vycrM
EPA/635/R-20/106a
Final Agency /Interagency Draft
www.epa.gov/iris
Toxicological Review of Ethyl Tertiary Butyl Ether
(CASRN 637-92-3]
August 2020
NOTICE
This document is a Final Agency and Interagency Draft. It has not been formally disseminated by
EPA. It does not represent and should not be construed to represent any Agency determination or
policy. It is being circulated for review of its technical accuracy and science policy implications.
Integrated Risk Information System
Center for Public Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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Toxicological Review of ETBE
1 DISCLAIMER
2 This document is a preliminary draft for review purposes only. This information is
3 distributed solely for the purpose of predissemination peer review under applicable information
4 quality guidelines. It has not been formally disseminated by EPA. It does not represent and should
5 not be construed to represent any Agency determination or policy. Mention of trade names or
6 commercial products does not constitute endorsement or recommendation for use.
7
This document is a draft for review purposes only and does not constitute Agency policy.
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CONTENTS
AUTHORS | CONTRIBUTORS | REVIEWERS viii
PREFACE xi
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS xiv
EXECUTIVE SUMMARY xxii
LITERATURE SEARCH STRATEGY | STUDY SELECTION AND EVALUATION xxviii
1. HAZARD IDENTIFICATION 1-1
1.1. OVERVIEW OF CHEMICAL PROPERTIES AND TOXICOKINETICS 1-1
1.1.1. Chemical Properties 1-1
1.1.2. Toxicokinetics 1-2
1.1.3. Description of Toxicokinetic Models 1-3
1.1.4. Related Chemicals that Provide Supporting Information 1-3
1.2. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM 1-4
1.2.1. Kidney Effects 1-4
1.2.2. Liver Effects 1-38
1.2.3. Reproductive Effects 1-59
1.2.4. Developmental Effects 1-92
1.2.5. Carcinogenicity (Other than in the Kidney or Liver) 1-104
1.2.6. Other Toxicological Effects 1-113
1.3. INTEGRATION AND EVALUATION 1-113
1.3.1. Effects Other Than Cancer 1-113
1.3.2. Carcinogenicity 1-114
1.3.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes 1-117
2. DOSE-RESPONSE ANALYSIS 2-1
2.1. ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER 2-1
2.1.1. Identification of Studies and Effects for Dose-Response Analysis 2-1
2.1.2. Methods of Analysis 2-3
2.1.3. Derivation of Candidate Values 2-5
2.1.4. Derivation of Organ/System-Specific Reference Doses 2-9
2.1.5. Selection of the Overall Reference Dose 2-10
This document is a draft for review purposes only and does not constitute Agency policy.
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2.1.6. Confidence Statement 2-10
2.1.7. Previous IRIS Assessment 2-10
2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER THAN CANCER 2-11
2.2.1. Identification of Studies and Effects for Dose-Response Analysis 2-11
2.2.2. Methods of Analysis 2-12
2.2.3. Derivation of Candidate Values 2-14
2.2.4. Derivation of Organ/System-Specific Reference Concentrations 2-18
2.2.5. Selection of the Overall Reference Concentration 2-18
2.2.6. Confidence Statement 2-19
2.2.7. Previous IRIS Assessment 2-19
2.2.8. Uncertainties in the Derivation of the Reference Dose and Reference Concentration..2-19
2.3. ORAL SLOPE FACTOR FOR CANCER 2-20
2.3.1. Analysis of Carcinogenicity Data Error! Bookmark not defined.
2.3.2. Dose-Response Analysis—Adjustments and Extrapolation Methods..Error! Bookmark not
defined.
2.3.3. Derivation of the Oral Slope Factor Error! Bookmark not defined.
2.3.4. Uncertainties in the Derivation of the Oral Slope Factor Error! Bookmark not defined.
2.3.5. Previous IRIS Assessment: Oral Slope Factor Error! Bookmark not defined.
2.4. INHALATION UNIT RISK FOR CANCER 2-20
2.4.1. Analysis of Carcinogenicity Data 2-20
2.4.2. Dose-Response Analysis—Adjustments and Extrapolation Methods 2-21
2.4.3. Inhalation Unit Risk Derivation 2-22
2.4.4. Uncertainties in the Derivation of the Inhalation Unit Risk 2-23
2.4.5. Previous IRIS Assessment: Inhalation Unit Risk 2-25
2.5. APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS 2-25
REFERENCES R-l
This document is a draft for review purposes only and does not constitute Agency policy.
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TABLES
Table ES-1. Organ-/system-specific RfDs and overall RfD for ETBE xxiv
Table ES-2. Organ-/system-specific RfCs and overall RfCfor ETBE xxv
Table LS-1. Details of the search strategy employed for ETBE xxxii
Table LS-2. Summary of additional search strategies for ETBE xxxiii
Table LS-3. Inclusion-exclusion criteria xxxiii
Table LS-4. Considerations for evaluation of experimental animal studies xxxv
Table LS-5. Summary of experimental animal database xxxvi
Table 1-1. Physicochemical properties and chemical identity of ETBE 1-1
Table 1-2. Evidence pertaining to kidney histopathology effects in animals following exposure to
ETBE 1-10
Table 1-3. Evidence pertaining to kidney biochemistry and urine effects in animals following
exposure to ETBE 1-13
Table 1-4. Evidence pertaining to kidney tumor effects in animals following exposure to ETBE 1-16
Table 1-5. Comparison of nephropathy and urothelial hyperplasia in individual male rats from 2-
year oral exposure (JPEC, 2010a) 1-18
Table 1-6. Comparison of nephropathy and urothelial hyperplasia in individual male rats from 2-
year inhalation exposure (JPEC, 2010b) 1-18
Table 1-7. Additional kidney effects potentially relevant to mode of action in animals exposed to
ETBE 1-24
Table 1-8. Summary of data informing whether the a2u-globulin process is occurring in male rats
exposed to ETBE 1-26
Table 1-9. Evidence pertaining to liver weight effects in animals exposed to ETBE 1-41
Table 1-10. Evidence pertaining to liver histopathology effects in animals exposed to ETBE 1-43
Table 1-11. Evidence pertaining to liver biochemistry effects in animals exposed to ETBE 1-47
Table 1-12. Evidence pertaining to liver tumor effects in animals exposed to ETBE 1-51
Table 1-13. Positive evidence of key characteristics of cancer for ETBE 1-53
Table 1-14. Evidence pertaining to male reproductive effects in animals exposed to ETBE 1-61
Table 1-15. Evidence pertaining to female reproductive effects in animals exposed to ETBE 1-81
Table 1-16. Evidence pertaining to developmental effects in animals following exposure to ETBE 1-95
Table 1-17. Evidence pertaining to ETBE promotion of mutagen-initiated tumors in animals 1-107
Table 1-18. Evidence pertaining to carcinogenic effects (in tissues other than liver or kidney) in
animals exposed to ETBE 1-108
Table 2-1. Summary of derivation of points of departure following oral exposure for up to 2
years 2-4
Table 2-2. Effects and corresponding derivation of candidate values 2-6
Table 2-3. Organ/system-specific RfDs and overall RfD for ETBE 2-10
Table 2-4. Summary of derivation of PODs following inhalation exposure 2-13
Table 2-5. Effects and corresponding derivation of candidate values 2-16
Table 2-6. Organ-/system-specific RfCs and overall RfCfor ETBE 2-18
Table 2-7. Summary of the oral slope factor derivation Error! Bookmark not defined.
Table 2-8. Summary of uncertainties in the derivation of the oral slope factor for ETBE.. Error! Bookmark
not defined.
Table 2-9. Summary of the inhalation unit risk derivation 2-23
Table 2-10. Summary of uncertainties in the derivation of the inhalation unit risk for ETBE 2-24
This document is a draft for review purposes only and does not constitute Agency policy.
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FIGURES
Figure LS-1. Summary of literature search and screening process for ETBE xxxi
Figure 1-1. Proposed metabolism of ETBE 1-3
Figure 1-2. Comparison of absolute kidney weight change in male and female rats across oral
and inhalation exposure based on internal blood concentration 1-8
Figure 1-3. Comparison of absolute kidney weight change in male and female mice following
inhalation exposure based on administered ETBE concentration 1-9
Figure 1-4. Exposure-response array of kidney effects following oral exposure to ETBE 1-19
Figure 1-5. Exposure-response array of kidney effects following inhalation exposure to ETBE 1-20
Figure 1-6. Temporal pathogenesis of a2u-globulin-associated nephropathy in male rats 1-23
Figure 1-7. ETBE oral exposure array of a2u-globulin data in male rats 1-28
Figure 1-8. ETBE inhalation exposure array of a2u-globulin data in male rats 1-29
Figure 1-9. Exposure-response array of noncancer liver effects following oral exposure to ETBE 1-49
Figure 1-10. Exposure-response array of noncancer liver effects following inhalation exposure to
ETBE 1-50
Figure 1-11. Exposure-response array of male reproductive effects following oral exposure to
ETBE 1-77
Figure 1-12. Exposure-response array of male reproductive effects following inhalation exposure
to ETBE 1-78
Figure 1-13. Exposure-response array of female reproductive effects following oral exposure to
ETBE 1-90
Figure 1-14. Exposure-response array of female reproductive effects following inhalation
exposure to ETBE 1-91
Figure 1-15. Exposure-response array of developmental effects following oral exposure to ETBE 1-103
Figure 1-16. Exposure-response array of carcinogenic effects following oral exposure to ETBE 1-111
Figure 1-17. Exposure-response array of carcinogenic effects following inhalation exposure to
ETBE 1-112
Figure 2-1. Candidate values with corresponding POD and composite UF. Each bar corresponds
to one data set described in Table 2-1 and Table 2-2 2-8
Figure 2-2. Candidate values with corresponding POD and composite UF 2-17
This document is a draft for review purposes only and does not constitute Agency policy.
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i ABBREVIATIONS
ACGIH
American Conference of Governmental
LCso
median lethal concentration
Industrial Hygienists
LD50
median lethal dose
AIC
Akaike's information criterion
LOAEL
lowest-observed-adverse-effect level
ATSDR
Agency for Toxic Substances and
MN
micronuclei
Disease Registry
MNPCE
micronucleated polychromatic
ALP
alkaline phosphatase
erythrocyte
ALT
alanine
MTD
maximum tolerated dose
aminotransferase/transaminase
MTBE
methyl tertiary butyl ether
AST
aspartate
NCEA
National Center for Environmental
aminotransferase/transaminase
Assessment
BMD
benchmark dose
NCI
National Cancer Institute
BMDL
benchmark dose lower confidence limit
NOAEL
no-observed-adverse-effect level
BMDS
Benchmark Dose Software
NTP
National Toxicology Program
BMR
benchmark response
ORD
Office of Research and Development
BUN
blood urea nitrogen
PBPK
physiologically based pharmacokinetic
BW
body weight
PCE
polychromatic erythrocytes
CA
chromosomal aberration
PCNA
proliferating cell nuclear antigen
CASRN
Chemical Abstracts Service Registry
PND
postnatal day
Number
POD
point of departure
CUT
Chemical Industry Institute of
POD [AD J]
duration-adjusted POD
Toxicology
QSAR
quantitative structure-activity
CL
confidence limit
relationship
CNS
central nervous system
RD
relative deviation
CPN
chronic progressive nephropathy
RfC
inhalation reference concentration
CYP450
cytochrome P450
RfD
oral reference dose
DAF
dosimetric adjustment factor
RNA
ribonucleic acid
DNA
deoxyribonucleic acid
SAR
structure activity relationship
EPA
Environmental Protection Agency
SCE
sister chromatid exchange
FDA
Food and Drug Administration
SD
standard deviation
FEVi
forced expiratory volume of 1 second
SE
standard error
GD
gestation day
SGOT
glutamic oxaloacetic transaminase, also
GDH
glutamate dehydrogenase
known as AST
GGT
y-glutamyl transferase
SGPT
glutamic pyruvic transaminase, also
GLP
Good Laboratory Practices
known as ALT
GSH
glutathione
UF
uncertainty factor
GST
glutathione-S-transferase
UFa
animal-to-human uncertainty factor
Hb/g-A
animal blood:gas partition coefficient
UFh
human variation uncertainty factor
Hb/g-H
human blood:gas partition coefficient
UFl
LOAEL-to-NOAEL uncertainty factor
HEC
human equivalent concentration
UFs
subchronic-to-chronic uncertainty
HED
human equivalent dose
factor
i.p.
intraperitoneal
UFd
database deficiencies uncertainty factor
IRIS
Integrated Risk Information System
U.S.
United States
JPEC
Japan Petroleum Energy Center
WT
wild type
KO
Knockout
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
Assessment Team
Kathleen Newhouse, MS (Chemical
Manager)
Andre Weaver, Ph.D. (Co-Chemical
Manager)
Janice Lee, Ph.D.
U.S. EPA
Office of Research and Development
Center for Public Health and Environmental
Assessment
Research Triangle Park, NC
Washington, DC
Keith Salazar, PhD.
Former Co-Chemical Manager
EPA/ORD/NCEA
Currently with U.S. EPA, Office of Chemical Safety
and Pollution Prevention, Office of Pollution
Prevention and Toxics
Washington, DC
Christopher Brinkerhoff, Ph.D.
Former ORISE Postdoctoral Fellow at U.S.
EPA/ORD/NCEA
Currently with U.S. EPA, Office of Chemical Safety
and Pollution Prevention, Office of Pollution
Prevention and Toxics
Washington, DC
Contributors
Andrew Hotchkiss, Ph.D.
Channa Keshava, Ph.D.
Jeff Gift, Ph.D.
Christine Cai, M.S.
Alan Sasso, Ph.D.
Paul Schlosser, Ph.D.
Karen Hogan, M.S.
Susan Makris, Ph.D.
Brandy Beverly, Ph.D.
Erin Yost, Ph.D.
U.S. EPA
Office of Research and Development
Center for Public Health and Environmental
Assessment
Research Triangle Park, NC
*Washington, DC
Vincent Cogliano, Ph.D.
Jason Fritz, Ph.D.
(previously with) U.S. EPA National Center for
Environmental Assessment
Charles Wood, Ph.D.
(previously with) U.S. EPA National Health and
Environmental Effects Research Lab
This document is a draft for review purposes only and does not constitute Agency policy.
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Production Team
Taukecha Cunningham U.S. EPA
Maureen Johnson Office of Research and Development
Terri Konoza Center for Public Health and Environmental
Vicki Soto Assessment
Dahnish Shams
Contractor Support
Robyn Blain, Ph.D.
Pam Ross, M.S.P.H.
Ami Gordon, M.P.H.
ICF
9300 Lee Highway
Fairfax, VA
Executive Direction
Wayne Cascio (CPHEA Director) U.S. EPA/ORD/CPHEA
Samantha Jones Ph.D. Research Triangle Park,
(CPHEA Associate Director)* NC
Kristina Thayer, Ph.D. (CPAD Director) *Washington, DC
Emma Lavoie, Ph.D. (CPHEA Senior #Cincinnati, OH
Science Advisor for Assessments)
Andrew Kraft, Ph.D. (CPAD Senior
Science Advisor)
Paul White, Ph.D. (CPAD Senior
Science Advisor)*
Belinda Hawkins, Ph.D. (CPAD Senior
Science Advisor)#
Ravi Subramaniam, Ph.D. (CPAD Branch
Chief)*
Internal Review Team
General Toxicology Workgroup U.S. EPA
Inhalation Workgroup Office of Research and Development
Neurotoxicity Workgroup Center for Public Health and Environmental Assessment
PK Workgroup
Reproductive and Developmental
Toxicology Workgroup
Statistical Workgroup
Toxicity Pathways Workgroup
Executive Review Committee
Reviewers
This assessment was provided for review to scientists in EPA's Program and Regional Offices.
Comments were submitted by:
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Office of Children's Health Protection, Washington, DC
Office of Policy, Washington, DC
Office of Solid Waste and Emergency Response, Washington, DC
Office of Air and Radiation, Washington, DC
Region 2, New York, NY
Region 8, Denver, CO
1 This assessment was provided for review to other federal agencies and the Executive Office of the
2 President Comments were submitted by:
Department of Health and Human Services/Agency for Toxic Substances and Disease Registry,
Department of Health and Human Services/National Institute of Environmental Health
Sciences/National Toxicology Program
Executive Office of the President/Office of Management and Budget
This document is a draft for review purposes only and does not constitute Agency policy.
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PREFACE
This Toxicological Review critically reviews the publicly available studies on ethyl tertiary
butyl ether (ETBE) to identify its adverse health effects and to characterize exposure-response
relationships. The assessment examined all effects by oral and inhalation routes of exposure and
includes an oral noncancer reference dose (RfD), an inhalation noncancer reference concentration
(RfC), a cancer weight of evidence descriptor, and a cancer dose-response assessment. It was
prepared under the auspices of the U.S. Environmental Protection Agency's (EPA's) Integrated Risk
Information System (IRIS) program.
This assessment updates a previous IRIS draft assessment of ETBE that went to peer review
in 2010. The previous draft assessment was suspended pending completion of several new studies
that were identified during the peer review and are now included in this document.
The Toxicological Reviews for ETBE and tert-butyl alcohol (tert-butanol) were developed
simultaneously because they have overlapping scientific aspects:
• tert-Butanol and acetaldehyde are the primary metabolites of ETBE, and some of the
toxicological effects of ETBE are likely attributed to tert-butanol. Therefore, data on tert-
butanol are considered informative for the hazard identification and dose-response
assessment of ETBE, and vice versa.
• The scientific literature for the two chemicals includes data on a2U-globulin-related
nephropathy; therefore, a common approach was used to evaluate the data as they relate to
the mode of action for kidney effects.
• A combined physiologically based pharmacokinetic (PBPK) model for ETBE and tert-
butanol in rats was applied to support the dose-response assessments for these chemicals
fBorghoffetal.. 20161.
Prior to the development of the IRIS assessment, a public meeting was held in December
2013 to obtain input on preliminary materials for ETBE, including draft literature searches and
associated search strategies, evidence tables, and exposure-response arrays. In June 2016, EPA
convened a public science meeting to discuss the public comment draft Toxicological Review of
tert-Butyl Alcohol (tert-butanol) during which time the Agency heard comments on "disentangling
mechanisms of kidney toxicity and carcinogenicity," an issue relevant to both tert-butanol and
ETBE. The complete set of public comments, including the slides presented at the June 2016 public
science meeting is available on the docket at http://www.regulations.gov (Docket ID No. EPA-HO-
QRD-2013-1111). In October 2016, a public science meeting was held to provide the public an
opportunity to engage in early discussions on the draft IRIS Toxicological Review of ETBE and the
draft charge to the peer review panel prior to release for external peer review. The complete set of
This document is a draft for review purposes only and does not constitute Agency policy.
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public comments, including the slides is available on the docket at http://www.regulations.gov
(Docket ID No. EPA-HQ-QRD-2009-02291.
Organ-/system-specific reference values are calculated where feasible (in this case only
representing kidney toxicity). These reference values could be useful for cumulative risk
assessments that consider the combined effect of multiple agents acting on the same biological
system.
This assessment was conducted in accordance with EPA guidance, which is cited and
summarized in the Preamble to IRIS Toxicological Reviews. Appendices for toxicokinetic
information, PBPK modeling, genotoxicity study summaries, dose-response modeling, and other
information are provided as Supplemental Information to this Toxicological Review. For additional
information about this assessment or for general questions regarding IRIS, please contact EPA's
IRIS Hotline at 202-566-1676 (phone), 202-566-1749 (fax), or hotline.iris@epa.gov.
Uses
ETBE has been used as a fuel oxygenate in the United States to improve combustion
efficiency and reduce pollutants in exhaust. From approximately 1990 to 2006, ETBE was
periodically added to gasoline at levels up to approximately 20%, but methyl tert-butyl ether
(MTBE) and other oxygenates were more commonly used. In 2006, use of ETBE and other ether fuel
additives ceased in the United States, and the use of ethanol increased dramatically fWeaver etal..
2010). ETBE is still registered with EPA for use as a fuel additive, but it is not used currently in the
United States. The use of ether fuel additives has been banned or limited by several states, largely in
response to groundwater contamination concerns.
The United States is a major exporter of ETBE, producing 25% of the world's ETBE in 2012.
Worldwide consumption of ETBE is concentrated in Western Europe (~70%). Use in Eastern
Europe and Japan also is relatively high. Japan's use increased dramatically in 2010 to fulfill its
2010 Kyoto Accord obligations (USDA. 2012).
Fate and Transport
ETBE is expected to be highly mobile in soil due to its high carbon-water partitioning
coefficient (HSDB. 2012). ETBE is not predicted to adsorb onto suspended particles and is unlikely
to undergo biodegradation in water fHSDB. 20121. ETBE is estimated to have a half-life of 2 days in
air fHSDB. 20121.
Occurrence in the Environment
ETBE can be released to the environment by gasoline leaks, evaporation, spills, and other
releases. ETBE degrades slowly in the environment and can move with water in soil. Monitoring
studies targeting groundwater near areas where petroleum contamination likely occurred detected
ETBE. For instance, a survey of states reported an average detection rate of 18% for ETBE in
groundwater samples associated with gasoline contamination fNEIWPCC. 20031. Nontargeted
This document is a draft for review purposes only and does not constitute Agency policy.
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studies, such as a 2006 U.S. Geological Survey (USGS) study (USGS. 20061 measuring volatile
organic compounds (VOCs) in general, have lower detection rates. The 2006 USGS study showed
detections of ETBE above 0.2 ng/L in five samples from two public drinking water wells,
corresponding to a 0.0013 rate of detection. The USGS study, which measured several VOCs, was
not targeted to sites that would be most vulnerable to ETBE contamination.
Fuel contamination cleanup is done largely by states, and information on the number of
private contaminated drinking water wells is not consistently available. The State of California
maintains an online database of measurements from contaminated sites (Cal/EPA. 20161. From
2010 to 2013, ETBE has been detected in California at 607 and 73 sites in groundwater and air,
respectively. Most of the contamination is attributed to leaking underground storage tanks, and
some contamination is associated with refineries and petroleum transportation. The contamination
was noted in approximately 48 counties, with higher-population counties (e.g., Los Angeles and
Orange) having more contaminated sites.
The occurrence of ETBE in other states was found using fewer and less-standardized data.
Currently, only 13 states routinely analyze for ETBE at fuel-contaminated sites (NEIWPCC. 20031.
Monitoring data associated with leaking storage tanks in Maryland show contamination in
groundwater affecting multiple properties fMarvland Department of the Environment. 20161.
General Population Exposure
ETBE exposure can occur in many different settings. Releases from underground storage
tanks could result in exposure to individuals who obtain their drinking water from wells. Due to its
environmental mobility and resistance to biodegradation, ETBE has the potential to contaminate
and persist in groundwater and soil fHSDB. 20121: therefore, exposure through ingestion of
contaminated drinking water is possible.
Other human exposure pathways of ETBE include inhalation and, to a lesser extent, dermal
contact ETBE inhalation exposure can occur due the chemical's volatility and release from
industrial processes and contaminated sites fHSDB. 2012).
Assessments by Other National and International Health Agencies
Toxicity information on ETBE has been evaluated by the National Institute for Public Health
and the Environment (Bilthoven, The Netherlands) fTiesiema and Baars. 20091. The results of this
assessment are presented in Appendix A of the Supplemental Information to this Toxicological
Review. Of importance to recognize is that earlier assessments could have been prepared for
different purposes and might use different methods. In addition, newer studies have been included
in the IRIS assessment
This document is a draft for review purposes only and does not constitute Agency policy.
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PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS
Note: The Preamble summarizes the
objectives and scope of the IRIS program,
general principles and systematic review
procedures used in developing IRIS
assessments, and the overall development
process and document structure.
1. Objectives and Scope of the IRIS
Program
Soon after EPA was established in 1970, it
was at the forefront of developing risk
assessment as a science and applying it in
support of actions to protect human health
and the environment. EPA's IRIS program1
contributes to this endeavor by reviewing
epidemiologic and experimental studies of
chemicals in the environment to identify
adverse health effects and characterize
exposure-response relationships. Health
agencies worldwide use IRIS assessments,
which are also a scientific resource for
researchers and the public.
IRIS assessments cover the hazard
identification and dose-response steps of
risk assessment Exposure assessment and
risk characterization are outside the scope of
IRIS assessments, as are political, economic,
and technical aspects of risk management An
IRIS assessment may cover one chemical, a
group of structurally or toxicologically
related chemicals, or a chemical mixture.
Exceptions outside the scope of the IRIS
program are radionuclides, chemicals used
35 only as pesticides, and the "criteria air
36 pollutants" (particulate matter, ground-level
37 ozone, carbon monoxide, sulfur oxides,
38 nitrogen oxides, and lead).
39 Enhancements to the IRIS program are
40 improving its science, transparency, and
41 productivity. To improve the science, the IRIS
42 program is adapting and implementing
43 principles of systematic review (i.e., using
44 explicit methods to identify, evaluate, and
45 synthesize study findings). To increase
46 transparency, the IRIS program discusses key
47 science issues with the scientific community
48 and the public as it begins an assessment
49 External peer review, independently
50 managed and in public, improves both
51 science and transparency. Increased
52 productivity requires that assessments be
53 concise, focused on EPA's needs, and
54 completed without undue delay.
55 IRIS assessments follow EPA guidance2
56 and standardized practices of systematic
57 review. This Preamble summarizes and does
58 not change IRIS operating procedures or EPA
59 guidance.
60 Periodically, the IRIS program asks for
61 nomination of agents for future assessment
62 or reassessment. Selection depends on EPA's
63 priorities, relevance to public health, and
64 availability of pertinent studies. The IRIS
65 multiyear agenda3 lists upcoming
66 assessments. The IRIS program may also
67 assess other agents in anticipation of public
68 health needs.
1 IRIS program website:
h ttp: II www, ep a. go v /i r i s /
2 EPA guidance documents:
http://www.epa.gov/iris/basic-information-
about-integrated-risk-information-
svstem# guidance /
This document is a draft for review purposes only and does not constitute Agency policy.
3 IRIS multiyear agenda: https: //www.epa.gov/
iris/iris-agenda
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2. Planning an Assessment:
Scoping, Problem Formulation,
and Protocols
Early attention to planning ensures that
IRIS assessments meet their objectives and
properly frame science issues.
Scoping refers to the first step of
planning, where the IRIS program consults
with EPA's program and regional offices to
ascertain their needs. Scoping specifies the
agents an assessment will address, routes
and durations of exposure, susceptible
populations and lifestages, and other topics of
interest
Problem formulation refers to the
science issues an assessment will address
and includes input from the scientific
community and the public. A preliminary
literature survey, beginning with secondary
sources (e.g., assessments by national and
international health agencies and
comprehensive review articles), identifies
potential health outcomes and science issues.
It also identifies related chemicals (e.g.,
toxicologically active metabolites and
compounds that metabolize to the chemical
of interest).
Each IRIS assessment comprises multiple
systematic reviews for multiple health
outcomes. It also evaluates hypothesized
mechanistic pathways and characterizes
exposure-response relationships. An
assessment may focus on important health
outcomes and analyses rather than expand
beyond what is necessary to meet its
objectives.
Protocols refer to the systematic review
procedures planned for use in an assessment
They include strategies for literature
searches, criteria for study inclusion or
exclusion, considerations for evaluating
study methods and quality, and approaches
to extracting data. Protocols may evolve as an
Toxicological Review of ETBE
44 assessment progresses and new agent-
45 specific insights and issues emerge.
46
47 3. Identifying and Selecting
48 Pertinent Studies
49 IRIS assessments conduct systematic
50 literature searches with criteria for inclusion
51 and exclusion. The objective is to retrieve the
52 pertinent primary studies (i.e., studies with
53 original data on health outcomes or their
54 mechanisms). PECO statements (Populations,
55 Exposures, Comparisons, Outcomes) govern
56 the literature searches and screening criteria.
57 "Populations" and animal species generally
58 have no restrictions. "Exposures" refers to
59 the agent and related chemicals identified
60 during scoping and problem formulation and
61 may consider route, duration, or timing of
62 exposure. "Comparisons" means studies that
63 allow comparison of effects across different
64 levels of exposure. "Outcomes" may become
65 more specific (e.g., from "toxicity" to
66 "developmental toxicity" to "hypospadias")
67 as an assessment progresses.
68 For studies of absorption, distribution,
69 metabolism, and elimination, the first
70 objective is to create an inventory of
71 pertinent studies. Subsequent sorting and
72 analysis facilitates characterization and
73 quantification of these processes.
74 Studies on mechanistic events can be
75 numerous and diverse. Here, too, the
76 objective is to create an inventory of studies
77 for later sorting to support analyses of related
78 data. The inventory also facilitates generation
79 and evaluation of hypothesized mechanistic
80 pathways.
81 The IRIS program posts initial protocols
82 for literature searches on its website and
83 adds search results to EPA's HERO database.4
84 Then the IRIS program takes extra steps to
85 ensure identification of pertinent studies: by
4 Health and Environmental Research Online:
https://hero.epa.gov/hero/
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
encouraging the scientific community and the
public to identify additional studies and
ongoing research; by searching for data
submitted under the Toxic Substances
Control Act or the Federal Insecticide,
Fungicide, and Rodenticide Act; and by
considering late-breaking studies that would
impact the credibility of the conclusions, even
during the review process.5
4. Evaluating Study Methods and
Quality
IRIS assessments evaluate study methods
and quality, using uniform approaches for
each group of similar studies. The objective is
that subsequent syntheses can weigh study
results on their merits. Key concerns are
potential bias (factors that affect the
magnitude or direction of an effect) and
insensitivity (factors that limit the ability of a
study to detect a true effect).
For human and animal studies, the
evaluation of study methods and quality
considers study design, exposure measures,
outcome measures, data analysis, selective
reporting, and study sensitivity. For human
studies, this evaluation also considers
selection of participant and referent groups
and potential confounding. Emphasis is on
discerning bias that could substantively
change an effect estimate, considering also
the expected direction of the bias. Low
sensitivity is a bias towards the null.
Study-evaluation considerations are
specific to each study design, health effect,
and agent Subject-matter experts evaluate
each group of studies to identify
characteristics that bear on the
informativeness of the results. For
carcinogenicity, neurotoxicity, reproductive
toxicity, and developmental toxicity, there is
EPA guidance for study evaluation (U.S. EPA.
2005a. 1998b. 1996. 1991b). As subject-
43 matter experts examine a group of studies,
44 additional agent-specific knowledge or
45 methodologic concerns may emerge and a
46 second pass become necessary.
47 Assessments use evidence tables to
48 summarize the design and results of
49 pertinent studies. If tables become too
50 numerous or unwieldy, they may focus on
51 effects that are more important or studies
52 that are more informative.
53 The IRIS program posts initial protocols
54 for study evaluation on its website, then
55 considers public input as it completes this
56 step.
57 5. Integrating the Evidence of
58 Causation for Each Health
59 Outcome
60 Synthesis within lines of evidence. For
61 each health outcome, IRIS assessments
62 synthesize the human evidence and the
63 animal evidence, augmenting each with
64 informative subsets of mechanistic data. Each
65 synthesis considers aspects of an association
66 that may suggest causation: consistency,
67 exposure-response relationship, strength of
68 association, temporal relationship, biological
69 plausibility, coherence, and "natural
70 experiments" in humans (U.S. EPA. 1994)
71 fU.S. EPA. 2005al.
72 Each synthesis seeks to reconcile
73 ostensible inconsistencies between studies,
74 taking into account differences in study
75 methods and quality. This leads to a
76 distinction between conflicting evidence
77 (unexplained positive and negative results in
78 similarly exposed human populations or in
79 similar animal models) and differing results
80 (mixed results attributable to differences
81 between human populations, animal models,
82 or exposure conditions) (U.S. EPA. 2005a).
83 Each synthesis of human evidence
84 explores alternative explanations (e.g.,
5 IRIS "stopping rules": https: //www.epa.gov/
sites/production/files/2 014-06/documents /
iris stoppingrules.pdf
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
chance, bias, or confounding) and determines
whether they may satisfactorily explain the
results. Each synthesis of animal evidence
explores the potential for analogous results in
humans. Coherent results across multiple
species increase confidence that the animal
results are relevant to humans.
Mechanistic data are useful to augment
the human or animal evidence with
information on precursor events, to evaluate
the human relevance of animal results, or to
identify susceptible populations and
lifestages. An agent may operate through
multiple mechanistic pathways, even if one
hypothesis dominates the literature (U.S.
EPA. 2005al
Integration across lines of evidence.
For each health outcome, IRIS assessments
integrate the human, animal, and mechanistic
evidence to answer the question: What is the
nature of the association between exposure to
the agent and the health outcome?
For cancer, EPA includes a standardized
hazard descriptor in characterizing the
strength of the evidence of causation. The
objective is to promote clarity and
consistency of conclusions across
assessments fU.S. EPA. 2005al
Carcinogenic to humans: convincing
epidemiologic evidence of a causal
association; or strong human evidence of
cancer or its key precursors, extensive animal
evidence, identification of mode-of-action
and its key precursors in animals, and strong
evidence that they are anticipated in humans.
Likely to be carcinogenic to humans:
evidence that demonstrates a potential
hazard to humans. Examples include a
plausible association in humans with
supporting experimental evidence, multiple
positive results in animals, a rare animal
response, or a positive study strengthened by
other lines of evidence.
Suggestive evidence of carcinogenic
potential: evidence that raises a concern for
humans. Examples include a positive result in
the only study, or a single positive result in an
extensive database.
49 Inadequate information to assess
50 carcinogenic potential: no other descriptors
51 apply. Examples include little or no pertinent
52 information, conflicting evidence, or negative
53 results not sufficiently robust for not likely.
54 Not likely to be carcinogenic to humans:
55 robust evidence to conclude that there is no
56 basis for concern. Examples include no effects
57 in well-conducted studies in both sexes of
58 multiple animal species, extensive evidence
59 showing that effects in animals arise through
60 modes-of-action that do not operate in
61 humans, or convincing evidence that effects
62 are not likely by a particular exposure route
63 or below a defined dose.
64 If there is credible evidence of
65 carcinogenicity, there is an evaluation of
66 mutagenicity, because this influences the
67 approach to dose-response assessment and
68 subsequent application of adjustment factors
69 for exposures early in life fU.S. EPA. 2005al
70 flJ.S. EPA. 200Sbl.
71 6. Selecting Studies for Derivation
72 of Toxicity Values
73 The purpose of toxicity values (slope
74 factors, unit risks, reference doses, reference
75 concentrations; see section 7) is to estimate
76 exposure levels likely to be without
77 appreciable risk of adverse health effects.
78 EPA uses these values to support its actions
79 to protect human health.
80 The health outcomes considered for
81 derivation of toxicity values may depend on
82 the hazard descriptors. For example, IRIS
83 assessments generally derive cancer values
84 for agents that are carcinogenic or likely to be
85 carcinogenic, and sometimes for agents with
86 suggestive evidence (U.S. EPA. 2005a).
87 Derivation of toxicity values begins with a
88 new evaluation of studies, as some studies
89 used qualitatively for hazard identification
90 may not be useful quantitatively for
91 exposure-response assessment Quantitative
92 analyses require quantitative measures of
93 exposure and response. An assessment
94 weighs the merits of the human and animal
95 studies, of various animal models, and of
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
different routes and durations of exposure
fU.S. EPA. 19941. Study selection is not
reducible to a formula, and each assessment
explains its approach.
Other biological determinants of study
quality include appropriate measures of
exposure and response, investigation of early
effects that precede overt toxicity, and
appropriate reporting of related effects (e.g.,
combining effects that comprise a syndrome,
or benign and malignant tumors in a specific
tissue).
Statistical determinants of study quality
include multiple levels of exposure (to
characterize the shape of the exposure-
response curve) and adequate exposure
range and sample sizes (to minimize
extrapolation and maximize precision) (U.S.
EPA. 20121.
Studies of low sensitivity may be less
useful if they fail to detect a true effect or
yield toxicity values with wide confidence
limits.
7. Deriving Toxicity Values
General approach. EPA guidance
describes a two-step approach to dose-
response assessment: analysis in the range of
observation, then extrapolation to lower
levels. Each toxicity value pertains to a route
(e.g., oral, inhalation, dermal) and duration or
timing of exposure (e.g., chronic, subchronic,
gestational) (U.S. EPA. 2002).
IRIS assessments derive a candidate
value from each suitable data set.
Consideration of candidate values yields a
toxicity value for each organ or system.
Consideration of the organ/system-specific
values results in the selection of an overall
toxicity value to cover all health outcomes.
The organ/system-specific values are useful
for subsequent cumulative risk assessments
that consider the combined effect of multiple
agents acting at a common anatomical site.
44 Analysis in the range of observation.
45 Within the observed range, the preferred
46 approach is modeling to incorporate a wide
47 range of data. Toxicokinetic modeling has
48 become increasingly common for its ability to
49 support target-dose estimation, cross-species
50 adjustment, or exposure-route conversion. If
51 data are too limited to support toxicokinetic
52 modeling, there are standardized approaches
53 to estimate daily exposures and scale them
54 from animals to humans (U.S. EPA. 19941.
55 fU.S. EPA. 2005al. fU.S. EPA. 2011. 20061.
56 For human studies, an assessment may
57 develop exposure-response models that
58 reflect the structure of the available data (U.S.
59 EPA. 2005al. For animal studies, EPA has
60 developed a set of empirical ("curve-fitting")
61 models6 that can fit typical data sets (U.S.
62 EPA. 2005a). Such modeling yields a point of
63 departure, defined as a dose near the lower
64 end of the observed range, without significant
65 extrapolation to lower levels (e.g., the
66 estimated dose associated with an extra risk
67 of 10% for animal data or 1% for human data,
68 or their 95% lower confidence limits) (U.S.
69 EPA. 2005al. ("U.S. EPA. 20121.
70 When justified by the scope of the
71 assessment, toxicodynamic ("biologically
72 based") modeling is possible if data are
73 sufficient to ascertain the key events of a
74 mode-of-action and to estimate their
75 parameters. Analysis of model uncertainty
76 can determine the range of lower doses
77 where data support further use of the model
78 CIJ.S. EPA. 2005al.
79 For a group of agents that act at a
80 common site or through common
81 mechanisms, an assessment may derive
82 relative potency factors based on relative
83 toxicity, rates of absorption or metabolism,
84 quantitative structure-activity relationships,
85 or receptor-binding characteristics (U.S. EPA.
86 2005a).
87 Extrapolation: slope factors and unit
88 risks. An oral slope factor or an inhalation
6 Benchmark Dose Software:
http://www.epa.gov/bmds/
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
unit risk facilitates subsequent estimation of
human cancer risks. Extrapolation proceeds
linearly (i.e., risk proportional to dose) from
the point of departure to the levels of interest.
This is appropriate for agents with direct
mutagenic activity. It is also the default if
there is no established mode-of-action (U.S.
EPA. 2005al.
Differences in susceptibility may warrant
derivation of multiple slope factors or unit
risks. For early-life exposure to carcinogens
with a mutagenic mode-of-action, EPA has
developed default age-dependent adjustment
factors for agents without chemical-specific
susceptibility data (U.S. EPA. 2005a). (U.S.
EPA. 2005bl.
If data are sufficient to ascertain the
mode-of-action and to conclude that it is not
linear at low levels, extrapolation may use the
reference-value approach (U.S. EPA. 2005a).
Extrapolation: reference values. An
oral reference dose or an inhalation reference
concentration is an estimate of human
exposure (including in susceptible
populations) likely to be without appreciable
risk of adverse health effects over a lifetime
fU.S. EPA. 20021. Reference values generally
cover effects other than cancer. They are also
appropriate for carcinogens with a nonlinear
mode-of-action.
Calculation of reference values involves
dividing the point of departure by a set of
uncertainty factors (each typically 1, 3, or 10,
unless there are adequate chemical-specific
data) to account for different sources of
uncertainty and variability fU.S. EPA. 20021.
fU.S. EPA. 20141.
Human variation: An uncertainty factor
covers susceptible populations and lifestages
that may respond at lower levels, unless the
data originate from a susceptible study
population.
Animal-to-human extrapolation: For
reference values based on animal results, an
uncertainty factor reflects cross-species
differences, which may cause humans to
respond at lower levels.
Subchronic-to-chronic exposure: For
chronic reference values based on subchronic
50 studies, an uncertainty factor reflects the
51 likelihood that a lower level over a longer
52 duration may induce a similar response. This
53 factor may not be necessary for reference
54 values of shorter duration.
55 Adverse-effect level to no-observed-
56 adverse-effect level: For reference values
57 based on a lowest-observed-adverse-effect
58 level, an uncertainty factor reflects a level
59 judged to have no observable adverse effects.
60 Database deficiencies: If there is concern
61 that future studies may identify a more
62 sensitive effect, target organ, population, or
63 lifestage, a database uncertainty factor
64 reflects the nature of the database deficiency.
65 8. Process for Developing and Peer-
66 Reviewing IRIS Assessments
67 The IRIS process (revised in 2009 and
68 enhanced in 2013) involves extensive public
69 engagement and multiple levels of scientific
70 review and comment IRIS program scientists
71 consider all comments. Materials released,
72 comments received from outside EPA, and
73 disposition of major comments (steps 3, 4,
74 and 6 below) become part of the public
75 record.
76 Step 1: Draft development. As outlined
77 in section 2 of this Preamble, IRIS program
78 scientists specify the scope of an assessment
79 and formulate science issues for discussion
80 with the scientific community and the public.
81 Next, they release initial protocols for the
82 systematic review procedures planned for
83 use in the assessment IRIS program
84 scientists then develop a first draft, using
85 structured approaches to identify pertinent
86 studies, evaluate study methods and quality,
87 integrate the evidence of causation for each
88 health outcome, select studies for derivation
89 of toxicity values, and derive toxicity values,
90 as outlined in Preamble sections 3-7.
91 Step 2: Agency review. Health scientists
92 across EPA review the draft assessment.
93 Step 3: Interagency science
94 consultation. Other federal agencies and the
95 Executive Office of the President review the
96 draft assessment.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
Step 4: Public comment, followed by
external peer review. The public reviews
the draft assessment. IRIS program scientists
release a revised draft for independent
external peer review. The peer reviewers
consider whether the draft assessment
assembled and evaluated the evidence
according to EPA guidance and whether the
evidence justifies the conclusions.
Step 5: Revise assessment. IRIS
program scientists revise the assessment to
address the comments from the peer review.
Step 6: Final agency review and
interagency science discussion. The IRIS
program discusses the revised assessment
with EPA's program and regional offices and
with other federal agencies and the Executive
Office of the President.
Step 7: Post final assessment. The IRIS
program posts the completed assessment
and a summary on its website.
9. General Structure of IRIS
Assessments
Main text. IRIS assessments generally
comprise two major sections: (1) Hazard
Identification and (2) Dose-Response
Assessment Section 1.1 briefly reviews
chemical properties and toxicokinetics to
describe the disposition of the agent in the
body. This section identifies related
chemicals and summarizes their health
outcomes, citing authoritative reviews. If an
assessment covers a chemical mixture, this
section discusses environmental processes
that alter the mixtures humans encounter
and compares them to mixtures studied
experimentally.
Section 1.2 includes a subsection for each
major health outcome. Each subsection
discusses the respective literature searches
and study considerations, as outlined in
Preamble sections 3 and 4, unless covered in
the front matter. Each subsection concludes
with evidence synthesis and integration, as
outlined in Preamble section 5.
Section 1.3 links health hazard
information to dose-response analyses for
48 each health outcome. One subsection
49 identifies susceptible populations and
50 lifestages, as observed in human or animal
51 studies or inferred from mechanistic data.
52 These may warrant further analysis to
53 quantify differences in susceptibility.
54 Another subsection identifies biological
55 considerations for selecting health outcomes,
56 studies, or data sets, as outlined in Preamble
57 section 6.
58 Section 2 includes a subsection for each
59 toxicity value. Each subsection discusses
60 study selection, methods of analysis, and
61 derivation of a toxicity value, as outlined in
62 Preamble sections 6 and 7.
63 Front matter. The Executive Summary
64 provides information historically included in
65 IRIS summaries on the IRIS program website.
66 Its structure reflects the needs and
67 expectations of EPA's program and regional
68 offices.
69 A section on systematic review methods
70 summarizes key elements of the protocols,
71 including methods to identify and evaluate
72 pertinent studies. The final protocols appear
73 as an appendix.
74 The Preface specifies the scope of an
75 assessment and its relation to prior
76 assessments. It discusses issues that arose
77 during assessment development and
78 emerging areas of concern.
79 This Preamble summarizes general
80 procedures for assessments begun after the
81 date below. The Preface identifies
82 assessment-specific approaches that differ
83 from these general procedures.
84 August 2016
85
86 10. Preamble References
87 U.S. EPA. (1991). Guidelines for
88 developmental toxicity risk assessment (pp.
89 1-83). (EPA/600/FR-91/001). Washington,
90 DC: U.S. Environmental Protection Agency,
91 Risk Assessment Forum.
This document is a draft for review purposes only and does not constitute Agency policy.
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46
47
48
49
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http://cfpub.epa.gov/ncea/cfm/recordispla
v. cfm? deid=2 316 2
U.S. EPA. (1994). Methods for derivation of
inhalation reference concentrations and
application of inhalation dosimetry [EPA
Report] (pp. 1-409). (EPA/600/8-90/066F).
Research Triangle Park, NC: U.S.
Environmental Protection Agency, Office of
Research and Development, Office of Health
and Environmental Assessment,
Environmental Criteria and Assessment
Office.
https://cfpub.epa.gov/ncea/risk/recordispl
ay.cfm?deid=71993&CFTD=51174829&CFT0
KEN=25006317
U.S. EPA. (1996). Guidelines for reproductive
toxicity risk assessment (pp. 1-143).
(EPA/630/R-96/009). Washington, DC: U.S.
Environmental Protection Agency, Risk
Assessment Forum.
U.S. EPA. (1998). Guidelines for neurotoxicity
risk assessment. Fed Reg 63: 26926-26954.
U.S. EPA. (2002). A review of the reference
dose and reference concentration processes
(pp. 1-192). (EPA/630/P-02/002F).
Washington, DC: U.S. Environmental
Protection Agency, Risk Assessment Forum.
http://www.epa.gov/osa/review-reference-
dose-and-reference-concentration-processes
U.S. EPA. (2005a). Guidelines for carcinogen
risk assessment [EPA Report] (pp. 1-166).
(EPA/630/P-03/001F). Washington, DC: U.S.
Environmental Protection Agency, Risk
Assessment Forum.
http://www2.epa.gov/osa/guidelines-
carcinogen-risk-assessment
U.S. EPA. (2005b). Supplemental guidance for
assessing susceptibility from early-life
exposure to carcinogens (pp. 1-125).
(EPA/630/R-03/003F). Washington, DC: U.S.
Environmental Protection Agency, Risk
Assessment Forum.
U.S. EPA. (2006). Approaches for the
application of physiologically based
pharmacokinetic (PBPK) models and
supporting data in risk assessment (Final
Report) [EPA Report] (pp. 1-123).
(EPA/600/R-05/043F). Washington, DC: U.S.
Environmental Protection Agency, Office of
50 Research and Development, National Center
51 for Environmental Assessment
52 http://cfpub.epa.gov/ncea/cfm/recordispla
53 v.cfm?deid=157668
54 U.S. EPA. (20111. Recommended use of body
55 weight 3/4 as the default method in
56 derivation of the oral reference dose (pp. 1-
57 50). (EPA/100/R11/0001). Washington, DC:
58 U.S. Environmental Protection Agency, Risk
59 Assessment Forum, Office of the Science
60 Advisor.
61 https://www.epa.gov/risk/recommended-
62 use-bodv-weight-34-default-method-
63 derivation-oral-reference-dose
64 U.S. EPA. (2012). Benchmark dose technical
65 guidance (pp. 1-99). (EPA/100/R-12/001).
66 Washington, DC: U.S. Environmental
67 Protection Agency, Risk Assessment Forum.
68 U.S. EPA. (2014). Guidance for applying
69 quantitative data to develop data-derived
70 extrapolation factors for interspecies and
71 intraspecies extrapolation. (EPA/100/R-
72 14/002F). Washington, DC: Risk Assessment
73 Forum, Office of the Science Advisor.
74 http://www, epa. gov/raf/D D E F/pdf/ddef-
75 final.pdf
76
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EXECUTIVE SUMMARY
Summation of Occurrence and Health Effects
Ethyl tert-butyl ether (ETBE) does not occur naturally; it is an ether oxygenate
produced by humans and primarily used as a gasoline additive. It was used until 2006
in the United States and is still used in Japan and the European Union. ETBE is
released into the environment because of gasoline leaks, evaporation, and spills.
Exposure to ETBE can occur by drinking contaminated groundwater or by inhaling
off-gases containing ETBE. Dermal exposure is possible in occupational settings
where the manufacture ofETBE occurs. The magnitude of human exposure to ETBE
depends on factors such as the distribution ofETBE in groundwater and the extent of
the contamination.
Animal studies demonstrate that exposure to ETBE is associated with noncancer
kidney effects following oral and inhalation exposure. Evidence is suggestive of
carcinogenic potential for ETBE based on liver tumors in rats following inhalation
exposure.
Effects Other Than Cancer Observed Following Oral Exposure
Kidney effects were identified as a potential human hazard ofETBE exposure. Although no
human studies are available to evaluate the effects ofETBE, oral exposure studies in animals have
consistently reported increased kidney weight in male and female rats accompanied by increased
chronic progressive nephropathy (CPN), urothelial hyperplasia of the renal pelvis (in males), and
increased blood concentrations of total cholesterol, blood urea nitrogen (BUN), and creatinine.
Overall, there was consistency across multiple measures of potential kidney toxicity, including
organ weight increases, exacerbated CPN, urothelial hyperplasia of the renal pelvis, and increases
in serum markers of kidney function. Additionally, effects were also observed across routes of
exposure, and sex (with the exception of urothelial hyperplasia of the renal pelvis which was
observed only in male rats).
The relevance of the kidney findings to humans was evaluated with respect to a2u-globulin
nephropathy, a disease process that occurs exclusively in the male rat kidney {Capen, 1999,
699905; U.S. EPA, 1991, 635839}. While ETBE binds to a2u-globulin and meets some criteria of the
a2u-globulin EPA and IARC frameworks {U.S. EPA, 1991, 635839; Capen, 1999, 699905}, it does not
meet all. With respect to male rats, US EPA 1991 notes that "[i] f a compound induces a2u-globulin
accumulation in hyaline droplets, the associated nephropathy in male rats is not an appropriate
endpoint to determine noncancer (systemic) effects potentially occuring in humans." However, as
a2u-globulin nephropathy is strictly a male rat phenomenon, the dose-related kidney effectsin
female rats are not confounded by a2u-globulin nephropathy.
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It has been observed that chemicals that bind to a2u-globulin also exacerbate the incidence
and/or severity of background chronic progressive nephropathy (CPN) in male rats {Travlos, 2011,
1239901}{U.S. EPA, 1991, 635839}{Frazier, 2012, 2919046}. While the etiology of CPN is unknown
{Hard, 2004, 782757}{NIEHS, 2019, 5098230}{Peter, 1986,194755} and it has no known analog in
the aging human kidney {Hard, 2009, 667590}{NIEHS, 2019, 5098230}, it cannot be ruled out that a
chemical which exacerbates CPN in rats could also exacerbate disease processes in the human
kidney (e.g. chronic kidney disease, diabetic nephropathy, glomerulonephritis, interstitial nephritis,
etc){NIEHS, 2019, 5098230}. Therefore, increased incidence of kidney effects with ETBE exposure
in the female rat (but not the male rat) are considered appropriate for identifying a hazard to the
kidney.
Evidence is suggestive that liver toxicity follows oral ETBE exposure. The strongest
supporting evidence is the increased liver weights and centrilobular hypertrophy in exposed male
and female rats consistently reported across studies evaluating oral exposures. No additional
histopathological findings were observed, however, and only one serum marker potentially
indicative of liver toxicity [gamma-glutamyl transferase (GGT)] was elevated, while other markers
[aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase
(ALP)] were unchanged. The magnitude of change for these noncancer effects were minimal and,
except for organ weight data, did not exhibit consistent dose-response relationships. Mechanistic
data suggest that ETBE exposure leads to activation of several nuclear receptors, but inadequate
evidence exists to establish a relationship between receptor activation and liver toxicity resulting
from ETBE exposure. In addition, mechanistic data suggest possibly greater susceptibility of toxic
effects related to reduced clearance of acetaldehyde, a metabolite of ETBE. Thus, even with the
consistently observed increases in rat liver weight and centrilobular hypertrophy, the evidence
remains suggestive that liver toxicity follows ETBE exposure because of the relatively small
magnitude of effects and lack of consistent dose response relationships.
