vycrM
EPA/635/R-20/105a
Final Agency and Interagency Draft
www.epa.gov/iris
Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
(CAS No. 75-65-0]
July 2020
NOTICE
This document is a Final Agency and Interagency Draft. It has not been formally disseminated by
EPA. It does not represent and should not be construed to represent any Agency determination or
policy. It is being circulated for review of its technical accuracy and science policy implications.
Integrated Risk Information System
Center for Public Health and Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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Toxicological Review oftert-Butyl Alcohol
1 DISCLAIMER
2 This document is a preliminary draft for review purposes only. This information is
3 distributed solely for the purpose of predissemination peer review under applicable information
4 quality guidelines. It has not been formally disseminated by EPA. It does not represent and should
5 not be construed to represent any Agency determination or policy. Mention of trade names or
6 commercial products does not constitute endorsement or recommendation for use.
7
This document is a draft for review purposes only and does not constitute Agency policy.
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CONTENTS
AUTHORS | CONTRIBUTORS | REVIEWERS viii
PREFACE xi
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS xv
EXECUTIVE SUMMARY xxiii
LITERATURE SEARCH STRATEGY | STUDY SELECTION AND EVALUATION 1-1
1 HAZARD IDENTIFICATION 1-10
1.1 OVERVIEW OF CHEMICAL PROPERTIES AND TOXICOKINETICS 1-10
1.1.1 Chemical Properties 1-10
1.1.2 Toxicokinetics 1-11
1.1.3 Description of Toxicokinetic Models 1-12
1.1.4 Chemicals Extensively Metabolized to ferf-Butanol 1-13
1.2 PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM 1-13
1.2.1 Kidney Effects 1-13
1.2.2 Thyroid Effects 1-52
1.2.3 Developmental Effects 1-61
1.2.4 Neurodevelopmental Effects 1-68
1.2.5 Reproductive Effects 1-71
1.2.6 Other Toxicological Effects 1-77
1.3 INTEGRATION AND EVALUATION 1-77
1.3.1 Effects Other Than Cancer 1-77
1.3.2 Carcinogenicity 1-79
1.3.3 Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes 1-82
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
This document is a draft for review purposes only and does not constitute Agency policy.
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2.1.6 Confidence Statement 2-9
2.1.7 Previous IRIS Assessment 2-9
2.2 INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER THAN CANCER 2-9
2.2.1 Identification of Studies and Effects for Dose-Response Analysis 2-9
2.2.2 Methods of Analysis 2-10
2.2.3 Derivation of Candidate Values 2-15
2.2.4 Derivation of Organ/System-Specific Reference Concentrations 2-17
2.2.5 Selection of the Overall Reference Concentration 2-18
2.2.6 Confidence Statement 2-19
2.2.7 Previous IRIS Assessment 2-19
2.2.8 Uncertainties in the Derivation of the Reference Dose and Reference Concentration..2-19
2.3 ORAL SLOPE FACTOR FOR CANCER 2-20
2.3.1 Analysis of Carcinogenicity Data 2-20
2.3.2 Dose-Response Analysis—Adjustments and Extrapolations Methods 2-22
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 IRIS Assessment: Oral Slope Factor 2-26
2.4 INHALATION UNIT RISK FOR CANCER 2-26
2.4.1 Previous IRIS Assessment: Inhalation Unit Risk 2-26
2.5 APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS 2-27
REFERENCES R-l
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review oftert-Butyl Alcohol
TABLES
Table ES-1. Organ/system-specific RfDs and overall RfD for tert-butanol xxiv
Table ES-2. Organ/system-specific RfCs and overall RfCfor ferf-butanol xxv
Table LS-1. Details of the search strategy employed for ferf-butanol 1-4
Table LS-2. Summary of additional search strategies for ferf-butanol 1-4
Table LS-3. Inclusion-exclusion criteria 1-5
Table LS-4. Considerations for evaluation of experimental animal studies 1-8
Table LS-5. Summary of experimental animal evidence base 1-8
Table 1-1. Physicochemical properties and chemical identity of ferf-butanol 1-10
Table 1-2. Changes in kidney histopathology in animals following exposure to ferf-butanol 1-21
Table 1-3. Changes in kidney tumors in animals following exposure to ferf-butanol 1-24
Table 1-4. Comparison of nephropathy and suppurative inflammation in individual male rats
from the 2-year NTP ferf-butanol bioassay 1-26
Table 1-5. Comparison of nephropathy and suppurative inflammation in individual female rats
from the 2-year NTP ferf-butanol bioassay 1-26
Table 1-6. Comparison of nephropathy and transitional epithelial hyperplasia in individual male
rats from the 2-year NTP ferf-butanol bioassay 1-26
Table 1-7. Comparison of nephropathy and transitional epithelial hyperplasia in individual
female rats from the 2-year NTP ferf-butanol bioassay 1-27
Table 1-8. Comparison of CPN and renal tubule hyperplasia with kidney adenomas and
carcinomas in male rats from the 2-year NTP ferf-butanol bioassay 1-27
Table 1-9. Summary of data on the alpha2u-globulin process in male rats exposed to ferf-
butanol 1-33
Table 1-10. International Agency for Research on Cancer (IARC) criteria for an agent causing
kidney tumors through an alpha2u-globulin associated response in male
rats 1-41
Table 1-11. Proposed empirical criteria for attributing renal tumors to CPN 1-47
Table 1-12. Evidence pertaining to thyroid effects in animals following oral exposure to ferf-
butanol 1-53
Table 1-13. Evidence pertaining to developmental effects in animals following exposure to ferf-
butanol 1-63
Table 1-14. Evidence pertaining to neurodevelopmental effects in animals following exposure to
ferf-butanol 1-70
Table 1-15. Evidence pertaining to reproductive effects in animals following exposure to ferf-
butanol 1-72
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 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 derivation of inhalation points of departure derived from route-to-route
extrapolation from oral exposures 2-13
Table 2-6. Effects and corresponding derivation of candidate values 2-15
Table 2-7. Organ-/system-specific 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
This document is a draft for review purposes only and does not constitute Agency policy.
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FIGURES
Figure LS-1. Summary of literature search and screening process for ferf-butanol 1-3
Figure 1-1. Biotransformation of ferf-butanol in rats and humans 1-12
Figure 1-2. Comparison of absolute kidney weight change in male and female rats across oral
and inhalation exposure based on internal blood concentration 1-19
Figure 1-3. Comparison of absolute kidney weight change in male and female mice following oral
exposure based on administered concentration 1-20
Figure 1-4. Comparison of absolute kidney weight change in male and female mice following
inhalation exposure based on administered concentration 1-20
Figure 1-5. Exposure response array for kidney effects following oral exposure to ferf-butanol 1-28
Figure 1-6. Exposure-response array of kidney effects following inhalation exposure to ferf-
butanol (13-week studies, no chronic studies available) 1-29
Figure 1-7. Temporal pathogenesis of alpha2u-globulin -associated nephropathy in male rats 1-32
Figure 1-8. Exposure-response array for effects potentially associated with alpha2u-globulin
renal tubule nephropathy and tumors in male rats after oral exposure to ferf-
butanol 1-35
Figure 1-9. Exposure-response array for effects potentially associated with alpha2u-globulin
renal tubule nephropathy and tumors in male rats after inhalation exposure to
ferf-butanol 1-36
Figure 1-10. Exposure-response array of thyroid follicular cell effects following chronic oral
exposure to ferf-butanol 1-56
Figure 1-11. Exposure-response array of developmental effects following oral exposure to ferf-
butanol 1-66
Figure 1-12. Exposure-response array of developmental effects following inhalation exposure to
ferf-butanol 1-67
Figure 1-13. Exposure-response array of reproductive effects following oral exposure to ferf-
butanol 1-75
Figure 1-14. Exposure-response array of reproductive effects following inhalation exposure to
ferf-butanol 1-76
Figure 2-1. Candidate values with corresponding POD and composite UF. Each bar corresponds
to one data set described in Table 2-1 and Table 2-2 2-7
Figure 2-2. Candidate RfC values with corresponding POD and composite UF 2-17
This document is a draft for review purposes only and does not constitute Agency policy.
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1 ABBREVIATIONS
2
AIC
Akaike's information criterion
MNPCE
micronucleated polychromatic
ALD
approximate lethal dosage
erythrocyte
ALT
alanine aminotransferase
MTD
maximum tolerated dose
AST
aspartate aminotransferase
NAG
N-acetyl-p-D-glucosaminidase
atm
atmosphere
NCEA
National Center for Environmental
ATSDR
Agency for Toxic Substances and
Assessment
Disease Registry
NCI
National Cancer Institute
BMD
benchmark dose
NOAEL
no-observed-adverse-effect level
BMDL
benchmark dose lower confidence limit
NTP
National Toxicology Program
BMDS
Benchmark Dose Software
NZW
New Zealand White (rabbit breed)
BMR
benchmark response
OCT
ornithine carbamoyl transferase
BW
body weight
ORD
Office of Research and Development
CA
chromosomal aberration
PBPK
physiologically based pharmacokinetic
CASRN
Chemical Abstracts Service Registry
POD
point of departure
Number
POD [AD J]
duration-adjusted POD
CBI
covalent binding index
QSAR
quantitative structure-activity
CHO
Chinese hamster ovary (cell line)
relationship
CL
confidence limit
RDS
replicative DNA synthesis
CNS
central nervous system
RfC
inhalation reference concentration
CPN
chronic progressive nephropathy
RfD
oral reference dose
CYP450
cytochrome P450
RGDR
regional gas dose ratio
DAF
dosimetric adjustment factor
RNA
ribonucleic acid
DEN
diethylnitrosamine
SAR
structure activity relationship
DMSO
dimethylsulfoxide
SCE
sister chromatid exchange
DNA
deoxyribonucleic acid
SD
standard deviation
EPA
Environmental Protection Agency
SDH
sorbitol dehydrogenase
FDA
Food and Drug Administration
SE
standard error
FEVi
forced expiratory volume of 1 second
SGOT
glutamic oxaloacetic transaminase, also
GD
gestation day
known as AST
GDH
glutamate dehydrogenase
SGPT
glutamic pyruvic transaminase, also
GGT
y-glutamyl transferase
known as ALT
GSH
glutathione
SSD
systemic scleroderma
GST
glutathione-S-transferase
TCA
trichloroacetic acid
Hb/g-A
animal blood:gas partition coefficient
TCE
trichloroethylene
Hb/g-H
human blood:gas partition coefficient
TWA
time-weighted average
HEC
human equivalent concentration
UF
uncertainty factor
HED
human equivalent dose
UFa
animal-to-human uncertainty factor
i.p.
intraperitoneal
UFh
human variation uncertainty factor
IRIS
Integrated Risk Information System
UFl
LOAEL-to-NOAEL uncertainty factor
IVF
in vitro fertilization
UFs
subchronic-to-chronic uncertainty
LC50
median lethal concentration
factor
LD50
median lethal dose
UFd
database deficiencies uncertainty factor
LOAEL
lowest-observed-adverse-effect level
U.S.
United States
MN
micronuclei
This document is a draft for review purposes only and does not constitute Agency policy.
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1
2
AUTHORS | CONTRIBUTORS | REVIEWERS
Assessment Team
Janice S. Lee, Ph.D. (Chemical Manager)
James A. Weaver (Co-Chemical
Manager)
Kathleen Newhouse, M.S.*
U.S. EPA
Office of Research and Development
Center for Public Health and Environmental
Assessment
Research Triangle Park, NC
*Seattle, WA
Keith Salazar, Ph.D.
Former Co-Chemical Manager
EPA/ORD/CPHEA
Currently with U.S. EPA, Office of Chemical Safety and
Pollution Prevention, Office of Pollution Prevention and
Toxics
Washington, DC
Chris Brinkerhoff, Ph.D.
Contributors
Andrew Hotchkiss, Ph.D.
Channa Keshava, Ph.D.
Amanda Persad, Ph.D.
Catherine Gibbons, Ph.D. *
Samantha Jones, Ph.D. *
Christine Cai, M.S.*
Karen Hogan, M.S.*
Paul Schlosser, Ph.D.
Alan Sasso, 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
U.S. EPA
Office of Research and Development
Center for Public Health and Environmental Assessment
Research Triangle Park, NC
*Washington, DC
Vincent Cogliano, Ph.D.
Jason Fritz, Ph.D.
(previously with) U.S. EPA National Center for
Environmental Assessment
Charles Wood, Ph.D.
(previously with) U.S. EPA National Health and
Environmental Effects Research Lab
Production Team
Hillary Hollinger
Ryan Jones
Samuel Thacker
Erin Vining
Vicki Soto
Dahnish Shams
Maureen Johnson
U.S. EPA
Office of Research and Development
Center for Public Health and Environmental
Assessment
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review oftert-Butyl Alcohol
Contractor Support
Robyn Blain, Ph.D. ICF
Michelle Cawley, M.L.S., M.A.* Fairfax, VA
William Mendez, Jr., Ph.D. *Research Triangle Park, NC
Pam Ross, M.S.P.H.
Cara Henning, Ph.D. *
Tao Hong, Ph.D.
Ami Gordon, M.P.H.
Executive Direction
Wayne Cascio, M.D. (CPHEA Director)
Samantha Jones, Ph.D. (CPHEA Associate
Director)*
Kristina Thayer, Ph.D. (CPAD Director)
Emma Lavoie, Ph.D. (CPHEA Senior Science
Advisor for Assessments)*
Andrew Kraft, Ph.D. (CPAD Senior Science
Advisor)*
Paul White, Ph.D. (CPAD Senior Science
Advisor)*
Belinda Hawkins, Ph.D. (CPAD Senior Science
Advisor)#
Ravi Subramaniam, Ph.D. (CPAD Branch
Chief)*
U.S. EPA/ORD/CPHEA
Research Triangle Park, NC
*Washington, DC
#Cincinnati, OH
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
U.S. EPA
Office of Research and Development
Center for Public Health and Environmental
Assessment
Washington, DC
Research Triangle Park, NC
Cincinnati, OH
This document is a draft for review purposes only and does not constitute Agency policy.
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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
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
Executive Office of the President/Office of Science and Technology Policy
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review oftert-Butyl Alcohol
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 an oral noncancer reference dose (RfD), an inhalation noncancer reference concentration
(RfC), a cancer weight of evidence descriptor, and a cancer dose-response assessment It was
prepared under the auspices of the U.S. Environmental Protection Agency's (EPA's) Integrated Risk
Information System (IRIS) program. This 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 alpha2u-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
(http://www.epa.gov/iris). Appendices for toxicokinetic information, PBPK modeling genotoxicity
This document is a draft for review purposes only and does not constitute Agency policy.
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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 primarily is an anthropogenic substance that is produced in large quantities
(HSDB. 2007) 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 (U.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 use in Europe and
Asia remains strong.1 Some states have banned MTBE in gasoline due to 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 As such, 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.
Cosmetic and food-related uses include the manufacture of flavors, and, because of its camphor-like
aroma, it also is used to create artificial musk, fruit essences, and perfume fHSDB. 20071. It is used
in coatings on metal and paperboard food containers (Cal/EPA. 1999) and industrial cleaning
compounds and can be used for chemical extraction in pharmaceutical applications fHSDB. 20071.
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
1 http://www.ihs.com/products/chemical/planning/ceh/gasoline-octane-improvers.aspx.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review oftert-Butyl Alcohol
(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 fHSDB. 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 with 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 has 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 at the facility to land, and disposed 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. 2012c). Additionally, tert-
butanol leaking from underground storage tanks could be a product of MTBE and ETBE, which can
degrade to form tert-butanol in soils fHSDB. 20071. The industrial chemical tert-butyl acetate also
can degrade to form tert-butanol in animals post exposure and in the environment.
Ambient outdoor air concentrations of tert-butanol vary according to proximity to urban
areas fHSDB. 20071.
2http://geotracker.waterboards.ca.gov/.
This document is a draft for review purposes only and does not constitute Agency policy.
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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
its high environmental mobility and resistance to biodegradation, tert-butanol has the potential to
contaminate and persist in groundwater and soil (HSDB. 2007).
Ingestion of contaminated food can be a source of tert-butanol exposure through its use as a
coating in metallic and paperboard food containers fCal/EPA. 19991. and tert-butanol has been
detected in food fHSDB. 2007], Internal exposure to tert-butanol also can occur as a result of
ingestion of MTBE or ETBE, as tert-butanol is a metabolite of these compounds fNSF International.
20031.
Other human exposure pathways include inhalation, lactation, and, to a lesser extent,
dermal contact Inhalation exposure can occur due to 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. 2007). the Occupational Safety and Health Administration
(OSHA. 2006). and the Food and Drug Administration (FDA. 2015. 2011). 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.
This document is a draft for review purposes only and does not constitute Agency policy.
