EPA/635/R-20/370Fa
www.epa.gov/iris
Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
[CASRN 75-65-0]
August 2021
Integrated Risk Information System
Center for Public Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection Agency
policy and approved for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
CONTENTS
AUTHORS | CONTRIBUTORS | REVIEWERS x
PREFACE xiv
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS xviii
EXECUTIVE SUMMARY xxvi
LITERATURE SEARCH STRATEGY | STUDY SELECTION AND EVALUATION xxxii
1. HAZARD IDENTIFICATION 1-1
1.1. OVERVIEW OF CHEMICAL PROPERTIES AND TOXICOKINETICS 1-1
1.1.1. Chemical Properties 1-1
1.1.2. Toxicokinetics 1-1
1.1.3. Description of Toxicokinetic Models 1-3
1.1.4. Chemicals Extensively Metabolized to £e/t-Butanol 1-4
1.2. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM 1-4
1.2.1. Kidney Effects 1-4
1.2.2. Thyroid Effects 1-45
1.2.3. Developmental Effects 1-54
1.2.4. Neurodevelopmental Effects 1-62
1.2.5. Reproductive Effects 1-66
1.2.6. Other Toxicological Effects 1-72
1.3. INTEGRATION AND EVALUATION 1-72
1.3.1. Effects Other Than Cancer 1-72
1.3.2. Carcinogenicity 1-74
1.3.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes 1-77
2. DOSE-RESPONSE ANALYSIS 2-1
2.1. ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER 2-1
2.1.1. Identification of Studies and Effects for Dose-Response Analysis 2-1
2.1.2. Methods of Analysis 2-2
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 Overall Reference Dose 2-8
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2.1.6. Confidence Statement 2-9
2.1.7. Previous Integrated Risk Information System (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 2-10
2.2.3. Derivation of Candidate Values 2-15
2.2.4. Derivation of Organ/System-Specific Reference Concentrations 2-18
2.2.5. Selection of the Overall Reference Concentration 2-18
2.2.6. Confidence Statement 2-19
2.2.7. Previous Integrated Risk Information System (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 2-23
2.3.4. Uncertainties in the Derivation of the Oral Slope Factor 2-24
2.3.5. Previous Integrated Risk Information System (IRIS) Assessment 2-26
2.4. INHALATION UNIT RISK FOR CANCER 2-26
2.4.1. Previous Integrated Risk Information System (IRIS) Assessment 2-27
2.5. APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS 2-27
REFERENCES R-l
SUPPLEMENTAL INFORMATION (see Volume 2)
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TABLES
Table ES-1. Organ/system-specific oral reference doses (RfDs) and overall RfD for ferf-butanol xxvii
Table ES-2. Organ/system-specific inhalation reference concentrations (RfCs) and overall RfCfor
ferf-butanol xxviii
Table LS-1. Details of the search strategy employed for ferf-butanol xxxvi
Table LS-2. Summary of additional search strategies for ferf-butanol xxxvii
Table LS-3. Inclusion-exclusion criteria xxxviii
Table LS-4. Considerations for evaluation of experimental animal studies xli
Table LS-5. Summary of experimental animal evidence base xlii
Table 1-1. Chemical identity and physicochemical properties of ferf-Butanol as curated by EPA's
CompTox Chemicals Dashboard 1-2
Table 1-2. Changes in kidney histopathology in animals following exposure to ferf-butanol 1-15
Table 1-3. Changes in kidney tumors in animals following exposure to ferf-butanol 1-19
Table 1-4. Comparison of nephropathy and suppurative inflammation in individual male rats
from the 2-year National Toxicology Program (NTP) ferf-butanol bioassay 1-21
Table 1-5. Comparison of nephropathy and suppurative inflammation in individual female rats
from the 2-year National Toxicology Program (NTP) ferf-butanol bioassay 1-21
Table 1-6. Comparison of nephropathy and transitional epithelial hyperplasia in individual male
rats from the 2-year National Toxicology Program (NTP) ferf-butanol bioassay 1-21
Table 1-7. Comparison of nephropathy and transitional epithelial hyperplasia in individual
female rats from the 2-year National Toxicology Program (NTP) ferf-butanol
bioassay 1-22
Table 1-8. Comparison of chronic progressive nephropathy and renal tubule hyperplasia with
kidney adenomas and carcinomas in male rats from the 2-year National
Toxicology Program (NTP) ferf-butanol bioassay 1-22
Table 1-9. Summary of data on the alpha 2u-globulin process in male rats exposed to ferf-
butanol 1-25
Table 1-10. International Agency for Research on Cancer (IARC) criteria for an agent causing
kidney tumors through an alpha 2u-globulin associated response in male rats 1-34
Table 1-11. Proposed empirical criteria for attributing renal tumors to chronic progressive
nephropathy 1-39
Table 1-12. Evidence pertaining to thyroid effects in animals following oral exposure to ferf-
butanol 1-46
Table 1-13. Evidence pertaining to developmental effects in animals following exposure to ferf-
butanol 1-56
Table 1-14. Evidence pertaining to neurodevelopmental effects in animals following exposure to
ferf-butanol 1-64
Table 1-15. Evidence pertaining to reproductive effects in animals following exposure to ferf-
butanol 1-67
Table 2-1. Summary of derivations of points of departure following oral exposure for up to 2
years 2-4
Table 2-2. Effects and corresponding derivation of candidate values 2-6
Table 2-3. Organ/system-specific oral reference doses (RfDs) and overall RfD for ferf-butanol 2-8
Table 2-4. Summary of derivation of PODs following inhalation exposure 2-12
Table 2-5. Summary of inhalation points of departure derived from route-to-route extrapolation
from oral exposures 2-14
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Table 2-6. Effects and corresponding derivation of candidate values 2-16
Table 2-7. Organ/system-specific inhalation reference concentrations (RfCs) and overall RfCfor
ferf-butanol 2-18
Table 2-8. Summary of the oral slope factor derivation 2-24
Table 2-9. Summary of uncertainties in the derivation of the oral slope factor for ferf-butanol 2-25
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FIGURES
Figure LS-1. Summary of literature search and screening process for ferf-butanol xxxv
Figure 1-1. Biotransformation of ferf-butanol in rats and humans 1-3
Figure 1-2. Exposure response array for kidney effects following oral exposure to ferf-butanol 1-11
Figure 1-3. Exposure-response array of kidney effects following inhalation exposure to ferf-
butanol (13-week studies, no chronic studies available) 1-12
Figure 1-4. Comparison of absolute kidney-weight change in male and female rats across oral
and inhalation exposure based on internal blood concentration 1-13
Figure 1-5. Comparison of absolute kidney-weight change in male and female mice following
oral exposure based on administered concentration 1-13
Figure 1-6. Comparison of absolute kidney-weight change in male and female mice following
inhalation exposure based on administered concentration 1-14
Figure 1-7. Temporal pathogenesis of alpha 2u-globulin-associated nephropathy in male rats 1-25
Figure 1-8. Exposure-response array for effects potentially associated with alpha 2u-globulin
renal tubule nephropathy and tumors in male rats after oral exposure to ferf-
butanol 1-28
Figure 1-9. Exposure-response array for effects potentially associated with alpha 2u-globulin
renal tubule nephropathy and tumors in male rats after inhalation exposure to
ferf-butanol 1-29
Figure 1-10. Exposure-response array of thyroid follicular cell effects following chronic oral
exposure to ferf-butanol 1-49
Figure 1-11. Exposure-response array of developmental effects following oral exposure to ferf-
butanol 1-60
Figure 1-12. Exposure-response array of developmental effects following inhalation exposure to
ferf-butanol 1-61
Figure 1-13. Exposure-response array of reproductive effects following oral exposure to ferf-
butanol 1-70
Figure 1-14. Exposure-response array of reproductive effects following inhalation exposure to
ferf-butanol 1-71
Figure 2-1. Candidate values with corresponding POD and composite uncertainty factor (UF) 2-7
Figure 2-2. Candidate inhalation reference concentration (RfC) values with corresponding point
of departure (POD) and composite uncertainty factor (UF) 2-17
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ABBREVIATIONS
ADAF
age-dependent adjustment factors
ETBE
ethyl tertiary butyl ether
ATH
acute tubule hyperplasia
F
female
AIC
Akaike's information criterion
FDA
Food and Drug Administration
Fe-EDTA
iron-catalyzed oxidation of ascorbic
GD
gestation day
acid
GLP
good laboratory practice
atm
atmosphere
Hb/g-A
animal blood:gas partition coefficient
BEC
blood ethanol concentration
Hb/g-H
human blood:gas partition coefficient
BMC
benchmark concentration
HBA
2 hydroxyisobutyrate
BMCL
benchmark concentration lower
HEC
human equivalent concentration
confidence level
HED
human equivalent dose
BMCLadj
duration-adjusted benchmark
HERO
Health and Environmental Research
concentration lower confidence level
Online
BMCLhec
benchmark concentration lower
HL-60
human promyelocytic leukemia
confidence limit, human equivalent
i.p.
intraperitoneal
concentration
i.v.
intravenous
BMD
benchmark dose
IARC
International Agency for Research on
BMDL
benchmark dose lower confidence limit
Cancer
BMDLbmr
benchmark dose lower confidence limit,
ICso
half-maximal inhibitory concentration
benchmark response
IRIS
Integrated Risk Information System
BMDS
Benchmark Dose Software
JPEC
Japan Petroleum Energy Center
BMDU
benchmark dose upper confidence limit
Km
Michaelis-Menten constant
BMR
benchmark response
LA
animal blood: air partition
BW
body weight
concentration
BW3/4
body weight to the % power
LH
human blood: air partition
BWa
animal body weight
concentration
BWh
human body weight
LOAEL
lowest-observed-adverse-effect level
CAAC
Chemical Assessment Advisory
M
male
Committee
Mavg
average rate of metabolism
CAR
constitutive androstane receptor
MFO
mixed function oxidase
CASRN
Chemical Abstracts Service registry
MPD
2 -methyl-1,2 -propanediol
number
mRNA
messenger ribonucleic acid
Cavg
average blood concentration
MTBE
methyl tert butyl ether
CFR
Code of Federal Regulations
NADPH
nicotinamide adenine dinucleotide
CHO
Chinese hamster ovary (cell line)
phosphate
CL
confidence limit
NA
not applicable
CL
liver concentration
MTD
maximum tolerated dose
CNS
central nervous system
MW
molecular weight
CPHEA
Center for Public Health and
NADPH
reduced form of nicotinamide adenine
Environmental Assessment
dinucleotide phosphate
CPN
chronic progressive nephropathy
NOAEL
no-observed-adverse-effect level
CSL
continuous simulation language
NTP
National Toxicology Program
CVL
concentration in the venous blood
OECD
Organisation for Economic
leaving the liver
Co-operation and Development
CYP450
cytochrome P450
•OH
hydroxyl radical
DAF
dosimetric adjustment factor
OSF
oral slope factor
df
degrees of freedom
PBPK
physiologically based pharmacokinetic
DMSO
dimethylsulfoxide
PECO
populations, exposures, comparators,
DNA
deoxyribonucleic acid
and outcomes
EHC
Environmental Health Criteria
PK
pharmacokinetic
ELISA
enzyme-linked immunosorbent assay
PND
postnatal day
EPA
Environmental Protection Agency
POD
point of departure
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PODadj
duration-adjusted POD
PODhec
point of departure, human equivalent
concentration
PODhed
point of departure, human equivalent
dose
QA
quality assurance
QSAR
quantitative structure-activity
relationship
QA
quality assurance
RfC
inhalation reference concentration
RfD
oral reference dose
rho
Spearman rank correlation coefficient
RTR
route-to-route
SAB
Science Advisory Board
SD
standard deviation
SULT1A1
sulfotransferase 1A1
T3
triiodothyronine
T4
thyroxine
TBA
tert-butyl alcohol; tert-butanol
tk
thymidine kinase
TRI
Toxics Release Inventory
TSCA
Toxic Substances Control Act
TSCATS
Toxic Substances Control Act Test
Submissions
TSH
thyroid-stimulating hormone
TWA
time-weighted average
UF
uncertainty factor
UFa
animal-to-human uncertainty factor
UFd
database deficiencies uncertainty factor
UFh
human variation uncertainty factor
UFl
LOAEL-to-NOAEL uncertainty factor
UFs
subchronic-to-chronic uncertainty
factor
U.S.
United States
w/v
weight by volume
WHO
World Health Organization
XMET
xenobiotic metabolizing enzyme and
transporter
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AUTHORS | CONTRIBUTORS | REVIEWERS
Assessment Team
Janice S. Lee, Ph.D. (Chemical Manager)
James A. Weaver (Co-Chemical Manager)
Kathleen Newhouse, M.S.*
EPA
Office of Research and Development
Center for Public Health and Environmental Assessment
Research Triangle Park, NC
*Washington, DC
Keith Salazar, Ph.D.
Former Co-Chemical Manager
EPA/ORD/NCEA
Currently with EPA, Office of Chemical Safety and Pollution
Prevention, Office of Pollution Prevention and Toxics, Risk
Assessment Division
Washington, DC
Chris Brinkerhoff, Ph.D.
Former ORISE Postdoctoral Fellow at EPA/ORD/NCEA
Currently with EPA, Office of Children's Health Protection,
Office of the Administrator
Washington, DC
Contributors
Christine Cai, M.S.*
Catherine Gibbons, Ph.D.:
Karen Hogan, M.S.*
Andrew Hotchkiss, Ph.D.
Samantha Jones, Ph.D.*
Channa Keshava, Ph.D.
Amanda Per sad, Ph.D.
Alan Sasso, Ph.D.*
Paul Schlosser, Ph.D.
Vincent Cogliano, Ph.D.
Jason Fritz, Ph.D.
Charles Wood, Ph.D.
EPA
Office of Research and Development
Center for Public Health and Environmental Assessment
Research Triangle Park, NC
*Washington, DC
(previously with) EPA National Center for Environmental
Assessment
(previously with) EPA National Health and Environmental
Effects Research Lab
Production Team
Hillary Hollinger
Maureen Johnson
Ryan Jones
Dahnish Shams
Vicki Soto
Samuel Thacker
Erin Vining
EPA
Office of Research and Development
Center for Public Health and Environmental Assessment
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Contractor Support
Robyn Blain, Ph.D. ICF
Michelle Cawley, M.L.S., M.A.* Fairfax, VA
Ami Gordon, M.P.H. *Research Triangle Park, NC
Cara Henning, Ph.D.*
Tao Hong, Ph.D.
William Mendez, Jr., Ph.D.
Pam Ross, M.S.P.H.
Executive Direction
Wayne E. Cascio, M.D. (CPHEA Director)
Samantha Jones, Ph.D. (CPHEA Associate
Director)*
Kristina Thayer, Ph.D. (CPAD Director)
Andrew Kraft, Ph.D. (CPAD Senior Science
Advisor)*
Paul White, Ph.D. (CPAD Senior Science
Advisor)*
Ravi Subramaniam, Ph.D. (CPAD Branch
Chief)*
Internal Review Team
General Toxicology Workgroup
Inhalation Workgroup
Neurotoxicity Workgroup
Pharmacokinetics Workgroup
Reproductive and Developmental
Toxicology Workgroup
Statistical Workgroup
Toxicity Pathways Workgroup
Executive Review Committee
Reviewers
This assessment was provided for review to scientists in EPA's Program and Region Offices.
Comments were submitted by:
Office of the Administrator/Office of Children's Health Protection
Office of Land and Emergency Management
Region 2, New York, NY
Region 8, Denver, CO
EPA/ORD/CPHEA
Research Triangle Park, NC
*Washington, DC
EPA
Office of Research and Development
Center for Public Health and Environmental Assessment
Washington, DC
Research Triangle Park, NC
Cincinnati, OH
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
This assessment was provided for review to other federal agencies and the Executive Office of the
President Comments were submitted by:
Department of Health and Human Services/Agency for Toxic Substances and Disease Registry
Department of Health and Human Services/National Institute of Environmental Health
Sciences/National Toxicology Program
Executive Office of the President/Office of Management and Budget
This assessment was released for public comment on May 16, 2016 and comments were due on July
15, 2016. The public comments are available on Regulations.gov. A summary and EPA's disposition
of the comments from the public is available in the revised external review draft assessment on the
IRIS website. Comments were received from the following entities:
Marcy Banton, DVM, Ph.D.
LyondellBasell
Patrick Beatty, Ph.D.
American Petroleum Institute
Nancy Beck, Ph.D.
American Chemistry Council
Susan Borghoff, Ph.D.
Tox Strategies, on behalf of LyondellBasell
James Bus, Ph.D.
Exponent, on behalf of LyondellBasell
Samuel Cohen, M.D., Ph.D.
University of Nebraska
Jeff Fowles, Ph.D.
Tox-Logic Consulting
Lawrence Lash, Ph.D.
Wayne State University
Fukumi Nishimaki, Ph.D.
Japan Petroleum Energy Center
A public science meeting was held on June 30, 2016 to obtain public input on the IRIS Toxicological
Review of tert-Butanol (Public Comment Draft). Public commenters, stakeholders, and members of
the scientific community were joined by independent experts identified by the National Academies'
National Research Council (identified by * below) in a discussion of key science topics. Discussants
and public commenters were:
Marcy Banton, DVM, Ph.D.
LyondellBasell
Nancy Beck, Ph.D.
American Chemistry Council
Susan Borghhoff, Ph.D.
ToxStrategies, Inc., on behalf of LyondellBasell
James Bus, Ph.D.
Exponent, on behalf of LyondellBasell
Sheue-yann Chang, Ph.D. *
NIH/National Cancer Institute
Samuel Cohen, M.D., Ph.D. *
University of Nebraska
Jeff Fowles, Ph.D.
Tox-Logic, LLC, on behalf of ExxonMobil
Gordon Hard, Ph.D., DSc
On behalf of LyondellBasell
Lawrence Lash, Ph.D. *
Wayne State University
Fukumi Nishimaki, Ph.D.
Japan Petroleum Energy Center
Thomas Rosol, DVM, Ph.D., MBA *
Ohio State University
Jerrold Ward, DVM *
Global Vet Pathology
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
This assessment was peer reviewed by independent, expert scientists external to EPA convened by
EPA's Science Advisory Board (SAB). A peer-review meeting was held on August 15-17, 2017. The
report of the SAB's review of the EPA's Draft Toxicological Review of tert-Butanol, dated February
27, 2019, is available on the IRIS website. A summary and EPA's disposition of the comments
received from the SAB is included in Appendix E.
Janice E. Chambers, Ph.D. (chair)
Hugh A. Barton, Ph.D.
Janet Benson, Ph.D.
Trish Berger, Ph.D.
John Budroe, Ph.D.
James V. Bruckner, Ph.D.
Karen Chou, Ph.D.
Harvey Clewell, Ph.D.
Deborah Cory-Slechta, Ph.D.
Bevin Engelward, Ph.D.
Jeffrey Fisher, Ph.D.
William Michael Foster, Ph.D.
Alan Hoberman, Ph.D.
Tamarra James-Todd, Ph.D.
Lawrence Lash, Ph.D.
Marvin Meistrich, Ph.D.
Maria Morandi, Ph.D.
Isaac Pessah, Ph.D.
Lorenz Rhomberg, Ph.D.
Stephen M. Roberts, Ph.D.
Alan Stern, Ph.D.
Mississippi State University, MS
Pfizer, Inc., Groton, CT
Lovelace Biomedical, Albuquerque, NM
University of California, Davis, Davis, CA
Office of Environmental Health Hazard Assessment,
Oakland, CA
University of Georgia, Athens, GA
Michigan State University, East Lansing, MI
Ramboll Environment and Health, Research Triangle
Park, NC
University of Rochester, Rochester, NY
Massachusetts Institute of Technology, Cambridge, MA
U.S. Food and Drug Administration, Jefferson, AR
Independent Consultant, Durham, NC
Charles River Laboratories, Inc., Horsham, PA
Harvard University, Boston, MA
Wayne State University, Detroit, MI
Anderson Cancer Center, University of Texas, Houston,
TX
Independent Consultant, Houston, TX
University of California, Davis, CA
Gradient, Cambridge, MA
University of Florida, Gainesville, FL
New Jersey Department of Environmental Protection/
University of Medicine and Dentistry of New Jersey-
Robert Wood Johnson Medical School, Trenton, NJ
The postexternal review draft of the assessment was provided for review to scientists in EPA's
Program and Regional Offices, and to other federal agencies and the Executive Office of the
President (EOP). A summary and EPA's disposition of major comments from the other federal
agencies and EOP is available on the IRIS website. Comments were submitted by:
EPA
Office of Water
EPA
Office of Land and Emergency Management
The National Institute for Occupational
Safety and Health
Office of Management and Budget
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PREFACE
This Toxicological Review critically reviews the publicly available studies on tert-butyl
alcohol (tert-butanol) to identify its adverse health effects and to characterize exposure-response
relationships. The assessment examined all effects by oral and inhalation routes of exposure and
includes a noncancer oral reference dose (RfD), a noncancer inhalation reference concentration
(RfC), a cancer weight-of-evidence descriptor, and a cancer dose-response assessment It was
prepared under the auspices of the U.S. Environmental Protection Agency's (EPA's) Integrated Risk
Information System (IRIS) Program. This is the first IRIS assessment for this chemical.
Toxicological Reviews for tert-butanol and ethyl tert-butyl ether (ETBE) were developed
simultaneously because they have several overlapping scientific aspects:
• tert-Butanol is one of the primary metabolites of ETBE, and some of the toxicological effects
of ETBE are attributed to tert-butanol. Therefore, data on ETBE are considered informative
for the hazard identification and dose-response assessment of tert-butanol, and vice versa.
• The scientific literature for the two chemicals includes data on alpha 2u-globulin-related
nephropathy; therefore, a common approach was employed to evaluate these data as they
relate to the mode of action for kidney effects.
• A combined physiologically based pharmacokinetic (PBPK) model for tert-butanol and
ETBE in rats was applied to support the dose-response assessments for these chemicals.
A public meeting was held in December 2013 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.
A public science meeting was held on June 30, 2016 to provide the public an opportunity to
engage in early discussions on the draft IRIS Toxicological Review and the draft charge to the peer
review panel prior to release for external peer review. The complete set of public comments,
including the slides presented at the June 2016 public science meeting is available on the docket at
http://www.regulations.gov (Docket ID No. EPA-HO-ORD-2013-1111).
Organ/system-specific reference values are calculated based on kidney and thyroid toxicity
data. These reference values could be useful for cumulative risk assessments that consider the
combined effect of multiple agents acting on the same biological system.
This assessment was conducted in accordance with EPA guidance, which is cited and
summarized in the Preamble to IRIS Toxicological Reviews. The findings of this assessment and
related documents produced during its development are available on the IRIS website
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f http://www.epa.gov/iris). Appendices for toxicokinetic information, PBPK modeling, genotoxicity
study summaries, dose-response modeling, and other information are provided as Supplemental
Information to this Toxicological Review. For additional information about this assessment or for
general questions regarding IRIS, please contact EPA's IRIS Hotline at 202-566-1676 (phone),
202-566-1749 (fax), or hotline.iris@epa.gov.
Uses
tert-Butanol is primarily an anthropogenic substance that is produced in large quantities
fHSDB. 20071 from several precursors, including 1-butene, isobutylene, acetyl chloride and
dimethylzinc, and tert-butyl hydroperoxide. The domestic production volume of tert-butanol,
including imports, was approximately 4 billion pounds in 2012 fU.S. EPA. 2014a!
tert-Butanol has been used as a fuel oxygenate, an octane booster in unleaded gasoline, and
a denaturant for ethanol. From 1997 to 2005, the annual tert-butanol volume found in gasoline
ranged from approximately 4 million to 6 million gallons. During that time, larger quantities were
used to make methyl tert-butyl ether (MTBE) and ETBE. MTBE and ETBE are fuel oxygenates that
were used in the United States prior to 2007 at levels of more than 2 billion gallons annually.
Current use levels of MTBE and ETBE in the United States are much lower, but the use in Europe
and Asia remains strong.1 Some states have banned MTBE in gasoline due to concerns of
groundwater contamination from gasoline leaks and spills.
tert-Butanol has been used for a variety of other purposes, including as a dehydrating agent
and solvent It is added to lacquers, paint removers, and nail enamels and polishes, tert-Butanol
also is used to manufacture methyl methacrylate plastics and flotation devices. It is used in the
manufacture of food flavoring, and, because of its camphor-like aroma, it also is used to create
artificial musk, fruit essences, and perfume (HSDB. 2007). tert-Butanol is used in coatings on metal
and paperboard food containers fCalEPA. 19991 and industrial cleaning compounds and can be
used for chemical extraction in pharmaceutical applications (HSDB. 2007).
Fate and Transport
Soil
tert-Butanol is expected to be highly mobile in soil due to its low affinity for soil organic
matter. Rainwater or other water percolating through soil is expected to dissolve and transport
most tert-butanol present in soil, potentially leading to groundwater contamination. Based on its
vapor pressure, tert-butanol's volatilization from soil surfaces is expected to be an important
dissipation process (HSDB. 2007). As a tertiary alcohol, tert-butanol is expected to degrade 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 months
1http://www.ihs.com/products/chemical/planning/ceh/gasoline-octane-improvers.aspx.
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(approximately 200 days). Microbial degradation rates are increased in soils supplemented with
nitrate and sulfate nutrients fHSDB. 20071.
Water
tert-Butanol 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. 2007). Biodegradation in
aerobic water occurs over weeks to months and in anaerobic aquatic conditions, the biodegradation
rate decreases. Bioconcentration of tert-butanol in aquatic organisms is low (HSDB. 2007).
Air
tert-Butanol primarily exists as a vapor in the ambient atmosphere. Vapor-phase
tert-butanol is degraded in the atmosphere by reacting with photochemically produced hydroxyl
radicals and has a half-life of 14 days (HSDB. 2007).
Occurrence in the Environment
The Toxics Release Inventory (TRI) program National Analysis Report estimated that more
than 1 million pounds of tert-butanol have been released into the soil from landfills, land treatment,
underground injection, surface impoundments, and other land disposal sources. In 2014, the TRI
program also reported 1,845,773 pounds of tert-butanol released into the air, discharged to bodies
of water, disposed of at the facility to land, and disposed of in underground injection wells (U.S.
EPA. 2016). Total off-site disposal or other releases of tert-butanol amounted to 67,060 pounds
(U.S. EPA. 2016). 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.
tert-Butanol has been identified in drinking water wells throughout the United States
fHSDB. 20071. California's Geotracker Database2 lists 3,496 detections of tert-butanol in
groundwater associated with contaminated sites in that state since 2011. tert-Butanol also has
been detected in drinking water wells in the vicinity of landfills (U.S. EPA. 2012b). Additionally,
MTBE and ETBE leaking from underground storage tanks can degrade to form tert-butanol in soils
fHSDB. 20071. The industrial chemical tert-butyl acetate also can degrade to form tert-butanol in
the environment and as a metabolite in animals post exposure.
Ambient outdoor air concentrations of tert-butanol vary according to proximity to urban
areas fHSDB. 20071.
General Population Exposure
tert-Butanol exposure can occur in many different settings. Releases from underground
storage tanks could result in exposure for people who get their drinking water from wells. Due to
2http: II geotracker .water boar ds. ca. go v /.
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its high environmental mobility and resistance to biodegradation, tert-butanol has the potential to
contaminate and persist in groundwater and soil fHSDB. 20071.
Ingestion of contaminated food can be a source of tert-butanol exposure through its use as a
coating in metallic and paperboard food containers fCalEPA. 19991. and tert-butanol has been
detected in food fHSDB. 20071. Internal exposure to tert-butanol also can occur by ingestion of
MTBE or ETBE because tert-butanol is a metabolite of these compounds (NSF International. 2003).
Other human exposure pathways include inhalation, breast milk, and, to a lesser extent,
dermal contact. Inhalation exposure can occur because of the chemical's volatility and release from
industrial processes, consumer products, and contaminated sites fHSDB. 20071. tert-Butanol has
been identified in mother's milk fHSDB. 20071. Dermal contact is a viable route of exposure
through handling consumer products containing tert-butanol fNSF International. 20031.
Assessments by Other National and International Health Agencies
Toxicity information on tert-butanol has been evaluated by the National Institute for
Occupational Safety and Health (NIOSH. 20071. the Occupational Safety and Health Administration
fOSHA. 20061. and the Food and Drug Administration fFDA. 2015a. b). The results of these
assessments are presented in Appendix A of the Supplemental Information to this Toxicological
Review. Of importance to recognize is that these earlier assessments could have been prepared for
different purposes and might use different methods. In addition, newer studies have been included
in the IRIS assessment.
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PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS
Note: The Preamble summarizes the objectives at
systematic review procedures used in developing
process and document structure.
1. Objectives and Scope of the IRIS
Program
Soon after EPA was established in 1970, it
was at the forefront of developing risk
assessment as a science and applying it in
support of actions to protect human health
and the environment EPA's IRIS program3
contributes to this endeavor by reviewing
epidemiologic and experimental studies of
chemicals in the environment to identify
adverse health effects and characterize
exposure-response relationships. Health
agencies worldwide use IRIS assessments,
which are also a scientific resource for
researchers and the public.
IRIS assessments cover the hazard
identification and dose-response steps of risk
assessment. Exposure assessment and risk
characterization are outside the scope of IRIS
assessments, as are political, economic, and
technical aspects of risk management. An IRIS
assessment may cover one chemical, a group
of structurally or toxicologically related
chemicals, or a chemical mixture. Exceptions
outside the scope of the IRIS program are
radionuclides, chemicals used only as
pesticides, and the "criteria air pollutants"
(particulate matter, ground-level ozone,
carbon monoxide, sulfur oxides, nitrogen
oxides, and lead).
Enhancements to the IRIS program are
improving its science, transparency, and
productivity. To improve the science, the IRIS
ope of the IRIS program, general principles and
assessments¦, and the overall development
program is adapting and implementing
principles of systematic review (i.e., using
explicit methods to identify, evaluate, and
synthesize study findings). To increase
transparency, the IRIS program discusses key
science issues with the scientific community
and the public as it begins an assessment
External peer review, independently managed
and in public, improves both science and
transparency. Increased productivity requires
that assessments be concise, focused on EPA's
needs, and completed without undue delay.
IRIS assessments follow EPA guidance4
and standardized practices of systematic
review. This Preamble summarizes and does
not change IRIS operating procedures or EPA
guidance.
Periodically, the IRIS program asks for
nomination of agents for future assessment or
reassessment. Selection depends on EPA's
priorities, relevance to public health, and
availability of pertinent studies. The IRIS
multiyear agenda5 lists upcoming
assessments. The IRIS program may also
assess other agents in anticipation of public
health needs.
2. Planning an Assessment: Scoping,
Problem Formulation, and
Protocols
Early attention to planning ensures that
IRIS assessments meet their objectives and
properly frame science issues.
3IRIS program website: http: //www.epa.gov/iris/.
4EPA guidance documents: http://www.epa.gov/iris/basic-information-about-integrated-risk-information-
svstem#guidance/.
5IRIS multiyear agenda: https: //www.epa.gov/iris/iris-agenda.
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Scoping refers to the first step of planning,
where the IRIS program consults with EPA's
program and regional offices to ascertain their
needs. Scoping specifies the agents an
assessment will address, routes and durations
of exposure, susceptible populations and
lifestages, and other topics of interest.
Problem formulation refers to the science
issues an assessment will address and
includes input from the scientific community
and the public. A preliminary literature
survey, beginning with secondary sources
(e.g., assessments by national and
international health agencies and
comprehensive review articles), identifies
potential health outcomes and science issues.
It also identifies related chemicals (e.g.,
toxicologically active metabolites and
compounds that metabolize to the chemical of
interest).
Each IRIS assessment comprises multiple
systematic reviews for multiple health
outcomes. It also evaluates hypothesized
mechanistic pathways and characterizes
exposure-response relationships. An
assessment may focus on important health
outcomes and analyses rather than expand
beyond what is necessary to meet its
objectives.
Protocols refer to the systematic review
procedures planned for use in an assessment
They include strategies for literature searches,
criteria for study inclusion or exclusion,
considerations for evaluating study methods
and quality, and approaches to extracting data.
Protocols may evolve as an assessment
progresses and new agent-specific insights
and issues emerge.
3. Identifying and Selecting
Pertinent Studies
IRIS assessments conduct systematic
literature searches with criteria for inclusion
and exclusion. The objective is to retrieve the
pertinent primary studies (i.e., studies with
6Health and Environmental Research Online:
https://hero.epa.gov/hero/.
original data on health outcomes or their
mechanisms). PECO statements (Populations,
Exposures, Comparisons, Outcomes) govern
the literature searches and screening criteria.
"Populations" and animal species generally
have no restrictions. "Exposures" refers to the
agent and related chemicals identified during
scoping and problem formulation and may
consider route, duration, or timing of
exposure. "Comparisons" means studies that
allow comparison of effects across different
levels of exposure. "Outcomes" may become
more specific (e.g., from "toxicity" to
"developmental toxicity" to "hypospadias") as
an assessment progresses.
For studies of absorption, distribution,
metabolism, and elimination, the first
objective is to create an inventory of pertinent
studies. Subsequent sorting and analysis
facilitate characterization and quantification
of these processes.
Studies on mechanistic events can be
numerous and diverse. Here, too, the objective
is to create an inventory of studies for later
sorting to support analyses of related data.
The inventory also facilitates generation and
evaluation of hypothesized mechanistic
pathways.
The IRIS program posts initial protocols
for literature searches on its website and adds
search results to EPA's HERO database.6 Then
the IRIS program takes extra steps to ensure
identification of pertinent studies: by
encouraging the scientific community and the
public to identify additional studies and
ongoing research; by searching for data
submitted under the Toxic Substances Control
Act or the Federal Insecticide, Fungicide, and
Rodenticide Act; and by considering
late-breaking studies that would impact the
credibility of the conclusions, even during the
review process.7
7IRIS "stopping rules":
https://www.epa.gov/sites/production/files/201
4-06 /documents /iris stoppingrules.pdf.
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4. Evaluating Study Methods and
Quality
IRIS assessments evaluate study methods
and quality, using uniform approaches for
each group of similar studies. The objective is
that subsequent syntheses can weigh study
results on their merits. Key concerns are
potential bias (factors that affect the
magnitude or direction of an effect) and
insensitivity (factors that limit the ability of a
study to detect a true effect).
For human and animal studies, the
evaluation of study methods and quality
considers study design, exposure measures,
outcome measures, data analysis, selective
reporting, and study sensitivity. For human
studies, this evaluation also considers
selection of participant and referent groups
and potential confounding. Emphasis is on
discerning bias that could substantively
change an effect estimate, considering also the
expected direction of the bias. Low sensitivity
is a bias towards the null.
Study-evaluation considerations are
specific to each study design, health effect, and
agent Subject-matter experts evaluate each
group of studies to identify characteristics that
bear on the informativeness of the results. For
carcinogenicity, neurotoxicity, reproductive
toxicity, and developmental toxicity, there is
EPA guidance for study evaluation (U.S. EPA.
2005b. 1998b. 1996. 1991b). As
subject-matter experts examine a group of
studies, additional agent-specific knowledge
or methodologic concerns may emerge and a
second pass become necessary.
Assessments use evidence tables to
summarize the design and results of pertinent
studies. If tables become too numerous or
unwieldy, they may focus on effects that are
more important or studies that are more
informative.
The IRIS program posts initial protocols
for study evaluation on its website, then
considers public input as it completes this
step.
5. Integrating the Evidence of
Causation for Each Health
Outcome
Synthesis within lines of evidence. For
each health outcome, IRIS assessments
synthesize the human evidence and the animal
evidence, augmenting each with informative
subsets of mechanistic data. Each synthesis
considers aspects of an association that may
suggest causation: consistency,
exposure-response relationship, strength of
association, temporal relationship, biological
plausibility, coherence, and "natural
experiments" in humans (U.S. EPA. 2005b.
19941.
Each synthesis seeks to reconcile
ostensible inconsistencies between studies,
taking into account differences in study
methods and quality. This leads to a
distinction between conflicting evidence
(unexplained positive and negative results in
similarly exposed human populations or in
similar animal models) and differing results
(mixed results attributable to differences
between human populations, animal models,
or exposure conditions) (U.S. EPA. 2005b).
Each synthesis of human evidence
explores alternative explanations (e.g., chance,
bias, or confounding) and determines whether
they may satisfactorily explain the results.
Each synthesis of animal evidence explores
the potential for analogous results in humans.
Coherent results across multiple species
increase confidence that the animal results are
relevant to humans.