Inadequate information exists to draw conclusions regarding reproductive effects,
developmental effects, or immune system effects.
Oral Reference Dose (RfD) for Effects Other Than Cancer
Kidney toxicity, represented by increased absolute kidney weight in female rats, was chosen
as the basis for the overall oral reference dose (RfD) (See Table ES-1). The chronic study by (TPEC.
2010a) [with selected data published as Suzuki etal. f20121] and the observed kidney effects were
used to derive the RfD. The endpoint of increased kidney weight was selected as the critical effect
because it is a specific and sensitive indicator of kidney toxicity and was induced in a dose-
responsive manner. Benchmark dose (BMD) modeling was used to derive the benchmark dose
lower confidence limit (BMDLi0o/o) of 120 mg/kg-day. The BMDL was converted to a human
equivalent dose (HED) of 28.8 mg/kg-day using body weight3/4 scaling fU.S. EPA. 20111. and this
value was used as the point of departure (POD) for RfD derivation.
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1 The overall RfD was calculated by dividing the POD for increased absolute kidney weight by
2 a composite uncertainty factor (UF) of 30 to account for extrapolation from animals to humans (3)
3 and interindividual differences in human susceptibility (10).
4 Table ES-1. Organ-/system-specific RfDs and overall RfD for ETBE
Hazard
Basis
Point of
departure*
(mg/kg-day)
UF
Chronic RfD
(mg/kg-day)
Study
exposure
description
Confidence
Kidney
Increased
absolute
kidney
weight
28.8
30
lx 10°
Chronic
High
Overall
RfD
Kidney
28.8
30
1 X 10°
Chronic
High
5
6 * Human equivalent dose (HED) PODs were calculated using body weight to the % power (BW3/4) scaling (U.S. EPA,
7 2011).
8 Effects Other Than Cancer Observed Following Inhalation Exposure
9 Kidney effects are a potential human hazard of inhalation exposure to ETBE. While no
10 human studies are available to evaluate the effects of exposure, studies in animals have observed
11 increases in kidney weight, altered kidney histopathology, as well as alterations in clinical
12 chemistry including serum cholesterol, BUN, and creatinine. While the histological lesion of
13 urothelial hyperplasia of the renal pelvis was a sensitive endpoint in male rats, it was not observed
14 in female rats or mice of either sex, whereas increased kidney weights were observed in multiple
15 studies in rats of both sexes and in mice. Changes in kidney weight in female rats, were dose-
16 dependant, consistent across multiple studies, and are not confounded by a2u-globulin
17 nephropathy, and therefore considered appropriate for identifying a hazard to the kidney.
18 Inhalation Reference Concentration (RfC) for Effects Other Than Cancer
19 Kidney toxicity, represented by increased absolute kidney weight, was chosen as the basis
20 for the overall inhalation reference concentration (RfC) (See Table ES-2). The chronic study by TPEC
21 (2010b) [selected data published as Saito etal. (2013)] and the observed kidney effects were used
22 to derive the RfC. The endpoint, increased absolute kidney weight, was selected as the critical effect
23 because it is a specific and sensitive indicator of kidney toxicity and was induced in a dose-
24 responsive manner. BMD modeling was attempted, but an adequate fit was not achieved.
25 Therefore, a NOAEL was used to derive the POD of 6270 mg/m3. The NOAEL was adjusted for
26 continuous exposure and converted to a human equivalent concentration (HEC) of 1110 mg/m3.
27 The overall RfC was calculated by dividing the POD for increased absolute kidney weight by
28 a composite UF of 30 to account for toxicodynamic differences between animals and humans (3)
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1 and interindividual differences in human susceptibility (10).
2 Table ES-2. Organ-/system-specific RfCs and overall RfC for ETBE
Hazard
Basis
Point of
departure*
(mg/m3)
UF
Chronic RfC
(mg/m3)
Study exposure
description
Confidence
Kidney
Increased
absolute
kidney
weight
1110
30
4x 101
Chronic
Medium
Overall RfC
Kidney
1110
30
4x 101
Chronic
Medium
3 *Continuous inhalation HEC was adjusted for continuous daily exposure and calculated by adjusting the duration-
4 adjusted POD (PODadj) by the dosimetric adjustment factor (DAF = 0.992) for a Category 3 gas.
5 Evidence of Human Carcinogenicity
6 Under EPA's cancer guidelines (U.S. EPA. 2005a). the evidence of carcinogenic potential for
7 ETBE is suggestive for inhalation exposure and inadequate for oral exposure. ETBE induced liver
8 tumors in male (but not female) rats in a 2-year inhalation exposure study. No significant effects
9 were observed in two chronic oral studies in male and female rats {JPEC, 2010,1517477}{Maltoni,
10 1999, 87642} (see Section 1.2.5). Data on tumorigenicity in mice following ETBE oral or inhalation
11 exposure was not available. However, supplementary evidence from 2-stage initiation-promotion
12 oral carcinogenesis bioassays indicate increased mutagen-initiated liver, as well as increased tumor
13 incidence in the thyroid, colon, urinary bladder.
14 Quantitative Estimate of Carcinogenic Risk from Oral Exposure
15 A quantitative estimate of carcinogenic potential from oral exposure to ETBE was not
16 derived as an increase in tumors was not observed in the two available chronic oral cancer
17 bioassays. A route to route extrapolation of cancer risk from the inhalation to oral route was not
18 carried out because there was no consistent dose-response relationship observed for liver tumors
19 when compared across oral and inhalation studies on the basis of PBPK modeled internal dose.
20 Quantitative Estimate of Carcinogenic Risk from Inhalation Exposure
21 A quantitative estimate of carcinogenic potential from inhalation exposure to ETBE was
22 based on the increased incidence of hepatocellular adenomas and carcinomas in male F344 rats
23 following 2-year inhalation exposure fSaito etal.. 2013: TPEC. 2010bl. The study included
24 histological examinations for tumors in many different tissues, contained three exposure levels and
25 controls, contained adequate numbers of animals per dose group (~50/sex/group), treated animals
26 for up to 2 years, and included detailed reporting of methods and results.
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Although ETBE was considered to have "suggestive evidence of carcinogenic potential," the
main study fSaito etal.. 2013:1PEC. 2010bl was conducted according to well-established guidelines
for examining potential carcinogenicity and considered suitable for quantitative analyses. An
inhalation unit risk was derived for liver tumors in male F344 rats. The modeled ETBE POD was
scaled to an HEC according to EPA guidance based on inhalation dosimetry for a Category 3 gas
(U.S. EPA. 1994). Using linear extrapolation from the BMCLio, a human equivalent inhalation unit
risk was derived (inhalation unit risk = 0.1/BMCLi0). The inhalation unit risk is
8 x 10"5 per mg/m3.
Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes
ETBE is metabolized to tert-butanol and acetaldehyde. Evidence is suggestive that genetic
polymorphism of aldehyde dehydrogenase (ALDH)—the enzyme that oxidizes acetaldehyde to
acetic acid—could affect ETBE toxicity. The virtually inactive form, ALDH2*2, is found in about one-
half of all East Asians (and by extension people of East Asian ancestry) (Brennan et al.. 2004).
Evidence is strong in humans that heterozygous ALDH2 increases the internal dose and the cancer
risks from acetaldehyde, especially in the development of alcohol-related cancers in the esophagus
and upper aerodigestive tract, but relevance of this finding in regards to liver tumorigenesis is less
clear flARC. 20101. Several in vivo and in vitro genotoxicity assays in AIdh2 knockout (KO) and
heterozygous mice reported that genotoxicity was significantly increased compared with wild-type
controls following ETBE exposure to similar doses associated with cancer and noncancer effects in
rodents {Weng, 2019, 5343910}(Wengetal.. 2014: Wengetal.. 2013: Wengetal.. 2012: Wengetal..
2011). Inhalation ETBE exposure increased blood concentrations of acetaldehyde in AIdh2 KO mice
compared with wild type {Weng, 2013, 2279880}. Thus, exposure to ETBE in individuals with the
ALDH2*2 variant would increase the internal dose of acetaldehyde and potentially increase risks
associated with acetaldehyde produced by ETBE metabolism.
Collectively, these data present evidence that people with diminished ALDH2 activity could
be considered a susceptible population that could be more sensitive to ETBE exposure.
Key Issues Addressed in Assessment
The human relevance of the kidney effects observed in male and female rats was analyzed
in the assessment, particularly as they relate to a2u-globulin nephropathy and the exacerbation of
chronic progressive nephropathy. An evaluation of whether ETBE caused a2U-globulin-associated
nephropathy was performed using the EPA 1991 and IARC 1999 a2U-globulin frameworks {Capen,
1999, 699905}{U.S. EPA, 1991, 635839}. ETBE induced an increase in hyaline droplet
accumulation and increased a2U-globulin deposition in male rats; however, most of the subsequent
steps in the pathological sequence were not observed. Although the conditions were not fully met
with either a2U-globulin framework, {U.S. EPA, 1991, 635839@@author-year} states that"[i]f a
compound induces a2U-globulin accumulation in hyaline droplets, the associated nephropathy in
male rats is not an appropriate endpointto determine noncancer (systemic) effects potentially
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Toxicological Review of ETBE
occuring in humans." However, as a2u-globulin nephropathy is strictly a male rat phenomenon,
dose-related kidney effects in female rats are not confounded by a2u-globulin nephropathy. CPN
also plays a role in the exacerbation of nephropathy in rats, however the MOA of CPN is unknown,
and therefore, its potential relevance to humans cannot be ruled outjNIEHS, 2019, 5098230}.
Dose-related changes in several indicators of kidney toxicity were observed, including increased
absolute kidney weight, histological changes, and increased blood biomarkers (Saito etal.. 2013:
Suzuki etal.. 2012: TPEC. 2010a. b). These specific effects are considered relevant to humans,
particularly the endpoints observed in female rats, as they are not confounded by a2u-globulin
related processes.
In addition, the human relevance of the observed liver tumors was discussed in the
assessment (see Sections 1.2.2 and 1.3.2). Briefly, a well conducted inhalation study demonstrated
a significant, positive exposure-response for hepatocellular adenomas and carcinomas in male rats
(Saito etal.. 2013: TPEC. 2010b). While the majority of liver tumors occurred at the highest
exposure, statistical tests conducted by the study authors found significant dose-response trend by
both the Peto test (incidental tumor test) and the Cochran-Armitage test However, two chronic
oral exposure studies (one with survival issues) were negative for liver tumors {Maltoni, 1999,
87642}{JPEC, 2010, 1517477}.
The potential MOA of for the observed liver tumors was evaluated in the assessement (see
Section 1.2.2). The available evidence base for the nuclear hormone receptor MOAs (i.e., PPARa,
PXR, and CAR) was inadequate to determine the role these pathways play, if any, in ETBE-induced
liver carcinogenesis. Acetaldehyde-mediated genotoxicity also was evaluated as a possible MOA,
and although evidence suggests that ALDH2 deficiency enhanced ETBE-induced genotoxicity in
exposed mice, the available database was inadequate to establish acetaldehyde-mediated
mutagenicity as an MOA for ETBE-induced liver tumors. No other MOAs for liver carcinogenesis
were identified, and the rat liver tumors observed following inhalation exposure are considered
relevant to humans (U.S. EPA. 2005a).
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1 LITERATURE SEARCH STRATEGY | STUDY
2 SELECTION AND EVALUATION
A literature search and screening strategy consisted of a broad search of online scientific
databases and other sources to identify all potentially pertinent studies. In subsequent steps,
references were screened to exclude papers not pertinent to an assessment of the health effects of
ETBE, and remaining references were sorted into categories for further evaluation. The original
chemical-specific search was conducted in four online scientific databases, PubMed, Toxline, Web of
Science, and TSCATS, through December 2016, using the keywords and limits described in Table
LS-1. The overall literature search approach is shown graphically in Figure LS-1. Another 114
citations were obtained using additional search strategies described in Table LS-2. After
electronically eliminating duplicates from the citations retrieved through these databases, 847
unique citations were identified. The resulting 847 citations were screened for pertinence and
separated into categories as presented in Figure LS-1
Figure LS-1 using the title and either abstract or full text, or both, to examine the health
effects of ETBE exposure. The inclusion and exclusion criteria used to screen the references and
identify sources of health effects data are provided in Table LS-3.
• 33 references were identified as potential "Sources of Health Effects Data" and were
considered for data extraction to evidence tables and exposure-response arrays.
• 70 references were identified as "Supporting Studies." These included 31 studies
describing physiologically based pharmacokinetic (PBPK) models and other
toxicokinetic information; 25 studies providing genotoxicity and other mechanistic
information; 9 acute, short-term, or preliminary toxicity studies; and 5 direct
administration (e.g., dermal) studies of ETBE. Although still considered sources of
health effects information, studies investigating the effects of acute and direct chemical
exposures are generally less pertinent for characterizing health hazards associated with
chronic oral and inhalation exposures. Therefore, information from these studies was
not considered for extraction into evidence tables. Nevertheless, these studies were still
evaluated as possible sources of supplementary health effects information.
• 29 references were identified as "Secondary Literature and Sources of Contextual
Information" (e.g., reviews and other agency assessments); these references were
retained as additional resources for development of the Toxicological Review.
• 715 references were identified as being not pertinent (not on topic) to an evaluation of
health effects for ETBE and were excluded from further consideration (see Figure LS-1)
for exclusion categories and Table LS-3 for exclusion criteria). For example, health effect
studies of gasoline and ETBE mixtures were not considered pertinent to the assessment
because the separate effects of gasoline components could not be determined.
Retrieving numerous references that are not on topic is a consequence of applying an
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Toxicological Review of ETBE
initial search strategy designed to cast a wide net and to minimize the possibility of
missing potentially relevant health effects data.
Figure LS-lThe complete list of references as sorted above can be found on the ETBE
project page of the HERO website at
https://hero.epa.gov/hero/index.cfm/project/page/project id/1376.
Post-Peer-Review Literature Search Update
A post-peer-review literature search update was conducted in PubMed, Toxline, TSCATS,
and DTIC for the period Dec 2016 to July 2019 using a search strategy consistent with previous
literature searches (see Table LS-1). Consistent with the IRIS Stopping Rules
(https://www.epa.gov/sites/production/files/2Q14-Q6/documents/iris stoppingrules.pdf).
manual screening of the literature search update focused on identifying new studies that might
change a major conclusion of the assessment. No animal bioassays or epideminological studies
were identified in the post-peer-review literature search which would change any major
conclusions in the assessment.
The documentation and results for the literature search and screen, including the specific
references identified using each search strategy and tags assigned to each reference based on the
manual screen, can be found on the HERO website on the ETBE project page at:
(https://hero.epa.gov/hero/mdex.cfin/project/page/project ).
Selection of Studies for Inclusion in Evidence Tables
To summarize the important information systematically from the primary health effects
studies in the ETBE evidence base, evidence tables were constructed in a standardized tabular
format as recommended by NRC f20111. Studies were arranged in evidence tables by route of
exposure and then alphabetized by author. Of the studies retained after the literature search and
screen, 33 were identified as "Sources of Health Effects Data" and considered for extraction into
evidence tables for the hazard identification in Section 1. Initial review of studies examining
neurotoxic endpoints did not find consistent effects to warrant a comprehensive hazard evaluation;
thus, the one subchronic study fDorman etal.. 19971 that examined neurotoxic endpoints only was
not included in evidence tables. Data from the remaining 32 studies were extracted into evidence
tables.
Supplementary studies that contain pertinent information for the Toxicological Review and
augment hazard identification conclusions, such as genotoxic and mechanistic studies, studies
describing the kinetics and disposition of ETBE absorption and metabolism, and pilot studies, were
not included in the evidence tables. One controlled human exposure toxicokinetic study was
identified, which is discussed in Appendix B.2 (Toxicokinetics). Short-term and acute studies did
not differ qualitatively from the results of the longer-term studies (i.e., >90-day exposure studies).
These were grouped as supplementary studies, however, because the evidence base of chronic and
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1 subchronic rodent studies was considered sufficient for evaluating chronic health effects of ETBE
2 exposure. Additionally, studies of effects from chronic exposure are most pertinent to lifetime
3 human exposure (i.e., the primary characterization provided by IRIS assessments) and are the focus
4 of this assessment Such supplementary studies can be discussed in the narrative sections of
5 Section 1 and are described in sections such as Mode of action analysis to augment the discussion or
6 presented in appendices, if they provide additional information.
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Database Searches
(see Table LS-1 for keywords and limits)
PubMed
n = 148
Web of Science
n = 518
Toxline
(inci. TSCATS)
n = 105
TSCATS 2
n = 1
Additional Search Strategies
(see Table LS-2 for methods and
results)
n = 114
Combined Dataset
(After duplicates removed electronically)
n = 847
Manual Screening For Pertinence
(Title/Abstract/Full Text)
Excluded/Not on Topic {n = 715)
50
Abstract only/comment/society
abstracts
82
Biodegradation/environmental fate
385
Chemical analysis/fuel chemistry
180
Other chemical/non ETBE
7
Exposure and biological monitoring
11
Methodology
Sources of Health Effects Data (n = 33)
0 Human health effects studies
33 Animal studies
Secondary Literature and Sources of
Contextual Information (n = 29)
1 QSAR
7 Mixtures
14 Reviews/editorials
5 Other agency assessments
2 Odor threshold
Supporting Studies
Sources of Supporting Health Effects Data
(n - 14)
5 Not relevant exposure paradigms (e.g.,
dermal, eye irritation)
9 Preliminary/acute data
Sources of Mechanistic and Toxicokinetic
Data (n = 56)
31 PBPK/ADME
13 Genotoxicity
12 Other mechanistic studies
1
2
Figure LS-1. Summary of literature search and screening process for ETBE.
3
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1
2 Table LS-1. Details of the search strategy employed for ETBE
Database
(Search date)
Keywords
Limits
PubMed
(03/31/2014)
Updated
(12/2016)
"ETBE" OR "Ethyl tert-butyl ether"
OR "2-ethoxy-2-methyl-propane" OR
"ethyl tertiary butyl ether" OR "ethyl
tert-butyl oxide" OR "tert-butyl ethyl
ether" OR "ethyl t-butyl ether" OR
"637-92-3"
None
Web of Science
(03/31/2014)
Updated
(12/2016)
"ETBE" OR "ethyl tert-butyl ether"
OR "2-ethoxy-2-methyl-propane" OR
"ethyl tertiary butyl ether" OR "ethyl
tert-butyl oxide" OR "tert-butyl ethyl
ether" OR "ethyl t-butyl ether" OR
"637-92-3"
Lemmatization on (e.g. the search term is reduced to
its lexical root)
Toxline
(includes
TSCATS)
(03/31/2014)
Updated
(12/2016)
"ETBE" OR "Ethyl tert-butyl ether"
OR "2-Ethoxy-2-methyl-propane" OR
"ethyl tertiary butyl ether" OR "ethyl
tert-butyl oxide" OR "tert-butyl ethyl
ether" OR "ethyl t-butyl ether" OR
"637-92-3"
Not PubMed
TSCATS2
(3/31/2014)
Updated
(12/2016)
637-92-3
01/2004 to 7/2019
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Table LS-2. Summary of additional search strategies for ETBE
Approach used
Source(s)
Date
performed
Number of additional
references identified
Electronic
backward search
through Web of
Science
Review article: Mcgregor (2007).
"Ethyl tertiary-butyl ether: a
toxicological review." Critical
Reviews in Toxicology 37(4):
287-312
3/2014
68 references
Review article: de Pevster (2010).
"Ethyl t-butyl ether: Review of
reproductive and developmental
toxicity." Birth Defects Research,
Part B: Developmental and
Reproductive Toxicology 89(3):
239-263
3/2014
26 references
Personal
communication
Japan Petroleum Energy Center
3/2014
Updated
(12/2016)
21 references
Table LS-3. Inclusion-exclusion criteria
Inclusion criteria
Exclusion criteria/Supplemental material*
Population
• Humans
• Standard mammalian animal models,
including rat, mouse, rabbit, guinea
pig, monkey, dog
• Ecological species*
• Nonmammalian species*
Exposure
• Exposure is to ETBE
• Exposure is measured in an
environmental medium (e.g., air,
water, diet)
• Exposure via oral or inhalation routes;
for supporting health effect studies,
exposure via oral or inhalation routes
• Study population is not exposed to ETBE
• Exposure to a mixture only (e.g., gasoline
containing ETBE)
• Exposure via injection (e.g., intravenous)
• Exposure paradigm not relevant (e.g., acute,
dermal, or ocular)
Outcome
• Study includes a measure of one or
more health effect endpoints,
including effects on the nervous,
kidney/urogenital, musculoskeletal,
cardiovascular, immune, and
gastrointestinal systems;
reproduction; development; liver;
eyes; and cancer
• Odor threshold studies*
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Inclusion criteria
Exclusion criteria/Supplemental material*
Other
Not on topic, including:
• Abstract only, editorial comments, policy
papers, were not considered further because
study was not potentially relevant
• Bioremediation, biodegradation, or
environmental fate of ETBE, including
evaluation of wastewater treatment
technologies and methods for remediation of
contaminated water and soil
• Chemical, physical, or fuel chemistry studies
• Analytical methods for measuring/detecting/
remotely sensing ETBE
• Not chemical specific: Studies that do not
involve testing of ETBE
• Quantitative structure activity relationship
studies
• Exposure studies without health effect
evaluation
*Studies that met this exclusion criterion were considered supplemental, e.g. not considered a primary source of
health effects data, but were retained as potential sources of contextual information.
1 Evidence base Evaluation
2 For this draft assessment, 33 experimental animal studies comprised the primary sources of
3 health effects data; no studies were identified that evaluated humans exposed to ETBE (e.g., cohort
4 studies, case reports, ecological studies). The animal studies were evaluated considering aspects of
5 design, conduct, or reporting that could affect the interpretation of results, overall contribution to
6 the synthesis of evidence, and determination of hazard potential as noted in various EPA guidance
7 documents fU.S. EPA. 2005a. 1998b. 1996.1991b). The objective was to identify the stronger, more
8 informative studies based on a uniform evaluation of quality characteristics across studies of
9 similar design. Studies were evaluated to identify their suitability based on:
• Study design
• Nature of the assay and validity for its intended purpose
• Characterization of the nature and extent of impurities and contaminants of ETBE
administered, if applicable
• Characterization of dose and dosing regimen (including age at exposure) and their
adequacy to elicit adverse effects, including latent effects
• Sample sizes to detect dose-related differences or trends
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• Ascertainment of survival, vital signs, disease or effects, and cause of death
• Control of other variables that could influence the occurrence of effects
Additionally, several general considerations, presented in Table LS-4, were used in evaluating the
animal studies (Table LS-5). Much of the key information for conducting this evaluation can be
determined based on study methods and how the study results were reported. Importantly, the
evaluation at this stage does not consider the direction or magnitude of any reported effects.
EPA considered statistical tests to evaluate whether the observations might be due to
chance. The standard for determining statistical significance of a response is a trend test or
comparison of outcomes in the exposed groups against those of concurrent controls. Studies that
did not report statistical testing were identified and, when appropriate, statistical tests were
conducted by EPA.
Information on study features related to this evaluation is reported in evidence tables and
documented in the synthesis of evidence. Discussions of study strengths and limitations are
included in the text where relevant. If EPA's interpretation of a study differs from that of the study
authors, the draft assessment discusses the basis for the difference.
Experimental Animal Studies
The 33 experimental animal studies, all of which were performed on rats, mice, and rabbits,
were associated with drinking water, oral gavage, or inhalation exposures to ETBE. Many of these
studies were conducted according to Organisation for Economic Co-operation and Development
Good Laboratory Practice (GLP) guidelines and used well established methods, were well-reported,
and evaluated an extensive range of endpoints and histopathological data. For the body of available
studies, detailed discussion of any identified methodological concerns precedes each endpoint
evaluated in the hazard identification section. Overall, the experimental animal studies of ETBE
involving repeated oral or inhalation exposure were considered acceptable quality, and whether
yielding positive, negative, or null results, were considered in assessing the evidence for health
effects associated with chronic exposure to ETBE.
Table LS-4. Considerations for evaluation of experimental animal studies
Methodological
feature
Considerations
(relevant information extracted into evidence tables)
Test animal
Suitability of species, strain, sex, and source of test animals
Experimental design
Suitability of animal age/lifestage at exposure and endpoint testing; periodicity and
duration of exposure (e.g., hr/day, day/week); timing of endpoint evaluations; and
sample size and experimental unit (e.g., animals, dams, litters)
Exposure
Characterization of test article source, composition, purity, and stability; suitability of
control (e.g., vehicle control); documentation of exposure techniques (e.g., route,
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chamber type, gavage volume); verification of exposure levels (e.g., consideration of
homogeneity, stability, analytical methods)
Endpoint evaluation
Suitability of specific methods for assessing endpoint(s) of interest
Results presentation
Data presentation for endpoint(s) of interest (including measures of variability) and for
other relevant endpoints needed for results interpretation (e.g., maternal toxicity,
decrements in body weight relative to organ weight)
Table LS-5. Summary of experimental animal evidence base
Study Category
Study duration, species/strain, and administration method
Chronic
2-vear studv in F344 rats (drinking water) JPEC (2010a)*; Suzuki et al. (2012)
2-year studv in F344 rats (inhalation) JPEC (2010b)*; Saito et al. (2013)
2-vear studv in Sprague-Dawlev rats (gavage) Maltoni et al. (1999)
Subchronic
13-week studv in F344 rats (inhalation) Medinskv et al. (1999); Bond et al. (1996b)
26-week studv in Sprague-Dawlev rats (gavage) JPEC (2008c)*; Mivata et al. (2013)
Fuiiietal. (2010); JPEC (2008e)
13-week studv in Sprague-Dawlev rats (inhalation) JPEC (2008b)*
23-week studv in F344 rats (gavage) Hagiwara et al. (2011); JPEC (2008d)
13-week studv in CD-I mice (inhalation) Medinskv et al. (1999); Bond et al. (1996a)
23-week studv in Wistar rats (gavage) Hagiwara et al. (2015)
31-week studv in F344/DuCrlCrli rats (drinking water) Hagiwara et al. (2013)
13-week studv in C57BL/6 mice (inhalation) Weng et al. (2012)
Reproductive
Two-generation reproductive toxicity studv on Sprague-Dawlev rats (gavage) Gaoua
(2004b)*
One-generation reproductive toxicity studv on Sprague-Dawlev rats (gavage) Fuiii et al.
(2010); JPEC (2008e)
2-week studv on Simonson albino rats (drinking water) Berger and Horner (2003)
9-week studv on C57BL/6 mice (inhalation) Weng et al. (2014)
14-dav studv on F344 rats (gavage) de Pevster et al. (2009)
Two-generation reproductive toxicity studv in Sprague-Dawlev rats (gavage) Gaoua
(2004b)*
Developmental
Developmental studv (GD6-27) on New Zealand rabbits (gavage) Asano et al. (2011);
JPEC (2008i)
Developmental studv (GD5-19) on Sprague-Dawlev rats (gavage) Aso et al. (2014); JPEC
(2008h)
Developmental studv (GD5-19) on Sprague-Dawlev rats (gavage) Gaoua (2004b)*
Developmental studv (GD5-19) on Sprague-Dawlev rats (gavage) Gaoua (2004a)*
Pharmacokinetic
Single-dose studv on Sprague-Dawlev rats (gavage) JPEC (2008g)
14-dav studv on Sprague-Dawlev rats (gavage) JPEC (2008f)
Single-dose studv on Sprague-Dawlev rats (gavage) JPEC (2008g)*
14-dav studv on Sprague-Dawlev rats (gavage) JPEC (2008f)*
2 *The IRIS program had this study peer reviewed.
This document is a draft for review purposes only and does not constitute Agency policy.
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i 1.HAZARD IDENTIFICATION
2 1.1. OVERVIEW OF CHEMICAL PROPERTIES AND TOXICOKINETICS
3 1.1.1. Chemical Properties
4 ETBE is a liquid at a temperature range of -94 to 72.6°C. It is soluble in ethanol, ethyl ether,
5 and water fDrogos and Diaz. 20011. ETBE has a strong, highly objectionable odor and taste at
6 relatively low concentrations. The chemical is highly flammable and reacts with strong oxidizing
7 agents. ETBE is stable when stored at room temperature in tightly closed containers (Drogos and
8 Diaz. 20011. Information on physiochemical properties for ETBE is available at U.S. Environmental
9 Protection Agency (EPA)'s Comptox Chemicals Dashboard (https://comptox.epa.gov/dashboard/)
10 and is summarized in Table 1-1.
11 Table 1-1. Chemical identity and physicochemical properties of ethyl tert-
12 butyl ether (ETBE) from EPA's CompTox Chemicals Dashboard
Characteristic or property
Value
Chemical structure
H,C
3\.CH3
K,C'^^0'X\
ch3
CASRN
637-92-3
Synonyms
Ethyl T-butyl ether; 2-Ethoxy-2-methylpropane; Propane, 2-ethoxy-2-methyl;
Ethyl tert-butyl ether; 2-Methyl-2-ethoxypropane
(see https://comptox.eDa.gov/dashboard for additional svnonvms)
Molecular formula
C6Hi40
Molecular weight
102.177
Average experimental value3
Average predicted value3
Flash point (°C)
—
-10.9
Boiling point (°C)
72.4
74.3
Melting point (°C)
-94
-90.8
Log Kow
1.72
Density (g/cm3)
0.768
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Henry's law constant
(atm-m3/mole)
1.64 x 10"3
1.66 x 10"3
Vapor pressure (mm Hg at 20°C)
124
124
atm = atmosphere; CASRN = Chemical Abstracts Service registry number.
aMedian values and ranges for physical chemical properties of ETBE are also provided on the CompTox Chemicals
Dashboard at https://comptox.epa.gov/dashboard/.
1.1.2. Toxicokinetics
ETBE is rapidly absorbed following exposure by oral and inhalation routes (see Appendix
B.l.l). Studies in experimental animals indicate that >90% of the compound was absorbed after
oral administration within 6-10 hours flPEC. 2008d. e). No data are available for oral absorption in
humans. ETBE is moderately absorbed following inhalation exposure in both rats and humans;
human blood levels of ETBE approached—but did not reach—steady-state concentrations within
2 hours, and a net respiratory uptake of ETBE was estimated to be 26% in a short term exposure
study conducted at light activity levels fNihlen et al.. 1998bl.
ETBE and its metabolite, tert-butanol, are distributed throughout the body following oral,
inhalation, and i.v. exposures (TPEC. 2008d. e; Poetetal.. 1997: Faulkner et al.. 1989: ARCO. 1983).
Following exposure to ETBE in rats, ETBE was found in kidney, liver, and blood. Comparison of
ETBE distribution in rats and mice demonstrated that concentrations of ETBE in the rat kidney and
mouse liver are proportional to the blood concentration.
A general metabolic scheme for ETBE, illustrating the biotransformation in rats and
humans, is shown in Figure 1-1 (see Appendix B.1.3).
Human data on the excretion of ETBE was measured in several studies (Nihlenetal.. 1998a.
c). The half-life of ETBE in urine was biphasic with half-lives of 8 minutes and 8.6 hours (Tohanson
etal.. 19951. These studies showed urinary excretion of ETBE to be less than 0.2% of the uptake or
absorption of ETBE (Nihlen etal.. 1998a. c). Ambergetal. (20001 observed a similar half-life of 1-6
hours after human exposure to ETBE of 170 mg/m3; however, the elimination for ETBE in rat urine
was considerably faster than in humans, and ETBE itself was undetectable in rat urine.
A more detailed summary of ETBE toxicokinetics is provided in Appendix B.l.
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glucuronide-0 ——ch3
CH3
t-butyl glucuronide
H0\^°
CYP2A6
CYP3A4
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effects observed in ETBE have been attributed to tert-butanol (Salazar etal.. 20151. Animal studies
demonstrate that chronic exposure to tert-butanol is associated with noncancer kidney effects,
including increased kidney weights in male and female rats accompanied by increased chronic
progressive nephropathy (CPN), urothelial hyperplasia/transitional epithelial hyperplasia (in males
and females), and increased suppurative inflammation in females fNTP. 1997.1995b).
Inhalation exposures to acetaldehyde were concluded to cause carcinomas of the nasal
mucosa in rats and carcinomas of the larynx in hamsters (IARC. 1999b). In addition, acetaldehyde
was concluded to be the key metabolite in cancer of the esophagus and aerodigestive tract
associated with ethanol consumption flARC. 20101.
MTBE is a structurally related compound that is metabolized to formaldehyde and
tert-butanol. In 1996, the U.S. Agency for Toxic Substances and Disease Registry's (ATSDR)
Toxicological Profile for MTBE fATSDR. 19961 identified cancer effect levels of MTBE based on
carcinogenicity data in animals. ATSDR reported that inhalation exposure resulted in kidney cancer
in rats and liver cancer in mice. ATSDR concluded that oral exposure to MTBE might cause liver and
kidney damage and nervous system effects in rats and mice. The chronic inhalation minimal risk
level was derived based on incidence and severity of chronic progressive nephropathy in female
rats fATSDR. 19961. In 1998, the International Agency for Research on Cancer (IARC) found
"limited" evidence of MTBE carcinogenicity in animals and classified MTBE in Group 3 (i.e., not
classifiable as to carcinogenicity in humans) (IARC. 1999d). Although some similar effects are seen
with tert-butanol and ETBE, the evidence from MTBE is confounded by its metabolite
formaldehyde, a known human carcinogen (as classified by IARC and NTP).
1.2. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM
1.2.1. Kidney Effects
Synthesis of Effects in Kidney
This section reviews the studies that investigated whether subchronic or chronic exposure
to ETBE can cause kidney toxicity in humans or animals. The evidence base examining kidney
effects following ETBE exposure contains no human data and 10 animal studies, predominantly in
rats. Exposures ranged from 13 weeks to 2 years and both inhalation and oral exposure routes are
well represented. Studies using short-term and acute exposures that examined kidney effects are
not included in the evidence tables; however, they are discussed in the text if they provided data to
inform mode of action (MOA) or hazard identification. Four unpublished technical reports relevant
to the kidney were externally peer reviewed at the request of EPA in August 2012 (Table LS-5):
TPEC f2010al. TPEC f2010bl. TPEC f2008cl. TPEC f2008bl. some of which were subsequently
published. These are TPEC f2010al [published as Suzuki etal. f20121], TPEC f2010bl [published as
Saito etal. f20131], and TPEC f2008cl [published as Mivataetal. f20131], Gaoua f2004bl was
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externally peer reviewed at the request of EPA in November 2008. Studies are arranged in evidence
tables by effect and alphabetical order by author.
The unpublished report by Cohen etal. f20111 was not peer reviewed externally. In Cohen
etal. (20111. a pathology working group reexamined kidney histopathology from the TPEC f2010al
[subsequently published as Suzuki etal. f20121] and TPEC f2007al studies. Cohen etal. f20111 did
not report incidences of carcinomas that differed from those in the original study (Suzuki etal..
2012: TPEC. 2010a): thus, these data have been presented only once and it was not considered
necessary to have the Cohen report peer reviewed externally. Histopathological interpretations
from both Cohen etal. f20111 and TPEC f2007bl are considered for hazard identification.
The design, conduct, and reporting of each study were reviewed, and each study was
considered adequate to provide information pertinent to this assessment Interpretation of non-
neoplastic kidney endpoints in rats, however, is complicated by the common occurrence of age-
related spontaneous lesions characteristic of CPN (NTP. 2015: Hard etal.. 2013: Melnick etal..
2012: U.S. EPA. 1991a): http://ntp.niehs.nih.gov/nnl/urinary/kidney/necp/index.html. CPN is
more severe in male rats than in females and is particularly common in the Sprague-Dawley and
Fischer 344 strains. Dietary and hormonal factors play a role in modifying CPN, although the
etiology is unknown (see further discussion below).
Kidney weight. Kidney weights (see Figure 1-2) exhibited strong dose-related increases
with estimates of the primary ETBE metabolite, tert-butanol, in blood in male rats following oral
exposures (Spearman's rank coefficient = 0.86, p < 0.01) of 16 weeks or longer (Mivataetal.. 2013:
Suzuki etal.. 2012: Fuiii etal.. 2010: TPEC. 2010a. 2008c: Gaoua. 2004b). and following inhalation
exposures (Spearman's rank coefficient = 0.71, p = 0.05) of 13 weeks or longer (Saito etal.. 2013:
TPEC. 2010b. 2008b: Medinskv et al.. 19991. Kidney weight also showed strong dose-related
increases following inhalation exposure (Spearman's rank coefficient = 0.82, p = 0.01) and
moderate dose-related increases following oral exposure (Spearman's rank coefficient = 0.42, p =
0.2). Short-term studies in rats also observed increased kidney weight (TPEC. 2008a). In utero
ETBE exposure induced greater increases in kidney weights in F1 male and female rats compared
to parental exposure in one unpublished study (independently peer reviewed via EPA contract, see
Table LS-5) but the magnitude of increases were comparable to those observed in other adult oral
studies fGaoua. 2004bl. A 13-week mouse inhalation study observed small increases in kidney
weight in both sexes, with a greater magnitude of effect in males, up to 10% as compared to 4-6%
increases in females, see Figure 1-3 {Bond, 1996, 74002}{Medinsky, 1999,10740}.
In most of the studies with data available for relative and absolute organ weight
comparisons, both relative and absolute kidney weights are increased (Mivataetal.. 2013: Saito et
al.. 2013: Suzuki etal.. 2012: TPEC. 2010b. 2008b. c; Gaoua. 2004b). Measures of relative, as
opposed to absolute, organ weight can account for the influence of body weight on some organ
weights fBailev etal.. 20041. For ETBE, body weight in exposed animals was consistently decreased
at several doses relative to controls in the oral and inhalation studies. Thus, use of relative organ
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weight change would not be a reliable measure of the kidney weight increase for this assessment.
Additionally, a recent analysis indicates that absolute, but not relative, subchronic kidney weights
are significantly correlated with chemically induced histopathological findings in the kidney in
chronic and subchronic studies fCraig etal.. 20141. Therefore, absolute weight was used as the
more appropriate measure of kidney weight change for determining ETBE hazard potential.
Absolute and relative kidney weight data are presented in Appendix B of the Supplemental
Information.
Interpretation of 2-year kidney weight data in male rats treated by inhalation is
complicated by increased mortality attributed to CPN which would be expected to bias) the analysis
of kidney weight (toward the null), as organ weight was not assessed in animals that did not
survive to study termination fSaito etal.. 2013: Suzuki etal.. 2012: TPEC. 2010a. b). CPN is an age-
associated disease characterized by cell proliferation and chronic inflammation that results in
increased kidney weight (Melnick et al.. 2 012: Travlos etal.. 2011). thus animals severely affected
by CPN, including those that died due to CPN, would be expected to have enlarged kidneys.
Although mortality in female rats in the 2-year inhalation study was also significantly increased, the
study authors attributed these deaths to pituitary tumors, which would not be expected to bias
measurement of kidney weight {JPEC, 2010,1517421}. Mortality of male and female rats in the 2
year drinking water studies was not significantly different from controls {JPEC, 2010,1517477}.
Kidney histopathology. Kidney lesions also were observed in several studies. The incidence
of nephropathy, which was characterized as CPN due to sclerosis of glomeruli, thickening of the
renal tubular basement membranes, inflammatory cell infiltration, and interstitial fibrosis, was not
increased in any chronic study because of ETBE exposure. However, the severity of CPN was
exacerbated by ETBE in male and female rats in a 2-year inhalation study, and the number of CPN
foci was increased in male rats in a 13-week drinking water study (see Table 1-2) f Cohen etal..
2011: TPEC. 2010b. 2007a). Increases in CPN graded as marked or severe were dose-related when
compared on an internal dose basis across routes of exposure in male and female rats (Salazar et
al.. 20151.
CPN is a common and well-established constellation of age-related lesions in the kidney of
rats, although the mode of action of CPN is not known. In addition, no known counterpart to CPN
has been identified in the aging human kidney. However, several individual lesions noted in CPN
(e.g. tubule atrophy, tubule dilation, thickening of tubular basement membranes,
glomerulosclerosis) also occur in the human kidney {Frazier, 2012, 2919046}{Lusco, 2016,
5926047}{Zoja, 2015, 5926046}{Satirapoj, 2012, 5926045}{NTP, 2019, 5926049}. Therefore,
exacerbation of one or more of these lesions following ETBE exposure may reflect some type of cell
injury or inflammatory process, which is relevant to the human kidney.
Increased incidence of urothelial hyperplasia (also known as transitional epithelial
hyperplasia) of the renal pelvis (graded as slight or minimal) was observed in male rats in 2-year
studies by both inhalation and oral exposure fSaito etal.. 2013: Suzuki etal.. 2012: TPEC. 2010a. b).
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However, urothelial hyperplasia was not observed in female rats following 2 years of oral or
inhalation exposure fSaito etal.. 2013: Suzuki etal.. 2012: TPEC. 2010a. b). The histological finding
of urothelial hyperplasia represents in an increase in the layers of urothelium and is typically
associated with inflammation or neoplasia {Peterson, 2019, 5374688}{NTP, 2019, 5934030}. The
increase in urothelial hyperplasia in male rats appeared to be dose related on an internal dose basis
across routes of exposure (Salazar etal.. 2015). Cohen etal. (2011). however, attributed this effect
to CPN rather than the "direct" result of ETBE treatment In addition, there is some confusion
regarding the terminology of "urothelial hyperplasia of the renal pelvis" as reported in the JPEC
studies {JPEC, 2010,1517477;JPEC, 2010,1517421}. Specifically, based on the pathological
description of this lesion, it may represent proliferation of the papillary lining epithelium and not
true "urothelial hyperplasia" {NIEHS, 2019, 5098230}. Hyperplasia of the epithelial lining of the
renal papilla has been associated with advanced CPN {NTP, 2019, 5926049}.
To determine if the severity of the hyperplasia was positively associated with the severity of
CPN, contingency tables comparing the occurrence of urothelial hyperplasia with CPN in individual
rats were arranged by severity and analyzed with Spearman's rank correlation tests to determine
strength of associations for each comparison (Table 1-25,1-6). Urothelial hyperplasia and severity
of CPN were weakly correlated (Spearman's rank coefficient = 0.36) in males following oral and
inhalation exposure to ETBE. The biological significance of urothelial hyperplasia and any
relationship with CPN is discussed in Mode of Action Analysis (see below).
The number and size of hyaline droplets were increased in the proximal tubules of male
rats, but not in females, and the hyaline droplets tested positive for the presence of a2u-globulin
(Mivata etal.. 2013: TPEC. 2008c. e, f; Medinskv etal.. 1999). The significance of this finding, along
with other potentially related histopathological effects, such as necrosis, linear tubule
mineralization, and tubular hyperplasia, are discussed in Mode of action analysis (see below).
Serum and urinary biomarkers. The increased kidney weight and CPN in male rats is
associated with several changes in urinary and serum biomarkers of renal function (see Table 1-2,
Table 1-3). CPN is proposed to be associated with several changes in urinary and serum measures
such as proteinuria, blood urea nitrogen (BUN), creatinine, and hypercholesterolemia (Hard etal..
2009). In general, ETBE exposure, increased serum measures at lower doses and in more studies
than were associated with increased CPN severity. Considering male rat blood concentrations in
both chronic and subchronic studies, total cholesterol was elevated in 3 of 4 studies, BUN was
elevated in 2 of 4 studies, and creatinine was elevated 1 of 4 studies fMivata etal.. 2013: Saito etal..
2013: Suzuki etal.. 2012: TPEC. 2010a. b, 2008c). In F344 female rats, cholesterol and BUN were
elevated at the highest dose in one chronic inhalation study, which corresponded with an elevated
severity of CPN in females (Saito etal.. 2013: TPEC. 2010b). The single reported instance of elevated
proteinuria occurred in female rats following chronic inhalation exposure; thus, no correlation of
elevated proteinuria with CPN in males was observed fSaito etal.. 2013: IPEC. 2010b],
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Kidney tumors. No increase in kidney tumor incidence was observed following chronic oral
or inhalation exposure in either F344 rats fSaito etal.. 2013: Suzuki etal.. 2012: TPEC. 2010a. b) or
SD rats {Maltoni, 1999, 87642}(see Table 1-4). However, animals in the {Maltoni, 1999,
87642@@author-year} study had extremely depressed survival in the control and treated groups
(approximately 25%-30%) after 104 weeks, which is much lower than anticipated for a 2-year
study, ultimately limiting the ability of this study to predict potential carcinogenicity (see
discussion in Section 1.2.5).
In two-stage ("initiation, promotion") cancer bioassays, 23 weeks of daily gavage ETBE
exposure did not increase kidney tumor incidence following 4 weeks of treatment with a
5-mutagens mixture (DMBDD) in male F344 rats fHagiwara etal.. 2011: IPEC. 2008d): however, a
moderate, but statistically significant dose-response trend in the incidence of renal tubular
adenoma or carcinoma incidence was observed with 19 weeks of daily gavage ETBE exposure
following 2 weeks of the mutagen (N-ethyl-N-hydroxyethylnitrosamine [EHEN]) administration in
male Wistar rats (Hagiwara etal.. 2015). In Hagiwara etal. (2011). kidney tumors were not
observed following 23 weeks of ETBE exposure without mutagen exposure (n=ll). An ETBE-only
exposure group was not evaluated in the later study in Wistar rats fHagiwara etal.. 20151.
Male rats
Female rats
30
rho= 0.75 (all)
rho= 0.46 (all)
rho= 0.86 (oral)
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rho= 0.71 (inh.)
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(/)
_Q
<
u -
tert-butanol blood concentration (mg/l)
0 20 40 60
tert-butanol blood concentration (mg/l)
• Oral exposure
O Inhalation exposure
Figure 1-2. Comparison of absolute kidney weight change in male and female
rats across oral and inhalation exposure based on metabolite internal blood
concentration. Spearman rank coefficient (rho) was calculated to evaluate the
direction of a monotonic association (e.g., positive value = positive association) and
the strength of association.
This document is a draft for review purposes only and does not constitute Agency policy.
1-8 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Mouse inhalation exposure
5000
10000
15000
20000
25000
Administered concentration (mg/m
•
Male mice
o
Female mice
1
2
3
4
5
Figure 1-3. Comparison of absolute kidney weight change in male and female
mice following 13 week inhalation exposure. Source: {Medinsky, 1999,
10740@@author-year}{Bond, 1996, 74002@@author-year}. No significant
relationships were calculated.
This document is a draft for review purposes only and does not constitute Agency policy.
1-9 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Table 1-2. Evidence pertaining to kidney histopathology effects in animals
following exposure to ETBE
Reference and study
Results
design
Cohen et al. (2011)
Male
Female
rat, F344/DuCrlCrlj
oral - water
male (50/group): 0, 625,
2,500, 10,000 ppm (0, 28,
Dose
(mg/kg-d)
Average Average
severity of Incidence of Dose severity of Incidence of
CPN CPN (mg/kg-d) CPN CPN
121, 542 mg/kg-d)a; female
0
2.08 49/50
0
1.14 45/50
(50/group): 0, 625, 2,500,
28
46
0.98 41/50
10,000 ppm (0, 46, 171,
560 mg/kg-d)a
121
-
171
1.2 46/50
reanalysis of histopathology
data from JPEC (2010a)
542
2.72* 50/50
560
1.36 46/50
study, for which animals
were dosed daily for 104 wk
Cohen et al. (2011)
Male
rat, F344/DuCrlCrlj
oral - water
male (10/group): 0, 250,
Dose
(mg/kg-d)
Number of CPN foci/rat Number of granular casts/rat
1,600, 4,000, 10,000 ppm
0
1.2
0
(0, 17, 40, 101, 259,
17
626 mg/kg-d)a
reanalysis of histopathology
40
-
-
data from JPEC 2007 (study
No. 0665) study, for which
101
-
-
animals were dosed daily for
259
-
-
13 wk
626
27.2
8.2
Miyata et al. (2013); JPEC
Male
Female
(2008c)
rat, CRL:CD(SD)
oral -gavage
male (15/group): 0, 5, 25,
Incidence of
Incidence of
Dose
(mg/kg-d)
papillary
mineralization
Dose
mg/kg-d)
papillary
mineralization
100, 400 mg/kg-d; female
0
0/15
0
0/15
(15/group): 0, 5, 25,100,
400 mg/kg-d
5
0/15
5
-
daily for 180 d
25
0/15
25
-
100
1/15
100
-
400
0/15
400
0/15
This document is a draft for review purposes only and does not constitute Agency policy.
1-10 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and study
Results
design
Saito etal. (2013); JPEC
Male
Average
Incidence of
(2010b)
severity of CPN
Incidence of
urothelial
rat, F344
as calculated
Incidence of
papillary
hyperplasia of
inhalation - vapor
Dose (mg/m
3) by EPAC
CPN
mineralization
the renal pelvis
male (50/group): 0, 500,
1,500, 5,000 ppm (0, 2,090,
0
2.4
49/50
0/50
2/50
6,270, 20,900 mg/m3)b;
2,090
2.6
50/50
0/50
5/50
female (50/group): 0, 500,
1,500, 5,000 ppm (0, 2,090,
6,270
2.7
49/49
1/49
16/49*
6,270, 20,900 mg/m3)b
20,900
3.1*
50/50
6/50*
41/50*
dynamic whole body
inhalation; 6 hr/d, 5 d/wk
Female
Average
for 104 wk; generation
severity of CPN
method, analytical
as calculated
Incidence of
concentration reported
Dose (mg/m
0
2,090
6,270
20,900
3) bv EPAC
0.9
1.3
1.3
1.6*
CPN
32/50
38/50
41/50
40/50
Atypical tubule hyperplasia not observed in males or females.
Papillary mineralization and urothelial hyperplasia of the renal pelvis not observed
in females.
Suzuki etal. (2012); JPEC
Male
Average
(2010a)
severity of CPN
Incidence of
rat, F344
Dose
Average
as calculated bv
atvoical tubule
Incidence of
oral - water
(mg/kg-d)
severity of CPN
EPAC
hyperplasia
CPN
male (50/group): 0, 625,
2,500, 10,000 ppm (0, 28,
0
2.1
2.1
0/50
49/50
121, 542 mg/kg-d)a; female
28
2.0
1.7
0/50
43/50
(50/group): 0, 625, 2,500,
10,000 ppm (0, 46, 171,
121
2.0
1.8
0/50
45/50
560 mg/kg-d)a
542
2.4*
2.3
1/50
48/50
daily for 104 wk
Incidence of
Incidence of
Incidence of
urothelial
Dose
papillary
Daoillarv
hyperplasia of
(mg/kg-d)
necrosis
mineralization
the renal pelvis
0
0/50
0/50
0/50
28
1/50
0/50
0/50
121
0/50
16/50*
10/50*
542
2/50
42/50*
25/50*
This document is a draft for review purposes only and does not constitute Agency policy.
1-11 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and study
design
Results
Female
Average
severity of CPN
Incidence of
Dose
Average
as calculated by
atypical tubule
Incidence of
(mg/kg-d)
severity of CPN
EPAC
hyperplasia
CPN
0
1.2
1.0
0/50
41/50
46
1.2
0.9
0/50
37/50
171
1.5
1.1
0/50
37/50
560
1.5*
1.2
2/50
39/50
Incidence of
Incidence of
Incidence of
urothelial
Dose
papillarv
Daoillarv
hyperplasia of
(mg/kg-d)
necrosis
mineralization
the renal pelvis
0
0/50
0/50
0/50
46
1/50
0/50
0/50
171
1/50
1/50
0/50
560
2/50
3/50
0/50
Conversion performed by study authors.
b4.18 mg/m3 = 1 ppm.
cAverage severity calculated as (grade x number of affected animals) -f total number of animals exposed.
*: result is statistically significant (p < 0.05) based on analysis of data by study authors.
for controls, no response relevant; for other doses, no quantitative response reported.
Percent change compared to controls calculated as 100 x [(treated value - control value) 4 control value].
This document is a draft for review purposes only and does not constitute Agency policy.
