£ CQA EPA/635/R-08/010F
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TOXICOLOGICAL REVIEW
OF
1,2,3-TRICHLOROPROPANE
(CAS No. 96-18-4)
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
September 2009
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
1,2,3-TRICHLOROPROPANE (CAS No. 96-18-4)
LIST OF TABLES vi
LIST OF FIGURES x
LIST OF ABBREVIATIONS AND ACRONYMS xi
FOREWORD xiii
AUTHORS, CONTRIBUTORS, AND REVIEWERS xiv
1. INTRODUCTION 1
2. CHEMICAL AND PHYSICAL INFORMATION 3
3. TOXICOKINETICS 4
3.1. ABSORPTION 4
3.2. DISTRIBUTION 5
3.3. METABOLISM 6
3.4. ELIMINATION 8
3.5. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELING 9
4. HAZARD IDENTIFICATION 10
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS 10
4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIO ASSAYS IN
ANIMALS—ORAL AND INHALATION 10
4.2.1. Oral Exposure 10
4.2.1.1. Subchronic Studies 10
4.2.1.2. Chronic Studies 23
4.2.2. Inhalation Exposure 34
4.2.2.1. Subchronic Studies 34
4.2.2.2. Chronic Studies 38
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION ..38
4.3.1. Oral Studies 38
4.3.2. Inhalation Studies 41
4.4. OTHER STUDIES 43
4.4.1. Acute Toxicity Data 43
4.4.2. Short-term Toxicity Data 43
4.4.3. Aquatic Species Studies 47
4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF
ACTION FOR CARCINOGENICITY 48
4.5.1. Mode of Action Studies 48
4.5.2. Genotoxicity Studies 53
4.5.3. Structural Analog Data—Relationship to l,2-Dibromo-3-chloropropane and 1,2-
Dibromoethane 59
4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS 61
4.6.1. Oral 61
4.6.2. Inhalation Exposure 65
4.7. EVALUATION OF CARCINOGENICITY 67
4.7.1. Summary of Overall Weight of Evidence 67
iii
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4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence 68
4.7.3. Mode of Action Analysis 68
4.7.3.1. Hypothesized Mode of Action 68
4.7.3.2. Experimental Support for the Hypothesized Mode of Action 69
4.7.3.3. Other Possible Modes of Action 74
4.7.3.4. Conclusions About the Hypothesized Mode of Action 74
4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 76
4.8.1. Possible Childhood Susceptibility 76
4.8.2. Possible Gender Differences 77
4.8.3. Other 77
5. DOSE RESPONSE ASSESSMENT 78
5.1. CHRONIC ORAL REFERENCE DOSE (RfD) 78
5.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification
78
5.1.2. Methods of Analysis—Including Models 79
5.1.3. Chronic RfD Derivation—Including Application of Uncertainty Factors (UFs)82
5.1.4. Chronic RfD Comparison Information 83
5.1.5. Previous Oral Assessment 86
5.2. CHRONIC INHALATION REFERENCE CONCENTRATION (RfC) 86
5.2.1. Choice of Principal Study and Critical Effect—with Rationale and Justification
86
5.2.2. Methods of Analysis—Including Models 87
5.2.3. Chronic RfC Derivation—Including Application of Uncertainty Factors (UFs) 89
5.2.4. Chronic RfC Comparison Information 90
5.3. UNCERTAINTIES IN CHRONIC ORAL REFERENCE DOSE AND INHALATION
REFERENCE CONCENTRATION 93
5.4. CANCER ASSESSMENT 96
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 Methods 101
5.4.4. Oral Slope Factor and Inhalation Unit Risk 103
5.4.5. Application of Age-Dependent Adjustment Factors 110
5.4.6. Uncertainties in Cancer Risk Values Ill
6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE
RESPONSE 116
6.1. HUMAN HAZARD POTENTIAL 116
6.2. DOSE RESPONSE 118
6.2.1. Noncancer—Oral 118
6.2.2. Noncancer—Inhalation 121
6.2.3. Cancer—Oral and Inhalation 124
7. REFERENCES 127
APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS
AND DISPOSITION 135
IV
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APPENDIX B: BENCHMARK DOSE MODELING RESULTS FOR THE DERIVATION OF
THERFD 163
APPENDIX C: BENCHMARK DOSE MODELING RESULTS FOR THE DERIVATION OF
THE RFC 190
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LIST OF TABLES
Table 2-1. Physical properties and chemical identity of 1,2,3-trichloropropane 3
Table 3-1. Distribution and excretion of radiolabeled 1,2,3-trichloropropane (30 mg/kg) 60
hours after oral (gavage) administration 5
Table 4-1. Relative liver weights (mg organ weight/g body weight) and percent change in
F344/N rats exposed to 1,2,3-trichloropropane by gavage for 17 weeks 12
Table 4-2. Absolute liver weights (g) and percent change in F344/N rats exposed to
1,2,3-trichloropropane by gavage for 17 weeks 12
Table 4-3. Relative kidney weights (mg organ weight/g body weight) and percent change in
F344/N rats exposed to 1,2,3-trichloropropane by gavage for 17 weeks 13
Table 4-4. Absolute kidney weights (g) and percent change in F344/N rats exposed to
1,2,3-trichloropropane by gavage for 17 weeks 13
Table 4-5. Incidence of liver, kidney, and nasal turbinate lesions in male and female F344/N rats
in a 17-week study 14
Table 4-6. Relative liver weights (mg organ weight/g body weight) and percent change in
B6C3Fi mice exposed to 1,2,3-trichloropropane by gavage for 17 weeks 17
Table 4-7. Absolute liver weights (g) and percent change in B6C3Fi mice exposed to
1,2,3-trichloropropane by gavage for 17 weeks 17
Table 4-8. Relative kidney weights (mg organ weight/g body weight) and percent change in
B6C3Fi mice exposed to 1,2,3-trichloropropane by gavage for 17 weeks 18
Table 4-9. Absolute kidney weights (g) and percent change in B6C3Fi mice exposed to
1,2,3-trichloropropane by gavage for 17 weeks 18
Table 4-10. Incidence of liver, lung, and forestomach lesions in male and female B6C3Fi mice
in a 17-week study 19
Table 4-11. Incidence of myocardial necrosis in male and female Sprague-Dawley rats
following 90-day 1,2,3-trichloropropane exposure 21
Table 4-12. Survival rates and percent probability of survival for F344/N rats exposed to
1,2,3-trichloropropane by gavage for 2 years 24
Table 4-13a. Relative liver weights (mg organ weight/g body weight) and percent change in
F344/N rats chronically exposed to 1,2,3-trichloropropane by gavage at the 15-month
interim evaluation 25
Table 4-13b. Absolute liver weights (g) and percent change in F344/N rats chronically exposed
to 1,2,3-trichloropropane by gavage at the 15-month interim evaluation 25
VI
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Table 4-14a. Relative right kidney weights (mg organ weight/g body weight) and percent
change in F344/N Rats chronically exposed to 1,2,3-trichloropropane by gavage at the 15-
month interim evaluation 26
Table 4-14b. Absolute right kidney weights (grams) and percent change in F344/N Rats
chronically exposed to 1,2,3-trichloropropane by gavage at the 15-month interim evaluation
26
Table 4-15. Incidence of neoplasms in F344/N rats chronically exposed to
1,2,3-trichloropropane by gavage 28
Table 4-16. Survival rates and percent probability of survival for B6C3Fi mice exposed to
1,2,3-trichloropropane by gavage for 2 years 30
Table 4-17'a. Relative liver weights (mg organ weight/g body weight) and percent change in
B6C3Fi mice chronically exposed to 1,2,3-trichloropropane by gavage 30
Table 4-17b. Absolute liver weights (g) and percent change in B6C3Fi mice chronically
exposed to 1,2,3-trichloropropane by gavage 31
Table 4-18a. Relative right kidney weights (mg organ weight/g body weight) and percent
change in B6C3Fi mice chronically exposed to 1,2,3-trichloropropane by gavage 31
Table 4-18b. Absolute right kidney weights (g) and percent change in B6C3Fi mice chronically
exposed to 1,2,3-trichloropropane by gavage 31
Table 4-19. Incidence of neoplasms in B6C3Fi mice chronically exposed to
1,2,3-trichloropropane by gavage 33
Table 4-20. Absolute and relative liver weights and percent change in CD rats exposed to
1,2,3-trichloropropane by inhalation, 6 hours/day, 5 days/week, for 13 weeks 35
Table 4-21. Absolute and relative lung weights and percent change in CD rats exposed to
1,2,3-trichloropropane by inhalation, 6 hours/day, 5 days/week, for 13 weeks 36
Table 4-22. Incidence of histopathologic lesions in CD rats exposed via inhalation to
1,2,3-trichloropropane, 6 hours/day, 5 days/week for 13 weeks 37
Table 4-23. Fertility indices and number of live pups/litter in breeding pairs of CD-I mice
exposed to 1,2,3-trichloropropane by gavage 39
Table 4-24. Decreased mating performance in female CD rats following inhalation of
1,2,3-trichloropropane for 6 hours/day, 5 days/week, for a 10-week pre-mating period, a
mating period (not to exceed 40 days), and gestation days 0-14 42
Table 4-25. Incidence and severity of decreased thickness and degeneration of the olfactory
epithelium in the nasal turbinates of F344/N rats exposed via inhalation to
1,2,3-trichloropropane 44
Table 4-26. Incidence and severity of inflammation of the olfactory epithelium in the nasal
turbinates ofF344/Nrats exposed via inhalation to 1,2,3-trichloropropane 45
vii
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Table 4-27. Incidence and severity of decreased thickness and degeneration of the olfactory
epithelium in the nasal turbinates in B6C3Fi mice exposed via inhalation to
1,2,3-trichloropropane 46
Table 4-28. Incidence and severity of inflammation of the olfactory epithelium in the nasal
turbinates ofB6C3Fi rats exposed via inhalation to 1,2,3-trichloropropane 46
Table 4-29. Comparison of tumor incidence and DNA-adduct formation in male F344/N rats
and B6C3Fi mice 51
Table 4-30. Formation of DNA adducts by [14C]-1,2,3- trichloropropane (6 mg/kg-day)
administered to B6C3Fi mice by gavage or drinking water 53
Table 4-31. Genotoxicity bioassays of 1,2,3-trichloropropane 54
Table 4-32. Observed effects and corresponding NOAELs and LOAELs for subchronic, chronic,
and reproductive toxicity studies following oral exposure to 1,2,3-trichloropropane 61
Table 5-1. Candidate BMDs for chronic and reproductive effects associated with oral exposure
to 1,2,3-trichloropropane 80
Table 5-2. BMD modeling results used in the derivation of the RfC 88
Table 5-3. Tumor incidence, (percent), and time of first occurrence in male and female F344/N
rats following gavage exposure to 1,2,3-trichloropropane 98
Table 5-4. Tumor incidence in male and female B6C3Fi mice following gavage exposure to
1,2,3-trichloropropane 99
Table 5-5. Dose-response modeling summary for tumors associated with oral exposure to
1,2,3-trichloropropane; rat and mouse tumor incidence data 106
Table 5-6. Dose-response modeling summary for oral cavity squamous cell neoplasia associated
with oral exposure to 1,2,3-trichloropropane; rat incidence data (NTP, 1993) 109
Table 5-7. Application of ADAFs for a 70-year exposure to 0.001 mg/kg-day
1,2,3-trichloropropane from ages 0 to 70 110
Table 5-8. Summary of uncertainty in the 1,2,3-trichloropropane cancer risk assessment Ill
Table B-l. BMD modeling used in the derivation of the RfD; final model selected for each
endpoint 164
Table C-l. BMD modeling used in the derivation of the RfC; final model selected for each
endpoint 191
Table D-l. Tumor incidence data, with time to death with tumor; male rats exposed by gavage
to 1,2,3-trichloropropane 197
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Table D-2. Tumor incidence data, with time to death with tumor; female rats exposed to
1,2,3-trichloropropane 207
Table D-3. Tumor incidence data, with time to death with tumor; male mice exposed by gavage
to 1,2,3-trichloropropane 218
Table D-4. Tumor incidence data, with time to death with tumor; female mice exposed by
gavage to 1,2,3-trichloropropane 224
Table D-5. Summary of human equivalent overall cancer risk values estimated by R/BMDR,
based on male and female rat and mouse tumor incidence 231
IX
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LIST OF FIGURES
Figure 2-1. 1,2,3-Trichloropropane 3
Figure 3-1. Possible metabolic pathways for 1,2,3-trichloropropane in rats 7
Figure 4-1. Structure of the DNA adduct S-[l-(hydroxymethyl)-2-(N7-guanyl)ethyl]glutathione.
52
Figure 5-1. Exposure-response array of selected subchronic, chronic, and reproductive toxicity
effects 84
Figure 5-2. PODs for selected endpoints (with critical effect circled) from Table 5-1 with
corresponding applied UFs and derived sample chronic oral reference values (RfVs).
85
Figure 5-3. PODs for selected endpoints (with critical effect circled) from Table 5-2 with
corresponding applied UFs and derived sample chronic inhalation RfVs 92
Figure 6-1. PODs for selected endpoints (with critical effect circled) from Table 5-1 with
corresponding applied UFs and derived sampe chronic RfVs 120
Figure 6-2. PODs for selected endpoints (with critical effect circled) from Table 5-2 with
corresponding applied UFs and derived sample chronic RfVs 123
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LIST OF ABBREVIATIONS AND ACRONYMS
ACPC
ADAF
AIC
ALT
AST
BMC
BMD
BMDL
BMDS
BMR
BSO
CASRN
CHO
CPC
CYP450
DAF
DCA
GMA
GSH
HEC
IRIS
i.p.
i.v.
J^ow
LDH
LOAEL
MLE
MTD
NADPH
NCI
NOAEL
NRC
NTP
PBPK
PBTK
RACE
RfC
RfD
RfV
SD
SDH
SMART
TSCA
UCL
UF
N-acetyl-S-(3-chloro-2-hydroxypropyl)-L-cysteine
age-dependent adjustment factor
Akaike Information Criterion
alanine aminotransferase
aspartate aminotransferase
benchmark concentration
benchmark dose
Benchmark dose, 95% lower bound
Benchmark Dose Software
benchmark response
l-buthionine-(R,S)-sulfoximine
Chemical Abstracts Service Registry Number
Chinese hamster ovary
S-(3-chloro-2-hydroxypropyl)-L-cysteine
cytochrome P-450
dosimetric adjustment factor
1,3 -di chl oroacetone
(S-glutathionyl)malonic acid
reduced glutathione
human equivalent concentration
Integrated Risk Information System
intraperitoneal
intravenous
octanol/water partition coefficient
lactate dehydrogenase
lowest-observed-adverse-effect-level
maximum likelihood estimate
maximum tolerated dose
reduced nicotinamide adenine dinucleotide phosphate
National Cancer Institute
no-observed-adverse-effect-level
National Research Council
National Toxicology Program
physiologically based pharmacokinetic
physiologically-based toxicokinetic
Reproductive Assessment by Continuous Breeding
reference concentration
reference dose
reference value
standard deviation
sorbitol dehydrogenase
somatic mutation and recombination test
Toxic Substances Control Act
upper confidence limit
uncertainty factor
XI
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U.S. EPA U.S. Environmental Protection Agency
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FOREWORD
The purpose of this Toxicological Review is to provide scientific support and rationale
for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to 1,2,3-
trichloropropane. It is not intended to be a comprehensive treatise on the chemical or
toxicological nature of 1,2,3-trichloropropane.
The intent of Section 6, Major Conclusions in the Characterization of Hazard and Dose
Response, is to present the major conclusions reached in the derivation of the reference dose,
reference concentration, and cancer assessment, where applicable, and to characterize the overall
confidence in the quantitative and qualitative aspects of hazard and dose response by addressing
the quality of the data and related uncertainties. The discussion is intended to convey the
limitations of the assessment and to aid and guide the risk assessor in the ensuing steps of the
risk assessment process.
For other general information about this assessment or other questions relating to IRIS,
the reader is referred to EPA's IRIS Hotline at (202) 566-1676 (phone), (202) 566-1749 (fax), or
hotline.iris@epa.gov (email address).
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AUTHORS, CONTRIBUTORS, AND REVIEWERS
CHEMICAL MANAGER/AUTHOR
Martin Gehlhaus, M.H.S
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
CONTRIBUTING AUTHORS
Stiven Foster, M.S.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Karen Hogan, M.S.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
George Holdsworth, Ph.D.
Oak Ridge Institute for Science and Education
Oak Ridge, TN
REVIEWERS
This document has been reviewed by 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
Bob Benson, Ph.D.
Region 8
Office of Partnerships and Regulatory Assistance (OPRA)
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
xiv
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U.S. Environmental Protection Agency
Washington, DC
Channa Keshava, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Elizabeth H. Margosches, Ph.D.
Office of Prevention, Pesticides and Toxic Substances (OPPTS)
Office of Pollution Protection and Toxics (OPPT)
Risk Assessment Division (RAD)
John Whalan
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
EXTERNAL PEER REVIEWERS
James V. Bruckner, Ph.D.
Department of Pharmaceutical and Biomedical Sciences
University of Georgia
Richard J. Bull, Ph.D.
MoBull Consulting
Dale Hattis, Ph.D.
George Perkins Marsh Institute
Clark University
Ralph L. Kodell, Ph.D.
University of Arkansas for Medical Sciences
Harihara M. Mehendale, Ph.D.
College of Pharmacy
University of Louisiana at Monroe
Helmut Zarbl, Ph.D.
Robert Wood Johnson Medical School
Environmental and Occupational Health Sciences Institute
Lauren Zeise, Ph.D.
California EPA Office of Environmental Health Hazard Assessment (OEHHA)
XV
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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 1,2,3-
trichloropropane. 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 1,2,3-
trichloropropane has followed the general guidelines for risk assessment as set forth by the
National Research Council (1983). EPA guidelines and Risk Assessment Forum Technical
Panel Reports that may have been used in the development of this assessment include the
following: Guidelines for the Health Risk Assessment of Chemical Mixtures (U.S. EPA, 1986a),
Guidelines for Mutagenicity Risk Assessment (U.S. EPA, 1986b), Recommendations for and
Documentation of Biological Values for Use in Risk Assessment (U.S. EPA, 1988), Guidelines
for Developmental Toxicity Risk Assessment (U.S. EPA, 1991), Interim Policy for Particle Size
1
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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 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 June 2009.
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2. CHEMICAL AND PHYSICAL INFORMATION
1,2,3-Trichloropropane (CASRN 96-18-4) is a three-carbon alkane with a single chlorine
atom attached to each carbon atom in the chain (Figure 2-1). Synonyms for the compound
include glyceryl trichlorohydrin, glycerol trichlorohydrin, and allyl trichloride. Some physical
and chemical properties are shown in Table 2-1 (HSDB, 2005).
Cl
Figure 2-1. 1,2,3-Trichloropropane.
Table 2-1. Physical properties and chemical identity of 1,2,3-trichloro-
propane
CASRN 96-18-4
Chemical formula
Molecular weight
Melting point
Boiling point
Density at 20°C
Water solubility at 25°C
Log Kow
Vapor pressure at 25°C
Henry's law constant
Conversion factors
C3H5C13
147.43
14.7°C
156.85°C
1.3889g/mL
1,750 mg/L
1.98/2.27
3. 1/3. 69 mm Hg
3.43 x l(T4 atm-nrVmol
1 ppm = 6.13 mg/m3; 1 mg/m3 = 0. 16 ppm
Sources: ATSDR (1992); HSDB (2005).
1,2,3-Trichloropropane is used in the chemical industry as a solvent for oils and fats,
waxes, and resins (HSDB, 2005; ATSDR, 1992). The compound has also been used in paint
thinner and varnish remover, and as a degreasing agent. 1,2,3-trichloropropane is generated as a
byproduct of the production of other chlorinated compounds such as epichlorohydrin (WHO,
2003). The compound is also used as an intermediate in the production of some pesticides and
polymers, such as polysulfide rubbers. The commercially available product is >98-99.9% pure.
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3. TOXICOKINETICS
No reports are available that address the toxicokinetics of 1,2,3-trichloropropane in
humans by any route of exposure. Experimental studies in rats and mice have demonstrated that
absorption of the compound via the oral route results in rapid distribution, extensive metabolism,
and clearance within 60 hours (Mahmood et al., 1991), with a half-life in male rats of 23 hours
(Gingell et al., 1987). The toxicokinetic data also demonstrate the ability of 1,2,3-
trichloropropane or metabolites to bind to intracellular macromolecules such as proteins and
nucleic acids (Mahmood et al., 1991; Weber and Sipes, 1990).
3.1. ABSORPTION
Data on the quantitative absorption of 1,2,3-trichloropropane from exposure via the
inhalation or dermal routes have not been reported. Quantitative data on the absorption,
distribution, and excretion following oral exposure to 1,2,3-trichloropropane were obtained from
a study in which rats and mice were treated with 14C-labeled compound by corn oil gavage
(Mahmood et al., 1991). Doses of 30 mg/kg (8-10 uCi) [14C]-l,2,3-trichloropropane were
administered to 9 male and 12 female Fischer rats, and either 30 or 60 mg/kg to B6C3F1 male
mice (three/group). By sacrificing the animals at intervals up to 60 hours, the researchers
collected information on the time-dependent distribution of radiolabel in urine, feces, breath, the
principal organs and tissues, and bile.
Estimates for the percent absorption of the oral dose can be made by summing the mean
values for the radiolabel recovered in the urine and exhaled as CC>2, as well as the radiolabel
distributed in the blood, liver, kidney, skin, adipose tissue, and muscle (Table 3-1). By this
approach, estimates of the absorbed oral load are 75% in male rats, 68% in female rats, and 84%
in male mice. The percent recovered from feces was not used in this calculation because it is
likely to contain both an absorbed and non-absorbed fraction. However, the true extent of
intestinal absorption is likely to have been greater than the presented 75-84%, because a portion
of the radiolabel that appeared in feces, which was not included in the above absorption
estimates, would also have been absorbed.
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Table 3-1. Distribution and excretion of radiolabeled 1,2,3-trichloropropane
(30 mg/kg) 60 hours after oral (gavage) administration
Tissue
Urine
Feces
C02
Volatiles
Blood
Liver
Kidney
Skin
Adipose tissue
Muscle
Male rats
57.1±6.2a
21.1 ±4.9
17.7 ±0.4
1.5 ±0.5
0.6 ±0.1
1.4 ±0.2
0.3 ±0.1
l.liO.l
0.4 ±0.1
1.1 ±0.3
Female rats
49.8±4.3a
19.4 ±2.2
18.5 ±0.6
1.4 ±0.8
0.9 ±0.2
1.2 ±0.3
0.3 ±0.1
l.OiO.l
0.6 ±0.3
1.0 ±0.4
Male mice
64.0 ± 5. 5a
16.0 ±6.0
20.2 ±1.8
0.6 ±0.4
0.1 ±0.04
0.6 ±0.03
0.1 ±0.01
0.5 ±0.1
0.2 ±0.1
1.0 ±0.2
"Percent of total dose (data are mean ± standard deviation [SD] from three rats or mice).
Source: Mahmoodetal. (1991).
3.2. DISTRIBUTION
Mahmood et al. (1991) examined the deposition of 30 mg/kg [2-14C]-l,2,3-
trichloropropane in rats and mice at three time points: 6, 24, and 60 hours post-administration in
corn oil gavage. After 6 hours, most of the radiolabel was found in the forestomach and
glandular stomach with smaller quantities in the intestines, adipose tissue, liver, and kidney. At
24 hours, the concentrations of radiolabel in the forestomach, intestines, liver, and kidney were
similar. By hour 60, the majority of the radiolabel had been excreted in the urine or feces with
some residual radioactivity sequestered predominantly in the liver, kidney, skin, muscle, and
adipose tissue (see Table 3-1). The radiolabel detected in tissues after 60 hours was generally
not extractable, suggesting that it was bound to macromolecules (Mahmood et al., 1991).
Volp et al. (1984) examined the time-dependent distribution of [1,3-14C]-1,2,3-
trichloropropane (2.1 mCi/mmol) in male Fischer rats (three rats per time point) following
intravenous (i.v.) injection of 3.6 mg/kg. Animals were maintained in metabolic cages and
sacrificed at the following time points: 15 and 30 minutes; 1, 2, 4, and 8 hours; and 1, 2, 4, and 6
days post-administration. Rapid distribution of the radiolabel was observed and 37% of the dose
was detected in adipose tissue 15 minutes after administration. After 4 hours, the largest portion
(maximum of 7.3% at 1 hour) of the radiolabel was sequestered in the liver, primarily as
metabolites, and the kidney contained a maximum of 2.8% of the dose at 2 hours post-
administration. The half-lives of phase 1 and phase 2 metabolism of 1,2,3-trichloropropane were
0.57 and 40 hours in the liver, 0.31 and 30 hours in the kidney, 1.8 and 44 hours in the adipose
tissue, and 0.29 and 23 hours in the blood, respectively (Volp et al., 1984).
-------
Weber and Sipes (1990) administered intraperitoneal (i.p.) injections of 30 mg/kg (100
uCi/kg) [2-14C]-l,2,3-trichloropropane in vegetable oil to male Fischer rats. Groups of four rats
were sacrificed after 1, 4, 24, 48, and 72 hours. Maximal covalent binding of radiolabel to
hepatic protein, approximately 600 pmol/mg, was observed at 4 hours post-administration.
Maximal covalent binding to hepatic DNA, approximately 250 pmol/mg, occurred at 24 hours.
After 72 hours, the amount of radiolabel covalently bound to both hepatic protein and DNA was
at or below the levels found 1 hour after administration.
3.3. METABOLISM
No studies of 1,2,3-trichloropropane metabolism in humans have been reported. In vitro
data indicate that human microsomes, in the presence of reduced nicotinamide adenine
dinucleotide phosphate (NADPH), are capable of forming the DNA- reactive chemical, 1,3-
dichloroacetone (DCA), from 1,2,3-trichloropropane (Weber and Sipes, 1992).
In rodents, 1,2,3-trichloropropane metabolism appears to involve oxidation catalyzed by
cytochrome P-450 (CYP450) or glutathione (GSH) conjugation, but specific details about the
metabolic process are unknown. Three potential routes for 1,2,3-trichloropropane metabolism
(Figure 3-1) have been proposed by Mahmood et al. (1991):
I) Nucleophilic displacement of a chlorine atom by GSH creates a p-chlorothio ether, and
internal displacement of another chlorine creates an episulfonium ion. This reactive ion
could hydrolyze to a GSH conjugate that can be cleaved to form N-acetyl-S-(3-chloro-
2-hydroxypropyl)-L-cysteine (ACPC) or S-(3-chloro-2-hydroxypropyl)-L-cysteine
(CPC). The reactive episulfonium ion could also react with water to form P-chlorothio
ether that could form a second episulfonium ion. This second episulfonium ion could
form 2-(S-glutathionyl)malonic acid (GMA) through hydrolysis to form a 1,3-
dihydroxypropyl GSH conjugate and subsequent oxidation.
II) Oxidation of 1,2,3-trichloropropane at the C2 position, possibly by CYP450 enzymes,
could lead to the formation of 1,3-dichloroacetone. Displacement of chlorine from 1,3-
dichloroacetone by GSH and reduction of the keto group can result in the formation of
ACPC and CPC.
Ill) Oxidation, possibly by CYP450 enzymes, of 1,2,3-trichloropropane at the Cl position
to form 2,3-dichloropropanal. This chlorohydrin could undergo loss of HC1 to form
chloroacrolein, and then rearrange with GSH to form an episulfonium ion. This ion
could then form GMA after the oxidation of the C2 and C3 carbon atoms to form
carboxylic acids.
-------
Cl
,ci 1,2,3-Trichloropropane
o
Ill
2,3-Dichl oropr opanal
I +GSH
II
Cl
Qn
S-G
Glutathionyl-2,3- Q,
dichloropropane
1,3-Dichloroacetone
O
G-S
Glutathionyl- °
3-chloroacetone
Cl
+GSH
Cl
g_G Glutathionyl-1-
choropropan-3- ol
S-G
'OH
Glutathionyl- OH
hydroxypropanal
GMA* O
(bile)
HO'
r^s+
r
I episulfonium ion
G-S
Glutathionyl-3-
choropropan-2-ol
'OH HO'
G-23-PD OH
S-G
S-G = S-glutathione
GSH= reduced glutathione
Nac = N-acetyl-L-cysteine
Cys = L-cysteine
* = Compund has been
detected in vivo
Nac
ACPC* OH
(urine)
CPC* OH
(urine)
ACPC = N-acetyl-S-(3-chloro-2-hydroxypropyl)-L-cysteine
CPC = S-(3-chloro-2-hydroxypropyl)-L-cysteine
GMA = 2-(S-glutathionyl)malonic acid
G-1,3-PD = Glutathionyl-l,3-propanediol
G-2,3-PD = Glutathionyl-2,3-propanediol
Sources: WHO, 2003; Mahmood et al., 1991.
Figure 3-1. Possible metabolic pathways for 1,2,3-trichloropropane in rats
-------
Evidence for the involvement of CYP450 in 1,2,3-trichloropropane metabolism is
provided by the in vitro formation of 1,3-dichloroacetone when isolated rat or human hepatic
microsomes were incubated with 1,2,3-trichloropropane (Weber and Sipes, 1992). The
formation of 1,3-dichloroacetone, an intermediate in the formation of ACPC and CPC, occurred
only in the presence of NADPH and was enhanced by the addition of such CYP450 inducers as
phenobarbital and dexamethasone. Conversely, formation of 1,3-dichloroacetone was blocked
by the CYP450 inhibitors SKF-525A and 1-aminobenzotriazol. This investigation also
demonstrated that rat hepatic microsomes formed 1,3-dichloroacetone from 1,2,3-
trichloropropane at a rate of 0.27 nmol/minute/mg protein, which was 10 times faster than the
rate of formation of 1,3-dichloroacetone by human hepatic microsomes (0.026 nmol/minute/mg
protein) (Weber and Sipes, 1992).
3.4. ELIMINATION
Mahmood et al. (1991) and Volp et al. (1984) demonstrated that urine is the primary
route of the excretion of 1,2,3-trichloropropane metabolites in rats and mice. Mahmood et al.
(1991) analyzed the urine of F344/N rats and male B6C3F1 mice treated with [2-14C]-l,2,3-
trichloropropane by corn oil gavage and found that the parent compound was extensively
metabolized to either ACPC or CPC. These investigators also documented that the principal
biliary metabolite was GMA. In rats, ACPC was the major urinary metabolite found 6 hours
after exposure, accounting for approximately 40% of the radiolabel recovered in males and 10%
in females. The urinary metabolite associated with the largest fraction of radiolabel in both
males and females 24 hours post-administration could not be identified. However, substantial
amounts of radiolabeled ACPC and CPC were detected in urine at 24 hours. In male mice,
ACPC accounted for only 3% of the radiolabel at 6 hours (females were not tested). The major
metabolites in male mice at both 6 and 24 hours were not identified.
Volp et al. (1984) examined the time-dependent distribution of [1,3-14C]-1,2,3-
trichloropropane in male Fischer rats (three rats per time point) following i.v. injection of 3.6
mg/kg. The data from this study demonstrated rapid excretion of the radiolabeled metabolite;
after 24 hours, 30% of the initial radiolabel had been exhaled, 40% had been released in the
urine, and 18% was excreted in feces. Unchanged 1,2,3-trichloropropane was not detected in the
urine.
Weber (1991) conducted a detailed analysis of urinary metabolites by employing proton
decoupled and two-dimensional homonuclear correlated nuclear magnetic resonance
spectroscopy following the i.p. coadministration of [l,2,3-13C]-trichloropropane and [2-14C]-
trichloropropane in soybean oil to male F344/N rats. This investigator identified N-acetyl-S-(2-
hydroxy-3-chloropropyl)cysteine, l,3-(2-propanol)-bis-S-(N-acetylcysteine), N-acetyl-S-(2-
hydroxy-2-carboxyethyl)cysteine, 2,3-dichloropropionic acid, 2-chloroethanol, ethylene glycol,
-------
and oxalic acid as potential urinary metabolites of 1,2,3-trichloropropane. It is unknown where
in the metabolic pathway these additional urinary metabolites may form.
3.5. PHYSIOLOGICALLY BASED TOXICOKINETIC MODELING
Volp et al. (1984) developed a physiologically based toxicokinetic (PBTK) model to
describe the time-dependent appearance of 1,2,3-trichloropropane and its metabolites in rat
tissues. The model consists of compartment-specific mass balance equations for tissues that
have physiological significance in storage, transport, and clearance. The model, which
accurately predicted the concentration versus time curves for the selected tissues, contains seven
compartments: blood, liver, kidney, adipose tissue, muscle, skin, and remaining distribution
volume. The model describes the rapid disappearance of 1,2,3-trichloropropane from the blood
with biotransformation products concurrently appearing in the urine, bile, and expired air. High
concentrations of metabolites were also found in the liver and kidney, and the half-lives for
trichloropropane clearance from blood and liver were 23 and 40 hours, respectively (Volp et al.,
1984). The tissue distribution of 1,2,3-trichloropropane and metabolites is presented in Section
3.2.
-------
4. HAZARD IDENTIFICATION
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS
Limited information from an acute inhalation study in humans (n = 12) demonstrated that
15-minute exposures to 100 ppm trichloropropane (purity unknown) resulted in irritation of the
nose, eyes, and throat of all subjects tested (Silverman et al., 1946). No occupational,
epidemiology, or case study data were identified that were applicable to 1,2,3-trichloropropane
exposure in humans.
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
Hazelton Laboratories (1983a, b) conducted a series of subchronic toxicity studies of
1,2,3-trichloropropane in F344/N rats and B6C3F1 mice. The findings of these subchronic
studies were included in the National Toxicology Program (NTP) technical report on the
toxicology and carcinogenesis of the compound and published in the peer-reviewed literature
(NTP, 1993).
The same protocol was used for both the rat and the mouse studies. 1,2,3-
Trichloropropane was administered by corn oil gavage 5 days/week for 120 days at doses of 0
(vehicle control), 8, 16, 32, 63, 125, or 250 mg/kg-day. Treatment groups contained 20
animals/sex and the vehicle control group contained 30 animals/ sex, and the animals were
approximately 6 weeks old when the studies began. Half of the animals in each group were
sacrificed after 8 weeks, and the rest were maintained until week 17. Animals were examined
twice daily for clinical signs of toxic stress. Animals were weighed at the start of the study and
at weekly intervals during the course of the study. Blood and urine samples were obtained from
animals during weeks 8 and 17. Blood samples were analyzed for hematocrit, hemoglobin, and
blood cell counts. A limited suite of clinical chemistry parameters was also evaluated. Specific
gravity of the urine specimens was determined. Necropsies were performed on all animals with
complete histopathologic examinations performed on all animals that had died during the study,
moribund animals that were sacrificed during the study, all rats receiving a dose of 125 mg/kg-
day, and all controls. A number of organs and tissues were excised and collected from all
animals. Tissue weights were reported for the 17-week study only.
In the rat study, 12 males that received 250 mg/kg-day died, or were sacrificed moribund,
during the first week of treatment. Six males died during the second week and the remaining
two animals were terminated in weeks 3 and 5. Sixteen females in the 250 mg/kg-day dose
10
-------
group died, or were sacrificed moribund, during the first week. The remaining four animals in
this treatment group died during the second week. One male and four female rats that received
125 mg/kg-day 1,2,3-trichloropropane died, or were sacrificed moribund, during the study.
During their brief survival period, rats in the 250 mg/kg-day treatment group were noted
to have been emaciated, lethargic, and debilitated. No clinical signs of toxicosis were observed
in any of the other treatment groups. Dose-dependent reductions in body weight gain were
observed in both males and females. Mean final body weights were significantly reduced for
male rats that received 63 and 125 mg/kg-day and females treated with 125 mg/kg-day 1,2,3-
trichloropropane. Whole-body and tissue weights were not reported for the 250 mg/kg-day
treatment groups. At the 17-week sacrifice, mean weight gain in the 125 mg/kg-day treatment
group was reduced by 43 and 60% for males and females, respectively, compared with controls.
A dose-dependent increase in relative liver and kidney weights (organ weight/body
weight) was observed in male and female rats, but increases in absolute liver weights and kidney
weights were not dose-dependent, other than absolute liver weight in female rats. Mean relative
liver weights were statistically (p < 0.01) significantly increased in males that received 32, 63, or
125 mg/kg-day by 24, 47, and 78%, respectively, compared with controls (Table 4-1), while
absolute liver weights statistically significantly (p<0.01) increased 10-26% in males receiving
8-125 mg/kg-day (Table 4-2). Mean relative liver weights, when compared with controls, were
statistically (p < 0.01) significantly increased by 12, 18, 37, and 105% in females receiving 16,
32, 63, or 125 mg/kg-day, respectively (Table 4-1), while absolute liver weights statistically
significantly (p<0.05) increased 17-61% in females receiving 16-125 mg/kg-day (Table 4-2).
Mean relative right kidney weights were statistically significantly increased in males that
received 32, 63, or 125 mg/kg-day by 12% (p < 0.05), 26% (p < 0.01), and 54% (p < 0.01),
respectively, compared with controls (Table 4-3), while absolute right kidney weights were
statistically significantly (p < 0.01) increased 5-19% in males receiving 32, 63, or 125 mg/kg-
day (Table 4-4). In females that received 63 and 125 mg/kg-day 1,2,3-trichloropropane, mean
relative right kidney weights were statistically (p < 0.01) significantly increased 32 and 43%,
respectively (Table 4-3), while absolute right kidney weights statistically significantly (p < 0.01)
increased 11-25% in females receiving 63-125 mg/kg-day (Table 4-4). Absolute heart weight
was statistically significantly (p < 0.01) decreased 14 and 21% in male rats at 63 and 125 mg/kg-
day, respectively. NTP (1993) considered the changes in relative brain and heart weights to be
associated with the change in body weight, and not with organ toxicity.
11
-------
Table 4-1. Relative liver weights (mg organ weight/g body weight) and
percent change in F344/N rats exposed to 1,2,3-trichloropropane by gavage
for 17 weeks
Dose (mg/kg-d)
0
8
16
32
63
125
250
n
10
10
10
10
10
9
-
Males
Mean ± SE
24.6 ±0.5
26.8 ±0.4
27.6 ±0.5
30.5±0.7C
36.2±1.9C
43.7±1.6C
Percentage change"
-
9%
12%
24%
47%
78%
NRb
n
10
10
10
10
10
6
-
Females
Mean ± SE
25.7 ±0.4
27.5 ±0.6
28.9±0.4C
30.2±0.6C
35.2±0.8C
52.6±2.3C
Percentage change"
-
7%
12%
18%
37%
105%
NR
"Calculated as the percent change from the control mean.
bNR = Due to the rapid onset of mortality, organ weights were not recorded for the high-dose group.
'Showing statistically significant differences (p < 0.01) from the control group by Williams' or Dunnett's test.
Source: NTP(1993).
Table 4-2. Absolute liver weights (g) and percent change in F344/N rats
exposed to 1,2,3-trichloropropane by gavage for 17 weeks
Dose (mg/kg-d)
0
8
16
32
63
125
250
n
10
10
10
10
10
9
Males
Mean ± SE
8.87 ±0.14
9.82 ±0.21
9.72 ±0.38
11.20 ±0.20
10.93 ±0.23
12.07 ±0.13
Percentage
change"
-
ll%b
10%b
26%b
23%b
19%b
NRd
n
10
10
10
10
10
6
Females
Mean ± SE
5. 14 ±0.1
5.49 ±0.09
6.07 ±0.16
6.00 ±0.09
6.79 ±0.17
8.25 ±0.2
Percentage
change"
-
7%
18%b
17%c
32%b
61%b
NR
""Calculated as the percent change from the control mean.
bShowing statistically significant differences (p < 0.01) from the control group by Williams' or Dunnett's test.
'Showing statistically significant differences (p < 0.05) from the control group by Williams' or Dunnett's test.
dNR = Due to the rapid onset of mortality, organ weights were not recorded for the high-dose group.
Source: NTP(1993).
12
-------
Table 4-3. Relative kidney weights (mg organ weight/g body weight) and
percent change in F344/N rats exposed to 1,2,3-trichloropropane by gavage
for 17 weeks
Dose (mg/kg-d)
0
8
16
32
63
125
250
n
10
10
10
10
10
9
-
Males
Mean ± SE
3.00 ±0.03
2.97 ±0.04
3. 14 ±0.04
3.37±0.03b
3.77±0.24C
4.63±0.16C
Percentage
change"
-
-1%
5%
12%
26%
54%
NRd
n
10
10
10
10
10
6
-
Females
Mean ± SE
3. 16 ±0.07
3.37±0.19
3.38 ±0.06
3.49 ±0.05
4.16±0.17C
4.52±0.19C
Percentage
change"
-
7%
7%
10%
32%
43%
NR
"Calculated as the percent change from the control mean.
bShowing statistically significant differences (p < 0.05) from the control group by Williams' or Dunnett's test.
'Showing statistically significant differences (p < 0.01) from the control group by Williams' or Dunnett's test.
dNR = Due to the rapid onset of mortality, organ weights were not recorded for the high dose group.
Source: NTP(1993).
Table 4-4. Absolute kidney weights (g) and percent change in F344/N rats
exposed to 1,2,3-trichloropropane by gavage for 17 weeks
Dose (mg/kg-d)
0
8
16
32
63
125
250
n
10
10
10
10
10
9
-
Males
Mean ± SE
1.08 ±0.03
1.09 ±0.02
1.10 ±0.03
1.24±0.02b
1.13±0.02b
1.28±0.02b
Percentage
change"
-
l%a
2%
15%
5%
19%
NRd
n
10
10
10
10
10
6
-
Females
Mean ± SE
0.64 ±0.01
0.67 ±0.03
0.71 ±0.02
0.70 ±0.02
0.80±0.03C
0.71±0.02C
Percentage
change"
-
5%a
11%
11%
25%
11%
NR
"Calculated as the percent change from the control mean.
bshowing statistically significant differences (p < 0.01) from the control group by Williams' or Dunnett's test.
'showing statistically significant differences (p < 0.05) from the control group by Williams' or Dunnett's test.
dNR = Due to the rapid onset of mortality, organ weights were not recorded for the high dose group.
Source: NTP(1993).
An increased incidence of lesions, as described below, was observed in the liver, kidney,
and nasal turbinates of rats receiving 125 mg/kg-day 1,2,3-trichloropropane for 120 days (Table
4-5). A time-dependent increase in the number of lesions was noted between the 8- and 17-week
13
-------
evaluations in the 125 mg/kg-day treatment group. This same pattern was not observed in the
250 mg/kg-day treatment group since the majority of animals did not survive more than 1 week.
Table 4-5. Incidence of liver, kidney, and nasal turbinate lesions in male and
female F344/N rats in a 17-week study
Endpoint
Dose (mg/kg-d)
0
8
16
32
63
125
Males
Liver necrosis3
Kidney necrosis3
Epithelial necrosis of nasal
turbinates3
0/20
0/20
0/20
0/10
0/10
0/10
0/10
0/10
0/10
1/10
0/10
0/10
1/10
0/10
0/10
1/10
1/10
3/9b
Females
Liver necrosis3
Kidney necrosis3
Epithelial necrosis of nasal
turbinates3
0/20
0/20
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
ll/llc
0/11
2/11
Incidence is the number of animals in which lesion was found/number of animals in which tissue was examined.
bShowing statistically significant differences (p < 0.05) from the control group by Fisher exact test.
'Showing statistically significant differences (p < 0.01) from the control group by Fisher exact test.
Source: NTP(1993).
The liver lesions in rats were characterized by multifocal, centrilobular hepatocellular
necrosis, with karyomegaly, hemorrhage, and bile duct hyperplasia. Hepatic necrosis was
observed in female rats (7/9) receiving 125 mg/kg-day and in all of the rats receiving 250 mg/kg-
day 1,2,3-trichloropropane (20/20 males and 20/20 females) at the time of their death. In the 17-
week evaluation, hepatic necrosis was observed at terminal sacrifice in 1/10 males and 11/11
females treated with a dose of 125 mg/kg-day, with liver necrosis also evident in 1/10 male rats
at 32 and 63 mg/kg-day.
The kidney lesions in the rats were characterized by early diffuse acute tubule necrosis or
regenerative hyperplasia, karyomegaly of epithelial cells, and multifocal necrosis. Renal tubular
necrosis was observed during the 8-week interim evaluation in 14/20 males and 20/20 females
treated with 250 mg/kg-day that died at or before the interim sacrifice. At the 17-week
evaluation, renal necrosis was observed in 1/10 males and 0/11 females treated with a dose of
125 mg/kg-day.
Lesions of the nasal turbinates included multifocal necrosis and epithelial attenuation,
subepithelial fibrosis, and inflammation. Epithelial necrosis of the nasal turbinates was observed
during the 8-week interim evaluation in 14/20 males and 19/20 females treated with 250 mg/kg-
day that died at or before the interim sacrifice. At the time of death or at the 17-week evaluation,
14
-------
epithelial necrosis of the nasal turbinates was observed in 3/9 males and 2/11 females treated
with 125 mg/kg-day.
A number of clinical chemistry parameters in rats were statistically significantly affected
upon exposure to 1,2,3-trichloropropane. Blood samples were not obtained from animals in the
250 mg/kg-day treatment group. Effects observed were predominantly biomarkers for liver
damage. At the 8-week interim evaluation, the activities of alanine aminotransferase (ALT),
sorbitol dehydrogenase (SDH), and aspartate aminotransferase (AST), were all statistically (p <
0.01) significantly elevated, 1,200, 433, and 1,000%, respectively, over controls in females that
received 125 mg/kg-day. Total bilirubin levels in female rats at the 8-week evaluation increased
50 and 150% at doses of 63 and 125 mg/kg-day, respectively. At the 17-week evaluation, ALT
and SDH activities were statistically [(p < 0.05) and (p < 0.01), respectively] significantly
elevated, 248 and 317%, respectively, over controls in females treated with 125 mg/kg-day.
The activity of ALT was statistically (p < 0.05) significantly elevated in males treated
with 125 mg/kg-day at week 8 but not at week 17, while the activity of SDH in males at 17
weeks was statistically significantly (p < 0.05) increased 25 and 12.5% at 63 and 125 mg/kg-day,
respectively. NTP (1993) stated that the increase in ALT and SDH was indicative of
hepatocellular damage with subsequent enzyme leakage. The only clinical chemistry parameter
that was consistently impacted in both males and females at both time points was
pseudocholinesterase (serum carboxylesterase). Activity of this hepatic enzyme decreased in
both species with increasing dose and NTP (1993) suggested that the depressed synthesis of
pseudocholinesterase was due to hepatocellular damage. A statistically significant decrease was
observed at both time points (8 and 17 weeks) evaluated in females at the lowest dose tested, 21
and 14% at 8 mg/kg-day (p < 0.01), and 9 and 8% in males that received 32 mg/kg-day (p <
0.05). The authors observed a dose-dependent decrease in pseudocholinesterase of 14-77% at 8
through 125 mg/kg-day in female rats and 8-21% at 32-125 mg/kg-day in male rats at 17 weeks.
In rats, hematocrit, hemoglobin, and erythrocyte counts were statistically significantly
decreased by 1,2,3-trichloropropane treatment. At the 8-week sacrifice, hematocrit and red
blood cell counts were significantly depressed by 13-23 and 10-18%, respectively, in males that
received doses of >16 mg/kg-day and in females that received doses of >8 mg/kg-day.
Hemoglobin was statistically significantly decreased 5-9% in male rats that received doses of
>16 mg/kg-day and female rats that received >63 mg/kg-day.
The 17-week, less-than-lifetime rat study was conducted to determine appropriate doses
for the 2-year, 1,2,3-trichloropropane study in rats (NTP, 1993), described later in this document.
NTP considered the dose-response of the increased liver and kidney weights to be consistent
with the clinical pathological and histopathological findings in the liver and kidney. The
NOAEL and LOAEL for hepatocellular necrosis in male rats at 17-weeks were 16 and 32 mg/kg-
day, and in females rats at 17-weeks were 63 and 125 mg/kg-day. The NOAEL and LOAEL for
15
-------
increased SDH in male rats were 32 and 63 mg/kg-day, respectively. A decrease in
pseudocholinesterase (serum carboxylesterase) activity in males presented a NOAEL of 16
mg/kg-day and LOAEL of 32 mg/kg-day, whereas females had a LOAEL of 8 mg/kg-day. The
NOAEL and LOAEL for renal tubular necrosis in male rats at 17 weeks were 63 and 125 mg/kg-
day, respectively, while the NOAEL for renal tubular necrosis in females was 125 mg/kg-day.
For epithelial necrosis of the nasal turbinates, the NOAEL and LOAEL in male and female rats
at 17 weeks were 63 and 125 mg/kg-day, respectively.
In the NTP (1993) subchronic, B6C3F1 mouse study, which used the same protocol as
the rat study above, 16 males that received 250 mg/kg-day 1,2,3- trichloropropane died, or were
sacrificed moribund, by week 4. Among the females that received a dose of 250 mg/kg-day,
seven died by week 2, and there was an additional death in week 17 (prior to the terminal
sacrifice). One male mouse and six female mice were sacrificed at the 8-week interim
evaluation. At the end of the 17-week evaluation, 2 out of 10 males at the highest dose were still
alive, whereas 7 out of 10 females, tallied before the death of single female during week 17,
survived the full evaluation period.
Mean body weight gain in male mice at 250 mg/kg-day was significantly reduced,
although the overall mean weight gains among male and female mice at the various doses were
similar. At week 17, a statistically significant (p < 0.01) increase in absolute and relative liver
weights was observed in males and females that received a dose of >125 mg/kg-day. Mean
relative liver weights were increased by 10 and 30% in males receiving 125 and 250 mg/kg-day,
respectively, compared to controls (Table 4-6), while absolute liver weights were statistically
significantly (p < 0.05) increased 14, 4, 22, and 25% at 32, 63, 125, and 250 mg/kg-day (Table 4-
7). Mean relative liver weights were increased by 12 and 22% in females receiving 125 and 250
mg/kg-day, respectively, compared to controls (Table 4-6), while absolute liver weights were
statistically significantly (p < 0.05) increased at 125 and 250 mg/kg-day by 24% at both doses
(Table 4-7). Mean relative right kidney weights in female mice were statistically significantly (p
< 0.01) decreased 17, 13, 11, 17, and 14% at 16, 32, 63, 125, and 250 mg/kg-day, respectively,
after 120 days (Table 4-8), while absolute right kidney weights were statistically significantly (p
< 0.05) decreased 13% at 250 mg/kg-day (Table 4-9). The changes in relative and absolute right
kidney weights in male mice did not follow a clear dose-response pattern.
16
-------
Table 4-6. Relative liver weights (mg organ weight/g body weight) and
percent change in B6C3Fi mice exposed to 1,2,3-trichloropropane by gavage
for 17 weeks
Dose (mg/kg-day)
0
8
16
32
63
125
250
n
10
10
10
10
10
8
2
Males
Mean ± SE
39.9 ± 1.0
40.3 ±0.8
38.8 ±0.6
42.0 ±0.5
39.9 ±0.8
44.0±1.3C
51.8 ± 1.0°
% Percentage
change"
—
1%
-3%
5%
0%
10%
30%
n
10
10
7
10
9
9
6
Females
Mean ± SE
43. 3 ±1.1
43.7 ±0.8
39.9 ± 1.8
44.5 ±0.5
45.2 ±0.7
48.7±1.0b
52.7±2.2b
% Percentage
change"
—
1%
-8%
3%
4%
12%
22%
"Calculated as the percent change from the control mean.
bShowing statistically significant differences (p < 0.01) from the control group by Williams' or Dunnett's test.
Source: NTP(1993).
Table 4-7. Absolute liver weights (g) and percent change in B6C3Fi mice
exposed to 1,2,3-trichloropropane by gavage for 17 weeks
Dose
(mg/kg-day)
0
8
16
32
63
125
250
n
10
10
10
10
10
8
2
Males
Mean ± SE
1.06 ±0.03
1.14 ±0.04
1.09 ±0.02
1.21 ± 0.03 b
1.10±0.03b
1.29 ± 0.04 c
1.32 ± 0.00 c
Percentage%
change"
—
8%
3%
14%
4%
22%
25%
n
10
10
7
10
9
9
6
Females
Mean ± SE
0.898 ±0.037
0.899 ±0.035
0.938 ±0.037
0.947 ±0.016
0.994 ±0.048
1.118±0.029C
1.112±0.053C
Percentage%
change"
—
0%
4%
5%
11%
24%
24%
"Calculated as the percent change from the control mean.
bShowing statistically significant differences (p < 0.05) from the control group by Williams' or Dunnett's test.
'Showing statistically significant differences (p < 0.01) from the control group by Williams' or Dunnett's test.
Source: NTP(1993).
17
-------
Table 4-8. Relative kidney weights (mg organ weight/g body weight) and
percent change in B6C3Fi mice exposed to 1,2,3-trichloropropane by gavage
for 17 weeks
Dose (mg/kg-day)
0
8
16
32
63
125
250
n
10
10
10
10
10
7
2
Males
Mean ± SE
8.75 ±0.25
8.97 ±0.15
8.85 ±0.22
9.19±0.14
7.86 ±0.31
8.44 ±0.58
8.83 ±0.37
% Percentage
change"
—
3%
1%
5%
-10%
-3%
1%
n
10
10
7
10
9
9
6
Females
Mean ± SE
8.23 ±0.18
8.07 ±0.17
6.86±0.54C
7.18±0.13C
7.31±0.23C
6.87±0.17C
7.04±0.30C
Percentage%
change"
—
-2%
-17%
-13%
-11%
-17%
-14%
""Calculated as the percent change from the control mean.
bShowing statistically significant differences (p < 0.05) from the control group by Williams' or Dunnett's test.
'Showing statistically significant differences (p < 0.01) from the control group by Williams' or Dunnett's test.
Source: NTP(1993).
Table 4-9. Absolute kidney weights (g) and percent change in B6C3Fi mice
exposed to 1,2,3-trichloropropane by gavage for 17 weeks
Dose (mg/kg-day)
0
8
16
32
63
125
250
n
10
10
10
10
10
7
2
Males
Mean ± SE
0.232 ± 0.006
0.253 ±0.007
0.248 ±0.008
0.265 ±0.008
0.215 ±0.008
0.247 ±0.011
0.225 ±0.005
% Percentage
change"
—
9%
7%
14%
-7%
6%
-3%
n
10
10
7
10
9
9
6
Females
Mean ± SE
0.170 ±0.005
0.166 ±0.004
0.164 ±0.010
0.153 ±0.005
0.160 ±0.007
0.158 ±0.005
0.148±0.006b
% Percentage
change"
—
-2%
-2%
-10%
-6%
-7%
-13%
""Calculated as the percent change from the control mean.
bShowing statistically significant differences (p < 0.05) from the control group by Williams' or Dunnett's test.
Source: NTP(1993).
Mean relative heart weights in males were statistically significantly (p < 0.05) decreased
14, 14, 11, 19, 22 and 22% at 8, 16, 32, 63, 125, and 250 mg/kg-day, respectively, compared to
controls. Absolute heart weights in males were statistically significantly (p < 0.01) reduced by
14-25% at >63 mg/kg-day and higher. Relative brain weights in male mice were statistically
significantly (p < 0.05) decreased at 16-125 mg/kg-day, with the decrease ranging from 6-11%.
Mean relative heart weights in females were statistically significantly (p < 0.05) reduced 19, 17,
18
-------
11, 19, and 27% at 16, 32, 63, 125, and 250 mg/kg-day. Absolute heart weights in females were
statistically significantly (p < 0.01) decreased 25% at 250 mg/kg-day. Absolute and relative
brain weights were statistically significantly (p < 0.01) decreased 6-15% in females receiving 16
mg/kg-day or more.
Complete histopathological examinations were conducted on all control animals and
mice receiving 125 or 250 mg/kg-day, and mice designated for the interim evaluation that died
during the study were included in the group of animals examined at the end of the 17-week
study. Forestomach and lung lesions in mice were observed at both the 8-week interim
evaluation and the 17-week terminal evaluation (Table 4-10). At the 8-week evaluation, male
mice displayed lung and forestomach lesions at 125 mg/kg-day in 1/8 and 6/8 mice, respectively,
whereas female mice displayed lung and forestomach lesions at 250 mg/kg-day in 5/6 and 6/6
mice, respectively. Lung and forestomach lesions were found in the one mouse from the 250
mg/kg-day dose group that was examined at the 8-week interim sacrifice.
Table 4-10. Incidence of liver, lung, and forestomach lesions in male and
female B6C3Fi mice in a 17-week study
Endpoint
Dose (mg/kg-d)
0
8
16
32
63
125
250
Males
Liver necrosis3
Liver karyomegaly3
Lung lesions-
regenerative3
Hyperkeratosis of the
forestomach3
1/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
1/12
1/12
9/12b
7/12b
14/19b
ll/19b
14/19b
4/19
Females
Liver necrosis3
Liver karyomegaly3
Lung lesions-
regenerative3
Hyperkeratosis of the
forestomach3
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/10
0/9
0/9
7/9b
7/9b
1/12
0/12
10/12b
9/12b
5/14c
1/14
7/14b
8/14b
Incidence is the number of animals in which lesion was found/number of animals in which tissue was examined.
bShowing statistically significant differences (p < 0.01) from the control group by Fisher exact test.
'Showing statistically significant differences (p < 0.05) from the control group by Fisher exact test.
