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www.epa.gov/iris
f/EPA
TOXICOLOGICAL REVIEW
OF
HEXACHLOROETHANE
(CAS No. 67-72-1)
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
April 2011
NOTICE
This document is a Final Agency/Interagency Science Discussion draft. This information is
distributed solely for the purpose of pre-dissemination peer review under applicable information
quality guidelines. It has not been formally disseminated by EPA. It does not represent and
should not be construed to represent any Agency determination or policy. It is being circulated
for review of its technical accuracy and science policy implications.
U.S. Environmental Protection Agency
Washington, DC
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DISCLAIMER
This document is a preliminary draft for review purposes only. This information is
distributed solely for the purpose of pre-dissemination peer review under applicable information
quality guidelines. It has not been formally disseminated by EPA. It does not represent and
should not be construed to represent any Agency determination or policy. Mention of trade
names or commercial products does not constitute endorsement or recommendation for use.
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CONTENTS—TOXICOLOGICAL REVIEW OF HEXACHLOROETHANE
(CAS No. 67-72-1)
LIST OF TABLES vi
LIST OF FIGURES viii
LIST OF ABBREVIATIONS AND ACRONYMS ix
FOREWORD xi
AUTHORS, CONTRIBUTORS, AND REVIEWERS xii
1. INTRODUCTION 1
2. CHEMICAL AND PHYSICAL INFORMATION 3
3. TOXICOKINETICS 5
3.1. ABSORPTION 5
3.2. DISTRIBUTION 5
3.3. METABOLISM 8
3.4. ELIMINATION 15
3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELS 16
4. HAZARD IDENTIFICATION 17
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL
CONTROLS 17
4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
ANIMALS—ORAL AND INHALATION 19
4.2.1. Oral 19
4.2.1.1. Subchronic Exposure 19
4.2.1.2. Chronic Exposure and Carcinogenicity 23
4.2.2. Inhalation 31
4.2.2.1. Subchronic Exposure 31
4.2.2.2. Chronic Exposure 33
4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND
INHALATION 34
4.3.1. Oral 34
4.3.2. Inhalation 36
4.4. OTHER DURATION- OR ENDPOINT-SPECIFIC STUDIES 37
4.4.1. Acute Exposure Studies 37
4.4.1.1. Oral 37
4.4.1.2. Inhalation 38
4.4.2. Short-term Exposure Studies 39
4.4.3. Neurological 42
4.4.3.1. Oral Studies 43
4.4.3.2. Inhalation Studies 44
4.4.4. Immunological 44
4.4.5. Dermatological 45
4.4.6. Eye Irritation 45
4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE
MODE OF ACTION 46
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4.5.1. Genotoxicity 46
4.5.2. In Vitro and Ex Vivo Studies Using Isolated Target Tissues/Organs or
Cells 52
4.5.3. Structure Activity Relationships 55
4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS 57
4.6.1. Oral 57
4.6.1.1. Nephrotoxicity 59
4.6.1.2. Hepatotoxicity 61
4.6.1.3. Developmental Toxicity 62
4.6.2. Inhalation 63
4.6.3. Mode-of-Action Information 64
4.7. EVALUATION OF CARCINOGENICITY 66
4.7.1. Summary of Overall Weight of Evidence 66
4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence 67
4.7.3. Mode-of-Action Information 69
4.7.3.1. Kidney Tumors 69
4.7.3.2. Liver Tumors 82
4.7.3.3. Pheochromocytomas 84
4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 84
4.8.1. Possible Childhood Susceptibility 85
4.8.2. Possible Gender Differences 85
4.8.3. Other 85
5. DOSE-RESPONSE ASSESSMENTS 86
5.1. ORAL REFERENCE DOSE (RfD) 86
5.1.1. Choice of Principal Study and Critical Effect—with Rationale and
Justification 86
5.1.2. Methods of Analysis—Including Models 90
5.1.3. RfD Derivation—Including Application of Uncertainty Factors (UFs) 93
5.1.4. RfD Comparison Information 94
5.1.5. Previous RfD Assessment 96
5.2. INHALATION REFERENCE CONCENTRATION (RfC) 96
5.2.1. Choice of Principal Study and Critical Effect—with Rationale and
Justification 96
5.2.2. Methods of Analysis—Including Models 99
5.2.3. RfC Derivation—Including Application of Uncertainty Factors (UFs) 101
5.2.4. RfC Comparison Information 102
5.2.5. Previous RfC Assessment 102
5.3. UNCERTAINTIES IN THE ORAL REFERENCE DOSE AND INHALATION
REFERENCE CONCENTRATION 102
5.4. CANCER ASSESSMENT 105
5.4.1. Choice of Study/Data—with Rationale and Justification 105
5.4.2. Dose-response Data 105
5.4.3. Dose Adjustments and Extrapolation Methods 106
5.4.4. Oral Slope Factor and Inhalation Unit Risk 109
5.4.5. Uncertainties in Cancer Risk Values 109
5.4.5.1. Sources of Uncertainty Ill
5.4.6. Previous Cancer Assessment 114
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6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND
DOSE RESPONSE 115
6.1. HUMAN HAZARD POTENTIAL 115
6.2. DOSE RESPONSE 116
6.2.1. Oral Noncancer 116
6.2.2. Inhalation Noncancer 117
6.2.3. Cancer 117
7. REFERENCES 120
APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC
COMMENTS AND DISPOSITION A-l
APPENDIX B: BENCHMARK DOSE MODELING OUTPUT B-l
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LIST OF TABLES
2-1. Physical properties of HCE 3
3-1. HCE, PERC, and pentachloroethane tissue concentrations in anesthetized sheep 8.5
hours after injection of 500 mg/kgHCE 6
3-2. Time course of HCE concentrations in male rat tissues after 57 days of dietary
exposure to 62 mg/kg-day 7
3-3. HCE concentrations in male and female rat tissues after 110 or 111 days of dietary
exposure 8
3-4. Disposition of HCE in male rats and mice during 48 hours following administration
of an MTD for 4 weeks 10
3-5. Metabolism of HCE measured in rats and mice 11
3-6. Product formation rates and relative ratios of the products formed by CYP450 1A2
metabolism of HCE 14
4-1. Body, kidney, and liver weights of rats exposed to HCE in the diet for 16 weeks 21
4-2. Histopathological results on kidney in rats exposed to HCE in the diet for 16 weeks3 21
4-3. Organ weight to body weight ratios for rats exposed to HCE for 13 weeks 22
4-4. Incidence and severity of nephropathy in male and female rats treated with HCE 24
4-5. Additional kidney effects in HCE-treated rats 26
4-6. Renal tubular hyperplasia and tumor incidences in HCE-treated male rats 26
4-7. Adrenal medullary lesions in HCE-treated male rats 27
4-8. Tumor incidences3 in male rats gavaged with HCE 29
4-9. Tumor incidences in female rats gavaged with HCE 30
4-10. Incidence of hepatocellular carcinomas in mice 31
4-11. Summary of HCE effects on pregnant Wi star rats and their fetuses 35
4-12. Summary of skeletal effects on fetuses from HCE-exposed rats 36
4-13. Summary of acute exposure data in rats, rabbits, and guinea pigs 38
4-14. Summary oftoxicity data from male rats exposed to HCE for 21 days 42
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4-15. Summary of genotoxicity studies of HCE 47
4-16. Number of enzyme-altered foci in rat liver of the promotion protocol 53
4-17. In vivo covalent binding of [14C]-HCE to DNA, RNA, and proteins from rat and
mouse organs 54
4-18. In vitro binding of [14C]-HCE to calf thymus DNA mediated by microsomal and/or
cytosolic phenobarbital-induced fractions of rat and mouse organs 55
4-19. Oral toxicity studies for HCE 58
4-20. Inhalation toxicity studies with HCE 64
4-21. Nephrotoxic effects characteristic of a2u-globulin nephropathy observed in male and
female rats administered HCE 71
5-1. Incidences of noncancerous kidney and liver effects in rats following oral exposure to
HCE 88
5-2. Summary of the BMD modeling results for the kidney 91
5-3. Potential PODs for nephrotoxicity in male rats with applied UFs and potential
reference values 94
5-4. Noncancerous effects observed in animals exposed to HCE via inhalation 99
5-5. Summary of incidence data in rodents orally exposed to HCE for use in cancer dose-
response assessment 106
5-6. Summary of BMD modeling results for oral cancer assessment of HCE 108
B-l. Dose-response modeling results using BMDS (version 2.0) based on non-cancerous
kidney and liver effects in rats following oral exposure to HCE B-l
B-2. Dose-response modeling results using BMDS (version 2.0) for BMRs of 1, 5, and
10% based on noncancerous kidney and liver effects in rats following oral exposure
to HCE B-5
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LIST OF FIGURES
2-1. Structure of HCE 3
3-1. Possible metabolic pathway of HCE 9
5-1. Array of potential PODs with applied UFs and potential reference values for
nephrotoxic effects of studies in Table 5-3 95
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LIST OF ABBREVIATIONS AND ACRONYMS
AIC Akaike's information criterion
ALD approximate lethal dosage
ALT alanine aminotransferase
AST aspartate aminotransferase
BMD benchmark dose
BMDL benchmark dose lower confidence limit
BMDS Benchmark Dose Software
BMR benchmark response
BUN blood urea nitrogen
BW body weight
CA chromosomal aberration
CAS Chemical Abstracts Service
CASRN Chemical Abstracts Service Registry Number
CBI covalent binding index
CHO Chinese hamster ovary
CL confidence limit
CNS central nervous system
CPN chronic progressive nephropathy
CYP450 cytochrome P450
DAF dosimetric adjustment factor
DEN diethylnitrosamine
DMSO dimethylsulfoxide
DNA deoxyribonucleic acid
FDA Food and Drug Administration
FEVi.o forced expiratory volume of 1 second
GD gestation day
GDH glutamate dehydrogenase
GGT y-glutamyl transferase
GSH glutathione
GST glutathione-S-transferase
Hb/g-A animal blood:gas partition coefficient
Hb/g-H human blood:gas partition coefficient
HCE hexachloroethane
HEC human equivalent concentration
HED human equivalent dose
i.p. intraperitoneal
IRIS Integrated Risk Information System
IVF in vitro fertilization
LCso median lethal concentration
LDso median lethal dose
LOAEL lowest-observed-adverse-effect level
MN micronuclei
MNPCE micrenucleated polychromatic erythrocyte
MTD maximum tolerated dose
NAG TV-acetyl-p-D-glucosaminidase
NCI National Cancer Institute
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NOAEL no-observed-adverse-effect level
NTP National Toxicology Program
OCT ornithine carbamoyl transferase
PBPK physiologically based pharmacokinetic
PCNA proliferating cell nuclear antigen
PERC tetrachloroethene, tetrachloroethylene, perchloroethylene
POD point of departure
POD[ADJ] duration-adjusted POD
QSAR quantitative structure-activity relationship
RDS replicative DNA synthesis
RfC inhalation reference concentration
RfD oral reference dose
RGDR regional gas dose ratio
RNA ribonucleic acid
SAR structure activity relationship
SCE sister chromatid exchange
SD standard deviation
SDH sorbitol dehydrogenase
SE standard error
SCOT glutamic oxaloacetic transaminase, also known as AST
SGPT glutamic pyruvic transaminase, also known as ALT
SSD systemic scleroderma
TCA trichloroacetic acid
TCE trichloroethylene
TWA time-weighted average
UF uncertainty factor
interspecies uncertainty factor
intraspecies uncertainty factor
subchronic-to-chronic uncertainty factor
database deficiencies uncertainty factor
U.S. EPA U.S. Environmental Protection Agency
UFH
UFs
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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
hexachloroethane. It is not intended to be a comprehensive treatise on the chemical or
toxicological nature of hexachloroethane.
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 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
John Cowden, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC
AUTHORS
Samantha Jones, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
CONTRIBUTORS
Ted Berner, M.S.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Glinda Cooper, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
Andrew A. Rooney, Ph.D.
Currently at National Toxicology Program
Center for the Evaluation of Risks to Human Reproduction
National Institute of Environmental Health Sciences
Research Triangle Park, NC
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CONTRACTOR SUPPORT
James Kim, Ph.D.
Sciences International, Inc.
Alexandria, VA
James Riddle, Ph.D.
Sciences International, Inc.
Alexandria, VA
Jay Turim, Ph.D.
Sciences International, Inc.
Alexandria, VA
Vera Jurgenson, M.S.
Sciences International, Inc.
Alexandria, VA
Sheila McCarthy, M.S.
Sciences International, Inc.
Alexandria, VA
Harriet McCollum
Sciences International, Inc.
Alexandria, VA
Bobette Nourse, Ph.D.
Oak Ridge Institute for Science and Education (ORISE)
Oak Ridge, TN
Lutz Weber, DABT
ORISE
Oak Ridge, TN
George Holdsworth, Ph.D.
ORISE
Oak Ridge, TN
Sheri Hester, M.S.
ORISE
Oak Ridge, TN
REVIEWERS
This document has been provided for review to EPA scientists, interagency reviewers from
other federal agencies and White House offices, and the public, and has been 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.
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INTERNAL EPA REVIEWERS
AmbujaBale, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Ghazi Dannan, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Kate Guyton, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Maureen Gwinn, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Jennifer Jinot, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Channa Keshava, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Allan Marcus, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
D. Charles Thompson, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Reeder Sams, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Debra Walsh
National Center for Environmental Assessment
Office of Research and Development
John Vandenberg, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
John Whalan
National Center for Environmental Assessment
Office of Research and Development
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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
hexachloroethane (HCE). 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 HCE 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'ChemicalMixtures (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, 199 la), Interim Policy for Particle Size and Limit Concentration
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Issues in Inhalation Toxicity (U.S. EPA, 1994a), Methods for Derivation of Inhalation Reference
Concentrations and Application of Inhalation Dosimetry (U.S. EPA, 1994b), Use of the
Benchmark Dose Approach in Health Risk Assessment (U.S. EPA, 1995), Guidelines for
Reproductive Toxicity Risk Assessment (U.S. EPA, 1996), Guidelines for Neurotoxicity Risk
Assessment (U.S. EPA, 1998), Science Policy Council Handbook. Risk Characterization (U.S.
EPA, 2000a), Benchmark Dose Technical Guidance Document (U.S. EPA, 2000b),
Supplementary Guidance for Conducting Health Risk Assessment of Chemical Mixtures (U.S.
EPA, 2000c), A Review of the Reference Dose and Reference Concentration Processes (U.S.
EPA, 2002), Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), Supplemental
Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens (U.S. EPA,
2005b), Science Policy Council Handbook: Peer Review (U.S. EPA, 2006a), and A Framework
for Assessing Health Risks of Environmental Exposures to Children (U.S. EPA, 2006b).
The literature search strategy employed for this compound was based on the Chemical
Abstracts Service Registry Number (CASRN) and at least one common name. Any pertinent
scientific information submitted by the public to the IRIS Submission Desk was also considered
in the development of this document. The relevant literature was reviewed through April 2011.
No new publications were identified since the release of the external peer review draft
Toxicological Review (May 2010).
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2. CHEMICAL AND PHYSICAL INFORMATION
Hexachloroethane (HCE; CASRN 67-72-1) is a halogenated hydrocarbon consisting of
six chlorines attached to an ethane backbone (Figure 2-1). Synonyms include
1,1,1,2,2,2-hexachloroethane, ethane hexachloride, ethylene hexachloride, perchloroethane,
carbon hexachloride, and carbon trichloride (ChemlDplus Advanced, 2005; ACGIH, 1991).
Certain physical and chemical properties are shown below in Table 2-1 (ACGIH, 2001; ATSDR,
1997a; Budavari, 1989; Howard, 1989; Weast, 1986; Spanggord etal., 1985; Verschueren, 1983;
U.S. EPA, 1982, 1979).
Cl Cl
ci—c—c—ci
Cl Cl
Figure 2-1. Structure of HCE.
Table 2-1. Physical properties of HCE
Name
CASRN
Synonyms
Molecular weight
Molecular formula
Melting point
Boiling point
Density
Water solubility3
Log Kow
Log Koc
Vapor pressure
Henry's law constant
Conversion factor
Hexachloroethane
67-72-1
1,1,1,2,2,2-hexachloroethane, ethane hexachloride, ethylene hexachloride,
perchloroethane, carbon hexachloride, carbon trichloride
236.74 g/mol
C2C16
Sublimes without melting
186.8°C
2.091g/mLat20°C
50 mg/L at 22°C; 14 mg/L at 25°C
3.82a, 3.34b, 4.14C
4.3
0.5 mniHg at 20°C; 1.0 mniHg at 32.7°C
2.8 x 10"3 atm-m3/mol at 20°C
1 ppm = 9.68 mg/m3; 1 mg/m3= 0.10 ppm
Sources: "Howard (1989); 'US. EPA (1979); cHansch et al. (1995).
HCE was produced in the United States for commercial distribution from 1921 to 1967,
but is currently not commercially distributed (ATSDR, 1997a; IARC, 1979). In the 1970s,
producers of HCE reported that HCE was not distributed, but was used in-house or recycled
(ATSDR, 1997a); distributors in the 1970s imported HCE from France, Spain, and the United
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Kingdom (ACGIH, 2001; ATSDR, 1997a). HCE and tetrachloroethane imports combined were
1.5 million pounds in 1989 and 612,000 pounds in 2000 (NTP, 2005). HCE production in 1977
was estimated between 2 and 20 million pounds; more recent information on production of HCE
was not located (NTP, 2005; ATSDR, 1997a). HCE is produced by the chlorination of
tetrachloroethylene (PERC) in the presence of ferric chloride at temperatures of 100-140°C
(ATSDR, 1997a; U.S. EPA, 1991b; Fishbein, 1979; IARC, 1979). HCE is primarily used in the
military for smoke pots, smoke grenades, and pyrotechnic devices (ACGIH, 2001; ATSDR,
1997a; U.S. EPA, 1991b; IARC, 1979). HCE was also identified in the headspace of
chlorine-bleach-containing household products (Odabasi, 2008). In the past, HCE was used as
an antihelminthic for the treatment of sheep flukes, but is no longer used for this purpose since
the U.S. Food and Drug Administration (FDA) withdrew approval for this use in 1971 (ATSDR,
1997a). HCE has also been used as a polymer additive, a moth repellant, a plasticizer for
cellulose esters, and an insecticide solvent, and in metallurgy for refining aluminum alloys
(ATSDR, 1997a; U.S. EPA, 1991b).
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3. TOXICOKINETICS
3.1. ABSORPTION
There are no studies that have systematically evaluated HCE absorption in humans by the
oral or inhalation routes of exposure. However, uptake was demonstrated by Younglai et al.
(2002) when HCE was identified in follicular fluid during an analysis for environmental
contaminants in 21 couples undergoing in vitro fertilization (IVF). These data identify the
potential for HCE absorption but not the source or route of exposure. No studies have been
reported that assess the inhalation absorption of HCE in humans. The dermal absorption rate of
HCE has been described as limited (ATSDR, 1997a). Based on physical properties, the
absorption of a saturated HCE solution across human skin was estimated to be
0.023 mg/cm2/hour (Fiserova-Bergerova et al., 1990).
Studies in animals via the oral route of exposure demonstrated that HCE is absorbed and
primarily distributed to fat (Gorzinski et al., 1985; Nolan and Karbowski, 1978; Fowler, 1969).
Fowler (1969) orally administered 500 mg/kg HCE to Scottish Blackface or Cheviot sheep and
found that maximal venous blood concentrations of HCE (10-28 ug/mL) were reached at
24 hours after HCE exposure, indicating slow absorption. Jondorf et al. (1957) reported that
rabbits fed [14C]-radiolabeled HCE at 500 mg/kg excreted only 5% of the applied radioactivity in
urine over a period of 3 days (fecal measurements were not conducted). During this 3-day
period, 14-24% of the applied radioactivity was detected in expired air, and the remainder was
present in the tissues and intestinal tract. The amount of HCE absorbed by the rabbits was not
determined; however, based on the amount of radioactivity present in urine and expired air,
approximately 19-29% of the HCE was absorbed. Studies in rats and mice (Mitoma et al., 1985)
using [14C]-radiolabeled HCE (500 mg/kg for rats; 1,000 mg/kg for mice) administered orally,
via corn oil, indicated that the amounts absorbed were 65-71 and 72-88%, respectively, based
on the amount of radiolabel detected in expired air and total excreta (i.e., both urine and fecal
excreta combined).
3.2. DISTRIBUTION
There are limited data on the distribution of HCE in humans (Younglai et al., 2002). The
animal studies evaluated (Gorzinski et al., 1985; Nolan and Karbowski, 1978; Fowler, 1969)
consistently demonstrated that HCE is distributed primarily to fat tissue followed by the kidney
and to a lesser extent the liver and the blood (Gorzinski et al., 1985; Nolan and Karbowski,
1978).
Younglai et al. (2002) evaluated the concentrations of various environmental
contaminants in follicular fluid, serum, and seminal plasma of 21 couples undergoing IVF. HCE
was one of the contaminants identified in >50% of follicular fluid samples, suggesting
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postabsorptive distribution to reproductive organs. The average HCE concentration in follicular
fluid was 232 + 27 pg/mL (mean + standard error [SE]). HCE was not detected in human female
serum obtained during oocyte retrieval for IVF. This study focused primarily on chemicals such
as pesticides and polychlorinated biphenyls, and the authors could not make any conclusions
with regards to the level of HCE in follicular fluid and its effect on fertility.
Fowler (1969) evaluated the tissue distribution of HCE, PERC (tetrachloroethylene), and
pentachloroethane in sheep. Two sheep were fasted for 24 hours and then anesthetized with
sodium pentobarbital. An HCE solution (15% w/v in olive oil) was injected for a total dose of
500 mg/kg directly into the rumen and lower duodenum (dose was divided). Anesthesia was
maintained for 8.5 hours, after which time the sheep were sacrificed and tissues were taken
within 10 minutes of death. Tissues that were evaluated include the brain, fat, kidney, liver, and
muscle. Bile and blood were also evaluated. HCE was widely distributed and the highest levels
were found in fat of one sheep. Fat from different sites did not show significant variation in
HCE concentration. The second sheep had only trace amounts of HCE in tissue (see Table 3-1).
Table 3-1. HCE, PERC, and pentachloroethane tissue concentrations in
anesthetized sheep 8.5 hours after injection of 500 mg/kg HCE
Tissue
Bile (4 hr)
Blood (6 hr)
Brain
Fat
Kidney
Liver
Muscle
Concentration (jig/g)
Sheep 1
HCE
1.7
0.2
0.2
1.1
0.1
0.2
0.04
PERC
0.3
0.4
0.9
2.1
1.2
0.9
0.5
Pentachloroethane
Trace
Trace
0.02
0.02
Trace
0.01
0.01
Sheep 2
HCE
2.2
0.2
Trace
Trace
Trace
Trace
Trace
PERC
0.5
0.2
Trace
0.6
0.6
2.8
Trace
Pentachloroethane
Nil
Nil
Trace
Nil
Trace
Trace
Trace
Source: Fowler (1969).
Nolan and Karbowski (1978) studied tissue clearance of HCE in rats. Male F344 rats
were placed on an HCE-containing diet that was calculated to deliver 100 mg/kg-day (later
determined to be 62 mg/kg-day by Gorzinski et al., 1985) for 57 days. After this exposure
period, the rats were returned to an HCE-free control diet and sacrificed (groups of three or four
rats) 0, 3, 6, 13, 22, and 31 days after this change in exposure. Samples of fat, liver, kidney, and
whole blood were collected for HCE analysis. The time-course related tissue HCE
concentrations are presented in Table 3-2. The highest tissue concentrations of HCE were in fat,
which were 3-fold greater than the concentration in the kidney and over 100-fold greater than
blood and liver concentrations. Fat concentrations decreased from 303 + 50 ug/g in a first-order
manner with a half-life of 2.7 days. Concentrations in blood and kidney also decreased in a first-
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order manner with half-lives of 2.5 and 2.6 days, respectively. Liver concentrations initially
increased in the first 3 days postexposure, but began to decrease by day 6. The half-life for liver
HCE was 2.3 days (calculated after peak levels were reached at day 3). These same results were
published in a follow-up study by Gorzinski et al. (1985) that included a toxicity assessment.
Table 3-2. Time course of HCE concentrations in male rat tissues after
57 days of dietary exposure to 62 mg/kg-day
Days after cessation of HCE exposure
0
o
5
6
13
22
31
HCE tissue concentrations (n = 3 or 4) (mean + SD jig/g tissue)
Blood
0.834+0.223
0.279 + 0.048
0.0835 + 0.0063
0.015+0.005
0.002 + 0.001
NDC
Liver
0.143+0.040
0.399 + 0.188
0.303 +0.1563
0.039+0.023
0.001+0.001
NDC
Kidney
81.8 + 5.3
41.0 + 1.4
18.5b
2.53 + 1.02
0.194 + 0.171
0.026+0.006
Fat
303+50
107.8+10.5
62.45 +3.043
6.56 + 0.52
0.472 + 0.232
0.125+0.020
"Values from one of the three rats was consistently low and not used to obtain the mean + standard deviation (SD).
bOne sample was lost and a mean + SD could not be calculated.
°ND: not detected (detection limit of 0.001 ug/g).
Sources: Gorzinski et al. (1985); Nolan and Karbowski (1978).
Nolan and Karbowski (1978) also evaluated tissue concentrations of HCE in both male
and female rats after an exposure period of 110-111 days (16 weeks) to doses of 3, 30, and
100 mg/kg-day via the diet. The actual doses were approximated as 1, 15, and 62 mg/kg-day
after factoring in volatility of the test material from the food and based on linear nighttime food
consumption rates (Gorzinski et al., 1985). The tissue concentrations are presented in Table 3-3.
Kidney concentrations of HCE were much higher in male rats compared with female rats,
particularly at the highest dose (47-fold greater in males) (Nolan and Karbowski, 1978). Kidney
concentrations of HCE proportionately increased with the doses in males, whereas the increase in
females was dose-dependent but not proportionate. The authors noted that the HCE kidney
concentrations and kidney toxicity were consistently different for the male and female rats.
Consequently, they speculated that the male rats would be 10-30 times more sensitive than
female rats to HCE toxicity, based on the relative HCE concentration measured in the rat kidney
(assuming that toxicity is due to HCE and not a metabolite). Both sexes exhibited comparable
levels (although levels in males were slightly greater) of HCE in blood, liver, and fat;
concentrations in fat were the highest for both sexes. Blood levels of HCE did not correlate well
to either the exposure dose or the dose at the major target organ, the kidney, indicating that blood
levels of HCE may not be a suitable metric for the estimation of exposure to HCE in rats.
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Table 3-3. HCE concentrations in male and female rat tissues after 110 or
111 days of dietary exposure
Dose (mg/kg-day)
1
15
62
Male
Female
Male
Female
Male
Female
HCE tissue concentration (n = 3 or 4) (mean + SD, jig/g tissue)
Blood
0.079 + 0.057
0.067 + 0.039
0.596 + 0.653
0.162 + 0.049
0.742 + 0.111
0.613+0.231
Liver
0.291+0.213
0.260 + 0.035
1.736+1.100
0.472 + 0.204
0.713+0.343
0.631+0.262
Kidney
1.356 + 0.286
0.369 + 0.505
24.33+5.73
0.688 + 0.165
95.12+11.56
2.01+0.66
Fat
3.09 + 0.33
2.59+0.72
37.90 + 6.10
45.27 + 11.33
176.1 + 14.5
162.1+7.1
Sources: Gorzinski et al. (1985); Nolan and Karbowski (1978).
3.3. METABOLISM
In vitro studies using liver microsomes indicated that the major enzymes involved in
HCE metabolism are phenobarbital-inducible cytochrome P450 (CYP450) enzymes (Salmon et
al., 1985; Town and Leibman, 1984; Nastainczyk et al., 1982, 1981; Salmon et al., 1981);
however, no specific (phenobarbital-inducible) enzymes have been identified. The enzymes
induced by phenobarbital include those from the 2A, 2B, 2C, and 3A subfamilies. One study
(Yanagita et al., 1997) found some evidence for CYP1A2 involvement in the metabolism of
HCE, although this was not supported by the results from in vitro studies with
3-methylcholanthrene, an inducer of the CYP450 1 subfamily (Nastainczyk et al., 1982, 1981;
Van Dyke and Wineman, 1971). Information regarding the roles of Aroclor 1254-inducible
enzymes other than 1A2 (including CYP 2A6, 2E1, 2C9, 2C19, 2D6, and 3A4) is not available
for HCE.
The metabolism data for HCE are limited because there are only three in vivo studies
available that provide information on metabolites: Mitoma et al. (1985) in rats and mice; Jondorf
et al. (1957) in rabbits; and Fowler (1969) in sheep. Each of these studies tends to support
limited metabolism for HCE. The data from the in vivo and in vitro studies support a conclusion
that metabolism of HCE is incomplete, with excretion of unmetabolized HCE in exhaled air and
possibly in urine. A variety of intermediary metabolites have also been identified in exhaled air
and urine (Fowler, 1969; Jondorf et al., 1957). Figure 3-1 provides a possible metabolic pathway
for HCE derived from the in vivo and in vitro data with ordering of metabolites based on
sequential dechlorination and oxidation state. The HCE metabolism information was
supplemented with data on the metabolism of the PERC (ATSDR, 1997b), trichloroethylene
(TCE; ATSDR, 1997c), and 1,1,2,2-tetrachloroethane (ATSDR, 2008) intermediary metabolites.
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Cl Cl
ci—c—c—
Cl Cl
Hexachloroethane
Cl
3 — C/Cxl.2*
Carbene intermediate ^ H
Cl Cl
\ /
C=C
/ \
Cl Cl
Tetrachloroethylene
Cl
Cl H
C — C— C— O
Cl H
Trichloroethanol
\
\
ci
ci
Cl
Cl — C — C
X
H—C—C—H
Cl Cl
1,1,2,2-
Tetrachloroethane
2C1-1
I
I
O Cl
Free radical reactions
Cl Cl
I I
Cl—C—C—H
Cl Cl
Pentachloroethane
2C1-^
I
Cl Cl
\ /
c=c
/ \
Cl H
Trichloroethylene
OH
ci
Trichloroacetic acid
HO
Dichloroacetic acid
HO Cl
X
H Cl
Dichloroethanol
O O
,V
ci
\
HO OH
Oxalic acid
CO,
\ O Cl
X
HO H
Monochloroacetic acid
One carbon pool
Sources: Adapted from ATSDR (1997a); Mitoma et al. (1985); Town and
Leibman (1984); Nastainczyk et al. (1982, 1981); Bonse and Henschler (1976);
Fowler (1969); Jondorf et al. (1957).
Figure 3-1. Possible metabolic pathway of HCE.
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Mitoma et al. (1985) examined the distribution of HCE in male Osborne-Mendel rats and
male B6C3Fi mice to evaluate the extent to which radiolabeled compound is metabolized in the
48 hours after administration of 125 or 500 mg/kg to the rats and 250 or 1,000 mg/kg to the
mice. These doses were selected based on the maximum tolerated dose (MTD) and Vi MTD of
HCE; the MTD in rats and mice is 500 mg/kg (2.11 mmol/kg) and 1,000 mg/kg (4.22 mmol/kg),
respectively. Four animals per dose were orally administered unlabeled HCE as a solution in
corn oil 5 days/week for 4 weeks, followed by a single dose of [14C]-radiolabeled HCE. The
48-hour observation period began after administration of the radiolabeled HCE. The animals
were then sacrificed, and urine and feces were collected from the cages. Table 3-4 summarizes
the metabolic disposition data (based on the detection of radiolabel) at the high dose in rats and
mice. The comparable data for the lower doses were not reported.
Table 3-4. Disposition of HCE in male rats and mice during 48 hours
following administration of an MTD for 4 weeks
Expired air
C02
Excreta
Carcass
Recovery
Total metabolism (CO2 + excreta + carcass)
Rat (500 mg/kg-day)
Mouse (1,000 mg/kg-day)
Percent of administered dose
64.55+6.67
2.37+0.76
6.33+2.39
20.02 + 3.70
93.28 + 6.23
28.72
71.51 + 5.09
1.84 + 0.94
16.21+3.76
5.90 + 1.60
95.47+23.95
23.95
Source: Mitoma etal. (1985).
Recovery of the radiolabel was >90% for both rats and mice. Total metabolism was
calculated by the authors as the sum of the radiolabel present in carbon dioxide, excreta, and the
carcass. This is an assumption by the authors and is not an accurate estimate of metabolism
since actual metabolites were not quantified. Data on the extent of metabolism for the
radiolabeled material are presented in Table 3-5. Based on the mass balance between dose and
the estimate for the sum of the metabolites, 30% of the parent compound was metabolized by
both the rats and mice. This is consistent with the 60-70% of the high dose that was reported to
be present unchanged in exhaled air. However, this assumes that all of the exhaled radiolabel
that was not identified as carbon dioxide was the unmetabolized parent compound. The major
urinary metabolites, determined qualitatively by high performance liquid chromatography, were
trichloroethanol and trichloroacetic acid (TCA) for both rats and mice. Trichloroethanol and
TCA were also qualitatively considered the major urinary metabolites for other halogenated
hydrocarbon compounds, including PERC, that were evaluated by Mitoma et al. (1985).
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Table 3-5. Metabolism of HCE measured in rats and mice
Species
Rat
Mouse
Dose (mmol/kg)
0.53
2.11
1.05
4.22
Metabolism (mmol/kg)
0.16
0.60
0.32
1.01
Percent metabolized"
30
28
30
24
aPercent metabolism was calculated from the dose and the reported sum of the metabolites. This calculation is
likely an underestimation of metabolism since the exhaled air was likely to include some volatile metabolites based
on the data from Jondorf et al. (1957).
Source: Mitomaetal. (1985).
Jondorf et al. (1957) reported that rabbits fed [14C]-radiolabeled HCE at 500 mg/kg (route
of administration not reported by study authors) excreted only 5% of the applied radioactivity in
urine over 3 days (72 hours), indicating slow metabolism. This is consistent with the results in
mice and rats reported by Mitoma et al. (1985) in which approximately 2-4% of the label was
found in urine after 48 hours. During this 3-day period, 14-24% of the radioactivity was
detected in expired air (a lower percentage than seen for rats at a comparable dose by Mitoma et
al., 1985), and the remainder was present in tissues and the intestinal tract. However, the authors
did not have the capability of quantifying HCE in tissues. Reported urinary metabolites include
trichloroethanol (1.3%), dichloroethanol (0.4%), TCA (1.3%), dichloroacetic acid (0.8%),
monochloroacetic acid (0.7%), and oxalic acid (0.1%). The expired air contained HCE, carbon
dioxide, PERC, and 1,1,2,2-tetrachloroethane (TCE was not found). Quantitative data on the
volatile metabolites in exhaled air were not reported.
The only other metabolite data come from the work of Fowler (1969) in sheep. HCE was
administered to four Scottish Blackface and six Cheviot cross sheep at three dose levels: 0 (two
sheep), 500 (six sheep), 750 (one sheep), and 1,000 (one sheep) mg/kg. Two HCE metabolites,
PERC and pentachloroethane, were detected in sheep blood 24 hours after oral HCE
administration by drenching bottle. Following administration of 500 mg/kg, blood
measurements were 10-28 ug/mL for HCE, 0.6-1.1 ug/mL for PERC, and 0.06-0.5 ug/mL for
pentachloroethane. Blood concentrations of HCE, PERC, and pentachloroethane were 2.3-
2.6 times greater than the corresponding concentrations in erythrocytes. Data were not reported
for the 750 and 1,000 mg/kg doses. In vitro experiments using fresh liver slices suspended in an
olive oil emulsion confirmed the presence of the metabolites PERC and pentachloroethane.
The metabolites identified in the in vivo studies (Mitoma et al., 1985; Fowler, 1969;
Jondorf et al., 1957) along with the in vitro studies (Town and Leibman, 1984; Nastainczyk et
al., 1982) and ATSDR (1997a) were used in the derivation of Figure 3-1. The proposed
metabolic pathway is based on limited information; therefore, it is likely that intermediate
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chemical reactions are not captured in the figure, which presents the formation of the various
metabolites as single-step reactions.
The in vivo data on HCE metabolism are supported by in vitro studies of hepatic
metabolism using liver microsomes. Nastainczyk et al. (1982, 1981) reported two studies that
provide evidence that HCE is metabolized by phenobarbital-inducible CYP450 enzymes that
catalyze their reductive dechlorination with NADPH, cytochrome bs, and NADH as electron
donors. HCE metabolism was measured using liver microsomes from male Sprague-Dawley rats
that were either pretreated with phenobarbital or 3-methylcholanthrene, or were not pretreated.
Only phenobarbital-induced rat liver microsomes demonstrated an increase in HCE metabolism
(27.0 ±1.1 nmol/mg protein/minute [mean + standard deviation or SD] compared with 8.0 +
1.2 nmol/mg protein/minute for controls). Oxidation of NADPH (under anaerobic conditions)
with an oxidation rate of 35 ±2 nmol/mg protein/minute (mean + SD) provided support for
reductive dehalogenation mediated by CYP450. Carbon monoxide inhibited the NADPH
oxidation rate, further indicating that CYP450 enzymes were involved in the reaction. The major
HCE metabolite of this reductive process was PERC. Nastainzcyk et al. (1982) determined that
the stoichiometry of the reaction was represented by the following equation:
NADPH + H+ + C13C—CC13 ^ NADP+ + C12C=CC12 + 2 H+ + 2 Cl
(HCE) (PERC)
Nastainczyk et al. (1982, 1981) proposed that since CYP450 is a one electron donor, the
two electrons would be transferred sequentially. The first electron reduction would result in a
carbon radical; the second electron reduction would result in a carbanion. From the carbanion,
three possible stabilization reactions are possible: (1) protonation by a hydrogen atom from the
milieu, forming pentachloroethane; (2) a-elimination of chloride to form the carbene, which
could be stabilized by the reduced CYP450; or (3) p-elimination of chloride to form PERC,
which is the major HCE metabolite. Nastainczyk et al. (1982) found that the products of
reductive dechlorination of HCE were 99.5% PERC and 0.5% pentachloroethane at
physiological pHs. At a higher pH (8.4-8.8), the ratio of pentachloroethane (one electron
reduction) to PERC (two electron reduction) increased since transfer of the second electron can
occur via cytochrome bs, which is influenced by pH. These reaction outcomes were proposed by
the authors to also apply to other polyhalogenated hydrocarbons.
To provide additional support for the reaction being catalyzed by CYP450, Nastainczyk
et al. (1982, 1981) inhibited CYP450 using carbon monoxide, metyrapone (CYP450 3A
inhibitor), or a-naphthoflavone (CYP450 1A and CYP450 IB inhibitor) (see Omiecinski et al.,
1999 for review). In vitro metabolism of HCE by phenobarbital-induced rat liver microsomes
was inhibited >99% when carbon monoxide was added to the incubation mixture. Metyrapone at
a concentration of 10"4 M inhibited PERC formation by 46 + 10% (mean + SD) and
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pentachloroethane formation by 41 + 8%. Treatment with 10~3 M metyrapone inhibited HCE
metabolism to a greater extent, reducing PERC and pentachloroethane formation 66 + 8 and
79 + 10%, respectively. a-Naphthoflavone (10~4 M) did not inhibit HCE metabolism as
effectively as metyrapone, inhibiting PERC formation by 13 + 2% and pentachloroethane
formation by 26 + 4%. These data indicate that CYP450 3 A inhibition partially attenuated HCE
metabolism, whereas inhibition of CYP450 1A and CYP450 IB did not attenuate HCE as much
as CYP450 3 A inhibition. Since metyrapone did not completely inhibit HCE metabolism by
phenobarbital-induced liver microsomes, the remainder of HCE metabolism may be accounted
for by the CYP450 enzymes whose inhibition was not evaluated in this study (i.e., CYP450 2A
and CYP450 2B subfamilies).
Town and Leibman (1984) prepared liver microsomes from phenobarbital-induced male
Holtzman rats to study the rate of metabolism of HCE to PERC. The formation of PERC was
favored in a low oxygen environment at observed metabolism rates of 50.2 + 0.45, 1.25 + 0.25,
and 0 nmol/minute/mg protein in atmospheres of N2, air, and C>2, respectively. When any part of
the NADPH-generating system, such as NADP+, glucose 6-phosphate, and glucose 6-phosphate
dehydrogenase, was omitted from the experiment, the metabolism of HCE to PERC was
inhibited (>91%). In addition, the use of carbon monoxide as a monooxygenase inhibitor
arrested HCE metabolism. Enzymes responsible for metabolism of HCE to PERC were located
in the microsomes, rather than the cytosol, of phenobarbital-treated rat livers. Formation of
malondialdehyde and conjugated dienes was statistically, significantly increased following
treatment with HCE (8 mM), indicating lipid peroxidation. The authors suggested the
involvement of a free radical. The Km and Vmax for the enzymatic formation of PERC from HCE
were 1.20 mM and 52.0 nmol/minute/mg, respectively. Phenobarbital-induced liver microsomes
from ICR mice were also studied and yielded Km and Vmax values of 3.34 mM and 30.2 nmol/
minute/mg, respectively. PERC formation was not detected in liver microsomes from
phenobarbital-induced New Zealand White rabbits, suggesting that HCE metabolism resulting in
the formation of PERC did not occur. These results support the hypothesis that rat liver
metabolism of HCE (reductive dehalogenation) occurs by CYP450. The report identifies PERC
as a metabolite of HCE; however, the metabolite was not quantitatively measured.
Salmon et al. (1981) used Aroclor 1254-induced Sprague-Dawley rats to quantify the
dechlorination of HCE. In this case, dechlorination was measured by the release of radioactive
Cr from the [36Cl]-radiolabeled HCE substrate during incubation with liver microsomes from
induced rats. The Km and Vmax were determined as 2.37 mM and 0.91 nmol/minute/mg protein,
respectively. A control group of noninduced rats was not included.
Salmon et al. (1985) reported a follow-up study that used liver microsomes from
noninduced rats (Wistar-derived Alderley Park strain) and a reconstituted CYP450 system from
noninduced and phenobarbital-induced New Zealand White rabbits. Metabolic experiments of
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HCE using liver microsomes from noninduced rats yielded a Km of 6.0 uM and a Vmax of
3.55 nmol NADPH/minute/mg protein (2.41 nmol NADPH/minute/nmol CYP450). These
results are not directly comparable to the previous study (Salmon et al., 1981) because of the use
of a different rat strain. A reconstituted CYP450 system from phenobarbital-induced New
Zealand White rabbits yielded Km and Vmax values of 50 uM and 2.39 nmol NADPH/minute/
nmol CYP450, respectively (Salmon et al., 1985). Microsomes from rabbits induced with
p-naphthoflavone did not metabolize HCE. These results provide further evidence that the
reductive dechlorination of HCE is catalyzed by phenobarbital-inducible CYP450 enzymes.
Yanagita et al. (1997) used recombinantly-expressed rat CYP450 1A2 in Saccharomyces
cerevisiae to evaluate the in vitro metabolism of several chlorinated ethylenes and ethanes,
including HCE. The metabolism of HCE by wild-type CYP450 1A2 under aerobic conditions
resulted in the formation of PERC (3.7 nmol/2.5 nmol CYP450/hour), pentachloroethane
(0.8 nmol/2.5 nmol CYP450/hour), and TCE (0.6 nmol/2.5 nmol CYP450/hour). CYP450 1A2
is a major hepatic CYP450 enzyme, but is not a phenobarbital-inducible enzyme; the major
phenobarbital-inducible CYP450 enzymes are the 2A and 2B subfamilies. A follow-up study
(Yanagita et al., 1998) that examined NADPH oxidation rates under anaerobic conditions found
that CYP450 1A2 wild type had a Vmax of 1.3 mol/mol CYP450/minute, a Km of 0.25 mM, and
an NADPH oxidation rate of 1.4 mol/mol CYP450/minute. Product formation rates and relative
ratios of the products formed by metabolism of HCE from the Yanagita et al., (1998) study are
shown in Table 3-6.
Table 3-6. Product formation rates and relative ratios of the products
formed by CYP450 1A2 metabolism of HCE
CYP450 1A2
Wild type
Product formation (nmol/nmol CYP450/minute)
PERC
0.68
Pentachloroethane
0.10
TCE
0.0034
Ratio of PERC:
pentachloroethane + TCE
6.6
Source: Yanagita etal. (1998).
Beurskens et al. (1991) used HCE as a reference compound to examine the metabolism of
three hexachlorocyclohexane isomers. Liver microsomes from male Wistar rats that were
induced with phenobarbital converted HCE to PERC and pentachloroethane at an initial
dechlorination rate of 12 nmol/minute/nmol CYP450 under anaerobic conditions.
Van Dyke (1977) and Van Dyke and Wineman (1971) evaluated the dechlorination
mechanisms of HCE and chlorinated olefins (alkenes) by using rat liver microsomes (a source of
CYP450 enzymes). An initial study with HCE and other chlorinated ethanes found that the
optimal configuration for dechlorination was a dichloromethyl group. HCE demonstrated a
considerable amount of dechlorination (3.9%) in this in vitro study; however, the authors
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determined that HCE was unstable in aqueous solution and that this dechlorination was
nonenzymatic based on the evidence of dechlorination in the absence of NADP.
Gargas and Andersen (1989) and Gargas et al. (1988) determined kinetic constants for
HCE metabolism in the rat using exhalation rates and a physiologically based pharmacokinetic
(PBPK) inhalation model described by Ramsey and Andersen (1984) for styrene. The Vmax
(scaled to a 1-kg rat) was 1.97 + 0.05 mg/hour, or 8.3 umol/hour. The Km was 0.80 mg/L, or
3.38uM.
3.4. ELIMINATION
No studies are available that evaluated the elimination of HCE in humans. Animal
studies indicated that the major routes of HCE elimination are either fecal or by expired air
(Mitoma et al., 1985; Fowler, 1969; Jondorf et al., 1957). The sheep studies (Fowler, 1969)
indicate that orally administered HCE is eliminated by the fecal route without absorption and
metabolism while the rodent studies (Mitoma et al., 1985) provided evidence that HCE is
absorbed and eliminated by exhalation. It is unknown why there is a discrepancy between the
studies in sheep and rodents.
Rabbits fed [14C]-radiolabeled HCE at 0.5 g/kg (Jondorf et al., 1957) eliminated 14-24%
of the radioactivity in expired air during a 3-day period following exposure. Only 5% of the
radiolabel was detected in urine. Fecal measurements were not conducted.
Fowler (1969) orally administered HCE to Scottish Blackface and Cheviot cross sheep.
Two Cheviot cross sheep were administered a single dose of 0.5 g/kg HCE and were confined to
metabolism cages; urine and feces were collected over a period of 4 days for HCE analysis.
More than 80% of the total fecal excretion of HCE occurred in the first 24 hours, and only small
amounts were detected in the urine. To assess bile concentrations of HCE, two Scottish
Blackface sheep were fasted for 24 hours and anaesthetized with sodium pentabarbital. The
hepatic duct was cannulated to collect bile; HCE was injected at a dose of 0.5 g/kg (15% w/v in
olive oil) into the rumen and lower duodenum. Bile was collected continuously, with 2 mL
retained every 30 minutes for analysis. HCE was detected in bile of anaesthetized sheep at
15 minutes, compared with 27 minutes for blood; at maximum, HCE was 8-10-fold greater in
bile.
Mitoma et al. (1985) evaluated excretion of HCE in Osborne-Mendel rats and B6C3Fi
mice following 4 weeks of administration of an MTD (500 mg/kg-day in rats, 1,000 mg/kg-day
in mice). Excretion of radiolabel was monitored for 48 hours following administration of a
tracer dose of [14C]-HCE. The findings are presented in Table 3-4. Most of the radiolabel was
detected in expired air, indicating this to be a major route of elimination. The authors did not
investigate whether the exhaled material was parent compound or volatile metabolite, and
assumed that it was the parent compound. A low percentage of the exhaled radioactivity was in
the form of CO2, with rats exhaling slightly more than mice. The amount of radioactivity in the
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excreta, on the other hand, was lower in rats than in mice (Table 3-4). The excreta contained
6.3 and 16.2% of the radiolabel in rats and mice, respectively.
3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELS
No physiologically based pharmacokinetic (PBPK) models for HCE have been developed
specifically for mammalian species. Models for waterborne chloroethanes have been reported
for rainbow trout and channel catfish; however, these are outside the scope of this toxicological
review and are not described.
Gargas and Andersen (1989) and Gargas et al. (1988) determined kinetic constants for
HCE metabolism in the rat using exhalation rates and a PBPK inhalation model described by
Ramsey and Andersen (1984) for the distribution and metabolism of inhaled gases and vapors.
These reports by Gargas and Andersen (1989) and Gargas et al. (1988) do not describe a PBPK
model for HCE, only kinetic constants for HCE metabolism by inhalation. During these breath
chamber experiments, adsorption of HCE on the fur of rats (fur loading) was observed to occur.
At an exposure concentration of 53.3 ppm HCE at 6 hours, the chemical mass in body tissues
was 7.29 mg and the chemical mass on fur was 0.6 mg (7.6% of total chemical mass).
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4. HAZARD IDENTIFICATION
4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL
CONTROLS
There are few published studies relating to the toxicology of HCE in humans. Case
reports of pneumonitis (Allen et al., 1992) and pneumonitis with evidence of liver abnormalities
(Loh et al., 2008, 2006) have been described in soldiers exposed to smoke bombs containing
HCE and zinc oxide. However, the smoke produced by this incineration is a mixture of
chemicals consisting primarily of zinc oxychloride and zinc chloride and the reported effects are
consistent with symptoms of zinc chloride exposure (NRC, 1997). Some aluminum production
processes involve the use of HCE in tablet or powder form, resulting in exposures to fumes
containing hexachlorobenzene, octachlorostyrene, dioxins, dibenzofurans, and other
organochlorinated compounds. A case report of a hepatocellular carcinoma (Selden et al., 1989)
and limited data concerning some clinical serologic measures (Selden et al., 1999, 1997) in
aluminum foundry workers involved in this process are available, but these data are not directly
relevant to the question of health effects of HCE in other settings. No epidemiologic studies of
the carcinogenicity of HCE were included in a 1985 review of cancer epidemiology with respect
to halogenated alkanes and alkenes (Axelson, 1985). A study of Swedish workers involved in
smoke bomb production has provided some information pertaining to exposure levels and
symptoms and clinical parameters relating primarily to liver and pulmonary function (Selden et
al., 1994, 1993).
Two separate studies were conducted on a small population of Swedish workers
occupationally exposed to HCE while producing military white smoke munitions. The first
study reported on biological exposure monitoring (Selden et al., 1993) and the second study
described health effects resulting from HCE exposure (Selden et al., 1994). The smoke
formulation was approximately 60% HCE, 30% titanium dioxide, 8% aluminum powder, 2%
cryolite, and a trace of zinc stearate. At the time this study was conducted in 1989, no HCE dust
was found in the air sample filters, but the integrated results of personal and stationary charcoal
tube samples revealed approximate HCE concentrations by location of 10-30 mg/m3 (milling/
mixing), 5-25 mg/m3 (pressing), <5 mg/m3 (assembly room), and nondetectable (storage room)
(Selden et al., 1993).
In the first study (Selden et al., 1993), the exposed group consisted of 12 people (six men
and six women) ranging in age from 23 to 57 (mean, 31.4 years; median, 30 years) (Selden et al.,
1993). The principal control group (n = 12) consisted of assembly line workers from the same
company who were unexposed to chlorinated hydrocarbons, but had some exposure to glass fiber
dust. They were matched to the exposure group by sex and age (± 5 years), except in the case of
one exposed male subject where only a younger control could be found. This latter-exposed
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male subject was excluded from the analysis of health effects (Selden et al., 1994). A second
control group of formerly HCE-exposed workers (3 males, 10 females; age range, 31-57 years;
mean, 43.6 years) was used in the biological exposure monitoring study.
Blood samples were collected for analysis of HCE concentration. For the exposed group,
samples were drawn 5 weeks into a temporary production break (the "baseline" period), and the
second samples were drawn 5 months later, after production had been underway for 5 weeks (the
"production" period). Analyses of blood plasma HCE indicated that all values for both control
groups (n = 25) were below the limit of detection (<0.02 ug/L).
Exposed subjects were stratified into three subgroups (n = 4) of perceived exposure (low,
medium, or high) based on information pertaining to work tasks, presence at work, and use of
protective equipment. At baseline, the HCE concentrations in 10 of the samples from exposed
workers were in the range of <0.02-0.06 ug/L, 1 sample was 0.15 ug/L, and 1 sample was
0.52 ug/L. The last sample was from an individual who had remained in an HCE-contaminated
area during the baseline period. Plasma HCE levels in the production period increased by nearly
100-fold over that of the baseline samples (mean of 7.30 ± 6.04 ug/L in the production sample
compared with 0.08 ± 0.14 ug/L in the baseline samples, p < 0.01). Although the magnitude of
individual increases varied considerably, there was a significant (p < 0.05) linear trend for values
in the low-, medium-, and high-exposure subgroups (means of 3.99, 7.14, and 10.75 ug/L,
respectively). These results suggest that a considerable increase in plasma HCE can occur after a
relatively brief occupational exposure, even though workers used fairly sophisticated personal
protective equipment.
As noted above, 11 of the subjects from the first study (Selden et al., 1993) and their
11 age- and sex-matched controls were included in the second health effects study (Selden et al.,
1994). Data pertaining to 15 clinical symptoms (including headaches, sleep quality, palpations,
difficulty concentrating, tension/restlessness, frequency of coughing, watery eyes/runny nose,
itching/other skin problems, shortness of breath/chest discomfort, and general health) were
obtained from self-administered questionnaires for the exposed workers and the company
controls. Similar data had been obtained in a previous study of 130 metal shop workers, and
these workers were used as a second, "historical" comparison group in the analysis of the
symptom data. Whole blood and serum samples from the 11 exposed and 11 matched company
controls were analyzed for routine clinical parameters. Spot urine samples were analyzed for
hemoglobin, protein, and glucose. Lung function was assessed by measuring vital capacity and
1-second forced expiratory volume (FEVi).
The matched company controls reported more symptoms of ill health than exposed
subjects, although the differences were not statistically significant. Although not statistically
significant, the exposed group reported a higher prevalence of "dry skin/dry mucous
membranes" (3/11 or 27%) than the matched controls (1/9, 9%) or historical controls (13/130,
10%), and a higher prevalence of "itching/other skin problems" (3/11, 27%) than the historical
18 DRAFT - DO NOT CITE OR QUOTE
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controls (16/130, 12%). The prevalence of "itching/other skin problems" in the matched controls
(3/11, 27%) was the same as that in the exposed group. These symptoms centered on the wrist
and neck areas, and the authors suggested that this could reflect exposure to HCE through joints
in the protective equipment, or could possibly be a "traumiterative effect of the equipment
itself." Clinical examination revealed no dermatological or respiratory mucous membrane
abnormalities in either group. The authors noted that a previous unpublished study of the plant
workers (but with primarily different workers) had also found dermatologic complaints in up to
90% of the exposed workers.
All of the spot urine tests were normal, and there was no evidence of an effect of HCE
exposure on pulmonary function as measured by vital capacity and FEVi. Exposed subjects had
significantly higher levels of serum creatinine, urate, and bilirubin than controls (p < 0.05),
although the group means were still in the normal range. One exposed subject had a marginally
elevated level of serum alanine aminotransferase (ALT) (70.5 U/L versus <41.1 U/L reference),
while one control subject displayed increased levels of serum ALT and aspartate
aminotransferase (AST) (67.6 and 186.4 U/L, respectively; 41.1 U/L reference for each). The
control individual's values returned to normal after 8 months, while the exposed subject's serum
ALT value worsened to 87.6 U/L 4 months later (Selden et al., 1994). Available data pertaining
to these liver function tests from 1982, when exposure levels at the worksite were higher than in
the current study, did not show elevations in these liver enzymes in this individual at that time.
