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£ CDA	EPA/635/R14/373a
%	Interagency Review Draft
www.epa.gov/iris
Toxicological Review of Ethyl Tertiary Butyl Ether
(CASRN 637-92-3]
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
September 2014
NOTICE
This document is an Interagency Science Consultation Review draft. This information is
distributed solely for the purpose of pre-dissemination peer review under applicable information
quality guidelines. It has not been formally disseminated by EPA. It does not represent and should
not be construed to represent any Agency determination or policy. It is being circulated for review
of its technical accuracy and science policy implications.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC

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Toxicological Review ofETBE
DISCLAIMER
This document is a preliminary draft for review purposes only. This information is
distributed solely for the purpose of pre-dissemination peer review under applicable information
quality guidelines. It has not been formally disseminated by EPA. It does not represent and should
not be construed to represent any Agency determination or policy. Mention of trade names or
commercial products does not constitute endorsement or recommendation for use.
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	x
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS	xv
EXECUTIVE SUMMARY	ES-1
LITERATURE SEARCH STRATEGY | STUDY SELECTION AND EVALUATION	LS-1
1.	HAZARD IDENTIFICATION	1-1
1.1.	PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM	1-1
1.1.1.	Kidney Effects	1-1
1.1.2.	Liver Effects	1-50
1.1.3.	Reproductive and Developmental Effects	1-84
1.1.4.	Carcinogenicity (other than in the kidney or liver)	1-111
1.1.5.	Other Toxicological Effects	1-121
1.2.	INTEGRATION AND EVALUATION	1-140
1.2.1.	Effects Other Than Cancer	1-140
1.2.2.	Carcinogenicity	1-140
1.2.3.	Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes	1-
142
2.	DOSE-RESPONSE ANALYSIS	2-1
2.1.ORAL REFERENCE DOSE FOR EFFECTS OTHERTHAN 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	2-9
2.1.4.	Derivation of Organ/System-Specific Reference Doses	2-15
2.1.5.	Selection of the Proposed Overall Reference Dose	2-16
2.1.6.	Confidence Statement	2-16
2.1.7.	Previous IRIS Assessment	2-16
2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER THAN CANCER	2-16
2.2.1.	Identification of Studies and Effects for Dose-Response Analysis	2-17
2.2.2.	Methods of Analysis	2-18
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2.2.3.	Derivation of Candidate Values	2-22
2.2.4.	Derivation of Organ/System-Specific Reference Concentrations	2-28
2.2.5.	Selection of the Proposed Overall Reference Concentration	2-29
2.2.6.	Confidence Statement	2-29
2.2.7.	Previous IRIS Assessment	2-29
2.2.8.	Uncertainties in the Derivation of the Reference Dose and Reference
Concentration	2-29
2.3.	ORAL SLOPE FACTOR FOR CANCER	2-30
2.3.1.	Analysis of Carcinogenicity Data	2-30
2.3.2.	Dose-Response Analysis—Adjustments and Extrapolations Methods	2-31
2.3.3.	Derivation of the Oral Slope Factor	2-32
2.3.4.	Uncertainties in the Derivation of the Oral Slope Factor	2-32
2.3.5.	Previous IRIS Assessment: Oral Slope Factor	2-34
2.4.	INHALATION UNIT RISK FOR CANCER	2-34
2.4.1.	Analysis of Carcinogenicity Data	2-34
2.4.2.	Dose-Response Analysis—Adjustments and Extrapolations Methods	2-35
2.4.3.	Inhalation Unit Risk Derivation	2-36
2.4.4.	Uncertainties in the Derivation of the Inhalation Unit Risk	2-37
2.4.5.	Previous IRIS Assessment: Inhalation Unit Risk	2-38
2.5.	APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS	2-38
REFERENCES	R
TABLES
Table ES-1. Summary of reference dose (RfD) derivation	ES-2
Table ES-2. Summary of reference concentration (RfC) derivation	ES-3
Table LS-1. Database search strategy for ETBE	LS-4
Table LS-2. Summary of additional search strategies for ETBE	LS-5
Table LS-3. Questions and relevant experimental information for evaluation of experimental
animal studies	LS-8
Table 1-1. Evidence pertaining to kidney weight effects in animals exposed to ETBE	1-3
Table 1-2. Evidence pertaining to kidney nephropathy and histopathological effects in animals
exposed to ETBE	1-13
Table 1-3. Evidence pertaining to kidney biochemistry effects in animals exposed to ETBE	1-20
Table 1-4. Evidence pertaining to kidney tumor effects in animals exposed to ETBE	1-32
Table 1-5. Evidence pertaining to kidney tumor promotion by ETBE in animals	1-33
Table 1-6. Additional kidney effects potentially relevant to mode of action in animals exposed to
ETBE	1-37
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Table 1-7. Summary of data informing whether the a2u-globulin process is occurring in male rats
exposed to ETBE	1-40
Table 1-8. Evidence pertaining to liver weight effects in animals exposed to ETBE	1-53
Table 1-9. Evidence pertaining to liver histopathology effects in animals exposed to ETBE	1-62
Table 1-10. Evidence pertaining to liver biochemistry effects in animals exposed to ETBE	1-68
Table 1-11. Evidence pertaining to liver tumor effects in animals exposed to ETBE	1-78
Table 1-12. Evidence pertaining to female reproductive effects in animals exposed to ETBE .. 1-86
Table 1-13. Evidence pertaining to male reproductive effects in animals exposed to ETBE	1-90
Table 1-14. Evidence pertaining to prenatal developmental effects in animals following exposure
to ETBE	1-101
Table 1-15. Evidence pertaining to postnatal developmental effects in animals following
exposure to ETBE	1-104
Table 1-16. Evidence pertaining to tumor promotion by ETBE in animals	1-113
Table 1-17. Evidence pertaining to carcinogenic effects (in tissues other than liver or kidney) in
animals exposed to ETBE	1-114
Table 1-18. Evidence pertaining to body weight effects in animals exposed to ETBE	1-123
Table 1-19. Evidence pertaining to adrenal effects in animals exposed to ETBE	1-128
Table 1-20. Evidence pertaining to immune effects in animals exposed to ETBE	1-129
Table 1-21. Evidence pertaining to mortality in animals exposed to ETBE	1-136
Table 2-1. Summary of derivation of PODs	2-5
Table 2-2. Summary of derivation of oral PODs derived from route-to-route extrapolation from
inhalation exposures	2-8
Table 2-3. Effects and corresponding derivation of candidate values	2-11
Table 2-4. Organ/system-specific RfDs and proposed overall RfD for ETBE	2-16
Table 2-5. Summary of derivation of PODs following inhalation exposure	2-19
Table 2-6. Summary of derivation of inhalation PODs derived from route-to-route extrapolation
from oral exposures	2-21
Table 2-7. Effects and corresponding derivation of candidate values	2-24
Table 2-8. Organ/system-specific RfCs and proposed overall RfCfor ETBE	2-28
Table 2-9. Summary of the oral slope factor derivation	2-32
Table 2-10. Summary of uncertainties in the derivation of cancer risk values for ETBE	2-33
Table 2-11. Summary of the inhalation unit risk derivation	2-37
Table 2-12. Summary of uncertainties in the derivation of cancer risk values for ETBE	2-37
FIGURES
Figure LS-1. Literature search approach for ETBE	LS-3
Figure 1-1. Exposure-response array of kidney effects following oral exposure to ETBE	1-30
Figure 1-2. Exposure-response array of kidney effects following inhalation exposure to ETBE.1-31
Figure 1-3. ETBE inhalation exposure array of a2u-globulin data in male rats	1-42
Figure 1-4. ETBE oral exposure array of a2u-globulin data in male rats	1-43
Figure 1-5. Exposure-response array of liver effects following oral exposure to ETBE	1-76
Figure 1-6. Exposure-response array of liver effects following inhalation exposure to ETBE.... 1-77
Figure 1-7. Exposure-response array of reproductive effects following oral exposure to ETBE 1-98
Figure 1-8. Exposure-response array of reproductive effects following inhalation exposure to
ETBE	1-99
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Figure 1-9. Exposure-response array of developmental effects following oral exposure to ETBE. 1-
110
Figure 1-9. Exposure-response array of carcinogenic effects following oral exposure to ETBE	1-
119
Figure 1-10. Exposure-response array of carcinogenic effects following inhalation exposure to
ETBE	1-120
Figure 1-11. Exposure-response array of body weight effects following oral exposure to ETBE... 1-
138
Figure 1-12. Exposure-response array of body weight effects following inhalation exposure to
ETBE	1-139
Figure 2-1. Candidate values with corresponding POD and composite UF	2-14
Figure 2-2. Candidate values with corresponding POD and composite UF	2-27
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ABBREVIATIONS
a2u-g
alpha2 u-globulin
LOAEL
lowest-observed-adverse-effect level
ACGIH
American Conference of Governmental
MN
micronuclei

Industrial Hygienists
MNPCE
micronucleated polychromatic
AIC
Akaike's information criterion

erythrocyte
ATSDR
Agency for Toxic Substances and
MTD
maximum tolerated dose

Disease Registry
MTBE
Methyl tertiary butyl ether
BMD
benchmark dose
NCFA
National Center for Environmental
BMDL
benchmark dose lower confidence limit

Assessment
BMDS
Benchmark Dose Software
NCI
National Cancer Institute
BMR
benchmark response
NOAI-I.
no-ohserved-adverse-effect level
BUN
blood urea nitrogen
NTI'
National Toxicology Program
BW
body weight
ORD
Office of Research and Development
CA
chromosomal aberration
I'BPK
physiologically based pharmacokinetic
CASRN
Chemical Abstracts Service Registry
I'CE
polychromatic erthyrocytes

Number
I'CNA
proliferating cell nuclear antigen
CUT
Chemical Industry Institute of
I'OD
point of departure

Toxicology
l'()|)| |.||
duration-adjusted I'OI)
CL
confidence limit
QSAK
quantitative structure-activity
CNS
central nervous system

