Toxicological Review of tert-Butyl Alcohol (tert-Butanol) (CAS No. 75-65-0) In Support of Summary Information on the Integrated Risk Information System (IRIS)


DRAFT DELIBERATIVE: FOR INTERAGENCY REVIEW ONLY.
DO NOT DISTRIBUTE OUTSIDE YOUR AGENCY.
EPA/635/R-14/378
Interagency Review Draft
www.epa.gov/iris
Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
(CAS No. 75-65-0]
In Support of Summary Information on the
Integrated Risk Information System (IRIS)
September 2014
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
vi-EPA

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Toxicological Review of tert-Butyl Alcohol
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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Toxicological Review of tert-Butyl Alcohol
CONTENTS
AUTHORS | CONTRIBUTORS | REVIEWERS	viii
PREFACE	x
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS	xvii
EXECUTIVE SUMMARY	ES-16
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.	Thyroid Effects	1-27
1.1.3.	Reproductive and Developmental Effects	1-32
1.1.4.	Carcinogenicity (other than in the kidney or thyroid)	1-46
1.1.5.	Other Toxicological Effects	1-46
1.2.	INTEGRATION AND EVALUATION	1-60
1.2.1.	Effects Other Than Cancer	1-60
1.2.2.	Carcinogenicity	1-61
1.2.3.	Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes	1-63
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	2-2
2.1.3.	Derivation of Candidate Values	2-4
2.1.4.	Derivation of Organ/System-Specific Reference Doses	2-8
2.1.5.	Selection of the Proposed Overall Reference Dose	2-8
2.1.6.	Confidence Statement	2-9
2.1.7.	Previous IRIS Assessment	2-9
2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER THAN CANCER	2-9
2.2.1.	Identification of Studies and Effects for Dose-Response Analysis	2-9
2.2.2.	Methods of Analysis	2-11
2.2.3.	Derivation of Candidate Values	2-15
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2.2.4.	Derivation of Organ/System-Specific Reference Concentrations	2-18
2.2.5.	Selection of the Proposed Overall Reference Concentration	2-18
2.2.6.	Confidence Statement	2-19
2.2.7.	Previous IRIS Assessment	2-19
2.2.8.	Uncertainties in the Derivation of the Reference Dose and Reference
Concentration	2-19
2.3.	ORAL SLOPE FACTOR FOR CANCER	2-20
2.3.1.	Analysis of Carcinogenicity Data	2-20
2.3.2.	Dose-Response Analysis—Adjustments and Extrapolations Methods	2-21
2.3.3.	Derivation of the Oral Slope Factor	2-22
2.3.4.	Uncertainties in the Derivation of the Oral Slope Factor	2-23
2.3.5.	Previous IRIS Assessment: Oral Slope Factor	2-24
2.4.	INHALATION UNIT RISK FOR CANCER	2-24
2.4.1.	Analysis of Carcinogenicity Data	2-25
2.4.2.	Dose Response Analysis - Adjustments and Extrapolation Methods	2-25
2.4.3.	Inhalation Unit Risk Derivation	2-26
2.4.4.	Uncertainties in the Derivation of the Inhalation Unit Risk	2-27
2.4.5.	Previous IRIS Assessment: Inhalation Unit Risk	2-29
2.5.	APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS	2-29
REFERENCES	R-l
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
TABLES
Table P-l. Physicochemical properties and chemical identity of ferf-butanol xiii
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. Details of the search strategy employed for ferf-butanol LS-5
Table LS-2. Summary of additional search strategies for ferf-butanol LS-6
Table LS-3. Questions and relevant experimental information for evaluation of experimental
animal studies LS-7
Table 1-1. Changes in kidney weight in animals following exposure to ferf-butanol 1-3
Table 1-2. Changes in kidney histopathology in animals following exposure to ferf-butanol 1-5
Table 1-3. Changes in kidney tumors in animals following exposure to ferf-butanol 1-9
Table 1-4. Additional kidney effects potentially relevant to mode of action in animals following
exposure to ferf-butanol 1-14
Table 1-5. Summary of data on the a2u-globulin process in male rats exposed to ferf-butanol 1-16
Table 1-6. Summary of additional data informing the contribution of the a^.-globulin process on
the renal tumor development in male rats exposed to ferf-butanol 1-19
Table 1-7. Evidence pertaining to thyroid effects in animals following oral exposure to ferf-
butanol 1-29
Table 1-8. Evidence pertaining to reproductive effects in animals following exposure to ferf-
butanol 1-34
Table 1-9. Evidence pertaining to developmental effects in animals following exposure to ferf-
butanol 1-37
Table 1-10. Evidence pertaining to neurodevelopmental effects in animals following exposure to
ferf-butanol 1-44
Table 1-11. Evidence pertaining to effects on body weight in animals following exposure to ferf-
butanol 1-49
Table 1-12. Changes in liver weight in animals following exposure to ferf-butanol 1-52
Table 1-13. Changes in liver histopathology in animals following exposure to ferf-butanol 1-54
Table 1-14. Changes in urinary bladder histopathology in animals following oral exposure to
ferf-butanol 1-56
Table 2-1. Summary of derivations of points of departure 2-4
Table 2-2. Effects and corresponding derivation of candidate RfDs 2-6
Table 2-3. Organ/system-specific RfDs and proposed overall RfD for ferf-butanol 2-8
Table 2-4. Summary of derivation of PODs following inhalation exposure 2-13
Table 2-5. Summary of derivation of inhalation points of departure derived from route-to-route
extrapolation from oral exposures 2-14
Table 2-6. Effects and corresponding derivation of candidate values 2-16
Table 2-7. Organ/system-specific RfCs and proposed overall RfC for ferf-butanol 2-18
Table 2-8. Summary of the oral slope factor derivations 2-22
Table 2-9. Summary of uncertainties in the derivation of cancer risk values for ferf-butanol 2-23
Table 2-10. Summary of the inhalation unit risk derivation 2-27
Table 2-11. Summary of uncertainties in the derivation of cancer risk values for ferf-butanol 2-28
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
FIGURES
Figure LS-1. Study selection strategy LS-4
Figure 1-1. Exposure response array for kidney effects following oral exposure to ferf-butanol 1-10
Figure 1-2. Exposure-response array of kidney effects following subchronic inhalation exposure
to ferf-butanol (no chronic studies available) 1-11
Figure 1-3. Exposure-response array for components of a2u-globulin nephropathy and renal
tumors in male rats after oral exposure to ferf-butanol 1-17
Figure 1-4. Exposure-response array for components of a2u-globulin nephropathy and renal
tumors in male rats after inhalation exposure to ferf-butanol 1-18
Figure 1-5. Exposure-response array of thyroid follicular cell effects following chronic oral
exposure to ferf-butanol 1-31
Figure 1-6. Exposure-response array of reproductive and developmental effects following oral
exposure to ferf-butanol 1-41
Figure 1-7. Exposure-response array of reproductive and developmental effects following
inhalation exposure to ferf-butanol 1-42
Figure 1-8. Exposure-response array of other effects following oral exposure to ferf-butanol 1-58
Figure 1-9. Exposure-response array of other effects following inhalation exposure to ferf-
butanol 1-59
Figure 2-1. Candidate RfD values with corresponding POD and composite UF 2-7
Figure 2-2. Candidate RfC values with corresponding POD and composite UF 2-17
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Toxicological Review of tert-Butyl Alcohol
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
ALD
approximate lethal dosage
MTD
maximum tolerated dose
ALT
alanine aminotransferase
NAG
N-acetyl-p-D-glucosaminidase
AST
aspartate aminotransferase
NCFA
National Center for Environmental
atm
atmosphere

Assessment
ATSDR
Agency for Toxic Substances and
NCI
Nalional Cancer Institute

Disease Registry
NOAFI.
110-ohserved-adverse-effect level
BMD
benchmark dose
NTI'
Nalional Toxicology Program
BMDL
benchmark dose lower confidence limit
NZW
New Zealand White (rabbit breed)
BMDS
Benchmark Dose Software
OCT
ornithine carbamoyl transferase
BMR
benchmark response
ORD
Office of Research and Development
BUN
blood urea nitrogen
I'BPK
physiologically based pharmacokinetic
BW
body weight
I'CNA
proliferating cell nuclear antigen
CA
chromosomal aberration
I'OI)
point of departure
CASRN
Chemical Abstracts Service Registry
POD| m|
tin ration-adjusted POD

Number
QSAIn
quantitative structure-activity
CBI
covalent binding index

relationship
CHO
Chinese hamster ovary (cell lint- cells)
RDS
implicative DNA synthesis
CL
confidence limit
KfC
inhalation reference concentration
CNS
central nervous system
KID
oral reference dose
CPN
chronic progressive nephropathy
KCDK
regional gas dose ratio
CYP450
cytochrome P4.~>0
UNA
ribonucleic acid
DAF
dosimetric adjustment factor
SAIn
structure activity relationship
DEN
diethylnitrosami 11c
sci-:
sister chromatid exchange
DMSO
dimethylsulloxide
SI)
standard deviation
DNA
deoxyribonucleic acid
SDN
sorbitol dehydrogenase
EPA
Fnvironmenlal Proleclion Agency
SI-
standard error
FDA
Food and Drug Administration
SGOT
glutamic oxaloacetic transaminase, also
FEVi
forced expiratory volume of 1 second

known as AST
GD
gestation day
SGPT
glutamic pyruvic transaminase, also
GDH
glulamale dehydrogenase

known as ALT
GGT
y-glulamyl transferase
SSD
systemic scleroderma
GSH
glutathione
TCA
trichloroacetic acid
GST
glutathione-S-transferase
TCE
trichloroethylene
Hb/g-A
animal blood:gas parlilion coefficient
TWA
time-weighted average
Hb/g-H
human blood:gas parlilion coefficient
UF
uncertainty factor
HEC
human equivalent concentration
UFa
animal-to-human uncertainty factor
HED
human equivalent dose
UFh
human variation uncertainty factor
i.p.
intraperitoneal
UFl
LOAEL-to-NOAEL uncertain factor
IRIS
Integrated Risk Information System
UFS
subchronic-to-chronic uncertainty
IVF
in vitro fertilization