Source: NTP(1993).
Regenerative lung lesions were observed in 9/12 male mice and 10/12 female mice, and
hyperkeratosis of the forestomach in 7/12 male mice and 9/12 female mice receiving 125 mg/kg-
day 1,2,3-trichloropropane at the 17-week evaluation. Lung lesions in male and female mice at
19
-------
250 mg/kg-day 1,2,3-trichloropropane were observed in 14/19 males and 7/14 females, while
forestomach lesions in the same dose group were observed in 4/19 males and 8/14 females. At
63 mg/kg-day, female mice displayed lung lesions (7/9) and forestomach lesions (7/9).
Hyperkeratosis of the forestomach was attributed to continued irritation resulting from the
gavage treatments and was not considered to be life-threatening (Hazelton Laboratories, 1983b).
Focal or multifocal desquamation of necrotic cells in the airways, flattened epithelium with loss
of differentiated cells, and thickened epithelium with an increase in goblet cells (hyperplasia)
were characteristic of the regenerative lung lesions (NTP, 1993).
Liver lesions were observed at both the 8-week interim and 17-week terminal sacrifice.
At the 8-week evaluation, liver lesions were not observed in the only examined male mouse that
received 250 mg/kg-day 1,2,3-trichloropropane, but hepatic necrosis was observed in 4/6
females that received this dose. Hepatic necrosis at the 8-week evaluation was observed in 6/8
males and 0/8 females that received 125 mg/kg-day. No liver lesions were observed in the 8-
week controls.
At the 17-week evaluation, liver necrosis was observed in 14/19 males, most of which
died prior to 8-week evaluation, and 5/14 females that received 250 mg/kg-day, and 1/10 male
and 0/10 female controls (Table 4-10). Hepatocelluar degeneration associated with fatty change
and karyomegaly was also observed in 11/19 males and 1/14 females of the high-dose group.
Also at the 17-week evaluation, liver lesions in mice at the 125 mg/kg-day dose occurred in 1/12
males and 1/12 females.
Differences in clinical chemistry parameters in mice administered 1,2,3-trichloropropane
for 17 weeks were not considered by the NTP investigators to be treatment related. Several
statistically significant changes were observed among hematological parameters; however, these
changes were not considered to be biologically significant and failed to follow a consistent dose-
response pattern. Hematocrit values were statistically significantly decreased at week 8 in
female mice that received 8 and 250 mg/kg-day. At week 17, hematocrit values were
statistically significantly decreased in female mice that received 16, 32, 125, or 250 mg/kg-day
1,2,3-trichloropropane. In male mice, a statistically significant decrease in hematocrit values
was observed only at week 8 in the 63 and 125 mg/kg-day treatment groups.
The 17-week, less-than-lifetime mouse study was conducted to determine appropriate
doses for the 2-year study in mice (NTP, 1993), described later in this document. The dose-
related increased liver weights were consistent with the histopathological results, while the
hematological data were not associated with 1,2,3-trichloropropane administration (NTP, 1993).
The NOAEL and LOAEL for regenerative lung lesions at the 17-week evaluation were 63 and
125 mg/kg-day for male mice and 32 and 63 mg/kg-day for female mice. The NOAEL and
LOAEL for liver lesions at the 17-week evaluation were 63 and 125 mg/kg-day for both male
20
-------
and female mice. A NOAEL of 63 mg/kg-day and LOAEL of 125 mg/kg-day for liver necrosis
in male and female mice were identified.
Merrick et al. (1991) administered 1,2,3-trichloropropane in corn oil to Sprague-Dawley
rats by gavage for 90 days. Groups of 10 males and 10 females received 0, 1.5, 7.4, 15, or 60
mg/kg-day. Animals that received 60 mg/kg-day exhibited a 14-19% reduction in mean body
weight gain when compared to controls. Relative liver weights were statistically (p < 0.05)
significantly increased after 90 days in animals that received 15 or 60 mg/kg-day, and relative
kidney weights were statistically (p < 0.05) significantly increased after 90 days in males that
received 60 mg/kg-day and females that received 15 or 60 mg/kg-day. Relative brain and testes
weights were statistically (p < 0.05) significantly increased in males from the high dose group.
Organ/body weight ratios were reported graphically.
Female rats that received 60 mg/kg-day 1,2,3-trichloropropane exhibited elevated ALT
and AST levels. Mean serum concentrations for these two enzymes appeared to be
approximately doubled in the high-dose females, but the actual magnitude of this effect could not
reliably be estimated from the graphical presentation of the data. Hematological parameters,
which included hemoglobin, hematocrit, and erythrocyte counts, were stated to be unremarkable
(data not provided).
An increased incidence of inflammation-associated myocardial necrosis was observed in
6/10 males and 7/10 females that received 60 mg/kg-day 1,2,3-trichloropropane (Table 4-11).
These lesions were marked by intense eosinophilic staining with necrotic cells containing
granulated or vacuolated cytoplasm and associated macrophages or polynuclear leukocytes.
Myocardial necrosis was also observed in a smaller number of animals from all other treatment
groups; no myocardial lesions were observed in the control group.
Table 4-11. Incidence of myocardial necrosis in male and female Sprague-
Dawley rats following 90-day 1,2,3-trichloropropane exposure
Endpoint
Myocardial
necrosis
Sex
Male
Female
Dose
0
0/10a
0/10
1.5
2/10
0/10
7.4
1/10
1/10
15
2/10
0/10
60
6/10
7/10
"Incidence is the number of animals in which lesion was found/number of animals in which tissue was examined.
Source: Merrick etal. (1991).
Bile duct hyperplasia was observed in the livers of one control male and 4/10 males and
8/10 females in the high-dose group. Other proliferative and neoplastic lesions observed in high-
dose animals included a forestomach squamous cell papilloma, forestomach squamous cell
hyperplasia, a hepatocellular adenoma, and plasma cell hyperplasia in the mandibular lymph
21
-------
node, with the latter lacking a dose-response relationship in male rats (2/10, 0/10, 1/10, 0/10,
9/10) and displaying an increased dose-response relationship in female rats (1/10, 1/10, 2/10,
3/10, 5/10).
Inflammation-associated myocardial necrosis was seen in all male dose groups, and the
LOAEL selected for this effect in male rats is 1.5 mg/kg-day. The NOAEL and LOAEL for bile
duct hyperplasia were 15 and 60 mg/kg-day. The NOAEL and LOAEL for plasma cell
hyperplasia in the mandibular lymph node was 1.5 and 7.4 mg/kg-day for male rats, while the
LOAEL was 1.5 mg/kg-day for female rats.
Villeneuve et al. (1985) administered 1,2,3-trichloropropane in drinking water to
Sprague-Dawley rats. Ten rats/sex/group were exposed 7 days/week for 90 days to 0, 1, 10, 100,
or 1,000 mg/L. Drinking water contained 0.5% Emulphor to assure adequate solubility of the
test chemical. Two groups of control animals were employed; one received tap water and the
other received a 0.5% Emulphor solution. Body weight and water intake values were used for
females in the 100 and 1,000 mg/L exposure groups to calculate delivered doses of 18 and 149
mg/kg-day, respectively. The delivered dose for males in the 1,000 mg/L exposure group was
calculated to be 113 mg/kg-day. Clinical signs were monitored daily, and body weights were
recorded weekly. At termination, the brain, liver, kidney, heart, and spleen were excised and
weighed. A number of hematological and clinical chemistry parameters were evaluated in blood
samples obtained at sacrifice. Each animal was subjected to a full necropsy, and tissues and
organs were obtained for histopathologic examination. In addition, the specific activities of
some mixed-function oxidases, including aniline hydroxylase and aminopyrine demethylase,
were measured in liver homogenates.
Three animals died during the course of the study, but their deaths were not considered to
be treatment-related. Mean body weight gain was reduced by approximately 30% in male and
female rats that were exposed to 1,000 mg/L 1,2,3-trichloropropane, when compared with both
controls (p < 0.05) and vehicle controls (p < 0.05). No difference in absolute organ weights was
observed. Relative liver and kidney weights were reportedly increased in males that were
exposed to 1,000 mg/L by 22 and 27%, respectively, when compared to vehicle controls. Mean
relative liver weights were apparently increased 6 and 17% in females that were exposed to 100
and 1,000 mg/L, respectively. Mean relative kidney weights in females were reportedly
increased 14 and 34% in the 100 and 1,000 mg/L treatment groups, respectively. Mean relative
brain weights for the 1,000 mg/L exposure groups were reportedly increased by 21 and 23% in
males and females, respectively. Mean serum cholesterol levels were apparently increased 55%
in female rats exposed to 1,000 mg/L and no effect on cholesterol was observed in males.
Hepatic aminopyrine demethylase activity was reportedly significantly increased in males and
females that were exposed to 1,000 mg/L. Aniline hydroxylase activity was apparently
significantly increased in males that received 1,000 mg/L.
22
-------
Mild, but significant, histomorphological changes were reported in the liver, including
anisokaryosis, accentuated zonation, and fatty vacuolation; kidney, including eosinophilic
inclusions, pyknosis, nuclear displacement, fine glomerular adhesions and interstitial reactions
and histologic proteinuria; and thyroid, including angular collapse of follicles, reduction in
colloid density, and increased epithelial height, of both sexes of rats in the highest exposure
group, although the number of affected animals was not reported. Biliary hyperplasia was also
noted in females at 1,000 mg/L. Treatment with 1,2,3-trichloropropane also caused liver and
kidney enlargement, as well as increased serum cholesterol levels and hepatic mixed-function
oxidase activity. Mean lymphocyte and neutrophil counts were depressed by approximately
40% in male rats exposed to 1,000 mg/L, but were still within the historical reference range for
Sprague-Dawley rats from the laboratory. A NOAEL of approximately 18 mg/kg-day and a
LOAEL of 113-149 mg/kg-day was identified for both increased relative liver weight in males
and females and serum cholesterol levels in females.
4.2.1.2. Chronic Studies
NTP (1993) conducted a 2-year study of the toxicity and carcinogenicity of 1,2,3-
trichloropropane in F344/N rats, the data of which were also published in Irwin et al. (1995).
The chemical was administered by corn oil gavage to 60 rats/sex/group. The rats were
approximately 6 weeks old when the study was initiated. Rats received doses of 0 (vehicle
control), 3, 10, or 30 mg/kg-day, and after 15 months (65-67 weeks), 8-10 rats per group were
sacrificed to allow an interim evaluation of all toxicological parameters and histopathology. Due
to high mortality in rats receiving 30 mg/kg at the interim evaluation, the remaining survivors in
that group were sacrificed at week 67 (females) and week 77 (males). Due to the early
termination of this treatment group, organ weights and hematology data were only obtained at
the 15-month interim sacrifices.
Clinical observations were made twice daily, while body weights were recorded weekly
for 13 weeks and then monthly (NTP, 1993). As mentioned above, up to 10 rats/group were
sacrificed at month 15. From this interim sacrifice blood samples were obtained for hematology
and clinical chemistry analyses. Hematological parameters included hematocrit, hemoglobin,
and counts of erythrocytes, leukocytes, and differential leukocytes. Clinical chemistry
parameters included the serum levels of ALT, AST, creatine kinase, lactate dehydrogenase
(LDH), sorbitol dehydrogenase (SDH), and 5'-nucleotidase. Whether at the planned sacrifice or
as each rat died or became moribund, all rats were subjected to a gross necropsy, and a full range
of organs and tissues was processed for histopathologic examination. Hematology, clinical
chemistry, and tissue weight data were obtained only from rats that were sacrificed at the 15-
month interim because the majority of treated animals died prior to the end of the study.
23
-------
Survival rates were statistically significantly reduced (p < 0.001) in rats that received 10
or 30 mg/kg-day 1,2,3-trichloropropane (Table 4-12). An effect on survival was apparent, as the
10 and 30 mg/kg-day groups of rats died or were sacrificed moribund prior to or soon following
the 15-month interim evaluation. The mortality in rats was attributed to cancer associated with
chemical exposure (NTP, 1993).
Table 4-12. Survival rates and percent probability of survival for F344/N
rats exposed to 1,2,3-trichloropropane by gavage for 2 years
F344/N rats
Dose
(mg/kg-d)
0
3
10
30
Males
34/49a
32/50
14/48
0/52
70b
64
30C
Oc
Females
31/50b
30/49
8/52
0/52
62a
62
16C
Oc
aAnimals surviving to study termination and number of animals in the treatment group. Accidental deaths were
excluded and censored from survival analysis.
bKaplan-Meier determinations of percent probability of survival at end of study.
>< 0.001.
Source: NTP (1993).
In rats, the mean body weights of males and females receiving doses of 3 or 10 mg/kg-
day, observed throughout the study, appeared similar to the mean body weights of corresponding
control rats; the mean body weights of the high-dose males and females, however, appeared
lower than the control rat body weights (NTP, 1993). Statistically significant increases (p <
0.05) in absolute liver weights were observed in male and female rats exposed for 15 months to
doses of >3 mg/kg-day 1,2,3-trichloropropane. Mean relative liver weights were significantly
increased by 15 and 28% in male rats that received doses of 10 or 30 mg/kg-day, respectively,
when compared with controls (Table 4-13a). Mean relative liver weights in female rats that
received doses of 10 or 30 mg/kg-day were increased 12 and 40%, respectively (Table 4-13a).
Absolute liver weights were significantly increased by 10, 18, and 28% in male rats and 14, 16,
and 34% in female rats that received doses of 3, 10, and 30 mg/kg-day, respectively (Table 4-
13b).
24
-------
Table 4-13a. Relative liver weights (mg organ weight/g body weight) and
percent change in F344/N rats chronically exposed to 1,2,3-trichloropropane
by gavage at the 15-month interim evaluation
F344/N
Dose
(mg/kg-d)
0
3
10
30
n
10
10
10
8
Males
Mean ± SE
31.2 ±0.6
33.1 ±0.7
36.0±0.7b
39.8±0.9b
Percentage
change"
-
6%
15%
28%
n
10
10
8
8
Females
Mean ± SE
30.8 ±0.8
30.9 ±0.6
34.6±1.0b
43.2±0.7b
Percentage
change"
-
0%
12%
40%
"Percent change relative to control.
V<0.01.
Source: NTP(1993).
Table 4-13b. Absolute liver weights (g) and percent change in F344/N rats
chronically exposed to 1,2,3-trichloropropane by gavage at the 15-month
interim evaluation
F344/N
Dose
(mg/kg-d)
0
3
10
30
n
10
10
10
8
Males
Mean ± SE
14.27 ±0.37
15.63 ±0.37b
16.8±0.48C
18.23 ±0.52C
Percentage
change"
-
10%
18%
28%
n
10
10
8
8
Females
Mean ± SE
7.79 ± 0.13
8.87±0.31C
9.00±0.28C
10.40 ±0.37C
Percentage
change"
-
14%
16%
34%
"Percent change relative to control.
V<0.01.
°p < 0.05 by Williams' or Dunnett's test.
Source: NTP(1993).
Statistically significant increases (p < 0.05) in absolute right kidney weights were
observed in male rats exposed for 15 months to doses of >3 mg/kg-day 1,2,3-trichloropropane
and female rats exposed to doses of >10 mg/kg-day. Mean relative kidney weights in males
from these treatment groups were increased by 4, 10 and 29%, respectively (Table 4-14a). Mean
relative kidney weights of females in the 10 and 30 mg/kg-day treatment groups were increased
by 8 and 31% (Table 4-14a). Absolute kidney weights were significantly increased by 8, 12, and
30% in male rats that received doses of 3, 10, and 30 mg/kg-day, and significantly increased by
11 and 24% in female rats that received doses of 10 and 30 mg/kg-day (Table 4-14b).
25
-------
Table 4-14a. Relative right kidney weights (mg organ weight/g body weight)
and percent change in F344/N Rats chronically exposed to 1,2,3-trichloro-
propane by gavage at the 15-month interim evaluation
F344/N
Dose
(mg/kg-day)
0
3
10
30
n
10
10
10
8
Males
Mean ± SE
2.96 ± 0.04
3.09 ±0.09
3.25±0.05b
3.82±0.05b
Percentage
change"
-
4%
10%
29%
n
10
10
8
8
Females
Mean ± SE
3.08 ±0.07
2.93 ±0.07
3.34±0.06C
4.04±0.12b
Percentage
change"
-
-5%
8%
31%
"Percent change relative to control.
V<0.01.
°p < 0.05 by Williams' or Dunnett's test.
Source: NTP(1993).
Table 4-14b. Absolute right kidney weights (grams) and percent change in
F344/N Rats chronically exposed to 1,2,3-trichloropropane by gavage at the
15-month interim evaluation
F344/N
Dose
(mg/kg-day)
0
3
10
30
n
10
10
10
8
Males
Mean ± SE
1.35 ±0.03
1.46±0.04b
1.51±0.03C
1.75±0.05C
Percentage
change"
-
8%
12%
30%
n
10
10
8
8
Females
Mean ± SE
0.786 ±0.015
0.839 ±0.023
0.869 ±0.019b
0.971 ±0.034C
Percentage
change"
-
7%
11%
24%
"Percent change relative to control.
V < 0.05 by Williams' or Dunnett's test.
°p<0.01.
Source: NTP(1993).
The data for clinical chemistry parameters was sporadic, with ALT and 5'-nucleotidase
levels statistically (p < 0.05) significantly decreased 31 and 13%, respectively, in males that
received 30 mg/kg-day.
Treatment-related effects were detected among the hematological parameters in rats;
however, the effects were not considered to be biologically relevant. Rats that received 30
mg/kg-day displayed mean hematocrit values that were statistically (p < 0.05) significantly
decreased by 5 and 7% for males and females, respectively, when compared with controls. The
26
-------
mean hemoglobin concentration was decreased by 4% in male rats that received either 3 (p <
0.01) or 30 (p < 0.05) mg/kg-day. Both males and female rats in the high dose group had
statistically (p < 0.01) significantly elevated counts of leukocytes and segmented neutrophils, but
not in the 10 mg/kg-day group. NTP (1993) stated that the decreased hematocrit may have been
associated with depressed erythropoeisis or with blood loss from neoplasms in the forestomach
or oral mucosa, and that the increase in leukocytes was likely due to inflammation associated
with the chemical-induced neoplasms (NTP, 1993).
At the 2-year evaluation, hepatocellular necrosis was observed in 1/49 and 4/52 female
rats at 3 and 30 mg/kg-day, respectively. Hepatocellular necrosis was not observed in male rats.
In addition, a dose-dependent increase in bile duct hyperplasia was observed in male rats at 30
mg/kg-day. Nonneoplastic effects were also observed in the forestomach, kidney, and pancreas
of male and female rats at the 2-year evaluation. The incidence of basal cell and squamous
hyperplasia of the forestomach was increased at 3, 10, and 30 mg/kg-day in both males (28/50,
13/49, and 6/52) and females (5/50, 8/49, and 7/52) (NTP, 1993). A dose-dependent increase in
the incidence of renal tubule hyperplasia was observed in males (1/50, 21/49, and 29/52)
exposed to 3, 10, or 30 mg/kg-day and in females (2/47, 3/52, and 10/51) exposed to 3, 10, or 30
mg/kg-day (NTP, 1993). Increased severity of nephropathy was also observed in males at 10
and 30 mg/kg-day, with mean severity scores of 2.6 and 2.4, respectively, compared to the
control and low dose, with mean severity scores of 2.0. In addition, a dose-dependent increase in
acinar hyperplasia of the pancreas was observed at 3, 10, and 30 mg/kg-day in male (44/50,
46/49, and 48/52) and in female rats (14/49, 24/52, and 9/52) (NTP, 1993).
An increase in the incidence of forestomach tumors was observed in all rat treatment
groups (Table 4-15), regardless of sex. However, the incidences of forestomach neoplasms were
generally higher in males than in females at the same dose levels. All male treatment groups
also had increased incidence of pancreatic tumors (Table 4-15). Male and female rats that
received doses of >10 mg/kg-day 1,2,3-trichloropropane had an increase in the incidence of oral
cavity tumors (Table 4-15). In each male group that received doses of >10 mg/kg-day, an
increased incidence of renal tumors was observed. An increase was observed in females at both
10 and 30 mg/kg-day for the clitoral gland tumors and at the 10 and 30 mg/kg-day for mammary
gland tumors (Table 4-15). In the 30 mg/kg-day treatment group, an increased incidence of
Zymbal's gland tumors was observed in females and an increased incidence of preputial gland
tumors was observed in males at 30 mg/kg-day (Table 4-15). Forestomach tumors were
described in the NTP (1993) report as follows:
The masses were squamous cell papillomas or squamous cell carcinomas arising
from the stratified squamous cell epithelium of the forestomach. Multiple
squamous cell papillomas or carcinomas often occurred in the same rat, and in
27
-------
some rats, the neoplasms were so extensive that it was difficult to discern if they
represented a single neoplasm or the confluent growth of multiple neoplasms.
Table 4-15. Incidence of neoplasms in F344/N rats chronically exposed to
1,2,3-trichloropropane by gavage
Tissue site/tumor type
Tumor incidence"
Males (mg/kg-d)
0
3
10
30
Females (mg/kg-d)
0
3
10
30
Oral cavity
Papillomas or carcinomas
1/60
4/60
19/59b
43/60b
1/60
6/59
28/60b
37/60b
Forestomach
Papillomas or carcinomas
0/60
35/60b
46/59b
51/60b
0/60
17/59b
42/59b
27/60b
Pancreas (acinar)
Adenomas or adenocarcinomas
5/60
21/60b
37/59b
31/60b
0/60
0/59
2/60
0/60
Kidney (renal tubules)
Adenomas or adenocarcinomas
0/60
2/60
20/59b
26/60b
0/60
0/57
0/60
1/59
Preputial gland
Adenomas or carcinomas
5/59
6/57
9/59
17/58C
-
-
-
-
Clitoral gland
Adenomas or carcinomas
-
-
-
-
5/56
11/56
18/58b
17/59C
Mammary gland
Adenocarcinomas
-
-
-
-
1/60
6/59
12/60b
22/60b
Zymbal's gland
Carcinomas
0/60
0/60
0/59
3/60
0/60
1/59
0/60
4/60c
aValues are pooled results from the outcome of histopathologic examinations of animals at the interim and terminal
sacrifices.
bp < 0.001 by life table or logistic regression test.
°p < 0.05 by life table or logistic regression test.
Source: NTP(1993).
Forestomach tumors were accompanied by an increased incidence of focal hyperplasia of
the stratified squamous cell epithelium. The hyperplasia, squamous cell papilloma, and
squamous cell carcinoma of the forestomach were said to constitute a morphological continuum
and the squamous cell papillomas and carcinomas were noted to be similar to those of the oral
mucosa(NTP, 1993).
The NOAEL and LOAEL for relative liver weight change in male and female rats is 3
and 10 mg/kg-day, respectively, while the LOAEL for absolute liver weight change in male and
female rats was 3 mg/kg-day. The NOAEL and LOAEL for relative right kidney weight in male
and female rats were 3 and 10 mg/kg-day, respectively, while the LOAEL for absolute right
kidney weight in male rats was 3 mg/kg-day and the NOAEL and LOAEL in female rats were 3
28
-------
and 10 mg/kg-day, respectively. Tumors were evident in the oral cavity, forestomach, pancreas,
kidney, and Zymbal's gland of male and female rats, along with preputial gland tumors in males
and clitoral gland and mammary gland tumors in females.
NTP (1993) conducted a 2-year study of the toxicity and carcinogenicity of 1,2,3-
trichloropropane in B6C3F1 mice. The chemical was administered by corn oil gavage to 60
mice/sex/group, and the mice were approximately 6 weeks old when the study began. Mice were
treated with 0 (vehicle control), 6, 20, or 60 mg/kg-day, and after 15 months (65-67 weeks), 8-
10 mice per group were sacrificed to allow an interim evaluation of all toxicological parameters
and histopathology. Due to high mortality in the mice receiving 60 mg/kg-day, surviving mice
were evaluated at week 73 (females) and week 79 (males). Due to the early termination of this
treatment group, organ weights and hematology data were only obtained at the 15-month interim
sacrifices.
Clinical observations were made twice daily, while body weights were recorded weekly
for 13 weeks and then monthly (NTP, 1993). As mentioned above, up to 10 mice/group were
sacrificed at month 15. From this interim sacrifice, blood samples were obtained for hematology
and clinical chemistry analyses. Hematological parameters included hematocrit, hemoglobin,
and counts of erythrocytes, leukocytes, and differential leukocytes. Clinical chemistry
parameters included the serum levels of ALT, AST, creatine kinase, LDH, SDH, and 5'-
nucleotidase. Whether at the planned sacrifice or as each mouse died or became moribund, all
mice were subjected to a gross necropsy, and a full range of organs and tissues was processed for
histopathologic examination. Hematology, clinical chemistry, and organ weight data were
obtained only from mice that were sacrificed at the 15-month interim because the majority of
treated mice died prior to the end of the study.
Survival rates were statistically significantly reduced (p < 0.001) in mice that received
doses of >6 mg/kg-day (Table 4-16). An effect on survival was apparent in all dose groups at the
15-month interim evaluation. The mortality in mice was attributed to cancer associated with
chemical exposure (NTP, 1993).
29
-------
Table 4-16. Survival rates and percent probability of survival for B6C3Fi
mice exposed to 1,2,3-trichloropropane by gavage for 2 years
Dose
(mg/kg-d)
0
6
20
60
Males
42/52a
18/51
0/54
0/56
81b
36C
Oc
Oc
Females
41/503
13/50
0/50
0/55
82b
26C
Oc
Oc
aAnimals surviving to study termination and number of animals in the treatment group. Accidental deaths were
excluded and censored from survival analysis.
bKaplan-Meier determinations of percent probability of survival at end of study.
>< 0.001.
Source: NTP(1993).
In mice, final mean body weights were significantly decreased by 17 and 18% in males
and females, respectively, after a dose of 60 mg/kg-day, when compared to controls. Mean
relative liver weights were increased by 32% in males and 40% in females that received 60
mg/kg-day (Table 4-17a). Other significant changes in organ weights among mice that received
this dose included increased relative kidney weights in females (21%) (Table 4-18a), and
increased relative brain weights in males (20%) and females (25%). Absolute liver and right
kidney weight changes were sporadic, and no consistent pattern of treatment-related effects was
apparent (Tables 4-17b and 4-18b).
Table 4-17a. Relative liver weights (mg organ weight/g body weight) and
percent change in B6C3Fi mice chronically exposed to 1,2,3-trichloro-
propane by gavage
Dose
(mg/kg-d)
0
6
20
60
n
10
9
8
5
Males
Mean ± SE
38.9 ±1.9
36.2 ±1.5
44.6 ±6.2
51.2±4.8b
Percentage
change"
-
-7%
15%
32%
n
10
10
9
5
Females
Mean ± SE
34.4 ±0.8
34.7 ±1.1
35.7 ±0.6
48.3±2.8C
Percentage
change"
-
1%
4%
40%
aPercent change relative to control.
bp < 0.05 by Williams' or Dunnett's test.
°p<0.01.
Source: NTP(1993).
30
-------
Table 4-17b. Absolute liver weights (g) and percent change in B6C3Fi mice
chronically exposed to 1,2,3-trichloropropane by gavage
Dose (mg/kg-d)
0
6
20
60
n
10
9
8
5
Males
Mean ± SE
1.72 ±0.09
1.63 ±0.08
1.76 ±0.19
1.92 ±0.14
Percentage
change"
-
-5%
2%
12%
n
10
10
9
5
Females
Mean ± SE
1.49 ±0.03
1.33±0.03b
1.50 ±0.04
1.69 ±0.18
Percentage
change"
-
-11%
1%
13%
aPercent change relative to control.
bp < 0.05 by Williams' or Dunnett's test.
Source: NTP(1993).
Table 4-18a. Relative right kidney weights (mg organ weight/g body weight)
and percent change in B6C3Fi mice chronically exposed to 1,2,3-trichloro-
propane by gavage
Dose
(mg/kg-d)
0
6
20
60
n
10
9
8
5
Males
Mean ± SE
8.0 ±0.25
7.67 ±0.41
7.81 ±0.18
8.4 ±0.59
Percentage
change"
-
-4%
-2%
5%
n
10
10
9
5
Females
Mean ± SE
4.99 ±0.09
5.27 ±0.14
5.19±0.14
6.02±0.11b
Percentage
change"
-
6%
4%
21%
aPercent change relative to control.
V<0.01.
Source: NTP(1993).
Table 4-18b. Absolute right kidney weights (g) and percent change in
B6C3Fi mice chronically exposed to 1,2,3-trichloropropane by gavage
Dose (mg/kg-d)
0
6
20
60
n
10
9
8
5
Males
Mean ± SE
0.353 ±0.011
0.344 ±0.019
0.314 ±0.013
0.3 17 ±0.022
Percentage
change"
-
-3%
-11%
-10%
n
10
10
9
5
Females
Mean ± SE
0.217 ±0.006
0.203 ± 0.006
0.217 ±0.006
0.210 ±0.015
Percentage
change"
-
-6%
0
-3%
"Percent change relative to control.
Source: NTP(1993).
31
-------
In mice, creatine kinase was statistically (p < 0.05) significantly elevated 235% in males
that received 60 mg/kg-day, and SDH was statistically (p < 0.05) significantly elevated 72% in
females that received the same dose. However, clinical chemistry differences between dose
groups and control animals were not considered to be directly related to 1,2,3-trichloropropane
administration (NTP, 1993).
Treatment-related effects were detected among the hematological parameters, but the
effects were indirectly related 1,2,3-trichloropropane toxicity. Mean hematocrit values were
decreased by 5 and 4% in male and female mice, respectively, that received 20 mg/kg-day.
Mean hematocrit values were statistically (p<0.01) decreased by 10 and 11% in males and
females, respectively, that received 60 mg/kg-day. Similar statistically (p < 0.01) significant
dose dependent changes in hemoglobin concentration and the number of erythrocytes were
observed in female mice that received doses of 20 or 60 mg/kg-day. Female mice in the high-
dose group also had statistically (p < 0.01) significantly elevated numbers of leukocytes,
segmented neutrophils, and lymphocytes. NTP stated that the decreased hematocrit may be
associated with depressed hematopoeisis or to blood loss from neoplasms in the forestomach,
and the increased number of leukocytes was likely due to inflammation associated with the
chemically-induced neoplasms (NTP, 1993).
At the 2-year evaluation, an increase in hepatocellular necrosis was observed in male
mice (1/52, 2/51, 11/54, and 8/56) and female mice (1/50, 6/50, 5/51, and 10/55) in the vehicle
control, 6, 20, and 60 mg/kg-day, respectively (NTP, 1993). In addition, a dose-dependent
increase in eosinophilic foci was observed in the livers of both males and females.
Nonneoplastic effects were also observed in the forestomach of male and female mice at the 15-
month and 2-year evaluation. The incidence of squamous hyperplasia in the forestomach
increased at 3, 10, and 30 mg/kg-day in male (29/51, 27/54, and 34/56) and female mice (15/49,
14/51, and 31/55), respectively (NTP, 1993).
In mice, the sites of statistically (p < 0.001) significant neoplasm formation for both
sexes were the forestomach and liver (Table 4-19). Incidences of Harderian gland tumors were
increased in males at 20 and 60 mg/kg-day, and the increase in incidence of oral cavity tumors
was statistically significant in females at the highest dose. The incidence of uterine/cervical
tumors in female mice was increased at 6, 20, and 60 mg/kg-day. The highest incidence of
neoplasms and most marked dose-response effect for both species was in the forestomach. A
97% incidence of tumors of the forestomach was evident in male mice at the lowest dose tested
(90% in females). These data suggest that an elevated incidence of tumors in the forestomach
might occur at doses lower than those employed in this study. NTP (1993) noted that:
In contrast to dosed rats, there were few neoplasms of the oral mucosa in dosed mice.
Nevertheless, squamous cell carcinomas arising from the pharyngeal or lingual mucosa
32
-------
were observed in one 20 mg/kg and five 60 mg/kg females, and none were seen in
controls.
Table 4-19. Incidence of neoplasms in B6C3Fi mice chronically exposed to
1,2,3-trichloropropane by gavage
Tissue site/tumor type
Tumor incidence"
Males (mg/kg-d)
0
6
20
60
Females (mg/kg-d)
0
6
20
60
Oral cavity
Papillomas or carcinomas
0/60
0/59
0/60
2/60
1/60
0/60
2/60
5/60b
Forestomach
Papillomas or carcinomas
3/60
57/59c
57/60c
59/60c
0/60
54/60c
59/60c
59/60c
Liver
Adenomas or carcinomas
14/60
24/59b
25/60b
33/60c
8/60
11/60
9/60
36/60c
Harderian gland
Adenomas
1/60
2/59
10/60b
ll/60b
3/60
6/60
7/60
10/60
Uterine/cervical
Adenomas or adenocarcinomas
-
-
-
-
0/50
5/50b
3/5 lb
9/54b
aValues are pooled results from the outcome of histopathologic examinations of animals at the interim and terminal
sacrifices.
bp < 0.05 by life table or logistic regression test.
°p < 0.001 by life table or logistic regression test.
Source: NTP(1993).
The exophytic, or outward growing, papillary or nodular masses in the forestomach of
mice were similar to those observed in rats. Moreover, the extensive neoplastic growth observed
in rats was also noted in mice. The study authors suggested that the hyperplasia, squamous cell
papilloma, and squamous cell carcinoma of the forestomach observed in the B6C3F1 mice were
on a morphological continuum as in the F344/N rats (NTP, 1993).
The NOAEL and LOAEL for relative liver weight change in male and female mice were
20 and 60 mg/kg-day, respectively; however, the NOAEL for absolute liver weight change was
60 mg/kg-day in male and female mice. The NOAEL and LOAEL for relative right kidney
weight change in female mice were 20 and 60 mg/kg-day, respectively; while the NOAEL in
male mice for relative right kidney weight change was 60 mg/kg-day. The NOAEL for absolute
right kidney weight was 60 mg/kg-day for both sexes. It should be noted that the high mortality
associated with chemical exposure led to the early termination of the 20 and 60 mg/kg-day dose
groups. Tumors were evident in the oral cavity, forestomach, liver, and Harderian gland of both
male and female mice, and in the uterine/cervical tissue in females. The critical effect for
noncancer data was weight change in the liver and right kidney, while the critical effect for the
cancer data was tumor development in the aforementioned organs of mice.
33
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4.2.2. Inhalation Exposure
4.2.2.1. Subchronic Studies
Johannsen et al. (1988) conducted a series of prechronic and subchronic inhalation
studies in 7-week-old CD rats. The initial subchronic study was followed by a second
subchronic study that used lower doses because lesions were observed in all three exposure
groups in the initial bioassay.
In a range-finding study, five CD rats/sex/group were exposed in 1 m3 stainless steel and
glass chambers to nominal concentrations of 0, 100, 300, 600, or 900 ppm 1,2,3-trichloropropane
vapor (0, 600, 1,800, 3,600, and 5,400 mg/m3) 6 hours/day, 5 days/week, for up to 4 weeks. At
the highest concentration, all but one of the rats died after a single exposure. Three animals
exposed to 600 ppm and one exposed to 300 ppm died prior to study termination. Surviving rats
exposed to 600 ppm trichloropropane became prostrated during exposure periods. Males that
were exposed to 600 ppm initially lost weight, but returned to their pre-exposure weights by the
end of the experiment. Females exposed to this concentration showed a similar pattern but did
not regain the initial weights. Weight gain was statistically significantly reduced (p < 0.05) in
rats exposed to 300 ppm and appeared depressed, but was not significantly different from
controls for animals exposed to 100 ppm. Relative and absolute liver weights were statistically
significantly elevated (p < 0.05) in males for all treatment groups and for females in the 300 and
600 ppm groups (p < 0.05) for relative liver weight and in the 300 ppm group for absolute liver
weight. Absolute brain and kidney weights and organ/body ratios were increased in the 300 and
600 ppm treatment groups. Absolute ovary weights and organ/weight ratios were decreased in
the 300 and 600 ppm groups, and absolute spleen weights and organ/weight ratios and absolute
testis weights were decreased in the 600 ppm treatment group. The magnitude of change in body
and tissue weights was not reported.
The results of the 4-week range finding study were used to establish target concentrations
0, 5, 15, or 50 ppm (0, 30, 90, or 300 mg/m3) as the exposure concentrations for a 13-week
study, with analytical concentrations of 4.5 ± 0.2, 15 ± 0.3, and 49 ± 1.0 ppm. Each exposure
group contained 15 CD rats/sex. Blood samples were taken for clinical chemistry and
hematological parameters at week 7 from controls and the animals that were exposed to 50 ppm,
and at termination from all surviving animals. A gross pathological examination was conducted
on all animals and the weights of all major organs were recorded. Portions of the major organs
and tissues were processed for histopathologic examination. The results of these examinations
are described in the following paragraphs.
There were no treatment-related deaths in the 13-week study. Daily observation of
treated animals revealed a general, dose-dependent pattern of respiratory tract and conjunctival
irritation, including red nasal discharge and excessive lacrimation. An increased incidence of
yellow staining of the anogenital fur was also observed.
34
-------
A number of statistically significant changes were reported for whole body and organ
weights; however, the magnitudes of change in the body and tissue weights was not reported by
Johannsen et al. (1988) but were provided by the initial investigating group, Biodynamics, Inc.
(1979). Statistically significant reductions in terminal body weight were observed in females
exposed to 15 (7%) and 50 (9%) ppm. No effect on body weight was observed in males. Mean
absolute and relative liver weights (Table 4-20) were statistically significantly elevated 13-21%
in the male rat exposure groups. Mean absolute liver weights were statistically significantly
elevated 10% in females exposed to 50 ppm (p< 0.01), and relative liver weights were
statistically significantly (p < 0.01) increased 8 and 20% in females at 15 and 50 ppm,
respectively. Relative lung weights (Table 4-21) were also statistically significantly (p < 0.01)
increased 14 and 13%, respectively, in female rats at doses of 15 and 50 ppm, although no effect
was evident in male rats. The mean relative kidney weight of males exposed to 50 ppm was
significantly increased approximately 10%.
Table 4-20. Absolute and relative liver weights and percent change in CD
rats exposed to 1,2,3-trichloropropane by inhalation, 6 hours/day,
5 days/week, for 13 weeks
Dose (ppm)
n
Absolute3
Relative13
Male
0
5
15
50
15
15
15
14
13.8 ± 1.06
16.7±1.58C
16.3±1.48C
16.4 ±1. 51 c
—
21%
18%
19%
3.14±0.128
3. 56 ± 0.258 c
3. 57 ± 0.207 c
3.79 ± 0.260 c
—
13%
14%
21%
Female
0
5
15
50
15
15
15
15
10.6 ±0.81
10.9 ±0.76
10.7 ±1.05
11.7 ±1.06°
—
3%
1%
10%
3.4 ±0.126
3.6 ±0.213
3.7±0.216C
4.1±0.266C
—
6%
8%
20%
aMean±SD.
bPercent increase relative to control.
Source: Biodynamics, Inc. (1979).
35
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Table 4-21. Absolute and relative lung weights and percent change in CD
rats exposed to 1,2,3-trichloropropane by inhalation, 6 hours/day,
5 days/week, for 13 weeks
Dose (ppm)
n
Absolute
Relative
Male
0
5
15
50
14
15
15
14
1.49±0.162a
1.62 ±0.192
1.58 ±0.100
1.51 ±0.102
—
9%b
6%
1%
0.340 ±0.029a
0.345 ±0.036
0.347 ±0.030
0.351 ±0.028
—
l%b
2%
3%
Female
0
5
15
50
15
15
15
15
1.27±0.126a
1.31 ±0.124
1.34 ±0.107
1.31 ±0.129
—
4%b
6%
3%
0.406 ± 0.03 la
0.430 ±0.040
0.461 ± 0.033 c
0.460 ± 0.051 c
—
6%b
14%
13%
aMean±SD.
bPercent increase relative to control.
Source: Biodynamics, Inc. (1979).
A number of histopathologic lesions were observed (Table 4-22), including an increased
incidence of mild to marked peribronchial lymphoid hyperplasia at 5, 15, and 50 ppm. The
peribronchial lymphoid hyperplasia in the 15-ppm male rats was of equal severity to the 50-ppm
group, but the hyperplasia in the 15-ppm female rats and that evident in the 5-ppm males and
females were less severe. Hepatocellular hypertrophy in males at 5, 15, and 50 ppm appeared to
be at mild centrilobular to midzonal levels, but was not evident in the highest dose group
females. Treated females appeared to show a dose-dependent increase in extramedullary
hematopoiesis of the spleen. Statistical analysis was not conducted on these results.
36
-------
Table 4-22. Incidence of histopathologic lesions in CD rats exposed via
inhalation to 1,2,3-trichloropropane, 6 hours/day, 5 days/week for 13 weeks
Response
0
0.5
1.5
5
15
50
Male rats (ppm via inhalation)
Peribronchial lymphoid hyperplasia
Hepatocellular hypertrophy
Hematopoiesis of the spleen
0/15
0/15
0/15
0/15
0/15
0/15
0/15
0/15
0/15
6/15a
13/15
No data
11/153
15/15
No data
10/153
15/15
5/15
Female rats (ppm via inhalation)
Peribronchial lymphoid hyperplasia
Hepatocellular hypertrophy
Hematopoiesis of the spleen
1/15
0/15
5/15
0/15
0/15
0/15
0/15
0/15
0/15
5/15
No data
7/15
4/15
No data
9/15
6/1 5b
0/15
13/15
"p < 0.0001, trend test conducted by EPA.
bp < 0.001, trend test comducted by EPA.
Sources: Biodynamics, Inc. (1979); Johannsen et al. (1988).
There were no significant dose-related changes in any of the hematological or clinical
chemistry parameters evaluated (Johannsen et al., 1988).
The NOAEL and LOAEL for decreased terminal body weight in female rats were 5 and
15 ppm, respectively, while the NOAEL for decreased terminal body weight in male rats was 50
ppm. The LOAEL for increased absolute and relative liver weight in male rats was 5 ppm, while
the NOAEL and LOAEL for increased absolute liver weight in female rats were 15 and 50 ppm,
respectively, and the NOAEL and LOAEL for increased relative liver weight in females were 5
and 15 ppm, respectively. The NOAEL and LOAEL for increased relative lung weights in
female rats is 5 and 15 ppm, respectively, and the NOAEL and LOAEL for increased relative
kidney weights in males is 15 and 50 ppm, respectively. A LOAEL of 5 ppm was designated for
peribronchial lymphoid hyperplasia in male CD rats, as well as for hepatocellular hypertrophy in
male rats and hematopoiesis of the spleen in female rats.
The presence of lesions in animals from all exposure groups of the 13-week study
prompted the initiation of a follow-up study using lower exposure concentrations (Johannsen et
al., 1988). In the second 13-week study, the investigators employed a very similar experimental
protocol with exposure concentrations of 0, 0.5, or 1.5 ppm (0, 3, or 9 mg/m3). The protocol for
the second study did not include urinalysis and the histopathological evaluation was limited to
bone, brain, gonads, kidneys, liver, lungs, lymph nodes, nasal turbinates, and spleen in control
and high-dose (1.5 ppm) rats. It also included two additional hematological and a few clinical
chemistry parameters.
Small increases in mean absolute and relative ovarian weights were observed in females
in the 1.5 ppm dose group, but microscopic results to support this as a treatment-related effect
were not found and this effect was not observed in the previous 13-week study with doses up to
37
-------
50 ppm. Treatment-related histopathological findings at 0.5 or 1.5 ppm were not observed in
any tissue examined (Table 4-22).
In the follow-up study sporadic changes were observed in some hematological and
clinical chemistry parameters, including apparently increased platelets in females exposed to 1.5
ppm for 7 weeks and increased fasting glucose levels in females exposed to 1.5 ppm for 13
weeks. In the absence of an apparent dose-response pattern these changes were considered by
the investigators to be unrelated to the 1,2,3-trichloropropane exposures. All other hematology
and clinical chemistry parameters measured were unremarkable and displayed no apparent effect
from 1,2,3-trichloropropane exposure.
This investigation by Johannsen et al. (1988) identified a NOAEL of 1.5 ppm, with
regards to body or organ weight changes and histopathological effects, such as those evident in
the first study by Johannsen et al.
4.2.2.2. Chronic Studies
No studies were identified that examined the chronic toxicity of 1,2,3-trichloropropane
via inhalation.
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION
4.3.1. Oral Studies
NTP (1990) conducted a reproduction and fertility assessment of 1,2,3-trichloropropane
in CD-I mice using the Reproductive Assessment by Continuous Breeding (RACE) protocol.
This assessment consisted of four tasks/studies: (1) a range-finding study, (2) a continuous
breeding study, (3) a determination of the affected sex, and (4) an offspring assessment. All
treatments were administered by corn oil gavage.
In Task 1, mice (eight/sex/group) received 0, 12.5, 25, 50, 100, and 200 mg/kg-day for 14
days. No effect on weight gain or clinical signs of toxicity was observed. One male in the high
dose group died. The results of this study were used to select the doses for Task 2.
Task 2 was a continuous breeding study in which 20 breeding pairs received 0, 30, 60, or
120 mg/kg-day for 126 days. Endpoints monitored for this task included clinical signs of
toxicity, parental body weight, water consumption, fertility, litters/pair, live pups/litter,
proportion of pups born alive, sex of live pups, and pup weights at birth. Pups were not
monitored for physical abnormalities. The last litter (Fi) born during the holding period
following the continuous breeding phase was reared by each dam until weaning, and was then
used in the assessment of second generation fertility in Task 4.
The parental body weights, both male and female, were within 10% of the corresponding
control values, except for the 120 mg/kg-day females, which exhibited an increase in body
weight greater than 10%. Water consumption was significantly increased in weeks 6, 10, and
38
-------
14; however, consumption was calculated per cage, and, up until week 14, male and female mice
were housed in the same cage. At terminal necropsy, absolute and relative liver weights were
statistically significantly increased in the 120 mg/kg-day male and female mice, but data analysis
for the intermediate dose groups is unavailable.
Statistically significant reduction in fertility was evident at the 4th and 5th breedings
(Table 4-23). A statistically significant (p < 0.05) reduction in fertility was evident from the
decrease in the number of pregnancies per fertile mouse pair at the fourth breeding (89%), but
not the fifth breeding (68%), at 60 mg/kg-day group, and the third, fourth, and fifth breedings
(89, 68, and 42%, respectively) at 120 mg/kg-day. A dose-related decrease in fertility from the
fourth to fifth breeding at 60 mg/kg-day was observed, but this decrease did not reach statistical
significance. A statistically significant (p < 0.05) reduction in the number of live mouse
pups/litter was observed, when compared with controls, in the second through the fifth breedings
at the highest dose (120 mg/kg-day) and at the fifth breeding at 60 mg/kg-day (Table 4-23). The
120 mg/kg-day group displays a dose- and time-related decrease in the number of live
pups/litter. However, the decrease in the number of pups/litter in the fifth breeding at 60 mg/kg-
day is statistically significant due to an increase in the number of pups/litter in the controls
during the fifth breeding. The number of live pups/litter increases from the fourth to the fifth
breeding at 60 mg/kg-day and does not follow a dose- or time-related response.
Table 4-23. Fertility indices and number of live pups/litter in breeding pairs
of CD-I mice exposed to 1,2,3-trichloropropane by gavage
Litter
1st
2nd
3rd
4th
5th
Dose group (mg/kg-d)
0
Fertility"
38/38 (100)
38/38 (100)
38/38 (100)
38/38 (100)
33/38 (87)
Live
pups/litter
11.1±0.6
12.6 ±0.4
12.4 ±0.5
11.8±0.6
12.8 ±0.4
30
Fertility"
18/18 (100)
18/18 (100)
18/18 (100)
17/18 (94)
14/18 (78)
Live
pups/litter
10.3 ±1.0
10.8 ±1.2
11.3 ±1.0
11.2 ±0.7
12.1 ±0.7
60
Fertility"
19/19 (100)
19/19 (100)
19/19 (100)
17/19b (89)
13/19 (68)
Live
pups/litter
10.5 ±0.9
10.7 ±1.1
11.0 ±1.2
9.9 ± 1.0
11.3±0.8b
120
Fertility"
18/19 (95)
18/19 (95)
17/19b (89)
13/19b (68)
8/1 9b (42)
Live
pups/litter
11. 5 ±0.8
5.2 ± 0.6b
6.7±1.0b
2.9±0.6b
2.5±0.6b
""Fertility data are the number of fertile pairs/number of cohabiting pairs (percent fertile).
bSignificantly different (p < 0.05) from the control group by Cochran-Armitage trend test.
Source: NTP(1990).
The cumulative days to litter were statistically significantly longer than control values for
the third breeding (12%) at 60 mg/kg-day and the fourth (6.5%) and fifth (3.3%) breedings at
120 mg/kg-day. The proportion of male pups born alive in the fifth breedings appeared to
decrease in a dose-dependent manner. The proportion of males in the fifth breeding of the 120
39
-------
mg/kg-day treatment group was 0.27 versus 0.53 for the controls, with proportions in the fifth
breeding at 30 and 60 mg/kg of 0.43 and 0.42, respectively. Live pup weights were slightly,
statistically significantly increased at the highest dose, 120 mg/kg-day. However, when adjusted
for average litter size ± standard error, the increase in live pup weights was eliminated in male
pups at 120 mg/kg-day, and a decrease, although not statistically significantly, in female pups
and combined pups was visible.
Task 3, a 1-week crossover mating trial, was conducted with the same adult mice from
the control and 120 mg/kg-day treatment groups from Task 2. Three groups of 20 breeding pairs
(control males x control females, control males x high-dose females, and control females x high-
dose males) were evaluated for fertility and the presence of morphological and histopathologic
changes to the reproductive organs. At termination, F0 mice were necropsied and major organs
were excised and weighed. Treated FO mice of both sexes displayed statistically significantly (p
< 0.05) increased absolute and relative liver weights, 19 and 20% in males and 25 and 22% in
females, respectively, compared with controls. The weights of the right epididymis and cauda
epididymis in F0 males were statistically significantly (p < 0.05) lower, 5 and 8%, respectively,
than those of controls. The absolute kidney weights of treated F0 females were statistically
significantly (p < 0.05) reduced (5%) compared with controls. All F0 males were evaluated for
epididymal sperm parameters, and no differences in motility, count, or abnormal sperm numbers
were detected. The 120 mg/kg-day treated females delivered fewer live pups (-50%) than
untreated females, with decreased body weight (9%) in male offspring and fewer live male pups
per litter than controls.
In Task 4, members of the last set of litters (Fi) to be born in Task 2 were reared, weaned,
and allowed to reach sexual maturity before being paired individually with a member of the
opposite sex from a separate litter but within the same treatment group. Breeding pairs were
assessed for the same mating endpoints as in Task 2 and the same terminal endpoints as in Task
3. There were statistically significant (p<0.05) decreases, 78 and 43% of controls, in the indices
for mating (number of females with plug/number of cohabiting pairs) and fertility (number of
fertile pairs/number of females with plug), respectively, for the 120 mg/kg-day group. The
estrous cycles for FI females of all treatment groups were statistically significantly longer than in
controls (p < 0.05), and may be associated with an increase in the infertile period of metestrus.
At necropsy, FI male and female terminal body weights were statistically significantly (p
< 0.05) increased, 5-11%, in the 60 and 120 mg/kg-day groups. There was a statistically
significant (p < 0.05) increase, 17-50%, in absolute and relative liver weights in males and
females at 60 and 120 mg/kg-day, and a statistically significant (p < 0.05) increase, 6-27%, in
absolute kidney weights in male and female mice at 60 and 120 mg/kg-day. A statistically
significant (p < 0.05) 34% decrease in absolute right ovary weight was evident at the highest
dose level, with a statistically significant (p < 0.05) decrease in relative ovary weight at 60 and
40
-------
120 mg/kg-day of 15 and 39%, respectively. Histopathological examination of tissues from 10
females from each group revealed no difference between the groups in the incidence and severity
of lesions.
Based on the decreased number of fertile pairs and live pups/litter among the cohabiting
pairs in the 120 mg/kg-day treatment group, the investigators concluded that 1,2,3-
trichloropropane treatment could impair fertility and reproduction (NTP, 1990). In Task 2, a
NOAEL and LOAEL of 30 and 60 mg/kg-day, respectively, were identified for a decrease in the
number of pregnancies per fertile mouse pair at the fourth and fifth breeding. A reduction in the
number of live mouse pups/litter was observed across doses in the second through the fifth
breedings from breeding pairs at the highest dose (120 mg/kg-day) and at the fifth breeding at 60
mg/kg-day; which provided a NOAEL of 30 mg/kg-day and a LOAEL of 60 mg/kg-day at the
fifth breeding and a NOAEL of 60 mg/kg-day and LOAEL of 120 mg/kg-day for the first
through the fourth breedings. The LOAEL for the decreased proportion of males in the fifth
breeding was 30 mg/kg-day. Task 3, a cross-over mating trial, identified a LOAEL of 120
mg/kg-day in treated females for decreased pups/litter, decreased male pup weight, and
decreased proportion of males/litter. A NOAEL and LOAEL of 60 and 120 mg/kg-day,
respectively, for decreased fertility and mating indices were identified from Task 4. A LOAEL
of 30 mg/kg-day for lengthened estrous cycle was also apparent.
4.3.2. Inhalation Studies
Johannsen et al. (1988) reported the results of a single-generation reproductive study
using 10 male and 20 female CD rats/group conducted in two dosing studies. In the first study,
animals in 1 m3 stainless steel and glass chambers were exposed to target vapor concentrations
of 0, 5, or 15 ppm (0, 30, or 90 mg/m3), with measured concentrations of 4.6 ± 0.2 and 15 ± 0.2,
1,2,3-trichloropropane 6 hours/day, 5 days/week, for a 10-week pre-mating period, a mating
period (not to exceed 40 days), and for gestation days 0-14 for females. Male and female rats
were housed in a ratio of 1:2, respectively, nightly during the mating period. Females that were
not impregnated after the 10 days were paired with a different male for 10 days until pregnant.
In the second study, the same numbers of rats were exposed to target concentrations of 0, 0.5, or
1.5 ppm (0, 3, or 9 mg/m3) using a similar protocol (mating period not to exceed 30 days).
Females delivered and all litters were weaned on postnatal day 21. Animals were examined
daily for clinical signs and received a weekly physical exam when body weights were recorded,
with mated females weighed through gestation and lactation. Pups were weighed at birth, on
postnatal days 4 and 14, and when they were sacrificed on postnatal day 21. At termination, all
F0 parents were necropsied, and sections of their reproductive organs were processed for
histopathologic examination.
41
-------
In the first study, females exposed to 15 ppm had lower body weights during gestation
and lactation, although weight gains were consistent with the controls. Both sexes exposed to 15
ppm exhibited decreases in weight and weight gain during the premating period of exposure. All
groups of female rats exhibited low mating performance, 16 females out of 20 mated at 5 ppm
and 10 females out of 20 mated at 15 ppm, compared with 37 females out of 40 mated in the
control group (Table 4-24); although fewer females in the high concentration group mated,
statistical significance was not demonstrated by Johannsen et al. (1988). The decrease in the
number of females that mated was statistically significant (p < 0.02) at 15 ppm compared to the
control group (Fisher Exact test conducted by EPA). The decrease in mating performance was
also statistically significant (p = 0.01) for linear trend (%2 test conducted by EPA). Male rats in
both treated and control groups displayed apparently lower mating performance, 4/10, 6/10 and
3/10 for control, 5 ppm and 15 ppm groups, respectively, but not statistically significant mating
indices. Fertility indices were unaffected by trichloropropane exposure. There was no
treatment-related effect on litter or pup data. Histopathological evaluation of the testes,
epididymis, and ovaries did not identify any treatment-related changes.
Table 4-24. Decreased mating performance in female CD rats following
inhalation of 1,2,3-trichloropropane for 6 hours/day, 5 days/week, for a
10-week pre-mating period, a mating period (not to exceed 40 days), and
gestation days 0-14
Mated
Failed to mate
n
Dose (ppm)
0
37
3
40
0.5
19
1
20
1.5
20
0
20
5
16
4
20
15
10a
10a
20
"Statistically significant (p < 0.02) compared to control group in the Fisher Exact test conducted by EPA.
In the second study, adverse effects on mating performance and fertility indices due to
1,2,3-trichloropropane were not observed (Table 4-24). Lesions of the testes, epididymides, and
ovaries were not evident. Consistent or obviously treatment-related reproductive effects were
not observed in any of the experimental groups in either generation.
This study identified a NOAEL of 15 ppm for low mating performance and fertility
indices. For decreased body weight in females during gestation and lactation and for decreased
body weights and weight gain in both sexes during the premating period, a NOAEL of 5 and
LOAEL of 15 were identified.
42
-------
4.4. OTHER STUDIES
4.4.1. Acute Toxicity Data
In the rat, oral LD50 values ranging from 150 to 500 mg/kg 1,2,3-trichloropropane have
been reported (Greim, 1993). A 4-hour LCso of approximately 500 ppm (3,000 mg/m3) has been
determined for rats and mice (Greim, 1993). McOmie and Barnes (1949) identified an LCso of
approximately 30 ppm in mice exposed to vapor for 20 minutes, while Reyna (1987) could not
determine an LCso in Sprague-Dawley rats, but suggested that the LCso was greater than 4.8
mg/L air.
Lag et al. (1991) conducted an acute study in rats that investigated the nephrotoxicity of
short-chain halogenated alkanes. 1,2,3-Trichloropropane was administered via a single, i.p.
injection to five male MOL:WIST rats per dose group at doses of 147, 294, and 441 mg/kg.
