rats > humans. Therefore, the toxicokinetic
component of the interspecies UF of 10 could be reduced, resulting in an interspecies
uncertainty factor of 3.
Response: The potential for difference between animal and human toxicokinetics following
1,1,2,2-tetrachloroethane exposure based on information from related halocarbons was added
to Section 5.3, Uncertainties in the Oral Reference Dose and Inhalation Reference
Concentration. Upon further evaluation, this information was not considered sufficient to
reduce the UF for 1,1,2,2-tetrachloroethane and the UF of 10 was retained.
Comment: A reviewer commented that Section 5.3 is a restatement of the features that
contributed to the valuation of the standard uncertainty factors, and recommended a
consideration of what additional uncertainties are present that might impact the results.
Response: Additional text was added to this section in response to the reviewer's comments.
C. Inhalation Reference Concentration (RfC) for 1,1,2,2-tetrachloroethane
1. An RfC for 1,1,2,2-tetrachloroethane has not been derived. Has the scientific
justification for not deriving an RfC been described in the document? Please identify
and provide the rationale for any studies that should be selected as the principal study.
Please identify and provide the rationale for any endpoints that should be considered in
the selection of the critical effect.
Comment: The reviewers agreed with the decision not to derive an RfC. One reviewer
commented that a comparison to metabolically-related compounds is useful and
recommended including this information in the discussion of the uncertainties associated
with not deriving an RfC.
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Response: Additional text related to uncertainties was added to Section 5.3.
D. Carcinogenicity of l,l?2,2-tetrachloroethane
1. Under EPA's 2005 Guidelines for carcinogen risk assessment (www.epa.gov/iris/backgr-
d.htm), the Agency concluded that l,l?2,2-tetrachloroethane is likely to be carcinogenic
to humans by all routes of exposure. Please comment on the cancer weight of the
evidence characterization. Is the cancer weight of evidence characterization
scientifically justified?
Comment: One reviewer did not concur with the conclusion that 1,1,2,2-tetrachloroethane is
likely to be carcinogenic to humans, and thought it would be more accurate to characterize
1,1,2,2-tetrachloroethane as a possible human carcinogen. This reviewer also commented
that the WOE characterization for 1,1,2,2-tetrachloroethane may not be applicable to all
routes of exposure based on general toxicokinetic differences between oral and inhalation
exposure. A second reviewer commented that the conclusion that 1,1,2,2-tetrachloroethane
is likely to be carcinogenic to humans is one of the weakest likely to be carcinogenic to
humans characterizations demonstrated when the data is singularly considered; in addition,
given the prevalence of and susceptibility to developing liver tumors in B6C3Fi mice, the
reviewer questioned whether a slope factor should be derived from this study. This reviewer
also advocated incorporating carcinogenicity information from dichloroacetic acid,
trichloroethylene, and perchloroethylene into the document to strengthen the WOE
characterization. A third reviewer agreed with the likely to be carcinogenic to humans
determination..
Response: The cancer weight of evidence descriptor for 1,1,2,2-tetrachloroethane is based
on the statistically significant increase in the incidence of hepatocellular carcinomas in both
male and female B6C3Fi mice, and the rare hepatocellular tumors observed in the male
Osborne-Mendel rats were considered a rare tumor by NCI (1978). According to the
Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), the likely to be carcinogenic
to humans descriptor is supported when an agent has tested positive in animal experiments in
more than one species, sex, strain, site, or exposure route with or without evidence of
carcinogenicity in humans, and in the case of 1,1,2,2-tetrachloroethane, a positive tumor
response was observed in both male and female mice. This descriptor is also supported when
a rare animal tumor is observed in a single experiment that is assumed to be relevant to
humans, and in the case of 1,1,2,2-tetrachloroethane, NCI (1978) considered the liver tumors
observed in male rats to be a rare tumor response. Goodman et al. (1980) observed
hepatocellular carcinomas in 2 male (n=975) and 4 female (n=970) Osborne-Mendel rats that
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were untreated, corn oil fed, or corn oil gavage controls, and stated that historical data
indicates the general rarity or prevalence of tumors and may be useful in assessing biological
significance.
According to the 2005 Cancer Guidelines, the cancer WOE characterization applies to
all exposure routes that have not been adequately tested at sufficient doses when tumors are
observed at a site other than the initial point of contact. In the case of 1,1,2,2-
tetrachoroethane, tumors were observed in the liver of both sexes of mice and in rats
following oral exposure, and the database for 1,1,2,2-tetrachloroethane lacks inhalation
studies that would be useful in determining a WOE characterization for the inhalation route.
Additional text was added to the discussion of the potential susceptibility of B6C3Fi
mice to developing hepatocellular carcinomas following 1,1,2,2-tetrachloroethane exposure
in Section 5.4.5, Uncertainties in Cancer Risk Values.
Section 4.7.1, Summary of Overall Weight of Evidence, presents the carcinogenicity
data available for 1,1,2,2-tetrachloroethane and describes the weight of the evidence cancer
descriptor. This section also includes a discussion of the carcinogenicity data available for
dichloroacetic acid, trichloroethylene, and perchloroethylene.
2. A two-year oral gavage cancer bioassay (NCI, 1978) was selected as the principal study
for the derivation of an oral slope factor. Please comment on the appropriateness of the
selection of the principal study.
Comment: The reviewers generally agreed with the selection of the NCI (1978) study as the
principal study for the development of an oral slope factor, although the reviewers
highlighted that this was the only study available for this purpose.
Response: Comment acknowledged.
Comment: One reviewer commented that the NCI study used poorly selected dose levels that
were adjusted during the course of the study, the exposure duration was 78 weeks as opposed
to the more standard 104 weeks, that there was also a concurrent disease (pneumonia)
observed, and that these deficiencies and resulting uncertainties need to be stated in the
document.
Response: Text was added to Sections 5.4.1., Choice of Study/Data—with Rationale and
Justification, and 5.4.5, Uncertainties in Cancer Risk Values, to address the concern
associated with the dose selection and modification, the exposure duration, and the increased
incidence of chronic murine pneumonia in the rats.
A-9
-------
Comment: A reviewer expressed concerns that gavage dosing may deliver the chemical in a
short term bolus dose and may not provide the same results as a dietary or other oral dosing
method that delivers the chemical more gradually over time.
Response: The potential effect of the corn oil vehicle, as well as the bolus nature of the
gavage dose, on the effects observed in the liver following 1,1,2,2-tetrachloroethane
exposure has been added to Section 5.4.5, Uncertainties in Cancer Risk Values.
3. An increased incidence of hepatocellular carcinomas in B6C3Fi mice was used to
estimate the oral cancer slope factor. Please comment on the scientific justification of
this analysis. Has the BMD modeling been appropriately conducted?
Comment: Several reviewers considered the modeling of the increased incidence of
hepatocellular tumors in B6C3Fi mice to be justified and appropriate. One reviewer
commented that maybe an oral slope factor should not be derived given the prevalence of and
susceptibility to developing liver tumors in this strain of mice. A reviewer commented that
both sexes of B6C3Fi mice have a high spontaneous cancer incidence and referenced a study
by Haseman et al. (1998) which reported that male B6C3Fi control mice have a 42% liver
cancer incidence. This reviewer recommended including a discussion addressing this in the
uncertainty section.
Response: The U.S. EPA considers liver tumors in mice to be relevant to humans unless
chemical-specific information is available to indicate otherwise. Text addressing this issue is
included in Section 5.4.5, Uncertainties in Cancer Risk Values.
Text was also added to Section 5.4.5, Uncertainties in Cancer Risk Values.,
addressing the high spontaneous cancer incidence of liver cancer in male B6C3Fi mice. The
42% liver cancer rate for male B6C3Fi mice was for liver adenomas and carcinomas
combined, but the NCI (1978) study reported only hepatocellular carcinomas. Haseman et al.
(1998) reported a lower spontaneous cancer incidence for hepatocellular carcinomas; 17.9
and 8.4% for male and female B6C3Fi mice, respectively.
Even though the B6C3Fi strain is frequently associated with a high spontaneous
cancer incidence, the incidence in the control mice in NCI (1978) was rather low; 1/18 in the
male vehicle controls and 0/20 in the female vehicle controls, and 3/36 and 1/40 in male and
female pooled-vehicle controls, respectively. Comparison of an experimental group with its
concurrent controls was considered to be the most appropriate comparison (Haseman et al.,
1992; Tarone et al., 1981; Gart et al., 1979 as cited in Haseman et al., 1998; Goodman et al.,
1980) and, in this case, the control values were considered low.
A-10
-------
Comment: One reviewer requested additional model output information in Appendix C
describing how the multi-stage model fit the data points, even if the reported goodness-of-fit
p-va\ue was provided as "NA" because of too many model parameters.
Response: In response to this comment, the incidence of hepatocellular carcinomas in male
and female mice was remodeled using the most recent version of BMDS (version 2.1.1). The
relevant information describing the fit of both the one- and two-stage multistage models to
these incidence data have now been included in Appendix C.
Comment: A reviewer requested additional analysis of the mode of action of carcinogenesis,
as the preponderance of genotoxicity data suggest that 1,1,2,2-tetrachloroethane is not
genotoxic and the data available indicate promotion potential. This reviewer recommended
an uncertainty factor approach for the cancer assessment. A second reviewer also
commented that it is more likely that 1,1,2,2-tetrachloroethane may act as a tumor promoter,
and that regenerative hyperplasia is a more likely mode of action, provided that the majority
of the in vitro and in vivo genotoxicity and mutagenicity studies yielded non-positive results.
Response: The two studies (Milman et al. [1988] and Story et al. [1986]) providing some
evidence to support the promotion potential of 1,1,2,2-tetrachloroethane were added to
Section 4.7.3, Mode of Action of Carcinogenicity Information. However, the key events
associated with any hypothesized mode of action of carcinogenesis of 1,1,2,2-tetra-
chloroethane, whether mutagenic or promotional, cannot be determined with the information
available.
Comment: A reviewer commented that mice and other rodents metabolize a considerably
larger portion of high doses of halocarbons than humans, and, therefore, experience more
severe hepatocellular injury, greater formation of covalent adducts, and higher cancer
incidences.
Response: Text was added to Section 5.4.5, Uncertainties in Cancer Risk Values, addressing
the potential difference between animal and human toxicokinetics following 1,1,2,2-
tetrachloroethane exposure, based on information from related halocarbons demonstrating
increased metabolic activation in mice compared with humans.