factor
LC50
median lethal concentration
UFd
database deficiencies uncertainty factor
LD50
median lethal dose
U.S.
United States of America
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
1
2	AUTHORS | CONTRIBUTORS | REVIEWERS
Assessment Team
Janice S. Lee, Ph.D. (Chemical Manager)
Keith Salazar, Ph.D*
Chris Brinkerhoff, Ph.D.
3		
Contributors
Andrew Hotchkiss, Ph.D.
Channa Keshava, Ph.D.
Amanda Persad, Ph.D.
4		
Production Team
Maureen Johnson
Vicki Soto
5		
Contractor Support
Rohyn llhiin, Ph.D.
Michelle Cawley*
William Meiulez, Jr., Ph.D.
Pam Ross
6		
Executive Direction
7 	
Internal Review Team
General Toxicology Workgroup
Inhalation Workgroup
Neurotoxicity Workgroup
PBPK Workgroup
Reproductive and Developmental
U.S. EPA
Office of Research and Development
National Center for Environmental Assessment
Research Triangle P;n k, NC
*Washington, DC
ORISE Postdoclornl Fellow ;il U.S.
epa/ord/nci:a
Currently with U.S. EPA, Office of Chemical Safety
and Pollution Prevention, Office of Pollution
Prevention and Toxics
Washington, DC
U.S. l-:i'A
Office of l\ese;irch ;ind Development
Nnlionnl Cenler for I jivi ion mental Assessment
Keseni ch Trinngle P:irk, NC
U.S. I-I'A
Office of Kesenrcli and Development
Nnlionnl Cenler for Environmental Assessment
U'nshinglon, DC
ICF Inlernnlionnl
Fairfax, VA
1 Research Triangle Park, NC
U.S. EPA/ORD/NCEA
Washington, DC
U.S. EPA
Office of Research and Development
National Center for Environmental Assessment
Washington, DC
Kenneth Olden, Ph.D., Sc.l)., LI 1.1). (Center Director)
John Vandenberg, Ph.D, (Nnlional 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, Ph.D. (Branch Chief)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
Toxicology Workgroup
Statistical Workgroup
Toxicity Pathways Workgroup
1		
Reviewers
2	This assessment was provided for review to scientists in EPA's Program and Region Offices.
3	Comments were submitted by:
Office of Children's Health Protection, Washington, DC
Office of Solid Waste and Emergency Response, Washington, DC
Region 2, New York, NY
Region 8, Denver, CO
4
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
PREFACE
This Toxicological Review critically reviews the publicly available studies on tert-butyl
alcohol (tert-butanol) in order to identify its adverse health effects and to characterize exposure-
response relationships. It was prepared under the auspices of EPA's Integrated Risk Information
System (IRIS) Program. The assessment covers an oral RfD, an inhalation RfC, and a cancer weight
of evidence descriptor.
The Toxicological Reviews for ethyl tert-butyl ether (liTI'li) and tert-butanol are being
developed simultaneously because they have a number ol overlapping scientific issues:
• tert-Butanol is a metabolite of ETBE, so some of the toxicological effects of ETBE may be due
to tert-butanol. Therefore, data on tert-butanol may inform hazard identification and dose-
response assessment of ETBE, and vice versa.
• The scientific literature for chemicals include data on u :il-globulin-related nephropathy.
Therefore, a common approach was employed to ev aluate 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 Rev iews. This is the first IRIS assessment for this
compound. The findings ol 111 is assessment and related documents produced during its
development are available on the IRIS Well site (http://www.epa.gov/irisl.
A pnhiic meeting was held in December 201o to obtain input on preliminary materials for
tert-butanol, 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 public comments are available on the docket at http://www.regulations.gov
(DocketID No. EPA-HQ-OKD-ZDIS-Olll).
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, the IRIS Program has made changes to
streamline the assessment development process, improve transparency, and create efficiencies in
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
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 that EPA has made substantial improvements to the
IRIS Program in a short amount of time.
The draft tert-butanol assessment represents a significant advancement in implementing
the NRC recommendations. This assessment is streamlined, and uses tables, 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 comprehensive, systematic, and
documented literature search and screening approach, provides llic database search strategy in a
table (databases, keywords), visually represents the inclusion and exclusion of studies in a flow
diagram, and all ofthe references are integrated within the Health and I jivironmental Research
Online (HERO) database. A study evaluation suction 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-specilie effects in which the evidence is synthesized within and
integrated across all evidence for each target organ/systems.
In the draft fiVf-liuUinol assessment, the IRIS Program has attempted to transparently and
uniformly identify strengths and limitations that would affect interpretation of results. All animal
studies of tert-butanol that were considered to lie oI acceptable quality, whether yielding positive,
negative, or null results, were considered in assessing the evidence for health effects associated
with chronic exposure to
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Toxicological Review of tert-Butyl Alcohol
weight-of-evidence approach to identify the potential human hazards associated with chemical
exposure.
The IRIS tert-butanol assessment provides a streamlined presentation of information,
integrated hazard identification of all toxic effects, and derivation of organ/system-specific
reference values. Additionally, consistent with the goal that 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 of study selection and
evaluation, as well as increased documentation of key assessment decisions.
For additional information aboutthis assessment, or for general questions regarding IRIS,
please contact EPA's IRIS Hotline at 202-566-1676 (phone), 202-5(>(>-1749 (fax), or
hotline.iris@epa.gov.
Chemical Properties
tert-Butanol is a white crystalline solid or colorless liquid (above 77 I") with a camphor-like
odor that is highly flammable (NIOSH. 2005: IPCS. l'U>7a). ^'/t-Rutanol contains a hydroxyl
chemical functional group and is misciblc with alcohol, ether, and other organic solvents and
soluble in water (IPCS. 1987a). Selected cliem ical and physical properties of tert-butanol are
presented in Table P-l.
This document is a draft for review purposes only and does not constitute Agency policy.
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1 Table P-l. Physicochemical properties and chemical identity of tert-butanol
Characteristic
Information
Reference
Chemical name
tert-Butanol
HSDB (2007)
Synonyms/Trade Names
t-butyl alcohol; tert-Butanol; tert-butyl
alcohol; t-Butyl hydroxide; 1,1-
Dimethylethanol; NCI-C55367; 2-
Methyl-2-propanol; tertiary butanol;
Trimethyl carbinol; Trimethyl
methanol, t-butyl alcohol, TBA
HSDB (2007)
IPCS (1987b)
Chemical Formula
C4H10O
HSDB (2007)
CASRN
75-65-0
HSDB (2007)
Molecular weight
74.12
HSDB (2007)
Melting point
25.7°C
HSDB (2007)
Boiling point
82.41°C
HSDB (2007)
Vapor pressure
40.7 mm Hg @ 25"C
HSDB (2007)
Density/Specific Gravity
0.78581
HSDB (2007)
Flashpoint
11°C (closed cup)
HSDB (2007)
Water solubility at 25°C
1 x 10s mg/L
HSDB (2007)
Octanol/Water Partition
Coefficient (Log Kow)
0.35
HSDB (2007)
Henry's Law Constant
9.05 x 10" atm-m /mole
HSDB (2007)
Odor threshold
219 mg/m3
HSDB (2007)
Conversion factors
1 ppm = 3.031 mg/m
1 mg/m3 = 0.324 ppm
IPCS (1987b)
Chemical structure
c
H.C
C
>LJ
'H3
QH
:h3
HSDB (2007)
2
3 Uses
4 tert-Butanol is primarily an anthropogenic substance that is produced in large quantities
5 fHSDB. 20071 from a number of precursors, including 1-butene, isobutylene, acetyl chloride and
6 dimethylzinc, and tert-butyl hydroperoxide. The domestic production volume of tert-butanol,
7 including imports, was approximately four billion pounds in 2012 (U.S. EPA. 20141.
8 tert-Butanol has been used as a fuel oxygenate, an octane booster in unleaded gasoline, and
9 a denaturant for ethanol. From 1997 to 2005, the annual tert-butanol volume found in gasoline
10 ranged from approximately 4 million to 6 million gallons. During that time, larger quantities were
11 used to make methyl tert-butyl ether (MTBE) and ETBE. MTBE and ETBE are fuel oxygenates that
This document is a draft for review purposes only and does not constitute Agency policy.
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were used in the U.S. prior to 2007 atlevels of more than 2 billion gallons annually. Current use
levels of MTBE and ETBE in the U.S. are much lower, but use in Europe and Asia remains strong1.
tert-Butanol has been used for a variety of other purposes including as a dehydrating agent
and solvent. As such, it is added to lacquers, paint removers, and nail enamels and polishes.
tert- Butanol is also used to manufacture methyl methacrylate plastics and flotation devices.
Cosmetic and food-related uses include the manufacture of flavors and, because of its camphor-like
aroma, it is also used to create artificial musk, fruit essences, and perfume (HSDB. 20071. It is also
used in coatings on metal and paperboard food containers (Cal/EPA. 19991. industrial cleaning
compounds, and can be used for chemical extractions in pharmaceutical application (HSDB. 20071.
Fate and Transport
Soil
The mobility of tert-butanol in soil is expected to be high due its low affinity for soil organic
matter. Rainwater or other water percolating through soil is expected to dissolve and transport
most tert-butanol presentin soil, potentially leading to groundwater contamination. Based on
tert-butanol's vapor pressure, volatilization from soil surfaces is expected to be an important
dissipation process fHSDB. 20071. terf-Butanol is a tertiary alcohol and this class of alcohols
generally degrades more slowly in the environment compared to primary (e.g., ethanol) or
secondary (e.g., isopropanol) alcohols. In anoxic soil conditions, the half-life of tert-butanol is
estimated to be on the order of months (approximately 200 (.lavs). Microbial degradation rates are
increased in soils supplemented with nitrate and sulfate nutrients (HSDB. 20071.
Water
/(.'/ MUilanol is expected to volatilize from water surfaces within 2 to 29 days and does not
readily adsorb to suspended solids and sediments in water (HSDB. 20071. Biodegradation in
aerobic water is on the magnitude of weeks to months and in anaerobic aquatic conditions, the
biodegradation rale decreases. I'ioconcenlration of tert-butanol in aquatic organisms is low
fHSDB. 20071.
Air
tert-Butanol exists primarily as a vapor in the ambient atmosphere. Vapor-phase tert-
butanol is degraded in the atmosphere by reacting with photochemically-produced hydroxyl
radicals with a half-life of 14 days fHSDB. 20071.
Occurrence in the Environment
The Toxics Release Inventory (TRI) Program National Analysis Report estimated that over
one million pounds of tert-butanol has been released into the soil from landfills, land treatment,
2
http://www.ihs.com/products/chemical/planning/ceh/gasoline-octane-improvers.aspx
This document is a draft for review purposes only and does not constitute Agency policy.
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underground injection, surface impoundments, and other land disposal sources. The TRI program
also estimated that 476,266 pounds of tert-butanol was released into the atmosphere from fugitive
emissions and point sources fU.S. EPA. 2012cl. In California, air emissions of tert-butanol from
stationary sources are estimated to be at least 27,000 pounds per year, based on data reported by
the state's Air Toxics Program fScorecard. 20141. The TRI National Analysis Report estimated
7,469 pounds of tert-butanol was released into surface waters from point and nonpoint sources in
2011 fU.S. EPA. 2012cl
tert-Butanol has been identified in drinking wells throughout the United States (HSDB.
20071. California's Geotracker Database2 lists 3496 detections o \ I erl-butanol in groundwater
associated with contaminated sites in that state since 2011. tert-Butanol has also been detected in
drinking water wells in the vicinity of landfills (U.S. EPA. 2012c). Additionally, tert- Butanol leaking
from underground storage tanks may be a product of MTBE and ETI'li, which can degrade to form
tert-butanol in soils fHSDB. 20071. The industrial chemical tert-butyl acetate also can degrade to
form tert-butanol in animals and in the environment.
Ambient outdoor air concentrations of tert-hulanol vary, seemingly according to proximity
to urban areas (HSDB. 20071.
General Population Exposure
tert-Butanol exposure can occur in many different sellings. Contamination resulting from
leaking underground storage tanks could potentially result in exposure to a large number of people
who get their drinking water from wells. Due to its high environmental mobility and resistance to
biodegradation, ter/-Imtanol has the potential to contaminate and persist in groundwater and soil;
therefore, exposure through ingestion ol contaminated drinking water is likely occurring (HSDB.
20071.
Contaminated food can also contribute to fert-butanol ingestion through its use as a coating
in metallic and paperboard food containers fCal/EPA. 19991. tert-Butanol has been detected in
food, namely beer and chickpeas, and identified in mother's milk (HSDB. 20071. Indirect exposure
to tert-butanol may also occur as a result of ingestion of MTBE or ETBE, as tert-butanol is a
metabolite of these compounds (NSF International. 20031.
Alternate human exposure pathways of tert-butanol include inhalation and, to a lesser
extent, dermal contact, f -1 Ui tanol inhalation exposure can occur due to the chemical's volatility
and release from industrial processes, consumer products and contaminated sites fHSDB. 20071.
Dermal contact is a viable route of exposure through handling consumer products containing
tert-butanol (NSF International. 20031.
2
http://geotracker.waterboards.ca.gov/
This document is a draft for review purposes only and does not constitute Agency policy.
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Assessments by Other Federal, State and International Health Agencies
Toxicity information on tert-butanol has been evaluated by the American Conference of
Governmental Industrial Hygienists fACGIH. 20121. the National Institute for Occupational Safety
and Health fNIOSH. 2007], the Occupational Safety and Health Administration fOSHA. 20061. and
Food and Drug Administration. The results of these assessments are presented in Appendix A of
the Supplemental Information. The California EPA carried out an expedited risk assessment for
tert-butanol in drinking water and calculated a cancer slope factor based on rat kidney tumors
observed in the NTP bioassays. It is important to recognize that these earlier assessments were
prepared for different purposes using different methods and could consider only the studies that
were available at the time.
This document is a draft for review purposes only and does not constitute Agency policy.
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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. 19831. IRIS
assessments cover the hazard identification
and (.lose-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
in air, water, soil, or sediment. Exceptions are
chemicals currently used exclusively as
pesticides, ionizing and non-ionizing
radiation, and criteria air pollutants listed
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 lor 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 lor adverse effects. Other agents
58 may also lie 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
75 studies and analytical approaches that might
76 contribute to their resolution.
77 Step 1. Development of a draft
78 Toxicological Review. The draft
79 assessment considers all pertinent
80 publicly available studies and applies
81 consistent criteria to evaluate study
82 quality, identify health effects, identify
83 mechanistic events and pathways,
This document is a draft for review purposes only and does not constitute Agency policy.
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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 El'A releases a dm ft for
external peer review. The peer review
meeting is open to the public and includes
time lor oral public comments. The peer
reviewers assess whether the evidence
has been assembled and evaluated
accordin» 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.
Toxicological Review of tert-Butyl Alcohol
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
f h 11 p: / / w w w. ep a. go v/ir is/1.
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. I-I'A. 2005a. b, 2000b. 1998b. 1996.
1')') 1 b. l'J86a. b) and other methods fU.S.
I-I'A. 2012a. b, 2011. 2006a. b, 2002.
l')')-l). 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.
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://hero.epa.gov/). Searches for
information on mechanisms of toxicity are
inherently specialized and may include
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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 Act or 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) l<>
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 lor subsequent
modeling.
In assessments of chemical mixtures.
mixture studies are preferred lor their ability
to reflect interactions among components.
The literature search seeks, in decreasing
order ol preference fU.S. EPA. 2f)f)f)h. §2.2:
1986b. §2.1):
Studies ol 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.
Ideological 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.
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
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Toxicological Review of tert-Butyl Alcohol
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.
Short-duration studies involving animals
or humans may provide toxicokinetic or
mechanistic information.
For developmental toxicity and
reproductive toxicity, irreversible effects may
result from a brief exposure (.luring a critical
period of development. Accordingly,
specialized study designs are used for these
effects fU.S. EPA. 2006b. 19Wh. 1996.
1991b).
4. Evaluating the quality of
individual studies
After the subsets of pcrli ncnl
epidemiologic and experimental studies have
been selected from the literature.- searches,
the assessment evaluates the quality of each
individual study. This (.¦valuation considers
the design, methods, conduct, and
documentation of each study, but not
whether the results are positive, negative, or
null. The objective is to identify the stronger,
more informative studies based on a uniform
evaluation of quality characteristics across
studies of similar design.
4.1. Evaluating the quality of
epidemiologic studies
The assessment evaluates design and
methodological aspects that can increase or
decrease the weight given to each
epidemiologic study in the overall evaluation
riJ.S. EPA. 2005a. 1998b. 1996.1994.1991b):
Documentation of study design,
methods, population characteristics,
and results.
Definition and selection of the study
group and comparison group.
Ascertainment of exposure to the
chemical or mixture.
- Ascertainment of disease or health
effect
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.
Measurement error (can lead to
misclassification of exposure, health
outcomes, and other factors) and
other types of information bias.
Potential confounding and other
sources of bias addressed in the study
design or in the analysis of results.
The basis for consideration of
confounding is a reasonable
expectation that the confounder is
related to both exposure and outcome
and is sufficiently prevalent to result
in bias.
For developmental toxicity, reproductive
toxicity, neurotoxicity, and cancer there is
further guidance on the nuances of evaluating
epidemiologic studies of these effects (U.S.
EPA. 2005a. 1998b. 1996.1991bl.
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Toxicological Review of tert-Butyl Alcohol
4.2. Evaluating the quality of
experimental studies
The assessment evaluates design and
methodological aspects that can increase or
decrease the weight given to each
experimental animal study, in-vitro study, or
human clinical study (U.S. EPA. 2005a. 1998b.
1996. 1991b). Research involving human
subjects is considered only if conducted
according to ethical principles.
Documentation of study design,
animals or study population, methods,
basic data, and results.
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 (.losing
regimen (including age at (.-xposn ix*)
and their adequacy to elicit adverse
effects, including latent effects.
Sample sizes and statistical power to
detect dose-related differences or
trends.
Ascertainment of survival, vital signs,
disease or effects, and cause of death.
Control of other variables that could
influence the occurrence of effects.
The assessment uses statistical tests to
evaluate whether the observations may be
due to chance. The standard lor determining
statistical significance of a response is a trend
test or comparison of outcomes in the
exposed groups against those of concurrent
controls. In some situations, examination of
historical control data from the same
laboratory within a few years of the study
may improve the analysis. For an uncommon
effect that is not statistically significant
compared with concurrent controls, historical
controls may show that the effect is unlikely
to be due to chance. For a response that
appears significant against a concurrent
control response that is unusual, historical
controls may offer a different interpretation
fU.S. EPA. 2005a. §2.2.2.1.31
For developmental toxicity, reproductive
toxicity, neurotoxicity, and cancer there is
further guidance on the nuances of evaluating
experimental studies of these effects fU.S.
EPA. 2005a. 1998b. 1996. 1991b). In multi-
generation studies, agents that produce
developmental effects at doses that are not
toxic to the maternal animal are of special
concern. Effects that occur at doses
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
lie permanent fU.S. KPA. 1998b. §3.1.2.4.5.4:
1991b. §3.1.1.41..
4.3. Reporting study results
The assessment uses evidence tables to
|iresent the design and key results of
pertinent studies. There may be separate
tallies lor 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.
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Toxicological Review of tert-Butyl Alcohol
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.
Positive, negative, and null results are given
weight according to study quality.
Causal inference involves scientific
judgment, and the considerations are
nuanced and complex. Several health
agencies have developed frameworks for
causal inference, among them the U.S.
Surgeon General fCDC. 2004: HEW. l'Kvl). tin.'
International Agency for Research on Cancer
(IARC. 2006). the Institute of Medicine (l()M.
20081. and the EPA C2010.S1.6: 2005a. S2.5).
Although developed lor different purposes,
the frameworks art.1 similar in nature and
provide an established structure and
language for causal inference. Each considers
aspects of an association llial suggest
causation, discussed liy Hill (Mill. l'K).r>) and
elaborated liy Kolhman and Greenland
fRothman and Greenland. 1998). and U.S. El'A
(200.r>a. §2.2.1.7: 1994. Appendix C).
Strength of association: The finding of a
large relative risk with narrow confidence
intervals strongly suggests that an
association is not due to chance, bias, or
other factors. Modest relative risks,
however, may reflect a small range of
exposures, an agent of low potency, an
increase in an effect that is common,
exposure misclassification, or other
sources of bias.
Consistency of association: An inference of
causation is strengthened if elevated risks
are observed in independent studies of
different populations and exposure
scenarios. Reproducibility of findings
constitutes one of the strongest
46 arguments for causation. Discordant
47 results sometimes reflect differences in
48 study design, exposure, or confounding
49 factors.
50 Specificity of association: As originally
51 intended, this refers to one cause
52 associated with one effect. Current
53 understanding that many agents cause
54 multiple effects and many effects have
55 multiple causes make this a less
56 informative aspect of causation, unless
57 the effect is rare or unlikely to have
58 ni ii I Li pic- causes.
59 Temporal relationship: A causal
60 interpretation requires that exposure
61 precede development of the effect.
62 Biologic gradient (exposure-response
63 relationship): Kxposure-response
64 relationships strongly suggest causation.
65 A monotonic increase is not the only
66 pattern consistent with causation. The
67 presence of an exposure-response
68 gradient also weighs against bias and
69 co n rounding as the source of an
70 association.
71 Biologic plausibility: An inference of
72 causation is strengthened by data
73 demonstrating plausible biologic
74 mechanisms, if available. Plausibility may
75 reflect subjective prior beliefs if there is
76 insufficient understanding of the biologic
77 process involved.
78 Coherence: An inference of causation is
79 strengthened by supportive results from
80 animal experiments, toxicokinetic studies,
81 and short-term tests. Coherence may also
82 be found in other lines of evidence, such
83 as changing disease patterns in the
84 population.
85 "Natural experiments": A change in
86 exposure that brings about a change in
87 disease frequency provides strong
88 evidence, as it tests the hypothesis of
89 causation. An example would be an
90 intervention to reduce exposure in the
91 workplace or environment that is
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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 fGuvatt et al..
2008b: Guvattetal.. 2008a).
5.2. Evaluating evidence in humans
For each effect, the assessment evaluates
the evidence from the epidemiologic studies
as a whole. The objective is l<> detenuino
whether a credible association has lieen
observed and, if so, whether that association
is consistent with causation. In doing this, the
assessment explores alternative explanations
(such as chance, Mas, 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 ma}' 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.
46 Suggestive epidemiologic evidence of an
47 association consistent with causation:
48 The evidence suggests a causal
49 association but chance, bias, or
50 confounding cannot be ruled out as
51 explaining the association.
52 Inadequate epidemiologic evidence to infer
53 a causal association: The available
54 studies do not permit a conclusion
55 regarding the presence or absence of an
56 association.
57 Epidemiologic evidence consistent with no
58 causal association: Several adequate
59 studies cov ering the full range of human
60 exposures and considering susceptible
61 populations, and for which alternative
62 explanations such as bias and
63 confounding can lie ruled out, are
64 mutually consistent in not finding an
65 association.
66 5.3. Evaluating evidence in animals
67 For each effect, the assessment evaluates
68 the evidence from the animal experiments as
69 a whole to determine the extent to which they
70 indicate a potential for effects in humans.
71 Consistent results across various species and
72 strains increase confidence that similar
73 results would occur in humans. Several
74 concepts discussed by Hill (Hill. 19651 are
75 pertinent to the weight of experimental
76 results: consistency of response, dose-
77 response relationships, strength of response,
78 biologic plausibility, and coherence fU.S. EPA.
79 2005a. §2.2.1.7: 1994. Appendix CI
80 In weighing evidence from multiple
81 experiments, U.S. EPA f2005a. §2.51
82 distinguishes:
83 Conflicting evidence (that is, mixed positive
84 and negative results in the same sex and
85 strain using a similar study protocol)
86 from
87 Differing results (that is, positive results and
88 negative results are in different sexes or
89 strains or use different study protocols).
This document is a draft for review purposes only and does not constitute Agency policy.
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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
example, physiologic or metabolic differences
across test systems) or methodological
differences (for example, relative sensitivity
of the tests, differences in dose levels,
insufficient sample size, or timing of dosing or
data collection).
It is well established that there are critical
periods for some developmental and
reproductive effects (U.S. EPA. 2006b. 2005a.
b, 1998b. 1996. 1991b). Accordingly, the
assessment determines whether critical
periods have been adequately investigated.
Similarly, the assessment determines
whether the database is adequate to evaluate
other critical sites and effects.
In evaluating evidence of genetic toxicity:
Demonstration of gene mutations,
chromosome aberrations, or
aneuploidy in humans or
experimental mammals (in vivo)
provides the strongest evidence.
- This is followed bv positive- results in
lower organisms or in cultured cells
[in vitro) or for other genetic events.
Negative results carry less weight,
partly because they cannot exclude
the possibility of effects in oilier
tissues flARC. 2006).
For germ-cell mutagenicity, The EPA has
defined categories of evidence, ranging from
positive results ul human germ-cell
mutagenicity to negative results lor all effects
of concern (U.S. EPA. 19t>(ia. $2./i).
5.4. Evaluating mechanistic data
Mechanistic data can be useful in
answering several questions.
- The biologic plausibility of a causal
interpretation of human studies.
- The generalizability of animal studies
to humans.
- The susceptibility of particular
populations or lifestages.
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.
Tosicodynamic 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. §2.5).
The assessment addresses several
questions about each hypothesized mode of
action fU.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.
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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 l<>
identify populations and lifestages that
might be susceptible to their occurrence.
Quantitative differences may result in
separate toxicity values lor susccplihlo
populations or lifestages.
The assessment discusses the likelihood
that an agent operates through multiple
modes of action. Ail uneven lev el 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 f(iuvatt etal.. 2008al.
For cancer, the assessment evaluates
evidence of a mutagenic mode of action to
guide extrapolation to lower (.loses 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
relationships to mutagenic carcinogens fU.S.
EPA. 2005a.§2.3.51.
Toxicological Review of tert-Butyl Alcohol
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. 1983). 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, liias, 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
database that includes negative results in
other species.
Inadequate information to assess
carcinogenic potential: No other
descriptors apply. Conflicting evidence can
be classified as inadequate information if
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all positive results are opposed by
negative studies of equal quality in the
same sex and strain. Differing results,
however, can be classified as suggestive
evidence or as likely to be carcinogenic.
Not likely to be carcinogenic to humans:
There is robust evidence for concluding
that there is no basis for concern. There
may be no effects in both sexes of at least
two appropriate animal species; positive
animal results and strong, consistent
evidence that each mode of action in
animals does not operate in humans; or
convincing evidence that effects are not
likely by a particular exposure route or
below a defined dose.
Multiple descriptors may be used if there
is evidence that carcinogenic effects differ by
dose range or exposure route fU.S. EPA.
2005a. §2.51.
Another example of standard descriptors
comes from the EPA's Integrated Science
Assessments, which evaluate causation for
the effects of the criteria pollutants in
ambient air fU.S. EPA. 2010. Sl.ol.
Causal relationship: Sufficient evidence to
conclude that there is a causal
relationship. Observational studies
cannot be explained by plausible
alternatives, or they are supported by
other lines of evidence, for example,
animal studies or mechanistic
information.
Likely to be a causal relationship: Sufficient
evidence that a causal relationship is
likely, but impo riant uncertainties
remain. For example, observational
studies show an association but co-
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
lev el ol exposure.
The KI'A is investigating and may on a
trial basis use these or other standard
descriptors to characterize the overall weight
of the evidence for e fleets 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
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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 I In.- reasons lor not
analyzing other groups ol studies. As a check
on the selection of studies for dose-response
analysis, the EPA asks peer reviewers l<>
identify studies thai won.' not adequately
considered.
7. Deriving toxicity values
7.1. General framework for dose-
response analysis
The EPA uses a two-step approach that
distinguishes analysis of I In.- observed dose-
response data from inferences about lower
doses (U.S. EPA. 2005a. §3).
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).
Toxicological Review of tert-Butyl Alcohol
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 ol 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.
200.r>a. §3.2.6: 2000b. H-IA).
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
il they can lie supported by robust data.
7.2. Modeling dose to sites of biologic
effects
The preferred approach for analysis of
(.lose 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
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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
effect occurs at the site of first contact
or after systemic circulation (U.S. El'A.
2012a: 1994. §3).
It can be informative to convert doses
across exposure routes. If this is done, the
assessment describes the underlying (.lata,
algorithms, and assumptions (M.S. li lJA.
2005a. §3.1.4).
In the absence of study-specific (.lata on,
for example, intake rates or hotly weight, the
EPA has developed recommended values for
use in dose-response analysis (U.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
Toxicological Review of tert-Butyl Alcohol
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. §3.2.2).
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://u'U'U'.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 (M.S. EPA. 2012b). Additional
judgment or alternative analyses are used if
the procedure fails to yield reliable results,
lor example, if the fit is poor, modeling may
he restricted to the lower doses, especially if
there is competing toxicity at higher doses
f 11.S. r:iJA. 2005a. §3.2.31.
Modeling is used to derive a point of
departure (U.S. EPA. 2012b: 2005a. §3.2.4).
(See Section 7.6 for alternatives if a point of
departure cannot be derived by modeling.):
If linear extrapolation is used,
selection of a response level
corresponding to the point of
departure is not highly influential, so
standard values near the low end of
the observable range are generally
used (for example, 10% extra risk for
cancer bioassay data, 1% for
epidemiologic data, lower for rare
cancers).
For nonlinear approaches, both
statistical and biologic considerations
are taken into account.
For dichotomous data, a response
level of 10% extra risk is generally
used for minimally adverse effects,
5% or lower for more severe effects.
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For continuous data, a response level
is ideally based on an established
definition of biologic significance. In
the absence of such definition, one
control standard deviation from the
control mean is often used for
minimally adverse effects, one-half
standard deviation for more severe
effects.
The point of departure is the 95% lower
bound on the dose associated with the
selected response level.
7.4. Extrapolating to lower doses and
response levels
The purpose of extrapolating to lower
doses is to estimate responses at exposures
below the observed data. Low-dose
extrapolation, typically used for cancer data,
considers what is known about modes of
action fU.S. EPA. 2005a. §3.3.1 and §3.3.2).
1) If a biologically based model has been
developed and validated for the agent,
extrapolation may use I Ik- lilted model
below the observed range if significant
model uncertainly can be ruled mil willi
reasonable confidence.1.
2) Linear extrapolation is used il Ihc dose-
response curve is expected l<> haw a
linear component below the point of
departure. This includes:
Agents or their metabolites that are
DNA-reactive and have direct
mutagenic activ ity.
- 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.
Toxicological Review of tert-Butyl Alcohol
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) I'olh 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
estimating the response at doses where a
high-dose mode of action would be less
important.
II linear extrapolation is used, the
assessment develops a candidate slope factor
or unit risk Ibr each suitable data set. These
results are arrayed, using common dose
metrics, to show the distribution of relative
potency across various effects and
experimental systems. The assessment then
derives or selects an overall slope factor and
an overall unit risk for the agent, considering
the various dose-response analyses, the study
preferences discussed in Section 6, and the
possibility of basing a more robust result on
multiple data sets.
7.5. Considering susceptible
populations and lifestages
The assessment analyzes the available
information on populations and lifestages
that may be particularly susceptible to each
effect. A tiered approach is used fU.S. EPA.
2005a. S3.51.
1) If an epidemiologic or experimental study
reports quantitative results for a
susceptible population or lifestage, these
data are analyzed to derive separate
toxicity values for susceptible individuals.
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2) If data on risk-related parameters allow
comparison of the general population and
susceptible individuals, these data are
used to adjust the general-population
toxicity values for application to
susceptible individuals.
3) In the absence of chemical-specific data,
the EPA has developed age-dependent
adjustment factors for early-life exposure
to potential carcinogens that have a
mutagenic mode of action. There is
evidence of early-life susceptibility to
various carcinogenic agents, but most
epidemiologic studies and cancer
bioassays do not include early-life
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 susccpli hlc
subgroups) lhal is likely lo he without an
appreciable risk ol''nilverse health effects over
a lifetime (M.S. EPA. 2002. §4.21 Reference
values are typically calculated lor effects
other than cancer and lor 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
flJ.S. EPA. 1998b. 1996.1991b).
To account for uncertainty and variability
in the derivation of a lifetime human
Toxicological Review of tert-Butyl Alcohol
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
lor variation in susceptibility across the
human population and the possibility that
the available data may not be
representative of individuals who are
most susceptible lo the effect A factor of
10 is generally used to account for this
variation. This factor is reduced only if
the point of departure is derived or
adjusted specifically for susceptible
individuals (not for a general population
lhal includes both susceptible and non-
susceptible individuals) (U.S. EPA.
2002. §4.4.5: 1998b. §4.2: 1996. §4:
lt)')4.§4.3.t).l: 1991b. §3.41.
Aninial-to-human extrapolation. If animal
results are used to make inferences about
humans, the assessment adjusts for cross-
species differences. These may arise from
differences in toxicokinetics or
toxicodynamics. Accordingly, if the point
of departure is standardized to equivalent
human terms or is based on toxicokinetic
or dosimetry modeling, a factor of 101/2
(rounded to 3) is applied to account for
the remaining uncertainty involving
toxicokinetic and toxicodynamic
differences. If a biologically based model
adjusts fully for toxicokinetic and
toxicodynamic differences across species,
this factor is not used. In most other
cases, a factor of 10 is applied (U.S. EPA.
2011: 2002. §4.4.5: 1998b. §4.2: 1996. §4:
1994. §4.3.9.1: 1991b. §3.41.
Adverse-effect level to no-observed-
adverse-effect level. If a point of
departure is based on a lowest-observed-
adverse-effect level, the assessment must
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infer a dose where such effects are not
expected. This can be a matter of great
uncertainty, especially if there is no
evidence available at lower doses. A
factor of 10 is applied to account for the
uncertainty in making this inference. A
factor other than 10 may be used,
depending on the magnitude and nature
of the response and the shape of the dose-
response curve fU.S. EPA. 2002. §4.4.5:
1998b. §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 he-
used, depending on the duration of the
studies and the nature of the response
flJ.S. EPA. 2002. §4.4..r>: l')')Hh. §4.2: 1994.
§4.3.9.11.
Incomplete chitiibiise. II an incomplete
database raises concern that further
studies might identity a more sensitive
effect, organ system, or lileslage, the
assessment may apply a database
uncertainty factor (U.S. lil'A. 2002. §4.4.5:
1998b. §4.2: 1996. §4: 1')')¦!. §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 (U.S. EPA. 2002. §4.4.5).
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
Toxicological Review of tert-Butyl Alcohol
occur across a range of exposures (U.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
studies, the most sensitive outcome, or a
clustering of values). By providing these
organ/system-specific reference values, IRIS
assessments facilitate subsequent cumulative
risk assessments that consider the combined
effect of multiple agents acting at a common
site or through common mechanisms (NRC.
20091.
The assessment then selects an overall
reference dose ami an overall reference
concentration for the agent to represent
lifetime human exposure levels where effects
are not anticipated to occur. This is generally
the most sensitive organ/system-specific
reference value, though consideration of
study quality and confidence in each value
may lend to a different selection.
7.7. Confidence and uncertainty in the
reference values
The assessment selects a standard
descriptor to characterize the level of
confidence in each reference value, based on
the likelihood that the value would change
with further testing. Confidence in reference
values is based on quality of the studies used
and completeness of the database, with more
weight given to the latter. The level of
confidence is increased for reference values
based on human data supported by animal
data flJ.S. EPA. 1994. §4.3.9.21.
High confidence: The reference value is not
likely to change with further testing,
except for mechanistic studies that might
affect the interpretation of prior test
results.
Medium confidence: This is a matter of
judgment, between high and low
confidence.
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Toxicological Review of tert-Butyl Alcohol
Low confidence: The reference value is
especially vulnerable to change with
further testing.
These criteria are consistent with
guidelines for systematic reviews that
evaluate the quality of evidence. These also
focus on whether further research would be
likely to change confidence in the estimate of
effect (Guvatt etal.. 2008b).
All assessments discuss the significant
uncertainties encountered in the analysis.
The EPA provides guidance on
characterization of uncertainty (U.S. EPA.
14 2005a. §3.61. For example, the discussion
15 distinguishes model uncertainty (lack of
16 knowledge about the most appropriate
17 experimental or analytic model) and
18 parameter uncertainty (lack of knowledge
19 about the parameters of a model).
20 Assessments also discuss human variation
21 (interpersonal differences in biologic
22 susceptibility or in exposures that modify the
23 effects of the agent).
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27
EXECUTIVE SUMMARY
Occurrence and Health Effects
/l'/MUiUiiioI (.Iocs not occur naturally, hut it is produced by humans for
multiple purposes, such as a solvent lor paints, a denaluranl lor elhanol and several
other alcohols, an octane booster in gasoline, a dehydrating agent, and the
manufacture of flotation agents, fruit essences, and perfumes. ^'/MUitanol is also a
primary metabolite of methyl tort-butyl ether (MTI'li) and ethyl tort-butyl ether
(l-Tlil-). Exposure to {(.'//-butanol primarily occurs through breathing air containing
fiVf-buUinol vapors, as well as consuming contaminated water (or breast milk) or
foods. Kxposure may also occur through direct skin contact.
Animal studies demonstrate that chronic oral exposure to {(.'//-butanol is
associated with kidney and thyroid effects. Dev elopmental effects (e.g., reduced letal
v iability) have been observed in short-term exposure to high levels of {c/7-bulanol
(via oral or inhalation exposure) in animals. No chronic inhalation exposure studies
have been conducted. There is suggestive evidence that
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Toxicological Review of tert-Butyl Alcohol
hyperplasia observed after 2 years of oral exposure. Additionally, increased suppurative
inflammation was noted in females after 2 years of oral exposure. Mode of action analysis
determined that male rat kidney effects were not mediated by a2U-globulin, and these effects are
concluded to be relevant for human health hazard assessment.
Oral Reference Dose (RfD) for Effects Other Than Cancer
Kidney toxicity, represented by kidney transitional epithelial hyperplasia, was chosen as the
basis for the proposed overall oral reference dose (RfD) (see Table ES-1), as it was the only
noncancer endpointfor which there is credible evidence ol an association with tert-butanol
exposure. The chronic study by NTP (19951 and the observed kidney effects were used to derive
the RfD. The endpoint of transitional epithelial hyperplasia was selected as the critical effect due to
its consistency in both sexes, its specificity and its sensitivity as an indicator of kidney toxicity, and
the observed dose-response relationship of effects across dose groups, lienchmark dose (BMD)
modeling was utilized to derive the BMDLioo/0 of 1 (> mg/kg-day. The BMD I, was converted to a
human equivalent dose using body weight^/'1 scaling, and this value of 3.84 mg/kg-day was used as
the point of departure (POD) for RfD derivation (U.S. l i lJA. 2011).
The proposed overall RfD was calculated by dividing the I'OD for kidney transitional
epithelial hyperplasia by a composite uncertainly factor (UF) ol 30 to account for the extrapolation
from animals to humans (3) and lor inlerindividual 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 incidence of kidney
transitional epithelial
hyperplasia
1 X 10
Chronic
HIGH
Proposed
overall RfD
Increased incidence of kidney
transitional epithelial
hyperplasia
1 x 10 1
Chronic
HIGH
Effects Other Than Cancer Observed Following Inhalation Exposure
EPA identified kidney effects as a human hazard of tert-butanol exposure. Both absolute
and relative kidney weights were increased in male and female rats. There was an increase in
nephropathy severity in male rats, which supported the increase in kidney weights. No available
human studies evaluated the effects of inhalation exposure. Mode of action analysis determined
that male rat kidney effects were not mediated by c^u-globulin, and these effects are concluded to
be relevant for human health hazard assessment
This document is a draft for review purposes only and does not constitute Agency policy.
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Inhalation Reference Concentration (RfC) for Effects Other Than Cancer
Kidney toxicity, represented by transitional epithelial hyperplasia, was chosen as the basis
for the proposed inhalation reference concentration (RfC) (see Table ES-2), as it was the only
noncancer endpoint for which there is credible evidence of an association with tert-butanol
exposure. The chronic oral exposure study in rats fNTP. 19951 was used to derive the overall RfC.
A PBPK model for tert-butanol in rats was developed internally, and route-to-route extrapolation
was used to derive equivalent inhalation PODs. The POD adjusted for the human equivalent
concentration (HEC) was 26.1 mg/m3 and based on transitional epithelial hyperplasia.
The RfC was calculated by dividing the POD by a composite I ip of 30 to account for
toxicodynamic differences between animals and humans (3) and inte [-individual 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 incidence of kidney
transitional epithelial
hyperplasia
9 x 10
Chronic
HIGH
Proposed
overall RfC
Increased incidence of kidney
transitional epithelial
hyperplasia
9 x 10"1
Chronic
HIGH
Evidence for Human Carcinogenicity
Milder KI'A's Caidelincs for Carcinogen l\isl< Assessment (U.S. EPA. 2005a). the database for
tert-butanol provides "suggestive evidence of carcinogenic potential." Human data are not available
to assess the carcinogenic potential of tert-butanol. In 2-year studies in F344 rats and B6C3Fi mice,
male rats exhibited dose-related increases in renal tubule adenoma and combined renal tubule
adenoma or carcinoma. Although (.lata support a2U-globulin deposition in the kidney of male rats,
there is insufficient evidence to support this as the only or primary mechanism for renal tumor
development in male rats. Therefore, the renal tumors are considered relevant to humans.
However, the observed renal tumors were predominantly benign, only occurred in a single
sex/species combination, and were not observed in studies that exposed the same strain of rat to
ETBE, which is rapidly metabolized to tert-butanol. In addition, a statistically significant increase in
the incidence of thyroid follicular cell adenoma was observed in a 2-year drinking water study in
female mice (NTP. 1995). These tumors were all benign and only a single sex/species combination
was affected. There are no studies examining the carcinogenic potential of tert-butanol after
inhalation exposure in animals. However, internal tumors developed after oral exposure and may
occur regardless of exposure route, as blood concentrations were found to be similar after oral or
This document is a draft for review purposes oniy and does not constitute Agency poiicy.
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Toxicological Review of tert-Butyl Alcohol
inhalation exposures. Genotoxicity data for tert-butanol are inconclusive, tert-Butanol was negative
in a variety of genotoxicity assays in different cell systems including gene mutations, sister
chromatid exchanges, micronucleus formation and chromosomal aberrations. However, DNA
adducts in male Kunming mice and DNA damage in human HL-60 leukemia cells have been
observed. Overall, the cancer descriptor "suggestive evidence of carcinogenic potential" is
plausible, as some concern is raised by the positive evidence of predominantly benign renal tumors
in male rats and benign thyroid tumors in female mice.
Quantitative Estimate of Carcinogenic Risk from Oral Exposure
Lifetime oral exposure to tert-butanol has been associated with increased renal tubule
adenomas and carcinoma in male F344 rats, increased thyroid follicular cell adenomas in female
B6C3Fi mice, and increased thyroid follicular cell adenomas and carcinomas in male B6C3Fimice.
The NTP (19951 study in rats and mice was the only available study for dose-response analysis. The
study included histological examinations for tumors in many different tissues, c< intained three
exposure levels and controls, contained adequate numbers of animals per dose group
(~50/sex/group), treated animals for up to 2 years, and included detailed reporting of methods
and results.
Although tert- butanol was considered to have "suggestive evidence of carcinogenic
potential," EPA concluded that the main study was well-conducted and quantitative analysis maybe
useful for providing a sense ol the magnitude of potential carcinogenic risk. For renal tumors, two
slope factors were deriv ed lor this (.Midpoint from the NTI' (1995 ) bioassay: one based on the
original reported incidences and one based on the I lard et al. f20111 reanalysis. The two estimates
differed by less than 20%, and rounded to the same number atone significant figure. However, the
Hard et al. (2011) reanalysis is considered preferable, as it is based on a Pathology Working Group
(PWG) analysis. A slope factor was also derived for thyroid tumors in female mice. The modeled
tert-bulanol I'ODs were scaled to I IKDs according to EPA guidance by converting the BMDLio on the
basis of (body weight)3/4 scaling (M.S. KI'A. 2011. 2005a). Using linear extrapolation from the
BMDLio, a human equivalent oral slope factor was derived (slope factor = 0.1/BMDLio). The more
sensitive endpoint of renal tu mors was used because there is no data to support neither renal nor
thyroid tumors most relev ant to humans. The oral slope factor of 1 x 10"2 per mg/kg-day, based
on the renal tubule tumor response in male F344 rats.
Quantitative Estimate of Carcinogenic Risk from Inhalation Exposure
No chronic inhalation exposure studies to tert-butanol are available. However, through the
oral route of exposure, lifetime exposure has been associated with increased renal tubule adenomas
and carcinoma in male F344 rats, increased thyroid follicular cell adenomas in female B6C3Fi mice,
and increased thyroid follicular cell adenomas and carcinomas in male B6C3Fi mice. The NTP
f!9951 study in rats and mice was the only available study for dose-response analysis. The study
included histological examinations for tumors in many different tissues, contained three exposure
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
levels and controls, contained adequate numbers of animals per dose group (~50/sex/group),
treated animals for up to 2 years, and included detailed reporting of methods and results.
Although tert-butanol was considered to have "suggestive evidence of carcinogenic
potential," EPA concluded that the main study was well-conducted and quantitative analysis may be
useful for providing a sense of the magnitude of potential carcinogenic risk. Since the available
evidence for tert-butanol carcinogenicity is from a 2 year oral exposure, route-to-route
extrapolation of the oral BMDL was performed to derive an inhalation equivalent BMCL. The BMCL
was then converted to a human equivalent concentration (HEC) according to the RfC guidelines
(U.S. EPA. 19941 by multiplying the BMCL by the blood:gas partition coefficient ratio. Using linear
extrapolation from the resulting BMCLlo-hec, a human equivalent inhalation unit risk was derived
(inhalation unit risk = 0.1/BMCLio-hec). Extrapolation from the oral study results for renal tubule
adenoma or carcinoma in male F44 rats gives a unit risk of 2 x l()-;: per ing/m3, associated with
lifetime inhalation exposure to tert-butanol.
Susceptible Populations and Lifestages for Cancer and Noncancer
No data were identified to indicate susceptible popukilioiis or lifestages.
Key Issues Addressed in Assessment
Due to the observation of kidney tumors and noncancer toxicity following chronic exposure
to tert-butanol, an ev aluation of whether/(.'/ /-butanol caused « u-globulin nephropathy was
performed. The presence ol a^u-globulin in the hyaline droplets was confirmed in male rats by ct2U
immunohistochemical staining. Linear mineralization and tubular hyperplasia were reported in
male rats, though only in the chronic study. Other subsequent steps in the pathological sequence,
including necrosis, exfoliation, and granular casts, were either absentor not consistently observed
across subchronic or chronic studies. None of the observed effects occurred in female rats or in
either sex of mice. Because the available data supports the occurrence of at least two of the
subsequent steps in the pathological sequence, these data are sufficientto conclude that
a2u-globulin nephropathy is occurring in the kidney of male rats following tert- butanol exposure.
Thus, the noncancer lesions associated with c^u-globulin nephropathy are not considered relevant
to humans.
However, tumors develop at doses lower than some precursors of a2U-globulin
nephropathy, such as granular casts and tubular hyperplasia fHard et al.. 2011: NTP. 19951.
Therefore, there is insufficient evidence to support a conclusion that a2U-globulin nephropathy is
the sole or primary contributor to renal tumor development Because carcinogenic processes other
than a2u-globulin nephropathy cannot be ruled out, the renal tumors are considered relevant to
humans.
In addition, some of the observed renal lesions in rats following exposure to tert-butanol
are effects commonly associated with chronic progressive nephropathy (CPN), an age-related renal
disease of laboratory rodents that occurs spontaneously. While it has been argued that CPN in rats
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is not relevant to humans, it is acknowledged that the mechanism regulating CPN in rats is not
understood. Moreover, no key events for the exacerbation of CPN have been identified, so no mode
of action analysis can be performed. Therefore, kidney effects from tert-butanol exposure
associated with CPN are considered relevant to humans.
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. The only endpoint available from the
subchronic inhalation study fNTP. 19971 was increased kidney weights, which is a less-specific
endpoint compared to other endpoints available for analysis from the oral study fNTP. 19951. In
regards to the carcinogenic effects, the 2-year oral study f was the only study to evaluate
lifetime carcinogenic effects and was selected for roule-lo-roule extrapolation.
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LITERATURE SEARCH STRATEGY | STUDY
SELECTION AND EVALUATION
A literature search and screening strategy were used to identify literature characterizing
the health effects of tert-butanol. 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 notpertinent l<> an assessment of the health
effects of tert-butanol, 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 April 2014, using the keywords and limits
described in Table LS-1. The overall literature search approach is shown graphically in Figure LS-1.
An additional 7 citations were obtained using additional search strategies described in Table LS-2.
After electronically eliminating duplicates from the citations retrieved through these databases,
2,532 unique citations were identified.
The resulting 2,532 citations were screened into categories as presented in Figure LS-1
using the title, abstract, and/or lull text for pertinence to examine the health effects of tert-butanol
exposure.
• 12 references were identified as potential "Sources of Health Effects Data" and were
considered lor (.lata extraction to evidence tallies and exposure-response arrays.
• l'H> references were identified as "Supporting Studies;" these included 39 studies
describing physiologically-based pharmacokinetic (PBPK) models and other toxicokinetic
information, 70 studies providing genotoxicity and other mechanistic information, 1 human
case report, 73 not relevant exposure paradigms (including acute, dermal, eye irritation,
and injection studies), (> preliminary toxicity studies, and 7 physical dependency studies.
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 exposure. 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.
• 63 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.
• 2,261 references were identified as not being pertinent to an evaluation of the health effects
of tert-butanol and were excluded from further consideration (see Figure LS-1 for exclusion
categories).
The complete list of references and the sorting of these materials can be found on the
HERO website at http: //hero.epa.gov.
This document is a draft for review purposes only and does not constitute Agency policy.
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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 that could affect the interpretation of results and the overall contribution to the
evidence for determination of hazard potential. Some general questions that were considered in
evaluating experimental animal studies are presented in Table LS-3. Much of the key information
for conducting this evaluation can generally be found in the study's methods section and in how the
study results are 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 Iliat systematically
summarize the important information from each study in a standardized tabular format as
recommended by the NRC (20111. Twelve studies identified as "Sources of Health Effects Data"
were considered for extraction into evidence tables lor hazard identification in Chapter 1. Initial
review of studies found two studies to be publications of the NTP fl')').r>) (.lata prior to the release of
the final NTP report (Cirvello etal.. 1995: Lindamood etal.. 19921. One publication in the
"Supporting Studies" category also was based on data from the NTP report (Takahashi etal.. 19931.
There were differences between the published reports and tin.' final NTP report; therefore, the
finalized NTP f 19951 report was included in evidence tallies. Data from the remaining 10 studies in
the "Sources of Health Effects Data" category were extracted into evidence tables.
Supporting studies that contain pertinent information lor the toxicological review and
augment hazard identification conclusions, such as genotoxic and mechanistic studies, studies
describing the kinetics and disposition of tert-butanol absorption and metabolism, pilot studies,
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 (.lata base for fiVf-liuLinol is comprised of animal toxicity studies containing one 2-year
bioassay that employs oral exposures in rats and mice; two oral subchronic studies in rats and one
in mice; one inhalation subchronic study in rats and mice; a re-evaluation of the NTP (19951 rat
data; two oral developmental studies; two inhalation developmental studies; and one one-
generation reproductive study that also evaluates other systemic effects. Several acute and short
term studies (including an 18-day inhalation study and a 14-day study by NTP) using oral and
inhalation exposures were performed mostly in rats, but were grouped as supporting studies since
the database of chronic and subchronic rat studies was considered sufficient No cohort studies,
case-control studies, or ecological studies exist in the published literature. There was one case
report available. Health effect studies of gasoline and tert-butanol mixtures were not considered
pertinent to the assessment since the separate effects of the gasoline components could not be
determined; thus, these studies were excluded during the manual screen.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
1 The "Sources of Health Effects Data" were comprised entirely of studies performed in rats
2 and mice with drinking water, oral gavage, and inhalation exposures to tert-butanol. These 12
3 sources were conducted according to OECD Good Laboratory Practice (GLP) guidelines, presented
4 extensive histopathological data, and/or clearly presented their methodology; thus, these are
5 considered high quality. Preliminary, acute, and short-term studies contained information that
6 supported and did not differ qualitatively from the results of the >30 day exposure studies; thus,
7 these studies are not included in the evidence tables. Some of these shorter duration studies are
8 presented in the text of the Toxicological Review and are used in sections such as "Mechanistic
9 Evidence" to augmentthe discussion. A more detailed discussion of methodological concerns that
10 were identified will precede each endpoint evaluated in the hazard identification section.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review oftert-Butyl Alcohol
Excluded (not pertinent) {n=2,261)
83
Biodegredation/environmental fate
80
Chemical analysis/fuel chemistry
1,230
Other chemical/non ferf-butanol
86
Method of detection/exposure and

biological monitoring
666
Methodology/solvent
39
Not relevant species/matrix (e.g.,

amphibians, fish, etc.)
62
Abstract only/comment/society

abstracts
14
QSAR
1
Mixtures
<
Sources of Health Effects
Data(n=12)
0 Human health effects
studies
12 Animal studies
Supporting Studies (n=196)
39
PBPK/ADME
22
Genotoxicity
48
Other mechanistic

studies
1
Human case reports
73
Not relevant exposure

paradigms (e.g., dermal.