Within the exposed group, there was no correlation between plasma HCE concentrations and the
clinical chemistry parameters, although the authors do not discuss the power limitations of this
exposure-response analysis (Selden et al., 1993). In summary, these studies demonstrated HCE
exposure in the smoke bomb production workers, but the sample size of health effects study is
too small to reach definitive conclusions. Based on the available data, the possible
dermatologic/mucosal effects and hepatic effects are the areas in most need of additional
research.
4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN
ANIMALS—ORAL AND INHALATION
4.2.1. Oral
4.2.1.1. Subchronic Exposure
Two subchronic toxicity assays for HCE were reported (NTP, 1989; Gorzinski et al.,
1985, 1980). The Gorzinski et al. (1985, 1980) study (16 weeks) reported histopathological
evaluations that found kidney degeneration in males, kidney degeneration in females, and
minimal hepatic effects. The NTP (1989) study (13 weeks) reported kidney effects in male rats
such as degeneration and necrosis of renal tubular epithelium, hyaline droplet formation, and
tubular regeneration and tubular casts. Female rats in this study exhibited a dose-response
increase in the incidence of hepatocellular necrosis of the centrilobular area. The NTP (1989)
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study suggested that male rats may be more susceptible to kidney effects, whereas female rats
may be more susceptible to liver effects.
Gorzinski et al. (1980) conducted a 16-week toxicity study in male and female F344 rats.
Rats were exposed via the diet to 3, 30, or 100 mg HCE/kg-day; however, due to sublimation of
HCE from the feed and feeding and diurnal eating patterns, actual doses were determined to be 1,
15, or 62 mg/kg-day, respectively (Gorzinski et al., 1985). Gorzinski et al. (1980) is a Research
and Development Report by Dow Chemical and is not publicly available. The data for this study
were published in the peer-reviewed literature by Gorzinski et al. (1985) and are presented in
detail below.
Gorzinski et al. (1985) fed 1, 15, or 62 mg/kg-day HCE (purity 99.4%) to F344 rats
(10 rats/sex/dose) for 16 weeks. As described in Section 3.2, HCE concentrations in male
kidneys were proportionately increased with administered dose, while the increases in females
were not proportionate. At the high dose, male rats displayed statistically significant increases in
absolute and relative kidney weights accompanied by macroscopically observed alterations.
Male rats displayed slight hypertrophy and/or dilation of proximal convoluted tubules of the
kidneys at incidences of 0/10, 1/10, 7/10, and 10/10 for the 0, 1, 15, and 62 mg/kg-day dose
groups, respectively. The increased incidence of slight hypertrophy and/or dilation of proximal
convoluted tubules was statistically significant in males at the 15 and 62 mg/kg-day doses. Male
rats displayed atrophy and degeneration of renal tubules at incidences of 1/10, 2/10, 7/10, and
10/10 for the 0, 1, 15, and 62 mg/kg-day dose groups, respectively. The increased incidence of
atrophy and degeneration of renal tubules was statistically significant in males at the 15 and
62 mg/kg-day doses. Female rats did not display hypertrophy and/or dilation of proximal
convoluted tubules of the kidneys at any dose, but did exhibit atrophy and degeneration of
proximal tubules (1/10, 1/10, 2/10, and 6/10 at the 0, 1, 15, and 62 mg/kg-day doses,
respectively). However, the increased incidence of atrophy and degeneration of proximal tubules
was only statistically significant in females at the 62 mg/kg-day dose. Male rats of the
62 mg/kg-day group exhibited statistically significant increases in absolute and relative liver
weights; histopathology revealed a slight swelling of the hepatocytes in this group. Although
female rats exhibited a statistically significant increase in relative liver weight at the high dose,
there was no evidence of hepatotoxicity in the histopathological examination. The data for liver
and kidney weights are presented in Table 4-1 and the data for the kidney effects are presented in
Table 4-2.
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Table 4-1. Body, kidney, and liver weights of rats exposed to HCE in the
diet for 16 weeks
Sex
Male3
Female3
Dose level
(mg/kg-day)
0
1
15
62
0
1
15
62
Fasted body
weight (g)
314.4+12.4
328.0 + 7.2
329.0 + 24.4
324.2+10.0
176.7 + 6.9
174.0+7.9
176.7+4.6
170.8 + 5.1
Liver
Absolute (g)
8.32 + 0.27
8.46 + 0.22
8.69 + 0.80
8.98 + 0.54b
4.65+0.26
4.74 + 0.22
4.79 + 0.21
4.71+0.23
Relative (g/100 g
body weight)
2.65+0.06
2.58 + 0.07
2.64+0.09
2.77 + 0.12b
2.63+0.06
2.73+0.11
2.69+0.09
2.76 + 0.10b
Kidney
Absolute (g)
2.28 + 0.08
2.31+0.09
2.40 + 0.15
2.51+0.12b
1.40 + 0.08
1.38 + 0.05
1.39 + 0.06
1.39 + 0.05
Relative (g/100 g
body weight)
0.73+0.04
0.70 + 0.02
0.73+0.01
0.77 + 0.02b
0.79 + 0.03
0.79 + 0.03
0.79 + 0.04
0.81+0.02
3Data are presented as means + SD of 10 rats/sex.
bStatistically significant from control using Dunnett's test (p = 0.05).
Source: Gorzinskietal. (1985).
Table 4-2. Histopathological results on kidney in rats exposed to HCE in the
diet for 16 weeks3
Organ
Kidney
Effect
Slight hypertrophy and/or dilation of proximal
convoluted tubules
Atrophy and degeneration of renal tubules0
Sex
Male
Female
Male
Female
Dose (mg/kg-day)
0
0
0
1
1
1
1
0
2
1
15
?b
0
?b
2
62
10b
0
10b
6b
3Data are presented as number of positive observations for 10 rats/sex/dose.
bEPA determined statistical significance from control using Fisher's Exact Test (p = 0.05).
°Graded as slight in 1 of 10 male control rats and very slight in 1 of 10 control female rats. Severity of nephropathy
was not reported for HCE-exposed rats.
Source: Gorzinskietal. (1985).
The authors concluded that the no-observed-effect level for both male and female rats
was 1 mg/kg-day. EPA considered 1 mg/kg-day as the male no-observed-adverse-effect level
(NOAEL) and 15 mg/kg-day as the lowest-observed-adverse-effect level (LOAEL), based on
renal tubule toxicity in male rats. For female rats, EPA considered the NOAEL as 15 mg/kg-day
and the LOAEL as 62 mg/kg-day, based on renal tubule toxicity.
NTP (1989) conducted a 13-week study of HCE oral toxicity in F344/N rats. Groups of
10 rats/sex/dose were administered 0, 47, 94, 188, 375, or 750 mg/kg (purity >99%) by corn oil
gavage, 5 days/week for 13 weeks. The time-weighted average (TWA) doses were 0, 34, 67,
134, 268, and 536 mg/kg-day, respectively. In the 536 mg/kg-day group, 5/10 male rats (only
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the 5 males that died were examined microscopically) and 2/10 female rats died before the end of
the study. Mean body weights of 536 mg/kg-day male and female rats were decreased 19 and
4%, respectively, compared with controls. Statistically significant increases in liver weights
were noted at doses of >67 mg/kg-day (females) and >134 mg/kg-day (males), and in kidney
weights at doses of >268 mg/kg-day (females) and >67 mg/kg-day (males). Organ weight to
body weight ratios (mg/g) generally increased in a dose-related manner for both male and female
rats exposed to HCE (Table 4-3).
Table 4-3. Organ weight to body weight ratios for rats exposed to HCE for
13 weeks
HCE dose by gavage (mg/kg-day)
0
34
67
134
268
536
Male3
Number
Body weight
Liver
Brain
Heart
Kidney
Lung
Right testis
Thymus
10
340+7.6
35.8 + 0.61
6.0+0.30
2.8+0.04
3.0+0.05
4.2+0.21
4.2+0.05
0.8+0.04
10
349 + 8.8
37.3+0.37
5.7+0.17
2.8+0.04
3.8+0.37
4.6+0.40
4.8+0.38
0.8+0.06
10
343+5.9
36.0 + 0.71
5.7+0.10
2.9+0.07
4.1+0.27C
4.4+0.48
4.3+0.10
0.6+0.02
10
348 + 5.9
39.1+0.62b
5.8 + 0.23
3.2 + 0.17c
4.7 + 0.44b
3.9 + 0.22
4.4 + 0.17
0.8 + 0.10
9
319 + 4.0
42.5+0.74b
6.3+0.21
3.3+0.18b
5.2+0.35b
3.9+0.15
4.7+0.05
0.7+0.04
5
262 + 13.5
46.3+0.95b
7.2 + 0.31b
3.2 + 0.10c
4.7 + 0.28b
4.9 + 0.50
5.3+0.21b
0.6 + 0.06
Female"
Number
Body weight
Liver
Brain
Heart
Kidney
Lung
Thymus
10
206 + 3.7
32.2 + 0.56
8.7+0.17
2.9+0.04
3.1+0.04
4.2+0.09
1.1+0.05
10
210 + 3.9
33.4+0.63
8.6+0.14
3.0+0.05
3.2+0.05
4.1+0.09
1.1+0.05
10
208+2.6
34.3+0.39c
8.6+0.10
3.0+0.03
3.2+0.07
4.2+0.10
1.1+0.04
10
200+2.9
36.3+0.44b
9.0 + 0.14
3.0 + 0.04
3.2 + 0.06
4.1+0.06
1.0 + 0.06
10
203+4.3
42.0+0.60b
9.0+0.15
3.1+0.07
3.6+0.05b
4.2+0.08
1.1+0.07
8
189 + 3.8
52.4 + 0.88b
9.5+0.17b
3.4 + 0.07b
4.1+0.10b
4.5+0.13
0.8 + 0.05b
"Data are presented as mean + SE in mg/g, except for body weight in grams.
bStatistically different from controls, p < 0.01
Statistically different from controls, p < 0.05
Source: NTP (1989).
Kidney effects (characterized by hyaline droplet formation, tubular regeneration, and
tubular casts), similar to the toxicity noted in the 16-day study also conducted by NTP (1989),
were observed in 90% of 34 mg/kg-day males and in males from all other HCE dose groups
(NTP reported incidence data only for the 34 mg/kg-day dose group). NTP (1989) reported that
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the severity of these effects increased with dose (data not presented by NTP). These kidney
effects were not observed in any of the treated females. At the 536 mg/kg-day dose, 5/10 males
died. Kidneys from these five animals were examined microscopically and revealed papillary
necrosis, degeneration, and necrosis of the renal tubular epithelium. Hepatocellular necrosis of
the centrilobular area was observed in 2/5 males and 8/10 females at the 536 mg/kg dose, 1/10
males and 4/10 females at the 268 mg/kg-day dose, and 2/10 females at the 134 mg/kg-day dose.
Additionally, males of the 536 mg/kg-day dose group exhibited hemorrhagic necrosis of the
urinary bladder. EPA considered the female rat NOAEL as 67 mg/kg-day and the LOAEL as
134 mg/kg-day, based on hepatocellular necrosis. A NOAEL could not be identified for male
rats since kidney effects were observed in >90% of the male rats at all tested doses (compared to
none of the controls). EPA considered the LOAEL for male rats as 34 mg/kg-day (lowest dose
tested), based on kidney lesions.
4.2.1.2. Chronic Exposure and Carcinogenicity
The National Toxicology Program (NTP) and National Cancer Institute (NCI) conducted
two chronic toxicity/carcinogenicity bioassays in rats and one in mice. Increased incidences of
renal tubular hyperplasia, renal adenoma or carcinoma, adrenal medulla hyperplasia,
pheochromocytomas, and malignant pheochromocytomas were noted in male F344/N rats;
female rats did not develop HCE-related tumors (NTP, 1989). Osborne-Mendel rats of both
sexes in the NCI (1978) study exhibited tumor types that have been previously identified as
spontaneous lesions in this strain, and do not provide evidence of carcinogenicity. B6C3Fi mice
of both sexes exhibited hepatocellular carcinomas, although only male mice demonstrated a dose
response with tumor incidence (NCI, 1978). Based on the body of evidence accumulated by
these studies, NTP and NCI concluded that there was evidence of HCE carcinogenicity in male
F344 rats and mice of both sexes, respectively, but there was no evidence of carcinogenicity in
female F344 or male and female Osborne-Mendel rats (NTP, 1989; NCI, 1978).
NTP (1989) conducted a chronic toxicity/carcinogenicity bioassay in F344/N rats.
Groups of 50 male rats/dose were administered 0, 10, or 20 mg/kg-day (TWA doses of 0, 7, or
14 mg/kg-day, respectively, after adjusting for continuous exposure) of HCE (purity >99%) by
corn oil gavage, 5 days/week for 103 weeks. Groups of 50 female rats/dose were administered 0,
80, or 160 mg HCE/kg by corn oil gavage, 5 days/week for 103 weeks (TWA doses of 0, 57, or
114 mg/kg-day, respectively, after adjusting for continuous exposure). These sex-specific doses
were selected based on the results of the 13-week study conducted by NTP (1989) that
demonstrated kidney lesions in male rats at the lower doses and liver lesions in female rats at the
higher doses. All animals were necropsied.
Mean body weights of the 14 mg/kg-day male rats were 5-6% lower than controls after
week 81. Mean body weights of the 114 mg/kg-day female rats were 5-9% lower between
weeks 41 and 101. Nephropathy, characterized by tubular cell degeneration and regeneration,
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tubular dilatation and atrophy, glomerulosclerosis, interstitial fibrosis, and chronic inflammation,
was observed in both treated and control rats. Incidences of male nephropathy were 48/50 in
controls, 48/50 in the 7 mg/kg-day dose group, and 47/50 in the 14 mg/kg-day dose group. The
mean severity scores for nephropathy in male rats increased with dose (2.34 ± 0.14, 2.62 ±0.15,
and 2.68 ± 0.16 in the 0, 7, and 14 mg/kg-day groups, respectively), with the 14 mg/kg-day
group being statistically significantly higher than the control group. While the mean severity
scores did not show more than a 15% increase over control in the high-dose group, an
examination of the various grades of severity revealed more moderate and marked nephrotoxicity
in treated male rats compared with controls, which predominantly exhibited mild nephropathy
(Table 4-4).
Incidences of female nephropathy were 22/50 for controls, 42/50 in the 57 mg/kg-day
dose group, and 44/49 in the 114 mg/kg-day dose group. The severity scores for nephropathy in
female rats were statistically significantly increased in both treated groups: 0.72 ±0.13 (mean ±
SE) in controls, 1.38 + 0.11 in the 57 mg/kg-day group, and 1.69 ± 0.12 in the 114 mg/kg-day
group. Examination of the various grades of severity showed mild and moderate nephropathy in
treated females compared with controls, which predominantly presented less than minimally
severe nephropathy. Females did not exhibit marked nephropathy in the control or treated
groups (Table 4-4).
Table 4-4. Incidence and severity of nephropathy in male and female rats
treated with HCE
Severity
None (0)
Minimal (1)
Mild (2)
Moderate (3)
Marked (4)
Total incidence (minimal to marked)
Total number of rats
Overall severity0
Dose (mg/kg-day)
0
7
14
Male
2
4
26
11
7
48
50
2.34 + 0.14
2
3
21
10
14
48
50
2.62+0.15
3
4
13
16
14
47
50
2.68 + 0.163
0
57
114
Female
28
10
10
2
0
22
50
0.72 + 0.13
8
17
23
2
0
42b
50
1.38+0.11b
5
12
25
7
0
44b
49
1.69 + 0.12b
"Authors reported as statistically significantly different from controls, p < 0.05.
bAuthors reported as statistically significantly different from controls, p < 0.01.
"Mean + SE.
Source: NTP(1989).
In light of these variations in severity, EPA considered the responses observed in both the
control and treated male rats associated with more severe (moderate and marked severity)
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nephropathy to better distinguish the HCE-related effects. Incidences of male nephropathy (that
were of moderate or marked severity) were 18/50, 24/50, and 30/50 in the control, 7, and
14 mg/kg-day dose groups, respectively. Similar to the male rats, the incidence of nephropathy
associated with the more severe (mild and moderate) responses was considered in the females
rats. Therefore, incidences of female nephropathy (that were of mild or moderate severity) were
12/50, 25/50, and 32/50 in the control, 57, and 114 mg/kg-day dose groups, respectively.
Additional kidney effects were noted in male rats (presented in Table 4-5). Linear
mineralization of the renal papillae was increased in a dose-dependent manner: 15/50 (30%) and
32/50 (64%) in the 7 and 14 mg/kg-day dose groups, respectively, compared with 2/50 (4%) in
controls. Hyperplasia of the pelvic transitional epithelium was increased in treated rats (14% in
7 and 14 mg/kg-day HCE dose groups) compared to 0% of control rats. Nonneoplastic lesions
such as casts (4%), cytomegaly (4%), chronic inflammation (4%), and focal necrosis (2%) were
observed in some of the male rats administered 14 mg/kg-day. An increased incidence of renal
tubule pigmentation was noted in 4/50 (8%) of the 7 mg/kg-day dose group and 5/50 (10%) of
the 14 mg/kg-day dose group, compared with 1/50 (2%) in the controls. Regeneration of the
renal tubule was observed in three males administered 14 mg/kg-day HCE.
Additional kidney effects in female rats included linear mineralization of the renal
papillae, although the incidence was not dose-dependent: 14/50 (28%) in vehicle controls,
22/50 (44%) in the 57 mg/kg-day dose, and 13/50 (26%) in the 114 mg/kg-day dose. Female rats
also exhibited casts (4% at 114 mg/kg-day) and chronic inflammation (2% at both 57 and
114 mg/kg-day). Pigmentation of the renal tubule was present in 4, 4, and 6% of control, 57, and
114 mg/kg-day females, respectively. Renal tubule regeneration was observed in treated females
(but not controls); 4% of the 57 mg/kg-day dose group and 2% of the 114 mg/kg-day dose group.
Only male rats demonstrated an increase in hyperplasia of the pelvic transitional epithelium and
a dose-dependent increase in incidences of mineralization along the renal papillae.
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Table 4-5. Additional kidney effects in HCE-treated rats
Renal tubule pigmentation
Linear mineralization of renal papillae
Hyperplasia of the pelvic transitional
epithelium
HCE Dose (mg/kg-day)
Males
Vehicle
control
1/50 (2%)
2/50 (4%)
0/50 (0%)
7
4/50 (8%)
15/50
(30%)a
7/50
(14%)a
14
5/50 (10%)
32/50
(64%)a
7/50
(14%)a
Females
Vehicle
control
2/50 (4%)
14/50 (28%)
Not
observed
57
2/50 (4%)
22/50 (44%)
Not
observed
114
3/50 (6%)
13/50
(26%)
Not
observed
aEPA determined statistical significance using Fisher's exact test, p < 0.05.
Source: NTP(1989).
EPA considered the male LOAEL as 7 mg/kg-day based on increased incidence of
moderate or marked nephropathy (Table 4-4), hyperplasia of the pelvic transitional epithelium
(Table 4-5), increased incidence of renal tubule pigmentation (Table 4-5), and linear
mineralization of the renal papillae (Table 4-5). EPA considered 57 mg/kg-day the female
LOAEL, based on dose-related increases in incidence and severity (minimal to moderate)
nephropathy. The male and female NOAELs could not be established as toxic effects were
observed at the lowest doses tested.
Renal tubular hyperplasia was observed at an increased incidence in treated male rats:
4/50 (8%) in the 7 mg/kg-day dose and 11/50 (22%; statistically significantly higher than
controls) in the 14 mg/kg-day dose, compared with 2/50 (4%) for control (Table 4-6). Only one
female rat, administered 57 mg/kg-day, exhibited renal hyperplasia. Dose-related increases in
the incidence of combined renal adenomas and carcinomas were observed in males rats
administered HCE at doses of 7 (4%) and 14 mg/kg-day (14%, statistically significantly higher
than controls) compared with controls (2%). No HCE-related tumors were observed in female
rats. NTP concluded that these data provided evidence of carcinogenicity in male rats based on a
comparison with the historical controls in the study laboratory (1/300; 0.3 ± 0.8%) and in NTP
studies (10/1,943; 0.5 ± 0.9%).
Table 4-6. Renal tubular hyperplasia and tumor incidences in HCE-treated
male rats
Hyperplasia
Adenoma
Carcinoma
Adenoma or carcinoma
Vehicle control
2/50 (4%)
1/50 (2%)
0/50 (0%)
1/50 (2%)
7 mg/kg-day HCE
4/50 (8%)
2/50 (4%)
0/50 (0%)
2/50 (4%)
14 mg/kg-day HCE
ll/50(22%)a
4/50 (8%)
3/50 (6%)
7/50 (14%)a
aSignificantly different from vehicle controls, p < 0.01.
Source: NTP (1989).
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This study demonstrates specificity for HCE-induced renal effects in male rats. The
males of both dose groups were administered 8 times less HCE than the corresponding females.
However, the treated male rats demonstrated more severe nephropathy than the treated female
rats. NTP (1989) also observed more severe nephropathy in control male rats (i.e., mild
nephropathy) than in control females (i.e., minimal nephropathy). Male rats, but not female rats,
also exhibited renal hyperplasia and tumors. NTP (1989) indicated that the renal hyperplasia and
tumors observed in the HCE-treated male rats represented a morphologic continuum.
Effects in the adrenal gland were also noted in HCE-treated rats. Hyperplasia of the
adrenal medulla was reported in 9 and 20% of male rats administered 7 and 14 mg/kg-day HCE,
respectively, compared with 12% of controls. Female rats in the control (10%) and
114 mg/kg-day (15%) groups exhibited hyperplasia of the adrenal medulla; this effect was not
observed in the 57 mg/kg-day dose group.
Adrenal medullary lesions were observed in male rats, but not female rats (Table 4-7).
Pheochromocytoma incidences were statistically significantly increased in the 7 mg/kg-day
group (26/45, 58%). The increase of pheochromocytomas in the 14 mg/kg-day group (19/49,
39%) was not statistically significant compared with controls (14/50, 28%). There were no
statistically significant differences in the incidences of malignant pheochromocytomas and
complex pheochromocytomas (defined as pheochromocytomas containing nervous tissue in
addition to the typical adrenal medullary cells) between controls and treated male rats. The
combined incidence of all three types of pheochromocytomas was statistically significantly
increased in males treated with 7 mg/kg-day HCE (62%) but not in males treated with
14 mg/kg-day HCE (43%) when compared with vehicle controls (30%) and historical controls in
the study laboratory (75/300; 25 ± 7%) and in NTP studies (543/1,937; 28 ± 11%). NTP
concluded that the increased incidences of pheochromocytomas in male rats were possibly
treatment-rel ated.
Table 4-7. Adrenal medullary lesions in HCE-treated male rats
Focal hyperplasia
Pheochromocytoma
Complex pheochromocytoma
Malignant pheochromocytoma
Combined pheochromocytoma
Control
6/50 (12%)
14/50 (28%)
0/50
1/50 (2%)
15/50 (30%)
7 mg/kg-day
4/45 (9%)
26/45 (58%)a
0/45
2/45 (4%)
28/45 (62%)a
14 mg/kg-day
10/49 (20%)
19/49 (39%)
2/49 (4%)
1/49 (2%)
21/49 (43%)
aSignificantly different from vehicle controls, p < 0.01.
Source: NTP (1989).
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NCI (1978; Weisburger, 1977) conducted a chronic toxicity/carcinogenicity bioassay in
Osborne-Mendel rats. HCE (purity >98%) at doses of 0, 250, or 500 mg/kg-day was
administered by corn oil gavage to 50 rats/sex/dose for 5 days/week for 78 weeks. Following
termination of exposure, animals were observed for 33-34 weeks for a total duration of 111-
112 weeks. Twenty rats/sex were used for the untreated and vehicle controls. Starting in
week 23, rats treated began a 5-week cyclic rotation that involved 1 week without exposure
followed by dosing for 4 weeks. After adjustment from 5 days/week for 78 weeks, with the
5-week cyclic rotation for part of the time, to continuous exposure over the standard 2 years for a
chronic bioassay, the TWA doses were 113 and 227 mg/kg-day.
Mortality was accelerated in the HCE-treated rats (NCI reported a statistically significant
association between increased dose and mortality). The 113 and 227 mg/kg-day males exhibited
survival rates of 24/50 (48%) and 19/50 (38%), respectively, compared with 14/20 (70%) in the
untreated controls and 11/20 (55%) in vehicle controls (seven rats in the vehicle control group
were sacrificed in week 60). Mortality in the treated groups occurred early in the bioassay.
Approximately 20% of the high- and low-dose males died by weeks 15 and 45, respectively,
compared with 90 weeks until 20% mortality for the controls. Survival rates for the female rats
were 14/20 (70%) for both the untreated and vehicle controls, and 27/50 (54%) and 24/50 (48%)
for the 113 and 227 mg/kg-day dose groups, respectively. Mortality also occurred early in the
bioassay for the female rats. Approximately 20% of the high- and low-dose females died by
weeks 25 and 30, respectively, compared with 110 weeks until 20% mortality for the controls.
Chronic inflammatory kidney lesions were observed in both control and treated rats:
male rats exhibited incidences of 15/20 (75%) in untreated controls, 14/20 (70%) in vehicle
controls, 32/49 (65%) in the 113 mg/kg-day dose group, and 25/50 (50%) in the 227 mg/kg-day
dose group; female rats exhibited incidences of 8/20 (40%) in untreated controls, 4/20 (20%) in
vehicle controls, 18/50 (36%) in the 113 mg/kg-day dose group, and 20/49 (41%) in the
227 mg/kg-day dose group. Tubular nephropathy (characterized by degeneration, necrosis, and
the presence of large hyperchromatic regenerative epithelial cells) was observed in 45 and 66%
of males and 18 and 59% of females in the 113 and 227 mg/kg-day dose groups, respectively.
These effects were not observed in the untreated or vehicle controls. EPA considered the
LOAEL as 113 mg/kg-day (lowest dose tested), based on a dose-related increase in the incidence
of nephropathy in both males and females. The NOAEL could not be identified.
Tumor types exhibited by male rats surviving at least 52 weeks included kidney tubular
cell adenoma, pituitary chromophobe adenoma, thyroid follicular cell adenoma or carcinoma,
and testicular interstitial cell tumors (Table 4-8). Due to the high mortality in the 227 mg/kg-day
males, statistical analyses of male rat tumors were based only on those rats surviving at least
52 weeks. Increased incidences of kidney tubular cell adenoma (4/37) and pituitary
chromophobe adenoma (4/32) were observed in the male rats of the 113 mg/kg-day dose group
but not in the 227 mg/kg-day group. Male vehicle controls did not exhibit kidney tubular cell
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adenomas, although 11% (2/18) exhibited pituitary chromophobe adenomas. Thyroid follicular
cell adenoma or carcinoma were observed in 11, 8, and 18% in vehicle control, 113, and
227 mg/kg-day males, respectively; high-dose males also demonstrated the shortest time to first
tumor of 60 weeks, compared with vehicle control (111 weeks) and low-dose males (92 weeks).
Testicular interstitial cell tumors were not observed in vehicle control or 113 mg/kg-day males,
but were observed in 10% of 227 mg/kg-day males.
Table 4-8. Tumor incidences" in male rats gavaged with HCE
Tumor type
Kidney tubular cell adenoma
Weeks to first tumor
Pituitary chromophobe adenoma
Weeks to first tumor
Thyroid follicular cell adenoma or carcinoma
Weeks to first tumor
Testis interstitial cell tumor
Weeks to first tumor
Vehicle control
0/18 (0%)
-
2/18(11%)
105
2/18(11%)
111
0/18 (0%)
-
113 mg/kg-day
4/37(11%)
86
4/32 (13%)
104
3/36 (8%)
92
0/36 (0%)
-
227 mg/kg-day
0/29 (0%)
-
0/24 (0%)
-
5/28 (18%)
60
3/29 (10%)
109
aDue to early accelerated mortality, the statistical analyses for the incidences of tumors are based on animals
surviving at least 52 weeks. Rat strain is Osborne-Mendel.
Source: NCI (1978).
Tumor types exhibited by female rats included kidney hamartoma (nonneoplastic
overgrowth), pituitary chromophobe adenoma, thyroid follicular cell adenoma or carcinoma,
mammary gland fibroadenoma, and ovary granulosa cell tumors (Table 4-9). Females
administered 227 mg/kg-day HCE had an incidence of 6% for kidney hamartoma, while none of
these tumors were observed in the vehicle control or 113 mg/kg-day female rats. The increased
incidences of the remaining tumor types observed in female rats were not dose-dependent.
Incidences of pituitary chromophobe adenomas, thyroid follicular cell adenoma or carcinomas,
and mammary gland fibroadenomas were lower in HCE-treated animals than in controls. Ovary
granulosa cell tumors were increased in the low-dose group, compared to controls, although none
of the female rats in the high-dose group exhibited this tumor. NCI (1978) noted that all these
tumor types had been encountered previously as spontaneous lesions in the Osborne-Mendel rat,
and the authors reported that no statistical differences in frequencies were observed between
treated and control rats. NCI concluded that there was no evidence of carcinogen!city in this rat
study.
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Table 4-9. Tumor incidences in female rats gavaged with HCE
Tumor type
Kidney hamartoma
Weeks to first tumor
Pituitary chromophobe adenoma
Weeks to first tumor
Thyroid follicular cell adenoma or carcinoma
Weeks to first tumor
Mammary gland fibroadenoma
Weeks to first tumor
Ovary granulosa cell tumor
Weeks to first tumor
Vehicle control
0/20 (0%)
-
7/20 (35%)
89
2/20 (10%)
111
6/20 (30%)
106
1/20 (5%)
111
113 mg/kg-day
0/50 (0%)
-
15/50 (30%)
89
3/47 (6%)
112
13/50 (26%)
57
4/48 (8%)
111
227 mg/kg-day
3/49 (6%)
112
6/46 (13%)
112
3/47 (6%)
109
9/50 (18%)
94
0/49 (0%)
-
Rat strain is Osborne-Mendel.
Source: NCI (1978).
NCI (1978; Weisburger, 1977) conducted a chronic oral study in 50 B6C3Fi mice/sex/
dose administered 0, 500, or 1,000 mg/kg-day HCE (purity >98%) via corn oil gavage for
5 days/week for 78 weeks. Following exposure termination, animals were observed for 12-
13 weeks for a total duration of 90-91 weeks. Twenty mice/sex were included as untreated and
vehicle controls. Starting in week 9, the doses were increased to 600 and 1,200 mg/kg-day; no
explanation was provided for this change in dose. After adjustment from 5 days/week for
78 weeks to continuous exposure, the TWA doses were 360 and 722 mg/kg-day. Survival rates
were unexpectedly low in males, particularly in the control and low-dose groups: 25 and 5% in
the vehicle and untreated control groups and 14 and 58% in the 360 and 722 mg/kg-day dose
group, respectively. NCI (1978) did not suggest a reason why more high-dose male mice
survived compared with the low-dose and control males. Individual animal data were not
available to make survival adjustments to the tumor incidence data discussed below. Survival
rates in females were 80 and 85% in vehicle and untreated control groups and 80 and 68% in the
360 and 722 mg/kg-day dose groups, respectively. As a result of the low survival rates in the
vehicle and untreated male control groups, NCI compared tumor incidences in the dosed males
and females to vehicle control data pooled from bioassays for hexachloroethane, trichloroethane,
and 1,1,2-trichloroethane. NCI reported that animals were all of the same strain, housed in the
same room, intubated with corn oil, tested concurrently for at least 1 year, and examined by the
same pathologists.
Chronic inflammation of the kidney was observed in control and treated male mice: 67,
80, 66, and 18% of untreated controls, pooled vehicle controls, low dose, and high dose,
respectively. Female mice in the pooled vehicle control group (15%) and 722 mg/kg-day (2%),
but not the untreated control and 360 mg/kg-day dose groups, exhibited chronic kidney
inflammation. Tubular nephropathy (characterized by degeneration of convoluted tubule
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epithelium at the junction of the cortex and medulla, enlarged dark staining regenerative tubular
epithelium, and infiltration of inflammatory cells, fibrosis, and calcium deposition) was not
observed in untreated or pooled vehicle controls of either sex, but was observed in mice treated
with HCE: 49/50 and 47/49 in males and 50/50 and 45/49 in females in the 360 and 722 mg/kg-
day dose groups, respectively. Information on the severity of these effects at the different dose
levels was not presented. No other HCE-related nonneoplastic effects were observed and no
renal tumors were observed in either sex. EPA considered 360 mg/kg-day as the LOAEL for this
study based on tubular nephropathy. EPA considered that a NOAEL was not established.
Increases in the incidence of hepatocellular carcinomas were observed in male and
female mice exposed to HCE (Table 4-10). Hepatocellular adenomas were not noted in the
report. NCI (1978) reported statistically significant increases in the incidence of hepatocellular
carcinomas in 30 and 63% of 360 and 722 mg/kg-day males, compared with 10 and 15% of
pooled vehicle and matched vehicle controls, respectively. Female mice also demonstrated an
increased tumor response, 40 and 31% of 360 and 722 mg/kg-day females compared with 3 and
10% of pooled vehicle and matched vehicle controls, respectively. While the increases in
HCE-treated females were not dose-dependent, a higher incidence of hepatocellular carcinomas
was observed at the low dose (20/50) compared with the high dose (15/49). NCI concluded that
HCE was carcinogenic in both sexes of B6C3Fi mice (1978).
Table 4-10. Incidence of hepatocellular carcinomas in mice
Males
Females
Pooled vehicle control"
6/60 (10%)
2/60 (3%)
Matched vehicle control
3/20 (15%)
2/20 (10%)
360 mg/kg-day
15/50 (30%)b
20/50 (40%)c
722 mg/kg-day
31/49(63%)c
15/49 (3 1%)C
aAs a result of the exceptionally low survival rates in the vehicle and untreated control groups, NCI used the pooled
vehicle control data derived from concurrently runbioassays for several other chemicals. Animals were all of the
same strain and housed in the same room. Incidences reported were not adjusted for survival.
bStatistically significant, p = 0.008.
"Statistically significant, p < 0.001.
Source: NCI (1978).
4.2.2. Inhalation
4.2.2.1. Subchronic Exposure
Only one study is available in the peer-reviewed literature that evaluated the subchronic
(Weeks et al., 1979) inhalation toxicity of HCE. Weeks et al. (1979) exposed Sprague-Dawley
rats, Beagle dogs, Hartley guinea pigs, and Coturnix japonica (Japanese quail) to HCE for 6
weeks. The effects observed in these species include neurotoxicity, reduced body weight gain,
increased organ weights, and some evidence of respiratory tract irritation.
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Weeks et al. (1979) exposed male, female, and pregnant female Sprague-Dawley rats
(21-25/sex/concentration) to control air, 15, 48, or 260 ppm HCE (145, 465, and 2,517 mg/m3,
respectively; purity 99.8%) for 6 hours/day, 5 days/week for 6 weeks. Postexposure observation
was carried out for 12 weeks. An oxygen consumption test was also conducted. The authors
reported that in the 2,517 mg/m3 group, body weight gain of male rats, but not the nonpregnant
female rats, was reduced beginning in the third week of exposure (although quantitative
information was not reported). All rats in the 2,517 mg/m3 group exhibited tremors, ruffled pelt,
and red exudates around the eyes following the fourth week of exposure. The authors reported
that in the male rats, relative kidney, spleen, and testes weights were significantly increased; in
the female rats, only relative liver weights were significantly increased (although quantitative
information was not reported). One male and one female rat in the 2,517 mg/m3 exposure group
died during the fourth week, but the authors did not report a cause of death. During the post-
exposure observation period, treatment related effects disappeared. No gross changes were
evident at necropsy after the 12 week postexposure observation period; however, male and
nonpregnant female rats of the 2,517 mg/m3group sacrificed immediately after the 6 week
inhalation exposure had a higher incidence and severity of mycoplasma-related lesions in nasal
turbinates, trachea, and lung compared with controls. The authors concluded that these lesions
were related to potentiation of an endemic mycoplasma infection rather than a direct effect of
HCE exposure However, no data were presented demonstrating the presence of mycoplasma in
the lung. There were no histopathological differences observed between control and exposed
rats sacrificed 12 weeks postexposure. No treatment-related effects were observed in the rats
exposed to 145 and 465 mg/m3 HCE.
In the oxygen consumption test, male rats (5/concentration) were tested prior to and
following exposure to 145, 465, or 2,517 mg/m3 HCE for 15 minutes, 3 days/week for the
duration of the study (6 weeks). The 2,517 mg/m3 rats exhibited significantly decreased mean
rates of consumption prior to (15%) and after (13%) exposure to HCE. The authors suggested
that this decrease in oxygen consumption, while nonspecific, is indicative of an alteration in
basal metabolic rate. No histopathological effects were observed at this concentration. EPA
considered 465 mg/m3 the NOAEL and 2,517 mg/m3 the LOAEL, based on reduced body weight
gain, and increased organ weights.
Weeks et al. (1979) also exposed male Sprague-Dawley rats (15/concentration) to 15, 48,
or 260 ppm HCE (145, 465, or 2,517 mg/m3) for 6 hours/day, 5 days/week for 6 weeks and
examined them for behavioral changes related to learned and unlearned responses (described in
detail in Section 4.4.3.2). Similar to the other treated rats, body weight gain was reduced. Final
mean body weight gain in male rats was reduced 2, 5, and 10% (statistically significant) in the
145, 465, and 2,517 mg/m3 dose groups, respectively, compared with controls. Additionally,
relative lung, liver, kidney, and testes weights were increased (quantitative information not
reported) compared with controls.
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Weeks et al. (1979) also exposed four male Beagle dogs/concentration to control air, 15,
48, or 260 ppm HCE (145, 465, and 2,517 mg/m3, respectively; purity 99.8%) for 6 hours/day,
5 days/week for 6 weeks. Postexposure observation was carried out for 12 weeks. Blood
samples were evaluated for blood chemistry parameters. In addition, the dogs underwent
pulmonary function tests prior to and following exposure. One dog died within 5 hours of
exposure to 2,517 mg/m3. The remaining animals in the 2,517 mg/m3 group exhibited signs of
neurotoxicity consisting of tremors, ataxia, hypersalivation, head bobbing, and facial
fasciculations. No blood parameters were significantly affected and no exposure-related
histopathological lesions were observed following necropsy on dogs sacrificed 12 weeks
postexposure. Dogs evaluated for pulmonary functions while anesthetized did not display any
significant effects. The HCE-exposed dogs did not display any treatment-related toxicity at
12 weeks postexposure. EPA considered 465 mg/m3 the NOAEL and 2,517 mg/m3 the LOAEL,
based on neurotoxic effects.
Weeks et al. (1979) also exposed male Hartley guinea pigs (10/concentration) to control
air, 15, 48, or 260 ppm HCE (145, 465, and 2,517 mg/m3, respectively; purity 99.8%) for
6 hours/day, 5 days/week for 6 weeks. Postexposure observation was carried out for 12 weeks.
Guinea pigs were also evaluated for sensitization potential following inhalation exposure to
HCE. Two guinea pigs died during each of the fourth and fifth weeks, resulting in four total
deaths. Guinea pigs of the 2,517 mg/m3 group displayed reductions in body weight beginning at
the second week of exposure and significantly increased liver to body weight ratios (quantitative
information was not reported). No treatment-related effects were observed in the other exposure
groups. EPA considered the NOAEL as 465 mg/m3 and the LOAEL as 2,517 mg/m3, based on
decreased body weight and significantly increased relative liver weight.
Weeks et al. (1979) also exposed male and female quail (C.japonica, 20/concentration)
to control air, 15, 48, or 260 ppm HCE (145, 465, and 2,517 mg/m3, respectively; purity 99.8%)
for 6 hours/day, 5 days/week for 6 weeks. Postexposure observation was carried out for
12 weeks. The only observed effect was excess mucus in nasal turbinates in 2/10 quail in the
2,517 mg/m3 group after 6 weeks. The authors considered the excess mucus to be transient based
on the lack of any inflammation or histopathological effects. Although the study authors
considered the excess mucus to be a transient effect, EPA notes that the lack of inflammation and
histopathological effects does not preclude the presence of more sensitive indicators of immune
response (e.g., antibodies or other immune signaling chemicals) unable to be detected with
methods available to the study authors. EPA considered 2,517 mg/m3 (highest exposure
concentration) as the NOAEL, while the LOAEL could not be established from this study.
4.2.2.2. Chronic Exposure
No inhalation chronic exposure studies were identified.
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4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION
4.3.1. Oral
Weeks et al. (1979) exposed 22 pregnant Sprague-Dawley rats/dose to 50, 100, or
500 mg/kg HCE (purity 99.8%) by gavage on gestation days (GDs) 6-16. Gavage controls
received corn oil and positive controls received 250 mg/kg aspirin. Dams orally administered
500 mg/kg HCE displayed tremors on GDs 15 and 16. Body weight gain of the 500 mg/kg dams
was significantly lower than controls beginning on GD 8. Rats in the 500 mg/kg group exhibited
an increased incidence of mucopurulent nasal exudates compared with controls. Approximately
70% of the orally exposed 500 mg/kg group had upper respiratory tract irritation; 20% had
subclinical pneumonitis, compared with 10% in controls.
The aspirin-positive control group produced fetuses with lower body weights and
malformations such as hydrocephalus, spina bifida, and cranioschesis. None of the fetuses
exhibited any significant skeletal or soft tissue anomalies, although fetuses from dams gavaged
with 500 mg/kg HCE displayed significantly lower gestation indices, lower numbers of viable
fetuses/dam, and higher fetal resorption rates compared with controls (data not reported in the
publication). It is unclear from the publication if the authors used a litter-based design for their
statistical analyses of the fetal gestational indices, nor do they provide incidence data on the fetal
gestational indices. EPA considered the maternal NOAEL and LOAEL as 100 and 500 mg/kg,
respectively, based on neurological effects (tremors) and body weight decreases. EPA
considered the developmental NOAEL and LOAEL to be the same as the maternal values, based
on decreased viability and increased resorption rates.
Shimizu et al. (1992) evaluated the teratogenicity of HCE (purity not specified) in
pregnant Wistar rats at doses of 0, 56, 167, or 500 mg/kg administered by gavage during GDs 7-
17 (20-21 rats/dose). The dams of the 500 mg/kg dose group exhibited significantly decreased
weight gain after the second day of HCE treatment (8th day of pregnancy); dams in the
167 mg/kg dose group displayed significantly decreased weight gain after the fourth day of
treatment (10th day of pregnancy), but not after the treatment ended on the 18th day of pregnancy.
Food intake was also significantly decreased in the 500 and 167 mg/kg dose groups after the
second and third days, respectively, of HCE treatment; however, intake was normal when
treatment ended. Dams in both the 167 and 500 mg/kg dose groups exhibited decreased motor
activity (incidence and method of analysis not reported); dams in the 500 mg/kg dose group also
exhibited piloerection and subcutaneous hemorrhage. These effects decreased or disappeared
when HCE exposure ended. An autopsy performed on dams on GD 20, 3 days post-HCE
exposure, revealed three rats with whitening of the liver in the 500 mg/kg dose group. The
significance of this observation is unknown. No deaths occurred in any of the dose groups.
There were no significant differences between the HCE treatment and control groups
with respect to the numbers of corpora lutea, implants, or live fetuses (Table 4-11). There was
no significant difference in the incidence of dead or resorbed fetuses, except for a significant
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increase during the late stage of pregnancy in the 500 mg/kg dose group (6.4% versus none in the
control). Fetuses in the 500 mg/kg dose group also displayed significantly decreased body
weight; 2.5 + 0.57 (mean + SD) and 2.3 + 0.45 g in male and female fetuses, compared with
3.3 + 0.20 and 3.1 + 0.24 g in male and female controls, respectively. The authors state that the
litter was used as the statistical unit for calculations of the fetal values.
Table 4-11. Summary of HCE effects on pregnant Wistar rats and their
fetuses
Number of dams
% of dead or resorbed fetuses
Early stage
Late stage
Body weight of live fetuses (g)b
Male
Female
Dose (mg/kg)
0
20
8.7
8.7
3.3+0.20
3.1+0.24
56
20
9.2
8.8
0.4
3.3+0.17
3.0 + 0.20
167
20
7.0
6.1
0.9
3.2 + 0.21
2.9 + 0.17
500
21
14.7
13.1
6.4a
2.5+0.573
2.3+0.453
Significantly different from control, p < 0.01.
Values are mean + SD.
Source: Shimizuetal. (1992).
The investigators (Shimizu et al., 1992) examined the fetuses for external anomalies and
found one case of acaudate in the 500 mg/kg dose group. Other anomalies included two fetuses
with subcutaneous hemorrhage in the 167 and 500 mg/kg dose groups and one case of hyposarca
in the 500 mg/kg dose group. No skeletal malformations were observed in any group, although a
statistically significant increase in skeletal variations was observed in the 500 mg/kg (60.3%)
group compared with controls (1.3%). Skeletal variations were significantly increased in the
500 mg/kg group (2 cases in the lumbar rib and 78 cases in the rudimentary lumbar rib) and
nonsignificantly increased in the 167 mg/kg group (6 cases in the rudimentary lumbar rib)
compared with controls (2 cases in the rudimentary lumbar rib) (Table 4-12). The degree of
ossification (including numbers of sternebrae, proximal and middle phalanges, and sacral and
caudal vertebrae) was significantly decreased in the 500 mg/kg dose group. No visceral
malformations were observed and no significant differences in visceral anomalies were noted.
The authors concluded that there was no indication of teratological effects in rats for dose levels
of HCE below 500 mg/kg. The authors state that the litter was used as the statistical unit for
calculations of the fetal values. Shimizu et al. (1992) established a NOAEL of 56 mg/kg for
dams and 167 mg/kg for fetuses. EPA considered the LOAEL for dams as 167 mg/kg-day, based
on decreased motor activity and significantly decreased body weight. EPA considered the
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LOAEL for fetuses as 500 mg/kg, based on significantly increased skeletal variations and
significantly decreased ossification and fetal body weight.
Table 4-12. Summary of skeletal effects on fetuses from HCE-exposed rats
Number of fetuses examined
Percent of fetal variations
Number of fetuses with variations
Lumbar rib
Rudimentary lumbar rib
Ossification13
Number of sternebrae
Number of proximal and middle
phalanges
Fore limb
Hind limb
Number of sacral and caudal vertebrae
Dose (mg/kg)
0
136
1.3
0
2
4.7+0.07
3.2+0.05
4.0+0.01
6.9+0.06
56
136
0
0
0
4.5+0.08
3.1+0.04
4.0 + 0.01
6.9 + 0.08
167
136
3.8
0
6
4.5+0.08
3.1+0.04
4.0+0.01
7.0+0.04
500
137
60.3a
2
78
3.4+0.273
2.9+0.11a
3.4 + 0.233
5.7+0.373
"Significantly different from control, p < 0.01.
bAs reported by Shimizu et al. (1992) the litter was used as the statistical unit for calculation of fetal values; thus,
these values represent the means + SD of litter means within each group.
Source: Shimizu etal. (1992).
4.3.2. Inhalation
Weeks et al. (1979) exposed 22 pregnant Sprague-Dawley rats/concentration to control
air, 15, 48, or 260 ppm HCE (145, 465, and 2,517 mg/m3, respectively; purity 99.8%) by
inhalation on GDs 6-16. Dams in the 2,517 mg/m3 group displayed tremors during GDs 12-16.
Body weight gain of the dams was significantly lower than controls beginning on GD 8 for the
2,517 mg/m3 group, and beginning on GD 14 for the 465 mg/m3 group. Rats in the 465 and
2,517 mg/m3 groups exhibited an increased incidence of mucopurulent nasal exudates compared
with controls. Inflammatory exudate was observed in the lumen of the nasal turbinates of 85%
of the 465 mg/m3 group and 100% of the 2,517 mg/m3 group. The authors attributed the
increased exudate to an endemic mycoplasma infection.
Fetuses of HCE-treated dams did not exhibit any significant skeletal or soft tissue
anomalies. It is unclear from the publication if the authors used a litter-based design for their
statistical analyses of the fetal gestational indices, nor do they provide incidence data of the fetal
gestational indices. EPA considered the NOAEL for the dams as 465 mg/m3 and the LOAEL as
2,517 mg/m3, based on neurological effects (tremors). EPA considered 2,517 mg/m3 (highest
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concentration tested) as a developmental NOAEL, based on the lack of treatment-related effects,
while a developmental LOAEL could not be established from this study.
4.4. OTHER DURATION- OR ENDPOINT-SPECIFIC STUDIES
4.4.1. Acute Exposure Studies
4.4.1.1. Oral
Several studies evaluated acute toxicity of HCE in animal species and reported lethal
dose concentrations. Oral lethal doses ranged from 2,332 to 8,640 mg/kg in rats, >1,000 mg/kg
in male rabbits, and 4,970 mg/kg in guinea pigs (Kinkead and Wolfe, 1992; Weeks et al., 1979).
According to the Hodge and Sterner Scale, these lethal doses place HCE in low toxicity range
(Hodge and Sterner, 1949). Reynolds (1972) administered a single dose of HCE (purity not
specified) at 26 mmol/kg (6,155 mg/kg) by gavage in mineral oil to male rats and reported that
liver function (assessed by microsomal protein concentration, antipyrine demethylase activity,
NADP-neotetrazolium reductase activity, glucose 6-phosphatase activity, and conjugated diene
concentration in microsomal lipids) was unaffected 2 hours after exposure. Kinkead and Wolfe
(1992) determined that the oral median lethal dose (LDso) for HCE (purity not specified) in male
and female Sprague-Dawley rats (5 rats/sex/dose) was 4,489 mg/kg (95% confidence limit [CL],
2,332-8,640 mg/kg). A study in sheep that was conducted at high doses (500-1,000 mg/kg)
found reduced hepatic function (Fowler, 1969).
Weeks et al. (1979) and Weeks and Thomasino (1978) determined acute oral toxicity
values for Sprague-Dawley rats, New Zealand White rabbits, and Hartley guinea pigs by
administering a single dose of HCE (99.8% purity) dissolved in corn oil (50% w/v) or
methylcellulose (5% w/v) via gavage. Approximate lethal dosages (ALD) or LDso values were
calculated after a 14-day observation period (Table 4-13). All LDso values were >1,000 mg/kg.
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Table 4-13. Summary of acute exposure data in rats, rabbits, and guinea
pigs
Species
Rabbit, male
Rat, male
Rat, male
Rat, female
Rat, male
Guinea pig, male
Rabbit, male
Treatment
OralALD
Intraperitoneal
(i.p.) ALD
OralALD
Oral LD50
Oral LD50
Oral LD50
Dermal LD50
Diluent
Methylcellulose
Corn oil
Corn oil
Corn oil
Methylcellulose
Corn oil
Methylcellulose
Corn oil
Water paste
Lethal value
mg/kg
>1,000
2,900
4,900
4,460
7,080
5,160
7,690
4,970
>32,000
95% CL
3,900-5,110
6,240-8,040
4,250-6,270
6,380-9,250
4,030-6,150
Slope
9.3
19.9
6.1
8.5
4.7
Sources: Weeks et al. (1979); Weeks and Thomasino (1978).
Fowler (1969) orally administered a single dose of HCE (purity not specified) through a
drenching bottle to Scottish Blackface and Cheviot cross sheep at three dose levels: 500 (six
sheep), 750 (one sheep), and 1,000 mg/kg (one sheep). Hepatotoxicity was assessed by
measurement of plasma enzyme activities and bromsulphthalein dye clearance tests, which are
widely-used indices of hepatic function in sheep. Plasma activities of glutamate dehydrogenase
(GDH), sorbitol dehydrogenase (SDH), ornithine carbamoyl transferase (OCT), and AST were
determined daily until they reached stable levels. Increases in these enzymes are indicative of
hepatic damage. HCE exposure resulted in a 3-6-fold increase in GDH, with the exception of
one sheep that exhibited a 55-fold increase. SDH was increased 3-6-fold and OCT was
increased 2-10-fold. GDH, SDH, and OCT levels peaked at 48 hours and returned to normal
within 4-5 days. AST increased only slightly. Bromsulphthalein dye clearance tests found a
reduction in transfer from liver cells to bile at 72 hours after HCE exposure, indicating reduced
hepatic function.
4.4.1.2. Inhalation
Median lethal concentration (LCso) values for HCE have not been reported. One study is
available in the peer-reviewed literature that evaluated acute inhalation exposure to HCE (Weeks
and Thomasino 1978). Six male rats/concentration (strain not specified, although one table in
the report indicated strain as Sprague-Dawley) were exposed to 260 or 5,900 ppm HCE (2,500 or
57,000 mg/m3) for 8 hours and to 1,000 ppm HCE (17,000 mg/m3) for 6 hours. Postexposure
observation was carried out for 14 days. Male rats exposed for 8 hours to 2,500 mg/m3 HCE
displayed no toxic signs during exposure or for 14 days thereafter. Body weight gain was
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slightly, but not statistically significantly, reduced over the 14-day exposure period. Male rats
exposed for 8 hours to 57,000 mg/m3 HCE displayed severe toxic signs including death. At 6
hours, one rat had a staggered gait. At 8 hours, 2/6 rats were dead. The surviving rats showed
statistically significant reductions in mean body weight on exposure days 0 (7%), 1 (21%), 3
(19%), 7 (15%), and 14 (15%), compared with controls. Necropsy did not reveal any gross
exposure-related lesions. Microscopy revealed that two of the four surviving rats had minimally
to moderately severe subacute diffuse interstitial pneumonitis and vascular congestion.
Additionally, a purulent exudate of the nasal turbinates was observed in one control and one
treated rat. The authors concluded that this effect was not exposure-related, but rather was
indicative of a low-grade endemic upper respiratory disease. The male rats exposed for 6 hours
to 17,000 mg/m3 showed slight reductions in body weight gain on postexposure days 1 (5%) and
3 (4%) and body weights similar to controls for the remaining 11 days of the postexposure
period. Two of the six rats demonstrated a staggered gait. No exposure-related gross or
histopathological changes were observed in tissues and organs.
4.4.2. Short-term Exposure Studies
Several studies evaluated short-term toxicity of HCE in animal species. A 12-day study
in male New Zealand White rabbits found liver degeneration and necrosis, as well as tubular
nephrosis in the kidney, indicating that both the liver and kidney are potential target tissues for
HCE-induced toxicity (Weeks et al., 1979). Short-term toxicity assays in rats (16 and 21 days)
demonstrated kidney effects in males (NTP, 1996, 1989) but not females (NTP, 1989).
Weeks et al. (1979) conducted a 12-day study of HCE in male New Zealand White
rabbits. Five rabbits/dose were administered a daily oral dose via a stomach tube of 100, 320, or
1,000 mg/kg HCE (purity 99.8%) suspended in 5% aqueous methylcellulose. Blood was drawn
from the central ear artery of the rabbits on treatment days 1, 4, 8, and 12, and on day 4
following termination of dosing. Serum was analyzed for the following parameters: glutamic
oxaloacetic transaminase (SGOT; also known as AST), glutamic pyruvic transaminase (SGPT;
also known as ALT), blood urea nitrogen (BUN), alkaline phosphatase, bilirubin, total protein,
potassium, and sodium. On the fourth day following the termination of dosing, rabbits were
necropsied and the following tissues were examined: eye, brain, lung, kidney, liver, spleen,
heart, stomach, pancreas, large intestine, skeletal muscle, bone, urinary bladder, small intestine,
and testes.