Comment: A reviewer recommended that the document highlight that dichloroacetic acid
and trichloroacetic acid are metabolites of both trichloroethylene and tetrachloroethylene, so
the hepatocarcinogenic effects of these metabolites should be additive; however, it is
important to point out that quantitative data on the quantities of these metabolites formed by
A-ll
-------
rodents or humans are unavailable. The reviewer recommended including a discussion
addressing this in the uncertainty section.
Response: The document was not modified because the hepatocarcinogenic effects of
dichloroacetic acid and trichloroacetic acid, whether they are additive or not, have no bearing
on the carcinogenicity observed for 1,1,2,2-tetrachloroethane.
Comment: A reviewer commented that the document should recognize that administration of
large quantities of corn oil promotes lipid accumulation and lipoperoxidative damage of
hepatocytes, and that corn oil is believed to be tumorigenic in rats and humans through
increased expression of protooncogenes, decreased apotosis, mitogenesis, etc. The reviewer
recommended including a discussion addressing this in the uncertainty section.
Response: EPA has included text in Section 5.4.5, Uncertainties in Cancer Risk Values, that
addresses the bolus administration of 1,1,2,2-tetrachloroethane was corn oil.
A-12
-------
APPENDIX B. BENCHMARK DOSE MODELING RESULTS FOR THE DERIVATION
OF THE RfD
Dichotomous Endpoints
Incidence of hepatocellular cytoplasmic vacuolization in male and female rats (NTP, 2004)
Table B-l. Incidences of hepatocellular cytoplasmic vacuolization in rats
exposed to dietary l,l?2,2-tetrachlorethane for 14 weeks
Nonneoplastic lesion
Dose (mg/kg-d)
Vehicle control
20
40
80
170
320
Males3
Hepatocellular cytoplasmic
vacuolization
0/10
7/10b
(1.3)
9/10b
(2.0)
10/10b
(1.9)
8/10b
(1.4)
0/10
Females3
Hepatocellular cytoplasmic
vacuolization
0/10
0/10
10/10b
(1.7)
10/10b
(2.2)
4/10b
(1.3)
0/10
3Values represent proportion of animals with the lesion; for those dose groups in which lesions were found, the
average severity score is in parentheses; severity grades were as follows: 1 = minimal, 2 = mild, 3 = moderate,
4 = severe.
bStatistically significantly different from vehicle control group.
Source: NTP (2004).
All available dichotomous models (except the "quantal-linear" and "quantal-quadratic")
in the EPA's BMDS (version 2.1.1) were fit to the incidence of hepatocellular cytoplasmic
vacuolization in male and female rats administered 1,1,2,2-tetrachloroethane in the diet for
14 weeks. Table B-l displays the incidence data for this endpoint for both males and females.
BMDs and their associated 95% lower confidence limits (i.e., BMDLs) at an extra risk of 10%
were estimated by each model. The results of this BMD modeling for male and female rats are
summarized in Tables B-2 and B-3, respectively, and the BMDS output from the selected model
is displayed following each table.
B-l
-------
Table B-2. Summary of BMD modeling results for the incidence of
hepatocellular cytoplasmic vacuolization in male rats
Model
DF
x2
/2 Goodness of
fit
/rvalue"
Scaled residuals
of interest1"
AIC
BMD10
(mg/kg-d)
BMDL10
(mg/kg-d)
All dose groups included
BMDS was unable to generate model outputs
Highest dose group dropped
Gamma0
Logistic
Log-logisticd'e
Log-probitd
Multistage (1 -degree/
Probit
Weibulf
4
3
4
4
4
3
4
57.61
22.78
6.78
36.46
57.61
20.45
57.61
0.001
0.001
0.15
0.001
0.001
0.001
0.001
0.00/1.66
-2.77/1.01
0.00/-0.06
0.00/0.85
0.00/1.66
3.00/0.94
0.00/1.66
47.97
57.05
36.14
41.77
47.97
58.24
47.97
3.64
10.59
0.91
4.70
3.64
13.29
3.64
2.60
6.70
0.40
3.03
2.60
8.99
2.60
Two highest dose groups dropped
Gamma0
Logistic
Log-logistic"1
Log-probitd
Multistage (1 -degree/'8
Multistage (2 -degree/
Multistage (3 -degree/
Probit
Weibull0
2
2
2
2
3
2
2
2
2
0.10
2.50
0.25
0.18
0.10
0.08
0.06
2.56
0.10
0.95
0.29
0.88
0.92
0.99
0.96
0.97
0.28
0.95
0.00/0.08
-0.82/0.81
0.00/0.09
0.00/0.10
0.00/-0.02
0.00/0.12
0.00/0.13
-0.81/1.03
0.00/0.10
22.87
25.51
23.09
22.98
20.89
22.83
22.80
25.71
22.86
2.47
6.78
6.16
5.49
1.73
1.99
1.89
6.45
2.32
1.12
3.67
0.31
1.80
1.12
1.12
1.13
3.73
1.12
AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the dose associated with the selected
BMR; BMDL = 95% lower confidence limit on the BMD; DF = degrees of freedom
aValues O.I fail to meet conventional goodness-of-fit criteria.
bScaled residuals at doses immediately below and immediately above the BMD.
°Power restricted to >1.
dSlope restricted to >1.
eBetas restricted to >0.
fAlthough the overall goodness of fit p-value suggested adequate fit of this model to the data, the model was
rejected because the very high residual at the high dose (-2.32) suggested that fit of the model to the data would be
improved by dropping that dose.
8Selected model is displayed in boldface type. BMDLs for models with adequate fit differed by greater than
threefold. However, the results from the log-logistic model were rejected as unreliable due to the large spread
between BMD and BMDL (20-fold) and because the BMDL from this model was an outlier in relation to the results
of the other models. After dropping this model, the results of the other models were within approximately
threefold. Among the remaining models, the 1-degree polynomial had the lowest AIC and also produced the lowest
BMDL and was therefore selected as the most suitable model for this dataset.
B-2
-------
As shown in Table B-2, in attempting to model the incidence of hepatocellular
cytoplasmic vacuolization in male rats with all six dose groups included, the BMDS failed to
generate any output because response was not a monotonically increasing function of dose (i.e.,
the response in the penultimate dose group was 80%, while the response in the highest dose
group was 0). A key underlying assumption for the fitting of the dichotomous models in BMDS
is that response must be a monotonically non-decreasing function of dose. Therefore, the highest
r\
dose group was dropped and the models were fit to the data again. In this instance, the x
goodness-of-fit test found that all models exhibited inadequate fit (i.e.,/? < 0.1). Finally, in an
attempt to find a model that fit, the two highest dose groups were dropped and the models were
refit to these data. In this case, all of the models exhibited adequate fit (p > 0.10).
Of these models exhibiting adequate fit, a "best-fit" model was selected consistent with
the EPA's Benchmark Dose Technical Guidance Document (U.S. EPA, 2000b) as follows. If the
BMDL estimates from the models exhibiting adequate fit are "sufficiently close," then the model
with the lowest AIC is to be used to estimate the BMDL from which the POD will be derived. In
this particular case, as explained in the footnote in Table B-2, BMDLs for models with adequate
fit differed by greater than threefold. However, the results from the log-logistic model were
rejected as unreliable due to the large spread between BMD and BMDL (20-fold) and because
the BMDL from this model was an outlier in relation to the results from the other models. After
dropping the log-logistic model, the BMDLs from the other models were within approximately
threefold. Among the remaining models, the one-stage multistage model had the lowest AIC and
also produced the lowest BMDL, and was, therefore, selected as the most suitable model for this
dataset. The BMDLio from this model (i.e., 1.12 mg/kg-day) was then selected as a possible
POD. The standard BMDS output from the one-stage multistage model is displayed below.
B-3
-------
Multistage Model with 0.95 Confidence Level
"o
ro
0.8
0.6
0.4
0.2
0 : •*
EMDL
11:41 03/302010
70
80
Multistage Model. (Version: 3.0; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\TCE\NTP\hepcytvac\male\mst_hepcytvacM2HDD_MS_l.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\TCE\NTP\hepcytvac\male\mst_hepcytvacM2HDD_MS_l.plt
Tue Mar 30 12:41:48 2010
HMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl)]
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 4
Total number of records with missing values
= 0
B-4
-------
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
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) = 1.28571e+018
Asymptotic Correlation Matrix of Parameter Estimates
Beta (1)
Beta(l)
Variable
Background
Beta(1)
- Indicates that this value is not calculated.
Parameter Estimates
Std. Err.
Model
Full model
Fitted model
Reduced model
AIC:
P-value
Goodness of Fit
Dose
0.0000
20.0000
40.0000
80.0000
Est. Prob.
0.0000
0.7034
0.9120
0.9923
Expected
0.000
7.034
9.120
9.923
Observed
0.000
7.000
9.000
10.000
Size
10
10
10
10
Scaled
Residual
0.000
-0.024
-0.134
0.279
ChiA2 = 0.10
d.f. = 3
P-value = 0.9922
Benchmark Dose Computation
B-5
-------
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 1.73382
BMDL = 1.11682
BMDU = 2.71595
Taken together, (1.11682, 2.71595) is a 90 % two-sided confidence
interval for the BMD
B-6
-------
Table B-3. Summary of BMD model results for the incidence of
hepatocellular cytoplasmic vacuolization in female rats
Model
DF
x2
/2 Goodness of fit
/rvalue"
Scaled
residuals of
interest1"
AIC
BMD10
(mg/kg-d)
BMDL10
(mg/kg-d)
All dose groups included
BMDS was unable to generate model outputs
Highest dose group dropped
Gamma0
Logistic
Log-logistic"1
Log-probitd
Multistage (1 -degree polynomial)6
Probit
Weibull0
4
3
4
4
4
3
4
45.13
38.70
31.61
49.11
45.13
38.70
45.13
0.001
0.001
0.001
0.001
0.001
0.001
O.001
0.00/-1.66
-2.52/3.63
0.00/-2.36
0.00/-1.61
0.00/-1.66
-2.50/3.65
0.00/-1.66
61.33
69.75
53.57
58.57
61.33
69.79
61.33
8.65
30.61
3.99
12.62
8.65
31.28
8.65
6.18
18.21
2.24
8.86
6.18
19.39
6.18
Two highest dose groups dropped
Gamma0
Logistic
Log-logisticd
Log-probitd
Multistage (1 -degree polynomial)6
Multistage (2 -degree polynomial)6
Multistage (3 -degree polynomial)6
Probit
Weibullc'f
o
J
2
o
5
3
o
J
o
J
o
J
2
3
1.56
0.00
0.04
0.00
13.83
7.48
4.41
0.00
0.00
0.67
1.00
1.00
1.00
0.003
0.06
0.22
1.00
1.00
-0.95/0.82
0.00/0.00
-0.14/0.14
0.00/0.00
0.00/-3.09
0.00/-2.24
0.00/-1.78
0.00/0.00
-0.02/0.01
5.00
4.00
2.08
2.00
22.89
14.54
9.85
4.00
2.00
20.59
29.46
25.03
26.36
3.14
10.17
14.53
28.77
30.68
17.05
19.38
19.51
19.56
2.05
5.95
9.15
19.85
19.16
AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the concentration associated with the
selected BMR; BMDL = 95% lower confidence limit on the BMD; DF = degrees of freedom
aValues O.I fail to meet conventional goodness-of-fit criteria.
bScaled residuals at doses immediately below and immediately above the BMD.