1-12 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
1 Table 1-3. Evidence pertaining to kidney biochemistry and urine effects in
2 animals following exposure to ETBE
Reference and study
Results
design
JPEC (2008b)
Male
rat, CRL:CD(SD)
inhalation - vapor
male (10/group): 0,150,
Dose (mg/m3)
Blood urea nitrogen
(BUN)
Cholesterol
Creatinine
500,1,500, 5,000 ppm
0
-
-
-
(0, 627, 2,090, 6,270,
20,900 mg/m3)a; female
627
-9%
8%
-13%
(10/group): 0,150, 500,
2,090
-5%
9%
-6%
1,500, 5,000 ppm (0, 627,
2,090, 6,270,
6,270
4%
26%
-6%
20,900 mg/m3)a
20,900
4%
15%
-3%
dynamic whole body
chamber; 6 hr/d, 5 d/wk for
Dose (mg/m3)
Proteinuria severitvb
Proteinuria incidence
Urinarv casts
13 wk; generation method,
0
0.5
3/6
0/6
analytical concentration,
and method reported
627
1.2
5/6
0/6
2,090
1.2
5/6
0/6
6,270
1.3
6/6
0/6
20,900
1.0
4/6
0/6
Female
Blood urea nitrogen
Dose (mg/m3)
0
627
(BUN)
Cholesterol
Creatinine
-5%
7%
0%
2,090
3%
9%
3%
6,270
-8%
11%
-9%
20,900
-4%
21%
-9%
Dose (mg/m3)
Proteinuria severitvb
Proteinuria incidence
Urinarv casts
0
0.2
1/6
0/6
627
0.3
1/6
0/6
2,090
0.2
1/6
0/6
6,270
0.5
2/6
0/6
20,900
0.3
2/6
0/6
This document is a draft for review purposes only and does not constitute Agency policy.
1-13 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and study
Results
design
Mivata et al. (2013); JPEC
Male
(2008c)
rat, CRL:CD(SD)
oral - gavage
Dose
(mg/kg-d)
Blood urea nitrogen
(BUN)
Cholesterol
Creatinine
male (15/group): 0, 5, 25,
0
-
-
-
100,400 mg/kg-d; female
(15/group): 0, 5, 25,100,
5
12%
-5%
0%
400 mg/kg-d
25
1%
21%
-10%
daily for approximately
26 wk
100
4%
12%
-3%
400
8%
53%*
0%
Dose
(mg/kg-d)
Proteinuria incidence
Proteinuria severitvb
Urinarv casts
0
10/10
1.5
0/10
5
10/10
1.6
-
25
10/10
1.6
-
100
10/10
1.3
-
400
10/10
1.5
0/10
Female
Dose
Blood urea nitrogen
(mg/kg-d)
0
5
(BUN)
Cholesterol
Creatinine
-5%
-7%
-19%
25
-7%
-7%
-12%
100
-1%
-2%
-16%
400
4%
3%
-16%
Dose
(mg/kg-d)
Proteinuria incidence
Proteinuria severity13
Urinarv casts
0
8/10
1.2
0/10
5
9/10
1.3
-
25
7/10
1.0
-
100
9/10
1.3
-
400
7/10
1.0
0/10
This document is a draft for review purposes only and does not constitute Agency policy.
1-14 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and study
Results
design
Saito etal. (2013); JPEC
Response relative to control
(2010b)
Male
rat, F344
inhalation - vapor
male (50/group): 0, 500,
1,500, 5,000 ppm (0, 2,090,
Dose
(mg/m3)
Blood urea
nitrogen
(BUN)
Cholesterol
Creatinine
Proteinuria
incidence
Proteinuria
severity13
6,270, 20,900 mg/m3)a;
0
-
-
-
44/44
3.7
female (50/group): 0, 500,
1,500, 5,000 ppm (0, 2,090,
2,090
41%*
10%
14%*
38/38
3.5
6,270, 20,900 mg/m3)a
6,270
45%*
29%*
29%*
40/40
3.6
dynamic whole body
inhalation; 6 hr/d, 5 d/wk
20,900
179%*
52%*
71%*
31/31
3.6
for 104 wk; generation
Female
method, analytical
concentration, and method
reported
Dose
Blood urea
nitrogen
Proteinuria
Proteinuria
(mg/m3)
(BUN)
Cholesterol
Creatinine
incidence
severitvb
0
-
-
-
33/38
2.8
2,090
10%
-3%
0%
39/39
3.1
6,270
4%
-4%
0%
30/30
3.3
20,900
30%*
53%*
0%
30/30
3.4*
This document is a draft for review purposes only and does not constitute Agency policy.
1-15 DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review of ETBE
Reference and study
Results
design
Suzuki etal. (2012); JPEC
Response relative to control
(2010a)
Male
rat, F344
oral - water
male (50/group): 0, 625,
2,500, 10,000 ppm (0, 28,
Blood urea
Dose
(mg/kg-d)
nitrogen
(BUN)
Cholesterol
Creatinine
Proteinuria
incidence
Proteinuria
severity13
121, 542 mg/kg-d)c; female
0
-
-
-
39/39
3.0
(50/group): 0, 625, 2,500,
10,000 ppm (0, 46, 171,
28
3%
-11%
0%
37/37
3.1
560 mg/kg-d)c
121
20%*
10%
17%
34/34
3.1
daily for 104 wk
542
Female
43%*
Blood urea
31%*
17%
35/35
3.1
Dose
nitrogen
Proteinuria
Proteinuria
(mg/kg-d)
(BUN)
Cholesterol
Creatinine
incidence
severitvb
0
-
-
-
37/37
2.8
46
-8%
-2%
0%
37/37
3.0
171
-5%
12%
-17%
38/38
3.0
560
-5%
8%
0%
38/38
3.1
a4.18 mg/m3 = 1 ppm.
Severity of proteinuria = (1 x number of animals with "1+") + (2 x number of animals with "2+") + (3 x number of
animals with "3+") + (4 x number of animals with "4+") 4- total number of animals in group.
Conversion performed by study authors.
*: result is statistically significant (p < 0.05) based on analysis of data by study authors.
for controls, no response relevant; for other doses, no quantitative response reported.
Percent change compared to controls calculated as 100 x [(treated value - control value) 4 control value],
1 Table 1-4. Evidence pertaining to kidney tumor effects in animals following
2 exposure to ETBE
Reference and study design
Results
{Saito, 2013, 2321101(®(®author-
Male
Female
vearMJPEC, 2010,1517421(5)(5)author-
vear}
rat, F344
Dose
Renal cell
Dose
Renal cell
(mg/m3)
carcinoma
(mg/m3)
carcinoma
inhalation - vapor
0
0/50
0
0/50
male (50/group): 0, 500,1,500,
5,000 ppm (0, 2,090, 6,270,
2,090
1/50
2,090
0/50
20,900 mg/m3)c; female (50/group): 0,
6,270
0/49
6,270
0/50
500,1,500, 5,000 ppm (0, 2,090, 6,270,
20,900 mg/m3)c
20,900
0/50
20,900
0/50
This document is a draft for review purposes only and does not constitute Agency policy.
1-16 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and study design
Results
{JPEC, 2010, 1517477(5)(5)author-
Male
Female
vearHSuzuki, 2012,1433129(®(®author-
vear}
rat, F344
Dose
Renal cell
Dose
Renal cell
(mg/kg-d)
carcinoma
(mg/kg-d)
carcinoma
oral - water
0
0/50
0
0/50
male (50/group): 0, 625, 2,500,
10,000 ppm (0, 28, 121, 542 mg/kg-d)b;
28
0/50
46
0/50
female (50/group): 0, 625, 2,500,
121
0/50
171
0/50
10,000 ppm (0, 46,171, 560 mg/kg-d)d
daily for 104 wk
542
1/50
560
1/50
{Hagiwara, 2011,1248019(®(®author-
Male
vearKJPEC, 2008,1517752(5)(5)author-
Renal tubular
vear}
Dose
Renal transitional
adenoma or
rat, F344
(mg/kg-d)
cell carcinoma
carcinoma
oral - gavage
male (12/group): 0,1,000 mg/kg-d
0
0/12
0/12
daily for 23 wk
1,000
0/12
0/12
Initiation Promotion Studies
{Hagiwara, 2011,1248019(®(®author-
Male
vear KJPEC, 2008,1517752(5)(5)author-
Renal tubular
vear}
Dose
adenoma or
Renal transitional
rat, Fischer 344
(mg/kg-d)
carcinoma
cell carcinoma
oral - gavage
male (30/group): 0, 300,1,000 mg/kg-d
0
11/30
1/30
daily for 23 wk following a 4-wk tumor
300
6/30
0/30
initiation by DMBDDC
1,000
13/30
2/30
{Hagiwara, 2015, 3046107(®(®author-
Male
year}
Renal tubular
rat, Wistar
Dose
adenoma or
oral - gavage
(mg/kg-d)
carcinomad
male (30/group): 0,100, 300, 500,
1,000 mg/kg-d
0
18/30
daily for 19 wk following a 2-wk tumor
100
23/30
initiation by N-ethyl-N-
hydroxyethylnitrosamine (EHEN)
300
500
1,000
25/30
26/30
26/30
1 a4.18 mg/m3 = 1 ppm.
2 bConversion performed by study authors.
3 cDiethylnitrosamine (DEN), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), N-methyl-N-nitrosourea (MNU),
4 1,2-dimethylhydrazine dihydrochloride (DMH), and N-bis(2-hydroxypropyl)nitrosamine (DHPN).
5 dAuthors report significant trend.
This document is a draft for review purposes only and does not constitute Agency policy.
1-17 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
2 Table 1-5. Comparison of nephropathy and urothelial hyperplasia in
3 individual male rats from 2-year oral exposure (IPEC. 2010a)
CPN
Urothelial
hyperplasia
None
Minimal
Mild
Moderate
Marked
None
15
21
105
23
1
Minimal
0
0
17
16
2
Mild
0
0
0
0
0
Moderate
0
0
0
0
0
Marked
0
0
0
0
0
Spearman's rank correlation test (1-sided), p < 0.0001, rs = 0.36
10
11
12
Table 1-6. Comparison of nephropathy and urothelial hyperplasia in
individual male rats from 2-year inhalation exposure (IPEC. 2010b)
CPN
Urothelial
hyperplasia
None
Minimal
Mild
Moderate
Marked
None
1
3
59
68
4
Minimal
0
0
14
29
21
Mild
0
0
0
0
0
Moderate
0
0
0
0
0
Marked
0
0
0
0
0
Spearman's rank correlation test (1-sided), p < 0.0001, rs = 0.36
This document is a draft for review purposes only and does not constitute Agency policy.
1-18 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
¦ = exposures at which the en dpoint was reported statistically significant by study authors
~ = exposures at which the en dpoint was reported not statistically significant by study authors
Increased Absolute
Kidney Weight
Incidence of Chronic
Progressive
Nephropathy
PO Male rat; 16tvk,s j B]
PO Feniaie rat; 17wks j Hi
PO Male rat; 18w ks jC(
PO Female rat; 18wks (C)
F1 Male I at, lepiuduLliit 1C)
F1 Female rat; reproductive fC(
Male rat; 26wkh. (Di
Female rat; 26wks fD)
Male rat; IWivkb | fc'|
Female rat; 104wks, ( E)
Male rat; 104 wks (A)
Female l'al; 104wks (A)
Male rat; 104wks (E)
Female rat; 104wks (E)
o a_
~—a—q
0—a-
-e-
Average Severity of Male rat; 104wks (A)
Chronic Progressive
Nephropathy
Female rat; 104wks j A)
Male rat; 104 wki fb)
Female rat; 104wks> f E)
~ ,—B-
n—¦—H—
Urothelial Hyperplasia. Ma!t. ral.l u4wk_s , b)
of t he Renal Pelvis
Female rat; 104wks j HI
-0—^—™~0
1 10 100 1,000 10,000
Dose (mg/kg-day)
Sources: (A) Cohen et al,, 2011 reanalvsis of 1 PEC 2010a; (B| Fujii et al., 2010; JPEC, 2008d; (C) Gaoua,
2004b; (D| Miyata et al., 2013; JPEC 2008b; (E) Suzuki et al., 2012; JPEC, 2010a
Figure 1-4. Exposure-response array of kidney effects following oral exposure
to ETBE.
This document is a draft for review purposes only and does not constitute Agency policy.
1-19
DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
= exposures at which the endpoint was reported statistically significant by study authors
= exposures at which the endpoint was reported not statistically significant bystudy authors
Increased Absolute
Kidney Weight
Male rat; 13wks (A)
Female rat; 13wks (A)
Male rat; 13 wks (C)
Female rat; 13 wks (C)
Male mouse; 13 wks (B)
Female mouse; 13 wks (B)
Male rat; 104wks (D)
Female rat; 104wks(D)
Incidence of Chronic
Progressive Male rat: 104wl
-------
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Toxicological Review of ETBE
Mode of Action Analysis—Kidney Effects
a) Toxicokinetic Considerations Relevant to Kidney Toxicity
ETBE is metabolized by cytochrome P450 (CYP) enzymes to an unstable hemiacetal that
decomposes spontaneously into tert-butanol and acetaldehyde (Bernauer etal.. 1998).
Acetaldehyde is metabolized further in the liver. The main circulating breakdown product of ETBE
metabolism is tert-butanol, which is filtered from the blood by the kidneys and excreted in urine.
Thus, following ETBE exposure, the kidney is exposed to significant concentrations of tert-butanol,
and kidney effects caused by tert-butanol (described in more detail in the draft IRIS assessment of
tert-butanol) also are relevant to evaluating the kidney effects observed after ETBE exposure. In
particular, similar to ETBE, tert-butanol has been reported to cause nephrotoxicity in rats, including
effects associated with a2U-globulin nephropathy
(https://cfpub.epa.gov/ncea/iris drafts/recordisplay.cfm?deid=2 62086). Unlike ETBE, however,
increased renal tumors, in the absence of an initiator, were reported following chronic drinking
water exposure to tert-butanol.
b) a?,,-Globulin-Associated Renal Tubule Nephropathy
One disease process to consider when interpreting kidney effects in rats is related to the
accumulation of a2U-globulin protein. a2U-globulin, a member of a large superfamily of low-
molecular-weight proteins, was first characterized in male rat urine. Such proteins have been
detected in various tissues and fluids of most mammals (including humans), but the particular
isoform of a2U-globulin commonly detected in male rat urine and associated with renal tubule
nephropathy (?) is considered specific to that sex and species. Exposure to chemicals that induce
a2u-globulin accumulation can initiate a sequence of histopathological events leading to kidney
tumorigenesis. Because a2U-globulin-related renal tubule nephropathy and carcinogenicity
occurring in male rats are presumed not relevant for assessing human health hazards (U.S. EPA.
1991a). evaluating the data to determine whether a2U-globulin plays a role is important The role of
a2u-globulin accumulation in the development of renal tubule nephropathy and carcinogenicity
observed following ETBE exposure was evaluated using the U.S. EPA f!991bl Risk Assessment
Forum Technical panel report, AIphct2U-GIobuIin: Association with Chemically Induced Renal Toxicity
and Neoplasia in the Male Rat as well as the IARC a2U-globulin criteria {Capen, 1999, 699905}. These
frameworks provide specific guidance for evaluating renal tubule tumors that are related to
chemical exposure for the purpose of risk assessment, based on an examination of the potential
involvement of a2u-globulin accumulation.
The hypothesized sequence of a2u-globulin renal tubule nephropathy, as described by U.S.
EPA fl991al. is as follows. Chemicals that induce a2U-globulin accumulation do so rapidly.
a2u-Globulin accumulating in hyaline droplets is deposited in the S2 (P2) segment of the proximal
tubule within 24 hours of exposure. Hyaline droplets are a normal constitutive feature of the
mature male rat kidney; they are particularly evident in the S2 (P2) segment of the proximal tubule
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and contain a2u-globulin (U.S. EPA. 1991a). Abnormal increases in hyaline droplets have more than
one etiology and can be associated with the accumulation of different proteins. As hyaline droplet
deposition continues, single-cell necrosis occurs in the S2 (P2) segment, which leads to exfoliation
of these cells into the tubule lumen within 5 days of chemical exposure. In response to the cell loss,
cell proliferation occurs in the S2 (P2) segment after 3 weeks and continues for the duration of the
exposure. After 2 or 3 weeks of exposure, the cell debris accumulates in the S3 (P3) segment of the
proximal tubule to form granular casts. Continued chemical exposure for 3 to 12 months leads to
the formation of calcium hydroxyapatite in the papilla, which results in linear mineralization. After
1 or more years of chemical exposure, these lesions can result in the induction of renal tubule
adenomas and carcinomas (Figure 1-6).
U.S. EPA fl991al identified two questions that must be addressed to determine the extent
to which a2u-globulin-mediated processes induce renal tubule nephropathy and carcinogenicity.
First, whether the a2U-globulin process occurs in male rats and influences renal tubule tumor
development must be determined. Second, whether the renal effects in male rats exposed to ETBE
are due solely to the a2u-globulin process must be determined.
U.S. EPA fl991al stated that the criteria for answering the first question in the affirmative
are as follows:
1) hyaline droplets are larger and more numerous in treated male rats,
2) the protein is present in the hyaline droplets in treated male rats is a2U-globulin (i.e.,
immunohistochemical evidence), and
3) several (but not necessarily all) additional steps in the pathological sequence appear in
treated male rats as a function of time, dose, and progressively increasing severity
consistent with the understanding of the underlying biology, as described above, and
illustrated in Figure 1-6.
The available data relevant to this first question are summarized in Table 1-7, Table 1-8,
Figure 1-7, and Table 1-10, and are evaluated below.
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1 Source: Adapted from Swenberg and Lehman-McKeeman (1999); U.S. EPA (1991a).
2 Figure 1-6. Temporal pathogenesis of a2U-globulin-associated nephropathy in
3 male rats. a2U-Globulin synthesized in the livers of male rats is delivered to the
4 kidney, where it can accumulate in hyaline droplets and be retained by epithelial
5 cells lining the S2 (P2) segment of the proximal tubules. Renal pathogenesis
6 following continued exposure and increasing droplet accumulation can progress
7 stepwise from increasing epithelial cell damage, death, and dysfunction, leading to
8 the formation of granular casts in the corticomedullary junction, and linear
9 mineralization of the renal papilla, in parallel with carcinogenesis of the renal
10 tubular epithelium.
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Table 1-7. Additional kidney effects potentially relevant to mode of action in
animals exposed to ETBE
Reference and study design
Results
JPEC (2008b)
rat, CRL:CD(SD)
inhalation - vapor
male (10/group): 0,150, 500,1,500,
5,000 ppm (0, 627, 2,090, 6,270,
20,900 mg/m3)a; female (10/group): 0,150,
500,1,500, 5,000 ppm (0, 627, 2,090, 6,270,
20,900 mg/m3)a
dynamic whole body chamber; 6 hr/d,
5 d/wk for 13 wk; generation method,
analytical concentration, and method
reported
Male
Incidence of
hyaline
droplets in
the proximal
Dose
tube
(mg/m3)
epithelium
0
0/10
627
3/10
2,090
8/10*
6,270
8/10*
20,900
8/10*
Unspecified representative samples reported as "weakly positive"
for a2u-globulin in males; no hyaline droplets observed in proximal
tubule of females; hyaline droplets positive for a2u-globulin not
examined in females.
JPEC (2008c); Mivata etal. (2013)
rat, CRL:CD(SD)
oral - gavage
male (15/group): 0, 5, 25,100,400 mg/kg-d;
female (15/group): 0, 5, 25,100,
400 mg/kg-d
daily for 180 d
Male
Dose
(mg/kg-d)
0
5
25
100
400
Incidence of
hyaline
droplets
0/15
0/15
0/15
4/15
10/15*
Incidence of
hyaline
droplets
positive for
cbu-globulin
0/1
2/2
1/1
Female
Dose
(mg/kg-d)
0
5
25
100
400
Incidence
of hyaline
droplets
0/15
0/15
Medinskv et al. (1999); Bond et al. (1996b)
rat, Fischer 344
inhalation - vapor
male (48/group): 0, 500,1,750, 5,000 ppm
(0, 2,090, 7,320, 20,900 mg/m3)a; female
(48/group): 0, 500, 1,750, 5,000 ppm
(0, 2,090, 7,320, 20,900 mg/m3)a
dynamic whole body chamber; 6 hr/d,
5 d/wk for 13 wk; generation method,
analytical concentration, and method
reported
Male
Dose
(mg/m3
0
Proximal tubule proliferation
Hyaline droplet
severity
1.8
1 week
4 weeks 13 weeks
2,090
3.0
39%
24%
137%
7,320
3.2
23%
-14%
274%
20,900
3.8
102%*
175%*
171%
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Reference and study design
Results
Female
Dose
(mg/m3)
0
2,090
7,320
20,900
1 week
60%*
88%*
49%*
Proximal tubule proliferation
4 weeks
3%
15%
31%*
13 weeks
73%
64%
47%
Saito et al. (2013); JPEC (2010b)
rat, Fischer 344
inhalation - vapor
male (50/group): 0, 500,1,500, 5,000 ppm
(0, 2,090, 6,270, 20,900 mg/m3)a; female
(50/group): 0, 500,1,500, 5,000 ppm
(0, 2,090, 6,270, 20,900 mg/m3)a
dynamic whole body inhalation; 6 hr/d,
5 d/wk for 104 wk; generation method,
analytical concentration, and method
reported
Male
No hyaline droplets observed.
Female
No hyaline droplets observed.
Suzuki et al. (2012); JPEC (2010a)
rat, Fischer 344
oral - water
male (50/group): 0, 625, 2,500,10,000 ppm
(0, 28,121, 542 mg/kg-d)b; female
(50/group): 0, 625, 2,500, 10,000 ppm (0, 46,
171, 560 mg/kg-d)b
daily for 104 wk
Male
No hyaline droplets observed.
Female
No hyaline droplets observed.
a4.18 mg/m3 = 1 ppm.
Conversion performed by study authors.
*: result is statistically significant (p < 0.05) based on analysis of data by study authors.
-: for controls, no response relevant; for other doses, no quantitative response reported.
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1 Table 1-8. Summary of data informing whether the a2u-globulin process is
2 occurring in male rats exposed to ETBE
Criterion
Duration
Results
Reference
(1) hyaline droplets are increased
in size and number
1 wk
(+)a
Medinskv et al. (1999)
4 wk
(+)a
Medinskv et al. (1999)
13 wk
(+)a
Medinskv et al. (1999)
13 wk
+
JPEC (2008b)
26 wk
+
Mivata et al. (2013); JPEC (2008c)
104 wk
-
Suzuki et al. (2012)
104 wk
-
Saito et al. (2013); JPEC (2010b)
(2) the protein in the hyaline
droplets is a2u-globulin
1 wk
(+)b
JPEC (2008b)
4 wk
(+)b
Medinskv et al. (1999)
13 wk
(+)b
Medinskv et al. (1999)
13 wk
(+)b
JPEC (2008b)
26 wk
(+)c
Mivata et al. (2013); JPEC (2008c)
(3) Several (but not necessarily all) additional steps in the pathological sequence are present in male rats, such as:
(a) single-cell necrosis
13 wk
-
JPEC (2008b)
13 wk
-
Medinskv et al. (1999)
26 wk
-
Mivata et al. (2013); JPEC (2008c)
104 wk
-
Suzuki et al. (2012); JPEC (2010a)
104 wk
-
Saito et al. (2013); JPEC (2010b)
(b) exfoliation of epithelial cells
into the tubular lumen
13 wk
-
JPEC (2008b)
13 wk
-
Medinskv et al. (1999)
26 wk
-
Mivata et al. (2013); JPEC (2008c)
104 wk
-
Suzuki et al. (2012); JPEC (2010a)
104 wk
-
Saito et al. (2013); JPEC (2010b)
(c) granular casts
13 wk
-
JPEC (2008b)
13 wk
(+)
Cohen et al. (2011); JPEC 2007a
13 wk
-
Medinskv et al. (1999)
26 wk
-
Mivata et al. (2013); JPEC (2008c)
104 wk
-
Suzuki et al. (2012); JPEC (2010a)
104 wk
-
Saito et al. (2013); JPEC (2010b)
(d) linear mineralization of tubules
in the renal papilla
13 wk
-
JPEC (2008b)
13 wk
-
Medinskv et al. (1999)
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Criterion
Duration
Results
Reference
26 wk
-
Mivata et al. (2013); JPEC (2008c)
104 wk
+
Suzuki et al. (2012); JPEC (2010a), Cohen et al. (2011)
104 wk
+
Saito et al. (2013); JPEC (2010b)
(e) Proliferation and foci of tubular
hyperplasia
13 wk
-
JPEC (2008b)
13 wk
+/-d
Medinskv et al. (1999)
26 wk
-
Mivata et al. (2013); JPEC (2008c)
104 wk
-
Suzuki et al. (2012); JPEC (2010a)
104 wk
-
Saito et al. (2013); JPEC (2010b)
1 + = Statistically significant change reported in one or more treated groups.
2 (+) = Effect reported in one or more treated groups, but statistics not reported.
3 - = No statistically significant change reported in any of the treated groups.
4 aDroplet severity.
5 bUnspecified "representative samples" examined.
6 Three samples from highest two dose groups examined.
7 dLabeling index statistically significantly increased, but no hyperplasia reported.
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¦ = exposures at which the endpoint was reported statistically significant by study authors
~ = exposures at which the endpoint was reported not statistically significant by study authors
9 = effect was observed but statistics not reported
+ = unspecified representative samples reported positive for tX2a-globulin
Miyata et a!, 2013; JPEC, 2008c - 26wks
Accumulation of
hyaline droplets
Suzuld et al, 2012; JPEC, 2010a - 104wfc
a -globulin in
hyaline Miyata et a 1,2013; JPEC, 2008c - 26wks
droplets
Granular
casts/dilation
Cohen et al„ 2011 -13 wks ¦
Miyata et al, 2013; JPEC, 2008c ¦ 26wks
Suzuki et al, 2012; JPEC, 2010a - 104wte
Miyata et al, 2013; JPEC, 2008c - 26wks
Linear
papillary
mineralization
Suzuki et al, 2012; JPEC, 2010a • 10-lwte ¦
Tubular
hyperplasia
Suzuki et al., 2012; JPEC, 2010a ¦ ICMwte
Renal
adenoma
or
carcinoma
Suzuki etal, 2012; JPEC, 2010a - ICMwfc
B-
-0 O-
O rB ~
+ +
43 0 0
~ B-
-0 B 0
Q-
~ B 0
a— o
10 100
Dose (nig/kg-day)
1,000
Figure 1-7. ETBE oral exposure array of a2u-globulin data in male rats.
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¦ = exposures at which the endpoint was reported statistically significant by study authors
~ = exposures at which the endpoint was reported not statistically significant by study authors
• = effect was observed but statistics not reported
+ = unspecified representative samples reported positive for a2ll-globulin
Medinsky et al., 1999; Bond et a], 1996 - Iwk
Medinsky et al., 1999; Bond et al, 1996 • 4wk
Accumulation
Of hyaline Medinsky et al., '1999; Bond et al. 1996 ¦ 13wk
droplets
J PEC, 2008b -13 wk
Saito et al., 2013; JPEC, 2010b -104wk
B—
• •-
• •-
~ •-
¦ ¦
B B—
•
•
•
¦
B
+
+
+
Medinsky et al., 1999; Bond et al, 1996 • Iwk
.globulin in Medinsl 2013. )PEq 20i0b - I04wk
or carcinoma
B B—
B
100 1,000 10,000 100,000
Exposure Concentration (mg/m3)
1 Figure 1-8. ETBE inhalation exposure array of a2u-globulin data in male rats.
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Question One: Is the a2u-globuIin process occurring in male rats exposed to ETBE?7
(1) The first criterion to consider is whether hyaline droplets are larger and more
numerous in male rats. The accumulation of hyaline droplets was observed in all three subchronic
ETBE exposure studies, but was not observed in two chronic ETBE studies (see Table 1-7 and Table
1-8). Failure to observe a2U-globulin and increased droplet accumulation in the 2-year studies is not
unusual because a2U-globulin naturally declines in males around 5 months of age (U.S. EPA. 1991a).
Accumulation of hyaline droplets in the proximal tubular epithelium of the kidney was observed in
male rats following 90-day inhalation exposure to 627, 2,090, 6,270, and 20,900 mg ETBE/m3
flPEC. 2008b], The increases at the three highest concentrations were statistically significant;
however, none of the animals had hyaline droplet grades over 1 flPEC. 2008bl Severity grade of the
hyaline droplets exhibited a dose-response after a 1-week exposure, as indicated by scores of 1.2,
3.4, 4.0, and 4.6 at 0, 2,090, 7,320, and 20,900 mg ETBE/m3, respectively, and 90 days of ETBE
inhalation exposure increased the severity grades of hyaline droplets from 1.8 in the control to 3.0,
3.2, and 3.8 (Medinskv etal.. 1999). In addition, the incidence of hyaline droplets statistically
significantly increased in a dose-related manner after 26 weeks of gavage exposure to 100 and
400 mg ETBE/kg-day fMivata etal.. 2013: TPEC. 2008cl. These data indicate consistent evidence of
hyaline droplets increasing both in a dose-responsive manner and within the expected timeframe.
Therefore, the available data are sufficient to fulfill the first criterion that hyaline droplets are
increased in size and number in male rats.
(2) The second criterion to consider is whether the protein in the hyaline droplets in male
rats is a2u-globulin. Immunohistological staining to ascertain the protein composition in the hyaline
droplets was performed only in ETBE exposure studies that observed accumulation of hyaline
droplets. At the two highest doses, Mivataetal. f20131: TPEC f2008cl identified hyaline droplets as
positive for a2U-globulin in 2/2 and 1/1 animals that were tested for the presence of a2U-globulin.
The other two studies also reported that unspecified samples were positive for a2U-globulin (TPEC.
2008b: Medinskv etal.. 1999). TPEC (2008b) reported that the samples stained weakly positive for
a2u-globulin and that positive a2u-globulin staining was observed only in male rats. No statistical
tests were performed on these results. The available studies that tested for a2u-globulin in hyaline
droplets did not test a sufficient number of samples within a dose group nor were enough dose
groups tested for a2U-globulin to perform dose-response analysis. Therefore, the available data are
minimally sufficient to fulfill the second criterion for a2U-globulin present in the hyaline droplets,
but suggest weak induction of a2U-globulin by ETBE.
(3) The third criterion considered is whether several (but not necessarily all) additional
steps in the histopathological sequence associated with a2U-globulin nephropathy appear in male
7 If the chemical meets the criteria for question one, then a second question is asked: Are the renal effects in
male rats exposed to this chemical due solely to the a2u globulin process?
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Toxicological Review of ETBE
rats in a manner consistent with the understanding of a2u-globulin pathogenesis (refer to Table
1-8). Of the remaining five endpoints in the pathological sequence, only linear papillary
mineralization and granular casts were observed. Papillary mineralization typically appears at
chronic time points, occurring after exposures of 3 months up to 2 years fU.S. EPA. 1991al. The
incidence of papillary mineralization was increased statistically significantly in both 2-year studies.
Papillary mineralization increased in a dose-related manner following oral ETBE exposure in male
rats at concentrations of 0, 28,121, and 542 mg/kg-day, respectively (Suzuki etal.. 2012: TPEC.
2010a], and in males at ETBE inhalation concentrations of 0, 2,090, 6,270, and 20,900 mg/m3 (Saito
etal.. 2013: TPEC. 2010bl. Hyaline droplet deposition was observed at a similar frequency as
mineralization following oral ETBE exposure fMivata etal.. 2013: Suzuki etal.. 2012: TPEC. 2010a.
2008c); however, hyaline droplet deposition was observed in 80% of animals at all three inhalation
exposure concentrations flPEC. 2008bl compared with mineralization rates of 0, 2, and 12%
(lowest to highest exposure concentration) (Saito etal.. 2013: TPEC. 2010b). A detailed evaluation
and analysis of all the evidence relevant to this criterion follows.
Detailed evaluation of the available evidence supporting the third criterion
a) Single cell death, exfoliation into the renal tubules, and necrosis were not observed in
any study flPEC. 2008b. c; Medinskv etal.. 19991. This observation might not be
inconsistent with the hypothesized MOA because cell death and exfoliation has been
observed to occur as early as 5 days post exposure, peak at 3 weeks, and then decline to
near background levels by 4-5 weeks (Kanerva et al.. 1987): this endpoint was not
examined in any study evaluating ETBE exposures less than 13 weeks. Thus, the lack of
exfoliation observations could be the result of both weak induction of a2u-globulin and a
lack of appropriately timed examinations.
b) Granular cast formation was observed in one study. The TPEC (2007a) study reported
that, at 13 weeks, granular casts were observed in high-dose males, while none were
observed in controls (no statistical tests performed). Other studies at similar time
points did not report the presence of granular casts (TPEC. 2008b. c; Medinskv etal..
19991 despite using similar exposure concentrations. Granular cast formation, however,
might not occur with weak inducers of a2u-globulin fShortetal.. 19861. which is
consistent with the weak staining of a2u-globulin, as discussed above flPEC. 2008bl.
c) Linear mineralization of tubules within the renal papilla was consistently observed in
male rats after 2 years f Saito etal.. 2013: Suzuki etal.. 20121. This lesion typically
appears at chronic time points, occurring after exposures of 3 months up to 2 years fU.S.
EPA. 1991al.
d) Cellular proliferation was increased after 1, 4, and 13 weeks in males and females;
however, the magnitude of effect was reduced in females compared to males.
Observation of proliferation in both sexes suggests that this effect is not male specific,
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and thus not solely due to a2u-globulin. Furthermore, renal tubule hyperplasia was not
observed in any 2-year study, suggesting that ETBE does not induce sustained
proliferation fSaito etal.. 2013: Suzuki etal.. 20121. Renal tubule hyperplasia is the
preneoplastic lesion associated with a2U-globulin nephropathy in chronic exposures that
leads to renal tubule tumors fU.S. EPA. 1991al.
The progression of histopathological lesions for a2U-globulin nephropathy is predicated on
the initial response of excessive hyaline droplet accumulation (containing a2U-globulin) leading to
cell necrosis and cytotoxicity, which in turn cause the accumulation of granular casts, linear
mineralization, and tubular hyperplasia resulting from sustained cellular proliferation. Therefore,
observations of temporal and dose-response concordance for these effects are informative for
drawing conclusions on causation.
As mentioned above (see Table 1-8), some steps in the sequence of a2U-globulin
nephropathy are observed at the expected time points following exposure to ETBE. Accumulation of
hyaline droplet severity was observed early, at 1 week following inhalation exposure (Medinskv et
al.. 19991. and increased incidence was subsequently observed at 90 days (TPEC. 2008b) or 26
weeks (TPEC. 2008c): a2U-globulin was identified as the protein in these droplets (Borghoffetal..
2001: Williams and Borghoff. 20011. Observations of the subsequent linear mineralization of
tubules fall within the expected timeframe of the appearance of these lesions. Granular cast
formation was reported in one oral study (Cohen etal.. 2011). while three other oral and inhalation
studies reported none (TPEC. 2008b. c; Medinskv etal.. 1999). which also could indicate weak
a2u-globulin induction. Neither a2u-globulin-mediated regenerative cell proliferation nor atypical
renal tubule hyperplasia were observed. Lack of necrosis and exfoliation might be due to the weak
induction of a2U-globulin and a lack of appropriately timed examinations..
Hyaline droplets were weakly induced in all male rats in the 13-week inhalation studies
flPEC. 2008b: Medinskv etal.. 19991. which did not result in increased linear mineralization at the
corresponding doses. The lack of increased linear mineralization at low doses also is consistent
with weak induction of hyaline droplets.
Overall, the histopathological sequence has numerous data gaps, such as the lack of
observable necrosis, cytotoxicity, and tubule hyperplasia at stages plausibly within the timeframe
of detectability. Furthermore, no explicit inconsistencies are present in the temporal appearance of
the histopathological lesions associated with the a2U-globulin nephropathy induced following ETBE
exposure; however, the data set would be bolstered by measurements at additional time points to
lend strength to the MOA evaluation. Therefore, the number of histopathological steps observed
was insufficient to fulfill the third criterion.
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Summary and conclusions for question one, Is the a2u-gIobuIin process occurring in male rats exposed
to ETBE?
The evidence suggests that ETBE causes hyaline droplets to increase in size and number.
The documentation of a2U-globulin staining is poor and provides weak evidence of a2U-globulin in
the hyaline droplets. Only one of the additional steps in the pathological sequence was consistently
observed (linear papillary mineralization), and the ETBE database lacks evidence of renal tubule
hyperplasia and adenomas or carcinomas, despite multiple chronic bioassays, exposure routes, and
durations ranging from 13 weeks to 2 years. Overall, the available data were insufficient to
conclude that the a2U-globulin process is exclusively operative.
Consideration of additional I ARC 1999 Criteria
An a2u-globulin framework was published by IARC in 1999 {Capen, 1999, 699905}. See
below for the criterion laid out in the IARC consensus document.
IARC criteria for an agent causing kidney tumors through an a2u-globulin associated
response in male rats:
• Lack of genotoxic activity (agent and/or metabolite) based on an overall evaluation of in-vitro
and in-vivo data
• Male rat specificity for nephropathy and renal tumorigenicity
• Induction of the characteristic sequence of histopathological changes in shorter-term studies,
of which protein droplet accumulation is obligatory
• Identification of the protein accumulating in tubule cells as a2u-globulin -
• Reversible binding of the chemical or metabolite to a2u-globulin
• Induction of sustained increased cell proliferation in the renal cortex
• Similarities in dose—response relationship of the tumor outcome with the histopathological
end-points (protein droplets, a2u-globulin accumulation, cell proliferation)
A few minor differences exist between the EPA and IARC criteria. The EPA framework
requires the observation of several (but not necessarily all) additional steps in the histopathological
sequence associated with a2u globulin nephropathy, whereas IARC requires the "induction of the
characteristic sequence of histopathological changes in shorter-term studies, of which protein
droplet accumulation is obligatory", but doesn't specify which or how many of the additional
histopathological changes must be observed to consider this criteria met In addition, the IARC
criteria have specific criteria pertaining to lack of genotoxicity of parent compound/metabolite and
male rat specificity for nephropathy and renal tumorigenicity whereas the EPA framework
considers these data as supplemental information (see Part 4, XVIIB. Additional Information Useful
for the Analysis). These additional criteria required by IARC (1999) are discussed below:
Lack of genotoxic action
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As discussed in Appendix B.2.2, limited data are available help inform the genotoxic
potential of ETBE. Most studies indicate that ETBE does not induce genotoxicity in the systems
tested, although several key types of assays are missing (e.g. studies investigating chromosomal
aberrations and DNA adducts). Regarding tert-butanol, the major metabolite of ETBE, while some
data suggest tert-butanol could be genotoxic, the overall evidence is inadequate to establish a
conclusion. Regarding the ETBE metabolite acetaldehyde, acetaldehyde has induced sister
chromatid exchanges in Chinese hamster ovary cells, gene mutations in mouse lymphomas, and
DNA strand breaks in human lymphocytes {IARC, 1999, 2342650}. In addition, increased
genotoxicity of ETBE is noted when tested in animals with polymorphisms in the acetaldehyde
dehydrogenase gene ALDH2, which decreases the ability to metabolize acetaldehyde {Wang, 2012,
1249293;Weng, 2014, 2321096;Weng, 2019, 5343910;Weng, 2013, 2279880;Weng, 2012,
1248016;Weng, 2011,1062385}. Approximately 8% of the world's population carries this variant
{Gross, 2015, 5353621}. Overall, this criterion has been weakly met for non-susceptible
populations but not met for the subset of the population which can't efficiently detoxify
acetaldehyde.
Male rat specificity for nephropathy
There is limited information to evaluate the potential for ETBE mediated kidney effects in
other species. Only one subchronic study in WT mice {Bond, 1996, 74002}{Medinsky, 1999,10740}
and no chronic studies are available which evaluated kidney effects in mice.
Increased absolute kidney weight and increased severity of chronic nephropathy was noted
in both sexes of rats exposed chronically to ETBE through inhalation and in drinking water (JPEC
2010a,b). Changes in clinical chemistry suggestive of kidney toxicity (e.g. increased BUN,
cholesterol, and protein urea) were also noted in both male and female rats. However, dose-related
increased incidence of urothelial hyperplasia of the renal pelvis was observed in male rats in
chronic oral and inhalation bioassays but was not found in female rats by either route of exposure.
In summary, while male rats appear to be more sensitive to ETBE meditated kidney toxicity,
indications of nephropathy were also observed in female rats. Therefore, this criterion has not
been met
Comparison of ETBE and tert-butanol a2U-gIobuIin data
Both EPA and IARC have accepted the biological plausibility of the a2U-globulin-mediated
hypothesis for inducing nephropathy and cancer in male rats (Swenberg and Lehman-McKeeman.
1999: U.S. EPA. 1991a). and those rationales will not be repeated here. A more recent retrospective
analysis indicating that several steps in the sequence of pathological events are not required for
tumor development has demonstrated this by evaluating several a2u-globulin-inducing chemicals
which fail to induce many of the pathological sequences in the a2U-globulin pathway fDoi etal..
20071. For instance, dose-response concordance was not observed for several endpoints such as
linear mineralization, tubular hyperplasia, granular casts, and hyaline droplets following exposure
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to chemicals that induce the a2u-globulin process such as d-limonene, decalin, propylene glycol
mono-t-butyl ether, and Stoddard Solvent IICA (SS IICA). Although some of these chemicals induced
dose-response effects for a few endpoints, all failed to induce a dose-response for at all of the
endpoints in the sequence. Furthermore, no endpoint in the pathological sequence was predictive
for tumor incidence when considering either the dose responsiveness or the severity. Tumor
incidence was not affected in a dose-related manner following either d-limonene or decalin
exposure. Tumor incidence was not correlated with the severity of any one effect in the a2U-globulin
sequence as demonstrated by SS IICA, which induced some of the most severe nephropathy relative
to the other chemicals, but did not significantly increase kidney tumors fDoi etal.. 20071. Thus, this
analysis suggests that another MOA could be operative for inducing kidney tumors in male rats.
As described above, ETBE is metabolized to tert-butanol, so kidney data following
tert-butanol exposure also are potentially relevant to evaluating the MOA of ETBE. In particular, the
effects of tert-butanol on the a2U-globulin process are relevant for evaluating the coherence of the
available data on ETBE-induced nephropathy.
Hyaline droplet deposition and linear mineralization were both observed following similar
exposure durations to tert-butanol and ETBE. After 13 weeks of exposure to tert-butanol or ETBE,
hyaline droplets were dose-responsively increased. ETBE exposure increased hyaline droplets at
lower internal concentrations of tert-butanol than did direct tert-butanol administration.
Tubule hyperplasia and renal tumors were both observed following 2-year exposure to
tert-butanol but not to ETBE, despite similar internal concentrations of tert-butanol following ETBE
exposure (Saito etal.. 2013: Suzuki etal.. 2012: TPEC. 2010bl. The failure of ETBE to induce several
histopathological lesions in the a2u-globulin pathological sequence, including renal tubule
hyperplasia and tumors, at similar internal tert-butanol concentrations as those that induced
hyperplasia and tumorigenesis following exposure to tert-butanol suggests a lack of coherence
across the two data sets.
c) Chronic Progressive Nephropathy (CPN)
Exacerbation of CPN has been proposed as another rat-specific mechanism of
nephrotoxicity that is not relevant to humans (Hard etal.. 20091. CPN is an age-related renal
disease that occurs in rats of both sexes (NTP. 2015. 2014: Hard etal.. 2013: Melnick etal.. 2012:
U.S. EPA. 1991al. CPN is more severe in males than in females and is particularly common in the
Sprague-Dawley and Fischer 344 strains. Dietary and hormonal factors play a role in modifying
CPN, though its etiology is unknown.
CPN has been suggested as a key event in the onset of renal tubule tumors, and a sequence
of key events in the MOA is as follows: (1) metabolic activation, (2) chemically exacerbated CPN, (3)
increased tubule cell proliferation, (4) tubule hyperplasia, and (5) adenomas (Hard etal.. 20131.
Arguments against this MOA also have been proposed (Melnick et al.. 20121. ETBE exposure
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increased CPN severity following 2-year inhalation and 13-week oral exposure but, unlike tert-
butanol, did not affect tubule hyperplasia or increase renal tubule tumor incidence.
Additional markers associated with CPN include elevated proteinuria and albumin in the
urine and increased BUN, creatinine, and cholesterol in the serum, of which proteinuria is the major
urinary effect and a very sensitive measure of CPN fHard etal.. 20091. In the case of ETBE exposure,
however, increased severity or incidence of proteinuria was not correlated with increased severity
of CPN in male rats possibly due to high background severity of CPN. In female rats, background
severity of CPN was much milder, thus increased proteinuria was observable only when CPN was
increased as in the 2-year inhalation exposure study fSaito etal.. 20131. Elevated BUN and
creatinine typically are not observed until very late in CPN progression. This was true for ETBE, as
most of these markers were elevated only after 2-year exposures.
Several of the CPN pathological effects are similar to—and can obscure the lesions
characteristic of—a2U-globulin- related hyaline droplet nephropathy (Webb etal.. 1990).
Additionally, renal effects of a2u-globulin accumulation can exacerbate the effects associated with
CPN fU.S. EPA. 1991allTravlos. 2011, 1239901}.
CPN often is more severe in males than in females. While background severity of CPN in
controls was higher in male rats, increased severity of CPN was reported in both male and female
rats with ETBE exposure, and was statistically significant at the highest exposure groups of both
sexes following chronic inhalation (see Table 1-2). Some of the observed renal lesions in male rats
following exposure to ETBE are effects commonly associated with CPN. A strong, statistically
significant, treatment-related relationship was observed between chronic ETBE exposure and
increased incidence of urothelial hyperplasia in male (but not female) rats in both the inhalation
and oral studies fSaito etal.. 2013: Suzuki etal.. 2012: TPEC. 2010a. b). Urothelial hyperplasia is
both increased by dose and weakly correlated with CPN, which is also dose-related (Table 1-5 and
Table 1-6). Thus, disentangling the contributions of dose and nephropathy in the development of
urothelial hyperplasia is not possible with the currently available information. Moreover, no
evidence is available to support that urothelial hyperplasia is not related toETBE treatment, given
the robust dose-response relationship in male rats treated with ETBE.
Finally, because tert-butanol is a major metabolite of ETBE and both chemicals induce
similar noncancer kidney effects, tert-butanol could be the active toxic moiety responsible for these
effects. The three noncancer kidney endpoints (kidney weights, urothelial hyperplasia, CPN) were
evaluated on an internal dose basis using PBPK modeling to compare these data from ETBE and
tert- butanol studies (Salazar etal.. 2015). The results demonstrate that noncancer kidney effects,
including kidney weight changes, urothelial hyperplasia, and exacerbated CPN, yielded consistent
dose-response relationships across routes of exposure and across ETBE and tert-butanol studies
using tert-butanol blood concentration as the dose metric. These results are consistent with the
hypothesis that tert-butanol mediates the noncancer kidney effects following ETBE administration,
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however contributing toxicity from the parent compound and other metabolites has not been ruled
out
Overall Conclusion on MOA for Kidney Effects
ETBE increases a2U-globulin deposition and hyaline droplet accumulation in male rat
kidneys, but only one of the five additional steps in the pathological sequence (linear
mineralization) was consistently observed (see Table 1-8). These data are insufficient to conclude
that ETBE induces a2U-globulin nephropathy, however, the observation of a2u-globulin
accumulation in hyaline droplets with ETBE exposure adds uncertainty regarding the human
relevance of the associated nephropathy in male rats CPN and the exacerbation of CPN could play a
role in the observed nephropathy. Currently, the MOA for the observed ETBE-induced exacerbation
of CPN is unknown, especially in female rats and in male and female mice which are not affected by
a2u-globulin nephropathy. Collectively, the evidence indicates other, unknown processes contribute
to the observed nephrotoxicity following ETBE exposure, particularly in female rats.
Integration of Kidney Effects
Kidney effects (increases in severity of nephropathy, increased kidney weight, alterations in
blood biomarkers, hyaline droplets, linear mineralization, and urothelial hyperplasia of the renal
pelvis) were observed across multiple studies, predominantly in rats; chronic bioassays found no
treatment-related increases in renal tumors. The available evidence indicates that multiple
processes induce the noncancer kidney effects.
Some endpoints in male rats (hyaline droplets, linear mineralization) are components of the
a2u-globulin process. U.S. EPA (1991a) states that"[i]f a compound induces a2U-globulin
accumulation in hyaline droplets, the associated nephropathy in male rats is not an appropriate
endpoint to determine noncancer (systemic) effects potentially occuring in humans." Therefore, in
the case of ETBE exposure, endpoints directly associated with a2U-globulin processes were not
considered an indication of human health hazard for noncancer kidney toxicity. Because a2u-
globulin nephropathy is strictly a male rat phenomenon, dose-related kidney effects in female rats
and mice are not confounded by a2u-globulin nephropathy.
It has been observed that chemicals that bind to a2U-globulin can lead to increased incidence
and/or severity of CPN {Travlos, 2011,1239901}{U.S. EPA, 1991, 635839}{Frazier, 2012,
2919046}. CPN is a common and well-established constellation of age-related lesions in the kidney
of male and female rats, and there is no known counterpart to CPN in aging humans. However, CPN
is not a specific diagnosis on its own but an aggregate term describing a spectrum of effects,
employed to reduce the time and effort required to grade each component of the disease. The
individual lesions associated with CPN (e.g. tubular degeneration, thickening of basement
membranes, glomerular sclerosis, etc.) also occur in the human kidney {Lusco, 2016,
5926047}{Frazier, 2012, 2919046}{Zoja, 2015, 5926046}{Abrass, 2000, 5426141}. Although CPN
has no known analog in the aging human kidney {Hard, 2009, 667590}{NIEHS, 2019, 5098230}, the
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etiology is unknown {NIEHS, 2019, 5098230}{Hard, 2004, 782757}{Peter, 1986,194755}. Given
that there is no definitive pathogenesis for CPN, it cannot be ruled out that a chemical which
exacerbates CPN in rats could also exacerbate existing disease processes in the human kidney
{NIEHS, 2019, 5098230}. Therefore, increased incidence of kidney effects with ETBE exposure in
the female rat (including increased kidney weight and increased severity of CPN) are considered
relevant to humans.
Several noncancer endpoints were concluded to result from ETBE exposure including
increased absolute kidney weight, histopathological changes, and increased blood biomarkers in
female rats, with the effects in males tending to be stronger than in females (potentially due to
confounding by a2U-globulin processes). A PBPK model-based analysis yielded consistent dose-
response relationships between kidney weight, urothelial hyperplasia, and chronic progressive
nephropathy (CPN) using tert-butanol blood concentration as the dose metric, consistent with the
hypothesis that tert-butanol mediates the noncancer kidney effects following ETBE administration
(Salazar 2015). Based on dose-related increases in these noncancer endpoints in rats, kidney
effects are a potential human hazard of ETBE exposure. The hazard and dose-response conclusions
regarding these noncancer endpoints associated with ETBE exposure are discussed further in
Section 1.3.1.
1.2.2. Liver Effects
Synthesis of Effects in Liver
This section reviews the studies that investigated whether exposure to ETBE can cause liver
noncancer or cancer effects in humans or animals. The database for ETBE-induced liver effects
includes nine studies conducted in animals, all but two of which were performed in rats. A
description of the studies comprising the database is provided in Section 1.2.1. Briefly, exposures
ranged from 13 weeks to 2 years and both inhalation and oral exposure routes are represented.
Studies using short-term and acute exposures that examined liver effects are not included in the
evidence tables; however, they are discussed in the text if they provide data informative of MOA or
hazard identification. Studies are arranged in evidence tables first by effect and then in alphabetical
order by author. The design, conduct, and reporting of each study were reviewed, and each study
was considered adequate to provide information pertinent to this assessment.
Liver weight Increased liver weight was observed with ETBE exposure in male and female
rats treated for various durations orally or by inhalation. Several factors associated with the 2-year
organ weight data could confound consideration for hazard identification. Proliferative lesions
(altered hepatocellular foci) were observed in rat livers, especially males, in both 2-year oral and
inhalation studies, which complicates interpretation of changes in organ weight. Furthermore,
inhalation exposure significantly increased liver adenomas and carcinomas in male rats at the
highest dose, corresponding to increased liver weights in those dose groups (Saito etal.. 2013:
TPEC. 2010bl. Organ weight data obtained from studies of shorter duration, however, are less
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complicated by these age-associated factors (e.g., tumors, mortality) and therefore could be
appropriate for hazard identification.