eye irritation, etc.)
6
Preliminary data
7
Physical dependency

studies
Secondary Sources of
Health Effects
Information (n=63)
37 Reviews/editorials
13 Other agency
assessments
13 Book chapter/section
1
2 Figure LS-1. Study selection strategy.
This document is a draft for review purposes only and does not constitute Agency policy,
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Toxicological Review of tert-Butyl Alcohol
Table LS-1. Details of the search strategy employed for tert-butanol
Database
(Search Date)
Keywords
Limits
PubMed
(12/20/2012)
(4/17/2014)
te/t-butanol OR 75-65-0[rn] OR "t-
butyl hydroxide" OR "2-methyl-2-
propanol" OR "trimethyl carbinol" OR
"t-butyl alcohol" OR te/t-butanol OR
"tert-butyl alcohol" OR tert-butyl
alcohol[mesh]
None
Web of Science
(12/20/2012)
(4/17/2014)
Topic = (te/t-butanol OR 75-65-0 OR
"t-butyl hydroxide" OR "2-methyl-2-
propanol" OR "trimethyl carbinol" OR
"t-butyl alcohol" OR "te/t-butanol"
OR "tert-butyl alcohol")
Refined by: Research Areas = (cell biology OR
respiratory system OR microscopy OR biochemistry
molecular biology OR gastroenterology hepatology OR
public environmental occupational health OR
oncology OR physiology OR cardiovascular system
cardiology or toxicology OR life sciences biomedicine
other topics OR hematology OR pathology OR
neurosciences neurology OR developmental biology)
Toxline (includes
TSCATS)
(1/11/2013)
(4/17/2014)
te/t-butanol OR 75-65-0 [rn] OR t-
butyl hydroxide OR 2-methyl-2-
propanol OR trimethyl carbinol OR t-
butyl alcohol OR te/t-butanol OR tert-
butyl alcohol OR tert-butyl alcohol
Not PubMed
TSCATS2
(1/4/2013)
(4/17/2014)
75-65-0
None
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
1 Table LS-2. Summary of additional search strategies for tert-butanol
Approach used
Source(s)
Date
performed
Number of additional references
identified
Manual search
of citations from
reviews
Review article: Mcgregor (2010).
Tertiary-butanol: A toxicological
review. Crit Rev Toxicol 40(8): 697-
727.
1/2013
5
Review article: Chen (2005). Amended
final report of the safety assessment
of t-butyl alcohol as used in
cosmetics." Int J Toxicol 24(2): 1-20.
1/2013
2
Manual search
of citations from
reviews
conducted by
other
international
and federal
agencies
IPCS (1987a). Butanols: Four isomers:
1-butanol, 2-butanol, tert-butanol,
isobutanol [WHO EHC], Geneva,
Switzerland: World Health
Organization.
1/2013
None
OSHA (1992). Occupational safety and
health guideline for te/t-butyl alcohol.
Cincinnati, OH: National Institute for
Occupational Safety and Health.
1/2013
None
2
3
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
1 Table LS-3. Questions and relevant experimental information for evaluation
2 of 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 timepoints
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
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Toxicological Review of tert-Butyl Alcohol
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 tert-butanol can
cause kidney effects in humans or animals. The database examining kidney effects following
tert-butanol exposure contains no human data, six studies performed in rats or mice, and one re-
evaluation of the rat data from NTP (19951. Studies employing short-term and acute exposures that
examined kidney effects are not included in the ev idence tables; however, they are discussed in the
text if they provide data to support mode of action or hazard identification. No methodological
concerns were identified that would lead one or more studies to lie considered less informative for
assessing human health hazard. A pathology working group (I lard etal., 20111 re-examined kidney
histopathology from the NTP T19951 13-week and 2-vear studios in rats to evaluate questions
involving MOAs for renal tubule development. All slides were analyzed in a blinded manner. Hard et
al. (20111 did report different incidences of adenomas or carcinomas compared with the original
NTP (19951 study; thus, these data were presented separately. Histopathological results from both
Hard and NTP will be considered for hazard identification.
fcrf-Fiutanol exposure resulted in a number of kidney effects after both oral (drinking
water) and inhalation exposure in both sexes of rats and mice. Kidney effects observed after oral
exposure (Talile l-l;Table 1-2; Figure 1-1) include increased kidney weight in female rats and in
male and female mice (13-week exposure), and kidney inflammation, kidney transitional epithelial
hyperplasia, and increased incidence and/or severity of kidney nephropathy in female rats (2-year
exposure) (NTP. l')')5). In a 2-vear oral exposure study in male rats, increased kidney weight,
increased hyaline droplets, kidney transitional epithelial hyperplasia, kidney mineralization, renal
tubule hyperplasia, and increased incidence and/or severity of kidney nephropathy were observed,
with some of these effects seen at earlier time periods (NTP. 19951. Other kidney effects in male
rats were observed in a 10-week oral exposure study (Acharva etal.. 1997: Acharva et al.. 19951.
No changes in clinical chemistry that would typically be indicative of kidney damage have been
observed with tert-butanol exposure. Although there were some changes in urinalysis parameters
(e.g., decreased urine volume and increased specific gravity), this was accompanied by reduced
water consumption and may not be related to an effect of kidney function.
The kidney is also the target organ for cancer effects (Table 1-3; Figure 1-1). Male F344 rats
had an increased incidence of renal tubule adenomas and combined renal tubule adenoma or
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Toxicological Review of tert-Butyl Alcohol
carcinoma in a 2-year oral bioassay (Hard etal.. 2011: NTP. 19951. The highest exposure group had
an increase in mortality, which may in part explain the apparent non-monotonicity in the observed
dose-response, in which the highest exposure group had a lower incidence of tumors than the
middle exposure group.
An Independent Pathology Working Group (PWG), sponsored by Lyondell Chemical
Company, re-evaluated the kidney changes in the NTP 2-year study fHard etal.. 20111. The PWG
consisted of senior pathologists with experience in chemically-induced nephrotoxicity and renal
neoplasia. In all cases, PWG members were blinded to treatment groups to preclude any possible
bias, and used guidelines published by the Society of Toxicologic Pathology. The PWG confirmed the
NTP findings of atypical tubule hyperplasia and renal tubule tumors in male rats at 2-years. In
particular, they reported very similar overall tumor incidences in I lie exposed groups. However,
the PWG evaluation of the control groups reported fewer renal tubule adenomas and carcinomas
than the original NTP study. As a result, based on the PWG evaluation, all treated groups had
statistically significant increases in renal tubule adenomas and carcinomas (combined) as
compared to controls. Additionally, the PWG considered fewer of the tumors lo he carcinomas as
compared to the original NTP study.
No chronic (2-year) inhalation exposure study is available, but minimal kidney effects were
observed in rats (mainly the males) alter /(.'//-butanol exposure by inhalation for 13 weeks at
concentrations ranging from 406-6,368 mg/m: fNTP. (Table 1 -1; Table 1-2; Figure 1-2).
Absolute kidney weights were elevated (9.8-11"..) in male rats exposed at >3,274 mg/m3 (not dose-
dependent); relative kidney weights were statistically elevated (~9'->u) in males at >3,274 mg/m3
and females at 6,368 mg/m :. Male rats exhibited an increase in the severity of chronic nephropathy
(characterized as number of foci of regenerativ e tulmles). Although the kidney effects were less
severe alter inhalation exposure, a direct comparison can only be made on the basis of internal
dose. ARCO (L983) found that blood levels of tert-butanol and its metabolites are equivalent after a
single oral (.lose of 350 mg/kg compared to a single 6-hour inhalation exposure to 6,164 mg/m3.
That would indicate, based on bolus exposures, that the inhalation exposures used in the NTP
(19971 study were in the range of the lower doses used in the NTP (19951 oral study. On the other
hand, based on I'I'I'K modeling, chronic exposure in the range of the NTP (19951 bioassay doses of
90-420 mg/kg-day lend to the same average blood concentration of tert-butanol as 6-hour/day, 5
day/week inhalation exposures to 860-4500 mg/m3, suggesting that the oral and inhalation
exposures in NTP (19951 and NTP (19971. respectively, overlap on the basis of internal dose.
Finally, the lack of either mortality or changes in body weight (both observed with oral exposure)
observed after the inhalation exposure suggests that a direct comparison cannot be made.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
1 Table 1-1. Changes in kidney weight in animals following exposure to
2 tert-butanol
Reference and study design
Results
Kidney weight (percent change as compared to control)
Lvondell Chemical Co. (2004)
Males

Sprague-Dawley rat;
Dose
Left absolute Left relative
Right absolute
Right relative
12/sex/treatment
(mg/kg-d)
weight
weight

weight
weight
Gavage 0, 64,160, 400, or
0
0
0

0
0
1,000 mg/kg-d

Males: 9 weeks beginning 4 weeks
64
+6
+8

+6
+8
prior to mating
160
+9
+14*

+6
+11*
Females: 4 weeks prior to mating

through PND21
400
+12*
+14*

+14*
+17*

1,000
+18*
+28*

+20*
+31*

Females

Dose
Left absolute Left relative
Right absolute
Right relative

(mg/kg-d)
weight
weight

weight
weight

0
0
0

0
0

64
-1
-2

+2
0

160
0
0

+1
0

400
+3
+2

+4
+2

1,000
+4
0

+7
+2
NTP (1995)
Males

Females

F344/N rat; 10/sex/treatment
Dose
Absolute
Relative
Dose
Absolute
Relative
Drinking water 0, 2.5, 5,10, 20,
(mg/kg-d)
weight
weight
(mg/kg-d) weight
weight
40 mg/mL
0
0
0
0
0
0
M: 0, 230, 490, 840, 1,520,

3,610a mg/kg-d
230
+12*
+19*
290
+19*
+17*
F: 0, 290, 590, 850, 1,560,

3,620a mg/kg-d
490
+17*
+26*
590
+16*
+15*
13 weeks

840
+16*
+32*
850
+29*
+28*

1,520
+26*
+54*
1,560
+39*
+40*

3,610
All dead
All dead
3,620
+36*
+81*
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
Table 1-1. Changes in kidney weight in animals following exposure to
tert-butanol (continued)
Reference and study design
Results
NTP (1995)

Males

Females

B6C3Fj mouse; 10/sex/treatment
Dose
Absolute
Relative

Dose
Absolute
Relative
Drinking water (0, 2.5, 5,10, 20,
(mg/kg-d)
weight
weight

(mg/kg-d)
weight
weight
40 mg/mL)
0
0
0

0
0
0
M: 0, 350, 640, 1,590, 3,940,

8,210a mg/kg-d
350
+1
+1

500
0
-3
F: 0, 500, 820, 1,660, 6,430,

11,620a mg/kg-d
640
+3
+2

820
-3
-1
13 weeks

1,590
+2
+8

1.660
+1
0

3,940
+6
+22*

6,430
+6
+15*

8,210
0
+48*

11,620
+12*
+35*
NTP (1995)

Males

Females

F344/N rat; 60/sex/treatment
Dose
Absolute
Relative

Dose
Absolute
Relative
(10/sex/treatment evaluated at 15
(mg/kg-d)
weight
weight

(mg/kg-d)
weight
weight
months)
0
0
0

0
0
0
Drinking water (0,1.25, 2.5, 5, or 10

mg/mL)
90
+4
+8

180
+8*
+14*
M: 0, 90, 200, or 420a mg/kg-d
200
+11
+15*

330
+18*
+21*

F: 0,180, 330, or 650 mg/kg-d

2 years
420
+7
+20*

650
+22*
+42*

Only animals sacrificed at 15 months were evaluated for organ weights. Organs were not

weighed in the 2-year mouse study

NTP (1997)

Males

Females

F344/N rat; 10/sex/treatment
Concentration
Absolute
Relative
Absolute
Relative
Analytical concentration: 0,134,
(mg/m )
weight

weight
weight
weight
272, 542, 1,080, or 2,101 ppm (0,
0
0

0
0
406, 824, 1,643, 3,273 or 6,368

mg/mB) (dynamic whole-body
406
+1

-4
-1
chamber)
824
-2

-1

0
+1
6 hr/d, 5 d/wk

13 weeks
1,643
+3

+4
+4
Generation method (Sonimist

Ultrasonic spray nozzle nebulizer),
3,273
+11*

+8*

+2
+2
analytical concentration and
6,368
+9.8*

+9*

+4
+9*
method were reported

This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
Table 1-1. Changes in kidney weight in animals following exposure to
tert-butanol (continued)
Reference and study design
Results
NTP (1997)

Males

Females

B6C3Fj mouse; 10/sex/treatment
Analytical concentration: 0,134,
Concentration
(mg/m3)
Absolute
weight
Relative
weight
Absolute
weight
Relative
weight
272, 542, 1,080, or 2,101 ppm (0,
0
0
0
0
0
406, 824, 1,643, 3,273 or 6,368

mg/mB) (dynamic whole-body
406
-6
-4
+1
-3
chamber)
6 hr/d, 5 d/wk
824
-1
+3
+5
+9
13 weeks
1,643
+4
+3
+1
-2
Generation method (Sonimist
Ultrasonic spray nozzle nebulizer),
3,273
-10
-3
0
+7
analytical concentration and
6,368
+3
+b
+3
+15*
method were reported

1 a The high-dose group had an increase in mortality.
2 * Statistically significant p < 0.05 as determined by the study authors.
3 Percentage change compared to control = (treated value - control value) 4- control value x 100.
4 Conversions from drinking water concentrations to mg/kg-d performed by study authors.
5 Conversion from ppm to mg/mB is 1 ppm = 3.031 mg/mB.
6
7 Table 1-2. Changes in kidney histopathology in animals following exposure to
8 tert-butanol
Reference and study design
Results
Acharva etal. (1997: 1995)
Wistar rat; 5-6 males/treatment
Drinking water (0 or 0.5%), 0 or
575 mg/kg-d
10 weeks
"T" tubular degeneration, degeneration of the basement membrane of the Bowman's
capsule, diffused glomeruli, and glomerular vacuolation (no incidences reported)
¦J, kidney glutathione (~40%)*
Lvondell Chemical Co. (2004)
Sprague-Dawley rat;
12/sex/treatment
Gavage 0, 64,160, 400, or
1,000 mg/kg-d
F0 males: 9 weeks beginning 4
weeks prior to mating
F0 females: 4 weeks prior to mating
through PND21
There were no changes in kidney histopathology observed.
9
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
Table 1-2. Changes in kidney histopathology in animals following exposure to
tert-butanol (continued)
Reference and study design
Results
NTP (1995)
Incidence (severity):

F344/N rat; 10/sex/treatment
Males

Females

Drinking water (0, 2.5, 5,10, 20, or
Dose

Dose

40 mg/mL)
M: 0, 230, 490, 840, 1,520,
(mg/kg-d)
Mineralization
Nephropathy
(mg/kg-d)
Mineralization
Nephropathy
3,610a mg/kg-d
0
0/10
7/10(1.0)
0
10/10(1.7)
2/10(1.0)
F: 0, 290, 590, 850, 1,560,

3,620a mg/kg-d
230
0/10
10/10(1.6*)
290
10/10 (2.0)
3/10(1.0)
13 weeks

490
2/10(1.5)
10/10(2.6*)
590
10/10 (2.0)
5/10(1.0)

840
8/10*(1.4)
10/10(2.7*)
850
10/10 (2.0)
7/10* (1.0)

1,520
4/10*(1.0)
10/10(2.6*)
1,560
10/10 (2.0)
8/10* (1.0)

3,610
4/10*(1.0)
7/10(1.1)
3,620
6/10(1.2)
7/10* (1.0)
NTP (1995)
B6C3Fj mouse; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or
40 mg/mL)
M: 0, 350, 640, 1,590, 3,940,
8,210a mg/kg-d
F: 0, 500, 820, 1,660, 6,430,
ll,620a mg/kg-d
13 weeks
Histopathology data for the 13-week study were not provided, but the kidney was
evaluated indicating that no changes in kidney histopathology were observed in the 13-
week study.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
Table 1-2. Changes in kidney histopathology in animals following exposure to
tert-butanol (continued)
Reference and study design
Results
NTP (1995)
F344/N rat; 60/sex/treatment
(10/sex/treatment evaluated at 15
months)
Incidence (severity):
Males
Dose
(mg/kg-d)
Mineralization
(interim)
Mineralization
(terminal)
Linear mineralization
(terminal)
Drinking water (0,1.25, 2.5, 5,10
mg/mL)
M: 0, 90, 200, 420a mg/kg-d
F: 0,180, 330, 650a mg/kg-d
2 years
0
90
200
420
1/10(1.0)
2/10(1.0)
5/10(1.8)
9/10* (2.3)
26/50(1.0)
28/50(1.1)
35/50(1.3)
48/50* (2.2)
0/50
5/50* (1.0)
24/50* (1.2)
46/50* (1.7)

Dose
(mg/kg-d)
Renal tubule
hyperplasia
(extended
evaluation)
Transitional
epithelium
hyperplasia
Nephropathy
severity

0
12/50(2.3)
25/50(1.7)
3.0

90
16/50(2.3)
32/50(1.7)
3.1

200
14/50(2.2)
36/50* (2.0)
3.1

420
23/50* (2.8)
40/50* (2.1)
3.3*

Females

Dose
(mg/kg-d)
Mineralization
Interim
Mineralization15
Terminal
Inflammation
(suppurative)
incidence

0
10/10 (2.8)
49/50 (2.6)
2/50

180
10/10 (2.9)
50/50 (2.6)
3/50

330
10/10 (2.9)
50/50 (2.7)
13/50*

650
10/10 (2.8)
50/50 (2.9)
17/50*

Dose
(mg/kg-d)
Renal tubule
hyperplasia
Transitional
epithelium
hyperplasia
Nephropathy
severity

0
0/50
0/50
1.6

180
0/50
0/50
1.9*

330
0/50
3/50 (1.0)
2.3*

650
1/50(1.0)
17/50*(1.4)
2.9*
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
Table 1-2. Changes in kidney histopathology in animals following exposure to
tert-butanol (continued)
Reference and study design
Results
NTP (1995)
B6C3Fj mouse; 60/sex/treatment
Drinking water (0, 5,10, or 20
mg/mL)
M: 0, 540,1,040, or 2,070a mg/kg-d
F: 0, 510,1,020, or 2,110 mg/kg-d
2 years
No changes in kidney related histopathology observed.0
NTP (1997)
F344/N rat; 10/sex/treatment
Analytical concentration: 0,134,
272, 542, 1,080, or 2,101 ppm (0,
406, 824, 1,643, 3,273 or
6,368 mg/mB) (dynamic whole-body
chamber)
6 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist
Ultrasonic spray nozzle nebulizer),
analytical concentration and
method were reported
Male
Concentration Average severity of
(ms/m3) chronic neohrooathv
0 1.0
406 1.4
824 1.4
1,643 1.6
3,273 1.9
6,368 2.0
Severity categories: 1= minimal, 2= mild. No results from statistical tests reported
NTP (1997)
B6C3Fj mouse; 10/sex/treatment
Analytical concentration: 0,134,
272, 542,1,080, or 2,101 ppm (0,
406, 824, 1,643, 3,273 or
6,368 mg/m3) (dynamic whole-body
chamber)
6 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist
Ultrasonic spray nozzle nebulizer),
analytical concentration and
method were reported
There were no kidney effects observed.
1 a The high-dose group had an increase in mortality.
2 b Linear mineralization not observed in female rats.
3 c Organs were not weighed in mice during the 2-year study. Relative organ weights refer to relative to body weight
4 * Statistically significant p < 0.05 as determined by the study authors.
5 Conversions from drinking water concentrations to mg/kg-d performed by study authors.
6 Conversion from ppm to mg/mB is 1 ppm = 3.031 mg/mB.
7
8
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1 Table 1-3. Changes in kidney tumors in animals following exposure to
2 tert-butanol
Reference and study design
Resu Its
NTP (1995)

Renal tubule

Male
Renal tubule
Renal tubule

adenoma (single
F344/N rat; 60/sex/treatment
Dose
adenoma
adenoma
Renal tubule
or multiple) or
(10/sex/treatment evaluated at 15
(mg/kg-d)
(single)
(multiole)
carcinoma
carcinoma
months)
Drinking water (0,1.25, 2.5, 5, or
0
7/50
1/50
0/50
8/50
10 mg/mL)
M: 0, 90, 200, or420a mg/kg-d
F: 0,180, 330, or 650a mg/kg-d
90
200
7/50
10/50
4/50
9/50*
2/50
1/50
13/50
19/50*
2 years
420
10/50
3/50
1/50
13/50
Renal tubule

Female
Renal tubule
Renal tubule

adenoma (single

Dose
adenoma
adenoma
Renal tubule
or multiple) or

(mg/kg-d)
(single)
(multiple)
carcinoma
carcinoma

0
0/50
0/50
0/50
0/50

180
0/50
0/50
0/50
0/50

330
0/50
0/50
0/50
0/50

650
0/50
0/50
0/50
0/50

Results do not include the animals sacrificed at 15 months.

