EPA/635/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
 
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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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 tert-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 OTHER THAN CANCER......................................................2-1
 

2.1.1. Identification of Studies and Effects for Dose-Response Analysis....................................2-1
 

2.1.2. Methods of Analysis ..........................................................................................................2-2
 

2.1.3. Derivation of Candidate Values.........................................................................................2-4
 

2.1.4. Derivation of Organ/System-Specific Reference Doses ....................................................2-8
 

2.1.5. Selection of the Overall Reference Dose...........................................................................2-8
 

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2.1.6. Confidence Statement.......................................................................................................2-9
 

2.1.7. Previous 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 Reference Dose and Reference 

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-1 

This document is a draft for review purposes only and does not constitute Agency policy.

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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 RfC for tert-butanol ............................................... ES-3
 

Table LS-1. Details of the search strategy employed for tert-butanol..................................................... LS-4
 

Table LS-2. Summary of additional search strategies for tert-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 tert-butanol..........................................1-1 

Table 1-2. Changes in kidney histopathology in animals following exposure to tert-butanol ................1-12 

Table 1-3. Changes in kidney tumors in animals following exposure to tert-butanol.............................1-15 

Table 1-4. Summary of data on the α2u-globulin process in male rats exposed to tert-butanol ............1-22 

Table 1-5. Proposed empirical criteria for attributing renal tumors to CPN ...........................................1-33 

Table 1-6. Evidence pertaining to thyroid effects in animals following oral exposure to tert

butanol...........................................................................................................................1-39 

Table 1-7. Evidence pertaining to developmental effects in animals following exposure to tert

butanol...........................................................................................................................1-48 

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Table 1-8. Evidence pertaining to neurodevelopmental effects in animals following exposure to 

tert-butanol....................................................................................................................1-54 

Table 1-9. Evidence pertaining to reproductive effects in animals following exposure to tert

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 tert-butanol ..................................................2-8 

Table 2-4. Summary of derivation of PODs following inhalation exposure.............................................2-12 

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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 RfC for tert-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 tert-butanol ...........2-22 

FIGURES 

Figure LS-1. Summary of literature search and screening process for tert-butanol. .............................. LS-3
 

Figure 1-1. Biotransformation of tert-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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Figure 1-7. Temporal pathogenesis of α2u-globulin-associated nephropathy in male rats. α2u

Globulin synthesized in the livers of male rats is delivered to the kidney, where 

it can accumulate in hyaline droplets and be retained by epithelial cells lining 

the S2 (P2) segment of the proximal tubules. Renal pathogenesis following 

continued tert-butanol (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 

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Figure 1-8. Exposure-response array for effects potentially associated with α2u -globulin renal 

tubule nephropathy and tumors in male rats after oral exposure to tert-butanol.......1-24 

Figure 1-9. Exposure-response array for effects potentially associated with α2u -globulin renal 

tubule nephropathy and tumors in male rats after inhalation exposure to 

tert-butanol....................................................................................................................1-25 

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.) ................................................................................................1-41 

Figure 1-11. Exposure-response array of developmental effects following oral exposure to tert

butanol...........................................................................................................................1-51 

Figure 1-12. Exposure-response array of developmental effects following inhalation exposure to 

tert-butanol....................................................................................................................1-52 

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Figure 1-13. Exposure-response array of reproductive effects following oral exposure to tert

butanol...........................................................................................................................1-59 

Figure 1-14. Exposure-response array of reproductive effects following inhalation exposure to 

tert-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
 

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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 tert-butanol. .........1-17
 

Figure 1-6. Exposure-response array of kidney effects following inhalation exposure to tertbutanol (13-week studies, no chronic studies available). .............................................1-18 

This document is a draft for review purposes only and does not constitute Agency policy.

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QSAR quantitative structure-activity

relationship 

ABBREVIATIONS 

AIC Akaike’s information criterion 

ALD approximate lethal dosage 

ALT alanine aminotransferase 

AST	 aspartate aminotransferase 

atm	 atmosphere 

ATSDR	 Agency for Toxic Substances and 

Disease Registry 

BMD benchmark dose 

BMDL	 benchmark dose lower confidence limit

BMDS Benchmark Dose Software 

BMR	 benchmark response 

BW	 body weight 

CA	 chromosomal aberration 

CASRN	 Chemical Abstracts Service Registry

Number 

CBI covalent binding index 

CHO	 Chinese hamster ovary (cell line) 

CL	 confidence limit 

CNS central nervous system 

CPN	 chronic progressive nephropathy 

CYP450	 cytochrome P450 

DAF	 dosimetric adjustment factor 

DEN	 diethylnitrosamine 

DMSO dimethylsulfoxide 

DNA deoxyribonucleic acid 

EPA Environmental Protection Agency 

FDA Food and Drug Administration 

FEV1 forced expiratory volume of 1 second 

GD gestation day 

GDH glutamate dehydrogenase 

GGT γ-glutamyl transferase 

GSH glutathione 

GST glutathione-S-transferase 

Hb/g-A animal blood:gas partition coefficient 

Hb/g-H human blood:gas partition coefficient 

HEC human equivalent concentration 

HED human equivalent dose 

i.p. intraperitoneal 

IRIS Integrated Risk Information System 

IVF in vitro fertilization 

LC50 median lethal concentration 

LD50 median lethal dose 

LOAEL lowest-observed-adverse-effect level 

MN micronuclei 

MNPCE micronucleated polychromatic

erythrocyte 

MTD maximum tolerated dose 

NAG N-acetyl-β-D-glucosaminidase 

NCEA National Center for Environmental 

Assessment 

NCI National Cancer Institute 

NOAEL no-observed-adverse-effect level 

NTP National Toxicology Program 

NZW New Zealand White (rabbit breed) 

OCT ornithine carbamoyl transferase 

ORD Office of Research and Development 

PBPK physiologically based pharmacokinetic 

POD point of departure 

POD[ADJ] duration-adjusted POD 

Toxicological Review of tert-Butyl Alcohol 

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RDS replicative DNA synthesis 

RfC inhalation reference concentration 

RfD oral reference dose 

RGDR regional gas dose ratio 

RNA ribonucleic acid 

SAR structure activity relationship 

SCE sister chromatid exchange 

SD standard deviation 

SDH sorbitol dehydrogenase 

SE standard error 

SGOT glutamic oxaloacetic transaminase, also 

known as AST 

SGPT glutamic pyruvic transaminase, also 

known as ALT 

SSD systemic scleroderma 

TCA trichloroacetic acid 

TCE trichloroethylene 

TWA time-weighted average 

UF uncertainty factor 

UFA animal-to-human uncertainty factor 

UFH human variation uncertainty factor 

UFL LOAEL-to-NOAEL uncertain factor 

UFS subchronic-to-chronic uncertainty

factor 

UFD database deficiencies uncertainty factor 

U.S. United States 
 

2 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 

Toxicological Review of tert-Butyl Alcohol 

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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)
 

Internal Review Team 

General Toxicology Workgroup

Inhalation Workgroup

Neurotoxicity Workgroup

Pharmacokinetics Workgroup

Reproductive and Developmental 

Toxicology Workgroup

Statistical Workgroup

Toxicity Pathways Workgroup

Executive Review Committee 

U.S. EPA 

Office of Research and Development

National Center for Environmental Assessment 

Washington, DC

Research Triangle Park, NC

Cincinnati, OH 

Reviewers 

This assessment was provided for review to scientists in EPA’s Program and Region Offices. 

Comments were submitted by: 

Office of the Administrator/Office of Children’s Health Protection 

Office of Land and Emergency Management

Region 2, New York, NY

Region 8, Denver, CO
 

This assessment was provided for review to other federal agencies and the Executive Office of the 

President. Comments were submitted by: 

Department of Health and Human Services/Agency for Toxic Substances and Disease Registry,

Department of Health and Human Services/National Institute of Environmental Health

Sciences/National Toxicology Program,

Executive Office of the President/Office of Management and Budget,

Executive Office of the President/Office of Science and Technology Policy 

This document is a draft for review purposes only and does not constitute Agency policy.

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Toxicological Review 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. 

A public meeting was held in December 2013 to obtain input on preliminary materials for 

• 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 α2u-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 et al., 2015). 

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 

(http://www.epa.gov/iris). Appendices for toxicokinetic information, PBPK modeling, genotoxicity 

study summaries, dose-response modeling, and other information are provided as Supplemental 

Information to this Toxicological Review. For additional information about this assessment or for 

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 (U.S. EPA, 2014). 

tert-Butanol has been used as a fuel oxygenate, an octane booster in unleaded gasoline, and 

a denaturant for ethanol. From 1997 to 2005, the annual tert-butanol volume found in gasoline 

ranged from approximately 4 million to 6 million gallons. During that time, larger quantities were 

used to make methyl tert-butyl ether (MTBE) and ETBE. MTBE and ETBE are fuel oxygenates that 

­

were used in the United States prior to 2007 at levels of more than 2 billion gallons annually. 

Current use levels of MTBE and ETBE in the United States are much lower, but use in Europe and 

Asia remains strong.1 

tert-Butanol has been used for a variety of other purposes, including as a dehydrating agent 

and solvent. As such, it is added to lacquers, paint removers, and nail enamels and polishes. 

tert-Butanol also is used to manufacture methyl methacrylate plastics and flotation devices. 

Cosmetic and food-related uses include the manufacture of flavors, and, because of its camphor-like 

aroma, it also is used to create artificial musk, fruit essences, and perfume (HSDB, 2007). It is used 

in coatings on metal and paperboard food containers (Cal/EPA, 1999) and industrial cleaning 

compounds, and can be used for chemical extraction in pharmaceutical applications (HSDB, 2007). 

Fate and Transport 

Soil 

tert-Butanol is expected to be highly mobile in soil due to its low affinity for soil organic 

matter. Rainwater or other water percolating through soil is expected to dissolve and transport 

most tert-butanol present in soil, potentially leading to groundwater contamination. Based on its 

vapor pressure, tert-butanol’s volatilization from soil surfaces is expected to be an important 

dissipation process (HSDB, 2007). As a tertiary alcohol, tert-butanol is expected to degrade more 

slowly in the environment compared to primary (e.g., ethanol) or secondary (e.g., isopropanol) 

alcohols. In anoxic soil conditions, the half-life of tert-butanol is estimated to be months 

(approximately 200 days). Microbial degradation rates are increased in soils supplemented with 

nitrate and sulfate nutrients (HSDB, 2007). 

1 http://www.ihs.com/products/chemical/planning/ceh/gasoline-octane-improvers.aspx. 

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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 (HSDB, 2007). Biodegradation in 

aerobic water occurs over weeks to months and in anaerobic aquatic conditions, the biodegradation 

rate decreases. Bioconcentration of tert-butanol in aquatic organisms is low (HSDB, 2007). 

Air
 

tert-Butanol primarily exists as a vapor in the ambient atmosphere. Vapor-phase tert

butanol is degraded in the atmosphere by reacting with photochemically produced hydroxyl 

radicals with a half-life of 14 days (HSDB, 2007). 

Occurrence in the Environment 

The Toxics Release Inventory (TRI) Program National Analysis Report estimated that more 

than 1 million pounds of tert-butanol has been released into the soil from landfills, land treatment, 

underground injection, surface impoundments, and other land disposal sources. The TRI program 

also estimated that 476,266 pounds of tert-butanol was released into the atmosphere from fugitive 

emissions and point sources (U.S. EPA, 2012c). In California, air emissions of tert-butanol from 

stationary sources are estimated to be at least 27,000 pounds per year, based on data reported by 

the state’s Air Toxics Program (Scorecard, 2014). The TRI National Analysis Report estimated 7,469 

pounds of tert-butanol was released into surface waters from point and nonpoint sources in 2011 

(U.S. EPA, 2012c). 

tert-Butanol has been identified in drinking water wells throughout the United States 

(HSDB, 2007). California’s Geotracker Database2 lists 3,496 detections of tert-butanol in 

groundwater associated with contaminated sites in that state since 2011. tert-Butanol also has been 

detected in drinking water wells in the vicinity of landfills (U.S. EPA, 2012c). Additionally, tertButanol leaking from underground storage tanks could be a product of MTBE and ETBE, which can 

degrade to form tert-butanol in soils (HSDB, 2007). The industrial chemical tert-butyl acetate also 

can degrade to form tert-butanol in animals post exposure and in the environment. 

Ambient outdoor air concentrations of tert-butanol vary according to proximity to urban 

areas (HSDB, 2007). 

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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1 Ingestion of contaminated food can be a source of tert-butanol exposure through its use as a 

coating in metallic and paperboard food containers (Cal/EPA, 1999), and tert-butanol has been 

detected in food (HSDB, 2007). Internal exposure to tert-butanol also can occur as a result of 

ingestion of MTBE or ETBE, as tert-butanol is a metabolite of these compounds (NSF International, 

2003). 

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 viable route of exposure through 

handling consumer products containing tert-butanol (NSF International, 2003). 

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Assessments by Other National and International Health Agencies 

Toxicity information on tert-butanol has been evaluated by the National Institute for 

Occupational Safety and Health (NIOSH, 2007), the Occupational Safety and Health Administration 

(OSHA, 2006), and the Food and Drug Administration (FDA, 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. 

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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, it was at the forefront of developing risk

assessment as a science and applying it in

decisions to protect human health and the

environment. The Clean Air Act, for example,

mandates that the EPA provide “an ample

margin of safety to protect public health”; the

Safe Drinking Water Act, that “no adverse 

effects on the health of persons may

reasonably be anticipated to occur, allowing

an adequate margin of safety.” Accordingly,

the EPA uses information on the adverse 

effects of chemicals and on exposure levels

below which these effects are not anticipated

to occur. 

IRIS assessments critically review the 

publicly available studies to identify adverse

health effects from exposure to chemicals and

to characterize exposure-response 

relationships. In terms set forth by the 

National Research Council (NRC, 1983), IRIS

assessments cover the hazard identification 

and dose-response assessment steps of risk

assessment, not the exposure assessment or

risk characterization steps that are conducted

by the EPA’s program and regional offices and 

by other federal, state, and local health 

agencies that evaluate risk in specific

populations and exposure scenarios. IRIS 

assessments are distinct from and do not 

address political, economic, and technical 

considerations that influence the design and

selection of risk management alternatives. 

An IRIS assessment may cover a single

chemical, a group of structurally or

toxicologically related chemicals, or a complex

mixture. These agents may be found in air,

water, soil, or sediment. Exceptions are 

chemicals currently used exclusively as

pesticides, ionizing and non-ionizing 

radiation, and criteria air pollutants listed 

under Section 108 of the Clean Air Act (carbon

monoxide, lead, nitrogen oxides, ozone,

particulate matter, and sulfur oxides).

Periodically, the IRIS Program asks other

EPA programs and regions, other federal 

agencies, state health agencies, and the

general public to nominate chemicals and 

mixtures for future assessment or

reassessment. Agents may be considered for

reassessment as significant new studies are

published. Selection is based on program and

regional office priorities and on availability of

adequate information to evaluate the potential

for adverse effects. Other agents may also be

assessed in response to an urgent public 

health need. 

2. Process for developing and peerreviewing IRIS assessments 

The process for developing IRIS 

assessments (revised in May 2009 and 

enhanced in July 2013) involves critical 

analysis of the pertinent studies, opportunities

for public input, and multiple levels of 

scientific review. The EPA revises draft 

assessments after each review, and external

drafts and comments become part of the 

public record (U.S. EPA, 2009).

Before beginning an assessment, the IRIS

program discusses the scope with other EPA 

programs and regions to ensure that the 

This document is a draft for review purp oses on ly and does not constitute Agency policy. 

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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. The draft

assessment is revised to address the 

comments from within the EPA. 

Step 3. Interagency science consultation 

with other federal agencies and the 

Executive Offices of the President. The 

draft assessment is revised to address the 

interagency comments. The science

consultation draft, interagency comments,

and the EPA’s response to major

comments become part of the public 

record. 

Step 4. Public review and comment, 

followed by external peer review. The 

EPA releases the draft assessment for 

public review and comment. A public 

meeting provides an opportunity to 

discuss the assessment prior to peer

review. Then the EPA releases a draft for 

external peer review. The peer review

meeting is open to the public and includes

time for oral public comments. The peer

reviewers assess whether the evidence 

has been assembled and evaluated 

according to guidelines and whether the

conclusions are justified by the evidence.

The peer review draft, written public 

comments, and peer review report

become part of the public record. 

Step 5. Revision of draft Toxicological 

Review and development of draft IRIS 

summary. The draft assessment is revised 

to reflect the peer review comments, 

public comments, and newly published

studies that are critical to the conclusions 

of the assessment. The disposition of peer

review comments and public comments 

becomes part of the public record. 

