EPA/635/R-09/007F
www. ep a. go v/iris
oEPA
TOXICOLOGICAL REVIEW
OF
HEXACHLOROETHANE
(CAS No. 67-72-1)
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
September 2011
U.S. Environmental Protection Agency
Washington, DC
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DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection Agency
policy and approved for publication. Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
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TABLE OF CONTENTS
ABBREVIATIONS AND ACRONYMS
FOREWORD
AUTHORS 1 CONTRIBUTORS 1 REVIEWERS
1
2
3
4
INTRODUCTION
CHEMICAL AND PHYSICAL INFORMATION
Table 2-1 Physical properties of HCE
TOXICOKINETICS
3.1 Absorption
3.2 Distribution
Table 3-1 HCE, PERC, and pentachloroethane tissue concentrations in
anesthetized sheep 8.5 hours after injection of 500 mq/kq HCE
Table 3-2 Time course of HCE concentrations in male rat tissues after 57 days
of dietary exposure to 62 mq/kq-day
Table 3-3 HCE concentrations in male and female rat tissues after 1 1 0 or 1 1 1
days of dietary exposure
3.3 Metabolism
Fiqure 3-1 Plausible metabolic pathway of HCE
Table 3-4 Disposition of HCE in male rats and mice during 48 hours following
administration of an MTD for 4 weeks
Table 3-5 Metabolism of HCE measured in rats and mice
Table 3-6 Product formation rates and relative ratios of the products formed by
CYP450 1 A2 metabolism of HCE
3.4 Elimination
3.5 Physiologically Based Pharmacokinetic Models
HAZARD IDENTIFICATION
4.1 Studies in Humans — Epidemioloqy, Case Reports, Clinical Controls
4.2 Subchronic and Chronic Studies and Cancer Bioassays in Animals — Oral and Inhalation
4.2.1 Oral
Table 4-1 Body, kidney, and liver weights of rats exposed to HCE in the diet for
16 weeks
Table 4-2 Histopathological results on kidney in rats exposed to HCE in the diet
for 16 weeks
Table 4-3 Organ weight to body weight ratios for rats exposed to HCE for 13
weeks
Table 4-4 Incidence and severity of nephropathy in male and female rats
exposed to HCE
Table 4-5 Additional kidney effects in HCE-exposed rats
Table 4-6 Renal tubular hyperplasia and tumor incidences in HCE-exposed
male rats
Table 4-7 Adrenal medullary lesions in HCE-exposed male rats
Table 4-8 Tumor incidences in male rats qavaqed with HCE
Table 4-9 Tumor incidences in female rats qavaqed with HCE
Table 4-10 Incidence of hepatocellular carcinomas in mice
4.2.2 Inhalation
4.3 Reproductive/Developmental Studies — Oral and Inhalation
4.3.1 Oral
Table 4-1 1 Summary of HCE effects on preqnant Wistar rats and their fetuses
Table 4-12 Summary of skeletal effects on fetuses from HCE-exposed rats
4.3.2 Inhalation
4.4 Other Duration- Or Endpoint-Specific Studies
4.4.1 Acute Exposure Studies
Table 4-13 Summary of acute exposure data in rats, rabbits, and quinea piqs
VI
VII
VIM
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15
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4.4.2 Short-term Exposure Studies 34
Table 4-14 Summary of toxicity data from male rats exposed to HCE for 21 days 37
4.4.3 Neurological 37
4.4.4 Immunological 39
4.4.5 Dermatological 39
4.4.6 Eye Irritation 39
4.5 Mechanistic Data and Other Studies in Support of the Mode of Action 40
4.5.1 Genotoxicity 40
Table 4-15 Summary of genotoxicity studies of HCE 41
Table 4-16 Number of enzyme-altered foci in rat liver: Promotion protocol 45
4.5.2 In Vitro and Ex Vivo Studies Using Isolated Target Tissues/Organs or Cells 45
Table 4-17 In vivo covalent binding of [14C]-HCE to DNA, RNA, and proteins from
rat and mouse organs 46
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 47
4.5.3 Structure Activity Relationships 47
4.6 Synthesis of Major Noncancer Effects 49
4.6.1 Oral 49
Table 4-19 Oral toxicity studies for HCE 50
4.6.2 Inhalation 54
Table 4-20 Summary of data from the Weeks et al. (1979) inhalation toxicity
study with HCE 55
4.6.3 Mode-of-Action Information 55
4.7 Evaluation of Carcinogenicity 57
4.7.1 Summary of Overall Weight of Evidence 57
4.7.2 Synthesis of Human, Animal, and Other Supporting Evidence 58
4.7.3 Mode-of-Action Information 59
Table 4-21 Nephrotoxic effects characteristic of a2u-globulin nephropathy
observed in male and female rats administered HCE 62
4.8 Susceptible Populations and Life Stages 71
4.8.1 Possible Childhood Susceptibility 71
4.8.2 Possible Gender Differences 72
4.8.3 Other 72
DOSE-RESPONSE ASSESSMENTS 73
5.
1 Oral Reference Dose (RfD)
5.1.1 Choice of Principal Study and Critical Effect — with Rationale and Justification
Table 5-1 Incidences of noncancerous kidney and liver effects in rats following
oral exposure to HCE
5.1.2 Methods of Analysis — Including Models
Table 5-2 Summary of the BMD modelinq results for the rat kidney
5.1.3 RfD Derivation — Including Application of Uncertainty Factors (UFs)
5.1.4 RfD Comparison Information
73
73
74
76
77
78
79
Table 5-3 Potential PODs for nephrotoxicity in male rats with applied UF values
and potential reference values 80
Figure 5-1 Array of potential PODs with applied UF values and potential
reference values for nephrotoxic effects in male rats, from the three
studies in Table 5-3. 81
5.1.5 Previous RfD Assessment 81
5.1.6 Confidence in the RfD 82
5.2 Inhalation Reference Concentration (RfC) 82
5.2.1 Choice of Principal Study and Critical Effect—with Rationale and Justification 82
Table 5-4 Noncancerous effects observed in animals exposed to HCE via
inhalation 83
5.2.2 Methods of Analysis—Including Models 84
5.2.3 RfC Derivation—Including Application of Uncertainty Factors (UFs) 85
5.2.4 RfC Comparison Information 86
5.2.5 Previous RfC Assessment 86
5.2.6 Confidence in the RfC 87
5.3 Uncertainties in the Oral Reference Dose and Inhalation Reference Concentration 87
5.4 Cancer Assessment 87
5.4.1 Choice of Study/Data—with Rationale and Justification 88
5.4.2 Dose-response Data 88
IV
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Table 5-5 Summary of incidence data in rodents orally exposed to HCE for use
in cancer dose-response assessment 89
5.4.3 Dose Adjustments and Extrapolation Methods 89
Table 5-6 Summary of BMD modeling results for oral cancer assessment of
HCE 91
5.4.4 Oral Slope Factor and Inhalation Unit Risk 91
5.4.5 Uncertainties in Cancer Risk Values 92
Table 5-7 Summary of uncertainties in the HCE cancer risk assessment 93
5.4.6 Previous Cancer Assessment 96
6 MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE
RESPONSE 97
6.1 Human Hazard Potential 97
6.2 Dose Response 98
6.2.1 Oral Noncancer 98
6.2.2 Inhalation Noncancer 99
6.2.3 Cancer 99
7 REFERENCES 101
APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC COMMENTS
AND DISPOSITION A-1
APPENDIX B: BENCHMARK DOSE MODELING OUTPUT B-1
Table B-1 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-1
Table B-2 Dose-response modeling results using BMDS (version 2.0) for BMRs
of 10, 5, and 1% based on noncancerous kidney and liver effects in
rats following oral exposure to HCE B-4
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ABBREVIATIONS AND ACRONYMS
a2u-g a2u-globulin
ACGIH American Conference of Governmental
Industrial Hygienists
AIC Akaike's information criterion
ALD approximate lethal dosage
ALT alanine aminotransferase
AST aspartate aminotransferase
atm atmosphere
ATSDR Agency for Toxic Substances and
Disease Registry
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 (cell line cells)
CL confidence limit
CNS central nervous system
CRN chronic progressive nephropathy
CYP450 cytochrome P450
DAF dosimetric adjustment factor
DEN diethylnitrosamine
DMSO dimethylsulfoxide
DNA deoxyribonucleic acid
EPA United States Environmental Protection
Agency
FDA Food and Drug Administration
FEV1 forced expiratory volume of 1 second
GD gestation day
GDH glutamate dehydrogenase
GGT Y-g|utamyl 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
LC50 median lethal concentration
LD50 median lethal dose
LOAEL lowest-observed-adverse-effect level
MN micronuclei
MNPCE micronucleated polychromatic
erythrocyte
MTD maximum tolerated dose
NAG N-acetyl-p-D-glucosaminidase
NCEA National Center for Environmental
Assessment
NCI National Cancer Institute
NOAEL no-observed-adverse-effect level
NTP National Toxicology Program
NZW New Zealand White (rabbit breed)
OCT ornithine carbamoyl transferase
ORD Office of Research and Development
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
UFA interspecies uncertainty factor
UFH intraspecies uncertainty factor
UFS subchronic-to-chronic uncertainty factor
UFD database deficiencies uncertainty factor
U.S. United States of America
VI
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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 (HCE).
It is not intended to be a comprehensive treatise on the chemical or toxicological nature of HCE.
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(g),epa.gov (email address).
VII
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AUTHORS I CONTRIBUTORS I REVIEWERS
Chemical Manager
John Cowden, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC
Authors
John Cowden, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, NC
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.
Formerly of the National Center for Environmental Assessment
Office of Research and Development
Technical Support
Ellen Lorang, M.S.
National Center for Environmental Assessment
Office of Research and Development; RTP, NC
James S. Lucy, B.A.
Student Services Authority
National Center for Environmental Assessment
Office of Research and Development; RTP, NC
Deborah Wales
National Center for Environmental Assessment
Office of Research and Development; RTP, NC
Vlll
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Contractor Support
Sheri Hester, M.S.
Oak Ridge Institute for Science and Education (ORISE)
Oak Ridge, TN
George Holdsworth, Ph.D.
Oak Ridge Institute for Science and Education (ORISE)
Oak Ridge, TN
James Kim, 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
James Riddle, Ph.D.
Sciences International, Inc.
Alexandria, VA
Jay Turim, Ph.D.
Sciences International, Inc.
Alexandria, VA
Lutz Weber, DABT
Oak Ridge Institute for Science and Education (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.
Internal EPA Reviewers
Ambuja Bale, 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
Lynn Flowers, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
IX
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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
Connie Meacham, M.S.
National Center for Environmental Assessment
Office of Research and Development
ReederSams II, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
D. Charles Thompson, Ph.D.
Formerly of the National Center for Environmental Assessment
Office of Research and Development
John Vandenberg, Ph.D.
National Center for Environmental Assessment
Office of Research and Development
Debra Walsh, M.S.
National Center for Environmental Assessment
Office of Research and Development
John Whalan
National Center for Environmental Assessment
Office of Research and Development
External Peer Reviewers
Jack B. Bishop, Ph.D.
National Institute of Environmental Health Sciences
Research Triangle Park, NC
Lucio G. Costa, Ph.D.
University of Washington
Seattle, WA
Lynne T. Haber, Ph.D., DABT
Toxicology Excellence for Risk Assessment (TERA)
Cincinnati, OH
Ralph L. Kodell, Ph.D.
University of Arkansas for Medical Sciences
Little Rock, AR
Lawrence H. Lash, Ph.D.
Wayne State University
Detroit, Ml
Edward A. Lock, Ph.D.
Liverpool John Moores University
Liverpool, UK
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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
Chemical Mixtures (U.S. EPA. 1986a). Guidelines for Mutagenicity Risk Assessment (U.S. EPA. 1986b).
Recommendations for and Documentation of Biological Values for Use in Risk Assessment (U.S. EPA.
1988). Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA. 1991c). Interim Policy for
Particle Size and Limit Concentration Issues in Inhalation Toxicity (U.S. EPA, \994b). Methods for
Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA.
1994a). 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. 2000c). Supplementary Guidance
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for Conducting Health Risk Assessment of Chemical Mixtures (U.S. EPA. 2000b). A Review of the
Reference Dose and Reference Concentration Processes (U.S. EPA. 2002). Guidelines for Carcinogen
Risk Assessment (U.S. EPA. 2005b), Supplemental Guidance for Assessing Susceptibility from Early-Life
Exposure to Carcinogens (U.S. EPA. 2005a). 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 HCE was based on the chemical name, Chemical
Abstracts Service Registry Number (CASRN), and multiple common synonyms. Any pertinent scientific
information submitted by the public to the IRIS Submission Desk was also considered in the development
of this document. Primary, peer-reviewed-literature was reviewed through May 2011, and was included
where that literature was determined to be critical to the assessment. The relevant literature included
publications on HCE which were identified through Toxicology Literature Online (TOXLINE), PubMed,
the Toxic Substance Control Act Test Submission Database (TSCATS), the Registry of Toxic Effects of
Chemical Substances (RTECS), the Chemical Carcinogenesis Research Information System (CCRIS), the
Developmental and Reproductive Toxicology/Environmental Teratology Information Center
(DART/ETIC), the Environmental Mutagens Information Center (EMIC) and Environmental Mutagen
Information Center Backfile (EMICBACK) databases, the Hazardous Substances Data Bank (HSDB), the
Genetic Toxicology Data Bank (GENE-TOX), Chemical abstracts, and Current Contents. Other peer-
reviewed information, including health assessments developed by other organizations, review articles, and
independent analyses of the health effects data were retrieved and may be included in the assessment
where appropriate. No new publications were identified since the release of the external peer review draft
Toxicological Review.
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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). 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, 1997c). HCE is primarily used
by the military for smoke pots, smoke grenades, and pyrotechnic devices (ACGIH. 2001; ATSDR. 1997c;
U.S. EPA. 1991b: IARC. 1979). 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. 1997c: U.S. EPA. 1991a). HCE was also identified in the headspace of
chlorine-bleach-containing household products (Odabasi. 2008). Certain physical and chemical properties
are shown below in Table 2-1 (ACGIH. 2001: ATSDR. 1997c: Vogel and Nivard. 1993: Budavari et al..
1989: 1989: Weastetal.. 1986: Spanggord et al.. 1985: Verschueren. 1983: Mabev et al.. 1982: Callahan
etal.. 1979).
Cl Ci
Cl C — C Cl
Cl Cl
Figure 2-1 Structure of HCE
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Table 2-1 Physical properties of HCE
Property Hexachloroethane
CASRN 67-72-1
„ a 1,1,1,2,2,2-hexachloroethane, ethane hexachloride, ethylene hexachloride,
ynonyms perchloroethane, carbon hexachloride, carbon trichloride
Molecular weight 236.74 g/mol
Molecular formula C2CI6
Melting point Sublimes without melting
Boiling point 186.8°C
Specific Gravity 2.091 at20°C
Water solubility3 50 mg/L. at 22°C; 14 mg/L at 25°C
Log KQW 3.82°, 3.34°, 4.14"
Log KQC 4.3
Vapor pressure 0.5 mmHg at 20°C; 1.0 mmHg at 32.7°C
Henry's law constant 2.8 x 1Q-3 (atm-mj)/mol at 20°C
Conversion factor 1 ppm = 9.68 mg/m ; 1 mg/m =0.10 ppm
Sources: a ChemlDplus Advanced Database (http://chem.sis.nlm.nih.gov/chemidplus/) and ACGIH (1991): "Howard (1989):
°Callahan et al. (1979): dHansch et al. (1995).
HCE is produced by the chlorination of tetrachloroethylene (PERC) in the presence of ferric
chloride (ATSDR. 1997c: U.S. EPA. 1991b: Fishbein. 1979; IARC. 1979). HCE was produced in the
United States (U.S.) for commercial distribution from 1921 to 1967, but is currently not commercially
distributed (ATSDR. 1997c: IARC. 1979). In the 1970s, U.S. producers of HCE reported that HCE was
not distributed, but was only used in-house or recycled (ATSDR. 1997c): U.S. distributors in the 1970s
imported HCE from France, Spain, and the United Kingdom (ACGIH. 2001; ATSDR. 1997c). U.S.
production plus imports of HCE totaled 10 million-50 million pounds in 1986, 1 million-10 million
pounds in 1990, 10 million-50 million pounds in 1994, 500,000-1 million pounds in 1998, 10,000-
500,000 pounds in 2002, and 1-10 million pounds in 2006 (NTP. 2011).
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3 TOXICOKINETICS
3.1 Absorption
No studies have evaluated HCE absorption in humans by oral or inhalation exposure. HCE was
identified in follicular fluid of women undergoing in vitro fertilization (IVF) during an analysis for
environmental contaminants (Younglai et al.. 2002). These data indicate the potential for HCE uptake, but
not the source or route of exposure. The dermal absorption rate of HCE has been described as limited
(ATSDR. 1997c): the absorption of a saturated HCE solution across human skin was estimated to be
0.023 mg/cm2-hour (Fiserova-Bergerova et al., 1990).
Oral exposure studies in animals have 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 in
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). Animal
studies (Gorzinski et al.. 1985; Nolan and Karbowski. 1978; Fowler. 1969) have consistently
demonstrated that HCE is distributed to fat ,kidney, liver, and 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 couples undergoing IVF. HCE was identified in >50% of
follicular fluid samples, suggesting post-absorptive 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 serum obtained from females during oocyte retrieval for IVF. The study authors did not make any
conclusions with regards to the level of HCE in follicular fluid and its effect on human fertility.
Fowler (1969) evaluated the tissue distribution of HCE, PERC, and pentachloroethane in sheep.
Brain, fat, kidney, liver, muscle, blood, and bile were evaluated for HCE (see Table 3-1). To assess bile
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concentrations of HCE, two of the HCE exposed Scottish Blackface sheep (Sheep 1 and Sheep 2) were
fasted for 24 hours and anaesthetized with sodium pentobarbital (Table 3-1). The hepatic duct was
cannulated to collect bile; HCE was injected at a dose of 500 mg/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.
Anesthesia was maintained for 8.5 hours, after which time the sheep were sacrificed and tissues were
taken within 10 minutes of death. HCE was widely distributed and the highest levels were found in fat of
Sheep 1. Fat from different sites did not show significant variation in HCE concentration. Sheep 2 had
only trace amounts of HCE in tissue. HCE was detected in bile of anaesthetized sheep at 15 minutes,
compared with detection at 27 minutes for blood; at maximum, HCE was 8-10-fold greater in bile.
Table 3-1 HCE, PERC, and pentachloroethane tissue concentrations in anesthetized sheep
8.5 hours after injection of 500 mg/kg HCE
Concentration (ug/g)
Tissue
Bile (4 hr)
Blood (6 hr)
Brain
Fat
Kidney
Liver
Muscle
Source: Fowler
HCE
1.7
0.2
0.2
1.1
0.1
0.2
0.04
(1969).
PERC
0.3
0.4
0.9
2.1
1.2
0.9
0.5
Sheep 1
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
Nolan and Karbowski (1978) studied tissue clearance of HCE in rats. Male F344 rats were placed
on an HCE-containing diet that delivered 100 mg/kg-day [later determined to be 62 mg/kg-day by
Gorzinski et al. (1985)] for 57 days. After exposure, 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 starting the HCE-free diet.
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 in a first-order manner with a half-life of 2.7 days.
Concentrations in blood and kidney also decreased in a first-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).
-------
Table 3-2 Time course of HCE concentrations in male rat tissues after 57 days of dietary
exposure to 62 mg/kg-day
HCE tissue concentrations (n = 3 or 4)
(mean + SD ug/g tissue)
Days after cessation of
HCE exposure
0
3
6
13
22
31
Blood
0.834
0.279
+ 0
+ 0
0.0835 +
0.015
0.002
NDC
+ 0
+ 0
.223
.048
0.006a
.005
.001
Liver
0.143
0.399
0.303
0.039
0.001
NDC
+ 0.
+ 0.
+ 0.
+ 0.
+ 0.
040
188
,156a
023
,001
Kidney
81.8 + 5
41.0 + 1
18.5°
2.53 + 1
0.194 +
0.026 +
.3
.4
.02
0.171
0.006
Fat
303 + 50
107.8 +
62.45 +
6.56 + 0
0.472 +
0.125 +
10.5
3.04a
.52
0.232
0.020
aValues 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: less than detection limit of 0.001 |jg/g
Sources: Gorzinski et al. (1985): Nolan and Karbowski (1978).
Nolan and Karbowski (1978) also evaluated tissue concentrations of HCE in male and female rats
after dietary exposure 3, 30, and 100 mg/kg-day of HCE for 110-111 days (16 weeks). The 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 higher in male rats compared with female
rats, particularly at the highest dose [47-fold greater in males (Nolan and Karbowski. 1978)1. Kidney
concentrations of HCE proportionately increased with increasing 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
hypothesized that 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.
-------
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
an=4
Male
Female
Male
Female
Male
Female
for each tissue/sex/dose
HCE tissue concentration3 (mean + SD, ug/g tissue)
Blood
0.079+0.057
0.067+0.039(3)
0.596+0.653
0.162+0.049(3)
0.742+0.111
0.613+0.231
group, except where
Liver
0.291 +0.213
0.260 + 0.035(2)
1.736 + 1.100
0.472 + 0.204
0.713 + 0.343
0.631 +0.262
noted in parentheses.
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
Data from 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. In vivo
metabolism data for HCE are limited to three studies: Mitoma et al. (1985) in rats and mice; Jondorf et al.
(1957) in rabbits; and Fowler (1969) in sheep. Each of these studies suggest limited metabolism for HCE.
A variety of intermediary metabolites have also been identified in exhaled air and urine (Fowler. 1969;
Jondorf et al.. 1957). In vitro studies using liver microsomes indicated that HCE metabolism involves
phenobarbital-inducible cytochrome P450 (CYP450) enzymes (Salmon et al.. 1985; Town and Leibman.
1984; Nastainczyk et al.. 1982a: Nastainczyk et al.. 1982b: Salmon etal.. 1981); however, no specific
enzymes have been identified. The CYP450 enzymes induced by phenobarbital include those from the
2A, 2B, 2C, and 3A subfamilies. One study (Yanagita et al.. 1997) found 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.. 1982a:
Nastainczyk et al.. 1982b; 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.
Figure 3-1 provides a plausible 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 metabolites
identified in in vivo (Mitoma et al.. 1985; Fowler. 1969; Jondorf et al.. 1957) and vitro studies (Town and
Leibman. 1984; Nastainczyk et al.. 1982a) were used in the derivation of Figure 3-1. The HCE
metabolism data was supplemented with data on the metabolism of the PERC (ATSDR. 1997a).
trichloroethylene (ATSDR, 1997b). and 1,1,2,2-tetrachloroethane (ATSDR, 2008) intermediary
metabolites.
The proposed metabolic pathway is based on limited information; therefore, it is likely that
intermediate chemical reactions are not captured in the figure, which presents the formation of the various
metabolites as single-step reactions.
-------
Cl Cl
Cl__c__c__
Cl Cl
Hexachioroethane
r*i
vxl
LCI
Cl
cci3 - cci2-
Carfaene
intermediate
Free radical reactions
Cl Cl
\-c/
/ \
Cl Cl
Tetrach Soroet hy iene
0,4
|
Cl H
1 1
Q Q^ OH
1 1
Cl H
Trichloroethanol
i
1
Cl
-Ls°
/ X
* %
,,
*"N
Cl Cl
H — C — C— H
1 1
w Cl Cl >
A"" Cl ^
1,1.2,2-
Tetrachioroeihane
2Cl4
T
|
1
T
O Cl
Cl Cl
1 1
I 1
ar* P w
W v> 1 1
1 I
Cl Cl
Pentachloroethane
2CI-X
|
Cl Cl
\ /
c=c
/ \
Cl H
Trfchloroethylene
Cl-^
4
HO Cl
- X
\
H
Cl
C|
OH
HO
Trichloroacetic acid
Dichloroacetic acid
O O
X
HO OH
Oxalic acid
ci
CO,
Oichloroetfianol
O Cl
X
HO H
Monochloroacetic acid
One carbon pool
Figure 3-1 Plausible metabolic pathway of HCE
Sources: Adapted from ATSDR (1997a).: Mitoma et al. (1985): Town & Leibman (1984): Nastainczyk et al. (1982a: 1982b): Bonse &
Henschler (1976): Fowler (1969): Jondorf (1957).