relationship
CPN
chronic progressive nephropathy
RD
Relative Deviation
CYP450
cytochrome P450
RfC
inhalation reference concentration
DAF
dosimetric adjustment factor
l
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AUTHORS | CONTRIBUTORS | REVIEWERS
Assessment Team
Keith Salazar, Ph.D. (Chemical
Manager)
Christopher Brinkerhoff, PhD
U.S. EPA
Office of Research and Development
National Center for Environmental Assessment
Washington, DC
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.
U.S. EPA
Office of Research and Development
National Center for Environmental Assessment
Research Triangle Park, NC
Production Team
Taukecha Cunningham
Maureen Johnson
Terri Konoza
Vicki Soto
U.S. EPA
Office of Research and Development
National Center for Environmental Assessment
Washington, DC
Contractor Support
Robyn Blain, PhD
Pam Ross, MSPH
ICF International
9300 Lee Highway
Fairfax, VA
Executive Direction
Kenneth Olden, Ph.D., Sc.D., L.H.D. (Center Director)
John Vandenberg, Ph.D, (National Program Director, HHRA)
Lynn Flowers, Ph.D., DABT (Associate Director for Health)
Vincent Cogliano, Ph.D. (IRIS Program Director—acting)
Samantha Jones, Ph.D. (IRIS Associate Director for Science)
Weihsueh A. Chiu, PhD (Toxicity Pathways Branch Chief)
U.S. EPA
Office of Research and
Development
National Center for
Environmental Assessment
Washington, DC
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Internal Review Team
General Toxicology Workgroup
Inhalation Workgroup
Neurotoxicity Workgroup
PBPK Workgroup
Reproductive and Developmental
Toxicology Workgroup
Toxicity Pathways Workgroup
Reviewers
1	This assessment was provided for review to scientists in K I'A's I'm"ram and Region Offices.
2	Comments were submitted by:
Office of Children's Health Protection, Washington, DC
Office of Policy, Washington, DC
Office of Solid Waste and Emergency Response, Washington, DC
Region 8, Denver, CO
Region 2, New York, NY
U.S. EPA
Office of Research and Development
National Center for Environmental Assessment
Washington, DC
3
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PREFACE
This Toxicological Review critically reviews the publicly available studies on ethyl tertiary
butyl ether (ETBE) in order to identify its adverse health effects and to characterize exposure-
response relationships. The assessment examined all effects by inhalation and oral routes of
exposure and covers 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 EPA's Integrated Risk Information System (IRIS)
program.
This assessment updates a previous IRIS draft assessment ofETBE that was peer reviewed
in 2010. The previous assessment was suspended pending completion of several 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 a number of overlapping scientific issues:
•	tert- Butanol is a metabolite ofETBE, thus some of the toxicological effects ofETBE
may be attributable to tert-butanol. Therefore, data on tert-butanol may inform the
hazard identification and dose-response assessment of ETBE, and vice versa.
•	The scientific literature for chemicals include data on a2U-globulin-related
nephropathy; therefore, a common approach was employed to evaluate those data as
they relate to the mode of action for kidney effects.
•	A combined PBPK model for ETBE and tert-butanol in rats was developed to support
the dose-response assessments for these chemicals.
This assessment was conducted in accordance with EPA guidance, which is cited and
summarized in the Preamble to IRIS Toxicological Reviews. The findings of this assessment and
draft materials produced during its development are available on the IRIS Web site
fhttp: //www.epa.gov/irisI Appendices for chemical and physical properties, toxicokinetic
information, and summaries of toxicity studies and other information are provided as Supplemental
Information to this 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 prior to the development of the IRIS assessment. All public comments
provided were taken into consideration in developing the draft assessment The complete set of
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public comments are available on the docket at http: //www.regulations.gov (Docket ID No. EPA-
HQ-ORD-2 009-02 29).
In April 2011, the National Research Council (NRC) released its Review of the Environmental
Protection Agency's Draft IRIS Assessment of Formaldehyde. In addition to offering comments
specifically about EPA's draft formaldehyde assessment, the NRC made several recommendations
to EPA for improving the development of IRIS assessments. EPA agreed with the recommendations
and is implementing them consistent with the Panel's "Roadmap for Revision," which viewed the
full implementation of their recommendations by the IRIS Program as a multi-year process.
In response to the NRC's 2011 recommendations, I In.- IRIS Program has made changes to
streamline the assessment development process, improve transparency, and create efficiencies in
the Program. The NRC has acknowledged EPA's successes in this area. In May 2014, the NRC
released their report Review of EPA's Integrated Risk Information System Process reviewing the IRIS
assessment development process and found thai KI'A has made substantial improvements to the
IRIS Program in a short amount of time.
The draft ETBE assessment represents a significant advancement in implementing the NRC
recommendations. This assessment is stream I ined, and uses tallies, figures, and appendices to
increase transparency and clarity. It is structured to have distinct sections for the literature search
and screening strategy, study selection and evaluation, hazard identification, and dose-response
assessment The assessment includes a comprehensiv e, systematic, and documented literature
search and screening approach, provides the database search strategy in a table (databases,
keywords), visually represents the inclusion and exclusion ol studies in a flow diagram, and all of
the references are integrated within the I lealth and Environmental Research Online (HERO)
database. A study ev aluation section provides a systematic review of methodological aspects of
epidemiology and experimental animal studies, including study design, conduct, and reporting, that
was subsequently taken into consideration in the evaluation and synthesis of data from these
studies. The evidence is presented in standardized evidence tables, and exposure-response arrays.
The hazard identification and dose-response sections include subsections based on organ/system-
specific effects in which the evidence is synthesized within and integrated across all evidence for
each target organ/systems.
In the draft ETBK assessment, the IRIS Program has attempted to transparently and
uniformly identify strengths and limitations that would affect interpretation of results. All animal
studies ofETBE that were considered to be of acceptable quality, whether yielding positive,
negative, or null results, were considered in assessing the evidence for health effects associated
with chronic exposure to ETBE. These studies were evaluated for aspects of design, conduct, and
reporting that could affect the interpretation of results and the overall contribution to the synthesis
of evidence for determination of human hazard potential using the study quality considerations
outlined in the Preamble. A brief summary of the evaluation is included in the section on methods
This document is a draft for review purposes only and does not constitute Agency policy.
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for study selection and evaluation. Information on study features related to this evaluation is
reported in evidence tables and documented in the synthesis of evidence. Discussion of study
strengths and limitations (that ultimately supported preferences for the studies and data relied
upon) were included in the text where relevant.
In this assessment, the IRIS Program is using existing guidelines to systematically approach
the integration of noncancer human, animal, and mechanistic evidence. In conducting this analysis
and developing the synthesis, the IRIS Program evaluates the data for the: strength of the
relationship between the exposure and response and the presence of a dose-response relationship;
specificity of the response to chemical exposure and whether the exposure precedes the effect;
consistency of the association between the chemical exposu re and response; and biological
plausibility of the response or effect and its relevance to humans. The IRIS Program uses this
weight-of-evidence approach to identify the potential liuman hazards associated with chemical
exposure.
The IRIS ETBE assessment provides a streamlined presentation of information, integrated
hazard identification of all toxic effects, and derivation ol organ/system-specific reference values.
Additionally, consistent with the goal thai assessments should provide a scientifically sound and
transparent evaluation of the relevant scientific literature and presentation of the analyses
performed, this assessment contains an expanded discussion ol study selection and evaluation, as
well as increased documentation of key assessment decisions.
For additional information aliout this assessment or lor general questions regarding IRIS,
please contactEPA's IRIS I lolline at 202-.r>(>6-1 (>7(> (phone), 202-566-1749 (fax), or
hotline, iris@epa.gov.
Chemical Properties and Uses
ETBE is volatile, relatively water soluble, stable under most conditions in soil and water,
and relatively short-lived in the atmosphere. It does not bind strongly to soil and has a low
potential to bioconcentrate in aquatic systems. ETBE does not occur naturally in the environment1
CH3
o
ch3
Ethyl Tertiary-Butyl Ether
i
http://www.epa.gov/oust/oxygenat/index.htm
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(C6Hi40; CAS # 637-92-3)
ETBE has been used as a fuel oxygenate in the U.S. 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 ofETBE and other ether fuel additives ceased
in the U.S., and the use of ethanol dramatically increased (Weaver etal.. 20101.2 ETBE is still
registered with EPA for use as a fuel additive, but its current use has not been documented. The use
of ether fuel additives has been banned or limited by several stales, largely in response to
groundwater contamination concerns.
The U.S. is a major exporter ofETBE, producing 2.r>";. ol the world's ETBE in 2012.
Worldwide consumption ofETBE is concentrated in Western Europe ( - 70%). Use in Eastern
Europe and Japan is also relatively high. Japan's use increased dramatically in 2010 in order to
fulfill its 2010 Kyoto Accord obligations (USDA. 2012).3
While it was used in the U.S., ETBE was released to the environment by gasoline leaks,
evaporation, spills, and other releases. I "I'lIE degrades slowly in the environment and can move
with water in soil. Monitoring studies targeting groundwater near areas where petroleum
contamination likely occurred commonly detect ETBE. For instance, a survey of states reported an
average detection rale of lor ETBE in groundwater samples associated with gasoline
contamination.4 Non-targeted studies, such as a 200(> U.S. Geological Survey (USGS) study5
measuring VOCs in general, have lower detection rales. The 2006 USGS study showed detections of
ETBE above 0.2 j.ig/1. in live samples from two public drinking water wells, corresponding to a
0.0013 rale ofSelection. The USGS study measured several VOCs and was not targeted to sites that
would lie most vulnerable to l"M!K contamination.
Fuel contamination cleanup is largely done by states, and information on the number of
private contaminated drinking water wells is not consistently available. The State of California
2	Gasoline Composition Regulations Affecting LUST Sites. EPA/600/R-10/001. January 2010.
3	USDA Foreign Agricultural Service Global Agricultural Information Network. Japan Biofuels
Annual: Japan Focuses on Next Generation Biofuels. 6/29/2012.
4	Summary Report on a Survey of State Experiences with MTBE and Other Oxygenate
Contamination at LUST Sites. New England Interstate Water Pollution Control Commission. 2003
http://www.neiwpcc.org/neiwpcc docs/2003mtbesum.pdf
5	http://water.usgs.gov/nawqa/vocs/national assessment/
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maintains an online database of measurements from contaminated sites6. 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 ofETBE in other states was found in fewer and less standardized data.
Presently, only 13 states routinely analyze for ETBE at fuel contaminated sites7. Monitoring data
associated with leaking storage tanks in Maryland show c< > n la in i nation in groundwater affecting
multiple properties8. A review from Georgia noted that ETI!K was detected at 6% of petroleum
cleanup sites and that it was the least-frequently detected ether oxygenate. New Hampshire has
noted two contaminated fuel sites with measured groundwater concentrations up to 190 ppb.
Assessments by Other National and International Health Agencies
Toxicity information on ETBE has been evaluated by the National Institute lor Public Health
and the Environment (Bilthoven, The Netherlands) (Tiesiema and Baars. 20091 and the American
Conference of Governmental Industrial I Ivgienists f AC Gill. 2001). ETBE has not been evaluated by
the International Agency for Research on (lancer (I ARC). The results of these assessments are
presented in Appendix A ol the Supplemental Information. It is important to recognize that these
assessments may have been prepared lor different purposes and may utilize different methods, and
that newer studies mav be included in the IRIS assessment.
6	http://geotracker.waterboards.ca.gov/
7	Summary Report on a Survey of State Experiences with MTBE and Other Oxygenate
Contamination at LUST Sites. New England Interstate Water Pollution Control Commission. 2003
http://www.neiwpcc.org/neiwpcc docs/2003mtbesum.pdf
8
http://www.mde.state.md.us/programs/Land/OilControl/RemediationSites/Pages/Programs/Lan
dPrograms/Oil Control/RemediationSites/index.aspx
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PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS
1. Scope of the IRIS Program
Soon after the EPA was established in
1970, it was at the forefront of developing
risk assessment as a science and applying it in
decisions to protect human health and the
environment. The Clean Air Act, for example,
mandates that the EPA provide "an ample
margin of safety to protect public health"; the
Safe Drinking Water Act, that "no adverse
effects on the health of persons may
reasonably be anticipated to occur, allowing
an adequate margin of safety." Accordingly,
the EPA uses information on the adverse
effects of chemicals and on exposure levels
below which these effects are not anticipated
to occur.
IRIS assessments critically review the
publicly available studies to identify adverse
health effects from exposure to chemicals and
to characterize exposure-response
relationships. In terms set forth by the
National Research Council fNRC. 1983], IRIS
assessments cover the hazard identification
and dose-response assessment steps of risk
assessment, not the exposure assessment or
risk characterization steps that are
conducted by the EPA's program and regional
offices and by other federal, state, and local
health agencies that evaluate risk in specific
populations and exposure scenarios. IRIS
assessments are distinct from and do not
address political, economic, and technical
considerations that influence the design and
selection of risk management alternatives.
An IRIS assessment may cover a single
chemical, a group of structurally or
toxicologically related chemicals, or a
complex mixture. These agents may be found
40	in air, water, soil, or sediment. Exceptions are
41	chemicals currently used exclusively as
42	pesticides, ionizing and non-ionizing
43	radiation, and criteria air pollutants listed
44	under Section 108 of the Clean Air Act
45	(carbon monoxide, lead, nitrogen oxides,
46	ozone, particulate matter, and sulfur oxides).
47	Periodically, the IRIS Program asks other
48	EPA programs and regions, other federal
49	agencies, state health agencies, and the
50	general public to nominate chemicals and
51	mixtures for future assessment or
52	reassessment. Agents may be considered for
53	reassessment as significant new studies are
54	published. Selection is based on program and
55	regional office priorities and on availability of
56	adequate information to evaluate the
57	potential for adverse effects. Other agents
58	may also be assessed in response to an urgent
59	public health need.
2. Process for developing and peer-
reviewing IRIS assessments
60	The process for developing IRIS
61	assessments (revised in May 2009 and
62	enhanced in July 2013) involves critical
63	analysis of the pertinent studies,
64	opportunities for public input, and multiple
65	levels of scientific review. The EPA revises
66	draft assessments after each review, and
67	external drafts and comments become part of
68	the public record (U.S. EPA. 2009).
69	Before beginning an assessment, the IRIS
70	Program discusses the scope with other EPA
71	programs and regions to ensure that the
72	assessment will meet their needs. Then a
73	public meeting on problem formulation
74	invites discussion of the key issues and the
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
studies and analytical approaches that might
contribute to their resolution.
Step 1. Development of a draft
Toxicological Review. The draft
assessment considers all pertinent
publicly available studies and applies
consistent criteria to evaluate study
quality, identify health effects, identify
mechanistic events and pathways,
integrate the evidence of causation for
each effect, and derive toxicity values. A
public meeting prior to the integration of
evidence and derivation of toxicity values
promotes public discussion of the
literature search, evidence, and key
issues.
Step 2. Internal review by scientists in
EPA programs and regions. The draft
assessment is revised to address the
comments from within the EPA.
Step 3. Interagency science consultation
with other federal agencies and the
Executive Offices of the President. The
draft assessment is revised to address the
interagency comments. The science
consultation draft, interagency
comments, and the EPA's response to
major comments become part of the
public record.
Step 4. Public review and comment,
followed by external peer review. The
EPA releases the draft assessment for
public review and comment. A public
meeting provides an opportunity to
discuss the assessment prior to peer
review. Then the EPA releases a draft for
external peer review. The peer review
meeting is open to the public and includes
time for oral public comments. The peer
reviewers assess whether the evidence
has been assembled and evaluated
according to guidelines and whether the
conclusions are justified by the evidence.
The peer review draft, written public
comments, and peer review report
become part of the public record.
Step 5. Revision of draft Toxicological
Review and development of draft IRIS
summary. The draft assessment is
revised to reflect the peer review
comments, public comments, and newly
published studies that are critical to the
conclusions of the assessment The
disposition of peer review comments and
public comments becomes part of the
public record.
Step 6. Final EPA review and interagency
science discussion with other federal
agencies and the Executive Offices of
the President The draft assessment and
summary are revised to address the EPA
and interagency comments. The science
discussion draft, written interagency
comments, and EPA's response to major
comments become part of the public
record.
Step 7. Completion and posting. The
Toxicological Review and IRIS summary
are posted on the IRIS website
(http://www.epa.gOv/iris/l.
The remainder of this Preamble addresses
step 1, the development of a draft
Toxicological Review. IRIS assessments
follow standard practices of evidence
evaluation and peer review, many of
which are discussed in EPA guidelines
fU.S. EPA. 2005a. b, 2000b. 1998. 1996.
1991b. 1986a. b) and other methods fU.S.
EPA. 2012a. b, 2011. 2006a. b, 2002.
19941. Transparent application of
scientific judgment is of paramount
importance. To provide a harmonized
approach across IRIS assessments, this
Preamble summarizes concepts from
these guidelines and emphasizes
principles of general applicability.
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Toxicological Review ofETBE
3. Identifying and selecting
pertinent studies
3.1. Identifying studies
Before beginning an assessment, the EPA
conducts a comprehensive search of the
primary scientific literature. The literature
search follows standard practices and
includes the PubMed and ToxNet databases
of the National Library of Medicine, Web of
Science, and other databases listed in the
EPA's HERO system (Health and
Environmental Research Online,
http://her0.epa.g0v/l. Searches for
information on mechanisms of toxicity are
inherently specialized and may include
studies on other agents that act through
related mechanisms.
Each assessment specifies the search
strategies, keywords, and cut-off dates of its
literature searches. The EPA posts the results
of the literature search on the IRIS web site
and requests information from the public on
additional studies and ongoing research.
The EPA also considers studies received
through the IRIS Submission Desk and
studies (typically unpublished) submitted
under the Toxic Substances Control Actor the
Federal Insecticide, Fungicide, and
Rodenticide Act Material submitted as
Confidential Business Information is
considered only if it includes health and
safety data that can be publicly released. If a
study that may be critical to the conclusions
of the assessment has not been peer-
reviewed, the EPA will have it peer-reviewed.
The EPA also examines the toxicokinetics
of the agent to identify other chemicals (for
example, major metabolites of the agent) to
include in the assessment if adequate
information is available, in order to more
fully explain the toxicity of the agent and to
suggest dose metrics for subsequent
modeling.
In assessments of chemical mixtures.
mixture studies are preferred for their ability
to reflect interactions among components.
The literature search seeks, in
decreasing order of preference fU.S. EPA.
2000b. §2.2: 1986b. S2.ll]:
Studies of the mixture being assessed.
Studies of a sufficiently similar
mixture. In evaluating similarity, the
assessment considers the alteration
of mixtures in the environment
through partitioning and
transformation.
Studies of individual chemical
components of the mixture, if there
are not adequate studies of
sufficiently similar mixtures.
3.2. Selecting pertinent epidemiologic
studies
Study design is the key consideration for
selecting pertinent epidemiologic studies
from the results of the literature search.
Cohort studies, case-control studies,
and some population-based surveys
(for example, NHANES) provide the
strongest epidemiologic evidence,
especially if they collect information
about individual exposures and
effects.
Ecological studies (geographic
correlation studies) relate exposures
and effects by geographic area. They
can provide strong evidence if there
are large exposure contrasts between
geographic areas, relatively little
exposure variation within study
areas, and population migration is
limited.
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Toxicological Review ofETBE
Case reports of high or accidental
exposure lack definition of the
population at risk and the expected
number of cases. They can provide
information about a rare effect or
about the relevance of analogous
results in animals.
The assessment briefly reviews
ecological studies and case reports but
reports details only if they suggest effects not
identified by other studies.
3.3. Selecting pertinent experimental
studies
Exposure route is a key design
consideration for selecting pertinent
experimental animal studies or human
clinical studies.
Studies of oral, inhalation, or dermal
exposure involve passage through an
absorption barrier and are
considered most pertinent to human
environmental exposure.
Injection or implantation studies are
often considered less pertinent but may
provide valuable toxicokinetic or
mechanistic information. They also may
be useful for identifying effects in animals
if deposition or absorption is problematic
(for example, for particles and fibers).
Exposure duration is also a key design
consideration for selecting pertinent
experimental animal studies.
Studies of effects from chronic
exposure are most pertinent to
lifetime human exposure.
Studies of effects from less-than-
chronic exposure are pertinent but
less preferred for identifying effects
from lifetime human exposure. Such
studies may be indicative of effects
from less-than-lifetime human
exposure.
43	Short-duration studies involving animals
44	or humans may provide toxicokinetic or
45	mechanistic information.
46	For developmental toxicity and
47	reproductive toxicity, irreversible effects
48	may result from a brief exposure during a
49	critical period of development Accordingly,
50	specialized study designs are used for these
51	effects fIJ.S. EPA. 2006b. 1998.1996. 1991bl
4. Evaluating the quality of
individual studies
52	After the subsets of pertinent
53	epidemiologic and experimental studies have
54	been selected from the literature searches,
55	the assessment evaluates the quality of each
56	individual study. This evaluation considers
57	the design, methods, conduct, and
58	documentation of each study, but not
59	whether the results are positive, negative, or
60	null. The objective is to identify the stronger,
61	more informative studies based on a uniform
62	evaluation of quality characteristics across
63	studies of similar design.
64	4.1. Evaluating the quality of
65	epidemiologic studies
66	The assessment evaluates design and
67	methodological aspects that can increase or
68	decrease the weight given to each
69	epidemiologic study in the overall evaluation
70	fIJ.S. EPA. 2005a. 1998. 1996.1994.1991b):
71	- Documentation of study design,
72	methods, population characteristics,
73	and results.
74	- Definition and selection of the study
75	group and comparison group.
76	- Ascertainment of exposure to the
77	chemical or mixture.
78	- Ascertainment of disease or health
79	effect
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Toxicological Review ofETBE
Duration of exposure and follow-up
and adequacy for assessing the
occurrence of effects.
Characterization of exposure during
critical periods.
Sample size and statistical power to
detect anticipated effects.
Participation rates and potential for
selection bias as a result of the
achieved participation rates.
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50
51
Nature of the assay and validity for its
intended purpose.
Characterization of the nature and
extent of impurities and
contaminants of the administered
chemical or mixture.
Characterization of dose and dosing
regimen (including age at exposure)
and their adequacy to elicit adverse
effects, including latent effects.
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11	- Measurement error (can lead to
12	misclassification of exposure, health
13	outcomes, and other factors) and
14	other types of information bias.
15	- Potential confounding and other
16	sources of bias addressed in the study
17	design or in the analysis of results.
18	The basis for consideration of
19	confounding is a reasonable
20	expectation that the confounder is
21	related to both exposure and
22	outcome and is sufficiently prevalent
23	to result in bias.
24	For developmental toxicity, reproductive
25	toxicity, neurotoxicity, and cancer there is
26	further guidance on the nuances of evaluating
27	epidemiologic studies of these effects fU.S.
28	EPA. 2005a. 1998.1996.1991bl
29	4.2. Evaluating the quality of
30	experimental studies
31	The assessment evaluates design and
32	methodological aspects that can increase or
33	decrease the weight given to each
34	experimental animal study, in-vitro study, or
35	human clinical study fU.S. EPA. 2005a. 1998.
36	1996. 1991b). Research involving human
37	subjects is considered only if conducted
38	according to ethical principles.
39	- Documentation of study design,
40	animals or study population,
41	methods, basic data, and results.
52	- Sample sizes and statistical power to
53	detect dose-related differences or
54	trends.
55	- Ascertainment of survival, vital signs,
56	disease or effects, and cause of death.
57	- Control of other variables that could
58	influence the occurrence of effects.
59	The assessment uses statistical tests to
60	evaluate whether the observations may be
61	due to chance. The standard for determining
62	statistical significance of a response is a trend
63	test or comparison of outcomes in the
64	exposed groups against those of concurrent
65	controls. In some situations, examination of
66	historical control data from the same
67	laboratory within a few years of the study
68	may improve the analysis. For an uncommon
69	effect that is not statistically significant
70	compared with concurrent controls,
71	historical controls may show that the effect is
72	unlikely to be due to chance. For a response
73	that appears significant against a concurrent
74	control response that is unusual, historical
75	controls may offer a different interpretation
76	(TJ.S. EPA. 2005a. §2.2.2.1.31
77	For developmental toxicity, reproductive
78	toxicity, neurotoxicity, and cancer there is
79	further guidance on the nuances of evaluating
80	experimental studies of these effects fU.S.
81	EPA. 2005a. 1998. 1996. 1991b). In multi-
82	generation studies, agents that produce
83	developmental effects at doses that are not
84	toxic to the maternal animal are of special
85	concern. Effects that occur at doses
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Toxicological Review ofETBE
associated with mild maternal toxicity are not
assumed to result only from maternal
toxicity. Moreover, maternal effects may be
reversible, while effects on the offspring may
be permanent fU.S. EPA. 1998. §3.1.2.4.5.4:
1991b. S3.1.1.41..
4.3. Reporting study results
The assessment uses evidence tables to
present the design and key results of
pertinent studies. There may be separate
tables for each site of toxicity or type of study.
If a large number of studies observe the
same effect, the assessment considers the
study quality characteristics in this section to
identify the strongest studies or types of
study. The tables present details from these
studies, and the assessment explains the
reasons for not reporting details of other
studies or groups of studies that do not add
new information. Supplemental information
provides references to all studies considered,
including those not summarized in the tables.
The assessment discusses strengths and
limitations that affect the interpretation of
each study. If the interpretation of a study in
the assessment differs from that of the study
authors, the assessment discusses the basis
for the difference.
As a check on the selection and evaluation
of pertinent studies, the EPA asks peer
reviewers to identify studies that were not
adequately considered.
5. Evaluating the overall evidence
of each effect
5.1. Concepts of causal inference
For each health effect, the assessment
evaluates the evidence as a whole to
determine whether it is reasonable to infer a
causal association between exposure to the
agent and the occurrence of the effect. This
inference is based on information from
pertinent human studies, animal studies, and
mechanistic studies of adequate quality.
42	Positive, negative, and null results are given
43	weight according to study quality.
44	Causal inference involves scientific
45	judgment, and the considerations are
46	nuanced and complex. Several health
47	agencies have developed frameworks for
48	causal inference, among them the U.S.
49	Surgeon General fCDC. 2004: HEW. 19641.
50	the International Agency for Research on
51	Cancer (IARC. 20061. the Institute of Medicine
52	flOM. 20081. and the EPA ("2010. §1.6:
53	2005a. §2.51. Although developed for
54	different purposes, the frameworks are
55	similar in nature and provide an established
56	structure and language for causal inference.
57	Each considers aspects of an association that
58	suggest causation, discussed by Hill (1965)
59	and elaborated by Rothman and Greenland
60	(1998), and U.S. EPA C2005a. §2.2.1.7:
61	1994. Appendix CI.
62	Strength of association: The finding of a
63	large relative risk with narrow
64	confidence intervals strongly suggests
65	that an association is not due to chance,
66	bias, or other factors. Modest relative
67	risks, however, may reflect a small range
68	of exposures, an agent of low potency, an
69	increase in an effect that is common,
70	exposure misclassification, or other
71	sources of bias.
72	Consistency of association: An inference of
73	causation is strengthened if elevated
74	risks are observed in independent studies
75	of different populations and exposure
76	scenarios. Reproducibility of findings
77	constitutes one of the strongest
78	arguments for causation. Discordant
79	results sometimes reflect differences in
80	study design, exposure, or confounding
81	factors.
82	Specificity of association: As originally
83	intended, this refers to one cause
84	associated with one effect. Current
85	understanding that many agents cause
86	multiple effects and many effects have
87	multiple causes make this a less
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Toxicological Review ofETBE
informative aspect of causation, unless
the effect is rare or unlikely to have
multiple causes.
Temporal relationship: A causal
interpretation requires that exposure
precede development of the effect.
Biologic gradient (exposure-response
relationship):	Exposure-response
relationships strongly suggest causation.
A monotonic increase is not the only
pattern consistent with causation. The
presence of an exposure-response
gradient also weighs against bias and
confounding as the source of an
association.
Biologic plausibility: An inference of
causation is strengthened by data
demonstrating plausible biologic
mechanisms, if available. Plausibility may
reflect subjective prior beliefs if there is
insufficient understanding of the biologic
process involved.
Coherence: An inference of causation is
strengthened by supportive results from
animal experiments, toxicokinetic
studies, and short-term tests. Coherence
may also be found in other lines of
evidence, such as changing disease
patterns in the population.
"Natural experiments": A change in
exposure that brings about a change in
disease frequency provides strong
evidence, as it tests the hypothesis of
causation. An example would be an
intervention to reduce exposure in the
workplace or environment that is
followed by a reduction of an adverse
effect.
Analogy: Information on structural
analogues or on chemicals that induce
similar mechanistic events can provide
insight into causation.
These considerations are consistent with
guidelines for systematic reviews that
evaluate the quality and weight of evidence.
Confidence is increased if the magnitude of
effect is large, if there is evidence of an
exposure-response relationship, or if an
association was observed and the plausible
biases would tend to decrease the magnitude
of the reported effect. Confidence is
decreased for study limitations,
inconsistency of results, indirectness of
evidence, imprecision, or reporting bias
(Guvattetal.. 2008b: Guvatt et al.. 2008al.
5.2. Evaluating evidence in humans
For each effect, the assessment evaluates
the evidence from the epidemiologic studies
as a whole. The objective is to determine
whether a credible association has been
observed and, if so, whether that association
is consistent with causation. In doing this, the
assessment explores alternative explanations
(such as chance, bias, and confounding) and
draws a conclusion about whether these
alternatives can satisfactorily explain any
observed association.
To make clear how much the
epidemiologic evidence contributes to the
overall weight of the evidence, the
assessment may select a standard descriptor
to characterize the epidemiologic evidence of
association between exposure to the agent
and occurrence of a health effect.
Sufficient epidemiologic evidence of an
association consistent with causation:
The evidence establishes a causal
association for which alternative
explanations such as chance, bias, and
confounding can be ruled out with
reasonable confidence.
Suggestive epidemiologic evidence of an
association consistent with causation:
The evidence suggests a causal
association but chance, bias, or
confounding cannot be ruled out as
explaining the association.
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Inadequate epidemiologic evidence to infer
a causal association: The available
studies do not permit a conclusion
regarding the presence or absence of an
association.
Epidemiologic evidence consistent with no
causal association: Several adequate
studies covering the full range of human
exposures and considering susceptible
populations, and for which alternative
explanations such as bias and
confounding can be ruled out, are
mutually consistent in not finding an
association.
5.3. Evaluating evidence in animals
For each effect, the assessment evaluates
the evidence from the animal experiments as
a whole to determine the extent to which they
indicate a potential for effects in humans.
Consistent results across various species and
strains increase confidence that similar
results would occur in humans. Several
concepts discussed by Hill (19651 are
pertinent to the weight of experimental
results: consistency of response, dose-
response relationships, strength of response,
biologic plausibility, and coherence fU.S. EPA.
2005a. §2.2.1.7: 1994. Appendix CI
In weighing evidence from multiple
experiments, U.S. EPA f2005a. §2.51
distinguishes:
Conflicting evidence (that is, mixed positive
and negative results in the same sex and
strain using a similar study protocol)
from
Differing results (that is, positive results and
negative results are in different sexes or
strains or use different study protocols).
Negative or null results do not invalidate
positive results in a different experimental
system. The EPA regards all as valid
observations and looks to explain differing
results using mechanistic information (for
44	example, physiologic or metabolic
45	differences across test systems) or
46	methodological differences (for example,
47	relative sensitivity of the tests, differences in
48	dose levels, insufficient sample size, or timing
49	of dosing or data collection).
50	It is well established that there are critical
51	periods for some developmental and
52	reproductive effects fU.S. EPA. 2006b. 2005a.
53	b, 1998. 1996. 1991b). Accordingly, the
54	assessment determines whether critical
55	periods have been adequately investigated.
56	Similarly, the assessment determines
57	whether the database is adequate to evaluate
58	other critical sites and effects.
59	In evaluating evidence of genetic toxicity:
60	- Demonstration of gene mutations,
61	chromosome aberrations,	or
62	aneuploidy in humans or
63	experimental mammals [in vivo)
64	provides the strongest evidence.
65	- This is followed by positive results in
66	lower organisms or in cultured cells
67	[in vitro) or for other genetic events.
68	- Negative results carry less weight,
69	partly because they cannot exclude
70	the possibility of effects in other
71	tissues flARC. 20061.
72 For germ-cell mutagenicity, The EPA has
73	defined categories of evidence, ranging from
74	positive results of human germ-cell
75	mutagenicity to negative results for all effects
76	of concern (U.S. EPA. 1986a. §2.3).
77	5.4. Evaluating mechanistic data
78	Mechanistic data can be useful in
79	answering several questions.
80	- The biologic plausibility of a causal
81	interpretation of human studies.
82	- The generalizability of animal studies
83	to humans.
84	- The susceptibility of particular
85	populations or lifestages.
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Toxicological Review ofETBE
The focus of the analysis is to describe, if
possible, mechanistic pathways that lead to a
health effect. These pathways encompass:
Toxicokinetic processes of absorption,
distribution, metabolism, and
elimination that lead to the formation
of an active agent and its presence at
the site of initial biologic interaction.
Toxicodynamic processes that lead to a
health effect at this or another site
(also known as a mode of action).
For each effect, the assessment discusses
the available information on its modes of
action and associated key events (key events
being empirically observable, necessary
precursor steps or biologic markers of such
steps; mode of action being a series of key
events involving interaction with cells,
operational and anatomic changes, and
resulting in disease). Pertinent information
may also come from studies of metabolites or
of compounds that are structurally similar or
that act through similar mechanisms.
Information on mode of action is not required
for a conclusion that the agent is causally
related to an effect (U.S. EPA. 2005a. §2.51.
The assessment addresses several
questions about each hypothesized mode of
actionfU.S. EPA. 2005a. §2.4.3.41.
1) Is the hypothesized mode of action
sufficiently supported in test animals?
Strong support for a key event being
necessary to a mode of action can come
from experimental challenge to the
hypothesized mode of action, in which
studies that suppress a key event observe
suppression of the effect Support for a
mode of action is meaningfully
strengthened by consistent results in
different experimental models, much
more so than by replicate experiments in
the same model. The assessment may
consider various aspects of causation in
addressing this question.
2)	Is the hypothesized mode of action
relevant to humans? The assessment
reviews the key events to identify critical
similarities and differences between the
test animals and humans. Site
concordance is not assumed between
animals and humans, though it may hold
for certain effects or modes of action.
Information suggesting quantitative
differences in doses where effects would
occur in animals or humans is considered
in the dose-response analysis. Current
levels of human exposure are not used to
rule out human relevance, as IRIS
assessments may be used in evaluating
new or unforeseen circumstances that
may entail higher exposures.
3)	Which populations or lifestages can be
particularly susceptible to the
hypothesized mode of action? The
assessment reviews the key events to
identify populations and lifestages that
might be susceptible to their occurrence.
Quantitative differences may result in
separate toxicity values for susceptible
populations or lifestages.
The assessment discusses the likelihood
that an agent operates through multiple
modes of action. An uneven level of support
for different modes of action can reflect
disproportionate resources spent
investigating them (U.S.	EPA.
2005a. §2.4.3.31. It should be noted that in
clinical reviews, the credibility of a series of
studies is reduced if evidence is limited to
studies funded by one interested sector
(Guvattetal.. 2008al.
For cancer, the assessment evaluates
evidence of a mutagenic mode of action to
guide extrapolation to lower doses and
consideration of susceptible lifestages. Key
data include the ability of the agent or a
metabolite to react with or bind to DNA,
positive results in multiple test systems, or
similar properties and structure-activity
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relationships to mutagenic carcinogens fU.S.
EPA. 2005a .§2.3.51.
5.5. Characterizing the overall weight
of the evidence
After evaluating the human, animal, and
mechanistic evidence pertinent to an effect,
the assessment answers the question: Does
the agent cause the adverse effect? fNRC.
2009. 19831. In doing this, the assessment
develops a narrative that integrates the
evidence pertinent to causation. To provide
clarity and consistency, the narrative
includes a standard hazard descriptor. For
example, the following standard descriptors
combine epidemiologic, experimental, and
mechanistic evidence of carcinogenicity fU.S.
EPA. 2005a. §2.51.
Carcinogenic to humans: There is
convincing epidemiologic evidence of a
causal association (that is, there is
reasonable confidence that the
association cannot be fully explained by
chance, bias, or confounding); or there is
strong human evidence of cancer or its
precursors, extensive animal evidence,
identification of key precursor events in
animals, and strong evidence that they
are anticipated to occur in humans.
Likely to be carcinogenic to humans: The
evidence demonstrates a potential
hazard to humans but does not meet the
criteria for carcinogenic. There may be a
plausible association in humans, multiple
positive results in animals, or a
combination of human, animal, or other
experimental evidence.
Suggestive evidence of carcinogenic
potential: The evidence raises concern
for effects in humans but is not sufficient
for a stronger conclusion. This descriptor
covers a range of evidence, from a
positive result in the only available study
to a single positive result in an extensive
44	database that includes negative results in
45	other species.
46	Inadequate information to assess
47	carcinogenic potential: No other
48	descriptors apply. Conflicting evidence
49	can be classified as inadequate
50	information if all positive results are
51	opposed by negative studies of equal
52	quality in the same sex and strain.
53	Differing results, however, can be
54	classified as suggestive evidence or as
55	likely to be carcinogenic.
56	Not likely to be carcinogenic to humans:
57	There is robust evidence for concluding
58	that there is no basis for concern. There
59	may be no effects in both sexes of at least
60	two appropriate animal species; positive
61	animal results and strong, consistent
62	evidence that each mode of action in
63	animals does not operate in humans; or
64	convincing evidence that effects are not
65	likely by a particular exposure route or
66	below a defined dose.
67	Multiple descriptors may be used if there
68	is evidence that carcinogenic effects differ by
69	dose range or exposure route fU.S. EPA.
70	2005a. 52.51.
71	Another example of standard descriptors
72	comes from the EPA's Integrated Science
73	Assessments, which evaluate causation for
74	the effects of the criteria pollutants in
75	ambient air CU.S. EPA. 2010. §1 .61.
76	Causal relationship: Sufficient evidence to
77	conclude that there is a causal
78	relationship. Observational studies
79	cannot be explained by plausible
80	alternatives, or they are supported by
81	other lines of evidence, for example,
82	animal studies or mechanistic
83	information.
84	Likely to be a causal relationship: Sufficient
85	evidence that a causal relationship is
86	likely, but important uncertainties
87	remain. For example, observational
88	studies show an association but co-
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exposures are difficult to address or other
lines of evidence are limited or
inconsistent; or multiple animal studies
from different laboratories demonstrate
effects and there are limited or no human
data.
Suggestive of a causal relationship: At least
one high-quality epidemiologic study
shows an association but other studies
are inconsistent
Inadequate to infer a causal relationship:
The studies do not permit a conclusion
regarding the presence or absence of an
association.
Not likely to be a causal relationship:
Several adequate studies, covering the
full range of human exposure and
considering susceptible populations, are
mutually consistent in not showing an
effect at any level of exposure.
The EPA is investigating and may on a
trial basis use these or other standard
descriptors to characterize the overall weight
of the evidence for effects other than cancer.
6. Selecting studies for derivation
of toxicity values
For each effect where there is credible
evidence of an association with the agent, the
assessment derives toxicity values if there
are suitable epidemiologic or experimental
data. The decision to derive toxicity values
may be linked to the hazard descriptor.
Dose-response analysis requires
quantitative measures of dose and response.
Then, other factors being equal:
Epidemiologic studies are preferred
over animal studies, if quantitative
measures of exposure are available
and effects can be attributed to the
agent
- Among experimental animal models,
those that respond most like humans
are preferred, if the comparability of
response can be determined.
Studies by a route of human
environmental exposure are
preferred, although a validated
toxicokinetic model can be used to
extrapolate across exposure routes.
Studies of longer exposure duration
and follow-up are preferred, to
minimize uncertainty about whether
effects are representative of lifetime
exposure.
Studies with multiple exposure levels
are preferred for their ability to
provide information about the shape
of the exposure-response curve.
Studies with adequate power to
detect effects at lower exposure
levels are preferred, to minimize the
extent of extrapolation to levels found
in the environment
Studies with non-monotonic exposure-
response relationships are not necessarily
excluded from the analysis. A diminished
effect at higher exposure levels may be
satisfactorily explained by factors such as
competing toxicity, saturation of absorption
or metabolism, exposure misclassification, or
selection bias.
If a large number of studies are suitable
for dose-response analysis, the assessment
considers the study characteristics in this
section to focus on the most informative data.
The assessment explains the reasons for not
analyzing other groups of studies. As a check
on the selection of studies for dose-response
analysis, the EPA asks peer reviewers to
identify studies that were not adequately
considered.
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7. Deriving toxicity values
7.1. General framework for dose-
response analysis
The EPA uses a two-step approach that
distinguishes analysis of the observed dose-
response data from inferences about lower
doses riJ.S. EPA. 2005a. §31.
Within the observed range, the preferred
approach is to use modeling to incorporate a
wide range of data into the analysis. The
modeling yields a point of departure (an
exposure level near the lower end of the
observed range, without significant
extrapolation to lower doses) (Sections 7.2-
7.3).
Extrapolation to lower doses considers
what is known about the modes of action for
each effect (Sections 7.4-7.5). If response
estimates at lower doses are not required, an
alternative is to derive reference values,
which are calculated by applying factors to
the point of departure in order to account for
sources of uncertainty and variability
(Section 7.6).
For a group of agents that induce an effect
through a common mode of action, the dose-
response analysis may derive a relative
potency factor for each agent A full dose-
response analysis is conducted for one well-
studied index chemical in the group, then the
potencies of other members are expressed in
relative terms based on relative toxic effects,
relative absorption or metabolic rates,
quantitative structure-activity relationships,
or receptor binding characteristics (U.S. EPA.
2005a. §3.2.6: 2000b. §4.41.
Increasingly, the EPA is basing toxicity
values on combined analyses of multiple data
sets or multiple responses. The EPA also
considers multiple dose-response
approaches if they can be supported by
robust data.
7.2. Modeling dose to sites of biologic
effects
The preferred approach for analysis of
dose is toxicokinetic modeling because of its
ability to incorporate a wide range of data.
The preferred dose metric would refer to the
active agent at the site of its biologic effect or
to a close, reliable surrogate measure. The
active agent may be the administered
chemical or a metabolite. Confidence in the
use of a toxicokinetic model depends on the
robustness of its validation process and on
the results of sensitivity analyses (U.S. EPA.
2006a: 2005a. §3.1: 1994. §4.31.
Because toxicokinetic modeling can
require many parameters and more data than
are typically available, the EPA has developed
standard approaches that can be applied to
typical data sets. These standard approaches
also facilitate comparison across exposure
patterns and species.
Intermittent study exposures are
standardized to a daily average over
the duration of exposure. For chronic
effects, daily exposures are averaged
over the lifespan. Exposures during a
critical period, however, are not
averaged over a longer duration (U.S.
EPA. 2005a. §3.1.1: 1991b. §3.21.
Doses are standardized to equivalent
human terms to facilitate comparison
of results from different species.
Oral doses are scaled allometrically
using mg/kg3/4-day as the equivalent
dose metric across species.
Allometric scaling pertains to
equivalence across species, not
across lifestages, and is not used to
scale doses from adult humans or
mature animals to infants or children
fU.S. EPA. 2011: 2005a. §3.1.31.
Inhalation exposures are scaled using
dosimetry models that apply species-
specific physiologic and anatomic
factors and consider whether the
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effect occurs at the site of first contact
or after systemic circulation (U.S.
EPA. 2012a: 1994.S31.
It can be informative to convert doses
across exposure routes. If this is done, the
assessment describes the underlying data,
algorithms, and assumptions (U.S. EPA.
2005a. §3.1.41.
In the absence of study-specific data on,
for example, intake rates or body weight, the
EPA has developed recommended values for
use in dose-response analysis fU.S. EPA.
19881.
7.3. Modeling response in the range
of observation
Toxicodynamic ("biologically based")
modeling can incorporate data on biologic
processes leading to an effect. Such models
require sufficient data to ascertain a mode of
action and to quantitatively support model
parameters associated with its key events.
Because different models may provide
equivalent fits to the observed data but
diverge substantially at lower doses, critical
biologic parameters should be measured
from laboratory studies, not by model fitting.
Confidence in the use of a toxicodynamic
model depends on the robustness of its
validation process and on the results of
sensitivity analyses. Peer review of the
scientific basis and performance of a model is
essential (U.S. EPA. 2005a. §3.2.21.
Because toxicodynamic modeling can
require many parameters and more
knowledge and data than are typically
available, the EPA has developed a standard
set of empirical ("curve-fitting") models
fhttp: //www.epa.gov/ncea/bmds/] that can
be applied to typical data sets, including those
that are nonlinear. The EPA has also
developed guidance on modeling dose-
response data, assessing model fit, selecting
suitable models, and reporting modeling
results (U.S. EPA. 2012b). Additional
judgment or alternative analyses are used if
46	the procedure fails to yield reliable results,
47	for example, if the fit is poor, modeling may
48	be restricted to the lower doses, especially if
49	there is competing toxicity at higher doses
50	fU.S. EPA. 2005a. §3.2.31.
51	Modeling is used to derive a point of
52	departure fU.S. EPA. 2012b: 2005a. §3.2.41.
53	(See Section 7.6 for alternatives if a point of
54	departure cannot be derived by modeling.):
55	- If linear extrapolation is used,
56	selection of a response level
57	corresponding to the point of
58	departure is not highly influential, so
59	standard values near the low end of
60	the observable range are generally
61	used (for example, 10% extra risk for
62	cancer bioassay data, 1% for
63	epidemiologic data, lower for rare
64	cancers).
65	- For nonlinear approaches, both
66	statistical and biologic considerations
67	are taken into account.
68	- For dichotomous data, a response
69	level of 10% extra risk is generally
70	used for minimally adverse effects,
71	5% or lower for more severe effects.
72	- For continuous data, a response level
73	is ideally based on an established
74	definition of biologic significance. In
75	the absence of such definition, one
76	control standard deviation from the
77	control mean is often used for
78	minimally adverse effects, one-half
79	standard deviation for more severe
80	effects.
81	The point of departure is the 95% lower
82	bound on the dose associated with the
83	selected response level.
84	7.4. Extrapolating to lower doses and
85	response levels
86	The purpose of extrapolating to lower
87	doses is to estimate responses at exposures
88	below the observed data. Low-dose
89	extrapolation, typically used for cancer data,
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considers what is known about modes of
action fU.S. EPA. 2005a. §3.3.1 and §3.3.21.
1) If a biologically based model has been
developed and validated for the agent,
extrapolation may use the fitted model
below the observed range if significant
model uncertainty can be ruled out with
reasonable confidence.
2) Linear extrapolation is used if the dose-
response curve is expected to have a
linear component below the point of
departure. This includes:
-	Agents or their metabolites that are
DNA-re active and have direct
mutagenic activity.
-	Agents or their metabolites for which
human exposures or body burdens
are near doses associated with key
events leading to an effect
Linear extrapolation is also used when
data are insufficient to establish mode of
action and when scientifically plausible.
The result of linear extrapolation is
described by an oral slope factor or an
inhalation unit risk, which is the slope of
the dose-response curve at lower doses
or concentrations, respectively.
3)	Nonlinear models are used for
extrapolation if there are sufficient data
to ascertain the mode of action and to
conclude that it is not linear at lower
doses, and the agent does not
demonstrate mutagenic or other activity
consistent with linearity at lower doses.
Nonlinear approaches generally should
not be used in cases where mode of action
has not ascertained. If nonlinear
extrapolation is appropriate but no
model is developed, an alternative is to
calculate reference values.
4)	Both linear and nonlinear approaches
may be used if there a multiple modes of
action. For example, modeling to a low
response level can be useful for
45	estimating the response at doses where a
46	high-dose mode of action would be less
47	important
48	If linear extrapolation is used, the
49	assessment develops a candidate slope factor
50	or unit risk for each suitable data set. These
51	results are arrayed, using common dose
52	metrics, to show the distribution of relative
53	potency across various effects and
54	experimental systems. The assessment then
55	derives or selects an overall slope factor and
56	an overall unit risk for the agent, considering
57	the various dose-response analyses, the
58	study preferences discussed in Section 6, and
59	the possibility of basing a more robust result
60	on multiple data sets.
61	7.5. Considering susceptible
62	populations and lifestages
63	The assessment analyzes the available
64	information on populations and lifestages
65	that may be particularly susceptible to each
66	effect A tiered approach is used (U.S. EPA.
67	2005a. S3.Sl.
68	1) If an epidemiologic or experimental study
69	reports quantitative results for a
70	susceptible population or lifestage, these
71	data are analyzed to derive separate
72	toxicity values for susceptible
73	individuals.
74	2) If data on risk-related parameters allow
75	comparison of the general population and
76	susceptible individuals, these data are
77	used to adjust the general-population
78	toxicity values for application to
79	susceptible individuals.
80	3) In the absence of chemical-specific data,
81	the EPA has developed age-dependent
82	adjustment factors for early-life exposure
83	to potential carcinogens that have a
84	mutagenic mode of action. There is
85	evidence of early-life susceptibility to
86	various carcinogenic agents, but most
87	epidemiologic studies and cancer
88	bioassays do not include early-life
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exposure. To address the potential for
early-life susceptibility, the EPA
recommends (U.S. EPA. 2005b. §51:
10-fold adjustment for exposures
before age 2 years.
3-fold adjustment for exposures
between ages 2 and 16 years.
7.6. Reference values and uncertainty
factors
An oral reference dose or an inhalation
reference concentration is an estimate of an
exposure (including in susceptible
subgroups) that is likely to be without an
appreciable risk of adverse health effects
over a lifetime fU.S. EPA. 2002. §4.21.
Reference values are typically calculated for
effects other than cancer and for suspected
carcinogens if a well characterized mode of
action indicates that a necessary key event
does not occur below a specific dose.
Reference values provide no information
about risks at higher exposure levels.
The assessment characterizes effects that
form the basis for reference values as
adverse, considered to be adverse, or a
precursor to an adverse effect For
developmental toxicity, reproductive toxicity,
and neurotoxicity there is guidance on
adverse effects and their biologic markers
(TJ.S. EPA. 1998.1996. 1991b).
To account for uncertainty and variability
in the derivation of a lifetime human
exposure where adverse effects are not
anticipated to occur, reference values are
calculated by applying a series of uncertainty
factors to the point of departure. If a point of
departure cannot be derived by modeling, a
no-observed-adverse-effect level or a lowest-
observed-adverse-effect level is used instead.
The assessment discusses scientific
considerations involving several areas of
variability or uncertainty.
Human variation. The assessment accounts
for variation in susceptibility across the
human population and the possibility
46	that the available data may not be
47	representative of individuals who are
48	most susceptible to the effect A factor of
49	10 is generally used to account for this
50	variation. This factor is reduced only if
51	the point of departure is derived or
52	adjusted specifically for susceptible
53	individuals (not for a general population
54	that includes both susceptible and non-
55	susceptible individuals) (U.S. EPA.
56	2002. §4.4.5: 1998. §4.2: 1996. §4:
57	1994. §4.3.9.1: 1991b. §3.41.
58	Animal-to-human extrapolation. If animal
59	results are used to make inferences about
60	humans, the assessment adjusts for
61	cross-species differences. These may
62	arise from differences in toxicokinetics or
63	toxicodynamics. Accordingly, if the point
64	of departure is standardized to
65	equivalent human terms or is based on
66	toxicokinetic or dosimetry modeling, a
67	factor of 101/2 (rounded to 3) is applied to
68	account for the remaining uncertainty
69	involving	toxicokinetic and
70	toxicodynamic differences. If a
71	biologically based model adjusts fully for
72	toxicokinetic and toxicodynamic
73	differences across species, this factor is
74	not used. In most other cases, a factor of
75	10 is applied (TJ.S. EPA. 2011:
76	2002. §4.4.5: 1998. §4.2: 1996. §4:
77	1994. §4.3.9.1: 1991b. §3.41.
78	Adverse-effect level to no-observed-
79	adverse-effect level. If a point of
80	departure is based on a lowest-observed-
81	adverse-effect level, the assessment must
82	infer a dose where such effects are not
83	expected. This can be a matter of great
84	uncertainty, especially if there is no
85	evidence available at lower doses. A
86	factor of 10 is applied to account for the
87	uncertainty in making this inference. A
88	factor other than 10 may be used,
89	depending on the magnitude and nature
90	of the response and the shape of the dose-
91	response curve fU.S. EPA. 2002. §4.4.5:
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1998. §4.2: 1996. §4: 1994. §4.3.9.1:
1991b. §3.41.
Subchronic-to-chronic exposure. If a point
of departure is based on subchronic
studies, the assessment considers
whether lifetime exposure could have
effects at lower levels of exposure. A
factor of 10 is applied to account for the
uncertainty in using subchronic studies
to make inferences about lifetime
exposure. This factor may also be applied
for developmental or reproductive effects
if exposure covered less than the full
critical period. A factor other than 10 may
be used, depending on the duration of the
studies and the nature of the response
(TJ.S. EPA. 2002. §4.4.5: 1998. §4.2: 1994.
§4.3.9.11.
Incomplete database. If an incomplete
database raises concern that further
studies might identify a more sensitive
effect, organ system, or lifestage, the
assessment may apply a database
uncertainty factor fU.S. EPA. 2002. §4.4.5:
1998. §4.2: 1996. §4: 1994. §4.3.9.1:
1991b. §3.41. The size of the factor
depends on the nature of the database
deficiency. For example, the EPA typically
follows the suggestion that a factor of 10
be applied if both a prenatal toxicity
study and a two-generation reproduction
study are missing and a factor of 101/2 if
either is missing fU.S. EPA. 2002. §4.4.51.
In this way, the assessment derives
candidate values for each suitable data set
and effect that is credibly associated with the
agent. These results are arrayed, using
common dose metrics, to show where effects
occur across a range of exposures fU.S. EPA.
1994. §4.3.91.
The assessment derives or selects an
organ- or system-specific reference value for
each organ or system affected by the agent.
The assessment explains the rationale for
each organ/system-specific reference value
(based on, for example, the highest quality
47	studies, the most sensitive outcome, or a
48	clustering of values). By providing these
49	organ/system-specific reference values, IRIS
50	assessments facilitate subsequent cumulative
51	risk assessments that consider the combined
52	effect of multiple agents acting at a common
53	site or through common mechanisms fNRC.
54	20091.
55	The assessment then selects an overall
56	reference dose and an overall reference
57	concentration for the agent to represent
58	lifetime human exposure levels where effects
59	are not anticipated to occur. This is generally
60	the most sensitive organ/system-specific
61	reference value, though consideration of
62	study quality and confidence in each value
63	may lead to a different selection.
64	7.7. Confidence and uncertainty in the
65	reference values
66	The assessment selects a standard
67	descriptor to characterize the level of
68	confidence in each reference value, based on
69	the likelihood that the value would change
70	with further testing. Confidence in reference
71	values is based on quality of the studies used
72	and completeness of the database, with more
73	weight given to the latter. The level of
74	confidence is increased for reference values
75	based on human data supported by animal
76	data fU.S. EPA. 1994. §4.3.9.21.
77	High confidence: The reference value is not
78	likely to change with further testing,
79	except for mechanistic studies that might
80	affect the interpretation of prior test
81	results.
82	Medium confidence: This is a matter of
83	judgment, between high and low
84	confidence.
85	Low confidence: The reference value is
86	especially vulnerable to change with
87	further testing.
88	These criteria are consistent with
89	guidelines for systematic reviews that
90	evaluate the quality of evidence. These also
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1	focus on whether further research would be
2	likely to change confidence in the estimate of
3	effect (Guvatt etal.. 2008b).
4	All assessments discuss the significant
5	uncertainties encountered in the analysis.
6	The EPA provides guidance on
7	characterization of uncertainty (U.S. EPA.
8	2005a. §3.61. For example, the discussion
9	distinguishes model uncertainty (lack of
10	knowledge about the most appropriate
11	experimental or analytic model) and
21
12	parameter uncertainty (lack of knowledge
13	about the parameters of a model).
14	Assessments also discuss human variation
15	(interpersonal differences in biologic
16	susceptibility or in exposures that modify the
17	effects of the agent).
18
19
20	August 2013
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EXECUTIVE SUMMARY
Occurrence and Health Effects
Ethyl tert-butyl ether (ETBE) is an ether oxygenate primarily used as a gasoline
additive. It was used until 2006 in the U.S., and continues to be used in Japan and the
European Union. ETBE is released into the environment as a result of gasoline leaks,
evaporation, and spills. Exposure to ETBE can occur by drinking contaminated
groundwater or by inhaling volatiles containing ETBE. Dermal exposure is possible in
occupational settings where the manufacture of ETBE occurs. The magnitude of
human exposure to ETBE depends on factors such as the distribution of ETBE in
groundwater and the extent of the contamination.
Animal studies demonstrate that exposure to ETBE is associated with kidney effects.
Available animal studies have not demonstrated ETBE to be associated with
reproductive or developmental effects. No epidemiological studies are available for
ETBE. Studies in rats suggest that ETBE may be carcinogenic in the liver. There are
no data in humans on carcinogenicity of ETBE. Studies in animals indicate that
deficient clearance of acetaldehyde, a metabolite of ETBE, could increase
susceptibility to ETBE toxicity or carcinogenicity.
Effects Other Than Cancer Observed Following Oral Exposure
EPA identified kidney effects as a human hazard ofETBE exposure, with increased kidney
weight in male and female rats accompanied by increased chronic progressive nephropathy (CPN),
urothelial hyperplasia (in males), and increased blood concentrations of total cholesterol, blood
urea nitrogen (BUN), and creatinine. Changes in kidney parameters were consistently observed, but
the magnitude of change was generally moderate, and males had greater severity of effects
compared with females. Overall, there was consistency across multiple measures of potential
kidney toxicity, including organ weight increases, exacerbated CPN, urothelial hyperplasia, and
increases in serum markers of kidney function. Additionally, effects were consistently observed
across routes of exposure, species, and sex; however, male rats appeared to be more sensitive to
exposure than female rats, and rats seemed to be more sensitive to exposure than mice. Mechanistic
data were insufficient to establish a mode of action; thus, kidney effects are considered relevant to
humans.
Increased liver weight and centrilobular hypertrophy in male and female rats were
consistently observed across studies. However, no additional histopathological findings were
observed, and only one serum marker 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
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effects was mild to moderate 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 a relationship between receptor activation and liver toxicity has not been
established for ETBE. However, mechanistic data suggest possible susceptibility related to
clearance of acetaldehyde, a metabolite ofETBE. Nonetheless, EPA concluded that the evidence
does not support liver effects as a potential human hazard ofETBE exposure.
No other noncancer effects were identified as adverse or exposure related; thus, EPA
concluded that the evidence does not support effects on the adrenals, the immune system, the
reproductive system, development, or mortality as potential human hazards ofETBE exposure.
Oral Reference Dose (RfD) for Effects Other Than Cancer
The chronic study by flPEC. 2010a) [selected data published as Suzuki etal. (20121] and the
observed increase incidences of urothelial hyperplasia were used to derive the RfD. The endpoint of
increased incidences of urothelial hyperplasia was selected as the critical effect due to its specificity
as an indicator of kidney toxicity, and the observed dose-response relationship of effects across
dose groups. Benchmark dose (BMD) modeling was utilized to derive the BMDLioo/0 of 60.5 mg/kg-
day. The BMDL was converted to a human equivalent dose of 14.5 mg/kg-day using body weight3/4
scaling, and this value was used as the point of departure (POD) for RfD derivation (U.S. EPA. 20111.
The proposed overall RfD was calculated by dividing the POD for increased absolute kidney
weight by a composite uncertainty factor (UF) of 30 to account for extrapolation from animals to
humans (10y2) and interindividual differences in human susceptibility (10).
Table ES-1. Summary of reference dose (RfD) derivation
Effect
Basis
RfD
(mg/kg-day)
Exposure
description
Confidence
Kidney toxicity
Increased urothelial hyperplasia
JPEC (2010b) [selected data
published as Saito et al. (2013)1
5 x 10 1
Chronic
HIGH
Proposed overall
RfD
Increased urothelial hyperplasia
JPEC (2010b) Selected data
oublished as Saito et al. (2013)1
5 x 10 1
Chronic
HIGH
Effects Other Than Cancer Observed Following Inhalation Exposure
EPA identified kidney effects as a human hazard ofETBE exposure. Studies in rats following
inhalation exposure have shown increases in kidney weights, nephropathy, mineralization,
urothelial hyperplasia, and increases in blood concentrations of cholesterol, BUN, and creatinine.
There were no available human studies that evaluated the effects ofETBE inhalation exposure.
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Mode-of-action analysis determined that kidney effects in male rats were not mediated by ct2u-
globulin, and these effects were concluded to be relevant for human health hazard assessment.
Inhalation Reference Concentration (RfC) for Effects Other Than Cancer
The chronic study by TPEC (2010bl [selected data published as Saito etal. (20131] and the
observed increase incidences of urothelial hyperplasia were used to derive the RfC. The endpoint of
increased incidences of urothelial hyperplasia was selected as the critical effect due to its specificity
as an indicator of kidney toxicity, and the observed dose-response relationship of effects across
dose groups. Benchmark dose (BMD) modeling was utilized to derive the BMCLioo/0 of 1498 mg/m3.
The BMCL was adjusted to a continuous exposure and converted to a human equivalent
concentration of 265 mg/m3.
The RfC was calculated by dividing the POD by a composite UF of 30 to account for
toxicodynamic differences between animals and humans (3) and interindividual differences in
human susceptibility (10).
Table ES-2. Summary of reference concentration (RfC) derivation
Effect
Basis
RfC
(mg/m3)
Exposure
Description
Confidence
Kidney toxicity
Increased urothelial hyperplasia
Saito et al. (2013); JPEC (2010b)
9x 10°
Chronic
HIGH
Proposed overall RfC
Increased urothelial hyperplasia
Saito et al. (2013): JPEC (2010b)
9x10°
Chronic
HIGH
Evidence for Carcinogenicity
Under EPA's Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005al. there is
"suggestive evidence of carcinogenic potential" for ETBE based on evidence in rats. The limited
evidence includes three bioassays in rats exposed via inhalation, drinking water, or gavage,
inadequate data in other experimental species or in humans, and limited mechanistic data. One 2-
year inhalation rat study observed a statistically significant increase in hepatocellular adenomas
and carcinomas in male rats at a single dose, but no other bioassay reported increased incidence of
liver tumors. Mechanistic data were inadequate to establish a mode of action. Mechanistic studies
reported that deficient enzyme function of aldehyde dehydrogenase 2 (ALDH2) enhanced ETBE-
induced genotoxicity in hepatocytes and leukocytes, suggestive of genotoxicity being mediated by
the ETBE metabolite acetaldehyde, which is directly genotoxic flARC. 20121. Overall, because a
statistically significant increase occurred at one dose only without a significant response at other
doses and no overall trends, and because the mechanistic data only provide some evidence of
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biological plausibility, ETBE is characterized as having "suggestive evidence of carcinogenic
potential."
Quantitative Estimate of Carcinogenic Risk from Oral Exposure
The main evidence ofETBE carcinogenicity consisted of the increased incidence of liver
tumors in male F344 rats following inhalation exposure fSaito etal.. 2013: TPEC. 2010b). This study
examined 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 methods
and results (including individual animal data).
Although ETBE was considered to have "suggestive evidence of carcinogenic potential," EPA
concluded that the main study was well-conducted and quantitative analyses may be useful for
providing a sense of the magnitude of potential carcinogenic risk. A PBPK model in rats for ETBE
and its metabolite, tert-butanol, was used for route-to-route extrapolation of the inhalation BMCLio
(described below) to an oral equivalent BMDLio, which was adjusted to a human equivalent BMDLio
on the basis of (body weight)3/4 scaling fU.S. EPA. 2011. 2005a). Using linear extrapolation from the
BMDLio, a human equivalent oral slope factor was derived (slope factor = 0.1/BMDLio). The oral
slope factor is 9 x 104 per mg/kg-day based on the liver tumor response in male rats (Saito etal..
2013: TPEC. 2010bl.
Quantitative Estimate of Carcinogenic Risk from Inhalation Exposure
Lifetime inhalation exposure to ETBE has been associated with increased liver adenomas
and carcinomas in male F344 rats. This is the only evidence of carcinogenicity following inhalation
exposure (Saito etal.. 2013: TPEC. 2010b): however, the biological plausibility of these data are
supported by mechanistic data on tumor promotion and genotoxicity in the absence of ALDH2, and
are analogous to the human carcinogenicity of acetaldehyde after consumption of ethanol. This
study examined 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
methods and results (including individual animal data).
Although ETBE was considered to have "suggestive evidence of carcinogenic potential," EPA
concluded that the main study was well-conducted and quantitative analyses may be useful for
providing a sense of the magnitude of potential carcinogenic risk. EPA used the multistage 1° model
for the derivation of the BMCLio, which was then adjusted to a human equivalent BMCLio on the
basis of inhalation dosimetry (U.S. EPA. 1994). Using linear extrapolation (inhalation unit risk =
0.1 /BMCLio), a human equivalent inhalation unit risk was derived. The inhalation unit risk is
8 x 10"5 per mg/m3 based on the liver tumor response in F344 male rats fSaito etal.. 2013: TPEC.
2010b).
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Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes
ETBE is metabolized to tert-butanol and acetaldehyde. There is suggestive evidence that
genetic polymorphisms of aldehyde dehydrogenase (ALDH)—the enzyme that oxidizes
acetaldehyde to acetic acid—may affect ETBE toxicity. The virtually inactive form, ALDH2*2, is
found in about one-half of all East Asians. 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. Several in vivo and in vitro genotoxic assays in
Aldh2 knockout (KO) mice reported that genotoxicity was significantly increased compared with
wild type controls following ETBE exposure to similar doses associated with cancer and noncancer
effects (Weng etal.. 2014: Wengetal.. 2013: Weng etal.. 2012: Wengetal.. 20111. Inhalation ETBE
exposure increased blood concentrations of acetaldehyde in Aldh2 knockout mice compared with
wild type. Altogether, these data present evidence that diminished ALDH2 activity could yield more
severe health effect outcomes in sensitive human populations.
Key Issues Addressed in Assessment
Sufficient data were available to develop a PBPK model in rats for both oral and inhalation
exposure that could be used to perform route-to-route extrapolation; therefore, rat studies from
both routes of exposure were considered for dose-response analysis. Analysis of the noncancer
endpoint available from the chronic inhalation and oral studies led to very similar PODs and
candidate references values when extrapolated across routes, so the route-specific chronic data
were used as the basis for the RfC and RfD. With respect to carcinogenic effects, the only available
inhalation 2-year study had the most robust evidence of carcinogenicity and was selected for route-
to-route extrapolation.
ETBE induced an increase in a2U-globulin deposition and increased hyaline droplet
accumulation in male rats; however, most of the subsequent steps in the pathological sequence
were not observed despite identical study conditions and doses in a number of experiments over a
2-year exposure period. These data fail to provide sufficient evidence that the a2U-globulin process
is operative. EPA finds that the data are insufficient to demonstrate a2U-globulin nephropathy due
to ETBE exposure; thus, the male rat kidney data are relevant for humans.
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LITERATURE SEARCH STRATEGY | STUDY
SELECTION AND EVALUATION
A literature search and screening strategy was used to identify literature characterizing the
health effects ofETBE. This strategy consisted of a broad search of online scientific databases and
other sources in order 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. This section describes the
literature search and screening strategy in detail.
The chemical-specific search was conducted in four online scientific databases, including
PubMed, Toxline, Web of Science, and TSCATS through March, 2014, 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,
808 unique citations were identified.
The resulting 808 citations were screened into categories as presented in Figure LS-1 using
the title, abstract, and/or full text for relevance in examining the health effects of ETBE exposure.
•	31 references were identified as potential "Sources of Health Effects Data" and were
considered for data extraction to evidence tables and exposure-response arrays.
•	51 references were identified as "Supporting Studies." These included 20 studies describing
physiologically-based pharmacokinetic (PBPK) models and other toxicokinetic information;
16 studies providing genotoxicity and other mechanistic information; 9 acute, short term, or
preliminary toxicity studies; 1 human toxicokinetic study; and 5 direct administration (e.g.,
dermal) studies ofETBE. While 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
supporting health effects information.
•	16 references were identified as secondary sources of health effects information (e.g.,
reviews and other agency assessments); these references were kept as additional resources
for development of the Toxicological Review.
•	710 references were identified as not being pertinent to an evaluation of health effects for
ETBE and were excluded from further consideration (see Figure LS-1 for exclusion
categories).
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1	The complete list of references as sorted above can be found on the HERO website at
2	http://hero.epa.gov/ETBE.
3
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Toxicological Review ofETBE
Pub me d
n=214
Database Searches
(see Table LS-1 fur keywords and limits)
Web of Science
n=532
Toxline
(incl. TSCATS)
n=il9
TSCATS 2
n=l
J
n = 114
Additional Search Strategies
(see Table LS-2 for methods arid
results)
Combined Dataset
(After duplicates removed electronically)
n=808
Manual Screening For Pertinence
(Title/Abstract/Full Text)
Supporting Studies (n=Sl
PBPK/ADME
Genotoxidty
Other mechanistic
studies
Preliminary /Acute
/short-term studies
Human "K study
Dermal or ocular
administration
Secondary Sources of
Health Effects
Information (n=16)
M Reviews, editorials
2 Health agency
assessments
Sources of Health Effects
Data (n=31)
Animal studies
Excluded (not pertinent) (n=710)
77 Biodegradation/ environ menial fate
362 Chemical analysis/Fuel chemistry
174 Other chemical/rwn L BL studies
31 Policy/Commentary
18 Abstract only
3 Exposure studies
S Analytical methods
2 Odor Lhres hold
. .1 . I , I . I ¦	¦¦¦¦¦; I , ¦ ,
33 Duplicates (identified by title, abstract,
or full text)
2	Figure LS-1. Literature search approach for ETBE
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1	Table LS-1. Database search strategy for ETBE
Database
(Search Date)
Keywords
Limits
PubMed
(03/31/2014)
"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)
"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
Toxline
(includes
TSCATS)
(03/31/2014)
"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)
637-92-3
01/01/2004 to 03/31/2014
2
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1 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
Japanese Petroleum Energy Center
3/2014
20 references
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Selection of Critical Studies for Inclusion in Evidence Tables
Each study retained after the literature search and screen was evaluated for aspects of its
design or conduct per the Preamble that could affect the interpretation of results and overall
contribution to the evidence for determination of hazard potential. 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.
To facilitate this evaluation, evidence tables were constructed that systematically
summarized the important information from each study in a standardized tabular format as
recommended by the NRC (20111. Thirty-one studies identified as "Sources of Health Effects" were
considered for extraction into evidence tables for hazard identification in Chapter 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 et al.. 19971 that
examined neurotoxic endpoints only was not included in evidence tables. Data from the remaining
30 studies were extracted into evidence tables.
Supporting 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, pilot studies, and
short-term or acute studies—were not included in the evidence tables. Such supporting studies
may be discussed in the narrative sections of Chapter 1 or presented in Appendices if they provide
additional or corroborating information.
Database Evaluation
The database for ETBE is comprised of animal toxicity studies containing three 2-year
bioassays that employ oral and inhalation exposures in rats, and several studies with oral and
inhalation exposures of >90 days in rats and mice. EPA externally peer-reviewed six unpublished
technical reports prior to their subsequent publication: TPEC f2010al. TPEC. 2010b. TPEC. 2008a.
TPEC. 2008c. and the pharmacokinetic studies TPEC (2008el and TPEC (2008dl. Several acute and
short-term studies using oral and inhalation exposures were performed in rats but were grouped as
supporting studies because the database of chronic and subchronic rat studies was considered most
relevant for characterizing chronic health effects. No cohort studies, case reports, or ecological
studies were found in the published literature. 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; thus, these studies were excluded during the manual screen.
One controlled human exposure toxicokinetic study was identified, and this is discussed in
Appendix B.2 (Toxicokinetics).
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Some general questions that were considered in evaluating experimental animal studies are
presented in Table LS-3. The "Sources of Health Effects Data" was comprised entirely of studies
performed in rats, mice, and rabbits associated with drinking water, oral gavage, or inhalation
exposures to ETBE. A large proportion of these 31 studies were conducted according to OECD Good
Laboratory Practice (GLP) guidelines, presented extensive histopathological data, and provided
clear presentation of the methodology; thus, these are considered high quality. Preliminary, acute,
and short term studies contained information that supported but did not differ qualitatively from
the results of the >90 day exposure studies; thus, these studies were not included in the evidence
tables. Some of these shorter duration studies are presented in the text of the Toxicological Review
and are described in sections such as the "Mechanistic Evidence" to augment the discussion. A more
detailed discussion of methodological concerns that were identified will precede each endpoint
evaluated in the hazard identification section.
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1	Table LS-3. Questions and relevant experimental information for evaluation of
2	experimental animal studies
Methodological
feature
Question(s) considered
Examples of relevant
information extracted
Test animal
Based on the endpoint(s) in question, are
concerns raised regarding the suitability
of the species, strain, or sex of the test
animals on study?
Test animal species, strain, sex
Experimental setup
Are the timing, frequency and duration of
exposure, as well as animal age and
experimental group allocation
procedures/ group size for each endpoint
evaluation, appropriate for the assessed
endpoint(s)?
Age/lifestage of test animals at exposure
and all endpoint testing time points
Timing and periodicity of exposure and
endpoint evaluations; duration of
exposure
Sample size for each experimental group
(e.g., animals; litters; dams) at each
endpoint evaluation
Exposure
Are the exposure conditions and controls
informative and reliable for the
endpoint(s) in question, and are they
sufficiently specific to the compound of
interest?
Exposure administration techniques (e.g.,
route; chamber type)
Endpoint evaluation
procedures
Do the procedures used to evaluate the
endpoint(s) in question conform to
established protocols, or are they
biologically sound? Are they sensitive for
examination of the outcome(s) of
interest?
Specific methods for assessing the
effect(s) of exposure, including related
details (e.g., specific region of
tissue/organ evaluated)
Endpoint evaluation controls, including
those put in place to minimize evaluator
bias
Outcomes and data
reporting
Were data reported for all pre-specified
endpoint(s) and study groups, or were any
data excluded from presentation/
analyses?
Data presentation for endpoint(s) of
interest
Note: "Outcome" refers to findings from an evaluation (e.g., hypertrophy), whereas "endpoint" refers to the
evaluation itself (e.g., liver histopathology).
3
4
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
1.HAZARD IDENTIFICATION
1.1. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM
1.1.1. Kidney Effects
Synthesis of Effects in Kidney
This section reviews the studies that investigated whether exposure to ETBE can cause
kidney toxicity or cancer in humans or animals. The database examining kidney effects following
ETBE exposure contains no human data, and 10 studies are performed in animals, predominantly
rats. Studies employing 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
support mode of action or hazard identification. EPA externally peer-reviewed six unpublished
technical reports prior to their subsequent publication: TPEC f2010al. TPEC. 2010b. TPEC. 2008a.
TPEC. 2008c. and the pharmacokinetic studies TPEC (2008g) and TPEC (2008f). No methodological
concerns were identified that would lead one or more studies to be considered less informative for
assessing human health hazard, although the report by Cohen etal. (20111 was not peer reviewed
externally. This report (Cohen etal.. 20111 consists of a pathology working group review
commissioned by the Lyondell Chemical Company to reexamine kidney histopathology from the
TPEC f2010al [subsequently published as Suzuki etal. f20121]and TPEC f20071 studies. Ail
reanalysis was conducted in a blinded manner with the exception of the analysis of 2-year tumor
data, data from low and intermediate doses in females, and data in all males from the control and
high doses. Cohen etal. (20111 did not report different incidences of carcinomas than the original
(Suzuki etal.. 2012: TPEC. 2010a) study; thus, these data will not be presented twice.
Histopathological results from both Cohen etal. (20111 and JPEC will be considered for hazard
identification.
The kidney effects observed were increased organ weight, increased severity of
histopathological lesions such as chronic progressive nephropathy (CPN), and urine and serum
biomarkers (see Table 1-1, Table 1-2, Table 1-3; Figure 1-1, Figure 1-2). No statistically significant
increases in renal tumors were observed in chronic bioassays (see Table 1-4). Kidney effects were
not observed in the lone mouse study; however, lack of additional mouse studies precludes a
conclusion on the species specificity of ETBE-induced kidney effects (Medinskv etal.. 19991.
In most of the studies with data available for relative and absolute organ weight
comparisons, relative kidney weights are increased to a greater extent than absolute kidney
weights fMivata etal.. 2013: Saito etal.. 2013: Suzuki etal.. 2012: TPEC. 2010b. 2008b. c; Gaoua.
2004b). Regression analysis indicates there is no discernible advantage to presenting absolute or
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
relative kidney weights fBailev etal.. 20041: thus, both absolute and relative weight were evaluated
to make a determination of hazard. Absolute and relative kidney weights were dose-responsively
increased in male and female rats following oral exposures of 16 weeks or longer fFuiii etal..
2010)(Miyata etal.. 2013: TPEC. 2008c)(Suzuki etal.. 2012: TPEC. 2010a). Absolute or relative
kidney weight increases in rats were also dose-responsive following inhalation exposures of 13
weeks or longer (TPEC. 2008b)(Medinsky etal.. 1999)(Saito etal.. 2013: TPEC. 2010b). Short-term
studies in rats also observed increased kidney weight flPEC. 2008al.
The number and size of hyaline droplets were increased in the proximal tubules of male
rats, but not females, and the hyaline droplets tested positive for the presence of a2U-globulin
fMivata etal.. 2013: TPEC. 2008c. e, f; Medinskv et al.. 19991. The significance of this effect, along
with other potentially related histopathological effects, such as necrosis, mineralization, and
tubular hyperplasia, will be discussed in the succeeding section on Mode of Action.
The incidence of CPN, which was characterized by sclerosis of glomeruli, thickening of the
renal tubular basement membranes, inflammatory cell infiltration and interstitial fibrosis, was not
increased in any study as a result of ETBE exposure; however, the severity of CPN was exacerbated
by ETBE in male and female rats in a 2-year inhalation study and in male rats in a 13-week drinking
water study (see Table l-21(Cohen etal.. 2011: (Saito etal.. 2013: TPEC. 2010b): (TPEC). 20071.
Increased incidence of urothelial hyperplasia was observed in male rats in two-year studies by both
inhalation and oral exposure (Suzuki etal.. 2012: TPEC. 2010a: (Saito etal.. 2013: TPEC. 2010b).
Cohen et al. (20111 attributed this effect to CPN rather than the "direct" result of ETBE treatment
The biological significance of this effect will be discussed in the succeeding Mode of Action Analysis.
The increased kidney weight and CPN in male rats is associated with several changes in
urinary and serum biomarkers of renal function (see Table 1-3). CPN elicits a number of changes in
urinary and blood serum measures such as proteinuria, blood urea nitrogen, creatinine, and
hypercholesterolemia (Hard etal.. 20091. Male rat blood concentrations of total cholesterol, blood
urea nitrogen (BUN), and creatinine were elevated in 3, 2, and 1 out of 4 chronic and subchronic
studies, respectively (Mivata etal.. 2013: Saito etal.. 2013: Suzuki etal.. 2012: TPEC. 2010a. b,
2008c)- With respect to female rats, cholesterol and BUN were elevated at the highest dose in one
chronic inhalation study, which corresponded with increased CPN (Saito etal.. 2013: TPEC. 2010b).
The single instance of elevated proteinuria in male and female rats occurred in a chronic inhalation
study (Saito etal.. 2013: TPEC. 2010b).
The 2-year kidney weight data are not appropriate for hazard identification due the
prevalence of age-associated confounders such as CPN and mortality that affect organ weight
analysis. CPN is an age-associated disease characterized by cell proliferation and chronic
inflammation that results in increased kidney weight fMelnick etal.. 2012: Travlos etal.. 20111. The
majority (64-100%) of the male and female rats in the 2-year oral and inhalation studies were
observed to have CPN regardless ofETBE administration f Saito etal.. 2013: Suzuki etal.. 2012:
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
1	TPEC. 2010a. b). In addition, mortality in the 2-year studies was significantly increased in ETBE-
2	treated male and female rats compared with controls following oral and inhalation exposure (see
3	Table 1-21). Causes of death were the result of age-associated diseases, such as CPN and tumors.
4	Using kidney weight data from these 2-year studies would impart bias by selecting animals that
5	survive to the end of the study for organ weight analysis. Thus, the 2-year organ weight data are not
6	appropriate for hazard identification.
7	Table 1-1. Evidence pertaining to kidney weight effects in animals exposed to
8	ETBE
Reference and Dosing Protocol
Results by Endpoint
Kidney: Absolute Weight
Fuiiietal. (2010); JPEC (2008e)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, male (24/group): 0,100, 300,1000 mg/kg-d
P0, Male
0
-
daily for 16 weeks beginning 10 weeks prior to

100
5%
mating

300
8%
P0, female (24/group): 0,100, 300,1000 mg/kg-d

1000
18%*
daily for 17 weeks beginning 10 weeks prior to

Dose(mg/kg-d)
Percent change
mating to lactation day 21


compared to



control

P0, Female
0
-


100
-2%


300
0%


1000
7%*
9
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-1. Evidence pertaining to kidney weight effects in animals exposed
to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Kidney: Absolute Weight (continued)
Gaoua (2004b)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, male (25/group): 0, 250, 500,1000 mg/kg-d
P0, Male
0
-
daily for a total of 18 weeks beginning 10 weeks

250
11%*
before mating until after weaning of the pups

500
15%*
P0, female (25/group): 0, 250, 500,1000 mg/kg-d

1000
21%*
daily for a total of 18 weeks beginning 10 weeks

Dose(mg/kg-d)
Percent change
before mating until PND 21


compared to
Fl, males and females (25/group/sex): via P0


control
dams in utero daily through gestation and
Fl, Male
0
-
lactation, then Fl doses beginning PND 22 until

250
10%
weaning of the F2 pups

500
22%*


1000
58%*


Dose(mg/kg-d)
Percent change



compared to



control

P0, Female
0
-


250
-1%


500
2%


1000
5%


Dose(mg/kg-d)
Percent change



compared to



control

Fl, Female
0
-


250
4%


500
3%


1000
11%*
Hagiwara et al. (2011); JPEC (2008d)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - gavage


control
male (12/group): 0,1000 mg/kg-d
Male
0
-
daily for 23 weeks

1000
19%*
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-1. Evidence pertaining to kidney weight effects in animals exposed
to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Kidney: Absolute Weight (continued)
Mivata et al. (2013);JPEC (2008c)

Dose(mg/kg-d)
Percent change
rat, CRL:CD(SD)


compared to
oral - gavage


control
female (15/group): 0, 5, 25,100, 400 mg/kg-d;
Male
0
-
male (15/group): 0, 5, 25,100, 400 mg/kg-d

5
1%
daily for 180 days

25
6%


100
5%


400
25%*


Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


5
1%


25
0%


100
7%


400
10%*
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - water


control
female (50/group): 0, 625, 2500,10,000 ppm (0,
Male
0
-
46,171, 560 mg/kg-day)a; male (50/group): 0,

28
-4%
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

121
5%
day)a

542
18%*
daily for 104 wks

Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


46
3%


171
10%*


560
14%*
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-1. Evidence pertaining to kidney weight effects in animals exposed
to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Kidney: Absolute Weight (continued)
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,
Male
0
-
2090, 6270, 20,900 mg/m3); male (NR): 0, 150,

627
10%
500, 1500, 5000 ppm (0, 627, 2090, 6270,

2090
11%
20,900 mg/m3)b

6270
18%*
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

20,900
16%*
13 wks; generation method, analytical

Dose(mg/m3)
Percent change
concentration and method were reported


compared to



control

Female
0
-


627
1%


2090
-1%


6270
4%


20,900
7%
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (6/group): 0, 5000 ppm (0,
Male
0
-
20,900 mg/m3)b; male (6/group): 0, 5000 ppm (0,