Step 6. Final EPA review and interagency 

science discussion with other federal 

agencies and the Executive Offices of 

the President The draft assessment and 

summary are revised to address the EPA

and interagency comments. The science

discussion draft, written interagency 

comments, and EPA’s response to major 

comments become part of the public 

record. 

Step 7. Completion and posting. The 

Toxicological Review and IRIS summary 

are posted on the IRIS website 

(http://www.epa.gov/iris/).

The remainder of this Preamble addresses step 1, 

the development of a draft Toxicological 

Review. IRIS assessments follow standard 

practices of evidence evaluation and peer

review, many of which are discussed in

EPA guidelines (U.S. EPA,

2005a, b, 2000b, 1998b, 1996, 1991b, 198 

6a, b) and other methods (U.S. EPA, 

2012a, b, 2011, 2006a, b, 2002, 1994).

Transparent application of scientific 

judgment is of paramount importance. To

provide a harmonized approach across

IRIS assessments, this Preamble 

summarizes concepts from these 

guidelines and emphasizes principles of

general applicability. 

3. Identifying and selecting 

pertinent studies 

3.1. Identifying studies 

Before beginning an assessment, the EPA

conducts a comprehensive search of the 

primary scientific literature. The literature 

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Toxicological Review of tert-Butyl Alcohol 

1 search follows standard practices and includes 

the PubMed and ToxNet databases of the 

National Library of Medicine, Web of Science,

and other databases listed in the EPA’s HERO 

system (Health and Environmental Research

Online, http://hero.epa.gov/). Searches for 

information on mechanisms of toxicity are

inherently specialized and may include

studies on other agents that act through 

related mechanisms. 

Each assessment specifies the search 

strategies, keywords, and cut-off dates of its

literature searches. The EPA posts the results 

of the literature search on the IRIS web site 

and requests information from the public on

additional studies and ongoing research.

The EPA also considers studies received 

through the IRIS Submission Desk and studies

(typically unpublished) submitted under the

Toxic Substances Control Act or the Federal 

Insecticide, Fungicide, and Rodenticide Act.

Material submitted as Confidential Business 

Information is considered only if it includes

health and safety data that can be publicly

released. If a study that may be critical to the

conclusions of the assessment has not been 

peer-reviewed, the EPA will have it peer

reviewed. 

The EPA also examines the toxicokinetics 

of the agent to identify other chemicals (for

example, major metabolites of the agent) 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.

48 

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 (U.S. EPA, 

2000b, §2.2; 1986b, §2.1): 

– Studies of the mixture being assessed. 

– Studies of a sufficiently similar

mixture. In evaluating similarity, the

assessment considers the alteration of 

mixtures in the environment through

partitioning and transformation. 

– Studies of individual chemical 

components of the mixture, if there are

not adequate studies of sufficiently 

similar mixtures. 

3.2. Selecting pertinent epidemiologic 

studies 

Study design is the key consideration for

selecting pertinent epidemiologic studies from

the results of the literature search. 

– Cohort studies, case-control studies, 

and some population-based surveys

(for example, NHANES) provide the

strongest epidemiologic evidence,

especially if they collect information

about individual exposures and 

effects. 

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correlation studies) relate exposures

and effects by geographic area. They

can provide strong evidence if there

are large exposure contrasts between

geographic areas, relatively little 

exposure variation within study areas,

and population migration is limited. 

– Case reports of high or accidental 

exposure lack definition of the 

population at risk and the expected

number of cases. They can provide

information about a rare effect or 

about the relevance of analogous

results in animals. 

The assessment briefly reviews ecological

studies and case reports but reports details

only if they suggest effects not identified by 

other studies. 

3.3. Selecting pertinent experimental 

studies 

Exposure route is a key design 

consideration for selecting pertinent

experimental animal studies or human clinical

studies. 

– Studies of oral, inhalation, or dermal

exposure involve passage through an

absorption barrier and are considered 

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xvi DRAFT—DO NOT CITE OR QUOTE 
 

1 most pertinent to human

environmental exposure. 

– Injection or implantation studies are

often considered less pertinent but

may provide valuable toxicokinetic or

mechanistic information. They also 

may be useful for identifying effects in

animals if deposition or absorption is

problematic (for example, for particles

and fibers). 

Exposure duration is also a key design

consideration for selecting pertinent

experimental animal studies. 

– Studies of effects from chronic

exposure are most pertinent to

lifetime human exposure. 

– Studies of effects from less-than

chronic exposure are pertinent but

less preferred for identifying effects

from lifetime human exposure. Such

studies may be indicative of effects

from less-than-lifetime human

exposure. 

Short-duration studies involving animals 

or humans may provide toxicokinetic or 

mechanistic information.
 

For developmental toxicity and

reproductive toxicity, irreversible effects may

result from a brief exposure during a critical

period of development. Accordingly,

specialized study designs are used for these

effects (U.S. EPA, 2006b, 1998b, 1996, 1991b). 

45 

4. Evaluating the quality of 

individual studies 

After the subsets of 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 

quality characteristics across studies of

similar design. 

4.1. Evaluating the quality of 

epidemiologic studies 

The assessment evaluates design and 

methodological aspects that can increase or

decrease the weight given to each

epidemiologic study in the overall evaluation

(U.S. EPA, 2005a, 1998b, 1996, 1994, 1991b): 

– Documentation of study design,

methods, population characteristics, 

and results. 

– Definition and selection of the study

group and comparison group. 

– Ascertainment of exposure to the 

chemical or mixture. 

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– Ascertainment of disease or health 

effect. 

– Duration of exposure and follow-up 

and adequacy for assessing the 

occurrence of effects. 

– Characterization of exposure during

critical periods.
 

– Sample size and statistical power to

detect anticipated effects. 

– Participation rates and potential for

selection bias as a result of the 

achieved participation rates. 

– Measurement error (can lead to 

misclassification of exposure, health

outcomes, and other factors) and other

types of information bias. 

– Potential confounding and other 

sources of bias addressed in the study

design or in the analysis of results. The

basis for consideration of confounding

is a reasonable expectation that the

confounder is related to both exposure

and outcome and is sufficiently 

prevalent to result in bias. 

For developmental toxicity, reproductive

toxicity, neurotoxicity, and cancer there is 

further guidance on the nuances of evaluating 

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1 epidemiologic studies of these effects (U.S. 

EPA, 2005a, 1998b, 1996, 1991b). 

4.2. Evaluating the quality of
 

experimental studies
 

The assessment evaluates design and 

methodological aspects that can increase or

decrease the weight given to each

experimental animal study, in-vitro study, or

human clinical study (U.S. EPA,

2005a, 1998b, 1996, 1991b). Research 

involving human subjects is considered only if

conducted according to ethical principles. 

– Documentation of study design,

animals or study population, methods,

basic data, and results. 

– Nature of the assay and validity for its

intended purpose. 

– Characterization of the nature and 

extent of impurities and contaminants

of the administered chemical or

mixture. 

– Characterization of dose and dosing

regimen (including age at exposure)

and their adequacy to elicit adverse

effects, including latent effects. 

– Sample sizes and statistical power to

detect dose-related differences or

trends. 

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disease or effects, and cause of death. 

– Control of other variables that could 

influence the occurrence of effects. 

The assessment uses statistical tests to 

evaluate whether the observations may be due

to chance. The standard for determining

statistical significance of a response is a trend

test or comparison of outcomes in the exposed

groups against those of concurrent controls. In 

some situations, examination of historical 

control data from the same laboratory within

a few years of the study may improve the

analysis. For an uncommon effect that is not

statistically significant compared with

concurrent controls, historical controls may 

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chance. For a response that appears significant

against a concurrent control response that is

unusual, historical controls may offer a 

different interpretation (U.S. EPA,

2005a, §2.2.2.1.3).

For developmental toxicity, reproductive

toxicity, neurotoxicity, and cancer there is 

further guidance on the nuances of evaluating

experimental studies of these effects (U.S. EPA, 

2005a, 1998b, 1996, 1991b). In multi

generation studies, agents that produce

developmental effects at doses that are not

toxic to the maternal animal are of special

concern. Effects that occur at doses associated 

with mild maternal toxicity are not assumed to 

result only from maternal toxicity. Moreover, 

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This document is a draft for review purposes only and does not constitute Agency policy.

xviii DRAFT—DO NOT CITE OR QUOTE 

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maternal effects may be reversible, while

effects on the offspring may be permanent

(U.S. EPA, 1998b, §3.1.2.4.5.4; 1991b, 

§3.1.1.4),. 

4.3. Reporting study results 

The assessment uses evidence tables to 

present the design and key results of pertinent

studies. There may be separate tables for each

site of toxicity or type of study.

If a large number of studies observe the 

same effect, the assessment considers the 

study quality characteristics in this section to

identify the strongest studies or types of study.

The tables present details from these studies,

and the assessment explains the reasons for

not reporting details of other studies or 

groups of studies that do not add new 

information. Supplemental information 

provides references to all studies considered,

including those not summarized in the tables.

The assessment discusses strengths and

limitations that affect the interpretation of

each study. If the interpretation of a study in

the assessment differs from that of the study

authors, the assessment discusses the basis for

the difference. 

As a check on the selection and evaluation 

of pertinent studies, the EPA asks peer

reviewers to identify studies that were not

adequately considered. 
 

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Toxicological Review of tert-Butyl Alcohol 

48 1 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 (CDC, 

2004; HEW, 1964), the International Agency 

for Research on Cancer (IARC, 2006), the 

Institute of Medicine (IOM, 2008), and the EPA 

(2010, §1.6; 2005a, §2.5). Although developed

for different purposes, the frameworks are

similar in nature and provide an established

structure and language for causal inference.

Each considers aspects of an association that

suggest causation, discussed by Hill (Hill, 

1965) and elaborated on by Rothman and

Greenland (Rothman and Greenland, 1998), 

and U.S. EPA (2005a, §2.2.1.7; 1994, Appendix 

C). 

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Strength of association: The finding of a large 

relative risk with narrow confidence 

intervals strongly suggests that an 

association is not due to chance, bias, or

other factors. Modest relative risks,

however, may reflect a small range of

exposures, an agent of low potency, an

increase in an effect that is common, 

exposure misclassification, or other 

sources of bias. 

Consistency of association: An inference of 

causation is strengthened if elevated risks

are observed in independent studies of

different populations and exposure 

scenarios. Reproducibility of findings

constitutes one of the strongest arguments 

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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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xix DRAFT—DO NOT CITE OR QUOTE 
 

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1 Analogy: Information on structural analogues 

2 or on chemicals that induce similar 

3 mechanistic events can provide insight

4 into causation. 

These considerations are consistent with 

6 guidelines for systematic reviews that 

7 evaluate the quality and weight of evidence.

8 Confidence is increased if the magnitude of

9 effect is large, if there is evidence of an 

exposure-response relationship, or if an 

11 association was observed and the plausible

12 biases would tend to decrease the magnitude

13 of the reported effect. Confidence is decreased

14 for study limitations, inconsistency of results,

indirectness of evidence, imprecision, or 

16 reporting bias (Guyatt et al., 2008b; Guyatt et 

17 al., 2008a). 

18 5.2. Evaluating evidence in humans 

19 For each effect, the assessment evaluates

the evidence from the epidemiologic studies as

21 a whole. The objective is to determine whether

22 a credible association has been observed and,

23 if so, whether that association is consistent

24 with causation. In doing this, the assessment

explores alternative explanations (such as 

26 chance, bias, and confounding) and draws a

27 conclusion about whether these alternatives 

28 can satisfactorily explain any observed 

29 association. 

To make clear how much the 

31 epidemiologic evidence contributes to the 

32 overall weight of the evidence, the assessment 

33 may select a standard descriptor to 

34 characterize the epidemiologic evidence of 

association between exposure to the agent and

36 occurrence of a health effect. 

37 Sufficient epidemiologic evidence of an 

38 association consistent with causation: 

39 The evidence establishes a causal 

association for which alternative 

41 explanations such as chance, bias, and 

42 confounding can be ruled out with 

43 reasonable confidence. 

44 Suggestive epidemiologic evidence of an 

association consistent with causation: 

46 The evidence suggests a causal association 

Toxicological Review of tert-Butyl Alcohol 

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 covering 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 finding 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 Hill (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 

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differences (for example, relative sensitivity of 

the tests, differences in dose levels,

insufficient sample size, or timing of dosing or

data collection).

It is well established that there are critical 

periods for some developmental and

reproductive effects (U.S. EPA,

2006b, 2005a, b, 1998b, 1996, 1991b). 

Accordingly, the assessment determines

whether critical periods have been adequately

investigated. Similarly, the assessment 

determines whether the database is adequate

to evaluate other critical sites and effects. 

In evaluating evidence of genetic toxicity: 

– Demonstration of gene mutations, 

chromosome aberrations, or

aneuploidy in humans or experimental

mammals (in vivo) provides the

strongest evidence. 

– This is followed by positive results in

lower organisms or in cultured cells

(in vitro) or for other genetic events. 

– Negative results carry less weight,

partly because they cannot exclude the

possibility of effects in other tissues

(IARC, 2006). 

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For germ-cell mutagenicity, The EPA has

defined categories of evidence, ranging from

positive results of human germ-cell

mutagenicity to negative results for all effects

of concern (U.S. EPA, 1986a, §2.3). 

5.4. Evaluating mechanistic data 

Mechanistic data can be useful in 

answering several questions. 

– The biologic plausibility of a causal

interpretation of human studies. 

– The generalizability of animal studies

to humans. 

– The susceptibility of particular

populations or lifestages. 

The focus of the analysis is to describe, if 

possible, mechanistic pathways that lead to a 

health effect. These pathways encompass: 

– Toxicokinetic processes of absorption, 

distribution, metabolism, and 

elimination that lead to the formation 

of an active agent and its presence at

the site of initial biologic interaction. 

– 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 

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may also come from studies of metabolites or

of compounds that are structurally similar or

that act through similar mechanisms. 

Information on mode of action is not required

for a conclusion that the agent is causally

related to an effect (U.S. EPA, 2005a, §2.5). 

The assessment addresses several 

questions about each hypothesized mode of

action (U.S. EPA, 2005a, §2.4.3.4). 

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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1 for certain effects or modes of action. 

Information suggesting quantitative

differences in doses where effects would 

occur in animals or humans is considered 

in the dose-response analysis. Current 

levels of human exposure are not used to

rule out human relevance, as IRIS 

assessments may be used in evaluating 

new or unforeseen circumstances that 

may entail higher exposures. 

3) Which populations or lifestages can be 

particularly susceptible to the

hypothesized mode of action? The 

assessment reviews the key events to 

identify populations and lifestages that

might be susceptible to their occurrence.

Quantitative differences may result in 

separate toxicity values for susceptible 

populations or lifestages. 

The assessment discusses the likelihood 

that an agent operates through multiple

modes of action. An uneven level of support

for different modes of action can reflect 

disproportionate resources spent 

investigating them (U.S. EPA, 2005a, §2.4.3.3). 

It should be noted that in clinical reviews, the

credibility of a series of studies is reduced if

evidence is limited to studies funded by one

interested sector (Guyatt et al., 2008a). 

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For cancer, the assessment evaluates 

evidence of a mutagenic mode of action to

guide extrapolation to lower doses and 

consideration of susceptible lifestages. Key

data include the ability of the agent or a

metabolite to react with or bind to DNA,

positive results in multiple test systems, or

similar properties and structure-activity

relationships to mutagenic carcinogens (U.S. 

EPA, 2005a ,§2.3.5). 

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? (NRC, 

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 (U.S. EPA, 2005a, 

§2.5). 

Carcinogenic to humans: There is convincing 

epidemiologic evidence of a causal 

association (that is, there is reasonable

confidence that the association cannot be 

fully explained by chance, bias, or 

confounding); or there is strong human

evidence of cancer or its precursors,

extensive animal evidence, identification

of key precursor events in animals, and

strong evidence that they are anticipated

to occur in humans. 

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Likely to be carcinogenic to humans: The 

evidence demonstrates a potential hazard

to humans but does not meet the criteria 

for carcinogenic. There may be a plausible

association in humans, multiple positive

results in animals, or a combination of

human, animal, or other experimental 

evidence. 

Suggestive evidence of carcinogenic 

potential: The evidence raises concern for 

effects in humans but is not sufficient for a 

stronger conclusion. This descriptor

covers a range of evidence, from a positive

result in the only available study to a single

positive result in an extensive database

that includes negative results in other 

species. 

Inadequate information to assess 

carcinogenic potential: No other 

descriptors apply. Conflicting evidence can 

be classified as inadequate information if 

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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1 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 (U.S. EPA, 2005a, 

§2.5).