Mitoma et al. (1985) examined HCE distribution in male Osborne-Mendel rats and male B6C3F]
mice to evaluate metabolism in the 48 hours after administration of 125 or 500 mg/kg radiolabeled HCE
to the rats and 250 or 1,000 mg/kg radiolabeled HCE to the mice. Doses were the maximum tolerated
dose (MTD) and 1A 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 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. 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
Rat (500 mg/kg-day) Mouse (1,000 mg/kg-day)
Percent of administered dose
Expired air 64.55+6.67 71.51 +5.09
CO2 2.37 + 0.76 1.84 + 0.94
Excreta 6.33 + 2.39 16.21 +3.76
Carcass 20.02+3.70 5.90 + 1.60
Recovery 93.28 +6.23 95.47 +9.59
Total metabolism
(CO2 + excreta + carcass)
Source: Reprinted with permission of Informa Healthcare©; Mitoma et al. (1985).
Recovery of the radiolabel was >90% for both rats and mice. The authors calculated total
metabolism as the sum of the radiolabel present in carbon dioxide, excreta, and the carcass. This
calculation is not an accurate estimate of metabolism because metabolites were not quantified. Data on
the extent of metabolism for the radiolabeled material are presented in Table 3-5. Both rats and mice
metabolized 30% of the parent compound, based on the mass balance between dose and the estimated
sum of metabolites. This finding is consistent with the 60-70% of the unmetabolized radiolabel present in
expired air. However, this conclusion assumed that all of the exhaled radiolabel in expired air 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.
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 metabolized3
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
Jondorfetal. (1957).
Source: Reprinted with permission of Informa Healthcare©; Mitoma et al. (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 5% of the applied radioactivity in urine over 72
10
-------
hours, indicating slow metabolism. This finding is consistent with the Mitoma et al. (1985) study in rats
and mice, in which approximately 2-4% of the label was found in urine after 48 hours. During the 72
hours, 14-24% of the radioactivity was detected in expired air, a lower percentage than seen for rats at a
comparable (Mitoma et al.. 1985). The remainder of the radioactivity was present in tissues and the
intestinal tract, although the authors unable to quantify 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 observed in expired air). Quantitative data on the
volatile metabolites in exhaled air were not reported.
Fowler (1969) orally administered HCE via drenching bottle 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 blood 24 hours after
exposure. Following administration of 500 mg/kg HCE, 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 confirmed
the presence of the metabolites PERC and pentachloroethane in liver slices.
The in vivo data on HCE metabolism are supported by in vitro studies of hepatic metabolism
using liver microsomes. Two studies by Nastainczyk et al. (1982a: 1982b) reported that HCE is
metabolized by phenobarbital-inducible CYP450 enzymes, which catalyze reductive dechlorination using
NADPH, cytochrome b5, 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 of HCE 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 HCE reduction was PERC. Nastainzcyk et al. (1982a) determined that the
stoichiometry of the reaction was represented by the following equation:
NADPH +H + + -3 NADP++ + 2H+
Because CYP450 is a one electron donor, Nastainczyk et al. (1982a: 1982b) proposed that 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 forming pentachloroethane; (2) a-elimination
of chloride to form the carbene, which could be stabilized by the reduced CYP450; or (3) (3-elimination of
chloride to form PERC, which is the major HCE metabolite. Nastainczyk et al. (1982a) found that the
products of HCE reductive dechlorination were 99.5% PERC and 0.5% pentachloroethane at
11
-------
physiological pH values. At a more basic 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 b5, which is influenced by pH.
To provide additional support for HCE reduction being catalyzed by CYP450, Nastainczyk et al.
(1982a: 1982b) inhibited CYP450 using carbon monoxide, metyrapone (CYP450 3A inhibitor), or a-
naphthoflavone (CYP450 1A and CYP450 IB inhibitor) [see Omiecinski (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 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 3A inhibition partially attenuated HCE metabolism, whereas
inhibition of CYP450 1A and CYP450 IB did not attenuate HCE as much as CYP450 3A 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 not
inhibited 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 HCE metabolism to PERC. The formation of PERC was favored in a
low oxygen (O2) environment, with observed metabolism rates of 50.2 ± 0.45, 1.25 ± 0.25, and 0
nmol/minute-mg protein in atmospheres of N2, air, and O2, respectively. When any part of the NADPH-
generating system was omitted from the experiment, the metabolism of HCE to PERC was inhibited (>
91%). In addition, the use of carbon monoxide (a monooxygenase inhibitor) arrested HCE metabolism.
Enzymes responsible for metabolism of HCE to PERC were located in the microsomes (not the cytosol)
of phenobarbital-treated rat livers. Formation of malondialdehyde and conjugated dienes was 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 (NZW) 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 identified PERC as a metabolite of HCE; however, the
metabolite was not quantitatively measured.
Salmon et al. (1981) used liver microsomes from Sprague-Dawley rats to quantify HCE
dechlorination. Dechlorination was measured by the release of radioactive Cl~ from the [36C1]-
radiolabeled HCE substrate during incubation with liver microsomes from Aroclor 1254-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.
12
-------
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 NZW rabbits. Metabolic experiments of 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 NZW 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 baker's yeast
(Saccharomyces cerevisiae) to evaluate the in vitro metabolism of HCE. CYP450 1A2 is not a
phenobarbital-inducible hepatic CYP450 enzyme. 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). A
follow-up study (Yanagita etal., 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
.».,„._„.._ Product formation (nmol/nmol CYP450-minute) _ .. ______ ± .. ±. T~.-
CYP450 1A2 —=7=7-: —-*-r\ — =^=* Ratio of PERC:Pentachloroethane+TCE
PERC Pentachloroethane TCE
Wild type 0.68 0.10 0.0034 6_6
Units expressed as ([nmol product content per nmol CYP450] per minute). Experiments were repeated three times. Experiment
errors were within 15%. Tetrachloroethylene (PERC); Pentachloroethylene; Trichlorethylene (TCE).
Source: Reprinted with permission of Elsevier©; Yanagita et al. (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 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 mechanisms of HCE
dechlorination using rat liver microsomes. HCE demonstrated a considerable amount of dechlorination
(3.9%) in this in vitro study; however, the authors 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)
13
-------
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 3.38 uM.
3.4 Elimination
No available studies evaluated the HCE elimination in humans. Animal studies indicated that the
major routes of HCE elimination are either by fecal matter or by expired air (Mitoma et al.. 1985; Fowler.
1969; Jondorf et al.. 1957). Sheep studies (Fowler. 1969) indicated that orally administered HCE is
eliminated by the fecal route without absorption and metabolism, while rodent studies (Mitoma et al..
1985) provided evidence that HCE is absorbed and eliminated by exhalation. It is unknown why there is a
difference in elimination between sheep and rodents.
Rabbits fed [14C]-radiolabeled HCE at 0.5 g/kg (Jondorf etal.. 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 a single 500 mg/kg dose of HCE to two Cheviot cross in
metabolism cages and collected urine and feces over 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.
Mitoma et al. (1985) evaluated excretion of radiolabeled HCE in Osborne-Mendel rats and
B6C3Fi mice following 4 weeks of administration of an MTD (see Section 3.3). In both rats and mice,
most of the radiolabel was detected in expired air, indicating this to be a major route of elimination. The
authors assumed the radiolabel in expired air was the parent compound and did not investigate whether
the exhaled air contained volatile HCE metabolites. Less than 2.5% he exhaled radioactivity was found in
CO2, with rats exhaling slightly more than mice. On the other hand, the amount of radioactivity in the
excreta was lower in rats than in mice (Table 3-4).
3.5 Physiologically Based Pharmacokinetic Models
No physiologically based pharmacokinetic (PBPK) models for HCE have been developed
specifically for mammalian species.
Gargas and Andersen (1989) and Gargas et al.(1988) determined kinetic constants for HCE
O V / O V /
metabolism in the rat using exhalation rates and a PBPK inhalation model originally developed for
styrene in rats (Ramsey and Andersen. 1984). The Gargas and Andersen (1989) and Gargas et al. (1988)
reports do not describe a PBPK model for HCE, only kinetic constants for HCE metabolism by
inhalation.
14
-------
4 HAZARD IDENTIFICATION
4.1 Studies in Humans—Epidemiology, Case Reports, Clinical
Controls
There are few studies related to HCE toxicology in humans. No epidemiology studies of HCE
carcinogenicity were identified. Case reports of pneumonitis alone (Allen et al.. 1992) and pneumonitis
with evidence of liver abnormalities (Loh et al.. 2008; Loh et al.. 2006) have been described in soldiers
exposed to smoke bombs containing HCE and zinc oxide. However, smoke bomb incineration produces a
mixture of chemicals consisting primarily of zinc oxychloride and zinc chloride, and the reported health
effects are consistent with zinc chloride exposure (NRC. 1997). Some aluminum production processes
involve HCE, resulting in exposures to fumes containing hexachlorobenzene, octachlorostyrene, dioxins,
dibenzofurans, and other organochlorinated compounds. A study of aluminum foundry workers provided
a hepatocellular carcinoma case report (Selden et al.. 1989) and data concerning some clinical serologic
measures (Selden et al.. 1997; Selden etal.. 1989). but these data are of limited use to inform health
effects of HCE exposure in other settings. Studies of Swedish workers involved in smoke bomb
production provided some information on exposure levels, as well as symptoms and clinical parameters
relating primarily to liver and pulmonary function (Selden et al.. 1994; Selden etal.. 1993).
Two studies were conducted on Swedish workers occupationally exposed to HCE while
producing military white smoke munitions. The smoke formulation was approximately 60% HCE, 30%
titanium dioxide, 8% aluminum powder, 2% cryolite, and a trace of zinc stearate. 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 area),
5-25 mg/m3 (pressing area), <5 mg/m3 (assembly room), and nondetectable (storage room) (Selden et al..
1993).The first study reported biological exposure monitoring (Selden et al.. 1993) and the second study
described health effects resulting from HCE exposure (Selden etal.. 1994).
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. The exposed male subject without an age-matched control 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, blood
samples were drawn at a time period 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
15
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(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. During
the production period, plasma HCE levels increased nearly 100-fold over the baseline-period plasma HCE
levels (mean of 7.30 ± 6.04 ug/L in the production samples compared with 0.08 ± 0.14 ug/L in the
baseline samples, p < 0.01). Although the magnitude of individual increases varied, 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 indicate that plasma HCE can increase after
occupational exposure, even though workers used 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 controls reported more symptoms 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 controls (16/130, 12%). The prevalence of "itching/other skin problems" in the
matched controls (3/11, 27%) was the same as in the exposed group. Skin symptoms centered on the wrist
and neck areas which the authors suggested could reflect HCE exposure through joints in the protective
equipment or a "traumiterative effect of the equipment itself." Clinical examination revealed no
dermatological or respiratory mucous membrane abnormalities in either group.
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 an elevated level of serum alanine aminotransferase
(ALT) (70.5 U/L versus < 41.1 U/L reference), while one control subject had increased levels of serum
ALT and aspartate aminotransferase (AST) (67.6 and 186.4 U/L, respectively; 41.1 U/L reference for
16
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each). The control individual's values returned to normal after 8 months, while the exposed subject's
serum ALT value increased to 87.6 U/L 4 months later (Selden et al.. 1994). Available data pertaining to
liver function tests in this individual from 1982, when exposure levels at the worksite were higher than in
the current study, did not show elevations in these liver enzymes at that time. Within the exposed group,
there was no correlation between plasma HCE concentrations and clinical chemistry parameters, although
the authors did 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 from the health effects study was too small to reach definitive conclusions. The possible
dermatologic/mucosal effects and hepatic effects 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;
Gorzinski et al., 1980). The Gorzinski et al. (1985; 1980) study (16 weeks) reported kidney degeneration
in male and female rats, as well as hepatic effects. The NTP (1989) study (13 weeks) reported
degeneration and necrosis of renal tubular epithelium, hyaline droplet formation, and tubular regeneration
and tubular casts in male rats. Female rats exhibited a dose-response increase in the incidence of
hepatocellular necrosis of the centrilobular area. The NTP (1989) 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 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.
The data from Gorzinski et al., (1980) were published in the peer-reviewed literature by Gorzinski et al.
(1985).
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 and gross pathological alterations (Table 4-1). Male rats of the 62 mg/kg-day group exhibited
statistically significant increases in absolute and relative liver weights (Table 4-1); histopathology
revealed a slight swelling of the hepatocytes in the 15 and 62 mg/kg-day dose groups. Female rats
17
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exhibited a statistically significant increase in relative liver weight at the high dose (Table 4-1), although
there was no evidence of hepatotoxicity in the histopathological examination.
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 (Table 4-2). 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 (Table 4-2). 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,
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). The increased incidence of atrophy and degeneration of proximal
tubules was statistically significant in females at the 62 mg/kg-day dose (Table 4-2).
18
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Table 4-1 Body, kidney, and liver weights of rats exposed to HCE in the diet for 16 weeks
Sex
i\/ioioa
a
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
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
Liver
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
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
Kidney
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
aData are presented as means ± SD in each treatment group (10 rats/sex/dose).
bStatistically significant from control using Dunnett's test (p = 0.05).
Source: Gorzinski et al. (1985).
Table 4-2 Histopathological results on kidney in rats exposed to HCE in the diet for 16 weeks
Organ
Kidney
Effect
Slight hypertrophy and/or dilation of
proximal convoluted tubules
Atrophy and degeneration
of renal tubulesc'd
Sex
Male
Female
Male
Female
0
0
0
1
1
Dose (mg/kg-day)a
1 15 62
1 7b 10b
00 0
2 T 10°
1 2 6b
aData 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.
dWith a degree of peritubular fibrosis in high dose males.
Source: Gorzinski et al. (1985).
The authors concluded the no-observed-effect level for both male and female rats was 1 mg/kg-
day. For male rats, EPA considered 1 mg/kg-day as no-observed-adverse-effect level (NOAEL) and 15
mg/kg-day as the lowest-observed-adverse-effect level (LOAEL), based on renal tubule toxicity. 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 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. At the highest dose, 5/10 male rats and 2/10 female rats died before the end of
the study. Mean body weight in the 536 mg/kg-day exposure group decreased 19% in male rats and 4% in
female rats when 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).
19
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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
Number0
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(9)
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(8)
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(3)
Female3
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(9)
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 + 0.17 (10)b
3.4 + 0.07b
4.1 +0.10b
4.5 + 0.13
0.8 + 0.05b
aData 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
dNumber animals, except where (noted).
Source: NTP (1989).
Kidney effects (characterized by hyaline droplet formation, tubular regeneration, and tubular
casts) were observed in 90% of 34 mg/kg-day males and in males from all other HCE dose groups. The
authors reported incidence data only for the 34 mg/kg-day dose group. NTP (1989) reported that the
severity of these effects increased with dose (data not presented by NTP). These kidney effects were not
observed in exposed females. Kidneys from the 5 male rats that died following exposure to 536 mg/kg-
day HCE underwent histopathological examination, which 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.
4212 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
20
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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). In the NCI (1978) study, Osborne-Mendel rats of both sexes exhibited tumor types
previously identified as spontaneous lesions in this strain, while B6C3Fi mice of both sexes exhibited
hepatocellular carcinomas, although only male mice demonstrated a dose response with tumor incidence
(NCI. 1978). NTP and NCI concluded there was evidence of HCE carcinogenicity in male F344/N rats
and mice of both sexes, respectively, but there was no evidence of carcinogenicity in female F344/N rats
or Osborne-Mendel rats (NTP. 1989: NCI. 1978).
NTP (1989) conducted a chronic toxicity/carcinogenicity bioassay in F344/N rats (Table 4-4).
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 based on the NTP (1989) subchronic study
(Section 4.2.1.1) 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, tubular dilatation and atrophy,
glomerulosclerosis, interstitial fibrosis, and chronic inflammation, was observed in both exposed 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 significantly higher than controls. Although mean severity
scores did not show more than a 15% increase over control in the high-dose group, examination of the
severity of nephropathy revealed more moderate and marked nephropathy in exposed male rats compared
with predominantly mild nephropathy in controls (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
significantly increased in both exposure 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 severity of
nephropathy showed more mild and moderate nephropathy in exposed females compared with
predominantly less than minimally severe nephropathy in controls. Females did not exhibit marked
nephropathy in the control or exposed groups (Table 4-4).
21
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Table 4-4 Incidence and severity of nephropathy in male and female rats exposed to HCE
Dose (mg/kg-day)
Severity
None (0)
Minimal (1)
Mild (2)
Moderate (3)
Marked (4)
Total incidence (minimal to marked)
Total number of rats
Overall severity0
0
2
4
26
11
7
48
50
2.34 + 0.14
7
Male
2
3
21
10
14
48
50
2.62 + 0.15
14
3
4
13
16
14
47
50
2.68 + 0.163
0
28
10
10
2
0
22
50
0.72 + 0.13
57
Female
8
17
23
2
0
42°
50
1.38 + 0.11b
114
5
12
25
7
0
44°
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.
cMean ± SE.
Source: NTP (1989).
To identify HCE-related kidney nephropathy, EPA compared the incidences of more severe
(moderate and marked severity) nephropathy between the control and exposed male rats. Incidences of
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. Similar to the male rats, the incidences of more severe (mild and moderate)
nephropathy were considered in female rats. Incidences of mild or moderate nephropathy in females 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 (Table 4-5). Linear mineralization of the renal
papillae 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 increased in exposed male rats (14% in both 7 and 14 mg/kg-day HCE dose groups) compared
to 0% of controls. Nonneoplastic lesions such as casts (4%), cytomegaly (4%), chronic inflammation
(4%), and focal necrosis (2%) were observed in 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 noted 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 exposed 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.
22
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Table 4-5 Additional kidney effects in HCE-exposed rats
HCE Dose (mg/kg-day)
Renal tubule
pigmentation
Linear mineralization of
renal papillae
Hyperplasia of the pelvic
transitional epithelium
Vehicle
control
1/50
(2%)
2/50
(4%)
0/50
(0%)
Males
7
4/50
(8%)
15/50
(30%)a
7/50
(14%)a
14
5/50
(10%)
32/50
(64%)a
7/50
(14%)a
Vehicle
control
2/50
(4%)
14/50
(28%)
Not
observed
Females
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 7 mg/kg-day as the male LOAEL, 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) of nephropathy. Male and female NOAELs could not be established
because renal effects were observed at the lowest doses tested.
Incidence of renal tubular hyperplasia increased in exposed male rats: 4/50 (8%) in the 7 mg/kg-
day dose and 11/50 (22%; 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%, 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-exposed 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
11/50(22%)a
4/50 (8%)
3/50 (6%)
7/50(14%)a
aSignificantly different from vehicle controls, p < 0.01.
Source: NTP (1989).
This study demonstrates specificity for HCE-induced renal effects in male rats. Although males
were exposed to less HCE than the corresponding females, exposed male rats demonstrated more severe
nephropathy than the exposed 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
23
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female rats, also exhibited renal hyperplasia and tumors. NTP (1989) indicated that the renal hyperplasia
and tumors observed in the HCE-exposed male rats represented a morphologic continuum.
Effects in the adrenal gland were also noted in HCE-exposed 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 (i.e.,
pheochromocytomas containing nervous tissue in addition to adrenal medullary cells) between controls
and exposed male rats. The combined incidence of all three types of pheochromocytomas was
significantly increased in males from the 7 mg/kg-day dose group (62%), but not in males from the
14 mg/kg-day dose group (43%), when compared with vehicle controls (30%), historical controls in the
study laboratory (75/300; 25 ± 7%), and historical controls in NTP studies (543/1,937; 28 ± 11%). NTP
concluded that the increased incidences of pheochromocytomas in male rats were possibly treatment-
related.
Table 4-7 Adrenal medullary lesions
Focal hyperplasia
Pheochromocytoma
Complex Pheochromocytoma
Malignant pheochromocytoma
Combined pheochromocytoma
in HCE-exposed
Control
6/50(12%)
14/50(28%)
0/50
1/50 (2%)
15/50(30%)
male rats
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).
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
un exposed and vehicle controls. Starting in week 23, exposed rats began a 5-week cyclic rotation that
involved 1 week without exposure followed by dosing for 4 weeks. After adjustment to continuous
exposure, the TWA doses were 113 and 227 mg/kg-day.
Mortality was accelerated in the HCE-exposed rats and the authors 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
24
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unexposed controls and 11/20 (55%) in vehicle controls (seven rats in the vehicle control group were
sacrificed in week 60). Mortality in the exposed groups occurred early in the bioassay. Approximately
20% of the high- and low-dose males died by week 15 and week 45, respectively, compared with 90
weeks to reach 20% mortality for the controls. Survival rates for the female rats were 14/20 (70%) for
both the unexposed 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 week 25 and week 30, respectively,
compared with 110 weeks to reach 20% mortality for the controls.
Chronic inflammatory kidney lesions were observed in both control and exposed rats: male rats
exhibited incidences of 15/20 (75%) in unexposed 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 unexposed 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 unexposed 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 established because renal
effects were observed at the lowest dose tested.
Tumor types exhibited by male rats 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
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.
25
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Table 4-8 Tumor incidences in male
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
rats gavaged with HCE
Vehicle control3
0/18(0%)
-
2/18(11%)
105
2/18(11%)
111
0/18(0%)
-
113 mg/kg-daya
4/37(11%)
86
4/32(13%)
104
3/36 (8%)
92
0/36 (0%)
-
227 mg/kg-daya
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 : 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). Kidney hamartomas were observed in 6% of
the females administered 227 mg/kg-day HCE, while no kidney hamartomas 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-exposed animals
than in controls. In the low-dose group, ovary granulosa cell tumors were increased compared to controls,
although none of the females in the high-dose group exhibited ovary granulose tumors. NCI (1978) noted
that all these tumor types had been encountered previously as spontaneous lesions in the Osborne-Mendel
rat, and the authors observed no statistical differences in frequencies between exposed and control rats.
NCI concluded that there was no evidence of carcinogenicity in this rat study.
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: Osborne-Mendel.
Source: NCI (1978).
NCI (1978; Weisburger. 1977) also conducted a chronic study in 50 B6C3FJ 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 termination of exposure, animals were observed for 12-13 weeks for atotal duration of
26
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90-91 weeks. Twenty mice/sex were included as unexposed 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 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 un exposed 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 unexposed 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 unexposed 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 exposed male mice: 67, 80, 66,
and 18% of unexposed 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 unexposed control
and 360 mg/kg-day dose groups, exhibited chronic kidney inflammation. Tubular nephropathy
(characterized by degeneration of convoluted tubule 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 unexposed or pooled vehicle controls of either sex, but was
observed in mice exposed 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. A NOAEL was not established because renal effects were observed at the lowest
dose tested.
The incidence of hepatocellular carcinomas increased 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 incidence of hepatocellular carcinomas, 40 and 31% of 360
and 722 mg/kg-day females compared with 3 and 10% of pooled vehicle and matched vehicle controls,
respectively. The increase in hepatocellular carcinoma in HCE-exposed females was not dose-dependent,
with a higher incidence of observed at the low dose (20/50) compared with the high dose (15/49). NCI
concluded that HCE was carcinogenic in both sexes of B6C3F] mice (NCI. 1978).
27
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Table 4-10 Incidence of hepatocellular carcinomas in mice
Males
Females
Pooled vehicle control3
6/60(10%)
2/60 (3%)
Matched vehicle control
3/20(15%)
2/20(10%)
360 mg/kg-day
15/50(30%)"
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 unexposed control groups, NCI used the pooled vehicle control
data derived from concurrently run bioassays for several other chemicals. Animals were all of the same strain (B6C3Fi) and
housed in the same room. Incidences reported were not adjusted for survival.
"Statistically significant, p = 0.008.
Statistically significant, p < 0.001.
Source: NCI (1978).
4.2.2 Inhalation
4221 Subchronic Exposure
Only one study in the peer-reviewed literature 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 included
neurotoxicity, reduced body weight gain, increased organ weights, and evidence of respiratory tract
irritation.