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PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS
Note: The Preamble summarizes the
objectives and scope of the IRIS program,
general principles and systematic review
procedures used in developing IRIS
assessments, and the overall development
process and document structure.
1. Objectives and Scope of the IRIS
Program
Soon after EPA was established in 1970, it
was at the forefront of developing risk
assessment as a science and applying it in
support of actions to protect human health
and the environment. EPA's IRIS 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
37 ozone, carbon monoxide, sulfur oxides,
38 nitrogen oxides, and lead).
39 Enhancements to the IRIS program are
40 improving its science, transparency, and
41 productivity. To improve the science, the IRIS
42 program is adapting and implementing
43 principles of systematic review (i.e., using
44 explicit methods to identify, evaluate, and
45 synthesize study findings). To increase
46 transparency, the IRIS program discusses key
47 science issues with the scientific community
48 and the public as it begins an assessment
49 External peer review, independently
50 managed and in public, improves both
51 science and transparency. Increased
52 productivity requires that assessments be
53 concise, focused on EPA's needs, and
54 completed without undue delay.
55 IRIS assessments follow EPA guidance4
56 and standardized practices of systematic
57 review. This Preamble summarizes and does
58 not change IRIS operating procedures or EPA
59 guidance.
60 Periodically, the IRIS program asks for
61 nomination of agents for future assessment
62 or reassessment Selection depends on EPA's
63 priorities, relevance to public health, and
64 availability of pertinent studies. The IRIS
65 multiyear agenda5 lists upcoming
66 assessments. The IRIS program may also
67 assess other agents in anticipation of public
68 health needs.
3 IRIS program website: http: //www.epa.gov/iris/
4 EPA guidance documents: http: //www.epa.gov/iris /basic-information-about-integrated-risk-information-
system#guidance/
5 IRIS multiyear agenda: https: //www.epa.gov/iris/iris-agenda
This document is a draft for review purposes only and does not constitute Agency policy.
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2. Planning an Assessment:
Scoping, Problem Formulation,
and Protocols
Early attention to planning ensures that
IRIS assessments meet their objectives and
properly frame science issues.
Scoping refers to the first step of
planning, where the IRIS program consults
with EPA's program and regional offices to
ascertain their needs. Scoping specifies the
agents an assessment will address, routes
and durations of exposure, susceptible
populations and lifestages, and other topics of
interest
Problem formulation refers to the
science issues an assessment will address
and includes input from the scientific
community and the public. A preliminary
literature survey, beginning with secondary
sources (e.g., assessments by national and
international health agencies and
comprehensive review articles), identifies
potential health outcomes and science issues.
It also identifies related chemicals (e.g.,
toxicologically active metabolites and
compounds that metabolize to the chemical
of interest).
Each IRIS assessment comprises multiple
systematic reviews for multiple health
outcomes. It also evaluates hypothesized
mechanistic pathways and characterizes
exposure-response relationships. An
assessment may focus on important health
outcomes and analyses rather than expand
beyond what is necessary to meet its
objectives.
Protocols refer to the systematic review
procedures planned for use in an assessment
They include strategies for literature
searches, criteria for study inclusion or
exclusion, considerations for evaluating
study methods and quality, and approaches
6 Health and Environmental Research Online:
https://hero.epa.gov/hero/
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
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 facilitates 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
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Toxicological Review oftert-Butyl Alcohol
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
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. 1998c. 1996a. 1991c). As subject-
44 matter experts examine a group of studies,
45 additional agent-specific knowledge or
46 methodologic concerns may emerge and a
47 second pass become necessary.
48 Assessments use evidence tables to
49 summarize the design and results of
50 pertinent studies. If tables become too
51 numerous or unwieldy, they may focus on
52 effects that are more important or studies
53 that are more informative.
54 The IRIS program posts initial protocols
55 for study evaluation on its website, then
56 considers public input as it completes this
57 step.
58 5. Integrating the Evidence of
59 Causation for Each Health
60 Outcome
61 Synthesis within lines of evidence. For
62 each health outcome, IRIS assessments
63 synthesize the human evidence and the
64 animal evidence, augmenting each with
65 informative subsets of mechanistic data. Each
66 synthesis considers aspects of an association
67 that may suggest causation: consistency,
68 exposure-response relationship, strength of
69 association, temporal relationship, biological
70 plausibility, coherence, and "natural
71 experiments" in humans fU.S. EPA. 1994b)
72 flJ.S. EPA. 2005b).
73 Each synthesis seeks to reconcile
74 ostensible inconsistencies between studies,
75 taking into account differences in study
76 methods and quality. This leads to a
77 distinction between conflicting evidence
78 (unexplained positive and negative results in
79 similarly exposed human populations or in
80 similar animal models) and differing results
81 (mixed results attributable to differences
82 between human populations, animal models,
83 or exposure conditions) (U.S. EPA. 2005b).
84 Each synthesis of human evidence
85 explores alternative explanations (e.g.,
7 IRIS "stopping rules": https: //www.epa.gov/sites/
production/files/2014-06/documents/
iris stoppingrules.pdf
This document is a draft for review purposes only and does not constitute Agency policy.
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chance, bias, or confounding) and determines
whether they may satisfactorily explain the
results. Each synthesis of animal evidence
explores the potential for analogous results in
humans. Coherent results across multiple
species increase confidence that the animal
results are relevant to humans.
Mechanistic data are useful to augment
the human or animal evidence with
information on precursor events, to evaluate
the human relevance of animal results, or to
identify susceptible populations and
lifestages. An agent may operate through
multiple mechanistic pathways, even if one
hypothesis dominates the literature (U.S.
EPA. 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
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. 2005bl
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
48 result in the only study, or a single
49 positive result in an extensive database.
50 Inadequate information to assess carcinogenic
51 potential: no other descriptors apply.
52 Examples include little or no pertinent
53 information, conflicting evidence, or
54 negative results not sufficiently robust
55 for not likely.
56 Not likely to be carcinogenic to humans:
57 robust evidence to conclude that there is
58 no basis for concern. Examples include no
59 effects in well-conducted studies in both
60 sexes of multiple animal species,
61 extensive evidence showing that effects
62 in animals arise through modes-of-action
63 that do not operate in humans, or
64 convincing evidence that effects are not
65 likely by a particular exposure route or
66 below a defined dose.
67 If there is credible evidence of
68 carcinogenicity, there is an evaluation of
69 mutagenicity, because this influences the
70 approach to dose-response assessment and
71 subsequent application of adjustment factors
72 for exposures early in life fU.S. EPA. 2005b ),
73 flJ.S. EPA. 2005c).
74 6. Selecting Studies for Derivation
75 of Toxicity Values
76 The purpose of toxicity values (slope
77 factors, unit risks, reference doses, reference
78 concentrations; see section 7) is to estimate
79 exposure levels likely to be without
80 appreciable risk of adverse health effects.
81 EPA uses these values to support its actions
82 to protect human health.
83 The health outcomes considered for
84 derivation of toxicity values may depend on
85 the hazard descriptors. For example, IRIS
86 assessments generally derive cancer values
87 for agents that are carcinogenic or likely to be
88 carcinogenic, and sometimes for agents with
89 suggestive evidence (U.S. EPA. 2005b).
90 Derivation of toxicity values begins with a
91 new evaluation of studies, as some studies
92 used qualitatively for hazard identification
93 may not be useful quantitatively for
This document is a draft for review purposes only and does not constitute Agency policy.
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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. 1994bl. 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. 2012bl.
Studies of low sensitivity may be less
useful if they fail to detect a true effect or
yield toxicity values with wide confidence
limits.
7. Deriving Toxicity Values
General approach. EPA guidance
describes a two-step approach to dose-
response assessment: analysis in the range of
observation, then extrapolation to lower
levels. Each toxicity value pertains to a route
(e.g., oral, inhalation, dermal) and duration or
timing of exposure (e.g., chronic, subchronic,
gestational) (U.S. EPA. 2002b).
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.
8 Benchmark Dose Software:
http://www.epa.gov/bmds/
Toxicological Review oftert-Butyl Alcohol
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. 1994b).
fU.S. EPA. 2005b"). CU.S. EPA. 2011b. 20061.
For human studies, an assessment may
develop exposure-response models that
reflect the structure of the available data (U.S.
EPA. 2005b). For animal studies, EPA has
developed a set of empirical ("curve-fitting")
models8 that can fit typical data sets fU.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
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. 2005bl. fU.S. EPA. 2012bl.
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
CIJ.S. EPA. 2005b").
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,
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Toxicological Review oftert-Butyl Alcohol
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. 2005bl.
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). (U.S.
EPA. 2005cl.
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
risk of adverse health effects over a lifetime
(U.S. EPA. 2002b). Reference values generally
cover effects other than cancer. They are also
appropriate for carcinogens with a nonlinear
mode-of-action.
Calculation of reference values involves
dividing the point of departure by a set of
uncertainty factors (each typically 1, 3, or 10,
unless there are adequate chemical-specific
data) to account for different sources of
uncertainty and variability fU.S. EPA. 2002b],
flJ.S. EPA. 2014bl
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-
50 species differences, which may cause
51 humans to respond at lower levels.
52 Subchronic-to-chronic exposure: For chronic
53 reference values based on subchronic
54 studies, an uncertainty factor reflects the
55 likelihood that a lower level over a longer
56 duration may induce a similar response.
57 This factor may not be necessary for
58 reference values of shorter duration.
59 Adverse-effect level to no-observed-adverse-
60 effect level: For reference values based on
61 a lowest-observed-adverse-effect level,
62 an uncertainty factor reflects a level
63 judged to have no observable adverse
64 effects.
65 Database deficiencies; If there is concern that
66 future studies may identify a more
67 sensitive effect, target organ, population,
68 or lifestage, a database uncertainty factor
69 reflects the nature of the database
70 deficiency.
71 8. Process for Developing and Peer-
72 Reviewing IRIS Assessments
73 The IRIS process (revised in 2009 and
74 enhanced in 2013) involves extensive public
75 engagement and multiple levels of scientific
76 review and comment IRIS program scientists
77 consider all comments. Materials released,
78 comments received from outside EPA, and
79 disposition of major comments (steps 3, 4,
80 and 6 below) become part of the public
81 record.
82 Step 1: Draft development. As outlined in
83 section 2 of this Preamble, IRIS program
84 scientists specify the scope of an
85 assessment and formulate science issues
86 for discussion with the scientific
87 community and the public. Next, they
88 release initial protocols for the
89 systematic review procedures planned
90 for use in the assessment IRIS program
91 scientists then develop a first draft, using
92 structured approaches to identify
93 pertinent studies, evaluate study
94 methods and quality, integrate the
95 evidence of causation for each health
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Toxicological Review oftert-Butyl Alcohol
1 outcome, select studies for derivation of
2 toxicity values, and derive toxicity values,
3 as outlined in Preamble sections 3-7.
4 Step 2: Agency review. Health scientists
5 across EPA review the draft assessment.
6 Step 3: Interagency science consultation.
7 Other federal agencies and the Executive
8 Office of the President review the draft
9 assessment.
10 Step 4: Public comment, followed by
11 external peer review. The public
12 reviews the draft assessment. IRIS
13 program scientists release a revised draft
14 for independent external peer review.
15 The peer reviewers consider whether the
16 draft assessment assembled and
17 evaluated the evidence according to EPA
18 guidance and whether the evidence
19 justifies the conclusions.
20 Step 5: Revise assessment. IRIS program
21 scientists revise the assessment to
22 address the comments from the peer
23 review.
24 Step 6: Final agency review and
25 interagency science discussion. The
26 IRIS program discusses the revised
27 assessment with EPA's program and
28 regional offices and with other federal
29 agencies and the Executive Office of the
30 President
31 Step 7: Post final assessment. The IRIS
32 program posts the completed assessment
33 and a summary on its website.
34 9. General Structure of IRIS
35 Assessments
36 Main text. IRIS assessments generally
37 comprise two major sections: (1) Hazard
38 Identification and (2) Dose-Response
39 Assessment Section 1.1 briefly reviews
40 chemical properties and toxicokinetics to
41 describe the disposition of the agent in the
42 body. This section identifies related
43 chemicals and summarizes their health
44 outcomes, citing authoritative reviews. If an
45 assessment covers a chemical mixture, this
46 section discusses environmental processes
47 that alter the mixtures humans encounter
48 and compares them to mixtures studied
49 experimentally.
50 Section 1.2 includes a subsection for each
51 major health outcome. Each subsection
52 discusses the respective literature searches
53 and study considerations, as outlined in
54 Preamble sections 3 and 4, unless covered in
55 the front matter. Each subsection concludes
56 with evidence synthesis and integration, as
57 outlined in Preamble section 5.
58 Section 1.3 links health hazard
59 information to dose-response analyses for
60 each health outcome. One subsection
61 identifies susceptible populations and
62 lifestages, as observed in human or animal
63 studies or inferred from mechanistic data.
64 These may warrant further analysis to
65 quantify differences in susceptibility.
66 Another subsection identifies biological
67 considerations for selecting health outcomes,
68 studies, or data sets, as outlined in Preamble
69 section 6.
70 Section 2 includes a subsection for each
71 toxicity value. Each subsection discusses
72 study selection, methods of analysis, and
73 derivation of a toxicity value, as outlined in
74 Preamble sections 6 and 7.
75 Front matter. The Executive Summary
76 provides information historically included in
77 IRIS summaries on the IRIS program website.
78 Its structure reflects the needs and
79 expectations of EPA's program and regional
80 offices.
81 A section on systematic review methods
82 summarizes key elements of the protocols,
83 including methods to identify and evaluate
84 pertinent studies. The final protocols appear
85 as an appendix.
86 The Preface specifies the scope of an
87 assessment and its relation to prior
88 assessments. It discusses issues that arose
89 during assessment development and
90 emerging areas of concern.
91 This Preamble summarizes general
92 procedures for assessments begun after the
93 date below. The Preface identifies
94 assessment-specific approaches that differ
95 from these general procedures.
This document is a draft for review purposes only and does not constitute Agency policy.
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10. Preamble References
U.S. EPA. (1991). 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/recordispla
v. cfm? deid=2 316 2
U.S. EPA. (1994). Methods for derivation of
inhalation reference concentrations and
application of inhalation dosimetry [EPA
Report] (pp. 1-409). (EPA/600/8-90/066F).
Research Triangle Park, NC: U.S.
Environmental Protection Agency, Office of
Research and Development, Office of Health
and Environmental Assessment,
Environmental Criteria and Assessment
Office.
https://cfpub.epa.gov/ncea/risk/recordispl
av.cfm?deid=71993&CFID=51174829&CFTO
KEN=25006317
U.S. EPA. (1996). Guidelines for reproductive
toxicity risk assessment (pp. 1-143).
(EPA/630/R-96/009). Washington, DC: U.S.
Environmental Protection Agency, Risk
Assessment Forum.
U.S. EPA. (1998). Guidelines for neurotoxicity
risk assessment. Fed Reg 63: 26926-26954.
U.S. EPA. (2002). A review of the reference
dose and reference concentration processes
(pp. 1-192). (EPA/630/P-02/002F).
Washington, DC: U.S. Environmental
Protection Agency, Risk Assessment Forum.
http://www.epa.gov/osa/review-reference-
dose-and-reference-concentration-processes
U.S. EPA. (2005a). Guidelines for carcinogen
risk assessment [EPA Report] (pp. 1-166).
(EPA/630/P-03/001F). Washington, DC: U.S.
Environmental Protection Agency, Risk
Assessment Forum.
http://www2.epa.gov/osa/guidelines-
carcinogen-risk-assessment
U.S. EPA. (2005b). Supplemental guidance for
assessing susceptibility from early-life
exposure to carcinogens (pp. 1-125).
(EPA/630/R-03/003F). Washington, DC: U.S.
Environmental Protection Agency, Risk
Assessment Forum.
U.S. EPA. (2006). Approaches for the
application of physiologically based
pharmacokinetic (PBPK) models and
supporting data in risk assessment (Final
Report) [EPA Report] (pp. 1-123).
(EPA/600/R-05/043F). Washington, DC: U.S.
Environmental Protection Agency, Office of
Research and Development, National Center
for Environmental Assessment
http://cfpub.epa.gov/ncea/cfm/recordispla
v.cfm?deid=157668
U.S. EPA. (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/recommended-
use-bodv-weight-34-default-method-
derivation-oral-reference-dose
U.S. EPA. (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. (2014). Guidance for applying
quantitative data to develop data-derived
extrapolation factors for interspecies and
intraspecies extrapolation. (EPA/100/R-
14/002F). Washington, DC: Risk Assessment
Forum, Office of the Science Advisor.
http://www, epa. gov /r af/D D E F /p df/ddef-
final.pdf
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EXECUTIVE SUMMARY
Summation of Occurrence and Health Effects
tert-Butanol does not occur naturally; it 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 primarily
occurs 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.