The 1,000 mg/kg dose group exhibited significantly reduced body weight (beginning on
treatment day 7) and increased relative liver and kidney weights. The 320 mg/kg dose group
exhibited significantly reduced body weight beginning on day 10. The 100 mg/kg dose group
did not display any changes. The 320 and 1,000 mg/kg dose groups displayed liver degeneration
and necrosis, including fatty degeneration, coagulation necrosis, hemorrhage, ballooning
degeneration, eosinophilic changes, and hemosiderin-laden macrophages and giant cells. These
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effects were not observed in controls or rabbits of the 100 mg/kg dose group. Liver lesions
increased in severity in a dose-related manner in which the effects were more severe in the
1,000 mg/kg group compared with the 320 mg/kg group. Tubular nephrosis of the convoluted
tubules in the corticomedullary region of the kidney was also observed in the rabbits of the
320 and 1,000 mg/kg dose groups. These animals also exhibited tubular nephrocalcinosis of a
minimal degree. The only blood chemistry parameters that were affected were significantly
decreased potassium and glucose levels in the 320 and 1,000 mg/kg groups. EPA considered the
NOAEL as 100 mg/kg and the LOAEL as 320 mg/kg, based on dose-related increases in severity
of liver and kidney lesions.
The NTP (1989) conducted a 16-day study of oral HCE toxicity in F344/N rats. Groups
of five rats/sex/dose were administered 0, 187, 375, 750, 1,500, or 3,000 mg HCE/kg (purity
>99%) for 12 doses over 16 days by corn oil gavage. TWA doses were 0, 140, 281, 563, 1,125,
and 2,250 mg/kg-day, respectively. Necropsy was performed on all rats; all organs and tissues
were examined for grossly visible lesions and histopathology. All rats of the 1,125 and
2,250 mg/kg-day dose groups and 1/5 males and 2/5 females from the 563 mg/kg-day dose group
died before the end of the study. Final mean body weights (statistical analyses were not
reported) were decreased by 25% in males of the 563 mg/kg-day dose group; female body
weights were decreased by 37% in the 563 mg/kg-day dose group. Microscopic observations of
the kidneys revealed hyaline droplet formation in the cytoplasm of renal tubular epithelium in all
treated males, and tubular cell regeneration and eosinophilic granular casts of cell debris in
tubule lumina of male rats administered 140 and 281 mg/kg-day. EPA considered
140 mg/kg-day (lowest dose tested) a male rat LOAEL based on kidney tubule lesions, while a
NOAEL could not be established for male rats. EPA considered the female rat LOAEL as
563 mg/kg-day, based on a dose-related decrease in body weight, and the female rat NOAEL as
281 mg/kg-day.
NTP (1996) conducted a 21-day study of oral HCE toxicity in male F344/N rats. Groups
of five rats/dose were administered 0.62 or 1.24 mmol HCE/kg-day (146 or 293 mg/kg-day,
respectively; purity 100%) by corn oil gavage. Necropsies were performed on all rats; the right
kidney, liver, and right testis were weighed and underwent histopathological evaluation. Urine
samples were collected during an overnight period that began 4 days before the end of the study.
Urinalysis included measurements of volume, specific gravity, creatinine, glucose, total protein,
AST, y-glutamyl transferase (GGT), and TV-acetyl-p-D-glucosaminidase (NAG). A Mallory-
Heidenhain stain was used for kidney sections to evaluate protein droplets, particularly hyaline
droplet formation. Cell proliferation analyses were performed on kidney sections and were
scored by a labeling index indicating the percentage of proximal and distal tubule epithelial cells
in S-phase.
Results from the measured endpoints/parameters are summarized in Table 4-14.
Absolute and relative kidney weights were significantly increased in both dose groups; absolute
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and relative (significant at high dose) liver weights were increased in both dose groups. Rats of
the 293 mg/kg-day group also exhibited significantly lower urinary creatinine and specific
gravity, while glucose and urine volumes were greater than controls. AST and NAG activities
were significantly higher than in controls. Nephropathy, consisting of hyaline droplet
accumulation, was observed in the male rats in addition to increased incidences of tubule
regeneration (3/5 and 4/5 for 146 and 293 mg/kg-day, respectively) and granular casts (4/5 and
3/5 for 146 and 293 mg/kg-day, respectively). The mean proliferating cell nuclear antigen
(PCNA) labeling index was significantly increased 5.7- and 9.2-fold, compared with controls, in
the 146 and 293 mg/kg-day dose groups. EPA did not identify a NOAEL because effects
(including increased absolute and relative kidney weight, increased AST and NAG activity,
increased PCNA labeling index, and nephropathy) were observed at the low dose level. EPA
considered 146 mg/kg-day a LOAEL based on statistically significant increases in kidney lesions
and urinalysis parameters.
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Table 4-14. Summary of toxicity data from male rats exposed to HCE for
21 days
Vehicle control
146 mg/kg-day HCE
293 mg/kg-day HCE
Right kidney weight3
Absolute (g)
Relative (mg/g)
1.009 + 0.025
3.19+0.04
1.157 + 0.011b
3.77+0.06b
1.250 + 0.022b
4.07 + 0.05b
Liver weight3
Absolute (g)
Relative (mg/g)
11.041+0.291
34.82 + 0.60
11.959+0.178
39.01+0.92
13.479 + 0.390
43.84 +0.64b
Right testis weight3
Absolute (g)
Relative (mg/g)
1.412 + 0.037
4.47 + 0.09
1.409+0.023
4.60 + 0.11
1.430 + 0.016
4.66 + 0.05
Urinalysis
Creatinine (mg/dL)
Glucose (ug/mg creatinine)
Protein (mU/mg creatinine)
AST (mU/mg creatinine)
GOT (mU/mg creatinine)
NAG (mU/mg creatinine)
Volume (mL/16 h)
Specific gravity (g/mL)
PCNA labeling index (mean + SE)
143.22 + 18.12
169 + 3
1,322 + 59
6 + 1
1,456 + 47
11+0
4.2+0.8
1.038 + 0.005
0.13+0.02
79.56 + 11.01
344 + 30
1,748 + 257
40 + 6C
1,547 + 66
23+2c
7.5 + 9
1.024 + 0.003
0.74 + 0.19C
54.48 + 3.06b
446 + 23b
2,980 + 103
66 + 5b
1,897 + 73
36 + lb
10.6+l.lb
1.020 + 0.001b
1.2 + 0.2C
3Data are mean + SE.
bSignificantly different from control (p < 0.01).
Significantly different from control (p < 0.05).
Source: NTP(1996).
4.4.3. Neurological
Neurological endpoints for HCE toxicity have been evaluated in several studies. The
studies listed below provide evidence that HCE produces neurological effects; however, it is
unknown if the central nervous system (CNS) effects are due to the parent compound or the
metabolites. Sheep exposed to high doses of HCE (500-1,000 mg/kg) developed facial muscle
tremors (Fowler, 1969; Southcott, 1951), and a staggering uncoordinated gait (Southcott, 1951).
Sprague-Dawley rats evaluated for HCE-induced effects on avoidance latency (i.e., learned
behavior) and spontaneous motor activity (i.e., unlearned behavior) did not show statistically
significant behavioral effects of HCE exposure. Male and female rats also exhibited tremors and
ruffled pelt at 2,517 mg/m3 (Weeks et al., 1979). Beagle dogs developed signs of neurotoxicity
such as tremors, ataxia, hypersalivation, and head bobbing following exposure to 2,517 mg/m3
HCE. Dogs showed similar signs of neurotoxicity intermittently throughout the HCE exposures,
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with signs disappearing overnight. During an observation period of 12 weeks following
exposure, these symptoms were not observed (Weeks et al., 1979).
4.4.3.1. Oral Studies
Fowler (1969) orally administered a single dose of HCE (purity not specified) to Scottish
Blackface and Cheviot cross sheep at three dose levels: 500 (10 sheep), 750 (1 sheep), and
1,000 mg/kg (1 sheep). Slight facial muscle tremors were noted in three sheep between 1 and
4 hours after dosages of 500-1,000 mg/kg HCE. The HCE dose level for the individual sheep
exhibiting facial tremors was not specified by the authors. Fowler (1969) also examined two
sheep administered 0.3 mL/kg PERC and two sheep administered 0.3 mL/kg pentachloroethane,
two proposed major metabolites of HCE. The sheep exposed to PERC exhibited no effects
following exposure, while the sheep exposed to pentachloroethane exhibited narcosis. One
pentachloroethane-exposed animal was recumbent within 30 minutes of exposure, exhibiting
flaccid limbs, depression of normal reflexes, and labial tremors. The sheep regained normal
posture 9 hours postexposure and appeared normal 72 hours postexposure. The second
pentachloroethane-treated sheep became recumbent within 20 minutes of exposure and exhibited
labial tremors. However, unlike the first sheep, this animal appeared normal 1.5 hours
postexposure. EPA considered the LOAEL as 500 mg/kg (lowest dose tested), based on
neurotoxic effects (tremors), while aNOAEL could not be established from these data.
Southcott (1951) treated 30 Merino Wethers sheep suffering from liver fluke infections
with 15 g HCE-bentonite dispersible powder (13.5 g HCE, 445 mg/kg; 15 sheep) or 30 g
HCE-bentonite (27 g HCE, 906 mg/kg; 15 sheep). The purity of the HCE was not specified.
One day after treatment, two sheep died and nine others were unable to rise and stand. One of
the severely affected sheep (i.e., unable to rise and stand) was from the 445 mg/kg HCE group
and the other eight were from the 906 mg/kg group. Some severely affected animals (two from
the 445 mg/kg group) could walk if placed on their feet, but displayed a staggering,
uncoordinated gait and fell again. The lips, face, neck, and forelegs were afflicted by fine
muscular tremors that were observed in most of the animals. EPA considered the LOAEL as
445 mg/kg (lowest dose tested), based on neurological effects consisting of tremors, staggering,
uncoordinated gait, and inability to stand, while a NOAEL could not be established from this
study.
As described in Section 4.3.1, Shimizu et al. (1992) reported decreased motor activity
(incidence and method of analysis not reported) in pregnant Wistar rats (20-21 rats/dose) at
doses of 167 and 500 mg/kg administered by gavage during GDs 7-17. These effects decreased
or disappeared when HCE exposure ended. Weeks et al. (1979) exposed 22 pregnant
Sprague-Dawley rats/dose to 50, 100, or 500 mg/kg HCE by corn oil gavage on GDs 6-16.
Dams orally administered 500 mg/kg HCE displayed tremors on GDs 15 and 16.
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4.4.3.2. Inhalation Studies
Weeks et al. (1979) exposed male Sprague-Dawley rats (15/concentration) to air, 15, 48,
or 260 ppm HCE (145, 465, or 2,517 mg/m3, respectively; purity 99.8%) for 6 hours/day,
5 days/week for 6 weeks. Learned behavior endpoints, evaluated using an avoidance latency
task by measuring the time it took the rats to avoid foot shock by escaping into a safe
compartment and unlearned behavior endpoints (spontaneous motor activity; evaluated by
photobeam interruptions) were measured in the animals. The avoidance latency task was
conducted prior to exposure, 1 day into exposure, after 3 weeks of exposure, and after 6 weeks of
exposure. Spontaneous motor activity was tested after 3 and 6 weeks of exposure.
Avoidance latency was increased in the 465 and 2,517 mg/m3 groups at 6 weeks (median
3.9 and 3.3 seconds, respectively) compared with control (median 2.2 seconds). Spontaneous
motor activity counts were also increased in the HCE-treated rats (mean + SD): 231 ±77 for
145 mg/m3, 183 + 109 for 465 mg/m3, and 201 + 102 for 2,517 mg/m3, compared with control
rats (163 + 74). Neither of these differences were satstistically significant. Weeks et al. (1979)
concluded that the rats did not display obvious signs of behavioral toxicity. However, tremors
and a ruffled pelt were noted in a separate experiment in male and female rats exposed to 2,517
mg/m3 HCE during the fourth week of exposure. Tremors are indicators of neurobehavioral
effects and lack of grooming could also be interpreted as an indicator of behavioral toxicity
(Kulig et al., 1996). The investigators sacrificed the rats 12 weeks after the last exposure and
reported that all measurable changes (e.g., brain histopathology, body weights) were comparable
to controls.
Weeks et al. (1979) also exposed 22 pregnant Sprague-Dawley rats/concentration and
4 Beagle dogs/concentration to 145, 465, and 2,517 mg/m3 HCE by inhalation. Rat dams in the
2,517 mg/m3 group displayed tremors during GDs 12-16, although the authors did not provide
any quantitative data. Dogs in the 2,517 mg/m3 exposure group developed tremors, ataxia,
hypersalivation, and displayed severe head bobbing, facial muscular fasciculations, and held
their eyelids closed during exposure. One dog experienced convulsions and died within 5 hours
after initial exposure. The surviving dogs exhibited less severe symptoms during exposure, but
recovered overnight after removal from exposure.
4.4.4. Immunological
Ten male Hartley guinea pigs/dose were exposed by inhalation to control air or three
concentrations of HCE (purity 99.8%): 15, 48, or 260 ppm (145, 465, and 2,517 mg/m3,
respectively; Weeks et al., 1979). Exposures were conducted for 6 hours/day, 5 days/week for
3 weeks. Following exposure, animals were allowed to rest for 2 weeks. The guinea pigs were
then challenged with a single intradermal injection of 0.1% HCE in saline. A sensitization
response was not produced.
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4.4.5. Dermatological
Yamakage and Ishikawa (1982) examined certain patients suffering from systemic
scleroderma (SSD) for potential exposure to solvents. These patients also presented with
localized scleroderma with bilateral distribution of multiple skin lesions reminiscent of those
observed in several cases of occupational or agent-induced scleroderma. Of nine such patients,
seven had had significant sub chronic/chronic exposure (5-44 years), while an eighth had had a
significant acute exposure (2 weeks). The solvents involved were reported as "variable and
mostly unidentified." As an experimental follow-up, groups of ddY mice received daily
intraperitoneal (i.p.) injections for 17 days with 1 of 10 experimental solvents, as well as with
0.9% saline to mitigate treatment lethality. For HCE, 17 mice were injected daily with 0.01 mL
of HCE (purity not specified) and 0.1 mL of 0.9% saline. Along with naphtha ("Esso No. 5")
and n-hexane, HCE was found, by double-blind histological examination and electron
microscopy, to be a significant inducer of sclerodermatous changes in skin taken from the
animals' backs, near the forelimbs. HCE treatment resulted in evident dermal sclerosis in five
mice, slight fibrosis in one mouse, and no change in nine mice; two mice died. PERC, a primary
metabolite of HCE, was similarly tested in 10 mice. Injections of 0.005 mL (+ saline) resulted in
evident dermal sclerosis in one mouse, slight fibrosis in two, no change in six, and death in one.
Even though this experimental route of exposure is generally irrelevant to humans, the skin
lesions produced by HCE were "fundamentally similar" to those produced by control reference
solvents that have been implicated in human occupational SSD. Thus, this study provides
indirect evidence that suggesting that HCE may be capable of inducing SSD-type conditions in
humans.
Weeks and Thomasino (1978) conducted two dermal studies in male New Zealand White
rabbits. A single 24-hour application of 500 mg of technical dry HCE to intact and abraded skin
of six rabbits did not result in primary irritation of intact or abraded skin when assessed at
24 hours, 72 hours, or 7 days after exposure. HCE was placed in Irritation Category IV (no
irritation). In the second study, HCE was applied as a paste in 0.5 mL of distilled water. Intact
skin displayed no edema and barely perceptible erythema at 24 hours. Abraded skin displayed
barely perceptible erythema in one rabbit with moderate to slight erythema reactions. HCE was
placed in Irritation Category III (mild or slight irritation).
4.4.6. Eye Irritation
Weeks and Thomasino (1978) applied a single, 24-hour dose of 100 mg dry HCE to one
eye of six male New Zealand White rabbits. Moderate corneal damage, iritis, and conjunctivitis
was observed in 5/6 rabbits 24, 48, and 72 hours after exposure. No effects were observed
7 days after exposure. HCE was placed in Irritation Category II for eye effects (corneal opacity
reversible within 7 days or persisting for 7 days).
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4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF
ACTION
4.5.1. Genotoxicity
In vivo genotoxicity studies have not been performed in humans exposed to HCE. In
vivo exposure to animals resulted in predominantly negative results. Similarly, in vitro
genotoxicity studies conducted in microorganisms, cultured mammalian cells, and insects
(Table 4-15) were largely negative both in the presence and absence of exogenous metabolic
activation. Ashby and Tennant (1988) examined genotoxic carcinogenesis in a set of
222 chemicals tested in rodents by NCI/NTP; HCE did not induce mutagenicity in Salmonella
typhimurium reverse mutation tester strains. NTP's technical report on the toxicity and
carcinogenicity of HCE in F344/N rats concluded that HCE (purity >99%) was not significantly
genotoxic, and that the increased incidence of tumors occurred through a mechanism other than
one involving the induction of mutations (NTP, 1989). In an examination of the available
mutagenicity and genotoxicity data (i.e., the ability to induce alterations in deoxyribonucleic
acid [DNA] structure or content, i.e., gene mutation, chromosomal aberrations [CAs], or
aneuploidy) from short-term tests with putative "nongenotoxic" carcinogens, HCE was
categorized as having insufficient mutagenicity data for evaluation (Jackson et al., 1993).
Studies conducted by Lohman and Lohman (2000) considering DNA damage, recombination,
gene mutation, sister chromatid exchange (SCE), micronuclei (MN), CA, aneuploidy, and cell
transformation as endpoints indicate that the genetic activity profile for HCE is predominantly
negative. However, some positive findings have been reported in assays for gene conversion,
somatic mutation/recombination, DNA adducts, and SCEs.
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Table 4-15. Summary of genotoxicity studies of HCE
Test system
Genetic endpoint
Strain/cells
Results
Reference
Comments
In vitro tests
Bacterial
Mammalian
Fungi
Gene reversion/
S. typhimurium
Forward mutations
SOS test
CAs
SCEs
MN
Cell
transformation
DNA adduct
formation
(nonhuman)
Mitotic
recombination
Aneuploidy
TA98, TA100, TA1535,
TA1537, TA1538
TA98, TA100, TA1535,
TA1537, TA1538
TA98, TA100, TA1535,
TA1537
TA98, TA100, TA1535,
TA1537
BA13
TA1535/pSK1002
Chinese hamster ovary
(CHO)
CHO
AHH-1
MCL-5
h2El
BALB/C-3T3
Wistar rats, calf thymus
DNA
BALB/c mice, calf
thymus DNA
S. cerevisiae D3
S. cerevisiae D4
S. cerevisiae D7
Aspergillus nidulans PI
diploid
-(+S9)
- (+S9)
- (+S9)
- (+S9)
- (+S9)
- (+S9)
- (+S9)
-(-S9),
+ (+S9)a
-
-
-
-
+ DNA binding
in liver, kidney,
lung, and
stomach
+ DNA binding
in liver, kidney,
lung, and
stomach
-(+S9)
-(+S9)
-(+S9)
Simmon and
Kauhanen
(1978)
Weeks et al.
(1979)
Haworth et al.
(1983)
Milman et al.
(1988)
Roldan-Arjona
etal. (1991)
Nakamura et al.
(1987)
Galloway et al.
(1987)
Galloway et al.
(1987)
Doherty et al.
(1996)
Doherty et al.
(1996)
Doherty et al.
(1996)
Milman et al.
(1988)
Lattanzi et al.
(1988)
Lattanzi et al.
(1988)
Simmon and
Kauhanen
(1978)
Weeks et al.
(1979)
Bronzetti et al.
(1990, 1989)
Crebelli et al.
(1995,1992,
1988)
Liquid
preincubation
protocol
Liquid
preincubation
protocol
umu test;
Liquid
preincubation
protocol
HCE
precipitation at
doses causing
positive results
Human cell line
Human cell line
Human cell line
DNA adducts
not identified
DNA adducts
not identified
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Table 4-15. Summary of genotoxicity studies of HCE
Test system
Genetic endpoint
Strain/cells
Results
Reference
Comments
In vivo tests
Rat
Mice
Human
lymphocytes
Drosophila
Rat liver foci
DNA adduct
formation
(nonhuman)
Micronucleus
induction
Replicative DNA
synthesis (RDS)
DNA strand
breaks
Mitotic
recombination
Osborne-Mendel
Wistar rats
CD-I mice
B6C3FJ mice
BALB/c mice
Isolated human
lymphocytes
Human lymphocyte
cultures
Drosophila
- (initiation)
+ (promotion)
Weakly + DNA
binding in liver
-
+
Moderately +
DNA binding in
liver
+ (+S9)
-
Weakly +
Milman et al.
(1988)
Lattanzi et al.
(1988)
Crebelli et al.
(1999)
Yoshikawa
(1996);
Miyagawa et al.
(1995)
Lattanzi et al.
(1988)
Tafazoli et al.
(1998)
Tafazoli et al.
(1998)
Vogel and
Nivard (1993)
Initiation or
promotion
protocols
Adducts not
identified
Hepatic cell
proliferation
Adducts not
identified
Comet assay
Eye mosaic
assay
Using the standard Ames assay for reversion of S. typhimurium histidine tester strains
(TA1535, TA1537, TA1538, TA98, and TA100), Simmon and Kauhanen (1978) found HCE to
be nonmutagenic at concentrations of 5,000 or 10,000 ug HCE/plate (purity not specified), both
in the absence and presence of an exogenous Aroclor 1254-stimulated rat liver S9 metabolic
activation system. HCE was reported to be slightly toxic at the 10,000 ug/plate concentration in
the absence of the S9 mix. Weeks et al. (1979) also reported negative results using the same
tester strains, test protocol, solvent, and metabolic activation system over a concentration range
of 0.1-500 ug HCE/plate (purity 99.8%). Further, as a part of NTP's mutagenicity screening
program, HCE was dissolved in dimethylsulfoxide (DMSO) and tested in two independent trials
in two separate laboratories over a collective concentration range of 1-10,000 ug/plate. HCE
was negative for induction of reverse mutation in S. typhimurium (tester strains TA1535,
TA1537, TA98, and TA100), with and without S9 metabolic activation (NTP, 1989; Haworth et
al., 1983). Finally, HCE (purity >97%) was reported to be negative in several Ames tester
strains, both with and without S9 from the Aroclor 1254-induced livers of both sexes of Osborne
Mendel rats and B6C3Fi mice (Milman et al., 1988).
Using a different S. typhimurium indicator strain, BA13, in a liquid preincubation
protocol of the Ara test, Roldan-Arjona et al. (1991) found HCE to be negative. This bacterial
assay examines the ability of an agent to induce forward mutations from L-arabinose sensitivity
to resistance, and theoretically might be expected to detect a broader range of mutagens than
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reverse-mutation assays. HCE (purity 98%) was dissolved in DMSO and tested over a
concentration range of 1.5-30.0 umol/plate (355-7,102 ug/plate), both with and without rat liver
S9 metabolic activation. Of the 16 chemicals tested in this study, HCE was the only one that did
not demonstrate any toxicity, which the authors speculated was probably related to its low
solubility in water. HCE (purity not specified) was negative when assayed in the umu test using
S. typhimurium tester strain TA1535/pSK1002 (Nakamura et al., 1987). This study also
employed a liquid preincubation protocol, and was conducted both with and without rat liver S9
metabolic activation up to a concentration of 42 ug/mL (the solvent, either water or DMSO, was
not specified for individual test agents). Although the available data indicate that HCE is not
mutagenic to Salmonella, Legator and Harper (1988) suggested that this may be related to
inadequate reductive dechlorination (i.e., if HCE is activated by metabolic pathways not present
in the in vitro system used).
HCE was assayed for its ability to induce mitotic recombination in tester strain D3 of the
yeast S. cerevisiae (Simmon and Kauhanen, 1978). No significant activity over a concentration
range of 0.1-5.0% HCE (1-50 mg/mL; purity not specified), with or without exogenous rat liver
S9 metabolic activation, was observed. In addition, negative findings for HCE were reported by
Weeks et al. (1979) using the S. cerevisiae D4 strain.
Bronzetti et al. (1989) evaluated HCE (purity not specified) for mitotic gene conversion
at the trp locus and reverse point mutation at the ilv locus in the S. cerevisiae D7 tester strain.
Two-hour liquid suspension exposures were conducted both on a logarithmic growth phase
culture having high levels of CYP450 metabolizing enzymes and on stationary growth phase
cultures either with or without exogenous liver S9 mix. Exposures were from 5 to 12.5 mM
(1.2-3.0 mg/mL) and were reportedly limited by solubility. HCE was inactive for both gene
conversion and reverse mutation in stationary cultures with or without S9, and for reverse
mutation in the logarithmic culture. However, statistically significant (p < 0.05-0.001) increases
in revertant frequency of more than twofold over background were observed at every
concentration (Bronzetti et al., 1989).
The ability of various halogenated hydrocarbons to induce aneuploidy in the PI diploid
strain of the mold Aspergillus nidulans has been reported (Crebelli et al., 1995, 1992, 1988).
Liquid suspension exposures (3 hours) to concentrations of 0.0025-0.04% HCE (0.005-
0.84 mg/mL; purity >98%) resulted in survival rates of 100-48%. Exposure to these
concentrations did not induce mitotic malsegregation of chromosomes.
A number of studies have evaluated the effects of in vivo and in vitro HCE exposures on
various cytogenetic endpoints in higher organisms (Crebelli et al., 1999; Tafazoli et al., 1998;
Doherty et al., 1996; Vogel andNivard, 1993; NTP, 1989; Galloway et al., 1987). Crebelli et al.
(1999) utilized the mouse bone marrow micronucleus test to investigate the in vivo induction of
micronucleated polychromatic erythrocytes (MNPCEs) by 10 aliphatic halogenated
hydrocarbons, including HCE. CD-I mice (5/sex/concentration) were injected i.p. with HCE
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doses of 2,000 or 4,000 mg/kg (purity >98%), representing approximately 40 and 70-80% of the
LDso, respectively. Animals were sacrificed and bone marrow cells were harvested at 24 and
48 hours post-treatment. At least 5,000 polychromatic erythrocytes/animal were analyzed. HCE
treatment induced clinical signs of general toxicity, but no significant increases in the frequency
of MNPCEs were noted in any treated group.
Vogel and Nivard (1993) utilized a Drosophila eye mosaic assay to monitor genetic
damage in somatic cells, predominantly interchromosomal mitotic recombination, caused by the
exposure of larvae to various chemicals. In the case of HCE (3% ethanol solvent; purity not
specified), adult flies of the C-l cross were permitted to lay eggs for 3 days on food
supplemented with 10 mM HCE. Examination for light spots in the normally colored eyes of the
resulting flies revealed what the authors classified as a weak positive response for HCE—a
reproducible increase of not more than a doubling of the spontaneous frequency at a dose
associated with toxicity. The authors suggested that the effect was unspecific and not likely
related to genotoxicity.
HCE was evaluated for its ability to induce MN and DNA damage in isolated human
lymphocytes from two donors (Tafazoli et al., 1998). Lymphocytes were exposed for 3 hours in
the presence of exogenous metabolic activation (S9 mix), or for 48 hours in the absence of S9.
Results using cells from one donor ("A") were reported for HCE (purity >99%) for exposures of
0.05-1.00 mM (0.012-0.24 mg/mL) in the presence of S9. Neither toxicity nor MN induction
was evident. Cells from the other donor ("D") were exposed to higher HCE concentrations of 1-
16 mM (a saturating concentration; 0.24-3.79 mg/mL), both with and without S9. Toxicity
(measured as a significant decrease in the relative division index) was still not observed, but
statistically positive results for percent cells with MN were recorded at HCE concentrations of
1 and 8 mM (0.24 and 1.89 mg/mL, respectively) in the absence of S9 (12 and 11%, respectively,
versus a control value of 5.5%,p < 0.05), and at 1 mM (0.24 mg/mL) in the presence of S9
(19.8% versus a control value of 9%,p < 0.01). In the second part of the study, lymphocyte
cultures exposed to test agents for 3 hours with and without S9 were assessed for DNA damage
(breaks, alkali-labile sites) using the Comet assay. HCE did not affect the measured DNA
damage parameters (tail length, fraction of total cellular DNA in the tail, and tail moment).
Doherty et al. (1996) examined in vitro induction of MN by HCE in three human cells
lines with metabolic competence; lymphoblastoid AHH-1 (native CYP1A1 activity), MCL-5
(transfected with cDNAs encoding human CYP1A2, 2A6, 3A4, 2E1, and microsomal epoxide
hydrolase), and h2El (with cDNA for human CYP2E1). Exponentially growing cultures were
exposed for approximately one cell cycle (18 hours for AHH-1, 24 hours for MCL-5 and h2El)
to 0, 0.01, 0.05, or 0.1 mM HCE (purity not specified; 0, 0.002, 0.012, or 0.024 mg/mL,
respectively), and then processed for scoring of kinetochore-positive and -negative MN. No MN
formation was observed in any of the three cell lines in response to HCE exposure. However,
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MN induction was enhanced by exposure to an HCE metabolite, PERC, in h2El and MCL-
5 cells.
Induction of CAs and SCEs in cultured Chinese hamster ovary (CHO) cells exposed to
HCE was investigated as part of an NTP screening program for genotoxicity (NTP, 1989;
Galloway et al., 1987). Concentrations for analysis were selected based on observations of cell
confluence and mitotic cell availability. HCE concentrations (purity >99%) ranged from 10 to
1,000 ug/mL (0.01-1.0 mg/mL). For both endpoints, linear regression was used to test for
dose-response trends. For individual doses, induction of CA was considered significant if
p values (adjusted by Dunnett's method to correct for multiple dose comparisons) relative to
controls were <0.05, while increases of SCEs/chromosome >20% over control values were
considered significant. For CAs, the durations of exposure were 8-10 hours in the absence of S9
metabolic activation and 2 hours in the presence of S9. For induction of SCEs, exposure
durations were 26 hours without S9 and 2 hours with S9 (followed by 24-hour incubation
without HCE). CAs were not observed in response to HCE exposure without S9. In the
presence of S9, the first study (0.15-0.50 mg/mL HCE) did not induce CAs; however, the second
study (0.20-0.40 mg/mL HCE) was judged equivocal due to a positive response at the low dose
(15.0% cells with CA versus 5.0% for the DMSO control). HCE (0.010-0.33 mg/mL) did not
induce SCE in the absence of S9; however, positive results were obtained in the presence of S9
(0.10-1.0 and 0.40-1.0 mg/mL HCE).
In vitro cell transformation studies were conducted to understand the effect of HCE in the
process of chemical carcinogenesis. In the absence of exogenous metabolic activation, a 3-day
exposure to concentrations of HCE (purity >97%) from 0.16 to 100.0 ug/mL (0.00016-
0.100 mg/mL) failed to induce morphological cell transformation in BALB/C-3T3 cells, as
measured by the incidence of Type III foci (characterized by the authors as an aggregation of
multilayered, densely stained cells that are randomly oriented and exhibit a criss-cross array at
the edge of the focus) (Milman et al., 1988; Tu et al., 1985). Milman et al. (1988) also examined
the capacity of HCE to initiate and promote tumors in a rat liver foci assay. To assess initiation
potential, 24 hours after partial hepatectomy, 10 young adult male Osborne-Mendel rats received
the MTD of HCE in corn oil by gavage. Six days later, the animals received a 0.05% dietary
exposure to the tumor promoter phenobarbital for 7 weeks. Following sacrifice, livers were
examined histopathologically for foci containing GGT, a putative preneoplastic indicator. To
assess promotion potential, animals were initiated 24 hours after partial hepatectomy with an i.p.
injection of 30 mg of the tumor initiator, diethylnitrosamine (DEN). Six days later, the animals
received the MTD of HCE in corn oil by gavage, 5 days/week for 7 weeks. The animals were
sacrificed and their livers were examined for the presence of GGT-positive foci. In these assays,
HCE failed to demonstrate any initiating activity, but did show significant (p < 0.05) promoting
I r\
capability (4.38 ± 1.04 GGT foci/cm , versus 1.77 ± 0.49 for the corn oil control).
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Yoshikawa and colleagues reported on the activity of HCE and other putative
nongenotoxic (i.e., Ames-negative) mouse hepatocarcinogens in an in vivo-in vitro hepatocyte
replicative DNA synthesis (RDS) assay (Yoshikawa, 1996; Miyagawa et al., 1995). Groups of
4-5 male B6C3Fi mice were exposed to single gavage doses of 0, 1,000, or 2,000 mg/kg HCE
(purity not specified). The hepatocytes were prepared at 24, 39, or 48 hours after exposure. The
1,000 mg/kg HCE-treated hepatocytes prepared 39 hours after exposure yielded a positive mean
RDS response of 1.21 ± 0.46% (the investigators noted that an RDS incidence rate of 0.4% for
any dose group was considered a positive response for the chemical). The remaining HCE
groups were negative with mean responses of 0.15-0.35%, while the solvent control mean was
0.26 ±0.17%.
4.5.2. In Vitro and Ex Vivo Studies Using Isolated Target Tissues/Organs or Cells
A study using a rat liver foci assay (Milman et al., 1988, Story et al., 1986) found that
HCE was a tumor promoter rather than an initiator. In vitro and in vivo assays were conducted
to assess the ability of HCE to bind to DNA, ribonucleic acid (RNA), and protein in several
mouse and rat tissues (Lattanzi et al., 1988). This study reported that binding of radiolabeled
carbon to DNA, RNA, and protein was observed following [14C]-HCE administration in both in
vitro and in vivo assays in mice and rats (Lattanzi et al., 1988), suggesting that either HCE or its
metabolites may bind to these macromolecules. The role of this binding in mediating HCE-
induced toxicity was not further evaluated.
Story et al. (1986) and Milman et al. (1988) conducted a rat liver foci assay to assess the
initiation and promotion potential of HCE, along with eight other chlorinated aliphatics. Male
Osborne-Mendel rats (10 rats/group) were given partial hepatectomies and then administered the
initiation protocol or the promotion protocol. In the initiation protocol, the rats were
administered by gavage the MTD of 2.1 mmol/kg (497 mg/kg) HCE (purity 98%), followed
6 days later with 7 weeks of phenobarbital in the diet at 0.05%. Control rats were administered
by gavage either corn oil (negative control) or 30 mg/kg DEN (positive control), followed by the
phenobarbital treatment. In the promotion protocol, rats were dosed with 30 mg/kg DEN by i.p.
injection, followed 6 days later with the MTD of 497 mg/kg HCE, 5 days/week for 7 weeks.
Phenobarbital was administered (in the same manner as HCE) as a positive control. Control rats
were either administered DEN or water, followed by corn oil for the promotion phase. Livers
were removed and stained for GGT activity. Results from the initiation protocol were negative,
with only a small number of GGT+ foci (1.0 foci/cm2 at most). However, initiation with DEN
followed by HCE or phenobarbital resulted in statistically significant increases in GGT+ foci
(Table 4-16). Absolute and relative liver weights were increased by HCE in the promotion
protocol. These results indicate that HCE is not an initiator in the rat liver foci assay, but is
capable of promotion.
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Table 4-16. Number of enzyme-altered foci in rat liver of the promotion
protocol
Promotion treatment
HCE
Phenobarbital
Corn oil
Total number of foci/cm2
+ DEN initiation
4.4 + 1.03
3.9 + 1.03
1.7+0.5
- DEN initiation
0.1+0.2
0.3+0.2
0.2 + 0.2
"Statistically different from DEN + corn oil control group, p < 0.05
Sources: Milman et al. (1988); Story et al. (1986).
Lattanzi et al. (1988) conducted in vivo and in vitro assays to assess the binding of
[14C]-HCE (specific activity 14.6 mCi/mmol, radiochemical purity 98%) to nucleic acids in
various organs from mice and rats following metabolic activation. For the in vivo studies,
6 male Wistar rats and 12 male BALB/c mice were injected i.p. with 127 uCi/kg HCE (purity
98%). The animals were fasted and sacrificed 22 hours after injection. The organs (liver,
kidney, lung, and stomach) were removed, pooled, and processed to obtain DNA, RNA, and
proteins. The in vitro studies examined microsomal and cytosolic fractions from these same
organs. The incubation mixture included 2.5 uCi [14C]-HCE, 1.5 mg calf thymus DNA or
polyribonucleotide, 2 mg microsomal proteins (plus 2 mg NADPH), and/or 6 mg of cytosolic
proteins (plus 9.2 mg glutathione [GSH]). Coenzymes were not utilized with the controls.
Measures for binding to macromolecules were determined by the presence of radiolabeled
carbon from [14C]-HCE in the DNA, RNA, and protein. The presence of radiolabeled carbon
may indicate HCE binding directly to the macromolecules or incorporation of radiolabeled
carbon from intermediate metabolites into these macromolecules.
In vivo binding data for HCE are presented in Table 4-17. Binding to macromolecules
was interpreted by the presence of radiolabeled carbon; however, HCE-specific metabolites were
not measured. In both rats and mice, binding values (in pmol HCE/mg) for RNA were
consistently much greater than those for DNA or protein. Greater binding to RNA was observed
in the kidneys of rats and mice (5-28 times greater) compared with the binding measured in the
livers, lungs, and stomachs. DNA exhibited the lowest amount of HCE binding. Species
differences were evident for all three macromolecule types (DNA, RNA, and protein) with the
mouse exhibiting much higher levels (9 times greater) of covalent binding for DNA in the liver
than the rat. The binding was 2 and 3 times greater for mice than rats with RNA and protein,
respectively, from the liver. The binding to DNA was similar between species, but slightly
greater in mice, for the kidney, lung, and stomach analyses. According to classifications
reported by Lutz (1986, 1979), the covalent binding index (CBI) values calculated on rat and
mouse liver indicate weak (rat) to moderate (mice) oncogenic potency in HCE-treated rodents.
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rl4x
Table 4-17. In vivo covalent binding of [14C]-HCE to DNA, RNA, and
proteins from rat and mouse organs
(pmol/mg)
DNA
(CBIb)
RNA
Protein
Liver3
Rat
0.43+0.05C
(15.1)b
46.59 + 7.23C
4.94 + 1.14c
Mouse
3.92 + 0.20d
(140)b
108.08 + 21.57d
14.99 + 0.83d
Kidney3
Rat
0.42
232.94
2.59
Mouse
0.50
564.98
4.91
Lung3
Rat
0.14
15.55
0.89
Mouse
0.35
60.10
3.42
Stomach3
Rat
0.26
8.33
0.80
Mouse
0.37
21.04
2.41
"Data are from pooled organs from 6 male Wistar rats or 12 male B ALB/c mice, except for liver (see indices).
bCBI calculated according to Lutz (1986, 1979), as cited in Lattanzi et al. (1988). Classification of CBI values for
oncogenic potency: strong, in the thousands; moderate, in the hundreds; weak, in the tens; and below one for
nongenotoxic oncogenes.
°Mean + SE of six individual values.
Mean + SE of four values, each obtained from three pooled livers.
Source: Lattanzi etal. (1988).
In vitro binding data for HCE are presented in Table 4-18. Liver microsomes from rats
and mice catalyzed HCE binding to DNA at comparable levels. Kidney microsomes from rats
and mice produced statistically significantly greater amounts of HCE binding to DNA when
compared with controls incubated in the absence of coenzymes. Kidney microsomes from mice
had a threefold increase in HCE binding to DNA when compared to controls, while kidney
microsomes from rats had a twofold increase in HCE binding to DNA when compared to
controls. Microsomes from lung and stomach in both species did not display increased DNA
binding activity over corresponding controls in the absence of coenzymes. Cytosolic fractions
from all organs in mice and rats exhibited higher levels of HCE binding to DNA than
microsomal fractions. Mouse liver cytosols produced much greater levels of HCE binding to
DNA than rat liver cytosols. When both microsomal and cytosolic fractions were in the
incubation mixture, HCE binding to DNA was decreased for liver and kidney. SKF 525-A, a
nonspecific CYP450 inhibitor, caused a 50.5% decrease in HCE binding to DNA (data not
included in report). Lattanzi et al. (1988) stated that addition of GSH to the microsomal fractions
also resulted in inhibition of HCE binding to DNA. When microsomal and cytosolic fractions
were heat-inactivated, HCE binding to DNA was similar to control. This study provided
evidence that HCE is metabolized by microsomal CYP450 enzymes and cytosolic GSH
transferases, and that DNA binding may be increased following HCE metabolism.
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rl4.
Table 4-18. In vitro binding of [14C]-HCE to calf thymus DNA mediated by
microsomal and/or cytosolic phenobarbital-induced fractions of rat and
mouse organs
Microsomes + NADPH
Rat
Mouse
Cytosol + GSH
Rat
Mouse
Microsomes + cytosol
(+ NADPH, + GSH)
Rat
Mouse
Liver
Standard3
Controls3
90.83 +5.31b
55.19 + 4.90
105.39+7.80b
46.96+4.19
195.51+21.44C
92.96+26.07
346.17 + 18.91b
128.56 + 8.92
95.06 +6.29C
52.85 + 12.93
133.44 + 2.42a
99.84 + 8.06
Kidney
Standard
Controls
395.84 +78.58C
136.26 + 9.04
78.86 +6.85C
39.12 + 5.34
246.85 +35.39C
88.82 + 30.91
251.42 +45.38C
81.91+9.93
247.99 + 3.40b
144.61 + 12.86
ND
ND
Lung
Standard
Controls
125.60+22.37
121.13 + 16.54
87.37+7.90
86.10 + 3.27
126.65 + 16.84b
40.23+7.34
168.52 + 19.41b
60.44+21.90
234.26 +28.35b
56.27 + 5.32
ND
ND
Stomach
Standard
Controls
94.41 + 14.38
93.20 + 15.24
47.67 + 17.00
47.12 + 11.20
289.58 + 31. 19b
130.51+4.01
228.74 + 20.42b
51.52 + 6.20
76.79 + 5.34b
44.77 + 2.28
ND
ND
"Data (total DNA binding in pmol/mg) are reported as mean + SE of three values; ND, not determined. Controls
were conducted in the absence of coenzymes.
bStatistically different from control, p < 0.01.
°Statistically different from control, p < 0.05.
Source: Lattanzi et al. (1988).
4.5.3. Structure Activity Relationships
Several studies were conducted with the objective of defining structure activity
relationships (SARs) of halogenated hydrocarbons and toxicity. NTP (1996) defined a group of
chlorinated ethanes that resulted in hyaline droplet nephropathy in male F344/N rats and a group
of halogenated ethanes that resulted in renal toxicity in the absence of hyaline droplet
nephropathy. In a series of studies, Crebelli et al. (1995, 1992, 1988) evaluated chlorinated and
halogenated hydrocarbons for their ability to induce chromosome malsegregation, lethality, and
mitotic growth arrest in the mold A. nidulans.
NTP (1996) conducted a 21-day oral toxicity study with halogenated ethanes in male
F344/N rats. Chemicals under investigation were 1,1,1,2-tetrachloroethane, 1,1,2,2-tetrachloro-
ethane, pentachloroethane, 1,1,2,2-tetrachloro-1,2-difluoroethane, 1,1,1 -trichloro-2,2,2-trifluoro-
ethane, l,2-dichloro-l,l-difluoroethane, 1,1,1-trichloroethane, 1,1,1,2-tetrabromoethane,
1,1,2,2-tetrabromoethane, pentabromoethane, and HCE (purity >98%). Groups of five male
rats/dose were administered 0.62 or 1.24 mmol/kg-day of the halogenated ethane (for HCE,
146 and 293 mg/kg-day, respectively). Increased kidney weights and evidence of renal toxicity
were observed in many of the rats administered halogenated ethanes; however, this was not
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always coincident with hyaline droplet nephropathy. Hyaline droplet nephropathy (assessed by
Mallory-Heidenhain staining, which allows for greater sensitivity in evaluating hyaline droplets
within the tubules of the kidney) was only observed in rats administered pentachloroethane,
1,1,1,2-tetrachloroethane, and HCE. RDS, indicated by PCNA labeling index, was increased in
male rats administered chemicals that induced hyaline droplet nephropathy (pentachloroethane,
1,1,1,2-tetrachloroethane, and HCE) as well as pentabromoethane and 1,1,2,2-tetrachloroethane,
compared with control rats. The increase in cell proliferation in the kidneys (as measured by the
PCNA labeling index) observed with some of the halogenated ethanes that did not induce hyaline
droplet nephropathy suggests the contribution of another toxic mechanism. NTP (1996)
concluded that the capacity to induce hyaline droplet nephropathy in male rats was restricted to
ethanes with four or more halogens, and only the chlorinated (compared with the fluorinated and
brominated) ethanes were active. This study also predicted that if hyaline droplet nephropathy is
the determining factor in the induction of renal tubule cell neoplasia, then chemicals such as
bromo- or chlorofluoroethanes would be negative for kidney neoplasia in 2-year cancer
bioassays of male rats.
Crebelli et al. (1988) evaluated three chloromethanes and eight chlorinated ethanes
(including HCE) for the induction of chromosome malsegregation in A. nidulans. Although 8 of
the 11 compounds tested provided positive results including the 3 chloromethanes and 5 out of
8 chlorinated ethanes, HCE was negative for chromosome malsegregation induction. Analyses
of relationships between biological and chemical variables indicate that the ability of a chemical
to induce chromosome malsegregation was not related to any of the chemical descriptors
examined, including molecular weight, melting point, boiling point, refractive index,
octanol/water partition coefficient, and the free energy of binding to biological receptors.
Because of the similarity of the chemical descriptors between the positive chlorinated ethanes,
aside from 1,1,1-trichloroethane which was negative, the authors argue against a previous
hypothesis that nonspecific interactions with hydrophobic cellular structures is the mechanism of
aneuploidy induction (Onfelt, 1987).
Crebelli et al. (1992) evaluated the ability of 24 chlorinated aliphatic hydrocarbons to
induce chromosome malsegregation, lethality, and mitotic growth arrest in the mold, A. nidulans.
Data were combined with previous data on 11 related compounds (Crebelli et al., 1988) to
generate a database for quantitative structure-activity relationship (QSAR) analysis. Physico-
chemical descriptors and electronic parameters for each chemical were included in the analysis.
Out of the 24 chemicals, 19 were negative for the induction of chromosome malsegregation;
5 chemicals produced reproducible increases in the frequency of euploid whole chromosome
segregants. HCE was negative for the induction of chromosome malsegregation. QSAR
analyses on these 35 chlorinated aliphatic hydrocarbons indicate that toxicity, such as the
induction of lethality, is primarily related to steric factors (the spatial orientation of reactive
centers within a molecule) and measures of the volume occupied by an atom or functional group
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(molar refract!vity). Measures of molar refractivity are a function of temperature, index of
refraction, and atmospheric pressure. Mitotic growth arrest was also primarily related to molar
refractivity. However, aneugenic activity was related to both molar refractivity and electronic
factors, such as the ease in accepting electrons (described by density and the energy of the lowest
unoccupied molecular orbital).
These QSAR studies (Crebelli et al., 1992, 1988) were expanded to include 20 additional
halogenated hydrocarbons (Crebelli et al., 1995). Chemicals in this study were also assayed for
lipid peroxidation in rat liver microsomes, and the authors reported that a partial coincidence was
found between the ability of a chemical to initiate lipid peroxidation and to disturb chromosome
segregation at mitosis. This updated study concluded that electronic and structural parameters
that determine the ease of homolitic cleavage of the carbon-halogen bond play a primary role in
the peroxidative properties of haloalkanes.
4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS
4.6.1. Oral
Table 4-19 summarizes the oral toxicity studies that have been reported in laboratory
animals. The primary noncancer effects observed in these studies include decreased body weight
or body weight gain, increased absolute and relative kidney weights, increased absolute and
relative liver weights, various effects associated with renal tubule toxicity in the kidney, and
hepatocellular necrosis. Developmental studies in rats did not consistently demonstrate fetal
effects, especially in those cases where maternal toxicity was absent.
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Table 4-19. Oral toxicity studies for HCE
Species
New Zealand
White Rabbits,
Male, (5/dose)
F344/N rats
(5/sex/dose)
F344/N rats.
Male, (5/dose)
F344/N rats
(10/sex/dose)
F344 rats
(10/sex/dose)
Osborne-
Mendel rats
(50/sex/dose)
B6C3FJ mice
(50/sex/dose)
F344/N rats
(50/sex/dose)
Pregnant
Sprague-Dawley
rats (22/dose)
Pregnant Wistar
rats (2 I/dose)
Dose
(mg/kg-d)/
duration
0, 100, 320 or
1,000 by oral;
12 d
0, 140, 281, 563,
1,125, or 2,250
by gavage; 16 d
0, 146, or 293 by
gavage; 21 d
0, 34, 67, 134,
268, or 536 by
gavage; 13 wks
0, 1, 15, or 62 by
diet; 16 wks
0, 113, or 227 by
gavage; 78 wks
0, 360, or 722 by
gavage; 78 wks
Male: 0, 7, or 14
Female: 0, 57, or
114 by gavage;
103 wks
0, 50, 100, or 500
by gavage on
CDs 6-16
0, 56, 167, or 500
by gavage on
CDs 7-17
NOAEL
(mg/kg-d)
100
Male: not
established
Female: 281
Not established
Male: not
established
Female: 67
Male: 1
Female: 15
Not established
Not established
Not established
Maternal: 100
Maternal: 56
Developmental:
167
LOAEL
(mg/kg-d)
320
Male: 140
Female: 563
146
Male: 34
Female: 134
Male: 15
Female: 62
113
360
Male: 7
Female: 57
Maternal: 500
Maternal: 167
Developmental:
500
Effect
Increased liver and kidney
weights; liver degeneration
and necrosis; tubular
nephrosis and
nephrocalcinosis
Male: kidney effects (hyaline
droplets, tubular cell
regeneration, granular casts)
Female: decreased body
weight
Increased kidney weight,
nephropathy (hyaline
droplets, tubule regeneration,
granular casts); effects on
urinalysis parameters
Male: decreased organ
weights, kidney effects in all
dose groups
Female: decreased organ
weights, hepatocellular
necrosis
Male: kidney atrophy,
proximal tubule degeneration
Female: proximal tubule
degeneration at highest dose
Tubular nephropathy in both
sexes
Tubular nephropathy in both
sexes
Male: tubular nephropathy;
renal tubular hyperplasia
Female: tubular nephropathy
Maternal: body weight
decreased; increased mucus
in nasal turbinates;
subclinical pneumonitis
Fetal: no effects
Maternal: decreased weight
gain and motor activity
Fetal: reduced body weight
increased incidence of
skeletal variations; decreased
ossification
Reference
Weeks et al.
(1979)
NTP (1989)
NTP (1996)
NTP (1989)
Gorzinski et
al. (1985)
NCI (1978);
Weisburger
(1977)
NCI (1978);
Weisburger
(1977)
NTP (1989)
Weeks et al.
(1979)
Shimizu et al.
(1992)
Acute and short-term toxicity tests in animals reported liver necrosis and tubular
nephrosis in male rabbits (Kinkead and Wolfe, 1992; Weeks et al., 1979; Weeks and Thomasino,
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1978), and evidence of kidney effects such as nephropathy with hyaline droplet formation and
tubular cell regeneration in male rats (NTP, 1996, 1989). Female rats in short-term toxicity tests
displayed only decreased body weights at the LOAEL of 563 mg/kg-day with a NOAEL of 281
mg/kg-day (NTP, 1989). Oral LD50 values in rats ranged from 4,460 to 7,690 mg/kg (Weeks et
al., 1979).
4.6.1.1. Nephrotoxicity
Two short-term studies in F344 rats (NTP, 1996; 1989; 21- and 16-day studies,
respectively) reported nephrotoxic effects at all administered doses in male rats. The formation
of hyaline droplets accompanied by cell regeneration and eosinophilic granular casts was
observed in the renal tubules of male rats administered 140-563 mg/kg-day HCE (NTP, 1989).
Female rats did not exhibit any renal toxicity. In the 21-day study by NTP (1996), male rats
exhibited increased absolute and relative kidney weights, tubular regeneration and granular casts,
and increased labeling index in kidneys at doses of 146 and 293 mg/kg-day HCE. Tubular
nephrosis, and to a minimal degree, tubular nephrocalcinosis were observed in the kidney of
male New Zealand White rabbits administered 320 and 1,000 mg/kg-day (but not 100 mg/kg-
day) HCE (Weeks et al., 1979). Compared with rabbits, the rats were more sensitive to renal
effects induced by HCE. A gender-specific response was demonstrated in the male rats (NTP,
1989). However, the use of only male rats (NTP, 1996) and male rabbits (Weeks et al., 1979) in
the other two studies makes it difficult to evaluate if the observed renal effects were gender-
specific.
Subchronic exposure (13 weeks) resulted in kidney effects including hyaline droplet
formation, tubular regeneration, and tubular casts in male F344/N rats administered HCE doses
of 34-536 mg/kg-day (NTP, 1989). Males in the 536 mg/kg-day dose group also exhibited renal
papillary necrosis and degeneration and necrosis of renal tubule epithelium. Female rats did not
display these kidney effects. These results suggest a sex-specific difference in HCE toxicity.
Another study (Gorzinski et al., 1985) in F344 rats reported slight hypertrophy and dilation of
the renal tubules in males and renal tubule atrophy and degeneration in male and female rats.
Evidence of kidney effects in female rats consisted of very slight renal tubular atrophy and
degeneration observed histopathologically at the highest dose tested. EPA considered the
NOAEL and LOAEL for male rats as 1 and 15 mg/kg-day, respectively, while the corresponding
values in the females were 15 and 62 mg/kg-day, indicating greater sensitivity of the males to the
renal effects of HCE. These data and tissue distribution information (see Section 3.2
Distribution) show that the male kidney accumulated higher HCE concentrations than the female
kidney, indicating that the kidney is the primary target organ following oral exposure to HCE
and that there are potential gender differences in the distribution and metabolism of HCE.
Consequently, male rats are likely more sensitive to the nephrotoxicity of HCE than female rats.
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Additionally, Gorzinski et al. (1985) is the only study of either short-term or subchronic duration
to report renal effects in female rats.
Chronic toxicity tests were conducted by NTP on F344/N rats and by NCI on Osborne-
Mendel rats and B6C3Fi mice (NTP, 1989; NCI, 1978). NTP (1989) administered much lower
doses of HCE (7 and 14 mg/kg-day in males; 57 and 114 mg/kg-day in females) to the F344 rats
compared with the Osborne-Mendel rats (113 and 227 mg/kg-day) in the NCI (1978) study. In
the NTP (1989) chronic study, nephropathy (characterized as tubular cell degeneration and
regeneration, dilation and atrophy, glomerulosclerosis, interstitial fibrosis, and chronic
inflammation) was observed in both male and female rats. In the case of the male rats, the
response was roughly equivalent across the control and treated groups, with nephropathy in more
than 94% of animals. The high incidence of nephropathy observed in control rats was likely a
result of a spontaneous syndrome known as chronic progressive nephropathy (CPN) that is
associated with aged rats, especially F344 and Osborne-Mendel strains (see Section 4.7.3.2.1 for
additional discussion). To examine the effects of chronic HCE exposure separate from CPN, the
nephropathy incidence in terms of severity was evaluated. The severity was increased in the
treated male rats compared with the controls. In considering severity, the increases in incidences
of nephropathy in males (that were of moderate or marked severity) were 18/50 (36%), 24/50
(48%), and 30/50 (60%) in the control, 7, and 14 mg/kg-day dose groups, respectively. In
females, both the incidence (44% of controls and approximately 84% of treated) and severity of
nephropathy were dose-related. When considering the severity, incidences of nephropathy in
females (that were of mild or moderate severity) were 12/50 (24%), 25/50 (50%), and 32/50
(64%) in the control, 57, and 114 mg/kg-day dose groups, respectively.