After 48 hours, the rats were weighed and euthanized, and their kidneys were removed, weighed,
and preserved. Dose-dependent increases in mortality, kidney/body weight ratio, and urea
excretion were evident. Histopathological examination detected moderate kidney necrosis in one
of the two surviving rats at the highest dose level tested.
4.4.2. Short-term Toxicity Data
Miller et al. (1987a, b) conducted two inhalation studies of male and female F344/N rats
and B6C3F1 mice. Following the results of the first investigation, the exposures were decreased
in the second bioassay. These unpublished studies were submitted to the EPA under the Toxic
Substances Control Act (TSCA). In the first rat and mouse study (Miller et al., 1987a),
5/sex/group were exposed to target concentrations of 0, 10, 30, and 100 ppm 6 hours/day, 5
days/week, for 9 days, with measured concentration of 0, 13 ± 0.5, 40 ± 0.4, or 132 ± 0.6 ppm (0,
78, 241, and 796 mg/m3). Endpoints evaluated included body weight, urinalysis, clinical
chemistry, hematology, and gross pathology and histopathology.
Rats in the high-exposure group were less active than controls and did not eat or drink
normally after treatment. An exposure and time-dependent reduction in weight gain was
observed in treated rats. Terminal body weights in rats were statistically significantly (p < 0.05)
decreased 14 and 10% in males and females, respectively, in the high-exposure group when
compared with controls. In male and female rats exposed to 40 ppm, relative liver weights were
statistically significantly (p < 0.05) increased 7 and 9%, respectively. At 132 ppm, absolute and
relative liver weights were statistically significantly (p < 0.05) increased 10 and 21%,
respectively, in males and 27 and 42%, respectively, in females.
The concentrations of serum albumin and total protein in male rats and serum albumin in
female rats were statistically significantly (p<0.05) increased in the high-exposure group, but
were not considered by the investigators to be lexicologically significant. No exposure-related
changes were observed among any of the hematology parameters, although a statistically
43
-------
significant (p < 0.05) increase (6%) in packed cell volume (hematocrit) and hemoglobin (5%),
were noted in female rats that were exposed to 40 ppm.
Several pathological changes in rats were associated with 1,2,3-trichloropropane
exposure. Gross observation suggested a decrease in thymus size among rats, but the study
authors considered this observation to be "secondary to stress." Very slight hepatocellular
necrosis and very slight depletion of lymphoid elements in the spleen were observed in all male
rats exposed to 132 ppm.
Miller et al. (1987a) also noted a dose-dependent increase in incidence and severity of
degeneration and decreased thickness of the olfactory epithelium in the nasal turbinates of rats
exposed to 13, 40, or 132 ppm 1,2,3-trichloropropane (Table 4-25). Inflammation in the
olfactory epithelium was also evident in rats exposed to 13, 40, or 132 ppm 1,2,3-
trichloropropane, and was accompanied by the exudation of inflammatory cells into the nasal
cavity lumen (Table 4-26).
Table 4-25. Incidence and severity of decreased thickness and degeneration
of the olfactory epithelium in the nasal turbinates of F344/N rats exposed via
inhalation to 1,2,3-trichloropropane
Severity3
Very slight
Slight
Moderate
Severe
Combined incidence
n
Males (ppm)
0
0
0
0
0
0
5
1
0
0
0
0
0
5
3
5
0
0
0
5
5
10
5
0
0
0
5
5
13
0
5
0
0
5
5
40
0
0
5
0
5
5
132
0
0
0
5
5
5
Females (ppm)
0
0
0
0
0
0
5
1
0
0
0
0
0
5
3
5
0
0
0
5
5
10
5
0
0
0
5
5
13
1
4
0
0
5
5
40
1
1
2
1
5
5
132
0
0
2
3
5
5
"Decreased thickness, bilateral, and multifocal, or degeneration, bilateral, and multifocal.
Sources: Miller et al. (1987a,b).
44
-------
Table 4-26. Incidence and severity of inflammation of the olfactory
epithelium in the nasal turbinates of F344/N rats exposed via inhalation to
1,2,3-trichloropropane
Severity3
Very slight
Slight
Moderate
Combined incidence
Exudate into lumen
n
Males (ppm)
0
3
0
0
3
0
5
1
1
0
0
1
0
5
3
0
0
0
0
0
5
10
5
0
0
5
2
5
13
2
3
0
5
2
5
40
0
4
1
5
o
J
5
132
0
1
4
5
1
5
Females (ppm)
0
3
0
0
3
0
5
1
3
0
0
3
0
5
3
2
0
0
2
0
5
10
5
0
0
5
2
5
13
4
1
0
5
4
5
40
1
4
0
5
4
5
132
0
1
4
5
5
5
Inflammation, bilateral, and multifocal.
Sources: Miller et al. (1987a,b).
Mice in the high-exposure group were less active than controls and did not eat or drink
normally after treatment; however, no effect on weight gain was observed in mice. Absolute
liver weights were statistically significantly (p < 0.05) increased 67 and 73% in male and female
mice, respectively, exposed to 132 ppm. Relative liver weights in the high-exposure group were
statistically significantly (p < 0.05) increased by 55 and 60% in males and females, respectively,
compared with controls. Male mice also displayed statistically significantly decreased absolute
and relative testes weights, 9 and 16%, respectively, in the highest exposure group, but
histopathological changes were not observed.
The concentrations of serum albumin and total protein were statistically significantly (p <
0.05) increased in both sexes of mice, but were not considered by the investigators to be
lexicologically significant. There were no dose-related changes among any of the hematological
parameters, but the number of platelets was statistically significantly (p < 0.05) increased 25 and
42% in male and female mice at 132 ppm.
Several pathological changes in mice were associated with 1,2,3-trichloropropane
exposure. A moderate increase in hepatocyte size was noted in all male and female mice
exposed to 132 ppm, and a slight or very slight depletion of lymphoid elements in the spleen was
also reported in all mice at this dose. A dose-dependent increase in the incidence and severity of
decreased thickness and degeneration of the olfactory epithelium in the nasal turbinates of mice
was also observed (Table 4-27). There was a dose-related increase in inflammation in the
olfactory epithelium of the nasal turbinates (Table 4-28) accompanied by the exudation of
inflammatory cells into the nasal cavity lumen.
45
-------
Table 4-27. Incidence and severity of decreased thickness and degeneration
of the olfactory epithelium in the nasal turbinates in B6C3Fi mice exposed
via inhalation to 1,2,3-trichloropropane
Severity3
Very slight
Slight
Moderate
Combined incidence
n
Males (ppm)
0
0
0
0
0
5
1
0
0
0
0
5
3
0
0
0
0
5
10
5
0
0
5
5
13
5
0
0
5
5
40
4
1
0
5
5
132
0
2
o
J
5
5
Females (ppm)
0
0
0
0
0
5
1
0
0
0
0
5
3
0
0
0
0
5
10
5
0
0
5
5
13
5
0
0
5
5
40
2
3
0
5
5
132
0
0
5
5
5
"Decreased thickness, bilateral, and multifocal, or degeneration, bilateral, and multifocal.
Sources: Miller et al. (1987a,b).
Table 4-28. Incidence and severity of inflammation of the olfactory
epithelium in the nasal turbinates of B6C3Fi rats exposed via inhalation to
1,2,3-trichloropropane
Severity3
Very slight
Slight
Moderate
Combined incidence
Exudate into lumen
n
Males (ppm)
0
0
0
0
0
0
5
1
0
0
0
0
0
5
3
0
0
0
0
0
5
10
2
0
0
2
1
5
13
2
0
0
2
1
5
40
4
1
0
5
1
5
132
0
2
3
5
5
5
Females (ppm)
0
0
0
0
0
0
5
1
0
0
0
0
0
5
3
0
0
0
0
0
5
10
5
0
0
5
0
5
13
1
0
0
1
0
5
40
3
2
0
5
2
5
132
0
0
5
5
5
5
"Inflammation, bilateral, and multifocal
Sources: Miller et al. (1987a,b).
Since changes to the nasal epithelium were observed in the 13, 40, and 132 ppm exposure
groups, a follow-up study (Miller et al., 1987b) was initiated using the same study protocol and
target exposure concentrations of 0, 1, 3, and 10 ppm, with measured concentrations of 0, 1.0 ±
0.0, 2.9 ± 0.2, or 9.7 ± 0.3 ppm (0, 6, 18, or 60 mg/m3). Body weights and organ weights of both
sexes of rats and mice were not affected at any concentration level. Very slight decreased
thickness and degeneration of the olfactory epithelium in the nasal turbinates was observed in
male and female rats that were exposed to 3 and 10 ppm (Table 4-25). Very slight inflammation
in the olfactory epithelium was also evident in rats exposed to 0, 1, 3, and 10 ppm (Table 4-26).
The exudation of inflammatory cells into the nasal cavity lumen was observed in two male and
two female rats at 10 ppm.
46
-------
A very slight decrease in thickness of the olfactory epithelium in the nasal turbinates was
observed in both sexes of mice that were exposed to 10 ppm (Table 4-27). Very slight
inflammation in the olfactory epithelium was also observed in mice at 10 ppm (Table 4-28). The
exudation of inflammatory cells into the nasal cavity lumen was observed in a single male mouse
at 10 ppm. No other exposure-related effects were detected.
4.4.3. Aquatic Species Studies
NTP (2005) conducted a toxicity study in 220 male and female guppies (Poecilia
reticulate) and 340 male and female medaka (Oryzias latipes) maintained in aquaria water
containing 0, 4.5, 9, or 18 mg/L. The guppies were exposed for 16 months and the medaka for
13 months. Ten of each species at each dose group were sacrificed at 9 months for
histopathologic analysis. Approximately one-third of the fish that survived until the 9-month
evaluation were transferred to chemical-free water at that time and evaluated at study
termination. These fish are described as the stop-exposure group.
In the medaka study, survival at 9 months was decreased in the 9 and 18 mg/L groups
(NTP, 2005). At the 9-month evaluation, the incidence of choliangiocarcinomas was
significantly increased in 9 and 18 mg/L males. The incidence of choliangiocarcinomas was
significantly increased in all male and female 1,2,3-trichloropropane treatment groups after 13
months, while the incidence of hepatocholangiocarcinomas was significantly increased only in
the fish exposed to 18 mg/L 1,2,3-trichloropropane. The incidence of papillary adenomas of the
gallbladder was significantly increased in the 9 and 18 mg/L males after the 13-month exposure.
In the stop exposure component of the study, the incidence of papillary adenomas in males was
significantly increased only at the highest exposure concentration.
Reduced survival was evident in the guppies at 6 months at the highest concentration
tested (18 mg/L) and at 7 months in the 4.5 and 9 mg/L concentrations as well. Survival was
significantly reduced in the 18 mg/L guppies at about 8 months (NTP, 2005). At the 9-month
interim evaluation, there was an increased incidence of bile duct and hepatocellular neoplasms in
the exposed male and female guppies at all concentrations tested. In the stop-exposure
component of the study, hepatocellular neoplasms were evident in 18 mg/L males, and bile duct
neoplasms were evident in 18 mg/L females.
1,2,3-Trichloropropane was characterized as carcinogenic at concentrations up to 18
mg/L in both sexes of guppies and medaka based on the increased incidence of liver neoplasms
and papillary adenoma of the gallbladder (NTP, 2005). Studies of toxicity in aquatic species
such as the medaka and guppy are increasingly being used as screening studies for tumor
formation and other endpoints of toxicity.
47
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4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF
ACTION FOR CARCINOGENICITY
4.5.1. Mode of Action Studies
Weber and Sipes (1990) conducted a series of experiments that examined the covalent
binding of 1,2,3-trichloropropane to hepatic macromolecules in male F344/N rats. In a
preliminary experiment, binding of [14C]-l,2,3-trichloropropane to hepatic protein, DNA, and
RNA was measured 4 hours after i.p. administration of 30 mg/kg (100 uCi/kg). Similar amounts
of radioactivity were bound to hepatic protein, 418 ± 19 pmol [14C]-l,2,3-trichloropropane
equivalents/mg, and RNA, 432 ± 74 pmol [14C] -1,2,3-trichloropropane equivalents/mg, and
approximately half as much was bound to DNA, 244 ± 29 pmol [14C] -1,2,3-trichloropropane
equivalents/mg. Because of methodological problems, the binding to RNA was not
characterized further in this investigation.
In a subsequent time-course study, male rats (4/group) were sacrificed at 1, 4, 24, 48, and
72 hours post i.p. administration of 30 mg/kg [14C]-l,2,3-trichloropropane (100 uQ/kg). To
examine the influence of various metabolic pathways, the investigators also administered 1,2,3-
trichloropropane to four additional groups (each containing four rats) that had been pretreated as
follows:
• 80 mg/kg-day phenobarbital, a CYP450 (CYP2B, CYP3A) inducer, in 0.9% NaCl (i.p.)
for 4 days with 1,2,3-trichloropropane treatment on day 5;
• 40 mg/kg-day p-naphthoflavone, a CYP450 inducer (CYP1 A), (i.p.) in vegetable oil for 3
days, followed by treatment with 1,2,3-trichloropropane on day 4;
• 75 mg/kg SKF 525-A, an inhibitor of CYP450, in phosphate-buffered saline (pH 5.0)
administered (i.p.) 2 hours prior to treatment with 1,2,3-trichloropropane;
• 2 g/kg l-buthionine-(R,S)-sulfoximine (BSO), which causes a depletion of hepatic GSH,
administered in two doses (i.p.) spaced 1.5 hours apart, followed by 1,2,3-
trichloropropane treatment 3 hours later.
All rats in the metabolic study were sacrificed 4 hours after treatment with 1,2,3-
trichloropropane.
In the time-course study, maximum trichloropropane-equivalent covalent binding to
hepatic proteins (approximately 600 pmol/mg) was observed 4 hours after trichloropropane
administration and was approximately 2.5-fold greater than at 1 hour post-administration.
Maximal covalent binding to hepatic DNA (approximately 250 pmol/mg) was observed 24 hours
after administration. By 72 hours, the amount of radioactivity bound to both protein and DNA
had returned to levels below those measured 1 hour post administration. At the point of maximal
48
-------
binding, the amount of [14C]-l,2,3-trichloropropane-derived radioactivity bound to hepatic
proteins was more than double the amount bound to hepatic DNA.
Administration of three consecutive doses each of 30 mg/kg 1,2,3-trichloropropane,
separated by 24 hours, produced a linear increase in the amount of [14C]-l,2,3-trichloropropane-
derived radioactivity bound to hepatic proteins. Repeated dosing did not affect the amount of the
chemical equivalent bound to DNA until the third dose at which point the amount of bound
radioactivity doubled.
In the metabolic study, induction of CYP450 (CYP) isozymes with phenobarbital
pretreatment significantly reduced chemical binding to hepatic protein and DNA by 70 and 64%,
respectively, when compared with controls. However, induction of CYP450 isozymes with P-
naphthoflavone pretreatment did not significantly alter binding to either macromolecule.
Depletion of GSH by BSO pretreatment increased binding to hepatic proteins by 342% and
decreased binding to DNA by 44% when compared with controls, with the increased covalent
binding due to decreased GSH conjugation of a 1,2,3-trichloropropane metabolite. Inhibition of
CYP450 isozymes with SKF 525-A significantly increased binding to hepatic protein and DNA
by 58 and 42%, respectively, compared with controls. The decrease in GSH appears to lead to
increased levels of a reactive metabolite that does not require GSH to bind with proteins, as
evidenced by the increased binding to hepatic proteins when GSH levels were reduced by BSO
pretreatment. 1,2,3-Trichloropropane metabolite(s) appear to conjugate with GSH and produce
compounds, such as episulfonium ions, that may covalently interact with hepatic DNA.
To further explore the effect of 1,2,3-trichloropropane on GSH, two additional
experiments were conducted by Weber and Sipes (1990): hepatic GSH levels were measured in
rats receiving 30, 100, and 300 mg/kg 1,2,3-trichloropropane (four rats per dose); GSH levels of
control and treated animals were evaluated with and without phenobarbital pretreatment. 1,2,3-
Trichloropropane treatment caused a dose-dependent, statistically significant decrease in GSH
levels 2 hours after exposure. Phenobarbital pretreatment, on the other hand, did not increase the
trichloropropane-induced reduction in hepatic GSH concentrations.
La et al. (1995) investigated the formation of DNA adducts in animals treated with 1,2,3-
trichloropropane by using the same route of administration and some of the doses used in the
NTP (1993) chronic bioassay. A single dose of either 3 or 30 mg/kg 1,2,3-trichloropropane
containing [14C]-l,2,3-trichloropropane (1 mCi) was administered by gavage to male F344/N rats
and 6 or 60 mg/kg [14C]-l,2,3-trichloropropane to male B6C3F1 mice. Animals were sacrificed
after 6 hours, and DNA adducts were hydrolyzed by neutral thermal or mild acid treatment and
separated by cation exchange high performance liquid chromatography. Peaks were
characterized by using electrospray ionization mass spectrometry, and their identity was verified
with synthesized standards.
49
-------
The elution profile of the labeled DNA indicated that a single, major DNA adduct was
formed (La et al., 1995). The adduct was determined to be S-[l-(hydroxymethyl)-2-(N7-guanyl)-
ethyljglutathione and was widely distributed among the organs examined (La et al., 1995). A
proposed formation pathway involves the biological activation, possibly by conjugation with
GSH, of 1,2,3-trichloropropane and intramolecular rearrangement to form episulfonium ions that
covalently bind to DNA. The formation of the S-[l-(hydroxymethyl)-2-(N7-guanyl)ethyl]-
glutathione adduct was detected in the forestomach, glandular stomach, kidney, liver, pancreas,
and tongue (oral) of F344/N rats, and in the forestomach, glandular stomach, kidney, and liver of
B6C3F1 mice. The concentrations of this adduct formed in the target organs (expressed as
umol/mol guanine) showed some correlation with the tumor incidence from the NTP (1993)
study (Table 4-29). For example, dose-dependent adduct formation was demonstrated in the
forestomach of F344/N rats and B6C3F1 mice, and the forestomach was a primary site of tumor
formation in both animal models in the NTP (1993) study. Conversely, dose-dependent adduct
formation was apparent in the liver and glandular stomach in both species, although NTP (1993)
detected no tumor formation at this tissue site. Adduct formation in the spleen of rats and mice,
when compared to other organs, appeared lower, and NTP (1993) did not detect tumors in the
spleens of rats and mice.
50
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Table 4-29. Comparison of tumor incidence and DNA-adduct formation in
male F344/N rats and B6C3Fi mice
Organ
Dose
Tumor incidence"
Adduct level (umol/mol guanine)b
Male rats
Forestomach0
Kidney"
Pancreas0
Preputial gland
Oralc
Glandular stomach
Liver
3
30
3
30
3
30
3
30
3
30
3
30
3
30
33/50
43/52
2/50
21/52
21/50
29/52
6/47
16/50
2/50
37/52
0/50
0/52
1/50
3/52
3.7
14.6
6.6 ±1.4
38.9 ±5.0
5.3 ±1.0
37.8 ±12.8
Not detected
Not detected
4.0
20.4
3.8
20.4
5.4 ±0.7
47.6 ±21.0
Male mice
Forestomach0
Liver0
Lung
Glandular stomach
Kidney
6
60
6
60
6
60
6
60
6
60
50/51
55/56
24/51
31/56
11/51
6/56
0/51
0/56
0/51
0/56
19.8
41.0
12.1 ±4.6
59.3 ±21.7
0.77 ±0.16
5. 3 ±0.2
28.1
208.1
4.4 ±2.9
32.5 ± 11.3
"From NTP (1993) and tallied in La et al. (1995).
bFrom La et al. (1995); expressed as mean ± standard deviation from four animals with statistical significance not
analyzed.
°Statistically significant increase in tumor formation from NTP (1993).
Source: Laetal. (1995).
The S-[l-(hydroxymethyl)-2-(N7-guanyl)ethyl]glutathione adduct indentified by La et al.
(1995) is an N7-guanyl adduct shown in Figure 4-1. The adduct crosslinks a physiological
oligopeptide, reduced GSH, to DNA by a chemical carcinogen (Ozawa and Guengerich, 1983).
The N7 position on the guanine is a highly electrophilic nitrogen atom that is located in an
accessible position on the DNA polymer (Gasparutto et al., 2005). N7-guanyl adducts generally
have an inhibitory effect on sequence-specific DNA binding by regulatory proteins, due to a
destabilization of the guanine nucleobase and spontaneous degradation (Gasparutto et al., 2005;
51
-------
Ezaz-Nikpay and Verdine, 1994). However, the exact role of the N7-guanyl adducts is unknown
(Gasparutto et al., 2005). This DNA adduct lends evidence of the involvement of the
episulfonium ion in DNA binding, as the episulfonium ion interacts with reduced GSH and binds
to DNA at the N7 of guanine. The formation of additional DNA adducts could potentially be
through the 1,3-dichloroacetone and 2-chloroacrolein pathways of metabolism.
OOC
COO
H2N
Sugar phosphate DNA backbone
Figure 4-1. Structure of the DNA adduct S-[l-(hydroxymethyl)-2-(N7-guanyl)-
ethyl] glutathione.
In a subsequent publication, the DNA adduct-forming capacity of 1,2,3-trichloropropane
in male B6C3F1 mice (n = 15) which received equivalent doses of [14C]-l,2,3-trichloropropane
via either corn oil gavage or drinking water was compared (La et al., 1996). The mice were
administered 6 mg/kg-day for 5 days via gavage or drinking water. As shown in Table 4-30, a
greater amount of DNA adduct was extracted from tissues of those animals receiving 1,2,3-
trichloropropane via gavage when compared to those exposed via drinking water, although the
only statistically significant differences were in the liver. Similarly, the authors observed little,
if any, cellular proliferation in the tissues of animals exposed to 1,2,3-trichloropropane in
drinking water. By contrast, cellular proliferation appeared to increase in a dose-dependent
manner in tissues of animals exposed to 1,2,3-trichloropropane by gavage.
52
-------
r14.
Table 4-30. Formation of DNA adducts by [ C]-l,2,3- trichloropropane
(6 mg/kg-day) administered to B6C3Fi mice by gavage or drinking water
Organ
DNA ad duct formation (umol/mol guanine)
Drinking water
Gavage
Target organs for tumor formation
Forestomach
Liver
86.8 ±73.2
185. 5 ±83.9
123.1 ±10.3
374.9 ±109.2a
Nontarget organs for tumor formation
Glandular stomach
Kidney
43.2 ±5.9
81.9±41.5
42.5 ±4.6
193.1±64.4a
"Indicates a statistically significant difference (p < 0.05) compared to values obtained in the same tissue of animals
receiving 1,2,3-trichloropropane via the alternative route of administration, as calculated by the authors.
Source: Laetal. (1996).
4.5.2. Genotoxicity Studies
Bacterial mutagenicity assays
Several in vitro genotoxicity studies have demonstrated 1,2,3-trichloropropane to be
mutagenic in various Salmonella strains in the presence of metabolic enzymes, also known as S9
fraction (Table 4-31). In a study of 250 individual chemicals, which included 1,2,3-
trichloropropane, Haworth et al. (1983) observed a dose-dependent increase in revertant colonies
in Salmonella typhimurium strains TA100 and TA1535 that were exposed to 10, 33, 100, and
333 ug 1,2,3-trichloropropane/ plate with activation by both rat and hamster S9 fractions. No
increases were observed in strains TA98 and TA1537. Shell Oil Co. (1979) observed dose-
dependent increases in revertant colonies in the presence of S9 fraction in tester stains: TA98, at
200 and 2,000 ug/plate; TA100, at 20, 200, and 2,000 ug/plate; and TA1537, at 20 and 200
ug/plate. These investigators also detected revertants, at 200 and 2,000 ug/plate in TA1535 both
in the presence and in the absence of an S9 fraction, with a greater number of revertants in the
plates with microsomal activation.
53
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Table 4-31. Genotoxicity bioassays of 1,2,3-trichloropropane
Test system
Cells/strain
Positive concentrations
Results
-S9
+S9
Reference
In vitro gene mutation assays
Bacterial assays
S. typhimurium (Ames
test)
E. coli (SOS chromotest)
E. coli (DNA-repair
deficient strain)
E. coli (DNA-repair-
proficient strain)
TA100, A1535
TA1537, TA98
TA98
TA100
TA1537
TA1535
TA1538
TA97, TA100,
TA1535
TA98
TA1537
TA100
TA100
TA1535, A100
TA98, TA1538,
TA1537
TA98, TA100,
TA1535
TA1537,
TA1538
PQ37
WP2 uvrA
WP2
10, 33, 100, 333 ug/plate
N/A
200, 2,000 jig/plate
20, 200, 2,000 jig/plate
20, 200 jig/plate
200, 2,000 ug
N/A
10, 33, 100, 333 ug/plate
100, 333 jig/plate
N/A
0. 1, 1 umol/plate
0.01,0.02,0.04,0.1
umol/plate
5, 10, 50, 100 jig/plate
N/A
0.02-1.0 mg/plate
N/A
N/A
2,000 jig/plate
N/A
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
—
-
-
+
-
+
+
+
+
+
+
+
NP
+
+
+
-
+
-
-
+
-
Haworth et al., 1983
Shell Oil Co., 1979
NTP, 1993
Stolzenberg and Hine,
1980
Lagetal., 1994
Ratpan and Plaumann,
1988
Kier, 1982
vonderHude etal.,
1988
Shell Oil Co., 1979
Lower eukaryote
Saccharomyces cerevisiae
(mitotic gene conversion)
Aspergillus nidulans
(abberrant mitotic
segregation)
JD1
PI
0.1,0.5, 1.0,5.0
mg/cm3
N/A
-
+
NP
Shell Oil Co., 1979
Crebelli et al., 1992
Mammalian cell assays
Mouse Lymphoma
L5178Y
L5178Y
0.01, 0.02, 0.03, 0.04,
0.05, 0.06 ug/mL
2.4,3.2,4.2,5.6,7.6, 10,
13, 18 ug/mL
-
NP
+
+
NTP, 1993
Shell Oil Co., 1982
54
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Table 4-31. Genotoxicity bioassays of 1,2,3-trichloropropane
Test system
Cells/strain
Positive concentrations
Results
-S9
+S9
Reference
In vitro chromosomal damage assays
Mammalian cells
Chromosomal aberrations
Micronucleus
Micro nucleus:
Unscheduled DNA
synthesis
DNA strand breaks
(Comet assay)
DNA Fragmentation
Sister chromatid
exchanges
Chinese hamster
ovary (CHO)
cells
Rat liver
epithelial
Human
lymphocytes
AHH-1
MCL-5
H2E1
Male rat
hepatocytes
(F344/N)
Human
lymphocytes
Wistar rat
hepatocytes
V79
CHO
V79
59.5, 69.4, 79.2 ug/mL
N/A
N/A
0.01, 1,2, 5mM
1, 2, 5 mM
0.01, 1,2, 5mM
N/A
2, 4mM
N/A
4, 5mM
14.2,39.7,49.6,59.5
ug/mL
0.3, 1.0 mM
-
-
+
+
+
+
-
+
-
-
NP
NP
NP
NP
+
NP
+a
-
-
+
+
NTP, 1993
Shell Oil Co., 1979
Tafazoli and Kirsch-
Volders, 1996
Dohertyetal., 1996
Williams et al., 1989
Tafazoli and Kirsch-
Volders, 1996
Holme etal., 1991
Eriksson et al., 1991
NTP, 1993
vonderHude etal.,
1987
In vivo bioassays
Chromosomal damage: mammalian
Micronucleus CD-I mice, bone
marrow
erythrocytes
DNA strand breaks F344/N male rat
(Comet assay) hepatocytes
Wistar male rat
Kidney
DNA adducts F344/N male rat
(multiple organs)
B6C3F! male mice
(multiple organs)
N/A
30, 100, 300 mg/kg
>375 umol/kg
3 or 30 mg/kg
6 or 60 mg/kg
+
+
+
+
Crebellietal., 1999
Weber and Sipes, 1991
Lag etal., 1991
La etal., 1995
55
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Table 4-31. Genotoxicity bioassays of 1,2,3-trichloropropane
Test system
Other in vivo assays
Dominant lethal
mutation
Wing spot test
Polyploidy
Cells/strain
Positive concentrations
Results
-S9 +S9
Reference
Sprague-Dawley
male rats, implants
and embryos
Drosophila
melanogaster
Albino male rat
hepatocytes
N/A
4.51 ug/L (inhalation)
0.8 mg/L (inhalation)
0.8, 2.16 mg/L
(inhalation)
+
+
+
Saito-Suzukietal.,
1982
Chroust et al., 2007
Belyaevaetal., 1974
Belyaeva et al., 1977
"Metabolic enzyme induction was not specified.
N/A = either chemical had no effect or information is not available (abstracts only); NP = assay was not performed
NTP (1993) tested strains at doses of 3, 10, 33, 100, or 333 ug/plate and 10, 33, 100, 333,
666, 667, or 1,000 ug/plate and observed a dose-dependent increase in the number of revertants
in colonies of TA97, TA100, and TA1535 treated with 1,2,3-trichloropropane in the presence of
either hamster or rat S9 fraction in repeated experiments. Mutagenic activity was observed in
TA98 in the presence of hamster and rat S9 fraction. No mutagenic activity was observed in the
TA1537 test strain in the presence of S9. In the absence of an S9 microsomal fraction, no
mutagenic activity was detected in any of the Salmonella strains. It should be noted that the
NTP (1993) report includes data from the Haworth et al. (1983) article. The descriptions
provided here are for trials not included in the earlier report.
Other groups also have demonstrated the mutagenic capability of 1,2,3-trichloropropane
by using the Ames test. Stolzenberg and Hine (1980) and Lag et al. (1994) found dose-
dependent increases in mutagenic activity in TA100 in the presence of S9 at doses of 14.7 (0.1
umol/plate) and 147 (1 umol/plate) ug/plate and -14.7 (0.1 umol/plate) ug/plate, respectively.
No increases were observed in the nonactivated cultures. A dose dependent, statistically
significant increase in mutagenic activity was also demonstrated by Ratpan and Plaumann (1988)
in TA1535 and TA100 in the presence of S9 at doses of 5, 10, 50, and 100 ug 1,2,3-
trichloropropane/plate, with no mutagenic activity in the same strains in the absence of S9 at the
same doses and no mutagenic activity in TA98, TA1537, or TA1538 in the presence and absence
of S9 at the same doses. Kier (1982) found mutagenic activity in TA100, TA1535, and TA98 in
the presence of S9 fraction at 20-1,000 ug/plate, 20-300 ug/plate, and 100-300 ug/plate,
respectively. No mutagenic activity was found in the same strains in the absence of S9 at the
same doses nor was mutagenic activity found in TA1537 and TA1538 in the presence and
absence of S9 at the same doses.
56
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The mutagenic effects of 1,2,3-trichloropropane have also been examined in other
microbial systems with mixed results, von der Hude et al. (1988) showed the compound to be
negative for DNA damage in the SOS chromotest using Escherichia coli PQ37. 1,2,3-Trichloro-
propane induced mutations in DNA repair-deficient E. coli WP2 uvr A at 2,000 ug/plate, but not
in the DNA repair-proficient strain WP2, and induced mitotic gene conversion in Saccharomyces
cerevisiae after exposure to 0.01, 0.1, 0.5, 1.0, or 5.0 mg/cm3 1,2,3-trichloropropane in the
presence of rat liver S9 (Shell Oil Co., 1979). Increases were not observed in the non-activated
cultures. 1,2,3-Trichloropropane tested negative in the Aspergillus nidulans diploid strain PI
assay for aberrant mitotic segregation at 0.1 % v:v with 5% survival (Crebelli et al., 1992).
Mammalian cell assays
1,2,3-Trichloropropane has also been shown to induce genotoxic effects in cultured
mammalian cells (Table 4-31). NTP (1993) conducted cytogenetic analysis in Chinese hamster
ovary (CHO) cells, and the results indicated that 1,2,3-trichloropropane induced both sister
chromatid exchanges, at 14.2, 39.7, 49.6, and 59.5 ug/plate and chromosomal aberrations at 59.5,
69.4, and 79.2 ug/plate, in the presence of rat liver S9 fraction. However, 1,2,3-trichloropropane
did not induce chromosomal damage in cultured rat liver epithelial cells at doses of 250, 500, or
1,000 ug/mL (Shell Oil Co., 1979), nor did it elicit micronucleus formation in isolated human
lymphocytes at doses of 0.1, 2, 4, 6, or 8 mM (0.015, 0.29, 0.59, 0.89, or 1.2 mg/L) (Tafazoli and
Kirsch-Volders, 1996). 1,2,3-Trichloropropane induced sister chromatid exchanges in Chinese
hamster V79 cells at 0.3 and 1.0 mM with microsomal activation, but did not induce sister
chromatic exchanges without microsomal activation (von der Hude et al., 1987). Eriksson et al.
(1991) observed DNA fragmentation in Chinese Hamster lung fibroblasts (V79) cells at 4 and 5
mM 1,2,3-trichloropropane, although induction levels were not provided.
1,2,3-Trichloropropane induced micronucleus formation in the mammalian cell lines,
AHH-1, MCL-5, and h2El, in a dose-dependent manner from 0.01 to 5.0 mM for each cell line
(Doherty et al., 1996). The human B lymphoblastoid AHH-1 cell line has native cytochrome
CYP1A1 activity, the MCL-5 cell line expresses cDNAs encoding human CYP1A2, 2A6, 3A4,
and microsomal epoxide hydrolase, and the h2El cell line contains CYP1A1 activity and a
cDNA for CYP2E1. The increase in micronuclei in AHH-1 and h2El was approximately eight-
fold, while the increase in MCL-5 was approximately four-fold. The micronuclei of all three cell
lines stained both positively and negatively for kinetochore antibody. Although the micronuclei
of the MCL-5 cell line stained primarily positive for kinetochore antibody, indicative of
aneugenic effects, those induced in the AHH-1 and h2El cell lines lacked kinetochore staining,
which is indicative of clastogenic effects. The difference in micronucleus formation between
AHH-1 and h2El and MCL-5 suggests the formation of a less genotoxic or further deactivated
metabolite in the MCL-5 line. The MCL-5 cell line endogenously expresses CYP1 Al and
contains cDNAs for CYP1A2, 2A6, 3A4, and 2E1, while AHH-1 and h2El contain CYP1A1 and
57
-------
CYP1A1 and 2E1, respectively. The MCL-5 cell line may be capable of metabolizing 1,2,3-
trichloropropane to less genotoxic metabolites or less potent inducer of micronuclei.
Use of an alkaline single cell gel electrophoresis test (Comet assay) demonstrated a
compound-related increase in the incidence of DNA strand breaks under cytotoxic conditions in
isolated human lymphocytes (Tafazoli and Kirsch-Volders, 1996). 1,2,3-Trichloropropane did
not induce DNA strand breaks, measured by alkaline elution, in male Wistar rat hepatocytes after
a 1-hour exposure to 50 uM (Holme et al., 1991). When tested for genotoxicity in the rat
hepatocyte unscheduled DNA synthesis assay, 1,2,3-trichloropropane (10"4% M) was negative
for unscheduled DNA synthesis, a general response to DNA damage (Williams et al., 1989).
NTP (1993) found a positive response to 1,2,3-trichloropropane in the mouse lymphoma
assay for induction of trifluorothymidine resistance in L5178Y cells in the presence of rat liver
S9 fraction; the lowest effective dose was 0.01 uL. Without S9 activation, no induction of
trifluorothymidine resistance was noted at doses below those that produced precipitation of
1,2,3-trichloropropane. Shell Oil Co. (1982) also demonstrated the capacity of the compound to
induce forward mutations to confer trifluorothymidine resistance in mouse lymphoma L5178Y
cells in the presence of S9 fraction, and an inability to induce forward mutations in the absence
of S9 fraction.
In vivo bioassays
In vivo assays provided both positive and negative evidence of genotoxicity (Table 4-31).
Chroust et al. (2007) investigated the genotoxic effects of 1,2,3-trichloropropane in the somatic
mutation and recombination test (SMART) using Drosophila melanogaster. In this bioassay,
72-hour-old larvae were administered 1,2,3-trichloropropane for 48 hours by inhalation, and the
wings of the adults were inspected for the presence of wing spots which were characterized as
small, large twin, and total spots. The induction of wing spots is caused by genotoxic effects
such as somatic mutation, chromosomal rearrangement, or nondisjunction. 1,2,3-
Trichloropropane caused a statistically significant (compared to control) increase in the number
of total wing spots.
Belyaeva et al. (1974) investigated the effect of 1,2,3-trichloropropane on the ploidy of
hepatocytes in rats. Male albino rats inhaled 0.8 mg/L 1,2,3-trichloropropane for 1 week. The
percentage of mononuclear tetraploid and octaploid cells was statistically significantly increased,
and an increase in ploidy of 16n was also evident. There was also a decrease in the percentage
of binuclear cells in concordance with the increase in tetraploid and octaploid. Belyaeve et al.,
(1977) conducted a similar investigation in order to compare the action of various concentrations
of 1,2,3-trichloropropane and 1,2-dichloropropane on the ploidy of hepatocytes. Male albino
rats inhaled 1,2,3-trichloropropane at 0.8 or 2.16 mg/L for 1 week, 0.08 mg/L for 2 weeks, and
0.002 mg/L for 3 months. After the 1-week exposure period, the 1,2,3-trichloropropane dosed
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animals demonstrated an increase in the number of mononuclear hepatocytes with a nucleus of
high ploidy with a decrease in the number of binuclear cells. Following the 2-week exposure,
however, the results in the experimental group and control group were indistinguishable. When
the exposure time was increased to 3 months and the dose decreased to 0.002 mg/L, a slight
increase in nuclei of intermediate ploidy was observed in the 1,2,3-trichloropropane-exposed
group. Additional positive evidence of genotoxicity was obtained by Weber and Sipes (1991),
who administered single, i.p. injections of 30, 100, or 300 mg/kg 1,2,3-trichloropropane to male
F344/N rats, which were then sacrificed 1, 2, 4, 8, 12, 24, and 48 hours post-administration.
Using alkaline elution to detect damaged hepatic DNA, they demonstrated that 1,2,3-
trichloropropane, or its metabolites, caused the formation of DNA strand breaks. La et al. (1995)
characterized the formation of DNA adducts in various organs in both B6C3F1 mice and F344/N
rats exposed to 6 or 60 and 3 or 30 mg/kg, respectively. High concentrations of DNA adducts
were evident in the tumor-forming organ tissues, including the forestomach, kidney, pancreas,
oral cavity, and liver in male rats and the forestomach and liver of male mice, from the NTP
(1993) study. DNA adducts were also found in tissues that did not develop tumors, although the
increased incidence of tumors and increased mortality in the NTP study may have precluded
tumor development in those tissues that formed DNA adducts without tumors. Male
MOL:WIST rats were killed 1 hour after receiving 1,2,3-trichloropropane by i.p. administration
and the kidney DNA damage was assessed by alkaline elution (Lag et al., 1991). 1,2,3-
Trichloropropane was observed to cause DNA breaks in the kidney DNA of rats at doses >375
umol/kg.
Negative results from in vivo assessments were obtained when the compound was
included in a survey of 10 aliphatic halogenated hydrocarbons using the CD-I mouse bone
marrow micronucleus test and 1,2,3-trichloropropane doses of 115 and 200 mg/kg (Crebelli et
al., 1999). Similarly, 1,2,3-trichloropropane did not induce dominant lethal mutations in male
Sprague-Dawley rats when administered by gavage in corn oil at 80 mg/kg-day for 5 days (Saito-
Suzukietal., 1982).
4.5.3. Structural Analog Data—Relationship to l,2-Dibromo-3-chloropropane and 1,2-
Dibromoethane
1,2,3-Trichloropropane is a halogenated propane, with a single chlorine atom attached to
each carbon atom in the chain. Halogenated propanes as a class of compounds are generally
found to be positive in assays that indicate mutagenicity (Lag et al., 1994; Ratpan and Plaumann,
1988), and there is clear evidence that members of this group, including l,2-dibromo-3-
chloropropane (DBCP) (NTP, 1982a; NCI, 1978) and 1,2-dibromoethane (NTP, 1982b), are
carcinogenic in whole animal models. In a study sponsored by the National Cancer Institute
(NCI), DBCP was found to be carcinogenic to Osborne-Mendel rats when administered by
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gavage in corn oil at 15 or 29 mg/kg-day for up to 78 weeks, and to B6C3F1 mice when
administered by gavage in corn oil at 114 and 110 or 219 and 209 mg/kg-day in male and female
mice, respectively, for up to 60 weeks (NCI, 1978). A statistically significant increase in the
incidence of adenocarcinomas of the mammary gland was observed in female rats. Squamous
cell carcinomas in the forestomach resulted in reduced survival in both species. These responses
are qualitatively similar to those produced by 1,2,3-trichloropropane.
In addition to the oral bioassay conducted by NCI (1978), an inhalation bioassay of
DBCP was conducted by NTP (1982a). NTP administered technical-grade DBCP, which
contained trace amounts of epichlorohydrin and 1,2-dibromoethane, to F344/N rats and B6C3F1
mice via inhalation at concentrations of 0.6 or 3.0 ppm for 6 hours/day, 5 days/week for 76-103
weeks. DBCP induced nasal cavity tumors and tumors of the tongue in male and female rats, as
well as cortical adenomas in the adrenal glands of female rats (NTP, 1982a). In mice, DBCP
induced nasal cavity tumors and lung tumors in both sexes (NTP, 1982a). NTP (1982a)
concluded that DBCP was carcinogenic in male and female F344/N rats and B6C3F1 mice.
DBCP also forms the same major DNA adduct, S-[l-(hydroxymethyl)-2-(N7-guanyl)ethyl]-
glutathione, as 1,2,3-trichloropropane (Humphreys et al., 1991).
NTP (1982b) also conducted an inhalation cancer bioassay in F344/N rats and B6C3F1
mice, in which test animals inhaled 10 or 40 ppm of 1,2-dibromoethane for 78-103 weeks. In
rats, 1,2-dibromoethane inhalation caused an increased incidence of carcinomas,
adenocarcinomas, and adenomas and adenomatous polyps of the nasal cavity;
hemangiosarcomas of the circulatory system; mesotheliomas of the tunica vaginalis;
fibroadenomas of the mammary glands; and alvoelar/bronchiolar adenomas and carcinomas
(NTP, 1982b). In mice, 1,2-dibromo-ethane inhalation caused an increased incidence of
alveolar/bronchiolar adenomas and carcinomas; hemangiosarcomas of the circulatory system;
fibrosarcomas in the subcutaneous tissue; carcinomas of the nasal cavity; and adenocarcinomas
of the mammary gland (NTP, 1982b).
In addition to the carcinogenicity data, mode-of-action data for similar compounds
support the proposed mode of action for 1,2,3-trichloropropane; specifically, the formation of
episulfonium ions and subsequent DNA binding. 1,2-Dibromoethane spontaneously forms the
episulfonium ion, thiiranium, following conjugation with GSH, which may then bind to DNA
(U.S. EPA, 2004). DNA binding of metabolites of 1,2-dibromoethane and DBCP have been
demonstrated in vitro in calf thymus DNA (Inskeep and Guengerich, 1984) and the binding of
metabolites of 1,2-dibromopropane to DNA in vivo has been demonstrated in rats following i.p.
injection (Kim and Guengerich, 1990).
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4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS
4.6.1. Oral
There are no data on the toxicological effects of exposure to 1,2,3-trichloropropane in
humans via ingestion. Three subchronic studies in rats and mice (NTP, 1993; Merrick et al.,
1991; Villeneuve et al., 1985), a single chronic study in rats and mice (NTP, 1993), and a
reproductive study in mice (NTP, 1990) have investigated the effects of oral exposure in animal
models.
The NTP (1993) toxicology and carcinogenesis studies conducted in F344/N rats and
B6C3F1 mice constitute the database of chronic oral toxicity studies for 1,2,3-trichloropropane.
The effects of subchronic oral exposure to 1,2,3-trichloropropane have been investigated by NTP
(1993), Merrick et al. (1991), and Villeneuve et al. (1985). A reproductive and fertility
assessment investigation of 1,2,3-trichloropropane was conducted with Swiss CD-I mice (NTP,
1990). 1,2,3-Trichloropropane was administered by corn oil gavage in all of these
investigations, except the study by Villeneuve et al. (1985), which provided 1,2,3-
trichloropropane to rats via drinking water. Table 4-32 provides the observed effects and
corresponding NOAELs and LOAELs for the subchronic, chronic, and reproductive toxicity
studies available for 1,2,3-trichloropropane.
Table 4-32. Observed effects and corresponding NOAELs and LOAELs for
subchronic, chronic, and reproductive toxicity studies following oral
exposure to 1,2,3-trichloropropane
Effect
Sex
NOAEL (mg/kg-d)
LOAEL (mg/kg-d)
Subchronic— NTP (1993)
F344/N rats
Increased relative liver weight
Increased absolute liver weight
Decreased pseudocholinesterase
Increased SDH
Increased ALT
Hepatocellular necrosis
Increased relative kidney weight
Increased absolute kidney weight
Kidney necrosis
Decreased absolute heart weight
Nasal turbinate necrosis
Female
Male
Female
Male
Female
Male
Male
Male
Male
Male
Male/female
8
-
-
32
63
16
16
16
63
32
63
16
8
8
63
125
32
32
32
125
63
125
B6C3Fi mice
Increased relative liver weight
Increased absolute liver weight
Hepatocellular necrosis
Hepatocellular karyomegaly
Male/female
Male
Male/female
Male
63
16
63
63
125
32
125
125
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Table 4-32. Observed effects and corresponding NOAELs and LOAELs for
subchronic, chronic, and reproductive toxicity studies following oral
exposure to 1,2,3-trichloropropane
Effect
Increased relative kidney weight
Increased absolute kidney weight
Decreased relative heart weight
Decreased absolute heart weight
Decreased relative brain weight
Decreased absolute brain weight
Regenerative lung lesions
Hyperkeratosis of the forestomach
Sex
Female
Female
Male
Male
Male/female
Female
Female
Female
NOAEL (mg/kg-d)
8
125
-
32
8
8
32
32
LOAEL (mg/kg-d)
16
250
8
63
16
16
63
63
Subchronic — Merrick et al. (1991)
F344/N rats
Increased ALT
Increased AST
Myocardial necrosis
Bile duct hyperplasia
Plasma cell hyperplasia in the mandibular
lymph node
Female
Female
Male
Male/female
Female
15
15
-
15
_
60
60
1.5
60
1.5
Subchronic — Villeneuve et al. (1985)
F344/N rats
Increased relative liver weight
Increased serum cholesterol
Increased hepatic aminopyrine demethylase
Aniline hydroxylase
Biliary hyperplasia
Increased relative kidney weight
Male/female
Female
Male/female
Male
Female
Female
-18
-18
-18
-18
-18
-1.8
113-149
149
149
113
149
-18
Chronic— NTP (1993)
F344/N rats
Increased relative liver weight
Increased absolute liver weight
Increased 5'-nucleotidase
Increased relative kidney weight
Increased absolute kidney weight
Hepatocellular necrosis
Forestomach hyperplasia
Male/female
Male/female
Male
Male/female
Male
Female
Male/female
3
-
10
3
-
3
-
10
3
30
10
o
5
30
o
3
B6C3Fj mice
Increased relative liver weight
Increased absolute liver weight
Increased relative kidney weight
Increased absolute kidney weight
Increased creatine kinase
Hepatocellular necrosis
Male/female
Male/female
Female
Male/female
Male
Female
20
60
20
60
20
-
60
-
60
-
60
6
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Table 4-32. Observed effects and corresponding NOAELs and LOAELs for
subchronic, chronic, and reproductive toxicity studies following oral
exposure to 1,2,3-trichloropropane
Effect
Forestomach hyperplasia
Sex
Male/female
NOAEL (mg/kg-d)
-
LOAEL (mg/kg-d)
6
Reproductive— NTP (1990)
CD-I mice
Decrease in number of pregnancies/fertile mouse
Decrease in number of live pups/litter
Increased cumulative d to litter
Decreased proportion of male pups
Decreased mating indices
Decreased fertility indices
30
30
30
60
60
60
60
60
60
120
120
120
The principal finding of the NTP (1993) chronic toxicity studies was a statistically
significant elevated incidence of tumors in both rats and mice at multiple sites. The tumorogenic
effects of 1,2,3-trichloropropane are discussed in greater detail in Section 4.7. The increased
incidence of tumors was accompanied by a significant decrease in survival. The percent
probability for survival was significantly decreased in rats receiving a dose of >10 mg/kg-day
1,2,3-trichloropropane, and in mice receiving a dose of >6 mg/kg-day. Because the decrease in
survival was associated with the increased incidence of tumors (NTP, 1993), it was
notconsidered a noncancer effect. However, it is important to note that the nonneoplastic
changes associated with chronic oral exposure to 1,2,3-trichloropropane occurred at doses that
also produced cancer and an associated decrease in the percent chance of survival.
Statistically significant increases in absolute and relative liver and right kidney weights
were observed in the subchronic and chronic studies. The increase in liver and kidney weights
may be associated with the metabolic role of these organs involving the induction of metabolic
enzymes and other proteins in metabolizing 1,2,3-trichloropropane. However, this metabolic
role may be combined with the binding of 1,2,3-trichloropropane metabolites to hepatic proteins
and DNA in the continuum to liver damage. Corn oil gavage has been shown to increase cell
proliferation in hepatocytes (Rusyn, et al., 1999); however, the NTP assay control animals, to
which the dose groups were compared, also received corn oil gavage. Organ weight increases
were proportionally greater in rats than mice, and increased organ weights were, generally, also
more pronounced in females than males. The variation in the effect on organ weights between
species and sexes indicates that there may be toxicokinetic and toxicodynamic differences that
affect the metabolism of 1,2,3-trichloropropane.
In male rats, a statistically significant decrease in ALT and 5'-nucleotidase was apparent
after chronic exposure to 30 mg/kg-day, while in female mice, a statistically significant increase
in SDH was evident after chronic exposure to 60 mg/kg-day. Hepatocellular necrosis was
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observed in female rats at 3 and 30 mg/kg-day and in male and female mice at all dose groups
including controls at the 15-month interim evaluation (not statistically significant).
In the subchronic studies, there was evidence of hepatocellular damage in both rats and
mice. Absolute and relative liver weight increases were observed in male and female rats in
several studies (NTP, 1993; Merrick et al., 1991; Villeneuve et al., 1985), and were also evident
in male and female mice (NTP, 1993). After subchronic exposure, an increase in the incidence
of hepatocellular necrosis was apparent in both rats and mice (NTP, 1993), and the serum
concentrations of ALT, AST, and SDH were increased in female rats (NTP, 1993; Merrick et al.,
1991). Increased serum cholesterol levels and hepatic aminopyrine demethylase activity were
also apparent after subchronic administration (Villeneuve et al., 1985). Serum concentrations of
pseudocholinesterase were decreased in male and female rats after subchronic 1,2,3-
trichloropropane exposure, and reflected a decrease in pseudocholinesterase synthesis (NTP,
1993). Taken as a whole, the increased incidence of hepatocellular necrosis, the increased ALT,
AST, SDH, hepatic aminopyrine demethylase activity, and cholesterol serum concentrations,
along with the decreases in pseudocholinesterase synthesis and concentration of 5'-nucleotidase,
is indicative of hepatocellular damage due to 1,2,3-trichloropropane exposure. Travlos et al.
(1996) reported that treatment-related alteration in clinical chemistry was highly associated with
histopathological changes.
Increased absolute and relative kidney weights in male and female rats were apparent in
several subchronic studies (NTP, 1993; Merrick et al., 1991; Villeneuve et al., 1985), with an
inconsistent dose-response pattern for absolute and relative kidney weight in mice (NTP, 1993).
The NTP (1993) chronic study showed an increase in absolute and relative right kidney weights
in rats and in relative right kidney weight in female mice, as well as an increased severity of
nephropathy and incidence of renal tubule hyperplasia in rats. Overt kidney damage was not
evident in these studies.
In addition to the liver and kidney effects, cardiac and respiratory system effects were
also observed. After subchronic exposure, a decrease in the absolute heart weight in male rats
and in the absolute and relative heart weight in mice was evident (NTP, 1993). Merrick et al.
(1991) reported an increased incidence of inflammation-associated myocardial necrosis in rats,
and an increase in creatine kinase, an indicator of myocardial damage, was evident in male mice
following chronic exposure (NTP, 1993). NTP (1993) also reported epithelial necrosis in the
nasal turbinates of rats and regenerative lung lesions in mice following subchronic exposure.
Hyperplasia was also observed in the forestomach (basal cell and squamous), kidney
(renal tubule), and pancreas (acinar) of rats and in the forestomach (squamous) of mice following
chronic exposure to 1,2,3-trichloropropane (NTP, 1993). However, the necrosis observed in the
liver, kidney, nasal turbinates, and heart of rats and liver, forestomach, and lungs of mice
following subchronic oral exposure to 1,2,3-trichloropropane was not observed in the chronic
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NTP (1993) studies, which employed doses lower than those reported to produce these effects in
the subchronic studies. The absence of observable necrosis in the chronic study may have been
due to decreased survival attributable to the onset of cancer in the chronic study, the observation
time points selected in the chronic NTP study, or the development of a tolerance to 1,2,3-
trichloropropane following chronic exposure.
Evidence of hematological effects, including decreased hematocrit values, hemoglobin
concentrations, erythrocyte counts, and elevated leukocytes and segmented neutrophils counts
were observed in both chronic and subchronic NTP (1993) studies; however, these effects were
not considered to be biologically relevant. NTP stated that the decreased hematocrit may be
associated with depressed hematopoeisis or to blood loss from neoplasms in the forestomach,
and the increased number of leukocytes may likely be due to inflammation associated with the
chemically-induced neoplasms (NTP, 1993).
A multigeneration fertility and reproduction assessment (NTP, 1990) found a significant
reduction in the number of fertile pairs of cohabiting Swiss CD-I mice exposed to 60 mg/kg-day
1,2,3-trichloropropane. The reduction in fertility was accompanied by a significant reduction in
the number of live pups per litter and in the proportion of male pups born alive in the fifth
breedings. The decrease in fertility may be related to the observed increase in metestrus, an
infertile period of estrous cycles that was reported during Task 4 of the NTP (1990) study. Male
reproductive performance and fertility were not affected.
4.6.2. Inhalation
No inhalation studies of 1,2,3-trichloropropane in humans have been identified. A single
study on the acute effects in humans found that all subjects (12/sex) reported irritation (eyes,
throat, and odor) following 15-minute exposures to 100 ppm trichloropropane (isomer and purity
not reported) (Silverman et al., 1946). The database of inhalation toxicity studies in animals
includes two 2-week studies submitted to EPA by Miller et al. (1987a, b), a 4-week range
finding study, two 13-week studies, and two single-generation reproductive assessments
(Johannsen et al., 1988; Biodynamics, Inc., 1979).
Inhalation exposure to 1,2,3-trichloropropane was associated with the following effects:
abnormal physical signs (increased lacrimation, discoloration of the anogenital fur), decreased
weight gain, increased organ weights, and increased incidences of nonneoplastic lesions in the
nasal epithelium, liver, lungs, and spleen (Johannsen et al., 1988; Miller et al., 1987a, b;
Biodynamics, Inc., 1979).
Decreased body weight and weight gain during the pre-mating period was observed in
both male and female rats in a single-generation reproductive study (Johannsen et al., 1988). In
addition, decreased body weight in female rats was observed during gestation and lactation. All
groups of female rats exhibited low mating performance, 16/20 females mated at 5 ppm and
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10/20 females mated at 15 ppm, compared with 17/20 females in the control group. Although
fewer females in the high-concentration group mated, statistical significance was not
demonstrated using the x2-square test (Johannsen et al., 1988). The decrease in the number of
females that mated was statistically significant (p < 0.02) at 15 ppm in the Fisher Exact test
conducted by EPA.
Similar to the oral toxicity database, the inhalation studies found statistically significant
increases in organ weights. Following the 13-week exposure to 1,2,3-trichloropropane,
increased absolute and relative liver weights were observed in male rats exposed to
concentrations of 5, 15, or 50 ppm and increased absolute and relative liver weights were
observed in female rats exposed to 50 ppm and 15 and 50 ppm, respectively (Johannsen et al.,
1988). Increased absolute and relative liver weights were observed following 2-week exposures
to concentrations of 40 or 132 ppm in rats, and 132 ppm in mice (Miller et al., 1987a). Increased
relative lung weights in female rats exposed to concentrations of 15 or 50 ppm for 13 weeks
(Biodynamics, Inc., 1979), and increased relative kidney weights were observed in male rats
exposed to concentrations of 50 ppm for 13 weeks (Johannsen et al., 1988).
Increased incidences of nonneoplastic lesions have been observed in the nasal epithelium,
liver, lung, and spleen of rats or mice following inhalation exposure to 1,2,3-trichloropropane
(Johannsen et al., 1988; Miller et al., 1987a, b; Biodynamics, Inc., 1979). Johanssen et al. (1988)
observed peribronchial lymphoid hyperplasia in the three high-dose treatment groups of male
and female rats, hepatocellular hypertrophy in the three highest male dose groups, and
hematopoiesis of the spleen in the highest dose group of male rats and in the three highest female
dose groups in female rats. Miller et al. (1987a, b) reported decreased thickness or degeneration
of the olfactory epithelium in rats exposed for 2 weeks to concentrations of 3, 10, 13, 40, or 132
ppm 1,2,3-trichloropropane (Tables 4-25 and 4-26). Similar effects were also observed in mice
that were exposed to 10, 13, 40, or 132 ppm concentrations (Tables 4-27 and 4-28).