Weeks et al. (1979) exposed male, female, and pregnant female Sprague-Dawley rats (21-
25/sex/concentration) to control air, 145, 465, and 2,517 mg/m3 HCE (purity 99.8%) for 6 hours/day, 5
days/week for 6 weeks. Postexposure observation was carried out for 12 weeks. An O2 consumption test
was also conducted in male rats. The authors reported that male rats in the 2,517 mg/m3 group had
reduced body weight gain of male rats beginning in the third week of exposure, although quantitative
information was not reported. The authors reported that nonpregnant female rats, in the 2,571 mg/m3 did
not reduced have body weight gain. 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. Although quantitative information
was not reported, the authors reported that the high dose male rats had significantly increased relative
kidney, spleen, and testes weights; in the high dose female rats, only relative liver weights were
significantly increased. One male and one female rat exposed to 2,517 mg/m3 HCE died during the fourth
week, but the authors did not report a cause of death. During the 12 week postexposure observation
period, exposure related effects disappeared. No gross changes were evident at necropsy after the
postexposure observation period; however, male and nonpregnant female rats from 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
28
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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 O2 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 examined male Sprague-Dawley rats (15/concentration) to 145, 465, or
2,517 mg/m3 HCE for 6 hours/day, 5 days/week for 6 weeks and examined them for behavioral changes
(see Section 4.4.3.2). 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 compared with controls,
although quantitative information not reported.
Weeks et al. (1979) also exposed four male Beagle dogs/concentration to control air, 145, 465,
and 2,517 mg/m3 HCE (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
and 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, 145,
465, and 2,517 mg/m3 HCE (purity 99.8%) for 6 hours/day, 5 days/week for 6 weeks. Postexposure
observation was carried out for 12 weeks. Guinea pigs were evaluated for sensitization potential
following inhalation exposure to HCE. Two guinea pigs died during the fourth week and two guinea pigs
died in the fifth week. 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, although
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, 10/sex/concentration) to
control air, 145, 465, and 2,517 mg/m3 HCE (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 after 6 weeks was
excess mucus in nasal turbinates in 2/10 quail in the 2,517 mg/m3 group. The authors considered the
29
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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 as the NOAEL, while the LOAEL
could not be established from this study because no effects were observed at highest dose tested.
4222 Chronic Exposure and Carcinogenicity
No inhalation chronic exposure studies were identified.
4.3 Reproductive/Developmental Studies—Oral and Inhalation
4i3/i 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, significantly reduced body weight gain beginning on GDS, and an increased
incidence of mucopurulent nasal exudates. Approximately 70% of the orally exposed 500 mg/kg group
had upper respiratory tract irritation; 20% had subclinical pneumonitis, compared with 10% in controls.
None of the fetuses exhibited significant skeletal or soft tissue anomalies. Fetuses from the
500mg/kg HCE exposure group 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 was unclear if the authors used a litter-based design for statistical analyses of fetal
gestational indices, and the authors did not 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 HCE teratogenicity by administering of 0, 56, 167, or 500 mg/kg
HCE (purity not specified) to pregnant Wistar rats (20-21 rats/dose) via gavage during GDs 7-17. Dams
in 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 exposure ended.
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 exposure ended. Dams in
both the 167 and 500 mg/kg dose groups exhibited decreased motor activity, although incidence and
method of analysis was not reported. Dams in the 500 mg/kg dose group also exhibited piloerection and
30
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subcutaneous hemorrhage. These effects decreased or disappeared when HCE exposure ended. An
autopsy performed GD20, 3 days post-HCE exposure, revealed three dams 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 exposure 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 increase during the late stage of
pregnancy in the 500 mg/kg dose group (Table 4-11). Male and female fetuses in the 500 mg/kg dose
group also displayed significantly decreased body weight compared to controls (Table 4-11). The authors
stated 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
Dose (mg/kg)
Number of dams
% of dead or resorbed fetuses
Early stage
Late stage
0
20
8.7
8.7
0
56
20
9.2
8.8
0.4
167
20
7.0
6.1
0.9
500
21
14.7
13.1
6.4a
Body weight of live fetuses (g)D
Male
Female
3.3 + 0.20
3.1 +0.24
3.3 + 0.17
3.0 + 0.20
3.2 + 0.21
2.9 + 0.17
2.5 + 0.573
2.3 + 0.453
"Significantly different from control, p < 0.01.
bValues are mean ± SD.
Source: Shimizu et al. (1992).
The investigators (Shimizu et al.. 1992) examined the fetuses for external anomalies. One fetus in
the 500 mg/kg dose group had no tail. Other external 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 group compared with controls (Table 4-12). Skeletal
variations were increased in the 500 mg/kg group and the 167 mg/kg group, with the increase in the 500
mg/kg group statistically significant compared with controls (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 stated that
the litter was used as the statistical unit for calculations of the fetal values. Shimizu et al. (1992)
established aNOAEL of 56 mg/kg for dams and 167 mg/kg for fetuses. EPA considered 167 mg/kg-day
the LOAEL for dams, based on decreased motor activity and significantly decreased body weight. EPA
considered 500 mg/kg LOAEL for fetuses, based on significantly increased skeletal variations,
significantly decreased ossification, and significantly decreased fetal body weight.
31
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Table 4-12 Summary of skeletal effects on fetuses from HCE-exposed rats
Dose (mg/kg)
Number of fetuses examined
Percent of fetal variations
0
136
1.3
56
136
0
167
136
3.8
500
127
60.3a
Number of fetuses with variations
Lumbar rib
Rudimentary lumbar rib
0
2
0
0
0
6
2
78
Ossification"
Number of sternebrae
4.7 + 0.07
4.5 + 0.08
4.5 + 0.08
3.4 + 0.273
Number of proximal and middle phalanges
Fore limb
Hind limb
Number of sacral
and caudal vertebrae
3.2 + 0.05
4.0 + 0.01
6.9 + 0.06
3.1 +0.04
4.0 + 0.01
6.9 + 0.08
3.1 +0.04
4.0 + 0.01
7.0 + 0.04
2.9 + 0.113
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 et al. (1992).
4.3.2 Inhalation
Weeks et al. (1979) exposed 22 pregnant Sprague-Dawley rats/concentration to control air, 145,
465, and 2,517 mg/m3 HCE (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 GDS for the 2,517 mg/m3 group, and beginning on GD14 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-exposed dams did not exhibit any significant skeletal or soft tissue anomalies. It
was unclear from the publication if a litter-based design was used for the statistical analysis of the fetal
gestational indices, and incidence data for the fetal gestational indices were not provided. EPA considered
465 mg/m3 the NOAEL for the dams and 2,517 mg/m3the LOAEL for the dams, based on neurological
effects (tremors). EPA considered 2,517 mg/m3 as a developmental NOAEL, while a developmental
LOAEL could not be established because no effects were observed at the highest dose tested.
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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 4,460 to 7,690 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 6,155 mg/kg HCE (purity not specified) by gavage
in mineral oil to male rats and reported that liver function was unaffected 2 hours after exposure. Kinkead
and Wolfe (1992) determined that the oral median lethal dose (LD50) 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).
Weeks et al. (1979) and Weeks and Thomasino (1978) determined acute oral toxicity values for
Sprague-Dawley rats, NZW 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 LD50 values were calculated after a 14-day observation period (Table 4-13). All
LD50 values were > 1,000 mg/kg.
Table 4-13 Summary of acute exposure data in rats, rabbits, and guinea pigs
Lethal value
Species
Rabbit, male
Rat, male
Rat, male
Rat, female
Rat, male
Guinea pig, male
Rabbit, male
Treatment
Oral ALD
Intraperitoneal (i.p.) ALD
Oral ALD
Oral LD5o
Oral LD50
Oral LD5o
Dermal LD5o
Diluent
Methylcellulose
Corn oil
Corn oil
Corn oil
Methylcellulose
Corn oil
Methylcellulose
Corn oil
Water paste
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
33
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function in sheep. Plasma activities of glutamate dehydrogenase (GDH), sorbitol dehydrogenase (SDH),
ornithine carbamoyl transferase (OCX), and AST were determined daily until they reached stable levels.
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 OCX was increased 2-10-fold. GDH, SDH, and OCX
levels peaked at 48 hours and returned to normal within 4-5 days. ASX increased only slightly. Increases
in these enzymes are indicative of hepatic damage. In addition, bromsulphthalein dye clearance tests
found a reduction in transfer from liver cells to bile at 72 hours after HCE exposure, indicating reduced
hepatic function.
4412 Inhalation
Median lethal concentration (LC50) values for HCE have not been reported. One study has
evaluated single acute inhalation exposures to HCE (Weeks and Xhomasino. 1978). Six male
rats/concentration (strain not specified, although one table in the report indicated strain as
Sprague-Dawley) were exposed to 2,500 or 57,000 mg/m3 HCE for 8 hours and to 17,000 mg/m3 HCE 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 effects either during exposure or for 14 days thereafter. Body weight gain was
reduced, but not statistically significantly, over the 14-day observation period. Male rats exposed for 8
hours to 57,000 mg/m3 HCE displayed effects, including death. At 6 hours, one rat had a staggered gait
and by 8 hours, 2 rats were dead. Xhe surviving rats showed statistically significant reductions in mean
body weight on postexposure days 0 (7%), 1 (21%), 3 (19%), 7 (15%), and 14 (15%), compared with
controls. Necropsy did not reveal any gross exposure-related lesions. Xwo 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 exposed rat.
Xhe authors concluded that this effect was not exposure-related, but rather was indicative of a low-grade
endemic upper respiratory disease. Xhe male rats exposed for 6 hours to 17,000 mg/m3 showed reductions
in body weight gain on postexposure day 1 (5%) and day 3 (4%) and body weights similar to controls for
the remaining 11 days of the postexposure period. Xwo 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
Xhree animal studies have evaluated short-term toxicity of HCE. A 12-day study in male NZW
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 etal.. 1979). Short-
term toxicity assays in rats (16 and 21 days) demonstrated kidney effects in males (NXP. 1996. 1989) but
not females (NXP. 1989).
Weeks et al. (1979) administered 100, 320, or 1,000 mg/kg-day HCE (purity 99.8%) via a
stomach tube to male NZW rabbits (5/dose) for 12 days. Blood was drawn from the central ear artery of
34
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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. Rabbits were necropsied on the fourth day
after termination of dosing, and the following tissues were examined: eye, brain, lung, kidney, liver,
spleen, heart, stomach, pancreas, large intestine, skeletal muscle, bone, urinary bladder, small intestine,
andtestes.
The 1,000 mg/kg dose group exhibited significantly reduced body weight (beginning on exposure
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 effects. 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 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, with more severe effects 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 100 mg/kg the NOAEL and 320 mg/kg the LOAEL, 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 died before the end of
the study, while 1 male and 2 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. Histopathology revealed hyaline droplet formation in the cytoplasm of renal tubular
epithelium in the kidneys of all exposed 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 the male rat LOAEL based on kidney tubule lesions, while a NOAEL for male rats could
not be established because effects were observed at the lowest dose. EPA considered 563 mg/kg-day the
female rat LOAEL, based on a dose-related decrease in body weight, and 281 mg/kg-day the female rat
NOAEL.
NTP (1996) administered 146 or 293 mg/kg-day HCE (purity 100%) via corn oil gavage to male
F344/N rats (5/dose) for 21-days. All rats were necropsied; 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 7V-acetyl-(3-D-
35
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glucosaminidase (NAG). A Mallory-Heidenhain stain was used to evaluate protein droplets, particularly
hyaline droplet formation, in kidney sections. 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 and relative liver
weights were increased in both dose groups (significant at high dose). 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 in both
dose groups (Table 4-14). Nephropathy (i.e., hyaline droplet accumulation) was observed in the male rats,
as were 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 in both dose groups compared with
controls (Table 4-14). EPA considered 146 mg/kg-day a LOAEL based on statistically significant
increases in kidney lesions and urinalysis parameters. A NOAEL could not be established because effects
were observed at the lowest dose tested (Table 4-14).
36
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Table 4-14 Summary of toxicity data from male rats exposed to HCE for 21 days
Vehicle control
146mg/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(4)
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)
GGT (mU/mg creatinine)
NAG (mU/mg creatinine)
Volume (ml_/16h)
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 + 0.9
1.024 + 0.003
0.74 + 0.19C
56.48 + 3.06b
446 + 23b
2,980 + 103
66 + 5b
1,897+73
36 + 1b
10.6 + 1. 1b
1.020+0.001b
1.2 + 0.2C
aData are mean ± SE. (For all groups, n=5, unless otherwise noted).
""Significantly different from control (p < 0.01, Dunnet's test)
""Significantly different from control (p < 0.05, Dunnet's test)
Source: NTP (1996).
4.4.3 Neurological
Several studies have provided evidence that oral and inhalation HCE exposure produced central
nervous system (CNS) effects; however, it is unknown if the neurological effects were due to the parent
compound or the metabolites. Sheep exposed to HCE developed facial muscle tremors (Fowler. 1969;
Southcott 1951) and a staggering uncoordinated gait (Southcott 1951). Sprague-Dawley rats did not
show statistically significant effects of HCE exposure on avoidance latency or spontaneous motor
activity. However, male rats, female rats, and pregnant rat dams exhibited tremors and/or ruffled pelt
following HCE exposure (Weeks etal.. 1979). Beagle dogs developed signs of neurotoxicity following
HCE exposure. (Weeks etal.. 1979).
4431 Oral Studies
Fowler (1969) orally administered acute doses of HCE to sheep (see Section 4.4.1.1) and reported
slight facial muscle tremors 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 in the study. EPA
considered the 500 mg/kg LOAEL, based on neurotoxic effects (tremors), while aNOAEL could not be
established from these data because effects were observed at the lowest dose tested.
37
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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.
Shimizu et al. (1992) reported decreased motor activity (incidence and method of analysis not
reported) in pregnant Wistar rats following gestational exposure to 167 or 500 mg/kg HCE(see Section
4.3.1). These effects decreased or disappeared when HCE exposure ended. Similarly, Weeks et al. (1979)
reported that pregnant Sprague-Dawley rats gestationally exposed to 500 mg/kg HCE displayed tremors
on GDs 15 and 16 (see Section 4.3.1).
4432 Inhalation Studies
Weeks et al. (1979) examined neurological effects in male Sprague-Dawley rats
(15/concentration) exposed to air, 145, 465, or 2,517 mg/m3 HCE (purity 99.8%) for 6 hours/day,
5 days/week for 6 weeks. Learned behavior was evaluated using an avoidance latency task by measuring
the time it took the rats to avoid foot shock by escaping into a safe compartment. Unlearned behavior (i.e.
spontaneous motor activity) was evaluated by photobeam interruptions. 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 and spontaneous motor activity counts were increased in the 465 and
2,517 mg/m3 groups at 6 weeks compared with control, but the differences were not statistically
significant. Weeks et al. (1979) concluded that the rats did not display signs of behavioral toxicity.
However, Weeks et al. (1979) noted tremors and a ruffled pelt in male and female rats exposed to 2,517
mg/m3 HCE during the fourth week of exposure in a separate experiment (see Section 4.2.2.1). In
addition, Weeks et al. (1979) reported in a developmental toxicity experiment (see Section that pregnant
Sprague-Dawley rat dams exposed to 2,517 mg/m3 group displayed tremors during GDs 12-16 (see
Section 4.3.2). Tremors are indicators of neurobehavioral effects and lack of grooming could be
interpreted as an indicator of behavioral toxicity (Kulig etal., 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) reported that Beagle dogs exposed to 2,517 mg/m3 HCE developed tremors,
ataxia, hypersalivation, and displayed severe head bobbing, facial muscular fasciculations, and held their
eyelids closed during exposure (see Section 4.2.2.1). One dog experienced convulsions and died within 5
38
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hours after initial exposure. The surviving dogs exhibited less severe symptoms during exposure, but
recovered overnight after removal from exposure.
4.4.4 Immunological
In a subchronic study, Weeks et al. (1979) exposed male Hartley guinea pigs to HCE via
inhalation (see Section 4.2.2.1). Two weeks after termination of exposure, guinea pigs were challenged
with a single intradermal injection of 0.1% HCE in saline. A sensitization response was not produced.
4.4.5 Dermatological
Yamakage and Ishikawa (1982) examined human patients suffering from systemic scleroderma
(SSD) and localized scleroderma with bilateral distribution of multiple skin lesions for potential solvent
exposure. Of nine such patients, seven had significant subchronic or chronic exposure (5-44 years) to
solvents, while an eighth had a significant acute exposure (2 weeks) to solvents. The solvents involved
were reported as "variable and mostly unidentified." As an experimental follow-up, Yamakage and
Ishikawa (1982) administered daily intraperitoneal (i.p.) injections with 0.01 mL of HCE (purity not
specified), as well as with 0.9% saline to mitigate exposure lethality, for 17 days in ddY mice (17 mice
total). 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. 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
provided indirect evidence that suggests that HCE may be capable of inducing SSD-type conditions in
humans.
Weeks and Thomasino (1978) conducted two dermal studies in male NZW rabbits. A single 24-
hour application of 500 mg of dry technical-grade 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, 500 mg 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 technical grade HCE
to one eye of each of six male NZW rabbits. Moderate corneal damage, iritis, and conjunctivitis was
39
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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).
4.5 Mechanistic Data and Other Studies in Support of the Mode of
Action
4.5.1 Genotoxicity
In vivo genotoxicity studies of HCE have not been performed in humans. In vivo HCE exposure
in animals resulted in predominantly negative results. Similarly, in vitro HCE 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. HCE did not induce mutagenicity in
Salmonella typhimurium reverse mutation tester strains (Ashby and Tennant 1988). The NTP toxicology
and carcinogenesis studies concluded that HCE (purity >99%) was not significantly genotoxic in F344/N
rats, and that increased tumors incidence occurred through a mechanism other than induction of mutations
(NTP. 1989). In an examination of available mutagenicity and genotoxicity data (i.e., the ability to induce
alterations in deoxyribonucleic acid [DNA] structure or content, 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 indicated 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.
40
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Table 4-1 5
Test
system
Summary of genotoxicity studies of HCE
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
DMA adduct
formation
(nonhuman)
M it otic
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
h2E1
BALB/C-3T3
Wistar rats,
calfthymus DMA
BALB/c mice,
calfthymus DMA
S. cerevisiae D3
S. cerevisiae D4
S. cerevisiae D7
Aspergillus nidulans
P1 diploid
- (±S9)
- (±S9)
- (±S9)
- (±S9)
- (±S9)
-(+S9)
- (±S9)
- (-S9),
+ (+S9)a
-
-
-
-
+ DMA binding in
liver, kidney, lung,
and stomach
+ DMA 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 et
al. (1991)
Nakamura et al.
(198Z)
Galloway et al.
(1987)
Galloway et al.
(198Z)
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.
(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
DMA adducts not
identified
DMA adducts not
identified
In vivo tests
Rat
Mice
Rat liver foci
DMA adduct
formation
(nonhuman)
Micronucleus
induction
Osborne-Mendel
Wistar rats
CD-1 mice
-(initiation)
+ (promotion)
Weakly + DMA
binding in liver
-
Milman et al.
(1988)
Lattanzi et al.
(1988)
Crebelli et al.
(1999)
Initiation or
promotion protocols
Adducts not
identified
41
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Human
lymphocytes
Drosophila
Replicative DMA DR^QC-I •
synthesis (RDS) B6C3F1 mice
BALB/c mice
Isolated human
lymphocytes
DMA strand Human lymphocyte
breaks cultures
Mit°tict. Drosophila
recombination r
-
Moderately + DMA
binding in liver
+ (+S9)
-
Weakly +
Yoshikawa
(1996):
Miyagawa et al.
(1995)
Lattanzi et al.
(1988)
Tafazoli et al.
(1998)
Tafazoli et al.
(1998)
Vogel and Nivard
(1993)
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 5,000 or 10,000 ug
HCE/plate (purity not specified) to be nonmutagenic, 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 that a
concentration range of 0.1-500 ug HCE/plate (purity 99.8%) yielded negative results using the same
tester strains, test protocol, solvent, and metabolic activation system. Further, as a part of NTP's
mutagenicity screening program, a concentration range of 1-10,000 ug/plate HCE was dissolved in
dimethylsulfoxide (DMSO) and tested in two independent trials in two separate laboratories. 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
metabolic activation (Milman et al.. 1988).
Using a different S. typhimurium indicator strain, BA13, that could theoretically detect a broader
range of mutagens than reverse-mutation assay, Roldan-Arjona et al. (1991) found HCE to be negative.
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. HCE 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 was also conducted both with and without rat liver
S9 metabolic activation up to a concentration of 42 ug/mL (the solvent was not specified). Although the
available data indicated 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 1-50
mg/mL HCE (purity not specified) was observed, either with or without exogenous rat liver S9 metabolic
activation. In addition, negative findings for HCE were reported by Weeks et al. (1979) using the S.
cerevisiae D4 strain.
42
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Bronzetti et al. (1989) evaluated HCE for mitotic gene conversion at the trp locus and reverse
point mutation at the ilv locus in the S. cerevisiae D7 tester strain. Exposures were from 1.2-3.0 mg/mL
HCE (purity not specified) 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).
HCE concentrations from 0.005-0.84 mg/mL HCE (purity >98%) to induce aneuploidy in the PI
diploid strain of the moldAspergillus nidulans has been evaluated (Crebelli et al., 1995; Crebelli et al..
1992; Crebelli etal.. 1988). Liquid suspension exposures (3 hours) resulted in survival rates of 100-48%.
Exposure to these concentrations did not induce mitotic malsegregation of chromosomes.
Studies have evaluated the effects of in vivo and in vitro HCE exposures on cytogenetic
endpoints in animals (Crebelli et al.. 1999; Tafazoli etal.. 1998; Doherty et al.. 1996; Vogel and Nivard.
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 HCE. CD-I mice (5/sex/concentration) were injected i.p. with 2,000 or 4,000 mg/kg HCE
(purity >98%), representing approximately 40 and 70-80% of the LD50, respectively. Animals were
sacrificed and bone marrow cells were harvested at 24 and 48 hours postexposure. HCE exposure resulted
in clinical signs of general toxicity, but no significant increases in the frequency of MNPCEs.
Vogel and Nivard (1993) utilized a Drosophila eye mosaic assay to monitor genetic damage
caused by the exposure of larvae to HCE. Drosophila larvae were exposed to food supplemented with 10
mM HCE (3% ethanol solvent; purity not specified) for 3 days. Based on their results, the authors
classified HCE as a weak positive —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. Lymphocytes
from one donor were exposed to 0.012-0.24 mg/mL HCE (purity >99%)in the presence of S9. Neither
toxicity nor MN induction was evident. Cells from the other donor were exposed to 0.24-3.79 mg/mL
HCE, both with and without S9. Although toxicity was not reported, statistically significant increases in
the percent of cells with MN were observed at 0.24 and 1.89 mg/mL HCE concentrations in the absence
of S9 (12 and 11%, respectively, versus a control value of 5.5%,/><0.05). In presence of S9, statistically
significant increased in MN were observed at 0.24 mg/mL (19.8% versus a control value of 9%,p <
0.01). In the second part of the study, lymphocyte cultures exposed to HCE for 3 hours with and without
S9 were assessed for DNA damage 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
43
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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, 0.002, 0.012, or 0.024 mg/mL HCE
(purity not specified), then scored for kinetochore-positive and -negative MN. No MN formation was
observed in any of the three cell lines in response to HCE exposure.
HCE was investigated for induction of CAs and SCEs in cultured Chinese hamster ovary (CHO)
cells as part of an NTP screening program for genotoxicity (NTP. 1989; Galloway et al.. 1987). HCE
concentrations, selected on the basis of cell confluence and mitotic cell availability, ranged from 0.01-1.0
mg/mL HCE (purity >99%). For CAs and SCEs, linear regression was used to test for dose-response
trends. Induction of CA was considered significant ifp values relative to controls were < 0.05, while
increases of SCEs/chromosome > 20% over controls 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, 0.15-0.50 mg/mL HCE did not induce CAs; however, 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). Exposure to 0.010-0.33 mg/mL HCE did not induce SCE in the absence of S9; however,
positive results for 0.10-1.0 and 0.40-1.0 mg/mL HCE were obtained in the presence of S9.