°Power restricted to >1.
dSlope restricted to >1.
6Betas restricted to >0.
Selected model is displayed in boldface type. BMDLs for models with adequate fit differed by less than threefold,
so the models with the lowest AIC (Log-probit and Weibull models) were initially selected as the best fitting. The
Weibull model had a slightly lower BMDL between the two models; thus, the Weibull was selected.
As shown in Table B-3, in attempting to model the incidence of hepatocellular
cytoplasmic vacuolization in female rats with all six dose groups included, the BMDS failed to
generate any output because response was not a monotonically increasing function of dose (i.e.,
the response in the penultimate dose group was 40%, while the response in the highest dose
group was 0). A key underlying assumption for the fitting of the dichotomous models in BMDS
is that response must be a monotonically non-decreasing function of dose. Therefore, the highest
r\
dose group was dropped, and the models were fit to the data again. In this instance, the x
goodness-of-fit test showed that all models exhibited inadequate fit (i.e.,/? < 0.1). Finally, in an
B-7
-------
attempt to find a model that fit, the two highest dose groups were dropped and the models were
refit to these data. In this case, all of the models exhibited adequate fit, except for the one- and
two-stage multistage models (p > 0.10).
Of the models exhibiting adequate fit, a "best-fit" model was selected consistent with the
EPA's Benchmark Dose Technical Guidance Document (U.S. EPA, 2000b) as follows. If the
BMDL estimates from the models exhibiting adequate fit are "sufficiently close," then the model
with the lowest AIC is to be used to estimate the BMDL from which the POD will be derived. In
this particular case, as explained in the footnote in Table B-3, BMDLs for models with adequate
fit differed by less than threefold. Among these models, the log-probit and Weibull models
shared the lowest AIC, and, thus, the average BMDLio from these two models (i.e., 19.36 mg/kg-
day) was used to derive a possible POD. The standard BMDS outputs from the log-probit and
Weibull models are displayed below.
B-S
-------
LogProbit Model with 0.95 Confidence Level
0)
-5
o
'
0.8
0.6
0.4
0.2
LogProbit
BMDL BMD
10 20 30 40 50 60 70 80
dose
11:5403/302010
Probit Model. (Version: 3.1; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\TCE\NTP\hepcytvac\female\lnp_hepcytvacF2HDD_logprobit.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\TCE\NTP\hepcytvac\female\lnp_hepcytvacF2HDD_logprobit.plt
Tue Mar 30 12:54:34 2010
HMDS Model Run
The form of the probability function is:
P[response] = Background
+ (1-Background) * CumNorm(Intercept+Slope*Log(Dose) ) ,
where CumNorm(.) is the cumulative normal distribution function
Dependent variable = incidence
Independent variable = dose
Slope parameter is restricted as slope >= 1
Total number of observations = 4
Total number of records with missing values
Maximum number of iterations = 250
= 0
B-9
-------
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
User has chosen the log transformed model
Default Initial (and Specified) Parameter Values
background = 0
intercept = -8.43383
slope = 2.43905
Asymptotic Correlation Matrix of Parameter Estimates
intercept
intercept
1
Parameter Estimates
- 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
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Goodness of Fit
P-value
Dose
0.0000
20.0000
40.0000
80.0000
Est. Prob.
0.0000
0.0000
1.0000
1.0000
Expected
0.000
0.000
10.000
10.000
Observed
0.000
0.000
10.000
10.000
Size
10
10
10
10
Scaled
Residual
0.000
-0.000
0.000
0.000
ChiA2 = 0.00
d.f. =3
P-value = 1.0000
Benchmark Dose Computation
Specified effect = 0.1
B-10
-------
Risk Type = Extra risk
Confidence level = 0.95
BMD = 26.3597
BMDL = 19.557
B-ll
-------
Weibull Model with 0.95 Confidence Level
-
"o
I
"o
ro
0.8
0.6
0.4
0.2
11:5403/302010
Weibull
BMDL
BMD
10
20
30
40
dose
50
60
70
80
Weibull Model using Weibull Model (Version: 2.12; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\TCE\NTP\hepcytvac\female\wei_hepcytvacF2HDD_weibull.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\TCE\NTP\hepcytvac\female\wei_hepcytvacF2HDD_weibull.plt
Tue Mar 30 12:54:37 2010
HMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(-slope*doseApower)]
Dependent variable = incidence
Independent variable = dose
Power parameter is restricted as power >=1
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
B-12
-------
Default Initial (and Specified) Parameter Values
Background = 0.0454545
Slope = 0.00369372
Power = 1.53227
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Power
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
Slope
Slope -1.$
Parameter Estimates
Variable
Background
Slope
Power
Std. Err.
NA
1.IQNAN
NA
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
AIC:
2.00103
P-value
1
Goodness of Fit
Dose
0.0000
20.0000
40.0000
80.0000
Est. Prob.
0.0000
0.0000
1.0000
1.0000
Expected
0.000
0.000
10.000
10.000
Observed
0.000
0.000
10.000
10.000
Size
10
10
10
10
Scaled
Residual
0.000
-0.022
0.006
0.000
ChiA2 = 0.00
d.f. =3
P-value = 1.0000
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
B-13
-------
BMD = 30.681
BMDL = 19.1631
B-14
-------
Continuous Endpoints
Organ weight and serum chemistry changes in male and female rats (NTP, 2004)
Table B-4. Selected organ weight and serum chemistry changes in male and
female F344 rats administered l,l?2,2-tetrachlroethane in the diet for
14 weeks
Endpoint
Absolute liver wt (g)
Relative liver wt (mg
organ wt/g body wt)
Serum ALT activity
(IU/L)
Serum SDH activity
(IU/L)
Serum bile acid
levels (umol/L)
Sex
Male
Female
Male
Female
Male
Female
Male
Female
Male
Female
Dose (mg/kg-d)
0
12.74 ± 0.26a
6.84 ±0.17
34.79 ±0.42
35.07 ±0.56
48 ±2
46 ±2
23 ±1
27 ±1
29.2 ±2.9
37.0 ±7.1
20
12.99 ±0.35
7.03 ±0.13
36.72 ±0.44
36.69 ±0.36
49 ±2
42 ±1
27 ±1
27 ±1
27.5 ±2.7
46.6 ±6.5
40
14.47 ±0.44
7.14 ±0.16
41.03 ±0.85
37.84 ±0.51
53 ±2
41±2
26 ±2
28 ±2
27.2 ±2.7
39.1 ±5.6
80
15.54 ±0.40
7.80 ±0.08
45.61 ±0.52
44.20 ± 0.27
69 ±3
49 ±2
31±1
25 ±1
35.9 ±3.9
36.3 ±3.9
170
11.60 ±0.44
6.66 ± 0.22
44.68 ±0.45
48.03 ±0.89
115±8
112±7
47 ±2
45 ±3
92.0 ± 16.6
39.3 ±7.9
320
6.57 ±0.18
4.94 ±0.12
52.23 ± 1.42
58.40 ±1.42
292 ±18
339 ±18
74 ±4
82 ±3
332.4 ±47.4
321. 5 ±50.6
aValues are means ± SE for 10 animals.
Source: NTP (2004).
All available continuous models in the EPA's BMDS (version 2.1.1) were fit to each of
the endpoints listed in Table B-4 for both male and female rats administered 1,1,2,2-tetrachloro-
ethane in the diet for 14 weeks. BMDs and their 95% lower confidence limits (i.e., BMDLs)
associated with a change in the response of 1 SD from the control were estimated by each model.
The results of this BMD modeling for male and female rats are summarized in Tables B-5
through B-14. Following each table is the BMDS output for the selected model.
The model fitting procedure for continuous data was as follows. The simplest model
(linear) is first applied to the data while assuming constant variance. If the data are consistent
with the assumption of constant variance (p > 0.1), then the fit of the linear model to the means is
evaluated and the polynomial, power, and Hill models are fit to the data while assuming constant
variance. In accordance with U.S. EPA (2000b) guidance, BMDs and BMDLs are estimated
employing a BMR that represents a change of 1 SD from the control. Adequate model fit is
judged primarily by the goodness-of-fit/>-value (p > 0.1), but visual inspection of the dose-
response curve and the examination of scaled residual at the data point (except the control)
closest to the predefined BMR also play a role. If the test for constant variance is negative, the
linear model is run again while applying the power model integrated into BMDS to account for
nonhomogeneous variance. If the nonhomogeneous variance model provides an adequate fit
(p > 0.1) to the variance data, then the fit of the linear model to the means is evaluated and the
B-15
-------
polynomial, power, and Hill models are fit to the data and evaluated while the variance model is
applied. If no model provides adequate fit to the data based on these criteria, then the highest
dose is dropped, if appropriate, and the continuous modeling procedure is repeated.
Absolute liver weights in male and female rats (Tables B-5 andB-6)
No adequate fit to the data for absolute liver weight in males or females was achieved
until the two highest doses were dropped. After dropping the two highest doses, the assumption
of constant variance was met, and all models provided adequate fit (except the Hill model, which
has too many parameters for the number of remaining data points). BMDL estimates across the
models with adequate fit differed by less than threefold. In accordance with U.S. EPA (2000b),
the model with the lowest AIC (linear, for both males and females) was selected as the basis for
the BMDiso and BMDLiso estimates for these endpoints (respectively, 30 and 23 mg/kg-day for
males, and 36 and 26 mg/kg-day for females).