Johannsen et al. (1988) (Biodynamics, Inc., 1979) found an increased incidence of
peribronchial lymphoid hyperplasia in male and female rats that were exposed to 5, 15, or 50
ppm 1,2,3-trichloropropane, but they did not examine epithelial tissue in their investigation.
Lesions remote from the respiratory tract were also observed (Table 4-22). Centrilobular to
midzonal hepatocellular hypertrophy was seen in nearly all male rats that were exposed for 13
weeks to concentrations of 5, 15, or 50 ppm 1,2,3-trichloropropane. However, no evidence of
hepatic effects was found in female rats that were exposed to 50 ppm 1,2,3-trichloropropane.
Conversely, a dose-dependent increase in the incidence and severity of extramedullary
hematopoiesis of the spleen was observed in female, but not male, rats, although this effect is not
biologically relevant. This differential expression of histopathic lesions suggests that for 1,2,3-
tri-chloropropane, there may be toxicokinetic or toxicodynamic differences between male and
female rats.
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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), 1,2,3-
trichloropropane is "likely to be carcinogenic to humans," based on a statistically significant and
dose-related increase in the formation of multiple tumors in both sexes of two species from an
NTP (1993) chronic oral bioassay. Statistically significant increases in incidences of tumors of
the oral cavity, forestomach, pancreas, kidney, preputial gland, clitoral gland, mammary gland,
and Zymbal's gland in rats, and the oral cavity, forestomach, liver, Harderian gland, and uterus
in mice were reported.
No human oral exposure studies are available. No information is available on the
carcinogenic effects of 1,2,3-trichloropropane via the inhalation route in humans or animals.
U.S. EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) indicate that for
tumors occurring at a site other than the initial point of contact, the weight of evidence for
carcinogenic potential may apply to all routes of exposure that have not been adequately tested at
sufficient doses. In addition, the data from the chronic oral study demonstrate that tumors occur
in tissues remote from the site of absorption, such as in the pancreas, kidney, preputial gland,
clitoral gland, and mammary gland. The presence of nonneoplastic lesions in the liver and
spleen of rats and mice following subchronic and shorter inhalation exposure to 1,2,3-
trichloropropane (Johannsen et al., 1988; Miller et al., 1987a, b) indicates that the chemical can
enter the blood stream from the respiratory tract, but the duration of the inhalation studies was
too short to show tumor development. Therefore, in the absence of information to indicate
otherwise, 1,2,3-trichloropropane is "likely to be carcinogenic to humans" by the inhalation
route of exposure. In addition, DBCP induced nasal cavity tumors and tumors of the tongue in
male and female F344/N rats, as well as cortical adenomas in the adrenal glands of female rats,
and nasal cavity tumors and lung tumors in both sexes of mice following inhalation exposure
(NTP, 1982a). 1,2-Dibromoethane caused an increased incidence of carcinomas,
adenocarcinomas, and adenomas and adenomatous polyps of the nasal cavity;
hemangiosarcomas of the circulatory system; mesotheliomas of the tunica vaginalis;
fibroadenomas of the mammary glands; and alvoelar/bronchiolar adenomas and carcinomas in
F344/N rats, as well as alveolar/bronchiolar adenomas and carcinomas; hemangiosarcomas of
the circulatory system; fibrosarcomas in the subcutaneous tissue; carcinomas of the nasal cavity;
and adenocarcinomas of the mammary gland in male and female B6C3F1 mice (NTP, 1982b).
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4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence
NTP (1993) conducted a 2-year study of the toxicity and carcinogenicity of 1,2,3-
trichloropropane in F344/N rats. The chemical was administered by corn oil gavage to 60
rats/sex/group. Rats received doses of 0, 3, 10, or 30 mg/kg-day, and after 15 months (65-67
weeks), 8-10 rats per group were sacrificed to allow an interim evaluation of all toxicological
parameters and histopathology. Due to high mortality in rats receiving 30 mg/kg at the interim
evaluation, the remaining survivors in that group were sacrificed at week 67 (females) and week
77 (males). In the rats, tumors were evident in the oral cavity, forestomach, pancreas, kidney,
Zymbal's gland of males and females, along with preputial gland tumors in males and clitoral
gland and mammary gland tumors in females. Tumors in the mice were evident in the oral
cavity, forestomach, liver, and Harderian gland of both males and females, and in the
uterine/cervical tissue in females.
In addition, 1,2,3-trichloropropane was characterized as carcinogenic at concentrations
up to 18 mg/L in both sexes of guppies and medaka based on the increased incidence of liver
neoplasms and papillary adenoma of the gallbladder (NTP, 2005). Other evidence that supports
the carcinogenic potential of 1,2,3-trichloropropane includes: (1) the demonstration that the
metabolically activated compound tested positive in a number of in vitro genotoxicity assays, (2)
the demonstrated ability of 1,2,3-trichloropropane metabolites to bind to intracellular protein and
DNA and form DNA adducts, (3) and the similar site-specific, multispecies carcinogenicity of a
structural analog of 1,2,3-trichloropropane, l,2-dibromo-3-chloropropane (NTP, 1982a), which
produces the same DNA adducts as 1,2,3-trichloropropane (Humphreys et al., 1991).
4.7.3. Mode of Action Analysis
4.7.3.1. Hypothesized Mode of A ction
It is hypothesized that 1,2,3-trichloropropane-induced carcinogenicity is through a
mutagenic mode of action. Specifically, the data suggest that bioactivated 1,2,3-
trichloropropane may bind directly to DNA resulting in a mutagenic event that may lead to
tumorigenicity in animals.
In vitro bacterial mutation assays have consistently demonstrated a mutagenic potential,
dependent on S9 activation, for 1,2,3-trichloropropane. Mammalian cell in vitro studies have
shown chromosomal damage, gene mutation, DNA breakage, and micronucleus formation after
1,2,3-trichloropropane exposure. In addition, in vivo assays have demonstrated the ability of
1,2,3-trichloropropane metabolites to bind to hepatic proteins, DNA, and RNA; form DNA
adducts in rats and mice; induce DNA strand breaks in the hepatocytes of rats; and induce wing
spots (caused by genotoxic alterations such as somatic mutation, chromosomal rearrangement, or
nondisjunction) in D. melanogaster. In vivo studies measuring dominant lethal induction or
micronucleus formation were nonpositive and limit the confidence in the hypothesized mode of
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action. Additional in vivo assays that would provide evidence of mutagenicity, such as
mutations in tumor suppressor genes or other mutagenic markers, are unavailable.
4.7.3.2. Experimental Support for the Hypothesized Mode of Action
Strength, consistency, specificity of association
The experimental support for mutagenicity of 1,2,3-trichloropropane is presented in
sequence, with the formation of DNA adducts first, followed by the in vitro and in vivo
evidence.
Evidence for the direct interaction of 1,2,3-trichloropropane metabolites with DNA was
presented in vivo (Weber and Sipes, 1990), in which the ability of 1,2,3-trichloropropane
metabolites to form covalent bonds with hepatic DNA, RNA, and proteins in rats following i.p.
administration was apparent. However, the levels of radioactivity bound to DNA at 72 hours
post administration, were below the level measured for 1 hour post administration, and may
reflect cytotoxicity and resultant DNA repair or DNA degradation. The administration of three
consecutive i.p. doses, 24 hours apart, of 30 mg/kg 1,2,3-trichloropropane resulted in a doubling
of the amount of radioactivity bound to DNA after the third dose. Weber and Sipes (1990)
conclude that this investigation demonstrates the ability of 1,2,3-trichloropropane or a reactive
metabolite to covalently bind to hepatic DNA, RNA, and protein, and that the covalent binding
increases with multiple doses. Weber and Sipes (1991) administered i.p. injections of 1,2,3-
trichloropropane to male F344/N rats. Following the extraction of hepatic DNA, they
demonstrated that 1,2,3-trichloropropane caused the formation of DNA strand breaks.
The involvement of GSH in the activation and binding of a metabolite of 1,2,3-
trichloropropane is supported by the pretreatment of Sprague-Dawley rats with BSO (Weber and
Sipes, 1990). BSO pretreatment causes a decrease in hepatic GSH in rats and a subsequent
decrease in binding of 1,2,3-trichloropropane or reactive metabolite to DNA. The study authors
suggested that an intermediate of 1,2,3-trichloropropane metabolism may rearrange to form an
episulfonium ion that may bind covalently to DNA.
In a subsequent study, La et al. (1995) characterized the formation of DNA adducts in
various organs in both B6C3F1 mice and F344/N rats, and found high concentrations of DNA
adducts in the tumor-forming organ tissues, including the forestomach, kidney, pancreas, and
liver in male rats and the forestomach, liver, lung and kidney of male mice, from the NTP (1993)
study (Table 4-29). The target organs of 1,2,3-trichloropropane toxicity (liver, kidney,
forestomach, and intestine) also contained the highest concentration of covalently bound 1,2,3-
trichloropropane and related metabolites (Mahmood et al., 1991). A dose-dependent formation
of DNA adducts was also evident in organs in which tumor formation was not observed.
However, the interpretation of the target organ specificity is complicated due to the high
mortality that was observed in the chronic bioassays. Early mortality may not have allowed
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tumors in some tissues to fully develop. The relationship between the adduct-forming tissues of
La et al. (1995) and the tumor-forming tissues of NTP (1993) support a mode of action involving
DNA adduct formation. However, the biological relevance of the major DNA adducts is not
known (La et al., 1995).
The S-[l-(hydroxymethyl)-2-(N7-guanyl)ethyl]glutathione adduct indentified by La et al.
(1995) is unusual in that it crosslinks a physiological oligopeptide, reduced GSH, to DNA by a
chemical carcinogen, in this case 1,2,3-trichloropropane (Ozawa and Guengerich, 1983). The
N7-guanyl adducts have an inhibitory effect on sequence-specific DNA binding by regulatory
proteins, due to a destabilization of the guanine nucleobase and spontaneous degradation
(Gasparutto et al., 2005; Ezaz-Nikpay and Verdine, 1994). However, the exact role of the N7-
guanyl adducts is unknown (Gasparutto et al., 2005).
The mutagenic activity of 1,2,3-trichloropropane has been demonstrated in bacterial and
mammalian cell systems treated with 1,2,3-trichloropropane and activated with an S9 fraction
from chemically-induced rat or hamster livers (Doherty et al., 1996; Tafazoli and Kirsch-
Volders, 1996; Lag et al., 1994; NTP, 1993; Ratpan and Plaumann, 1988; von der Hude et al.,
1987; Haworth et al., 1983; Kier, 1982; Shell Oil Co., 1982, 1979; Stolzenburg and Hine, 1980).
In the absence of the enzyme-rich S9 fraction mutagenic activity is typically not observed.
1,2,3-Trichloropropane was positive in primarily S. typhimuium strains that detect base pair
mutations (TA1535 and TA100) and frame shift mutations (TA1537 [one assay] and TA98) in
the presence of S9 fraction (Lag et al., 1994; NTP, 1993; Ratpan and Plaumann, 1988; Haworth
et al., 1983; Kier, 1982; Stolzenburg and Hine, 1980; Shell Oil Co., 1979). Mutagenicity was
also evident in E. coli WP2 uvr A, in the presence of S9 fraction, after exposure to 1,2,3-
trichloropropane (Shell Oil Co., 1979). Chromosomal aberrations and sister chromatid
exchanges were evident in CHO cells or V79 assays (NTP, 1993; von der Hude et al., 1987), and
trifluorothymidine resistance was induced in mouse lymphoma assays, after 1,2,3-
trichloropropane exposure and in the presence of S9 fraction (NTP, 1993; Shell Oil Co., 1982).
DNA strand breakage caused by 1,2,3-trichloropropane was measured by the Comet assay
(single gel electrophoresis test) in isolated human lymphocytes (Tafazoli and Kirsch-Volders,
1996), and 1,2,3-trichloropropane induced micronucleus formation in the mammalian cell lines,
AHH-1, MCL-5, and h2El (Doherty et al., 1996). The data also demonstrate that the
metabolism of 1,2,3-trichloropropane is necessary to activate the chemical's mutagenic potential.
In an in vivo bioassay in D. melanogaster, Chroust et al. (2007) investigated the
genotoxic effects of 1,2,3-trichloropropane in the SMART. 1,2,3-Trichloropropane caused a
statistically significant (compared to control) increase in the number of total wing spots, which is
evidence for genotoxic effects such as somatic mutation, chromosomal rearrangement, or
nondisjunction. Belyaeva et al. (1977, 1974) observed an increase in the number of mononuclear
hepatocytes with a nucleus of high ploidy and a decrease in the number of binuclear cells
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following exposure to 1,2,3-trichloropropane. 1,2,3-Trichloropropane also caused DNA breaks
in the DNA from isolated kidney nuclei of rats exposed to 1,2,3-trichloropropane (Lag et al.,
1991).
1,2,3-Trichloropropane tested nonpositive in bacterial systems not activated with S9
fraction (NTP, 1993; Ratpan and Plaumann, 1988), in the SOS chromotest in E. coli (von der
Hude et al., 1988), in the DNA-repair proficient K coli WP2 (Shell Oil Co., 1979), and in the A.
nidulans diploid strain PI assay for aberrant mitotic segregation (Crebelli et al., 1992).
Mammalian cell in vitro assays in which 1,2,3-trichloropropane tested nonpositive for
genotoxicity included: the induction of trifluorothymidine resistance in mouse lymphoma cells
not activated with S9 fraction (NTP, 1993; Shell Oil Co., 1982); the induction of chromosomal
damage in Carworth Farm E rat liver epithelial cells (Shell Oil Co., 1979); the micronucleus
formation assay in human lymphocytes, although numerous chlorinated aliphatics failed to
induce a clear dose-dependent increase (Tafazoli and Kirsch-Volders, 1996); the unscheduled
DNA synthesis assay in rat hepatocytes (Williams et al., 1989); and the induction of DNA strand
breaks in Wistar rat hepatocytes (Holme et al., 1991). The in vivo assays in which 1,2,3-
trichloropropane tested nonpositive included the bone marrow micronucleus formation assay in
CD-I mice (Crebelli et al., 1999) and the dominant lethal induction assay in male Sprague-
Dawley rats (Saito-Suzuki et al., 1982).
An in vitro assay conducted by Weber and Sipes (1992), utilizing rat and human hepatic
cells, demonstrated a dose-dependent increase in the formation of the intermediate metabolite,
DCA, which the study authors characterized as a direct-acting mutagen. In this study, the
formation of DCA was 10-times faster in in vitro systems with rat hepatic microsomes than in in
vitro systems with human hepatic microsomes (Weber and Sipes, 1992). DCA, also referred to
as 1,3-dichloropropanone or l,3-dichloro-2-propanone, has shown mutagenicity in S.
typhimurium TA100 without microsomal activation (Meier et al., 1985). DCA was also shown
to be mutagenic in TA1535 and TA100 with and without metabolic activation, with increased
mutagenicity in strains TA1535 and TA100 without microsomal activation compared to the same
strains with activation (Merrick et al., 1987).
DCA initiated skin tumors after both single and repeated topical treatment of female
SENCAR mice followed by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA)
(Robinson et al., 1989). The percentages of tumor-bearing mice after a single initiating dose of
37.5, 75, or 150 mg/kg DCA was 47, 47, and 68%, respectively. The percentages of tumor-
bearing mice after repeated doses of 300, 450, or 600 mg/kg DCA was 48, 45, and 32%,
respectively. In control mice receiving ethanol, the percentage of tumor-bearing mice observed
was 12%. The inverted dose response observed in mice under the repeated dosing regimen may
have been the result of localized cellular toxicity, which prevented initiated cells from
progressing to papilloma (Robinson et al., 1989). The association between this cellular injury
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and the increased incidence of carcinomas in animals receiving repeated doses is uncertain and
needs to be investigated (Robinson et al., 1989).
Dose-response concordance
The in vitro studies were positive for genotoxicity or mutagenicity at concentrations
ranging from 0.001 to 1,000 ug/plate, and indicate that point mutations are the most consistent
type of genetic alteration induced by 1,2,3-trichloropropane and are detectable above
background and at lower concentrations than the chromosomal damage.
La et al. (1995) characterized the formation of DNA adducts in various organs in both
B6C3F1 mice and F344/N rats, and found high concentrations of DNA adducts at 6 hours
postadministration in the tumor-forming organ tissues, including the forestomach, kidney,
pancreas, and liver, in male rats at 3 or 30 mg/kg-day and in the forestomach, liver, lung, and
kidney of male mice at 6 or 60 mg/kg-day, from the NTP (1993) study. The formation of DNA
adducts displayed a dose-dependent increase in the same organs that displayed a similar dose-
dependent increase in tumor incidence from the NTP (1993) study.
The binding of 1,2,3-trichloropropane or related metabolites to DNA increased with
multiple doses of 30 mg/kg-day administered 24 hours apart (Weber and Sipes, 1990). The
binding to DNA did not increase after the second dose, but was approximately doubled after the
third dose. Polyploidy was apparent in the hepatocytes of male albino rats dosed with 0.8 and
2.16 mg/L for 2 hours (Belyaeva et al., 1974), and a dose-dependent increase in DNA strand
breaks was evident in hepatocytes from F344/N rats at 30-100 mg/kg (Weber and Sipes, 1991)
and in kidney cells from male Wistar rats at >375 mmol/kg (Lag et al., 1991). A dose-dependent
increase in the incidence of tumors was observed in rats dosed with 3-30 mg/kg-day and in mice
dosed with 6-60 mg/kg-day (NTP, 1993). The in vivo data demonstrate an increase in DNA-
binding capability, DNA strand breaks, and DNA adducts at doses of 1,2,3-trichloropropane that
are similar to the dose levels administered in the NTP (1993) bioassay in which an increased
incidence of tumors in multiple organs was observed at all dose levels tested.
Temporal relationship
The temporal relationship for mutagenicity and tumorigenicity has not been adequately
studied. However, data indicate that metabolism of 1,2,3-trichloropropane to its metabolites is a
necessary event in the mutagenic mode of action. 1,2,3-Trichloropropane metabolism follows
three potential routes, each of which involves GSH at different steps in the metabolism process.
Two primary routes of metabolism involve the formation of an episulfonium ion, while the third
involves the intermediate metabolite, DCA (Mahmood 1991), which is a reported mutagen
(Weber and Sipes, 1992).
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In addition, there are in vitro and in vivo data that demonstrate metabolism of 1,2,3-
trichloropropane, followed by binding of reactive metabolites to DNA, and the ultimate
formation of DNA adducts. This sequence of events has been demonstrated in the bacterial and
mammalian cell systems assays in which activation with an S9 fraction from chemically-induced
rat or hamster livers may be necessary for genotoxicity and potential mutagenicity (Tafazoli and
Kirsch-Volders, 1996; Lag et al., 1994; NTP, 1993; Ratpan and Plaumann, 1988; von der Hude
et al., 1987; Haworth et al., 1983; Kier, 1982; Shell Oil Co., 1982, 1979; Stolzenburg and Hine,
1980).
Evidence for the direct interaction of 1,2,3-trichloropropane metabolites with DNA,
RNA, and hepatic proteins was observed 4 hours following i.p. administration of 1,2,3-
trichloropropane (Weber and Sipes, 1990). This investigation demonstrates the ability of 1,2,3-
trichloropropane metabolites to bind to hepatic DNA, RNA, and protein, and that the binding
increases with multiple doses. DNA strand breaks were evident in the extracted hepatic DNA of
male F344/N rats administered 1,2,3-trichloropropane by i.p. injection, thus demonstrating that
1,2,3-trichloropropane metabolites cause the formation of DNA strand breaks (Weber and Sipes,
1991).
La et al. (1995) characterized the formation of DNA adducts in various organs in both
B6C3F1 mice and F344/N rats 6 hours following a single dose of 1,2,3-trichloropropane, and
found high concentrations of DNA adducts in the tumor-forming organ tissues, including the
forestomach, kidney, pancreas, and liver in male rats and the forestomach, liver, lung, and
kidney of male mice.
Biological plausibility and coherence
Mutagenicity as a mode of action for carcinogenicity in humans is generally accepted and
is a biologically plausible mechanism for tumor induction. The formation of DNA adducts in
organs that also displayed an increase in the tumor incidence in rats and mice indicates
coherence of the effects and is evidence supporting a mutagenic mode of action (Table 4-29).
The proposed mode of action includes bioactivation of 1,2,3-trichloropropane leading to DNA
adduct formation, followed by the induction of mutations in cancer-related genes, and eventually
resulting in tumor formation. However, the formation of DNA adducts of 1,2,3-trichloropropane
in tissues other than those where tumors formed (La et al., 1995) is an area of uncertainty
associated with the suggested mutagenic mode of action. DNA adduct formation for some tumor
types may be necessary but not sufficient for the induction of tumors and is not an uncommon
occurrence as DNA adducts of known direct-acting carcinogens (e.g., benzo[a]pyrene) have been
observed in tissues where tumors were not found. The formation of DNA adducts in nontumor
forming tissues and organs may signify that DNA adducts by themselves are insufficient to cause
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tumors or that the increased mortality in the rats and increased tumor incidence in other organs
precluded tumor formation in the nontumor-forming organs.
In addition, the formation of episulfonium ions and subsequent DNA binding by similar
compounds supports a mutagenic mode of action of carcinogenesis for 1,2,3-trichloropropane.
Specifically, 1,2-dibromoethane spontaneously forms the episulfonium ion, thiiranium,
following conjugation with GSH, which may then bind to DNA (U.S. EPA, 2004). DNA binding
of metabolites of 1,2-dibromoethane and DBCP has also been demonstrated in vitro in calf
thymus DNA (Inskeep and Guengerich, 1984). Binding of metabolites of 1,2-dibromopropane in
vivo in rats following i.p. injection (Kim and Guengerich, 1990) also provides support for the
proposed mode of action. Holme et al. (1989) found that DBCP induced DNA damage in liver
cells at concentrations much lower than concentrations that resulted in cytotoxicity and bacterial
(S. typhimuriuni) mutagenicity.
4.7.3.3. Other Possible Modes of A ction
Data are not available to make a determination about whether other modes of action, such
as cytotoxicity with tissue repair due to DNA degradation or disruption of cell signaling, are
associated with the carcinogenic activity of 1,2,3-trichloropropane. However, the mode of action
of 1,2,3-trichloropropane-induced forestomach tumors may include promotion. Specifically, the
use of corn oil as a vehicle for the administration of carcinogenic chemicals has been shown to
increase the incidence and severity of epithelial cell proliferation of the forestomach in rats
(Ghanayem et al., 1986). However, no data demonstrating proliferation in the forestomach
following corn oil gavage administration of 1,2,3-trichloropropane are available.
4.7.3.4. Conclusions About the Hypothesized Mode of Action
The proposed mode of action for 1,2,3-trichloropropane tumorigenicity involves
mutagenicity via reactive metabolites. The data supporting a mutagenic mode of action include:
• Mutagenic response in short-term bacterial assays (with microsomal activation),
indicative of base-pair substitutions and frameshift mutations, and induced
chromosomal damage, gene mutations, DNA breakage, micronucleus formation,
and enhanced DNA viral transformation in mammalian cell assays;
• Covalent binding of 1,2,3-trichloropropane metabolites to hepatic protein, DNA,
and RNA and the induction of DNA strand breaks in the hepatocytes of rats
following in vivo exposure, and induced wing spot formation in the SMART in D.
melanogaster;
• Dose-dependent formation of DNA adducts, including the major adduct S-[l-
(hydroxymethyl)-2-(N7-guanyl)ethyl]glutathione, in various organs of both
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B6C3F1 mice and F344/N/N rats, with DNA adducts present in tumor-forming
organs of male rats and mice; and
• Dose-dependent increase in the formation of the intermediate metabolite, and
reported mutagen and tumor initiator, DCA, and the formation of reactive
episulfonium ion metabolites.
The available in vitro and in vivo data also indicate that metabolites of 1,2,3-
trichloropropane have an affinity for certain nucleic acids and a capacity to form DNA adducts,
although in vivo assays that directly measure mutagenicity are unavailable. For example, regular
test batteries for different genetic endpoints in vitro and, especially, in vivo, such as
micronucleus formation, chromosomal aberrations, unscheduled DNA synthesis, sister chromatid
exchanges, Comet assay, and DNA adduct analysis, are limited.
A number of assays have tested nonpositive for DNA reactivity and mutagenicity of
1,2,3-trichloropropane. 1,2,3-Trichloropropane tested nonpositive in studies investigating
mutagenic potential (without microsomal activation), micronucleus formation, unscheduled
DNA synthesis, and chromosomal damage in vitro. Nonpositive in vivo assays included
dominant lethal induction and micronucleus formation. Despite these nonpositive results, other
chlorinated aliphatics, while showing a weak response, failed to induce a clear dose-dependent
increase in miconucleus formation, which suggests that the Comet assay, in which 1,2,3-
trichloropropane induced DNA damage, may be a more suitable and sensitive method for this
chemical class (Tafazoli and Kirsch-Volders, 1996). In addition, Crebelli et al. (1999) stated that
micronucleus formation in mouse bone marrow is weakly sensitive to the genotoxic effects
induced by halogenated hydrocarbons in other test systems, and a negative bone marrow
micronucleus assay should not offset the consistently positive in vitro results (Dearfield and
Moore, 2005).
Is the hypothesized mode of action sufficiently supported in test animals!
The covalent binding of bioactivated 1,2,3-trichloropropane to hepatic DNA, RNA, and
protein was evident in male F344/N rats (Weber and Sipes, 1990). A dose-dependent increase in
the amount of 1,2,3-trichloropropane equivalents bound to hepatic DNA and protein was
demonstrated.
Weber and Sipes (1991) administered i.p. injections to male F344/N rats. Following the
extraction of hepatic DNA, they demonstrated that 1,2,3-trichloropropane, or its metabolites,
caused the formation of DNA strand breaks.
La et al. (1995) characterized the formation of DNA adducts in various organs both in
B6C3F1 mice and F344/N rats, and found high concentrations of DNA adducts in organ tissues
in which tumor formation was observed by NTP (1993). The investigators characterized the
DNA adduct, indicating that a single, major 1,2,3-trichloropropane-derived DNA adduct was
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formed irrespective of the tissue type, and determined the adduct to be S-[l-(hydroxymethyl)-2-
(N7-guanyl)ethyl]glutathione. The formation of this adduct was detected in the forestomach,
glandular stomach, kidney, liver, pancreas, and tongue of F344/N rats, and in the forestomach,
glandular stomach, kidney, and liver of B6C3F1 mice. The concentrations of adduct formed in
the target organs showed correlation with the tumor incidence from the NTP (1993) study.
The target organs of 1,2,3-trichloropropane toxicity (liver, kidney, forestomach, and
intestine) also contain the highest concentration of covalently-bound 1,2,3-trichloropropane and
related metabolites (Mahmood et al., 1991), which supports a role for metabolic activation and
binding in the early stages of carcinogenesis.
In addition to the experimental data for 1,2,3-trichloropropane, halogenated propanes, as
a class of compounds, are generally considered to be mutagenic (Lag et al., 1994; Ratpan and
Plaumann, 1988).
Is the hypothesized mode of action relevant to humans!
The postulated key events, the metabolism of 1,2,3-trichloropropane to a DNA-reactive
compound and the alteration of the genetic material leading to tumor-inducing mutations, are
both possible in humans. Mutagenicity as a mode of action for carcinogenicity in humans is
generally accepted and is a biologically plausible mechanism for tumor induction. The
toxicokinetic and toxicodynamic processes that would enable reactive metabolites to produce
mutations in animal models are biologically plausible in humans.
Which populations or life stages can be particularly susceptible to the hypothesized mode of
action!
According to the Supplemental Guidance for Assessing Susceptibility from Early-Life
Exposure to Carcinogens (U.S. EPA, 2005b), children exposed to carcinogens with a mutagenic
mode of action are assumed to have increased early-life susceptibility. The Supplemental
Guidance (U.S. EPA, 2005b) recommends the application of age-dependent adjustment factors
(ADAFs) for carcinogens that act through a mutagenic mode of action. Given the weight of the
available evidence, 1,2,3-trichloropropane acts through a mutagenic mode of carcinogenic action
and the ADAFs should be applied.
4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES
4.8.1. Possible Childhood Susceptibility
No studies are available that address the possible adverse effects of 1,2,3-
trichloropropane in children. However, there is evidence that 1,2,3-trichloropropane is
mutagenic and, therefore, may act through a mutagenic mode of action for carcinogenicity. In
accordance with the Supplemental Guidance (U.S. EPA, 2005b), the mutagenic mode of
carcinogenic action for 1,2,3-trichloropropane would indicate an increased carcinogenic
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susceptibility for early-life exposures. Although developmental toxicity studies for 1,2,3-
trichloropropane are unavailable, developmental toxicity is a concern due to the genotoxicity of
1,2,3-trichloropropane and the possibility for genetic damage to the germ cells of the Fl
generation that could be transmitted to the F2 generation. In addition, the two-generation
reproductive assessment by gavage indicates that the developing fetus may be a target of toxicity
due to an observed reduction in the number of live mouse pups/litter and in the proportion of
male pups born alive following oral exposure.
4.8.2. Possible Gender Differences
The extent to which men and women differ in susceptibility to 1,2,3-trichloropropane is
unknown. However, some data may exist that imply a difference between male and female rats
in their response to inhalation of the compound. For example, 15/15 male CD rats exposed to
1,2,3-trichloropropane via inhalation (6 hours/day, 5 days/week, for 13 weeks) at a concentration
of 50 ppm displayed histopathological lesions in the liver, while 0/15 females displayed this
effect at the same concentration (Johannsen et al., 1988). A clear-cut dose-dependent response
in this effect was seen in the males, but females showed no response. The biological
significance of this finding for lower doses and for other species is unclear.
4.8.3. Other
GSH appears to be necessary for the formation of the DNA adduct, S-[l-
(hydroxymethyl)-2-(N7-guanyl)ethyl]glutathione. Individuals with a GSH deficiency may be
less susceptible to the carcinogenic effects of 1,2,3-trichloropropane. Conversely, individuals
with increased expression of GSH may have an increased susceptibility to the genotoxic effects
of 1,2,3-trichloropropane.
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5. DOSE RESPONSE ASSESSMENT
5.1. CHRONIC ORAL REFERENCE DOSE (RfD)
5.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification
Data on the health effects of oral exposure to 1,2,3-trichloropropane in humans are not
available. The database of chronic and subchronic animal studies included a 2-year gavage
study in F344/N rats and B6C3F1 mice (Irwin et al., 1995; NTP, 1993), a 90-day gavage study in
Sprague-Dawley rats (Merrick et al., 1991), a 90-day drinking water study in Sprague-Dawley
rats (Villeneuve et al., 1985), a 17-week gavage study in F344/N rats (NTP, 1993; Hazleton
Laboratories, 1983a), a 17-week gavage study in B6C3F1 mice (NTP 1993; Hazleton
Laboratories, 1983b), and a two-generation reproductive/fertility assessment in Swiss CD-I mice
(NTP, 1990). The subchronic (i.e., 90-day study or less) study data were not considered in the
selection of a principal study for deriving the chronic RfD because the database contains reliable
dose-response data from a chronic study of two species and a two-generation reproductive
assessment. The data from the subchronic studies were, however, used to corroborate the
findings of the chronic studies.
The dose-dependent, noncancer effects associated with oral exposure to 1,2,3-
trichloropropane include increased liver weights (subchronic and chronic); increased kidney
weights (subchronic and chronic); hepatic, renal, myocardial, lung, and nasal turbinate epithelial
necrosis (subchronic); decreased synthesis of pseudocholinesterase (subchronic); decreased ALT
and 5'-nucleotidase levels (chronic); increased ALT, AST, and SDH levels (subchronic and
chronic); increased hepatic aminopyrine demethylase and aniline hydroxylase activity
(subchronic); elevated creatine kinase (chronic); decreased number of pregnancies per fertile
pair, reduced number of live pups/litter; and decreased proportion of male pups born alive (NTP,
1993, 1990; Merrick et al., 1991; Villeneuve et al., 1985).
The NTP (1993) study was selected as the principal study because it was a well-designed
chronic study, conducted in both sexes of 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. Increased liver weight was selected as the critical effect because liver
toxicity appeared to be the most sensitive effect. There is evidence of hepatocellular damage,
including increased incidence of hepatocellular necrosis and decreased synthesis of pseudo-
cholinesterase, from the subchronic NTP (1993) studies, and increased serum concentrations of
hepatocellular enzymes (ALT and SDH), decreased concentration of 5'-nucleotidase, and
increased incidence of histopathologic liver lesions, including hepatocellular necrosis, from the
chronic NTP (1993) studies. Increased liver weight was selected as the critical effect because it
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represents the most sensitive effect observed in the liver and occurs early in the process of liver
toxicity associated with oral exposure to 1,2,3-trichloropropane. The designation of the liver as
a target organ for noncancer effects is also supported by the mechanistic data from Weber and
Sipes (1990) that demonstrated the binding of 1,2,3-trichloropropane metabolites to hepatic
proteins and nucleic acids.
Other possible critical effects include kidney, respiratory, myocardial, or reproductive
toxicity. In the kidney, an increase in organ weight after both subchronic and chronic exposure
was accompanied by an increased severity of nephropathy and incidence of renal tubule
hyperplasia in rats in the chronic NTP (1993) study. The subchronic NTP (1993) study also
demonstrated epithelial necrosis in the nasal turbinates of rats and regenerative lung lesions in
mice. Hyperplasia was also observed in the forestomach (basal cell and squamous) and pancreas
(acinar) of rats and in the forestomach (squamous) of mice following chronic exposure to 1,2,3-
trichloropropane (NTP, 1993). Merrick et al. (1991) showed an increased incidence of
inflammation-associated myocardial necrosis in rats, and increased levels of creatine kinase were
apparent in the chronic NTP study. NTP (1990) demonstrated a decrease in the number of
pregnancies per fertile pair, a reduction in the number of live pups/litter, and a decrease in the
proportion of male pups born alive. Although the liver appeared to be the most sensitive
indicator of 1,2,3-trichloropropane-induced toxicity, RfDs for the changes in kidney weight,
fertility, and pups/litter were also derived for comparison purposes.
5.1.2. Methods of Analysis—Including Models
Benchmark dose (BMD) modeling was conducted using the EPA's Benchmark Dose
Software (BMDS, version 1.4.1) to analyze the changes in liver and kidney weight, fertility, and
number of pups/litter associated with chronic exposure to 1,2,3-trichloropropane (see Appendix
B for details). The software was used to calculate potential points of departure (PODs) for
deriving the chronic RfD by estimating the effective dose at a specified level of response
(BMDX) and its 95% lower bound (BMDLX). For continuous endpoints, the Benchmark Dose
Technical Guidance Document (U.S. EPA, 2000c) states that a minimal level of change in an
endpoint that is generally considered to be biologically significant may be used to define the
benchmark response (BMR). For analysis of absolute and relative liver and kidney weight
changes in both rats and mice, a BMR of 10% was selected, as it is analogous to the 10% change
in body weight used to identify maximum tolerated doses (MTDs). A BMR of 1 standard
deviation (SD) from the control mean was also included for comparisons with other chemicals in
the IRIS database that affect absolute and relative liver weights. In the reproductive toxicity
study, a 10% change in fertility rate was selected as the BMR, in accordance with the Benchmark
Dose Technical Guidance Document (US EPA, 2000c) and a 1% change in mean live pups/litter
for the 4th and 5th litters was selected as the BMR due to the frank toxicity of this endpoint.
79
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Absolute and relative liver weight changes were also modeled using a BMR of 1 SD, as
recommended by the Benchmark Dose Technical Guidance Document (US EPA, 2000) for
continuous endpoints for comparison purposes.
Table 5-1 presents BMDs and their corresponding lower 95% confidence limits
(BMDLs) for each observed effect that was considered and amenable to modeling. The
candidate BMD for each endpoint was identified by comparing the BMDS outputs from the
fitted models for each of the four data sets: male rats, female rats, male mice, and female mice.
Adequacy of each model fit was determined by assessing the %2 goodness-of-fit statistic using a
significance level of a = 0.1. The best-fitting models were selected from those exhibiting
adequate fit by considering the Akaike Information Criterion (AIC) value of each model and
how well the model visually fit the data (see Appendix B).
Table 5-1. Candidate BMDs for chronic and reproductive effects associated
with oral exposure to 1,2,3-trichloropropane
Endpoint
Absolute liver weight
Absolute liver weight
Relative liver weight
Relative liver weight
Absolute kidney weight
Relative kidney weight
Fertility generating 4th litter
Fertility generating 5th litter
Live pups/litter- 4th litter
Live pups/litter- 5th litter
Species/sex
Rat/male
Rat/male
Rat/male
Rat/male
Rat/female
Rat/male
Mice
Mice
Mice
Mice
Model
Hill
Hill
Hill
Hill
Hill
Hill
Log-probit (slope > 1)
Probit
Polynomial
Polynomial
BMDa
(mg/kg-d)
3.8
3.2
5.5
3.2
9.0
10.5
52.6
31.2
13.8
13.6
BMDLa
(mg/kg-d)
1.6
1.4
3.1
1.8
3.4
6.4
37.3
23.3
3.2
5.6
BMR
10% change in
mean organ
weight
1 SD
10% change in
mean organ
weight
1 SD
10% change in
mean organ
weight
10% change in
mean organ
weight
10% change in
fertility rate
10% change in
fertility rate
1% change in
mean live
pups/litter
1% change in
mean live
pups/litter
aBMDs and BMDLs from the best-fitting models for each endpoint (see Appendix B).
80
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The increases in both absolute and relative liver weights in male rats were fit adequately
by the Hill model. Increase in liver weight was chosen as the critical effect and, more
specifically, absolute liver weight was selected to represent this increase in liver weight because
it was the most sensitive endpoint. The selection of the liver as the critical target organ and
increased liver weight as the critical effect representative of this hepatotoxicity is supported by
increased serum liver enzyme levels, increased incidence of hepatic necrosis, and decreased
pseudocholinesterase. Increased liver weight was selected as the critical effect because it
represents the most sensitive endpoint in a spectrum of liver effects and occurs early in the
process of liver toxicity associated with oral exposure to 1,2,3-trichloropropane.
The 10% increase in absolute liver weight is a more sensitive endpoint than the 1%
decrease in the number of live pups/litter in the 4th and 5th litters. Statistically significant
reductions, relative to controls, in the number of live pups/litter were observed in mice in the
second through the fifth breedings at the highest dose tested (120 mg/kg-day) and at the fifth
breeding at a dose of 60 mg/kg-day. When comparing the BMD modeling results, the lower
POD identified for the increase in absolute liver weight (BMDLi0% of 1.6 mg/kg-day) is thus
thought to represent a more sensitive endpoint than the decrease in the number of live pups/litter
(BMDLio/0 of 3.2 mg/kg-day), even though the decrease in live pups/litter is considered a frank
effect.
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 2-year gavage study in Fischer rats (NTP, 1993) and increased absolute
liver weight in males as the principal study and critical effect, respectively, for deriving the
chronic RfD for 1,2,3-trichloropropane. The dose-response relationships between oral exposure
to 1,2,3-trichloropropane and impaired fertility in CD-I mice are also suitable for deriving a
chronic RfD, but these endpoints yielded higher BMDLs than the selected critical effect
(absolute liver weight) and corresponding BMDL.
The BMDL corresponds to the 95% lower bound on dose associated with a 10% increase
in mean absolute liver weight. The BMDio estimated from the Hill model using absolute liver
weight change in male F344/N rats is 3.8 mg/kg-day and the corresponding BMDLio is 1.6
mg/kg-day. A BMR of a 10% change in the mean was used in the modeling of absolute and
relative liver and kidney weight because the Benchmark Dose Technical Guidance Document
(U.S. EPA, 2000c) recommends using the minimal amount of change in the endpoint that is
considered to be biologically significant to define the BMR. Duration-adjustment of the PODs
was done to approximate continuous daily exposures by multiplying the BMDio and BMDLio by
(5 days)/(7 days) = 0.71; resulting in a BMDADJ of 2.70 mg/kg-day and a BMDLADJ of 1.1
mg/kg-day.
81
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5.1.3. Chronic RfD Derivation—Including Application of Uncertainty Factors (UFs)
A BMDLAoj of 1.1 mg/kg-day for increased absolute liver weight in male rats
chronically exposed to 1,2,3-trichloropropane by gavage (NTP, 1993) was used as the POD to
calculate the chronic RfD. A total UF of 300 was applied to this effect level: 10 for uncertainty
associated with interspecies differences (UFA: animal to human), 10 to account for intraspecies
variation (UFn: human variability), and 3 for database deficiencies (UFo: database deficiency).
The rationale for application of these UFs is described below.
A 10-fold UFA was used to account for uncertainty in extrapolating from laboratory
animals to humans (i.e., interspecies variability) because information was unavailable to
quantitatively assess toxicokinetic or toxicodynamic differences between animals and humans.
A 10-fold UFH was used to account for variation in susceptibility among members of the
human population (i.e., interindividual variability) because information is unavailable to predict
potential variability in human susceptibility.
An UFs was not needed to account for extrapolation from subchronic-to-chronic
exposure because a chronic study was used to derive the chronic RfD.
An UFL 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 benchmark dose
modeling. In this case, a BMR of a 10% change in absolute liver weight was selected under an
assumption that it represents a minimal biologically significant change.
A 3-fold UFD was selected to account for database deficiencies. The database of chronic
and subchronic animal studies includes a 2-year gavage study in F344/N rats and B6C3F1 mice
(Irwin et al., 1995; NTP, 1993), a 90-day gavage study in Sprague-Dawley rats (Merrick et al.,
1991), a 90-day drinking water study in Sprague-Dawley rats (Villeneuve et al., 1985), a 17-
week gavage study in F344/N rats (NTP, 1993; Hazleton Laboratories, 1983a), a 17-week
gavage study in B6C3F1 mice (NTP 1993; Hazleton Laboratories, 1983b), and a two-generation
reproductive/fertility assessment in Swiss CD-I mice (NTP, 1990). A threefold UFo for
database deficiencies was applied because the database lacks information on developmental
toxicity associated with 1,2,3-trichloropropane. In addition, the two-generation reproductive
toxicity study indicates that the developing fetus may be a target of toxicity. The lack of a
reproductive toxicity study that extends beyond two generations and the absence of a
developmental toxicity study are of particular concern due to the genotoxicity of 1,2,3-
trichloropropane, which may mean that any resulting genetic damage to the germ cells of the Fl
generation may not be detected until the F2 generation.
82
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The chronic RfD for 1,2,3-trichloropropane was calculated as follows:
RfD = BMDLADJ + UF
= 1.1 mg/kg-day - 300
= 4 x 10"3 mg/kg-day (rounded to one significant figure)
5.1.4. Chronic 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, some of which were considered candidates
for chronic RfD derivation, from subchronic, chronic, and reproductive toxicity studies. The
health effects from the subchronic NTP (1993) study included decreased synthesis of
pseudocholinesterase and hepatic necrosis. The health effects from the chronic NTP study
included increased absolute and relative liver and kidney weights, and the effects from the NTP
reproductive toxicity study included a decrease in the number of pregnancies per fertile pair and
a decrease in the number of live pups per litter.
Figure 5-2 presents the POD, applied uncertainty factors, and candidate chronic RfDs for
additional endpoints that were modeled using the EPA's BMDS (version 1.4.1) and which
appear in Table 5-1. This figure is intended to provide information on additional health effects
associated with varying levels of 1,2,3-trichloropropane exposure.
PODs and candidate chronic RfDs that could be derived from the additional health effects
identified in Table 5-1 are presented in Figure 5-1 to allow a comparison with the critical effect.
For increased relative liver weight, increased absolute and relative kidney weights, decreased
fertility generating the 4th and 5th litters, and decreased live pups/litter, the uncertainty factors
applied were a 10-fold UF to account for uncertainty in extrapolating from laboratory animals to
humans, a 10-fold UF to account for variation in susceptibility among members of the human
population, and a threefold UF for database deficiencies.
A change in liver weight is the most sensitive endpoint in a spectrum of liver effects
following oral exposure to 1,2,3-trichloropropane, and increased serum liver enzymes and an
increased incidence of hepatic necrosis, as well as a decrease in pseudocholinesterase, all
indicators of liver damage, provide support for the selection of the liver as the critical target
organ. The dose-response relationships between oral exposure to 1,2,3-trichloropropane and
impaired fertility in CD-I mice are also suitable for deriving a chronic RfD, but yield higher
BMDLs than the selected critical effect. Thus, the RfD based on absolute liver weight is likely
to be protective of any impaired fertility effects. 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 2-year gavage study in Fischer
rats (NTP, 1993) and increased absolute liver weight in males as the principal study and critical
effect, respectively, for deriving the chronic RfD for 1,2,3-trichloropropane.
83
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140
120
100
% 80
60
40
20
[
<
]
>
decreased synthesis of
pseudocholinesterase;
male rats; (NTP, 1 993)
[
<
]
decreased synthesis of
pseudochol i nesterase ;
M female rats; (NTP,
| 1993)
>
D
<
o
D
o hepatic necrosis; male
rats; (NTP, 1993)
P
>
[
hepatic necrosis;
female rats; (NTP,
1993)
ONOAEL
D LOAEL
]
increase absolute liver
weight; male rats;
(NTP, 1993)
[
<
increase relative liver
The vertical
lines represent
the range of
doses tested in
a given study.
]
> [
g
Chr
]
o
^
° increase absolute
kidney weight; male
rats; (NTP, 1993)
[
<
<
IT1
>
<
>
}
>
_a> ^
CO * 5-
<
decrease in
pregnancies per fertile
pair at 4th and 5th
breedings; (NTP,
1990)
o> reduction in live pups
o per litter in 1 st through
i" 4th breedings; (NTP,
| 1990)
(D
reduction in live pups
per litter in 5th
breeding; (NTP, 1990)
Figure 5-1. Exposure-response array of selected subchronic, chronic, and reproductive toxicity effects.
84
-------
100
10
re
73
0)
0)
tn
o
•a
0.1
0.01
0.001
0.0001
Increased
relative liver
weight, <$ rats
(NTP, 1993)
Increased
relative
kidney
weight, <$ rats
(NTP, 1993)
Fertility
generating
the 4th litter,
mice (NTP,
1990)
Live
pups/litter -
4th litter, mice
(NTP, 1990)
Fertility
generating 5th
litter, mice
(NTP, 1990)
Increased
absolute liver
weight, c? rats
(NTP, 1993)
Increased
absolute kidney
weight, $ rats
(NTP, 1993)
Live
pups/litter -
5th litter, mice
(NTP, 1990)
Point of Departure
RfD
UF, animal-to-human
UF, human variability
UF, database
Figure 5-2. PODs for selected endpoints (with critical effect circled) from Table 5-1 with corresponding applied UFs and derived
candidate chronic oral RfDs.
85
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5.1.5. Previous Oral Assessment
The previous IRIS assessment for 1,2,3-trichloropropane was entered into the database in
1987 and contains an oral chronic RfD of 6 x 10"3 mg/kg-day. The chronic RfD was based on a
duration-adjusted NOAEL of 5.71 mg/kg-day for alterations in clinical chemistry and reduced
red blood cell mass in female F344/N rats following a 17-week gavage exposure (NTP, 1983;
Hazleton Laboratories, 1983a). A total UF of 1000 was used to account for interspecies
extrapolation, human variability, and extrapolation from a subchronic study. This assessment
was last updated in 1990 before the publication of the NTP (1993) Technical Report used for this
assessment.
5.2. CHRONIC INHALATION REFERENCE CONCENTRATION (RfC)
5.2.1. Choice of Principal Study and Critical Effect—with Rationale and Justification
Inhalation studies of 1,2,3-trichloropropane in humans are limited. A single report
(Silverman et al., 1946) on the effects in humans found that all subjects (12/sex) experienced
irritation (eyes, throat, and odor) following 15-minute exposures to air concentrations of 100
ppm trichloropropane (isomer and purity not reported). The database of inhalation toxicity
studies in animals includes two 2-week studies submitted to EPA by Miller et al. (1987a, b), a 4-
week range-finding study, two 13-week studies, and two single-generation reproductive
assessments (Johannsen et al., 1988; Biodynamics, Inc., 1979).
Increased organ weights and histopathological lesions in rodents have been associated
with subchronic inhalation exposure to 1,2,3-trichloropropane. Concentration-dependent
increases in absolute and relative liver weight were observed in males and female rats
(Johannsen et al., 1988; Miller et al., 1987a; Biodynamics, Inc., 1979). An increase in relative
lung weight was also observed in female rats (Biodynamics, Inc., 1979). The histology data
demonstrated that 1,2,3-trichloropropane is both a local irritant affecting the nasal epithelium
(Miller et al., 1987a, b) and a systemic toxicant producing effects remote from the site of entry,
including peribronchial lymphoid hyperplasia, hepatocellular hypertrophy, and extramedullary
hematopoiesis (Johannsen et al., 1988; Biodynamics, Inc., 1979).
Johannsen et al. (1988) also conducted two single-generation reproductive toxicity
studies using 10 male and 20 female CD rats/group. Female rats exhibited decreased mating
performance at 5 ppm, where 16 out of 20 females mated, and at 15 ppm, where 10 out of 20
females mated, compared with 17 out of 20 mated females in the control group. The decrease in
the proportion of females that mated was found to be statistically significant (p < 0.02) at 15 ppm
in the Fisher Exact Test conducted by EPA.
The Johannsen et al. (1988) study was selected as the principal study. The number of test
animals allocated among five dose levels and an untreated control group was acceptable, with
examination of appropriate toxicological endpoints in both sexes. The critical effect selected for
the derivation of the chronic RfC is the development of peribronchial lymphoid hyperplasia in
86
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the lungs of CD rats, with a NOAEL of 1.5 ppm and a LOAEL of 5 ppm, which is supported by
the occurrence of this effect in both male and female rats and the possible correlation between
the hyperplasia and the observed increased lung weight. Peribronchial lymphoid hyperplasia,
also defined as lymphoid hyperplasia of the bronchus-associated lymphoid tissue, is
histologically characterized by the presence of hyperplastic lymphoid follicles with reactive
germinal centers distributed along the bronchioles and bronchi (Howling et al., 1999; Myers and
Kurtin, 1995; Fortoul et al., 1985; Yousem et al., 1985). A NOAEL of 5 ppm and a LOAEL of
15 ppm were identified for the increase in lung weight.. Although an increase in liver and
kidney weights was apparent, lesions and serum enzyme levels indicative of liver and kidney
damage were not evident. The only pathological endpoint observed in the liver was
hepatocellular hypertrophy in male rats at 5, 15, and 50 ppm. The observed liver effects occurred
at doses higher than those found for lung effects and , as such, were not considered further for
the derivation fo the POD. The hematopoiesis of the spleen in female rats was not considered
biologically relevant as there was no change in the clinical chemistry and hematology
parameters.
5.2.2. Methods of Analysis—Including Models
Benchmark dose (BMD) modeling was conducted using the EPA's BMDS (version
1.4.1) to analyze the increased incidence of peribronchial lymphoid hyperplasia in CD rats and,
for purposes of comparison, the decreased mating performance in female CD rats (see Appendix
C for details). The software was used to calculate potential PODs for deriving the chronic RfC
by estimating the effective dose at a specified level of response (BMCX) and its 95% lower
bound (BMCLX). For dichotomous endpoints, the Benchmark Dose Technical Guidance
Document (US EPA, 2000c) states that an excess risk of 10% is the generally recommended
BMR in the absence of any specific data on the change in the critical effect that would be
considered biologically significant. Therefore, for the analysis of increased incidence of
peribronchial lymphoid hyperplasia, a BMR of 10% is selected. In modeling the decreased
mating performance, a BMR of 10% was selected, in accordance with the Benchmark Dose
Technical Guidance Document (US EPA, 2000c).
Table 5-2 presents benchmark concentrations (BMC) and the corresponding lower 95%
confidence limits (BMCLs) for each observed effect that was considered and amenable to
modeling. The candidate BMCs for each endpoint were identified by examining the BMDS
outputs from the fitted models for each of the data sets. Adequacy of model fit was determined
by evaluating the %2 goodness-of-fit statistic using a significance level of a = 0.1. Of the models
that exhibited adequate fit, the best-fitting models were selected based on AIC values and how
well the model fit the data visually (see Appendix C).
87
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Table 5-2. BMD modeling results used in the derivation of the RfC
Endpoint
Peribronchial lymphoid
hyperplasia
Decreased mating
performance
Species/sex
Rat/male
Rat/female
Model
Log-logistic (slope > 1)
Log-probit (slope > 1)
BMC (ppm)
1.6
4.5
BMCLa
(ppm)
0.84
3.0
BMR
10% extra risk
10% extra risk
aBMCs and BMCLs from the best-fitting models for each endpoint (see Appendix B).
The 13-week inhalation study in CD rats (Johannsen et al., 1988) was selected as the
principal study and increased incidence of peribronchial lymphoid hyperplasia in males was
selected as the critical effect for deriving the chronic RfC for 1,2,3-trichloropropane. The dose-
response relationship between inhalation exposure to 1,2,3-trichloropropane and decreased
mating performance in female CD rats is also suitable for deriving a chronic RfC, but this
relationship yields higher BMCLs than the critical effect selected.
The BMCL corresponds to the 95% lower bound on the dose associated with a 10%
increase in the incidence of peribronchial lymphoid hyperplasia. The BMDio calculated from the
log-logistic model (slope > 1) using the incidence of peribronchial lymphoid hyperplasia in male
CD rats is 1.6 mg/m3 and the corresponding BMCLio is 0.84 mg/ m3.
Human equivalent concentrations (HECs) were calculated from the candidate PODs.
PODs were converted to mg/m3, adjusted to continuous exposure (7 days/week, 24 hours/day),
and multiplied by a dosimetric adjustment factor (DAF) to calculate the HEC. A DAF is a ratio
of animal and human physiologic parameters. The specific DAF used depends on the nature of
the contaminant (particle or gas) and the target site (e.g., respiratory tract or remote to the portal-
of-entry).
The RfC methodology (U.S. EPA, 1994b) classifies gases into three categories based on
their water solubility and reactivity with respiratory tract tissue. 1,2,3-Trichloropropane is
considered a category 2 gas because it is relatively insoluble in water and demonstrates systemic
toxicity. For category 2 gases, HEC values are calculated using methods for both category 1
(portal-of-entry effects) and category 3 (systemic effects) gases (U.S. EPA, 1994b). The DAF
for a category 1 gas is based on the animal-to-human ratio of the minute volume (Ve) divided by
the surface area (SA) of the region of the respiratory tract where the effect occurs. The DAF for
a category 3 gas is based on the ratio of the animal blood:gas partition coefficient (Hb/g.animai) and
the human blood:gas partition coefficient (Hb/g-human).
The critical effect for the chronic RfC is considered a systemic effect because the critical
effect is located beyond the lung tissue in the bronchus-associated lymphoid tissue. The HEC
for increased incidence of peribronchial lymphoid hyperplasia in rats exposed to 1,2,3-
trichloropropane (a category 3 gas) for 6 hours/day, 5 days/week for 13 weeks was calculated
-------
from a BMDLio of 0.84 ppm (0.84 ppm x molecular weight[147.43] / 24.45 = 5.07 mg/m3).
Conversionto a continuous exposure was accomplished as follows:
BMCLADj = BMCLio x (6 hours)/(24 hours) x (5 days)/(7 days)
= 5.07 mg/m3 x 0.25 x 0.71
= 0.90 mg/m3
The DAF for an extra-respiratory effect of a gas is the ratio of the animal/human blood:
air partition coefficients [(Hb/g)A/(Hb/g)H]. However, the human and rat blood partition
coefficients for 1,2,3-trichloropropane are not known. In accordance with the RfC methodology
(U.S. EPA, 1994b), when these partition coefficients are unknown, a ratio of 1 is used. This
allows a BMDLnEc to be derived as follows:
BMCLHEC = BMCLADJ (mg/m3) x (Hb/g)A/(Hb/g)H
= BMCLADJ (mg/m3) x 1
= 0.90 mg/m3
Application of the inhalation dosimetry methods to the incidence of peribronchial
lymphoid hyperplasia in the lung resulted in a BMCLHEc of 0.90 mg/m3.
5.2.3. Chronic RfC Derivation—Including Application of Uncertainty Factors (UFs)
The BMCLHEc value of 0.90 mg/m3 based on increased incidence of peribronchial
lymphoid hyperplasia in the lungs of male CD rats exposed to 1,2,3-trichloropropane via
inhalation (Johannsen et al., 1988) was used as the POD to derive the chronic RfC for 1,2,3-
trichloropropane. A total UF of 3000 was applied to this POD: 3 for extrapolation from rats to
humans (UFA: animal to human), 10 for consideration of intraspecies variation (UFH: human
variability), 10 for extrapolation from a subchronic to a chronic exposure (UFS), and 10 for
database deficiencies. The rationale for application of these UFs is described below. Figure 5-3
presents the POD, applied UFs, and quantified chronic RfC for the critical effect selected, an
increased incidence of peribronchial lymphoid hyperplasia in the lungs of male rats, as well as
decreased mating performance in female rats.