In vitro cell transformation studies have examined the effect of HCE on the process of chemical
carcinogenesis. In the absence of exogenous metabolic activation, a 3-day exposure to 0.00016-
0.100 mg/mL HCE (purity >97%) did not induce morphological cell transformation in BALB/C-3T3 cells
(Milman et al.. 1988; Story etal.. 1986; Tuetal.. 1985). In a rat liver foci assay, Story et al. (1986) and
Milman et al. (1988) examined the capacity of HCE to initiate and promote tumors. To assess initiation
potential, 10 young adult male Osborne-Mendel rats received 500 mg/kg HCE (the MTD) by corn oil
gavage 24 hours after partial hepatectomy. 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 GOT, a putative preneoplastic indicator. To assess promotion
potential, animals were i.p. injected with 30 mg of the tumor initiator, diethylnitrosamine (DEN) 24 hours
after partial hepatectomy. Six days later, the animals received 500 mg/kg of HCE (the MTD) in corn oil
by gavage, 5 days/week for 7 weeks. Animals were sacrificed and livers were examined for the presence
of GGT-positive foci. In these assays, HCE failed to demonstrate any initiating activity, but did show
statistically significant promoting capability (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.
44
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Table 4-16 Number of enzyme-altered foci in rat liver: Promotion protocol
Promotion treatment — Total number of foci/cm
+ DEN initiation - DEN initiation
HCE 4.38 + 1.07b(7) 0.1+0.15(8)
Phenobarbital 3.89+ 0.98b(10) 0.3 + 0.19(9)
Corn oil 1.77 + 0.49C(10) 0.2 + 0.15(10)
aMeans ± SE (with the number of animals in parentheses)
""Statistically different from DEN + corn oil control group, p < 0.05
°Statistically different from corn-oil-only (-DEN) control group, p < 0.05
Sources: Milman et al. (1988): Story et al. (1986).
Yoshikawa and colleagues reported on the effect of HCE exposure in an in vivo-in vitro
hepatocyte replicative DNA synthesis (RDS) assay (Yoshikawa. 1996; Miyagawa et al., 1995). Groups of
4-5 male B6C3F] mice were administered single gavage doses of 0, 1,000, or 2,000 mg/kg HCE (purity
not specified). Hepatocytes were prepared 24, 39, or 48 hours after exposure. Hepatocytes prepared 39
hours after exposure to 1,000 mg/kg HCE yielded a positive mean RDS response of 1.21 ± 0.46% (the
investigators noted that an RDS incidence rate of 0.4% was considered a positive response). The
remaining HCE exposure 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
In vitro and in vivo assays have assessed the ability of HCE to bind DNA, ribonucleic acid
(RNA), and protein in several mouse and rat tissues (Lattanzi et al.. 1988). In vitro and in vivo assays in
mice and rats have demonstrated binding of radiolabeled carbon to DNA, RNA, and protein following
[14C]-HCE administration (Lattanzi et al.. 1988). suggesting that either HCE or its metabolites bind these
macromolecules. The role of macromolecule binding in mediating HCE toxicity was not further
evaluated.
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 i.p. injected with 127 uCi/kg HCE (purity 98%; ~1 mg /kg HCE). The animals were fasted and
sacrificed 22 hours after injection. 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. Measures for binding to macromolecules were determined by the presence of
radiolabeled carbon from [14C]-HCE in the DNA, RNA, and protein; however, HCE-specific metabolites
were not measured. Therefore, the presence of radiolabeled carbon may indicate HCE binding directly to
the macromolecules or incorporation of radiolabeled carbon from intermediate metabolites into these
macromolecules.
45
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In vivo binding data for HCE are presented in Table 4-17. In both rats and mice, binding values
(in pmol HCE/mg) for RNA were consistently greater than binding values for DNA or protein. Greater
RNA binding 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 higher levels (9 times greater) of covalent binding for DNA in the liver than the rat.
Binding to liver RNA and liver protein was 2 and 3 times greater in mice than rats, respectively. DNA
binding was similar between species, but slightly greater in mice, for the kidney, lung, and stomach.
According to Lutz (1986. 1979). the covalent binding index (CBI) values indicate weak (rat liver) to
moderate (mice liver) oncogenic potency in HCE-exposed rodents.
Table 4-17 In vivo covalent binding of [14C]-HCE to DNA, RNA, and proteins from rat and mouse
organs
Liver Kidney Lung Stomach
DNA
(CBIb)
RNA
0.43
(1!
46.59
+ 0.05C
5.1)b
+ 7.23C
3.
108
Mouse
92 + 0.20d
(140)b
.08 + 21.57d
Rat
0.42
232.94
Mouse
0.50
564.98
Rat
0.14
15.55
Mouse
0.35
60.10
Rat
0,
8
.26
.33
Mouse
0.37
21.04
Protein 4.94 + 1.14° 14.99+0.83° 2.59 4.91 0.89 3.42 0.80 2.41
"Data are from pooled organs from 6 male Wistar rats or 12 male BALB/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.
dMean ± SE of four values, each obtained from three pooled livers.
Source: Reprinted with permission of Taylor & Francis©; Lattanzi et al. (1988).
In vitro binding data for HCE are presented in Table 4-18. Coenzymes were not utilized in the
controls. Liver microsomes from rats and mice catalyzed HCE binding to DNA at comparable levels.
Kidney microsomes from rats and mice produced significantly greater amounts of HCE binding to DNA.
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 compared to
controls. Microsomes from lung and stomach in both species did not display increased DNA binding
activity over corresponding controls. Cytosolic fractions from all organs in mice and rats exhibited higher
levels of HCE binding to DNA than microsomal fractions, except for rat lung cystol. 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 compared to
cytosolic fractions alone for liver. 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.
46
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Table 4-18 In vitro binding of [ C]-HCE to calf thymus DMA mediated by microsomal and/or
cytosolic phenobarbital-induced fractions of rat and mouse organs
Microsomes
Rat
+ NADPH
Mouse
Cytosol
Rat
+ GSH
Mouse
Microsomes
(+ NADPH,
Rat
+ cytosol
+ GSH)
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
1 33.44 +2.423
99.84 + 8.06
Kidney
Standard
Controls
395.84 + 78.58°
136.26 + 9.04
78.86 +6.85C
39.12+5.34
246.85 + 35.39°
88.82+30.91
251.42 + 45.38°
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 DMA binding in pmol/mg) are reported as mean ± SE of three values; ND, not determined. Controls were conducted in
the absence of coenzymes.
"Statistically different from control, p < 0.01.
°Statistically different from control, p < 0.05.
Source: Reprinted with permission of Taylor & Francis©; Lattanzi et al. (1988).
4.5.3 Structure Activity Relationships
Several studies were conducted with the objective of defining structure activity relationships
(SARs) between 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
(see Section 4.4.2). 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-trifluoroethane,
1,2-dichloro-l, 1 -difluoroethane, 1,1,1 -trichloroethane, 1,1,1,2-tetrabromoethane,
1,1,2,2-tetrabromoethane, pentabromoethane, and HCE (purity >98%). Increased kidney weights and
evidence of renal toxicity were observed in many of the rats administered halogenated ethanes; however,
this was not always coincident with hyaline droplet nephropathy. Hyaline droplet nephropathy (assessed
by Mallory-Heidenhain staining) was observed in rats administered pentachloroethane, 1,1,1,2-
tetrachloroethane, and HCE. RDS, indicated by PCNA labeling index, was increased in male rats
administered pentachloroethane, 1,1,1,2-tetrachloroethane, HCE, pentabromoethane, and 1,1,2,2-
tetrachloroethane. The increase in cell proliferation in the kidneys observed with halogenated ethanes that
did not induce hyaline droplet nephropathy suggests the contribution of another toxic mechanism. NTP
47
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(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.
Crebelli et al. (1988) evaluated three chloromethanes and eight chlorinated ethanes (including
HCE) forthe induction of chromosome malsegregation in the fungus A. nidulans (see Section 4.5.1).
Although 8 of the 11 compounds tested provided positive results, HCE was negative for chromosome
malsegregation induction. Analyses of relationships between biological and chemical variables indicated
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, the authors argued 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 fungus, A. nidulans (see Section
4.5.1). Out of the 24 chemicals, 19 were negative forthe 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. 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. 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 (molar refractivity). 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; Crebelli et al.. 1988) were expanded to include 20
additional halogenated hydrocarbons (Crebelli etal.. 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.
48
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4.6 Synthesis of Major Noncancer Effects
4.6.1 Oral
No epidemiology studies of HCE carcinogenicity were identified. Case studies in humans have
demonstrated HCE exposure; however limitations of these studies include lack of information on the
source and route of HCE exposure, co-exposures, and small sample size (Loh et al.. 2008; Loh et al.,
2006: Younglai et al.. 2002: Selden et al.. 1997: Selden et al.. 1994: Selden etal.. 1993: Allen etal. 1992:
Selden etal.. 1989). Oral toxicity studies in laboratory animals are summarized in Table 4-19. The
primary noncancer effects observed in animal studies included decreased body weight or body weight
gain, increased absolute and relative kidney weights, increased absolute and relative liver weights, 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.
49
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Table 4-1 9 Oral toxicity studies for HCE
Species
NZW 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)
B6C3F-I mice
(50/sex/dose)
F344/N rats
(50/sex/dose)
Pregnant
Sprague-
Dawley rats
(22/dose)
Pregnant Wistar
rats (21 /dose)
Concentration
(mg/m3)/
duration3
0, 100, 320
or 1,000
by oral;
12 days
0, 140,281, 563,
1,125, or 2,250
by gavage;
16 days
0, 146, or 293
by gavage;
21 days
0, 34, 67,
134,268,
or 536
by gavage;
13 weeks
0, 1, 15,
or 62
by diet;
16 weeks
0, 113, or 227
by gavage;
78 weeks
0, 360, or 722
by gavage;
78 weeks
Male: 0, 7, or 14
Female: 0, 57, or
114
by gavage;
103 weeks
0, 50, 100,
or 500
by gavage
on CDs 6-16
0, 56, 167,
or 500
by gavage
on CDs 7-17
NOAEL
(mg/m3)
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/m3)
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 (Weeks et al.. 1979; Weeks and Thomasino. 1978). and evidence of kidney effects such as
50
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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 (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
Three short-term studies have reported nephrotoxic effects following oral HCE exposure (see
Section 4.4.2). In a 16-day study in F344/N rats, 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 a 21-day study in male F344/N
rats, increased absolute and relative kidney weights, tubular regeneration and granular casts, and
increased PCNA labeling index in kidneys at doses of 146 and 293 mg/kg-day HCE (NTP. 1996). In a 12-
day study with male NZW rabbits, tubular nephrosis and tubular nephrocalcinosis were observed
following exposure to 320 and 1,000 mg/kg-day HCE (Weeks et al.. 1979). Compared with rabbits, 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 short-term studies makes it difficult to evaluate if the observed renal effects were
sex-specific. In addition, subchronic and chronic exposure studies reported nephrotoxic effects of HCE
exposure in female rats.
Two subchronic exposure studies have reported nephrotoxic effects of oral HCE exposure (see
Section 4.2.1.1). Male F344/N rats administered 34-536 mg/kg-day HCE for 13 weeks (NTP. 1989)
exhibited hyaline droplet formation, tubular regeneration, and tubular casts. Male rats 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 kidney effects. Another subchronic exposure study (Gorzinski et
al.. 1985) in F344 rats reported slight hypertrophy and dilation of the renal tubules in males rats, as well
as renal tubule atrophy and degeneration in male and female rats, following exposure to 15-62 mg/kg-day
HCE for 16 weeks. Kidney effects in female rats were observed only at the 62 mg/kg-day HCE dose.
Two chronic studies have reported nephrotoxic effects of oral HCE exposure (see Section
4.2.1.2). Chronic toxicity studies were conducted by NTP on F344/N rats and by NCI on Osborne-Mendel
rats and B6C3FJ 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. The chronic toxicity test in
B6C3Fi mice (NCI. 1978) was the only study conducted in this species.
Nephropathy (characterized as tubular cell degeneration and regeneration, dilation and atrophy,
glomerulosclerosis, interstitial fibrosis, and chronic inflammation) was observed in both male and female
F344/N rats following HCE exposure for 103 weeks (NTP. 1989). 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.
Nephropathy was reported in both control and exposed groups, likely as result of a spontaneous syndrome
known as chronic progressive nephropathy (CPN) associated with aged rats (see Section 4.7.3.1). To
51
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examine the effects of chronic HCE exposure separate from CPN, nephropathy incidence in terms of
severity was evaluated. Male rats exposed to HCE showed a dose-related increase in the severity of
nephropathy compared to controls: 36%, 48%, and 60% in control, 7, and 14 mg/kg-day HCE dose
groups, respectively. Additional dose-related nephrotoxic effects in male rats included linear
mineralization of the renal papillae and hyperplasia of pelvic transitional epithelium in the kidney (see
Table 4-5). In female rats, increases in both incidence and severity of nephropathy were dose-related (see
Table 4-4). Increased linear of renal papillae was also noted in female rats, but only at 57 mg/kg-day dose
(see Table 4-5).
Osborne-Mendel rats of both sexes displayed chronic inflammatory kidney lesions in both control
and exposed groups following 78 weeks of HCE exposure, 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 (NCI. 1978). There were dose-related increases
in incidences of nephropathy in males and females administered 113 and 227 mg/kg-day HCE (see
Section 4.2.1.2). B6C3FJ mice of both sexes displayed chronic kidney inflammation and tubular
nephropathy following HCE exposure for 78 weeks (see Section 4.2.1.2). In male B6C3F] mice, chronic
kidney inflammation was observed at higher levels in the controls and low dose group than in the high
dose mice; 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. In female B6C3F] mice, chronic kidney
inflammation was observed only in vehicle controls and the 227 mg/kg-day HCE dose group (2%),
whereas tubular nephropathy was observed in both exposure groups of both sexes at high incidences (see
Section 4.2.1.2).
The available information for HCE-induced nephrotoxicity indicates that the male rat is the most
sensitive sex/species to HCE-induced renal toxicity. Limited information available in other species
indicates that nephrotoxic doses 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. Similarly, subchronic and chronic exposure studies in rats
(NTP. 1989; Gorzinski et al.. 1985) indicated that nephrotoxic doses in female rats were at least 4-fold
greater than the lowest nephrotoxic dose in male rats. These toxicity data are consistent with the tissue
distribution data (see Section 3.2) and demonstrating that the male kidney accumulated higher HCE
concentrations than the female kidney and potential sex differences in the distribution and metabolism of
HCE and support the kidney as the primary target organ following oral exposure to HCE.
4612 Hepatotoxicity
Short-term studies in rats (NTP. 1996). male rabbits (Weeks et al.. 1979). and sheep (Fowler.
1969) reported hepatotoxicity at doses > 300 mg/kg-day HCE (see Section 4.4.2). Male F344 rats
exhibited significantly increased relative liver weights at 293 mg/kg-day, and significantly increased AST
and NAG serum activity. Liver degeneration and necrosis, including fatty degeneration, coagulation
necrosis, hemorrhage, ballooning degeneration, eosinophilic changes, and hemosiderin-laden
52
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macrophages and giant cells were observed in male NZW rabbits administered 320 and 1,000 mg/kg-day
HCE 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 OCX that were increased at least twofold more controls,
indicating reduced hepatic function.
Two subchronic studies (NTP. 1989; Gorzinski et al.. 1985) reported hepatotoxic effects in male
and female rats exposed to HCE (see Section 4.2.1.1). Females were more sensitive than males, with
severity and statistically significant increases in hepatoxicity occurring at lower doses than male rats.
Liver weight to body weight ratios (mg/g) increased in a dose-related manner for both male and female
rats exposed to HCE (Table 4-3). Hepatocellular necrosis was noted in females exposed to 134-536
mg/kg-day and in males exposed to 268-536 mg/kg-day (NTP. 1989). Gorzinski et al. (1985) reported a
slight swelling of the hepatocytes in the 15 and 62 mg/kg-day dose groups. 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. Other than a statistically significant increase in liver weight at
62 mg/kg-day HCE, the females were not affected. This result contrasts the hepatocellular effects noted in
female rats in the 13-week NTP study (1989). However, the highest dose used by Gorzinski et al. (1985).
62 mg/kg-day, is below the NOAEL(67 mg/kg-day) for female rats in the NTP (1989) study, indicating
that exposure doses in Gorzinski et al. (1985) may have been too low to cause hepatotoxicity in female
rats.
Although hepatocellular necrosis was observed in the subchronic study, no noncancerous liver
effects observed in the rats and mice administered HCE for chronic durations. The range of doses in the
subchronic assay [34 - 536 mg/kg-day in F344 rats (NTP. 1989)] encompassed the doses used in the
chronic assays for female F344 rats and both sexes of Osborne-Mendel rats (57-227 mg/kg-day) (NTP.
1989: NCI. 1978). The LOAEL for hepatocellular necrosis in female F344/N rats [134 mg/kg-day (NTP.
1989)1 in the subchronic study exceeded the highest dose of the chronic study (NTP. 1989) of F344/N
rats, 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 at 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.
HCE-induced liver effects were only observed in animals in short-term and subchronic studies.
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. Female rats exhibited
a greater sensitivity to liver effects as evidenced by the effects observed at lower doses compared with
males (NTP. 1989). These data suggest that the female rat is the most sensitive sex/species to HCE
hepatotoxicity.
53
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4613 Developmental Toxicity
Two developmental studies in rats (Shimizu et al., 1992; Weeks etal.. 1979) indicated that oral
HCE exposure induced teratogenicity in the presence of maternal toxicity (see Section 4.3.1). In the
Shimizu et al. (1992) study, pregnant Wistar rats (gavaged with HCE) displayed decreased motor activity,
piloerection, and subcutaneous hemorrhage. Fetuses from dams exposed to 500 mg/kg HCE displayed
decreased body weight, skeletal variations such as rudimentary lumbar ribs, and ossification effects, but
no skeletal malformations were observed. In Weeks et al. (1979). pregnant Sprague-Dawley rats gavaged
with 500 mg/kg HCE displayed pulmonary effects, but fetuses did not exhibit any skeletal or soft tissue
anomalies. These results indicate that oral exposure HCE resulted in teratogenic effects at doses that were
also maternally toxic.
4.6.2 Inhalation
The database of inhalation toxicity studies on HCE is limited. 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 HCE exposure in smoke bomb production workers, but the
sample sizes were too small to provide definitive conclusions on health effects. The inhalation toxicity
database of HCE in animals is limited to a single publication (Weeks etal.. 1979). This study conducted
acute exposure (see Section 4.4.1.2), subchronic (see Section 4.2.2.1), and developmental (see Section
4.3.2) toxicity studies with HCE. The data from this study are summarized in Table 4-20. Neurological
effects, such as tremors and ataxia, were observed in Beagle dogs and in pregnant and nonpregnant
female 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. 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). Overall, changes in body or organ weight and neurotoxic
effects (e.g., tremors) were consistently observed across several species at the highest exposure dose
(2,517mg/m3HCE).
54
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Table 4-20 Summary of data from the Weeks et al
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)/
duration3
0, 145, 465, or
2,517; 6 weeks
0, 145, 465, or
2,517; 6 weeks
0, 145, 465, or
2,517; 6 weeks
0, 145, 465, or
2,517; 6 weeks
0, 145, 465, or
2,517; CDs 6-16;
11 days
0, 145, 465, or
2,517; 6 weeks
NOAEL
(mg/m3)
465
465
465
2,517
Maternal: 465
Developmental:
2,517
465
a145, 465, and 2,517 mg/m3 correspond to concentrations reported
. (1979) inhalation toxicity study with HCE
LOAEL
(mg/m3)
2,517
2,517
2,517
Not established
Maternal: 2,51 7
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",
decreased body weight
gain
Fetal: no effects
Behavioral tests:
avoidance latency and
spontaneous motor
activity
by Weeks et al. (1979) as 15, 48, and 260 ppm,
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)
respectively
Incidence data on tremors was not reported by the study authors
4.6.3 Mode-of-Action Information
The mode of action for HCE-induced toxicity is unknown. The available data on mode of action
for HCE toxicity is limited to animal studies. 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 (see Figure 3-1). It is unknown whether 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 is metabolized (Gorzinski et al., 1985). This study did not quantify metabolite
concentrations, so these estimations are of limited.
Neurological effects have been consistently observed following oral and inhalation exposure to
HCE (Weeks etal.. 1979) (Shimizu et al.. 1992; Fowler. 1969; Southcott 1951); however, these data for
neurological effects of HCE exposure are limited are inadequate to determine a mode of action. Thus, the
mode of action for HCE-induced neurotoxicity is unknown
Although HCE-induced nephropathy has been observed in both sexes of rats and mice (see
Section 4.6.1.1), the mode of action for HCE-induced kidney toxicity is unknown. Some data suggest an
55
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a2u-globulin mode of action could contribute to HCE-induced nephropathy (see Section 4.7.3.1).
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
immunohistochemical data demonstrating a2u-globulin in the hyaline droplets, (2) the hyaline droplet
accumulation (caused by excessive protein load) may not be exclusively related to a2u-globulin
accumulation, and (3) the existence of renal toxicity in female rats, as well as 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 (see Section 4.7.3.1) could contribute to the observed
nephrotoxicity following HCE exposure. However, changes in the severity of the nephropathy were
greater in HCE exposed male rats 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 in the HCE-exposed female
rats, suggesting that CPN is not solely responsible for the nephropathy observed by NTP (1989).
Insufficient data are available that support an a2u-globulin mode of action and the data indicate that CPN
is not solely responsible for the observed nephropathy. Therefore, the data are unavailable to support
exacerbation of CPN by a2u-globulin nephropathy as a mode of action for renal nephropathy following
HCE exposure.
The liver has been demonstrated to be a target organ of HCE exposure (see Section 4.6.1.2), but
the mode of action of the hepatotoxicity of HCE is unknown. Studies of TCA (a potential metabolite of
HCE) indicate that free radical generation may play a role in mediating toxicity, particularly in the liver.
Town and Leibman (1984) reported lipid peroxidation following treatment with HCE, which the authors
suggested involved a free radical. However, no data were available that demonstrated generation of free
OO 5 O
radicals following exposure to HCE.
Although in vitro and in vivo experiments on HCE genotoxicity were predominantly negative, in
vivo binding studies suggested that HCE can bind to DNA, RNA, and protein (see Section 4.5). In the rat,
higher levels of DNA, RNA, and protein binding were observed in the kidney and liver. 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 indicated 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 free
radicals. Binding HCE to macromolecules was interpreted by the presence of radiolabeled carbon;
however, radiolabeled carbon may have been incorporated into these macromolecules from intermediary
HCE metabolites.
56
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4.7 Evaluation of Carcinogenicity
4.7.1 Summary of Overall Weight of Evidence
Under the U.S. EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005b). HCE is
"likely to be carcinogenic to humans" based on evidence of statistically significant increased incidences
of multiple tumor types in male rats and both sexes of mice (NTP. 1989; NCI. 1978). Specifically, NTP
(1989) reported dose-dependent increases in the combined incidence of renal adenomas or carcinomas in
male F344/N rats (see Table 4-6). NTP (1989) also reported increases in the incidence of
pheochromocytomas in male F344/N rats, although the increase was not dose-related (see Table 4-7). NCI
(1978) observed statistically significant increases in the incidence of hepatocellular carcinomas in male
and female B6C3F] mice (see Table 4-10). The male mice demonstrated a dose-related increase in
hepatocellular carcinomas, although increases in hepatocellular carcinomas in female mice were not dose-
related.
Some data suggest that HCE-induced kidney tumors in male rats may involve a male rat-specific
a2u-globulin-mediated mode of action. As this mode of action is unique to the male rats, there is some
uncertainty regarding the human relevance of these tumors for human health assessment. The available
data on the role of a2u-globulin-mediated mode of action in the carcinogenic effects of HCE were
considered (see Section 4.7.3.1). EPA concluded that there is insufficient evidence to attribute HCE-
induced kidney tumors in male rats to an a2u-globulin mode of action and that the mode of action for renal
tumors is unknown.
The available data are considered insufficient to describe the mode of action for the carcinogenic
effects of HCE in the liver (see Section 4.7.3.2). 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 office radicals during HCE metabolism. These processes could potentially lead to
cytotoxicity, inflammation, and regenerative cell proliferation. However, these potential key events have
not been evaluated for HCE.