Mechanistic data are useful to augment the
human or animal evidence with information
on precursor events, to evaluate the human
relevance of animal results, or to identify
susceptible populations and lifestages. An
agent may operate through multiple
mechanistic pathways, even if one hypothesis
dominates the literature fU.S. EPA. 2005bl.
Integration across lines of evidence. For
each health outcome, IRIS assessments
integrate the human, animal, and mechanistic
evidence to answer the question: What is the
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nature of the association between exposure to
the agent and the health outcome?
For cancer, EPA includes a standardized
hazard descriptor in characterizing the
strength of the evidence of causation. The
objective is to promote clarity and consistency
of conclusions across assessments fU.S. EPA.
2005b).
Carcinogenic to humans: convincing
epidemiologic evidence of a causal
association; or strong human evidence of
cancer or its key precursors, extensive
animal evidence, identification of
mode-of-action and its key precursors in
animals, and strong evidence that they are
anticipated in humans.
Likely to be carcinogenic to humans: evidence
that demonstrates a potential hazard to
humans. Examples include a plausible
association in humans with supporting
experimental evidence, multiple positive
results in animals, a rare animal response,
or a positive study strengthened by other
lines of evidence.
Suggestive evidence of carcinogenic potential:
evidence that raises a concern for humans.
Examples include a positive result in the
only study, or a single positive result in an
extensive database.
Inadequate information to assess carcinogenic
potential: no other descriptors apply.
Examples include little or no pertinent
information, conflicting evidence, or
negative results not sufficiently robust for
not likely.
Not likely to be carcinogenic to humans: robust
evidence to conclude that there is no basis
for concern. Examples include no effects in
well-conducted studies in both sexes of
multiple animal species, extensive
evidence showing that effects in animals
arise through modes-of-action that do not
operate in humans, or convincing evidence
that effects are not likely by a particular
exposure route or below a defined dose.
If there is credible evidence of
carcinogenicity, there is an evaluation of
mutagenicity, because this influences the
approach to dose-response assessment and
subsequent application of adjustment factors
for exposures early in life (U.S. EPA. 2005b. c).
6. Selecting Studies for Derivation of
Toxicity Values
The purpose of toxicity values (slope
factors, unit risks, reference doses, reference
concentrations; see section 7) is to estimate
exposure levels likely to be without
appreciable risk of adverse health effects. EPA
uses these values to support its actions to
protect human health.
The health outcomes considered for
derivation of toxicity values may depend on
the hazard descriptors. For example, IRIS
assessments generally derive cancer values
for agents that are carcinogenic or likely to be
carcinogenic, and sometimes for agents with
suggestive evidence (U.S. EPA. 2005b).
Derivation of toxicity values begins with a
new evaluation of studies, as some studies
used qualitatively for hazard identification
may not be useful quantitatively for
exposure-response assessment Quantitative
analyses require quantitative measures of
exposure and response. An assessment weighs
the merits of the human and animal studies, of
various animal models, and of different routes
and durations of exposure fU.S. EPA. 19941.
Study selection is not reducible to a formula,
and each assessment explains its approach.
Other biological determinants of study
quality include appropriate measures of
exposure and response, investigation of early
effects that precede overt toxicity, and
appropriate reporting of related effects (e.g.,
combining effects that comprise a syndrome,
or benign and malignant tumors in a specific
tissue).
Statistical determinants of study quality
include multiple levels of exposure (to
characterize the shape of the
exposure-response curve) and adequate
exposure range and sample sizes (to minimize
extrapolation and maximize precision) (U.S.
EPA. 2012al.
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Studies of low sensitivity may be less
useful if they fail to detect a true effect or yield
toxicity values with wide confidence limits.
7. Deriving Toxicity Values
General approach. EPA guidance
describes a two-step approach to
dose-response assessment: analysis in the
range of observation, then extrapolation to
lower levels. Each toxicity value pertains to a
route (e.g., oral, inhalation, dermal) and
duration or timing of exposure (e.g., chronic,
subchronic, gestational) fU.S. EPA. 20021.
IRIS assessments derive a candidate value
from each suitable data set Consideration of
candidate values yields a toxicity value for
each organ or system. Consideration of the
organ/system-specific values results in the
selection of an overall toxicity value to cover
all health outcomes. The organ/system-
specific values are useful for subsequent
cumulative risk assessments that consider the
combined effect of multiple agents acting at a
common anatomical site.
Analysis in the range of observation.
Within the observed range, the preferred
approach is modeling to incorporate a wide
range of data. Toxicokinetic modeling has
become increasingly common for its ability to
support target-dose estimation, cross-species
adjustment, or exposure-route conversion. If
data are too limited to support toxicokinetic
modeling, there are standardized approaches
to estimate daily exposures and scale them
from animals to humans (U.S. EPA. 2011.2006.
2005b. 19941.
For human studies, an assessment may
develop exposure-response models that
reflect the structure of the available data (U.S.
EPA. 2005bl. For animal studies, EPA has
developed a set of empirical ("curve-fitting")
models8 that can fit typical data sets (U.S. EPA.
2005b). Such modeling yields a point of
departure, defined as a dose near the lower
end of the observed range, without significant
extrapolation to lower levels (e.g., the
benchmark Dose Software:
http://www.epa.gov/bmds/.
estimated dose associated with an extra risk of
10% for animal data or 1% for human data, or
their 95% lower confidence limitslfU.S. EPA.
2012a. 2005bl.
When justified by the scope of the
assessment, toxicodynamic ("biologically
based") modeling is possible if data are
sufficient to ascertain the key events of a
mode-of-action and to estimate their
parameters. Analysis of model uncertainty can
determine the range of lower doses where
data support further use of the model (U.S.
EPA. 2005bl.
For a group of agents that act at a common
site or through common mechanisms, an
assessment may derive relative potency
factors based on relative toxicity, rates of
absorption or metabolism, quantitative
structure-activity relationships, or
receptor-binding characteristics (U.S. EPA.
2005b).
Extrapolation: slope factors and unit
risks. An oral slope factor or an inhalation unit
risk facilitates subsequent estimation of
human cancer risks. Extrapolation proceeds
linearly (i.e., risk proportional to dose) from
the point of departure to the levels of interest.
This is appropriate for agents with direct
mutagenic activity. It is also the default if there
is no established mode-of-action (U.S. EPA.
2005b).
Differences in susceptibility may warrant
derivation of multiple slope factors or unit
risks. For early-life exposure to carcinogens
with a mutagenic mode-of-action, EPA has
developed default age-dependent adjustment
factors for agents without chemical-specific
susceptibility data (U.S. EPA. 2005b. c).
If data are sufficient to ascertain the
mode-of-action and to conclude that it is not
linear at low levels, extrapolation may use the
reference-value approach (U.S. EPA. 2005b).
Extrapolation: reference values. An oral
reference dose or an inhalation reference
concentration is an estimate of human
exposure (including in susceptible
populations) likely to be without appreciable
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risk of adverse health effects over a lifetime
(U.S. EPA. 20021. Reference values generally
cover effects other than cancer. They are also
appropriate for carcinogens with a nonlinear
mode-of-action.
Calculation of reference values involves
dividing the point of departure by a set of
uncertainty factors (each typically 1, 3, or 10,
unless there are adequate chemical-specific
data) to account for different sources of
uncertainty and variability (U.S. EPA. 2014b.
20021.
Human variation: An uncertainty factor covers
susceptible populations and lifestages that
may respond at lower levels, unless the
data originate from a susceptible study
population.
Animal-to-human extrapolation: For reference
values based on animal results, an
uncertainty factor reflects cross-species
differences, which may cause humans to
respond at lower levels.
Subchronic-to-chronic exposure: For chronic
reference values based on subchronic
studies, an uncertainty factor reflects the
likelihood that a lower level over a longer
duration may induce a similar response.
This factor may not be necessary for
reference values of shorter duration.
Adverse-effect level to no-observed-adverse-
effect level: For reference values based on
a lowest-observed-adverse-effect level, an
uncertainty factor reflects a level judged to
have no observable adverse effects.
Database deficiencies: If there is concern that
future studies may identify a more
sensitive effect, target organ, population,
or lifestage, a database uncertainty factor
reflects the nature of the database
deficiency.
8. Process for Developing and
Peer-Reviewing IRIS Assessments
The IRIS process (revised in 2009 and
enhanced in 2013) involves extensive public
engagement and multiple levels of scientific
review and comment IRIS program scientists
consider all comments. Materials released,
comments received from outside EPA, and
disposition of major comments (steps 3,4, and
6 below) become part of the public record.
Step 1: Draft development. As outlined in
section 2 of this Preamble, IRIS program
scientists specify the scope of an
assessment and formulate science issues
for discussion with the scientific
community and the public. Next, they
release initial protocols for the systematic
review procedures planned for use in the
assessment. IRIS program scientists then
develop a first draft, using structured
approaches to identify pertinent studies,
evaluate study methods and quality,
integrate the evidence of causation for
each health outcome, select studies for
derivation of toxicity values, and derive
toxicity values, as outlined in Preamble
sections 3-7.
Step 2: Agency review. Health scientists
across EPA review the draft assessment.
Step 3: Interagency science consultation.
Other federal agencies and the Executive
Office of the President review the draft
assessment.
Step 4: Public comment, followed by
external peer review. The public reviews
the draft assessment. IRIS program
scientists release a revised draft for
independent external peer review. The
peer reviewers consider whether the draft
assessment assembled and evaluated the
evidence according to EPA guidance and
whether the evidence justifies the
conclusions.
Step 5: Revise assessment. IRIS program
scientists revise the assessment to address
the comments from the peer review.
Step 6: Final agency review and
interagency science discussion. The IRIS
program discusses the revised assessment
with EPA's program and regional offices
and with other federal agencies and the
Executive Office of the President
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Step 7: Post final assessment. The IRIS
program posts the completed assessment
and a summary on its website.
9. General Structure of IRIS
Assessments
Main text. IRIS assessments generally
Main text IRIS assessments generally
comprise two major sections: (1) Hazard
Identification and (2) Dose-Response
Assessment. Section 1.1 briefly reviews
chemical properties and toxicokinetics to
describe the disposition of the agent in the
body. This section identifies related chemicals
and summarizes their health outcomes, citing
authoritative reviews. If an assessment covers
a chemical mixture, this section discusses
environmental processes that alter the
mixtures humans encounter and compares
them to mixtures studied experimentally.
Section 1.2 includes a subsection for each
major health outcome. Each subsection
discusses the respective literature searches
and study considerations, as outlined in
Preamble sections 3 and 4, unless covered in
the front matter. Each subsection concludes
with evidence synthesis and integration, as
outlined in Preamble section 5.
Section 1.3 links health hazard
information to dose-response analyses for
each health outcome. One subsection
identifies susceptible populations and
lifestages, as observed in human or animal
studies or inferred from mechanistic data.
These may warrant further analysis to
quantify differences in susceptibility. Another
subsection identifies biological considerations
for selecting health outcomes, studies, or data
sets, as outlined in Preamble section 6.
Section 2 includes a subsection for each
toxicity value. Each subsection discusses study
selection, methods of analysis, and derivation
of a toxicity value, as outlined in Preamble
sections 6 and 7.
Front matter. The Executive Summary
provides information historically included in
IRIS summaries on the IRIS program website.
Its structure reflects the needs and
expectations of EPA's program and regional
offices.
A section on systematic review methods
summarizes key elements of the protocols,
including methods to identify and evaluate
pertinent studies. The final protocols appear
as an appendix.
The Preface specifies the scope of an
assessment and its relation to prior
assessments. It discusses issues that arose
during assessment development and
emerging areas of concern.
This Preamble summarizes general
procedures for assessments begun after the
date below. The Preface identifies
assessment-specific approaches that differ
from these general procedures.
10.References
U.S. EPA (U.S. Environmental Protection
Agency). (1991b). Guidelines for
developmental toxicity risk
assessment (pp. 1-83). (EPA/600/FR-
91/001). Washington, DC: U.S.
Environmental Protection Agency,
Risk Assessment Forum.
http://cfpub.epa.gov/ncea/cfm/recor
displav.cfm?deid=23162
U.S. EPA (U.S. Environmental Protection
Agency). (1994). Methods for
derivation of inhalation reference
concentrations and application of
inhalation dosimetry [EPA Report]
(pp. 1-409). (EPA/600/8-90/066F).
Research Triangle Park, NC: U.S.
Environmental Protection Agency,
Office of Research and Development,
Office of Health and Environmental
Assessment, Environmental Criteria
and Assessment Office.
https://cfpub.epa.gov/ncea/risk/reco
rdisplav.cfm?deid=71993&CFID=511
74829&CFTQKEN=25006317
U.S. EPA (U.S. Environmental Protection
Agency). (1996). Guidelines for
reproductive toxicity risk assessment
[EPA Report], (EPA/630/R-96/009).
Washington, DC.
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http://www.epa.gov/raf/publications
/pdfs/REPR051.PDF
U.S. EPA (U.S. Environmental Protection
Agency). (1998b). Guidelines for
neurotoxicity risk assessment [EPA
Report], (EPA/630/R-95/001F).
Washington, DC.
http://www.epa.gov/raf/publications
/pdfs/NEI JROTOX.PDF
U.S. EPA (U.S. Environmental Protection
Agency). (2002). A review of the
reference dose and reference
concentration processes (pp. 1-192).
(EPA/630/P-02/002F). Washington,
DC: U.S. Environmental Protection
Agency, Risk Assessment Forum.
http://www.epa.gov/osa/review-
reference-dose-and-reference-
concentration-processes
U.S. EPA (U.S. Environmental Protection
Agency). (2005a). Guidelines for
carcinogen risk assessment [EPA
Report] (pp. 1-166). (EPA/630/P-
03/001F). Washington, DC: U.S.
Environmental Protection Agency,
Risk Assessment Forum.
http://www2.epa.gov/osa/guidelines
-carcinogen-risk-assessment
U.S. EPA (U.S. Environmental Protection
Agency). (2005b). Supplemental
guidance for assessing susceptibility
from early-life exposure to
carcinogens [EPA Report] (pp. 1125-
1133). (EPA/63 0/R-03/003F).
Washington, DC.
http://www.epa.gov/cancerguideline
s/guidelines-carcinogen-
supplementhtm
U.S. EPA (U.S. Environmental Protection
Agency). (2006). Approaches for the
application of physiologically based
pharmacokinetic (PBPK) models and
supporting data in risk assessment
(Final Report) [EPA Report],
(EPA/600/R-05/043F). Washington,
DC.
http://cfpub.epa.gov/ncea/cfm/recor
displav.cfm?deid=l 57668
U.S. EPA (U.S. Environmental Protection
Agency). (2011). Recommended use of
body weight 3/4 as the default method
in derivation of the oral reference dose
(pp. 1-50). (EPA/100/R11/0001).
Washington, DC: U.S. Environmental
Protection Agency, Risk Assessment
Forum, Office of the Science Advisor.
https://www.epa.gov/risk/recomme
nded-use-bodv-weight-34-default-
method-derivation-oral-reference-
dose
U.S. EPA (U.S. Environmental Protection
Agency). (2012). Benchmark dose
technical guidance (pp. 1-99).
(EPA/100/R-12/001). Washington,
DC: U.S. Environmental Protection
Agency, Risk Assessment Forum.
U.S. EPA. (U.S. Environmental Protection
Agency). (2014). Guidance for
applying quantitative data to develop
data-derived extrapolation factors for
interspecies and intraspecies
extrapolation [EPA Report],
(EPA/100/R-14/002F). Washington,
DC: Risk Assessment Forum, Office of
the Science Advisor.
https://www.epa.gov/risk/guidance-
applving-quantitative-data-develop-
data- derive d- extr ap olatio n- facto r s-
interspecies-and
August 2016
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
EXECUTIVE SUMMARY
Summation of Occurrence and Health Effects
tert-Butanol is produced by humans for multiple purposes, such as a solvent for
paints, a denaturant for ethanol and several other alcohols, an agent for dehydrating,
and in the manufacture of flotation agents, fruit essences, and perfumes. tert-Butanol
also is a primary metabolite of methyl tert-butyl ether (MTBE] and ethyl tert-butyl
ether (ETBE). Exposure to tert-butanol occurs primarily through breathing air
containing tert-butanol vapors and consuming contaminated water or foods.
Exposure can also occur through direct skin contact.
Animal studies demonstrate that chronic oral exposure to tert-butanol is associated
with kidney and thyroid effects. No chronic inhalation exposure studies have been
conducted. Evidence is suggestive of carcinogenic potential for tert-butanol, based
on thyroid tumors in male and female mice and renal tumors in male rats.
ES.l EFFECTS OTHER THAN CANCER OBSERVED FOLLOWING ORAL EXPOSURE
Kidney effects are a potential human hazard of oral exposure to tert-butanol. Kidney
toxicity was observed in males and females in two strains of rats. Kidney weights were increased in
male and female rats after 13 weeks or 15 months of treatment Histopathological examination in
male and female rats showed increased incidence or severity of nephropathy after 13 weeks of oral
exposure, increases in severity of nephropathy after 2 years of oral exposure, and increased
transitional epithelial hyperplasia after 2 years of oral exposure. Additionally, increased
suppurative inflammation was noted in females after 2 years of oral exposure. In one strain of
mice, the only kidney effect observed was an increase in kidney weight (absolute or relative) in
female mice after 13 weeks, but no treatment-related histopathological lesions were reported in the
kidneys of male or female mice at 13 weeks or 2 years. A mode of action (MOA) analysis
determined that tert-butanol exposure induces a male rat-specific alpha 2u-globulin-associated
nephropathy. tert-Butanol, however, is a weak inducer of alpha 2u-globulin nephropathy, which is
not the sole process contributing to renal tubule nephropathy. Chronic progressive nephropathy
(CPN) might also be involved in some noncancer effects, but the data are complicated by alpha
2u-globulin nephropathy in males. Effects attributable to alpha 2u-globulin nephropathy in males
were not considered for kidney hazard identification. Females are not affected by alpha 2u-globulin
nephropathy, so changes in kidney weights, transitional epithelial hyperplasia, suppurative
inflammation, and severity and incidence of nephropathy in female rats are considered to result
from tert-butanol exposure and are appropriate for identifying a hazard to the kidney.
At this time, evidence of selective developmental toxicity, neurodevelopmental toxicity, and
reproductive system toxicity following tert-butanol exposure is inadequate with minimal effects
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
observed at otherwise toxic dose levels. The available information also is inadequate to draw
conclusions regarding liver and urinary bladder toxicity because of lack of consistency and lack of
progression, respectively.
ES.2 ORAL REFERENCE DOSE (RFD) FOR EFFECTS OTHER THAN CANCER
Kidney toxicity, represented by increases in severity of nephropathy in female rats, was
chosen as the basis for the overall RfD (see Table ES-1). The kidney effects observed in female rats
in the chronic study by NTP (19951 were used to derive the RfD. Increased severity of nephropathy
was selected as the critical effect because it was observed in female rats consistently, it is an
indicator of kidney toxicity, and it was induced in a dose-responsive manner. While dose-response
modeling was technically feasible, there was uncertainty related to BMR type and values to use for
this type of endpoint; accordingly, the point of departure (POD) was derived from the
lowest-observed-adverse-effect level (LOAEL) of 43 mg/kg-day (NTP. 1995).
The overall RfD was calculated by dividing the POD for increases in severity of nephropathy
by a composite uncertainty factor (UF) of 100 to account for the extrapolation from animals to
humans (3), derivation from a LOAEL (3), and for interindividual differences in human
susceptibility (10).
Table ES-1. Organ/system-specific oral reference doses (RfDs) and overall
RfD for tert-butanol
Hazard
Basis
Point of
departure3
(mg/kg-d)
UF
Chronic RfD
(mg/kg-d)
Study
exposure
description
Confidence
Kidney
Increases in
severity of
nephropathy
43.2
100
4 x 10"1
Chronic
Medium
Overall RfD
Kidney
43.2
100
4 x 10"1
Chronic
Medium
aHuman equivalent dose PODs were calculated using body weight to the % power (BW3/4) scaling (U.S. EPA, 2011)
ES.3 EFFECTS OTHER THAN CANCER OBSERVED FOLLOWING INHALATION EXPOSURE
Kidney effects are a potential human hazard of inhalation exposure to tert-butanol.
Although no effects were observed in mice, kidney weights were increased in male and female rats
following 13 weeks of inhalation exposure. In addition, the severity of nephropathy increased in
male rats. No human studies are available to evaluate the effects of inhalation exposure. As
discussed above for oral effects, endpoints in males specifically related to alpha 2u-globulin
nephropathy were not considered for kidney hazard identification. Changes in kidney weights and
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
severity of nephropathy in females, however, are considered a result of tert-butanol exposure and
are appropriate for identifying a hazard to the kidney.
ES.4 INHALATION REFERENCE CONCENTRATION (RFC) FOR EFFECTS OTHER THAN
CANCER
Kidney toxicity, represented by increases in severity of nephropathy, was chosen as the
basis for the RfC (see Table ES-2). Although endpoints from a route-specific study were considered,
the availability of a physiologically based pharmacokinetic (PBPK) model for tert-butanol in rats
fBorghoffetal.. 20161 allowed for more specific and sensitive equivalent inhalation PODs derived
from a route-to-route (RTR) extrapolation from the PODs of the oral NTP T19951 study. The POD
adjusted for the human equivalent concentration (HEC) was 491 mg/m3 based on increases in
severity of nephropathy.
As discussed in Section 2.2.2, it is recognized that there is uncertainty in RTR extrapolation
because actual risk may not correlate exactly with the internal dose metric used for the
extrapolation (in this case, average blood concentration of tert-butanol). EPA is not aware of a
quantitative analysis of such uncertainty; it would involve comparing cross-route extrapolation to
toxicity data for a number of chemicals and endpoints sufficient to characterize the accuracy of the
approach. Such an analysis is beyond the scope of this assessment However, it is EPA's judgment
that this uncertainty is less than the uncertainty of the alternative, which would be to base the RfC
on the subchronic toxicity data. In particular, toxicity to the kidney requires that tert-butanol be
systemically distributed in the blood, hence the toxicity must be correlated with some measure of
blood concentration. The uncertainty in the extrapolation occurs because the metric used might
not accurately predict the effect, versus other possible metrics such as peak concentration.
The RfC was calculated by dividing the POD by a composite UF of 100 to account for
toxicodynamic differences between animals and humans (3), derivation from a LOAEL (3), and
interindividual differences in human susceptibility (10).
Table ES-2. Organ/system-specific inhalation reference concentrations (RfCs)
and overall RfC for tert-butanol
Hazard
Basis
Point of
departure3
(mg/m3)
UF
Chronic RfC
(mg/m3)
Study
exposure
description
Confidence
Kidney
Increases in severity
of nephropathy
491
100
5 x 10°
Chronic
Medium
Overall RfC
Kidney
491
100
5 x 10°
Chronic
Medium
BMDL = benchmark dose lower confidence limit.
Continuous inhalation HEC that leads to the same average blood concentration of tert-butanol as drinking water
exposure to the rat at the BMDL.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
ES.5 EVIDENCE OF HUMAN CARCINOGENICITY
Under EPA Cancer Guidelines fU.S. EPA. 2005al. there is suggestive evidence of carcinogenic
potential for tert-butanol. tert-Butanol induced kidney tumors in male (but not female) rats and
thyroid tumors (primarily benign) in male and female mice following long-term administration in
drinking water (NTP. 1995). The potential for carcinogenicity applies to all routes of human
exposure.
ES.6 QUANTITATIVE ESTIMATE OF CARCINOGENIC RISK FROM ORAL EXPOSURE
In accordance with EPA's guidance on alpha 2u-globulin fU.S. EPA. 1991bl. rat kidney
tumors are unsuitable for quantitative analysis because not enough data are available to determine
the relative contribution of alpha 2u-globulin nephropathy and other processes to the overall
kidney tumor response. A quantitative estimate of carcinogenic potential from oral exposure to
tert-butanol was based on the increased incidence of thyroid follicular cell adenomas in female
B6C3F1 mice and thyroid follicular cell adenomas and carcinomas in male B6C3F1 mice (NTP.
19951. The study included histological examinations for tumors in many different tissues,
contained three exposure levels and controls, contained adequate numbers of animals per dose
group (~50/sex/group), treated animals for up to 2 years, and included detailed reporting of
methods and results.
Although tert- butanol was considered to have only suggestive evidence of carcinogenic
potential, the National Toxicology Program (NTP) study was well conducted and suitable for
quantitative analysis. Slope factors were derived for thyroid tumors in female or male mice. The
modeled tert-butanol POD was scaled to human equivalent doses (HEDs) according to EPA
guidance by converting the benchmark dose lower confidence limit corresponding to 10% extra
risk (BMDLio) on the basis of body weight scaling to the % power (BW3/4) fU.S. EPA. 2011. 2005b).
Using linear extrapolation from the BMDLio, a human equivalent oral slope factor was derived
(slope factor = 0.1/BMDLio). The resulting oral slope factor is 5 x 10"4 per mg/kg-day.
ES.7 QUANTITATIVE ESTIMATE OF CARCINOGENIC RISK FROM INHALATION EXPOSURE
No chronic inhalation studies of exposure to tert-butanol are available. Although the mouse
thyroid tumors served as the basis for the oral slope factor, RTR extrapolation is not possible for
these thyroid effects in mice because the only PBPK model available is for rats. Therefore, no
quantitative estimate of carcinogenic risk could be determined for inhalation exposure.
ES.8 SUSCEPTIBLE POPULATIONS AND LIFESTAGES FOR CANCER AND NONCANCER
OUTCOMES
Information is inadequate to identify any populations or lifestages that might be especially
susceptible to tert-butanol.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
ES.9 KEY ISSUES ADDRESSED IN ASSESSMENT
This document assesses the human relevance of the kidney effects observed in male and
female rats, particularly as the effects relate to alpha 2u-globulin nephropathy and the exacerbation
of chronic progressive nephropathy. EPA 1991 and International Agency for Research on Cancer
(IARC) 1999 frameworks were used to evaluate whether tert-butanol caused alpha
2u-globulin-associated nephropathy (Capen etal.. 1999: U.S. EPA. 1991a). The presence of alpha
2u-globulin in the hyaline droplets was confirmed in male rats by alpha 2u-globulin
immunohistochemical staining. Linear mineralization and tubular hyperplasia were reported in
male rats, although only in the chronic study. Other subsequent steps in the pathological sequence,
including necrosis, exfoliation, and granular casts, either were absent or inconsistently observed
across subchronic or chronic studies. None of these effects occurred in female rats or in either sex
of mice, although these endpoints were less frequently evaluated in these models. Evidence implies
that an alpha 2u-globulin MOA is operative, although it is relatively weak in response to
tert-butanol and is not solely responsible for the renal tubule nephropathy observed in male rats.
Chronic progressive nephropathy (CPN) also plays a role in exacerbating nephropathy in both male
and female rats. While the etiology of CPN is unknownfNIEHS. 2019: Hard and Khan. 2004: Peter et
al.. 19861 and it has no known analog in the aging human kidney fNIEHS. 2019: Hard etal.. 20091. it
cannot be ruled out that a chemical which exacerbates CPN in rats could also exacerbate disease
processes in the human kidney (e.g. chronic kidney disease, diabetic nephropathy,
glomerulonephritis, interstitial nephritis, etc.) (NIEHS. 20191 Several other effects in the kidney
unrelated to alpha 2u-globulin were observed in female rats, including suppurative inflammation,
transitional epithelial hyperplasia, and increased kidney weights (NTP. 1997.1995). These specific
effects observed in female rats, not confounded by alpha 2u-globulin-related processes, are
considered the result of tert-butanol exposure, and therefore, relevant to humans.
Concerning cancer, alpha 2u-globulin accumulation is indicated as relatively weak in
response to tert-butanol exposure and not the sole mechanism responsible for the renal tubule
carcinogenicity observed in male rats. CPN and other effects induced by both alpha 2u-globulin
processes and tert-butanol play a role in renal tubule nephropathy, and the evidence indicates that
CPN augments the renal tubule tumor induction associated with tert-butanol exposure in male rats.
Poor dose-response relationships between alpha 2u-globulin processes and renal tumors in male
rats and a lack of renal tumors in female rats despite increased CPN severity, however, suggest that
other, unknown processes contribute to renal tumor development. Based on this analysis of
available MOA data, these renal tumors are considered relevant to humans.
In addition, an increase in the incidence of thyroid follicular cell adenomas was observed in
male and female mice in a 2-year drinking water study (NTP. 1995). Thyroid follicular cell
hyperplasia was considered a preneoplastic effect associated with the thyroid tumors, and the
incidences of follicular cell hyperplasias were elevated in both male and female B6C3F1 mice
following exposure. U.S. EPA f!998al describes the procedures the Agency uses in evaluating
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
chemicals that are animal thyroid carcinogens. The available evidence base is inadequate for
concluding that an antithyroid MOA is operating in mouse thyroid follicular cell tumorigenesis. No
other MOAs for thyroid tumors were identified, and the mouse thyroid tumors are considered
relevant to humans fU.S. EPA. 1998al.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
LITERATURE SEARCH STRATEGY | STUDY
SELECTION AND EVALUATION
A literature search and screening strategy was 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 using the most common synonyms and trade names to identify all
potentially pertinent studies. In subsequent steps, references were screened to exclude papers not
pertinent to an assessment of the health effects of tert-butanol, and the 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 core online scientific databases,
PubMed, Web of Science, and Toxline, as well as Toxic Substances Control Act Test Submissions
(TSCATS) through December 2016, using the keywords and limits described in Table LS-1. The
overall literature search approach is shown graphically in Figure LS-1. Eight more citations were
obtained using additional search strategies described in Table LS-2. After electronically eliminating
duplicates from the citations retrieved through these databases, 3,138 unique citations were
identified.
The resulting 3,138 citations were screened for pertinence and separated into categories as
presented in Figure LS-1 using the title and either abstract or full text, or both, to examine the
health effects of tert-butanol exposure. The inclusion and exclusion criteria used to screen the
references and identify sources of health effects data are provided in Table LS-3:
• 12 references were identified as "Sources of Health Effects Data" and were considered for
data extraction to evidence tables and exposure-response arrays.
• 202 references were identified as "Sources of Mechanistic and Toxicokinetic Data" and
"Sources of Supporting Health Effects Data"; these included 41 studies describing
physiologically based pharmacokinetic (PBPK) models and other toxicokinetic information,
73 studies providing genotoxicity and other mechanistic information, 1 human case report,
74 irrelevant exposure paradigms (including acute, dermal, eye irritation, and injection
studies), 6 preliminary toxicity studies, and 7 physical dependency studies. Information
from these studies was not extracted into evidence tables; however, these studies were
considered as support for assessing tert-butanol health effects, for example, evaluation of
mode of action and extrapolation of experimental animal findings to humans. Additionally,
although still considered sources of health effects information, studies investigating the
effects of acute and direct chemical exposures are generally less pertinent for characterizing
health hazards associated with chronic oral and inhalation exposure. Therefore,
information from these studies was not considered for extraction into evidence tables.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Nevertheless, these studies were still evaluated as possible sources of supplementary health
effects information.
• 128 references were identified as "Secondary Literature and Sources of Contextual
Information" (e.g., reviews and other agency assessments); these references were retained
as additional resources for developing the Toxicological Review.
• 2,796 references were identified as not being pertinent (not on topic) to an evaluation of
the health effects of tert-butanol and were excluded from further consideration (see
Figure LS-1 for exclusion categories and Table LS-3 for exclusion criteria). For example,
health effect studies of gasoline and tert-butanol mixtures were not considered pertinent to
the assessment because the separate effects of the gasoline or other chemical components
could not be determined. Retrieving a large number of references that are not on topic is a
consequence of applying an initial search strategy designed to cast a wide net and to
minimize the possibility of missing potentially relevant health effects data.
The complete list of references and the sorting of these materials can be found on the
tert-butanol project page of the Health and Environmental Research Online (HERO) website at
https://hero.epa.gov/index.cfm/project/page/project id/1543.
SELECTION OF STUDIES FOR INCLUSION IN EVIDENCE TABLES
To summarize the important information systematically from the primary health effects
studies in the tert-butanol evidence base, evidence tables were constructed in a standardized
tabular format as recommended by NRC f2011I Studies were arranged in the evidence tables by
effect, species, duration, and design, and not by quality. Of the studies retained after the literature
search and screen, 12 studies were identified as "Sources of Health Effects Data" and were
considered for extraction into evidence tables for hazard identification in Section 1. Initial review
found two references (Cirvello etal.. 1995: Lindamood et al.. 1992) to be publications of the NTP
f!9951 data prior to the release of the final National Toxicology Program (NTP) report One
publication fTakahashi etal.. 19931 in the "Supplementary Studies" category also was based on data
from the NTP report The interim publications and the final NTP report differed. The finalized NTP
f!9951 report was considered the more complete and accurate presentation of the data; therefore,
this report was included in the evidence tables, and the Cirvello etal. (1995). T akahashi et al.
(1993). and Lindamood et al. (1992) papers were not Data from the remaining 10 references in the
"Sources of Health Effects Data" category were extracted into evidence tables.
Supplementary studies that contain pertinent information for the Toxicological Review and
augment hazard identification conclusions, such as genotoxic and mechanistic studies, studies
describing the kinetics and disposition of tert-butanol absorption and metabolism, pilot studies,
and one case report, were not included in the evidence tables. Short-term and acute studies
(including an 18-day study and a 14-day study by NTP), which used oral and inhalation exposures
performed primarily in rats, did not differ qualitatively from the results of the longer term studies
(i.e., >30-day exposure studies). These were grouped as supplementary studies, however, because
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
the evidence base of chronic and subchronic rodent studies was considered sufficient for evaluating
chronic health effects of tert-butanol exposure. Additionally, studies of effects from chronic
exposure are most pertinent to lifetime human exposure (i.e., the primary characterization
provided by Integrated Risk Information System [IRIS] assessments) and are the focus of this
assessment. Such supplementary studies are discussed in the narrative sections of Section 1 and
are described in sections such as the "Mode of Action Analysis" to augment the discussion or
presented in appendices, if they provide additional information.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Database Searches
(See Table LS-lfor keywords and limits)
PubMed
n = 1,896
-¦
Web of Science
n = 823
Toxline
(incl. TSCATS)
n = 964
f
TSCATS 2
n = 2
(After duplicates removed electronically)
n = 3,130
Additional Search Strategies
(See Table LS-2 for methods and results)
n = 8
Combined Dataset
(After all duplicates removed)
n = 3,138
Manual Screening for Pertinence
(Title/Abstract/Full Text)
1
Excluded/Not on Topicfn = 2,796)
Secondary Literature and Sources of
62 Abstract only/comment/society
Contextual Information (n = 128)
abstracts
42
Not relevant species/matrix (e.g.,
104 Biodegradation/environmentalfate
amphibians, fish)
ISO Chemical analysis/fuel chemistry
14
QSAR
1,467 Other chemical/non t-butanol
S
Mixtures
87 Method of detection/exposure and
38
Reviews/editorials
biological monitoring
13
Other agency assessments
896 Methodology/solvent
13
Book chapter/section
Sources of Health Effects Data (n = 12)
o
12
Human health effects studies
Animal studies
Supporting Studies
Sources of Supporting Health Effects Data
(n = 88)
1
74
Human case reports
Not relevant exposure paradigms (e.g.,
dermal, eye irritation, acute)
Preliminary data
Physical dependency studies
Sources of Mechanistic and Toxicokinetic
Data (n = 114)
41 PBPK/ADME
22 Genotoxicity
51 Other mechanistic studies
Figure LS-1. Summary of literature search and screening process for
tert-butanol.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Table LS-1. Details of the search strategy employed for tert-butanol
Database
(search date)
Keywords
Limits
PubMed
(12/20/2012)
(4/17/2014)
(5/13/2015)
(12/31/2016)
(7/5/2019)a
tert-butanol OR 75-65-0[rn] OR
"t-butyl hydroxide" OR
"2-methyl-2-propanol" OR
"trimethyl carbinol" OR "t-butyl
alcohol" OR tert-butanol OR
"tert-butyl alcohol" OR tert-butyl
alcohol[mesh]
None
Web of Science
(12/20/2012)
(4/17/2014)
(5/13/2015)
(12/31/2016)
(7/5/2019)a
Topic = (tert-butanol OR 75-65-0 OR
"t-butyl hydroxide" OR
"2-methyl-2-propanol" OR
"trimethyl carbinol" OR "t-butyl
alcohol" OR "tert-butanol" OR
"tert-butyl alcohol")
Refined by: Research Areas = (cell biology OR
respiratory system OR microscopy OR biochemistry
molecular biology OR gastroenterology OR 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)
(5/13/2015)
(12/31/2016)
(7/5/2019)a
tert-butanol OR 75-65-0 [rn] OR
t-butyl hydroxide OR
2-methyl-2-propanol OR trimethyl
carbinol OR t-butyl alcohol OR
tert-butyl alcohol
Not PubMed
TSCATS2
(1/4/2013)
(4/17/2014)
(5/13/2015)
(12/31/2016)
75-65-0
None
aSee post-peer-reviewed literature search update section.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
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 and public
comments
Review article: McGregor (2010).