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

(U.S. EPA, 2010, §1.6). 

Causal relationship: Sufficient evidence to 

conclude that there is a causal

relationship. Observational studies cannot

be explained by plausible alternatives, or

they are supported by other lines of 

evidence, for example, animal studies or

mechanistic information. 

Likely to be a causal relationship: Sufficient 

evidence that a causal relationship is

likely, but important uncertainties remain.

For example, observational studies show 

an association but co-exposures are

difficult to address or other lines of 

evidence are limited or inconsistent; or

multiple animal studies from different 

laboratories demonstrate effects and

there are limited or no human data. 

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Suggestive of a causal relationship: At least 

one	 high-quality epidemiologic study

shows an association but other studies are 

inconsistent.	 

Inadequate to infer a causal relationship:

The studies do not permit a conclusion

regarding the presence or absence of an

association. 

Not likely to be a causal relationship: Several 

adequate studies, covering the full range of 

human exposure and considering

susceptible populations, are mutually 

consistent in not showing an effect at any 

level of exposure. 

The EPA is investigating and may on a trial

basis use these or other standard descriptors

to characterize the overall weight of the 

evidence for effects other than cancer. 

6. Selecting studies for derivation of 

toxicity values 

For each effect where there is credible 

evidence of an association with the agent, the

assessment derives toxicity values if there are

suitable epidemiologic or experimental data.

The decision to derive toxicity values may be

linked to the hazard descriptor. 

Dose-response analysis requires quantitative 

measures of dose and response. Then, other 

factors being equal: 

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– Epidemiologic studies are preferred 

over animal studies, if quantitative

measures of exposure are available

and effects can be attributed to the 

agent. 

– Among experimental animal models,

those that respond most like humans

are preferred, if the comparability of

response can be determined. 

– Studies by a route of human 

environmental exposure are

preferred, although a validated 

toxicokinetic model can be used to 

extrapolate across exposure routes. 

– Studies of longer exposure duration

and follow-up are preferred, to 

minimize uncertainty about whether

effects are representative of lifetime 

exposure. 

– Studies with multiple exposure levels 

are preferred for their ability to 

provide information about the shape

of the exposure-response curve. 

– Studies with adequate power to detect

effects at lower exposure levels are

preferred, to minimize the extent of 

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1 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 doseresponse analysis 

The EPA uses a two-step approach that

distinguishes analysis of the observed doseresponse data from inferences about lower

doses (U.S. EPA, 2005a, §3).

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Within the observed range, the preferred

approach is to use modeling to incorporate a

wide range of data into the analysis. The 

modeling yields a point of departure (an 

exposure level near the lower end of the 

observed range, without significant

extrapolation to lower doses) (Sections 7.2-

7.3).

Extrapolation to lower doses considers

what is known about the modes of action for 

each effect (Sections 7.4-7.5). If response

estimates at lower doses are not required, an

alternative is to derive reference values, which

are calculated by applying factors to the point

of departure in order to account for sources of

uncertainty and variability (Section 7.6).

For a group of agents that induce an effect

through a common mode of action, the doseresponse analysis may derive a relative 

potency factor for each agent. A full dose

response analysis is conducted for one well

studied index chemical in the group, then the

potencies of other members are expressed in

relative terms based on relative toxic effects,

relative absorption or metabolic rates, 

quantitative structure-activity relationships,

or receptor binding characteristics (U.S. EPA, 

2005a, §3.2.6; 2000b, §4.4).

Increasingly, the EPA is basing toxicity

values on combined analyses of multiple data

sets or multiple responses. The EPA also 

considers multiple dose-response approaches

if they can be supported by robust data. 

7.2. Modeling dose to sites of biologic 

effects 

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The preferred approach for analysis of

dose is toxicokinetic modeling because of its

ability to incorporate a wide range of data. The

preferred dose metric would refer to the 

active agent at the site of its biologic effect or

to a close, reliable surrogate measure. The

active agent may be the administered chemical

or a metabolite. Confidence in the use of a 

toxicokinetic model depends on the 

robustness of its validation process and on the

results of sensitivity analyses (U.S. EPA, 

2006a; 2005a, §3.1; 1994, §4.3). 

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, §3.2).

– Doses are standardized to equivalent

human terms to facilitate comparison

of results from different species. 

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1 – 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, §3.1.3). 

– Inhalation exposures are scaled using

dosimetry models that apply species

specific physiologic and anatomic 

factors and consider whether the 

effect occurs at the site of first contact 

or after systemic circulation (U.S. EPA, 

2012a; 1994, §3). 

It can be informative to convert doses 

across exposure routes. If this is done, the 

assessment describes the underlying data, 

algorithms, and assumptions (U.S. EPA, 

2005a, §3.1.4).

In the absence of study-specific data on,

for example, intake rates or body weight, the

EPA has developed recommended values for 

use in dose-response analysis (U.S. EPA, 

1988). 

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7.3. Modeling response in the range of 

observation 

Toxicodynamic (“biologically based”)

modeling can incorporate data on biologic

processes leading to an effect. Such models

require sufficient data to ascertain a mode of

action and to quantitatively support model

parameters associated with its key events.

Because different models may provide 

equivalent fits to the observed data but 

diverge substantially at lower doses, critical

biologic parameters should be measured from

laboratory studies, not by model fitting. 

Confidence in the use of a toxicodynamic 

model depends on the robustness of its 

validation process and on the results of 

sensitivity analyses. Peer review of the 

scientific basis and performance of a model is

essential (U.S. EPA, 2005a, §3.2.2). 

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 are used if the procedure fails to yield

reliable results, for example, if the fit is poor,

modeling may be restricted to the lower doses,

especially if there is competing toxicity at

higher doses (U.S. EPA, 2005a, §3.2.3). 

Modeling is used to derive a point of 

departure (U.S. EPA, 2012b; 2005a, §3.2.4).

(See Section 7.6 for alternatives if a point of

departure cannot be derived by modeling.): 

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– If linear extrapolation is used, 

selection of a response level 

corresponding to the point of

departure is not highly influential, so

standard values near the low end of 

the observable range are generally

used (for example, 10% extra risk for 

cancer bioassay data, 1% for 

epidemiologic data, lower for rare 

cancers). 

– For nonlinear approaches, both 

statistical and biologic considerations

are taken into account. 

– For dichotomous data, a response level

of 10% extra risk is generally used for

minimally adverse effects, 5% or

lower for more severe effects. 

– 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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1 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 (U.S. EPA, 2005a, §3.3.1 and §3.3.2). 

1) If a biologically based model has been

developed and validated for the agent,

extrapolation may use the fitted model

below the observed range if significant

model uncertainty can be ruled out with

reasonable confidence. 

2) Linear extrapolation is used if the dose

response curve is expected to have a

linear component below the point of 

departure. This includes: 

– Agents or their metabolites that are

DNA-reactive and have direct

mutagenic activity. 

– Agents or their metabolites for which

human exposures or body burdens are

near doses associated with key events

leading to an effect. 

46 

Linear extrapolation is also used when

data are insufficient to establish mode of 

action and when scientifically plausible. 

The result of linear extrapolation is 

described by an oral slope factor or an

inhalation unit risk, which is the slope of

the dose-response curve at lower doses or

concentrations, respectively. 

3) Nonlinear models are used for

extrapolation if there are sufficient data to

ascertain the mode of action and to 

conclude that it is not linear at lower 

doses, and the agent does not demonstrate

mutagenic or other activity consistent 

with linearity at lower doses. Nonlinear

approaches generally should not be used

in cases where mode of action has not 

ascertained. If nonlinear extrapolation is 

appropriate but no model is developed, an

alternative is to calculate reference values. 

4) Both linear and nonlinear approaches may

be used if there a multiple modes of action.

For example, modeling to a low response

level can be useful for estimating the

response at doses where a high-dose mode

of action would be less important. 

If linear extrapolation is used, the 

assessment develops a candidate slope factor

or unit risk for each suitable data set. These 

results are arrayed, using common dose 

metrics, to show the distribution of relative 

potency across various effects and 

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experimental systems. The assessment then

derives or selects an overall slope factor and

an overall unit risk for the agent, considering

the various dose-response analyses, the study

preferences discussed in Section 6, and the

possibility of basing a more robust result on

multiple data sets. 

7.5. Considering susceptible 

populations and lifestages 

The assessment analyzes the available 

information on populations and lifestages that

may be particularly susceptible to each effect.

A tiered approach is used (U.S. EPA, 

2005a, §3.5). 

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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1 various carcinogenic agents, but most 

epidemiologic studies and cancer

bioassays do not include early-life 

exposure. To address the potential for 

early-life susceptibility, the EPA 

recommends (U.S. EPA, 2005b, §5): 

– 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 (U.S. 

EPA, 2002, §4.2). Reference values are

typically calculated for effects other than 

cancer and for suspected carcinogens if a well

characterized mode of action indicates that a 

necessary key event does not occur below a

specific dose. Reference values provide no

information about risks at higher exposure

levels. 

The assessment characterizes effects that 

form the basis for reference values as adverse,

considered to be adverse, or a precursor to an

adverse effect. For developmental toxicity,

reproductive toxicity, and neurotoxicity there

is guidance on adverse effects and their 

biologic markers (U.S. EPA,

1998b, 1996, 1991b).

48 

To account for uncertainty and variability

in the derivation of a lifetime human exposure

where adverse effects are not anticipated to

occur, reference values are calculated by 

applying a series of uncertainty factors to the 

point of departure. If a point of departure 

cannot be derived by modeling, a noobserved-adverse-effect level or a lowestobserved-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 

individuals) (U.S. EPA,

2002, §4.4.5; 1998b, §4.2; 1996, §4; 1994, 

§4.3.9.1; 1991b, §3.4). 

Animal-to-human extrapolation. If animal 

results are used to make inferences about 

humans, the assessment adjusts for crossspecies differences. These may arise from

differences in toxicokinetics or 

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toxicodynamics. Accordingly, if the point

of departure is standardized to equivalent

human terms or is based on toxicokinetic 

or dosimetry modeling, a factor of 101/2 

(rounded to 3) is applied to account for the

remaining uncertainty involving

toxicokinetic and toxicodynamic

differences. If a biologically based model

adjusts fully for toxicokinetic and

toxicodynamic differences across species,

this factor is not used. In most other cases,

a factor of 10 is applied (U.S. EPA, 

2011; 2002, §4.4.5; 1998b, §4.2; 1996, §4;

1994, §4.3.9.1; 1991b, §3.4). 

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 doseresponse curve (U.S. EPA,

2002, §4.4.5; 1998b, §4.2; 1996, §4; 1994, 

§4.3.9.1; 1991b, §3.4).

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Subchronic-to-chronic exposure. If a p oint 

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, §4.3.9.1). 

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 (U.S. EPA,

2002, §4.4.5; 1998b, §4.2; 1996, §4; 1994, 

§4.3.9.1; 1991b, §3.4). The size of the 

factor depends on the nature of the 

database deficiency. For example, the EPA

typically follows the suggestion that a 

factor of 10 be applied if both a prenatal 

toxicity study and a two-generation 

reproduction study are missing and a 

factor of 101/2 if either is missing (U.S. 

EPA, 2002, §4.4.5). 

In this way, the assessment derives 

candidate values for each suitable data set and 

effect that is credibly associated with the 

agent. These results are arrayed, using

common dose metrics, to show where effects

occur across a range of exposures (U.S. EPA, 

1994, §4.3.9). 

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The assessment derives or selects an 

organ- or system-specific reference value for 

each organ or system affected by the agent.

The assessment explains the rationale for each

organ/system-specific reference value (based

on, for example, the highest quality studies,

the most sensitive outcome, or a clustering of

values). By providing these organ/system

specific reference values, IRIS assessments 

facilitate subsequent cumulative risk

assessments that consider the combined effect 

of multiple agents acting at a common site or

through common mechanisms (NRC, 2009).

The assessment then selects an overall 

reference dose and an overall reference 

concentration for the agent to represent

lifetime human exposure levels where effects

are not anticipated to occur. This is generally

the most sensitive organ/system-specific 

reference value, though consideration of study

quality and confidence in each value may lead

to a different selection. 

7.7. Confidence and uncertainty in the 

reference values 

The assessment selects a standard 

descriptor to characterize the level of 

confidence in each reference value, based on

the likelihood that the value would change

with further testing. Confidence in reference

values is based on quality of the studies used

and completeness of the database, with more

weight given to the latter. The level of 

confidence is increased for reference values 

based on human data supported by animal

data (U.S. EPA, 1994, §4.3.9.2). 

High confidence: The reference value is not 

likely to change with further testing,

except for mechanistic studies that might

affect the interpretation of prior test 

results. 

Medium confidence: This is a matter of 

judgment, between high and low 

confidence. 

Low confidence: The reference value is 

especially vulnerable to change with 

further testing. 

These criteria are consistent with 

guidelines for systematic reviews that 

evaluate the quality of evidence. These also

focus on whether further research would be 

likely to change confidence in the estimate of

effect (Guyatt et al., 2008b). 

All assessments discuss the significant

uncertainties encountered in the analysis. The

EPA provides guidance on characterization of

uncertainty (U.S. EPA, 2005a, §3.6). For 

example, the discussion distinguishes model 

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1 uncertainty (lack of knowledge about the 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

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Toxicological Review of tert-Butyl Alcohol 

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effects of the agent). 

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Toxicological Review of tert-Butyl Alcohol 

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 α2u-globulin-associated nephropathy. tertButanol, however, is a weak inducer of α2u-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 α2u-globulin-nephropathy or CPN were not considered for kidney hazard 

identification. Changes in kidney weights, transitional epithelial hyperplasia, suppurative 

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Toxicological Review of tert-Butyl Alcohol 

1 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 epithelial hyperplasia, was chosen as the 

basis for the overall oral reference dose (RfD) (see Table ES-1). The chronic study by NTP (1995) 

and the observed kidney effects were used to derive the RfD. The endpoint of transitional epithelial 

hyperplasia was selected as the critical effect because it was observed in both rat sexes 

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responsive manner. Benchmark dose (BMD) modeling was used to derive the benchmark dose 

lower confidence limit (BMDL10%) of 16 mg/kg-day. The BMDL was converted to a human 

equivalent dose using body weight3/4 scaling, and the value of 3.84 mg/kg-day was used as the 

point of departure (POD) for RfD derivation (U.S. EPA, 2011). 

The overall RfD was calculated by dividing the POD for kidney transitional epithelial 

hyperplasia by a composite uncertainty factor (UF) of 30 to account for the extrapolation from 

animals to humans (3) and for interindividual differences in human susceptibility (10). 

Table ES-1. Organ/system-specific RfDs and overall RfD for tert-butanol 

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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 × 10-1 Chronic High 

Overall RfD Kidney 3.8 30 1 × 10-1 Chronic High 

*HED PODs were calculated using BW3/4 scaling (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 α2uglobulin nephropathy or CPN were 

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1 not considered for kidney hazard identification. Changes in kidney weights and severity of 

nephropathy, however, are considered a result of tert-butanol exposure and are appropriate for
 

identifying a hazard to the kidney.
 

Inhalation Reference Concentration (RfC) for Effects Other Than Cancer 

Kidney toxicity, represented by transitional epithelial hyperplasia, was chosen as the basis 

for the inhalation reference concentration (RfC) (see Table ES-2). Although endpoints from a route

specific study were considered, the availability of a physiologically based pharmacokinetic (PBPK) 

model for tert-butanol in rats [modified by Salazar et al. (2015)] allowed for more specific and 

sensitive equivalent inhalation PODs derived from a route-to-route extrapolation from the PODs of 

the oral NTP (1995) study. The POD adjusted for the human equivalent concentration (HEC) was 

26.1 mg/m3 based on transitional epithelial hyperplasia. 

The RfC was calculated by dividing the POD by a composite UF of 30 to account for 

toxicodynamic differences between animals and humans (3) and interindividual differences in 

human susceptibility (10). 

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Hazard Basis 

Point of 

departure* 

(mg/m3) UF 

Chronic RfC 

(mg/m3) 

Study exposure 

description Confidence 

Kidney Transitional epithelial 

hyperplasia 26.1 30 9 × 10-1 Chronic High 

Overall RfC Kidney 26.1 30 9 × 10-1 Chronic High 

*Continuous inhalation human equivalent concentration that leads to the same average blood concentration of 

tert-butanol as continuous oral exposure at the BMDL. 

Evidence of Human Carcinogenicity 

Under the EPA’s cancer guidelines (U.S. EPA, 2005a), there is suggestive evidence of 

carcinogenic potential for tert-butanol. tert-Butanol induced kidney tumors in male (but not female) 

rats and thyroid tumors (primarily benign) in male and female mice following long-term 

administration in drinking water (NTP, 1995). The potential for carcinogenicity applies to all routes 

of human exposure. 