An UFA of 3 was selected to account for uncertainties in extrapolating from rats to
humans. This value is adopted by convention where an adjustment from an animal-specific
BMCLADj to a BMCLHEc has been incorporated. An UF of 10 is comprised of two components
of uncertainty (i.e., toxicokinetic and toxicodynamic uncertainties). In this assessment, the
toxicokinetic component is mostly addressed through the application of a human equivalent
concentration (HEC) as described in the RfC methodology (U.S. EPA, 1994b). The
89
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toxicodynamic uncertainty is also accounted for to a certain degree by the use of the applied
dosimetry method. However, a threefold UF is retained to address this component.
A 10-fold UFH was used to account for variation in susceptibility among members of the
human population (i.e., interindividual variability) because insufficient information is available
to predict potential variability in susceptibility among the population.
A 10-fold UFs was used to account for uncertainty in extrapolating from a subchronic to
chronic exposure duration. A 10-fold UFD was used to account for deficiencies in the
database. The database of 1,2,3-trichloropropane inhalation studies, which included two 2-week
studies submitted to EPA by Miller et al. (1987a, b), a 4-week range finding study, two 13-week
studies, and a single-generation reproductive toxicity study (Johannsen et al., 1988;
Biodynamics, Inc., 1979), provides reliable dose-response data from subchronic studies in two
species and from a single-generation reproductive toxicity study. However, the database is
lacking a multigenerational reproductive toxicity study and a developmental toxicity study. The
database deficiencies are of particular concern due to the genotoxicity of 1,2,3-trichloropropane,
because genetic damage to the germ cells of the Fl generation may not be detected until the F2
generation.
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 of a 10% change in the incidence of peribronchial lyphoid hyperplasia was
selected as the BMR.
The chronic RfC for 1,2,3-trichloropropane was calculated as follows:
RfC =BMCLHEc-UF
= 0.90 mg/m3 - 3,000
= 3 x 10"4 mg/m3 (rounded to one significant figure)
5.2.4. Chronic RfC Comparison Information
Similar to the oral toxicity studies, the inhalation studies found statistically significant
increases in organ weights. An increase in absolute and relative liver weights were observed in
male and female rats following subchronic inhalation exposure and in male and female mice
following a 2-week exposure to 1,2,3-trichloropropane. Additionally, an increase in relative
lung weights was observed in female rats and an increase in relative kidney weights was
observed in male rats following subchronic exposure to 1,2,3-trichloropropane. An increased
incidence of peribronchial lymphoid hyperplasia was observed in male and female rats exposed
to 5, 15, or 50 ppm 1,2,3-trichloropropane, but the investigators did not examine epithelial tissue
in this study. Centrilobular to midzonal hepatocellular hypertrophy was seen in nearly all male
rats that were exposed for 13 weeks via inhalation to concentrations of 5, 15, or 50 ppm 1,2,3-
90
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trichloropropane. However, no evidence of hepatic effects was found in female rats that were
exposed via inhalation to 50 ppm 1,2,3-trichloropropane. Conversely, a dose-dependent increase
in the incidence and severity of extramedullary hematopoiesis of the spleen was observed in
female, but not male rats, although this effect is not biologically relevant.
The critical effect selected for the derivation of the chronic RfC is the incidence of
peribronchial lymphoid hyperplasia in the lungs of male CD rats due to the occurrence of this
effect in both male and female rats, and the possible correlation between the hyperplasia and the
observed increased lung weight. In addition, the decreased mating performance in female CD
rats was considered a potential critical effect and this endpoint was subjected to BMD modeling.
PODs and chronic reference concentrations (RfCs) that could be derived from the additional
health effects identified in Table 5-2 are presented in Figure 5-3 to allow a comparison with the
critical effect. For the increased incidence of peribronchial lymphoid hyperplasia and decreased
mating performance, the UFs applied were a 10-fold UF to account for uncertainty in
extrapolating from laboratory animals to humans, a 10-fold UF to account for variation in
susceptibility among members of the human population, a 10-fold UF to account for subchronic-
to-chronic extrapolation, and a 3-fold UF for database deficiencies.
91
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10
1 --L-1
0.1
O)
E
0.01
0.001
0.0001
1
fri
.\\
.\\
| UF, animal-to-human
;: UF, human variability
v UF, subchronic-to-chronic ,'~\
•" / *
UF, database
/ • \
i
i
i
i
i
* Point of Departure
• RfC ;
i
1
1
I
t
1
t
I
t
Peribronchial lymphoid \
hyperplasia, male rats, *
(Johannsen et al. 1988) \
m
\\\\\\\\^
\ /
\ X
1
1
t
t
1
t
1
1
1
111:
•.\\V\\\\
•.\\V\\\\
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1
I Decreased mating
/ performance, female rats,
/ (Johannsen etal., 1988)
' 1
/ j
Figure 5-3. PODs for selected endpoints (with critical effect circled) from Table 5-2 with corresponding applied UFs and derived
candidate chronic inhalation RfCs.
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5.2.5. Previous Inhalation Assessment
A RfC is not available in the current IRIS assessment, which was completed in 1987
(U.S. EPA, 2007).
5.3. UNCERTAINTIES IN CHRONIC ORAL REFERENCE DOSE AND INHALATION
REFERENCE CONCENTRATION
Risk assessments need to portray associated uncertainty. The following discussion
identifies uncertainties associated with the chronic RfD and chronic RfC for 1,2,3-
trichloropropane. As presented earlier in this chapter (Sections 5.1.2 and 5.1.3; 5.2.2 and 5.2.3),
the UF approach, following EPA practices and RfC and RfD guidance (U.S. EPA, 1994b), was
applied to a POD, a BMDLHEc for the RfD and a BMCLHEc for the chronic RfC. 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 population of
varying susceptibilities, and database deficiencies. These extrapolations are carried out with
default approaches given the paucity of experimental 1,2,3-trichloropropane data to inform
individual steps.
An adequate range of animal toxicology data is available for the hazard assessment of
1,2,3-trichloropropane, as described throughout the previous section (Chapter 4). The database
of oral toxicity studies includes a chronic gavage study in rats and mice, multiple subchronic
gavage and drinking water studies conducted in rats and mice, and a two-generation
reproductive/fertility assessment in mice. Toxicity associated with oral exposure to 1,2,3-
trichloropropane is observed in the liver, kidney, and reproductive endpoints, including
decreased fertility in generating the 4th and 5th litters and decreased number of live pups/litter in
the 4th and 5th litters. The database of inhalation toxicity studies in animals includes two 2-week
studies submitted to EPA, a 4-week range-finding study, two 13-week studies, and two single-
generation reproductive assessments. The inhalation database, however, is lacking a chronic
exposure study. Toxicity associated with inhalation exposure to 1,2,3-trichloropropane is
observed in the respiratory system as an increased incidence of peribronchial lymphoid
hyperplasia. In addition to the oral and inhalation data, there are numerous absorption,
distribution, metabolism, and excretion studies, although information on internal or target organ
doses of 1,2,3-trichloropropane is not available. Mode of action and genotoxicity studies are
also available. Critical data gaps have been identified and uncertainties associated with data
deficiencies are more fully discussed below.
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 2-year gavage study in Fischer rats (NTP, 1993) and increased liver
weight in males as the principal study and critical effect, respectfully, for deriving the chronic
RfD for 1,2,3-trichloropropane. The dose-response relationships between oral exposure to 1,2,3-
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trichloropropane and impaired fertility in CD-I mice are also suitable for use in deriving a
chronic RfD, but yields higher BMDLs than the selected critical effect. Thus, the RfD based on
an increase in absolute liver weight should be protective of impaired fertility. It should be noted
that mice exposed via gavage demonstrated higher DNA adduct formation and cellular
proliferation than mice exposed via drinking water (La et al., 1996). Thus, the utilization of the
gavage study to derive the RfD, rather than a drinking water or dietary study, may lead to the
derivation of a conservative RfD.
In addition, studies have demonstrated that the use of corn oil as a vehicle may increase
chemically-induced hepatotoxicity. For example, use of corn oil gavage led to increased
hepatotoxicity, measured by altered liver weight, serum chemistry, and histopathological
examination, of chloroform when compared to administration via drinking water in F344/N rats
(Larson et al., 1995) and B6C3F1 mice (Bull et al., 1986). The role of the corn oil vehicle in the
observed hepatotoxicity following gavage exposure to 1,2,3-trichloropropane is unknown. .
The critical effect selected for the derivation of the chronic RfC is based on the increased
incidence of peribronchial lymphoid hyperplasia in the lungs of male CD rats. Support for the
selection of this critical effect was observed in both male and female rats along with increased
lung weight. Although an increase in liver and kidney weights was apparent, serum enzyme
level alterations and an increased incidence of histopathological lesions indicative of liver and
kidney damage were not observed and the weight changes were observed at doses higher than
those observed for the lung effects. The hepatocellular hypertrophy reported in male rats and the
hematopoiesis of the spleen in female rats was considered to be of questionable biological
significance in the absence of additional overt toxicity in the liver and spleen, as there was no
change in the clinical chemistry or hematology parameters. It is important to recognize that the
critical effect selected for the derivation of the RfD was increased absolute liver weight, which
was supported by additional evidence of hepatotoxicity (i.e., increased serum enzyme levels,
cholinesterase levels, and necrosis), and that endpoints indicative of hepatotoxicity were not
observed following inhalation of 1,2,3-trichloropropane. In addition, there are toxicokinetic and
possible toxicodynamic differences between the two routes of exposure that may account for
these differences. A portal-of-entry effect is expected following 1,2,3-trichloropropane exposure
via inhalation and a first-pass effect is expected following oral exposure.
The selection of the BMD model for the quantitation of the chronic RfD does not lead to
significant uncertainties in estimating the POD since benchmark effect levels were within the
range of experimental data. However, the selected model, the Hill 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.
Similarly, the selection of the BMD model for the quantitation of the chronic RfC does
not lead to significant uncertainties in estimating the POD since benchmark effect levels were
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within the range of experimental data. However, the selected model, the log-logistic model, does
not represent all possible models one might fit, and other models could be selected to yield
results that may be either higher or lower than those included in this assessment.
Extrapolating from animals to humans yields further uncertainties. The effect and the
magnitude associated with the concentration at the POD in rodents are extrapolated to human
response. Pharmacokinetic models are useful to examine species differences in pharmacokinetic
processing; however, dosimetric adjustment using pharmacokinetic modeling was not possible
for the toxicity observed following oral and inhalation exposure to 1,2,3-trichloropropane.
Information was unavailable to quantitatively assess toxicokinetic or toxicodynamic differences
between animals and humans, so the 10-fold UF was used to account for uncertainty in
extrapolating from laboratory animals to humans in the derivation of the chronic RfD. For the
chronic RfC, a factor of 3 was adopted by convention where an adjustment from an animal-
specific BMCLADJ to a BMCLnEc has been incorporated. AnUF of 10 is comprised of two areas
of uncertainty (i.e., toxicokinetic and toxicodynamic uncertainties). In this assessment, the
toxicokinetic component is mostly addressed through the use of a HEC as described in the RfC
methodology (U.S. EPA, 1994b). The toxicodynamic uncertainty is also accounted for to a
certain degree by the use of the applied dosimetry method, but a UF of 3 is retained to account
for this component.
Heterogeneity among humans is another uncertainty associated with extrapolating doses
from animals to humans. Uncertainty related to human variation needs consideration. In the
absence of 1,2,3-trichloropropane-specific data on human variation, a factor of 10 was used in
the derivation of both the chronic RfD and the chronic RfC. Human variation may be larger or
smaller; however, 1,2,3-trichloropropane-specific data to examine the potential magnitude of
over- or under-estimation are unavailable.
Data gaps have been identified that yield uncertainties associated with deficiencies
regarding the developmental toxicity of 1,2,3-trichloropropane following oral exposure. The
two-generation reproductive toxicity study indicated that the developing fetus may be a target of
toxicity. In addition, the lack of a multigenerational study, beyond two generations, is of
particular concern due to the genotoxicity of 1,2,3-trichloropropane, as genetic damage to the
germ cells of the Fl generation may not be detected until the F2 generation. Thus, the absence
of a study specifically evaluating developmental toxicity represents an area of uncertainty or gap
in the database. Likewise, the database of inhalation studies is lacking a multigenerational
reproductive toxicity study and a developmental toxicity study.
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5.4. CANCER ASSESSMENT
There are no available studies on cancer in humans associated with exposure to 1,2,3-
trichloropropane. NTP (1993) observed 1,2,3-trichloropropane-induced benign and malignant
tumors in male and female F344/N rats and male and female B6C3Fi mice in a 2-year gavage
cancer bioassay. 1,2,3-Trichloropropane has been reported to be a mutagen in S. typhimurium
assays (Lag et al., 1994; NTP, 1993; Ratpan and Plaumann, 1988; Haworth et al., 1983; Kier,
1982; Stolzenberg and Hine, 1980; Shell Oil Co., 1979). Studies have also demonstrated the
induction of chromosomal aberrations and sister chromatid exchanges in CHO cell assays (NTP,
1993), trifluorothymidine resistance induction in mouse lymphoma assays (NTP, 1993; Shell Oil
Co., 1982), DNA strand breakage measured by the Comet assay (single gel electrophoresis test)
in isolated human lymphocytes (Tafazoli and Kirsch-Volders, 1996), and the induction of
micronucleus formation in the mammalian cell lines, AHH-1, MCL-5, and h2El (Doherty et al.,
1996).
Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), 1,2,3-
trichloropropane is "likely to be carcinogenic to humans," based on a statistically significant and
dose-related increase in the formation of multiple tumors in both sexes of two species from an
NTP (1993) chronic oral bioassay. Statistically significant increases in incidences of tumors of
the oral cavity, forestomach, pancreas, kidney, preputial gland, clitoral gland, mammary gland,
and Zymbal's gland in rats, and the oral cavity, forestomach, liver, Harderian gland, and uterus
in mice, were reported. In the absence of any data on the carcinogenicity of 1,2,3-
trichloropropane via the inhalation route, no inhalation unit risk has been derived in this
evaluation.
5.4.1. Choice of Study/Data with Rationale and Justification
The study by NTP (1993) was used for development of an oral slope factor. This was a
well-designed study, conducted in both sexes in two species, and included examination of
appropriate toxicological endpoints in both sexes of rats and mice. Tumor incidences were
elevated with increasing exposure level at numerous sites across all sex/species combinations,
involving point of contact in the alimentary system and more distant locations. Due to the
increased carcinogenic response at all dose levels and the increased mortality in the two high-
dose groups in both rats and mice, NTP stated that carcinogenic activity might have been
detected at doses lower than those tested in the chronic study (NTP, 1993). The early mortality
observed in rats and mice was associated with the development of chemical-related neoplasms,
especially in the forestomach (NTP, 1993).
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5.4.2. Dose-Response Data
In the NTP (1993) study, groups of 60 male and female F344/N rats and B6C3Fi mice
were administered 0, 3, 10, or 30 and 6, 20, or 60 mg/kg-day 1,2,3-trichloropropane,
respectively, by gavage, 5 days/week, for 2 years. Ten male and 10 female rats and mice from
each dose group were designated for evaluation at 15 months. High mortality in both species in
all high-dose groups necessitated early termination of the rat high-dose groups at weeks 77
(males) and 67 (females). All other groups of rats were sacrificed after 2 years (104 weeks). For
the mice, mid-dose groups were sacrificed at week 89, and high-dose male and female mice were
sacrificed at weeks 79 and 73, respectively. All other groups of mice were sacrificed after week
104.
Dose-related, statistically significant increasing trends in tumors were noted at the
following sites:
• Squamous cell carcinomas or papillomas of the alimentary system in male and female
rats and mice;
• Zymbal's gland carcinomas in male and female rats;
• Pancreatic acinar cell adenomas or adenocarcinomas, preputial gland adenomas or
carcinomas, and kidney tubular cell adenomas in male rats;
Clitoral gland adenomas or carcinomas, and mammary gland adenocarcinomas in female
rats;
• Hepatocellular adenomas or carcinomas, and harderian gland adenomas in male and
female mice; and
• Uterine adenomas or adenocarcinomas in female mice.
The tumors generally appeared earlier and showed statistically significantly increasing
trends with increasing exposure level (by life table test or logistic regression, p<0.001). The data
are summarized in Tables 5-3 (male and female rats) and 5-4 (male and female mice). The
alimentary system tumors are presented as the combined incidence of squamous papillomas or
squamous cell carcinomas of the pharynx/palate, tongue, or forestomach. Additionally,
incidences of oral cavity tumors only (squamous papillomas or squamous cell carcinomas of the
pharynx/palate or tongue) are presented. Data are not available to indicate whether the
malignant tumors developed specifically from progression of the benign tumors. However,
etiologically similar tumor types, i.e., benign and malignant tumors of the same cell type, were
combined for these tabulations because of the possibility that the benign tumors could progress
to the malignant form, as outlined in the 2005 Cancer Guidelines (U.S. EPA, 2005a).
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Table 5-3. Tumor incidence and time of first occurrence in male and female
F344/N rats following gavage exposure to 1,2,3-trichloropropane
Site
0 mg/kg-d
3 mg/kg-d
10 mg/kg-d
30 mg/kg-d
Trend test
/7-valuea
Male rats
Alimentary
system, squamous
neoplasms
Totalb
Oral cavity
only
Pancreas: acinar adenoma or
adenocarcinoma
Kidney tubular cell: adenoma
Preputial gland: adenoma or
carcinoma
Hepatocellular adenoma or
carcinoma
Skin: squamous cell papilloma or
carcinoma
Zymbal's gland, carcinoma
1/59C (2%)
104d
1/59C (2%)
104
5/59 (8%)
104
0/59 (0%)
5/58 (8%)
72
1/59 (2%)
105
0/59 (0%)
0/59 (0%)
39/60 (65%)
64
4/60 (7%)
99
20/60e (33%)
98
2/60 (3%)
104
6/57(11%)
93
1/60 2%
105
2/60 (3%)
98
0/60 (0%)
48/57 (84%)
58
19/57 (33%)
58
36/57 (63%)
67
18/57e (35%)
94
9/57 (16%)
58
4/57 7%
96
1/57 (2%)
86
0/57 (0%)
58/60 (97%)
47
43/60 (72%)
47
31/58(53%)
60
26/58 (45%)
60
17/56 (30%)
55
3/58 5%
65
6/57 (10%)
64
3/58 (5%)
56
O.001
0.001
O.001
0.001
O.001
O.001 (life)
0.011 (log)
O.001 (life)
0.014 (log)
0.005 (life)
0.058 (log)
Female rats
Alimentary
system, squamous
neoplasms
Totalb
Oral cavity
only
Clitoral gland, adenoma or
carcinoma
Mammary gland, adenoma or
adenocarcinoma
Zymbal's gland, carcinoma
1/60 (2%)
104
1/60 (2%)
104
5/56 (9%)
102
2/57 (4%)
64
0/60 (0%)
22/59 (37%)
73
6/59 (10%)
95
11/56(20%)
66
6/56 (10%)
67
1/59 (2%)
102
49/59 (83%)
58
28/59 (47%)
58
18/57 (32%)
62
14/52 (27%)
61
0/59 (0%)
44/58 (76%)
33
37/58 (64%)
33
17/51 (33%)
44
23/47 (48%)
34
4/45 (9%)
48
0.001
0.001
O.001
O.001
O.001
aBy both life table test ("live") and logistic regression ("log") unless otherwise noted.
bSquamous cell papillomas or carcinomas of the pharynx/palate, tongue, or forestomach.
'Numbers of animals at risk (denominators) vary due to missing tissues, or due to deaths either occurring before the
first incidence of tumor in that group or before wk 52, whichever was earlier.
dWeek of first incidence.
eNTP (1993) summary tables reported slightly higher incidences—21 low-dose males with pancreatic acinar cell
tumors, 20 mid-dose males with kidney tubule adenomas—than noted in individual animal histopathology tables.
Source: NTP(1993).
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Table 5-4. Tumor incidence in male and female B6C3Fi mice following
gavage exposure to 1,2,3-trichloropropane
Site
0 mg/kg-d
6 mg/kg-d
20 mg/kg-d
60 mg/kg-d
Trend test
p-value?
Male mice
Alimentary
system,
squamous
neoplasms
Totalb
Oral cavity
only
Liver: adenoma or carcinoma
Harderian gland adenoma
3/59c (5%)
69d
0/59 (0%)
14/59 (23%)
65
1/59 (2%)
104
57/59c (97%)
61
0/59 (0%)
24/59 (41%)
74
2/59 (3%)
91
57/60c (95%)
55
0/60 (0%)
25/60 (42%)
59
10/60 (17%)
72
59/60c (98%)
46
2/58 (3%)
68
33/60 (55%)
46
11/60(20%)
65
O.001
P>0.05
<0.001
0.001
Female mice
Alimentary
system,
squamous
neoplasms
Totalb
Oral cavity
only
Liver: adenoma or carcinoma
Harderian gland adenoma
Uterus: adenoma or adenocarcinoma
0/59C (0%)
d
1/59 (0%)
104
8/59 (13%)
66
3/59 (5%)
66
0/59 (0%)
54/60c (90%)
59
0/59 (0%)
11/60(18%)
77
6/59 (10%)
80
5/59 (8%)
100
59/60c (98%)
45
2/60 (3%)
79
9/60 (15%)
65
7/60 (12%)
78
3/59 (5%)
83
59/60c (98%)
42
5/60 (8%)
61
36/58 (60%)
60
10/60 (17%)
64
11/57(19%)
66
O.001
O.001 (life)
0.008 (log)
0.001
O.001 (life)
0.004 (log)
<0.001
aBy life table test ("life") and logistic regression ("log") unless otherwise noted.
bSquamous papillomas or squamous cell carcinomas of the pharynx/palate, tongue, or forestomach.
'Numbers of animals at risk (denominators) vary due to missing tissues, or due to deaths occurring before
the first incidence of tumor in that group or before wk 52, whichever was earlier.
dWeek of first incidence.
Source: NTP(1993).
Risk estimates are generally calculated from the incidence of rodents of the most
sensitive species, strain, and sex bearing tumors at any of the sites displaying treatment-
attributable increases. For 1,2,3-trichloropropane, mice were the more sensitive species, with
90% of females and 97% of males developing tumors. However, with such a high response,
extrapolation to lower, environmentally relevant exposures is more uncertain. Consequently,
dose-response modeling was considered for all four species/sex combinations.
NTP noted additional tumor sites with apparent dose-related increases in squamous cell
papillomas and carcinomas of the skin and hepatocellular adenomas and carcinomas, both in
male rats. NTP concluded that because the incidence in no one group was statistically
significantly higher than control, the overall trends were not treatment-related. On the other
hand, both endpoints show some consistency with other effects observed in the NTP study.
First, the squamous cell papillomas and carcinomas of the skin are the same type as observed in
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the alimentary system. While all of these animals also had either squamous cell papillomas or
carcinomas of the forestomach, neither site was noted as a metastasis of the other. The skin
tumors may reflect a systemic component rather than a strictly site of contact mode of action for
this tumor type. Consequently, both of these sites were carried through the dose-response
modeling analysis. Statistically significant increases in incidences of tumors of the alimentary
system (oral cavity and forestomach), pancreas, kidney, preputial gland, clitoral gland, mammary
gland, and Zymbal's gland in F344/N rats and the alimentary system (oral cavity and
forestomach), liver, Harderian gland, and uterus in B6C3F1 mice were included in the dose-
response analysis to derive the cancer oral slope factor for 1,2,3-trichloropropane.
However, some of the external peer review panel members (see Appendix A: Summary
of External Peer Review and Public Comments and Disposition) recommended removing the
forestomach tumors observed in rats and mice from the quantitative cancer analysis as humans
do not have a forestomach or an organ that is homologous to the rodent forestomach. In
addition, several panel members indicated that the bolus dose of the chemical administered by
gavage, coupled with the slow emptying of the forestomach lends uncertainty to the actual dose
that should be used for quantification.
As noted by the peer reviewers, humans do not have a forestomach; however, squamous
epithelial tissues in the oral cavity and the upper two-thirds of the esophagous in humans are
comparable to the rodent forestomach (IARC, 2003). In addition, forestomach carcinogens in
rodents may affect other tissues in humans. It has been suggested that most genotoxic
forestomach carcinogens appear to act through a mutagenic mode of action (IARC, 2003). For
multi-site carcinogens that induce forestomach tumors and are genotoxic, these tumors are likely
relevant to human carcinogenesis (IARC, 2003; Proctor et al., 2007).
Therefore, in the absence of data to indicate otherwise, EPA considers forestomach
tumors to be relevant to humans but recognizes that there is some uncertainty associated with the
quantification of the tumors with respect to estimating the dose given the gavage dosing
regimen. EPA has included the data for these tumors in the quantitative carcinogenic dose-
response analysis for the derivation of the oral slope factor (Section 5.4.4). However, in
response to the recommendations of some of the external peer review panel members, Section
5.4.4 also includes the derivation of oral slope factors for rats and mice in which forestomach
tumors were excluded from the analysis. In addition, uncertainties associated with the
quantification of forestomach tumors as noted by some of the external peer review panelists are
discussed in Section 5.4.6 (Uncertainties in Cancer Risk Values).
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5.4.3. Dose Adjustments and Extrapolation Methods
The EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) recommend that
the method used to characterize and quantify cancer risk from a chemical is determined by what
is known about the mode of action of the carcinogen and the shape of the cancer dose-response
curve. The dose response is assumed to be linear in the low dose range, when evidence supports
a mutagenic mode of action because of DNA reactivity, or if another mode of action that is
anticipated to be linear is applicable. A linear-low-dose extrapolation approach was used to
estimate human carcinogenic risk associated with 1,2,3-trichloropropane exposure due to the
mutagenic mode of carcinogenic action of 1,2,3-trichloropropane.
Due to the occurrence of multiple tumor types, earlier occurrence with increasing
exposure, and early termination of at least one dose group, methods that can reflect the influence
of competing risks and intercurrent mortality on site-specific tumor incidence rates are preferred.
EPA has generally used a model which incorporates the time at which death-with-tumor
occurred as well as the dose; the multistage-Weibull model is multistage in dose and Weibull in
time, and has the form:
P(d) = 1 - exp[-(q0 + qid + q2d + ... + q*) x ft ± t0f],
where P(d) represents the lifetime risk (probability) of cancer at dose d(i.e., human equivalent
exposure in this case); parameters qt > 0, for i = 0, 1, ..., k; t is the time at which the tumor was
observed; and z is a parameter which characterizes the change in response with age. The
parameter to represents the time between when a potentially fatal tumor becomes observable and
when it causes death, and is generally set to 0 either when all tumors are considered incidental or
because of a lack of data to estimate the time reliably. The dose-response analyses were
conducted using the computer software program TOX_RISK, version 5.2 (property of ICF,
Fairfax, VA), which is based on Weibull models drawn from Krewski et al. (1983). Parameters
were estimated using the method of maximum likelihood.
Other characteristics of the observed tumor types were considered prior to modeling,
including allowance for different, although possibly unidentified, modes of action, and for
relative severity of tumor types. First, etiologically different tumor types were not combined
across sites prior to modeling, in order to allow for the possibility that different tumor types can
have different dose-response relationships because of varying time courses or other underlying
mechanisms or factors. Consequently, all of the tumor types listed separately in Tables 5-3 and
5-4 were modeled separately.
A further consideration allowed by the software program is the distinction between tumor
types as being either fatal or incidental, in order to adjust for competing risks. Incidental tumors
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are those tumors thought not to have caused the death of an animal, while fatal tumors are
thought to have resulted in animal death. NTP (1993) stated that neoplasms of the forestomach
and oral mucosa in rats and mammary tumors in female rats were the principal cause of death of
most animals dying or killed moribund before the end of the study, but did not report individual
causes of death, which would be preferable for time-to-tumor analysis. However, because the
likely causes of death were relatively evident in this study, a bounding exercise was carried out
for each these two malignant tumor types. For the first analysis, all tumors at these sites were
considered incidental (an "incidental" analysis). In the second, the tumors observed at
unscheduled deaths (including early group termination because those animals were thought to be
in extremis) were considered as fatal and the rest for that site considered incidental (a "fatal"
analysis). Analyses treating some tumors as fatal permit estimation of incidental or fatal risk; for
the purposes of slope factor estimation, estimating the risk of developing a tumor (incidental
risk) is of greater interest than estimating the risk of dying with a tumor (fatal risk). Note that
there was a slight overlap of squamous cell carcinomas and mammary adenocarcinomas in
female rats, involving one rat in the mid-dose group and six rats in the high-dose group. For all
other tumor sites, all tumors were treated as incidental. The data modeled are provided in Tables
D-l through D-4 (Appendix D).
For the fatal analyses, it was feasible to estimate to, the time between when a potentially
fatal tumor becomes observable and when it causes death, because some early deaths were not
accompanied by the malignant form of the tumor being analyzed. In addition, the NTP study
had the additional feature of an interim sacrifice of 10 animals per sex and species in each dose
group at about week 65, which permitted the observation of tumors at these sites before
becoming fatal. For the incidental analyses, t0 was set to zero.
Specific n-stage Weibull models were selected for the individual tumor types for each sex
based on the values of the log-likelihoods according to the strategy used by EPA (U.S. EPA,
2002). If twice the difference in log-likelihoods was less than a %2 with degrees of freedom
equal to the difference in the number of stages included in the models being compared, the
models were considered comparable and the most parsimonious model (i.e., the lowest-stage
model) was selected. For tumors treated as incidental, plots of model fits compared with Hoel-
Walburg estimates of cumulative incidence were also examined for goodness of fit in the lower
exposure region of the observed data (Gart et al., 1986). If a model with one more stage fitted
the low-dose data better than the most parsimonious model, then the model with one higher stage
was selected.
PODs for estimating low-dose risk were identified at doses at the lower end of the
observed data, generally corresponding to 10% extra risk, where extra risk is defined as [P(d) -
P(0)]/[l - P(0)]. Lower risks were used for responses demonstrating less than a 10% response
throughout the data range. The lifetime oral cancer slope factor for humans is defined as the
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slope of the line from the lower 95% bound on the exposure at the POD. This 95% upper
confidence limit (UCL) represents a plausible upper bound on the true risk.
Adjustments for approximating human equivalent slope factors applicable for continuous
exposure were also carried out by the dose-response software program. Consistent with the
Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), an adjustment for cross-species
scaling was applied by the software program, to address toxicological equivalence across
species, after the model-fitting phase. Following EPA's cross-species scaling methodology, the
time-weighted daily average doses were converted to human equivalent doses on the basis of
(body weight)374 (U.S. EPA, 1992). It was not necessary to adjust the administered doses for
lifetime exposure prior to modeling for the groups terminated early, because the software
program used characterizes the tumor incidence as a function of time, from which it provides an
extrapolation to lifetime exposure. In addition, TOX_RISK estimated continuous daily exposure
by multiplying each slope factor by (5 days)/(7 days) = 0.71.
5.4.4. Oral Slope Factor and Inhalation Unit Risk
The results of applying the multistage-Weibull models to the tumor incidence data for the
four sex and species combinations in the NTP study are provided in Table 5-5. An oral slope
factor for each of the tumor sites was calculated by dividing the BMR level (usually 10%) by its
corresponding BMDL. In the absence of any data on the carcinogenicity of 1,2,3-
trichloropropane via the inhalation route, no inhalation unit risk has been derived in this
evaluation.
The highest slope factor for each of the four data sets corresponded to squamous cell
neoplasms of the alimentary system, whether or not malignant tumors observed at unscheduled
deaths were considered incidental or fatal. The incidental analyses led to higher slope factors
than did the fatal analysis for female rats and mice, and to generally similar results in the male
rats and mice. While the fatal analyses may tend to overestimate the risk estimates, because the
number of deaths due to these tumors may have been overestimated, the slope factors in this
assessment were derived from the fatal analyses because this tumor context describes these data
sets as well as possible given the available information.
The mouse data led to higher risk estimates than did the rat data, with the female mice
demonstrating the highest slope factor of all, at 26 per mg/kg-day for the fatal analysis of
alimentary system tumors. The highest slope factor based on the male mice data was 5.6 per
mg/kg-day for the fatal analysis of alimentary system tumors. For rats, the corresponding
estimates were 3.1 and 1.1 per mg/kg-day for males and females, respectively.
Although the time-to-tumor modeling does help account for competing risks associated
with decreased survival times and other tumors, considering the tumor sites individually does not
convey the total amount of risk potentially arising from the sensitivity of multiple sites. To get
103
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some indication of the total unit risk from multiple tumor sites, assuming the multiple sites are
mechanistically independent, several approaches are available. One approach suggested in the
2005 Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) would be to estimate cancer
risk from tumor-bearing animals. EPA traditionally used this approach until the NRC document
Science and Judgment (1994) made a case that this approach would tend to underestimate overall
risk when tumor types occur in a statistically independent manner. In addition, application of
one model to a composite data set does not accommodate biologically relevant information that
may vary across sites or may only be available for a subset of sites. For instance, the time
courses of the multiple tumor types evaluated varied, as is suggested by the variation in estimates
of z (see Table 5-5), from 1.0 (e.g., male rat Zymbal's gland tumors), indicating relatively little
effect of age on tumor incidence, to 10 (e.g., female mouse uterine tumors), indicating a much
more rapidly increasing response with increasing exposure level. The result of fitting a model
with underlying mechanism-related parameters, such as z in the multistage-Weibull model,
would be difficult to interpret with composite data. A simpler model could be used for the
composite data, such as the multistage model, but available biological information would then be
ignored.
Following the recommendations of the NRC (1994) and the 2005 Guidelines for
Carcinogen Risk Assessment (U.S. EPA, 2005a), a statistically appropriate upper bound on total
risk was estimated in order to gain some understanding of the total risk from multiple tumor sites
for each sex/species combination. Note that this upper bound estimate of overall risk describes
the risk of developing any combination of the tumor types considered, not just the risk of
developing all three simultaneously. Statistical methods which can accommodate the underlying
distribution of slope factors are optimal, such as through maximum likelihood estimation or
through bootstrapping or Bayesian analysis. However, these methods have not yet been
extended to models such as the multistage-Weibull model. Consequently, this analysis used the
same method as in the IRIS assessments for 1,3-butadiene (U.S. EPA, 2000d) and 1,2-
dibromoethane (U.S. EPA, 2004), which involves assuming that slope factors can be
characterized by a normal distribution. Using the results in female mice to illustrate, the overall
risk estimate involved the following steps:
1) It was assumed that the tumor types associated with 1,2,3-trichloropropane
exposure were statistically independent - that is, that the occurrence of a liver
tumor, say, was not dependent upon whether there was a forestomach tumor.
This assumption cannot currently be verified, and if not correct could lead to an
overestimate of risk from summing across tumor sites. However, NRC (1994)
argued that a general assumption of statistical independence of tumor-type
occurrences within animals was not likely to introduce substantial error in
assessing carcinogenic potency from rodent bioassay data.
104
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2) The models previously fitted to estimate the BMDs and BMDLs were used to
extrapolate to a low level of risk (R), in order to reach the region of each
estimated dose-response function where the slope was reasonably constant and
upper bound estimation was still numerically stable. For these data, a 10"3 risk
was generally the lowest risk necessary. The oral slope factor for each site was
then estimated by R/BMDLR, as for the estimates for each tumor site above.
3) The maximum likelihood estimates (MLE) of unit potency (that is, risk per unit
of exposure) estimated by R/BMDR, were summed across the alimentary
system, liver, Harderian gland, and uterus in female mice.
4) An estimate of the 95% upper bound on the summed oral slope factor was
calculated by assuming a normal distribution for the individual risk estimates,
and deriving the variance of the risk estimate for each tumor site from its 95%
upper confidence limit (UCL) according to the formula:
95% UCL = MLE + 1.645 x s.d.,
rearranged to:
s.d. = (UCL-MLE)/1.645,
where 1.645 is the t-statistic corresponding to a one-sided 95% confidence
interval and >120 degrees of freedom, and the standard deviation (s.d.) is the
square root of the variance of the MLE. The variances (variance = s.d.2) for each
site-specific estimate were summed across tumor sites to obtain the variance of
the sum of the MLEs. The 95% UCL on the sum of MLEs was calculated from
the expression above for the UCL, using the variance of the sum of the MLE to
obtain the relevant s.d (s.d. = variance172).
Tables 5-5 and D-5 provides a summary of combined risk estimates for all four data sets.
The resulting combined upper bound slope factor for female mice was 28 per mg/kg-day,
compared with 26 per mg/kg-day for just alimentary system tumors. The difference between
these two estimates is insignificant given the approximate nature of low dose extrapolation and
because details of the algorithms differ slightly, with the risk values being estimated at a 10%
extra risk, and the combined risk estimates being estimated at 10"3 extra risk. More importantly,
the alimentary system tumors were clearly a much more sensitive response in female mice, and
both estimates converge on 30 per mg/kg-day when rounded to one significant digit. Combined
risks for the other data sets ranged from 1.6 per mg/kg-day for female rats (an increase of about
50% from 1.1 per mg/kg-day for alimentary system tumors only) to 6.8 per mg/kg-day for male
mice (an increase of about 15% from 5.9 per mg/kg-day for alimentary system tumors only).
Interestingly, despite the greater number of sites with dose-related increases in male rats, the
combined risk of 4.1 per mg/kg-day essentially reflects only the alimentary system and
pancreatic tumors (see Tables 5-5 and D-5, proportion of total variance column).
Based on the analyses discussed above, the recommended upper bound estimate on
human extra cancer risk from continuous lifetime oral exposure to 1,2,3-trichloropropane is
105
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30 per mg/kg-day. The oral slope factor based on incidences of tumors in the alimentary system
(including oral cavity and forestomach tumors), liver, harderian gland and uterus of female mice
was recommended because female mice are the most sensitive to tumor induction following
exposure to 1,2,3-trichloropropane. The recommended estimate reflects the time-to-tumor
dimension of the responses as well as the exposure-response relationships for the multiple tumor
sites. This slope factor should not be used with exposures greater than 0.6 mg/kg-day, the
human equivalent dose corresponding to the POD for the female mouse alimentary system
tumors, because the observed dose-response relationships do not continue linearly above this
level and the fitted dose-response models better characterize what is known about the
carcinogenicity of 1,2,3-trichloropropane.
Table 5-5. Dose-response modeling summary for tumors associated with
oral exposure to 1,2,3-trichloropropane; rat and mouse tumor incidence
data
Tumor type and context"
Multistage-
Weibull model
coefficients
(MLE)b
Human equivalent
continuous PODC, mg/kg-
d
BMD10
BMDL10
Slope factord,
(mg/kg-d)1
Overall
slope factor,
(mg/kg-d)1
Male rats
Alimentary
system, total
squamous
neoplasms
Incidental
Fatal
Pancreas: acinar adenoma or
adenocarcinoma
Preputial gland adenoma or
carcinoma
Kidney tubular cell adenoma
Hepatocellular adenomas or
carcinomas
Skin: squamous cell
papillomas or carcinomas
Zymbal's gland carcinoma
qo=l.l x 10'12
q1 = 1.9 x lO'11
q2 = 2.1 x 10'12
z = 5.1
q0 = 2.9x ID'15
ql = 5.9 x lO'14
q4 = 2.5 x lO'17
z = 6.4
to = 29
q0 = 4.5 x 1Q-19
ql = 2.4 x ID'19
q2=1.2x ID'19
z = 8.7
q0=l.l x ID'4
ql = 2.7 x ID'5
z= 1.4
q2 = 2.5 x 10'15
z = 6.2
q0 = 4.6 x ID'19
q2 = 3.5 x ID'20
z = 8.2
q1 = 3.6 x ID'6
7.= 1.6
q1 = 1.6 x ID'5
z=1.0
0.050
0.041
0.20
1.3
0.49
0.85f
3.4
6.1f
0.033
0.032
0.10
0.59
0.32
0.53f
1.4
2.5f
3.0
3.1
1.0
0.17
0.16
0.010
0.070
0.021
4.1e
(3.9)
106
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Table 5-5. Dose-response modeling summary for tumors associated with
oral exposure to 1,2,3-trichloropropane; rat and mouse tumor incidence
data
Tumor type and context"
Multistage-
Weibull model
coefficients
(MLE)b
Human equivalent
continuous PODC, mg/kg-
d
BMD10
BMDL10
Slope factor11,
(mg/kg-d)1
Overall
slope factor,
(mg/kg-d)1
Female rats
Alimentary
system, total
squamous
neoplasms
Incidental
Fatal
Clitoral gland adenoma or
carcinoma
Mammary
gland adeno-
carcinoma
Incidental
Fatal
Zymbal's gland carcinoma
qo = 2.5 x ID'12
q! = 8.1 x 1Q-12
q2 = 5.6 x ID'12
z = 4.9
q0=1.7x ID'11
qj = 5.7 x lO'11
q2 = 2.7 x lO'11
z = 4.5
to = 27
q0 = 3.1 x 10'7
qj = 6.5 x 10"7
z = 2.4
qo = 3.5 x ID'4
ql = 2.9 x ID'4
z=l
q0 = 9.8x ID'13
ql = 3.3 x ID'13
q3 = 7.5 x ID'15
z = 5.3
to = 4.7
qj = 1.4 x 10'5
z= 1.2
0.15
0.17
0.31
0.61
0.72
4.9
0.055
0.09
0.24
0.43
0.34
1.6
1.8
1.1
0.41
0.24
0.29
0.063
1.5
(2.4)
Male mice
Alimentary
system, total
squamous
neoplasms
Incidental
Fatal
Liver: adenoma or carcinoma
Harderian gland adenoma
q0 = 6.5 x ID'9
ql = 6.5 x 10'8
z = 3.5
q0 = 2.6 x ID'10
qj = 2.8 x ID'9
z = 4.2
to = 32
q0 = 3.4x ID'10
ql = 8.9 x lO'11
z = 4.5
q0 = 2.0 x ID'10
q2 = 2.2 x ID'10
z = 3.9
0.030
0.022
0.22
1.1
0.017
0.017
0.14
0.57
5.9
5.9
0.73
0.17
6.8e
(6.8)
107
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Table 5-5. Dose-response modeling summary for tumors associated with
oral exposure to 1,2,3-trichloropropane; rat and mouse tumor incidence
data
Tumor type and context"
Multistage-
Weibull model
coefficients
(MLE)b
Human equivalent
continuous PODC, mg/kg-
d
BMD10 BMDL10
Slope factor11,
(mg/kg-d)1
Overall
slope factor,
(mg/kg-d)1
Female mice
Alimentary
system, total
squamous
neoplasms
Incidental
Fatal
Liver: adenoma or carcinoma
Harderian gland adenoma
Uterus: adenoma or
carcinoma
q1 = 3.3 x ID'12
z = 6.0
q1 = 7.3 x ID'16
q2 = 9.6 x 10'17
z = 7.5
to = 24
q0 = 5.6x ID'18
q1 = 9.2 x ID'19
q3=5.5 x 10'21
z = 8.2
q0 = 6.9x ID'12
q1 = 3.0 x ID'12
z = 4.9
q1 = 6.0 x ID'23
q2 = 2.4 x ID'23
z= 10
0.0032
0.0095
0.30
0.42
0.42
0.00065
0.0039
0.14
0.20
0.21
150
26
0.73
0.50
0.47
28
(160)
""'Incidental" denotes models treating all tumors of the type listed as incidental to the death of the animal. "Fatal"
denotes models treating the tumors (among the type listed) present at unscheduled deaths as causing the death, with
the remaining tumors considered incidental. If no context is listed, all tumors were considered incidental.
bMultistage-Weibull model: P(d) = 1 - exp[-(q0 + qid + q2d2 + ... + qkdk) x (t ±to)z], with coefficients estimated in
terms of mg/kg-d as administered inbioassay; lower or intermediate stage q; not listed were estimated to be zero.
°POD adjusted to estimate human equivalent continuous exposure, using BW3/4 cross-species scaling and by
multiplying by (5 d)/(7 d).
dSlope factors estimated by dividing the BMR (10% unless specified otherwise) by the BMDL.
eOverall slope factor including fatal context for tumors considered under both possibilities. Overall slope factor in
parentheses represents incidental context for all tumor types.
fBMR = 5%.
BMD10 = Concentration at 10% extra risk; BMDL10 = 95% lower bound on concentration at 10% extra risk.
Source: NTP(1993).
Oral slope factors derived from tumor incidence data excluding forestomach tumors (see
Tables 5-3 and 5-4 for oral cavity tumor incidence data) are presented for purposes of
comparision.
Male and female rats and female mice showed statistically significant increasing trends in
oral cavity tumor rates.. Male mice did not demonstrate a statistically significant increase in oral
cavity tumors, but there was an increasing trend that was modeled for comparison purposes. For
time to tumor modeling, the oral cavity tumors were all considered to be incidental to animal
108
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mortality. All of the carcinomas in the oral cavity in the four species/sex combinations occurred
concurrently with carcinomas in the forestomach, which the NTP had considered to be largely
responsible for the early mortality seen across the study. In all other respects the modeling was
performed as described above in this section. Table 5-6 summarizes the results of modeling the
oral cavity tumors.
As discussed earlier in this section, considering the tumor sites individually does not
convey the total amount of risk potentially arising from the sensitivity of multiple sites.
Therefore, the same procedure described previously was used to estimate the total risk for all
tumors, excluding those of the forestomach, for each species/sex combination,. The results are
shown in the last column of Table 5-6. Higher risk estimates were calculated for the overall
slope factors that included analyses of the forestomach tumors in rats and mice as shown in
Tables 5-5 and 5-6.
Table 5-6. Dose-response modeling summary for oral cavity squamous cell
neoplasia associated with oral exposure to 1,2,3-trichloropropane (NTP, 1993)
Sex, species
Male rats
Female rats
Male mice
Female mice
Multistage-
Weibull model
Coefficients
(MLE)a
qo = 2.2 x 10°
ql = 3.4 x ID'5
q2 = 2.5 x ID'6
z =1.4
qo = 4.3 x I0'y
ql = 2.8 x ID'9
q2 = 2.1 x ID'9
z = 3.3
q3 = 4.6 x 10'19
z =6.2
q0= 1.1 x 10"'
q1 = 2.4 x 10'6
z = 1.5
Human Equivalent
Continuous Point of
departure1", mg/kg-day
BMD10
0.68
0.44
7.7
4.9
BMDL10
0.41
0.24
2.9
1.1
Slope factor0,
(mg/kg-day)1
for oral
cavity tumors
0.24
0.44
0.034
0.092
Combined
slope factor11,
(mg/kg-day) *
1.3
0.9
0.9
1.3
a Multistage-Weibull model: P(d) = 1 - exp[-(q0 + qid + q2d2 + ... + qkdk) * (t ±to)z], with coefficients estimated
in terms of mg/kg-day as administered in bioassay ; lower or intermediate stage q; not listed were estimated to
be zero.
b Point of departure adjusted to estimate human equivalent continuous exposure, using BW3/4 cross-species
scaling and by multiplying by (5 days)/(7 days).
BMD10 = Concentration at 10% extra risk; BMDL10 = 95% lower bound on concentration at 10% extra risk.
0 Slope factors estimated by dividing the BMR (10% unless specified otherwise) by the BMDL.
d Slope factor for oral cavity tumors combined with the non-alimentary system tumors listed in Table 5-
5, for each species/sex combination.
Therefore, the recommended upper bound estimate on human extra cancer risk from
continuous lifetime oral exposure to 1,2,3-trichloropropane is 30 per mg/kg-day.
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5.4.5. Application of Age-Dependent Adjustment Factors
Because a mutagenic mode of action for 1,2,3-trichloropropane carcinogenicity is
sufficiently supported in laboratory animals and relevant to humans (Section 4.7.3.4), and in the
absence of chemical-specific data to evaluate differences in susceptibility, increased early-life
susceptibility is assumed and the age-dependent adjustment factors (ADAFs) should be applied,
as appropriate, in accordance with the Supplemental Guidance for Assessing Susceptibility from
Early-Life Exposure to Carcinogens (U.S. EPA, 2005b). The oral slope factor of 30 per mg/kg-
day, calculated from data for adult exposures, does not reflect presumed early-life susceptibility
for this chemical. Example evaluations of cancer risks based on age at exposure are given in
Section 6 of the Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to
Carcinogens (U.S. EPA, 2005b).
The Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to
Carcinogens establishes ADAFs for three specific age groups. The current ADAFs and their age
groupings are 10 for <2 years, 3 for 2 to <16 years, and 1 for 16 years and above (U.S. EPA,
2005b). The 10-fold and 3-fold adjustments in slope factor are to be combined with age specific
exposure estimates when estimating cancer risks from early life (<16 years age) exposure to
1,2,3-trichloropropane.
To illustrate the use of the ADAFs established in the Supplemental Guidance for
Assessing Susceptibility from Early-Life Exposure to Carcinogens (U.S. EPA, 2005b), sample
calculations are presented for three exposure duration scenarios, including full lifetime,
assuming a constant 1,2,3-trichloropropane exposure of 0.001 mg/kg-day (Table 5-7).
Table 5-7. Application of ADAFs for a 70-year exposure to 0.001 mg/kg-day
1,2,3-trichloropropane from ages 0 to 70
Age group
0-<2 yrs
2-<16yrs
>16yrs
ADAF
10
3
1
Unit risk
(per mg/kg-d)
30
30
30
Exposure
concentration
(mg/kg-d)
0.001
0.001
0.001
Duration
adjustment
2 yrs/
70 yrs
14 yrs/
70 yrs
54 yrs/
70 yrs
Total risk
Partial risk
0.01
0.02
0.02
0.05
Note that the partial risk for each age group is the product of the values in columns 2-5
[e.g., 10 x 30 x 0.001 x 2/70 = 0.01], and the total risk is the sum of the partial risks. Thus, a 70-
year risk estimate for a constant exposure of 0.001 mg /kg-day starting at birth is 0.05, or 5%.
If calculating the cancer risk for a 30-year exposure to a constant 1,2,3-trichloropropane
exposure level of 0.001 mg/kg-day from ages 0 to 30, the duration adjustments would be 2/70,
110
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14/70, and 14/70, and the partial risks would be 0.01, 0.02, and 0.01, resulting in a total risk
estimate of 0.03, or 3%.
If calculating the cancer risk for a 30-year exposure to a constant 1,2,3-trichloropropane
exposure level of 0.001 mg/kg-day from ages 20 to 50, the duration adjustments would be 0/70,
0/70, and 30/70, and the partial risks would be 0, 0, and 0.01, resulting in a total risk estimate of
0.01, or 1%.
5.4.6. Uncertainties in Cancer Risk Values
As in most risk assessments, extrapolation of study data to estimate potential risks to
human populations from exposure to 1,2,3-trichloropropane has engendered some uncertainty in
the results. Some types of uncertainty, but not all, may be considered quantitatively. Principal
uncertainties are summarized below and in Table 5-8.
Table 5-8. Summary of uncertainty in the 1,2,3-trichloropropane cancer
risk assessment
Consideration/
approach
Low-dose
extrapolation
procedure
Dose metric
Cross-species
scaling
Statistical
uncertainty at
POD
Impact on oral slope
factor
Alternatives could J, or
t slope factor by an
unknown extent
Alternatives could t or
J, slope factor by an
unknown extent
Alternatives could J, or
t slope factor [e.g.,
sixfold J, (scaling by
BW) or t twofold
(scaling by BW2/3)]
J, slope factor 2. 5 -fold
if MLE used rather
than lower bound on
POD
Decision
Multistage-Weibull
model to determine
POD, linear low-
dose extrapolation
from POD (due to
mutagenic mode of
carcinogenic
action)
Used administered
exposure
BW3/4 (default
approach)
BMDL (default
approach for
calculating
reasonable upper
bound slope factor)
Justification
A linear-low-dose extrapolation approach was used
to estimate human carcinogenic risk associated
with 1,2,3-trichloropropane exposure due to the
mutagenic mode of carcinogenic action. Linear
extrapolation is generally considered to be a health-
protective approach (U.S. EPA, 2005a).
Experimental evidence supports a role for
metabolism in toxicity, but actual responsible
metabolites are not clearly identified. If the
responsible metabolites are generated in proportion
to administered concentration, the estimated slope
factor is an unbiased estimate.
There are no data to support alternatives. Because
the dose metric was not an area under the curve,
BW3/4 scaling was used to calculate equivalent
cumulative exposures for estimating equivalent
human risks (U.S. EPA, 1992).
Size of bioassay results in sampling variability;
lower bound is 95% confidence interval on
administered exposure.
Ill
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Table 5-8. Summary of uncertainty in the 1,2,3-trichloropropane cancer
risk assessment
Consideration/
approach
Bioassay-
exposure issues
Species/gender
combination
Human relevance
of rodent tumor
data
Human
population
variability in
metabolism and
response/
sensitive
subpopulations
Impact on oral slope
factor
Alternative oral
exposures better
approximating likely
human exposure
patterns (without corn
oil vehicle, regular/
constant exposure
rather than daily bolus
gavage) could J, slope
factor, by an unknown
extent
Human risk could J, or
t, depending on
relative sensitivity
Lack of human
relevance of tumor
data would J, slope
factor
Low-dose risk f or J,
to an unknown extent
Decision
NTP study
Female mouse
tumors
(forestomach,
pharynx/palate,
tongue; liver,
Harderian gland,
uterus)
Tumors with
significant dose-
response
considered for
estimating potential
human cancer
response
Considered
qualitatively
Justification
Alternative bioassays were unavailable.
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.
1,2,3-Trichloropropane is carcinogenic through a
mutagenic mode of action and is a multisite
carcinogen in rodents; therefore, the
carcinogenicity observed in the rodent studies is
relevant to human exposure.
No data to support range of human
variability/sensitivity .
Choice of low-dose extrapolation approach. The mode of action is a key consideration
in clarifying how risks should be estimated for low-dose exposure. A linear, low-dose
extrapolation approach was used to estimate human carcinogenic risk associated with 1,2,3-
trichloropropane exposure due to the mutagenic mode of carcinogenic action of 1,2,3-
trichloropropane. Linear extrapolation is, generally, considered to be a health-protective
approach, and, in some cases, may lead to an overestimation of risk, as stated in the 2005 Cancer
Guidelines (U.S. EPA, 2005a).
The multistage-Weibull model was used to model the cancer data because it incorporates
the time at which death-with-tumor occurred; however, it is unknown how well this model or the
linear low-dose extrapolation predicts low-dose risks for 1,2,3-trichloropropane. The selected
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model does not represent all possible models one might fit, and other models could conceivably
be selected to yield more extreme results consistent with the observed data, both higher and
lower than those included in this assessment. Etiologically different tumor types were not
combined across sites prior to modeling, in order to allow for the possibility that different tumor
types can have different dose-response relationships because of varying time courses or other
underlying mechanisms or factors. The human equivalent oral slope factors estimated from the
tumor sites with statistically significant increases ranged from 0.010 to 30 per mg/kg-day, a
range of about three orders of magnitude.
However, given the multiplicity of tumor sites, basing the oral slope factor on one tumor
site may underestimate the carcinogenic potential of 1,2,3-trichloropropane. Following the
recommendations of the NRC (1994) and the 2005 Guidelines for Carcinogen Risk Assessment
(U.S. EPA, 2005a), a statistically appropriate upper bound on total risk was estimated in order to
gain some understanding of the total risk from multiple tumor sites in male and female F344/N
rats and B6C3F1 mice (Table 5-5). Note that this estimate of overall risk describes the risk of
developing any combination of the tumor types considered, not just the risk of developing all
simultaneously. The estimates of the overall oral slope factor ranged from 2 to 30 per mg/kg-
day.
Dose metric. 1,2,3-Trichloropropane is known to be metabolized to intermediates with
carcinogenic potential. However, it is unknown whether a metabolite or some combination of
metabolites is responsible for the observed toxicity. If the actual carcinogenic moiety(ies) is
(are) 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 most relevant dose metric,
then the impact on the human equivalent slope factor is unknown; the low-dose cancer risk value
may be higher or lower than that estimated, by an unknown amount.
Cross-species scaling. Without data to the contrary, it was assumed that equal risks
result from equivalent constant lifetime exposures. An adjustment for cross-species scaling
(BW3/4) was therefore applied to address toxicological equivalence of internal doses between
each rodent species and humans, consistent with the 2005 Guidelines for Carcinogen Risk
Assessment (U.S. EPA, 2005a). Because it is unknown whether there are differences in the
pharmacokinetic pathways and pharmacodynamic processes in animals and humans following
1,2,3-trichloropropane exposure, it is not possible to estimate the full impact of this uncertainty
beyond that associated with other arbitrary choices for default cross-species scaling factors (such
as BW2/3 or assuming equivalence on a mg/kg-day basis).
Statistical uncertainty at the point of departure. Measures of statistical uncertainty
require assuming that the underlying model and associated assumptions are valid for the data
under consideration. For the multistage-Weibull model applied to the female mice alimentary
tumor data, there is a reasonably typical degree of uncertainty at the 10% extra risk level (the
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POD for linear low-dose extrapolation). The BMDLio for female mice is approximately 2.5-fold
lower than the BMDio.
Bioassay selection. The study by NTP (1993) was used for development of an oral slope
factor. This was a well-designed study, conducted in both sexes in two species. However, the
bolus nature of the 1,2,3-trichloropropane gavage exposures in NTP (1993) may lead to more
pronounced irritation, inflammation, cell death, and an eventual increase in tumor incidence at
portals of entry because of direct contact of the test chemical with the gastroinstestinal tissues.
The number of test animals allocated among three dose levels and an untreated control group
was greater than the norm at 60 per group, with examination of appropriate toxicological
endpoints in both sexes of rats and mice. Alternative bioassays were unavailable. Overall
responses across the four species/sex combinations were similarly robust, all involving the
alimentary system in particular, and multiple tumor sites generally.
