*>EPA
EPA/63 5/R-16/079a
Public Comment Draft
www.epa.gov/iris
Toxicological Review of tert-Butyl Alcohol (tert-Butanol)
(CAS No. 75-65-0]
April 2016
NOTICE
This document is a Public Comment Draft. This information is distributed solely for the purpose of
pre-dissemination peer review under applicable information quality guidelines. It has not been
formally disseminated by EPA. It does not represent and should not be construed to represent any
Agency determination or policy. It is being circulated for review of its technical accuracy and
science policy implications.
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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Toxicological Review of tert-Butyl Alcohol
DISCLAIMER
This document is a preliminary draft for review purposes only. This information is
distributed solely for the purpose of pre-dissemination peer review under applicable information
quality guidelines. It has not been formally disseminated by EPA. It does not represent and should
not be construed to represent any Agency determination or policy. Mention of trade names or
commercial products does not constitute endorsement or recommendation for use.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
CONTENTS
AUTHORS | CONTRIBUTORS | REVIEWERS viii
PREFACE x
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS xiv
EXECUTIVE SUMMARY ES-1
LITERATURE SEARCH STRATEGY | STUDY SELECTION AND EVALUATION LS-1
1. HAZARD IDENTIFICATION 1-1
1.1. Overview of Chemical Properties and Toxicokinetics 1-1
1.1.1. Chemical Properties 1-1
1.1.2. Toxicokinetics 1-2
1.1.3. Description of Toxicokinetic Models 1-3
1.1.4. Chemicals Extensively Metabolized to ferf-Butanol 1-4
1.2. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM 1-4
1.2.1. Kidney Effects 1-4
1.2.2. Thyroid Effects 1-38
1.2.3. Developmental Effects 1-46
1.2.4. Neurodevelopmental Effects 1-53
1.2.5. Reproductive Effects 1-56
1.2.6. Other Toxicological Effects 1-61
1.3. INTEGRATION AND EVALUATION 1-61
1.3.1. Effects Other Than Cancer 1-61
1.3.2. Carcinogenicity 1-63
1.3.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes 1-65
2. DOSE-RESPONSE ANALYSIS 2-1
2.1.ORAL REFERENCE DOSE FOR EFFECTS OTHERTHAN CANCER 2-1
2.1.1. Identification of Studies and Effects for Dose-Response Analysis 2-1
2.1.2. Methods of Analysis 2-2
2.1.3. Derivation of Candidate Values 2-4
2.1.4. Derivation of Organ/System-Specific Reference Doses 2-8
2.1.5. Selection of the Overall Reference Dose 2-8
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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 2-13
2.2.4. Derivation of Organ/System-Specific Reference Concentrations 2-16
2.2.5. Selection of the Overall Reference Concentration. 2-16
2.2.6. Confidence Statement 2-17
2.2.7. Previous IRIS Assessment 2-17
2.2.8. Uncertainties in the Derivation of the . ence Dose ark ference
Concentration 2-17
2.3. ORAL SLOPE FACTOR FOR CANCER 2-18
2.3.1. Analysis of Carcinogenicity Data 2-18
2.3.2. Dose-Response Analysis—Adjustments and Extrapolations Methods 2-19
2.3.3. Derivation of the Oral Slope Factor 2-20
2.3.4. Uncertainties in the Derivation of the Oral Slope Factor 2-21
2.3.5. Previous IRIS Assessment: Oral Slope Factor 2-23
2.4. INHALATION UNIT RISK FOR CANCER 2-23
2.4.1. Previous IRIS Assessment: Inhalation Unit Risk 2-24
2.5. APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS 2-24
REFERENCES R-l
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Toxicological Review of tert-Butyl Alcohol
TABLES
Table ES-1. Organ/system-specific RfDs and overall RfD for tert-butanol ES-2
Table ES-2. Organ/system-specific RfCs and overall RfCfor ferf-butanol ES-3
Table LS-1. Details of the search strategy employed for ferf-butanol LS-4
Table LS-2. Summary of additional search strategies for ferf-butanol LS-4
Table LS-3. Inclusion-exclusion criteria LS-5
Table LS-4. Considerations for evaluation of experimental animal studies LS-8
Table LS-5. Summary of experimental animal database LS-8
Table 1-1. Physicochemical properties and chemical identity of terf inol 1-1
Table 1-2. Changes in kidney histopathology in animals followin- jsure to ferf-butanol 1-12
Table 1-3. Changes in kidney tumors in animals following exr -> ferf-butanol 1-15
Table 1-4. Summary of data on the a2u-globulin process in _¦ rat-., ^osed to ferf-butanol 1-22
Table 1-5. Proposed empirical criteria for attributing rf i tumors to C 1-33
Table 1-6. Evidence pertaining to thyroid effects in " .•is following oral isure to ferf-
butanol 1-39
Table 1-7. Evidence pertaining to developmental et, - in animals following ex, 're to ferf-
butanol 1-48
Table 1-8. Evidence pertaining to neurodevelopmental effects in animals following exposure to
ferf-butanol 1-54
Table 1-9. Evidence pertaining to reproductive effects in animals following exposure to ferf-
butanol 1-56
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 2-14
Table 2-7. Organ/system-specific RfCs and overall RfCfor ferf-butanol 2-16
Table 2-8. Summary of the oral slope factor derivation 2-21
Table 2-9. Summary of uncertainties in the derivation of the oral slope factor for ferf-butanol 2-22
FIGURES
Figure LS-1. Summary of literature search and screening process for ferf-butanol LS-3
Figure 1-1. Biotransformation of ferf-butanol in rats and humans 1-3
Figure 1-2. Comparison of absolute kidney weight change in male and female rats across oral
and inhalation exposure based on internal blood concentration. 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 1-10
Figure 1-3. Comparison of absolute kidney weight change in male and female mice following oral
exposure based on administered concentration. Spearman rank correlation
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Toxicological Review of tert-Butyl Alcohol
coefficient (rho) was calculated to evaluate the direction of a monotonic
association (e.g., positive value = positive association) and the strength of
association 1-11
Figure 1-4. Comparison of absolute kidney weight change in male and female mice following
inhalation exposure based on administered 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 1-11
Figure 1-5. Exposure response array for kidney effects following oral exposure to ferf-butanol 1-17
Figure 1-6. Exposure-response array of kidney effects following inhalation exposure to ferf-
butanol (13-week studies, no chronic studies available) 1-18
Figure 1-7. Temporal pathogenesis of a2u-globulin-associated nephropathy in male rats. a2u-
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 ferf-butanol (TBA) exposure and increasing droplet accumulation can
progress step-wise 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 (Adapted from Swenberg and Lehman-McKeeman
(1999) and U.S. EPA (1991a) 1-21
Figure 1-8. Exposure-response array for effects potentially associated with a2u-globulin renal
tubule nephropathy and tumors in male rats after oral exposure to ferf-butanol 1-24
Figure 1-9. Exposure-response array for effects potentially associated with a2u-globulin renal
tubule nephropathy and tumors in male rats after inhalation exposure to
ferf-butanol 1-25
Figure 1-10. Exposure-response array of thyroid follicular cell effects following chronic oral
exposure to ferf-butanol. (Note: Only one carcinoma was observed in male mice
at the high-dose group.) 1-41
Figure 1-11. Exposure-response array of developmental effects following oral exposure to ferf-
butanol 1-51
Figure 1-12. Exposure-response array of developmental effects following inhalation exposure to
ferf-butanol 1-52
Figure 1-13. Exposure-response array of reproductive effects following oral exposure to ferf-
butanol 1-59
Figure 1-14. Exposure-response array of reproductive effects following inhalation exposure to
ferf-butanol 1-60
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 2-15
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
ABBREVIATIONS
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
ornilhine carbamoyl transferase
BW
body weight
ORD
Office of Research and Development
CA
chromosomal aberration
I'BPK
physiologically based pharmacokinetic
CASRN
Chemical Abstracts Service Registry
POD
point of departure
Number
POD [ADJ]
duration-ailjusleil POD
CBI
covalent binding index
QSAR
quantitative structure-activity
CHO
Chinese hamster ovary (cell line)
relationship
CL
confidence limit
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Toxicological Review of tert-Butyl Alcohol
AUTHORS | CONTRIBUTORS | REVIEWERS
Assessment Team
Janice S. Lee, Ph.D. (Chemical Manager)
Keith Salazar, Ph.D.* (Co-Chemical
Manager)
Chris Brinkerhoff, Ph.D.
U.S. EPA
Office of Research and Development
National Center for Environmental Assessment
Research Triangle Park, NC
*Washington, DC
ORISE Postdoctoral Fellow at U.S.
EPA/ORD/NCEA
Currently with U.S. EPA, Office of Chemical Safety
and Pollution Prevention, Office of Pollution
Prevention and Toxics
Washington, DC
Contributors
Andrew Hotchkiss, Ph.D.
Channa Keshava, Ph.D.
Amanda Persad, Ph.D.
Vincent Cogliano, Ph.D.*
Jason Fritz, Ph.D.*
Catherine Gibbons, Ph.D. *
Samantha Jones, Ph.D. *
Kathleen Newhouse *
Karen Hogan *
U.S. EPA
Office of Research and Development
National Center for Environmental Assessment
Research Triangle Park, NC
*Washington, DC
Production Team
Maureen Johnson
Vicki Soto
U.S. EPA
Office of Research and Development
National Center for Environmental Assessment
Washington, DC
Contractor Support
Robyn Blain, Ph.D.
Michelle Cawley*
William Mendez, Jr., Ph.D.
Pam Ross
ICF International
Fairfax, VA
*Research Triangle Park, NC
Executive Direction
Kenneth Olden, Ph.D., Sc.D., L.H.D. (Center Director)
John Vandenberg, Ph.D,# (National Program Director, Human
Health Risk Assessment)
Lynn Flowers, Ph.D., DABT (Associate Director for Health)
Vincent Cogliano, Ph.D. (IRIS Program Director)
Samantha Jones, Ph.D. (IRIS Associate Director for Science)
Weihsueh A. Chiu, Ph.D. (Branch Chief, Toxicity Pathways
Branch) formerly with the U.S. EPA
U.S. EPA/ORD/NCEA
Washington, DC
Cincinnati, OH
# Research Triangle Park, NC
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
Andrew Hotchkiss, Ph.D.# (Acting Branch Chief, Toxicity
Pathways Branch)
Jason Lambert, Ph.D., DABT* (Acting Branch Chief, Biological
Risk Assessment Branch)
Ted Berner (Assistant Center Director)
Karen Hogan (former Acting Branch Chief, Toxicity Effects
Branch)
1
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
2
Reviewers
3 This assessment was provided for r >v to scientists ' > I'rogram and Reg, n Offices.
4 Comments were submitted by:
5 Office of the Administrator/Office ol :klrei, >lth Prolei
6 Office of Land and Emercency Manage nl
7 Region 2, New Yd1-1
8 Region 8, Deny
9 This assessment was |. 'ded for iinvlooll, '(.-deral agencies and the Executive Office of the
10 President. Commeills uv. ilinr ' Ui-:
11 I' .niL'iUi. 'Miami. inn Servile -no. ~>r Toxic Substances and Disease Registry,
12 .arUiK'iil of I k. nul III. ' Services/iv lal Institute of Environmental Health
13 'ccs/Nnlionnl Ti. logy h mi,
14 I-\l 'e Office of the i ident/(. ¦ of Management and Budget,
15 l lxecu. Mflice of llie l'i\ 'ent/OflK f Science and Technology Policy
U.S. EPA
Office of Research and Development
National Center for f> unental Assessment
Washington, DC
Research Triaii!'' ¦ NC
Cincinnati, 01'
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
PREFACE
This Toxicological Review critically reviews the publicly available studies on tert-butyl
alcohol (tert-butanol) 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 a2U-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 modified to support the dose-response assessments for these chemicals
(Salazar etal.. 20151.
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. The
complete set of public comments is available on the docket at http: //www.regulations.gov (Docket
ID No. EPA-HQ-ORD-2013-0111).
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
fhttp:/ /www.epa.gov/irisl. Appendices for toxicokinetic information, PBPK modeling genotoxicity
study summaries, dose-response modeling and other information are provided as Supplemental
Information to this Toxicological Review. For additional information about this assessment or for
This document is a draft for review purposes only and does not constitute Agency policy.
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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 iiPA. 2014).
tert-Butanol has been used as a fuel oxygenate, an <• booster in unleaded gasoline, and
a denaturant for ethanol. From 1997 to 2005, the annui'' in. <>l volume found in gasoline
ranged from approximately 4 million to 6 million ga1' r.s. iHiring 11. ;me, larger quantities were
used to make methyl tert-butyl ether (MTBE) an-' .E. MTBE and E'l l ."ire fuel oxygenates that
were used in the United States prior to 2007 al. -Is of more than 2 billio. lions annually.
Current use levels of MTBE and ETBE in the Uniteu 'I'Siiivi' :h lower, bik ¦ in Europe and
Asia remains strong.1
tert-Butanol has been used loi 1 v of oilier pi »ses, including as a dehydrating agent
and solvent. As such, it is added to Iaequ \ |"u. ¦movers, a. viiI enamels and polishes.
tert-Butanol also is used to manufacture n. 'ivl mei. "lale pla. ¦: and flotation devices.
Cosmetic and food-rela1 'lude the i. hi fuel ¦ <>rs, a.„~, because of its camphor-like
aroma, it also is usei! reale arl. ' il musk, i '' slices, ai. jrfumc (HSDB. 2007). It is used
in coatings on metal a in., ¦lerlioar lodeonlaii s (Cal/EPA. 1999) and industrial cleaning
compounds, f.nn lu-usi.\ ''iJ viraclio. • pharmaceutical applications (HSDB. 2007).
Fate and Transport
Soil
te/ t-IUilanol is expei'led lo lie highly mobile in soil due to its low affinity for soil organic
matter. Rainwater or oilier waler percolaling through soil is expected to dissolve and transport
most tert-butanol p resell I in soil, polenlially leading to groundwater contamination. Based on its
vapor pressure, tert-liuLinol's volalili/.alion from soil surfaces is expected to be an important
dissipation process f ilSDlj. 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
(approximately 200 days). Microbial degradation rates are increased in soils supplemented with
nitrate and sulfate nutrients (HSDB. 2007).
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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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 fHSDB. 20071. 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. 20071.
Air
tert-Butanol primarily exists as a vapor in the amliient sphere. Vapor-phase tert-
butanol is degraded in the atmosphere by reacting with ph- ¦.¦in ically produced hydroxyl
radicals with a half-life of 14 days fHSDB. 20071.
Occurrence in the Environment
The Toxics Release Inventory (TRI) Pro. n National Analysis Rc| estimated that more
than 1 million pounds of tert- butanol has been rek. I into llv oil from lain.. \ land treatment,
underground injection, surface impoundments, and oli .sposal sources. 11 TRI program
also estimated that 476,266 pounds o[ ^'/7-lnilanol was k ed into the atmosphere from fugitive
emissions and point sources fU.S. K I'A. 2012c). In California, missions of tert- butanol from
stationary sources are estimated to be al least 27,000 pounds pei t, based on data reported by
the state's Air Toxics I'r 'recard. 20.1 ll. The TRI National a. ..-lysis Report estimated 7,469
pounds of tert-butan as release nto surface waters from point and nonpoint sources in 2011
fU.S. EPA. 2012cl.
tert-Butanol has been ii k-r : ¦irinking water wells throughout the United States
fHSDB. 2007). California's Geou er Dalai '1 ists !-!,496 detections of tert- butanol in
groundwater associated with conk -laled sites , .iat state since 2011. tert-Butanol also has been
detected in drinking water wells in the vicinity of landfills fU.S. EPA. 2012cl. Additionally, tert-
Butanol leaking from underground storage .nks could be a product of MTBE and ETBE, which can
degrade to form /c*/7-hiilanol in soils (HSDB. 2007). The industrial chemical tert-butyl acetate also
can degrade to form {e/7-bulanol in animals postexposure and in the environment
Ambient outdoor air concentrations of tert-butanol vary according to proximity to urban
areas fHSDB. 20071.
General Population Exposure
tert-Butanol exposure can occur in many different settings. Releases from underground
storage tanks could potentially 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).
2 http://geotracker.waterboards.ca.gov/.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
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. 20071. 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 (HSDB. 2007). tert-Butanol has been
identified in mother's milk (HSDB. 2007). Dermal contact is a v: route of exposure through
handling consumer products containing tert-butanol I N SI¦' I • .ational. 20031.
Assessments by Other National and International Health Agencies
Toxicity information on tert-butanol has lu-en evaluated by the National Institute for
Occupational Safety and Health fNIOSH. 2007). the Occupational Safety and I lealth Administration
fOSHA. 2006). and the Food and Drug Administration (FDA. 2011a. b). The results of these
assessments are presented in Appendix A of the Supplemental Information to this Toxicological
Review. Of importance to recognize is that these earlier assessments could have been prepared for
different purposes and might use different methods. In addition, newer studies have been included
in the IRIS assessment.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS
Note: The Preamble to IRIS assessments is
being revised based on comments received
from external peer reviewers and the
public, and based on IRIS Program
experience with the implementation of
systematic review methods. Subsequent
drafts of the tert-butanol assessment will
include the revised Preamble.
1. Scope of the IRIS Program
Soon after the EPA was established in
1970, itwas atthe forefront of developing risk
assessment as a science and applying it in
decisions to protect human health and the
environment. The Clean Air Act, for example,
mandates that the EPA provide "an ample
margin of safety to protect public health"; the
Safe Drinking Water Act, that "no adverse
effects on the health of persons may
reasonably be anticipated to occur, allowing
an adequate margin of safety." Accordingly,
the EPA uses information on the adverse
effects of chemicals and on exposure levels
below which these effects are not anticipated
to occur.
IRIS assessments critically review the
publicly available studies to identify adverse
health effects from exposure to chemicals and
to characterize exposure-response
relationships. In terms set forth by the
National Research Council fNRC. 19831. IRIS
assessments cover the hazard identification
and dose-response assessment steps of risk
assessment, not the exposure assessment or
risk characterization steps that are conducted
by the EPA's program and regional offices and
by other federal, state, and local health
agencies that evaluate risk in specific
populations and exposure scenarios. IRIS
assessments are distinct from and do not
42 address political, economic, and technical
43 considerations that influence the design and
44 selection of risk management alternatives.
45 An IRIS assessment may cover a single
46 chemical, a group of structurally or
47 toxicologically related chemicals, or a complex
48 mixture. These agents may be found in air,
49 water, soil, or sediment Exceptions are
50 chemicals currently used exclusively as
51 pesticides, ionizing and non-ionizing
52 radiation, and criteria air pollutants listed
53 under Section 108 of the Clean Air Act (carbon
54 monoxide, lead, nitrogen oxides, ozone,
55 particulate matter, and sulfur oxides).
56 Periodically, the IRIS Program asks other
57 EPA programs and regions, other federal
58 agencies, state health agencies, and the
59 general public to nominate chemicals and
60 mixtures for future assessment or
61 reassessment Agents may be considered for
62 reassessment as significant new studies are
63 published. Selection is based on program and
64 regional office priorities and on availability of
65 adequate information to evaluate the potential
66 for adverse effects. Other agents may also be
67 assessed in response to an urgent public
68 health need.
69 2. Process for developing and peer-
70 reviewing IRIS assessments
71 The process for developing IRIS
72 assessments (revised in May 2009 and
73 enhanced in July 2013) involves critical
74 analysis of the pertinent studies, opportunities
75 for public input, and multiple levels of
76 scientific review. The EPA revises draft
77 assessments after each review, and external
78 drafts and comments become part of the
79 public record (U.S. EPA. 2009).
80 Before beginning an assessment, the IRIS
81 program discusses the scope with other EPA
82 programs and regions to ensure that the
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
assessment will meet their needs. Then a
public meeting on problem formulation
invites discussion of the key issues and the
studies and analytical approaches that might
contribute to their resolution.
Step 1. Development of a draft
Toxicological Review. The draft
assessment considers all pertinent
publicly available studies and applies
consistent criteria to evaluate study
quality, identify health effects, identify
mechanistic events and pathways,
integrate the evidence of causation for
each effect, and derive toxicity values. A
public meeting prior to the integration of
evidence and derivation of toxicity values
promotes public discussion of the
literature search, evidence, and key issues.
Step 2. Internal review by scientists in EPA
programs and regions. T' ¦ I raft
assessment is revised to adtli
comments from within the EPA.
Step 3. Interagency science consulta. 'i
with other federa1 :es and t.
Executive Office* .ne h. "'lent. TIil
draft assessment -vised lo t 'ressthe
interagency comii. ' s. Tin. science-
consultation draft, ink-. 'Ill'V -"ills,
and i1 rcs|i<, .i
cum .s lu-Oi. 'xii'l <. he pulilk
rei
Step 4. . 'lie review I coii >nt,
followed external pei. 'eview. e
El'A release. 'lie draft as sment for
public review -I com me A public
meeting provide in or lunity to
discuss the assess. U jr to peer
review. Then the EPA i .ses a draft for
external peer review. The peer review
meeting is open to the public and includes
time for oral public comments. The peer
reviewers assess whether the evidence
has been assembled and evaluated
according to guidelines and whether the
conclusions are justified by the evidence.
The peer review draft, written public
comments, and peer review report
become part of the public record.
49 Step 5. Revision of draft Toxicological
50 Review and development of draft IRIS
51 summary. The draft assessment is revised
52 to reflect the peer review comments,
53 public comments, and newly published
54 studies that are critical to the conclusions
55 of the assessment. The disposition of peer
56 review comments and public comments
57 becomes part of the public record.
58 Step 6. Firil EPA review and interagency
59 scier iscussion with other federal
60 aj» _s and the Executive Offices of
61 'sident The draft assessment and
62 .innit. ire revised to address the EPA
6? nlid inlei ncy comments. The science
d iscussion 'II, written interagency
j comments, aiu. ''A's response to major
comments bean, oarl of the public
6, reco1
68 Step 7. Completion and posting. The
69 Toxicological Review and IRIS summary
"'O are posted on the IRIS website
(litlp://u'ww.epa.gov/iris/l.
72 The remainder of this Preamble addresses step 1,
7? ilic development of a draft Toxicological
Review. IRIS assessments follow standard
practices of evidence evaluation and peer
V review, many of which are discussed in
77 EPA guidelines (U.S. EPA.
¦"S 2005a. b. 2000b. 1998b. 1996. 1991b. 198
i 6a, b) and other methods (U.S. EPA.
80 2012a. b, 2011. 2006a. b, 2002. 19941.
81 Transparent application of scientific
82 judgment is of paramount importance. To
83 provide a harmonized approach across
84 IRIS assessments, this Preamble
85 summarizes concepts from these
86 guidelines and emphasizes principles of
87 general applicability.
88 3. Identifying and selecting
89 pertinent studies
90 3.1. Identifying studies
91 Before beginning an assessment, the EPA
92 conducts a comprehensive search of the
93 primary scientific literature. The literature
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
search follows standard practices and includes
the PubMed and ToxNet databases of the
National Library of Medicine, Web of Science,
and other databases listed in the EPA's HERO
system (Health and Environmental Research
Online, http://hero.epa.gov/1. Searches for
information on mechanisms of toxicity are
inherently specialized and may include
studies on other agents that act through
related mechanisms.
Each assessment specifies the search
strategies, keywords, and cut-off dates of its
literature searches. The EPA posts the results
of the literature search on the IRIS web site
and requests information from the public on
additional studies and ongoing research.
The EPA also considers studies received
through the IRIS Submission Desk and studies
(typically unpublished) submitted under the
Toxic Substances Control Act or the Federal
Insecticide, Fungicide, and Rodenticide Act.
Material submitted as Confidential business
Information is considered only if it includes
health and safety data that can be publicly
released. If a study that may be critical l<> the
conclusions of the assessment has not lieen
peer-reviewed, the lil'A will have il peer-
reviewed.
The EPA also examines the toxicokinetics
of the agent to identify other chemicals (for
example, major metabolites of the agent) to
include in the assessment if adequate
information is available, in order to more fully
explain the toxicity of the agent and to suggest
dose metrics for subsequent modeling.
In assessments of chemical mixtures.
mixture studies are preferred for their ability
to reflect interactions among components.
The literature search seeks, in decreasing
order of preference fU.S. ili'A.
2000b. §2.2: 1986b. §2.11:
Studies of the mixture being assessed.
Studies of a sufficiently similar
mixture. In evaluating similarity, the
assessment considers the alteration of
mixtures in the environment through
partitioning and transformation.
Studies of individual chemical
components of the mixture, if there are
not adequate studies of sufficiently
similar mixtures.
3.2. Selecting pertinent epidemiologic
studies
Study design is the key consideration for
selecting pertinent epidemiologic studies from
the results of the literature search.
Cohort studies, case-control studies,
and some population-based surveys
(lor example, NHANES) provide the
strongest epidemiologic evidence,
especially if they collect information
about individual exposures and
effects.
Ideological studies (geographic
correlation studies) relate exposures
and effects by geographic area. They
can provide strong evidence if there
are large exposure contrasts between
geographic areas, relatively little
exposu re v ariation within study areas,
and population migration is limited.
Case reports of high or accidental
exposure lack definition of the
population at risk and the expected
number of cases. They can provide
information about a rare effect or
about the relevance of analogous
results in animals.
The assessment briefly reviews ecological
studies and case reports but reports details
only if they suggest effects not identified by
other studies.
3.3. Selecting pertinent experimental
studies
Exposure route is a key design
consideration for selecting pertinent
experimental animal studies or human clinical
studies.
Studies of oral, inhalation, or dermal
exposure involve passage through an
absorption barrier and are considered
48
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86
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89
90
91
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
most pertinent to human
environmental exposure.
Injection or implantation studies are
often considered less pertinent but
may provide valuable toxicokinetic or
mechanistic information. They also
may be useful for identifying effects in
animals if deposition or absorption is
problematic (for example, for particles
and fibers).
Exposure duration is also a key design
consideration for selecting pertinent
experimental animal studies.
Studies of effects from chronic
exposure are most pertinent to
lifetime human exposure.
Studies of effects from less-than-
chronic exposure are pertinent but
less preferred for identifyir 1 Iccts
from lifetime human exposu 11
studies may be indicative of i.
from less-than-lifetime In. in
exposure.
Short-duration si1 in\» animal,
or humans may |i. de toxk\ letic or
mechanistic informatioi
For developmental loxi' and
reproducti,:,; irrovoi. '
result fn . brier e> = ; ire l)i. • a critic,...
periou «>f develop. '. ;» rdingly,
special i/>. tudy designs t used i 'hese
effects (U.S. 5 \2flfl6b. jj b).