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1 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 B6C3F1 mice, and
 

thyroid follicular cell adenomas and carcinomas in male B6C3F1 mice (NTP, 1995). The study
 

included histological examinations for tumors in many different tissues, contained three exposure 

levels and controls, contained adequate numbers of animals per dose group (~50/sex/group), 

treated animals for up to 2 years, and included detailed reporting of methods and results. 

Although tert-butanol was considered to have “suggestive evidence of carcinogenic 

potential,” the NTP study was well conducted and quantitative analysis could be useful for 

providing a sense of the magnitude of potential carcinogenic risk (U.S. EPA, 2005a). A slope factor 

was derived for thyroid tumors in female and male mice. The modeled tert-butanol POD was scaled 

to HEDs according to EPA guidance by converting the BMDL10 on the basis of (body weight) 3/4 

scaling (U.S. EPA, 2011, 2005a). Using linear extrapolation from the BMDL10, a human equivalent 

oral slope factor was derived (slope factor = 0.1/BMDL ). The resulting oral slope factor is 5 × 10-4 10

per mg/kg-day. 

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Susceptible Populations and Lifestages for Cancer and Noncancer 

In vitro studies suggest that cytochrome P-450 (CYP450) (Cederbaum et al., 1983; 

Cederbaum and Cohen, 1980), plays a role in the metabolism of tert-butanol. No studies, however, 

have identified the specific CYPs responsible for the biotransformation of tert-butanol. Various 

CYPs are under-expressed in the mouse fetus and neonate (Lee et al., 2011) and decreased in older 

mice (Lee et al., 2011) and rats (Lee et al., 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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No chronic inhalation exposure studies to tert-butanol are available. Lifetime exposure to 

tert-butanol has been associated with increased renal tubule adenomas and carcinomas as well as 

thyroid follicular cell adenomas and carcinomas. As stated above, the rat kidney tumors are 

unsuitable for quantitative analysis as there is not enough data to determine the relative 

contribution of α2u-globulin 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-toroute extrapolation is not possible for these thyroid effects in mice because the only PBPK model 

available is for rats. Therefore, no quantitative estimate of carcinogenic risk could be determined
 

for inhalation exposure. 

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1 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 α2u-globulin-associated nephropathy was 

performed. The presence of α2u-globulin in the hyaline droplets was confirmed in male rats by 

α2u-globulin immunohistochemical staining. Linear mineralization and tubular hyperplasia were 

reported in male rats, although only in the chronic study. Other subsequent steps in the 

pathological sequence, including necrosis, exfoliation, and granular casts, either were absent or 

inconsistently observed across subchronic or chronic studies. None of these effects occurred in 

female rats or in either sex of mice, although these endpoints were less frequently evaluated in 

these models. Evidence implies an α2u-globulin MOA is operative, although it is relatively weak in 

response to tert-butanol and is not solely responsible for the renal tubule nephropathy observed in 

male rats. CPN also is instrumental in renal tubule nephropathy, in both male and female rats. 

Several other effects in the kidney unrelated to α2u-globulin or CPN, however, were observed in 

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female or male rats (U.S. EPA, 1991a), including suppurative inflammation in female rats, 

transitional epithelial hyperplasia in male and female rats, and increased kidney weights in both 

sexes of rats (NTP, 1997, 1995). These specific effects are considered the result of tert-butanol 

exposure and therefore, relevant to humans. 

Concerning cancer, α2u-globulin accumulation is indicated as relatively weak in response to 

tert-butanol exposure and not the sole mechanism responsible for the renal tubule carcinogenicity 

observed in male rats. Although CPN and other effects induced by both α2u-globulin processes and 

tert-butanol play a role in renal tubule nephropathy, the evidence indicates that CPN does not 

induce the renal tubule tumors associated with tert-butanol exposure in male rats, suggesting that 

other, unknown processes contribute to renal tumor development. Based on this analysis of 

available MOA data, these renal tumors are considered relevant to humans (U.S. EPA, 1991a). 

In addition, an increase in the incidence of thyroid follicular cell adenomas was observed in 

male and female mice in a 2-year drinking water study (NTP, 1995). Thyroid follicular cell 

hyperplasia was considered a preneoplastic effect associated with the thyroid tumors, and the 

incidences of follicular cell hyperplasias were elevated in both male and female B6C3F1 mice 

following exposure. U.S. EPA (1998a) describes the procedures the Agency uses in evaluating 

potential human cancer hazard and dose-response assessments for chemicals that are animal 

thyroid carcinogens. The available database is inadequate in four of the five required areas (U.S. 

EPA, 1998a), suggesting that an antithyroid MOA is not operating in mouse thyroid follicular cell 

tumorigenesis. No other MOAs for thyroid tumors were identified, and the mouse thyroid tumors 

are considered relevant to humans (U.S. EPA, 2005a, 1998a). 

 

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Toxicological Review of tert-Butyl Alcohol 

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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1 • 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 designed to cast a wide net and to

minimize the possibility of missing potentially relevant health effects data. 

The complete list of references and the sorting of these materials can be found on the tert

butanol project page of the HERO website at 

https://hero.epa.gov/index.cfm/project/page/project_id/1543. 

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(Takahashi et al., 1993) in the “Supplementary Studies” category also was based on data from the 

NTP report. The interim publications and the final NTP report differed. The finalized NTP (1995) 

report was considered the more complete and accurate presentation of the data; therefore, this 

report was included in evidence tables and Cirvello et al. (1995), Takahashi et al. (1993), and 

Lindamood et al. (1992) 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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Selection of Studies for Inclusion in Evidence Tables 

To summarize the important information systematically from the primary health effects 

studies in the tert-butanol database, evidence tables were constructed in a standardized tabular 

format as recommended by NRC (2011). Studies were arranged in evidence tables by effect, species, 

duration, and design, and not by quality. Of the studies that were retained after the literature search 

and screen, 12 studies were identified as “Sources of Health Effects Data” and were considered for 

extraction into evidence tables for hazard identification in Chapter 1. Initial review found two 

references (Cirvello et al., 1995; Lindamood et al., 1992) to be publications of the NTP (1995) data 

prior to the release of the final National Toxicology Program (NTP) report. One publication 

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1 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. 

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3 

4 

5 

6 

Figure LS-1. Summary of literature search and screening process for 

tert-butanol. 

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Toxicological Review of tert-Butyl Alcohol 

Table LS-1. Details of the search strategy employed for tert-butanol 

Database 

(Search Date) Keywords Limits 

PubMed tert-butanol OR 75-65-0[rn] OR “t- None 

(12/20/2012) butyl hydroxide” OR “2-methyl-2-

(4/17/2014) propanol” OR “trimethyl carbinol” 

(5/13/2015) OR “t-butyl alcohol” OR tert-butanol 

OR "tert-butyl alcohol" OR tert-butyl 

alcohol[mesh] 

Web of Science Topic = (tert-butanol OR 75-65-0 OR Refined by: Research Areas = (cell biology OR 

(12/20/2012) "t-butyl hydroxide" OR "2-methyl-2- respiratory system OR microscopy OR biochemistry 

(4/17/2014) propanol" OR "trimethyl carbinol" molecular biology OR gastroenterology OR hepatology 

(5/13/2015) OR "t-butyl alcohol" OR "tertbutanol" OR "tert-butyl alcohol") 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 tert-butanol OR 75-65-0 [rn] OR t- Not PubMed 

TSCATS) butyl hydroxide OR 2-methyl-2-

(1/11/2013) propanol OR trimethyl carbinol OR t-

(4/17/2014) butyl alcohol OR tert-butanol OR 

(5/13/2015) tert-butyl alcohol OR tert-butyl 

alcohol 

TSCATS2 75-65-0 None 

(1/4/2013) 

(4/17/2014) 

(5/13/2015) 

Table LS-2. Summary of additional search strategies for tert-butanol 

Approach used Source(s) 

Date 

performed 

Number of additional references 

identified 

Manual search of 

citations from 

reviews 

Review article: McGregor (2010). 

Tertiary-butanol: A toxicological 

review. Crit Rev Toxicol 40(8): 697­

727. 

1/2013 5 

Review article: Chen (2005). Amended 

final report of the safety assessment 

of t-butyl alcohol as used in 

cosmetics. Int J Toxicol 24(2): 1-20. 

1/2013 2 

Manual search of 

citations from 

reviews conducted 

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 safety and 

health guideline for tert-butyl alcohol. 

Cincinnati, OH: Occupational Safety 

and Health Administration. 

1/2013 None 

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 tert-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 tert-butanol 

• Exposure to a mixture only (e.g., gasoline containing 

tert-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 tert-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 tert-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 tert-butanol 

• Use of tert-butanol as a solvent or methodology for 

testing unrelated to tert-butanol 

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Inclusion criteria Exclusion criteria 

• Not chemical specific: Studies that do not involve 

testing of tert-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. 

Database Evaluation 

For this draft assessment, 12 references reported on experimental animal studies that 

comprised the primary sources of health effects data; no studies were identified that evaluated 

humans exposed to tert-butanol (e.g., cohort studies, ecological studies). The animal studies were 

evaluated using the study quality considerations outlined in the Preamble, considering aspects of 

design, conduct, or reporting that could affect the interpretation of results, overall contribution to 

the synthesis of evidence, and determination of hazard potential as noted in various EPA guidance 

documents (U.S. EPA, 2005a, 1998b, 1996, 1991b). The objective was to identify the stronger, more 

informative studies based on a uniform evaluation of quality characteristics across studies of 

similar design. As stated in the Preamble, studies were evaluated to identify the suitability of the 

study based on: 

• Study design 

• Nature of the assay and validity for its intended purpose 

• Characterization of the nature and extent of impurities and contaminants of tert-butanol 

administered, if applicable 

• Characterization of dose and dosing regimen (including age at exposure) and their

adequacy to elicit adverse effects, including latent effects 

• Sample sizes and statistical power to detect dose-related differences or trends 

• Ascertainment of survival, vital signs, disease or effects, and cause of death 

• Control of other variables that could influence the occurrence of effects 

Additionally, several general considerations, presented in Table LS-4, were used in 

evaluating the animal studies. Much of the key information for conducting this evaluation can be 

determined based on study methods and how the study results were reported. Importantly, the 

evaluation at this stage does not consider the direction or magnitude of any reported effects. 

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Toxicological Review of tert-Butyl Alcohol 

1 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 difference. 

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Experimental Animal Studies 

The experimental animal studies, comprised entirely of studies performed in rats and mice, 

were associated with drinking water, oral gavage, liquid diets (i.e., maltose/dextrin), and inhalation 

exposures to tert-butanol. With the exception of neurodevelopmental studies, these sources were 

conducted according to Organisation for Economic Co-operation and Development Good 

Laboratory Practice (GLP) guidelines, presented extensive histopathological data, or clearly 

presented their methodology; thus, these studies are considered high quality. These studies include 

2-year bioassays using oral exposures in rats and mice; two subchronic drinking water studies in 

rats and one in mice; an inhalation subchronic study in rats and mice; a reevaluation of the NTP 

(1995) rat data; two oral developmental studies; two inhalation developmental studies; and a 

single one-generation reproductive study that also evaluates other systemic effects (Table LS-5). A 

more detailed discussion of any methodological concerns that were identified precedes each 

endpoint evaluated in the hazard identification section. Overall, the experimental animal studies of 

tert-butanol involving repeated oral or inhalation exposure were considered to be of acceptable 

quality, and whether yielding positive, negative, or null results, were considered in assessing the 

evidence for health effects associated with chronic exposure to tert-butanol. 

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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-year study in F344 rats (drinking water) NTP (1995) 

2-year study in B6C3F1 mice (drinking water) NTP (1995) 

Subchronic 13-week study in B6C3F1 mice (drinking water) NTP (1995) 

13-week study in F344 rats (drinking water) NTP (1995) 

13-week study in F344 rats (inhalation) NTP (1997) 

13-week study in B6C3F1 mice (inhalation) NTP (1997) 

10-week study in Wistar rats (drinking water) Acharya et al. (1997), Acharya et al. (1995) 

Reproductive One-generation reproductive toxicity study in Sprague-Dawley rats (gavage) Lyondell 

Chemical Co. (2004) 

Developmental Developmental study (GD 6–20) in Swiss Webster mice (diet) Daniel and Evans (1982) 

Developmental study (GD 6–18) in CBA/J mice (drinking water) Faulkner et al. (1989) 

Developmental study (GD 6–18) in C57BL/6J mice (drinking water) Faulkner et al. (1989) 

Developmental study (GD 1–19) in Sprague-Dawley rats (inhalation) Nelson et al. (1989) 

Neurodevelopmental Neurodevelopmental study (GD 6–20) in Swiss Webster mice (diet) Daniel and Evans 

(1982) 

Neurodevelopmental study (GD 1–19) in Sprague-Dawley rats (inhalation) Nelson et al. 

(1991) 

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1. HAZARD IDENTIFICATION 

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1.1. Overview of Chemical Properties and Toxicokinetics 

1.1.1. Chemical Properties 

tert-Butanol is a white crystalline solid or colorless, highly flammable liquid (above 25.7°C) 

with a camphor-like odor (NIOSH, 2005; IPCS, 1987a). tert-Butanol contains a hydroxyl chemical 

functional group; is miscible with alcohol, ether, and other organic solvents; and is soluble in water 

(IPCS, 1987a). Selected chemical and physical properties of tert-butanol are presented in Table 1-1. 

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; tButyl 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 × 106 mg/L HSDB (2007) 

Octanol/Water Partition 

Coefficient (Log KOW ) 0.35 HSDB (2007) 

Henry’s Law Constant 9.05 × 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 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 of tert-butanol and its metabolites
 

have also been observed after acute oral or inhalation exposures in rats (ARCO, 1983). In another
 

study (Faulkner et al., 1989), blood concentrations indicated that absorption was complete at 1.5
 

hours following oral gavage doses of tert-butanol in female mice.
 

tert-Butanol is distributed throughout the body following oral, inhalation, and i.v. exposures
 

(Poet et al., 1997; Faulkner et al., 1989; ARCO, 1983). Following exposure to tert-butanol in rats,
 

tert-butanol was found in kidney, liver, and blood, with male rats retaining more tert-butanol than 

female rats (Williams and Borghoff, 2001). 

A general metabolic scheme for tert-butanol, illustrating the biotransformation in rats and
 

humans, is shown in Figure 1-1 (see Appendix B.1.3).
 

Human data on the excretion of tert-butanol comes from studies of methyl tert-butyl ether 

(MTBE) and ethyl tert-butyl ether (ETBE) (Nihlén et al., 1998a, 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 of tert-butanol in urine following ETBE exposure was 7.9 ± 2.7 hours (average 

of 104- and 210-mg/m3 ETBE doses). These studies reported urinary levels of tert-butanol (not 

including downstream metabolites) to be less than 1% of administered MTBE or ETBE 

concentrations (Nihlén et al., 1998a, b). Amberg et al. (2000) also observed a similar half-life of
 

9.8 ± 1.4 hours after human exposure to ETBE of 170 mg/m3. The half-life for tert-butanol in rat
 

urine was 4.6 ± 1.4 hours at ETBE levels of 170 mg/m3. 

A more detailed summary of tert-butanol toxicokinetics is provided in Appendix B,
 

Section B.1. 

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CH3 

glucuronide–O CH3 

CH3 HO O 

t-butyl glucuronide
 

HO
 CH3 

rats, humans [O] CH3

CH3 OH O 2-hydroxyisobutyric acid 

CYP450 

HO CH3 H3C H OH rats, 3C O CH3 humans CH OH 

3
 

t-butanol
 2-methyl-1,2-propanediol 

rats 

- O
 O
 

CH3
 

CH3 

3CH CH3 

O 

acetone 

H2C 

formaldehyde 

Source: NSF International (2003), ATSDR (1996), Bernauer et al. (1998), Amberg et al. (1999), 

S 

O
 O
 CH3 

CH3
 

t-butyl sulfate
 

and Cederbaum and Cohen (1980).
 

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Figure 1-1. Biotransformation of tert-butanol in rats and humans. 

1.1.3.
 Description of Toxicokinetic Models 

No physiologically based pharmacokinetic (PBPK) models have been developed specifically 

for administration of tert-butanol. Some models have been used to study tert-butanol as the 

primary metabolite after 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 

α2u-globulin (Borghoff et al., 2010; Leavens and Borghoff, 2009). A more-detailed summary of the 

toxicokinetic models is provided in Appendix B, Section B.1.5. 