Dose-related increases (30 and 64% at 7 and 14 mg/kg-day, respectively) in linear
mineralization of the renal papillae and treatment-related increases (14% at 7 and 14 mg/kg-day)
in hyperplasia of pelvic transitional epithelium in the kidney were observed in the male rats. In
females, an increased incidence of mineralization was only noted at the low dose (44% at
57 mg/kg-day compared with 28% in controls). The low dose for the females was 8 times
greater than that for the males, yet the signs of nephropathy were more severe in the males.
In the NCI (1978) study, Osborne-Mendel rats of both sexes displayed chronic
inflammatory kidney lesions in both control and treated groups, although tubular nephropathy
(characterized by degeneration, necrosis, and the presence of large hyperchromatic regenerative
epithelial cells) was observed only in the HCE-exposed male and female rats. There were
dose-related increases in incidences of nephropathy in males (45 and 66%, respectively) and
females (15 and 59%, respectively) administered 113 and 227 mg/kg-day HCE. The chronic
toxicity test in B6C3Fi mice (NCI, 1978) is the only study conducted in this species. Male mice
experienced low survival in the control and 360 mg/kg-day (low-dose) groups. Chronic kidney
inflammation was observed in 67 and 80% of males in the vehicle and untreated control groups,
respectively, as well as in 66 and 18% of the 360 and 722 mg/kg-day HCE males, respectively.
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The report did not provide an explanation for the large response in the control and low-dose mice
and the relatively small response in the high-dose group. Female mice exhibited chronic kidney
inflammation only in vehicle controls (15%) and the high-dose group (2%). Tubular
nephropathy was observed in both dose groups of both sexes at high incidences (92-100%), and
was characterized by degeneration of convoluted tubule epithelium with some hyaline casts.
Enlarged dark staining regenerative tubular epithelium was also observed, with the kidney
exhibiting infiltration of inflammatory cells, fibrosis, and calcium deposition. The response in
the treated male and female mice compared with the absence of nephropathy in the controls
suggests that the doses used in this study were too high.
The available information for HCE-induced nephropathy in rats, mice, male rabbits, and
sheep indicates that the male rat is the most sensitive sex/species to the renal toxicity of HCE.
Limited, if any, information is available for species other than the rat; however, the doses that
elicited toxic responses in mice (NCI, 1978), male rabbits (Weeks et al., 1979), and sheep
(Fowler, 1969) were at least 45-fold greater than the lowest dose (7 mg/kg-day; NTP, 1989) that
induced a statistically significant response in rats.
4.6.1.2. Hepatotoxicity
Short-term studies in rats (NTP, 1996), male rabbits (Weeks et al., 1979), and sheep
(Fowler, 1969) reported hepatotoxicity at doses approaching >300 mg/kg-day. Male F344 rats
exhibited significantly increased relative liver weights at the highest dose of 293 mg/kg-day.
AST and NAG serum activities were also significantly higher than in controls. These effects
were not observed at 146 mg/kg-day HCE (NTP, 1996). Liver degeneration and necrosis,
including fatty degeneration, coagulation necrosis, hemorrhage, ballooning degeneration,
eosinophilic changes, and hemosiderin-laden macrophages and giant cells were observed in male
New Zealand White rabbits administered 320 and 1,000 mg/kg-day HCE (but not
100 mg/kg-day), increasing in severity with increasing dose. Sheep given single oral doses of
500-1,000 mg/kg of HCE exhibited plasma levels of GDH, SDH, and OCT that were increased
twofold or more than levels in controls, indicating reduced hepatic function.
Effects in the liver of animals treated with HCE were observed in male and female rats in
two subchronic studies (NTP, 1989; Gorzinski et al., 1985). Liver weight increased in a
dose-related fashion from the lowest dose (34 mg/kg-day) to the highest (536 mg/kg-day).
Females were more sensitive than males; severity and statistical significance increased in
females at doses lower than those eliciting toxicity in male rats. Hepatocellular necrosis was
noted in females at doses ranging from 134 to 156 mg/kg-day and in males at the two highest
doses, 268 and 536 mg/kg-day (NTP, 1989). Gorzinski et al. (1985) reported slight swelling of
hepatocytes in control and treated males, although there were dose-related increases in
incidences of swelling at the two highest doses (15 and 62 mg/kg-day). Other than a statistically
significant increase (5%) in liver weight at 62 mg/kg-day HCE, the females were not affected.
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This is in contrast to the hepatocellular effects noted in female rats in the NTP study (NTP,
1989). However, the highest dose used by Gorzinski et al. (1985), 62 mg/kg-day, is below the
67 mg/kg-day NOAEL for females of the NTP (1989) study, indicating that sufficient doses may
not have been reached in the Gorzinski et al. (1985) study to cause hepatotoxicity in female rats.
There were no liver effects observed in the animals administered HCE for chronic
durations. The range of doses in the subchronic assay (0, 34, 67, 134, 268, and 536 mg/kg-day
on F344 rats; NTP, 1989) encompassed the doses used in the chronic assays for female F344 rats
(57 and 114 mg/kg-day; NTP, 1989) and Osborne-Mendel rats (113 and 227 mg/kg-day; NCI,
1978). Hepatocellular necrosis was observed in female rats in the subchronic study, but not the
chronic study. The LOAEL for female F344/N rat hepatocellular necrosis, 134 mg/kg-day, in
the subchronic study (NTP, 1989) occurred at a dose that exceeded the highest dose of the
chronic study (NTP, 1989), suggesting that a sufficiently high dose may have not been achieved
to elicit hepatocellular necrosis despite the longer exposure period. The NCI (1978) study in
Osborne-Mendel rats was conducted with doses above the LOAEL for hepatocellular necrosis in
female F344/N rats (NTP, 1989), but hepatocellular effects were not observed. Osborne-Mendel
rats may not be as sensitive to HCE-induced hepatotoxicity as F344/N rats. The only study in
mice (NCI, 1978; chronic) did not report any hepatotoxic effects other than the development of
hepatocellular tumors.
HCE-induced liver effects were only observed in animals in short-term and subchronic
studies. Female rats exhibited a greater sensitivity to liver effects as evidenced by the effects
observed at lower doses compared with males (NTP, 1989). The implications of the slight
swelling of hepatocytes in the absence of other histopathological effects at 15 and 62 mg/kg-day
in male rats (Gorzinski et al., 1985) are unknown. Rabbits (males) and sheep demonstrated
hepatic effects at doses at least fourfold greater than the lowest dose (67 mg/kg-day) that induced
a statistically significant response in female rats.
4.6.1.3. Developmental Toxicity
Two developmental studies in rats indicated that HCE induced teratogenicity in the
presence of maternal toxicity (Shimizu et al., 1992; Weeks et al., 1979). In the Shimizu et al.
(1992) study, maternal rats gavaged with 167 and 500 mg/kg HCE displayed decreased motor
activity. At the high dose, dams also exhibited piloerection and subcutaneous hemorrhage.
Fetuses of the 500 mg/kg dose displayed decreased body weight, skeletal variations such as
rudimentary lumbar ribs, and ossification effects, but no skeletal malformations were observed.
The NOAEL for this study was 56 mg/kg for the dams and 167 mg/kg for the fetuses. In Weeks
et al. (1979), maternal rats gavaged with 500 mg/kg HCE displayed pulmonary effects such as
increased incidence of mucopurulent nasal exudates, upper respiratory tract irritation, and
subclinical pneumonitis. The fetuses did not exhibit any skeletal or soft tissue anomalies. The
maternal LOAEL and NOAEL were 500 and 100 mg/kg, respectively.
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4.6.2. Inhalation
Inhalation toxicity has only been evaluated in a single 6-week repeated exposure study in
multiple species performed by Weeks et al. (1979). There is some uncertainty regarding the
exposure to HCE vapor because HCE would remain a vapor only when surrounded by heated air.
However, as soon as the hot HCE vapor was mixed with room temperature air, most (but not all)
vapor in the airstream would condense into fine particles (a solid aerosol). The data from this
study are summarized in Table 4-20. The study authors reported NOAELs and LOAELs for
Beagle dogs, guinea pigs, and rats of 48 ppm (465 mg/m3) and 260 ppm (2,517 mg/m3),
respectively. Neurological effects, such as tremors and ataxia, were observed in Beagle dogs and
in pregnant and nonpregnant Sprague-Dawley rats. Rats and guinea pigs exhibited reduced body
weight gain and increased relative liver weight. Male rats also displayed increased relative
spleen and testes weights. Behavioral tests were conducted in male Sprague-Dawley rats at the
same exposure concentrations, and no significant effects were observed. Overall, the
information on the inhalation toxicity of HCE is limited.
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Table 4-20. Inhalation toxicity studies with HCE
Species
Male Beagle dogs
(4/concentration)
Male Hartley guinea
pigs (10/concentration)
Sprague-Dawley rats
(25/sex/concentration)
C. Japonica (Japanese
quail)
(20/concentration)
Pregnant Sprague-
Dawley rats
(22/concentration)
Male Sprague-Dawley
rats (15/concentration)
Concentration
(mg/m3)/durationa
0, 145, 465, or
2,517;6wks
0, 145, 465, or
2,517;6wks
0, 145, 465, or
2,517;6wks
0, 145, 465, or
2,517;6wks
0, 145, 465, or
2,5 17; CDs 6-16
0, 145, 465, or
2,517;6wks
NOAEL
(mg/m3)
465
465
465
2,517
Maternal: 465
Developmental:
2,517
465
LOAEL
(mg/m3)
2,517
2,517
2,517
Not established
Maternal: 2,5 17
Developmental:
Not established
2,517
Effect
Tremors, ataxia,
hypersalivation,
head bobbing, facial
muscular
fasciculations
Reduced body
weight, increased
relative liver weight
Males: reduced
body weight gain,
increased relative
kidney, spleen, and
testes weights
Females: increased
relative liver weight
No effects
Maternal: tremors'3,
decreased body
weight
Fetal: no effects
Behavioral tests:
avoidance latency
and spontaneous
motor activity
Reference
Weeks et al.
(1979)
Weeks et al.
(1979)
Weeks et al.
(1979)
Weeks et al.
(1979)
Weeks et al.
(1979)
Weeks et al.
(1979)
a!45, 465, and 2,517 mg/m3 correspond to concentrations reported by Weeks et al. (1979) as 15, 48, and 260 ppm,
respectively
Incidence data on tremors was not reported by the study authors
4.6.3. Mode-of-Action Information
Reports on HCE-induced human health effects are limited and confounded by co-
exposure to multiple solvents or other toxicants (e.g., HCE-zinc oxide smoke). Studies observed
substantial HCE exposure in smoke bomb production workers, but the sample sizes were too
small to provide definitive conclusions on health effects.
Animal studies suggest that HCE is primarily metabolized to PERC and
pentachloroethane by CYP450 enzymes of the liver, with likely subsequent metabolism to TCE.
Metabolites identified in the urine include TCA, trichloroethanol, oxalic acid, dichloroethanol,
dichloroacetic acid, and monochloroacetic acid. However, only 5% of a radiolabeled compound
was measured in the urine, indicating that all of the urinary metabolites account for a small
percentage of the dose. It is unknown whether unchanged HCE or its metabolites are responsible
for the liver and kidney toxicities observed in animal studies. Only one study attempted to assess
the extent of HCE metabolism in rats and mice and estimated that 24-29% of administered HCE
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is metabolized (Mitoma et al., 1985). This study did not quantify actual metabolite
concentrations, so these estimations are of questionable accuracy.
The mode of action for HCE-induced kidney toxicity is unknown. HCE-induced
nephropathy has been observed in both sexes of rats and mice. Specifically, short-term assays in
male rats showed nephropathy characterized by hyaline droplet accumulation and increased
incidences of tubule regeneration and granular casts (NTP, 1996, 1989). Cell proliferation of
kidney sections using PCNA labeling analysis was also increased (NTP, 1996). Subchronic and
chronic animal bioassays confirmed these renal effects (NTP, 1989; Gorzinski et al., 1985; NCI,
1978). Chronic inflammatory kidney lesions and tubular nephropathy were observed in rats, and
tubular nephropathy was also observed in mice (NCI, 1978).
Some data suggest that an a2U-globulin mode of action could contribute to HCE-induced
nephropathy. However, there is insufficient evidence to conclude that the kidney effects
observed following HCE exposure (NTP, 1989) are related to an a2U-globulin mode of action for
the following reasons: (1) the lack of a2U-globulin immunohistochemical data for HCE-induced
nephrotoxicity and carcinogenicity, (2) the hyaline droplet accumulation is caused by excessive
protein load that may not be exclusively related to a2U-globulin accumulation, and (3) the
existence of renal toxicity in female rats and male and female mice indicates that the nephrotoxic
effects are not limited to an a2U-globulin-induced sequence of lesions.
It is also possible that advanced CPN, an age-related renal disease of laboratory rodents
that occurs spontaneously, may contribute to the observed nephrotoxicity following HCE
exposure. However, changes in the severity of the nephropathy were observed to be greater in
male rats exposed to HCE compared with controls, indicating that HCE exposure exacerbated
effects in the kidney. Additionally, HCE-exposed male rats demonstrated dose-dependent
increases in incidences of mineralization of the renal papillae and hyperplasia of pelvic
transitional epithelium. Neither of these effects increased in a dose-related manner in the
controls or the HCE-exposed female rats, suggesting that CPN is not solely responsible for the
nephropathy observed by NTP (1989).
The liver has been demonstrated to be a target organ in several animal species. Sheep
(Fowler, 1969) and male rabbits (Weeks et al., 1979) exhibited hepatotoxicity characterized by
clinical chemistry parameters that indicated impaired hepatic function and showed
histopathological findings including hepatocellular necrosis. Subchronic studies showed
statistically significant decreases in relative and absolute liver weight (Gorzinski et al., 1985) and
statistically significant increases in relative liver weight and hepatocellular necrosis (NTP, 1989)
in female F344/N rats. Studies of TCA (a potential metabolite of HCE) indicate that free radical
generation may play a role in mediating toxicity, particularly in the liver. However, no data are
available demonstrating generation of free radicals following exposure to HCE, and it is
unknown whether unchanged HCE or its metabolites are responsible for the liver and kidney
toxicities observed in animal studies. Town and Leibman (1984) reported lipid peroxidation (as
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indicated by a statistically significant increase in the formation of malondialdehyde and
conjugated dienes) following treatment with HCE (8 mM). The authors suggested the
involvement of a free radical. However, this mode of action has not been explored or further
addressed in the literature for HCE.
The presence of radiolabeled carbon measured by in vivo binding studies suggested that
HCE can bind to DNA, RNA, and protein (Lattanzi et al., 1988). Binding to macromolecules
was interpreted by the presence of radiolabeled carbon; however, radiolabeled carbon may have
been incorporated into these macromolecules from intermediary HCE metabolites. In the rat,
higher levels of DNA, RNA, and protein binding were observed in the kidney and liver
compared with the lung and stomach. The mouse demonstrated the highest levels of DNA and
protein binding in the liver and RNA binding in the liver and kidney. Studies using CYP450
indicate that HCE must be metabolized to reactive intermediates prior to binding to
macromolecules. Therefore, renal toxicity and hepatotoxicity may also involve HCE binding to
DNA, RNA, or protein, resulting in cytotoxicity and contributing to the cytotoxic damage from
radicals.
The neurological effects observed in Beagle dogs (Weeks et al., 1979) and sheep (Fowler,
1969; Southcott, 1951) are commonly observed effects of chlorinated hydrocarbons. These
effects have not been extensively studied for HCE, and data are inadequate to determine a mode
of action.
4.7. EVALUATION OF CARCINOGENICITY
4.7.1. Summary of Overall Weight of Evidence
Under EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), HCE is
"likely to be carcinogenic to humans" based on evidence of carcinogenicity from two chronic
bioassays in F344/N rats and B6C3Fi mice (NTP, 1989; NCI, 1978). Tumors that have been
observed include renal adenomas and carcinomas and pheochromocytomas/malignant
pheochromocytomas in male F344/N rats (NTP, 1989), and hepatocellular carcinomas in male
and female B6C3Fi mice (NCI, 1978). Human data are not available to assess the carcinogenic
potential of HCE.
NTP (1989) reported dose-dependent increases (statistically significant at the high dose)
in the combined incidence of renal adenomas or carcinomas and increases (statistically
significant at the low dose) in the incidence of pheochromocytomas in male F344/N rats. For
male rat kidney tumors, when the mode-of-action evidence demonstrates that the response is
secondary to a2U-globulin accumulation, the tumor data are not used in the cancer assessment
(U.S. EPA, 1991b). Immunohistochemical evidence does not exist to conclude that the renal
adenomas and carcinomas observed in male rats administered HCE (NTP, 1989) are related to an
a2u-globulin mode of action (see Section 4.7.3.1 and Section 5.4.5.1); therefore, the renal
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adenomas and carcinomas observed in male rats administered HCE (NTP, 1989) were
considered relevant to humans. The relevance of rodent pheochromocytomas as a model for
human cancer risk has been the subject of discussion in the scientific literature (e.g., Greim et al.,
2009; Powers et al., 2008). Although more common in laboratory rats, evidence suggests that rat
pheochromocytomas may have similarity to human pheochromocytomas and that they may be
produced by the same mechanism of action. Therefore, adrenal gland tumors in rodents are
considered relevant to humans.
In addition, NCI (1978) observed statistically significant increases in the incidence of
hepatocellular carcinomas in male and female B6C3Fi mice. The male mice demonstrated a
statistically significantly increased tumor response for hepatocellular carcinomas that was dose-
related. The female mice displayed a statistically significantly elevated incidence of
hepatocellular carcinomas at both doses, although no dose-related increase in tumor response
was evident.
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. An exception occurs when there is convincing information (e.g., toxicokinetic
data) that absorption does not occur by other routes. Information available on the carcinogenic
effects of HCE via the oral route demonstrates that tumors occur in tissues remote from the site
of absorption. Information on the carcinogenic effects of HCE via the inhalation and dermal
routes in humans or animals is absent. Based on the observance of systemic tumors following
oral exposure, and in the absence of information to indicate otherwise, it is assumed that an
internal dose will be achieved regardless of the route of exposure. Therefore, HCE is "likely to
be carcinogenic to humans" by all routes of exposure.
4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence
There are currently no data from human studies pertaining to the carcinogen! city of HCE.
NTP (1989) conducted a chronic toxicity/carcinogenicity bioassay in F344/N rats. Groups of
50 male rats/dose were administered TWA doses of 7 and 14 mg/kg-day of HCE (purity >99%)
by corn oil gavage, 5 days/week for 103 weeks. Groups of 50 female rats/dose were
administered, by corn oil gavage, 5 days/week for 103 weeks, TWA doses of 57 and
114 mg/kg-day. Male rats exhibited a dose-related, statistically significant increase in the
incidence of combined renal adenomas or carcinomas at the highest dose. Combined renal
adenomas or carcinomas were observed in 2, 4, and 14% of controls, 7, and 14 mg/kg-day males,
respectively. No HCE-related renal tumors were observed in female rats.
Combinedpheochromocytomas (benign, malignant, and complex pheochromocytomas) were
observed in 62 and 43 % of males treated with 7 mg/kg-day and 14 mg/kg-day HCE,
respectively, when compared with vehicle controls (30%) and historical controls in the study
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laboratory (75/300; 25 ± 7%) and in NTP studies (543/1,937; 28 ± 11%). Only the mid-dose
increases were statistically significant. No HCE-related adrenal gland tumors were observed in
female rats.
NCI (1978; Weisburger, 1977) conducted a chronic toxicity/carcinogenicity bioassay in
Osborne-Mendel rats. HCE (purity >98%) at doses of 0, 250, or 500 mg/kg-day was
administered by corn oil gavage to 50 rats/sex/dose for 5 days/week for 78 weeks. Following
termination of exposure, rats were observed for 33-34 weeks for a total duration of 111-
112 weeks. Twenty rats/sex were used for the untreated and vehicle controls. Starting in
week 23, rats in the exposure groups began a 5-week cyclic rotation that involved 1 week
without exposure followed by dosing for 4 weeks. After adjustment from 5 days/week for
78 weeks, with the 5-week cyclic rotation for part of the time, to continuous exposure over the
standard 2 years for a chronic bioassay, the TWA doses were 113 and 227 mg/kg-day. Mortality
was increased in the 113 and 227 mg/kg-day males with survival rates of 24/50 (48%) and
19/50 (38%), respectively, compared with 14/20 (70%) in the untreated controls. Survival rates
for the female rats were 14/20 (70%) for both the untreated and vehicle controls, and
27/50 (54%) and 24/50 (48%) for the 113 and 227 mg/kg-day dose groups, respectively.
All of the tumor types observed had been encountered previously as spontaneous lesions
in the Osborne-Mendel rat, and no statistical differences in frequencies were observed between
treated and control rats. NCI concluded that there was no evidence of carcinogen!city in this rat
study. Notably, the doses used in the Osborne-Mendel rats of the NCI (1978) study were
approximately 16 times greater than those doses administered to F344 male rats by NTP (1989).
In a B6C3Fi mouse study conducted by NCI (1978; Weisburger, 1977), HCE (purity
>98%) was administered by corn oil gavage at TWA doses of 360 and 722 mg/kg-day for
5 days/week for 78 weeks, followed by 12-13 weeks of an observation period (total 91 weeks).
Survival rates in males were 5/20 (25%), 1/20 (5%), 7/50 (14%), and 29/50 (58%) in the vehicle
control, untreated control, and 360 and 722 mg/kg-day dose groups, respectively. Survival rates
in females were 80, 85, 80, and 68% in vehicle control, untreated control, 360 and 722 mg/kg-
day groups, respectively. Both male and female mice exhibited statistically significantly
increased incidences of hepatocellular carcinomas. The treated males demonstrated an increased
tumor response for hepatocellular carcinomas that was dose-related: 30 and 63% in the 360 and
722 mg/kg-day dose groups, respectively, compared with 10% in pooled vehicle controls and
15% in matched vehicle controls. Females demonstrated an increased tumor response that was
not dose related in that a higher incidence of hepatocellular carcinomas occurred at the low dose
(40%) compared with the high dose (31%); pooled vehicle and matched vehicle controls had
incidences of 3 and 10%, respectively. NCI concluded that HCE was carcinogenic in both sexes
of B6C3Fi mice.
Evidence of HCE's promotion (following treatment with DEN), but not initiation,
potential was observed in the liver of male Osborne-Mendel rats administered a single gavage
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dose of 497 mg/kg HCE (Milman et al., 1988; Story et al., 1986). Lattanzi et al. (1988) reported
in vivo and in vitro binding of HCE to DNA, RNA, and protein in mice and rats. In both rats and
mice administered single i.p. injections of 127 uCi/kg [14C]-HCE, in vivo covalent binding of
HCE for RNA was consistently much greater than that for DNA or protein. DNA exhibited the
lowest amount of HCE binding. Species differences were evident for all three macromolecule
types (DNA, RNA, and protein), with the mouse exhibiting much higher levels (9 times greater)
of covalent binding for DNA in the liver than the rat. The binding was 2 and 3 times greater for
mice than rats with RNA and protein, respectively, from the liver. The binding was similar
between species, but slightly greater in mice, for the kidney, lung, and stomach analyses. In
vitro covalent binding to DNA was observed at comparable levels in liver microsomes from both
rats and mice following exposure to HCE. Kidney microsomes from rats and mice produced
statistically significantly greater amounts of DNA binding compared with controls, with greater
amounts of DNA binding from mice (threefold increase) compared with rats (twofold increase).
Microsomes from the lungs and stomachs in both species did not display increased DNA binding
activity over corresponding controls.
4.7.3. Mode-of-Action Information
Hepatocellular and renal adenomas and carcinomas and pheochromocytomas were
observed in rats and mice following oral exposure to HCE (NTP, 1989; NCI, 1978). The
mechanistic data available for HCE is limited; and the mode(s) of carcinogenic action of HCE in
the liver, kidney, and adrenal gland is unknown. However, there are data suggesting that the
induction of kidney tumors in male rats involves the accumulation of a2U-globulin in the kidney
and the induction of liver tumors in male and female mice may involve increased cytotoxicity,
inflammation, and regenerative cell proliferation in the liver, respectively.
4.7.3.1. Kidney Tumors
Description of the Hypothesized Mode of Action
Hypothesized mode of action. Generally, kidney tumors observed in cancer bioassays are
assumed to be relevant for assessment of human carcinogenic potential. However, male rat-
specific kidney tumors that are caused by the accumulation of a2U-globulin are not generally
considered relevant to humans. Accumulation of a2U-globulin in hyaline droplets initiates a
sequence of events that leads to renal nephropathy and, eventually, renal tubular tumor
formation. The phenomenon is unique to the male rat since female rats and other laboratory
mammals administered the same chemicals do not accumulate a2U-globulin in the kidney and do
not subsequently develop renal tubule tumors (Doi et al., 2007; IARC, 1999; U.S. EPA, 1991c).
Some experimental data suggest that development of kidney tumors in male rats
following exposure to HCE may involve an a2U-globulin-mediated mode of action. However, an
analysis of the data as outlined below indicates that there is insufficient evidence to establish the
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role of a2u-globulin in HCE-induced kidney tumors. Specifically, the key events leading to
development of kidney tumors in male rats exposed to HCE have not been adequately
characterized. For example, no immunohistochemical data are available that demonstrate the
presence of a2U-globulin in hyaline droplets. Furthermore, reported renal toxicity in female rats
and male and female mice exposed to HCE suggests a mode of action other than a2U-globulin-
associated nephropathy. In the absence of sufficient information demonstrating the involvement
of a2U-globulin processes, male rat renal toxicity/tumors are considered relevant for risk
assessment purposes.
Identification of key events.
The role of a2U-globulin accumulation in the development of renal nephropathy and
carcinogenicity observed following HCE exposure was evaluated using the U.S. EPA (1991c)
Risk Assessment Forum Technical panel report. This report (U.S. EPA, 1991c) provides specific
guidance for evaluating chemical exposure-related male rat renal tubule tumors for the purpose
of risk assessment, based on an examination of the potential involvement of a2U-globulin
accumulation.
The protein, a2U-globulin, is a member of a large superfamily of low-molecular-weight
proteins and was first characterized in male rat urine. It has been detected in various tissues and
fluids of most mammals, including humans. However, the particular isoform of a2U-globulin
commonly detected in male rat urine is considered specific for the male rat; moreover, the urine
and kidney concentrations detected in the mature male rat are several orders of magnitude greater
than in any other age, sex, or species tested (Doi et al., 2007; IARC, 1999; U.S. EPA, 1991c).
The hypothesized mode of action ascribed to a2U-globulin-associated nephropathy is
defined by a progressive sequence of effects in the male rat kidney, often culminating in renal
tumors. The involvement of hyaline droplet accumulation in the early stages of nephropathy
associated with a2U-globulin-binding chemicals is an important difference from the sequence of
events observed with classical carcinogens. The pathological changes that precede the
proliferative sequence for classical renal carcinogens also include early nephrotoxicity (e.g.,
cytotoxicity and cellular necrosis) but no apparent hyaline droplet accumulation. Furthermore,
the nephrotoxicity that can ensue from hyaline droplet accumulation is novel because it is
associated with excessive a2U-globulin accumulation. This a2U-globulin accumulation is
proposed to result from reduced renal catabolism of the a2U-globulin chemical complex and is
thought to initiate a sequence of events leading to chronic proliferation of the renal tubule
epithelium. The histopathological sequence of events in mature male rats consists of the
following (see Table 4-21 summarizing available data on HCE for each step of this sequence):
• Excessive accumulation of hyaline droplets in renal proximal tubules
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• Immunohistochemical evidence that a2U-globulin is the protein accumulating in the
hyaline droplets
• Subsequent cytotoxicity and single-cell necrosis of the tubule epithelium;
• Sustained regenerative tubule cell proliferation;
• Development of intralumenal granular casts from sloughed cellular debris associated
with tubule dilatation and papillary mineralization;
• Foci of tubule hyperplasia in the convoluted proximal tubules; and
• Renal tubule tumors.
Table 4-21. Nephrotoxic effects characteristic of am-globulin nephropathy
observed in male and female rats administered HCE
Study, dose,
duration, and
sex
Accumulation of
hyaline droplets
Accumulation of
a2u-globulin in
hyaline droplets
Necrosis/
degeneration
Tubular
regeneration
Granular
casts/dilatation
Papillary
mineralization
Tubular
hyperplasia
NTP, 1989
7 or 14
mg/kg-d (M);
57 or 114
mg/kg-d (F)
103 wks
M
NT
X
X
X
X
X
F
NT
X
X
X
NCI, 1978
113 or 227
mg/kg-d
104 wks
M
NT
X
X
X
F
NT
X
X
X
Gorzinski et
al., 1985
1,15, or 62
mg/kg-d
16 wks
M
NT
X
X
F
NT
X
NTP, 1989
34, 67, 134,
268,
or 536 mg/kg-d
13 wks
M
X
NT
X
X
X
F
NT
NTP, 1996
146 or 293
mg/kg-d
3 wks
M
X
NT
X
X
X
F
NT
NT
NT
NT
NT
NT
NT
NTP, 1989
140, 281, or
563 mg/kg-d
16 d
M
X
NT
X
X
F
NT
NT = not tested; X = presence of effect; M = male; F = female
In addition to this histopathological sequence, U.S. EPA (1991c) provides more specific
guidance for evaluating chemically induced male rat renal tubule tumors for the purpose of risk
assessment. To determine the appropriateness of the data for use in risk assessment, chemicals
inducing renal tubule tumors in the male rat are examined in terms of three categories:
• The a2U-globulin sequence of events accounts for the renal tumors.
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• Other potential carcinogenic processes account for the renal tumors.
• The a2U-globulin-associated events occur in the presence of other potential
carcinogenic processes, both of which result in renal tumors.
Therefore, it is important to determine whether the a2U-globulin process is involved in
nephrotoxicity and carcinogenicity following HCE exposure and, if so, to what extent
a2U-globulin-associated events, rather than other processes, account for the tumor increase.
Determination of these elements requires a database of bioassay data not only from male
rats, but also from female rats and mice, and such toxicity studies should demonstrate whether or
not a2U-globulin processes are operative. In the absence of sufficient information demonstrating
the involvement of a2U-globulin processes, it should be assumed that any male rat renal
toxicity/tumors are relevant for risk assessment purposes.
As outlined in the U.S. EPA Risk Assessment Forum Technical Panel report (U.S. EPA,
1991c), the following information from studies of male rats is used for demonstrating that the
a2U-globulin process may be a factor in any observed renal effects—an affirmative response in
each of the three categories is desired. The three categories of information and criteria are as
follows:
• Increased number and size of hyaline droplets in the renal proximal tubule cells of
treated male rats. The abnormal accumulation of hyaline droplets in the P2 segment
helps differentiate a2U-globulin inducers from chemicals that produce renal tubule
tumors by other modes of action.
• Accumulating protein in the hyaline droplets is v.2u-globulin. Hyaline droplet
accumulation is a nonspecific response to protein overload; thus, it is necessary to
demonstrate that the protein in the droplet is, in fact, a2U-globulin.
• Additional aspects of the pathological sequence of lesions associated with
u.2n-globulin nephropathy are present. Typical lesions include single-cell necrosis,
exfoliation of epithelial cells into the proximal tubular lumen, formation of granular
casts, linear mineralization of papillary tubules, and tubule hyperplasia. If the
response is mild, not all of these lesions may be observed. However, some elements
consistent with the pathological sequence must be demonstrated to be present.
Experimental Support for the Hypothesized Mode of Action
Strength, consistency, and specificity of association
NTP (1989)—16-day study
In a short-term exposure study, NTP (1989) administered 140, 281, 563, 1,125, or
2,250 mg/kg-day HCE to F344/N rats via gavage for 16 days. All of the surviving HCE-exposed
male rats exhibited hyaline droplets in the cytoplasm of the renal tubular epithelium.
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Additionally, male rats exposed to 140 and 281 mg/kg-day HCE demonstrated tubular cell
regeneration and eosinophilic granular casts of cell debris in the tubule lumina at the
corticomedullary junction. NTP (1989) did not report regeneration or granular casts in the
surviving males of the 563 mg/kg-day dose group. NTP (1989) did not report the incidence or
severity of the lesions observed in the treated males. None of the nephrotoxic effects were
observed at any HCE dose in the female rats or in the controls.
NTP (1996)—21-day study
In a second short-term exposure study, NTP (1996) administered 146 or 293 mg/kg-day
HCE by gavage to male F344/N rats for 21 days. Marked hyaline droplet accumulation was
observed and categorized by severity in relation to controls. The hyaline droplet accumulation
exhibited by HCE-exposed male rats was characterized as two severity grades above the control
rats. A Mallory-Heidenhain stain allowed for greater sensitivity in evaluating hyaline droplets
within the tubules of the kidney and further supported the presence of the hyaline droplets in the
kidney tubules. Increased incidence of tubular regeneration (60 and 100% in the 146 and
293 mg/kg-day dose groups, respectively) was also observed in male rats following HCE
exposure. The severity of the tubular lesions was considered mild at both doses. Eosinophilic
granular casts, of minimal to mild severity, were identified in the outer medullary tubules in male
rats exposed to HCE: 80 and 60% in the 146 and 293 mg/kg-day HCE, respectively. There was
a dose-related, statistically significant increase in the PCNA labeling index in HCE-treated male
rats. The percentage of replicating proximal and distal tubule epithelial cells was increased
5.7-fold over controls in the 146 mg/kg-day dose group and 9.2-fold over the controls in
293 mg/kg-day dose group. The nephrotoxic effects reported by NTP (1996) were not noted in
the control animals. Female rats were not included in this study; therefore, gender specificity of
the nephrotoxic effect was not examined.
NTP (1989)—13-week study
In a subchronic exposure study, NTP (1989) administered 34, 67, 134, 268, or
536 mg/kg-day HCE via gavage to F344/N rats for 13 weeks. Male rats from all dose groups
exposed to HCE exhibited exposure-related kidney effects, although incidence data were only
reported for the 34 mg/kg-day dose group. These kidney effects were characterized by hyaline
droplet formation in the renal tubular epithelium, eosinophilic granular casts of cell debris in the
tubular lumina at the corticomedullary region (with associated tubular dilatation), and tubular
cell regeneration. The severity of these lesions increased with HCE exposure dose, although the
severity grades were not reported. Furthermore, as the HCE exposure dose increased, the
animals developed additional lesions. Renal papillary necrosis and renal tubule epithelium
degeneration and necrosis were observed in all 536 mg/kg-day males (only the five male rats that
died before the end of the study were analyzed microscopically).
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Urinalysis in male rats administered HCE showed fine and course granules, cellular casts,
and epithelial cells, findings that were consistent with the histopathological changes observed in
the male rats. Kidney weights of HCE-exposed males were increased 27, 37, 57, 73, and 57% in
34, 67, 134, 268, and 536 mg/kg-day males, respectively (increases were statistically significant,
compared with control kidney weights except the low-dose group). Female kidney weight was
increased following HCE exposure: 16 and 32% (statistically significant) in the 268 and
536 mg/kg-day dose groups, respectively. Treated females showed no other HCE-exposure-
related kidney effects.
Gorzinski et al. (1985)—16-week study
Gorzinski et al. (1985) observed dose-related levels of HCE in the kidneys of male F344
rats fed 1, 15, or 62 mg/kg-day HCE for 16 weeks. HCE was also detected in the kidneys of
female rats, although at much lower levels and did not increase proportionally with dose. Renal
tubular atrophy and degeneration was observed in male rats: 20, 70, and 100% in the 1,15, and
62 mg/kg-day dose groups, respectively. These renal degenerative effects were also noted in
10% of the male controls, although the authors noted that these lesions were graded as slight.
Slight hypertrophy and/or dilation of the proximal convoluted tubules were noted in 10, 70, and
100% of the HCE-exposed male rats in the 1,15, and 62 mg/kg-day dose groups, respectively.
Slight hypertrophy and dilation of the proximal convoluted tubules were not observed in the
male control rats. Peritubular fibrosis was also noted in the high-dose group males. Renal
tubular atrophy and degeneration were observed in 10, 20, and 60% of female rats in the 1,15,
and 62 mg/kg-day dose groups, respectively. These lesions were seen in one female control rat
(10%), although the authors characterized the severity grade of the lesions as very slight.
Male rat sensitivity was evident in the histopathological changes seen in the
HCE-exposed male rats compared with the female rats. Renal effects were either observed in
more male rats than female rats (statistical analyses were not reported) or did not occur in
females. Additionally, kidney concentrations of HCE were much higher in male rats compared
with female rats. Gorzinski et al. (1985) noted that the differences in HCE concentrations
measured in male rat and female rat kidneys may explain the differences observed in the kidney
effects (i.e., male sensitivity to HCE exposure).
NCI (1978)—78-week study
NCI (1978) conducted a carcinogenicity bioassay in Osborne-Mendel rats administered
113 and 227 mg/kg-day HCE via gavage for 5 days/week for 78 weeks. Chronic inflammatory
kidney lesions were observed in both control and HCE-exposed rats. Male rats exhibited chronic
inflammation in the kidney: 75, 70, 65, and 50% of untreated control, vehicle control, 113, and
227 mg/kg-day dose groups, respectively. Similarly, female rats showed an incidence of
inflammatory lesions in 40, 20, 36, and 41% in the untreated control, vehicle control, 113, and
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227 mg/kg-day dose groups, respectively. The control and HCE-exposed male rats exhibited
greater sensitivity to the chronic inflammation compared with the female rats. NCI (1978) noted
that these lesions observed in the control and HCE-exposed animals of both sexes were
characteristic of age-related renal lesions. Some renal lesions observed in older rats could be
related to a spontaneous syndrome known as CPN. CPN is associated with aged rats, especially
F344, Sprague-Dawley, and Osborne-Mendel strains. CPN is frequently more severe in males
compared with females. Hard et al. (1993) reported the pathologic features attributed to CPN
including:
• Thickening of tubular and glomerular basement membranes;
• Basophilic segments of proximal convoluted tubules with sporadic mitoses indicative
of tubule cell proliferation;
• Tubular hyaline casts of proteinaceous material originating in the more distal portion
of the nephron, mainly in the medulla, and later plugging a considerable length of the
tubule;
• Focal interstitial aggregations of mononuclear inflammatory cells within areas of
affected tubules;
• Glomerular hyalinization and sclerosis;
• Interstitial fibrosis and scarring;
• Tubular atrophy involving segments of proximal tubule;
• Occasional hyperplastic foci in affected tubules (chronically in advanced cases); and
• Accumulation of protein droplets in sporadic proximal tubules (in some advanced
cases).
Several of the CPN pathological effects are similar to and can obscure the lesions
characteristic of a2U-globulin-related hyaline droplet nephropathy (Hard et al., 1993).
Additionally, renal effects of a2U-globulin accumulation can exacerbate the effects associated
with CPN (U.S. EPA, 1991c). However, Webb et al. (1989) suggested that exacerbated CPN
was one component of the nephropathy resulting from exposure to chemicals that induce
a2U-globulin nephropathy. Male rat sensitivity has been noted with both CPN and a2U-globulin
nephropathy.
With the exception of atrophy of the proximal tubule, tubular cell proliferation, and
hyaline casts of proteinaceous material, the histopathological effects associated with CPN are
distinctive from those of a2U-globulin nephropathy. The urinalysis and serum chemistry of CPN
rats show albuminuria, hypoalbuminemia, and hypocholesterolemia as well as increased serum
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creatinine and urea nitrogen levels, whereas these changes in a2U-globulin nephropathy are
minimal (Hard et al., 1993).
NCI (1978) reported tubular nephropathy in HCE-exposed rats, but not in untreated or
vehicle controls. Increased incidence of nephropathy described as tubular degeneration and
necrosis and the presence of large hyperchromatic regenerative epithelial cells was observed in
45 and 66% of male rats exposed to 113 and 227 mg/kg-day HCE, respectively. Female rats also
exhibited tubular nephropathy following HCE exposure: 18 and 59% in the 113 and 227 mg/kg-
day dose groups, respectively. In addition to the tubular nephropathy, observed effects overlying
these lesions included focal pyonephritis, tubular ectasia, cast formation, chronic interstitial
nephritis and fibrosis, and focal glomerulosclerosis. Renal tubular cell adenomas were observed
in four male rats (11% incidence rate) exposed to 113 mg/kg-day HCE. Similar renal tumors
were not observed in males from the high-dose group, males from the vehicle control, males
from the untreated control, or female rats. NCI (1978) concluded that there was no evidence of
HCE-exposure-related carcinogenicity in Osborne-Mendel rats based on the lack of statistical
significance and dose-response in the tumor incidence rate. However, it is possible that the
truncated duration of HCE treatment (78 weeks, cyclical) and the significantly accelerated
mortality in the male rats did not allow enough time for the renal tubule tumors to develop.
According to Goodman et al. (1980), the incidences of spontaneous renal tubule tumors in
control male and female Osborne-Mendel rats (as recorded in the NCI Carcinogenesis Testing
Program) were 0.3 and 0%, respectively. The incidence of renal adenomas (11%, first observed
at 86 weeks; 8 weeks after the treatment period ended) following administration of 113 mg/kg-
day HCE exceeded both the concurrent (0%) and historical (0.3%) controls in males.
NTP (1989)—103-week study
NTP (1989) administered 7 or 14 mg/kg-day HCE in corn oil via gavage to male F344/N
rats for 103 weeks. Kidney effects consisting of tubular cell degeneration and atrophy, tubular
dilatation, tubular cell regeneration, glomerulosclerosis, interstitial fibrosis, and chronic
inflammation were observed in >94% of the HCE-exposed male rats. The incidence of
nephropathy in male control rats was 96%. The mean severity of the kidney effects in male rats
increased following HCE exposure: 2.34 + 0.14, 2.62 + 0.15, and 2.68 + 0.16 (statistically
significant) in the control, 7, and 14 mg/kg-day dose groups, respectively. Kidney effect severity
was considered mild for the controls and mild to moderate for the HCE-exposed male rats.
While the mean severity scores do not show more than a 15% increase over control in the high-
dose group, more moderate and marked nephropathy was observed in HCE-exposed male rats
compared with controls. The incidences of severe (moderate or marked) nephropathy in males
were 18/50, 24/50, and 30/50 in the control, 7, and 14 mg/kg-day dose groups, respectively.
Additionally, the male rats exhibited increased incidences in linear mineralization of the renal
papillae: 4, 30, and 64% in the control, 7, and 14 mg/kg-day dose groups, respectively. Pelvic
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epithelium hyperplasia was also observed in 14% of male rats exposed to either 7 or 14 mg/kg-
day HCE. These hyperplastic effects were not observed in either the controls or the treated
females.
NTP (1989) administered 57 or 114 mg/kg-day HCE in corn oil via gavage to female
F344/N rats for 103 weeks. The incidences of nephropathy in female rats following chronic
HCE exposure were 44, 84, and 90% for the control, 57, and 114 mg/kg-day dose groups,
respectively. The severity scores for nephrotoxicity in female rats were statistically significantly
increased in both treated groups: 0.72 + 0.13, 1.38 + 0.11, and 1.69 + 0.12 in the control, 57, and
114 mg/kg-day dose groups, respectively. The average severity of nephropathy was considered
minimal for the controls and minimal to mild for the HCE-exposed female rats. Examination of
the various grades of nephropathy severity shows more mild and moderate nephrotoxicity in
HCE-exposed females compared with controls. In females, the incidences of severe (mild or
moderate) nephropathy were 12/50, 25/50, and 32/50 in the control, 57, and 114 mg/kg-day dose
groups, respectively (statistical analysis was not reported). Female rats also showed an increase
in linear mineralization at 57 (44%) and 114 mg/kg-day (26%) compared with relatively high
response in the controls (28%). This increase in linear mineralization was not dose-related. The
HCE-exposed male rats also exhibited renal tubular hyperplasia, renal tubule adenomas, and
renal tubule carcinomas. The combined renal adenoma or carcinoma incidences were 2, 4, and
14% (3, 6, and 24% after adjusting for intercurrent mortality) in the control, 7, and 14 mg/kg-day
dose groups, respectively. There were no HCE-related neoplasms observed in female rats treated
with 57 or 114 mg/kg-day HCE. NTP (1989) noted that the hyperplasia and tumors of the renal
tubules represented a morphologic continuum. The observed hyperplasia incidences were 4, 8,
and 22% of the control, 7, and 14 mg/kg-day dose groups, respectively. The incidence of renal
tubule neoplasia in male rats also exceeded historical controls (0.5%). Female rats did not
exhibit renal tubule hyperplasia.
A sex difference was noted in the observed nephropathy, as males were more sensitive to
HCE-exposure-related nephropathy than females. This sex specificity is apparent for the
nephrotoxicity and grades of nephropathy severity in both control and HCE-treated groups.
Although administered only one-eighth of the dose given to the female rats, the male rats
demonstrated a greater incidence of nephropathy that was more severe and included additional
kidney effects (i.e., increases in incidence of mineralization of the renal papillae and hyperplasia
of pelvic transitional epithelium) compared with the female rats.
With the exceptions of glomerulosclerosis, interstitial fibrosis, and chronic inflammation,
the observed nephrotoxic effects in the male rats are characteristic of a2U-globulin nephropathy.
However, NTP (1989) did not report accumulation of hyaline droplets containing the
a2U-globulin protein in the proximal tubule. It is possible that hyaline droplets were present,
considering that the 16-day and 13-week rats examined by NTP (1989) exhibited hyaline
droplets; however, the hyaline droplets were likely obscured by the prevalence of the other
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lesions. Evidence of these effects in almost all of the control males and in treated and control
female rats also complicates the characterization of the mode of action. Considering that
a2u-globulin nephropathy is typically male rat-specific, the appearance of nephrotoxic effects in
the female rats as well as the male and female controls and the identification of other effects not
specifically associated with a2U-globulin (i.e., glomerulosclerosis and interstitial fibrosis) suggest
that the at least some of effects may not be the result of a2U-globulin accumulation.
Considering the strain and age of the rats in the chronic (103 weeks) NTP study (1989), it
is also possible that the rats were affected by CPN (i.e., increased incidence of nephrotoxicity
inaging rats). However, changes in severity of the nephropathy that are greater in the HCE-
exposed animals indicate chemical-related effects. Additionally, HCE-exposed male rats
demonstrated dose-dependent increases in incidence of mineralization of the renal papillae and
hyperplasia of pelvic transitional epithelium. Neither of these effects increased in a dose-related
manner in the controls or the HCE-exposed female rats. Therefore, the treatment-related effects
in male and female rats indicate that CPN is not likely to be solely responsible for the
nephropathy observed by NTP (1989).
Limitations in the available studies. These studies describe the effects associated with
HCE exposure using a general, nonspecific term: tubular nephropathy (Weeks et al., 1979; NCI,
1978). This general term does not provide information on the specific histopathological changes
characterizing the nephropathy. Additionally, the reported incidences of effects were grouped
and measured as nephropathy rather than individual effects. Effects described in this way are
difficult to interpret with regards to a2U-globulin nephropathy. One study (NTP, 1996) was
limited in its usefulness because only male rats were exposed and the experimental design sought
to draw conclusions about SARs involved in the induction of hyaline droplet nephropathy of
11 halogenated ethanes. The study focused predominantly on the kidneys and the purpose of the
study was to compare chlorinated ethanes, not to examine the mode of action of HCE. The
divergence in doses used for male and females in the NTP (1989) chronic exposure experiment
highlighted the male sensitivity to HCE-induced nephrotoxicity. However, this study design
made it difficult to otherwise compare the sexes. Additionally, three of the six HCE exposure
studies utilized only two dose groups, limiting the ability to characterize the dose response of
HCE-exposure-related nephropathy.
Summary of evidence for strength, specificity, and consistency. Generally, kidney tumors
observed in cancer bioassays are assumed to be relevant for assessment of human carcinogenic
potential. However when the mode-of-action evidence demonstrates that kidney tumors in male
rats result from an accumulation of a2U-globulin, the tumor data are considered to not be relevant
to humans, and are not suitable for use in risk assessment (IARC, 1999; U.S. EPA, 1991c). The
criteria for demonstrating the a2U-globulin-related mode of action for risk assessment purposes
have been defined (U.S. EPA, 1991c). Three criteria are considered to be desirable: (1) an
increase in hyaline droplets in the renal proximal tubule cells; (2) the determination that the
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accumulating protein in the droplets is a2U-globulin; and (3) the presence of additional
pathological lesions associated with a2U-globulin. The key event in the histopathological
sequence for the a2U-globulin-related mode of action is excessive accumulation of hyaline
droplets containing a2U-globulin in renal proximal tubules.
None of the HCE studies performed immunohistochemical assays to confirm the
presence of a2U-globulin protein within the hyaline droplets observed following administration of
HCE (NTP, 1996, 1989). It is unclear whether HCE is binding to a2u-globulin or to other
proteins during the formation of hyaline droplets, or if another mechanism is operating. This
represents an important data gap.
In addition, the data on female rats and mice of both sexes from chronic exposure studies
(NTP, 1989; NCI, 1978) suggest an a2U-globulin independent mode of action for HCE-exposure
related nephropathy. NCI (1978) reported dose-related nephropathy in female rats that was not
apparent in the controls. Nephropathy was also reported in male and female mice chronically
administered HCE (NCI, 1978). NCI (1978) reported the appearance of renal tubular effects in
almost all (>92%) of the HCE-treated male and female mice following chronic HCE exposure,
but the mice did not develop renal tubule tumors. The presence of kidney effects in HCE-
exposed female rats and male and female mice, which generally do not accumulate the
a2U-globulin protein, suggests a mode of action other than a2U-globulin-associated nephropathy.
Dose-response concordance. The initial key event in the histopathological sequence for
the a2u-globulin-related mode of action is excessive accumulation of hyaline droplets containing
a2U-globulin in renal proximal tubules. The accumulation of a2U-globulin in hyaline droplets
must occur at lower doses than subsequent a2U-globulin-related effects. None of the HCE studies
performed the necessary immunohistochemical assays to confirm the presence of a2U-globulin
protein within the hyaline droplets observed following administration of HCE (NTP, 1996,
1989). Therefore, dose-response concordance of the accumulation of a2U-globulin in hyaline
droplets cannot be demonstrated from the available data.
Most of the effects characterizing the histopathological sequence of events in epithelial
cells of the proximal tubules leading to renal tumors (Doi et al., 2007; IARC, 1999; U.S. EPA,
1991c) increased in incidence with increasing doses of HCE in the short-term and subchronic
exposure studies. Dose-related increases in nephrotoxicity and renal carcinogenicity were noted
in the two chronic HCE exposure studies. The short-term and subchronic exposure studies did
not report evidence of carcinogenicity in rats administered HCE. In the NTP (1989) study, male
rats administered 7 or 14 mg/kg-day HCE for 2 years exhibited a dose-related increased
incidence of renal tubule adenomas and carcinomas. Histopathological effects associated with
a2u-globulin nephropathy (tubular cell degeneration and atrophy, tubular dilatation, and tubular
cell regeneration) were noted in almost all of the treated and untreated animals. A dose-response
relationship was difficult to detect considering the number of animals affected by nephrotoxicity.
However, dose-related increases over controls for toxic kidney effects such as linear
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mineralization, severity of nephrotoxicity, and renal tubule hyperplasia were observed. NTP
(1989) did not report interim data; therefore, examinations were performed at study termination.
Consequently, the nephrotoxicity (generally attributed to leading up to the formation of renal
tubular tumors associated with a2U-globulin) is reportedly increased at doses similar to those that
induce tumor formation.
Overall, dose-related kidney effects were noted for almost all of the male rats
administered HCE at doses ranging from 1 to 563 mg/kg-day. Even at the lowest HCE dose
administered in the studies, renal effects were observed in male rats. Animals treated with
greater amounts of HCE exhibited dose-related increases in incidence and severity of effect
when compared with those of the lower dose groups. It is difficult to establish dose-response
concordance between the noncancer nephropathy and the renal tubule tumors reported by NTP
(1989). Renal tubule tumors were observed at 7 mg/kg-day HCE, the lowest dose administered
for a chronic duration, which also induced significant nephropathy in HCE-exposed animals.
The other studies that administered doses within an order of magnitude of 7 mg/kg-day were the
NTP (1989) study (34 or 67 mg/kg-day for 13 weeks) and the Gorzinski et al. (1985) study (1,
15, or 62 mg/kg-day for 16 weeks). Although nephropathy was noted in the shorter duration
studies (NTP, 1996, 1989; Gorzinski et al., 1985), the only evidence of carcinogenicity was from
the chronic exposure studies (NTP, 1989; NCI, 1978).
Temporal relationship. The initial key event in the histopathological sequence for the
a2u-globulin-related mode of action is excessive accumulation of hyaline droplets containing
a2U-globulin in renal proximal tubules. The accumulation of a2U-globulin in hyaline droplets
must occur first in the sequela leading to a2U-globulin-related nephrotoxicity and tumor
formation. None of the HCE studies performed the necessary immunohistochemical assays to
confirm the presence of a2U-globulin protein within the hyaline droplets observed following
administration of HCE (NTP, 1996, 1989). Therefore, this key event and the important temporal
relationship for the accumulation of a2U-globulin cannot be demonstrated from the available data.
Histopathological effects associated with a2U-globulin-related nephropathy were observed
in animals treated with HCE in studies that varied in exposure duration from 16 days to 2 years.
The sequence of histopathological events characteristic of the a2U-globulin-related mode of
action was noted in the chronic exposure study NTP (1989) that reported renal tubule adenomas
and carcinomas. All of the studies (NTP, 1996, 1989; Gorzinski et al., 1985; NCI, 1978) that
administered HCE for shorter durations than the NTP (1989) study reported similar
histopathological changes, although an increase in renal tubule tumors was not observed. It is
unknown if the nephropathy observed by NTP (1989) led to the reported renal tubule tumors
because the animals were only examined at the end of the 103-week study period. A temporal
relationship cannot be distinguished from reported data.
Biological plausibility and coherence. The kidney toxicity and tumor formation that was
observed in rats and mice are biologically plausible effects that could potentially occur in
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humans. If the tumor formation in male rats, however, is due to accumulation of a2u-globulin
protein in the renal tubules, then these tumors would not be considered to be relevant to humans.
The sequence of events including accumulation of a2u-globulin protein in the renal tubules of
male rats initiating a sequence of nephrotoxic events leading to renal tubule tumor formation
(Doi et al., 2007; IARC, 1999; U.S. EPA, 1991c) was evaluated as a hypothesized mode of
action for HCE-induced carcinogenicity and nephropathy. These a2u-globulin related effects are
typically not observed in female rats or other species due to the absence or minimal presence of
the a2u-globulin protein in these animals (Hard et al., 1993). Evidence of nephrotoxic effects in
female rats in two chronic studies (NTP, 1989; NCI, 1978) and in male and female mice in one
chronic study (NCI, 1978) precludes the conclusion that HCE is acting through an a2u-globulin-
associated mode of carcinogenic action.
Other Possible Modes of Action
There is insufficient evidence to support an a2U-globulin-related mode of action for renal
tumors following HCE exposure. It is possible that advanced CPN may play a role in the
incidence of nephrotoxicity and kidney tumors in male rats. CPN is an age-related renal disease
of laboratory rodents that occurs spontaneously. The observed renal lesions in male rats
following exposure to HCE are effects commonly associated with CPN. Nephropathy (described
as tubular cell degeneration and regeneration, tubular dilatation and atrophy, glomerulosclerosis,
interstitial fibrosis, and chronic inflammation) was also observed in female rats (NTP, 1989), as
well as in male and female mice (NCI, 1978). However, changes in severity of the nephropathy
were observed to be greater in male rats exposed to HCE compared to controls, indicating that
HCE exposure exacerbated effects in the kidney. Additionally, HCE-exposed male rats
demonstrated dose-dependent increases in incidence of mineralization of the renal papillae and
hyperplasia of pelvic transitional epithelium. Neither of these effects increased in a dose-related
manner in the controls or the HCE-exposed female rats. The treatment-related effects in male
and female rats serve as evidence that CPN is not solely responsible for the nephropathy
observed by NTP (1989).