Hard etal. (2011)

Renal tubule

Male
Renal tubule
Renal tubule

adenoma (single
reanalysis of the slides from male
Dose
adenoma
adenoma
Renal tubule
or multiple) or
rats in the NTP (1995) study (see
(mg/kg-d)
(single)
(multiple)
carcinoma
carcinoma
above)
0
3/50
1/50
0/50
4/50

90
9/50
3/50
1/50
13/50*

200
9/50
9/50
0/50
18/50*

420
9/50
3/50
1/50
12/50*
NTP (1995)
No changes in kidney-related tumors

B6C3Fj mouse; 60/sex/treatment

Drinking water (0, 5,10, or

20 mg/mL)

M: 0, 540,1,040, or 2,070a mg/kg-

F: 0, 510,1,020, or 2,110 mg/kg-d

2 years

3 a The high-dose group had an increase in mortality.
4 * Statistically significant p < 0.05 as determined by the study authors.
5 Conversions from drinking water concentrations to mg/kg-d performed by study authors.
6
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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
x = exposures at which all animals were dead and unable to be examined for the endpoint
Kidney
Weight
Absolute weight; M Rat; Reproductive (C)
Relative weight; M Rat; Reproductive (C)
Absolute weight; F Rat; Reproductive (C)
Relative weight; F Rat; Reproductive (Q
Absolute weight; M Rat; 13wk (D)
Relative weight; M Rat; 13wk (D)
Absolute weight; F Rat; 13wk (D)
Relative weight; F Rat; 13wk (D)
Absolute weight; M Mouse; 13wk (D)
Relative weight; M Mouse; 13wk (D)
Absolute weight; F Mouse; 13wk (D)
Relative weight; F Mouse; 13wk (D)
Absolute weight; M Rat; 15mo (D)
Relative weight; M Rat; 15mo (D)
Absolute weight; F Rat; ISmo (D)
Relative weight; F Rat; 15mo (D)
Kidney
Histopatholugy
Decreased glutathione; M Rat; lOwk [A)
Inflammation; F Rat; 2yr (D)
Nephropathy severity; M Rat; 13wk (D)
Nephropathy incidence; F Rat; 13wk (D)
Mineralization; M Rat; 13wk (0)
Mineralization; F Rat; 1.3wk (D)
Nephropathy severity; M Rat; 2yr(D)
Nephropathy severity; F Rat; 2yrf0)
Linear mineralization; M Rat; 2yr (D)
Interim/terminal mineralization; M Rat; 2yr(D)
Interim/terminal mineralization; F Rat; 2yr(D)
Transitional epithelium hyperplasia; M Rat; 2yr (D)
Transitional epithelium hyperplasia; F Rat; 2yr (D)
Renal tubular hyperplasia; M Rat; 2yr (D)
Renal tubule hyperplasia; F Rat; 2yr (D)
Kidney Rena' tubular adenoma or carcinoma; M Rat; 2yr (D)
Tumors Renal tubular adenoma or carcinoma; M Rat; 2yr [B)
Renal tubular adenoma or carcinoma; F Rat; 2yr (D)
Renal tubular adenoma or carcinoma; M Mouse; 2yr(D)
Renal tubular adenoma or carcinoma; F Mouse; 2yr (D)
10 100 1,000 10,000 100,000
Dose (mg/kg-day)
Sources: (A) Acharya etal. (1997; 1995); (B) Hard etal. 120111*: (C) I.vondRll Chemical Co. f 20041 (D) NTP f!99Sl: * reanalysis of NTP
fl9951
Figure 1-1. Exposure response array for kidney effects following oral
exposure to tert-butanol.
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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
Absolute weight; M Rat
Relative weight; M Rat
Absolute weight; F Rat
Relative weight. I ' Rai
Absolute ivlali\o woiuhl. \l Mouse
Absolute woiuhl. I' Mouse
Relative weight; F Mouse
~ B B-
~ B B-
B B B B-
B B B B-
100 1,000
Concentration (mg/m3)
~ B B B ~
~ B B B ~
-B
10,000
Source: NTP ft 9971
Figure 1-2. Exposure-response array of kidney effects following subchronic
inhalation exposure to tert-butanol (no chronic studies available).
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Mode of Action Analysis—Kidney Effects
Mode of Action Analysis for a?,,-globulin-associated nephropathy
Description of the hypothesized MOA
Several studies were identified that evaluated the role of a2U-globulin in tert-butanol-
induced renal tumor development (Borghoffetal.. 2001: Williams and Borghoff. 2001: Takahashi et
al.. 19931. ct2u- Globulin is a member of a large superfamily of low-molecular-weight proteins and
was first characterized in male rat urine. Such proteins have been detected in various tissues and
fluids of most mammals (including humans), but the particular isolorm of a2U-globulin commonly
detected in male rat urine is considered specific to the male ral.
The hypothesized sequence of a2u-globulin-associated nephropathy, as described by U.S.
EPA (1991a). is as follows. Chemicals that induce u u-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 ol exposure. Hyaline droplets are a normal
constitutive feature of the mature male rat kidney; they are particularly evident in the P2 segment
of the proximal tubule and contain u ¦„-»lobulin fU.S. El'A. !')')! a). Abnormal increases in hyaline
droplets have more than one etiology and can lie associated with the accumulation of different
proteins. As hyaline droplet deposition continues, single-cell necrosis occurs in the P2 segment
which leads to exfoliation of these cells into the tulmle lumen within f> (.lays of chemical exposure. In
response to the cell loss, cell proliferation is observed in the V2 segmentafter 3 weeks and
continues for the duration ol the exposure. Alter 2 or 3 weeks of exposure, the cell debris
accumulates in the P3 segment ol the proximal lulmle to form granular casts. Continued chemical
exposure for 3 to 12 months leads to the formation ol calcium hydroxyapatite in the papilla which
results in linear mineralization. Alter 1 or more years ol chemical exposure, these lesions may
resultin the induction ol renal adenomas and carcinomas.
U.S. K I'A (1991a) states that two questions must be addressed to determine the extent to
which a2u-gl<>bulin mediated processes induce renal tumors and nephropathy. First, itmustbe
determined whether the cfeu-glolnilin process is occurring in male rats and therefore could be a
factor in renal effects. M.S. K I'A f l')'*lal states that the criteria for answering this question in the
affirmative are as follows:
1) hyaline droplets are increased in size and number in male rats,
2) the protein in the hyaline droplets in male rats is cfeu-globulin, and
3) if 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,
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(c) granular casts,
(d) linear mineralization, and
(e) tubule hyperplasia.
The available data relevant to this question in male rats are summarized in Table 1-5 and
Figure 1-3 and Figure 1-4, and will be evaluated below in accordance with the mode of action
(MOA) framework from the EPA cancer guidelines (U.S. EPA. 2005a).
If the a2u-globulin process is operative, then U.S. EPA ("!')') kO states that a second question
must be answered as to whether the renal effects are solely due l<> the a2U-globulin process, are a
combination of the a2U-globulin process and other carcinogenic processes, or are due primarily to
other processes. U.S. EPA f!991al states thatthe following types ol data may be useful for
answering this question:
1) Hypothesis-testing data
2) Biochemical information
3) Sustained cell division in the proximal tubulcol the male rat
4) Structure-activity relationships
5) Covalentbinding to macromolecules
6) Genotoxicity
7) Nephrotoxicity
8) Animal Moassay (.lata in other species-, sex-comhinations
9) Other information not specifically listed
The available (.lata relev ant to this question are summarized in Table 1-6, and will be
evaluated below in accordance with the MOA framework from the EPA cancer guidelines fU.S. EPA.
2005a).
From these two questions. U.S. EPA (1991a) states that one of three possible conclusions
can be made:
• If renal tumors in male rats are attributable solely to the a2U-globulin process, then U.S. EPA
(1991a) states that such tumors will not be used for human cancer hazard identification or
for dose-response extrapolations.
• If renal tumors in male rats are not linked to the a2U-globulin process, then U.S. EPA (1991a)
states that such tumors are an appropriate endpoint for human hazard identification and
are considered, along with other appropriate endpoints, for quantitative risk estimation.
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• If some renal tumors in male rats are attributable to the a2U-globulin process and some
attributable to other carcinogenic processes, then U.S. EPA (1991a) states that such tumors
remain relevant for purposes of hazard identification, but a dose-response estimate based
on such tumors in male rats should not be performed unless there is enough information to
determine the relative contribution of each process to the overall renal tumor response.
Additionally, U.S. EPA (1991a) 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. In such a case, the characterization
of human health hazard for renal toxicity would need to rd v on oIIkt types of nephrotoxic effect
data in male rats and/or on nephrotoxic effect data in fL-mak- mis or other species.
Table 1-4. Additional kidney effects potentially relevant to mode of action in
animals following exposure to tert-butanol
Reference and study design
Results
Williams and Borehoff (2001)
F344 rats; 4/sex
Single gavage dose: 500 mg/kg
Males: 'T* binding of tert-butanol to a2u-globulin compared to females*
Females: no change in binding observed
NTP (1995)
F344/N rat; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or
40 mg/mL)
M: 0, 230, 490, 840, 1,520, 3,610
mg/kg-d
F: 0, 290, 590, 850, 1,560, 3,620a
mg/kg-d
13 weeks
Accumulation of hyaline droplets:
Male
Dose
(mg/kg-d)
Hvaline
droolet accumulation
0
0/10
230
+e
490
++
840
++
1,520
++
3,610
0/10
No information provided on females. No results from statistical tests reported.
Hard etal. (2011)
Reanalysis of the slides in the NTP
(1995) study
Males: Confirmed accumulation of hyaline droplets increased with increasing dose-levels
in 13 week study above. No incidence data available.
Females: not evaluated
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Table 1-4. Additional kidney effects potentially relevant to mode of action in
animals following exposure to tert-butanol (continued)
Reference and study design
Results
Borghoff et al. (2001)
F344 rat; 5/sex/treatment
Analytical concentration^, 250,
450, 1,750 ppm (0,771, 1,387 or
5,395mg/ms) 6hr/d
10 days
Males: positive trend for accumulation of protein droplets (p < 0.05), significant
increase in accumulation of a2u-globulin at 5,395 mg/mB as compared to controls (no
incidence data provided)
Females: No positive staining for a2u-globulin was observed in exposed female rats.
a The high-dose group had an increase in mortality.
b Linear mineralization not observed in female rats.
c Organs were not weighed in mice during the 2-year study.
d Standard & extended evaluation combined.
0 + or ++ indicated an increased accumulation relative to controls, as reported by the authors; no additional incidence data and
no results from statistical tests available.
* Statistically significant p < 0.05 as determined by the study authors.
Conversion from ppm to mg/mB is 1 ppm = 3.031 mg/mB.
Percentage change compared to control = (treated value - control value) 4- control value x 100.
Conversions from drinking water concentrations to mg/kg-d performed by study authors.
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1 Table 1-5. Summary of data on the a2u-globulin process in male rats exposed
2 to tert-butanol
Criterion
Duration
Resu Its
Reference
(1) hyaline droplets are
increased in size and number
10 d
+
Borghoff et al. (2001)
13 wk
(+)
NTP(1995)
13 wk
-
NTP(1997)
13 wk
(+)a
Hard et al. (2011)
(2) the protein in the hyaline
droplets is a2u-globulin
12 hr
+
Williams and Borghoff (2001)
10 d
+
Borghoff et al. (2001)
(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
-
NTP(1995)
2 yr
-
NTP(1995)
2 yr
-
Hard et al. (2011)
(b) exfoliation of
epithelial cells into
the tubular lumen
13 wk
-
NTP(1995)
2 yr
-
NTP (1995)
2 yr
-
Hard et al. (2011)
(c) granular casts
13 wk
-
NTP(1995)
13 wk
(+r
Hard et al. (2011)
13 wk
-
NTP(1997)
2 yr
b
NTP(1995)
(d) linear mineralization
13 wk
-
NTP(1995)
13 wk
-
NTP(1997)
2 yr
+
NTP(1995)
2 yr
(+)a
Hard et al. (2011)
(e) tubule hyperplasia
2 yr
+
NTP(1995)
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 a Re-analysis of one control and one treated group from NTP (1995)
7 b Protein casts reported, not granular casts
8 c Precursors to granular casts reported
9
10
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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
x = exposures at which all animals were dead and unable to be examined for the endpoint
• = exposures at which effect was observed but statistics not reported
NTP (1995); 13 wk
Accumulation of
hyaline droplets
Hard et al, (2011); 13 wk

•

tx2u-globuIinin
hyaline Williams and Borghoff (2001); single dose
droplets
NTP (1995); 13 wk
Granular *
casts/dilation Hard et a1'{20n*13 wk '
NTP (1995); 2 yr
~
11 [] ¦
n I
—B *
•

**NTP (1995); 13 wk -
Linear
papillary NTP (1995); 2 yr -
mineralization
Hard et al, (2011); 2 yr
¦
" T
• ¦ n
[]
—B k
NTP (1995); 2 yr
Tubular
hyperplasia
Hard et al, (2011); 2 yr
~
B ¦
~

Renal
adenoma Hard et a! (2011); 2 yr
or
carcinoma
¦
¦ ¦

~ Hard et al. poll) reported presence of "precursor 10 100 1,000 10,000
granular casts"
**NTP (1995) "13-wk study reported kidney Dose (mg/kg-day)
mineralization but not linear mineralization
Figure 1-3. Exposure-response array for components of a2u-globulin
nephropathy and renal tumors in male rats after oral exposure to
tert- butanol.
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Toxicological Review of tert-Butyl Alcohol
¦ = 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
Borghoffet al. (2001) -10 d
Accumulation
of hyaline
droplets
NTP (1997) -13 wks
~ B-
-B B B
a2u-globulin in
hyaline Borghoffet al, (2001) -10 d
droplets
Linear
papillary
mineralization
NTP (1997) -13 wks
~ &
-a b b
Tubular
hyperplasia
NTP (1997) - 13 wks
B B-
-B B B
100
1,000 10,000
Exposure Concentration (mg/m3)
2 Figure 1-4. Exposure-response array for components of a2u-globulin
3 nephropathy and renal tumors in male rats after inhalation exposure to
4 tert-butanol.
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1 Table 1-6. Summary of additional data informing the contribution of the
2 a2u-globulin process on the renal tumor development in male rats exposed to
3 tert-butanol
Type of data
Description
Reference
(1) Hypothesis-testing data
No data

(2) Biochemical information
Reversible, non-covalent binding of tert-butanol to a2u-globulin.
Williams and Borghoff (2001)
(3) Sustained cell division in the proximal tubule of the male rat
Hyperplasia at 2 yr reported in both male and female rats, attributed to
CPN.
Hard etal. (2011)
No effect on proliferation at 13 wk
NTP (1997)
Increased proliferation at 13 wk based on PCNA assay
NTP (1995)
Increased proliferation of the P2 segment at 10 days based on Brdll
labeling
Borghoff et al. (2001)
(4) Structure-activity relationships
No data

(5) Covalent binding to macromolecules
No data

(6) Genotoxicity
Limited database to conclude tert-butanol is genotoxic or non-
genotoxic
See Appendix B.3.
(7) Nephrotoxicity
Increased tubular regeneration and intratubule protein cast formation
at 2 yr in males and females, with effects in females occurring at lower
dose.
NTP (1995)
Increased severity of CPN in male rats after 13 wk inhalation exposure
NTP (1997)
Increased CPN in male and female F344/N rats following drinking water
exposure for 13 wk.
NTP(1995)
Increased CPN in male and female F344/N rats following drinking water
exposure for 2 yr.
NTP(1995)
(8) Animal bioassay data in other species-, sex-combinations
Two renal tubular adenocarcinomas (not statistically significant as
compared to concurrent controls) reported in male mice following
drinking water exposure for 2 yr. These tumors are very rare in mice.
NTP(1995)
(9) Other data
Dose-response and temporal concordance (see Figures 1-3 and 1-4).

4
5
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Strength, consistencyspecificity of association
Is the a2u-gIobuIin process occurring in male rats exposed to tert-butanol?
The first criterion considered is whether hyaline droplets are increased in size and number
in male rats. Protein droplet accumulation was statistically significantly increased in the kidneys of
male rats exposed to 5,305 mg/m3 tert-butanol for 6 hr/day for 10 days (Borghoff etal.. 20011. Data
from drinking water studies (NTP. 1995: T akahashi et al.. 1993: Lindamood etal.. 19921
demonstrated a statistically significant increase, except at the highest dose, in hyaline droplet
formation and severity in the proximal tubule of male rats following oral exposure to tert-butanol
for 13 weeks. Treated males had large hyaline droplets with crystal accumulation, butthe controls
had small droplets without crystals. NTP (19971 stained lor hyaline droplet formation in male rats
exposed to 0, 3,273, or 6,368 mg/m3 tert-butanol via inhalation lor 13 weeks, and there was no
difference between the controls and treatment groups.
The second criterion considered is whether the protein in the hyaline droplets in male rats
is a2u-globulin. Two studies measured a2U-globulin i in nui no reactivity in the hyaline droplets of the
renal proximal tubular epithelium fBorghoff et al.. 2001: Williams and Borghoff. 20011. Borghoff et
al. (20011 observed a2U-globulin imnuinoreactivity present in the hyaline droplets in the male rats.
No a2u-globulin immunostaining was observed in the kidneys of'the female rats. Williams and
Borghoff (20011 found the content oli< ¦„-»loliulin statistically significantly elevated in 72% of the
kidneys of male rats treated with tert-butanol compared with controls treated with corn oil.
The third criterion considered is whether sev eral (but not necessarily all) additional steps
in the pathological sequence are present in male rats. Several, but not all, of the subsequent
histopathological lesions were observed in the available subchronic or chronic tert-butanol
exposure studies. I,inear mineralization was the lesion most consistently observed in male rats and
was found to be statistically significantly increased in male rats after 2 years of oral exposure (NTP.
1995) (see Table 1-2). The 13-week study in rats (NTP. 19951 reported mineralization, butit was
not characterized as linear mineralization. Additionally, although the inhalation study by NTP
(19971 did not report linear mineralization at 13 weeks, this maybe due to the lower internal dose
as compared to the oral studies. Atypical tubule hyperplasia was statistically significantly increased
at the highest dose following 2 years of oral exposure fNTP. 19951. Granular casts were increased at
the 13 week time point (N'l'll')951. though statistical significance was not determined. The
reanalysis of the 13-week data by Hard etal. (20111 concluded that the lesions were precursors to
granular casts and not the casts themselves. Other studies did not observe granular casts at 13
weeks or 2 years fNTP. 1997.19951. The formation of protein casts were observed, but these may
be part of the pathology of CPN and are not thought to be related to a2U-globulin nephropathy (NTP.
19951. Neither necrosis nor epithelial exfoliation was reported in any study.
In summary, the evidence supports the conclusion that tert-butanol causes increases in the
size and number of hyaline droplets, and the accumulating protein in the hyaline droplets is
a2u-globulin. Additionally, several, but not all, of the additional steps in the pathological sequence
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were observed, and not always consistently across studies. Therefore, the overall strength,
consistency, and specificity of the association between tert-butanol and the hypothesized key
events is moderate.
Are the renal effects in male rats exposed to tert-butanol solely due to the a2U-gIobuIin process?
As summarized in Table 1-6, there are many potential sources of additional data that may
inform the contribution of the a2U-globulin process on renal tumor development No hypothesis-
testing data, structure-activity relationships, or covalent binding data were located, so these types
of data are not discussed. Additional data related to close- response.' concordance and temporal
relationships are discussed in subsequent sections.
In terms of biochemical information, Williams and lloruholl (20011 report that tert- butanol
reversibly and non-covalently binds to a2U-globulin. This provides additional support to the
evidence thatthe a2U-process is occurring, butit does not inform the relativ e contribution to renal
tumor development.
Sustained cell division in the proximal tubule of the male ratis consistent with, though not
specific to, the a2U-process. Proliferation of the proximal tubule was significantly increased in male
rats after 10 days of inhalation exposure to /<.'//-butanol at concentrations of 771-5,395 mg/m3
(Borghoff etal.. 20011 and after 13 weeks olOral exposure to 1520 mg/kg-day fNTP. 1995:
Takahashi etal.. 1993: Lindamood el al.. l')')2 ). but not alter 13 week inhalation exposure up to
6368 mg/m3 fNTP. l')')7). Therefore, it is unclear the extent to which increased cell division is
sustained. While hyperplasia was reported in chronic studies [I lard etal.. 20111. it was observed in
both male and female rats, and attributed to CPN.
There are a limited number ofsludies available to assess the genotoxic potential of tert-
butanol (see Appendix B.3 in Supplemental Information for further details). tert-Butanol was
generally negative in a variety ol genotoxicitv assays and cell systems including Salmonella
typhimiirium. Escherichia coli and Neurospora crassa fMcgregor etal.. 2005: Zeiger etal.. 1987:
Dickey et al.. I'M91. Studies also demonstrate negative results for gene mutations, sister chromatid
exchanges, niicronuclcus formation, and chromosomal aberrations fNTP. 1995: McGregor etal..
19881. However, DNA adducls were found in male Kunming mice fYuan etal.. 20071. and DNA
damage was observed in human 111,-60 leukemia cells fTangetal.. 19971. In another study by
Sgambato etal. f20091. an initial increase in DNA damage was observed as measured by nuclear
fragmentation, but the damage declined drastically following 4 hours of exposure and disappeared
entirely after 12 hours of exposure to tert-butanol.
In terms of nephrotoxicity, a number of renal effects have been reported in female rats
and/or in mice. Kidney transitional epithelial hyperplasia and inflammation were significantly
increased in both male and female F344 rats exposed for 2 years via oral exposure. F344 rats
exposed to tert-butanol at dose ranges of 230-1520 mg/kg-day and 850-3,620 mg/kg-day in males
and females, respectively, exhibited a statistically significant increase in the incidence of
nephropathy compared with controls after 13 weeks of exposure fNTP. 19951. Nephropathy
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severity was also significantly increased at 420 mg/kg-day in males and 180-650 mg/kg-day in
females after 2 years of exposure fNTP. 19951. Average severity of chronic nephropathy was
minimal to mild in males after a 13-week inhalation exposure fNTP. 19971. Female rats also had
lesions associated with nephropathy fNTP. 19951. but none of the lesions were similar to those
observed in the male rat that are associated with a2U-globulin nephropathy.
With respect to renal tumors, no statistically significant increases in renal tumors were
reported in tert-butanol-exposed female rats or mice compared with concurrent controls. Two
renal tubular adenocarcinomas were reported in male mice following drinking water exposure for
2 years (NTP. 19951 (one each in the low and high dose groups). Although such tumors are very
rare in mice, with historical control incidences of 2/1351 ("0.15%) in feeding studies and 4/1093
(0.37%) in chamber studies (Haseman et al.. 19981. these data are not sufficient to indicate that the
kidney tumors observed in mice exposed to tert-butanol are treatment-related.
Overall, the strength, consistency, and specificity of the data supporting a tert- butanol-
induced a2U-globulin process as the sole actor for renal effects in male rats is weak to moderate.
Dose-response concordance
Is the a2u-gIobuIin process occurring in mule rats exposed to lurl-hutunol?
As shown in Figure 1-3 and Figure 1-4, the (.lose-response concordance among hypothesized
key events is mixed.
Borghofl et al. (2001) exposed male and female F344 rats to / ert-butanol at concentrations
of 758,1,364, or 5,305 mg/m : lor (> hr/ilav lor 10 (.lavs to assess the role of a2U-globulin
nephropathy and renal cell proliferation. Significant lulmlar proliferation in males was observed at
all exposure levels, but accumulation of it :u-glolnilin-posilive hyaline droplets was increased only at
the highest dose (Horuholl et al.. 2001). These data suggest that cell proliferation may be related to
the a u-glolnilinprocess only at the highest exposure concentration.
The dose-response relationships observed after 13 weeks and 2-years were also only
moderately concordant. Data from a drinking water study (NTP. 1995: Takahashi etal.. 1993:
Lindamood etal.. 1()')21 demonstrated hyaline droplet formation in the proximal tubule of male rats
at all tested doses (except al the highest dose where all rats died during weeks 5-12) following oral
exposure to tert-bulanol lor 13 weeks. PWG reevaluation by Hard etal. f20111 reported observing
precursors to granular casts at the only dose level evaluated (1,520 mg/kg-day). Spontaneous
mineralization was observed, but the linear mineralization characteristic of this MOA was not
observed at any dose. At the 2-year timepoint, linear mineralization was observed at all exposure
levels from 90-420 mg/kg-day, and renal tubular hyperplasia was observed at the highest dose of
420 mg/kg-day fNTP. 19951. consistent with the expected dose-response relationship. Notably,
however, granular casts were not observed.
Overall, the dose-response concordance of the association between tert-butanol and the
hypothesized key events is moderate.
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Are the renal effects in male rats exposed to tert-butanol solely due to the a2u-globulin process?
Dose-response concordance between the hypothesized key events and the occurrence of
renal tumors can inform whether carcinogenesis is solely due to the a2U-globulin process. Male
F344 rats exhibited an increased incidence of renal tubule adenomas and combined renal tubule
adenoma or carcinoma in a 2-year oral bioassay fHard etal.. 2011: NTP. 19951. Increased tumors
were observed at 90, 200, and 420 mg/kg-day. Although some effects related to a2U-globulin
nephropathy, including hyaline droplets and linear mineralization, were observed at all doses,
tubule hyperplasia was not observed at doses lower than 420 mg/kg-day in any study and only
precursor granular casts were observed at a much higher dost- <>1 1,520 mg/kg-day. Moreover, the
middle dose of tert-butanol induced the greatest incidence ol luniors, so increasing the dose from
200 to 420 mg/kg-day led to additional markers of a2u-glolmlin nephropathy in the form of tubule
hyperplasia, but without any increase in tumor burden. Thus, te/7-liutanol induced tumors at lower
doses than for other precursor effects such as hyperplasia and granular casts, suggesting a weak
dose response concordance with the incidence ol luniors.
Therefore, on the basis of weak dose-response concordance, the data suggest that the
observed tumors are not solely due to u u-globulin and lhal oilier processes are primarily
responsible for tumors.
Temporal relationship
Is the a2U-globulin process ocean iiuj in mule ruIs exposed to lerl-butanol?
As shown in Til Me 1-2 and Table 1-4, hyaline droplets and a2u-globulin accumulation were
observed after a single (.lose or 10-day exposure: precursors to granular casts were observed at 13
weeks: and tubular hyperplasia was observed al 2 years. The observations are consistent with the
expected temporal relationship (I lard et al.. 2011: Borghoff etal.. 2001: Williams and Borghoff.
2001: NTI'. l')')5). I lowever, the absence of other key events such as necrosis, exfoliation, and
granular casts in inostother studies attlie anticipated time points weaken the case for a2u-globulin
MOA. Additionally, the NTP's 13-week study in rat reported kidney mineralization, but it was not
characterized as linear mineralization.
Are the renal effects in mule ruts exposed to tert -butanol solely due to the a2u-globulin process?
This question cannot lie answered from data on temporality.
Biological plausibility and coherence
Both U.S. EPA and IARC have accepted the general biological plausibility and coherence of
role of a2U-globulin-mediated nephropathy in renal tumor induction fSwenberg and Lehman-
McKeeman. 1999: U.S. EPA. 1991a). and those rationales will not be repeated here.
However, a retrospective analysis has suggested that a number of a2u-globulin inducing
chemicals fail to induce many of the pathological sequences in the a2u-globulin pathway fDoi etal..
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20071. For instance, dose-response concordance was not observed for several endpoints such as
linear mineralization, tubular hyperplasia, granular casts, and hyaline droplets following exposure
to a2u-globulin-inducing chemicals such as d-limonene, decalin, propylene glycol mono-t-butyl
ether, and Stoddard solvent IIC (SSIIC). Although some of these chemicals induced
histopathological lesions that exhibited a dose response, all of them failed to induce a dose-
response trend for at least one of the endpoints in the sequence. Furthermore, no endpoint in the
pathological sequence was predictive for tumor incidence. Tumor incidence did not exhibit a dose
response following either d-limonene or decalin exposure. Finally, tumor incidence was not
predicted by the severity of a particular effect in the ctarglolmlin sequence as demonstrated by SS
IIC which induced some of the most severe nephropathy precursors relative to the other chemicals
but did not significantly increase kidney tumors fDoi et al.. 2007). Thus, these analyses suggest that
another MOA may be operative for inducing tumors.
Moreover, renal tumors were not observed following exposure to ETBE, which is rapidly
metabolized to tert-butanol. Specifically, Suzuki et al. (20121 and Saito et al. (20131 reported no
increase in renal tumors in both sexes of Fischer !vH nils following 2-year oral or inhalation
exposures to ETBE at doses thatyield similar internal concentrations (based on HBPK modeling) of
tert- butanol compared with the concentrations of the tert-butanol bioassays. After 13 weeks of
exposure to tert-butanol or ETBE, hyaline droplets were increased in a dose-response manner.
ETBE exposure increased hyaline droplets al 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 a tert-butanol exposure. I!v contrast, tulmle hyperplasia and renal tumors were both observed
following a 2-year exposure to /erMiutanol but not following ETBE exposure. Renal tumors and
tubule hyperplasia were not observed following any ETBE exposure despite achieving similar blood
concentrations of tert-butanol as the N'l'l' (1995 ) study. The failure of internal tert-butanol
concentrations to induce histopathological lesions early in the oi2u-globulin pathological sequence
at blood levels that later induced hyperplasia and tumors suggests alack of coherence across the
two data sets.
Conclusions about the hypol hcsi/al MOA for
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With respect to the second question, male rats are more sensitive to the kidney effects of
tert-butanol, and the available data indicate that male rats accumulate a2U-globulin in the kidney,
which is a specific MOA for male rats. Many of the steps in the pathological sequence of lesions
related to a2U-globulin-associated nephropathy were observed exclusively in male rats but not in
female rats, or mice of either sex, and renal tumors occurred only in male rats. However, there is
insufficient evidence to support a conclusion that a2U-globulin nephropathy is the sole or primary
contributor to renal tumor development. Given the inconsistencies and limitations of the
genotoxicity database, the effect of tert-butanol with respect to genotoxicity cannot be ruled out
Additionally, tert-butanol induced tumors at lower doses than < precursor effects such as
hyperplasia and granular casts, with no further increase in1 , i ncidence coinciding with the
additional markers of a2U-globulin nephropathy. Thus, r ai. -s observed in male rats are
unlikely to be confounded by the presence of c^irglo1 Hn nephrop^ \ Therefore, on the basis of a
weak dose-response concordance, the data supp< conclusion thai |. "sses other than
a2u-globulin nephropathy are likely responsible renal tumor develop m
Is the hypothesized MOA relevant to humans?
Based on the conclusion thai processes other Lha. globulin nephropathy are likely
responsible for renal tumor develop in en I induced by lerl-bu 'I. U.S. EPA (1991a) states thatthe
following conclusion will be made:
• If renal tumors in male mis are nol linked lo llie « ^-globulin process, then fU.S. EPA.
1991a) states that such lumors are an appropriate endpoint for human hazard
identification and are considered, along willi other appropriate endpoints, for quantitative
risk estimation.
Therefore, kidney lumors are relevant lo humans for purposes of hazard identification and
dose-response assessment, liecause female rats and both sexes of mice do not have a2U-globulin
present, kidney effects in these animals are considered relevant to humans for both hazard
identification and dose-response.
Which populations or lifcslaijcs can he particularly susceptible to the hypothesized MOA?
This question is not applicable.
Alternative MOA hypotheses with inadequate data for analysis
Other nephrotoxic responses, such as exacerbation of CPN, inflammation, transitional
epithelial hyperplasia, and increased kidney weight, are observed in rats and/or mice, suggesting
other possible processes are operative. It has been proposed that enhanced chronic progressive
nephropathy (CPN) is a mode of action for chemically-induced kidney tumors in male rats and that
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renal tubule tumors induced by chemicals that concomitantly exacerbate CPN are not relevant to
humans (Hard and Khan. 20041.
CPN is an age-related renal disease of unknown etiology that occurs spontaneously in rats,
especially the F344, Sprague-Dawley, and Osborne-Mendel strains. Additional markers associated
with CPN include elevated protein and albumin in the urine and increased BUN, creatinine, and
cholesterol in the serum fHard etal.. 20091. CPN is often 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 ("Webb etal.. 19901. Additionally,
renal effects of c^u-globulin accumulation can exacerbate I lie e Heels associated with CPN fU.S. EPA.
1991a). However. Webb etal. f!9901 suggestedthatexacerbated CI'N was one component of the
nephropathy resulting from exposure to chemicals that induce « :il-globulin nephropathy. Male rat
sensitivity has been noted with both CPN and c^u-gloliulin nephropathy.
Increased severity of CPN occurred in both male and female rats as a result of tert-butanol
exposure. Some of the observed renal lesions in male rats following exposure.- to tert- butanol are
effects commonly associated with CPN. Hard et al. (2011) concluded thatthe observation of
transitional epithelial hyperplasia in the 2-year drinking study conducted by NTP (19951 was
associated with CPN, and not a direct effect of tert-butanol exposu re. However, there was a strong,
statistically-significant, treatment-related, dose-response relationship between chronic tert-butanol
exposure and increased incidence of transitional epithelial hyperplasia in both male and female rats
in the NTP (19951 study. The severity ol CPN also increased with tert-butanol exposure, although
the dose-response relationship in males was very weak (only a 10% increase in mean severity at
the highest dose). The very different dose-response relationships argue against a close association.
Moreover, even if transitional epithelial hyperplasia were associated with CPN, there is no evidence
to support that the effect is independent of tert-butanol treatment, given the robust dose-response
relationships. Therefore, the (.lata are insufficient to dismiss transitional epithelial hyperplasia as
causally related to tert-butanol exposure.
Additionally, there have been a lew research groups who have discussed the role of CPN and
a2u-globulin accumulation on the renal tumors observed in male rats exposed to tert-butanol.
Cruzan etal. (20071 concluded that a2U-globulin, exacerbation of CPN, or a combination of both
were the MOAs for the kidney lu mors in males. Hard etal. f20111 also concluded that both a2u-
globulin-induced nephropathy and exacerbated CPN were MOAs for the kidney tumors observed in
the male rats in the 2-year drinking study conducted by NTP (19951. However, 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 (Melnick etal.. 2012:
Hard etal.. 20091. Moreover, no key events for the exacerbation of CPN have been identified, so no
MOA analysis can be performed under the EPA Cancer Guidelines MOA framework (U.S. EPA.
2005a)- Therefore, kidney effects from tert-butanol exposure associated with CPN are considered
relevant to humans.
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Summary of kidney toxicity
Kidney toxicity was consistently observed after oral exposure in two strains of rats and in
one strain of mice and in both sexes. Absolute and relative kidney weights also were increased in
male and female rats in both the 13-week and 2-year studies. In male and female rats,
histopathological examination of the kidneys indicated kidney lesions exhibiting a dose-response
trend, increased incidence of nephropathy after 13 weeks and 2 years, and increased transitional
epithelium hyperplasia and suppurative inflammation (females only) after 2 years. In mice, the
only kidney effect observed was an increase in kidney weight ("absolute and/or relative) in both
sexes of mice in the 13-week study. Organs were not weighed in I In.- 2-year mouse study, so no
determination can be made. Furthermore, there were no treatment-related histopathological
lesions in the kidneys of mice at 13 weeks or 2 years.
Male rats are more sensitive to the kidney effects of/i.*/"<-liuliinol, and the available data
indicate that male rats accumulate a2U-globulin in the kidney, which is a specific MOA for male rats.
MOA analysis determined that the renal tumors observed in male rats are mediated by other
processes besides c^u-globulin. Therefore, in the absence of a known MOA, ]¦ I'A u
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Toxicological Review of tert-Butyl Alcohol
1 doses in male mice, but only a marginal increase in follicular cell adenomas in the mid-dose group.
2 One high-dose male mouse developed a follicular cell carcinoma. The lower tumor incidence in
3 males may be due to the increased mortality seen in the high-dose group. NTP T19951 noted that
4 thyroid follicular cell tumorigenesis follows a progression from hyperplasia to adenoma and
5 carcinoma, suggesting that hyperplasia is a preneoplastic lesion in the thyroid.
6
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Toxicological Review of tert-Butyl Alcohol
1 Table 1-7. Evidence pertaining to thyroid effects in animals following oral
2 exposure to tert-butanol
Reference and study design
Resu Its
Follicular cell hyperplasia
NTP (1995)
Incidence15