A PBPK model for tert-butanol was modified by adapting previous models for MTBE and 

tert-butanol (Leavens and Borghoff, 2009; Blancato et al., 2007). The addition of a sequestered 

blood compartment for tert-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 et al., 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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In 1996, the U.S. Agency for Toxic Substances and Disease Registry’s (ATSDR) Toxicological 

Profile for MTBE (ATSDR, 1996) identified cancer effect levels of MTBE based on data on 

carcinogenicity in animals. ATSDR reported that inhalation exposure was associated with kidney 

cancer in rats and liver cancer in mice. ATSDR concluded that oral exposure to MTBE might cause 

liver and kidney damage, and nervous system effects in rats and mice. The chronic inhalation 

minimal risk level was derived based on incidence and severity of chronic progressive nephropathy 

in female rats (ATSDR, 1996). In 1997, EPA’s Office of Water concluded that MTBE is carcinogenic 

to animals and poses a potential carcinogenic potential to humans based on an increased incidence 

of Leydig cell adenomas of the testes, kidney tumors, lymphomas, and leukemia in exposed rats 

(U.S. EPA, 1997). In 1998, the International Agency for Research on Cancer (IARC) found “limited 

evidence” of MTBE carcinogenicity in animals and placed MTBE in Group 3 (i.e., not classifiable as 

to carcinogenicity in humans) (IARC, 1999). IARC reported that oral exposure in rats resulted in 

testicular tumors in males and lymphomas and leukemias (combined) in females; inhalation 

exposure in male rats resulted in renal tubule adenomas; and inhalation exposure in female mice 

resulted in hepatocellular adenomas (IARC, 1999). 

No assessments by national or international agencies or chronic studies for tert-butyl 

acetate are available. 

1.2. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM 

1.2.1. Kidney Effects 

Synthesis of Effects in Kidney 

This section reviews the studies that investigated whether subchronic or chronic exposure 

to tert-butanol can affect kidneys in humans or animals. The database examining kidney effects 

following tert-butanol exposure contains eight studies from 5 references performed in rats or mice 

(Lyondell Chemical Co., 2004; Acharya et al., 1997; NTP, 1997; Acharya et al., 1995; NTP, 1995), and 

a reevaluation of the rat data from NTP (1995), published by Hard et al. (2011); no human data are 

1 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 et al., 2013; Suzuki et 

al., 2012). 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 et al., 2013). 

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1 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 nonneoplastic kidney endpoints in rats, however, is somewhat complicated by the common occurrence 

of age-related, spontaneous lesions characteristic of chronic progressive nephropathy (CPN) (NTP, 

2015; Hard et al., 2013; Melnick et al., 2012; U.S. EPA, 

1991a);http://ntp.niehs.nih.gov/nnl/urinary/kidney/necp/index.htm). CPN is more severe in male 

rats than in females and is particularly common in the Sprague-Dawley and Fischer 344 strains. 

Dietary and hormonal factors play a role in modifying CPN, although the etiology is largely 

unknown (see further discussion below). 

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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) (NTP, 

1997) and 15 months (oral) (NTP, 1995). Lyondell Chemical Co. (2004) also reported increases in 

absolute and relative kidney weight in Sprague-Dawley rats 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). Of 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 

(Bailey et al., 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 (1995). In 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 (Craig et al., 2014). 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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1 Kidney histopathology. Treatment-related histopathological changes were observed in the 

kidneys of male and female F344 rats following 13-week and 2-year oral exposures (NTP, 1995) 

and male F344 rats following a 13-week inhalation exposure (NTP, 1997). Similarly, male Wistar 

rats exposed for approximately 10 weeks exhibited an increase in histopathological kidney lesions 

(Acharya et al., 1997; Acharya et al., 1995). B6C3F1 mice, however, did not exhibit histopathological 

changes when exposed for 13 weeks and 2 years via the oral route (NTP, 1995) and 13 weeks via 

the inhalation route (NTP, 1997). More specific details on the effects observed in rats, reported by 

NTP (1997, 1995) and Acharya et al. (1997); (1995) are described below. 

Nephropathy and severity of nephropathy were reported in male and female rats in the 

13-week oral studies (NTP, 1995). The nephropathy was characterized as “...a spontaneous 

background lesion...typically consist[ing] of scattered renal tubules lined by basophilic 

regenerating tubule epithelium.” (NTP, 1995). NTP (1995) noted that the increase in severity of 

nephropathy was related to tert-butanol and “characterized by an increase in the number and size 

of foci of regeneration.” The severity of nephropathy increased, compared with controls, in the 

13-week male rats, which exhibited nephropathy in 94% of all exposed animals and 70% of 

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from minimal to mild with increasing dose as “evidenced by an increased number of foci.” No 

treatment-related kidney histopathology was reported in the female rats exposed through 

inhalation (NTP, 1997). 

In the 2-year oral study by NTP (1995), 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 of interstitial fibrosis and scarring, and glomerular sclerosis.” At 15 

months, male and female rats (30/30 treated; 10/10 controls) had nephropathy, and the severity 

scores ranged from minimal to mild. At 2 years, male and female rats (149/150 treated; 49/50 

controls) also had nephropathy, and although the severity was moderate in the control males and 

minimal to mild in the control females, severity increased with tert-butanol exposure in both sexes 

(NTP, 1995). 

The lesions collectively described by NTP (1997, 1995) as nephropathy and noted 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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incidence significantly increased with tert-butanol exposure. In the 13-week inhalation study (NTP, 

1997), nephropathy was present in all but two male rats, including controls. NTP (1997) 

characterized the reported chronic nephropathy in control male rats as “1 to 3 scattered foci of 

regenerative tubules per kidney section. Regenerative foci were characterized by tubules with 

cytoplasmic basophilia, increased nuclear/cytoplasmic ratio, and occasionally thickened basement 

membranes and intraluminal protein casts.” In exposed groups, the severity generally increased 

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1 [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 (NTP, 

1997), 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 (1995) 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 α2u-globulin nephropathy (for further 

discussion of this particular lesion, see Mode of Action Analysis—Kidney Effects). The mineralization 

is characterized as distinct linear deposits along radiating medullary collecting ducts. An increased 

incidence of linear mineralization was limited to exposed males in the 2-year oral study (NTP, 

1995). 

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control and low-dose groups of 13-week males and the dose-response relationships the tertbutanol-exposed males exhibited in the 13-week (NTP, 1997, 1995) and 2-year studies (NTP, 

1995). Furthermore, due to the overwhelming presence of mineralization in the control and treated 

female rats, the contribution, if any, of tert-butanol to the formation of this lesion in females could 

not be determined. Thus, the mineralization could be related to both aging of the animals and tert

butanol exposure. 

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durations. A dose-related, increased incidence of mineralization was reported in male rats at the 

13-week, 15-month, and 2-year oral evaluations (NTP, 1995). NTP (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 of this lesion in females. This description, however, is inconsistent with 

the observation in this and other databases that age-related nephropathy (i.e., CPN) is generally 

more prevalent and more severe 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 

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inflammation and transitional epithelial hyperplasia. These lesions were observed in the 2-year 

oral NTP (1995) study. Although NTP (1995) 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 

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1 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 (NTP, 1995) or inhalation study (NTP, 1997), 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 (NTP, 1995). In the control males, 50% of the animals exhibited transitional epithelial 

hyperplasia and the incidence and severity increased with dose. Only the mid- and high-dose 

females, however, exhibited dose-related increases in incidence and severity of transitional 

epithelial hyperplasia; this lesion was not reported in the control or low-dose females. NTP (1995) 

described transitional epithelial hyperplasia as increased layers of the transitional epithelial lining 

of the renal pelvis; study authors noted no progression of this hyperplastic lesion to neoplasia. The 

relatively high background in male controls (i.e., 50%) suggests some potential influence, other 

than tert-butanol treatment, on this effect. The absence of this effect in female control and low-dose 

animals and the dose-related increases in both males and females, however, indicate that similar to 

the suppurative inflammation, the transitional epithelial hyperplasia is predominantly treatment 

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study authors noted as potentially indicative of oxidative damage. Acharya et al. (1997); Acharya et 

al. (1995) used one dose and a control group and did not report incidences. The increased tubule 

degeneration and glomerular vacuolation could be characterized as tubular atrophy and glomerular 

hyalinization, respectively, consistent with CPN; however, without quantitative information, 

examining the differences between the control and treated animals to determine if CPN plays a role 

in development of these effects is not possible. Although based on the noted appearance of the 

effects in the treated animals compared with controls, the effects likely are treatment related. 

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13 

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16 related. Transitional epithelial hyperplasia should not be 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 tubular degeneration, 

degeneration of the basement membrane of the Bowman’s capsule, diffused glomeruli, and 

glomerular vacuolation were noted in a 10-week study in male Wistar rats (Acharya et al., 1997; 

Acharya et al., 1995). A decrease in glutathione in the kidney accompanied these changes, which the 

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Serum or urinary biomarkers informative of kidney toxicity were not measured in the 

studies discussed above. Some changes occurred in urinalysis parameters (e.g., decreased urine 

volume and increased specific gravity), accompanied by reduced water consumption, and thus
 

might not be related to an effect of kidney function (NTP, 1995).
 

Kidney tumors. The kidney is also a target organ for cancer effects (Table 1-3, Figure 1-1).
 

Male F344 rats had an increased incidence of combined renal tubule adenomas or carcinomas in 

the 2-year oral bioassay (Hard et al., 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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1 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 (Hard, 

1986).” 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 (NTP, 1995); no increase in severity was observed. This effect in females, which was not 

considered toxicologically significant, is not discussed further. Two renal tubular adenocarcinomas 

in male mice also were reported (NTP, 1995), one each in the low- and high-dose groups, but were 

not considered by NTP to be “biologically noteworthy changes”; thus the tumors in mice are not 

discussed further. 

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13 A Pathology Working Group, sponsored by Lyondell Chemical Company, reevaluated the 

kidney changes in the NTP 2-year study to determine if additional histopathological changes could 

be identified to inform the MOA for renal tubule tumor development (Hard et al., 2011). In all cases, 

working group members were blinded to treatment groups, and used guidelines published by Hard 

and Wolf (1999) and refinements reported by (Hard and Seely, 2006); Hard and Seely (2005) and 

Hard (2008). The group’s report and analysis by Hard et al. (2011) confirmed the NTP findings of 

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19 renal tubule hyperplasia and renal tubule tumors in male rats at 2 years. In particular, they 

reported similar overall tumor incidences in the exposed groups. Hard et al. (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 et al. (2011) considered 

fewer tumors to be carcinomas than did the original NTP study. Results of both NTP (1995) and the 

reanalysis by Hard et al. (2011) are included in Table 1-3 and Figure 1-1. 

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Male rats 

1 10 100 1000 

Absolute kidney weight (% change) 

0 

10 

20 

30 

40 

50 

Oral exposure 

Inhalation exposure 

rho= 0.78 (all) 

rho= 0.89 (oral) 

rho= 0.80 (inhalation) 

Female rats 

tert-butanol blood concentration (mg/l) 

1 10 100 1000 10000 

rho= 0.78 (all) 

rho= 0.72 (oral) 

rho= 0.9 (inhalation) 

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Figure 1-2. Comparison of absolute kidney weight change in male and female 

rats across oral and inhalation exposure based on internal blood 

concentration. Spearman rank correlation coefficient (rho) was calculated to 

evaluate the direction of a monotonic association (e.g., positive value = 

positive association) and the strength of association. 
 

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Male mice 

Administered dose (mg/kg-day) 

0 2000 4000 6000 8000 

Absolute kidney w eight (% change) 

-4 

-2 

0 

2 

4 

6 

8 

10 

12 

14 

Female mice 

Administered dose (mg/kg-day) 

0 2000 4000 6000 8000 10000 12000 

rho= -0.1 rho= 0.9 

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 

Administered dose (mg/m3

) 

0 1000 2000 3000 4000 5000 6000 

Absolute kidney w eight (% change) 

-12 

-10 

-8 

-6 

-4 

-2 

0 

2 

4 

6 

Female mice 

Administered dose (mg/m3

) 

0 1000 2000 3000 4000 5000 6000 7000 

rho= 0.2 rho= -0.1 

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. 

Toxicological Review of tert-Butyl Alcohol 

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Table 1-2. Changes in kidney histopathology in animals following exposure to 

tert-butanol 

Reference and study design Results 

Acharya et al. (1997) ↑ tubular degeneration, degeneration of the basement membrane of the 

Acharya et al. (1995) Bowman’s capsule, diffused glomeruli, and glomerular vacuolation (no 

incidences reported) 

Wistar rat; 5–6 males/treatment 

Drinking water (0 or 0.5%), 0 or ↓ kidney glutathione (~40%)* 

575 mg/kg-d 

10 weeks 

NTP (1995) Incidence (severity): 

F344/N rat; 10/sex/treatment Males Females 

Drinking water (0, 2.5, 5, 10, 20, Dose Minerali- Nephro- Dose Minerali- Nephro­ or 40 mg/mL) (mg/kg-d) zationb pathyc (mg/kg-d) zationb pathyc 

M: 0, 230, 490, 840, 1,520, 

3,610a mg/kg-d 0 0/10 7/10 (1.0) 0 10/10 (1.7) 2/10 (1.0) 

F: 0, 290, 590, 850, 1,560, 

3,620a mg/kg-d 

13 weeks 

230 0/10 10/10 

(1.6*) 

290 10/10 (2.0) 3/10 (1.0) 

490 2/10 (1.5) 10/10 

(2.6*) 

590 10/10 (2.0) 5/10 (1.0) 

840 8/10*(1.4) 10/10 

(2.7*) 

850 10/10 (2.0) 7/10* (1.0) 

1,520 4/10*(1.0) 10/10 

(2.6*) 

1,560 10/10 (2.0) 8/10* (1.0) 

3,610a 4/10*(1.0) 7/10 (1.1) 3,620a 6/10 (1.2) 7/10* (1.0) 

NTP (1995) Study authors indicated no treatment-related changes in kidney 

histopathology (histopathological data not provided for the 13-week study) 

B6C3F1 mouse; 10/sex/treatment 

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

or 40 mg/mL) 

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

8,210a mg/kgd 

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

11,620a mg/kgd 

13 weeks 

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Reference and study design Results 

NTP (1995) 

F344/N rat; 60/sex/treatment 

(10/sex/treatment evaluated at 

15 months interim) 

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

Incidence (severity): 

Males 

Dose 

(mg/kg-d) 

Mineralizationb 

(interim) 

Mineralizationb 

(terminal) 

Linear 

mineralizationb 

(terminal) 

mg/mL) 

M: 0, 90, 200, 420a mg/kg-d 

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

2 years 

0 1/10 (1.0) 26/50 (1.0) 0/50 

90 2/10 (1.0) 28/50 (1.1) 5/50* (1.0) 

200 5/10 (1.8) 35/50 (1.3) 24/50* (1.2) 

420a 9/10* (2.3) 48/50* (2.2) 46/50* (1.7) 

Dose 

(mg/kg-d) 

Transitional 

epithelial 

hyperplasia 

Nephropathyc 

severity 

0 25/50 (1.7) 3.0 

90 32/50 (1.7) 3.1 

200 36/50* (2.0) 3.1 

420a 40/50* (2.1) 3.3* 

Females 

Dose 

(mg/kg-d) 

Mineralizationb 

Interim 

Mineralizationb 

Terminal 

Inflammation 

(suppurative) 

incidence 

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

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

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

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

Dose 

(mg/kg-d) 

Transitional 

epithelial 

hyperplasia 

Nephropathyc 

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) 

B6C3F1 mouse; 60/sex/treatment 

Drinking water (0, 5, 10, or 20 

mg/mL) 

M: 0, 540, 1,040, or 2,070a 

mg/kg-d 

No treatment-related changes in kidney related histopathology observed 

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Reference and study design Results 

F: 0, 510, 1,020, or 2,110 mg/kgd 

2 years 

NTP (1997) Male 

F344/N rat; 10/sex/treatment 

Inhalation analytical 

concentration: 0, 134, 272, 542, 

1,080, or 2,101 ppm (0, 406, 824, 

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 

Concentration 

(mg/m3) 

Incidence of 

chronic 

nephropathyd 

Average severity 

of chronic 

nephropathy 

0 9/10 1.0 

406 8/10 1.4 

824 9/10 1.4 

1,643 10/10 1.6 

3,273 10/10 1.9 

6,368 10/10 2.0 

Females: no treatment-related changes in kidney related histopathology 

observed 

Severity categories: 1= minimal, 2= mild. No results from statistical tests 

reported 

NTP (1997) 

B6C3F1 mouse; 10/sex/treatment 

Inhalation analytical 

concentration: 0, 134, 272, 542, 

1,080, or 2,101 ppm (0, 406, 824, 

1,643, 3,273 or 6,368 mg/m3

) 

(dynamic whole-body chamber) 

6 hr/d, 5 d/wk 

13 weeks 

Generation method (Sonimist 

Ultrasonic spray nozzle 

nebulizer), analytical 

concentration and method were 

reported 

No treatment-related changes in kidney related histopathology observed 

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* Statistically significant p ≤ 0.05 as determined by the study authors. a The high-dose group had an increase in mortality. b Mineralization defined in NTP (1995) as focal mineral deposits, primarily at the corticomedullary junction. Linear 

mineralization was defined as foci of distinct linear deposits along radiating medullary collecting ducts; linear 

mineralization not observed in female rats. 
Toxicological Review of tert-Butyl Alcohol 

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c

 

Nephropathy defined in NTP (1995) as lesions including thickened tubule and glomerular basement membranes, 

basophilic foci of regenerating tubule epithelium, intratubule protein casts, focal mononuclear inflammatory cell 

aggregates within areas of interstitial fibrosis and scarring, and glomerular sclerosis. d Nephropathy characterized in NTP (1997) as scattered foci of regenerative tubules (with cytoplasmic basophilia, 

increased nuclear/cytoplasmic ratio, and occasionally thickened basement membranes and intraluminal protein 

casts). 