Tert/ory-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
Public comment article: Borghoffet
al. (2016)
10/2016
1
Manual search of
citations from
reviews conducted
by other
international and
federal agencies
IPCS (1987). Butanols: Four isomers:
1-butanol, 2-butanol, te/t-butanol,
isobutanol [WHO EHC], Geneva,
Switzerland: World Health
Organization.
1/2013
None
OSHA(1992). Occupational safetv
and health guideline for te/t-butyl
alcohol. Cincinnati, OH: Occupational
Safety and Health Administration.
1/2013
None
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Table LS-3. Inclusion-exclusion criteria
Inclusion criteria
Exclusion criteria/Supplemental material3
Population
• Humans.
• Standard mammalian animal
models, including rat, mouse,
rabbit, guinea pig, monkey, dog.
• Ecological species.3
• Nonmammalian species.3
Exposure
• Exposure is to te/t-butanol.
• Exposure is measured in an
environmental medium (e.g., air,
water, diet).
• Exposure via oral, inhalation, or
dermal routes.
• Study population is not exposed to
te/t-butanol.
• Exposure to a mixture only (e.g., gasoline
containing te/t-butanol).
• Exposure via injection (e.g., intravenous).
• Exposure pattern less relevant to chronic
health effects (e.g., acute).
Outcome
• Study includes a measure of one or
more health effect endpoints,
including effects on the nervous,
musculoskeletal, cardiovascular,
immune, hematological,
endocrine, respiratory, urinary,
and gastrointestinal systems;
reproduction; development; liver;
kidney; eyes; skin; and cancer.
• Physical dependency studies where
withdrawal symptoms were
evaluated after removal of
te/t-butanol treatment.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Table LS-3. Inclusion-exclusion criteria (continued)
Inclusion criteria
Exclusion criteria/Supplemental material3
Other
Not on topic, including:
• Abstract only, editorial comments were not
considered further.
• Bioremediation, biodegradation, or
environmental fate of te/t-butanol, including
evaluation of wastewater treatment
technologies and methods for remediation of
contaminated water and soil.
• Chemical, physical, or fuel chemistry studies.
• Analytical methods for measuring/detecting/
remotely sensing te/t-butanol.
• Use of te/t-butanol as a solvent or
methodology for testing unrelated to
te/t-butanol.
• Not chemical specific: Studies that do not
involve testing of te/t-butanol.
• Foreign language studies that were not
considered further because, based on title or
abstract, judged not potentially relevant.
• QSAR studies.
EHC = Environmental Health Criteria; WHO = World Health Organization.
aStudies that met this exclusion criterion were considered supplemental (i.e., not considered a primary source of
health effects data but were retained as potential sources of contextual information).
DATABASE EVALUATION
For this draft assessment, 12 references reported on experimental animal studies that
comprised the primary sources of health effects data; no studies were identified that evaluated
humans exposed to tert-butanol (e.g., cohort studies, ecological studies). The animal studies were
evaluated using the study quality considerations outlined in the Preamble, considering aspects of
design, conduct, or reporting that could affect the interpretation of results, overall contribution to
the synthesis of evidence, and determination of hazard potential as noted in various EPA guidance
documents fU.S. EPA. 2005b. 1998a. 1996.1991b). The objective was to identify the stronger, more
informative studies based on a uniform evaluation of quality characteristics across studies of
similar design. As stated in the Preamble, these studies were evaluated to identify the suitability of
the study based on:
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
• Study design.
• Nature of the assay and validity for its intended purpose.
• Characterization of the nature and extent of impurities and contaminants of tert-butanol
administered, if applicable.
• Characterization of dose and dosing regimen (including age at exposure) and their
adequacy to elicit adverse effects, including latent effects.
• Sample sizes 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.
Additionally, several general considerations, presented in Table LS-4, were used in
evaluating the animal studies. Much of the key information for conducting this evaluation can be
determined based on study methods and how the study results were reported. Importantly, the
evaluation at this stage does not consider the direction or magnitude of any reported effects.
EPA considered statistical tests to evaluate whether the observations might be due to
chance. The standard for determining statistical significance of a response is a trend test or
comparison of outcomes in the exposed groups against those of concurrent controls. Studies that
did not report statistical testing were identified and, when appropriate, statistical tests were
conducted by EPA.
Information on study features related to this evaluation is reported in evidence tables and
documented in the synthesis of evidence. Discussion of study strengths and limitations are
included in the text, where relevant If EPA's interpretation of a study differs from that of the study
authors, the draft assessment discusses the basis for the difference.
Experimental Animal Studies
The experimental animal studies, comprised entirely of studies performed in rats and mice,
were associated with drinking water, oral gavage, liquid diets (i.e., maltose/dextrin), and inhalation
exposures to tert-butanol. With the exception of neurodevelopmental studies, these studies were
conducted according to Organisation for Economic Co-operation and Development (OECD) Good
Laboratory Practice (GLP) guidelines; they used well-established methods, were well reported, and
evaluated an extensive range of endpoints and histopathological data. These studies include 2-year
bioassays using oral exposures in rats and mice, two subchronic drinking water studies in rats and
one in mice, an inhalation subchronic study in rats and mice, a reevaluation of the NTP (1995) rat
data, two oral developmental studies, two inhalation developmental studies, and a single
one-generation reproductive study that also evaluates other systemic effects (see Table LS-5). For
the body of available studies, detailed discussion of any identified methodological concerns
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
precedes each endpoint evaluated in the hazard identification section. Overall, the experimental
animal studies of tert-butanol involving repeated oral or inhalation exposure were considered to be
of acceptable quality, and whether yielding positive, negative, or null results, were considered in
assessing the evidence for health effects associated with chronic exposure to tert-butanol.
Table LS-4. Considerations for evaluation of experimental animal studies
Methodological
feature
Considerations
(relevant information extracted into evidence tables)
Test animal
Suitability of the species, strain, sex, and source of the test animals
Experimental design
Suitability of animal age/lifestage at exposure and endpoint testing; periodicity and
duration of exposure (e.g., h/d, d/wk); timing of endpoint evaluations; and sample size
and experimental unit (e.g., animals, dams, litters)
Exposure
Characterization of test article source, composition, purity, and stability; suitability of the
control (e.g., vehicle control); documentation of exposure techniques (e.g., route,
chamber type, gavage volume); verification of exposure levels (e.g., consideration of
homogeneity, stability, analytical methods)
Endpoint evaluation
Suitability of specific methods for assessing the endpoint(s) of interest
Results presentation
Data presentation for endpoint(s) of interest (including measures of variability) and for
other relevant endpoints needed for interpreting results (e.g., maternal toxicity,
decrements in body weight relative to organ weight)
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Table LS-5. Summary of experimental animal evidence base
Study category
Study duration, species/strain, and administration method
Chronic
2-vr studv in F344 rats (drinking water); NTP (1995)
2-vr studv in B6C3F1 mice (drinking water); NTP (1995)
Subchronic
13-wk studv in B6C3F1 mice (drinking water); NTP (1995)
13-wk studv in F344 rats (drinking water); NTP (1995)
13-wk studv in F344 rats (inhalation); NTP (1997)
13-wk studv in B6C3F1 mice (inhalation); NTP (1997)
10-wk studv in Wistar rats (drinking water); Acharva et al. (1997), Acharva et al. (1995)
Reproductive
One-generation reproductive toxicity studv in Sprague-Dawlev rats (gavage); Huntingdon
Life Sciences (2004)
Developmental
Developmental studv (GD 6-20) in Swiss Webster mice (diet); Daniel and Evans (1982)
Developmental studv (GD 6-18) in CBA/J mice (drinking water); Faulkner et al. (1989)
Developmental studv (GD 6-18) in C57BL/6J mice (drinking water); Faulkner et al. (1989)
Developmental studv (GD 1-19) in Sprague-Dawlev rats (inhalation); Nelson et al. (1989)
Neurodevelopmental
Neurodevelopmental studv (GD 6-20) in Swiss Webster mice (diet); Daniel and Evans
(1982)
Neurodevelopmental studv (GD 1-19) in Sprague-Dawlev rats (inhalation); Nelson et al.
(1991)
GD = gestation day.
Post-Peer-Review Literature Search Update
A post-peer-review literature search update was conducted in PubMed, Web of Science, and
Toxline for the period January 2017 to July 2019 using a search strategy consistent with previous
literature searches (see Table LS-1). The documentation and results for the literature search and
screen, including the specific references identified using each search strategy and tags assigned to
each reference based on the manual screen, can be found on the HERO website on the tert-butanol
project page at: fhttps: //hero.epa.gov/hero/index.cfm/proiect/page/project id/15431.
Consistent with the IRIS Stopping Rules f https: //www.epa.gov/sites/production/files/
2014-06 /documents /iris stoppingrules.pdfl. manual screening of the literature search update
focused on identifying new studies that might change a major conclusion of the assessment The
last formal literature search was in 2019 while the draft was in external peer review, after which
the literature was monitored in PubMed through January 2021. No animal bioassays or
epidemiological studies were identified in the post-peer-review literature searches that would
change any major conclusions in the assessment.
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1.HAZARD IDENTIFICATION
1.1. OVERVIEW OF CHEMICAL PROPERTIES AND TOXICOKINETICS
1.1.1. Chemical Properties
tert-Butanol is a white crystalline solid or colorless, highly flammable liquid (above 25.7°C)
with a camphor-like odor fNIOSH. 2005: IPCS. 19871. tert-Butanol contains a hydroxyl chemical
functional group; is miscible with alcohol, ether, and other organic solvents; and is soluble in water
(IPCS. 1987). Chemical and physical properties of tert-butanol are presented in Table 1-1.
1.1.2. Toxicokinetics
tert-Butanol is rapidly absorbed following exposure through oral and inhalation routes (see
Appendix B, Section B.l.l). Studies in experimental animals indicate that 99% of the compound is
absorbed after oral administration. Comparable blood levels of tert-butanol and its metabolites
also have been observed after acute oral or inhalation exposures in rats fARCO. 19831. In another
study (Faulkner et al.. 1989). blood concentrations indicated that absorption was complete at
1.5 hours following oral gavage doses of tert-butanol in female mice.
tert-Butanol is distributed throughout the body following oral, inhalation, and intravenous
exposures fPoetetal.. 1997: Faulkner etal.. 1989: ARCO. 19831. Following exposure to tert-butanol
in rats, tert-butanol was found in kidney, liver, and blood, with male rats retaining more
tert-butanol than female rats fWilliams and Borghoff. 20011.
A general metabolic scheme for tert-butanol, illustrating the biotransformation in rats and
humans, is shown in Figure 1-1 (see Appendix B, Section B.1.3).
Human data on the excretion of tert-butanol comes from studies of methyl tert-butyl ether
(MTBE) and ethyl tertiary butyl ether [ETBE; Nihlen etal. (1998a. 1998b)]. The half-life of
tert-butanol in urine following MTBE exposure was 8.1 ± 2.0 hours (average of the 90.1- and
757-mg/m3 MTBE doses); the half-life of tert-butanol in urine following ETBE exposure was
7.9 ± 2.7 hours (average of 104- and 210-mg/m3 ETBE doses). These studies reported urinary
levels of tert-butanol (not including downstream metabolites) to be less than 1% of administered
MTBE or ETBE concentrations (Nihlen etal.. 1998a. b). Ambergetal. (2000) observed a similar
half-life of 9.8 ± 1.4 hours after human exposure to ETBE of 170 mg/m3. The half-life for
tert-butanol in rat urine was 4.6 ± 1.4 hours at ETBE levels of 170 mg/m3.
A more detailed summary of tert-butanol toxicokinetics is provided in Appendix B,
Section B.l.
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Table 1-1. Chemical identity and physicochemical properties of tert-Butanol
as curated by EPA's CompTox Chemicals Dashboard
Characteristic or property
Value
Chemical structure
(
h3c
(
=h3
OH
~ LJ
-m3
CASRN
75-65-0
Synonyms
2-Propanol, 2-methyl-, 1,1-dimethylethanol, 2-methylpropan-2-ol,
2-methylpropan-2-ol (other name: te/t-butylalcohol), 2-methylpropane-2-ol,
2-metilpropan-2-ol, t-butanol, trimethyl carbinol, trimethylcarbinol,
trimethylmethanol, arconol, t-butyl hydroxide, te/t-butanol, (see
https://comptox.epa.gov/dashboard for additional svnonvms)
Molecular formula
C4H10O
Molecular weight (g/mol)
74.123
Average experimental value3
Average predicted value3
Flash point (°C)
—
17.5
Boiling point (°C)
82.8
79.7
Melting point (°C)
25.2
-30.4
Log Kow
3.50
3.78
Water solubility (mol/L)
13.5
3.61
Density (g/cm3)
—
0.833
Henry's law constant
(atm-m3/mole)
9.05 x 10"6
9.01 x 10"6
Vapor pressure (mm Hg at 20°C)
40.7
52.7
atm = atmosphere; CASRN = Chemical Abstracts Service registry number.
aMedian values and ranges for physicochemical properties are also provided on the CompTox Chemicals
Dashboard at https://comptox.epa.gov/dashboard/.
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cm
glucuronide-O-
-CH,
CH,
t-butyl glucuronide
rats, humans
CH,
HO-
-CH,
CH3
t-butanol
CYP450
I
rats,
humans
OH
"Y
CH3 oh
2-methyl-1,2-propanediol
-OH
CH,
rats
\^0 CH,
O^ \
-CH,
CH,
t-butyl sulfate
HO^O
HO-
-CH,
[O]
CH,
2-hydroxyisobutyric acid
formaldehyde
CH,
H3C
acetone
Source: NSF International (2003), ATSDR (1996), Bernauer et al. (1998), Ambers et al. (1999), and Cederbaum and
Cohen (1980).
Figure 1-1. Biotransformation of tert-butanol in rats and humans.
1.1.3. Description of Toxicokinetic Models
While no models of tert-butanol have been created independently of other chemicals from
which it arises as a metabolite (e.g., MTBE, ETBE), tert-butanol submodels have been adapted
specifically to estimate internal doses for administration of tert-butanol. In particular, some of
these tert-butanol submodels were parameterized using pharmacokinetic studies with tert-butanol
exposures. Three physiologically based pharmacokinetic (PBPK) models [Leavens and Borghoff
(20091: Salazaretal. (20151. and Borghoff et al. (20161] have been developed that can be used to
simulate direct administration of tert-butanol, as well as its parent compound, in rats. Other
models have incorporated tert-butanol as a submodel following MTBE administration but were not
considered further because they do not include terms for direct exposure to tert-butanol (e.g., Rao
and Ginsberg f!99711. In Leavens and Borghoff f20091. tert-butanol is incorporated as a metabolite
of MTBE; in Salazaretal. (2015) and Borghoff et al. (2016). it is incorporated as a metabolite of
ETBE. With all three models, inhalation and oral exposure to tert-butanol can be simulated in rats
(i.e., with exposure to the parent MTBE or ETBE set to zero). A more detailed summary and
evaluation of the toxicokinetic models is provided in Appendix B of the Supplemental Information
(see Sections B.1.5 and B.1.7).
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1.1.4. Chemicals Extensively Metabolized to tert-Butanol
tert-Butanol is a metabolite of other compounds, including ETBE, MTBE, and tert-butyl
acetate. Some of the toxicological effects observed for these compounds are attributed to
tert-butanol. Animal studies demonstrate that chronic exposure to ETBE is associated with
noncancer kidney effects, including increased kidney weights in male and female rats accompanied
by increased chronic progressive nephropathy (CPN), urothelial hyperplasia (in males), and
increased blood concentrations of total cholesterol, blood urea nitrogen, and creatinine (Saito etal..
2013: Suzuki etal.. 20121. In these studies, increased liver weight and centrilobular hypertrophy
also were observed in male and female rats exposed to ETBE. Liver adenomas and carcinomas
were increased in male rats following 2-year inhalation exposure fSaito etal.. 20131.
In 1996, the U.S. Agency for Toxic Substances and Disease Registry's (ATSDR) Toxicological
Profile for MTBE (ATSDR. 1996) identified cancer effect levels of MTBE based on carcinogenicity
data in animals. ATSDR reported that inhalation exposure was associated with kidney cancer in
rats and liver cancer in mice. ATSDR concluded that oral exposure to MTBE might cause liver and
kidney damage and nervous system effects in rats and mice. The chronic inhalation minimal risk
level was derived based on incidence and severity of chronic progressive nephropathy in female
rats fATSDR. 19961. In 1997, EPA's Office of Water concluded that MTBE is carcinogenic to animals
and poses a potential carcinogenic potential to humans based on an increased incidence of Leydig
cell adenomas of the testes, kidney tumors, lymphomas, and leukemia in exposed rats (U.S. EPA.
1997). In 1998, the International Agency for Research on Cancer (IARC) found "limited evidence"
of MTBE carcinogenicity in animals and placed MTBE in Group 3 [i.e., not classifiable as to
carcinogenicity in humans; IARC (1999)]. IARC reported that oral exposure in rats resulted in
testicular tumors in males and lymphomas and leukemias (combined) in females; inhalation
exposure in male rats resulted in renal tubule adenomas; and inhalation exposure in female mice
resulted in hepatocellular adenomas (IARC. 1999).
No assessments by national or international agencies or chronic studies for tert-butyl
acetate are available.
1.2. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM
1.2.1. Kidney Effects
Synthesis of Effects in Kidney
This section reviews the studies that investigated whether subchronic or chronic exposure
to tert-butanol can affect kidneys in humans or animals. The evidence base examining kidney
effects following tert-butanol exposure contains eight studies (from five references) performed in
rats or mice fHuntingdon Life Sciences. 2004: Acharva etal.. 1997: NTP. 1997: Acharva etal.. 1995:
NTP. 19951 and a reevaluation of the rat data from NTP f!9951. published by Hard etal. f20111 and
Hard etal. (2019): no human data are available. Studies using short-term and acute exposures that
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examined kidney effects are not included in the evidence tables; they are discussed in the text,
however, if they provide data to inform mode of action (MOA) or hazard identification. tert-Butanol
exposure resulted in kidney effects after both oral (drinking water) and inhalation exposure in both
sexes of rats (see Table 1-2, Table 1-3, Figure 1-2, and Figure 1-3); studies are arranged in the
evidence tables first by effect, then by route, and then duration.
The design, conduct, and reporting of each study were reviewed, and each study was
considered adequate to provide information pertinent to this assessment. Interpretation of
nonneoplastic kidney endpoints in rats, however, is complicated by the common occurrence of
age-related, spontaneous lesions characteristic of chronic progressive nephropathy [NTP f20151:
Hard et al. C20131: Melnick et al. C20121: U.S. FPA fl 991 al:
http: //ntp.niehs.nih.gov/nnl/urinary/kidney/necp/index.htm]. CPN is more severe in male rats
than in females and is particularly common in the Sprague-Dawley and Fischer 344 strains. Dietary
and hormonal factors play a role in modifying CPN, although the etiology is largely unknown (see
further discussion below).
Kidney weight. Kidney weight was observed to increase in male and female F344 rats
following exposures of 13 weeks [oral and inhalation; NTP T19971] and 15 months [oral; NTP
f!9951], Huntingdon Life Sciences f20041 also reported increased kidney weight in
Sprague-Dawley rats administered tert-butanol orally for approximately 10 weeks (tabular data on
relative and absolute kidney weights are presented in the Supplemental Information to this
Toxicological Review). A dose-related increase in absolute kidney weight was also observed in both
male and female rats (Spearman's rank coefficient [rho] > 0.72) following either oral or inhalation
exposures (see Figure 1-4, Figure 1-5, and Figure 1-6), and in female mice following inhalation
exposure (rho = 0.9).
Measures of relative, as opposed to absolute, organ weight are sometimes preferred
because they account for changes in body weight that might influence changes in organ weight
(Bailey etal.. 2004). although the potential impact of both should be evaluated. However, for
tert-butanol, body weight in exposed animals noticeably decreased at the high doses relative to
controls in the oral 13-week and 2-year studies (NTP. 19951. Thus, use of relative organ-weight
change would not be a reliable measure of kidney-weight change for this assessment. Although
relative and absolute kidney-weight data are both presented in exposure-response arrays (and in
evidence tables in Appendix B of the Supplemental Information), the absolute measures were
considered more informative for determining tert-butanol hazard potential. Support for this
judgement can be found in a 2014 analysis indicating that increased absolute, but not relative,
subchronic kidney weights are significantly correlated with chemically induced histopathological
findings in the kidney in chronic and subchronic studies (Craig etal.. 20151.
Kidney histopathology. Treatment-related histopathological changes were observed in the
kidneys of male and female F344 rats following 13-week and 2-year oral exposures fNTP. 19951
and in male F344 rats following a 13-week inhalation exposure fNTP. 19971. Similarly, male Wistar
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rats exposed for approximately 10 weeks exhibited an increase in histopathological kidney lesions
fAcharva et al.. 1997: Acharva et al.. 19951. For B6C3F1 mice, however, the study authors did not
report histopathological changes for mice exposed for 13 weeks and 2 years to tert-butanol via the
oral route fNTP. 19951 and 13 weeks via the inhalation route fNTP. 19971. More specific details on
the effects observed in rats, reported by NTP T1997.19951 and Acharva etal. f 19971: (1995.) are
described below.
Nephropathy and severity of nephropathy were reported in male and female rats in the
13-week oral studies fNTP. 19951. The nephropathy was characterized as "...a spontaneous
background lesion...typically consisting] of scattered renal tubules lined by basophilic
regenerating tubule epithelium" fNTP. 19951. NTP T19951 noted that the increase in severity of
nephropathy was related to tert-butanol and "characterized by an increase in the number and size
of foci of regeneration." Compared with controls, the severity of nephropathy increased in the
13-week male rats, which exhibited nephropathy in 94% of all exposed animals and 70% of
controls. Conversely, lesion severity was unchanged in the females, although nephropathy
incidence significantly increased with tert-butanol exposure. In the 13-week inhalation study (NTP.
19971. nephropathy was present in all but two male rats, including controls. NTP T19971
characterized the reported chronic nephropathy in control male rats as "1 to 3 scattered foci of
regenerative tubules per kidney section. Regenerative foci were characterized by tubules with
cytoplasmic basophilia, increased nuclear/cytoplasmic ratio, and occasionally thickened basement
membranes and intraluminal protein casts." In exposed groups, the severity generally increased
from minimal to mild with increasing dose as "evidenced by an increased number of foci." No
treatment-related kidney histopathology was reported in the female rats exposed through
inhalation fNTP. 19971.
In the 2-year oral study by NTP T19951. nephropathy was reported at 15 months and
2 years. The NTP T19951 characterization of nephropathy following chronic exposure included
multiple lesions: "thickened tubule and glomerular basement membranes, basophilic foci of
regenerating tubule epithelium, intratubule protein casts, focal mononuclear inflammatory cell
aggregates within areas of interstitial fibrosis and scarring, and glomerular sclerosis." At
15 months, male and female rats (30/30 treated; 10/10 controls) had nephropathy, and the
severity scores ranged from minimal to mild. At 2 years, male and female rats (149/150 treated;
49/50 controls) also had nephropathy, and although the severity was moderate in the control
males and minimal to mild in the control females, severity increased with tert-butanol exposure in
both sexes (NTP. 1995).
The lesions collectively described by NTP (1997.1995) as nephropathy and noted as
common spontaneous lesions in rats are consistent with CPN. CPN is not a specific diagnosis per se
but rather an aggregate term describing a spectrum of effects. In addition, no known counterpart to
CPN has been identified in the aging human kidney. However, several individual lesions noted in
CPN (e.g., tubule atrophy, tubule dilation, thickening of tubular basement membranes,
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glomerulosclerosis) also occur in the human kidney (Lusco etal.. 2016: Zoiaetal.. 2015: Frazier et
al.. 2012: Satirapoi etal.. 20121. Therefore, exacerbation of one or more of these lesions following
tert-butanol exposure may reflect some type of cell injury or inflammatory process that is relevant
to the human kidney.
Several factors, including genetic predisposition, increased glomerular permeability,
elevated protein loads, and hemodynamic changes in the kidney, may play a role in the progression
of CPN; however, no etiological factors have been clearly identified (NIEHS. 20191. The effects
characterized as CPN are related to age (increased severity and incidence) and strain (higher in
Sprague-Dawley rats compared with other strains); they are not considered histopathological
manifestations of chemically induced toxicity fNIEHS. 20191 [see U.S. EPA f!991al. p. 35 for further
details and a list of the typical, observable histopathological features of CPN], These lesions,
however, are frequently exacerbated by tert-butanol treatment fNTP. 19971. as evidenced by the
dose-related increases in severity of the nephropathy compared with female and male rat controls.
The chemical-related changes in increased severity of nephropathy are included in the
consideration of hazard potential.
NTP f!9951 observed other kidney lesions, described as being associated with nephropathy
but diagnosed separately. Renal mineralization is defined by NTP f!9951 as "focal mineral deposits
primarily at the corticomedullary junction." Renal (corticomedullary) mineralization was observed
in essentially all female rats at all reported treatment durations. NTP (1995) described focal,
medullary mineralization as being associated with CPN but noted that focal mineralization is
"usually more prominent in untreated females than in untreated males," which is consistent with
the widespread appearance of this lesion in females. Corticomedullary mineralization (also
referred to as nephrocalcinosis) in the rat is a common (especially in females)
background/incidental finding that is not generally considered to be clinically important to rats or
relevant to human health fFrazier etal.. 20121. Thus, corticomedullary mineralization was not
included in the consideration of hazard potential.
A dose-related, increased incidence of renal mineralization was reported in male rats at the
end of the 13-week, 15-month, and 2-year oral evaluations (NTP. 19951. This mineralization is
distinct from linear mineralization, which is considered a lesion characteristic of alpha 2u-globulin
nephropathy (for further discussion of this particular lesion, see Mode of Action Analysis—Kidney
Effects). Linear mineralization is characterized as distinct linear deposits along radiating medullary
collecting ducts. An increased incidence of linear mineralization was limited to exposed males in
the 2-year oral study (NTP. 1995). Linear mineralization was not included in the consideration of
hazard potential.
Two other histological kidney lesions observed in male and female rats are suppurative
inflammation and transitional epithelial hyperplasia (also known as urothelial hyperplasia). These
lesions were observed in the 2-year oral NTP f!9951 study. NTP f!9951 and Frazier etal. f20121
described these lesions as related to the nephropathy (characterized above as common and
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spontaneous and considered CPN). However, suppurative inflammation and urothelial hyperplasia
are typically not related to CPN or are noted as secondary changes to CPN and not a direct result of
CPN fNIEHS. 20191. Incidence of suppurative inflammation in female rats was low in the control
group and increased with dose, with incidences >24% in the two highest dose groups, compared
with controls. In comparison, 20% of the control males exhibited suppurative inflammation, and
the changes in incidence were not dose related (incidences ranging from 18 to 36%). To determine
whether the severity of these lesions was positively associated with the severity of nephropathy,
contingency tables comparing the occurrence of suppurative inflammation with nephropathy in
individual rats were arranged by severity and analyzed with Spearman's rank correlation tests to
determine strength of associations for each comparison (see Table 1-4 and Table 1-5). Suppurative
inflammation and nephropathy were moderately correlated in females (rho = 0.47) and weakly
correlated in males (rho = 0.17). The data indicate that CPN correlates with the induction of
suppurative inflammation; however, the inflammation in female rats is also treatment related.
Given that CPN is also dose-dependently increased in male and female rats (Salazar etal.. 2015).
disentangling the relative contribution of CPN and tert-butanol in the exacerbation of suppurative
inflammation is problematic.
Transitional epithelial hyperplasia (also known as urothelial hyperplasia) was observed in
both male and female rats exposed orally fNTP. 19951. In the control males, 50% of the animals
exhibited transitional epithelial hyperplasia and the incidence and severity increased with dose.
Only the mid- and high-dose females, however, exhibited dose-related increases in incidence and
severity of transitional epithelial hyperplasia. This type of lesion was not reported in the control or
low-dose females. NTP (1995) described transitional epithelial hyperplasia as increased layers of
the transitional epithelial lining of the renal pelvis; the study authors noted no progression of this
hyperplastic lesion to neoplasia. To determine whether the severity of the hyperplasia was
positively associated with the severity of nephropathy, contingency tables comparing the
occurrence of transitional epithelial hyperplasia with nephropathy in individual rats were arranged
by severity and analyzed with Spearman's rank correlation tests to determine strength of
associations for each comparison (see Table 1-6 and Table 1-7). Transitional epithelial hyperplasia
and nephropathy were strongly correlated (rho = 0.66) in males and moderately correlated
(rho = 0.44) in females. The transitional epithelial hyperplasia observed in male and female rats is
consistent with advanced CPN fFrazier etal.. 20121. Similar to suppurative inflammation,
transitional epithelial hyperplasia is both increased by dose and correlated with nephropathy,
which is also dose related. Thus, disentangling the contributions of dose and nephropathy in the
development of transitional epithelial hyperplasia is not possible. Transitional epithelial
hyperplasia should not be confused with another lesion noted in the 2-year evaluation, renal tubule
hyperplasia, which was considered preneoplastic (for further details regarding this type of
hyperplasia, see the discussion under Kidney tumors, below).
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Additional histopathological changes, including increased tubular degeneration,
degeneration of the basement membrane of the Bowman's capsule, diffused glomeruli, and
glomerular vacuolation were noted in a 10-week study in male Wistar rats fAcharva et al.. 1997:
Acharva et al.. 19951. A decrease in glutathione in the kidney accompanied these changes, which
the study authors noted as potentially indicative of oxidative damage. Acharva et al. f!9971 and
Acharva et al. (1995) used one dose and a control group and did not report incidences. The
increased tubule degeneration and glomerular vacuolation could be characterized as tubular
atrophy and glomerular hyalinization, respectively, consistent with CPN; however, without
quantitative information, examining the differences between the control and treated animals to
determine whether CPN plays a role in development of these effects is not possible. Although based
on the noted appearance of the effects in the treated animals compared with controls, the effects
likely are treatment related.
Serum or urinary biomarkers informative of kidney toxicity were not measured in the
studies discussed above. Some changes occurred in urinalysis parameters (e.g., decreased urine
volume and increased specific gravity), accompanied by reduced water consumption, and thus,
might not be related to an effect of kidney function fNTP. 19951.
Kidney tumors. The kidney is also a target organ for cancer effects following long-term
exposure to tert-butanol (see Table 1-3, Figure 1-2). Male F344 rats had an increased incidence of
combined renal tubule adenomas or carcinomas in the 2-year oral bioassay (Hard etal.. 2011: NTP.
1995). The increase in tumors from control was similar in the low- and high-dose groups and
highest in the mid-dose group. Overall, tumor increases were statistically significant in trend
testing, which accounted for mortality (p < 0.018). At the highest exposure group in male rats, the
mean body weight decreased by 24%, raising some question as to whether the kidney tumors
observed in the mid-dose group in male rats were solely the result of excessive toxicity rather than
the carcinogenicity of tert-butanol. EPA Cancer Guidelines fU.S. EPA. 2005al discusses the
determination of an "excessively high dose" as compared to an "adequate high dose" and describes
the process as one of expert judgment, which requires that "adequate data demonstrate that the
effects are solely the result of excessive toxicity rather than the carcinogenicity of the tested agent"
In the 2-year oral bioassay (NTP. 1995). the study authors did not report exposure-related overt
toxicity in male rats or any changes in toxicokinetics at the middle or high doses. Furthermore, the
tumor incidence at the high dose in male rats, which had a final body-weight reduction of 24%, was
not significantly different from controls. Mortality increased with increasing exposure (p = 0.001)
over the 2-year exposure period; however, increased mortality does not correlate with the highest
tumor incidence occurring at the middle dose.
Increases in incidence and severity of renal tubule hyperplasia also were observed in male
rats. NTP (1995) stated that"[t]he pathogenesis of proliferative lesions of renal tubule epithelium
is generally considered to follow a progression from hyperplasia to adenoma to carcinoma fHard.
19861." Similarly, EPA considered the renal tubule hyperplasia to be a preneoplastic effect
1-9
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
associated with the renal tubule tumors. Renal tubule hyperplasia was found in one high-dose
female fNTP. 19951: no increase in severity was observed and no tumors were reported in female
rats. Therefore, this effect in females, which was not considered toxicologically significant, is not
discussed further. Two renal tubular adenocarcinomas in male mice also were reported fNTP.
19951. one each in the low- and high-dose groups, but were not considered by National Toxicology
Program (NTP) to be "biologically noteworthy changes"; thus, the tumors in mice are not discussed
further.
A Pathology Working Group, sponsored by Lyondell Chemical Company, reevaluated the
kidney changes in the NTP 2-year study to determine whether additional histopathological changes
could be identified to inform the MOA for renal tubule tumor development fHard etal.. 20111.
Working group members were blinded to treatment groups and used guidelines published by Hard
and Wolf f 19991 and refinements reported by Hard and Seelv f20051. Hard and Seelv f20061. and
Hard (2008). The group's report and analysis by Hard etal. (2011) confirmed the NTP findings of
renal tubule hyperplasia and renal tubule tumors in male rats at 2 years. Not all findings were
considered nephrotoxic responses; for example, the study authors considered transitional
hyperplasia secondary to CPN. In particular, they reported similar overall tumor incidences in the
exposed groups. Hard etal. f20111. however, reported fewer renal tubule adenomas and
carcinomas in the control group than in the original NTP study. As a result, all treated groups had
statistically significant increases in renal tubule adenomas and carcinomas (combined) compared
with controls. Additionally, Hard etal. (2011) considered fewer tumors to be carcinomas than did
the original NTP study. Results of both NTP (1995) and the reanalysis by Hard etal. (2011) are
included in Table 1-3 and Figure 1-2.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
¦ = 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 (CJ
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; 15rao (D)
Absolute weight; F Rat; 15mo (D)
Relative weight; F Rat; 15mo (D)
Decreased glutathione; M Rat; lOwk (A]
Inflammation; F Rat; 2yr (D)
Nephropathy severity; M Rat; 13wk CD]
Nephropathy incidence; F Rat; 13wl< (D)
Mineralization; M Rat; 13wl< (D)
Mineralization; F Rat; 13wk (D)
Nephropathy severity; M Rat; 2yr (D)
Nephropathy severity; F Rat; 2yr (DJ
Linear mineralization; M Rat; 2yr (D)
Interim/terminal mineralization; M Rat; 2yr (D)
Interim/tenninal 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)
0—B-
~ ~ ~ ~
~ ~ ~ ~
¦ ¦ ¦
-¦—x
® A
~-B-
-B
~ ~ ~
-B-m
~ ~ ~
~ ~ ~
Kidney
Histopathology
~-Si
B-e-B
~—BB-B—~
~ ~ ¦
~ ~ ¦
~-B-B
~-B-
~ ~ ¦
~-B-B
~B~ i ~D~ 1
~-B-B
B-B-B
B-B-B
10 100 1,000 10,000 100,000
Dose (mg/kg-day]
Sources: (A) Acharva et al, (1997): (1995): (B) Hard et al. (2011)a; (C) Huntingdon Life Sciences (2004):
(D) NTP (1995).
aReanalysis of NTP (1995).
Figure 1-2. Exposure response array for kidney effects following oral
exposure to Cert-butanol.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
¦ = 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; F Rat
Absolute/relative weight; M Mouse
Absolute weight; F Mouse
Relative weight; F Mouse
~ B
~ B
~ B
~ B
~ B
~ B
~ B
-B B B
-B B-
-B B B
-B B B
-B B-
1 1 1 1 1—I I I | 1 1 1 1 1—I I I
100 1,000 10,000
Concentration (mg/m3)
Source: NTP (1997).
Figure 1-3. Exposure-response array of kidney effects following inhalation
exposure to tert-butanol (13-week studies, no chronic studies available).
1-12
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Male rats Female rats
rho= 0.84 (all)
rho= 0.78 (all)
rho= 0.92 (oral)
rho= 0.72 (oral)
rho= 0.80 (inhalation)
rho= 0.9 (inhalation) 9
•
•
•
•
••
•
• • O •
• o
• O
• •
•
•
0
o
• o
0 .° .
o
o
1 10 100 1000 1 10 100 1000 10000
tert-butanol blood conc. (mg/l) tert-butanol blood concentration (mg/l)
• Oral exposure
O Inhalation exposure
Sources: NTP (1997); NTP (1995).