Chronic and subchronic studies by both oral and inhalation routes reported consistent,
statistically significant, dose-related increases in liver weights (see Figure 1-9, Figure 1-10, Table
1-9). Liver weight and body weight have been demonstrated to be proportional, and liver weight
normalized to body weight was concluded to be optimal for data analysis (Bailey etal.. 2004): thus,
only relative liver weight is considered in the determination of hazard. Relative liver weights were
consistently increased at similar exposure concentrations in four of five studies for males and three
of four studies for females; however, statistically significant increases often occurred only at the
highest tested concentration with increases in relative liver weight ranging from 17 to 27% in
males and 8 to 18% in females. Relative liver weights in rats were increased at only the highest
dose following oral exposures of 16 weeks or longer fMivataetal.. 2013: Fuiii etal.. 2010: TPEC.
2008c: Gaoua. 2004b). In utero exposure yielded similar effects on F1 liver weights, in terms of the
magnitude of percent change, from adult exposure (Gaoua. 2004b). Inhalation exposure increased
liver weight at the highest dose in female rats, but not in males, following 13-week exposure (TPEC.
2008b). Following a 28-day recovery period, male but not female liver weights were increased
fTPEC. 2008bl. Short-term studies observed similar effects on liver weight fTPEC. 2008a: White et
al.. 19951
Liver histopathology. Centrilobular hypertrophy and acidophilic (eosinophilic) and
basophilic focal lesions were the only dose-related types of pathological lesions observed in the
liver. Centrilobular hypertrophy was inconsistently increased throughout the evidence base, but
also was observed at the same concentrations that induced liver weight changes in rats of both
sexes after 13-week inhalation and 26-week oral exposures (see Table 1-10; Figure 1-9, Figure
1-10). A 26-week oral gavage study fMivataetal.. 2013: TPEC. 2008cl in rats and three 13-week
inhalation studies in mice and rats fWeng etal.. 2012: TPEC. 2008b: Medinskv etal.. 19991
demonstrated a statistically significant increase in centrilobular hypertrophy at the highest dose.
In addition, 2-year oral and inhalation studies in rats reported increased liver weight in male and
female rats.
Acidophilic (eosinophilic) and basophilic preneoplastic lesions were increased in male, but
not female rats, at the highest tested dose following a 2-year inhalation exposure to ETBE fSaito et
al.. 2013: TPEC. 2010bl. Following 2-year drinking water exposure to ETBE, an increasing, but not
statistically significant, trend in basophilic preneoplastic lesions was observed in the liver of male
rats, while incidence of these lesions decreased in female rats (Suzuki etal.. 2012: TPEC. 2010a).
Serum liver enzymes. Serum liver enzymes were inconsistently affected across exposure
routes (see Table 1-11; Figure 1-9, Figure 1-10). No enzyme levels were affected in studies of
exposure durations less than 2 years fMivataetal.. 2013: TPEC. 2008b). Gamma-glutamyl
transpeptidase (GGT) was significantly increased in male rats at one intermediate dose following
oral exposure and the two highest doses following inhalation exposure in 2-year studies fTPEC.
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2010a. b). GGT was not significantly affected in female rats in any study. No consistent dose-related
changes were observed in aspartate aminotransferase (AST), alanine aminotransferase (ALT), or
alkaline phosphatase (ALP) liver enzymes following either oral or inhalation exposure of any
duration. With the exception of a dose-related increase in serum GGT in male rats and an increase
in AST at the highest dose in females, no other dose-related changes in liver enzyme levels were
observed that were directionally consistent with the liver weight and hypertrophy effects.
Liver tumors. Data on liver tumor induction by ETBE are presented in Table 1-12. Liver
tumors were statistically significantly increased in male F344 rats at the high dose, but not in
females, following 2-year inhalation exposure fSaito etal.. 2013: TPEC. 2010bl. The incidence of
combined adenomas and carcinomas of 0/50, 2/50,1/50 and 10/50 at 0, 2,090, 6,270, 20,900
mg/m3, also resulted in a statistically significant, positive exposure-response trend (Peto's testp <
0.001). In this study, preneoplastic lesions, acidophilic (eosinophilic) and basophilic foci, were also
increased in male rats following a similar exposure pattern.
At the highest exposure dose in male rats, the dose at which the majority of liver tumors
were observed, a 25% reduction in body weight was seen, raising some question as to whether the
liver tumors observed in the highest exposure group in male rats are solely the result of excessive
toxicity rather than carcinogenicity of the tested agent. EPA's 2005 Cancer Guidelines discuss the
determination of an "excessively high dose" as compared to an "adequately high dose" and describe
the process as one of expert judgment which requires that "...adequate data demonstrate that the
effects are solely the result of excessive toxicity rather than carcinogenicity of the tested agent"
{U.S. EPA, 2005, 86237}. In the case of the 2-year inhalation study, the study authors did not report
any overt toxicity or altered toxicokinetics atthe high dose {JPEC, 2010,1517421}. In addition, the
high-dose female rats had a similar reduction in body weight (22%) and no liver tumors (or
increase in preneoplastic foci) were observed.
No significant increase in tumors was observed following two chronic oral bioassays
fSuzuki etal.. 2012: TPEC. 2010a: Maltoni et al.. 19991. However, one bioassay {Maltoni, 1999,
87642} was confounded by extremely low survival in controls (25-28% at 2 years), potentially due
to widespread respiratory infections (see discussion in Section 1.2.5). This extreme depression in
survival likely impacts this study's power to detect potential carcinogenicity. The other available
two year oral cancer bioassay was well designed, conducted, and reported and did not observed
significant increases in liver tumors, however an increased, but not statistically significant, trend in
basophilic preneoplastic lesions was observed in the liver of male rats, while incidence of these
lesions decreased in female rats (Suzuki etal.. 2012: TPEC. 2010a).
Two-stage "initiation, promotion" studies in male F344 and Wistar rats administered
mutagens for 2-4 weeks reported statistically significant increases in liver adenomas, carcinomas,
or total neoplasms after 19-23 weeks of ETBE exposure via oral gavage (Hagiwara et al.. 2 015:
Hagiwara etal.. 20111. Liver tumors were not observed in male F344 rats exposed to ETBE for 23
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1 weeks (n=12) without prior mutagen exposure fHagiwara etal.. 20111. while liver tumorigenesis
2 without prior mutagen exposure was not evaluated in Wistar rats fHagiwara etal.. 20151.
3 Table 1-9. Evidence pertaining to liver weight effects in animals exposed to
4 ETBE
Reference and study design
Results
Fuiiietal. (2010); JPEC (2008e)
Response relative to control
rat, Sprague-Dawley
P0, Male
P0, Female
oral - gavage
P0, male (24/group): 0,100, 300,1,000 mg/kg-d
daily for 16 wk beginning 10 wk prior to mating
Dose
(mg/kg-d)
Relative
weight
Dose
(mg/kg-d)
Relative
weight
P0, female (24/group): 0,100, 300,1,000 mg/kg-d
0
-
0
-
daily for 17 wk beginning 10 wk prior to mating to
lactation day (LD) 21
100
1%
100
-1%
300
2%
300
3%
1,000
21%*
1,000
9%*
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Reference and study design
Results
Gaoua (2004b)
Response relative to control
rat, Sprague-Dawley
oral - gavage
P0, Male
P0, Female
PO, male (25/group): 0, 250, 500,1,000 mg/kg-d
Dose
(mg/kg-d)
Relative
weight
Dose
(mg/kg-d)
Relative
weight
daily for a total of 18 wk beginning 10 wk before
mating until after weaning of the pups
P0, female (25/group): 0, 250, 500,1,000 mg/kg-d
0
-
0
-
daily for a total of 18 wk beginning 10 wk before
mating until PND 21
250
3%
250
10%
Fl, male (25/group): 0, 250, 500,1,000 mg/kg-d
500
6%
500
8%
P0 dams dosed daily through gestation and
lactation, then Fl doses beginning PND 22 until
1,000
24%*
1,000
4%
weaning of the F2 pups
Fl, Male
Fl, Female
Fl, female (24-25/group): 0, 250, 500,
1,000 mg/kg-d
P0 dams dosed daily through gestation and
Dose
(mg/kg-d)
Relative
weight
Dose
(mg/kg-d)
Relative
weight
lactation, then Fl dosed beginning PND 22 until
0
-
0
-
weaning of F2 pups
250
0%
250
3%
500
11%*
500
6%
1,000
25%*
1,000
9%*
Hagiwara et al. (2011); JPEC (2008d)
Response relative to control
rat, Fischer 344
Male
oral - gavage
male (12/group): 0,1,000 mg/kg-d
daily for 23 wk
Dose
(mg/kg-d)
0
1,000
Relative
weight
27%*
JPEC (2008b)
Response relative to control
rat, CRL:CD(SD)
Male
Female
inhalation - vapor
male (NR): 0, 150, 500,1,500, 5,000 ppm (0, 627,
2,090, 6,270, 20,900 mg/m3)b; female (NR): 0,150,
Dose
(mg/m3)
Relative
weight
Dose
(mg/m3)
Relative
weight
500,1,500, 5,000 ppm (0, 627, 2,090, 6,270,
0
-
0
-
20,900 mg/m3)
dynamic whole body chamber; 6 hr/d, 5 d/wk for
627
5%
627
4%
13 wk; generation method, analytical
2,090
5%
2,090
-1%
concentration, and method reported
6,270
5%
6,270
6%
20,900
10%
20,900
18%*
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Reference and study design
Results
JPEC (2008b)
Response relative to control
rat, CRL:CD(SD)
Male
Female
inhalation - vapor
male (6/group): 0, 5,000 ppm (0, 20,900 mg/m3)b;
female (6/group): 0, 5,000 ppm (0,
Dose
(mg/m3)
Relative
weight
Dose
(mg/m3)
Relative
weight
20,900 mg/m3)b
0
-
0
-
dynamic whole body chamber; 6 hr/d, 5 d/wk for
13 wk followed by a 28-d recovery period;
20,900
9%*
20,900
7%
generation method, analytical concentration, and
method reported
Mivata et al. (2013); JPEC (2008c)
Response relative to control
rat, CRL:CD(SD)
Male
Female
oral - gavage
male (15/group): 0, 5, 25,100,400 mg/kg-d;
female (15/group): 0, 5, 25,100, 400 mg/kg-d
Dose
(mg/kg-d)
Relative
weight
Dose
(mg/kg-d)
Relative
weight
daily for 26 wk
0
-
0
-
5
5%
5
1%
25
7%
25
1%
100
9%
100
4%
400
17%*
400
12%*
1 Conversion performed by study authors.
2 b4.18 mg/m3 = 1 ppm.
3 NR: not reported; *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
4 for controls, no response relevant; for other doses, no quantitative response reported.
5 Percent change compared to controls calculated as 100 x [(treated value - control value) -f control value],
6 Table 1-10. Evidence pertaining to liver histopathology effects in animals
7 exposed to ETBE
Reference and study design
Results
Gaoua (2004b)
P0, Male
P0, Female
rat, Sprague-Dawley
oral - gavage
P0, male (25/group): 0, 250, 500,1,000 mg/kg-d
daily for a total of 18 wk beginning 10 wk before
Dose
(mg/kg-d)
Incidence of
centrilobular
hvoertrophv
Dose
(mg/kg-d)
Incidence of
centrilobular
hvoertrophv
mating until after weaning of the pups
0
0/25
0
0/25
P0, female (25/group): 0, 250, 500,1,000 mg/kg-d
daily for a total of 18 wk beginning 10 wk before
250
0/25
250
0/25
mating until PND 21
500
0/25
500
0/25
1,000
3/25
1,000
0/25
This document is a draft for review purposes only and does not constitute Agency policy.
1-43 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and study design
Results
JPEC (2008b)
Male
Female
rat, CRL:CD(SD)
inhalation - vapor
male (NR): 0,150, 500, 1,500, 5,000 ppm (0, 627,
2,090, 6,270, 20,900 mg/m3)b; female (NR): 0,150,
Dose
Incidence of
centrilobular
Dose
Incidence of
centrilobular
(mg/m3)
hvoertrophv
(mg/m3)
hvpertrophv
500,1,500, 5,000 ppm (0, 627, 2,090, 6,270,
0
0/10
0
0/10
20,900 mg/m3)
dynamic whole body chamber; 6 hr/d, 5 d/wk for
627
0/10
627
0/10
13 wk; generation method, analytical
2,090
0/10
2,090
0/10
concentration, and method reported
6,270
0/10
6,270
0/10
20,900
4/10*
20,900
6/10*
JPEC (2008b)
Male
Female
rat, CRL:CD(SD)
inhalation - vapor
male (6/group): 0, 5,000 ppm (0, 20,900 mg/m3)b;
female (6/group): 0, 5,000 ppm (0,
Dose
(mg/m3)
Incidence of
centrilobular
hvoertrophv
Dose
(mg/m3)
Incidence of
centrilobular
hvpertrophv
20,900 mg/m3)b
0
0/6
0
0/6
dynamic whole body chamber; 6 hr/d, 5 d/wk for
13 wk followed by a 28-d recovery period;
20,900
0/6
20,900
0/6
generation method, analytical concentration, and
method reported
Medinskv et al. (1999); Bond et al. (1996b)
Male
Female
rat, Fischer 344
inhalation - vapor
male (48/group): 0, 500,1,750, 5,000 ppm (0,
2,090, 7,320, 20,900 mg/m3)b; female (48/group):
Dose
(mg/m3)
Incidence of
centrilobular
hvoertrophv
Dose
(mg/m3)
Incidence of
centrilobular
hvpertrophv
0, 500,1,750, 5,000 ppm (0, 2,090, 7,320,
0
0/11
0
0/10
20,900 mg/m3)b;
dynamic whole body chamber; 6 hr/d, 5 d/wk for
2,090
0/11
2,090
0/11
13 wk; generation method, analytical
7,320
0/11
7,320
0/11
concentration, and method reported
20,900
0/11
20,900
0/11
Medinskv et al. (1999); Bond et al. (1996a)
Male
Female
mice, CD-I
inhalation - vapor
male (40/group): 0, 500,1,750, 5,000 ppm (0,
2,090, 7,320, 20,900 mg/m3)b; female (40/group):
Dose
(mg/m3)
Incidence of
centrilobular
hypertrophy
Dose
(mg/m3)
Incidence of
centrilobular
hypertrophy
0, 500,1,750, 5,000 ppm (0, 2,090, 7,320,
0
0/15
0
0/13
20,900 mg/m3)b
dynamic whole body chamber; 6 hr/d, 5 d/wk for
2,090
0/15
2,090
2/15
13 wk; generation method, analytical
7,320
2/15
7,320
1/15
concentration, and method reported
20,900
8/10*
20,900
9/14*
This document is a draft for review purposes only and does not constitute Agency policy.
1-44 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and study design
Results
Mivata et al. (2013); JPEC (2008c)
Male
Female
rat, CRL:CD(SD)
oral - gavage
male (15/group): 0, 5, 25,100,400 mg/kg-d;
female (15/group): 0, 5, 25,100, 400 mg/kg-d
Dose Incidence of Dose
(mg/kg-d) centrilobular (mg/kg-d)
hvoertrophv
Incidence of
centrilobular
hvoertrophv
daily for 26 wk
0
0/15
0
0/15
5
0/15
5
0/15
25
0/15
25
0/15
100
0/15
100
0/15
400
6/15*
400
6/15*
Saito et al. (2013); JPEC (2010b)
Male
rat, Fischer 344
inhalation - vapor
male (50/group): 0, 500,1,500, 5,000 ppm (0,
Dose
(mg/m3)
Acidophilic Basophilic
foci in liver foci in liver
Bile duct
hvoerplasia
Centrilobular
hvoertroohv
2,090, 6,270, 20,900 mg/m3)b; female (50/group):
0
31/50 18/50
48/50
0/50
0, 500,1,500, 5,000 ppm (0, 2,090, 6,270,
20,900 mg/m3)b
2,090
28/50 10/50
44/50
0/50
dynamic whole body inhalation; 6 hr/d, 5 d/wk for
6,270
36/49 13/49
46/49
0/49
104 wk; generation method, analytical
concentration, and method reported
20,900
Female
39/50* 33/50*
41/50
0/50
Dose
Acidophilic Basophilic
Bile duct
Centrilobular
(mg/m3)
foci in liver foci in liver
hvoerplasia
hvoertroohv
0
2/50 36/50
5/50
0/50
2,090
1/50 31/50
8/50
0/50
6,270
4/50 32/50
7/50
0/50
20,900
2/50 28/50
6/50
0/50
This document is a draft for review purposes only and does not constitute Agency policy.
1-45 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and study design
Results
Suzuki et al. (2012); JPEC (2010a)
Male
rat, Fischer 344
oral - water
male (50/group): 0, 625, 2,500,10,000 ppm (0, 28,
Dose
(mg/kg-
dl
Acidophilic
foci in liver
Basophilic Bile duct
foci in liver hyperplasia
Centrilobular
hvpertrophv
121, 542 mg/kg-d)a; female (50/group): 0, 625,
2,500, 10,000 ppm (0, 46,171, 560 mg/kg-d)a
0
14/50
14/50
49/50
0/50
daily for 104 wk
28
12/50
18/50
47/50
0/50
121
17/50
20/50
48/50
0/50
542
13/50
22/50
47/50
0/50
Female
Dose
Acidophilic
Basophilic Bile duct
Centrilobular
(mg/kg-
d)
foci in liver
foci in liver hyperplasia
hvpertrophv
0
2/50
36/50
1/50
0/50
46
2/50
25/50*
4/50
0/50
171
1/50
31/50
4/50
0/50
560
0/50
30/50*
3/50
0/50
Weng et al. (2012)
Male
Female
mice, C57BL/6
inhalation - vapor
male (5/group): 0, 500,1,750, 5,000 ppm (0,
2,090, 7,320, 20,900 mg/m3)b; female (5/group):
Dose
(mg/m3
Incidence of
) centrilobular
hypertrophy
Dose
(mg/m3)
Incidence of
centrilobular
hvpertrophv
0, 500,1,750, 5,000 ppm (0, 2,090, 7,320,
0
1/5
0
0/5
20,900 mg/m3)b
dynamic whole body chamber, 6 hr/d, 5 d/wk for
2,090
0/5
2,090
0/5
13 wk; generation methods not reported, but
7,320
0/5
7,320
1/5
analytical methods (gas chromatograph) and
concentration reported
20,900
5/5*
20,900
5/5*
Weng et al. (2012)
Male
Female
mice, Aldh2-/-
inhalation - vapor
male (5/group): 0, 500,1,750, 5,000 ppm (0,
2,090, 7,320, 20,900 mg/m3)b; female (5/group):
Dose
(mg/m3
Incidence of
) centrilobular
Dose
(mg/m3)
Incidence of
centrilobular
hvoertrophv
hvpertrophv
0, 500,1,750, 5,000 ppm (0, 2,090, 7,320,
0
0/5
0
0/5
20,900 mg/m3)b
dynamic whole body chamber, 6 hr/d, 5 d/wk for
2,090
3/5
2,090
0/5
13 wk; generation methods were not reported,
7,320
2/5
7,320
0/5
but analytical methods (gas chromatograph) and
concentration reported
20,900
5/5*
20,900
4/5*
1 Conversion performed by study authors.
2 b4.18 mg/m3 = 1 ppm.
3 NR: not reported; *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
4 for controls, no response relevant; for other doses, no quantitative response reported.
5
This document is a draft for review purposes only and does not constitute Agency policy.
1-46
DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
1 Table 1-11. Evidence pertaining to liver biochemistry effects in animals
2 exposed to ETBE
Reference and study design
Results
JPEC (2008b)
rat, CRL:CD(SD)
inhalation - vapor
male (NR): 0,150, 500,1,500, 5,000 ppm
(0, 627, 2,090, 6,270, 20,900 mg/m3)b;
Response relative to control
Male
Dose
(mg/m3) ALT
ALP
AST
GGT
female (NR): 0,150, 500,1,500,
0
-
-
-
-
5,000 ppm (0, 627, 2,090, 6,270,
627
9%
13%
3%
11%
20,900 mg/m3)
dynamic whole body chamber; 6 hr/d,
5 d/wk for 13 wk; generation method,
2,090
6,270
0%
5%
12%
-12%
1%
-7%
0%
11%
analytical concentration, and method
20,900
12%
-9%
4%
-100%
reported
Female
Dose
(mg/m3)
0
627
ALT
ALP
AST
GGT
-1%
-3%
2%
25%
2,090
11%
-12%
-95%
12%
6,270
-5%
-7%
12%
25%
20,900
26%
5%
0%
25%
Mivata et al. (2013); JPEC (2008c)
Response relative to control
rat, CRL:CD(SD)
oral - gavage
Male
male (15/group): 0, 5, 25,100,
Dose
400 mg/kg-d; female (15/group): 0, 5, 25,
(mg/kg-d)
ALT
ALP
AST
GGT
100,400 mg/kg-d
0
daily for 180 d
5
10%
2%
16%
25%
25
48%
12%
19%
50%
100
13%
-7%
20%
25%
400
35%
27%
23%
100%
Female
Dose
(mg/kg-d)
0
5
ALT
ALP
AST
GGT
11%
6%
10%
40%
25
21%
-21%
13%
20%
100
46%
-18%
19%
0%
400
21%
-19%
4%
-20%
This document is a draft for review purposes only and does not constitute Agency policy.
1-47 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and study design
Results
Saito et al. (2013); JPEC (2010b)
rat, Fischer 344
inhalation - vapor
male (50/group): 0, 500,1,500, 5,000 ppm
(0, 2,090, 6,270, 20,900 mg/m3)b; female
(50/group): 0, 500, 1,500, 5,000 ppm (0,
2,090, 6,270, 20,900 mg/m3)b
dynamic whole body inhalation; 6 hr/d,
5 d/wk for 104 wk; generation method,
analytical concentration, and method
reported
Response relative to control
Male
Dose
(mg/m3) ALT
0
2,090 53%
6,270 -3%
20,900 24%
Female
Dose
(mg/m3) MI ME ASI GGT
0 - - - -
2,090 2% 12%
6,270 -5% -4%
20,900 4%* 4%
ALP
0%
-21%*
-5%
AST
29%
-16%
-2%*
22%
10%
18%*
GGT
33%
50%*
200%*
50%
0%
150%
Suzuki et al. (2012); JPEC (2010a)
rat, Fischer 344
oral - water
male (50/group): 0, 625, 2,500,
10,000 ppm (0, 28, 121, 542 mg/kg-d)a;
female (50/group): 0, 625, 2,500,
10,000 ppm (0, 46, 171, 560 mg/kg-d)a;
daily for 104 wk
Response relative to control
Male
Dose
(mg/kg-d) MI ME ASI GGT
0
-
-
-
-
28
-17%
-5%
-21%
0%
121
2%
3%
-3%
43%*
542
-4%
0%
-1%
29%
Female
Dose
(mg/kg-d)
ALT
ALP
AST
GGT
0
-
-
-
-
46
-10%
-16%
-19%
0%
171
-15%
2%
-17%
0%
560
-26%
-15%
-46%*
33%
1 Conversion performed by study authors.
2 b4.18 mg/m3 = 1 ppm.
3 NR: not reported; *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
4 -: for controls, no response relevant; for other doses, no quantitative response reported.
5 (n): number evaluated from group.
6 Percent change compared to controls calculated as 100 x [(treated value - control value) -f control value],
7 Abbreviations: ALT = alanine aminotransferase, ALP = alkaline phosphatase, AST = aspartate aminotransferase,
8 GGT = gamma-glutamyl transferase.
9
This document is a draft for review purposes only and does not constitute Agency policy.
1-48 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
¦ = exposures at which the endpoint was reported statistically significant by study authors
~ =exposures at which the endpoint was reported not statistically significant by study authors
Relative Liver
Weight
PO Male rat; 16wks (A)
PO Female rat; 16wks [A]
PO Male rat; 18wks (B)
PO Female rat; IBwks (B)
F1 Male rat; GD 0-adult (B)
F1 Female rat; GD 0-adult (B)
Male rat; 23wks (C)
Male rat; 26wks (D)
Female rat; 26wks (D)
-B-
B—B-
~ ~ m
B—B-
B-
B-
-B-
Centrilobular
Hypertrophy
PO Male rat; 18wks (B)
PO Female rat; IBwks (B)
Male rat; 26wks (D)
Female rat; 26wks (D)
Male rat; 104wks (E)
Female rat; 104wks (Ej
B—D—a
~ ~ HI
Qh
B-
-B-
-B B ¦
B IB B
B B B
Serum
Liver
Enzymes
Male rat; ALT, AST, ALP, GGT;26wks (D)
Female rat; ALT, AST, ALP, GGT;26wks (D)
Male rat; ALT, AST, ALP;lQ4wks (E)
Male rat; t GGT;104w'--
Fem ale rat; ALT, ALP, GGT;10 ! I (E)
Female rat; i AST;104wks (E)
B-
B-
-B B-
-0
B-
10 100
Dose (mg/kg-day)
1,000
10,000
Sources; [A] Fujii et al„ 2010; JPEC,2O08e (B) Gaoua, 2004b (C) Hagiwara et al, 2011 (D) Miyata et al, 2013;
JPEG, 2008c (E) Suzuki et al, 2012; JPEC, 2010a
Figure 1-9. Exposure-response array of noncancer liver effects following oral
exposure to ETBE.
This document is a draft for review purposes only and does not constitute Agency policy.
1-49 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
¦ = exposures at which the endpoint was reported statistically significant by study authors
~ =exposures at which the endpoint was reported not statistically significant by study authors
Relative Liver
Weight
Male rats; 13wks (A)
Female rats; 13wks (A)
Male rats; 13wks, 28d recovery (A)
Female rats; 13wks, 28d recovery (A)
Centrilobular
Hypertrophy
Male rats; 13wks (A)
Female rats; 13wks (A)
Male rats; 13wks, 28d recovery (AJ
Female rats; 13wks, 28d recovery (A)
Male rats; 13 wks (B)
Female rats; 13 wks (B)
Male mice; 13 wks (B)
Female mice; 13 wks (B)
Male mice; 13 wks (D)
Female mice; 13 wks (D)
Male Aldh2-/- mice; 13 wks (D)
Female AIdh2-/- mice; 13 wks (D)
Male rats; 104wks (C)
Female rats; 104wks (C)
Male rats; ALT, AST, ALP, GGT; 13wks (A)
Female rats; ALT, AST, ALP, GGT; 13wks (A)
Male rats; ALT; 104wks (C)
Male rats; I AST; 104wks (C)
Serum Liver Male rats; iALP; 104wks (C)
Enzymes
Male rats; TGGT; 104wks (C)
Female rats; ALT, ALP, GGT; 104wks (C)
Female rats; TAST; 104wks (C)
100 1,000 10,000
Exposure Concentration (mg/ra1)
100,000
Sources: (A) IPEC, 2008b (B) Medmsky et al, 1999; Bond et al„ 1996 (C) Saito et al., 2013; JPEC, 2010b (D) Weng
etal.,2012
Figure 1-10. Exposure-response array of noncancer liver effects following
inhalation exposure to ETBE.
This document is a draft for review purposes only and does not constitute Agency policy.
1-50 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Table 1-12. Evidence pertaining to liver tumor effects in animals exposed to
ETBE
Reference and study design
Results
Hepatocellular Adenoma and Carcinoma
Suzuki et al. (2012); JPEC (2010a)
Male
rat, Fischer 344
Dose
Adenoma or
oral - drinking water
(mg/kg-d)
Adenoma
Carcinoma
carcinoma
male (50/group): 0, 625, 2,500,10,000 ppm (0,
28,121, 542 mg/kg-d)a; female (50/group): 0, 625,
0
2/50
2/50
4/50
2,500, 10,000 ppm (0, 46, 171, 560 mg/kg-d)a
28
0/50
0/50
0/50
daily for 104 wk
121
0/50
0/50
0/50
542
0/50
0/50
0/50
Female
Dose
Adenoma or
(mg/kg-d)
Adenoma
Carcinoma
carcinoma
0
0/50
0/50
0/50
46
0/50
0/50
0/50
171
0/50
0/50
0/50
560
1/50
0/50
1/50
Maltoni et al. (1999)
Male
Female
rat, Sprague-Dawley
Dose
Adenoma or
Dose
Adenoma or
oral - gavage in olive oil
male (60/group): 0, 250,1,000 mg/kg-d; female
(60/group): 0, 250,1,000 mg/kg-d
4 d/wk for 104 wk; observed until natural death
(mg/kg-d)
0
250
carcinoma
0/60
0/60
(mg/kg-d)
0
250
carcinoma
0/60
0/60
(depressed survival 25-28% seen in controls at
1,000
0/60
1,000
0/60
104 wks)
NOTE: Tumor data not reanalyzed bv Malarkev
and Bucher (2011).
This document is a draft for review purposes only and does not constitute Agency policy.
1-51 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and study design
Results
Saito et al. (2013); JPEC (2010b)
rat, Fischer 344
inhalation - vapor
male (50/group): 0, 500,1,500, 5,000 ppm (0,
2,090, 6,270, 20,900 mg/m3)b; female (50/group):
0, 500,1,500, 5,000 ppm (0, 2,090, 6,270,
20,900 mg/m3)b
dynamic whole body inhalation; 6 hr/d, 5 d/wk for
104 wk; generation method, analytical
concentration, and method reported
Male
Dose
(mg/m3)
0
2,090
6,270
20,900
Female
Adenoma
0/50
2/50
1/50
9/50*
Carcinoma
0/50
0/50
0/50
1/50
Adenoma or
carcinoma
0/50
2/50
1/50
10/50*
Dose
(mg/m3)
Adenoma
Carcinoma
Adenoma or
carcinoma
0
1/50
0/50
1/50
2,090
0/50
0/50
0/50
6,270
1/50
0/50
1/50
20,900
1/50
0/50
1/50
Initiation-Promotion Studies
Hagiwara et al. (2011); JPEC (2008d)
rat, Fischer 344
oral - gavage in olive oil
male (30/group): 0, 300,1,000 mg/kg-d
daily for 23 wk following a 4-wk tumor initiation
by DMBDDC
+ no DMBDD initiation
Male
Dose
(mg/kg-d)
0
300
Adenoma
0/30
1/30
Carcinoma
1/30
0/30
1,000
6/30*
0/30
0+
0/12
0/12
1,000+
0/12
0/12
Hagiwara et al. (2015)
rat, Wistar
oral - gavage in olive oil
male (30/group): 0,100, 300, 500,1,000 mg/kg-d
daily for 19 wk following 2-wk tumor initiation by
N-ethyl-N-hydroxyethylnitrosamine (EHEN)
Male
Dose
(mg/kg-d)
0
100
Adenoma
4/30
5/30
Carcinoma
0/30
2/30
Adenoma or
carcinoma
4/30
7/30
300
8/30
0/30
8/30
500
8/30
3/30
10/30
1,000
15/30*
5/30*
17/30*
1 Conversion performed by study authors.
2 b4.18 mg/m3 = 1 ppm.
3 cDiethylnitrosamine (DEN), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), N-methyl-N-nitrosourea (MNU), 1,2-
4 dimethylhydrazine dihydrochloride (DMH), and N-bis(2-hydroxypropyl)nitrosamine (DHPN).
5 *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
6 for controls, no response relevant; for other doses, no quantitative response reported.
This document is a draft for review purposes only and does not constitute Agency policy.
1-52
DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
1 Mode of Action Analysis - Liver Effects
2 Key characteristics of carcinogens
3 Mechanistic information was grouped into 10 "key characteristics" useful for summarizing
4 and organizing the mechanistic data relevant to carcinogens (Smith etal.. 2016). The evidence
5 available for each characteristic is summarized in Table 1-13. Altogether, experimental evidence
6 informing several of the key characteristics of carcinogens was identified in the available literature..
7 ETBE was found to have the potential for the formation of electrophilic metabolites, but it was
8 concluded that there was inadequate evidence that ETBE induced any of the remaining key
9 characteristics.
10 Table 1-13.Evidence of key characteristics of carcinogens for ETBE.
Characteristic
Evidence
1. Is electrophilic or can be metabolically activated
to electrophiles
Metabolized to acetaldehyde in the liver.1,3
2. Is genotoxic
Inadequate evidence to draw a conclusion from 12
studies examining micronucleus, DNA strand breaks,
chromosomal aberration, and gene mutation
assays2,3
3. Alters DNA repair or causes genomic instability
No pertinent studies identified
4. Induces epigenetic alterations
No pertinent studies identified
5. Induces oxidative stress
Inadequate evidence to draw a conclusion from 3
studies examining 8-OHdG, 8-hOGGl formation3,4
6. Induces chronic inflammation
No pertinent studies identified
7. Is immunosuppressive
No pertinent studies identified
8. Modulates receptor-mediated effects
Inadequate evidence to draw a conclusion from 2
studies examining PPAR, CAR, and PXR activation5
9. Causes immortalization
No pertinent studies identified
10. Alters cell proliferation, cell death, or nutrient
supply
Inadequate evidence to draw a conclusion from 3
studies examining basophilic, acidophilic foci and
cellular proliferation5
11 1See Supplemental Information section B.1.3.
12 2See Supplemental Information section B.2.2.
13 3See Acetaldehyde-mediated liver toxicity and genotoxicity in this section.
14 4See Oxidative stress in this section.
15 5See Receptor-mediated effects in this section.
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Toxicokinetic considerations relevant to liver toxicity and tumors
ETBE is metabolized by cytochrome P450 (CYP) enzymes to an unstable hemiacetal that
decomposes spontaneously into tert-butanol and acetaldehyde fBernauer etal.. 19981.
Acetaldehyde is further metabolized in the liver by ALDH2, while tert-butanol undergoes systemic
circulation and ultimate excretion in urine. Thus, following ETBE exposure, the liver is exposed to
both acetaldehyde and tert-butanol, so the liver effects caused by tert-butanol (described in the
more detail in the IRIS assessment of tert-butanol) and acetaldehyde are relevant to evaluating the
MOA for liver effects observed after ETBE exposure.
tert-Butanol induces thyroid tumors in mice and kidney tumors in male rats, but has not
been observed to affect the incidence of rodent liver tumors following a 2-year oral exposure.
Although some data suggest tert-butanol could be genotoxic, the overall evidence is inadequate to
establish a conclusion. One study reported that tert-butanol might induce centrilobular
hypertrophy in mice after 2 weeks f Blanck et al.. 20101: however, no related liver pathology was
observed in other repeat-exposure rodent studies including both subchronic and 2-year bioassays.
Although Blanck etal. (20101 reported some limited induction of mouse liver enzymes following
short-term tert-butanol exposure, no corresponding evidence exists in rats following any exposure
duration. Therefore, a role for tert-butanol in liver carcinogenesis of ETBE appears unlikely.
In comparison, acetaldehyde associated with the consumption of alcoholic beverages is
genotoxic and mutagenic (IARC. 1999a). and acetaldehyde produced in the liver as a result of
ethanol metabolism has been suggested to be a contributor to ethanol-related liver toxicity and
cancer (Setshedi etal.. 2010). Additional discussion on the potential role of acetaldehyde in the
liver carcinogenesis of ETBE is provided below.
Receptor-mediated effects
ETBE exposure consistently increased relative liver weights in male and female rats and
increased hepatocellular adenomas and carcinomas in males (Saito etal.. 2013: TPEC. 2010b). In
addition to the increased centrilobular hypertrophy, which is one possible indication of liver
enzyme induction, chronic exposure induced focal proliferative lesions (including basophilic and
acidophilic foci) that could be more directly related to tumorigenesis. The centrilobular
hypertrophy was increased in rats of both sexes via both oral and inhalation exposure at
subchronic time points; not at 2 years, although significantly increased liver weight was observed.
Liver tumors were only observed in one sex (males) following one route of exposure (inhalation),
however, indicating that subchronic hypertrophy may not be associated with later tumor
development This process was investigated in several studies to determine whether nuclear
receptor activation is involved.
Centrilobular hypertrophy is induced through several possible mechanisms, many of which
are via activation of nuclear hormone receptors such as peroxisome proliferator-activated receptor
a (PPARa), pregnane X receptor (PXR), and the constitutive androstane receptor (CAR). The
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sequence of key events hypothesized for PPARa induction of liver tumors is as follows: activation of
PPARa, upregulation of peroxisomal genes, induction of gene expression driving PPARa-mediated
growth and apoptosis, disrupted cell proliferation and apoptosis, peroxisome proliferation,
preneoplastic foci, and tumors fKlaunig et al.. 20031. The sequence of key events hypothesized for
CAR-mediated liver tumors is as follows: CAR activation, altered gene expression as a result of CAR
activation, increased cell proliferation, clonal expansion leading to altered foci, and liver adenomas
and carcinomas (Elcombe etal.. 20141. PXR, which has no established MOA, is hypothesized to
progress from PXR activation to liver tumors in a similar manner as CAR. This progression would
include PXR activation, cell proliferation, hypertrophy, CYP3A induction, and clonal expansion
resulting in foci development One study that orally exposed male rats to low and high
concentrations of ETBE reported that several key sequences in the PPARa, PXR, and CAR pathways
were affected fKakehashi et al.. 2 0131.
PPAR
Limited evidence suggests that ETBE could activate PPAR-mediated events (Kakehashi et
al.. 20131. For instance, mRNA expression was significantly elevated for PPARa and PPARy after 1
week of exposure but not after 2 weeks. In addition, several PPARa-mediated proteins involved in
lipid and xenobiotic metabolism were upregulated in the liver after 2 weeks of exposure such as
AC0X1, CYP4A2, and ECH1. Additional effects in the PPAR pathway such as DNA damage (8-OHdG)
and apoptosis (ssDNA) also were significantly increased after 2 weeks at the highest concentration
of ETBE. Cell proliferation was increased after 3 days (Kakehashi et al.. 2 0151. unchanged after 1
week, significantly decreased after 2 weeks (Kakehashi etal.. 20131 and increased after 28 days
f Kakehashi etal.. 20151. The number of peroxisomes per hepatocyte was increased greater than
fivefold after 2 weeks of treatments. Finally, the incidences of preneoplastic basophilic and
acidophilic foci were significantly increased in males after 2 years of inhalation exposure to ETBE
fSaito et al.. 2013: TPEC. 201 Obi.
PPARa mediated genes were investigated in one study (Kakehashi et al., 2013). The high
dose of ETBE (2,000 mg/kg-day) which induced the most consistent changes in PPARa, Cyp4a,
Cypla, and Cyp3a in the oral gavage study (Kakehashi et al.. 20131 yielded a higher internal
metabolic rate in the liver (3.98 mg ETBE/hr) than from the 20,700 mg ETBE/m3 inhalation dose
(3.34 mg ETBE/hr) that increased liver tumors in the 2-year inhalation study (Saito etal.. 2013:
TPEC. 2010bl. Only Cyp2b genes associated with PPARa expression were affected at the low gavage
dose (3 00mg/kg-day), thus demonstrating poor dose-response relationships between PPAR-
mediated genes and downstream effects. Finally, PPAR agonists typically decrease rates of
apoptosis early in the process, which is in contrast to the increased rate of apoptosis observed after
2 weeks of ETBE exposure (Kakehashi etal.. 2013).However. several measures required for a full
evaluation of the PPAR MOA were absent. Selective clonal expansion and gap junction intercellular
communication were not examined in any study. No evidence is available in wild-type or PPARa-
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null mice to demonstrate if PPARa gene expression changes in KO mice. Overall, these data are
inadequate to conclude that ETBE induces liver tumors via a PPARa MOA.
CAR/PXR
Kakehashi etal. T20131 reported several CAR- and PXR-mediated events following ETBE
exposure. After 2 weeks of exposure at the high dose of ETBE, CAR- and PXR-regulated xenobiotic
metabolic enzymes were upregulated, including Cyp2bl, Cyp2b2, Cyp3al, and Cyp3a2 as determined
by mRNA or protein expression. Other PXR/CAR-regulated genes such as Sultldl, Ugt2b5, and
Ugtlal also had elevated mRNA expression after 1 and 2 weeks of exposure, which all suggest
activation of CAR and PXR. However, with the exception of Cyp2b, these genes were only increased
at the high dose, which yielded an internal rate of ETBE metabolism (3.98 mg/hr) that was greater
than the metabolism rate (3.34 mg/hr) associated with liver tumors in the 2-year inhalation study
(Saito etal.. 2013: TPEC. 2010b). Histological evidence (preneoplastic foci) supporting increased
liver cell proliferation is available following chronic, but not subchronic exposures (Saito etal..
2013: TPEC. 2010b). Several data gaps were not evaluated, such as a lack of clonal expansion and
gap junction communication. These data provide evidence that CAR and PXR are activated at high
concentrations in the liver following acute ETBE exposure; however, due to crosstalk of CAR and
PXR on downstream effects such as cell proliferation, preneoplastic foci, and apoptosis..
Furthermore, the data do not provide enough information to determine dose-response
relationships or temporal associations, which are helpful for establishing an MOA. Altogether, these
data are inadequate to conclude that ETBE induces liver tumors via a CAR/PXR MOA.
In summary, several gaps in the receptor mediated effects data (for PPARa, CAR, and PXR)
are noted such as evidence in only one species, lack of any studies in PPAR KO mice, lack of dose
response concordance between receptor mediated gene changes and tumors, and lack of any
receptor mediated data outside of the 1 and 2 week time points, which preclude establishing
temporal associations. Overall, these data are inadequate to conclude that ETBE induces liver
tumors via a PPARa or CAR/PXR MOA.
Acetaldehvde-mediated liver toxicity and genotoxicity
Another possible MOA for increased tumors could be due to genotoxicity and mutagenicity
resulting from the production of acetaldehyde in the liver, the primary site for ETBE metabolism.
Acetaldehyde produced as a result of metabolism of alcohol consumption is considered
carcinogenic to humans, although evidence is not sufficient to show that acetaldehyde formed in
this manner causes liver carcinogenesis (IARC. 2012). Acetaldehyde administered directly has been
demonstrated to increase the incidence of carcinomas following inhalation exposure in the nasal
mucosa and larynx of rats and hamsters. Furthermore, acetaldehyde has induced sister chromatid
exchanges in Chinese hamster ovary cells, gene mutations in mouse lymphomas, and DNA strand
breaks in human lymphocytes IARC fl999al. Acetaldehyde has been shown to have an inhibitory
effect on PPARa transcriptional activity fVenkata et al.. 20081. although no effect of acetaldehyde on
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CAR or PXR activation has been established. Additionally, the acetaldehyde metabolic enzyme
aldehyde dehydrogenase 2 (ALDH2) is polymorphic in the human population, which contributes to
enhanced sensitivity to the effects of acetaldehyde among some subpopulations such as people of
East Asian origin flARC. 2012: Brennan et al.. 20041. IARC f20121 found that ALDH2 status was
associated with increased esophageal cancer. Although IARC f20121 found inconclusive evidence
for a contribution of ALDH2 to liver cancer, Eriksson (2015) concluded that reduced aldehyde
metabolism is associated with liver cancer by further analyzing the ALDH2 compositions of the
controls in the case-control studies.
Several studies have examined the role of acetaldehyde and the metabolizing enzyme
ALDH2 in genotoxicity and centrilobular hypertrophy following ETBE exposure. Ninety-day
inhalation exposure to ETBE significantly increased the incidence of centrilobular hypertrophy in
male Aldh2 knockout (KO) mice compared with wild type (WT), while females appeared to be less
sensitive, similar to controls (Weng etal.. 2012). Hepatocyte DNA damage as determined by DNA
strand breaks and oxidative base modification was increased at the highest concentration of ETBE
exposure in the WT males, but not in WT females. Measures of DNA damage were all statistically
significantly increased in both male and female Aldh2 KO mice fWeng etal.. 20121. Further
demonstrating enhanced genotoxic sensitivity in males compared with females, erythrocyte
micronucleus assays and oxidative DNA damage (8-hOGGl) in leukocytes were observed to be
statistically significantly increased and dose responsive only in male Aldh2 KO mice (Weng etal..
2013). Together, although these data suggest a potential role for acetaldehyde in the increased liver
tumor response observed in male rats exposed to ETBE, the available data are inadequate to
conclude that ETBE induces liver tumors via acetaldehyde-mediated mutagenicity.
Oxidative Stress
Studies with pertinent information on the evaluation of oxidative stress are limited to a few
studies measuring oxidative DNA damage in leukoyctes and hepatocytes in mice (Weng etal.. 2012)
and one study in the liver of rats (Kakehashi etal.. 2013). Hepatocytes in male mice had increased
levels of 8-OHdG after 13 weeks of inhalation exposure to the concentration of ETBE that induced
liver tumors following 2 years of inhalation exposure. No significant dose response was reported.
Similarly, 8-OHdG was increased after 2 weeks of oral gavage in rats f Kakehashi etal.. 20131 at a
concentration two-fold greater than that inducing rat liver tumors in two-stage initiation-
promotion assays fHagiwara etal.. 2015: Hagiwara etal.. 20111. In addition, as discussed in the
previous paragraph, oxidative DNA damage was also induced in AIdh2 KO mice (Weng etal.. 2013)
and AIdh2 heterozygous mice {Weng, 2019, 5343910}. Overall, these data are inadequate to
conclude that ETBE induces liver tumors via oxidative stress.
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Overall Conclusions on MO A for Liver Effects
Several reviews of the available mechanistic data suggest that the PPAR, PXR, and CAR
pathways induce liver tumors in a manner not relevant to humans fElcombe et al.. 2014: Klaunig et
al.. 20031. although this conclusion has been questioned fGuvton et al.. 20091. The database is
inadequate to determine if nuclear receptor-mediated pathways (i.e., PPAR and CAR/PXR)
contribute to the tumorigenesis observed in ETBE-treated male rats. Furthermore, centrilobular
hypertrophy was observed at the same concentrations that induced liver weight changes in rats of
both sexes after 13-week inhalation and 26-week oral exposure, yet liver tumors were observed
only following oral exposure in male rats. This observation suggests that these effects are not
associated with the observed rat liver tumorigenesis. Therefore, given the available data, ETBE-
induced liver tumors in male rats are relevant to human hazard identification and are scientifically
supported.
Evidence suggests that metabolism of ETBE to acetaldehyde could contribute to ETBE-
induced liver carcinogenesis. For instance, enhancement of ETBE-induced liver toxicity and
genotoxicity has been reported inv4/d/j2-deficient mice, which have an impaired ability to
metabolize acetaldehyde fWeng etal.. 2013: Wengetal.. 20121. The database, however, is
inadequate to conclude that ETBE induces liver tumors via acetaldehyde-mediated mutagenic MOA.
Integration of Liver Effects
Liver effects were observed in oral and inhalation studies with exposure durations of
13 weeks to 2 years. Evidence for ETBE-induced noncancer liver effects is available from rat and
mouse studies that include centrilobular hypertrophy, increased liver weight, and changes in serum
liver enzyme levels. Based on dose-related increases in relative liver weight and increases in
hepatocellular hypertrophy in male and female rats, and considering the poor temporal correlation
of serum biomarkers and pathological lesions indicative of accumulating damage, evidence of liver
effects associated with ETBE exposure is suggestive. The hazard and dose-response conclusions
regarding these noncancer endpoints associated with ETBE exposure are further discussed in
Section 1.3.1.
The carcinogenic effects observed include increased hepatocellular adenomas and
carcinomas in males in a 2-year bioassay and ETBE-promoted liver tumorigenesis after 23 weeks
following mutagen pretreatment. Although only one carcinoma was observed, rodent liver
adenomas could progress to malignancy, eventually forming carcinomas (Liau etal.. 2013:
McConnell etal.. 1986). Mechanistic data on the role of PPAR, PXR, and CAR activation in liver
tumorigenesis were inadequate to conclude that these pathways mediate tumor formation.
Additional mechanistic studies in transgenic mice suggest that lack of Aldh2 enhances ETBE-
induced liver toxicity and genotoxicity, which is consistent with the observed genotoxicity being
mediated by the ETBE metabolite acetaldehyde, although the database is inadequate to conclude
that ETBE induces liver tumors via acetaldehyde-mediated mutagenic MOA. The hazard and dose-
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response conclusions regarding the liver tumors associated with ETBE exposure are further
discussed as part of the overall weight of evidence for carcinogenicity in Section 1.3.2.
1.2.3. Reproductive Effects
Synthesis of Effects Related to Male Reproduction
The database examining male reproductive effects following ETBE exposure contains no
human data but is comprised of animal data from rats and mice. Effects on male reproduction,
including fertility, male reproductive organ weights, histopathology, sperm parameters, and
hormone levels were evaluated in a one-generation oral study fFuiii etal.. 20101. a two-generation
oral study fGaoua. 2004bl. 13- and 9-week inhalation studies fWeng etal.. 20141. and a 14-day oral
study fde Pevster etal.. 20091. Additional data on male reproductive organ weights and
histopathology were obtained from two 2-year carcinogenicity studies [oral: Suzuki etal. (2012):
TPEC (2010a): inhalation: Saito etal. (2013): TPEC (2010b)]. a medium term carcinogenicity study
(23-week oral exposure) (Hagiwara etal.. 2011: TPEC. 2008d). a 180-day oral study (Mivata et al..
2013: TPEC. 2008cl. a 90-day inhalation study flPEC. 2008bl. and a 13-week inhalation study
(Medinskv et al.. 1999). These studies were conducted in Sprague-Dawley rats, Fischer 344 rats,
CD-I mice, and C57BL/6 mice, and the design, conduct, and reporting of each study were of
sufficient quality to inform human health hazard assessment. Selected endpoints from these studies
are summarized in
Table 1-14.
The one- and two-generation reproductive toxicity studies found no effects on copulation,
fertility, or sperm parameters in adult male Sprague-Dawley rats exposed to ETBE by oral gavage at
concentrations up to 1,000 mg/kg-day for 10 weeks prior to mating fFuiii etal.. 2010: Gaoua.
2004b). nor in F1 male offspring exposed during gestation, lactation, and post-weaning in the diet
fGaoua. 2004b). No dose-related changes in testicular histopathology were observed in F0 or F1
males fGaoua. 2004b). Furthermore, no dose-related histopathological changes or significant
changes in absolute male reproductive organ weight were observed in the 2-year carcinogenicity
studies in Fischer 344 rats at oral doses up to 542 mg/kg-day (Suzuki etal.. 2012: TPEC. 2010a) or
at inhalation exposure concentrations up to 20,900 mg/m3 f Saito etal.. 2013: TPEC. 2010bl: in the
medium term carcinogenicity study in Fischer 344 rats (Hagiwara etal.. 2011: TPEC. 2008d): in the
180-day oral study in Sprague-Dawley rats at doses up to 400 mg/kg-day (Mivata etal.. 2013: TPEC.
2008c): in the 90-day inhalation study in Sprague-Dawley rats at doses up to 20,900 mg/m3 (TPEC.
2008b): or in the 14-day oral study in Fischer 344 rats at doses up to 1,800 mg/kg-day fde Pevster
etal.. 20091. In some cases, dose-related increases in relative organ weights were observed,
including significant increases in relative testis weight fFuiii etal.. 2010: TPEC. 2010b: Gaoua.
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2004b 1 and relative prostate weight (Gaoua. 2004b 1 at the highest doses tested, which may have
been attributable to reduced body weight gain in these groups.
In contrast, testicular degeneration was observed in two 13-week ETBE inhalation studies
in which rats and mice were exposed to concentrations ranging from 2,090-20,900 mg/m3. In
Fischer 344 rats, a statistically significant increase in the percentage of seminiferous tubules with
spermatocyte degeneration was observed; however, there were no significant microscopic findings
in CD-I mice under these same exposure conditions and no changes in male reproductive organ
weights in rats or mice (Medinskv etal.. 19991. In C57BL/6 wild type and AIdh2 KO mice, there was
a dose-related increase in the incidence of atrophy of seminiferous tubules (described by the
authors as "slight" or "extremely slight" atrophy), with a greater incidence of atrophy occurring in
Aldh2 KO mice compared to wild type fWengetal.. 20141. ETBE-exposed mice also had significant
decreases in sperm head numbers and sperm mobility (expressed as the percentage of motile
sperm, percentage of static sperm, and percentage of sperm with rapid movement) and a significant
increase in sperm DNA damage (expressed as strand breaks and oxidative DNA damage), with
effects on sperm parameters reaching statistical significance at lower exposure concentrations in
AIdh2 KO mice (2,090 mg/m3) compared to wild type (7,320-20,090 mg/m3). Significantly
decreased epididymis weight was observed in AIdh2 KO mice but not wild type mice.