20,900
19%
20,900 mg/m3)b

Dose(mg/m3)
Percent change
dynamic whole body chamber; 6 hrs/d, 5 d/wk for


compared to
13 wks followed by a 28 day recovery period;


control
generation method, analytical concentration and
Female
0
-
method were reported

20,900
8%
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-1. Evidence pertaining to kidney weight effects in animals exposed
to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Kidney: Absolute Weight (continued)
Medinskv et al. (1999); Bond et al. (1996b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor


control
female (48/group): 0, 500,1750, 5000 ppm (0,
Male
0
-
2090, 7320, 20,900 mg/m3)b; male (48/group): 0,

2090
7%
500, 1750, 5000 ppm (0, 2090, 7320,

7320
10%*
20,900 mg/m3)b

20,900
19%*
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

Dose(mg/m3)
Percent change
13 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


2090
4%


7320
12%*


20,900
21%*
Medinskv et al. (1999); Bond et al. (1996a)

Dose(mg/m3)
Percent change
mice, CD-I


compared to
inhalation - vapor


control
female (40/group): 0, 500,1750, 5000 ppm(0,
Male
0
-
2090, 7320, 20,900 mg/m3)b; male (40/group): 0,

2090
9%
500, 1750, 5000 ppm (0, 2090, 7320,

7320
10%
20,900 mg/m3)b

20,900
5%
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

Dose(mg/m3)
Percent change
13 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


2090
0%


7320
6%


20,900
4%
1
2
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-1. Evidence pertaining to kidney weight effects in animals exposed
to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Kidney: Absolute Weight (continued)
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor


control
female (50/group): 0, 500,1500, 5000 ppm (0,
Male
0
-
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

2090
8%*
500, 1500, 5000 ppm (0, 2090, 6270,

6270
17%*
20,900 mg/m3)b

20,900
22%*
dynamic whole body inhalation; 6 hrs/d, 5 d/wk

Dose(mg/m3)
Percent change
for 104 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


2090
5%


6270
6%*


20,900
18%*
Kidney: Relative Weight
Fuiiietal. (2010); JPEC (2008e)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, male (24/group): 0,100, 300,1000 mg/kg-d
P0, Male
0
-
daily for 16 weeks beginning 10 weeks prior to

100
8%*
mating

300
12%*
P0, female (24/group): 0,100, 300,1000 mg/kg-d

1000
26%*
daily for 17 weeks beginning 10 weeks prior to

Dose(mg/kg-d)
Percent change
mating to lactation day 21


compared to



control

P0, Female
0
-


100
-3%


300
-1%


1000
2%
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-1. Evidence pertaining to kidney weight effects in animals exposed
to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Kidney: Relative Weight (continued)
Gaoua (2004b)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, male (25/group): 0, 250, 500,1000 mg/kg-d
P0, Male
0
-
daily for a total of 18 weeks beginning 10 weeks

250
11%*
before mating until after weaning of the pups

500
18%*
P0, female (25/group): 0, 250, 500,1000 mg/kg-d

1000
28%*
daily for a total of 18 weeks beginning 10 weeks

Dose(mg/kg-d)
Percent change
before mating until PND 21


compared to
Fl, males and females (25/group/sex): via P0


control
dams in utero daily through gestation and
Fl, Male
0
-
lactation, then Fl doses beginning PND 22 until

250
10%*
weaning of the F2 pups

500
19%*


1000
58%*


Dose(mg/kg-d)
Percent change



compared to



control

P0, Female
0
-


250
9%


500
5%


1000
3%


Dose(mg/kg-d)
Percent change



compared to



control

Fl, Female
0
-


250
6%


500
6%


1000
10%*
Hagiwara et al. (2011); JPEC (2008d)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - gavage


control
male (12/group): 0,1000 mg/kg-d
Male
0
-
daily for 23 weeks

1000
25%*
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-1. Evidence pertaining to kidney weight effects in animals exposed
to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Kidney: Relative Weight (continued)
Mivata et al. (2013); JPEC (2008c)

Dose(mg/kg-d)
Percent change
rat, CRL:CD(SD)


compared to
oral - gavage


control
female (15/group): 0, 5, 25,100, 400 mg/kg-d;
Male
0
-
male (15/group): 0, 5, 25,100, 400 mg/kg-d

5
8%
daily for 180 days

25
6%


100
12%*


400
21%*


Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


5
7%


25
4%


100
11%*


400
15%*
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - water


control
female (50/group): 0, 625, 2500,10,000 ppm (0,
Male
0
-
46,171, 560 mg/kg-day)a; male (50/group): 0,

28
0%
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

121
12%*
day)a

542
31%*
daily for 104 wks

Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


46
13%*


171
22%*


560
37%*
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-1. Evidence pertaining to kidney weight effects in animals exposed
to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Kidney: Relative Weight (continued)
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,
Male
0
-
2090, 6270, 20,900 mg/m3); male (NR): 0, 150,

627
10%
500, 1500, 5000 ppm (0, 627, 2090, 6270,

2090
9%
20,900 mg/m3)b

6270
20%*
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

20,900
24%*
13 wks; generation method, analytical

Dose(mg/m3)
Percent change
concentration and method were reported


compared to



control

Female
0
-


627
8%


2090
7%


6270
12%*


20,900
20%*
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (6/group): 0, 5000 ppm (0,
Male
0
-
20,900 mg/m3)b; male (6/group): 0, 5000 ppm (0,

20,900
15%*
20,900 mg/m3)b

Dose(mg/m3)
Percent change
dynamic whole body chamber; 6 hrs/d, 5 d/wk for


compared to
13 wks followed by a 28 day recovery period;


control
generation method, analytical concentration and
Female
0
-
method were reported

20,900
5%
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-1. Evidence pertaining to kidney weight effects in animals exposed
to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Kidney: Relative Weight (continued)
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor


control
female (50/group): 0, 500,1500, 5000 ppm (0,
Male
0
-
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

2090
19%*
500, 1500, 5000 ppm (0, 2090, 6270,

6270
26%*
20,900 mg/m3)b

20,900
66%*
dynamic whole body inhalation; 6 hrs/d, 5 d/wk

Dose(mg/m3)
Percent change
for 104 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


2090
11%*


6270
16%*


20,900
51%*
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
7
8
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
1	Table 1-2. Evidence pertaining to kidney nephropathy and histopathological
2	effects in animals exposed to ETBE
Reference and Dosing Protocol
Results by Endpoint
Incidence of Chronic Nephropathy
Cohen et al. (2011)

Dose(mg/kg-d)
Response
rat, F344/DuCrlCrlj


(incidence)
oral - water
Male
0
49/50
female (50/group): 0, 625, 2500,10,000 ppm (0,

28
-
46,171, 560 mg/kg-d)a; male (50/group): 0, 625,

121
-
2500, 10,000 ppm (0, 28, 121, 542 mg/kg-d)a

542
50/50
reanalysis of the histopathology from JPEC

Dose(mg/kg-d)
Response
(2010a) studv where animals were dosed dailv for


(incidence)
104 wks
Female
0
45/50


46
41/50


171
46/50


560
46/50
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Response
rat, Fischer 344


(incidence)
oral - water
Male
0
49/50
female (50/group): 0, 625, 2500,10,000 ppm (0,

28
43/50
46,171, 560 mg/kg-day)a; male (50/group): 0,

121
45/50
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

542
48/50
day)a

Dose(mg/kg-d)
Response
daily for 104 wks


(incidence)

Female
0
41/50


46
37/50


171
37/50


560
39/50
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Response
rat, Fischer 344


(incidence)
inhalation - vapor
Male
0
49/50
female (50/group): 0, 500,1500, 5000 ppm (0,

2090
50/50
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

6270
49/49
500, 1500, 5000 ppm (0, 2090, 6270,

20,900
50/50
20,900 mg/m3)b

Dose(mg/m3)
Response
dynamic whole body inhalation; 6 hrs/d, 5 d/wk


(incidence)
for 104 wks; generation method, analytical
Female
0
32/50
concentration and method were reported

2090
38/50


6270
41/50


20,900
40/50
This document is a draft for review purposes only and does not constitute Agency policy.
1-13	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-2. Evidence pertaining to kidney nephropathy and histopathological
effects in animals exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Average Severity of Chronic Nephropathy
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Response
rat, Fischer 344


(severity)
oral - water
Male
0
2.1
female (50/group): 0, 625, 2500,10,000 ppm (0,

28
2
46,171, 560 mg/kg-day)a; male (50/group): 0,

121
2
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

542
2.4
day)a

Dose(mg/kg-d)
Response
daily for 104 wks


(severity)

Female
0
1.2


46
1.2


171
1.5


560
1.5
Cohen et al. (2011)

Dose(mg/kg-d)
Response
rat, F344/DuCrlCrlj


(severity)
oral - water
Male
0
2.08
female (50/group): 0, 625, 2500,10,000 ppm (0,

28
-
46,171, 560 mg/kg-d)a; male (50/group): 0, 625,

121
-
2500, 10,000 ppm (0, 28, 121, 542 mg/kg-d)a

542
2.72
reanalysis of the histopathology from JPEC 2010

Dose(mg/kg-d)
Response
(HERO ID 1561279) study where animals were


(severity)
dosed daily for 104 wks
Female
0
1.14


46
0.98


171
1.2


560
1.36
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Response
rat, Fischer 344


(severity)
inhalation - vapor
Male
0
2.4
female (50/group): 0, 500,1500, 5000 ppm (0,

2090
2.6
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

6270
2.7
500, 1500, 5000 ppm (0, 2090, 6270,

20,900
3.1*
20,900 mg/m3)b

Dose(mg/m3)
Response
dynamic whole body inhalation; 6 hrs/d, 5 d/wk


(severity)
for 104 wks; generation method, analytical
Female
0
0.9
concentration and method were reported

2090
1.3


6270
1.3


20,900
1.6*
This document is a draft for review purposes only and does not constitute Agency policy.
1-14	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-2. Evidence pertaining to kidney nephropathy and histopathological
effects in animals exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Average Severity of Chronic Nephropathy as Calculated by EPA
Suzuki et al. (2012); JPEC (2010a)
rat, Fischer 344
oral - water
female (50/group): 0, 625, 2500,10,000 ppm (0,
46,171, 560 mg/kg-day)a; male (50/group): 0,
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-
day)a
daily for 104 wks
Dose(mg/kg-d) Response
(severity)
Male 0 2.1
28 1.7
121 1.8
542 2.3
Average severity calculated as (grade x # of affected
animals)/total # of animals exposed
Dose(mg/kg-d) Response
(severity)
Female 0 1
46 0.9
171 1.1
560 1.2
Average severity calculated as (grade x # of affected
animals)/total # of animals exposed
Number of CPN Foci
Cohen et al. (2011)
rat, F344/DuCrlCrlj
oral - water
male (10/group): 0, 250,1600, 4000,10000 ppm
reanalysis of the histopathology from JPEC 2006
(study No. 0665) study where animals were dosed
daily for 13 weeks
Dose(ppm) Response
(foci/rat)
Male 0 1.2
250
1600
4000
10000 27.2
This document is a draft for review purposes only and does not constitute Agency policy.
1-15	DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofETBE
Table 1-2. Evidence pertaining to kidney nephropathy and histopathological
effects in animals exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Slight Urothelial Hyperplasia of the Renal Pelvis
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Response
rat, Fischer 344


(incidence)
oral - water
Male
0
0/50
female (50/group): 0, 625, 2500,10,000 ppm (0,

28
0/50
46,171, 560 mg/kg-day)a; male (50/group): 0,

121
10/50*
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-d)a

542
25/50*
daily for 104 wks
Female



urothelial hyperplasia of the renal pelvis not observed
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Response
rat, Fischer 344


(incidence)
inhalation - vapor
female (50/group): 0, 500,1500, 5000 ppm (0,
Male
0
2090
2/50
5/50
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

6270
16/49*
500, 1500, 5000 ppm (0, 2090, 6270,
20,900 mg/m3)b
dynamic whole body inhalation; 6 hrs/d, 5 d/wk
for 104 wks; generation method, analytical

20,900
41/50*
Female
urothelial hyperplasia of the renal pelvis not observed
concentration and method were reported



Incidence of Atypical Tubule Hyperplasia
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Response
rat, Fischer 344


(incidence)
oral - water
Male
0
0/50
female (50/group): 0, 625, 2500,10,000 ppm (0,

28
0/50
46,171, 560 mg/kg-day)a; male (50/group): 0,

121
0/50
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

542
1/50
day)a
daily for 104 wks

Dose(mg/kg-d)
Response
(incidence)

Female
0
46
171
560
0/50
0/50
0/50
2/50
This document is a draft for review purposes only and does not constitute Agency policy.
1-16	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-2. Evidence pertaining to kidney nephropathy and histopathological
effects in animals exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Incidence of Atypical Tubule Hyperplasia (continued)
Saito et al. (2013); JPEC (2010b)
rat, Fischer 344
inhalation - vapor
female (50/group): 0, 500,1500, 5000 ppm (0,
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,
500, 1500, 5000 ppm (0, 2090, 6270,
20,900 mg/m3)b
dynamic whole body inhalation; 6 hrs/d, 5 d/wk
for 104 wks; generation method, analytical
concentration and method were reported
Male
atypical tubule hyperplasia not observed
Female
atypical tubule hyperplasia not observed
Incidence of Papillary Mineralization
Mivata et al. (2013); JPEC (2008c)
rat, CRL:CD(SD)
oral - gavage
female (15/group): 0, 5, 25,100, 400 mg/kg-d;
male (15/group): 0, 5, 25,100, 400 mg/kg-d
daily for 180 days
Dose(mg/kg-d) Response
(incidence)
Male 0 0/15
5 0/15
25 0/15
100 1/15
400 0/15
Dose(mg/kg-d) Response
(incidence)
Female 0 0/15
5
25
100
400 0/15
Suzuki et al. (2012); JPEC (2010a)
rat, Fischer 344
oral - water
female (50/group): 0, 625, 2500,10,000 ppm (0,
46,171, 560 mg/kg-day)a; male (50/group): 0,
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-
day)a
daily for 104 wks
Dose(mg/kg-d) Response
(incidence)
Male 0 0/50
28 0/50
121 16/50*
542 42/50*
Dose(mg/kg-d) Response
(incidence)
Female 0 0/50
46 0/50
171 1/50
560 3/50
This document is a draft for review purposes only and does not constitute Agency policy.
1-17	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-2. Evidence pertaining to kidney nephropathy and histopathological
effects in animals exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Incidence of Papillary Mineralization (continued)
Saito et al. (2013); JPEC (2010b)
rat, Fischer 344
inhalation - vapor
female (50/group): 0, 500,1500, 5000 ppm (0,
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,
500, 1500, 5000 ppm (0, 2090, 6270,
20,900 mg/m3)b
dynamic whole body inhalation; 6 hrs/d, 5 d/wk
for 104 wks; generation method, analytical
concentration and method were reported
Dose(mg/m3) Response
(incidence)
Male 0 0/50
2090 0/50
6270 1/49
20,900 6/50*
Incidence of Papillary Necrosis
Suzuki et al. (2012); JPEC (2010a)
rat, Fischer 344
oral - water
female (50/group): 0, 625, 2500,10,000 ppm (0,
46,171, 560 mg/kg-day)a; male (50/group): 0,
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-
day)a
daily for 104 wks
Dose(mg/kg-d) Response
(incidence)
Male 0 0/50
28 1/50
121 0/50
542 2/50
Dose(mg/kg-d) Response
(incidence)
Female 0 0/50
46 1/50
171 1/50
560 2/50
Proximal Tubule Proliferation
Medinsky et al. (1999); Bond et al. (1996b)
rat, Fischer 344
inhalation - vapor
female (48/group): 0, 500,1750, 5000 ppm (0,
2090, 7320, 20,900 mg/m3)b; male (48/group): 0,
500, 1750, 5000 ppm (0, 2090, 7320,
20,900 mg/m3)b
dynamic whole body chamber; 6 hrs/d, 5 d/wk for
13 wks; generation method, analytical
concentration and method were reported
Dose(mg/m3) Percent change
compared to
control
Male 0
2090 137%*
7320 274%*
20,900 171%*
Dose(mg/m3) Percent change
compared to
control
Female 0
2090 73%
7320 64%
20,900 47%
1 Conversion performed by study authors.
This document is a draft for review purposes only and does not constitute Agency policy.
1-18	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
1	b4.18 mg/m3 = 1 ppm.
2	NR: not reported; *: result is statistically significant (p<0.05) based on analysis of data by study authors
3	for controls, no response relevant; for other doses, no quantitative response reported
4	Percent change compared to controls calculated as 100 x ((treated value - control value) -f control value).
5
6
This document is a draft for review purposes only and does not constitute Agency policy.
1-19	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
1	Table 1-3. Evidence pertaining to kidney biochemistry effects in animals
2	exposed to ETBE
Reference and Dosing Protocol
Results by Endpoint
Blood Urea Nitrogen (BUN)
Mivata et al. (2013);JPEC (2008c)

Dose(mg/kg-d)
Percent change
rat, CRL:CD(SD)


compared to
oral - gavage


control
female (15/group): 0, 5, 25,100, 400 mg/kg-d;
Male
0
-
male (15/group): 0, 5, 25,100, 400 mg/kg-d

5
12%
daily for 180 days

25
1%


100
4%


400
8%


Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


5
-5%


25
-7%


100
-1%


400
4%
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - water


control
female (50/group): 0, 625, 2500,10,000 ppm (0,
Male
0
-
46,171, 560 mg/kg-day)a; male (50/group): 0,

28
3%
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

121
20%*
day)a

542
43%*
daily for 104 wks

Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


46
-8%


171
-5%


560
-5%
This document is a draft for review purposes only and does not constitute Agency policy.
1-20	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-3. Evidence pertaining to kidney biochemistry effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Blood Urea Nitrogen (BUN) (continued)
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,
Male
0
-
2090, 6270, 20,900 mg/m3); male (NR): 0, 150,

627
-9%
500, 1500, 5000 ppm (0, 627, 2090, 6270,

2090
-5%
20,900 mg/m3)b

6270
4%
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

20,900
4%
13 wks; generation method, analytical

Dose(mg/m3)
Percent change
concentration and method were reported


compared to



control

Female
0
-


627
-5%


2090
3%


6270
-8%


20,900
-4%
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor


control
female (50/group): 0, 500,1500, 5000 ppm (0,
Male
0
-
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

2090
41%*
500, 1500, 5000 ppm (0, 2090, 6270,

6270
45%*
20,900 mg/m3)b

20,900
179%*
dynamic whole body inhalation; 6 hrs/d, 5 d/wk

Dose(mg/m3)
Percent change
for 104 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


2090
10%


6270
4%


20,900
30%*
This document is a draft for review purposes only and does not constitute Agency policy.
1-21	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-3. Evidence pertaining to kidney biochemistry effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Cholesterol
Mivata et al. (2013);JPEC (2008c)
rat, CRL:CD(SD)
oral - gavage
female (15/group): 0, 5, 25,100, 400 mg/kg-d;
male (15/group): 0, 5, 25,100, 400 mg/kg-d
daily for 180 days
Dose(mg/kg-d) Percent change
compared to
control
Male 0
5 -5%
25 21%
100 12%
400 53%*
Dose(mg/kg-d) Percent change
compared to
control
Female 0
5 -7%
25 -7%
100 -2%
400 3%
Suzuki et al. (2012); JPEC (2010a)
rat, Fischer 344
oral - water
female (50/group): 0, 625, 2500,10,000 ppm (0,
46,171, 560 mg/kg-day)a; male (50/group): 0,
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-
day)a
daily for 104 wks
Dose(mg/kg-d) Percent change
compared to
control
Male 0
28 -11%
121 10%
542 31%*
Dose(mg/kg-d) Percent change
compared to
control
Female 0
46 -2%
171 12%
560 8%
This document is a draft for review purposes only and does not constitute Agency policy.
1-22	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-3. Evidence pertaining to kidney biochemistry effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Cholesterol (continued)
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,
Male
0
-
2090, 6270, 20,900 mg/m3); male (NR): 0, 150,

627
8%
500, 1500, 5000 ppm (0, 627, 2090, 6270,

2090
9%
20,900 mg/m3)b

6270
26%
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

20,900
15%
13 wks; generation method, analytical

Dose(mg/m3)
Percent change
concentration and method were reported


compared to



control

Female
0
-


627
7%


2090
9%


6270
11%


20,900
21%
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor


control
female (50/group): 0, 500,1500, 5000 ppm (0,
Male
0
-
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

2090
10%
500, 1500, 5000 ppm (0, 2090, 6270,

6270
29%*
20,900 mg/m3)b

20,900
52%*
dynamic whole body inhalation; 6 hrs/d, 5 d/wk

Dose(mg/m3)
Percent change
for 104 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


2090
-3%


6270
-4%


20,900
53%*
This document is a draft for review purposes only and does not constitute Agency policy.
1-23	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-3. Evidence pertaining to kidney biochemistry effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Creatinine
Mivata et al. (2013);JPEC (2008c)

Dose(mg/kg-d)
Percent change
rat, CRL:CD(SD)


compared to
oral - gavage


control
female (15/group): 0, 5, 25,100, 400 mg/kg-d;
Male
0
-
male (15/group): 0, 5, 25,100, 400 mg/kg-d

5
0%
daily for 180 days

25
-10%


100
-3%


400
0%


Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


5
-19%


25
-12%


100
-16%


400
-16%
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - water


control
female (50/group): 0, 625, 2500,10,000 ppm (0,
Male
0
-
46,171, 560 mg/kg-day)a; male (50/group): 0,

28
0%
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

121
17%
day)a

542
17%
daily for 104 wks

Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


46
0%


171
-17%


560
0%
This document is a draft for review purposes only and does not constitute Agency policy.
1-24	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-3. Evidence pertaining to kidney biochemistry effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Creatinine (continued)
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,
Male
0
-
2090, 6270, 20,900 mg/m3); male (NR): 0, 150,

627
-13%
500, 1500, 5000 ppm (0, 627, 2090, 6270,

2090
-6%
20,900 mg/m3)b

6270
-6%
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

20,900
-3%
13 wks; generation method, analytical

Dose(mg/m3)
Percent change
concentration and method were reported


compared to



control

Female
0
-


627
0%


2090
3%


6270
-9%


20,900
-9%
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor


control
female (50/group): 0, 500,1500, 5000 ppm (0,
Male
0
-
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

2090
14%*
500, 1500, 5000 ppm (0, 2090, 6270,

6270
29%*
20,900 mg/m3)b

20,900
71%*
dynamic whole body inhalation; 6 hrs/d, 5 d/wk

Dose(mg/m3)
Percent change
for 104 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


2090
0%


6270
0%


20,900
0%
This document is a draft for review purposes only and does not constitute Agency policy.
1-25	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-3. Evidence pertaining to kidney biochemistry effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Incidence of Proteinuria
Mivata et al. (2013);JPEC (2008c)

Dose(mg/kg-d)
Response
rat, CRL:CD(SD)
Male
0
10/10
oral - gavage

5
10/10
female (15/group): 0, 5, 25,100, 400 mg/kg-d;

25
10/10
male (15/group): 0, 5, 25,100, 400 mg/kg-d

100
10/10
daily for 180 days

400
10/10


Dose(mg/kg-d)
Response

Female
0
8/10


5
9/10


25
7/10


100
9/10


400
7/10
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Response
rat, Fischer 344
Male
0
39/39
oral - water

28
37/37
female (50/group): 0, 625, 2500,10,000 ppm (0,

121
34/34
46,171, 560 mg/kg-day)a; male (50/group): 0,

542
35/35
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

Dose(mg/kg-d)
Response
day)a
Female
0
37/37
daily for 104 wks

46
37/37


171
38/38


560
38/38
JPEC (2008b)

Dose(mg/m3)
Response
rat, CRL:CD(SD)
Male
0
3/6
inhalation - vapor

627
5/6
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,

2090
5/6
2090, 6270, 20,900 mg/m3); male (NR): 0,150,

6270
6/6
500, 1500, 5000 ppm (0, 627, 2090, 6270,

20,900
4/6
20,900 mg/m3)b

Dose(mg/m3)
Response
dynamic whole body chamber; 6 hrs/d, 5 d/wk for
Female
0
1/6
13 wks; generation method, analytical

627
1/6
concentration and method were reported

2090
1/6


6270
2/6


20,900
2/6
This document is a draft for review purposes only and does not constitute Agency policy.
1-26	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-3. Evidence pertaining to kidney biochemistry effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Incidence of Proteinuria (continued)
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Response
rat, Fischer 344


(incidence)
inhalation - vapor
Male
0
44/44
female (50/group): 0, 500,1500, 5000 ppm (0,

2090
38/38
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

6270
40/40
500, 1500, 5000 ppm (0, 2090, 6270,

20,900
31/31
20,900 mg/m3)b

Dose(mg/m3)
Response
dynamic whole body inhalation; 6 hrs/d, 5 d/wk


(incidence)
for 104 wks; generation method, analytical
Female
0
33/38
concentration and method were reported

2090
39/39


6270
30/30


20,900
30/30
Severity of Proteinuriac
Mivata et al. (2013);JPEC (2008c)

Dose(mg/kg-d)
Percent change
rat, CRL:CD(SD)


compared to
oral - gavage


control
female (15/group): 0, 5, 25,100, 400 mg/kg-d;
Male
0
-
male (15/group): 0, 5, 25,100, 400 mg/kg-d

5
7%
daily for 180 days

25
7%


100
-13%


400
0%


Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


5
8%


25
-17%


100
8%


400
-17%
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-3. Evidence pertaining to kidney biochemistry effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Severity of Proteinuria (continued)c
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - water


control
female (50/group): 0, 625, 2500,10,000 ppm (0,
Male
0
-
46, 171, 560 mg/kg-day)a; male (50/group): 0,

28
3%
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

121
3%
day)a

542
3%
daily for 104 wks

Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


46
7%


171
7%


560
11%
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,
Male
0
-
2090, 6270, 20,900 mg/m3); male (NR): 0,150,

627
140%
500, 1500, 5000 ppm (0, 627, 2090, 6270,

2090
140%
20,900 mg/m3)b

6270
160%
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

Dose(mg/m3)
Percent change
13 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


627
50%


2090
0%


6270
150%


20,900
50%
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-3. Evidence pertaining to kidney biochemistry effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Severity of Proteinuria (continued)c
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor


control
female (50/group): 0, 500,1500, 5000 ppm (0,
Male
0
-
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

2090
-5%
500, 1500, 5000 ppm (0, 2090, 6270,

6270
-3%
20,900 mg/m3)b

20,900
-3%
dynamic whole body inhalation; 6 hrs/d, 5 d/wk

Dose(mg/m3)
Percent change
for 104 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


2090
11%


6270
18%


20,900
21%*
1	Conversion performed by study authors.
2	b4.18 mg/m3 = 1 ppm.
3	Severity of proteinuria= (1* number of animals with "1+") + (2*number of animals with "2+") + (3 * number of
4	animals with "3+") + (4 * number of animals with "4+")/ total number of animals in group
5	NR: not reported; *: 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	Percent change compared to controls calculated as 100 x ((treated value - control value) -f control value).
8
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
¦ = 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
Increased Relative P0 Male rat;l6wks (B)
Kidney Weight
PO Female rat;16wks (B)
PO Male rat;reproductive (C)
PO Female rat;reproductive (C)
F1 Male rat;reproductive [C]
F1 Female rat;reproductive [C)
Male rat;23wks (D)
Male rat;26wks (E)
Female rat;26wks (E)
Male rat;*104wks (F)
Female rat;104wks (F)
E	B	B
~—B—B
B—B-
B-
-B	¦	¦
O-
Incidence of Chronic
Progressive
Nephropathy
Average Severity of
Chronic Progressive
Nephropathy
Male rat;104wks (A)
Female rat;104wks (A)
Male rat;104wks (F)
Female rat;104wks (F)
Male rat;104wks (A)
Female rat;104wks (A)
Male rat;104wks (F)
Female rat;104wks (F)
B	!—B-
B-
-JB-
B	h-B-
-B-
Q	—B-
D
-B
-B
-B
~
-B
-O
-B
Urothelial Hyperplasia
of the Renal Pelvis
Male rat;lQ4wks (F)
Female rat;104wks (F)
B	I—B
-0
1	10	100	1,000 10,000
Dose (mg/kg-day)
Sources: (A) Cohen et al, 2011 reanalysisof JPEC, 2010a; (B) Fujiiet al., 2010; JPEC, 2008e; (C) Gaoua, 2004b;
(D) Hagiwara et al, 2011; (E) Miyata et al, 2013; JPEC, 2008c; [F) Suzuki et al., 2012; JPEC, 2010a
Figure 1-1. 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.
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Toxicological Review ofETBE
¦ = 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
Increased Relative
Kidney Weight
Male rat;13wks (A) -
Female rat;13wks (A)
Male rat;13wks, 2Bd recovery (A)
Female rat;13wks, 28d recovery (A)
Male rat;104wks (B) ¦
Female rat;104wks (B)
Incidence of Chronic
Progressive	Male rat;104wks (B)
Nephropathy
Female rat;104wks (B)
Average Severity of
Chronic Progressive Male rat;l04wlcs W
Nephropathy
Female rat;l04wks (8)

Female rat;104wks (BJ
~—	B-
~	B——0
~	B-
B-
Q	B—J	0
10	100	1,000	10,000 100,000
Exposure Concentration (mR/m:i)
Sources: (A) JPEG, 2008b; (B) Saito et al, 2013; JPEC, 2010b
Figure 1-2. Exposure-response array of kidney effects following inhalation
exposure to ETBE.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
1	Table 1-4. Evidence pertaining to kidney tumor effects in animals exposed to
2	ETBE
Reference and Dosing Protocol
Results by Endpoint
Renal Cell Carcinoma
Maltoni et al. (1999)

Dose(mg/kg-d)
Response
rat, Sprague-Dawley


(incidence)
oral - gavage
Male
0
0/60
female (60/group): 0, 250,1000 mg/kg-d; male

250
0/60
(60/group): 0, 250,1000 mg/kg-d

1000
0/60
4 d/wk for 104 wks; observed until natural death

Dose(mg/kg-d)
Response
(incidence)

Female
0
250
1000
0/60
0/60
0/60
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Response
rat, Fischer 344


(incidence)
oral - water
Male
0
0/50
female (50/group): 0, 625, 2500,10,000 ppm (0,

28
0/50
46,171, 560 mg/kg-day)a; male (50/group): 0,

121
0/50
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-d)a

542
1/50
daily for 104 wks

Dose(mg/kg-d)
Response
(incidence)

Female
0
46
171
560
0/50
0/50
0/50
1/50
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Response
rat, Fischer 344


(incidence)
inhalation - vapor
Male
0
0/50
female (50/group): 0, 500,1500, 5000 ppm (0,

2090
1/50
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

6270
0/49
500, 1500, 5000 ppm (0, 2090, 6270,

20,900
0/50
20,900 mg/m3)b



dynamic whole body inhalation; 6 hrs/d, 5 d/wk
Female


for 104 wks; generation method, analytical
none were observed

concentration and method were reported



3
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
1	Table 1-5. Evidence pertaining to kidney tumor promotion by ETBE in animals
Reference and Dosing Protocol
Results by Endpoint
Renal Transitional Cell Carcinoma
Hagiwara et al. (2011); JPEC (2008d)
rat, Fischer 344
oral - gavage
male (12/group): 0,1000 mg/kg-d
daily for 23 weeks
+ no DMBB initiation
Dose(mg/kg-d) Response
(incidence)
Male 0 1/30
300 0/30
1000 2/30
0+ 0/12
1000+ 0/12
Renal Tubular Adenoma or Carcinoma
Hagiwara et al. (2011); JPEC (2008d)
rat, Fischer 344
oral - gavage
male (30/group): 0, 300,1000 mg/kg-d
daily for 23 weeks following a 4 week tumor
initiation by DMBDD
+ no DMBB initiation
Dose(mg/kg-d) Response
(incidence)
Male 0 11/30
300 6/30
1000 13/30
0+ 0/12
1000+ 0/12
2	Conversion performed by study authors.
3	b4.18 mg/m3 = 1 ppm.
4	NR: not reported; *: result is statistically significant (p<0.05) based on analysis of data by study authors
5	for controls, no response relevant; for other doses, no quantitative response reported
6	(n): number evaluated from group
7	Mode of Action Analysis-Kidney Effects
8	Toxicokinetic considerations relevant to kidney toxicity
9	ETBE is metabolized by cytochrome P450 (CYP) enzymes to an unstable hemiacetal that
10	decomposes spontaneously into tert-butanol and acetaldehyde fBernauer et al.. 19981.
11	Acetaldehyde is further metabolized in the liver and is not thought to play a role in extrahepatic
12	toxicity. The main circulating metabolite is tert-butanol, which is filtered from the blood by the
13	kidneys and excreted in urine. Thus, following ETBE exposure, the kidney is exposed to significant
14	concentrations of tert-butanol, and kidney effects caused by tert-butanol (described in the more
15	detail in the draft IRIS assessment of tert-butanol) are also relevant to evaluating the kidney effects
16	observed after ETBE exposure. In particular, similar to ETBE, tert-butanol has been reported to
17	causes nephrotoxicity in rats, including effects associated with a2U-globulin nephropathy. However,
18	unlike ETBE, increased renal tumors were reported following chronic drinking water exposure to
19	tert-butanol.
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
Toxicological Review ofETBE
a?,,-Globulin-related nephropathy
Description of the hypothesized MOA
In the case of male rats treated with ETBE, a2U-globulin was confirmed in the hyaline
droplets from multiple studies (Mivata etal.. 2013: TPEC. 2008b. c; Medinskv etal.. 19991.
a2u-Globulin is derived from hepatic synthesis and can be chemically induced to accumulate in the
proximal tubule as the result of impaired renal catabolism fU.S. EPA. 1991al. In the context of
noncancer kidney toxicity observed after ETBE exposure, this accumulation could lead to various
types of nephropathy, including chronic proliferation of the renal tubule epithelium and possibly
exacerbation of CPN (U.S. EPA. 1991a).
U.S. EPA fl991al has described the hypothesized sequence of events in a2U-globulin-
associated nephropathy. Chemicals that induce a2U-globulin accumulation do so rapidly. The
accumulation of a2U-globulin in the hyaline droplets results in hyaline droplet deposition in the P2
segment of the proximal tubule within 24 hours of exposure. As hyaline droplet deposition
continues, single-cell necrosis occurs in the 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
is observed in the 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 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 may result in the induction of renal adenomas and carcinomas.
U.S. EPA fl991al states that two questions must be addressed to determine the extent to
which a2u-globulin-mediated processes induce renal effects. First, it must be determined whether
or not the a2u-globulin process is occurring in male rats, and therefore could be a factor in renal
effects. Because ETBE has not been found to cause kidney tumors in male rats, the second question
as to whether the renal effects are solely due to the a2U-globulin process, are a combination of the
a2u-globulin process and other carcinogenic processes, or are due primarily to other processes, is
not pertinent to this MOA analysis. However, U.S. EPA f!991al states that if the a2U-globulin process
is occurring in male rats, then the associated nephropathy in male rats (described above) would not
be an appropriate endpointto determine noncancer effects occurring in humans due to the
specificity of the protein to male rats. In such a case, the characterization of human health hazard
for renal toxicity would need to rely on data on other types of nephrotoxic effects in male rats
and/or on nephrotoxic effects in female rats or other species.
Based on the information above, the MOA analysis for ETBE-induced renal effects are
focused only on the first question of whether or not the a2U-globulin process is occurring in male
rats. U.S. EPA f!991al describes the criteria for determining this as follows:
(1) hyaline droplets are increased in size and number in male rats,
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
Toxicological Review ofETBE
(2)	the protein in the hyaline droplets in male rats is a2u-globulin, and
(3)	several (but not necessarily all) additional steps in the pathological sequence are
present in male rats, such as:
(a)	single-cell necrosis,
(b)	exfoliation of epithelial cells into the tubular lumen,
(c)	granular casts,
(d)	linear mineralization, and
(e)	tubule hyperplasia.
The available data in male rats will be evaluated in accordance with the MOA
framework from the EPA cancer guidelines (U.S. EPA. 2005al. These data are
summarized in
This document is a draft for review purposes only and does not constitute Agency policy.
1-35	DRAFT—DO NOT CITE OR QUOTE
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1	Table 1-7 and Figure 1-3 and Figure 1-4.
2
Toxicological Review ofETBE
This document is a draft for review purposes only and does not constitute Agency policy.
1-36	DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofETBE
1	Table 1-6. Additional kidney effects potentially relevant to mode of action in
2	animals exposed to ETBE
Reference and Dosing Protocol
Results by Endpoint
Incidence of Hyaline Droplets
Mivata et al. (2013); JPEC (2008c)
rat, CRL:CD(SD)
oral - gavage
female (15/group): 0, 5, 25,100, 400 mg/kg-d;
male (15/group): 0, 5, 25,100, 400 mg/kg-d
daily for 180 days
Dose(mg/kg-d) Response
(incidence)
Male 0 0/15
5 0/15
25 0/15
100 4/15*
400 10/15*
Dose(mg/kg-d) Response
(incidence)
Female 0 0/15
5
25
100
400 0/15
Suzuki et al. (2012); JPEC (2010a)
rat, Fischer 344
oral - water
female (50/group): 0, 625, 2500,10,000 ppm (0,
46,171, 560 mg/kg-day)a; male (50/group): 0,
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-
day)a
daily for 104 wks
Male
no hyaline droplets observed
Female
no hyaline droplets observed
Saito et al. (2013); JPEC (2010b)
rat, Fischer 344
inhalation - vapor
female (50/group): 0, 500,1500, 5000 ppm (0,
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,
500, 1500, 5000 ppm (0, 2090, 6270,
20,900 mg/m3)b
dynamic whole body inhalation; 6 hrs/d, 5 d/wk
for 104 wks; generation method, analytical
concentration and method were reported
Male
no hyaline droplets observed
Female
no hyaline droplets observed
This document is a draft for review purposes only and does not constitute Agency policy.
1-37	DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofETBE
Table 1-6. Additional kidney effects potentially relevant to mode of action in
animals exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Incidence of Hyaline Droplets in the Proximal Tube Epithelium
JPEC (2008b)
rat, CRL:CD(SD)
inhalation - vapor
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,
2090, 6270, 20,900 mg/m3); male (NR): 0, 150,
500, 1500, 5000 ppm (0, 627, 2090, 6270,
20,900 mg/m3)b
dynamic whole body chamber; 6 hrs/d, 5 d/wk for
13 wks; generation method, analytical
concentration and method were reported
Dose(mg/m3) Response
(incidence)
Male 0 0/10
627 3/10
2090 8/10*
6270 8/10*
20,900 8/10*
Female
no hyaline droplets observed in proximal tubule
Average Hyaline Droplet Severity
Medinskv et al. (1999); Bond et al. (1996b)
rat, Fischer 344
inhalation - vapor
female (48/group): 0, 500,1750, 5000 ppm (0,
2090, 7320, 20,900 mg/m3)b; male (48/group): 0,
500, 1750, 5000 ppm (0, 2090, 7320,
20,900 mg/m3)b
dynamic whole body chamber; 6 hrs/d, 5 d/wk for
13 wks; generation method, analytical
concentration and method were reported
Dose(mg/m3) Response
(severity)
Male 0 1.8
2090 3
7320 3.2
20,900 3.8
Female
no hyaline droplets observed
Incidence of Hyaline Droplets Positive for a2u-globulin
Mivata et al. (2013); JPEC (2008c)
rat, CRL:CD(SD)
oral - gavage
female (15/group): 0, 5, 25,100, 400 mg/kg-d;
male (15/group): 0, 5, 25,100, 400 mg/kg-d
daily for 180 days
Dose(mg/kg-d) Response
(incidence)
Male 0 0/1
5
25
100 2/2
400 1/1
Female
Incidence of hyaline droplets positive for c^u-globulin not
examined in females
JPEC (2008b)
rat, CRL:CD(SD)
inhalation - vapor
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,
2090, 6270, 20,900 mg/m3); male (NR): 0,150,
Male
unspecified representative samples reported as "weakly
positive" for a2u-globulin
Female
This document is a draft for review purposes only and does not constitute Agency policy.
1-38	DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofETBE
Table 1-6. Additional kidney effects potentially relevant to mode of action in
animals exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
500, 1500, 5000 ppm (0, 627, 2090, 6270,
20,900 mg/m3)b
dynamic whole body chamber; 6 hrs/d, 5 d/wk for
13 wks; generation method, analytical
concentration and method were reported
hyaline droplets positive for ci2u -globulin not examined in
females
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
7
This document is a draft for review purposes only and does not constitute Agency policy.
1-39	DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofETBE
1	Table 1-7. 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
1 wk
(+)
Medinskv et al. (1999)
in size and number
4 wk
(+)
Medinskv et al. (1999)

13 wk
(+)
Medinsky 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
1 wk
(+)a
JPEC (2008b)
droplets is a2u-globulin
4 wk
(+)a
Medinskv et al. (1999)

13 wk
(+)a
Medinskv et al. (1999)

13 wk
(+)a
JPEC (2008b)

26 wk
(+)b
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
13 wk
-
JPEC (2008b)
into the tubular lumen
13 wk
-
Medinsky 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)

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
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)
Cohen et al. (2011)

104 wk
+
Saito et al. (2013); JPEC (2010b)
(e) tubule hyperplasia
13 wk
-
JPEC (2008b)

13 wk
+/-c
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)
3	+ = Statistically significant change reported in one or more treated groups.
4	(+) = Effect was reported in one or more treated groups, but statistics not reported.
5	- = No statistically significant change reported in any of the treated groups.
6	aUnspecified "representative samples" examined.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
1	bThree samples from highest two dose groups examined.
2	labeling index statistically significantly increased, but no hyperplasia reported.
3
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
¦ = 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 a2u-globulm
Medinsky et al., 1999; Bond et al., 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; (PEC, 2010b - 104wk
ta-
•	•-
•	
•	•-
—¦	m—
Q	B—
	•
	•
	•
	¦
	0
—
Medinsky et al., 1999; Bond et al., 1996 - Iwk -
a globulin In Medinsky et ¦l-1-999; Bond et al., 1996 - 4wk
hyaline
droplets Medinsky et al, 1999; Bond et al, 1996 - 13wk
J PEC, 2008b -13 wk
nk—
l
|
1
	+
	+
	+
	+
Medinsfy et al., 1999; Bond et al, 1996 - 13wk
Granular
casts/dilation )PEC, 2008b - i3wk-
Saito et al., 2013; JPEC, 2010b-104wk
~—
	~	B-j	~
	B	B—!	~
	~	B—|	~
Medinsky et al., '1999; Bond et al, "1996 - 13wk
Linear
papillary JPEC, 2008b - 13wk
mineralization
Saito et al., 2013; JPEC, 2010b - 104wk
~—
	~	B-)	~
	B	B-j	~
	~	B—|	¦

Tubular
, , . Saito et al„ 2013; JPEC, 2010b- 104wk
hyperplasia

~	B—|	~
—
Renal adenoma Sajt0 et aL_ 2013, )pEC 2010b.} 04wk
or carcinoma

a	B—
	~
100	1,000	10,000	100,000
Exposure Concentration (mg/m3)
Figure 1-3. ETBE inhalation 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
+ = unspecified representative samples reported positive for a2u-globulin (3 samples examined)
Miyata et al, 2013; JPEC, 2008c - 26wks
Accumulation of
hyaline droplets
Suzuki et al., 2012; JPEC, 2010a - 104wks
~	
	B	1
~	
i	¦
-a	a
h	+
—
aZll-globulinin
hyaline Miyata et al, 2013; JPEC, 2008c - 26wks -
droplets

H
Miyata et al, 2013; JPEC, 2008c - 26wks -
Granular
casts/dilation
Suzuki ct al., 2012; JPEC, 2010a - 104wks -
~	
	B	E
~	
[]
~
~

Miyata et al, 2013; JPEC, 2008c - 26wks
Linear
papillary
mineralization
Suzuki et al, 2012; JPEC, 2010a - 104wks
~	
~ ! ~ n
	 !	 	r t ....
3	~
-B	B
-B	~
_
Tubular
hyperplasia Suzuki et al"2012; 'PEC'2010a"104wks