Figure 1-4. Comparison of absolute kidney-weight change in male and female
rats across oral and inhalation exposure based on internal blood
concentration. Spearman's rank correlation coefficient (rho) was calculated to
evaluate the direction of a monotonic association (e.g., positive value = positive
association) and the strength of association.
Male mice Female mice
rtio= -0.1
rtio= 0.9
~
•
•
•
•
•
•
•
*
0 2000 4000 6000 8000 0 2000 6000 10000 12000
Admnstered Administered dose (mgYg-day)
Source: NTP (1995).
Figure 1-5. Comparison of absolute kidney-weight change in male and female
mice following oral exposure based on administered concentration.
Spearman's rank correlation coefficient (rho) was calculated to evaluate the
direction of a monotonic association (e.g., positive value = positive association) and
the strength of association.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Male mice Female mice
rho= 0.2 #
o
'
II
o
-E
*
*
i *
*
•
*
*
0 1 000 2000 5000 40CC 5000 6C00 0 1000 2000 3000 4000 5000 SMC TOW
Administered dose{rnj''nf) Admiiistered dose {mg.Trr1}
Source: NTP (1997).
Figure 1-6. Comparison of absolute kidney-weight change in male and female
mice following inhalation exposure based on administered concentration.
Spearman's rank correlation coefficient (rho) was calculated to evaluate the
direction of a monotonic association (e.g., positive value = positive association) and
the strength of association.
1-14
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Table 1-2. Changes in kidney histopathology in animals following exposure to
tert- butanol
Reference and study design
Results
Acharva et al. (1997)
Acharva et al. (1995)
Wistar rat; 5-6 males/treatment
Drinking water (0 or 0.5%), 0 or
575 mg/kg-d
10 wk
1" tubular degeneration, degeneration of the basement membrane of the
Bowman's capsule, diffused glomeruli, and glomerular vacuolation (no
incidences reported)
4/ kidney glutathione (~40%)a
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, or
3,610b mg/kg-d
F: 0, 290, 590, 850,1,560, or
3,620b mg/kg-d
13 wk
Incidence (severity):
Male
Female
Dose
(mg/kg-d)
Mineral-
ization
Nephrop-
athyd
Dose
(mg/kg-d)
Mineral-
ization0
Nephrop-
athyd
0
0/10
7/10 (1.0)
0
10/10 (1.7)
2/10 (1.0)
230
0/10
10/10 (1.6a)
290
10/10 (2.0)
3/10 (1.0)
490
2/10 (1.5)
10/10 (2.6a)
590
10/10 (2.0)
5/10 (1.0)
840
8/10a(1.4)
10/10 (2.7a)
850
10/10 (2.0)
7/10a (1.0)
1,520
4/10a(1.0)
10/10 (2.6a)
1,560
10/10 (2.0)
8/10a (1.0)
3,610b
4/10a(1.0)
7/10 (1.1)
3,620b
6/10 (1.2)
7/10a (1.0)
NTP (1995)
B6C3F1 mouse;
10/sex/treatment
Drinking water (0, 2.5, 5,10, 20,
or 40 mg/mL)
M: 0, 350, 640, 1,590, 3,940, or
8,210b mg/kg-d
F: 0, 500, 820, 1,660, 6,430, or
ll,620b mg/kg-d
13 wk
Study authors indicated no treatment-related changes in kidney-related
histopathology (histopathological data not provided for the 13-wk study)
1-15
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
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; 60/sex/treatment
(10/sex/treatment evaluated at
15 mo interim)
Drinking water (0,1.25, 2.5, 5, or
Male
Dose
(mg/kg-d)
Mineralization0
(interim)
Mineralizationc
(terminal)
Linear mineralizationc
(terminal)
10 mg/mL)
M: 0, 90, 200, or 420b mg/kg-d
0
1/10 (1.0)
26/50 (1.0)
0/50
F: 0,180, 330, or 650b mg/kg-d
90
2/10 (1.0)
28/50(1.1)
5/50a (1.0)
2 yr
200
5/10 (1.8)
35/50 (1.3)
24/50a (1.2)
420b
9/10a (2.3)
48/50a (2.2)
46/50a (1.7)
Dose
(mg/kg-d)
Transitional
epithelial
hyperplasia
Nephropathyd
severity
Inflammation
(suppurative) incidence
0
25/50 (1.7)
3.0
10/50
90
32/50 (1.7)
3.1
18/50
200
36/50a (2.0)
3.1
12/50
420b
40/50a (2.1)
3.3a
9/50
Female
Dose
(mg/kg-d)
Mineralizationc
(interim)
Mineralization0
(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)
13/50a
650b
10/10 (2.8)
50/50 (2.9)
17/50a
Dose
(mg/kg-d)
Transitional
epithelial
hyperplasia
Nephropathyd
severity
0
0/50
1.6
180
0/50
1.9a
330
3/50 (1.0)
2.3a
650b
17/50a (1.4)
2.9a
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Table 1-2. Changes in kidney histopathology in animals following exposure
to tert-butanol (continued)
Reference and study design
Results
NTP (1995)
B6C3F1 mouse;
60/sex/treatment
Drinking water (0, 5,10, or
20 mg/mL)
M:0, 540, 1,040, or
2,070b mg/kg-d
F: 0, 510,1,020, or
2,110 mg/kg-d
2 yr
No treatment-related changes in kidney-related histopathology observed
NTP (1997)
F344/N rat; 10/sex/treatment
Inhalation 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 h/d, 5 d/wk
13 wk
Generation method (Sonimist
ultrasonic spray nozzle
nebulizer), analytical
concentration and method were
reported
Male
Concentration
(mg/m3)
Incidence of chronic
nephropathy®
Average severity of chronic
nephropathy
0
9/10
1.0
406
8/10
1.4
824
9/10
1.4
1,643
10/10
1.6
3,273
10/10
1.9
6,368
10/10
2.0
Females: no treatment-related changes in kidney-related histopathology
observed
Severity categories: 1 = minimal, 2 = mild. No results from statistical tests
reported
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Table 1-2. Changes in kidney histopathology in animals following exposure
to tert-butanol (continued)
Reference and study design
Results
NTP (1997)
B6C3F1 mouse;
10/sex/treatment
Inhalation 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 h/d, 5 d/wk
13 wk
Generation method (Sonimist
ultrasonic spray nozzle
nebulizer), analytical
concentration and method were
reported
No treatment-related changes in kidney-related histopathology observed
statistically significant p < 0.05, as determined by the study authors.
bThe high-dose group had an increase in mortality.
cMineralization defined in NTP (1995) as focal mineral deposits, primarily at the corticomedullary junction.
Linear mineralization was defined as foci of distinct linear deposits along radiating medullary collecting ducts;
linear mineralization not observed in female rats.
Nephropathy defined in NTP (1995) as lesions, including thickened tubule and glomerular basement
membranes, basophilic foci of regenerating tubule epithelium, intratubule protein casts, focal mononuclear
inflammatory cell aggregates within areas of interstitial fibrosis and scarring, and glomerular sclerosis.
Nephropathy characterized in NTP (1997) as scattered foci of regenerative tubules (with cytoplasmic basophilia,
increased nuclear:cytoplasmic ratio, and occasionally thickened basement membranes and intraluminal protein
casts).
Note: Conversions from drinking water concentrations to mg/kg-d were performed by the study authors.
Conversion from ppm to mg/m3 is 1 ppm = 3.031 mg/m3.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Table 1-3. Changes in kidney tumors in animals following exposure to
tert- butanol
Reference and study design
Results
NTP (1995)
Male
F344/N rat; 60/sex/treatment
(10/sex/treatment evaluated at
15 mo)
Drinking water (0,1.25, 2.5, 5, or
10 mg/mL)
M: 0, 90, 200, or 420a mg/kg-d
F: 0,180, 330, or 650a mg/kg-d
2 yr
Dose
(mg/kg-d)
Renal tubule
hyperplasia
(standard and
extended
evaluation
combined)
Renal tubule adenoma
(single)
Renal tubule
adenoma
(multiple)
0
14/50 (2.3)
7/50
1/50
90
20/50 (2.3)
7/50
4/50
200
17/50 (2.2)
10/50
9/50b
420a
25/50b (2.8)
10/50
3/50
Dose
(mg/kg-d)
Renal tubule
carcinoma
Renal tubule adenoma
(single or multiple) or
carcinoma
0
0/50
8/50
90
2/50
13/50
200
1/50
19/50b
420a
1/50
13/50
Female
Dose
(mg/kg-d)
Renal tubule
hyperplasia
Renal tubule adenoma
(single)
Renal tubule
adenoma
(multiple)
0
0/50
0/50
0/50
180
0/50
0/50
0/50
330
0/50
0/50
0/50
650a
1/50 (1.0)
0/50
0/50
Dose
(mg/kg-d)
Renal tubule
carcinoma
Renal tubule adenoma
(single or multiple) or
carcinoma
0
0/50
0/50
180
0/50
0/50
330
0/50
0/50
650a
0/50
0/50
Based on standard and extended evaluations (combined). Results do not
include the animals sacrificed at 15 mo.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Table 1-3. Changes in kidney tumors in animals following exposure to
tert-butanol (continued)
Reference and study design
Results
Hard etal. (2011)
Reanalysis of the slides from
male rats (all slides in control and
high-dose groups of males and
females, and slides from all other
males with renal tumors) in the
NTP (1995) studv (see above)
Male
Dose
(mg/kg-d)
Renal tubule
adenoma
(single)
Renal tubule
adenoma
(multiple)
Renal tubule
carcinoma
Renal tubule
adenoma
(single or
multiple) or
carcinoma
0
3/50
1/50
0/50
4/50
90
9/50
3/50
1/50
13/50b
200
9/50
9/50
0/50
18/50b
420
9/50
3/50
1/50
12/50b
NTP (1995)
B6C3F1 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 yr
No increases in kidney-related tumors. Two renal tubule adenocarcinomas,
one in the low-dose and one in the high-dose groups, were observed in male
mice. These tumors were not considered treatment related.
aThe high-dose group had an increase in mortality.
Statistically significant p < 0.05, as determined by the study authors.
Note: Conversions from drinking water concentrations to mg/kg-d were performed by the study authors.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Table 1-4. Comparison of nephropathy and suppurative inflammation in
individual male rats from the 2-year National Toxicology Program (NTP)
tert-butanol bioassay
Suppurative
inflammation
Nephropathy
None
Minimal
Mild
Moderate
Marked
None
2
1
55
82
51
Minimal
0
0
3
23
16
Mild
0
0
1
4
2
Moderate
0
0
0
0
0
Marked
0
0
0
0
0
Spearman's rank correlation test (1-sided), p = 0.0015, rho = 0.17.
Table 1-5. Comparison of nephropathy and suppurative inflammation in
individual female rats from the 2-year National Toxicology Program (NTP)
tert-butanol bioassay
Suppurative
inflammation
Nephropathy
None
Minimal
Mild
Moderate
Marked
None
7
67
90
37
4
Minimal
0
1
5
14
13
Mild
0
0
0
1
1
Moderate
0
0
0
0
0
Marked
0
0
0
0
0
Spearman's rank correlation test (1-sided), p < 0.0001, rho = 0.47.
Table 1-6. Comparison of nephropathy and transitional epithelial hyperplasia
in individual male rats from the 2-year National Toxicology Program (NTP)
tert-butanol bioassay
Transitional epithelial
hyperplasia
Nephropathy
None
Minimal
Mild
Moderate
Marked
None
2
1
51
52
l
Minimal
0
0
4
26
9
1-21
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Transitional epithelial
hyperplasia
Nephropathy
None
Minimal
Mild
Moderate
Marked
Mild
0
0
2
25
42
Moderate
0
0
2
6
17
Marked
0
0
0
0
0
Spearman's rank correlation test (1-sided), p < 0.0001, rho = 0.66.
Table 1-7. Comparison of nephropathy and transitional epithelial hyperplasia
in individual female rats from the 2-year National Toxicology Program (NTP)
tert-butanol bioassay
Transitional epithelial
hyperplasia
Nephropathy
None
Minimal
Mild
Moderate
Marked
None
7
68
95
43
7
Minimal
0
0
0
8
6
Mild
0
0
0
1
5
Moderate
0
0
0
0
0
Marked
0
0
0
0
0
Spearman's rank correlation test (1-sided), p < 0.0001, rho = 0.437.
Table 1-8. Comparison of chronic progressive nephropathy and renal tubule
hyperplasia with kidney adenomas and carcinomas in male rats from the
2-year National Toxicology Program (NTP) tert-butanol bioassay
CPN
Renal tumors
absent
Renal tumors
present
Renal tubule
hyperplasia
Renal tumors
absent
Renal tumors
present
None
2
0
None
133
29
Minimal
1
0
Minimal
17
2
Mild
57
2
Mild
17
13
Moderate
93
16
Moderate
10
3
Marked
34
35
Marked
10
6
Spearman's rank correlation test (1-sided): CPN, p < 0.0001, rho = 0.430; renal tubule hyperplasia, p = 0.01,
rho = 0.161.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Mode of Action Analysis—Kidney Effects
Alpha 2u-globulin-associated renal tubule nephropathy and carcinogenicity
One disease process to consider when interpreting kidney effects in rats is related to the
accumulation of alpha 2u-globulin protein. Alpha 2u-globulin, a member of a large superfamily of
low-molecular-weight proteins, was first characterized in male rat urine. Such proteins have been
detected in various tissues and fluids of most mammals (including humans), but the particular
isoform of alpha 2u-globulin commonly detected in male rat urine is considered specific to that sex
and species. Exposure to chemicals that induce alpha 2u-globulin accumulation can initiate a
sequence of histopathological events leading to kidney tumorigenesis. Because alpha
2u-globulin-associated renal tubule nephropathy and carcinogenicity occurring in male rats are
presumed not relevant for assessing human health hazards (U.S. EPA. 1991a). evaluating the data to
determine whether alpha 2u-globulin plays a role is important The role of alpha 2u-globulin
accumulation in the development of renal tubule nephropathy and carcinogenicity observed
following tert-butanol exposure was evaluated using the U.S. EPA (1991a) Risk Assessment Forum
Technical panel report, Alpha 2u-GIobuIin: Association with Chemically Induced Renal Toxicity and
Neoplasia in the Male Rat as well as the IARC framework fCapenetal.. 19991. These frameworks
provide specific guidance for evaluating renal tubule tumors in male rats that are related to
chemical exposure for the purpose of risk assessment, based on an examination of the potential
involvement of alpha 2 u-globulin accumulation.
Studies in the tert-butanol evidence base evaluated and reported effects on the kidney,
providing some evidence to evaluate this MOA. Additionally, several studies were identified that
specifically evaluated the role of alpha 2u-globulin in tert-butanol-induced renal tubule
nephropathy and carcinogenicity fBorghoffetal.. 2001: Williams and Borghoff. 2001: Takahashi et
al.. 19931. Because the evidence reported in these studies is specific to alpha 2u-globulin
accumulation, it is presented in this section; it was not included in the animal evidence tables in the
previous section.
The hypothesized sequence of alpha 2u-globulin renal tubule nephropathy, as described by
U.S. EPA (1991a). is as follows. Chemicals that induce alpha 2u-globulin accumulation do so
rapidly. Alpha 2u-globulin accumulating in hyaline droplets is deposited in the S2 (P2) segment of
the proximal tubule within 24 hours of exposure. Hyaline droplets are a normal constitutive
feature of the mature male rat kidney; they are particularly evident in the S2 (P2) segment of the
proximal tubule and contain alpha 2u-globulin (U.S. EPA. 1991a). Abnormal increases in hyaline
droplets have more than one etiology and can be associated with the accumulation of different
proteins. As hyaline droplet deposition continues, single-cell necrosis occurs in the S2 (P2)
segment, which leads to exfoliation of these cells into the tubule lumen within 5 days of chemical
exposure. In response to the cell loss, cell proliferation occurs in the S2 (P2) segment after 3 weeks
and continues for the duration of the exposure. After 2 or 3 weeks of exposure, the cell debris
accumulates in the S3 (P3) segment of the proximal tubule to form granular casts. Continued
1-23
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
chemical exposure for 3 to 12 months leads to the formation of calcium hydroxyapatite in the
papillae, which results in linear mineralization. After 1 or more years of chemical exposure, these
lesions can result in the induction of renal tubule adenomas and carcinomas (see Figure 1-7).
U.S. EPA fl991al identified two questions that must be addressed to determine the extent
to which alpha 2u-globulin-mediated processes induce renal tubule nephropathy and
carcinogenicity. First, whether the alpha 2u-globulin process occurs in male rats and influences
renal tubule tumor development must be determined. Second, whether the renal effects in male
rats exposed to tert-butanol are due solely to the alpha 2u-globulin process must be determined.
U.S. EPA f!991al stated the criteria for answering the first question in the affirmative are as
follows:
1) Hyaline droplets are larger and more numerous in treated male rats;
2) The protein in the hyaline droplets in treated male rats is alpha 2u-globulin (i.e.,
immunohistochemical evidence); and
3) Several (but not necessarily all) additional steps in the pathological sequence appear in
treated male rats as a function of time, dose, and progressively increasing severity
consistent with the understanding of the underlying biology, as described above, and
illustrated in Figure 1-7.
The available data relevant to this first question are summarized in Table 1-9, Figure 1-8,
and Figure 1-9, and are evaluated below.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Source: Adapted from Swenberg and Lehman-McKeeman (1999) and U.S. EPA (1991a).
Figure 1-7. Temporal pathogenesis of alpha 2u-globulin-associated
nephropathy in male rats. Alpha 2u-globulin synthesized in the livers of male rats
is delivered to the kidney where it can accumulate in hyaline droplets and be
retained by epithelial cells lining the S2 (P2) segment of the proximal tubules. Renal
pathogenesis following continued tert-butanol exposure and increasing droplet
accumulation can progress stepwise from increasing epithelial cell damage, death,
and dysfunction leading to the formation of granular casts in the corticomedullary
junction, linear mineralization of the renal papillae, and carcinogenesis of the renal
tubular epithelium.
Table 1-9. Summary of data on the alpha 2u-globulin process in male rats
exposed to tert-butanol
Duration
Dose
Results
Comments
Reference
1) Hyaline droplets are increased in size and number
10 d (inhalation)
0, 758, 1,364,
5,304 mg/m3
+
stat sig at 5,304 mg/m3;
stat sig trend
Borghoff et al. (2001)
13 wk (inhalation)
0, 3,273, 6,368 mg/m3
-
NTP (1997)a
13 wk (oral)
0, 230, 490, 840, 1,520,
3,610 mg/kg-d
(+)
observed in all but
highest dose group
NTP(1995)
2) The protein in the hyaline droplets is alpha 2u-globulin
10 d (inhalation)
0, 758, 1,364,
5,304 mg/m3
+
stat sig at 5,304 mg/m3;
stat sig trend
Borghoff et al. (2001)
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Duration
Dose
Results
Comments
Reference
12 h (elapsed time
following single
oral dose)
0, 500 mg/kg
+
Williams and Borghoff
(2001)
3)
Several (but not necessarily all) additional steps in the pathological sequence are present in male rats,
such as:
a) Subsequent cytotoxicity and single-cell necrosis of tubule epithelium, with exfoliation of degenerate
epithelial cells
10 wk (oral)
0, 575 mg/kg-d
(+)
degeneration of renal
tubules reported
Acharva et al. (1997)
13 wk (oral)
0, 230, 490, 840, 1,520,
3,610 mg/kg-d
-
NTP(1995)
b) Sustained regenerative tubule cell proliferation (NOTE: The positive studies below reported cell
proliferation but did not show necrosis or cytotoxicity; therefore, it cannot be assumed that the results
indicate regenerative proliferation is occurring.)
10 wk (oral)
0, 575 mg/kg-d
-
Acharva et al. (1997)
10 d (inhalation)
0, 758, 1,364,
5,304 mg/m3
+
stat sig at all doses; stat
sig trend
Borghoff et al. (2001)
13 wk (oral)
0, 230, 490, 840, 1,520,
3,610 mg/kg-d
+
elevated at 840 mg/kg-d;
stat sig at 1,520 mg/kg-d
NTP(1995)
c) Development of intraluminal granular casts from sloughed cellular debris, with consequent tubule
dilation
13 wk (oral)
0, 230, 490, 840, 1,520,
3,610 mg/kg-d
(+)b
NTP (1995); Hard etal.
(2011)°
2 yr (oral)
0, 90, 200,
420 mg/kg-d
-
NTP (1995); Hard etal.
(2011)d
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Table 1-9. Summary of data on the alpha 2u-globulin process in male rats
exposed to tert-butanol (continued)
Duration
Dose
Results
Comments
Reference
d) Linear mineralization of tubules in the renal papilla
13 wk (oral)
0, 230, 490, 840, 1,520,
3,610 mg/kg-d
-
NTP (1995); Hard etal.
(2011)°
2 yr (oral)
0, 90, 200,
420 mg/kg-d
+; (+)
all doses stat sig
NTP (1995); Hard etal.
(2011)d
e) Foci of tubular hyperplasia
2 yr (oral)
0, 90, 200,
420 mg/kg-d
+
stat sig trend at all doses;
stat sig at 420 mg/kg-d
NTP (1995)
+ = Statistically significant change reported in one or more treated groups; (+) = Effect was reported in one or
more treated groups, but statistics not reported; - = No statistically significant change reported in any of the
treated groups; stat sig = statistically significant.
aNTP (1997) did not observe any effects consistent with alpha 2u-globulin nephropathy.
Precursors to granular casts reported.
cReanalysis of hematoxylin and eosin-stained kidney sections from all male control and 1,520-mg/kg-d groups and
a representative sample of kidney sections stained with Mallory-Heidenhain stain, from the 13-wk study from
NTP (1995).
dReanalysis of slides for all males in the control and 420-mg/kg-d dose groups and all animals with renal tubule
tumors from 2-yr (NTP, 1995). Protein casts reported, not granular casts.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
¦ = 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
I = exposures at which all animals were dead and unable to be examined for the endpoint
a = exposures at which effect was observed but statistics not reported
T Hyaline droplet NTP (1995]; 13 wk
size/number
• •—~
—• x
Identification of ct2u- Williams and Borghoff (2001);
globulin in hyaline x 2 hr after single dose
droplets
¦
Acharya et al. (1997]; 10 wk
Cytotoxicity/single-cell
necrosis of tubule epithelium;
epithelial cell exfoliation
NTP (1995]; 13 wk
•
~—&-e
-a—x
Acharya et al. (1997]; 10 wk
Tubule cell
proliferation
NTP (1995]; 13 wk
~
~ B—B
—¦ X
NTP (1995]; Hard et al. (2011]*; 13 wk
Granular
casts/tubule
dilation
NTP (1995]; Hard et al. (2011]; 2 yr
~
~ B—B
B B
—B X
NTP (1995]**; Hard et al. (2011]; 13 wk
Linear papillary
mineralization
NTP (1995]; Hard et al. (2011]; 2 yr
¦
~ B-B
¦ ¦
-a—x
Foci of
tubular NTP (1995]; 2 yr
hyperplasia
~
—b—a
* Hard et al. (2011) reported presence of "precursor
granular casts" 10 100 1,000 10,000
**NTP (1995) 13-wk study reported kidney
mineralization but not linear mineralization Dose (mg/kg-day)
*Hard et al. (2011) reported presence of "precursor granular casts."
**NTP (1995i 13-wk study reported kidney mineralization but not linear mineralization.
Figure 1-8. Exposure-response array for effects potentially associated with
alpha 2u-globulin renal tubule nephropathy and tumors in male rats after oral
exposure to tert-butanol.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
¦ = 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
Borghoff et al. [2001) -10 d
t Hyaline
droplet
size/number
NTP (1997) -13 wks
~—
—~ ¦
~ ~
Identification
of a2u-
globulin in Borghoff ct al. (2001) -10d
hyaline
droplets
~—
—~ a
Tubule cell Borghoff ct al. (2001) -10 d
proliferation
¦—
—¦ ¦
100 1,000 10,000
Exposure Concentration (mg/m1)
Figure 1-9. Exposure-response array for effects potentially associated with
alpha 2u-globulin renal tubule nephropathy and tumors in male rats after
inhalation exposure to tert-butanol.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Question One: is the alpha 2u-gIobuIin process occurring in male rats exposed to tert-butanol?9
(1) The first criterion to consider is whether hyaline droplets are larger and more
numerous in male rats exposed to tert-butanol. As noted above, the excessive accumulation of
hyaline droplets can appear quickly, within 1 or 2 days, and persist throughout chronic exposures,
although the severity begins to decline around 5 months (U.S. EPA. 1991a). A statistically
significant positive trend in the accumulation of large protein droplets with crystalloid protein
structures was observed in kidneys of male rats exposed to inhalation concentrations of 758,1,364,
and 5,304 mg/m3 tert-butanol for 6 hour/day for 10 days fBorghoffetal.. 20011. These droplets
were small and minimally present in control male rats and were not observed in female rats.
Similarly, data from the 13-week NTP oral study fNTP. 1995: Takahashi etal.. 1993: Lindamood et
al.. 19921 demonstrated an increase in the accumulation of hyaline droplets. The lowest dose
group, 230 mg/kg-day, had minimal hyaline droplet formation compared with controls, although
the next three dose groups (490, 840, and 1,520 mg/kg-day) had a higher accumulation of droplets
with angular, crystalline structures that was similar in incidence and severity among these dose
groups. No droplets were observed in female rats or in mice.
NTP f!9971. however, found no difference between the control and treatment groups
stained for hyaline droplet formation in male rats exposed to 0-, 3,273-, or 6,368-mg/m3
tert-butanol via inhalation for 13 weeks; in fact, this study reported no other lesions that could be
specifically associated with alpha 2u-globulin nephropathy in male rats. These results from NTP
(1997). which are inconsistent with the findings of both Borghoff et al. (2001) and NTP (1995). do
not appear to be due to differences in dose. Comparison of the oral and inhalation studies on the
basis of tert-butanol blood concentration (see Supplemental Information) showed that an exposure
in the range of the NTP T19951 doses of 490-840 mg/kg-day for 13 weeks leads to the same
average blood concentration as inhalation exposures to 3,273-6,368 mg/m3 for 6 hour/day,
5 day/week. The absence of similar histopathological findings in the 13-week inhalation NTP
(1997) study compared with those reported in the two oral studies is not understood, but might be
indicative of the strength of tert-butanol to induce, consistently, alpha 2u-globulin nephropathy.
Also, it is possible that differences in the route of exposure may influence unknown mechanisms of
action, The results from the two other studies fBorghoffetal.. 2001: NTP. 1995) indicate that
hyaline droplets increase in size and number in male rats following tert-butanol exposures. Despite
the inconsistency, the findings from NTP T19951 and Borghoff et al. f20011.are considered as
sufficient evidence to fulfill the first criterion that hyaline droplets are increased in size and number
in male rats.
(2) The second criterion to consider is whether the protein in the hyaline droplets in male
rats is alpha 2u-globulin. Accumulated hyaline droplets with an alpha 2u-globulin etiology can be
confirmed by using immunohistochemistry to identify the alpha 2u-globulin protein. Two
9If the chemical meets the criteria for question one, then a second question is asked: Are the renal effects in
male rats exposed to this chemical due solely to the alpha 2u-globulin process?
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
short-term studies measured alpha 2u-globulin immunoreactivity in the hyaline droplets of the
renal proximal tubular epithelium fBorghoffetal.. 2001: Williams and Borghoff. 20011. Following
10 days of inhalation exposure, Borghoff et al. f20011 did not observe an exposure-related increase
in alpha 2u-globulin using immunohistochemical staining. However, when using an enzyme-linked
immunosorbent assay (ELISA), considered by Borghoff et al. (20011 to be a more sensitive method
of detecting alpha 2u-globulin, a statistically significant positive correlation of alpha 2u-globulin
concentration with dose of tert-butanol was observed. The accumulation of alpha 2u-globulin
protein was statistically significant by pairwise comparison only in the highest dose group. No
positive staining for alpha 2u-globulin was observed in exposed female rats. In a follow-up study,
Williams and Borghoff f20011 used a single gavage dose of 500 mg/kg [selected on the basis of
results by NTP T19951 for induction of hyaline droplet accumulation] and reported a statistically
significantly higher renal concentration of alpha 2u-globulin (by ELISA) in treated male rats than in
controls 12 hours after exposure. Further, equilibrium dialysis methods determined that the
binding of tert-butanol to alpha 2u-globulin was reversible. These data indicate the presence of
alpha 2u-globulin in tert-butanol-treated male rats, although requiring a more sensitive method of
detection for alpha 2u-globulin than is typically used could indicate that tert-butanol is not a strong
inducer of alpha 2u-globulin accumulation. Therefore, the available data are sufficient to fulfill the
second criterion for alpha 2u-globulin present in the hyaline droplets but suggest weak induction of
alpha 2u-globulin by tert-butanol.
(3) The third criterion considered is whether several (but not necessarily all) additional
events in the histopathological sequence associated with alpha 2u-globulin nephropathy appear in
male rats in a manner consistent with the understanding of alpha 2u-globulin pathogenesis.
Evidence of cytotoxicity and single-cell necrosis of the tubule epithelium subsequent to the
excessive accumulation of hyaline droplets, with exfoliation of degenerate epithelial cells, should be
observable after ~5 days of continuous exposure, peaking at ~19 days [reviewed in U.S. EPA
(1991a)]. The formation and accumulation of granular casts from the exfoliated cellular debris
would follow, causing tubule dilation at the junction of the S3 (P3) segment of the proximal tubule
and the descending thin loop of Henle, and the commencement of compensatory cell proliferation
within the S2 (P2) segment, both occurring after 3 weeks of continuous exposure. Following
chronic exposures, this regenerative proliferation could result in focal tubular hyperplasia, and
eventually progress to renal adenoma and carcinoma (see Figure 1-7).
Several of these steps were observed following tert-butanol exposure in male rats, most
notably linear papillary mineralization and foci of tubular hyperplasia, consistent with the expected
disease progression. Some lack of consistency and dose-related concordance, however, was evident
across the remaining steps in the histopathological sequence. First, the accumulation of hyaline
droplets and the concentrations of alpha 2u-globulin in the hyaline droplets at doses that induced
significant tumor formation in male rats were not remarkable. Next, necrosis or cytotoxicity was
absent, and only precursors to granular casts at stages well within the expected time frame of
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
detectability were present. Finally, a 13-week inhalation study found no evidence of alpha
2u-globulin nephropathy fNTP. 19971. despite evaluating exposure concentrations predicted to
result in similar blood tert-butanol levels as for the 13-week oral study fNTP. 19951. which
reported increases in droplet accumulation and sustained regenerative tubule cell proliferation.
A detailed evaluation and analysis of all the evidence relevant to this criterion follows.
Detailed evaluation of the available evidence supporting the third criterion:
a. Single cell death and exfoliation into the renal tubules might logically be expected to
accompany the occurrence of CPN, but this result was inconsistently observed. Single
cell death or necrosis was not associated with tert-butanol exposure in male rat kidneys
after 10 or 13 weeks (Acharva et al.. 1997: NTP. 1995). Acharvaetal. (1997) reported
degeneration of renal tubules, one pathological consequence of single cell necrosis, in
male rats exposed to tert-butanol in drinking water for 10 weeks. Because renal tubule
epithelial cell death and epithelial degeneration should occur as early as 5 days post
exposure and persist for up to 48 weeks (Swenbergand Lehman-McKeeman. 1999:
Short etal.. 1989). the lack of consistency in these observations could be due to both
weak induction of alpha 2u-globulin and a lack of later examinations.
b. Sustained regenerative cell proliferation also might be logically expected to accompany
the occurrence of CPN, but this result also was inconsistent Acharva et al. (1997) did
not observe tert-butanol-induced proliferation following 10 weeks of oral exposure but
did observe renal tubule proliferation following another chemical exposure
(trichloroacetic acid) in the same study. Therefore, the inference is that tert-butanol
treatment did not induce regenerative tubule cell proliferation in male rats from this
study. Borghoff et al. (2001). however, reported a dose-related increase in epithelial
cell proliferation within the proximal tubule as measured by BrdU (bromodeoxyuridine)
labeling indices in all male rats exposed to tert-butanol via inhalation for 10 days. The
study did not report cytotoxicity and combined with the early time point makes it
unlikely that the cell proliferation was compensatory. NTP T19951 also observed
increased cell proliferation in the renal tubule epithelium following 13-week oral
exposures in male rats [only male rats were studied in the retrospective analysis by
Takahashi etal. f 19931 reported in NTP T19951]. Proliferation was elevated at
840-1,520 mg/kg-day, a range higher than the single 575-mg/kg-day dose that elicited
epithelial degeneration (Acharva et al.. 1997) and a result that could be consistent with
a compensatory proliferative effect NTP (1995). however, reported that no necrosis or
exfoliation was observed. Altogether, proliferation and necrosis or degeneration were
not observed within the same study despite several attempts to measure both effects.
Thus, these data provide inadequate evidence to conclude that the proliferation was
compensatory.
c. Granular cast formation was not observed, although one study noted precursors to cast
formation. NTP T19951 did not observe the formation of granular casts or tubular
dilation; however, Hard etal. (2011) reanalyzed the 13-week oral NTP data from male
rats treated with 0 or 1,520 mg/kg-day and identified precursors to granular casts in
5/10 animals in the treated group. The significance of these granular cast precursors,
described as sporadic basophilic tubules containing cellular debris, is unknown because
13 weeks of exposure is within the expected time frame of frank formation and
accumulation of granular casts (>3 weeks). Granular cast formation, however, might
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
not be significantly elevated with weak inducers of alpha 2u-globulin (Short etal..
19861. which is consistent with the reported difficulty in measuring alpha 2u-globulin in
hyaline droplets associated with tert-butanol exposure.
d. Linear mineralization of tubules within the renal papillae was consistently observed in
male rats. This lesion typically appears at chronic time points, occurring after
exposures of 3 months up to 2 years fU.S. EPA. 1991al. Consistent with this description,
2-year oral exposure to tert-butanol induced a dose-related increase in linear
mineralization, but not following 13-week exposure [NTP (1995): see Table 1-2].
e. Renal tubule hyperplasia was observed in the only available 2-year study. Renal tubule
hyperplasia is the preneoplastic lesion associated with alpha 2u-globulin nephropathy
in chronic exposures that leads to renal tubule tumors (U.S. EPA. 1991a). A dose-related
increase in renal tubule hyperplasia was observed in male rats following 2-year oral
exposures fNTP. 19951. By comparison, renal tubule hyperplasia was observed in only
one high-dose female.
The progression of histopathological lesions for alpha 2u-globulin nephropathy is
predicated on the initial response of excessive hyaline droplet accumulation (containing alpha
2u-globulin) leading to cell necrosis and cytotoxicity, which in turn cause the accumulation of
granular casts, linear mineralization, and tubular hyperplasia. Therefore, observations of temporal
and dose-response concordance for these effects are informative for drawing conclusions on
causation.
As mentioned above, most steps in the sequence of alpha 2u-globulin nephropathy are
observed at the expected time points following exposure to tert-butanol. Accumulation of hyaline
droplets was observed early, at 12 hours following a single bolus exposure (Williams and Borghoff.
2001) and at 10 days (Borghoff et al.. 2001) or 13 weeks (NTP. 1995) following continuous
exposure; alpha 2u-globulin was identified as the protein in these droplets fBorghoffetal.. 2001:
Williams and Borghoff. 2001). Lack of necrosis and exfoliation might be due to the weak induction
of alpha 2u-globulin and a lack of later examinations. Granular cast formation was not reported in
any of the available studies, which could also indicate weak alpha 2u-globulin induction.
Regenerative cell proliferation, which was not observed, is discussed in more detail below.
Observations of the subsequent linear mineralization of tubules and focal tubular hyperplasia fall
within the expected time frame of the appearance of these lesions. Overall, no explicit
inconsistencies are present in the temporal appearance of the histopathological lesions associated
with alpha 2u-globulin nephropathy; however, the data set would be bolstered by measurements at
additional time points to lend strength to the MOA evaluation.