The relevance of rodent pheochromocytomas as a model for human cancer risk has been the
subject of discussion in the scientific literature (Greim et al., 2009; Powers etal.. 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 (Greim et al.,
2009; Eisenhoferetal.. 2004: Lehnert et al.. 2004; Elder etal.. 2003; Goldstein et al.. 1999). Data are
lacking to describe the mode of action for pheochromocytomas following HCE exposure (see Section
4.7.3.3).
The descriptor'likely to be carcinogenic to humans" is appropriate when the weight of the
evidence is adequate to demonstrate carcinogenic potential to humans but does not reach the weight of
evidence for the descriptor "carcinogenic to humans." An example provided in the U.S. EPA Guidelines
for Carcinogen Risk Assessment (U.S. EPA. 2005b) is "an agent that has tested positive in animal
57
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experiments in more than one species, sex, strain, site, or exposure route, with or without evidence of
carcinogenicity in humans." As is discussed in Section 4.2.1.2 of this assessment the results from several
rodent bioassays indicate that HCE exposure can cause tumors in two species, both sexes of animals, and
multiple sites. On this basis, these data support the cancer descriptor "likely to be carcinogenic to
humans." However, there are uncertainties associated with relating the observed tumors in animals
following exposure to HCE to human carcinogenicity. Additional mechanistic data, particularly related to
the formation of the renal tumors in male rats, would inform the uncertainty associated with the
assumption that these tumors are relevant to humans. If these tumors were determined to not be relevant
to humans, then the weight of evidence regarding human carcinogenic potential would be reduced.
U.S. EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005b) 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
demonstrated that tumors occurred 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 was absent. Based
on the observance of systemic tumors following oral exposure, and in the absence of information to
indicate otherwise, it was assumed that an internal dose will be achieved regardless of the route of
exposure. Therefore, the data are sufficient to conclude that 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 on HCE carcinogenicity. Carcinogenic effects of
chronic oral HCE exposure have been reported in animal bioassays (see Section 4.2.1.2). These animal
bioassays provided evidence of renal adenomas and carcinomas and pheochromocytomas in male rats
and hepatocellular carcinomas in male and female mice following HCE exposure. The available
information indicates that the male rat kidney is the most sensitive sex/species/organ for HCE-induced
carcinogenicity. In vitro data provide evidence of macromolecule binding of HCE and suggests that HCE
is capable of tumor promotion, but not tumor initiation in the liver.
Male F344/N rats chronically exposed to 7 or 14 mg/kg-day HCE (NTP. 1989) exhibited a dose-
related increase in the incidence of combined renal adenomas or carcinomas (see Table 4-6). In addition,
NTP (NTP. 1989) reported increased incidence of combined pheochromocytomas (benign, malignant, and
complex pheochromocytomas) at all exposure doses in male F344/N rats, although only the increased
incidence was not dose-related (see Table 4-7). NTP (NTP. 1989) did not observe renal or adrenal tumors
in female rats chronically exposed to 57 or 114 mg/kg-day HCE for 103 weeks. The study authors
concluded that the renal adenomas and carcinomas in male rats were evidence of carcinogenicity, based
on incidence in historical controls. NTP (NTP. 1989) also stated that the increased incidence of
pheochromocytomas male rats were possibly exposure related. NCI (1978; Weisburger. 1977) conducted
58
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chronic exposure studies in Osborne-Mendel rats and B6C3FJ mice. Male and female Osborne-Mendel
rats chronically exposed to 113 or 227 mg/kg-day HCE had increased incidences of several tumor types
including kidney tubular cell adenoma and hartoma, pituitary chromophobe adenoma, thyroid follicular
cell adenoma or carcinoma, testicular interstitial cell tumors, mammary gland fibroadenoma, and ovary
granulosa cell tumors in rats (see Table 4-8 and Table 4-9); however, no statistical differences in tumor
frequencies were observed between exposed and control rats. In addition, these tumor types are
considered to be spontaneous lesions in Osborne-Mendel rats. Therefore NCI concluded that there was no
evidence of carcinogenicity in this rat study. Male and female B6C3Fi mice chronically exposed to 360 or
722 mg/kg-day HCE exhibited statistically significant increases in incidences of hepatocellular
carcinomas (see Table 4-10). Male mice demonstrated a dose-related increased tumor response for
hepatocellular carcinomas, whereas, female mice demonstrated an increased tumor response that was not
dose related. NCI concluded that HCE was carcinogenic in both sexes of B6C3Fi mice.
The animal carcinogenicity bioassays have provided evidence of renal tumors in male rats and
liver tumors in male and female mice following HCE exposure. Notably, the HCE dose resulting in a
statistically significant increase in hepatocellular carcinoma in B6C3Fi mice (NCI. 1978) was
approximately 26 times greater than doses producing a statistically different incidence in renal tumors in
F344 male rats (NTP. 1989). In vitro experiments also provide some evidence of HCE carcinogenicity.
Evidence of HCE carcinogenic promotion, but not initiation, potential was observed in the liver of male
Osborne-Mendel rats (Milman et al., 1988; Story et al., 1986). Lattanzi et al. (1988) reported species
differences for binding to DNA, RNA, and protein (see Table 4-17), with the mouse exhibiting higher
levels of DNA binding in the liver than the rat.
4.7.3 Mode-of-Action Information
Hepatocellular carcinoma, renal adenomas and carcinomas, and pheochromocytomas were
observed in rats and mice following oral exposure to HCE (NTP. 1989; NCI. 1978). The mode(s) of
carcinogenic action of HCE in the liver, kidney, and adrenal gland is unknown. The mechanistic data
available for HCE is limited; however, there are data suggesting that induction of kidney tumors in male
rats involves the accumulation of a2u-globulin in the kidney and induction of liver tumors in male and
female mice may involve increased cytotoxicity, inflammation, and regenerative cell proliferation in the
liver.
4731 Kidney Tumors
Some data suggested that HCE-induced kidney tumors in male rats may involve an a2u-globulin-
mediated mode of action. This mode of action is unique to the male rat; 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. 1991b). An analysis
of the data, outlined below, indicates that there is insufficient evidence to attribute HCE-induced kidney
59
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tumors in male rats to an a2u-globulin mode of action. Specifically, no immunohistochemical data
demonstrated 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.
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
caused by the a2u-globulin accumulation are not 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.
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 (1991a) Risk
Assessment Forum Technical panel report. This report (U.S. EPA. 1991a) 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 to 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. 1991a).
The hypothesized mode of action ascribed to a2u-globulin-associated nephropathy is defined by a
progressive sequence of events in the male rat kidney, often culminating in renal tumors. The
involvement of hyaline droplet accumulation in renal tubules, in the early stages of nephropathy
(associated with excessive accumulation of the urinary protein, a2u-globulin) is an important difference
from the sequence of events observed with classic carcinogens. The pathological changes that precede the
proliferative sequence for classic 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 unique 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
60
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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
• 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 (with continued exposure);
• 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
61
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Table 4-21 Nephrotoxic effects characteristic of a2u-globulin nephropathy observed in male and
female rats administered HCE
Study,
Dose (mg/kg-day [Sex]),
Exposure Duration (weeks or days)
Progressive sequence
of events leading to
renal tubule tumors
Accumulation of hyaline droplets
Accumulation of a2u-globulin
in hyaline droplets
Necrosis/
Degeneration
Tubular regeneration
Granular casts/dilatation
Papillary mineralization
Tubular hyperplasia
Renal tubule tumors
NTP
(1989)
7 or
14 [M];
57 or
114 [F]
103
weeks
M F
NT NT
X X
X X
X X
X
X
X
NCI
(1978)
113
or 227
111-112
weeks
M F
NT NT
X X
X X
X X
Gorzinski
et al.,
(1985)
1; 15;
or 62
16 weeks
M F
NT NT
X X
X
NTP
(1989)
34; 67;
134; 268;
or 536
13 weeks
M F
X
NT NT
X
X
X
NTP
(1996)
146
or 293
21 days
M F
X NT
NT NT
NT
X NT
X NT
NT
X NT
NTP
(1989)
140; 281;
or 563
16 days
M F
X
NT NT
X
X
NT = not tested; X = presence of effect; M = male; F = female
In addition to this histopathological sequence, U.S. EPA (1991a) 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.
• 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.
As outlined in the U.S. EPA Risk Assessment Forum Technical Panel report (U.S. EPA. 199la),
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 exposed male rats. The abnormal accumulation of hyaline droplets in the P2
62
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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 a2u-globulm. 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
a2U-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.
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.
Experimental Support for the Hypothesized Mode of Action: Strength, consistency, and
specificity of association
The oral database for HCE consists of short-term exposure studies in male and female rats (NTP.
1989. 1986). subchronic exposure studies in male and female rats (NTP. 1989; Gorzinski et al.. 1985).
chronic exposure studies in male and female rats (NTP. 1989; NCI. 1978). and chronic exposure studies
in male and female mice (NCI. 1978). These studies have reported the accumulation of hyaline droplets in
male rats, as well as aspects of the pathological sequence associated with a2u-globulin nephropathy.
However, there is insufficient evidence to attribute HCE-induced kidney tumors in male rats to an a2u-
globulin mode of action because none of the available studies demonstrated the accumulation of
a2u-globulin in the hyaline droplets.
Accumulation of hyaline droplets in male rats has been demonstrated following short-term and
subchronic exposure to HCE. In one short-term exposure study, NTP (1989) reported hyaline droplets in
the cytoplasm of the renal tubular epithelium in all males surviving exposure to 140-563 mg/kg-day HCE
for 16 days. Female rats exposed to HCE at the same doses did not display accumulation of hyaline
droplets. In a second short-term exposure study, NTP (1996) reported marked hyaline droplet
accumulation in male rats, characterized as two severity grades above the control rats, following exposure
to 146 or 293 mg/kg-day HCE for 21 days. Hyaline droplet accumulation was also reported in male rats,
but not female rats, following subchronic exposure to 34-536 mg/kg-day HCE (NTP. 1989). These data
provide evidence that HCE exposure leads to hyaline droplet accumulation in male rats.
Although hyaline droplet accumulation has been demonstrated following HCE exposure, hyaline
droplet accumulation is a nonspecific response to protein overload. Based upon the criteria for evaluating
the role of a2u-globulin mode of action in male rat nephropathy (U.S. EPA. 1991a). it is necessary to
demonstrate that the protein in the droplet is a2u-globulin. None of the available studies demonstrated that
the protein in the hyaline droplets was a2u-globulin (see Table 4-21). This lack of a2u-globulin
immunohistochemical data represents an important data gap.
63
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The available short-term, subchronic, and chronic HCE exposure studies have reported
histopathological effects consistent with an a2u-globulin mode of action. Tubular cell regeneration and
eosinophilic granular casts of cell debris in the tubule lumina at the corticomedullary junction male, but
not female, rats exposed to 140 or 281 mg/kg-day HCE for 16 days (NTP. 1989). Increased incidence of
tubular regeneration and eosinophilic granular casts in the outer medullary tubules were also reported in
male rats exposed to 146 and 293 mg/kg-day HCE for 21 days (NTP (1996). Tubular cell regeneration,
eosinophilic granular casts of cell debris in the tubular lumina at the corticomedullary region (with
associated tubular dilatation), and renal degeneration and necrosis were reported in male, but not female,
rats exposed to 34-536 mg/kg-day HCE 13 weeks (NTP (1989). Similarly, Gorzinski et al. (1985)
reported increased incidences of renal degeneration and slight hypertrophy/dilation of the renal proximal
tubules in male rats following exposure to 15 or 62 mg/kg-day HCE for 16 weeks. Interestingly,
Gorzinski et al. (1985) observed renal degeneration in female rats subchronically exposed to 62 mg/kg-
day HCE. Chronic exposure studies also reported histopathological effects in both male and female rats
(see Table 4-21), although the male rats were more sensitive to HCE exposure. Both sexes of rats
chronically exposed to 113 or 227 mg/kg-day HCE for 78 weeks (NCI. 1978) had increased incidence of
nephropathy (described as tubular degeneration and necrosis and the presence of large hyperchromatic
regenerative epithelial cells). NCI (1978) also reported additional histopathological effects in male and
female rats, some of which are consistent with an a2u-globulin mode of action: focal pyelonephritis,
tubular ectasia, cast formation, chronic interstitial nephritis and fibrosis, and focal glomerulosclerosis. In
another chronic HCE exposure study, nephropathy (characterized as tubular cell degeneration and
atrophy, tubular dilatation, tubular cell regeneration, glomerulosclerosis, interstitial fibrosis, and chronic
inflammation) was reported in both male and female rats (NTP. 1989). Cast formation was also reported
in both male and female rats. Additional histopathological effects consistent with an a2u-globulin mode of
action were also reported in male rats, including linear mineralization of the renal papillae, pelvic
epithelium hyperplasia, and renal tumors. NTP (1989) noted that the hyperplasia and tumors of the renal
tubules represented a morphologic continuum. A sex difference was noted in (NTP (1989). as males were
more sensitive to HCE-exposure-related nephropathy than females . This sex specificity is associated with
both the incidence of nephrotoxicity and severity of nephropathy observed in both control and HCE-
exposed groups. 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 to female rats at a dose one eighth of the dose
administered to female rats.
Summary of evidence for strength, specificity, and consistency
The criteria for demonstrating the a2u-globulin-related mode of action for risk assessment
purposes have been defined (U.S. EPA. 1991a): (1) an increase in hyaline droplets in the renal proximal
tubule cells; (2) the determination that the accumulating protein in the droplets is a2u-globulin; and (3) the
presence of additional pathological lesions associated with a2u-globulin.
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Hyaline droplets in renal proximal tubule cells were reported following short-term and subchronic
exposure to HCE. However, hyaline droplets accumulation was not reported following chronic exposure
(NTP. 1989). It is possible that hyaline droplets were present, but were obscured by the prevalence of the
other lesions. Therefore, the available data on hyaline droplet accumulation are consistent with an
a2u-globulin mode of action for HCE.
Although hyaline droplets observed following administration of HCE (NTP. 1989. 1986). none of
the HCE studies performed immunohistochemical assays to confirm the presence of a2u-globulin protein
within. It is unclear whether HCE is binding to a2u-globulin or to other proteins during the formation of
hyaline droplets. Therefore, there is insufficient immunohistochemical data to determine if HCE renal
nephropathy occurs through a2u-globulin accumulation or if another mechanism is operating.
Pathological lesions consistent with an a2u-globulin mode of action have been reported following
short-term, subchronic, and chronic HCE exposure. In male rats, each histopathological event in the
progressive sequence leading to renal tubule tumors has been observed (see Table 4-21). In addition, data
are available indicating histopathological events in female rats and male and female mice following
chronic HCE exposure (NTP. 1989; NCI. 1978). NTP (1989) reported dose-dependent increases
incidence and severity of renal nephropathy in female rats, while NCI (1978) reported dose-related
nephropathy in female rats that was not apparent in the controls (see Section 4.2.1.2). 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- exposed male and female mice
following chronic HCE exposure, but the mice did not develop renal tubule tumors (see Section 4.2.1.2).
Considering that a2u-globulin nephropathy is male rat-specific, the appearance of nephrotoxic effects in
the female rats, as well as the male and female mice, and the identification of other histopathological
effects not specifically associated with a2u-globulin (i.e., glomerulosclerosis and interstitial fibrosis)
suggest that at least some of renal effects of HCE may not be the result of a2u-globulin accumulation.
Therefore, although some of the histopathological data in male rats is consistent with an a2u-globulin
protein, the available data in HCE-exposed female rats and male and female mice is inconsistent with an
a2u-globulin mode of action.
Limitations do exist in studies describing 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
65
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two dose groups, limiting the ability to more fully characterize the dose response of HCE-exposure-
related nephropathy.
Dose-response concordance
The accumulation of a2u-globulin in hyaline droplets is expected to occur at lower doses than
subsequent a2u-globulin-related effects. Because none of the HCE studies confirmed the presence of a2u-
globulin protein within the hyaline droplets observed following administration of HCE (NTP. 1996.
1989). dose-response concordance of the a2u-globulin accumulation in hyaline droplets cannot be
determined.
Most of the histopathological events in epithelial cells of the proximal tubules leading to renal
tumors (Doi et al.. 2007; IARC. 1999; U.S. EPA. 1991a) increased in incidence with increasing doses of
HCE in the short-term and subchronic exposure studies. Histopathological effects associated with
a2u-globulin nephropathy (i.e., tubular cell degeneration and atrophy, tubular dilatation, and tubular cell
regeneration) were noted in almost all of the exposed and unexposed animals. Dose-related increases over
controls for toxic kidney effects such as linear 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. 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 subchronic studies (Gorzinski et al.. 1985).
Although nephropathy was noted in the shorter duration studies (NTP. 1989. 1986; 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. As immunohistochemical evidence of a2u-globulin protein
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within the hyaline droplets was available for HCE, the temporal relationship for the accumulation of
a2u-globulin could not be determined.
Histopathological effects associated with a2u-globulin-related nephropathy were observed in
animals exposed 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: 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 between renal nephropathy and renal carcinogenicity cannot be determined from
available data.
Biological plausibility and coherence
Generally, the kidney toxicity and tumor formation was observed in rats and mice are biologically
plausible effects that could potentially occur in humans. If the tumor formation in male rats is due to
accumulation of a2u-globulin mode of action, then these tumors would not be considered to be relevant to
human health risk assessment. An a2u-globulin mode of action was evaluated as a hypothesized mode of
action for HCE-induced carcinogenicity and nephropathy (U.S. EPA. 1991c). The 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 ((Doi et al.. 2007: IARC. 1999: Hardetal. 1993: U.S. EPA.
1991a). Evidence of nephrotoxic effects in female rats in two chronic studies (NTP. 1989: NCI. 1978). in
female rats in a subchronic study (Gorzinski etal.. 1985). 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
in male rats following HCE exposure. It is possible that advanced CPN may play a role in the incidence of
nephrotoxicity and kidney tumors in aged male rats. CPN is associated with aged rats, especially F344,
Sprague-Dawley, and Osborne-Mendel strains. CPN is frequently more severe in males compared with
females. Several of the CPN histopathological effects are similar to, and can obscure, 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. 1991a).
However, Webb et al. (1990) 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
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noted with both CPN and a2u-globulin nephropathy. Hard et al. (1993) reported the histopathologic
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).
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 creatinine and urea nitrogen
levels, whereas these changes in a2u-globulin nephropathy are minimal (Hard et al.. 1993).
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).
Another potential mode of action is the exacerbation of CPN by a2u-globulin nephropathy. Insufficient
data are available that support an a2u-globulin mode of action and the data indicate that CPN is not solely
responsible for the observed nephropathy. Furthermore, additional data gaps identified prevent attributing
the renal effects of HCE to the exacerbation of chronic progressive nephropathy by a2u-globulin
accumulation (i.e., categorization of end stage renal failure in either the control or HCE-exposed animals,
presences of foci of atypical hyperplasia were present, or if renal adenomas were within the areas of
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chronic progressive nephropathy). Therefore, there is insufficient data for this potential mode of action for
the renal effects of HCE.
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. As tumors were observed in the male rat kidney following
chronic HCE exposure (NTP. 1989). the available data were evaluated for a potential a2u-globulin mode
of action. The lack of a2u-globulin immunohistochemical data, as well as data demonstrating kidney
effects in female rats and mice of both sexes (NTP. 1989; NCI. 1978). prevented attribution of the renal
tumors to an a2u-globulin mode of action. Although the age and strain of rats suggested a potential role for
CPN in the observed histopathological effects, treatment-related effects in male and female rats suggest
that CPN is not solely responsible for the observed nephropathy. While it is possible that an a2u-globulin-
related mode of action may be responsible for the male rat kidney tumors whereas more than one mode of
action may be responsible for the nephropathy, there is insufficient data to support this hypothesized
mode of action. Therefore, the mode(s) of action for the renal adenomas and carcinomas in male rats is
unknown.
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 results from a2u-globulin accumulation, the tumor data are not
used in the cancer assessment (U.S. EPA. 1991a). 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. Therefore, the renal adenomas and carcinomas observed in male rats
administered HCE (NTP. 1989) were considered relevant for human health risk assessment.
4732 Liver Tumors
Significantly increased incidences of hepatocellular carcinomas were observed in male and
female B6C3F] mice in a chronic oral bioassay conducted by NCI (1978). Tumor incidences in male mice
demonstrated a dose response, whereas female mice did not demonstrate a dose response. 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-exposed mice. The investigators did not find nonneoplastic liver effects (such as organized
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thrombus, inflammation, fibrosis, necrosis, infarctions, amyloidosis, or hyperplasia) in either sex.
However, hepatocellular necrosis of the centrilobular area was observed in rats following subchronic
HCE exposure (NTP, 1989). It is unknown if hepatocellular necrosis could be a key event in the
carcinogenic process because rats in chronic exposure studies (NTP. 1989; NCI. 1978) have not displayed
hepatocellular neoplastic endpoints
The mode of action for the carcinogenic effects of HCE in the liver is unknown. Metabolism
studies of HCE indicated that the major enzymes involved are phenobarbital-inducible CYP450s, which
are primarily localized in the liver, and the majority of HCE metabolism is presumed to occur in the liver.
Comparisons of HCE metabolism rates indicated that mice metabolize HCE at twice the rate of rats
(Mitoma et al., 1985). In vivo assays have demonstrated that macromolecule binding of HCE (or its
metabolites) is consistently higher in mice than in rats, with moderate oncogenic potential in the liver (see
Section 4.5.2). Cellular damage leading to cytotoxicity, inflammation, and regenerative cell proliferation
is a possible consequence of this binding in the liver. Regenerative cell proliferation has been evaluated in
the kidney, but not in the liver of HCE-exposed rats (NTP. 1996). RDS in hepatocytes was evaluated in
mice exposed to 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 provided support for the hypothesis
that HCE acts as a tumor promoter, not as a tumor initiator (Milman et al.. 1988; Story et al.. 1986).
The radiolabel binding studies provided a possible explanation for the difference in carcinogenic
target organs between rats and mice. The in vivo radiolabel binding data suggest that HCE (or its
metabolites) 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, 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.
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. While some data suggest 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. Therefore, the mode of action for liver carcinogenicity is
unknown.
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4733 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, adrenal gland tumors in rodents are considered relevant to human
health risk assessment.
No studies were identified to determine a mode of action for HCE-induced tumors of the adrenal
gland. The mode of action for pheochromocytomas observed following oral exposure to HCE is
unknown.
4.8 Susceptible Populations and Life Stages
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 have addressed possible childhood susceptibility to HCE-induced toxicity or
carcinogenicity. CYP450 enzymes of the 2A, 2B, and 3A subfamilies, and CYP450 1A2 are involved in
HCE metabolism, suggesting that age-related differences in CYP450 activity could lead to age-related
susceptibility in HCE toxicity. 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; Vieiraetal.. 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 may be responsible for age-related differences. Therefore, the extent to which
variable age-related expression of CYP450 contributes to childhood susceptibility is unknown.
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4.8.2 Possible Gender Differences
Male rats were more sensitive to HCE-induced nephrotoxicity than females (NTP. 1989;
Gorzinski et al.. 1985; NCI. 1978). whereas 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 (see Table 3-3), sex hormone
differences, and/or sex-specific differences in CYP450 activities. No additional studies were located that
addressed possible sex-specific differences for HCE-induced toxicity or carcinogenicity.
4.8.3 Other
CYP450 enzymes, which have been shown to metabolize HCE, are polymorphic in the human
population. Polymorphisms result in CYP450 enzymes with variant catalytic activity for substrates such
as HCE. This enzyme polymorphism 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. No studies were located that addressed possible interindividual differences in
CYP450 polymorphisms in HCE-induced toxicity or carcinogenicity.
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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 were not available. The oral exposure
database for HCE includes a 103-week gavage study in F344 rats (NTP. 1989). a 111-112-week gavage
study in Osborne-Mendel rats (NCI. 1978). a 111-112-week gavage study in B6C3FJ 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). Reported effects included 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 etal.. 1985). linear mineralization of renal tubules (NTP.
1989). hyperplasia of the renal pelvic transitional epithelium (NTP. 1989). and hepatocellular necrosis
(NTP. 1989). Chronic and subchronic studies in rats and mice indicated that the kidney and liver are
target organs of HCE oral toxicity in rodents. The incidence of kidney and liver effects from the studies
considered for selection as the principal study are summarized in Table 5-1.
Specifically, nephropathy was observed in both chronic studies (NTP. 1986; NCI. 1978);
however, the animals in the chronic NTP study (1989) 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.