Conclusions about the Hypothesized Mode of Action
Support for the hypothesized mode of action in animals. The mode of action for the
carcinogenic effects of HCE in the kidney is unknown, although there are data to indicate that
a2u-globulin accumulation may play a role in the observed tumors in male rats. Studies
following short-term, subchronic, and chronic exposure of male rats have reported renal lesions
(NTP, 1996, 1989; Gorzinski et al., 1985; NCI, 1978) and formation of renal tubule adenomas
and carcinomas (preceded by hyperplasia) following chronic HCE exposure (NTP, 1989),
suggesting an a2U-globulin-related mode of action. However, the key event in the
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histopathological sequence of events demonstrating an a2U-globulin-related mode of action
(excessive accumulation of hyaline droplets containing a2U-globulin in renal proximal tubules)
leading to the development of kidney tumors in male rats exposed to HCE has not been
characterized. None of the HCE studies performed immunohistochemical assays to confirm the
presence of a2U-globulin protein within the hyaline droplets observed following administration of
HCE (NTP, 1996, 1989). It is unknown if HCE is binding to a2U-globulin or to other proteins
during the formation of hyaline droplets. This represents an important data gap. On the other
hand, it is possible that an a2U-globulin-associated mode of action may, in fact, be responsible for
the tumors observed in male rats and that more than one mode of action may be operating to
induce the nephropathy observed across species and sexes.
In addition, data are available that demonstrate kidney effects in female rats and mice of
both sexes from chronic exposure studies (NTP, 1989; NCI, 1978). The NCI (1978) study
reported dose-related nephropathy in female rats that was not apparent in the controls.
Nephropathy was also reported in male and female mice chronically-administered HCE (NCI,
1978). The presence of kidney effects in HCE-exposed male and female mice, which generally
do not accumulate the a2U-globulin protein, suggests a mode of action other than a2U-globulin
nephropathy.
Relevance of the Hypothesized Mode of Action to Humans
Generally, kidney tumors observed in cancer bioassays are assumed to be relevant for
assessment of human carcinogenic potential. However, for male rat kidney tumors, when the
mode-of-action evidence demonstrates that the response is secondary to a2U-globulin
accumulation, the tumor data are not used in the cancer assessment (U.S. EPA, 1991b). There is
insufficient evidence to conclude that the renal adenomas and carcinomas observed in male rats
administered HCE (NTP, 1989) are related to an a2U-globulin mode of action for the following
reasons: (1) there is a lack of a2U-globulin immunohistochemical data for HCE-induced
nephrotoxicity and carcinogenicity; (2) the hyaline droplet accumulation is caused by excessive
protein load that may not be exclusively related to a2U-globulin accumulation; and (3) the
existence of renal toxicity in female rats and male and female mice indicates that the nephrotoxic
effects are not limited to an a2U-globulin-induced sequence of lesions. Therefore, the renal
adenomas and carcinomas observed in male rats administered HCE (NTP, 1989) were
considered relevant to humans.
4.7.3.2. Liver Tumors
Hepatocellular carcinomas were observed in male and female B6C3Fi mice administered
360 or 722 mg/kg-day HCE, via gavage, in a chronic oral bioassay conducted by NCI (1978).
Tumor incidences in males of both dose groups were statistically significantly elevated compared
with control groups, and demonstrated a dose response. Both dose groups of female mice
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presented statistically significantly elevated incidences of hepatocellular carcinoma compared
with control groups, but a dose response was not observed. The investigators did not find
nonneoplastic liver effects (such as organized thrombus, inflammation, fibrosis, necrosis,
infarctions, amyloidosis, or hyperplasia) in either sex.
The mode of action for the carcinogenic effects of HCE in the liver is unknown.
Metabolism studies of HCE indicate that the major enzymes involved are phenobarbital-
inducible CYP450s. These are primarily localized in the liver. Although tissue-specific
metabolism of HCE has not been studied extensively, the majority of HCE metabolism is
presumed to occur in the liver. HCE is proposed to metabolize to PERC and pentachloroethane
and is likely subsequently metabolized to TCE. It is possible that the HCE-induced
hepatocellular carcinomas in mice occur as a result of the binding of HCE metabolites to liver
macromolecules and the generation of free radicals during HCE metabolism, causing key events
in the carcinogenic process such as cytotoxicity, inflammation, and regenerative cell
proliferation. However, these potential key events have not been systematically evaluated for
HCE.
In a 13-week study, hepatocellular necrosis of the centrilobular area was observed in rats
(NTP, 1989). It is unknown if this could be considered a key event in the carcinogenic process
because rats in the available studies (NTP, 1989; NCI, 1978) have not displayed hepatocellular
neoplastic endpoints. Although mice demonstrated hepatocellular carcinoma, nonneoplastic
effects such as hepatocellular necrosis were not observed (NCI, 1978). HCE-induced
hepatocellular carcinomas in mice varied in microscopic appearance (NCI, 1978). Some
carcinomas were characterized by well-differentiated hepatic cells with uniform cord
arrangement, while others had anaplastic liver cells with large hyperchromatic nuclei, often with
inclusion bodies and vacuolated pale cytoplasm. Arrangement of neoplastic liver cells also
varied from short stubby cords to nests of cells and occasional pseudo-acinar formations.
Neoplasms in control mice did not vary in appearance from those in HCE-treated mice.
In vivo binding of radiolabeled carbon to DNA, RNA, and protein from liver, kidney,
lung, and stomach following administration of [14C]-HCE was consistently greater in mice
compared with rats (Lattanzi et al., 1988). Binding to macromolecules was interpreted by the
presence of radiolabeled carbon; however, radiolabeled carbon may have been incorporated into
these macromolecules from intermediary HCE metabolites. In vitro binding studies using calf
thymus DNA demonstrated that mouse liver cytosol (induced by phenobarbital) mediated more
extensive DNA binding than rat liver cytosol (Lattanzi et al., 1988). Comparisons of HCE
metabolism rates indicated that mice metabolize HCE at twice the rate of rats (Mitoma et al.,
1985).
Cellular damage leading to cytotoxicity, inflammation, and regenerative cell proliferation
is a possible consequence of this binding in the liver. The binding studies provide a line of
evidence as to why the liver is the major carcinogenic target in the mouse, but not the rat.
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Regenerative cell proliferation has been evaluated in the kidney, but not in the liver of
HCE-treated rats (NTP, 1996). RDS in hepatocytes was evaluated in mice treated with HCE
(Yoshikawa, 1996; Miyagawa et al., 1995). This study reported ambiguous results; the lower
HCE dose caused a statistically significant increase in RDS, whereas the higher dose did not
(Yoshikawa, 1996; Miyagawa et al., 1995). Rat liver foci experiments provide support for the
hypothesis that HCE acts as a tumor promoter, not as a tumor initiator (Milman et al., 1988;
Story etal., 1986).
The in vivo binding data suggest that HCE is sequestered in the liver of mice and rats and
metabolic data suggest that mice metabolize HCE at a greater rate compared with rats.
Considering the greater potential for metabolism in mice compared with rats and the proposed
increase in DNA binding following metabolism of HCE (Lattanzi et al., 1988), the increased
incidence of hepatocellular carcinomas in mice, but not rats, may be related to DNA binding.
However, the DNA binding measurements were based solely on the presence of radiolabeled
carbon; specific HCE metabolites were not identified. Therefore, this process does not take into
account the possibility of normal biological mechanisms in which the radiolabeled carbon can be
incorporated into the macromolecules via anabolic processes. All together, while it is possible
that metabolism and binding in mice are involved in the development of liver tumors, the role of
DNA binding in the mode of action for HCE-induced hepatotoxicity and carcinogenesis is not
known and, as such, the mode of action is not known.
4.7.3.3. Pheochromocytomas
Pheochromocytomas are catecholamine-producing neuroendocrine tumors. The
relevance of rodent pheochromocytomas as a model for human cancer risk has been the subject
of discussion in the scientific literature (e.g., Greim et al., 2009; Powers et al., 2008). In humans,
pheochromocytomas are rare and usually benign, but may also present as or develop into a
malignancy (Eisenhofer et al., 2004; Lehnert et al., 2004; Elder et al., 2003; Goldstein et al.,
1999). Hereditary factors in humans have been identified as important in the development of
pheochromocytomas (Eisenhofer et al., 2004). Pheochromocytomas are more common in
laboratory rats, though evidence suggests that certain rat pheochromocytomas may have
similarity to human pheochromocytomas (Powers et al., 2008). Furthermore, mechanisms of
action inducing pheochromocytomas in rats are expected to occur in humans as well (Greim et
al., 2009). Therefore, in the absence of information indicating otherwise, adrenal gland tumors
in rodents are considered relevant to humans.
No studies were identified to determine a mode of action for HCE-induced tumors of the
adrenal gland. Therefore, the mode of action for pheochromocytomas observed following oral
exposure to HCE is unknown.
4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES
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No studies were located that address the susceptibility of populations or life stages to
HCE-induced toxicity or carcinogenicity in humans.
4.8.1. Possible Childhood Susceptibility
No studies were located that addressed possible childhood susceptibility to HCE-induced
toxicity or carcinogenicity. Although it is unknown if HCE toxicity is mediated by parent
compound or its metabolites, CYP450 enzymes of the 2A, 2B, and 3 A subfamilies and CYP450
1A2 are involved in HCE metabolism. Many drugs reportedly exhibit a higher systemic
clearance in children than in adults (Evans et al., 1989). Although Dome (2004) reported that
Phase I (including CYP450 activities) and Phase II enzymatic activities are 1.3-1.5-fold higher
in children (aged 1-16 years) compared with adults , studies of fetal and neonatal livers indicate
that CYP450 expression is similar to adult levels by a few months of age (Lacroix et al., 1997;
Vieira et al., 1996; Cazeneuve et al., 1994; Treluyer et al., 1991). Similarly, Blanco et al. (2000)
compared liver microsomal CYP450 activities of humans <10 years old with those >10-60 years
old and concluded that factors other than maximal CYP450 catalytic activities, such as
reductions in hepatic blood flow, hepatic size, and oxygen supply in the elderly, may be
responsible for age-related changes in drug clearance. Therefore, the extent to which variable
age-related expression of CYP450 contributes to childhood susceptibility is unknown.
4.8.2. Possible Gender Differences
Toxicity studies in rats indicate that male rats are more sensitive to HCE-induced
nephrotoxicity than females (NTP, 1989; Gorzinski et al., 1985, 1980; NCI, 1978). Evidence
suggests that female rats are more sensitive to HCE-induced hepatotoxicity. The reasons for
these sex-specific differences are unknown, but may be related to sex-specific differences in
tissue concentrations following HCE administration (i.e., higher concentrations observed in male
rat tissues when compared with female rats, see Table 3-3), sex hormone differences, and/or
gender differences in CYP450 activities. No additional studies were located that addressed
possible gender differences for HCE-induced toxicity or carcinogenicity.
4.8.3. Other
CYP450 enzymes are polymorphic in the human population. Polymorphisms result in
CYP450 enzymes with variant catalytic activity for substrates such as HCE. This could
potentially result in decreased HCE detoxification or increased HCE bioactivation.
Detoxification enzymes such as the glutathione-S-transferase (GST) family are also polymorphic
in the human population, with variant catalytic activities that could affect the detoxification of
HCE.
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5. DOSE-RESPONSE ASSESSMENTS
5.1. ORAL REFERENCE DOSE (RfD)
5.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification
Data on the health effects of oral HCE exposure in humans are not available. The oral
exposure database for HCE includes a 103-week gavage study in F344 rats (NTP, 1989), a
78-week gavage study in Osborne-Mendel rats (NCI, 1978), a 91-week gavage study in B6C3Fi
mice (NCI, 1978), a 16-week feeding study in F344 rats (Gorzinski et al., 1985), and a 13-week
gavage study in F344 rats (NTP, 1989). The short-term study data were not considered in the
selection of the principal study for the derivation of the RfD because the database contains dose-
response data from studies of subchronic and chronic durations. However, short-term studies in
rats (NTP, 1996, 1989) were used to support findings in the subchronic and chronic studies. The
available oral exposure studies identified kidney or liver effects associated with exposure to
HCE. Reported effects include tubular nephropathy (NTP, 1989; NCI, 1978), atrophy and
degeneration of renal tubules (NTP, 1989; Gorzinski et al., 1985), slight hypertrophy and/or
dilation of proximal convoluted renal tubules (Gorzinski et al., 1985), linear mineralization of
renal tubules (NTP, 1989), hyperplasia of the renal pelvic transitional epithelium (NTP, 1989),
and hepatocellular necrosis (NTP, 1989).
In the NTP (1989) chronic study, HCE was administered via gavage at doses of 7 and
14 mg/kg-day in male F344 rats and 57 and 114 mg/kg-day in female F344 rats for 103 weeks.
Nephropathy (characterized by tubular cell degeneration and regeneration, tubular dilatation and
atrophy, glomerulosclerosis, interstitial fibrosis, and chronic inflammation) was observed in
HCE-treated rats of both sexes. Nephropathy was also reported in control rats of both sexes.
Although a high incidence of nephropathy was observed in control rats, the study authors
reported that the incidence of more severe nephropathy increased in dosed rats relative to
controls (NTP, 1989). EPA considered the increase in severity of nephropathy in male rats by
analyzing the incidence of greater than mild nephropathy. EPA determined that the increased
incidence of moderate or marked nephropathy in males was statistically significant at the
14 mg/kg-day dose (see Table 5-1). EPA considered the increased severity of nephropathy in
female rats by analyzing the incidence of nephropathy that was greater than minimal
nephropathy. EPA determined that the increased incidences of mild to moderate nephropathy
were statistically significant in females at the 57 and 114 mg/kg-day doses (see Table 5-1).
Linear mineralization of the renal papillae and hyperplasia of the renal pelvic epithelium were
increased in a dose-dependent, statistically significant manner in the treated male rats. EPA
determined that the increased incidences of linear mineralization of the renal papillae and
hyperplasia of the renal pelvic epithelium were statistically significant in males at the 7 and
14 mg/kg-day doses (see Table 5-1). The increased severity of nephropathy and dose-dependent
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increases in the incidence of mineralization of the renal papillae and hyperplasia of renal pelvic
transitional epithelium in male rats suggest that HCE exposure exacerbated the nephropathy
observed in the NTP (1989) study. The NTP (1989) chronic study did not identify NOAELs for
male or female rats as kidney effects were observed at the lowest doses tested. EPA considered
the male rat LOAEL as 7 mg/kg-day based on increased incidence in moderate or marked tubular
nephropathy (characterized by degeneration, necrosis, and regenerative epithelial cells),
hyperplasia of the pelvic transitional epithelium, and linear mineralization of the renal papillae in
the NTP (1989) study. EPA considered the female rat LOAEL as 57 mg/kg-day, based on dose-
related increases in incidence and severity of nephropathy in the NTP (1989) study.
In the NCI (1978) chronic rat study, HCE was administered via gavage to groups of
50 male and 50 female Osborne-Mendel rats for 5 days/week, cyclically for 66 of the 78 weeks,
followed by an observation period of 33-34 weeks (total of 112 weeks). The TWA doses of
HCE were 113 and 227 mg/kg-day. Tubular nephropathy was observed in all groups of treated
animals, but was not observed in either untreated or vehicle controls. Statistically significant
increases in incidence of tubular nephropathy were observed at 113 and 227 mg/kg-day HCE in
both male and female rats (see Table 5-1). The NCI (1978) study did not identify a NOAEL for
tubular nephropathy in rats. EPA considered the LOAEL as 113 mg/kg-day, based on a dose-
related increase in incidence of nephropathy in both male and female rats.
In the NCI (1978) chronic mouse study, HCE was administered via corn oil gavage to
groups of 50 male and 50 female B6C3Fi mice for 5 days/week for 78 weeks followed by an
observation period of 12-13 weeks (total of 90 weeks). Starting in week 9, the HCE doses were
increased, though no explanation for the increase was provided. The TWA doses of HCE were
360 and 722 mg/kg-day. Because of low survival rates in the vehicle and untreated male control
groups, NCI (1978) compared tumor incidences in the dosed males and females to the pooled
vehicle control data derived from concurrently run bioassays for several other chemicals. NCI
(1978) reported chronic kidney inflammation (i.e., tubular nephropathy characterized by
degeneration of the convoluted tubule epithelium at the junction of the cortex and medulla and
hyaline casts) in male and female B6C3Fi mice administered 360 and 721 mg/kg-day HCE.
EPA considered the LOAEL for this study as 360 mg/kg-day based on tubular nephropathy,
while a NOAEL could not established from these data.
In the Gorzinski et al. (1985) study, HCE was administered (in feed) to groups of 10 male
and 10 female F344 rats at doses of 0, 1, 15, or 62 mg/kg-day for a period of 16 weeks. Kidney
effects consisted of slight hypertrophy and/or dilation of proximal convoluted renal tubules and
atrophy and degeneration of renal tubules. Slight hypertrophy and/or dilation of the proximal
convoluted renal tubules was not observed in the control rats of either sex or in HCE exposed
female rats. EPA determined that increases in slight hypertrophy and/or dilation of the proximal
convoluted renal tubules were statistically significant in male rats treated with 15 or
62 mg/kg-day HCE (see Table 5-1). Atrophy and degeneration of renal tubules was observed in
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both male and female rats. EPA determined that increases in incidences of atrophy and
degeneration of renal tubules were statistically significant in male rats treated with 15 or
62 mg/kg-day HCE and in female rats fed 62 mg/kg-day HCE (see Table 5-1). EPA considered
the male rat LOAEL as 15 mg/kg-day and the male rat NOAEL as 1 mg/kg-day, based on
increased incidence of the renal tubule effects. EPA considered the female rat LOAEL as
62 mg/kg-day and the female rat NOAEL as 15 mg/kg-day, based on increased incidence of
renal tubule effects.
In the NTP (1989) subchronic study, HCE was administered via gavage to groups of
10 male and 10 female F344 rats at TWA doses of 0, 34, 67, 134, 268, and 536 mg/kg-day for
13 weeks. Kidney effects (i.e., hyaline droplet formation, renal tubular regeneration, and renal
tubular casts) were observed in male rats from all HCE exposure groups, though incidence data
were only provided for the 34 mg/kg-day dose group. NTP (1989) reported that the severity of
kidney effects in male rats increased with dose, but no data on severity were presented. No
kidney effects were reported in female F344 rats exposed to HCE. Liver effects were observed
in male and female rats at higher doses of HCE and EPA determined that statistically significant
increases in hepatocellular necrosis were observed in female rats exposed to 268 or
536 mg/kg-day HCE (see Table 5-1).
The incidence of kidney and liver effects from the studies considered for selection as the
principal study are summarized in Table 5-1. As incidence data on kidney effects reported in the
13-week subchronic study (NTP, 1989) were limited to males in the 34 mg/kg-day dose group,
these data are not presented in Table 5-1.
Table 5-1. Incidences of noncancerous kidney and liver effects in rats
following oral exposure to HCE
Study
Duration
(route)
Strain/sex/species
Endpoint
Dose
(mg/kg-day)
Incidence
Kidney Effects
NCI (1978)
NTP (1989)
NTP (1989)
78wks
(gavage)
103 wks
(gavage)
103 wks
Osborne-Mendel male
rat
Osborne-Mendel
female rat
F344 male rat
F344 female rat
F344 male rat
Tubular nephropathy
Tubular nephropathy
Moderate to marked tubular
nephropathy
Mild to moderate tubular
nephropathy
Linear mineralization
0
113
227
0
113
227
0
7
14
0
57
114
0
0/20 (0%)
22/49a (45%)
33/503 (66%)
0/20 (0%)
9/50a (18%)
29/49a (59%)
18/50 (36%)
24/50 (48%)
30/503 (60%)
12/50 (24%)
25/503 (50%)
32/49a (65%)
2/50 (4%)
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Table 5-1. Incidences of noncancerous kidney and liver effects in rats
following oral exposure to HCE
Study
NTP (1989)
Gorzinski
etal. (1985)
Gorzinski
etal. (1985)
Duration
(route)
(gavage)
103 wks
(gavage)
16 wks
(dietary)
16 wks
(dietary)
Strain/sex/species
F344 male rat
F344 male rat
F344 male rat
F344 female rat
Endpoint
Hyperplasia of the renal pelvic
transitional epithelium
Slight hypertrophy and/or
dilation of proximal convoluted
renal tubules
Atrophy and degeneration of
renal tubules
Atrophy and degeneration of
renal tubules
Dose
(mg/kg-day)
7
14
0
7
14
0
1
15
62
0
1
15
62
0
1
15
62
Incidence
15/503 (30%)
32/50a (64%)
0/50 (0%)
7/50a (14%)
7/50a (14%)
0/10 (0%)
1/10 (10%)
7/10a (70%)
10/103
(100%)
1/10 (10%)
2/10 (20%)
7/10a (70%)
10/103
(100%)
1/10 (10%)
1/10 (10%)
2/10 (20%)
6/10a (60%)
Liver Effects
NTP (1989)
13 weeks
(gavage)
F344 male rat
F344 female rat
Hepatocellular necrosis
Hepatocellular necrosis
0
33.5
67.1
134.3
267.8
535.7
0
33.5
67.1
134.3
267.8
535.7
0/10 (0%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
1/10 (10%)
2/5 (40%)
0/10 (0%)
0/10 (0%)
0/10 (0%)
2/10 (20%)
4/10a (40%)
8/10a (80%)
aEPA determined statistical significance using Fisher's Exact Test (p < 0.05).
These chronic and subchronic studies in rats and mice indicate that the kidney and liver
are both target organs of HCE oral toxicity in rodents. Given the number of effects reported in
the kidney and the greater sensitivity of these effects in available studies, the kidney is
considered the primary target of oral HCE exposure toxicity in rodents. HCE exposure resulted
in a number of kidney effects: atrophy and degeneration of renal tubules in male and female
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F344 rats (Gorzinski et al., 1985), slight hypertrophy and/or dilation of proximal convoluted
renal tubules in male F344 rats (Gorzinski et al., 1985), linear mineralization in male F344 rats
(NTP, 1989), tubular nephropathy in male and female F344 rats (NTP, 1989), hyperplasia of the
renal pelvic transitional epithelium in male F344 rats (NTP, 1989), and tubular nephropathy in
male and female Osborne-Mendel rats (NCI, 1978). Further consideration was given to these
endpoints as candidate critical effects for the determination of the point of departure (POD) for
derivation of the oral RfD.
Although the doses associated with hepatic effects were more than 10-fold higher than
doses associated with kidney effects, data from the NTP (1989) study on incidence of
hepatocellular necrosis from the female rats were also considered as candidate critical effects for
comparison purposes. The data on the male rat liver effects from the NTP (1989) study were not
considered because the incidence of hepatocellular necrosis was not significantly elevated above
controls at any HCE dose. The kidney effects reported in the 13-week subchronic study (NTP,
1989) were not further considered because the lack of the incidence data for the control groups
made it uncertain whether the 34 mg/kg-day HCE dose represented a LOAEL. In addition, the
HCE doses administered were more than fourfold higher than those doses associated with kidney
effects in other subchronic (Gorzinski et al., 1985) and chronic (NTP, 1989) studies. The ability
of the chronic NTP (1989) study to inform the effects observed at the lowest dose tested in the
Gorzinski et al. (1985) study is limited because the lowest dose tested in the chronic exposure
study represented a LOAEL and Gorzinski et al. (1985) did not provide severity data to compare
with the NTP (1989) study. The chronic study in B6C3Fi mice (NCI, 1978) was not considered
for selection as the principal study because the HCE doses that induced kidney effects were more
than sevenfold higher than doses associated with kidney effects in rats following subchronic
(Gorzinski et al., 1985) or chronic (NTP, 1989; NCI, 1978) exposure.
5.1.2. Methods of Analysis—Including Models
The benchmark dose (BMD) modeling approach (U.S. EPA, 2000b) was employed to
identify the candidate POD for each of the endpoints described above. A benchmark response
(BMR) of 10% extra risk was considered appropriate for derivation under the assumption that it
represents a minimally biologically significant response level. All of the dichotomous dose-
response models available in the EPA benchmark dose software (BMDS), version 2.0, were fit to
the incidence data for kidney effects in male and female rats reported by NTP (1989), NCI
(1978), and Gorzinski et al. (1985), as well as the incidence data for hepatocellular necrosis in
female rats reported by NTP (1989). Details of the BMD dose-response modeling reported in
Table 5-2 are presented in Appendix B (Table B-l). In addition, the BMD and 95% lower bound
confidence limit on the BMD (BMDL) modeling outcomes for a BMR of 5 and 1% are presented
in Appendix B (Table B-2) for comparison with the 10% BMR. From the BMD modeling
analysis results presented in Table B-l, candidate PODs were selected. Table 5-2 summarizes
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the BMD modeling results of the available data and the BMR levels and the candidate PODs are
identified for each effect.
Table 5-2. Summary of the BMD modeling results for the kidney
Study
Gorzinski et al.
(1985)
Gorzinski et al.
(1985)
NCI (1978)
NTP (1989)
NTP (1989)
NTP (1989)
Endpoint
Slight hypertrophy
and/or dilation of
proximal convoluted
renal tubules
Atrophy and
degeneration of renal
tubules
Tubular
nephropathy
Increased incidence of
moderate to marked
tubular
nephropathy
Increased incidence of
mild to moderate
tubular
nephropathy
Linear mineralization
Hyperplasia of the
pelvic transitional
epithelium
Sex/species
(group size)
Male rats
(n=10)
Male rats
(n=10)
Female rats
(n=10)
Male rats
11
Female rats
(n ~ 3U)
Male rats
(n~50)
Female rats
(n~50)
Male rats
(n~50)
Male rats
Duration
(route)
16 wks
(dietary)
16 wks
(dietary)
78 wks
(gavage)
103 wks
(gavage)
103 wks
(gavage)
103 wks
(gavage)
"Best-fit"
model
Gamma
Quantal-
linear, and
Weibull
Gamma,
Multistage 1°,
and Quantal-
linear
Probit
Gamma,
Multistage 1°,
and Weibull
Multistage 2°
Probit 1°
Gamma,
Quantal-
linear, and
Weibull
Probit
LogLogistic
BMD
(mg/kg-d)
1 22
1 34
16.10
21.22
80.63
3.81
15 17
3.98
7.05
BMDL10
(mg/kg-d)
0 710
0 728
10.51
16.99
41.89
2.60
10 72
3.22
4.48
The range of candidate PODs (approximately 0.7-40 mg/kg-day) is about 60-fold.
Kidney effects (i.e., tubular nephropathy, linear mineralization of the renal tubules, hyperplasia
of the pelvic transitional epithelium, atrophy and degeneration of renal tubules, and slight
hypertrophy and/or dilation of the proximal convoluted renal tubules) observed in male rats
resulted in lower candidate PODs than comparable effects in female rats.
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The most sensitive effect observed in male rats exposed to HCE is slight hypertrophy
and/or dilation of proximal convoluted renal tubules (Gorzinski et al., 1985), although the
candidate POD for slight hypertrophy and/or dilation of proximal convoluted renal tubules (i.e.,
0.710 mg/kg-day) is nearly identical to the candidate POD for atrophy and degeneration of renal
tubules (i.e., 0.728 mg/kg-day). As tubular nephropathy in the chronic studies (NTP, 1989; NCI,
1978) was characterized as atrophy and degeneration of renal tubules, this endpoint has been
consistently observed following HCE exposure in several studies. Therefore, atrophy and
degeneration of renal tubules was selected as the candidate critical effect for male rats exposed to
HCE. As shown in Appendix B, the gamma, multistage 1°, logistic, probit, Weibull models in
BMDS (version 2.0) provided adequate fits to the incidence data for atrophy and degeneration of
renal tubules in male rats from the Gorzinski et al. (1989) 16-week study (Table B-l), as
assessed by a %2 goodness-of-fit p-values. BMDio and BMDLio estimates from these models
were within a factor of three of each other, suggesting no appreciable model dependence. The
models with the lowest Akaike's information criterion (AIC; a measure of the deviance of the
model fit that allows for comparison across models for a particular endpoint) values were for the
gamma, multistage 1°, and quantal-linear models; therefore, the model with the lowest BMDLio
was selected. These models had identical BMDio and BMDLio values. Therefore, the BMDLio
of 0.728 mg/kg-day associated with a 10% extra risk for nephropathy in male rats was selected
as the candidate POD for these data.
The tubular nephropathy in male rats observed in the chronic exposure studies (NCI,
1978; NTP, 1989) resulted in higher PODs than the atrophy and degeneration of renal tubules in
male rats observed following 16 weeks of HCE exposure (Gorzinski et al., 1985). The ability of
the chronic studies (NCI, 1978; NTP, 1989) to inform the effects observed at the lowest dose
tested in the Gorzinski et al. (1985) study is limited because the lowest dose tested in the chronic
exposure studies represented a LOAEL and Gorzinski et al. (1985) did not provide severity data
for comparison with NTP (1989). Therefore, the Gorzinski et al. (1985) study was selected as
the principal study and atrophy and degeneration of renal tubules in male rats was selected as the
critical effect. The BMDLio of 0.728 mg/kg-day was selected as the POD and serves as the basis
for the derivation of the oral RfD for HCE. This endpoint is supported by additional kidney
effects associated with oral exposure to HCE and supports the weight of evidence for HCE-
associated nephrotoxicity.
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5.1.3. RfD Derivation—Including Application of Uncertainty Factors (UFs)
The derivation of the RfD for atrophy and degeneration of renal tubules in male F344 rats
from the Gorzinski et al. (1985) 16-week toxicity study was calculated from the BMDLio of
0.728 mg/kg-day. The composite UF of 1,000 was comprised of the following:
• A default interspecies UF (UFA) of 10 was applied to account for the variability in
extrapolating from rats to humans. Although the toxicokinetics have been minimally
evaluated in animals, the toxicokinetics of HCE have not been sufficiently
characterized in either rats or humans to identify the active compound or determine
dose metrics.
• A default intraspecies UF (UFH) of 10 was applied to adjust for potentially sensitive
human subpopulations in the absence of information on the variability of response to
HCE in the human population. Current information is unavailable to assess human-
to-human variability in HCE toxicokinetics and toxicodynamics.
• The study selected as the principal study was a 16-week study by Gorzinski et al
(1985), a study duration that is minimally past the standard subchronic (90-day) study
and falls well short of a standard lifetime study (i.e., two year chronic bioassay).
Some data (NTP, 1989; Gorzinski et al., 1985; NCI, 1978) are available to inform the
nature and extent of effects that would be observed with a longer duration of exposure
to HCE. The chronic data identify the kidney is the target organ of HCE toxicity,
consistent with the findings from the Gorzinski et al. (1985) study. In addition, data
from the NCI (1978) chronic study suggest that an increase in duration of HCE
exposure may not increase the incidence of nephropathy. As the Gorzinski et al.
(1985) study did not report severity data for the renal effects, there are insufficient
data to exclude the possibility that chronic exposure could increase the severity of the
observed kidney effects. However, increases in severity of tubular nephropathy in the
NTP (1989) chronic study was reported at similar doses as atrophy and degeneration
of renal tubules in the Gorzinski et al. (1985) subchronic study, suggesting
consistency in dose response relationships between chronic and subchronic studies.
For these reasons, a subchronic-to-chronic UF (UFS) of 3 was used to account for the
extrapolation from subchronic-to-chronic exposure duration.
• An UF for a LOAEL to a NOAEL extrapolation was not applied because the current
approach is to address this extrapolation as one of the considerations in selecting a
BMR for BMD modeling. In this case, a BMR of a 10% increase in the incidence of
renal tubule atrophy and degeneration was selected under an assumption that it
represents a minimal biologically significant change.
• An UF of 3 was applied to account for deficiencies in the HCE toxicity database,
including the lack of a multigenerational reproductive study. The database includes
studies in laboratory animals, including chronic and subchronic dietary exposure
studies and two oral developmental toxicity studies. Therefore, in consideration of
the oral database for HCE, a database UF of 3 was applied to account for the lack of a
two-generational reproductive study.
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Given the UFs established above, the RfD for HCE was calculated employing the
following equation:
RfD = POD -H UF
= 0.728 mg/kg-day -1,000
= 7 x 10"4 mg/kg-day
5.1.4. RfD Comparison Information
The predominant noncancer effect of acute, short-term, subchronic, and chronic oral
exposure to HCE is renal toxicity. Table 5-3 presents the potential PODs for nephrotoxicity in
male rats with applied UFs and potential reference values. Figure 5-1 provides a graphical
display of dose-response information from three studies that reported kidney toxicity in male rats
following chronic and subchronic oral exposure to HCE, focusing on potential PODs that could
be considered in deriving the oral RfD. As discussed in Sections 5.1.1 and 5.1.2, among those
studies that demonstrated kidney toxicity, atrophy and degeneration of renal tubules in male
F344 rats from the Gorzinski et al. (1985) study provided the POD for deriving the RfD (see
dotted box in Figure 5-1). Potential reference values that might be derived from other studies are
also presented. Only endpoints observed in male rats are presented because the database for
HCE consistently showed that male rats exhibited greater sensitivity to HCE toxicity compared
with females.
Table 5-3. Potential PODs for nephrotoxicity in male rats with applied UFs
and potential reference values
Potential PODs (mg/kg-day)
Tubular nephropathy; BMDL (2-yr)
Hyperplasia of pelvic transitional
epithelium; BMDL (2-yr)
Linear mineralization; BMDL (2-yr )
Moderate to marked tubular
nephropathy; BMDL (2-yr)
Slight hypertrophy and/or dilation of
proximal convoluted renal tubules;
BMDL (16-wk)
Atrophy and degeneration of renal
tubules; BMDL (16-wk)
16.99
4.48
3.22
2.60
0.710
0.728
Total
TIF
300
300
300
300
1,000
1,000
UFA
10
10
10
10
10
10
UFH
10
10
10
10
10
10
UFS
1
1
1
1
3
3
UFD
3
3
3
3
3
3
Potential
reference values
(mg/kg-d)
0.0566
0.0149
0.0107
0.0087
0.0007
0.0007
Reference
NCI (1978)
NTP
(1989)
Gorzinski
etal.
(1985)
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Figure 5-1. Array of potential PODs with applied UFs and potential
reference values for nephrotoxic effects of studies in Table 5-3.
Point of Departure
ITFA, Interspecies
UFa Intraspecies
UFs, Subchronic to
Chronic
UFD, Database
Reference Dose
0.0001
Increased
incidence of
moderate to
marked
tubular
nephropathv
BMDL
NTP, 1989
2-yr
Increased
incidence of
hyperplasia of
the pelvic
transitional
epithelium
BMDL
NTP. 1989
2-yr
Increased
incidence
linear
mineralization
BMDL
NTP, 1989
2-yr
Increased
incidence of
tubular
nephropathy
BMDL
NO. 1978
91 -wk
Increased
incidence of
slight
hypertrophy
and/or dilation
of convoluted
renal tubules
BMDL
Goranski et
al., 1985
16-wk
Increased
incidence of
atrophy and
degeneration of
tubules
BMDL
Gorzinski el
al., 1985
16-wk
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The nephropathy observed by NCI (1978) was similar to that reported by NTP (1989);
however, the animals in the NTP study were exposed to and exhibited effects at a lower range of
doses of HCE than those in the NCI study (Table 5-1). NTP (1989) described tubular
nephropathy characterized by degeneration, necrosis, and regenerative epithelial cells in rats.
Gorzinski et al. (1985) described similar renal effects characterized by atrophy and degeneration
of renal tubules and slight hypertrophy and/or dilation of proximal convoluted tubules. Linear
mineralization of the renal tubules, hyperplasia of the pelvic transitional epithelium, slight
hypertrophy and/or dilation of the proximal convoluted tubules, increased severity of tubular
nephropathy, and atrophy and degeneration or renal tubules were all reported in male rats
exposed to HCE (NTP, 1989; Gorzinski et al., 1985). Additionally, nephropathy was observed
in both male and female rats, whereas linear mineralization was only observed in male rats.
Kidney effects were observed in male rats in the Gorzinski et al. (1985) study at doses below the
range of exposure tested in the NTP (1989) study. In addition, the ability of the chronic studies
to inform the effects observed at the low dose in the Gorzinski et al. (1985) study is limited
because the lowest dose tested in the NTP (1989) chronic exposure study represented a LOAEL
and Gorzinski et al. (1985) did not provide severity data to compare with the NTP (1989) study.
The potential POD associated with atrophy and degeneration of renal tubules from the Gorzinski
et al. (1985) study was lower than the POD based on increased severity of tubular nephropathy
from NTP (1989). Therefore the POD based on atrophy and degeneration of renal tubules from
the Gorzinski et al. (1985) study was selected to serve as the basis for the derivation of the RfD.
5.1.5. Previous RfD Assessment
In the previous RfD assessment for HCE, completed in 1987, the Gorzinski et al. (1985)
study was employed in deriving the RfD using a NOAEL/LOAEL approach. In this study, the
identified LOAEL for atrophy and degeneration of renal tubules was 15 mg/kg-day, with a
corresponding NOAEL of 1 mg/kg-day. A composite UF of 1,000 was employed to account for
the following three limitations or uncertainties: (1) interspecies extrapolation (UFA = 10);
(2) intraspecies variation (UFH = 10); and (3) subchronic-to-chronic extrapolation (UFS = 10).
An RfD of 1 x 10~3 mg/kg-day was derived. For the current assessment, the atrophy and
degeneration of renal tubules in rats reported by Gorzinski et al. (1985) also served as the basis
for the RfD; however, BMD modeling was used to derive a POD and, in accordance with current
risk assessment practices, an additional UF of 3 for database deficiencies was applied.
5.2. INHALATION REFERENCE CONCENTRATION (RfC)
5.2.1. Choice of Principal Study and Critical Effect—with Rationale and Justification
The database of inhalation toxicity studies on HCE is limited. Human studies
demonstrated HCE exposure in smoke bomb production workers, but the sample sizes are too
small to reach definitive conclusions regarding health effects and the exposure was likely a
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mixture of HCE and zinc oxide. There are no chronic studies available, and only a single
subchronic inhalation study (in four species) that included a developmental toxicity experiment
is available. Weeks et al. (1979) exposed Sprague-Dawley rats, male Beagle dogs, male Hartley
guinea pigs, and Japanese quail to HCE air concentrations of 145, 465, or 2,517 mg/m3 for
6 hours/day, 5 days/week, for 6 weeks. Postexposure observations were carried out for
12 weeks.
As discussed in Section 4.4.3.2, toxic effects observed in treated rats, dogs, and guinea
pigs (the quail did not show signs of toxicity) were at the highest exposure level, 2,517 mg/m3,
except for dams in the 465 mg/m3 exposure group in the developmental study, which exhibited
significantly decreased body weight gain and an increased incidence (85%) of mucopurulent
nasal exudate. This inflammatory exudate was observed in 100% of the dams treated with 2,517
mg/m3. Similar to the dams, male and female rats exposed to 2,517 mg/m3 HCE for 6 weeks
exhibited mucopurulent exudate in the nasal turbinates. Excess mucus in the nasal turbinates
was also observed in 2/10 quail in the 2,517 mg/m3 concentration group. Effects of this nature
were not observed in the 465 or 145 mg/m3 rats and quail or in the treated guinea pigs and dogs.
Weeks et al. (1979) concluded that the excess mucus in two of the 2,517 mg/m3 quails
was a transient effect of the HCE exposure because there was no evidence of inflammatory cells
or tissue damage. The authors attributed the increased incidence of respiratory lesions in rats to
an endemic mycoplasma infection as evidenced by the histopathological observation of an
increased incidence and severity of mycoplasma-related lesions in the nasal turbinates
(mucopurulent exudate), trachea (lymphoid hyperplasia in the lamina propria), and lung
(pneumonitis) of 2,517 mg/m3 male and female rats. Similar lesions characteristic of respiratory
mycoplasmosis in rodents were detected in an oral developmental study in rats that paralleled the
inhalation developmental study described above (both conducted by Weeks et al., 1979).
Irritation of the upper respiratory tract was observed in approximately 70% of the pregnant rats
(20% diagnosed with subclinical pneumonitis) orally exposed to 500 mg/kg HCE, compared
with 10% of controls showing irritation and pneumonitis.
The presence of the infection in the rats in both the oral and inhalation studies and in the
controls of the oral study suggests that respiratory tract effects are a potentiation of the
underlying mycoplasma infection rather than a direct result of HCE exposure. Additionally, the
reduced weight gain in the rats could be related to the condition of the infected animals,
considering that mycoplasma-infected rodents generally gain less weight or lose weight
compared with noninfected rodents (Xu et al., 2006; Sandstedt et al., 1997). Reduced weight
gain was also observed in the 2,517 mg/m3 guinea pigs, but mycoplasma infection was not
reported (Weeks et al., 1979). Like rats and mice, guinea pigs can carry the mycoplasma
organism; however, they are not clinically affected (Fox et al., 1984; Holmes, 1984). No data
were presented demonstrating the presence of mycoplasma in the lungs; therefore, the respiratory
tract effects cannot be excluded from consideration as a potential critical effect.
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As discussed in Section 4.4.3, neurobehavioral effects were consistently observed in the
rats and dogs exposed to 2,517 mg/m3. The male and female rats in the 6-week study exhibited
tremors and ruffled pelt. The pregnant rats developed tremors on GDs 12-16. Similarly, the
dams exposed to 500 mg/kg HCE in the concurrent oral developmental study by Weeks et al.
(1979) experienced tremors on GDs 15 and 16 of the 11-day exposure period. The HCE-exposed
dogs showed tremors, ataxia, and hypersalivation, severe head bobbing, facial muscular
fasciculations, and closed eyelids. These effects were noted in the dogs throughout the study,
although they disappeared overnight during nonexposure time periods.
Effects of inhalation exposure to HCE were also reported in an acute study by Weeks and
Thomasino (1978), in which a single 8-hour inhalation exposure to 2,500 or 57,000 mg/m3 HCE
and a single 6-hour exposure to 17,000 mg/m3 HCE in male Sprague-Dawley rats resulted in
neurological and lung effects. The male rats exposed to 57,000 mg/m3 HCE had reduced body
weight gain compared with controls over the 14 days postexposure. By 6 hours of exposure, one
rat had a staggered gait. Necropsy did not reveal any gross exposure-related lesions, although
microscopy revealed that two of these rats had subacute diffuse interstitial pneumonitis of
minimal to moderate severity and vascular congestion associated with these lung effects.
Following 6 hours of exposure to 17,000 mg/m3, the six rats in this group showed reduced
weight gain compared with controls and two of these rats exhibited a staggered gait. No
exposure-related gross or histopathological changes were observed in tissues and organs. These
effects were not noticeable 14 days postexposure.
The subchronic inhalation study by Weeks et al. (1979), as the only repeated exposure
study available, was selected as the principal study for the derivation of the RfC. This study is a
well-conducted subchronic bioassay which used three concentrations and incorporated a variety
of endpoints (e.g., lexicological, teratological, neurological, pulmonary) across a range of
species (see Table 5-4). The authors evaluated portal of entry effects by gross examination of
lungs, trachea, and nasal turbinates following necroscopy on animals that died during the study
or were sacrificed at 12 weeks postexposure. In addition, Weeks et al. (1979) evaluated upper
respiratory effects by examining histological sections of the nasal turbinates and evaluated upper
respiratory inflammation by the presence of polymorphonuclear leukocytes in close association
with excess mucus within the lumens of the nasal passages. The primary limitation of Weeks et
al. (1979) is the minimal amount of quantitative information provided characterizing the reported
effects. Several experiments only utilized one sex, and additional exposure concentration(s)
between the mid- and high concentration would have allowed for better characterization of the
exposure-response curve. However, this study identified neurotoxicity, statistically significant
decreases in body weight gain, and upper and lower respiratory tract irritation. The responses
were generally observed following exposure to the highest concentration, and not in the two
lower concentrations. Considering the consistent observation of neurotoxic effects across
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experiments in rats and dogs, these effects following inhalation exposure to HCE were selected
as the critical effect.
Table 5-4. Noncancerous effects observed in animals exposed to HCE via
inhalation
Species
Sprague-Dawley rats
(25/sex/dose)
Male Beagle dogs (4/dose)
Male Hartley guinea pigs
(10/dose)
Japanese quail (20/dose)
Pregnant Sprague-Dawley
rats (22/dose)
Dose/duration
0, 145, 465, or
2,517mg/m3;
6 wks
0 145 465
or2,517mg/m3;
CDs 6-16
NOAEL
(mg/m3)
465 mg/m3
465 mg/m3
465 mg/m3
2,517 mg/m3
Maternal:
465 mg/m3
LOAEL
(mg/m3)
2,5 17 mg/m3
2,5 17 mg/m3
2,5 17 mg/m3
Not
established
Maternal:
2,517 mg/m3
Effect
Males: neurotoxic effects (tremors
and ruffled pelt), reduced body
weight gain, increased relative,
spleen, and testes weights
Females: neurotoxic effects (tremors
and ruffled pelt), increased relative
liver weight
Tremors, ataxia, hypersalivation,
head bobbing, facial muscular
fasciculations
Reduced body weight, increased
relative liver weight
No effects observed
Maternal: tremors
Developmental: no effects
Source: Weeks etal. (1979).
5.2.2. Methods of Analysis—Including Models
The Weeks et al. (1979) study included three exposure groups (145, 465, and
2,517 mg/m3) plus a control. Neurological effects were observed in male and female Sprague-
Dawley rats, male Beagle dogs, and pregnant Sprague-Dawley rats only at the highest dose
tested. Incidence data were not reported, which precluded application of BMD modeling.
Therefore, a NOAEL served as the POD. The NOAEL of 465 mg/m3, identified in Weeks et al.
(1979), was selected as the POD for the derivation of the RfC based on effects in male and
female rats and male dogs exposed to HCE for 6 weeks and pregnant rats exposed on GDs 6-16.
Although the NOAELs are the same, the male and female rats exposed to HCE for 6 weeks were
selected as the study animals upon which to base the POD, as the duration of exposure for the
dams in the teratology study was only 11 days and only four male dogs were exposed to HCE in
the 6-week study.
The NOAEL is based on intermittent HCE inhalation exposures in male and female rats
for 6 hours/day, 5 days/week. Thus, prior to deriving the RfC, this POD was adjusted for
continuous exposure (24 hours/day, 7 days/week). The duration-adjusted POD (POD[ADJ]) is
derived using the following equation (U.S. EPA, 1994b):
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POD[ADJ] = (POD) x (hours of exposure/24 hours) x (days of exposure/7 days)
= (465 mg/m3) x (6/24 hours) x (5/7 days)
= 83.0mg/m3
The Methods for Derivation of Inhalation Reference Concentrations and Application of
Inhalation Dosimetry (hereafter referred to as the RfC Methodology) recommends converting the
POD[ADj] to a human equivalent concentration (HEC) (U.S. EPA, 1994b). The RfC
Methodology separates gases into three categories based on their water solubility and reactivity
with tissues in the respiratory tract. Category 1 gases are highly water soluble and/or rapidly
irreversibly reactive in the surface-liquid/tissue of the respiratory tract, such that they do not
significantly accumulate in blood. Category 2 gases are moderately water soluble and rapidly
reversibly reactive or moderately to slowly irreversibly metabolized in respiratory tract tissue,
such that they have the potential for significant accumulation in the blood and potential for
respiratory and systemic toxicity. Category 3 gases are relatively water insoluble and unreactive
in the surface-liquid/tissue of the respiratory tract.
Categorizing HCE into one of these three gas categories is difficult because data
regarding the inhalation effects of HCE are limited. HCE is a slightly water soluble, non-directly
reactive gas, and has an unknown blood:air partition coefficient. Inhalation exposure to HCE
produces a variety of systemic effects and no noted respiratory tract effects. HCE has been
observed in blood following oral exposures to HCE, but it is unknown whether HCE
accumulates in blood following inhalation exposure. Thus, HCE appears to exhibit
characteristics most concordant with Category 3 Gases whose uptake occurs primarily in the
pulmonary region and site of toxicity is generally remote to the site of absorption. In view of the
fact that neurotoxicity is a systemic effect, the methods for Category 3 gases were used to derive
the HEC.
Consequently, for dosimetric purposes, the human equivalent concentration (HEC) for
HCE was calculated by applying the appropriate dosimetric adjustment factor (DAF) for
systemic acting gases (i.e. Category 3 gases) to the duration-adjusted exposure level (POD[ADJ]),
in accordance with the U.S. EPA RfC methodology (U.S. EPA, 1994). The DAF for a
Category 3 gas is based on the regional gas dose ratio (RGDR), where the RGDR is the ratio of
the animal blood:gas partition coefficient (Hb/g)A and the human blood:gas partition coefficient
(Hb/g)n-
POD[HEc] = POD[ADj] x (Hb/g)A/(Hb/g)H
However, the human and animal blood:gas partition coefficients for HCE are not known.
In accordance with the RfC Methodology (U.S. EPA, 1994b) when the partition coefficients are
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unknown or (Hb/g)A is greater than (Hb/g)n, a RGDR of 1 is used. The partition coefficients are
unknown for HCE resulting in aNOAELjHEq of 83.0 mg/m3.
POD[HEC] = POD[ADJ] X (Hb/g)A/(Hb/g)H
= 83.0 mg/m3 x 1
= 83.0 mg/m3
5.2.3. RfC Derivation—Including Application of Uncertainty Factors (UFs)
The NOAEL[HEC] value of 83 mg/m3 for evidence of neurotoxicity in Sprague-Dawley
rats was used as the POD to derive the RfC for HCE. A composite UF of 3,000 was applied as
follows:
• For animal-to-human interspecies differences (UFA), a UF of 3 was applied to
account for the uncertainty in extrapolating from laboratory animals to humans. This
value is adopted by convention, where an adjustment from an animal-specific
NOAELADJ to a NOAELnEC has been incorporated. Application of an UF of
10 would depend on two areas of uncertainty (i.e., toxicokinetic and toxicodynamic
uncertainties). In this assessment, the toxicokinetic component associated with HCE
is mostly addressed by the determination of an HEC as described in the RfC
methodology (U.S. EPA, 1994b). Insufficient data exist to inform the toxicodynamic
uncertainty component; therefore, an UF of 3 is retained to account for uncertainty
regarding the toxicodynamic differences between rats and humans.
• A default intraspecies UF (UFH) of 10 was applied to account for potentially sensitive
human subpopulations in the absence of information on the variability of response to
HCE in the human population. Information is currently unavailable to assess human-
to-human variability in HCE toxicokinetics and toxicodynamics.
• The subchronic inhalation study by Weeks et al. (1979), as the only repeated
exposure study available, was selected as the principal study. No chronic inhalation
studies were identified for HCE; therefore, there are no data to inform the effects that
might be observed with increased exposure duration. Therefore, a subchronic-to-
chronic UF (UFs) of 10 was applied to account for the use of the POD selected
following a subchronic duration of exposure to HCE to estimate a chronic exposure
RfC.
• An UF for a LOAEL to a NOAEL extrapolation was not applied because this
assessment utilized a NOAEL as the POD.
• A 10-fold UF was used to account for deficiencies in the toxicity database for
inhalation exposure to HCE. The toxicity data for inhalation exposure to HCE is
limited and largely restricted to one subchronic (6-week) inhalation study (Weeks et
al., 1979) in rats, male dogs, male rabbits, and quail. The same investigators
performed a developmental study and an acute study in rats. Maternal toxicity was
observed at both doses. Fetuses of HCE-treated dams did not exhibit any significant
skeletal or soft tissue anomalies. The toxic effects observed in the dams in the
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developmental study were similar to those observed in the rats exposed for 6 weeks,
although additional effects were observed in the rats exposed for a longer duration.
The absence of teratogenic effects does not abrogate concern for other fetal effects,
given that only developmental toxicity studies in a single species are available in the
inhalation database for HCE. The database lacks a long-term study, and a
multigeneration reproductive toxicity study. In addition, the database lacks studies of
neurotoxicity and developmental neurotoxicity, endpoints of concern based on the
available inhalation data. Therefore, in consideration of the inhalation database for
HCE, a database UF of 10 was applied.
Given the UFs established above, the RfC for HCE was calculated employing the
following equation:
RfC = NOAEL[HEc]
= 83 mg/m3 - 3,000
= 0.028 mg/m3 or 3 x 10"2 mg/m3
5.2.4. RfC Comparison Information
The predominant noncancer effect of subchronic inhalation exposure to HCE based on
the available data is neurotoxicity. The other effects noted by Weeks et al. (1979) at the same
dose level were decreases in body weight and increases in organ (liver or kidney) weights in
male guinea pigs, male and female rats, and pregnant rats. As discussed in Sections 5.2. 1 and
5.2.2, the neurotoxicity reported in the available inhalation study (Weeks et al., 1979) was
selected for the RfC derivation because of the consistent observation of the neurotoxicity. Based
on the lack of alternative endpoints to be considered for the basis of the RfC, a graphical display
of dose-response information from the subchronic inhalation study was not provided. For the
reasons discussed above and in Section 5.2.1, neurotoxic effects in male and female rats,
pregnant rats, and male dogs reported by Weeks et al. (1979) are considered the most sensitive
effects and were selected to serve as the basis for the derivation of the RfC for HCE.
5.2.5. Previous RfC Assessment
An RfC for HCE was not previously developed by the U.S. EPA. In the 1987 IRIS
Summary, Weeks et al. (1979) was briefly summarized in the Additional Studies/Comments
section for the oral RfD. The IRIS Summary (1987) stated that Weeks et al. (1979) administered
HCE to rats by inhalation at 0, 145, 465, or 2,520 mg/mg3, 6 hours/day during gestation. At the
two highest doses, maternal toxicity was observed, but there was no evidence of fetotoxicity or
teratogenicity. No discussion was presented in the IRIS Summary (1987) describing why this
study was not used to develop an RfC.
5.3. UNCERTAINTIES IN THE ORAL REFERENCE DOSE AND INHALATION
REFERENCE CONCENTRATION
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The following discussion identifies uncertainties associated with the quantification of the
RfD and RfC for HCE. Following EPA practices and guidance (U.S. EPA, 1994b), the UF
approach was applied to the chosen PODs to derive an RfD and RfC (see Sections 5.1.3 and
5.2.3). Factors accounting for uncertainties associated with a number of steps in the analyses
were adopted to account for extrapolating from an animal study to human exposure, a diverse
human population of varying susceptibilities, and database deficiencies.
The oral database includes short-term, subchronic, and chronic studies in rats, and a
chronic study in mice, and developmental studies in rats. Toxicity associated with oral exposure
to HCE is predominantly reported as kidney toxicity, specifically, renal tubule nephropathy. The
inhalation database includes a subchronic study in rats, pregnant rats, male dogs, male guinea
pigs, and quail. Toxicity associated with inhalation exposure to HCE in this study is mainly
neurotoxicity. Critical data gaps have been identified in Section 4 and uncertainties associated
with data deficiencies are more fully discussed below.