Inconsistencies do occur in the dose-response among lesions associated with the alpha
2u-globulin nephropathy progression. Hyaline droplets were induced in the proximal tubule of all
surviving male rats in the 13-week NTP oral study (NTP. 1995: Takahashi etal.. 1993: Lindamood
etal.. 1992). although the incidence at the lowest dose was minimal, whereas the incidence at the
three higher doses was more prominent These results are discordant with the tumor results, given
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
that all treated groups of male rats in the NTP 2-year oral bioassay had increased kidney tumor
incidence, including the lowest dose of 90 mg/kg-day [according to the reanalysis by Hard et al.
£201JJ.]. This lowest dose was less than the 230 mg/kg-day in the 13-week oral study that showed
only minimal hyaline droplet formation. Furthermore, although the incidence of renal tubule
hyperplasia had a dose-related increase fNTP. 19951. a corresponding dose-related increase in the
severity of tubular hyperplasia did not result Severity of tubule hyperplasia was increased only at
the highest dose, which was not consistent with renal tumor incidence.
Although the histopathological sequence has data gaps, such as the lack of observable
necrosis or cytotoxicity or granular casts at stages within the time frame of detectability, overall, a
sufficient number of steps (e.g., linear papillary mineralization, foci of tubular hyperplasia) were
observed to fulfill the third criterion.
Consideration of additional International Agency for Research on Cancer (IARC] 1999 criteria
An alpha 2u-globulin framework was published by IARC in 1999 (Capen etal.. 19991. See
Table 1-10 for criteria laid out in the IARC consensus document.
Table 1-10. International Agency for Research on Cancer (IARC) criteria for an
agent causing kidney tumors through an alpha 2u-globulin associated
response in male rats
IARC criteria (Capen etal.. 19991
• Lack of genotoxic activity (agent and/or metabolite) based on an overall evaluation of in
vitro and in vivo data.
• Male rat specificity for nephropathy and renal tumorigenicity.
• Induction of the characteristic sequence of histopathological changes in shorter term
studies, of which protein droplet accumulation is obligatory.
• Identification of the protein accumulating in tubule cells as alpha—alpha 2u-globulin.
• Reversible binding of the chemical or metabolite to alpha 2u-globulin.
• Induction of sustained increased cell proliferation in the renal cortex.
• Similarities in dose-response relationship of the tumor outcome with the
histopathological end-points (protein droplets, alpha 2u-globulin accumulation, cell
proliferation).
A few minor differences exist between the EPA and IARC criteria. The EPA framework
requires the identification of several (but not necessarily all) additional steps in the
histopathological sequence associated with alpha 2u-globulin nephropathy, whereas IARC requires
the "induction of the characteristic sequence of histopathological changes in shorter-term studies,
of which protein droplet accumulation is obligatory" but doesn't specify which or how many of the
additional histopathological changes must be observed to consider this criteria met In addition,
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
IARC has specific criteria pertaining to lack of genotoxicity of parent compound/metabolite and
male rat specificity for nephropathy and renal tumorigenicity whereas the EPA framework
considers these data as supplemental information (see Part 4, XVIIB. Additional Information Useful
for Analysis). Additional criteria required by IARC which are not considered essential in the EPA's
framework are discussed below.
Lack ofgenotoxic action
There are a limited number of studies available to assess the genotoxic potential of
tert-butanol (see Appendix B.2.2. in Supplemental Information for further details). tert-Butanol
was generally negative in a variety of genotoxicity assays and cell systems, including Salmonella
typhimurium, Escherichia coli, andNeurospora crassa fMcGregor etal.. 2005: Zeiger etal.. 1987:
Dickey etal.. 19491. Studies also demonstrated negative results for gene mutations, sister
chromatid exchanges, micronucleus formation, and chromosomal aberrations (NTP. 1995:
Mcgregor etal.. 19881. However, deoxyribonucleic acid (DNA) adducts were found in male
Kunming mice (Yuan et al.. 20071 and DNA damage in human promyelocytic leukemia (HL-60) cells
fTang etal.. 19971. In another study fSgambato etal.. 20091. an initial increase in DNA damage was
observed as measured by nuclear fragmentation, but the damage reduced drastically following
4 hours of exposure and entirely disappeared after 12 hours of exposure to tert-butanol.
Overall, the evidence base is limited in terms of either the array of genotoxicity tests
conducted or the number of studies within the same type of test. In addition, the results are either
conflicting or inconsistent. The test strains, solvents, or control for volatility used in certain studies
are variable and could influence results. Furthermore, in some studies, the specificity of the
methodology used has been challenged. Given the inconsistencies and limitations of the evidence
base in terms of the methodology used, number of studies in the overall evidence base, coverage of
studies across the genotoxicity battery, and the quality of the studies, the weight-of-evidence
analysis is inconclusive.
Male rat specificity for nephropathy and tumors
Kidney tumors were observed only in male rats and not in female rats or mice of either sex.
Because an alpha 2u-globulin MOA is specific to male rats, the endpoints would not expected in
female rats or mice of either sex and none were observed (see Table 1-2). No treatment related
changes in kidney histopathology following oral or inhalation exposures were observed in male or
female mice (NTP. 1997.1995). No protein droplets, alpha 2u-globulin immunostaining, or
increases in cell proliferation were observed in the kidneys of female rats, but they were seen in the
male rats (Borghoffetal.. 2001). Cell proliferation increased in male, but not female rats, exposed
to tert- butanol via inhalation for 10 days (Borghoffetal.. 2001) and via drinking water for 90 days
(Lindamood etal.. 1992). Increased kidney weights were observed in female rats exposed to 1,364
or 5,304 mg/m3 tert-butanol after 10-days fBorghoffetal.. 20011: in female F344 rats exposed to
290, 590, 850,1,560, or 3,620 mg/kg-day tert-butanol in a 13-week drinking water study fNTP.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
19951: in female B6C3F1 mice exposed to 11,620 mg/kg-day tert-butanol in a 13-week drinking
water study fNTP. 19951: and in female F344 rats exposed to 6,368 mg/m3 tert-butanol in a
13-week inhalation study fNTP. 19971. Kidney transitional epithelial hyperplasia and inflammation
were observed in female F344 rats exposed to 850,1,560, or 3,620 mg/kg-day for 13 weeks, as well
as 180, 330, or 650 mg/kg-day for 2 years fNTP. 19951. Female F344 rats exposed to 850,1,560, or
3,620 mg/kg-day tert-butanol had a dose-related increase in the incidence of nephropathy and the
incidences were greater than that of controls (NTP. 19951. Female rats also had lesions associated
with nephropathy fNTP. 19951. but none of the lesions were similar to those observed in the male
rat, which were associated with alpha 2u-globulin nephropathy. Therefore, the results indicate that
there are some kidney effects in female rats and mice, but that the characteristic changes that occur
with alpha 2u-globulin accumulation are only observed in male rats. This criterion of male rat
specificity is met.
Summary and conclusions for Question One: Is the a2u-g!obuIin process occurring in male rats
exposed to tert-butanol?
Oral exposure of tert-butanol to male F344 rats resulted in an increased incidence of renal
tubule tumors in a 2-year oral bioassay fHard etal.. 2011: NTP. 19951. Several histopathological
observations in exposed male rats were consistent with an alpha 2u-globulin MOA. This evidence
includes the increased size and number of hyaline droplets and the accumulated alpha 2u-globulin
protein in the hyaline droplets. Additionally, several subsequent steps in the histopathological
sequence were observed. Overall, available data are sufficient for all three required EPA criteria,
suggesting that the alpha 2u-globulin process is operative. Furthermore, the available data is
sufficient to fulfill the IARC criteria for establishing the role for alpha 2u-globulin in male rats, with
the exception of genotoxic potential because of a limited genotoxicity evidence base. Although the
evidence indicates a role for alpha 2u-globulin accumulation in the etiology of kidney tumors
induced by exposure to tert-butanol in male rats, it is plausible that tert-butanol is a weak inducer
of alpha 2u-globulin considering the available histopathological observations and uncertainty
regarding the temporal and dose concordance of the lesions.
Question Two: Are the renal effects in male rats exposed to tert-butanol due solely to the alpha
2u-gIobuIin process?
If the alpha 2u-globulin process is operative, U.S. EPA (1991a) identified a second question
that must be answered regarding whether the renal effects are due solely to the alpha 2u-globulin
process, a combination of the alpha 2u-globulin process and other carcinogenic processes, or
primarily due to other processes. U.S. EPA f!991al stated that additional data can help inform
whether the alpha 2u-globulin process is the sole contributor to renal tubule tumor development in
male rats. These additional data are considered and discussed in detail below.
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(a) Hypothesis-testing of the alpha 2u-gIobuIin sequence of effects and structure-activity
relationships that might suggest the chemical belongs in a different class of suspected carcinogens: No
data are available to evaluate these considerations.
(b) Biochemical information regarding binding of the chemical to the alpha 2u-gIobuIin
protein: Williams and Borghoff f20011 reported that tert-butanol reversibly and noncovalently
binds to alpha 2u-globulin in the kidneys of male rats. This provides additional support to the
involvement of the alpha 2u-globulin process although EPA could not determine the relative
contribution of alpha 2u-globulin to kidney tumors in male rats.
(c) Presence of sustained cell replication in the S2 (P2) segment of the renal tubule at doses
used in the cancer bioassay and a dose-related increase in hyperplasia of the renal tubule: Sustained
cell division in the proximal tubule of the male rat is consistent with, although not specific to, the
alpha 2u-globulin process. Cell proliferation was observed in two studies [13-week, NTP f!9951
and 10-day, Borghoff et al. (2001)] but whether the proliferation was compensatory is unknown
because cytotoxicity was not observed in these studies. Although the data do not support sustained
occurrence of cell division subsequent to cytotoxic cell death, renal tubule hyperplasia in male rats
was reported after 2 years of exposure fNTP. 19951. Thus, although some evidence of sustained cell
replication is available, it does not specifically support alpha 2u-globulin protein accumulation.
(d) Covalent binding to DNA or other macromolecules, suggesting another process leading to
tumors andgenotoxicity (alpha 2u-gIobuIin inducers are essentially nongenotoxic): One study (Yuan
etal.. 2007) reported an increase in tert-butanol-DNA adducts in the liver, kidney, and lung of mice
administered a single low dose of tert-butanol (<1 mg/kg) in saline via gavage (see Appendix B,
Section B.3 in Supplemental Information for further details). An extremely sensitive method of
detection was used (accelerator mass spectrometry), but the DNA adduct species were not
identified, and no replication of these results has been identified in the literature. The few studies
available to assess the direct genotoxic potential of tert-butanol had primarily negative results,
although a few studies report DNA damage induced by oxidative stress. DNA damage induced by
oxidative stress is consistent with the decreased levels of glutathione in male rat kidneys reported
by Acharva etal. (1995) after 10 weeks of tert- butanol exposure. This type of genetic damage
would not necessarily preclude a role for alpha 2u-globulin, but not enough information is available
to determine whether oxidative stress could initiate or promote kidney tumors in concert with
alpha 2u-globulin accumulation in male rat kidneys.
(e) Nephrotoxicity in the male rat not associated with the alpha 2u-gIobuIin process or CPN,
suggesting the possibility of other processes leading to renal tubule nephrotoxicity and
carcinogenicity: Nephropathy reported in the 13-week oral and inhalation and 2-year oral studies
was considered CPN and these effects were exacerbated by treatment with tert-butanol. At
13 weeks (NTP. 1997.1995) and 2 years (NTP. 1995). oral and inhalation exposure increased the
severity of nephropathy in male rats fNTP. 19951. Similarly, the severity of nephropathy was
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increased in females at 2 years, but only the incidence of nephropathy was increased in females
following a 13-week oral exposure fNTP. 19951.
Increased incidences of suppurative inflammation and kidney transitional epithelial
hyperplasia were observed in female rats orally exposed to tert-butanol for 2 years. NTP f!9951
and Frazier etal. f20121 characterized these endpoints as associated with CPN, and an analysis of
the individual animals indicates these endpoints are moderately correlated with CPN. However,
most cases of suppurative inflammation and transitional epithelial hyperplasia are spontaneous
changes whose cause is unknown and are typically unrelated to CPN or have been noted as
secondary changes to CPN fNIEHS. 20191. At 2 years, the male rats also exhibited a dose-related
increase in transitional epithelial hyperplasia, and the correlation of this endpoint with CPN was
stronger than in female rats.
Kidney weights were increased in male and female rats in the 13-week oral and inhalation
evaluations (NTP. 1997.1995) and 15-month oral evaluation (NTP. 1995). The dose-related
increases observed in both male and female rats suggest that the kidney-weight changes are
indicative of treatment-related molecular processes primarily unrelated to alpha 2u-globulin
protein accumulation. Given that CPN also was increased at these time points, however, the
influence of CPN on kidney weights cannot be ruled out.
Overall, the nephrotoxicity observed in the male rat is difficult to disentangle from CPN and
alpha 2u-globulin processes. The moderate correlation between CPN severity and renal tumor
incidence in male rats (rho = 0.45) and the very weak correlation between renal tubule hyperplasia
and renal tumors (rho = 0.16; see Table 1-8) suggest that alpha 2u-globulin nephropathy is not
solely responsible for the renal tumors. Furthermore, considering that the treatment-related
exacerbation of CPN severity in female rats occurs without the subsequent induction of renal
tumors, this suggests that other processes besides alpha 2u-globulin and CPN in males might be
responsible for the renal tubule tumors.
Summary and conclusions for Question Two
Although the evidence suggests that tert-butanol induces alpha 2u-globulin nephropathy,
the data indicate that it is a weak inducer and the relative contribution of alpha 2u-globulin and
other processes to tumors observed in male rats cannot be determined.
The lack of compensatory cell proliferation in male rats and evidence of nephrotoxicity in
female rats suggest that other processes, in addition to the alpha 2u-globulin process, are operating.
Furthermore, the accumulation of hyaline droplets and the induction of renal tubule hyperplasia
occurred at higher doses than those inducing renal tubule tumors. Collectively, these data suggest
that tert-butanol induces the alpha 2u-globulin pathway at high doses (>420 mg/kg-day), resulting
in tumor formation in male rats. However, other unknown pathways that may contribute to renal
tumor induction could be operative at lower doses (<420 mg/kg-day).
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Chronic progressive nephropathy and renal carcinogenicity
Scientists disagree about the extent to which CPN can be characterized as a carcinogenic
MOA suitable for analysis under EPA Cancer Guidelines fHard etal.. 2013: Melnick etal.. 2013:
Melnick etal.. 20121. The etiology of CPN is unknown, and CPN is both a spontaneous and complex
disease whose processes are affected by aging and strain specificity (NIEHS. 2019). Therefore, it is
difficult to separate the effects of spontaneously occurring CPN from those effects on CPN induced
by chemical exposure. Proponents of CPN as an MOA have developed an evolving series of
empirical criteria for attributing renal tubule tumors to CPN. Hard and Khan (20041 proposed
criteria for concluding that a chemical is associated with renal tubule tumors through an interaction
with CPN. Hard etal. f20131 slightly revised and restated their criteria for considering
exacerbation of CPN as an MOA for renal tubule tumors in rats. Table 1-11 lists these sets of
proposed empirical criteria for attributing renal tubule tumors to CPN.
Table 1-11. Proposed empirical criteria for attributing renal tumors to
chronic progressive nephropathy
Hard and Khan C20041
Hard etal. C20131
• First and foremost, the chemical must have
been shown to exacerbate CPN to very
advanced stages of severity, especially
end-stage kidney disease, in comparison to
control rats in a 2-yr carcinogenicity study.
• The tumors should occur in very low
incidence and, for the most part, be
minimal-grade lesions conforming to small
adenomas or lesions borderline between
atypical tubule hyperplasia (ATH) and
adenoma.
• Such tumors should be associated only with
the highest grades of CPN severity.
• The tumors and any precursor foci of ATH
must be restricted to CPN-affected
parenchyma and are usually observed only
toward the end of the 2-yr studies.
• Careful microscopic examination of renal
parenchyma not involved in the CPN process
should reveal no evidence of
compound-induced cellular injury or other
changes that would suggest alternative
modes of action.
• Genotoxic activity based on overall
evaluation of in vitro and in vivo data is
absent.
• Tumor incidence is low, usually <10%.
• Tumors are found toward the end of 2-yr
studies.
• Lesions are usually ATH or adenomas
(carcinomas occasionally can occur).
• Chemical exacerbates CPN to most advanced
stages, including end-stage kidney disease.
• ATH and tumors occur in rats with advanced
CPN and in CPN-affected tissue.
• Cytotoxicity in CPN-unaffected tubules, in
rats with lower grades of CPN, and in
subchronic studies is absent.
Hard etal. (20131 maintain that knowing the detailed etiology or underlying mechanism for
CPN is unnecessary. Instead, identifying increased CPN with its associated increase in tubule cell
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proliferation as the key event is adequate. Nonetheless, Hard etal. (20131 also postulated a
sequence of key events for renal tumorigenesis involving exacerbation of CPN:
• Exposure to chemical (usually at high concentrations);
• Metabolic activation (if necessary);
• Exacerbated CPN, including increased number of rats with end-stage renal disease;
• Increased tubule cell proliferation because more kidney is damaged due to CPN
exacerbation;
• Hyperplasia; and
• Adenoma (infrequently carcinoma).
Evaluation of the mode of action (MOA) proposed by Hard et al. (20131
Setting aside the question of whether CPN is (Hard etal.. 2013: Hard and Khan. 2004) or is
not f Melnick et al.. 2 013: Melnick etal.. 20121 an MOA suitable for analysis, this section provides an
analysis of the mechanistic data pertinent to CPN. EPA Cancer Guidelines fU.S. EPA. 2005bl define a
framework for judging whether available data support a hypothesized MOA; the analysis in this
section follows the structure presented in the cancer guidelines.
Description of the hypothesized MOA. Under the EPA framework, toxicokinetic studies are
important for identifying the active agent, but toxicokinetic events per se are not key events of an
MOA; specifically, chemical exposure and metabolic activation considered in Hard etal. (2013) as
the first two key events were not considered in EPA's evaluation of the proposed MOA. Thus, the
EPA analysis of the MOA proposed by Hard etal. f20131 begins with (1) exacerbated CPN, including
increased number of rats with end-stage renal disease, and proceeds via (2) increased tubule cell
proliferation, (3) hyperplasia, and (4) adenoma, or infrequently, carcinoma.
Strength, consistencyspecificity of association. The relationship between exacerbated CPN
and renal tumors is moderate in male rats in the NTP f!9951 study. According to the NTP f!9951
analysis, the mean CPN grades (same as "severity of nephropathy" reported by NTP) presented on a
scale 1-4 for male rats with renal tumors were 3.5, 3.6, 3.7, and 3.4 at doses 0,1.25, 2.5, and
5 mg/mL, respectively. The mean CPN grades for male rats without renal tumors were 2.9, 2.8, 2.8,
and 3.2 for the same dose groups. The reanalysis of the NTP data by Hard etal. (2011) yielded
similar numbers. Analysis of the individual occurrence of CPN and renal tumors demonstrated a
moderately positive correlation (rho = 0.43; see Table 1-8). The relationship between CPN and
renal tumors, however, is neither consistent nor specific in the NTP T19951 study: No female rats
developed renal tumors regardless of the presence of relatively low-grade or relatively high-grade
CPN. For example, in female rats surviving more than 700 days, the mean CPN grades were 1.7 and
3.2 at doses of 0 and 10 mg/mL, respectively, but no tumors developed in either group.
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Dose-response concordance. The dose-response relationships for CPN, renal tubule
hyperplasia, and renal tubule tumors somewhat differ between the two analyses. According to the
NTP T19951 analysis, at doses of 0,1.25, 2.5, and 5 mg/mL, the mean CPN grades for all male rats
were 3.0, 3.1, 3.1, and 3.3; the incidences of renal tubule hyperplasia (standard and extended
evaluation combined) were 14/50, 20/50,17/50, and 25/50; and the incidences of renal tubule
adenomas or carcinomas were 8/50,13/50,19/50, and 13/50 (see Table 1-3). The reanalysis by
Hard etal. (2011) reported similar tumor incidences (4/50,13/50,18/50, and 12/50), except that
four fewer rats in the controls and one fewer rat in the group exposed to 2.5 mg/mL had tumors.
The lower control incidence (4/50 vs. 14/50 in the NTP study) observed in this reanalysis
accentuates the differences in these dose-response relationships. For example, the maximal tumor
response (4/50 in controls vs. 18/50 at the middle dose) does not parallel the marginal change in
CPN severity (i.e., group average of 3.0 to 3.1). That a marginal increase in CPN severity would be
associated with significant tumor induction seems inconsistent Furthermore, CPN severity is
nearly as great in the female rats, yet no females developed tumors, as noted above.
Temporal relationship. The severity of CPN progressed over time. According to the NTP
f!9951 analysis, the mean CPN grades in the 13-week study of male rats were 1.0,1.6, 2.6, 2.7, 2.6,
and 1.1 at doses of 0, 2.5, 5,10, 20, and 40 mg/mL. At the 15-month interim evaluation of the
2-year study, the mean CPN grades were 2.4, 2.8, 2.7, and 2.6 at doses of 0,1.25, 2.5, and 5 mg/mL
and, at 2 years, had increased to 3.0, 3.1, 3.1, and 3.3. Similarly, the severity of neoplastic lesions
increased at the end of life. At the 15-month interim evaluation, only two rats had developed renal
tubule hyperplasia and one other had a renal tubule adenoma; at 2 years, the incidences of these
two lesions were much higher in all dose groups (see previous paragraph). These results are
consistent with CPN as an age-related disease and with hyperplasia and tumors appearing near the
end of life.
Biological plausibility and coherence. In general, the relationship between exacerbated CPN
and renal tubule tumors in male rats seems plausible and coherent. Some patterns in the
dose-response relationships for CPN, hyperplasia, and tumors are discrepant Perhaps more
importantly, the patterns also are discrepant for the relationships between CPN grades and renal
tubule tumors in female rats. In addition, the increased incidences in renal tubule tumors in all
exposed male rats exceed the 10% criterion proposed by Hard etal. f20131 fsee Table 1-11), even
more so when making comparisons with the lower control tumor incidence from the Hard et al.
f20111 reanalysis.
Conclusions about the hypothesized chronic progressive nephropathy (CPN)-related mode of action
(MOA)
As recommended by EPA Cancer Guidelines (U.S. EPA. 2005b). conclusions about the
hypothesized MOA can be clarified by answering three questions presented below.
(a) Is the hypothesized MOA sufficiently supported in the test animals? Exacerbated CPN
leading to renal tubule tumors in male rats late in life appears to have some support Consistency is
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lacking, however, between males and females and in the dose-response relationships between CPN,
hyperplasia, and adenomas, Melnicketal. f20121 and Melnick etal. f20131 concluded, based on an
analysis of 60 NTP studies, no consistent association exists between exacerbated CPN and the
incidence of renal tubule tumors in rats. Without a consistent association and an understanding of
key events, the authors maintain that determining the human relevance of processes that might be
occurring in rats is not possible. In an earlier analysis of 28 NTP studies, Seelv etal. (2002) found a
slight but statistically significant increase in CPN severity in animals with renal tubule tumors, but
did not determine that this relationship is causal. They suggested that the number of tumors due to
chemically exacerbated CPN would be few.
These inconsistencies make it difficult to attribute all renal tumors to either CPN or to alpha
2u-globulin-related nephropathy (see previous section on alpha 2u-globulin), raising the likelihood
of another, yet unspecified MOA.
(b) Is the hypothesized MOA relevant to humans? CPN is a common and well-established
constellation of age-related lesions in the kidney of rats, and no counterpart to CPN in aging
humans is known (NIEHS. 20191. Scientists disagree, however, on the relevancy of the CPN MOA to
humans. Hard etal. f20091 and Hard etal. f20131 cited several differences in pathology between
rat CPN and human nephropathies in their arguments that CPN-related renal tumors in rats are not
relevant to humans. On the other hand, Melnicketal. f20121 and Melnick etal. f20131 argued that
the etiology of CPN and the mechanisms for its exacerbation by chemicals are unknown and fail to
meet fundamental principles for defining an MOA and for evaluating human relevance. While the
morphological spectrum observed in CPN in male rats does not have a human analogue in the aging
kidney (NIEHS. 20191. these individual lesions or processes could occur in a human kidney, and
their occurrence as a group in the aged rat kidney does not make each one rat-specific if a
treatment effect occurs for one or more of them. Given that the etiology of CPN is unknown and the
disease process is complex, it is plausible that any chemical that causes CPN in rats may have the
potential to exacerbate disease processes in the human kidney (NIEHS. 2019). This issue is
unresolved.
(c) Which populations or lifestages can be particularly susceptible to the hypothesized MOA?
It is unknown whether certain human populations or lifestages are especially susceptible to tumors
induced through exacerbated CPN.
In summary, the renal tubule tumors are partially attributed to CPN in male rats and not in
female rats, considering discrepant patterns in the dose-response relationships for CPN,
hyperplasia, and renal tubule tumors; the moderately strong correlation between CPN grades and
renal tubule tumors in male rats; and the lack of relationships between CPN severity and renal
tumors in female rats together with the lack of a generally accepted MOA for CPN.
This position can be reconciled with that of Melnick etal. (2012) and Melnick etal. (2013).
who argued against dismissing renal tubule tumors in rats that can be related to exacerbated CPN.
It also can be reconciled with Hard etal. f20131. who, while maintaining these tumors are not
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relevant to humans, also allowed that there is no generally accepted MOA for CPN akin to that for
alpha 2u-globulin-related nephropathy. Hard etal. f20131 made this statement after reporting on
the collective experience of national and international health agencies worldwide with the use of
CPN as an MOA. Of 21 substances that exacerbated CPN and caused renal tumors, most were
multisite carcinogens, and other tumor sites contributed to the evaluations. Only two assessments
explicitly considered CPN as a renal tumor mechanism. One was the assessment of ethylbenzene by
the German Federal Institute for Occupational Safety and Health, in which the agency concluded
that the kidney tumors were associated with the high, strain-specific incidence of CPN unknown in
humans [as discussed in Hard etal. f20131]. The other was the Integrated Risk Information System
(IRIS) assessment of tetrahydrofuran, for which EPA found the evidence insufficient to conclude
that the kidney tumors are mediated solely by the hypothesized MOAs fU.S. EPA. 2012cl. Hard et al.
f20131 attributed these different conclusions to either different data for the two chemicals or the
lack of a generally accepted MOA akin to alpha 2u-globulin-related nephropathy.
Relevant to this last point, IARC (19991 developed a consensus statement that listed
considerations for evaluating alpha 2u-globulin-related nephropathy in rats, which was based on
the work of 22 scientists, including 3 who were coauthors of Hard etal. (2 0131 and 2 who were
coauthors of Melnick etal. f20121 and Melnick etal. f20131. A similar broad-based consensus that
defines a sequence of key events for exacerbated CPN, distinguishes it more clearly from alpha
2u-globulin-related nephropathy, and evaluates its relevance to humans would be helpful in
advancing the understanding of these issues.
Overall Conclusions on Mode of Action for Kidney Effects
tert-Butanol increases alpha 2u-globulin deposition and hyaline droplet accumulation in
male rat kidneys and affects several of the subsequent steps in that pathological sequence. These
data provide evidence that the alpha 2u-globulin process is operating. tert-Butanol appears to be a
weak inducer of alpha 2u-globulin nephropathy, and the relative contribution of this process to
renal tubule nephropathy and carcinogenicity cannot be determined with the information available.
CPN and the exacerbation of CPN (likely due to both the alpha 2u-globulin process and tert-butanol
exposure) play a role in renal tubule nephropathy. The available evidence indicates that CPN might
be involved in the induction of renal tubule tumors in male rats, likely by providing proliferative
stimulus in the form of compensatory regeneration following toxicity to the renal tubule
epithelium, although these effects were not observed in some studies. Additionally, several
endpoints in female rats indicate that renal tubule nephrotoxicity and increased kidney weights
related to tert-butanol exposure cannot be explained by the alpha 2u-globulin process.
Integration of Kidney Effects
Kidney effects (increases in nephropathy, severity of nephropathy, hyaline droplets, linear
mineralization, suppurative inflammation, transitional epithelial hyperplasia, mineralization, and
kidney weight) were observed, predominantly in male and female rats across multiple tert-butanol
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studies. The available evidence indicates that multiple processes induce the noncancer kidney
effects. Two endpoints in male rats (hyaline droplets, linear mineralization) are components of the
alpha 2u-globulin process. U.S. EPA f!991al states that if the alpha 2u-globulin process were
occurring in male rats, the renal tubule effects associated with this process in male rats would not
be relevant to humans for the purposes of hazard identification. In cases such as these, the
characterization of human health hazard for noncancer kidney toxicity would rely on effects not
specifically associated with the alpha 2u-globulin process in male rats.
The group of lesions generally reported as "nephropathy," is related to CPN. CPN is a
common and well-established constellation of age-related lesions in the kidney of rats; for which no
known counterpart to CPN exists in aging humans, and the mode of action is unknown fNIEHS.
20191. CPN is not, inherently, a specific diagnosis but an aggregate term describing a spectrum of
effects. The individual lesions associated with CPN (tubular degeneration, glomerular sclerosis,
etc.) also occur in the human kidney. (Zojaetal.. 2015: Gorriz and Martinez-Castelao. 2012). Thus,
it cannot be ruled out that chemicals that exacerbate CPN in rats may have the potential to
exacerbate disease processes in the human kidney (NIEHS. 2019).
Because female rats are not affected by alpha 2u-globulin nephropathy, lesions associated
with CPN in female rats are informative for human hazard characterization. Several other
noncancer endpoints resulted from tert-butanol exposure and are appropriate for consideration of
a kidney hazard, specifically: suppurative inflammation in female rats, transitional epithelial
hyperplasia in female rats, severity of nephropathy in female rats, incidence of nephropathy in
female rats, and increased kidney weights in rats but not mice. Based on dose-related increases in
these noncancer endpoints in rats, kidney effects are a potential human hazard of tert-butanol
exposure. The hazard and dose-response conclusions regarding these noncancer endpoints
associated with tert-butanol exposure are discussed further in Section 1.3.1.
The carcinogenic effects observed following tert-butanol exposure include increased
incidences of renal tubule hyperplasia (considered a preneoplastic effect) and tumors in male rats.
EPA concluded that the three criteria were met to indicate that an alpha 2u-globulin process is
operating. Because renal tubule tumors in male rats did not arise solely from the alpha 2u-globulin
and CPN processes and because some of the tumors are attributable to other carcinogenic
processes, such tumors remains relevant for purposes of hazard identification fU.S. EPA. 1991al.10
10When the alpha 2u-globulin process is occurring, U.S. EPA f!991a) states that one of the following
conclusions will be made: (a) if renal tumors in male rats are attributable solely to the alpha 2u-globulin
process, such tumors will not be used for human cancer hazard identification or for dose-response
extrapolations; (b) if renal tumors in male rats are not linked to the alpha 2u-globulin process, such tumors
are an appropriate endpoint for human hazard identification and are considered, along with other
appropriate endpoints, for quantitative risk estimation; or (c) if some renal tumors in male rats are
attributable to the alpha 2u-globulin process and some are attributable to other carcinogenic processes, 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 enough information is available to determine the relative
contribution of each process to the overall renal tumor response.
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The hazard and dose-response conclusions regarding the renal tubule hyperplasia and tumors
associated with tert-butanol exposure are further discussed as part of the overall weight of
evidence for carcinogenicity in Section 1.3.2.
1.2.2. Thyroid Effects
Synthesis of Effects in the Thyroid
The evidence base on thyroid effects following tert-butanol exposure contains no human
data, two oral subchronic and two oral chronic studies [one of each duration in rats and in mice;
NTP f 19951]. and two inhalation subchronic studies [one in rats and one in mice; NTP f 19971].
Studies employing short-term and acute exposures that examined thyroid effects are not included
in the evidence table. These studies are discussed, however, in the text if they provide data
informative of MOA or hazard identification. No gross thyroid effects were reported in the 13-week
evaluations of mice or rats following oral or inhalation exposure (NTP. 1997.1995). and therefore,
subchronic studies were not included in the evidence table. The two available chronic studies are
arranged in the evidence table by effect and then by species (see Table 1-12 and Figure 1-10).
Thyroid effects, specifically follicular cell hyperplasia and adenomas, were observed in mice
of both sexes after 2 years of oral exposure via drinking water fNTP. 19951. NTP f!9951 noted,
"[proliferation of thyroid gland follicular cells is generally considered to follow a progression from
hyperplasia to adenoma and carcinoma." Both male and female mice exhibited a dose-related
increase in the incidence of hyperplasia, and the average severity across all dose groups was
minimal to mild with scores ranging from 1.2 to 2.2 (out of 4). Increased incidence of adenomas
also was observed in the tert-butanol-treated female mice at the high dose, with the only carcinoma
observed in high-dose males. At the highest dose, mean body weights of female mice were 10 to
15% lower than control animals from Week 13 to the end of the study, with a final average
body-weight reduction of 12% raising some question that the thyroid tumors were the result of
excessive toxicity in female mice rather than carcinogenicity of tert-butanol. EPA Cancer Guidelines
(U.S. EPA. 2005a) discusses the determination of an "excessively high dose" as compared to an
"adequate high dose" and describes the process as one of expert judgment which requires that
"...adequate data demonstrate that the effects are solely the result of excessive toxicity rather than
the carcinogenicity of the tested agent" (U.S. EPA. 2005a). In the 2-year oral bioassay (NTP. 1995).
study authors noted that water consumption by exposed females was similar to controls and that
no overt toxicity was observed. Furthermore, female mice in the high-dose group had higher rates
of survival than did the control animals. No treatment-related thyroid effects were reported in rats
of either sex following 2 years of oral exposure (NTP. 1995).
The tumor response in male mice, adjusted for early mortality, showed a statistically
significant increasing trend (Cochran-Armitage trend test, p = 0.041; analysis performed by EPA).
Although the response appeared nonmonotonic, with a slightly lower response at the high-dose
level than at the mid-dose level, the increased mortality reported in the high-dose group occurred
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before tumors appeared; about 40% of the high-dose males died before the first tumor
(a carcinoma) appeared in this group at Week 83. By comparison, only ~10% of the control group
had died by this time, and the single tumor in the control group was observed at study termination.
Mortality in the exposed female mice was similar to controls.
Table 1-12. Evidence pertaining to thyroid effects in animals following oral
exposure to tert-butanol
Reference and study design
Results
Follicular cell hyperplasia
NTP (1995)
F344/N rat; 60/sex/treatment
(10/sex/treatment evaluated at
15 mo)
Drinking water (0,1.25, 2.5, 5, or
10 mg/mL)
M: 0, 90, 200, or 420a mg/kg-d
F: 0,180, 330, or 650a mg/kg-d
2 yr
Incidence15
Male
Female
Dose
(mg/kg-d)
Follicular cell
hyperplasia
Dose
(mg/kg-d)
Follicular cell
hyperplasia
0
3/50
0
0/50
90
0/49
180
0/50
200
0/50
330
0/50
420a
0/50
650a
0/50
NTP (1995)
B6C3F1 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 yr
Incidence (severity)
Male
Female
Dose
(mg/kg-d)
Follicular cell
hyperplasia
Dose
(mg/kg-d)
Follicular cell
hyperplasia
0
5/60 (1.2)
0
19/58 (1.8)
540
18/59° (1.6)
510
28/60 (1.9)
1,040
15/59° (1.4)
1,020
33/59° (1.7)
2,070a
18/57° (2.1)
2,110
47/59° (2.2)
Follicular cell tumors
NTP (1995)
F344/N rat; 60/sex/treatment
(10/sex/treatment evaluated at
15 mo)
Drinking water (0,1.25, 2.5, 5, or
10 mg/mL)
M: 0, 90, 200, or 420a mg/kg-d
F: 0,180, 330, or 650a mg/kg-d
2 yr
Incidence15
Dose (mg/kg-d)
Follicular cell adenoma
Follicular cell carcinoma
Male
0
2/50
2/50
90
0/49
0/49
200
0/50
0/50
420a
0/50
0/50
Female
0
1/50
1/50
180
0/50
0/50
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Reference and study design
Results
330
1/50
1/50
650a
0/50
0/50
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Table 1-12. Evidence pertaining to thyroid effects in animals following oral
exposure to tert-butanol (continued)
Reference and study design
Results
NTP (1995)
Incidence
B6C3F1 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 yr
Dose
(mg/kg-d)
Follicular
cell
adenoma
Follicular cell
carcinoma
Follicular cell
adenoma or
carcinoma
(mortality
adjusted
rates)de
Animals
surviving to
study
termination
Male
0
1/60
0/60
1/60 (3.6%)
27/60
540
0/59
0/59
0/59 (0.0%)
36/60
1,040
4/59
0/59
4/59 (10.1%)
34/60
2,070a
1/57
1/57
2/57 (8.7%)
17/60
Female
0
2/58
0/58
2/58 (5.6%)
36/60
510
3/60
0/60
3/60 (8.6%)
35/60
1,020
2/59
0/59
2/59 (4.9%)
41/60
2,110
9/59°
0/59
9/59° (19.6%)
42/60
Survival in the high-dose group significantly decreased.
bResults do not include the animals sacrificed at 15 mo.