Renal
adenoma SuzuW et a)v 2oi2; JPEC, 2010a - I04wte
or
carcinoma

~	
-B	B

1	10	100	1,000
Dose (mg/kg-day)
1
2	Figure 1-4. ETBE oral exposure array of a2u-globulin data in male rats
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Strength, consistencyand specificity of association
The first criterion to consider in determining if the a2U-globulin process is occurring is
whether or not hyaline droplets are increased in size and number 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 1-6). Accumulation of hyaline droplets in the proximal
tubular epithelium of the kidney was observed in 8 of 10 male rats at the 3 highest exposure
concentrations ofETBE compared with 0 of 10 in control rats following 90-day inhalation exposure.
The increases at these 3 doses were statistically significant; however, none of the animals had
hyaline droplet grades over 1 flPEC. 2008b], Hyaline droplets were statistically significantly
increased in 4 of 15 (all grade 1 severity) and 10 of 15 (5 of each grade 1 and 2 severity) male rats
at the two highest doses of ETBE, respectively, compared with 0 of 15 controls following oral
exposure for 180 days (Mivata etal.. 2013: TPEC. 2008c). Finally, a 90-day inhalation ETBE
exposure study reported an increase in the grade of hyaline droplets as indicated by severity grades
of 1.8, 3.0, 3.2, and 3.8 in the control and 3 ETBE dose groups, respectively fMedinskv et al.. 19991.
The second criterion in determining occurrence of the a2U-globulin process 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 only performed in ETBE exposure studies that
observed accumulation of hyaline droplets. At the two highest doses, (Mivata etal. (2013): TPEC.
2008c) 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 flPEC. 2008b: Medinskv etal.. 19991. TPEC f2008bl reported
that the samples stained weakly positive for a2U-globulin and that positive a2U-globulin staining was
only observed in male rats. No statistical tests were performed on any of these results.
The third criterion in determining occurrence of the a2u-globulin process considers the
presence of additional steps in the pathological sequence in male rats (refer to
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Table 1-7). The incidence of papillary mineralization was statistically significantly increased
in both of the 2-year studies. In the drinking water study, incidence of mineralization was increased
from 0/50 in the control animals to 16/50 and 42/50 in the 121- and 542-mg/kg-day dose groups,
respectively (Suzuki etal.. 2012: IPEC. 2010a], Cohen etal. (2011) further reported that the
observed mineralization in fSuzuki etal.. 2012: IPEC. 2010al was linear mineralization. In the
inhalation study, incidence of mineralization was 6/50 in the 20,900-mg/m3 group compared with
0/50 in the control group (Saito etal.. 2013: IPEC. 2010b). However, single-cell necrosis, exfoliation
of epithelial cells into the tubular lumen, granular casts, and tubule hyperplasia were either absent
or not consistently observed across studies. Cohen etal. (2011) 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 did not report the presence of granular casts. Medinsky et al. (1999)
reported increased labeling indices indicative of tubular proliferation, but no hyperplasia, after 1 to
13 weeks of exposure. However, both males and females showed statistically significant increases
at shorter durations, and both sexes had elevated labeling indices at 13 weeks, though only the
males were statistically significantly increased. Moreover, increased hyperplasia was not observed
in any other studies.
In summary, the evidence supports ETBE causing hyaline droplets to be increased in size
and number and the accumulating protein being a2U-globulin, but only one of the additional steps in
the pathological sequence was consistently observed (linear papillary mineralization), and only
after exposure for 2 years. Overall, the strength, consistency, and specificity of the association
between ETBE and the hypothesized key events is weak.
Dose-response concordance
The accumulation of hyaline droplets was dose responsive in the 90-day inhalation ETBE
exposure study. Hyaline droplets were observed in 0/10, 3/10, 8/10, 8/10, and 8/10 at 0, 627,
2,090, 6,270, and 20,900 mg ETBE/m3, respectively fTPEC. 2008bl In addition, the incidence of
hyaline droplets was dose responsive after a 26-week gavage as indicated by droplets in 0/15,
0/15, 0/15, 4/15, and 10/15 at 0, 5, 25,100, and 400 mg ETBE/kg-day, respectively fMivata etal..
2013: IPEC. 2008c). Finally, 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, 2090, 7320, and 20,900 mg
ETBE/m3, respectively (Medinsky et al.. 1999).
The available studies that tested for a2U-globulin in the hyaline droplets did not test a
sufficient number of samples within a dose group nor were enough dose groups tested for ct2u-
globulin to perform dose-response analysis. All three studies that tested for a2U-globulin failed to
report the actual number of positive samples. For these reasons, no dose response concordance can
be established between accumulation of hyaline droplets and a2u-globulin accumulation.
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Papillary mineralization was dose-responsively increased following oral ETBE exposure in
0/50, 0/50,16/50, and 42/50 male rats atdoses of 0, 28,121, and 542 mg/kg-day, respectively
fSuzuki etal.. 2012: TPEC. 2010al. and in 0/50, 0/50,1/49, and 6/50 males at ETBE inhalation
concentrations of 0, 2090, 6270, and 20,900 mg/m3 fSaito etal.. 2013: TPEC. 2010bl. Based on the
above data, hyaline droplet deposition was observed at a similar frequency as mineralization
following oral ETBE exposure ((Suzuki etal.. 2012: TPEC. 2010al: Mivataetal.. 2013: TPEC. 2008cl:
however, hyaline droplet deposition was observed in 80% of animals at the 3 highest inhalation
exposure concentrations (TPEC. 2008bl compared with mineralization rates of 0, 2, and 12% at the
corresponding doses fSaito etal.. 2013: TPEC. 2010bl.
Although these results suggest that mineralization is dose responsive following either oral
or inhalation ETBE exposure, a stronger dose-response concordance between mineralization and
hyaline droplet deposition was observed for oral exposures. Furthermore, as discussed above, the
additional steps in the pathological sequence were not observed, so overall there is only weak
evidence of dose-response concordance among the hypothesized key events.
Temporal relationship
The accumulation of hyaline droplets is the first endpoint that is observed in a2U-globulin-
mediated nephropathy that may occur within 24 hours post-exposure. Droplets were increased
after 1, 4,13, and 26 weeks of exposure (Mivata etal.. 2013: TPEC. 2008b. c; Medinskv etal.. 19991.
Confirmation of a2U-globulin in the droplets was reported after 13 weeks (TPEC. 2008bl. 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.
Of the other endpoints in the pathological sequence, only papillary mineralization was
observed. Mineralization was reported after 2-year oral and inhalation exposures but not in any
study employing a shorter exposure. Endpoints such as necrosis, exfoliation of epithelial cells into
the tubular lumen, granular casts, and hyperplasia were not observed at the expected subchronic
and chronic time points. Due to the absence of the other key effects at the critical time points in the
a2U-globulin-mediated pathological sequence, the evidence for temporal relationship among the
hypothesized key events is weak.
Biological plausibility and coherence
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. 1991al. and those rationales will not be repeated here. More recent retrospective
analysis indicates that several steps in the sequence of pathological events are not required for
tumor development.
A retrospective analysis has demonstrated that a number of ct2u-globulin-inducing chemicals
fail to induce many of the pathological sequences in the a2u-globulin pathway (Doi etal.. 20071. For
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instance, dose-response concordance was not observed for several endpoints such as linear
mineralization, tubular hyperplasia, granular casts, and hyaline droplets following exposure to ct2u-
globulin-inducing chemicals such as d-limonene, decalin, propylene glycol mono-t-butyl ether, and
Stoddard solvent IICA (SSIICA). Although some of these chemicals induced dose-responsive effects
for a few endpoints, all of them failed to induce a dose response for at least one 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 dose responsive 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 IIC
which induced some of the most severe nephropathy relative to the other chemicals, but did not
significantly increase kidney tumors (Doi etal.. 20071. Thus, this analysis suggests that another
MOA may be operative for inducing tumors in male rats.
As described above, ETBE is metabolized to tert-butanol, so kidney data following
tert- butanol exposure is also potentially relevant to evaluating the MOA ofETBE. In particular, the
effects of tert-butanol on a2U-globulin 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 by direct tert-butanol administration. Similar to
hyaline droplets, linear mineralization was increased at an internal tert-butanol concentration
approximately tenfold lower following ETBE exposure than tert-butanol exposure.
Tubule hyperplasia and renal tumors were both observed following 2-year exposure to
tert-butanol but not ETBE. Tubule hyperplasia occurred at an internal concentration of tert-butanol
that was similar to the blood concentrations of tert- butanol following ETBE exposure fSaito etal..
2013: Suzuki etal.. 2012: TPEC. 2010b). Similarly, the incidence of renal tumors was increased at
three internal concentrations of tert-butanol that were achieved in two separate ETBE studies. The
failure of internal tert-butanol concentrations to induce histopathological lesions early in the
a2u-globulin pathological sequence at blood levels that later induced hyperplasia and tumors
suggests a lack of coherence across the two data sets.
With regard to the discrepancy in renal tumors between ETBE and tert-butanol, it should be
noted that the background renal tumor rate in the tert-butanol exposure study was high compared
with historical values. Renal tumors in the NTP (19951 chronic bioassay of tert-butanol, as re-
analyzed by Hard etal. (20111 were reported in 4/50 of control male rats, which is much greater
than would be expected from historical NTP F344 rat data (0/450) fDinse and Peddada. 20111.
Thus, it is possible that tert-butanol treatment served as a promoter of background tumorigenic
processes occurring in that experiment and that, had background renal tumor rates in the ETBE
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bioassays been higher, renal tumors would have been observed. However, key events in such a
"promotion" MOA have not been identified (proliferation does not appear to be a likely key event
because ETBE only induces transient increases in cell proliferation).
Conclusions about the hypothesized MOAfora.2u-globulin -associated nephropathy
Is the hypothesized MOA sufficiently supported in test animals?
Although ETBE induced an increase in a2U-globulin deposition and increased hyaline droplet
accumulation, most of the subsequent steps in the pathological sequence were not observed despite
identical study conditions and doses in a number of experiments over a 2-year exposure period.
These data failed to provide sufficient evidence that the a2U-globulin process is operative. Since
these data do not suggest that a2u-globulin process is operative for ETBE exposures, the extent to
which that a2U-globulin is operative will not be examined further. Considering that a retrospective
analysis found poor concordance of tumor incidence with the severity of any of the key pathological
steps fDoi etal.. 20071. the observation that ETBE does not induce renal tumors is not unexpected.
Is the hypothesized MOA relevant to humans?
Because EPA finds that the data are insufficient to demonstrate a2u-globulin nephropathy,
the male rat kidney data are relevant for humans.
Which populations or lifestages can be particularly susceptible to the hypothesized MOA?
This question is not applicable.
Alternative MOA hypotheses
Other nephrotoxic responses, such as exacerbation of CPN, urothelial hyperplasia, elevated
biochemical markers, and increased kidney weight, are observed in male and/or female rats,
suggesting other possible processes are operative for kidney toxicity. Exacerbation of CPN has been
proposed to be a rat-specific mechanism of nephrotoxicity that is not relevant to humans (Hard et
al.. 20091.
CPN is an age-related renal disease of laboratory rodents of unknown etiology that occurs
spontaneously in rats, especially the F344, Sprague-Dawley, and Osborne-Mendel strains (Hard et
al.. 20091. Additional markers associated with CPN include elevated proteinuria and albumin in the
urine and increased BUN, creatinine, and cholesterol in the serum (Hard etal.. 20091. CPN is
frequently more severe in males compared with females. Several of the CPN pathological effects are
similar to and can obscure the lesions characteristic of a2U-globulin-related hyaline droplet
nephropathy fWebb etal.. 19901. Additionally, renal effects of a2U-globulin accumulation can
exacerbate the effects associated with CPN fU.S. EPA. 1991al. However, fWebb etal.. 19901
suggested that exacerbated CPN was one component of the nephropathy resulting from exposure to
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chemicals that induce a2U-globulin nephropathy. Male rat sensitivity has been noted with both CPN
and ct2u- globulin nephropathy.
Increased severity of CPN occurred in both male and female rats as a result ofETBE
exposure, but was statistically significant only in the highest exposure group in the chronic
inhalation study. Some of the observed renal lesions in male rats following exposure to ETBE are
effects commonly associated with CPN. Cohen etal. (20111 concluded that the observation of slight
(or mild) urothelial hyperplasia in the 2-year drinking study conducted by f Suzuki etal.. 2012:
TPEC. 2010a) was associated with CPN, and not a direct effect of ETBE exposure. However, there
was a strong, statistically-significant, treatment-related, dose-response relationship between
chronic ETBE exposure and increased incidence of urothelial hyperplasia in male rats in both the
inhalation and oral studies (Suzuki etal.. 2012: TPEC. 2010a). (Saito etal.. 2013: TPEC. 2010bl). The
severity of CPN also increased with ETBE exposure, although the dose-response relationship is very
weak (only statistically significant at the highest dose in the inhalation study; trend test was not
significant). The very different dose-response relationships argue against their being a close
association. Moreover, even if urothelial hyperplasia were associated with CPN, there is no
evidence to support that it is independent of ETBE treatment, given the robust dose-response
relationships. Therefore, the data are insufficient to dismiss urothelial hyperplasia as causally
related to ETBE exposure.
The underlying mechanisms regulating CPN and its exacerbation are not well understood,
and to date, there is no scientific consensus on the relevance of CPN in rats to human health hazard
fMelnick et al.. 2 012: Hard etal.. 20091. Moreover, no key events for the exacerbation of CPN have
been identified, so no MOA analysis can be performed. Therefore, kidney effects from ETBE
exposure associated with CPN are considered relevant to humans.
Summary of Kidney Toxicity
The data that report kidney effects following oral and inhalation ETBE exposure are entirely
from experimental rodent studies. Several noncancer effects in the kidney have been observed
across multiple studies; chronic bioassays did not find treatment-related increases in renal tumors.
Kidney weights were consistently increased in male and female rats at several doses
following subchronic and chronic gavage and inhalation exposures fMivata etal.. 2013: TPEC.
2008b. c; Medinskv et al.. 19991. Regarding oral exposure, male kidney weights were more
consistently increased across all exposure durations than females; however, both sexes responded
similarly following inhalation exposures. The magnitude of the increases in kidney weight was
moderate, with maximal changes in relative or absolute weights that were less than twofold.
Several studies observing statistically significant increases at multiple exposure levels are
consistent with a monotonic dose-response relationship. In mice, only one subchronic study was
available, and it reported no changes in kidney weights fMedinskv et al.. 19991. but the lack of
additional mouse studies precludes a conclusion on the species specificity of ETBE-induced kidney
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weight changes. In rats, chronic kidney weights were increased similarly to subchronic studies but
were not considered for hazard assessment due to age-associated confounding factors (e.g., CPN);
therefore a temporal relationship cannot be determined for this endpoint.
Histopathological analysis observed increased CPN lesions in male rats after a 13-week oral
exposure and increased CPN severity in male and female rats after a 2-year inhalation exposure
(Cohen etal.. 2011: TPEC. 2010b): however, this was only observed at the highest tested doses.
Urothelial hyperplasia was observed in male rats after 2-year inhalation or oral exposures fSuzuki
etal.. 2012: TPEC. 2010a). (Saito etal.. 2013: IPEC. 2010b]]. Although Cohen etal. (2011) attributed
this finding to CPN, independent ofETBE exposure, the robust dose-response relationship
(especially as compared to that for CPN) suggests it is a treatment-related effect.
Additional evidence of altered kidney function included elevated blood concentrations of
total cholesterol, BUN, and creatinine in rats (Mivata etal.. 2013: TPEC. 2010a. b, 2008c)- These
biochemistry markers were increased more consistently in males than females. Males had dose-
related increases at several biochemistry endpoints, and these increases in biochemistry markers
occurred at lower doses than lesions of nephropathy, consistent with the expected relationship
between early markers of altered function and observable histopathology. Elevations in
biochemical markers of kidney disease were greater in males than females, consistent with males'
greater sensitivity to changes in kidney weights and histopathological changes, further adding to
the biological coherence of the available data on kidney toxicity.
MOA analysis determined that the data are insufficient to conclude that the nephropathy
observed in male rats is mediated by a2U-globulin. The available data also precluded establishing
any other MOA for ETBE-induced kidney toxicity. Therefore, in the absence of information
indicating otherwise, EPA considered the male and female kidney effects observed in experimental
animals to be relevant to assessing human health hazard. EPA identified kidney effects as a human
hazard ofETBE exposure.
1.1.2. Liver Effects
Synthesis of Effects in Liver
This section reviews the studies that investigated whether exposure to ETBE can cause liver
toxicity or cancer in humans or animals. The database for ETBE-induced liver effects includes 10
studies conducted in animals, all but one performed in rats. Studies employing 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 provided data to support mode of action or hazard identification. No
methodological concerns were identified that would lead one or more studies to be considered less
informative for assessing human health hazard.
Chronic and subchronic studies by both the oral and inhalation routes reported consistent
statistically-significant, dose-related increases in liver weights (see
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Table 1-8; Figure 1-5, Figure 1-6). Liver weight and body weight have been demonstrated to
be proportional and liver weight normalized to body weight is optimal for data analysis fBailev et
al.. 20041: thus, only relative liver weight is presented and considered in the determination of
hazard. Relative liver weights were consistently increased in males in 8 of 9 studies and 6 of 8
studies for females; however, statistically significant increases frequently occurred only at the
highest tested concentration with modest increases in relative liver weight ranging from 17-27% in
males and 8-18% in females. Relative liver weights in rats were increased at the only highest dose
following oral exposures of 16 weeks or longer fMivata etal.. 2013: Fuiii etal.. 2010: TPEC. 2008c).
Inhalation exposure increased liver weight at the highest dose in female rats following 13 week
exposure flPEC. 2008bl and was dose responsively increased following 2 year exposure fSaito et
al.. 2013: TPEC. 2010b). Short-term studies observed similar effects on liver weight (TPEC. 2008a:
White etal.. 19951.
Centrilobular hypertrophy was inconsistently increased throughout the database (see Table
1-9;	Figure 1-5, Figure 1-6). A 26-week oral gavage study fMivata etal.. 2013: TPEC. 2008cl in rats
and three 13-week inhalation studies in mice and rats fWeng etal.. 2012: TPEC. 2008b: Medinskv et
al.. 19991 demonstrated a statistically significant increase in centrilobular hypertrophy at the
highest dose, but 2-year oral or inhalation studies in rats failed to induce a similar effect Following
a 2-year inhalation exposure to ETBE, acidophilic and basophilic preneoplastic lesions were
increased in males, but not females, at the highest tested dose fSaito etal.. 2013: TPEC. 2010b). After
2-year	drinking water exposure to ETBE, an increasing, but not significant, trend in basophilic
preneoplastic lesions was observed in the liver of male rats, but not in female rats f Suzuki etal..
2012: TPEC. 2010al.
Analysis of serum liver enzymes demonstrated inconsistent results across exposure routes
(see Table 1-10; Figure 1-5, Figure 1-6). Gamma-glutamyl transpeptidase (GGT) was significantly
increased in male rats at one dose following oral exposure and the two highest doses following
inhalation exposure in 2-year studies (TPEC. 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.
Data on liver tumor induction by ETBE are presented in Table 1-11. Liver adenomas and
carcinomas (combined) were increased in male rats, but not females, following 2-year inhalation
exposure (Saito etal.. 2013: TPEC. 2010b). No significant increase in tumors was observed following
2 year oral exposure (Suzuki etal.. 2012: TPEC. 2010a: Maltoni etal.. 1999). An initiation-
promotion study by gavage in male F344 rats suggest tumor promotion activity by ETBE (Hagiwara
etal.. 20111.
Several factors associated with the 2-year organ weight data confound consideration for
hazard identification. As mentioned previously in the discussion of kidney effects, mortality was a
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confounding factor in 2-year studies. In addition, neoplastic and non-neoplastic lesions were
observed in the livers of all treatment groups in both oral and inhalation studies which further
confound organ weight data. For instance, the non-neoplastic lesion bile duct hyperplasia was
observed at varying levels of severity in 100% of males surviving to 104 weeks (Suzuki etal.. 2012:
TPEC. 2010a). Inhalation exposure significantly increased adenomas and carcinomas at the highest
dose which corresponded to increased liver weights fSaito etal.. 2013: TPEC. 2010b). Altogether,
these observations preclude including 2-year liver weight data for hazard identification. However,
organ weight data obtained from studies of shorter duration that are not confounded by these age-
associated factors may be appropriate for hazard identification.
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1	Table 1-8. Evidence pertaining to liver weight effects in animals exposed to
2	ETBE
Reference and Dosing Protocol
Results by Endpoint
Liver: Absolute Weight
Fuiiietal. (2010); JPEC (2008e)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, male (24/group): 0,100, 300,1000 mg/kg-d
P0, Male
0
-
daily for 16 weeks beginning 10 weeks prior to

100
-3%
mating

300
-1%
P0, female (24/group): 0,100, 300,1000 mg/kg-d

1000
13%*
daily for 17 weeks beginning 10 weeks prior to

Dose(mg/kg-d)
Percent change
mating to lactation day 21


compared to



control

P0, Female
0
-


100
-1%


300
3%


1000
14%*
3
This document is a draft for review purposes only and does not constitute Agency policy.
1-53	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-8. Evidence pertaining to liver weight effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Liver: Absolute Weight (continued)
Gaoua (2004b)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, male (25/group): 0, 250, 500,1000 mg/kg-d
P0, Male
0
-
daily for a total of 18 weeks beginning 10 weeks

250
2%
before mating until after weaning of the pups

500
2%
P0, female (25/group): 0, 250, 500,1000 mg/kg-d

1000
17%*
daily for a total of 18 weeks beginning 10 weeks

Dose(mg/kg-d)
Percent change
before mating until PND 21


compared to
Fl, male (25/group): 0, 250, 500,1000 mg/kg-d


control
P0 dams dosed daily through gestation and
Fl, Male
0
-
lactation, then Fl doses beginning PND 22 until

250
0%
weaning of the F2 pups

500
14%*
Fl, female (24-25/group): 0, 250, 500,1000

1000
27%*
mg/kg-d

Dose(mg/kg-d)
Percent change
P0 dams dosed daily through gestation and


compared to
lactation, then Fl dosed beginning PND 22 until


control
weaning of the F2 pups
P0, Female
0
-


250
-1%


500
4%


1000
6%


Dose(mg/kg-d)
Percent change



compared to



control

Fl, Female
0
-


250
1%


500
3%


1000
10%*
Hagiwara et al. (2011); JPEC (2008d)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - gavage


control
male (12/group): 0,1000 mg/kg-d
Male
0
-
daily for 23 weeks

1000
21%*
This document is a draft for review purposes only and does not constitute Agency policy.
1-54	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-8. Evidence pertaining to liver weight effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Liver: Absolute Weight (continued)
Mivata et al. (2013); JPEC (2008c)

Dose(mg/kg-d)
Percent change
rat, CRL:CD(SD)


compared to
oral - gavage


control
female (15/group): 0, 5, 25,100, 400 mg/kg-d;
Male
0
-
male (15/group): 0, 5, 25,100, 400 mg/kg-d

5
-2%
daily for 180 days

25
7%


100
4%


400
19%


Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


5
-4%


25
-1%


100
2%


400
9%
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - water


control
female (50/group): 0, 625, 2500,10,000 ppm (0,
Male
0
-
46,171, 560 mg/kg-day)a; male (50/group): 0,

28
-11%*
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

121
-4%
day)a

542
2%
daily for 104 wks

Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


46
-5%


171
-2%


560
-10%
This document is a draft for review purposes only and does not constitute Agency policy.
1-55	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-8. Evidence pertaining to liver weight effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Liver: Absolute Weight (continued)
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,
Male
0
-
2090, 6270, 20,900 mg/m3); male (NR): 0, 150,

627
5%
500, 1500, 5000 ppm (0, 627, 2090, 6270,

2090
6%
20,900 mg/m3)b

6270
4%
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

20,900
2%
13 wks; generation method, analytical

Dose(mg/m3)
Percent change
concentration and method were reported


compared to



control

Female
0
-


627
-3%


2090
-8%


6270
-2%


20,900
5%
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (6/group): 0, 5000 ppm (0,
Male
0
-
20,900 mg/m3)b; male (6/group): 0, 5000 ppm (0,

20,900
13%
20,900 mg/m3)b

Dose(mg/m3)
Percent change
dynamic whole body chamber; 6 hrs/d, 5 d/wk for


compared to
13 wks followed by a 28 day recovery period;


control
generation method, analytical concentration and
Female
0
-
method were reported

20,900
11%
Medinsky et al. (1999); Bond et al. (1996b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor


control
female (48/group): 0, 500,1750, 5000 ppm (0,
Male
0
-
2090, 7320, 20,900 mg/m3)b; male (48/group): 0,

2090
6%
500, 1750, 5000 ppm (0, 2090, 7320,

7320
14%*
20,900 mg/m3)b

20,900
32%*
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

Dose(mg/m3)
Percent change
13 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


2090
2%


7320
9%
This document is a draft for review purposes only and does not constitute Agency policy.
1-56	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-8. Evidence pertaining to liver weight effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint


20,900
26%*
Liver: Absolute Weight (continued)
Medinskv et al. (1999); Bond et al. (1996a)

Dose(mg/m3)
Percent change
mice, CD-I


compared to
inhalation - vapor


control
female (40/group): 0, 500,1750, 5000 ppm(0,
Male
0
-
2090, 7320, 20,900 mg/m3)b; male (40/group): 0,

2090
4%
500, 1750, 5000 ppm (0, 2090, 7320,

7320
13%*
20,900 mg/m3)b

20,900
18%*
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

Dose(mg/m3)
Percent change
13 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


2090
2%


7320
19%*


20,900
33%*
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor


control
female (50/group): 0, 500,1500, 5000 ppm (0,
Male
0
-
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

2090
1%
500, 1500, 5000 ppm (0, 2090, 6270,

6270
11%*
20,900 mg/m3)b

20,900
10%
dynamic whole body inhalation; 6 hrs/d, 5 d/wk

Dose(mg/m3)
Percent change
for 104 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


2090
-3%


6270
-8%


20,900
1%
This document is a draft for review purposes only and does not constitute Agency policy.
1-57	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-8. Evidence pertaining to liver weight effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Liver: Relative Weight
Fuiiietal. (2010); JPEC (2008e)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, male (24/group): 0,100, 300,1000 mg/kg-d
P0, Male
0
-
daily for 16 weeks beginning 10 weeks prior to

100
1%
mating

300
3%
P0, female (24/group): 0,100, 300,1000 mg/kg-d

1000
21%*
daily for 17 weeks beginning 10 weeks prior to

Dose(mg/kg-d)
Percent change
mating to lactation day 21


compared to



control

P0, Female
0
-


100
-2%


300
2%


1000
8%*
Gaoua (2004b)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, male (25/group): 0, 250, 500,1000 mg/kg-d
P0, Male
0
-
daily for a total of 18 weeks beginning 10 weeks

250
3%
before mating until after weaning of the pups

500
6%
P0, female (25/group): 0, 250, 500,1000 mg/kg-d

1000
24%*
daily for a total of 18 weeks beginning 10 weeks

Dose(mg/kg-d)
Percent change
before mating until PND 21


compared to
Fl, male (25/group): 0, 250, 500,1000 mg/kg-d


control
dams dosed daily through gestation and lactation,
Fl, Male
0
-
then Fl doses beginning PND 22 until weaning of

250
0%
the F2 pups

500
11%*
Fl, female (24-25/group): 0, 250, 500,1000

1000
25%*
mg/kg-d

Dose(mg/kg-d)
Percent change
dams dosed daily through gestation and lactation,


compared to
then Fl dosed beginning PND 22 until weaning of


control
the F2 pups
P0, Female
0
-


250
10%


500
8%


1000
4%


Dose(mg/kg-d)
Percent change



compared to



control

Fl, Female
0
-


250
3%
This document is a draft for review purposes only and does not constitute Agency policy.
1-58	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-8. Evidence pertaining to liver weight effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint


500
6%


1000
9%*
Liver: Relative Weight (continued)
Hagiwara et al. (2011); JPEC (2008d)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - gavage


control
male (12/group): 0,1000 mg/kg-d
Male
0
-
daily for 23 weeks

1000
27%*
Mivata et al. (2013); JPEC (2008c)

Dose(mg/kg-d)
Percent change
rat, CRL:CD(SD)


compared to
oral - gavage


control
female (15/group): 0, 5, 25,100, 400 mg/kg-d;
Male
0
-
male (15/group): 0, 5, 25,100, 400 mg/kg-d

5
5%
daily for 180 days

25
7%


100
9%


400
17%*


Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


5
1%


25
1%


100
4%


400
12%*
This document is a draft for review purposes only and does not constitute Agency policy.
1-59	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-8. Evidence pertaining to liver weight effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Liver: Relative Weight (continued)
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - water


control
female (50/group): 0, 625, 2500,10,000 ppm (0,
Male
0
-
46,171, 560 mg/kg-day)a; male (50/group): 0,

28
-8%
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

121
3%*
day)a

542
12%*
daily for 104 wks
Study authors stated that increased relative liver weights

were due to significantly lowered final body weights of

treated groups; individual animal data were not available

to confirm statistical analysis conducted by study authors

(e.g., 3% statistically significant increase in males at the

mid-dose).




Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


46
4%


171
9%


560
8%
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,
Female
0
-
2090, 6270, 20,900 mg/m3); male (NR): 0, 150,

627
4%
500, 1500, 5000 ppm (0, 627, 2090, 6270,

2090
-1%
20,900 mg/m3)b

6270
6%
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

20,900
18%*
13 wks; generation method, analytical

Dose(mg/m3)
Percent change
concentration and method were reported


compared to



control

Male
0
-


627
5%


2090
5%


6270
5%


20,900
10%
This document is a draft for review purposes only and does not constitute Agency policy.
1-60	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-8. Evidence pertaining to liver weight effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Liver: Relative Weight (continued)
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (6/group): 0, 5000 ppm (0,
Female
0
-
20,900 mg/m3)b; male (6/group): 0, 5000 ppm (0,

20,900
7%
20,900 mg/m3)b

Dose(mg/m3)
Percent change
dynamic whole body chamber; 6 hrs/d, 5 d/wk for


compared to
13 wks followed by a 28 day recovery period;


control
generation method, analytical concentration and
Male
0
-
method were reported

20,900
9%*
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor


control
female (50/group): 0, 500,1500, 5000 ppm (0,
Male
0
-
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

2090
9%*
500, 1500, 5000 ppm (0, 2090, 6270,

6270
19%*
20,900 mg/m3)b

20,900
49%*
dynamic whole body inhalation; 6 hrs/d, 5 d/wk
Study authors stated that increased relative liver weights
for 104 wks; generation method, analytical
were due to significantly lowered final body weights of
concentration and method were reported
treated groups; individual animal data were not available

to confirm statistical analysis conducted by study authors

(e.g., 1% statistically significant increase in females at the


mid-dose).



Dose(mg/m3)
Percent change



compared to



control

Female
0
-


2090
3%


6270
1%*


20,900
30%*
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).
This document is a draft for review purposes only and does not constitute Agency policy.
1-61	DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofETBE
1	Table 1-9. Evidence pertaining to liver histopathology effects in animals
2	exposed to ETBE
Reference and Dosing Protocol
Results by Endpoint
Acidophilic Foci in Liver
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Response
rat, Fischer 344


(incidence)
oral - water
Male
0
14/50
female (50/group): 0, 625, 2500,10,000 ppm (0,

28
12/50
46,171, 560 mg/kg-day)a; male (50/group): 0,

121
17/50
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

542
13/50
day)a

Dose(mg/kg-d)
Response
daily for 104 wks


(incidence)

Female
0
2/50


46
2/50


171
1/50


560
0/50
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Response
rat, Fischer 344


(incidence)
inhalation - vapor
Male
0
31/50
female (50/group): 0, 500,1500, 5000 ppm (0,

2090
28/50
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

6270
36/49
500, 1500, 5000 ppm (0, 2090, 6270,

20,900
39/50*
20,900 mg/m3)b

Dose(mg/m3)
Response
dynamic whole body inhalation; 6 hrs/d, 5 d/wk


(incidence)
for 104 wks; generation method, analytical
Female
0
2/50
concentration and method were reported

2090
1/50


6270
4/50


20,900
2/50
This document is a draft for review purposes only and does not constitute Agency policy.
1-62	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-9. Evidence pertaining to liver histopathology effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Basophilic Foci in Liver
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Response
rat, Fischer 344


(incidence)
oral - water
Male
0
14/50
female (50/group): 0, 625, 2500,10,000 ppm (0,

28
18/50
46,171, 560 mg/kg-day)a; male (50/group): 0,

121
20/50
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

542
22/50
day)a

Dose(mg/kg-d)
Response
daily for 104 wks


(incidence)

Female
0
36/50


46
25/50*


171
31/50


560
30/50*
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Response
rat, Fischer 344


(incidence)
inhalation - vapor
Male
0
18/50
female (50/group): 0, 500,1500, 5000 ppm (0,

2090
10/50
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

6270
13/49
500, 1500, 5000 ppm (0, 2090, 6270,

20,900
33/50*
20,900 mg/m3)b

Dose(mg/m3)
Response
dynamic whole body inhalation; 6 hrs/d, 5 d/wk


(incidence)
for 104 wks; generation method, analytical
Female
0
36/50
concentration and method were reported

2090
31/50


6270
32/50


20,900
28/50
Bile Duct Hyperplasia
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Response
rat, Fischer 344


(incidence)
oral - water
Male
0
49/50
female (50/group): 0, 625, 2500,10,000 ppm (0,

28
47/50
46,171, 560 mg/kg-day)a; male (50/group): 0,

121
48/50
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

542
47/50
day)a

Dose(mg/kg-d)
Response
daily for 104 wks


(incidence)

Female
0
1/50


46
4/50


171
4/50


560
3/50
This document is a draft for review purposes only and does not constitute Agency policy.
1-63	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-9. Evidence pertaining to liver histopathology effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Bile Duct Hyperplasia (continued)
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Response
rat, Fischer 344


(incidence)
inhalation - vapor
Male
0
48/50
female (50/group): 0, 500,1500, 5000 ppm (0,

2090
44/50
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

6270
46/49
500, 1500, 5000 ppm (0, 2090, 6270,

20,900
41/50
20,900 mg/m3)b

Dose(mg/m3)
Response
dynamic whole body inhalation; 6 hrs/d, 5 d/wk


(incidence)
for 104 wks; generation method, analytical
Female
0
5/50
concentration and method were reported

2090
8/50


6270
7/50


20,900
6/50
Centrilobular Hypertrophy
Gaoua (2004b)

Dose(mg/kg-d)
Response
rat, Sprague-Dawley


(incidence)
oral - gavage
P0, Male
0
0/25
P0, male (25/group): 0, 250, 500,1000 mg/kg-d

250
0/25
daily for a total of 18 weeks beginning 10 weeks

500
0/25
before mating until after weaning of the pups

1000
3/25
P0, female (25/group): 0, 250, 500,1000 mg/kg-d

Dose(mg/kg-d)
Response
daily for a total of 18 weeks beginning 10 weeks
P0, Female
0
0/25
before mating until PND 21

250
0/25


500
0/25


1000
0/25
Mivata et al. (2013); JPEC (2008c)

Dose(mg/kg-d)
Response
rat, CRL:CD(SD)


(incidence)
oral - gavage
Male
0
0/15
female (15/group): 0, 5, 25,100, 400 mg/kg-d;

5
0/15
male (15/group): 0, 5, 25,100, 400 mg/kg-d

25
0/15
daily for 180 days

100
0/15


400
6/15*


Dose(mg/kg-d)
Response



(incidence)

Female
0
0/15


5
0/15


25
0/15


100
0/15


400
6/15*
This document is a draft for review purposes only and does not constitute Agency policy.
1-64	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-9. Evidence pertaining to liver histopathology effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Centrilobular Hypertrophy (continued)
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Response
rat, Fischer 344


(incidence)
oral - water
Male
0
0/50
female (50/group): 0, 625, 2500,10,000 ppm (0,

28
0/50
46,171, 560 mg/kg-d)a; male (50/group): 0, 625,

121
0/50
2500, 10,000 ppm (0, 28, 121, 542 mg/kg-d)a

542
0/50
daily for 104 wks

Dose(mg/kg-d)
Response



(incidence)

Female
0
0/50


46
0/50


171
0/50


560
0/50
JPEC (2008b)

Dose(mg/m3)
Response
rat, CRL:CD(SD)


(incidence)
inhalation - vapor
Male
0
0/10
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,

627
0/10
2090, 6270, 20,900 mg/m3); male (NR): 0, 150,

2090
0/10
500, 1500, 5000 ppm (0, 627, 2090, 6270,

6270
0/10
20,900 mg/m3)b

20,900
4/10*
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

Dose(mg/m3)
Response
13 wks; generation method, analytical


(incidence)
concentration and method were reported
Female
0
0/10


627
0/10


2090
0/10


6270
0/10


20,900
6/10*
JPEC (2008b)

Dose(mg/m3)
Response
rat, CRL:CD(SD)
Male
0
0/6
inhalation - vapor

20,900
0/6
female (6/group): 0, 5000 ppm (0,

Dose(mg/m3)
Response
20,900 mg/m3)b; male (6/group): 0, 5000 ppm (0,
Female
0
0/6
20,900 mg/m3)b

20,900
0/6
dynamic whole body chamber; 6 hrs/d, 5 d/wk for



13 wks followed by a 28 day recovery period;



generation method, analytical concentration and



method were reported



This document is a draft for review purposes only and does not constitute Agency policy.
1-65	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-9. Evidence pertaining to liver histopathology effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Centrilobular Hypertrophy (continued)
Medinskv et al. (1999); Bond et al. (1996b)

Dose(mg/m3)
Response
rat, Fischer 344


(incidence)
inhalation - vapor
Male
0
0/11
female (48/group): 0, 500,1750, 5000 ppm (0,

2090
0/11
2090, 7320, 20,900 mg/m3)b; male (48/group): 0,

7320
0/11
500, 1750, 5000 ppm (0, 2090, 7320,

20,900
0/11
20,900 mg/m3)b

Dose(mg/m3)
Response
dynamic whole body chamber; 6 hrs/d, 5 d/wk for


(incidence)
13 wks; generation method, analytical
Female
0
0/10
concentration and method were reported

2090
0/11


7320
0/11


20,900
0/11
Medinskv et al. (1999); Bond et al. (1996a)

Dose(mg/m3)
Response
mice, CD-I


(incidence)
inhalation - vapor
Male
0
0/15
female (40/group): 0, 500,1750, 5000 ppm (0,

2090
0/15
2090, 7320, 20,900 mg/m3)b; male (40/group): 0,

7320
2/15
500, 1750, 5000 ppm (0, 2090, 7320,

20,900
8/10*
20,900 mg/m3)b

Dose(mg/m3)
Response
dynamic whole body chamber; 6 hrs/d, 5 d/wk for


(incidence)
13 wks; generation method, analytical
Female
0
0/13
concentration and method were reported

2090
2/15


7320
1/15


20,900
9/14*
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Response
rat, Fischer 344


(incidence)
inhalation - vapor
Male
0
0/50
female (50/group): 0, 500,1500, 5000 ppm (0,

2090
0/50
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

6270
0/49
500, 1500, 5000 ppm (0, 2090, 6270,

20,900
0/50
20,900 mg/m3)b

Dose(mg/m3)
Response
dynamic whole body inhalation; 6 hrs/d, 5 d/wk


(incidence)
for 104 wks; generation method, analytical
Female
0
0/50
concentration and method were reported

2090
0/50


6270
0/50


20,900
0/50
This document is a draft for review purposes only and does not constitute Agency policy.
1-66	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-9. Evidence pertaining to liver histopathology effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Centrilobular Hypertrophy (continued)
Weng etal. (2012)

Dose(mg/m3)
Response
mice, C57BL/6


(incidence)
inhalation - vapor
Male
0
1/5
female (5/group): 0, 500,1750, 5000 ppm (0,

2090
0/5
2090, 7320, 20,900 mg/m3)b; male (5/group): 0,

7320
0/5
500, 1750, 5000 ppm (0, 2090, 7320,

20,900
5/5*
20,900 mg/m3)b

Dose(mg/m3)
Response
dynamic whole body chamber, 6 hr/d, 5 d/wk for


(incidence)
13 wks; generation methods were not reported,
Female
0
0/5
but analytical methods (gas chromatograph) and

2090
0/5
concentration were reported

7320
1/5


20,900
5/5*
Weng etal. (2012)

Dose(mg/m3)
Response
mice, ALDH2-/-


(incidence)
inhalation - vapor
Male
0
0/5
female (5/group): 0, 500,1750, 5000 ppm (0,

2090
3/5
2090, 7320, 20,900 mg/m3)b; male (5/group): 0,

7320
2/5
500, 1750, 5000 ppm (0, 2090, 7320,

20,900
5/5*
20,900 mg/m3)b

Dose(mg/m3)
Response
dynamic whole body chamber, 6 hr/d, 5 d/wk for


(incidence)
13 wks; generation methods were not reported,
Female
0
0/5
but analytical methods (gas chromatograph) and

2090
0/5
concentration were reported

7320
0/5


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	(n): number evaluated from group
6
7
This document is a draft for review purposes only and does not constitute Agency policy.
1-67	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
1	Table 1-10. Evidence pertaining to liver biochemistry effects in animals
2	exposed to ETBE
Reference and Dosing Protocol
Results by Endpoint
Alanine Aminotransferase (ALT)
Mivata et al. (2013); JPEC (2008c)

Dose(mg/kg-d)
Percent change
rat, CRL:CD(SD)


compared to
oral - gavage


control
female (15/group): 0, 5, 25,100, 400 mg/kg-d;
Male
0
-
male (15/group): 0, 5, 25,100, 400 mg/kg-d

5
10%
daily for 180 days

25
48%


100
13%


400
35%


Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


5
11%


25
21%


100
46%


400
21%
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - water


control
female (50/group): 0, 625, 2500,10,000 ppm (0,
Male
0
-
46,171, 560 mg/kg-day)a; male (50/group): 0,

28
-17%
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

121
2%
day)a

542
-4%
daily for 104 wks

Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


46
-10


171
-15


560
-26
This document is a draft for review purposes only and does not constitute Agency policy.
1-68	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-10. Evidence pertaining to liver biochemistry effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Alanine Aminotransferase (ALT) (continued)
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,
Male
0
-
2090, 6270, 20,900 mg/m3); male (NR): 0, 150,

627
9%
500, 1500, 5000 ppm (0, 627, 2090, 6270,

2090
0%
20,900 mg/m3)b

6270
5%
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

20,900
12%
13 wks; generation method, analytical

Dose(mg/m3)
Percent change
concentration and method were reported


compared to



control

Female
0
-


627
-1%


2090
11%


6270
-5%


20,900
26%
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor


control
female (50/group): 0, 500,1500, 5000 ppm (0,
Male
0
-
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

2090
53%
500, 1500, 5000 ppm (0, 2090, 6270,

6270
-3%
20,900 mg/m3)b

20,900
24%
dynamic whole body inhalation; 6 hrs/d, 5 d/wk

Dose(mg/m3)
Percent change
for 104 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


2090
2%


6270
-5%


20,900
4%*
This document is a draft for review purposes only and does not constitute Agency policy.
1-69	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-10. Evidence pertaining to liver biochemistry effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Alkaline Phosphatase (ALP)
Mivata et al. (2013); JPEC (2008c)

Dose(mg/kg-d)
Percent change
rat, CRL:CD(SD)


compared to
oral - gavage


control
female (15/group): 0, 5, 25,100, 400 mg/kg-d;
Male
0
-
male (15/group): 0, 5, 25,100, 400 mg/kg-d

5
2%
daily for 180 days

25
12%


100
-7%


400
27%


Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


5
6%


25
-21%


100
-18%


400
-19%
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - water


control
female (50/group): 0, 625, 2500,10,000 ppm (0,
Male
0
-
46,171, 560 mg/kg-day)a; male (50/group): 0,

28
-5%
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

121
3%
day)a

542
0%
daily for 104 wks

Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


46
-16%


171
2%


560
-15%
This document is a draft for review purposes only and does not constitute Agency policy.
1-70	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-10. Evidence pertaining to liver biochemistry effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Alkaline Phosphatase (ALP) (continued)
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,
Male
0
-
2090, 6270, 20,900 mg/m3); male (NR): 0, 150,

627
13%
500, 1500, 5000 ppm (0, 627, 2090, 6270,

2090
12%
20,900 mg/m3)b

6270
-12%
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

20,900
-9%
13 wks; generation method, analytical

Dose(mg/m3)
Percent change
concentration and method were reported


compared to



control

Female
0
-


627
-3%


2090
-12%


6270
-7%


20,900
5%
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor


control
female (50/group): 0, 500,1500, 5000 ppm (0,
Male
0
-
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

2090
0%
500, 1500, 5000 ppm (0, 2090, 6270,

6270
-21%*
20,900 mg/m3)b

20,900
-5%
dynamic whole body inhalation; 6 hrs/d, 5 d/wk

Dose(mg/m3)
Percent change
for 104 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


2090
12%


6270
-4%


20,900
4%
This document is a draft for review purposes only and does not constitute Agency policy.
1-71	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-10. Evidence pertaining to liver biochemistry effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Aspartate Aminotransferase (AST)
Mivata et al. (2013); JPEC (2008c)

Dose(mg/kg-d)
Percent change
rat, CRL:CD(SD)


compared to
oral - gavage


control
female (15/group): 0, 5, 25,100, 400 mg/kg-d;
Male
0
-
male (15/group): 0, 5, 25,100, 400 mg/kg-d

5
16%
daily for 180 days

25
19%


100
20%


400
23%


Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


5
10%


25
13%


100
19%


400
4%
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - water


control
female (50/group): 0, 625, 2500,10,000 ppm (0,
Male
0
-
46,171, 560 mg/kg-d)a; male (50/group): 0, 625,

28
-21%
2500, 10,000 ppm (0, 28, 121, 542 mg/kg-d)a

121
-3%
daily for 104 wks

542
-1%


Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


46
-19%


171
-17%


560
-46%*
This document is a draft for review purposes only and does not constitute Agency policy.
1-72	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-10. Evidence pertaining to liver biochemistry effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Aspartate Aminotransferase (AST) (continued)
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,
Male
0
-
2090, 6270, 20,900 mg/m3); male (NR): 0, 150,

627
3%
500, 1500, 5000 ppm (0, 627, 2090, 6270,

2090
1%
20,900 mg/m3)b

6270
-7%
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

20,900
4%
13 wks; generation method, analytical

Dose(mg/m3)
Percent change
concentration and method were reported


compared to



control

Female
0
-


627
2%


2090
-95%


6270
12%


20,900
0%
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor


control
female (50/group): 0, 500,1500, 5000 ppm (0,
Male
0
-
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

2090
29%
500, 1500, 5000 ppm (0, 2090, 6270,

6270
-16%
20,900 mg/m3)b

20,900
-2%*
dynamic whole body inhalation; 6 hrs/d, 5 d/wk

Dose(mg/m3)
Percent change
for 104 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


2090
22%


6270
10%


20900
18%*
This document is a draft for review purposes only and does not constitute Agency policy.
1-73	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-10. Evidence pertaining to liver biochemistry effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Gamma-Glutamyl Transpeptidase (GGT)
Mivata et al. (2013); JPEC (2008c)

Dose(mg/kg-d)
Percent change
rat, CRL:CD(SD)


compared to
oral - gavage


control
female (15/group): 0, 5, 25,100, 400 mg/kg-d;
Male
0
-
male (15/group): 0, 5, 25,100, 400 mg/kg-d

5
25%
daily for 180 days

25
50%


100
25%


400
100%


Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


5
40%


25
20%


100
0%


400
-20%
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - water


control
female (50/group): 0, 625, 2500,10,000 ppm (0,
Male
0
-
46,171, 560 mg/kg-d)a; male (50/group): 0, 625,

28
0%
2500, 10,000 ppm (0, 28, 121, 542 mg/kg-d)a

121
43%*
daily for 104 wks

542
29%


Dose(mg/kg-d)
Percent change



compared to



control

Female
0
-


46
0%


171
0%


560
33%
This document is a draft for review purposes only and does not constitute Agency policy.
1-74	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-10. Evidence pertaining to liver biochemistry effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Gamma-Glutamyl Transpeptidase (GGT) (continued)
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,
Male
0
-
2090, 6270, 20,900 mg/m3); male (NR): 0, 150,

627
11%
500, 1500, 5000 ppm (0, 627, 2090, 6270,

2090
0%
20,900 mg/m3)b

6270
11%
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

20,900
-100%
13 wks; generation method, analytical

Dose(mg/m3)
Percent change
concentration and method were reported


compared to



control

Female
0
-


627
25%


2090
12%


6270
25%


20,900
25%
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor


control
female (50/group): 0, 500,1500, 5000 ppm (0,
Male
0
-
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

2090
33%
500, 1500, 5000 ppm (0, 2090, 6270,

6270
50%*
20,900 mg/m3)b

20,900
200%*
dynamic whole body inhalation; 6 hrs/d, 5 d/wk

Dose(mg/m3)
Percent change
for 104 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