Note: Conversions from drinking water concentrations to mg/kg-day performed by study authors. 

Conversion from ppm to mg/m3 is 1 ppm = 3.031 mg/m3

. 

Table 1-3. Changes in kidney tumors in animals following exposure to 

tert-butanol 

Results 

Renal tubule 

hyperplasia Male (standard and 

extended Renal tubule 

Dose evaluation Renal tubule adenoma 

(mg/kg-d) combined) adenoma (single) (multiple) 

0 14/50 (2.3) 7/50 1/50 

90 20/50 (2.3) 7/50 4/50 

200 17/50 (2.2) 10/50 9/50* 

420a 25/50* (2.8) 10/50 3/50 

Renal tubule 

adenoma (single 

Dose Renal tubule or multiple) or 

(mg/kg-d) carcinoma carcinoma 

0 0/50 8/50 

90 2/50 13/50 

200 1/50 19/50* 

420a 1/50 13/50 

Female 

Renal tubule 

Dose Renal tubule Renal tubule adenoma 

(mg/kg-d) hyperplasia adenoma (single) (multiple) 

0 0/50 0/50 0/50 

180 0/50 0/50 0/50 

330 0/50 0/50 0/50 

650a 1/50 (1.0) 0/50 0/50 

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Reference and study design 

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 

2 years 
 

 

 

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Toxicological Review of tert-Butyl Alcohol 

Reference and study design Results 

Dose 

(mg/kg-d) 

Renal tubule 

carcinoma 

Renal tubule 

adenoma (single 

or multiple) or 

carcinoma 

0 0/50 0/50 

180 0/50 0/50 

330 0/50 0/50 

650a 0/50 0/50 

Based on standard and extended evaluations (combined). Results do not 

include the animals sacrificed at 15 months. 

Hard et al. (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 

(single) 

Renal tubule 

adenoma 

(multiple) 

Renal tubule 

carcinoma 

Renal tubule 

adenoma 

(single or 

multiple) or 

carcinoma 

Dose 

(mg/kg-d) 

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) 

B6C3F1 mouse; 60/sex/treatment 

Drinking water (0, 5, 10, or 

20 mg/mL) 

M: 0, 540, 1,040, or 2,070a 

mg/kg-d 

F: 0, 510, 1,020, or 2,110 mg/kgd 

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. 

* Statistically significant p ≤ 0.05 as determined by the study authors.
	 a The high-dose group had an increase in mortality.
 

Note: Conversions from drinking water concentrations to mg/kg-d performed by study authors.
 

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Sources: (A) Acharya et al. (1997); (1995); (B) Hard et al. (2011)*; (C) Lyondell 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. 

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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 

Relative weight; F Rat 

Absolute/relative weight; M Mouse 

Absolute weight; F Mouse 

Relative weight; F Mouse 

100 1,000 10,000

Concentration (mg/m3) 

Absolute weight; M Rat 

Relative weight; M Rat 

Absolute weight; F Rat 

Source: NTP (1997). 

Figure 1-6. Exposure-response array of kidney effects following inhalation 

exposure to tert-butanol (13-week studies, no chronic studies available). 

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Mode of Action Analysis—Kidney Effects 

a) α2u-Globulin-Associated Renal Tubule Nephropathy and Carcinogenicity 

One disease process to consider when interpreting kidney effects in rats is related to the 

accumulation of α2u-globulin protein. α2u-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 α2u-globulin commonly detected in male rat urine is considered specific to that sex and 

species. Exposure to chemicals that induce α2u-globulin accumulation can initiate a sequence of 

histopathological events leading to kidney tumorigenesis. Because α2u-globulin-associated renal 

tubule nephropathy and carcinogenicity occurring in male rats are presumed not to be relevant for 

assessing human health hazards (U.S. EPA, 1991a), evaluating the data to determine if α2u-globulin 

plays a role is important. The role of α2u-globulin accumulation in the development of renal tubule 

nephropathy and carcinogenicity observed following tert-butanol exposure was evaluated using the 

U.S. EPA (1991a) Risk Assessment Forum Technical panel report, Alpha2u-Globulin: Association with 

the evidence reported in these studies is specific to α2u-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 α2u-globulin renal tubule nephropathy, as described by U.S. 

EPA (1991a), is as follows. Chemicals that induce α2u -globulin accumulation do so rapidly. 

α2u-Globulin accumulating in hyaline droplets is deposited in the S2 (P2) segment of the proximal 

tubule within 24 hours of exposure. Hyaline droplets are a normal constitutive feature of the 

mature male rat kidney; they are particularly evident in the S2 (P2) segment of the proximal tubule 

and contain α2u-globulin (U.S. EPA, 1991a). Abnormal increases in hyaline droplets have more than 

one etiology and can be associated with the accumulation of different proteins. As hyaline droplet 

deposition continues, single-cell necrosis occurs in the S2 (P2) segment, which leads to exfoliation 

of these cells into the tubule lumen within 5 days of chemical exposure. In response to the cell loss, 

cell proliferation occurs in the S2 (P2) segment after 3 weeks and continues for the duration of the 

exposure. After 2 or 3 weeks of exposure, the cell debris accumulates in the S3 (P3) segment of the 

proximal tubule to form granular casts. Continued chemical exposure for 3 to 12 months leads to 

the formation of calcium hydroxyapatite in the papillae which results in linear mineralization. After 

Chemically Induced Renal Toxicity and Neoplasia in the Male Rat. This report provides specific 

guidance for evaluating renal tubule tumors in male rats that are related to chemical exposure for 

the purpose of risk assessment, based on an examination of the potential involvement of 

α2u-globulin accumulation. 

Studies in the tert-butanol database evaluated and reported effects on the kidney, providing 

some evidence to evaluate this MOA. Additionally, several studies were identified that specifically 

evaluated the role of α2u-globulin in tert-butanol-induced renal tubule nephropathy and 

carcinogenicity (Borghoff et al., 2001; Williams and Borghoff, 2001; Takahashi et al., 1993). Because 

Toxicological Review of tert-Butyl Alcohol 

­

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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 (1991a) identified two questions that must be addressed to determine the extent 

to which α2u-globulin-mediated processes induce renal tubule nephropathy and carcinogenicity. 

First, whether the α2u-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 α2u-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 treated male rats, 

2) the protein in the hyaline droplets in treated male rats is α2uglobulin (i.e., 

immunohistochemical evidence), and 

3) several (but not necessarily all) additional steps in the pathological sequence appear in

treated male rats as a function of time, dose, and progressively increasing severity

consistent with the understanding of the underlying biology, as described above, and

illustrated in Figure 1-7. 

The available data relevant to this first question are summarized in Table 1-4, Figures 1-8 

and 1-9, and are evaluated below. 

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Figure 1-7. Temporal pathogenesis of α2u -globulin-associated nephropathy in 

male rats. α2u-Globulin synthesized in the livers of male rats is delivered to the 

kidney, where it can accumulate in hyaline droplets and be retained by epithelial 

cells lining the S2 (P2) segment of the proximal tubules. Renal pathogenesis

following continued tert-butanol exposure and increasing droplet accumulation can 

progress 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). 
 

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Table 1-4. Summary of data on the α2u-globulin process in male rats exposed 

to tert-butanol 

Duration Dose Results Comments Reference 

1) Hyaline droplets are increased in size and number 

10 d (inhalation) 0, 758, 1,364, 5,304 

mg/m3 

+ stat sig at 5,304 mg/m3; 

stat sig trend 

Borghoff et al. (2001) 

13 wk (inhalation) 0, 3,273, 6,368 mg/m3 – NTP (1997)a 

13 wk (oral) 0, 230, 490, 840, 

1,520, 3,610 mg/kg-d 

(+) observed in all but 

highest dose group 

NTP (1995) 

2) The protein in the hyaline droplets is α2uglobulin 

10 d (inhalation) 0, 758, 1,364, 5,304 

mg/m3 

+ stat sig at 5,304 mg/m3; 

stat sig trend 

Borghoff et al. (2001) 

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 

Acharya 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 – Acharya et al. (1997) 

10 d (inhalation) 0, 758, 1,364, 5,304 

mg/m3 

+ stat sig at all doses; stat 

sig trend 

Borghoff et al. (2001) 

13 wk (oral) 0, 230, 490, 840, 

1,520, 3,610 mg/kg-d 

+ elevated at 840 mg/kg-d; 

stat sig at 1,520 mg/kg-d 

NTP (1995) 

c) Development of intraluminal granular casts from sloughed cellular debris, with consequent tubule 

dilation 

13 wk (oral) 0, 230, 490, 840, 

1,520, 3,610 mg/kg-d 

–; (+)b NTP (1995); Hard et al. 

(2011)c 

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 

– 

2 yr (oral) 0, 90, 200, 420 

mg/kg-d 

+; (+) all doses stat sig 

NTP (1995); Hard et al. 

(2011)c 

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) 

+ = Statistically significant change reported in one or more treated groups.
 

(+) = Effect was reported in one or more treated groups, but statistics not reported.
 

– = No statistically significant change reported in any of the treated groups.
 a NTP (1997) did not observe any effects consistent with α2u-globulin nephropathy.
 b Precursors to granular casts reported. 

c Reanalysis of hematoxylin and eosin-stained kidney sections from all male control and 1,520 mg/kg-d groups, as
 

well as a representative sample of kidney sections stained with Mallory Heidenhain stain, from the 13-wk study 

from NTP (1995).
 d Reanalysis of slides for all males in the control and 420 mg/kg-day dose group and all animals with renal tubule
 

tumors from 2-yr NTP (1995). Protein casts reported, not granular casts. 

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Toxicological Review of tert-Butyl Alcohol 

 = 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 

× = 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 

*Hard et al. (2011) reported presence of “precursor granular casts.”
 

**NTP (1995) 13-wk study reported kidney mineralization but not linear mineralization.
 

Figure 1-8. Exposure-response array for effects potentially associated with 

α2u-globulin renal tubule nephropathy and tumors in male rats after 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 

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Figure 1-9. Exposure-response array for effects potentially associated with 

α2u -globulin renal tubule nephropathy and tumors in male rats after 

inhalation exposure to tert-butanol. 
 

Question One: Is the α2uglobulin 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 (U.S. EPA, 1991a). A statistically significant positive trend in the 

accumulation of large protein droplets with crystalloid protein structures was observed in kidneys 

of male rats exposed to inhalation concentrations of 758, 1,364, and 5,304 mg/m3 tert-butanol for 6 

hr/day for 10 days (Borghoff et al., 2001). These droplets were small and minimally present in 

control male rats and were not observed in female rats. Similarly, data from the 13-week NTP oral 

study (NTP, 1995; Takahashi et al., 1993; Lindamood et al., 1992) demonstrated an increase in the 

accumulation of hyaline droplets. The lowest dose of 230 mg/kg-day had minimal hyaline droplet 

formation compared to controls, although the next three doses (490, 840, and 1,520 mg/kg-day) 

NTP (1997), 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 α2u-globulin nephropathy in male rats. These results from NTP (1997), 

which are inconsistent with the findings of both Borghoff et al. (2001) and NTP (1995), do not 

appear to be due to differences in dose. Comparison of the oral and inhalation studies on the basis 

of tert-butanol blood concentration (see Supplemental Information) showed that a 13-week 

exposure in the range of the NTP (1995) doses of 490–840 mg/kg-day leads to the same average 

blood concentration as 6-hr/day, 5 day/week inhalation exposures to 3,273–6,368 mg/m3. The 

absence of similar histopathological findings in the 13-week inhalation NTP (1997) study compared 

to those reported in the two oral studies is not understood, but might be indicative of the strength 

of tert-butanol to induce, consistently, α2u-globulin nephropathy. The results from the two other 

studies (Borghoff et al., 2001; NTP, 1995) indicate that hyaline droplets increase in size and number 

in male rats following tert-butanol 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 α2u-globulin. Accumulated hyaline droplets with an α2u-globulin etiology can be confirmed 

by using immunohistochemistry to identify the α2u-globulin protein. Two short-term studies 

measured α2u-globulin immunoreactivity in the hyaline droplets of the renal proximal tubular 

epithelium (Borghoff et al., 2001; Williams and Borghoff, 2001). Following 10 days of inhalation 

exposure, Borghoff et al. (2001) did not observe an exposure-related increase in α2u-globulin using 

immunohistochemical staining. When using an enzyme-linked immunosorbent assay (ELISA), a 

more sensitive method of detecting α2u-globulin, however, a statistically significant positive 

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. 

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(3) The third criterion considered is whether several (but not necessarily all) additional 

events in the histopathological sequence associated with α2u-globulin nephropathy appear in male 

rats in a manner consistent with the understanding of α2u-globulin pathogenesis. Evidence of 

cytotoxicity and single-cell necrosis of the tubule epithelium subsequent to the excessive 

accumulation of hyaline droplets, with exfoliation of degenerate epithelial cells, should be 

observable after five days of continuous exposure, peaking at 19 days [reviewed in U.S. EPA 

(1991a)]. The formation and accumulation of granular casts from the exfoliated cellular debris 

would follow, causing tubule dilation at the junction of the S3 (P3) segment of the proximal tubule 

and the descending thin loop of Henle, and the commencement of compensatory cell proliferation 

within the S2 (P2) segment, both occurring after three weeks of continuous exposure. Following 

chronic exposures, this regenerative proliferation could result in focal tubular hyperplasia, and 

eventually progress to renal adenoma and carcinoma (Figure 1-7). 

Several of these steps were observed following tert-butanol exposure in male rats, most 

notably linear papillary mineralization and foci of tubular hyperplasia, consistent with the expected 

disease progression. Some lack of consistency and dose-related concordance, however, was evident 

across the remaining steps in the histopathological sequence. First, the accumulation of hyaline 

droplets and the concentrations of α2u-globulin in the hyaline droplets at doses that induced 

significant tumor formation in male rats were not significant. Next, necrosis or cytotoxicity was 

absent and only precursors to granular casts at stages well within the expected timeframe of 

detectability were present. Finally, a 13-week inhalation study found no evidence of α2u-globulin 

nephropathy (NTP, 1997), despite evaluating exposure concentrations predicted to result in similar 

blood tert-butanol levels as for the 13-week oral study (NTP, 1995), which reported increases in 

droplet accumulation and sustained regenerative tubule cell proliferation. A detailed evaluation 

and analysis of all the evidence relevant to this criterion follows. 

Toxicological Review of tert-Butyl Alcohol 

1 correlation of α2u-globulin concentration with dose of tert-butanol (determined by correlating with 

cell proliferation labeling indices) was observed, with accumulation of α2u-globulin protein 

statistically significant by pairwise comparison only in the highest dose group. No positive staining 

for α2u-globulin was observed in exposed female rats. In a follow-up study, Williams and Borghoff 

(2001) used a single gavage dose of 500 mg/kg [selected on the basis of results by NTP (1995) for 

induction of hyaline droplet accumulation], and reported a statistically significantly higher renal 

concentration of α2u-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 

α2u-globulin was reversible. These data indicate the presence of α2u-globulin in tert-butanol-treated 

male rats, although requiring a more sensitive method of detection of α2u-globulin than is typically 

used could indicate that tert-butanol is not a strong inducer of α2u-globulin accumulation. 

Therefore, the available data are sufficient to fulfill the second criterion for α2u-globulin present in 

the hyaline droplets, but suggest weak induction of α2u-globulin by tert-butanol. 