B-16
-------
Table B-5. Summary of BMD modeling results for absolute liver weight in
male rats
Model
Test for
significant
difference
/7-valuea
Variance
/7-valueb
Mean
/7-valueb
Scaled
residuals of
interest0
AIC
BMD1SD
(mg/kg-d)
BMDL1SD
(mg/kg-d)
All dose groups included
Constant variance
Lineard
O.0001
0.07
O.0001
NA
198.13
NA
3,925.92
Non-constant variance
Hill6
Lineard
Polynomial (2-degree)d
Polynomial (3 -degree)d
Polynomial (4-degree)d
Polynomial (5-degree)d
Power6
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.39
0.39
0.39
0.39
0.39
0.39
0.39
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
-0.7/1.81
NA
NA
NA
NA
NA
-1.43/0.08
160.48
200.13
200.13
200.13
200.13
200.13
106.77
36.49
NA
NA
NA
NA
NA
173.92
NA
10.43
10.45
733.03
595.06
533.37
141.52
Highest dose group dropped
Constant variance
Hill"
Lineard
Polynomial (2-degree)d
Polynomial (3-degree)d
Polynomial (4-degree)d
Powere
0.0001
0.0001
0.0001
O.0001
O.0001
O.0001
0.49
0.49
0.49
0.49
0.49
0.49
0.0001
0.0001
0.0001
O.0001
O.0001
O.0001
3.3/0.00
NA
NA
NA
NA
3.3/0.00
100.95
112.67
112.67
112.67
112.67
98.95
165.58
NA
NA
NA
NA
166.09
94.36
606.09
416.42
326.66
282.11
145.65
Two highest dose groups dropped
Constant variance
Hill"
Lineard'f
Power6
O.0001
<0.0001
O.0001
0.41
0.41
0.41
NA
0.32
0.13
0.00/0.00
-1.07/0.97
-1.03/1.01
57.97
56.26
58.25
32.10
30.40
31.30
20.62
22.92
22.93
AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the dose associated with the selected
BMR; BMDL = 95% lower confidence limit on the BMD;; NA = not applicable (BMD/BMDL computation failed
or insufficient DF)
aValues >0.05 fail to meet conventional goodness-of-fit criteria.
bValues O.10 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals at doses immediately below and immediately above the BMD.
dCoefficients restricted to be positive.
ePower restricted to >1.
fBest-fitting model is displayed in boldface type. BMDLs for models providing adequate fit differed by less than
threefold, so the model with the lowest AIC was selected.
B-17
-------
o
Q.
£=
ro
0)
16
15
14
13
12
Linear Model with 0.95 Confidence Level
Linear
BMDL
BMD
10
20
30
40
dose
50
60
70
80
14:1203/262010
B-18
-------
Polynomial Model. (Version: 2.13; Date: 04/08/2008)
Input Data File:
C:\USEPA\IRIS\TCE\NTP\abslivwt\male\lin_abslivwtM2HDD_linear.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\TCE\NTP\abslivwt\male\lin_abslivwtM2HDD_linear.plt
Fri Mar 26 15:12:39 2010
HMDS Model Run
The form of the response function is:
Y[dose] = beta 0 + beta l*dose + beta 2*doseA2 + ...
Dependent variable = mean
Independent variable = dose
rho is set to 0
The polynomial coefficients are restricted to be positive
A constant variance model is fit
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 = 1.35605
rho = 0 Specified
beta_0 = 12.626
beta 1 = 0.0374
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -rho
have been estimated at a boundary point, or have been
specified by the user,
and do not appear in the correlation matrix )
alpha beta_0 beta_l
alpha 1 -6.9e-010 -4.8e-011
beta_0 -6.9e-010 1 -0.76
beta 1 -4.8e-011 -0.76 1
Parameter Estimates
Variable
alpha
beta 0
B-19
-------
beta 1 0.0374
12.7
13
14.5
15.5
12 . 6
13.4
14.1
15. 6
0.82
1.11
1.39
1.26
1.14
1.14
1.14
1.14
0.317
-1.07
0. 968
-0.217
Model Descriptions for likelihoods calculated
Model Al: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)} = SigmaA2
Likelihoods of Interest
Model Log(likelihood) # Param's AIC
Al -23.984311 5 57.968622
A2 -22.556035 8 61.112070
A3 -23.984311 5 57.968622
fitted -25.129323 3 56.258645
R -38.455553 2 80.911106
Explanation of Tests
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.
B-20
-------
Tests of Interest
-2*log(Likelihood Ratio) Test df
p-value
31.799
2.85655
2.85655
2.29002
6
3
3
2
<.0001
0.4143
0.4143
0.3182
Test
Test 1
Test 2
Test 3
Test 4
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is greater than .1. A homogeneous variance
model appears to be appropriate here
The p-value for Test 3 is greater than .1.
to be appropriate here
The p-value for Test 4 is greater than .1.
to adequately describe the data
The modeled variance appears
The model chosen seems
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 30.3962
BMDL =
22.9198
B-21
-------
Table B-6. Summary of BMD modeling results for absolute liver weight in
female rats
Model
Test for
significant
difference
/7-valuea
Variance
^-valueb
Mean
/7-valueb
Scaled
residuals of
interest0
AIC
BMD1SD
(mg/kg-d)
BMDL1SD
(mg/kg-d)
All dose groups included
Constant variance
Lineard
O.0001
0.05
O.0001
NA
62.98
NA
3,632.46
Non-constant variance
Lineard
0.0001
0.02
0.0001
NA
64.98
NA
24.07
Highest dose group dropped
Constant variance
Lineard
0.0001
0.04
0.0001
NA
5.69
NA
377.10
Non-constant variance
Hill"
Lineard
Polynomial (2 -degree)d
Polynomial (3-degree)d
Polynomial (4-degree)d
Power6
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
0.84
0.84
0.84
0.84
0.84
0.84
0.0001
0.0001
0.0001
0.0001
0.0001
0.0001
o.oof
NA
NA
NA
NA
o.oof
4.52
7.69
7.69
7.69
7.69
2.52
170.20
NA
NA
NA
NA
170.19
NA
397.23
343.87
290.54
67.91
153.95
Two highest dose groups dropped
Constant variance
Hill"
Lineard'g
Polynomial (2-degree)d
Polynomial (3-degree)d
Power6
O.0001
<0.0001
O.0001
O.0001
O.0001
0.11
0.11
0.11
0.11
0.11
NA
0.55
0.63
0.71
0.57
-0.30/0.05
0.05/-0.91
-0.28/0.05
-0.19/0.02
-0.30/0.05
-19.17
-22.27
-21.25
-21.35
-21.17
48.28
35.62
48.21
49.83
48.28
25.37
26.10
27.58
27.77
27.44
AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the dose associated with the selected
BMR; BMDL = 95% lower confidence limit on the BMD; NA = not applicable (BMD/BMDL computation failed
or insufficient DF to fit model)
aValues >0.05 fail to meet conventional goodness-of-fit criteria.
bValues O.10 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals at doses immediately below and immediately above the BMD.
dCoefficients restricted to be positive.
ePower restricted to >1.
fResidual at highest dose tested.
8Best-fitting model displayed in boldface type. BMDLs for models providing adequate fit differed by less than
threefold, so the model with the lowest AIC was selected.
B-22
-------
Linear Model with 0.95 Confidence Level
7.8
7.6
o
Q.
£=
ro
0)
6.4
BMD
10
20
30 40
dose
50
60
70
80
14:5803/262010
Polynomial Model. (Version: 2.13; Date: 04/08/2008)
Input Data File:
C:\USEPA\IRIS\TCE\NTP\abslivwt\female\lin_abslivwtF2HDD_linear.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\TCE\NTP\abslivwt\female\lin_abslivwtF2HDD_linear.plt
Fri Mar 26 15:58:54 2010
HMDS Model Run
The form of the response function is:
Y[dose] = beta 0 + beta l*dose + beta 2*doseA2 + ...
Dependent variable = mean
Independent variable = dose
rho is set to 0
The polynomial coefficients are restricted to be positive
A constant variance model is fit
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
B-23
-------
Default Initial Parameter Values
alpha = 0.195575
rho = 0 Specified
beta_0 = 6.784
beta 1 = 0.0119571
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -rho
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
alpha
beta 0
beta 1
alpha
1
-8e-009
8.2e-009
beta 0
-8e-009
1
-0.76
beta 1
8.2e-009
-0.76
1
Parameter Estimates
beta 1
Model Descriptions for likelihoods calculated
Model Al: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e (i) } = SigmaA2
B-24
-------
Likelihoods of Interest
Model
Al
A2
A3
fitted
R
Log(likelihood)
14.743437
17.781442
14.743437
14.137196
3.648385
# Param' s
5
8
5
3
2
AIC
-19.486874
-19.562884
-19.486874
-22.274391
-3.296770
Explanation of Tests
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
Test 1
Test 2
Test 3
Test 4
-2*log(Likelihood Ratio) Test df
28.2661
6.07601
6.07601
1.21248
6
3
3
2
p-value
<.0001
0.108
0.108
0.5454
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is greater than .1.
model appears to be appropriate here
A homogeneous variance
The p-value for Test 3 is greater than .1.
to be appropriate here
The p-value for Test 4 is greater than .1.
to adequately describe the data
The modeled variance appears
The model chosen seems
B-25
-------
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 35.6232
BMDL = 26.1046
B-26
-------
Relative liver weights in male and female rats (Tables B-7 andB-8)
No model provided an adequate fit to the relative liver weight data in male rats even after
dropping the two highest dose groups. Therefore, these data are considered unsuitable for BMD
modeling. For the relative liver weight data in females, the assumption of constant variance was
satisfied and the power and 2- and 3-degree polynomial models provided adequate fit to the data
after the highest two dose groups were dropped. BMDL estimates across these models differed
by less than threefold. In accordance with U.S. EPA (2000b), the model with the lowest AIC
(3-degree polynomial) was selected as the basis for the BMDiso and BMDLiso estimates of
22 and 15 mg/kg-day, respectively, for this endpoint.