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 alpha2u-globulin -associated nephropathy.
tert-Butanol, however, is a weak inducer of alpha2u-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 alpha2u-globulin
nephropathy in males. Effects attributable to alpha2u-globulin nephropathy in males were not
considered for kidney hazard identification. Females are not affected by alpha2u-globulin
nephropathy, so changes in kidney weights in female rats, transitional epithelial hyperplasia in
female rats, suppurative inflammation in female rats, 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.
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1 At this time, evidence of selective developmental toxicity, neurodevelopmental toxicity, and
2 reproductive system toxicity following tert-butanol exposure is inadequate with minimal effects
3 observed at otherwise toxic dose levels. The available information also is inadequate to draw
4 conclusions regarding liver and urinary bladder toxicity, respectively, because of lack of
5 consistency and lack of progression.
6 Oral Reference Dose (RfD) for Effects Other Than Cancer
7 Kidney toxicity, represented by increases in severity of nephropathy in female rats, was
8 chosen as the basis for the overall oral reference dose (RfD) (see Table ES-1). The kidney effects
9 observed in female rats in the chronic study by NTP (19951 were used to derive the RfD. Increased
10 severity of nephropathy was selected as the critical effect because it was observed in female rats
11 consistently, it is an indicator of kidney toxicity, and it was induced in a dose-responsive manner.
12 Dose-response data were not amenable to modeling; accordingly, the point of departure was
13 derived from the lowest-observed-adverse-effect level (LOAEL) of 43 mg/kg-day (U.S. EPA. 2011a).
14 The overall RfD was calculated by dividing the POD for increases in severity of nephropathy
15 by a composite uncertainty factor (UF) of 100 to account for the extrapolation from animals to
16 humans (3), derivation from a LOAEL (3), and for interindividual differences in human
17 susceptibility (10).
18 Table ES-1. Organ/system-specific RfDs and overall RfD for tert-butanol
Hazard
Basis
Point of
departure*
(mg/kg-day)
UF
Chronic RfD
(mg/kg-day)
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 101
Chronic
Medium
19
20 *Human equivalent dose (HED) PODs were calculated using body weight to the % power (BW3/4) scaling (U.S. EPA,
21 2011a).
22 Effects Other Than Cancer Observed Following Inhalation Exposure
23 Kidney effects are a potential human hazard of inhalation exposure to tert-butanol.
24 Although no effects were observed in mice, kidney weights were increased in male and female rats
25 following 13 weeks of inhalation exposure. In addition, the severity of nephropathy increased in
26 male rats. No human studies are available to evaluate the effects of inhalation exposure. As
27 discussed above for oral effects, endpoints in males specifically related to alpha2u-globulin
28 nephropathy were not considered for kidney hazard identification. Changes in kidney weights and
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severity of nephropathy in females, however, are considered a result of tert-butanol exposure and
are appropriate for identifying a hazard to the kidney.
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
(Borghoffetal.. 20161 allowed for more specific and sensitive equivalent inhalation PODs derived
from a route-to-route 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 route-to-route
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). The U.S. EPA is not
aware of a quantitative analysis of such uncertainty, which would involve comparison of 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 the U.S. 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 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, vs. 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 RfCs and overall RfC for tert-butanol
Hazard
Basis
Point of
departure*
(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
*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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Evidence of Human Carcinogenicity
Under EPA's 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.
Quantitative Estimate of Carcinogenic Risk from Oral Exposure
In accordance with EPA's guidance on alpha2u-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 alpha2u-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
B6C3Fi mice and thyroid follicular cell adenomas and carcinomas in male B6C3Fi mice (NTP. 1995).
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 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 HEDs according to EPA guidance by converting the BMDLio on the basis of (body weight)3/4
scaling fU.S. EPA. 2011a. 2005a)- 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 KM
per mg/kg-day.
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, route-to-route 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.
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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Key Issues Addressed in Assessment
The human relevance of the kidney effects observed in male and female rats was analyzed
in the assessment, particularly as they relate to alpha2u-globulin nephropathy and the exacerbation
of chronic progressive nephropathy. An evaluation of whether tert-butanol caused alpha2u-
globulin -associated nephropathy was performed using the EPA 1991 and IARC 1999 frameworks
evaluated (U.S. EPA. 1991a: Capen. 1999. 699905). The presence of alpha2u-globulin in the hyaline
droplets was confirmed in male rats by alpha2u-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 alpha2u-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. CPN also plays a role in the exacerbation of
nephropathy in both male and female rats, however the MOA of CPN is unknown, and therefore, its
potential relevance to humans cannot be ruled out fNIEHS. 20191. Several other effects in the
kidney unrelated to alpha2u-globulin were observed in female rats, including suppurative
inflammation, transitional epithelial hyperplasia, and increased kidney weights fNTP. 1997.19951.
These specific effects observed in female rats, not confounded by alpha2u-globulin related
processes, are considered the result of tert-butanol exposure and therefore relevant to humans.
Concerning cancer, alpha2u-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 alpha2u-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 alpha2u-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 fNTP. 19951. 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 B6C3Fi mice
following exposure. U.S. EPA f!998bl describes the procedures the Agency uses in evaluating
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 f!998al.
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1
2 LITERATURE SEARCH STRATEGY | STUDY
3 SELECTION AND EVALUATION
4 A literature search and screening strategy was used to identify literature characterizing the
5 health effects of tert-butanol. This strategy consisted of a broad search of online scientific databases
6 and other sources using the most common synonyms and trade names to identify all potentially
7 pertinent studies. In subsequent steps, references were screened to exclude papers not pertinent to
8 an assessment of the health effects of tert-butanol, and remaining references were sorted into
9 categories for further evaluation. This section describes the literature search and screening strategy
10 in detail.
11 The chemical-specific search was conducted in four core online scientific databases,
12 PubMed, Web of Science, and Toxline. as well as TSCATS through December 2016, using the
13 keywords and limits described in Table LS-1. The overall literature search approach is
14 shown graphically in Figure LS-1. Eight more citations were obtained using additional
15 search strategies described in Table LS-2. After electronically eliminating duplicates from
16 the citations retrieved through these databases, 3,138 unique citations were identified.
17 The resulting 3,138 citations were screened for pertinence and separated into categories as
18 presented in Figure LS-1 using the title and either abstract or full text, or both, to examine the
19 health effects of tert-butanol exposure. The inclusion and exclusion criteria used to screen the
20 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.
Nevertheless, these studies were still evaluated as possible sources of supplementary health effects
information.
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• 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 development of 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 HERO website at
https://hero.epa.gov/index.cfm/proiect/page/proiect 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 (2011). Studies were arranged in 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 Chapter 1. Initial review found two
references fCirvello etal.. 1995: Lindamood et al.. 19921 to be publications of the NTP T19951 data
prior to the release of the final National Toxicology Program (NTP) report. One publication
(Takahashi etal.. 1993) 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 (1995)
report was considered the more complete and accurate presentation of the data; therefore, this
report was included in evidence tables and Cirvello etal. (1995). Takahashi etal. (1993). and
Lindamood et al. T19921 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 studies (i.e.,
>3 0-day exposure studies). These were grouped as supplementary studies, however, because 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
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1 provided by IRIS assessments) and are the focus of this assessment Such supplementary studies
2 are discussed in the narrative sections of Chapter 1 and are described in sections such as the "Mode
3 of Action Analysis" to augment the discussion or presented in appendices, if they provide additional
4 information.
5 Figure LS-1. Summary of literature search and screening process for
6 tert-butanol.
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1 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)*
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)*
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)*
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
2 *: See post-peer-review literature search update section
3 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
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Approach used
Source(s)
Date
performed
Number of additional references
identified
Public comment article: (Borghoff et
al„ 2016)
10/2016
l
Manual search of
citations from
reviews conducted
by other
international and
federal agencies
IPCS (1987a). 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
1 Table LS-3. Inclusion-exclusion criteria
Inclusion criteria
Exclusion criteria/Supplemental material*
Population
• Humans
• Standard mammalian animal models,
including rat, mouse, rabbit, guinea pig,
monkey, dog
• Ecological species*
• Non-mammalian species*
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
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
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Inclusion criteria
Exclusion criteria/Supplemental material*
• 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
*Studies that met this exclusion criterion were considered supplemental, e.g., not considered a primary source of
health effects data but were retained as potential sources of contextual information.
Evidence base 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 (U.S. EPA. 2005a. 1998d. 1996b. 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, studies were evaluated to identify the suitability of the
study based on:
• Study design
• Nature of the assay and validity for its intended purpose
• Characterization of the nature and extent of impurities and contaminants of 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
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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 Good
Laboratory Practice (GLP) guidelines, and 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 T19951 rat
data; two oral developmental studies; two inhalation developmental studies; and a single one-
generation reproductive study that also evaluates other systemic effects (Table LS-5). For the body
of available studies, detailed discussion of any identified methodological concerns precedes each
endpoint evaluated in the hazard identification section. Overall, the experimental animal studies of
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.
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1 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., hr/day, day/week); timing of endpoint evaluations; and
sample size and experimental unit (e.g., animals, dams, litters)
Exposure
Characterization of test article source, composition, purity, and stability; suitability of 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 results interpretation (e.g., maternal toxicity,
decrements in body weight relative to organ weight)
Table LS-5. Summary of experimental animal evidence base
Study category
Study duration, species/strain, and administration method
Chronic
2-vear studv in F344 rats (drinking water) NTP (1995)
2-vear studv in B6C3Fi mice (drinking water) NTP (1995)
Subchronic
13-week studv in B6C3Fi mice (drinking water) NTP (1995)
13-week studv in F344 rats (drinking water) NTP (1995)
13-week studv in F344 rats (inhalation) NTP (1997)
13-week studv in B6C3Fi mice (inhalation) NTP (1997)
10-week studv in Wistar rats (drinking water) Acharya et al. (1997), Acharya 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)
Post-Peer-Review Literature Search Update
3 A post-peer-review literature search update was conducted in PubMed, Web of Science, and
4 Toxline for the period January 2017 to July 2019 using a search strategy consistent with previous
5 literature searches (see LS-1).
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Consistent with the IRIS Stopping Rules
fhttps://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. No references were identified in the post-peer-
review literature search which would change any major conclusions in the assessment
The documentation and results for the literature search and screen, including the specific
references identified using each search strategy and tags assigned to each reference based on the
manual screen, can be found on the HERO website on the tert-butanol project page at:
fhttps://hero.epa.gov/hero/index.cfm/proiect/page/project id/15431.
This document is a draft for review purposes only and does not constitute Agency policy.
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i 1 HAZARD IDENTIFICATION
2 1.1 OVERVIEW OF CHEMICAL PROPERTIES AND TOXICOKINETICS
3 1.1.1 Chemical Properties
4 tert-Butanol is a white crystalline solid or colorless, highly flammable liquid (above 25.7°C)
5 with a camphor-like odor fNIOSH. 2005: IPCS. 1987al. tert-Butanol contains a hydroxyl chemical
6 functional group; is miscible with alcohol, ether, and other organic solvents; and is soluble in water
7 (IPCS. 1987a). Chemical and physical properties of tert-butanol are presented in Table 1-1.
8 Table 1-1. Chemical identity and physicochemical properties of tert-Butanol
9 as curated by EPA's CompTox Chemicals Dashboard
Characteristic or property
Value
Chemical structure
(
h3c
(
:h3
OH
:h3
CASRN
75-65-0
Synonyms
2-Propanol, 2-methyl-, 1,1-Dimethylethanol, 2-Methylpropan-2-ol, 2-Methylpropan-2-
ol(other name: tert-Butylalcohol), 2-methylpropane-2-ol, 2-metilpropan-2-ol, t-Butanol,
Trimethyl carbinol, Trimethylcarbinol, Trimethylmethanol, Arconol, t-Butyl hydroxide,
tert-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
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Vapor pressure (mm Hg at 20°C)
40.7
52.7
atm = atmosphere; CASRN = Chemical Abstracts Service registry number.
aMedian values and ranges for physical chemical properties are also provided on the CompTox Chemicals
Dashboard at https://comptox.epa.gov/dashboard/.
1.1.2 Toxicokinetics
tert-Butanol is rapidly absorbed following exposure by oral and inhalation routes (see
Appendix B, Section B.l.l). Studies in experimental animals indicate that 99% of the compound was
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 (ARCO. 19831. In another study
(Faulkner et al.. 19891. 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 i.v. exposures
fPoet etal.. 1997: Faulkner et al.. 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 (Williams and Borghoff. 2001).
A general metabolic scheme for tert-butanol, illustrating the biotransformation in rats and
humans, is shown in Figure 1-1 (see Appendix B.1.3).
Human data on the excretion of tert-butanol comes from studies of methyl tert-butyl ether
(MTBE) and ethyl tert- butyl ether (ETBE) fNihlen 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 et al.. 1998a. 1998b). 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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cm
glucuronide-O-
-CH,
CH,
t-butyl glucuronide
HO^O
HO-
rats, humans
-CH,
[O]
CH,
CH,
HO-
-CH,
CH3
t-butanol
CYP450
I
rats,
humans
OH
"Y
CH3 oh
2-methyl-1,2-propanediol
r^
-OH
CH,
rats
\^° CH,
\
2-hydroxyisobutyric acid
formaldehyde
0
h3c^ ^ch,
acetone
-CH,
CH,
t-butyl sulfate
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 sub-models have been adapted
specifically to estimate internal doses for administration of tert-butanol. In particular, some of these
tert-butanol sub-models were parameterized using pharmacokinetic studies with tert-butanol
exposures and three physiologically based pharmacokinetic (PBPK) models have been developed.
These PBPK models can be used to simulate direct administration of tert-butanol in rats, in addition
to exposure to the parent compound for each: Leavens and Borghoff f20091: Salazaretal. f20151.
and Borghoff et al. (20161. Other models have incorporated tert-butanol as a sub-model 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 1007). In Leavens and Borghoff f20091. tert-
butanol is incorporated as a metabolite of MTBE; in Salazar etal. (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 (Sections B.1.5. and B.I.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 (Table 1-1, Table 1-2, Figure 1-1, and Figure 1-2); 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 non-
neoplastic kidney endpoints in rats, however, is somewhat complicated by the common occurrence
of age-related, spontaneous lesions characteristic of chronic progressive nephropathy (CPN) fNTP.
2015: Hard etal.. 2013: Melnick etal.,2012: U.S. EPA. 1991al:
(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) fNTP. 19971 and 15 months (oral) fNTP.
1995). Huntingdon Life Sciences (2004) also reported increased kidney weight in Sprague-Dawley
rats administered tert-butanol orally for approximately 10 weeks (tabular data presented in the
Supplemental Information to this Toxicological Review). Dose-related increase in kidney weight
was also observed in both male and female rats (Spearman's rank coefficient > 0.72) following
either oral or inhalation exposures (Figure 1-3), and in female mice following inhalation exposure
(Spearman's rank coefficient = 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 potential impact 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 fNTP. 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 which indicates 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 fCraig etal.. 20141.
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 (NTP. 1995)
and male F344 rats following a 13-week inhalation exposure (NTP. 1997). Similarly, male Wistar
This document is a draft for review purposes only and does not constitute Agency policy.
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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. B6C3Fi mice, however, did not exhibit histopathological
changes when exposed for 13 weeks and 2 years 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. f19971: (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." The severity of nephropathy increased, compared with controls, 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 fNTP.
19971. nephropathy was present in all but two male rats, including controls. NTP (19971
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 (NTP. 19971.
In the 2-year oral study by NTP f!9951. 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
fNTP. 19951.
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. The morphological spectrum
observed in CPN in male rats does not appear to have a human analogue in the aging kidney (NTP
2019). However, several individual lesions noted in CPN (e.g. tubule atrophy, tubule dilation,
thickening of tubular basement membranes, glomerulosclerosis) also occur in the human kidney
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(Lusco etal.. 2016: Zoia etal.. 2015: Frazier etal.. 2012: Satirapoi etal.. 2012: NIEHS. 20191.
Therefore, exacerbation of one or more of these lesions following tert-butanol exposure may reflect
some type of cell injury or inflammatory process, which 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. 2019). The effects
characterized as CPN which are related to age (increased severity and incidence) and strain (higher
in Sprague-Dawley rats compared to other strains) incidence 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 to female and male rat controls.
The chemical-related changes in increased severity of nephropathy are included in the
consideration of hazard potential.
NTP (1995) 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 f!9951 describes focal,
medullary mineralization as being associated with CPN but notes 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. 1995). This mineralization is
distinct from linear mineralization, which is considered a lesion characteristic of alpha2u-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 fNTP. 19951. 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 (1995) study. NTP (1995) and Frazier etal. (2012)
describe these lesions as related to the nephropathy (characterized above as common and
spontaneous and considered CPN). However, suppurative inflammation and urothelial hyperplasia
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Toxicological Review oftert-Butyl Alcohol
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
if 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 (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 lesion was not reported in the control or low-
dose females. NTP (19951 described transitional epithelial hyperplasia as increased layers of the
transitional epithelial lining of the renal pelvis; study authors noted no progression of this
hyperplastic lesion to neoplasia. To determine if 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 (Table 1-6 and Table 1-7). Transitional epithelial hyperplasia and nephropathy were
strongly correlated (Spearman's rank coefficient = 0.66) in males and moderately correlated
(Spearman's rank coefficient = 0.44) in females. The transitional epithelial hyperplasia observed in
male and female rats is consistent with advanced CPN fFrazier et al.. 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).