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1 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 (Acharya et al., 1997; NTP, 1995). Acharya 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 et al., 1989), the lack of consistency in these 

observations could be the result of both weak induction of α2u-globulin and a lack of later 

examinations. 

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11 a. Sustained regenerative cell proliferation also was not observed. Acharya et al. (1997) 

did not observe tert-butanol-induced proliferation following 10 weeks of oral exposure, 

but renal tubule proliferation was observed following another chemical exposure 

(trichloroacetic acid) in the same study. Therefore, the inference is that tert-butanol 

treatment did not induce regenerative tubule cell proliferation in male rats from this 

study. Borghoff et al. (2001) reported a dose-related increase in epithelial cell 

proliferation within the proximal tubule as measured by BrdU labeling indices in all

male rats exposed to tert-butanol via inhalation for 10 days. The study did not report

cytotoxicity, however, which, combined with the early time point makes unlikely that 

the cell proliferation was compensatory. NTP (1995) also observed increased cell 

proliferation in the renal tubule epithelium following 13-week oral exposures in male 

rats [only male rats were studied in the retrospective analysis by Takahashi et al. 

(1993) reported in NTP (1995)]. Proliferation was elevated at 840–1,520 mg/kg-day, a 

range higher than the single 575-mg/kg-day dose eliciting no such proliferative effect

(Acharya et al., 1997), as described above. NTP (1995) reported, however, that no

necrosis was observed, suggesting the proliferation was not regenerative. 

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b. Granular cast formation was not observed, although one study noted precursors to cast

formation. NTP (1995) did not observe the formation of granular casts or tubular 

dilation; however, Hard et al. (2011) reanalyzed the 13-week oral NTP data from male 

rats treated with 0 or 1,520 mg/kg-day and identified precursors to granular casts in

5/10 animals in the treated group. The significance of these granular cast precursors, 

described as sporadic basophilic tubules containing cellular debris, is unknown, because 

13 weeks of exposure is within the expected timeframe of frank formation and

accumulation of granular casts (≥3 weeks). Granular cast formation, however, might not

be significantly elevated with weak inducers of α2u-globulin (Short et al., 1986), which is 

consistent with the reported difficulty in measuring α2u-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 (U.S. EPA, 1991a). 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]. 

Toxicological Review of tert-Butyl Alcohol 

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1 d. Renal tubule hyperplasia was observed in the only available 2-year study. Renal tubule 

hyperplasia is the preneoplastic lesion associated with α2u-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. 

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7 The progression of histopathological lesions for α2u-globulin nephropathy is predicated on 

the initial response of excessive hyaline droplet accumulation (containing α2u-globulin) leading to 

cell necrosis and cytotoxicity, which in turn cause the accumulation of granular casts, linear 

mineralization, and tubular hyperplasia. Therefore, observations of temporal and dose-response 

concordance for these effects are informative for drawing conclusions on causation. 

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mineralization of tubules and focal tubular hyperplasia fall within the expected timeframe of the 

appearance of these lesions. Overall, no explicit inconsistencies are present in the temporal 

appearance of the histopathological lesions associated with α2u-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 

α2u-globulin nephropathy progression. Hyaline droplets were induced in the proximal tubule of all 

surviving male rats in the 13-week NTP oral study (NTP, 1995; Takahashi et al., 1993; Lindamood 

et al., 1992), although the incidence at the lowest dose was minimal, while the incidence at the 

three higher doses was more prominent. These results are discordant with the tumor results, given 

that all treated groups of male rats in the NTP 2-year oral bioassay had increased kidney tumor 

incidence, including the lowest dose of 90 mg/kg-day [according to the reanalysis by Hard et al. 

(2011)]. This lowest dose was less than the 230 mg/kg-day in the 13-week oral study that had only 

minimal hyaline droplet formation. Furthermore, although the incidence of renal tubule 

hyperplasia had a dose-related increase (NTP, 1995), a corresponding dose-related increase in the 

severity of tubular hyperplasia did not result. Severity of tubule hyperplasia was increased only at 

the highest dose, which was not consistent with renal tumor incidence. 

Although the histopathological sequence has data gaps, such as the lack of observable 

necrosis or cytotoxicity or granular casts at stages within the timeframe of detectability, overall, a 

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12 As mentioned above, most steps in the sequence of α2u-globulin nephropathy are observed 

at the expected time points following exposure to tert-butanol. Accumulation of hyaline droplets 

was observed early, at 12 hours following a single bolus exposure (Williams and Borghoff, 2001), 

and at 10 days (Borghoff et al., 2001) or 13 weeks (NTP, 1995) following continuous exposure; 

α2u-Globulin was identified as the protein in these droplets (Borghoff et al., 2001; Williams and 

Borghoff, 2001). Lack of necrosis and exfoliation might be due to the weak induction of α2u-globulin 

and a lack of later examinations. Granular cast formation was not reported by any of the available 

studies, which could also indicate weak α2u-globulin induction. Regenerative cell proliferation, 

which was not observed, is discussed in more detail below. Observations of the subsequent linear 

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1 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 et al., 2011; NTP, 1995). Several histopathological observations in 

exposed male rats were consistent with an α2u-globulin MOA. This evidence includes the increased 

size and number of hyaline droplets and the accumulated α2u-globulin protein in the hyaline 

droplets. Additionally, several subsequent steps in the histopathological sequence were observed. 

Overall, available data are sufficient for all three required criteria, suggesting that the α2u-globulin 

process is operative. Although the evidence indicates a role for α2u-globulin accumulation in the 

etiology of kidney tumors induced by exposure to tert-butanol in male rats, that tert-butanol is a 

weak inducer of α2u-globulin is plausible, considering the limited histopathological observations 

and uncertainty regarding the temporal and dose concordance of the lesions. 

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that might suggest the chemical belongs in a different class of suspected carcinogens: No data are 

available to evaluate these considerations. 

Biochemical information regarding binding of the chemical to the α2u-globulin protein: 

Williams and Borghoff (2001) report that tert-butanol reversibly and noncovalently binds to 

α2u-globulin in the kidneys of male rats. This provides additional support to the involvement of the 

α2u-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 α2u-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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14	 Question Two: Are the renal effects in male rats exposed to tert-butanol solely due to the α2u-globulin 

process? 

If the α2u-globulin process is operative, U.S. EPA (1991a) identifies a second question that 

must be answered regarding whether the renal effects are (a) solely due to the α2u-globulin 

process, (b) a combination of the α2u-globulin process and other carcinogenic processes, or (c) 

primarily due to other processes. U.S. EPA (1991a) states that additional data can help inform 

whether the α2u-globulin process is the sole contributor to renal tubule tumor development in male 

rats. These additional considerations are highlighted and discussed, where possible, in detail below. 

Hypothesis-testing of the α2u-globulin sequence of effects and structure-activity relationships 

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Toxicological Review of tert-Butyl Alcohol 

1 sustained cell replication is available, it does not specifically support α2u-globulin protein
 

accumulation.
 

Covalent binding to DNA or other macromolecules, suggesting another process leading to
 

tumors and genotoxicity (α2u-globulin-inducers are essentially nongenotoxic): One study (Yuan et al.,
 

2007) 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 literature. The few studies available to assess 

the genotoxic potential of tert-butanol primarily are negative, although a few studies report DNA 

damage induced by oxidative stress. DNA damage induced by oxidative stress is consistent with the 

decreased levels of glutathione in male rat kidneys reported by Acharya et al. (1995) after 10 weeks 

of tert-butanol exposure. This type of genetic damage would not necessarily preclude a role for 

α2u-globulin, but not enough information is available to determine whether oxidative stress could 

initiate or promote kidney tumors in concert with α2u-globulin accumulation in male rat kidneys. 

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were observed in female rats orally exposed to tert-butanol for 2 years. Although NTP (1995) 

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 

endpoint in males can be attributed to CPN or α2u-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 α2u-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 α2u-globulin 

indicate that tert-butanol induces renal tubule nephrotoxicity partially independently of 

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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. At 13 weeks (NTP, 1997, 1995) and 2 years (NTP, 

1995), oral and inhalation exposure increased the severity of nephropathy in male rats (NTP, 

1995). Similarly, the severity of nephropathy was increased in females at 2 years, but only the 

incidence of nephropathy was increased in females following a 13-week oral exposure (NTP, 1995). 

Increased incidences of suppurative inflammation and kidney transitional epithelial hyperplasia 

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1 α2u-globulin. The evidence that other processes might be responsible for the renal tubule 

nephrotoxicity thereby decreases the likelihood that α2u-globulin accumulation is solely 

responsible for the renal tubule tumors. 

Positive tubule tumor responses in female rats and other species implying that α2u-globulinrelated 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 

α2u-globulin process in tumor development. 

Summary and Conclusions for Question Two: 

Although the evidence suggests that tert-butanol induces α2u-globulin nephropathy, the 

data indicate that tert-butanol is a weak inducer of α2u-globulin and that this process is not solely 

responsible for the renal tubule nephropathy and carcinogenicity observed in male rats. The lack of 

compensatory cell proliferation in male rats and evidence of nephrotoxicity in female rats suggest 

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that other processes, in addition to the α2u-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 α2u-globulin pathway at high doses (>420 mg/kg-day), which results in 

tumor formation. Other, unknown pathways, however, could be operative at lower doses 

(<420 mg/kg-day), which contribute to renal tumor induction. 

b) Chronic Progressive Nephropathy and Renal Carcinogenicity 

There is scientific disagreement regarding the extent to which CPN can be characterized as 

a carcinogenic MOA suitable for analysis under the EPA’s cancer guidelines. Proponents of CPN as 

an MOA have developed an evolving series of empirical criteria for attributing renal tubule tumors 

to CPN. Hard and Khan (2004) proposed criteria for concluding that a chemical is associated with 

renal tubule tumors through an interaction with CPN. Hard et al. (2013) slightly revised and 

restated their criteria for considering exacerbation of CPN as an MOA for renal tubule tumors in 

rats. Table 1-5 lists these sets of 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. 

Source: Hard and Khan (2004) 

• 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) 

Hard et al. (2013) maintain knowing the detailed etiology or underlying mechanism for CPN 

is not necessary. Instead, identifying increased CPN with its associated increase in tubule cell 

proliferation as the key event is adequate. Nonetheless, Hard et al. (2013) also postulated a 

sequence of key events for renal tumorigenesis involving exacerbation of CPN: 

• Exposure to chemical (usually at high concentrations);
 

• Metabolic activation (if necessary);
 

• Exacerbated CPN, including increased number of rats with end-stage renal disease;
 

• Increased tubule cell proliferation because more kidney is damaged due to CPN

exacerbation;
 

• Hyperplasia; and
 

• Adenoma (infrequently carcinoma). 

In contrast to Hard et al. (2013); Hard and Khan (2004),Melnick et al. (2013); Melnick et al. 

(2012) concluded, based on an analysis of 60 NTP studies, no consistent association exists between 

exacerbated CPN and the incidence of renal tubule tumors in rats. Without a consistent association 

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1	 and an understanding of its key events, they maintain that determining the human relevance of 

processes that might be occurring in rats is not possible. An earlier analysis of 28 NTP studies 

(Seely et al., 2002) 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 et al. (2013)
 

Setting aside the question of whether CPN is (Hard et al., 2013; Hard and Khan, 2004) or is
 

not (Melnick et al., 2013; Melnick et al., 2012) an MOA suitable for analysis, this section provides an 

analysis of the mechanistic data pertinent to CPN. EPA’s cancer guidelines (U.S. EPA, 2005a) define 

a framework for judging whether available data support a hypothesized MOA; the analysis in this 

section follows the structure presented in the cancer guidelines. 

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11 

12 Description of the hypothesized MOA. Under the EPA framework, toxicokinetic studies are 

important for identifying the active agent, but toxicokinetic events per se are not key events of an 

MOA. Thus, the EPA analysis of the MOA proposed by Hard et al. (2013) begins with 

(1) exacerbated CPN, including increased number of rats with end-stage renal disease, and 

proceeds via (2) increased tubule cell proliferation, (3) hyperplasia, and (4) adenoma, or 

infrequently, carcinoma. 

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16 

17 

18 Strength, consistency, specificity of association. The relationship between exacerbated CPN 

and renal tumors is moderate to strong in male rats in the NTP (1995) study. According to the NTP 

(1995) analysis, the mean CPN grades (same as “severity of nephropathy” reported by NTP) 

presented on a scale 1–4 for male rats with renal tumors were 3.5, 3.6, 3.7, and 3.4 at doses 0, 1.25, 

2.5, and 5 mg/mL. The mean CPN grades for male rats without renal tumors were 2.9, 2.8, 2.8, and 

3.2 for the same dose groups. The reanalysis of the NTP data by Hard et al. (2011) yielded similar 

numbers. The relationship between CPN and renal tumors, however, is neither consistent nor 

specific in the NTP (1995) study: No female rats developed renal tumors regardless of the presence 

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26 of relatively low-grade or relatively high-grade CPN. For example, in female rats surviving more 

than 700 days, the mean CPN grades were 1.7 and 3.2 at doses of 0 and 10 mg/mL, respectively, but 

no tumors developed in either group. 

Dose-response concordance. The dose-response relationships for CPN, renal tubule 

hyperplasia, and renal tubule tumors somewhat differ. 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 et al. (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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1 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 of 0, 1.25, 2.5, and 5 mg/mL and at 

2 years, increased to 3.0, 3.1, 3.1, and 3.3. Similarly, the severity of neoplastic lesions increased at 

the end of life. At the 15-month interim evaluation, only two rats had developed renal tubule 

hyperplasia and one other had a renal tubule adenoma; at 2 years, the incidences of these two 

lesions were much higher in all dose groups (see previous paragraph). These results are consistent 

with CPN as an age-related disease and with hyperplasia and tumors appearing near the end of life. 

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Biological plausibility and coherence. In general, the relationship between exacerbated CPN 

and renal tubule tumors in male rats appears plausible and coherent. Some patterns in the dose

response relationships for CPN, hyperplasia, and tumors are discrepant. Perhaps more importantly, 

the patterns also are discrepant for the relationships between CPN grades and renal tubule tumors 

in male and female rats. In addition, the increased incidences in renal tubule tumors in all exposed 

male rats exceed the 10% criterion proposed by Hard et al. (2013) (Table 1-5), even more so when 

making comparisons with the lower control tumor incidence from the Hard et al. (2011) reanalysis. 

Conclusions about the hypothesized CPN-related MOA 

As recommended by EPA’s cancer guidelines (U.S. EPA, 2005a), conclusions about the 

hypothesized MOA can be clarified by answering three questions presented below. 

(a) Is the hypothesized MOA sufficiently supported in the test animals? Exacerbated CPN 

leading to renal tubule tumors in male rats late in life appears to have some support. There is lack 

of consistency, however, between males and females and in the dose-response relationships 

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28 between CPN, hyperplasia, and adenomas. These inconsistencies make difficult attributing all renal 

tumors to either CPN or to α2u-globulin-related nephropathy (see previous section on α2u-globulin), 

raising the likelihood of another, yet unspecified MOA. 

(b) Is the hypothesized MOA relevant to humans? There is scientific disagreement on this 

question. Hard et al. (2013); Hard et al. (2009)cite several differences in pathology between rat CPN 

and human nephropathies in their arguments that CPN-related renal tumors in rats are not relevant 

to humans. On the other hand, Melnick et al. (2013); Melnick et al. (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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1 (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 Melnick et al. (2013); Melnick et al. (2012), who 

argued against dismissing renal tubule tumors in rats that can be related to exacerbated CPN. It also 

can be reconciled with Hard et al. (2013), who, while maintaining that these tumors are not 

relevant to humans, also allow that there is no generally accepted MOA for CPN akin to that for α2u

globulin-related nephropathy. Hard et al. (2013) made this statement after reporting on the 

collective experience of national and international health agencies worldwide with the use of CPN 

as an MOA. Of 21 substances that exacerbated CPN and caused renal tumors, most were multisite 

carcinogens, and other tumor sites contributed to the evaluations. Only two assessments explicitly 

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Relevant to this last point, IARC (1999) developed a consensus statement that listed 

considerations for evaluating α2u-globulin-related nephropathy in rats, which was based on the 

work of 22 scientists, including three who were co-authors of Hard et al. (2013) and two who were 

co-authors of Melnick et al. (2013); Melnick et al. (2012). A similar broad-based consensus that 

defines a sequence of key events for exacerbated CPN, distinguishes it more clearly from α2u

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 α2uglobulin 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 α2uglobulin process is operating, although 

based on further analysis this chemical appears to be a weak inducer of α2uglobulin-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 α2u-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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16 considered CPN as a renal tumor mechanism. One was the assessment of ethylbenzene by the 

German Federal Institute for Occupational Safety and Health, in which the agency concluded that 

the kidney tumors were associated with the high, strain-specific incidence of CPN that is unknown 

for humans [as discussed in Hard et al. (2013)]. The other was the IRIS assessment of 

tetrahydrofuran, for which EPA found the evidence insufficient to conclude that the kidney tumors 

are mediated solely by the hypothesized MOAs (U.S. EPA, 2012d). Hard et al. (2013) attributed 

these different conclusions to either different data for the two chemicals or the lack of a generally 

accepted MOA akin to α2u-globulin-related nephropathy. 