B-27
-------
Table B-7. Summary of BMD modeling results for relative liver weight in
male rats
Model
Test for
significant
difference />-valuea
Variance
/7-valueb
Mean
/7-valueb
Scaled
residuals
of interest0
AIC
BMD1SD
(mg/kg-d)
BMDL1SD
(mg/kg-d)
All dose groups included
Constant variance
Lineard
O.0001
O.0001
O.0001
1.6/4.15
208.74
68.02
56.64
Non-constant variance
Lineard
O.0001
0.03
O.0001
1.93/4.36
208.89
55.05
37.77
Highest dose group dropped
Constant variance
Lineard
<0.0001
0.09
O.0001
1.84/4.25
165.27
51.62
40.95
Non-constant variance
Lineard
<0.0001
0.06
O.0001
-0.79/-0.95
157.11
12.93
8.10
Two highest dose groups dropped
Constant variance
Lineard
<0.0001
0.07
0.15
0.25/-1.24
94.60
13.14
10.76
Non-constant variance
Lineard
0.0001
0.08
0.09
0.35/-1.32
95.74
10.97
7.77
Three highest doses dropped
Constant variance
Lineard
0.0001
0.03
0.10
0.66/-1.32
74.39
12.16
9.27
Non-constant variance
Hill6
Lineard
Polynomial (2-degree)d
Power6
NA
0.0001
0.0001
0.0001
0.52
0.52
0.52
0.05
NA
NA
0.45/-1.32
-0.07/0.12
-0.07/0.12
71.18
69.32
69.32
8.47
15.27
15.50
6.05
8.46
9.02
AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the dose associated with the selected
BMR; BMDL = 95% lower confidence limit on the BMD; NA = not applicable (insufficient DF to fit the model)
aValues >0.05 fail to meet conventional goodness-of-fit criteria.
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals at doses immediately below and immediately above the BMD.
dCoefficients restricted to be positive.
ePower restricted to >1.
B-28
-------
Table B-8. Summary of BMD modeling results for relative liver weight in
female rats
Model
Test for significant
difference />-valuea
Variance
/7-valueb
Mean
/7-valueb
Scaled
residuals of
interest0
AIC
BMD1SD
(mg/kg-d)
BMDL1SD
(mg/kg-d)
All dose groups included
Constant variance
Lineard
O.0001
O.0001
0.01
-0.66/-1.01
181.20
36.16
30.95
Non-constant variance
Lineard
O.0001
0.01
O.0001
<-10/<-10
6.00
0.003
NA
Highest dose group dropped
Constant variance
Lineard
O.0001
0.002
O.0001
-0.52/-1.19
129.06
26.16
21.87
Non-constant variance
Lineard
O.0001
0.01
0.001
-0.12/-0.30
123.73
16.52
12.39
Two highest dose groups dropped
Constant variance
Hill"
Lineard
Polynomial (2 -degree)d
Polynomial (3-
degree)d'f
Power6
<0.0001
0.0001
0.0001
<0.0001
0.0001
0.11
0.11
0.11
0.11
0.11
NA
0.005
0.22
0.38
0.15
1.12/-0.72
1.31/-0.09
0.94/-0.70
0.69/-0.43
1.12/-0.72
74.32
78.98
71.76
70.98
72.32
25.33
13.20
23.57
21.90
25.31
17.12
10.81
15.68
14.78
17.12
AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the dose associated with the selected
BMR; BMDL = 95% lower confidence limit on the BMD; NA= not applicable (BMD/BMDL computation failed or
insufficient DF to fit model)
"Values >0.05 fail to meet conventional goodness-of-fit criteria.
bValues O.10 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals at doses immediately below and immediately above the BMD.
dCoefficients restricted to be positive.
ePower restricted to >1.
fBest-fitting model is displayed in boldface type. BMDLs for models providing adequate fit differed by less than
threefold, so the model with the lowest AIC was selected.
B-29
-------
Polynomial Model with 0.95 Confidence Level
o
Q.
£=
ro
0)
44
42
40
38
36
34
Polynomial
BMDL
BMD
10
20
30
40
dose
50
60
70
80
08:3403/292010
Polynomial Model. (Version: 2.13; Date: 04/08/2008)
Input Data File:
C:\USEPA\IRIS\TCE\NTP\rellivwt\female\ply_rellivwtF2HDD_Poly_3.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\TCE\NTP\rellivwt\female\ply_rellivwtF2HDD_Poly_3.plt
Mon Mar 29 09:34:20 2010
HMDS Model Run
The form of the response function is:
Y[dose] = beta 0 + beta l*dose + beta 2*doseA2 + ...
Dependent variable = mean
Independent variable = dose
rho is set to 0
The polynomial coefficients are restricted to be positive
A constant variance model is fit
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
B-30
-------
Default Initial Parameter Values
alpha
rho
beta_0
beta_l
beta_2
beta 3
1.93677
0
35.07
0.115542
0
2.84896e-005
Specified
Asymptotic Correlation Matrix of Parameter Estimates
alpha
beta 0
beta 1
beta 3
alpha
1
-6e-009
3.2e-009
-1.7e-009
beta 0
-6e-009
1
-0.76
0.56
beta 1
3.2e-009
-0.76
1
-0.92
beta 3
-1.7e-009
0.56
-0.92
1
Parameter Estimates
Variable
alpha
beta_0
beta_l
beta_2
beta 3
Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std De
Model Descriptions for likelihoods calculated
Model Al: Yij
Var{e(ij) }
Mu(i) + e(i j '
S i gma A 2
Model A2:
Yij = Mu(i)
B-31
-------
Var{e(ij)} = Sigma(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)} = SigmaA2
Likelihoods of Interest
Model Log(likelihood)
Al -31.113274
A2 -28.050020
A3 -31.113274
fitted -31.491356
R -72.394938
# Param' s
5
8
5
4
2
AIC
72.226548
72.100041
72.226548
70.982711
148.789876
Explanation of Tests
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.
Test
Test 1
Test 2
Test 3
Test 4
Tests of Interest
-2*log(Likelihood Ratio) Test df
88.6898
6.12651
6.12651
0.756163
6
3
3
1
p-value
<.0001
0.1056
0.1056
0.3845
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is greater than .1.
model appears to be appropriate here
A homogeneous variance
The p-value for Test 3 is greater than .1.
to be appropriate here
The p-value for Test 4 is greater than .1.
to adequately describe the data
The modeled variance appears
The model chosen seems
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
B-32
-------
Confidence level = 0.95
BMD = 21.8955
BMDL = 14.7785
B-33
-------
Serum ALT activity in male and female rats (Tables B-9 and B-10)
All doses were retained in the BMD modeling of serum ALT in males and females. The
assumption of constant variance was not upheld for either dataset, but in each case, the power
model for variance built into the BMDS provided adequate fit to the variance data. With the
variance model applied, adequate fit to the means was provided by the Hill, power, and 2- and
5-degree polynomial models for the males, and by the Hill model alone for the females. For the
males, estimated BMDLs from the adequately fitting models differed by less than threefold. In
accordance with U.S. EPA (2000b), the model with the lowest AIC (i.e., 2-degree polynomial)
was selected as the basis for the BMDiso and BMDLiso estimates of 41 and 26 mg/kg-day. For
the females, BMDiso and BMDLiso estimates of 82 and 69 mg/kg-day were based on the Hill
model.
B-34
-------
Table B-9. Summary of BMD modeling results for serum ALT activity in
male rats
Model
Test for
significant
difference
/7-valuea
Variance
/7-valueb
Mean
^-valueb
Scaled
residuals of
interest0
AIC
BMD1SD
(mg/kg-d)
BMDL1SD
(mg/kg-d)
All dose groups included
Constant variance
Lineard
O.0001
O.0001
O.0001
-0.19/-1.55
486.88
43.91
37.37
Non-constant variance
Hill"
Lineard
Polynomial (2-degree)d'f
Polynomial (3-degree)d
Polynomial (4-degree)d
Polynomial (5-degree)d
Powere
<0.0001
<0.0001
<0.0001
<0.0001
O.0001
<0.0001
<0.0001
0.72
0.72
0.72
0.72
0.72
0.72
0.72
0.51
<0.0001
0.84
<0.0001
O.0001
0.47
0.73
0.10/0.77
>10
-0.21/1.00
>10
NA
-0.14/1.06
-0.11/0.76
370.02
6.00
366.08
10.00
606.63
370.17
367.96
42.19
0.00
40.98
0.00
NA
40.62
41.97
34.33
NA
26.35
NA
28.22
26.19
32.24
Akaike Information Criterion; BMD = maximum likelihood estimate of the dose associated with the selected BMR;
BMDL = 95% lower confidence limit on the BMD; NA= not applicable (BMD/BMDL computation failed)
"Values >0.05 fail to meet conventional goodness-of-fit criteria.
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals at doses immediately below and immediately above the BMD.
dCoefficients restricted to be positive.
ePower restricted to >1.
fBest-fitting model is displayed in boldface type. BMDLs for models providing adequate fit differed by less than
threefold, so the model with the lowest AIC was selected.
B-35
-------
Polynomial Model with 0.95 Confidence Level
0)
en
o
Q.
£=
ro
0)
350
300
250
200
150
100
50
300
09:5903/292010
Polynomial Model. (Version: 2.13; Date: 04/08/2008)
Input Data File: C:\USEPA\IRIS\TCE\NTP\ALT\male\ply_ALTM_poly_2.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\TCE\NTP\ALT\male\ply_ALTM_poly_2.plt
Mon Mar 29 10:59:45 2010
HMDS Model Run
The form of the response function is:
Y[dose] = beta 0 + beta l*dose + beta 2*doseA2 + ...
Dependent variable = mean
Independent variable = dose
The polynomial coefficients are restricted to be positive
The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)
Total number of dose groups = 6
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
B-36
-------
Default Initial Parameter Values
lalpha =
rho =
beta_0 =
beta_l =
beta 2 =
6.52437
0
48.8991
0.00912505
0.00233971
!!! Warning: optimum may not have been found. !!!
!!! You may want to try choosing different initial values. !!!