2090
50%


6270
0%


20,900
150%
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).
This document is a draft for review purposes only and does not constitute Agency policy.
1-75	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
¦ = 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
Centrilobular
Hypertrophy
Serum Liver
Enzymes (ALT,
ALP)
AST,
PO Male rat; 16wks [A)
PO Female rat; 16wks (A)
PO Male rat; 18wks (B)
PO Female rat; 18wks (B)
F1 Male rat; GD 0-adult (B)
F1 Female rat; GD 0-adult (B)
Male rat; 23wks (C)
Female rat; 26wks (D)
Male rat; 26wks (D)
Female rat; 104wks [E)
Male rat; 104wks (E)
PO Male rat; 18wks (B)
PO Female rat; 18wl
-------
Toxicological Review ofETBE
¦ = 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
Centrllobular
Hypertrophy
Female rats; 13wks (A]
Male rats; 13wks (A)
Female rats; 13wks, 28d recovery (A)
Male rats; 13wks, 2Sd recovery (A)
Female rats; 104wks (C)
Male rats; 104wks (C)
Female rats; 13wks (A)
Male rats; 13wks (A)
Female rats; 13wks, 28d recovery (A)
Male rats; 13wks, 28d recovery [A)
Female rats; 13 wks (B)
Male rats; 13 wks (B)
Female mice; 13 wks (B]
Male mice; 13 wks (B)
Female mice; 13 wks [D]
Male mice; 13 wks (D)
Female Aldh2-/- mice; 13 wks (D)
Male Aldh2-/- mice; 13 wks (D)
Female rats; 104wks (C)
Male rats; 104wks (Q
-B	B-
-B	B-
B-
-B	B-
-B
~
~
~
B	B—B
B-
-Bi—B
~ i ~
B-
-B
Q	B-
B	B-
Serum Liver
Enzymes (ALT, AST,
ALP)
Female rats; 13wks (A)
Male rats; 13wks [A]
Female rats; 104wks (C)
Male rats; 104wks (C)
-B-
-B	B-
-B-
B	B-
-B
-B
-B
-B
1	10	100 1,000 10,000 100,000
Exposure Concentration (mg/m3)
Sources: (A) JPEC, 2008b (B) Medinsky et al, 1999; Bondet al„ 1996(C) Saito et al„ 2013; JPEC,2010b (D) Weng
et at 2012
Figure 1-6. Exposure-response array of liver effects following inhalation
exposure to ETBE.
This document is a draft for review purposes only and does not constitute Agency policy.
1-77	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
1	Table 1-11. Evidence pertaining to liver tumor effects in animals exposed to
2	ETBE
Reference and Dosing Protocol
Results by Endpoint
Hepatocellular Adenoma and Carcinoma
Suzuki et al. (2012); JPEC (2010a)
Incidence



Adenoma
rat, Fischer 344

Dose


or
oral - water

(mg/kg-d)
Adenoma
Carcinoma
Carcinoma
female (50/group): 0, 625, 2500,10,000 ppm (0,
Male
0
2/50
2/50
4/50
46,171, 560 mg/kg-day)a; male (50/group): 0,

28
0/50
0/50
0/50
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

121
0/50
0/50
0/50
day)a

542
0/50
0/50
0/50
daily for 104 wks

Dose


Adenoma
or


(mg/kg-d)
Adenoma
Carcinoma
Carcinoma

Female
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
Saito et al. (2013); JPEC (2010b)
Incidence



Adenoma
rat, Fischer 344

Dose


or
inhalation - vapor

(mg/m3)
Adenoma
Carcinoma
Carcinoma
female (50/group): 0, 500,1500, 5000 ppm (0,
Male
0
0/50
0/50
0/50
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

2090
2/50
0/50
2/50
500, 1500, 5000 ppm (0, 2090, 6270,

6270
1/50
0/50
1/50
20,900 mg/m3)b

20,900
9/50*
1/50
10/50*
dynamic whole body inhalation; 6 hrs/d, 5 d/wk




Adenoma
for 104 wks; generation method, analytical

Dose


or
concentration and method were reported

(mg/m3)
Adenoma
Carcinoma
Carcinoma

Female
0
1/50
0/50
1/50


2090
0/50
0/50
0/50


6270
1/50
0/50
1/50


20,900
1/50
0/50
1/50
This document is a draft for review purposes only and does not constitute Agency policy.
1-78	DRAFT—DO NOT CITE OR QUOTE
-------
Toxicological Review ofETBE
Table 1-11. Evidence pertaining to liver tumor effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Liver Neoplasm
Hagiwara et al. (2011); JPEC (2008d)
rat, Fischer 344
oral - gavage
male (30/group): 0, 300,1000 mg/kg-d
daily for 23 weeks following a 4 week tumor
initiation by DMBDD
+ no DMBB initiation
Dose(mg/kg-d) Response (incidence)
Male 0 1/30
300 1/30
1000 6/30*
0+ 0/12
1000+ 0/12
Maltoni et al. (1999)
rat, Sprague-Dawley
oral - gavage
female (60/group): 0, 250,1000 mg/kg-d; male
(60/group): 0, 250,1000 mg/kg-d
4 d/wk for 104 wks; observed until natural death
NOTE: These tumor data were not re-analyzed by
Malarkev and Bucher (2011)
Dose(mg/kg-d) Response (incidence)
Male 0 0/60
250 0/60
1000 0/60
Dose(mg/kg-d) Response (incidence)
Female 0 0/60
250 0/60
1000 0/60
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
7	Mode of Action Analysis- Liver Effects
8	Toxicokinetic considerations relevant to liver toxicity and tumors
9	ETBE is metabolized by cytochrome P450 (CYP) enzymes to an unstable hemiacetal that
10	decomposes spontaneously into tert-butanol and acetaldehyde fBernauer et al.. 19981.
11	Acetaldehyde is further metabolized in the liver by ALDH2, whereas tert-butanol undergoes
12	systemic circulation and is ultimately excreted in urine. Thus, following ETBE exposure, the liver is
13	exposed to both acetaldehyde and tert-butanol, so the liver effects caused by tert-butanol
14	(described in the more detail in the draft IRIS assessment of tert-butanol) and acetaldehyde are also
15	relevant to evaluating the liver effects observed after ETBE exposure.
16	tert-Butanol induces thyroid and kidney tumors in rodents, but has not been observed to
17	affect the incidence of liver tumors following a 2-year oral exposure. Whereas there are some data
18	suggesting tert-butanol may be genotoxic, the overall evidence is inadequate to establish a
19	conclusion. No study has reported that tert-butanol causes centrilobular hypertrophy or that it
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activates nuclear receptors. Therefore, a role for tert-butanol in liver carcinogenesis ofETBE does
not appear likely. No mode of action information is available for tert-butanol-induced noncancer
liver effects.
On the other hand, acetaldehyde is genotoxic and mutagenic flARC. 1999a], and
acetaldehyde produced in the liver as a result of ethanol metabolism has been suggested as a
contributor to ethanol-related liver toxicity and cancer (Setshedi etal.. 2010). Additional discussion
on the potential role of acetaldehyde in the liver carcinogenesis ofETBE is provided below.
Receptor-mediated effects
ETBE exposure consistently increased both relative and absolute liver weights in male and
female rats. In addition, ETBE increased hepatocellular adenomas and carcinomas in males exposed
via inhalation for 2 years (Saito etal.. 2013: TPEC. 2010b). These studies did not report consistent
effects on liver function as demonstrated by a lack of concordant changes in serum liver enzyme
levels. However, several studies have demonstrated that ETBE increases centrilobular hypertrophy
and preneoplastic lesions, which may lead to tumorigenesis. This process was investigated in
several studies to determine whether nuclear receptor activation is involved.
Centrilobular hypertrophy is induced through a number of possible mechanisms, of which
many are via nuclear hormone receptors such as peroxisome proliferator-activated receptor a
(PPARa), pregnane X receptor (PXR), and the constitutive androstane receptor (CAR). The
sequence of key events hypothesized for PPARa induction of liver tumors is as follows: activation of
PPARa, upregulation of peroxisomal genes, expression of PPARa-mediated growth and apoptosis,
disrupted cell proliferation and apoptosis, peroxisome proliferation, preneoplastic foci, and tumors
fKlaunig etal.. 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.. 2014). PXR does not have an established MOA but is hypothesized to progress from PXR
activation to liver tumors in a similar manner as CAR, which would include PXR activation, cell
proliferation, hypertrophy, CYP3A induction, and clonal expansion resulting in foci development
One study that exposed male rats to a high and low concentration of ETBE via gavage twice per day
for 2 weeks reported that several key sequences in these aforementioned pathways were affected
(Kakehashi etal.. 2013).
PPAR
The data suggest that PPAR may be involved in ETBE-induced liver tumors (Kakehashi etal..
20131. For instance, mRNA expression was statistically significantly elevated for PPARa and PPARy
after 1 week of exposure but not after 2 weeks. In addition, a number of PPARa-mediated proteins
involved in lipid and xenobiotic metabolism were upregulated in the liver after 2 weeks of exposure
such as ACOX1, CYP4A2, and ECH1. DNA damage (8-OHdG) and apoptosis (ssDNA) were also
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statistically significantly increased after 2 weeks at the highest concentration ofETBE. Cell
proliferation was unchanged after 1 week and significantly decreased after 2 weeks. The number of
peroxisomes per hepatocyte was increased greater than fivefold after 2 weeks of treatments.
Finally, the incidences of basophilic and acidophilic foci were significantly increased in males after
2 years of inhalation exposure to ETBE (Saito etal.. 2013: TPEC. 2010bl.
Altogether, a number of key sequences in the PPAR pathway were observed in the
Kakehashi etal. f20131 and f Saito etal.. 2013: TPEC. 2010bl studies; however, several steps in the
pathway were either not observed or not examined. For instance, selective clonal expansion was
not examined in any study. Furthermore, the cell proliferation and apoptosis data were contrary to
what would be expected if a PPAR MOA were operative. Cell proliferation was decreased after 2
weeks of exposure; no other time points in the data set were available (Kakehashi etal.. 20131. In
addition, 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 ofETBE exposure (Kakehashi et
al.. 20131. Perturbation of cell proliferation and apoptosis are both required steps for MOA and
future studies with longer exposures could address this data gap. Overall, these data are suggestive
but not adequate for establishing a PPAR MOA for liver tumorigenesis.
CAR/PXR
Kakehashi etal. (20131 reported a number of CAR and PXR-mediated events following ETBE
exposure. After 2 weeks of exposure at the high dose ofETBE, PXR- and CAR-regulated xenobiotic
metabolic enzymes were upregulated, including Cyp2bl, Cyp2b2, Cyp3al, and Cyp3a2 as
determined by mRNA and/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 PXR and CAR. As described above for Kakehashi etal. (20131. cell proliferation
was reduced, and apoptosis was increased following ETBE exposure, in contrast to what is expected
during the CAR/PXR sequence of events. There were several data gaps that were not evaluated such
as a lack of clonal expansion and gap junction communication. These data provide evidence that
PXR and CAR are activated in the liver following ETBE exposure; however, due to crosstalk of PXR
and CAR on downstream effects such as cell proliferation, preneoplastic foci, and apoptosis, it is not
possible to determine the relative contribution of each pathway in tumorigenesis. The data do not
provide enough information to determine dose-response concordance or temporal associations,
which are critical for establishing a MOA. Furthermore, the available data from this study do not
allow for parsing which effects are induced by PPAR or CAR/PXR activation. Altogether, these data
are inadequate to establish a CAR/PXR MOA for inducing liver tumors.
Acetaldehvde-mediated liver toxicity and genotoxicitv
Another possible MOA for increased tumors could be due to the production of acetaldehyde
in the liver, the primary site for ETBE metabolism. Acetaldehyde produced as a result of
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metabolism of alcohol consumption is considered carcinogenic to humans by IARC f!999al. though
there is not sufficient evidence that acetaldehyde formed in this manner causes liver carcinogenesis
flARC. 20121. 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 f!999al.
Acetaldehyde has been shown to have an inhibitory effect on PPARa transcriptional activity
fVenkata etal.. 20081. The effect of acetaldehyde on CAR or PXR activation has not 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, particularly esophageal cancer, among some subpopulations such as East Asians
(IARC. 2012: Brennan etal.. 20041. However, the importance of this polymorphism for
hepatocarcinogenesis is unclear.
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
Aldh2 KO mice compared with wild type (WT) (Wengetal.. 20121. 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 inWT females. Measures of DNA damage
were all statistically significantly exacerbated in both male and female Aldh2 KO mice (Weng etal..
20121. Further demonstrating enhanced genotoxic sensitivity in males compared with females,
erythrocyte micronucleus assays and oxidative DNA damage in leukocytes were only observed to
be statistically significantly increased and dose responsive in male Aldh2 KO mice fWeng etal..
20131. Altogether, while these data are suggestive of a potential role for acetaldehyde in the
increased liver tumor response observed in male rats exposed to ETBE, the available data are
inadequate to establish acetaldehyde-mediated mutagenicity as a MOA for ETBE-induced liver
tumors.
Summary of mode of action analysis
The available mechanistic data provide some evidence that two nuclear receptor-mediated
pathways (PPAR and CAR/PXR) may contribute to both the hypertrophy and tumorigenesis
observed in ETBE-treated males. These studies do not provide any evidence on the relative
contributions of either of these pathways in the development of liver tumors. Several reviews
suggest that the PPAR, PXR, and/or CAR pathways induce liver tumors in a manner that is not
relevant to humans (Elcombe etal.. 2014: Klaunigetal.. 20031 although this conclusion has been
questioned fGuvton etal.. 20091. The available data are inadequate to conclude that the liver
tumors observed in rats are caused by one of these nuclear receptor-mediated pathways.
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Therefore, given the available data, ETBE-induced liver tumors in male rats are considered relevant
to humans.
Evidence also suggests that metabolism ofETBE to acetaldehyde may contribute to ETBE-
induced liver carcinogenesis. For instance, enhancement of ETBE-induced liver toxicity and
ge no toxicity has been reported in Aldh2-deficient mice, which have an impaired ability to
metabolize acetaldehyde (Weng etal.. 2013: Wengetal.. 20121. Additionally, lack of ALDH2 is
directly relevant to the substantial human subpopulation that is deficient in the ALDH2 isozyme.
Given the known genotoxicity and carcinogenicity of acetaldehyde flARC. 2012], these data are
suggestive of a role for acetaldehyde in ETBE-induced liver tumorigenesis. However, the available
data are inadequate to establish acetaldehyde-mediated mutagenicity as a MOA for ETBE-induced
liver tumors.
Summary of Liver Toxicity
Evidence for ETBE-induced noncancer liver effects is available from rat and mouse studies.
Several endpoints such as increased liver weight and liver enzymes were more severely affected in
males compared with females fSaito etal.. 2013: Suzuki etal.. 2012: TPEC. 2010a. b). Noncancer
effects were observed in subchronic oral and inhalation studies. One chronic inhalation study
observed increased hepatocellular tumors in male rats f Suzuki etal.. 2012: TPEC. 2010al.
Relative liver weights were consistently increased in males in 8 of 9 studies and 6 of 8
studies for females; however, statistically significant increases frequently occurred only at the
highest tested concentration with modest increases in relative liver weight ranging from 17-27% in
males and 8-18% in females. Centrilobular hypertrophy also was observed at the same high doses
in males and females after 13-week and 26-week inhalation and oral exposure, respectively. No
other accompanying pathologies were observed. A significant dose-related increase in GGT was
only observed in one 2-year inhalation study in male rats; no other consistent changes in liver
enzymes were observed in males or females.
Given the modest organ weight changes, lack of dose response with other liver endpoints,
and poor temporal correlation indicative of accumulating damage, EPA concluded that the evidence
does not support liver effects as a potential human hazard of ETBE exposure.
With respect to liver carcinogenicity, one 2-year inhalation rat study observed increased
hepatocellular adenomas and carcinomas in males at the highest tested dose (Saito etal.. 2013:
TPEC. 2010bl. Although only one carcinoma was observed, the adenomas have the potential to
transform into malignant carcinomas (McConnell etal., 1986). However, increases in liver tumors
were not observed either in a 2-year oral drinking water bioassay in rats in the same laboratory or
in an additional cancer bioassay in rats performed by oral gavage. A mechanistic study conducted
by gavage in rats observed ETBE-related increases in liver tumors following initiation by DMBDD,
suggesting that ETBE exposure can promote liver tumors fHagiwara etal.. 20111. Additional
mechanistic data on the role of PPAR, PXR, and CAR activation in liver tumorigenesis were
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inadequate to conclude that these pathways mediate tumor formation. Additional mechanistic
studies reported that lack of ALDH2 enhanced ETBE-induced liver toxicity and genotoxicity fWeng
etal.. 2013: Wengetal.. 20121. These findings are consistent with genotoxicity being mediated by
the ETBE metabolite acetaldehyde, which is genotoxic and considered carcinogenic when produced
as a result of metabolism from ingested ethanol (IARC. 20121. Overall, available mechanistic data
provide some biological plausibility to the liver carcinogenicity ofETBE. Section 1.2.2 discusses the
overall weight of evidence for ETBE carcinogenicity.
1.1.3. Reproductive and Developmental Effects
Synthesis of reproductive and developmental toxicity
This section reviews the studies that investigated whether exposure to ETBE can cause
reproductive or developmental toxicity in humans or animals. The database examining
reproductive or developmental effects following ETBE exposure contains no human data, but is
comprised of animal data primarily from rats. Three studies evaluated reproductive effects: a one-
generation study, two-generation study, and subchronic study. In addition, there were two short-
term studies evaluating effects on reproductive hormones and effects on oocytes. Reproductive
organs were also evaluated in a subchronic study and four chronic studies that evaluated
reproductive organs with no significant effects observed. Five studies evaluated developmental
effects (three developmental studies, a one-generation reproductive study, and a two-generation
reproductive study). One preliminary reproductive and developmental study is not discussed
because it was superseded by two later studies within the same laboratory. Methodological
concerns were identified with the Wengetal.. 2014 study and included the lack of reported
experimental blinding for histophathological examinations and the lack of standard terminology for
reporting sperm effects which reduced confidence in these endpoints. No other methodological
concerns were identified that would lead one or more studies to be considered less informative for
assessing human health hazard.
Reproductive effects
Reproductive endpoints that were reported include oocyte viability, sex hormones,
seminiferous tubules, and sperm effects. Sperm parameters in rats were not affected by ETBE in
either generation of the two-generation study (Gaoua. 2004b) or in wild-type mice fWeng etal..
20141 (see Table 1-13; Figure 1-7, Figure 1-8). Sperm effects as measured by percent change in
sperm heads and sperm motility (number of sperm that were mobile, number of sperm that were
static, sperm with rapid movement) were observed in Aldh2 knockout or heterozygous mice but
not in wild type fWeng etal.. 20141. Lack of data on the biological relevance of reduced sperm
motility reduced the possibility that this finding is a potential hazard. Short-term studies did not
observe any effects on the number of oocytes recovered from ovulating female rats or in the ability
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1	of the oocytes to be fertilized fBerger and Horner. 20031 nor was there an effect on testosterone
2	levels fde Pevster et al.. 20091: however, male rats had a statistically significant increase in
3	estradiol levels fde Pevster et al.. 20091. No effects from ETBE were observed on the seminiferous
4	tubules fWengetal.. 20141. No additional reproductive effects have been reported.
5
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1	Table 1-12. Evidence pertaining to female reproductive effects in animals
2	exposed to ETBE
Reference and Dosing Protocol
Results by Endpoint
Delivery Index (pups delivered/implantations)
Fuiiietal. (2010); JPEC (2008e)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, female (24/group): 0,100, 300,1000 mg/kg-d
P0, Female
0
-
daily for 17 weeks beginning 10 weeks prior to

100
-7%
mating to lactation day 21

300
-4%


1000
-3%
Fertility Index
Fuiiietal. (2010); JPEC (2008e)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, male (24/group): 0,100, 300,1000 mg/kg-d
P0, Male
0
-
daily for 16 weeks beginning 10 weeks prior to

100
14%
mating

300
9%
P0, female (24/group): 0,100, 300,1000 mg/kg-d

1000
5%
daily for 17 weeks beginning 10 weeks prior to

Dose(mg/kg-d)
Percent change
mating to lactation day 21


compared to



control

P0, Female
0
-


100
14%


300
9%


1000
5%
3
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Table 1-12. Evidence pertaining to female reproductive effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Fertility Index (continued)
Gaoua (2004b)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, male (25/group): 0, 250, 500,1000 mg/kg-d
P0, Male
0
-
daily for a total of 18 weeks beginning 10 weeks

250
-9%
before mating until after weaning of the pups

500
-4%
P0, female (25/group): 0, 250, 500,1000 mg/kg-d

1000
9%
daily for a total of 18 weeks beginning 10 weeks

Dose(mg/kg-d)
Percent change
before mating until PND 21


compared to
Fl, male (25/group): 0, 250, 500,1000 mg/kg-d


control
dams dosed daily through gestation and lactation,
Fl, Male
0
-
then Fl doses beginning PND 22 until weaning of

250
0%
the F2 pups

500
-4%
Fl, female (24-25/group): 0, 250, 500,1000

1000
4%
mg/kg-d

Dose(mg/kg-d)
Percent change
dams dosed daily through gestation and lactation,


compared to
then Fl dosed beginning PND 22 until weaning of


control
the F2 pups
P0, Female
0
-


250
-9%


500
-4%


1000
9%


Dose(mg/kg-d)
Percent change



compared to



control

Fl, Female
0
-


250
5%


500
0%


1000
9%
Postimplantation Loss
Gaoua (2004b)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, female (25/group): 0, 250, 500,1000 mg/kg-d
P0, Female
0
-
daily for a total of 18 weeks beginning 10 weeks

250
33%
before mating until PND 21

500
14%


1000
51%
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Table 1-12. Evidence pertaining to female reproductive effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Litter Size
Gaoua (2004b)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, female (25/group): 0, 250, 500,1000 mg/kg-d
P0, Female
0
-
daily for a total of 18 weeks beginning 10 weeks

250
-1%
before mating until PND 21

500
4%
Fl, female (24-25/group): 0, 250, 500,1000

1000
-1%
mg/kg-d

Dose(mg/kg-d)
Percent change
dams dosed daily through gestation and lactation,


compared to
then Fl dosed beginning PND 22 until weaning of


control
the F2 pups
Fl, Female
0
250
500
1000
0%
0%
2%
Oocytes Fertilized
Berger and Horner (2003)

Dose(mg/kg-d)
Percent change
rat, Simonson albino


compared to
oral - water


control
P0, female (NR): 0, 0.3 % (estimated to be 0,1887
P0, Female
0
-
mg/kg-d)

1887
-2%
daily for 2 weeks; then oocytes fertilized in vitro
Treatment with ETBE did not affect the percentage of

oocytes fertilized.


Oocytes Recovered Per Ovulating Female
Berger and Horner (2003)

Dose(mg/kg-d)
Percent change
rat, Simonson albino


compared to
oral - water


control
P0, female (NR): 0, 0.3 % (estimated to be 0,1887
P0, Female
0
-
mg/kg-d)

1887
-3%
daily for 2 weeks; then oocytes fertilized in vitro
ETBE had no effect on the percentage of females ovulating
or number of oocytes per ovulating female.
Estradiol
de Peyster et al. (2009)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - gavage


control
P0, male (12/group): 0, 600,1200,1800 mg/kg-d
P0, Male
0
-
daily for 14 days

600
1200
1800
29%
106%*
105%*
1	*: result is statistically significant (p<0.05) based on analysis of data by study authors.
2	for controls, no response relevant; for other doses, no quantitative response reported.
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1	(n): number evaluated from group.
2	Percent change compared to controls calculated as 100 x ((treated value - control value) -f control value).
3
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1	Table 1-13. Evidence pertaining to male reproductive effects in animals
2	exposed to ETBE
Reference and Dosing Protocol
Results by Endpoint
Sperm Heads (Testicular)
Gaoua (2004b)
rat, Sprague-Dawley
oral - gavage
P0, male (25/group): 0, 250, 500,1000 mg/kg-d
daily for a total of 18 weeks beginning 10 weeks
before mating until after weaning of the pups
Fl, male (25/group): 0, 250, 500,1000 mg/kg-d
dams dosed daily through gestation and lactation,
then Fl doses beginning PND 22 until weaning of
the F2 pups
Dose(mg/kg-d) Percent change
compared to
control
P0, Male 0
250 -5%
500 -6%
1000 -4%
Dose(mg/kg-d) Percent change
compared to
control
Fl, Male 0
250 -3%
500 5%
1000 -1%
Weng et al. (2014)
mice, C57BL/6
inhalation - vapor
male (NR): 0, 50, 200, 500 ppm (209, 836,
2090 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
9 wk; methods were stated to be described in
Weng et al., 2012
Dose(mg/m3) Percent change
compared to
control
Male 0
209 -13%
836 -15%
2090 -13%
Weng et al. (2014)
mice, Aldh2-/-
inhalation - vapor
male (NR): 0, 50, 200, 500 ppm (209, 836,
2090 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
9 wk; methods were stated to be described in
Weng et al., 2012
Dose(mg/m3) Percent change
compared to
control
Male 0
209 -8%
836 -16%*
2090 -23%*
Weng et al. (2014)
mice, Aldh2 heterogeneous
inhalation - vapor
male (NR): 0, 50, 200, 500 ppm (209, 836,
2090 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
9 wk; methods were stated to be described in
Weng et al., 2012
Dose(mg/m3) Percent change
compared to
control
Male 0
209 0%
836 -46%*
2090 -53%*
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Table 1-13. Evidence pertaining to male reproductive effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Sperm Heads
Testicular) (continued)
Weng et al. (2014)
mice, C57BL/6
inhalation - vapor
male (5/group): 0, 500,1750, 5000 ppm (0, 2090,
7320, 20,900 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
13 wk; methods were stated to be described in
Weng et al., 2012
Dose(mg/m3) Percent change
compared to
control
Male 0
2090 1%
7320 1%
20,900 -9%
Weng et al. (2014)
mice, Aldh2-/-
inhalation - vapor
male (5/group): 0, 500,1750, 5000 ppm (0, 2090,
7320, 20,900 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
13 wk; methods were stated to be described in
Weng et al., 2012
Dose(mg/m3) Percent change
compared to
control
Male 0
2090 -25%*
7320 -26%*
20,900 -26%*
Sperm Motility (Epididymal)
Gaoua (2004b)
rat, Sprague-Dawley
oral - gavage
P0, male (25/group): 0, 250, 500,1000 mg/kg-d
daily for a total of 18 weeks beginning 10 weeks
before mating until after weaning of the pups
Fl, male (25/group): 0, 250, 500,1000 mg/kg-d
dams dosed daily through gestation and lactation,
then Fl doses beginning PND 22 until weaning of
the F2 pups
Dose(mg/kg-d) Percent change
compared to
control
P0, Male 0
250 0%
500 -1%
1000 -2%
Dose(mg/kg-d) Percent change
compared to
control
Fl, Male 0
250 3%
500 10%
1000 4%
Weng et al. (2014)
mice, C57BL/6
inhalation - vapor
male (NR): 0, 50, 200, 500 ppm (0, 209, 836,
2090 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
9 wk; methods were stated to be described in
Weng et al., 2012
Male
no significant change (results in figure only)
This document is a draft for review purposes only and does not constitute Agency policy.
1-91	DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofETBE
Table 1-13. Evidence pertaining to male reproductive effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Sperm Motility
Epididymal) (continued)
Weng et al. (2014)
mice, Aldh2-/-
inhalation - vapor
male (NR): 0, 50, 200, 500 ppm (0, 209, 836,
2090 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
9 wk; methods were stated to be described in
Weng et al., 2012
Male
significantly decreased at 500 ppm (2090 mg/m3) (results
in figure only)
Weng et al. (2014)
mice, Aldh2 heterogeneous
inhalation - vapor
male (NR): 0, 50, 200, 500 ppm (0, 209, 836,
2090 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
9 wk; methods were stated to be described in
Weng et al., 2012
Male
significantly decreased at >=200 ppm (836 mg/m3) (results
in figures only)
Weng et al. (2014)
mice, C57BL/6
inhalation - vapor
male (5/group): 0, 500,1750, 5000 ppm (0, 2090,
7320, 20,900 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
13 wk; methods were stated to be described in
Weng et al., 2012
Male
no significant change (results in figure only)
Weng et al. (2014)
mice, Aldh2-/-
inhalation - vapor
male (5/group): 0, 500,1750, 5000 ppm (0, 2090,
7320, 20,900 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
13 wk; methods were stated to be described in
Weng et al., 2012
Male
significantly decreased at all doses (results in figure only)
This document is a draft for review purposes only and does not constitute Agency policy.
1-92	DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofETBE
Table 1-13. Evidence pertaining to male reproductive effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Sperm Normal Morphology (Epididymal)
Gaoua (2004b)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, male (25/group): 0, 250, 500,1000 mg/kg-d
P0, Male
0
-
daily for a total of 18 weeks beginning 10 weeks

250
0%
before mating until after weaning of the pups

500
4%
Fl, male (25/group): 0, 250, 500,1000 mg/kg-d

1000
3%
dams dosed daily through gestation and lactation,

Dose(mg/kg-d)
Percent change
then Fl doses beginning PND 22 until weaning of


compared to
the F2 pups
Fl, Male
0
250
500
1000
control
2%
2%
5%
Sperm Production (Daily, Testicular)
Gaoua (2004b)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, male (25/group): 0, 250, 500,1000 mg/kg-d
P0, Male
0
-
daily for a total of 18 weeks beginning 10 weeks

250
-5%
before mating until after weaning of the pups

500
-6%
Fl, male (25/group): 0, 250, 500,1000 mg/kg-d

1000
-4%
dams dosed daily through gestation and lactation,

Dose(mg/kg-d)
Percent change
then Fl doses beginning PND 22 until weaning of


compared to
the F2 pups
Fl, Male
0
250
500
1000
control
-3%
5%
-1%
Sperm with Rapid Movement
Weng et al. (2014)



mice, C57BL/6
Male


inhalation - vapor
no significant change (results in figure only)
male (NR): 0, 50, 200, 500 ppm (0, 209, 836,



2090 mg/m3)a



dynamic whole body inhalation; 6 h/d, 5 d/wk for



9 wk; methods were stated to be described in



Weng et al., 2012



This document is a draft for review purposes only and does not constitute Agency policy.
1-93	DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofETBE
Table 1-13. Evidence pertaining to male reproductive effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Sperm with Rapid Movement (continued)
Weng et al. (2014)
mice, Aldh2-/-
inhalation - vapor
male (NR): 0, 50, 200, 500 ppm (0, 209, 836,
2090 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
9 wk; methods were stated to be described in
Weng et al., 2012
Male
significantly decreased at 500 ppm (2090 mg/m3) (results
in figure only)
Weng et al. (2014)
mice, Aldh2 heterogeneous
inhalation - vapor
male (NR): 0, 50, 200, 500 ppm (0, 209, 836,
2090 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
9 wk; methods were stated to be described in
Weng et al., 2012
Male
significantly decreased at >=200 ppm (836 mg/m3) (results
in figure only)
Weng et al. (2014)
mice, C57BL/6
inhalation - vapor
male (5/group): 0, 500,1750, 5000 ppm (0, 2090,
7320, 20,900 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
13 wk; methods were stated to be described in
Weng et al., 2012
Male
significant decrease in the 5000 ppm (20,900 mg/m3)
group (results in figure only)
Weng et al. (2014)
mice, Aldh2-/-
inhalation - vapor
male (5/group): 0, 500,1750, 5000 ppm (0, 2090,
7320, 20,900 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
13 wk; methods were stated to be described in
Weng et al., 2012
Male
significantly decreased at all doses (results in figure only)
Sperm, Static
Weng et al. (2014)
mice, C57BL/6
inhalation - vapor
male (NR): 0, 50, 200, 500 ppm
dynamic whole body inhalation; 6 h/d, 5 d/wk for
9 wk; methods were stated to be described in
Weng et al., 2012
Male
no significant change (results in figure only)
This document is a draft for review purposes only and does not constitute Agency policy.
1-94	DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofETBE
Table 1-13. Evidence pertaining to male reproductive effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Sperm, Static (continued)
Weng et al. (2014)
mice, Aldh2-/-
inhalation - vapor
male (NR): 0, 50, 200, 500 ppm (0, 209, 836,
2090 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
9 wk; methods were stated to be described in
Weng et al., 2012
Male
significantly increased at 500 ppm (2090 mg/m3) (results in
figure only)
Weng et al. (2014)
mice, Aldh2 heterogeneous
inhalation - vapor
male (NR): 0, 50, 200, 500 ppm (0, 209, 836,
2090 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
9 wk; methods were stated to be described in
Weng et al., 2012
Male
significantly increased at >=200 ppm (836 mg/m3) (results
in figure only)
Weng et al. (2014)
mice, C57BL/6
inhalation - vapor
male (5/group): 0, 500,1750, 5000 ppm (0, 2090,
7320, 20,900 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
13 wk; methods were stated to be described in
Weng et al., 2012
Male
no significant change (results in figure only)
Weng et al. (2014)
mice, Aldh2-/-
inhalation - vapor
male (5/group): 0, 500,1750, 5000 ppm (0, 2090,
7320, 20,900 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
13 wk; methods were stated to be described in
Weng et al., 2012
Male
significantly increased at all doses (results in figure only)
This document is a draft for review purposes only and does not constitute Agency policy.
1-95	DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofETBE
Table 1-13. Evidence pertaining to male reproductive effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Spermatozoa Count (Epididymal)
Gaoua (2004b)
rat, Sprague-Dawley
oral - gavage
P0, male (25/group): 0, 250, 500,1000 mg/kg-d
daily for a total of 18 weeks beginning 10 weeks
before mating until after weaning of the pups
Fl, male (25/group): 0, 250, 500,1000 mg/kg-d
dams dosed daily through gestation and lactation,
then Fl doses beginning PND 22 until weaning of
the F2 pups
Dose(mg/kg-d) Percent change
compared to
control
P0, Male 0
250 2%
500 1%
1000 -1%
Dose(mg/kg-d) Percent change
compared to
control
Fl, Male 0
250 -7%
500 -3%
1000 -5%
Testosterone
de Pevster et al. (2009)
rat, Fischer 344
oral - gavage
P0, male (12/group): 0, 600,1200,1800 mg/kg-d
daily for 14 days
Dose(mg/kg-d) Percent change
compared to
control
P0, Male 0
600 50%
1200 26%
1800 -34%
Atrophy of the Seminiferous Tubules in the Right Testis
Weng et al. (2014)
mice, C57BL/6
inhalation - vapor
male (NR): 0, 50, 200, 500 ppm (0, 209, 836,
2090 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
9 wk; methods were stated to be described in
Weng et al., 2012
Male
no effects were observed (data not provided)
Weng et al. (2014)
mice, Aldh2-/-
inhalation - vapor
male (NR): 0, 50, 200, 500 ppm (0, 209, 836,
2090 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
9 wk; methods were stated to be described in
Weng et al., 2012
Male
no effects observed (data not provided)
This document is a draft for review purposes only and does not constitute Agency policy.
1-96 DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofETBE
Table 1-13. Evidence pertaining to male reproductive effects in animals
exposed to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Atrophy of the Seminiferous Tubules in the Right Testis (continued)
Weng et al. (2014)
mice, Aldh2 heterogeneous
inhalation - vapor
male (NR): 0, 50, 200, 500 ppm (0, 209, 836,
2090 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
9 wk; methods were stated to be described in
Weng et al., 2012
Male
no effects observed (data not provided)
Weng et al. (2014)
mice, C57BL/6
inhalation - vapor
male (5/group): 0, 500,1750, 5000 ppm (0, 2090,
7320, 20,900 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
13 wk; methods were stated to be described in
Weng et al., 2012
Dose(mg/m3) Response
(incidence)
Male 0 1/5
2090 0/5
7320 2/5
20,900 3/5
Weng et al. (2014)
mice, Aldh2-/-
inhalation - vapor
male (5/group): 0, 500,1750, 5000 ppm (0, 2090,
7320, 20,900 mg/m3)a
dynamic whole body inhalation; 6 h/d, 5 d/wk for
13 wk; methods were stated to be described in
Weng et al., 2012
Dose(mg/m3) Response
(incidence)
Male 0 2/5
2090 5/5
7320 5/5
20,900 5/5
1	a4.18 mg/m3 = 1 ppm.
2	*: result is statistically significant (p<0.05) based on analysis of data by study authors.
3	for controls, no response relevant; for other doses, no quantitative response reported.
4	(n): number evaluated from group.
5	Percent change compared to controls calculated as 100 x ((treated value - control value) -f control value).
This document is a draft for review purposes only and does not constitute Agency policy.
1-97	DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofETBE
¦ = 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
PO Female rat; delivery index 16wks (B)
PO Male rat; fertility index 16wks (B)
PO Female rat; fertility index 16wks (B)
PO Male rat; fertility index IBwks (C)
Pregnancy
Outcomes	pQ FemaIe rat. fcrtillty in(jex tswks (C)
F1 Male rat; fertility index GD 0-adult (C)
I-1 Female rat; fertility index GD 0-adult (C)
PO Female rat; postimplantation loss 18wks (C)
F0 Female rat; litter size 18wks (C)
F1 Female rat; litter size GD 0-adult (C)
Female rat; oocytes fertilized 2wks (A)
Oocytes
Female rat; oocytes recovered per ovulating female Zwlcs (A)
PO Male rat; sperm heads (testicular), motility, morphology,
production, spermatozoa count 18wks (C)
Sperm
F1 Male rat; sperm heads (testicular), motility, morphology,
production, spermatozoa count 18wte (C)
Testosterone
and Estradiol
Male rat; testosterone 14d (D)
Male rat; estradiol 14d (D)
B-
-B	El
-0
~—a—ep
0—B—0
~—a—q
O	B	"El
0—B—El
a—-•¦a—ci

Q—H—El
0-
o-
e-s
10	100	1,000
Dose (rtig/ki; 
-------
Toxicological Review ofETBE
¦ = 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 mice; 13wks (A)
Mate Aldh2-/- mice; 13wks (A)
Decreased Sperm
Heads
Male mice; 9wks (A)
Male Aldh2-/- mice; 9wks (A)
Male Aldh2+/- mice; 9wks (A)
Male mice; 13wks (A)
Mate Aldh2-/- mice; 13wks (A)
Decreased Sperm
Motility
Male mice; 9wk$ (A)
Male Aldh2-/- mice; 9wks (A)
Male Aldh2+/- mice; 9wks (A)
Male mice; 13wks [A]
Male Aldh2-/- mice; 13wks (A)
Decreased No. of
Rapidly-moving
Sperm
Male mice; 9wks (A)
Mule Aldh2-/- mice; 9wks (A)
Male Aldh2+/- mice; 9wks (A)
Increased Static
Sperm
Male mice; 13wks (A)
Male Aldh2-/- mice; 13wks (A)
Male mice; 9wks (A)
Male Aldh2-/- mice; 9wks (A)
Male Aldh2+/- mice; 9wks (A)
Atrophy of
Seminiferous
Tubules
Male mice; 13wks (A)
Male Aldh2-/- mice; 13wks (A)
Male mice; 9wks (A)
Male AldhZ-/- mice; 9wks (A)
Male Aklh2+/- mice; 9wks (A)
Q-
B-
~	B	El
~	&—Q
-Bi	O
-S—Q
------Hi
Q.			
-a
	~	Bi	E3
	~	Q	~
-b4—a
S3	B-
G	B
~	eh—o
¦	¦-(—¦
Q	B-1	0
~	B-
10	100	1,000	10,000 100,000
ExpoMitvCont'i-ntivilion (m^/m')
Source: (A) Weng et al,, 2014
Figure 1-8. Exposure-response array of reproductive effects following
inhalation exposure to ETBE
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
Toxicological Review ofETBE
Developmental effects
Developmental endpoints that were evaluated include pup survival and growth of fetus and
pups. Two studies indicated maternal toxicity associated with exposure to ETBE based on
decreases in maternal body weight (Asano etal.. 2011: Gaoua. 2004a). However, one of the studies
was in rabbits, and EPA's (1991b) developmental guidelines indicate that body weight change is
not a useful indicator of maternal toxicity in rabbits. In addition, this same dose did not cause
maternal toxicity in rat studies fAso etal.. 2014: Asano etal.. 2011: Fuiii etal.. 2010: Gaoua. 2004bl
There was no significant effects of ETBE on pup survival as measured by pre- or post-
implantation loss fAso etal.. 2014: Asano etal.. 2011: Gaoua. 2004al. number of live births (Asano
etal.. 2011: IPEC. 2008h]. pup viability at PND 4 including total litter loss (Fuiii etal.. 2010: Gaoua.
2004b], or lactational index (also called viability index on PND 21) (Fuiii etal.. 2010: Gaoua.
20Mb).
Fetal and pup growth were also not affected by ETBE treatment fAso etal.. 2014: Asano et
al.. 2011: Fuiii etal.. 20101. Fuiii etal. f20101 did not observe any effects in physical development or
reflex ontogeny in the F1 offspring in a one-generation reproductive study nor was there an effect
on sexual maturity observed in a two-generation study fGaoua. 2004bl In section 1.1.1, increased
kidney weights in F1 offspring are discussed. No differences were observed in external, skeletal, or
visceral variations or malformations (Aso etal.. 2014: Asano etal.. 2011). Aso etal. (2014) reported
a significant increase in rudimentary lumbar ribs, but the result was within the historical control
range and vanished after birth.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
1	Table 1-14. Evidence pertaining to prenatal developmental effects in animals
2	following exposure to ETBE
Reference and Dosing Protocol
Results by Endpoint
Maternal Body Weight Gain (GD0-20)
Fuiiietal. (2010); JPEC (2008e)
rat, Sprague-Dawley
oral - gavage
Fl, combined (NR): 0,100, 300,1000 mg/kg-d; P0,
female (24/group): 0,100, 300,1000 mg/kg-d
daily for 17 weeks beginning 10 weeks prior to
mating to lactation day 21
Dose(mg/kg-d) Percent change
compared to
control
P0, Female 0
100 -4%
300 8%
1000 12%*
Gaoua (2004b)
rat, Sprague-Dawley
oral - gavage
P0, female (25/group): 0, 250, 500,1000 mg/kg-d
daily for a total of 18 weeks beginning 10 weeks
before mating until PND 21
Fl, female (24-25/group): 0, 250, 500,1000
mg/kg-d
dams dosed daily through gestation and lactation,
then Fl dosed beginning PND 22 until weaning of
the F2 pups
Dose(mg/kg-d) Percent change
compared to
control
P0, Female 0
250 2%
500 3%
1000 3%
Dose(mg/kg-d) Percent change
compared to
control
Fl, Female 0
250 -1%
500 -3%
1000 -6%
Aso et al. (2014); JPEC (2008h)
rat, CRL:CD(SD)
oral - gavage
Fl, combined (251-285/group): 0,100, 300,1000
mg/kg-d; Fl, female (119-159/group): 0,100, 300,
1000 mg/kg-d; P0, female (24/group): 0,100, 300,
1000 mg/kg-d; Fl, male (126-136/group): 0,100,
300,1000 mg/kg-d
dams treated daily from GD5 to GD19
Dose(mg/kg-d) Percent change
compared to
control
P0, Female 0 ;
100 -7%
300 -4%
1000 -7%
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-14. Evidence pertaining to prenatal developmental effects in
animals following exposure to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Maternal Body Weight Gain (GDO-28)
Asano et al. (2011); JPEC (2008i)

Dose(mg/kg-d)
Percent change
rabbit, New Zealand


compared to
oral - gavage


control
Fl, combined (24/group): 0,100, 300,1000
P0, Female
0
-
mg/kg-d; P0, female (24/group): 0,100, 300,1000

100
-13%
mg/kg-d

300
0%
dams exposed from GD6 to GD27

1000
-38%*
Maternal Body Weight Gain (GD5-20)
Gaoua (2004a)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, female (24/group): 0, 250, 500,1000 mg/kg-d
P0, Female
0
-
dams exposed from GD5 to GD19

250
500
1000
-4%
-3%
-17%*
Postimplantation Loss3
Gaoua (2004a)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, female (24/group): 0, 250, 500,1000 mg/kg-d
P0, Female
0
-
dams exposed daily from GD5 to GD19

250
500
1000
27%
38%
44%
Postimplantation Loss (Resorptions/lmplantations)
Aso et al. (2014); JPEC (2008h)

Dose(mg/kg-d)
Percent change
rat, CRL:CD(SD)


compared to
oral - gavage


control
Fl, combined (251-285/group): 0,100, 300,1000
P0, Female
0
-
mg/kg-d; Fl, female (119-159/group): 0,100, 300,

100
24%
1000 mg/kg-d; P0, female (24/group): 0,100, 300,

300
-28%
1000 mg/kg-d; Fl, male (126-136/group): 0,100,

1000
-14%
300,1000 mg/kg-d



dams treated daily from GD5 to GD19



This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-14. Evidence pertaining to prenatal developmental effects in
animals following exposure to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Postimplantation Loss Per Litter
Asano et al. (2011); JPEC (2008i)

Dose(mg/kg-d)
Percent change
rabbit, New Zealand


compared to
oral - gavage


control
Fl, combined (24/group): 0,100, 300,1000
P0, Female
0
-
mg/kg-d; P0, female (24/group): 0,100, 300,1000

100
3%
mg/kg-d

300
-36%
dams exposed from GD6 to GD27

1000
-21%
Preimplantation Lossb
Aso et al. (2014); JPEC (2008h)

Dose(mg/kg-d)
Percent change
rat, CRL:CD(SD)


compared to
oral - gavage


control
Fl, combined (251-285/group): 0,100, 300,1000
P0, Female
0
-
mg/kg-d; Fl, female (119-159/group): 0,100, 300,

100
38%
1000 mg/kg-d; P0, female (24/group): 0,100, 300,

300
21%
1000 mg/kg-d; Fl, male (126-136/group): 0,100,

1000
82%
300,1000 mg/kg-d



dams treated daily from GD5 to GD19



Gaoua (2004a)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, female (24/group): 0, 250, 500,1000 mg/kg-d
P0, Female
0
-
dams exposed daily from GD5 to GD19

250
500
1000
-15%
-17%
-5%
1	aPost-implantation loss = (resorptions + dead fetus/ total implantations) x 100, calculated per litter.
2	bPre-implantation loss = (corpora lutea-implantations/corpora lutea) x 100, calculated per litter.
3	*: 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
8
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
1	Table 1-15. Evidence pertaining to postnatal developmental effects in animals
2	following exposure to ETBE
Reference and Dosing Protocol
Results by Endpoint
Live Births
Aso et al. (2014); JPEC (2008h)
rat, CRL:CD(SD)
oral - gavage
Fl, combined (251-285/group): 0,100, 300,1000
mg/kg-d; Fl, female (119-159/group): 0,100, 300,
1000 mg/kg-d; P0, female (24/group): 0,100, 300,
1000 mg/kg-d; Fl, male (126-136/group): 0,100,
300,1000 mg/kg-d
dams treated daily from GD5 to GD19
Dose(mg/kg-d) Percent change
compared to
control
P0, Female 0
100 -8%
300 -12%
1000 -5%
Live Fetuses Per Litter
Asano et al. (2011); JPEC (2008i)
rabbit, New Zealand
oral - gavage
Fl, combined (24/group): 0,100, 300,1000
mg/kg-d; P0, female (24/group): 0,100, 300,1000
mg/kg-d
dams exposed from GD6 to GD27
Dose(mg/kg-d) Percent change
compared to
control
P0, Female 0
100 1%
300 8%
1000 -12%
Viability Index PND 4
Fuiiietal. (2010); JPEC (2008e)
rat, Sprague-Dawley
oral - gavage
Fl, combined (NR): 0,100, 300,1000 mg/kg-d; P0,
female (24/group): 0,100, 300,1000 mg/kg-d
daily for 17 weeks beginning 10 weeks prior to
mating to lactation day 21
Dose(mg/kg-d) Percent change
compared to
control
Fl, Combined 0
100 -1%
300 2%
1000 -10%
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-15. Evidence pertaining to postnatal developmental effects in
animals following exposure to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Viability Index PND 4 (continued)
Gaoua (2004b)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, female (25/group): 0, 250, 500,1000 mg/kg-d
P0, Female
0
-
daily for a total of 18 weeks beginning 10 weeks