After consideration of the candidate PODs, the RfD of 7 x io~4 mg/kg-day was derived
from a BMDLio of 0.728 mg/kg-day, which was based on the observation of atrophy and
degeneration of renal tubules in male F344 rats from the Gorzinski et al. (1985) 16-week toxicity
study. The dose-response relationships for oral exposure to HCE and nephropathy in other
studies of rats are also available for deriving an RfD, but are associated with higher
NOAELs/LOAELs/BMDLios that are less sensitive than the selected critical effect and
corresponding POD. The derived RfD was quantified using a BMDLio for the POD. The
selection of the BMD model for the quantitation of the RfD does not lead to significant
uncertainty in estimating the POD since benchmark effect levels were within the range of
experimental data. However, the selected models do not represent all possible models one might
fit, and other models could be selected to yield different results, both higher and lower than those
included in this assessment. Uncertainty exists in the selection of the BMR level utilized in the
BMD modeling of the critical effect (atrophy and degeneration of renal tubules in male F344
rats) to estimate the POD. In the absence of information to identify the level of change in
atrophy and degeneration of renal tubules in male F344 rats related to a biologically significant
change, a BMR of 10% was selected for the modeling of the increased incidence to represent a
minimally biologically significant change.
The RfC was derived from a NOAELjHEC] value of 83 mg/m3 for evidence of
neurotoxicity in Sprague-Dawley rats from a subchronic (6-week) inhalation study by Weeks et
al. (1979). A POD based on a NOAEL or LOAEL is, in part, a reflection of the particular
exposure concentration or dose at which a study was conducted. It lacks characterization of the
dose-response curve and for this reason is less informative than a POD obtained from benchmark
dose-response modeling. As the only repeat exposure study, the subchronic inhalation study in
rats (Weeks et al., 1979) was selected as the principal study and neurotoxicity was identified as
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the critical effect. A NOAEL of 465 mg/m was selected to serve as the POD and the basis for
derivation of the RfC.
Extrapolating from animals to humans adds further uncertainty. The effect and its
magnitude at the POD in rats are extrapolated to a human response. Pharmacokinetic models are
useful for examining species differences in pharmacokinetic processing; however, dosimetric
adjustment using pharmacokinetic modeling was not available for oral exposure to HCE.
Information was unavailable to quantitatively assess toxicokinetic or toxicodynamic differences
between animals and humans, so a 10-fold UF was used to account for uncertainty in
extrapolating from laboratory animals to humans in the derivation of the RfD. For the RfC, a
factor of 3 was adopted by convention where an adjustment from an animal-specific NOAELADJ
to a NOAELnEc has been incorporated. Application of an UF of 10 would depend on two areas
of uncertainty (i.e., toxicokinetic and toxicodynamic uncertainties). In this assessment, the
toxicokinetic component is mostly addressed by the determination of a HEC as described in the
RfC methodology (U.S. EPA, 1994b). Insufficient data exist to inform the toxicodynamic
uncertainty component; therefore, an 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
extrapolating dose from a subset or smaller sized population, say of one sex or a narrow range of
life stages typical of occupational epidemiologic studies, to a larger, more diverse population. In
the absence of HCE-specific data on human variation, a factor of 10 was used to account for
uncertainty associated with human variation in the derivation of both the RfD and RfC.
HCE-specific data to examine the potential magnitude of over- or under-estimation of this factor
of 10 are unavailable.
Uncertainties associated with data gaps in the HCE database have been identified. Data
more fully characterizing potential multigenerational reproductive effects associated with both
oral and inhalation HCE exposure are lacking. The oral database includes studies in laboratory
animals, including chronic and subchronic dietary exposure studies and two oral developmental
toxicity studies. The developmental studies show effects at doses higher than those observed to
induce renal toxicity in the subchronic and chronic toxicity studies. Therefore, in consideration
of the entire oral database for HCE, a database UF of 3 was considered appropriate to account for
the lack of a two-generational reproductive study. There are no available human occupational or
epidemiological studies of inhalation exposure to HCE. There are no standard chronic toxicity
or multigeneration reproductive toxicity animal studies available for inhalation exposure to HCE.
The toxicity data on inhalation exposure to HCE is limited and largely restricted to one
subchronic (6-week) inhalation study (Weeks et al., 1979) in rats, male dogs, male rabbits, and
quail. The same investigators performed a developmental study and an acute study in rats. The
developmental study in rats did not provide any evidence of teratogenic effects. However, these
data do not abrogate concern for other fetal effects given the paucity of the inhalation database
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for HCE. In addition, the inhalation database lacks studies of developmental neurotoxicity,
endpoints of concern based on the available inhalation data (critical effect for the RfC).
Therefore, in consideration of the inhalation database for HCE, a database UF of 10 is was
applied.
5.4. CANCER ASSESSMENT
There are no available studies on cancer in humans associated with exposure to HCE.
NTP (1989) provided evidence of renal adenomas and carcinomas and pheochromocytomas and
malignant pheochromocytomas in male F344/N rats in a 2-year cancer bioassay. NCI (1978)
provided evidence of hepatocellular carcinomas in male and female B6C3Fi mice in a 91-week
cancer bioassay. Additionally, HCE was shown to be a promoter, but not an initiator, in an
Osborne-Mendel rat liver foci assay (Milman et al., 1988; Story et al., 1986). Binding of
radiolabeled carbon to DNA, RNA, and protein was observed following [14C]-HCE
administration in both in vitro and in vivo assays in mice and rats (Lattanzi et al., 1988).
The carcinogenic data reported in chronic animal studies include: (1) dose-dependent,
statistically significant increases in the incidence of renal adenoma or carcinoma combined in
male F344/N rats; (2) statistically significant increases in the incidence of
pheochromocytomas/malignant pheochromocytomas combined in male F344/N rats (NTP,
1989); and (3) statistically significant increases in the incidence of hepatocellular carcinomas in
male and female B6C3Fi mice (NCI, 1978).
5.4.1. Choice of Study/Data—with Rationale and Justification
Two animal studies were selected for BMD analysis and subsequent quantitative cancer
assessment. In the first study, NTP (1989) reported statistically significantly elevated incidences
of renal adenomas and carcinomas combined and pheochromocytomas, malignant
pheochromocytomas, and complex pheochromocytomas combined in male F344 rats
administered HCE via gavage for 2 years. Female rats in this study did not exhibit any
HCE-related tumors. In the second study, NCI (1978) reported statistically significantly elevated
incidences of hepatocellular carcinomas in both sexes of B6C3Fi mice administered HCE via
gavage for 78 weeks. However, male mice in this study demonstrated a dose-response
relationship, while female mice did not.
5.4.2. Dose-response Data
NTP (1989) administered, via gavage, TWA doses of 7 or 14 mg/kg-day HCE to male
F344/N rats and TWA doses of 57 or 114 mg/kg-day HCE to female F344/N rats for 103 weeks.
No HCE-related tumors were observed in female rats. Renal adenomas and carcinomas
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combined were observed in 2, 4, and 14% (statistically significant) of male rats administered 0
(controls), 7, and 14 mg/kg-day HCE, respectively. Male rats also exhibited increased
incidences of pheochromocytomas and malignant pheochromocytomas combined; 28, 58
(statistically significant), and 39% in the control, 7 and 14 mg/kg-day dose groups, respectively
(NTP, 1989). The NCI (1978) gavage study administered TWA doses of 0, 360, and 722 mg/kg-
day HCE to male and female B6C3Fi mice for 91 weeks. Statistically significant increases in
the incidence of hepatocellular carcinomas were observed in 15, 30, and 63% of males and 10,
40, and 31% of females in the control, 360, and 722 mg/kg-day dose groups, respectively.
Both NTP (1989) and NCI (1978) are well-designed studies, conducted in both sexes of
two species with 50 animals/sex/dose. Each study utilized two dose groups of HCE and an
untreated control group, with examination of a wide range of toxicological endpoints in both
sexes of the rodents. Tumor incidences were elevated over controls at two sites in rats (NTP,
1989) and at one site in mice (NCI, 1978). Some limitations associated with the NCI (1978)
study in mice include changes to the dosing regimen 9 weeks into the study, cyclical dosing
periods, and decreased survival in all study groups for the male mice. Individual animal data
were unavailable to perform time-to-tumor modeling or adjust the tumor incidences for survival
before BMD modeling. The cancer incidence data are summarized in Table 5-5.
Table 5-5. Summary of incidence data in rodents orally exposed to HCE for
use in cancer dose-response assessment
Study
NTP (1989)
NTP (1989)
NCI (1978)
NCI (1978)
Sex/strain/species
Male F344 rats
Male F344 rats
Male B6C3FJ mice
Female B6C3FJ mice
Endpoint
Kidney adenoma or
carcinoma
Pheochromocytomas/
malignant
pheochromocytomas
Hepatocellular
carcinoma
Hepatocellular
carcinoma
HCE dose
(mg/kg-day)
0
7.1
14.3
0
7.1
14.3
0
360
722
0
360
722
Incidence
1/50 (2%)
2/50 (4%)
7/50 (14%)b
14/50 (28%)
26/45 (58%)b
19/49 (39%)
3/20 (15%) a
15/50 (30%)b
31/49(63%)b
2/20 (10%) a
20/50 (40%)b
15/49 (3 l%)b
"Incidence data are for the matched vehicle controls rather than the pooled controls from NCI (1978).
bDenotes statistical significance.
5.4.3. Dose Adjustments and Extrapolation Methods
The HCE doses administered to laboratory animals were scaled to human equivalent
doses (HEDs) according to EPA guidance (U.S. EPA, 2005a, 1992). More specifically, animal
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doses were converted to HEDs by assuming that doses in animals and humans are lexicologically
equivalent when scaled by body weight raised to the % power, as follows:
Dose(mg I day] \_ammai\ _ Dose(mg I day] [human]
BW3/\ammal] BW3/\human]
The body weights for the laboratory animals used in the scaled human dose conversions
are the mean body weights reported in the studies for each dose group. The following formula
was used for the conversion of oral animal doses to oral HEDs:
Scaled human dose (HED) = animal dose x (animal body weight/human body weight)'74
Therefore, the HCE doses of 7 and 14 mg/kg-day employed by NTP (1989) in rats were
converted to HEDs, as follows:
Scaled human dose (HED) = 7 mg/kg-day x (0.483 kg/70 kg)1/4
= 2.05 mg/kg-day
Scaled human dose (HED) = 14 mg/kg-day x (0.471 kg/70 kg)'/4
= 4.10 mg/kg-day
Similarly, the HCE doses of 360 and 722 mg/kg-day employed by NCI (1978) in mice
were converted to HEDs, as follows:
Scaled human dose (HED) = 360 mg/kg-day x (0.033 kg/70 kg)
= 53.05 mg/kg-day
Scaled human dose (HED) = 722 mg/kg-day x (0.030 kg/70 kg)'/4
= 103.88 mg/kg-day
These scaled human doses are used in the dose-response modeling described below.
The multistage model was the primary model considered for fitting the dose-response
data and is given by:
P(d) = 1 - exp[-(q0 + qid + q2d2 + ... + qkdk)],
where:
P(d) = lifetime risk (probability) of cancer at dose d
q; = parameters estimated in fitting the model, i = 1, ..., k
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And extra risk is defined as (P(d) -P(0))/(1-P(0)).
The multistage model in BMDS (version 2.0) (U.S. EPA, 2008) was fit to the incidence
data summarized in Table 5-5 using the calculated HEDs in order to derive an oral slope factor
for HCE. In the NCI (1978) data, the low survival rates in the vehicle and untreated male control
groups led the authors to compare tumor incidences in the dosed males and females to vehicle
control data pooled from bioassays for hexachloroethane, trichloroethane, and 1,1,2-
trichloroethane. For BMD modeling, the incidence of hepatocellular carcinoma in the exposed
group was compared to the incidence of hepatocellular carcinoma in the matched vehicle
controls rather than the pooled controls. The BMR selected was the default value of 10% extra
risk recommended for dichotomous models (U.S. EPA, 2000b). No data were excluded from the
BMD multistage modeling.
As stated above, the multistage model was fit to the incidences of renal adenomas or
carcinomas combined in male rats and hepatocellular carcinomas in male mice. In all cases, the
2° multistage model provided the best fit. The multistage model was also fit to the incidence of
pheochromocytomas or malignant pheochromocytomas in male rats and the incidence of
hepatocellular carcinomas in female mice. The model exhibited a significant lack of fit for the
pheochromocytomas and hepatocellular carcinomas in female mice (according to the %2 statistic
with/? < 0.1, see Appendix B for modeling outputs). Thus, these datasets were not useful for
dose-response assessment because the tumor incidences are not a monotonic increasing function
of dose, as demonstrated by the Cochran-Armitage Trend Test. Therefore, the BMD modeling
results for the kidney and liver tumors in male rats and male mice, respectively, are summarized
in Table 5-6, with more detailed results contained in Appendix B.
Table 5-6. Summary of BMD modeling results for oral cancer
assessment of HCE
Study
NTP
(1989)
NCI
(1978)
Sex/strain/
species
Male F344
rats
Male
B6C3FJ
mice
Endpoint
Renal
adenomas/carcinomas
combined
Hepatocellular
carcinomas
"Best-fit"
model
2° Multistage
2° Multistage
BMR
0.1
0.1
BMD10
3.74
38.09
BMDL10
or POD
2.45
13.80
Oral slope
factor
(mg/kg-d)-1
0.041
0.007
The U.S. 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 linear approach is used as a default option if the mode of action of carcinogenicity is
not understood (U.S. EPA, 2005a). There are data to indicate that the mode of carcinogenic
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action of HCE in the rat kidney,may be associated with male rat-specific a2U-globulin
accumulation and toxicity. As described in Section 4.7.3.1, two principal factors contribute to
the conclusion that the available data are insufficient support an a2U-globulin mode of action for
the development of renal tumors: (1) the lack of information identifying the a2U-globulin protein
in HCE-treated rats, and (2) evidence of nephropathy in female rats as well as male and female
mice (because the a2U-globulin-related mode of action is specific for male rats). Therefore, a
linear low-dose extrapolation approach was used to estimate human carcinogenic risk associated
with HCE exposure. It is recognized that an a2U-globulin-associated mode of action may, in fact,
be responsible for the tumors observed in male rats and that more than one mode of action may
be operating to induce the nephropathy observed across species and sexes. In that case, the renal
tumors would be utilized for quantitation of cancer risk as they would be characterized as not
relevant to humans.
5.4.4. Oral Slope Factor and Inhalation Unit Risk
The candidate oral slope factors were derived by linear extrapolation to the origin from
the POD by dividing the BMR by the BMDLio (the lower bound on the exposure associated with
a 10% extra cancer risk). The oral slope factor represents an upper bound estimate on cancer risk
associated with a continuous lifetime exposure to HCE. In accordance with the U.S. EPA
guidelines (2005a), an oral slope factor for renal tumors in male rats of 0.04 (mg/kg-day)"1 was
calculated by dividing the BMR of 0.1 by the human equivalent BMDLio of 2.45 mg/kg-day
(Appendix B). An oral slope factor for hepatocellular tumors in male mice of 0.007 (mg/kg-
day)"1 was calculated by dividing the BMR of 0.1 by the human equivalent BMDLio of 13.80
mg/kg-day (Appendix B). The rats exhibited greater sensitivity to HCE-induced carcinogenicity
than the mice Thus, the risk estimate associated with the male rats that developed renal
adenomas or carcinomas was selected as the oral slope factor of 0.04 (mg/kg-day)"1 for
HCE.
In the absence of data on the carcinogenicity of HCE via the inhalation route, an
inhalation unit risk has not been derived.
5.4.5. Uncertainties in Cancer Risk Values
Extrapolation of data from animals to estimate potential cancer risks to human
populations from exposure to HCE yields uncertainty. Several types of uncertainty may be
considered quantitatively, whereas others can only be addressed qualitatively. Thus, an overall
integrated quantitative uncertainty analysis cannot be developed. Major sources of uncertainty in
the cancer assessment for HCE are summarized in Section 5.4.5.1 and in Table 5-7.
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Table 5-7. Summary of uncertainties in the HCE cancer risk assessment
Consideration/
approach
Human relevance
of rodent tumor
data
Bioassay
Species/gender
choice
Dose metric
Low-dose
extrapolation
procedure
Cross-species
scaling
Statistical
uncertainty at
POD
Human
population
variability in
metabolism and
response/sensitive
subpopulations
Impact on oral slope
factor
Human risk could | or |,
depending on relative
sensitivity; if rodent
tumors proved not to be
relevant to humans, oral
cancer risk estimate
would not apply (i.e.,
human risk would |)
Alternatives could t or J,
oral slope factor by an
unknown extent
Human risk could t or J,,
depending on relative
sensitivity
Alternatives could t or J,
oral slope factor by an
unknown extent
Alternatives could t or J,
oral slope factor by an
unknown extent
Alternatives could | or |
the oral slope factor
(e.g., 3.5-fold I [scaling
by body weight] or f
2-fold [scaling by
BW2/3])
I oral slope factor
1.5-fold if BMD used as
the POD rather than
lower bound on POD
Low-dose risk f or J, to
an unknown extent
Decision
Kidney and adrenal
gland tumors in male
rats and liver tumors in
male and female mice
are relevant to human
exposure
NTP study
Incidence of renal
adenoma/carcinoma in
male rats
Used administered
exposure
Multistage model to
determine POD, linear
low-dose extrapolation
from POD (default
approach)
BW3/4 (default
approach)
BMDL (preferred
approach for
calculating reasonable
upper bound slope
factor)
Considered
qualitatively
Justification
It was assumed that rodent tumors are relevant
to humans; tumor correspondence is
unknown. The carcinogenic response occurs
across species. HCE is a multi-site
carcinogen, although direct site concordance
is generally not assumed (U.S. EPA, 2005a).
Alternative bioassays in rats were unavailable.
A NCI (1978) bioassay in mice was available,
although mice were less sensitive than rats to
HCE carcinogenicity and were not utilized in
estimating carcinogenic risk to humans.
It was assumed that humans are as sensitive as
the most sensitive rodent gender/species
tested; true correspondence is unknown.
Increased tumor incidence in mice resulted in
a lower risk estimate than rats. No increase of
kidney tumors was observed in female rats.
Experimental evidence supports a role for
metabolism in toxicity, but actual responsible
metabolites are not identified. If the
responsible metabolites are generated in
proportion to administered dose, the estimated
slope factor is an unbiased estimate.
Available mode-of-action data do not inform
selection of dose-response model; linear
approach employed in absence of support for
an alternative approach.
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.
Limited size of bioassay results in sampling
variability; lower bound is 95% confidence
interval on administered exposure.
No data to support range of human
variability /sensitivity, including whether
children are more sensitive.
t = increase; J, = decrease
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5.4.5.1. Sources of Uncertainty
Relevance to humans. The modes of action for the kidney (adenomas/carcinomas) and
adrenal gland tumors (pheochromocytomas) in male rats and liver tumors (hepatocellular
carcinomas) in male and female mice are unknown. There are some data in experimental
animals evaluating a2U-globulin accumulation and toxicity in the kidney. As described in
Section 4.7.3, two principal factors contribute to the conclusion that there are insufficient data to
support an a2U-globulin mode of action for the development of renal tumors. First, the presence
of kidney effects in HCE-exposed male and female mice, which generally do not accumulate the
a2U-globulin protein, suggests a mode of action other than a2U-globulin nephropathy. Second,
none of the HCE studies performed the necessary immunohistochemical assays to confirm the
presence of a2U-globulin protein within the hyaline droplets observed following administration of
HCE (NTP, 1996, 1989). This represents a data gap.
The relevance of the mode of action of liver tumor induction to humans was considered
in Section 4.7.2. There is no available information regarding hepatic cancer associated with
HCE exposure in humans. The experimental animal literature, however, shows that oral
exposure to HCE induces liver tumors in male and female mice. It is possible that the HCE-
induced hepatocellular carcinomas in mice occur as a result of the binding of HCE metabolites to
liver macromolecules and the generation of free radicals during HCE metabolism, causing key
events in the carcinogenic process such as cytotoxicity, inflammation, and regenerative cell
proliferation. Limited information exists to distinguish the similarities and differences between
experimental animals and humans in terms of HCE metabolism or toxicity. However, these
potential key events have not been evaluated for HCE.
Pheochromocytomas are catecholamine-producing neuroendocrine tumors. The
relevance of rodent pheochromocytomas as a model for human cancer risk has been the subject
of discussion in the scientific literature (e.g., Greim et al., 2009; Powers et al., 2008). In humans,
pheochromocytomas are rare and usually benign, but may also present as or develop into a
malignancy (Eisenhofer et al., 2004; Lehnert et al., 2004; Elder et al., 2003; Goldstein et al.,
1999). Hereditary factors in humans have been identified as important in the development of
pheochromocytomas (Eisenhofer et al., 2004). Pheochromocytomas are more common in
laboratory rats, though evidence suggests that certain rat pheochromocytomas may have
similarity to human pheochromocytomas (Powers et al., 2009). Furthermore, mechanisms of
action inducing pheochromocytomas in rats are expected to occur in humans as well (Greim et
al., 2009). Therefore, in the absence of information indicating otherwise, the kidney and adrenal
gland tumors in male rats and liver tumors in male and female mice are considered relevant to
humans.
Bioassay selection. The study by NTP (1989) was used for the development of an oral
slope factor. This study was conducted in both sexes of F344/N rats and used 50 male and
50 female rats per dose group. Test animals were allocated among two dose levels of HCE and
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an untreated control group. Animals were observed twice daily and examined weekly (for
14 weeks) then monthly for body weight and monthly for feed consumption. Animals were
necropsied and all organs and tissues were examined grossly and microscopically for
histopathological lesions for a comprehensive set of toxicological endpoints in both sexes.
Choice of species/gender. The oral slope factor for HCE was quantified using the tumor
incidence data for male rats, which were found to be more sensitive than male or female mice to
the carcinogenicity of HCE. The oral slope factor calculated from male rats was higher than the
slope factors calculated from male and female mice. As there is no information to inform which
species or gender of animals would be most applicable to humans, the most sensitive group was
selected for the basis of the oral slope factor. Though the mode of action for observed kidney
tumors in rodents is unknown, the evidence suggesting the kidney as a target organ of HCE
toxicity in both species lends strength to the concern for human carcinogenic potential.
Dose metric. HCE is potentially metabolized to PERC and pentachloroethane; however,
it is unknown whether a metabolite or some combination of parent compound and metabolites is
responsible for the observed toxicity and carcinogenicity of HCE. If the actual carcinogenic
moiety(ies) is(are) proportional to administered exposure, then use of administered exposure as
the dose metric provides an unbiased estimate of carcinogenicity. On the other hand, if
administered exposure is not the most relevant dose metric, then the impact on the human
equivalent slope factor is unknown. Consequently; the low-dose cancer risk value may be higher
or lower than that estimated, by an unknown amount. In the absence of data identifying the
carcinogenic moiety for HCE, the administered exposure was selected as the dose metric.
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 HCE exposure, in the
absence of information to inform the dose-response at low doses. The extent to which the
overall uncertainty in low-dose risk estimation could be reduced if the mode of action for HCE
were known is of interest, but data on the mode of action of HCE are limited and the mode of
action is not known. If an a2U-globulin-associated mode of action is, in fact, responsible for male
rat tumor formation, then these tumors would not have been utilized for quantitation of cancer
risk as they would have been characterized as not relevant to humans.
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 0.007 to 0.04 per mg/kg-day, a range less than one order of magnitude,
with greater risk coming from the male rat kidney data.
Choice of model. All risk assessments involve uncertainty, as study data are extrapolated
to make inferences about potential effects in humans from environmental exposure. The largest
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sources of uncertainty in the HCE cancer risk estimates are interspecies extrapolation and low-
dose extrapolation. There are no human data from which to estimate human cancer risk;
therefore, the risk estimate must rely on data from studies of rodents exposed to levels greater
than would occur from environmental exposures.
Without human cancer data or better mechanistic data, the relevance of the rodent cancer
results to humans is uncertain. The occurrence of increased incidences of kidney and adrenal
gland tumors in male rats, and liver tumors in male and female mice exposed to HCE from the
oral route of exposure suggests that HCE is potentially carcinogenic to humans as well.
However, the lack of concordance in tumor sites between the two rodent species makes it more
difficult to quantitatively estimate human cancer risk.
Regarding low-dose extrapolation, in the absence of mechanistic data for biologically
based low-dose modeling or mechanistic evidence supporting a nonlinear approach (see the
discussion at the beginning of Section 5.4.3), a linear low-dose extrapolation was carried out
from the BMDLio. It is expected that this approach provides an upper bound on low-dose cancer
risk for humans. The true low-dose risks cannot be known without additional data.
With respect to uncertainties in the dose-response modeling, the two-step approach of
modeling only in the observable range (U.S. EPA, 2005a) and extrapolating from a POD in the
observable range is designed in part to minimize model dependence. Measures of statistical
uncertainty require assuming that the underlying model and associated assumptions are valid for
the data under consideration. The multistage model used provided an adequate fit to all the
datasets for kidney and liver tumors. For the multistage model applied to the incidence of
tumors, the BMDLs should generally be within a factor of 3 of the BMDs. This indicates that
there is a reasonably typical degree of uncertainty at the 10% extra risk level. A large difference
between the BMD and BMDL raises concern that the algorithm for the calculation of the BMDL
is not accurate (U.S. EPA, 2000b). The ratios of the BMDio values to the BMDLio values did
not exceed a value of 2.6, indicating that the estimated risk is not influenced by any unusual
variability in the model and associated assumptions.
Cross-species scaling. An adjustment for cross-species scaling (BW3/4) was applied to
address toxicological equivalence of internal doses between rats and humans, consistent with the
Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a). It is assumed that equal risks
result from equivalent constant lifetime exposures.
Human population variability. The extent of inter-individual variability or sensitivity to
the potential carcinogen!city of HCE is unknown. There are no data exploring whether there is
differential sensitivity to HCE carcinogenicity across life stages. In addition, neither the extent
of interindividual variability in HCE metabolism nor human variability in response to HCE has
been characterized. Factors that could contribute to a range of human responses to HCE include
variations in CYP450 levels because of age-related differences or other factors (e.g., exposure to
other chemicals that induce or inhibit microsomal enzymes), nutritional status, alcohol
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consumption, or the presence of underlying disease that could alter metabolism of HCE or
antioxidant protection systems. This lack of understanding about potential susceptibility
differences across exposed human populations thus represents a source of uncertainty. Humans
are expected to be more genetically heterogeneous than inbred strains of laboratory animals
(Calderon, 2000), and this variability is likely to be influenced by ongoing or background
exposures, diseases, and biological processes.
5.4.6. Previous Cancer Assessment
The previous HCE cancer assessment was based on the incidence of hepatocellular
carcinomas in male mice in the NCI (1978) study. The current risk value is derived from the
incidence of renal adenomas or carcinomas in male rats (NTP, 1989), resulting in an oral slope
factor approximately 2.8-fold higher than the one derived in the previous assessment.
In addition, the scaled human doses were calculated using a slightly different formula
than is current practice:
Scaled human dose = animal dose x (animal weight/human body weight)13 x (546/637)
The difference in the animal-to-human dose scaling procedure is due to the fact that
current practice bases dose equivalence on the % power of body weight instead of the previous
% power of body weight.
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6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE
RESPONSE
6.1. HUMAN HAZARD POTENTIAL
HCE is a halogenated hydrocarbon consisting of six chlorines attached to an ethane
backbone. HCE was produced in the United States from 1921 to 1967, but is currently not
commercially distributed. HCE is primarily used in the military for smoke pots, smoke
grenades, and pyrotechnic devices. In the past, HCE was used as antihelminthic for the
treatment of sheep flukes, but is no longer used for this purpose since the FDA withdrew
approval for this use in 1971. HCE has also been used as a polymer additive, a moth repellant, a
plasticizer for cellulose esters, and an insecticide solvent, and in metallurgy for refining
aluminum alloys.
There is limited information on the toxicity of HCE in humans. Current understanding of
HCE toxicology is based on the limited database of animal studies. After absorption by oral
exposure, HCE is primarily distributed to fat tissue. Toxicokinetic studies in animals indicated
that HCE is also localized and metabolized in the liver and kidney. Kidney concentrations of
HCE were higher in male rats than female rats (Gorzinski et al., 1985; Nolan and Karbowski,
1978). Studies of HCE metabolism indicated that the major CYP450 enzymes involved are
phenobarbital-inducible, which include the 2A, 2B, and 3A subfamilies (Salmon et al., 1985,
1981; Town and Leibman, 1984; Nastainczyk et al., 1982, 1981). HCE is putatively metabolized
via a pentachloroethyl free radical to PERC and pentachloroethane. Pentachloroethane is then
metabolized to TCE. TCE and PERC are further metabolized by hepatic oxidation to several
urinary metabolites including TCA, trichloroethanol, oxalic acid, dichloroethanol, dichloroacetic
acid, and monochloroacetic acid (Mitoma et al., 1985; Nastainczyk et al., 1982, 1981; Bonse and
Henschler, 1976; Fowler, 1969; Jondorf et al., 1957). Metabolism is minimal based on the few
studies that provided quantitative data on metabolites. However, several of these metabolites
have demonstrated liver and kidney toxicities similar to HCE.
The kidney has consistently been shown as the target for toxicity in acute, subchronic,
and chronic toxicity bioassays in animals (NTP, 1996, 1989; Gorzinski et al., 1985; NCI, 1978).
Noncancer effects include kidney degeneration (tubular nephropathy, necrosis of renal tubular
epithelium, hyaline droplet formation, tubular regeneration, and tubular casts) and hepatocellular
necrosis. Hepatotoxicity was noted in animals exposed to HCE, although endpoints of this
nature have not been evaluated in laboratory animals as fully as the renal effects. Hepatocellular
necrosis was reported in female rats (NTP, 1989), but was not evaluated in a chronic exposure
study of mice (NCI, 1978). The mouse study (NCI, 1978) focused on tumorigenic endpoints
rather than noncancer effects.
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There is no information available describing the metabolism of HCE following exposure
via inhalation. The inhalation database for HCE contains one acute (Weeks and Thomasino,
1978) and one subchronic (Weeks et al., 1979) study. Neurological effects, such as tremors and
ataxia, were observed in male Beagle dogs, male and female rats, and pregnant rats. Other
effects included reduced body weight gain and increased relative liver weight in rats and guinea
pigs exposed to HCE via inhalation. Male rats also displayed increased relative spleen and testes
weights.
Under EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), HCE is
"likely to be carcinogenic to humans" because HCE induced kidney and adrenal gland tumors in
male rats and liver tumors in male and female mice. Studies evaluating the carcinogenicity in
humans exposed to HCE are unavailable. The carcinogenicity incidence data in male rats (NTP,
1989) were used to develop a quantitative cancer risk assessment for HCE. The consistency of
the kidney and liver as target organs in different species for HCE distribution and metabolism,
and both noncancer and cancer endpoints, provides support for the evaluation of these endpoints
as relevant to humans.
6.2. DOSE RESPONSE
6.2.1. Oral Noncancer
Subchronic and chronic bioassays in rats and mice have identified the following
endpoints after exposure to HCE: tubular nephropathy, atrophy and degeneration of renal
tubules, and hepatocellular necrosis. In female rats, tubular nephropathy, atrophy and
degeneration of the renal tubules, and hepatocellular necrosis were observed in a statistically
significant dose-response manner (NTP, 1989; Gorzinski et al., 1985; NCI, 1978). Tubular
nephropathy, severity of nephropathy, and atrophy and degeneration of the renal tubules in male
rats demonstrated a statistically significant dose response. Although mice were evaluated in a
chronic exposure study (NCI, 1978), noncancer effects were not reported because this study was
focused on tumorigenic endpoints.
The most sensitive endpoint identified for HCE by oral exposure relates to kidney
toxicity in the 16-week feeding study by Gorzinski et al. (1985) in male rats. Gorzinski et al.
(1985) was selected as the principal study and atrophy and degeneration of renal tubules in male
rats were chosen as the critical effect for the derivation of the oral RfD. This study included both
sexes of F344 rats, 10 animals/sex/dose, and three dose groups plus controls (0, 1,15, and 62
mg/kg-day). Dose-response analyses of the noncancer endpoint, atrophy and degeneration of
renal tubules (Gorzinski et al., 1985), using EPA's BMDS, resulted in a POD of
0.728 mg/kg-day. A composite UF of 1,000 was applied to the POD to derive an oral RfD of
7 x 10"4 mg/kg-day.
Confidence in the principal study, Gorzinski et al. (1985), is high. The 16-week study is
a well-conducted study that used three dose groups plus a control. NTP (1989) also conducted
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16-day, 13-week, and 103-week studies that supported the results observed in the 16-week study.
Application of BMD modeling provided a POD upon which to base the derivation of the RfD.
The critical effect on which the RfD is based is well-supported by other oral short-term,
subchronic, and chronic studies. Confidence in the database is low to medium because the
database includes acute, short-term, subchronic, and chronic toxicity studies and developmental
toxicity studies in rats and chronic carcinogenicity bioassays in rats and mice. The database
lacks a multigenerational reproductive study and studies in other species. Overall confidence in
the RfD is low to medium.
6.2.2. Inhalation Noncancer
The inhalation toxicity database is limited to a single 6-week repeat-exposure study by
Weeks et al. (1979). This study reported a NOAEL of 465 mg/m3 and a LOAEL of 2,517 mg/m3
in several species including Sprague-Dawley rats, male Beagle dogs, and male Hartley guinea
pigs. The effects described in this report include neurotoxicity, reduced body weight gain, and
increased relative liver, spleen, and testes weights. Based on neurological effects in Sprague-
Dawley rats, the NOAEL of 465 mg/m3 was selected to serve as the POD. Adjustments for
continuous exposure and for the HEC, resulted in the POD[HEC] of 83 mg/m3. An UF of 3,000
was applied to derive an inhalation RfC of 3 x 10"2 mg/m3. Confidence in the principal study,
Weeks et al. (1979), is low. The 6-week study was conducted in several species (including male
dogs, male and female rats, male guinea pigs, and quail). The study used three exposure groups
(145, 465, and 2,517 mg/m3) plus a control. The study is limited by the relatively short exposure
duration (6 weeks) and minimal reporting of effects, especially quantitative changes.
Application of BMD modeling was precluded based on a 100% response in animals for the
neurological effects and the lack of quantitative information. Therefore, a NOAEL served as the
POD. The critical effect on which the RfD is based is supported by the oral short-term study
conducted by the same investigators and two oral subchronic studies. Confidence in the database
is low because the database includes one acute and one subchronic toxicity study in multiple
species and one developmental toxicity study in rats. The database lacks studies by another
laboratory and a multigenerational reproductive study. Overall confidence in the RfC is low.
6.2.3. Cancer
Under EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), HCE is
"likely to be carcinogenic to humans" by all routes of exposure. This descriptor is based on
evidence of carcinogenicity from animal studies. HCE induced statistically significant increases
in the incidence of kidney and adrenal gland tumors in male rats and liver tumors in male and
female mice. The NTP (1989) rat study was selected for dose-response assessment based on
statistically significant increased incidences of renal adenomas and carcinomas and adrenal
pheochromocytomas and malignant pheochromocytomas in male rats. This study was used for
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development of an oral slope factor. This was a well-designed study, conducted in both sexes of
F344 rats with 50 rats/sex/dose, typical of carcinogenicity bioassays. Test animals were
allocated among two dose levels (7 and 14 mg/kg-day) and an untreated control group. Animals
were observed twice daily and examined weekly (for 14 weeks) and then monthly for body
weight and monthly for feed consumption. Animals were necropsied and all organs and tissues
were examined grossly and microscopically for histopathological lesions for a comprehensive set
of toxicological endpoints in both sexes.
Renal adenomas and carcinomas and pheochromocytomas and malignant
pheochromocytomas observed in male rats (NTP, 1989) were not seen in female rats or other
species orally-exposed to HCE. Hepatocellular carcinomas were observed in male and female
mice, but not in the rats. The male B6C3Fi mice tumor incidence data (NCI, 1978)
demonstrated evidence of carcinogenicity and a low-dose quantitative risk estimate was derived.
The cancer risk associated with mice exposed to HCE was less sensitive than that of rats. Thus,
the oral slope factor derived for HCE is based on the increased incidence of kidney tumors in
male rats.
A linear approach was applied in the dose-response assessment for HCE, consistent with
U.S. EPA's (2005a) Guidelines for Carcinogen Risk Assessment. The guidelines recommend the
use of a linear extrapolation as a default approach when the available data are insufficient to
establish a mode of action for a tumor site. As discussed in Section 4.7, while there are data to
indicate that the the mechanism leading to the formation of the kidney tumors may be due to
a2U-globulin accumulation, important information is lacking and data indicating nephrotoxicity in
other species and sexes confound any conclusions. The database for HCE lacks information on
the mode of action and the shape of the curve in the region below the POD; therefore, a linear
extrapolation was performed in determining the oral slope factor in the derivation of a
quantitative estimate of cancer risk for ingested HCE.
Increased incidence of renal adenomas and carcinomas in a 2-year rat bioassay (NTP,
1989) served as the basis for the oral cancer dose-response analysis. A multistage model using
linear extrapolation from the POD was performed to derive an oral slope factor of
9 1
4 x 10" (mg/kg-day)" for HCE. Extrapolation of the experimental data to estimate potential
cancer risk in human populations introduces uncertainty in the risk estimation for HCE.
Uncertainty can be considered quantitatively; however, some uncertainty can only be addressed
qualitatively. For this reason, an overall integrated quantitative uncertainty analysis cannot be
developed. However, EPA's development of the cancer quantitative assessment for HCE
included consideration of potential areas of uncertainty.
A biologically-based model was not supported by the available data; therefore, a multistage
model was the preferred model. The multistage model can accommodate a wide variety of dose-
response shapes and provides consistency with previous quantitative dose-response assessments
for cancer. Linear low-dose extrapolation from a POD determined by an empirical fit of tumor
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data has been judged to lead to plausible upper bound risk estimates at low doses for several
reasons. However, it is unknown how well this model or the linear low-dose extrapolation
predicts low dose risks for HCE. An adjustment for cross-species scaling (BW3/4) was applied to
address toxicological equivalence of internal doses between rats and humans based on the
assumption that equal risks result from equivalent constant lifetime exposures.
An inhalation unit risk was not derived in this assessment. Data on the carcinogenicity of
HCE via the inhalation route are unavailable, and route-to-route extrapolation was not possible
due to the lack of a PBPK model. However, it is proposed that HCE is likely to be carcinogenic
to humans by the inhalation route since the compound is absorbed and, in oral studies, induces
tumors at sites other than the portal of entry.
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7. REFERENCES
ACGIH (American Conference of Governmental Industrial Hygienists). (1991) Documentation of the threshold limit
values and biological exposure indices. 6th edition. Cincinnati, OH: American Conference of Governmental
Industrial Hygienists.
ACGIH (2001) Hexachloroethane. In: TLV chemical substances 7th edition. Cincinnati, OH: American Conference
of Governmental Industrial Hygienists.
Allen, MB; Crisp, A; Snook, N; et al. (1992) Smoke-bomb pneumonitis. RespirMed 86:165-166.
Ashby, J; Tennant, RW. (1988) Chemical structure, salmonella mutagenicity and extent of carcinogenicity as
indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the U.S. NCI/NTP. Mutat Res
204:17-115.
ATSDR (Agency for Toxic Substances and Disease Registry). (1997c) Toxicological profile for hexachloroethane.
Atlanta, GA: U.S. Department of Health and Humans Services. Available from
http://www.atsdr.cdc.gov/toxprofiles/tp97.pdf.
ATSDR (1997b) Toxicological profile for tetrachloroethylene. Atlanta, GA: U.S. Department of Health and
Humans Services. Available online at http://www.atsdr.cdc.gov/.
ATSDR (1997a) Toxicological profile for trichloroethylene. Atlanta, GA: U.S. Department of Health and Humans
Services. Available online at http://www.atsdr.cdc.gov/.
ATSDR (2008) Toxicological profile for 1,1,2,2-tetrachloroethane. Atlanta, GA: U.S. Department of Health and
Humans Services. Available online at http://www.atsdr.cdc.gov/.
Axelson, O. (1985) Halogenated alkanes and alkenes and cancer: epidemiological aspects. In: Fishbein, L; O'Neill,
IK, eds. Environmental carcinogens: selected methods of analysis. Vol. 7. Lyon, France: International Agency for
Research on Cancer, pp. 5-20.
Beurskens, JE; Stams, AJ; Zehnder, AJ; et al. (1991) Relative biochemical reactivity of three
hexachlorocyclohexane isomers. Ecotoxicol Environ Saf 21:128-136.
Blanco, JG; Harrison, PL; Evans, W; et al. (2000) Human cytochrome P450 maximal activities in pediatric versus
adult liver. Drug Metab Disp 28(4):379-382.
Bonse, G; Henschler, D. (1976) Chemical reactivity, biotransformation, andtoxicity of poly chlorinated aliphatic
compounds. Crit Rev Toxicol 4:395-409.
Bronzetti, G; Morichetti, E; Del Carratore, R; et al. (1989) Tetrachloroethane, pentachloroethane, and
hexachloroethane: genetic and biochemical studies. Teratog Carcinog Mutagen 9:349-357.
Bronzetti, G; Morichetti, E; Vellosi, R; et al. (1990) Genotoxicity and effects on microsomal enzymes of three
chlorinated ethanes. Mutat Res 234:429-430.
Budavari, S; O'Neil, MJ; Smith, A; et al., eds. (1989) The Merck index: an encyclopedia of chemicals, drugs, and
biologicals. 11th edition. Rahway, NJ: Merck & Co., Inc., p. 740.
Bull, RJ; Sanchez, IM; Nelson, MA; et al. (1990) Liver tumor induction in B6C3F1 mice by dichloroacetate and
trichloroacetate. Toxicology 63:34lB359.
Calderon, RL. (2000) Measuring Risks in Humans: the Promise and Practice of Epidemiology. Food Chem Toxicol
38: S59-S63.
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-------
Cal EPA (California Environmental Protection Agency). (2001) Public health goal for tetrachloroethylene in
drinking water. Office of Environmental Health Hazard Assessment. Available online at
http://oehha.ca.gov/water/phg/pdf/PCEAug2001 .pdf.
Cazeneuve, C; Pons, G; Rey, E; et al. (1994) Biotransformation of caffeine in human liver microsomes from
foetuses, neonates, infants and adults. Br J Clin Pharmacol 37:405^412.
ChemlDplus Advanced. (2005) Hexachloroethane. ChemFinder.com database & internet searching. Available
online at http://chem.sis.nlm.nih.gov/chemidplus/.
Crebelli, R; Benigni, R; Franekic, J; et al. (1988) Induction of chromosome malsegregation by halogenated organic
solvents mAspergillue nidulans: unspecific or specific mechanism? Mutat Res 201:401^411.
Crebelli, R; Andreoli, C; Carere, A; et al. (1992) The induction of mitotic chromosome malsegregation in
Aspergillus nidulans. Quantitative structure activity relationship (QSAR) analysis with chlorinated aliphatic
hydrocarbons. Mutat Res 266:117-13 4.
Crebelli, R; Andreoli, C; Carere, A; et al. (1995) Toxicology of halogenated aliphatic hydrocarbons: structural and
molecular determinants for the disturbance of chromosome segregation and the induction of lipid peroxidation.
Chem Biol Interact 98(2): 113-129.
Crebelli, R; Carere, A; Leopardi, P; et al. (1999) Evaluation of 10 aliphatic halogenated hydrocarbons in the mouse
bone marrow micronucleus test. Mutagenesis 14(2):207-215.
Doherty, AT; Ellard, S; Parry, EM; et al. (1996) An investigation into the activation and deactivation of chlorinated
hydrocarbons to genotoxins in metabolically competent human cells. Mutagenesis 11(3):247-274.
Doi, AM; Hill, G; Seely, J; et al. (2007) o2u-Globulin nephropathy and renal tumors in national toxicology program
studies. ToxicolPathol35(4): 533-540.
Dome, JLCM. (2004) Impact of inter-individual difference in drug metabolism and pharmacokinetics on safety
evaluation. Fundam Clin Pharmacol 18:609-620.
Elder, EE; Xu, D; Hoog, A; et al. (2003) KI-67 AND hTERT expression can aid in the distinction between
malignant and benign pheochromocytoma and paraganglioma. Mod Pathol 16(3):246-255.
Eisenhofer, G; Huynh, TT; Pacak, K; et al. (2004) Distinct gene expression profiles in norepinephrine- and
epinephrine-producing hereditary and sporadic pheochromocytomas: activation of hypoxia-driven angiogenic
pathways in von Hippel-Lindau syndrome. EndocrRelat Cancer 11(4):897-911.
Evans, WE; Relling, MF; de Graaf, S; et al. (1989) Hepatic drug clearance in children: studies with indocyanine
green as a model substrate. J Pharmacol Exp Ther 78:452^456.
Fiserova-Bergerova, V; Pierce, JT; Droz, PO. (1990) Dermal absorption potential of industrial chemicals: criteria for
skin notation. Am J Ind Med 17:617-635.
Fishbein, L. (1979) Potential halogenated industrial carcinogenic and mutagenic chemicals. II. Halogenated
saturated hydrocarbons. Sci Total Environ 11:163-195.
Fowler, JS. (1969) Some hepatotoxic action of hexachloroethane and its metabolites in sheep. Br J Pharmacol
35:530-542.
Fox, JG; Cohen, BJ; Loew, FM; eds. (1984) Laboratory animal medicine. New York, NY: Academic Press..
Galloway, SM; Armstrong, MJ; Reuben, C; et al. (1987) Chromosome aberrations and sister chromatid exchanges in
Chinese hamster ovary cells: evaluations of 108 chemicals. Environ Mol Mutagen 10:1-175.
Gargas, ML; Andersen, ME. (1989) Determining kinetic constants of chlorinated ethane metabolism in the rat from
rates of exhalation. Toxicol Appl Pharmacol 99:344-353.
121 DRAFT - DO NOT CITE OR QUOTE
-------
Gargas, ML; Seybold, PG; Andersen, ME. (1988) Modeling the tissue solubilities and metabolic rate constant Vmax
of halogenated methanes, ethanes and ethylenes: symposium on quantitative toxicology held at the 17th conference
on toxicology; November 3-5; Dayton, Ohio, USA. Toxicol Lett 43:235-256.
Goldstein, RE; O'Neill, JA, Jr; Holcomb, GW, III; et al. (1999) Clinical experience over 48 years with
pheochromocytoma. Ann Surg 229(6):755-764.
Goodman, DG.; Ward, JM; Squire, RA; et al. (1980) Neoplastic and non-neoplastic lesions in aging Osborne-
Mendel rats. Toxicol Appl Pharmacol 55:433-447.
Gorzinski, SJ; Nolan, RJ; McCollister, SB; et al. (1985) Subchronic oral toxicity, tissue distribution and clearance of
hexachloroethane in the rat. Drug Chem Toxicol 8:155-169.
Gorzinski, SJ; Wade, CE; McCollister, SB; et al. (1980) Hexachloroethane: results of a 16 week toxicity study in the
diet of CDF Fischer 344 rats. Midland, MI: Dow Chemical Company.
Greim, H: Hartwig, A; Reuter, U; et al. (2009) Chemically induced pheochromocytomas in rats: mechanisms and
relevance for human risk assessment. Critical Rev Toxicol 39(8):695-718.
Hard, GS; Rodgers, IS; Baetcke, KP; et al. (1993) Hazard evaluation of chemicals that cause accumulation of
a2u-globulin, hyaline droplet nephropathy, and tubule neoplasia in the kidneys of male rats. Environ Health Perspect
99:313-349.
Haworth, S; Lawlor, T; Mortelmans, K; et al. (1983) Salmonella mutagenicity test results for 250 chemicals.
Environ Mutagen 5(Suppl 1):3-142.
Hodge, HC; Sterner, JH. (1949) Tabulation of toxicity classes. Am Ind Hyg Assoc Q 10:93-96.
Holmes, DD. (1984) Clinical laboratory animal medicine. Ames, IA: Iowa State University Press.
Howard, PH; ed. (1989) Handbook of environmental fate and exposure data for organic chemicals. Vol. I. Large
production and priority pollutants. Chelsea, MI: Lewis Publishers.
IARC (International Agency for Research on Cancer). (1979) IARC monographs on the evaluation of the
carcinogenic risk of chemicals to humans. Vol. 20. Some halogenated hydrocarbons. Lyon, France: International
Agency for Research on Cancer, p. 467.
IARC. (1999) IARC scientific publications no. 147. Species differences in thyroid, kidney and urinary bladder
carcinogenesis. Lyon, France.
Jackson, MA; Stack, HF; Waters, MD. (1993) The genetic toxicology of putative nongenotoxic carcinogens. Mutat
Res 296:241-277.
Jondorf, WR; Parke, DV; Williams, RT. (1957) The metabolism of [14C]hexachloroethane. Biochem J 65:14-15.
JISA (Japan Industrial Safety Association). (1993) Carcinogenicity study of tetrachloroethylene by inhalation in rats
and mice. Data No. 3-1. Kanagawa, Japan. Available from: IRIS Information Desk, U.S. Environmental Protection
Agency, Washington, DC.
Kinkead, ER; Wolfe, RE. (1992) Single oral toxicity of various organic compounds. J Am Coll Toxicol 11(6):713.
Kulig, B; Alleva, E; Bignami, G; et al. (1996) Animal behavioral methods in neurotoxicity assessment: SGOMSEC
joint report. Environ Health Perspect 104(Suppl 2): 193-204.
Lacroix, D; Sonnier, M; Moncion, A; et al. (1997) Expression of CYP3 A in the human liver-evidence that the shift
between CYP3 A7 and CYP3 A4 occurs immediately after birth. Eur J Biochem 247:625-634.
122 DRAFT - DO NOT CITE OR QUOTE
-------
Lattanzi, G; Colacci, A; Grilli, S; et al. (1988) Binding of hexachloroethane to biological macromolecules from rat
and mouse organs. J Toxicol Environ Health 24:403-411.
Legator, MS; Harper, BL. (1988) Mutagenicity screening/in vitro testing—the end of an era; animal and human
studies—the direction for the future. Ann NY Acad Sci 534:833-844.
Lehnert, H; Mundschenk, J; Hahn, K. (2004) Malignant pheochromocytoma. Front Horm Res 31:155-162.
Loh, CH; Chang, YW; Liou, SH; et al. (2006) Case report: hexachloroethane smoke inhalation: a rare cause of
severe hepatic injuries. Environ Health Perspect 114(5):763-765.
Loh, CH; Liou, SH; Chang, YW; et al. (2008) Hepatic injuries of hexachloroethane smoke inhalation: the first
analytical epidemiological study. Toxicology 247(2-3): 119-122.
Lohman, PHM; Lohman, WJA. (2000) Genetic activity profiles 2000 (program version 1.3.0), data record for
hexachloroethane. Data base and software are a joint effort of the U.S. Environmental Protection Agency and the
International Agency for Research on Cancer.
Lutz, WK. (1979) In vivo covalent binding of organic chemicals to DNA as a quantitative indicator in the process of
chemical carcinogenesis. MutatRes 65:289-356.
Lutz, WK. (1986) Quantitative evaluation of DNA binding data for risk estimation and for classification of direct
and indirect carcinogens. J Cancer Res Clin Oncol 112:85-91.
Mather, GG; Exon, JH; Koller, LD. (1990) Subchronic 90-day toxicity of dichloroacetic and trichloroacetic acid in
rats. Toxicology 64:71-80.
Milman, HA; Story, DL; Riccio, ES; et al. (1988) Rat liver foci and in vitro assays to detect initiating and promoting
effects of chlorinated ethanes and ethylenes. Ann NY Acad Sci 534:521-530.
Mitoma, C; Steeger, T; Jackson, SE; et al. (1985) Metabolic disposition study of chlorinated hydrocarbons in rats
and mice. Drug Chem Toxicol 8:183-194.
Miyagawa, M; Takasawa, H; Sugiyama, A; et al. (1995) The in vivo-in vitro replicative DNA synthesis (RDS) test
with hepatocytes prepared from male B6C3F1 mice as an early prediction assay for putative nongenotoxic (Ames-
negative) mouse hepatocarcinogens. Mutat Res 343:157-183.
Nakamura, S; Oda, Y; Shimada, T; et al. (1987) SOS-inducing activity of chemical carcinogens and mutagens in
Salmonella typhimurium TA1535/pSK1002: examination with 151 chemicals. Mutat Res 192:239-246.
Nastainczyk, W; Ahr, H; Uhich, V; et al. (1981) The mechanism of the reductive dehalogenation of polyhalogenated
compounds by microsomal cytochrome P450. Adv Exp Med Biol 136(A):799-808.
Nastainczyk, W; Ahr, HJ; Ullrich, V. (1982) The reductive metabolism of halogenated alkanes by liver microsomal
cytochrome P450. Biochem Pharmacol 131:391-396.
NCI (National Cancer Institute). (1976) Carcinogenesis bioassay of trichloroethylene (CAS No. 79-01-6). Public
Health Service, U.S. Department of Health and Human Services; NTP TR-2. Available from: National Institute of
Environmental Health Sciences, Research Triangle Park, NC. Available online at
http://ntp.niehs.nih.gov/index.cfm?objectid=07028C7F-AB6E-6D29-3FClCC9D48574701.
NCI. (1977) Bioassay of tetrachloroethylene for possible carcinogenicity. Public Health Service, U.S. Department
of Health, Education, and Welfare; NTP TR-13. Available from: National Cancer Institute, Bethesda, MD.
Available online at http://ntp.niehs.nih.gov/index.cfm?objectid=0702B823-CEA9-1089-DFBDC6F9207C56F2.
NCI. (1978) Bioassay of hexachloroethane for possible carcinogenicity. Public Health Service, U.S. Department of
Health, Education, and Welfare; NTP TR-68. Available from: National Cancer Institute, Bethesda, MD. Available
online at http://ntp.niehs.nih.gov/ntp/htdocs/LT_rpts/tr068.pdf.
123 DRAFT - DO NOT CITE OR QUOTE
-------
Nolan, RJ; Karbowski, RJ. (1978) Hexachloroethane: tissue clearance and distribution in Fischer 344 rats. Midland,
MI: Dow Chemical Company.
NRC (National Research Council) (1983) Risk assessment in the federal government: managing the process.
Washington, DC: National Academy Press.
NRC (1987) Hexachloroethane smoke. Toxicity of Military Smoke and Obscurants, vol.1. Washington, DC:
National Academy Press.
NTP (National Toxicology Program). (1983) Carcinogenesis studies of pentachloroethane (CAS No. 76-01-7) in
F344/N rats and B6C3F1 mice (gavage study). Public Health Service, U.S. Department of Health and Human
Services; NTP TR-232. Available from National Institute of Environmental Health Sciences, Research Triangle
Park, NC. Available online at http://ntp.niehs.nih.gov/ntp/htdocs/LT_rpts/tr232.pdf.
NTP. (1986) Toxicology and carcinogenesis studies of tetrachloroethylene (perchloroethylene) (CAS No. 127-18-4)
in F344/N rats and B6C3F! mice (inhalation studies). Public Health Service, U.S. Department of Health and Human
Services; NTP TR-311. Available from National Institute of Environmental Health Sciences, Research Triangle
Park, NC. Available online at http://ntp.niehs.nih.gov/ntp/htdocs/LT_rpts/tr311.pdf.
NTP. (1988) Toxicology and carcinogenesis studies of trichloroethylene (CAS No. 79-01-6) infour strains of rats
(ACI, August, Marshall, Osborne-Mendel)(gavage studies). Public Health Service, U.S. Department of Health and
Human Services; NTP TR-273. Available from National Institute of Environmental Health Sciences, Research
Triangle Park, NC. Available online at http://ntp.niehs.nih.gov/ntp/htdocs/LT_rpts/tr273.pdf.