Asymptotic Correlation Matrix of Parameter Estimates
lalpha
beta 0
beta 1
beta 2
lalpha
1
-0.0021
-0.015
0.027
beta 0
-0.0021
1
-0.71
0.49
beta 1
-0.015
-0.71
1
-0.86
beta 2
0.027
0.49
-0.86
1
Parameter Estimates
Variable
lalpha
rho
beta_0
beta_l
beta 2
Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std De
Model Descriptions for likelihoods calculated
Model Al: Yij = Mu(i) + e(ij;
Var{e(ij)} = SigmaA2
B-37
-------
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = exp(lalpha + rho*ln(Mu(i)))
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)} = SigmaA2
Likelihoods of Interest
Model
Al
A2
A3
fitted
R
Log (likelihood)
-222.570247
-177.293103
-178.329731
-179.039110
-300.315008
# Param's
7
12
8
4
2
AIC
459.140493
378.586206
372.659462
366.078220
604.630016
Explanation of Tests
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.
Test
Test 1
Test 2
Test 3
Test 4
Tests of Interest
-2*log(Likelihood Ratio) Test df
246.044
90.5543
2.07326
1.41876
10
5
4
4
p-value
<.0001
<.0001
0.7223
0.8409
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is less than .1. A non-homogeneous variance
model appears to be appropriate
The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
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 = Estimated standard deviations from the control mean
B-38
-------
Confidence level = 0.95
BMD = 40.9754
BMDL = 26.3459
B-39
-------
Table B-10. Summary of BMD modeling results for serum ALT activity in
female rats
Model
Test for
significant
difference
/7-valuea
Variance
/7-valueb
Mean
^-valueb
Scaled
residuals of
interest0
AIC
BMD1SD
(mg/kg-d)
BMDL1SD
(mg/kg-d)
All dose groups included
Constant variance
Lineard
0.0001
O.0001
O.0001
-0.12/2.54
512.92
45.04
38.30
Non-constant variance
HiUe'f
Lineard
Polynomial (2-degree)d
Polynomial (3-degree)d
Polynomial (4-degree)d
Polynomial (5-degree)d
Power"
<0.0001
<0.0001
<0.0001
<0.0001
0.0001
0.0001
0.0001
0.23
0.23
0.23
0.23
0.23
0.23
0.23
0.16
O.0001
O.0001
O.0001
O.0001
O.0001
0.02
0.09/-0.29
0.79/3.84
-0.91/-0.16
-0.95/-0.20
-0.77/-0.40
-0.85/-0.14
-0.26/-1.58
351.50
444.14
413.32
415.39
392.73
432.77
355.84
82.49
142.23
65.95
71.30
71.75
79.16
64.07
68.61
12.12
19.55
15.90
22.50
13.16
55.45
AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the dose associated with the selected
BMR; BMDL = 95% lower confidence limit on the BMD
"Values >0.05 fail to meet conventional goodness-of-fit criteria.
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals at doses immediately below and immediately above the BMD.
dCoefficients restricted to be positive.
ePower restricted to >1.
fBest-fitting model is displayed in boldface type. In this case, Hill model was the only model that provided an
adequate fit to the data.
B-40
-------
Hill Model with 0.95 Confidence Level
0)
en
o
Q.
£=
ro
0)
400
350
300
250
200
150
100
50
Hill
50
100
150
dose
200
250
300
10:0803/292010
Hill Model. (Version: 2.14; Date: 06/26/2008)
Input Data File: C:\USEPA\IRIS\TCE\NTP\ALT\female\hil_ALTF_Hill.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\TCE\NTP\ALT\female\hil_ALTF_Hill.plt
Mon Mar 29 11:08:43 2010
HMDS Model Run
The form of the response function is:
Y[dose] = intercept + v*doseAn/(kAn + doseAn)
Dependent variable = mean
Independent variable = dose
Power parameter restricted to be greater than 1
The variance is to be modeled as Var(i) = exp(lalpha
rho * In(mean(i))
Total number of dose groups = 6
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
B-41
-------
Default Initial Parameter Values
lalpha =
rho =
intercept =
v =
n =
k =
6.46604
0
46
293
2.07344
416.806
lalpha
rho
intercept
Asymptotic Correlation Matrix of Parameter Estimates
rho intercept v n
-0.99 -0.12 0.1 -0.0074
1 0.098 -0.11 0.0073
0.098 1 -0.41 0.49
-0.11 -0.41 1 -0.9
0.0073 0.49 -0.9 1
-0.052 -0.42 0.98 -0.95
Parameter Estimates
Variable
lalpha
rho
intercept
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
-7.80242
1.82615
41.7428
202.612
2.41747
177.378
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.
Model Descriptions for likelihoods calculated
Model Al:
Model A2:
Yij =
Var{e(ij)
Yij =
Var{e(ij)
Mu(i) + e (i j '
SigmaA2
Mu(i) + e(i j '
Sigma(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = exp(lalpha + rho*ln(Mu(i)))
Model A3 uses any fixed variance parameters that
B-42
-------
were specified by the user
Model R: Yi = Mu + e(i;
Var{e(i)} = SigmaA2
Likelihoods of Interest
Model
Al
A2
A3
fitted
R
Log(likelihood)
-220.820465
-165.059425
-167.889045
-169.749216
-312.021870
# Param' s
7
12
8
6
2
AIC
455.640931
354.118851
351.778089
351.498431
628.043741
Explanation of Tests
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
Test 1
Test 2
Test 3
Test 4
-2*log(Likelihood Ratio) Test df
293.925
111.522
5.65924
3.72034
10
5
4
2
p-value
<.0001
<.0001
0.2261
0.1556
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is less than .1,
model appears to be appropriate
A non-homogeneous variance
The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
The p-value for Test 4 is greater than .1. The model chosen seems
to adequately describe the data
B-43
-------
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 82.493
BMDL = 68.6138
B-44
-------
Serum SDH activity in male and female rats (Tables B-ll and B-12)
No model provided an adequate fit to the data for changes in serum SDH activity in male
rats. This was due to the difficulty in modeling the reported variances. As a result, these data
are considered unsuitable for BMD modeling. For females, only the power model provided an
adequate fit to the serum SDH activity data after the highest dose was dropped and the variance
was modeled using the non-constant variance model included in BMDS. This model served as
the basis for the BMDiso and BMDLiso estimates of 157 and 113 mg/kg-day for this endpoint.
B-45
-------
Table B-ll. Summary of BMD modeling results for serum SDH activity in
male rats
Model
Test for
significant
difference
/7-valuea
Variance
/7-valueb
Mean
/7-valueb
Scaled residuals of
interest0
AIC
BMD1SD
(mg/kg-d)
BMDL1SD
(mg/kg-d)
All dose groups included
Constant variance
Lineard
<0.0001
Non-constant variance
Lineard
<0.0001
O.0001
0.19
-0.75/-1.42
293.96
41.70
35.55
0.05
O.0001
-0.92/0.60
307.18
62.52
11.14
Highest dose group dropped
Constant variance
Lineard
O.0001
Non-constant variance
Lineard
<0.0001
0.02
0.08
1.33/-1.16
212.18
34.45
28.37
0.03
0.05
1.09/-1.28
212.07
32.47
19.12
Two Highest dose groups dropped
Constant variance
Lineard
0.0004
Non-constant variance
Lineard
0.0004
0.04
0.26
-0.92/0.15
159.19
45.73
31.69
0.03
0.17
-0.91/0.13
161.04
42.28
25.15
Three highest dose groups dropped
Constant variance
Lineard
0.03
Non-constant variance
Lineard
0.03
0.04
0.14
-0.60e
125.02
58.79
27.97
0.05
0.64
1.20/-0.82
122.10
27.88
13.75
AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the dose associated with the selected
BMR; BMDL = 95% lower confidence limit on the BMD
"Values >0.05 fail to meet conventional goodness-of-fit criteria.
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals at doses immediately below and immediately above the BMD.
dCoefficients restricted to be positive.
eResidual reported for highest dose tested.
B-46
-------
Table B-12. Summary of BMD modeling results for serum SDH activity in
female rats
Model
Test for
significant
difference /rvalue"
Variance
/7-valueb
Mean
/7-valueb
Scaled
residuals of
interest0
AIC
BMD1SD
(mg/kg-
d)
BMDL1SD
(mg/kg-d)
All dose groups included
Constant variance
Lineard
O.0001
<0.0001
O.OOOl
0.18/-3.60
321.64
47.70
40.47
Non-constant variance
Lineard
O.0001
0.04
O.OOOl
NA
432.91
NA
24.11
Highest dose group dropped
Constant variance
Lineard
O.OOOl
0.0002
0.0001
-0.05/-3.48
244.99
63.45
48.93
Non-constant variance
Hill"
Lineard
Polynomial (2-degree)d
Polynomial (3 -degree)d
Polynomial (4-degree)d
Powere'f
<0.0001
<0.0001
<0.0001
<0.0001
0.0001
<0.0001
0.18
0.18
0.18
0.18
0.18
0.18
0.05
0.00
0.00
0.01
0.04
0.10
-1.34/0.00
-0.09/-2.36
-2.77/1.04
-2.19/0.42
-1.78/0.17
-1.34/0.00
217.37
229.76
224.39
219.90
217.52
215.37
153.80
67.45
87.97
106.18
118.22
156.52
NA
38.00
66.87
87.33
102.34
113.49
AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the dose associated with the selected
BMR; BMDL = 95% lower confidence limit on the BMD; NA = not applicable (BMD/BMDL computation failed)
aValues >0.05 fail to meet conventional goodness-of-fit criteria.
bValues O.10 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals at doses immediately below and immediately above the BMD.
dCoefficients restricted to be positive.
ePower restricted to >1.
fBest-fitting model is displayed in boldface type. Power model was the only model that provided an adequate fit to
the data.
B-47
-------
Power Model with 0.95 Confidence Level
0)
en
o
Q.
£=
ro
0)
50
45
40
35
30
25
20
Power
BMDL
BMD
20
40
60
80 100
dose
120
140
160
14:2003/292010
Power Model. (Version: 2.15; Date: 04/07/2008)
Input Data File:
C:\USEPA\IRIS\TCE\NTP\SDH\female\pow_SDHFHDD_power.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\TCE\NTP\SDH\female\pow_SDHFHDD_power.plt
Mon Mar 29 15:20:23 2010
HMDS Model Run
The form of the response function is:
Y[dose] = control + slope * doseApower
Dependent variable = mean
Independent variable = dose
The power is restricted to be greater than or equal to 1
The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)
Total number of dose groups = 5
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
B-48
-------
Default Initial Parameter Values
lalpha = 3.46985
rho = 0
control = 25
slope = 0.0617409
power = 1.1118
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -power
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
lalpha
rho
control
slope
lalpha
1
-1
-0.15
0.37
rho
-1
1
0.14
-0.37
control
-0
0
-0
.15
.14
1
.22
slope
0
-0
-0
.37
.37
.22
1
Parameter Estimates
Variable
lalpha
rho
control
slope
power
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std De
Model Descriptions for likelihoods calculated
Model Al: Yij = Mu(i) + e(ij;
Var{e(ij)} = SigmaA2
B-49
-------
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = exp(lalpha + rho*ln(Mu(i)))
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)} = SigmaA2
Likelihoods of Interest
Model Log(likelihood) # Param's AIC
Al -109.112298 6 230.224595
A2 -98.178926 10 216.357851
A3 -100.610596 7 215.221192
fitted -103.685379 4 215.370759
R -135.518801 2 275.037602
Explanation of Tests
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.