Wengetal. f20141 also conducted a 9-week inhalation study using lower ETBE exposure
concentrations (209-2,090 mg/m3) and three mouse genotypes (wild type, Aldh2 KO, and AIdh2
heterozygous; n=5/group). Wild type mice had little to no change in male reproductive organ
weights or sperm parameters at any of the tested concentrations, whereas significant effects were
observed on sperm count, sperm mobility, and sperm DNA damage in AIdh2 KO and heterozygous
mice at exposure concentrations as low as 836 mg/m3 ETBE. Aldh2 heterozygous mice had
significantly decreased relative testis and epididymis weight in the 20,090 mg/m3 exposure group.
However, for unknown reasons, several reproductive effects were noted to be more pronounced in
the heterozygous mice as compared to the Aldh2 KO mice. Taken together, the results ofWengetal.
(2014) indicate that populations with inactive Aldh2 variants may be more susceptible to male
reproductive toxicity following exposure to ETBE. However, these effects are considered to be
preliminary due to the small sample size (n=5) in one species, in one study, and the unconvincing
magnitude of many of the statistically significant effects (including the observation that the
heterozygotes exhibited more robust changes than the knockouts).
Although testicular lesions were not found in the 14-day oral study in Fischer 344 rats fde
Pevster et al.. 2009). plasma estradiol levels in these animals were increased by up to 106%
compared to controls. Plasma testosterone in the 1,800 mg/kg-day dose group was decreased by
34% compared to controls, but the difference was not statistically significant and was not observed
in any other ETBE dose group. The authors conducted a separate in vitro experiment to evaluate
testosterone production in isolated Sprague-Dawley rat Leydig cells and found reduced
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1 testosterone production in ETBE-treated cells compared to controls (data not shown in evidence
2 table).
3
4
5 Table 1-14. Evidence pertaining to male reproductive effects in animals
6 exposed to ETBE
Reference and Study Design
Results
Male Fertility
Fuiiietal. (2010); JPEC (2008e)
F0 Generation-Parent
rat, Sprague-Dawley
oral - gavage
F0, male and female
(24/sex/group): 0,100, 300,
Dose (mg/kg-
dl
Copulation
index (%)
Absolute
change from
control (%)
Fertility index
i%l
Absolute
change from
control (%)
1,000 mg/kg-d; dosed daily for
0
100
-
87.5
-
17 wks, from 10 wks premating
100
91.7
-8.3
100
12.5
to lactation day 21
300
95.8
-4.2
95.7
8.2
1,000
100
0
91.7
4.2
Gaoua (2004b)
F0 Generation-Parent
rat, Sprague-Dawley
oral - gavage
F0, male and female
(25/sex/group): 0, 250, 500,
Dose (mg/kg-
Male mating
Absolute
change from
Male fertility
Absolute
change from
dl
index3 (%)
control (%)
index" (%)
control (%)
1,000 mg/kg-d
0
100
-
92
-
dosed daily for 18 wks from 10
250
100
0
84
-8
wks premating until weaning of
F1 pups
500
100
0
88
-4
Fl, male and female (24—
25/group): 0, 250, 500, 1,000
1,000
100
0
100
8
mg/kg-d
dosed daily from PND 22 until
Fl Generation-Offspring
weaning of F2 pups
F0 Generation-Parent
Dose (mg/kg-
Male mating
Absolute
change from
Male fertility
Absolute
change from
dl
index3 (%)
control (%)
index" (%)
control (%)
0
96
-
92
-
250
96
0
92
0
500
100
4
88
-4
1,000
96
0
96
4
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Reference and Study Design
Results
Testicular Histopathology
Medinskv et al. (1999); Bond et
Incidence of spermatocyte
Incidence of sloughed
al. (1996b)
Dose (mg/m3)
degeneration
epithelium
rat, Fischer 344
inhalation - vapor
0
11/11
7/11
male (48/group): 0, 500,1,750,
2,090
11/11
3/11
5,000 ppm
(0, 2,090, 7,320,
7,320
11/11
3/11
20,900 mg/m3)a; female
20,900
10/11
7/11
(48/group): 0, 500, 1,750,
5,000 ppm
(0, 2,090, 7,320,
20,900 mg/m3)a
Dose (mg/m3)
Mean seminiferous tubules with
spermatocyte degeneration (%)
Absolute change from control
i%l
dynamic whole body chamber;
0
2.1
-
6 hr/d, 5 d/wk for 13 wk
2,090
2.4
0
7,320
7.8*
6
20,900
12.7*
Mean seminiferous tubules with
11
Absolute change from control
Dose (mg/m3)
lumenal debris (%)
i%l
0
2.1
-
2,090
0.7
-1
7,320
2.8
1
20,900
1
-1
This document is a draft for review purposes only and does not constitute Agency policy.
1-62 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and Study Design
Results
Weng et al. (2014)
Wild Type Mice; 13-week Exposure
mice, C57BL/6
inhalation - vapor
male (5/group): 0, 500,1,750,
5,000 ppm (0, 2,090, 7,320,
Incidence of
Total incidence of
Dose
(mg/m3)
"extremelv slight"
atrophv
Incidence of atrophv of
"slight" atrophv seminiferous tubules
20,900 mg/m3)a
0
1/5
0/5
1/5
dynamic whole body
inhalation; 6 h/d, 5 d/wk for 13
2,090
0/5
0/5
0/5
wk; methods described in
7,320
2/5
0/5
2/5
Weng et al. (2012)
20,900
3/5
0/5
3/5
Knockout Mice (Aldh2-/-); 13-week Exposure
Incidence of
Total incidence of
Dose
"extremelv slight"
Incidence of
atrophv of
(mg/ m3)
atrophv
"slight" atrophv seminiferous tubules
0
2/5
0/5
2/5
2,090
2/5
3/5
5/5
7,320
4/5
1/5
5/5
20,900
3/5
2/5
5/5
Sperm Parameters
Gaoua (2004b)
F0 Males
rat, Sprague-Dawley
oral - gavage
F0, male and female
(25/sex/group): 0, 250, 500,
Dose
Mean epididvmal
spermatozoa
% change
Mean epididvmal
sperm motilitv
Absolute
change from
(mg/kg-d)
count (n) ± SD
from control
(%) ± SD
control (%)
1,000 mg/kg-d
0
923 ± 200
-
99.7 ± 1.5
-
dosed daily for 18 wks from
10 wks premating until
250
938 ± 205
2
100 ±0
0
weaning of F1 pups
500
935 ±159
1
98.6 ±4
-1
Fl, male and female (24—
25/group): 0, 250, 500,
1,000
918 ±194
-1
97.6 ±6.6
-2
1,000 mg/kg-d
Mean epididvmal
dosed daily from PND 22 until
sperm with
Mean testicular
weaning of F2 pups
normal
Absolute
sperm heads
Dose
morphology
change from
(10s/gram of
% change from
(mg/kg-d)
(%) ± SD
control (%)
testis) ± SD
control
0
93 ± 19
-
114.8 ± 18.7
-
250
93 ±19
0
109 ± 13.1
-5
500
97 ±2
4
108.1 ± 18.6
-6
1,000
96 ±2
3
109.8 ± 16.5
-4
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
Reference and Study Design
Results
Gaoua (2004b) (continued)
Mean dailv
testicular sperm
production
Dose
(106/gram of
% change
N (epididvmal
N (other sperm
(mg/kg-d)
testis)
from control
sperm count)
parameters)
0
18.8 ±3.1
-
25
25
250
17.9 ±2.2
-5
25
25
500
17.7 ±3.1
-6
25
25
1,000
18 ± 2.7
-4
24
25
F1 Males
Mean epididvmal
spermatozoa
Mean epididvmal
Absolute
Dose
count (n)
% change
sperm motility
change from
(mg/kg-d)
±SD
from control
(%) ± SD
control (%)
0
725 ±150
-
84.6 ±34.1
-
250
673 ±197
-7
87.1 ±31.6
3
500
701 ± 97
-3
93.3 ±22
9
1,000
688 ±177
-5
88.3 ±29.4
4
Mean epididvmal
sperm with
Mean testicular
normal
Absolute
sperm heads
Dose
morphology
change from
(106/gram of
% change from
(mg/kg-d)
(% ) ± SD
control (%)
testis) ± SD
control
0
84 ±30
-
100.6 ± 36.7
-
250
86 ±28
2
97.8 ±32.3
-3
500
86 ±27
2
105.3 ± 27.2
5
1,000
88 ±24
4
99.8 ±38.9
-1
Mean dailv
testicular sperm
production
Dose
(106/gram of
% change
N (epididvmal
N (other sperm
(mg/kg-d)
testis)
from control
sperm count)
parameters)
0
16.5 ±6
-
22
24
250
16 ± 5.3
-3
24
25
500
17.3 ±4.5
5
23
24
1,000
16.4 ±6.4
-1
24
25
This document is a draft for review purposes only and does not constitute Agency policy.
1-64 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and Study Design
Results
Weng et al. (2014)
Wild Type Mice; 13-week Exposure
mice, C57BL/6
inhalation - vapor
male (5/group): 0, 500,1,750,
5,000 ppm (0, 2,090, 7,320,
20,900 mg/m3)a
Dose
(mg/m3)
Mean sperm
head numbers
(testis)
(x 106/g) ± SD
% change
from control
Motile sperm
(epididvmal) ± SE
Absolute
change from
control (%)
dynamic whole body
0
166.62 ±21.9
-
67.34 ± 3.45
-
inhalation; 6 hr/d, 5 d/wk for
13 wk; methods described in
2,090
167.74 ± 28.02
1
69.64 ± 3.45
2
Weng et al. (2012)
7,320
167.78 ±25.52
1
62.73 ± 1.73
-5
20,900
150.94 ± 23.07
-9
Absolute
58.13 ±2.30
% Sperm with
-9
Absolute
Dose
% Static sperm
change from
rapid movement
change from
(mg/m3)
(epididvmal)
control (%)
(epididvmal)
control (%)
0
32.57 ± 3.00
-
55.00 ± 3.75
-
2,090
30.86 ± 3.86
-2
56.25 ±3.13
1
7,320
37.29 ± 1.71
5
49.38 ±3.13
-6
20,900
42.43 ± 2.57
Epididvmal
sperm DNA
breaks (tail
10
46.25* ±2.50
Epididvmal
sperm DNA
damage
(measurement of
-9
Dose
intensity in
% change
8-OH-dG in
% change from
(mg/m3)
comet assav)
from control
comet assav)
control
0
4.91 ±0.34
-
3.46 ± 0.45
-
2,090
5.91 ±0.35
20
4.23 ±0.22
23
7,320
7.60* ±0.69
55
5.16* ±0.46
49
20,900
7.91* ±0.52
61
6.55 ± 1.13
89
Knockout Mice (Aldh2-/-); 13-week Exposure
Mean sperm
head numbers
Absolute
Dose
(testis)
% change
Motile sperm
change from
(mg/m3)
(x 106/g) ± SD
from control
(epididvmal) ± SE
control (%)
0
169.15 ±28.33
-
75.07 ± 2.88
-
2,090
127.08 ± 17.32
-25
61.23 ±5.03
-14
7,320
124.6* ± 11.96
-26
61.05* ±5.75
-16
20,900
124.72* ± 18.72
-26
57.27* ±5.77
-20
This document is a draft for review purposes only and does not constitute Agency policy.
1-65 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and Study Design
Results
Weng et al. (2014) (continued)
Absolute
% Sperm with
Absolute
Dose
% Static sperm
change from
rapid movement
change from
(mg/m3)
(epididymal)
control (%)
(epididvmal)
control (%)
0
25.46 ±2.56
-
66.74 ±2.17
-
2,090
40.34 ±5.14
15
51.54 ±2.84
-15
7,320
41.51* ±5.57
16
47.74* ±5.66
-19
20,900
45.27* ±5.58
Epididvmal
sperm DNA
breaks (tail
20
45.03* ±3.97
Epididvmal
sperm DNA
damage
(measurement of
-22
Dose
intensity in
% change
8-OH-dG in
% change from
(mg/m3)
comet assay)
from control
comet assay)
control
0
4.90 ±0.52
-
3.64 ±0.61
-
2,090
7.71 ±0.69
58
5.45 ±0.15
50
7,320
10.44* ± 0.78
113
7.65* ±0.61
110
20,900
9.46* ±0.69
93
7.95* ± 1.52
119
Weng et al. (2014)
Wild Type Mice; 9-Week Exposure
mice, C57BL/6
inhalation - vapor
male (NR): 0, 50, 200, 500 ppm
(0, 209, 836, 2,090 mg/m3)a
dynamic whole body
Dose
(mg/m3)
Mean sperm
head numbers
(testis)
(x 106/g) ± SD
% change
from control
Motile sperm
(epididvmal) ± SE
Absolute
change from
control (%)
inhalation; 6 hr/d, 5 d/wk for 9
0
199.62 ±27.22
-
85.82 ±4.26
-
wk; methods described in
Weng et al. (2012)
209
173.35 ±23.35
-13
78.72 ± 1.42
-7
836
170.47 ± 25.37
-15
82.27 ±2.13
-4
2,090
173.13 ± 16.28
-13
Absolute
80.14 ± 1.42
% Sperm with
-6
Absolute
Dose
% Static sperm
change from
rapid movement
change from
(mg/m3)
(epididvmal)
control (%)
(epididvmal)
control (%)
0
13.02 ± 3.38
-
71.11 ±2.78
-
209
21.74 ±2.96
9
65.56 ±2.22
-6
836
17.78 ±2.11
5
67.22 ±2.22
-4
2,090
16.36 ± 1.68
3
67.22 ±2.78
-4
This document is a draft for review purposes only and does not constitute Agency policy.
1-66 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and Study Design
Results
Weng et al. (2014) (continued)
Epididvmal
Epididvmal
sperm DNA
sperm DNA
damage
breaks (tail
(measurement of
Dose
intensity in
% change
8-OH-dG in
% change from
(mg/m3)
comet assav)
from control
comet assav)
control
0
4.10 ±0.26
-
3.88 ±0.30
-
209
4.04 ±0.10
-2
3.73 ±0.15
-4
836
4.40 ± 0.26
7
4.25 ±0.30
10
2,090
4.59 ±0.26
12
4.48 ±0.37
15
Knockout Mice (Aldh29-week Exposure
Mean sperm
head numbers
Absolute
Dose
(testis)
% change
Motile sperm
change from
(mg/m3)
(x 106/g) ± SD
from control
(epididvmal) ± SE
control (%)
0
216.19 ± 12.46
-
84.17 ±2.88
-
209
198.21 ± 20.54
-8
83.45 ± 2.88
-1
836
180.71* ± 23.5
-16
77.70 ±2.88
-6
2,090
165.8* ±43.52
-23
69.06 ± 6.47
-15
Absolute
% Sperm with
Absolute
Dose
% Static sperm
change from
rapid movement
change from
(mg/m3)
(epididvmal)
control (%)
(epididvmal)
control (%)
0
14.57 ± 1.71
-
69.79 ±2.84
-
209
16.29 ±4.29
2
68.65 ±3.97
-1
836
21.43 ± 3.00
7
63.55 ±2.27
-6
2,090
30.00* ± 6.00
15
52.20* ±5.11
-18
Epididvmal
Epididvmal
sperm DNA
sperm DNA
damage
breaks (tail
(measurement of
Dose
intensity in
% change
8-OH-dG in
% change from
(mg/m3)
comet assav)
from control
comet assav)
control
0
4.65 ±0.17
-
3.66 ±0.30
-
209
4.67 ±0.09
0
3.96 ±0.30
8
836
5.71* ±0.34
23
4.48 ± 0.30
22
2,090
7.01* ±0.26
51
4.85* ±0.22
33
This document is a draft for review purposes only and does not constitute Agency policy.
1-67 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and Study Design
Results
Weng et al. (2014) (continued)
Haplotype Mice (Aldh2 heterozygous); 9-week Exposure
Mean sperm
head numbers
Absolute
Dose
(testis)
% change
Motile sperm
change from
(mg/m3)
(x 106/g) ± SD
from control
(epididvmal) ± SE
control (%)
0
202.76 ± 14.59
-
85.61 ±2.16
-
209
202.26 ±26.31
0
85.61 ±2.16
0
836
109.53* ±21.56
-46
73.38* ±3.60
-12
2,090
96.31* ±33.4
-53
76.98* ±3.60
-9
Absolute
% Sperm with
Absolute
Dose
% Static sperm
change from
rapid movement
change from
(mg/m3)
(epididvmal)
control (%)
(epididvmal)
control (%)
0
15.00 ± 1.71
-
70.14 ±2.24
-
209
15.00 ±2.14
0
68.59 ±2.24
-2
836
27.43* ±3.86
12
49.42* ±6.24
-21
2,090
24.00* ± 3.00
9
58.08* ± 1.69
-12
Epididvmal
Epididvmal
sperm DNA
sperm DNA
damage
breaks (tail
(measurement of
Dose
intensity in
% change
8-OH-dG in
% change from
(mg/m3)
comet assav)
from control
comet assav)
control
0
3.51 ±0.25
-
4.04 ± 0.22
-
209
3.70 ±0.34
5
4.45 ±0.14
10
836
5.32* ±0.43
52
4.86 ± 0.43
20
2,090
5.86* ±0.42
67
5.34* ±0.50
32
Organ Weights
Fuiiietal. (2010); JPEC (2008e)
F0 Parents-Absolute Organ Weights
rat, Sprague-Dawley
oral - gavage
Dose
Mean testis
% change
Mean epididymis
% change from
FO, male and female
(mg/kg-d)
weight (g) ± SD
from control
weight (mg) ± SD
control
(24/sex/group): 0,100, 300,
1,000 mg/kg-d; dosed daily for
0
3.47 ±0.31
-
1371±136
-
17 wks, from 10 wks premating
100
3.48 ±0.28
0
1360 ± 83
-1
to lactation day 21
300
3.57 ±0.24
3
1381± 73
1
1,000
3.57 ±0.31
3
1349 ± 95
-2
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
Reference and Study Design
Results
Fuiiietal. (2010); JPEC (2008e)
Mean prostate
Mean seminal
(continued)
Dose
weight (mg)
% change
vesicle weight (g)
% change from
(mg/kg-d)
±SD
from control
± SD
control
0
787 ± 180
-
2.16 ±0.23
-
100
778 ±158
-1
2.1 ±0.32
-3
300
752 ±172
-4
2.19 ±0.24
1
1,000
816 ±136
4
2.19 ±0.23
1
F0 Parents-Relative Organ Weights
Mean
Mean testis:
Absolute
epididymis: bodv
Absolute
Dose
body weight
change from
weight ratio (%)
change from
(mg/kg-d)
ratio (%) ± SD
control (%)
± SD
control (%)
0
0.554 ±0.065
-
219 ±30
-
100
0.572 ±0.062
0.02
223 ± 18
4
300
0.589 ±0.076
0.03
228 ±25
9
1,000
0.61* ±0.074
0.06
230 ± 24
Mean seminal
11
Mean prostate:
Absolute
vesicle: bodv
Absolute
Dose
bodv weight
change from
weight ratio (%)
change from
(mg/kg-d)
ratio (%) ± SD
control (%)
± SD
control (%)
0
125 ± 28
-
0.345 ± 0.054
-
100
128 ± 30
3
0.343 ±0.051
0.00
300
124 ± 30
-1
0.361 ±0.052
0.02
1,000
139 ± 23
14
0.373 ± 0.042
0.03
Gaoua (2004b)
F0 Parents-Absolute Organ Weights
rat, Sprague-Dawley
oral - gavage
FO, male and female
(25/sex/group): 0, 250, 500,
Dose
(mg/kg-d)
Mean testis
weight (left) (g)
± SD
% change
from control
Mean testis
weight (right) (g)
± SD
% change from
control
1,000 mg/kg-d
0
1.78 ±0.116
-
1.76 ±0.105
-
dosed daily for 18 wks from
10 wks premating until
250
1.73 ±0.181
-3
1.76 ±0.179
0
weaning of F1 pups
500
1.78 ±0.142
0
1.76 ±0.13
0
Fl, male and female (24—
25/group): 0, 250, 500,
1,000
1.75 ±0.237
-2
1.79 ±0.126
2
1,000 mg/kg-d
dosed daily from PND 22 until
weaning of F2 pups
This document is a draft for review purposes only and does not constitute Agency policy.
1-69 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and Study Design
Results
Gaoua (2004b) (continued)
Dose
Mean epididymis
weight (left) (g) ±
% change
Mean epididymis
weight (right) (g)
% change from
(mg/kg-d)
SD
from control
± SD
control
0
0.77008 ± 0.054
-
0.78148 ± 0.053
-
250
0.77092 ±0.077
0
0.78698 ±0.092
1
500
0.77784 ±0.067
1
0.77492 ± 0.062
-1
1,000
0.80988 ±0.189
5
0.77528 ±0.056
-1
Dose
(mg/kg-d)
Mean prostate
weight ± SD
% change
from control
Mean seminal
vesicle weight ±
SD
% change
from
control N
0
1.41 ±0.272
-
2.06 ±0.309
25
250
1.63 ±0.32
16
2.26 ±0.595
10 25
500
1.37 ±0.285
-3
2.19 ±0.439
6 25
1,000
1.62 ±0.396
15
2.28 ±0.574
11 25
FO Parents-Relative Organ Weights
Dose
(mg/kg-d)
Mean testis
weight: body
weight ratio
(left) (g) ± SD
Absolute
change from
control (%)
Mean testis
weight: bodv
weight ratio
(right) (g) ± SD
Absolute
change from
control (%)
0
0.297488 ± 0.029
-
0.29488 ± 0.029
-
250
0.29005 ± 0.025
-0.01
0.29427 ± 0.025
0.00
500
0.307 ± 0.033
0.01
0.30321 ±0.033
0.01
1,000
0.31052 ± 0.049
0.01
0.31497* ±0.029
0.02
Dose
(mg/kg-d)
Mean epididymis
weight (left):
bodv weight
ratio (g) ± SD
Absolute
change from
control (%)
Mean
epididymis: bodv
weight ratio
(right) (%) ± SD
Absolute
change from
control (%)
0
0.12886 ±0.014
-
0.13072 ±0.013
-
250
0.12947 ±0.013
0.00
0.13245 ± 1.014
0.00
500
0.13434 ±0.016
0.01
0.13383 ±0.015
0.00
1,000
0.14209 ±0.027
0.01
0.1367 ±0.012
0.01
This document is a draft for review purposes only and does not constitute Agency policy.
1-70 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and Study Design
Results
Gaoua (2004b) (continued)
Dose
(mg/kg-d)
Mean prostate
weight: bodv
weight ratio ± SD
Absolute
change from
control (%)
Mean seminal
vesicle: bodv
weight ratio ± SD
Absolute
change
from
control
1%1
N
0
0.23582 ±0.054
-
0.34605 ± 0.066
-
25
250
0.27279 ±0.053
0.04
0.37895 ±0.098
0.03
25
500
0.23656 ±0.054
0.00
0.37615 ±0.073
0.03
25
100
0.28593* ±0.069
0.05
0.40207 ±0.1
0.06
25
F1 Offspring-Absolute Organ Weights
Dose
(mg/kg-d)
Mean testis
weight (left) (g)
± SD
% change
from control
Mean testis
weight (right) (g)
± SD
% change from
control
0
1.79 ±0.11
-
1.84 ±0.137
-
250
1.77 ±0.39
-1
1.75 ±0.337
-5
500
1.84 ±0.21
3
1.86 ±0.226
1
1,000
1.84 ±0.171
3
1.82 ±0.255
-1
Dose
Mean epididymis
weight (left) (g) ±
% change
Mean epididymis
weight (right) (g)
% change from
(mg/kg-d)
SD
from control
± SD
control
0
0.71683 ±0.11
-
0.75575 ±0.041
-
250
0.69636 ±0.123
-3
0.70512 ±0.148
-7
500
0.71904 ±0.123
0
0.75008 ±0.113
-1
1,000
0.6898 ±0.12
-4
0.71244 ±0.127
-6
Dose
(mg/kg-d)
Mean prostate
weight ± SD
% change
from control
Mean seminal
vesicle weight ±
SD
% change
from
control
N
0
1.470 ±0.311
-
1.71 ±0.295
-
24
250
1.48 ± 0.249
1
1.94 ±0.567
13
25
500
1.38 ±0.23
-6
1.86 ±0.422
9
24
1,000
1.41 ±0.279
-4
1.92 ±0.436
12
25
This document is a draft for review purposes only and does not constitute Agency policy.
1-71 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of ETBE
Reference and Study Design
Results
Gaoua (2004b) (continued)
F1 Offspring-Relative Organ Weights
Mean testis
weight: body
Mean testis
weight ratio
Absolute
weight: body
Absolute
Dose
(left) (g)
change from
weight ratio
change from
(mg/kg-d)
±SD
control (%)
(right) (g) ± SD
control (%)
0
0.30842 ± 0.065
-
0.31441 ±0.036
-
250
0.30222 ±0.067
-0.01
0.29746 ± 0.059
-0.02
500
0.30679 ±0.037
0.00
0.31004 ± 0.04
0.00
1,000
0.31198 ±0.042
Mean epididymis
0.00
0.30958 ± 0.05
0.00
weight (left):
Absolute
Mean epididymis
Absolute
Dose
bodv weight
change from
weight (right) (g)
change from
(mg/kg-d)
ratio (g) ± SD
control (%)
± SD
control (%)
0
0.12299 ±0.023
-
0.12915 ±0.012
-
250
0.11863 ±0.021
0.00
0.12002 ±0.025
-0.01
500
0.1198 ±0.021
0.00
0.12492 ±0.018
0.00
1,000
0.11693 ±0.021
-0.01
0.12065 ±0.022
-0.01
Absolute
change
Mean prostate
Absolute
Mean seminal
from
Dose
weight: body
change from
vesicle: body
control
(mg/kg-d)
weight ratio ± SD
control (%)
weight ratio ± SD
1%) N
0
0.25136 ±0.057
-
0.29278± 0.055
24
250
0.25239 ±0.043
0.00
0.33038 0.085
0.04 25
500
0.23059 ± 0.043
-0.02
0.3165 ±0.113
0.02 24
1,000
0.2374 ± 0.04
-0.01
0.32424 ± 0.073
0.03 25
Weng et al. (2014)
Wild Type Mice; 13-Week Exposure
mice, C57BL/6
inhalation - vapor
male (5/group): 0, 500,1,750,
5,000 ppm (0, 2,090, 7,320,
20,900 mg/m3)a
Dose
(mg/m3)
Mean testis:
bodv weight
ratio (%)
± SD
Absolute
change from
control (%)
Mean
epididymis: bodv
weight ratio (%)
± SD
Absolute
change from
control (%)
dynamic whole body
0
0.7 ±0.06
-
0.24 ±0.02
-
inhalation; 6 hr/d, 5 d/wk for
13 wk; methods described in
2,090
0.74 ± 0.04
0.04
0.26 ±0.02
0.02
Weng et al. (2012)
7,320
0.67 ±0.09
-0.03
0.25 ±0.01
0.01
20,900
0.7 ±0.02
0.00
0.24 ±0.02
0.00
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
Reference and Study Design
Results
Weng et al. (2014)
Knockout Mice (Aldh213-week Exposure
(continued)
Mean testis:
Mean
body weight
Absolute
epididymis: bodv
Absolute
Dose
ratio (%)
change from
weight ratio (%)
change from
(mg/m3)
±SD
control (%)
±SD
control (%)
0
0.76 ± 0.04
-
0.26 ±0.01
-
2,090
0.71 ±0.11
-0.05
0.24 ±0.02
-0.02
7,320
0.72 ±0.05
-0.04
0.24* ±0.02
-0.02
20,900
0.71 ±0.07
-0.05
0.23* ±0.02
-0.03
Weng et al. (2014)
Wild Type Mice; 9-Week Exposure
mice, C57BL/6
inhalation - vapor
male (NR): 0, 50, 200, 500 ppm
(209, 836, 2,090 mg/m3)a
dynamic whole body
Dose
(mg/m3)
Mean testis:
body weight
ratio (%)
±SD
Absolute
change from
control (%)
Mean
epididymis: bodv
weight ratio (%)±
SD
Absolute
change from
control (%)
inhalation; 6 hr/d, 5 d/wk for 9
0
0.8 ±0.12
-
0.26 ±0.03
-
wk; methods described in
Weng et al. (2012)
209
0.77 ±0.09
-0.03
0.25 ±0.03
-0.01
836
0.77 ±0.09
-0.03
0.25 ±0.02
-0.01
2,090
0.78 ±0.08
-0.02
0.25 ±0.02
-0.01
Knockout
Mice (Aldh2-/-); 9-week Exposure
Mean testis:
Mean
bodv weight
Absolute
epididymis: bodv
Absolute
Dose
ratio (%)
change from
weight ratio (%)±
change from
(mg/m3)
±SD
control (%)
SD
control (%)
0
0.8 ±0.06
-
0.27 ±0.02
-
209
0.76 ±0.05
-0.04
0.26 ±0.02
-0.01
836
0.79 ±0.07
-0.01
0.27 ±0.01
0.00
2,090
0.74 ±0.01
-0.06
0.25 ±0.03
-0.02
Haplotype Mice (Aldh2 heterozygous); 9-week Exposure
Mean testis:
Mean
bodv weight
Absolute
epididymis: bodv
Absolute
Dose
ratio (%)
change from
weight ratio (%)±
change from
(mg/m3)
±SD
control (%)
SD
control (%)
0
0.82 ± 0.07
-
0.26 ±0.02
-
209
0.8 ±0.06
-0.02
0.26 ±0.01
0.00
836
0.81 ±0.09
-0.01
0.26 ±0.02
0.00
2,090
0.73 ±0.03
-0.09
0.24 ±0.01
-0.02
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
Reference and Study Design
Results
de Pevster et al. (2009)
Absolute Organ Weights
rat, Fischer 344
oral - gavage
PO, male (12/group): 0, 600,
Dose
(mg/kg-d)
Mean testis
weight (g) ± SD
% change
from control
Mean epididymis
weight (mg) ± SD
% change from
control
1,200,1,800 mg/kg-d
0
2.55 ±0.09
-
0.696 ±0.016
-
daily for 14 days
600
2.53 ±0.05
-1
0.693 ±0.027
0
1,200
2.49 ± 0.07
-2
0.701 ±0.026
1
1,800
2.47 ±0.1
Mean prostate
-3
0.663 ±0.029
Mean seminal
-5
Dose
weight (g)
% change
vesicle weight (g)
% change from
(mg/kg-d)
± SD
from control
± SD
control
0
0.238 ±0.018
-
0.781 ±0.022
-
600
0.309 ± 0.034
30
0.733 ±0.024
-6
1,200
0.252 ±0.018
6
0.749 ± 0.037
-4
1,800
0.269 ±0.036
13
0.701 ±0.041
-10
Mean weight of
Dose
combined accessory sex
(mg/kg-d)
organs (g)±SD % change from control
0
1.712 ±0.041
-
600
1.735 ±0.057
1
1,200
1.702 ± 0.063
-1
1,800
1.633 ±0.059
-5
Relative Organ Weights
Mean
Mean testis:
Absolute
epididymis: bodv
Absolute
Dose
bodv weight
change from
weight ratio (%)
change from
(mg/kg-d)
ratio (%) ± SD
control (%)
± SD
control (%)
0
0.997 ±0.036
-
0.272 ± 0.007
-
600
1.014 ± 0.027
0.02
0.275 ± 0.009
0.00
1,200
1.097 ± 0.03
0.10
0.308 ± 0.009
0.04
1,800
1.097 ± 0.045
0.10
0.294 ±0.014
0.02
Mean prostate:
Absolute
Mean seminal
Absolute
Dose
bodv weight
change from
vesicle: bodv wt.
change from
(mg/kg-d)
ratio (%) ± SD
control (%)
ratio (%) ± SD
control (%)
0
0.092 ± 0.007
-
0.304 ± 0.008
-
600
0.124 ±0.015
0.03
0.292 ±0.012
-0.01
1,200
0.111 ±0.076
0.02
0.328 ±0.012
0.02
1,800
0.123 ±0.021
0.03
0.31 ±0.017
0.01
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
Reference and Study Design
Results
de Pevster et al. (2009)
(continued)
Mean combined
accessory sex
Dose organs:bodv weight ratio Absolute change from
(mg/kg-d) (%)±SD control (%)
0 0.668 ±0.018
600 0.691 ±0.026 0.02
1,200 0.746 ± 0.019 0.08
1,800 0.727 ± 0.035 0.06
Medinskv et al. (1999); Bond et
al. (1996b)
rat, Fischer 344
inhalation - vapor
male (48/group): 0, 500,1,750,
5,000 ppm
(0, 2,090, 7,320,
20,900 mg/m3)a; female
(48/group): 0, 500, 1,750,
5,000 ppm
(0, 2,090, 7,320,
20,900 mg/m3)a
dynamic whole body chamber;
6 hr/d, 5 d/wk for 13 wk
Organ weights of Fisher 344 rats and CD-I mice were not altered by exposure to
ETBE.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
Reference and Study Design
Results
Testosterone and Estradiol
de Pevster et al. (2009)
Dose
Mean plasma testosterone
rat, Fischer 344
(mg/kg-d)
N
(ng/ml) ± SE
% change from control
oral - gavage
P0, male (12/group): 0, 600,
0
12
2.07 ± 42
-
1,200,1,800 mg/kg-d
600
12
3.1 ±0.78
50
daily for 14 days
1,200
11
2.61 ±0.55
26
1,800
10
1.36 ±0.39
-34
Dose
Mean plasma estradiol
(mg/kg-d)
N
(pg/ml)
% change from control
0
12
1.085 ±0.1
-
600
12
1.395 ± 0.403
29
1,200
11
2.238* ±0.377
106
1,800
9
2.224* ±0.611
105
1 a4.18 mg/m3 = 1 ppm.
2 bConversion performed by study authors.
3 cMale mating index (%) = (No. males able to mate with at least one female / Total males) x 100.
4 dMale fertility index (%) = (No. males with at least one pregnant partner / Males that mated at least once) x 100
5 *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
6 for controls, no response relevant; for other doses, no quantitative response reported.
7 % change from control = [(treated group value -control value)/control value] x 100.
8 Absolute change from control (%) = control value (%) - treated group value (%).
9
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
¦ = exposures at which the endpoint was reported statistically significant by study authors
~ = exposures at which the endpoint was reported not statistically significant by study authors
Male Fertility
PC! Male rat; copulation; one-gen repro (A)
PO Male rat; copulation; two-gen repro (B)
PI Male rat; copulation; two-gen repro (B)
F0 Male rat; fertility; one-gen repro (A)
FO Male rat; fertility; two-gen repro (B)
F1 Male rat; fertility; two-gen repro (B)
Sperm FQ Male rat; epididymal sperm count; one-gen repro {A} -
Parameters
FO Male rat; epididymal sperm count; two-gen repro (B)
F'l Male rat; epididymal sperm count; two-gen repro (B)
FO Male rat; epididymal sperm motility; one-gen repro (A)
FO Male rat: epididymal sperm motility; two-gen repro (B)
F1 Male rat; epididymal sperm motility; two-gen repro (B)
FO Mate rat; epididymal sperm morphology; one-gen repro
(A)
FO Male rat; epididymal sperm morphology; two-gen repro
(B)
F1 Male rat; epididymal sperm morphology; two-gen repro
(B)
FO Male rat; testicular sperm heads; one-gen repro (A)
FO Male rat; testicular sperm heads; two-gen repro (B)
PI Male rat; testicular sperm heads; two-gen repro (B)
FO Male rat; daily sperm production; two-gen repro (B) -
Ft Male rat; daily sperm production; two-gen repro (B) -
Testosterone
and Estradiol
Male rat; testosterone; 14 d (C)
Male rat; estradiol; 14 d (C)
~ ~ ~
~ Q Q
n 0 ' q
O—B—Q
s q—B—0
~ B E3
~—a—Ei
~—B—0
a—b—a
b b a
Q—B—0
Q B El
O—B—B
~—B—Q
~—n—Fi
IMM tn. I li i|ll
o—a—a
B—B—B
~ ~
~ ¦¦
10 100
Dose (mg/kg-day)
Sources; (A) Fuji! et al, 2010; JPEC, 2008e (B) Gaoua, 2004b (C) de Peysteretal, 2009
1,000
10,000
Figure 1-11. Exposure-response array of male reproductive effects following
oral exposure to ETBE.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
¦ = exposures at which the endpoint was reported statistically significant by study authors
~ = exposures at which the endpoint was reported not statistically significant by study authors
Testicular
Histopathology Male nit, spermatocyte degeneration; 13 wis (A)
Male mouse; atrophy of seminiferous tubules; 13 wks (B)
Male Aidh2-/- mouse; atrophy ofsemmiferaus tubules; 13 wks (B)
Mate i phy of seminiferous tubules; 9 wks (8)
Male Aldti2-/-: phy ofsemmiferous tubules; 9 wks (B)
Male Aldh2+/- mouse U>ophy <>s svmit\«tennis tuhule\ wits 113
M si * irniM, (H'( r sps nn heads, l.\ wks !.
Sperm
Parameters
M.nt AitihJ /- mouse, Jeer. .sp<. • m sit'ads; 1.3 wks (R
Male mouse; deer, set nr. heads; 9 wks (B
Male Aldh2-/- mouse; deer, sperm heads; 9 wks I,
Mate Aldh2+/~ mouse; deer, sperm heads; 9 u k, \ U
Male mouse; deer, sperm motility; 13 u ks 1
Male Alcih2-/- mouse; deer, sperm motility; 13 wks 11]
Male mouse; deer sperm » ot t-cy v, ks ¦ '
Male Aklh2 -/- mouse; decs; sperm moii uv ') u ks t»!
Male Aldh2+/- mouse; deer, sperm motility, 9 w Ks B
Male mouse; incr. no. ofstatic sperm; 13 uhs
Male Aldh2-/- mouse; iticr, no, of static sperm; 13 k^ l .0
Male mouse; men no. nUtatu ^perm; 9 wks f n
Male Aldh2-/- "iuiim r a no oi statu sp? nn ^uks/'
Male Akih2 *-/ - ntous<\ toer nu ot statu sperm, wks 1 H
Male mouse; di < r <>o o! r.jpaiiv moving sperm, « J wks ri
Male Aldh2-/- mouse; dt er no ol rapidly moving : -i wks 11!
Male mouse; detr no at rapidly moving spi s in, " wks i H
Male A!dh2-/~ mouse; de<, !¦ no ot rapidly movim; spr rm wks 11
Male Aldh2+/- mouse; deer no or rap.dlv niovim', spt nn '* wks (li
Organ
Weights
Male mouse dei i U-stt> wt i relative I Li wks 1 H
Male A!dh2-/- mtn.se. dec r teMi> wt i relative |, 1 wks i |i
Mali ifoH'St dtx l asUs Wl | i i \H 11 | m u ks I H
Mule Aldh J / mouse ds,u testis vu ! k lativi j wks 1?
M ue AUt'k »/ HMus* dett ustisut t uvi'ivt i 9wk>il.
Maie mousi at a r pitl.dym4- ut idutru | ^uksjH
Male Aldh2 -/" nioust du t «. puhdymt-. u t Mekuiv*) lUvhji
Male mouse; dett <; di l\uiswt. lit Unl 4» wl
-------
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Toxicological Review of ETBE
Mechanistic Evidence
No mechanistic evidence for male reproductive effects was identified by the literature
search.
Integration of Male Reproductive Effects
The male reproductive endpoints examined in this database were not consistently affected
across studies or across doses in wildtype animals. The 13-week and 9-week inhalation studies
conducted in rats and mice fWeng etal.. 2014: Medinskv etal.. 19991 provide suggestive evidence
of ETBE-induced testicular degeneration and effects on sperm count, sperm mobility, and sperm
DNA damage. In contrast, no male reproductive toxicity was observed in any of the other studies
examined in this database, including one- and two-generation reproductive toxicity studies, 2-year
carcinogenicity studies, and sub-chronic studies. For example, the 2-year inhalation carcinogenicity
study (Saito etal.. 2013: TPEC. 2010b) used the same rat strain, same route of exposure, and similar
range of exposure concentrations as Medinskv et al. (19991 and did not observe any dose-related
effects on testicular histopathology. Wengetal. f20141. however, found that Aldh2 KO and
heterozygous mice had consistently reduced numbers of sperm heads and sperm motility as well as
reductions in male reproductive organ weights, suggesting that populations with ALDH2
polymorphisms could be more susceptible to effects from ETBE exposure (discussed in Section
1.3.3). The 14-day study by de Pevster etal. (2009) observed increased estradiol and decreased
testosterone in ETBE-exposed rats, which is a potential mechanism for testicular degeneration;
however, no effects on testicular histopathology or organ weight were observed in this study.
Collectively, although minimal effects were observed at otherwise toxic dose levels, the available
evidence is considered insufficient to identify male reproductive effects as a potential human
hazard of ETBE, and male reproductive effects are not carried forward as a hazard. While the
ALDH2 knock out data suggest a potential sensitive subpopulation for male reproductive effects,
these findings are considered preliminary.
Synthesis of Effects Related to Female Reproduction
The available evidence for ETBE-induced effects on the female reproductive system
includes no human data. The evidence was obtained primarily from a one-generation reproductive
toxicity study fFuiii etal.. 2010: TPEC. 2008el. a two-generation reproductive toxicity study fGaoua.
2004b), and three developmental toxicity studies fAso etal.. 2014: Asano etal.. 2011: TPEC. 2008h.
i; Gaoua. 2004a). In addition, some evidence was obtained from two 90-day toxicity studies (TPEC.
2008b: Medinskv etal.. 1999: Bond etal.. 1996a). one subchronic (180-day) study (Mivata etal..
2013: TPEC. 2008c). two 2-year carcinogenicity studies (Saito etal.. 2013: Suzuki etal.. 2012: TPEC.
2010a. b), and a short-term study evaluating ETBE-induced oocyte effects fBerger and Horner.
2003). These studies evaluated the effects of ETBE exposure on maternal body weight change (Aso
etal.. 2014: Asano etal.. 2011: Fuiii etal.. 2010: TPEC. 2008e. h, i; Gaoua. 2004a. b), fertility, mating,
and pregnancy parameters (Fujii etal.. 2010: TPEC. 2008e: Gaoua. 2004b: Berger and Horner.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
20031. fecundity (Aso etal.. 2014: Asano etal.. 2011: Fuiii etal.. 2010: TPEC. 2008e. h, i; Gaoua.
2004a. b), estrous cyclicity fFuiii etal.. 2010: TPEC. 2008e: Gaoua. 2004bl. and organ weights fAso
etal.. 2014: Mivata etal.. 2013: Saito etal.. 2013: Suzuki etal.. 2012: Asano etal.. 2011: Fuiii etal..
2010: TPEC. 2010a. b, 2008b. c, e, h, i; Gaoua. 2004b: Medinskv et al.. 1999: Bond etal.. 1996al.
ETBE-induced effects were examined in pregnant rats and rabbits and non-pregnant female rats
after oral or whole body inhalation exposures, and the design, conduct, and reporting of each study
were of sufficient quality to inform human health hazard assessment. Selected female reproductive
toxicity endpoints from these studies are summarized in Table 1-15.
The one- and two-generation reproductive toxicity studies and developmental studies
evaluated maternal toxicity and several endpoints related to fertility, pregnancy, and pregnancy
outcomes in rats and rabbits up to 1,000 mg/kg-day ETBE. Maternal toxicity, as shown by
decreased maternal body weight and corrected (for the gravid uterus) body weight, was observed
following gestational exposure to 1,000 mg/kg-day ETBE from GD 5-19; however, this effect was
not observed in another developmental exposure study in which ETBE was administered at the
same dose and exposure duration (Aso etal.. 2014: TPEC. 2008h). Further, administration of ETBE
during the pre-mating through lactation periods in parental and F1 generations fFuiii etal.. 2010:
TPEC. 2008e: Gaoua. 2 004b 1 did not affect maternal body weight parameters in rats. Maternal body
weight during the entire pregnancy (GD 0-28) and corrected body weight change were decreased
in rabbits administered 1,000 mg/kg-day ETBE (Asano etal.. 2011: TPEC. 2008i): however, the lack
of change in body weight during the treatment period (GD 6-27), the lack of a dose-related
response, and the inherent variability in body weight parameters during pregnancy in rabbits (U.S.
EPA. 1991b) limit the interpretation of this effect. ETBE did not affect indices of mating or fertility,
and pre-coital times and gestation lengths were similarly unaffected in rats in the parental fFuiii et
al.. 2010: TPEC. 2008e: Gaoua. 2004bl and the F1 generation fGaoua. 2004bl. In addition, the
number of corpora lutea in pregnant rats and rabbits fAso etal.. 2014: Asano etal.. 2011: TPEC.
2008h. i), the average estrous cycle length, and the percent of females with normal estrous cycles
(Fujii etal.. 2010: TPEC. 2008e) were not significantly affected by ETBE when compared to control
values. Further supporting these findings, oocyte quality and fertilizability was shown to be
unaffected by ETBE (Berger and Horner. 2003). Litter size was evaluated by Fuiii etal. (2010). TPEC
(~2008e"). Gaoua r2004bl. Aso etal. C20141. TPEC f2008hl. and Asano etal. r20in TPEC r2008il. and
no significant, dose-related effects were observed in rats or rabbits following ETBE exposure.
Reproductive organ weights were also reported after oral and inhalation exposures to
ETBE. Gravid uterine weights were not affected following ETBE exposure during gestation in
rabbits (Asano etal.. 2011: TPEC. 2008i) nor were ovary and uterine weights affected after exposure
during pre-mating through lactation periods in rats fFuiii etal.. 2010: TPEC. 2008e). Consistent with
these findings, ovary and uterine weights in non-pregnant females were not affected by ETBE after
90-day inhalation flPEC. 2008b: Medinskv etal.. 1999: Bond etal.. 1996al. 180-day oral fMivata et
al.. 2013: TPEC. 2008cl. and 2-year oral fSuzuki etal.. 2012: TPEC. 2010al exposure assessments. In
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
1 a 2-year inhalation study in rats (Saito etal.. 2013: IPEC. 2010b], however, a significant increase in
2 relative (but not absolute) ovary weight was observed at exposures of 1,500 and 5,000 ppm ETBE.
3 It is possible the finding of increased relative ovary weight was influenced by concurrent decreases
4 in bodyweight (9-22%) at these exposures.
5
6 Table 1-15. Evidence pertaining to female reproductive effects in animals
7 exposed to ETBE
Reference and study design
Results
Maternal Body Weight
Gaoua (2004a)
Net bodv wt
rat, Sprague-Dawley
Body wt change ±
% change
change ± SD
% change from
oral - gavage
Dose (mg/kg-d)
SD, GD 5-20 (g)
from control
Isl
control
PO, female (24/group): 0, 250,
0
132 ± 22
61.8 ± 13
500,1,000 mg/kg-d
dams exposed from GD 5 to
250
132 ± 12
-2
59.4 ±8.1
-4
GD 19
500
134 ± 19
-1
60 ± 11.3
-3
1,000
120* ±15
-11
51.5* ± 10.3
-17
Gaoua (2004b)
Fl:Bodv wt
rat, Sprague-Dawley
F0: Bodv wt
% change
change ± SD
% change from
oral - gavage
Dose (mg/kg-d)
change ± SD (g)
from control
isl
control
F0, male and female
0
132 ± 15
146 ±21
(25/sex/group): 0, 250, 500,
1,000 mg/kg-d
250
134 ± 14
2
145 ± 15
-1
dosed daily for 18 wks from
10 wks premating until weaning
500
136 ± 25
3
141 ±21
-3
of F1 pups
1,000
136 ± 12
3
137 ± 12
-6
Fl, male and female (24—
25/group): 0, 250, 500, 1,000
mg/kg-d
dosed daily from PND 22 until
weaning of F2 pups
Aso et al. (2014); JPEC (2008h)
Bodv wt
rat, Sprague-Dawley
Bodv wt ± SD,
Bodv wt ± SD,
change ± SD,
% change from
oral - gavage
Dose (mg/kg-d)
GD 5 (g)
GD 20 (g)
GD 5-20 (g)
control
female (24/group): 0,100, 300,
1,000 mg/kg-d
0
280.9 ± 16.7
394.4 ±26.9
113.5
-
dams dosed daily from GD 5 to
100
273.4 ± 10.8
380.3 ±23.9
106.9
-6
GD 19
C-section GD 20
300
280 ± 13.4
389.8 ±25.9
109.8
-3
1,000
277.7 ± 15.9
382.4 ±27.1
104.7
-8
Fuiiietal. (2010); JPEC (2008e)
F0: Bodv wt
rat, Sprague-Dawley
change ± SD,
% change
oral - gavage
Dose (mg/kg-d)
GD 5-20 (g)
from control
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Toxicological Review of ETBE
Reference and study design
Results
FO, male and female
0
124.9 ± 22
-
(24/sex/group): 0,100, 300,
1,000 mg/kg-d; dosed daily for
100
119.6 ±20.3
-4
17 wks, from 10 wks premating
300
135.2 ±21.5
8
to lactation day 21
1,000
140.2* ± 19.1
12
Asano et al. (2011); JPEC (2008i)
Bodv wt
Adjusted
rabbit, New Zealand White
change ± % change
Bodv wt
bodv wt
% change
oral - gavage
SD, GD 6-28
from
change ± SD,
change ±
from
female (24/group): 0,100, 300,
Dose (mg/kg-d)
M
control
GD 0-28 (kg)
SD (kg)
control
1,000 mg/kg-d
dams dosed daily from GD 6 to
0
0.26 ±0.12
-
0.40 ±0.12
0.02 ±0.14
-
GD 27
100
0.23 ±0.12
-12
0.35 ±0.12
-0.06 ±0.12
-400
C-section GD 28
300
0.28 ±0.08
8
0.40 ± 0.08
0±0.1
-100
1,000
0.12 ±0.19
-54
0.25* ±0.21
-0.07 ±0.19
-450
Fertility, Mating, and Pregnancy
Fuiiietal. (2010); JPEC (2008e)
Copulation
Fertility
rat, Sprague-Dawley
Dose (mg/kg-d)
index0 (%) indexd (%)
oral - gavage
F0, male and female
0
100
87.5
(24/sex/group): 0,100, 300,
1,000 mg/kg-d; dosed daily for
100
95.8
100
17 wks, from 10 wks premating
300
100
95.8
to lactation day 21
1,000
100
91.7
Gaoua (2004b)
Pregnant/ma
Fertilitv
rat, Sprague-Dawley
Dose
Pregnant/mated
Fertilitv index, F0 ted females.
index, Fl
oral - gavage
(mg/kg-d)
females, F0
1%1
£1
(%)
F0, male and female
(25/sex/group): 0, 250, 500,
0
23/25
92
22/25
88
1,000 mg/kg-d
250
21/25
84
22/24
92
dosed daily for 18 wks from
10 wks premating until weaning
500
22/25
88
22/25
88
of F1 pups
1,000
25/25
100
22/23
96
Fl, male and female (24—
25/group): 0, 250, 500, 1,000
mg/kg-d
dosed daily from PND 22 until
weaning of F2 pups
Aso et al. (2014); JPEC (2008h)
rat, Sprague-Dawley
Dose Mean no. corpora
(mg/kg-d) luteal SD
% change from
control
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Reference and study design
Results
oral -gavage
0
15.5 ± 1.54
-
female (24/group): 0,100, 300,
1,000 mg/kg-d
100
14.1 ± 1.48
-9
dams dosed daily from GD 5 to
300
14.4 ± 1.85
-7
GD 19
C-section GD 20
1,000
14.6 ± 2.44
-6
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Toxicological Review of ETBE
Reference and study design
Results
Litter Size
Fuiiietal. (2010); JPEC (2008e)
Dose
Mean no. duds % change from
rat, Sprague-Dawley
(mg/kg-d)
delivered ± SD (kg) control
oral - gavage
FO, male and female
0
11.8 ±3.2
(24/sex/group): 0,100, 300,
100
10.4 ±3.4 -12
1,000 mg/kg-d; dosed daily for
300
12.1 ±2.3 3
17 wks, from 10 wks premating
to lactation day 21
1,000
13.0 ±1.9 10
Gaoua (2004b)
% change
rat, Sprague-Dawley
Dose
Litter size at birth, from control.