4. Evaluating «. quality c
individual stuc. ~
After the subset . pertinent
epidemiologic and experimental studies have
been selected from the literature searches, the
assessment evaluates the quality of each
individual study. This evaluation considers the
design, methods, conduct, and documentation
of each study, but not whether the results are
positive, negative, or null. The objective is to
identify the stronger, more informative
studies based on a uniform evaluation of
45 quality characteristics across studies of
46 similar design.
47 4.1. Evaluating the quality of
48 epidemiologic studies
49 The assessment evaluates design and
50 methodological aspects that can increase or
51 decrease the weight given to each
52 epidemiologic study in the overall evaluation
53 fU.S. EPA. 200Sa. 1998b. 1996. 1994. 1991 hi:
54
.mentation of study design,
55
¦lh<>ds, population characteristics,
56
iii -suits.
57
Deliiii n and selection of the study
58
group an- Miiparison group.
59
- Ascertaining of exposure to the
60
chemical or mi..
61
Ascertainment of disease or health
62
el led.
63
Duration of exposure and follow-up
64
and adequacy for assessing the
65
occurrence of effects.
66
Characterization of exposure during
67
critical periods.
68
Sample size and statistical power to
69
detect anticipated effects.
70
Participation rates and potential for
71
selection bias as a result of the
72
achieved participation rates.
73
Measurement error (can lead to
74
misclassification of exposure, health
75
outcomes, and other factors) and other
76
types of information bias.
77
Potential confounding and other
78
sources of bias addressed in the study
79
design or in the analysis of results. The
80
basis for consideration of confounding
81
is a reasonable expectation that the
82
confounder is related to both exposure
83
and outcome and is sufficiently
84
prevalent to result in bias.
85
For developmental toxicity, reproductive
86 toxicity, neurotoxicity, and cancer there is
87 further guidance on the nuances of evaluating
This document is a draft for review purposes only and does not constitute Agency policy.
xvii DRAFT—DO NOT CITE OR QUOTE
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Toxicological Review of tert-Butyl Alcohol
1 epidemiologic studies of these effects (U.S.
2 EPA. 2005a. 1998b. 1996.1991bl
3 4.2. Evaluating the quality of
4 experimental studies
5 The assessment evaluates design and
6 methodological aspects that can increase or
7 decrease the weight given to each
8 experimental animal study, in-vitro study, or
9 human clinical study fU.S. EPA.
10 2005a. 1998b. 1996. 1991b). Research
11 involving human subjects is considered only if
12 conducted according to ethical principles.
13
Documentation of study design,
14
animals or study population, methods,
15
basic data, and results.
16
Nature of the assay and validity for its
17
intended purpose.
18
Characterization of the nature and
19
extent of impurities and contaminants
20
of the administered chemical or
21
mixture.
22
Characterization of (.lose and dosing
23
regimen (including age at exposure)
24
and their adequacy to elicit adverse
25
effects, including latent effects.
26
Sample sizes and statistical power to
27
detect dose-related differences or
28
trends.
29
Ascertainment ol survival, vital signs,
30
disease or effects, and cause of death.
31
Control of other variables that could
32
influence the occurrence of effects.
33
The assessment uses statistical tests to
34 evaluate whether the observations niaybedue
35 to chance. The standard for determining
36 statistical significance of a response is a trend
37 test or comparison of outcomes in the exposed
38 groups against those of concurrent controls. In
39 some situations, examination of historical
40 control data from the same laboratory within
41 a few years of the study may improve the
42 analysis. For an uncommon effect that is not
43 statistically significant compared with
44 concurrent controls, historical controls may
45 show that the effect is unlikely to be due to
46 chance. For a response that appears significant
47 against a concurrent control response that is
48 unusual, historical controls may offer a
49 different interpretation (U.S. EPA.
50 2005a. §2.2.2.1.31.
51 For developmental toxicity, reproductive
52 toxicity, neurotoxicity, and cancer there is
53 further guidance on the nuances of evaluating
54 experimental studies of these effects fU.S. EPA.
55 2005a. i')98b. 1996. 1991bl. In multi-
56 generation studies, agents that produce
57 developmental effects at doses that are not
58 toxic to the maternal animal are of special
59 concern. Effects that occur at doses associated
60 with mild maternal toxicity are not assumed to
61 result only from maternal toxicity. Moreover,
62 maternal effects may lie reversible, while
63 effects on the offspring may be permanent
64 fU.S. r'I'A. 1998n.b. 1.2.4.5.4: 1991b.
65 &LLL1)..
66 4.3. Reporting study results
67 The assessment uses evidence tables to
68 present the des ign and key results of pertinent
69 studies. There maybe separate tables for each
70 site of toxicity or type of study.
71 If a large number of studies observe the
72 same effect, the assessment considers the
73 study quality characteristics in this section to
74 ide ntify the strongest studies or types of study.
75 The tables present details from these studies,
76 and the assessment explains the reasons for
77 not reporting details of other studies or
78 groups of studies that do not add new
79 information. Supplemental information
80 provides references to all studies considered,
81 including those not summarized in the tables.
82 The assessment discusses strengths and
83 limitations that affect the interpretation of
84 each study. If the interpretation of a study in
85 the assessment differs from that of the study
86 authors, the assessment discusses the basis for
87 the difference.
88 As a check on the selection and evaluation
89 of pertinent studies, the EPA asks peer
90 reviewers to identify studies that were not
91 adequately considered.
This document is a draft for review purposes only and does not constitute Agency policy.
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5. Evaluating the overall evidence of
each effect
5.1. Concepts of causal inference
For each health effect, the assessment
evaluates the evidence as a whole to
determine whether it is reasonable to infer a
causal association between exposure to the
agent and the occurrence of the effect This
inference is based on information from
pertinent human studies, animal studies, and
mechanistic studies of adequate quality.
Positive, negative, and null results are given
weight according to study quality.
Causal inference involves scientific
judgment, and the considerations are nuanced
and complex. Several health agencies have
developed frameworks for causal inference,
among them the U.S. Surgeon General fCDC.
2004: HEW. 19641. the Internatiom "vy
for Research on Cancer (IARC. 20i
Institute of Medicine (IQM. 2008). and lli 'I'A
f2010. §1.6: 2005a. §2.51 Although devek d
for different purposes, '' meworks
similar in nature ami n.ie . stablishei.
structure and langik lor caus;i 'ference.
Each considers aspects >n assoc on thai
suggest causation, discu. 1 by " ("Hill.
1965) and 'on I. mm.. 1
Greenlar .othrria.; I Cn-i 'id. i'>9b;
and IJ.. A f2005a. §2.. 1 ')')¦. inendix
C)-
Strength of association: The lindingola large
relative risk with narrow confidence
intervals strongly suggests that an
association is not due to chance, bias, or
other factors. Modest relative risks,
however, may reflect a small range of
exposures, an agent of low potency, an
increase in an effect that is common,
exposure misclassification, or other
sources of bias.
Consistency of association: An inference of
causation is strengthened if elevated risks
are observed in independent studies of
different populations and exposure
scenarios. Reproducibility of findings
constitutes one of the strongest arguments
Toxicological Review of tert-Butyl Alcohol
for causation. Discordant results
sometimes reflect differences in study
design, exposure, or confounding factors.
Specificity of association: As originally
intended, this refers to one cause
associated with one effect Current
understanding that many agents cause
multiple effects and many effects have
multiple causes make this a less
informative aspect of causation, unless the
effect is rare or unlikely to have multiple
causes.
Temporal relationship: A causal
interpretation requires that exposure
precede development of the effect.
Biologic gradient (exposure-response
relationship): Exposure-response
relationships strongly suggest causation. A
monotonic increase is not the only pattern
consistent with causation. The presence of
an exposure-response gradient also
weighs against bias and confounding as
the source of an association.
Biologic plausibility: An inference of
causation is strengthened by data
demonstrating plausible biologic
mechanisms, if available. Plausibility may
reflect subjective prior beliefs if there is
insufficient understanding of the biologic
process involved.
Coherence: An inference of causation is
strengthened by supportive results from
animal experiments, toxicokinetic studies,
and short-term tests. Coherence may also
be found in other lines of evidence, such as
changing disease patterns in the
population.
"Natural experiments": A change in exposure
that brings about a change in disease
frequency provides strong evidence, as it
tests the hypothesis of causation. An
example would be an intervention to
reduce exposure in the workplace or
environment that is followed by a
reduction of an adverse effect
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This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
Analogy: Information on structural analogues
or on chemicals that induce similar
mechanistic events can provide insight
into causation.
These considerations are consistent with
guidelines for systematic reviews that
evaluate the quality and weight of evidence.
Confidence is increased if the magnitude of
effect is large, if there is evidence of an
exposure-response relationship, or if an
association was observed and the plausible
biases would tend to decrease the magnitude
of the reported effect Confidence is decreased
for study limitations, inconsistency of results,
indirectness of evidence, imprecision, or
reporting bias fGuvatt et al.. 2008b: Guvatt et
al.. 2008al.
5.2. Evaluating evidence in humans
For each effect, the assessment evaluates
the evidence from the epidemiologic studies as
a whole. The objective is to determine whether
a credible association has been observed and,
if so, whether that association is consistent
with causation. In doing this, the assessment
explores alternative explanations (such as
chance, bias, and confounding) and draws a
conclusion about whether these alternatives
can satisfactorily explain any observed
association.
To make clear how much the
epidemiologic evidence contributes to the
overall weight of the evidence, the assessment
may select a standard descriptor to
characterize the epidemiologic evidence of
association between exposure to the agent and
occurrence of a health el led.
Sufficient epidemiologic evidence of an
association consistent with causation:
The evidence establishes a causal
association for which alternative
explanations such as chance, bias, and
confounding can be ruled out with
reasonable confidence.
Suggestive epidemiologic evidence of an
association consistent with causation:
The evidence suggests a causal association
47 but chance, bias, or confounding cannot be
48 ruled out as explaining the association.
49 Inadequate epidemiologic evidence to infer
50 a causal association: The available
51 studies do not permit a conclusion
52 regarding the presence or absence of an
53 association.
54 Epidemiologic evidence consistent with no
55 causal association: Several adequate
56 studies cov ering the full range of human
57 exposures and considering susceptible
58 populations, and for which alternative
59 explanations such as bias and confounding
60 can be ruled out, are mutually consistent
61 in not findin» an association.
62 5.3. Evaluating evidence in animals
63 For each effect, the assessment evaluates
64 the evidence from the animal experiments as a
65 whole to determine the extent to which they
66 indicate a potential for effects in humans.
67 Consistent results across various species and
68 strains increase confidence that similar results
69 would occur in humans. Several concepts
70 discussed by Mill (Hill. 1965) are pertinent to
71 the weight of experimental results:
72 consistency of response, dose-response
73 relationships, strength of response, biologic
74 plausibility, and coherence (U.S. EPA.
75 2005a. §2.2.1.7: 1994. Appendix C).
76 In weighing evidence from multiple
77 experiments, U.S. EPA (2005a. §2.5)
78 distinguishes:
79 Conflicting evidence (that is, mixed positive
80 and negative results in the same sex and
81 strain using a similar study protocol) from
82 Differing results (that is, positive results and
83 negative results are in different sexes or
84 strains or use different study protocols).
85 Negative or null results do not invalidate
86 positive results in a different experimental
87 system. The EPA regards all as valid
88 observations and looks to explain differing
89 results using mechanistic information (for
90 example, physiologic or metabolic differences
91 across test systems) or methodological
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
differences (for example, relative sensitivity of
the tests, differences in dose levels,
insufficient sample size, or timing of dosing or
data collection).
It is well established that there are critical
periods for some developmental and
reproductive effects fU.S. EPA.
2006b. 2005a. b, 1998b. 1996. 1991 hi
Accordingly, the assessment determines
whether critical periods have been adequately
investigated. Similarly, the assessment
determines whether the database is adequate
to evaluate other critical sites and effects.
In evaluating evidence of genetic toxicity:
Demonstration of gene mutations,
chromosome aberrations, or
aneuploidy in humans or experimental
mammals [in vivo) provides the
strongest evidence.
- This is followed by positive results in
lower organisms or in cultured colls
[in vitro) or for other genetic eve ills.
Negative results carry less weight,
partly because I hoy cannot exclude the
possibility of effects in other tissues
flARC. 2006).
For germ-cell mutagenicity, The KI'A has
defined categories of evidence, ranging from
positive results of human germ-cell
mutagenicity to negativ e results for all effects
of concern (M.S. EPA. S2./5).
5.4. Evaluating mechanistic data
Mechanistic (.lata can lie useful in
answering severaI tjnestions.
- The biologic plausibility of a causal
interpretation ol human studies.
- The generalizability of animal studies
to humans.
- The susceptibility of particular
populations or lifestages.
The focus of the analysis is to describe, if
possible, mechanistic pathways that lead to a
health effect. These pathways encompass:
Toxicokinetic processes of absorption,
distribution, metabolism, and
elimination that lead to the formation
of an active agent and its presence at
the site of initial biologic interaction.
Toxicodynamic processes that lead to a
health effect at this or another site
(also known as a mode of action).
For each effect, the assessment discusses
the available information on its modes of
action and associated key events [key events
being empirically observable, necessary
precursor steps or biologic markers of such
steps; mode of action being a series of key
events involving interaction with cells,
operational and anatomic changes, and
resulting in disease]. Pertinent information
may also come from studies of metabolites or
of compounds that are structurally similar or
that act through similar mechanisms.
Information on mode of action is not required
for a conclusion that the agent is causally
related to an effect (U.S. EPA. 2005a. 52.5).
The assessment addresses several
questions about each hypothesized mode of
action fU.S. EPA. 2005a. §2.4.3.41.
1) Is the hypothesized mode of action
sufficiently supported in test animals?
Strong support for a key event being
necessary to a mode of action can come
from experimental challenge to the
hypothesized mode of action, in which
studies that suppress a key event observe
suppression of the effect Support for a
mode of action is meaningfully
strengthened by consistent results in
different experimental models, much
more so than by replicate experiments in
the same model. The assessment may
consider various aspects of causation in
addressing this question.
2) Is the hypothesized mode of action
relevant to humans? The assessment
reviews the key events to identify critical
similarities and differences between the
test animals and humans. Site
concordance is not assumed between
animals and humans, though it may hold
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for certain effects or modes of action.
Information suggesting quantitative
differences in doses where effects would
occur in animals or humans is considered
in the dose-response analysis. Current
levels of human exposure are not used to
rule out human relevance, as IRIS
assessments may be used in evaluating
new or unforeseen circumstances that
may entail higher exposures.
3) Which populations or lifestages can be
particularly susceptible to the
hypothesized mode of action? The
assessment reviews the key events to
identify populations and lifestages that
might be susceptible to their occurrence.
Quantitative differences may result in
separate toxicity values for susceptible
populations or lifestages.
The assessment discusses the 1' ''mod
that an agent operates through
modes of action. An uneven level oi s> ioi
for different modes of action can n. cl
disproportionate re^ ^es sp '
investigating them (M.1-' .. " i. S2.4.3.S
It should be noted tl' . clinical , iews, (.In-
credibility of a series . 'udies is luced if
evidence is limited to sli s luiu' hv one-
interested sc"' f^-uvatteU ''''
For i, "sst'ssn. evaluiu
eviden; . a mutage, iiode 'clion l<>
guide e isolation to 'er ill ¦ and
considerai. of susceptibk festagi. \ey
data include.- ¦ ability of I. agent l a
metabolite lo . 1 with or i I to DNA,
positive results in 1 Itiplc les rstems, or
similar properties I sir1 re-activity
relationships to mulagc. <• .ogens (U.S.
EPA. 2005a .52.3.51.
5.5. Characterizing the overall weight
of the evidence
After evaluating the human, animal, and
mechanistic evidence pertinent to an effect,
the assessment answers the question: Does
the agent cause the adverse effect? fNRC.
2009. 1983). In doing this, the assessment
develops a narrative that integrates the
evidence pertinent to causation. To provide
clarity and consistency, the narrative includes
a standard hazard descriptor. For example, the
following standard descriptors combine
epidemiologic, experimental, and mechanistic
evidence of carcinogenicity fU.S. EPA. 2005a.
§2£).
Carcinogenic to humans: There is convincing
epidemiologic evidence of a causal
associn^on (that is, there is reasonable
confid that the association cannot be
fu1' explained by chance, bias, or
xling); or there is strong human
iden 'if cancer or its precursors,
extensive ;mal evidence, identification
of key prei^ -r events in animals, and
strong evident 'nl Ihey are anticipated
to occur in humai,
Likely to be carcinogenic to humans: The
evidence demonstrates a potential hazard
lo hum a lis but does not meet the criteria
lor carcinogenic. There may be a plausible
association in humans, multiple positive
results in animals, or a combination of
human, animal, or other experimental
evidence.
Suggestive evidence of carcinogenic
potential: The evidence raises concern for
effects in humans but is not sufficient for a
stronger conclusion. This descriptor
covers a range of evidence, from a positive
result in the only available study to a single
positive result in an extensive database
that includes negative results in other
species.
Inadequate information to assess
carcinogenic potential: No other
descriptors apply. Conflicting evidence can
be classified as inadequate information if
all positive results are opposed by
negative studies of equal quality in the
same sex and strain. Differing results,
however, can be classified as suggestive
evidence or as likely to be carcinogenic.
Not likely to be carcinogenic to humans:
There is robust evidence for concluding
that there is no basis for concern. There
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may be no effects in both sexes of at least
two appropriate animal species; positive
animal results and strong, consistent
evidence that each mode of action in
animals does not operate in humans; or
convincing evidence that effects are not
likely by a particular exposure route or
below a defined dose.
Multiple descriptors may be used if there
is evidence that carcinogenic effects differ by
dose range or exposure route fU.S. EPA. 2005a.
§Z5).
Another example of standard descriptors
comes from the EPA's Integrated Science
Assessments, which evaluate causation for the
effects of the criteria pollutants in ambient air
(TJ.S. EPA. 2010. §1.6").
Causal relationship: Sufficient evidence to
conclude that there is a causal
relationship. Observational studi 'nnot
be explained by plausible altermi
they are supported by other J i. ¦ i.
evidence, for example, animal studk ->r
mechanistic informnti^"
Likely to be a causal . Sulficien.
evidence that j usal rela iship is
likely, but importa iu ¦¦erUiinli remain.
For example, observai 'I si- ¦¦how
an as' bill ct. .. i e.-.
diffir io aciu. or ik 1 Iilies o.
evi ce art,1 limited ¦ incoi. ''.-lit; or
mulli, animal sludk 'mm <. -rent
laborali. dcmonslrai effects nd
there are I. -(.I or no hum, data.
Suggestive of a ci. */ relation p; At least
one high-qualiU oidenr gic study
shows an association studies are
inconsistent
Inadequate to infer a causal relationship:
The studies do not permit a conclusion
regarding the presence or absence of an
association.
Not likely to be a causal relationship: Several
adequate studies, covering the full range of
human exposure and considering
susceptible populations, are mutually
consistent in not showing an effect at any
level of exposure.
The EPA is investigating and may on a trial
basis use these or other standard descriptors
to characterize the overall weight of the
evidence for effects other than cancer.
6. Selecting studies for derivation of
toxicity values
For effect where there is credible
ex iLlc" i an association with the agent, the
ass 'le rives toxicity values if there are
sii .ile q. iiiologic or experimental data.
he decision '"rive toxicity values may be
linked to the ha>.. ' 'lescriptor.
Dose-response a. sis requires quantitative
measures of dose one s|-ioiise. Then, other
¦rlors b equal:
F|iklemiologic studies are preferred
over animal studies, if quantitative
measures of exposure are available
and effects can be attributed to the
agent.
Among experimental animal models,
those that respond most like humans
are preferred, if the comparability of
response can be determined.
Studies by a route of human
environmental exposure are
preferred, although a validated
toxicokinetic model can be used to
extrapolate across exposure routes.
Studies of longer exposure duration
and follow-up are preferred, to
minimize uncertainty about whether
effects are representative of lifetime
exposure.
Studies with multiple exposure levels
are preferred for their ability to
provide information about the shape
of the exposure-response curve.
Studies with adequate power to detect
effects at lower exposure levels are
preferred, to minimize the extent of
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extrapolation to levels found in the
environment
Studies with non-monotonic exposure-
response relationships are not necessarily
excluded from the analysis. A diminished
effect at higher exposure levels may be
satisfactorily explained by factors such as
competing toxicity, saturation of absorption or
metabolism, exposure misclassification, or
selection bias.
If a large number of studies are suitable for
dose-response analysis, the assessment
considers the study characteristics in this
section to focus on the most informative data.
The assessment explains the reasons for not
analyzing other groups of studies. As a check
on the selection of studies for dose-response
analysis, the EPA asks peer reviewers to
identify studies that were not adequately
considered.
7. Deriving toxicity values
7.1. General framework for dose-
response analv
The EPA uses a '-step ;i|i| lch that
distinguishes analysis i. -,e obsei J dose-
response data from inleu \s 1 'wr
doses fU.S ' .'-r - §31
With .(_¦ olisoi . "-uige, nreferrei.,
approi. s l<> use modi. l<> im 'irate a
wide rai,t of (.lata into anak. The
modeling \ "s a point o/ epurtui\ in
exposure level -ar the lowe ?nd of uie
observed raiiL, without significant
extrapolation to Im. doses] 1 :tions 7.2-
7.3).
Extrapolation to low ,es considers
what is known about the niuues of action for
each effect (Sections 7.4-7.5). If response
estimates at lower doses are not required, an
alternative is to derive reference values, which
are calculated by applying factors to the point
of departure in order to account for sources of
uncertainty and variability (Section 7.6).
For a group of agents that induce an effect
through a common mode of action, the dose-
response analysis may derive a relative
Toxicological Review of tert-Butyl Alcohol
potency factor for each agent A full dose-
response analysis is conducted for one well-
studied index chemical in the group, then the
potencies of other members are expressed in
relative terms based on relative toxic effects,
relative absorption or metabolic rates,
quantitative structure-activity relationships,
or receptor binding characteristics (U.S. EPA.
2005a. §3.2.6: 2000b. §4.41.
Increasingly, the EPA is basing toxicity
values on Wiined analyses of multiple data
sets or .liple responses. The EPA also
cons: multiple dose-response approaches
if .a, supported by robust data.
.2. ModelihL 'ose to sites of biologic
effects
The preferred approach for analysis of
(.lose is loxicokinetic modeling because of its
a hi lily lo incorporate a wide range of data. The
preferred (.lose metric would refer to the
activ e agent at the site of its biologic effect or
to a close, reliable surrogate measure. The
activ e agent may be the administered chemical
or a metabolite. Confidence in the use of a
loxicokinetic model depends on the
robustness of its validation process and on the
results of sensitivity analyses (U.S. EPA.
2006a: 2005a. §3.1: 1994. §4.31.
because toxicokinetic modeling can
require many parameters and more data than
are typically available, the EPA has developed
standard approaches that can be applied to
typical data sets. These standard approaches
also facilitate comparison across exposure
patterns and species.
Intermittent study exposures are
standardized to a daily average over
the duration of exposure. For chronic
effects, daily exposures are averaged
over the lifespan. Exposures during a
critical period, however, are not
averaged over a longer duration (U.S.
EPA. 2005a. §3.1.1: 1991b. §3.21.
Doses are standardized to equivalent
human terms to facilitate comparison
of results from different species.
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Toxicological Review of tert-Butyl Alcohol
Oral doses are scaled allometrically
using mg/kg3/4-day as the equivalent
dose metric across species. Allometric
scaling pertains to equivalence across
species, not across lifestages, and is
not used to scale doses from adult
humans or mature animals to infants
or children (U.S. EPA.
2011: 2005a. §3.1.31
Inhalation exposures are scaled using
dosimetry models that apply species-
specific physiologic and anatomic
factors and consider whether the
effect occurs at the site of first contact
or after systemic circulation fU.S. EPA.
2012a: 1994. 331.
It can be informative to convert doses
across exposure routes. If this is done, the
assessment describes the underlying data,
algorithms, and assumptions (i' ! i I' /\.
2005a. 33.1.41.
In the absence of study-specific iK <>i.
for example, intake rates or body weigh. V
EPA has developed recoi^ •'ded values
use in dose-respons< .. fU.S. El',
19881.
7.3. Modeling respon. 'n the ngeof
obser' " ¦*
Toy: mimic '>logia. based't
model. can incoipoi't. lata <. 'Mologic
processes >ding to an d with its key events.
Because different >dels r provide
equivalent fits to Uk bse ea data but
diverge substantially at i> ioses, critical
biologic parameters should be measured from
laboratory studies, not by model fitting.
Confidence in the use of a toxicodynamic
model depends on the robustness of its
validation process and on the results of
sensitivity analyses. Peer review of the
scientific basis and performance of a model is
essential (U.S. EPA. 2005a. 33.2.2).
Because toxicodynamic modeling can
require many parameters and more
knowledge and data than are typically
available, the EPA has developed a standard
set of empirical ("curve-fitting") models
(http://www.epa.gov/ncea/bmds/) that can
be applied to typical data sets, including those
that are nonlinear. The EPA has also developed
guidance on modeling dose-response data,
assessing model fit, selecting suitable models,
and reporting modeling results (U.S. EPA.
2012b). Additional judgment or alternative
analyses a. . s'sed if the procedure fails to yield
reliable its, for example, if the fit is poor,
modr nay he restricted to the lower doses,
es .11\ there is competing toxicity at
'""igiidr dose;., j: S. EPA. 2005a. 33.2.3).
Modeling i< 'sed to derive a point of
departure fU.S. i ' 2012b: 2005a. 33.2.41.
(See Section 7.6 loi Tiiatives if a point of
¦le|iartur< -annot be d<. -d hy modeling.):
II linear extrapolation is used,
selection of a response level
corresponding to the point of
departure is not highly influential, so
standard values near the low end of
the observable range are generally
used (lor example, 10% extra risk for
cancer bioassay data, 1% for
epidemiologic data, lower for rare
cancers).
For nonlinear approaches, both
statistical and biologic considerations
are taken into account.
For dichotomous data, a response level
of 10% extra risk is generally used for
minimally adverse effects, 5% or
lower for more severe effects.
For continuous data, a response level
is ideally based on an established
definition of biologic significance. In
the absence of such definition, one
control standard deviation from the
control mean is often used for
minimally adverse effects, one-half
standard deviation for more severe
effects.
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Toxicological Review of tert-Butyl Alcohol
The point of departure is the 95% lower
bound on the dose associated with the
selected response level.
7.4. Extrapolating to lower doses and
response levels
The purpose of extrapolating to lower
doses is to estimate responses at exposures
below the observed data. Low-dose
extrapolation, typically used for cancer data,
considers what is known about modes of
action fU.S. EPA. 2005a. 33.3.1 and §3.3.21.