Test
Test 1
Test 2
Test 3
Test 4
Tests of Interest
-2*log(Likelihood Ratio) Test df
74.6798
21.8667
4.86334
6.14957
p-value
<.0001
0.000213
0.1821
0.1046
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is less than .1,
model appears to be appropriate
A non-homogeneous variance
The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
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 = Estimated standard deviations from the control mean
Confidence level = 0.95
B-50
-------
BMD = 156.523
BMDL = 113.491
B-51
-------
Serum bile acids in male and female rats (Tables B-13 and B-14)
All doses were retained in the modeling of serum bile acids in males and females. The
assumption of constant variance was not upheld for either dataset, but in each case, the power
model for variance included in BMDS provided adequate fit to the variance data. With the
variance model applied, adequate fit to the mean data was provided by several models for each
sex, and for both datasets, BMDL estimates across models with adequate fit differed by less than
threefold. In accordance with U.S. EPA (2000b), the models with the lowest AIC (power model
for males and 5-degree polynomial model for females) were selected as the basis for the BMDiso
and BMDLiso estimates for these endpoints (respectively, 72 and 57 mg/kg-day for males and
188 and 170 mg/kg-day for females).
B-52
-------
Table B-13. Summary of BMD results for serum bile acid levels in male rats
Model
Test for
significant
difference
/7-valuea
Variance
/7-valueb
Mean
/7-valueb
Scaled
residuals of
interest0
AIC
BMD1SD
(mg/kg-d)
BMDL1SD
(mg/kg-d)
All dose groups included
Constant variance
Lineard
0.0001
0.0001
0.002
-0.10/-1.38
578.68
76.00
62.75
Non-constant variance
Hill"
Lineard
Polynomial (2-degree)d
Polynomial (3-degree)d
Polynomial (4-degree)d
Polynomial (5-degree)d
Powere'f
O.0001
<0.0001
<0.0001
<0.0001
O.0001
<0.0001
<0.0001
0.77
0.77
0.77
0.77
0.77
0.77
0.77
0.69
0.0001
0.21
0.32
0.32
O.0001
0.46
0.17/-0.74
0.48/2.69
-0.88/-1.16
-0.65/-0.56
-0.65/-0.56
-1.08/0.17
-0.56/-0.43
427.84
454.67
428.95
428.58
428.58
449.32
427.70
82.84
115.63
58.37
69.21
69.21
76.72
72.45
66.69
36.05
50.80
54.31
54.31
25.65
57.17
AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the dose associated with the selected
BMR; BMDL = 95% lower confidence limit on the BMD
aValues >0.05 fail to meet conventional goodness-of-fit criteria.
bValues O.10 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals at doses immediately below and immediately above the BMD.
dCoefficients restricted to be positive.
ePower restricted to >1.
fBest-fitting model is displayed in boldface type. BMDLs for models providing adequate fit differed by less than
threefold, so the model with the lowest AIC was selected.
B-53
-------
Power Model with 0.95 Confidence Level
0)
en
o
Q.
£=
ro
0)
400
300
200
100
Power
50
100
150
dose
200
250
300
14:3903/292010
Power Model. (Version: 2.15; Date: 04/07/2008)
Input Data File: C:\USEPA\IRIS\TCE\NTP\bile\male\pow_BileM_power.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\TCE\NTP\bile\male\pow_BileM_power.plt
Mon Mar 29 15:39:39 2010
HMDS Model Run
The form of the response function is:
Y[dose] = control + slope * doseApower
Dependent variable = mean
Independent variable = dose
The power is restricted to be greater than or equal to 1
The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)
Total number of dose groups = 6
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
B-54
-------
Default Initial Parameter Values
lalpha = 8.35885
rho = 0
control = 27.2
slope = 0.000160062
power = 2.50584
Asymptotic Correlation Matrix of Parameter Estimates
lalpha
rho
control
slope
power
lalpha
1
-0.98
-0.31
-0.17
0.22
rho
-0.98
1
0.25
0.18
-0.23
control
-0.31
0.25
1
-0.3
0.28
slope
-0.17
0.18
-0.3
1
-1
power
0.22
-0.23
0.28
-1
1
Parameter Estimates
Variable
lalpha
rho
control
slope
power
95.0% Wald Confidence Interval
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.
Model Descriptions for likelihoods calculated
Model Al: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = exp(lalpha + rho*ln(Mu(i)))
Model A3 uses any fixed variance parameters that
B-55
-------
were specified by the user
Model R: Yi = Mu + e(i;
Var{e(i)} = SigmaA2
Likelihoods of Interest
Model
Al
A2
A3
fitted
R
Log(likelihood)
-277.604668
-206.636351
-207.553828
-208.851786
-320.497188
# Param's
7
12
8
5
2
AIC
569.209336
437.272702
431.107657
427.703572
644.994376
Explanation of Tests
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
Test 1
Test 2
Test 3
Test 4
-2*log(Likelihood Ratio) Test df
227.722
141.937
1.83495
2.59591
10
5
4
3
p-value
<.0001
<.0001
0.7661
0.4582
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is less than .1.
model appears to be appropriate
A non-homogeneous variance
The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
The p-value for Test 4 is greater than .1. The model chosen seems
to adequately describe the data
B-56
-------
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 72.4471
BMDL = 57.1682
B-57
-------
Table B-14. Summary of BMD modeling results for serum bile acid levels in
female rats
Model
Test for
significant
difference
/7-valuea
Variance
/7-valueb
Mean
/7-valueb
Scaled
residuals of
interest0
AIC
BMD1SD
(mg/kg-d)
BMDL1SD
(mg/kg-d)
All dose groups included
Constant variance
Lineard
0.0001
0.0001
0.0001
-1.13/-3.83
596.57
101.36
81.28
Non-constant variance
Hill"
Lineard
Polynomial (2-degree)d
Polynomial (3-degree)d
Polynomial (4-degree)d
Polynomial (5-degree)d'g
Power6
O.0001
0.0001
O.0001
0.0001
O.0001
<0.0001
0.0001
0.47
0.47
0.47
0.47
0.47
0.47
0.47
0.38
0.0001
O.0001
0.003
0.08
0.33
0.38
-0.51/0.02
3.70f
3.09f
-0.71/-2.18
-0.42/-1.95
-1.34/0.34
-0.50/0.02
466.68
505.52
485.36
477.39
469.90
466.14
466.68
186.94
343.48
344.76
149.70
168.35
187.71
216.74
177.64
139.12
145.95
129.07
152.78
169.55
177.00
AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the dose associated with the selected
BMR; BMDL = 95% lower confidence limit on the BMD
aValues >0.05 fail to meet conventional goodness-of-fit criteria.
bValues O.10 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals at doses immediately below and immediately above the BMD.
dCoefficients restricted to be positive.
ePower restricted to >1.
fResidual at highest dose tested.
8Best-fitting model is displayed in boldface type. BMDLs for models providing adequate fit differed by less than
threefold, so the model with the lowest AIC was selected.
B-58
-------
Polynomial Model with 0.95 Confidence Level
450
400
350
0)
en
o
Q.
£=
ro
0)
300
14:4703/292010
Polynomial Model. (Version: 2.13; Date: 04/08/2008)
Input Data File:
C:\USEPA\IRIS\TCE\NTP\bile\female\ply_BileF_Poly_5.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\TCE\NTP\bile\female\ply_BileF_Poly_5.plt
Mon Mar 29 15:47:49 2010
HMDS Model Run
The form of the response function is:
Y[dose] = beta 0 + beta l*dose + beta 2*doseA2 + ...
Dependent variable = mean
Independent variable = dose
The polynomial coefficients are restricted to be positive
The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)
Total number of dose groups = 6
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
B-59
-------
Default Initial Parameter Values
lalpha =
rho =
beta_0 =
beta_l =
beta_2 =
beta_3 =
beta_4 =
beta 5 =
8.43454
0
37
0
0
0
0
0
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -beta_l -beta_2 -beta_3 -beta_4
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
lalpha
rho
beta_0
beta 5
lalpha
1
-0.98
-0.049
0.16
rho
-0.98
1
0.049
-0.16
beta_0
-0.049
0.049
1
-0.15
beta_5
0.16
-0.16
-0.15
1
Parameter Estimates
Std. Err.
1.00675
0.245366
2.76802
NA
NA
NA
NA
1. 43294e-011
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
-3.55517 0.391218
1.55634 2.51816
32.7849 43.6353
B-60
-------
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.
Model Descriptions for likelihoods calculated
Model Al: Yij
Var{e(ij) }
Model A2: Yij
Var{e(ij) }
Mu(i) + e(i j '
SigmaA2
Mu(i) + e(i j '
Sigma(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = exp(lalpha + rho*ln(Mu(i)))
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)} = SigmaA2
Likelihoods of Interest
Model
Al
A2
A3
fitted
R
Log(likelihood)
-279.875470
-224.999384
-226.787639
-229.071113
-318.845182
# Param's
7
12
8
4
2
AIC
573.750939
473.998768
469.575277
466.142225
641.690364
Explanation of Tests
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
Test 1
Test 2
Test 3
Test 4
-2*log(Likelihood Ratio) Test df
187.692
109.752
3.57651
4.56695
10
5
4
4
p-value
<.0001
<.0001
0.4663
0.3347
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
B-61
-------
It seems appropriate to model the data
The p-value for Test 2 is less than .1. A non-homogeneous variance
model appears to be appropriate
The p-value for Test 3 is greater than .1. The modeled variance appears
to be appropriate here
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 = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 187.713
BMDL =
169.553
B-62
-------
Fetal body weights in Sprague-Daw ley rats (Tables B-15 and B-16)
Fetal body weight data from Gulati et al. (1991a) in Sprague-Dawley rats administered
1,1,2,2-tetrachloroethane in the diet on GD 4-20 are shown in Table B-15. BMD modeling
results based on these data are shown in Table B-16. Adequate model fit was achieved for the
fetal body weight data only after the highest two dose groups were dropped. This was due to
difficulty in modeling the reported variances. After dropping the two highest dose groups, the
remaining dose groups satisfied the assumption of constant variance. Assuming constant
variance, the linear model provided adequate fit to the mean fetal body weight data. The higher
order models either did not fit (p < 0.1: higher order polynomial, power) or failed due to too
many parameters for the available data points (Hill). The linear model is the basis for the
BMDiso and BMDLiso estimates of 83 and 60 mg/kg-day, respectively, for this endpoint shown
in Table B-16.