F344/N rat; 60/sex/treatment
(10/sex/treatment evaluated at 15
months)
Drinking water (0,1.25, 2.5, 5, or 10
mg/mL)
M: 0, 90, 200, or420a mg/kg-d
F: 0,180, 330, or 650a mg/kg-d
2 years
Males
Dose
(mg/kg-d)
0
90
200
420
Follicular cell
hvoerolasia
3/50
0/49
0/50
0/50
Females
Dose
(mg/kg-d)
0
180
330
650
Follicular cell
hvoerolasia
0/50
0/50
0/50
0/50
NTP (1995)
Incidence (severity)

B6C3Fj mouse; 60/sex/treatment
Drinking water (0, 5,10, or 20 mg/mL)
M: 0, 540,1,040, or 2,070a mg/kg-d
F: 0, 510,1,020, or 2,110 mg/kg-d
2 years
Males
Dose
(mg/kg-d)
0
540
Follicular cell
hvoerolasia
5/60(1.2)
18/59* (1.6)
Females
Dose
(mg/kg-d)
0
510
Follicular cell
hvoerolasia
19/58 (1.8)
28/60 (1.9)

1,040
15/59* (1.4)
1,020
33/59* (1.7)

2,070
18/57* (2.1)
2,110
47/59* (2.2)
Tumors
NTP (1995)
Incidence15

90
200
420
Female
0/49
0/50
0/50
0/49
0/50
0/50

0
1/50
1/50

180
0/50
0/50

330
1/50
1/50

650
0/50
0/50

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Table 1-7. Evidence pertaining to thyroid effects in animals following oral
exposure to tert-butanol (continued)
Reference and study design
Resu Its
NTP (1995)
Incidence

B6C3Fj mouse; 60/sex/treatment
Drinking water (0, 5,10, or 20 mg/mL)
M: 0, 540,1,040, or 2,070a mg/kg-d
F: 0, 510,1,020, or 2,110 mg/kg-d
2 years
Dose
(mg/kg-d)
Male
Follicular
cell
adenoma
Mortalitv-
adiusted
rate (%)
Follicular
cell
carcinoma
or adenoma
Mortalitv-
adiusted
rate
(%}
Follicular
cell
carcinoma0

0
1/60
3.6
1/60
3.6
0/60

540
0/59
0.0
0/59
0.0
0/59

1,040
4/59
10.1
4/59
10.1
0/59

2,070
1/57
5.9
2/57
8.7
1/57

Female

0
2/58
5.6
2/58
5.6
0/58

510
3/60
8.6
3/60
8.6
0/60

1.020
2/59
4.9
2/59
4.9
0/59

2,110
9/59*
19.6
9/59*
19.6
0/59
1 aThere was a significant decrease in survival in the high-dose group.
2 bResults do not include the animals sacrificed at 15 months.
3 cMortality-adjusted rates were not calculated by study authors for follicular cell carcinoma.
4 * Statistically significant p < 0.05 as determined by the study authors.
5 Conversions from drinking water concentrations to mg/kg-d performed by study authors.
6
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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
Hyperplasia; M mouse
Hyperplasia; F mouse
NONCANCER
Hyperplasia; M rat
Hyperplasia; F rat
-B 0
~ B ~
Adenoma. M mouse
Adenoma. I' mouse
CAiNCi: u
Adenoma. M ral
Adenoma; 1' ral
~ B B
~ B-
B
-b a
B B ~
10 100 1 000 10,000
Dose (mg/kg-day)
Source: NTP f 19951
Figure 1-5. Exposure-response array of thyroid follicular cell effects following
chronic oral exposure to tert-butanol.
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Mode of Action Analysis—Thyroid Effects
There are inadequate data to determine the MOA for tert-butanol-induced thyroid follicular
cell lesions. The mechanism of formation of these lesions resulting from tert-butanol exposure has
not been specifically studied; however, Blanck etal. (20101 conducted a short-term study
examining the hepatic and thyroid effects of tert-butanol exposure to provide additional data on the
thyroid tumors observed in the chronic NTP (19951 study, tert-Butanol did not have any effect on
liver weight when compared to the control group, but the livers were visibly enlarged, in some
cases accompanied by centrilobular hepatocellular hypertrophy, in some treated groups. There
were no treatment-related histological alterations in the thyroid in /crt-butanol treated mice. Only a
slight statistically nonsignificant increase in thyroid stimu kiting hormone (TSH) was observed after
3 days of exposure, but both thyroxine (T4) and triiodothyronine (T :) levels were decreased.
Sustained increases in TSH, resulting in sustained thyroid follicular cell proliferation, may
eventually resultin progression of hyperplasia to adenoma and carcinoma (U.S. EPA. 1998al. Based
on alterations in hepatic phase I and phase II 011 ¦/vim.1 activities and gene expression, the data from
Blanck etal. (20101 suggest a possible role for increased thyroid hormone clearance in the liver in
tert-butanol-induced thyroid tumors. The available support lor this hypothesis, however, is weak.
In particular, Blanck etal. f20101 did not find any significant changes in TSH levels, though the
study duration was short (<14 days), and there are 110 data regarding thyroid cell proliferation after
exposure to tert-liutanol.
Summary of thyroid toxicity
EPA identified thyroid effects as a potential human hazard of tert-butanol exposure. The
thyroid endpoints reported following chronic exposure to tert-butanol include follicular cell
hyperplasia, follicular cell adenoma, and follicular cell carcinoma. Together with the evidence of
significantly increased incidence of thyroid follicular cell adenomas in high-dose females, these
observations support the finding that the increased hyperplasia in male mice is a preneoplastic
effect rather than an adaptive response. There is no conclusive MOA for the development of thyroid
follicular cell adenomas, although there is some evidence supporting greater clearance of thyroid
hormones by the liver causing continual secretion of TSH by the pituitary leading to follicular cell
hyperplasia and tumors. Data 011 thyroid tumors associated with tert-butanol exposure are
discussed further as part of the overall weightof evidence for carcinogenicity in Section 1.2.2.
1.1.3. Reproductive and Developmental Effects
Synthesis of reproductive and developmental toxicity
This section reviews the studies that investigated whether exposure to tert-butanol can
cause reproductive and developmental effects in humans or animals. This section contains
information on reproductive effects, systemic developmental effects, as well as
neurodevelopmental effects following tert-butanol exposure. The database examining reproductive
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Toxicological Review of tert-Butyl Alcohol
or developmental effects following tert-butanol exposure contains no human data and 7 studies
performed in rats and mice. Three studies evaluating reproductive effects included changes in
reproductive organs (one one-generation reproductive study and two subchronic studies). In
addition, there was one two-year oral study in rats and mice that evaluated reproductive
histopathology and did not find any treatment-related effects. The studies selected for discussion
below exposed animals via oral gavage, drinking water, and inhalation for >63 days. The collection
of reproductive studies on tert-butanol is limited by the absence of any two-generation
reproductive oral or inhalation studies. Four studies evaluated developmental effects (three
developmental studies and one one-generation reproductive.- study). As mentioned in the Study
Selection, the studies selected for discussion below exposed aninulls to tert-butanol via inhalation,
gavage, and drinking water. No methodological concerns were identified thatwould lead one or
more studies to be considered less informative for assessing human health hazard, but the Faulkner
etal. (19891 study did notprovide results in the dam that could be used to adequately determine if
fetal effects occurred due to maternal toxicity. Three studies evaluated neurodevelopmental effects
following tert-butanol exposure in rats and mice. These studies selected for discussion below
exposed animals via liquid diet and inhalation. The collection ol neurodevelopmental studies on
tert-butanol is limited in thatall studies were conducted prior to Dev elopmental Toxicity
Guidelines being available from the M.S. KIJA (!')')! hi and OK CD: as such, there are study design
considerations for each of the studies. Daniel and Kvans f l')821 hud a small number of animals per
treatment group, presentation of results provided limited use ol the (.lata with no comparisons to
controls, and there was no long term neurod eve lop mental testing. Nelson etal. (19911 evaluated
neurodevelopmental effects alter either paternal or maternal exposure. Although the study authors
used two different exposure concentrations, the exposures were not run concurrently nor was
there information provided on exposure methods to indicate the studies were conducted similarly.
Reproductive effects
Reproductive endpoints, such as sex organ weights, estrous cycle length, and sperm effects
were examined following either oral or inhalation exposure in three subchronic studies (Lvondell
Chemical Co.. 2004: NTP. l')')7. l'lQSlfTable l-8;Figure l-6;Figure 1-7). The only reproductive
effect noted was an increase in estrous cycle length. Estrous cycle length was increased (28%
increase, p < 0.01) in female mice orally exposed to 11,620 mg/kg-day. No significant changes in
estrous cycle length were observed following oral exposure in rats, or inhalation exposure in mice
or rats.
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Toxicological Review of tert-Butyl Alcohol
1 Table 1-8. Evidence pertaining to reproductive effects in animals following
2 exposure to tert-butanol
Reference and study design
Resu Its
Male reproductive effects
Lvondell Chemical Co. (2004)
Sprague-Dawley rat; 12/sex/treatment
Gavage 0, 64,160, 400, or 1,000 mg/kg-d
F0 males: 9 weeks beginning 4 weeks prior to
mating
F0 females: 4 weeks prior to mating through
PND21
F0 reproductive effects
No significant effect on weights of male reproductive organs or sperm
observed
NTP (1995)
F344/N rat; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or 40 mg/mL)
M: 0, 230, 490, 840, 1,520, 3,610a mg/kg-d
F: 0, 290, 590, 850, 1,560, 3,620a mg/kg-d
13 weeks
No significant effect on weights of male reproductive organs or sperm
observed
NTP (1995)
B6C3FJ mouse; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or 40 mg/mL)
M: 0, 350, 640, 1,590, 3,940, 8,210a mg/kg-d
F: 0, 500, 820, 1,660, 6,430, ll,620a mg/kg-d
13 weeks
No significant effect on weights of male reproductive organs or sperm
observed
NTP (1997)
F344/N rat; 10/sex/treatment
Analytical concentration: 0,134, 272, 542,
1,080, or 2,101 ppm (0, 406, 824,1,643, 3,273
or 6,368 mg/mB) (dynamic whole body
chamber)
6 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist Ultrasonic spray
nozzle nebulizer), analytical concentration and
method were reported
No significant effect on weights of male reproductive organs or sperm
observed
Evaluations were performed only for concentrations >542 ppm
(1,643 mg/mB)
NTP (1997)
B6C3Fj mouse; 10/sex/treatment
Analytical concentration: 0,134, 272, 542,
1,080, or 2,101 ppm (0, 406, 824,1,643, 3,273
or 6,368 mg/mB) (dynamic whole body
chamber)
6 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist Ultrasonic spray
nozzle nebulizer), analytical concentration and
method were reported
No significant effect on weights of male reproductive organs or sperm
observed
Evaluations were performed only for concentrations >542 ppm
(1,643 mg/mB)
3
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
Table 1-8. Evidence pertaining to reproductive effects in animals following
exposure to tert-butanol (continued)
Reference and study design
Results
Female reproductive effects
Lvondell Chemical Co. (2004)
Sprague-Dawley rat; 12/sex/treatment
Gavage 0, 64,160, 400, or 1,000 mg/kg-d
F0 males: 9 weeks beginning 4 weeks prior to mating
F0 females: 4 weeks prior to mating through PND21
Pregnancy index
91.7% 91.7% 100% 100% 91.7%
NTP (1995)
F344/N rat; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or 40 mg/mL)
M: 0, 230, 490, 840, 1,520, 3,610a mg/kg-d
F: 0, 290, 590, 850, 1,560, 3,620a mg/kg-d
13 weeks
No significant effect on female estrous cycle (0, -2, -4, 0, +8 %
change relative to control)
NTP (1995)
B6C3Fj mouse; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or 40 mg/mL)
M: 0, 350, 640, 1,590, 3,940, 8,210a mg/kg-d
F: 0, 500, 820, 1,660, 6,430, ll,620a mg/kg-d
13 weeks
'T* length of estrous cycle
Response relative to control: 0, +5, +5, +5, +6, +28*%
NTP (1997)
F344/N rat; 10/sex/treatment
Analytical concentration: 0,134, 272, 542,1,080, or
2,101 ppm (0, 406, 824, 1,643, 3,273 or 6,368 mg/m3)
(dynamic whole body chamber)
6 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist Ultrasonic spray nozzle
nebulizer), analytical concentration and method were
reported
No significant effect on female estrous cycle (0, -4, +2, +4 % change
relative to control)
Evaluations were performed only for concentrations >542 ppm
(1,643 mg/m3)
NTP (1997)
B6C3Fj mouse; 10/sex/treatment
Analytical concentration: 0,134, 272, 542,1,080, or
2,101 ppm (0, 406, 824, 1,643, 3,273 or 6,368 mg/m3)
(dynamic whole body chamber)
6 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist Ultrasonic spray nozzle
nebulizer), analytical concentration and method were
reported
No significant effect on female estrous cycle (0, -3, -9, -5 % change
relative to control)
Evaluations were only performed for concentrations >542 ppm
(1,643 mg/m3)
1 * Statistically significant p < 0.05 as determined by the study authors.
2 Conversions from drinking water concentrations to mg/kg-d performed by study authors.
3 Conversion from ppm to mg/mB is 1 ppm = 3.031 mg/mB.
4 Percentage change compared to control = (treated value - control value) 4- control value x 100.
5
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Toxicological Review of tert-Butyl Alcohol
Developmental effects
Data from three developmental studies (two oral, one inhalation) suggest that fetal effects
are generally observed at doses that cause toxicity in the dams as measured by clinical signs (e.g.,
decreased body weight gain, and/or food consumption) (Table 1-9; Figure 1-6; Figure 1-7).
Developmental effects of tert-butanol observed after oral exposure (liquid diets or gavage)
in several mouse strains and one rat strain include measures of fetal loss or viability (e.g., increased
number of resorptions, decreased numbers of neonates per litter) and decreased fetal body weight
(Lvondell Chemical Co.. 2004: Faulkner etal.. 1989: Daniel and Evans. 1982). Daniel and Evans
£1982} also observed decreases in body weight gain during PND 2-10; however, data suggest that
the effect may be due to maternal behavior or nutritional status. I n addition, one study reported
increased incidence of variations of the skull or sternebrae in two mouse strains, but the difference
was not statistically significant fFaulkner et al.. 1()8')). Similar developmental effects were also
observed after whole-body inhalation exposure.- in Sprague-Dawley nils for 7 hours/day on GDs 1-
19 (Nelson etal.. 1989). Fetal effects included concentration-related reductions in body weight in
male and female fetuses and higher incidence of skeletal variations when analyzed on the basis of
individual fetuses (but not on a per litter basis). In contrast to the oral exposure studies in mice
and rats, however, there was no effect on measures of letal loss.
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Toxicological Review of tert-Butyl Alcohol
Table 1-9. Evidence pertaining to developmental effects in animals following
exposure to tert-butanol
Reference and study design
Resu Its
Daniel and Evans (1982)
Swiss Webster (Cox) mouse; 15 pregnant
dams/treatment
Liquid diet (0, 0.5, 0.75, 1.0%, w/v)
0 (isocaloric amounts of maltose/dextrin),
3,324, 4,879, 6,677 mg/kg-d
GD 6-20
No statistical analysis was conducted on any of these data
Maternal
Percent change compared to control:
Food

consumotion

Number of litters
Dose
(mean
Body weight
(% pregnant
(mg/kg-d)
g/animal/dav)
gain
dams)
0
0
0
11 (77%)
3,324
+2
-3
12 (80%)
4,879
-3
-19
8 (53%)
6,677
-4
-20
7 (47%)
Authors note that lower food consumption in higher tert-butanol dose groups
reflects problems with pair feeding and maternal sedation.
Fetal
Percent change compared to control:
Dose
Number of
Fetal body weight
(mg/kg-d)
neonates/litter
on PND2
0
0
0
3,324
-1
-7
4,879
-29
-19
6,677
-49
-38
Number of stillborn also increased with dose (3, 6,14, and 20, respectively), but
the number of stillborn per litter was not provided. The high dose also caused a
delay in eye opening and a lag in weight gain during PND 2-10 (information was
only provided in text or figures)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
Table 1-9. Evidence pertaining to developmental effects in animals following
exposure to tert-butanol (continued)
Reference and study design
Resu Its
Faulkner etal. (1989)
Maternal results not reported.

CBA/J mouse; 7 pregnant females in
control, 12 pregnant females in treated
Gavage (10.5 mmoles/kg twice a day);
0 (tap water), 1,556 mg/kg-d
GD 6-18
Fetal
Dose
(mg/kg-d)
Percent change compared to
control:
Live Fetal
fetuses/litter weight
Incidence:
Sternebral
variations
Skull
variations

0
0 0
4/28
1/28

1,556
1
*
r—1
1
7/30
3/30

Sternal variations: misaligned or unossified sternebrae
Skull variations: moderate reduction in ossification of supraoccipital bone

Number of total resorptions (10 resorptions/66 implants in controls, 37/94
implants in treated) and resorptions per litter (+118%) increased (p < 0.05)
Faulkner etal. (1989)
Maternal results not reported.

C57BL/6J mouse; 5 pregnant females in
controls, 9 pregnant females treated
Gavage (10.5 mmoles/kg twice a day)
0 (tap water), 1,556 mg/kg-d
GD 6-18
Fetal
Dose
(mg/kg-d)
Percent change compared to
control:
Live Fetal
fetuses/litter weight
Incidence:
Sternal
variations
Skull variations

0
0 0
5/21
1/21

1,556
-58%* -4
9/16
7/16

Sternal variations: misaligned or unossified sternebrae
Skull variations: moderate reduction in ossification of supraoccipital bone

Number of total resorptions (4 resorptions/44 implants in controls, 38/68
implants in treated) and resorptions per litter (+428%) increased (p < 0.05)
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
Table 1-9. Evidence pertaining to developmental effects in animals following
exposure to tert-butanol (continued)
Reference and study design
Resu Its
Lvondell Chemical Co. (2004)
Response relative to control

OECD guideline 421 study:
Dose
(mg/kg-d) 0
64
160
400
1000
Sprague-Dawley rat; 12/sex/treatment
Gavage 0, 64,160, 400, or 1,000 mg/kg-d
F0 males: 9 weeks beginning 4 weeks prior
to mating
F0 females: 4 weeks prior to mating
through PND21
F1 Males and Females: 7 weeks
(throughout gestation and lactation; 1
male and 1 female from each litter was
dosed directly from PND 21-28)
Maternal effects
Body weight gain GD 0-20
0
Food consumption GD 0-20
0
Body weight gain PND 1-21
0
Food consumption LD1-14
-3
0
+3
-4
0
-10
0
+4
+3
-16*
0
+100*

0
-2
-6
0
-16

Live pups/litter response relative to control

0
-9
11
-7
-33*

Dams dosed with 1000 or
400 ms/ks/d showed CNS effects
e.s.. ataxia.

lethargy) which became undetectable by 4-weeks of exposure
in animals

exposed to 400 mg/kg/d but not those in the higher dose group.

F1 effects

Viability index (pup survival to PND4)

96.4%
98.7%
98.2%
99.4%
74.1%*

Lactation index (pup survival to PND21)

100%
100%
100%
99.2%
98.8%

Sex ratio (% males)

54.4
52.3
50.9
53.4
52.1

Pup weight/litter PND 1 relative to control (%)

0
+6
+4
+7
-10

Pup weight PND 28 relative to control (%)

M: 0
+2
0
0
-12*

F: 0
0
-4
-2
-8
Nelson et al. (1989)
Sprague-Dawley rat; 15 pregnant
dams/treatment
Maternal: Unsteady gait (no statistical tests reported), dose-dependent \|/ in
body weight gain (results presented in figure only), dose-dependent \|/ in food
consumption ranging from 7-36% depending on dose and time
Fetal
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Toxicological Review of tert-Butyl Alcohol
Table 1-9. Evidence pertaining to developmental effects in animals following
exposure to tert-butanol (continued)
Reference and study design
Resu Its
Analytical concentration: 0, 2,200, 3,510,
5,030 ppm (0, 6,669, 10,640,
15,248 mg/mB), (dynamic whole body
chamber)
7 hr/d
GD 1-19
Generation method, analytical
concentration and method were reported
Percent change compared to control:
Dose
(mg/m3)
Number of
live
fetuses/litter
Resorptions
oer litter

0
0
0

6,669
0
+9

10,640
+15
-18

15,248
+8
0

Percent change compared to
control:
Incidence:

Dose
(mg/m3)
Fetal weight
(males)
Fetal weight
(females)
Skeletal
variation
bv litter
Skeletal
variation
bv fetus
0
0
0
10/15
18/96
6,669
—9*
—9*
14/17
35/104
10,640
-12*
-13*
14/14
53/103*
15,248
-32*
-31*
12/12
76/83*
Skeletal variation by litter refers to the number of variations observed in the
number of litters examined. Skeletal variation by fetus refers to the number of
variations observed in the total number of fetuses examined. Fetuses are not
categorized by litter.
1 * Statistically significant p < 0.05 as determined by study authors.
2 Conversions from diet concentrations to mg/kg-d performed by study authors.
3 Conversion from ppm to mg/mB is 1 ppm = 3.031 mg/mB.
4 Percentage change compared to control = (treated value - control value) -f control value x 100.
5
6
7
8
9
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Toxicological Review of tert-Butyl Alcohol
¦ = 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
Reproductive organs or sperm; M rat (C
Reproductive organs or sperm; M rat (B
Number of live pups per litter; F rat (B
Estrous cycle length; F rat (C
REPRODUCTIVE
Pregnancy index; F rat (B
Reproductive organs or sperm; M mouse (C
Estrous cycle length; F mouse (C
Maternal body weight gain (CDs 0-20); F rat(B
Maternal body weight gain (IDs 1-21); F rat (B
Pup weight per litter (PND 28); M rat (B
Pup weight per litter (PND 28); F rat (B
Viability index; M+F rat (B
Pup weight per litter (PND1); M+F rat (B
DEVELOPMENTAL Sex ratio; m+F rat (B
lactation index; M+F rat (B
Number of resorptions; M+F mouse (A
Number of live fetuses per litter; M+F mouse (A
Pup weight; M+F mouse (A
Skeletal variations; M+F mouse (A
B—e-B-a—a
0 B
D ~~ ~—0
B— -B-—B 0
B—B B B—Qi
B~B B B
0 B-
~ I ~ B ¦
~—1—S B-
~ ~ B B
Q——S B-
B—B B—B

10 100 1,000 10,000 100,000
Dose (mg/kg-day)
Sources: (A) Faulkner etal. 119891: (B) I.vondfill Chemical Co. 120041: (C) NTP 119951
Figure 1-6. Exposure-response array of reproductive and developmental
effects following oral exposure to tert-butanol.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
¦ = 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
REPRODUCTIVE
Reproductive organs or sperm; M rat (A)
Estrous cycle; F rat (A)
Reproductive organs or sperm; M mouse (A)
Estrous cycle; F mouse (A)
DEVELOPMENTAL
Fetal weight; M rat (B)
Fetal weight; F rat (B)
Skeletal variation; M+F rat (B)
Number of resorptions per litter; M+F rat(B)
Number of live pups per litter; M+F rat (B)
Q B—Q
-a—a
~ B—E3