Pregnant/mated % change from
oral - gavage
(mg/kg-d)
IS
IS
females, Fl control, Fl
F0, male and female
0
14.3
13.7
(25/sex/group): 0, 250, 500,
1,000 mg/kg-d
250
14.1 -1
13.7 0
dosed daily for 18 wks from
500
14.9 4
13.7 0
10 wks premating until weaning
of F1 pups
1,000
14.2 -1
14 2
Fl, male and female (24—
25/group): 0, 250, 500, 1,000
mg/kg-d
dosed daily from PND 22 until
weaning of F2 pups
Aso et al. (2014); JPEC (2008h)
Dose
rat, Sprague-Dawley
(mg/kg-d)
Mean no. live fetuses ± SD (kg)
% change from control
oral - gavage
female (24/group): 0,100, 300,
0
13.6 ± 1.5
-
1,000 mg/kg-d
100
12.0 ±2.65
-12
dams dosed daily from GD 5 to
GD 19
300
12.6 ±2.58
-7
C-section GD 20
1,000
12.3 ±2.8
-10
Asano et al. (2011); JPEC (2008i)
Dose
rabbit, New Zealand White
(mg/kg-d)
Mean no. live fetuses ± SD (kg)
% change from control
oral - gavage
female (24/group): 0,100, 300,
0
7.8 ±3.1
-
1,000 mg/kg-d
100
7.9 ±3.2
1
dams dosed daily from GD 6 to
GD 27
300
8.4 ±2.0
8
C-section GD 28
1,000
6.9 ±3.2
-12
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Toxicological Review of ETBE
Reference and study design
Results
Gestation Length
Fuiiietal. (2010); JPEC (2008e)
Dose
rat, Sprague-Dawley
(mg/kg-d)
Mean gestation length ± SD (davs) % change from control
oral - gavage
FO, male and female
0
22.2 ±0.4
-
(24/sex/group): 0,100, 300,
100
22.1 ±0.4
0
1,000 mg/kg-d; dosed daily for
300
22.2 ±0.4
0
17 wks, from 10 wks premating
to lactation day 21
1,000
22.6 ±0.5
2
Gaoua (2004b)
Dose
Gestation length % change from Gestation length
% change from
rat, Sprague-Dawley
(mg/kg-d)
(davs), F0 control, F0
(davs), Fl
control, Fl
oral - gavage
F0, male and female
0
21.7
21.5
-
(25/sex/group): 0, 250, 500,
250
21.5 -1
21.6
0
1,000 mg/kg-d
dosed daily for 18 wks from
500
21.5 -1
21.6
0
10 wks premating until weaning
1,000
21.8 0
21.6
0
of F1 pups
Fl, male and female (24—
25/group): 0, 250, 500, 1,000
mg/kg-d
dosed daily from PND 22 until
weaning of F2 pups
Estrous Cyclicity
Fuiiietal. (2010); JPEC (2008e)
Mean estrous
rat, Sprague-Dawley
Dose
% Females w/normal estrous
cvcle length ± SD
% change
oral - gavage
(mg/kg-d)
cvcles, F0
(davs)
from control
F0, male and female
0
91.7
4.03 ± 0.09
(24/sex/group): 0,100, 300,
1,000 mg/kg-d; dosed daily for
100
97.1
4.1 ±0.29
2
17 wks, from 10 wks premating
to lactation day 21
300
97.1
4.06 ±0.17
1
1,000
95.8
4.29 ±0.61
6
Organ Weights
Fuiiietal. (2010); JPEC (2008e)
Absolute Weight
rat, Sprague-Dawley
oral - gavage
F0, male and female
Dose
(mg/kg-d)
Mean ovarv wt ± % change
SD (mg) from control
Mean uterus wt
± SD (mg)
% change from
control
(24/sex/group): 0,100, 300,
0
98.8 ± 14.9
468 ± 68
-
1,000 mg/kg-d; dosed daily for
100
92.5 ± 16.6 -6
513 ±151
10
17 wks, from 10 wks premating
to lactation day 21
300
95.3 ±11.1 -4
523 ±157
12
1,000
100.9 ± 16.9 2
516 ±136
10
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Toxicological Review of ETBE
Reference and study design
Results
Fuiiietal. (2010); JPEC (2008e)
Relative Weight
(continued)
Dose
Mean ovarv wt ±
% change
Mean uterus wt
% change from
(mg/kg-d)
SD(mg)
from control
±SD(mg)
control
0
30.7 ±4.7
-
146 ± 27
-
100
28.6 ± 6
-7
158 ±49
8
300
29.3 ±3.6
-5
162 ± 53
11
1,000
29.9 ±4.9
-3
154 ± 46
5
Gaoua (2004b)
% change
rat, Sprague-Dawley
Dose
Mean ovarv Wt. ±
from
Mean uterus Wt. ±
% change
oral - gavage
(mg/kg-d)
SD (e)
control
SD(g)
from control
FO, male and female (19-
25/sex/group): 0, 250, 500,
Absolute Weight, F0
1,000 mg/kg-d
0
0.168 ±0.025
-
0.54 ±0.096
-
dosed daily for 18 wks from
10 wks premating until weaning
250
0.167 ±0.027
-1
0.587 ±0.231
9
of F1 pups
500
0.167 ±0.022
-1
0.483 ±0.102
-11
Fl, male and female (19—
25/group): 0, 250, 500,
1,000
0.164 ±0.023
-2
0.576 ±0.218
7
1,000 mg/kg-d
dosed daily from PND 22 until
Absolute Weight, Fl
weaning of F2 pups
0
0.164 ±0.027
-
0.557 ±0.13
-
250
0.172 ±0.028
5
0.577 ±0.161
4
500
0.168 ±0.031
2
0.538 ±0.141
-3
1,000
0.163 ±0.049
-1
0.547 ±0.122
-2
Medinskv et al. (1999); Bond et
Dose
al. (1996b)
(mg/m3)
Mean ovarv wt
±SD(g)
% change from control
rat, Fischer 344
inhalation - vapor
0
0.085 ± 0.022
-
male (48/group): 0, 500,1,750,
2,090
0.095 ± 0.016
12
5,000 ppm
(0, 2,090, 7,320,
7,320
0.088 ±0.12
4
20,900 mg/m3)a; female
20,900
0.090 ±0.19
6
(48/group): 0, 500, 1,750,
5,000 ppm
(0, 2,090, 7,320, 20,900 mg/m3)a
dynamic whole body chamber;
6 hr/d, 5 d/wk for 13 wk
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Reference and study design
Results
Asano et al. (2011); JPEC (2008i)
Dose
rabbit, New Zealand White
(mg/kg-d)
Gravid uterus wt ± SD (g)
% change from control
oral - gavage
female (24/group): 0,100, 300,
0
383 ± 98
-
1,000 mg/kg-d
100
398±128
4
dams dosed daily from GD 6 to
GD 27
300
403 ± 91
5
C-section GD 28
1,000
323 ±128
-16
Mivata et al. (2013); JPEC
Dose
Mean absolute ovarv wt ± SD
(2008c)
(mg/kg-d)
(mg)
% change from control
rat, Sprague-Dawley
oral - gavage
0
70.0 ± 18.7
-
male (15/group): 0, 5, 25,100,
5
71.0 ±21.7
1
400 mg/kg-d; female
25
73.8 ± 16.6
(15/group): 0, 5, 25,100,
O
400 mg/kg-d
100
67.7 ± 17.7
-3
daily for 180 days
400
Dose
76.6 ± 18.2
Mean relative ovarv wt ± SD
9
(mg/kg-d)
(mg/lOOg)
% change from control
0
20.4 ± 5.4
-
5
21.4 ±5
5
25
21.8 ±4.8
7
100
20.0 ±4.9
-2
400
22.8 ±5.5
12
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Toxicological Review of ETBE
Reference and study design
Results
JPEC (2008b)
% change
% change
rat, Sprague-Dawley
Dose
Mean ovarv wt
from
Mean uterus
from
inhalation - vapor
(mg/m3)
N
±SD (mg)
control
wt ± SD (g)
control
male (10/group): 0,150, 500,
1,500, 5,000 ppm
Absolute Weight, Day 92
(0, 627, 2,090, 6,270,
0
10
91.47 ± 10.26
-
0.709 ±0.222
-
20,900 mg/m3)a; female
(10/group): 0, 150, 500, 1,500,
627
10
87.36 ± 15.83
0
0.819 ±0.38
16
5,000 ppm (0, 627, 2,090, 6,270,
2,090
10
84.92 ± 16.91
0
0.654 ±0.159
-8
20,900 mg/m3)a
dynamic whole body chamber;
6,270
10
78.39 ±9.83
0
0.712 ±0.198
0
6 hr/d, 5 d/wk for 13 wk;
20,900
10
91.94 ±21.84
0
0.702 ± 0.205
-1
generation method, analytical
concentration, and method
Absolute Weight, Day 120
reported
0
627
2,090
6,270
6
82.82 ± 17.89
-
0.965 ±0.332
-
20,900
6
90.38 ± 15.88
9
0.818 ±0.286
-15
Relative Weight, Day 92
0
10
27.19 ±3.8
-
0.21 ±0.066
-
627
10
27.58 ±4.35
1
0.269 ±0.151
28
2,090
10
27.03 ±4.55
0
0.211 ±0.055
0
6,270
10
25 ±2.67
-6
0.228 ±0.061
9
20,900
10
30.39 ± 6.46
9
0.231 ±0.071
10
Relative Weight, Day 120
0
6
25.02 ±4.03
-
0.298 ±0.107
-
627
-
-
-
-
-
2,090
-
-
-
-
-
6,270
-
-
-
-
-
20,900
6
26.72 ±4.79
7
0.24 ± 0.089
-19
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Toxicological Review of ETBE
Reference and study design
Results
JPEC (2010a)
rat, Fischer 344
oral - water
male (50/group): 0, 625, 2,500,
10,000 ppm (0, 28, 121, 542
mg/kg-d)b; female (50/group): 0,
625, 2,500, 10,000 ppm (0, 46,
171, 560 mg/kg-d)b
daily for 104 wk
Dose
(mg/kg-d) Mean ovarv wt ± SD (g) % change from control
0 0.194 ±0.238
46 0.18 ±0.146 -7.21649
171 0.153 ±0.035 -21.134
560 0.147 ±0.023 -24.2268
1
2 a4.18 mg/m3 = 1 ppm.
3 bConversion performed by study authors.
4 Population index (%) = (no. of rats with successful copulation/no. of rats paired) x 100.
5 fertility index (%) = (no. females pregnant or no. of males sired/no. of rats with successful copulation) x 100.
6 *: result is statistically significant (p < 0.05) based on analysis of data by study authors.
7 for controls, no response relevant; for other doses, no quantitative response reported.
8 % change from control = (control value - treated group value)/control value] x 100.
9 Absolute change from control (%) = control value (%) - treated group value (%).
10
11
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Toxicological Review of ETBE
I - exposures at which the ondpmnf was reported statistically significant by study authors
J - exposures at which the Midpoint was reported not statistically significant hv study authors
Pregnancy Outcomes
FD Female rat; copulation; one-gert repro (C)
FO Female rat; fertility; one-gen repro (C)
FO Female rat; fertility; two-gen repro (E)
F1 Female rat; fertility; two-gen repro jE)
Female rat; maternal body weight gain; GD 5-19 (D)
FO Female rat; maternal body weight gain; two-gen
repro (E)
F1 Female rat; maternal body weight gain; two-gen
repro (E)
Female rat; maternal body weight gain; GD 5-19 (B)
FO Female rat; maternal body weight gain; one-gen
repro (C)
Female rabbit; maternal body weight gain; 60 6-27
(A)
Female rabbit; gravid uterine weight; GD 8-27 (A)
FO Female rat; gestation length; one-gert repro (C)
FO female rat; gestation length; two-gen repro (E)
F1 female rat; gestation length; two-gen repro (E)
FO Female rat; titter size; one-gen repro (C)
FO Female rat; litter size; two-gen repro (E)
F1 Female rat; litter size; two-gen repro (E)
Female rat; litter size; GD 5-19 (B)
Female rabbit; litter size; GD 6-27 (A)
FO Female rat; estrous cyclicity; one-gen repro (C)
' maternal weight gain significantly increased in this
stmly whereas other studies showed a significant
decrease
4
10
o-
B-
-B-
Q—B-
~ 0 rJ
I Ii^1liinnriiiiiii|7rri7|inw
~ H f.
0-
B-
-B-
GH
-e_
Or
-B-
Q B 51
~ Q f ]
B-
_Q_
Q—B 'J
~ p
D
Q-
-B-
-B-
100 1,000
Dose (mg/kg-day)
Seuiws (A) As.iiw et j|,>0ll IPBf 2008h(B)Asoetal,2014;JPiC,2008g(C| Fujiirt *1 2010;lPEC.2008e
(D) tuwiu, 2004,t(_E) C,w»ia, 200 U»
10,000
Figure 1-13. Exposure-response array of female reproductive effects following
oral exposure to ETBE.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
¦ = exposures at which the Midpoint was reported statistically significant by study authors
~ = exposures at which the endpoint was reported not statistically significant by study authors
Female Reproductive Organ Weights
Female rat; ovary wt, (absolute); 13 wks (B)
Female rat; ovary wt. (absolute); 13 wks (A)
Female rat; ovary wt (relative); 13 wks (A)
Female rat; uterine wt. (absolute); 13 wks (A)
Q B B Q
~ B B—i B
O 0 B 0
Female rat; uterine wt. (relative); 13 wks (A)
-B-
-B-
100 1,000 10,000 100,000
l"x|>ostsrt'< onceutration (ing/in-*)
Source: (A) JPEC 2008b (B) Medinsky et al, 1999; Bond etal, 1996b
Figure 1-14. Exposure-response array of female reproductive effects following
inhalation exposure to ETBE.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Toxicological Review of ETBE
Mechanistic Evidence
No mechanistic evidence for female reproductive effects was identified by the literature
search.
Integration of Female Reproductive Effects
The available evidence to assess female reproductive effects consists of one- and two-
generation reproductive toxicity studies, developmental toxicity studies, and 90-day through 2-year
oral and inhalation exposure studies that adequately evaluate the relevant female reproductive
endpoints. These studies show that ETBE does not adversely affect maternal body weight gain,
fertility, mating, pregnancy parameters, or reproductive organ weights in all but one study up to
1,000 mg/kg-day (oral exposure) or 5,000 ppm (whole body inhalation exposure) in the female rat
or rabbit. Relative, but not absolute ovary weights were significantly increased following ETBE
inhalation exposure in one 2-year study but not observed in other 2-year, 180-/90-day,
reproductive, or developmental studies. Collectively, although minimal effects were observed at
otherwise toxic dose levels, the available evidence is considered insufficient to identify female
reproductive effects as a potential human hazard of ETBE, and female reproductive effects are not
carried forward as a hazard.
1.2.4. Developmental Effects
Synthesis of Effects Related to Development
The database examining developmental effects following ETBE exposure includes no
human data; it is composed of data from toxicology studies conducted in Sprague-Dawley rats or
New Zealand White rabbits in which ETBE was administered via oral gavage. These consisted of
three prenatal developmental toxicity studies [two in rats: fAso etal.. 2014: TPEC. 2008hl and
(Gaoua. 2004a) and one in rabbits: (Asano etal.. 2011: TPEC. 2008i)]. a one-generation reproductive
toxicity study in rats (Fujii etal.. 2010: TPEC. 2008e). and a two-gene ration reproductive toxicity
study in rats (Gaoua. 2004a). The design, conduct, and reporting of all five studies were of sufficient
quality to inform human health hazard assessment. The highest dose level tested in each study was
1,000 mg/kg-d, the recommended limit dose for prenatal developmental toxicology studies (OECD.
2001: U.S. EPA. 1998cl
Developmental endpoints evaluated after ETBE exposure include prenatal and postnatal
survival, growth, and morphological development In addition, limited assessments of postnatal
neurological functional development were conducted. Selected developmental toxicity data are
summarized in
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Toxicological Review of ETBE
Table 1-16.
Evidence of effects of ETBE treatment on pre- or postnatal survival was minimal. In the
developmental toxicity study in rats by fAso etal.. 2014: IPEC. 2008h). increased preimplantation
loss was observed in the treated groups. The percent preimplantation loss in the 1,000 mg/kg-day
dams was 81.8% greater than control, while it was increased 37.9% at 100 mg/kg-day and 21.2%
at 300 mg/kg-day. Statistical significance was not reported. Increased preimplantation loss was not
observed in the other available developmental toxicity studies in rats or rabbits [fGaoua. 2004a)
and (Asano etal.. 2011: IPEC. 20080. respectively]. Postnatal survival was not affected by ETBE
treatment in either the first or second generation of the reproductive toxicity study by Gaoua
f2004bl Viability indices throughout the lactation period were similar between control and treated
groups during both generations of this study. In the one-generation reproductive toxicity study
fFuiii etal.. 2010: IPEC. 2008el. there was evidence of a non-significant decrease (10.5% as
compared to control) in the PND 4 viability index at 1,000 mg/kg-day. Examination of the
individual animal data indicated that total litter loss in three litters had resulted in the majority of
pup deaths that occurred from PND 0-4. For two of these litters, severe maternal toxicity had led to
moribund sacrifice of the dams in early lactation; this is the only evidence in the available ETBE
data where adverse outcomes in the offspring were definitively associated with maternal toxicity.
The third dam with total litter loss had no evidence of treatment-related toxicity.
Neither prenatal nor postnatal growth were affected by ETBE treatment. Mean fetal weights
were comparable between control and ETBE-treated groups in the prenatal developmental toxicity
studies in rats and rabbits (Aso etal.. 2014: Asano etal.. 2011: IPEC. 2008h. ij Gaoua. 2004a).
Similarly, pup weights from PND 0-21 were not affected by treatment in the reproductive toxicity
studies fFuiii etal.. 2010: IPEC. 2008e: Gaoua. 2004bl. Additionally, fFuiii etal.. 2010: IPEC. 2008el
no effects were observed in the rate of completion of development landmarks in male and female
F1 offspring, specifically pinna detachment on PND 3, incisor eruption on PND 11, and eye opening
on PND 15. Organ weights (brain, spleen, and thymus) were evaluated in PND 21 pups in the one-
and two-generation reproduction studies (Fujii etal.. 2010: IPEC. 2008e: Gaoua. 2004b): no
significant differences were observed between control and treated groups (not shown in evidence
table). At the termination of adult animals in the reproductive toxicity studies, a number of organ
weights were measured. Sections 1.2.1 and 1.2.2 discuss increased mean kidney and liver weights,
respectively, observed in the adult F1 offspring of the two-generation reproduction study fGaoua.
2004b). The findings in the F1 adults were similar to those in the P adults, indicating an absence of
life stage-related susceptibility for these outcomes.
No evidence existed of treatment-related effects on postnatal morphological assessments
that consisted of PND 1 anogenital distance measurements in F1 and F2 pups fGaoua. 2004b) and
the age of F1 sexual maturation (preputial separation in males and vaginal opening in females)
fFuiii etal.. 2010: IPEC. 2008e: Gaoua. 2004bl.
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
Toxicological Review of ETBE
In the prenatal developmental toxicity studies with ETBE (Aso etal.. 2014: Asano etal..
2011:1PEC. 2008h. ij Gaoua. 2004al. the evidence of treatment-related alterations in fetal
development at 1,000 mg/kg-day were sporadic, and there was no consistent pattern of effect.
In Aso etal. f20141. a >3-fold increase in the number and percent of rat fetuses with skeletal
variations was noted at 1,000 mg/kg-day compared to control. Examination of the individual litter
data revealed that this increase was primarily attributable to a statistically significant >6-fold
increase in the number of fetuses (and >3-fold increase in the number of litters) with rudimentary
lumbar rib at that dose. The study authors dismissed the relevance of this finding, reporting that it
is within a historical control range (1.1-21.2%) for the strain of ratused in the study and because
the effect has sometimes been viewed as transient [e.g., fChernoff etal.. 19911], Nevertheless, the
incidence of this finding is significantly increased as compared to the concurrent control, which is
considered more relevant and preferable to historical control and the finding might have been the
result of an alteration of vertebral development; therefore, it is considered potentially treatment-
related.
In Gaoua (2004a). a statistically significant 37% increase in the number of fetuses with
unossified 4th metacarpal as compared to control was observed at 1,000 mg/kg-day. Further
evaluation of the fetuses, which were double-stained with alcian blue, revealed that a cartilage
precursor was present, suggesting that the finding represented a treatment-related delay in
development rather than a malformation.
An increase in the number of rabbit fetuses and litters with visceral malformations at 1,000
mg/kg-day was noted in Asano etal. (2011) and TPEC (2008i). This was specifically attributed to
observations of fetuses with absent right atrioventricular valve of the heart. The incidences of this
finding did not achieve statistical significance. Also in Asano etal. f20111 and TPEC f2008il. a 66%
increase in the number of rabbit fetuses with skeletal variations at 1,000 mg/kg-day as compared
to control was found to be primarily attributed to incidences of unossified talus (in 12 fetuses, 6
litters).
Limited evaluation of postnatal functional neurological development in F1 male and female
offspring in reproductive toxicity studies were conducted by Fujii etal. (2010). TPEC (2008e). and
Gaoua (2004b). No treatment-related effects were found in assessments of reflex ontogeny, which
included surface righting reflex on PND 5 fFuiii etal.. 2010: TPEC. 2008e: Gaoua. 2004bl. negative
geotaxis on PND 8 fFuiii etal.. 2010: TPEC. 2008el. cliff avoidance on PND 11 f Gaoua. 2004bl. and
air righting reflex on PND 17 fGaoua. 2004bl or PND 18 fFuiii etal.. 2010: TPEC. 2008el. Gaoua
(2004b) also conducted tests in F1 males and females of acoustic startle response [postnatal week
(PNW) 4], pupil constriction (PNW 4), and motor activity (PNW 7 and 8). The motor activity testing
was performed using an automated device that measured the number of movements within the
front or back of the cage, back and forth movements, and vertical movements. Two 10-minute trials
were conducted 1 week apart. No treatment-related effects were found.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
1
2
3
4 Table 1-16. Evidence pertaining to developmental effects in animals following
5 exposure to ETBE
Reference and study design
Results
Prenatal Survival
Aso et al. (2014); JPEC (2008h)
% change % change
rat, Sprague-Dawley
Dose
No. No. preimplan-
from % Preimplan- from
oral -gavage
(mg/kg-d)
Litters tation loss
control tation loss3 control
female (24/group): 0,100, 300,
1,000 mg/kg-d
0
21
22
6.6
dams dosed daily from GD 5 to
100
22
25
13.6 9.1 37.9
GD 19
C-section GD 20
300
20
25
13.6 8.0 21.2
1,000
22
39
77.3 12.0 81.8
Dose
(mg/kg-d)
No. resorptions
% Postimplantation lossb
0
18
5.8
100
22
7.2
300
12
4.2
1,000
13
5
Gaoua (2004a)
Dose
No. preimplantation loss
rat, Sprague-Dawley
(mg/kg-d)
No. Litters
% Preimplantation loss3
oral - gavage
female (24/group): 0, 250, 500,
0
21
48 17.8
1,000 mg/kg-d
250
19
36 14.9
dosed daily from GD 5 to GD 19
C-section GD 20
500
20
38 14.3
1,000
22
47 16.8
Dose
(mg/kg-d)
No. Postimplantation loss % Postimplantation lossb
0
14
5.2
250
16
6.6
500
19
7.2
1,000
21
7.5
Asano et al. (2011); JPEC (2008i)
Dose
% Postimplantation
rabbit, New Zealand White
(mg/kg-d)
No. litters
% Preimplantation loss3 lossb
oral - gavage
0
22
19.6 11.0
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
Reference and study design
Results
female (24/group): 0,100, 300,
1,000 mg/kg-d
dams dosed daily from GD 6 to
GD 27
C-section GD 28
100 22 15.3 11.3
300 20 10.7 7.0
1,000 23 22.9 8.7
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
Reference and study design
Results
Postnatal Survival
Fuiiietal. (2010); JPEC (2008e)
% change
rat, Sprague-Dawley
from
oral -gavage
Dose
Viability index
Viabilitv index
control
Total litter loss
FO, male and female
(mg/kg-d)
PND0±SD
PND 4 ± SD
(PND 4)
(PND 0-4)c
(24/sex/group): 0,100, 300,
1,000 mg/kg-d; dosed daily for
0
98.9 ±3.7
97.4 ±4.7
-
0
17 wks, from 10 wks premating
100
97.9 ±5.6
96.7 ±8.1
-0.7
0
to lactation day 21
300
99.5 ±2.6
99.6 ± 1.9
2.3
0
1,000
93.6 ± 15.5
87.2 ±29.8
-10.5
3
Dose
(mg/kg-d)
Viabilitv Index - PND 21 ± SD
0
97 ± 11.1
100
95.8 ± 11.4
300
95.7 ± 11.1
1,000
92.5 ±23.1
Gaoua (2004b)
Dose
Viabilitv index
Viabilitv index Total litter loss
Viabilitv index
rat, Sprague-Dawley
(mg/kg-d)
PND 0
PND 4
(PND 0-4)
PND 21
oral - gavage
Fl
F0, male and female
(25/sex/group): 0, 250, 500,
0
100
97.6
0
94.6
1,000 mg/kg-d
250
100
92.9
91.7
dosed daily for 18 wks from
1
10 wks premating until weaning
500
100
82.3
0
96.1
of F1 pups
Fl, male and female (24—
1,000
100
97.7
1
99.5
25/group): 0, 250, 500,
F2
1,000 mg/kg-d
dosed daily from PND 22 until
0
100
97.6
0
97.6
weaning of F2 pups
250
100
94.8
0
98.8
500
100
97.0
3
100
1,000
100
92.9
0
99.3
Prenatal Growth
Aso et al. (2014); JPEC (2008h)
Dose
Mean fetal weight ± SD Mean fetal weight ± SD
rat, Sprague-Dawley
(mg/kg-d)
No. litters
male (g)
female (g)
oral - gavage
female (24/group): 0,100, 300,
0
21
4.1 ±0.3
3.89 ±0.25
1,000 mg/kg-d
100
22
4.14 ±0.33
3.92 ±0.23
dams dosed daily from GD 5 to
GD 19
300
20
4.23 ±0.22
4.01 ±0.22
C-section GD 20
1,000
22
4.14 ±0.34
3.91 ±0.39
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
Reference and study design
Results
Gaoua (2004b)
Dose
Mean fetal weight ± SD Mean fetal weight ± SD
rat, Sprague-Dawley
(mg/kg-d)
No. litters
male (g)
female (g)
oral -gavage
FO, male and female
0
21
3.92 ±0.58
3.77 ±0.5
(25/sex/group): 0, 250, 500,
250
19
4.03 ±0.32
3.82 ±0.33
1,000 mg/kg-d
500
20
3.94 ±0.35
3.75 ±0.32
dosed daily for 18 wks from
10 wks premating until weaning
1,000
22
3.91 ±0.33
3.66 ±0.39
of F1 pups
Fl, male and female (24—
25/group): 0, 250, 500, 1,000
mg/kg-d
dosed daily from PND 22 until
weaning of F2 pups
Asano et al. (2011); JPEC (2008i)
Dose
Mean fetal weight ± SD Mean fetal weight ± SD
rabbit, New Zealand White
(mg/kg-d)
No. litters
male (g)
female (g)
oral - gavage
female (24/group): 0,100, 300,
0
22
33.5 ±4.1
31.5 ±3.7
1,000 mg/kg-d
100
22
33.4 ±6.2
31.5 ±4.8
dams dosed daily from GD 6 to
GD 27
300
20
33.9 ±2.5
32.0 ±3.6
C-section GD 28
1,000
23
32.3 ±6.5
30.1 ±6.0
Postnatal Growth
Fuiiietal. (2010); JPEC (2008e)
Dose
No.
Mean±SD Mean±SD
Mean ± SD
rat, Sprague-Dawley
(mg/kg-d)
litters
PND 0(g) PND 4 precull (g)
PND 21(g)
oral - gavage
F0, male and female
Fl-Male Pup Weight
(24/sex/group): 0,100, 300,
0
21
6.9 ±0.7 11.0 ±2.0
61.3 ±6.3
1,000 mg/kg-d; dosed daily for
17 wks, from 10 wks premating
100
22
6.9 ±0.8 11.0 ±1.8
61.0 ±7.0
to lactation day 21
300
23
6.9 ±0.6 10.8 ±1.4
61.6 ±4.6
1,000
22
7.0 ±0.7 10.4 ±1.7
61.6 ±6.4
Fl-Female Pup Weight
0
21
6.5 ±0.7 10.4 ±1.8
59.3 ±6.4
100
22
6.5 ±0.6 10.4 ±1.6
58.5 ±6.4
300
23
6.5 ±0.6 10.2 ±1.4
58.5 ±6.4
1,000
22
6.6 ±0.6 10.0 ±1.8
59.7 ±5.2
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
Reference and study design
Results
Gaoua (2004b)
Dose
Mean ± SD
Mean ± SD
Mean ± SD
rat, Sprague-Dawley
(mg/kg-d)
PND 1(h)
PND 4 precull (g)
PND 21(g)
oral - gavage
FO, male and female
Fl-Male Pup Weight
(25/sex/group): 0, 250, 500,
0
6.8 ±0.7
9.1 ± 1.4
50.1 ±4.9
1,000 mg/kg-d
dosed daily for 18 wks from
250
6.7 ±0.6
9.0 ± 1.6
51.7 ±4.1
10 wks premating until weaning
500
6.5 ±0.7
8.7 ± 1.3
50.5 ±6.7
of F1 pups
Fl, male and female (24—
1,000
7.0 ±0.7
9.3 ± 1.2
52.4 ±4.5
25/group): 0, 250, 500,
Fl-Female Pup Weight
1,000 mg/kg-d
dosed daily from PND 22 until
0
6.4 ±0.6
8.6 ± 1.4
48.1 ±6.1
weaning of F2 pups
250
6.4 ±0.6
8.5 ± 1.6
49.5 ±4.3
500
6.0 ±0.6
8.1 ± 1.2
48.2 ±5.9
1,000
6.5 ±0.6
F2-Male Pup Weight
8.9 ± 1.2
50.6 ±4.4
0
6.9 ±0.6
9.5 ± 1.5
51.5 ±7.2
250
6.7 ±0.6
9.3 ± 1.0
52.1 ±4.4
500
6.4 ±0.5
9.2 ± 1.0
50.3 ±5.8
1,000
6.3 ±0.6
F2-Female Pup Weight
9.2 ± 1.4
51.2 ±3.6
0
6.5 ±0.6
8.9 ± 1.3
49.6 ±6.2
250
6.3 ±0.6
8.8 ± 1.0
49.9 ±3.6
500
6.4 ±0.5
8.9 ±0.9
49.0 ±5.5
1,000
6.3 ±0.6
8.7 ± 1.4
49.1 ±3.7
Prenatal Morphology
Aso et al. (2014); JPEC (2008h)
rat, Sprague-Dawley
oral -gavage
female (24/group): 0,100, 300,
1,000 mg/kg-d
dams dosed daily from GD 5 to
GD 19
C-section GD 20
No. fetuses
examined for
No. fetuses with
No. fetuses
Dose
No. fetuses
visceral
visceral
with visceral
(mg/kg-d)
(litters)d
anomalies
malformations
variations
0
285(21)
146
3(3)
6(6)
100
263(22)
137
2(2)
8(7)
300
251(20)
132
2(2)
4(4)
1,000
270(22)
139
0
8(7)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
Reference and study design
Results
Aso et al. (2014); JPEC (2008h)
No. fetuses
% fetuses
(continued)
examined
No. fetuses with No. fetuses
(litters) with
Dose
for skeletal
skeletal
with skeletal
skeletal
(mg/kg-d)
anomalies
malformations variations
variations
0
139
0
9(8)
6.5(38.1)
100
126
0
3(3)
2.4(13.6)
300
119
0
3(3)
2.5(15.0)
1,000
131
0
29(13)
22.1(59.1)
No. fetuses (litters) % fetuses (litters)
Dose
with rudimentary with rudimentary
(mg/kg-d)
lumbar rib
lumbar rib
0
4(4)
2.9(19.0)
100
0
0(0)
300
2(2)
1.7(10.0)
1,000
25*(11)
19.1*(50.0)
Gaoua (2004a)
No. fetuses
rat, Sprague-Dawley
No. fetuses with
examined for
No. fetuses with
oral -gavage
Dose
No. fetuses
external
visceral
visceral
female (24/group): 0, 250, 500,
(mg/kg-d)
(litters)d malformations
anomalies
malformations
1,000 mg/kg-d
255(21)
dosed daily from GD 5 to GD 19
0
0
120
0
C-section GD 20
250
226(19)
1(1)
109
0
500
246(20)
0
116
0
1,000
258(22)
0
122
1(1)
No. fetuses
No. fetuses
examined for
No. fetuses with No. fetuses
Dose
with visceral
skeletal
skeletal
with skeletal
(mg/kg-d)
variations
anomalies
malformations
variations
0
1(1)
135
1(1)
125(21)
250
2(2)
117
2(2)
101(19)
500
1(1)
130
1(1)
116(20)
1,000
3(3)
136
2(2)
112(22)
No. fetuses with
% fetuses with
unossified 4th
unossified 4th
metacarpal
% change from control
metacarpal
0
27(9)
-
20.0(42.9)
250
21(10)
-22.2
17.9(52.6)
500
24(9)
-11.1
18.5(45.0)
1,000
43*(12)
37.2
31.6(54.5)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of ETBE
Reference and study design
Results
Asano et al. (2011); JPEC (2008i)
No.
No. fetuses with No. fetuses with No. fetuses
rabbit, New Zealand White
Dose
fetuses
external visceral with skeletal
oral -gavage
(mg/kg-d)
(litters)d
malformations malformations malformations
female (24/group): 0,100, 300,
0
171(22)
0
1(1) 5(4)
1,000 mg/kg-d
dams dosed daily from GD 6 to
100
174(22)e
1(1)
1(1) 4(4)
GD 27
C-section GD 28
300
167(20)
0
1(1) 3(2)
1,000
159(23)e
1(1)
3(2) 8(5)
Absent right
Dose
No. fetuses with atrioventricular
% change from
(mg/kg-d)
skeletal variations valve
control
0
9(7) 0
-
100
11(9) 0
0(0)
300
6(6) 1(1)
0.6(5.0)
1,000
15(8) 3(2)
1.9(8.7)
Postnatal Morphology
Fuiiietal. (2010); JPEC (2008e)
Male preputial separation -
Female vaginal opening -
rat, Sprague-Dawley
Dose
No.
age (davs)
age (davs)
oral - gavage
(mg/kg-d)
litters
mean ± SD
mean ± SD
F0, male and female
(24/sex/group): 0,100, 300,
rl
1,000 mg/kg-d; dosed daily for
0
21
41.0 ± 1.7
31.2 ± 1.4
17 wks, from 10 wks premating
to lactation day 21
100
22
41.4 ± 1.1
30.9 ± 1.7
300
23
40.6 ± 1.5
30.5 ±2.2
1,000
19
41.2 ± 1.6
30.3 ±2.1
Gaoua (2004b)
Anogenital distance' - males
Anogenital distance'-
rat, Sprague-Dawley
Dose
No.
(PND 1)
females (PND 1)
oral - gavage
(mg/kg-d)
litters
mean ± SD
mean ± SD
F0, male and female
(25/sex/group): 0, 250, 500,
1,000 mg/kg-d
0
21
2.48 ±0.18
1.53 ±0.18
dosed daily for 18 wks from
250
22
2.45 + 0.17
1.5 + 0.14
10 wks premating until weaning
of F1 pups
500
23
2.4 ±0.21
1.45 ±0.14
Fl, male and female (24—
1,000
20
2.43 + 0.15
1.44 + 0.2
25/group): 0, 250, 500,
1,000 mg/kg-d
F2
dosed daily from PND 22 until
0
21
2.41 ±0.18
1.51 ±0.18
weaning of F2 pups
250
22
2.42 ±0.25
1.47 ±0.19
500
23
2.42 ±0.23
1.51 ±0.17
1,000
20
2.45 ±0.21
1.57 ±0.22
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
Toxicological Review of ETBE
Reference and study design
Results
Gaoua (2004b)
Dose
No.
Male preputial separation -
Female vaginal opening -
(continued)
(mg/kg-d)
litters
age (davs) - mean ± SD
age (davs) - mean ± SD
F1
0
25
35 ±2
34 ±3
250
25
34 ±2
34 ±3
500
25
35 ±2
35 ±2
1,000
25
35 ±2
33 ±2
aPercent preimplantation loss = (no. preimplantation embryonic loss/no. corpora lutea) xlOO.
bPercent postimplantation loss = (no. resorptions and dead fetuses/no. implantations) xlOO.
cTwo 1,000 mg/kg-d dams were killed in a moribund condition on PND 2 and 4, thus compromising the survival of
their litters. In a third litter, all pups died between PND 1-4 although there was no evidence of maternal toxicity
throughout the study.
dThe parenthetical number following fetal incidence indicates the associated litter incidence for all findings.
eNo. of fetuses examined for visceral and skeletal anomalies at 100 and 1,000 mg/kg-d were 173 and 158,
respectively, because fetuses with external malformations were excluded.
fAGD/cube root of body weight.
*: result is statistically significant (p < 0.05) based on analysis of data by study authors.
-: for controls, no response relevant; for other doses, no quantitative response reported.
% change from control = (control value - treated group value)/control value] x 100.
Absolute change from control (%) = control value (%) - treated group value (%).
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE—Volume 1 of 2
¦ = exposures at which the endpoint was reported statistically significant by study authors
~ = exposures at which the endpoint was reported not statistically significant by study authors
Survival -
Prenatal
Rat; preimplantation loss; GD 5-19 (B)
Rat; preimplantation loss; GD 5-19 (D)
Rabbit; preimplantation loss; GD6-27 (A)
Rat; postimplantation loss; GD 5-19 (B)
Rat; postimplantation loss; GD 5-19 (D)
Rabbit; postimplantation loss; GIH> (A)
Survival - Hat; decreased viability, l'ND4 orPNI) 21, one sen icpto (f)
Postnatal
Rat; decreased viability, I'NI) 4 or l*ND21; two-gen repro (K)
(HOWtll -
I'lCIUll.ll
Rat; decreased mean total wt.; GD 5-1't (P)
Rat; decreased mean fetal wt,; GO 5-19 (ii)
Rabbit;decreased mean fetal \rt, GDft 27 (A)
Growth -
Postnatal
Rat; decreased mean pup wt.; one «en i < pro (C)
Rat; decreased mean pup wt,; two-gen repro (E) -
Morphology-
Prenatal
Rat; fetal external or visceral anomalies; GD 5-19 (B) •
Rat; fetal external or visceral anomalies; GD 5-19 (D)
Rabbit; fetal external or visceral anomalies; GD 6-27 (A) -
Rat; skeletal variation; rudimentary lumbar ribs; GD 5-19 (13)
Rat, ski letal % ai lation: rudimentary lumbar ribs; GD 5-19 (D)
Rabbi 1, skeletal vat ration: rudimentary lumbar ribs; GD6-27
(A)
Rat; skeletal variation: unossified 4th metatarsal; GD 5-19 (B)
Rat; skeletal variation; unossified 4th metatarsal; GD 5-19 (D)
Rabbit; skeletal variation: unossitied I'll metatarsal; GD6-27
(A)
Ral; alteied F1 or i-"2 anoRcnital distance (I'ND 1); one-gen
Morphology- repro (C)
Postnatal Rat; altered F1 or F2 anogenital distance (RND 1); two-gen
repro (B)
Rat; altered F1 age of puberty (male or female); one-gen repro
(C)
Rat; altered F1 age of puberty (male or female); two-gen repro
' (K)
Rat; altered F1 reflex ontogeny, acoustic startle response.
Functional pupil constriction, motor aciivity; one-gen repro (C)
(Neurological) Rat; altered F1 reflex ontogeny, aroustic startle response,
Development pupil constriction, motor activity; two-gen repro (E)
B B-
-B
[3—B—ED
B B-
B-
-B
~ ~ ~
Q B B
Q—B—B
~ B El
B B~
-a
a—b-
-a
~ ~ B
-B~
O—B—B
B B-
B B-
o-e—a
B B-
-B
B B-
-B
O—B-
B B-
-B
B-
-B
~ ~ ~
B B-
~—B—B
10 100
Dose (mg/kg-day)
1,000
10,000
Sources: (A) Asano et ai. 2011; JPEC, 20Q8h (B)Aso et al. 2014; JPEG,2008g(C) Fuji! et al, 2010;[PEC, 2008e
(D) Gaoua, 2004a (E) Gaoua, 2004b
Figure 1-15. Exposure-response array of developmental effects following oral
exposure to ETBE.
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Mechanistic Evidence
No mechanistic evidence for developmental effects was identified by the literature search
Integration of Developmental Effects
The evidence to assess developmental toxicity for ETBE consists of two prenatal
developmental toxicity studies in rats and one in rabbits, a one-generation reproductive toxicity
study in rats, and a two-generation reproductive toxicity study in rats. These studies included
assessments of pre- and postnatal survival, growth, morphology, and functional neurological
development following oral (gavage) administration during sensitive periods of development
Slight evidence of effects of ETBE treatment on prenatal or postnatal survival consisted of
preimplantation loss in a developmental toxicity study in rats and decreased PND 0-4 pup viability
that was associated with severe maternal toxicity. Pre- and postnatal growth (body weights and
developmental landmarks), anogenital distance, sexual maturation, and evaluation of neurological
function (including reflex ontogeny and assessments of acoustic startle response, pupil constriction,
and motor activity in offspring) were not affected by treatment. Evidence of incidental structural
(visceral and skeletal) fetal anomalies following in utero exposures to ETBE were observed at the
highest dose tested (1,000 mg/kg-day). The findings were limited to increased incidences of
rudimentary lumbar rib fAso etal.. 2014: TPEC. 2008hl and unossified 4th metatarsal fGaoua.
2004b) in two rat studies and unossified talus and absent right atrioventricular valve in a rabbit
study (Asano etal.. 2011: TPEC. 2008i). The fetal, but not litter, incidences of skeletal findings in rats
(rudimentary lumbar rib and unossified 4th metatarsal) were statistically significant at the highest
dose tested (1,000 mg/kg-day). These skeletal observations were not confirmed in other species.
No inhalation prenatal developmental or reproductive toxicity studies were conducted, thus
potential effects of inhalation exposure on pre- and postnatal development have not been
characterized. Overall, the available evidence is considered inadequate to draw conclusions
regarding the development toxicity of ETBE, and developmental effects are not carried forward as a
hazard.
1.2.5. Carcinogenicity (Other than in the Kidney or Liver)
Synthesis of Carcinogenicity Data (Other than in the Kidney or Liverj
This section reviews the studies that investigated whether exposure to ETBE can cause
cancers (other than in the kidney or liver) in humans or animals. The evidence pertaining to
tumorigenicity in the kidney and liver was previously discussed in Sections 1.2.1 and 1.2.2,
respectively. The database for ETBE carcinogenicity consists of only animal data: three 2-year
studies (two oral, one inhalation), and two "initiation, promotion" cancer bioassays performed in
rats (Hagiwara etal.. 2013: Saito etal.. 2013: Suzuki etal.. 2012: Hagiwara etal.. 2011: Malarkev
and Bucher. 2011: TPEC. 2010a. b; Maltoni etal.. 19991 (see Table 1-17, Table 1-18; Figure 1-16,
Figure 1-17). Interpretation of the study results reported by Maltoni et al. f 19991 is complicated by
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the nonstandard histopathological diagnoses used and the greater than expected mortality in
treated groups and controls compared with other laboratories. Survival was reduced at the highest
exposure in males and females after 72 weeks (data not shown) and after 104 weeks, survival in the
controls was approximately 25% in males and 28% in females which is much lower than
anticipated for a 2-year study fMaltoni etal.. 19991. The survival data in this study was potentially
attributable to chronic respiratory infections in the controls and treated groups (Malarkev and
Bucher. 20111. ultimately limiting the ability of this study to predict potential carcinogenicity.
In response to the concerns regarding high mortality and the utilization of nonstandard
histopathological diagnoses, a pathology working group sponsored by EPA and the National
Toxicology Program (NTP) reviewed the histopathological data fMalarkev and Bucher. 20111. In
addition to recalculating tumor incidences, the working group found that the respiratory infections
in the study animals confound interpretation of leukemia and lymphoma. Thus, the Malarkev and
Bucher (2011) data were used when considering carcinogenicity in place of the published Maltoni
etal. (1999) study, and leukemia and lymphoma incidences from this study were not considered.
Following 2-year exposure to ETBE, the incidence of leiomyomas was increased in the
uterus of Sprague-Dawley rats in the high-dose group fMaltoni et al.. 19991. Malignant
schwannomas in the uterus were increased only at the lowest dose, and no significant trend was
observed. These neoplasms arise from nervous tissue and are not specific to uterine tissue.
Leiomyomas and a carcinoma were observed in uterine/vaginal tissue, but no significant trend was
observed (Malarkev and Bucher. 2011).
Several initiation-promotion studies have been conducted with ETBE {Hagiwara, 2011,
1248019;Hagiwara, 2015, 3046107;Hagiwara, 2013, 2321105}. While chronic cancer bioassays are
considered key data for the evaluation of carcinogenicity, other types of studies, such as initiation-
promotion studies, are considered supplemental lines of information which can aid in the
interpretation of more standard toxicological evidence (e.g., rodent chronic bioassays), especially
regarding potential modes of action {U.S. EPA, 2005, 86237}. A statistically significant and dose-
dependent increase in incidence of neoplastic lesions was observed in the thyroid of F344 male rats
following subchronic exposure to ETBE after a 4-week tumor initiation exposure to DMBDD
(Hagiwara etal.. 2011): incidences of colon and urinary bladder neoplasms also were statistically
significantly increased fHagiwara etal.. 20131. Forestomach papilloma or hyperplasia incidence
was elevated statistically significantly, while no cases were reported in control animals receiving 4
weeks of mutagenic treatment This finding is consistent with the rarity of forestomach squamous
cell papillomas in untreated animals (historical control rate = 0.08% in untreated male F344/N rats
after 2 years; (NTP. 2011): comparability with JPEC controls unknown). While increased tumors
were observed with ETBE following administration of tumor initiators, it is important to recognize
the limitations of these experimental protocols. Such limitations include experimental manipulation
of the carcinogenic process, a generally less than chronic exposure duration, and smaller groups of
animals. Male F344 rats (n=12), exposed to ETBE via gavage for 23 weeks (in the absence of
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DMBDD treatment) did not significantly induce tumor development in any organs evaluated
fHagiwara etal.. 20111. Increased tumorigenesis in these tissues was not reported following 2 years
of exposure to ETBE alone via drinking water or inhalation in male or female F344 rats fSaito etal..
2013: Suzuki etal.. 2012: TPEC. 201 Obi.
Mechanistic Evidence
The available mechanistic evidence was previously discussed in the context of kidney and
liver tumors (Sections 1.1.1 and 1.1.2). Aside from (predominantly negative) genotoxicity testing
results, generally relevant to tumorigenesis in any tissue location (discussed in the Supplemental
Information), no further mechanistic evidence was identified relevant to uterine, thyroid, colon,
forestomach, or urinary bladder carcinogenesis.
Integration of Carcinogenicity Evidence
The evidence for carcinogenic effects other than liver or kidney is solely from rat studies.
ETBE exposure following mutagen administration increased the incidence of thyroid adenomas or
carcinomas, colon adenomas or carcinomas, forestomach papillomas, and urinary bladder
carcinomas in male rats. Confidence in the data demonstrating an increase in the incidence of
schwannomas is reduced due to the lack of a dose-response in Sprague-Dawley rats and lack of a
similar effect reported in F344 rats from two other well-conducted 2-year studies, or in F344 or
Wistar rats from the two-stage subchronic cancer bioassays. The hazard and dose-response
conclusions regarding these carcinomas and adenomas associated with ETBE exposure are further
discussed as part of the overall weight of evidence for carcinogenicity in Section 1.3.2.
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Table 1-17. Evidence pertaining to carcinogenic effects (in tissues other than
liver or kidney) in animals exposed to ETBE
Reference and study design
Resu Its
Thyroid adenomas/adenocarcinomas
JPEC (2010a); Suzuki et al.
(2012)
rat, Fischer 344
oral - water
male (50/group): 0, 625, 2,500,
10,000 ppm (0, 28, 121,
542 mg/kg-d)a; female
(50/group): 0, 625, 2,500,
10,000 ppm (0, 46, 171,
560 mg/kg-d)a
daily for 104 wk
Male
Female
Thyroid
Dose
Thyroid
Dose
follicular
Thyroid
Thyroid follicular
follicular
adenocarcino
follicular
[mg/kg-d)
adenocarcinoma
adenoma
(mg/kg-d)
ma
adenoma
0
0/50
1/50
0
0/50
0/50
28
1/50
0/50
46
1/50
0/50
121
0/50
0/50
171
0/50
0/50
542
0/50
0/50
560
0/50
0/50
\/lale
Female
Thyroid
Thyroid
follicular
Thyroid
Dose
Thyroid follicular
follicular
Dose
adenocarcino
follicular
(mg/m3)
adenocarcinoma
adenoma
(mg/m3)
ma
adenoma
0
0/50
1/50
0
1/50
0/50
2,090
0/50
0/50
2,090
1/50
0/50
6,270
0/50
1/50
6,270
1/50
0/50
20,900
0/50
2/50
20,900
0/50
0/50
JPEC (2010b);Saito et al. (2013)
rat, Fischer 344
inhalation - vapor
male (50/group): 0, 500,1,500,
5,000 ppm (0, 2,090, 6,270,
20,900 mg/m3)b; female
(50/group): 0, 500,1,500,
5,000 ppm (0, 2090, 6270,
20,900 mg/m3)b
dynamic whole body
inhalation; 6 hr/d, 5 d/wk for
104 wk; generation method,
analytical concentration, and
method reported
Maltoni et al. (1999)
rat, Sprague-Dawley
oral -gavage
male (60/group): 0, 250,
1,000 mg/kg-d; female
(60/group): 0, 250,
1,000 mg/kg-d
4 d/wk for 104 wk; observed
until natural death
NOTE: Tumor data not
reanalyzed by Malarkey and
Bucher (2011).
Male
Female
Dose
Dose
(mg/kg-d)
Thyroid adenocarcinoma
(mg/kg-d)
Thyroid adenocarcinoma
0
0/60
0
0/60
250
0/60
250
0/60
1,000
0/60
1,000
1/60
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Reference and study design
Resu Its
Endometrial/Uterine carcinogenic effects
JPEC (2010a);Suzuki et al.
(2012)
Female
rat, Fischer 344
Dose
Endometrial
Uterine
Uterine
female (50/group): 0, 625,
(mg/kg-d)
stromal sarcoma adenocarcinoma
fibroma
2,500, 10,000 ppm (0, 46, 171,
0
6/50
1/50
1/50
560 mg/kg-d)a
46
9/50
0/50
0/50
daily for 104 wk
171
3/50
2/50
0/50
560
7/50
2/50
0/50
JPEC (2010b);Saito et al. (2013)
Female
rat, Fischer 344
Dose
Endometrial
Uterine
inhalation - vapor
(mg/m3)
stromal sarcoma
adenocarcinoma
female (50/group): 0, 500,
2/50
2/50
0
1,500, 5,000 ppm (0, 2,090,
6,270, 20,900 mg/m3)b
2,090
2/50
3/50
dynamic whole body
6,270
3/50
1/50
inhalation; 6 hr/d, 5 d/wk for
20,900
2/50
4/50
104 wk; generation method,
analytical concentration, and
method reported
Malarkev and Bucher (2011);
Female
Maltoni et al. (1999)
Carcinoma of
Schwannoma
rat, Sprague-Dawley
Dose
the uterus/ Uterine Uterine
of the
Uterine
oral -gavage
(mg/kg-d)
vagina leiomvoma leiomvosarcoma
uterus/vagina
carcinoma
female (60/group): 0, 250,
0/60
0/60 1/60
0/60
0/60
1,000 mg/kg-d
0
reanalvsis of data from Maltoni
250
1/60
0/60 0/60
7/60
1/60
et al. (1999) for which animals
1,000
0/60
3/60 0/60
2/60
0/60
were dosed 4 d/wk for 104 wk
1
2 Conversion performed by study authors.
3 b4.18 mg/m3 = 1 ppm.
4 ^Statistically significant (p < 0.05) based on analysis of data conducted by study authors.