The impact of the corn oil vehicle on the effects observed in the forestomach following
1,2,3-trichloropropane exposures is unknown. The corn oil, combined with the bolus dosing,
may enhance cellular proliferation in the forestomach. An increased incidence and severity of
epithelial cell proliferation of the forestomach has been demonstrated in rats following the
administration of reported forestomach carcinogens in corn oil (Ghanayem et al., 1986).
However, forestomach lesions were not observed in vehicle (corn oil) controls for male and
female rats and female mice, and were observed only in male mice (3/50). Evidence of irritation,
inflammation, or necrosis localized in the forestomach of rats or mice was not observed
following the oral administration of 1,2,3-trichloropropane. In addition, while the administration
of 1,2,3-trichloropropane in corn oil may increase the residency time in the forestomach, the
effect of this increased residency time in the forestomach is unknown. The tumors observed in
the forestomach may be the result of direct interaction of the 1,2,3-trichloropropane with the
squamous epithelium lining the forestomach, or the result of 1,2,3-trichloropropane absorbed
from the intestine back into the forestomach.
For the dose-response analysis, etiologically similar tumor types (i.e., benign and
malignant tumors of the same cell type) were combined because of the possibility that the benign
tumors could progress to the malignant form as outlined in the 2005 Cancer Guidelines (U.S.
EPA, 2005a).
Choice of species/gender. The oral slope factor for 1,2,3-trichloropropane was quantified
using the tumor incidence data for female mice, which were thought to be more sensitive than
the other experimental rodents to the carcinogenicity of 1,2,3-trichloropropane. The male and
female mice tumor incidence data, while clearly demonstrating carcinogenicity, unfortunately
missed nearly all of the relevant dose-response range for mice, with both male and female mice
having nearly 100% responses at the lowest exposure level. While these responses were higher
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than those of the rats at a comparable exposure level, suggesting greater sensitivity of the mice, it
is unknown which animal species would be more sensitive at lower exposures.
Relevance to humans. The human relevance of the forestomach tumors has been noted as
a concern because humans lack a forestomach, which serves as a food storage organ. However,
the Guidelines for Carcinogen Risk Assessment (2005) state that site concordance is not a
prerequisite for evaluating the implications of animal study results for humans. The oral cavity,
pharynx, and glandular stomach are histologically similar to the rat forestomach, but the tissue
dose in these human organs is likely different than the tissue dose in the rodent forestomach, due
to prolonged exposure from the food storage function of the forestomach in rodents (Proctor et
al., 2007).
Chemicals that are genotoxic and cause tumors at multiple sites in the absence of
forestomach irritation are likely relevant to human carcinogenesis. Additionally, it has been
suggested that most genotoxic forestomach carcinogens appear to act through a mutagenic mode
of action (IARC, 2003). 1,2,3-Trichloropropane is carcinogenic through a mutagenic mode of
action and is a multisite carcinogen in rodents. Considering all of the available information, the
carcinogenicity observed in the rodent studies is considered relevant to human exposure. In
addition, the concordance of the alimentary system tumors across rats and mice lends strength to
the concern for human carcinogenic potential. The impact of gavage dosing and the potential
delayed emptying time in the forestomach lends uncertainty to the derivation of the oral slope
factor the extent of which is unknown.
Human population variability. The extent of inter-individual variability in 1,2,3-
trichloropropane metabolism has not been characterized. The human variability in response to
1,2,3-trichloropropane is also unknown. Although a mutagenic mode of action would indicate
increased early-life susceptibility, the data exploring whether there is differential sensitivity to
1,2,3-trichloropropane carcinogenicity across life stages is unavailable. This lack of
understanding about potential differences in metabolism and susceptibility across exposed
human populations thus represents a source of uncertainty. The uncertainties associated with
this lack of data and knowledge about human variability can, at present, only be discussed in
qualitative terms; however, EPA has developed ADAFs to quantitatively account for some of the
potential differences in age-dependent response to carcinogens with a mutagenic mode of action.
ADAFs are to be applied to the slope factors when assessing cancer risks that include childhood
exposures (U.S. EPA, 2005b, also see Section 5.4.5).
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6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF
HAZARD AND DOSE RESPONSE
6.1. HUMAN HAZARD POTENTIAL
1,2,3-Trichloropropane (CAS No. 96-18-4) is used in the chemical industry as a solvent
for oils and fats, waxes, and resins. The compound also is used industrially in the production of
polymers, such as polysulfide rubbers, and of some pesticides. Significant amounts of 1,2,3-
trichloropropane are produced as byproducts during the manufacture of other chlorinated
compounds, such as epichlorohydrin. The compound is found in consumer products, such as
paint thinner and varnish remover.
Toxicokinetic studies in mice and rats have examined the absorption, distribution,
metabolism, and elimination of the compound. These studies have documented the rapid
metabolism and excretion of the metabolic products in urine or feces, or on the breath
(Mahmood et al., 1991; Volp et al., 1984). The absorbed fraction of an administrated dose is
almost completely metabolized by a combination of both the phase I and phase II metabolic
pathways. Most of the metabolites are rapidly cleared from the body, although a small fraction
of the metabolites have been found to bind to intracellular proteins and nucleic acids (Weber,
1991; Weber and Sipes, 1991, 1990).
No epidemiology studies, case reports, or other studies have documented the effects of
oral exposure to 1,2,3-trichloropropane in humans. Data from a chronic toxicity test in F344/N
rats and B6C3F1 mice (NTP, 1993) and several subchronic studies (NTP, 1993; Merrick et al.,
1991; Villeneuve et al., 1985) have identified the liver as a principal target organ for noncancer
effects. All non-neoplastic changes reported following chronic oral exposure to 1,2,3-
trichloropropane occurred at doses that also produced increased incidences of tumors. A
spectrum of hepatic effects has been reported, ranging from cellular necrosis at high doses to
significantly increased organ weights at lower doses. An increase in kidney weight was
observed following subchronic (NTP, 1993; Merrick et al., 1991; Villeneuve et al. 1985) and
chronic exposure (NTP, 1993); however, overt kidney damage was not evident in these studies.
Treatment-related effects were detected in rats and mice among the hematological parameters,
but the effects were not biologically relevant or related to direct 1,2,3-trichloropropane toxicity
(NTP, 1993). Oral exposure has also been shown to reduce fertility in female CD-I mice (NTP,
1990).
There are very limited data on the effects of 1,2,3-trichloropropane inhalation in humans.
An acute inhalation study from the 1940s found that subjects exposed to 5 ppm trichloropropane
(isomer and purity not reported) for 15 minutes found the odor objectionable and complained of
irritation of the eyes and throat (Silverman et al., 1946). Likewise, there is a limited database of
inhalation toxicity studies in animals, which includes two 2-week studies submitted to EPA by
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Miller et al. (1987a, b), a 4-week range finding study, two 13-week studies, and two single-
generation reproductive assessments (Johannsen et al., 1988; Biodynamics, Inc., 1979).
Increased incidences of nonneoplastic lesions were observed in the nasal epithelium,
liver, lungs, and spleen of rats or mice following subchronic inhalation exposure to 1,2,3-
trichloropropane (Johannsen et al., 1988; Miller et al., 1987a, b; Biodynamics, Inc., 1979).
Miller et al. (1987a, b) reported decreased thickness or degeneration of the olfactory epithelium
in rats exposed for 2 weeks to concentrations of >3 ppm 1,2,3-trichloropropane (Table 4-25).
Similar effects were also observed in mice that were exposed to concentrations of >10 ppm
1,2,3-trichloropropane (Table 4-27).
Inhalation exposure to 1,2,3-trichloropropane was also associated with significant
increases in organ weights. Increased absolute and relative liver weights were observed in male
rats exposed to concentrations of >5 ppm 1,2,3-trichloropropane for 13 weeks (Johannsen et al.,
1988). Increased liver weights were observed following 2-week exposures to >40 ppm in rats
and 132 ppm in mice (Miller et al., 1987a). Other organ weight changes included increased
relative lung weights in female rats that were exposed to concentrations of >15 ppm for 13
weeks (Johannsen et al., 1988), and increased relative kidney and brain weights in male mice
exposed to 50 ppm for 13 weeks (Johannsen et al., 1988).
There are no reports of cancer in humans associated with exposure to 1,2,3-
trichloropropane. Increased incidence of tumors was observed in rats and mice following oral
exposure to 1,2,3-trichloropropane (NTP, 1993). Dose-related increasing trends in tumors were
noted at the following sites:
squamous cell carcinomas or papillomas of the alimentary system in male and
female rats and mice;
• Zymbal's gland carcinomas in male and female rats;
• pancreatic acinar cell adenomas or adenocarcinomas, preputial gland adenomas or
carcinomas, and kidney tubular cell adenomas in male rats;
• clitoral gland adenomas or carcinomas, and mammary gland adenocarcinomas in
female rats;
• hepatocellular adenomas or carcinomas, harderian gland adenomas in male and
female mice; and
• uterine/cervical adenomas or adenocarcinomas in female mice.
All of these tumor sites showed statistically significantly positive trends with increasing
exposure level (Cochran-Armitage test for trend, p<0.05, most with p^.OOl) and generally
appeared earlier with increasing exposure levels.
The hypothesized mode of action for 1,2,3-trichloropropane induced carcinogenicity is
through a mutagenic mode of action. Specifically, the data suggest that bioactivated 1,2,3-
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trichloropropane may bind directly to DNA resulting in a mutagenic event that may lead to
tumorigenicity in animals.
In vitro bacterial mutation assays have consistently demonstrated a mutagenic potential,
dependent on S9 activation, for 1,2,3-trichloropropane. Mammalian cell in vitro studies have
shown chromosomal damage, gene mutation, DNA breakage, and micronucleus formation after
1,2,3-trichloropropane exposure. In addition, in vivo assays have demonstrated the ability of
1,2,3-trichloropropane metabolites to bind to hepatic proteins, DNA, and RNA; form DNA
adducts in rats and mice; induce DNA strand breaks in the hepatocytes of rats; and to induce
wing spots (caused by genotoxic alterations such as somatic mutation, chromosomal
rearrangement, or nondisjunction) in D. melanogaster. In vivo studies measuring dominant
lethal induction or micronucleus formation were non-positive and limit the confidence in the
hypothesized mode of action. Additional in vivo assays which would provide evidence of
mutagenicity, such as mutations in tumor suppressor genes or other mutagenic markers, are
unavailable.
Given the weight of the available evidence, 1,2,3-trichloropropane acts through a
mutagenic mode of carcinogenic action andage-dependent adjustment factors (ADAFs) should
be applied.
6.2. DOSE RESPONSE
6.2.1. Noncancer—Oral
The NTP (1993) study is selected as the principal study because it was a well-designed
chronic study, conducted in both sexes in two 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. Increased liver weight is chosen as the critical effect because liver
toxicity appeared to be the most sensitive effect. There is evidence of hepatocellular damage,
including increased incidence of hepatic necrosis and decreased synthesis of pseudo-
cholinesterase, from the subchronic NTP (1993) study, and increased serum concentrations of
hepatocellular enzymes, decreased concentration of 5'-nucleotidase, and increase incidence of
histopathologic liver lesions, from the chronic NTP (1993) study. Thus, increased liver weight
represents the most sensitive endpoint in a spectrum of liver effects and occurs early in the
process of liver toxicity associated with oral exposure to 1,2,3-trichloropropane.
Other effects considered in the selection of the critical effect included kidney, respiratory,
myocardial, or reproductive toxicity endpoints. The increase in kidney weights after both
subchronic and chronic exposure was accompanied by renal tubular necrosis following
subchronic exposure and renal tubule hyperplasia following chronic exposure (NTP, 1993). In
addition, the NTP (1993) study demonstrated epithelial necrosis in the nasal turbinates of rats
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and regenerative lung lesions in mice following subchronic exposure to 1,2,3-trichloropropane.
Pulmonary toxicity including an increased incidence of inflammation-associated myocardial
necrosis in rats and increased levels of creatine kinase were also observed (NTP, 1993; Merrick
et al., 1991). NTP (1990) demonstrated a decrease in the number of pregnancies per fertile pair,
a reduction in the number of live pups/litter, and a decrease in the proportion of male pups born
alive. Although the liver appeared to be the most sensitive indicator of 1,2,3-trichloropropane-
induced toxicity, RfDs for the changes in kidney weight, fertility, and pups/liter were quantified
for comparison purposes.
BMD modeling was conducted to calculate potential PODs for deriving the chronic RfD
by estimating the effective dose at a specified level of response (BMDX) and its 95% lower
bound (BMDLX) for the changes in liver and kidney weight, fertility, and live pups/litter
associated with chronic exposure to 1,2,3-trichloropropane. A BMR of 10% was selected for the
derivation of the BMDL for liver and kidney weight increases, and the BMR of 1 SD was
modeled for comparison purposes. In the developmental study, a 10% decrease in fertility and a
1% change in mean live pups/litter for the 4th and 5th litters were selected as the BMRs due to the
frank toxicity of the reproductive toxicity endpoint.
The chronic RfD of 4 x 10"3 mg/kg-day was calculated from a BMDLADJ of 1.1 mg/kg-
day for increased absolute liver weight in male rats chronically exposed to 1,2,3-
trichloropropane by gavage (NTP, 1993). A total UF of 300 was used: 10 for interspecies
variability, 10 for interindividual variability, and 3 for database uncertainties. 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. In addition, a 3-fold database UF was applied due to the
lack of information addressing the potential developmental toxicity associated with 1,2,3-
trichloropropane. The RfD comparison figure (Figure 6-1) presents the potential PODs, applied
UFs, and derived chronic RfD and comparison RfDs for the critical effect and additional
endpoints, respectively, from Table 5-1 in Chapter 5.
The overall confidence in the chronic RfD is medium-to-high. Confidence in the
principal study (NTP, 1993) is high. Confidence in the database is medium-to-high even though
the database lacks a multigenerational developmental toxicity study. The lack of a
multigenerational study is of particular concern due to the genotoxicity of 1,2,3-
trichloropropane, because genetic damage to the germ cells of the Fl generation may not be
detected until the F2 generation. Reflecting high confidence in the principal study and medium-
to-high confidence in the database, confidence in the RfD is medium-to-high.
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100
10
CO
T3
cn
"5) 0.1
w
o
0.01
0.001
0.0001
Increased
relative liver
weight, <$ rats
(NTP, 1993)
Increased
relative
kidney
weight, c? rats
(NTP, 1993)
Fertility
generating
4th litter,
mice (NTP,
1990)
Fertility
generating
the 5th litter,
mice (NTP,
1990)
Increased
absolute liver
weight, <$ rats
(NTP, 1993)
Increased
absolute kidney
weight, $ rats
(NTP, 1993)
Live
pups/litter -
4th litter,
mice (NTP,
1990)
Live
pups/litter -
Splitter,
mice (NTP,
1990)
Point of Departure
RfD
UF, animal-to-human
UF, human variability
UF, database
Figure 6-1. PODs for selected endpoints (with critical effect circled) from Table 5-1 with corresponding applied UFs and
derived candidate chronic oral RfDs.
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6.2.2. Noncancer—Inhalation
The Johannsen et al. (1988) study is selected as the principal study because it was a well-
designed subchronic study with a sufficient number of animals per dose group. The number of
test animals allocated among five dose levels and an untreated control group was acceptable,
with examination of appropriate toxicological endpoints in both sexes of rats and mice. The
critical effect selected for the derivation of the chronic RfC is the development of peribronchial
lymphoid hyperplasia in the lungs of male CD rats, with a NOAEL of 1.5 ppm and a LOAEL of
5 ppm 1,2,3-trichloropropane, due to the occurrence of this adverse effect in both male and
female rats and the possible correlation between the hyperplasia and the observed increased lung
weight. Although an increase in liver and kidney weights was apparent, lesions and serum
enzyme levels indicative of liver and kidney damage were not evident. The only pathological
endpoint observed in the liver was hepatocellular hypertrophy in male rats at 5, 15, and 50 ppm.
In the absence of additional toxic effects in the liver (i.e., serum enzyme levels, necrosis), liver
weight and hypertrophy observed following inhalation exposure to 1,2,3-trichloropropane were
not considered biologically significant.
Johannsen et al. (1988) also conducted two single-generation reproductive toxicity
studies using 10 male and 20 female CD rats/group. Female rats exhibited decreased mating
performance at 5 ppm, where 16 out of 20 females mated, and at 15 ppm, where 10 out of 20
females mated, compared with 17 out of 20 mated females in the control group. The decrease in
the proportion of females that mated was found to be statistically significant (p<0.02) at 15 ppm
in the Fisher Exact Test conducted by EPA.
BMD modeling was conducted using EPA BMDS version 1.4.1 to analyze the increased
incidence of peribronchial lymphoid hyperplasia in CD rats and, for purposes of comparison, the
decreased mating performance in female CD rats (see Appendix C for details). The software
was used to calculate potential PODs for deriving the chronic RfC by estimating the effective
dose at a specified level of response (BMCX) and its 95% lower bound (BMCLX). For
dichotomous endpoints, the Benchmark Dose Technical Guidance Document (US EPA, 2000c)
states that an excess risk of 10% is generally the default BMR.
HECs were calculated from the candidate POD. HECs were converted to mg/m3,
adjusted to continuous exposure (7 days/week, 24 hours/day), and multiplied by a dosimetric
adjustment factor (DAF), a ratio of animal and human physiologic parameters. The specific
DAF used depends on the nature of the contaminant (particle or gas) and the target site (e.g.,
respiratory tract or remote to the portal-of-entry). The DAF for an extra-respiratory effect of a
gas is the ratio of the animal/human blood:air partition coefficients [(Hb/g)A/(Hb/g)n]. However,
the human and rat blood partition coefficients for 1,2,3-trichloropropane are not known. In
accordance with the RfC methodology (U.S. EPA, 1994b) when the partition coefficients are
unknown a ratio of 1 is used. The unknown human and rat blood partition coefficients for 1,2,3-
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trichloropropane represent a significant data gap, in which the availability of this information
would provide for a more accurate HEC calculation.
The chronic RfC of 3 x 10'4 mg/m3 was calculated from a BMCLHEc of 0.90 mg/m3 for
increased incidence of peribronchial lymphoid hyperplasia in the lungs of male CD rats
(Johannsen et al., 1988). A total UF of 3,000 was used: 3 for interspecies variability, 10 for
interindividual variability, 10 for extrapolating from a subchronic to chronic exposure duration,
and 10 for database deficiencies. A factor of 3 was selected to account for uncertainties in
extrapolating from rats to humans, which is adopted by convention where an adjustment from an
animal specific NOAELAoj to a NOAELHEc has been incorporated. Insufficient information is
available to predict potential variability in susceptibility among the population; thus, the human
variability UF of 10 was applied. A 10-fold UF was used to account for uncertainty in
extrapolating from a subchronic to chronic exposure duration. A 10-fold UF was used to
account for deficiencies in the database. The database of 1,2,3-trichloropropane inhalation
studies is lacking a multigenerational reproductive study and a developmental toxicity study.
The lack of the multigenerational study is of particular concern due to the genotoxicity of 1,2,3-
trichloropropane, because genetic damage to the germ cells of the Fl generation may not be
detected until the F2 generation. Figure 6-2 presents the potential PODs, applied UFs, and
derived chronic RfC and comparison RfC for the critical effect and additional endpoint,
respectively, from Table 5-2 in Chapter 5.
The overall confidence in the chronic RfC is low. Confidence in the principal study
(Johannsen et al., 1988) is low-to-medium. Confidence in the database is low as the database
lacks a chronic inhalation bioassay and multigenerational reproductive and developmental
toxicity studies. The lack of a multigenerational developmental study is of particular concern
due to the genotoxicity of 1,2,3-trichloropropane, because genetic damage to the germ cells of
the Fl generation may not be detected until the F2 generation. Reflecting low-to-medium
confidence in the principal study and low confidence in the database, confidence in the chronic
RfC is low.
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10
0.1
^
en
E
0.01
0.001
0.0001
HH UF. animal-to-human
i ; i ; i ; i UF , human variability
:™^ UF, subchronic-to-chronic
UF, database
i
* Point of Departure
• RfC
i
f
i
\
i
t
t
\
\
\
\
\
\
\
\
Peribronchial lymphoid \
hyperplasia, male rats, ^
(Johannsen et al. 1988)
\
\
\
nil
sss
^ /
\
\
\
\
\
\
\
\
\
i
i
t
i
t
i
i
i
1
HE
••
|
i
I Decreased mating
/ performance, female rats,
(Johannsen etal., 1988)
t |
Figure 6-2. PODs for selected endpoints (with critical effect circled) from Table 5-2 with corresponding applied UFs and derived
candidatechronic inhalation RfCs.
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6.2.3. Cancer—Oral and Inhalation
Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), 1,2,3-
trichloropropane is likely to be carcinogenic to humans, based on the existence of compelling
evidence of the compound's turn origeni city in a single, well-carried-out bioassay in two animal
species (Irwin et al., 1995; NTP, 1993). There are no studies that examine the potential
carcinogenicity of 1,2,3-trichloropropane in humans. While the use of gavage studies in
experimental animals to extrapolate to human exposure to the compound in drinking water may
introduce quantitative uncertainty, the consistent dose-dependent formation of tumors, at and
remote from the site-of-entry in two animal models, suggests a tumorigenic capacity of 1,2,3-
trichloropropane in humans.
A dose-related, statistically significant increasing trend in tumors was observed in the
following sites:
• squamous cell carcinomas or papillomas of the alimentary system in male and female rats
and mice;
• Zymbal's gland carcinomas in male and female rats;
• pancreatic acinar cell adenomas or adenocarcinomas, preputial gland adenomas or
carcinomas, and kidney tubular cell adenomas in male rats;
clitoral gland adenomas or carcinomas, and mammary gland adenocarcinomas in female
rats;
• hepatocellular adenomas or carcinomas, and harderian gland adenomas in male and
female mice; and
• uterine/ cervical adenomas or adenocarcinomas in female mice.
These tumors generally appeared earlier with increasing exposure levels, and showed
statistically significantly increasing trends with increasing exposure level. Etiologically similar
tumor types, benign and malignant tumors of the same cell type, were combined for these
tabulations because of the possibility that the benign tumors could progress to the malignant
form (U.S. EPA, 2005a). This assumption, if incorrect, has some limited potential to
overestimate the carcinogenic potential of 1,2,3-trichloropropane, and is an accepted practice
(McConnell et al., 1986).
The mode of action is a key consideration in clarifying how risks should be estimated for
low-dose exposure. A linear-low-dose extrapolation approach was used to estimate human
carcinogenic risk associated with 1,2,3-trichloropropane exposures. This approach is supported
by the positive evidence of genotoxicity and a mutagenic mode of action.
Due to the occurrence of multiple tumor types, earlier occurrence with increasing
exposure, and early termination of at least one dose group, dose-response methods which can
reflect the influence of competing risks, and intercurrent mortality on site-specific tumor
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incidence rates are preferred. EPA has generally used the multistage-Weibull model in this type
of situation, because it incorporates the time at which death-with-tumor occurred and can
account for differences in mortality observed between the exposure groups in the rat bioassay.
Additionally, etiologically different tumor types were not combined across sites prior to
modeling, in order to allow for the possibility that different tumor types can have different dose-
response relationships because of varying time courses or other underlying mechanisms or
factors.
PODs for estimating low-dose risk were identified at doses at the lower end of the
observed data, generally corresponding to 10% extra risk, defined as the extra risk over the
background tumor rate. The lifetime oral cancer slope factor for humans is defined as the slope
of the line from the lower 95% bound on the exposure at the POD. This 95% UCL represents a
plausible upper bound on the true risk.
Adjustments for approximating human equivalent slope factors applicable for continuous
exposure were calculated. Following EPA's cross-species scaling methodology, the time-
weighted daily average doses were converted to human equivalent doses on the basis of (body
weight)374 (U.S. EPA, 1992) and the estimated continuous daily exposures were calculated by
multiplying each slope factor by (5 days)/(7 days) = 0.71. The impact of applying these
adjument factors to the slope factor is unknown. The human equivalent oral slope factors
estimated from the tumor sites with statistically significant increases ranged from 0.02 to 3.0 per
mg/kg-day.
However, given the multiplicity of tumor sites, basing the oral slope factor on one tumor
site may underestimate the low-dose carcinogenic potential of 1,2,3-trichloropropane. Following
the recommendations of the NRC (1994) and the 2005 Guidelines for Carcinogen Risk
Assessment (U.S. EPA, 2005a), a statistically appropriate upper bound on total risk was
estimated in order to gain some understanding of the total risk from multiple tumor sites in male
F344/N rats (Table 5-7). Note that this estimate of overall risk describes the risk of developing
any combination of the tumor types considered, not just the risk of developing all three
simultaneously.
The recommended estimate for an upper bound on human extra cancer risk from lifetime
oral exposure to 1,2,3-trichloropropane is 30 per mg/kg-day, derived from female mice
alimentary system tumors. This estimate reflects the time-to-tumor response as well as the
exposure-response relationships for the multiple tumor sites in female rats. The value based on
female rats is recommended because female rats are the most sensitive to tumor induction
following exposure to 1,2,3-trichloropropane and yield the highest slope factor. Note that this
slope factor should not be used with human exposures greater than 0.6 mg/kg-day, since the
observed dose-response does not continue linearly above this level. Cancer risk estimates
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derived from the other available datasets ranged from 2 (female rats) to 7 per mg/kg-day (male
mice).
Because a mutagenic mode of action for 1,2,3-trichloropropane carcinogenicity is
sufficiently supported in laboratory animals and relevant to humans (Section 4.7.3.4), and in the
absence of chemical-specific data to evaluate differences in susceptibility, increased early-life
susceptibility is assumed and the ADAFs should be applied to the slope factor, as appropriate, in
accordance with the Supplemental Guidance for Assessing Susceptibility from Early-Life
Exposure to Carcinogens (U.S. EPA, 2005b).
An inhalation unit risk was not derived in this assessment. Data on the carcinogenicity of
the compound via the inhalation route are unavailable, and route-to-route extrapolation was not
possible due to the lack of an adequate physiologically based pharmacokinetic model. However,
1,2,3-trichloropropane is likely to be carcinogenic to humans by the inhalation route since the
compound is well-absorbed, and induces tumors at sites other than the portal of entry in oral
studies.
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APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC
COMMENTS AND DISPOSITION
The Toxicological Review of 1,2,3-trichloropropane has undergone a formal external
peer review performed by scientists in accordance with EPA guidance on peer review (U.S.
EPA, 2006a, 2000a). 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 also received scientific comments from the public. These
comments and EPA's responses are included in a separate section of this appendix.
On April 10, 2008, EPA introduced revisions to the IRIS process for developing chemical
assessments. As part of the revised process, the disposition of peer reviewer and public
comments, as found in this Appendix, and the revised IRIS Toxicological Review were provided
to the external peer review panel members for their comment on April 24, 2009 The external
peer reviewers did not provide any additional comments.
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 accurately, clearly and
objectively represented and synthesized the scientific evidence for noncancer and cancer
hazard??
Comments: Several reviewers agreed that the document is logical, clear, and concise.
Other reviewers considered the document redundant and confusing due to the repetition
of the toxicological data throughout the document instead of presenting a scientific basis
for using the described data to make a decision. Additionally, a reviewer questioned
whether the evidence has been completely and logically synthesized. Some of the
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reviewers recommended improving the presentation of the evidence for hazard by
reducing the redundancy of Section 4 and Section 5.
Response: The content of the Toxicological Review was consistent with the current
outline for IRIS toxicological reviews, although an effort has been made to streamline the
document and reduce the redundancy in Chapter 4 and Chapter 5.
2. Please identify any additional studies that should be considered in the assessment of the
noncancer and cancer health effects of 1,2,3-trichloropropane.
Comments: Several reviewers did not find any additional studies. Studies on the health
effects of 1,2,3-trichloropropane that were identified by the reviewers for consideration
are presented below. Additionally, a reviewer identified carcinogenesis studies for the
structurally related chemicals, ethylene dibromide and dibromochloropropane, that may
provide support for the 1,2,3-trichloropropane cancer assessment. One reviewer
commented that the IARC (1995) cancer classification for 1,2,3-trichloropropane should
be included in the document.
The following studies were identified by the external peer reviewers for
consideration:
Chroust, K., M. Pavlova, Z. Prokop, J. Mendel, K. Bozkova, Z. Kubat, V. Zajickova,
and J. Damborsky. 2007. Quantitative structure-activity relationships for toxicity
and genotoxicity of halogenated aliphatic compounds: wing spot test ofDrosophila
melanogastor. Chemosphere 67(1): 152-9
Glutathione-mediated binding of dibromoalkanes to DNA: specificity of rat
glutathione-S-transferases and dibromoalkane structure.
Inskeep PB, Guengerich FP. Carcinogenesis. 1984 Jun;5(6):805-8
Carcinogenesis Bioassay of l,2-Dibromo-3-chloropropane (CAS No. 96-12-8) in
F344/N Rats and B6C3F1 Mice (Inhalation Study). National Toxicology Program.
Natl Toxicol Program Tech Rep Ser. 1982 Mar;206:1-174
Formation of the DNA adduct S-[2-(N7-guanyl)ethyl]glutathione from ethylene
dibromide: effects of modulation of glutathione and glutathione S-transferase levels
and lack of a role for sulfation. Kim DH, Guengerich FP. Carcinogenesis. 1990
Mar;ll(3):419-24.
Direct-acting alkylating and acylating agents. DNA adduct formation, structure-
activity, and carcinogenesis. Van Duuren BL. Ann N Y Acad Sci. 1988;534:620-34
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Induction of DNA repair in rat spermatocytes and hepatocytes by 1,2-dibromoethane:
the role of glutathione conjugation. Working PK, Smith-Oliver T, White RD,
Butterworth BE. Carcinogenesis. 1986 Mar;7(3):467-72
Comparative in vivo genotoxicity and acute hepatotoxicity of three 1,2-dihaloethanes.
StorerRD, ConollyRB. Carcinogenesis. 1983 Nov;4(ll): 1491-4
Carcinogenesis Bioassay of 1,2-Dibromoethane (CAS No. 106-93-4) in F344/N Rats
and B6C3F1 Mice (Inhalation Study). National Toxicology Program. Natl Toxicol
Program Tech Rep Ser. 1982 Mar;210:l-163
Report on Carcinogenesis bioassay of 1,2-dibromoethane (EDB).
[No authors listed]
Am Ind Hyg Assoc J. 1979 Feb;40(2):A31-5
Carcinogenesis in rats of combined ethylene dibromide and disulfiram.
PlotnickHB. JAMA. 1978 Apr 21;239(16):1609
Ginsberg, G. L., Pepelko, W. E., Goble, R. L., and Hattis, D. B. "Comparison of
Contact Site Cancer Potency Across Dose Routes: Case Study with
Epichlorohydrin," Risk Analysis Vol. 16, pp. 667-681, 1996
NTP. (2005) Carcinogenesis studies of 2,2-bis(bromomethyl)-l,3-propanediol,
nitromethane, and 1,2,3-trichloropropane (CAS Nos. 3296-90-0, 75-52-5, and 96-18-
4) in guppies (Poecilia reticulate) and medaka (Oryzias latipes) (waterborne studies).
Public Health Service, U.S. Department of Health and Human Services; NTP TR 528.
NIH Publication No. 06-4464. Available from: National Institute of Environmental
Health Sciences, Research Triangle Park, NC
International Agency for Research on Cancer. 1995. IARC Monographs on the
Evaluation of Carcinogenic Risks to Humans. Volume 63. Dry cleaning, Some
Chlorinated Solvents and Other Industrial Chemicals, World Health Organization,
IARC, Lyon
Response: Several of the recommended studies were already included in the
Toxicological Review. Chroust et al. (2007) and NTP (2005) were previously included
in Sections 4.5.2, Genotoxicity Studies, and 4.4.3, Aquatic Species Studies, respectively.
In addition, the 1982 NTP inhalation study on l,2-dibromo-3-chloropropane has been
expanded in Section 4.5.3, Structural Analog Data. The references that have not been
added to the Toxicological Review include; Van Duuren, BL (1988), Working et al.
(1986), Storer and Conolly (1983), Plotnick, HB (1978), and Ginsberg et al. (1996), as
these references do not contribute significant information to the discussion and analysis
in the document. With regard to the IARC classification, EPA does not typically include
cancer characterizations of other health agencies in IRIS assessments.
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Comment: One reviewer stated that the dermal administration of 1,3-dichloroacetone
study should be included in the animal cancer section and considered in the weight of
evidence evaluation.
Response: The dermal administration study of 1,3-dichloroacetone was included as
supporting evidence of the mode of action of carcinogenicity in Section 4.7.3.2,
Experimental Support for the Hypothesized Mode of Action.
3. Please discuss research that you think would be likely to reduce uncertainty in the toxicity
values for future assessments of 1,2,3-trichloropropane.
Comments: Several reviewers suggested additional research to address the data gaps for
1,2,3-trichloropropane. Specifically, several reviewers suggested that studies addressing
the identity and role of the metabolic pathways that produce cytotoxic and/or
carcinogenic metabolites that could be utilized in the development of a PBPK model. In
addition, the reviewers suggested that PBPK modeling studies that investigate the
comparative dosimetry in animals and humans would be useful. Mode of action studies,
especially gene mutation studies, could also reduce the uncertainty in the cancer
assessment. Another reviewer highlighted the need for a mouse study that captures the
correct dose range for assessing carcinogenicity. Toxicity studies performed by
administering 1,2,3-trichloropropane in drinking water or other vehicle besides corn oil
would allow for a more accurate assessment of the toxicological effects resulting from an
exposure that resembles the potential human exposure. In addition, an inhalation cancer
bioassay and multigenerational reproductive toxicity study, as well as an oral
developmental study, were also recommended.
Response: EPA agrees that the above research recommendations would improve future
hazard identifications of 1,2,3-trichloropropane.
4. Please comment on the identification and characterization of sources of uncertainty in
sections 5 and 6 of the assessment document. Please comment on whether the key sources of
uncertainty have been adequately discussed. Have the choices and assumptions made in the
discussion of uncertainty been transparently and objectively described? Has the impact of the
uncertainty on the assessment been transparently and objectively described?
The reviewers generally agreed that the uncertainty has been clearly, transparently, and
adequately discussed, but several reviewers offered suggestions to more completely
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characterize the uncertainty associated with the 1,2,3-trichloropropane database. These
comments are presented as follows:
Comment: Two reviewers suggested more complete discussion of the relevance of
forestomach tumors in the cancer assessment, especially the potential role of hyperplasia
of the forestomach epithelium in the development of cancer.
Response: Additional discussion of the relevance of forestomach tumors in the cancer
assessment has been included in Sections 5.4.2, 5.4.4, and 5.4.6. Please also see response
to comment under questions D.3 and D.4.
Comment: One reviewer commented that the report underestimates the potential for the
corn oil vehicle to influence the carcinogenicity of 1,2,3-trichloropropane.
Response: EPA recognizes that the administration of 1,2,3-trichloropropane in corn oil
may enhance the proliferative mechanisms that may follow the genetic changes that were
the result of the mutagenic mode of action, and has included text in Section 5.4.6,
Uncertainties in Cancer Risk Values.
Comment: Several reviewers highlighted the need for consideration and discussion of
the mouse tumor data with respect to the most sensitive species being selected for the
cancer quantification.
Response: EPA originally excluded the mouse data from the cancer quantitation because
the tumor response in the lowest dose was close to a maximum response; however, based
on the external peer review comments, EPA has modeled the mouse data using the same
methods as were used with the rat data. The analysis of the mouse tumor data (added to
Section 5.4) is now used in the derivation of the OSF because the mouse is the most
sensitive species.
Comment: One reviewer stated that more discussion of the pharmacokinetic
uncertainties would be beneficial.
Response: Text was added to Section 5.4.6, Uncertainties in Cancer Risk Values.,
addressing pharmacokinetic uncertainty.
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Comment: One reviewer commented that the uncertainty needs to be based on an
improved discussion and utilization of the information that is available. More
specifically, data is available that provides estimates of relative rates of formation of a
particular adduct with DNA at varying doses that were not fully exploited. This data can
be used to develop better insight into the dosimetric aspects of the cancer risk
assessment.
Response: Sections 5.3, Uncertainties in Chronic Oral Reference Dose and Inhalation
Reference Concentration., and 5.4.6, Uncertainties in Cancer Risk Values, have been
expanded upon and modified. Also, please see response to the third comment under D.2.
B. Oral Reference Dose (RfD) for 1,2,3-Trichloropropane
1. A chronic RfD for 1,2,3-trichloropropane has been derived from a 2-year oral gavage study
(NTP, 1993) in rats and mice. Please comment on whether the selection of this study as the
principal study has been scientifically justified. Has this study been transparently and
objectively described in the document? 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 (1993) report as
the principal study was scientifically justified.
Response: No response.
Comment: One reviewer expressed concern that it is problematic to have such a large
prediction of cancer risk at the RfD. The reviewer stated that the cancer risk estimated at
the RfD of 0.004 mg/kg-day using the proposed EPA potency of 4 per mg/kg-day is
0.016 or 2 in 100. Another reviewer also raised concern over deriving an RfD at doses
that are carcinogenic.
Response: Under current Agency practice, an RfD is derived based solely on noncancer
effects observed in animal or human studies, while an oral cancer slope factor is derived
based solely on cancer effects seen in animal or human studies. In addition, these two
toxicity values are typically derived using different qualitative and quantitative analyses.
2. Increased liver weight was selected as the critical effect. Please comment on whether the
rationale for the selection of this critical effect has been scientifically justified. Is the rationale
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for this selection transparently and objectively described in the document? Please provide
detailed explanation. Please identify and provide the rationale for any other endpoints that
should be considered in the selection of the critical effect. Please comment on the use of
increased absolute liver weight instead of relative liver weight to describe the liver weight
change.
Comment: Several reviewers commented that the increase in liver weight as the critical
effect has not been scientifically justified and may be considered an adaptive response to
1,2,3-tichloropropane exposures. A reviewer stated that EPA's conclusion that increased
liver weight may be part of a continuum of adverse hepatic effects was highly
speculative.
Response: EPA considered that, given the available data, increased liver weight
represents the most sensitive effect observed in the liver and occurs early in the process
of liver toxicity associated with oral exposure to 1,2,3-trichloropropane. In addition to
increased liver weight following both subchronic and chronic exposures to 1,2,3-
trichloropropane, there is evidence of hepatocellular damage, including increased
incidence of hepatocellular necrosis and decreased synthesis of pseudocholinesterase
from the subchronic studies at higher doses. Also, increased serum concentrations of
hepatocellular enzymes (ALT and SDH), decreased concentration of 5'-nucleotidase, and
increased incidence of histopathologic liver lesions, including hepatocellular necrosis,
were observed in the chronic study at higher doses. Thus, EPA concluded that increased
liver weight may represent the most sensitive effect that occurs early in the process of
1,2,3-trichloropropane-induced hepatoxicity following oral exposure. Additionally, the
statement that "increased liver weight may be part of a continuum of adverse hepatic
effects" has been modified in Section 5.1.1, Choice of Principal Study and Critical Effect
—with Rationale and Justification, and now reads: "Increased liver weight was selected
as the critical effect because it represents the most sensitive effect observed in the liver
and occurs early in the process of liver toxicity associated with oral exposure to 1,2,3-
trichloropropane."
Comment: A reviewer questioned whether the corn oil gavage may be affecting the
pathology observed in the liver of male and female rats and mice following subchronic
exposure, and referenced the synergistic affect of corn oil and chloroform on liver
pathology.
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Response: Text has been added to Section 5.3, Uncertainties in Chronic Oral Reference
Dose and Inhalation Reference Concentration, of the draft document addressing the
effect the corn oil vehicle may have on the derivation of the RfD.
Comment: Reviewers also commented that the data presentation and discussion could be
improved by including additional non-neoplastic effects noted by NTP and a side-by-side
comparison of the dose response for liver weight increases with the development of
pathology or indirect indicators of such pathology.
Response: The sections of the document that present the observed effects of 1,2,3-
trichloropropane exposure (Section 4.2, Subchronic and Chronic Studies and Cancer
Bioassays in Animals—Oral and Inhalation), as well as the analysis of the observed
effects (Section 4.6, Synthesis of Major Noncancer Effects) and the selection of the
critical effect (Section 5.1.1, Choice of Principal Study and Critical Effect—with
Rationale and Justification) have been modified in an effort to improve the presentation
and discussion of the data. Text was added to Section 4.2 and Section 5.1.1 describing
additional non-neoplastic effects observed, and a table has been added to Section 4.6.1
that presents the observed effects and corresponding NOAELs and LOAELs for the
subchronic, chronic, and reproductive toxicity studies.
Comment: Two reviewers commented that Benchmark Dose modeling should be
conducted on additional liver endpoints, such as pseudocholinesterase levels. A reviewer
suggested that the modeling of these endpoints would be beneficial to selecting a critical
effect by considering all hepatic endpoints together and rounding to derive the point of
departure.
Response: EPA concluded that increased liver weight represents the most sensitive effect
observed in the liver and occurs early in the process of liver toxicity associated with oral
exposure to 1,2,3-trichloropropane. Additional liver endpoints following subchronic
exposure in rats were not modeled using Benchmark Dose Software. However, the
additional hepatotoxicity endpoints observed were incorporated into Chapter 5 as
supporting evidence of the selected critical effect. The supporting evidence of
hepatocellular damage includes increased incidence of hepatocellular necrosis and
decreased synthesis of pseudocholinesterase, from the subchronic NTP (1993) study, and
increased serum concentrations of hepatocellular enzymes (ALT and SDH), decreased
concentration of 5'-nucleotidase, and increase incidence of histopathologic liver lesions,
including hepatocellular necrosis, from the chronic NTP (1993) study.
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Comment: A reviewer recommended that additional consideration be provided for other
endpoints that were not modeled, but were considered by NTP to be important. These
effects included the increased severity of nephropathy, hyperplasia in the forestomach,
pancreas, and kidney.
Response: EPA recognizes that these additional effects were observed in the chronic
NTP study, but believes that the modeled endpoints, including increased liver and kidney
weight, decreased fertility, and decreased live pups/litter, are representative of the most
sensitive toxicological effects observed. Additional effects observed are utilized in the
qualitative discussion of the toxicity of 1,2,3-trichloropropane following oral exposure.
The incidence of hyperplasia in the forestomach (basal cell and squamous), kidney (renal
tubule), and pancreas (acinar) of rats and the incidence of hyperplasia in the forestomach
(squamous) of mice following chronic exposure to 1,2,3-trichloropropane (NTP, 1993)
were added to Sections 4.2.1.2, Chronic Studies; 4.6, Synthesis of Major Noncancer
Effects; and 5.1.1, Choice of Principal Study and Critical Effect—with Rationale and
Justification.
3. The chronic RfD has 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 absolute and relative kidney weight,
fertility generating the 4th and 5th litter, and the number of live pups/litter in the 4th and 5th
litters. Please provide comments with regards to whether BMD modeling is the best approach
for determining the point of departure. Has the BMD modeling been appropriately conducted
and adequately described? Is the benchmark response selected for use in deriving the POD
scientifically justified and has it been transparently and objectively described? Please identify
and provide rationale for any alternative approaches (including the selection of BMR, model,
etc.) for the determination of the point of departure, and if such approaches are preferred to
EPA's approach.
Comment: One reviewer commented that a 10% weight change was too low to use as a
benchmark response level in this modeling exercise because BMD modeling is a
conservative approach to selecting a point of departure and the critical effect selected has
a questionable toxicological significance. Another reviewer stated that the choice of the
BMR of 10% was unclear.
Response: The BMR of 10% is analogous to the 10% change in body weight used to
identify maximum tolerated doses and is considered to be the minimal level of change
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that is biologically significant. The modeling results with a BMR of 1 SD are provided
for comparison purposes.
Comment: Two reviewers commented that the benchmark response level of 1% for mean
live pups per litter was too low and clearly well below a level of change that could be
measured experimentally.
Response: The BMR selected for the mean live pups per litter was selected as the BMR
due to the frank toxicity of the reproductive toxicity endpoint, not because the 1% level
would be a level of detection for the experiment. The BMR was selected because of the
severity of the effect.
Comment: A reviewer commented that comparing the NOAELs and LOAELs for an
endpoint of interest to the Benchmark Dose Modeling results would be informative.
Response: Table 4-32 presents the NOAELs and LOAELs identified for the endpoints of
interest from the NTP (1993) study and has been added to Section 4.6, Synthesis of Major
Noncancer Effects. A direct comparison of the NOAELs and LOAELs with the BMDLs
was not conducted in Chapter 5 because a comparison of the values from a
NOAEL/LOAEL approach and a BMD modeling approach is inappropriate and does not
provide confidence in the reference value.
Comment: One reviewer requested that the modeling inputs and results be more
comprehensively described. Additionally, a reviewer commented that the modeling
outputs for the organ weight changes in the mice should be included in Appendix B.
Response: Appendix B, Benchmark Dose Modeling Results for the Derivation of the
RfD, has been expanded to include tables for each endpoint modeled and presents the
results for each model output.
4. 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 and transparently and
objectively described in the document?
Comment: One reviewer commented that the 10-fold interspecies uncertainty factor is
not justified (a 3-fold uncertainty factor is justified in the absence of human and animal
toxicodynamic data) because the livers of mice and rats have substantially higher
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CYP450 and glutathione-S-transferase activities than humans. One reviewer commented
that the 10-fold interspecies uncertainty factor was not justified and should be reduced to
3 because the higher DNA adduct formation and cell proliferation observed in mice
following oral gavage compared to drinking water.
Response: While the activities of CYP450 and glutathione-S-transferase may be
increased in rats compared to humans, we do not know enough about the mode of action
or toxicodynamics of 1,2,3-trichloropropane-induced hepatotoxicity to confidently
decrease the interspecies uncertainty factor from 10 to 3.
The interspecies uncertainty factor is applied to account for the uncertainty in
extrapolating laboratory animal data to average healthy humans. The comparison of
DNA adducts formation and cell proliferation in mice following oral gavage and drinking
water does not inform the interspecies differences between mice and humans. The DNA
adduct data was characterized as an uncertainty in the assessment in Section 5.3,
Uncertainties in Chronic Oral Reference Dose and Inhalation Reference Concentration.
Comment: One reviewer commented that because the observations in the NTP study
were made at 15 months, the point of departure should be adjusted from 15 to 24 months.
If an adjustment is not made, a subchronic-to-chronic uncertainty factor should be
applied.
Response: EPA considers the 15-month exposure period of the NTP (1993) study to be a
chronic exposure.
Comment: A reviewer commented that the BMR of 10% used for the analysis of the
liver weight change data should be interpreted as a LOAEL response and that the UFL
should be increased to a value of 3.
Response: The current approach is to address this factor as one of the considerations in
selecting a BMR for benchmark dose modeling. In this case, a BMR of a 10% change in
absolute liver weight was selected under an assumption that it represents a minimal
biologically significant change. When BMD modeling is used to derive the point of
departure, a LOAEL-to-NOAEL uncertainty factor is not applied.
5. Please comment on the transparency and scientific rationale and justification for the selection
of the database uncertainty factor. Please comment on whether the application of the database
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uncertainty factor adequately represents the gap in oral reproductive and developmental toxicity
data for 1,2,3-trichloropropane.
Comment: One reviewer commented that the 3-fold database uncertainty factor should
be applied to a point of departure from the reproductive/developmental toxicity studies
and not applied to a point of departure for liver weight changes. The reviewer
commented that application of the 3-fold database uncertainty factor to the POD for
reproductive/developmental toxicity is logical because it is a clear effect and could be
exacerbated in the next generation. Another reviewer commented that the database UF
was not justified because the point of departure for the derived RfD should be protective
of the developing fetus (when comparing BMDs and BMDLs for both endpoints). One
reviewer suggested that a 10-fold database uncertainty factor be applied because
developmental toxicity data is unavailable.
Response: EPA recognizes the adequacy of the available subchronic, chronic, and
reproductive toxicity studies following oral exposure to 1,2,3-trichloropropane, and that
the critical effect selected may be protective of developmental toxicity. However, the
database uncertainty factor is applied to account for the potential for deriving an under-
protective reference value as a result of data gaps in the characterization of the
chemical's toxicity, and is applied to the entire database of effects unless mode of action
information states otherwise. In this instance, the database uncertainty factor of 3 was
applied because the available database of subchronic, chronic, and reproductive toxicity
studies is lacking a developmental toxicity study and a reproductive toxicity study
extended beyond two-generations, due to concern for genetic damage to germ cells. EPA
believes that a database uncertainty factor of 3 accounts for the lack of a developmental
study and takes into consideration the availability of a two-generation reproductive
toxicity study.
C. Inhalation Reference Concentration (RfC) for 1,2,3-Trichloropropane
1. A chronic RfC for 1,2,3-trichloropropane has been derived from the 13 week inhalation study
(Johannsen et al., 1988) in rats. Please comment on whether the selection of this study as the
principal study is scientifically justified. Is the rationale for this selection transparently and
objectively described in the document? Please identify and provide the rationale for any other
studies that should be selected as the principal study.
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Comment: Reviewers commented that the Johannsen et al. (1988) study has not been
peer-reviewed.
Response: The Johannsen et al. (1988) study has been peer-reviewed and published in
the Journal of Toxicology and Environmental Health, Volume 25, 1988.
Comment: One reviewer commented that the decreased mating performance, which was
described by Johannsen et al. (1988) as not statistically significant, was actually
statistically significant in a two-group comparison and should be considered further as a
potential critical effect. This reviewer also stated that there are other toxicological
endpoints from Johannsen et al. (1988) that should be given more consideration as
potential critical effects.
Response: Text was added to Section 5.2.1, Choice of Principal Study and Critical
Effect-with Rationale and Justification describing the decreased mating performance
observed in the females exposed to 15 ppm 1,2,3-trichloropropane. EPA conducted a
Fisher Exact test and has included the results in Section 5.2.1. With regards to the
critical effect selected and additional toxicological effects, please see response to
Question 2 below.
2. Peribronchial lymphoid hyperplasia in the lungs of male rats was selected as the critical
toxicological effect. Please comment on whether the selection of this critical effect has been
scientifically justified. Is the rationale for this selection transparently and objectively described
in the document? Please provide detailed explanation. Please identify and provide the rationale
for any other endpoints that should be considered in the selection of the critical effect.
Comment: Several reviewers commented that the rationale for selecting peribronchial
lymphoid hyperplasia over liver weight change was not well justified, and that it would
seem that the liver weight/hepatocellular hypertrophy should be selected as the critical
effect. Specifically, when related to achieving consistency between the derivation of the
RfD and RfC. A reviewer also questioned the toxicological significance of peribronchial
lymphoid hyperplasia. Another reviewer commented that the justification was
reasonable, but the argument for its selection is not sufficiently compelling.
Response: In the case of 1,2,3-trichloropropane, the increase in liver weights observed
following the inhalation exposure was not dose-related. In the absence of additional
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effects in the liver (i.e., serum enzyme levels, necrosis), liver weight and hypertrophy
were not considered biologically significant.
In addition, there are toxicokinetic, and as a result possible toxicodynamic,
differences between the two routes of exposure. A first-pass effect by the liver is
expected for the metabolism of 1,2,3-trichloropropane following oral exposure. The
Methods for Derivation of Inhalation Reference Concentrations and Application of
Inhalation Dosimetry (U.S. EPA, 1994b) states that a route-to-route extrapolation, in this
case a qualitative extrapolation, should not be conducted when a first-pass effect by the
liver is expected.
Peribronchial lymphoid hyperplasia, also defined as lymphoid hyperplasia of the
bronchus-associated lymphoid tissue, is histologically characterized by the presence of
hyperplastic lymphoid follicles with reactive germinal centers distributed along the
bronchioles and bronchi (Howling et al., 1999; Myers and Kurtin, 1995; Fortoul et al.,
1985; Yousem et al, 1985). The peribronchial lymphoid hyperplasia is a portal-of-entry
effect that is more sensitive than any observed liver effects.
Text was reorganized and added to Section 5.2.1, Choice of Principal Study and
Critical Effect—with Rationale and Justification, addressing the selection of the critical
effect.
Comment: One reviewer commented that the argument that characterizing the liver
weight changes observed following inhalation exposure as adaptive is in conflict with
selecting increased liver weight as the critical effect following oral exposure.
Response: The increased liver weight following inhalation exposure to 1,2,3-trichloro-
propane was described as follows: "Although an increase in liver and kidney weights was
apparent, lesions and serum enzyme levels indicative of liver and kidney damage were
not evident. The only pathological endpoint observed in the liver was hepatocellular
hypertrophy in male rats at 5, 15, and 50 ppm. In the absence of a dose-related increase
in liver weight and the lack of additional effects in the liver (i.e. serum enzyme levels,
necrosis), liver weight and hypertrophy were not considered biologically significant."
The liver weight changes following oral exposure to 1,2,3-trichloropropane were
considered biologically significant as the change in liver weight was accompanied by
increased serum liver enzymes, increased incidence of hepatic necrosis, and a decrease in
pseudocholinesterase. All of these effects are indicative of liver damage and provide
support for the selection of the liver as the critical target organ. Additionally, a portal-of-
entry effect is expected following 1,2,3-trichloropropane exposure via inhalation and a
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first-pass effect is expected following oral exposure; indicating that direct comparison
between routes is not possible.
3. The chronic RfC has been derived utilizing the NOAEL/LOAEL approach to define the point
of departure. Please provide comments with regards to whether this is the best approach for
determining the point of departure. Please identify and provide rationale for any alternative
approaches (including the selection of BMR, model, etc.) for the determination of the point of
departure, and if such approaches are preferred to EPA's approach.
Comment: Several reviewers commented that benchmark dose modeling should have
been attempted, and that, in an effort to establish a good model fit, the highest-dose may
be dropped from the modeling.
Response: The increased incidence of peribronchial lymphoid hyperplasia in male and
female rats and the decreased mating performance in female rats, following inhalation
exposure to 1,2,3-trichloropropane, were modeled using Benchmark Dose Software
(1.4. Ic). The BMD analysis is now the basis for the POD used in the derivation of the
RfC and the model outputs are included in Appendix C.
Comment: One reviewer commented that BMDs and BMDLs or NOAELs and LOAELs
should be compared to the NOAEL (or BMDL) for peribronchial lymphoid hyperplasia,
in a similar manner as was done for the RfD in Section 5.1.4.
Response: Additional text and Figure 5-3 have been added to Section 5.2.4, Chronic RfC
Comparison Information, comparing the RfC derived from the BMDL for peribronchial
lymphoid hyperplasia in male rats with the RfC derived from the BMDL for decreased
mating performance in female rats.
4. Please comment on the selection of the uncertainty factors applied to the POD for the
derivation of the RfCs. For instance, are they scientifically justified and transparently and
objectively described in the document?
Comment: One reviewer stated that the use of interspecies UF of 3 was not well
justified.
Response: In this assessment, the toxicokinetic component of the interspecies
uncertainty factor is addressed by the determination of a human equivalent concentration
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as described in the RfC methodology (U.S. EPA, 1994b). However, the toxicodynamic
component of the interspecies uncertainty factor is only partially accounted for by the use
of the applied dosimetry method. The application of this uncertainty factor in the
Toxicological Review of 1,2,3-trichloropropane is in line with the guidance outlined in
the RfC methodology.
5. EPA concluded that a database uncertainty factor of 10 was appropriate for the derivation of
the RfC to account for the lack of a two-generation reproductive toxicity study and a
developmental toxicity study. Please comment on whether the selection of the database
uncertainty factor for the RfC is scientifically justified and has been transparently and
objectively described in the document.
Comment: Several reviewers commented that the 10-fold database uncertainty factor
was too excessive and stated that the oral 2-generation reproductive toxicity study should
provide adequate information addressing 1,2,3-trichloropropane's ability to alter
reproduction. A reviewer also commented that the point of departure selected would
probably be protective of reproductive and developmental effects. Conversely, a
reviewer questioned whether the database uncertainty was large enough given the lack of
a chronic inhalation study of 1,2,3-trichlororpropane.
Response: A qualitative, or quantitative, route-to-route comparison of toxicological
effects is not appropriate in this case because a portal-of-entry effect is expected
following 1,2,3-trichloropropane exposure via inhalation and a first-pass effect is
expected following oral exposure. As such, there are toxicokinetic, and as a result
possible toxicodynamic, differences between the two routes of exposure. Thus, the
utility of the oral reproductive toxicity study to decrease the database uncertainty factor is
limited as there is not enough information regarding the toxicokinetics and
toxicodynamics following both inhalation and oral exposure to make this comparison.
The point of departure from the chronic study, increased liver weight, may be
protective of reproductive and developmental effects, but the lack of an inhalation two-
generation reproductive toxicity study and a developmental toxicity study still represents
a major data gap and uncertainty.
The lack of a chronic inhalation study of 1,2,3-trichloropropane is addressed by
the application of the subchronic-to-chronic uncertainty factor.
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D. Carcinogenicity of 1,2,3-trichloropropane
1. Under the EPA's 2005 Guidelines for carcinogen risk assessment (www.epa.gov/iris/backgr-
d.htm), 1,2,3-trichloropropane is likely to be carcinogenic to humans. Please comment on the
cancer weight of the evidence characterization. Do the available data support the conclusion that
1,2,3-trichloropropane is a likely human carcinogen? Has the scientific justification for the
weight of evidence characterization been sufficiently, transparently, and objectively described?