NTP. (1989) Toxicology and carcinogenesis studies of hexachloroethane (CAS No. 67-72-1) inF344/N rats (gavage
studies). Public Health Service, U.S. Department of Health and Human Services; NTP TR-361. Available from
National Institute of Environmental Health Sciences, Research Triangle Park, NC. Available online at
http://ntp.niehs.nih. gov/ntp/htdocs/LT_rpts/tr361.pdf.
NTP. (1990) Carcinogenesis studies of trichloroethylene (without epichlorohydrin) (CAS No. 79-01-6) inF344/N
rats and B6C3F1 mice (gavage study). Public Health Service, U.S. Department of Health and Human Services; NTP
TR-243. Available from National Institute of Environmental Health Sciences, Research Triangle Park, NC.
Available online at http://ntp.niehs.nih.gov/index.cfm?objectid=07067B36-09A5-8398-7E70FB2C35377215.
NTP. (1996) NTP technical report on renal toxicity studies of selected halogenated ethanes administered by gavage
to F344/N rats. Public Health Service, U.S. Department of Health and Human Services; NTP TOX-45. Available
from National Institute of Environmental Health Sciences, Research Triangle Park, NC. Available online at
http://ntp.niehs.nih.gov/ntpweb/index.cfm?objectid=D1512B41-FlF6-975E-7FBA3D4A2132FlCl.
NTP. (2005) 11th report on carcinogens. Public Health Service, U.S. Department of Health and Human Services,
Research Triangle Park, NC. Available online at http://ntp-server.niehs.nih.gov.
Odabasi, M. (2008) Halogenated volatile organic compounds from the use of chlorine-bleach-containing household
products. Environ Sci Technol 42(5):1445-1451.
Omiecinski, CJ; Remmel, RP; Hosagrahara, VP. (1999) Concise review of the cytochrome P450s and their roles in
toxicology. ToxicolSci48:151-156.
Onfelt, A. (1987) Spindle disturbances in mammalian cells. III. Toxicity, c-mitosis and aneuploidy with 22 different
compounds. Specific and unspecific mechanisms. MutatRes 182(3): 135-154.
Powers, JF; Picard, KL; Nyska, A; et al. (2008) Adrenergic Differentiation and Ret Expression in Rat
Pheochromocytomas. EndocrPathol 19:9-16.
Ramsey, JC; Andersen, ME. (1984) A physiologically based description of the inhalation pharmacokinetics of
styrene monomer in rats and humans. Toxicol Appl Pharmacol 73:159-175.
124 DRAFT - DO NOT CITE OR QUOTE
-------
Reynolds, ES. (1972) Comparison of early injury to liver endoplasmic reticulumby halomethanes,
hexachloroethane, benzene, toluene, bromobenzene, ethionine, thioacetamide and dimethylnitrosamine. Biochem
Pharmacol 21:2555-2561.
Roldan-Arjona, T; Garcia-Pedrajas, MD; Luque-Romero, FL; et al. (1991) An association between mutagenicity in
rodents for 16 halogenated aliphatic hydrocarbons. Mutagenesis 6:199-205.
Salmon, AG; Jones, RB; Mackrodt, WC. (1981) Microsomal dechlorination of chloroethanes: structure reactivity
relationships. Xenobiotica 11:723-734.
Salmon, AG; Nash, JA; Walkin, CM; et al. (1985) Dechlorination of halocarbons by microsomes and vesicular
reconstituted cytochrome P-450 systems under reductive conditions. Br J Ind Med 42:305-311.
Sandstedt, K; Berglof, A; Feinstein, R; et al. (1997) Differential susceptibility to Mycoplasma pulmonis intranasal
infection in X-linked immunodeficient (xid), severe combined immunodeficient (scid), and immunocompetent mice.
ClinExpImmunol 108:490^96.
Selden, A; Jacobson, G; Berg, P; et al. (1989) Hepatocellular carcinoma and exposure to hexachlorobenzene: a case
report. BrJ Ind Med 46:138-140.
Selden, A; Nygren, M; Kvamlof, A; et al. (1993) Biological monitoring of hexachloroethane. Int Ach Occup
Environ Health 65(Suppl 1):S 111-S114.
Selden, A; Kvamlof, A; Bodin, L; et al. (1994) Health effects of low level occupational exposure to
hexachloroethane. J Occup Med Toxicol 3(10):73-79.
Selden, Al; Nygren, Y; Westberg, HB; et al. (1997) Hexachlorobenzene and octachlorostyrene in plasma of
aluminium foundry workers using hexachloroethane for degassing. Occup Environ Med 54(8):613-618.
Selden, Al; Floderus, Y; Bodin, LS; et al. (1999) Porphyrin status in aluminum foundry workers exposed to
hexachlorobenzene and octachlorostyrene. Arch Environ Health 54(4):248-253.
Shimizu, M; Noda, T; Yamano, T; et al. (1992) Safety evaluation of chemicals for use in household products
(XVII). A teratological study on hexachloroethane in rats. Osaka City Institute of Public Health and Environmental
Sciences 54:70-75. (Japanese)
Simmon, VF; Kauhanen, K. (1978) In vitro microbiological mutagenicity assays of hexachloroethane. SRI
International, Menlo Park, CA. Prepared for U.S. Environmental Protection Agency, National Environmental
Research Center, Water Supply Research Laboratory, Cincinnati, OH.
Southcott, WH. (1951) The toxicity and antihelmintic efficiency of hexachloroethane in sheep. Aust Vet J 27:18-
21.
Spanggord, RJ; Chou, TW; Mill, T; et al. (1985) Environmental fate of nitroguanidine, diethyleneglycol dinitrate,
and hexachloroethane smoke. SRI International, Menlo Park, CA. Prepared for U.S. Army Medical Research and
Development Command.
Story, DL; Meierhenry, EF; Tyson, CA; et al. (1986) Differences in rat liver enzyme-altered foci produced by
chlorinated aliphatics and phenobarbital. Toxicol Ind Health 2:351-362.
Tafazoli, M; Baeten, A; Geerlings, P; et al. (1998) In vitro mutagenicity and genotoxicity study of a number of
short-chain chlorinated hydrocarbons using the micronucleus test and the alkaline single cell gel electrophoresis
technique (Comet assay) in human lymphocytes: a structure-activity relationship (QSAR) analysis of the genotoxic
and cytotoxic potential. Mutagenesis 13(2): 115-126.
Town, C; Leibman, KC. (1984) The in vitro dechlorination of some polychlorinated ethanes. Drug Metab Disp
12:4-8.
125 DRAFT - DO NOT CITE OR QUOTE
-------
Treluyer, JM; Jacqz-Aigrain, E; Alvarez, F; et al. (1991) Expression of CYP2D6 in developing human liver. Eur J
Biochem 202:583-588.
Tu, AS; Murray, TA; Hatch, KM; et al. (1985) In vitro transformation of BALB/C-3T3 cells by chlorinated ethanes
and ethylenes. Cancer Lett 28:85-92.
U.S. EPA (Environmental Protection Agency). (1979) Water-related environmental fate of 129 priority pollutants.
Vol. II. Monitoring and Data Support Division, Washington, DC; EPA440/4-79-029b. PB80-204381.
U.S. EPA. (1982) Aquatic fate process data for organic priority pollutants. Monitoring and Data Support Division,
Washington, DC; EPA 440/4-81-014.
U.S. EPA. (1986a) Guidelines for the health risk assessment of chemical mixtures. Federal Register
51(185):34014-34025. Available online at http://www.epa.gov/iris/backgrd.html.
U.S. EPA. (1986b) Guidelines for mutagenicity risk assessment. Federal Register 51(185):34006-34012. Available
online at http://www.epa.gov/iris/backgrd.html.
U.S. EPA. (1988) Recommendations for and documentation of biological values for use in risk assessment.
Prepared by the Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment,
Cincinnati, OH for the Office of Solid Waste and Emergency Response, Washington, DC; EPA 600/6-87/008.
Available online at http://www.epa.gov/iris/backgrd.html.
U.S. EPA. (1991a) Guidelines for developmental toxicity risk assessment. Federal Register 56(234):63798-63826.
Available online at http://www.epa.gov/iris/backgrd.html.
U.S. EPA. (1991b) Health advisory for hexachloroethane. Office of Water, Washington, DC; EPA/625/3-91/019F;
PB91-159657.
U.S. EPA. (1991c) Alpha2u-globulin: association with chemically induced renal toxicity and neoplasia in the male
rat. Risk Assessment Forum, Washington, DC; EPA/625/3-91/019F; PB92-143668.
U.S. EPA. (1992) Draft report: a cross-species scaling factor for carcinogen risk assessment based on equivalence of
mg/kg3/4/day. Federal Register 57(109):24152-24173.
U.S. EPA. (1994a) Interim policy for particle size and limit concentration issues in inhalation toxicity studies.
Federal Register 59(206):53799. Available online at http://www.epa.gov/iris/backgrd.html.
U.S. EPA. (1994b) Methods for derivation of inhalation reference concentrations and application of inhalation
dosimetry. Office of Research and Development, Washington, DC; EPA/600/8-90/066F. Available online at
http://www.epa.gov/iris/backgrd.html.
U.S. EPA. (1995) Use of the benchmark dose approach in health risk assessment. Risk Assessment Forum,
Washington, DC; EPA/630/R-94/007. Available online at
http://cfpub.epa. gov/ncea/raf/recordisplay.cfm?deid=42601.
U.S. EPA. (1996) Guidelines for reproductive toxicity risk assessment. Federal Register 61(212):56274-56322.
Available online at http://www.epa.gov/iris/backgrd.html.
U.S. EPA. (1998) Guidelines for neurotoxicity risk assessment. Federal Register 63(93):26926-26954.
U.S. EPA. (2000a) Science policy council handbook: risk characterization. Office of Science Policy, Office of
Research and Development, Washington, DC; EPA 100-B-00-002. Available online at
http://www.epa.gov/iris/backgrd.html.
U.S. EPA. (2000b) Benchmark dose technical guidance. External review draft. Risk Assessment Forum,
Washington, DC; EPA/630/R-00/001. Available online at http://www.epa.gov/iris/backgrd.html.
126 DRAFT - DO NOT CITE OR QUOTE
-------
U.S. EPA. (2000c) Supplementary guidance for conducting health risk assessment of chemical mixtures. Risk
Assessment Forum, Washington, DC; EPA/630/R-00/002. Available online at
http://www.epa.gov/iris/backgrd.html.
U.S. EPA. (2002) A review of the reference dose and reference concentration processes. Risk Assessment Forum,
Washington, DC; EPA/630/P-02/0002F. Available online at http://www.epa.gov/iris/backgrd.html.
U.S. EPA. (2005a) Guidelines for carcinogen risk assessment. Risk Assessment Forum, Washington, DC;
EPA/630/P-03/001F. Available online at http://www.epa.gov/iris/backgrd.html.
U.S. EPA. (2005b) Supplemental guidance for assessing susceptibility from early-life exposure to carcinogens. Risk
Assessment Forum, Washington, DC; EPA/630/R-03/003F. Available online at
http://www.epa.gov/iris/backgrd.html.
U.S. EPA. (2006a) Science policy council handbook: peer review. Third edition. Office of Science Policy, Office of
Research and Development, Washington, DC; EPA/ 100/B-06/002. Available online at
http://www.epa.gov/iris/backgrd.html.
U.S. EPA. (2006b) A framework for assessing health risk of environmental exposures to children. National Center
for Environmental Assessment, Washington, DC, EPA/600/R-05/093F. Available online at
http://cfpub.epa. gov/ncea/cfm/recordisplay.cfm?deid=158363.
U.S. EPA. (2008) Benchmark dose software (BMDS) version 2.0. Available online at
http://www.epa.gov/ncea/bmds.html.
Van Dyke, RA. (1977) Dechlorination mechanisms of chlorinated olefms. Environ Health Perspect 21:121-124.
Van Dyke, RA; Wineman, CG. (1971) Enzymatic dechlorination of chloroethanes andpropanes in vitro. Biochem
Pharmacol 20:463-470.
Verschueren, K. (1983) Handbook of environmental data on organic chemicals. 2nd ed. New York, NY: Van
Nostrand Reinhold Company.
Vieira, I; Sonnier, M; Cresteil, T. (1996) Developmental expression of CYP2E1 in the human liver:
hypermethylation control of gene expression during the neonatal period. Eur J Biochem 238:476-483.
Vogel, EW; Nivard, MJ. (1993) Performance of 181 chemicals in a Drosophila assay predominantly monitoring
interchromosomal mitotic recombination. Mutagenesis 8:57-81.
Weast, RC, ed. (1986) CRC handbook of chemistry and physics. 67th ed. Boca Raton, FL: CRC Press.
Webb, DR; Ridder, GM; Alden, CL. (1989) Acute and subchronic nephrotoxicity of d-limonene in dogs. Food
Chem Toxicol 28:669-675.
Weeks, MH; Thomasino, JA. (1978) Assessment of acute toxicity of hexachloroethane in laboratory animals. U.S.
Army Environmental Hygiene Agency, Aberdeen Proving Ground, MD; Report No. 51-0075-78.
Weeks, MH; Angerhofer, RA; Bishop, R; et al. (1979) The toxicity of hexachloroethane in laboratory animals. Am
IndAssoc 140:187-199.
Weisburger, EK. (1977) Carcinogenicity studies on halogenated hydrocarbons. Environ Health Perspect 21:7-16.
Xu, X; Zhang, D; Lyubynska, N; et al. (2006) Mast cells protect mice from mycoplasma pneumonia. Am J Respir
Crit Care Med 173:219-225.
Yamakage, A; Ishikawa, H. (1982) Generalized morphea-like scleroderma occurring in people exposed to organic
solvents. Dermatologica 165:186-193.
127 DRAFT - DO NOT CITE OR QUOTE
-------
Yanagita, K; Sagami, I; Shimizu, T. (1997) Distal site and surface mutations of cytochrome P450 1A2 markedly
enhance dehalogenation of chlorinated hydrocarbons. Arch Biochem Biophys 346(2):269-276.
Yanagita, K; Sagami, I; Daff, S; et al. (1998) Marked enhancement in the reductive dehalogenation of
hexachloroethane by a Thr319Ala mutation of cytochrome P450 1A2. Biochem Biophys Res Commun 249(3):678-
682.
Yoshikawa, K. (1996) Anomalous nonidentity between Salmonella genotoxicants and rodent carcinogens and
genotoxic noncarcinogens. Environ Health Perspect 104:40-46.
Younglai, EV; Foster, WG; Hughes, EG; et al. (2002) Levels of environmental contaminants in human follicular
fluid, serum, and seminal plasma of couples undergoing in vitro fertilization. Arch Environ Contam Toxicol
43(1):121-126.
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APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC
COMMENTS AND DISPOSITION
The Toxicological Review of Hexachloroethane (dated January, 2010) has undergone a
formal external peer review performed by scientists in accordance with EPA guidance on peer
review (U.S. EPA, 2006). An external peer-review workshop was held September 21, 2010.
There were six external peer reviewers. The external peer reviewers were tasked with providing
written answers to general questions on the overall assessment and on chemical-specific
questions in areas of scientific controversy or uncertainty. A summary of significant comments
made by the external reviewers and EPA's responses to these comments arranged by charge
question follow. In many cases the comments of the individual reviewers have been synthesized
and paraphrased in development of Appendix A. EPA did not receive any scientific comments
from the public on the Toxicological Review of HCE.
EXTERNAL PEER REVIEW 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.
When the external peer reviewers commented on decisions and analyses in the
Toxicological Review under multiple charge questions, these comments were organized under
the most appropriate charge question. In addition, the external peer reviewers made numerous
specific comments that were organized and responded to in a separate section of the section of
this appendix. When multiple reviewers provided specific comments on the same subject, or
suggested similar revisions to the document, their comments were combined, as appropriate.
General Charge Questions
Charge Question 1. Is the Toxicological Review logical, clear and concise? Has EPA clearly
presented and synthesized the scientific evidence for noncancer and cancer hazards?
Comment 1: The majority of the reviewers commented that the Toxicological Review was
comprehensive and logically presented; however, all of the reviewers commented that the
Toxicological Review was repetitious. The reviewers recommended including more synthesis,
particularly in Section 5, as a way to improve clarity and conciseness of the Toxicological
Review. Individual reviewers provided suggestions for improving clarity. One reviewer
commented that the Toxicological Review did not provide adequate justification for using a
NOAEL/LOAEL approach to model the inhalation effects. One reviewer disagreed with the
rationale for the application of some of the uncertainty factors. One reviewer requested
additional consideration of sublimation on the estimates of oral exposure dose when comparing
the subchronic dietary exposure study (Gorzinski et al., 1985) and the subchronic gavage study
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(NTP, 1989). One reviewer commented that the available data on renal cancer following HCE
exposure is consistent with a mode of action that is a combination of a2U-globulin nephropathy
and exacerbation of chronic progressive nephropathy. One reviewer recommended that
conclusions regarding the evaluation of the a2U-globulin mode of action be stated in a more
positive manner (e.g., indicate that data met 6 of the 7 criteria).
Response: Section 5 summarizes the dose, exposure, and effects of the evaluated studies to
facilitate study comparisons. Although presented elsewhere in the Toxicological Review, and
thus repetitious, this study information is necessary to support the selection of principal study
and critical effect, as well as the application of relevant uncertainty factors.
A NOAEL/LOAEL approach was selected for the inhalation data because the Weeks et
al. (1979) study did not provide incidence data for the neurological effects, which precluded
application of BMD modeling. Text in Section 5.2.2 has been modified to clarify this
justification.
The rationale for the application of uncertainty factors is presented in Section 5.1.3 and
Section 5.2.3. Text has been added to Section 5.1.3 and Section 5.2.3 to clarify the rationale for
applying individual uncertainty factors. Further explanation of the rationale for applying
individual uncertainty factors is provided in response to Charge Question 6 and Charge Question
10.
The effect of sublimation on dietary HCE dose has been considered in Gorzinski et
al. (1989); however, potential inhalation effects from sublimation were not discussed by the
study authors. While the NTP (1989) 13 week study administered HCE by gavage, thus
eliminating potential inhalation effects, the study had limitations. The NTP (1989) 13 week
study did not identify a NOAEL or provide incidence data for the kidney effects. In addition, the
NTP (1989) 13 week study administered higher doses of HCE than the Gorzinski et al. (1989)
study and the NTP (1989) chronic study. Therefore, the NTP (1989) 13 week study was
considered, but not selected as the principal study.
Chronic progressive nephropathy and the potential exacerbation of chronic progressive
nephropathy by a2U-globulin accumulation are discussed in Section 4.7.3.1. EPA concludes that
HCE-related effects in male and female rats indicate that chronic progressive nephropathy is not
solely responsible for the reported effects. In addition, EPA concludes that there is insufficient
evidence to attribute the kidney effects of HCE exposure to an a2U-globulin mode of action.
There is also insufficient data to determine if the HCE-related nephropathy results from the
exacerbation of chronic progressive nephropathy by a2U-globulin accumulation.
The criteria for attributing the kidney effects of HCE exposure to an a2U-globulin mode of
action are outlined in Section 4.7.3.1. Although there are data suggesting an a2U-globulin mode
of action for HCE-related nephropathy, none of the available studies identified a2U-globulin in
the hyaline droplets (see Table 4-21). Lack of immunohistochemical data prevents attributing
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the kidney effects of HCE exposure to an a2U-globulin mode of action. Text has been modified
in Section 5.4.3 and Section 5.4.5.1 to reiterate the conclusion reached in Section 4.6.3 and
Section 4.7.3.1 that the available data are insufficient to support the a2U-globulin mode of action.
Charge Question 2. Please identify any additional studies that would make a significant impact
on the conclusions of the Toxicological Review.
Comment 2: Four of the reviewers were unaware of published studies that would significantly
impact the Toxicological Review. Two reviewers commented that an immunohistochemical
assessment of kidneys from the NTP 90-day study animals (NTP, 1989) would inform the mode
of action for male rat kidney lesions. One reviewer provided a review of the exacerbation of
chronic progressive nephropathy following chemical exposure as support for a combination of
a2U-globulin accumulation and exacerbation of chronic progressive nephropathy as the mode of
action for renal tumors following HCE exposure. One reviewer provided a reference on the
cytotoxicity, genotoxicity, and irritation potency of two red phosphorus-based pyrotechnic
smokes, but commented that this study was unlikely to provide significant insight into
HCE-induced toxicity.
Response: The lack of immunohistochemical evidence of a2U-globulin in the hyaline droplets is
identified in Section 4.7.3.1 and Section 5.4.5.1 as a data gap. This data gap would be addressed
by the experiments recommended by the reviewers; however, immunohistochemical data are
unavailable.
Section 4.7.3.1 discusses both chronic progressive nephropathy and the potential for
exacerbation of chronic progressive nephropathy by a2U-globulin accumulation. EPA concludes
that (1) chronic progressive nephropathy is not solely responsible for the reported kidney effects
of HCE exposure and (2) lack of immunohistochemical data prevents attributing the kidney
effects of HCE exposure to an a2U-globulin mode of action. Therefore, the available is
insufficient to support an exacerbation of chronic progressive nephropathy by a2U-globulin
accumulation as a mode of action for the renal effects of HCE exposure.
The human health effects of pyrotechnic smokes are considered in Section 4.1. These
studies demonstrate HCE exposure in the smoke bomb production workers, but the sample sizes
of the health effects studies are too small to reach definitive conclusions. Furthermore, the
smoke produced by pyrotechnic smoke bombs is a mixture of chemicals consisting primarily of
zinc oxychloride and zinc chloride. Therefore, this study is unlikely to provide insight into
HCE-induced toxicity and was not included in the Toxicological Review.
Chemical Specific Charge Questions
A. Oral Reference Dose (RfD) for HCE
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Charge Question A.I. A 16-week dietary exposure study of HCE in F344 rats by Gorzinski et
al. (1985) was selected as the basis for the derivation of the RfD. Kidney effects were observed
in male rats in this study at doses below the range of exposure tested in the available chronic
NTP (1989) study. Please comment on the scientific justification for the use of the subchronic
Gorzinski et al. (1985) study as the principal study for the derivation of the RfD. Is the rationale
for this selection clearly described? Please identify and provide the rationale for any other
studies that should be selected as the principal study.
Comment: All of the reviewers agreed with the selection of Gorzinski et al. (1985) as the
principal study; however, some reviewers suggested clarifications to improve transparency in the
selection of the Gorzinski et al. (1985) study. One reviewer requested additional discussion
regarding the observation that kidney effects in the subchronic study were observed at doses
lower than estimated by BMD modeling of data from the chronic studies. One reviewer
requested further discussion of the selection of atrophy and degeneration of renal tubules from
the subchronic study over other kidney effects (i.e., increased severity of tubular nephropathy or
linear mineralization) reported in the chronic study. One reviewer recommended that the
rationale for selecting the subchronic Gorzinski et al. (1985) study over the chronic NTP (1989)
should include discussion of both duration and the need for extrapolation below the lowest dose
tested. One reviewer suggested that a better rationale for selecting the subchronic study was that
kidney toxicity was observed after only 16 weeks of exposure and the subchronic study produced
the lowest BMD/BMDL values.
Response: Kidney effects in the subchronic study (Gorzinski et al., 1985) were observed at
doses lower than estimated by BMD modeling of the available chronic data; however, these
reported kidney effects were not statistically different from controls. The subchronic doses
causing statistically significant increases in kidney effects are consistent with the BMDLio for
the kidney effect data from chronic studies.
Atrophy and degeneration of renal tubules was consistently observed in both subchronic
and chronic studies, leading to its selection as the candidate critical effect for male rats exposed
to HCE. The subchronic study (Gorzinski et al., 1985) was selected as the principal study
because it identified a NOAEL for kidney effects and BMD modeling led to a lower POD than
the chronic studies (see Section 5.1.2).
Study duration was considered during the application of uncertainty factors, as described
in Section 5.1.3. Section 5.1.2 describes the selection of the subchronic Gorzinski et al. (1985)
study over the chronic NTP (1989) and NCI (1978) studies, including consideration of dose. As
stated in the Toxicological Review, the Gorzinski et al. (1985) study derived a NOAEL for the
kidney effects, whereas lowest doses tested in the chronic exposure studies (NCI, 1978: NTP,
1989) represented LOAELs. EPA selected the Gorzinski et al. (1985) study as the principal
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study for derivation of the RfD because it identified a NOAEL and the chronic exposure studies
resulted in higher PODs for tubular nephropathy in male rats.
Charge Question A.2. Nephrotoxicity as indicated by atrophy and degeneration of renal tubules
in male rats (Gorzinski et al., 1985) was selected as the critical effect for the RfD. Please
comment on whether the selection of this critical effect is scientifically justified and clearly
described. Please identify and provide the rationale for any other endpoints that should be
selected as the critical effect.
Comment: All of the reviewers supported the selection of atrophy and degeneration of renal
tubules in male rats as the critical effect. One reviewer commented that the moderate-to-marked
renal nephropathy from the chronic NTP (1989) study should be considered during the
application of the subchronic-to-chronic uncertainty factor. One reviewer commented that
spontaneous chronic progressive nephropathy may be a confounding factor and noted that the
Gorzinski et al. (1985) study did not score the extent of chronic progressive nephropathy. This
reviewer questioned if it was possible to distinguish effects of chronic progressive nephropathy
and effects related to HCE exposure, suggesting that chemical-specific renal injury separate from
chronic progressive nephropathy may occur at higher doses.
Response: Section 5.1.3 has been revised to discuss the available chronic data in the application
of the subchronic-to-chronic uncertainty factor. The application of the subchronic-to-chronic
uncertainty factor is further discussed in response to Charge Question 6.
Section 4.7.3.1 discusses chronic progressive nephropathy and acknowledges that chronic
progressive nephropathy can obscure the lesions characteristic of a2U-globulin-related
nephropathy. Although the Gorzinski et al. (1985) study did not provide data on chronic
progressive nephropathy, the authors reported dose-dependent renal effects in both male and
female rats and identified NOAELs for the renal effects in both sexes. These data suggest that
chronic progressive nephropathy is not solely responsible for the reported renal effects (see
Section 4.6.3 and Section 4.7.3.1).
Charge Question A.3. Benchmark dose (BMD) modeling was applied to the atrophy and
degeneration of renal tubules data in male rats to derive the point of departure (POD) for the
RfD. Has the BMD modeling been appropriately conducted and clearly described? Is the
benchmark response (BMR) selected for use in deriving the POD (i.e., a 10% increase in the
incidence of atrophy and degeneration of renal tubules) scientifically justified and clearly
described?
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Comment: The majority of reviewers commented that BMD modeling was appropriately
conducted and clearly described. One reviewer commented that mode of action considerations
could lead to a higher point of departure.
Response: EPA concludes that while there are data to indicate that the mode of action for renal
effects may be specific for male-rats, there are confounding issues as indicated in Section 4.6.3.
Charge Question A.4. Please comment on the rationale for the selection of the uncertainty
factors (UFs) applied to the POD for the derivation of the RfD. Are the UFs scientifically
justified and clearly described? If changes to the selected UFs are proposed, please identify and
provide a rationale.
Comment: All of the reviewers agreed with the application of an uncertainty factor of 10 for the
interspecies extrapolation. One reviewer recommended expanding the justification for the
application of this uncertainty factor to include availability of data on the active form of HCE
and appropriate dose metrics.
All of the reviewers agreed with the application of an uncertainty factor of 10 for the
intraspecies extrapolation.
One reviewer agreed with the application of an uncertainty factor of 10 for the
subchronic-to-chronic extrapolation, while the remaining reviewers recommended changes to
this uncertainty factor. One reviewer commented that the findings from the chronic NTP (1989)
study supported the findings from the subchronic Gorzinski et al. (1985) study and, therefore,
application of an uncertainty factor of 2-4 for the subchronic-to-chronic extrapolation would be
adequate. One reviewer recommended applying an uncertainty factor of 1 for the subchronic-to-
chronic uncertainty factor because the RfD is unchanged whether the chronic or subchronic data
are selected. Two reviewers recommended application of a subchronic-to-chronic uncertainty
factor of 3 because the available chronic data do not suggest that prolonged exposure would
exacerbate the renal tubule effects observed in the subchronic Gorzinski et al. (1985) study. One
reviewer questioned whether the renal effects observed in the chronic NTP (1989) study were
more severe than the renal effects in the subchronic Gorzinski et al. (1985) study and commented
that a subchronic-to-chronic uncertainty factor of 10 was debatable; however, the reviewer did
not recommend a value for the uncertainty factor.
The majority of reviewers agreed with the application of an uncertainty factor of 1 for the
LOAEL-to-NOAEL extrapolation because the Gorzinski et al. (1985) study identified aNOAEL.
One reviewer commented that the BMDLio is more reflective of a LOAEL than a NOAEL and,
therefore, suggested application of an uncertainty factor of 3 for LOAEL-to-NOAEL
extrapolation.
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The majority of the reviewers agreed with the application of an uncertainty factor of 3 for
the database uncertainty factor because of the lack of a multigeneration reproductive toxicity
study. One reviewer recommended expanding the justification for the application of the database
uncertainty factor to more transparently describe which studies were missing from the database
and include considerations of related chemicals and metabolites. One reviewer objected to the
practice of using of database uncertainty factors and recommended applying an uncertainty
factor of 1.
Response: The discussion of the interspecies uncertainty factor in Section 5.1.3 was modified to
indicate that the available toxicokinetic data for HCE was insufficient to identify the active
compound or determine dose metrics for extrapolation.
The available data were reconsidered in the selection of the subchronic-to-chronic
uncertainty factor and as a result, the subchronic-to-chronic uncertainty factor has been reduced
from 10 to 3. The Gorzinski et al. (1985) study duration was minimally longer the standard
subchronic (90-day) study and falls well short of a standard lifetime study (i.e., two year chronic
bioassay), although chronic data are available for comparison. These chronic data suggest: (1)
incidence of nephropathy may not increase with prolonged exposure and (2) consistency in dose
response relationships between chronic and subchronic studies. However, as the Gorzinski et al.
(1985) study did not report severity data for the renal effects, there are no data to exclude the
possibility that chronic exposure could increase the severity of the observed kidney effects or
could result in similar effects at lower doses. Reduction of the subchronic-to-chronic uncertainty
factor is consistent with the majority of the comments. Section 5.1.3 has been modified to reflect
the reduction of the subchronic-to-chronic uncertainty factor.
Section 5.1.3 has been modified to indicate that a two-generation reproduction study is
absent from the database, resulting in the application of a database uncertainty factor of 3.
Available data on HCE metabolism are limited and there is insufficient evidence to determine if
the reported effects are due to the parent compound or the metabolites. In the absence of
metabolism data, additional discussion of potential HCE metabolites and the impact on the
database uncertainty factor is not warranted. Application of a database uncertainty factor does
not indicate that the available data are insufficient to derive a reference value, as suggested by
one reviewer. Rather, the database uncertainty factor accounts for the potential to underestimate
noncancer hazard as a result of data gaps. The database for HCE does not contain a two-
generation reproductive study, indicating an incomplete characterization of HCE toxicity.
Therefore, the database uncertainty factor of 3 was applied in the derivation of the RfD.
B Chronic Inhalation Reference Concentration (RfC) for HCE
Charge Question B.I. A 6-week inhalation exposure study in rats by Weeks et al. (1979) was
selected as the basis for the derivation of the RfC. Please comment on whether the selection of
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this study as the principal study is scientifically justified. Is the rationale for this selection
clearly described? Please identify and provide the rationale for any other studies that should be
selected as the principal study.
Comment: All the reviewers commented that in light of the limited database, the Weeks et al.
(1979) data was the most appropriate study for deriving the RfC. One reviewer requested
additional discussion regarding the adequacy of the Weeks et al. (1979) for consideration as a
principal study. One reviewer recommended an expanded discussion of why the Weeks et al.
(1979) study was not used for RfC derivation in the previous IRIS assessment (U.S. EPA, 1987).
One reviewer requested additional discussion of the human relevance of the reported
neurobehavioral effects, given that these effects were observed only at a high dose of HCE.
Response: The Weeks et al. (1979) study is a well-conducted subchronic inhalation bioassay
that evaluated an array of endpoints and established NOAELs and LOAELs for HCE in a
number of different species. The authors evaluated portal of entry effects on lungs, trachea, and
nasal turbinates by gross examination as well as histological sectioning. Weeks et al. (1979)
examined sections of the nasal turbinates for upper respiratory effects and evaluated upper
respiratory inflammation by the presence of polymorphonuclear leukocytes in close association
with excess mucus within the lumens of the nasal passages. Section 5.2.1 has been revised to
indicate the examination of the portal of entry effects by the study authors.
An RfC for HCE was not previously derived. In the 1987 IRIS Summary, Weeks et al.
(1979) was briefly summarized in the Additional Studies/Comments section for the oral RfD.
The 1987 IRIS Summary concludes that the Gorzinski et al. (1985) study is a better basis for the
oral RfD. The 1987 IRIS Summary does not discuss why an inhalation RfC was not derived;
therefore, it is unclear if the Weeks et al. (1979) data were considered for the derivation of the
inhalation RfC. This information has been added to the Toxicological Review.
The Weeks et al. (1979) study identified neurobehavioral effects in animals, but did not
provide sufficient data on pharmacokinetic or mechanistic considerations to inform the human
relevance of these effects. In the absence of pharmacokinetic or mechanistic data, the
neurobehavioral effects in animals were assumed to be relevant to humans.
Charge Question B.2. Neurobehavioral effects in Sprague-Dawley rats (Weeks et al., 1979)
were selected as the critical effect for the RfC. Please comment on whether the selection of this
critical effect is scientifically justified and clearly described. Please identify and provide the
rationale for any other endpoints that should be selected as the critical effect.
Comment: All of the reviewers agreed that neurobehavioral effects were supported by the
available data and were appropriately chosen as the critical effect. One reviewer recommended
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clarifications to the discussion of critical effect selection, including (1) considerations of
structure-activity relationships with related chemicals to support the selection of neurobehavioral
effects as the critical effect; (2) discussion of the differences in target organ between oral and
inhalation exposure; and (3) clarification that the respiratory effects were not selected as the
critical effect because these effects were considered by the authors (Weeks et al, 1979) as
attributable to mycoplasma infection. Two other reviewers also requested additional discussion
on the difference in effects between oral and inhalation exposure to HCE, particularly the
absence of nephrotoxicity following inhalation exposure. One reviewer commented that body
weight changes were also observed in multiple species after exposure to the highest dose and
could have been selected as the critical effect. One reviewer recommended more discussion of
dose relevance with respect to human exposures, as well as the implications of neurobehavioral
effects that only occurred at the high exposure doses.
Response: A literature search did not identify any structure-activity relationships relevant to
neurobehavioral effects of HCE exposure. Although oral and inhalation exposure to HCE affects
different target organs, data are unavailable to inform the observed differences. As stated in
Section 4.2.2.1, Weeks et al. (1979) attributed the increased incidence of respiratory lesions in
rats to an endemic mycoplasma infection; however, the Weeks et al. (1979) did not provide data
demonstrating the presence of mycoplasma in the lungs. This data gap prevented exclusion of
the respiratory tract effects from consideration as a potential critical effect. Rather, the
consistent observation of neurotoxic effects across experiments was the rationale for selecting
neurobehavioral effects as the critical effect.
The Weeks et al. (1979) study identified neurotoxicity, statistically significant decreases
in body weight gain, and upper and lower respiratory tract irritation as effects of inhalation
exposure to HCE. Of these effects, neurobehavioral effects and changes in body weight gain
were consistently observed in multiple species. Neurobehavioral effects were assumed to pose a
potential hazard to humans and therefore selected as the critical effect.
The Weeks et al. (1979) study did not provide data to inform pharmacokinetic
considerations of the human relevance of the exposure dose; however, the available human
exposure data for HCE (see Section 4.1) report HCE levels lower than the neurotoxic dose
reported in the Weeks et al. (1979) study. In the absence of pharmacokinetic or mechanistic
data, the neurobehavioral effects in animals were assumed to pose a potential hazard to humans.
Charge Question B.3. The NOAEL/LOAEL approach was used to derive the POD for the RfC.
Please comment on whether this approach is scientifically justified and clearly described.
Comment 9: All of the reviewers agreed that the NOAEL approach was justified for deriving the
POD for the RfC. Two reviewers recommended clarification that it was the lack of individual
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responses at all exposure doses, not the 100% response at the high dose, which prevented BMD
modeling of the Weeks et al. (1979) data. One reviewer suggested additional discussion of the
human equivalent concentration derivation, particularly the categorization of HCE as a Category
2 gas.
Response: Section 5.2.2 has been modified to indicate that the lack of incidence data prevented
BMD modeling of the Weeks et al. (1979) neurotoxicity data.
Section 5.2.2 has been modified to clarify the gas categories for deriving a human
equivalent concentration and the classification of HCE as a Category 2 gas.
Charge Question B.4. Please comment on the rationale for the selection of the UFs applied to
the POD for the derivation of the RfC. Are the UFs scientifically justified and clearly described?
If changes to the selected UFs are proposed, please identify and provide a rationale.
Comment: Three reviewers agreed with the application of an uncertainty factor of 3 for
interspecies extrapolation, whereas the remaining three reviewers recommended an interspecies
uncertainty factor of 10. One reviewer commented that the derivation of a human equivalent
concentration did not adequately cover the interspecies toxicity uncertainty. One reviewer
commented that derivation of a human equivalent concentration partially addressed
toxicokinetics, but requested additional explanation for how the human equivalent concentration
derivation addresses toxicodynamic considerations. Two reviewers recommended additional
discussion for reducing the interspecies uncertainty factor to 3 when the regional gas dose ratio
defaulted to 1 because of the absence of data. One reviewer suggested additional discussion of
the blood:air partition coefficients in rats and humans for related chemicals, as well as discussion
that the regional gas dose ratio defaults to 1 because the animal coefficient is usually larger than
the human value. One reviewer requested additional clarification for the human equivalent
concentration derivation.
All of the reviewers agreed with the application of an uncertainty factor of 10 for the
intraspecies extrapolation.
All of the reviewers agreed with the application of an uncertainty factor of 10 for the
subchronic-to-chronic exposure extrapolation. One reviewer recommended clarifying that the
subchronic-to-chronic uncertainty factor was applied in the absence of any longer-term studies.
All of the reviewers agreed that no uncertainty factor was necessary for LOAEL-to-
NOAEL extrapolation.
Two reviewers agreed with the application of an uncertainty factor of 10 for the
limitations in the inhalation database, whereas the reminaing three reviewers recommended a
database uncertainty factor of 3. Comments received from individual reviewers included the
following: (1) that arguments could be made to support both an uncertainty factor of 3 for the
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database limitations as well as a database uncertainty factor of 10; (2) the available toxicity
studies were sufficiently diverse and supported a database uncertainty factor of 3; (3) even
though a chronic inhalation study was not available, the availability of a developmental study
would support an uncertainty factor of 3; (4) the justification for application of the database
uncertainty factor could be improved by discussing the available studies and key concerns; and
(5) the lack of a developmental neurotoxicity study and multigenerational reproductive toxicity
was of concern, but recommended a database uncertainty factor of 3 because the available
literature included exposure in multiple species, a general toxicity study, a reproductive study,
and a neurobehavioral study.
One reviewer noted that the overall uncertainty factor for the RfC was similar to the RfD,
despite the comparatively larger data gaps in the inhalation database.
Response: As described in Section 5.2.3, an interspecies uncertainty factor of 3 is applied when
incorporating an animal-specific NOAELADJ to a human equivalent NOAELnEC dosimetric
adjustment. This dosimetric adjustment reduces uncertainty by accounting for the variability in
toxicokinetics; however, this dosimetric adjustment does not account for species differences in
toxicodynamics. Therefore, in the absence of sufficient toxicodynamic data, an interspecies
uncertainty factor of 3 is retained to account for toxicodynamic differences between animals and
humans. Text has been modified in Section 5.2.3 to clarify that toxicokinetic component of
interspecies uncertainty is addressed by the dosimetric adjustment, whereas insufficient data
exist to inform the toxicodynamic component of the intraspecies uncertainty factor. A regional
gas dose ratio of 1 is also recommended if the animal blood:gas coefficient is greater than the
human blood:gas coefficient or the animal and human partition coefficients are unknown. In
accordance with current practices, a regional gas dose ratio of 1 was used because the animal and
human blood:gas partition coefficients are unknown. Text has been added to the Section 5.2.2 to
clarify the application of the default regional gas dose ratio as well as the derivation of the
human equivalent concentration.
Text has been added in Section 5.2.3 to clarify the subchronic Weeks et al. (1979) was
the only repeat exposure study available.
Weeks et al. (1979) is a subchronic inhalation bioassay that evaluated an array of
endpoints and established NOAELs and LOAELs. In applying the database uncertainty factor,
Section 5.2.3 indicates the deficiencies for the inhalation database. Specifically, the database is
lacking long-term studies, a multigeneration reproductive toxicity study, a developmental study
in a second species, and neurotoxicity and developmental neurotoxicity studies. Because of
these data gaps, a database uncertainty factor of 10 was applied for the RfC derivation. Text has
been modified in Section 5.2.3 to clarify these data gaps.
Generally, uncertainty factors for the RfD and RfC are independently determined. The
available information for the RfD and RfC (e.g., resulting in database deficiencies, use of
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NOAEL/LOAEL approach versus BMD modeling) was taken into account in the application of
the individual uncertainty factors.
C. Carcinogenicity of HCE
Charge Question C.I. Under the EPA's 2005 Guidelines for Carcinogen Risk Assessment
(www.epa.gov/iris/backgrd.html), HCE is likely to be carcinogenic to humans by all routes of
exposure. Is the cancer weight of evidence characterization scientifically justified and clearly
described?
Comment 11: Five of the reviewers agreed with the cancer descriptor of "likely to be
carcinogenic to humans," whereas one of the reviewers did not agree with this descriptor. One
reviewer noted that the available evidence minimally met the criteria for the likely to be
carcinogenic to humans. Some reviewers suggested that the overall weight of evidence should
address uncertainties in the data (i.e., including the level of evidence for the a2U-globulin mode of
action) and further discuss the human relevance of the renal, hepatocellular, and adrenal tumor
types, observed following HCE exposure. One reviewer commented that concerns over human
relevance over the three observed tumor types and the limited genotoxicity evidence should be
given further consideration for the cancer descriptor. One reviewer proposed a combination of
a2u-globulin accumulation and exacerbation of chronic progressive nephropathy as the mode of
action for renal tumors following HCE exposure, which would not be relevant for human health
considerations. Two reviewers recommended collecting additional data to help evaluate the
human relevance of the renal tubule tumors, including: the incidence of end stage renal failure or
high severe nephropathy for controls and HCE-exposed animals, the presence of foci of atypical
hyperplasia, the location of renal adenomas were within the areas of chronic progressive
nephropathy, and the presence of a2U-globulin protein in the hyaline droplets. One reviewer
commented that pentachloroethane, a potential metabolite of HCE, causes a2U-globulin
nephropathy. One reviewer commented that the incidence of pheochromocytomas was not dose-
related and questioned the relevance of the reported increase of pheochromocytomas.
Response: Animal studies of chronic oral exposure to HCE have reported: (1) dose-dependent,
statistically significant increases in the incidence of renal adenoma or carcinoma combined in
male F344/N rats; (2) statistically significant increases in the incidence of
pheochromocytomas/malignant pheochromocytomas combined in male F344/N rats; and
(3) statistically significant increases in the incidence of hepatocellular carcinomas in male and
female B6C3Fi mice. The data are positive for tumor formation in more than one animal
species, more than one sex, and for more than one site of cancer which are all lines of evidence
supporting the cancer descriptor "likely to be carcinogenic to humans" for HCE.
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Uncertainties in the HCE cancer risk assessment are discussed in Section 5.4.5 and
summarized in Table 5-7. The potential role for an a2U-globulin mode of action for the renal
effects of HCE exposure is evaluated in Section 4.7.3.1 and Section 5.4.5.1. Section 5.4.5.1
summarizes the data gaps that prevent attributing the development of renal tumors to an
a2U-globulin mode of action. Section 5.4.5.1 also evaluates the human relevance of the renal,
hepatic, and adrenal gland tumors. Text in Section 4.7.1 has been revised to discuss
uncertainties in the data, including the insufficient evidence for an a2U-globulin mode of action in
the kidney effects as well as the human relevance of the kidney, adrenal gland, and liver tumors.
Chronic progressive nephropathy and the potential exacerbation of chronic progressive
nephropathy by a2U-globulin accumulation are discussed in Section 4.7.3.1. The severity of
nephropathy was considered in the evaluation of the NTP (1989) study in Section 4.2.1.2
(summarized in Table 4-4); however, data are unavailable to categorize end stage renal failure in
either the control or HCE-exposed animals. Similarly, data were unavailable to determine if foci
of atypical hyperplasia were present, if renal adenomas were within the areas of chronic
progressive nephropathy, or confirming the presence of a2U-globulin protein in the hyaline
droplets. These data gaps prevent attributing the renal effects of HCE to the exacerbation of
chronic progressive nephropathy by a2U-globulin accumulation. Text in Section 5.4.5.1 has been
revised to indicate the additional data gaps that would inform the human relevance of the kidney
tumors.
Also, one reviewer suggested that data for pentachloroethane may have potential
relevance to the toxicity of HCE. Pentachloroethane is a putative metabolite of HCE (see Figure
3-1), but the available data on HCE metabolism are limited. The putative metabolites are briefly
discussed in the Toxicological Review as support for the toxicological effects reported following
HCE exposure; however, there is insufficient evidence to determine if the reported effects are
due to the parent compound or the metabolites.
Lastly, pheochromocytomas were statistically significantly increased in the low dose
group, but not the high dose group (see Table 4-7). This dataset was not used in the
dose-response assessment because the tumor incidence was not a monotonic increasing function
of dose. Pheochromocytomas were considered relevant to humans (see Section 4.7.3.3);
therefore, the observation of pheochromocytomas was considered as supporting evidence for the
cancer descriptor of "likely to be carcinogenic to humans" for HCE.
Charge Question C.2. A two-year oral gavage cancer bioassay in F344 rats (NTP, 1989) was
selected for the derivation of an oral slope factor. Please comment on whether the selection of
this study for quantitation is scientifically justified and clearly described. Please identify and
provide the rationale for any other studies that should be selected.
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Comment: Five reviewers agreed with the selection of the NTP (1989) study for the derivation
of an oral slope factor, based on the sensitivity of male rats to HCE exposure. One reviewer
recommended characterizing the available data as "insufficient" to support an a2U-globulin mode
of action. One reviewer requested the BMD modeling output for hepatocellular carcinomas in
female mice be added to Appendix B and recommended to not include the hepatocellular
carcinoma endpoint in Table 5-6. One reviewer questioned the use of linear low-dose
extrapolation in the derivation of the oral slope factor. One reviewer noted that measurements of
a2U-globulin in the NTP (1989) study and consideration of the a2U-globulin exacerbation of
chronic progressive nephropathy mode of action would inform the human relevance of the
observed renal tumors in male rats.
One reviewer disagreed with the derivation of an oral slope factor based on renal tubule
tumors in male rats from the NTP (1989). This reviewer recommended deriving an oral slope
factor based on hepatocellular carcinomas in male mice reported in the NCI (1978) study.
Response: Sections 5.4.3 and 5.4.5.1 have been modified to reiterate the conclusions in Section
4.6.3 and Section 4.7.3.1 that the available data were insufficient to support an a2U-globulin mode
of action.
The multistage cancer BMD modeling output for the hepatocellular carcinomas in female
mice has been added to Appendix B of the Toxicological Review. Also, because the BMD
modeling output indicates that the multistage model exhibited significant lack of fit for the
hepatocellular carcinomas in female mice, the data have been removed from Table 5-6 and the
corresponding text has been modified.
Determination of a2U-globulin protein in the hyaline droplets could inform the role of an
a2U-globulin mode of action in the renal effects of HCE exposure reported in the NTP (1989)
study; however, these data are unavailable. In addition, the nephrotoxic effects observed of HCE
in male and female mice confounds the determination and indicates that there may be more than
one mode of action for renal toxicity. Similarly, data are unavailable to inform the potential for
exacerbation of chronic progressive nephropathy by a2U-globulin accumulation. Currently, there
are insufficient data to conclude that the renal effects observed following HCE exposure are
attributable to either an a2U-globulin mode of action or exacerbation of chronic progressive
nephropathy by a2U-globulin accumulation. The oral slope factor was derived based on the renal
tubule tumors in male rats because (1) the renal effects were considered relevant to humans, and
(2) the rats exhibited greater sensitivity to HCE-induced carcinogenicity than the mice.
Charge Question C.3. The renal tubule tumor data in male rats from the NTP (1989) two-year
oral gavage cancer bioassay were selected to serve as the basis for the quantitative cancer
assessment. Please comment on whether this selection is scientifically justified and clearly
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described. Please identify and provide the rationale for any other endpoints that should be
selected to serve as the basis for the quantitative cancer assessment.
Comment: Four reviewers agreed with the selection of renal tubule tumor data in male rats from
the NTP (1989) study as the basis for the quanitative cancer assessment. Two reviewers
questioned the human relevance of the renal tubule tumors and recommended selecting
hepatocellular carcinomas in mice as the basis for the quanitative cancer assessment.
Response: As summarized in Section 5.4.5.1, two principal factors contribute to the conclusion
that there are insufficient data to support an a2U-globulin mode of action for the development of
renal tumors. First, the presence of kidney effects in HCE-exposed male and female mice and
female rats suggests a mode of action other than a2U-globulin nephropathy. Second, none of the
HCE studies confirmed the presence of a2U-globulin protein within the hyaline droplets. The
renal tubule tumors were selected for the basis of the cancer slope factor because the rats
exhibited greater sensitivity to HCE-induced carcinogenicity than the mice.
Charge Question C.4. EPA concluded that the mode of action for renal tubule tumors observed
following oral exposure to HCE is unknown. An analysis of the mode of action data for renal
tumors is presented in the Toxicological Review. Based on this analysis, EPA determined that
HCE-induced renal tumors could not be attributed to the accumulation of a2U-globulin. Please
comment on the scientific support for these conclusions. Please comment on whether the analysis
is scientifically justified and clearly described.
Comment: Four reviewers agreed with the determination that HCE-induced renal tumors could
not be attributed to the accumulation of a2U-globulin. Two reviewers disagreed with the
conclusion that HCE-induced renal tumors were not attributable to a2U-globulin accumulation.
One reviewer requested additional discussion of the mice hepatic tumors and
pheochromocytomas in the overall cancer risk assessment of HCE, specifically in the context of
determining the cancer descriptor. One reviewer requested discussion of the a2U-globulin mode
of action conclusions reached by several recent reviews on HCE carcinogenicity. Several
reviewers suggested revisions to the mode of action information. These suggested revisions
included the following: that the cancer mode of action discussion compare HCE and
tetrahydrofuran discussing renal proliferative lesions produced by low grade a2U-globulin
nephropathy and advanced chronic progressive nephropathy; more synthesis in the dose
response section and a table showing at which doses the key events in the a2U-globulin mode of
action occur; the available data support key events in the a2U-globulin mode of action occurring
before tumors occur and at the same or lower doses necessary for tumor formation and
recommended discussion of these points in the dose-response section; and since renal tubule
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tumors were only observed in male rats, the occurrence of nephrotoxicity in female rats is not
contributing evidence for excluding the a2U-globulin mode of action for HCE carcinogen!city.
Several reviewers commented on the mode of action conclusion. One reviewer
commented that even if the renal tubule tumors are not attributable to an a2U-globulin mode of
action, the other established mechanism for renal tubule tumors in rats is not relevant to humans
either; therefore, the evaluation of the a2U-globulin mode of action data for HCE-related renal
tubule tumors may not be critical for determining the human relevance of these tumors. One
reviewer requested clarification on the mode of action conclusion, suggesting that the
a2U-globulin mode of action may partially explain the renal tubule tumors. Another reviewer
disagreed with the conclusion that HCE renal carcinogenicity cannot be attributed to an
a2u-globulin mode of action, arguing that the available data support 6 out of 7 necessary criteria.
One reviewer commented that weight of evidence from chemicals structurally related to HCE
support a male rat-specific mode of action for HCE renal carcinogenicity. Lastly, a reviewer
suggested that exacerbation of chronic progressive nephropathy by HCE further supports a mode
of action for HCE renal carcinogenicity that is not relevant to humans.
Response: The presence of statistically significant increases in the incidence of
pheochromocytomas/malignant pheochromocytomas combined in male F344/N rats and
statistically significant increases in the incidence of hepatocellular carcinomas in male and
female B6C3Fi mice provide support for the cancer descriptor "likely to be carcinogenic to
humans" for HCE. Section 5.4 has been modified to clarify the selection of the cancer descriptor
"likely to be carcinogenic to humans" for HCE. With respect to the conclusions of review
manuscripts on HCE carcinogenicity, EPA conducted an independent mode of action analysis of
the relevant primary literature as the basis for the conclusions presented in the Toxicological
Review. The available review manuscripts relevant to the a2U-globulin mode of action in renal
nephropathy and renal tumors were used as references for the evaluation of the available renal
effects data for HCE.
Unlike the tetrahydrofuran studies (Chhabra et al. Toxicol. Sci. 41: 183-188, 1998, and
Bruner et al. Regul. Pharmacol. Toxicol. 2010, in press), none of the available HCE studies
performed immunohistochemical analysis to identify a2U-globulin in the hyaline droplets (see
Table 4-21). Therefore, consideration of the tetrahydrofuran data would not inform the mode of
action for the renal effects of HCE exposure. Because of the absence of immunohistochemical
data, the HCE dose at which a2U-globulin accumulates hyaline droplets is unknown.
Consequently, there is insufficient data to determine if accumulation of a2U-globulin in hyaline
droplets occurs at lower HCE doses than subsequent a2U-globulin-related effects. In the absence
of immunohistochemical data, a table showing the HCE doses at which key events in the
a2U-globulin mode of action occur was not added to the Toxicological Review. Text has been
added to Section 4.7.3.1 to clarify that dose-response concordance of the accumulation of
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a2U-globulin in hyaline droplets cannot be demonstrated from the available data. As discussed in
Section 4.7.3.1, the temporal relationship between renal tumor tubules and the a2U-globulin mode
of action could not be established because none of the HCE studies confirmed the presence of
a2U-globulin protein within the hyaline droplets. Although renal tubule tumors were only
reported in male rats, the occurrence of nephrotoxicity in female rats is supporting evidence that
the renal effect of HCE may not be attributable to a2U-globulin accumulation. Accumulation of
a2U-globulin is unique to the male rat, as female rats and other laboratory mammals do not
accumulate a2U-globulin in the kidney and do not subsequently develop renal tubule tumors.
Therefore, the evidence of nephropathy in female rats, as well as male and female mice, suggests
that the renal tumors may not be attributable to an a2U-globulin mode of action or that more than
one mode fo action may be operating. The sex-specific difference in carcinogenic effects of
HCE exposure may reflect sex-specific differences in the kidney concentrations of HCE
following oral exposure (see Table 3-3).
Some data suggest that the male rat-specific a2U-globulin mode of action could contribute
to HCE-induced nephropathy. As summarized in Section 5.4.5.1, the data are insufficient to
support an a2U-globulin mode of action in the development of renal tumors following HCE
exposure. Chronic progressive nephropathy was discussed in Section 4.7.3.1. There is
insufficient evidence to attribute the kidney effects of HCE exposure to exacerbation of chronic
progressive nephropathy. Lastly, a literature search did not identify any structure-activity
relationships relevant to carcinogenic effects of HCE exposure.
Charge Question C.5. The oral cancer slope factor was calculated by linear extrapolation from
the POD (i.e., the lower 95% confidence limit on the dose associated with 10% extra risk for
renal tumors in male rats). Has the modeling approach been appropriately conducted and clearly
described?