B-M
0—B-B

100 1,000 10,000 100,000
Concentration (mig/m3)
Sources: (A) NTP (1997): (B) Nelson etal. f 19891
Figure 1-7. Exposure-response array of reproductive and developmental
effects following inhalation exposure to tert-butanol.
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Neurodevelopmental Effects
In addition to the developmental effects noted above, neurodevelopmental effects also have
been observed. This includes changes in rotarod performance following oral or inhalation
exposures, as well as decreases in open field behavior and cliff avoidance following oral exposure,
and reduced time hanging on wire after inhalation exposure during gestation (Table 1-10; Figure
1-6; Figure 1-7).
Rotarod performance
Looking across studies, not all the findings were consistent. While Daniel and Evans T19821
found decreased rotarod performance in mouse pups ol dams orally exposed during gestation,
Nelson etal. T19911 observed an increase in rotarod perlorma nee in rat pups of dams exposed via
inhalation during gestation.
Neurochemical measurements
In addition to behavioral effects, one study evaluated biochemical or physiological changes
in the brain of offspring exposed during gestation or early in the postnatal period. Nelson etal.
fl9911 found statistically significant changes in neurochemical measurements in the brain in
offspring of dams exposed via inhalation (.luring gestation; however, the two concentrations tested
were not run concurrently, and very little (.lata were provided.
Physiological and psychomotor development
Data also suggest that neurodevelopmental effects were not solely due to in utero exposure
fDaniel and Evans. Daniel and Evans f 19821 cross-fostered half of the mouse pups born to
treated mother with untreated surrogate females to lest the effects ofmaternal nutrition and
behavioral factors on the pups' physiological and psychomotor development. Results indicated that
pups fostered with control dams performed significantly better than those maintained with treated
dams(Talile 1-10) fDaniel and Kvans. 1982). Results were only presented in figures and were not
compared with controls.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
1 Table 1-10. Evidence pertaining to neurodevelopmental effects in animals
2 following exposure to tert-butanol
Reference and study design
Resu Its
Daniel and Evans (1982)
Liquid diet (0, 0.5, 0.75, or 1.0%, w/v); GD6-20;
Swiss Webster (Cox) mouse; 15 pregnant
dams/treatment; after birth half the pups were
nursed with their treated dams and the other
half were fostered by untreated dams who
recently gave birth
0 (isocaloric amounts of maltose/dextrin),
3,324, 4,879, or 6,677 mg/kg-d
• a dose-dependent increase righting reflex time, with more time
needed in animals maintained with maternal dams
• a dose-dependent decrease in open field behavior, with less activity
in pups maintained with maternal dams
• a dose-dependent decrease in rotarod performance with the pups
from maternal dams having lower performances
• a dose-dependent decrease in the amount of time the pups were
able to avoid a cliff, with animals maintained with their maternal
dams having less avoidance time
Nelson et al. (1991)
Sprague-Dawley rat; 15 pregnant
dams/treatment
Analytical concentration: 0, 6,000, or 12,000
mg/mB; (dynamic whole body chamber)
7 hr/d
GD 1-19
Generation method, analytical concentration
and method were reported
Data were not presented specifically by dose nor were any tables or figures
of the data provided
Maternal toxicity was noted by decreased food consumption and body
weight gains
Results in offspring
• increase in rotarod performance in high-dose group (16 versus 26
revolutions/min for controls and 12,000 mg/mB animals,
respectively)
• decreased time held on wire in the performance ascent test in the
low-dose group (16 sec versus 10 sec for controls and 1,750 mg/mB
animals, respectively)
The following differences in neurochemical measurements in the brain
between control and treated offspring were observed,
• 53% decrease in norepinephrine in the cerebellum at
12,000 mg/mB
• 57% decrease in met-enkephalin in the cerebrum at
12,000 mg/mB and 83% decrease at 6,000 mg/mB
• 61% decrease in (3-endorphin in the cerebellum at 12,000 mg/mB
• 67% decrease in serotonin in the midbrain at 6,000 mg/mB
3
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
Table 1-10. Evidence pertaining to neurodevelopmental effects in animals
following exposure to tert-butanol (continued)
Reference and study design
Resu Its
Nelson et al. (1991)
adult male Sprague-Dawley rats (18/treatment)
mated to untreated females
Analytical concentration: 0, 6,000, or
12,000 mg/mB; (dynamic whole body chamber)
7 hr/d for 6 wk
Generation method, analytical concentration
and method were reported
Data were not presented specifically by dose nor were any tables or figures
of the data provided
Results (generally only specified as paternally treated versus controls) in
offspring indicate
• increase in rotarod performance (16 versus 20 revolutions/min for
controls and 12,000 mg/m3 animals, respectively)
• decreased time in open field (less time to reach the outer circle of
the field, 210 sec versus 115 seconds for controls and 12,000 mg/mB
animals, respectively)
The following differences in neurochemical measurements in the brain
between control and treated offspring were observed
• 39% decrease in norepinephrine in the cerebellum at
12,000 mg/mB
• 40% decrease in met-enkephalin in the cerebrum at
12,000 mg/mB and 75% decrease at 6,000 mg/mB
• 71% decrease in (3-endorphin in the cerebellum at 12,000 mg/mB
• 47% decrease in serotonin in the midbrain at 6,000 mg/mB
1 * Statistically significant p < 0.05 as determined by study authors.
2 Conversions from diet concentrations to mg/kg-d performed by study authors.
3 Percentage change compared to control = (treated value - control value) 4- control value x 100.
4
5 Mechanistic Evidence
6 No mechanistic evidence is available lor reproductive or developmental effects, including
7 neurodevelopmental effects.
8 Summary of Reproductive and Developmental Toxicity
9 El'A concluded that the evidence does not support reproductive effects as a potential
10 human hazard of ftrf-butanol exposure. There are no two-generation reproductive studies
11 available by oral or inhalation exposure. Two oral exposure studies fLvondell Chemical Co.. 2004:
12 NTP. 19951 and one sulichronic inhalation study fNTP. 19971 are available. Overall, reproductive
13 effects observed due to exposure to tert-butanol were limited to altered length of estrous cycle
14 fNTP. 19951. but there is no available information to infer how this effect may influence
15 reproductive ability.
16 EPA identified suggestive evidence of developmental effects as a potential human hazard of
17 tert-butanol exposure. Exposure during gestation resulted in increased fetal loss, decreased fetal
18 body weight, and possible increases in skeletal variations in exposed offspring or pups, although
19 effects were not always consistent across exposure routes (oral and inhalation). Dams had
20 decreased body weight and/or body weight gains, decreased food consumption, and/or clinical
21 signs of intoxication at the same doses that tert-butanol caused fetal effects. Neurodevelopmental
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
Toxicological Review of tert-Butyl Alcohol
effects including decreased brain weight, changes in brain biochemistry, and changes in behavioral
performances have also been observed. Each of the neurodevelopmental studies, however, had
limitations in the study design and/or reporting. In addition, results from the neurodevelopmental
studies were not always consistent between studies or across dose.
1.1.4. Carcinogenicity (other than in the kidney or thyroid)
Synthesis of Carcinogenicity Data (Other than in the Kidney or ThyroidJ
This section reviews the studies that investigated whether exposure to tert-butanol can
cause cancers (other than in the kidney or thyroid) in humans or animals. The database examining
carcinogenicity following tert-butanol exposure contains no human (.lata and two chronic studies,
one in rats and one in mice. As mentioned in the Study Selection, the studies providing data on
carcinogenicity exposed animals via drinking water for >30 days. Shorter duration studies do not
generally evaluate carcinogenicity, but any shorter duration studies that examined carcinogenicity
are discussed in the text if they provide data to support mode of action or hazard identification. No
methodological concerns were identified that would lead one or more studies to lie considered less
informative for assessing human health hazard.
Kidney and thyroid tumors are presented above with the specific organ hazard
identification. No other treatment-related changed in tumors in other organs were noted in the 2-
year oral rator mouse studies conducted by N'l'l' (l')').r>). which ev aluated a comprehensive setof
tissues/organs. There is no 2-year inhalation study.
Mechanistic Evidence
Available mechanistic evidence was previously discussed in the context of kidney and
thyroid tumors (Sections 1.1.1 and 1.1.2).
Summary of Carcinogenicity Evidence
There are limited (.lata av ailable on the carcinogenicity of tert-butanol. There are 2-year
oral studies in one strain of rats and one strain of mice, butno 2-year inhalation studies. EPA
identified suggestive evidence of kidney and thyroid tumors as a potential human hazard.
1.1.5. Other Toxicological Effects
Synthesis of Other Toxicity Data
The database for effects other than kidney, thyroid, reproductive, developmental (including
neurodevelopmental) and cancer contain only 14 rodent studies. As previously mentioned in the
Study Selection, all selected studies employed inhalation, oral gavage, or drinking water exposures
for >30 days. Studies employing short term and acute exposures that examined other toxicological
effects are not included in the evidence tables; however, they are discussed in the text if they
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
21
22
23
24
25
26
27
28
29
30
31
32
33
34
Toxicological Review of tert-Butyl Alcohol
provide data to support mode of action or hazard identification. No studies were removed for
methodological concerns.
tert-Butanol also has been found to have CNS effects similar to ethanol in terms of animals
appearing intoxicated and having withdrawal symptoms after cessation of oral or inhalation
exposure. Severity of CNS symptoms such as withdrawal increased with dose and duration of
exposure. However, study quality concerns (e.g., short exposure durations, lack of data reporting,
small number of animals per treatment group) associated with all of the studies in the database
preclude a clear understanding of potential neurotoxicity following tert-butanol exposure, and
therefore, CNS studies are not presented in evidence tabk-s.
Effects in other tissues were observed with less consislf nt'v. These include decreased body
weight, liver effects, and urinary bladder effects.
Body weight
Body weightwas decreased by >10% in hoth rats and mice with suhdironic and chronic
exposure, with males generally more affected than females (Table 1-11). The font-titrations used
in the subchronic inhalation study did not decrease body wt-iglils. However, a short-term (i.e., 18-
day) inhalation study in rats observed a >10",, tiff rfast- in body weight at concentrations about
threefold higher (in nig/111:) limn llit- hight-sl onio.-nlralion ust-d in the subchronic study. The same
concentrations did nol haw any (_¦ Heel on Ihc hotly wcighl in miff with short-term inhalation
exposure.
Liver effects
Although sonif rotlf ill slutlifs ohsf rvt-tl sliilisliuilly significant changes in relative liver
weighl willi ) suhfhronic and chronic studies did not
observe Irfalnit1 nt-rflalfd t-1 Iff Is on livt- r hislo|ialhologv in both sexes of F344 rats, but in a 10-
week study in a tlil lf rent rat slrain (Wistar rats), several liver lesions (including necrosis) and
increased liver gl vcogf n wert- st-f n in male rats (no females were included in the study) with the
only dose used (Acharya fl al.. l')')7: Acharva etal.. 1995). The study did not provide any incidence
or severity data. The dost- usftl in this study was in the range of the lower doses used in the NTP
f!9951 study. In the developmental study by Lvondell Chemical Co. f20041. the Fl SD rats treated
by tert-butanol for at least 9 weeks did not show any liver effects. An increased incidence of fatty
liver was observed in the male mice of the highest dose group in the 2-year mouse bioassay, but no
histopathologic changes were seen in the subchronic mouse study. No changes in liver
histopathology were observed in the NTP (1997) subchronic inhalation study.
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
Toxicological Review of tert-Butyl Alcohol
Urinary bladder effects
Several studies also reported effects in the urinary bladder (Table 1-14). Transitional
epithelial hyperplasia was observed in male rats and mice after 13 weeks of exposure at doses of
3,610 mg/kg-day (male rats) and >3940 mg/kg-day (male mice). Male mice exposed at doses of
2,070 mg/kg-day for 2 years of also exhibited transitional epithelial hyperplasia. Neither female
rats nor female mice showed increased incidences of transitional epithelial hyperplasia. Both sexes
of mice demonstrated incidence of inflammation in the urinary bladder after both subchronic and
chronic exposures, with a greater incidence in males compared to females.
An exposure-response array of these effects in body weight, I iver, and urinary bladder is
provided in Figure 1-8 and Figure 1-9 for oral and inhalation studies, respectively.
Mechanistic Evidence
No mechanistic evidence is available for these other toxicological (.-fleets.
Summary of Other Toxicity Data
EPA concluded that the evidence docs not support hotly weight changes, I iver effects, and
urinary bladder effects as potential human hazards oi tc/V-hulanol exposure.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
1 Table 1-11. Evidence pertaining to effects on body weight in animals
2 following exposure to tert-butanol
Reference and study design
Resu Its
Acharva et al. (1995)
Wistar rat; 5-6 males/treatment
Drinking water (0 or 0.5%), 0 or 575 mg/kg-d
10 weeks
Body weight in treated animals lower than controls by
only provided in a figure)
~7% (p< 0.05); (results
Lvondell Chemical Co. (2004)
Percent change compared to control:

Sprague-Dawley rat; 12/sex/treatment
Gavage 0, 64,160, 400, or 1,000 mg/kg-d
F0 males: 9 weeks beginning 4 weeks prior to
mating
F0 females: 4 weeks prior to mating through
PND21
F0 Males
Dose
(mg/kg-d)
0
64
Bodv weight
0
-2
F0 Females
Dose
(mg/kg-d)
0
64
Bodv weight
0
0

160
-4
160
-2

400
+2
400
+1

1,000
-7
1,000
+4
NTP (1995)
Percent change compared to control:

F344/N rat; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or 40 mg/mL)
M: 0, 230, 490, 840, 1,520, 3,610a mg/kg-d
F: 0, 290, 590, 850, 1,560, 3,620a mg/kg-d
13 weeks
Males
Dose
(mg/kg-d)
0
230
Bodv weight
0
-4
Females
Dose
(mg/kg-d)
0
290
Bodv weight
0
+2

490
-5*
590
+1

840
-12*
850
+1

1,520
*
1^
T—1
1
1,560
-2

3,610
All dead
3,620
-21*
NTP (1995)
Percent change compared to control:

B6C3Fi mouse; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or 40 mg/mL)
M: 0, 350, 640, 1,590, 3,940, 8,210a mg/kg-d
F: 0, 500, 820, 1,660, 6,430, ll,620a mg/kg-d
13 weeks
Males
Dose
(mg/kg-d)
0
350
Bodv weight
0
-1
Females
Dose
(mg/kg-d)
0
500
Bodv weight
0
+3

640
+1
820
-1

1,590
-4
1,660
+4

3,940
*
-------
Toxicological Review of tert-Butyl Alcohol
Table 1-11. Evidence pertaining to effects on body weight in animals
following exposure to tert-butanol (continued)
Reference and study design
Resu Its
NTP (1995)
F344/N rat; 60/sex/treatment
(10/sex/treatment evaluated at 15 months)
Drinking water (0,1.25, 2.5, 5,10 mg/mL)
M: 0, 90, 200, 420a mg/kg-d
F: 0,180, 330, 650a mg/kg-d
2 years
Percent change compared to control:
Males

Females

Dose

(mg/kg-d)
Body weight
(mg/kg-d)
Body weight
0
0
0
0
90
-15
180
-2
200
-18
330
-5
420
-24
650
-21
Only animals that survived at the end of 2 years were evaluated for body
weight. Note: statistical significance not determined by study authors.
NTP (1995)
B6C3Fj mouse; 60/sex/treatment
Drinking water (0, 5,10, 20 mg/mL);
M: 0, 540,1,040, 2,070a mg/kg-d
F: 0, 510,1,020, 2,110 mg/kg-d
2 years
Percent change compared to control:
Males
Dose
(mg/kg-d)
0
540
1.040
2,070
Body weight
0
+1
-2
-1
Females
Dose
(mg/kg-d)
0
510
1,020
2,110
Body weight
0
-2
-3
-12
Only animals that survived at the end of 2 years were evaluated for body
weight. Note: statistical significance not determined by study authors.
NTP (1997)
F344/N rat; 10/sex/treatment
Analytical concentration: 0,134, 272, 542,
1,080, or 2,101 ppm (0, 406, 824,1,643, 3,273
or 6,368 mg/mB) (dynamic whole body
chamber)
6 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist Ultrasonic spray
nozzle nebulizer), analytical concentration and
method were reported
Percent change compared to control:
Concentration
(mg/mB)
0
406
824
1,643
3,273
6,368
Males
Body weight
Females
Body weight
0
0
-1
-5
-2
-1
+2
0
+3
0
+2
-3
NTP (1997)
B6C3Fj mouse; 10/sex/treatment
Analytical concentration: 0,134, 272, 542,
1,080, or 2,101 ppm (0, 406, 824,1,643, 3,273
or 6,368 mg/mB) (dynamic whole body
chamber)
6 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist Ultrasonic spray
nozzle nebulizer), analytical concentration and
Percent change compared to control:
Concentration Males
(me/m3) Body weight
0 0
406 +4
824 -2
1,643 0
Females
Body weight
0
+3
-3
+3
3,273
-4
-6
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
Table 1-11. Evidence pertaining to effects on body weight in animals
following exposure to tert-butanol (continued)
Reference and study design
Resu Its
method were reported
6,368 0 -8
1 aThere was a significant decrease in survival in the high-dose group.
2 * Statistically significant p < 0.05 as determined by study authors.
3 Conversions from drinking water concentrations to mg/kg-d performed by study authors.
4 Percentage change compared to control = (treated value - control value) -f control value x 100.
5
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Toxicological Review of tert-Butyl Alcohol
1 Table 1-12. Changes in liver weight in animals following exposure to
2 tert-butanol
Reference and study design
Results
Acharva et al. (1995)
Wistar rat; 5-6 males/treatment
Drinking water (0 or 0.5%), 0 or 575 mg/kg-d
10 weeks
No significant treatment-related effects (results were only provided in a
figure)
Lvondell Chemical Co. (2004)
Percent change compared to control:

Sprague-Dawley rat; 12/sex/treatment
Gavage 0, 64,160, 400, or 1,000 mg/kg-d
Males: 9 weeks beginning 4 weeks prior to
mating
Females: 4 weeks prior to mating through
PND21
Males
Dose
(mg/kg-d)
0
64
Absolute
weight
0
-1
Relative
weight
0
0
Females
Dose
(mg/kg-d)
0
64
Absolute
weight
0
-4
Relative
weight
0
-4

160
-3
+1
160
-7
-5

400
-2
-1
400
+2
+1

1,000
+8
+16*
1,000
+8
+3
NTP (1995)
Percent change compared to control:

F344/N rat; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or 40 mg/mL)
M: 0, 230, 490, 840, 1,520, 3,610a mg/kg-d
F: 0, 290, 590, 850, 1,560, 3,620a mg/kg-d
13 weeks
Males
Dose
(mg/kg-d)
0
Absolute
weight
0
Relative
weight
0
Females
Dose
(mg/kg-d)
0
Absolute
weight
0
Relative
weight
0

230
-2
+4
290
+11*
+9*

490
+1
+8*
590
+10*
+9*

840
+5
+20*
850
+12*
+11*

1,520
+8
+31*
1,560
+15*
+16*

3,610
All dead
All dead
3,620
+9*
+41*
NTP (1995)
Percent change compared to control:

B6C3FJ mouse; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or 40 mg/mL)
M: 0, 350, 640, 1,590, 3,940, 8,210a mg/kg-d
F: 0, 500, 820, 1,660, 6,430, ll,620a mg/kg-d
13 weeks
Males
Dose
(mg/kg-d)
0
Absolute
weight
0
Relative
weight
0
Females
Dose
(mg/kg-d)
0
Absolute
weight
0
Relative
weight
0

350
+2
+3
500
-1
-4

640
-1
-2
820
-5
-3

1,590
-1
+5
1,660
-8

3,940
0
+14*
6,430
-2
+6

8,210
-16
+22*
11,620
-6
+13*
3
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
Table 1-12. Changes in liver weight in animals following exposure to
tert-butanol (continued)
Reference and study design
Results
NTP (1995)
F344/N rat; 60/sex/treatment
(10/sex/treatment evaluated at 15 months)
Drinking water (0,1.25, 2.5, 5 or 10 mg/mL)
M: 0, 90, 200, or420a mg/kg-d
F: 0,180, 330, or 650a mg/kg-d
2 years
Percent change compared to control:
Males

Females

Dose
(mg/kg-d)
Absolute
weight
Relative Dose
weight (mg/kg-d)
Absolute
weight
Relative
weight
0
0
0
0
0
0
90
+2
+7
180
*
-------
Toxicological Review of tert-Butyl Alcohol
1 Conversion from ppm to mg/mB is 1 ppm = 3.031 mg/mB.
2 Percentage change compared to control = (treated value - control value) 4- control value x 100.
3
4 Table 1-13. Changes in liver histopathology in animals following exposure to
5 tert-butanol
Reference and study design
Results
Acharva etal. (1997: 1995)
'T* liver glycogen (~ 7 fold)*
Wistar rat; 5-6 males/treatment
Drinking water (0, 0.5%), 0, 575 mg/kg-d
10 weeks
^incidence of centrilobular necrosis, vacuolation of hepatocytes, loss of
hepatocyte architecture, peripheral proliferation, and lymphocyte
infiltration (incidences and results of statistical tests not reported)
Lvondell Chemical Co. (2004)
No treatment-related effects observed.
Sprague-Dawley rat; 12/sex/treatment
Gavage 0, 64,160, 400, or 1,000 mg/kg-d

Males: 9 weeks beginning 4 weeks prior to
mating
Females: 4 weeks prior to mating through
PND21

NTP (1995)
F344/N rat; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or 40 mg/mL)
M: 0, 230, 490, 840, 1,520, 3,610a mg/kg-d
F: 0, 290, 590, 850, 1,560, 3,620a mg/kg-d
13 weeks
Histopathology data for the 13-week study were not provided, but the liver
was evaluated indicating that no changes in liver histopathology were
observed in the 13-week study.
NTP (1995)
B6C3F;l mouse; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, 40 mg/mL)
M: 0, 350, 640, 1,590, 3,940, 8,210a mg/kg-d
F: 0, 500, 820, 1,660, 6,430, ll,620a mg/kg-d
13 weeks
Histopathology data for the 13-week study were not provided, but the liver
was evaluated indicating that no changes in liver histopathology were
observed in the 13-week study.
NTP NTP (1995)
No treatment-related effects observed.
F344/N rat; 60/sex/treatment
(10/sex/treatment evaluated at 15 months)
Drinking water (0,1.25, 2.5, 5,10 mg/mL)
M: 0, 90, 200, or 420a mg/kg-d
F: 0,180, 330, or 650a mg/kg-d
2 years

6
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
Table 1-13. Changes in liver histopathology in animals following exposure to
tert-butanol (continued)
Reference and study design
Results
NTP (1995)
Males Females
B6C3FJ mouse; 60/sex/treatment
Drinking water (0, 5,10, 20 mg/mL)
M: 0, 540,1,040, or 2,070a mg/kg-d
Dose Incidence of fatty Dose Incidence of fatty
(mg/kg-d) change (mg/kg-d) change
F: 0, 510,1,020, or 2,110 mg/kg-d
0 12/59 0 11/60
2 years
540 5/60 510 8/60
1,040 8/59 1,020 8/60
2,070 29/59* 2,110 6/60
NTP (1997)
Histopathology data for the 13-week study were not provided, but the liver
was evaluated in control and high-dose group indicating that no changes in
F344/N rat; 10/sex/treatment
liver histopathology were observed in the 13-week study.
Analytical concentration: 0,134, 272, 542,

1,080, or 2,101 ppm (0, 406, 824,1,643, 3,273

or 6,368 mg/mB) (dynamic whole body

chamber)

6 hr/d, 5 d/wk

13 weeks

Generation method (Sonimist Ultrasonic spray

nozzle nebulizer), analytical concentration and

method were reported

NTP (1997)
Authors stated that there were no treatment-related microscopic
observations, but data were not provided.
B6C3Fj mouse; 10/sex/treatment

Analytical concentration: 0,134, 272, 542,

1,080, or 2,101 ppm (0, 406, 824,1,643, 3,273

or 6,368 mg/mB) (dynamic whole body

chamber)

6 hr/d, 5 d/wk

13 weeks

Generation method (Sonimist Ultrasonic spray

nozzle nebulizer), analytical concentration and

method were reported

1 aThe high-dose group had an increase in mortality.
2 * Statistically significant p < 0.05 as determined by study authors.
3 Conversions from drinking water concentrations to mg/kg-d performed by study authors.
4 Conversion from ppm to mg/mB is 1 ppm = 3.031 mg/mB.
5
6
7
8
9
10
11
12
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Toxicological Review of tert-Butyl Alcohol
Table 1-14. Changes in urinary bladder histopathology in animals following
oral exposure to tert-butanol
Reference and study design
Results
NTP (1995)
Incidence (severity):

F344/N rat; 10/sex/treatment
Males

Females

Drinking water (0, 2.5, 5,10, 20, 40

Transitional

Transitional
mg/mL)

epithelial

epithelial
M: 0, 230, 490, 840, 1,520, 3,610a
Dose (ms/ks-d)
hyperplasia
Dose (ms/ks-d)
hyperplasia
mg/kg-d
F: 0, 290, 590, 850, 1,560, 3,620a
0
0/10
0
0/10
mg/kg-d
230
not evaluated
290
not evaluated
13 weeks
490
not evaluated
590
not evaluated

840
0/10
850
not evaluated

1,520
1/10 (3.0)
1,560
0/10

3,610
7/10* (2.9)
3,620
3/10 (2.0)
Severity: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked
NTP (1995)
B6C3Fj mouse; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, 40
mg/mL)
M: 0, 350, 640, 1,590, 3,940,
8,210a mg/kg-d
F: 0, 500, 820, 1,660, 6,430,
ll,620a mg/kg-d
13 weeks
Incidence (severity):
Males
Dose
(mg/kg-d)
0
350
640
1,590
3,940
8,210
Transitional
epithelial
hyperplasia
0/10
Inflam-
mation
0/10
not evaluated
not evaluated
0/10 0/10
6/10* (1.3) 6/10* (1.3)
10/10* (2.0) 10/10* (2.3)
Severity: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked
Females
Dose
(mg/kg-d)
0
500
820
I,660
6,430
II,620
Transitional

epithelial
Inflam-
hyperplasia
mation
0/10
0/10
0/1
0/1
not evaluated
not evaluated
0/10 0/10
3/9 (2.0) 6/9* (1.2)
NTP (1995)
F344/N rat; 60/sex/treatment
(10/sex/treatment evaluated at 15
months)
Drinking water (0,1.25, 2.5, 5, or 10
mg/mL)
M: 0, 90, 200, 420a mg/kg-d
F: 0,180, 330, 650a mg/kg-d
2 years
No treatment-related effects observed
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
Table 1-14. Evidence pertaining to urinary bladder effects in animals
following oral exposure to tert-butanol (continued)
Reference and study design
Results
NTP (1995)
Incidence (severity):

B6C3Fj mouse; 60/sex/treatment
Males

Females

Drinking water (0, 5,10, or 20 mg/mL)

Transitional

M: 0, 540,1,040, 2,070a mg/kg-d
Dose
epithelial
Inflam-
Dose
epithelial
Inflam-
F: 0, 510,1,020, 2,110 mg/kg-d
(mg/kg-d)
hyperplasia
mation
(mg/kg-d)
hyperplasia
mation
2 years
0
1/59 (2.0)
0/59
0
0/59
0/59

540
3/59 (1.7)
3/59 (1.7)
510
0/60
0/60

1,040
1/58 (1.0)
1/58 (1.0)
1,020
0/59
0/59

2,070
17/59* (1.8)
37/59* (2.0)
2,110
3/57(1.0)
4/57* (2.0)

Severity: 1 =
minimal, 2 = mild, 3 = moderate, 4 = marked

1 aThe high-dose group had an increase in mortality.
2 * Statistically significant p < 0.05 as determined by study authors.
3 Conversions from drinking water concentrations to mg/kg-d performed by study authors.
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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
x = exposures at which all animals were dead and unable to be examined for the endpoint
H
*3
0
HnSubchronic
W
£
£5 — —
M rat (D)
M rat (A)
F rat (D)
M mouse (D)
F mouse (D)
O
CO Reproductive
M rat (C)
F rat (C)
-X
~ ~~ ~
~—B B-
~ ~ ~
—El
B-^
Transitional epithelium hyperplasia; M rat (D)
Transitional epithelium hyperplasia; F rat (D)
p Transitional epithelium hyperplasia; M mouse (D)
0L , , . Inflammation; M mouse (D)
¦®5»ubcnronic
g Inflammation; F mouse (D)
Transitional epithelium hyperplasia; F mouse (D)
-B—Q
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Toxicological Review of tert-Butyl Alcohol
¦ = 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
BODY WEIGHT EFFECTS
Body weight; M rat (A) B B 0 B El
Body weight; F rat (A) ~ B B B ~
Body weight; M mouse (A) B B- B B ~
Body weight; F mouse (A) B 0 B B B
Absolute liver weight; M rat (A) ~ —B B— B———H
Relative liver weight; M rat (A) B ——B- B B 0
Absolute liver weight; F rat (A) B—B-- B ---a— B
Relative liver weight; F rat (A) B~—-B- B~ ——¦
Absolute liver weight; M mouse (A) Q B- B B B
Relative liver weight; M mouse (A) Q ~0 B B ¦—B
LIVER EFFECTS
Absolute liver weight; F mouse (A) O 1—B B B B
Relative liver weight; F mouse (A) B B B ¦ ¦
Liver histopathology; M rat (A) ~ B- B B B
Liver histopathology; F rat (A) B B B B B
Liver histopathology; M mouse (A) B 0— B B B
Liver histopathology; F mouse (A] Q B B B B
100 1,000 10,000
Concentration (mg/m3)
Source: (A) NTP 119971
Figure 1-9. Exposure-response array of other effects following inhalation
exposure to tert-butanol.
-B B
B-
-B B B
-B B B
B-
_g H Q
_g_ B_ B— ~
B-
B-
-B B B
-B ———B—¦—O