Additional histopathological changes, including increased tubular degeneration,
degeneration of the basement membrane of the Bowman's capsule, diffused glomeruli, and
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Toxicological Review oftert-Butyl Alcohol
glomerular vacuolation were noted in a 10-week study in male Wistar rats (Acharva 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: Acharva et
al. f!9951 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 if 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 (NTP. 1995).
Kidney tumors. The kidney is also a target organ for cancer effects (Table 1-3, Figure 1-1).
Male F344 rats had an increased incidence of combined renal tubule adenomas or carcinomas in
the 2-year oral bioassay fHard etal.. 2011: NTP. 19951. 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's 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" fU.S. EPA. 2005al. In the 2-year oral bioassay fNTP. 19951.
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 account for 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 (Hard.
19861." Similarly, EPA considered the renal tubule hyperplasia to be a preneoplastic effect
associated with the renal tubule tumors. Renal tubule hyperplasia was found in one high-dose
female fNTP. 19951: no increase in severity was observed. This effect in females, which was not
considered toxicologically significant, is not discussed further. Two renal tubular adenocarcinomas
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Toxicological Review oftert-Butyl Alcohol
in male mice also were reported (NTP. 19951. one each in the low- and high-dose groups, but were
not considered by 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 if additional histopathological changes could
be identified to inform the MOA for renal tubule tumor development (Hard etal.. 2011). Working
group members were blinded to treatment groups and used guidelines published by Hard and Wolf
(19991 and refinements reported by (Hard and Seelv. 20061: Hard and Seelv (20051 and Hard
f20081. The group's report and analysis by Hard etal. f20111 confirmed the NTP findings of renal
tubule hyperplasia and renal tubule tumors in male rats at 2 years. 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) when compared to 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 et al. f20111 are included in Table 1-3 and Figure 1-1.
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) #
•
•
•
•
••
• .*
•
•* o •
• °
• O
• •
° o •
• o
•
o
•
o
o
o
10 100 1000
tert-butanol blood conc. (mg/l)
10 100 1000
tert-butanol blood concentration (mg/l)
10000
• Oral exposure
O Inhalation exposure
Figure 1-2. Comparison of absolute kidney weight change in male and female
rats across oral and inhalation exposure based on internal blood
concentration. Spearman 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 oftert-Butyl Alcohol
Male mice Female mice
ftio= -0.1
rtios 0.9
•
#
•
•
~
•
m
*
0 JOOO S003 6 000 9000 0 5000 <000 6 000 SOOO 10000 1 2000
Admnstered ctose{Administered dose {mgr*g»day)
1
2 Figure 1-3. Comparison of absolute kidney weight change in male and female
3 mice following oral exposure based on administered concentration. Spearman
4 rank correlation coefficient (rho) was calculated to evaluate the direction of a
5 monotonic association (e.g., positive value = positive association) and the
6 strength of association.
Male mice Female mice
rho= 0.2 #
CD
i
ii
o
-E
*
*
¦ *
*
*
0 1OOO I0CO 5000 4000 5000 6000 0 1000 2000 3000 4000 5000 SOOO 7000
Ad ministered dose {my'ni^ Admiiistered dose {mgmf}
7 Figure 1-4. Comparison of absolute kidney weight change in male and female
8 mice following inhalation exposure based on administered concentration.
9 Spearman rank correlation coefficient (rho) was calculated to evaluate the
10 direction of a monotonic association (e.g., positive value = positive
11 association) and the strength of association.
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Table 1-2. Changes in kidney histopathology in animals following exposure to
tert- butanol
Reference and study design
Results
Acharva et al. (1997)
1" tubular degeneration, degeneration of the basement membrane of the
Acharva et al. (1995)
Bowman's capsule, diffused glomeruli, and glomerular vacuolation
(no
Wistar rat; 5-6 males/treatment
incidences reported)
Drinking water (0 or 0.5%), 0 or
575 mg/kg-d
4/ kidney glutathione (~40%)*
10 weeks
NTP (1995)
Incidence (severity):
F344/N rat; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20,
Males
Females
or 40 mg/mL)
Dose
Minerali-
Nephro-
Dose
Minerali-
Nephro-
M: 0, 230, 490, 840, 1,520,
3,610a mg/kg-d
F: 0, 290, 590, 850, 1,560,
(mg/kg-d)
0
zation15
0/10
pathy0
7/10 (1.0)
(mg/kg-d)
0
zation15
10/10 (1.7)
pathy0
2/10 (1.0)
3,620a mg/kg-d
13 weeks
230
0/10
10/10
(1.6*)
290
10/10 (2.0)
3/10 (1.0)
490
2/10 (1.5)
10/10
(2.6*)
590
10/10 (2.0)
5/10 (1.0)
840
8/10*(1.4)
10/10
(2.7*)
850
10/10 (2.0)
7/10* (1.0)
1,520
4/10*(1.0)
10/10
(2.6*)
1,560
10/10 (2.0)
8/10* (1.0)
3,610a
4/10*(1.0)
7/10 (1.1)
3,620a
6/10 (1.2)
7/10* (1.0)
NTP (1995)
Study authors indicated no treatment-related changes in kidney-related
B6C3Fi mouse; 10/sex/treatment
histopathology (histopathological data not provided for the 13-week study)
Drinking water (0, 2.5, 5,10, 20,
or 40 mg/mL)
M: 0, 350, 640, 1,590, 3,940,
8,210a mg/kg-d
F: 0, 500, 820, 1,660, 6,430,
ll,620a mg/kg-d
13 weeks
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Toxicological Review oftert-Butyl Alcohol
Reference and study design
Results
NTP (1995)
Incidence (severity):
F344/N rat; 60/sex/treatment
Males
(10/sex/treatment evaluated at
Linear
mineralization15
(terminal)
15 months interim)
Drinking water (0,1.25, 2.5, 5,
10 mg/mL)
Dose
(mg/kg-d)
Mineralization15
(interim)
Mineralization15
(terminal)
M: 0, 90, 200, 420a mg/kg-d
0
1/10 (1.0)
26/50 (1.0)
0/50
F: 0,180, 330, 650a mg/kg-d
2 years
90
2/10 (1.0)
28/50 (1.1)
5/50* (1.0)
200
5/10 (1.8)
35/50 (1.3)
24/50* (1.2)
420a
9/10* (2.3)
Transitional
48/50* (2.2)
46/50* (1.7)
Inflammation
Dose
epithelial
Nephropathy0
(suppurative)
(mg/kg-d)
hyperplasia
severity
incidence
0
25/50 (1.7)
3.0
10/50
90
32/50 (1.7)
3.1
18/50
200
36/50* (2.0)
3.1
12/50
420a
40/50* (2.1)
3.3*
9/50
Females
Inflammation
Dose
Mineralization15
Mineralization15
(suppurative)
(mg/kg-d)
Interim
Terminal
incidence
0
10/10 (2.8)
49/50 (2.6)
2/50
180
10/10 (2.9)
50/50 (2.6)
3/50
330
10/10 (2.9)
50/50 (2.7)
13/50*
650a
10/10 (2.8)
Transitional
50/50 (2.9)
17/50*
Dose
epithelial
Nephropathy0
(mg/kg-d)
hyperplasia
severity
0
0/50
1.6
180
0/50
1.9*
330
3/50 (1.0)
2.3*
650a
17/50*(1.4)
2.9*
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Toxicological Review oftert-Butyl Alcohol
Reference and study design
Results
NTP (1995)
No treatment-related changes in kidney-related histopathology observed
B6C3Fi mouse; 60/sex/treatment
Drinking water (0, 5,10, or
20 mg/mL)
M: 0, 540, 1,040, or
2,070a mg/kg-d
F: 0, 510,1,020, or
2,110 mg/kg-d
2 years
NTP (1997)
Male
F344/N rat; 10/sex/treatment
Inhalation analytical
concentration: 0,134, 272, 542,
1,080, or 2,101 ppm (0, 406, 824,
Incidence of Average severity
Concentration chronic of chronic
(mg/m3) nephropathvd nephropathy
1,643, 3,273 or 6,368 mg/m3)
0 9/10 1.0
(dynamic whole-body chamber)
6 hr/d, 5 d/wk
406 8/10 1.4
13 weeks
824 9/10 1.4
Generation method (Sonimist
Ultrasonic spray nozzle
1,643 10/10 1.6
nebulizer), analytical
3,273 10/10 1.9
concentration and method were
reported
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
NTP (1997)
No treatment-related changes in kidney-related histopathology observed
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 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist
Ultrasonic spray nozzle
nebulizer), analytical
concentration and method were
reported
1 ^Statistically significant p < 0.05, as determined by the study authors.
2 aThe high-dose group had an increase in mortality.
3 bMineralization defined in NTP (1995) as focal mineral deposits, primarily at the corticomedullary junction. Linear
4 mineralization was defined as foci of distinct linear deposits along radiating medullary collecting ducts; linear
5 mineralization not observed in female rats.
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1 Nephropathy defined in NTP (1995) as lesions, including thickened tubule and glomerular basement membranes,
2 basophilic foci of regenerating tubule epithelium, intratubule protein casts, focal mononuclear inflammatory cell
3 aggregates within areas of interstitial fibrosis and scarring, and glomerular sclerosis.
4 Nephropathy characterized in NTP (1997) as scattered foci of regenerative tubules (with cytoplasmic basophilia,
5 increased nuclear/cytoplasmic ratio, and occasionally thickened basement membranes and intraluminal protein
6 casts).
7
8 Note: Conversions from drinking water concentrations to mg/kg-d performed by study authors.
9 Conversion from ppm to mg/m3 is 1 ppm = 3.031 mg/m3.
10 Table 1-3. Changes in kidney tumors in animals following exposure to
11 tert-butanol
Reference and study design
Results
NTP (1995)
F344/N rat; 60/sex/treatment
(10/sex/treatment evaluated at
15 months)
Drinking water (0,1.25, 2.5, 5, or
Male
Dose
Renal tubule
hyperplasia
(standard and
extended
evaluation
Renal tubule
Renal tubule
adenoma
10 mg/mL)
M: 0, 90, 200, or 420a mg/kg-d
(mg/kg-d)
combined)
adenoma (single)
(multiple)
F: 0,180, 330, or 650a mg/kg-d
0
14/50 (2.3)
7/50
1/50
2 years
90
20/50 (2.3)
7/50
4/50
200
17/50 (2.2)
10/50
9/50*
420a
25/50* (2.8)
10/50
Renal tubule
adenoma (single
3/50
Dose
Renal tubule
or multiple) or
(mg/kg-d)
carcinoma
carcinoma
0
0/50
8/50
90
2/50
13/50
200
1/50
19/50*
420a
1/50
13/50
Female
Renal tubule
Dose
Renal tubule
Renal tubule
adenoma
(mg/kg-d)
hyperplasia
adenoma (single)
(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
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Reference and study design
Results
Dose
(mg/kg-d)
0
180
330
650a
Renal tubule
carcinoma
0/50
0/50
0/50
0/50
Renal tubule
adenoma (single
or multiple) or
carcinoma
0/50
0/50
0/50
0/50
Based on standard and extended evaluations (combined). Results do not
include the animals sacrificed at 15 months.
Hard etal. (2011)
Reanalysis of the slides from
male rats (all slides in controls
and high-dose groups of males
and females, and slides from all
other males with renal tumors) in
the NTP (1995) study (see above)
Male
Renal tubule
adenoma
Dose
Renal tubule
adenoma
Renal tubule
adenoma
Renal tubule
(single or
multiple) or
(mg/kg-d)
(single)
(multiple)
carcinoma
carcinoma
0
3/50
1/50
0/50
4/50
90
9/50
3/50
1/50
13/50*
200
9/50
9/50
0/50
18/50*
420
9/50
3/50
1/50
12/50*
NTP (1995)
B6C3Fi mouse; 60/sex/treatment
Drinking water (0, 5,10, or
20 mg/mL)
M: 0, 540, 1,040, or
2,070a mg/kg-d
F: 0, 510,1,020, or
2,110 mg/kg-d
2 years
No 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.
1 ^Statistically significant p < 0.05, as determined by the study authors.
2 aThe high-dose group had an increase in mortality.
3 Note: Conversions from drinking water concentrations to mg/kg-d performed by study authors.
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1 Table 1-4. Comparison of nephropathy and suppurative inflammation in
2 individual male rats from the 2-year 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
3 Spearman's rank correlation test (1-sided), p = 0.0015, rs = 0.17
4 Table 1-5. Comparison of nephropathy and suppurative inflammation in
5 individual female rats from the 2-year 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
6 Spearman's rank correlation test (1-sided), p < 0.0001, rs = 0.47
7 Table 1-6. Comparison of nephropathy and transitional epithelial hyperplasia
8 in individual male rats from the 2-year 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
Mild
0
0
2
25
42
Moderate
0
0
2
6
17
Marked
0
0
0
0
0
9 Spearman's rank correlation test (1-sided), p < 0.0001, rs = 0.66
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1 Table 1-7. Comparison of nephropathy and transitional epithelial hyperplasia
2 in individual female rats from the 2-year 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
3 Spearman's rank correlation test (1-sided), p < 0.0001, rs = 0.437
4 Table 1-8. Comparison of CPN and renal tubule hyperplasia with kidney
5 adenomas and carcinomas in male rats from the 2-year NTP tert-butanol
6 bioassay
Renal Tumors
Renal Tumors
Renal tubule
Renal Tumors
Renal Tumors
CPN
Absent
Present
hyperplasia
Absent
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
7 Spearman's rank correlation test (1-sided): CPN, p < 0.0001, rs = 0.430; renal tubule hyperplasia, p = 0.01, rs = 0.161
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¦ = exposures at which the endpoint was reported statistically significant by study authors
~ = exposures at which the endpoint was reported not statistically significant by study authors
x = exposures at which all animals were dead and unable to be examined for the endpoint
Kidney
Weight
Absolute weight; M Rat; Reproductive (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/terminal mineralization; F Rat; 2yr (D)
Transitional epithelium hyperplasia; M Rat; 2yr (D)
Transitional epithelium hyperplasia; F Rat; 2yr (D)
Renal tubular hyperplasia; M Rat; 2yr (D)
Renal tubule hyperplasia; F Rat; 2yr (D)
Kidney Rena' tubular adenoma or carcinoma; M Rat; 2yr (D)
Tumors Renal tubular adenoma or carcinoma; M Rat; 2yr (B)
Renal tubular adenoma or carcinoma; F Rat; 2yr (D)
Renal tubular adenoma or carcinoma; M Mouse; 2yr (D)
Renal tubular adenoma or carcinoma; F Mouse; 2yr (D)
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)*: (C) Huntingdon Life Sciences (2004) (D)
NTP (1995): Preanalysis of NTP (1995).
Figure 1-5. Exposure response array for kidney effects following oral exposure
to tert-butanol.
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¦ = exposures at which the endpoint was reported statistically significant by study authors
~ = exposures at which the endpoint was reported not statistically significant by study authors
Absolute weight; M Rat
Relative weight; M Rat
Absolute weight; F Rat
Relative weight; 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 1-6. Exposure-response array of kidney effects following inhalation
exposure to tert-butanol (13-week studies, no chronic studies available).
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Mode of Action Analysis—Kidney Effects
a) alpha2u-globulin -Associated Renal Tubule Nephropathy and Carcinogenicity
One disease process to consider when interpreting kidney effects in rats is related to the
accumulation of alpha2u-globulin protein. alpha2u-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 alpha2u-globulin commonly detected in male rat urine is considered specific to that sex
and species. Exposure to chemicals that induce alpha2u-globulin accumulation can initiate a
sequence of histopathological events leading to kidney tumorigenesis. Because alpha2u-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 if
alpha2u-globulin plays a role is important The role of alpha2u-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,
AIphct2U-GIobuIin: Association with Chemically Induced Renal Toxicity and Neoplasia in the Male Rat as
well as the IARC 1999 framework fCapen. 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 alpha2u-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 alpha2u-globulin in tert-butanol-induced renal tubule
nephropathy and carcinogenicity fBorghoff etal.. 2001: Williams and Borghoff. 2001: Takahashi et
al.. 1993: Hard etal.. 20191. Because the evidence reported in these studies is specific to alpha2u-
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 alpha2u-globulin renal tubule nephropathy, as described by
U.S. EPA (1991a). is as follows. Chemicals that induce alpha2u-globulin accumulation do so rapidly.
alpha2u-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 alpha2u-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
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1 chemical exposure for 3 to 12 months leads to the formation of calcium hydroxyapatite in the
2 papillae which results in linear mineralization. After 1 or more years of chemical exposure, these
3 lesions can result in the induction of renal tubule adenomas and carcinomas (Figure 1-7).