1) If a biologically based model has been
developed and validated for the agent,
extrapolation may use the fitted model
below the observed range if significant
model uncertainty can be ruled out with
reasonable confidence.
2) Linear extrapolation is used if t,_~' dose-
response curve is expected ti a
linear component below the |i u
departure. This includes:
- Agents or their p' " 'elites that >
DNA- reactive e dirtx
mutagenic ai /.
- Agents or theii 'taholiles r which
human exposures 'vxly li lens are
near 1 '«ssociat<.. 11' 's
If1 £ LU UJ ''.'I.
Lii. extrapolation also i 1 when
data . insufficient l<> 'lilish . k- of
actional "hen scientilk. plaush
The result »r linear ex I isolation is
described by t, ^ral slope lor or an
inhalation unit risk vhich : .e slope of
the dose-response cy ¦ ¦ v'ur doses or
concentrations, respect
3) Nonlinear models are used for
extrapolation if there are sufficient data to
ascertain the mode of action and to
conclude that it is not linear at lower
doses, and the agent does not demonstrate
mutagenic or other activity consistent
with linearity at lower doses. Nonlinear
approaches generally should not be used
in cases where mode of action has not
ascertained. If nonlinear extrapolation is
appropriate but no model is developed, an
alternative is to calculate reference values.
4) Both linear and nonlinear approaches may
be used if there a multiple modes of action.
For example, modeling to a low response
level can be useful for estimating the
response at doses where a high-dose mode
of action would be less important.
II I iiv extrapolation is used, the
assessn- .iL-velops a candidate slope factor
or in- i< lor each suitable data set. These
rec t. arrayed, using common dose
me ics, to w the distribution of relative
otency aci various effects and
experimental systems. The assessment then
derives or selects an overall slope factor and
an overall unit risk lor the agent, considering
the various dose-response analyses, the study
preferences discussed in Section 6, and the
possibility of basing a more robust result on
multiple (.lata sets.
7.5. Considering susceptible
populations and lifestages
The assessment analyzes the available
information on populations and lifestages that
may be particularly susceptible to each effect.
A tiered approach is used fU.S. EPA.
20115a. §3.51.
1) If an epidemiologic or experimental study
reports quantitative results for a
susceptible population or lifestage, these
data are analyzed to derive separate
toxicity values for susceptible individuals.
2) If data on risk-related parameters allow
comparison of the general population and
susceptible individuals, these data are
used to adjust the general-population
toxicity values for application to
susceptible individuals.
3) In the absence of chemical-specific data,
the EPA has developed age-dependent
adjustment factors for early-life exposure
to potential carcinogens that have a
mutagenic mode of action. There is
evidence of early-life susceptibility to
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Toxicological Review of tert-Butyl Alcohol
various carcinogenic agents, but most
epidemiologic studies and cancer
bioassays do not include early-life
exposure. To address the potential for
early-life susceptibility, the EPA
recommends fU.S. EPA. 2005b. 551:
10-fold adjustment for exposures
before age 2 years.
3-fold adjustment for exposures
between ages 2 and 16 years.
7.6. Reference values and uncertainty
factors
An oral reference dose or an inhalation
reference concentration is an estimate of an
exposure (including in susceptible subgroups)
that is likely to be without an appreciable risk
of adverse health effects over a lifetime fU.S.
EPA. 2002.54.21. Reference values are
typically calculated for effects ot' ' han
cancer and for suspected carcinogens .
characterized mode of action indicates it t.
necessary key event does not occur beli a
specific dose. Reference ' -s provide
information about ris1 . , ¦¦ exposui
levels.
The assessment elk ¦tcri/es <. cts that
form the basis for refereiK ikies nlverse,
considered' diverse, oi ¦¦ -n
adverse i. iu "elopn. ll toxin.,
rep roc' . e toxicity, a. "uroli ;tv there
is guida ¦ on adverse -cts their
biologic markers S. !)A.
1998b. 1996. 'in).
To account iv ¦ncerUiinlv ;i variability
in the derivation oi 'etinie Inn [exposure
where adverse effect. ¦¦ not cipated to
occur, reference valuer ¦¦ iiculated by
applying a series of uncerU. y factors to the
point of departure. If a point of departure
cannot be derived by modeling, a no-
observed-adverse-effect level or a lowest-
observed-adverse-effect level is used instead.
The assessment discusses scientific
considerations involving several areas of
variability or uncertainty.
Human variation. The assessment accounts
for variation in susceptibility across the
human population and the possibility that
the available data may not be
representative of individuals who are
most susceptible to the effect A factor of
10 is generally used to account for this
variation. This factor is reduced only if the
point of departure is derived or adjusted
specifically for susceptible individuals
(not for a general population that includes
both susceptible and non-susceptible
indivir! "s) fU.S. EPA.
200" .4.5: 1998b. 54.2: 1996. 54: 1994.
5 1: 1991b. 53.41.
A. 'mm extrapolation. If animal
results ai ed to make inferences about
humans, the •'jssment adjusts for cross-
species differe. ¦ These may arise from
differences in toxicokinetics or
toxicodvnamics. Accordingly, if the point
ol departure is standardized to equivalent
human terms or is based on toxicokinetic
or dosimetry modeling, a factor of 101/2
(rounded to 3) is applied to accountfor the
remaining uncertainty involving
toxicokinetic and toxicodynamic
differences. If a biologically based model
adjusts fully for toxicokinetic and
toxicodynamic differences across species,
this factor is not used. In most other cases,
a factor of 10 is applied (U.S. EPA.
2011: 2002.54.4.5: 1998b. 54.2: 1996. 54:
1994.54.3.9.1: 1991b. 53.41.
Adverse-effect level to no-observed-
adverse-effect level. If a point of
departure is based on a lowest-observed-
adverse-effect level, the assessment must
infer a dose where such effects are not
expected. This can be a matter of great
uncertainty, especially if there is no
evidence available at lower doses. A factor
of 10 is applied to account for the
uncertainty in making this inference. A
factor other than 10 may be used,
depending on the magnitude and nature of
the response and the shape of the dose-
response curve (U.S. EPA.
2002.54.4.5: 1998b. 54.2: 1996. 54: 1994.
54.3.9.1: 1991b. 53.41.
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Subchronic-to-chronic exposure. If a point
of departure is based on subchronic
studies, the assessment considers whether
lifetime exposure could have effects at
lower levels of exposure. A factor of 10 is
applied to account for the uncertainty in
using subchronic studies to make
inferences about lifetime exposure. This
factor may also be applied for
developmental or reproductive effects if
exposure covered less than the full critical
period. A factor other than 10 may be used,
depending on the duration of the studies
and the nature of the response (U.S. EPA.
2002. §4.4.5: 1998b. §4.2: 1994. 54.3.9.11.
Incomplete database. If an incomplete
database raises concern that further
studies might identify a more sensitive
effect, organ system, or lifestage, the
assessment may apply a database
uncertainty factor fU.S. I ¦. I 'A.
2002. §4.4.5: 1998b. §4.2: 1996. §1:
§4.3.9.1: 1991b. §3.41 The size of the
factor depends on the nature of Hit-
database deficiency. I ¦'or example, the KI'A
typically follows the suggestion thai a
factor of 10 be applied if both a prenatal
toxicity study and a two-generation
reproduction study are missing and a
factor of 10'" if either is missing (I S
EPA. 2002. g4 4 5).
In this way, the assessment derives
candidate values for each suitable (.lata setand
effect that is credibly associated with the
agent These results are arrayed, using
common dose metrics, to show where effects
occur across a range of exposures fU.S. EPA.
1994. §4.3.91
The assessment derives or selects an
organ- or system-specific reference value for
each organ or system affected by the agent.
The assessment explains the rationale for each
organ/system-specific reference value (based
on, for example, the highest quality studies,
the most sensitive outcome, or a clustering of
values). By providing these organ/system-
specific reference values, IRIS assessments
facilitate subsequent cumulative risk
assessments that consider the combined effect
50 of multiple agents acting at a common site or
51 through common mechanisms (NRC. 20091.
52 The assessment then selects an overall
53 reference dose and an overall reference
54 concentration for the agent to represent
55 lifetime human exposure levels where effects
56 are not anticipated to occur. This is generally
57 the most sensitive organ/system-specific
58 reference value, though consideration of study
59 quality and confidence in each value may lead
60 to a different selection.
61 7.7. Confidence and uncertainty in the
62 reference values
63 The assessment selects a standard
64 descriptor to characterize the level of
65 confidence in each reference value, based on
66 the likelihood that the value would change
67 with further testing. Confidence in reference
68 values is based on quality of the studies used
69 and completeness of the database, with more
70 weight given to the latter. The level of
71 confidence is increased for reference values
72 based on human data supported by animal
73 data fU.S. I-I'A. 1994. §4.3.9.21
74 High confidence: The reference value is not
75 likely to change with further testing,
76 except for mechanistic studies that might
77 affect the interpretation of prior test
78 results.
79 Medium confidence: This is a matter of
80 judgment, between high and low
81 confidence.
82 Low confidence: The reference value is
83 especially vulnerable to change with
84 further testing.
85 These criteria are consistent with
86 guidelines for systematic reviews that
87 evaluate the quality of evidence. These also
88 focus on whether further research would be
89 likely to change confidence in the estimate of
90 effect (Guvatt etal.. 2008bl
91 All assessments discuss the significant
92 uncertainties encountered in the analysis. The
93 EPA provides guidance on characterization of
94 uncertainty (U.S. EPA. 2005a. §3.61 For
95 example, the discussion distinguishes model
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uncertainty (lack of knowledge aboutthe most
appropriate experimental or analytic model)
and parameter uncertainty (lack of knowledge
about the parameters of a model).
Assessments also discuss human variation
(interpersonal differences in biologic
7 susceptibility or in exposures that modify the
8 effects of the agent).
9
10
11 August 2013
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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, a
dehydrating agent, 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. Developmental effects (e.g., reduced fetal viability) have
been observed in short-term exposure to high levels of tert-butanol (via oral or inhalation
exposure) in animals. Neurodevelopmental effects also have been observed, but results
were inconsistent. No chronic inhalation exposure studies have been conducted. There is
suggestive evidence that tert-butanol is carcinogenic to humans based on renal tumors in
male rats and thyroid tumors in female mice.
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 observed increased incidence or severity of nephropathy after 13 weeks of oral
exposure, increased severity of nephropathy after a 2-year 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 a2U-globulin-associated nephropathy, tert-
Butanol, however, is a weak inducer of a2u-globulin-nephropathy, and is not the sole process
contributing to renal tubule nephropathy. Chronic progressive nephropathy (CPN) may also be
involved in some of the noncancer effects, but the evidence is inconclusive. Endpoints specifically
related to either a2U-globulin-nephropathy or CPN were not considered for kidney hazard
identification. Changes in kidney weights, transitional epithelial hyperplasia, suppurative
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inflammation, and severity and incidence of nephropathy, however, are considered to result from
tert-butanol exposure and are appropriate for identifying a hazard to the kidney.
There is suggestive evidence of developmental toxicity following tert-butanol exposure.
Developmental effects include increased fetal loss, decreased fetal body weight, and increased
skeletal variations. At this time, no conclusions were drawn in regard to reproductive system
toxicity. There is inadequate information at this time to draw conclusions regarding
neurodevelopmental toxicity, liver, and urinary bladder toxicity.
Oral Reference Dose (RfD) for Effects Other Than Cancer
Kidney toxicity, represented by kidney transitional c . i ml hyperplasia, was chosen as the
basis for the overall oral reference dose (RfD) (see Table j. ¦¦ chronic study by NTP Q9951
and the observed kidney effects were used to derive 1' e kiD. The l Miint of transitional epithelial
hyperplasia was selected as the critical effect liec ,c was observed n '¦>oth rat sexes
consistently, it is a specific and sensitive indicator of kidney toxicity, and was induced in a dose-
responsive manner. Benchmark dose (BMD) modeling was used to derive the bench mark dose
lower confidence limit (BMDLio%) of 1 ^ mg/kg-day. The I1MDI, was converted to a human
equivalent dose using body weight3/ ¦ 1 and the value of /i.!M mg/kg-day was used as the
point of departure (POD) for RfD deriv;. n i !,'I'A. 2!) i!).
The overall RfD was calculated by iding . ,)f)|) for kidney transitional epithelial
hyperplasia by a composite uncertainly fact. 'HI') of "count lor the extrapolation from
animals to humans (!-!) and for interindividual '< _os in !¦ 'i susceptibility (10).
Table ES-1. Organ/system-specific RfDs d overall RfD for tert-butanol
Hazard
Basis
Point of
departure*
(mg/kg-day)
UF
Chronic RfD
(mg/kg-day)
Study
exposure
description
Confidence
Kidney
Transitional epithelial
hyperplasia
3.8
30
1 X 10 1
Chronic
High
Overall RfD
Kidney
3.8
30
1 X 10 1
Chronic
High
*HED PODs were calculated using BW3/4scaling (U.S. EPA, 2011).
Effects Other Than Cancer Observed Following Inhalation Exposure
Kidney effects are a potential human hazard of inhalation exposure to tert-butanol.
Although no effects were observed in mice, kidney weights were increased in male and female rats
following 13 weeks of inhalation exposure. In addition, nephropathy severity increased in male
rats. No human studies are available to evaluate the effects of inhalation exposure. As discussed
above for oral effects, endpoints specifically related to either a2uglobulin nephropathy or CPN were
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Toxicological Review of tert-Butyl Alcohol
1 not considered for kidney hazard identification. Changes in kidney weights and severity of
2 nephropathy, however, are considered a result of tert-butanol exposure and are appropriate for
3 identifying a hazard to the kidney.
4 Inhalation Reference Concentration (RfC) for Effects Other Than Cancer
5 Kidney toxicity, represented by transitional epithelial hyperplasia, was chosen as the basis
6 for the inhalation reference concentration (RfC) (see Table ES-2). Although endpoints from a route-
7 specific study were considered, the availability of a physiologically based pharmacokinetic (PBPK)
8 model for tert-butanol in rats [modified by Salazar et al. 121 i i .r>)1 wed for more specific and
9 sensitive equivalent inhalation PODs derived from a rouk'-l' extrapolation from the PODs of
10 the oral NTP f 19951 study. The POD adjusted for the hur ^ 'k-nt concentration (HEC) was
11 26.1 mg/m3 based on transitional epithelial hyperplasia.
12 The RfC was calculated by dividing the POD by a composite Ui '-!(> to account for
13 toxicodynamic differences between animals and humans (3) and interinu. ''ial differences in
14 human susceptibility (10).
15 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
Transitional epithelial
hyperplasia
26.1
30
9 x 10 1
Chronic
High
Overall RfC
Kidney
26.1
30
9 x 101
Chronic
High
16 Continuous inhalation human equivalent concentration that leads to the same average blood concentration of
17 te/t-butanol as continuous oral exposure at the BMDL.
18 Evidence of Human Carcinogenicity
19 Under I In.- K I'A's ainccr guidelines (U.S. EPA. 2005a). there is suggestive evidence of
20 carcinogenic potential for fiVf-liuUmol. tert-Butanol induced kidney tumors in male (but not female)
21 rats and thyroid tumors (primarily benign) in male and female mice following long-term
22 administration in drinking water (NTP. 1995). The potential for carcinogenicity applies to all routes
23 of human exposure.
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Quantitative Estimate of Carcinogenic Risk from Oral Exposure
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 fNTP. 19951. The study
included histological examinations for tumors in many different tissues, contained three exposure
levels and controls, contained adequate numbers of animals per dose group (~50/sex/group),
treated animals for up to 2 years, and included detailed reporting of methods and results.
Although tert-butanol was considered to have "suggestive evidence of carcinogenic
potential," the NTP study was well conducted and quantitative .n ^ sis could be useful for
providing a sense of the magnitude of potential carcinogen* 1 fl'.S. EPA. 2005al. A slope factor
was derived for thyroid tumors in female and male mice . m> 'cd fert-butanol POD was scaled
to HEDs according to EPA guidance by converting th MDLio on ti. isis of (body weight)3/4
scaling (U.S. EPA. 2011. 2005a). Using linear exlr jtion from the I'lv. a human equivalent
oral slope factor was derived (slope factor = 0. i, 1HLio). The resulting <>i 'ope factor is 5 x KM
per mg/kg-day.
Quantitative Estimate of Carcinogenic Risk from Inhalation Exposure
No chronic inhalation exposure.1 studies lo <(.*/¦<-butanol art.1 available. Lifetime exposure to
tert-butanol has been associated with increased renal tuliule ink'nomas and carcinomas as well as
thyroid follicular cell adenomas and cam nomas. As stated aliove, I lie rat kidney tumors are
unsuitable for quantitative analysis as there is not enough data Lo determine the relative
contribution of a2u-glolmlin nephropathy and other processes to the overall kidney tumor
response. Although the mouse thyroid tumors served as the basis for the oral slope factor, route-to-
route extrapolation is not possible lor these thyroid effects in mice because the only PBPK model
available is lor rats. The re lore, no quantitative estimate of carcinogenic risk could be determined
for inhalation exposure.
Susceptible Populations and Lifestages for Cancer and Noncancer
In vitro studies suggest that cytochrome P-450 (CYP450) (Cederbaum etal.. 1983:
Cederbaum and Cohen. 1'UiO). plays a role in the metabolism of tert-butanol. No studies, however,
have identified the specific CYl's responsible for the biotransformation of tert-butanol. Various
CYPs are under-expressed in the mouse fetus and neonate fLee etal.. 20111 and decreased in older
mice (Lee etal.. 2011) and rats (Lee etal.. 2008). Decreased ability to detoxify and transport tert-
butanol out of the body could result in increased susceptibility to tert-butanol.
With regard to cancer, differences in lifestage sensitivity to chemically induced thyroid
carcinogenesis are unknown (U.S. EPA. 1998a). An increased incidence of thyroid tumors was
identified in mice after tert-butanol exposure, and human studies have demonstrated that children
are more sensitive than adults are to thyroid carcinogenesis resulting from ionizing radiation.
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Collectively, there is little evidence on tert-butanol itself to identify any populations or lifestages
that may be especially susceptible.
Key Issues Addressed in Assessment
An evaluation of whether tert-butanol caused a2U-globulin-associated nephropathy was
performed. The presence of a2U-globulin in the hyaline droplets was confirmed in male rats by
a2u-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 gra it -asts, either were absent or
inconsistently observed across subchronic or chronic studie . ic of these effects occurred in
female rats or in either sex of mice, although these endp< n less frequently evaluated in
these models. Evidence implies an a2U-globulin MOA r ^pc.ativc. aio 'gh it is relatively weak in
response to tert-butanol and is notsolely respond or the renal tin. nephropathy observed in
male rats. CPN also is instrumental in renal tub icphropathy, in both h and female rats.
Several other effects in the kidney unrelated to ct; h
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LITERATURE SEARCH STRATEGY | STUDY
SELECTION AND EVALUATION
A literature search and screening strategy were used to identify literature characterizing
the health effects of tert-butanol. This strategy consisted of a broad search of online scientific
databases and other sources to identify all potentially pertinent studies. In subsequent steps,
references were screened to exclude papers not pertinent to an assessment of the health effects of
tert-butanol, and remaining references were sorted into categories for further evaluation. This
section describes the literature search and screening strategy in detail.
The chemical-specific search was conducted in four online scientific databases, including
PubMed, Web of Science, Toxline, and TSCATS through May 2015, using the keywords and limits
described in Table LS-1. The overall literature search approach is shown graphically in Figure LS-1.
An additional seven citations were obtained using additional search strategies described in Table
LS-2. After electronically eliminating duplicates from the citations retrieved through these
databases, 2,648 unique citations were identified.
The resulting 2,648 citations were screened for pertinence and separated into categories as
presented in Figure LS-1 using the title and either abstract or full text, or both, to examine the
health effects of tert-butanol exposure. The inclusion and exclusion criteria used to screen the
references and identify sources of health effects data are provided in Table LS-3.
• 12 references were identified as "Sources of Health Effects Data" and were considered for
data extraction to evidence tables and exposure-response arrays.
• 200 references were identified as "Sources of Mechanistic and Toxicokinetic Data" and
"Sources of Supplementary Health Effects Data"; these included 39 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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• 63 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.
• 2,373 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 design 1 o cast a wide net and to
minimize the possibility of missing potentially releva'' ,ith effects data.
The complete list of references and the sorting c' ,e rials can be found on the tert-
butanol project page of the HERO website at
https://hero.epa.gov/index.cfm/project/page/fv . id / i 543.
Selection of Studies for Inclusion in Evidence Tables
To summarize the important information systematically li'om the primary health effects
studies in the tert-butanol database, evidence tables were constructed in a standardized tabular
formatas recommended by NRC (2011). Studies were arranged in evidence tables by effect, species,
duration, and design, and not by quality. 01 the studies thai were retained after the literature search
and screen, 12 studies were identified as "Sources of I leallh Kfleets Data" and were considered for
extraction into evidence tallies lor hazard identification in Chapter 1. Initial review found two
references fCirvello et al.. i'>'>5: l-indamood ct a!.. l')')21 to be publications of the NTP Q9951 data
prior to the release of the final National Toxicology Program (NTP) report One publication
(Takahashi et al.. l')')3) 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 et al. (1995). Takahashi etal. (1993). and
Lindamood et al. f 19921 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) using oral and inhalation exposures were
performed primarily in rats and did not differ qualitatively from the results of the longer studies
(i.e., >30-day exposure studies). These were grouped as supplementary studies, however, because
the database 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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provided by IRIS assessments) and are the focus of this assessment. Such supplementary studies
may be discussed in the narrative sections of Chapter 1 and are described in sections such as the
"Mode of Action Analysis" to augment the discussion or presented in appendices, if they provide
additional information.
Supporting Studies
Additional Search Strategies
(See Table LS-2 for methods and results)
n = 7
Combined Dataset
(After all duplicates removed)
n = 2,648
Sources of Health Effects Data (n = 12)
12
Human health effects studies
Animal studies
Manual Screening for Pertinence
(Title/Abstract/Full Text)
Sources of Mechanistic and Toxicokinetic
Data (n = 112)
PBPK/ADME
Genotoxicity
51 Other mechanistic studies
39
Sources of Supporting Health Effects Data
(n = 88)
Human case reports
Not relevant exposure paradigms (e.g.,
dermal, eye irritation, acute)
Preliminary data
Physical dependency studies
74
62
87
85
1,286
87
703
Excluded/Not on Topic (n = 2,310)
Abstract only/comment/society
abstracts
Biodegradation/environmental fate
Chemical analysis/fuel chemistry
Other chemical/non t-butanol
Method of detection/exposure and
biological monitoring
Methodology/solvent
Secondary Literature and Sources of
Contextual Information (n = 126)
Not relevant species/matrix (e.g.,
amphibians, fish)
14 QSAR
Mixtures
Reviews/editorials
Other agency assessments
Book chapter/section
41
37
13
(After duplicates removed electronically)
n=2/641
Database Searches
(See Table LS-1 for keywords and limits)
Figure LS-1. Summary of literature search and screening process for
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)
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)
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)
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
Not PubMed
TSCATS2
(1/4/2013)
(4/17/2014)
(5/13/2015)
75-65-0
None
2 Table LS-2. Summary of additional search strategies for tert-butanol
Approach used
Source(s)
Date
performed
Number of additional references
identified
Manual search of
citations from
reviews
Review article: McGregor (2010).
7ert7o/j/-butanol: A toxicological
review. Crit Rev Toxicol 40(8): 697-
727.
1/2013
5
Review article: Chen (2005). Amended
final report of the safety assessment
of t-butyl alcohol as used in
cosmetics. Int J Toxicol 24(2): 1-20.
1/2013
2
Manual search of
citations from
reviews conducted
IPCS (1987a). Butanols: Four isomers:
1-butanol, 2-butanol, tert-butanol,
isobutanol [WHO EHC], Geneva,
1/2013
None
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Approach used
Source(s)
Date
performed
Number of additional references
identified
by other
international and
federal agencies
Switzerland: World Health
Organization.
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
Population
• Humans
• Standard mammalian animal models,
including rat, mouse, rabbit, guinea pig,
monkey, dog
• Ecological species *
• Nonmammalian 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 because study was not
potentially relevant
• Bioremediation, biodegradation, or environmental
fate of te/t-butanol, including evaluation of
wastewater treatment technologies and methods
for remediation of contaminated water and soil
• Chemical, physical, or fuel chemistry studies
• Analytical methods for measuring/detecting/
remotely sensing te/t-butanol
• Use of te/t-butanol as a solvent or methodology for
testing unrelated to te/t-butanol
This document is a draft for review purposes only and does not constitute Agency policy.
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Inclusion criteria
Exclusion criteria
• 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 not considered a source of health effects data or supplementary
health effects data/mechanistic and toxicokinetic data, but were considered as sources of contextual
information.
1 Database Evaluation
2 For this draft assessment, 12 references reported on experimental animal studies that
3 comprised the primary sources of health effects data; no studies were identified that evaluated
4 humans exposed to tert- butanol (e.g., cohort studies, ecological studies). The animal studies were
5 evaluated using the study quality considerations outlined in the Preamble, considering aspects of
6 design, conduct, or reporting that could affect the interpretation of results, overall contribution to
7 the synthesis of evidence, and detenu i nation of hazard pole ill ial as noted in various EPA guidance
8 documents (U.S. EPA. 2005a. 1998b. 1')') i b). The objective was to identify the stronger, more
9 informative studies based on a uniform evaluation of quality characteristics across studies of
10 similar design. As stated in the Preamble, studies were evaluated to identify the suitability of the
11 study based on:
12 • Study design
13 • Nature ol the assay and validity lor its intended purpose
14 • Characterization ol the nature and extent of impurities and contaminants of tert- butanol
15 administered, if applicable
16 • Characterization of dose and (.losing regimen (including age at exposure) and their
17 adequacy to elicitadverse effects, including latent effects
18 • Sample sizes and statistical power to detect dose-related differences or trends
19 • Ascertainment of survival, vital signs, disease or effects, and cause of death
20 • Control of other variables that could influence the occurrence of effects
21 Additionally, several general considerations, presented in Table LS-4, were used in
22 evaluating the animal studies. Much of the key information for conducting this evaluation can be
23 determined based on study methods and how the study results were reported. Importantly, the
24 evaluation at this stage does not consider the direction or magnitude of any reported effects.