Statistically significant p < 0.05 as determined by the study authors.
dMortality-adjusted rates were not calculated by the study authors for follicular cell carcinoma. The
mortality-adjusted rates for the incidence of adenomas are the same as the combined rates, with the exception
of the male high-dose group, in which the rate for adenomas alone was 5.9%.
eCochran-Armitage trend test was applied to mortality-adjusted thyroid tumor incidences, by applying the NTP
adjusted rates to the observed numbers of tumors to estimate the effective number at risk in each group. For
male mice, p = 0.041; for female mice, p = 0.028.
Note: Conversions from drinking water concentrations to mg/kg-d were performed by the study authors.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
¦ = 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
~
-a ~
~ 0 ~
Adenoma; M mouse
Adenoma; F mouse
CANCER
Adenoma; M rat
Adenoma; F rat
10
Source: NTP (1995).
~ 13 ~
~ IJ
-a a
~—b ~
100 1,000
Dose (mg/kg-day)
10,000
Figure 1-10. Exposure-response array of thyroid follicular cell effects
following chronic oral exposure to tert-butanol. (Note: Only one carcinoma was
observed in male mice in the high-dose group.)
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Mode of Action Analysis—Thyroid Effects
Few studies have examined the MOA responsible for tert-butanol-induced thyroid effects.
One hypothesis is that tert-butanol increases liver metabolism of thyroid hormones, triggering a
compensatory increase in pituitary thyroid-stimulating hormone (TSH) production. Such sustained
increases in TSH could induce elevated thyroid follicular cell proliferation and hyperplasia and lead
to follicular cell adenoma and carcinoma. This enhancement of liver metabolism and excretion of
thyroid hormones is one of several potential antithyroid MOAs identified in EPA's guidance on the
assessment of thyroid follicular cell tumors (U.S. EPA. 1998a).
To determine whether the thyroid follicular cell tumors result from a chemically induced
antithyroid MOA, U.S. EPA f!998al requires that the available evidence base demonstrate
(1) increases in thyroid cell growth, (2) thyroid and pituitary hormone changes consistent with the
antithyroid MOA, (3) site(s) of the antithyroid action, (4) dose correlation among the various
effects, and (5) reversibility of effects in the early stages of disruption. In addition, another critical
element is the determination of the mutagenic potential. The available evidence for each of these
aspects of antithyroid activity following tert-butanol exposure is discussed below.
11 Increases in cell growth (required)
U.S. EPA f!998al considers increased absolute or relative thyroid weights, histological
indicators of cellular hypertrophy and hyperplasia, DNA labeling, and other measurements
(e.g., Ki-67 or proliferating cell nuclear antigen expression) to be indicators of increased cell
growth. Only a few studies (NTP. 1997.1995) have evaluated the thyroid by routine histological
examination following tert-butanol exposure, and none have investigated specific molecular
endpoints. None of the available long-term studies measured thyroid weight in mice, likely due to
the technical limitations involved, and no thyroid effects were attributed to tert-butanol exposure
in rats treated up to 2 years fNTP. 1997.19951. Given the mouse findings above fNTP. 19951. the
absence of treatment-related thyroid effects in rats is unusual, as chemically induced thyroid
tumorigenesis is observed more frequently in rats than in mice (Hurley. 1998: U.S. EPA. 1998a).
Although the short-term female mouse study by Blanck etal. (2010) stated that thyroids were
weighed, no results were reported.
An increase in thyroid follicular cell hyperplasia was observed in both female and male mice
after a 2-year drinking water exposure to tert-butanol fNTP. 19951. The increase was dose
dependent in female mice, with a slight increase in severity in the highest dose, while male mice
experienced a similar magnitude of hyperplasia induction at all doses evaluated, with increased
severity at the highest dose (NTP. 1995). Thyroid follicular cell hyperplasia was not observed in
any mouse study with less than 2 years of exposure: No treatment-related histological alterations in
the thyroid of tert-butanol-treated (2 or 20 mg/mL) female mice after 3 or 14 days of drinking
water exposure f Blanck etal.. 20101 were reported, in male or female mice after 13 weeks of
drinking water exposure fNTP. 19951. or in male or female mice following 18-day or 13-week
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
inhalation studies (NTP. 19971. The observation of increased hyperplasia in male and female mice
after 2 years of exposure is sufficient evidence to support increased thyroid cell growth.
21 Changes in thyroid and relevant pituitary hormones frequiredl
Evidence of hormonal changes, including decreases in triiodothyronine (T3) and thyroxine
(T4) and increases in TSH, are required to demonstrate a disruption in the thyroid-pituitary
signaling axis (U.S. EPA. 1998a). Blancketal. (2010) evaluated serum thyroid hormones in mice
after 3 or 14 days of exposure to tert-butanol. No tert-butanol-related effects were observed in T3,
T4, or TSH levels after 3 days. Although both T3 and T4 levels were significantly decreased by
approximately 10-20% after 14 days of treatment with tert-butanol, TSH levels remained
unaffected. Similar results were reported with the positive control (phenobarbital). The limited
evidence available from this single study suggests that although T3 and T4 levels were decreased
after 14 days, this perturbation likely did not exceed the range of homeostatic regulation in female
B6C3F1 mice and thus was not likely to induce compensatory thyroid follicular cell proliferation.
Multiple lines of evidence support this observation: (1) TSH levels were unaffected, indicating that
the decrease in T3 and T4 levels was not severe enough to stimulate increased TSH secretion by the
pituitary in this time frame; (2) thyroid hyperplasia was not induced in this study, or any others
exposing mice to similar or greater concentrations for 2.5-13 weeks, suggesting that thyroid
proliferation was either not induced by the hormone fluctuations or that any follicular cell
proliferation during this period was too slight to be detected by routine histopathological
examination; and (3) the maximal decrease in T3 or T4 hormone levels induced by tert-butanol
exposure after 14 days (i.e., ~20%) was well within the range of fluctuation in T3 and T4 hormone
levels reported to occur between the 3- and 14-day control groups [15-40%; Blancketal. f20101].
Although the study authors later attributed the lower T3 and T4 levels following tert-butanol
exposure to an increase in liver metabolism (see next section), these changes could, alternatively,
be due to a variety of other possible, yet uninvestigated, molecular interactions with tert-butanol.
Such interactions might include (1) inhibition of iodide transport into thyroid follicular cells,
(2) thyroid peroxidase inhibition, (3) thyroid follicular cell dysfunction leading to inhibition of
thyroid hormone production or release, or (4) inhibition of 5'-monodeiondinase (Hurley. 1998: U.S.
EPA. 1998al.
The absence of information regarding thyroid hormone levels in male mice and lack of
molecular studies evaluating exposures >2 weeks in female mice are significant deficiencies in the
available evidence base. Together, although small decreases in some thyroid hormone levels have
been reported in female mice, the available evidence is inadequate to determine whether
tert-butanol negatively affects the pituitary-thyroid signaling axis in female mice; furthermore, no
evidence was available to evaluate this effect in male mice.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
31 Sitefs) of antithyroid action (required)
The thyroid and liver are two of several potential sites of antithyroid action, with the liver
the most common because increased microsomal enzyme activity there could enhance thyroid
hormone metabolism and removal fU.S. EPA. 1998al. Rats are thought to be more sensitive than
mice to this aspect of antithyroid activity (Rogues etal.. 2013: Oatanani etal.. 2005: U.S. EPA.
1998a): however, rats exposed to tert-butanol for 2 years exhibited no treatment-related thyroid
effects, while mice did. Typically, chronic induction of liver microsomal enzyme activity resulting
from repeated chemical exposure would manifest some manner of liver histopathology, such as
hepatocellular hypertrophy or hyperplasia fU.S. EPA. 1998a: NTP. 19951. In a 14-day mechanistic
investigation, tert-butanol had no effect on liver weight when compared with the control group, but
centrilobular hepatocellular hypertrophy was reported in 2/5 livers from high-dose mice versus
0/6 in control and 0/5 in low-dose mice (Blanck etal.. 2010). Relative liver weights increased in
male and female mice after 13 weeks of oral exposure (NTP. 1995) to higher doses than those
evaluated by Blanck etal. (2010). although absolute liver-weight measurements in treated animals
showed little change from controls suggesting that the relative measures could have been related to
decreases in body weight rather than specific liver effects. Relative (and absolute) liver weights
were increased in female mice (only) after 13 weeks of inhalation exposure at the two highest
concentrations fNTP. 19971: liver weight was not reported in mice orally exposed for 2 years fNTP.
1995). No increase in mouse hepatocellular hypertrophic or hyperplastic histopathology was
reported following 2.5 weeks to 2 years of exposure (NTP. 1997.1995). In fact, the only liver
pathology associated with tert-butanol exposure in either rats or mice from these studies was an
increase in fatty liver in male mice in the high-dose group after 2 years of oral exposure (NTP.
19951. Although increased fatty liver could indicate some nonspecific metabolic alteration, the
absence of a similar treatment-related effect in livers from female mice, which were sensitive to
both thyroid follicular cell hyperplasia and tumor induction, suggests that it might not be related to
the thyroid tumorigenesis.
One study evaluated liver enzyme expression and found highly dose-responsive induction
of a single phase I cytochrome p450 (CYP450) enzyme (CYP2B10) following 14 days of tert-butanol
exposure in female mice, with much smaller increases in the expression of another phase I enzyme,
CYP2B9, and the phase II thyroid hormone-metabolizing enzyme, sulfotransferase 1A1 [SULT1A1;
Blanck etal. f 20101]. CYP2B enzyme induction is commonly used as an indication of constitutive
androstane receptor (CAR) activation; CAR can induce expression of a wide range of hepatic
enzymes, including several CYPs along with thyroid hormone-metabolizing sulfotransferases
(Rogues etal.. 2013). The only thyroid hormone-metabolizing enzyme induced by tert- butanol,
however, was SULT1A1, which has been reported to be inducible in a CAR-independent manner in
mice (Oatanani etal.. 2005). Based on alterations in hepatic phase I and phase II enzyme activities
and gene expression, the above data suggest a possible role for increased thyroid hormone
clearance in the liver following repeated tert-butanol exposure; however, the expression changes in
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
these few enzymes are not supported by any liver histopathological effects in mice exposed for
longer durations, so whether this enzyme induction is transient, or simply insufficient to induce
liver pathology after >2 weeks of exposure, is unknown. As noted above, no evidence is available to
evaluate the potential for intrathyroidal or any other extrahepatic effects in female mice or for any
of these molecular endpoints in male mice; therefore, the available evidence is inadequate to
determine whether major site(s) of antithyroid action are affected.
41 Dose correlation (required)
Confidence in the disruption of the thyroid-pituitary function is enhanced when dose
correlation is present among the hormone levels producing various changes in thyroid
histopathology, including thyroid tumors fU.S. EPA. 1998al. Furthermore, if thyroid hormone
levels were affected by liver enzyme induction, confidence would be increased by a concordance
among liver effects, thyroid hormone levels, and thyroid pathology. Thyroid hormone levels were
evaluated only in female mice exposed to tert-butanol; after 2 weeks of exposure, both T3 and T4
were decreased with both doses (2 and 20 mg/L), and TSH was unaffected at either dose (Blanck et
al.. 20101. Liver expression of CYP2B10 was increased in a dose-responsive manner, while
SULT1A1 messenger ribonucleic acid (mRNA) was induced by 20-30% at both doses (Blanck etal..
20101. As described above, induction of liver microsomal enzyme activity would manifest some
manner of liver histopathology fMaronpot etal.. 2010: U.S. EPA. 1998a: NTP. 19951. and consistent
with this expected association, centrilobular hepatocellular hypertrophy was reported in
2/5 high-dose mice exposed for 2 weeks (Blanck etal.. 20101. No liver histopathology, however,
was attributed to tert-butanol exposure in female mice exposed for 2.5 weeks to 2 years to
comparable tert-butanol concentrations fNTP. 1997.19951. Although liver enzyme levels and
activity were not specifically evaluated following subchronic to chronic exposure, the lack of liver
pathology suggests a comparable lack of enzyme induction. Conversely, no histopathological
alterations were reported in the thyroids of female mice after 2 weeks of oral exposure at doses
that elevated some liver enzyme levels (Blanck etal.. 2010).
Following 2 years of oral exposure, both follicular cell hyperplasia and follicular cell tumor
incidence were increased in mice, despite a lack of treatment-related liver pathology (NTP. 1995).
Any associations relating hormone changes to thyroid pathology or liver enzyme induction are
limited because of the inadequate evidence base (described above); the available evidence suggests
little concordance among reports of liver, pituitary, and thyroid effects in female mice, and no
evidence was available to evaluate these associations in male mice.
5) Reversibility (required)
Chemicals acting via an antithyroid MOAhave effects (e.g., increased TSH levels, thyroid
follicular cell proliferation) that are reversible after cessation of treatment (U.S. EPA. 1998a).
Although increased TSH levels have not been demonstrated following tert-butanol exposure,
thyroid follicular cell proliferation was observed following chronic exposure. Because no studies
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
have evaluated changes in thyroid hormones or thyroid histopathology after cessation of
tert-butanol treatment, the available evidence is inadequate to evaluate reversibility of these
effects.
In summary, the available evidence base sufficiently supports only (1)—increases in thyroid
cell growth. The existing data are inadequate to evaluate (2) thyroid and pituitary hormone
changes consistent with the antithyroid MOA, (3) site(s) of the antithyroid action, or
(4) reversibility of effects in the early stages of disruption. Although these inadequacies also limit
the evaluation of (5) dose correlation among the various effects, the available evidence suggests
that little correlation exists among reported thyroid, pituitary, and liver endpoints. An additional
consideration is the evaluation of genotoxic potential. As summarized in Appendix B, there is
limited evidence to suggest that thyroid tumors following tert-butanol exposure are due to
mutagenic changes. Together, the evidence base is inadequate to determine whether an antithyroid
MOA is operating in mice. In the absence of information to indicate otherwise, the thyroid tumors
observed in mice are considered relevant to humans.
Integration of Thyroid Effects
The thyroid endpoints reported following chronic exposure to tert-butanol include
increases in follicular cell hyperplasia and tumors in male and female mice. As discussed above,
due to inadequacies in four of the five required areas fU.S. EPA. 1998al. the evidence is inadequate
to determine whether an antithyroid MOA is operating in mice; therefore, the MOA(s) for thyroid
tumorigenesis has not been identified. EPA considers the thyroid follicular cell hyperplasia to be an
early event in the neoplastic progression of thyroid follicular cell tumors, and no other noncancer
effects on the thyroid were observed. Thus, the hazard and dose-response conclusions regarding
the thyroid follicular cell hyperplasia and tumors associated with tert-butanol exposure are
discussed as part of the overall weight of evidence for carcinogenicity in Section 1.3.2.
1.2.3. Developmental Effects
Synthesis of Effects Related to Development
Four studies evaluated developmental effects [three oral or inhalation developmental
studies fFaulkner et al.. 1989: Nelson etal.. 1989: Daniel and Evans. 19821 and a one-generation,
oral reproductive study fHuntingdon Life Sciences. 20041] in animals exposed to tert-butanol via
liquid diet (i.e., maltose/dextrin), oral gavage, or inhalation. No developmental epidemiological
studies are available for tert-butanol. The animal studies are arranged in the evidence tables by
species, strain, and route of exposure. The design, conduct, and reporting of each study were
reviewed, and each study was considered adequate to provide information pertinent to this
assessment. Two studies, however, were considered less informative: (1) Faulkner et al. (19891.
because it did not provide sufficient information on the dams to determine if fetal effects occurred
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
due to maternal toxicity and (2) Daniel and Evans (19821 due to the use of individual data instead
of litter means as the statistical unit of analysis.
Developmental effects of tert-butanol observed after oral exposure (liquid diets or gavage)
in several mouse strains and one rat strain include measures of embryo-fetal loss or viability
(e.g., increased number of resorptions, decreased numbers of neonates per litter) and decreased
fetal body weight (Huntingdon Life Sciences. 2004: Faulkner et al.. 1989: Daniel and Evans. 1982).
Daniel and Evans (1982) observed decreases in pup body-weight gain during postnatal days
(PNDs) 2-10; however, the data suggest that this effect might be due to altered maternal behavior
or nutritional status. In addition, a single-dose study reported a small increase in the incidence of
variations of the skull or sternebrae in two mouse strains fFaulkner et al.. 19891. Although
variations in skeletal development were noted in the study, no malformations were reported.
Similar developmental effects were observed after whole-body inhalation exposure in
Sprague-Dawley rats for 7 hours/day on gestation days (GDs) 1-19 (Nelson et al.. 1989). Fetal
effects included dose-related reductions in body weight in male and female fetuses and a higher
incidence of skeletal variations when analyzed based on individual fetuses (but not on a per litter
basis).
In these studies, fetal effects were generally observed at high doses that cause toxicity in the
dams as measured by clinical signs (e.g., decreased [—7—36%] body-weight gain and food
consumption and reported ataxia and lethargy; see Table 1-13; Figure 1-11; Figure 1-12). As stated
in the Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA. 1991b). "an integrated
evaluation must be performed considering all maternal and developmental endpoints....[W]hen
adverse developmental effects are produced only at doses that cause minimal maternal toxicity; in
these cases, the developmental effects are still considered to represent developmental toxicity and
should not be discounted." Although, at doses of "excessive maternal toxicity ...information on
developmental effects may be difficult to interpret and of limited value." In considering the
observed fetal and maternal toxicity data following tert-butanol exposure and the severity of the
maternal effects, the role of maternal toxicity in the developmental effects observed at the doses
used remains unclear. Specifically, discerning from the available data whether the fetal effects are
directly related to tert-butanol treatment or are secondary to maternal toxicity is not possible.
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Table 1-13. Evidence pertaining to developmental effects in animals following
exposure to tert-butanol
Reference and study
design
Results
Huntingdon Life Sciences
(2004)
Sprague-Dawley rat;
12/sex/treatment
Gavage 0, 64,160, 400, or
1,000 mg/kg-d
F0 males: 9 wk beginning
4 wk prior to mating
F0 females: 4 wk prior to
mating through PND 21
F1 males and females:
7 wk (throughout gestation
and lactation; 1 male and
1 female from each litter
were dosed directly from
PND 21-28)
Response relative to control
Maternal effects
Percentage change compared to control:
Dose
(mg/kg-d)
Body-weight
gain GD
0-20
Food
consump-
tion GD
0-20
Body-weight
gain
PND 1-21
Food
consumption
LD 1-14
Live
pups/litter
response
0
-
-
-
-
-
64
-3
0
3
-2
-9
160
-4
0
-10
-6
-11
400
0
4
3
0
-7
1,000
-16a
0
100ab
-16
-33a
Dams dosed with 400 or 1,000 mg/kg-d showed CNS effects (e.g., ataxia, lethargy)
that were undetectable by 4 wk of exposure in animals exposed to 400 mg/kg-d but
not those in the higher dose group
F1 effects
Dose
(mg/kg-d)
Viability index
(pup survival
to PND 4)
Lactation
index (pup
survival to
PND 21)
Sex ratio
(% males)
Pup
weight/litter
PND 1
relative to
control (%)
Pup weight
PND 28
relative to
control (%)
Male
Female
0
96.4
100
54.4
-
-
-
64
98.7
100
52.3
6
2
0
160
98.2
100
50.9
4
0
-4
400
99.4
99.2
53.5
7
0
-2
1,000
74. la
98.8
52.1
-10
-12a
-8
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Table 1-13. Evidence pertaining to developmental effects in animals
following exposure to tert-butanol (continued)
Reference and study
design
Results
Daniel and Evans (1982)
Swiss Webster (Cox) mouse;
15 pregnant dams/treatment
Liquid diet (0, 0.5, 0.75, or
1.0%, w/v)
0 (isocaloric amounts of
maltose/dextrin), 3,324,
4,879, or 6,677 mg/kg-d
GD 6-20
No statistical analysis was conducted on any of these data.
Maternal
Percentage change compared to control:
Dose
(mg/kg-d)
Food consumption
(mean g/animal/d)
Body-weight
gain
Number of litters
(% pregnant dams)
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
Percentage change compared to control:
Dose
(mg/kg-d)
Number of
neonates/litter
Fetal body weight on
PND 2
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
provided only in text or figures).
Faulkner et al. (1989)
CBA/J mouse; 7 pregnant
females in control, 12
pregnant females in treated
Gavage (10.5 mmoles/kg
twice a day);
0 (tap water) or
1,556 mg/kg-d
GD 6-18
Maternal results not reported.
Fetal
Dose
(mg/kg-d)
Resorptions/
litter
Live
fetuses/
litter
Fetal
weight
Sternebral
variations
Skull
variations
0
-
-
-
4/28
1/28
1,556
118a
-41a
-4
7/30
3/30
Sternebral 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) increased (p < 0.05).
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Table 1-13. Evidence pertaining to developmental effects in animals
following exposure to tert-butanol (continued)
Reference and study
design
Results
Faulkner et al. (1989)
C57BL/6J mouse; 5 pregnant
females in controls, 9
pregnant females treated
Gavage (10.5 mmoles/kg
twice a day)
0 (tap water) or
1,556 mg/kg-d
GD 6-18
Maternal results not reported.
Fetal
Dose
(mg/kg-d)
Resorptions/
litter
Live
fetuses/
litter
Fetal
weight
Sternebral
variations
Skull
variations
0
-
-
-
5/21
1/21
1,556
428a
-58a
-4
9/16
7/16
Sternebral 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) increased (p < 0.05).
Nelson et al. (1989)
Sprague-Dawley rat; 15
pregnant dams/treatment
Inhalation analytical
concentration: 0, 2,200, 3,510,
or 5,030 ppm (0, 6,669,
10,640, or 15,248 mg/m3),
dynamic whole-body chamber
7 h/d
GD 1-19
Maternal: Unsteady gait (no statistical tests reported), dose-dependent 4, in
body-weight gain (results presented in figure only), dose-dependent 4' in food
consumption ranging from 7 to 36%, depending on dose and time.
Fetal
Percentage change compared to control (mean ± standard error):
Dose
(mg/m3)
Number of live fetuses/litter
Resorptions per litter
0
-(13 ± 2)
-(1.1 ± 1.2)
6,669
0(13 ±4)
9 (1.2 ±1.1)
10,640
15 (15 ± 2)
-18 (0.9 ± 1.0)
15,248
8 (14 ± 2)
0(1.1 ±0.9)
Dose
(mg/m3)
Fetal weight
(males)
Fetal weight
(females)
Skeletal
variation
by litter
Skeletal
variation
by fetus
0
-
-
10/15
18/96
6,669
-9a
-9a
14/17
35/104
10,640
-12a
-13a
14/14
53/1033
15,248
-32a
-31a
12/12
76/83a
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.
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Table 1-13. Evidence pertaining to developmental effects in animals
following exposure to tert-butanol (continued)
Reference and study
design
Results
CNS = central nervous system; w/v = weight by volume.
statistically significant p < 0.05, as determined by the study authors. Conversions from diet concentrations to
mg/kg-d were performed by the study authors. Conversion from ppm to mg/m3 is 1 ppm = 3.031 mg/m3.
bLarge body weight gain from PND 1-21 was noted by study authors, who stated that during the first 2 weeks of
lactation "F0 dams showed reduced feed consumption, but then in late lactation they showed weight gain
instead of the more usual slight weight loss, a pattern of effects that suggested deficient lactation."
Note: Percentage change compared to control = [(treated value - control value) -f control value] x 100.
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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
DEVELOPMENTAL
~ B B I
-B-
B B B-
l Maternal body weight gain
(GD 0-20); F rat (C)
T Maternal body weight gain
(LD 1-21); F rat (C)
.1 Number of live pups per litter; M+F
rat (C)
^Viability index; M+F rat(C)
Lactation index; M+F rat (C) - B B B H
Sex ratio; M+F rat (C) B B B 0
B B B B
B B B-
B——B B B
I Pup weight per litter
(PND 1); M+F rat (C)
iPup weight per litter
(PND 28); M rat (C)
iPup weight per litter
(PND 28); F rat (C)
I Maternal body weight gain; F
mouse (A) *
4-Numberof neonates/litter, fetal
body weight; M+F mouse (A)*
TNumberof resorptions per litter;
M+F mouse (B)
JNumberof live fetuses per litter;
M+F mouse (B)
IFetal weight; M+F mouse (B)
Skeletal variations; M+F mouse (B) - B
a-m-m
*
The study authors did not conduct statistical analysis on these endpoints, but EPA has determined the
results to be biologically significant.
Sources: (A) Daniel and Evans (1982); (B) Faulkner et al. (19891; (C) Huntingdon Life Sciences (2004K
Figure 1-11. Exposure-response array of developmental effects following oral
exposure to terfc-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
DEVELOPMENTAL
Number of live fetuses per litter; M+F rat
(Nelson et al., 1989)
Number of resorptions per litter; M+F rat
(Nelson et al., 1989)
J-Fetal weight; M rat
(Nelson et al., 1989)
iFetal weight; F rat
(Nelson et al., 1989)
Skeletal variation by litter; M+F rat
(Nelson et al., 1989)
Skeletal variation by fetus; M+F rat
(Nelson et al., 1989)
1,000 10,000
Exposure Concentration (mg/m3)
100,000
Figure 1-12. Exposure-response array of developmental effects following
inhalation exposure to tert-butanol.
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Mechanistic Evidence
No mechanistic evidence for developmental effects was identified by the literature search.
Integration of Developmental Effects
Although minimal effects were observed at otherwise toxic dose levels, the available
evidence is considered insufficient to identify selective developmental effects as a potential human
health hazard of tert-butanol exposure. Exposure to tert-butanol during gestation resulted in
increased fetal loss, decreased fetal body weight, and increases in skeletal variations in exposed
offspring. Dams, however, had body-weight losses or gains (or both), decreased food consumption,
and clinical signs of intoxication at the same doses of tert-butanol causing fetal effects. Therefore,
determining whether tert-butanol exposure results in specific developmental toxicity or the fetal
effects are due to maternal toxicity is difficult, if not impossible, from the available data. However,
selective developmental toxicity of tert-butanol at the higher doses examined cannot be ruled out.
Furthermore, no adverse effects were reported in one- and two-generation
reproductive/developmental studies on ETBE fGaoua. 2004a. b), further supporting the lack of
evidence for developmental effects as possible human hazards following tert-butanol exposure.
1.2.4. Neurodevelopmental Effects
Synthesis of Effects Related to Neurodevelopment
Three studies evaluated neurodevelopmental effects [one in female mice, one in male rats,
one in female rats; Nelson etal. (19911: Daniel and Evans (19821] following tert-butanol exposure
via liquid diet (maltose/dextrin) or inhalation. No epidemiological studies on neurodevelopment
are available. The animal studies evaluating neurodevelopmental effects of tert-butanol contain
study design limitations. The Daniel and Evans T19821 study had few animals per treatment group,
lacked comparison of treatment-related effects to controls for all endpoints investigated, and
performed no long-term neurodevelopmental testing. Further, animals in this study had lower
dietary intake than the ad libitum control animals. The authors addressed this issue with a pair-fed
experimental design, but a slight decrease in maternal dietary intake remained. This decrease was
likely due to difficulties in the pair feeding or increased maternal sedation (Daniel and Evans.
19821. The two studies by Nelson et al. f 19911 evaluated neurodevelopmental effects after either
paternal or maternal exposure but did not run the exposures concurrently. The studies are
arranged in the evidence tables by species and sex.
Various neurodevelopmental effects have been observed in the available studies. Effects
include changes in rotarod performance following oral or inhalation exposures, decreases in open
field behavior and cliff avoidance following oral exposure, and reduced time hanging on wire after
inhalation exposure during gestation (see Table 1-14).
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Rotarod performance
Inconsistent results were observed across exposure routes and species. Although Daniel
and Evans T19821 found decreased rotarod performance in mouse pups of dams orally exposed
during gestation, Nelson etal. f 19911 observed an increase in rotarod performance in rat pups of
dams exposed via inhalation during gestation.
Neurochemical measurements
Biochemical or physiological changes in the brain of offspring exposed during gestation or
early in the postnatal period were examined only in the Nelson etal. fl9911 study. The authors
reported statistically significant changes in neurochemical measurements (norepinephrine,
met-enkephalin, (3-endorphin, serotonin) in the brain in offspring of both dams exposed via
inhalation during gestation and treated adult males mated with untreated dams. The strength of
these results is compromised, however, because the two concentrations tested (in both
experiments) were not run concurrently, and only the data on statistically significant effects were
reported. Therefore, comparison across doses or trend analysis for the effects is not feasible.
Physiological and psychomotor development
Daniel and Evans T19821 cross-fostered half the mouse pups born to treated mothers with
untreated surrogate females to test the effects of maternal nutrition and behavioral factors on pup
physiological and psychomotor development. The results indicated that pups fostered to control
dams performed significantly better than those maintained with treated dams [see Table 1-14;
Daniel and Evans fl9821]. These data suggest that neurodevelopmental effects were not solely due
to in utero exposure to tert-butanol (Daniel and Evans. 1982). Interpretation of these results is
limited, however, because the neurodevelopmental data were presented only in figures and could
not be compared with controls.
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Table 1-14. Evidence pertaining to neurodevelopmental effects in animals
following exposure to tert-butanol
Reference and study design
Results
Daniel and Evans (1982)
Swiss Webster (Cox) mouse; 15
pregnant dams/treatment (3 or 4
dams/treatment group for
neurodevelopmental endpoints)
Liquid diet (0, 0.5, 0.75, or 1.0%,
w/v); GD 6-20; 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 in 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 (no. of litters
born not reported)
Inhalation analytical
concentration: 0, 6,000, or
12,000 mg/m3; dynamic
whole-body chamber
7 h/d
GD 1-19
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 vs.
26 revolutions/min for the control and 12,000 mg/m3 animals,
respectively).3
• Decreased time held on wire in the performance ascent test in the
low-dose group (16 vs. 10 sec for the control and 6,000 mg/m3
animals, respectively).3
• For the high-dose group, no effects were noted for ascent on a wire
mesh screen, open field activity, automated motor activity,
avoidance conditioning, operant conditioning.
• For the low-dose group, no effects were observed on rotarod, open
field activity, automated motor activity, avoidance conditioning,
operant conditioning.
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/m3.3
• 57% decrease in met-enkephalin in the cerebrum at 12,000 mg/m3
and 83% decrease at 6,000 mg/m3.3
• 61% decrease in p-endorphin in the cerebellum at 12,000 mg/m3.3
• 67% decrease in serotonin in the midbrain at 6,000 mg/m3.3
• No effects were observed for other neurotransmitter levels
(acetylcholine, dopamine, substance P) at both low and high doses.
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Table 1-14. Evidence pertaining to neurodevelopmental effects in animals
following exposure to tert-butanol (continued)
Reference and study design
Results
Nelson et al. (1991)
Adult male Sprague-Dawley rats
(18/treatment) mated to
untreated females
Inhalation analytical
concentration: 0, 6,000, or
12,000 mg/m3; dynamic
whole-body chamber
7 h/d for 6 wk
Data were not presented specifically by dose nor were any tables or figures
of the data provided.
Results (generally only specified as paternally treated vs. controls) in
offspring indicate:
• Increase in rotarod performance (16 vs. 20 revolutions/min for the
control and 12,000 mg/m3 animals, respectively).3
• Decreased time in open field (less time to reach the outer circle of
the field, 210 vs. 115 sec for controls and 12,000 mg/m3 animals,
respectively).3
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/m3.3.
• 40% decrease in met-enkephalin in the cerebrum at 12,000 mg/m3
and 75% decrease at 6,000 mg/m3.3
• 71% decrease in p-endorphin in the cerebellum at 12,000 mg/m3.3
• 47% decrease in serotonin in the midbrain at 6,000 mg/m3.3
w/v = weight by volume.
statistically significant p < 0.05, as determined by the study authors.
Note: Conversions from diet concentrations to mg/kg-d were performed by the study authors.
Percentage change compared to control = [(treated value - control value) -f control value] x 100.
Mechanistic Evidence
No mechanistic evidence for neurodevelopmental effects was identified by the literature
search. The available mechanistic information for tert-butanol is limited to three studies examining
muscarinic acetylcholine receptor function, and what, if any, relationship these effects might have
pertaining to developmental neurotoxicity effects remains unclear fBale and Lee. 20161.
Integration of Neurodevelopmental Effects
Neurodevelopmental effects, including decreased brain weight, changes in brain
biochemistry, and changes in behavioral performances, have been observed. Each study evaluating
neurodevelopmental effects, however, had limitations in study design, reporting, or both. In
addition, results were not always consistent between studies or across dose. Although minimal
effects were observed at otherwise toxic dose levels, the available evidence is considered
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insufficient to identify neurodevelopmental effects as a potential human health hazard of
tert-butanol exposure.
1.2.5. Reproductive Effects
Synthesis of Effects Related to Reproduction
Several studies evaluated reproductive effects [a one-generation, oral reproductive study
(Huntingdon Life Sciences. 20041 and subchronic effects in rats and mice following oral and
inhalation exposure (NTP. 1997.19951] in animals exposed to tert-butanol via oral gavage, drinking
water, or inhalation for >63 days. The studies are arranged in the evidence tables by sex, route of
exposure, duration of exposure, and species. The collection of studies evaluating reproductive
effects of tert-butanol is limited by the absence of two-generation reproductive oral or inhalation
studies and by the lack of human studies on reproduction. The design, conduct, and reporting of
each study were reviewed, and each study was considered adequate to provide information
pertinent to this assessment
Reproductive endpoints, such as reproductive organ weights, estrous cycle length, and
sperm effects were examined following either oral or inhalation exposure [Huntingdon Life
Sciences f20041: NTP f!997.19951: see Table l-15;Figure 1-13; Figure 1-14]. In males, the only
significant effect observed was a slight decrease in sperm motility for F0 males treated with
1,000 mg/kg-day tert-butanol (Huntingdon Life Sciences. 2004). No significant changes in sperm
motility were reported following oral exposure in other rat studies or via inhalation exposure in
mice or rats. In addition, the reduced motility in treated animals falls within the range of historical
control data, and therefore, its biological significance is uncertain. In female B6C3F1 mice, estrous
cycle length was increased 28% following oral exposure to 11,620 mg/kg-day (NTP. 19951. No
significant changes in estrous cycle length were observed following oral exposure in rats or
inhalation exposure in mice or rats. However, there was some evidence of increased numbers of
animals with long, unclear, or absent cycles in tert-butanol-exposed mice (oral/inhalation) and rats
(oral; see Table 1-15). It is noteworthy that these effects were limited to the highest doses tested
with some doses accompanied by body-weight loss or lethality.
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Table 1-15. Evidence pertaining to reproductive effects in animals following
exposure to tert-butanol
Reference and study design
Results
Male reproductive effects
Huntingdon Life Sciences (2004)
Sprague-Dawley rat; 12/sex/treatment
Gavage 0, 64,160, 400, or 1,000 mg/kg-d
F0 males: 9 wk beginning 4 wk prior to
mating
PND21
F0 reproductive effects
Sperm motility (only control and high-dose groups examined)
0: 94% 1,000: 91%a
No other 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, or
3,610b mg/kg-d
13 wk
No significant effect on weights of male reproductive organs or
sperm observed.
NTP (1995)
B6C3F1 mouse; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or
40 mg/mL)
M: 0, 350, 640, 1,590, 3,940, or
8,210b mg/kg-d
13 wk
No significant effect on weights of male reproductive organs or
sperm observed.
Note: NTP results unclear in regard to testis weight—Table F3 shows
a significant decrease in testis weight at 8,210 mg/kg-d (0.115 to
0.096 mg) but Table H2 shows the same dose decreasing testis
weight nonsignificantly from 0.115 to 0.101 mg.
NTP (1997)
F344/N rat; 10/sex/treatment
Inhalation 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 h/d, 5 d/wk
13 wk
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/m3).
NTP (1997)
B6C3Fi mouse; 10/sex/treatment
Inhalation 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 h/d, 5 d/wk
13 wk
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/m3).