4 U.S. EPA fl991al identified two questions that must be addressed to determine the extent
5 to which alpha2u-globulin -mediated processes induce renal tubule nephropathy and
6 carcinogenicity. First, whether the alpha2u-globulin process occurs in male rats and influences
7 renal tubule tumor development must be determined. Second, whether the renal effects in male
8 rats exposed to tert-butanol are due solely to the alpha2u-globulin process must be determined.
9 U.S. EPA f!991al stated the criteria for answering the first question in the affirmative are as
10 follows:
11 1) hyaline droplets are larger and more numerous in treated male rats,
12 2) the protein in the hyaline droplets in treated male rats is alpha2u-globulin (i.e.,
13 immunohistochemical evidence), and
14 3) several (but not necessarily all) additional steps in the pathological sequence appear in
15 treated male rats as a function of time, dose, and progressively increasing severity consistent with
16 the understanding of the underlying biology, as described above, and illustrated in Figure 1-7.
17 The available data relevant to this first question are summarized in Table 1-9, Figure 1-8,
18 and Figure 1-9, and are evaluated below.
19
20
21
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Source: Adapted from Swenberg and Lehman-McKeeman (1999) and U.S. EPA (1991a).
Figure 1-7. Temporal pathogenesis of alpha2u-globulin -associated
nephropathy in male rats. alpha2u-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.
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Table 1-9. Summary of data on the alpha2u-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 + stat sig at 5,304 mg/m3;
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, (+) observed in all but
1,520, 3,610 mg/kg-d highest dose group
NTP(1995)
2)
The protein in the hyaline droplets is a2uglobulin
10 d (inhalation)
0, 758, 1,364, 5,304 + stat sig at 5,304 mg/m3;
mg/m3 stat sig trend
Borghoff et al. (2001)
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 observe necrosis or cytotoxicity; therefore, that the results indicate
regenerative proliferation is occurring cannot be assumed.)
10 wk (oral)
0, 575 mg/kg-d -
Acharva et al. (1997)
10 d (inhalation)
0, 758,1,364, 5,304 + stat sig at all doses; stat
mg/m3 sig trend
Borghoff et al. (2001)
13 wk (oral)
0,230,490, 840, + elevated at 840 mg/kg-d;
1,520, 3,610 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, -; (+)b
1,520, 3,610 mg/kg-d
NTP (1995); Hard et al.
(2011)°
2 yr (oral)
0, 90, 200, 420
mg/kg-d
NTP (1995); Hard et al.
(2011)d
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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 et al.
(2011)°
2 yr (oral)
0, 90, 200, 420 +; (+)
mg/kg-d
all doses stat sig
NTP (1995); Hard et al.
(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)
1 + = Statistically significant change reported in one or more treated groups.
2 (+) = Effect was reported in one or more treated groups, but statistics not reported.
3 - = No statistically significant change reported in any of the treated groups.
4 aNTP (1997) did not observe any effects consistent with alpha2u-globulin nephropathy.
5 Precursors to granular casts reported.
6 cReanalysis of hematoxylin and eosin-stained kidney sections from all male control and 1,520-mg/kg-d groups and
7 a representative sample of kidney sections stained with Mallory Heidenhain stain, from the 13-wk study from NTP
8 (1995).
9 dReanalysis of slides for all males in the control and 420-mg/kg-d dose groups and all animals with renal tubule
10 tumors from 2-yr NTP (1995). Protein casts reported, not granular casts.
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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
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 12 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)
1 *Hard et al. (2011) reported presence of "precursor granular casts."
2 **NTP (1995) 13-wk study reported kidney mineralization but not linear mineralization.
3 Figure 1-8. Exposure-response array for effects potentially associated with
4 alpha2u-globulin renal tubule nephropathy and tumors in male rats after oral
5 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
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)
1 Figure 1-9. Exposure-response array for effects potentially associated with
2 alpha2u-globulin renal tubule nephropathy and tumors in male rats after
3 inhalation exposure to tert-butanol.
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Question One: Is the a2ugIobuIin 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 hr/day for 10 days (Borghoff etal.. 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 et al.. 1993: Lindamoodetal.. 19921
demonstrated an increase in the accumulation of hyaline droplets. The lowest dose, 230 mg/kg-day,
had minimal hyaline droplet formation compared to controls, although the next three doses (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 alpha2u-globulin nephropathy in male rats. These results from NTP
(1997). which are inconsistent with the findings of both Borghoffetal. (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 6hr/day, 5
day/week. The absence of similar histopathological findings in the 13-week inhalation NTP (1997)
study compared to those reported in the two oral studies is not understood, but might be indicative
of the strength of tert-butanol to induce, consistently, alpha2u-globulin nephropathy. The results
from the two other studies (Borghoffetal.. 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 fBorghoff etal.. 2001: NTP. 19951 ,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 alpha2u-globulin. Accumulated hyaline droplets with an alpha2u-globulin etiology can be
confirmed by using immunohistochemistry to identify the alpha2u-globulin protein. Two short-
term studies measured alpha2u-globulin immunoreactivity in the hyaline droplets of the renal
9 If the chemical meets the criteria for question one, then a second question is asked: Are the renal effects in male rats
exposed to this chemical due solely to the a2u globin process?
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proximal tubular epithelium (Borghoff etal.. 2001: Williams and Borghoff. 20011. Following 10
days of inhalation exposure, Borghoff et al. f20011 did not observe an exposure-related increase in
alpha2u-globulin using immunohistochemical staining. However, when using an enzyme-linked
immunosorbent assay (ELISA), a more sensitive method of detecting alpha2u-globulin, a
statistically significant positive correlation of alpha2u-globulin concentration with dose of tert-
butanol was observed. The accumulation of alpha2u-globulin protein was statistically significant by
pairwise comparison only in the highest dose group. No positive staining for alpha2u-globulin was
observed in exposed female rats. In a follow-up study, Williams and Borghoff (20011 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
alpha2u-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 alpha2u-globulin was
reversible. These data indicate the presence of alpha2u-globulin in tert-butanol-treated male rats,
although requiring a more sensitive method of detection for alpha2u-globulin than is typically used
could indicate that tert-butanol is not a strong inducer of alpha2u-globulin accumulation.
Therefore, the available data are sufficient to fulfill the second criterion for alpha2u-globulin
present in the hyaline droplets but suggest weak induction of alpha2u-globulin by tert-butanol.
(3) The third criterion considered is whether several (but not necessarily all) additional
events in the histopathological sequence associated with alpha2u-globulin nephropathy appear in
male rats in a manner consistent with the understanding of alpha2u-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
fl991al]. 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 (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 alpha2u-globulin in the hyaline droplets at doses that induced
significant tumor formation in male rats were not significant. Next, necrosis or cytotoxicity was
absent, and only precursors to granular casts at stages well within the expected timeframe of
detectability were present. Finally, a 13-week inhalation study found no evidence of alpha2u-
globulin nephropathy fNTP. 19971. despite evaluating exposure concentrations predicted to result
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in similar blood tert-butanol levels as for the 13-week oral study (NTP. 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 fAcharva etal.. 1997: NTP. 19951. Acharva et al. T19971 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. As 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 the result of both weak induction of alpha2u-
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 was also inconsistently observed. Acharva etal. T19971
did not observe tert-butanol-induced proliferation following 10 weeks of oral exposure, but
renal tubule proliferation was observed 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 (1995) 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. (1993)
reported in NTP (1995)]. 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 fAcharva et
al.. 19971 which could be consistent with a compensatory proliferative effect. NTP T19951
reported, however, 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 (1995) did not observe the formation of granular casts or tubular dilation;
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however, Hard etal. (20111 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 timeframe of frank formation and accumulation of granular casts
(>3 weeks). Granular cast formation, however, might not be significantly elevated with
weak inducers of alpha2u-globulin (Short etal.. 19861. which is consistent with the
reported difficulty in measuring alpha2u-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. 19951: 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 alpha2u-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 (NTP. 1995). By comparison, renal tubule hyperplasia was observed in only one
high-dose female.
The progression of histopathological lesions for alpha2u-globulin nephropathy is
predicated on the initial response of excessive hyaline droplet accumulation (containing alpha2u-
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 alpha2u-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; alpha2u-globulin was identified as the protein in these droplets fBorghoffetal.. 2001:
Williams and Borghoff. 20011. Lack of necrosis and exfoliation might be due to the weak induction
of alpha2u-globulin and a lack of later examinations. Granular cast formation was not reported in
any of the available studies, which could also indicate weak alpha2u-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 timeframe of the appearance of these lesions. Overall, no explicit
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inconsistencies are present in the temporal appearance of the histopathological lesions associated
with alpha2u-globulin nephropathy; however, the dataset 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 alpha2u-
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.. 19921. although the incidence at the lowest dose was minimal, while the incidence at the
three higher doses was more prominent These results are discordant with the tumor results, given
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.
f20111]. This lowest dose was less than the 230 mg/kg-day in the 13-week oral study that had only
minimal hyaline droplet formation. Furthermore, although the incidence of renal tubule
hyperplasia had a dose-related increase (NTP. 1995). 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 timeframe 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 IARC1999 Criteria
An alpha2u-globulin framework was published by IARC in 1999 (Capen etal.. 1999). See
Table 1-10 for criterion 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 alpha2u-globulin associated response in male rats (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-a2U globulin
Reversible binding of the chemical or metabolite to ct2U 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)
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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 alpha2u-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,
IARC (Capen etal.. 19991 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 fCapen et al.. 19991 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, Escherichiacoli and Neurospora crassa fMcGregor etal.. 2005: Zeiger etal.. 1987:
Dickey etal.. 19491. Studies also demonstrate negative results for gene mutations, sister chromatid
exchanges, micronucleus formation and chromosomal aberrations fNTP. 1995: McGregor etal..
1988). However, DNA adducts were found in male Kunming mice (Yuan etal.. 2007) and DNA
damage in human HL-60 leukemia cells (Tang etal.. 1997). In another study (Sgambato etal.. 2009).
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 were not observed in either female rats
or either sex of mice. Because an alpha2u-globulin MOA is specific to male rats, the endpoints would
not be expected in female rats or either sex of mice and none were observed (see Table 1-2). No
treatment related changes in kidney histopathology following oral or inhalation exposures were
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observed in male or female mice fNTP. 1997.19951. No protein droplets, alpha2u-globulin
immunostaining, or increases in cell proliferation were observed in the kidneys of female rats, but
they were seen in the male rats fBorghoff et al.. 20011. Cell proliferation increased in male, but not
female rats exposed to t-butanol via inhalation exposure for 10 days fBorghoffetal.. 20011 and
drinking water exposure for 90 days fLindamood etal.. 19921. Increased kidney weights were
observed in female rats exposed to 1,364 or 5,304 mg/m3 t-butanol after 10-days (Borghoffetal..
20011. in female F344 rats exposed to 290, 590, 850,1,560, and 3,620 mg/kg-day t-butanol in a 13-
week drinking water study fNTP. 19951. in female B6C3Fi mice exposed to 11,620 mg/kg-day t-
butanol in a 13-week drinking water study fNTP. 19951. and female F344 rats exposed to 6,368
mg/m3 t-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, and 3,620
mg/kg-day for 13 weeks, as well as 180, 330, and 650 mg/kg-d for 2 years fNTP. 19951. Female
F344 rats exposed to 850,1,560, and 3,620 mg/kg-day t-butanol had a dose-related increase in the
incidence of nephropathy and the incidences were greater than that of controls fNTP. 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 that are associated with alpha2u-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 alpha2u-globulin accumulation are only observed in male
rats. This criterion of male rat specificity is met
Summary and Conclusions for Question One: Is the a!pha2u-gIobuIin process occurring in male rats
exposed to tert-butanol?
Oral exposure 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 alpha2u-globulin MOA. This evidence includes the
increased size and number of hyaline droplets and the accumulated alpha2u-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 alpha2u-globulin process is operative. Furthermore, the available data is sufficient to fulfill the
IARC criteria for establishing the role for alpha2u-globulin in male rats, with the exception of
genotoxic potential because of a limited genotoxicity evidence base. Although the evidence
indicates a role for alpha2u-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 alpha2u-
globulin considering the available histopathological observations and uncertainty regarding the
temporal and dose concordance of the lesions.
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Question Two: Are the renal effects in male rats exposed to tert-butanol due solely to the alpha2u-
globulin process?
If the alpha2u-globulin process is operative, U.S. EPA f!991al identifies a second question
that must be answered regarding whether the renal effects are solely due to the alpha2u-globulin
process, a combination of the alpha2u-globulin process and other carcinogenic processes, or
primarily due to other processes. U.S. EPA (1991a) states that additional data can help inform
whether the alpha2u-globulin process is the sole contributor to renal tubule tumor development in
male rats. These additional data are considered and discussed in detail below.
(a) Hypothesis-testing of the a!pha2u-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 a!pha2u-gIobuIin
protein: Williams and Borghoff (20011 report that tert-butanol reversibly and noncovalently binds
to alpha2u-globulin in the kidneys of male rats. This provides additional support to the involvement
of the alpha2u-globulin process.
(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 alpha2u-globulin process. Cell proliferation was observed in two studies [13-week,
NTP (1995) and 10-day, Borghoff et al. (20011] but whether the proliferation was compensatory is
unknown, as 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 alpha2u-globulin protein
accumulation.
(d) Covalent binding to DNA or other macromolecules, suggesting another process leading to
tumors andgenotoxicity (a!pha2u-gIobuIin -inducers are essentially nongenotoxic): One study (Yuan
etal.. 20071 observed a dose-related increase in tert-butanol-DNA adducts in liver, kidney, and lung
of mice administered a single low dose of tert-butanol (<1 mg/kg) in saline via gavage (see
Appendix 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 primarily are negative, 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 et al. T19951 after 10 weeks of tert-butanol exposure. This type of genetic damage would
not necessarily preclude a role for alpha2u-globulin, but not enough information is available to
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determine whether oxidative stress could initiate or promote kidney tumors in concert with
alpha2u-globulin accumulation in male rat kidneys.
(e) Nephrotoxicity in the male rat not associated with the a!pha2u-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. 1997.19951 and 2 years (NTP. 19951. oral and inhalation exposure increased the
severity of nephropathy in male rats fNTP. 19951. Similarly, the severity of nephropathy was
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 T19951
and Frazier etal. (2012) 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. 1997.19951 and 15-month oral evaluation (NTP. 19951. 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 alpha2u-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
alpha2u-globulin processes. The moderate correlation between CPN severity and renal tumor
incidence in male rats (Spearman's rank coefficient = 0.45) and the very weak correlation between
renal tubule hyperplasia and renal tumors (Spearman's rank coefficient = 0.16) (Table 1-8)
suggests that alpha2u-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
alpha2u-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 alpha2u-globulin nephropathy,
the data indicate that tert-butanol is a weak inducer of alpha2u-globulin and that this process is not
solely responsible for the renal tubule nephropathy and carcinogenicity observed in male rats. The
lack of compensatory cell proliferation in male rats and evidence of nephrotoxicity in female rats
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suggest that other processes, in addition to the alpha2u-globulin process, are operating.
Furthermore, the accumulation of hyaline droplets and the induction of renal tubule hyperplasia
were affected at higher doses compared to those inducing renal tubule tumors. Collectively, these
data suggest that tert-butanol induces the alpha2u-globulin pathway at high doses (>42 0 mg/kg-
day), which results in tumor formation. Other, unknown pathways, however, could be operative at
lower doses (<420 mg/kg-day), which may contribute to renal tumor induction.
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 the EPA's cancer guidelines (Hard 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 fNIEHS. 20191. 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. (20131 slightly revised and restated their criteria for considering exacerbation of CPN as
a MOA for renal tubule tumors in rats. Table 1-11 lists these sets of proposed empirical criteria for
attributing renal tubule tumors to CPN.
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Table 1-11. Proposed empirical criteria for attributing renal tumors to CPN
• 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-year
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-year 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-year
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.
Source: Hard et al. (2013)
Source: Hard and Khan (2004)
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
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).
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Evaluation of the MOA Proposed by Hard et al. (20131
Setting aside the question of whether CPN is fHard etal.. 2013: Hard and Khan. 20041 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's cancer guidelines fU.S. EPA. 2005al 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. f20131 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 et al. (2013) 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. 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 (Spearman's rank coefficient rs = 0.43) (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.
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 (1995) 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 (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 versus 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 versus 18/50 at the middle dose) does notparallel 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
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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, 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 appears 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 et al. f20131 (Table 1-10), even more so when making
comparisons with the lower control tumor incidence from the Hard etal. f20111 reanalysis.