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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 were
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 differc
Experimental Animal Studies
The experimental animal studies, comprised entirely ol 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 ol lieun(developmental studies, these sources were
conducted according to Organisation for Economic Co-ope ration and Development Good
Laboratory Practice (GLP) guidelines, presented extensiv e histopathological data, or clearly
presented their methodology; thus, these studies are considered high quality. These studies include
2-year bioassays using oral exposures in ratsand mice; two sulichronic drinking water studies in
rats and one in mice; an inhalation subchronic study in rats and mice: a reevaluation of the NTP
(1995) rat data: two oral developmental studies: two inhalation developmental studies; and a
single one-generation reproductive study that also ev aluates other systemic effects (Table LS-5). A
more detailed discussion ol any methodological concerns that were identified precedes each
endpointevaluated in the hazard identification section. Overall, the experimental animal studies of
tert-butanol involving repealed 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 lor 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 database
Study category
Study duration, species/strain, and administration method
Chronic
2-vear studv in F344 rats (drinking water) NIP (1995)
2-vear studv in B6C3Fi mice (drinking water) NIP (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 B6C3F: mice (inhalation) NTP (1997)
10-week studv in Wistar rats (drinking water) Acharva et al. (1997), Acharva et al. (1995)
Reproductive
One-generation reproductive toxicity studv in Sprague-Dawlev rats (gavage) Lyondell
Chemical Co. (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)
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1 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 flPCS. 1987al. Selected chemical and physical properties of tert-butanol are presented in Table 1-1.
8 Table 1-1. Physicochemical properties and chemical identity of tert-butanol
Characteristic
Information
Reference
Chemical name
tert-Butanol
HSDB (2007)
Synonyms/Trade names
t-Butyl alcohol; tert-Butanol; tert-Butyl alcohol; t-
Butyl hydroxide; 1,1-Dimethylethanol; NCI-C55367;
2-Methyl-2-propanol; tertiary Butanol; Trimethyl
carbinol; Trimethyl methanol; t-butyl alcohol; TBA
HSDB (2007)
IPCS (1987b)
Chemical formula
C4H10O
HSDB (2007)
CASRN
75-65-0
HSDB (2007)
Molecular weight
74.12
HSDB (2007)
Melting point
25.7°C
HSDB (2007)
Boiling point
82.41°C
HSDB (2007)
Vapor pressure
40.7 mm Hg @ 25°C
HSDB (2007)
Density/Specific gravity
0.78581
HSDB (2007)
Flashpoint
11°C (closed cup)
HSDB (2007)
Water solubility at 25°C
1 x 106 mg/L
HSDB (2007)
Octanol/Water Partition
Coefficient (Log Kow)
0.35
HSDB (2007)
Henry's Law Constant
9.05 x 10"6 atm-m3/mole
HSDB (2007)
Odor threshold
219 mg/m3
HSDB (2007)
Conversion factors
1 ppm = 3.031 mg/m3
1 mg/m3 = 0.324 ppm
IPCS (1987b)
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Characteristic
Information
Reference
Chemical structure
C
H3c—
c
;h3
OH
ih3
HSDB (2007)
1.1.2. Toxicokinetics
tert-Butanol is rapidly absorbed following exposure by oral and inhalation routes (see
Appendix B, Section B. 1.1). Studies in experimental animals indicate that 99% of the compound was
absorbed after oral administration. Comparable blood levels ol'' <(.*/¦<-butanol and its metabolites
have also been observed after acute oral or inhalation exposures in rats fARCO. 1983). In another
study fFaulkner etal.. 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
(Poet etal.. 1997: Faulkner etal.. 1989: ARCO. 1983). Following exposure to <(.*/¦<-butanol in rats,
tert-butanol was found in kidney, liver, and blood, with male rats retaining more <(.*/¦<-butanol than
female rats fWilliams and Borghoff. 2001).
A general metabolic scheme lor fivf-butanol, illustrating the biotransformation in rats and
humans, is shown in Figure 1-1 (see Appendix I!. 1.3).
Human data on the excretion ol /(.'//-butanol comes Irom studies of methyl tert-butyl ether
(MTBE) and ethyl te/7-bulvl ether (l"M!E) (Nihlen et al.. i'>'>8a. b). 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 ol /(.'//-bulanol in urine following ITBE exposure was 7.9 ± 2.7 hours (average
of 104- and 2 10-mg/m : l "M!F (.loses). These studies reported urinary levels of tert-butanol (not
including downstream metabolites) to be less than l"u of administered MTBE or ETBE
concentrations (Nihlen etal.. l')'>8a. b). Ambergetal. (2000) also observed a similar half-life of
9.8 ±1.4 hours alter human exposure to ETBE of 170 mg/m3. The half-life for tert-butanol in rat
urine was 4.0 ± 1.4 hours al l"M!F 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
\
rats,
humans
OH
"Y
CH3 oh
2-methyl-1,2-propanediol
-OH
CH,
2-hydroxyisobutyric acid
formaldehyde
o
rats
CH,
\
h3c
acetone
CH,
-CH,
CH,
t-butyl sulfate
Source: NSF International (2003), /¦ "*96), Bernauer ' (1998), Ambers et al. (1999),
and Cederbaum and Cohen (1980).
Figure 1-1. Biotransformation offerf-butanol in rats and humans.
1.1.3. Description of'Toxicokinetic Models
No physiologically based pharmacokinetic (I'liPK) models have been developed specifically
for administration of tert-bulanol. Some models have heen used to study tert-butanol as the
primary metabolite alter oral or inhalation exposure to MTBE or ETBE. The most recent models for
MTBE oral and inhalation exposure include a component for the binding of tert-butanol to
a2u-glo bu I i n (liorghoffetal.. 2010: Leavens and Borghoff. 20091. A more-detailed summary of the
toxicokinetic models is provided in Appendix B, Section B.1.5.
A PBPK model lor tert-bulanol was modified by adapting previous models for MTBE and
tert- butanol (Leavens and BoruholT. 2009: Blancato etal.. 20071. The addition of a sequestered
blood compartment lor /(.'//-butanol substantially improved the model fit. The alternative
modification of changing to diffusion-limited distribution between blood and tissues also improved
the model fit, but was considered less biologically plausible. Physiological parameters and partition
coefficients were obtained from published measurements. The rate constants for tert-butanol
metabolism and elimination were from a published PBPK model of MTBE with a tert-butanol
subcompartment (Blancato etal.. 2007). Additional model parameters were estimated by
calibrating to data sets for i.v., oral, and inhalation exposures as well as repeated dosing studies for
tert-butanol. Overall, the model produced acceptable fits to multiple rat time-course datasets of
tert-butanol blood levels following either inhalation or oral gavage exposures.
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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 in these compounds are attributed to tert-
butanol. There are no assessments by national or international health agencies for ETBE. 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 et
al.. 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 (Saito el al.. 2013).
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 MT151: based on data on
carcinogenicity in animals. ATSDR reported thai inhalation exposure was associated with kidney
cancerin rats andliver cancerin mice. ATSDR concluded thai oral exposure lo MTI'li mightcause
liver and kidney damage, and nervous system effects in rals and mice. The chronic inhalation
minimal risk level was derived based on incidence and severity of chronic progressive nephropathy
in female rats (ATSDR. 1996). In 1()')7, KIWs Office of Water concluded that MTBE is carcinogenic
to animals and poses a potential carcinogenic potential lo humans based on an increased incidence
ofLeydigcell adenomas ol the testes, kidney tumors, lymphomas, and leukemia in exposed rats
(U.S. EPA. 1997). In l'J'W, the International Agency lor 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) MARC. IARC reported that oral exposure in rats resulted in
testicular tumors in males and lymphomas and leukemias (combined) in females; inhalation
exposu re 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 database examining kidney effects
following tert-butanol exposure contains eight studies from 5 references performed in rats or mice
(Lvondell Chemical Co.. 2004: Acharva etal.. 1997: NTP. 1997: Acharva et al.. 1995: NTP. 1995). and
a reevaluation of the rat data from NTP Q995I published by Hard etal. f20111: no human data are
This document is a draft for review purposes only and does not constitute Agency policy.
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available. Studies using short-term and acute exposures that 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 con- .led by the common occurrence
of age-related, spontaneous lesions characteristic of chroni ^ressive nephropathy (CPN) fNTP.
2015: Hard etal.. 2013: Melnick etal.. 2012: U.S. FPA.
1991 a):http://ntp.niehs.nih.gov/nnl/urinarv/kidnc qcp/index.!. ' CPN is more severe in male
rats than in females and is particularly common ' _¦ Sprague-Dawle\ I Fischer 344 strains.
Dietary and hormonal factors play a role in mm, ng CPN, although the ei »v is largely
unknown (see further discussion below).
Kidney weight. Changes in kidney weight (absolute and relative to body weight) were
observed in male and female F344 rats following exposures of 13 weeks (oral and inhalation) fNTP.
1997) and 15 months (oral) (NTP. 1995). I.yondell Chemical Co. (2004) also reported increases in
absolute and relative kidney weight in Sprague-Dawlev mis administered tert-butanol orally for
approximately 10 weeks (tabular data presented in Supplemental Information to this Toxicological
Review). Changes were observed in both male and female rats, which exhibited strong dose-related
increases in absolute kidney weight (Spearman's rank coefficient > 0.78) following either oral or
inhalation exposures (Figure 1-3). 01 the oral (Figure 1-4 and inhalation (Figure 1-5) mouse
studies, only inhalation exposure in female mice induced a strong dose-related increase
(Spearman's rank coefficient = 0.9) in absolute kidney weights.
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
fBailev etal.. 2004). although potential impact should be evaluated. For tert-butanol, body weight in
exposed animals noticeably decreased at the high doses relative to controls in the oral 13-week and
2-year studies NTP f!9')5). I n this case, the decreased body weight of the animals affects the
relative kidney weight measures, resulting in an artificial exaggeration of changes. Thus, absolute
weight was determined the more reliable measure of kidney weight change for this assessment
Additionally, a recent analysis 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 f Craig etal.. 20141. Although relative and absolute kidney
weight data are both presented in exposure-response arrays (and in evidence tables in
Supplemental Information), the absolute measures were considered more informative for
determining tert-butanol hazard potential.
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Kidney histopathology. Treatment-related histopathological changes were observed in the
kidneys of male and female F344 rats following 13-week and 2-year oral exposures fNTP. 19951
and male F344 rats following a 13-week inhalation exposure fNTP. 19971. Similarly, male Wistar
rats exposed for approximately 10 weeks exhibited an increase in histopathological kidney lesions
(Acharva et al.. 1997: Acharva et al.. 19951. B6C3F| 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 (1997.19951 and Acharva etal. (19971: (19951 are described below.
Nephropathy and severity of nephropathy were report' ,nale and female rats in the
13-week oral studies fNTP. 19951. The nephropathy was ch ori/.ed as "...a spontaneous
background lesion...typically consisting] of scattered re ,il< ¦ lined by basophilic
regenerating tubule epithelium." fNTP. 19951. NT!' f ' ^5 i noted li. he increase in severity of
nephropathy was related to tert-butanol and "civ prized by an inci. -in the number and size
of foci of regeneration." The severity of nephro( -v increased, comparer h controls, in the
13-week male rats, which exhibited nephropathy 11 '"..olall' 'losed aninu. 'id 70% of
controls. Conversely, lesion severity was unchanged in I ho females, although nephropathy
incidence significantly increased with ^'/MiuUinol 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. Regenerativ e foci were characterized by tubules with
cytoplasmic basophilia, increased nuclear/cytoplasm ic ratio, and occasionally thickened basement
membranes and intraluminal protein casts." In exposed groups, the severity generally increased
from minimal to mild with increasing (.lose as "evidenced by an increased number of foci." No
treatment-related kidney histopathology was reported inthe female rats exposed through
inhalation (NTP. 19'>7).
In the 2-year oral study by NTP (19951. nephropathy was reported at 15 months and 2
years. The NTP (1995) 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 ol interstitial fibrosis and scarring, and glomerular sclerosis." At 15
months, male and female rats (30/30 treated; 10/10 controls) had nephropathy, and the severity
scores ranged from minimal to mild. At 2 years, male and female rats (149/150 treated; 49/50
controls) also had nephropathy, and although the severity was moderate in the control males and
minimal to mild in the control females, severity increased with tert-butanol exposure in both sexes
(NTP. 19951.
The lesions collectively described by NTP (1997.1995) as nephropathy and noted to be
common spontaneous lesions in rats, are consistent with CPN. The effects characterized as CPN are
related to age and not considered histopathological manifestations of chemically induced toxicity
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[see U.S. EPA (1991a). p. 35 for further details and a list of the typical observable histopathological
features of CPN], These lesions, however, are frequently exacerbated by chemical 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 nephropathy severity are included in
the consideration of hazard potential.
NTP Q9951 observed other kidney lesions, described as being associated with nephropathy
but diagnosed separately. Renal mineralization is defined by NTP (1995) as "focal mineral deposits
primarily at the corticomedullary junction." This mineralization is distinct from linear
mineralization, which is considered a lesion characteristic of it bulin nephropathy (for further
discussion of this particular lesion, see Mode of Action Anuh• Kidney Effects). The mineralization
is characterized as distinct linear deposits along radiati"' . iK "V collecting ducts. An increased
incidence of linear mineralization was limited to exr -il males in ii ?- vear oral study fNTP.
19951.
Renal mineralization was observed in essentially all female rats at all reported treatment
durations. A dose-related, increased incidence of mineralization was reported in male rats atthe
13-week, 15-month, and 2-year oral evaluations (N'i'l'. l')')5). NTI' (1995) 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 ofthis lesion in females. This description, however, is inconsistent with
the observation in this anil other databases that age-related nephropathy (i.e., CPN) is generally
more prevalentand more sev ere in male rats compared to females (U.S. EPA. 1991a). The
association of mineralization with CPN is unclear, considering the lack of spontaneous lesions in the
control and low-dose groups of 13-week males anil the dose-response relationships the tert-
butanol-exposeil males exhibited in the 13-week f NTP. 1997. 19951 and 2-year studies fNTP.
1995.). Furthermore, due to the overwhelming presence of mineralization in the control and treated
female rats, the contribution, il any, ol /<.'/ /-butanol to the formation ofthis lesion in females could
not be determined. Thus, the mineralization could be related to both aging of the animals and tert-
butanol exposure.
Two other histological kidney lesions observed in male and female rats are suppurative
inflammation and transitional epithelial hyperplasia. These lesions were observed in the 2-year
oral NTP Q9951 study. Although NTP Q9951 describes these lesions as related to the nephropathy
(characterized above as common and spontaneous, and considered CPN), that suppurative
inflammation and transitional epithelial hyperplasia exhibited incidence patterns different from
those reported for nephropathy is notable. 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%). The data for males suggest that CPN plays a role in the induction of suppurative
This document is a draft for review purposes oniy and does not constitute Agency poiicy.
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Toxicological Review of tert-Butyl Alcohol
inflammation; considering the responses in the females, however, the effect appears to be
predominantly treatment related. Suppurative inflammation was not observed in the animals of the
13-week oral fNTP. 19951 or inhalation study fNTP. 19971. which both reported nephropathy (as
CPN), providing further support that this lesion is not specifically related to the nephropathy.
Transitional epithelial 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 conlr low-dose females. NTP (19951
described transitional epithelial hyperplasia as increased l;>' .)l the transitional epithelial lining
of the renal pelvis; study authors noted no progression < > L 'plastic lesion to neoplasia. The
relatively high background in male controls (i.e., 50" suggests soi Potential influence, other
than tert- butanol treatment, on this effect The a1' e of this effect iii Kile control andlow-dose
animals and the dose-related increases in both . '\s and females, howevi. nlicate that similar to
the suppurative inflammation, the transitional epii. ' il hyper >sia is predo. nitly treatment
related. Transitional epithelial hyperplasia should not he confused with another lesion noted at 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 luhular degeneration,
degeneration of the hasemenl membrane ol the l!owman's capsule, diffused glomeruli, and
glomerular vacuolation were noted in a 10-week study in male Wistar rats (Acharva etal.. 1997:
Acharva et al.. 1995). A decrease in glutathione in the kidney accompanied these changes, which the
study authors noted as potentially indicativ e ol oxidative damage. Acharva etal. (19971: Acharva et
al. f!995 i used one (.lose 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 ol 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 hiomarkers informative of kidney toxicity were not measured in the
studies discussed above. Some changes occurred in urinalysis parameters (e.g., decreased urine
volume and increased specific gravity), accompanied by reduced water consumption, and thus
might not be related to an effect of kidney function fNTP. 19951.
Kidney tumors. The kidney is also a target organ for cancer effects (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 (Hard etal.. 2011: NTP. 1995). The increase in tumors from control was
similar in the low- and high-dose groups and highest in the mid-dose group. Overall, tumor
increases were statistically significant in trend testing, which accounted for mortality (p < 0.018).
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Toxicological Review of tert-Butyl Alcohol
Mortality increased with increasing exposure (p = 0.001); increased mortality alone, however, 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 fHard.
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,enal tubular adenocarcinomas
in male mice also were reported fNTP. 19951. one each in 11- v- and high-dose groups, but were
not considered by NTP to be "biologically noteworthy cl- . s . >s the tumors in mice are not
discussed further.
A Pathology Working Group, sponsored by Ia oikMI Chemical Company, reevaluated the
kidney changes in the NTP 2-year study to determine if additional histopathological changes could
be identified to inform the MOAfor renal tubule tumor development (Hard el al.. 20 LI). In all cases,
working group members were blinded to treatment groups, and used guidelines published by Hard
and Wolf T19991 and refinements reported by fHard and Seely. 20061: Hard and Seelv f20051 and
Hard (2008). The group's report and analysis by I lard et al. (201 i) confirmed the NTP findings of
renal tubule hyperplasia and renal tubule tumors in male rats al 2 years. In particular, they
reported similar overall tumor incidences in the exposed groups. Hard etal. (2011). 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 f 19951 and the
reanalysis by I lard et al. f 20 i 1 ; are included in Table 1-3 and Figure 1-1.
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Toxicological Review of tert-Butyl Alcohol
Male rats
Female rats
rho= 0.78 (all)
rho= 0.78 (all)
rho= 0.89 (oral)
rho= 0.72 (oral)
rho= 0.80 (inhalation)
rho= 0.9 (inhalation)
•
•
•
•
•
••
• •
•
• • o*
• O
• °
• •
• o
° O. •
o
o
o ,° .
1000 1
tert-butanol blood concentration (mg/l)
• Oral exposure
o Inhalation exposure
2
3 Figure 1-2. Comparison of absolute kidney weight change in male and female
4 rats across oral and inhalation exposure based on internal blood
5 concentration. Spearman rank correlation coefficient (rho) was calculated to
6 evaluate the direction of a monotonic association (e.g., positive value =
7 positive association) and the strength of association.
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Toxicological Review of tert-Butyl Alcohol
Male mice Female mice
3"
O
II
O
3"
O
II
O
Id
•
•
•
•
•
•
•
•
•
•
0 2000 4000 6000 8000 0 2000 4000 6000 8000 10000 12000
Administered dose (mg/kg-day) Administered dose (mg/kg-day)
Figure 1-3. Comparison of absolute kidney weight change in male and female
mice following oral exposure based on administered 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.
Male mice Female mice
•
CM
O
II
O
€
o
i
ii
o
_c
•
•
•
• •
•
•
•
•
0 1000 2000 3000 4000 5000 6000 0 1000 2000 3000 4000 5000 6000 7000
Administered dose (mg/m3) Administered dose (mg/m3)
Figure 1-4. Comparison of absolute kidney weight change in male and female
mice following inhalation exposure based on administered 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.
This document is a draft for review purposes only and does not constitute Agency policy.
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Toxicological Review of tert-Butyl Alcohol
1 Table 1-2. Changes in kidney histopathology in animals following exposure to
2 tert-butanol
Reference and study design
Results
Acharva et al. (1997)
Acharva et al. (1995)
Wistar rat; 5-6 males/treatment
Drinking water (0 or 0.5%), 0 or
575 mg/kg-d
10 weeks
1" tubular degeneration, degeneration of the basement membrane of the
Bowman's capsule, diffused glomeruli, and glomerular vacuolation (no
incidences reported)
4/ kidney glutathione (~40%)*
NTP (1995)
F344/N rat; 10/sex/treatment
Drinking water (0, 2.5, 5, 10, 20,
or 40 mg/mL)
M: 0, 230, 490, 840, 1,520,
3,610a mg/kg-d
F: 0, 290, 590, 850, 1,560,
3,620a mg/kg-d
13 weeks
Incidence (severity):
Males
Females
Dose
(mg/kg-d)
Minerali-
zation1
Neohro-
pathy£
Dose
(mg/kg-d)
Minerali-
zation-
Nephro-
pathy£
0
0/10
7/10 (1.0)
0
10/10 (1.7)
2/10 (1.0)
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)
3,610
4/10'(1.0)
7/10(1.1)
3,620a
6/10 (1.2)
7/10* (1.0)
NTP (1995)
B6C3F 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/kgd
F: 0, 500, 820, 1,660, 6,430,
ll,620a mg/kgd
13 weeks
Study authors indicated no treatment-related changes in kidney
histopathology (histopathological data not provided for the 13-week study)
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Toxicological Review of tert-Butyl Alcohol
Reference and study design
Results
NTP (1995)
Incidence (severity):
Males
F344/N rat; 60/sex/treatment
Linear
mineralization-
(terminal)
(10/sex/treatment evaluated at
15 months interim)
Drinking water (0,1.25, 2.5, 5, 10
Dose
(mg/kg-d)
Mineralization-
(interim)
Mineralization-
(terminal)
mg/mL)
0
1/10 (1.0)
26/50 (1.0)
0/50
M: 0, 90, 200, 420a mg/kg-d
F: 0,180, 330, 650a mg/kg-d
90
2/10 (1.0)
28/50 (1.1)
5/50* (1.0)
2 years
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)
Dose
epithelial
Nephropathy2
(mg/kg-d)
hyperplasia
severity
0
25/50 (1.7)
3.0
90
32/50 (1.7)
3.1
200
36/50* (2.0)
3.1
420
40/50* (2.1)
3.3*
Females
Inflammation
Dose
Mineralization-
Mineralization-
(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*
650"
10/10 (2.8)
Transitional
50/50 (2.9)
17/50*
Dose
epithelial
Nephropathy2
(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*
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
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Toxicological Review of tert-Butyl Alcohol
Reference and study design
Results
F: 0, 510,1,020, or 2,110 mg/kg-
d
2 years
NTP (1997)
Male
F344/N rat; 10/sex/treatment
Incidence of Average severity
Inhalation analytical
Concentration chronic of chronic
concentration: 0,134, 272, 542,
(mg/m3) nephropathy-1 nephropathy
1,080, or 2,101 ppm (0, 406, 824,
0 9/10 1.0
1,643, 3,273 or 6,368 mg/m3)
406 8/10 1.4
(dynamic whole-body chamber)
6 hr/d, 5 d/wk
824 9/10 1.4
13 weeks
Generation method (Sonimist
1,643 10/10 1.6
Ultrasonic spray nozzle
3,273 10/10 1.9
nebulizer), analytical
concentration and method were
6,368 10/10 2.0
reported
Females: no treatment-related changes in kidney related histopathology
observed
Severity categories: 1= minimal, 2= mild. \n results from statistical tests
repurled
NTP (1997)
No treatment-related changes in kidney related histopathology observed
B6C3F 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 a The high-dose group had an increase in mortality.
3 b Mineralization 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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Toxicological Review of tert-Butyl Alcohol
1 c 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 d 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 Note: Conversions from drinking water concentrations to mg/kg-day performed by study authors.
8 Conversion from ppm to mg/m3 is 1 ppm = 3.031 mg/m3.
9 Table 1-3. Changes in kidney tumors in animals foil' g exposure to
10 tert-butanol
Reference and study design
Rfc. *
NTP (1995)
.lal tubule
F344/N rat; 60/sex/treatment
Male
hyperplasia
(10/sex/treatment evaluated at
~andard and
15 months)
*ended
Renal tubule
Drinking water (0,1.25, 2.5, 5, or
r">se
ei_ -tk
Renal tubule
adenoma
10 mg/mL)
(r,
comi.
adenoma (single)
(multiple)
M: 0, 90, 200, or 420a mg/kg-d
14/50 (2-
7/50
1/50
F: 0,180, 330, or 650a mg/kg-d
2 years
90
""-0 (2.3)
7/50
4/50
200
10/50
9/50*
4201
. 50* (2.8)
10/50
3/50
Renal tubule
adenoma (single
DOSt
h 1 tubule
or multiple) or
mg/kg-d)
carcinoma
carcinoma
0/50
8/50
9o
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)
hvoerplasia
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
Renal tubule
Renal tubule
(single or
Dose
adenoma
adenoma
Renal tubule
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 2,070
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 a The high-dose group had an increase in mortality.
3
4 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
x = exposures at which all animals were dead and unable to be examined for the endpoint
Kidney
Weight
Absolute weight; M Rat; Reproductive (C)
Relative weight; M Rat; Reproductive (C)
Absolute weight; F Rat; Reproductive (C
Relative weight; F Rat; Reproductive (C
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; 1 Smo (D
Relative weight; M Rat; ISrao (D
Absolute weight; F Rat; 15mo (D
Relative weight; F Rat; ISmo (D
Kidney
Histopathology
_
Decreased glutathione; M Rat; IQwk (A
Inflammation; F Rat; 2yr (D
Nephropathy severity; M Rat; 13wk (D
Nephropathy incidence; F Rat; 13wk (D
Mineralization; M Rat; 13wk CD
Mineralization; F Rat; 13wk [D
Nephropathy severity; M Rat; 2yr (d;
Nephropathy severity; F Rat; 2yr (D
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
~ ~ qi
~ ~ ~
¦—¦
¦¦ ¦ ¦
bb-b—B-p
~—B—0
Hi m —
LJ ¦ ¦
Q-Bi
n mn n
ra n ¦¦
UJI O ¦
rai... .n....--
L| d ¦
M~€)
Q-0-
~ - O"
B-B-0
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
~ ¦ ~
B-B-O
B-B-O
B-B-O
±
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) Lvondell Chemical Co. (2004) (D) NTP
(1995); *reanalysis of NTP (1995).
Figure 1-5. Exposure response array for kidney effects following oral exposure
to tert-butanol.
This document is a draft for review purposes only and does not constitute Agency policy.
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¦ = exposures at which the endpoint was reported statistically significant by study authors
~ = exposures at which the endpoint was reported not statistically significant by study authors
Absolute weight; M Rat
Relative weight; M Rat
Absolute weight; F Rat
Relative weight. I ' Rai
Absolute ivlali\e \\oiuhl. \l Mouse
Absolute uei^lil. I Mouse
Relative weight; F Mouse
Source: NTP (1997).