B-63
-------
Table B-15. Fetal body weight in Sprague-Dawley rats administered
1,1,2,2-tetrachloroethane in the diet on GDs 4-20
Dose (mg/kg-d)
0
34
98
180
278
330
Number of animals
9
8
8
9
9
5
Mean (g)
2.28
2.17
2.19
1.99
2.04
1.81
Standard error
0.04
0.04
0.03
0.05
0.14
0.12
Source: Gulatietal. (1991a).
B-64
-------
Table B-16. Summary of BMD modeling results for fetal body weight
following exposure of pregnant Sprague-Dawley rats on GDs 4-20
Model
Test for
significant
difference
/7-valuea
Variance
/7-valueb
Mean
p-valueb
Scaled residuals of
interest0
AIC
BMD1SD
(mg/kg-
d)
BMDL1SD
(mg/kg-d)
All dose groups included
Constant variance
Lineard
O.0001
O.OOOl
0.40
-0.92/1.23
-91.54
201.09
139.17
Non constant variance
Lineard
O.0001
0.07
0.20
-1.25/0.88
-112.47
84.64
56.25
Highest dose group dropped
Constant variance
Lineard
O.0001
O.OOOl
0.40
-1.24/0.70
-83.65
238.24
147.87
Non constant variance
Lineard
0.0001
0.05
0.18
-1.27/0.83
-105.40
84.31
53.36
Two highest dose groups dropped
Constant variance
Hill6
Lineard'f
Polynomial (2-degree)d
Polynomial (3 -degree)d
Power6
0.0002
0.0002
0.0002
0.0002
0.0002
0.35
0.35
0.35
0.35
0.35
NA
0.12
0.06
0.08
0.06
0.38/-0.06
-1.19/1.46
0.87/-0.20
0.65/-0.09
0.38/-0.06
-101.33
-104.84
-103.53
-103.98
-103.33
129.74
83.10
110.21
118.06
129.71
61.35
59.73
62.16
64.06
61.40
AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the dose associated with the selected
BMR; BMDL = 95% lower confidence limit on the BMD
"Values >0.05 fail to meet conventional goodness-of-fit criteria.
bValues <0.10 fail to meet conventional goodness-of-fit criteria.
°Scaled residuals at doses immediately below and immediately above the BMD.
dCoefficients restricted to be negative.
ePower restricted to >1.
fBest-fitting model is displayed in boldface type. The linear model is the only model providing an adequate fit to
the data.
B-65
-------
Linear Model with 0.95 Confidence Level
0)
en
o
Q.
£=
ro
0)
2.4
2.3
2.2
2.1
1.9
Linear
BMDL
BMD
20
40
60
80 100
dose
120
140
160
180
15:0203/292010
Polynomial Model. (Version: 2.13; Date: 04/08/2008)
Input Data File:
C:\USEPA\IRIS\TCE\gulati\fetalbdwt\lin_fetalbdwt2HDD_linear.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\TCE\gulati\fetalbdwt\lin_fetalbdwt2HDD_linear.plt
Mon Mar 29 16:02:57 2010
HMDS Model Run
The form of the response function is:
Y[dose] = beta 0 + beta l*dose + beta 2*doseA2 + ...
Dependent variable = mean
Independent variable = dose
rho is set to 0
The polynomial coefficients are restricted to be negative
A constant variance model is fit
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
B-66
-------
Default Initial Parameter Values
alpha = 0.0141567
rho = 0 Specified
beta_0 = 2.26747
beta 1 = -0.0014099
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -rho
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
alpha
beta 0
beta 1
alpha
1
-1.3e-010
2e-010
beta 0
-1.3e-010
1
-0.75
beta 1
2e-010
-0.75
1
Parameter Estimates
beta 1
Model Descriptions for likelihoods calculated
Model Al: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)A2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = SigmaA2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)} = SigmaA2
B-67
-------
Likelihoods of Interest
Model
Al
A2
A3
fitted
R
Log(likelihood)
57.506457
59.148779
57.506457
55.418685
46.282389
# Param' s
5
8
5
3
2
AIC
-105.012914
-102.297557
-105.012914
-104.837369
-88.564779
Explanation of Tests
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
Test 1
Test 2
Test 3
Test 4
-2*log(Likelihood Ratio) Test df
25.7328
3.28464
3.28464
4.17554
6
3
3
2
p-value
0.0002497
0.3498
0.3498
0.124
The p-value for Test 1 is less than .05. There appears to be a
difference between response and/or variances among the dose levels
It seems appropriate to model the data
The p-value for Test 2 is greater than .1.
model appears to be appropriate here
A homogeneous variance
The p-value for Test 3 is greater than .1.
to be appropriate here
The p-value for Test 4 is greater than .1.
to adequately describe the data
The modeled variance appears
The model chosen seems
B-68
-------
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 83. 0965
BMDL = 59.7345
B-69
-------
APPENDIX C. BENCHMARK DOSE MODELING RESULTS FOR THE DERIVATION
OF THE ORAL SLOPE FACTOR
Hepatocellular carcinomas in male and female B6C3Fj mice (Tables C-l and C-2)
The incidence data for hepatocellular carcinomas in male and female B6C3Fi mice
exposed via gavage to 1,1,2,2-tetrachloroethane 5 days/week for 78 weeks are shown in
Table C-l (NCI, 1978).
Table C-l. Incidence of hepatocellular carcinomas in B6C3Fi mice
administered l,l?2,2-tetrachloroethane by gavage for 78 weeks
Endpoint
Hepatocellular carcinomas
Sex
Male
Female
Dose (mg/kg-d)a
Ob
3/36
1/40
8.22
13/50
30/48
16.5
44/49
43/47
aHED as calculated in Section 5.4.3 and shown in Table 5-5.
bPooled vehicle controls.
Source: NCI (1978).
The BMD modeling results from the data in Table C-l are summarized in Tables C-2 (for
males) and C-3 (for females) followed by the standard BMDS output for the selected models
from version 2.1.1 of the software. The multistage cancer model did not provide an adequate fit
to the incidence data for hepatocellular carcinomas in male mice; these data are considered
unsuitable for BMD modeling. The one-stage multistage model provided the best fit to the
incidence data for hepatocellular carcinomas in females, and this model was used as the basis for
the BMDio and BMDLio estimates (0.81 and 0.65 mg/kg-day, respectively, as HEDs) for this
endpoint.
C-l
-------
Table C-2. Summary of BMD modeling results for the incidence of
hepatocellular carcinomas in male mice
Model
Multistage (1 -degree polynomial)0
Multistage (2 -degree polynomial)0
DF
1
1
x2
18.30
5.24
/2 Goodness
of fit
/rvalue"
O.001
0.02
Scaled
residuals of
interest11
0.51/-3.27
0.53/-1.83
AIC
134.58
119.87
BMDio[HED]
(mg/kg-d)
1.42
4.10
BMDLio[HED]
(mg/kg-d)
1.11
3.08
AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the dose associated with the selected
BMR; BMDL = 95% lower confidence limit on the BMD; DF = degrees of freedom
aValues <0.1 fail to meet conventional goodness-of-fit criteria.
bScaled residuals at doses immediately below and immediately above the BMD.
°Betas restricted to >0.
Table C-3. Summary of BMD modeling results for the incidence of
hepatocellular carcinomas in female mice
Model
Multistage (1-degree polynomial)0'11
Multistage (2 -degree polynomial)0
DF
1
0
x2
0.74
0.00
X2 Goodness
of fit
/7-valuea
0.39
NA
Scaled
residual of
interest1"
0.04/-0.61
0.00/0.00
AIC
104.99
106.22
BMDio[HED]
(mg/kg-d)
0.81
1.18
BMDLio[HED]
(mg/kg-d)
0.65
0.67
AIC = Akaike Information Criterion; BMD = maximum likelihood estimate of the dose associated with the selected
BMR; BMDL = 95% lower confidence limit on the BMD; DF = degrees of freedom; NA= not applicable (p-value
was not generated due to insufficient DF)
"Values <0.1 fail to meet conventional goodness-of-fit criteria.
bScaled residuals at doses immediately below and immediately above the BMD.
°Betas restricted to >0.
dSelected model is displayed in boldface type.
C-2
-------
Multistage Cancer Model with 0.95 Confidence Level
Multistage Cancer
Linear extrapolation
0)
-5
£=
g
'
0.8
0.6
0.4
0.2
8 10 12 14
16
15:11 03/292010
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File:
C:\USEPA\IRIS\TCE\NCI\hepcarc\female\msc_hepcarcF_MS_l.(d)
Gnuplot Plotting File:
C:\USEPA\IRIS\TCE\NCI\hepcarc\female\msc_hepcarcF_MS_l.pit
Mon Mar 29 16:11:43 2010
HMDS Model Run
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl)]
The parameter betas are restricted to be positive
Dependent variable = incidence
Independent variable = dose
Total number of observations = 3
Total number of records with missing values
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
= 0
C-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) = 0.147828
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.54
Beta(l) -0.54 1
Parameter Estimates
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
Goodness of Fit
P-value
Dose
0.0000
8.2200
16.5000
Est. Prob.
0.0241
0.6664
0.8869
Expected
0.964
31.988
41.682
Observed
1.000
30.000
43.000
Size
40
48
47
Scaled
Residual
0.037
-0.608
0.607
ChiA2 = 0.74
d.f. = 1
P-value = 0.3897
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
C-4
-------
BMD = 0. 806812
BMDL = 0.648049
BMDU = 1.01577
Taken together, (0.648049, 1.01577) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.154309
C-5
------- |