Conclusions about the hypothesized CPN-related MOA
As recommended by EPA's cancer guidelines (U.S. EPA. 2005a). 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
lacking, however, between males and females and in the dose-response relationships between CPN,
hyperplasia, and adenomas, Melnick etal. (2013): Melnick et al. (2012) 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
its key events, they maintain that determining the human relevance of processes that might be
occurring in rats is not possible. An earlier analysis of 28 NTP studies fSeelv etal.. 20021 found a
slight but statistically significant increase in CPN severity in animals with renal tubule tumors,
without determining that this relationship is causal. They suggested that the number of tumors due
to chemically exacerbated CPN would be few.
These inconsistencies make difficult attributing all renal tumors to either CPN or to
alpha2u-globulin -related nephropathy (see previous section on alpha2u-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 fNIEHS. 20191. Scientists disagree, however, on the relevancy of the CPN MOA to
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humans. Hard etal. (20131: Hard etal. (20091 cite 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, Melnick etal. f20131: Melnicketal. f20121 argue 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 appear to 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 fNIEHS. 20191. 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 et al. f20131: Melnicketal. f20121. 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 relevant to
humans, also allow there is no generally accepted MOA for CPN akin to that for alpha2u-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 that is unknown for humans [as
discussed in Hard etal. f20131]. The other was the IRIS assessment of tetrahydrofuran, for which
EPA found the evidence insufficient to conclude that the kidney tumors are mediated solely by the
hypothesized MOAs (U.S. EPA. 2012d). Hard etal. (2013) attributed these different conclusions to
either different data for the two chemicals or the lack of a generally accepted MOA akin to alpha2u-
globulin -related nephropathy.
Relevant to this last point, IARC T19991 developed a consensus statement that listed
considerations for evaluating alpha2u-globulin -related nephropathy in rats, which was based on
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the work of 22 scientists, including 3 who were co-authors of Hard etal. (20131 and 2 who were co-
authors of Melnicketal. f20131: Melnicketal. f20121. A similar broad-based consensus that defines
a sequence of key events for exacerbated CPN, distinguishes it more clearly from alpha2u-globulin -
related nephropathy, and evaluates its relevance to humans would be helpful in advancing the
understanding of these issues.
Overall Conclusions on MO A for Kidney Effects
tert-Butanol increases alpha2u-globulin deposition and hyaline droplet accumulation in
male rat kidneys and several of the subsequent steps in that pathological sequence. These data
provide some evidence that the alpha2u-globulin process is operating. This chemical appears to be
a weak inducer of alpha2u-globulin nephropathy and this induction is not the sole contributor to
renal tubule nephropathy and carcinogenicity. CPN and the exacerbation of CPN (likely due to both
alpha2u-globulin and tert-butanol) 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 alpha2u-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 the multiple tert-
butanol 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 alpha2u-globulin process. U.S. EPA f!991al states that if the alpha2u-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 alpha2u-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 (NIEHS.
2019). CPN is not, inherently, a specific diagnosis, however, 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. ((Zoiaetal.. 2015: Gorriz and Martinez-Castelao.
2012). Thus, it cannot be ruled out that chemicals which exacerbate CPN in rats may have the
potential to exacerbate disease processes in the human kidney fNIEHS. 20191.
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Because female rats are not affected by alpha2u-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 alpha2u-globulin process is
operating. Because renal tubule tumors in male rats did not arise solely due to the alpha2u-globulin
and CPN processes and some of the tumors are attributable to other carcinogenic processes, such
tumors remain relevant for purposes of hazard identification (U.S. EPA. 1991a).10 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 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)
fNTP. 19951. and two inhalation subchronic studies (one in rats and one in mice) fNTP. 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. 1997.19951. 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 (Table 1-12; Figure 1-10).
i°When the alpha2u-globulin process is occurring, U.S. EPA f1991a) states that one of the following conclusions will be
made: (a] if renal tumors in male rats are attributable solely to the alpha2u-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 0C2U 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 alpha2u-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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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%, and the final average body weight reduction in male mice was 5% to 10%,
raising some question that the thyroid tumors were the result of excessive toxicity in mice rather
than carcinogenicity of tert-butanol. EPA's 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"
fU.S. EPA. 2005al. In the 2-year oral bioassay fNTP. 19951. study authors noted that water
consumption by exposed females and males 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
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
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
months)
Drinking water (0,1.25, 2.5, 5, or 10
mg/mL)
M: 0, 90, 200, or 420a mg/kg-d
Incidence15
Males Females
Dose Follicular cell Dose Follicular cell
(mg/kg-d) hyperplasia (mg/kg-d) hyperplasia
0 3/50 0 0/50
90 0/49 180 0/50
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Reference and study design
Results
F: 0,180, 330, or 650a mg/kg-d
200
0/50
330
0/50
2 years
420a
0/50
650a
0/50
NTP (1995)
B6C3Fi 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
Incidence (severity)
Males
Dose
(mg/kg-d)
Follicular cell
hyperplasia
Females
Dose
(mg/kg-d)
Follicular cell
hyperplasia
2 years
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)
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Reference and study design
Results
Follicular cell tumors
NTP (1995)
Incidence15
F344/N rat; 60/sex/treatment
(10/sex/treatment evaluated at 15
months)
Dose (ms/ks-d)
Follicular cell
adenoma
Follicular cell
carcinoma
Drinking water (0,1.25, 2.5, 5, or 10
Male
mg/mL)
M: 0, 90, 200, or 420a mg/kg-d
F: 0,180, 330, or 650a mg/kg-d
0
90
2/50
0/49
2/50
0/49
2 years
200
420a
Female
0
180
330
650a
0/50
0/50
1/50
0/50
1/50
0/50
0/50
0/50
1/50
0/50
1/50
0/50
NTP (1995)
Incidence
B6C3Fi mouse; 60/sex/treatment
Drinking water (0, 5,10, or 20 mg/mL)
M: 0, 540,1,040, or 2,070a mg/kg-d
Follicular cell
adenoma or
Animals
survivins to
study
F: 0, 510,1,020, or 2,110 mg/kg-d
2 years
Dose Follicular cell Follicular cell
(mortality
(ms/ks-d) adenoma carcinoma
adjusted rates)c,d
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
1
2 aSurvival in the high-dose group significantly decreased.
3 bResults do not include the animals sacrificed at 15 months.
4 cMortality-adjusted rates were not calculated by study authors for follicular cell carcinoma. The mortality-adjusted rates for the
5 incidence of adenomas are the same as the combined rates, with the exception of the male high-dose group, where the rate
6 for adenomas alone was 5.9%.
7 dCochran-Armitage trend test was applied to mortality-adjusted thyroid tumor incidences, by applying the NTP adjusted rates
8 to the observed numbers of tumors to estimate the effective number at risk in each group. For male mice, p = 0.041; for
9 female mice, p = 0.028.^Statistically significant p < 0.05 as determined by the study authors.
10 Note: Conversions from drinking water concentrations to mg/kg-d performed by study authors.
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¦ = exposures at which the endpoint was reported statistically significant by study authors
~ = exposures at which the endpoint was reported not statistically significant by study authors
Hyperplasia; M mouse
Hyperplasia; F mouse
NONCANCER
Hyperplasia; M rat
Hyperplasia; F rat
B
-B ~
~ B B
Adenoma; M mouse
Adenoma; F mouse
CANCER
Adenoma; M rat
Adenoma; F rat
10
Source: NTP (1995)
~ B B
~ B
B
-B B
~ B B
i 1—i—i—r~
—i 1 1 1—i—i i i
100 1,000
Dose (mg/kg-day)
10,000
2
3
4
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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Mode of Action Analysis—Thyroid Effects
The MOA responsible for tert-butanol-induced thyroid effects has been the subject of little
study. 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, as
identified in EPA's guidance on the assessment of thyroid follicular cell tumors (U.S. EPA. 1998b).
To determine if the thyroid follicular cell tumors result from a chemically-induced
antithyroid MOA, U.S. EPA f!998bl 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 pertaining to each
of these aspects of antithyroid activity following tert-butanol exposure is discussed below.
11 Increases in cell growth (required)
U.S. EPA f!998bl 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 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, 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. 1998b). 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 fBlanck 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 inhalation
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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. 1998b). Blanck etal. (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
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
B6C3Fi 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 timeframe; (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; (3) the maximal decrease in T3 or T4hormone 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%; fBlanck etal.. 20101],
Although the lower T3 and T4 levels following tert-butanol were later attributed by the study
authors to an increase in liver metabolism (see next section), alternatively, they could be due to a
variety of other possible, yet uninvestigated, molecular interactions of 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.
1998b).
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 if 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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31 Site f si of antithyroid action (required)
The thyroid and liver are two of several potential sites of antithyroid action, with the liver
the most common, where increased microsomal enzyme activity could enhance thyroid hormone
metabolism and removal fU.S. EPA. 1998bl. Rats are thought to be more sensitive than mice to this
aspect of antithyroid activity (Rogues et al.. 2 013: 0atanani et al.. 2 005: U.S. EPA. 1998b): 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. 1998b: NTP. 19951. In a 14-day mechanistic investigation,
tert-butanol had no effect on liver weight when compared to 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 et
al. (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. 19951. 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. 1995). 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 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 et
al. f20101]. 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 fOatanani 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 these few enzymes
are not supported by any liver histopathological effects in mice exposed for longer durations, so
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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 if major site(s)
of antithyroid action are affected.
4) 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 (U.S. EPA. 1998b). 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 fBlanck et
al.. 20101. Liver expression of CYP2B10 was increased in a dose-responsive manner, while
SULT1A1 mRNA was induced by 20-30% at both doses fBlanck etal.. 20101. As described above,
induction of liver microsomal enzyme activity would manifest some manner of liver histopathology
fMaronpotetal.. 2010: U.S. EPA. 1998b: 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.. 2010). 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 (NTP.
1997.1995). 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 fBlanck etal.. 20101.
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 due to 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.
51 Reversibility frequiredl
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. 1998b).
Although increased TSH levels have not been demonstrated following tert-butanol exposure,
thyroid follicular cell proliferation was observed following chronic exposure. As no studies have
evaluated changes in thyroid hormones or thyroid histopathology after cessation of tert-butanol
treatment, however, the available evidence is inadequate to evaluate reversibility of these effects.
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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 (5)
reversibility of effects in the early stages of disruption. Although these inadequacies also limit the
evaluation of (4) 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 if 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. 1998b! the evidence is inadequate to
determine if 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 (Faulkner et al.. 1989: Nelson etal.. 1989: Daniel and Evans. 1982) and a one-generation,
oral reproductive study (Huntingdon 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: Faulkner etal. (1989).
because it did not provide sufficient information on the dams to determine if fetal effects occurred
due to maternal toxicity, and Daniel and Evans (1982) 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.,
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increased number of resorptions, decreased numbers of neonates per litter) and decreased fetal
body weight fHuntingdon Life Sciences. 2004: Faulkner etal.. 1989: Daniel and Evans. 19821. Daniel
and Evans f!9821 observed decreases in pup body weight gain during post-natal days (PNDs) 2-10;
data suggest, however, 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 (Faulkner et al.. 1989). 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 fNelson et al.. 19891. Fetal effects included dose-related
reductions in body weight in male and female fetuses and higher incidence of skeletal variations
when analyzed based on individual fetuses (but not on a per litter basis).
In these studies, fetal effects are 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) (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 weeks beginning 4
weeks prior to mating
F0 females: 4 weeks prior to
mating through PND 21
F1 males and females: 7 weeks
(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
Percent change compared to control:
Dose
Body
weight
Food
Body
weight
Food
(mg/kg- gain GD consumption
d) 0-20 GD 0-20
0
64 -3 0
160 -4 0
400 0 4
1000 -16* 0
gain consumption Live pups/litter
PND 1-21 LP 1-14 response
3
-10
3
100*a
-2
-6
0
-16
-9
-11
-7
-33*
Dams dosed with 400 or 1000 mg/kg-d showed CNS effects (e.g., ataxia,
lethargy) that were undetectable by 4 weeks of exposure in animals exposed
to 400 mg/kg-d but not those in the higher dose group.a Large body weight
gain from PND 1-21 potentially due to make up from gestational losses and
smaller litter sizes during lactation.
F1 effects
Viability Lactation
Dose index (pup index (pup
Pup weight
weight/litter pND28 relative
PND 1
Sex ratio relative to
to control (%)
dl
PND 4)
PND 21)
(% males)
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
1000
74.1*
98.8
52.1
-10
-12*
-8
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Reference and study design
Results
Daniel and Evans (1982)
No statistical analysis was conducted on any of these data.
Swiss Webster (Cox) mouse; 15
Maternal
pregnant dams/treatment
Liquid diet (0, 0.5, 0.75,1.0%, w/v)
Percent change compared to control:
0 (isocaloric amounts of
maltose/dextrin), 3,324, 4,879,
6,677 mg/kg-d
GD 6-20
Dose
(mg/kg-d)
Food consumption
(mean g/animal/dav)
Number of
Bodv weight litters (%
gain 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 te/t-butanol dose groups
reflects problems with pair feeding and maternal sedation.
Fetal
Percent change compared to control:
Fetal bodv
Dose
Number of
weight on PND
(mg/kg-d)
0
3,324
neonates/litter
2
-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)
Maternal results not reported.
CBA/J mouse; 7 pregnant females
Fetal
in control, 12 pregnant females in
treated
Percent change compared to control: Incidence:
Gavage (10.5 mmoles/kg twice a
day);
0 (tap water), 1,556 mg/kg-d
GD 6-18
Dose
(mg/kg-d)
0
Live
fetuses/
Resorptions/litter litter
Fetal Sternebral Skull
weight variations variations
4/28 1/28
1,556
118* -41*
-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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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), 1,556 mg/kg-d
GD 6-18
Maternal results not reported.
Fetal
Percent change compared to control: Incidence:
Dose
(mg/kg-d)
0
1,556
Live
fetuses/
Resorptions/litter litter
Fetal Sternebral
Skull
weight variations variations
428*
-58*
-4
5/21
9/16
1/21
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,
5,030 ppm (0, 6,669, 10,640,
15,248 mg/m3), dynamic whole-
body chamber
7 hr/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
Dose
(mg/m3)
0
6,669
10,640
15,248
Percent change compared to control (mean ± standard error):
Number of live
Resorptions
fetuses/litter
per litter
-(13 ±2)
-(1.1 ± 1.2)
0(13 ±4)
9 (1.2 ± 1.1)
15 (15 ± 2)
-18 (0.9 ±
1.0)
8 (14 ±2)
0(1.1 ±0.9)
Percent change compared to
control:
Incidence:
Skeletal
Skeletal
Dose
Fetal weight
variation
variation
(mg/m3)
Fetal weight (males)
(females)
bv litter
bv fetus
0
-
-
10/15
18/96
6,669
_9*
_9*
14/17
35/104
10,640
-12*
-13*
14/14
53/103*
15,248
-32*
-31*
12/12
76/83*
Skeletal variation by litter refers to the number of variations observed in the
number of litters examined. Skeletal variation by fetus refers to the number of
variations observed in the total number of fetuses examined. Fetuses are not
categorized by litter.
1
2
3
4
^Statistically significant p < 0.05, as determined by study authors. Conversions from diet concentrations to mg/kg-d
performed by study authors. Conversion from ppm to mg/m3 is 1 ppm = 3.031 mg/m3.
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
¦IMaternal body weight gain
(GD 0-20); F rat (C)
TMaternal body weight gain
(LD 1-21); F rat (C)
INumberof live pups perlitter; M+F
rat (C)
I Viability index; M+F rat(C)
Lactation index; M+F rat (C)
Sex ratio; M+F rat (C)
!Pup weight per litter
(PND 1); M+F rat(C)
I Pup weight per litter
[PND 28); M rat (C)
IPup weight per litter
(PND 28); F rat (C)
1 Maternal body weight gain; F
mouse (A) *
iNumberof neonates/litter, fetal
body weight; M+F mouse (A)*
INumberof resorptions per litter;
M+F mouse (B)
INumberof live fetuses per litter;
M+F mouse (B)
I Fetal weight; M+F mouse (B)
Skeletal variations; M+F mouse (B)
~ B 0-
-B El
-a ~
-B ED
-a ~
1
2
3
4
10 100 1,000 10,000
Dose (mg/kg-day)
*Study authors did not conduct statistical analysis on these endpoints, but results are determined by EPA
to be biologically significant.
Sources: (A) Daniel and Evans (1982); (B) Faulkner et al. (1989); (C) Huntingdon Life Sciences (2004)
5
6
Figure 1-11. Exposure-response array of developmental 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
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. Selective
developmental toxicity of tert-butanol at the higher doses examined, however, cannot be ruled out.