Has the scientific justification for deriving a quantitative cancer assessment been transparently
and objectively described?
Comment: The reviewers agreed with EPA's conclusion that 1,2,3-trichloropropane is
likely to be carcinogenic to humans. The reviewers recommended improving the
transparency and objectivity of the cancer assessment by reducing the redundancy in
Section 4.7. Reviewers also requested the inclusion of structurally-similar chemicals and
identified carcinogenesis studies for ethylene dibromide and dibromochloropropane that
may provide an important group of comparisons for the cancer assessment for 1,2,3-
trichloropropane.
Response: An effort has been made to streamline and reduce the redundancy in Section
4.7. The carcinogenesis bioassays for 1,2-dibromoethane (ethylene dibromide) and 1,2,-
dibromo-3-chloropropane (DBCP) were added to Section 4.5.3, Structural Analog Data.
2. Evidence indicating the mode of action of carcinogenicity of 1,2,3-trichloropropane was
considered. The proposed mode of action includes bioactivation of 1,2,3-trichloropropane
leading to the induction of mutations in cancer-related genes. A conclusion was reached that it is
possible that this chemical is operating through a mutagenic mode of action, but the database
contains limited evidence of in vivo mutagenic events that could lead to the observed cancer.
Please comment on whether the weight of the scientific evidence supports this conclusion.
Please comment on whether the rationale for this conclusion has been transparently and
objectively described. Please comment on data available for 1,2,3-trichloropropane that may
support an alternative mode of action.
Comment: The reviewers generally indicated that the weight of evidence supports
mutagenesis as the primary mode of carcinogenic action. Specifically, the reviewer's
comments were:
• A reviewer commented that the weight of evidence supports mutagenesis as the
primary mode of action for trichloropropane; mutagenesis and cytotoxicity were
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due to reactive metabolites, and chronic irritation and cell death likely play a role
in carcinogenesis at the initial portals of entry.
• Another reviewer commented that the basic argument was not adequately
justified, although there are sufficient data to indicate that 1,2,3-TCP should be
considered a genotoxic carcinogen based upon criteria established under the
current risk assessment guidelines.
• Another reviewer commented that the conclusion could be strengthened to "very
likely" because of the known mutagenic properties of the episulfonium activated
metabolite, the dose response data on the DNA adducts in relation to
carcinogenesis, and the analogy with other mutagenic carcinogens [ethylene
dibromide (1,2-dibromoethane) and dibromochloropropane] that produce similar
or the same type of episulfonium activated intermediates via reactions with
glutathione.
• Another reviewer stated that the weight of evidence supports the conclusion that it
is possible that 1,2,3-trichloropropane is acting through a mutagenic mode of
action, but the database contains limited evidence of in vivo mutagenic events
that could lead to the observed cancer. In addition, the reviewer stated that data
are unavailable to make a determination that other modes of action, such as
cytotoxicity followed by regenerative cell proliferation, are plausible.
• A reviewer commented that there is sufficient converging scientific evidence for a
mutagenic mode of action.
• A reviewer commented that the data in support of a genotoxic mechanism of
action are limited; however, the lines of evidence presented would seem to make
a strong case for genotoxicity.
• A reviewer stated that, although the hypothesis of a likely mutagenic mode of
action is strongly supported by the available evidence, the hypothesis had yet to
be proven. This reviewer stated that there is much stronger evidence that a
mutagenic mode of action is "likely" than evidence that would suggest that
1,2,3-trichloropropane is not acting through a mutagenic mode of action.
Response: Taking into consideration the comments from the external peer reviewers and
a reevaluation of the available mode of action data, EPA concluded that 1,2,3-
trichloropropane acts through a mutagenic mode of action for carcinogenesis. The
evidence supporting a mutagenic mode of carcinogenic action includes: mutagenic
response, chromosomal damage, DNA breakage, micronucleus formation, and enhanced
DNA viral transformation in in vitro studies, covalent binding of 1,2,3-trichloropropane
metabolites to hepatic DNA, RNA, and hepatic proteins, the induction of DNA strand
152
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breaks in hepatocytes, induced wing spot formation, dose-dependent formation of DNA
adducts, and the dose-dependent increase in 1,3-dichloroacetone, a reported mutagen and
tumor initiator. The text in Section 4.7.3, Mode of Action Analysis, was revised.
Comment: A reviewer suggested an improvement in the organization of the mode of
action analysis section and that alternative modes of action should be considered,
especially with respect to forestomach tumors.
Response: The text in Section 4.7, Evaluation ofCarcinogenicity, has been reorganized.
Alternative modes of action have not been addressed in this document because the
available data for 1,2,3-trichloropropane does not support a mode of action other than
mutagenicity. Text addressing the potential for enhanced carcinogenic response due to
the use of corn oil as the vehicle has been added to Section 4.7.3.3, Other Possible Modes
of Action.
Comment: One reviewer also proposed modifying the dose response analysis to reflect
the likely saturation of the activating metabolism pathway via either depletion of
glutathione or the glutathione transferase enzymes. This reviewer stated that the DNA
adduct observations from La et al. (1995) indicated some degree of saturation.
Response: Modifying the dose response analysis to reflect the saturation of the activating
metabolic pathway was not included in the draft document. Saturation of the metabolic
pathway of 1,2,3-trichloropropane may occur, but the evidence for saturation is not
sufficient to make this type of dose conversion. The DNA adduct formation data is from
an acute gavage dose of 1,2,3-trichloropropane with sacrifice of the exposed rodents 6
hours post-exposure. The comparison of the DNA adduct formation data from the acute
study to the incidence of tumors from the chronic NTP study can not be scientifically
supported and does not contribute to the development of an oral slope factor with
improved confidence. This type of inference is not supported by the available data and
would incur additional uncertainty than what is already present.
Comment: One reviewer stated that the decision to not apply the age-dependent
adjustment factors (ADAFs), as referenced at the end of the cancer uncertainty section,
needed additional explanation. In particular, the data supporting the application of the
ADAFs based upon a mutagenic mode of action seems justified based on the Agency's
cancer risk assessment guidelines. Another reviewer recommended incorporating the
ADAFs because of the very strong likelihood of a mutagenic mode of action. One
153
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reviewer recommended not applying the ADAFs because the data are inconclusive
regarding the postulated mode of carcinogenic action for 1,2,3-trichloropropane.
Response: Upon reanalysis of the mode of action data, EPA concluded that 1,2,3-
trichloropropane is carcinogenic through a mutagenic mode of action and recommends
applying the ADAFs. The recommendation for the application of ADAFs follows the
Supplemental Guidance (U.S. EPA, 2005b). Section 5.4.5, Application of Age-
Dependent Adjustment Factors, was added to the document.
Comment: One reviewer commented that the absence of mutations in ras genes that are
consistent with the one DNA adduct known to be formed from 1,2,3-trichloropropane
provides no substantive insight into the question of whether trichloropropane is
carcinogenic via a mutagenic mode of action.
Response: The abstract in which the ras mutations were observed was removed from the
document due to a lack of adequate study documentation.
3. A two-year oral gavage cancer bioassay (NTP, 1993) was selected as the principal study for
the development of an oral slope factor (OSF). Please comment on the appropriateness of the
selection of the principal study. Has the rationale for this choice been transparently and
objectively described?
Comment: A reviewer again commented that because of the DNA adduct and cell
proliferation results of La et al. (1996), an adjustment could be made to the doses from
the NTP (1993) study to better estimate drinking water exposure.
Response: The utilization of DNA adduct formation in tumor-forming organ tissues from
an acute exposure to 1,2,3-trichloropropane to transform the administered doses in the
NTP (1993) study to "low-dose equivalents" imparts another level of uncertainly in the
derivation of the oral slope factor. See response to the third comment under question
D.2.
Comment: The reviewers generally agreed with the selection of the NTP (1993) 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: No response.
154
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Comment: One reviewer commented that the NTP (1993) cancer bioassay was limited in
characterizing the carcinogenic potency in the test species due to a high incidence of
mortality in rats and mice. This reviewer also commented that a major limitation of the
NTP (1993) bioassay is that the experimental doses exceed the "maximum tolerated
dose".
Response: The mortality in both rats and mice was attributed to cancer associated with
1,2,3-trichloropropane exposures and supports the use of the cancer bioassay for
quantitative analysis. Text was added to Section 5.4.1, Choice of Study/Data with
Rationale and Justification, addressing the increased mortality. The use of time-to-tumor
modeling makes greater use of the available data than quantal dose-response models.
Comment: Several reviewers commented that the corn oil vehicle may synergize with
carcinogens by acting as a co-carcinogen or tumor promoter, thus leading to an
overestimation of the cancer risk. One reviewer commented that the bolus nature of the
gavage dose used in the NTP (1993) bioassay should be discussed in the document.
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 forestomach following 1,2,3-trichloropropane
exposure has been added to Section 5.4.6, Uncertainties in Cancer Risk Values.
Comment: Several reviewers commented that the high frequency of tumors observed in
the forestomach, and the questionable significance of these tumors, could lead to an
overestimation of the cancer risk.
Response: EPA considers the forestomach tumors observed in rodents to be relevant to
humans. Text was added to Sections 5.4.2 and 5.4.4 to further support this conclusion.
However, in response to the recommendations of some of the external peer review panel
members, Section 5.4.4 also includes the derivation of oral slope factors for rats and mice
in which forestomach tumors were excluded from the analysis. Additionally, the
uncertainties noted by the reviewers are discussed in Section 5.4.6, Uncertainties in
Cancer Risk Values.
Comment: Reviewers commented that tumors observed in organs with no human
homolog were not relevant to human exposure and could lead to an overestimation of the
cancer risk.
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Response: EPA considers the tumor incidences for the forestomach, Zymbal's gland,
Harderian gland, and preputial gland tumors observed in the NTP (1993) study to be
relevant to human exposure. This conclusion is based on a lack of data to indicate
otherwiseand the Guidelines for Carcinogen Risk Assessment (2005) which state that site
concordance is not a prerequisite for evaluating the implications of animal study results
for humans. Additional text was added to Section 5.4.6, Uncertainties in Cancer Risk
Values.
4. Data on tumors in multiple organs in F344/N rats were used to estimate the oral cancer slope
factor. Please comment on the scientific justification and transparency of this analysis. Please
comment on the combination of etiologically similar tumor types, benign and malignant tumors
of the same cell type, for quantitative purposes. Please specifically comment on EPA's inclusion
of the data on forestomach tumors for cancer quantitation in rats following the administration of
1,2,3-trichloropropane. Please comment on the estimation of a statistically appropriate upper
bound on total risk (combined slope factor), which describes the risk of developing any
combination of tumor types considered, and the quantitative process used to calculate the
combined slope factor.
Comment: The reviewers generally agreed with the decision to combine benign and
malignant tumors of the same cell type of the same organ for quantitative purposes. A
reviewer stated that the inclusion of premalignant lesions may lead to an overestimation
of risk because the frequency with which premalignant lesions progress to carcinomas
can be quite low.
Response: Text was added to the Bioassay selection subsection of Section 5.4.6,
Uncertainties in Cancer Risk Values, that addresses the assumption that benign tumors
observed following 1,2,3-trichloropropane exposures progress to malignancy.
Comment: Four reviewers disagreed with the inclusion of forestomach tumors in the
cancer quantification, as humans do not have a forestomach or an organ that is
homologous to the rodent forestomach and that the quantification should be conducted
without forestomach tumors. One reviewer also stated that the absence of any lesions in
the forestomach of vehicle controls could alleviate concerns that the use of the corn oil
vehicle may have influenced the tumor data.
Two reviewers commented that a different mode of action may be operative for
the forestomach because of the absence of DNA adducts in the forestomach and the
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mutations found in the forestomach were not consistent with the miscoding properties of
the major adduct. Another reviewer stated that in the absence of strong evidence that the
effects in the forestomach following gavage exposure overpredicts activities local to the
site of compound administration, the forestomach tumors should not be excluded from
the assessment.
Response: EPA considers forestomach tumors to be relevant to humans and included the
data for these tumors in the quantitative carcinogenic dose-response analysis for the
derivation of the oral slope factor. Detailed discussions of human relevance were added
to Sections 5.4.2 and 5.4.4 to further support this conclusion. However, as recommended
by some of the external peer review panelists, Section 5.4.4 was revised to include the
derivation of oral slope factors for rats and mice in which forestomach tumors were
excluded from the analyses.
EPA has included text in Section 5.4.6, Uncertainties in Cancer Risk Values, that
addresses that the bolus administration of 1,2,3-trichloropropane in corn oil. In addition,
data supporting an alternative mode of action for the forestomach tumors observed in
rodents are lacking, and the effect that the corn oil vehicle and bolus dosing may have on
the carcinogenicity of 1,2,3-trichloropropane has been addressed in 5.4.6, Uncertainties
in Cancer Risk Values. As a reviewer stated, the absence of forestomach lesions in the
vehicle controls may alleviate concerns about the use of the corn oil vehicle. EPA agrees
and has noted in Section 5.4.6 that forestomach lesions were not observed in vehicle
controls for male and female rats and female mice, and were observed only in male mice
(3/50), further supporting the inclusion of the forestomach tumors in the cancer
assessment.
DNA adducts were identified in the forestomachs of both rats and mice 6 hours
following a single oral dose and demonstrated a dose-dependent increase, however, the
level of DNA adduct observed was not statistically significantly increased from the low
to high dose (La et al., 1995). The study investigating the mutations in the forestomach
was removed from the document because only the study abstract was available and
critical study information could not be ascertained. In addition, as stated by two
reviewers, the absence of mutations in the ras gene that are consistent with an identified
DNA adduct does not provide substantive insight into the question of whether or not the
compound is a mutagenic carcinogen.
Comment: One reviewer commented that the oral bolus dosing with a corn oil gavage is
not relevant to actual human exposures. Another reviewer stated that confounding by
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gavage, judging by studies of similar compounds, is unlikely to explain the large cancer
effect observed.
Response: Additional text regarding the potential effect of the corn oil vehicle on the
forestomach following 1,2,3-trichloropropane exposure has been included in Section
5.4.6, Uncertainties in Cancer Risk Values.
Comment: Six reviewers disagreed with the exclusion of the mouse data from the cancer
quantification. A reviewer commented that excluding the evidence of carcinogen!city in
mice from the oral slope factor quantification was not appropriate, and recommended
analysis of the mouse data to provide perspective on the uncertainty regarding the
differences in sensitivity of the rats and mice. Additionally, a reviewer commented that a
lack of adequate dose-response information was available from the mouse tumor
incidence data.
Response: EPA originally excluded the mouse data from the cancer quantitation because
the tumor response in the lowest dose was close to a maximum response; however, based
on the external peer review comments, EPA has modeled the mouse data using the same
methods that were used for the rat data. The analysis of the mouse tumor data (added to
Section 5.4) is now used in the derivation of the OSF because the mouse is the most
sensitive species.
Text was added to Section_5.4.6, Uncertainties in Cancer Risk Values, addressing
the near maximal tumor response observed in mice at the lowest dose.
Comment: A reviewer suggested including an analysis using Michaelis-Menten
modeling to transform the administered doses to multiples of "low-dose equivalents"
when projecting low dose risks. By calculating "low dose equivalents", the effect of high
dose metabolic saturation would be removed from the dose response model.
Response: Modifying the dose response analysis to reflect the saturation of the activating
metabolism pathway was not included in the draft document. In this case, the DNA
adduct formation in tumor-forming organ tissues from an acute exposure to 1,2,3-
trichloropropane can not be utilized to transform the administered doses in the NTP
(1993) study to "low-dose equivalents." See response to the third comment under
question D.2.
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Comment: Two reviewers disagreed with the classification of all tumors as 'incidental'
and stated that the specific tumor types in the dead and sacrificed moribund animals
ought to be modeled as fatal tumors.
Response: EPA has included additional analyses classifying the alimentary system
sqamous cell carcinomas (in all four sex-species groups) and mammary adenocarcinomas
in female rats as fatal in the unscheduled deaths.
Comment: One reviewer stated that the development of an inhalation unit risk should be
considered. One approach would be to consider structurally similar compounds that have
similar tumor findings and studies by inhalation routes.
Response: The need for an inhalation unit risk is recognized, but carcinogenicity data
following inhalation exposure to 1,2,3-trichloropropane are not available. A route-to
route extrapolation of the oral slope factor to an inhalation unit risk would be one method
to derive an inhalation unit risk, but this method is not appropriate in this case because a
portal-of-entry effect is expected following 1,2,3-trichloropropane exposure via
inhalation and a first-pass effect is expected following oral exposure. As such, there are
toxicokinetic, and as a result possible toxicodynamic, differences between the two routes
of exposure.
In addition, analyses relying on structure-activity relationships with similar
chemicals are not typically incorporated in IRIS assessments; thus, this approach was not
considered for this assessment.
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PUBLIC COMMENTS
A. Oral Reference Dose (RfD) for 1,2,3-Trichloropropane
Comment: One commenter stated that the application of the database uncertainty factor to
account for limitations in reproductive and developmental studies did not seem warranted,
and recommended a 3-fold lower UF be applied in the derivation of the RfD and RfC.
Response: The application of the database uncertainty factor in the Toxicological Review of
1,2,3-trichloropropane is in concordance with the guidance outlined in the RfC Methodology
(US EPA, 1994b) and the Review of the Reference Dose and Reference Concentration
Processes (U.S. EPA, 2002). Please see response to comment under Charge question B.5
above.
C. Carcinogenicity of 1,2,3-trichloropropane
Comment: One commenterstated that 1,2,3-trichloropropane is carcinogenic due to the
induction of tumors in multiple sites.
Response: Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), 1,2,3-
trichloropropane is "likely to be carcinogenic to humans", based on a statistically significant
and dose-related increase in the formation of multiple tumors in both sexes of two species
from an NTP (1993) chronic oral bioassay.
Comment: One commenter questioned the applicability of the gavage data in assessing
human exposures. The commenter highlighted the data investigating gavage versus drinking
water exposures and the possibility for overestimating the cancer risk in humans by using the
gavage data.
Response: As discussed above under D.3., the potential affect of the bolus nature of the
gavage dose on the tumorigenesis observed following 1,2,3-trichloropropane exposure has
been added to the Bioassay selection subsection of Section 5.4.6, Uncertainties in Cancer
Risk Values.
Comment: The commenter also stated that some of the tumors attributed to 1,2,3-
trichloropropane exposure are not relevant to humans. The commenter highlighted that the
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Zymbal's gland, Harderian gland, preputial gland, and forestomach lack human tissue
homologues, and are, therefore, not useful for quantitative dose-response assessment.
Response: As discussed above, EPA considers the tumor incidences for the forestomach,
Zymbal's gland, Harderian gland, and preputial gland tumors observed in the NTP (1993)
study are relevant to human exposure.. Please see response to comments under question D.3
and D.4.
Comment: The commenter also stated that the corn oil gavage exposures to 1,2,3-
trichloropropane utilized in the NTP (1993) bioassay are likely to overstate the cancer
potency.
Response: Please see reponses to comments under questions D.3 and D.4.
Comment: The commenter also stated that the consideration of the mode of action of
carcinogenesis of 1,2,3-trichloropropane justifies decreases in the cancer potency. The
commenter also inferred that if EPA's underlying conclusion that 1,2,3-trichloropropane is
operating through a mutagenic mode of carcinogenic action is incorrect, other nonlinear,
low-dose models are also plausible and should be acknowledged in the document.
Response: EPA disagrees that adequate data are available suggesting or indicating additional
modes of action. The mode of action data available supports a mutagenic mode of action.
Thus, the presentation of a nonlinear mode of action is not supported by the available data.
Comment: The commenter recommended the inclusion of 1,2,3-trichloropropane
metabolism and kinetics information into the estimate of cancer potency. Specifically, that
1,3-dichloroacetone is generated at a rate ten times faster by rat microsomes than by human
microsomes.
Response: The metabolic and kinetic information available for 1,2,3-trichloropropane is
suitable for qualitative analysis, but it is not sufficient for use in the quantitative derivation of
the oral slope factor for cancer. The in vitro investigation provides useful qualitative
information, thus the results of the Weber and Sipes (1992) study that demonstrated that 1,3-
dichloroacetone was generated at a rate ten-times faster by rat microsomes than by human
microsomes was added to Sections 3.3, Metabolism, and 4.7.3.2, Experimental Support for
the Hypothesized Mode of Action.
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Comment: The commenter also stated that the allometric scaling factor used to extrapolate
doses from the rats in the NTP bioassay to humans is inconsistent with the presumed mode of
action, and recommends applying an allometric scaling factor of one.
Response: The (body weight)374 adjustment for cross-species scaling (from rodent to human)
was applied in concordance with the Guidelines for Carcinogen Risk Assessment (U.S. EPA,
2005a), which follows EPA's cross-species scaling methodology which states that the time-
weighted daily average doses are converted to human equivalent doses on the basis of (body
weight)374 (U.S. EPA, 1992).
Comment: The commenter also stated that the dose-response model used in the derivation of
the oral slope factor is inappropriate for analyzing the 1,2,3-trichloropropane tumor data.
Accounting for competing causes of death with the time-to-tumor model is not needed when
assessing forestomach tumors because nearly all of the tested animals developed forestomach
tumors and these tumors were the primary cause of death.
Response: EPA agrees that competing causes of death are less of an issue at the dose levels
tested in the study. However, the analysis was undertaken in order to consider what could be
inferred about cancer risks at lower exposures, where forestomach tumors were less often the
primary cause of death, especially in rats.
Comment: The commenter also stated that the poor quality of the NTP (1993) bioassay
should be articulated. Specifically, the high incidence of early mortality due to point-of-
contact tumors in both rats and mice should be noted.
Response: Text was added to Section 5.4.1, Choice of Study/Data with Rationale and
Justification, highlighting the increased mortality in rats and mice at the intermediate and
high doses associated with the development of chemical-related neoplasms in the
forestomach.
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APPENDIX B: BENCHMARK DOSE MODELING RESULTS FOR THE
DERIVATION OF THE RFD
Benchmark dose (BMD) modeling was performed to identify the POD for the derivation
of the chronic RfD for 1,2,3-trichloropropane. The modeling was conducted in accordance with
the draft EPA guidelines (U.S. EPA, 2000b) using BMDS version 1.4.1. The BMD modeling
results for the derivation of the chronic RfD are summarized in Table B-l. In addition, the
model output results for all of the models per endpoint are presented in the corresponding tables.
The model outputs for the selected models for each endpoint are also presented. A brief
discussion of the modeling results is presented below.
The following critical effects were modeled using the BMDS version 1.4.1: increased
absolute and relative liver weight, increased absolute and relative kidney weight, decreased
fertility in the 4th litter, decreased fertility in the 5th litter, pups/litter in the 4th litter, and
pups/litter in the 5th litter. The endpoint being modeled specified which set of models,
continuous (liner, polynomial, power, and Hill) or dichotomous (gamma, logistic, multi-stage,
probit, and Weibull), would be utilized. Model eligibility was determined by assessing the
goodness-of-fit using a value of a = 0.1 (when appropriate), visual fit, and ranking by AIC.
For absolute liver weight, the male rat data using the Hill model (BMR of 10% change in
mean organ weight) was selected. The male rat data using the Hill model (BMR of 10% change
in mean organ weight) was selected as the best fit for the relative liver weight changes. Absolute
and relative liver weight changes were also modeled using a BMR of 1 SD, as recommended by
the Benchmark Dose Technical Guidance Document (U.S. EPA, 2000b). For absolute kidney
weight, the female rat data using the Hill model (BMR of 10% change in mean organ weight)
was the best fit. The male rat data using the Hill model (BMR of 10% change in mean organ
weight) was selected as the best fit for the change in relative kidney weight. The best model fit
for decreased fertility in the 4th litter was the log-Probit model (slope > 1) with a BMP of 10%
extra risk. The best model fit for decreased fertility in the 5th litter was the Probit model (BMR
of 10% extra risk). The best model fit for the number of pups/litter in the 4th litter, as well as for
the number of live pups/litter in the 5th litter, was the polynomial model (BMR of 1% change in
mean live pups/litter). The BMD results for the best fit models are summarized in Table B-l.
The critical endpoint selected for the derivation of the chronic RfD was increased liver
weight with increased absolute liver weight in male rats as the best representation of this critical
effect. The Hill model provided the best fit for this data set. The increase in absolute liver
weight was selected as the best representation of the critical effect, as opposed to relative liver
weight, which provided a BMDL very similar to the change in absolute liver weight, because it
is a more direct measure of liver weight change.
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Table B-l. BMD modeling used in the derivation of the RfD; final model
selected for each endpoint
Endpoint
Absolute liver
weight
Relative liver weight
Absolute kidney
weight
Relative kidney
weight
Decreased fertility in
the 4th litter
Decreased fertility in
the 5th litter
Pups/litter-4th litter
Pups/litter-5th litter
Species/sex
Rat/male
Rat/male
Rat/female
Rat/male
Mice
Mice
Mice
Mice
Model
Hill
Hill
Hill
Hill
Log-Probit
(slope > 1)
Probit
Polynomial
Polynomial
Goodness-of-fit
/7-value
0.677
0.986
0.359
0.549
0.9458
0.9953
0.8157
0.337
BMD
3.8
3.2
5.5
3.2
9.0
10.5
52.6
31.2
13.8
13.6
BMDL
1.6
1.4
3.1
1.8
3.4
6.4
37.3
23.3
3.2
5.6
BMR
10% extra risk
ISO
10% extra risk
ISO
10% extra risk
10% extra risk
10% extra risk
10% extra risk
1% change in
mean live
pups/litter
1% change in
mean live
pups/litter
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Absolute liver weight change
Data set
Female rat
Male rat
Female mouse
Male mouse
Model
Polynomial (linear);
power
Hill
Polynomial (linear);
power
Hill
Linear
Polynomial
(degrees = 2)
Power
Polynomial (linear);
power
Hill
Goodness-of-fit
/7-value
0.005
0.037
0.046
0.677
0.001
0.004
0.001
-0.069
0.245
AIC
31.6
27.2
67.9
63.9
-88.6
-91.3
-89.3
-29.8
-31.2
-29.8
-31.8
BMD10
10.6
1.7
12.6
3.8
46.4
40.7
39.9
42.0
Failed
BMDL10
7.8
0.6
9.6
1.6
22.0
28.7
25.4
16.1
Failed
Hill Model. (Version: 2.12; Date: 02/20/2007)
Input Data File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS
MOVED\M_R_ABLIVWT.(d)
Gnuplot Plotting File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS
MOVED\M_R_ABLIVWT.pit
Mon Apr 16 12:20:13 2007
BMDS MODEL RUN
The form of the response function is:
Y[dose] = intercept + v*dose*n/(k*n + dose^n)
Dependent variable = MEAN
Independent variable = Dose
rho is set to 0
Power parameter restricted to be greater than 1
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
rho
intercept
v
n
k
1.78118
0
14 .27
3 .96
0.217686
13 .2906
Specified
Asymptotic Correlation Matrix of Parameter Estimates
165
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the user,
*** The model parameter(s) -rho -n
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
alpha
intercept
V
k
3.
9.
9.
alpha
1
,6e-007
,4e-007
,9e-007
intercept
3 .6e-007
1
-0 .0082
0.53
V
9.4e-007
-0.0082
1
0.78
k
9 .9e-007
0.53
0 .78
1
Parameter Estimates
Variable
alpha
intercept
V
n
k
Estimate
1.60099
14 .3111
5.12912
1
9.74696
Std. Err.
0.367293
0.393288
1.17736
NA
6 .65395
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
0.881113 2.32088
13.5403 15.082
2.82153 7.43672
-3.29454
Model Descriptions for likelihoods calculated
22.7885
NA - 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 Dev Scaled Res.
0
3
10
30
10
10
10
8
14.3
15.6
16 . 8
18 .2
14 .3
15.5
16 .9
18 .2
1.17
1.17
1 .52
1 .47
1.27
1.27
1.27
1.27
-0.103
0.279
-0.271
0. 106
Model Al : Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma*2
Model A2 : Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma (i)*2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma*2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e (i) }
Likelihoods of Interest
Model Log(likelihood)
Al -27.854952
A2 -27.294744
A3 -27.854952
fitted -27.941868
R -43.424328
# Param's
5
8
5
4
2
AIC
65.709904
70.589488
65. 709904
63 . 883737
90.848657
Explanation of Tests
Test 1: Do responses and/or variances differ among Dose levels?
(A2 vs. R)
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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
Tests of Interest
-2*log(Likelihood Ratio) Test df
Test
Test
Test 4
32 .2592
1. 12042
1. 12042
0.173833
p-value
<.0001
0.7721
0.7721
0.6767
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. The modeled variance appears
to be appropriate here
The p-value for Test 4 is greater than .1.
to adequately describe the data
The model chosen seems
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
20
19
18
17
a:
CO
16
15
14
13
0.1
Relative risk
0. 95
3 .77203
1.60397
Hill Model with 0.95 Confidence Level
Hill
BMD Lower Bound
BMDL
BIV|D
0
10
15
dose
20
25
30
12:2004/162007
167
-------
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 3.19188
BMDL = 1.42159
20
19
18
o 17
Q. ' '
a:
CO
a>
16
15
14
13
Hill
BIVIDL
Hill Model with 0.95 Confidence Level
BMP
10
15
dose
20
25
30
14:1805/072007
168
-------
Relative liver weight change
Data set
Female rat
Male rat
Female mouse
Male mouse
Model
Linear
Polynomial
Power
Linear
Polynomial
Power
Hill
Linear
Polynomial
Power
Linear
Polynomial
Power
Goodness-of-fit
^j-value
0.479
0.255
0.297
0.062
0.018
0.018
0.986
0.033
0.629
O.00001
0.026
0.007
O.00001
AIC
97.7
99.5
101.3
100.3
100.3
104.3
98.8
121.9
115.3
116.9
184.0
184.0
185.7
BMD10
7.0
7.9
8.4
11.8
11.8
11.8
5.5
15.9
29.7
34.9
12.8
13.4
14.0
BMDL10
6.1
6.2
6.2
9.7
9.7
9.7
3.1
12.4
20.7
21.9
7.0
7.0
7.1
Hill Model. (Version: 2.12; Date: 02/20/2007)
Input Data File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS
MOVED\M_R_REL_LIVERWT.(d)
Gnuplot Plotting File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS
MOVED\M_R_REL_LIVERWT.pit
Mon Apr 16 15:05:35 2007
HMDS MODEL RUN
The form of the response function is:
Y[dose] = intercept + v*dose*n/(k*n + dose^n)
Dependent variable = MEAN
Independent variable = Dose
rho is set to 0
Power parameter restricted to be greater than 1
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
rho
intercept
v
n
k
4.47912
0 Specified
31.2
8 . 6
0 .478123
11.2069
Asymptotic Correlation Matrix of Parameter Estimates
( *** xhe model parameter(s) -rho -n
169
-------
the user,
alpha
intercept
v
k
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
alpha intercept v k
1 -1.8e-008 -3.6e-008 -2.7e-008
-l.Se-008 1 0.25 0.55
-3.6e-008 0.25 1 0.91
-2.7e-008 0.55 0.91 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable
alpha
intercept
v
n
k
Estimate
4 .00767
31.2041
14 .2018
1
19.5753
Std. Err.
0.919422
0.591154
3 .58574
NA
11.3509
Lower Conf. Limit
2 .20563
30.0455
7.17388
-2 .67211
Upper Conf . Lim
5.8097
32 .3627
21.2297
41.8227
NA - 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 Dev Scaled Res.
0
3
10
30
10
10
10
8
31.2
33.1
36
39. 8
31 .2
33 .1
36
39 .8
1. 9
2.2
1.9
2 . 5
2
2
2
2
-0. 00647
0.0137
-0.00949
0. 00258
Model Descriptions for likelihoods calculated
Model Al: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma*2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)*2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma*2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)}
Likelihoods of Interest
Model
Al
A2
A3
fitted
R
Log (likelihood)
-45.375808
-44.937444
-45.375808
-45.375971
-68.896353
# Param's
5
8
5
4
2
AIC
100.751617
105.874888
100.751617
98.751942
141.792706
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)
170
-------
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
47.9178
0.876729
0 .876729
0 .00032515
p-value
<.0001
0.831
0 .831
0.9856
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 = 0.1
Risk Type = Relative risk
Confidence level = 0.95
BMD = 5.51221
BMDL = 3.14799
42
40
| 38
§36
CO
32
30
Hill
BMDL
Hill Model with 0.95 Confidence Level
BMD
10
15
dose
20
25
30
15:0504/162007
171
-------
Benchmark Dose Computation
Specified effect = 1
Risk Type = Estimated standard deviations from the control mean
Confidence level = 0.95
BMD = 3.21217
BMDL = 1.83718
Hill Model with 0.95 Confidence Level
42
40
CD O Q
(/) OO
O
cS 36
or
c
cc
i 34
32
30
~-
r
r
r
r
!
; B
Hill
i i i i -
•:
^___ <> :
^^~—^~ j
^~- -_
/
>
MDL
^X
S^
BMP ;
0 5 10 15 20 25 30
dose
14:5505/072007
172
-------
Absolute kidney weight
Data set
Female rat
Male rat
Female mouse
Male mouse
Model
Polynomial (linear);
power
Hill
Polynomial (linear);
power
Hill
Goodness-of-fit
^j-value
0.444
0.359
0.320
0.174
AIC
-153.0
-151.8
-123.6
-122.0
BMD10
14.2
9.0
11.2
8.6
BMDL10
10.8
3.4
9.0
4.1
Data not modeled due to lack of dose-response trend
Polynomial (linear);
power
0.342
-162.9
55.0
28.2
Hill Model. (Version: 2.12; Date: 02/20/2007)
Input Data File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS
MOVED\F_R_ABSKIDNEYWT.(d)
Gnuplot Plotting File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS
MOVED\F_R_ABSKIDNEYWT.pit
Thu Apr 19 13:10:47 2007
BMDS MODEL RUN
The form of the response function is:
Y[dose] = intercept + v*dose*n/(k*n + dose^n)
Dependent variable = MEAN
Independent variable = Dose
rho is set to 0
Power parameter restricted to be greater than 1
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 =
rho =
intercept =
v =
n =
k =
0 .00477394
0
0.786
0. 185
0 .229976
48 .1373
Specified
the user,
Asymptotic Correlation Matrix of Parameter Estimates
( *** xhe model parameter(s) -rho -n
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
alpha intercept v k
173
-------
alpha
intercept
V
k
1
-le-008
-5.8e-008
-5. 5e-008
-le-008
1
0.47
0 .61
5.8e-008
0.47
1
0. 97
-5.5e-008
0 .61
0.97
1
Parameter Estimates
Variable
alpha
intercept
v
n
k
Estimate
0.00434387
0.793675
0.366433
1
32.6862
Std. Err.
0.00102386
0.0198246
0.275832
NA
46 .6525
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
0.00233714 0.0063506
0.754819 0.83253
-0.174189 0.907054
-58.7509
124.123
Table of Data and Estimated Values of Interest
Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev
Scaled Res.
0
3
10
30
10
10
8
8
0.786
0. 839
0. 869
0.971
0.794
0 .824
0. 88
0.969
0.047
0. 073
0. 054
0.096
0.0659
0. 0659
0. 0659
0.0659
-0.368
0. 697
-0.451
0.0841
Model Descriptions for likelihoods calculated
Model Al: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma*2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)*2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma*2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)} =
Likelihoods of Interest
Model
Al
A2
A3
fitted
R
Log (likelihood)
80.322604
82.968318
80.322604
79.901816
67.518029
# Param's
5
8
5
4
2
AIC
-150.645208
-149.936636
-150.645208
-151.803632
-131.036058
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
174
-------
Test
-2*log(Likelihood Ratio) Test df
p-value
Test 1
Test 2
Test 3
Test 4
30 .9006
5.29143
5.29143
0 .841576
<.0001
0.1517
0.1517
0.3589
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 modeled variance appears
The p-value for Test 4 is greater than .1. The model chosen seems
to adequately describe the data
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Relative risk
Confidence level = 0.95
BMD = 9.03706
BMDL = 3.3571
Hill Model with 0.95 Confidence Level
8.
CO
CD
a:
c
CD
1.05
0.95
0.9
0.85
0.8
0.75
Hill
BMDL
BMP
10
15
dose
20
25
30
13:1004/192007
175
-------
Relative kidney weight
Data set
Female rat
Male rat
Female mouse
Male mouse
Model
Linear
Polynomial
Power
Linear
Polynomial
Power
Hill
Linear
Polynomial
Power
Linear
Polynomial
Power
Goodness-of-fit
^j-value
0.052
0.020
0.027
0.985
0.871
0.865
0.549
0.169
0.096
0.085
0.575
0.403
0.430
AIC
-60.3
-58.8
-57.3
-85.8
-83.8
-83.8
-84.1
-30.3
-29.1
-26.9
31.1
30.7
32.6
BMD10
8.4
10.7
11.1
10.6
10.8
10.7
10.5
32.5
41.4
43.9
92.4
70.3
61.3
BMDL10
7.0
7.1
7.2
9.2
9.2
9.2
6.4
23.8
24.9
24.6
34.5
45.3
36.8
Hill Model. (Version: 2.12; Date: 02/20/2007)
Input Data File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS
MOVED\M_R_REL_KIDNEYWT.(d)
Gnuplot Plotting File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS
MOVED\M_R_REL_KIDNEYWT.pit
Thu Apr 19 13:56:54 2007
HMDS MODEL RUN
The form of the response function is:
Y[dose] = intercept + v*dose*n/(k*n + dose^n)
Dependent variable = MEAN
Independent variable = Dose
rho is set to 0
Power parameter restricted to be greater than 1
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
rho
intercept
v
n
k
0. 0360382
0
2 .96
0 .86
0 .542711
45.0877
Specified
Asymptotic Correlation Matrix of Parameter Estimates
( *** xhe model parameter(s) -rho -n
176
-------
the user,
alpha
intercept
v
k
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
alpha intercept v k
1 0.0005 0.00077 0.00077
0.0005 1 0.65 0.65
0.00077 0.65 1 1
0.00077 0.65 1 1
Parameter Estimates
Variable
alpha
intercept
v
n
k
Estimate
0.032551
2 .97723
49.0294
1
1717.72
Std. Err.
0.00746772
0.0512988
1180.31
NA
42099
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
0.0179146 0.0471875
2.87668 3.07777
-2264.34 2362.4
-80794 .(
Model Descriptions for likelihoods calculated
84230.2
NA - 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 Dev Scaled Res.
0
3
10
30
10
10
10
8
2 .96
3 .09
3 .25
3 .82
2 . 98
3.06
3.26
3 . 82
0 .13
0.28
0.16
0 .14
0. 18
0.18
0.18
0. 18
-0.302
0.478
-0.193
0 .0184
Model Al: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma*2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)*2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma*2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e(i)}
Likelihoods of Interest
Model
Al
A2
A3
fitted
R
Log (likelihood)
46.253609
50.301116
46.253609
46.073978
19.835849
# Param's
5
8
5
4
2
AIC
-82 . 507217
-84.602232
-82.507217
-84 . 147957
-35.671698
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)
177
-------
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
60.9305
8.09501
8.09501
0.35926
p-value
<.0001
0.04409
0 .04409
0. 5489
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. Consider running a
non-homogeneous variance model
The p-value for Test 3 is less than .1. You may want to consider a
different variance model
The p-value for Test 4 is greater than .1. The model chosen seems
to adequately describe the data
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Relative risk
Confidence level = 0.95
BMD = 10.4943
BMDL = 6.39915
Hill Model with 0.95 Confidence Level
4
3.8
8> 3-6
I
I 3.4
CO
CD
3.2
2.8
Hill
BMDL
BMD
10
15
dose
20
25
30
13:5604/192007
178
-------
th
Decreased fertility in the 4 litter
Model
log-probit (slope > 1)
Multistage (degree =1)
Weibull (power > 1)
Gamma (power > 1)
Log-logistic (slope > 1)
Probit
Logistic
Goodness-of-fit
/7-value
0.9458
0.9058
0.9509
0.9426
0.9372
0.6667
0.6003
AIC
46.54
48.23
48.31
48.33
48.34
49.37
49.72
BMD10
52.6
54.6
51.7
51.2
51.2
68.8
73.5
BMDL10
37.3
26.1
25.9
25.8
23.6
51.7
55.9
Probit Model. (Version: 2.8; Date: 02/20/2007)
Input Data File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS MOVED\BMD
2\FERTILITY_FOURTH_LITTER.(d)
Gnuplot Plotting File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS
MOVED\BMD 2\FERTILITY_FOURTH_LITTER.pit
Mon Apr 23 10:51:16 2007
BMDS 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 = Infertile
Independent variable = Dose
Slope parameter is restricted as slope >= 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
User has chosen the log transformed model
Default Initial (and Specified) Parameter Values
background = 0
intercept = -5.20395
slope = 1
Asymptotic Correlation Matrix of Parameter Estimates
( *** xhe model parameter(s) -background -slope
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
intercept
intercept 1
the user,
Parameter Estimates
179
-------
Variable
background
intercept
slope
Estimate
0
-5.24473
1
Std. Err.
NA
0.214728
NA
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
-5.66559
NA - Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
-4.82387
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-22 . 1049
-22.2676
-29.6693
46.5353
# Param's
4
1
1
Deviance Test d.f.
0.325422
15.1288
P-value
0.9552
0.00171
Goodness of Fit
Dose
0 .0000
30 .0000
60.0000
120.0000
Est. Prob.
0. 0000
0. 0326
0.1250
0.3237
Expected
0 .000
0 .587
2 .375
6 .151
Observed
0
1
2
6
Size
38
18
19
19
Scaled
Residual
0. 000
0. 548
-0.260
-0.074
Chi*2 = 0.37
d.f. = 3
P-value = 0.9458
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 52.6244
BMDL = 37.3271
180
-------
0.6
0.5
si
< 0.3
c
o
5 0.2
0.1
Probit Model with 0.95 Confidence Level
Probit
BMD Lower Bound
BMDL
BMD
20
40
60
dose
80
100
120
10:51 04/232007
181
-------
th
Decreased fertility in the 5 litter
Model
Multistage (degree =1)
Weibull (power > 1)
Gamma (power > 1)
Log-logistic (slope > 1)
Log-probit (slope > 1)
Probit
Logistic
Goodness-of-fit
/7-value
0.8078
0.8516
0.8244
0.7913
0.7351
0.9953
0.9925
AIC
102.66
104.26
104.27
104.30
104.34
102.23
102.24
BMD10
20.1
32.8
33.1
34
34.4
31.2
33
BMDL10
12.6
13
13
10.3
22.3
23.3
24.6
Probit Model. (Version: 2.8; Date: 02/20/2007)
Input Data File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS
MOVED\MICE_INFERTILITY.(d)
Gnuplot Plotting File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS
MOVED\MICE_INFERTILITY.pit
Mon Apr 23 10:26:34 2007
HMDS MODEL RUN
The form of the probability function is:
P [response] = CumNorm(Intercept + Slope*Dose),
where CumNorm(.) is the cumulative normal distribution function
Dependent variable = infertile
Independent variable = Dose
Slope parameter is not restricted
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
Default Initial (and Specified) Parameter Values
background = 0 Specified
intercept = -1.10027
slope = 0.0107802
the user,
intercept
slope
Asymptotic Correlation Matrix of Parameter Estimates
( *** xhe model parameter(s) -background
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
intercept slope
1 -0.74
-0.74 1
Parameter Estimates
95.0% Wald Confidence Interval
182
-------
Variable
intercept
slope
Estimate
-1.11544
0.0109181
Std. Err.
0.214289
0.00314451
Lower Conf. Limit
-1.53544
0.00475495
Upper Conf. Limit
-0.695445
0.0170812
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-49. 1124
-49.1172
-55.4327
102 .234
# Param's Deviance Test d.f. P-value
4
2 0.00946155 2 0.9953
1 12.6405 3 0.005482
Goodness of Fit
Dose
0 .0000
30 .0000
60.0000
120.0000
Est. Prob.
0. 1323
0.2154
0.3226
0.5772
Expected
5 .029
3 .877
6 .130
10.967
Observed
5
4
6
11
Size
38
18
19
19
Scaled
Residual
-0. 014
0. 071
-0.064
0.015
Chi'
0. 01
d.f.
P-value = 0.9953
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
o.i
Extra risk
0.95
31.1591
23.2749
Probit Model with 0.95 Confidence Level
0.8
0.7
0.6
1 °'5
0 °'4
£ 0.3
u_
0.2
0.1
0
Pro
BMD Lower Bou
-
-
-
-
!
-
[
^^^^
Biyipu
0 20
bit
nd
-"''
^^+
,^^\^~~^~~^
^_^--^^
^^^'
-_
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^
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'-.
BMD, , , , \
40 60 80 100 120
dose
10:2604/232007
183
-------
th
Live pups per litter in the 4 litter
Model
Linear
Polynomial
Power
Goodness-of-fit /7-value
0.006
0.816
0.666
AIC
305.4
295.6
297.4
BMD1%
1.7
13.8
18.7
BMDL1%
1.5
3.2
6.2
Polynomial Model. (Version: 2.12; Date: 02/20/2007)
Input Data File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS MOVED\BMD
2\LIVE_PUPS_4TH_LITTER.(d)
Gnuplot Plotting File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS
MOVED\BMD 2\LIVE_PUPS_4TH_LITTER.pit
Wed May 02 08:54:44 2007
BMDS MODEL RUN
The form of the response function is:
Y[dose] = beta 0 + beta l*dose + beta 2*dose*2 +
Dependent variable = MEAN
Independent variable = Dose
The polynomial coefficients are restricted to be negative
The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)
Total number of dose groups = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
lalpha = 2.4801
rho = 0
beta_0 = 11.7373
beta_l = 0
beta 2 = -0.000679293
the user,
Asymptotic Correlation Matrix of Parameter Estimates
( *** xhe model parameter(s) -beta_l
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
beta_2
0 .0025
-0.0044
-0 .66
1
lalpha
rho
beta 0
beta 2
lalpha
1
-0.98
-0. 0063
0.0025
rho
-0 .98
1
0. 007
-0.0044
beta_0
-0. 0063
0.007
1
-0.66
Variable
lalpha
rho
Estimate
0.737776
0.745657
Parameter Estimates
95.0% Wald Confidence Interval
Std. Err. Lower Conf. Limit Upper Conf. Limit
0.731853 -0.696628 2.17218
0.324171 0.110295 1.38102
184
-------
beta_0
beta_l
beta 2
11.8652
0
-0.00062153
0.454377
NA
5.41197e-005
10.9746
-0.000727603
12.7557
-0.000515458
NA - 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 Dev
0 38
30 17
60 17
120 13
Scaled Res.
11.8
11.2
9. 9
2.9
11.9
11 .3
9. 63
2.92
3.7
2 .88
4 .12
2 .17
3.64
3 . 57
3 .36
2.15
-0.11
-0. 122
0.334
-0.0253
Model Descriptions for likelihoods calculated
Model Al:
Model A2:
Yil =
Var{e(ij)} = Sigma*2
Yi
Var{e(ij)
= Mu(i) + e(
= Sigma(i)*2
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)}
Likelihoods of Interest
Model
Al
A2
A3
fitted
R
Log (likelihood)
-145.855593
-142.282446
-143.607572
-143.811261
-171.536421
# Param's
5
8
6
4
2
AIC
301.711185
300.564892
299.215144
295.622522
347.072841
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
Tests of Interest
-2*log(Likelihood Ratio) Test df
Test
Test
Test 4
58 .5079
7. 14629
2 . 65025
0.407378
p-value
<.0001
0.06738
0.2658
0.8157
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
185
-------
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 = 0.01
Risk Type = Relative risk
Confidence level = 0.95
BMD = 13.8167
BMDL =
3 .22598
14
12
10
Polynomial Model with 0.95 Confidence Level
o
Q.
o:
I Q
Polynomial
3.MPL BMP
0
08:54 05/02 2007
20
40
60
dose
80
100
120
186
-------
th
Live pups per litter in the 5 litter
Model
Linear
Polynomial
Power
Goodness-of-fit /7-value
O.0001
0.337
0.507
AIC
210.4
193.0
193.3
BMD1%
1.8
13.6
24.5
BMDL1%
1.6
5.5
11.6
Polynomial Model. (Version: 2.12; Date: 02/20/2007)
Input Data File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS MOVED\BMD
2\LIVE_PUPS_5TH_LITTER.(d)
Gnuplot Plotting File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS
MOVED\BMD 2\LIVE_PUPS_5TH_LITTER.pit
Wed May 02 09:00:47 2007
BMDS MODEL RUN
The form of the response function is:
Y[dose] = beta 0 + beta l*dose + beta 2*dose*2 +
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
Default Initial Parameter Values
alpha = 5.92144
rho =
beta_0 = 12.6118
beta_l = 0
beta 2 = -0.000926768
Specified
the user,
alpha
beta_0
beta 2
Asymptotic Correlation Matrix of Parameter Estimates
( *** xhe model parameter(s) -rho -beta_l
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
alpha beta_0 beta_2
1 1.5e-009 4.5e-009
1.5e-009 1 -0.49
4.5e-009 -0.49 1
Variable
Parameter Estimates
Estimate
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
187
-------
alpha
beta_0
beta_l
beta 2
5.75447
12.9649
0
-0.000703957
0.986884
0.33453
NA
6 .433316-005
3.82022
12.3092
-0.000830048
7.68873
13.6205
-0.000577866
NA - 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 Dev
0 33
30 14
60 13
120 8
Scaled Res.
12 . 8
12 . 1
11.3
2.5
13
12 .3
10.4
2.83
2 .3
2 .62
2 .89
1.7
2 .4
2 .4
2 .4
2 .4
-0.395
-0.361
1.31
-0.387
Model Descriptions for likelihoods calculated
Model Al: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma*2
Model A2: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma(i)*2
Model A3: Yij = Mu(i) + e(ij)
Var{e(ij)} = Sigma*2
Model A3 uses any fixed variance parameters that
were specified by the user
Model R: Yi = Mu + e(i)
Var{e (i)}
Likelihoods of Interest
Model
Al
A2
A3
fitted
R
Log(likelihood)
-92.410493
-90.930911
-92.410493
-93.499230
-128 . 027125
# Param's
5
8
5
3
2
AIC
194.820986
197.861821
194.820986
192.998461
260.054251
Explanation of Tests
Test 1: Do responses and/or variances differ among Dose levels?
(A2 vs. R)
2: Are Variances Homogeneous? (Al vs A2)
3: Are variances adequately modeled? (A2 vs. A3)
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
Test
Test
Test 1
Test 2
Test 3
Test 4
Tests of Interest
-2*log(Likelihood Ratio) Test df
74 .1924
2.95916
2.95916
2.17747
p-value
<.0001
0 .398
0.398
0.3366
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
model appears to be appropriate here
.1. A homogeneous variance
188
-------
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 = 0.01
Risk Type = Relative risk
Confidence level = 0.95
BMD = 13 .571
BMDL =
5. 5772
Polynomial Model with 0.95 Confidence Level
o>
14
12
10
o
°- R
w o
OL
a 6
Polynomial
BMDL
BMP
20
40
60
dose
80
100
120
09:00 05/02 2007
189
-------
APPENDIX C: BENCHMARK DOSE MODELING RESULTS FOR THE
DERIVATION OF THE RFC
Benchmark dose (BMD) modeling was conducted to identify the POD for the derivation
of the chronic RfC for 1,2,3-trichloropropane. The modeling was conducted in accordance with
the draft EPA guidelines (U.S. EPA, 2000b) using HMDS version 1.4.1. The BMD modeling
results for the derivation of the chronic RfC are summarized in Table C-l. In addition, the
model output results for all of the models per endpoint are presented in the corresponding tables,
and the model outputs for the selected models for each endpoint are also presented. A brief
discussion of the modeling results is presented below.
The following critical effects were modeled using the BMDS version 1.4.1: increased
incidence of peribronchial lymphoid hyperplasia in male and female CD rats and decreased
mating performance in female CD rats. The endpoints were modeled using the dichotomous
models (gamma, logistic, multi-stage, probit, and Weibull). Model eligibility was determined by
assessing the goodness-of-fit using a value of a = 0.1 (when appropriate), visual fit, and ranking
by AIC. The decrease in mating performance in female CD rats was presented as the incidence
of females that mated compared to total females, but when modeled the incidence data were
modified to females that did not mate compared to total females. This was done because the
dichotomous models can have difficulty modeling incidence data that decreases with increasing
exposure.
Adequate model fits were not available for the increased incidence of peribronchial
lymphoid hyperplasia in female rats. An adequate model fit was available for a single
constrained model, log-logistic (slope > 1), following BMD modeling of increased incidence of
peribronchial lymphoid hyperplasia in male rats.
The BMD modeling of the decrease in mating performance in female CD rats resulted in
adequate model fits from all of the dichotomous models. The log-probit model was selected to
represent the decreased mating performance because it provided an adequate model fit and the
lowest AIC.
190
-------
Table C-l. BMD modeling used in the derivation of the RfC; final model
selected for each endpoint
Endpoint
Peribronchial
lymphoid
hyperplasia
Decreased mating
performance
Species/sex
Rat/male
Rat/female
Model
Log-logistic
(slope > 1)
Log-probit
Goodness-of-
fit/7-value
0.1081
0.3933
AIC
69.7
86.0
BMC
1.6
4.5
BMCL
0.84
3.0
BMR
10% extra
risk
10% extra
risk
191
-------
Peribronchial lymphoid hyperplasia
Male CD rats
Model
Log-logistic (slope > 1)
Multistage (degree = 1),
Weibull (power > 1),
gamma (power > 1)
log-probit (slope > 1)
Goodness-of-fit
/7-value
0.1081
0.0065
0.0012
AIC
69.7
73.7
72.3
BMD
1.6
2.5
3.3
BMDL10
0.84
1.8
2.4
Female CD rats
Model
Log-logistic (slope > 1)
Multistage (degree =1)
Weibull (power > 1)
Gamma (power > 1)
Log-probit (slope > 1)
Goodness-of-fit
/7-value
0.0697
0.0414
0.0414
0.0414
0.0133
AIC
77
78.1
78.1
78.1
81.4
BMD
5.7
8.2
8.2
8.2
16.8
BMDL10
2.9
4.7
4.7
4.7
8.9
Logistic Model. (Version: 2.10; Date: 09/23/2007)
Input Data File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS
MOVED\PERI_LYMPHOID_HYPERPLASIA_MALES050608.(d)
Gnuplot Plotting File: C:\DOCUMENTS AND SETTINGS\MGEHLHAU\DESKTOP\BMDS
MOVED\PERI_LYMPHOID_HYPERPLASIA_MALES050608.pit
Tue May 06 14:56:25 2008
HMDS MODEL RUN
The form of the probability function is:
P [response] = background+(1-background)/[1+EXP(-intercept-siope*Log(dose))]
Dependent variable = perilymphyper
Independent variable = exposure
Slope parameter is restricted as slope >= 1
Total number of observations = 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
User has chosen the log transformed model
Default Initial Parameter Values
background = 0
intercept = -2.86665
slope = 1.10359
the user,
Asymptotic Correlation Matrix of Parameter Estimates
( *** xhe model parameter(s) -background
have been estimated at a boundary point, or have been specified by
192
-------
intercept
slope
and do not appear in the correlation matrix
intercept slope
1 -0.87
-0.87 1
Parameter Estimates
Variable
background
intercept
slope
Estimate
0
-2.69161
1.08968
Std. Err.
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
* - Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-28.3416
-32.8595
-54.9778
69.7191
# Param's Deviance Test d.f.
9 .03583
53.2723
P-value
0. 06021
<.0001
Goodness of Fit
Dose
0.0000
0.5000
1 .5000
5 .0000
15.0000
50.0000
Est. Prob.
0.0000
0.0309
0. 0954
0.2813
0.5645
0.8280
Expected
0.000
0.463
1 .431
4 .220
8 .467
12 .419
Observed
0
0
0
6
11
10
Size
15
15
15
15
15
15
Scaled
Residual
0.000
-0.691
-1.258
1. 022
1.319
-1.655
Chi*2 = 7.58
d.f. = 4
P-value = 0.1081
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 1.57411
BMDL = 0.836756
193
-------
Log-Logistic Model with 0.95 Confidence Level
0.8
0.6
0.4
0.2
Log-Logistic
gMD.L BMP
0
14:5605/062008
10
20 30
dose
40
50
194
-------
Decreased Mating Performance
Female CD rats
Model
Log-probit (slope > 1)
Log-logistic (slope > 1)
Gamma (power > 1)
Weibull (power > 1)
Multistage (degree = 2)
Goodness-of-fit
^j-value
0.3933
0.3529
0.3462
0.3325
0.2955
AIC
86.0
86.3
86.4
86.5
86.7
BMD
4.5
4.6
4.8
4.8
5.3
BMDL10
3.0
2.1
2.1
2.1
2.1
Probit Model. (Version: 2.9; Date: 09/23/2007)
Input Data File: C:\BMDS\UNSAVED1.(d)
Gnuplot Plotting File: C:\BMDS\UNSAVEDl.plt
Tue May 27 12:32:29 2008
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 = failedmating
Independent variable = dose
Slope parameter is restricted as slope >= 1
Total number of observations = 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
User has chosen the log transformed model
Default Initial (and Specified) Parameter Values
background = 0.075
intercept = -2.72992
slope = 1
Asymptotic Correlation Matrix of Parameter Estimates
background intercept slope
background 1 -0.18 0.11
intercept -0.18 1 -0.96
slope 0.11 -0.96 1
Parameter Estimates
Variable
background
intercept
slope
Estimate
0.0521339
-2.84071
1.0388
Std. Err.