5
6 Table 1-18. Supplemental Evidence pertaining to ETBE promotion of mutagen-
7 initiated tumors in animals
Reference and Dosing Protocol
Results by Endpoint
Colon Adenoma or Carcinoma
Hagiwara et al. (2011); JPEC (2008d)
Dose (mg/kg-d)
Response
rat, Fischer 344
(incidence)
oral - gavage
Male 0
25/30
male (30/group): 0, 300,1,000 mg/kg-d
300
21/30
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Reference and Dosing Protocol
Results by Endpoint
daily for 23 wk following a 4-wk tumor initiation by
DMBDDa
+no DMBDD initiation
1,000 28/30*
0+ 0/12
1,000+ 0/12
Forestomach Papillomas or Hyperplasia
Hagiwara et al. (2011); JPEC (2008d)
rat, Fischer 344
oral - gavage
male (30/group): 0, 300,1,000 mg/kg-d
daily for 23 wk following a 4-wk tumor initiation by
DMBDD3
+no DMBDD initiation
Dose (mg/kg-d) Response
(incidence)
Male 0 0/30
300 6/30*
1,000 6/30*
0+ 0/12
1,000+ 0/12
Thyroid Gland Adenoma or Carcinoma
Hagiwara et al. (2011); JPEC (2008d)
rat, Fischer 344
oral - gavage
male (30/group): 0, 300,1,000 mg/kg-d
daily for 23 wk following a 4-wk tumor initiation by
DMBDD3
+no DMBDD initiation
Dose (mg/kg-d) Response
(incidence)
Male 0 8/30
300 17/30*
1,000 20/30*
0+ 0/12
1,000+ 0/12
Urinary Bladder Carcinoma
Hagiwara et al. (2013)
rat, F344/DuCrlCrlj
oral - water
male (30/group): 0,100, 300, 500,1,000 mg/kg-d
daily for 31 wk beginning 1 wk after a 4-wk
exposure to BBN
Dose (mg/kg-d) Response
(incidence)
Male 0 5/30
100 7/30
300 6/30
500 14/30*
1,000 9/26
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Reference and Dosing Protocol
Results by Endpoint
Urinary Bladder Papilloma
Hagiwara et al. (2013)
rat, F344/DuCrlCrlj
oral - water
male (30/group): 0,100, 300, 500,1,000 mg/kg-d
daily for 31 wk beginning 1 wk after a 4-wk
exposure to N-butyl-N-(4-hydroxybutyl) (BBN)
Response
Dose (mg/kg-d) (incidence)
Male 0 21/30
100 13/30
300 17/30
500 17/30
1,000 21/26
Urinary Bladder Papilloma or Carcinoma
Hagiwara et al. (2013)
rat, F344/DuCrlCrlj
oral - water
male (30/group): 0,100, 300, 500,1,000 mg/kg-d
daily for 31 wk beginning 1 wk after a 4-wk
exposure to N-butyl-N-(4-hydroxybutyl) (BBN)
Response
Dose (mg/kg-d) (incidence)
Male 0 24/30
100 18/30
300 20/30
500 25/30
1,000 21/26
Urinary Bladder Papillomatosis
Hagiwara et al. (2011); JPEC (2008d)
rat, F344
oral - gavage
male (12/group): 0,1,000 mg/kg-d
daily for 23 wk following a 4-wk tumor initiation by
DMBDDa
+no DMBDD initiation
Response
Dose (mg/kg-d) (incidence)
Male 0 0/30
300 0/30
1,000 10/30*
0+ 0/12
1,000+ 2/12
1 aDiethylnitrosamine (DEN), N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), N-methyl-N-nitrosourea (MNU), 1,2-
2 dimethylhydrazine dihydrochloride (DMH), and N-bis(2-hydroxypropyl)nitrosamine (DHPN).
3
4
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¦ = exposures at which the endpoint was reported statistically significant by study authors
~ = exposures at which the endpoint was reported not statistically significant by study authors
Male rat oral cavity;104wks (C)
Female rat uterine malignancies;'!04wks (C)
Male rat all tissues;'104wks (D)
Female rat all tissues;!. 04wlcs (D)
Male rat all tlssues;23wks without DMBDD initiation (A)
Male rat colon;23wks following 4wk initiation with DMBDD
CA)
Male rat forestomach or hyperplasla;23wks following 4wk
initiation with DMBDD (A)
Male rat thyroid;23wks following 4wk initiation wi th
DMBDD (A)'
Male rat urinary bladder carcinoma;31wks following 4wk
initiation with BBN [B]
Male rat urinary bladder papilloma;31wks following 4wk
initiation with BBN (B)
Male rat: urinary bladder papillamatosis;23wks following
4wk initiation with DMBDD (A)
~ -a
I3_ g
O——tB— 0
Q b Q
~
~ B—¦ Q
Q B—B El
O-
10 100 1,000
Dose (mg/kg-day)
10,000
Sources: (A) Hagiwara etal, 2011; JPEC 2008d (B) Hagiwara et al, 2013 (C) Malarkey and Bucher, 2011
(reanalysis of Maltoni et al, 1999) Maltoni et al, 1999; (D) Suzuki et al, 2012; JPEC, 2010a
Figure 1-16. Exposure-response array of carcinogenic effects following oral
exposure to ETBE.
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¦ = exposures at which the endpoint was reported statistically significant by study authors
~ =exposures at which the endpoint was reported not statistically significant by study authors
Female rat; thyroid adenoma/adenocarcinoma;
104wks (A)
Male rat; thyroid adenoma/adenocarcinoma;
104wks (A)
Female rat; uterine malignancies; 104wks (A)
100 1,000 10,000 100,000
Exposure Concentration (mg/m')
Source: (A) Saito etal, 2013; JPEC, 2010b
1 Figure 1-17. Exposure-response array of carcinogenic effects following
2 inhalation exposure to ETBE.
3
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1.2.6. Other Toxicological Effects
The evidence base for other effects includes 11 rodent studies, some of which reported
decreased body weight, increased adrenal weights, altered spleen weights, and increased mortality.
The available subchronic or chronic studies used inhalation, oral gavage, or drinking water routes
of exposure for 90 days or more. Shorter-duration, multiple-exposure studies that examined
immunological endpoints also were included. The design, conduct, and reporting of each study
were reviewed, and each study was considered adequate.
At this time, the available evidence is considered inadequate to draw conclusions regarding
these other toxic effects following ETBE exposure. For more information, see Appendix B.3.
1.3. INTEGRATION AND EVALUATION
1.3.1. Effects Other Than Cancer
Kidney effects were identified as a potential human hazard of ETBE exposure based on
several affected endpoints in male and female rats, including kidney weight increases, urothelial
hyperplasia (in male rats only), and—to a lesser extent—exacerbated severity of CPN, and
increases in serum markers of kidney function such as cholesterol, BUN, and creatinine. These
effects are similar to the kidney effects observed with the ETBE metabolite tert-butanol (e.g., CPN
and transitional epithelial hyperplasia) and a related compound, MTBE (e.g., CPN and
mineralization) (ATSDR. 1996). Changes in kidney parameters were consistently observed but the
magnitude of change was generally moderate, while males had greater severity of effects compared
to females. While the ETBE metabolite tert-butanol meets the criteria for a2U-globulin nephropathy
(https://cfpub.epa.gov/ncea/iris_drafts/recordisplay.cfm?deid=262086), ETBE binds to ct2u-
globulin and meets some but not all the criteria in the EPA and IARC a2U-globulin frameworks {U.S.
EPA, 1991, 635839}{Capen, 1999, 699905}, see Section 1.2.1. {U.S. EPA, 1991, 635839@@author-
year} notes that "[i]f a compound induces a2U-globulin accumulation in hyaline droplets, the
associated nephropathy in male rats is not an appropriate endpointto determine noncancer
(systemic) effects potentially occuring in humans" (Section XVIII, p. 89). However, as a2u-globulin
nephropathy is strictly a male rat phenomenon, dose-related kidney effects in female rats are not
confounded by a2U-globulin nephropathy.
It has been observed that chemicals that bind to a2U-globulin also exacerbate the incidence
and/or severity of background chronic progressive nephropathy (CPN) in male rats {Travlos, 2011,
1239901}{U.S. EPA, 1991, 635839}{Frazier, 2012, 2919046}. CPN has no known analog in the aging
human kidney {Hard, 2009, 667590}{NIEHS, 2019, 5098230} and the etiology is unknown {Hard,
2004, 782757}{NIEHS, 2019, 5098230}{Peter, 1986,194755}{Frazier, 2012, 2919046}. However,
many of the same lesions observed in CPN (e.g. thickening of tubule basement membranes, tubule
atrophy, tubule dilation, and glomerular sclerosis) are also observed in chronic kidney disease in
humans {Lusco, 2016, 5926047}{Frazier, 2012, 2919046}{Zoja, 2015, 5926046}{Abrass, 2000,
5426141}. Because the mode of action is unknown, it cannot be ruled out that a chemical which
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exacerbates CPN in rats could also exacerbate existing disease processes in the human kidney
{NIEHS, 2019, 5098230}. Therefore, increased incidence of kidney effects with ETBE exposure,
particularly in the female rat (including increased kidney weight and increased severity of CPN) are
considered relevant to humans and were carried forward for dose-response analysis. Kidney
effects in male rats were also modeled and presented for comparison.
Evidence is suggestive that liver effects are associated with ETBE exposure. Increased liver
weight in male and female rats was consistently observed across studies. Centrilobular
hypertrophy was observed at the same concentrations that induced liver weight changes in rats of
both sexes after 13-week inhalation and 26-week oral exposures. No other histopathological
findings were observed, and only one serum marker of liver toxicity (GGT) was elevated, although
other markers (AST, ALT, and ALP) were not The magnitude of change for these noncancer liver
effects was considered modest and, except for organ weight data, did not exhibit consistent dose-
response relationships. Mechanistic data suggest ETBE exposure leads to activation of several
nuclear receptors, but evidence that nuclear receptor-mediated pathways contribute to the
tumorigenesis observed in ETBE-treated males is inadequate, thus these data remain relevant for
human noncancer hazard identification. Due to the lack of data supporting the adversity of the liver
weight increases with ETBE exposure (e.g. alterations in histology or clinical chemistry), liver
effects were not considered further for dose-response analysis and the derivation of reference
values.
At this time, there is insufficient information to draw conclusions regarding male
reproductive effects, female reproductive effects, developmental effects, or other toxic effects as
human hazards of ETBE exposure.
1.3.2. Carcinogenicity
Summary of Evidence
In F344 rats, administration of ETBE via inhalation increased hepatocellular adenomas in
males in an exposure-dependent manner, as indicated by a significant positive trend (p <0.001
with Peto's test). Hepatocellular tumors were not increased in female rats (Saito etal.. 2013). A
significantly increased incidence of hepatocellular adenomas or carcinomas (only one carcinoma
observed) was observed at the highest dose tested in males, and three hepatocellular adenomas
were observed at the two lower concentrations. Significant increases in preneoplastic foci
(basophilic and eosinophilic foci) were also observed in male rats(Saito etal.. 2013). Following 2
year gavage or drinking water exposure, liver tumors were not increased in Sprague-Dawley or
F344 rats of either sex (Suzuki etal.. 2012: Maltoni etal.. 1999). although an apparent, but non-
statistically significant increase in preneoplastic foci (basophilic) was observed in F344 male rats
f Suzuki etal.. 20121. Regarding the 2 year oral gavage study by {Maltoni, 1999, 87642@@author-
year}, depressed survival (25-28% of male and female control rats survived to week 104) may have
confounded the ability to detect carcinogenicity.
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{Saito, 2013, 2321101@@author-year} compared the doses achieved in the 2 year drinking
water bioassay compared with the 2 year inhalation bioassay {Suzuki, 2012,1433129}{JPEC, 2010,
1517477} and calculated the highest intake in male rats to be 542 mg/kg-day as compared to the
highest inhalation intake estimate of 3015 mg/kg-day (after adjusting for 6 hour/day, 5 day a week
exposure, minute volume of 561 ml/min, and 100% ETBE absorption). However, the ranges of the
internal dose metrics of ETBE expressed as either metabolized ETBE or metabolized t-butanol (one
of the two primary breakdown products of ETBE) as computed by toxicokinetic modeling were
similar for the oral and inhalation exposures of ETBE and the oral exposure of t-butanol in these
three studies (see the range of the internal dose variables in Figure B-3 of Supplemental
Information). Nonetheless, the incidence of liver tumors was not consistently correlated with any
internal dose measure and the lack of a consistent dose-response relationship using any of these
internal metrics suggest that differences in liver tumor responses between oral and inhalation
exposures is not likely due to pharmacokinetic factors alone. Statistically significant increases in
liver tumor incidence, were observed in the livers of male F344 and Wistar rats in supplemental
initiation-promotion studies, after 19-23 weeks of ETBE exposure via oral gavage, only following an
initial 2-4 week mutagen exposure fHagiwara etal.. 2015: Hagiwara etal.. 20111: along with
increases in, colon, thyroid, forestomach, and urinary bladder tumorigenesis in male F344 rats
fHagiwara etal.. 2013: Hagiwara etal.. 20111. No studies have evaluated chronic ETBE exposure in
mice via any route.
The EPA Cancer Guidelines (U.S. EPA. 2005a) emphasize that knowledge of the biochemical
and biological changes preceding tumor development could inform whether a cancer hazard exists
and might help in understanding events relevant to potential mode of carcinogenic action.
However, as discussed in Section 1.2.2, an MOA for liver carcinogenesis could not be established,
and in the absence of information to indicate otherwise fU.S. EPA. 2005bl. the liver tumors induced
by ETBE following inhalation exposure are considered relevant to human hazard identification.
The EPA Cancer Guidelines (2005) indicate that information on metabolites can help inform
the weight of evidence for carcinogenicity. ETBE is primarily metabolized into acetaldehyde and
tert-butanol. Regarding the ETBE metabolite tert-butanol, drinking water exposure in F344 rats did
not cause an increase in liver tumors, but resulted in renal tubule tumors, mostly adenomas, in
males; drinking water exposure also increased the incidence of thyroid follicular cell adenomas in
female B6C3Fi mice and adenomas or carcinomas in males {NTP, 1995, 91022}. Regarding the
ETBE metabolite acetaldehyde, inhalation exposures to acetaldehyde were concluded to cause
carcinomas of the nasal mucosa in rats and carcinomas of the larynx in hamsters (IARC. 1999b).
IARC classifies acetaldehyde as possibly carcinogenic to humans (Group 2B) based on sufficient
evidence in experimental animals for the carcinogenicity of acetaldehyde. In addition, acetaldehyde
associated with the consumption of alcoholic beverages is considered by IARC to be carcinogenic to
humans (Group 1) flARC. 1999al and was concluded to be the key metabolite in cancer of the
esophagus and aerodigestive tract flARC. 20101. Acetaldehyde produced in the liver as a result of
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ethanol metabolism has been suggested to be a contributor to liver toxicity and cancer (Setshedi et
al.. 20101.
Integration of evidence
The descriptor suggestive evidence of carcinogenic potential is appropriate when the
evidence raises "a concern for potential carcinogenic effects in humans" but is not sufficient for a
stronger conclusion and covers a spectrum of evidence associated with varying levels of concern for
carcinogenicity. Such evidence can range from a positive cancer result in the only study on an agent
to a single positive cancer result in an extensive database that includes negative studies in other
species. The results for ETBE raise a concern for cancer, however, the effects were limited to
tumors in one tissue (liver), primarily at the high dose, by one route of exposure (inhalation) in
male rats (females were negative and no ETBE bioassays are available in mice).
Regarding the animal database for carcinogenicity, EPA considers chronic bioassays as key
evidence (i.e., the three chronic cancer bioassays, one inhalation and two oral), and other types of
studies, including initiation promotion studies, as supplemental lines of information which can aid
in the interpretation of more standard toxicological evidence, especially regarding potential modes
of action {U.S. EPA, 2005, 86237}. Across three initiation-promotion studies, orally administered
ETBE enhanced tumorigenesis in multiple tissues in male rats pre-exposed to mutagens, including
kidney, liver, forestomach, thyroid, colon, and urinary bladder. Furthermore, no MOA has been
identified for ETBE which could explain the potentiation of mutagen-induced carcinogenesis in the
forestomach, thyroid, colon, and urinary bladder. This suggests that the available database is
limited with regard to informing molecular mechanisms of ETBE carcinogenesis. The available
evidence suggests that populations exposed to mutagenic agents prior to, or concomitant with, oral
ETBE exposure might be more susceptible to chemically induced carcinogenesis than predicted by
the results of ETBE 2-year rodent oral bioassays alone.
The carcinogenicity of ETBE appears to be route dependent, therefore multiple cancer
descriptors are used in accordance with the Cancer Guidelines {U.S. EPA, 2005, 86237}. The
evidence of carcinogenic potential for ETBE is determined to be suggestive for exposure via the
inhalation route and inadequate for exposure via the oral route. These weight of evidence
descriptors are based primarily on a positive carcinogenic response following inhalation exposure
in the liver in a single animal study, along with significant increases in pre-neoplastic liver lesions
and mechanistic data (i.e. the metabolism of ETBE to the genotoxic compound acetaldehyde in the
liver) for the inhalation route and no increased liver tumors detected in two chronic oral studies in
Sprague-Dawley or F344 rats for the oral route {Maltoni, 1999, 87642}{Suzuki, 2012,
1433129}{JPEC, 2010, 1517477}.
Biological considerations for dose-response analysis
This section addresses the cancer hazards to bring forward to Section 2 for dose-response
analysis. The observed liver tumors in male rats following inhalation exposure are deemed
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relevant for estimating human cancer hazard. The Saito etal. (20131 inhalation study observed a
positive exposure-response trend in the incidence of hepatocellular tumors in male rats (although
the majority of tumors were observed at the highest dose). This study was considered suitable for
dose-response analysis, as it is part of a well-designed study that evaluated multiple dose levels
flPEC. 2010bl. The study included histological examinations for tumors in many different tissues,
contained three exposure levels and controls, contained adequate numbers of animals per dose
group (~50/sex/group), treated animals for up to 2 years, and included detailed reporting of
methods and results. Although decreased body weight gain and survival was noted in the high dose
males and females, the study authors did not detect changes to the animals' general condition (e.g.
abnormal behavior or clinical signs) associated with ETBE. Similar decreases in body weight were
observed in male (75% of control) and female animals (78% of control), although significantly
increased liver tumors were only observed in male rats. Some concern exists that decreased
survival in ETBE treated dose groups (primarily from CPN in the male rats) could bias the cancer
findings toward the null. This study was deemed appropriate for dose-response as there is no clear
indication that overt toxicity or altered toxicokinetics {as discussed in \U.S. EPA, 2005, 86237} are
responsible for the significantly increased incidence of liver tumors in male rats.
The results from MOA analysis can potentially inform dose-response analysis and
extrapolation approaches fU.S. EPA. 2005al. however, for ETBE, no clear MOA was identified. As
discussed above, the evidence was inadequate to determine the role of nuclear receptor activation
in liver carcinogenesis, due in part to a lack of coherence between nuclear receptor activation and
proliferation or apoptosis, key events in these pathways. Evidence also was inadequate to conclude
that ETBE induces liver tumors via acetaldehyde-mediated mutagenic MOA, due in part to a paucity
of evidence specifically evaluating intermediate key events following ETBE exposure in rats. No
other systemic cancer MOAs were identified. In the absence of MOA information to indicate
otherwise, dose-response analysis should use linear extrapolation fU.S. EPA. 2005al.
1.3.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes
Genetic polymorphisms of ALDH2, the enzyme that oxidizes acetaldehyde to acetic acid,
might affect potential ETBE liver toxicity. The virtually inactive form, ALDH2*2, is responsible for
alcohol intolerance and is found in about one-half of East Asian populations fBrennan. 20021. This
variant is associated with slow metabolism of acetaldehyde and, hence, extended exposure to a
genotoxic compound. Other studies also have linked ALDH2 polymorphisms to hepatocellular
cancers in humans (Eriksson. 2015). With respect to ETBE exposure, the ALDH2*2 variant should
increase any type of risk associated with acetaldehyde produced by ETBE metabolism because it
will prolong internal exposure to this metabolite. As demonstrated in several in vivo and in vitro
genotoxic assays in AIdh2 KO mice or cells, genotoxicity was significantly increased compared with
wild-type controls following ETBE exposure to similar doses where both cancer and noncancer
effects were observed following chronic rodent exposure bioassays {Weng, 2011,1062385;Weng,
2012, 1248016;Weng, 2013, 2279880;Weng, 2014, 2321096;Weng, 2019, 5343910}. Studies in
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AIdh2 KO mice observed elevated blood concentrations of acetaldehyde following ETBE exposure
compared with wild-type mice fWeng etal.. 20131. increased alterations to sperm and male
reproductive tissue fWeng etal.. 20141. and increased incidence of centrilobular hypertrophy
fWeng etal.. 2013: Weng etal.. 20121. Similar effects on genotoxicity and liver histopathology have
also been observed in AIdh2 heterozygous animals {Weng, 2019, 5343910}. Notably, a consistent
finding in these studies was increased severity of genotoxicity in males compared with females,
which corresponds with increased incidence of hepatic tumors only in male rats (Saito etal.. 2013:
TPEC. 2010bl.
No MOA information exists to account for the sex discrepancies in genotoxic effects. Finally,
IARC f!999al and IARC T20121 identified acetaldehyde produced as a result of ethanol metabolism
as contributing to human carcinogenesis in the upper aerodigestive tract and esophagus following
ethanol ingestion, with effects amplified by slower acetaldehyde metabolism. Altogether, these data
present plausible evidence that diminished ALDH2 activity yields health effect outcomes that are
more severe than those organisms with fully functional ALDH2. It is also plausible that
individuals with non-coding region variants in ALDH2 (which could potentially affect gene
expression), as well as individuals with other variants in alcohol metabolism may also be
disproportionately impacted by ETBE exposure. No other specific potential polymorphic-related
susceptibility issues were reported in the literature. CYP2A6 is likely to be the P450 isoenzyme in
humans to cleave the ether bond in ETBE. It also exists in an array of variants, and at least one
variant (2A6*4) clearly has no catalytic activity fFukami et al.. 20041: however, the effect of this
variability on ETBE toxicity is unknown. In addition, the data on ETBE-induced mutagenicity are
inconclusive.
Regarding lifestages particularly susceptible to ETBE exposure, while certain lifestages (e.g.
development) are generally thought to have heightened vulnerability to most chemical exposures,
no specific data was identified to support heightened periods of susceptibility to ETBE exposure in
the available database of developmental and reproductive studies. It should be noted that the
majority of developmental and reproductive studies were performed by the oral route, so
differential susceptibility from inhalation exposure cannot be ruled out, however, in general, the
effects observed from ETBE exposure (except for liver tumors) appeared to be consistent across
routes of exposure (e.g. kidney effects, liver hypertrophy).
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2. DOSE-RESPONSE ANALYSIS
2.1. ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER
The reference dose (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. It can be derived from a no-observed-adverse-effect level (NOAEL), lowest-
observed-adverse-effect level (LOAEL), or the 95% lower bound on the benchmark dose (BMDL),
with uncertainty factors (UF values) generally applied to reflect limitations of the data used.
2.1.1. Identification of Studies and Effects for Dose-Response Analysis
Studies were evaluated using general study quality characteristics as discussed in
Section 1.1.1; see also U.S. EPA f20021 to help inform the selection of studies from which to derive
toxicity values.
Human studies are preferred over animal studies when quantitative measures of exposure
are reported and the reported effects are determined to be associated with exposure. No human
occupational or epidemiological studies of oral exposure to ETBE, however, are available.
Animal studies were evaluated to determine which studies provided (1) the most relevant
routes and durations of exposure, (2) multiple exposure levels that informed the shape of the dose-
response curve, and (3) sufficient sample size to detect effects at low exposure levels fU.S. EPA.
2002). The database for ETBE includes several chronic and subchronic studies, mostly in rats,
showing effects in the kidney that are suitable for use in deriving oral reference values. In general,
lifetime exposures are preferred over subchronic exposures.
Kidney Toxicity
Kidney effects were identified as a potential human hazard of ETBE-induced toxicity based
on findings in male and female rats (summarized in Section 1.3.1). Kidney toxicity was observed
across several chronic and subchronic studies following oral and inhalation exposure, based on
findings of organ weight changes, histopathology (urothelial hyperplasia in males), and altered
serum biomarkers (cholesterol, creatinine, BUN) in rats. The strongest and most consistent findings
across oral exposure routes and durations were for absolute kidney weight changes and urothelial
hyperplasia in male rats; thus, only these endpoints were analyzed for dose-response. Kidney
effects observed after chronic exposure, such as urothelial hyperplasia, could affect the ability of the
kidney to filter waste, and changes in kidney weight could serve as a general indication of renal
toxicity. In the case of kidney weight changes, numerous chronic and subchronic studies
investigated this endpoint following oral and inhalation exposure (Mivata etal.. 2013: Saito etal..
2013: Suzuki etal.. 2012: Hagiwara etal.. 2011: Fuiii etal.. 2010: TPEC. 2010b. 2008b. c; Gaoua.
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2004b: Medinskv etal.. 19991. Chronic studies of oral exposure reported urothelial hyperplasia to
be increased with treatment in male rats fSaito etal.. 2013: Suzuki etal.. 2012: TPEC. 2010a. b).
Hagiwara et al. f20111. with only one dose group, was not considered further given its
concordance with several other rat studies that had multiple groups. Additionally, as discussed in
Section 1.1.1, 2-year organ weight data in male rats was not considered suitable due to the
prevalence of age-associated confounders (e.g., CPN-related early deaths in male rats). Therefore,
the urothelial hyperplasia data (observed in male rats only) and absolute kidney weight (female
rats, only) were the only endpoints from the 2-year studies [TPEC. 2010a: with selected data
published as Suzuki etal. f20121], and absolute kidney weight was the only endpoint from the 13-
to 26-week studies that were considered for dose-response analysis. These data and the absolute
kidney weights from the remaining studies, TPEC [2008c: selected data published as Mivata et al.
(2013)]. Gaoua (2004b). Fuiii etal. (2010). are discussed further below.
In the 2-year drinking water study (Suzuki etal.. 2012: TPEC. 2010a). male and female F344
rats (50/sex/dose group) were exposed to doses of 0, 28,121, or 542 mg/kg-day. Increased
incidence of urothelial hyperplasia was observed only in males and significantly increased at 121
and 542 mg/kg-day.
In the TPEC f2008cl 26-week gavage study, male and female Crl:CD(SD) rats (15/sex/dose
group) were exposed to daily doses of 0, 5, 25,100, or 400 mg/kg-day. Absolute kidney weight was
significantly increased in males and females treated with 400 mg/kg-day. Abnormal
histopathological findings in the kidney (basophilic tubules and hyaline droplets) were observed in
male rats, but not in female rats.
In the Gaoua f2004bl two-generation reproductive toxicity study, Sprague-Dawley rats
(25/sex/dose group) were exposed via gavage to doses of 0, 250, 500, or 1,000 mg/kg-day;
treatment commenced 10 weeks before mating and continued throughout the 2-week mating
period, gestation, and the end of lactation (PND 21) for 18 weeks. Absolute kidney weights were
significantly increased in all dose groups in P0 males, but not in P0 females, which was associated
with the presence of acidophilic globules in renal tissue from 5/6 males examined. In addition,
tubular basophilia (4/6), peritubular fibrosis (3/6), and proteinaceous casts (1/6) were observed
in kidneys of male rats at the high dose. Similar microscopic effects in females were not observed,
thus P0 female kidney weights were not modeled. Absolute kidney weights were also increased in
F1 animals, however, F1 animals appeared to be less impacted than P0 animals.
In the Fujii etal. (2010) one-generation reproductive toxicity study, male and female
Crl:CD(SD) rats (24/sex/dose group) were exposed via gavage to doses of 0,100, 300, or
1,000 mg/kg-day beginning 10 weeks prior to F0 mating and continuing throughout the
reproductive period (mating, gestation, lactation). Treatment durations were stated to be
approximately 16 weeks for males and 17 weeks for females but ranged up to 20 weeks in animals
that took longer to mate. Kidney weights were significantly increased in F0 males and females at
1,000 mg/kg-day.
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2.1.2. Methods of Analysis
No biologically based dose-response models are available for ETBE. In this situation, a range
of dose-response models was evaluated to determine how best to model the dose-response
relationship empirically in the range of the observed data. The models in EPA's Benchmark Dose
Software (BMDS) were applied. Consistent with EPA's Benchmark Dose Technical Guidance
Document (U.S. EPA. 20121. the BMD and the BMDL are estimated using a benchmark response
(BMR) to represent a minimal, biologically significant level of change. In the absence of information
regarding what level of change is considered biologically significant, a BMR of 10% change from the
control mean (relative deviation; RD) for kidney weight and a BMR of 10% extra risk on incidences
of urothelial hyperplasia data were used to estimate the BMD and BMDL and to facilitate a
consistent basis of comparison across endpoints, studies, and assessments. When modeling was
feasible, the estimated BMDLs were used as points of departure (PODs); the PODs are summarized
in Table 2-1. Details, including the modeling output and graphical results for the model selected for
each endpoint are presented in Appendix C of the Supplemental Information to this Toxicological
Review.
Human equivalent doses (HEDs) for oral exposures were derived from the PODs according
to the hierarchy of approaches outlined in EPA's Recommended Use of Body Weight3/4 as the Default
Method in Derivation of the Oral Reference Dose (U.S. EPA. 2011). The preferred approach is
physiologically based pharmacokinetic (PBPK) modeling. Other approaches include using chemical-
specific information in the absence of a complete PBPK model. As discussed in Appendix B of the
Supplemental Information, several rat PBPK models for ETBE have been developed and published,
but a validated human PBPK model for ETBE for extrapolating doses from animals to humans is not
available. In lieu of chemical-specific models or data to inform the derivation of human equivalent
oral exposures, body-weight scaling to the % power (BW3/4) is applied to extrapolate toxicologically
equivalent doses of orally administered agents from adult laboratory animals to adult humans to
derive an oral RfD. BW3/4 scaling was not used for deriving HEDs from studies in which doses were
administered directly to early postnatal animals because of the absence of information on whether
allometric (i.e., body weight) scaling holds when extrapolating doses from neonatal animals to adult
humans due to presumed toxicokinetic or toxicodynamic differences between lifestages fU.S. EPA.
2011: Hattis etal.. 2004).
Consistent with EPA guidance (U.S. EPA. 2011). the PODs estimated based on effects in adult
animals are converted to HEDs using a standard dosimetric adjustment factor (DAF) derived as
follows:
DAF = (BWa1/4 / BWh1/4)
where:
BWa = animal body weight
BWh = human body weight
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1 Using a standard BWa of 0.25 kg for rats and a BWh of 70 kg for humans fU.S. EPA. 19881.
2 the resulting DAF for rats is 0.24. Applying the DAF to the POD identified for effects in adult rats
3 yields a PODhed as follows (see Table 2-1):
4
5 PODhed = Duration-adjusted laboratory animal dose (mg/kg-day) x DAF
6
7 Table 2-1 summarizes the sequence of calculations leading to the derivation of a human-
8 equivalent POD for each data set discussed above.
9 Table 2-1. Summary of derivation of points of departure following oral
10 exposure for up to 2 years
Endpoint and Reference
Species/
Sex
Model3
BMR
BMD
(mg/kg-d)
BMDL
(mg/kg-d)
PODadj15
(mg/kg-d)
PODhed0
(mg/kg-d)
Kidney
Increased urothelial
hyperplasia; 2-year
Suzuki etal. (2012); JPEC
(2010a)
Male F344
rats
Quantal-
Linear
10%
ER
79.3
60.5
60.5
14.5
Increased absolute kidney
weight; 2-year
Suzuki etal. (2012); JPEC
(2010a)
Male F344
rats
NOAELde 121 mg/kg-d
5% 1" in kidney weight
121
29.0
Increased absolute kidney
weight; 2-year
Suzuki etal. (2012); JPEC
(2010a)
Female F344
rats
Exponential
(M4)
10%
RD
204
120
120
28.8
Increased absolute kidney
weight; 26-week
JPEC (2008c); Miyata et al.
(2013)
Male
Sprague-
Dawley rats
Linear
10%
RD
176
115
115
27.6
Increased absolute kidney
weight; 26-week
JPEC (2008c); Mivata et al.
(2013)
Female
Sprague-
Dawley rats
Exponential
(M4)
10%
RD
224
57
57
13.7
Increased absolute kidney
weight (P0 generation);
18-week
Gaoua (2004b)
Male
Sprague-
Dawley rats
Hill
10%
RD
244
94
94
22.6
Increased absolute kidney
weight (P0 generation); 16-
week
Fuiii et al. (2010)
Male
Sprague-
Dawley rats
Hill
10%
RD
435
139
139
33.4
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Endpoint and Reference
Species/
Sex
Model3
BMR
BMD
(mg/kg-d)
BMDL
(mg/kg-d)
PODadj15
(mg/kg-d)
PODhed0
(mg/kg-d)
Increased absolute kidney
weight (PO generation); 17-
week
Fuiii et al. (2010)
Female
Sprague-
Dawley rats
Polynomial
2°
10%
RD
1,094
905
905
217
1 aFor modeling details, see Appendix C of the Supplemental Information.
2 bFor studies in which animals were not dosed daily, administered doses were adjusted to calculate the TWA daily
3 doses prior to BMD modeling. This adjustment, however, was not required for the studies evaluated.
4 CHED PODs were calculated using BW3/4scaling (U.S. EPA, 2011).
5 ER = extra risk, RD = relative deviation.
6 dNOAEL was used due to lack of suitable model fit (see Appendix C).
7 e 18% increase in kidney weight at LOAEL
8 2.1.3. Derivation of Candidate Values
9 Consistent with EPA's A Review of the Reference Dose and Reference Concentration Processes
10 fU.S. EPA. 2002: Section 4.4.51. five possible areas of uncertainty and variability were considered
11 when determining the application of UF values to the PODs presented in Table 2-1. An explanation
12 follows.
13 An intraspecies uncertainty factor, UFH, of 10 was applied to all PODs to account for
14 potential differences in toxicokinetics and toxicodynamics in the absence of information on the
15 variability of response in the human population following oral exposure to ETBE fU.S. EPA. 20021.
16 An interspecies uncertainty factor, UFa, of 3 (10°5 = 3.16, rounded to 3) was applied to PODs
17 that used BW3/4 scaling to extrapolate oral doses from laboratory animals to humans. Although
18 BW3/4 scaling addresses some aspects of cross-species extrapolation of toxicokinetic and
19 toxicodynamic processes, some residual uncertainty remains. In the absence of chemical-specific
20 data to quantify this uncertainty, EPA's BW3/4 guidance (U.S. EPA. 20111 recommends using an
21 uncertainty factor of 3. For PODs that did not use BW3/4 such as early-life effects, an interspecies
22 uncertainty factor, UFa, of 10 was applied fU.S. EPA. 20111.
23 A subchronic-to-chronic uncertainty factor, UFs, differs depending on the exposure
24 duration. For studies of 16- to 2 6-week duration, the magnitude of change observed in kidney
25 weights was similar to the effect observed at 104 weeks. This suggests a maximum effect could
26 have been reached by 16-26 weeks. The 104-week kidney data, however, are confounded due to
27 age-associated factors, so this comparison might not be completely reliable. Additionally, some but
28 not all markers of kidney toxicity appear more severely affected by ETBE at 2 years compared with
29 observations at 16-26 weeks (e.g., histopathology, BUN) fSuzuki etal.. 2012: TPEC. 2010al. Thus, a
30 UFs of 3 was applied for studies of 16- to 26-week duration to account for this uncertainty, and a
31 UFs of 1 was applied to 2-year studies.
32 A LOAEL-to-NOAEL uncertainty factor, UFl, of 1 was applied to all PODs derived because the
33 current approach is to address this factor as one of the considerations in selecting a BMR for
34 benchmark dose modeling. In this case, BMRs of a 10% change in absolute kidney weight and a
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10% extra risk of urothelial hyperplasia were selected assuming that they represent minimal
biologically significant response levels.
A database uncertainty factor, UFd, of 1 was applied to all PODs. The ETBE oral toxicity data
set includes a 2-year toxicity study in rats (Suzuki etal.. 2012: IPEC. 2010a). a 26-week toxicity
study in rats (Mivata etal.. 2013). prenatal developmental toxicity studies in rats and rabbits (Aso
etal.. 2014: Asano etal.. 2011). and both single- and multigeneration reproductive studies and
developmental studies in rats {Fujii, 2010,1248027;Gaoua, 2004, 87678;Gaoua, 2004, 87676}. The
ETBE data set does not indicate immunotoxicity fBanton etal.. 2011: Li etal.. 20111. Additionally,
the available mouse study observed less severe effects than those in rats, suggesting that mice are
less sensitive than rats. Although most of the studies are in rats, the ETBE oral database adequately
covers all major systemic effects, including reproductive and developmental effects, and the
available evidence does not raise concern that additional studies would likely lead to identification
of a more sensitive endpoint or a lower POD. Furthermore, the effects observed in inhalation
studies support the noncancer effects observed in the oral studies. Therefore, an uncertainty factor
for the database, UFd, of 1 was applied.
Figure 2-1 graphically presents the candidate values, UFs, and PODhed values, with each bar
corresponding to one data set described in Table 2-1 and Table 2-2.
Table 2-2. Effects and corresponding derivation of candidate values
Endpoint and Reference
PODhed
(mg/kg-d)
POD
type
UFa
UFh
UFl
UFs
UFd
Composite
UF
Candidate
value
(mg/kg-d)
Kidney
Increased urothelial hyperplasia;
male rat; 2-year
Suzuki et al. (2012); JPEC (2010a)
14.5
BMDLio%
3
10
1
1
1
30
5 x 10 1
Increased absolute kidney weight;
male rat; 2-year
Suzuki et al. (2012); JPEC (2010a)
29.0
NOAEL
3
10
1
1
1
30
lx 10°
Increased absolute kidney weight;
female rat; 2-year
Suzuki et al. (2012); JPEC (2010a)
28.8
BMDLio%
3
10
1
1
1
30
lx 10°
Increased absolute kidney weight;
male rat; 26-week
JPEC (2008c); Mivata et al. (2013)
27.6
BMDLio%
3
10
1
3
1
100
3 x 10 1
Increased absolute kidney weight;
female rat; 26-week
JPEC (2008c); Mivata et al. (2013)
13.7
BMDLio%
3
10
1
3
1
100
1 x 10 1
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Endpoint and Reference
PODhed
(mg/kg-d)
POD
type
ufa
UFh
UFl
UFS
UFd
Composite
UF
Candidate
value
(mg/kg-d)
Increased absolute kidney weight;
PO male rat; 18-week
Gaoua(2004b)
22.6
BMDLio%
3
10
1
3
1
100
2 x 10 1
Increased absolute kidney weight;
male rat; 16-week
Fuiii et al. (2010)
33.4
BMDLio%
3
10
1
3
1
100
3 x 10 1
Increased absolute kidney weight;
female rat; 17-week
Fuiii et al. (2010)
217
BMDLio%
3
10
1
3
1
100
2x 10°
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Increased urothelial hyperplasia; male
rat; 2 year
Suzuki et al. (2012); JPEC (2010a]
Increased absolute kidney weight;
male rat; 2 year
Suzuki et al. (2012); JPEC (2010a)
Increased absolute kidney weight;
female rat; 2 year
Suzuki et al. (2012); JPEC (2010a)
Increased absolute kidney weight;
male rat; 26 week
JPEC (2008c)
Increased absolute kidney weight;
female rat; 26 week
JPEC (2008c)
Increased absolute kidney weight;
male rat; 18 week
Gaoua (2004b)
Increased absolute kidney weight;male
rat; 16 week
Fujii (2010)
Increased absolute kidney weight;
female rat; 17 week
Fujii (2010)
0.1 1 10 100 1000
mg/kg-day
~ Candidate RfD
® PODhb,
Composite l(F
Figure 2-1. Candidate values with corresponding POD and composite UF.
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2.1.4. Derivation of Organ/System-Specific Reference Doses
Table 2-3 distills the candidate values from Table 2-2 into a single value for each organ or
system. Organ- or system-specific RfDs are useful for subsequent cumulative risk assessments that
consider the combined effect of multiple agents acting at a common site.
Kidney Toxicity
For ETBE, candidate values were derived for increases in urothelial hyperplasia or absolute
kidney weight in male or female rats, spanning a range from 1 x 101 to 2 x 10° mg/kg-day, for an
overall 20-fold range. Selection of a point estimate considered multiple aspects, including study
design and consistency across estimates. As stated previously, reference values based on lifetime
exposure are preferred over subchronic exposures. The only candidate reference values based on
data from a 2-year oral study are those for increased absolute kidney weight in female rats and
urothelial hyperplasia in male rats (Saito etal.. 2013: Suzuki etal.. 2012: TPEC. 2010a. b). While
urothelial hyperplasia in male rats was the most sensitive candidate value, this endpoint was not
observed in female rats or mice of either sex whereas increased kidney weights were observed in
multiple studies in rats of both sexes and in mice. A lower candidate value based on increased
kidney weights in female rats was derived from a smaller (n=15), shorter duration study (26
weeks; JPEC 2008c), however, based on comparison of BMD values (204 vs 224 mg/kg-d) and
composite UFs (30 vs 100), this lower value reflects greater variability and uncertainty in the data.
The composite UF for the candidate value based on increased kidney weight in female rats treated
for 2 years was the lowest uncertainty, providing greater certainty in the selection of this candidate
value. In addition, the candidate values from female rats are not potentially confounded by ct2u-
globulin related processes.
Collectively, these observations indicate that the most appropriate basis for a kidney-
specific RfD would be the increased absolute kidney weight in female rats from the 2-year oral
study (Suzuki etal.. 2012: TPEC. 2010a). Therefore, the candidate value for increased absolute
kidney weight in female rats (1x10° mg/kg-day) was selected as the kidney-specific reference
dose for ETBE. Confidence in this RfD is high. The candidate value is derived from a well-conducted
GLP study, involving approximately 50 animals per group, assessing a wide range of kidney
endpoints. In addition, the POD is based on benchmark dose modeling, with the POD within the
range of tested doses (e.g., not requiring extrapolation well beyond the experimental range), and
the reference value is associated with less relative uncertainty (as illustrated by the smaller
composite UF). Furthermore, several additional studies demonstrate quantitatively similar PODs
and candidate values for kidney effects (see Table 2-2 and Figure 2-1).
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Table 2-3. Organ/system-specific RfDs and overall RfD for ETBE
Effect
Basis
RfD
(mg/kg-day)
Study exposure
description
Confidence
Kidney
Increased absolute kidney
weight in female rats Suzuki et
al. (2012); JPEC (2010a)
lx 10°
Chronic
High
Overall RfD
Kidney
1 X 10°
Chronic
High
2.1.5. Selection of the Overall Reference Dose
For ETBE, kidney effects were identified as the strongest hazard and carried forward for
dose-response analysis; thus, only one organ/system-specific reference dose was derived.
Therefore, the kidney-specific RfD of 1 x 10° mg/kg-day is the overall RfD for ETBE. This value is
based on increased absolute kidney weight in female rats exposed to ETBE.
The overall reference dose is derived to be protective of all types of effects for a given
duration of exposure and is intended to protect the population as a whole, including potentially
susceptible subgroups fU.S. EPA. 20021. Decisions concerning averaging exposures over time for
comparison with the RfD should consider the types of toxicological effects and specific lifestages of
concern. Fluctuations in exposure levels that result in elevated exposures during these lifestages
could lead to an appreciable risk, even if average levels over the full exposure duration were less
than or equal to the RfD. In the case of ETBE, no specific potential for early lifestage susceptibility to
ETBE exposure was identified, as discussed in Section 1.3.3.
2.1.6. Confidence Statement
A confidence level of high, medium, or low is assigned to the study used to derive the RfD,
the overall database, and the RfD, as described in Section 4.3.9.2 of EPA's Methods for Derivation of
Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA. 19941. The
overall confidence in this RfD is high. Confidence in the principal study (Suzuki etal.. 2012: TPEC.
2010a) is high. This study was well conducted, complied with OECD guidelines for GLP studies,
involved a sufficient number of animals per group (including both sexes), and assessed a wide
range of tissues and endpoints Confidence in the database is high. The available studies evaluated a
comprehensive array of endpoints, and the existing evidence base does not raise concerns that
additional studies would be likely to lead to the identification of a more sensitive endpoint.
Furthermore, multiple studies demonstrate quantitatively similar PODs and candidate values for
kidney effects (see Table 2-2 and Figure 2-1), providing additional support Reflecting high
confidence in the principal study and high confidence in the database, confidence in the RfD is high.
2.1.7. Previous IRIS Assessment
No previous oral assessment for ETBE is available in IRIS.
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2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER
THAN CANCER
The inhalation RfC (expressed in units of mg/m3) is defined as an estimate (with
uncertainty spanning perhaps an order of magnitude) of a continuous inhalation exposure to the
human population (including sensitive subgroups) that is likely to be without an appreciable risk of
deleterious effects during a lifetime. It can be derived from a NOAEL, LOAEL, or the 95% lower
bound on the benchmark concentration (BMCL), with UF values generally applied to reflect
limitations of the data used.
2.2.1. Identification of Studies and Effects for Dose-Response Analysis
Kidney effects were identified as a potential human hazard of ETBE exposure based on
studies in experimental animals (summarized in Section 1.3.1). These studies were evaluated using
general study quality characteristics as discussed in Section 6 of the Preamble and in Section 1.1.1;
see also U.S. EPA (2002) to help inform the selection of studies from which to derive toxicity values.
Rationale for selection of studies and effects representative of this hazard is summarized below.
Human studies are generally preferred over animal studies as the basis for reference values
when quantitative measures of exposure are reported and the reported effects are determined to
be associated with exposure. Data on the effects of inhaled ETBE in humans is limited to a small
number of 2-hour inhalation studies at doses up to 208.9 mg/m3 fNihlen etal.. 1998b: Vetrano.
1993). These studies were not considered for dose-response assessment because they are of acute
duration and investigated toxicokinetics.
The database for ETBE includes inhalation studies and data sets that are potentially suitable
for use in deriving inhalation reference values. Specifically, effects associated with ETBE exposure
in animals include observations of organ weight and histological changes in the kidney in chronic
and subchronic studies in male and female rats.
Kidney Toxicity
Evidence exists supporting kidney effects following ETBE exposure in rats, including organ
weight changes, histopathology (urothelial hyperplasia and exacerbation of CPN), and altered
serum biomarkers (creatinine, BUN, cholesterol). The most consistent, dose-related findings across
multiple studies, in both sexes were for kidney weight changes and increased CPN severity. In the
case of kidney weight changes, one chronic and numerous subchronic studies investigated this
endpoint following inhalation exposure f Suzuki etal.. 2012: Hagiwara etal.. 2011: Fuiii etal.. 2010:
TPEC. 2010b. 2008b. c; Gaoua. 2004b: Medinskv et al.. 1999). A 2-year study by inhalation (Saito et
al.. 2013: TPEC. 2010b) exposure reported increased urothelial hyperplasia in male rats only, and
increased kidney weight and CPN severity in both sexes. Increased kidney weights from the 13-
week studies were also considered for dose-response analysis (Saito etal.. 2013: TPEC. 2010b).
In the Saito etal. f20131 2-year inhalation study, male and female F344 rats (50/sex/dose
group) were exposed to concentrations of 0, 2,090, 6,270, or 20,900 mg/m3 flPEC. 2010bl.
Increased incidence of urothelial hyperplasia was only observed in males and significantly
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increased at 6,270 and 20,900 mg/m3. Increased severity of CPN was significantly increased in
males and females at 20,900 mg/m3. Absolute kidney weight was significantly increased in males
at concentrations > 2,090 mg/m3 and in females at > 6,270 mg/m3.
In the TPEC (2008b) 13-week whole-body inhalation study, male and female Crl:CD(SD) rats
were exposed to concentrations of 0, 627, 2,090, 6,270, or 20,900 mg/m3 for 6 hours/day,
5 days/week. Significant increases in absolute kidney weights occurred in male rats exposed to
6,270 or 20,900 mg/m3 ETBE compared with controls, while changes in female rats were not
statistically significant, and were not modeled.
In the Medinskv et al. f 19991 13-week whole-body inhalation study, male and female F344
rats were exposed to concentrations of 0, 2,090, 7,320, or 20,900 mg/m3 for 6 hours/day,
5 days/week. Kidney weights were increased at the highest two doses in both male and females.
Slight, but statistically significant, increases in various clinical chemistry parameters were
observed; however, these effects were reported to be of uncertain toxicological significance and
were not modeled.
2.2.2. Methods of Analysis
No biologically based dose-response models are available for ETBE. In this situation, dose-
response models thought to be consistent with underlying biological processes were evaluated to
determine how best to model the dose-response relationship empirically in the range of the
observed data. Consistent with this approach, all models available in EPA's BMDS were evaluated.
Consistent with EPA's Benchmark Dose Technical Guidance Document fU.S. EPA. 20121. the BMC and
the 95% BMCL were estimated using BMR to represent a minimal, biologically significant level of
change. As noted in Section 2.1.2, a 10% relative change from the control mean (relative deviation;
RD) was used as a BMR for absolute kidney weight, and a BMR of 10% extra risk was considered
appropriate for the quantal data on incidences of urothelial hyperplasia. When modeling was
feasible, the estimated BMCLs were used as points of departure (PODs); the PODs are summarized
in Table 2-4. Further details including the modeling output and graphical results for the model
selected for each endpoint are found in Appendix C of the Supplemental Information to this
T oxicological Review.
Because the RfC is applicable to a continuous lifetime human exposure but is derived from
animal studies featuring intermittent exposure, EPA guidance (U.S. EPA. 1994) provides
mechanisms for: (1) adjusting experimental exposure concentrations to a value reflecting
continuous exposure duration (ADJ) and (2) determining a human equivalent concentration (HEC)
from the animal exposure data. The former employs an inverse concentration-time relationship to
derive a health-protective duration adjustment to time-weight the intermittent exposures used in
the studies. The modeled benchmark concentration from the animal exposures the inhalation
studies flPEC. 2008b: Medinskv et al.. 19991{1PEC. 2010,1517421} were adjusted to reflect a
continuous exposure by multiplying concentration by (6 hours/day) 4- (24 hours/day) and
(5 days/week) 4 (7 days/week) as follows:
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BMCLadj = BMCL (mg/m3) x (6 -h 24) x (5 -h 7)
BMCL (mg/m3) x (0.1786)
The RfC methodology provides a mechanism for deriving an HEC from the duration-
adjusted POD (BMCLadj) determined from the animal data. The approach takes into account the
extra-respiratory nature of the toxicological responses and accommodates species differences by
considering blood:air partition coefficients for ETBE in the laboratory animal (rat or mouse) and
humans. According to the RfC guidelines (U.S. EPA. 1994). ETBE is a Category 3 gas because extra-
respiratory effects were observed. Therefore, the duration-adjusted BMCLadj is multiplied by the
ratio of animal/human blood:air partition coefficients (LA/LH). As detailed in Appendix B.2.2 of the
Supplemental Information, the values reported in the literature for these parameters include an La
of 11.6 for Wistar rats (Kaneko etal.. 2000) and an Lh in humans of 11.7 (Nihlen et al.. 1995). This
allowed a BMCLhec to be derived as follows:
BMCLhec = BMCLadj (mg/m3) x (La ^ Lh) (interspecies conversion)
= BMCLadj (mg/m3) x (11.6 -h 11.7)
= BMCLadj (mg/m3) x (0.992)
Table 2-4 summarizes the sequence of calculations leading to the derivation of a human-
equivalent POD (PODhec) for each inhalation data set discussed above.
Table 2-4. Summary of derivation of PODs following inhalation exposure
Endpoint and
Reference
Species/
Sex
Model3
BMR
BMC
(mg/m3)
BMCL (mg/m3)
PODADJb
(mg/m3)
PODHEcc
(mg/m3)
Kidney
Increased urothelial
hyperplasia; 2-year
Saito et al. (2013);
JPEC (2010b)
Male F344
rats
Gamma
10%
2,734
1,498
268
265
Increased CPN
severity; 2-year
Saito et al. (2013);
JPEC (2010b)
Male and
female F344
rats
NOAELd: 6270 mg/m3
1,120
1,110
Increased absolute
kidney weight; 2-year
Saito et al. (2013);
JPEC (2010b)
Female F344
rats
NOAELde: 6270 mg/m3
6% 1" in kidney weight
1,120
1,110
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Endpoint and
Reference
Species/
Sex
Model3
BMR
BMC
(mg/m3)
BMCL (mg/m3)
PODadj15
(mg/m3)
PODhec0
(mg/m3)
Increased absolute
kidney weight;
13- week
JPEC (2008b)
Male
Sprague-
Dawley rats
NOAELd: 627 mg/m3
10% 1" in kidney weight
112
ill
Increased absolute
kidney weight;
13-week
JPEC (2008b)
Female
Sprague-
Dawley rats
Linear
10% RD
28,591
16,628
2,969
2,946
Increased absolute
kidney weight;
13-week
Medinskv et al.