Furthermore, no adverse effects were reported in one- and two-generation
reproductive/developmental studies on ETBE fGaoua. 2004a. 2004b), 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 (Nelson etal.. 1991: Daniel and Evans.
1982)[one in female mice; one in male rats; one in female rats] 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. Daniel and Evans f 19821 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 decreased dietary
intake compared to 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 (1982).
The two studies by Nelson etal. fl9911 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 (Table 1-14).
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Rotarod performance
Inconsistent results were observed across exposure routes and species Although Daniel
and Evans f!9821 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 in one study. In this study, Nelson etal. f!9911
reported statistically significant changes in neurochemical measurements 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 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 f!9821 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. Results indicated that pups fostered to control dams
performed significantly better than those maintained with treated dams (Table l-13]fDaniel and
Evans. 19821. These data suggest that neurodevelopmental effects were not solely due to in utero
exposure to tert-butanol (Daniel and Evans. 19821. Interpretation of these results is limited,
however, as the neurodevelopmental data were presented only in figures and could not be
compared with controls.
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1 Table 1-14. Evidence pertaining to neurodevelopmental effects in animals
2 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 hr/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 versus 26
revolutions/min for controls and 12,000 mg/m3 animals, respectively)
• decreased time held on wire in the performance ascent test in the
low-dose group (16 sec versus 10 sec for controls and 1,750 mg/m3 animals,
respectively)
• 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
• 57% decrease in met-enkephalin in the cerebrum at
12,000 mg/m3 and 83% decrease at 6,000 mg/m3
• 61% decrease in (3-endorphin in the cerebellum at 12,000 mg/m3
• 67% decrease in serotonin in the midbrain at 6,000 mg/m3
• no effects were observed for other neurotransmitter levels
(acetylcholine, dopamine, substance P) at both low and high doses
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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 hr/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 versus controls) in
offspring indicate
• increase in rotarod performance (16 versus 20 revolutions/min
for controls and 12,000 mg/m3 animals, respectively)
• decreased time in open field (less time to reach the outer circle of
the field, 210 sec versus 115 seconds for controls and 12,000 mg/m3
animals, respectively)
The following differences in neurochemical measurements in the brain
between control and treated offspring were observed:
• 39% decrease in norepinephrine in the cerebellum at
12,000 mg/m3
• 40% decrease in met-enkephalin in the cerebrum at
12,000 mg/m3 and 75% decrease at 6,000 mg/m3
• 71% decrease in (3-endorphin in the cerebellum at 12,000 mg/m3
• 47% decrease in serotonin in the midbrain at 6,000 mg/m3
1 ^Statistically significant p < 0.05, as determined by study authors.
2
3 Note: Conversions from diet concentrations to mg/kg-d performed by study authors.
4 Percentage change compared to control = (treated value - control value) 4- control value x 100.
5 Mechanistic Evidence
6 No mechanistic evidence for neurodevelopmental effects was identified by the literature
7 search. The available mechanistic information for tert-butanol is limited to three studies examining
8 muscarinic acetylcholine receptor function, and what, if any, relationship these effects might have
9 pertaining to developmental neurotoxicity effects remains unclear fBale and Lee. 20161.
10 Integration of Neurodevelopmental Effects
11 Neurodevelopmental effects, including decreased brain weight, changes in brain
12 biochemistry, and changes in behavioral performances, have been observed. Each study evaluating
13 neurodevelopmental effects, however, had limitations in study design, reporting, or both. In
14 addition, results were not always consistent between studies or across dose. Although minimal
15 effects were observed at otherwise toxic dose levels, the available evidence is considered
16 insufficient to identify neurodevelopmental effects as a potential human health hazard of tert-
17 butanol exposure.
18 1.2.5 Reproductive Effects
19 Synthesis of Effects Related to Reproduction
20 Several studies evaluated reproductive effects [a one-generation, oral reproductive study
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(Huntingdon Life Sciences. 20041 and subchronic effects in rats and mice following oral and
inhalation exposure fNTP. 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 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 fHuntingdon Life
Sciences. 2004: NTP. 1997.19951 (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 1000
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 B6C3Fi mice, estrous cycle
length was increased 28% following oral exposure to 11,620 mg/kg-day fNTP. 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) (Table
1-14). It is noteworthy that these effects were limited to the highest doses tested with some doses
accompanied by body weight loss or lethality.
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 weeks beginning 4 weeks prior to
mating
PND21
F0 reproductive effects
Sperm motility (only control and high-dose groups examined)
0: 94% 1000: 91%*
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, 3,610a mg/kg-d
13 weeks
No significant effect on weights of male reproductive organs or sperm
observed
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Reference and study design
Results
NTP (1995)
B6C3Fi mouse; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or 40 mg/mL)
M: 0, 350, 640, 1,590, 3,940, 8,210a mg/kg-d
13 weeks
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-day (0.115 mg to 0.096
mg) but Table H2 shows the same dose decreasing testis weight non-
significantly from 0.115 mg 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 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist Ultrasonic spray
nozzle nebulizer), analytical concentration and
method were reported
No significant effect on weights of male reproductive organs or sperm
observed
Evaluations were performed only for concentrations >542 ppm
(1,643 mg/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 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist Ultrasonic spray
nozzle nebulizer), analytical concentration and
method were reported
No significant effect on weights of male reproductive organs or sperm
observed
Evaluations were performed only for concentrations >542 ppm
(1,643 mg/m3)
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 weeks prior to mating through
PND21
Pregnancy index
91.7% 91.7% 100% 100% 91.7%
NTP (1995)
F344/N rat; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or 40 mg/mL)
F: 0, 290, 590, 850, 1,560, 3,620a mg/kg-d
13 weeks
No significant effect on female estrous cycle length (0, -2, -4, 0, 8% change
relative to control)
Note: Number of animals that had > 7-day cycle length, unclear cycles, or
no cycles
0: 0 1,560: 2/10 3,620: 4/4"
NTP (1995)
B6C3Fi mouse; 10/sex/treatment
Drinking water (0, 2.5, 5,10, 20, or 40 mg/mL)
F: 0, 500, 820, 1,660, 6,430, ll,620a mg/kg-d
13 weeks
'T* length of estrous cycle
Response relative to control: 0, 5, 5, 5, 6, 28*%
Note: Animals with > 7-day 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: 4/6*"
1
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Reference and study design
Results
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 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist Ultrasonic spray
nozzle nebulizer), analytical concentration and
method were reported
No significant effect on female estrous cycle 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)
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 hr/d, 5 d/wk
13 weeks
Generation method (Sonimist Ultrasonic spray
nozzle nebulizer), analytical concentration and
method were reported
No significant effect on female estrous cycle 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-day cycle length, unclear cycles, or no
cycles
0:0/10 542: 2/10 1,080: 1/10 2,101: 3/10
1 ^Statistically significant p < 0.05, as determined by the study authors.
2 **Statistically significant p < 0.01, as determined by EPA.
3 ***Statistically significant p < 0.05, as determined by EPA.
4
5 Notes: Conversions from drinking water concentrations to mg/kg-d performed by study authors.
6 Conversion from ppm to mg/m3 is 1 ppm = 3.031 mg/m3.
7 Percent change compared to control = (treated value - control value) 4- 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 fGaoua. 2004a.
2004b). 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. Due to a lack of consistency in the liver effects and minimal-to-
mild effects with a lack of progression in urinary bladder, however, inadequate information is
available 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. Due to 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.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) fATSDR. 19961.
Based on mechanistic evidence indicating that an alpha2u-globulin -related process is
operating in male rats fHard etal.. 2011: Cirvello etal.. 1995: NTP. 1995: Lindamood et al.. 19921.
any kidney effects associated with alpha2u-globulin nephropathy are not considered relevant for
human hazard identification. Because alpha2u-globulin nephropathy contributes to CPN, CPN and
CPN-associated lesions in male rats were not considered 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 alpha2u-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). Dams had body weight losses or gains (or both), decreased food consumption, and
clinical signs of intoxication, however, 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, due to the uncertainty regarding whether
fetal effects were due to direct effects of tert-butanol or indirect effects of maternal toxicity and the
lack of consistency across some endpoints.
Although minimal effects were observed at otherwise toxic dose levels, the available
evidence is considered insufficient to identify neurodevelopmental effects as a potential human
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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
across exposure routes and species, and 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 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 B6C3Fi 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 fNTP. 19951. as discussed in Section 1.2.2. According to EPA's thyroid tumor
guidance (U.S. EPA. 1998b). 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
alpha2u-globulin nephropathy, an MOA considered specific to the male rat fU.S. EPA. 1991al.
Evidence in support of this hypothesized MOA includes the accumulation of hyaline droplets in
renal tubule cells, the presence of alpha2u-globulin in the hyaline droplets, and additional aspects
associated with alpha2u-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 alpha2u-globulin were low at doses that induced tumors;
and no significant necrosis or cytotoxicity was associated with compensatory regenerative
proliferation or induction of granular casts observed within a timeframe consistent with alpha2u-
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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 alpha2u-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 (U.S. EPA. 1991a). 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 T1999IIARC 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 oral gavage in male and female Sprague-Dawley rats (U.S. EPA, 2017).
Integration of evidence
This evidence leads to consideration of two hazard descriptors under EPA's cancer
guidelines (U.S. EPA. 2005a). 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
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 B6C3Fi mice.
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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 was strong or
coherent with the results for ETBE, which 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's 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. 1998b) 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 not adequate or are not supportive. There is
little correlation among 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. 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.
EPA's 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 the magnitude and uncertainty
of potential risks from tert-butanol exposure, including worker or consumer exposures, an analysis
of thyroid tumors is presented in Section 2.
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EPA's guidance on renal tumors in male rats (U.S. EPA. 1991al advises that, unless the
relative contribution of alpha2u-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 (MFO) 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, sulfotransferase 1A1 [(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 to 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 (Lee etal.. 20111 and decreased in older mice (Lee etal.. 20111 and rats (Lee et
al.. 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. 1998b). 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 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 95% lower bound on the benchmark dose (BMDL),
with uncertainty factors (UF values) generally applied to reflect limitations of the data used.
2.1.1 Identification of Studies and Effects for Dose-Response Analysis
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 f2002al] 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 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 observed after chronic exposure, such as suppurative inflammation and transitional
epithelial hyperplasia, 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 (2004) conducted a
reproductive study in Sprague-Dawley rats that was of shorter duration, and reported changes in
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kidney weight but did not examine changes in histopathology. 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 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 alpha2u-
globulin, male kidney effects are not considered. 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 (U.S. EPA. 2012a). the BMD and the
BMDL are estimated using a benchmark response (BMR) to represent a minimal, biologically
significant level of change. In the absence of information regarding 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
confidence in the absolute kidney weight measure, it was considered more appropriate for dose-
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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 because the data was not amenable to modeling.
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 fU.S. EPA. 2011al. 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 ETBE or inhalation and dermal
exposure to 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 oral RfD.
Consistent with EPA guidance (U.S. EPA. 2011a). the PODs estimated based on effects in
adult animals were converted to HEDs employing a standard dosimetric adjustment factor (DAF)
derived as follows:
DAF = (BWaV4 / BWh1/4),
where
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 (U.S. EPA. 1988).
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):
PODhed = Laboratory animal dose (mg/kg-day) x DAF
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.
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1 Table 2-1. Summary of derivations of points of departure following oral
2 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
months
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
3 aFor modeling details, see Appendix C in Supplemental Information.
4 bFor studies in which animals were not dosed daily, EPA would adjust administered doses to calculate the time-
5 weighted average daily doses prior to BMD modeling. This adjustment was not required for the NTP (1995) study.
6 CHED PODs were calculated using BW3/4scaling (U.S. EPA, 2011a).
7 dPOD calculated from the LOAEL (lowest dose tested had a significant increase in severity).
8 NA= not applicable
9 2.1.3 Derivation of Candidate Values
10 Consistent with EPA's A Review of the Reference Dose and Reference Concentration Processes
11 [fU.S. EPA. 2002al: Section 4.4.5], also described in the Preamble, five possible areas of uncertainty
12 and variability were considered when determining the application of UF values to the PODs
13 presented in Table 2-1. An explanation follows.
14 An intraspecies uncertainty factor, UFh, of 10 was applied to all PODs to account for
15 potential differences in toxicokinetics and toxicodynamics in the absence of information on the
16 variability of response in the human population following oral exposure to tert-butanol fU.S. EPA.
17 2002a).
18 An interspecies uncertainty factor, UFa, of 3 (10°5 = 3.16, rounded to 3) was applied to all
19 PODs because BW3/4 scaling was used to extrapolate oral doses from laboratory animals to humans.
20 Although BW3/4 scaling addresses some aspects of cross-species extrapolation of toxicokinetic and
21 toxicodynamic processes, some residual uncertainty in the extrapolation remains. In the absence of
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chemical-specific data to quantify this uncertainty, EPA's BW3/4 guidance fU.S. EPA. 2011al
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, UF, 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 etal..
1997: Acharva etal.. 1995: NTP. 19951 and developmental toxicity studies in rats and mice
fHuntingdon Life Sciences. 2004: Faulkner etal.. 1989: Daniel and Evans. 19821. In the
developmental studies, no effects were observed at exposure levels below 1000 mg/kg-day, and
effects observed at >1000 mg/kg-day were accompanied by evidence of maternal toxicity. These
exposure levels are much higher than the PODs 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 consideration of the
gaps in the tert-butanol oral evidence base. The evidence base for ETBE does not indicate
immunotoxicity fBanton etal.. 2011: Li etal.. 20111. 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 (Gaoua. 2004a. 2004b). 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. 20041 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 database UFd
of 1 was applied.
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
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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 months
NTP (1995)
22
BMDLio%
3
10
1
1
1
30
7 x 101
Kidney inflammation (suppurative);
female rat at 2 vears NTP (1995)
48
BMDLio%
3
10
1
1
1
30
2 x 10°
Kidney transitional epithelial
hyperplasia; female rat at 2 years
NTP (1995)
81
BMDLio%
3
10
1
1
1
30
3 x 10°
Increases in severity of
nephropathy; female rat at 2 years
NTP (1995)
43.2
LOAEL
3
10
3
1
1
100
4 x 101
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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
100
mg/kg-day
Figure 2-1. Candidate values with corresponding POD and composite UF. Each
bar corresponds to one data set described in Table 2-1 and Table 2-2.
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2.1.4 Derivation of Organ/System-Specific Reference Doses
Table 2-3 distills the candidate values from Table 2-2 into a single value for each organ or
system. Organ- or system-specific RfDs are useful for subsequent cumulative risk assessments that
consider the combined effect of multiple agents acting at a common site.
Kidney Toxicity
For tert-butanol, candidate values were for several different kidney effects in female rats,
spanning a range from 4 x 101 to 3 x 10° mg/kg-day, for an overall 7.5-fold range. 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. This indicator of kidney toxicity is
more specific 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 uncertainty surrounding human
relevance of CPN which remains unresolved.. The POD for increases in severity of nephropathy is
based on a LOAEL, and 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 RfDs and overall RfD for tert-butanol
Effect
Basis
RfD (mg/kg-day)
Study exposure
description
Confidence
Kidney
Increases in severity of
nephropathy (NTP, 1995)
4 x 101
Chronic
Medium
Overall RfD
Kidney
4 x 101
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-i 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
susceptible subgroups (U.S. EPA. 2002al 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
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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, 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 (U.S. EPA.
1994a], Confidence in the principal study fNTP. 1995] 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 evidence base is medium.
Reflecting high confidence in the principal study and medium confidence in the evidence base,
confidence in the RfD is medium.
2.1.7 Previous 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 RfC (expressed in units of mg/m3) is defined as an estimate (with
uncertainty spanning perhaps an order of magnitude) of a continuous inhalation exposure to the
human population (including sensitive subgroups) that is likely to be without an appreciable risk of
deleterious effects during a lifetime. It can be derived from a NOAEL, LOAEL, or the 95% lower
bound on the benchmark concentration (BMCL), with UF values generally applied to reflect
limitations of the data used.
2.2.1 Identification of Studies and Effects for Dose-Response Analysis
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 (NTP. 1997). A rat PBPK
model was available for both oral and inhalation exposure, which was suitable for a route-to-route
extrapolation fBorghoffetal.. 20161. As a result, rat studies from both routes of exposure were
considered for dose-response analysis.
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
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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 alpha2u-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
(19971 inhalation study is the only route-specific study available, and was carried forward for
further analysis. For oral studies considered for route-to-route 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 (1995) 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 route-to-route
extrapolation. This study was discussed previously in Section 2.1.1 as part of the derivation of the
oral reference dose, so is not reviewed here again. The NTP (19971 subchronic inhalation study
shares many strengths with the 2-year drinking water study fNTP. 19951 and is described in more
detail below.
NTP (1997) 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 (1995) 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 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.