Comment: All of the reviewers agreed that the modeling approach was appropriately conducted.
One reviewer requested clarification that EPA used the matched vehicle control data when
modeling the hepatocellular carcinoma data.
Response: Text has been modified in the Section 5.4.3 and Table 5-5 to indicate that the
matched vehicle control data is presented and is used for BMD modeling.
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APPENDIX B: BENCHMARK DOSE MODELING OUTPUT
Table B-l. Dose-response modeling results using BMDS (version 2.0) based
on non-cancerous kidney and liver effects in rats following oral exposure to
HCE
Study
Endpoint
Sex/species
Fitted model"
/7-Value
AIC
BMD10
(mg/kg-d)
BMDL10
(mg/kg-d)
Kidney effects
NCI
(1978)
NTP
(1989)
NTP
(1989)
NTP
(1989)
Gorzinski
etal.
(1985)
Tubular nephropathy
Moderate to marked
Tubular nephropathy
Mild to moderate
Tubular nephropathy
Linear mineralization
Hyperplasia of the
pelvic transitional
epithelium
Atrophy and
degeneration of renal
tubules
Male rat
Female rat
Male rat
Female rat
Male rat
Male rat
Male rat
Female rat
Gamma
Multistage 1°
Weibull
Gamma
Multistage 2°
Logistic
Probit
Weibull
Logistic
Multistage 1°
Probit
Quantal-linear
Gamma
Logistic
Multistage 1°
Probit
Quantal-
linear
Weibull
Logistic
Multistage 1°
Probit
Gamma
LogLogistic
Multistage 2°
Weibull
Quantal-linear
Gamma
Multistage 1°
Logistic
Probit
Quantal-
linear
Weibull
Gamma
Multistage 1°
0.93
0.93
0.93
1.00
0.94
0.42
0.53
1.00
0.99
0.87
0.99
0.87
0.86
0.46
0.78
0.47
0.86
0.86
0.36
0.20
0.51
0.42
0.48
0.42
0.42
0.42
0.70
0.93
0.89
0.89
0.93
0.69
0.99
0.93
133.68
133.66
133.68
117.47
116.09
118.61
118.14
117.47
205.88
205.90
205.88
205.90
191.90
192.42
192.96
192.40
191.90
191.90
148.11
148.90
147.66
84.64
84.42
84.64
84.64
84.64
32.94
32.94
32.97
32.95
32.94
34.92
42.47
40.61
21.22
21.25
21.22
87.24
80.63
95.19
91.25
84.22
3.84
3.20
3.81
3.20
15.17
23.06
15.91
22.55
15.17
15.17
4.30
1.75
3.98
7.33
7.05
7.33
7.33
7.33
1.34
1.34
3.30
3.08
1.34
1.72
13.80
8.54
16.99
17.01
16.99
50.63
41.89
73.25
69.20
48.62
2.62
1.88
2.60
1.88
10.72
18.33
11.14
18.04
10.72
10.72
3.45
1.40
3.22
4.87
4.48
4.87
4.87
4.87
0.728
0.728
1.98
1.95
0.728
0.729
4.56
4.49
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Table B-l. Dose-response modeling results using BMDS (version 2.0) based
on non-cancerous kidney and liver effects in rats following oral exposure to
HCE
Study
Gorzinski
etal.
(1985)
Endpoint
Slight hypertrophy
and/or dilation of
proximal convoluted
tubules
Sex/species
Male rat
Fitted model3
Logistic
Probit
Quantal-linear
Weibull
Gamma
Logistic
LogLogistic
LogProbit
Multistage 2°
Probit
Weibull
Quantal-
linear
/7-Value
0.98
0.99
0.93
0.98
0.99
0.66
0.68
0.54
0.94
0.67
0.99
0.99
AIC
40.51
40.49
40.61
42.47
20.88
23.91
23.89
24.26
22.84
23.85
20.88
20.88
BMD10
(mg/kg-d)
17.40
16.10
8.54
13.71
1.22
4.85
1.23
2.11
1.33
4.28
1.22
1.22
BMDL10
(mg/kg-d)
11.07
10.51
4.49
4.56
0.710
2.71
0.308
1.01
0.713
2.54
0.710
0.710
Liver effects
NTP
(1989)
Hepatocellular
necrosis
Female rat
Gamma
Multistage 1°
Logistic
Probit
Weibull
0.93
0.68
0.55
0.61
0.91
38.62
40.56
41.58
40.95
38.91
118.04
53.82
156.22
148.49
114.68
60.18
35.19
107.49
102.71
56.75
Tor all models, a BMR of 0.1 was employed in deriving the estimates of the benchmark dose (BMD10) and its
95% lower CL (BMDL10). Modeling output is provided for models that represent the POD for each of the kidney
endpoints; these models are highlighted in bold font.
Table B-l presents the dose-response modeling results using BMDS (version 2.0) based
on non-cancerous kidney and liver effects in rats following oral exposure to HCE. Based on the
incidence of tubular nephropathy in male rats (NCI, 1978), the logistic and probit models
exhibited significant lack-of-fit (p < 0.1), while the gamma, multistage (1°) and Weibull models
had/>-values > 0.1. All three of these models that showed adequate fit yielded the same AIC
values, as well as nearly equivalent BMDio and BMDLio estimates of 21.22 and 16.99 mg/kg-
day, respectively. Therefore, the candidate POD selected for this dataset is 16.99 mg/kg-day.
Based on the incidence of tubular nephropathy in female rats (NCI, 1978), only the
1° multistage model exhibited significant lack-of-fit. Of the models that did not show significant
lack-of-fit (i.e., gamma, multistage 2°, logistic, probit, and Weibull models), the BMDLio
estimates were within a factor of three of each other, suggesting no appreciable model
dependence. As the BMDLio values did not show large variation, the model with the lowest AIC
value was selected. Therefore, the multistage 2° model BMDLio of 41.89 mg/kg-day was
selected as the candidate POD for this dataset.
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In fitting the available dichotomous dose-response models to the incidence of moderate to
marked tubular nephropathy in male rats (NTP, 1989), the gamma and Weibull models exhibited
significant lack-of-fit (p < 0.1). The models that did not show significant lack-of-fit (i.e.,
logistic, multistage 1°, probit, and quantal-linear) yielded BMDLio estimates that were within a
factor of three of each other, suggesting no appreciable model dependence. As the BMDLio
values did not show large variation, the model with the lowest AIC value was selected. The AIC
values for the logistic and probit models were the lowest (and identical); therefore, the probit
model with the lowest BMDLio, of 2.60 mg/kg-day was selected as the candidate POD for this
dataset.
Based on the incidence of mild to moderate tubular nephropathy in female rats (NTP,
1989), none of the models exhibited significant lack-of-fit. These models (i.e., gamma, logistic,
multistage 1°, probit, quantal-linear, and Weibull models) yielded BMDLio estimates that were
within a factor of three of each other, suggesting no appreciable model dependence. As the
BMDLio values did not show large variation, the model with the lowest AIC value was selected.
The gamma, quantal-linear, and Weibull models had identical AIC values; therefore, the model
with the lowest BMDLio was selected. The BMDLio values for these models were identical;
therefore, the BMDLio of 10.72 mg/kg-day was selected as the candidate POD for this dataset.
In fitting the available dichotomous dose-response models to the incidence of linear
mineralization in male rats (NTP, 1989), the gamma and the Weibull models exhibited
significant lack-of-fit (p < 0.1). Of the models that did not show significant lack-of-fit (i.e.,
logistic, multistage 1°, and probit), the resulting BMDLio estimates were within a factor of three
of each other, suggesting no appreciable model dependence. As the BMDLio values did not
show large variation, the model with the lowest AIC value was selected. Therefore, the probit
model BMDLio of 3.22 mg/kg-day was selected as the candidate POD for this dataset.
In fitting the available dichotomous dose-response models to the incidence of hyperplasia
of the pelvic transitional epithelium in male rats (NTP, 1989), the logistic, logprobit, and probit
models exhibited significant lack-of-fit (p < 0.1). Of the models that did not show significant
lack-of-fit (i.e., gamma, loglogistic, multistage 2°, Weibull, and quantal-linear), the resulting
BMDLio estimates were within a factor of three of each other, suggesting no appreciable model
dependence. As the BMDLio values did not show large variation, the model with the lowest AIC
value was selected. Therefore, the loglogistic model BMDLio of 4.48 mg/kg-day was selected as
the candidate POD for this dataset.
In fitting the available dichotomous dose-response models to the incidence of atrophy and
degeneration of renal tubules in male and female rats (Gorzinski et al., 1985), none of the models
exhibited a significant lack-of-fit in either sex. For male rats, these models (i.e., gamma,
multistage 1°, logistic, probit, quantal-linear, and Weibull) yielded BMDLio estimates that were
within a factor of three of each other, suggesting no appreciable model dependence. As the
BMDLio values did not show large variation, the model with the lowest AIC value was selected.
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The AIC values for the gamma, multistage 1°, and quantal-linear were identical; therefore, the
model with the lowest BMDLio was selected. All of the BMDLio values were identical for these
models; therefore, the BMDLio of 0.728 mg/kg-day was selected as the candidate POD for this
dataset.
For female rats, these models (i.e., gamma, multistage 1°, logistic, probit, quantal-linear,
and Weibull) yielded BMDLio estimates that were within a factor of three of each other,
suggesting no appreciable model dependence. As the BMDLio values did not show large
variation, the model with the lowest AIC value was selected. The probit BMDLio of 10.51
mg/kg-day was selected as the candidate POD for this dataset.
In fitting the available dichotomous dose-response models to the incidence of slight
hypertrophy and/or dilation of proximal convoluted tubules in male rats (Gorzinski et al., 1985),
none of the models exhibited a significant lack-of-fit. For male rats, these models (i.e., gamma,
logistic, loglogistic, logprobit, multistage 2°, probit, Weibull, and quantal-linear) yielded
BMDLio estimates that were within a factor of three of each other, suggesting no appreciable
model dependence. As the BMDLio values did not show large variation, the model with the
lowest AIC value was selected. The gamma, Weibull, and quantal-linear models yielded the
lowest ( and identical) AICs. All of the BMDLio values were identical for these models;
therefore, the BMDLio of 0.710 mg/kg-day was selected as the candidate POD for this dataset.
Based on the incidence of hepatocellular necrosis in female rats (NTP, 1989), none of the
dichotomous dose-response models exhibited a significant lack-of-fit. All of these models (i.e.,
gamma, multistage 1°, logistic, probit, and Weibull) yielded BMDLio estimates that were within
a factor of three of each other, suggesting no appreciable model dependence. As the BMDLio
values did not show large variation, the model with the lowest AIC value was selected.
Therefore, the gamma model BMDLio of 60.18 mg/kg-day was selected as the candidate POD
for this dataset.
For comparison purposes, BMD modeling for the above endpoints was also conducted
using BMRs of 5 and 1%. The modeling results are included in Table B-2.
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Table B-2. Dose-response modeling results using BMDS (version 2.0) for
BMRs of 1, 5, and 10% based on noncancerous kidney and liver effects in
rats following oral exposure to HCE
Study
Endpoint
Sex/
species
Fitted
model3
BMD10
(mg/
kg-d)
BMDL10
(mg/
kg-d)
BMDOS
(mg/
kg-d)
BMDLos
(mg/
kg-d)
BMD01
(mg/
kg-d)
BMDL01
(mg/
kg-d)
Kidney effects
NCI
(1978)
NTP
(1989)
NTP
(1989)
NTP
(1989)
Gorzinski
etal.
(1985)
Gorzinski
etal.
(1985)
Tubular
nephropathy
Moderate to
marked
tubular
nephropathy
Mild to
moderate
tubular
nephropathy
Linear
mineralization
Hyperplasia of
the pelvic
transitional
epithelium
Atrophy and
degeneration
of renal
tubules
Slight
hypertrophy
and/or dilation
of proximal
convoluted
tubules
Male
rat
Female
rat
Male
rat
Female
rat
Male
rat
Male
rat
Male
rat
Female
rat
Male
rat
Gamma and
Weibull
Multistage 1°
Multistage 2°
Probit
Gamma,
Quantal-
linear, and
Weibull
Probit
LogLogistic
Gamma,
Multistage
1°, and
Quantal-
linear
Probit
Gamma,
Weibull, and
Quantal-
linear
21.22
21.25
80.63
3.81
15.17
3.98
7.05
1.34
16.10
1.22
16.99
17.01
41.89
2.60
10.72
3.22
4.48
0.73
10.51
0.71
10.33
10.35
56.26
1.93
7.39
2.36
3.34
0.66
8.89
0.60
8.27
8.28
21.18
1.32
5.22
1.80
2.12
0.35
5.60
0.35
2.02
2.03
24.90
0.39
1.45
0.58
0.64
0.13
1.97
0.12
1.62
1.62
4.28
0.27
1.02
0.40
0.41
0.07
1.18
0.07
Liver effects
NTP
(1989)
Hepatocellular
necrosis
Female
rat
Gamma
118.03
60.18
84.66
33.34
41.75
8.60
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Modeling for Noncancer Assessment
NCI (1978) Tubular Nephropathv in Male Rats
Gamma Model
Gamma Model. (Version: 2.13; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmpCDF.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmpCDF.plt
Thu Apr 09 14:55:06 2009
BMDS Model Run NCI 1978 Tubular Nephropathy Male Rat - Gamma Model
The form of the probability function is:
P[response]= background+(1-background)*CumGamma[siope*dose,power],
where CumGamma(.) is the cumulative Gamma distribution function
Dependent variable = PercentPositiveModerateMarkedTubularNephropathy
Independent variable = ularNephropathy
Power parameter is restricted as power >=1
Total number of observations = 3
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 = 0.0238095
Slope = 0.00474439
Power = 1.01848
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Power
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Slope
Slope 1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background 0 NA
Slope 0.00496352 0.000693669 0.00360396
0.00632309
Power 1 NA
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NA - Indicates that this parameter has hit a bound
implied by some inequality constraint and thus
has no standard error.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-65.7706
-65.8419
-82.1514
133.684
# Param' s
3
1
1
Deviance
0.142715
32.7616
Test d.f.
2
2
P-value
0.9311
<.0001
Goodness of Fit
Dose Est._Prob. Expected Observed Size
Scaled
Residual
0.0000
113.0000
227.0000
ChiA2 = 0.14
0.0000
0.4293
0.6759
d.f.
0.000 0.000 20
21.035 22.050 49
33.795 33.000 50
P-value = 0.9308
0.000
0.293
-0.240
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 21.227
BMDL = 16.9904
B-7
DRAFT - DO NOT CITE OR QUOTE
-------
Gamma Multi-Hit Model with 0.95 Confidence Level
I
£=
O
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Gamma Multi-Hit
50
100
150
200
dose
14:5504/092009
B-S
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage 1'
Multistage Model. $Revision: 2.1 $ $Date: 2000/08/21 03:38:21 $
Input Data File: C:\BMDS\UNSAVED1.(d)
Gnuplot Plotting File: C:\BMDS\UNSAVEDl.plt
Thu Sep 14 09:09:29 2006
BMDS Model Run NCI 1978 Tubular Nephropathy Male Rat - Multistage 1 degree Model
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
Dependent variable = PercentPositiveModerateMarkedTubularNephropathy
Independent variable = ularNephropathy
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0.0201528
Beta(l) = 0.00475168
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Beta(l)
Beta(l) 1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background 0 * * *
Beta(l) 0.00495719 * * *
* - Indicates that this value is not calculated.
Analysis of Deviance Table
B-9 DRAFT - DO NOT CITE OR QUOTE
-------
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood) # Param's Deviance Test d.f. P-value
-65.7706
-65.8277
-82.1514
133.655
0.114158
32.7616
0.9445
<.0001
Goodness of Fit
Dose
0.0000
113.0000
227.0000
Est. Prob.
0.0000
0.4289
0.6754
Expected
0.000
21.015
33.772
Observed
0.000
22.050
33.000
Size
20
49
50
Scaled
Residual
0.000
0.299
-0.233
ChiA2 = 0.14
d.f.
P-value = 0.9307
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
21.2541
17.0107
26.9612
Taken together, (17.0107, 26.9612) is a 90
interval for the BMD
two-sided confidence
0.8
0.7
0.6
J> 0.5
o
< 0.4
o
tj 0.3
5
"~ 0.2
0.1
0
Multistage Model with 0.95 Confidence Lexel
Multistage
BMR
0
50
09:0909/142006
100
150
dose
200
250
300
B-10
DRAFT - DO NOT CITE OR QUOTE
-------
Weibull
Weibull Model $Revision: 2.2 $ $Date: 2000/03/17 22:27:16 $
Input Data File: C:\BMDS\UNSAVED1.(d)
Gnuplot Plotting File: C:\BMDS\UNSAVEDl.plt
Thu Sep 14 09:13:24 2006
BMDS Model Run NCI 1978 Tubular Nephropathy Male Rat - Weibull Model
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(-slope*dose/xpower)]
Dependent variable = PercentPositiveModerateMarkedTubularNephropathy
Independent variable = ularNephropathy
Power parameter is restricted as power >=1
Total number of observations = 3
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 = 0.0238095
Slope = 0.00453277
Power = 1.00295
the user,
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Power
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
Slope
Slope 1
Parameter Estimates
Estimate
0
0.00496352
1
Std. Err.
NA
0.000693669
NA
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
0.00360396
Interval
Variable
Limit
Background
Slope
0.00632309
Power
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
B-ll
DRAFT - DO NOT CITE OR QUOTE
-------
Full model -65.7706 3
Fitted model -65.8419 1 0.142715 2 0.9311
Reduced model -82.1514 1 32.7616 2 <.0001
AIC: 133.684
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.0000 0.000 0.000 20 0.000
113.0000 0.4293 21.035 22.050 49 0.293
227.0000 0.6759 33.795 33.000 50 -0.240
ChiA2 = 0.14 d.f. = 2 P-value = 0.9308
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 21.227
BMDL = 16.9904
B-12 DRAFT - DO NOT CITE OR QUOTE
-------
Weibull Model with 0.95 Confidence Lexel
i Affected
o
tj
5
LJ_
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
E \A/_:u.. .11 :
: VVeiDUM :
I
-------
NCI (1978) Tubular Nephropathy in Female Rats
Multistage 2°
Multistage Model. $Revision: 2.1 $ $Date: 2000/08/21 03:38:21 $
Input Data File: C:\BMDS\UNSAVED1.(d)
Gnuplot Plotting File: C:\BMDS\UNSAVEDl.plt
Thu Apr 09 16:18:29 2009
BMDS Model Run - NCI 1978 Tubular Nephropathy Female Rat - Multistage 2 degree Model
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/xl-beta2*dose/x2) ]
The parameter betas are restricted to be positive
Dependent variable = PercentPositiveModerateMarkedTubularNephropathy
Independent variable = ularNephropathy
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0
Beta(l) = 0
Beta(2) = 1.74381e-005
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Beta(l)
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Beta(2)
Beta(2) 1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background 0 * * *
Beta(l) 0 * * *
Beta(2) 1.62048e-005 * * *
* - Indicates that this value is not calculated.
B-14 DRAFT - DO NOT CITE OR QUOTE
-------
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-56.7357
-57.0429
-74.4688
116.086
# Param's
3
1
1
Deviance Test d.f.
P-value
0.614339
35.466
0.7355
<.0001
Goodness of Fit
Dose Est._Prob. Expected Observed Size
Scaled
Residual
0.0000
113.0000
227.0000
ChiA2 =0.13
0.0000
0.1869
0.5661
d.f.
0.000 0.000 20
9.346 9.000 50
27.741 28.910 49
P-value = 0.9374
0.000
-0.125
0.337
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
80.6338
41.8864
93.2552
Taken together, (41.8864, 93.2552) is a 90
interval for the BMD
two-sided confidence
B-15
DRAFT - DO NOT CITE OR QUOTE
-------
0.7
0.6
| 0.4
•2 0.3
o
ro
^ 0.2
0.1
0
Multistage Model with 0.95 Confidence Lexel
Multistage
BMD
0
09:21 09/142006
50
100
150
dose
200
250
300
B-16
DRAFT - DO NOT CITE OR QUOTE
-------
NTP (1989) Male Rat Nephropathv
Probit Model
Probit Model. (Version: 3.1; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmpAOE.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmpAOE.plt
Wed Apr 08 13:27:38 2009
BMDS Model Run NTP 1989 Tubular Nephropathy Male Rat - Probit Model
The form of the probability function is:
P[response] = CumNorm(Intercept+Slope*Dose),
where CumNorm(.) is the cumulative normal distribution function
Dependent variable = PercentPositiveModerateMarkedTubularNephropathy
Independent variable = ularNephropathy
Slope parameter is not restricted
Total number of observations = 3
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 = -0.354714
slope = 0.0433259
Asymptotic Correlation Matrix of Parameter Estimates
the user,
( *** The 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
intercept 1 -0.78
slope -0.78 1
Parameter Estimates
Interval
Variable
Limit
intercept
0.0341335
slope
0.0794134
Estimate
-0.35763
0.0436991
Std. Err.
0.165052
0.0182219
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
-0.681127
0.00798493
B-17
DRAFT - DO NOT CITE OR QUOTE
-------
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-100.939
-100.939
-103.852
205.878
# Param's Deviance Test d.f.
3
2 0.000120944 1
1 5.82641 2
P-value
0.9912
0.0543
Goodness of Fit
Dose Est._Prob. Expected Observed Size
Scaled
Residual
0.0000
7.0000
14.0000
0.3603
0.4794
0.6003
= 0.00
d.f. = 1
18.016 18.000 50 -0.005
23.968 24.000 50 0.009
30.016 30.000 50 -0.005
P-value = 0.9912
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
£=
g
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Extra risk
0.95
3.81407
2.59812
Probit Model with 0.95 Confidence Level
Probit
BMDL
BMD
10
12
14
dose
13:2704/082009
B-18
DRAFT - DO NOT CITE OR QUOTE
-------
NTP (1989) Female Rat Nephropathv
Gamma Model
Gamma Model. (Version: 2.13; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmpD9.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmpD9.plt
Fri Apr 10 10:19:37 2009
BMDS Model Run NTP 1989 Tubular Nephropathy Female Rat - Gamma Model
The form of the probability function is:
P[response]= background+(1-background)*CumGamma[siope*dose,power] ,
where CumGamma(.) is the cummulative Gamma distribution function
Dependent variable = PercentPositiveModerateMarkedTubularNephropathy
Independent variable = ularNephropathy
Power parameter is restricted as power >=1
Total number of observations = 3
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 = 0.245098
Slope = 0.0111213
Power = 1.3
Asymptotic Correlation Matrix of Parameter Estimates
the user,
( *** The model parameter(s) -Power
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
Background Slope
Background 1 -0.55
Slope -0.55 1
Interval
Variable
Limit
Background
0.358621
Slope
0.0102497
Power
Estimate
0.242452
0.00694477
1
Parameter Estimates
Std. Err.
0.0592711
0.0016862
NA
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
0.126283
0.00363988
NA - Indicates that this parameter has hit a bound
B-19
DRAFT - DO NOT CITE OR QUOTE
-------
implied by some inequality constraint and thus
has no standard error.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log (likelihood)
-93.9362
-93.9519
-102.85
# Param' s
3
2
1
Deviance
0.0312372
17.8276
AIC:
Test d.f.
1
2
191.904
P-value
0.8597
0.0001345
Goodness of Fit
Dose Est._Prob. Expected Observed Size
Scaled
Residual
0.0000 0.2425
57.0000 0.4901
114.0000 0.6568
12.123 12.000
24.504 25.000
32.182 31.850
50
50
49
-0.040
0.140
-0.100
=0.03
d.f. = 1
P-value = 0.8596
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 15.1712
BMDL = 10.7248
B-20
DRAFT - DO NOT CITE OR QUOTE
-------
Quantal Linear Model with 0.95 Confidence Level
o
t3
ro
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
12:21 12/162008
Quantal Linear
BMD Lower Bound
100
B-21
DRAFT - DO NOT CITE OR QUOTE
-------
Quantal-linear Model
Quantal Linear Model using Weibull Model (Version: 2.12; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmpE4.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmpE4.plt
Fri Apr 10 10:36:29 2009
BMDS Model Run NTP 1989 Tubular Nephropathy Female Rat - Quantal-linear Model
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(-slope*dose)]
Dependent variable = PercentPositiveModerateMarkedTubularNephropathy
Independent variable = ularNephropathy
Total number of observations = 3
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 = 0.245098
Slope = 0.00666772
Power = 1 Specified
the user,
Background
Slope
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Power
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
Background Slope
1 -0.55
-0.55 1
Interval
Variable
Limit
Background
0.358621
Slope
0.0102497
Estimate
0.242451
0.00694478
Parameter Estimates
Std. Err.
0.0592711
0.0016862
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
0.126282
0.00363989
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -93.9362 3
Fitted model -93.9519 2 0.0312372 1 0.8597
B-22
DRAFT - DO NOT CITE OR QUOTE
-------
Reduced model
AIC:
-102.85
191.904
17.8276
0.0001345
Goodness of Fit
Dose
0.0000
57.0000
114.0000
Est. Prob.
0.2425
0.4901
0. 6568
Expected
12.123
24.504
32.182
Observed
12.000
25.000
31.850
Size
50
50
49
Scaled
Residual
-0.040
0.140
-0.100
ChiA2 =0.03
d.f. = 1
P-value = 0.8596
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 15.1712
BMDL = 10.7248
I
£=
O
'
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
10:3604/102009
Quantal Linear Model with 0.95 Confidence Level
100
B-23
DRAFT - DO NOT CITE OR QUOTE
-------
B-24 DRAFT - DO NOT CITE OR QUOTE
-------
Weibull Model
Weibull Model using Weibull Model (Version: 2.12; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmpE3.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmpE3.plt
Fri Apr 10 10:34:27 2009
BMDS Model Run NTP 1989 Tubular Nephropathy Female Rat - Weibull Model
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(-slope*dose/xpower)]
Dependent variable = PercentPositiveModerateMarkedTubularNephropathy
Independent variable = ularNephropathy
Power parameter is restricted as power >=1
Total number of observations = 3
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 = 0.245098
Slope = 0.00666772
Power = 1
Asymptotic Correlation Matrix of Parameter Estimates
the user,
( *** The model parameter(s) -Power
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
Background Slope
Background 1 -0.55
Slope -0.55 1
Interval
Variable
Limit
Background
0.358621
Slope
0.0102497
Power
Estimate
0.242451
0.00694478
1
Parameter Estimates
Std. Err.
0.0592711
0.0016862
NA
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
0.126282
0.00363989
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
B-25
DRAFT - DO NOT CITE OR QUOTE
-------
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-93.9362
-93.9519
-102.85
191.904
# Param's Deviance Test d.f. P-value
3
2 0.0312372 1 0.8597
1 17.8276 2 0.0001345
Goodness of Fit
Dose
0.0000
57.0000
114.0000
Est. Prob.
0.2425
0.4901
0.6568
Expected
12.123
24.504
32.182
Observed
12.000
25.000
31.850
Size
50
50
49
Scaled
Residual
-0.040
0.140
-0.100
ChiA2 =0.03
d.f. = 1
P-value = 0.8596
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 15.1712
BMDL = 10.7248
B-26
DRAFT - DO NOT CITE OR QUOTE
-------
Weibull Model with 0.95 Confidence Level
I
£=
O
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
10:3404/102009
100
B-27
DRAFT - DO NOT CITE OR QUOTE
-------
NTP (1989) Linear Mineralization in Male Rats
Probit Model
Probit Model. (Version: 3.1; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmpA33.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmpA33.plt
Wed Apr 08 14:24:02 2009
BMDS Model Run NTP 1989 Linear Mineralization Male Rat - Probit Model
The form of the probability function is:
P[response] = CumNorm(Intercept+Slope*Dose),
where CumNorm(.) is the cumulative normal distribution function
Dependent variable = PercentPositiveLinearMineralization
Independent variable = ion
Slope parameter is not restricted
Total number of observations = 3
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 = -1.67551
slope = 0.149038
Asymptotic Correlation Matrix of Parameter Estimates
the user,
( *** The 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
intercept 1 -0.87
slope -0.87 1
Parameter Estimates
Interval
Variable
Limit
intercept
1.14919
slope
0.191579
Estimate
-1. 62793
0.144885
Std. Err.
0.244257
0.0238239
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
-2.10666
0.0981906
B-28
DRAFT - DO NOT CITE OR QUOTE
-------
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-71.6113
-71.8283
-94.7689
147.657
# Param's
3
2
1
Deviance Test d.f.
P-value
0.433989
46.3152
0.51
<.0001
Goodness of Fit
Dose Est._Prob. Expected Observed Size
Scaled
Residual
0.0000
7.0000
14.0000
0.0518
0.2697
0.6556
= 0.43
d.f. = 1
2.589 2.000 50 -0.376
13.485 15.000 50 0.483
32.780 32.000 50 -0.232
P-value = 0.5129
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 3.98089
BMDL = 3.21773
I
£=
O
'
0.1
Probit Model with 0.95 Confidence Level
10
12
14
dose
14:24 04/08 2009
B-29
DRAFT - DO NOT CITE OR QUOTE
-------
B-30 DRAFT - DO NOT CITE OR QUOTE
-------
NTP (1989) Male Rat Hyperplasia of Pelvic Transitional
Epithelium
LogLogistic Model
Logistic Model. (Version: 2.12; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmp4D5.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmp4D5.plt
Wed Aug 12 14:26:53 2009
BMDS Model Run - NTP 989 - Male Rat - Hyperplasia - LogLogistic Model
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-siope*Log(dose)) ]
Dependent variable = Effect
Independent variable = DOSE
Slope parameter is restricted as slope >= 1
Total number of observations = 3
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 = -3.7612
slope = 1
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -background -slope
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
intercept
intercept 1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Limit
background 0 * * *
intercept -4.15077 * * *
slope 1 * * *
* - Indicates that this value is not calculated.
B-31 DRAFT - DO NOT CITE OR QUOTE
-------
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-40.4963
-41.2103
-46.5274
84.4207
# Param's
3
1
1
Deviance Test d.f.
1.42796
12.0622
P-value
0.4897
0.002403
Dose
0.0000
7.0000
14.0000
Est. Prob.
0.0000
0.0993
0.1807
Goodness of Fit
Expected Observed Size
0.000
4.966
9.034
0.000
7.000
7.000
50
50
50
Scaled
Residual
0.000
0.962
-0.748
= I.-
d.f. = 2
P-value = 0.4761
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 7.05365
BMDL = 4.48322
"o
I
0.3
0.25
0.2
0.15
0.1
0.05
Log-Logistic Model with 0.95 Confidence Level
Log-Logistic
BMDL
BMD
10 12 14
dose
14:2608/122009
B-32
DRAFT - DO NOT CITE OR QUOTE
-------
B-33 DRAFT - DO NOT CITE OR QUOTE
-------
Gorzinski (1985) Atrophy and Degeneration of renal tubules in
Male Rats
Gamma Model
Gamma Model. (Version: 2.13; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmpF14.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmpF14.plt
Thu Oct 08 08:59:00 2009
ndDegenRenalTubulesDataNoSeverityMaleRat.dax
The form of the probability function is:
P[response]= background+(1-background)*CumGamma[siope*dose,power],
where CumGamma(.) is the cummulative Gamma distribution function
Dependent variable = Effect
Independent variable = DOSE
Power parameter is restricted as power >=1
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial (and Specified) Parameter Values
Background = 0.136364
Slope = 0.0871864
Power = 1.3
Asymptotic Correlation Matrix of Parameter Estimates
Background Slope Power
Background 1 0.52 0.64
Slope 0.52 1 0.93
Power 0.64 0.93 1
Interval
Variable
Limit
Background
0.320747
Slope
0.244756
Power
3.0996
Parameter Estimates
95.0% Wald Confidence
Estimate Std. Err. Lower Conf. Limit Upper Conf.
0.110626 0.107207 -0.0994949
0.0787607 0.0846932 -0.0872348
1.00164 1.07041 -1.09632
Analysis of Deviance Table
B-34
DRAFT - DO NOT CITE OR QUOTE
-------
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-14.3635
-14.4712
-27.7259
34.9424
# Param's
4
3
1
Deviance Test d.f.
0.215359
26.7248
P-value
0.6426
<.0001
Goodness of Fit
Dose
0.0000
1.0000
15.0000
62.0000
Est. Prob.
0.1106
0.1777
0.7265
0.9932
Expected
1.106
1.777
7.265
9.932
Observed
1.000
2.000
7.000
10.000
Size
10
10
10
10
Scaled
Residual
-0.107
0.185
-0.188
0.261
=0.15
d.f. = 1
P-value = 0.6994
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 1.34399
BMDL = 0.727509
Gamma Multi-Hit Model with 0.95 Confidence Level
g
t3
ro
0.8
0.6
0.4
0.2
BMDL
BMD
0
08:5910/082009
Gamma Multi-Hit —
10 20 30 40 50 60
dose
B-35
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage 1 degree Model
Multistage Model. (Version: 3.0; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmpF17.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmpF17.plt
Thu Oct 08 09:00:57 2009
ndDegenRenalTubulesDataNoSeverityMaleRat.dax
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*doseAl) ]
The parameter betas are restricted to be positive
Dependent variable = Effect
Independent variable = DOSE
Total number of observations = 4
Total number of records with missing values = 0
Total number of parameters in model = 2
Total number of specified parameters = 0
Degree of polynomial = 1
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial Parameter Values
Background = 0
Beta(l) = 1.66732e+018
Asymptotic Correlation Matrix of Parameter Estimates
Background Beta(l)
Background 1 -0.4
Beta(l) -0.4 1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background 0.11052 * * *
Beta(l) 0.0786399 * * *
Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
B-36 DRAFT - DO NOT CITE OR QUOTE
-------
Full model
Fitted model
Reduced model
AIC:
-14.3635
-14.4712
-27.7259
32.9424
0.215361
26.7248
0.8979
<.0001
Goodness of Fit
Dose
0.0000
1.0000
15.0000
62.0000
Est. Prob.
0.1105
0.1778
0.7266
0.9932
Expected
1.105
1.778
7.266
9.932
Observed
1.000
2.000
7.000
10.000
Size
10
10
10
10
Scaled
Residual
-0.106
0.184
-0.189
0.261
ChiA2 =0.15
d.f.
P-value = 0.9283
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
1.33978
0.727509
2.66189
Taken together, (0.727509, 2.66189) is a 90
interval for the BMD
two-sided confidence
B-37
DRAFT - DO NOT CITE OR QUOTE
-------
Multistage Model with 0.95 Confidence Level
o
t3
ro
0.8
0.6
0.4
0.2
Multistage
BMDLBMD
0
09:0010/082009
10 20 30 40 50 60
dose
B-3 8 DRAFT - DO NOT CITE OR QUOTE
-------
Quantal-linear Model
Quantal Linear Model using Weibull Model (Version: 2.12; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmpF18.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmpF18.plt
Thu Oct 08 09:02:11 2009
ndDegenRenalTubulesDataNoSeverityMaleRat.dax
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(-slope*dose)]
Dependent variable = Effect
Independent variable = DOSE
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial (and Specified) Parameter Values
Background = 0.136364
Slope = 0.047491
Power = 1 Specified
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Power
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Background Slope
Background 1 -0.29
Slope -0.29 1
Parameter Estimates
Interval
Variable
Limit
Background
0.271199
Slope
0.139505
Estimate
0.11052
0.0786399
Std. Err.
0.0819804
0.0310542
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
-0.0501583
0.0177749
Model
Full model
Fitted model
Analysis of Deviance Table
Log(likelihood)
-14.3635
-14.4712
# Param's Deviance Test d.f. P-value
4
2 0.215361 2 0.8979
B-39
DRAFT - DO NOT CITE OR QUOTE
-------
Reduced model
AIC:
-27.7259
32.9424
26.7248
<.0001
Goodness of Fit
Dose
0.0000
1.0000
15.0000
62.0000
Est. Prob.
0.1105
0.1778
0.7266
0.9932
Expected
1.105
1.778
7.266
9.932
Observed
1.000
2.000
7.000
10.000
Size
10
10
10
10
Scaled
Residual
-0.106
0.184
-0.189
0.261
=0.15
d.f. = 2
P-value = 0.9283
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 1.33978
BMDL = 0.727509
I
o
ro
0.8
0.6
0.4
0.2
BMDLBMD
0
09:0210/082009
Quantal Linear Model with 0.95 Confidence Level
Quantal Linear
10
20
30 40
dose
50
60
B-40
DRAFT - DO NOT CITE OR QUOTE
-------
Gorzinski (1985) Atrophy and Degeneration of renal tubules in
Female Rats
Probit Model
Probit Model. (Version: 3.1; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmpFOE.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmpFOE.plt
Thu May 06 10:06:12 2010
ubulesDataNoSeverityFemaleRat.dax
The form of the probability function is:
P[response] = CumNorm(Intercept+Slope*Dose),
where CumNorm(.) is the cumulative normal distribution function
Dependent variable = Effect
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 = -1.21184
slope = 0.0236401
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -background
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
intercept slope
intercept 1 -0.69
slope -0.69 1
Interval
Variable
Limit
intercept
0.628881
slope
0.0417261
Estimate
-1.26508
0.0246481
Parameter Estimates
Std. Err.
0.324595
0.00871343
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
-1.90127
0.00757005
B-41
DRAFT - DO NOT CITE OR QUOTE
-------
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-18.2358
-18.2465
-22.4934
40.493
# Param's
4
2
1
Deviance Test d.f.
P-value
0.0214055
8.51521
0.9894
0.03648
Goodness of Fit
Dose
0.0000
1.0000
15.0000
62.0000
Est. Prob.
0.1029
0.1074
0.1853
0. 6038
Expected
1.029
1.074
1.853
6.038
Observed
1.000
1.000
2.000
6.000
Size
10
10
10
10
Scaled
Residual
-0.030
-0.076
0.120
-0.024
=0.02
d.f. = 2
P-value = 0.9893
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 16.0998
BMDL = 10.5128
I
o
ro
0.8
0.6
0.4
0.2
Probit Model with 0.95 Confidence Level
10:0605/062010
B-42
DRAFT - DO NOT CITE OR QUOTE
-------
B-43 DRAFT - DO NOT CITE OR QUOTE
-------
Gorzinski et al. (1985) Male Rat Hypertrophy and/or Dilation of
Proximal Tubules
Gamma Model
Gamma Model. (Version: 2.13; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmp4D6.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmp4D6.plt
Wed Aug 12 14:31:38 2009
HMDS Model Run - Gorzinski et al (1985) - Male Rat - Hypertrophy/Dilation of Proximal
Tubules - Gamma Model
The form of the probability function is:
P[response]= background+(1-background)*CumGamma[siope*dose,power],
where CumGamma(.) is the cummulative Gamma distribution function
Dependent variable = Effect
Independent variable = DOSE
Power parameter is restricted as power >=1
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial (and Specified) Parameter Values
Background = 0.0454545
Slope = 0.0907614
Power = 1.3
the user,
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Power
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
Slope
Slope 1
Parameter Estimates
Interval
Variable
Limit
Background
Slope
0.143889
Power
Estimate
0
0.0860249
1
Std. Err.
NA
0.029523
NA
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
0.0281609
B-44
DRAFT - DO NOT CITE OR QUOTE
-------
has no standard error.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log (likelihood)
-9.35947
-9.44226
-27.5256
# Param' s
4
1
1
Deviance
0.165576
36.3322
Test d.f.
3
3
P-value
0.9829
<.0001
AIC: 20.8845
Goodness of Fit
Dose
0.0000
1.0000
15.0000
62.0000
Est. Prob.
0.0000
0.0824
0.7248
0.9952
Expected
0.000
0.824
7.248
9.952
Observed
0.000
1.000
7.000
10.000
Size
10
10
10
10
Scaled
Residual
0.000
0.202
-0.176
0.220
=0.12 d.f. = 3 P-value = 0.9893
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 1.22477
BMDL = 0.710032
B-45 DRAFT - DO NOT CITE OR QUOTE
-------
Gamma Multi-Hit Model with 0.95 Confidence Level
o
t3
ro
0.8
0.6
0.4
0.2
Gamma Multi-Hit
BMDLBMD
0
14:31 08/122009
10 20 30 40 50 60
dose
B-46 DRAFT - DO NOT CITE OR QUOTE
-------
Weibull Model
Weibull Model using Weibull Model (Version: 2.12; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmp4D9.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmp4D9.plt
Wed Aug 12 14:35:51 2009
BMDS Model Run - Gorzinski et al (1985) - Male rats - Hypertrophy/Dilation of
Proximal Tubules - Weibull Model using Weibull Model (Version: 2.12; Date:
05/16/2008)
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(-slope*dose/xpower)]
Dependent variable = Effect
Independent variable = DOSE
Power parameter is restricted as power >=1
Total number of observations = 4
Total number of records with missing values = 0
Maximum number of iterations = 250
Relative Function Convergence has been set to: le-008
Parameter Convergence has been set to: le-008
Default Initial (and Specified) Parameter Values
Background = 0.0454545
Slope = 0.0491052
Power = 1
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Power
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Slope
Slope 1
Parameter Estimates
95.0% Wald Confidence
Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.
Limit
Background 0 NA
Slope 0.086025 0.0295231 0.0281608
0.143889
Power 1 NA
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
B-47 DRAFT - DO NOT CITE OR QUOTE
-------
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-9.35947
-9.44226
-27.5256
20.8845
# Param's
4
1
1
Deviance Test d.f.
P-value
0.165576
36.3322
0.9829
<.0001
Goodness of Fit
Dose
0.0000
1.0000
15.0000
62.0000
Est. Prob.
0.0000
0.0824
0.7248
0.9952
Expected
0.000
0.824
7.248
9.952
Observed
0.000
1.000
7.000
10.000
Size
10
10
10
10
Scaled
Residual
0.000
0.202
-0.176
0.220
=0.12
d.f. = 3
P-value = 0.9893
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 1.22477
BMDL = 0.710032
I
o
ro
Weibull Model with 0.95 Confidence Level
0.8
0.6
0.4
0.2
0 :
BMDLBMD
60
14:3508/122009
B-48
DRAFT - DO NOT CITE OR QUOTE
-------
Quantal-linear Model
antal-linear Model using Weibull Model (Version: 2.12; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmp4DA.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmp4DA.plt
Wed Aug 12 14:37:26 2009
BMDS Model Run - Gorzinski et al (1985) - Male rats - Hypertrophy/Dilation Proximal
Tubules - Quantal-linear Model using Weibull Model (Version: 2.12; Date: 05/16/2008)
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(-slope*dose/xpower)]
Dependent variable = Effect
Independent variable = DOSE
Power parameter is set to 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
Default Initial (and Specified) Parameter Values
Background = 0.0454545
Slope = 0.0491052
Power = 1 Specified
the user,
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background -Power
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
Slope
Slope 1
Parameter Estimates
Interval
Variable
Limit
Background
Slope
0.143889
Estimate
0
0.0860249
Std. Err.
NA
0.029523
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
Analysis of Deviance Table
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
0.0281608
B-49
DRAFT - DO NOT CITE OR QUOTE
-------
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-9.35947
-9.44226
-27.5256
20.8845
# Param's
4
1
1
Deviance Test d.f.
0.165576
36.3322
P-value
0.9829
<.0001
Goodness of Fit
Dose
0.0000
1.0000
15.0000
62.0000
Est. Prob.
0.0000
0.0824
0.7248
0.9952
Expected
0.000
0.824
7.248
9.952
Observed
0.000
1.000
7.000
10.000
Size
10
10
10
10
Scaled
Residual
0.000
0.202
-0.176
0.220
=0.12
d.f. = 3
P-value = 0.9893
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 1.22477
BMDL = 0.710032
I
o
ro
Weibull Model with 0.95 Confidence Level
0.8
0.6
0.4
0.2
0 :
BMDLBMD
60
14:3708/122009
B-50
DRAFT - DO NOT CITE OR QUOTE
-------
NTP (1989) Female Rat Hepatocellular Necrosis
Gamma Model
Gamma Model. (Version: 2.13; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmpB62.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmpB62.plt
Thu Apr 09 09:14:08 2009
BMDS Model Run NTP 1989 Hepatocellular Necrosis Female Rat - Gamma Model
The form of the probability function is:
P[response]= background+(1-background)*CumGamma[siope*dose,power],
where CumGamma(.) is the cummulative Gamma distribution function
Dependent variable = PercentPositiveHepatocellularNecrosis
Independent variable = rosis
Power parameter is restricted as power >=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
Default Initial (and Specified) Parameter Values
Background = 0.0454545
Slope = 0.00743289
Power = 2.82109
the user,
Slope
Power
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Background
have been estimated at a boundary point, or have been specified by
and do not appear in the correlation matrix )
Slope Power
1 0.95
0.95 1
Parameter Estimates
Interval
Variable
Limit
Background
Slope
0.0150393
Power
4.823
Estimate
0
0.00723384
2.58447
Std. Err.
NA
0.00398244
1.14213
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
-0.000571608
0.345944
B-51
DRAFT - DO NOT CITE OR QUOTE
-------
has no standard error.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log(likelihood)
-16.7382
-17.3091
-32.5964
38.6182
Param's
6
2
1
Deviance Test d.f.
1.14186
31.7164
P-value
0.8876
<.0001
ChiA2 = O.i
Goodness of Fit
0
33
67
134
267
535
Dose
.0000
.5000
.1000
.3000
.8000
.7000
Est
0.
0.
0.
0.
0.
0.
. Prob.
0000
0059
0300
1289
4095
8159
Expe
0,
0,
0,
1.
4,
8,
scted
.000
.059
.300
.289
.095
.159
0
0
0
0
2
4
8
bserved
.000
.000
.000
.000
.000
.000
Size
10
10
10
10
10
10
Scaled
Residual
0.000
-0.244
-0.556
0. 671
-0.061
-0.130
d.f. = 4
P-value = 0.9331
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 118.037
BMDL = 60.1812
B-52
DRAFT - DO NOT CITE OR QUOTE
-------
Gamma Multi-Hit Model with 0.95 Confidence Level
I
£=
O
0.8
0.6
0.4
0.2
Gamma Multi-Hit
BMDL BMD
100
200 300
dose
400 500
09:1404/092009
B-53 DRAFT - DO NOT CITE OR QUOTE
-------
Modeling for Cancer Assessment
NTP (1989) BMP Modeling of Renal Adenoma/Carcinoma in Male Rats
Multistage 2°Model
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmp6E8.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmp6E8.plt
Mon Apr 13 14:38:06 2009
BMDS Model Run NTP 1989 Kidney Adenoma-Carcinoma Male Rat - Multistage Cancer 2
degree Model
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/xl-beta2*dose/x2) ]
The parameter betas are restricted to be positive
Dependent variable = PercentAdenomaCarcinoma
Independent variable = DOSE
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
**** the web sight for model updates which will eventually ****
**** incorporate these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background = 0.014541
Beta(l) = 0
Beta(2) = 0.00799069
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta(l)
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Background Beta (2)
Background 1 -0.67
Beta(2) -0.67 1
B-54 DRAFT - DO NOT CITE OR QUOTE
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Parameter Estimates
Interval
Variable
Limit
Background
Beta(l)
Beta(2)
Estimate
0.0177261
0
0.00751246
Std. Err.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
Indicates that this value is not calculated.
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-33.5473
-33.6008
-36.7395
71.2015
# Param's Deviance Test d.f.
3
0.106829
6.38433
P-value
0.7438
0.04108
Goodness of Fit
Dose Est.
i
i
i
: 1
0.0000
: 2
2.0400
: 3
4.0900
Chi-square =
0.
0.
0.
Prob. Expected Observed Size
0177
0481
1343
0.10
0.
2.
6.
DF=
887
407
717
1
1
2
7
P-value = 0.
Scaled
Residual
50
50
50
,7510
0.129
-0.178
0.049
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
3.74496
2.45283
9.24921
Taken together, (2.45283, 9.24921) is a 90
interval for the BMD
two-sided confidence
Multistage Cancer Slope Factor =
0.0407692
B-55
DRAFT - DO NOT CITE OR QUOTE
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Multistage Cancer Model with 0.95 Confidence Level
I
£=
O
0.3
0.25
0.2
0.15
0.1
0.05
Multistage Cancer
Linear extrapolation
BMDL
0 0.5 1
1.5 2 2.5
dose
BMD
3.5
14:3804/132009
B-56 DRAFT - DO NOT CITE OR QUOTE
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NCI (1978) BMP Modeling of Hepatocellular Carcinoma in Male Mice
Multistage 2°
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmp7B8.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmp7B8.plt
Tue Apr 14 08:30:03 2009
HMDS Model Run NCI 1978 Hepatocellular Carcinoma Male Mice - Multistage Cancer 2
degree Model
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/xl-beta2*dose/x2) ]
The parameter betas are restricted to be positive
Dependent variable = PercentHepatocellularCarcinoma
Independent variable = DOSE
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
**** the web sight for model updates which will eventually ****
**** incorporate these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background = 0.141096
Beta(l) = 0
Beta(2) = 7.77012e-005
Asymptotic Correlation Matrix of Parameter Estimates
( *** The model parameter(s) -Beta(l)
have been estimated at a boundary point, or have been specified by
the user,
and do not appear in the correlation matrix )
Background Beta (2)
Background 1 -0.73
Beta(2) -0.73 1
Parameter Estimates
B-57 DRAFT - DO NOT CITE OR QUOTE
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Interval
Variable
Limit
Background
Beta(l)
Beta(2)
Estimate
0.146344
0
7.26074e-005
Std. Err.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
Indicates that this value is not calculated.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
AIC:
Log (likelihood)
-71.2862
-71.7199
-80.5752
147.44
# Param's Deviance Test d.f. P-value
3
2 0.867331 1 0.3517
1 18.5779 2 <.0001
Goodness of Fit
Dose
0.0000
53.0500
103.8800
Est. Prob.
0.1463
0.3041
0. 6101
Expected
2.927
15.206
29.892
Observed
3.000
15.000
30.870
Size
20
50
49
Scaled
Residual
0.046
-0.063
0.286
= 0.09
d.f. = 1
P-value = 0.7666
Benchmark Dose Computation
Specified effect =
Risk Type
Confidence level =
BMD =
BMDL =
BMDU =
0.1
Extra risk
0.95
38.0933
13.8018
49.5091
Taken together, (13.8018, 49.5091) is a 90
interval for the BMD
two-sided confidence
Multistage Cancer Slope Factor =
0.00724545
B-58
DRAFT - DO NOT CITE OR QUOTE
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Multistage Cancer Model with 0.95 Confidence Level
I
£=
O
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Multistage Cancer
Linear extrapolation
BMDL
BMD
20
40 60
dose
80
100
08:3004/142009
B-59
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NCI (1978) BMP Modeling of Hepatocellular Carcinoma in Female
Mice
Multistage 2°
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmp303.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmp303.plt
Wed May 20 14:37:03 2009
BMDS Model Run - NCI 1978 Hepatocellular Carcinoma Female Mice - Multistage Cancer 2
degree Model
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/xl-beta2*dose/x2) ]
The parameter betas are restricted to be positive
Dependent variable = PercentHepatocellularCarcinoma
Independent variable = DOSE
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
**** the web sight for model updates which will eventually ****
**** incorporate these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background = 0.178486
Beta(l) = 0.000367312
Beta(2) = 0
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 )
Background Beta(l)
Background 1 -0.89
Beta(l) -0.89 1
B-60 DRAFT - DO NOT CITE OR QUOTE
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Parameter Estimates
Interval
Variable
Limit
Background
Beta(l)
Beta(2)
Estimate
0.189829
0.000368083
0
Std. Err.
* - Indicates that this value is not calculated.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
Analysis of Deviance Table
Model
Full model
Fitted model
Reduced model
Log (likelihood)
-70.4882
-72.8848
-73.9112
# Param' s
3
2
1
AIC:
149.77
Deviance Test d.f.
4.79332
6.84615
P-value
0.02857
0.03261
Goodness of Fit
Dose Est._Prob. Expected Observed Size
Scaled
Residual
0.0000 0.1898
360.0000 0.2904
722.0000 0.3789
ChiA2 =4.95
3.797 2.000
14.519 20.000
18.566 15.190
20
50
49
d.f. = 1
P-value = 0.0260
-1.024
1.708
-0.994
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 286.241
BMDL = 136.877
BMDU did not converge for BMR = 0.100000
BMDU calculation failed
BMDU = 1.40207e+009
Multistage Cancer Slope Factor = 0.000730581
B-61
DRAFT - DO NOT CITE OR QUOTE
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Multistage Cancer Model with 0.95 Confidence Level
I
£=
O
0.6
0.5
0.4
0.3
0.2
0.1
Multistage Cancer
Linear extrapolation
100 200 300 400 500 600 700
14:3705/202009
B-62 DRAFT - DO NOT CITE OR QUOTE
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NTP (1989) BMP Modeling of Pheochromocvtoma/Malignant
Pheochromocvtomas in Male Rats
Multistage 2°
Multistage Cancer Model. (Version: 1.7; Date: 05/16/2008)
Input Data File: C:\USEPA\BMDS2\Temp\tmp70C.(d)
Gnuplot Plotting File: C:\USEPA\BMDS2\Temp\tmp70C.plt
Mon Apr 13 15:55:38 2009
tomaMaleRat.dax
The form of the probability function is:
P[response] = background + (1-background)*[1-EXP(
-betal*dose/xl-beta2*dose/x2) ]
The parameter betas are restricted to be positive
Dependent variable = PercentPheochromocytomaMalignantPheochromocytoma
Independent variable = Pheochromocytoma
Total number of observations = 3
Total number of records with missing values = 0
Total number of parameters in model = 3
Total number of specified parameters = 0
Degree of polynomial = 2
Maximum number of iterations = 250
Relative Function Convergence has been set to: 2.22045e-016
Parameter Convergence has been set to: 1.49012e-008
**** We are sorry but Relative Function and Parameter Convergence ****
**** are currently unavailable in this model. Please keep checking ****
**** the web sight for model updates which will eventually ****
**** incorporate these convergence criterion. Default values used. ****
Default Initial Parameter Values
Background = 0.381549
Beta(l) = 0.0404371
Beta(2) = 0
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 )
Background Beta(l)
Background 1 -0.78
Beta(l) -0.78 1
Parameter Estimates
B-63 DRAFT - DO NOT CITE OR QUOTE
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Interval
Variable
Limit
Background
Beta(l)
Beta(2)
Estimate
0.341708
0.055345
0
Std. Err.
Indicates that this value is not calculated.
95.0% Wald Confidence
Lower Conf. Limit Upper Conf.
Model
Full model
Fitted model
Reduced model
AIC:
Analysis of Deviance Table
Log(likelihood)
-93.0295
-96.701
-97.5291
197.402
# Param' s
3
2
1
Deviance Test d.f.
7.34302
8.99926
P-value
0.006732
0.01111
Goodness of Fit
Dose Est._Prob. Expected Observed Size
Scaled
Residual
0.0000 0.3417
2.0500 0.4123
4.1000 0.4753
= 7.50
17.085 14.000
18.554 26.100
23.292 19.110
50
45
49
-0.920
2.285
-1.196
d.f. = 1
P-value = 0.0062
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 1.9037
BMDL = 0.811704
BMDU did not converge for BMR = 0.100000
BMDU calculation failed
BMDU = Inf
B-64
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Multistage Cancer Model with 0.95 Confidence Level
0.7
0.6
0.5
I
£=
O
t> 0.4
CO
0.3
0.2
Multistage Cancer
Linear extrapolation
BMDL
BMD
0 0.5 1 1.5
2
dose
2.5
3.5
15:5504/132009
B-65 DRAFT - DO NOT CITE OR QUOTE
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