250
-5%
before mating until PND 21

500
-16%
Fl, female (24-25/group): 0, 250, 500,1000

1000
0%
mg/kg-d

Dose(mg/kg-d)
Percent change
dams dosed daily through gestation and lactation,


compared to
then Fl dosed beginning PND 22 until weaning of


control
the F2 pups
Fl, Female
0
-


250
-3%


500
-1%


1000
-5%
Total Litter Loss PND 4
Fuiiietal. (2010); JPEC (2008e)

Dose(mg/kg-d)
Response (litters)
rat, Sprague-Dawley
P0, Female
0
0/21
oral - gavage

100
0/22
Fl, combined (NR): 0,100, 300,1000 mg/kg-d; P0,

300
0/23
female (24/group): 0,100, 300,1000 mg/kg-d

1000
3/22
daily for 17 weeks beginning 10 weeks prior to



mating to lactation day 21



Gaoua (2004b)

Dose(mg/kg-d)
Response
rat, Sprague-Dawley
P0, Female
0
0/23
oral - gavage

250
1/21
P0, female (25/group): 0, 250, 500,1000 mg/kg-d

500
3/22
daily for a total of 18 weeks beginning 10 weeks

1000
0/25
before mating until PND 21

Dose(mg/kg-d)
Response
Fl, female (24-25/group): 0, 250, 500,1000


(incidence)
mg/kg-d
Fl, Female
0
0/21
dams dosed daily through gestation and lactation,

250
1/21
then Fl dosed beginning PND 22 until weaning of

500
0/22
the F2 pups

1000
1/20
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-15. Evidence pertaining to postnatal developmental effects in
animals following exposure to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Lactation lndexa
Fuiiietal. (2010); JPEC (2008e)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
Fl, combined (NR): 0,100, 300,1000 mg/kg-d; P0,
P0, Female
0
-
female (24/group): 0,100, 300,1000 mg/kg-d

100
-1%
daily for 17 weeks beginning 10 weeks prior to

300
-1%
mating to lactation day 21

1000
-5%
Gaoua (2004b)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, female (25/group): 0, 250, 500,1000 mg/kg-d
P0, Female
0
-
daily for a total of 18 weeks beginning 10 weeks

250
-3%
before mating until PND 21

500
2%
Fl, female (24-25/group): 0, 250, 500,1000

1000
5%
mg/kg-d

Dose(mg/kg-d)
Percent change
dams dosed daily through gestation and lactation,


compared to
then Fl dosed beginning PND 22 until weaning of


control
the F2 pups
Fl, Female
0
250
500
1000
1%
2%
2%
Gravid Uterus Weight
Asano et al. (2011); JPEC (2008i)

Dose(mg/kg-d)
Percent change
rabbit, New Zealand


compared to
oral - gavage


control
Fl, combined (24/group): 0,100, 300,1000
P0, Female
0
-
mg/kg-d; P0, female (24/group): 0,100, 300,1000

100
4%
mg/kg-d

300
5%
dams exposed from GD6 to GD27

1000
-16%
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-15. Evidence pertaining to postnatal developmental effects in
animals following exposure to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Fetal Body Weight
Aso et al. (2014); JPEC (2008h)

Dose(mg/kg-d)
Percent change
rat, CRL:CD(SD)


compared to
oral - gavage


control
Fl, combined (251-285/group): 0,100, 300,1000
Fl, Male
0
-
mg/kg-d; Fl, female (119-159/group): 0,100, 300,

100
1%
1000 mg/kg-d; P0, female (24/group): 0,100, 300,

300
3%
1000 mg/kg-d; Fl, male (126-136/group): 0,100,

1000
1%
300,1000 mg/kg-d

Dose(mg/kg-d)
Percent change
dams treated daily from GD5 to GD19


compared to



control

Fl, Female
0
-


100
0%


300
2%


1000
5%
Asano et al. (2011); JPEC (2008i)

Dose(mg/kg-d)
Percent change
rabbit, New Zealand


compared to
oral - gavage


control
Fl, combined (24/group): 0,100, 300,1000
Fl, Males
0
-
mg/kg-d; P0, female (24/group): 0,100, 300,1000

100
0%
mg/kg-d

300
1%
dams exposed from GD6 to GD27

1000
-4%


Dose(mg/kg-d)
Percent change



compared to



control

Fl, Females
0
-


100
1%


300
3%


1000
-4%
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-15. Evidence pertaining to postnatal developmental effects in
animals following exposure to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Body Weight (PND 21)
Fuiiietal. (2010); JPEC (2008e)
rat, Sprague-Dawley
oral - gavage
Fl, male (84-92/group): 0,100, 300,1000 mg/kg-
d
dams exposed daily from GD0 to lactational day
21; Fl weanlings selected for observation of
sexual development continued treatment for
approximately 4 weeks
Dose(mg/kg-d) Percent change
compared to
control
Fl, Male 0
100 0%
300 0%
1000 0%
Dose(mg/kg-d) Percent change
compared to
control
Fl, Female 0
100 -1%
300 -1%
1000 1%
External Malformation
Aso et al. (2014); JPEC (2008h)
rat, CRL:CD(SD)
oral - gavage
Fl, combined (251-285/group): 0,100, 300,1000
mg/kg-d; Fl, female (119-159/group): 0,100, 300,
1000 mg/kg-d; P0, female (24/group): 0,100, 300,
1000 mg/kg-d; Fl, male (126-136/group): 0,100,
300,1000 mg/kg-d
dams treated daily from GD5 to GD19
Dose(mg/kg-d) Response
(incidence)
Fl, Combined 0 0/285
100 0/263
300 0/251
1000 0/270
Skeletal Variation or Malformation
Aso et al. (2014); JPEC (2008h)
rat, CRL:CD(SD)
oral - gavage
Fl, combined (251-285/group): 0,100, 300,1000
mg/kg-d; Fl, female (119-159/group): 0,100, 300,
1000 mg/kg-d; P0, female (24/group): 0,100, 300,
1000 mg/kg-d; Fl, male (126-136/group): 0,100,
300,1000 mg/kg-d
dams treated daily from GD5 to GD19
Dose(mg/kg-d) Response
(incidence)
Fl, Combined 0 9/139
100 3/126
300 3/119
1000 29/131
mostly rudimentary lumbar rib, incidence was within
historical range
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-15. Evidence pertaining to postnatal developmental effects in
animals following exposure to ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Skeletal Variation or Malformation (continued)
Asano et al. (2011); JPEC (2008i)


rabbit, New Zealand
Fl, Combined

oral - gavage
There were no significant differences in the incidence of
Fl, combined (24/group): 0,100, 300,1000
skeletal malformations or variations.

mg/kg-d; P0, female (24/group): 0,100, 300,1000


mg/kg-d


dams exposed from GD6 to GD27


Visceral Variation or Malformation
Asano et al. (2011); JPEC (2008i)


rabbit, New Zealand
Fl, Combined

oral - gavage
There was no significant difference in the incidence of
Fl, combined (24/group): 0,100, 300,1000
fetuses with visceral malformations or variations, but there
mg/kg-d; P0, female (24/group): 0,100, 300,1000
was a slight (dose-related) increase in the incidence of an
mg/kg-d
absent right atrioventricular valve.

dams exposed from GD6 to GD27


Aso et al. (2014); JPEC (2008h)
Dose(mg/kg-d)
Response
rat, CRL:CD(SD)

(incidence)
oral - gavage
Fl, Combined 0
6/146
Fl, combined (251-285/group): 0,100, 300,1000
100
8/137
mg/kg-d; Fl, female (119-159/group): 0,100, 300,
300
4/132
1000 mg/kg-d; P0, female (24/group): 0,100, 300,
1000
8/139
1000 mg/kg-d; Fl, male (126-136/group): 0,100,


300,1000 mg/kg-d


dams treated daily from GD5 to GD19


1	aLactation index = (pups alive at day 21/pups at day 4) x 100; LI is the same as viability index on day 21.
2	NR: not reported; *: result is statistically significant (p<0.05) based on analysis of data by study authors.
3	for controls, no response relevant; for other doses, no quantitative response reported.
4	(n): number evaluated from group.
5	Percentage change compared to control = (treated value - control value) -f control value x 100.
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
2
3
4
5
Prenatal Developmental
Effects
Decreased Maternal Body
Weight Gain
Postimplantation Loss
PO Female rat; 16wks(C)
PO Female rat; 18wks (Ej
FX Female rat; GO 0-adult [E]
PO Female rat; GD5-19(B)
PO Female rat; GD5-19 [D]
PO Female rabbit GD6-27(Aj
PO Female rat; GD5-19[D)
PO Female rat; GD5-19(B)
PQ Female rabbit CD6-27(A]
Freim plantation Loss
PO Female rat; GD5-19 (D)
PO Female rat; GDS-19 f
Postnatal
Developmental Effects
Live Births/Fetuses
Viability Index, Total
Litter Loss, Lactation
Index
PO Female rat; CD5-1 9 (B)
PO Female rabbit; GQ6-27(A)
PO Female rat; 16wl
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Mechanistic Evidence
No mechanistic evidence is available for reproductive or developmental effects.
Summary of reproductive and developmental toxicity
The evidence for reproductive and developmental effects is entirely from animal studies.
Reproductive endpoints were not consistently affected across studies. Subchronic but not chronic
exposures to ETBE decreased rapid sperm movement at the highest tested dose. However, Aldh2
knockout or heterozygous mice had reduced number of sperm heads and sperm motility effects
(i.e., number of sperm that were mobile, number of sperm that were static, sperm with rapid
movement) associated with ETBE (Wengetal.. 20141. These effects suggest that populations with
Aldh2 polymorphism may be sensitive to reproductive effects (discussed in section 1.2.3). A single
short-term exposure study reported an increase in estradiol levels in male rats that did not exhibit
a dose responsefde Pevster etal.. 20091.
Of the endpoints assessed in two studies evaluating developmental effects, reduced
maternal body weight was the only statistically significant effect reported fAsano etal.. 2011:
Gaoua. 2004al. This effect was not dose-responsive, was inconsistently observed, and did not
correspond to any other maternal effects or effects in offspring.
EPA concluded that the evidence does not support reproductive or developmental effects as
a potential human hazard ofETBE exposure.
1.1.4. 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. Tumorigenicity in the liver and
kidney were previously discussed in the relevant organ-specific section and will not be discussed in
this section. The database for ETBE carcinogenicity consists of only animal data: three 2-year
studies, one 23-week initiation study, and one 31-week initiation study performed in rats
fHagiwara etal.. 2013: Saito etal.. 2013: Suzuki etal.. 2012: Hagiwara etal.. 2011: Malarkev and
Bucher. 2011: TPEC. 2010a. b; Maltoni etal.. 19991 (see Table 1-16, Table 1-17; Figure 1-9, Figure
1-10). One study conducted by Maltoni et al. (19991 had several methodological limitations such as
only two treatment groups, nonstandard histopathological diagnoses, a nonstandard 4-day dosing
schedule, and greater than expected mortality in treated groups and controls compared with other
laboratories. In response to these concerns, a pathology working group (PWG) sponsored by U.S.
EPA and the National Toxicology Program (NTP) reviewed the histopathological data fMalarkev
and Bucher. 20111. In addition to recalculating tumor incidences, the PWG found that the
respiratory infections in the study animals confound interpretation of leukemia and lymphoma.
Thus, U.S. EPA will use the Malarkev and Bucher (20111 data when considering carcinogenicity in
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
place of the published Maltoni et al. (19991 study and will not consider leukemia and lymphoma
from this study.
Following 2-year exposure to ETBE, the incidence of leiomyomas was increased in the
uterus of rats in the high-dose group Maltoni etal. (19991. Malignant schwannomas in the uterus
were increased only at the lowest dose and no significant trend was observed. Leiomyomas and a
carcinoma were observed in uterine/vaginal tissue, but no significant trend was observed
fMalarkev and Bucher. 20111. A statistically significant increase in incidence of neoplastic lesions
was observed in the thyroid of male rats following subchronic exposure to ETBE after a 4-week
tumor initiation exposure to DMBDD f Hagiwara etal.. 20111. An increase in carcinomas of the
urinary bladder also occurred f Hagiwara etal.. 20131: however, subchronic exposure to ETBE via
gavage without initiation using DMBDD treatment did not result in tumor development in any of
the organs that previously demonstrated tumorigenicity (Hagiwara etal.. 20111. The incidence of
neoplastic lesions in the thyroid was dose-dependently increased, which demonstrate that ETBE
possesses tumor promotion potential f Hagiwara etal.. 20111. While increased incidences of
tumorigenicity were observed in Hagiwara et al. (20111. a chronic drinking water study and chronic
inhalation study failed to demonstrate significant increases in the incidence of tumors in any of
these tissues (Saito etal.. 2013: Suzuki etal.. 2012: TPEC. 2010b).
Mechanistic Evidence
Available mechanistic evidence was previously discussed in the context of kidney and liver
tumors (Sections 1.1.1 and 1.1.2).
Summary of Carcinogenicity Evidence
The evidence for carcinogenic effects not of the liver or kidney is all from rat studies. Tumor
initiation increased the incidence of thyroid adenomas and carcinomas and urinary bladder
carcinomas in male rats (Hagiwara etal.. 20111: however, these results were not observed in the
three 2-year bioassays. A statistically significant increase in the trend of uterine leiomyomas and
leimyosarcomas was not observed fMalarkev and Bucher. 20111. Malignant schwannomas were
increased at the lowest dose in the uterus/vagina in one study but these neoplasms arise from
nervous tissue and are not specific to uterine tissue fMalarkev and Bucher. 20111. Low survival
rates at 104 weeks (approximately 25%) in control groups confounds these data because it cannot
be determined if tumors in the control group were not observed due to premature death. In
addition, these results differed from two other 2-year bioassays, one oral and one inhalation (Saito
etal.. 2013: Suzuki etal.. 2012: TPEC. 2010a. b). No methodological problems that could lead to false
negative outcomes were identified in these two bioassays.
Confidence in the data demonstrating an increase in the incidence of schwannomas is low
due to the lack of a similar effect in two other well-conducted studies. No mechanistic evidence is
available to suggest that nervous tissue or uterine tissue are targets for ETBE carcinogenicity.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
1	Table 1-16. Evidence pertaining to tumor promotion by ETBE 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, 300,1000 mg/kg-d

300
21/30
daily for 23 weeks following a 4 week tumor

1000
28/30*
initiation by DMBDD

0+
0/12
+no DMBDD initiation

1000+
0/12
Forestomach Papillomas
Hagiwara et al. (2011); JPEC (2008d)

Dose(mg/kg-d)
Response
rat, Fischer 344


(incidence)
oral - gavage
Male
0
0/30
male (30/group): 0, 300,1000 mg/kg-d

300
4/30
daily for 23 weeks following a 4 week tumor

1000
3/30
initiation by DMBDD

0+
0/12
+no DMBDD initiation

1000+
0/12
Thyroid Gland Adenoma or Carcinoma
Hagiwara et al. (2011); JPEC (2008d)

Dose(mg/kg-d)
Response
rat, Fischer 344


(incidence)
oral - gavage
Male
0
8/30
male (30/group): 0, 300,1000 mg/kg-d

300
17/30*
daily for 23 weeks following a 4 week tumor

1000
20/30*
initiation by DMBDD

0+
0/12
+no DMBDD initiation

1000+
0/12
Urinary Bladder Carcinoma
Hagiwara et al. (2013)

Dose(mg/kg-d)
Response
rat, F344/DuCrlCrlj


(incidence)
oral - water
Male
0
5/30
male (30/group): 0,100, 300, 500,1000 mg/kg-d

100
7/30
daily for 31 weeks beginning one week after a 4

300
6/30
wk exposure to BBN

500
1000
14/30*
9/26
Urinary Bladder Papilloma
Hagiwara et al. (2013)

Dose(mg/kg-d)
Response
rat, F344/DuCrlCrlj


(incidence)
oral - water
Male
0
21/30
male (30/group): 0,100, 300, 500, 1000 mg/kg-d

100
13/30
daily for 31 weeks beginning one week after a 4

300
17/30
wk exposure to BBN

500
1000
17/30
21/26
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-16. Evidence pertaining to tumor promotion by ETBE in animals
(continued)
Reference and Dosing Protocol
Results by Endpoint
Urinary Bladder Papilloma or Carcinoma
Hagiwara et al. (2013)

Dose(mg/kg-d)
Response
rat, F344/DuCrlCrlj


(incidence)
oral - water
Male
0
24/30
male (30/group): 0,100, 300, 500,1000 mg/kg-d

100
18/30
daily for 31 weeks beginning one week after a 4

300
20/30
wk exposure to BBN

500
1000
25/30
21/26
Urinary Bladder Papillamotosis
Hagiwara et al. (2011); JPEC (2008d)

Dose(mg/kg-d)
Response
rat, Fischer 344


(incidence)
oral - gavage
Male
0
0/30
male (12/group): 0,1000 mg/kg-d

300
0/30
daily for 23 weeks following a 4 week tumor

1000
10/30*
initiation by DMBDD

0+
0/12
+no DMBDD initiation

1000+
2/12
1
2	Table 1-17. Evidence pertaining to carcinogenic effects (in tissues other than
3	liver or kidney) in animals exposed to ETBE
Reference and Dosing Protocol

Results by Endpoint

Papillomas of the Oral Mucosa/Tongue
Malarkev and Bucher (2011); Maltoni et al. (1999)

Dose(mg/kg-d)
Response
rat, Sprague-Dawley


(incidence)
oral - gavage
Male
0
0/60
female (60/group): 0, 250,1000 mg/kg-d; male

250
0/60
(60/group): 0, 250,1000 mg/kg-d

1000
0/60
reanalysis of data from Maltoni et al. (1999)

Dose(mg/kg-d)
Response
where animals were dosed 4 d/wk for 104 weeks


(incidence)

Female
0
0/60


250
0/60


1000
0/60
Squamous Cell Carcinoma of Oral Mucosa/Tongue
Malarkev and Bucher (2011); Maltoni et al. (1999)

Dose(mg/kg-d)
Response
rat, Sprague-Dawley


(incidence)
oral - gavage
Male
0
0/60
female (60/group): 0, 250,1000 mg/kg-d; male

250
0/60
(60/group): 0, 250,1000 mg/kg-d

1000
0/60
reanalysis of data from Maltoni et al. (1999)

Dose(mg/kg-d)
Response
where animals were dosed 4 d/wk for 104 weeks


(incidence)

Female
0
0/60


250
0/60


1000
0/60
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-17. Evidence pertaining to carcinogenic effects (in tissues other than
liver or kidney) in animals exposed to ETBE (continued)
Reference and Dosing Protocol

Results by Endpoint

Thyroid Follicular Adenocarcinoma
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Response
rat, Fischer 344


(incidence)
oral - water
Male
0
0/50
female (50/group): 0, 625, 2500,10,000 ppm (0,

28
1/50
46,171, 560 mg/kg-day)a; male (50/group): 0,

121
0/50
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-
day)a
daily for 104 wks

542
0/50

Dose(mg/kg-d)
Response
(incidence)

Female
0
46
171
560
0/50
1/50
0/50
0/50
Saito et al. (2013);JPEC (2010b)

Dose(mg/m3)
Response
rat, Fischer 344


(incidence)
inhalation - vapor
Male
0
0/50
female (50/group): 0, 500,1500, 5000 ppm (0,

2090
0/50
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

6270
0/50
500, 1500, 5000 ppm (0, 2090, 6270,

20,900
0/50
20,900 mg/m3)b
dynamic whole body inhalation; 6 hrs/d, 5 d/wk
for 104 wks; generation method, analytical
Female
Dose(mg/m3)
0
2090
6270
20,900
Response
(incidence)
1/50
1/50
1/50
0/50
concentration and method were reported

Thyroid Adenocarcinoma
Maltoni et al. (1999)

Dose(mg/kg-d)
Response
rat, Sprague-Dawley


(incidence)
oral - gavage
Male
0
0/60
female (60/group): 0, 250,1000 mg/kg-d; male

250
0/60
(60/group): 0, 250,1000 mg/kg-d

1000
0/60
4 d/wk for 104 wks; observed until natural death;

Dose(mg/kg-d)
Response
NOTE: These tumor data were not re-analyzed by
Female
0
(incidence)
0/60
Malarkev and Bucher (2011)

250
1000
0/60
1/60
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-17. Evidence pertaining to carcinogenic effects (in tissues other than
liver or kidney) in animals exposed to ETBE (continued)
Reference and Dosing Protocol

Results by Endpoint

Thyroid Follicular Adenoma
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Response
rat, Fischer 344


(incidence)
oral - water
Male
0
1/50
female (50/group): 0, 625, 2500,10,000 ppm (0,

28
0/50
46,171, 560 mg/kg-day)a; male (50/group): 0,

121
0/50
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

542
0/50
day)a

Dose(mg/kg-d)
Response
daily for 104 wks
Female
0
46
171
560
(incidence)
0/50
0/50
0/50
0/50
Saito et al. (2013);JPEC (2010b)

Dose(mg/m3)
Response
rat, Fischer 344


(incidence)
inhalation - vapor
Male
0
1/50
female (50/group): 0, 500,1500, 5000 ppm (0,

2090
0/50
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

6270
1/50
500, 1500, 5000 ppm (0, 2090, 6270,

20,900
2/50
20,900 mg/m3)b
dynamic whole body inhalation; 6 hrs/d, 5 d/wk
for 104 wks; generation method, analytical
Female
Dose(mg/m3)
0
2090
6270
20,900
Response
(incidence)
0/50
0/50
0/50
0/50
concentration and method were reported

Endometrial Stromal Sarcoma
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Response
rat, Fischer 344


(incidence)
oral - water
Female
0
6/50
female (50/group): 0, 625, 2500,10,000 ppm (0,

46
9/50
46,171, 560 mg/kg-day)a; male (50/group): 0,

171
3/50
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

560
7/50
day)a



daily for 104 wks



Saito et al. (2013);JPEC (2010b)

Dose(mg/m3)
Response
rat, Fischer 344


(incidence)
inhalation - vapor
Female
0
2/50
female (50/group): 0, 500,1500, 5000 ppm (0,

2090
2/50
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,
500, 1500, 5000 ppm (0, 2090, 6270,

6270
20,900
3/50
2/50
20,900 mg/m3)b



dynamic whole body inhalation; 6 hrs/d, 5 d/wk



for 104 wks; generation method, analytical



concentration and method were reported



This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-17. Evidence pertaining to carcinogenic effects (in tissues other than
liver or kidney) in animals exposed to ETBE (continued)
Reference and Dosing Protocol

Results by Endpoint

Carcinoma of the Uterus/Vagina
Malarkev and Bucher (2011); Maltoni et al. (1999)

Dose(mg/kg-d)
Response
rat, Sprague-Dawley


(incidence)
oral - gavage
Female
0
0/60
female (60/group): 0, 250,1000 mg/kg-d; male

250
1/60
(60/group): 0, 250,1000 mg/kg-d

1000
0/60
reanalysis of data from Maltoni et al. (1999)



where animals were dosed 4 d/wk for 104 weeks



Uterine Leiomyosarcoma
Malarkev and Bucher (2011); Maltoni et al. (1999)

Dose(mg/kg-d)
Response
rat, Sprague-Dawley


(incidence)
oral - gavage
Female
0
1/60
female (60/group): 0, 250,1000 mg/kg-d; male

250
0/60
(60/group): 0, 250,1000 mg/kg-d

1000
0/60
reanalysis of data from Maltoni et al. (1999)



where animals were dosed 4 d/wk for 104 weeks



Uterine Leiomyoma
Malarkev and Bucher (2011); Maltoni et al. (1999)

Dose(mg/kg-d)
Response
rat, Sprague-Dawley


(incidence)
oral - gavage
Female
0
0/60
female (60/group): 0, 250,1000 mg/kg-d; male

250
0/60
(60/group): 0, 250,1000 mg/kg-d

1000
3/60
reanalysis of data from Maltoni et al. (1999)



where animals were dosed 4 d/wk for 104 weeks



Schwannoma of the Uterus/Vagina
Malarkev and Bucher (2011); Maltoni et al. (1999)

Dose(mg/kg-d)
Response
rat, Sprague-Dawley


(incidence)
oral - gavage
Female
0
0/60
female (60/group): 0, 250,1000 mg/kg-d; male

250
7/60
(60/group): 0, 250,1000 mg/kg-d

1000
2/60
reanalysis of data from Maltoni et al. (1999)



where animals were dosed 4 d/wk for 104 weeks



Uterine Adenocarcinoma
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Response
rat, Fischer 344


(incidence)
oral - water
Female
0
1/50
female (50/group): 0, 625, 2500,10,000 ppm (0,

46
0/50
46,171, 560 mg/kg-day)a; male (50/group): 0,

171
2/50
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

560
2/50
day)a



daily for 104 wks



This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-17. Evidence pertaining to carcinogenic effects (in tissues other than
liver or kidney) in animals exposed to ETBE (continued)
Reference and Dosing Protocol

Results by Endpoint

Uterine Adenocarcinoma (continued)
Saito et al. (2013);JPEC (2010b)

Dose(mg/m3)
Response
rat, Fischer 344


(incidence)
inhalation - vapor
Female
0
2/50
female (50/group): 0, 500,1500, 5000 ppm (0,

2090
3/50
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

6270
1/50
500, 1500, 5000 ppm (0, 2090, 6270,

20,900
4/50
20,900 mg/m3)b



dynamic whole body inhalation; 6 hrs/d, 5 d/wk



for 104 wks; generation method, analytical



concentration and method were reported



Uterine Fibroma
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Response
rat, Fischer 344


(incidence)
oral - water
Female
0
1/50
female (50/group): 0, 625, 2500,10,000 ppm (0,

46
0/50
46,171, 560 mg/kg-day)a; male (50/group): 0,

171
0/50
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

560
0/50
day)a



daily for 104 wks



Uterine Carcinoma
Malarkev and Bucher (2011); Maltoni et al. (1999)

Dose(mg/kg-d)
Response
rat, Sprague-Dawley


(incidence)
oral - gavage
Female
0
0/60
female (60/group): 0, 250,1000 mg/kg-d; male

250
1/60
(60/group): 0, 250,1000 mg/kg-d

1000
0/60
4 d/wk for 104 wks; observed until natural death



1	Conversion performed by study authors.
2	b4.18 mg/m3 = 1 ppm.
3	'Statistically significant (p < 0.05) based on analysis of data conducted by study authors.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
I = 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 ;104wks (C)
Male rat ;104wks (C)
Female rat; uterine m align anctes;104wks (B)
Male rat; mouth ep»thelium;104wks (B)
-0-	—a
€3	E3
-EJ
Male rat; thyroid;23wks following 4wk initiation
with DMBDD (A)
Male rat; forestomach;23wks following 4wk
initiation with DMBDD (A)
Male rat; colon;23wks following 4wk initiation with
DMBDD (A)
Male rat; liver;23wks following 4wk initiation with
DMBDD [A)
Male rat; kidney;23wks following 4wk initiation
with DMBDD (A)
B-
{3	0
B-
10
100	1,000
Dose (mg/kg-day)
10,000
Sources: (A) Hagiwara et al, 2011; JPEC 2008d (B) Maltoni et al, 1999; Malarkey et al, 2011 (reanalysis of
Maltoni et al, 1999} (C) Suzuki et al, 2012; JPEC, 2010a
2
3
4
Figure 1-9. Exposure-response array of carcinogenic 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 ofETBE
¦ = 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/adenocarcinoraa;
104wks (A)
Male rat; thyroid adenoma/adenocarcinoma;
104-wks (A)
Female rat; uterine malignancies; 104wks (A)
100	1,000	10,000	100,000
Exposure Concentration (nig/m ')
Source: (A) Saito et al., 2013; JPEC, 2010b
1
2	Figure 1-10. Exposure-response array of carcinogenic effects following
3	inhalation exposure to ETBE
4
5
6
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
Toxicological Review ofETBE
1.1.5. Other Toxicological Effects
Synthesis of other toxicity data
The database for effects other than kidney, liver, reproductive, and cancer contain only 11
rodent studies. All selected studies employed inhalation, oral gavage, or drinking water exposures
for >90 days. Shorter duration multiple exposure studies that examined immunological endpoints
were also included. No studies were removed for methodological concerns.
Body weight
As presented in Table 1-18, body weights were significantly reduced compared with vehicle
controls following 2-year oral and inhalation exposures to ETBE fSaito etal.. 2013: Suzuki etal..
2012: TPEC. 2010a. b). Reductions were also reported in studies of exposure durations shorter than
2 years (Hagiwaraetal.. 2011: Banton etal.. 2011: Fuiiietal.. 2010: Gaoua. 2004b: TPEC. 2008b. c;
Medinskv etal.. 19991: however, these effects were frequently not statistically significant. Food
consumption did not correlate well with body weight fSaito etal.. 2013: Suzuki etal.. 2012: TPEC.
2010a. b). Water consumption was reduced in the 2-year oral exposure study (TPEC. 2010a).
Palatability and reduced water consumption due to ETBE exposure may contribute to the reduced
body weight, particularly for oral exposures. Ptyalism, which is frequently observed with
unpalatable chemicals following gavage, was observed in rats gavaged for 18 weeks (Gaoua.
2004b). Body weight changes are poor indicators of systemic toxicity but are important when
evaluating relative organ weight changes. Because body weight was most severely affected in 2-
year studies, and 2-year organ weights are not appropriate for analysis as stated in Sections 1.1.1
and 1.1.2, this endpoint will not be considered further.
Adrenal weight
Adrenal weights were increased in 13-week and 26-week studies (see Table 1-19). For
instance, a 13-week drinking water study found that relative adrenal weights were increased in
male and female rats fMedinskv etal.. 19991. In another study, absolute adrenal weights were
increased in male rats (Hagiwara etal.. 20111. None of the observed organ weight changes
corresponded with functional or histopathological changes.
Immune system
Immunological endpoints yielded inconsistent results in a number of studies (see Table
1-20). Relative spleen weights were increased in male rats following 2-year oral and inhalation
exposures to ETBE (Suzuki etal.. 2012: TPEC. 2010b). CD3+, CD4+, and CD8+ T cells were reduced
in male mice after 6 or 13 weeks ofETBE exposure via inhalation fLi etal.. 20111. An analysis of
antibody response reported that the number of IgM+ splenic antibody forming cells was not
significantly affected after a 28-day oral exposure to ETBE followed by sheep red blood cell
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
Toxicological Review ofETBE
immunization (Banton etal.. 20111. No other indicators of histopathological or functional changes
were reported with a single chemical exposure.
Mortality
Mortality was significantly increased in male and female rats following a 2-year ETBE
inhalation exposure (Saito etal.. 2013: TPEC. 2010bl but not significantly affected following a 2-year
drinking water exposure (Suzuki etal.. 2012: TPEC. 2010al. Increased mortality in male rats
correlated with increased CPN severity in the kidney. Increased mortality in females was attributed
to pituitary tumors by the study authors; however, pituitary tumors were not dose responsively
increased by ETBE exposure. Survival was also reduced in a chronic gavage study at the highest
exposure in males and females at 72 weeks (data not shown); however, by 104 weeks survival in
controls was approximately 25% in males and 28% in females which is much lower than
anticipated for a 2-year study (Maltoni etal.. 19991. Thus, additional confounding factors such as
chronic respiratory infections may have contributed to the reduced survival. These data do not
suggest that mortality was increased in these studies due to excessively high exposure
concentrations ofETBE.
Mechanistic Evidence
No relevant mechanistic data are available for these endpoints.
Summary of other toxicity data
EPA concluded that the evidence does not support body weight changes, adrenal and
immunological effects, and mortality as potential human hazards ofETBE exposure.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
1	Table 1-18. Evidence pertaining to body weight effects in animals exposed to
2	ETBE
Reference and Dosing Protocol
Results by Endpoint
Body Weight
Banton et al. (2011)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
female (10/group): 0, 250, 500,1000 mg/kg-d
Female
0
-
daily for 28 consecutive days

250
3%


500
5%


1000
-1%
Fuiiietal. (2010); JPEC (2008e)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, female (24/group): 0,100, 300,1000 mg/kg-d
P0, Male
0
-
daily for 17 weeks beginning 10 weeks prior to

100
-4%
mating to lactation day 21

300
-4%
P0, male (24/group): 0,100, 300,1000 mg/kg-d

1000
-7%
daily for 16 weeks beginning 10 weeks prior to

Dose(mg/kg-d)
Percent change
mating


compared to



control

P0, Female
0
-


100
1%


300
1%


1000
5%
3
4
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-18. Evidence pertaining to body weight effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Body Weight (continued)
Gaoua (2004b)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, male (25/group): 0, 250, 500,1000 mg/kg-d
P0, Male
0
-
daily for a total of 18 weeks beginning 10 weeks

250
-1%
before mating until after weaning of the pups

500
-3%
P0, female (25/group): 0, 250, 500,1000 mg/kg-d

1000
-5%*
daily for a total of 18 weeks beginning 10 weeks

Dose(mg/kg-d)
Percent change
before mating until PND 21


compared to
Fl, male (25/group): 0, 250, 500,1000 mg/kg-d


control
dams dosed daily through gestation and lactation,
Fl, Male
0
-
then Fl doses beginning PND 22 until weaning of

250
0%
the F2 pups

500
3%
Fl, female (24-25/group): 0, 250, 500,1000

1000
1%
mg/kg-d

Dose(mg/kg-d)
Percent change
P0 dams dosed daily through gestation and


compared to
lactation, then Fl dosed beginning PND 22 until


control
weaning of the F2 pups
P0, Female
0
-


250
-7%


500
-2%


1000
0%


Dose(mg/kg-d)
Percent change



compared to



control

Fl, Female
0
-


250
-2%


500
-3%


1000
2%
Hagiwara et al. (2011); JPEC (2008d)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - gavage


control
male (12/group): 0,1000 mg/kg-d
Male
0
-
daily for 23 weeks

1000
-5%*
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-18. Evidence pertaining to body weight effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Body Weight (continued)
Mivata et al. (2013);JPEC (2008c)

Dose(mg/kg-d)
Percent change
rat, CRL:CD(SD)


compared to
oral - gavage


control
female (15/group): 0, 5, 25,100, 400 mg/kg-d;
Male
0
-
male (15/group): 0, 5, 25,100, 400 mg/kg-d

5
-6%
daily for 180 days

25
100
400
0%
-5%
2%


Dose(mg/kg-d)
Percent change
compared to
control

Female
0
5
25
100
400
-5%
-2%
-2%
-3%
Maltoni et al. (1999)



rat, Sprague-Dawley
Male


oral - gavage
no significant difference at any dose

female (60/group): 0, 250,1000 mg/kg-d; male



(60/group): 0, 250,1000 mg/kg-d
Female


4 d/wk for 104 wks; observed until natural death
no significant difference at any dose

Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - water


control
female (50/group): 0, 625, 2500,10,000 ppm (0,
Male
0
-
46,171, 560 mg/kg-d)a; male (50/group): 0, 625,

28
-4%
2500, 10,000 ppm (0, 28, 121, 542 mg/kg-d)a

121
-7%*
daily for 104 wks

542
-9%*


Dose(mg/kg-d)
Percent change
compared to
control

Female
0
46
171
560
-10%*
-11%*
-17%*
This document is a draft for review purposes only and does not constitute Agency policy.
1-125	DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review ofETBE
Table 1-18. Evidence pertaining to body weight effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Body Weight (continued)
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,
Male
0
-
2090, 6270, 20,900 mg/m3); male (NR): 0,150,

627
0%
500, 1500, 5000 ppm (0, 627, 2090, 6270,

2090
1%
20,900 mg/m3)b
dynamic whole body chamber; 6 hrs/d, 5 d/wk for
13 wks; generation method, analytical

6270
20,900
-1%
-7%

Dose(mg/m3)
Percent change
concentration and method were reported


compared to
control

Female
0
627
2090
6270
20,900
-6%
-7%
-7%
-11%
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (6/group): 0, 5000 ppm (0,
Male
0
-
20,900 mg/m3)b; male (6/group): 0, 5000 ppm (0,

20,900
3%
20,900 mg/m3)b
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

Dose(mg/m3)
Percent change
compared to
13 wks followed by a 28 day recovery period;


control
generation method, analytical concentration and
Female
0
-
method were reported

20,900
4%
Medinskv et al. (1999); Bond et al. (1996b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor


control
female (48/group): 0, 500,1750, 5000 ppm (0,
Male
0
-
2090, 7320, 20,900 mg/m3)b; male (48/group): 0,

2090
2%
500, 1750, 5000 ppm (0, 2090, 7320,

7320
4%
20,900 mg/m3)b

20,900
2%
dynamic whole body chamber; 6 hrs/d, 5 d/wk for
13 wks; generation method, analytical

Dose(mg/m3)
Percent change
compared to
concentration and method were reported


control

Female
0
2090
7320
20,900
-3%
3%
6%*
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-18. Evidence pertaining to body weight effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Body Weight (continued)
Medinskv et al. (1999); Bond et al. (1996b)

Dose(mg/m3)
Percent change
mice, CD-I


compared to
inhalation - vapor


control
female (40/group): 0, 500,1750, 5000 ppm(0,
Male
0
-
2090, 7320, 20,900 mg/m3)b; male (40/group): 0,

2090
0%
500, 1750, 5000 ppm (0, 2090, 7320,

7320
-1%
20,900 mg/m3)b

20,900
-3%
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

Dose(mg/m3)
Percent change
13 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


2090
-2%


7320
-1%


20,900
2%
Saito et al. (2013);JPEC (2010b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor


control
female (50/group): 0, 500,1500, 5000 ppm (0,
Male
0
-
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

2090
-7%*
500, 1500, 5000 ppm (0, 2090, 6270,

6270
-7%*
20,900 mg/m3)b

20,900
-26%*
dynamic whole body inhalation; 6 hrs/d, 5 d/wk

Dose(mg/m3)
Percent change
for 104 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
-


2090
-6%*


6270
-10%*


20,900
-23%*
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
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
1	Table 1-19. Evidence pertaining to adrenal effects in animals exposed to ETBE
Reference and Dosing Protocol
Results by Endpoint
Adrenal Gland: Absolute Weight
Hagiwara et al. (2011); JPEC (2008d)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - gavage


control
male (12/group): 0,1000 mg/kg-d
Male
0
-
daily for 23 weeks

1000
16%*
Medinskv et al. (1999); Bond et al. (1996b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor
Male
0
2090
7320
20,900
control
female (48/group): 0, 500,1750, 5000 ppm (0,
11%
9%
34%*
2090, 7320, 20,900 mg/m3)b; male (48/group): 0,
500, 1750, 5000 ppm (0, 2090, 7320,

20,900 mg/m3)b
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

Dose(mg/m3)
Percent change
compared to
13 wks; generation method, analytical


control
concentration and method were reported
Female
0
2090
7320
20,900
7%
7%
18%*
Medinskv et al. (1999); Bond et al. (1996a)

Dose(mg/m3)
Percent change
mice, CD-I


compared to
inhalation - vapor
Male
0
2090
7320
20,900
control
female (40/group): 0, 500,1750, 5000 ppm(0,
0%
50%
0%
2090, 7320, 20,900 mg/m3)b; male (40/group): 0,

500, 1750, 5000 ppm (0, 2090, 7320,

20,900 mg/m3)b
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

Dose(mg/m3)
Percent change
compared to
13 wks; generation method, analytical


control
concentration and method were reported
Female
0
2090
7320
20,900
-8%
8%
-8%
Adrenal Gland: Relative Weight
Hagiwara et al. (2011); JPEC (2008d)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - gavage


control
male (12/group): 0,1000 mg/kg-d
Male
0
-
daily for 23 weeks

1000
19%*
2
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
1	Table 1-20. Evidence pertaining to immune effects in animals exposed to ETBE
Reference and Dosing Protocol
Results by Endpoint
Sheep red blood cell- specific IgM Antibody Forming Cells/10A6 Spleen Cells
Banton et al. (2011)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
female (10/group): 0, 250, 500,1000 mg/kg-d
Female
0
-
daily for 28 consecutive days

250
-21%
immunized i.v. 4 days prior to sacrifice with sheep

500
42%
red blood cells

1000
8%
Sheep red blood cell-specific IgM Antibody Forming Cells/Spleen
Banton et al. (2011)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
female (10/group): 0, 250, 500,1000 mg/kg-d
Female
0
-
daily for 28 consecutive days

250
-20%
immunized i.v. 4 days prior to sacrifice with sheep

500
36%
red blood cells

1000
8%
Number of CD3+ T cells
Li et al. (2011)

Dose(mg/m3)
Percent change
mice, C57BL/6


compared to
inhalation - vapor


control
male (6/group): 0, 500,1,750, 5,000 ppm(0, 2,090,
Male
0
-
7,320, 20,900 mg/m3)a

2090
-14%
whole body, 6 hrs/d for 5 d /wk over 6 wks;

7320
-13%
generation method not reported; analytical

20900
-24%*
concentration and method were reported



Li et al. (2011)

Dose(mg/m3)
Percent change
mice, 129/SV


compared to
inhalation - vapor


control
male (6/group): 0, 500,1,750, 5,000 ppm(0, 2,090,
Male
0
-
7,320, 20,900 mg/m3)a

2090
-18%*
whole body, 6 hrs/d for 5 d/wk over 6 wks;

7320
-16%
generation method not reported; analytical

20900
-21%*
concentration and method were reported



This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-20. Evidence pertaining to immune effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Number of CD4+ T cells
Li etal. (2011)
mice, C57BL/6
inhalation - vapor
male (6/group): 0, 500,1,750, 5,000 ppm(0, 2,090,
7,320, 20,900 mg/m3)a
whole body, 6 hrs/d for 5 d/wk over 6 wks;
generation method not reported; analytical
concentration and method were reported
Dose(mg/m3) Percent change
compared to
control
Male 0
2090 -15%
7320 -11%
20900 -23%*
Li etal. (2011)
mice, 129/SV
inhalation - vapor
male (6/group): 0, 500,1,750, 5,000 ppm(0, 2,090,
7,320, 20,900 mg/m3)a
whole body, 6 hrs/d for 5 d/wk over 6 wks;
generation method not reported; analytical
concentration and method were reported
Dose(mg/m3) Percent change
compared to
control
Male 0
2090 -16%
7320 -11%
20900 -17%*
Li etal. (2011)
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
whole body, 6 hrs/d for 5 d/wk over 13 wks;
generation method not reported; analytical
concentration and method were reported
Dose(mg/m3) Percent change
compared to
control
Male 0
2090 -9%
7320 -17%*
20900 -24%*
Li etal. (2011)
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
whole body, 6 hrs/d for 5 d/wk over 13 wks;
generation method not reported; analytical
concentration and method were reported
Dose(mg/m3) Percent change
compared to
control
Male 0
2090 -11%
7320 -28%*
20900 -37%*
Number of CD8+ T cells
Li etal. (2011)
mice, C57BL/6
inhalation - vapor
male (6/group): 0, 500,1,750, 5,000 ppm(0, 2,090,
7,320, 20,900 mg/m3)a
whole body, 6 hrs/d for 5 d/wk over 6 wks
Dose(mg/m3) Percent change
compared to
control
Male 0
2090 -12%
7320 -13%*
20900 -23%*
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-20. Evidence pertaining to immune effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Number of CD8+ T cells (continued)
Li etal. (2011)
mice, 129/SV
inhalation - vapor
male (6/group): 0, 500,1,750, 5,000 ppm(0, 2,090,
7,320, 20,900 mg/m3)a
whole body, 6 hrs/d for 5 d/wk over 6 wks;
generation method not reported; analytical
concentration and method were reported
Dose(mg/m3) Percent change
compared to
control
Male 0
2090 -13%
7320 -14%
20900 -25%
Li etal. (2011)
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
whole body, 6 hrs/d for 5 d/wk over 13 wks;
generation method not reported; analytical
concentration and method were reported
Dose(mg/m3) Percent change
compared to
control
Male 0
2090 -8%
7320 -12%
20900 -20%
Spleen: Absolute Weight
Banton et al. (2011)
rat, Sprague-Dawley
oral - gavage
female (10/group): 0, 250, 500,1000 mg/kg-d
daily for 28 consecutive days
Dose(mg/kg-d) Percent change
compared to
control
Female 0
250 -3%
500 -15%
1000 -9%
Fuiiietal. (2010); JPEC (2008e)
rat, Sprague-Dawley
oral - gavage
P0, male (24/group): 0,100, 300,1000 mg/kg-d
daily for 16 weeks beginning 10 weeks prior to
mating
P0, female (24/group): 0,100, 300,1000 mg/kg-d
daily for 17 weeks beginning 10 weeks prior to
mating to lactation day 21
Dose(mg/kg-d) Percent change
compared to
control
P0, Male 0
100 -4%
300 -2%
1000 0%
Dose(mg/kg-d) Percent change
compared to
control
P0, Female 0
100 0%
300 -2%
1000 -1%
Hagiwara et al. (2011); JPEC (2008d)
rat, Fischer 344
oral - gavage
male (12/group): 0,1000 mg/kg-d
daily for 23 weeks
Dose(mg/kg-d) Percent change
compared to
control
Male 0
1000 -5%
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-20. Evidence pertaining to immune effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Spleen: Absolute Weight (continued)
Suzuki et al. (2012); JPEC (2010a)
rat, Fischer 344
oral - water
female (50/group): 0, 625, 2500,10,000 ppm (0,
46,171, 560 mg/kg-day)a; male (50/group): 0, 625,
2500,10,000 ppm (0, 28,121, 542 mg/kg-day)a
daily for 104 wks
Dose(mg/kg-d) Percent change
compared to
control
Male 0
628 -3%
121 19%
542 39%
Dose(mg/kg-d) Percent change
compared to
control
Female 0
46 -35%
171 -1%
560 -50%*
JPEC (2008b)
rat, CRL:CD(SD)
inhalation - vapor
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,
2090, 6270, 20,900 mg/m3); male (NR): 0, 150,
500, 1500, 5000 ppm (0, 627, 2090, 6270,
20,900 mg/m3)b
dynamic whole body chamber; 6 hrs/d, 5 d/wk for
13 wks; generation method, analytical
concentration and method were reported
Dose(mg/m3) Percent change
compared to
control
Male 0
627 0%
2090 1%
6270 -1%
20,900 -9%
Dose(mg/m3) Percent change
compared to
control
Female 0
627 -9%
2090 -2%
6270 -5%
20,900 1%
JPEC (2008b)
rat, CRL:CD(SD)
inhalation - vapor
female (6/group): 0, 5000 ppm (0, 20,900 mg/m3)b;
male (6/group): 0, 5000 ppm (0, 20,900 mg/m3)b
dynamic whole body chamber; 6 hrs/d, 5 d/wk for
13 wks followed by a 28 day recovery period;
generation method, analytical concentration and
method were reported
Dose(mg/m3) Percent change
compared to
control
Male 0
20,900 10%
Dose(mg/m3) Percent change
compared to
control
Female 0
20,900 6%
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-20. Evidence pertaining to immune effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Spleen: Absolute Weight (continued)
Medinskv et al. (1999); Bond et al. (1996b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor
Male
0
2090
7320
20,900
control
female (48/group): 0, 500,1750, 5000 ppm (0,
6%
3%
5%
2090, 7320, 20,900 mg/m3)b; male (48/group): 0,