~ BH B-
~-
~-
-B ~
~ BH B B-
-B ~
-B ~
B B B B-
100 1,000 10,000
Concentration (mg/m3)
Figure 1-6. Exposure-response array of kidney effects following inhalation
exposure to tert-butanol (13-week studies, no chronic studies available).
This document is a draft for review purposes only and does not constitute Agency policy.
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Mode of Action Analysis—Kidney Effects
a) a?,,-Globulin-Associated Renal Tubule Nephropathy and Carcinogenicity
One disease process to consider when interpreting kidney effects in rats is related to the
accumulation of a2U-globulin protein. a2u-Globulin, a member of a large superfamily of low-
molecular-weight proteins, was first characterized in male rat urine. Such proteins have been
detected in various tissues and fluids of most mammals (including humans), but the particular
isoform of a2u-globulin commonly detected in male rat urine is considered specific to that sex and
species. Exposure to chemicals that induce a2U-globulin accumul- in can initiate a sequence of
histopathological events leading to kidney tumorigenesis. I !e- . « ^-globulin-associated renal
tubule nephropathy and carcinogenicity occurring in mal- ¦¦ presumed not to be relevant for
assessing human health hazards fU.S. EPA. 1991a"). cvnlu ,ng tin. 'i l<> determine if a2U-globulin
plays a role is important. The role of a2U-globulin ;• nu la lion in llic ¦elopment of renal tubule
nephropathy and carcinogenicity observed foil fert-butanol expose 'vas evaluated using the
U.S. EPA (1991a) Risk Assessment Forum Technu. ^mcl report. Alpha2u-(n. 'in: Association with
Chemically Induced Renal Toxicity and Neoplasia in the Male Hat. This report provides specific
guidance for evaluating renal tubule tumors in male nils that are related to chemical exposure for
the purpose of risk assessment, based on an examination ollhe potential involvement of
a2u-globulin accumulation.
Studies in the fivf-liutanol database evaluated and reported effects on the kidney, providing
some evidence to evaluate this MOA. Additionally, sev eral studies were identified that specifically
evaluated the role olK ^-globulin in ^'/7-lnilanol-iiuluced renal tubule nephropathy and
carcinogenicity fBonjh'ii! el al.. 201) 1: Williams and liorghoff. 2001: Takahashi etal.. 19931. Because
the evidence reported in these studies is specific to « ^-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 a2u-globulin renal tubule nephropathy, as described by U.S.
EPA (1991a). is as follows. Chemicals that induce a2u-globulin accumulation do so rapidly.
a2u-Globulin accumulating in hyaline droplets is deposited in the S2 (P2) segmentofthe 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) segmentofthe proximal tubule
and contain a2U-globulin f U.S. El'A. 1991al. Abnormal increases in hyaline droplets have more than
one etiology and can be associated with the accumulation of different proteins. As hyaline droplet
deposition continues, single-cell necrosis occurs in the S2 (P2) segment, which leads to exfoliation
of these cells into the tubule lumen within 5 days of chemical exposure. In response to the cell loss,
cell proliferation occurs in the S2 (P2) segment after 3 weeks and continues for the duration of the
exposure. After 2 or 3 weeks of exposure, the cell debris accumulates in the S3 (P3) segment of the
proximal tubule to form granular casts. Continued chemical exposure for 3 to 12 months leads to
the formation of calcium hydroxyapatite in the papillae which results in linear mineralization. After
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1 or more years of chemical exposure, these lesions can result in the induction of renal tubule
adenomas and carcinomas (Figure 1-7).
U.S. EPA f l991al identified two questions that must be addressed to determine the extent
to which a2u-globulin-mediated processes induce renal tubule nephropathy and carcinogenicity.
First, whether the a2U-globulin process is occurring in male rats and is involved in renal tubule
tumor development must be determined. Second, whether the renal effects in male rats exposed to
tert-butanol are solely due to the a2u-globulin process also must be determined.
U.S. EPA (1991a) stated the criteria for answering the first question in the affirmative are as
follows:
1) hyaline droplets are increased in size and number in Irealed male rats,
2) the protein in the hyaline droplets in treated male nils is « uglolmlin (i.e.,
immunohistochemical evidence), and
3) several (but not necessarily all) additional steps in the pathological sequence appear in
treated male rats as a function of time, dose, and progressively increasing severity
consistent with the understands °f the underlying biology, as described above, and
illustrated in Figure 1-7.
The available data relevantto th¦ tsI ' 'on are summarized in Table 1-4, Figures 1-8
and 1-9, and are evaluated l-^lnw.
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Male rat liver
Male rat kidney
Synthesis of aJU-globulin
¦4" -¦ TBA binding
Resorption of poorly digestible
protein-chemical complex
> 1 days
Hyaline droplet accumulation
within lysosomes
1 -150 days
5 days - 48 weeks
te death and exfonation
Granular cast
formation
Sustained eel i
> 3 weeks
proliferation
Linear
local tuoular
hyoerplasia
> 3 months
mineralization
Renal adenoma,
carcinoma
3-48 weeks
>12 months
12 months
Figure 1-7. Temporal pathogenesis oraj„-globulin-associated nephropathy in
male rats. a^u-Globulin synthesized in the livers ol male nils is delivered to the
kidney, where il can accumulate in hyaline droplets and lie retained by epithelial
cells lining the S2 (lJ2) segment of the proximal tubules. Renal pathogenesis
following continued tert-butanol exposure and increasing droplet accumulation can
progress step-wise 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 ofthe renal tubular
epithelium. Adapted from Swenberg and Lehman-McKeeman (1999) and U.S. EPA
filial.
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1 Table 1-4. Summary of data on the a2u -globulin process in male rats exposed
2 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 ct2uglobulin
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, it cannot be assumed that the
results indicate regenerative proliferation is occurring.)
10 wk (oral)
0, 575 mg/kg-d -
Acharva et al. (1997)
10 d (inhalation)
0, 758, 1,364, 5,304 + stat sig at all doses; stat
mg/m 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 gianular 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 a NTP (1997) did not observe any effects consistent with a -globulin nephropathy.
5 b Precursors to granular casts reported.
6 c Reanalysis of hematoxylin and eosin-stained kidney sections from all male control and 1,520 mg/kg-d groups, as
7 well as a representative sample of kidney sections stained with Mallory Heidenhain stain, from the 13-wk study
8 from NTP (1995).
9 d Reanalysis of slides for all males in the con '20 mg/kg-day dose group and all animals with renal tubule
10 tumors from 2-vr NTP (1995). Protein casts r vtt. * 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
x = exposures at which all animals were dead and unable to be examined for the endpoint
• = exposures at which effect was observed but statistics not reported
T Hyaline droplet NTP (1995); 13 wk
•—• • > X
size/number
Identification of cr211- Williams and Borghoff (2001);
globulin in hyaline 12 hr after single dose
droplets
Achatya et al. (1997); 10 wk
•
Cytotoxicity/single-cell
necrosis of tubule epithelium,
epithelial eel exfoliation
NTP (1995); 13 wk
J ~—~ ~ ~ X
Achatya et al. (1997); 10 wk
!
!
I O 1
Tubule cell
! i
proliferation
i
NTP (1995); 13 wk"
1 ~—~ ~ ¦ X
NTP (1995); Hani et al. (2011)*; 13 wk
j
~
[J
n
n j
Granular
i !
casts/tubule
dilation
i !
NTP (1995); Hard et al. (2011); 2 yr
~ i
[]
cr
NTP (1995)**; Hard et al. (2011); 13 wk
I
1 ~—~ ~ ~ X
Li near papillary
mineralization
;
[
NTP (1995); Hard et al. (2011); 2 yr
¦ ¦ ¦
Foci of
tubular NTP (1995); 2 yr
Q B ¦ i
hyperplasia
i
* Hard et al. (2011) reported presence of "precursor
granular casts" 10 100 1,000 10,000
**NTP (1995] 13-wkstudy 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 a2u-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) - lOd
t Hyaline
droplet
size/number
NTP (1997) -13 wks
~—
—B ¦
~ El
Identification
of o2u-
globulin in Borghoff et al. (2001) -10 d
hyaline
droplets
Tubule cell Borghoff et al. (2001) - lOd
proliferation
~—
—B ¦
—
¦—
—¦ ¦
100 1,000 10,000
Exposure Concentration (mg/m3)
Figure 1-9. Exposure-response array for effects potentially associated with
a2u-globulin renal tubule nephropathy and tumors in male rats after
inhalation exposure to tert-butanol.
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Question One: Is the a2ugIobuIin process occurring in male rats exposed to tert-butanol?
(1) The first criterion to consider is whether hyaline droplets are increased in size and
number in male rats. 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 fU.S. EPA. 1991al. 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 (Borghoffetal.. 2001). These droplets were small and minimally present in
control male rats and were not observed in female rats. Simiki1'' .ila from the 13-week NTP oral
study fNTP. 1995: Takahashi etal.. 1993: Lindamood etai. J demonstrated an increase in the
accumulation of hyaline droplets. The lowest dose of 2!-' ,/kj, v had minimal hyaline droplet
formation compared to controls, although the next 11 s (.loses (4'K 1 (). 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 fl9971. 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 did not report any other lesions that could be
specifically associated with « u-globulin nephropathy in male rats. These results from NTP (1997).
which are inconsistent with the findings of both I'mpjIiqI'I'et al. f200!) 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 thata 13-week
exposure in the range ol the NTP f 19951 doses ol -I'M)—840 mg/kg-day leads to the same average
blood concentration as (>-hr/dav, 5 day/week inhalation exposures to 3,273-6,368 mg/m3. The
absence ol similar histopathologic^! 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, a^u-globulin nephropathy. The results from the two other
studies (Borgho'iet ai.. 200 i: N'l'l'. 1.995) indicate that hyaline droplets increase in size and number
in male rats following ^'/7-lnitanol exposures. Therefore, the available data are sufficient to fulfill
the first criterion that hyaline droplets are increased in size and number in male rats.
(2) The second criterion to consider is whether the protein in the hyaline droplets in male
rats is a2U-globulin. Accumulated hyaline droplets with an a2U-globulin etiology can be confirmed
by using immunohistochemistry to identify the a2U-globulin protein. Two short-term studies
measured a2u-globulin immunoreactivity in the hyaline droplets of the renal proximal tubular
epithelium (Borghoffetal.. 2001: Williams and Borghoff. 2001). Following 10 days of inhalation
exposure, Borghoffetal. (2001) did not observe an exposure-related increase in a2U-globulin using
immunohistochemical staining. When using an enzyme-linked immunosorbent assay (ELISA), a
more sensitive method of detecting a2U-globulin, however, a statistically significant positive
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correlation of a2u-globulin concentration with dose of tert-butanol (determined by correlating with
cell proliferation labeling indices) was observed, with accumulation of a2U-globulin protein
statistically significant by pairwise comparison only in the highest dose group. No positive staining
for a2u-globulin was observed in exposed female rats. In a follow-up study, Williams and Borghoff
f2 0011 used a single gavage dose of 500 mg/kg [selected on the basis of results by NTP Q9951 for
induction of hyaline droplet accumulation], and reported a statistically significantly higher renal
concentration of a2u-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
a2u-globulin was reversible. These data indicate the presence of it u-globulin in tert-butanol-treated
male rats, although requiring a more sensitive method ol detection
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Detailed evaluation of the available evidence supporting the third criterion
Single cell death and exfoliation into the renal tubules 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. (1997) reported degeneration of renal
tubules, one pathological consequence of single cell necrosis, however, 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
(Swenberg and Lehman-McKeeman. 1999: Short etal.. 198Q). the lack of consistency in these
observations could be the result of both weak induction of it -globulin and alack of later
examinations.
a. Sustained regenerative cell proliferation also was not observed. Acharva et al. f 19971
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. (20011 reported a dose-related increase in epithelial cell
proliferation within the proximal tubule as measured hy BrdU labeling indices in all
male rats exposed to te/7-butanol via inhalation lor 10 days. The study did not report
cytotoxicity, however, which, combined with the early lime point makes unlikely that
the cell proliferation was compensatory. NT!' f i'>'>5) 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 bvTakahashi etal.
f 19931 reported in NTI' (l')')5 )|. Proliferation was elevated at 840-1,520 mg/kg-day, a
range higher than the single 575-mg/kg-ilay dose eliciting no such proliferative effect
fAcharva etal.. 19971. as described above. NTP f 19951 reported, however, that no
necrosis was observed, suggesting the proliferation was not regenerative.
b. (Iranular cast formation was not observed, although one study noted precursors to cast
formation. NTP (19951 did not observe the formation of granular casts or tubular
dilation: however. Hard etal. f20111 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 exposu re is within the expected timeframe of frank formation and
accumulation ol granular casts (>3 weeks). Granular cast formation, however, might not
be significantly elev ated with weak inducers of ct2U-globulin fShortetal.. 1986). which is
consistent with the reported difficulty in measuring ct2U-globulin in hyaline droplets
associated with tert-butanol exposure.
c. Linear mineralization of tubules within the renal papillae was consistently observed in
male rats. This lesion typically appears at chronic time points, occurring after exposures
of 3 months up to 2 years fU.S. EPA. 1991al. Consistent with this description, 2-year oral
exposure to tert-butanol induced a dose-related increase in linear mineralization, but
not following 13-week exposure [(NTP. 1995): Table 1-2],
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d. Renal tubule hyperplasia was observed in the only available 2-year study. Renal tubule
hyperplasia is the preneoplastic lesion associated with a2U-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 a2U-globulin nephropathy is predicated on
the initial response of excessive hyaline droplet accumulation (containing ct2U-globulin) leading to
cell necrosis and cytotoxicity, which in turn cause the accumulation of granular casts, linear
mineralization, and tubular hyperplasia. Therefore, observati< . temporal and dose-response
concordance for these effects are informative for drawing - sions on causation.
As mentioned above, most steps in the sequence of it u-glohulin nephropathy are observed
atthe expected time points following exposure to {ivf-butanol. Accumulation of hyaline droplets
was observed early, at 12 hours following a single bolus exposure fWilliams and Borghoff. 20011.
and at 10 days (Borghoff etal.. 2001) or 13 weeks (NTP. 1995) following continuous exposure;
ct2U-Globulin was identified as the protein in these droplets f Borghoff etal.. 2001: W illiams and
Borghoff. 2001). Lack of necrosis anil exfoliation might he due to the weak induction of ct2u-globulin
and a lack of later examinations. Granular cast formation was not reported by any of the available
studies, which could also indicate weak it u-glohulin induction. Regenerative cell proliferation,
which was not observed, is discussed in more detail below. Observations of the subsequent linear
mineralization of tuhules anil local tubular hyperplasia lall within the expected timeframe ofthe
appearance ofthese lesions. Overall, no explicit inconsistencies are present in the temporal
appearance ofthe histopathological lesions associated with ct2u-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
ct2U-globulin nephropathy progression. I (valine droplets were induced in the proximal tubule of all
surviving male rats in the 13-week NTI' oral study fNTP. 1995: Takahashi etal.. 1993: Lindamood
etal.. 1992). although the incidence atthe lowest dose was minimal, while the incidence atthe
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.
f 2 0111 ]. 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 fNTP. 19951. a corresponding dose-related increase in the
severity of tubular hyperplasia did not result Severity of tubule hyperplasia was increased only at
the highest dose, which was not consistent with renal tumor incidence.
Although the histopathological sequence has data gaps, such as the lack of observable
necrosis or cytotoxicity or granular casts at stages within the timeframe of detectability, overall, a
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sufficient number of steps (e.g., linear papillary mineralization, foci of tubular hyperplasia) were
observed to fulfill the third criterion.
Summary and Conclusions for Question One:
Oral exposure to male F344 rats resulted in an increased incidence of renal tubule tumors in
a 2-year oral bioassay (Hard etal.. 2011: NTP. 19951. Several histopathological observations in
exposed male rats were consistent with an a2U-globulin MOA. This evidence includes the increased
size and number of hyaline droplets and the accumulated a2u-globulin protein in the hyaline
droplets. Additionally, several subsequent steps in the hislopiill" ",ical sequence were observed.
Overall, available data are sufficient for all three required cr suggesting that the a2U-globulin
process is operative. Although the evidence indicates a r i -globulin accumulation in the
etiology of kidney tumors induced by exposure to ter' biuanol in i. rats, that tert-butanol is a
weak inducer of a2U-globulin is plausible, considc .ne limited histi ' hological observations
and uncertainty regarding the temporal anddi. .mcordance of the lesu
Question Two: Are the renal effects in male rats exposed to terl-hulunol solely due to the a2U-gIobulin
process?
If the a2u-globulin process is operative, M.S. ili'A f i')') la) identifies a second question that
must be answered regarding whether the renal effects are (aj solely due to the a2U-globulin
process, (b) a combination of the a-u-globulin process and other carcinogenic processes, or (c)
primarily due to other processes. M.S. EPA f i')')la) stales that additional data can help inform
whether the a2U-gloliulin process is the sole contributor lo renal tubule tumor development in male
rats. These additional considerations are highlighted and discussed, where possible, in detail below.
Hypothesis-testing of the a ¦ -globulin sequence of effects and structure-activity relationships
that miij lit suggest the chemical belongs in a different class of suspected carcinogens: No data are
available to ev aluate these considerations.
Biochemical information regarding binding of the chemical to the a2U-gIobulin protein:
Williams and lloruhoff (2001) report that tert-butanol reversibly and noncovalently binds to
a2u-globulin in the kidneys of male rats. This provides additional support to the involvement of the
a2u-globulin process.
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 a2U-globulin process. Cell proliferation was observed in two studies [13-week, NTP
(1995) and 10-day, Borghoff et al. (2001)] but whether the proliferation was compensatory is
unknown, as cytotoxicity was not observed in these studies. Although the data do not support
sustained cell division occurring subsequent to cytotoxic cell death, renal tubule hyperplasia in
male rats was reported after 2 years of exposure (NTP. 1995). Thus, although some evidence of
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sustained cell replication is available, it does not specifically support a2u-globulin protein
accumulation.
Covalent binding to DNA or other macromolecules, suggesting another process leading to
tumors andgenotoxicity (a2U-gIobulin-inducers are essentially nongenotoxic): One study fYuan 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
validation of these results has been identified in the litenilu re.jw studies available to assess
the genotoxic potential of tert-butanol primarily are negate ..lough a few studies report DNA
damage induced by oxidative stress. DNA damage indue <>. -live stress is consistent with the
decreased levels of glutathione in male rat kidneys r orted by Aci. "i et al. T19951 after 10 weeks
of tert-butanol exposure. This type of genetic dar vvould not neces. 'v preclude a role for
a2u-globulin, but not enough information is ava 'e l<> determine whethi. illative stress could
initiate or promote kidney tumors in concert with i "Jobulin cumulation . \ile rat kidneys.
Nephrotoxicity not associated with I he a2u-glohulin 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, but these effects
were exacerbated by treatment with tert-butanol. Al 13 weeks f NTI'. 1997.19951 and 2 years fNTP.
19951. oral and inhalation (.-xposnre increased the sev erity of nephropathy in male rats (NTP.
1995). Similarly, the sev erity 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 (NTP. 1995).
Increased incidences of suppurative inflammation and kidney transitional epithelial hyperplasia
were observed in female rats orally exposed to /i.'//-butanol for 2 years. Although NTP f 19951
characterized these endpoints as associated with CPN, the low background incidence in the controls
combined with the dose-related increase in incidences indicate that these effects were not related
to an age-associated, spontaneous induction of nephropathy. At 2 years, the male rats also exhibited
dose-related increases in focal mineralization and transitional epithelial hyperplasia, although the
background incidence in the controls was high (i.e., approximately 50%) (NTP. 1995). Neither
endpointin males can be attributed to CPN or a2u-globulin.
Kidney weights also were increased in male and female rats in the 13-week oral and
inhalation evaluations (NTP. 1997.1995) and 15-month oral evaluation (NTP. 1995). The dose-
related increases observed in both male and female rats suggest that the kidney weight changes are
indicative of treatment-related molecular processes primarily unrelated to either a2U-globulin
protein accumulation or CPN. The exacerbation of CPN and the appearance of kidney effects in
female (i.e., suppurative inflammation, transitional epithelial hyperplasia) and male rats (i.e., focal
mineralization, transitional epithelial hyperplasia) that are not attributed to CPN or a2U-globulin
indicate that tert-butanol induces renal tubule nephrotoxicity partially independently of
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a2u-globulin. The evidence that other processes might be responsible for the renal tubule
nephrotoxicity thereby decreases the likelihood that a2U-globulin accumulation is solely
responsible for the renal tubule tumors.
Positive tubule tumor responses in female rats and other species implying that a2U-gIobuIin-
related processes alone do not account for the renal tubule tumor response: No increase in renal
tubule tumor incidence was reported in tert-butanol-exposed female rats or mice compared with
concurrent controls. Renal tubule tumors were observed only in male rats, providing support for an
a2u-globulin process in tumor development.
Summary and Conclusions for Question Two:
Although the evidence suggests that tert-butanol ^ - ¦ ¦„-globulin nephropathy, the
data indicate that tert-butanol is a weak inducer of« giuoulin ar. -at this process is not solely
responsible for the renal tubule nephropathy am1 .nogenicity ohs«. d in male rats. The lack of
compensatory cell proliferation in male rats an idence of nephrotoxicity It-male rats suggest
that other processes, in addition to the a2u-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 a2U-globulin pathway at high doses ( >420 mg/kg-day), which results in
tumor formation. Other, unknown pathways, however, could lie operative atlower doses
(<420 mg/kg-day), which contribute to renal tumor induction.
b) Chronic Progressiv e Nephropathy and Renal Carcinogenicity
There is scientific disagreement regarding the extent to which CPN can be characterized as
a carcinogenic MOA suitable lor analysis under the KI'A's cancer guidelines. Proponents of CPN as
an MOA have developed an evok ing series ol empirical criteria for attributing renal tubule tumors
to CPN. I kird and Khan (20041 proposed criteria for concluding that a chemical is associated with
renal tubule tumors through an interaction with CPN. Hard etal. (2013) slightly revised and
restated their criteria lor considering exacerbation of CPN as an MOA for renal tubule tumors in
rats. Table 1-5 lists these sets ol proposed empirical criteria for attributing renal tubule tumors to
CPN.
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Table 1-5. 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.
Lack of genotoxic activity based on overall
evaluation of in vitro and in vivo data.
Tumor incidence is low, usually <10%.
Tumors are found toward the end of 2-year
studies.
Lesions are usually ATH or adenomas (carcinomas
can occasionally 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.
Absence of cytotoxicity in CPN-unaffected
tubules, in rats with lower grades of CPN, and in
subchronic studies.
Source: Hard et al. (2013)
Source: Hard and Khan (2004)
Hard etal. (2013 1 maintain knowing I In.- detailed etiology or underlying mechanism for CPN
is notnecess—~~ T'^tead, idenlilying increased CI'N with its associated increase in tubule cell
probler;1 iik- . "l-iiI is adequate. Nonetheless. Hard etal. (20131 also postulated a
sequel. »f key events h. nal luniorigeiiesis involving exacerbation of CPN:
• Exposure lo chemical (usually al high concentrations);
• Metabolic activ ation (if necessary);
• Exacerbated CI'N, 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).
In contrast to Hard etal. (2013): Hard and Khan (2004).Melnick etal. (2013): Melnick et al.
(20121 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
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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 2 8 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.
Evaluation of the MOA Proposed by Hard etal f20131
Setting aside the question of whether CPN is (Hard etal.. 2013: Hard and Khan. 2004) or is
not (Melnick etal.. 2013: Melnick etal.. 2012) an MOA suitable' lalysis, this section provides an
analysis of the mechanistic data pertinent to CPN. EPA's can- .iidelines (U.S. EPA. 2005a) define
a framework for judging whether available data support ,» -sized MOA; the analysis in this
section follows the structure presented in the cancer uk, Jines.
Description of the hypothesized MOA. Under the KPA framework, toxicokinetic studies are
important for identifying the active agent, but loxicokinetic events per se are not key events of an
MOA. Thus, the EPA analysis of the MOA proposed by i lard el al. T2013) begi lis with
(1) exacerbated CPN, including increased number of rals 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 mode rule lo strong in male rals in the NTP f 1995) study. According to the NTP
(1995) analysis, the mean CI'N grades (same as "sev erity of nephropathy" reported by NTP)
presented on a scale 1-4 lor male rals with renal tumors were 3.5, 3.6, 3.7, and 3.4 atdoses 0,1.25,
2.5, and 5 mg/mL. The mean CI'N grades lor male rals without renal tumors were 2.9, 2.8, 2.8, and
3.2 for the same (.lose groups. The reanalysis of the NTI' data by Hard etal. (2011) yielded similar
numbers. The relationship between CI'N and renal tumors, however, is neither consistent nor
specific in the NTP f 19951 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 atdoses 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. 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
observed in this reanalysis accentuates the differences in these dose-response relationships. In
examining the various lesions at the mid-dose—the dose with the greatest increase in renal tubule
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tumors in male rats—a minor increase (14/50 in controls versus 17/50 in the mid-dose group) in
renal tubule hyperplasia incidence was observed, with a marginal change in CPN severity (i.e.,
group average of 3.0 to 3.1). That a minor increase in hyperplasia and marginal increase in CPN
severity would be associated with significant tumor induction seems inconsistent Furthermore,
CPN severity is nearly as great in the female rats, yet no females developed tumors, as noted above.
Temporal relationship. The severity of CPN progressed over time. According to the NTP
(1995) 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 - 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 sow if neoplastic lesions increased at
the end of life. Atthe 15-month interim evaluation, only ' . >d developed renal tubule
hyperplasia and one other had a renal tubule adenor at 2 years,„ :ncidences of these two
lesions were much higher in all dose groups (see .mis paragraph). \se results are consistent
with CPN as an age-related disease and with hj, ilasia and tumors appe<; '<• 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 CI'N grades and renal tubule tumors
in male and female rats. In addition, the increased incidences in renal tubule tumors in all exposed
male rats exceed the 1 criterion proposed by I lard et a I. (2013) (Table 1-5), even more so when
making comparisons with the lower control tumor incidence from the Hard etal. (2011) reanalvsis.
Conclusions about the hypothesized CI'N-reluted MOA
As recommended by K I'A's cancer guidelines (U.S. EPA. 2005a). conclusions aboutthe
hypothesized MOA can be clarified by answering three questions presented below.
(a) Is I he 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 There is lack
of consistency, howev er, between males and females and in the dose-response relationships
between CPN, hyperplasia, and adenomas. These inconsistencies make difficult attributing all renal
tumors to either CPN or to « u-globulin-related nephropathy (see previous section on a2U-globulin),
raising the likelihood ol another, yet unspecified MOA.