-a a B
-b b——a

S B
~B B
-a b b
-B B B
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1.2. INTEGRATION AND EVALUATION
1.2.1. Effects Other Than Cancer
The strongest evidence following tert-butanol exposure is for kidney, with toxicity observed
after oral exposure in two strains of rats and in one strain of mice and in both sexes. In mice, the
only kidney effect observed was an increase in kidney weight (absolute and/or relative) in both
sexes of mice in the 13-week study, but no treatment-related histopathological lesions were
reported in the kidneys of mice at the 13-week or 2-year time points (NTP. 19951. In male rats,
effects related to the accumulation of a2U-globulin in the kidney have been reported, including
precursors to granular casts, linear mineralization, and tubular hyperplasia, but these are not
considered relevant to humans (Hard etal.. 2011: Cirvello et al.. NTP. 1995: Lindamood etal..
19921. However, several other effects in the kidney unrelated to a ¦„-»!<>hulin were observed in
female and/or male rats. Absolute and relative kidney weights were increased in both male and
female rats after both 13 weeks and 15 months of treatment (NTP. 1995). Histopathological
examination also indicated kidney toxicity in both male and female rats, with increased incidence of
nephropathy after 13 weeks of oral exposure and transitional epithelium hyperplasia observed
after 2 years of oral exposure fNTP. l')')5). Additionally, increased inflammation (suppurative) was
noted in females after 2 years oral exposure (NTP. l')')5). EPA identified kidney effects as a human
hazard of tert-butanol oral exposure.
Fewer and less sev ere kidney effects were observed via inhalation than via oral exposure,
likely due to the differing levels of internal (.loses achieved via the different routes. Specifically,
available inhalation studies (NTP. were conducted at concentrations that are comparable, in
terms of tert- butanol blood concentration, to the lower range of doses in oral studies. Moreover,
there is convincing loxicokinelic (.lata to indicate that fert-butanol is absorbed by both routes, and
kidney effects are remote from the site of absorption. EPA identified kidney effects as a human
hazard of/(.'//-butanol inhalation exposure.
Thyroid follicular cell hyperplasia was observed in the mice after 2 years of exposure via
drinking water (NTP. 19951: and EPA identified thyroid effects as a potential human hazard of tert-
butanol exposure. I lowever, this endpoint most likely reflects early events in the neoplastic
progression ofthyroid follicular cell tumors following tert-butanol exposure (see Section 1.1.2) and
was not considered further lor dose-response analysis and derivation of noncancer reference
values.
EPA identified suggestive evidence of developmental effects as a potential human hazard of
tert-butanol exposure. Exposure to high doses of tert-butanol during gestation resulted in some
effects in exposed offspring or pups, although the effects were not always consistent across
exposure routes (oral and inhalation). Dams exhibit effects at the same doses as fetal effects.
Neurodevelopmental effects have also been observed; however, the neurodevelopmental studies
had limitations in the study design and/or reporting and results were inconsistent between studies
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or across dose. Thus, these effects were not considered further for dose-response analysis and
derivation of reference values.
EPA concluded that the evidence does not support reproductive effects, body weight
changes, liver effects, and urinary bladder effects as potential human hazards of tert-butanol
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 (M.S. I'PA. 2005a). the database for
tert- butanol provides "suggestive evidence of carcinogenic potential," based on a statistically
significant increase in renal tumors (renal tubule adenomas and carcinomas) in male F344 rats and
a statistically significant increase in thyroid follicular cell adenomas in female B6C3Fi mice, all
exposed to tert-butanol in drinking water for 2 years (Cirvello etal.. NTP. 19951. There are
no available studies of cancer in humans associated with exposure to te/Miutanol.
In the NTP (19951 rodent bioassay, te/i-buUinol-cxposcd male rats had a significant
increase in renal tumors compared to controls, a result confirmed hy a PWG revaluation (Hard et
al.. 20111. Although mechanistic data show that it u-glolnilin-rclaled processes occur with tert-
butanol exposure, there is insufficient ev idence to support a conclusion thatc^u-globulin
nephropathy is the sole or primary contributor to renal tumor development Specifically,
tert-butanol induced tumors al lower doses than those for other precursor effects such as
hyperplasia and granular casts, with no further increase in tumor incidence coinciding with the
induction of additional markers of it u-gloliulin nephropathy. Based on analysis of available mode
of action data, these tumors are not allrilnileil to it ^-globulin and are considered relevant in
humans (M.S. li lJA. !')')! a). /t'/MUilanol was negative in a variety of genotoxicity assays in different
cell systems including gene mutations, sister chromatid exchanges, micronucleus formation, and
chromosomal aberrations. However, I )N A adducts in male Kunming mice and DNA damage in
human HI,-(>0 leukemia cells have been observed. Overall, the mode(s) of carcinogenic action for
tert-butanol in the kidney anil the thyroid are not known, and these tumor data are considered
relevant to humans.
As emphasized in the Cancer Guidelines (U.S. EPA. 2005a). selection of the cancer descriptor
follows a full evaluation of the available evidence. The carcinogenicity evidence for tert-butanol
could be considered a borderline case between two cancer descriptors—"suggestive evidence of
carcinogenic potential" and "likely to be carcinogenic to humans." 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 not sufficient for a stronger conclusion. Exposure to
tert-butanol produced a positive tumor response at more than one site (kidney and thyroid) and in
more than one species (rat and mouse). These data appear to correspond closely to one of the
examples in the Cancer Guidelines (U.S. EPA. 2005a) for the descriptor of "likely to be carcinogenic
to humans;" i.e., "an agent that has tested positive in animal experiments in more than one species,
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sex, strain, site, or exposure route, with or without evidence of carcinogenicity in humans." Several
aspects of the data support the conclusion that these data are not sufficient to characterize tert-
butanol as "likely to be carcinogenic to humans."
First, the renal tumors associated with tert-butanol exposure in the NTP (19951 rodent
bioassay were predominantly benign. Based on the PWG reevaluation fHard etal.. 20111. among
the three treated groups, only two of the 43 animals with tumors has carcinomas (there were no
carcinomas among the 4 control animals with tumors). Additionally, no kidney tumors were
observed in female rats or in either sex of mice. Furthermore, ETBE, which is rapidly metabolized
to tert-butanol, did not induce kidney tumors in the same strain of rats at doses that resulted in
similar internal concentrations of tert-butanol. Therefore, the level of concern raised by renal
tumors associated with tert- butanol exposure is reduced hased on the predominance of benign
tumors, an increase in renal tumors in a single sex/species combination only, and the lack of
coherence with the metabolically-related compound ITBE.
The thyroid tumors associated with te/7-liuLinol exposure were also predominantly benign.
In the NTP (19951 rodent bioassay. only female mice had a statistically significant increase in
thyroid tumors; none of these were carcinomas. In males, decreased survival complicates the
interpretation of thyroid tumors because.- male mice had an increased incidence of thyroid follicular
cell hyperplasia at all exposure levels, hut there was no significant increase in thyroid tumors at any
exposure. Interestingly, one thyroid follicular cell carcinoma occurred in a high-dose male, but
limited conclusions can he drawn from this single observation. Thyroid tumors were notobserved
in either sex of the rat exposed chronically to ^'/7-hutanol. Additionally, ETBE did not induce
thyroid tumors, although only rats and not mice were tested. Therefore, the level of concern raised
by thyroid tumors associated with /(.'//-hutanol exposure is reduced based on the predominance of
benign tumors and an increase in thyroid tumors in a single sex/species combination only.
Ov erall, the cancer descriptor "suggestive evidence of carcinogenic potential" was selected,
as some concern is raised by the positive evidence of predominantly benign renal tumors in male
rats and thyroid tumors in female mice.
The Cancer Guidelines (M.S. EPA. 2005al indicate that for tumors occurring at a site other
than the initial point ol contact, the weight of evidence for carcinogenic potential may apply to all
routes of exposure that have not heen adequately tested at sufficient doses. An exception occurs
when there is convincing loxicokinetic data that absorption does not occur by other routes.
Information available on the carcinogenic effects of tert-butanol via the oral route demonstrates
that tumors occur in tissues remote from the site of absorption. Information on the carcinogenic
effects of tert-butanol via the inhalation and dermal routes in humans or animals is not available.
Based on the observation of systemic tumors following oral ingestion, and in the absence of
information to indicate otherwise, it is assumed that an internal dose will be achieved regardless of
the route of exposure. Therefore, there is "suggestive evidence of carcinogenic potential" from
exposure to tert-butanol by all routes of exposure.
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1 1.2.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes
2 No data were identified to indicate any possible susceptible populations or lifestages.
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2. DOSE-RESPONSE ANALYSIS
2.1. ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER
The RfD (expressed in units of mg/kg-day) is defined as an estimate (with uncertainty
spanning perhaps an order of magnitude) of a daily exposure to the human population (including
sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a
lifetime. 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 of tert-butanol exposure. Studies within
this effect category 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. Rationales for selecting the studies and effects to represent each of these hazards are
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 tert-
butanol.
Animal studies were evaluated to determine which studies provided: (a) the most relevant
routes and durations of exposure; (b) multiple exposure levels to provide information about the
shape of the dose-response curve; and (c) power to detect effects at low exposure levels (U.S. EPA.
2002). 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. The database for tert-butanol includes
several studies and data sets that are potentially suitable for use in deriving reference values.
Specifically, effects associated with tert-butanol exposure in animals include observations of organ
weight and/or histological changes in the kidney observed in several chronic and subchronic
studies.
Kidney Toxicity
EPA identified kidney effects as a human hazard of tert-butanol-induced toxicity based on
findings of organ weight changes in rats and mice, as well as histopathology in rats. These findings
were consistent across multiple chronic, subchronic, and short-term studies following oral and
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inhalation exposure. Acharya et al. (1997: 19951 used a single exposure group and did not provide
incidence or severity data, and thus was not considered for dose-response assessment Lvondell
Chemical Co. f20041 and NTP f!9971 were of subchronic or shorter duration, and so were set aside
given the availability of a longer duration study. Therefore, the NTP 2-year drinking water study
fNTP. 19951 was identified most suitable for dose-response assessment considering the study
duration, comprehensive reporting of outcomes, multiple species tested, and multiple doses tested.
In the NTP (19951 drinking water study, male F344 rats were exposed to approximate
doses of 0, 90, 200, or 420 mg/kg-day; female F344 rats were exposed to approximate doses of 0,
180, 330, or 650 mg/kg-day; male B6C3Fi mice were exposed l<> approximate doses of 0, 540,
1,040, or 2,070 mg/kg-day; and female B6C3Fi mice were imposed l<> approximate doses of 0, 510,
1,020, or 2,110 mg/kg-day. Reduced body weights and survival won.1 observed and reflected in
some of the effects. Kidney effects including changes in organ weight and/or histopathology were
observed in both sexes in rats and mice. Effects were also observed alter 13 weeks, 15 months, and
2 years of treatment (NTP. 19951. Effects were more consistent and occurred al lower doses in rats
as compared to mice, so as a result, only data in the more sensitiv e species of rats were used for
dose-response assessment Endpoints potentially confounded by the presence of a^u-globulin
nephropathy in male rats, such as linear mineralization and renal tubule hyperplasia, were notused
for dose-response analysis. Specific endpoints chosen for analysis were absolute and relative
kidney weight (observed in males and females), kidney inflammation (observed only in females),
and kidney transitional epithelial hyperplasia (observed in males and females). For most
endpoints, the data al the longestduralion of 2 years were selected. However, as discussed in
Section 1.1.1, 2-year kidney weight (.lata were not considered because organs were only weighed at
15 months.
2.1.2. Methods of Analysis
No biologically based dose-response models are available for tert-butanol. In this situation,
EPA evaluates a range of dose-response models thought to be consistent with underlying biological
processes to determine how to best 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
mean for organ weight data in the absence of information regarding the level of change that is
considered biologically significant Furthermore, the BMD and BMDL were estimated 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 kidney
inflammation and kidney transitional epithelial hyperplasia. For each endpoint, the BMDL estimate
(95% lower confidence limit on the BMD, as estimated by the profile likelihood method) and Akaike
Information Criterion (AIC) value were used to select a best-fit model among models exhibiting
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adequate fit If the BMDL estimates were "sufficiently close," that is, differed by at most 3-fold, the
model selected was the one that yielded the lowest AIC value. If the BMDL estimates were not
sufficiently close, the lowest BMDL was selected as the POD. 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 (Bailey etal.. 20041. However, in the NTP (19951 2-year drinking water
study, there was a noticeable decrease in body weight in exposed animals relative to controls at the
15 month interim sacrifice (see Table 1-1). In such a case, relative kidney weights are the
preferred, so changes in absolute kidney weights were not analyzed.
Human equivalent doses (HEDs) for oral exposures were derived from the PODs estimated
from the laboratory animal data as described in E I'A's Recommended Use of Body Weight3/4 as the
Default Method in Derivation of the Oral Reference Dose (U.S. EPA. 2011). I n this guidance, EPA
advocates a hierarchy of approaches for deriving I lEDs from data in laboratory animals, with the
preferred approach being physiologically-based toxicokinetic modeling. Othor approaches can
include using chemical-specific information in the absence ol a complete physiologically-based
toxicokinetic model. As discussed in Appendix B of the Supplemental Information, several rat
physiologically based pharmacokinetic (I'I'I'K) models for te/7-buUinol have been developed and
published, but a validated human PBPK model for tert-butanol lor 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 lor deriving I Mills Irom studies in which doses were administered
directly to early postnatal animals because ol 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 Loxicodynamic differences between lifestages (U.S. EPA.
2011: Hattis etal.. 20041.
Consistent, with KI'A guidance (U.S. EPA. 20111. the PODs estimated based on effects in adult
animals are converted to 111iI)s employing a standard dosimetric adjustment factor (DAF) derived
as follows:
DAF = (BWa1/4 / BWh1/4),
where
BWa = animal body weight
BWh = human body weight
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1 Using a standard BWa of 0.25 kg for rats and a BWh of 70 kg for humans (U.S. EPA. 19881.
2 the resulting DAFs for rats is 0.24. Applying this DAF to the POD identified for effects in adult rats
3 yields a PODhed as follows (see Table 2-1):
4
5 PODhed = Laboratory animal dose (mg/kg-day) x DAF
6
7 Table 2-1 summarizes the sequence of calculations leading to the derivation of a human-
8 equivalent POD for each data set discussed above.
9
10 Table 2-1. Summary of derivations of points of departure
Endpoint and
Reference
Species/
sex
Model3
BMR
BMD
(mg/kg-d)
BMDL
(mg/kg-d)
PODADJb
(mg/kg-d)
PODhedc
(mg/kg-d)
Kidney

Increased relative
kidney weight
NTP (1995)
Rat/M
Exponential
(M4)
10%
117
48
48
11.5
Increased relative
kidney weight
NTP (1995)
Rat/F
Linear
10'x,
158
133
133
31.9
Kidney inflammation
NTP (1995)
Rat/F
Log-probit
10'-'..
254
200
200
48
Kidney transitional
epithelial
hyperplasia
NTP (1995)
Rat/M
Log-logistic
10'-'..
30
16
16
3.84
Kidney transitional
epithelial
hyperplasia
NTP (1995)
Rat/F
Multistage,
3-degree
10%
412
339
339
81.4
11 aFor modeling details, see Appendix C in Supplemental Information.
12 bFor studies in which animals were not dosed daily, administered doses were adjusted to calculate the TWA daily
13 doses prior to BMD modeling.
14 CHED PODs were calculated using BW scaling (U.S. EPA, 2011).
15
16 2.1.3. Derivation of Candidate Values
17 Under EPA's A Review of the Reference Dose and Reference Concentration Processes fU.S. EPA.
18 2002: Section 4.4.51. also described in the Preamble, five possible areas of uncertainty and
19 variability were considered. An explanation follows:
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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 tert-butanol.
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, <>l 1 was applied to all PODs since the
endpoints examined were all observed following chronic exposure.
A LOAEL to NOAEL uncertainty factor, UFi, of 1 was applied to all PODs because the current
approach is to address this factor as one of the- considerations in selecting a I5MR for benchmark
dose modeling. In this case, BMRs of a 10% change in relative kidney weight, a 10% extra risk of
kidney inflammation, and a 10% extra risk of transitional cell hyperplasia were selected under an
assumption that they represent minimal biologically significant changes.
A database uncertainty factor, UFp, of 1 was applied to all I'ODs. The tert-butanol toxicity
database includes a chronic toxicity study in rats and mice fN'I'IJ. 1Q951. a subchronic toxicity study
in rats and mice fNTP. 10071. and developmental toxicity studies in rats and mice fLvondell
Chemical Co.. 2004: Faulkner et al.. 1989: Daniel and Fvans. 19821. In the developmental studies,
no effects were observ ed al exposure lev els lielow 1000 mg/kg-day, and effects observed at
>1000 mg/kg-day were accompanied by evidence of maternal toxicity. These exposure levels are
much higher than the PODs lor kidney effects, suggesting developmental toxicity is not a sensitive
endpoint. The /c/Mwtanol database contains a one-generation reproductive toxicity study in rats
fLvondell Chemical Co.. 20041. though no mulligenerational reproductive study has been
performed. There are no immunotoxicity studies for tert-butanol. Information provided by studies
onETBE, which is rapidly metabolized to systemically-available tert-butanol, can help in
considering the lack ol a tert-bulaiml multigenerational reproductive study or an immunotoxicity
study. No adverse effects were reported in one- and two-generation reproductive/developmental
studies on ETBE (Gaoua. 2004a. hj, and the database for ETBE does not indicate immunotoxicity
fBanton etal.. 2011: Li et al.. 20111. Thus, although there are some gaps in the toxicity database for
tert-butanol, the available data on tert-butanol, informed by the data on ETBE, do 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 2-2 is a continuation of Table 2-1 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 oral reference value for a specific
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1 hazard and subsequent overall RfD for tert-butanol.
2 Figure 2-1 presents graphically the candidate values, UFs, and PODs, with each bar
3 corresponding to one data set described in Table 2-1 and Table 2-2.
4
5 Table 2-2. Effects and corresponding derivation of candidate RfDs
Endpoint and Reference
PODhed
(mg/kg-d)
POD type
ufa
UFh
ufl
UFS
ufd
Composite
UF
Candidate
value
(mg/kg-d)
Kidney
Increased relative kidney weight;
male rat
NTP (1995)
12
BMDL
3
10
1
1
1
30
4 x 10 1
Increased relative kidney weight;
female rat
NTP (1995)
32
BMDL
3
10
1
1
1
30
lx 10°
Kidney inflammation; female rat
NTP (1995)
48
BMDL
3
10
1
1
1
30
2 x 10°
Kidney transitional epithelial
hyperplasia; male rat
NTP (1995)
3.8
BMDL
3
10
1
1
1
30
1 x 10 1
Kidney transitional epithelial
hyperplasia; female rat
NTP (1995)
81
BMDL
3
10
1
1
1
30
3 x 10°
6
7
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T Mean relative kidney
weight; male rat (NTP,
1995)
T Mean relative kidney
weight; female rat (NTP,
1995)
Kidney inflammation;
female rat (NTP, 1995)
Kidney transitional
epithelial hyperplasia;
male rat (NTP, 1995)
Kidney transitional
epithelial hyperplasia;
female rat (NTP, 1995)
0.1
10
100
~ Candidate RfD
# P0Dhed
Composite UF
mg/kg-day
Figure 2-1. Candidate RfD values with corresponding POD and composite UF.
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1 2.1.4. Derivation of Organ/System-Specific Reference Doses
2 Table 2-3 distills the candidate values from Table 2-2 into a single value for the kidney.
3 Organ or system-specific reference values may be useful for subsequent cumulative risk
4 assessments that consider the combined effect of multiple agents acting at a common site.
5 Kidney Toxicity
6 For tert-butanol, candidate values were for several different effects in both sexes, spanning
7 a range from 1 x 101 to 3 x 10° mg/kg-day, for an overall thirtyfold range. To estimate an exposure
8 level below which kidney toxicity from tert-butanol exposure is not expected to occur, the RfD for
9 increased incidence of transitional epithelial hyperplasia in male rats (1 x 10"1 mg/kg-day) is
10 proposed as the kidney-specific reference dose for tert-bulanol. 11 ill ike kidney inflammation, this
11 effect was observed in both sexes, with males appearing to be more sensitive than females.
12 Additionally, it is a more specific and more sensitive indicator of kidney toxicity than the relatively
13 non-specific endpoint of kidney weight changes. Confidence in this kidney-specific RfD is high. The
14 PODs are based on modeled benchmark dose estimates, and the candidate values are derived from
15 a well-conducted study, involving a sufficient number ol animals per group, includingboth sexes,
16 and assessing a wide range of kidney end points.
17 Table 2-3. Organ/system-specific RIDs and proposed overall RfD for tert-
18 butanol
Effect
Basis
RfD
(mg/kg-day)
Exposure
description
Confidence
Kidney toxicity
Increased incidence of
transitional epithelial
hyperplasia
lx 10
Chronic
HIGH
Proposed
overall RfD
Increased incidence of
transitional epithelial
hyperplasia
1 X 10"1
Chronic
HIGH
19
20 2.1.5. Selection of the Proposed Overall Reference Dose
21 For tert-butanol, only kidney effects were identified as a hazard; thus a single
22 organ/system-specific reference dose was derived. Therefore, the kidney-specific RfD of
23 1 x 10"1 mg/kg-day is also proposed as an estimated exposure level below which deleterious
24 effects from tert-butanol exposure are not expected to occur.
25 The overall reference dose is derived to be protective of all types of effects for a given
26 duration of exposure and is intended to protect the population as a whole including potentially
27 susceptible subgroups fU.S. EPA. 20021. Decisions concerning averaging exposures over time for
28 comparison with the RfD should consider the types of toxicological effects and specific lifestages of
29 concern. Fluctuations in exposure levels that result in elevated exposures during these lifestages
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could potentially lead to an appreciable risk, even if average levels over the full exposure duration
were less than or equal to the RfD. In the case of tert-butanol, no specific lifestages have been
identified as a potentially susceptible subgroup.
2.1.6. Confidence Statement
A confidence level of high, medium, or low is assigned to the study used to derive the RfD,
the overall database, and the RfD 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 RfD is high. Confidence in Ihc principal study (NTP. 19951 is
high. This study was well-conducted, complied with FDA (II,I' regulations, involved a sufficient
number of animals per group (including both sexes), and assessed a wide range of tissues and
endpoints. Although there are some gaps in the toxicity database for {<.'/¦{-butanol, these areas are
informed by the data on ETBE, a parent compound <>l /ert-butanol. Therefore, the confidence in the
database is high. Reflecting high confidence in the principal study and high confidence in the
database, confidence in the RfD is high.
2.1.7. Previous IRIS Assessment
An oral assessment for tert-butanol was not previously av ailable on IRIS.
2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER
THAN CANCER
The inhalation reference concentration (RIC) (expressed in units ofmg/m3) is defined as an
estimate (with uncertainly spanning perhaps an order of magnitude) of a continuous inhalation
exposure to the human population (including sensitive subgroups) that is likely to be without an
appreciable risk ol''deleterious effects (.luring a lifetime. It can be derived from a NOAEL, LOAEL, or
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 of tert-butanol exposure. Studies within
this effect category were ev aluated 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. Rationales for selecting the studies and effects to represent this hazard are 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 inhalation exposure to
tert- butanol.
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Animal studies were evaluated to determine which study provided: (a) the most relevant
routes and durations of exposure; (b) multiple exposure levels to provide information about the
shape of the dose-response curve; and (c) power to detect effects at low exposure levels fU.S. EPA.
20021. 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. The database for tert-butanol includes a
several studies and data sets that are potentially suitable for use in deriving reference values.
Specifically, effects associated with tert-butanol exposure in animals include observations of organ
weight and histological changes in the kidney in several chronic and subchronic studies.
Kidney Toxicity
EPA identified kidney effects as a human hazard oI tert-butanol exposure based on findings
of organ weight changes in rats and mice and histopathology in rats. These findings were
consistent across multiple chronic, subchronic, and short-term studies following oral and inhalation
exposure. Acharya etal. (1997: 19951 used a single exposure group and did not provide incidence
or severity data, so was not considered for dose-response assessment Lvondell Chemical Co.
(20041 was of shorter than subchronic du ration, and so was set aside given the availability of a
longer duration studies. Given the availability of a chronic study, the subchronic studies of NTP
f!9951 and NTP T19971 would normally also lie set aside lor (.lose-response analysis. NTP T19971 is
the longest duration study via the inhalation route, not requiring route-to-route extrapolation, so
was kept for comparison purposes. Ov erall, the NTI' 2-year drinking water study NTP (19951 was
identified as the study most suitable lor (.lose-response assessment, given the study duration,
comprehensive reporting ol Outcomes, use of multiple species tested, multiple doses tested, and
availability ol a I'I'I'K model lor route-to-route extrapolation. This study was discussed previously
in Section 2.1.1 as part of the derivation ol the oral reference dose, so will not be reviewed here
again. The NTP T19971 subchronic inhalation study is described in more detail below.
NTI' (l')')71 was a wel l-designed subchronic study that evaluated the effect of tert- butanol
exposure on multiple species at multiple inhalation doses. Briefly, groups of F344 rats and B6C3Fi
mice (10 per sex per species) were exposed to tert-butanol (>99% pure) at concentrations of 0,
409, 819,1,637, 3,27-1 or 6,3(>(> mg/m3 by inhalation for 6 hours per day, 5 days per week, for 13
weeks (NTP. 19971. Absolute kidney weights were elevated (10-11%) in male rats exposed at
>3,274 mg/m3; relative kidney weights were statistically significantly elevated (~9%) in males at
>3,274 mg/m3 and females at 6,366 mg/m3. Male rats exhibited an increase in the severity of
chronic nephropathy (characterized as number of foci of regenerative tubules). There were few
endpoints available for consideration in the subchronic study, but changes in kidney weights were
also observed in the oral studies, such as the NTP (19951 2-year drinking water study.
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2.2.2. Methods of Analysis
No biologically based dose-response models are available for tert-butanol. 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 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
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.- (l!M K) of 10% extra risk was
considered appropriate for the quantal data on incidences of kidney inflammation and kidney
transitional epithelial hyperplasia. The estimated I'M DLs were used as points of departure (PODs).
Further details including the modeling output a nd graphical results for the hest-fit model for each
endpoint can be found in Appendix C of the Supplemental Information.
In general, absolute and relative kidney weight (.lata may both be considered appropriate
endpoints for analysis fBailev etal.. 2004). I lowever, in the N'l'l' f 19951 2-year drinking water
study, there was a noticeable decrease in hotly weight in exposed animals relative to controls atthe
15 month interim sacrifice (see Talile 1-1). In such a case, relative kidney weights are preferred, so
changes in absolute kidney weights from N'I'IJ (l'>'>5) were not analyzed. However, body weights
were not impacted in the N'l'l' (1997) suhchronic inhalation study. Based on a historical review of
26 studies of coi Urol rats from 1-month hioassays. Hailev etal. (20041 concluded that neither
absolute kidney weight nor relativ e kidney:hody (or kidney:brain) weight are optimal for
evaluating organ weight changes. Since neither approach is preferred, both were considered to be
appropriate for I'MI) analysis of the N'l'l' (l')'>7) data set.
PODs from Inhalation Studies
Because the RfC is applicable to a continuous lifetime human exposure but derived from
animal studies featuring intcrmiltentexposure, EPA guidance (U.S. EPA. 19941 provides
mechanisms for: (1) adjusting experimental exposure concentrations to a value reflecting
continuous exposure duration (ADJ) and (2) determining a human equivalent concentration (HEC)
from the animal exposure data. The former employs an inverse concentration-time relationship to
derive a health-protective duration adjustment to time-weight the intermittent exposures used in
the studies. The modeled benchmark concentration from the inhalation study (NTP. 19971 was
adjusted to reflect a continuous exposure by multiplying it by (6 hours per day) 4- (24 hours per
day) and (5 days per week) 4 (7 days per week) as follows:
BMC Lad j = BMCL (mg/m^) x (6 - 24) x (5 - 7)
BMCL (mg/m3) x (0.1786)
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The RfC methodology provides a mechanism for deriving a HEC from the duration-adjusted
POD (BMCLadj) determined from the animal data. The approach takes into account the extra-
respiratory nature of the toxicological responses and accommodates species differences by
considering blood:air partition coefficients for tert-butanol in the laboratory animal (rat or mouse)
and humans. According to the RfC guidelines fU.S. EPA. 19941. tert-butanol is a Category 3 gas
because extra-respiratory effects were observed. Kaneko etal. f20001 measured a blood:gas
partition coefficient of 531 ± 102 for tert-butanol in the male Wistar rat, while Borghoff et al.
(19961 measured a value of 481 ± 29 in male F344 rats. A blood:gas partition coefficient of 462 was
reported for tert-butanol in humans (Nihlen et al.. 19951. The calculation (Hb/g)A ^ (Hb/g)H was used
to calculate a blood:gas partition coefficient ratio to apply to the delivered concentration. Because
F344 rats were used in the study, the blood:gas partition cocf f icicnl lor F344 rats was used. Thus,
the calculation was: 481 4- 462 = 1.04. Therefore, a ratio of 1.04 was used to calculate the HEC. This
allowed a BMCLhec to be derived as follows:
BMCLhec = BMCLadj (mg/m3) x (interspecies conversion)
= BMCLadj (mg/ill:) ¦ ('M>1 4(>2)
= BMCLadj (mg/m :) ¦( l.O'l )
Table 2-4 summarizes the sequence ol calculations leading to the derivation of a human-
equivalent point of departure for each inhalation (.lata set discussed aliove.
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1 Table 2-4. Summary of derivation of PODs following inhalation exposure
Endpoint and
Reference
Species/
Sex
Model3
BMR
BMCb
(mg/m3)
BMCLb
(mg/m3)
PODADJb
(mg/m3)
PODHEcC
(mg/m3)
Kidney
Increased relative
kidney weight
NTP (1997)
Male F344
rats
Linear
10%
6309
4821
861
861
Increased absolute
kidney weight
NTP (1997)
Male F344
rats
Hill
10%
1931
1705
304
304
Increased relative
kidney weight
NTP (1997)
Female F344
rats
No model
selected
10%

Increased absolute
kidney weight
NTP (1997)
Female F344
rats
No model
selected
10'x,