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Table 1-15. Evidence pertaining to reproductive effects in animals following
exposure to tert-butanol (continued)
Reference and study design
Results
Female reproductive effects
Huntingdon Life Sciences (2004)
Sprague-Dawley rat; 12/sex/treatment
Gavage 0, 64,160, 400, or 1,000 mg/kg-d
F0 females: 4 wk 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)
F: 0, 290, 590, 850,1,560, or
3,620b mg/kg-d
13 wk
No significant effect on female estrous cycle length (0, -2, -4,0, 8%
change relative to control).
Note: Number of animals that had >7-d cycle length, unclear cycles,
or no cycles.
0: 0
1,560: 2/10
3,620: 4/4°
NTP (1995)
B6C3F1 mouse; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or
40 mg/mL)
F: 0, 500, 820, 1,660, 6,430, or
ll,620b mg/kg-d
13 wk
1" length of estrous cycle
Response relative to control: 0, 5, 5, 5, 6, 28a%
Note: Animals with >7-d cycle length, unclear cycles, or no cycles.
0: 0/10
500: 0/9
820:1/10
1,660: 1/10
6,430: 1/9
11,620: 4/6d
NTP (1997)
F344/N rat; 10/sex/treatment
Inhalation 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 h/d, 5 d/wk
13 wk
Generation method (Sonimist ultrasonic
spray nozzle nebulizer), analytical
concentration and method were reported
No significant effect on female estrous cycle length (0, -4, 2, 4%
change relative to control) or the number of animals cycling.
Evaluations were performed only for concentrations >542 ppm
(1,643 mg/m3).
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Table 1-15. Evidence pertaining to reproductive effects in animals following
exposure to tert-butanol (continued)
Reference and study design
Results
NTP (1997)
B6C3F1 mouse; 10/sex/treatment
Inhalation analytical concentration: 0,134,
272, 542, 1,080, or 2,101 pm (0, 406, 824,
1,643, 3,273, or 6,368 mg/m3), dynamic
whole-body chamber
6 h/d, 5 d/wk
13 wk
Generation method (Sonimist ultrasonic
spray nozzle nebulizer), analytical
concentration and method were reported
No significant effect on female estrous cycle length (0, -3, -9, -5%
change relative to control).
Evaluations were only performed for concentrations >542 ppm
(1,643 mg/m3).
Note: Number of animals with >7-d cycle length, unclear cycles, or no
cycles.
0:0/10
542: 2/10
1,080:1/10
2,101: 3/10
Statistically significant p < 0.05, as determined by the study authors.
bThe high-dose group had an increase in mortality.
Statistically significant p < 0.01, as determined by EPA.
Statistically significant p < 0.05, as determined by EPA.
Notes: Conversions from drinking water concentrations to mg/kg-d were performed by the study authors.
Conversion from ppm to mg/m3 is 1 ppm = 3.031 mg/m3.
Percentage change compared to control = [(treated value - control value) -f control value] x 100.
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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
REPRODUCTIVE EFFECTS
Male reproductive effects
Reproductive organs or sperm; M
rat (A)
B-
-B-
Reproductive organs or sperm; M
rat(B)
~ ~ ~
Reproductive organs or sperm; M
mouse (B)
~—B-
-B B ~
Female reproductive effects
Pregnancy index; F rat (A]
Estrous cycle length; F rat (B)
~ B-Br
-B ~
1 Estrous cycle length; F mouse (B)
~—B-
10 100 1,000 10,000 100,000
Dose (mg/kg-day)
Sources: (A) Huntingdon Life Sciences (2004); (B) NTP (1995).
Figure 1-13. Exposure-response array of reproductive 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
REPRODUCTIVE EFFECTS
Male reproductive effects
Reproductive organs or sperm; M rat
(NTP, 1997)
Reproductive organs or sperm; M mouse
(NTP, 1997)
Female reproductive effects
Estrous cycle; F rat (NTP, 1997)
Estrous cycle; F mouse (NTP, 1997)
1,000 10,000
Exposure Concentration (mg/m3)
Figure 1-14. Exposure-response array of reproductive effects following
inhalation exposure to tert-butanol.
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Mechanistic Evidence
No mechanistic evidence for reproductive effects was identified by the literature search.
Integration of Reproductive Effects
Although minimal effects were observed at otherwise toxic dose levels, the available
evidence is considered insufficient to identify reproductive effects as a potential human health
hazard of tert-butanol exposure. The evidence base is limited to a one-generation study
(Huntingdon Life Sciences. 2004: NTP. 19951. No two-generation reproductive studies are
available that evaluate oral or inhalation exposure. In males, the only observed effect was a slight
decrease in sperm motility for F0 males in the highest dose group of rats treated with tert-butanol.
This effect was not observed, however, in other studies with orally treated rats and mice or in rats
exposed via inhalation. In females, NTP (1995) reported an increased length of the estrous cycle in
the highest dose group of orally exposed mice. This effect was not observed in similarly treated
rats or in mice and rats exposed via inhalation. In addition, there was limited evidence of increased
numbers of animals with long, unclear, or absent cycles in exposed rats and mice. However, these
effects were limited to the highest doses tested (some with accompanying body-weight loss or
lethality) and were not consistent across species or route of exposure. Furthermore, no adverse
effects were reported in one- and two-generation reproductive/developmental studies on ETBE
(Gaoua. 2004a. b), providing additional support for the lack of evidence supporting reproductive
effects as possible human hazards following tert-butanol exposure.
1.2.6. Other Toxicological Effects
Effects other than those related to kidney, thyroid, reproductive, developmental, and
neurodevelopmental toxicity were observed in some of the available rodent studies; these include
liver and urinary bladder effects. However, because of a lack of consistency in the liver effects and
the minimal to mild effects with a lack of progression in urinary bladder, the available information
is inadequate to draw conclusions regarding liver or urinary bladder toxicity at this time.
Additionally, central nervous system (CNS) effects similar to those caused by ethanol
(animals appearing intoxicated and having withdrawal symptoms after cessation of oral or
inhalation exposure) were observed. Because of study quality concerns (e.g., lack of data reporting,
small number of animals per treatment group), however, adequate information to assess CNS
toxicity is unavailable at this time. For more information on these other toxicological effects, see
Appendix B, Section B.3.
1.3. INTEGRATION AND EVALUATION
1.3.1. Effects Other Than Cancer
Kidney effects were identified as a potential human hazard of tert-butanol exposure based
on several endpoints in female rats, including suppurative inflammation, transitional epithelial
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hyperplasia, severity and incidence of nephropathy, and increased kidney weights. These effects
are similar to the kidney effects observed with ETBE exposure (e.g., CPN and urothelial
hyperplasia) and MTBE [e.g., CPN and mineralization; ATSDR f 19961],
Based on mechanistic evidence indicating that an alpha 2u-globulin-related process is
operating in male rats fHard etal.. 2011: Cirvello etal.. 1995: NTP. 1995: Lindamood et al.. 19921.
any kidney effects may be confounded by the association with alpha 2u-globulin. However, it is
difficult to determine the relevant contribution of alpha 2u-globulin and chemically induced or
spontaneously occurring CPN. Thus, nephropathy in male rats is not considered relevant for
human hazard identification. Furthermore, mineralization in male rats was not considered
clinically important to rats or relevant to human health and was not considered for dose-response
analysis.
CPN played a role in the renal tubule nephropathy observed following tert-butanol
exposure in female rats. Because female rats were not affected by alpha 2u-globulin nephropathy
and the individual lesions associated with the spectrum of toxicities collectively described as CPN
can occur in the human kidney (NIEHS. 20191. exacerbation of one or more of these lesions might
reflect a type of injury relevant to the human kidney. Effects associated with such nephropathy are
considered relevant for human hazard identification and suitable for derivation of reference values.
Overall, the female rat kidney effects (suppurative inflammation, transitional epithelial hyperplasia,
increased severity of CPN, and increased kidney weights) are considered the result of tert-butanol
exposure and relevant to human hazard characterization. These effects therefore are suitable for
consideration for dose-response analysis and derivation of reference values, in Section 2.
Although minimal effects were observed at otherwise toxic dose levels, the available
evidence is considered insufficient to identify developmental effects as a potential human health
hazard of tert-butanol exposure. Increased fetal loss, decreased fetal body weight, and increases in
skeletal variations in exposed offspring were observed following exposure to relatively high doses
of tert-butanol during gestation. These effects are similar to the developmental effects observed
with MTBE exposure [e.g., decreased fetal body weight and increases in skeletal variations; ATSDR
(1996)]. However, dams had body-weight losses or gains (or both), decreased food consumption,
and clinical signs of intoxication at the same doses of tert-butanol causing fetal effects. Therefore,
determining whether tert-butanol exposure results in specific developmental toxicity or the fetal
effects are due to maternal toxicity is difficult, if not impossible, from the available data.
Nevertheless, selective developmental toxicity of tert-butanol at the higher doses examined cannot
be ruled out
No mechanistic evidence is available for developmental effects of tert-butanol. There is
inadequate evidence of selective developmental toxicity because of the uncertainty about whether
fetal effects were due to direct effects of tert-butanol or indirect effects of maternal toxicity and
because of the lack of consistency across some endpoints.
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Although minimal effects were observed at otherwise toxic dose levels, the available
evidence is considered insufficient to identify neurodevelopmental effects as a potential human
health hazard of tert-butanol exposure. While neurodevelopmental effects have been observed, the
studies had limitations in design or reporting, or both, and results were inconsistent between and
across exposure routes and species. Also, the limited available mechanistic information is unclear.
Therefore, neurodevelopmental effects were not considered further for dose-response analysis and
derivation of reference values.
Although minimal effects were observed at otherwise toxic dose levels, the available
evidence is considered insufficient to identify reproductive effects as a potential human health
hazard of tert-butanol exposure. The only reproductive effect observed due to tert-butanol
exposure was increased length of estrous cycle fNTP. 19951 in the highest dose group of orally
exposed mice, and this effect was not observed in orally exposed rats or in mice and rats exposed
via inhalation. Further, the evidence base was limited and contained only two oral exposure studies
and one subchronic inhalation study. No mechanistic or MOA information is available for
reproductive effects of tert-butanol. These effects were not considered further for dose-response
analysis and the derivation of reference values.
At this time, information is inadequate to draw conclusions regarding liver or urinary
bladder toxicity due to lack of consistency of effects and minimal/mild effects showing a lack of
progression, respectively. No mechanistic evidence is available for these effects. The liver and
urinary bladder effects were not considered further for dose-response analysis and the derivation
of reference values.
1.3.2. Carcinogenicity
Summary of Evidence
In B6C3F1 mice, administration of tert-butanol in drinking water increased the incidence of
thyroid follicular cell adenomas in females and adenomas or carcinomas (only one carcinoma
observed) in males (NTP. 1995). as discussed in Section 1.2.2. According to EPA's thyroid tumor
guidance (U.S. EPA. 1998a). chemicals that produce thyroid tumors in rodents might pose a
carcinogenic hazard to humans.
In F344/N rats, administration of tert-butanol in drinking water increased the incidence of
renal tubule tumors, mostly adenomas, in males; no renal tumors in females were reported (Hardet
al.. 2011: NTP. 1995). As discussed in Section 1.2.1, some of these tumors might be associated with
alpha 2u-globulin nephropathy, an MOA considered specific to the male rat (U.S. EPA. 1991a).
Evidence in support of this hypothesized MOA includes the accumulation of hyaline droplets in
renal tubule cells, the presence of alpha 2u-globulin in the hyaline droplets, and additional aspects
associated with alpha 2u-globulin nephropathy, including linear papillary mineralization and foci of
tubular hyperplasia. Other evidence, however, is not supportive: the accumulation of hyaline
droplets was minimal, concentrations of alpha 2u-globulin were low at doses that induced tumors,
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and no significant necrosis or cytotoxicity was associated with compensatory regenerative
proliferation or induction of granular casts observed within a time frame consistent with alpha
2u-globulin-mediated nephropathy. Renal tumors also are associated with chronic progressive
nephropathy, but the data on CPN are not coherent; dose-response relationships for CPN, renal
tubule hyperplasia, and renal tubule tumors differed. In addition, CPN was nearly as severe in
female rats as in male rats, yet no female rats developed renal tumors. Thus, some renal tumors
might be attributable to alpha 2u-globulin nephropathy augmented by CPN, and some to other, yet
unspecified, processes. Taken together, and according to EPA's guidance on renal tumors in male
rats fU.S. EPA. 1991al. renal tumors induced by tert-butanol are relevant for human hazard
identification.
In addition, as mentioned in Section 1.1.4, tert-butanol is a primary metabolite of MTBE and
of ETBE, two compounds tested in rats and mice that could provide supplementary information on
the carcinogenicity of tert-butanol. For MTBE, the most recent cancer evaluation by a national or
international health agency is from IARC (1999). IARC reported that oral gavage exposure in
Sprague-Dawley rats resulted in testicular tumors in males and lymphomas and leukemias
(combined) in females. Inhalation exposure in male and female F344 rats resulted in renal tubule
adenomas in males, and inhalation exposure in male and female CD-I mice resulted in
hepatocellular adenomas in females flARC. 19991. For ETBE, the IRIS assessment developed
concurrently with this assessment reports that inhalation exposure in male and female F344 rats
resulted in hepatocellular tumors, primarily adenomas, in males. No significant tumor increases,
including kidney tumors, were reported for 2-year studies by drinking water exposure in male and
female F344 rats or by gavage in male and female Sprague-Dawley rats (NTP. 1997).
Integration of evidence
This evidence leads to consideration of two hazard descriptors under EPA Cancer
Guidelines (U.S. EPA. 2005b). The descriptor likely to be carcinogenic to humans is appropriate
when the evidence is "adequate to demonstrate carcinogenic potential to humans" but does not
support the descriptor carcinogenic to humans. One example from the cancer guidelines is "an
agent that has tested positive in animal experiments in more than one species, sex, strain, site, or
exposure route, with or without evidence of carcinogenicity in humans." tert-Butanol matches the
conditions of this example, having increased tumor incidences in two species, in both sexes, and at
two sites.
Alternatively, the descriptor suggestive evidence of carcinogenic potential is appropriate
when the evidence raises "a concern for potential carcinogenic effects in humans" but is not
sufficient for a stronger conclusion. The results for tert-butanol raise a concern for cancer but none
of the effects is particularly strong. The thyroid tumors induced in male and female mice were
almost entirely benign. The kidney tumors resulted, in part, from an MOA that is specific to male
rats, while no kidney tumors occurred in female rats. In addition, while MTBE was also associated
with male rat kidney tumorigenesis, results between tert-butanol- and ETBE-associated
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tumorigenesis in rats have little coherence. MTBE or ETBE effects following chronic oral exposure
in mice have not been investigated, however, so no evidence exists to evaluate the coherence of the
thyroid tumorigenesis observed following tert-butanol exposure in B6C3F1 mice.
These considerations, interpreted in light of the cancer guidelines, support the conclusion,
suggestive evidence of carcinogenic potential for tert- butanol. Although increased tumor incidences
were reported for two species, two sexes, and two sites, none of the tumor responses were strong
or coherent with the results for ETBE, and this was decisive in selecting a hazard descriptor.
The descriptor suggestive evidence of carcinogenic potential applies to all routes of human
exposure. Oral administration of tert-butanol to rats and mice induced tumors at sites beyond the
point of initial contact, and inhalation exposure for 13 weeks resulted in absorption and
distribution of tert-butanol into the systemic circulation, as discussed in Section 1.2.1. Therefore, in
agreement with EPA Cancer Guidelines, this information provides sufficient basis to apply the
cancer descriptor developed from oral studies to other exposure routes.
Biological considerations for dose-response analysis
Regarding hazards to bring forward to Section 2 for dose-response analysis, EPA's guidance
on thyroid tumors (U.S. EPA. 1998a) advise that, for thyroid tumors resulting from thyroid-pituitary
disruption, dose-response analysis should use nonlinear extrapolation, in the absence of MOA
information to indicate otherwise. As discussed in Section 1.2.2, increases in thyroid follicular cell
hyperplasia in male and female mice provide partial support for thyroid-pituitary disruption. Other
necessary data on tert-butanol, however, are either not adequate or not supportive. There is little
correlation between thyroid, pituitary, and liver effects in female mice, and no data are available to
evaluate the potential for antithyroid effects in male mice. Data are not adequate to conclude that
thyroid hormone changes exceed the range of homeostatic regulation or to evaluate effects on
extrahepatic sites involved in thyroid-pituitary disruption. Also, no data are available to evaluate
reversibility of effects upon cessation of exposure. Thus, according to EPA's thyroid tumor
guidance, concluding that the thyroid tumors result from thyroid-pituitary disruption is premature,
and dose-response analysis should use linear extrapolation.
As discussed in Section 1.2.2, the available data do not demonstrate that the thyroid tumors
are the result of excessive toxicity in female mice rather than the carcinogenicity of tert-butanol.
The final average body-weight reduction in female mice was 12% fNTP. 1995], but water
consumption by exposed females was similar to controls and no overt toxicity was observed.
Furthermore, female mice in the high-dose group had higher rates of survival than control animals.
EPA Cancer Guidelines (U.S. EPA. 2005a) also states that when there is suggestive evidence of
carcinogenicity and when the evidence includes a well-conducted study, "quantitative analysis may
be useful for some purposes, for example, providing a sense of magnitude and uncertainty of
potential risk, ranking potential hazards, or setting research priorities." Given that the data are well
suited to dose-response analysis, coming from an NTP study that tested multiple dose levels, and
because quantitative analysis may be useful in providing a sense of magnitude and uncertainty of
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potential risks from tert-butanol exposure, including worker or consumer exposures, an analysis of
thyroid tumors is presented in Section 2.
EPA's guidance on renal tumors in male rats fU.S. EPA. 1991al advises that, unless the
relative contribution of alpha 2u-globulin nephropathy and other process can be determined,
dose-response analysis should not be performed. As discussed in Section 1.2.1, the available data
do not allow such determination, and so an analysis of kidney tumors does not appear in Section 2.
1.3.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes
No chemical-specific data that would allow for the identification of populations with
increased susceptibility to tert-butanol exposure are available. In vitro studies have implicated the
liver microsomal mixed function oxidase system, namely CYP450 fCederbaum etal.. 1983:
Cederbaum and Cohen. 19801. as playing a role in the metabolism of tert-butanol. One study
evaluated liver enzyme expression and found a dose-responsive induction of CYP2B10 following
14 days of tert-butanol exposure in female mice, with much smaller increases in the expression of
CYP2B9 and the thyroid hormone-metabolizing enzyme SULT1A1 (Blanck etal.. 20101. No studies,
however, have identified the specific CYPs responsible for the biotransformation of tert-butanol.
Pharmacokinetic differences among the fetus, newborns, children, and the aged might alter
responses to chemicals compared with adults, resulting in differences in health effects. In the
presence of environmental chemicals, metabolic homeostasis is maintained by the liver's ability to
detoxify and eliminate xenobiotics. This process is accomplished, in part, by the expression of
xenobiotic metabolizing enzymes and transporters (XMETs), which metabolize and transport
xenobiotics and determine whether exposure will result in altered responses. XMETs, including
various CYPs, have been found to be underexpressed in the mouse fetus and neonate fLee etal..
20111 and decreased in older mice fLee etal.. 20111 and rats fLee etal.. 20081. Decreased ability to
detoxify and transport tert-butanol out of the body could result in increased susceptibility to
tert-butanol in the young and old.
In regard to cancer, although children are more sensitive than adults to thyroid
carcinogenesis resulting from ionizing radiation, relative differences in lifestage sensitivity to
chemically induced thyroid carcinogenesis are unknown (U.S. EPA. 1998al. In addition, the data on
tert-butanol mutagenicity are inconclusive.
Collectively, evidence on tert-butanol is minimal for identifying susceptible populations or
lifestages.
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2. DOSE-RESPONSE ANALYSIS
2.1. ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER
The oral reference dose (RfD, expressed in units of mg/kg-day) is defined as an estimate
(with uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human
population (including sensitive subgroups) that is likely to be without an appreciable risk of
deleterious effects during a lifetime. The RfD can be derived from a no-observed-adverse-effect
level (NOAEL), lowest-observed-adverse-effect level (LOAEL), or the benchmark dose lower
confidence limit (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 potential human hazard of tert-butanol exposure (see
Section 1.2.1). Studies within this effect category were evaluated using general study quality
characteristics [as discussed in Section 4 of the Preamble; see also U.S. EPA f20021] to help inform
the selection of studies from which to derive toxicity values. No other hazards were identified for
further consideration in the derivation of reference values.
Human studies are preferred over animal studies when quantitative measures of exposure
are reported and the effects are determined to be associated with exposure. No human
occupational or epidemiological studies of oral exposure to tert-butanol, however, are available.
Animal studies were evaluated to determine which studies provided the most relevant
routes and durations of exposure, and multiple exposure levels covering a broad range were used
to provide information about the shape of the dose-response curve. The evidence base for
tert-butanol includes both chronic and subchronic studies showing effects in the kidney that are
suitable for deriving reference values.
Kidney Toxicity
EPA identified kidney effects as a potential human hazard of tert-butanol-induced toxicity
based on findings in female rats (summarized in Section 1.3.1). Kidney toxicity was observed
across multiple chronic, subchronic, and short-term studies following oral and inhalation exposure.
Kidney effects, such as suppurative inflammation and transitional epithelial hyperplasia, observed
after chronic exposure could influence the ability of the kidney to filter waste. Exacerbated
nephropathy also would affect kidney function. Observed changes in kidney weight also could
indicate toxic effects in the kidney. For the oral tert-butanol evidence base, several studies that
evaluated these kidney effects are available. Huntingdon Life Sciences f20041 conducted a
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reproductive study in Sprague-Dawley rats that was of shorter duration; the study reported
changes in kidney weight but did not examine changes in histopathology. National Toxicology
Program (NTP) conducted a 2-year drinking water study fNTP. 1995] in F344 rats that evaluated
multiple doses in both males and females and reported on all three endpoints highlighted above.
NTP f!9951 was identified as the study most suitable for dose-response assessment considering the
study duration, comprehensive reporting of outcomes, and multiple doses tested.
In the NTP (1995) 2-year drinking water study, female F344 rats were exposed to
approximate doses of 0,180, 330, or 650 mg/kg-day. Reduced body weights and survival were
observed and reflected in some of the effects. Kidney effects, including changes in organ weight,
histopathology, or both, were observed in both sexes of rats after 13 weeks, 15 months, and 2 years
of treatment fNTP. 19951. Because the kidney effects in male rats are complicated by alpha
2u-globulin, male kidney effects are not considered in developing toxicity values. Specific
endpoints in female rats chosen for dose-response analysis were absolute kidney weight, kidney
suppurative inflammation, kidney transitional epithelial hyperplasia, and increases in severity of
nephropathy. For absolute kidney weight, data from 15-month duration were selected as described
in Section 1.2.1; for the other endpoints, data at the longest duration of 2 years were selected.
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 best to empirically model the dose-response relationship in the range
of the observed data. The models in EPA's Benchmark Dose Software (BMDS) were applied.
Consistent with EPA's Benchmark Dose Technical Guidance fU.S. EPA. 2012al. the benchmark dose
(BMD) and the BMDL are estimated using a benchmark response (BMR) to represent a minimal,
biologically significant level of change. In the absence of information regarding the level of change
considered biologically significant, a BMR of 1 standard deviation from the control mean for
continuous data or a BMR of 10% extra risk for dichotomous data is used to estimate the BMD and
BMDL and to facilitate a consistent basis of comparison across endpoints, studies, and assessments.
Endpoint-specific BMRs, where feasible, are described further below. When modeling was feasible,
the estimated BMDLs were used as points of departure (PODs); the PODs are summarized in
Table 2-1. Details including the modeling output and graphical results for the model selected for
each endpoint are presented in Appendix C of the Supplemental Information to this Toxicological
Review. When modeling was not feasible, the study NOAEL or LOAEL was used as the POD.
Kidney weights were analyzed as absolute weights rather than weights relative to body
weight. In general, both absolute and relative kidney-weight data are considered appropriate
endpoints for analysis (Bailey etal.. 2004). In the NTP (1995) 2-year drinking water study, body
weight in exposed animals noticeably decreased relative to controls at the 15-month interim
sacrifice, but this decrease in body weight disproportionately influenced the measure of relative
kidney weight, resulting in exaggerated kidney-weight changes. Because there was greater
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confidence in the absolute kidney-weight measure, it was considered more appropriate for
dose-response analysis, and changes in relative kidney weights were not analyzed. A 10% relative
change from control was used as a BMR for absolute kidney weight, analogous to a 10% change in
body weight as an indicator of toxicity. A BMR of 10% extra risk was considered appropriate for
the quantal data on incidences of kidney suppurative inflammation and kidney transitional
epithelial hyperplasia. Dose-response modeling was not conducted on the increases in severity of
nephropathy due to uncertainty regarding BMR selection. That is, it was not clear what critical
values, including BMR type as well as values, to use for a common, high background outcome with
severity levels as high as moderate even in controls. EPA relied on the interpretation NTP provided
fNTP. 19951 and identified the lowest dose, associated with a statistically significantly elevated
average severity score, as a LOAEL. Human equivalent doses (HEDs) for oral exposures were
derived from the PODs according to the hierarchy of approaches outlined in EPA's Recommended
Use of Body Weight3/4 as the Default Method in Derivation of the Oral Reference Dose (U.S. EPA. 2011).
The preferred approach is physiologically based pharmacokinetic (PBPK) modeling. Other
approaches include using chemical-specific information in the absence of a complete PBPK model.
As discussed in Appendix B of the Supplemental Information, human PBPK models for inhalation of
ethyl tert-butyl ether (ETBE) or inhalation and dermal exposure to methyl tert-butyl ether (MTBE)
have been published, which include tert-butanol submodels. A validated human PBPK model for
tert-butanol, however, is not available for extrapolating doses from animals to humans. In lieu of
either chemical-specific models or data to inform the derivation of human equivalent oral
exposures, body-weight scaling to the % power (BW3/4) is applied to extrapolate toxicologically
equivalent doses of orally administered agents from adult laboratory animals to adult humans for
the purpose of deriving an RfD.
Consistent with EPA guidance fU.S. EPA. 20111. the PODs estimated based on effects in adult
animals were converted to HEDs employing a standard dosimetric adjustment factor (DAF) derived
as follows:
BWa = animal body weight
BWh = human body weight
Using a standard BWa of 0.25 kg for rats and a BWh of 70 kg for humans fU.S. EPA. 19881.
the resulting DAF is 0.24 for rats. Applying this DAF to the POD identified for effects in adult rats
yields a PODhed as follows (see Table 2-1):
DAF
(BW.1/* - BWh1/4),
(2-1)
where
PODhed = Laboratory animal dose (mg/kg-day) x DAF
(2-2)
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Table 2-1 summarizes all PODs and the sequence of calculations leading to the derivation of
a human equivalent POD for each endpoint discussed above.
Table 2-1. Summary of derivations of points of departure following oral
exposure for up to 2 years
Endpoint and
reference
Species/
sex
Model3
BMR
BMD
(mg/kg-d)
BMDL
(mg/kg-d)
PODadj15
(mg/kg-d)
PODhed0
(mg/kg-d)
Kidney
Increased absolute
kidney weight at
15 mo
NTP (1995)
Rat/F
Exponential
(M4)
(constant
variance)
10%
164
91
91
22
Kidney inflammation
(suppurative)
NTP (1995)
Rat/F
Log-Pro bit
10%
254
200
200
48
Kidney transitional
epithelial
hyperplasia
NTP (1995)
Rat/F
Multistage
(3 degree)
10%
412
339
339
81.4
Increases in severity
of nephropathy
NTP (1995)
Rat/F
NA
NA
NA
NA
180d
43.2
ADJ = adjusted; BMR = benchmark response; HED = human equivalent dose; NA = not applicable.
aFor modeling details, see Appendix C in Supplemental Information.
bFor studies in which animals were not dosed daily, EPA would adjust administered doses to calculate the
time-weighted average daily doses prior to BMD modeling. This adjustment was not required for the NTP
(1995) study.
CHED PODs were calculated using BW3/4 scaling (U.S. EPA, 2011).
dPOD calculated from the LOAEL (lowest dose tested had a significant increase in severity).
2.1.3. Derivation of Candidate Values
Consistent with EPA's A Review of the Reference Dose and Reference Concentration Processes
[U.S. EPA (2002): see Section 4.4.5], also described in the Preamble, five possible areas of
uncertainty and variability were considered when determining the application of UF values to the
PODs presented in Table 2-1. An explanation follows.
An intraspecies uncertainty factor, UFh, of 10 was applied to all PODs to account for
potential differences in toxicokinetics and toxicodynamics in the absence of information on the
variability of response in the human population following oral exposure to tert-butanol fU.S. EPA.
20021.
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An interspecies uncertainty factor, UFA, of 3 (10°5 = 3.16, rounded to 3) was applied to all
PODs because BW3/4 scaling was 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 in the extrapolation 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, of 1 was applied to all PODs because all
endpoints were observed following chronic exposure.
A LOAEL-to-NOAEL uncertainty factor, UFl, of 1 was applied to most PODs derived because
the current approach is to address this factor as one of the considerations in selecting a BMR for
benchmark dose modeling. In this case, BMRs of a 10% relative change in absolute kidney weight, a
10% extra risk of kidney suppurative inflammation, and a 10% extra risk of transitional cell
hyperplasia were selected, assuming they represent minimal biologically significant response
levels. A LOAEL-to-NOAEL uncertainty factor of 3 was applied to the increases in severity of
nephropathy. Although a LOAEL was used to derive the POD, the severity of 1.9 was only slightly
higher than the control value of 1.6, indicating that the LOAEL was close to the result in controls.
A database uncertainty factor, UFd, of 1 was applied to all PODs. The tert-butanol oral
toxicity evidence base includes chronic and subchronic toxicity studies in rats and mice fAcharva et
al.. 1997: Acharva etal.. 1995: NTP. 1995) and developmental toxicity studies in rats and mice
(Huntingdon Life Sciences. 2004: Faulkner etal.. 1989: Daniel and Evans. 1982). In the
developmental studies, no effects were observed at exposure levels below 1,000 mg/kg-day, and
effects observed at >1,000 mg/kg-day were accompanied by evidence of maternal toxicity. These
exposure levels are much higher than the PODs for kidney effects, suggesting any selective
developmental toxicity is not as sensitive an endpoint as kidney effects. No immunotoxicity or
multigenerational reproductive studies are available for tert-butanol. Studies on ETBE, which is
rapidly metabolized to systemically available tert-butanol, are informative for considering the gaps
in the tert-butanol oral evidence base. The evidence base for ETBE does not indicate
immunotoxicity (Banton etal.. 2011: Li etal.. 2011). suggesting immune system effects would not
be a sensitive target for tert-butanol. No adverse effects were reported in one- and two-generation
reproductive/developmental studies on ETBE fGaoua. 2004a. b), indicating that
reproductive/developmental effects would not be a sensitive target for tert-butanol. Additionally, a
one-generation, reproductive toxicity study in rats from a Toxic Substances Control Act submission
(Huntingdon Life Sciences. 2004) is available for tert-butanol. This study did not observe
reproductive effects. Although the oral toxicity evidence base for tert-butanol has some gaps, 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 UFd of 1 was
applied.
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Table 2-2 is a continuation of Table 2-1 and summarizes the application of UF values to each
POD to derive a candidate value for each data set, preliminary to the derivation of the
organ/system-specific RfDs. These candidate values are considered individually in selecting a
representative oral reference value for a specific hazard and subsequent overall RfD for
tert-butanol. Figure 2-1 presents graphically the candidate values, UF values, and PODhed values,
with each bar corresponding to one data set described in Table 2-1 and Table 2-2.
Table 2-2. Effects and corresponding derivation of candidate values
Endpoint and reference
PODhed
(mg/kg-d)
POD
type
ufa
UFh
UFl
UFS
UFd
Composite
UF
Candidate
value
(mg/kg-d)
Kidney
Increased absolute kidney
weight; female rat at 15 mo
NTP (1995)
22
BMDLio
3
10
1
1
1
30
7 x 10"1
Kidney inflammation
(suppurative); female rat at
2 vr NTP (1995)
48
BMDLio
3
10
1
1
1
30
2 x 10°
Kidney transitional epithelial
hyperplasia; female rat at 2 yr
NTP (1995)
81
BMDLio
3
10
1
1
1
30
3 x 10°
Increases in severity of
nephropathy; female rat at 2 yr
NTP (1995)
43.2
LOAEL
3
10
3
1
1
100
4 x 10"1
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
T Absolute kidney
weight; female rat at 15
months (NTP, 1995)
Kidney inflammation
(suppurative); female rat
(NTP, 1995)
Kidney transitional
epithelial hyperplasia;
female rat (NTP, 1995)
Nephropathy severity;
female rat (NTP, 1995)
~ Candidate RfD
• PODhed
Composite UF
0.1
10
100
mg/kg-day
Figure 2-1. Candidate values with corresponding POD and composite
uncertainty factor (UF). Each bar corresponds to one data set described in
Table 2-1 and Table 2-2.
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2.1.4. Derivation of Organ/System-Specific Reference Doses
Table 2-3 distills the candidate values from Table 2-2 into a single value for each organ or
system. Organ- or system-specific RfDs are useful for subsequent cumulative risk assessments that
consider the combined effect of multiple agents acting at a common site.
Kidney Toxicity
To estimate an exposure level below which kidney toxicity from tert-butanol exposure is
not expected to occur, the RfD for greater increases in severity of nephropathy in female rats
(4 x 10"1 mg/kg-day) was selected as the kidney-specific reference dose for tert-butanol. The
database for tert-butanol is relatively small ;therefore, a limited number of endpoints were
considered for kidney toxicity including absolute kidney weight, suppurative inflammation,
transition epithelial hyperplasia, and increased severity of nephropathy. Increased severity of
nephropathy is considered a specific marker of kidney function and more sensitive than the
relatively nonspecific endpoint of absolute kidney-weight changes, and more sensitive than the
endpoints of inflammation and transitional epithelial hyperplasia. Confidence in this
kidney-specific RfD is medium, in part, due to the scientific disagreement on the human relevance
of CPN which remains unresolved. The POD for increases in severity of nephropathy is within 2-fold
of other endpoints and is based on a LOAEL Although the use of a LOAEL adds some uncertainty,
only an UFl of 3 was applied based on the magnitude of change observed at the LOAEL All of the
candidate values are derived from a well-conducted, long-term study involving a sufficient number
of animals per group, including both sexes, and assessing a wide range of kidney endpoints.
Table 2-3. Organ/system-specific oral reference doses (RfDs) and overall RfD
for tert-butanol
Effect
Basis
RfD (mg/kg-d)
Study exposure
description
Confidence
Kidney
Increases in severity of
nephropathy (NTP, 1995)
4 x 10"1
Chronic
Medium
Overall RfD
Kidney
4 x 10"1
Chronic
Medium
2.1.5. Selection of the Overall Reference Dose
For tert-butanol, only kidney effects were identified as a hazard and carried forward for
dose-response analysis; thus, only one organ/system-specific reference dose was derived.
Therefore, the kidney-specific RfD of 4 x 10_1 mg/kg-day is the overall RfD for tert-butanol. This
value is based on greater increases in severity of nephropathy in female rats exposed to
tert- butanol.
The overall reference dose is derived to be protective of all types of effects for a given
duration of exposure and is intended to protect the population as a whole, including potentially
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susceptible subgroups fU.S. EPA. 20021. Decisions concerning averaging exposures over time for
comparison with the RfD should consider the types of toxicological effects and specific lifestages of
concern. Fluctuations in exposure levels that result in elevated exposures during these lifestages
could lead to an appreciable risk, even if average levels over the full exposure duration were less
than or equal to the RfD. In the case of tert-butanol, the potential exists for early lifestage
susceptibility to tert-butanol exposure, as discussed in Section 1.3.3.
2.1.6. Confidence Statement
A confidence level of high, medium, or low is assigned to the study used to derive the RfD,
the overall evidence base, and the RfD, as described in Section 4.3.9.2 of EPA's Methods for
Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry fU.S. EPA.
19941. Confidence in the principal study fNTP. 19951 is high. This study was well conducted,
complied with Food and Drug Administration (FDA) Good Laboratory Practice (GLP) regulations,
involved 50 animals per dose group (including both sexes), and assessed a wide range of tissues
and endpoints. The toxicity evidence base for tert-butanol has some gaps such as a lack of human
studies and limited reproductive/development toxicity data, despite the inclusion of data on ETBE,
a parent compound of tert-butanol. Therefore, the confidence in the RfD is medium based on the
high confidence in the principal study and medium confidence in the evidence base.
2.1.7. Previous Integrated Risk Information System (IRIS) Assessment
No previous oral assessment for tert-butanol is available in IRIS.