(b) Is the hypothesized MOA relevant to humans? There is scientific disagreement on this
question. Hard etal. f20131: Hard et al. f20091cite 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. (2013): Melnick etal. (2012) 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. This issue is
unresolved.
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(c) Which populations or lifestages can be particularly susceptible to the hypothesized MOA?
There are no indications of a human population or lifestage that is especially susceptible to tumors
induced through exacerbated CPN.
In summary, considering discrepant patterns in the dose-response relationships for CPN,
hyperplasia, and renal tubule tumors and the lack of relationships between CPN grades and renal
tubule tumors in male and female rats, together with the lack of a generally accepted MOA for CPN,
the renal tubule tumors in rats cannot be attributed to CPN.
This position can be reconciled with that of Mel nick etal. ("20131: Melnicketal. (2012). who
argued against dismissing renal tubule tumors in rats thai can |_ ated to exacerbated CPN. It also
can be reconciled with Hard etal. (2013). who, while mainl- ^ I hat these tumors are not
relevant to humans, also allow that there is no generally ,)U * K)A for CPN akin to that for ct2u-
globulin-related nephropathy. Hard etal. (20131 ma-' this statemi. 'Iter reporting on the
collective experience of national and internatioiv . ith agencies woi 'ide with the use of CPN
as an MOA. Of 21 substances that exacerbated (. uid caused renal tumo. nost were multisite
carcinogens, and other tumor sites contributed to t ¦valuatir Only two a. sments explicitly
considered CPN as a renal tumor mechanism. One was I lie assessment of ethylben/.ene by the
German Federal Institute for Occupational Safety and I leallh, 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. f2013 )|. The other was the IRIS assessment of
tetrahydrofuran, for which KI'A found the evidence insufficient to conclude that the kidney tumors
are mediated solely by the hypothesized MOAs (M.S. KI'A. 2012d). Hard etal. (2013) attributed
these different conclusions to either different (.lata lor the two chemicals or the lack of a generally
accepted MOA akin to a^u-glolmlin-relaled nephropathy.
Relevant to this last point. IAKC (1999) developed a consensus statement that listed
considerations for evaluating a^u-globulin-related nephropathy in rats, which was based on the
work of 22 scientists, including three who were co-authors of Hard etal. T20131 and two who were
co-authors olM el nick etal. (2013): Meinick etal. (2012). A similar broad-based consensus that
defines a sequence of key ev ents lor exacerbated CPN, distinguishes it more clearly from ct2u-
globulin-related nephropathy, and evaluates its relevance to humans would be helpful in advancing
the understanding of these issues.
Overall Conclusions on MOA for Kidney Effects
tert-Butanol increases a2Uglobulin deposition and hyaline droplet accumulation in male rat
kidneys, as well as several of the subsequent steps in that pathological sequence. These data
provide sufficient evidence (albeit minimal) that the a2uglobulin process is operating, although
based on further analysis this chemical appears to be a weak inducer of a2uglobulin-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 a2U-globulin and tert-butanol) play a role in renal
tubule nephropathy. Although CPN was indicated in the induction of renal tubule nephropathy, the
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available evidence indicates that it does not induce the renal tubule tumors in male rats.
Additionally, several endpoints indicate renal tubule nephrotoxicity and increased kidney weights
related to tert-butanol exposure cannot be explained by the a2Uglobulin or CPN processes.
Collectively, the evidence indicates other, unknown processes contribute to renal tubule
nephrotoxicity and carcinogenicity.
Integration of kidney effects
Kidney effects (increases in nephropathy, severity of nephropathy, hyaline droplets, linear
mineralization, suppurative inflammation, transitional epithet1' lerplasia, mineralization, and
kidney weight) were observed, predominantly in male ami I- rats across the multiple tert-
butanol studies. The available evidence indicates that mi j , "esses induce the noncancer
kidney effects. The group of lesions generally reporli • 'as nephro, "'v." is related to CPN. Because
this disease is considered to be spontaneous and ¦ elated in rats, ti 'id points associated with
CPN would not be relevant to humans for purp • of hazard identification \lditionally, two
endpoints in male rats (hyaline droplets, linear mineralization) are components of the a2u-globulin
process. U.S. EPA (1991a) states that if the a2U-globulin process is occurring in male rats, the renal
tubule effects associated with this process in male rats would not lie relevant to humans for
purposes of hazard identification. I n cases such as these, the characterization of human health
hazard for noncancer kidney toxicity would rely on effects not specifically associated with CPN or
the a2u-globulin-process in male rats.
Several other noncancer endpoints resulted from /c*/7-hntanol exposure and are appropriate
for consideration of a kidney hazard, specifically: suppurative inflammation in female rats,
transitional epithelial hyperplasia in male and female rats, severity of nephropathy in male and
female rats, incidence of nephropathy in female rats, incidence oI mineralization in male rats, and
increased kidney weights in rats but not mice, liased 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 luliule hyperplasia (considered a preneoplastic effect) and tumors in male rats.
EPA concluded that the three criteria were met to indicate that an a2U-globulin process is operating.
Because renal tubule tumors in male rats did not arise solely due to the a2U-globulin process and
some of the tumors are attributable to other carcinogenic processes, such tumors remain relevant
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for purposes of hazard identification U.S. EPA (1991a).3 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 database on thyroid effects following tert-buLinol (.'xposn ix* contains no human data,
two oral subchronic and two oral chronic studios (oik.- of each du ration in rats and in mice) (NTP.
1995). and two inhalation subchronic studies (oik- in nils and one in mice) (NTP. 1997). Studies
employing short-term and acute exposures thai examined thyroid effects an.- nol included in the
evidence table; they are discussed, however, in the lexl il lliev provide data informative of MOAor
hazard identification. No gross thyro' ''"its were reported in the 13-week evaluations of mice or
rats following oral or inhalation expos ¦ y_ '¦ 1997.19'>5). and therefore subchronic studies
were notincluded in the evidence table. ¦ Uvi :liiblc chronic studies are arranged in the
evidence table by effect an^ ^,rin by specie, 'lie dt-si^ -nduct, and reporting of each study were
reviewed, each study '¦ -d adec|iia '<> pr nation pertinent to this assessment
(Table 1-6 and Figure '()).
Thyroid effects, specifically follicular cell hyperplasia and adenomas, were observed in mice
of both sexes after 2 years ol Oral exposure via drinking water (NTP. 1995). NTP (1995) noted that
"[proliferation oI thyroid gland follicular cells is generally considered to follow a progression from
hyperplasia lo adenoma and carcinoma." Similarly, EPA considered the thyroid follicular cell
hyperplasia lo lie a preneoplastic effect associated with the thyroid tumors. 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 were also observed in the tert-butanol-treated mice, with the only
carcinoma observed in h igh-dose males. No treatment-related thyroid effects were reported in rats
of either sex following 2 years of oral exposure (NTP. 1995).
Although the tumor response in male mice showed a statistically significant increasing
trend (Cochran-Armitage trend test, p = 0.041) (analysis performed by EPA using the mortality-
adjusted rates), the response was non-monotonic, with a slightly lower response at the high-dose
3 When the a2u-globulin process is occurring, U.S. EPA (1991a) states that one of the following conclusions will
be made: (a) if renal tumors in male rats are attributable solely to the a2u-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 a2u 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 a2u-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 there is enough
information to determine the relative contribution of each process to the overall renal tumor response.
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1 level than at the mid-dose level. The reason for the non-monotonicity is unclear, although it could
2 be related to the increased mortality in the high-dose group (17/60 animals survived compared
3 with 27/60 animals in the control group). The decreased survival of male mice might have affected
4 the thyroid tumor incidences because animals could have died before tumors could develop. High
5 mortality in the high-dose group occurred before tumors appeared; about 40% of the high-dose
6 males died before the first tumor (a carcinoma) appeared in this group at week 83. By comparison,
7 only ~10% of the control group had died by this time, and the single tumor in the control group
8 was observed at study termination. Mortality in the exposed female mice was similar to controls.
9 Table 1-6. Evidence pertaining to thyroid effects in animals following oral
10 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
F: 0,180, 330, or 650a mg/kg-d
Incidence15
Males
Dose
(mg/kg-d)
0
90
Follicular cell
hyperplasia
3/50
0/49
Females
Dose
(mg/kg-d)
0
180
Follicular cell
hyperplasia
0/50
0/50
2 years
200
0/50
330
0/50
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)
Follicular cell tumors
NTP (1995)
Incidence15
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Reference and study design
Results
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
F: 0,180, 330, or 650a mg/kg-d
2 years
Dose (mg/kg-d)
Male
0
90
200
420a
Female
0
180
330
650a
Follicular cell
adenoma
2/50
0/49
0/50
0/50
1/50
0/50
1/50
0/50
Follicular cell
carcinoma
2/50
0/49
0/50
0/50
1/50
0/50
1/50
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
2 years
Incidence
Dose
(mg/kg-d)
Male
0
540
1,040
2,070a
Female
0
510
1.020
2,110
Follicular cell Follicular cell
adenoma carcinoma
1/60 0/60
0/59 0/59
4/59 0/59
1/57 1/57
2/58 0/58
3/60 0/60
2/59 0/59
9/59* 0/59
Follicular cell
adenoma or
carcinoma
(mortality
adjusted rates)-^
1/60 (3.6%)
0/59 (0.0%)
4/59 (10.1%)
2/57 (8.7%)
2/58 (5.6%)
3/60 (8.6%)
2/59 (4.9%)
9/59* (19.6%)
Animals
survivine to
study
termination
27/60
36/60
34/60
17/60
36/60
35/60
41/60
42/60
1 aThere was a significant decrease in survival in the high-dose group.
2 bResults do not include the animals sacrificed at 15 months.
3 cMortality-adjusted rates were not calculated by study authors for follicular cell carcinoma. The mortality-adjusted rates for the
4 incidence of adenomas are the same as the combined rates, with the exception of the male high-dose group, where the rate
5 for adenomas alone was 5.9%.
6 dCochran-Armitage trend test was applied to mortality-adjusted thyroid tumor incidences, by applying the NTP adjusted rates
7 to the observed numbers of tumors to estimate the effective number at risk in each group. For male mice, p = 0.041; for
8 female mice, p = 0.028.* Statistically significant p < 0.05 as determined by the study authors.
9 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 ral
Hyperplasia; F ral
Adenoma. \1 mouse
Ulenonu. I- mouse
\denoma. M ral
CANCKU
Adenoma, F rat
100 1,000
Dose (mg/kg-day)
10,000
Source: NTP (1995)
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 at the high-dose group.)
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Mode of Action Analysis—Thyroid Effects
The MOA responsible for tert-butanol-induced thyroid effects has not been the subject of
much 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, that, an antithyroid MOA, as
identified in U.S. EPA's guidance on the assessment of thyroid follicular cell tumors (U.S. EPA.
1998a").
To determine if the thyroid follicular cell tumors result from a chemically induced
antithyroid MOA, U.S. EPA f!998a) requires thatthe available.- database 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) dost.1 correlation among I In.- various effects, and (5)
reversibility of effects in the early stages of disruption. The available evidence pertaining to each of
these aspects of antithyroid activity following /(.'//-butanol exposure is discussed below.
1) Increases in cell growth (required")
U.S. EPA (1998a) considers increased absolute or relativ e thyroid weights, histological
indicators of cellular hypertrophy and hyperplasia, DN A 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. l')')7. l')').r>) 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 fi.'/7-butanol exposure in rats treated up to 2
years I'N'i'l'. 1997. l')').r>). Although the short-term female mouse study by Blanck et al. (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 driliking water exposure to / (.rtbutanol- (NTP. 1995). The increase was dose
dependentin 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, with increased severity at
the highest dose fN'l'i'. Thyroid follicular cell hyperplasia was not observed in any mouse
study with less than 2 years oI 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
(Blanck etal.. 2010) were reported, in male or female mice after 13 weeks of drinking water
exposure (NTP. 1995) or in male or female mice following 18-day or 13-week inhalation studies
(NTP. 1997). The observation of increased hyperplasia in male and female mice after 2 years of
exposure is sufficient evidence to support increased thyroid cell growth.
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2) Changes in thyroid and relevant pituitary hormones (required)
Evidence of hormonal changes, including decreases in thyroxine (T4) and triiodothyronine
(T3) and increases in TSH, are required to demonstrate a disruption in the thyroid-pituitary
signaling axis (U.S. EPA. 1998a). 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, and 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 althoui.'1 .1 ud T4 levels were decreased
after 14 days, this perturbation likely was not in excess oi l1 -ige of homeostatic regulation in
female B6C3Fi mice and thus not likely to induce comp^ ,.>r\ roid follicular cell proliferation.
Multiple lines of evidence support this observation: ' 1 TSH levels v ¦ unaffected, indicating that
the decrease inT3 and T4 levels was not seven.- < ^11 lo stimulate iiK sed TSH secretion by the
pituitary; (2) thyroid hyperplasia was not indue : 11 111 is study, or any othc --xposing mice for
2.5-13 weeks, suggesting that thyroid proliferation was either notinduced by I In.- hormone
fluctuations or thatany follicular cell proli feral ion during this period was too slight to be detected
by routine histopathological examination: (3) the maximal decrease in T 3 orT4hormone levels
induced by tert-butanol exposure alter 14 days (i.e., - 20"..) was well within the range of fluctuation
inT3 and T4 hormone levels reported to occur between the 3- and 14-day control groups [15-40%;
(Blanck etal.. 2010)|. Although the lower T : andTi lev els following tert-butanol were later
attributed by the study authors to an increase in liver metabolism (see next section), they could in
fact be due to a decrease in thyroid hormone production, resulting from some, as ofyet,
uninvestigated molecular interactions ol''fiVf-buLinol in the thyroid, pituitary, or hypothalamus.
The absence ol 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 database. Together, although small decreases in some thyroid hormone levels have been
reported in female mice, the av ailable 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.
3) Sitefs) of antithyroid action (required)
The thyroid and liver are two of several potential sites of antithyroid action, with the liver
the most common site of action, where increased microsomal enzyme activity could enhance
thyroid hormone metabolism and removal (U.S. EPA. 1998a). Rats are thought to be more sensitive
than mice to this aspect of antithyroid activity (Rogues etal.. 2013: Qatanani etal.. 2005: U.S. EPA.
1998a): however, rats exposed to tert-butanol for 2 years did not exhibit 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
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hepatocellular hypertrophy or hyperplasia (U.S. EPA. 1998a: NTP. 1995). 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 fBlanck etal.. 20101. Relative liver weights increased in
male and female mice after 13 weeks of oral exposure fNTP. 19951 to higher doses than those
evaluated by Blanck etal. (20101. 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 inhalati .posure at the two highest
concentrations fNTP. 19971: liver weight was not reported ; .e orally exposed for 2 years fNTP.
1995). No increase in mouse hepatocellular hypertroph1 i\, ilastic histopathology was
reported following 2.5 weeks to 2 years of exposn re ' TI-. 1(>'>T. j V I n fact, the only liver
pathology associated with tert-butanol exposure1 jse studies was ^crease in fatty liver in
male mice in the high-dose group after 2 years > ''ill exposure (NTP. 199:;» \lihough increased
fatty liver could indicate some non-specific metabolic alteration, the absence ol a similar treatment-
related effect in livers from female mice, which were sensitiv e 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 (CYP2I! 10) following 1 'I- days of tert- butanol exposure
in female mice, with much smaller increases in the expression olanother phase I enzyme CYP2B9,
and the phase II thyroid hormone-metabolizing enzyme, sulfotransferase 1A1 [(SULT1A1; Blanck et
al. (2010)]. 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 CYl's along with thyroid hormone-metabolizingsulfotransferases fRoques etal.. 20131. 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 /(.'//-butanol exposure; however, the expression changes in these few enzymes
are not supported by any liver hisli(pathological effects in mice exposed for longer durations, so
whether this enzyme induction is transient, or simply insufficient to induce liver pathology after >2
weeks of exposure, is unknown. 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
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histopathology, including thyroid tumors (U.S. EPA. 1998a). 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 T 4 and T 3
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
(Maronpotetal.. 2010: U.S. EPA. 1998a: NTP. 1995). Toxicol I';'' 18:776-795), and consistent
with this expected association, centrilobular hepatocellular' rtrophy was reported in 2/5 high-
dose mice exposed for 2 weeks fBlanck etal.. 2010). No 1 .11. \ithology, however, was
attributed to tert-butanol exposure in female mice (.'xposc'Ll lor 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 ol 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 al (.loses that elevated some liver
enzyme levels fBlanck etal.. 20101.
Following 2 years of oral exposure, both follicular cell hyperplasia and follicular cell tumor
incidence was increased in mice despite a lack of Irealmenl-relaled liver pathology fNTP. 19951
(Table 1-6). Any associations relating hormone changes to thyroid pathology or liver enzyme
induction are limited due to the inadequate database (described above); the available evidence
suggests little concordance among reports ol liver, pituitary, and thyroid effects in female mice, and
no evidence was available to evaluate these associations in male mice.
51 Reversibility f requiredl
Chemicals actingvia an antithyroid MOAhave effects (e.g., increased TSH levels, thyroid
follicular cell proliferation) that are reversible after cessation of treatment (U.S. EPA. 1998a).
Although increased TSTT 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.
In summary, the available database 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. Together, the database 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.
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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 (U.S. EPA. 1998a). 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 associa ted u,: ,7-butanol exposure are
discussed as part of the overall weight of evidence for carc' licitv in Section 1.3.2.
1.2.3. Developmental Effects
Synthesis of effects related to development
Four studies evaluated developmental (.'Heels |three oral or inhalation developmental
studies fFaulkner etal.. 1989: Nelson etal.. 1989: Daniel and Evans. 19821 a nil a one-generation,
oral reproductive study fLvondell Chemical Co.. 2004)| in animals exposed to tert-butanolvialiquid
diet (i.e., maltose/dextrin), oral gavage, or inhalation. No developmental epidemiology 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 ol each study were reviewed, and each
study was considered adequate to provide information pertinent to this assessment. One study was
considered less informative. Faulkner et al. f 1')?»')). because it did not provide sufficient information
on the dams to determine if fetal effects occurred due to maternal toxicity.
Dev elopmental effects ol'te/Mnilanol observed after oral exposure (liquid diets or gavage)
in several mouse strains and one rat strain include measures of fetal loss or viability (e.g., increased
number of resorptions, decreased numbers of neonates per litter) and decreased fetal body weight
fLvondell Chemical Co.. 200-!: Faulkner etal.. 1989: Daniel and Evans. 1982). Daniel and Evans
(1982) also observed decreases in 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 (.lose study reported a small increase in the incidence of variations of the skull or
sternebrae in two mouse strains (Faulkner etal.. 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 etal.. 1989). 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 doses that cause toxicity in the dams
as measured by clinical signs (e.g., decreased body weight gain, food consumption) (Table 1-7;
Figure 1-11; Figure 1-12). As stated in the Guidelines for Developmental Toxicity Risk Assessment
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1 (U.S. EPA. 1991b). "an integrated evaluation must be performed considering all maternal and
2 developmental endpoints." "[W]hen adverse developmental effects are produced only at doses that
3 cause minimal maternal toxicity; in these cases, the developmental effects are still considered to
4 represent developmental toxicity and should not be discounted." Although, at doses of "excessive
5 maternal toxicity...information on developmental effects may be difficult to interpret and of limited
6 value." In considering the fetal and maternal toxicity data following tert-butanol exposure, the
7 severity of the maternal effects were minimal and therefore the developmental effects in the fetuses
8 should not be discounted (U.S. EPA. 1991b). The observed fetal effects occurred, however, at doses
9 resulting in maternal toxicity across all available studies. TIktc' whether the fetal effects are
10 directly related to tert-butanol treatment or are secondary 1 .uTiial toxicity remains unclear.
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1 Table 1-7. Evidence pertaining to developmental effects in animals following
2 exposure to tert-butanol
Reference and study design
Results
Lvondell Chemical Co. (2004)
Response relative to control
Sprague-Dawley rat; 12/sex/treatment
Gavage 0, 64,160, 400, or 1,000 mg/kg-d
Dose
(mg/kg-d) 0
64
160
400
1000
FO males: 9 weeks beginning 4 weeks prior
Maternal effects
to mating
F0 females: 4 weeks prior to mating
Body weight gain GD 0-20
through PND21
F1 Males and Females: 7 weeks
0
-3
-4
0
-16*
(throughout gestation and lactation; 1 male
Food consumption GD 0-20
and 1 female from each litter was dosed
0
Body weight gain PND 1-21
0
0
+4
0
directly from PND 21-28)
0
+3
-10
+3
+100*
Food consumption LD1-14
0
-2
-6
0
-16
Live pups/litter response relative to control
0
-9
-11
-7
-33*
Dams dosed with 400 or 1000 ms/ks-d showed CNS effects (e.g.. ataxia, letharsv)
which were undetectable bv 4-weeks of exposure in animals exoosed to 400
mg/kg-d but not those in the higher dose group.
F1 effects
Viability index (pup survival to PND4)
96.4%
98.7%
98.2%
99.4%
74.1%*
Lactation index (pup survival to PND21)
100%
100%
100%
99.2%
98.8%
Sex ratio (% males)
54.4
52.3
50.9
53.4
52.1
Pup weight/litter PND 1 relative to control (%)
0
+6
+4
+7
-10
Pup weight PND 28 relative to control (%)
M: 0
+2
0
0
-12*
F: 0
0
-4
-2
-8
3
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Reference and study design
Results
Daniel and Evans (1982)
Swiss Webster (Cox) mouse; 15 pregnant
dams/treatment
Liquid diet (0, 0.5, 0.75, 1.0%, w/v)
0 (isocaloric amounts of maltose/dextrin),
3,324, 4,879, 6,677 mg/kg-d
GD 6-20
No statistical analysis was conducted on any of these data
Maternal
Percent change compared to control:
Number of litters
Dose
Food consumption
Body weight
(% pregnant
(mg/kg-d)
(mean g/animal/dav)
gain
dams)
0
0
0
11 (77%)
3,324
+2
-3
12 (80%)
4,879
-3
-19
8 (53%)
6,677
-4
-20
7 (47%)
Authors note that lower food consumption in higher te/t-butanol dose groups
reflects problems with pair feeding and maternal sedation.
Fetal
Percent change compared to control:
Dose Number of
(mg/kg-d)
0
3,324
4,879
6,677
neonates/litter
0
-1
-29
-49
Fetal body weight
on PND 2
0
-7
-19
-38
Number of stillborn also increased with dose (3, 6,14, and 20, respectively), but
the number of stillborn per litter was not provided. The high dose also caused a
delay in eye opening and a lag in weight gain during PND 2-10 (information was
only provided in text or figures)
Faulkner etal. (1989)
CBA/J mouse; 7 pregnant females in
control, 12 pregnant females in treated
Gavage (10.5 mmoles/kg twice a day);
0 (tap water), 1,556 mg/kg-d
GD 6-18
Maternal results not reported.
Fetal
Percent change compared to control:
Dose
(mg/kg-d)
0
1,556
Live
fetuses/
Resorptions/litter litter
0
+118*
0
-41*
Fetal
weight
0
-4
Incidence:
Sternebral
variations
4/28
7/30
Skull
variations
1/28
3/30
Sternal variations: misaligned or unossified sternebrae
Skull variations: moderate reduction in ossification of supraoccipital bone
Number of total resorptions (10 resorptions/66 implants in controls, 37/94
implants in treated) increased (p < 0.05)
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Reference and study design
Results
Faulkner etal. (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:
Dose
(mg/kg-d)
0
1,556
Live
fetuses/
Resorptions/litter litter
0
+428*
0
-58*
Fetal
weight
0
-4
Incidence:
Sternebral
variations
5/21
9/16
Skull
variations
1/21
7/16
Sternal variations: misaligned or unossified sternebrae
Skull variations: moderate reduction in ossification of supraoccipital bone
Number of total resorptions (4 resorptions/44 implants in controls, 38/68
implants in treated) 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 \|/ in
body weight gain (results presented in figure only), dose-dependent \|/ in food
consumption ranging from 7-36% depending on dose and time
Fetal
Percent change compared to control
(mean ± standard error):
Dose
(mg/mS)
Number of live
fetuses/litter
Resorotions
per litter
0
0(13±2)
0 (1.1±1.2)
6,669
0(13±4)
+9 (1.2±1.1)
10,640
+15(15±2)
-18 (0.9±1.0)
15,248
+8(14±2)
0 (1.1±0.9)
Percent change compared to
control:
Incidence:
Dose
(mg/mS)
Fetal weight
(males)
Fetal weight
(females)
Skeletal
variation
bv litter
Skeletal
variation
bv fetus
0
0
0
10/15
18/96
6,669
—9*
—9*
14/17
35/104
10,640
-12*
-13*
14/14
53/103*
15,248
-32*
-31*
12/12
76/83*
Skeletal variation by litter refers to the number of variations observed in the
number of litters examined. Skeletal variation by fetus refers to the number of
variations observed in the total number of fetuses examined. Fetuses are not
categorized by litter.
1
2 * Statistically significant p < 0.05 as determined by study authors. Conversions from diet concentrations to mg/kg-d
3 performed by study authors. Conversion from ppm to mg/m3 is 1 ppm = 3.031 mg/m3.
4
5 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 per litter; M+F
rat (C)
^Viability index; M+F rat (C)
Lactation index; M+F rat (C)
Sex ratio; M+F rat (C)
IPup weight per litter
(PNDt); M+F rat (C)
•I Pup weight per litter
(PND 28); M rat (C)
IPup weight per litter
(PND 28); F rat (C)
J. Maternal body weight gain; F
mouse (A) •
iNumberof neonates/litter, fetal
body weight; M+F mouse (A)*
t Number of resorptions per litter;
M+F mouse (B)
INumberof live fetuses per litter;
M+F mouse (B)
IFetal weight; M+F mouse (B)
Skeletal variations; M+F mouse (B)
~—I—B B ¦
~—|—B B ¦
B ! B B ¦
~—|—B B ¦
B ! B B B
B ! B B H
u
B—
B—
1)
n—
B
—B—
—F1
a-m-m"
~
~
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): Lvondell Chemical Co. (2004)
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)
4-Fetal weight; F rat
(Nelson et al,, 1989)
Skeletal variation by li tter; M+F rat
(Nelson et al,, 1989)
Skeletal variation by fetus; M+F rat
(Nelson et al,, 1989)
~—B—Q
~—6—E3
~—B—~
D-
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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Integration of developmental effects
There is suggestive evidence of developmental effects associated with 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 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 whether the fetal effects are due to maternal toxicity is
difficult The observed maternal effects are minimal, however, and thus, the developmental effects
observed in the fetuses are not discounted as being secondary l<> maternal toxicity fU.S. EPA.