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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 fU.S. EPA. 1994al provides
mechanisms for (1) adjusting experimental exposure concentrations to a value reflecting
continuous exposure duration (ADJ) and (2) determining a human equivalent concentration (HEC)
from the animal exposure data. The former employs an inverse concentration-time relationship to
derive a health-protective duration adjustment to time weight the intermittent exposures used in
the studies. The modeled benchmark concentration (BMCL) from the inhalation study (NTP. 19971
was adjusted to reflect a continuous exposure by multiplying it by (6 hours per day) 4- (24 hours
per day) and (5 days per week) 4 (7 days per week) as follows:
BMCLadj = BMCL (mg/m3) x (6 -h 24) x (5 4 7)
= BMCL (mg/m3) x (0.1786)
The RfC methodology provides a mechanism for deriving an HEC from the duration-
adjusted POD (BMCLadj) determined from the animal data. The approach takes into account the
extra-respiratory nature of the toxicological responses and accommodates species differences by
considering blood:air partition coefficients for tert-butanol in the laboratory animal (rat or mouse)
and humans. According to the RfC guidelines (U.S. EPA. 1994a). tert-butanol is a Category 3 gas
because extrarespiratory effects were observed. Kaneko etal. (2000) measured a blood:gas
partition coefficient [(Hb/g)A] of 531 ± 102 for tert-butanol in the male Wistar rat, while Borghoff et
al. f!9961 measured a value of 481 ± 29 in male F344 rats. A blood:gas partition coefficient [(Hb/g)H]
of 462 was reported for tert-butanol in humans fNihlen et al.. 19951. The calculation, (Hb/g)A +
(Hb/g)H, was used to calculate a blood:gas partition coefficient ratio to apply to the delivered
concentration. Because F344 rats were used in the study, the blood:gas partition coefficient for
F344 rats was used. Thus, the calculation was 481 4 462 = 1.04. Therefore, a ratio of 1.04 was used
to calculate the HEC. This allowed a BMCLhec to be derived as follows:
BMCLhec = BMCLadj (mg/m3) x (interspecies conversion)
= 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
weeks
NTP (1997)
Female F344
rats
No model
selectedd
10%
1137
1137
aFor modeling details, see Appendix C in Supplemental Information.
bBMCs, BMCLs, and PODs were adjusted for continuous daily exposure by multiplying by (hours exposed per day /
24 hr) x (days exposed per week / 7 days).
cPODHec calculated by adjusting the PODadj by the DAF (= 1.0, rounded from 1.04) for a Category 3 gas (U.S. EPA.
1994a).
dBMD modeling failed to calculate a BMD value successfully (see Appendix C); POD calculated from NOAEL of
6368 mg/m3.
PODs from oral studies - use ofPBPK model for route-to-route 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 route-to-route 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 that suggest that metabolites of
tert-butanol mediate its renal toxicity. Without evidence that suggests otherwise, tert-butanol is
assumed 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 vs. concentration, and
this differs for oral vs. 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/d 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
there isn't a sub-model for the key metabolite(s) which can be used to estimate 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.
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Using the PBPK model, route-to-route 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 non-
continuous 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, since 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 human equivalent
concentration POD (PODhec) using the methodology previously described in the section, PODs from
inhalation studies:
PODhec = POD (mg/m3) x (interspecies conversion)
POD (mg/m3) x (481 -h 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.
Table 2-5. Summary of derivation 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 dosea
(mg/L)
Equivalent
PODb (mg/m3)
Equivalent
PODHEcc (mg/m3)
Kidney
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Endpoint and
reference
Species/sex
BMR
BMDL
(mg/kg-d)
Internal dosea
(mg/L)
Equivalent
PODb (mg/m3)
Equivalent
PODHEcc (mg/m3)
Mean absolute kidney
weight at 15 months
NTP (1995)
Rat/F
10%
91
21.5
238.9
248
Kidney inflammation
(suppurative) at 2
vears NTP (1995)
Rat/F
10%
200
61.9
523.7
545
Kidney transitional
epithelial hyperplasia
at 2 vears NTP (1995)
Rat/F
10%
339
127
883.9
919
Species/sex
POD (LOAEL; mg/kg-d)
Internal dosea
(mg/L)
Equivalent
PODb (mg/m3)
Equivalent
PODHEcc (mg/m3)
Increases in severity of
nephropathy at 2
years
NTP (1995)
Rat/F
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 route-to-route (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 response with internal dose irrespective of
exposure route, including combined inhalation and oral exposures
fhttps: //heronet.epa.gov/heronet/index.cfm/reference/details/referenee id/19361081 On the
other hand, 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
fhttps://he ronet.epa.gov/heronet/index.cfm/reference/details/reference id/26343091. 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 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 have an effect on internal tissues. Therefore, the toxicological activity must be related to
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blood concentration, although it may not be exactly predicted by a particular metric. In the case of
tert-butanol, there is toxicological uncertainty due to the fact that 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 sub-chronic 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 U.S. 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 [fU.S. EPA.
2002a): 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
route-to-route extrapolation from the PODs estimated from the chronic oral toxicity study in rats
(NTP. 1995) in the derivation of the oral RfD (Section 2.1). With the exception of the subchronic
inhalation (NTP. 1997) study, the UF values selected and applied to PODs derived from the chronic
oral (NTP. 1995) study for route-to-route extrapolation are the same as those for the RfD for tert-
butanol (see Section 2.1.3). The model used to perform this route-to-route extrapolation is a well-
characterized model considered appropriate for the purposes of this assessment.
For the PODs derived from the subchronic inhalation fNTP. 19971 study, 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 the selection of 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.
Table 2-6. Effects and corresponding derivation of candidate values
Endpoint (sex and species) and
reference
PODhec3
(mg/m3)
POD
type
UFa
UFh
ufl
UFS
UFd
Composite
UF
Candidate
value
(mg/m3)
Kidney
Increased absolute kidney weight
at 13 weeks; female rat
1137
NOAEL
3
10
1
10
1
300
4x 10°
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Endpoint (sex and species) and
reference
PODhec3
(mg/m3)
POD
type
ufa
UFh
ufl
UFS
ufd
Composite
UF
Candidate
value
(mg/m3)
NTP (1997)
Increased absolute kidney weight
at 15 months; female rat
NTP (1995)
248
BMCLio%
3
10
1
1
1
30
8 x 10° *
Kidney inflammation
(suppurative); female rat at 2
years
NTP (1995)
546
BMCLio%
3
10
1
1
1
30
2 x 101*
Kidney transitional epithelial
hyperplasia; female rat at 2 years
NTP (1995)
920
BMCLio%
3
10
1
1
1
30
3 x 101*
Increases in severity of
nephropathy; female rat at 2
years
NTP (1995)
491
LOAEL
3
10
3
1
1
100
5 x 10° *
1 These candidate values are derived using route-to-route extrapolated PODs based on NTP's chronic drinking
2 water study.
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X Absolut* kidnty svtdfht
at 13 wmVji f*m*W r*t
[NTPr 1997]
T Absolute kidney weight
at 15 months: female rat
[NTP, 1995]
Kidney Liiflauiina tio u
(j uppuritlv#]i f*mal* rat
(NTP, 1995]
Kidney tra lisitia ua 1
•pi t h *! I*\ hyptrpUs la i
female rat [NTP, 1995]
Nephropathy severity:
famuli rat (NTP, 1995]
^ Candidate RfC
• PODbk
Composite UF
ID
100
1000
10000
mg/itr
1 Figure 2-2. Candidate RfC values with corresponding POD and composite UF.
2 2.2.4 Derivation of Organ/System-Specific Reference Concentrations
3 Table 2-7 distills the candidate values from Table 2-6 into a single value for the kidney.
4 Organ-/system-specific reference values can be useful for subsequent cumulative risk assessments
5 that consider the combined effect of multiple agents acting at a common site.
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Kidney Toxicity
For the derivation of candidate values, whether PODs from the subchronic inhalation study
of NTP T19971 would provide a better basis than the route-to-route extrapolated PODs based on the
chronic oral study of NTP T19951 must be considered. Candidate values were derived for increased
kidney weight observed in the subchronic inhalation study (NTP. 1997) and several kidney effects
observed in the chronic oral study (NTP. 1995) in female rat, spanning a range from 4 x 10° to
3 x 101 mg/m3, for an overall 7.5-fold range. To estimate an exposure level below which kidney
toxicity from tert-butanol exposure is not expected to occur, the RfC for increased 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). This
endpoint is based on a longer (chronic) duration and a more specific and sensitive indicator of
kidney toxicity than the relatively nonspecific endpoint of kidney weight change and less sensitive
endpoints of inflammation and hyperplasia. Confidence in this kidney-specific RfC is medium. The
POD for increases in severity of nephropathy is based on a LOAEL, and 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, and availability of a PBPK
model for route-to-route extrapolation.
Table 2-7. Organ-/system-specific RfCs and overall RfC for tert-butanol
Effect
Basis
RfC
(mg/m3)*
Study exposure
description
Confidence
Kidney
Increases in severity of
nephropathy (NTP, 1995)
5 x 10°
Chronic
Medium
Overall RfC
Kidney
5 x 10°
Chronic
Medium
* Derived from oral study, by route-to-route 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 (U.S. EPA. 2002a). 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
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to the RfC. In the case of tert-butanol, the potential exists for early lifestage susceptibility to tert-
butanol exposure, as discussed in Section 1.3.3.
2.2.6 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 (U.S. EPA.
1994a). A PBPK model was used to perform a route-to-route extrapolation to determine a POD for
the derivation of the RfC from the NTP T19951 oral study and corresponding critical effect.
Confidence in the principal study fNTP. 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 a route-to-route extrapolation, the
overall confidence in the RfC for tert-butanol is medium.
2.2.7 Previous 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 fU.S. EPA. 2000.1994b) 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
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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
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 were found to appear more susceptible than mice,
and males appear 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 MOA analysis conducted for the kidney
effects. The assessment concluded that tert-butanol is a weak inducer of alpha2u-globulin, which is
operative in male kidney tumors; therefore, noncancer effects related to alpha2u-globulin were
considered not relevant for hazard identification and, therefore, not suitable for dose response
consideration. If this conclusion was incorrect and the noncancer effects characterized in this
assessment as being related to alpha2u-globulin were relevant to humans, the RfD and RfC values
could underestimate toxicity. The assessment also used noncancer effects related to CPN in
derivation of 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. 2005a) state:
When there is suggestive evidence, the Agency generally would not attempt a dose-
response assessment, as the nature of the data generally would not support one; however
when the evidence includes a well-conducted study, quantitative analysis may be useful for
some purposes, for example, 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.
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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
fNTP. 19951. which reported renal tumors in male rats and thyroid tumors in both male and female
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 fHard 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
exact test, p < 0.05) and by trend test (Cochran-Armitage trend test, p < 0.05). Based on a mode of
action analysis, the alpha2u-globulin process was concluded to be at least partially responsible for
the male rat renal tumors, in addition to other, unknown, processes. Because the relative
contribution of each process to tumor formation cannot be determined (U.S. EPA. 1991a). the male
rat renal tumors are not considered suitable for quantitative analysis. Conversely, the mouse
thyroid tumors are suitable for dose-response analysis and unit risk estimation, as described in
Section 1.3.2.. 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 at the highest dose was 12% (NTP, 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.
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. While 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 due to the suggestive nature of the tumorigenic response
flJ.S. FPA. 2005al.
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2.3.2 Dose-Response Analysis—Adjustments and Extrapolations Methods
The EPA Guidelines for Carcinogen Risk Assessment fU.S. EPA. 2005al recommend that
determining the method to use 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 two-step approach that distinguishes analysis of the observed dose-
response data from inferences about lower doses (U.S. EPA. 2005al. 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. 2005a). Otherwise, linear low-dose extrapolation is recommended if the MOA of
carcinogenicity is mutagenic or has not been established fU.S. EPA. 2005al 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 male mice 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. 2011a. 2005a). In particular, the
BMDL was converted to an HED by assuming that doses in animals and humans are toxicologically
equivalent when scaled by body weight raised to the 3/4 power. 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 the
conversion of 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
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= (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.
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/BMDLi0). 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 104 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 '1 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 B6C3Fi 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 /[0.14 x (2002 mg/kg-day)] =
4 x 10"4 per mg/kg-day.
11 This value is 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 fU.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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1 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-day)1
Thyroid follicular
cell adenoma
B6C3Fi
mouse/Female
3°
Multistage
10%
2002
1437
201
5 x 10"4
Thyroid follicular
cell adenoma or
carcinoma
B6C3Fi
mouse/Male
All dose
groups: 1°
Multistage
5%c
1788
787
110
5 x 10"4
High dose
omitted: 2°
Multistage
5%c
1028
644
90
6 x 10"4
2 aHED PODs were calculated using BW3/4scaling (U.S. EPA, 2011a).
3 bHuman equivalent slope factor = 0.1/BMDLiohed; see Appendix C of the Supplemental Information for details of
4 modeling results. These values are uncertain because it is based on a determination of suggestive evidence of
5 carcinogenic potential; however, the slope factors may be useful for some decision purposes such as providing a
6 sense of the magnitude of potential risks or ranking potential hazards (U.S. EPA. 2005al The uncertainties in the
7 data leading to this suggestive weight of evidence determination for carcinogenicity are detailed in Section 2.3.4.
8 below.
9 cBecause the observed responses were <10%, a BMR of 5% was used to represent the observed response range for
10 low-dose extrapolation; human equivalent slope factor = 0.05/BMDL5hed-
11 2.3.4 Uncertainties in the Derivation of the Oral Slope Factor
12 There is uncertainty when extrapolating data from animals to estimate potential cancer
13 risks to human populations from exposure to tert-butanol.
14 Table 2-9 summarizes several uncertainties that could affect the oral slope factor. There are
15 no other chronic studies to replicate these findings or that examined other animal models, no data
16 in humans to confirm a cancer response in general or the specific tumors observed in the NTP
17 (1995) bioassay, and no other data (e.g., MOA) to support alternative approaches for deriving the
18 oral slope factor.
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1 Table 2-9. Summary of uncertainties in the derivation of the oral slope factor
2 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, as
the fraction of rat kidney tumors not
attributed to the male rat specific
Ob^-globulin process could not be
determined. Alternatively,
quantifying rat kidney tumors could
T* slope factor to 1 x 10"2 mg/kg-day
(see Appendix C, Supplemental
Information)
Thyroid tumors in female
and male mice were
selected U.S. EPA (1998b),
U.S. EPA (1991a)
MOA data suggested that mouse thyroid
tumors were relevant to humans.
Quantitation of thyroid tumors in male mice,
which was impacted 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 add support to
the findings, facilitate determination of what
fraction of kidney tumors are not attributable
to the ob^-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 impact 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 T* 2-fold [scaling by
BW 2/3])
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 neither to 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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Consideration and
impact on cancer risk value
Decision
Justification
Low-dose extrapolation:
4/ cancer risk estimate would be
expected with the application of
nonlinear low-dose extrapolation
Linear extrapolation of risk
in low-dose region used
U.S. EPA (1998b)
Linear low-dose extrapolation for agents
without a known MOA is supported (U.S.
EPA, 2005a) and recommended for rodent
thyroid tumors arising from an unknown
MOA (U.S. EPA, 1998b).
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% CI 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 IRIS Assessment: Oral Slope Factor
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 (IUR) 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 B6C3Fi mice, and increased thyroid follicular
cell adenomas and carcinomas in male B6C3Fi mice. Because only a rat PBPK model exists,
however, route-to-route extrapolation cannot be performed for thyroid tumors in mice at this time.
The NTP f!9951 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 T19951 drinking
water study were not used for route-to-route extrapolation because enough information to
determine the relative contribution of alpha2u-globulin nephropathy and other processes to the
overall renal tumor response fU.S. EPA. 1991a) is not available.
2.4.1 Previous IRIS Assessment: Inhalation Unit Risk
An inhalation cancer assessment for tert-butanol was not previously available on IRIS.
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1 2.5 APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS
2 As discussed in the Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to
3 Carcinogens fU.S. EPA. 2005cl. either default or chemical-specific age-dependent adjustment factors
4 (ADAFs) are recommended to account for early-life exposure to carcinogens that act through a
5 mutagenic MOA. Because chemical-specific lifestage susceptibility data for cancer are not available,
6 and because the MOA for tert-butanol carcinogenicity is not known (see Section 1.3.2), application
7 of ADAFs is not recommended.
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This document is a draft for review purposes only and does not constitute Agency policy.
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This document is a draft for review purposes only and does not constitute Agency policy.
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trimethylpentane in the male rat kidney. Toxicol Pathol 14: 194-203.
This document is a draft for review purposes only and does not constitute Agency policy.
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tox-risk-assessment.htm
This document is a draft for review purposes only and does not constitute Agency policy.
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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.
This document is a draft for review purposes only and does not constitute Agency policy.
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This document is a draft for review purposes only and does not constitute Agency policy.
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