500, 1750, 5000 ppm (0, 2090, 7320,

20,900 mg/m3)b
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

Dose(mg/m3)
Percent change
compared to
13 wks; generation method, analytical


control
concentration and method were reported
Female
0
2090
7320
20,900
-3%
3%
0%
Medinskv et al. (1999); Bond et al. (1996a)

Dose(mg/m3)
Percent change
mice, CD-I


compared to
inhalation - vapor
Male
0
2090
7320
control
female (40/group): 0, 500,1750, 5000 ppm(0,
-5%
0%
2090, 7320, 20,900 mg/m3)b; male (40/group): 0,

500, 1750, 5000 ppm (0, 2090, 7320,

20,900
-15%
20,900 mg/m3)b
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

Dose(mg/m3)
Percent change
compared to
13 wks; generation method, analytical


control
concentration and method were reported
Female
0
2090
7320
20,900
-11%
-2%
-11%
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor
Male
0
2090
6270
20,900
control
female (50/group): 0, 500,1500, 5000 ppm (0,
4%
32%
17%
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

500, 1500, 5000 ppm (0, 2090, 6270,

20,900 mg/m3)b
dynamic whole body inhalation; 6 hrs/d, 5 d/wk

Dose(mg/m3)
Percent change
compared to
for 104 wks; generation method, analytical


control
concentration and method were reported
Female
0
2090
6270
20,900
5%
-39%
-43%*
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-20. Evidence pertaining to immune effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Spleen: Relative Weight
Banton et al. (2011)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
female (10/group): 0, 250, 500,1000 mg/kg-d
Female
0
-
daily for 28 consecutive days

250
500
1000
0%
-18%
0%
Fuiiietal. (2010); JPEC (2008e)

Dose(mg/kg-d)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage


control
P0, male (24/group): 0,100, 300,1000 mg/kg-d
P0, Male
0
-
daily for 16 weeks beginning 10 weeks prior to

100
-1%
mating

300
2%
P0, female (24/group): 0,100, 300,1000 mg/kg-d

1000
8%
daily for 17 weeks beginning 10 weeks prior to

Dose(mg/kg-d)
Percent change
mating to lactation day 21


compared to
control

P0, Female
0
100
300
1000
-2%
-3%
-5%
Hagiwara et al. (2011); JPEC (2008d)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - gavage


control
male (12/group): 0,1000 mg/kg-d
Male
0
-
daily for 23 weeks

1000
0%
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - water
Male
0
628
121
542
control
female (50/group): 0, 625, 2500,10,000 ppm (0,
2%
28%
55%*
46,171, 560 mg/kg-day)a; male (50/group): 0, 625,

2500,10,000 ppm (0, 28,121, 542 mg/kg-day)a

daily for 104 wks

Dose(mg/kg-d)
Percent change
compared to
control

Female
0
46
171
560
-35%
3%*
-45%
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Table 1-20. Evidence pertaining to immune effects in animals exposed to
ETBE (continued)
Reference and Dosing Protocol
Results by Endpoint
Spleen: Relative Weight
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)
inhalation - vapor
Male
0
627
2090
6270
compared to
control
female (NR): 0,150, 500,1500, 5000 ppm (0, 627,
0%
5%
1%
2090, 6270, 20,900 mg/m3); male (NR): 0, 150,

500, 1500, 5000 ppm (0, 627, 2090, 6270,

20,900 mg/m3)b

20,900
-2%
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

Dose(mg/m3)
Percent change
13 wks; generation method, analytical


compared to
concentration and method were reported


control

Female
0
627
2090
6270
20,900
-3%
5%
1%
12%
JPEC (2008b)

Dose(mg/m3)
Percent change
rat, CRL:CD(SD)


compared to
inhalation - vapor


control
female (6/group): 0, 5000 ppm (0, 20,900 mg/m3)b;
Male
0
-
male (6/group): 0, 5000 ppm (0, 20,900 mg/m3)b

20,900
6%
dynamic whole body chamber; 6 hrs/d, 5 d/wk for

Dose(mg/m3)
Percent change
13 wks followed by a 28 day recovery period;


compared to
generation method, analytical concentration and


control
method were reported
Female
0
20,900
0%
Saito et al. (2013); JPEC (2010b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor
Male
0
2090
6270
20,900
control
female (50/group): 0, 500,1500, 5000 ppm (0,
15%
43%*
66%*
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

500, 1500, 5000 ppm (0, 2090, 6270,

20,900 mg/m3)b
dynamic whole body inhalation; 6 hrs/d, 5 d/wk

Dose(mg/m3)
Percent change
compared to
for 104 wks; generation method, analytical


control
concentration and method were reported
Female
0
2090
6270
20,900
30%
-31%
-25%
Conversion performed by study authors.
b4.18 mg/m3 = 1 ppm.
NR: not reported; *: 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
(n): number evaluated from group
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
1
2	Table 1-21. Evidence pertaining to mortality in animals exposed to ETBE
Reference and Dosing Protocol

Results by Endpoint

Survival at 104 wks
Maltoni et al. (1999)

Dose(mg/m3)
Percent change
rat, Sprague-Dawley


compared to
oral - gavage
female (60/group): 0, 250,1000 mg/kg-d; male
Male
0
250
1000
control
(60/group): 0, 250,1000 mg/kg-d
-8%
-54%
4 d/wk for 104 wks; observed until natural death



Dose(mg/m3)
Percent change
compared to
control

Female
0
250
1000
-8%
18%
Suzuki et al. (2012); JPEC (2010a)

Dose(mg/kg-d)
Percent change
rat, Fischer 344


compared to
oral - water


control
female (50/group): 0, 625, 2500,10,000 ppm (0,
Male
0
-
46,171, 560 mg/kg-day)a; male (50/group): 0,

628
-3%
625, 2500, 10,000 ppm (0, 28, 121, 542 mg/kg-

121
-11%
day)a

542
-11%
daily for 104 wks
Female
Dose(mg/kg-d)
0
46
171
560
Percent change
compared to
control
3%
6%
6%
Saito et al. (2013);JPEC (2010b)

Dose(mg/m3)
Percent change
rat, Fischer 344


compared to
inhalation - vapor


control
female (50/group): 0, 500,1500, 5000 ppm (0,
Male
0
-
2090, 6270, 20,900 mg/m3)b; male (50/group): 0,

2090
-14%
500, 1500, 5000 ppm (0, 2090, 6270,

6270
-9%
20,900 mg/m3)b

20,900
-32%*
dynamic whole body inhalation; 6 hrs/d, 5 d/wk

Dose(mg/m3)
Percent change
for 104 wks; generation method, analytical


compared to
concentration and method were reported
Female
0
2090
control
3%
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Reference and Dosing Protocol
Results by Endpoint