(1999)
Male F344
rats
Hill
10% RD
6,968
2,521
450
447
Increased absolute
kidney weight;
13-week
Medinskv et al.
(1999)
Female F344
rats
Exponenti
al (M4)
10% RD
5,610
3,411
609
604
1 aFor modeling details, see Appendix C of the Supplemental Information.
2 bPODs were adjusted for continuous daily exposure: PODadj = POD x (hours exposed per day -f 24 hr) x (days
3 exposed per week 4 7 days).
4 cPODHec calculated by adjusting the PODadj by the DAF (=0.992) for a Category 3 gas (U.S. EPA, 1994).
5 dNOAELwas used due to lack of suitable model fit (see Appendix C).
6 eAbsolute kidney weight was increased 5, 6, and 18% at 2090, 6270, and 20,900 mg/m3. A NOAEL was selected
7 based on the dose closest to a 10% change in order to more closely approximate a minimally biologically
8 significant change.
9 2.2.3. Derivation of Candidate Values
10 In EPA's A Review of the Reference Dose and Reference Concentration Processes fU.S. EPA.
11 2002: Section 4.4.5). also described in the Preamble, five possible areas of uncertainty and
12 variability were considered. An explanation follows:
13 An intraspecies uncertainty factor, UFH, of 10 was applied to all PODs to account for
14 potential differences in toxicokinetics and toxicodynamics in the absence of information on the
15 variability of response in the human population following inhalation exposure to ETBE fU.S. EPA.
16 20021.
17 An interspecies uncertainty factor, UFa, of 3 (10°5 = 3.16, rounded to 3) was applied to all
18 PODs to account for residual uncertainty in the extrapolation from laboratory animals to humans in
19 the absence of information to characterize toxicodynamic differences between rodents and humans
20 after inhalation exposure to ETBE. This value is adopted by convention where an adjustment from
21 animal to a human equivalent concentration has been performed as described in EPA's Methods for
22 Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry fU.S. EPA.
23 19941.
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A subchronic to chronic uncertainty factor, UFs, differs depending on the exposure duration.
For rodent studies, exposure durations of 90 days (or 13 weeks) are generally considered
subchronic. Furthermore, the magnitude of change in absolute kidney weights appeared to increase
in male and female rats exposed for 26 weeks compared with 13-18 weeks, when results across
oral and inhalation exposures were evaluated based upon internal blood concentrations (see Figure
1-2), suggesting that toxicity would be expected to increase with exposure durations greater than
13 weeks. Therefore, a UFs of 10 was applied for studies of 13 weeks. A UFs of 1 was applied to 2-
year studies.
A LOAEL to NOAEL uncertainty factor, UFl, of 1 was applied to all PODs derived because the
current approach is to address this factor as one of the considerations in selecting a BMR for
benchmark dose modeling. In this case, BMRs of a 10% change in absolute kidney weight and a
10% extra risk of histological lesions were selected assuming that they represent minimal
biologically significant response levels. In some cases, the data were not amenable to modeling,
thus a NOAEL was selected to represent a minimal biologically significant response.
A database uncertainty factor, UFd, of 1 was applied to all PODs. The ETBE inhalation
toxicity database includes a 2-year toxicity study in rats fSaito etal.. 2013: TPEC. 2010bl and
13-week toxicity studies in mice and rats flPEC. 2008b: Medinskv et al.. 19991. Generally, if the RfD
or RfC is based on animal data, a factor of 3 is often applied if either a prenatal toxicity study or a
two-generation reproduction study is missing, or a factor of 10 if both are missing {U.S. EPA, 2002,
88824}. There are no developmental or multi-generation reproductive studies by the inhalation
route; however, the oral studies of prenatal developmental toxicity in rats and rabbits fAso etal..
2014: Asano etal.. 20111. and single- and multi-generation reproductive toxicity and
developmental toxicity in rats fFuiii etal.. 2010: Gaoua. 2004a. b) are available to inform the
inhalation database. In addition, systemic effects are anticipated to be similar via oral or inhalation
exposure to ETBE, first pass effects are not indicated by the available data, and no evidence is
available to suggest that untransformed ETBE would have a significant role in toxicity. Similarly, the
oral ETBE data set does not indicate immunotoxicity and differences in outcome would not be
anticipated for inhalation exposures fBanton etal.. 2011: Li etal.. 20111. Although most of the
studies are in rats, the available mouse study observed effects that were less severe than those in
rats, suggesting that mice are not more sensitive than rats. The ETBE inhalation database,
supported by the information from the oral database, adequately covers all major systemic effects,
including reproductive, developmental, immunological and neurological effects, and the available
evidence does not raise concern that additional studies would likely lead to identification of a more
sensitive endpoint or a lower POD. Therefore, a database UFD of 1 was applied.
Table 2-5 is a continuation of Table 2-4, and summarizes the application of UF values to
each POD to derive a candidate value for each data set. The candidate values presented in the table
below are preliminary to the derivation of the organ/system-specific reference values. These
candidate values are considered individually in the selection of a representative inhalation
reference value for a specific hazard and subsequent overall RfC for ETBE.
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1 Figure 2-2 presents graphically the candidate values, UF values, and PODs, with each bar
2 corresponding to one data set described in Table 2-4 and Table 2-5.
3 Table 2-5. Effects and corresponding derivation of candidate values
Endpoint (Sex and species) and
Reference
PODhec
(mg/m3)
POD
type
ufa
UFh
UFl
UFs
UFd
Composite
UF
Candidate
value
(mg/m3)
Kidney
Increased urothelial hyperplasia;
male rat; 2-year
Saito et al. (2013); JPEC (2010b)
265
BMCLio%
3
10
1
1
1
30
9x 10°
Increased CPN severity; male and
female rats; 2-year
Saito et al. (2013); JPEC (2010b)
1,110
NOAEL
3
10
1
1
1
30
4x 101
Increased absolute kidney weight;
female rats; 2-year
Saito et al. (2013); JPEC (2010b)
1,110
NOAEL
3
10
1
1
1
30
4x 101
Increased absolute kidney weight;
male rat; 13-week
JPEC (2008b)
111
NOAEL
3
10
1
10
1
300
4 x 101
Increased absolute kidney weight;
female rat; 13-week
JPEC (2008b)
2,946
BMCLio%
3
10
1
10
1
300
lx 101
Increased absolute kidney weight;
male rat; 13-week
Medinskv et al. (1999)
447
BMCLio%
3
10
1
10
1
300
2x 10°
Increased absolute kidney weight;
female rat; 13-week
Medinskv et al. (1999)
604
BMCLio%
3
10
1
10
1
300
2x 10°
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Increased urothelial hyperplasia;
male rat; 2 year
Sato et al. (2013); JPEC (2010b]
Increased CPN severity;
male and female rats; 2 year
Saito et al, (2013); JPEC 2010b
Increased absolute kidney weight;
female rats; 2-year Saito et al.
(2013); JPEC (2010b)
Increased absolute kidney weight;
male rat; 13 week
JPEC (2008b)
Increased absolute kidney weight;
female rat; 13 week
JPEC (2008b)
Increased absolute kidney weight;
male rat; 13 week
Medinsky et al. (1999)
Increased absolute kidney weight;
female rat; 13 week
Medinsky et al. (1999)
0.1 1 10 100 1000 10000
img/m3
~
Candidate RfC
•
podhec
Composite UF
1 Figure 2-2. Candidate values with corresponding POD and composite UF.
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2.2.4. Derivation of Organ/System-Specific Reference Concentrations
Table 2-6 distills the candidate values from Table 2-5 into a single value for the kidney.
Organ- or system-specific reference values can be useful for subsequent cumulative risk
assessments that consider the combined effect of multiple agents acting at a common site.
Kidney Toxicity
For ETBE, candidate values were derived for increased kidney weight in both sexes of rats,
and urothelial hyperplasia in males, spanning a range from 4 x 101 to 4 x 101 mg/m3, for an overall
100-fold range. While the increase in urothelial hyperplasia in male rats resulted in the lowest
candidate value, this endpoint was observed only in male rats and thus the biological relevance of
this lesion is more uncertain than the kidney endpoints observed in both sexes. The candidate RfC
for increased absolute kidney weight in female rats (4 x 101 mg/m3) was ultimately selected as the
kidney-specific RfC for ETBE, consistent with the selection of the kidney-specific RfD (see Section
2.1.4). As discussed in Section 2.1.4, this lesion is a general indicator of kidney toxicity, which has
been observed in both sexes of rats (and to a lesser extent in mice). This RfC is identical to the
candidate RfC for the increased severity of CPN in male and female rats, however in this case the
kidney weight was considered a more certain indication of kidney toxicity. Confidence in this
kidney-specific RfC is medium. The candidate value is derived from a well-conducted study,
involving a sufficient number of animals per group, including both sexes, and assessing a wide
range of kidney endpoints, however, the inability to model the selected endpoint resulted in some
reduction in confidence.
Table 2-6. Organ-/system-specific RfCs and overall RfC for ETBE
Effect
Basis
RfC (mg/m3)
Study exposure
description
Confidence
Kidney
Increased absolute kidney
weight in female rats Saito et
al. (2013); JPEC (2010b)
4x 101
Chronic
Medium
Overall RfC
Kidney
4x 101
Chronic
Medium
2.2.5. Selection of the Overall Reference Concentration
For ETBE, kidney effects were identified as the primary hazard; thus, a single
organ-/system-specific RfC was derived. Therefore, the kidney-specific RfC of 4 x 101 mg/m3 is
selected as the overall RfC, representing an estimated exposure level below which deleterious
effects from ETBE exposure are not expected to occur.
The overall RfC is derived to be protective for all types of effects for a given duration of
exposure and is intended to protect the population as a whole including potentially susceptible
subgroups (U.S. EPA. 2002). Decisions concerning averaging exposures over time for comparison
with the RfC should consider the types of toxicological effects and specific lifestages of concern.
Fluctuations in exposure levels that result in elevated exposures during these lifestages could lead
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to an increased level of concern,, even if average levels over the full exposure duration were less
than or equal to the RfC. In the case of ETBE, no specific potential for early lifestage susceptibility to
ETBE exposure was identified, as discussed in Section 1.3.3.
2.2.6. Confidence Statement
A confidence level of high, medium, or low is assigned to the study used to derive the RfC,
the overall database, and the RfC itself, as described in Section 4.3.9.2 of EPA's Methods for
Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry fU.S. EPA.
19941. The overall confidence in this RfC is medium. Confidence in the principal study, Saito et al.
f20131: TPEC f2010bl. is medium This study was well conducted, following GLP guidelines that
involved a sufficient number of animals per group (including both sexes), and assessed a wide
range of tissues and endpoints, however, the inability to model the POD resulted in some reduction
in confidence. Confidence in the database is high; the available studies evaluated a comprehensive
array of endpoints, and that additional studies would lead to identification of a more sensitive
endpoint is not indicated. Reflecting medium confidence in the principal study and high confidence
in the database, overall confidence in the RfC for ETBE is medium.
2.2.7. Previous IRIS Assessment
No previous inhalation assessment for ETBE is available in IRIS.
2.2.8. Uncertainties in the Derivation of the Reference Dose and Reference Concentration
The following discussion identifies uncertainties associated with the RfD and RfC for ETBE.
To derive the RfD and RfC, the UF approach fU.S. EPA. 2000.19941 was applied to a POD based on
kidney toxicity in rats treated chronically. UFs were applied to the PODs to account for
extrapolating from an animal bioassay to human exposure and for the likely existence of a diverse
human population of varying susceptibility. Default approaches are used for these extrapolations,
given the lack of data to inform individual steps.
The database for ETBE contains no human data on adverse health effects from subchronic
or chronic exposure, and the PODs were calculated from data on the effects of ETBE reported by
studies in rats. The database for ETBE exposure includes three lifetime bioassays in rats, several
reproductive/developmental studies in rats and rabbits, several subchronic studies in rats and
mice, and immunotoxicity assays.
Although the database is adequate for reference value derivation, some uncertainty
associated with the database remains, such as the lack of chronic studies in a species other than rats
(e.g., mice), the lack of developmental/reproductive inhalation studies, and limited or no
information available regarding effects in humans or animals with deficient ALDH2 activity.
The toxicokinetic and toxicodynamic differences for ETBE between the animal species from
which the POD was derived and humans are unknown. Although sufficient information is available
to develop a PBPK model in rats to evaluate differences across routes of exposure, the ETBE
database lacks an adequate model that would inform potential interspecies differences. Generally,
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male rats appear more susceptible than females to ETBE toxicity. The underlying mechanistic basis
of this apparent difference, however, is not understood (except in the case of kidney effects, in
which effects in males are likely exacerbated by a2u-globulin nephropathy). Most importantly,
which animal species and sexes are more comparable to humans is unknown.
Overall, the ETBE data are insufficient to conclude that the a2U-globulin process is operative;
however, key noncancer effects related to a2u-globulin (including exacerbation of CPN in male rats)
were observed and considered not appropriate for hazard identification and, therefore, not suitable
for dose-response consideration. Instead, candidate values in female rats were prioritized to avoid
confounding by a2U-globulin processes. Only one subchronic study is available in mice, which are
not affected by a2U-globulin. This study seemed to indicate male mice were more sensitive to
kidney weight changes than female mice, although these changes did not reach statistical
significance {Bond, 1996, 74002}{Medinsky, 1999,10740}. Therefore, there is uncertainty
regarding whether other factors (unrelated to a2u-globulin) may increase the susceptibility of male
rats to ETBE related kidney effects.
2.3. RAL SLOPE FACTOR FOR CANCER
The oral slope factor (OSF) is a plausible upper bound on the estimate of risk per
mg/kg-day of oral exposure. The OSF can be multiplied by an estimate of lifetime exposure (in
mg/kg-day) to estimate the lifetime cancer risk.
As noted in Section 1.3.2, EPA concluded that there is "inadequate evidence of carcinogenic
potential" for oral exposure to ETBE since the two available chronic oral bioassays for ETBE were
negative in rats {Maltoni, 1999, 87642}{JPEC, 2010,1517477}{Suzuki, 2012,1433129}, and no
chronic oral bioassays are available in mice. Furthermore, PBPK analysis indicated the absence of a
consistent dose-response relationship for liver tumors when compared across oral and inhalation
exposures; therefore, a route to route extrapolation was not performed and no oral slope factor is
derived.
2.4. INHALATION UNIT RISK FOR CANCER
The carcinogenicity assessment provides information on the carcinogenic hazard potential
of the substance in question, and quantitative estimates of risk from inhalation exposure can be
derived. Quantitative risk estimates can be derived from the application of a low-dose extrapolation
procedure. If derived, the inhalation unit risk is a plausible upper bound on the estimate of risk per
|ig/m3 air breathed.
2.4.1. Analysis of Carcinogenicity Data
As noted in Section 1.3.2, there is "suggestive evidence of carcinogenic potential" for
inhalation exposure to ETBE. The Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a) state:
When there is suggestive evidence, the Agency generally would not attempt a
dose-response assessment, as the nature of the data generally would not support
one; however, when the evidence includes a well-conducted study, quantitative
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analysis may be useful for some purposes, for example, providing a sense of the
magnitude and uncertainty of potential risks, ranking potential hazards, or setting
research priorities. In each case, the rationale for the quantitative analysis is
explained, considering the uncertainty in the data and the suggestive nature of the
weight of evidence. These analyses generally would not be considered Agency
consensus estimates.
In the case of ETBE, an inhalation unit risk is derived. A description of the carcinogenicity
data is presented in the discussions of biological considerations for cancer dose-response analysis
(see Section 1.3.2). Briefly, a well conducted study demonstrated a significant, positive exposure-
response for hepatocellular adenomas and carcinomas in male rats. While the majority of liver
tumors occurred at the high dose, statistical tests conducted by the study authors found significant
dose-response trend by both the Peto test (incidental tumor test) and the Cochran-Armitage test.
The available data do not demonstrate that the liver tumors are the result of excessive toxicity in
male rats rather than the carcinogenicity of ETBE. Although decreased body weight gain and
survival was noted in the high dose males and females, the study authors did not detect changes to
the animals' general condition (e.g. abnormal behavior or clinical signs) associated with ETBE. In
addition, the study provided no indication that altered to toxicokinetics was responsible for the
significantly increased incidence of liver tumors in male rats. Considering these data, along with
the uncertainty associated with the suggestive nature of the weight of evidence, quantitative
analysis of the tumor data may be useful for providing a sense of the magnitude of potential
carcinogenic risk (including workers and consumers). Therefore, the hepatocellular adenomas and
carcinomas in male rats were considered for dose response modeling and calculation of a
quantitative risk estimate. Because of the suggestive nature of the tumorigenic response fU.S. EPA.
2005a), there is increased uncertainty in this risk estimate, and this is noted below where relevant.
2.4.2. Dose-Response Analysis—Adjustments and Extrapolation Methods
The EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a) recommend that the
method used to characterize and quantify cancer risk from a chemical be determined by what is
known about the MOA of the carcinogen and the shape of the cancer dose-response curve. EPA uses
a two-step approach that distinguishes analysis of the observed dose-response data from
inferences about lower doses (U.S. EPA. 2005a). Within the observed range, the preferred approach
is to use modeling to incorporate a wide range of data into the analysis, such as through a
biologically based model, if supported by substantial data. Without a biologically based model, as in
the case of ETBE, a standard model is used to curve-fit the data and to estimate a POD. EPA uses the
multistage model in IRIS dose-response analyses for cancer (Gehlhaus etal.. 2011) because it
parallels the multistage carcinogenic process and fits a broad array of dose-response patterns.
The second step, extrapolation to lower exposures from the POD, considers what is known
about the modes of action for each effect As above, a biologically based model is preferred fU.S.
EPA. 2005al. Otherwise, linear low-dose extrapolation is recommended if the MOA of
carcinogenicity is mutagenic or has not been established (U.S. EPA. 2005a). For ETBE, the mode(s)
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of carcinogenic action for liver tumors has not been established (see Section 1.3.2). Therefore,
linear low-dose extrapolation was used to estimate human carcinogenic risk.
Details of the modeling and the model selection process can be found in Appendix C of the
Supplemental Information. A POD for estimating low-dose risk was identified at the lower end of
the observed data, corresponding to 10% extra risk.
Because the inhalation unit risk is applicable to a continuous lifetime human exposure but
derived from animal studies featuring intermittent exposure, EPA guidance fU.S. EPA. 19941
provides mechanisms for (1) adjusting experimental exposure concentrations to a value reflecting
continuous exposure duration and (2) determining a human equivalent concentration (HEC) from
the animal exposure data. The former uses an inverse concentration-time relationship to derive a
health-protective duration adjustment to time weight the intermittent exposures used in the study.
The animal BMCL (Table 2-7) estimated from the inhalation study (Saito etal.. 2013: TPEC. 2010b)
was adjusted to reflect continuous exposure by multiplying it by (6 hours/day) 4- (24 hours/day)
and (5 days/week) 4 (7 days/week) as follows:
BMCLadj = BMCL (mg/m^) x (6 -h 24) x (5 4 7)
= 7,118 mg/m3 x 0.25 x 0.71
= 1,271 mg/m3
The approach to determine the HEC accounts for the extrarespiratory nature of the
toxicological responses and accommodates species differences by considering blood:air partition
coefficients for ETBE in the laboratory animal (rat) and humans. According to the RfC guidelines
(U.S. EPA. 1994). ETBE is a Category 3 gas because extrarespiratory effects were observed. The
values reported in the literature for these parameters include a blood:air partition coefficient of
11.6 for rats, La fKaneko etal.. 20001 and a blood:air partition coefficient for humans of 11.7, Lh
(Nihlen et al.. 1995). This allowed a BMCLhec to be derived as follows:
BMCLhec = BMCLadj (mg/m3) x (LA 4 LH) (interspecies conversion)
BMCLadj (mg/m3) x (11.6 4 11.7)
= BMCLadj (mg/m3) x (0.992)
1,271 mg/m3 x (0.992)
= 1,261 mg/m3
2.4.3. Inhalation Unit Risk Derivation
The POD estimate based on the male rat liver tumor data (Saito etal.. 2013: TPEC. 2010b) is
summarized in Table 2-7779. The lifetime inhalation unit risk for humans is defined as the slope of
the line from the lower 95% bound on the exposure at the POD to the control response (inhalation
unit risk = 0.1 4 BMCLio). This slope represents a plausible upper bound on the true risk. Using
linear extrapolation from the BMCLio, a human-equivalent inhalation unit risk was derived as
presented in Table 2-7.
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1 A single inhalation unit risk was derived. Therefore, the recommended inhalation unit risk
2 for providing a sense of the magnitude of potential carcinogenic risk associated with lifetime
3 inhalation exposure to ETBE is 8 x 10"5 per mg/m3, based on the liver tumor response in male
4 F344 rats (Saito etal.. 2013: TPEC. 2010b). This unit risk should not be used with continuous
5 exposures exceeding 1,271 mg/m3 (the POD) because above this level the dose-response
6 relationship is nonlinear. If risk estimates are needed for exposure corresponding to expected
7 overall cancer risks greater than 10%, the full dose-response model as should be consulted.. The
8 slope of the linear extrapolation from the central estimate BMDio is 0.1 4- 0.992 x (1,944 mg/kg-
9 day)] = 5 x 10"5 per mg/m3.
10 Table 2-7. Summary of the inhalation unit risk derivation
Tumor
Species/Sex
Selected
Model
BMR
BMCadj
(mg/m3)
POD=
BMCLadj
(mg/m3)
BMCLhec
(mg/m3)
Slope
factor3-b
(mg/m3)1
Hepatocellular
adenomas or
carcinomas
Saito et al. (2013);
Male F344 rat
1°
Multistage
10%
1,944
1,271
1,261
8 x 10"5
JPEC (2010b)
11 aHuman equivalent slope factor = 0.1/BMCLlohec; see Appendix C of the Supplemental Information for details of
12 modeling results.
13 bThis value is uncertain because it is based on a determination of suggestive evidence of carcinogenic potential;
14 however, the III R may be useful for some decision purposes such as providing a sense of the magnitude of
15 potential risks or ranking potential hazards (U.S. EPA, 2005a). The uncertainties in the data leading to this
16 suggestive weight of evidence determination for carcinogenicity are detailed in Sections 1.3.2., 2.4.1, and 2.4.4.
17 2.4.4. Uncertainties in the Derivation of the Inhalation Unit Risk
18 Uncertainty exists when extrapolating data from animals to estimate potential cancer risks
19 to human populations from exposure to ETBE.
20 Table 2-8 summarizes several uncertainties that could affect the inhalation unit risk.
21 Although the chronic studies did not report an increase in liver tumorigenesis following oral
22 exposure in rats, no other inhalation studies are available to replicate these findings and none
23 examined other animal models (e.g. mice). In addition, no data in humans are available to confirm a
24 general cancer response or the specific tumors observed in the rat bioassay (Saito etal.. 2013: TPEC.
25 2010b). Although changing the methods used to derive the inhalation unit risk could change the
26 results, standard practices were used due to the lack of a human PBPK model, and no other data
27 (e.g., MOA) supported alternative derivation approaches.
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1 Table 2-8. Summary of uncertainties in the derivation of the inhalation unit
2 risk for ETBE
Consideration and
Impact on Cancer Risk Value
Decision
Justification and Discussion
Selection of tumor type and
relevance to humans:
Rat liver tumors are the basis for
estimating human cancer risk.
The liver was selected as
the target organ (U.S. EPA,
2005a).
An MOA for liver carcinogenicity could not be
established, in absence to indicate otherwise
rat liver tumors were considered relevant to
humans (U.S. EPA, 2005a).
Selection of data set:
No other studies are available.
Saito etal. (2013UPEC
(2010b) was selected to
derive cancer risks for
humans.
Saito et al. (2013), JPEC (2010b) was a well-
conducted inhalation study which reported a
positive exposure-response trend for liver
tumors in male rats. Additional bioassays
might add support to the findings or provide
results for different (possibly lower) doses.
Selection of dose metric:
Alternative could T* inhalation unit
risk.
Administered
concentration was used.
Modeling based on the PBPK-derived internal
dose metric of ETBE metabolism decreased
the BMCL by 35%. However, PBPK modeling
was not used for the dose-response because
there was no human model.
Interspecies extrapolation of
dosimetry and risk:
Alternatives could 4^ or T*
inhalation unit risk.
The default approach for a
Category 3 gas was used.
No data suggest an alternative approach.
Although the true human correspondence is
unknown, this overall approach is expected
to neither overestimate nor underestimate
human equivalent risks.
Dose-response modeling:
Alternatives could 4^ or T* slope
factor.
Used multistage dose-
response model to derive a
BMC and BMCL
No biologically based models for ETBE were
available. The multistage model has
biological support and is the model most
consistently used in EPA cancer assessments.
Low-dose extrapolation:
4/ cancer risk estimate would be
expected with the application of
nonlinear low-dose extrapolation.
Linear extrapolation of risk
in low-dose region was
used.
Linear low-dose extrapolation for agents
without a known MOA is supported (U.S.
EPA, 2005a).
Statistical uncertainty at POD:
4/ inhalation unit risk 1.4-fold if
BMC used as the POD rather than
BMCL
BMCL (preferred approach
for calculating slope factor)
was used.
Limited size of bioassay results in sampling
variability; lower bound is 95% CI on
administered exposure at 10% extra risk of
liver tumors.
Sensitive subpopulations
T* inhalation unit risk to unknown
extent.
Individuals deficient in
ALDH2 are potentially
more sensitive.
Experiments showed enhanced liver toxicity
and genotoxicity in mice when ALDH2 was
absent. Human subpopulations deficient in
ALDH2 are known to be at enhanced risk of
ethanol-induced cancer mediated by
acetaldehyde, discussed in Section 1.3.3. No
chemical-specific data are available,
however, to determine the extent of
enhanced sensitivity due to ETBE-induced
carcinogenicity. Beyond ALDH deficiency, no
chemical-specific data are available to
determine the range of human
toxicodynamic variability or sensitivity,
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Consideration and
Impact on Cancer Risk Value
Decision
Justification and Discussion
including the susceptibility of children.
Because determination of a mutagenic MOA
has not been made, an age-specific
adjustment factor is not applied.
1 2.4.5. Previous IRIS Assessment: Inhalation Unit Risk
2 No previous cancer assessment for ETBE is available in IRIS.
3 2.5. APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS
4 As discussed in the Supplemental Guidance for Assessing Susceptibility from Early-Life
5 Exposure to Carcinogens fU.S. EPA. 2005bl either default or chemical-specific age-dependent
6 adjustment factors (ADAFs) are recommended to account for early-life exposure to carcinogens
7 that act through a mutagenic MOA. Because chemical-specific lifestage susceptibility data for cancer
8 are not available, and because the MOA for ETBE carcinogenicity is not known (see Section 1.3.2),
9 application of ADAFs is not recommended.
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ARCO (ARCO Chemical Company). (1983). Toxicologist's report on metabolism and
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03/24/1994. (8EHQ86940000263). Newton Square, PA.
Asano. Y: Ishikura. T: Kudoh. K: Haneda. R: Endoh. T. (2011). Prenatal developmental toxicity study
of ethyl tertiary-butyl ether in rabbits. Drug Chem Toxicol. 34: 311-317.
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Aso. S: Mivata. K: Takakura. S: Hoshuvama. S: Muroi. T: Kusune. Y: Aiimi. S: Furukawa. K. (2014).
Prenatal developmental toxicity study of ethyl tertiary-butyl ether in rats. Drug Chem
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ATSDR (Agency for Toxic Substances and Disease Registry). (1996). Toxicological profile for
methyl-tert-butyl ether [ATSDR Tox Profile], Atlanta, GA: U.S. Department of Health and
Human Services, Public Health Service. http: //www.atsdr.cdc.gov/ToxProfiles/tp91.pdf.
Bailey. SA: Zidell. RH: Perry. RW. (2004). Relationships between organ weight and body/brain
weight in the rat: What is the best analytical endpoint? Toxicol Pathol. 32: 448-466.
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Banton. MI: Peachee. VL: White. KL: Padgett. EL. (2011). Oral subchronic immunotoxicity study of
ethyl tertiary butyl ether in the rat. J Immunotoxicol. 8: 298-304.
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Berger. T: Horner. CM. (2003). In vivo exposure of female rats to toxicants may affect oocyte quality.
Reprod Toxicol. 17: 273-281. http://dx.doi.Org/10.1016/S0890-6238r03100009-l.
Bernauer. U: Amberg. A: Scheutzow. D: Dekant. W. (1998). Biotransformation of 12C- and 2-13C-
labeled methyl tert-butyl ether, ethyl tert-butyl ether, and tert-butyl alcohol in rats:
Identification of metabolites in urine by 13C nuclear magnetic resonance and gas
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Blanck. 0: Fowles. I: Schorsch. F: Pallen. C: Espinasse-Lormeau. H: Schulte-Koerne. E: Totis. M:
Banton. M. (2010). Tertiary butyl alcohol in drinking water induces phase I and II liver
enzymes with consequent effects on thyroid hormone homeostasis in the B6C3F1 female
mouse. J Appl Toxicol. 30: 125-132. http://dx.doi.org/10.1002/)at. 1478.
Bond. TA: Medinskv. MA: Wolf. DC: Cattlev. R: Farris. G: Wong. B: Tanszen. D: Turner. Ml: Sumner.
SCI. (1996a). Ethyl tertiary butyl ether (ETBE): ninety-day vapor inhalation toxicity study in
CD-l(R) mice. Bond, JA; Medinsky, MA; Wolf, DC; Cattley, R; Farris, G; Wong, B; Janszen, D;
Turner, MJ; Sumner, SCJ.
Bond. TA: Medinsky. MA: Wolf. DC: Dorman. DC: Cattley. R: Farris. G: Wong. B: Morgan. K: Tanszen. D:
Turner. Ml: Sumner. SCI. (1996b). Ethyl tertiary butyl ether (ETBE): ninety-day vapor
This document is a draft for review purposes only and does not constitute Agency policy.
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inhalation toxicity study with neurotoxicity evaluations in Fischer 344 rats [TSCA
Submission] (pp. 1-90). (89970000047). Research Triangle Park, NC: Chemical Industry
Institute of Toxicology under contract to ARCO Chemical Company.
http://yosemite.epa.gov/oppts/epatscat8.nsf/by+Service/1332F4B209355DC785256F9E0
06B7EA0/$File/89970000047.pdf.
Borghoff. ST: Parkinson. H: Leavens. TL. (2010). Physiologically based pharmacokinetic rat model
for methyl tertiary-butyl ether; comparison of selected dose metrics following various
MTBE exposure scenarios used for toxicity and carcinogenicity evaluation. Toxicology. 275:
79-91. http://dx.doi.org/10.1016/i.tox.2010.06.003.
Borghoff. ST: Prescott. IS: Tanszen. DB: Wong. BA: Everitt. II. (2001). alpha2u-Globulin nephropathy,
renal cell proliferation, and dosimetry of inhaled tert-butyl alcohol in male and female F-
344 rats. Toxicol Sci. 61: 176-186. http://dx.doi.Org/10.1093/toxsci/61.l.176.
Borghoff. ST: Ring. C: Banton. MI: Leavens. TL. (2016). Physiologically based pharmacokinetic model
for ethyl tertiary-butyl ether and tertiary-butyl alcohol in rats: Contribution of binding to
a2u-globulin in male rats and high-exposure nonlinear kinetics to toxicity and cancer
outcomes. J Appl Toxicol, http://dx.doi.org/10.1002/iat.3412.
Brennan. P. (2002). Gene-environment interaction and aetiology of cancer: what does it mean and
how can we measure it? Carcinogenesis. 23: 381-387.
Brennan. P: Lewis. S: Hashibe. M: Bell. DA: Boffetta. P: Bouchardv. C: Caporaso. N: Chen. C: Coutelle.
C: Diehl. SR: Hayes. RB: Olshan. AF: Schwartz. SM: Sturgis. EM: Wei. 0: Zavras. AI:
Benhamou. S. (2004). Pooled analysis of alcohol dehydrogenase genotypes and head and
neck cancer: a HuGE review. Am J Epidemiol. 159: 1-16.
Cal/EPA (California Environmental Protection Agency). (2016). GeoTracker. Available online at
http: //geotracker.waterboards.ca.gov/ (accessed
Cederbaum. AI: Cohen. G. (1980). Oxidative demethylation of t-butyl alcohol by rat liver
microsomes. Biochem Biophys Res Commun. 97: 730-736.
Chernoff. N: Rogers. TM: Turner. CI: Francis. BM. (1991). SIGNIFICANCE OF SUPERNUMERARY RIBS
IN RODENT DEVELOPMENTAL TOXICITY STUDIES - POSTNATAL PERSISTENCE IN RATS
AND MICE. Fundam Appl Toxicol. 17: 448-453. http: //dx.doi.org/10.1016/0272-
0590C91190196-B.
Cohen. SM: Hard. GC: Regan. KS: Seelv. TC: Bruner. RH. (2011). Pathology working group review of
selected histopathologic changes in the kidneys of rats assigned to toxicology and
carcinogenicity studies of ethyl tertiary butyl ether (ETBE): Japan Bioassay Research Center
studies no.: 0065 and 0691 [Unpublished report] (pp. 1-30). Research Triangle Park, NC:
Research Pathology Associates under contract to Lyondell Chemical Company.
Craig. EA: Yan. Z: Zhao. 01. (2014). The relationship between chemical-induced kidney weight
increases and kidney histopathology in rats. J Appl Toxicol. 35: 729-736.
http: / /dx. doi. or g/10.10 0 2 /i at 3 0 3 6.
de Pevster. A. (2010). Ethyl t-butyl ether: Review of reproductive and developmental toxicity
[Review], Birth Defects Res B Dev Reprod Toxicol. 89: 239-263.
http://dx.doi.org/10.1002/bdrb.20246.
de Pevster. A: Stanard. B: Westover. C. (2009). Effect of ETBE on reproductive steroids in male rats
and rat Leydig cell cultures. Toxicol Lett 190: 74-80.
http://dx.doi.Org/10.1016/j.toxlet.2009.06.879.
This document is a draft for review purposes only and does not constitute Agency policy.
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Dekant. W: Bernauer. U: Rosner. E: Amberg. A. (2001). Toxicokinetics of ethers used as fuel
oxygenates [Review], Toxicol Lett. 124: 37-45. http://dx.doi.org/10.1016/s0378-
4274f00100284-8.
Doi. AM: Hill. G: Seelv. 1: Hailev. 1R: Kissling. G: Bucher. TR. (2007). a2u-Globulin Nephropathy and
Renal Tumors in National Toxicology Program Studies. Toxicol Pathol. 35: 533-540.
Dorman. DC: Struve. MF: Wong. BA: Morgan. KT: Tanszen. DB: Gross. EB: Bond. TA. (1997).
Neurotoxicological evaluation of ethyl tertiary-butyl ether following subchronic (90-day)
inhalation in the Fischer 344 rat J Appl Toxicol. 17: 235-242.
http://dx.doi.org/10.1002 /fsiciH 099-1263C199707H 7:4<235::aid-iat435>3.0.co:2-4.
Drogos. PL: Diaz. AF. (2001). Oxygenates in Gasoline Appendix A: Physical properties of fuel
oxgenates and addititves. In ACS Symposium Series. Washington, DC: American Chemical
Society, http://dx.doi.org/10.1021/bk-2002-0799.ch018.
ECHA (European Chemicals Agency). (2016). Registered substances. 2-ethoxy-2-methylpropane. EC
number: 211-309-7. CAS number: 637-92-3. https://echa.europa.eu/registration-dossier/-
/registered-dossier/15520.
Elcombe. CR: Peffer. RC: Wolf. DC: Bailey. 1: Bars. R: Bell. D: Cattlev. RC: Ferguson. SS: Geter. D:
Goetz. A: Goodman. II: Hester. S: Tacobs. A: Omiecinski. CI: Schoenv. R: Xie. W: Lake. BG.
(2014). Mode of action and human relevance analysis for nuclear receptor-mediated liver
toxicity: A case study with phenobarbital as a model constitutive androstane receptor (CAR)
activator [Review], Crit Rev Toxicol. 44: 64-82.
http://dx.doi. org/10.3109 /10408444.2013.835786.
Eriksson. CI. (2015). Genetic-epidemiological evidence for the role of acetaldehyde in cancers
related to alcohol drinking [Review], Adv Exp Med Biol. 815: 41-58.
http://dx.doi.org/10.1007/978-3-319-09614-8 3.
Faulkner. TP: Wiechart. TP: Hartman. DM: Hussain. AS. (1989). The effects of prenatal tertiary
butanol administration in CBA/J and C57BL/6J mice. Life Sci. 45: 1989-1995.
Fuiii. S: Yabe. K: Furukawa. M: Matsuura. M: Aovama. H. (2010). A one-generation reproductive
toxicity study of ethyl tertiary butyl ether in rats. Reprod Toxicol. 30: 414-421.
http://dx.doi.Org/10.1016/j.reprotox.2010.04.013.
Fukami. T: Nakaiima. M: Yoshida. R: Tsuchiva. Y: Fuiiki. Y: Katoh. M: Mcleod. HL: Yokoi. T. (2004). A
novel polymorphism of human CYP2A6 gene CYP2A6*17 has an amino acid substitution
(V365M) that decreases enzymatic activity in vitro and in vivo. Clin Pharmacol Ther. 76:
519-527. http://dx.d0i.0rg/l 0.1016/i.clpt.2004.08.014.
Gaoua. W. (2004a). Ethyl tertiary butyl ether (ETBE): Prenatal developmental toxicity study by the
oral route (gavage) in rats (pp. 1-280). (CIT Study No. 24860 RSR). unpublished study for
Totalfinaelf on behalf of the ETBE Producers' Consortium. An external peer review was
conducted by EPA in November 2008 to evaluate the accuracy of experimental procedures,
results, and interpretation and discussion of the findings presented. A report of this peer
review is available through EPA's IRIS Hotline, at (202) 566-1676 (phone), (202) 56-1749
(fax), or hotline.iris@epa.gov (e-mail address) and at www.epa.gov/iris.
Gaoua. W. (2004b). Ethyl tertiary butyl ether (ETBE): Two-generation study (reproduction and
fertility effects) by the oral route (gavage) in rats. (CIT Study No. 24859 RSR). unpublished
study for Totalfinaelf on behalf of the ETBE Producers' Consortium. An external peer review
was conducted by EPA in November 2008 to evaluate the accuracy of experimental
procedures, results, and interpretation and discussion of the findings presented. A report of
this peer review is available through EPA's IRIS Hotline, at (202) 566-1676 (phone), (202)
56-1749 (fax), or hotline.iris@epa.gov (e-mail address) and at www.epa.gov/iris.
This document is a draft for review purposes only and does not constitute Agency policy.
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Gehlhaus. MW. Ill: Gift. TS: Hogan. KA: Kopvlev. L: Schlosser. PM: Kadrv. A. -R. (2011). Approaches to
cancer assessment in EPA's Integrated Risk Information System [Review], Toxicol Appl
Pharmacol. 254: 170-180. http://dx.doi.Org/10.1016/j.taap.2010.10.019.
Guvton. KZ: Chiu. WA: Bateson. TF: linot. I: Scott. CS: Brown. RC: Caldwell. TC. (2009). A
reexamination of the PPAR-alpha activation mode of action as a basis for assessing human
cancer risks of environmental contaminants [Review], Environ Health Perspect. 117: 1664-
1672. http: //dx.d0i.0rg/l 0.1289/ehp. 0900758.
Hagiwara. A: Doi. Y: Imai. N: Nakashima. H: Ono. T: Kawabe. M: Furukawa. F: Tamano. S: Nagano. K:
Fukushima. S. (2011). Medium-term multi-organ carcinogenesis bioassay of ethyl tertiary-
butyl ether in rats. Toxicology. 289: 160-166. http://dx.doi.Org/10.1016/j.tox.2011.08.007.
Hagiwara. A: Doi. Y: Imai. N: Suguro. M: Kawabe. M: Furukawa. F: Tamano. S: Nagano. K: Fukushima.
S. (2015). Promotion of liver and kidney carcinogenesis by ethyl tertiary-butyl ether (ETBE)
in male Wistar rats. J Toxicol Pathol. 28: 189-195. http://dx.doi.org/10.1293/tox.TTP-2015-
0023.
Hagiwara. A: Imai. N: Doi. Y: Suguro. M: Kawabe. M: Furukawa. F: Nagano. K: Fukushima. S. (2013).
No Promoting Effect of Ethyl Tertiary-butyl Ether (ETBE) on Rat Urinary Bladder
Carcinogenesis Initiated with N-Butyl-N-(4-hydroxybutyl)nitrosamine. J Toxicol Pathol. 26:
351-357. http://dx.doi.org/10.1293 /tox.2013-0027.
Hard. GC: Banton. MI: Bretzlaff. RS: Dekant. W: Fowles. I. R.: Mallett. AK: Mcgregor. DB: Roberts. KM:
Sielken. RL: Valdez-Flores. C: Cohen. SM. (2013). Consideration of rat chronic progressive
nephropathy in regulatory evaluations for carcinogenicity. Toxicol Sci. 132: 268-275.
http ://dx. doi. or g/10.109 3 /toxsci/kfs3 0 5.
Hard. GC: Tohnson. KT: Cohen. SM. (2009). A comparison of rat chronic progressive nephropathy
with human renal disease-implications for human risk assessment [Review], CritRev
Toxicol. 39: 332-346. http://dx.d0i.0rg/l 0.1080/10408440802368642.
Hattis. D: Goble. R: Russ. A: Chu. M: Ericson. I. (2004). Age-related differences in susceptibility to
carcinogenesis: A quantitative analysis of empirical animal bioassay data. Environ Health
Perspect 112: 1152-1158. http://dx.doi.org/10.1289/ehp.6871.
HSDB (Hazardous Substances Database). (2012). Ethyl tert-butyl ether - CASRN: 637-92-3.
Washington D.C.. https://toxnetnlm.nih.gov/cgi-
bin/sis/search/a?dbs+hsdb:@term+@DQCN0+7867.
I ARC (International Agency for Research on Cancer). (1999a). Acetaldehyde [IARC Monograph] (pp.
319-335). Lyon, France. http://monographs.iarc.fr/ENG/Monographs/vol71/mono71-
88.pdf.
IARC (International Agency for Research on Cancer). (1999b). IARC monographs on the evaluation
of carcinogenic risks to humans: Re-evaluation of some organic chemicals, hydrazine and
hydrogen peroxide [IARC Monograph], Lyon, France: World Health Organization.
IARC (International Agency for Research on Cancer). (1999c). Methyl tert-butyl ether (group 3) (pp.
339-383). Lyon, France.
IARC (International Agency for Research on Cancer). (1999d). Some chemicals that cause tumours
of the kidney or urinary bladder in rodents and some other substances: Methyl tert-butyl
ether. In IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Lyon,
France: World Health Organization.
IARC (International Agency for Research on Cancer). (2010). Alcohol consumption and ethyl
carbamate [IARC Monograph], Lyon, France.
http://monographs.iarc.fr/ENG/Monographs/vol96/mono96.pdf.
This document is a draft for review purposes only and does not constitute Agency policy.
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IARC (International Agency for Research on Cancer). (2012). Consumption of Alcoholic Beverages
[IARC Monograph], Lyon, France. http://monographs.iarc.fr/ENG/Monographs/vollOOE/.
IARC (International Agency for Research on Cancer). (2015). Preamble to the IARC monographs.
Lyon, France: International Agency for Research on Cancer, World Health Organization.
http://monographs.iarc.fr/ENG/Preamble/.
Tohanson. G: Nihlen. A: Lof. A. (1995). Toxicokinetics and acute effects of MTBE and ETBE in male
volunteers. Toxicol Lett 82/83: 713-718. http://dx.doi.org/10.1016/0378-4274r95103589-
3.
TPEC (Japan Petroleum Energy Center). (2007a). 13-Week toxicity test of 2-ethoxy-2-
methylpropane in F344 rats (drinking water study) [Preliminary study for the
carcinogenicity test], (Study No. 0665). Kanagawa, Japan: Japan Industrial Safety and
Health.
TPEC (Japan Petroleum Energy Center). (2007b). Micronucleus test of ETBE using bone marrow of
rats of the "13-week toxicity study of 2-ethoxy-2-methylpropane in F344 rats (drinking
water study) [preliminary carcinogenicity study]". (Study Number: 7046). Japan Bioassay
Research Center, Japan Industrial Safety and Health Association.
TPEC (Japan Petroleum Energy Center). (2008a). [28-day ETBE repeated dose full-body inhalation
toxicity test in rats (preliminary test)]. (Study No. B061828). Japan: Mitsubishi Chemical
Safety Institute Ltd.
TPEC (Japan Petroleum Energy Center). (2008b). A 90-day repeated dose toxicity study of ETBE by
whole-body inhalation exposure in rats. (Study Number: B061829). Mitsubishi Chemical
Safety Institute Ltd. An external peer review was conducted by EPA in August 2012 to
evaluate the accuracy of experimental procedures, results, and interpretation and
discussion of the findings presented. A report of this peer review is available through EPA's
IRIS Hotline, at (202) 566-1676 (phone), (202) 56-1749 (fax), or hotline.iris@epa.gov (e-
mail address) and at www, epa. gov/ir is.
TPEC (Japan Petroleum Energy Center). (2008c). A 180-Day repeated dose oral toxicity study of
ETBE in rats. (Study Number: D19-0002). Japan: Hita Laboratory, Chemicals Evaluation and
Research Institute (CERI). An external peer review was conducted by EPA in August 2012 to
evaluate the accuracy of experimental procedures, results, and interpretation and
discussion of the findings presented. A report of this peer review is available through EPA's
IRIS Hotline, at (202) 566-1676 (phone), (202) 56-1749 (fax), or hotline.iris@epa.gov fe-
mail address) and at www.epa.gov/iris.
TPEC (Japan Petroleum Energy Center). (2008d). Medium-term mutli-organ carcinogenesis bioassay
of 2-ethoxy-2-methylpropane (ETBE) in rats. (Study Number: 0635). Ichinomiya, Japan:
DIMS Institute of Medical Science.
TPEC (Japan Petroleum Energy Center). (2008e). A one-generation reproduction toxicity study of
ETBE in rats. (Study Number: SR07060). Safety Research Institute for Chemical Compounds.
TPEC (Japan Petroleum Energy Center). (2008f). Pharmacokinetic study in rats treated with [14c]
ETBE repeatedly for 14 days. (P070497). Japan: Kumamoto Laboratory, Mitsubishi
Chemical Safety Institute Ltd. An external peer review was conducted by EPA in August
2012 to evaluate the accuracy of experimental procedures, results, and interpretation and
discussion of the findings presented. A report of this peer review is available through EPA's
IRIS Hotline, at (202) 566-1676 (phone), (202) 56-1749 (fax), or hotline.iris@epa.gov (e-
mail address) and at www.epa.gov/iris.
TPEC (Japan Petroleum Energy Center). (2008g). Pharmacokinetic study in rats treated with single
dose of [14C] ETBE. (P070496). Japan: Kumamoto Laboratory, Mitsubishi Chemical Safety
This document is a draft for review purposes only and does not constitute Agency policy.
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Institute Ltd. An external peer review was conducted by EPA in August 2012 to evaluate the
accuracy of experimental procedures, results, and interpretation and discussion of the
findings presented. A report of this peer review is available through EPA's IRIS Hotline, at
(202) 566-1676 (phone), (202) 56-1749 (fax), or hotline.iris@epa.gov fe-mail address) and
at www.epa.gov/iris.
TPEC (Japan Petroleum Energy Center). (2008h). A prenatal developmental toxicity study of ETBE in
rats. (Study Code Number E09-0006). Hita Research Laboratories, Chemicals Evaluation
and Research Institute (CERI).
TPEC (Japan Petroleum Energy Center). (2008i). Study for effects on embryo-fetal development in
rabbits treated orally with ETBE. (Study No. R-965). Shizuoka, Japan: Kannami Laboratory,
Bozo Research Center Inc.
TPEC (Japan Petroleum Energy Center). (2010a). Carcinogenicity test of 2-Ethoxy-2-methylpropane
in rats (Drinking water study). (Study No: 0691). Japan Industrial Safety and Health
Association, Japan Bioassay Research Center. An external peer review was conducted by
EPA in August 2012 to evaluate the accuracy of experimental procedures, results, and
interpretation and discussion of the findings presented. A report of this peer review is
available through EPA's IRIS Hotline, at (202) 566-1676 (phone), (202) 56-1749 (fax), or
hotline.iris@epa.gov (e-mail address) and at www.epa.gov/iris.
TPEC (Japan Petroleum Energy Center). (2010b). Carcinogenicity test of 2-Ethoxy-2-methylpropane
in rats (Inhalation study). (Study No: 0686). Japan: Japan Industrial Safety and Health
Association.. An external peer review was conducted by EPA in August 2012 to evaluate the
accuracy of experimental procedures, results, and interpretation and discussion of the
findings presented. A report of this peer review is available through EPA's IRIS Hotline, at
(202) 566-1676 (phone), (202) 56-1749 ffaxl. or hotline.iris@epa.gov fe-mail address) and
at www.epa.gov/iris.
Kakehashi. A: Hagiwara. A: Imai. N: Nagano. K: Nishimaki. F: Banton. M: Wei. M: Fukushima. S:
Wanibuchi. H. (2013). Mode of action of ethyl tertiary-butyl ether hepatotumorigenicity in
the rat: evidence for a role of oxidative stress via activation of CAR, PXR and PPAR signaling
pathways. Toxicol Appl Pharmacol. 273: 390-400.
http://dx.doi.Org/10.1016/i.taap.2013.09.016.
Kakehashi. A: Hagiwara. A: Imai. N: Wei. M: Fukushima. S: Wanibuchi. H. (2015). Induction of cell
proliferation in the rat liver by the short-term administration of ethyl tertiary-butyl ether. J
Toxicol Pathol. 28: 27-32. http://dx.doi.org/10.1293/tox.2014-0056.
Kaneko. T: Wang. P. -Y: Sato. A. (2000). Partition coefficients for gasoline additives and their
metabolites. J Occup Health. 42: 86-87. http://dx.doi.org/10.1539/ioh.42.86.
Kanerva. RL: Ridder. GM: Stone. LC: Alden. CL. (1987). Characterization of spontaneous and decalin-
induced hyaline droplets in kidneys of adult male rats. Food Chem Toxicol. 25: 63-82.
Klaunig. IE: Babich. MA: Baetcke. KP: Cook. TC: Corton. TC: David. RM: Deluca. TG: Lai. DY: Mckee. RH:
Peters. TM: Roberts. RA: Fenner-Crisp. PA. (2003). PPARalpha agonist-induced rodent
tumors: Modes of action and human relevance [Review], Crit Rev Toxicol. 33: 655-780.
http://dx.doi.org/10.1080/713608372.
Leavens. TL: Borghoff. ST. (2009). Physiologically based pharmacokinetic model of methyl tertiary
butyl ether and tertiary butyl alcohol dosimetry in male rats based on binding to alpha2u-
globulin. Toxicol Sci. 109: 321-335. http://dx.doi.org/10.1093/toxsci/kfp049.
Li. 0: Kobavashi. M: Inagaki. H: Hirata. Y: Hirata. K: Shimizu. T: Wang. R. -S: Suda. M: Kawamoto. T:
Nakaiima. T: Kawada. T. (2011). Effects of subchronic inhalation exposure to ethyl tertiary
This document is a draft for review purposes only and does not constitute Agency policy.
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butyl ether on splenocytes in mice. Int J Immunopathol Pharmacol. 24: 837-847.
http://dx.doi. org/10.1177/03946320110240040.
Liau. SS: Oureshi. MS: Praseedom. R: Huguet. E. (2013). Molecular pathogenesis of hepatic
adenomas and its implications for surgical management [Review], 17: 1869-1882.
http://dx.doi. org/10.1007/sl 1605-013-2274-6.
Malarkev. DE: Bucher. TR. (2011). Summary report of the National Toxicology Program and
Environmental Protection Agency-sponsored review of pathology materials from selected
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