1991b) and the evidence is considered suggestive of developmental toxicity.
1.2.4. Neurodevelopmental Effects
Synthesis of effects related to neurodevelopinent
Three studies evaluated neurodevelopmental effects ("Nelson etal.. 1')') i: Daniel and Evans.
19821[one in male rats; one in female rats] following /c/Mmlanol exposure via liquid diet (i.e.,
maltose/dextrin) or inhalation. No epidemiology studies on neumdevelopmentare available. The
animal studies evaluating neurodevelopmental effects of fi'/'f-liuLinol contain study design
limitations. Daniel and Evans f!982) hud a small number of animals per treatment group, lacked
comparison of treatment-related effects to controls for all endpoints investigated, and did notuse
long-term neurodevelopmental testing. The two studies by Nelson et al. (1991) evaluated
neurodevelopmental effects alter either paternal or maternal exposure butdid notrunthe
exposures concurrently or provide exposure methods to indicate the studies were conducted
similarly. The studies are arranged in the evidence tables by species and sex.
Various neurodevelopmental effects have been observed in the available studies. These
include changes in rota rod performance following oral or inhalation exposures and decreases in
open field behavior and el illAvoidance following oral exposure, and reduced time hanging on wire
after inhalation exposure during gestation (Table 1-8).
Rotarod performance
Inconsistent results were observed across studies. Although Daniel and Evans Q9821 found
decreased rotarod performance in mouse pups of dams orally exposed during gestation, Nelson et
al. (1991) 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. Q9911
reported statistically significant changes in neurochemical measurements in the brain in offspring
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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 (1982) cross-fostered half the mouse pups born to treated mothers with
untreated surrogate females to test the effects of maternal null''' and behavioral factors on the
pups' physiological and psychomotor development Results ; .iled that pups fostered to control
dams performed significantly better than those maintain ''.-ated dams (Table l-8)(DanieI
and Evans. 19821. Data suggest that neurodevelopin< tai effects w not solely due to in utero
exposure to tert-butanol (Daniel and Evans. V>
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Toxicological Review of tert-Butyl Alcohol
Reference and study design
Results
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 P-endorphin in the cerebellum at 12,000 mg/m3
• 67% decrease in serotonin in the midbrain at 6,000 mg/m3
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 P-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 in klc-ncc is available for reproductive, developmental, or
7 neurodevelopmental effects.
8 Integration of neurodevelopmental effects
9 Neurodevelopmental effects, including decreased brain weight, changes in brain
10 biochemistry, and changes in behavioral performances, have been observed. Each study evaluating
11 neurodevelopmental effects, however, had limitations in study design, reporting, or both. In
12 addition, results were not always consistent between studies or across dose. At this time, there is
13 inadequate information to draw conclusions regarding neurodevelopmental toxicity.
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1.2.5. Reproductive Effects
Synthesis of effects related to reproduction
Several studies evaluated reproductive effects [a one-generation, oral reproductive study
fLvondell Chemical Co.. 20041 and subchronic evaluations in rats and mice following oral and
inhalation exposure (NTP. 1997.1995)] 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-ge neratio' -productive oral or inhalation
studies and by having no human studies on reproduction. Tlv ,gn, conduct, and reporting of
each study were reviewed, and each study was considere- >te 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- (I.yondell Chemical Co..
2004: NTP. 1997.19951 (Table l-9;Figure 1-13; Figure 1-14). In males, the only significant effect
observed was a slight decrease in sperm motility lor I'd males treated with 1000 mg/kg-day of tert-
butanol fLvondell Chemical Co.. 20041. 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 (nils within the range of historical control data and,
therefore, its biological s1 'ice is uncertain. In female B6C3F i mice, estrous cycle length was
increased 28% follow' ,ral ex, ire to 11,620 mg/kg-day (NTP. 1995). No significant changes in
estrous cycle length u. -iliserveu lowing oral exposure in rats, or inhalation exposure in mice
or rats.
Table 1 -9. Evidence perti. 'ig to repn. ctive effects in animals following
exposure to terf-butanol
Reference and study design
Results
Male reproductive effects
Lvondell Chemical Co. (2004)
Sprague-Dawley rat; 12/sex/treatment
Gavage 0, 64,160, 400, or 1,000 mg/kg-d
F0 males: 9 weeks beginning 4 weeks prior to
mating
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
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
Lvondell Chemical Co. (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 (0, -2, -4, 0, +8 % change
relative to control)
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*%
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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 (0, -4, +2, +4 % change relative
to control)
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 U estrous cycle (0, -3, -9, -5 % change relative
to control)
Evaluations were only fjerformea \ concentrations >542 ppm
(1,643 mg/m3)
1 * Statistically significant p < 0.05 as determined by the study authors.
2 Notes: Conversions from drinking water concentrations to mg/kg-d performed by study authors.
3 Conversion from ppm to mg/m3 is 1 ppm = 3.031 mg/m3.
4 Percentage 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)
Reproductive organs or sperm; M
rat(B)
Reproductive organs or sperm; M
mouse (B)
Female reproductive effects
Pregnancy index; F rat (A)
Estrous cycle length; F rat (B)
t Estrous cycle length; F mouse (B)
~ B B 0
~ ~ ~ ~
~—B B B B
~ ~ B B
~—~~ ~ B
10 100 1,000 10,000 100,000
Dose (mg/kg-day)
Sources: (A) Lvondell Chemical Co. (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
(OTP, 199?)
Reproductive organs or sperm; M mouse
(NTP, 1997)
B-
O
-B
Female reproductive effects
Estrous cycle; F rat (NTP, 1997)
Estrous cycle; F mouse (NTP, 1997)
B-
_e ~
B-
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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Integration of reproductive effects
At this time, no conclusions are drawn in regard to reproductive toxicity. The database is
limited to a one-generation study fLvondell Chemical Co.. 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 nils e jd via inhalation.
1.2.6. Other Toxicological Effects
Effects other than those related to kidney, thyroid, rcprotlncliv c. developmental, and
neurodevelopmental toxicity were observed in souk.1 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 alack of progression in urinary Madder, however, inadequale information is
available to draw conclusions regarding liver or urinary Madder toxicity atthis time.
Additionally, central nervous system ((INS) 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 lime. For more information on these other toxicological effects, see
Appendix B.3.
1.3. INTEGRATION AND EVALUATION
1.3.1. Effects Other Tluin Ciincer
Kidney effects were identified as a potential human hazard of tert-butanol exposure based
on several endpoints, including suppurativ e inflammation in female rats, transitional epithelial
hyperplasia in male and female rats, severity of nephropathy in male and female rats, incidences of
nephropathy in female rats, mineralization in male rats, and increased kidney weights in both male
and female rats. 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.
Several effects were observed in the kidneys of rats. Based on mechanistic evidence
indicating that an a2u-globulin-related process is operating in male rats (Hard etal.. 2011: Cirvello
etal.. 1995: NTP. 1995: Lindamood etal.. 1992). any kidney effects associated with a 2U-globulin
nephropathy are not considered relevant for human hazard identification. In addition, CPN played a
role in the renal tubule nephropathy observed following tert-butanol exposure, and effects
associated with such nephropathy are not considered relevant for human hazard identification.
Although increases in severity (males and females) or incidence (females) of nephropathy were
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related to tert-butanol exposure and could have arisen from chemical-specific processes
independent from CPN, the association of these effects with CPN makes this measure less suitable
for dose-response analysis, and therefore these effects were not considered for the derivation of
reference values. Furthermore, some uncertainty exists regarding whether mineralization is also
associated with CPN in male rats; due to this uncertainty, and because other kidney effects were
identified as being associated with tert-butanol exposure and yet independent from CPN,
mineralization in male rats was not considered for dose-response analysis. The remaining effects
(suppurative inflammation, transitional epithelial hyperplasia, and increased kidney weights) are
considered the result of tert-butanol exposure and relevant l<> I- n hazard characterization.
These effects therefore are suitable for consideration for do spouse analysis and derivation of
reference values, in Section 2.
There is suggestive evidence of developmental effects associated with tert-butanol
exposure. Increased fetal loss, decreased fetal hotly weight, and increases in skeletal variations in
exposed offspring were observed following exposure lo relatively high doses ol /ert-butanol during
gestation.. These effects are similar to the develop in en la I effects observed with MTI'E exposure
(e.g., decreased fetal body weight and increases in skeletal v ariations) (ATSDR. 199b).
No mechanistic evidence is available for developmental effects of tert-butanol. Although the
evidence is suggestive of developmental toxicity, due to the uncertainty as to 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, developmental effects were not considered for dose-response
analysis and derivation of reference values in Section 2. furthermore, no adverse effects were
reported in one- ami two-generation reproductive/developmental studies on ETBE (Gaoua. 2004a.
b), providing further support lor the lack ol evidence supporting reproductive or developmental
effects as possible human hazards following it'/V-hulanol exposure.
At this time, there is inadequate information to draw conclusions regarding
neurudevelopmental effects as a human hazard of tert- butanol exposure. Although
neurodevelopmenlal effects have heen observed, the studies had limitations in design or reporting,
or both, and results were inconsistent between studies and across dose groups. No mechanistic
evidence is available to inform the MOA for neurodevelopmental effects of tert- butanol. These
effects were not considered further for dose-response analysis and derivation of reference values.
At this time, no conclusions are drawn regarding reproductive effects as a human 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 database 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.
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At this time, there is inadequate information 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 F344/N rats, administration of tert-butanol in drink' .ater increased the incidence of
renal tubule tumors, mostly adenomas, in males; no renal 1 ¦; in females were reported (Hardet
al.. 2011: NTP. 19951. As discussed in Section 1.2.1, son'. Jies^ "liors might be associated with
a2u-globulin nephropathy, an MOA considered spiv' .o the male- 11 ' i.S. EPA. 1991a). Evidence
in support of this hypothesized MOA includes ll- . umulation of hyali. 'mplets in renal tubule
cells, the presence of a2u-globulin in the hyaline-. 'lk-ts, and additional as, ' s associated with
a2u-globulin nephropathy, including linear papillary mineralization and foci oltulmlar hyperplasia.
Other evidence, however, is not supportive-: The accumulation ol hyaline droplets was minimal;
concentrations ofa2U-globulinwere low at doses that inckie'e-il tumors; and no significant necrosis
or cytotoxicity was associated with compensatory regenerative proliferation or induction of
granular casts observed within a lime-lranie- consistent with i< u-glolmlin-mediated nephropathy.
Renal tumors also could lie- associate-el with chronic progressive- nephropathy, butthe data on CPN
are notcoherent: Dose-response- relationships for CPN, renal tubule hyperplasia, and renal tubule
tumors were different: in addition, CPN was nearly as sev ere in female rats as in male rats, yetno
female- rats el eve-1 ope-il renal tumors. Thus, some- renal tumors maybe attributable to a2U-globulin
nephropathy anil some- to other, vet unspe-cilie-el, processes. Taken together, and according to EPA's
guidance- on renal tumors in male- rats f M.S. I1PA. 1991a). renal tumors induced by tert-butanol are
relevant for human hazard identification.
In BbC:-!l' i mice, administration of tert-butanol in drinking water increased the incidence of
thyroid follicular cell adenomas in females, and adenomas or carcinomas (only one carcinoma
observed) in males (NTP. as discussed in Section 1.2.2. According to EPA's thyroid tumor
guidance (U.S. EPA. l')'>?>a). chemicals that produce thyroid tumors in rodents might pose a
carcinogenic hazard to humans.
In addition, as mentioned in Section 1.1.4, tert-butanol is a primary metabolite of MTBE and
of ETBE, two compounds tested in rats and mice that could provide supplementary information on
the carcinogenicity of tert-butanol. For MTBE, the most recent cancer evaluation by a national or
international health agency is from IARC (1999). IARC reported that oral gavage exposure in
Sprague-Dawley rats resulted in testicular tumors in males and lymphomas and leukemias
(combined) in females; inhalation exposure in male and female F344 rats resulted in renal tubule
adenomas in males; and inhalation exposure in male and female CD-I mice resulted in
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hepatocellular adenomas in females (IARC. 1999). For ETBE, a draft IRIS assessment under
development concurrently with this assessment reports that inhalation exposure in male and
female F344 rats resulted in hepatocellular tumors, mostly adenomas, in males; no significant
tumor increases 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.
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 carcinoi to humans is appropriate when
the evidence is "adequate to demonstrate carcinogenic pole1 .<> luimans" but does not support
the descriptor carcinogenic to humans. One example fror l -t guidelines is "an agentthathas
tested positive in animal experiments in more than (>¦ 1 species, sc. ' ruin, site, or exposure route,
with or without evidence of carcinogenicity in lur tert-Butanol m 'lies the conditions of this
example, having increased tumor incidences in . species, in both sexes, I 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" hut is not
sufficient for a stronger conclusion. The results for te/7-huUinol raise a concern for cancer but none
of the effects is particularly strong. The kidney tumors resulted, in part, from an MOAthatis specific
to male rats, while no kidney tumors occu rred in female rats. The thyroid tumors induced in male
and female mice were almost entirely benign. In addition, while MTI!E was also associated with
male rat kidney tumorigenesis, there is little coherence of results between tert-butanol and ETBE
associated tumorigenesis in rats. MTI'E or ETI!K 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 ^'/Mnitanol exposure inB6C3Fi mice.
These considerations, interpreted in light of the cancer guidelines, support the conclusion
that there is 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, and this was decisive in selecting a hazard
descriptor.
The descriptor suggestive evidence of carcinogenic potential applies to all routes of human
exposure. Oral administration ol'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. According to
the 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 renal tumors in male rats fU.S. EPA. 199 lal advises that unless the relative contribution of
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a2u-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.
EPA's guidance on thyroid tumors and EPA's cancer guidelines (U.S. EPA. 1998al advises
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 a1 , thyroid, pituitary, and liver
effects in female mice, and no data are available to evaluate otential for antithyroid effects in
male mice. Data are not adequate to conclude thatthyrc: rn. ¦ changes exceed the range of
homeostatic regulation or to evaluate effects on exlr ?patic sites u -Ived 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 lu mors result from
thyroid-pituitary disruption is premature, and dose-response analysis should use linear
extrapolation. The data are well suited to dose-response analysis, coming from an NTP study that
tested multiple dose levels.
1.3.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes
No chemical-specific (.lata that would allow for the identification of populations with
increased susceptibility to /(.'//-butanol exposure exist. In vitro studies have implicated the liver
microsomal mixed function oxidase (MI'O) system, namely CYP450 (Cederbaum etal.. 1983:
Cederbaum and Cohen. !'){{()). as playing a role in the metabolism of tert-butanol. No studies,
however, have identified the specific CYl's responsible for the biotransformation of tert-butanol.
Pharmacokinetic differences among the letus, newborns, children, and the aged might alter
responses to chemicals compared to adults, resulting in differences in health effects. In the
presence ol 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. The expression of
XMETs, including various CYl's, has been found to be underexpressed in the mouse fetus and
neonate fLee etal.. 20111 and decreased in older mice fLee etal.. 20111 and rats fLee etal.. 20081.
Decreased ability to detoxify and transport tert-butanol out of the body could result in increased
susceptibility to tert-butanol in the young and old.
In regard to cancer, although children are more sensitive than adults to thyroid
carcinogenesis resulting from ionizing radiation, relative differences in lifestage sensitivity to
chemically induced thyroid carcinogenesis are unknown (U.S. EPA. 1998al. In addition, the data on
tert-butanol mutagenicity are inconclusive.
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1 Collectively, there is little evidence on tert-butanol itself to identify susceptible populations
2 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 (UFs) generally applied to reflect limitations of the data used.
2.1.1. Identification of Studies and Effects for Dose-Response Analysis
EPA identified kidney effects as a potential human hazard of tert-butanol exposure (see
Section 1.2.1). Studies within this effect category were evaluated using general study quality
characteristics [as discussed in Section 6 of the Preamble; see also U.S. EPA (2002)] to help inform
the selection of studies from which to derive toxicity values. No other hazards were identified for
further for consideration in the derivation of reference values.
Human studies are preferred over animal studies when quantitative measures of exposure
are reported and the reported effects are determined to be associated with exposure. No human
occupational or epidemiological studies of oral exposure to tert-butanol, however, are available.
Animal studies were evaluated to determine which studies provided: (1) the most relevant
routes and durations of exposure, (2) multiple exposure levels to provide information about the
shape of the dose-response curve, and (3) power to detect effects at low exposure levels. The
database 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 male and 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, may impact the ability of the kidney to filter waste. Observed
changes in kidney weight could also indicate toxic effects in the kidney. For the oral tert-butanol
database, there are several studies available that evaluated these kidney effects. Lvondell Chemical
Co. (2004) conducted a reproductive study in Sprague-Dawley rats that was of shorter duration,
and reported changes in kidney weight but did not examine changes in histopathology. NTP
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conducted a 2-year drinking water study (NTP. 1995) in F344 rats that evaluated multiple doses in
both males and females, and reported on all three endpoints highlighted above. NTP (1995) was
identified as most suitable for dose-response assessment considering the study duration,
comprehensive reporting of outcomes, and multiple doses tested.
In the NTP (19951 2-year drinking water study, male F344 rats were exposed to
approximate doses of 0, 90, 200, or 420 mg/kg-day; female F344 rats were exposed to approximate
doses of 0,180, 330, or 650 mg/kg-day. 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 .lis, and 2 years of treatment
fNTP. 19951. Specific endpoints chosen for dose-response a .is were absolute kidney weight
(observed in males and females), kidney suppurative in1' ,ia. (observed in females), and
kidney transitional epithelial hyperplasia (observed ' males and k les). For absolute kidney
weight, data from 15 months was selected as des d in Section 1.2. i ¦¦ the oilier endpoints,
data at the longest duration of 2 years were sek ¦ I.
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 lo empirically model the dose-response relationship in the range
ofthe observed data. The models in KI'A's Benchmark Dose Software (BMDS) were applied.
Consistent with EPA's Henchmurk Dose Technical (iuidance ( U.S. EPA. 2012b). the BMD and the
BMDL are estimated using a benchmark response (I'MK) lo represent a minimal, biologically
significant level ol change. In the absence ofInformation regarding the level of change that is
considered biologically significant, a I'MK of 1 standard deviation from the control mean for
continuous (.lata or a I'MK of 10".. extra risk for dichotomous data is used to estimate the BMD and
BMDL and also to facilitate a consistent basis of comparison across endpoints, studies, and
assessments. I julpoint-specilic I'MKs, 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. Further details including the modeling output and graphical results for
the model selected lor each endpointcanbe found inAppendixC ofthe Supplemental Information
to this Toxicological Review.
Kidney weights were analyzed as absolute weights rather than relative to body weight In
general, absolute and relative kidney weight data can both be 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 (see
Table 1-1), but this decrease in body weight impacted the measure of relative kidney weight
resulting in an exaggeration of the kidney weight change. There was greater confidence in the
absolute kidney weight measure; thus, it was considered more appropriate for dose-response
analysis, and changes in relative kidney weights were not analyzed. A 10% relative change from
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control was used as a BMR for absolute kidney weight by analogy with 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.
Human equivalent doses (HEDs) for oral exposures were derived from the PODs according
to the hierarchy of approaches outlined in EPA's Recommended Use of Body Weight3/4 as the Default
Method in Derivation of the Oral Reference Dose (U.S. EPA. 20111. The preferred approach is
physiologically based toxicokinetic modeling (PBPK). Other approaches include using chemical-
specific information in the absence of a complete PBPK model. ' ;cussed in Appendix B of the
Supplemental Information, human PBPK models for inhalal: . I "M !E or inhalation and dermal
exposure to MTBE have been published, which include I ju 1 submodels. A validated human
PBPK model for tert-butanol, however, is not avai lah' for extrapoi. <• doses from animals to
humans. In lieu of either chemical-specific mode1 ..ata to inform Uk nvation of human
equivalent oral exposures, body weight scaling -it- '¦% power (i.e., BW: ¦, -lpplied to extrapolate
toxicologically equivalent doses of orally adminislt 'agents l- -n adult laho. tv animals to
adult humans for the purpose of deriv' 'q an oral RfD.
Consistent with EPA guidance.- f M.S. I j'A. 2011). tin.- I'ODs estimated based on effects in adult
animals were converted to HEDs employing a standard dosimetric adjustment factor (DAF) derived
as follows:
l)AI; = (i:W,' '/IIUV ').
where
l!W., = animal hotly weight
l!Wi, = human hotly weight
Msing a standard l!W., ol ().2.r) kg lor rats and a BWh of 70 kg for humans (U.S. EPA. 19881.
the resulting DAI; is 0.24 lor rats. Applying this DAF to the POD identified for effects in adultrats
yields a I'ODm |. as follows (st-f Tahlt- 2-1):
PODiii |. = l.ahoralorv 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)
PODADjb(m
g/kg-d)
PODHEDc(mg/
kg-d)
Kidney
Increased absolute
kidney weight at 15
months
NTP (1995)
Rat/M
Linear
(constant
variance)
10%
657
296
296
71
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-probit
10%
254
200
200
48
Kidney transitional
epithelial
hyperplasia
NTP (1995)
Rat/M
Log-logistic
10%
30
16
16
3.84
Kidney transitional
epithelial
hyperplasia
NTP (1995)
Rat/F
Multistage,
3-degree
10v-„
412
339
339
81.4
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 TWA
5 daily doses prior to BMD modeling. However, this adjustment was not required for the NTP (1995) study.
6 CHED PODs were calculated using BW scaling (U.S. EPA, 201i).
7 NA= not applicable
8 2.1.3. Derivation of Candidate Values
9 Consistent with K I'A's ,1 Review of the Reference Dose and Reference Concentration Processes
10 [(U.S. EPA. 2002): Suction -1.-1 .f> |. also described in the Preamble, five possible areas of uncertainty
11 and variability were considered when determining the application of UFs to the PODs presented in
12 Table 2-1. An explanation follows:
13 An intraspecies uncertainty factor, UFh, of 10 was applied to all PODs to account for
14 potential differences in toxicokinetics and toxicodynamics in the absence of information on the
15 variability of response in the human population following oral exposure to tert- butanol fU.S. EPA.
16 20021.
17 An interspecies uncertainty factor, UFa, of 3 (10°5 = 3.16, rounded to 3) was applied to all
18 PODs because BW3/4 scaling was used to extrapolate oral doses from laboratory animals to humans.
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Although BW3/4 scaling addresses some aspects of cross-species extrapolation of toxicokinetic and
toxicodynamic processes, some residual uncertainty in the extrapolation remains. In the absence of
chemical-specific data to quantify this uncertainty, EPA's BW3/4 guidance fU.S. EPA. 20111
recommends use of an uncertainty factor of 3.
A subchronic to chronic uncertainty factor, UFS, of 1 was applied to all PODs because the
endpoints were all observed following chronic exposure.
A LOAEL to NOAEL uncertainty factor, UFl, of 1 was applied to all PODs derived because the
current approach is to address this factor as one of the considerations in selecting a BMR for
benchmark dose modeling. In this case, BMRs of a 10% relative ,ge in absolute kidney weight, a
10% extra risk of kidney suppurative inflammation, and a 1' xtra risk of transitional cell
hyperplasia were selected assuming they represent min' .>i. 'icallv significant response levels.
A database uncertainty factor, UF, of 1 was ;r Heu to all Pi- The tert-butanol oral toxicity
database includes chronic and subchronic toxic M" .lies in rats and i. f Acharva et al.. 1997:
Acharva etal.. 1995: NTP. 1995) and developnu -I toxicity studies in rat. ¦ I mice (Lvondell
Chemical Co.. 2004: Faulkner et al.. 1989: Daniel ai. '"ans. 1')' '1. In the dew mental studies, no
effects were observed at exposure levels below 1000 mg/kg-dav, and effects observ ed 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 developmental toxicity is not as sensitive
an endpoint as kidney effects. No immuiK (toxicity or multige lie rational reproductive studies are
available for tert-butanol. Studieson KTBE, which is rapidly metabolized to systemically available
tert-butanol, are informativ e for consideration of the gaps in the LerL-butanol oral database. The
database for ETBE does not indicate immunotoxicity (13anton etal.. 2011: Li etal.. 2011). suggesting
immune system effects would not be a sensitiv e target for tert-butanol. No adverse effects were
reported in one- and two-generation reproductive/developmental studies on ETBE fGaoua. 2004a.
b), indicating that reproductive/developmental effects would not be a sensitive target for tert-
butanol. Additionally, a one-generation, reproductive toxicity study in rats from a Toxic Substances
Control Act submission (Lvondell Chemical Co.. 2004) is available for tert-butanol. This study did
not observe reproductive effects. Although the oral toxicity database for tert-butanol has some
gaps, the available (.lata on fivf-bulatiol, 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 U l'|. ol 1 was applied.
Figure 2-1 presents graphically the candidate values, UFs, and PODhed values, with each bar
corresponding to one data set described in Tables 2-1 and 2-2.
Table 2-2 is a continuation of Table 2-1 and summarizes the application of UFs to each POD
to derive a candidate value for each data set, preliminary to the derivation of the organ/system-
specific RfDs. These candidate values are considered individually in the selection of a
representative oral reference value for a specific hazard and subsequent overall RfD for tert-
butanol. Figure 2-1 presents graphically the candidate values, UFs, and PODhed values, with each
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1 bar corresponding to one data set described in Tables 2-1 and 2-2.
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;
male rat at 15 months
NTP (1995)
71
BMDLio0/
3
10
1
1
1
30
2 x 10°
Increased absolute kidney weight;
female rat at 15 months
NTP (1995)
22
BMDL
3
10
1
1
1
30
7 x 10 1
Kidney inflammation (suppurative);
female rat NTP (1995)
48
BMDL
3
10
1
1
1
30
2 x 10°
Kidney transitional epithelial
hyperplasia; male rat
NTP (1995)
3.8
BMDL„„
3
10
1
1
1
30
1 x 10 1
Kidney transitional epithelial
hyperplasia; female rat
NTP (1995)
81
BMDL
3
10
1
1
1
30
3 x 10°
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T Absolute kidney
weight; male rat
(NTP, 1995}
I Absolute kidney
weight; female rat
(NTP, 1995)
Kidney inflammation;
female rat (NTP, 1995)
Kidney transitional
epithelial hyperplasia;
male rat (NTP, 199S)
Kidney transitional
epithelial hyperplasia;
female rat (NTP, 1995}
0.1
10
100
~ Candidate RfD
® PODheb
Composite UF
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 both sexes,
spanning a range from 1 x 101 to 3 x 10° mg/kg-day, for an overall 30-fold range. To estimate an
exposure level below which kidney toxicity from tert- Ini la no I exposure is not expected to occur, the
RfD for increased incidence of transitional epithelial liypcrpkisiii in male rats (1 x 10"1 mg/kg-day)
was selected as the kidney-specific reference dose for /e/Mnilanol. Unlike kidney suppurative
inflammation, this effect was observed in both sexes, with males appearing to be more sensitive
than females. Additionally, this indicator of kidney toxicity is more specific and more sensitive than
the relatively non-specific endpoint of absolute kidney weight changes. Confidence in this kidney-
specific RfD is high. The PODs are based on benchmark (.lose modeling, and the candidate values are
derived from a well-conducted long-term study, invok ing a sufficient number of animals per group,
including both sexes, and assessing a wide range of kidney (.Midpoints.