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Table 5-1 Incidences of
HCE
Study
Duration
(route)
noncancerous kidney and liver effects in rats following oral exposure to
Strain/
sex/species
Endpoint
Dose
(mg/kg day)
Incidence
Kidney Effects
NCI (1978)
NTP (1989)
NTP (1989)
NTP (1989)
Gorzinski,
et al. (1985)
Gorzinski,
et al. (1985)
78 weeks
(gavage)
103 weeks
(gavage)
103 weeks
(gavage)
103 weeks
(gavage)
16 weeks
(dietary)
16 weeks
(dietary)
Osborne-
Mendel
male rat
Osborne-
Mendel
female rat
F344
male rat
F344
female rat
F344
male rat
F344
male rat
F344
male rat
F344
male rat
F344
female rat
Tubular
nephropathy
Tubular
nephropathy
Moderate to
marked tubular
nephropathy
Mild to moderate
tubular
nephropathy
Linear
mineralization
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
0
113
227
0
113
227
0
7
14
0
57
114
0
7
14
0
7
14
0
1
15
62
0
1
15
62
0
1
15
62
0/20 (0%)
22/49a (45%)
33/50a (66%)
0/20 (0%)
9/50a(18%)
29/49a (59%)
18/50(36%)
24/50 (48%)
30/50a (60%)
12/50(24%)
25/50a (50%)
32/49a (65%)
2/50 (4%)
15/50a(30%)
32/50a (64%)
0/50 (0%)
7/50a(14%)
7/50a(14%)
0/10(0%)
1/10(10%)
7/10a(70%)
10/10a(100%)
1/10(10%)
2/10(20%)
7/10a(70%)
10/10a(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
34
67
134
268
536
0
34
67
134
268
536
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).
The short-term studies 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
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chronic durations. However, data from short-term studies in rats (NTP. 1996. 1989) were used to support
findings in the chronic (see Section 4.2.1.2) and subchronic (see Section 4.2.1.1) studies.
The subchronic exposure studies (NTP. 1989; Gorzinski etal.. 1985) and chronic exposure
studies (NTP. 1989; NCI. 1978) are well designed studies, with animals exposed to at least two HCE
doses and an unexposed control group, and examined for a wide range of toxicological endpoints in both
sexes of the rodents. One limitation of the Gorzinski et al. (1985) study is potential sublimation of HCE
from the food. While the authors did consider HCE sublimation and eating patterns when reporting the
administered doses, the potential for inhalation exposure was not considered.
Limitations of the NTP (1989) subchronic study include the lack of incidence and severity data
for the reported kidney effects in male rats and the need for additional exposure concentration(s) between
the control and low dose for better characterization of the exposure-response curve in male rats.
Limitations of the NTP (1989) chronic study are the need for additional exposure concentration(s)
between the control and low dose for better characterization of the exposure-response curve for renal
effects in male and female rats, as well as the high incidence of renal nephropathy observed in both the
control and exposure groups.
Limitations of the NCI (1978) chronic exposure study in Osborne-Mendel rats include alterations
in the dosing regimen during the study, cyclical dosing periods, and the need for additional exposure
concentration(s) between the control and low dose for better characterization of the exposure-response
curve in male and female rats. Limitations of the NCI (1978) chronic exposure study in B6C3FJ mice
include alterations in the dosing regimen during the study, cyclical dosing periods, low survival rates in
control and low dose males, and the need for additional exposure concentration(s) between the control
and low dose for better characterization of the exposure-response curve in male and female rats.
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 were not further considered for POD
determination because of the lack of incidence data for the control groups. 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 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; NCL 1978) exposure.
Further consideration was given to the renal endpoints including, atrophy and degeneration of
renal tubules in male and female 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) 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 in male rats, data from the NTP (1989)
13-week subchronic study on incidence of hepatocellular necrosis from the female rats were also
75
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considered as candidate critical effects for comparison purposes. The hepatocellular necrosis data on the
male rat liver effects were not considered for comparison purposes because incidence was not
significantly elevated above controls at any HCE dose.
5.1.2 Methods of Analysis—Including Models
The benchmark dose (BMD) modeling approach (U.S. EPA. 2000c) was employed to identify the
candidate POD following subchronic (Gorzinski et al.. 1985) and chronic (NTP. 1989; NCI. 1978) HCE
exposure (Table 5-2 and B-l). 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
(NTP. 1989; Gorzinski et al.. 1985; NCI. 1978). as well as the incidence data for hepatocellular necrosis
in female rats (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 (U.S. EPA. 2009) modeling outcomes for a BMR of 5 and 1% are also 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 the BMD modeling results of the
available kidney effects data at the 10% BMR level and the candidate PODs (BMDLio values) are
identified for each effect.
76
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Table 5-2 Summary of the BMD modeling results for the rat 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
(n = 50)
Female rats
(n = 50)
Male rats
(n = 50)
Female rats
(n = 50)
Male rats
(n = 50)
Male rats
(n = 50)
Duration
(route)
16 weeks
(dietary)
16 weeks
(dietary)
78 weeks
(gavage)
103 weeks
(gavage)
103 weeks
(gavage)
103 weeks
(gavage)
"Best-fit" model
Gamma Quantal-
linear,
and Weibull
Gamma,
Multistage 1°, and
Quantal-linear
Probit
Gamma,
Multistage 1°,
and Weibull
Multistage 2°
Probit
Gamma,
Quantal-linear,
and Weibull
Probit
Log Logistic
BMD
(mg/kg-
day)
1.22
1.34
16.10
21.23
80.63
3.81
15.17
3.98
7.05
BMDL10
(mg/kg-
day)
0.710
0.728
10.51
16.99
41.89
2.60
10.72
3.22
4.48
The most sensitive effect observed in male rats exposed to HCE was slight hypertrophy and/or
dilation of proximal convoluted renal tubules (Gorzinski et al.. 1985): however, 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. The tubular nephropathy in male rats observed in the chronic
exposure studies (NTP. 1989; NCI. 1978) 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).
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.
As shown in Appendix B, the gamma, multistage 1°, logistic, probit, quantal-linear, and 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.. 1985) 16-week study (Table B-l), as assessed by a
%2goodness-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 BMDL10 was selected. These models had identical BMD10 and
77
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BMDL10 values. Therefore, the BMDL10 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 BMDLio of 0.728 mg/kg-
day 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.
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 BMDL10 of 0.728 mg/kg-day.
Based on EPA's A Review of the Reference Dose and Reference Concentration Processes (U.S. EPA.
2002; Section 4.4.5), uncertainty factors, addressing five areas of uncertainty resulting in a composite UF
of 1,000, were applied to the selected POD to derive an RfD.
• An interspecies uncertainty factor, UFA, of 10 was applied to account for
uncertainty in extrapolating from laboratory animals to humans in the absence of
information to characterize the toxicokinetic or toxicodynamic differences
between rats and humans after oral HCE exposure. 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.
• An intraspecies uncertainty factor, UFH, of 10 was applied to account for
potentially susceptible individuals in the absence of data evaluating variability of
response to oral HCE exposure in the human population.
• A subchronic-to-chronic UF (UFS) of 3 was applied. The study selected as the
principal study was the 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
chronic data (NTP. 1989: Gorzinski et al.. 1985: NCI. 1978) were available to
inform the nature and extent of effects that would be observed with a longer
duration of exposure to HCE. The chronic data identified the kidney is the target
organ of HCE toxicity, consistent with the findings from the Gorzinski et al.
(1985) study. 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. In addition,
data from the NCI (1978) chronic study suggested that an increase in duration of
HCE exposure may not increase the incidence of nephropathy. However, the
lowest dose tested in the chronic exposure studies (NTP. 1989: NCI. 1978)
represented a LOAEL, limiting the ability of these studies to inform the impact of
increased exposure duration on renal effects observed at the lowest dose in the
subchronic study (Gorzinski et al.. 1985). For these reasons, aUFs of 3 was used
to account for extrapolation from subchronic-to-chronic exposure duration.
• A LOAEL to NOAEL uncertainty factor, UFL of 1 was applied because the
current approach is to address this factor as one of the considerations in selecting
a BMR for BMD modeling. In this case, a BMR of a 10% increase in the
78
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incidence of renal tubule atrophy and degeneration was selected under an
assumption that it represents a minimal biologically significant change.
• A database uncertainty factor, UFD of 3was applied to account for database
deficiencies due to 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.
The chronic RfD of 7 x 10~4 mg/kg-day for HCE was calculated as follows:
RfD = BMDL10 - 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
UF values and potential reference values. 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. Figure 5-1 provides a graphical display of dose-response information from three
studies considered in the selection of a POD for the oral RfD. As discussed in Section 5.1.1 and Section
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 derived from the other studies are presented for comparison
purposes.
79
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Table 5-3 Potential PODs for nephrotoxicity in male rats with applied UF values and potential
reference values
Tubular nephropathy;
BMDL(111-112week)
Potential PODs
(mg/kg-day)
16.99
Total
UF
300
UFA
10
UFH
10
UFS
1
UFD
3a
Potential
reference
values
(mg/kg-day)
0.0566
Reference
NCI (1978)
Hyperplasia of pelvic transitional
epithelium; BMDL (103 week)
4.48 300 10 10 1
Linear mineralization;
BMDL (103 week)
3.22 300 10 10 1
Moderate to marked tubular
nephropathy; BMDL (103 week)
2.60 300 10 10 1
0.0149
0.0107
0.0087
NTP (1989)
Slight hypertrophy and/or dilation of
proximal convoluted renal tubules;
BMDL (16-week)
0.710 1,000 10 10 3a 3a
Atrophy and degeneration
of renal tubules;
BMDL (16-week)
0.728 1,000 10 10 3a 3a
0.0007
Gorzinski et al.
(1985)
0.0007
a3:(10'/2 = 3.16, rounded to 3)
80
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1 -
II IHKtl
,
§$
1
t#*
»»*
. p
HIM
**H|
**«
^
1
!***
C^
**
'»*
^r
|
W l*tii
Increased
incidence of
moderate to
marked tubular
nephropathy
BMDL
(NTP, 1989)
103 week
Increased
incidence of
hyperplasia
of the pelvic
transitional
epithelium
BMDL
(NTP. 1989)
103 week
Increased
incidence of
linear
mineralization
BMDL
(NTP. 1989)
103 week
Increased
incidence of
tubular
nephropathy
BMDL
(NCI. 1978)
78 week
Increased
incidence of
slight
hypertrophy
and/or dilation
of convoluted
renal tubules
BMDL
(Gorzinski et
al.. 1985)
16-week
Increased
incidence of
atrophy and
degeneration
of tubules
BMDL
(Gorzinski et
al.. 1985)
16-week
Figure 5-1 Array of potential PODs with applied UF values and potential reference values
for nephrotoxic effects in male rats, from the three studies in Table 5-3.
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. 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. In 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 EPA practices and guidance (U.S. EPA. 2002). an additional uncertainty factor
was applied to account for database deficiencies.
81
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5.1.6 Confidence in the RfD
Confidence in the principal study (Gorzinski etal.. 1985). is high. The 16-week diet study was a
well-conducted study that used three dose groups plus a control. NTP (1989) also conducted 16-day, 13-
week, and 103-week gavage studies that supported the results observed in the 16-week diet study.
Application of BMD modeling provided a POD upon which to base the derivation of the RfD. The critical
effect on which the RfD was based is supported by other oral short-term, subchronic, and chronic studies.
Confidence in the database was low to medium because the database included acute, short-term,
subchronic, and chronic toxicity studies and developmental/teratogenic 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.
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. Data on HCE-induced human
health effects are limited and confounded by co-exposure (e.g., HCE-zinc oxide smoke). Studies observed
HCE exposure in smoke bomb production workers, but the sample sizes were too small to provide
definitive conclusions on health effects. No chronic inhalation exposure studies were available, and only a
single subchronic inhalation study in animals was identified. Weeks et al. (1979) exposed Sprague-
Dawley rats, male Beagle dogs, male Hartley guinea pigs, and Japanese quail to HCE, and examined a
number of endpoints including toxicology, neurotoxicity, pulmonary, and teratology (see Section 4.2.2,
Section 4.3.2, Section 4.4.1.2, Section 4.4.3.2, and Section 4.4.4). The 6-week subchronic inhalation
study by Weeks et al. (1979) was considered for derivation of an RfD.
Weeks et al. (1979) was a well-conducted subchronic bioassay which used three concentrations of
HCE plus an unexposed control, and incorporated a variety of endpoints across a range of species. 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 post-exposure.
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 exposure concentration would have allowed for better
characterization of the exposure-response curve. As the only repeat exposure study available, the Weeks
et al. (1979) study was selected as the principal study for the derivation of the RfC.
82
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Weeks et al. (1979) identified excess mucopurulent exudate, upper and lower respiratory tract
irritation, statistically significant decreases in body weight gain, and neurotoxicity following inhalation
exposure to HCE (see Table 5-4). The responses were generally observed only following exposure to the
highest concentration. Body weight changes, pulmonary effects, and neurological effects were also
reported following acute exposure to HCE (Weeks and Thomasino. 1978).
Table 5-4 Noncancerous effects observed in animals exposed to HCE via inhalation
Species
Sprague-Dawley rats
(25/sex/dose)
Subchronic
Male Beagle dogs (4/dose)
Subchronic
Male Hartley guinea pigs
(10/dose) Subchronic
Pregnant Sprague-Dawley
rats (22/dose)
Teratogenic/
Developmental
Dose/
duration
0,
145,
465, or
2,517
mg/m3
6 weeks
0, 145,
465,
or 2,517
mg/m3
CDs 6-1 6
(11 days)
NOAEL
(mg/m3)
465
465
465
Maternal:
465
LOAEL
(mg/m3)
2,517
2,517
2,517
Maternal:
2,517
Effect
Males: neurotoxic effects (tremors and
ruffled pelt), reduced body weight gain,
increased relative, spleen, and testes
weights
Non-pregnant 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
Maternal: neurotoxic effects (tremors
[GD12-16]); reduced body weight gain
Developmental: no effects
Source: Weeks et al. (1979).
Weeks et al. (1979) attributed the increased incidence of respiratory lesions in rats to an endemic
mycoplasma infection, based on 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) in male and female rats. Lesions characteristic
of respiratory mycoplasmosis in rodents were also detected in the oral developmental/teratogenic study in
pregnant rats. The presence of mycoplasma infection in the rats in both the oral and inhalation studies and
in the controls of the oral study suggested that respiratory tract effects were due to a potentiation of the
underlying infection rather than a result of HCE exposure.
Reduced weight gain in the rats could also be related to mycoplasma, as infected rodents
generally gain less weight or lose weight compared with noninfected rodents (Xu et al.. 2006; Sandstedt
etal.. 1997). Reduced weight gain was also observed in guinea pigs, but mycoplasma infection was not
reported (Weeks etal.. 1979). Like rats and mice, guinea pigs can carry the mycoplasma organism;
however, they are not clinically affected (Foxet 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.
83
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As discussed in Section 4.4.3, neurobehavioral effects were consistently observed in the rats and
dogs exposed to HCE. The male and non-pregnant female rats exhibited tremors and ruffled pelt. The
inhalation-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. Considering the consistent
observation of neurotoxic effects across experiments in rats and dogs, neurotoxic effects were selected as
the critical effect for determination of the POD for the RfC.
5.2.2 Methods of Analysis—Including Models
The subchronic Weeks et al. (1979) inhalation study included three exposure groups (145, 465,
and 2,517 mg/m3) plus a control. Neurological effects were observed in male and non-pregnant 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, the
NOAEL of 465 mg/m3 served as the POD. Although the NOAELs for neurological effects in dogs and
rats are the same, the male and female rats were selected as the study animals upon which to base the
POD because the pregnant dams in the teratology study were only exposed for 11 days and only four male
dogs were exposed to HCE.
The POD from this study was based on repeated, intermittent HCE inhalation exposures (in male
and non-pregnant female rats) for 6 hours/day, 5 days/week, for 6 weeks. Thus, prior to deriving the RfC,
this POD was adjusted for continuous exposure (24 hours/day, 7 days/week). The duration-adjusted POD
(POD[ADi]) was derived using the following equation (U.S. EPA. 1994a):
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)
= 83.0 mg/m3
The Methods for Derivation of Inhalation Reference Concentrations and Application of
Inhalation Dosimetry (referred to as the RfC Methodology) recommends converting the POD[ADi] to a
human equivalent concentration (HEC) (U.S. EPA. 1994a). 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
84
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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 (1994a). 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)H.
PODpEcj = POD[ADJ] x (Hb/g)A/(Hb/g)H
However, the animal and human blood:gas partition coefficients for HCE are not known. In
accordance with the RfC Methodology (U.S. EPA. 1994a) when the partition coefficients are unknown or
(Hb/g)A is greater than (Hb/g)H, a RGDR of 1 is used. The partition coefficients were unknown for HCE;
resulting in a POD[HEC] 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. Based on EPA's A Review of the Reference Dose and
Reference Concentration Processes (U.S. EPA. 2002; Section 4.4.5), uncertainty factors, addressing five
areas of uncertainty resulting in a composite UF of 3,000, were applied to the selected POD to derive an
RfC.
• An interspecies uncertainty factor, UFA, of 3 was applied to account for
uncertainty in extrapolating from laboratory animals to humans in the absence of
information to characterize the toxicodynamic differences between rats and
humans after oral HCE exposure. This value is adopted by convention, where an
adjustment from an animal-specific PODADJ to a PODnEc has been incorporated
as described in the RfC methodology (U.S. EPA. 1994a).
• An intraspecies uncertainty factor, UFH, of 10 was applied to account for
potentially susceptible individuals in the absence of data evaluating variability of
response to oral HCE exposure in the human population.
• A subchronic-to-chronic, UFS, of 10 was applied to account for extrapolation
from a subchronic exposure duration study to a chronic RfD. The study selected
85
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as the principal study was a 6 week study by Weeks et al. (1979). No chronic
inhalation studies were identified for HCE; therefore, there were no data to
inform the effects that might be observed with increased exposure duration.
• A LOAEL to NOAEL uncertainty factor, UFL, of 1 was applied because this
assessment utilized a NOAEL as the POD.
• A database uncertainty factor, UFD, of 10 was applied 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 guinea pigs,
and quail. The same investigators performed a developmental/teratogenic study
and an acute study (single 6 or 8 hour inhalation exposures) in rats. Although
maternal toxicity was reported in the developmental/teratogenic study, fetuses of
HCE-exposed dams did not exhibit any significant skeletal or soft tissue
anomalies. The toxic effects observed in the dams in the
developmental/teratogenic study (11-day exposure) were similar to those
observed in the rats exposed for 6 weeks, although additional effects were
observed in the rats exposed for the longer duration. 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 demonstrating
neurotoxicity in rats and dogs.
The chronic RfC of 3 x 10~2 mg/m3 for HCE was calculated as follows:
RfC = NOAEL[HEc] - UF
= 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 was 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. Because of the consistent observation across species, neurotoxic
effects were considered the most sensitive effects and were selected to serve as the basis for the derivation
of the RfC for HCE (Weeks etal.. 1979). Based on the lack of alternative endpoints that could be
considered for the basis of the RfC, a graphical display of dose-response information from the subchronic
inhalation study was not provided.
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.
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However, no discussion was presented in the 1987 IRIS Summary describing why this study was not used
to develop an RfC.
5.2.6 Confidence in the RfC
Confidence in the principal study, Weeks et al. (1979). is low. The study was limited by the
relatively short exposure duration (6 weeks) and minimal reporting of effects, especially quantitative
changes. Confidence in the database is low because the database included one acute and one subchronic
inhalation toxicity study in multiple species and one inhalation developmental/teratogenic toxicity study
in rats. The database lacks studies by another laboratory and a multigenerational reproductive study.
Overall confidence in the RfC is low.
5.3 Uncertainties in the Oral Reference Dose and Inhalation
Reference Concentration
The following discussion identifies uncertainties in the quantification of the RfD and RfC for
HCE beyond those discussed during application of the UFs (U.S. EPA. 1994b) to the POD values for
derivation of the RfD (see Section 5.1.3) and RfC (see Section 5.2.3).
The RfD was quantified using a BMDL10 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 that might provide adequate fit, and other models could be selected to yield different
results, both higher and lower than those included in this assessment. Uncertainty also exists in the
selection of the BMR level utilized in the BMD modeling of the critical effect 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 based on the NOAEL from a subchronic inhalation study. 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 BMD modeling.
5.4 Cancer Assessment
There were no available studies on cancer in humans associated with exposure to HCE. The
carcinogenic data reported in chronic animal studies included: (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)
87
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in male F344/N rats (NTP. 1989); and (3) statistically significant increases in the incidence of
hepatocellular carcinomas in male and female B6C3F] mice (NCI. 1978). Additionally, HCE was shown
to be a tumor promoter, but not an initiator, in an Osborne-Mendel rat liver foci assay (Milman et al..
1988; Story etal.. 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). Under the Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005b). data indicating cancer
in more than one animal species, more than one sex, and more than one site of cancer supported the
cancer descriptor "likely to be carcinogenic to humans" for HCE; even in the absence of evidence of
carcinogenicity in humans.
5.4.1 Choice of Study/Data—with Rationale and Justification
Two chronic 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 or carcinomas (combined), and pheochromocytomas/malignant pheochromocytomas and
complex pheochromocytomas (combined) in male F344 rats administered HCE for 103 weeks. Female
rats received higher doses in this study, yet 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 for 78 weeks. Male mice in this study demonstrated a dose-
response relationship, whereas female mice did not.
Both NTP (1989) and NCI (1978) were well-designed studies, conducted in both sexes of two
species with 50 animals/sex/dose. Each study utilized two dosage groups of HCE and an unexposed
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 included 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.
5.4.2 Dose-response Data
The cancer incidence data are summarized in Table 5-5. Dose-related increases were reported in
renal adenomas or carcinomas (combined) following chronic exposure to HCE (NTP. 1989). Male rats
also exhibited increased incidences of pheochromocytomas/malignant pheochromocytomas (combined),
although these increases were not dose-related (NTP. 1989). No HCE-related tumors were observed in
female rats. In male mice, dose-related increases in hepatocellular carcinomas were reported following
chronic exposure to HCE (NCI. 1978). Statistically significant increases in the incidence of hepatocellular
carcinomas were also reported in female mice following chronic exposure to HCE, although these
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increases were not dose-related (NCI. 1978). These data indicate that male rats are more sensitive to
HCE-related carcinogenicity than male or female mice.
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
B6C3F-,
mice
Female
B6C3F-,
mice
Endpoint
Kidney adenoma
or carcinoma
Pheochromocytomas/
malignant
pheochromocytomas
Hepatocellular
carcinoma
Hepatocellular
carcinoma
HCE dose
(mg/kg-day)
0
7
14
0
7
14
0
360
722
0
360
722
Incidence
1/50 (2%)
2/50 (4%)
7/50(14%)'
14/50(28%)
26/45 (58%)b
19/49(39%)
3/20 (1 5%) a
15/50(30%)'
31/49 (63%)b
2/20(10%)a
20/50 (40%)b
15/49 (3 1%)b
"Incidence data are for the matched vehicle controls rather than the pooled controls from NCI (1978),
""Denotes 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. 2011. 2005b). More specifically, animal 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\
The body weights for the laboratory animals used in the scaled human dose conversions were 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)%
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)%
= 2.05 mg/kg-day
Scaled human dose (HED) = 14 mg/kg-day x (0.471 kg/70 kg)'7'
= 4.10 mg/kg-day
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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)%
= 103.88 mg/kg-day
These scaled human doses were 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, / = 1, ..., k
and extra risk is defined as (P(d) -P(0))/(1-P(0)).
The multistage model in BMDS (U.S. EPA. 2009) was fit to the incidence data (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 unexposed 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. 2000c). No data were
excluded from the BMD multistage modeling.
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/malignant
pheochromocytomas (combined) in male rats and the incidence of hepatocellular carcinomas in female
mice. The multistage model exhibited a significant lack of fit for the pheochromocytomas (in male rats)
and the 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, only 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.