0.0252193
0.843497
0.354414
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
0.00270489 0.101563
-4.49394 -1.18749
0.344159 1.73344
195
-------
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-38.4967
-39.9962
-50.7251
85.9924
# Param's
5
3
1
Deviance Test d.f.
P-value
2 .99908
24 .4568
0 .2232
<.0001
Goodness of Fit
Dose
0.0000
0.5000
1 .5000
5 .0000
15.0000
Est. Prob.
0.0521
0.0523
0. 0595
0. 1670
0.5156
Expected
2 .085
1.046
1 .190
3 .341
10.313
Observed
3
1
0
4
10
Size
40
20
20
20
20
Scaled
Residual
0.651
-0.046
-1. 125
0.395
-0.140
Chi*2 = 1.87
d.f. = 2
P-value = 0.3933
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
0 .1
Extra risk
0. 95
4 .48585
2.98577
Probit Model with 0.95 Confidence Level
0.7
0.6
"8 0.5
t3
| 0.4
I 0.3
CO
£ 0.2
0.1
0
Probit
BMD
0 2
12:3205/272008
6 8
dose
10
12
14
196
-------
APPENDIX D: DERIVATION OF THE ORAL SLOPE FACTOR USING
THE MULTISTAGE-WEIBULL MODEL
Table D-l. Tumor incidence data, with time to death with tumor; male rats
exposed by gavage to 1,2,3-trichloropropane
Dose group,
mg/kg-d
0
3
10
Wkof
death
49
64
69
72
84
86
87
88
90
93
95
97
99
104
105
64
82
84
86
89
93
94
95
97
98
99
100
101
104
4
32
58
64
67
73
74
75
77
84
87
88
91
92
93
94
95
96
97
Total
examined
1
10
1
1
2
1
1
3
1
1
2
1
1
11
23
10
1
1
3
1
1
1
1
1
3
3
1
1
32
1
1
2
11
1
1
1
1
3
2
1
1
1
1
2
2
2
1
1
Number of animals with
Squamous cell
neoplasia3
Incidenta
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
1
1
0
0
1
1
1
1
25
0
0
1
4
0
1
0
0
2
0
0
0
1
1
0
1
0
0
1
Fatal
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
1
1
0
0
0
0
2
2
0
0
0
0
0
1
0
1
0
0
0
1
2
1
1
0
0
2
1
2
1
0
Pancreas
tumors
0
0
0
0
0
0
0
0
0
0
0
0
0
3
2
0
0
0
0
0
0
0
0
0
2
1
0
0
17
0
0
0
1
1
0
0
1
1
1
1
1
1
0
1
2
1
1
1
Kidney
adenomas
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
0
Preputial
gland
tumors
0
0
0
1
0
0
0
0
0
0
0
0
0
2
2
0
0
0
0
0
1
0
0
0
1
1
1
0
2
0
0
1
1
0
1
0
0
0
0
0
1
0
0
1
0
0
0
0
Zymbal's
gland
tumors
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Liver
tumors
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Skin,
squamous
cell
neoplasia
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
197
-------
Table D-l. Tumor incidence data, with time to death with tumor; male rats
exposed by gavage to 1,2,3-trichloropropane
Dose group,
mg/kg-d
30
Wkof
death
98
100
101
103
104
47
48
52
53
55
56
57
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
Total
examined
4
2
1
1
15
1
1
1
3
2
1
1
1
2
2
1
9
1
1
2
3
5
5
2
1
3
2
1
9
Number of animals with
Squamous cell
neoplasia3
Incidenta
1
1
1
0
0
15
0
1
0
0
1
0
0
0
2
0
1
0
8
0
1
1
2
2
1
0
0
0
0
9
Fatal
3
1
1
1
0
1
0
0
3
1
0
1
1
0
2
0
1
1
1
1
2
3
3
1
1
3
2
1
0
Pancreas
tumors
4
1
1
1
15
0
0
0
0
0
0
0
1
0
1
0
2
0
0
1
3
4
3
2
1
3
1
1
8
Kidney
adenomas
4
2
0
0
9
0
0
0
0
0
0
0
1
0
0
0
5
1
0
2
1
3
4
1
0
1
1
0
6
Preputial
gland
tumors
0
0
1
0
3
0
0
0
0
1
0
0
0
0
1
1
1
0
1
1
2
1
2
0
1
1
1
0
3
Zymbal's
gland
tumors
0
0
0
0
0
0
0
0
0
0
1
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Liver
tumors
0
0
1
0
2
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
1
Skin,
squamous
cell
neoplasia
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
3
0
0
1
0
0
0
0
0
0
2
a"Incidental" denotes incidence of papillomas, or of carcinomas observed at scheduled death. "Fatal" denotes incidence of
carcinomas at unscheduled deaths. Some papillomas were present with carcinomas in both categories.
Source: NTP(1993).
198
-------
Male Rat; Squamous Papillomas, Carcinomas
A. All tumors treated as incidental to death of animal
Dataset: G:\_ToxRiskData\Trichloropropane\MR Sq-inc kh.ttd
Model: Two Stage Weib
Functional form: 1 - EXP[( -QO - Ql * D - Q2 * D^2) * (T - TO)'
Maximum Log-Likelihood = -5.624514e+001
Z]
Parameter Estimates :
Q 0 = 1.087183E-012
Q 1 = 1.914937E-011
Q 2 = 2.116410E-012
Z = 5.126149E+000
TO = O.OOOOOOE+000
Set by User
Avg. Doses
(mg/kg/day)
0
3
10
30
of animals
60
60
59
60
J.>J ULLU^C i.
with fatal
tumors
0
0
0
0
with incidental
tumors
1
39
48
58
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk
l.OOOOE-006
l.OOOOE-005
0.0001
0.0010
0.01
0.10
Time
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
3.1642E-004
3.1642E-003
3.1643E-002
3.1656E-001
3.1784E+000
3.3146E+001
MLE
4 .8863E-004
4 .8864E-003
4 .8864E-002
4 .8874E-001
4 .8973E+000
5.0061E+001
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
1
0.8
0.6
0.4
0.2
0
Incidental Graph
MR Sq-inc.ttd - TCP male rat oral route squamous pap, carcinomas
Model: Two Stage Weib
Dose (mg/kg/day)=3
Dose (mg/kg/day)=10
Dose (mg/kg/day)=30
Hoel Walburg (3)
HoelWalburg (10)
Hoel Walburg (30)
20
40
60
Time (wks)
80
100
120
199
-------
Male Rat; Squamous Cell Papillomas, Carcinomas
B. Carcinomas occurring at unscheduled deaths treated as cause of death:
Dataset: M:\_ToxRiskData\Trichloropropane\MR Sq-F.ttd
Model: Four Stage Weib
Functional form: 1 - EXP[( -QO - Ql * D - Q2 * D^2 ... - Q4 * D^4 )
* (T - TO)*Z]
Maximum Log-Likelihood = -3.000199e+002
Parameter Estimates :
Q 0 =
Q 1 =
Q 2 =
Q 3 =
Q 4 =
Z
TO
2 .864368E-015
5.913534E-014
= O.OOOOOOE+000
= O.OOOOOOE+000
= 2.482747E-017
= 6.413765E+000
= 2.901108E+001
Avg. Doses
(mg/kg/day)
0
3
10
30
of animals
60
60
59
60
J.>J ULLU^C i.
with fatal
tumors
1
3
18
32
with incidental
tumors
0
36
30
26
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk Time (yr)
l.OOOOE-006
l.OOOOE-005
0.0001
0.0010
0.01
0.10
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
3.0422E-004
3.0422E-003
3.0423E-002
3.0437E-001
3.0575E+000
3.2053E+001
MLE
3 .9173E-004
3.9173E-003
3.9175E-002
3 .9193E-001
3.9370E+000
4.1273E+001
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Fatal Graph
MR Sq-F.ttd - TCP male rat oral route squamous pap, carcinomas
Model: Four Stage Weib
ir:
1
0.8
0.6
0.4
0.2
Dose (mg/kg/day)=3
Dose (mg/kg/day)=10
Dose (mg/kg/day)=30
Kaplan Meier (3)
Kaplan Meier (10)
Kaplan Meier (30)
30 40 50 60 70 80
Time (wks)
90
100
110
200
-------
Male Rat; Pancreas Acinar Tumors
Dataset: G:\_ToxRiskData\Trichloropropane\MR pane kh.ttd
Model: Two Stage Weib
Functional form: 1 - EXP[( -QO - Ql * D - Q2 * D^2) * (T - TO)'
Maximum Log-Likelihood = -9.484725e+001
Z]
Parameter Estimates :
Q 0 = 4 .471590E-019
Q 1 = 2 .430231E-019
Q 2 = 1.162004E-019
Z = 8.663144E+000
TO = O.OOOOOOE+000
Set by User
Avg. Doses
(mg/kg/day)
0
3
10
30
of animals
60
60
59
60
J.>J ULLU^C i.
with fatal
tumors
0
0
0
0
with incidental
tumors
5
20
36
31
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk
l.OOOOE-006
l.OOOOE-005
0.0001
0.0010
0.01
0.10
Time
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
9.9894E-004
9.9894E-003
9.9894E-002
9.9901E-001
9.9965E+000
1.0077E+002
MLE
2 .8518E-003
2 .8517E-002
2 .8500E-001
2.8338E+000
2.6906E+001
2.0173E+002
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
1
0.8
0.6
0.4
0.2
0
Incidental Graph
MR pane kh.ttd - TCP male rat pancreas acinar tumors
Model: Two Stage Weib
Dose (mg/kg/day)=3
Dose (mg/kg/day)=10
Dose (mg/kg/day)=30
Hoel Walburg (3)
HoelWalburg(IO)
Hoel Walburg (30)
20
40
60
Time (wks)
80
100
120
201
-------
Male Rat; Kidney Tubule Adenomas
Dataset: G:\_ToxRiskData\Trichloropropane\MR kidney.ttd
Model: Two Stage Weib
Functional form: 1 - EXP[( -QO - Ql * D - Q2 * D^2) * (T - TO)'
Maximum Log-Likelihood = -6.953871e+001
Z]
Parameter Estimates :
Q 0 = O.OOOOOOE+000
Q 1 = O.OOOOOOE+000
Q 2 = 2.539769E-015
Z = 6.217551E+000
TO = O.OOOOOOE+000
Set by User
Avg. Doses
(mg/kg/day)
0
3
10
30
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
of animals
60
60
59
60
with fatal
tumors
0
0
0
0
with incidental
tumors
0
2
18
26
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk
l.OOOOE-006
l.OOOOE-005
0.0001
0.01
0.05
0.10
Time
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
9.2297E-003
9.2286E-002
9.2177E-001
8 .2580E+001
3.1744E+002
5.2586E+002
MLE
2.1530E+000
6 .8083E+000
2.1530E+001
2.1584E+002
4.8760E+002
6.9883E+002
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Incidental Graph
MR kidney.ttd - TCP male rat kidney tubule tumors
Model: Two Stage Weib
1
0.8
0.6
0.4
0.2
0
Dose (mg/kg/day)=3
Dose (mg/kg/day)=10
Dose (mg/kg/day)=30
Hoel Walburg (3)
Hoel Walburg (10)
Hoel Walburg (30)
/H
20
40
60
Time (wks)
80
100
120
202
-------
Male Rat; Hepatocellular Adenomas and Carcinomas
Q 0 = 4.588266E-019
Q 1 = O.OOOOOOE+000
Q 2 = 3.549482E-020
Z = 8.244602E+000
TO = O.OOOOOOE+000 Set by User
Human Equivalent Dose Estimates (ug/kg/day)
MLE
1.6447E+001
5.2012E+001
1.6451E+002
5.2141E+002
1.1779E+003
1.6882E+003
1
0.8
0.6
I
'0.4
0.2
Incidental Graph
MR liver.ttd - TCP male rat liver tumors
Model: Two Stage Weib
uose
Dose
Dose
— ' Hoel
— ' Hoel
— ' Hoel
^mg/Kg;aay;-o
(mg/kg/day)=10 "
(mg/kg/day)=30
Walburg (3) |
Walburg (10) ;
Walburg (30) ;
/ / "
20
40 60 80 100 120
Time (wks)
203
-------
Male Rat; Squamous Cell Papillomas or Carcinomas, Skin
Dataset: M:\_ToxRiskData\Trichloropropane\MR skin.ttd
Model: One Stage Weib
Functional form: 1 - EXP[( -QO - Ql * D ) * (T - TO)*Z]
Maximum Log-Likelihood = -3.408636e+001
Parameter Estimates :
Q 0 = O.OOOOOOE+000
Q 1 = 3 .644870E-006
Z = 1.606192E+000
TO = O.OOOOOOE+000
Set by User
Avg. Doses
(mg/kg/day)
0
3
10
30
of animals
60
60
59
60
J.>J ULLU^C i.
with fatal
tumors
0
0
0
0
with incidental
tumors
0
2
1
6
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk Time (yr)
OOOOE-006
l.OOOOE-005
0.0001
0.0010
0.01
0.10
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
1.3601E-002
1.3601E-001
1.3602E+000
1.3608E+001
1.3669E+002
1.4330E+003
MLE
3 .1922E-002
3 .1922E-001
3.1924E+000
3.1938E+001
3.2083E+002
3.3634E+003
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
U)
ir:
1
0.8
0.6
0.4
0.2
Incidental Graph
MR skin.ttd - TCP male skin squamous cell neoplasia
Model: One Stage Weib
Dose (mg/kg/day)=3
Dose (mg/kg/day)=10
Dose (mg/kg/day)=30
Hoel Walburg (3)
HoelWalburg(IO)
Hoel Walburg (30)
20
40
60
Time (wks)
80
100
120
204
-------
Male Rat; Preputial Gland Tumors
Dataset: G:\_ToxRiskData\Trichloropropane\MR preput.ttd
Model: One Stage Weib
Functional form: 1 - EXP[( -QO - Ql * D ) * (T - TO)*Z]
Maximum Log-Likelihood = -1.086836e+002
Parameter Estimates
Q 0 = 1.054336E-004
Q 1 = 2.704366E-005
Z = 1.371929E+000
TO = O.OOOOOOE+000
Set by User
Avg. Doses
(mg/kg/day)
0
3
10
30
of animals
60
60
59
60
with fatal
tumors
0
0
0
0
with incidental
tumors
5
6
9
17
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk
l.OOOOE-006
l.OOOOE-005
0.0001
0.0010
0.01
0.10
Time
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
5.5682E-003
5.5682E-002
5.5685E-001
5.5710E+000
5.5962E+001
5.8667E+002
MLE
1.2735E-002
1.2735E-001
1.2736E+000
1.2741E+001
1.2799E+002
1.3418E+003
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Incidental Graph
MR preput.ttd - TCP male rat preputial gland tumors
Model: One Stage Weib
1
0.8
0.6
0.4
0.2
Dose (mg/kg/day)=3
Dose (mg/kg/day)=10
Dose (mg/kg/day)=30
HoelWalburg(S)
HoelWalburg(IO)
HoelWalburg(SO)
20
40
60
Time (wks)
80
100
120
205
-------
Male Rat; Zymbal's Gland Carcinomas
Dataset: G:\_ToxRiskData\Trichloropropane\MR Zymbal gl.ttd
Model: One Stage Weib
Functional form: 1 - EXP[( -QO - Ql * D ) * (T - TO)*Z]
Maximum Log-Likelihood = -1.360128e+001
Parameter Estimates
Q 0 = O.OOOOOOE+000
Q 1 = 1.632672E-005
Z = l.OOOOOOE+000
TO = O.OOOOOOE+000
Set by User
Avg. Doses
(mg/kg/day)
0
3
10
30
of animals
60
60
59
60
with fatal
tumors
0
0
0
0
with incidental
tumors
0
0
0
3
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk
l.OOOOE-006
l.OOOOE-005
0.0001
0.0010
0.01
0.10
Time
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
4.8346E-002
4.8347E-001
4 .8349E+000
4.8371E+001
4.8590E+002
5.0938E+003
MLE
1.1804E-001
1.1804E+000
1 .1805E+001
1.1810E+002
1.1864E+003
1.2437E+004
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
or
0.8
0.6
0.4
0.2
0
Incidental Graph
MR Zymbal gl.ttd - TCP male rat Zymbal's gland tumors
Model: One Stage Weib
Dose (mg/kg/day)=3
Dose (mg/kg/day)=10
Dose (mg/kg/day)=30
Hoel Walburg (30)
20
40
60
Time (wks)
80
100
120
206
-------
Table D-2. Tumor incidence data, with time to death with tumor; female
rats exposed to 1,2,3-trichloropropane
Dose
group,
mg/kg-d
0
3
Wkof
observation
61
64
66
68
75
78
79
85
86
89
92
93
96
98
100
101
102
105
106
62
66
67
73
78
83
84
86
95
96
97
99
101
102
104
105
Total
examined
1
1
10
1
1
1
2
1
1
1
2
1
1
1
1
1
2
18
13
1
11
1
1
1
1
1
1
1
1
1
3
1
2
2
30
Number of animals with
Squamous cell neoplasia
Incidental3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
0
0
0
1
0
1
0
1
1
1
14
Fatal3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
Mammary gland tumors
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4
Fatal
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
1
0
0
Clitoral
gland tumors
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
2
2
0
1
0
0
0
0
0
0
1
0
1
0
0
0
1
7
Zymbal's
gland tumors
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
207
-------
Table D-2. Tumor incidence data, with time to death with tumor; female
rats exposed to 1,2,3-trichloropropane
Dose
group,
mg/kg-d
10
30
Wkof
observation
36
58
61
62
64
66
68
72
73
74
77
79
80
81
82
83
85
86
87
90
91
92
96
97
98
100
101
103
104
105
12
26
33
34
36
42
44
46
47
48
49
50
51
52
53
54
55
57
58
59
60
62
63
Total
examined
1
2
1
1
2
8
1
1
3
2
2
1
1
2
1
2
1
2
3
1
2
3
1
1
1
2
1
1
2
8
2
1
1
1
1
2
3
1
3
3
5
1
1
3
4
1
2
4
2
3
3
1
2
Number of animals with
Squamous cell neoplasia
Incidental3
0
1
0
0
1
5
1
1
3
1
2
1
1
2
1
2
1
2
2
1
2
2
1
1
1
2
1
1
2
8
0
0
1
0
0
2
2
1
1
2
2
1
1
2
2
1
2
4
2
3
3
1
2
Fatal3
0
0
0
0
0
0
0
0
1
1
2
0
1
2
0
1
1
1
1
1
1
2
0
1
0
1
0
1
1
0
0
0
0
0
0
2
1
1
1
0
1
1
1
1
0
1
1
3
2
3
3
0
0
Mammary gland tumors
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Fatal
0
0
1
0
0
0
1
0
1
1
1
1
0
0
1
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
1
0
1
2
2
0
1
1
3
0
2
3
0
0
0
1
1
Clitoral
gland tumors
0
0
0
1
2
1
0
1
1
0
1
0
0
0
0
0
0
1
2
0
0
0
0
1
0
1
0
0
0
6
0
0
0
0
0
0
1
1
2
1
3
0
1
1
0
1
0
1
1
1
0
0
0
Zymbal's
gland tumors
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
1
208
-------
Table D-2. Tumor incidence data, with time to death with tumor; female
rats exposed to 1,2,3-trichloropropane
Dose
group,
mg/kg-d
30
Wkof
observation
64
66
Total
examined
1
9
Number of animals with
Squamous cell neoplasia
Incidental3
1
9
Fatal3
0
1
Mammary gland tumors
Incidental
0
0
Fatal
1
2
Clitoral
gland tumors
1
2
Zymbal's
gland tumors
0
2
a"Incidental" denotes incidence of papillomas only, or of carcinomas observed at scheduled sacrifice. "Fatal" denotes incidence
of carcinomas at unscheduled deaths. Some papillomas were present with carcinomas in both categories.
Source: NTP(1993).
209
-------
Female Rat; Alimentary System Squamous Papillomas, Carcinomas
A. All tumors treated as incidental to death of animal:
Dataset: G:\_ToxRiskData\Trichloropropane\FR ST-inc kh.ttd
Model: Two Stage Weib
Functional form: 1 - EXP[( -QO - Ql * D - Q2 * D^2) * (T - TO)*Z]
Maximum Log-Likelihood = -9.100477e+001
Parameter Estimates :
Q 0 = 2 .485425E-012
Q 1 = 8.109448E-012
Q 2 = 5.601264E-012
Z = 4.940580E+000
TO = O.OOOOOOE+000
Set by User
Avg. Doses
(mg/kg/day)
0
3
10
30
of animals
60
59
60
60
with fatal
tumors
0
0
0
0
with incidental
tumors
1
22
49
44
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk Time (yr)
l.OOOOE-006
l.OOOOE-005
0.0001
0.0010
0.01
0.10
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
5.2769E-004
5.2769E-003
5.2771E-002
5.2793E-001
5.3009E+000
5.5310E+001
MLE
2 .2831E-003
2 .2829E-002
2 .2810E-001
2 .2622E+000
2.1036E+001
1.4711E+002
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
1
0.8
0.6
0.4
0.2
0
Incidental Graph
FR ST-inc.ttd - TCP Female Rats Fstomach tumors
Model: Two Stage Weib
Dose (mg/kg/day)=3
Dose (mg/kg/day)=10
Dose (mg/kg/day)=30
Hoel Walburg (3)
HoelWalburg(IO)
Hoel Walburg (30)
20
40
60
Time (wks)
80
100
120
210
-------
Female Rat; Alimentary System Squamous Papillomas, Carcinomas (cont.)
B. Carcinomas occurring at unscheduled deaths treated as cause of death:
Model: Two Stage Weib
Dataset: M:\_ToxRiskData\Trichloropropane\FR ST fatal.ttd
Functional form: 1 - EXP[( -QO - Ql * D - Q2 * D^2) * (T - TO)'
Maximum Log-Likelihood = -2.626680e+002
•z]
Parameter Estimates :
Q 0 = 1.708057E-011
Q 1 = 5.693671E-011
Q 2 = 2 .666721E-011
Z = 4.532096E+000
TO = 2.708322E+001
Avg. Doses
(mg/kg/day)
0
3
10
30
of animals
60
59
60
60
with fatal
tumors
0
1
18
16
with incidental
tumors
1
21
31
28
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk
l.OOOOE-006
l.OOOOE-005
0.0001
0.0010
0.01
0.10
Time (yr)
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
9.7449E-004
9.7449E-003
9.7446E-002
9.7417E-001
9.7131E+000
9.4856E+001
MLE
2 .3918E-003
2 .3917E-002
2 .3902E-001
2.3752E+000
2.2449E+001
1.6553E+002
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Fatal Graph
FR ST fatal.ttd - TCP Female Rats Fstomach tumors
Model: Two Stage Weib
ir:
1
0.8
0.6
0.4
0.2
Dose (mg/kg/day)=3
Dose (mg/kg/day)=10
Dose (mg/kg/day)=30
Kaplan Meier (3)
Kaplan Meier (10)
Kaplan Meier (30)
30 40 50 60 70 80
Time (wks)
90
100
110
211
-------
Female Rat; Mammary Adenomas, Adenocarcinomas1
A. All tumors treated as incidental to death of animal:
Multistage Weibull Model. (Version: 1.3; Date: 08/30/2007)
Input Data File: FRmamm_M=U_mswlI.msw
Mon Sep 14 16:27:55 2009
TCP: NTP Female Rats mammary adenocarcinomas, M=U
The form of the probability function is:
P[response] = l-EXP{-(t - t_0)*c *
(beta_0+beta_l*dose^l)}
The parameter betas are restricted to be positive
Dependent variable = CLASS
Independent variables = DOSE, TIME
Total number of observations = 125
Total number of records with missing values = 0
Data Summary (N Totals)
CLASS
C F I U Total
DOSE
0 55 0 2 3 60
3 50 0 6 3 59
10 39 0 14 7 60
30 28 0 23 9 60
Total number of parameters in model = 4
Total number of specified parameters = 1
Degree of polynomial = 1
User specifies the following parameters:
t_0 = 0
Maximum number of iterations = 16
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
c = 1.11111
t_0 = 0 Specified
beta_0 = 0.000201751
beta_l = 0.000184682
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -c ~t_0
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
beta_0 beta_l
beta_0 1 -0.35
beta_l -0.35 1
Parameter Estimates
95.0% Wald Confidence Interval
Lower Conf. Limit Upper Conf. Limit
-0.000279565 0.00097004
1 Note: 22 female rats across the dose groups had missing tissues. While the multistage Weibull model can take advantage of the
length of time that the animals were on study without developing tumors, the version of ToxRisk used in this assessment did not
provide correct estimates. The program used in this case (MSW, currently under development by EPA) provides correct
estimates.
212
Variable
c
beta_0
Estimate
1
0.000345237
Std. Err.
NA
0.000318783
-------
beta 1
0.000292263
7.43911e-005
0.000146459
0.000438067
NA - Indicates that this parameter has hit a
bound implied by some inequality constraint
and thus has no standard error.
Model
Full Model
Fitted Model
Reduced Model
AIC:
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f. P-value
0 6
-100.346 4 02 <.0001
03 03 <.0001
204.693
Benchmark Dose Computation
Specified effect =
Time
Risk Response
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
104
Incidental
Extra
0.9
3 .46634
2.58643
4.64231
The BMD and BMD above were then adjusted to estimate human equivalent continuous exposure using BW3/4
cross-species scaling and by multiplying by (5 days)/(7 days): BMDHED = 0.605, BMDnED = 0.426 mg/kg-day.
Incidental Graph
FR mamm inc.ttd - TCP Female Rats mammary adenocarcinomas
Model: One Stage Weib
Dose (mg/kg/day)=3
Dose (mg/kg/day)=10
Dose (mg/kg/day)=30
Hoel Walburg (3)
HoelWalburg(IO)
Hoel Walburg (30)
60
Time (wks)
213
-------
Female Rat; Mammary Adenomas, Adenocarcinomas
B. Carcinomas occurring at unscheduled deaths treated as cause of death:
Multistage Weibull Model. (Version: 1.3; Date: 08/30/2007)
Input Data File: FRmamm_M=U_msw3f.msw
Mon Sep 14 17:03:49 2009
TCP: NTP Female Rats mammary adenocarcinomas, M=U
The form of the probability function is:
P[response] = l-EXP{-(t - t_0)*c *
(beta_0+beta_l*dose^l+beta_2*dose^2+beta_3*dose^3)}
The parameter betas are restricted to be positive
Dependent variable = CLASS
Independent variables = DOSE, TIME
Total number of observations = 125
Total number of records with missing values = 0
Data Summary (N Totals)
CLASS
C F I U Total
DOSE
0 55 1 1 3 60
3 50 2 4 3 59
10 39 9 5 7 60
30 28 23 0 9 60
Minimum observation time for F tumor context = 34
Total number of parameters in model = 6
Total number of specified parameters = 0
Degree of polynomial = 3
Maximum number of iterations = 16
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
c = 3.33333
t_0 = 6.8
beta_0 = 8.44695e-009
beta_l = 5.32978e-009
beta_2 = 7.59254e-011
beta_3 = 2.12067e-011
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -beta_2
have been estimated at a boundary point, or have been specified by the user,
and do not appear in the correlation matrix )
c t_0 beta_0 beta_l beta_3
c 1 -0.52 -0.98 -0.97 -1
t_0 -0.52 1 0.51 0.5 0.53
beta_0 -0.98 0.51 1 0.92 0.98
beta_l -0.97 0.5 0.92 1 0.96
beta_3 -1 0.53 0.98 0.96 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
c 5.32444 0.954401 3.45384 7.19503
t_0 4.70501 2.2168 0.36016 9.04986
beta_0 9.83773e-013 4.32656e-012 -7.49612e-012 9.46367e-012
beta_l 3.34357e-013 1.699e-012 -2.99562e-012 3.66433e-012
214
-------
beta_2
beta 3
7.55424e-015
NA
2 .97886-014
NA - Indicates that this parameter has hit a
bound implied by some inequality constraint
and thus has no standard error.
-5.08292e-014
6.593776-014
Model
Full Model
Fitted Model
Reduced Model
AIC:
Analysis of Deviance Table
Log(likelihood) # Param's Deviance Test d.f. P-value
-230.308 6
-230.308 6 0 0 NA
03 03 <.0001
468.616
Benchmark Dose Computation
Specified effect =
Time
Risk Response
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
104
Incidental
Extra
0.95
4.13847
1.95917
7.01446
The BMD and BMD above were then adjusted to estimate human equivalent continuous exposure using BW3/4
cross-species scaling and by multiplying by (5 days)/(7 days): BMDHED = 0.723, BMDnED = 0.342 mg/kg-day.
a:
0.4
0.2
Fatal Graph
FR mamm fatal M=U.ttd - TCP Female Rats mammary adenocarcinomas
Model: Three Stage Weib
Dose (mg/kg/day)=3
Dose (mg/kg/day)=10
Dose (mg/kg/day)=30
Kaplan Meier (3)
Kaplan Meier (10)
Kaplan Meier (30)
100 110
215
-------
Female Rat; Clitoral Gland Adenomas, Carcinomas
Model: One Stage Weib
Dataset: G:\_ToxRiskData\Trichloropropane\FR cl gland.ttd
Functional form: 1 - EXP[( -QO - Ql * D ) * (T - TO)*Z]
Maximum Log-Likelihood = -1.422177e+002
Parameter Estimates :
Q 0 = 3.143833E-007
Q 1 = 6.526662E-007
Z = 2.445897E+000
TO = O.OOOOOOE+000
Set by User
Avg. Doses
(mg/kg/day)
0
3
10
30
of animals
60
58
58
60
J.>J ULLU^C i.
with fatal
tumors
0
0
0
0
with incidental
tumors
5
11
18
17
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk Time
.OOOOE-006 70.00
l.OOOOE-005
0.0001
0.0010
0.01
0.10 70.00
95.00 %
(yr) Lower Bound
2.2971E-003 2.
70.00 2.2971E-002
70.00 2.2973E-001
70.00 2.2983E+000
70.00 2.3087E+001
2.4203E+002
95.00 %
MLE Upper Bound
9484E-003 Not Reqstd
2.9484E-002 Not Reqstd
2.9485E-001 Not Reqstd
2.9499E+000 Not Reqstd
2.9632E+001 Not Reqstd
3.1064E+002 Not Reqstd
Incidental Graph
FR cl gland.ttd - TCP Female Rats cl gland tumors
Model: One Stage Weib
0.8
0.6
0.4
0.2
0
Dose (mg/kg/day)=3
Dose (mg/kg/day)=10
Dose (mg/kg/day)=30
Hoel Walburg (3)
HoelWalburg(IO)
Hoel Walburg (30)
H
20
40
60
Time (wks)
80
100
120
216
-------
Female Rat; Zymbal's Gland Carcinomas
Dataset: G:\_ToxRiskData\Trichloropropane\FR Zymbal gl.ttd
Model: One Stage Weib
Functional form: 1 - EXP[( -QO - Ql * D ) * (T - TO)*Z]
Maximum Log-Likelihood = -2.101568e+001
Parameter Estimates :
Q 0 = O.OOOOOOE+000
Q 1 = 1.393807E-005
Z = 1.198267E+000
TO = O.OOOOOOE+000
Set by User
Avg. Doses
(mg/kg/day)
0
3
10
30
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
of animals
60
59
60
60
with fatal
tumors
0
0
0
0
with incidental
tumors
0
1
0
4
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk Time (yr)
l.OOOOE-006
l.OOOOE-005
0.0001
0.0010
0.01
0.10
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
1.4957E-002
1.4958E-001
1.4958E+000
1.4965E+001
1.5033E+002
1.5759E+003
MLE
4 .6884E-002
4 .6884E-001
4.6886E+000
4.6907E+001
4.7120E+002
4.9397E+003
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
U)
ir:
1
0.8
0.6
0.4
0.2
0
Incidental Graph
FR Zymbal gl.ttd - TCP Female Rats Zymbal's gland tumors
Model: One Stage Weib
Dose (mg/kg/day)=3
Dose (mg/kg/day)=10
Dose (mg/kg/day)=30
Hoel Walburg (3)
Hoel Walburg (30)
20
40
60
Time (wks)
80
100
120
217
-------
Table D-3. Tumor incidence data, with time to death with tumor; male mice
exposed by gavage to 1,2,3-trichloropropane
Dose group,
mg/kg-d
0
6
20
Wk of death
3
65
66
70
71
77
85
88
94
98
105
58
61
65
66
75
77
78
81
82
83
84
85
89
91
92
93
94
95
97
98
99
101
105
55
59
63
64
65
66
67
68
69
72
73
74
76
77
78
80
81
83
84
85
Total examined
1
2
8
1
1
1
1
1
1
1
42
1
1
1
9
2
1
2
2
1
2
2
1
1
1
3
1
1
1
3
1
2
2
18
2
1
1
2
1
8
1
2
4
2
2
1
2
1
3
1
4
2
2
3
Number of animals with
Squamous cell neoplasia"
Incidental
0
0
0
1
0
0
0
0
0
0
2
0
0
0
8
0
0
0
0
0
0
0
0
1
0
0
1
0
0
1
0
0
0
18
0
0
0
0
1
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Fatal
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
2
1
2
2
1
2
2
1
0
1
3
0
1
1
2
1
2
2
0
2
1
1
2
0
2
1
1
4
2
2
1
1
1
2
1
4
2
2
3
Liver tumors
0
0
1
1
0
0
1
0
1
1
9
0
0
0
0
1
0
0
2
0
2
0
0
1
0
1
0
0
1
3
1
1
1
10
0
1
1
0
0
1
0
1
2
0
2
1
0
1
0
0
0
2
1
2
Harderian
gland adenomas
0
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
1
1
0
1
0
1
2
218
-------
Table D-3. Tumor incidence data, with time to death with tumor; male mice
exposed by gavage to 1,2,3-trichloropropane
Dose group,
mg/kg-d
60
Wk of death
86
88
89
46
50
53
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
Total examined
1
3
11
1
1
2
1
3
1
1
1
1
1
2
3
3
3
6
1
4
1
3
1
2
1
1
1
1
2
2
1
9
Number of animals with
Squamous cell neoplasia"
Incidental
0
0
11
1
0
0
0
3
0
0
0
0
0
2
0
0
0
6
0
0
0
0
0
0
0
0
0
0
0
0
0
9
Fatal
1
3
0
0
1
2
1
2
1
1
1
1
1
1
3
3
3
2
1
4
1
3
1
2
1
1
1
1
2
2
1
0
Liver tumors
0
3
7
1
1
0
0
1
0
0
0
0
1
0
0
1
1
3
0
3
0
3
0
1
0
1
1
1
2
2
1
9
Harderian
gland adenomas
0
1
2
0
0
0
0
0
0
0
0
0
0
0
0
0
2
2
1
1
0
0
0
1
0
0
0
0
1
0
1
2
^'Incidental" denotes incidence of papillomas or of carcinomas observed at scheduled death. "Fatal" denotes incidence of
carcinomas at unscheduled deaths. Papillomas present with carcinomas in both categories are not indicated.
Source: NTP(1993).
219
-------
Male Mouse; Alimentary System Squamous Cell Papillomas, Carcinomas
A. All tumors treated as incidental to death of animal:
Model: One Stage Weib Dataset: M:\_ToxRiskData\Trichloropropane\MM ST inc.ttd
Functional form: 1 - EXP[( -QO - Ql * D ) * (T - TO)*Z]
Maximum Log-Likelihood = -4.395030e+001
Parameter Estimates :
Q 0 = 6 .451129E-009
Q 1 = 6.539700E-008
Z = 3 .481792E+000
TO = O.OOOOOOE+000
Set by User
Avg. Doses
(mg/kg/day)
0
6
20
60
of animals
60
59
60
60
J.>J ULLU^C i.
with fatal
tumors
0
0
0
0
with incidental
tumors
3
57
55
60
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk Time (yr)
l.OOOOE-006
l.OOOOE-005
0.0001
0.0010
0.01
0.10
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
1.6127E-004
1.6127E-003
1.6127E-002
1.6135E-001
1.6208E+000
1 .6991E+001
MLE
2 .8611E-004
2 .8611E-003
2 .8612E-002
2 .8625E-001
2.8755E+000
3.0145E+001
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
1
0.8
0.6
0.4
0.2
0
Incidental Graph
MM ST inc.ttd - TCP male mouse forestomach tumor
Model: One Stage Weib
uose ^mg/Kg;aay;-o t t t -_
Dose (mg/kg/day)-20^-^ ^— — ' ^ ;
Dose
— ' Hoel
1 ' Hoel
1 ' Hoel
3 (mg/kg/day)=60 >^ :
Walburg (6) / ^^
Walburg (20) /
WalburgJ60) / ;
/ / ;
/ / :
/ /
/ /
/ s/ '-
20
40
60
Time (wks)
80
100
120
220
-------
Male Mouse; Alimentary System Squamous Cell Papillomas, Carcinomas (cont.)
B. Carcinomas occurring at unscheduled deaths treated as cause of death:
Dataset: M:\_ToxRiskData\Trichloropropane\MM ST fatal.ttd
Model: One Stage Weib
Functional form: 1 - EXP[( -QO - Ql * D ) * (T - TO)*Z]
Maximum Log-Likelihood = -5.550902e+002
Parameter Estimates :
Q 0 = 6.129572E-012
Q 1 = 8.676377E-011
Z = 4.972905E+000
TO = 2.678381E+001
Avg. Doses
(mg/kg/day)
0
6
20
60
of animals
60
59
60
60
J.>J ULLU^C i.
with fatal
tumors
0
28
50
51
with incidental
tumors
3
29
5
9
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk Time (yr)
l.OOOOE-006
l.OOOOE-005
0.0001
0.0010
0.01
0.10
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
1.6037E-004
1.6037E-003
1.6038E-002
1.6045E-001
1.6118E+000
1.6897E+001
MLE
2 .1199E-004
2.1200E-003
2.1200E-002
2 .1210E-001
2.1306E+000
2 .2336E+001
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
U)
ir:
1
0.8
0.6
0.4
0.2
Fatal Graph
ST fatal.ttd - TCP male mouse forestomach tumor
Model: One Stage Weib
Dose (mg/kg/day)=6
Dose (mg/kg/day)=20
Dose (mg/kg/day)=60
Kaplan Meier (6)
Kaplan Meier (20)
Kaplan Meier (60)
30 40 50 60 70 80
Time (wks)
90
100
110
221
-------
Male Mouse; Liver Tumors
Dataset: M:\_ToxRiskData\Trichloropropane\MM liver kh.ttd
Model: One Stage Weib
Functional form: 1 - EXP[( -QO - Ql * D ) * (T - TO)*Z]
Maximum Log-Likelihood = -1.400104e+002
Parameter Estimates :
Q 0 = 3 .361501E-010
Q 1 = 8.911625E-011
Z = 4 .466842E+000
TO = O.OOOOOOE+000
Set by User
Avg. Doses
(mg/kg/day)
0
6
20
60
of animals
60
59
60
60
with fatal
tumors
0
0
0
0
with incidental
tumors
14
24
25
33
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk Time (yr)
l.OOOOE-006
l.OOOOE-005
0.0001
0.0010
0.01
0.10
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
1.2990E-003
1.2990E-002
1.2991E-001
1.2997E+000
1.3055E+001
1.3686E+002
MLE
.0840E-003
.0840E-002
.0841E-001
.0850E+000
.0944E+001
2.1957E+002
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
1
0.8
0.6
0.4
0.2
Incidental Graph
MM liver kh.ttd - TCP male mouse liver tumors
Model: One Stage Weib
Dose (mg/kg/day)=6
Dose (mg/kg/day)=20
Dose (mg/kg/day)=60
Hoel Walburg (6)
Hoel Walburg (20)
Hoel Walburg (60)
40
60
Time (wks)
80
100
120
222
-------
Male Mouse; Harderian Gland Tumors
Dataset: M:\_ToxRiskData\Trichloropropane\MM harderian. ttd
Model: One Stage Weib
Functional form: 1 - EXP [ ( -QO - Ql * D ) * (T - TO)*Z]
Maximum Log-Likelihood = -6 . 696811e+001
Parameter Estimates :
Q 0 = 1.980258E-010
Q 1 = 2.197164E-010
Z = 3.927133E+000
TO = O.OOOOOOE+000
Set by User
Avg. Doses
(mg/kg/day)
0
6
20
60
of animals
60
59
60
60
with fatal
tumors
0
0
0
0
with incidental
tumors
1
2
10
11
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk Time (yr)
OOOOE-006
l.OOOOE-005
0.0001
0.0010
0.01
0.10
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
5.4492E-003
5.4492E-002
5.4495E-001
5.4519E+000
5.4766E+001
5.7413E+002
MLE
0720E-002
0720E-001
0720E+000
0725E+001
0774E+002
1.1294E+003
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
ir:
1
0.8
0.6
0.4
0.2
0
Incidental Graph
MM harder!an.ttd - TCP male mouse harderian gland
Model: One Stage Weib
Dose (mg/kg/day)=6
Dose (mg/kg/day)=20
Dose (mg/kg/day)=60
Hoel Walburg (6)
Hoel Walburg (20)
Hoel Walburg (60)
20
40
60
Time (wks)
80
100
120
223
-------
Table D-4. Tumor incidence data, with time to death with tumor; female
mice exposed by gavage to 1,2,3-trichloropropane
Dose group,
mg/kg-d
0
6
20
Wkof
death
10
66
69
73
90
99
100
104
105
23
30
59
64
66
74
77
80
81
82
83
84
86
87
89
90
91
92
93
96
97
98
99
100
101
103
104
105
2
45
55
63
64
65
66
67
68
71
72
73
75
76
77
78
Total
examined
1
10
2
1
1
1
2
1
41
1
1
1
1
10
1
1
2
1
2
3
1
3
2
2
1
1
1
1
1
2
2
1
1
1
2
1
13
1
1
1
2
1
2
12
2
1
2
4
2
2
1
1
1
Number of animals with
Squamous cell neoplasia3
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
0
6
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
13
0
0
0
0
0
0
10
0
0
0
0
0
0
0
0
0
Fatal
0
0
0
0
0
0
0
0
0
0
0
1
1
0
1
1
2
1
2
3
1
3
2
2
1
1
1
1
1
2
2
1
1
1
2
1
0
0
1
1
2
1
2
2
2
1
2
4
2
2
1
1
1
Liver tumors
0
1
0
0
0
0
0
0
7
0
0
0
0
0
0
1
0
0
0
1
0
0
0
1
0
0
0
0
0
0
1
0
0
0
1
0
6
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
Harderian
gland adenomas
0
1
0
0
0
0
0
0
2
0
0
0
0
0
0
0
1
0
0
0
0
0
1
0
0
0
0
1
0
0
1
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
Uterine
adenomas or
carcinomas
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
3
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
224
-------
Table D-4. Tumor incidence data, with time to death with tumor; female
mice exposed by gavage to 1,2,3-trichloropropane
Dose group,
mg/kg-d
60
Wkof
death
79
80
81
82
83
84
86
87
88
89
42
49
54
55
56
57
58
59
60
61
62
63
64
66
67
68
69
70
71
72
73
Total
examined
1
2
2
2
3
1
1
1
1
10
1
1
4
2
1
1
2
1
2
2
2
2
3
6
6
3
2
4
3
4
8
Number of animals with
Squamous cell neoplasia3
Incidental
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
0
1
0
5
0
0
0
1
0
0
1
Fatal
1
2
2
2
3
1
1
1
1
10
1
0
4
2
0
1
2
1
1
2
2
1
3
1
6
3
2
3
3
4
7
Liver tumors
0
0
0
0
0
0
0
0
1
6
0
0
0
0
0
0
0
0
1
1
1
1
1
5
4
1
1
4
3
4
7
Harderian
gland adenomas
0
1
0
2
0
1
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
1
0
1
0
1
1
2
2
2
Uterine
adenomas or
carcinomas
0
0
0
0
1
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
2
1
2
4
a"Incidental" denotes incidence of papillomas or of carcinomas observed at scheduled death (interim sacrifice, or terminal
sacrifice). "Fatal" denotes incidence of carcinomas at unscheduled deaths or groups terminated early. Papillomas present with
carcinomas in both categories are not indicated.
Source: NTP(1993).
225
-------
Female Mouse; Alimentary System Squamous Cell Papillomas, Carcinomas
A. All tumors treated as incidental to death of animal:
TITLE: TCP - female mouse/gavage/forestomach tumors
Dataset: M:\_ToxRiskData\Trichloropropane\FM FS inc.TTD
Model: One Stage Weib
Functional form: 1 - EXP[( -QO - Ql * D ) * (T - TO)*Z]
Maximum Log-Likelihood = -1.291538e+001
Parameter Estimates :
Q 0 = O.OOOOOOE+000
Q 1 = 3 .331328E-012
Z = 5.958858E+000
TO = O.OOOOOOE+000
Set by User
Avg. Doses
(mg/kg/day)
0
6
20
60
of animals
60
60
60
60
with fatal
tumors
0
0
0
0
with incidental
tumors
0
54
59
59
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk
OOOOE-006
OOOOE-005
0001
0010
01
Time
70.
70.
70.
70.
70.
(yr)
00
00
00
00
00
95.00 %
Lower Bound
6.1978E-006
6.1978E-005
6.1981E-004
6 .2009E-003
6.2290E-002
MLE
.9932E-005
.9932E-004
.9934E-003
.9947E-002
.0083E-001
0.10
70.00
6.5300E-001
3.1537E+000
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Incidental Graph
FM FS inc.TTD - TCP - female mouse/gavage/forestomach tumors
Model: One Stage Weib
1
0.8
0.6
0.4
0.2
E
Dose (mg/kg/day)=
Dose (mg/kg/day)=2U
Dose (mg/kg/day)=60
Hoel Walburg (6)
Hoel Walburg (20)
Hoel Walburg (60)
-H-
20
40
60
Time (wks)
80
100
120
226
-------
Female Mouse; Alimentary System Squamous Cell Papillomas, Carcinomas (cont.)
B. Carcinomas occurring at unscheduled deaths treated as cause of death:
Dataset: M:\_ToxRiskData\Trichloropropane\FM FS fatal.TTD
Model: Two Stage Weib
Functional form: 1 - EXP[( -QO - Ql * D - Q2 * D^2) * (T - TO)'
Maximum Log-Likelihood = -5.423304e+002
Parameter Estimates :
Q 0 = O.OOOOOOE+000
Q 1 = 7.259298E-016
Q 2 = 9.569303E-017
Z = 7.510793E+000
TO = 2.356030E+001
Z]
Avg. Doses
(mg/kg/day)
0
6
20
60
of animals
60
60
60
60
J.>J ULLU^C i.
with fatal
tumors
0
35
49
49
with incidental
tumors
0
19
10
10
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk Time (yr)
l.OOOOE-006
l.OOOOE-005
0.0001
0.0010
0.01
0.10
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
3 .7108E-005
3.7108E-004
3.7110E-003
3.7126E-002
3.9005E-001
3.9064E+000
MLE
9.2044E-005
9.2044E-004
9.2047E-003
9.2076E-002
9.2366E-001
9.5469E+000
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
ir:
Fatal Graph
FM FS fatal.TTD - TCP - female mouse/gavage/forestomach tumors
Model: Two Stage Weib
1
0.8
0.6
0.4
0.2
Dose (mg/kg/day)=6
Dose (mg/kg/day)=20
Dose (mg/kg/day)=60
Kaplan Meier (6)
Kaplan Meier (20)
Kaplan Meier (60)
30 40 50 60 70 80
Time (wks)
90
100
110
227
-------
Female Mouse; Liver Tumors
Dataset: M:\_ToxRiskData\Trichloropropane\FM liver.TTD
Model: Three Stage Weib
Functional form: 1 - EXP[( -QO - Ql * D - Q2 * D^2 - Q3
* (T - TO)*Z]
Maximum Log-Likelihood = -9.623458e+001
Parameter Estimates :
Q 0
Q 1
Q 2
Q 3
Z
TO
= 5.629531E-018
= 9.202992E-019
= O.OOOOOOE+000
= 5.479834E-021
= 8.197183E+000
= O.OOOOOOE+000
Set by User
Avg. Doses
(mg/kg/day)
0
6
20
60
of animals
60
60
60
60
with fatal
tumors
0
0
0
0
with incidental
tumors
8
11
9
36
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk Time (yr)
OOOOE-006
l.OOOOE-005
0.0001
0.0010
0.01
0.10
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
1.3081E-003
1.3081E-002
1.3082E-001
1.3088E+000
1.3146E+001
1.3711E+002
MLE
.0192E-003
.0192E-002
.0193E-001
.0207E+000
.0328E+001
3.0250E+002
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
(fl
or
1
0.8
0.6
0.4
0.2
Incidental Graph
FM liver.TTD - TCP - female mouse/gavage/liver tumors
Model: Three Stage Weib
Dose (mg/kg/day)=6
Dose (mg/kg/day)=20
Dose (mg/kg/day)=60
Hoel Walburg (6)
Hoel Walburg (20)
Hoel Walburg (60)
20
40
60
Time (wks)
80
100
120
228
-------
Female Mouse; Harderian Gland Tumors
Dataset: M:\_ToxRiskData\Trichloropropane\FM harderian.TTD
Model: One Stage Weib
Functional form: 1 - EXP[( -QO - Ql * D ) * (T - TO)*Z]
Maximum Log-Likelihood = -7.460252e+001
Parameter Estimates :
Q 0 = 6.904433E-012
Q 1 = 3.003880E-012
Z = 4 .928402E+000
TO = O.OOOOOOE+000
Set by User
Avg. Doses
(mg/kg/day)
0
6
20
60
of animals
60
60
60
60
with fatal
tumors
0
0
0
0
with incidental
tumors
3
6
7
10
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Human Equivalent Dose Estimates (ug/kg/day)
Incid Extra Risk
l.OOOOE-006
l.OOOOE-005
0.0001
0.0010
0.01
0.10
Time (yr)
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
1.8987E-003
1.8987E-002
1.8987E-001
1.8996E+000
1.9082E+001
2 .0004E+002
MLE
.9648E-003
.9648E-002
.9650E-001
.9668E+000
.9848E+001
4.1774E+002
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Incidental Graph
FM harderian.TTD - TCP - female mouse/gavage/harderian gland
Model: One Stage Weib
1
0.8
0.6
0.4
0.2
0
Dose (mg/kg/day)=6
Dose (mg/kg/day)=20
Dose (mg/kg/day)=60
Hoel Walburg (6)
Hoel Walburg (20)
Hoel Walburg (60)
20
40
60
Time (wks)
80
100
120
229
-------
Female Mouse; Uterus Adenomas or Carcinomas
Dataset: M:\_ToxRiskData\Trichloropropane\FM uterus.TTD
Model: Two Stage Weib
Functional form: 1 - EXP[( -QO - Ql * D - Q2 * D^2) * (T - TO)*Z]
Maximum Log-Likelihood = -4.567713e+001
Parameter Estimates :
Q 0 = O.OOOOOOE+000
Q 1 = 5.955970E-023
Q 2 = 2.395006E-023
Z = l.OOOOOOE+001
TO = O.OOOOOOE+000
Avg. Doses
(mg/kg/day)
0
6
20
60
of animals
60
60
60
60
with fatal
tumors
0
0
0
0
with incidental
tumors
0
5
3
11
Animal to human conversion method: MG/KG BODY WEIGHT(3/4)/DAY
Incid Extra Risk
l.OOOOE-006
l.OOOOE-005
0.0001
0.0010
0.01
0.10
Human Equivalent Dose Estimates (ug/kg/day)
Time (yr)
70.00
70.00
70.00
70.00
70.00
70.00
95.00 %
Lower Bound
2.2635E-003
2.2635E-002
2 .2634E-001
2 .2617E+000
2.2459E+001
2 .1205E+002
MLE
0593E-002
0589E-001
0550E+000
0192E+001
0875E+001
4.2138E+002
95.00 %
Upper Bound
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Not Reqstd
Incidental Graph
FM uterus.TTD - TCP - female mouse, uterus
Model: Two Stage Weib
U)
ir:
1
0.8
0.6
0.4
0.2
Dose (mg/kg/day)=6
Dose (mg/kg/day)=20
Dose (mg/kg/day)=60
Hoel Walburg (6)
Hoel Walburg (20)
Hoel Walburg (60)
H
=F
20
40
60
Time (wks)
80
100
120
230
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Table D-5. Summary of human equivalent overall cancer risk values estimated by R/BMDR, based on male and female
rat and mouse tumor incidence
Tumor site
Tumor
context
Risk, R
BMDR,
mg/kg-day
BMDLR,
mg/kg-d
Risk value at
BMDRa, per
mg/kg-d
Oral slope
factor1", per
mg/kg-d
SD
SD2
Proportion of
total variance
Male rats
Oral route squamous
papillomas, carcinomas
Pancreas acinar tumors
Preputial gland tumors
Kidney tubule adenomas
Skin: squamous papillomas,
carcinomas
Zymbal's gland carcinomas
Hepatocellular tumors
Incidental
Fatal
Incidental
Incidental
Incidental
Incidental
Incidental
Incidental
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.001
0.00049
0.00039
0.0028
0.0127
0.6988
0.0319
0.1181
0.1645
0.00032
0.00030
0.0010
0.0056
0.5259
0.0136
0.0484
0.0115
Sum, MLE cancer risks calculated at R=0.001C:
2.046
2.551
0.353
0.078
0.015
0.031
0.008
0.006
3.04C (2.54)
Upper bound on sum of risk estimates":
3.16
3.29
1.00
0.18
0.11
0.07
0.02
0.09
4.04 (3.84)
0.6765
0.4462
0.3940
0.0614
0.0578
0.0256
0.0074
0.0491
Sum, SD2:
Overall SDd:
0.4577
0.1991
0.1552
0.0038
0.0033
0.0007
0.00006
0.0024
0.365 (0.623)
0.603 (0.789)
0.55
0.43
0.01
0.01
0.00
0.00
0.00
0.73
0.25
0.01
0.01
0.00
0.00
0.00
Female rats
Oral route squamous
papillomas, carcinomas
Mammary adenocarcinomas
Clitoral gland adenomas,
carcinomas
Zymbal's gland carcinomas
Incidental
Fatal
Incidental
Fatal
Incidental
Incidental
0.001
0.001
0.001
0.001
0.001
0.001
0.00226
0.00238
0.00575
0.00952
0.00295
0.04691
0.00053
0.00097
0.00405
0.00332
0.00230
0.01497
Sum, MLE cancer risks calculated at R=0.001C:
0.442
0.421
0.174
0.105
0.339
0.021
0.91C(1.01)
Upper bound on sum of risk estimates6:
1.894
1.027
0.247
0.302
0.435
0.067
0.001
1.53 (2.47)
0.8828
0.3681
0.0442
0.1194
0.0584
0.0277
Sum, SD2:
Overall SDd:
0.7793
0.1355
0.0020
0.0143
0.0034
0.0008
0.155 (0.786)
0.394 (0.886)
0.880
0.093
0.022
0.005
0.992
0.003
0.004
0.001
231
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Table D-5. Summary of human equivalent overall cancer risk values estimated by R/BMDR, based on male and female
rat and mouse tumor incidence
Tumor site
Tumor
context
Risk,
R
BMDR,
mg/kg-d
BMDLR,
mg/kg-d
Risk value at
BMDRa, per
mg/kg-d
Oral slope
factor1", per
mg/kg-d
SD
SD2
Proportion of total
variance
Male mice
Oral route squamous
papillomas, carcinomas.
Liver adenoma or carcinoma
Harderian gland
Incidental
Fatal
Incidental
Incidental
0.001
0.001
0.001
0.001
0.00029
0.0030
0.0021
0.0107
0.00016
0.0013
0.0013
0.0055
Sum, MLE cancer risks calculated at R=0.001C:
3.49
4.71
0.48
0.09
5.29C (4.07)
Upper bound on sum of risk estimates6:
6.20
6.23
0.77
0.18
6.84 (6.79)
1.6439
0.9226
0.1762
0.0548
Sum, SD2:
Overall SDd:
2.703
0.851
0.031
0.003
0.885 (2.737)
0.94 (1.65)
0.96
0.04
0.00
0.99
0.01
0.00
Female mice
Oral route squamous
papillomas, carcinomas.
Liver adenoma or carcinoma
Harderian gland
Uterus
Incidental
Fatal
Incidental
Incidental
Incidental
0.001
0.001
0.001
0.001
0.001
0.000030
0.000092
0.0030
0.0040
0.0102
0.000006
0.000037
0.0013
0.0019
0.0023
Sum, MLE cancer risks calculated at R=0.001C:
33.392
10.861
0.331
0.252
0.098
11.5C(34.1)
Upper bound on sum of risk estimates6:
161.267
26.935
0.764
0.526
0.442
27.6 (162)
77.7353
9.7719
0.2632
0.1668
0.2091
Sum, SD2:
Overall SDd:
6042.78
95.49
0.07
0.03
0.04
95.63 (6042.9)
9.8 (77.7)
1.00
0.00
0.00
0.00
1.00
0.00
0.00
0.00
aR/BMDR
bR/BMDLR
'Summary statistics in bold were calculated using the bolded table entries, including the "fatal" entries for the oral route (and mammary gland for female rats). The
summary statistics in parentheses were calculated using the "incidental" entries for the oral route and the bolded entries for the other tumor sites.
dOverall SD = (Sum, SD2)05
eUpper bound on the overall risk estimate = sum of MLE cancer risks + 1.645 x overall SD
Source: NTP(1993).
232
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