Table 2-3. Organ/system-specific RfDs and overall RfD for tert-butanol
Effect
Basis
RfD (mg/kg-day)
Study exposure
description
Confidence
Kidney
Incidence of transitional
epithelial hyperplasia (NTP
(1995)
lx 10 1
Chronic
High
Overall RfD
Kidney
1 x 10 1
Chronic
High
2.1.5. Selection of the Overall Reference Dose
For tert-bulanol, 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 specilie RID of (1 x 101 mg/kg-day) is the overall RfD for tert-butanol. This
value is based on increased incidence of transitional epithelial hyperplasia in male 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. 2002). Decisions concerning averaging exposures over time for
comparison with the RfD should consider the types of toxicological effects and specific lifestages of
concern. Fluctuations in exposure levels that result in elevated exposures during these lifestages
could lead to an appreciable risk, even if average levels over the full exposure duration were less
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than or equal to the RfD. In the case of tert-butanol, there is potential 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 database, and the RfD, as described in Section 4.3.9.2 of EPA's Methods for Derivation of
Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA. 19941. The
overall confidence in this RfD is high. Confidence in the principal study (NTP. 1995) is high. This
study was well conducted, complied with Food and Drug Admin' lion (FDA) Good Laboratory
Practice (GLP) regulations, involved a sufficient number ol ;> .s per dose group (including both
sexes), and assessed a wide range of tissues and endpoin' i. the toxicity database for tert-
butanol has some gaps, they are informed by the ikilr >n l f BE, a |. 'il compound of tert-butanol.
Therefore, the confidence in the database is high. ' cting high conli re in the principal study
and high confidence in the database, confident-. Jie RfD is high.
2.1.7. Previous IRIS Assessment
No previous oral assessment for /(.'//-butanol is available in IRIS.
2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER
THAN CANCER
The inhalation RIC (expressed in units ol mg/m :) is defined as an estimate (with
uncertainty spanning perhaps an order ol magnitude) ol a continuous inhalation exposure to the
human population (including sensitive subgroups) that is likely to be without an appreciable risk of
deleterious effects (.luring a lifetime. II can lie derived from a NOAEL, LOAEL, or the 95% lower
bound on the benchmark concentration (l!MCI,J, with Ill's 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, KI'A 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; there is only one 13-week study in rats and mice (NTP. 1997). Sufficient data
were available to modify and utilize a PBPK model in rats for both oral and inhalation exposure in
order to perform a route-to-route extrapolation, so rat studies from both routes of exposure were
considered for dose-response analysis.
The database for tert-butanol includes oral and inhalation studies and data sets that are
potentially suitable for use in deriving inhalation reference values. Specifically, effects associated
with tert-butanol exposure in animals include observations of organ weight and histological
changes in the kidney in chronic and subchronic studies in male and female rats.
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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. These findings were
observed across multiple chronic, subchronic, and short-term studies following oral and inhalation
exposure. The subchronic NTP f19971 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 f19971
subchronic inhalation study shares many strengths with the 2-year drinking water study (NTP.
19951. and is described in more detail below.
NTP (1997) was a well-designed subchronic study lluil ev aluated the effect of tert-butanol
exposure on multiple species at multiple inhalation (.loses. Absolute kidney weights were elevated
(10-11%) in male rats exposed at >3,273 nig/111:: relativ e kidney weights were elevated (~9%) in
males at >3,273 mg/m3 and in females at (>,3(>f$ ing/ni:. Male rats exhibited an increase in the
severity of chronic nephropathy (charade ri/ed as number of loci of regenerative tubules). Few
endpoints were available for consideration in the subchronic inhalation study, butchanges 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 (.lose 'lonse mou... .ire available for tert-butanol. In this situation,
EPA evaluates a range of dose-res|ion. lodels considered consistent with underlying biological
processes to determine how best to modi. : e dose-response relationship empirically in the range
of the observed (.lata. Consistent with this approach, all models available in EPA's BMDS were
evaluated. Consistent with EPA's Ik'nchmark Dose Technical Guidance (U.S. EPA. 2012b). the
benchmark dose or concentration (I5MD/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. Where
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, can be found in Appendix C of the Supplemental Information.
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PODsfrom 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. 19941 provides
mechanisms for (1) adjusting experimental exposure concentrations to a value reflecting
continuous exposure duration (ADJ) and (2) determining a human equivalent concentration (HEC)
from the animal exposure data. The former employs an inverse concentration-time relationship to
derive a health-protective duration adjustment to time weight the intermittent exposures used in
the studies. The modeled benchmark concentration from the inhalation study fNTP. 19971 was
adjusted to reflect a continuous exposure by multiplying il by ((> 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 - 24) ¦ (f. - 7)
= BMCL (mg/m3) ¦ (0.178(>)
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 ftrf-butanol in the laboratory animal (rator mouse)
and humans. According to the RfC guidelines (U.S. KI'A. 10Q-1-). fivf-butanol is a Category 3 gas
because extra-respiratory effects were observed. Kaneko el ai. f2000 I measured a blood:gas
partition coefficient |(I h, | olT>>! 1 ± 102 for fivf-liuUinol in the male Wistar rat, while Borghoff et
al. (19961 measured a value of 181 ± 2') in male I'ivl l rats. A blood:gas partition coefficient
[(Hb/g)H] of 462 was reported for ^'/7-bulanol in humans fNihlen et al.. 19951. The calculation
(Hb/g) •, 4 (111, ¦¦ )m was used lo calculate a blood:gas partition coefficient ratio to apply to the
delivered concentration, liecause I ¦'311 rats were used in the study, the blood:gas partition
coefficient lor l;314 rats was used. Thus, the calculation was 481 4 462 = 1.04. Therefore, a ratio of
1.04 was used to calculate the IILC. This allowed a BMCLhec to be derived as follows:
BMCLim . = IIMCI -jadj (mg/m3) x (interspecies conversion)
= HMCLadj (mg/m3) x (481 4 462)
= HMCLadj (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)
PODadj13
(mg/m3)
PODhecc(
mg/m3)
Kidney
Increased absolute
kidney weight
NTP (1997)
Male F344
rats
Hill
10%
1931
1705
304
304
Increased absolute
kidney weight
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,
1994).
dBMD modeling failed to calculate a BMD value successfully (see Appendix C); POD calculated from no-observed
adverse effect level (NOAEL) of 6368 mg/m .
PODs from oral studies - use ofPBPK model for route-to-route extrapolation
APBPKmodel for tert-butanol in nils has hecn moililk'il. as described in Appendix B of the
Supplemental Information. Using this model, r< hi te-to-route extra polation of the oral BMDLs to
derive inhalation PODs was performed as follows. First, the internal (.lose in the ratateach oral
BMDL (assuming continuous exposure) was estimated using the PBPK model, to derive an "internal
dose BMDL." Then, the inhalation air concentration (again, assuming continuous exposure) that led
to the same internal dose in the rat was estimated using the PBPK model. The resulting BMCL was
then converted to a human equivalent concentration POD (PODhec) using the methodology
previously described in "PODs from inhalation studies":
BMC Li M. = l!MCI, m.| (mg/m3) x (interspecies conversion)
= HMCLadj (mg/m3) x (481 4- 462)
= I'MCLadj (mg/m3) x (1.04)
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 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
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1 toxicological agent. Therefore, the concentration of tert-butanol in blood was selected as the dose
2 metric.
3 Table 2-5 summarizes the sequence of calculations leading to the derivation of a human-
4 equivalent inhalation POD from each oral data set discussed above.
5 Table 2-5. Summary of derivation of inhalation points of departure derived
6 from route-to-route extrapolation from oral exposures
Endpoint and reference
Species/sex
BMR
BMDL
(mg/kg-d)
Internal dosea
(mg/L)
Equivalent
PODhec15
(mg/m3)
Kidney
Mean absolute kidney weight
at 15 months NTP (1995)
Rat/M
10%
296
22.4
551
Mean absolute kidney weight
at 15 months NTP (1995)
Rat/F
10"„
91
4.76
155
Kidney inflammation
(suDDurative) NTP (1995)
Rat/F
10°<.
200
12.6
359
Kidney transitional epithelial
hyperplasia
NTP (1995)
Rat/M
lO'.'-r,
16
0.745
26.1
Kidney transitional epithelial
hyperplasia
NTP (1995)
Rat/F
lOw.
339
27.9
638
7 a Average blood concentration of te/t-butanol under continuous oral exposure at the BMDL
8 b Continuous inhalation human equivalent concentration that leads to the same average blood concentration of
9 te/t-butanol as continuous oral exposure at the BMDL.
10 2.2.3. Derivation of Candidate Values
11 In EPA's ,1 Review of the Reference Dose and Reference Concentration Processes [fU.S. EPA.
12 20021: Section 4.1..r> J, also described in the Preamble, five possible areas of uncertainty and
13 variability were considered. Sev eral I'ODs for the candidate inhalation values were derived using a
14 route-to-route extrapolation from the PODs estimated from the chronic oral toxicity study in rats
15 (NTP. 1995) in the derivation of the oral RfD (Section 2.1). With the exception of the subchronic
16 inhalation fNTP. 19971 study, the uncertainty factors (UFs) selected and applied to PODs derived
17 from the chronic oral fNTP. 19951 study for route-to-route extrapolation are the same as those for
18 the RfD for tert-butanol (see Section 2.1.3). The model used to perform this route-to-route
19 extrapolation is a well-characterized model considered appropriate for the purposes of this
20 assessment One source of uncertainty regarding the route-to-route extrapolation is the assumption
21 of that 100% of inhaled tert-butanol reaches the gas-exchange region, that is, 100% of the inhaled
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tert-butanol could be absorbed and distributed to the rest of the body in rats. If not all of the
compound is bioavailable for the rat, a lower blood concentration would be expected compared to
the current estimate, and thus, a higher RfC would be calculated.
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
UFs to each POD to derive a candidate value for each data set. The candidate values presented in the
table below are preliminary to the derivation of the organ/system-specific reference values. These
candidate values are considered individually in the selection <>l a representative inhalation
reference value for a specific hazard and subsequent overall RIC lor tert-butanol.
Figure 2-2 presents graphically the candidate values, I' I"s. and PODhec values, with each bar
corresponding to one data set described in Tables 2-4, 2-5, and 2-(>.
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; male rat
NTP (1997)
304
BMCL
3
10
1
10
1
300
lx 10°
Increased absolute kidney weight
at 13 weeks; female rat
NTP (1997)
1137
NOAEL
3
10
1
10
1
300
4x 10°
Increased absolute kidney weight
at 15 months; male rat
NTP (1995)
551
BMCL
3
10
1
1
1
30
2 x 101*
Increased absolute kidney weight
at 15 months; female rat
NTP (1995)
155
BMCLio%
3
10
1
1
1
30
5 x 10° *
Kidney inflammation
(suppurative); female rat
NTP(1995)
359
BMCL io%
3
10
1
1
1
30
1 x 101*
Kidney transitional epithelial
hyperplasia; male rat
NTP(1995)
26.1
BMCL io%
3
10
1
1
1
30
9 x 10 1 *
Kidney transitional epithelial
hyperplasia; female rat
NTP (1995)
638
BMCL io%
3
10
1
1
1
30
2 x 101*
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These candidate values are derived using route-to-route extrapolated PODs based on NTP's chronic drinking
water study.
T Absolute kidneyweight;
male rat (N TP, 1997)
TAbsolute kidneyweight;
female rat (NTP, 1997)
t Absolute kidney weight;
male rat (NTP, 1995)
TAbsolute kidneyweight;
female rat (NTP, 1995)
Kidney inflammation;
female rat [NTP, 1995)
Kidney transitional
epithelial hyperplasia;
male rat (NTP, 1995)
Kidney transitional
epithelial hyperplasia;
female rat (NTP, 1995)
Candidate Rft
0 PGDjjec
Composite UF
0.1
10
100 1000 10000
mg/m3
Figure 2-2. Candidate RfC values with corresponding POD and composite UF.
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2.2.4. Derivation of Organ/System-Specific Reference Concentrations
Table 2-7 distills the candidate values from Table 2-6 into a single value for the kidney.
Organ- or system-specific reference values can be useful for subsequent cumulative risk
assessments that consider the combined effect of multiple agents acting at a common site.
Kidney Toxicity
For the derivation of candidate values, whether PODs from the subchronic inhalation study
of NTP (1997) would provide a better basis than the route-to-route extrapolated PODs based on the
chronic oral study of NTP Q9951 must be considered. Candidal .ies were derived for increased
kidney weight observed in the subchronic inhalation study 1 . ! '>'>71 and several kidney effects
observed in the chronic oral study fNTP. 19951 in both v >>¦ spanning a range from 9 x 101
to 2 x 101 mg/m3, for an overall 20-fold range. To os1 ' 'ate an expo ¦¦ level below which kidney
toxicity from tert-butanol exposure is not expect' occur, the RfC l<. Teased incidence of
transitional epithelial hyperplasia in male rats v 1 01 mg/m3) was sekv 1 as the kidney-specific
RfC for tert-butanol, consistent with the selection (. "kidney ¦>ecificRfD (.\ Section 2.1.4). As
discussed in Section 2.1.4, unlike kidn^' suppurative inflammation, this effect was observed in both
sexes, with males appearing to be mo :| ive than females. Additionally, it is based on a longer
(chronic) duration and a more specific' so. " indicator of kidney toxicity than the relatively
non-specific endpoint of kidney weight ch> >o. Coi,. '<-o in this kidney-specific RfC is high. The
PODs are based on 1!MI) modeling, and the i. lidale' -o derived from a well-conducted
study, involving a sufficient number of animals ¦¦ up, inciu . j both sexes, assessing a wide
range of kidney endpoinls. and availability of a I'. \ model for route-to-route extrapolation.
Table 2-7. Organ/system-specific RfCs am. 'erall RfC for tert-butanol
Effect
Basis
RfC
(mg/m3)
Study exposure
description
Confidence
Kidney
Incidence of transitional
epithelial hyperplasia (NTP,
1995)
9 x 10 1
Chronic
High
Overall RfC
Kidney
9 x 10 1
Chronic
High
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 9 x 10-i 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
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subgroups (U.S. EPA. 2002). Decisions concerning averaging exposures over time for comparison
with the RfC should consider the types of toxicological effects and specific lifestages of concern.
Fluctuations in exposure levels that result in elevated exposures during these lifestages could
potentially lead to an appreciable risk, even if average levels over the full exposure duration were
less than or equal to the RfC. In the case of tert-butanol, there is potential 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 lc study used to derive the RfC,
the overall database, and the RfC itself, as described in Seel i' ,M2 of EPA's Methods for
Derivation of Inhalation Reference Concentrations and Ap .i. •/ Inhalation Dosimetry fU.S. EPA.
19941. APBPK model was utilized to perform a route -ouie extra |. ' ion to determine a POD for
the derivation of the RfC from the NTP (1995) on-' ay and correspi. ng critical effect.
Confidence in the principal study (NTP. 1995) . ^h. This study was wei, ¦ulucted, complied
with FDA GLP regulations, involved a sufficient mi. Tofanim 's per group -In ding both
sexes), and assessed a wide range of tissues and endpoinls. Although there are some gaps in the
toxicity database for tert-butanol, these areas are informed liy the (.lata on ETBE, a parent
compound of tert-butanol. Therefore, the confidence in the database is high. Reflecting high
confidence in the principal study, high confidence in the database, and minimal uncertainty
surrounding the application ol'lhc modified I'lil'K model lor the purposes of a route-to-route
extrapolation, the overall confidence in the RIC lor fivf-liulanol is high.
2.2.7. Previous IRIS Assessment
No prev ious inhalation assessment for fivf-liutanol 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 KID, the UF approach (U.S. EPA. 2000a. 1994) was applied to a POD
based on kidney toxicity in rats treated chronically. UFs were applied to the POD to account for
extrapolating from an animal hioassay 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 database 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 database for tert-butanol exposure includes one lifetime
bioassay, several reproductive/developmental studies, and several subchronic oral studies.
Although the database is adequate for reference value derivation, there is uncertainty
associated with the lack of a comprehensive multigeneration reproductive toxicity study.
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Additionally, only subchronic and short-term inhalation studies have been conducted, and no
chronic inhalation studies are available. Developmental studies identified significant increases in
fetal loss, decreases in fetal body weight, and possible increases in skeletal variations in exposed
offspring or pups. However, effects were not always consistent across exposure routes, 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 database 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, it was found that mis appear more susceptible than mice, and
males appear more susceptible than females to tert-buUinol toxicity. However, the underlying
mechanistic basis of these apparent differences is nol understood. Mosl importantly, it is unknown
which animal species and/or sexes may be more comparable to humans.
Another uncertainty to consider relates l<> Ihc MOA analysis conduced lor the kidney
effects. The assessment concluded that tert-butanol is a weak inducer of c^u-globulin which is
operative in male kidney tumors; therefore, noncancer (.¦Ifecls related to a2u-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 lo « u-glolmlin were relev ant to humans, then the RfD and RfC values
could be underestimating toxicity. Similarly, the renal effects characterized as CPN and dismissed as
not being treatment related, ilConsidered relev ant, would likewise contribute to the hazard
potential and dose-response analysis lor the kidney-specific RfD and RfC.
2.3. ORAL SLOPE FACTOR FOR CANCER
The oral slope factor (OSI") is a plausible upper bound on the estimate of risk per mg/kgday
of oral exposure. The OSI' can lie 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.'-5.2, that there is "suggestive evidence of carcinogenic potential" for
tert- butanol. The Guidelines for Carcinogen Risk Assessment fU.S. EPA. 2005al state:
When there is suggestive evidence, the Agency generally would not attempt a dose-
response assessment, as the nature of the data generally would not support one; however
when the evidence includes a well-conducted study, quantitative analysis may be useful for
some purposes, for example, providing a sense of the magnitude and uncertainty of
potential risks, ranking potential hazards, or setting research priorities.
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
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(NTP. 1995). 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 (Hard etal.. 20111.
Based on a mode of action analysis, it was concluded llv . a2U-globulin process was at
least partially responsible for the male ratrenal tumors, in • .on lo other, unknown, processes.
Because the relative contribution of each process to tuir n ¦. »n cannot be determined (U.S.
EPA. 1991a). the male ratrenal tumors are notcons1 Ten suitable quantitative analysis.
Conversely, the mouse thyroid tumors are suitah' dose-response ai vsis and unit risk
estimation, as described in Section 1.3.2.
2.3.2. Dose-Response Analysis—Adjustments and Extrapolations Methods
The EPA Guidelines for Carcinoijcn l\isl< Assessment (M.S. !¦ I'A. 2005a] recommend that the
method used to characterize and quanlilv cancer risk from a chemical be determined by what is
known about the MOAofthe carcinogen and I lie shape of the cancer dose-response curve. EPA
uses atwo-step approach that distinguishes analysis of the observed dose-response data from
inferences about lower doses ( M.S. EPA. 2005a ) illiin 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 ol ^'/7-liulanol, a standard model is used to curve-fit the data and estimate a POD. EPA uses
the multistage model in IRIS dose-response analyses for cancer fGehlhaus etal.. 20111 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 ol action for each effect As above, a biologically based model is preferred (U.S.
EPA. 2005a). Otherwise, linear low-dose extrapolation is recommended if the MOA of
carcinogenicity is m utagenic or has not been established (U.S. EPA. 2005a). For tert-butanol, the
mode(s) of carcinogenic action lor 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
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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 fU.S. EPA. 2011. 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/i power. Standard body weights of 0.025 kg
for mice and 70 kg for humans were used (U.S. EPA. 1988). The wing formula was used for the
conversion of oral BMDL to oral HED for mouse endpoints:
HED in mg/kgday- = (BMDL in mg/kc' i\ , x (aniii,, 'iody weight/70)1/4
= (BMDLii- Kgday-) x O.l-i
PODs for estimating low-dose risk were k 1 ilied atdoses atthe l|x V line li 'he lower 95% bound on the
exposure at the POD lc '-esponse ( >pe f - ""/BMuLbmr = 0.1/BMDLio). This
slope represents a pk 'ilc uppci ¦indonlh population verage risk. Using linear
extrapolation from the 11. 1.i... hni l ei|iiiviileh "ill slope factors were derived for male and
female mice - ' "¦ listed ii. '¦>'
T . <.11 sk j,. "<>rlxi.\ mlheiiK. '"oi .,iyroid follicular cell adenomas in female
mice u x l n i per mg/ lav. I)l 'e high moi ^ility in high-dose male mice, estimating slope
factors usi. he poly-3 meli. was fe. !e for addressing competing risks. Whether using the full
data set (inclu the only thy. I follicuu cell carcinoma observed at the highest dose) or
omitting the high- e group alh ther (under the assumption that mortality in this group was too
extensive to interpret ¦ result' ral slope factors based on the incidence of thyroid follicular cell
adenomas or carcinomas - .nice were similar when rounded to one significant digit—5 x 10 4
per mg/kg-day or 6 x lu 1 pti mg/kg-day, respectively.
The recommended slope factor for lifetime oral exposure to tert-butanol is
5 x KM 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
1400 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.
This document is a draft for review purposes only and does not constitute Agency policy.
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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)
i
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
aHED PODs were calculated using BW3/4scaling (U.S. EPA, 2011).
bHuman equivalent slope factor = 0.1/BMDLiohed; see Appendix C of the Supplemental Information for details of
modeling results.
cBecause the observed responses were <10'.'-.., a BMR of 5'.'-.. was used to represent the observed response range for
low-dose extrapolation; human equivalent slope factor = 0.05/BMDL
2.3.4. Uncertainties in the Derivation of the Oral Slope Factor
There is uncertainty'"'hen extrapolating data from animals to estimate potential cancer
risks to human popula1 uosure lo ^'/7-lniUinol.
Table 2-9 sui, ¦ ri/.cs sovl uncertainties thai could affect the oral slope factor. There are
no other chronic studies "plicali .esc findings or thai examined other animal models, no data
in humans U- r:,'macani. "¦ "lie ml or I ho specific tumors observed in the NTP
f 19951 h .iiy, an^ 'Hutu. 'e.g., MO,. i p purl alternative approaches for deriving the
oralsli 'actor.
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Toxicological Review of tert-Butyl Alcohol
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
a2n-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 (1998a),
U.S. EPA (1991a).
MOA data suggested that mouse thyroid
tumors were relevant to humans.
Quantitation of thyroid tumors in male mice
was impacted only slightly by high mortality
in the high-dose group, and 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 u2n-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 #r 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 '|* slope
factor (e.g., 3.5-fold 4, [scaling by
body weight] or T* 2-fold [scaling by
BW 2/3])
The 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 (1998a).
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, 1998a).
Statistical uncertainty at POD:
4/ oral slope factor 1.4-fold if BMD
used as the POD rather than BMDL
BMDL (preferred approach
for calculating slope factor)
Limited size of bioassay results in sampling
variability; lower bound is 95% 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
has not been made, an age-specific
adjustment factor is not applied.
2.3.5. Previous IRIS Assessment: Oml Slope Fiictor
No previous cancer assessment lor fivf-butanol is available in TRIS.
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
may be derived. Quantitative risk estimates may lie 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 tig/m : air breathed.
No chronic inhalation exposure studies to ^'/7-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 Q9951 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 Q9951 drinking
water study were not used for route-to-route extrapolation because enough information to
determine the relative contribution of a2u-globulin nephropathy and other processes to the overall
renal tumor response (U.S. EPA. 1991a) is not available. Alternatively, if kidney tumors were
considered acceptable for quantitation, then route-to-route extrapolation could be conducted to
calculate an IUR (see Appendix C in Supplemental Information).
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Toxicological Review of tert-Butyl Alcohol
1 2.4.1. Previous IRIS Assessment: Inhalation Unit Risk
2 An inhalation cancer assessment for tert-butanol was not previously available on IRIS.
3 2.5. APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS
4 As discussed in the Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to
5 Carcinogens (U.S. EPA. 2005b). either default or chemical-specific age-dependent adjustment
6 factors (ADAFs) are recommended to account for early-life exposure to carcinogens that act
7 through a mutagenic MOA. Because chemical-specific lifestage susceptibility data for cancer are not
8 available, and because the MOA for tert-butanol carcinogenicM .ot known (see Section 1.3.2),
9 application of ADAFs is not recommended.
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http://www.inchem.org/documents/ehc/ehc/ehc65.htm.
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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localization of accumulated "alpha"2u-globul: in .ivl-l rai. 'ring chronic exposure to
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Suzuki. M: Yamazaki. K: Kano. H: Aiso. S: Nagan- . ukushima. S. f201 ^ '<> carcinogenicity of
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This document is a draft for review purposes only and does not constitute Agency policy.
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assessment [El'A Report], (EPA/630/P-03/001F). Washington, DC: U.S. Environmental
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carcinogen-risk-assessment
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susceptibility from early-life exposure to carcinogens. (EPA/630/R-03/003F). Washington,
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This document is a draft for review purposes only and does not constitute Agency policy.
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environmental exposures to children. (EPA/600/R-05/093F). Washington, DC: U.S.
Environmental Protection Agency, Office of Research and Development, National Center for
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http://epa.gov/iris/process.htm.
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the default method in derivation of the r elerence dose. (El';. 'O/Rll/OOOl).
Washington, DC: U.S. Environmental Pro ' ion Agency, Risk Asses. nt Forum, Office of
the Science Advisor, http://www.epa.gov/. 'mibliCcil i' 's/interspec. extrapolation.htm.
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(EPA/100/R-12/001). Washingto ')C: M.. ironmen Protection Agency, Risk
Assessment Foru1' " //www.ep -uv/imI/i ' •; lions/ ichmarkdose.htm.
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in the Chemical Data Access Tool. Available online at
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globulin in F-344 rats. Toxicol Sci 62: 228-235. http://dx.doi.Org/10.1093/toxsci/62.2.228.
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DNA with MT]!F and TI!A in mice studied by accelerator mass Spectrometry. Environ
Toxicol 22: 630-635. http://dx.doi.org/10.1002/tox.20295.
This document is a draft for review purposes only and does not constitute Agency policy.
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