2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER
THAN CANCER
The inhalation reference concentration (RfC; expressed in units of mg/m3) is defined as an
estimate (with uncertainty spanning perhaps an order of magnitude) of a continuous inhalation
exposure to the human population (including sensitive subgroups) that is likely to be without an
appreciable risk of deleterious effects during a lifetime. It can be derived from a NOAEL, LOAEL, or
the benchmark concentration lower confidence level (BMCL), with UF values generally applied to
reflect limitations of the data used.
2.2.1. Identification of Studies and Effects for Dose-Response Analysis
As for oral exposure, EPA identified kidney effects as a potential human hazard of
tert-butanol inhalation exposure (summarized in Section 1.3.1). No chronic inhalation study for
tert-butanol is available; only one 13-week study in rats and mice is available fNTP. 19971. A rat
PBPK model was available for both oral and inhalation exposure, which was suitable for a
route-to-route (RTR) extrapolation (Borghoff etal.. 2016). As a result, rat studies from both routes
of exposure were considered for dose-response analysis.
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The evidence base for tert-butanol includes oral and inhalation studies and data sets that
are potentially suitable for use in deriving inhalation reference values. Specifically, effects
associated with tert-butanol exposure in animals include observations of organ weight and
histological changes in the kidney in chronic and subchronic studies in female rats.
Kidney Toxicity
EPA identified kidney effects as a potential human hazard of tert-butanol exposure based on
findings of organ-weight changes and histopathology primarily in male rats; however, the kidney
effects in male rats are complicated by the presence of alpha 2u-globulin. Therefore, kidney effects
in male rats are not considered. The kidney findings were observed across multiple chronic,
subchronic, and short-term studies following oral and inhalation exposure. The subchronic NTP
f!9971 inhalation study is the only route-specific study available, and was carried forward for
further analysis. For oral studies considered for RTR extrapolation, see Section 2.1.1 for a summary
of considerations for selecting oral studies for dose-response analysis. Overall, the NTP 2-year
drinking water study (NTP. 19951 was identified as the study most suitable for dose-response
assessment, given the study duration, comprehensive reporting of outcomes, use of multiple
species tested, multiple doses tested, and availability of a PBPK model for RTR extrapolation. This
study was discussed previously in Section 2.1.1 as part of the derivation of the oral reference dose,
so it is not reviewed here again. The NTP f!9971 subchronic inhalation study shares many
strengths with the 2-year drinking water study (NTP. 1995) and is described in more detail below.
NTP (19971 was a well-designed subchronic study that evaluated the effect of tert-butanol
exposure on multiple species at multiple inhalation doses. Relative kidney weights were elevated
in females at 6,368 mg/m3. Few endpoints were available for consideration in the subchronic
inhalation study, but changes in kidney weights also were observed in the oral studies, such as the
NTP T19951 2-year drinking water study.
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 considered consistent with underlying biological
processes to determine how best to model the dose-response relationship empirically in the range
of the observed data. Consistent with this approach, all models available in EPA's BMDS were
evaluated. Consistent with EPA's Benchmark Dose Technical Guidance fU.S. EPA. 2012al. the
benchmark dose or concentration (BMD/C) and the 95% lower confidence limit on the BMD/C
(BMD/CL) were estimated using a BMR of 10% change from the control mean for absolute
kidney-weight changes (as described in Section 2.1.2). As noted in Section 2.1.2, a BMR of 10%
extra risk was considered appropriate for the quantal data on incidences of kidney suppurative
inflammation and kidney transitional epithelial hyperplasia. The estimated BMD/CLs were used as
PODs. When dose-response modeling was not feasible, NOAELs or LOAELs were identified and
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summarized in Table 2-4. Further details, including the modeling output and graphical results for
the best-fit model for each endpoint, are found in Appendix C of the Supplemental Information.
Points of Departure (PODs) from Inhalation Studies
Because the RfC is applicable to a continuous lifetime human exposure but derived from
animal studies featuring intermittent exposure, EPA guidance (U.S. EPA. 1994) provides
mechanisms for (1) adjusting experimental exposure concentrations to a value reflecting
continuous exposure duration (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 BMCL from the inhalation study fNTP. 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:
BMCLadj = BMCL (mg/m3) x (6 -h 24) x (5 4 7) (2-3)
BMCL (mg/m3) x (0.1786)
The RfC methodology provides a mechanism for deriving an HEC from the
duration-adjusted POD (BMCLadj) determined from the animal data. The approach takes into
account the extrarespiratory 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 extrarespiratory effects were observed. Kaneko etal. f20001 measured an
animal blood:gas partition coefficient (Hb/g-A) of 531 ± 102 for tert-butanol in male Wistar rats,
while Borghoff et al. (1996) measured a value of 481 ± 29 in male F344 rats. A human blood:gas
partition coefficient (Hb/g-H) of 462 was reported for tert-butanol (Nihlen et al.. 1995). The
calculation Hb/g-A 4 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
coefficient for F344 rats was used. Thus, the calculation was 481 4 462 = 1.04. 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) (2-4)
= BMCLadj (mg/m3) x (481 4 462)
= BMCLadj (mg/m3) x (1.04)
Table 2-4 summarizes the sequence of calculations leading to the derivation of a human
equivalent POD for each inhalation data set discussed above.
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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 absolute
kidney weight at
13 wk
NTP (1997)
Rat (F344)/
female
No model
selectedd
10%
1,137
1,137
BMC = benchmark concentration.
aFor modeling details, see Appendix C in Supplemental Information.
bBMCs, BMCLs, and PODs were adjusted for continuous daily exposure by multiplying by (h exposed per
d/24 h) x (d exposed per wk/7 d).
cPODHec calculated by adjusting the PODAdj by the DAF (= 1.0, rounded from 1.04) for a Category 3 gas (U.S. EPA,
1994).
dBMD modeling failed to calculate a BMD value successfully (see Appendix C); POD calculated from NOAEL of
6,368 mg/m3.
Points of Departure (PODs) from Oral Studies—Use of Physiologically Based Pharmacokinetic
(PBPK) Model for Route-to-Route (RTR) Extrapolation
A PBPK model for tert-butanol in rats has been modified, as described in Appendix B of the
Supplemental Information. A critical decision in the RTR extrapolation is selection of the internal
dose metric that establishes "equivalent" oral and inhalation exposures. For tert-butanol-induced
kidney effects, the two options are the concentration of tert-butanol in blood and the 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 because the distribution
from blood to kidney is independent of route. Data are not available to suggest that metabolites of
tert-butanol mediate its renal toxicity. Without evidence suggesting otherwise, tert-butanol is
assumed to be the active toxicological agent Moreover, since extrapolation is within the same
species, use of the rate of metabolism as the metric (the alternate possibility) will only result in a
different value to the extent that there is nonlinearity (saturation) in the metabolism versus
concentration, and this differs for oral versus inhalation exposure. For example, for the internal
dose of 61.9 mg/L average blood concentration (CaVg, associated with a BMDL of 200 mg/kg-day
oral exposure), the corresponding average rate of metabolism is 0.83 mg/h (Mavg). If using CaVg as
the metric, the corresponding continuous inhalation concentration for the rat is 523.7 mg/m3. If
using Mavg as the metric, the corresponding continuous rat inhalation concentration is 439.9 mg/m,
only 16% lower. Hence, from a practical standpoint the choice between these two possible metrics
has little impact On the other hand, the use of metabolic rate has a higher degree of qualitative
uncertainty in that a submodel does not exist for the key metabolite(s) that can be used to estimate
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
its (their) internal concentration under different scenarios. The rate of metabolism is only inferred
by observing the rate of tert-butanol clearance; tert-butanol blood concentrations after various
exposures have been measured directly. Therefore, the concentration of tert-butanol in blood was
selected as the dose metric.
Using the PBPK model fU.S EPA. 20161. RTR extrapolation of the oral BMDLs or LOAEL to
derive inhalation PODs was therefore performed as follows. First, the internal dose in the rat at
each oral BMDL or LOAEL (assuming oral exposure by a circadian drinking water pattern) was
estimated using the PBPK model to derive an "internal dose BMDL or LOAEL." More specifically, for
noncontinuous exposures, the PBPK model was run for a number of days or weeks such that the
predicted time course of tert-butanol in blood did not change with further days or weeks simulated
(e.g., until blood concentration during the 2nd-to-last day of exposure was predicted to be the same
as the last day of exposure). This state is referred to as "periodicity." The average blood
concentration of tert-butanol was calculated during the final periodic exposure for oral exposure at
the BMDL for a given endpoint. For uniformity, all model scripts calculated the average from
episodic exposures on the basis of the final week of exposure, regardless of whether exposure is
daily or 5 times per week, because either exposure profile will be fully captured by averaging a
1-week time period.
For continuous inhalation exposures (24 hours/day, 7 days/week), the steady-state blood
concentration at the end of a simulation is equal to the average blood concentration for the last
week. Therefore, the continuous inhalation exposure equivalent to an oral BMDL was identified by
using the PBPK model to identify the inhalation concentration for which the final (steady-state)
blood concentration was equal to the average blood concentration for the last week of oral
exposure at the oral BMDL. The resulting POD then was converted to a PODhec using the
methodology previously described in the section, PODs from Inhalation Studies:
PODhec = POD (mg/m3) x (interspecies conversion) (2-5)
= POD (mg/m3) x (481 4- 462)
= POD (mg/m3) x (1.04)
Table 2-5 summarizes the sequence of calculations leading to the derivation of a human
equivalent inhalation POD from each oral data set discussed above.
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Table 2-5. Summary of inhalation points of departure derived from route-to-
route extrapolation from oral exposures
Endpoint and
reference
Species/sex
BMR
BMDL
(mg/kg-d)
Internal
dose3
(mg/L)
Equivalent
PODb
(mg/m3)
Equivalent
PODhec0
(mg/m3)
Kidney
Mean absolute
kidney weight at
15 mo NTP (1995)
Rat/female
10%
91
21.5
238.9
248
Kidney inflammation
(suppurative) at 2 yr
NTP (1995)
Rat/female
10%
200
61.9
523.7
545
Kidney transitional
epithelial hyperplasia
at 2 vr NTP (1995)
Rat/female
10%
339
127
883.9
919
Species/sex
POD (LOAEL;
mg/kg-d)
Internal
dose3
(mg/L)
Equivalent
PODb
(mg/m3)
Equivalent
PODhec0
(mg/m3)
Increases in severity
of nephropathy at
2 vr NTP (1995)
Rat/female
180
53.6
471.8
491
aAverage rodent blood concentration of te/t-butanol under circadian drinking water ingestion at the BMDL
Continuous inhalation equivalent concentration that leads to the same average blood concentration of
te/t-butanol as circadian drinking water ingestion at the BMDL in the rat.
Continuous inhalation human equivalent concentration that leads to the same average blood concentration of
te/t-butanol as continuous oral exposure at the BMDL Calculated as the rodent POD x 1.04.
To our knowledge, a meta-analysis of the accuracy of RTR extrapolation using PBPK models
has not been conducted. Ideally one would evaluate results for multiple chemicals, for which a
PBPK model and both oral and inhalation toxicity studies have been conducted, to determine the
accuracy of RTR extrapolation by comparing a predicted point of departure (e.g., BMD) with actual
data for the alternate route. For chloroform, use of a PBPK model has been shown to be successful
at correlating a response with internal dose irrespective of exposure route, including combined
inhalation and oral exposures fSasso etal.. 20131. Conversely, prenatal exposure of rats to inhaled
ethanol did not result in the degree of teratological effects expected, even though the internal dose
achieved (blood ethanol concentration [BEC]) was in the range associated with those effects when
ethanol is orally ingested (Oshiro etal.. 2014). In the latter case, although a PBPK model could
successfully predict the inhalation concentration of ethanol that yields a similar BEC to oral
exposure, the RTR extrapolation effectively failed because the same level of effect did not occur.
Thus, even for a well-studied compound like ethanol, the internal dose metric presumed to be
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
correct may in fact be inadequate for accurate extrapolation. Together these results indicate both
promise and uncertainty in RTR extrapolation.
Despite the uncertainty, a chemical must enter the body and be distributed through the
blood to affect internal tissues. Therefore, the toxicological activity must be related to blood
concentration, although it may not be exactly predicted by a particular metric. In the case of
tert-butanol, there is toxicological uncertainty because a chronic inhalation bioassay has not been
conducted. Thus, one must make a judgment as to whether it is more uncertain to extrapolate from
a subchronic inhalation bioassay or a chronic oral bioassay. Because a quantitative analysis of RTR
extrapolation across chemicals has not been conducted, it is not possible to quantitatively compare
the uncertainty of these two options. The EPA has assumed in this assessment that extrapolation
across study duration is more uncertain than extrapolation across exposure routes, given that
toxicity must be related to the concentration of tert-butanol in the blood.
2.2.3. Derivation of Candidate Values
In EPA's A Review of the Reference Dose and Reference Concentration Processes [U.S. EPA
f20021: see Section 4.4.5], also described in the Preamble, five possible areas of uncertainty and
variability were considered. Several PODs for the candidate inhalation values were derived using a
RTR extrapolation from the PODs estimated from the chronic oral toxicity study in rats fNTP. 19951
in deriving the RfD (see Section 2.1). Except for the subchronic inhalation study fNTP. 19971. the
UF values selected and applied to PODs derived from the chronic oral study (NTP. 1995) for RTR
extrapolation are the same as those for the RfD for tert-butanol (see Section 2.1.3). The model used
to perform this RTR extrapolation is a well-characterized model considered appropriate for the
purposes of this assessment
For the PODs derived from the subchronic inhalation study (NTP. 1997). a UFs of 10 was
applied to account for extrapolation from subchronic-to-chronic duration.
Table 2-6 is a continuation of Table 2-4 and Table 2-5, and summarizes the application of UF
values to each POD to derive a candidate value for each data set. The candidate values presented in
the table below are preliminary to the derivation of the organ/system-specific reference values.
These candidate values are considered individually in selecting a representative reference value for
inhalation for a specific hazard and subsequent overall RfC for tert-butanol.
Figure 2-2 presents graphically the candidate values, UF values, and PODhec values, with
each bar corresponding to one data set described in Table 2-4, Table 2-5, and Table 2-6.
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Table 2-6. Effects and corresponding derivation of candidate values
Endpoint (sex and
species) and reference
PODhec
(mg/m3)
POD
type
ufa
UFh
UFl
UFS
UFd
Composite
UF
Candidate
value
(mg/m3)
Kidney
Increased absolute kidney
weight at 13 wk; female rat
NTP (1997)
1,137
NOAEL
3
10
1
10
1
300
4x 10°
Increased absolute kidney
weight at 15 mo; female rat
NTP (1995)
248
BMCLio
3
10
1
1
1
30
8 x 100a
Kidney inflammation
(suppurative); female rat at
2 yr
NTP (1995)
546
BMCLio
3
10
1
1
1
30
2 x 10la
Kidney transitional
epithelial hyperplasia;
female rat at 2 yr
NTP (1995)
920
BMCLio
3
10
1
1
1
30
3 x 10la
Increases in severity of
nephropathy; female rat at
2 yr
NTP (1995)
491
LOAEL
3
10
3
1
1
100
5 x 100a
aThese candidate values are derived using RTR extrapolated PODs based on NTP's chronic drinking water study.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
T Absolut* kidnty w*d|ht
at 13 wmIui ftmal*
(NTP, 1997)
T Absolute kidney weight
at 15 months; female rat
[NTP, 1995]
Kidney inflammatio ti
[suppurativa] i f*nval* rat
[NTP, 1995]
Kidney tra national
•plth#l lal hypurplai la;
female rat [NTP, 1995]
Nephropathy severity;
fanaW rat [NTP, 1995]
^ Candidate RfC
• PODhk
Composite UF
ID
100
1000
10000
ing/m3
Figure 2-2. Candidate inhalation reference concentration (RfC) values with
corresponding point of departure (POD) and composite uncertainty factor
(UF).
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
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/system-specific reference values can be useful for subsequent cumulative risk assessments
that consider the combined effect of multiple agents acting at a common site.
Kidney Toxicity
For the derivation of candidate values, whether PODs from the subchronic inhalation study
of NTP (19971 would provide a better basis than the RTR extrapolated PODs based on the chronic
oral study of NTP f!9951 must be considered. To estimate an exposure level below which kidney
toxicity from tert-butanol exposure is not expected to occur, the RfC for increases in severity of
nephropathy in female rats (5 x 10° mg/m3) was selected as the kidney-specific RfC for
tert-butanol, consistent with the selection of the kidney-specific RfD (see Section 2.1.4).
Justification for the selection of increased severity of nephropathy is discussed in Section 2.1.4.
Similar to the RfD confidence in this kidney-specific RfC is medium.
Table 2-7. Organ/system-specific inhalation reference concentrations (RfCs)
and overall RfC for tert-butanol
Effect
Basis
RfC
(mg/m3)a
Study exposure
description
Confidence
Kidney
Increases in severity of
nephropathy (NTP, 1995)
5x 10°
Chronic
Medium
Overall RfC
Kidney
5x10°
Chronic
Medium
aDerived from oral study, by RTR extrapolation.
2.2.5. Selection of the Overall Reference Concentration
For tert-butanol, kidney effects were identified as the primary hazard; thus, a single
organ/system-specific RfC was derived. The kidney-specific RfC of 5 x 10° mg/m3 is selected as
the overall RfC, representing an estimated exposure level below which deleterious effects from
tert-butanol exposure are not expected to occur.
The overall RfC is derived to be protective of all types of effects for a given duration of
exposure and is intended to protect the population as a whole, including potentially susceptible
subgroups fU.S. EPA. 20021. Decisions concerning averaging exposures over time for comparison
with the RfC should consider the types of toxicological effects and specific lifestages of concern.
Fluctuations in exposure levels that result in elevated exposures during these lifestages could lead
to an appreciable risk, even if average levels over the full exposure duration were less than or equal
to the RfC. The potential exists for early lifestage susceptibility to tert-butanol exposure, as
discussed in Section 1.3.3.
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2.2.6. Confidence Statement
A confidence level of high, medium, or low is assigned to the study used to derive the RfC,
the overall evidence base, and the RfC itself, as described in Section 4.3.9.2 of EPA's Methods for
Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry fU.S. EPA.
1994). A PBPK model was used to perform an RTR extrapolation to determine a POD for deriving
the RfC from the NTP (1995) oral study and corresponding critical effect. Confidence in the
principal study (NTP. 1995) is high. This study was well conducted, complied with FDA GLP
regulations, involved 50 animals per group (including both sexes), and assessed a wide range of
tissues and endpoints. Although the toxicity evidence base for tert-butanol contains some gaps,
these areas are partially informed by the data on ETBE, a parent compound of tert-butanol.
Therefore, the confidence in the evidence base is medium. Reflecting high confidence in the
principal study, medium confidence in the evidence base, and minimal uncertainty surrounding the
application of the modified PBPK model for the purposes of an RTR extrapolation, the overall
confidence in the RfC for tert-butanol is medium.
2.2.7. Previous Integrated Risk Information System (IRIS) Assessment
No previous inhalation assessment for tert-butanol is available in IRIS.
2.2.8. Uncertainties in the Derivation of the Reference Dose and Reference Concentration
The following discussion identifies uncertainties associated with the RfD and RfC for
tert-butanol. To derive the RfD, the UF approach (U.S. EPA. 2000.1994) was applied to a POD based
on kidney toxicity in rats treated chronically. UF values were applied to the POD to account for
extrapolating from an animal bioassay to human exposure, and the likely existence of a diverse
human population of varying susceptibilities. These extrapolations are carried out with default
approaches, given the lack of data to inform individual steps. To derive the RfC, this same approach
was applied, but a PBPK model was used to extrapolate from oral to inhalation exposure.
The evidence base for tert-butanol contains no human data on adverse health effects from
subchronic or chronic exposure, and the PODs were calculated from data on the effects of
tert-butanol reported by studies in rats. The evidence base for tert-butanol exposure includes one
lifetime bioassay, several reproductive/developmental studies, and several subchronic oral studies.
Although the evidence base is adequate for reference value derivation, uncertainty is
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
fetal loss, decreases in fetal body weight, and possible increases in skeletal variations in exposed
offspring or pups. Effects were not always consistent across exposure routes, however, and
maternal 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. The tert-butanol evidence base
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lacks an adequate model that would inform potential interspecies differences (a limited data set
exists for tert-butanol appearing as a metabolite from ETBE exposure in humans, but none for
direct exposure to tert-butanol). Generally, rats seemed more susceptible than mice, and males
more susceptible than females to tert-butanol toxicity. The underlying mechanistic basis of these
apparent differences, however, is not understood. Most importantly, which animal species or sexes
might be more comparable to humans is unknown.
Another uncertainty to consider relates to the mode-of-action (MOA) analysis conducted for
the kidney effects. The assessment concluded that tert-butanol is a weak inducer of alpha
2u-globulin, which is operative in male kidney tumors; therefore, noncancer effects related to alpha
2u-globulin were considered not relevant for hazard identification and, therefore, not suitable for
dose-response consideration. If this conclusion were incorrect and the noncancer effects
characterized in this assessment as being related to alpha 2u-globulin were relevant to humans, the
RfD and RfC values could underestimate toxicity. The assessment also used noncancer effects
related to CPN in deriving the reference values. If noncancer effects characterized in this
assessment as being related to CPN were not relevant to humans, the RfD value (0.4 mg/kg-day)
could slightly overestimate toxicity compared with an alternative endpoint (increased absolute
kidney weight [0.7 mg/kg-day]), while the RfC value would be similar (5 mg/m3 compared with
4 mg/m3).
2.3. ORAL SLOPE FACTOR FOR CANCER
The oral slope factor (OSF) is a plausible upper bound on the estimate of risk per
mg/kg-day of oral exposure. The OSF can be multiplied by an estimate of lifetime exposure (in
mg/kg-day) to estimate the lifetime cancer risk.
2.3.1. Analysis of Carcinogenicity Data
As noted in Section 1.3.2, there is suggestive evidence of carcinogenic potential for
tert- butanol. The Guidelines for Carcinogen Risk Assessment (U.S. EPA. 2005b) state:
When there is suggestive evidence, the Agency generally would not attempt a
dose-response assessment, as the nature of the data generally would not support
one; however, when the evidence includes a well-conducted study, quantitative
analysis may be useful for some purposes, for example, providing a sense of the
magnitude and uncertainty of potential risks, ranking potential hazards, or setting
research priorities. In each case, the rationale for the quantitative analysis is
explained, considering the uncertainty in the data and the suggestive nature of the
weight of evidence. These analyses generally would not be considered Agency
consensus estimates.
No human data relevant to an evaluation of the carcinogenicity of tert-butanol were
available. The cancer descriptor was based on the 2-year drinking water study in rats and mice by
NTP (1995). which reported renal tumors in male rats and thyroid tumors in both male and female
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mice. This study was considered suitable for dose-response analysis. It was conducted in
accordance with FDA 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 reexamined by a Pathology Working
Group (Hard etal.. 20111.
Dose-related increasing trends in tumors were noted at the following sites:
• Renal tubule adenomas and carcinomas in male rats and
• Thyroid follicular adenomas in female mice and thyroid follicular adenomas and carcinoma
in male mice.
These tumors were statistically significantly increased by pairwise comparison (Fisher's
exact test, p < 0.05) and by trend test (Cochran-Armitage trend test, p < 0.05). Based on a mode of
action analysis, the alpha 2u-globulin process, in addition to other unknown processes, was
concluded to be at least partially responsible for the male rat renal tumors. Because the relative
contribution of each process to tumor formation cannot be determined fU.S. EPA. 1991al. the renal
tumors in male rats are not considered suitable for quantitative analysis. Conversely, the thyroid
tumors in mice are suitable for dose-response analysis and unit risk estimation, as described in
Section 1.3.2, and the available data do not demonstrate that the thyroid tumors are the result of
excessive toxicity in female mice. The final average body-weight reduction in female mice at the
highest dose was 12% fNTP. 19951. but water consumption by exposed females was similar to
controls and no overt toxicity was observed. Furthermore, female mice in the high-dose group had
higher rates of survival than control animals. The final average body weight reduction in male mice
at the highest dose was 5% to 10% (NTP. 1995) and water consumption by exposed males was
similar to controls, but survival was reduced at the highest dose and the tumor response in male
mice was adjusted for early mortality. Considering these data, along with the uncertainty associated
with the suggestive nature of the weight of evidence, quantitative analysis of the tumor data may be
useful for providing a sense of the magnitude of potential carcinogenic risk from tert-butanol
exposure, including worker and consumer exposures. Although this assessment determined that
the female mouse thyroid data set is suitable for dose-response modeling and calculation of a
quantitative risk estimate, there is increased uncertainty in this risk estimate owing to the
suggestive nature of the tumorigenic response (U.S. EPA. 2005a).
2.3.2. Dose-Response Analysis—Adjustments and Extrapolations Methods
The EPA Guidelines for Carcinogen Risk Assessment fU.S. EPA. 2005bl recommend the
method for characterizing and quantifying cancer risk from a chemical be based on what is known
about the MOA of the carcinogen and the shape of the cancer dose-response curve. EPA uses a
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
two-step approach that distinguishes analysis of the observed dose-response data from inferences
about lower doses fU.S. EPA. 2005bl Within the observed range, the preferred approach is to use
modeling to incorporate a wide range of data into the analysis, such as through a biologically based
model, if supported by substantial data. Without a biologically based model, as in the case of
tert-butanol, a standard model is used for curve fitting the data and estimating a POD. EPA uses the
multistage model in IRIS dose-response analyses for cancer (Gehlhaus etal.. 2011) because it
parallels the multistage carcinogenic process and fits a broad array of dose-response patterns.
The second step, extrapolation to lower exposures from the POD, considers what is known
about the modes of action for each effect As above, a biologically based model is preferred fU.S.
EPA. 2005bl Otherwise, linear low-dose extrapolation is recommended if the MOA of
carcinogenicity is mutagenic or has not been established fU.S. EPA. 2005bl For tert-butanol, the
mode(s) of carcinogenic action for thyroid follicular cell tumors has not been established (see
Section 1.3.2). Therefore, linear low-dose extrapolation was used to estimate human carcinogenic
risk.
The dose-response modeling used administered dose because a PBPK model to characterize
internal dosimetry in mice was not available. For the analysis of thyroid tumors, the incidence data
were adjusted to account for the increased mortality in high-dose male mice, relative to the other
groups, that reduced the number of mice at risk for developing tumors. The Poly-3 method (Bailer
and Portier. 1988) was used to estimate the number at risk of developing tumors, by weighting the
length of time each animal was on study (details in Appendix C of the Supplemental Information).
This method was not applied to the female mice data because a difference in survival with
increasing exposure was not appreciable and only one tumor, in the high-dose group, occurred
before study termination.
The data modeled and other details of the modeling are provided in Appendix C. The BMDs
and BMDLs recommended for each data set are summarized in Table 2-8. The modeled
tert-butanol PODs were scaled to HEDs according to EPA guidance (U.S. EPA. 2011. 2005b). In
particular, the BMDL was converted to an HED by assuming that doses in animals and humans are
toxicologically equivalent when scaled by BW3/4. Standard body weights of 0.025 kg for mice and
70 kg for humans were used (U.S. EPA. 1988). The following formula was used for converting an
oral BMDL to oral HED for mouse endpoints:
HED in mg/kg-day = (BMDL in mg/kg-day) x (animal body weight/70)1/4 (2-6)
= (BMDL in mg/kg-day) x 0.14
PODs for estimating low-dose risk were identified at doses at the lower end of the observed
data, corresponding to 10% extra risk in female mice and 5% extra risk in male mice.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
2.3.3. Derivation of the Oral Slope Factor
The PODs estimated for each tumor data set are summarized in Table 2-8. The lifetime oral
cancer slope factor 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 (slope factor = BMR/BMDLbmr = 0.1/BMDLio). This
slope represents a plausible upper bound on the true population average risk. Using linear
extrapolation from the BMDLio, human equivalent oral slope factors were derived for male and
female mice and are listed in Table 2-8.
The oral slope factor based on the incidence of thyroid follicular cell adenomas in female
mice was 5 x 10~4 per mg/kg-day. Despite high mortality in high-dose male mice, estimating slope
factors using the Poly-3 method was feasible for addressing competing risks. Whether using the
full data set (including the only thyroid follicular cell carcinoma observed at the highest dose) or
omitting the high-dose group altogether (under the assumption that mortality in this group was too
extensive to interpret the results), oral slope factors based on the incidence of thyroid follicular cell
adenomas or carcinomas in male mice were similar when rounded to one significant digit
(5 x 10"4 per mg/kg-day or 6 x 10~4 per mg/kg-day, respectively).
The recommended slope factor11 for lifetime oral exposure to tert-butanol is
5 x 10-4 per mg/kg-day, based on the thyroid follicular cell adenoma or carcinoma response in
male or female B6C3F1 mice. This slope factor should not be used with exposures exceeding
1,400 mg/kg-day, the highest POD from the two data sets, because above this level the cancer risk
might not increase linearly with exposure. The slope of the linear extrapolation from the central
estimate BMDiohed derived from the female mouse data set is
0.1 4- [0.14 x (2,002 mg/kg-day)] = 4 x 10"4per mg/kg-day.
1 'This value is notably uncertain because it is based on a determination of suggestive evidence of carcinogenic
potential; however, the value may be useful for some decision purposes such as providing a sense of the
magnitude of potential risks or ranking potential hazards (U.S. EPA. 2005a). The uncertainties in the data
leading to this suggestive weight of evidence determination for carcinogenicity are detailed in Section 2.3.4.
below.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Table 2-8. Summary of the oral slope factor derivation
Tumor
Species/sex
Selected
model
BMR
BMD
(mg/kg-d)
POD =
BMDL
(mg/kg-d)
BMDLhed3
(mg/kg-d)
Slope factor15
(mg/kg-d)"1
Thyroid follicular
cell adenoma
B6C3F1
mouse/female
3°
Multistage
10%
2,002
1,437
201
5 x 10"4
Thyroid follicular
cell adenoma or
carcinoma
B6C3F1
mouse/male
All dose
groups: 1°
Multistage
5%c
1,788
787
110
5 x 10"4
High dose
omitted: 2°
Multistage
5%c
1,028
644
90
6 x 10"4
aHED PODs were calculated using BW3/4 scaling (U.S. EPA, 2011).
bHuman equivalent slope factor = 0.1/BMDLiohed; see Appendix C of the Supplemental Information for details of
modeling results. These values are notably uncertain because it is based on a determination of suggestive
evidence of carcinogenic potential; however, the slope factors may be useful for some decision purposes such as
providing a sense of the magnitude of potential risks or ranking potential hazards (U.S. EPA, 2005a). The
uncertainties in the data leading to this suggestive weight of evidence determination for carcinogenicity are
detailed in Section 2.3.4. below.
cBecause the observed responses were <10%, a BMR of 5% was used to represent the observed response range
for low-dose extrapolation; human equivalent slope factor = 0.05/BMDL5hed-
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.
Table 2-9 summarizes several uncertainties that could affect the oral slope factor. There are
no other chronic studies to replicate these findings or that examined other animal models, no data
in humans to confirm a cancer response in general or the specific tumors observed in the NTP
f!9951 bioassay, and no other data (e.g., MOA) to support alternative approaches for deriving the
oral slope factor.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Table 2-9. Summary of uncertainties in the derivation of the oral slope factor
for tert-butanol
Consideration and
impact on cancer risk value
Decision
Justification
Selection of tumor type and
relevance to humans:
Mouse thyroid tumors are the basis
for estimating human cancer risk,
because the fraction of rat kidney
tumors not attributed to the male
rat-specific alpha 2n-globulin
process could not be determined.
Alternatively, quantifying rat kidney
tumors could T* slope factor to
1 x 10"2 mg/kg-d (see Appendix C,
Supplemental Information)
Thyroid tumors in female
and male mice were
selected, U.S. EPA (1998a),
U.S. EPA (1991a)
MOA data suggested that mouse thyroid
tumors were relevant to humans.
Quantitation of thyroid tumors in male mice,
which was affected only slightly by high
mortality in the high-dose group, supports
the estimate based on female mice.
Selection of data set:
No other studies are available
NTP (1995), oral (drinking
water) study, was selected
to derive cancer risks for
humans
NTP (1995), the onlv chronic bioassav
available, was a well-conducted study.
Additional bioassays might support the
findings, help determine what fraction of
kidney tumors are not attributable to the
alpha 2n-globulin process, or provide results
for different (possibly lower) doses, which
would affect (possibly increase) the oral slope
factor.
Selection of dose metric:
Alternatives could 4^ or T* slope
factor
Used administered dose
For mice, PBPK-estimated internal doses
could affect the OSF value for thyroid tumors
if the carcinogenic moiety is not proportional
to administered dose, but no PBPK model
was available, and no information is available
to suggest if any metabolites elicit
carcinogenic effects.
Interspecies extrapolation of
dosimetry and risk:
Alternatives could 4^ or T* slope
factor (e.g., 3.5-fold 4^ [scaling by
body weight] or 2-fold T* [scaling by
BW2/3])
Default approach of body
weight3'4 was used
No data to suggest an alternative approach
for tert-butanol. 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. Although the true
human correspondence is unknown, this
overall approach is expected to neither
over- or underestimate human equivalent
risks.
Dose-response modeling:
Alternatives could 4/ or T* slope
factor
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.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
Table 2-9. Summary of uncertainties in the derivation of the oral slope factor
for tert-butanol (continued)
Consideration and
impact on cancer risk value
Decision
Justification
Low-dose extrapolation:
4/ cancer risk estimate would be
expected by applying nonlinear
low-dose extrapolation
Linear extrapolation of risk
in low-dose region used
U.S. EPA (1998a)
Linear low-dose extrapolation for agents
without a known MOA is supported (U.S. EPA,
2005b) and recommended for rodent thvroid
tumors arising from an unknown MOA (U.S.
EPA, 1998a).
Statistical uncertainty at POD:
4/ oral slope factor 1.4-fold if BMD
used as the POD rather than BMDL
BMDL (preferred approach
for calculating slope factor)
Limited size of bioassay results in sampling
variability; lower bound is 95% confidence
interval on administered exposure at 10%
extra risk of thyroid 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 is not
known, an age-specific adjustment factor is
not applied.
2.3.5. Previous Integrated Risk Information System (IRIS) Assessment
No previous cancer assessment for tert-butanol is available in IRIS.
2.4. INHALATION UNIT RISK FOR CANCER
The carcinogenicity assessment provides information on the carcinogenic hazard potential
of the substance in question, and quantitative estimates of risk from oral and inhalation exposure
can be derived. Quantitative risk estimates can be derived from the application of a low-dose
extrapolation procedure. If derived, the inhalation unit risk is a plausible upper bound on the
estimate of risk per |J.g/m3 air breathed.
No chronic inhalation exposure studies to tert-butanol are available. Lifetime oral exposure
has been associated with increased renal tubule adenomas and carcinoma in male F344 rats,
increased thyroid follicular cell adenomas in female B6C3F1 mice, and increased thyroid follicular
cell adenomas and carcinomas in male B6C3F1 mice. Although an oral slope factor was derived for
tert-butanol based on increased thyroid tumors in mice; no mouse PBPK model exists for
tert-butanol; therefore, RTR extrapolation (oral to inhalation) cannot be performed for thyroid
tumors in mice at this time. The NTP (1995) drinking water study in rats and mice was the only
chronic bioassay available for dose-response analysis. Still, the rat PBPK model and kidney tumors
from the NTP (1995) drinking water study were not used for RTR extrapolation or the derivation
of an OSF because enough information to determine the relative contribution of alpha 2u-globulin
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
nephropathy and other processes to the overall renal tumor response fU.S. EPA. 1991a1 is not
available. As stated in fU.S. EPA. 1991al "if some tumors are attributable to the alpha 2u-globulin
process and some are attributable to other processes, 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 enough information is available to determine the relative contribution of each
process to the overall tumor response."
2.4.1. Previous Integrated Risk Information System (IRIS) Assessment
An inhalation cancer assessment for tert-butanol was not previously available on IRIS.
2.5. APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS
As discussed in the Supplemental Guidance for Assessing Susceptibility from Early-Life
Exposure to Carcinogens (U.S. EPA. 2005c). either default or chemical-specific age-dependent
adjustment factors (ADAFs) are recommended to account for early-life exposure to carcinogens
that act through a mutagenic MOA. Because chemical-specific lifestage susceptibility data for
cancer are not available, and because the MOA for tert-butanol carcinogenicity is not known (see
Section 1.3.2), application of ADAFs is not recommended.
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Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
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