2 aFor modeling details, see Appendix C in Supplemental Information.
3 bBMCs, BMCLs, and PODs were adjusted for continuous daily exposure by multiplying by (hours exposed per day /
4 24 hrs) x (days exposed per week / 7 days).
5 cPODHEc calculated by adjusting the PODad by the DAF (=1.0) for a category 3 gas (U.S. EPA, 1994).
6 dBMD modeling failed to successfully calculate a BMD value (see Appendix C).
7
8 PODs from oral studies - use ofPBPK model for route-to-route extrapolation
9 A PBPK model for tcrf-butanol in rats lias Ix-en developed, as described in Appendix B.
10 Using this model, route-to-mule.- extrapolation of Ihc oral BMDLs to derive inhalation PODs was
11 performed as follows. First, the internal dose in the rat at each oral BMDL (assuming continuous
12 exposure) was eslimated using the PBPK model, to derive an "internal dose BMDL." Then, the
13 inhalation air concentration (again, assuming continuous exposure) thatledto the same internal
14 dose in the ral was estimated using the PBPK model. The resulting BMCL was then converted to a
15 human equivalent concentration I'OD using the methodology previously described in "PODs from
16 inhalation studies":
17 BMCLhec = I!MCI.\i.i (mg/m3) x (interspecies conversion)
18 = BMCLadj (mg/m3) x (481 4- 462)
19 = BMCLadj (mg/m3) x (1.04)
20 A critical decision in the route-to-route extrapolation is the selection of the internal dose
21 metric that establishes "equivalent" oral and inhalation exposures. For tert-butanol-induced kidney
22 effects, the two options are the concentration of tert-butanol in blood and rate of tert-butanol
23 metabolism. Note that using the kidney concentration of tert-butanol will lead to the same route-
24 to-route extrapolation relationship as tert-butanol in blood, since the distribution from blood to
25 kidney is independent of route. There are no data to suggest that metabolites of tert-butanol
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Toxicological Review of tert-Butyl Alcohol
1 mediate its renal toxicity. In the absence of evidence that would suggest otherwise, it is assumed
2 that tert-butanol itself is the active toxicological agent. Therefore, the concentration of tert-butanol
3 in blood was selected as the dose metric.
4 Table 2-5 summarizes the sequence of calculations leading to the derivation of a human-
5 equivalent point of departure for each oral data set discussed above.
6 Table 2-5. Summary of derivation of inhalation points of departure derived
7 from route-to-route extrapolation from oral exposures
Endpoint and reference
Species/sex
BMR
BMDL
(mg/kg-d)
Internal dosea
(mg/L)
Equivalent
PODhec15 (mg/m3)
Kidney
Mean relative kidney weight
NTP (1995)
Rat/M
10v-„
48
2.34
79.6
Mean relative kidney weight
NTP (1995)
Rat/F
10v-„
133
7.46
231
Kidney inflammation
NTP (1995)
Rat/F
10%
200
12.6
359
Kidney transitional epithelial
hyperplasia
NTP (1995)
Rat/M
10%
16
0.745
26.1
Kidney transitional epithelial
hyperplasia
NTP (1995)
Rat/F
10%
339
27.9
638
8 a Average blood concentration of fe/t-butanol under continuous oral exposure at the BMDL
9 b Continuous inhalation human equivalent concentration that leads to the same average blood concentration of
10 te/t-butanol as continuous oral exposure at the BMDL
11
12 PODs carried forth to derivation of candidate values
13 For the derivation ol candidate values, itmustbe considered whether PODs from the
14 inhalation study oI NTI' f 1QQ71 would provide a better basis than the route-to-route extrapolated
15 PODs based on the oral study ol N'l'l' (19951. The only endpoint available from NTP (19971 is
16 increased kidney weights. The corresponding PODs from this subchronic inhalation study are
17 substantially higher than those for the same endpoint derived by route-to-route extrapolation from
18 the chronic study (NTP. 19951. consistent with longer duration requiring a lower dose to elicit an
19 effect Additionally, as discussed in Section 2.1.3, kidney weight is a less-specific endpoint
20 compared to some of the other endpoints available for analysis from the oral study fNTP. 19951.
21 Therefore, the PODs derived from PBPK model-based route-to-route extrapolation are the
22 preferred basis for deriving kidney-specific candidate RfCs, as they are based on a longer (chronic)
23 duration and a more specific endpoint.
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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 tert-butanol.
An interspecies uncertainty factor, UFa, of 3 (101 /? = 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 tert-butanol. This value is adopted hv 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 (U.S. EPA. 19941.
Asubchronic to chronic uncertainty factor, 111\, of 1 was applied to I In.- PODs derived from
the NTP f!9951 study, as the endpoints were observed following chronic exposure. For the PODs
derived from the subchronic NTP f!9971 study, a UFs of 10 was applied to account for extrapolation
from subchronic to chronic duration.
A LOAEL to NOAEL uncertainty factor, UFi, of 1 was iippliccl lo all PODs because the current
approach is to address this factor as one of the considerations in selecting a BMRfor benchmark
dose modeling. In this case, BMRs of a 10% change in kidney weight, a 10% extra risk of kidney
inflammation, and a 10% extra risk of transitional cell hyperplasia were selected under an
assumption that they represent minimal biologically significant changes.
A database uncertainly factor, UFp, of 1 was applied to all PODs. The tert-butanol toxicity
database includes a chronic toxicity study in rats and mice fNTP. 19951. a subchronic toxicity study
in rats and mice (NTP. l')')7). and developmental toxicity studies in rats and mice fLvondell
Chemical Co.. 200-1: Faulkner et al.. L9U9: Daniel and Evans. 19821. In the developmental studies,
no effects were observ ed atexposure levels below 1000 mg/kg-day, and effects observed at
>1000 mg/kg-day were accompanied by evidence of maternal toxicity. These exposure levels are
much higher than the PODs lor kidney effects, suggesting developmental toxicity is not a sensitive
endpoint The tert-butanol database contains a one-generation reproductive toxicity study in rats
fLvondell Chemical Co.. 20041. though no multigenerational reproductive study has been
performed. There are no immunotoxicity studies for tert-butanol. Information provided by studies
on ETBE, which is rapidly metabolized to systemically-available tert-butanol, can help in
considering the lack of a tert-butanol multigenerational reproductive study or an immunotoxicity
study. No adverse effects were reported in one- and two-generation reproductive/developmental
studies on ETBE fGaoua. 2004a. b), and the database for ETBE does not indicate immunotoxicity
fBanton etal.. 2011: Li etal.. 20111. Thus, although there are some gaps in the toxicity database for
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1 tert-butanol, the available data on tert-butanol, informed by the data on ETBE, do not suggest that
2 additional studies would lead to identification of a more sensitive endpoint or a lower POD.
3 Therefore, a database UFd of 1 was applied.
4 Table 2-6 is a continuation of Table 2-4 and Table 2-5, and summarizes the application of
5 UFs to each POD to derive a candidate value for each data set. The candidate values presented in
6 the table below are preliminary to the derivation of the organ/system-specific reference values.
7 These candidate values are considered individually in the selection of a representative inhalation
8 reference value for a specific hazard and subsequent overall RfC for tert-butanol.
9 Table 2-6. 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 relative kidney
weight; male rat
NTP (1997)
861
BMCL10o/o
3
10
1
10
1
300
3x 10°
Increased absolute kidney
weight; male rat
NTP (1997)
304
BMCL
3
10
1
10
1
300
lx 10°
Increased relative kidney
weight; female rat
NTP (1997)
1137
NOAEL
3
10
1
10
1
300
4x 10°
Increased absolute kidney
weight; female rat
NTP (1997)
1137
NOAEL
3
10
1
10
1
300
4x 10°
Increased relative kidney
weight; male rat
NTP (1995)
79.6
BMCL
3
10
1
1
1
30
3x 10°
Increased relative kidney
weight; female rat
NTP (1995)
231
BMCL10o/o
3
10
1
1
1
30
8x 10°
Kidney inflammation; female
rat
NTP (1995)
359
BMCL10o/o
3
10
1
1
1
30
lx 101
Kidney transitional epithelial
hyperplasia; male rat
NTP (1995)
26.1
BMCL10o/o
3
10
1
1
1
30
9 x 10 1
Kidney transitional epithelial
hyperplasia; female rat
NTP (1995)
638
BMCL10%
3
10
1
1
1
30
2x 101
10
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TRelative kidney weight;
male rat [NTP, 1997)
t Absolute kidney weight;
male rat (NTP, 1997)
TRelative kidney weight;
female rat (NTP, 1997)
TAbsolute kidney weight;
female rat (NTP, 1997)
TRelative kidney weight;
male rat (NTP, 1995)
TRelative kidney weight;
female rat (NTP, 1995)
Kidney inflammation;
female rat (NTP, 1995)
Kidney transitional
epithelial hyperplasia;
male rat (NTP, 1995)
Kidney transitional
epithelial hyperplasia;
female rat (NTP, 1995)
^ Candidate RfC
% PODhec
Composite UF
0.1
10
100
1000
10000
mg/m3
Figure 2-2. Candidate RfC 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 tert-butanol, candidate values were for several different effects in both sexes, spanning
a range from 9 x 101 to 2 x 101 mg/m3, for an overall twenty-fold range. To estimate an exposure
level below which kidney toxicity from tert-butanol exposure.- is not expected to occur, the RfC for
increased incidence of transitional epithelial hyperplasia in male.- nils (9 x 10"1 mg/m3) is proposed
as the kidney-specific reference concentration for tert-lnitanol, consistent with the selection of the
kidney-specific RfD (see Section 2.1.4). As discussed previously, unlike kidney inflammation, this
effect was observed in both sexes, with males ^ippe*^i ring to be more sensitive than females.
Additionally, it is a more specific and more sensitive- indicator of kidney toxicity than the relatively
non-specific endpoint of kidney weight changes. Confidence in this kidney-specific RfC is medium.
The PODs are based on modeled benchmark dose estimates, and the candidate values are derived
from a well-conducted study, involving a sufficient number ol animals per group, including both
sexes, assessing a wide range of kidney (.Midpoints, and availability of a PBPK model for route-to-
route extrapolation.
Table 2-7. Organ/system-specific RfCs and proposed overall RfC for
tert- butanol
Effect
Basis
RfC
(mg/m3)
Exposure
description
Confidence
Kidney toxicity
Increased incidence of
transitional epithelial
hyperplasia
9 x 10
Chronic
HIGH
Proposed
overall RfC
Increased incidence of
transitional epithelial
hyperplasia
9 x 101
Chronic
HIGH
2.2.5. Selection of the Proposed Overall Reference Concentration
For tert-butanol, 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 x 10"1 mg/m3 is also proposed as an estimated exposure level below which deleterious effects
from tert-butanol exposure are not expected to occur.
The overall reference concentration is derived to be protective of all types of effects for a
given duration of exposure and is intended to protect the population as a whole including
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potentially susceptible subgroups (U.S. EPA. 20021. Decisions concerning averaging exposures over
time for comparison with the RfC should consider the types of toxicological effects and specific
lifestages of concern. Fluctuations in exposure levels that result in elevated exposures during these
lifestages could potentially lead to an appreciable risk, even if average levels over the full exposure
duration were less than or equal to the RfC. In the case of tert-butanol, no specific lifestages have
been identified has a potentially susceptible subgroup.
2.2.6. Confidence Statement
A confidence level of high, medium, or low is assigned l<> Iht,* study used to derive the RfC,
the overall database, and the RfC itself, as described in Suction 4./!.').2 of EPA's Methods for
Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry fU.S. EPA.
19941. The overall confidence in this RfC is high. Confidence in the.- principal study fNTP. 19951 is
high. This study was well-conducted, compiled with I'DA GLP regulations, involved a sufficient
number of animals per group (including both sexes), and assessed a wide range of tissues and
endpoints. Although there are some gaps in the toxicity database for tert-butanol, these areas are
informed by the data on ETBE, a parent compound of fivf-liutanol. Therefore, tin.1 confidence in the
database is high. Reflecting high con fide no.- in the principal study and high confidence in the
database, confidence in the RfC is high.
2.2.7. Previous IRIS Assessment
An inhalation assessment for /c*/7-hiitanol was not prev iously 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 for
tert-butanol. To derive the KID, the UF approach (U.S. EPA. 2000a. 19941 was applied to a POD
based on kidney toxicity in rats treated chronically. To derive the RfC, this same approach was
applied, hut a I'I'I'K model was used to extrapolate from oral to inhalation exposure. UFs were
applied to the I'OI) to account lor 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 tert-butanol contains no human data on adverse health effects from
subchronic or chronic exposure. Data on the effects of tert-butanol are derived from a small
database of studies in rats and mice. The database for tert-butanol exposure includes one lifetime
bioassay, several reproductive/developmental studies, and several subchronic studies.
Although the database is adequate for reference value derivation, there is uncertainty
associated with the lack of a comprehensive multigeneration reproductive toxicity study.
Additionally, only subchronic and short-term inhalation studies have been conducted, and no
chronic inhalation studies are available. Developmental studies identified significant increases in
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fetal loss, decreases in fetal body weight, and possible increases in skeletal variations in exposed
offspring or pups. However, effects were not always consistent across exposure routes, and
significant material toxicity was present whenever developmental effects were observed.
The toxicokinetic and toxicodynamic differences for tert-butanol between the animal
species in which the POD was derived and humans are unknown. Although sufficient information is
available to develop a PBPK model in rats to evaluate difference across routes of exposure, the
tert-butanol database lacks an adequate model that would inform potential interspecies differences.
Generally, it was found that rats appear more susceptible than mice, and males appear more
susceptible than females to tert- butanol toxicity. However, I In.- underlying mechanistic basis of
these apparent differences is not understood. Most importantly, it is unknown which animal
species and/or sexes maybe more comparable to humans.
23. 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
thatmaybe derived. Quantitative risk estimates may lie derived from the application ofalow-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. KI'A concluded that there is "suggestive evidence of carcinogenic
potential" for te/Mnitanol. The (iuidclincs for (Aircinoijcn Risk Assessment (U.S. EPA. 2005al state:
When there is suggestiv e evidence, the Agency generally would not attempt a dose-
response assessment, as the nature of the (.lata 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.
The only data av ailable on potential carcinogenicity was derived from the 2-year drinking
water study in rats and mice by (NTP. 19951. This study was considered suitable for dose-response
analysis. It was conducted in accordance with Food and Drug Administration (FDA) Good
Laboratory Practice (GLP) Regulations, 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. Additionally, the renal tumors were re-examined by a Pathology Working
Group (Hard etal.. 20111.
Dose-related increasing trends in tumors were noted at the following sites:
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• Renal tubule adenomas and carcinomas in male rats; and
• Thyroid follicular adenomas in female mice.
These tumors were statistically significantly increased by pairwise comparison (Fisher
exact test, p < 0.05) and by trend test (Cochran-Armitage trend test, p < 0.05). Based on analysis of
mode of action data, it was concluded that processes other than a2U-globulin nephropathy are likely
responsible for the male rat renal tumors, so these tumors may be suitable for quantitative analysis
(U.S. EPA. 1991a). Additionally, a thyroid follicular carcinoma was observed in male mice, so it is
possible thatthe thyroid follicular adenomas in female mi a.- could progress to malignant form.
Therefore, the thyroid follicular adenomas in female mice may also lie considered suitable for
quantitative analysis. Considering these data along with I lie u ncerla i nty 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.
2.3.2. Dose-Response Analysis—Adjustments and Extrapolations Methods
The U.S. EPA Guidelines for Carcinogen Risk Assessment (M.S. EPA. 2005a) recommends that
the method used to characterize and quantify cancer risk from a chemical is determined by what is
known about the MOAofthe carcinogen and the shape of the cancer dose-response curve. The
linear approach is recommended if the MOA ol carcinogenicity has not been established fU.S. EPA.
2005a). In the case ol ^'/Mnitanol, the modes oI carcinogenic action lor renal tubule and thyroid
follicular tumors are not lullv understood (see Section 1.2.2). Therefore, a linear low-dose
extrapolation approach was used to estimate human carcinogenic risk associated with tert-butanol
exposure.
The modeled
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Toxicological Review of tert-Butyl Alcohol
1 was derived after dropping the highest exposure group (U.S. EPA. 2012bl. The highest exposure
2 group also had increased mortality, which may in part explain the observed non-monotonicity.
3 2.3.3. Derivation of the Oral Slope Factor
4 The PODs estimated for each tumor site are summarized in Table 2-8. The lifetime oral
5 cancer slope factor for humans is defined as the slope of the line from the lower 95% bound on the
6 exposure at the POD to the control response (slope factor = 0.1/BMDLio). This slope, a 95% upper
7 confidence limit, represents a plausible upper bound on the true risk. Using linear extrapolation
8 from the BMDLio, human equivalent oral slope factors were derived lor each species/tumor site
9 combination and are listed in Table 2-8.
10 The oral slope factors derived from the NTP fl')')5) hioassay differ by twenty-fold,
11 depending on the species and tumor site. The most sensitive endpoinl of renal tumors was used to
12 derive the oral slope factor because there are no data lo support any one result as most relevant for
13 extrapolating to humans. Two slope factors were derived for this endpoinl from the NTP (19951
14 bioassay, one based on the original reported incidences and the other based on the Hard etal.
15 (20111 reanalysis. The two estimates differed by less than 20"n, and rounded to the same number
16 atone significant figure. However, the Hard el al. (201 1) reanalysis is considered preferable, as it is
17 based on a PWG analysis. Therefore, the recommended slope factor for providing a sense of the
18 magnitude of potential carcinogenic risk associated with lifetime oral exposure to tert- butanol is
19 1 x 1()-'- per mg/kg-day, based on the renal tubule tumor response in male F344 rats.
20 Table 2-H. Summary of the oral slope factor derivations
Tumor
Species/Sex
Selected Model
BMR
BMD
(mg/kg-
d)
POD=
BMDL
(mg/kg-d)
BMDLHEDa
(mg/kg-d)
Slope factor13
(mg/kg-day)"1
Renal tubule
adenoma or
carcinoma
Male F344
rat; dose as
administered
1" Multistage
(high dose
dropped)
10%
70
42
10.1
1 X 10"2
Renal tubule
adenoma or
carcinoma (Hard et
al. (2011) reanalvsisl
Male F344
rat; dose as
administered
1" Multistage
(high dose
dropped)
10%
54
36
8.88
1 x 10"2
Thyroid follicular cell
adenoma
Female
B6C3F1
mouse
3° Multistage
10%
2002
1437
201
5 x 10"4
21
22 aHED PODs were calculated using BW3/4 scaling (U.S. EPA, 2011).
23 bHuman equivalent slope factor = 0.1/BMDLiOHed; see Appendix C of the Supplemental Information for details of
24 modeling results.
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2.3.4. Uncertainties in the Derivation of the Oral Slope Factor
There is uncertainty when extrapolating data from animals to estimate potential cancer
risks to human populations from exposure to tert-butanol (see Table 2-9). Uncertainty in the
magnitude of the recommended oral slope factor is reflected to some extent in the range of slope
factors; the oral slope factor based on the male rat data was about twenty-fold higher than the oral
slope factor based on female mouse data (Table 2-9). These comparisons show that the selection of
target organ, animal species, and interspecies extrapolation can impact the oral cancer risk
estimate. Although the thyroid follicular cell tumors occurred in male and female mice, high
mortality in high-dose male mice limited the usefulness of the data. Renal tubule tumors occurred
in male rats, but not female rats. Therefore, only the data in male rats and female mice were
available for deriving the oral slope factor. There are no other chronic studies to replicate these
findings or that examined other animal models. There are no data in humans to support the tumors
observed in animals. Although changing the methods used to derive the oral slope factor could
change the results, standard practices were used due to the lack of a mouse or human PBPK model
or specific MOA to indicate other methods would be preferable. Additionally, considering the
uncertainty associated with the suggest ive nature of the weight of evidence, the oral slope factor is
recommended only for providing a sense of the magnitude of potential carcinogenic risk.
Table 2-9. Summary of uncertainties in the derivation of cancer risk values for
tert- butanol
Consideration and
Impact on Cancer Risk Value
Decision
Justification and Discussion
Selection of target organ
4/ oral slope factor, up to twenty-
fold, if renal tumors not selected.
The kidney was selected as
the target organ.
As there are no data to support any one
result as most relevant for extrapolating to
humans, the most sensitive result for kidney
renal tubular adenomas and carcinomas was
used to derive the oral slope factor.
However, the overall evidence for
carcinogenicity was considered "suggestive."
Selection of data set
Unknown change in oral slope
factor, since no other studies are
available.
NTP (1995) as principal oral
(drinking water) study to
derive cancer risks for
humans.
NTP (1995) was a well-conducted studv. It
was also the only bioassay available.
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
(Selection of extrapolation approach
could change the recommended
cancer risk values.)
Oral data used for OSF.
No extrapolation methods were used.
Selection of dose metric
Alternatives could 4, or T* slope
factor
Used administered dose
converted to HED units.
Additional runs using the administered dose
without conversion to HED units were also
conducted, resulting in a similar oral slope
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Consideration and
Impact on Cancer Risk Value
Decision
Justification and Discussion

factor. For rats, a PBPK model of internal
dose was available, but the POD changed by
less than 1.2-fold when modeling was based
on internal doses. For mice, no PBPK model
was available, so using a PBPK model for
determining internal doses could have an
unknown effect on the estimated OSF value.
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, BW3/4 scaling 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 te/t-butanol
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 used.
Linear low-dose extrapolation for agents
without a known MOA is supported.
Statistical uncertainty at POD
4/ oral slope factor 1.7-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
renal tumors.
Sensitive subpopulations
1" oral slope factor to unknown
extent
No sensitive populations
have been identified.
No chemical-specific data are available to
determine the range of human
toxicodynamic variability or sensitivity,
including the susceptibility of children.
Because determination of a mutagenic MOA
has not been made, an age-specific
adjustment factor is not applied.
1
2 2.3.5. Previous IRIS Assessment: Oral Slope Factor
3 A cancer assessment for tert-butanol 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
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extrapolation procedure. If derived, the inhalation unit risk is a plausible upper bound on the
estimate of risk per |J.g/m3 air breathed.
2.4.1. Analysis of Carcinogenicity Data
As noted in Section 1.2.2, EPA concluded that there is "suggestive evidence of carcinogenic
potential" for tert- butanol. The Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005a) state:
When there is suggestive evidence, the Agency generally would not attempt a dose-
response assessment, as the nature of the data generally would not support one; however,
when the evidence includes a well-conducted study, quantitative analysis may be useful for
some purposes. For example, it could provide a sense of the magnitude and uncertainty of
potential risks, rank potential hazards, or set research priorities.
The only data available on potential carcinogenicity were from the 2-year drinking water
study in rats and mice by NTP f!9951. discussed prev iously in Section 2.o.1. I!ecause a PBPK model
for the rat is available to conduct route-to-route extrapolation ("discussed below), the male rat renal
tubule adenoma and carcinoma data are suitable for quantitative analysis to support an inhalation
unit risk. Considering these data and uncertainty associated with the suggestive nature ofthe
weight of evidence, EPA concluded that quantitative analyses may lie useful for providing a sense of
the magnitude of potential carcinogenic risk.
2.4.2. Dose Response Analysis - Adjustments and Extrapolation Methods
Details ofthe modeling and the model selection process can be found in Appendix C ofthe
Supplemental Information. A POD lor estimating low-dose risk was identified at doses at the lower
end of the observed (.lata corres|ionding to 1 extra risk.
A I'I'I'K model lor /<.'//-butanol in rats has been developed, as described in Appendix B.
Usinglhis model, route-to-route extrapolation ofthe oral BMDL to derive an inhalation POD was
performed as follows. First, the internal dose in the ratatthe 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, resulting in a route-to-route extrapolated
BMCL.
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 tert-butanol-induced
kidney effects, the two options are the concentration of tert-butanol in blood and rate of tert-
butanol metabolism. Note that using the kidney concentration of tert-butanol will lead to the same
route-to-route extrapolation relationship as tert-butanol in blood, since the distribution from blood
to kidney is independent of route. There are no data that suggest metabolites of tert-butanol
mediate its renal toxicity. In the absence of evidence that would suggest otherwise, it is assumed
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that tert-butanol itself is the active toxicological agent. Therefore, the concentration of tert-butanol
in blood was selected as the dose metric to derive the BMCL.
The RfC methodology provides a mechanism for deriving a HEC from the BMCL 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 tert-butanol in the laboratory animal (rat or mouse) and humans. According to the
RfC guidelines (U.S. EPA. 19941. tert-butanol is a Category 3 gas because extra-respiratory effects
were observed.Kaneko etal. (20001 measured a blood:gas partition coefficient of 531 ±102 for tert-
butanol in the male Wistar rat, while Borghoff et al. (19961 measured a value of 481 ± 29 in male
F344 rats. A blood:gas partition coefficient of 462 was reported for tert-butanol in humans (Nihlen
etal.. 19951. The calculation (Hb/g)A ^ (Hb/g)H was used to calculate a lilood:gas partition coefficient
ratio to apply to the delivered concentration. Because P344 rats were used in the study, the
blood:gas partition coefficient for F344 rats was used. Thus, the calculation was: 481 4- 462 = 1.04.
Therefore, a ratio of 1.04 was used to calculate the IIKC. This allowed a BMC Lin. to be derived as
follows:
BMCLhec = BMCLadj (mg/m :) ¦ (interspecies conversion)
= BMCLadj (mg/m :) ¦ (-M>1 ¦ 4(>2)
= BMCL-,|.| (mg/m :) ¦ (1.0-1)
The U.S. EPA (iuiik'lincs for Carcinoiicn Risk Assessment f M.S. K PA. 2005al recommend that
the method used to characterize and t|uanlilv cancer risk Irom a chemical is determined by what is
known about the MOA ol the carcinogen and the shape of the cancer dose-response curve. The
linear approach is recommended il the MOA ol carcinogenicity has notbeen established fU.S. EPA.
2005a). In the case ol''fiVf-liuLinol, the mode ol carcinogenic action for renal tubule tumors is not
fully understood (see Section 1.2.2). Therefore, a linear low-dose extrapolation approach was used
to estimate human carcinogenic risk associated with tert-butanol exposure.
2.4.3. Inhalation Unit Risk Derivation
The results Irom route-to-route extrapolation of the male rat renal tubule tumor data are
summarized in Table 2-10. 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 listed inTable 2-10.
Two inhalation unit risks were derived from the NTP (19951 bioassay: one based on the
original reported incidences and one based on the Hard etal. (20111 reanalysis. The two estimates
differ by less than 20%, but the Hard etal. (20111 reanalysis is considered preferable, as it is based
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1 on a PWG analysis. Therefore, the recommended inhalation unit risk for providing a sense of the
2 magnitude of potential carcinogenic risk associated with lifetime inhalation exposure to
3 tert-butanol is 2 x 103 per mg/m3, or 2 x 106 per pg/m3, based on the renal tubule tumor
4 response in male F344 rats.
5 Table 2-10. Summary of the inhalation unit risk derivation
Tumor
Species/Sex
BMR
BMDL
(mg/kg-d)
Internal Dosea
(mg/L)
POD=
BMCLHEcC
(mg/m3)
Unit Riskb
(mg/m3)1
Renal tubule adenoma or
carcinoma
Male F344
rat
10%
41.6
2.01
68.7
1 X 10"3
Renal tubule adenoma or
carcinoma fHard et al.
(2011) reanalvsisl
Male F344
rat
10%
36.3
1.74
59.8
2 x 10"3
6 a Average blood concentration of te/t-butanol under continuous oral exposure at the BMDL
7 b Continuous inhalation human equivalent concentration that leads to the same average blood concentration of
8 te/t-butanol as continuous oral exposure at the BMDL
9 cHuman equivalent inhalation unit risk = 0.1/BMCLHEc-
10
11 2.4.4. Uncertainties in the Derivation of the Inhalation Unit Risk
12 There is uncertainty when extrapolating data from animals to estimate potential cancer
13 risks to human populations Ironi exposure to tert-butanol (see Table 2-11). Uncertainty in the
14 magnitude of the recommended inhalation unit risk can be inferred to some extent from the range
15 of oral slope factors: the oral slope factor based on the male rat data was about twenty-fold higher
16 than the oral slope factor based on female mouse data (Table 2-9). These comparisons showthat
17 the selection ol target organ, animal species, and interspecies extrapolation can impact the
18 inhalation unit risk estimate. Although the thyroid follicular cell tumors occurred in male and
19 female mice, high mortality in high-dose male mice limited the usefulness of the data. Additionally,
20 no PBPK model was available in mice for use in route-to-route extrapolation, so these data could
21 not be used to estimate an inhalation unit risk. Renal tubule tumors occurred in male rats, but not
22 female rats. Therefore, only the (.lata in male rats were available for deriving the inhalation unit
23 risk. There are no other chronic studies to replicate these findings or that examined other animal
24 models. There are no data in humans to support the tumors observed in animals. Although
25 changing the methods used to derive the inhalation unit risk could change the results, standard
26 practices were used due to the lack of a mouse or human PBPK model or specific MOA to indicate
27 other methods which would be preferable. Additionally, considering the uncertainty associated
28 with the suggestive nature of the weight of evidence, the inhalation unit risk is recommended only
29 for providing a sense of the magnitude of potential carcinogenic risk.
This document is a draft for review purposes only and does not constitute Agency policy.
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1 Table 2-11. Summary of uncertainties in the derivation of cancer risk values
2 for tert-butanol
Consideration and
Impact on Cancer Risk Value
Decision
Justification and Discussion
Selection of target organ
Inhalation unit risk may change by
an unknown amount if a PBPK
model to extrapolate mouse thyroid
tumors to the inhalation route were
available.
The kidney was selected as
the target organ.
No PBPK model to extrapolation mouse
thyroid tumors was available. Additionally,
the overall evidence for carcinogenicity was
considered "suggestive."
Selection of data set
Unknown change in inhalation unit
risk, since no other studies are
available.
NTP (1995) as principal oral
(drinking water) study to
derive cancer risks for
humans.
NTP (1995) was a well-conducted studv. It
was also the only bioassay available.
Additional bioassays 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
Different PBPK model could 4^ or T*
inhalation unit risk.
PBPK model-based
extrapolation of oral data
used for inhalation unit
risk.
PBPK model accurately predicted tert-
butanol toxicokinetics. Data and model
predictions were within 2-fold of each other.
Selection of dose metric
Alternatives could 4^ or T*
inhalation unit risk.
Used tert-butanol
concentration in blood as
the dose metric for route-
to-route extrapolation,
converted to HEC.
In the absence of evidence that would
suggest that metabolites of tert-butanol are
responsible for carcinogenicity, it is assumed
that tert-butanol itself is the active
toxicological agent. An alternative dose
metric of tert-butanol metabolism would
result in a 1.2-fold decrease in the inhalation
unit risk.
Interspecies extrapolation of
dosimetry and risk
Alternatives could 4' or '|*
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- nor
underestimate human equivalent risks.
Dose-response modeling
Alternatives could 4' or
inhalation unit risk.
Used multistage dose-
response model to derive a
BMD and BMDL
No biologically based models for tert-butanol
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 used.
Linear low-dose extrapolation for agents
without a known MOA is supported.
Statistical uncertainty at POD
4/ inhalation unit risk 1.7-fold if the
BMD used to derive the inhalation
POD rather than BMDL.
BMDL (preferred approach
for calculating plausible
upper bound)
Limited size of bioassay results in sampling
variability; lower bound is 95% CI on
administered exposure at 10% extra risk of
renal tumors.
Sensitive subpopulations
No sensitive populations
No chemical-specific data are available to
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
Consideration and
Impact on Cancer Risk Value
Decision
Justification and Discussion
1" inhalation unit risk to unknown
extent.
have been identified.
determine the range of human
toxicodynamic variability or sensitivity,
including the susceptibility of children.
Because determination of a mutagenic MOA
has not been made, an age-specific
adjustment factor is not applied.
1
2 2.4.5. Previous IRIS Assessment: Inhalation Unit Risk
3 A cancer assessment for tert-butanol was notproviously av ailable on IRIS.
4 2.5. APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS
5 As discussed in the Supplemental Guidance [or Assessing Susceplihility from Early-Life
6 Exposure to Carcinogens fU.S. EPA. 2005bl either do limit or chemical-specific age-dependent
7 adjustment factors (ADAFs) are applied lo account for early-lilc exposure to carcinogens thatact
8 through a mutagenic mode of action, I localise chemical-specific life-stage susceptibility data for
9 cancer are not available, and because the mode of action for tc/7-liutanol carcinogenicity is not
10 known (see Section 1.1.4), ADAFs were not applied.
11
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This document is a draft for review purposes only and does not constitute Agency policy.
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