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Table 5-6 Summary of BMD modeling
Study
NTP
(1989)
NCI
(1978)
Sex/strain/
species
Male
F344
rats
Male
B6C3F-,
mice
Endpoint
Renal adenomas/
carcinomas
combined
Hepatocellular
carcinomas
results for oral cancer assessment of
~' BMRBMD,.™^"
2° Multistage 0.1 3.74 2.45
2° Multistage 0.1 38.09 13.80
HCE
• Oral slope factor
(mg/kg-day)~1
0.041
0.007
The U.S. EPA Guidelines for Carcinogen Risk Assessment (2005b) 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. 2005b). As
summarized in Section 4.7.3.1 and 4.7.3.2, the mode of carcinogenic action of HCE in the rat kidney and
mouse liver is unknown. Although data was evaluated for with the involvement of the male rat-specific
a2u-globulin mode of action in the kidney, two principal factors contributed to the conclusion that the
available data were insufficient support an a2u-globulin mode of action for the development of renal
tumors in male rats: (1) the lack of information identifying the a2u-globulin protein in HCE-exposed rats,
and (2) evidence of nephropathy in female rats as well as male and female mice (see Section 4.7.3.1).
Some data suggest that metabolism and binding in mice are involved in the development of liver tumors.
However, the role of DNA binding in the mode of action for HCE-induced hepatotoxicity and
carcinogenesis is not known. Therefore, a linear low-dose extrapolation approach was used to estimate
human carcinogenic risk associated with HCE exposure.
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 BMDL10 (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 for Carcinogen Risk
Assessment (2005b). 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 BMDL10 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 BMDL10 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)' for HCE.
In the absence of data on the carcinogenicity of HCE via the inhalation route, an inhalation unit
risk was not derived.
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5.4.5 Uncertainties in Cancer Risk Values
The largest sources of uncertainty in the HCE cancer risk estimates are interspecies extrapolation
and low-dose extrapolation. 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
Impact on oral
slope factor
Decision
Justification
Human relevance of
rodent tumor data
Human risk could J, or
t, 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 |)
Kidney and adrenal gland
tumors in male rats and liver
tumors in male and female
mice are relevant to human
exposure
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, 2005b)
Bioassay
Alternatives could f or
I oral slope factor by
an unknown extent
NTP study (1989)
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.
Species/gender choice
Human risk could f or
J,, depending on
relative sensitivity
Incidence of renal
adenoma/carcinoma in male
rats
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.
Dose metric
Alternatives could f or
4 oral slope factor by
an unknown extent
Used administered exposure 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.
Low-dose extrapolation Alternatives could f or
procedure J, oral slope factor by
an unknown extent
Multistage model to
determine POD, linear low-
dose extrapolation from
POD (default approach)
Available mode-of-action data do not
inform selection of dose-response
model; linear approach employed in
absence of support for an alternative
approach.
Cross-species scaling
Alternatives could J, or
f the oral slope factor
(e.g., 3.5-fold J, [scaling
by body weight] or f
2-fold [scaling by
BW2/3])
(default 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.
Statistical uncertainty at
POD
4 oral slope factor
1.5-fold ifBMD used as
the POD rather than
lower bound on POD
BMDL (preferred approach
for calculating reasonable
upper bound slope factor)
Limited size of bioassay results in
sampling variability; lower bound is
95% confidence interval on admin-
istered exposure.
Human population
variability in metabo-
lism and response/-
sensitive
subpopulations
Low-dose risk f or J, to
an unknown extent
Considered qualitatively
No data to support range of human
variability/sensitivity, including whether
children are more sensitive.
| = increase; [ = decrease
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5451 Sources of Uncertainty
Relevance to humans. As described in Section 4.7.3, 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. The human relevance of the renal
tumor mode of action was considered in Section 4.7.3.1. An evaluation of the available data concluded
that there were insufficient data to support an a2u-globulin mode of action for the development of renal
tumors in male rats. Additional information on key data gaps (e.g., immunohistochemical data identifying
a2u-globulin in the hyaline droplets, data on the incidence of end stage renal failure or high severe
nephropathy for controls and HCE-exposed animals, presence of foci of atypical hyperplasia, and if the
location of renal adenomas were within the areas of chronic progressive nephropathy) would inform the
human relevance of the observed kidney tumors.
The human relevance of the liver tumor mode of action was considered in Section 4.7.3.2.
Experimental animal studies have demonstrated that oral exposure to HCE induces liver tumors in male
and female mice. A potential mode of action for HCE-induced hepatocellular carcinomas in mice was 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 evaluated for HCE.
Additional data distinguishing the similarities and differences between experimental animals and humans
in terms of HCE metabolism or toxicity would inform the human relevance of the reported liver tumors.
The human relevance of the adrenal gland tumor mode of action was considered in Section
4.7.3.3. Pheochromocytomas occur in both humans and rats, although they are more common in
laboratory rats. 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 (Greimetal.. 2009). The relevance
of rodent pheochromocytomas as a model for human cancer risk has been the subject of discussion in the
scientific literature (Greim et al., 2009; Powers et al., 2008). Additional data distinguishing the
similarities and differences between pheochromocytoma induction in animals and humans would inform
the human relevance of the reported adrenal gland tumors.
Bioassay selection. Of the two chronic animal bioassays selected for BMD analysis and
subsequent quantitative cancer assessment, the NTP (1989) study was used for the development of an oral
slope factor because male rats exhibited greater sensitivity to HCE-induced carcinogenicity than mice.
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 were 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. Evidence suggesting the kidney is a target organ of HCE toxicity in both species lends
strength to the concern for human carcinogenic potential.
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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. In the absence of mode of action
information to inform the dose-response at low doses, a linear-low-dose extrapolation approach was used
to estimate human carcinogenic risk associated with HCE exposure. The overall uncertainty in low-dose
risk estimation would be reduced if the mode of action for HCE was more fully characterized.
Etiologically different tumor types were not combined across sites prior to modeling, 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. 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 additional mechanistic data, the human
relevance of the rodent cancer results 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 suggested that HCE is potentially carcinogenic to humans. 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, a linear low-dose extrapolation
was carried out from the BMDLi0. 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. 2005b) 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 BMDL values 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%
95
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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. 2005b). The ratios of the BMDio values to the
BMDL10 values did not exceed a value of 2.6, indicating that the estimated risk was 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 U.S. EPA
Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2011. 2005b). 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 carcinogenicity 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 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 1987 HCE cancer assessment was based on the incidence of hepatocellular
carcinomas in male mice in the NCI (1978) study. The current risk value was derived from the incidence
of renal adenomas or carcinomas in male rats (NTP. 1989) and resulted in an oral slope factor
approximately 2.8-fold higher than the previous assessment.
In addition, the scaled human doses were previously calculated using a slightly different formula
than is current practice:
Scaled human dose = animal dose x (animal weight/human body weight) 1/3 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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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; Town and Leibman. 1984; Nastainczvk et al.. 1982a; Nastainczyk et al..
1982b; Salmon et al.. 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
etal.. 1982a; Nastainczvk et al.. 1982b; 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.
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
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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 (2005b), 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 in 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 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
98
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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. 2005b). 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 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.
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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 B6C3F]
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 Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005b). 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 4 x ICT^mg/kg-day)"1 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 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 (U.S. EPA. 2011. 2005b) 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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APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW
AND PUBLIC COMMENTS AND DISPOSITION
The Toxicological Review of Hexachloroethane (dated May, 2010) has undergone a formal
external peer review performed by scientists in accordance with EPA guidance on peer review (U.S. EPA.
2006a). An external peer-review workshop was held September 21, 2010. The external peer reviewers
were tasked with providing written answers to general questions on the overall assessment and on
chemical-specific questions in areas of scientific controversy or uncertainty. A summary of significant
comments made by the external reviewers, and EPA's responses to these comments arranged by charge
question follow. In many cases the comments of the individual reviewers have been synthesized and
paraphrased in the 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 (NTP. 1989). One reviewer commented that the available data on renal cancer following
A-l
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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.
Response The Toxicological Review in general, and Section 5 in particular, have been revised to include more
synthesis and less repetition in the text to improve clarity and conciseness.
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 A-4 and Charge Question B-4.
Text has been revised in Section 5.1.1 to clarify the selection of the principal study for the derivation
of the RfD. The effect of sublimation on dietary HCE exposure has been considered in Gorzinski et
al. (1985): however, potential inhalation effects from sublimation were not discussed by the study
authors. The potential for inhalation effects from sublimation has been identified as a weakness in the
study in Section 5.1.1. 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 provide incidence data for the kidney effects and administered higher doses of HCE than the
subchronic study (Gorzinski etal. 1985) 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 were discussed in Section 4.6.3 and Section 4.7.3.1. EPA concluded
that HCE-related effects in male and female rats indicated that chronic progressive nephropathy is not
solely responsible for the reported effects. In addition, EPA concluded that there is insufficient
evidence to attribute the kidney effects of HCE exposure to an a2u-globulin mode of action. Therefore,
there was insufficient data to determine if the HCE-related nephropathy results from the exacerbation
of chronic progressive nephropathy by a2u-globulin accumulation.
The evidence needed to attribute the kidney effects of HCE exposure to an a2u-globulin mode of
action were outlined in Section 4.7.3.1. Although there were 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 prevented attributing the kidney
effects of HCE exposure to an a2u-globulin mode of action. Text was modified in Section 4.6.3,
Section 5.4.3, and Section 5.4.5.1 to reiterate the conclusion reached in Section 4.7.3.1 that the
available data were 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.
A-2
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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 on 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 [Hemmila et al., Mutat. Res 701(2), 137-144] 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 was 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 were unavailable.
Section 4.6.3 and 4.7.3.1 discussed both chronic progressive nephropathy and potential for the
exacerbation of chronic progressive nephropathy by a2u-globulin accumulation. EPA concluded that
(1) chronic progressive nephropathy was not solely responsible for the reported kidney effects of
HCE exposure and (2) lack of immunohistochemical data prevented attributing the kidney effects of
HCE exposure to an a2u-globulin mode of action. Therefore, the available data was 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. EPA appreciates the identification of this review
article; however the review describes other primary literature demonstrating exacerbation of chronic
progressive nephropathy following exposures to other chemicals. In the case of HCE, nephropathy
was also observed in female rats (NTP. 1989). as well as in male and female mice (NCI. 1978).
Changes in severity of the nephropathy were also observed to be greater in male rats exposed to HCE
compared to controls, indicating that HCE exposure exacerbated effects in the kidney of male rats.
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). The study recommended by the peer reviewer does not
specifically address HCE-induced renal toxicity and therefore was not included in the Toxicological
Review.
The human health effects of pyrotechnic munition smokes were briefly considered in Section 4.1.
These studies (Seldenetal.. 1994; SeldenetaL 1993) demonstrated HCE exposure in the smoke
bomb production workers, but the sample sizes of the health effects studies were 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, the study
recommended by the peer reviewer is unlikely to provide insight into HCE-induced toxicity and was
not included in the Toxicological Review.
A-3
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Chemical Specific Charge Questions
Oral Reference Dose (RfD)for HCE
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) study 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 16 week subchronic study (Gorzinski etal.. 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 doses utilized in the subchronic study which
reported statistically significant increases in kidney effects are consistent with the BMDL10 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. 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, U.S. EPA selected the Gorzinski et al. (1985) study as the principal study for derivation of
the RfD because kidney effects were observed in male rats at doses below the range of exposure
tested in the NTP (1989) study and the tubular nephropathy in male rats in the chronic exposure
studies (NTP. 1989; NCI. 1978) resulted in higher PODs than the subchronic study (Gorzinski et al..
1985).
A-4
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Study duration was considered during the application of uncertainty factors, as described in Section
5.1.3.
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 was 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 A-4.
Section 4.7.3.1 discussed chronic progressive nephropathy and acknowledged 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 suggested that chronic progressive
nephropathy was not solely responsible for the reported renal effects.
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?
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.
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Response EPA concluded that while there were data to indicate that the mode of action for renal effects may be
specific for male rats, there were 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 stated that the
application of an uncertainty factor of 10 to account for subchronic-to-chronic extrapolation was
debatable. Specifically, this reviewer provided a comparison of RfDs derived from both the chronic
and subchronic data. This reviewer stated that if EPA were to apply a composite UF of 300 (keeping
the currently applied UFs, except 1 for subchronic-to-chronic extrapolation) to the subchronic
Gorzinski et al. (1985) study and a composite UF of 1000 (10 for inter- and intraspecies differences,
10 for LOAEL to NOAEL extrapolation, 1 for subchronic-to-chronic extrapolation, and 3 for database
deficiencies) to the chronic NTP (1989) study the RfDs would be the same . 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 a NOAEL. One reviewer
commented that the BMDL10 is more reflective of a LOAEL than a NOAEL and, therefore, suggested
application of an uncertainty factor of 3 for LOAEL-to-NOAEL extrapolation.
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
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considerations of related chemicals and metabolites. One recommended either not applying the
database uncertainty factor or equivalently applying a database uncertainty factor of 1. This reviewer
stated that in practice either the database is adequate to derive an RfD or it is inadequate and no RfD
is developed. This reviewer also suggested that the toxic effects observed in the developmental
toxicity studies for HCE were at higher doses than the doses that induce renal toxicity in the
subchronic and chronic studies.
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 than the standard subchronic (90-day)
study and falls well short of a standard lifetime study (i.e., two year chronic bioassay), although
chronic data were available for comparison. These chronic data suggest: (1) incidence of nephropathy
may not increase with prolonged exposure and (2) consistency in dose response relationships with the
subchronic studies. However, the lowest dose tested in the chronic exposure studies (NTP. 1989: NCI.
1978) represented a LOAEL, limiting the ability of these studies to inform the impact of increased
exposure duration on renal effects observed at the lowest dose in the subchronic study (Gorzinski et
al.. 1985). Reduction of the subchronic-to-chronic uncertainty factor resulted in a composite
uncertainty factor of 1,000. 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.1
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 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.
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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) study 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 1987 IRIS assessment. 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 concluded that the Gorzinski et al. (1985) study is a better basis for the oral RfD. The 1987
IRIS Summary did 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 Section 5.2.5.
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 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 etal.. 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
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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 were 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) study 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. Text has been modified in Section 5.2.1 to clarify these conclusions.
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) reported 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 be relevant 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 NO AEL approach was justified for deriving the POD for the RfC.
Two reviewers recommended clarification that it was the lack of individual 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 also been modified to clarify the gas categories for deriving a human equivalent
concentration and the classification of HCE as a Category 3 gas.
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Charge Question E.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 addressed 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 remaining three reviewers recommended a database uncertainty
factor of 3. One of the reviewers stated that the available toxicity studies were sufficiently diverse and
supported a database uncertainty factor of 3. Another reviewer commented that arguments could be
made to support both an uncertainty factor of 3 for the database limitations as well as a database
uncertainty factor of 10. Specifically, this reviewer recommended that EPA describe the key study
types and remaining uncertainty in greater depth. This reviewer stated that a factor of 3 is sufficient
given the lack of a multi-generation reproductive toxicity study. However, this reviewer believed that
an argument could be made for the application of a factor of 10 due to the observed neurotoxicity
(tremors) and lack of evaluation of more sensitive measures of neurological effects at the NOAEL
(citing lack of post exposure sacrifice and histopathology). A third reviewer believed that even though
a chronic inhalation study was not available, the availability of a developmental study supports an
uncertainty factor of 3. Another reviewer recognized 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.
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Response As described in Section 5.2.3, an interspecies uncertainty factor of 3 is applied when incorporating an
animal-specific NOAELMjj to a human equivalent NOAELHEc 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 was 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 also 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) was a subchronic inhalation bioassay that evaluated an array of endpoints and
established NOAELs and LOAELs. The Weeks et al. (1979) study represents the minimum database
for deriving an RfC. In applying the database uncertainty factor, Section 5.2.3 indicated the
deficiencies for the inhalation database. Specifically, the database is lacking a long-term study, a
multigeneration reproductive toxicity study, and neurotoxicity and developmental neurotoxicity
studies. Because of these data gaps, a database uncertainty factor of 10 was applied for the RfC
derivation. Text was 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 NOAEL/LOAEL
approach versus BMD modeling) was taken into account in the application of the individual
uncertainty factors.
C. Carcinogenicity ofHCE
Charge Question C.1
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 Five of the six reviewers agreed with the cancer descriptor "likely to be carcinogenic to humans."
Several reviewers provided qualifying comments related to the carcinogenic mode of action and
potential human relevance. One reviewer suggested that the narrative should capture the uncertainties
associated with the cancer descriptor including more discussion of the human relevance of the kidney
tumors and pheochromocytomas in the male rats and liver tumors in mice. This reviewer stated that
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while the data indicate that HCE is "likely to be carcinogenic to humans," the weight of the evidence
is on the low end of the spectrum for this descriptor. Another reviewer stated that the classification of
HCE as "likely carcinogenic to humans" appeared to be excessive, but when evaluating the data and
the U.S. EPA Guidelines for Carcinogen Risk Assessment the descriptor was inevitable.
One of the six reviewers did not comment specifically on the choice of the cancer descriptor but
stated that in his opinion both an a2u-globulin mechanism and chronic progressive nephropathy are
involved in kidney tumor development, the mechanism for liver tumor development appears to be
unknown, and the relevance of the increase in pheochromocytomas at low dose was questionable
because the response was not dose-related.
A reviewer 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, if 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.
Response EPA agrees with the majority of the reviewers that the cancer descriptor of "likely to be carcinogenic
to humans" is an appropriate characterization of the weight of the evidence and is in accordance with
the Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a). Text in Section 4.7.1 was revised
to discuss uncertainties in the data, including the insufficient evidence for an a2u-globulin mode of
action for the kidney effects, as well as the human relevance of the kidney, adrenal gland, and liver
tumors. Additionally, text related to these and other uncertainties is discussed in Section 5.4.5 and
summarized in Table 5-7.
The specific comments on the mode of action for kidney, liver and adrenal tumors are addressed in
the response to Charge Question C.4. Regarding the pheochromocytoma dose-response comment,
these tumors were 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.
Chronic progressive nephropathy and the potential exacerbation of chronic progressive nephropathy
by a2u-globulin accumulation were 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 were 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 was revised to indicate the additional data gaps that would
inform the human relevance of the kidney tumors.
EPA acknowledges that additional mechanistic data, as well as additional human carcinogenicity data,
would inform the uncertainty in the human relevance of renal and adrenal tumors following HCE
exposure of the observed tumors and potentially impact selection of the cancer descriptor.
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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 lexicological 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.
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.
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 were 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 was
added to Appendix B of the Toxicological Review. Also, because the BMD modeling output
indicated 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 of HCE observed in male and female mice
confounded the determination and indicates that there may be more than one mode of action for renal
toxicity. Similarly, data were 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.
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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 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 quantitative 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 quantitative cancer assessment.
Response As discussed in Section 4.7.3.1, two principal factors contributed to the conclusion that there are
insufficient data to support an a2u-globulin mode of action for the development of renal tumors in
male rats. 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. As stated in Section 5.4.4,
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. Human relevance of the renal
tubule tumors is discussed in Section 4.7.3.1. EPA concluded that in the absence of sufficient
information demonstrating the involvement of a2u-globulin processes, male rat renal toxicity/tumors
were considered relevant for risk assessment purposes.
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 mouse hepatic tumors and pheochromocytomas
in rats in the overall cancer risk assessment of HCE, particularly in the context of determining the
cancer descriptor and the U.S. EPA Guidelines for Carcinogen Risk Assessment. This reviewer also
recommended inclusion of a discussion of the similarities and differences related to the data for renal
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proliferative lesions produced by a2u-globulin nephropathy and advanced chronic progressive
nephropathy for tetrahydrofuran.
One reviewer commended EPA on the clarity and concise presentation of Section 4.7.3.1. This
reviewer stated that EPA correctly concluded that the criteria for an a2u-globulin mode of action for
HCE-induced renal tumors were not met under EPA's guidance. This reviewer suggested that the data
do indicate a dose-response and temporal relationship for key events in an a2u-globulin mode of action
and requested that EPA include more synthesis in the dose response section (i.e., table showing at
which doses the key events in the a2u-globulin mode of action occur). This reviewer also requested
discussion of the a2u-globulin mode of action conclusions reached by several recent reviews on HCE
carcinogenicity.
Another reviewer noted that since renal tubule 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 carcinogenicity. This reviewer also commented that they did not ultimately disagree
with EPA's conclusions regarding mode of action for renal tumors, but stated that the document
should be clearer about the rationale and explanation for the conclusion. Specifically, this reviewer
suggested that EPA state that the a2u-globulin mode of action for HCE carcinogenicity may in part
explain the renal effects, but other modes of action also exist that would not exclude these tumors
from consideration in risk assessment. This reviewer did not specifically state what other modes of
action may exist.
One reviewer also disagreed with the conclusion that HCE renal carcinogenicity cannot be attributed
to an a2u-globulin mode of action, arguing that the available data provide evidence of 6 out of 7 steps
in the a2u-globulin mode of action. This reviewer commented that weight of evidence from other
chemicals structurally related to HCE support a male rat specific mode of action for HCE renal
carcinogenicity. Lastly, the 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 Section 4.7 has been revised to include additional consideration of the hepatocellular carcinomas and
pheochromocytomas in the determination of the cancer descriptor. The presence of statistically
significant increases in the incidence of pheochromocytomas/malignant pheochromocytomas
(combined) and renal tubule tumors in male F344/N rats and statistically significant increases in the
incidence of hepatocellular carcinomas in male and female B6C3F! mice provided evidence 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 (see Section 4.7.3.1). 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 (Burner et al.. 2010: Chhabra et al.. 1998). 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,
A-15
-------
the HCE dose at which a2u-globulin accumulates hyaline droplets is unknown. Consequently, there
was 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 these 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 was added to Section 4.7.3.1 to clarify that dose-response concordance
(of the accumulation of a2u-globulin in hyaline droplets) could not 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 contributed to the 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, suggested
that the HCE-induced renal tumors may not be attributable to an a2u-globulin mode of action, or that
more than one mode of action may be operating. The sex-specific differences in carcinogenic effects
of HCE exposure may also reflect sex-specific differences in the kidney concentrations of HCE
following oral exposure (see Table 3-3).
Some data suggested that the male rat-specific a2u-globulin mode of action could contribute to HCE-
induced nephropathy. As summarized in Section 4.7.3.1, the data were insufficient to support an
a2u-globulin mode of action in the development of renal tumors in male rats following HCE exposure.
Chronic progressive nephropathy was also discussed in Section 4.7.3.1. There was 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 about whether or not EPA used the matched vehicle control data when
modeling the hepatocellular carcinoma data.
Response Text was modified in the Section 5.4.3 and Table 5-5 to indicate that the matched vehicle control data
were presented and were used for BMD modeling.
A-16
-------
APPENDIX B: BENCHMARK DOSE MODELING OUTPUT
Table B-1 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 model3
p-Value
AIC
BMD10
(mg/kg-
day)
BMDL10
(mg/kg-
day)
Kidney effects
NCI (1978)
78 weeks
Gavage
NTP (1989)
103 weeks
Gavage
NTP (1989)
103 weeks
Gavage
NTP (1989)
103 weeks
Gavage
Gorzinski et al.
(1985)
16 weeks
Diet
Gorzinski et al.
(1985)
16 weeks
Diet
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
Osborne-
Mendel
Female
rat
Osborne-
Mendel
Male rat
F344
Female
rat
F344
Male rat
F344
Male rat
F344
Male rat
F344
Female
rat
F344
Male rat
F344
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
Log Logistic
Multistage 2°
Weibull
Quantal-linear
Gamma
Multistage 1°
Logistic
Probit
Quantal-linear
Weibull
Gamma
Multistage 1°
Logistic
Probit
Quantal-linear
Weibull
Gamma
Logistic
LogLogistic
LogProbit
Multistage 2°
Probit
Weibull
Quantal-linear
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
0.98
0.99
0.93
0.98
0.99
0.66
0.68
0.54
0.94
0.67
0.99
0.99
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
34.94
32.94
32.97
32.95
32.94
34.92
42.47
40.61
40.51
40.49
40.61
42.47
20.88
23.91
23.89
24.26
22.84
23.85
20.88
20.88
21.23
21.25
21.23
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
17.40
16.10
8.54
13.71
1.22
4.85
1.23
2.11
1.33
4.28
1.22
1.22
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
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
Female
Gamma
0.93
38.62
118.04
60.18
B-1
-------
13 weeks necrosis rat Multistage 1° 0.68 40.56 53.82 35.19
Gavage F344 Logistic 0.55 41.58 156.22 107.49
Probit 0.61 40.95 148.49 102.71
Weibull 0.91 38.91 114.68 56.75
aFor all models, a BMR of 0.1 was employed in deriving the estimates of the benchmark dose (BMDio) and its 95% lower CL
(BMDLio). 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 BMD10 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 BMDL10 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.