6270 -21%*
20,900 -21%*
Conversion performed by study authors.
b4.18 mg/m3 = 1 ppm.
NR: not reported; *: 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
(n): number evaluated from group
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
¦ = 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
Decreased Body
Weight
subchronic/
reproductive
chronic
Female rat; 28d (A)
PO Female rat; 16wks (B)
PO Male rat; 16wks (B)
PO Male rat; 18wks (C)
PO Female rat; 18wks [€)
F1 Male rat; GD 0-adult (CJ
F1 Female rat; GD 0-adult (C)
Male rat; 23wks (D)
Female rat; 26wks (F)
Male rat; 26wks (F)
Female rat; 104-wks (G)
Male rat; 104wks (G)
Female rat; 104wks (E)
Male rat; 104wks [E]
0	0	a
G—b	a
~	a	a	a
a a a
a—e-
a a a
a a a
~—b—a
e	a	a
a	a
~	a
10	100	1,000	10,000
Dose (mg/kg-day)
Sources: (A) Banton et al, 2011 (B] Fujiiet al, 2010; JPEC, 2008e (C) Gaoua, 2004b (D) Hagiwara et al, 2011
(E) Maltoni et al., 1999 (F) Miyata et al., 2013; JPEC, 2008c (G) Suzuki et al, 2012; JPEC, 2010a
Figure 1-11. Exposure-response array of body weight 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 ofETBE
¦ = 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
Decreased Body
Weight
Female rats; 13wks (A)
Male rats; 13wks (A)
Female rats; 13wks, 28d recovery (A)
Male rats; 13wks, 28d recovery (A)
subchronic
chronic
Female rats; 13 wks (B)
Male rats; 13 wks (B)
Female mice; 13 wks (B)
Male mice; 13 wks (B)
Female rats; 104wks (C)
Male rats; 104wks [C)
~ ~	B-
13	El	B-
13	B
~	B
B	B
B	B
-0
1	10	100 1,000 10,000 100,000
Exposure Concentration (mg/m3)
•significantly increased body weight
Sources: (A) JPEC, 2008b (B) Medinsky et al„ 1999; Bond et al, 1996 (C) Saito et al„ 2013; JPEC,2010b
Figure 1-12. Exposure-response array of body weight effects following
inhalation exposure to ETBE
This document is a draft for review purposes only and does not constitute Agency policy.
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2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
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23
24
25
26
27
28
29
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36
Toxicological Review ofETBE
1.2. INTEGRATION AND EVALUATION
1.2.1.	Effects Other Than Cancer
The evidence for noncancer effects associated with ETBE is entirely from rodent studies.
Kidney and liver were the most frequently affected endpoints following oral and inhalation
exposure to ETBE.
Changes in kidney parameters were consistently observed but the magnitude of change was
generally moderate while males had greater severity of effects compared with females. Overall,
there was consistency across multiple measures of potential kidney toxicity, including organ weight
increases, exacerbated CPN, urothelial hyperplasia, and increases in serum markers of kidney
function such as cholesterol, BUN, and creatinine. Additionally, effects were consistently observed
across routes of exposure, species, and sex although male rats appear more sensitive than female
rats, and rats in general appear more sensitive than mice. Mechanistic data were insufficient to
establish a mode of action, and thus these effects are considered relevant to humans. EPA identified
kidney effects as a human hazard ofETBE exposure.
Increased liver weight and centrilobular hypertrophy in male and female rats were
consistently observed across studies. However, no additional histopathological findings were
observed, and only one serum marker of liver toxicity (GGT) was elevated, while other markers
(AST, ALT, and ALP) were not. The magnitude of change for these noncancer effects was mild to
moderate 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 a
relationship between receptor activation and liver toxicity has not been established for ETBE.
Additionally, mechanistic data suggest possible susceptibility related to reduced clearance of
acetaldehyde, a metabolite ofETBE, as discussed below in Section 1.2.3. EPA concluded that the
evidence does not support liver effects as a potential human hazard ofETBE exposure. Thus, these
effects were not considered further for dose-response analysis and the derivation of reference
values. Potential for liver carcinogenicity is discussed in the following section.
EPA concluded that the evidence does not support body weight changes, adrenal,
immunological, reproductive and developmental effects, and mortality as potential human hazards
ofETBE exposure. Thus, these effects were not considered further for dose-response analysis and
the derivation of reference values.
1.2.2.	Carcinogenicity
Under EPA's Guidelines for Carcinogen Risk Assessment fU.S. EPA. 2005al the database for
ETBE provides "suggestive evidence of carcinogenic potential." This is based on induction of
hepatocellular adenomas and carcinomas (combined) at the highest dose in male F344 rats by
inhalation (Saito etal.. 2013: TPEC. 2010b). but not in female rats in the same study or in either sex
of two strains of rats exposed orally to ETBE (Suzuki etal.. 2012: Malarkev and Bucher. 2011: TPEC.
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2010a: Maltoni et al.. 19991. Additionally, there is an absence of data in other experimental species
or in humans, and limited mechanistic data.
EPA evaluated the available mechanistic data and concluded that the evidence related to
putative pathways PPAR, PXR, and CAR was insufficient to determine the role these pathways play,
if any, in tumor formation. Genotoxicity data for ETBE and its metabolite tert-butanol are
inadequate to form a conclusion about ETBE's potential for genotoxicity. Additional mechanistic
studies reported that deficient function of Aldh2 enhanced ETBE-induced genotoxicity in
hepatocytes and leukocytes (Wengetal.. 2013: Wengetal.. 20121. These findings are consistent
with genotoxicity being mediated by the ETBE metabolite acetaldehyde, which is directly genotoxic
(IARC, 1999) and considered carcinogenic when produced as a result of metabolism from ingested
ethanol (IARC. 20121. A mechanistic study conducted by gavage in rats reported ETBE-related
increases in thyroid, urinary bladder, and liver tumors following initiation by DMBDD, suggesting
that ETBE exposure promotes tumors (Hagiwara etal.. 20111. Thus, these mechanistic data provide
some biological plausibility to the carcinogenicity ofETBE.
The chronic gavage bioassay reported an increased incidence of schwannomas (Malarkev
and Bucher. 2011: Maltoni etal.. 19911. but confidence in these data are low as the increase was
small, only observed at the lowest dose, and not accompanied by any mechanistic data supporting
their biological plausibility.
As emphasized in the Cancer Guidelines (U.S. EPA. 2005a). selection of the cancer descriptor
followed a full evaluation of the available evidence. The descriptor of "suggestive evidence of
carcinogenic potential" is appropriate when a concern for potential carcinogenic effects in humans
is raised, but the data are judged to be insufficient for a stronger conclusion. Exposure to ETBE
produced a clearly positive tumor response at only one tissue (liver), one dose (highest), and one
sex/species combination (male rats). Thus, these data correspond most closely to one of the
examples in the Cancer Guidelines (U.S. EPA. 2005a) for the descriptor of "suggestive evidence of
carcinogenic potential;" i.e., "a small, and possibly not statistically significant, increase in tumor
incidence observed in a single animal or human study that does not reach the weight of evidence
for the descriptor 'likely to be carcinogenic to humans'." Overall, the cancer descriptor "suggestive
evidence of carcinogenic potential" is plausible given that some concern for carcinogenic effects in
humans is raised by the presence of a single positive result at one dose in one study and some
biological plausibility provided by the available mechanistic data, including the metabolism of ETBE
to acetaldehyde.
The Cancer Guidelines (U.S. EPA. 2005a) indicate that for tumors occurring at a site other
than the initial point of contact, the weight of evidence for carcinogenic potential may apply to all
routes of exposure that have not been adequately tested at sufficient doses. An exception occurs
when there are convincing toxicokinetic data that absorption does not occur by other routes. In the
case ofETBE, the positive tumor response was in a tissue (liver) remote from the site of absorption
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(respiratory tract). Although both oral and inhalation routes have been tested, all the bioassays
were in a single species (rats). Absorption ofETBE via inhalation, oral, or dermal routes either has
been demonstrated experimentally or is expected based on chemical properties. Therefore, the
conclusion that ETBE presents "suggestive evidence of carcinogenic potential" applies to all routes
of exposure.
1.2.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes
Genetic polymorphisms of ALDH, the enzyme that oxidizes acetaldehyde to acetic acid, may
also affect potential ETBE liver toxicity. The virtually inactive form, ALDH2*2, is responsible for
alcohol intolerance and is found in about one-half of all East Asians (Brennan. 2002). This variant is
associated with slow metabolism of acetaldehyde and, hence, extended exposure to a genotoxic
compound. 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
Aldh2 knockout mice, genotoxicity was significantly increased compared with wild type controls
following ETBE exposure to similar doses where both cancer and noncancer effects were observed
(Wengetal.. 2014: Wengetal.. 2013: Weng etal.. 2012: Wengetal.. 2011). Studies inAIdh2
knockout mice observed elevated blood concentrations of acetaldehyde following ETBE exposure
compared with wild type mice (Wengetal.. 2013) as well as increased alterations to sperm and
male reproductive tissue (Weng etal.. 2014) and increased severity of centrilobular hypertrophy
(Wengetal.. 2013: Wengetal.. 2012). 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 fSaito etal.. 2013: TPEC. 2010bl. No mode-of-action
information exists to account for the sex discrepancies in genotoxic effects. Finally, (IARC. 2012:
IARC (1999b)) identified acetaldehyde produced as a result of ethanol metabolism as the
predominant cause of carcinogenesis in the upper aerodigestive tract and esophagus following
ethanol ingestion, with effects amplified by deficient acetaldehyde metabolism in humans.
Altogether, these data present plausible evidence that diminished Aldh2 activity yields health effect
outcomes that are more severe than those in wild type counterparts. It is reasonable to assume
similar outcomes could occur in sensitive human populations.
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 it is clear that at least one variant (2 A6*4) has no catalytic
activity (Fukami et al.. 2004): however, the effect of this variability on ETBE toxicity is unknown.
Finally, specific age-related susceptibility to ETBE is not indicated by the data.
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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 (UFs) generally applied to reflect limitations of the data used.
2.1.1. Identification of Studies and Effects for Dose-Response Analysis
EPA identified kidney effects as a human hazard ofETBE exposure. Studies were evaluated
using general study quality characteristics (as discussed in Section 6 of the Preamble) 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 preferred over animal studies when quantitative measures of exposure
are reported and the reported effects are determined to be associated with exposure. However,
there are no available human occupational or epidemiological studies of oral exposure to ETBE.
Animal studies were evaluated to determine which studies provided: (a) the most relevant
routes and durations of exposure; (b) multiple exposure levels that informed the shape of the dose-
response curve; and (c) the power to detect effects at low exposure levels (U.S. EPA. 2002). The
database for ETBE includes several studies and data sets that are suitable for use in deriving
reference values. Specifically effects associated with ETBE exposure in animals included
observations of organ weight and histological changes in the kidney in several chronic and
subchronic studies, mostly in rats. Sufficient data were available to develop a PBPK model in rats
for both oral and inhalation exposure in order to perform route-to-route extrapolation, so rat
studies from both routes of exposure were considered for dose-response analysis.
Kidney Toxicity
The kidney was identified as the only human hazard ofETBE exposure based on findings of
organ weight changes, histopathology (nephropathy, urothelial hyperplasia), and altered serum
biomarkers (cholesterol, creatinine, BUN) in rats. The most consistent findings across studies were
for kidney weight changes and urothelial hyperplasia. 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..
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2010: TPEC. 2010b. 2008b. c; Gaoua. 2004b: Medinskv etal.. 19991. For urothelial hyperplasia,
chronic studies by both inhalation and oral exposure reported this effect to be increased with
treatment in male rats fSaito etal.. 2013: Suzuki etal.. 2012: TPEC. 2010a. b). Changes in serum
biomarkers lacked consistency and strength of association and were not considered for modeling.
Hagiwara etal. (20111. with only one dose group, was not considered further given its
concordance with multiple other rat studies that had multiple groups. Additionally, as discussed in
Section 1.1.1, 2-year organ weight data were not considered suitable due to the prevalence of age-
associated confounders. Therefore, only the urothelial hyperplasia data from the TPEC (2010a)
[selected data published as Suzuki etal. T20121] and TPEC f2010bl [selected data published as Saito
etal. T20131] studies were considered for dose-response analysis. These and the remaining studies,
TPEC (2008c) [selected data published as Mivataetal. (20131]. Gaoua (2004b). Fuiii etal. (20101.
TPEC (2008b). Medinskv etal. (19991. and Suzuki etal. (20121. are discussed further below.
Oral studies
The f Suzuki etal.. 2012: TPEC. 2010al study treated male and female F344 rats
(50/sex/dose group) with ETBE via drinking water at dose levels of 0, 28,121, or 542 mg/kg-day in
males for 104 consecutive weeks. Increased incidence of slight urothelial hyperplasia was only
observed in males and significantly increased at 121 and 542 mg/kg-day. Similar effects were not
observed in females.
The TPEC (2008c) study treated male and female Crl:CD(SD) rats (15/sex/dose group) with
ETBE via gavage at dose levels of 0, 5, 25,100, or 400 mg/kg-day daily for 180 consecutive days
(26 weeks). Relative kidney weight was significantly increased in males and females treated with
100 or 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. As discussed in Section 1.1.1.,
although an increase in a2U-globulin was measured by immunohistochemical staining, there was
inadequate evidence to conclude that the observed kidney effects are the result of a2U-globulin
accumulation.
A two-generation reproductive toxicity study of ETBE was conducted in rats by Gaoua
f2004bl. Sprague-Dawley rats (25/sex/dose group) were administered ETBE via gavage for 18
weeks at dose levels of 0, 250, 500, or 1000 mg/kg-day that commenced 10 weeks before mating
and continued throughout the 2-week mating period, gestation, and end of lactation (PND 21) for a
total of 18 weeks. Absolute and relative kidney weights were increased in all dose groups in males,
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.
A one-generation reproductive toxicity study of ETBE was conducted in rats by Fuiii etal.
(20101. Male and female Crl:CD(SD) rats (24/sex/dose group) were administered ETBE via gavage
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at dose levels of 0,100, 300, or 1000 mg/kg-day beginning 10 weeks prior to F0 mating and
continuing throughout the reproduction period (mating, gestation, and 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 1000 mg/kg-day. F0 males had a dose-dependent increase in
relative kidney weight with statistically significant increases in all three dose groups.
Inhalation studies
The fSaito etal.. 2013: TPEC. 2010bl study treated male and female F344 rats (50/sex/dose
group) with ETBE via inhalation at dose levels of 0, 2090, 6270, or 20,900 mg/m3 in males and
females for 104 consecutive weeks. Increased incidences of slight urothelial hyperplasia were only
observed in males and significantly increased at 6270 and 20,900 mg/m3. Similar effects were not
observed in females.
In a subchronic-duration inhalation study, TPEC (2008b) exposed male and female
Crl:CD(SD) rats to ETBE via whole-body inhalation exposure at 0, 626.8, 2089, 6268, or
20,894 mg/m3 for 6 hours/day, 5 days/week, for 13 weeks (65 exposures total). There were no
significant differences in body weight throughout the study period for males or females. Significant
increases in relative kidney weights occurred in male and female rats exposed to 6268 or
20,894 mg/m3 ETBE compared with controls. After a recovery period of 28 days, the only
remaining effect observed was an increase in kidney weight in high-dose males.
Medinskv et al. (1999) exposed male and female F344 rats in whole-body chambers to 0,
2089, 8358, or 16,717 mg/m3 ETBE 6 hours/day, 5 days/week, for 13 weeks. At termination, body
weights of female rats in the 16,717-mg/m3 group were significantly higher than controls, but body
weights of other groups, both male and female, did not differ significantly from those of controls.
Slight, but statistically significant, increases in various clinical chemistry parameters were
observed, but these effects were reported to be of uncertain toxicological significance.
Medinskv et al. (1999) also exposed male and female CD-I mice in whole-body chambers to
0, 2089, 7313, or 20,894 mg/m3 ETBE for 6 hours/day, 5 days/week, for 13 weeks. No statistically
significant effects were noted in the kidney.
2.1.2. Methods of Analysis
No biologically based dose-response models are available for ETBE. In this case, EPA
evaluates a range of dose-response models thought to be consistent with underlying biological
processes to determine how best to empirically model the dose-response relationship in the range
of the observed data. Consistent with this approach, all models available in EPA's Benchmark Dose
Software (BMDS) were evaluated. Consistent with EPA's Benchmark Dose Technical Guidance
Document (U.S. EPA. 2012b). the benchmark dose (BMD) and the 95% lower confidence limit on the
BMD (BMDL) were estimated using a benchmark response (BMR) of 10% change from the control
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mean (Relative Deviation; RD) for organ weight data in the absence of information regarding what
level of change is considered biologically significant, and also to facilitate a consistent basis of
comparison across endpoints, studies, and assessments. A benchmark response (BMR) of 10%
extra risk was considered appropriate for the quantal data on incidences of slight urothelial
hyperplasia. The estimated BMDLs were used as points of departure (PODs). Further details
including the modeling output and graphical results for the best fit model for each endpoint can be
found in Appendix C of the Supplemental Information.
In general, absolute and relative kidney weight data may both be considered appropriate
endpoints for analysis. Body weight, which may impact interpretation of relative organ weights,
was not significantly affected in the studies chosen. Based on a historical review of 26 studies of 1-
month exposed control rats, Bailey etal. (20041 concluded that neither absolute kidney weight nor
relative kidney:body (or kidney:brain) weight are optimal for evaluating organ weight changes. As
neither approach is preferred, both were considered to be appropriate for BMD analysis.
PODs from Oral Studies
Human equivalent doses (HEDs) for oral exposures were derived from the PODs estimated
from the laboratory animal data as described in EPA's Recommended Use of Body Weight3/4 as the
Default Method in Derivation of the Oral Reference Dose (U.S. EPA. 20111. In this guidance, EPA
advocates a hierarchy of approaches for deriving HEDs from data in laboratory animals, with the
preferred approach being physiologically based toxicokinetic modeling. Other approaches can
include using chemical-specific information in the absence of a complete physiologically based
toxicokinetic model. As discussed in Appendix D of the Supplemental Information, several rat
physiologically based pharmacokinetic (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 either chemical-specific models or data to inform the derivation
of human equivalent oral exposures, a body weight scaling to the % power (i.e., BW3/4) approach is
applied to extrapolate toxicologically equivalent doses of orally administered agents from adult
laboratory animals to adult humans for the purpose of deriving an oral RfD. BW3/4 scaling was not
employed 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 and/or toxicodynamic differences betweenlifestages (U.S. EPA. 2011: Hattis etal..
20041.
Consistent with EPA guidance (U.S. EPA. 20111. the PODs estimated based on effects in adult
animals are converted to HEDs employing a standard dosimetric adjustment factor (DAF) derived
as follows:
DAF = (BWa1/4 / BWh1/4)
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1	where:
2	BWa = animal body weight
3	BWh = human body weight
4
5	Using a standard BWa of 0.25 kg for rats and a BWh of 70 kg for humans (U.S. EPA. 19881.
6	the resulting DAFs for rats is 0.24. The DAF would be applied to the POD identified for effects in
7	adult rats as follows to yield a PODhed (see Table 2-1):
8
9	PODhed = Laboratory animal dose (mg/kg-day) x DAF
10
11	Table 2-1 summarizes the sequence of calculations leading to the derivation of a human-
12	equivalent POD for each data set discussed above.
13	Table 2-1. Summary of derivation of PODs
Endpoint and Reference
Species/
Sex
Model3
BMR
BMD
(mg/kg-d)
BMDL
(mg/kg-d)
PODadj"
(mg/kg-d)
PODhed0
(mg/kg-d)
Kidney
Increased urothelial hyperplasia
(Suzuki et al., 2012; JPEC,
2010a)
Male Fischer
rats
Quanta 1-
Linear
10%
79.3
60.5
60.5
14.5
Increased absolute kidney
weight
JPEC (2008c); Mivata et al.
(2013)
Male
Sprague-
Dawley rats
Linear
10%
RD
176
115
115
27.6
Increased relative kidney weight
JPEC (2008c); Mivata et al.
(2013)
Male
Sprague-
Dawley rats
NOAEL (25 mg/kg-d)
(6% 1" in kidney weight)
25
6.0
Increased absolute kidney
weight
JPEC (2008c); Mivata et al.
(2013)
Female
Sprague-
Dawley rats
Exponential
(M4)
10%
RD
224
57
57
13.7
Increased relative kidney weight
JPEC (2008c); Mivata et al.
(2013)
Female
Sprague-
Dawley rats
Hill
10%
RD
191
20
20
4.8
14
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Table 2-1. Summary of derivation of PODs (continued)
Endpoint and
Reference
Species/
Sex
Model3
BMR
BMD
(mg/kg-d)
BMDL
(mg/kg-d)
PODadj"
(mg/kg-d)
PODhed0
(mg/kg-d)
Increased absolute
kidney weight (PO
generation)
Gaoua (2004b)
Male
Sprague-
Dawley rats
Hill
10%
RD
244
94
94
22.6
Increased relative
kidney weight (PO
generation)
Gaoua (2004b)
Male
Sprague-
Dawley rats
Hill
10%
RD
224
137
137
32.9
Increased absolute
kidney weight (PO
generation)
Gaoua (2004b)
Female
Sprague-
Dawley rats
Exponential
(M2)
10%
RD
1734
1030
1030
247
Increased relative
kidney weight (P0
generation)
Gaoua (2004b)
Female
Sprague-
Dawley rats
NOAEL (1000 mg/kg-d)
(5% 1" in kidney weight)
1000
240
Increased absolute
kidney weight (F1
generation)
Gaoua (2004b)
Male
Sprague-
Dawley rats
Polynomial
3°
10%
RD
318
235
235
56.4
Increased relative
kidney weight (F1
generation)
Gaoua (2004b)
Male
Sprague-
Dawley rats
LOAEL (250 mg/kg-d)
(10% 1" in kidney weight)
250
60
Increased absolute
kidney weight (F1
generation)
Gaoua (2004b)
Female
Sprague-
Dawley rats
Exponential
(M2)
10%
RD
978
670
670
161
Increased relative
kidney weight (F1
generation)
Gaoua (2004b)
Female
Sprague-
Dawley rats
NOAEL (500 mg/kg-d)
(6% 1" in kidney weight)
500
120
Increased absolute
kidney weight (P0
generation)
Fuiii et al. (2010)
Male
Sprague-
Dawley rats
Hill
10%
RD
435
139
139
33.4
Increased relative
kidney weight (P0
generation)
Fuiii et al. (2010)
Male
Sprague-
Dawley rats
Hill
10%
RD
243
129
129
31.0
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Table 2-1. Summary of derivation of PODs (continued)
Endpoint and
Reference
Species/
Sex
Model3
BMR
BMD
(mg/kg-d)
BMDL
(mg/kg-d)
PODadj"
(mg/kg-d)
PODhed0
(mg/kg-d)
Increased absolute
kidney weight (P0
generation)
Fuiii et al. (2010)
Female
Sprague-
Dawley rats
Polynomial
2°
10%
RD
1094
905
905
217
Increased relative
kidney weight (P0
generation)
Fuiii et al. (2010)
Female
Sprague-
Dawley rats
Polynomial
2°
10%
RD
1751
1254
1254
301
aFor modeling details, see Appendix C of the Supplemental Information.
bFor studies in which animals were not dosed daily, administered doses were adjusted to calculate the TWA daily
doses prior to BMD modeling.
CHED PODs were calculated using BW3/4scaling (U.S. EPA, 2011).
dBMD modeling failed to successfully calculate a BMD value (see Appendix C of the Supplemental Information).
RD = relative deviation; NA = not applicable
PODs from Inhalation Studies - Use ofPBPK Model for Route-to-route Extrapolation
A PBPK model for ETBE and its metabolite tert-butanol in rats has been developed, as
described in Appendix B of the Supplemental Information. Using this model, route-to-route
extrapolation of the inhalation BMCLs to derive oral PODs was performed as follows. First, the
internal dose in the rat at each inhalation BMCLadj (already adjusted to continuous exposure) was
estimated using the PBPK model to derive an "internal dose BMDL." Then, the oral dose
concentration (assuming continuous exposure) that led to the same internal dose in the rat was
estimated using the PBPK model. The resulting BMDL already reflects a continuous exposure so it is
equivalent to a PODadj, described above. This value was then converted to a human equivalent dose
POD using the formula previously described in "PODs from oral studies":
PODhed = PODadj (mg/kg-day) x DAF
A critical decision in the route-to-route extrapolation is the selection of the internal dose
metric to use that established "equivalent" oral and inhalation exposures. For ETBE-induced kidney
effects, the four options are the concentration of tert-butanol in blood, the rate of tert-butanol
metabolism, the rate ofETBE metabolism, and the concentration ofETBE in blood. Note that using a
kidney concentration for ETBE or tert-butanol will lead to the same route-to-route extrapolation
relationship as using blood concentration ofETBE or tert- butanol, respectively, because the
distribution from blood to kidney is independent of route. The major systemically available
metabolite ofETBE is tert- butanol, which has also been shown to cause kidney toxicity, so
tert-butanol is a plausible dose metric. There are no data to suggest that metabolites of tert-butanol
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1	mediate its renal toxicity, so the rate of tert-butanol metabolism is not a supported dose metric. The
2	other metabolite of ETBE is acetaldehyde, but it is largely produced in the liver, and its systemic
3	availability is limited due to its rapid clearance. Therefore, the rate of metabolism of ETBE is not
4	supported as a dose metric. The final dose metric option is ETBE blood concentration. Although it is
5	possible that tert-butanol contributes to the kidney effects of ETBE, it is clear that ETBE alone
6	cannot fully account for the kidney effects, given the presence of systemically available tert-butanol
7	following ETBE exposure. Therefore, tert-butanol in blood was selected as the best available dose
8	metric for route-to-route extrapolation, while recognizing that some uncertainty remains as to
9	whether it can fully account for the kidney effects ofETBE.
10	Table 2-2 summarizes the sequence of calculations leading to the derivation of a human-
11	equivalent POD for each inhalation data set discussed above.
12	Table 2-2. Summary of derivation of oral PODs derived from route-to-route
13	extrapolation from inhalation exposures
Endpoint and reference
Species/sex
BMR
BMCLadj
(mg/m3)
Internal
dose3
(mg/L)
Equivalent
PODadj
(mg/kg-d)
Equivalent
PODhed15
(mg/kg-d)
Kidney
Increased urothelial hyperplasia
(Saito et al., 2013; JPEC, 2010b)
Male F344 rats
10%
268
3.40
93.7
22.5
Increased absolute kidney weight
JPEC (2008b)
Male Sprague-
Dawley rats
10%
12
0.12
4.24
1.02
Increased relative kidney weight
JPEC (2008b)
Male Sprague-
Dawley rats
10%
99
1.19
34.9
8.38
Increased absolute kidney weight
JPEC (2008b)
Female Sprague-
Dawley rats
10%
2969
103
1110
266
Increased relative kidney weight
JPEC (2008b)
Female Sprague-
Dawley rats
10%
236
2.96
82.8
19.9
Increased absolute kidney weight
Medinskv et al. (1999)
Male F344 rats
10%
450
6.06
158
37.9
Increased absolute kidney weight
Medinskv et al. (1999)
Female F344 rats
10%
609
8.60
213
51.1
14	aAverage blood concentration of te/t-butanol under continuous inhalation exposure to ETBE at the BMDL (from
15	Table 2-1).
16	Continuous ETBE oral human equivalent dose that leads to the same average blood concentration of te/t-butanol
17	as continuous inhalation exposure to ETBE at the BMCL (see text for details).
18
19
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2.1.3. Derivation of Candidate Values
Under EPA's A Review of the Reference Dose and Reference Concentration Processes fU.S. EPA.
2002: Section 4.4.51. also described in the Preamble, five possible areas of uncertainty and
variability were considered. An explanation follows.
An intraspecies uncertainty factor, UFh, of 10 was applied to all PODs to account for
potential differences in toxicokinetics and toxicodynamics in the absence of information on the
variability of response in the human population following oral exposure to ETBE.
An interspecies uncertainty factor, UFa, of 3 (101/2 = 3.16, rounded to 3) was applied to all
PODs because BW3/4 scaling is used to extrapolate oral doses from laboratory animals to humans.
Although BW3/4 scaling addresses some aspects of cross-species extrapolation of toxicokinetic and
toxicodynamic processes, some residual uncertainty remains. In the absence of chemical-specific
data to quantify this uncertainty, EPA's BW3/4 guidance fU.S. EPA. 20111 recommends use of an
uncertainty factor of 3.
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, so a UFs of 10 was applied for studies of 13 weeks. In the case of the studies of 16-26
week duration, the magnitude of change observed in kidney weights was similar to the effect
observed at 104 weeks. This suggests a maximum effect may have been reached by 16-26 weeks.
However, the 104 week kidney data are confounded due to age-associated factors, so this
comparison may not be completely reliable. Additionally, some, but not all, markers of kidney
toxicity appear to be more severely affected by ETBE at 2 years (e.g., BUN). Thus, a UFs of 3 was
applied for studies of 16-26 week duration to account for this uncertainty and a UFs of 1 was
applied to 2 year studies.
A LOAEL to NOAEL uncertainty factor, UFl, of 1 was applied because either the POD was a
NOAEL or a BMDL. When the POD is a BMDL, 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 or relative kidney weight and a 10% extra risk of urothelial hyperplasia were
selected under an assumption that they represent minimal biologically significant changes. When
the POD was a LOAEL, a UFl of 10 was applied.
A database uncertainty factor, UFd, of 1 was applied to all PODs. The ETBE toxicity database
includes two chronic toxicity studies in rats (Suzuki etal.. 2012: TPEC. 2010al(Saito etal.. 2013:
TPEC. 2010b). several 13-26 week toxicity studies in mice and rats (Mivata etal.. 2013: Medinskv et
al.. 1999: TPEC. 2008b). prenatal developmental toxicity studies in rats and rabbits (Aso etal.. 2014:
Asano etal.. 20111. and both single- and multi-generation reproductive studies and developmental
studies in rats fFuiii etal.. 2010: Gaoua. 2004a: Gaoua. 2004bl. Additionally, the available mouse
study observed effects that were less severe than those in rats, suggesting that mice are not more
sensitive than rats. Although most of the studies are in rats, the ETBE database adequately covers
all major systemic effects, including reproductive and developmental effects, and does not suggest
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1	that additional studies would lead to identification of a more sensitive endpoint or a lower POD.
2	Therefore, a database UFd of 1 was applied.
3	Table 2-3 is a continuation of Tables 2-1 and 2-2 and summarizes the application of UFs to
4	each POD to derive a candidate value for each data set The candidate values presented in the table
5	below are preliminary to the derivation of the organ/system-specific reference values. These
6	candidate values are considered individually in the selection of a representative oral reference
7	value for a specific hazard and subsequent overall RfD for ETBE.
8	Figure 2-1 presents graphically the candidate values, UFs, and PODs, with each bar
9	corresponding to one data set described in Table 2-3.
10
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1	Table 2-3. Effects and corresponding derivation of candidate values
Endpoint and Reference
PODhed3
(mg/kg-d)
POD
type
UFa
UFh
UFl
UFs
UFd
Composite
UF
Candidate
value
(mg/kg-d)
Kidney
Increased urothelial hyperplasia;
male rat
Suzuki et al. (2012); JPEC (2010a)
14.5
BMDLio%
3
10
1
1
1
30
5 x 10 1
Increased urothelial hyperplasia;
male rat
Saito et al. (2013); JPEC (2010b)
22.5
BMDLio%
3
10
1
1
1
30
8 x 10 1
Increased absolute kidney weight;
male rat
JPEC (2008c); Mivata et al. (2013)
28
BMDLio%
3
10
1
3
1
100
3 x 10 1
Increased relative kidney weight;
male rat
JPEC (2008c); Mivata et al. (2013)
6.0
NOAEL
3
10
1
3
1
100
6 x 10"2
Increased absolute kidney weight;
female rat
JPEC (2008c); Mivata et al. (2013)
14
BMDLio%
3
10
1
3
1
100
1 x 10 1
Increased relative kidney weight;
female rat
JPEC (2008c); Mivata et al. (2013)
4.8
BMDLio%
3
10
1
3
1
100
5 x 10"2
Increased absolute kidney weight;
PO male rat
Gaoua (2004b)
23
BMDLio%
3
10
1
3
1
100
2 x 10 1
Increased relative kidney weight;
PO male rat
Gaoua (2004b)
33
BMDLio%
3
10
1
3
1
100
3 x 10 1
Increased absolute kidney weight;
PO female rat
Gaoua (2004b)
250
BMDLio%
3
10
1
3
1
100
3x 10°
Increased relative kidney weight;
PO female rat
Gaoua (2004b)
240
NOAEL
3
10
1
3
1
100
2x 10°
Increased absolute kidney weight;
F1 male rat
Gaoua (2004b)
56.4
BMDLio%
3
10
1
3
1
100
6 x 10 1
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Endpoint and Reference
PODhed3
(mg/kg-d)
POD
type
UFa
UFh
UFl
UFs
UFd
Composite
UF
Candidate
value
(mg/kg-d)
Increased relative kidney weight;
F1 male rat
Gaoua (2004b)
60
LOAEL
3
10
10
3
1
1000
6 x 10"2
Increased absolute kidney weight;
F1 female rat
Gaoua (2004b)
161
BMDLio%
3
10
1
3
1
100
2x 10°
Increased relative kidney weight;
F1 female rat
Gaoua (2004b)
120
NOAEL
3
10
1
3
1
100
lx 10°
Increased absolute kidney weight;
male rat
Fuiii et al. (2010)
33
BMDLio%
3
10
1
3
1
100
3 x 10 1
Increased relative kidney weight;
male rat
Fuiii et al. (2010)
31
BMDLio%
3
10
1
3
1
100
3 x 10 1
Increased absolute kidney weight;
female rat
Fuiii et al. (2010)
220
BMDLio%
3
10
1
3
1
100
2x 10°
Increased relative kidney weight;
female rat
Fuiii et al. (2010)
300
BMDLio%
3
10
1
3
1
100
3x 10°
Increased absolute kidney weight;
male rat
JPEC (2008b)
1.02
BMDLio%
3
10
1
10
1
300
3 x 10"3
Increased relative kidney weight;
male rat
JPEC (2008b)
8.38
BMDLio%
3
10
1
10
1
300
3 x 10"2
Increased absolute kidney weight;
female rat
JPEC (2008b)
266
BMDLio%
3
10
1
10
1
300
9 x 10 1
Increased relative kidney weight;
female rat
JPEC (2008b)
19.9
BMDLio%
3
10
1
10
1
300
7 x 10"2
Increased absolute kidney weight;
male rat
Medinsky et al. (1999)
37.9
BMDLio%
3
10
1
10
1
300
1 x 10 1
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Endpoint and Reference
PODhed3
(mg/kg-d)
POD
type
UFa
UFh
UFl
UFS
UFd
Composite
UF
Candidate
value
(mg/kg-d)
Increased absolute kidney weight;
female rat
Medinskv et al. (1999)
51.1
BMDLio%
3
10
1
10
1
300
2 x 10 1
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Increased urothelial hyperplasia; male rat
Suzuki et al. (2012); JPEG (2010a)
Increased urothelial hyperplasia; male rat
Saitoetal. (2013); JPEC (2010b)
Increased absolute kidney weight; male rat
JPEC (2008c)
Increased relative kidney weight; male rat
JPEC (2008c)
Increased absolute kidney weight; female rat
JPEC (2008c)
Increased relative kidney weight; female rat
JPEC (2008c)
Increased absolute kidney weight; male rat
Gaoua(2004b)
Increased relative kidney weight male rat
Gaoua(2004b)
Increased absolute kidney weight; female rat
Gaoua(2004b)
Increased relative kidney weight, female rat
Gaoua(2004b)
Increased absolute kidney weight; F1 male rat
Gaoua(2004b)
Increased relative kidney weight; F'l male rat
Gaoua(2004b)
Increased absolute kidney weight; F1 female rat
Gaoua(2004b)
Increased relative kidney weight; F1 female rat
Gaoua(2004b)
Increased absolute kidney weight;male rat
Fujii (2010)
Increased relative kidney weight; male rat
Fujii (2010)
Increased absolute kidney weight; female rat
Fujii (2010)
Increased relative kidney weight female rat
Fujii (2010)
Increased absolute kidney weight; male rat
JPEC(2008b)
Increased relative kidney weight; male rat
JPEC(2008b)
Increased absolute kidney weight; female rat
JPEC(2008b)
Increased relative kidney weight; female rat
JPEC(2008b)
Increased absolute kidney weight; male rat
Medinsky et al. (1999)
Increased absolute kidney weight; female rat
Medinsky etal. (1999)
^ Candidate RfD
• podhed
Composite UF
0.001
0.01
0.1	1
mg/kg-day
10
100
1000
2
3
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 2-4 distills the candidate values from Table 2-3 into a single value for the kidney.
Organ-specific reference values may 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 reference values were for several different effects in both sexes,
spanning a range from 3 x 10 3 to 3 x 10° mg/kg-day, for an overall thousand range. Selection of a
point estimate considered multiple aspects, including study design and consistency across
estimates. The only data from a chronic study are for urothelial hyperplasia in male rats, exposed
via inhalation or oral routes (Suzuki etal.. 2012: TPEC. 2010a)(Saito etal.. 2013: TPEC. 2010bl. This
is a specific indicator of kidney toxicity, and is synonymous with the transitional epithelial
hyperplasia observed after chronic tert-butanol exposure NTP fl9951. Additionally, estimated
benchmark doses are consistent between the two chronic ETBE studies, with the benchmark dose
estimated from the oral study within less than twofold of the benchmark dose derived by PBPK
model-based route-to-route extrapolation from the inhalation study. On the other hand, data on
kidney weight changes are limited to studies of 13-26 week duration, and the estimated benchmark
doses are highly variable across studies.
Taken together, these observations suggest that the most appropriate basis for a kidney-
specific RfD would be the results in male rats from the chronic studies f Suzuki etal.. 2012: TPEC.
2010airSaito etal.. 2013: TPEC. 2010bl. For the RfD, the results from the oral study (Suzuki etal..
2012: TPEC. 2010al are preferred, though it is notable that the two candidate values are very
similar. Therefore, to estimate an exposure level below which kidney toxicity from ETBE exposure
is not expected to occur, the candidate value for increased incidence of urothelial hyperplasia in
male rats from (Suzuki etal.. 2012: TPEC. 2010a) of 5 x 10-i mg/kg-day is proposed as the kidney-
specific reference dose for ETBE. Confidence in this kidney-specific RfD is high. The POD is based on
modeled benchmark dose estimates, and the candidate value is derived from a well-conducted GLP
study, involving a sufficient number of animals per group, assessing a wide range of kidney
endpoints. A candidate value for the same endpoint of urothelial hyperplasia based on route-to-
route extrapolation from the inhalation study fSaito etal.. 2013: TPEC. 2010bl is 8 x 101 mg/kg-day,
differing from the recommended kidney-specific RfD by less than twofold.
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1	Table 2-4. Organ/system-specific RfDs and proposed overall RfD for ETBE
Effect
Basis
RfD
(mg/kg-day)
Exposure
description
Confidence
Kidney toxicity
Increased urothelial
hyperplasia
5 x 10 1
Chronic
HIGH
Proposed
overall RfD
Increased urothelial
hyperplasia
5 x 101
Chronic
HIGH
2
3	2.1.5. Selection of the Proposed Overall Reference Dose
4	For ETBE, only kidney effects were identified as a hazard; thus a single organ/system-
5	specific reference dose was derived. Therefore, the kidney-specific RfD of 5 x 101 mg/kg-day is also
6	proposed as an estimated exposure level below which deleterious effects from ETBE exposure are
7	not expected to occur. The overall reference dose is derived to be protective of all types of effects
8	for a given duration of exposure and is intended to protect the population as a whole including
9	potentially susceptible subgroups (U.S. EPA. 20021.
10	2.1.6. Confidence Statement
11	A confidence level of high, medium, or low is assigned to the study used to derive the RfD,
12	the overall database, and the RfD itself, as described in Section 4.3.9.2 of EPA's Methods for
13	Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA.
14	19941. The overall confidence in this RfD is high. Confidence in the principal study TPEC (2008c) is
15	high. This study was well conducted, complied with OECD guidelines for GLP studies, involved a
16	sufficient number of animals per group (including both sexes), and assessed a wide range of tissues
17	and endpoints. Confidence in the database is high; the available studies evaluated a comprehensive
18	array of endpoints and there is no indication that additional studies would lead to identification of a
19	more sensitive endpoint Reflecting high confidence in the principal study and high confidence in
20	the database, confidence in the overall RfD for ETBE is high.
21	2.1.7. Previous IRIS Assessment
22	An oral assessment for ETBE was not previously available on IRIS.
23	2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER
24	THAN CANCER
25	The inhalation reference concentration (RfC) (expressed in units of mg/m3) is defined as an
26	estimate (with uncertainty spanning perhaps an order of magnitude) of a continuous inhalation
27	exposure to the human population (including sensitive subgroups) that is likely to be without an
28	appreciable risk of deleterious effects during a lifetime. It can be derived from a NOAEL, LOAEL, or
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the 95% lower bound on the benchmark concentration (BMCL), with UFs generally applied to
reflect limitations of the data used.
2.2.1. Identification of Studies and Effects for Dose-Response Analysis
EPA identified kidney effects as a human hazard ofETBE exposure. Studies were evaluated
using general study quality characteristics (as discussed in Section 6 of the Preamble) 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 preferred over animal studies 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 few 2-hour inhalation studies at doses up to
208.9 mg/m3 (Nihlen etal.. 1998: Vetrano. 19931. These studies were not considered for dose-
response assessment, because they are of acute duration and did not investigate effects in the
kidney.
Animal studies were evaluated to determine which provided, (a) the most relevant routes
and durations of exposure, (b) multiple exposure levels to inform the shape of the dose-response
curve, and (c) the power to detect effects at low exposure levels (U.S. EPA. 20021. Sufficient data
were available to develop a PBPK model in rats for both oral and inhalation exposure to perform
route-to-route extrapolation, so rat studies from both routes of exposure were considered for dose-
response analysis. The database for ETBE includes several studies and data sets that are suitable for
use in deriving reference values. Specifically, effects associated with ETBE exposure in animals
included observations of organ weight and histological changes in the kidney reported in several
chronic and subchronic studies, mostly in rats.
Kidney Effects
The kidney was identified as the only human hazard ofETBE exposure based on findings of
organ weight changes, histopathology (nephropathy, urothelial hyperplasia), and altered serum
biomarkers (creatinine, BUN, cholesterol) in rats. The most consistent findings across studies were
for kidney weight changes and urothelial hyperplasia. In the case of kidney weight changes,
numerous chronic and subchronic studies investigated this endpoint following oral and inhalation
exposure fSuzuki etal.. 2012: Hagiwara etal.. 2011: Fuiii etal.. 2010: TPEC. 2010b. 2008b. c; Gaoua.
2004b: Medinskv etal.. 19991. For urothelial hyperplasia, chronic studies by both inhalation and
oral exposure reported this effect to be increased with treatment in male rats.
Hagiwara etal. (20111. with only one dose group, was not considered further given its
concordance with several other rat studies that had multiple dose groups. Additionally, as
discussed in Section 1.1.1, 2-year organ weight data were not considered suitable due to the
prevalence of age-associated confounders. Therefore, only the urothelial hyperplasia data from the
(Suzuki etal.. 2012: TPEC. 2010a) (Saito etal.. 2013: TPEC. 2010b) studies were considered for dose-
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response analysis. These and the remaining studies were discussed previously in Section 2.1.1 as
part of the derivation of the oral reference dose, so they will not be reviewed here again.
2.2.2. Methods of Analysis
No biologically based dose-response models are available for ETBE. In this situation, EPA
evaluates a range of dose-response models thought to be consistent with underlying biological
processes to determine how best to empirically model the dose-response relationship in the range
of the observed data. Consistent with this approach, all models available in EPA's Benchmark Dose
Software (BMDS) were evaluated. Consistent with EPA's Benchmark Dose Technical Guidance
Document (U.S. EPA. 2012b). the benchmark concentration (BMC) and the 95% lower confidence
limit on the BMD (BMDL) were estimated using a benchmark response (BMR) of 10% change from
the control mean for organ weight data in the absence of information regarding what level of
change is considered biologically significant, and also to facilitate a consistent basis of comparison
across endpoints, studies, and assessments. A benchmark response (BMR) of 10% extra risk was
considered appropriate for the quantal data on incidences of slight urothelial hyperplasia. The
estimated BMCLs were used as points of departure (PODs). Further details including the modeling
output and graphical results for the best fit model for each endpoint can be found in Appendix C of
the Supplemental Information.
In general, absolute and relative kidney weight data may both be considered appropriate
endpoints for analysis. Body weight, which may impact interpretation of relative organ weights,
was not significantly affected in the studies chosen as discussed in Section 2.1.2.
PODs from Inhalation Studies
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 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 animal exposures in both inhalation studies (TPEC. 2008b: Medinskv etal.. 1999) were
adjusted to reflect a continuous exposure by multiplying concentration by
(6 hours/day)/(24 hours/day) and (5 days/week)/(7 days/week) as follows:
BMCLadj = BMCL (mg/m^) x (6 -h 24) x (5 -h 7)
BMCL (mg/m3) x (0.1786)
The RfC methodology provides a mechanism for deriving a human equivalent concentration
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
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mouse) and humans. According to the RfC guidelines (U.S. EPA. 19941. ETBE is a Category 3 gas
because it is largely inactive in the respiratory tract, is rapidly transferred between the lungs and
blood, and the toxicological effects observed are extra-respiratory. 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 Supplementary Information, the values reported in the literature
for these parameters include an La of 11.6 for Wistar rats (Kaneko etal.. 20001 and an Lh in humans
of 11.7 (Nihlen etal.. 19951. 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 2-5 summarizes the sequence of calculations leading to the derivation of a human-
equivalent POD for each inhalation data set discussed above.
Table 2-5. Summary of derivation of PODs following inhalation exposure
Endpoint and
Reference
Species/
Sex
Model3
BMR
BMC
(mg/m3)
BMCL
(mg/m3)
PODadj"
(mg/m3)
PODhec0
(mg/m3)
Kidney
Increased urothelial
hyperplasia
(Saito et al., 2013;
JPEC, 2010b)
Male F344
rats
Gamma
10%
RD
2734
1498
268
265
Increased absolute
kidney weight
JPEC (2008b)
Male
Sprague-
Dawley rats
Hill
10%
RD
911
68
12
11.9
Increased relative
kidney weight
JPEC (2008b)
Male
Sprague-
Dawley rats
Hill
10%
RD
1965
556
99
98
Increased absolute
kidney weight
JPEC (2008b)
Female
Sprague-
Dawley rats
Linear
10%
RD
28,591
16,628
2969
2945
Increased relative
kidney weight
JPEC (2008b)
Female
Sprague-
Dawley rats
Hill
10%
RD
5559
1321
236
234
Increased absolute
kidney weight
Medinskv et al. (1999)
Male F344
rats
Hill
10%
RD
6968
2521
450
446
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Endpoint and
Reference
Species/
Sex
Model3
BMR
BMC
(mg/m3)
BMCL
(mg/m3)
PODadj"
(mg/m3)
PODhec0
(mg/m3)
Increased absolute
kidney weight
Medinskv et al. (1999)
Female
F344rats
Exponential
(M4)
10%
RD
5610
3411
609
604
aFor modeling details, see Appendix C of the Supplemental Information.
bPODs were adjusted for continuous daily exposure: PODadj = POD x (hours exposed per day / 24 hrs) x (days
exposed per week / 7 days).
cPODhec calculated by adjusting the PODadj by the DAF for a Category 3 gas (U.S. EPA, 1994).
PODs from Oral Studies - Use ofPBPK Model for Route-to-route Extrapolation
Since tert-butanol is the primary metabolite of ETBE and the evidence suggests it is
involved in kidney toxicity, a PBPK model for ETBE and its metabolite tert-butanol in rats was
developed, as described in Appendix B. Using this model, route-to-route extrapolation of the oral
BMDLs to derive inhalation PODs was performed as follows. First, the internal dose in the rat at
each oral BMDL (assuming continuous exposure) was estimated using the PBPK model to derive an
"internal dose BMDL." Then, the inhalation air concentration (again assuming continuous exposure)
that led to the same internal dose in the rat was estimated using the PBPK model. The resulting
BMCL already reflects a continuous exposure so it is equivalent to a BMCLadj, described above. This
value was then converted to a human equivalent dose POD using the formula previously described
in "PODs from inhalation studies":
BMCLhec = BMCLadj (mg/m3) x (LaLh) (interspecies conversion)
= BMCLadj (mg/m3) x (11.6 -h 11.7)
= BMCLadj (mg/m3) x (0.992)
A critical decision in the route-to-route extrapolation is the selection of the internal dose
metric to use that established "equivalent" oral and inhalation exposures. For ETBE-induced kidney
effects, the four options are the concentration of tert-butanol in blood, the rate of tert-butanol
metabolism, the rate ofETBE metabolism, and the concentration ofETBE in blood. Note that using a
kidney concentration for ETBE or tert-butanol will lead to the same route-to-route extrapolation
relationship as using blood concentration ofETBE or tert- butanol, respectively, because the
distribution from blood to kidney is independent of route. The major systemically available
metabolite ofETBE is tert- butanol, which has also been shown to cause kidney toxicity, so
tert-butanol is a plausible dose metric. There are no data to suggest that metabolites of tert-butanol
mediate its renal toxicity, so the rate of tert-butanol metabolism is not a supported dose metric. The
other metabolite of ETBE is acetaldehyde, but it is largely produced in the liver, and its systemic
availability is limited due to its rapid clearance. Therefore, the rate of metabolism ofETBE is not
supported as a dose metric. The final dose metric option is ETBE blood concentration. It is clear that
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1	ETBE alone cannot fully account for the kidney effects, given the presence of systemically available
2	tert-butanol following ETBE exposure and the relatively small concentrations of ETBE measured in
3	the urine. Therefore, tert-butanol in blood was selected as the best available dose metric for route-
4	to-route extrapolation, while recognizing that some uncertainty remains as to whether it can fully
5	account for the kidney effects of ETBE.
6	Table 2-6 summarizes the sequence of calculations leading to the derivation of a human-
7	equivalent POD for each inhalation data set discussed above.
8	Table 2-6. Summary of derivation of inhalation PODs derived from route-to-
9	route extrapolation from oral exposures
Endpoint and reference
Species/sex
BMR
BMDL
(mg/kg-d)
Internal
dose3
(mg/L)
Equivalent
PODhec15
(mg/m3)
Kidney
Increased urothelial hyperplasia
(Suzuki et al., 2012; JPEC, 2010a)
Male F344 rats
10%
60.5
2.11
171
Increased absolute kidney weight
JPEC (2008c); Mivata et al. (2013)
Male Sprague-
Dawley rats
10%
115
4.25
326
Increased relative kidney weight
JPEC (2008c); Mivata et al. (2013)
Male Sprague-
Dawley rats
NA
25°
1.99
70
Increased absolute kidney weight
JPEC (2008c); Mivata et al. (2013)
Female Sprague-
Dawley rats
10%
57
1.99
161
Increased relative kidney weight
JPEC (2008c); Mivata et al. (2013)
Female Sprague-
Dawley rats
10%
20
0.670
56
Increased absolute kidney weight
(P0 generation) Gaoua (2004b)
Male Sprague-
Dawley rats
10%
94
3.41
266
Increased relative kidney weight
(P0 generation) Gaoua (2004b)
Male Sprague-
Dawley rats
10%
137
5.17
388
Increased absolute kidney weight
(P0 generation) Gaoua (2004b)
Female Sprague-
Dawley rats
10%
1030
90.2
2770
Increased relative kidney weight
(P0 generation) Gaoua (2004b)
Female Sprague-
Dawley rats
NA
1000°
85.5
2700
Increased absolute kidney weight
(F1 generation) Gaoua (2004b)
Male Sprague-
Dawley rats
10%
235
9.7
667
Increased relative kidney weight
(F1 generation) Gaoua (2004b)
Male Sprague-
Dawley rats
NA
250°
10.4
710
Increased absolute kidney weight
(F1 generation) Gaoua (2004b)
Female Sprague-
Dawley rats
10%
670
42.4
1900
Increased relative kidney weight
(F1 generation) Gaoua (2004b)
Female Sprague-
Dawley rats
NA
500°
26.7
1440
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Endpoint and reference
Species/sex
BMR
BMDL
(mg/kg-d)
Internal
dose3
(mg/L)
Equivalent
PODhec15
(mg/m3)
Increased absolute kidney weight
(PO generation)
Fuiii et al. (2010)
Male Sprague-
Dawley rats
10%
139
5.25
394
Increased relative kidney weight
(PO generation)
Fuiii et al. (2010)
Male Sprague-
Dawley rats
10%
129
4.83
365
Increased absolute kidney weight
(P0 generation)
Fuiii et al. (2010)
Female Sprague-
Dawley rats
10%
905
71.5
2480
Increased relative kidney weight
(P0 generation)
Fuiii et al. (2010)
Female Sprague-
Dawley rats
10%
1254
127
3230
aAverage blood concentration of te/t-butanol under continuous oral exposure to ETBE at the BMDL (from
Table 2-1).
Continuous ETBE inhalation human equivalent concentration that leads to the same average blood
concentration of te/t-butanol as continuous oral exposure to ETBE at the BMDL (see text for details).
CBMD modeling failed to successfully calculate a BMD value (see Appendix C of the Supplemental Information).
NOAELor LOAELwas used for route-to-route extrapolation.
NA = not applicable
2.2.3. Derivation of Candidate Values
Under EPA's A Review of the Reference Dose and Reference Concentration Processes fU.S. EPA.
2002: Section 4.4.51. also described in the Preamble, five possible areas of uncertainty and
variability were considered. An explanation follows:
An intraspecies uncertainty factor, UFh, of 10 was applied to all PODs to account for
potential differences in toxicokinetics and toxicodynamics in the absence of information on the
variability of response in the human population following inhalation exposure to ETBE.
An interspecies uncertainty factor, UFa, of 3 (101/2 = 3.16, rounded to 3) was applied to all
PODs to account for residual uncertainty in the extrapolation from laboratory animals to humans in
the absence of information to characterize toxicodynamic differences between rodents and humans
after inhalation exposure to ETBE. This value is adopted by convention where an adjustment from
animal to a human equivalent concentration has been performed as described in EPA's Methods for
Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry fU.S. EPA.
19941.
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, so a UFs of 10 was applied for studies of 13 weeks. In the case of the studies of 16-26
week duration, the magnitude of change observed in kidney weights was similar to the effect
observed at 104 weeks. This suggests a maximum effect may have been reached by 16-26 weeks.
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However, the 104 week kidney data are confounded due to age-associated factors, so this
comparison may not be completely reliable. Additionally, some, but not all markers of kidney
toxicity appear to be more severely affected by ETBE at 2 years (e.g., BUN). Thus a UFs of 3 was
applied for studies of 16-26 week duration to account for this uncertainty and a UFs of 1 was
applied to 2 year studies.
A LOAEL to NOAEL uncertainty factor, UFl, of 1 was applied because either the POD was a
NOAEL or a BMCL. When the POD is a BMCL, 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 or relative kidney weight and a 10% extra risk of urothelial hyperplasia were
selected under an assumption that they represent minimal biologically significant changes. When
the POD was a LOAEL, a UFl of 10 was applied.
A database uncertainty factor, UFd, of 1 was applied to all PODs. The ETBE toxicity database
includes two chronic toxicity studies in rats (Suzuki etal.. 2012: IPEC. 2010a]fSaito etal.. 2013:
TPEC. 2010bl. several 13-26 week toxicity studies in mice and rats fMivata etal.. 2013: Medinskv et
al.. 1999: IPEC. 2008b). prenatal developmental toxicity studies in rats and rabbits (Aso etal.. 2014:
Asano etal.. 20111. and both single- and multi-generation reproductive studies and developmental
studies in rats (Fuiii etal.. 2010: Gaoua. 2004a: Gaoua. 2004b). Additionally, the available mouse
study observed effects that were less severe than those in rats, suggesting that mice are not more
sensitive than rats. Although most of the studies are in rats, the ETBE database adequately covers
all major systemic effects, including reproductive and developmental effects, and does not suggest
that additional studies would lead to identification of a more sensitive endpoint or a lower POD.
Therefore, a database UFd of 1 was applied.
Table 2-7 is a continuation of Tables 2-5 and 2-6, and summarizes the application of UFs 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.
Figure 2-2 presents graphically the candidate values, UFs, and PODs, with each bar
corresponding to one data set described in Table 2-7.
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1	Table 2-7. Effects and corresponding derivation of candidate values
Endpoint (Sex and species) and
Reference
PODhec3
(mg/m3)
POD
type
UFa
UFh
UFl
UFs
UFd
Composite
UF
Candidate
value
(mg/m3)
Kidney
Increased urothelial hyperplasia;
male rat
Suzuki et al. (2012); JPEC (2010a)
171
BMCLio%
3
10
1
1
1
30
6x 10°
Increased urothelial hyperplasia;
male rat
Saito et al. (2013); JPEC (2010b)
265
BMCLio%
3
10
1
1
1
30
9x 10°
Increased absolute kidney weight;
male rat
JPEC (2008c); Mivata et al. (2013)
326
BMCLio%
3
10
1
3
1
100
3x 10°
Increased relative kidney weight;
male rat
JPEC (2008c); Mivata et al. (2013)
70
NOAEL
3
10
1
3
1
100
7 x 101
Increased absolute kidney weight;
female rat
JPEC (2008c); Mivata et al. (2013)
161
BMCLio%
3
10
1
3
1
100
2x 10°
Increased relative kidney weight;
female rat
JPEC (2008c); Mivata et al. (2013)
56
BMCLio%
3
10
1
3
1
100
6 x 101
Increased absolute kidney weight;
PO male rat
Gaoua (2004b)
266
BMCLio%
3
10
1
3
1
100
3x 10°
Increased relative kidney weight;
PO male rat
Gaoua (2004b)
388
BMCLio%
3
10
1
3
1
100
4x 10°
Increased absolute kidney weight;
PO female rat
Gaoua (2004b)
2770
BMCLio%
3
10
1
3
1
100
3x 101
Increased relative kidney weight;
PO female rat
Gaoua (2004b)
2700
NOAEL
3
10
1
3
1
100
3x 101
Increased absolute kidney weight;
F1 male rat
Gaoua (2004b)
667
BMCLio%
3
10
1
3
1
100
7x 10°
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Endpoint (Sex and species) and
Reference
PODhec3
(mg/m3)
POD
type
UFa
UFh
UFl
UFs
UFd
Composite
UF
Candidate
value
(mg/m3)
Increased relative kidney weight;
F1 male rat
Gaoua (2004b)
710
LOAEL
3
10
10
3
1
1000
7 x 101
Increased absolute kidney weight;
F1 female rat
Gaoua (2004b)
1900
BMCLio%
3
10
1
3
1
100
2x 101
Increased relative kidney weight;
F1 female rat
Gaoua (2004b)
1440
NOAEL
3
10
1
3
1
100
lx 101
Increased absolute kidney weight;
PO male rat
Fuiii et al. (2010)
394
BMCLio%
3
10
1
3
1
100
4x 10°
Increased relative kidney weight;
PO male rat
Fuiii et al. (2010)
365
BMCLio%
3
10
1
3
1
100
4x 10°
Increased absolute kidney weight;
PO female rat
Fuiii et al. (2010)
2480
BMCLio%
3
10
1
3
1
100
2x 101
Increased relative kidney weight;
PO female rat
Fuiii et al. (2010)
3230
BMCLio%
3
10
1
3
1
100
3x 101
Increased absolute kidney weight;
male rat
JPEC (2008b)
11.9
BMCLio%
3
10
1
10
1
300
4 x 10"2
Increased relative kidney weight;
male rat
JPEC (2008b)
98
BMCLio%
3
10
1
10
1
300
3 x 10 1
Increased absolute kidney weight;
female rat
JPEC (2008b)
2945
BMCLio%
3
10
1
10
1
300
lx 101
Increased relative kidney weight;
female rate
JPEC (2008b)
234
BMCLio%
3
10
1
10
1
300
8 x 10 1
Increased absolute kidney weight;
male rat
Medinsky et al. (1999)
446
BMCLio%
3
10
1
10
1
300
lx 10°
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Endpoint (Sex and species) and
Reference
PODhec3
(mg/m3)
POD
type
UFa
UFh
UFl
UFS
UFd
Composite
UF
Candidate
value
(mg/m3)
Increased absolute kidney weight;
female rat
Medinskv et al. (1999)
604
BMCLio%
3
10
1
10
1
300
2x 10°
1	a PODh ecs from JPEC (2008c), Gaoua (2004b), and Fujii et al. (2010) derived from route-to-route extrapolation using
2	a dose metric of average blood concentration of te/t-butanol under continuous oral exposure to ETBE at the
3	BMDL.
4
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Increased urothelial hyperplasia; male rat
Suzuki etal. (2012); JPEC(2010a)
Increased urothelial hyperplasia; male rat
Saitoetal. (2013); JPEC (2010b)
Increased absolute kidney weight; male rat
JPEC (2008c)
Increased relative kidney weight; male rat
JPEC (2008c)
Increased absolute kidney weight; female rat
jPEC (2008c)
Increased relative kidney weight; female rat
JPEC (2008c)
Increased absolute kidney weight; male rat
Gaoua(2004b)
Increased relative kidney weight; male rat
Gaoua(2004b)
Increased absolute kidney weight; female rat
Gaoua(2004b)
Increased relative kidney weight female rat
Gaoua(2004b)
Increased absolute kidney weight; F1 male rat
Gaoua(2004b)
Increased relative kidney weight; F1 male rat
Gaoua(2004b)
Increased absolute kidney weight; F1 female rat
Gaoua(2004b)
Increased relative kidney weight; F1 female rat
Gaoua(2004b)
Increased absolute kidney weight;male rat
Fujii (2010)
Increased relative kidney weight; male rat
Fujii (2010)
Increased absolute kidney weight; female rat
Fujii (2010)
Increased relative kidney weight; female rat
Fujii (2010)
Increased absolute kidney weight; male rat
JPEC(2008b)
Increased relative kidney weight; male rat
JPEC(2008b)
Increased absolute kidney weight; female rat
JPEC(2008b)
Increased relative kidney weight; female rat
JPEC(2008b)
Increased absolute kidney weight; male rat
Medinsky etal. (1999)
Increased absolute kidney weight; female rat
Medinsky etal. (1999)
^ Candidate RfC
% podhec
Composite UF
0.01
0.1
10	100
mg/m3
1000
10000
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 2-7 distills the candidate values from Table 2-6 into a single value for the kidney.
Organ- or system-specific reference values may 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 reference values were for increased kidney weight in both sexes,
spanning a range from 4 x 10 2 to 3 x 101 mg/m3, for an overall 750-fold range. Selection of a point
estimate considered multiple aspects, including study design and consistency across estimates. The
only data from a chronic study are for urothelial hyperplasia in male rats, exposed via inhalation or
oral routes (Suzuki etal.. 2012: TPEC. 2010alfSaito etal.. 2013: TPEC. 2010b). This is a specific
indicator of kidney toxicity and is synonymous with the transitional epithelial hyperplasia observed
after chronic tert-butanol exposure NTP T1995I Additionally, estimated benchmark doses are
consistent between the two chronic ETBE studies, with the benchmark dose estimated from the
oral study within less than twofold of the benchmark dose derived by PBPK model-based route-to-
route extrapolation from the inhalation study. On the other hand, data on kidney weight changes
are limited to studies of 13-26 week duration, and the estimated benchmark doses are highly
variable across studies. Based on the previous discussion in Section 2.1.4, the results in male rats
from the chronic studies fSuzuki etal.. 2012: TPEC. 2010alfSaito etal.. 2013: TPEC. 2010bl. For the
RfC, the results from the inhalation study fSaito etal.. 2013: TPEC. 2010bl are preferred, though it is
notable that the two candidate values are very similar.
Therefore, to estimate an exposure level below which kidney toxicity from ETBE exposure
is not expected to occur, the candidate RfC of 9 mg/m3 for increased incidence of urothelial
hyperplasia in male rats from fSaito etal.. 2013: TPEC. 2010b) is proposed as the kidney-specific
reference concentration for ETBE. Confidence in this kidney-specific RfC is high. The POD is based
on modeled benchmark dose estimates, and the candidate value is derived from a well-conducted
GLP study, involving a sufficient number of animals per group, and assessing a wide range of kidney
endpoints. A candidate RfC for the same endpoint of urothelial hyperplasia based on route-to-route
extrapolation from the oral study fSuzuki etal.. 2012: TPEC. 2010al is 6 mg/kg-day, differing from
the recommended kidney-specific RfC by less than twofold.
Table 2-8. Organ/system-specific RfCs and proposed overall RfC for ETBE
Effect
Basis
RfC (mg/m3)
Exposure
description
Confidence
Kidney toxicity
Increased urothelial
hyperplasia
9 x 10°
Chronic
HIGH
Proposed overall RfC
Increased urothelial
hyperplasia
9 x 10°
Chronic
HIGH
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2.2.5.	Selection of the Proposed Overall Reference Concentration
For ETBE, only kidney effects were identified as a hazard; thus a single organ/system-
specific reference concentration was derived. Therefore, the kidney-specific RfC of 9 mg/m3 is
proposed as an estimated exposure level below which deleterious effects from ETBE exposure are
not expected to occur. The overall reference concentration 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. 20021.
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 (U.S. EPA.
19941. The overall confidence in this RfC is high. Confidence in the principal study TPEC (2008cl:
Mivata etal. (20131 is high. The study was well conducted following OECD GLP Guideline 452 that
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 there is no indication that additional studies would lead to
identification of a more sensitive endpoint Reflecting high confidence in the principal studies and
high confidence in the database, confidence in the overall RfC is high.
2.2.7.	Previous IRIS Assessment
An RfC for ETBE was not previously available on 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 values
derived for ETBE. To derive the RfD and RfC, the UF approach (U.S. EPA. 2000a. 19941 was applied
to a POD based on renal changes in rats treated chronically. UFs were applied to the PODs to
account for extrapolating from an animal bioassay to human exposure, the likely existence of a
diverse population of varying susceptibilities, and database deficiencies. These extrapolations are
carried out with default approaches 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. Data on the effects ofETBE are derived from a small, but high-quality database
of studies in animal models, primarily rats. The database for ETBE exposure includes three lifetime
bioassays in rats, several reproductive/developmental studies in rats and rabbits, and several
subchronic studies in rats and mice.
Although the database is adequate for reference value derivation, there is uncertainty
associated with the database, including the lack of chronic studies in a species other than rats, such
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as mice. Additionally, there are no available developmental/reproductive inhalation studies.
Finally, the database lacks adequate studies that examine the effect on kidney or liver in animals
with deficient Aldh2.
The toxicokinetic and toxicodynamic differences between the animal species from which
the POD was derived and humans are unknown for ETBE. 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, it was found that males appear more susceptible than females to ETBE toxicity. However,
the underlying mechanistic basis of this apparent difference is not understood. Most importantly, it
is unknown which animal species and/or sexes may be more comparable to humans.
2.3. ORAL SLOPE FACTOR FOR CANCER
The carcinogenicity assessment provides information on the carcinogenic hazard potential
of the substance in question, and quantitative estimates of risk from oral and inhalation exposure
may be derived. Quantitative risk estimates may be derived from the application of a low-dose
extrapolation procedure. If derived, the oral slope factor is a plausible upper bound on the estimate
of risk per mg/kg-day of oral exposure.
2.3.1. Analysis of Carcinogenicity Data
As noted in Section 1.2.2, EPA concluded that there is "suggestive evidence of carcinogenic
potential" for ETBE. The Guidelines for Carcinogen Risk Assessment fU.S. EPA. 2005al 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 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 this case, the carcinogenicity ofETBE has been evaluated in three oral and inhalation
cancer bioassays in rats fSaito etal.. 2013: Suzuki etal.. 2012: Malarkev and Bucher. 2011: TPEC.
2010a. b). The strongest evidence of carcinogenicity is the increased incidence of liver tumors in
male F344 rats fSaito etal.. 2013: TPEC. 2010bl. Mechanistic data on liver tumor promotion and
enhanced genotoxicity in the absence of Aldh2 provide some biological plausibility for liver
carcinogenicity. Considering these data along with the uncertainty associated with the suggestive
nature of the weight of evidence, EPA concluded that quantitative analyses may be useful for
providing a sense of the magnitude of potential carcinogenic risk. Because the data are from an
inhalation study and ETBE induces systemic toxicity independent of exposure route, a PBPK model
is used to conduct route-to-route extrapolation to the oral route. Description of analysis of
carcinogenicity data is contained in the section on the inhalation unit risk, Section 2.4.1.
This document is a draft for review purposes only and does not constitute Agency policy.
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2.3.2. Dose-Response Analysis—Adjustments and Extrapolations Methods
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 doses at the lower
end of the observed data corresponding to 10% extra risk.
A PBPK model for ETBE in rats has been developed as described in Appendix B of the
Supplemental Information. Using this model, route-to-route extrapolation of the inhalation BMCL to
derive an oral POD was performed as follows. First, the internal dose in the rat at the inhalation
BMCLadj (i.e., adjusted to continuous exposure) was estimated using the PBPK model to derive an
"internal dose BMDL." Then, the oral dose (again assuming continuous exposure) that led to the
same internal dose in the rat was estimated using the PBPK model, resulting in a route-to-route
extrapolated BMDL.
A critical decision in the route-to-route extrapolation is the selection of the internal dose
metric for establishing "equivalent" oral and inhalation exposures. For ETBE-induced liver tumors,
the four options are the concentration of tert-butanol in blood, the rate of tert-butanol metabolism,
the concentration ofETBE in blood, and the rate ofETBE metabolism. The major systemically
available metabolite ofETBE is tert- butanol, which has not been shown to cause liver toxicity, so
tert-butanol and ETBE metabolism to tert-butanol are not plausible dose metrics. ETBE in the blood
is not supported as a dose metric either because liver concentrations ofETBE are more proximal to
the site of interest. However, liver concentration for ETBE will lead to the same route-to-route
extrapolation relationship as using metabolism ofETBE because the metabolism is proportional to
the liver concentration in a manner independent of route. Therefore, the rate of metabolism of
ETBE is a plausible dose metric based on the possibility that ETBE itself is responsible for potential
liver carcinogenicity in addition to acetaldehyde, the other metabolite ofETBE produced in the
liver, and a genotoxic carcinogen. Therefore, the rate of metabolism ofETBE was selected as the
best available basis for route-to-route extrapolation.
The route-to-route extrapolated ETBE BMDL is scaled to HED according to EPA guidance
fU.S. EPA. 2011. 2005a). In particular, the BMDL was converted to an HED assuming that doses in
animals and humans are toxicologically equivalent when scaled by body weight raised to the
power. Standard body weights of 0.25 kg for rats and 70 kg for humans were used (U.S. EPA. 1988).
The following formula was used for the conversion of oral BMDL to oral HED:
Scaled HED in mg/kg-d = (BMDL in mg/kg-d) x (0.25/70)1/4
= (BMDL in mg/kg-d) x 0.24
The U.S. EPA Guidelines for Carcinogen Risk Assessment fU.S. EPA. 2005al recommend that
the method used to characterize and quantify cancer risk from a chemical is determined by what is
known about the MOA of the carcinogen and the shape of the cancer dose-response curve. The
linear approach is recommended if the MOA of carcinogenicity has not been established fU.S. EPA.
This document is a draft for review purposes only and does not constitute Agency policy.
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1	2005a). In the case of ETBE, the mode of carcinogenic action for liver tumors is not understood (see
2	Section 1.2.2). Therefore, a linear low-dose extrapolation approach was used to estimate human
3	carcinogenic risk associated with ETBE exposure.
4	2.3.3. Derivation of the Oral Slope Factor
5	The results from route-to-route extrapolation of the male rat liver tumor data (Saito etal..
6	2013: TPEC. 2010b) are summarized in Table 2-9. The lifetime oral cancer slope factor for humans is
7	defined as the slope of the line from the lower 95% bound on the exposure at the POD to the control
8	response (slope factor = 0.1/BMDLio). This slope, a 95% upper confidence limit, represents a
9	plausible upper bound on the true risk. Using linear extrapolation from the BMDLio, a human
10	equivalent oral slope factor was derived as presented in Table 2-9.
11	A single oral slope factor was derived. The recommended oral slope factor for providing a
12	sense of the magnitude of potential carcinogenic risk associated with lifetime oral exposure to
13	ETBE is 9 x KM per mg/kg-day based on the liver tumor response in male F344 rats (Saito etal..
14	2013: TPEC. 201 Obi
15	Table 2-9. Summary of the oral slope factor derivation
Tumor
Species/Sex
BMR
BMCLadj
(mg/m3)
Internal Dosea
(mg/h)
BMDLb
(mg/kg-d)
POD=
BMDLhed0
(mg/kg-d)
Slope
Factord
(mg/kg-d)1
Hepatocellular
adenomas and
carcinomas
Male F344
rat
10%
1,271
4.00
455
ill
9 x 10"4
16	aAverage rate of ETBE metabolism in rats under continuous inhalation exposure at the BMCLadj.
17	Continuous oral exposure in rats that leads to the same average rate of ETBE metabolism as continuous inhalation
18	exposure in rats at the BMCL
19	Continuous oral exposure human equivalent dose = BMDL x (0.25/70)^.
20	dHuman equivalent oral slope factor = 0.1/BMDLhed.
21	2.3.4. Uncertainties in the Derivation of the Oral Slope Factor
22	There is uncertainty when extrapolating data from animals to estimate potential cancer
23	risks to human populations from exposure to ETBE (see Table 2-10). There are no data in humans
24	to support the tumors observed in animals. Although changing the methods used to derive the oral
25	slope factor could change the results, standard practices were used due to the lack of a human
26	PBPK model or specific MOA to indicate other methods would be preferable. Additionally,
27	considering the uncertainty associated with the suggestive nature of the weight of evidence, the
28	oral slope factor is recommended only for providing a sense of the magnitude of potential
29	carcinogenic risk.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
1	Table 2-10. Summary of uncertainties in the derivation of cancer risk values
2	for ETBE
Consideration and
Impact on Cancer Risk Value
Decision
Justification and Discussion
Selection of target organ
4/ oral slope factor by unknown
amount if liver not selected.
The liver was selected as
the target organ.
The liver was the best supported target site
based on a single bioassay result in male rats,
one data set on tumor promotion, and
mechanistic data providing biological
plausibility. However, the overall evidence
for carcinogenicity was considered
"suggestive."
Selection of data set
4/ oral slope factor by unknown
amount if different data set
selected.
Saito etal. (2013UPEC
(2010b) was selected.
Saito et al. (2013), JPEC (2010b) was a well-
conducted study. It was also the only
bioassay that reported increased liver
tumors. Additional bioassays might add
support to the findings or provide results for
different (possibly lower) doses, which may
affect the oral slope factor.
Selection of extrapolation approach
Different PBPK model could 4^ or T*
oral slope factor.
PBPK model-based
extrapolation of inhalation
data was used for oral
slope factor.
PBPK model accurately predicted ETBE
toxicokinetics. Data and model predictions
were within twofold of each other.
Selection of dose metric
Alternatives could 4/ or T* oral
slope factor.
ETBE metabolism rate as
the dose metric for route-
to-route extrapolation was
converted to HED.
ETBE metabolized is the best supported dose
metric. It is consistent with a hypothesis of
acetaldehyde playing a role in liver
carcinogenesis of ETBE. It is also consistent
with ETBE concentration in the liver being
the mediator of carcinogenesis (metabolism
is proportional to ETBE liver concentration).
Alternative dose metrics of ETBE
concentration, te/t-butanol concentration, or
te/t-butanol metabolism would result in a
range of 2.4-fold decrease to 1.04-fold
increase in the oral slope factor.
Interspecies extrapolation of
dosimetry and risk
Alternatives could 4^ or T* slope
factor (e.g., 3.5-fold 4^ [scaling by
body weight] or T* 2-fold [scaling by
BW2/3]).
The default approach of
body weight3'4 was used.
There are no data to suggest an alternative
approach. Because the dose metric was not
an area under the curve, BW3/4scaling was
used to calculate equivalent cumulative
exposures for estimating equivalent human
risks. While the true human correspondence
is unknown, this overall approach is expected
to neither over- nor underestimate human
equivalent risks.
Dose-response modeling
Alternatives could 4^ or T* slope
factor.
Used multistage dose-
response model to derive a
BMD and BMDL.
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.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Consideration and
Impact on Cancer Risk Value
Decision
Justification and Discussion
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 used.
Linear low-dose extrapolation for agents
without a known MOA is supported.
Statistical uncertainty at POD
4/ oral slope factor 1.5-fold if BMD
used as the POD rather than BMDL.
BMDL (preferred approach
for calculating plausible
upper bound slope factor).
Limited size of bioassay results in sampling
variability; lower bound is 95% CI on
administered exposure at 10% extra risk of
liver.
Sensitive subpopulations
1" oral slope factor 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. However, no chemical-specific
data are available to determine the extent of
enhanced susceptibility due to ETBE-induced
carcinogenicity. Because determination of a
mutagenic MOA has not been made, an age-
specific adjustment factor is not applied.
1
2	2.3.5. Previous IRIS Assessment: Oral Slope Factor
3	A cancer assessment for ETBE was not previously available on IRIS.
4	2.4. INHALATION UNIT RISK FOR CANCER
5	The carcinogenicity assessment provides information on the carcinogenic hazard potential
6	of the substance in question, and quantitative estimates of risk from oral and inhalation exposure
7	may be derived. Quantitative risk estimates may be derived from the application of a low-dose
8	extrapolation procedure. If derived, the inhalation unit risk is a plausible upper bound on the
9	estimate of risk per |J.g/m3 air breathed.
10	2.4.1. Analysis of Carcinogenicity Data
11	As noted in Section 1.2.2, EPA concluded that there is "suggestive evidence of carcinogenic
12	potential" for ETBE. The Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a) state:
13
14	When there is suggestive evidence, the Agency generally would not attempt a dose-
15	response assessment, as the nature of the data generally would not support one; however,
16	when the evidence includes a well-conducted study, quantitative analysis may be useful for
17	some purposes. For example, it could provide a sense of the magnitude and uncertainty of
18	potential risks, rank potential hazards, or set research priorities.
19
This document is a draft for review purposes only and does not constitute Agency policy.
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In this case, the carcinogenicity ofETBE has been evaluated in three cancer bioassays in rats
fSaito etal.. 2013: Suzuki etal.. 2012: Malarkev and Bucher.2011: TPEC. 2010a. b). Considering
these data and uncertainty associated with the suggestive nature of the weight of evidence, EPA
concluded that quantitative analyses may be useful for providing a sense of the magnitude of
potential carcinogenic risk.
The most robust evidence of carcinogenicity is the increased incidences of liver tumors in
male F344 rats fSaito etal.. 2013: IPEC. 2010b], These data have additional support due to the
biological plausibility of mechanistic data on tumor promotion and genotoxicity in the absence of
Aldh2, and analogy to the human carcinogenicity of acetaldehyde after consumption of ethanol. The
Saito etal. f20131. flPEC. 2010bl study was considered suitable for dose-response analysis. It was
conducted in accordance with GLP (OECD Guideline 451), and all aspects were subjected to
retrospective quality assurance audits. 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. With respect to hepatocellular adenomas and
carcinomas, statistical tests conducted by the study authors found significant dose-response trends
by both the Peto test (incidental tumor test) and the Cochran-Armitage test; a significant increase in
the 20,894-mg/m3 group compared with controls was calculated by Fisher's exact test In females,
no exposure-related neoplastic lesions were observed. Therefore, the hepatocellular adenomas and
carcinomas in male rats were considered suitable for quantitative analysis.
2.4.2. Dose-Response Analysis—Adjustments and Extrapolations Methods
The U.S. EPA Guidelines for Carcinogen Risk Assessment fU.S. EPA. 2005al recommend that
the method used to characterize and quantify cancer risk from a chemical is determined by what is
known about the MOA of the carcinogen and the shape of the cancer dose-response curve. The
linear approach is recommended if the MOA of carcinogenicity has not been established (U.S. EPA.
2005a). In the case ofETBE, the modes of carcinogenic action for liver tumors are not fully
understood (see Section 1.2.2). Therefore, a linear low-dose extrapolation approach was used to
estimate potential human carcinogenic risk associated with ETBE exposure. 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 a dose at the lower end of the observed data,
generally 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 (U.S. EPA. 1994)
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 employs an inverse concentration-time relationship to derive
a health-protective duration adjustment to time-weight the intermittent exposures used in the
This document is a draft for review purposes only and does not constitute Agency policy.
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study. The animal BMCL estimated from the inhalation study Saito etal. (20131. flPEC. 2010b] was
adjusted to reflect a continuous exposure by multiplying it by (6 hours/day)/(24 hours/day) and
(5 days/week)/(7 days/week) as follows:
BMCLadj = BMCL (mg/m3) x 6/24 x 5/7
= 7,118 mg/m3 x 0.25 x 0.71
= 1,271 mg/m3
The approach to determine the HEC 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) and humans. According to the RfC guidelines
(U.S. EPA. 1994). ETBE is a Category 3 gas because extra-respiratory effects were observed. The
values reported in the literature for these parameters include an La of 11.6 for rats (Kaneko etal..
2000). and an Lh in humans of 11.7 (Nihlen etal.. 1995). This allowed a BMCLhec to be derived as
follows:
BMCLhec = BMCLadj (mg/m3) x (La/Lh) (interspecies conversion)
BMCLadj (mg/m3) x (11.6/11.7)
= 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 liver tumor data (Saito etal.. 2013: TPEC. 2010b) is
summarized in Table 2-11. 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/BMCLio). This slope, a 95% upper confidence limit, 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 2-11
A single inhalation unit risk was derived. Therefore, the recommended inhalation unit risk
for providing a sense of the magnitude of potential carcinogenic risk associated with lifetime
inhalation exposure to ETBE is 8 x 10"5 per mg/m3, based on the liver tumor response in male
F344 rats (Saito etal.. 2013: TPEC. 2010b).
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
1	Table 2-11. Summary of the inhalation unit risk derivation
Tumor
Species/Sex
Selected
Model
BMR
BMC
(mg/m3)
POD=
BMCL
(mg/m3)
Slope factor3
(mg/m3)1
Hepatocellular adenomas
and carcinomas
Male F344 rat
1° Multistage
10%
1928
1261
8 x 105
2	aHuman equivalent slope factor = 0.1/BMCLiohec; see Appendix C of the Supplemental Information for details of
3	modeling results.
4
5	2.4.4. Uncertainties in the Derivation of the Inhalation Unit Risk
6	There is uncertainty when extrapolating data from animals to estimate potential cancer
7	risks to human populations from exposure to ETBE. There are no data in humans to support the
8	tumors observed in animals. Although changing the methods used to derive the inhalation unit risk
9	could change the results, standard practices were used due to the lack of a human PBPK model or
10	specific MOA to indicate other methods would be preferable. Additionally, considering the
11	uncertainty associated with the suggestive nature of the weight of evidence, the inhalation unit risk
12	is recommended only for providing a sense of the magnitude of potential carcinogenic risk.
13	Table 2-12. Summary of uncertainties in the derivation of cancer risk values
14	for ETBE
Consideration and
Impact on Cancer Risk Value
Decision
Justification and Discussion
Selection of target organ
4/ inhalation unit risk by unknown
amount if liver not selected.
The liver was selected as
the target organ.
The liver was the best supported target site,
based on a single bioassay result in male rats,
one data set on tumor promotion, and
mechanistic data providing biological
plausibility. However, the overall evidence
for carcinogenicity was considered
"suggestive."
Selection of data set
4/ or 1" inhalation unit risk by
unknown amount if different data
set selected.
Saito etal. (2013), JPEC
(2010b) was selected.
Saito et al. (2013), JPEC (2010b) was a well-
conducted study, and it was also the only
bioassay that reported increased liver
tumors. Using other bioassays (and hence
other target organs) would decrease the
inhalation unit risk. Additional bioassays
(e.g., in mice) might add support to the
findings or provide results for different
(possibly lower) doses, which may affect the
inhalation unit risk.
Selection of extrapolation approach
Inhalation data used for
inhalation unit risk.
No extrapolation methods were used.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
Consideration and
Impact on Cancer Risk Value
Decision
Justification and Discussion
Selection of dose metric
Alternatives could 4^ or T*
inhalation unit risk.
Administered
concentration was used.
Modeling based on the best supported PBPK
model-based internal dose metric of ETBE
metabolism decreased the BMCL by 2.1-fold.
Interspecies extrapolation of
dosimetry and risk
Alternatives could 4^ or T*
inhalation unit risk.
The default approach for a
Category 3 gas was used.
There are no data to suggest an alternative
approach. While the true human
correspondence is unknown, this overall
approach is expected to neither over- or
underestimate human equivalent risks.
Dose-response modeling
Alternatives could 4^ or T* slope
factor.
Multistage dose-response
model to derive a BMC and
BMCL was used.
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.
Statistical uncertainty at POD
4/ oral slope factor 1.5-fold if BMC
used as the POD rather than BMCL
BMCL (preferred approach
for calculating plausible
upper bound 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* oral slope factor 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. However, no chemical-specific
data are available to determine the extent of
enhanced sensitivity due to ETBE-induced
carcinogenicity. Because determination of a
mutagenic MOA has not been made, an age-
specific adjustment factor is not applied.
1
2	2.4.5. Previous IRIS Assessment: Inhalation Unit Risk
3	A cancer assessment for ETBE was not previously available on IRIS.
4	2.5. APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS
5	As discussed in the Supplemental Guidance for Assessing Susceptibility from Early-Life
6	Exposure to Carcinogens fU.S. EPA. 2005c). either default or chemical-specific age-dependent
7	adjustment factors (ADAFs) are applied to account for early-life exposure to carcinogens that act
8	through a mutagenic mode of action. Because chemical-specific life-stage susceptibility data for
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review ofETBE
cancer are not available, and because the mode of action for ETBE carcinogenicity is not known (see
Section 1.1.4), ADAFs were not applied.
This document is a draft for review purposes only and does not constitute Agency policy.
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Aso. S: Mivata. K: Takakura. S: Hoshuvama. S: Muroi. T: Kusune. Y: Aiimi. S: Furukawa. K. (2014).
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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.
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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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This document is a draft for review purposes only and does not constitute Agency policy.
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This document is a draft for review purposes only and does not constitute Agency policy.
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