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
B-2
-------
appreciable model dependence. As the BMDL10 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 BMDL10
values did not show large variation, the model with the lowest AIC value was selected. Therefore, the
loglogistic model BMDL10 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 BMDL10 values did not show large
variation, the model with the lowest AIC value was selected. The AIC values for the gamma, multistage
1°, and quantal-linear were identical; therefore, the model with the lowest BMDL10 was selected. All of
the BMDLio values were identical for these models; therefore, the BMDL10 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 BMDL10 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 BMDL10 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 , 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 BMDL10 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.
B-3
-------
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.
Table B-2 Dose-response modeling results using BMDS (version 2.0) for BMRs of 10, 5, and 1%
based on noncancerous kidney and liver effects in rats following oral exposure to
HCE
Study
Endpoint
Sex/
species
Fitted
model
BMD10
kg-day)
BMDL10
kg-day)
BMD05
(mg/
kg-day)
BMDL05
(mg/
kg-day)
BMD0i
(mg/
kg-day)
BMDL0i
(mg/
kg-day)
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
21.23
21.25
80.63
3.81
,,17
3.98
7.05
1.34
16.10
1.22
16.99
17.01
41.89
2.60
,„.*
3.22
4.48
0.73
10.51
0.71
10
10
56
1.
7.
2.
3.
0.
8.
0.
.33
.35
.26
93
39
36
34
66
89
60
8.27
8.28
21.18
1.32
5.22
1.80
2.12
0.35
5.60
0.35
2.
2.
24
0.
1.
0.
0.
0.
1.
0.
02
03
.90
39
45
58
64
13
97
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
118.04
60.18
84
.66
33.34
41
.75
8.60
B-4
-------
Modeling for Noncancer Assessment
Gamma Model
NCI (1978) Tubular Nephropathy in Male Rats
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
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
Analysis of Deviance Table
B-5
-------
Model Log(likelihood) # Param's Deviance Test d.f. P-value
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-6
-------
Gamma Multi-Hit Model with 0.95 Confidence Level
2
o
o
"o
0.7
0.6
0.5
0.4
0.3
0.2
0.1
14:55 04/09 2009
50
100
150
200
dose
Figure B-1 Male rats, Gamma model, Kidney effect (noncancerous):
Tubular Nephropathology. NCI (1978). Osborne-Mendel Strain,
78 weeks exposure by gavage.
B-7
-------
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
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -65.7706 3
B-8
-------
Fitted model -65.8277 1 0.114158 2 0.9445
Reduced model -82.1514 1 32.7616 2 <.0001
AIC: 133.655
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.0000 0.000 0.000 20 0.000
113.0000 0.4289 21.015 22.050 49 0.299
227.0000 0.6754 33.772 33.000 50 -0.233
= 0.14 d.f. = 2 P-value = 0.9307
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 21.2541
BMDL = 17.0107
BMDU = 26.9612
Taken together, (17.0107, 26.9612) is a 90 % two-sided confidence
interval for the BMD
B-9
-------
Multistage Model with 0.95 Confidence Level
50
09:0909/142006
100
150
dose
200
250
300
Figure B-2 Male rats, Multistage 1° model, Kidney effect (noncancerous):
Tubular Nephropathology. NCI (1978). Osborne-Mendel Strain,
78 weeks exposure by gavage.
B-10
-------
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
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
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
Full model -65.7706 3
Fitted model -65.8419 1 0.142715 2 0.9311
Reduced model -82.1514 1 32.7616 2 <.0001
B-ll
-------
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
-------
Weibull Model with 0.95 Confidence Level
50
09:1309/142006
100
150
dose
200
250
300
Figure B-3 Male rats, Weibull model, Kidney effect (noncancerous):
Tubular Nephropathology. NCI (1978). Osborne-Mendel Strain,
78 weeks exposure by gavage.
B-13
-------
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.
Analysis of Deviance Table
B-14
-------
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -56.7357 3
Fitted model -57.0429 1 0.614339 2 0.7355
Reduced model -74.4688 1 35.466 2 <.0001
AIC: 116.086
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.0000 0.000 0.000 20 0.000
113.0000 0.1869 9.346 9.000 50 -0.125
227.0000 0.5661 27.741 28.910 49 0.337
ChiA2 = 0.13 d.f. = 2 P-value = 0.9374
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 80.6338
BMDL = 41.8864
BMDU = 93.2552
Taken together, (41.8864, 93.2552) is a 90 % two-sided confidence
interval for the BMD
B-15
-------
Multistage Model with 0.95 Confidence Level
50
100
150
dose
200
250
09:21 09/142006
Figure B-4 Female rats, Multistage 2° model, Kidney effect
(noncancerous): Tubular Nephropathology. NCI (1978).
Osborne-Mendel Strain, 78 weeks exposure by gavage.
300
B-16
-------
NTP (1989) Male Rat Nephropathy
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 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 -0.78
slope -0.78 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
intercept -0.35763 0.165052 -0.681127 -0.0341335
slope 0.0436991 0.0182219 0.00798493 0.0794134
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -100.939 3
Fitted model -100.939 2 0.000120944 1 0.9912
B-17
-------
Reduced model -103.852 1 5.82641 2 0.0543
AIC: 205.878
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.3603 18.016 18.000 50 -0.005
7.0000 0.4794 23.968 24.000 50 0.009
14.0000 0.6003 30.016 30.000 50 -0.005
ChiA2 = 0.00 d.f. = 1 P-value = 0.9912
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 3.81407
BMDL = 2.59812
B-18
-------
Probit Model with 0.95 Confidence Level
2
o
o
"o
0.7
0.6
0.5
0.4
0.3
0.2
13:27 04/08 2009
10 12
14
dose
Figure B-5 Male rats, Probit model, Kidney effect (noncancerous): Tubular
Nephropathology (moderate to marked). NTP (1989). F344
Strain, 103 weeks exposure by gavage.
B-19
-------
NTP (1989) Female Rat Nephropathy
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[slope*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 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 -0.55
Slope -0.55 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0.242452 0.0592711 0.126283 0.358621
Slope 0.00694477 0.0016862 0.00363988 0.0102497
Power 1 NA
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
B-20
-------
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
Reduced model -102.85 1 17.8276 2 0.0001345
AIC: 191.904
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.2425 12.123 12.000 50 -0.040
57.0000 0.4901 24.504 25.000 50 0.140
114.0000 0.6568 32.182 31.850 49 -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-21
-------
Gamma Multi-Hit Model with 0.95 Confidence Level
2
o
o
"o
0.7
0.6
0.5
0.4
0.3
0.2
0.1
10:19 04/10 2009
100
Figure B-6 Female rats, Gamma model, Kidney effect (noncancerous):
Tubular Nephropathology (mild to moderate). NTP (1989). F344
Strain, 103 weeks exposure by gavage.
B-22
-------
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
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 -0.55
Slope -0.55 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0.242451 0.0592711 0.126282 0.358621
Slope 0.00694478 0.0016862 0.00363989 0.0102497
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
Reduced model -102.85 1 17.8276 2 0.0001345
AIC: 191.904
B-23
-------
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size
Residual
0.0000 0.2425 12.123 12.000 50 -0.040
57.0000 0.4901 24.504 25.000 50 0.140
114.0000 0.6568 32.182 31.850 49 -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
2
~o
p
<
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
Figure B-7 Female rats, Quantal-linear model, Kidney effect
(noncancerous): Tubular Nephropathology (mild to moderate).
NTP (1989). F344 Strain, 103 weeks exposure by gavage.
B-24
-------
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 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 -0.55
Slope -0.55 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0.242451 0.0592711 0.126282 0.358621
Slope 0.00694478 0.0016862 0.00363989 0.0102497
Power 1 NA
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
Analysis of Deviance Table
B-25
-------
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
Reduced model -102.85 1 17.8276 2 0.0001345
AIC: 191.904
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.2425 12.123 12.000 50 -0.040
57.0000 0.4901 24.504 25.000 50 0.140
114.0000 0.6568 32.182 31.850 49 -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
-------
Weibull Model with 0.95 Confidence Level
2
o
o
"o
0.7
0.6
0.5
0.4
0.3
0.2
0.1
10:34 04/10 2009
100
Figure B-8 Female rats, Weibull model, Kidney effect (noncancerous):
Tubular Nephropathology (mild to moderate). NTP (1989). F344
Strain, 103 weeks exposure by gavage.
B-27
-------
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 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 -0.87
slope -0.87 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
intercept -1.62793 0.244257 -2.10666 -1.14919
slope 0.144885 0.0238239 0.0981906 0.191579
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -71.6113 3
Fitted model -71.8283 2 0.433989 1 0.51
B-28
-------
Reduced model -94.7689 1 46.3152 2 <.0001
AIC: 147.657
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.0518 2.589 2.000 50 -0.376
7.0000 0.2697 13.485 15.000 50 0.483
14.0000 0.6556 32.780 32.000 50 -0.232
ChiA2 = 0.43 d.f. = 1 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
B-29
-------
Probit Model with 0.95 Confidence Level
2
o
o
"o
0.7
0.6
0.5
0.4
0.3
0.2
0.1
14:24 04/08 2009
10
12
14
dose
Figure B-9 Male rats, Probit model, Kidney effect (noncancerous): Linear
minerialization. NTP (1989). F344 Strain, 103 weeks exposure
by gavage.
B-30
-------
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 1989 - Male Rat - Hyperplasia - LogLogistic Model
The form of the probability function is:
P[response] = background+(1-background)/[1+EXP(-intercept-slope*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.
Analysis of Deviance Table
B-31
-------
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -40.4963 3
Fitted model -41.2103 1 1.42796 2 0.4897
Reduced model -46.5274 1 12.0622 2 0.002403
AIC: 84.4207
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.0000 0.000 0.000 50 0.000
7.0000 0.0993 4.966 7.000 50 0.962
14.0000 0.1807 9.034 7.000 50 -0.748
ChiA2 = 1.48 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
B-32
-------
Log-Logistic Model with 0.95 Confidence Level
o
o
0.3
0 25
0.2
0.15
0.1
0.05
14:2608/122009
Log-Logistic
BMDL
BMD
10 12
14
dose
Figure B-10 Male rats, Log-logistic model, Kidney effect (noncancerous):
Hyperplasia of the pelvic transitional epithelium. NTP (1989).
F344 Strain, 103 weeks exposure by gavage.
B-33
-------
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
Gamma AtropyandDegenRenalTubulesDataNoSeverityMaleRat.dax
The form of the probability function is:
P[response]= background+(1-background)*CumGamma[slope*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
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0.110626 0.107207 -0.0994949 0.320747
Slope 0.0787607 0.0846932 -0.0872348 0.244756
Power 1.00164 1.07041 -1.09632 3.0996
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model
Fitted model
Reduced model
-14.3635
-14.4712
-27.7259
0.215359
26.7248
0.6426
<.0001
B-34
-------
AIC:
34.9424
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size
Residual
0.0000 0.1106
1.0000 0.1777
15.0000 0.7265
62.0000 0.9932
1.106 1.000 10 -0.107
1.777 2.000 10 0.185
7.265 7.000 10 -0.188
9.932 10.000 10 0.261
ChiA2 =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
0.8
06
0.4
0.2
HMDL
BMD
Gamma Multi-Hit Model with 0.95 Confidence Level
Gamma Multi-Hit
10
20
30
dose
40
50
60
::59 10/082009
Figure B-11 Male rats, Gamma model, Kidney effect (noncancerous):
Atrophy and degeneration of renal tubules. Gorzinski et al.
(1985). F344 Strain, 16 weeks exposure by diet.
B-35
-------
Multistage 1° 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
MS1° AtropyandDegenRenalTubulesDataNoSeverityMaleRat.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
Full model -14.3635 4
Fitted model -14.4712 2 0.215361 2 0.8979
B-36
-------
Reduced model -27.7259 1 26.7248 3 <.0001
AIC: 32.9424
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.1105 1.105 1.000 10 -0.106
1.0000 0.1778 1.778 2.000 10 0.184
15.0000 0.7266 7.266 7.000 10 -0.189
62.0000 0.9932 9.932 10.000 10 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
BMDU = 2.66189
Taken together, (0.727509, 2.66189) is a 90 % two-sided confidence
interval for the BMD
B-37
-------
Multistage Model with 0.95 Confidence Level
o
o
0.6
0.4
0.2
0 -
09:00 10/082009
Multistage
10
30
dose
40
50
00
Figure B-12 Male rats, Multistage 1° model, Kidney effect (noncancerous):
Atrophy and degeneration of renal tubules. Gorzinski et al.
(1985). F344 Strain, 16 weeks exposure by diet.
B-38
-------
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
QL AtropyandDegenRenalTubulesDataNoSeverityMaleRat.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
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0.11052 0.0819804 -0.0501583 0.271199
Slope 0.0786399 0.0310542 0.0177749 0.139505
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model
Fitted model
Reduced model
AIC:
-14.3635
-14.4712
-27.7259
32.9424
0.215361
26.7248
0.8979
<.0001
B-39
-------
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size
Residual
0.0000 0.1105
1.0000 0.1778
15.0000 0.7266
62.0000 0.9932
1.105 1.000 10 -0.106
1.778 2.000 10 0.184
7.266 7.000 10 -0.189
9.932 10.000 10 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
o
0.6
0.4
0.2
HMDL
BMD
Quantal Linear Model with 0.95 Confidence Level
Quantal Linear
10
20
30
dose
40
50
60
09:02 10/08 2009
Figure B-13 Male rats, Quantal-linear model, Kidney effect (noncancerous):
Atrophy and degeneration of renal tubules. Gorzinski et al.
(1985). F344 Strain, 16 weeks exposure by diet.
B-40
-------
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
Probit AtropyandDegenRenalTubulesDataNoSeverityFemaleRat.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
Variable
intercept
slope
Parameter Estimates
Estimate
-1.26508
0.0246481
95.0% Wald Confidence Interval
Std. Err. Lower Conf. Limit Upper Conf. Limit
0.324595 -1.90127 -0.628881
0.00871343
0.00757005
0.0417261
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -18.2358 4
Fitted model -18.2465 2 0.0214055 2 0.9894
Reduced model
-22.4934
i.51521
0.03648
B-41
-------
AIC:
40.493
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size
Residual
0.0000
1.0000
15.0000
62.0000
0.1029
0.1074
0.1853
0.6038
1.029
1.074
1.853
6.038
1.000
1.000
2.000
6.000
10
10
10
10
-0.030
-0.076
0.120
-0.024
ChiA2 =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
B-42
-------
Probit Model with 0.95 Confidence Level
o
o
0.6
0.4
0.2
60
10:0605/062010
Figure B-14 Female rats, Probit model, Kidney effect (noncancerous):
Atrophy and degeneration of renal tubules. Gorzinski et al.
(1985). F344 Strain, 16 weeks exposure by diet.
B-43
-------
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[slope*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
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 0.0860249 0.029523 0.0281609 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.
Analysis of Deviance Table
B-44
-------
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -9.35947 4
Fitted model -9.44226 1 0.165576 3 0.9829
Reduced model -27.5256 1 36.3322 3 <.0001
AIC: 20.8845
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000
1.0000
15.0000
62.0000
0.0000
0.0824
0.7248
0.9952
0.000
0.824
7.248
9.952
0.000
1.000
7.000
10.000
10
10
10
10
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
-------
Gamma Multi-Hit Model with 0.95 Confidence Level
o
o
0.6
0.4
0.2
0 -
HMDL BMD
14:31 08/122009
Gamma Multi-Hit
10
30
dose
40
50
00
Figure B-15 Male rats, Gamma model, Kidney effect (noncancerous): Slight
hypertrophy and/or dilation of proximal convoluted tubules.
Gorzinski et al. (1985). F344 Strain, 16 weeks exposure by diet.
B-46
-------
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.
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -9.35947 4
Fitted model -9.44226 1 0.165576 3 0.9829
B-47
-------
Reduced model -27.5256 1 36.3322 3 <.0001
AIC: 20.8845
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.0000 0.000 0.000 10 0.000
1.0000 0.0824 0.824 1.000 10 0.202
15.0000 0.7248 7.248 7.000 10 -0.176
62.0000 0.9952 9.952 10.000 10 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-48
-------
Weibull Model with 0.95 Confidence Level
o
o
0.6
0.4
0.2
0 - -* -
14:3508/122009
60
Figure B-16 Male rats, Weibull model, Kidney effect (noncancerous): Slight
hypertrophy and/or dilation of proximal convoluted tubules.
Gorzinski et al. (1985). F344 Strain, 16 weeks exposure by diet.
B-49
-------
Quantal-linear Model
Quantal-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
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 0.0860249 0.029523 0.0281608 0.143889
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
Full model -9.35947 4
B-50
-------
Fitted model -9.44226 1 0.165576 3 0.9829
Reduced model -27.5256 1 36.3322 3 <.0001
AIC: 20.8845
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000
1.0000
15.0000
62.0000
0.0000
0.0824
0.7248
0.9952
0.000
0.824
7.248
9.952
0.000
1.000
7.000
10.000
10
10
10
10
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-51
-------
Weibull Model with 0.95 Confidence Level
o
o
0.6
0.4
0.2
0 - -* -
14:37 08/12 2009
60
Figure B-17 Male rats, Quantal-linear model, Kidney effect (noncancerous):
Slight hypertrophy and/or dilation of proximal convoluted
tubules. Gorzinski et al. (1985). F344 Strain, 16 weeks exposure
by diet.
B-52
-------
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[slope*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
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 )
Slope Power
Slope 1 0.95
Power 0.95 1
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0 NA
Slope 0.00723384 0.00398244 -0.000571608 0.0150393
Power 2.58447 1.14213 0.345944 4.823
NA - Indicates that this parameter has hit a bound
implied by some ineguality constraint and thus
has no standard error.
Analysis of Deviance Table
B-53
-------
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model
Fitted model
Reduced model
AIC:
-16.7382
-17.3091
-32.5964
38.6182
1.14186
31.7164
0.8876
<.0001
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size
Residual
0.0000
33.5000
67.1000
134.3000
267.8000
535.7000
0.0000
0.0059
0.0300
0.1289
0.4095
0.8159
0.000
0.059
0.300
1.289
4.095
8.159
0.000
0.000
0.000
2.000
4.000
8.000
10
10
10
10
10
10
0.000
-0.244
-0.556
0. 671
-0.061
-0.130
ChiA2 = 0.84
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-54
-------
2
O
(I)
"o
(li
0.6
0.4
0.2
BMDL
Gamma Multi-Hit Model with 0.95 Confidence Level
Gamma Multi-Hit
BMD
100
200 300
dose
400
500
09:14 04/09 2009
Figure B-18 Female rats, Gamma model, Liver effect (noncancerous):
Heptocellular necrosis. NTP (1989). F344 Strain, 13 weeks
exposure by gavage.
B-55
-------
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-56
-------
Parameter Estimates
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0.0177261 * * *
Beta(l) 0 * * *
Beta(2) 0.00751246 * * *
Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -33.5473 3
Fitted
Reduced
AIC
model -33
model -36
.6008 2 0.106829 1
.7395 1 6.38433 2
0.7438
0.04108
: 71.2015
Goodness of Fit
Dose
i: 1
0.0000
i: 2
2.0400
i: 3
4.0900
Chi-squ
Est. Prob.
0.0177
0.0481
0.1343
are = 0.
Scaled
Expected Observed Size
0.887 1 50
2.407 2 50
6.717 7 50
10 DF= 1 P-value =
Residual
0.129
-0.178
0.049
0.7510
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 3.74496
BMDL = 2.45283
BMDU = 9.24921
Taken together, (2.45283, 9.24921) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.0407692
B-57
-------
Multistage Cancer Model with 0.95 Confidence Level
2
o
o
"o
0.3
0 25
0.2
0.15
0.1
0 05
Multistage Cancer
Linear extrapolation
0 0.5 1 1.5
3.5
14:38 04/13 2009
Figure B-19 Male rats, Multistage 2° model, Kidney effect (cancerous):
Renal Adenoma/Carcinoma. NTP (1989). F344 Strain, 103
weeks exposure by gavage.
B-58
-------
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
95.0% Wald Confidence Interval
B-59
-------
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0.146344 * * *
Beta(l) 0 * * *
Beta(2) 7.26074e-005 * * *
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -71.2862 3
Fitted model -71.7199 2 0.867331 1 0.3517
Reduced model -80.5752 1 18.5779 2 <.0001
AIC: 147.44
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.1463 2.927 3.000 20 0.046
53.0500 0.3041 15.206 15.000 50 -0.063
103.8800 0.6101 29.892 30.870 49 0.286
ChiA2 = 0.09 d.f. = 1 P-value = 0.7666
Benchmark Dose Computation
Specified effect = 0.1
Risk Type = Extra risk
Confidence level = 0.95
BMD = 38.0933
BMDL = 13.8018
BMDU = 49.5091
Taken together, (13.8018, 49.5091) is a 90 % two-sided confidence
interval for the BMD
Multistage Cancer Slope Factor = 0.00724545
B-60
-------
Multistage Cancer Model with 0.95 Confidence Level
2
o
o
"o
0.7
0.6
0.5
0.4
0.3
0.2
0.1
08:30 04/14 2009
BMDL
20
Multistage Cancer
Linear extrapolation
BMD
40 60
dose
80
100
Figure B-20 Male mice, Multistage 2° model, Liver effect
(cancerous):Hepatocellular carcinoma. NCI (1978). B6C3F1
Strain, 78 weeks exposure by gavage.
B-61
-------
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
Parameter Estimates
B-62
-------
95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0.189829 * * *
Beta(l) 0.000368083 * * *
Beta(2) 0 * * *
* - Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -70.4882 3
Fitted model -72.8848 2 4.79332 1 0.02857
Reduced model -73.9112 1 6.84615 2 0.03261
AIC: 149.77
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.1898 3.797 2.000 20 -1.024
360.0000 0.2904 14.519 20.000 50 1.708
722.0000 0.3789 18.566 15.190 49 -0.994
ChiA2 = 4.95 d.f. = 1 P-value = 0.0260
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-63
-------
Multistage Cancer Model with 0.95 Confidence Level
2
o
o
"o
0.6
0.5
0.4
0.3
0.2
0.1
Multistage Cancer
Linear extrapolation
100 200 300 400 500 600 700
14:37 05/20 2009
Figure B-21 Female mice, Multistage 2° model, Liver effect
(cancerous):Hepatocellular carcinoma. NCI (1978). B6C3F1
Strain, 78 weeks exposure by gavage.
B-64
-------
NTP (1989) BMP Modeling of Pheochromocytoma/Malignant Pheochromocytomas 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
BMDS Model Run - NTP 1989 Pheochromocytoma/Malignant Pheochromocytomas Male Mice -
Multistage Cancer 2 degree Model
PheochromocytomaMaleRat.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-65
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95.0% Wald Confidence Interval
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit
Background 0.341708 * * *
Beta(l) 0.055345 * * *
Beta(2) 0 * * *
Indicates that this value is not calculated.
Analysis of Deviance Table
Model Log(likelihood) # Param's Deviance Test d.f. P-value
Full model -93.0295 3
Fitted model -96.701 2 7.34302 1 0.006732
Reduced model -97.5291 1 8.99926 2 0.01111
AIC: 197.402
Goodness of Fit
Scaled
Dose Est._Prob. Expected Observed Size Residual
0.0000 0.3417 17.085 14.000 50 -0.920
2.0500 0.4123 18.554 26.100 45 2.285
4.1000 0.4753 23.292 19.110 49 -1.196
ChiA2 = 7.50 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-66
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Multistage Cancer Model with 0.95 Confidence Level
0.7
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0.4
0.3
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Linear extrapo
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BMDL
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BMD :
0 0.5 1 1.5 2 2.5 3 3.5 4
dose
15:5504/132009
Figure B-22 Male rats, Multistage 2° model, Adrenal effect (cancerous):
Pheochromocytoma/Malignant Pheochromocytomas. NTP
(1989). F344 Strain, 103 weeks exposure by gavage.
B-67
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