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1 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 α2uglobulin 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 epithelial hyperplasia, mineralization, and 

kidney weight) were observed, predominantly in male and female rats across the multiple tert

butanol studies. The available evidence indicates that multiple processes induce the noncancer 

kidney effects. The group of lesions generally reported as “nephropathy,” is related to CPN. Because 

this disease is considered to be spontaneous and age-related in rats, the endpoints associated with 

CPN would not be relevant to humans for purposes of hazard identification. Additionally, two 

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endpoints in male rats (hyaline droplets, linear mineralization) are components of the α2u-globulin 

process. U.S. EPA (1991a) states that if the α2u-globulin process is occurring in male rats, the renal 

tubule effects associated with this process in male rats would not be relevant to humans for 

purposes of hazard identification. In cases such as these, the characterization of human health 

hazard for noncancer kidney toxicity would rely on effects not specifically associated with CPN or 

the α2u-globulin-process in male rats. 

Several other noncancer endpoints resulted from -butanol 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 of mineralization in male rats, and 

increased kidney weights in rats but not mice. Based on dose-related increases in these noncancer 

endpoints in rats, kidney effects are a potential human hazard of tert-butanol exposure. The hazard 

and dose-response conclusions regarding these noncancer endpoints associated with tert-butanol 

exposure are discussed further in Section 1.3.1. 

tert

The carcinogenic effects observed following -butanol exposure include increased 

incidences of renal tubule hyperplasia (considered a preneoplastic effect) and tumors in male rats. 

EPA concluded that the three criteria were met to indicate that an α2u-globulin process is operating. 

Because renal tubule tumors in male rats did not arise solely due to the α2u-globulin process and 

some of the tumors are attributable to other carcinogenic processes, such tumors remain relevant 

tert

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Toxicological Review of tert-Butyl Alcohol 

1 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-butanol exposure contains no human data, 

two oral subchronic and two oral chronic studies (one of each duration in rats and in mice) (NTP, 

1995), and two inhalation subchronic studies (one in rats and one in mice) (NTP, 1997). Studies 

employing short-term and acute exposures that examined thyroid effects are not included in the 

evidence table; they are discussed, however, in the text if they provide data informative of MOA or 

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hazard identification. No gross thyroid effects were reported in the 13-week evaluations of mice or 

rats following oral or inhalation exposure (NTP, 1997, 1995), and therefore subchronic studies 

were not included in the evidence table. The two available chronic studies are arranged in the 

evidence table by effect and then by species. The design, conduct, and reporting of each study were 

reviewed, each study was considered adequate to provide information pertinent to this assessment 

(Table 1-6 and Figure 1-10). 

 

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Thyroid effects, specifically follicular cell hyperplasia and adenomas, were observed in mice 

of both sexes after 2 years of oral exposure via drinking water (NTP, 1995). NTP (1995) noted that 

“[p]roliferation of thyroid gland follicular cells is generally considered to follow a progression from 

hyperplasia to adenoma and carcinoma.” Similarly, EPA considered the thyroid follicular cell 

hyperplasia to be 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 high-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 α2u-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 α2u-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 α2u 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 α2u-globulin process and some are attributable to

other carcinogenic processes, such tumors remain relevant for purposes of hazard identification, but a doseresponse 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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Toxicological Review of tert-Butyl Alcohol 

1 level than at the mid-dose level. The reason for the non-monotonicity is unclear, although it could 

be related to the increased mortality in the high-dose group (17/60 animals survived compared 

with 27/60 animals in the control group). The decreased survival of male mice might have affected 

the thyroid tumor incidences because animals could have died before tumors could develop. High 

mortality in the high-dose group occurred before tumors appeared; about 40% of the high-dose 

males died before the first tumor (a carcinoma) appeared in this group at week 83. By comparison, 

only ~10% of the control group had died by this time, and the single tumor in the control group 

was observed at study termination. Mortality in the exposed female mice was similar to controls. 

Table 1-6. Evidence pertaining to thyroid effects in animals following oral 

exposure to tert-butanol 

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Reference and study design Results 

Follicular cell hyperplasia 

NTP (1995) Incidenceb 

F344/N rat; 60/sex/treatment 

(10/sex/treatment evaluated at 15 

Males Females 

months) Dose Follicular cell Dose Follicular cell 

Drinking water (0, 1.25, 2.5, 5, or 10 (mg/kg-d) hyperplasia (mg/kg-d) hyperplasia 

mg/mL) 0 3/50 0 0/50 

M: 0, 90, 200, or 420a mg/kg-d 

F: 0, 180, 330, or 650a mg/kg-d 90 0/49 180 0/50 

2 years 200 

420a 

0/50 

0/50 

330 

650 a 

0/50 

0/50 

NTP (1995) Incidence (severity) 

B6C3F1 mouse; 60/sex/treatment 

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

Males Females 

M: 0, 540, 1,040, or 2,070a mg/kg-d Dose Follicular cell Dose Follicular cell 

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

2 years 0 

540 

1,040 

2,070 a 

5/60 (1.2) 

18/59* (1.6) 

15/59* (1.4) 

18/57* (2.1) 

0 

510 

1,020 

2,110 

19/58 (1.8) 

28/60 (1.9) 

33/59* (1.7) 

47/59* (2.2) 

Follicular cell tumors 

NTP (1995) Incidenceb 

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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) 

Follicular cell 

adenoma 

Follicular cell 

carcinoma 

Male 

0 2/50 2/50 

90 0/49 0/49 

200 0/50 0/50 

420 a 0/50 0/50 

Female 

0 1/50 1/50 

180 0/50 0/50 

330 1/50 1/50 

650 a 0/50 0/50 

NTP (1995) Incidence 

B6C3F1 mouse; 60/sex/treatment 

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

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

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

2 years 

Dose 

(mg/kg-d) 

Follicular cell 

adenoma 

Follicular cell 

carcinoma 

Follicular cell 

adenoma or 

carcinoma 

(mortality 

adjusted rates) c,d 

Animals 

surviving to 

study 

termination 

Male 

0 1/60 0/60 1/60 (3.6%) 

540 0/59 0/59 0/59 (0.0%) 

1,040 4/59 0/59 4/59 (10.1%) 

2,070 a 1/57 1/57 2/57 (8.7%) 

Female 

0 2/58 0/58 2/58 (5.6%) 

510 3/60 0/60 3/60 (8.6%) 

1,020 2/59 0/59 2/59 (4.9%) 

2,110 9/59* 0/59 9/59* (19.6%) 

27/60 

36/60 

34/60 

17/60 

36/60 

35/60 

41/60 

42/60 

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aThere was a significant decrease in survival in the high-dose group. bResults do not include the animals sacrificed at 15 months. cMortality-adjusted rates were not calculated by study authors for follicular cell carcinoma. The mortality-adjusted rates for the 

incidence of adenomas are the same as the combined rates, with the exception of the male high-dose group, where the rate 

for adenomas alone was 5.9%. 

dCochran-Armitage trend test was applied to mortality-adjusted thyroid tumor incidences, by applying the NTP adjusted rates 

to the observed numbers of tumors to estimate the effective number at risk in each group. For male mice, p = 0.041; for 

female mice, p = 0.028.* Statistically significant p ≤ 0.05 as determined by the study authors. 

Note: Conversions from drinking water concentrations to mg/kg-d performed by study authors. 
 

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 = exposures at which the endpoint was reported statistically significant by study authors 

 = exposures at which the endpoint was reported not statistically significant by study authors 

Hyperplasia; M mouse 

Hyperplasia; F mouse 

NONCANCER 

Hyperplasia; M rat 

Hyperplasia; F rat 

10 100 1,000 10,000

Dose (mg/kg-day) 

Adenoma; M mouse 

Adenoma; F mouse 

Adenoma; M rat 

Adenoma; F rat 

CANCER 

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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1 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). 

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To determine if the thyroid follicular cell tumors result from a chemically induced 

antithyroid MOA, U.S. EPA (1998a) requires that the 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) dose correlation among the 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 tert-butanol exposure is discussed below. 

1) Increases in cell growth (required) 

U.S. EPA (1998a) considers increased absolute or relative thyroid weights, histological 

indicators of cellular hypertrophy and hyperplasia, DNA labeling, and other measurements (e.g., Ki

67 or proliferating cell nuclear antigen expression) to be indicators of increased cell growth. Only a 

few studies (NTP, 1997, 1995) have evaluated the thyroid by routine histological examination 

following tert-butanol exposure, and none investigated specific molecular endpoints. None of the 

available long-term studies measured thyroid weight in mice, likely due to the technical limitations 

involved, and no thyroid effects were attributed to tert-butanol exposure in rats treated up to 2 

years (NTP, 1997, 1995). 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 drinking water exposure to tertbutanol- (NTP, 1995). The increase was dose 

dependent in female mice with a slight increase in severity in the highest dose, while male mice 

experienced a similar magnitude of hyperplasia induction at all doses, with increased severity at 

the highest dose (NTP, 1995). Thyroid follicular cell hyperplasia was not observed in any mouse 

study with less than 2 years of exposure: no treatment-related histological alterations in the thyroid 

of tert-butanol-treated (2 or 20 mg/mL) female mice after 3 or 14 days of drinking water exposure 

(Blanck et al., 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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1 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 et al. (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 although T3 and T4 levels were decreased 

after 14 days, this perturbation likely was not in excess of the range of homeostatic regulation in 

female B6C3F1 mice and thus not likely to induce compensatory thyroid follicular cell proliferation. 

Multiple lines of evidence support this observation: (1) TSH levels were unaffected, indicating that 

the decrease in T3 and T4 levels was not severe enough to stimulate increased TSH secretion by the 

pituitary; (2) thyroid hyperplasia was not induced in this study, or any others exposing mice for 

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fact be due to a decrease in thyroid hormone production, resulting from some, as of yet, 

uninvestigated molecular interactions of tert-butanol in the thyroid, pituitary, or hypothalamus. 

The absence of information regarding thyroid hormone levels in male mice and lack of 

molecular studies evaluating exposures >2 weeks in female mice are significant deficiencies in the 

available database. Together, although small decreases in some thyroid hormone levels have been 

reported in female mice, the available evidence is inadequate to determine if tert-butanol 

negatively affects the pituitary-thyroid signaling axis in female mice; furthermore, no evidence was 

available to evaluate this effect in male mice. 

3) Site(s) 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 (Roques et al., 2013; Qatanani et al., 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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2.5–13 weeks, suggesting that thyroid proliferation was either not induced by the hormone 

fluctuations or that any follicular cell proliferation during this period was too slight to be detected 

by routine histopathological examination; (3) the maximal decrease in T3 or T4 hormone levels 

induced by tert-butanol exposure after 14 days (i.e., ~20%) was well within the range of fluctuation 

in T3 and T4 hormone levels reported to occur between the 3- and 14-day control groups [15–40%; 

(Blanck et al., 2010)]. Although the lower T3 and T4 levels following tert-butanol were later 

attributed by the study authors to an increase in liver metabolism (see next section), they could in 

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1 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 (Blanck et al., 2010). Relative liver weights increased in
 

male and female mice after 13 weeks of oral exposure (NTP, 1995) to higher doses than those 

evaluated by Blanck et al. (2010), although absolute liver weight measurements in treated animals 

showed little change from controls suggesting that the relative measures could have been related to 

decreases in body weight rather than specific liver effects. Relative (and absolute) liver weights 

were increased in female mice (only) after 13 weeks of inhalation exposure at the two highest 

concentrations (NTP, 1997); liver weight was not reported in mice orally exposed for 2 years (NTP, 

1995). No increase in mouse hepatocellular hypertrophic or hyperplastic histopathology was 

reported following 2.5 weeks to 2 years of exposure (NTP, 1997, 1995). In fact, the only liver 

pathology associated with tert-butanol exposure in these studies was an increase in fatty liver in 

male mice in the high-dose group after 2 years of oral exposure (NTP, 1995). Although increased 

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8 

receptor (CAR) activation; CAR can induce expression of a wide range of hepatic enzymes, including 

several CYPs along with thyroid hormone-metabolizing sulfotransferases (Roques et al., 2013). The 

only thyroid hormone-metabolizing enzyme induced by tert-butanol, however, was SULT1A1, 

which has been reported to be inducible in a CAR-independent manner in mice (Qatanani et al., 

2005). Based on alterations in hepatic phase I and phase II enzyme activities and gene expression, 

the above data suggest a possible role for increased thyroid hormone clearance in the liver 

following repeated tert-butanol exposure; however, the expression changes in these few enzymes 

are not supported by any liver histopathological effects in mice exposed for longer durations, so 

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fatty liver could indicate some non-specific metabolic alteration, the absence of a similar treatment

related effect in livers from female mice, which were sensitive to both thyroid follicular cell 

hyperplasia and tumor induction, suggests that it might not be related to the thyroid tumorigenesis. 

One study evaluated liver enzyme expression and found highly dose-responsive induction 

of a single phase I cytochrome p450 enzyme (CYP2B10) following 14 days of tert-butanol exposure 

in female mice, with much smaller increases in the expression of another phase I enzyme CYP2B9, 

and the phase II thyroid hormone-metabolizing enzyme, sulfotransferase 1A1 [(SULT1A1; Blanck et 

al. (2010)]. CYP2B enzyme induction is commonly used as an indication of constitutive androstane 

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31 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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Toxicological Review of tert-Butyl Alcohol 

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Toxicological Review of tert-Butyl Alcohol 

1 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 T4 and T3 

were decreased with both doses (2 and 20 mg/L), and TSH was unaffected at either dose (Blanck et 

al., 2010). Liver expression of CYP2B10 was increased in a dose-responsive manner, while 

SULT1A1 mRNA was induced by 20–30% at both doses (Blanck et al., 2010). As described above, 

induction of liver microsomal enzyme activity would manifest some manner of liver histopathology 

(Maronpot et al., 2010; U.S. EPA, 1998a; NTP, 1995). Toxicol Pathol 38:776-795), and consistent 

with this expected association, centrilobular hepatocellular hypertrophy was reported in 2/5 high

dose mice exposed for 2 weeks (Blanck et al., 2010). No liver histopathology, however, was 

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attributed to tert-butanol exposure in female mice exposed for 2.5 weeks to 2 years to comparable 

tert-butanol concentrations (NTP, 1997, 1995). Although liver enzyme levels and activity were not 

specifically evaluated following subchronic to chronic exposure, the lack of liver pathology suggests 

a comparable lack of enzyme induction. Conversely, no histopathological alterations were reported 

in the thyroids of female mice after 2 weeks of oral exposure at doses that elevated some liver 

enzyme levels (Blanck et al., 2010). 

Following 2 years of oral exposure, both follicular cell hyperplasia and follicular cell tumor 

incidence was increased in mice despite a lack of treatment-related liver pathology (NTP, 1995) 

(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 of liver, pituitary, and thyroid effects in female mice, and 

no evidence was available to evaluate these associations in male mice. 

5) Reversibility (required) 

Chemicals acting via an antithyroid MOA have effects (e.g., increased TSH levels, thyroid 

follicular cell proliferation) that are reversible after cessation of treatment (U.S. EPA, 1998a). 

Although increased TSH levels have not been demonstrated following tert-butanol exposure, 

thyroid follicular cell proliferation was observed following chronic exposure. As no studies have 

evaluated changes in thyroid hormones or thyroid histopathology after cessation of tert-butanol 

treatment, however, the available evidence is inadequate to evaluate reversibility of these effects. 

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31 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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1 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 associated with tert-butanol exposure are 

discussed as part of the overall weight of evidence for carcinogenicity in Section 1.3.2. 

1.2.3. Developmental Effects 

Synthesis of effects related to development 

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8 

Developmental effects of tert-butanol observed after oral exposure (liquid diets or gavage) 

in several mouse strains and one rat strain include measures of fetal loss or viability (e.g., increased 

number of resorptions, decreased numbers of neonates per litter) and decreased fetal body weight 

(Lyondell Chemical Co., 2004; Faulkner et al., 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 dose study reported a small increase in the incidence of variations of the skull or 

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13 Four studies evaluated developmental effects [three oral or inhalation developmental 

studies (Faulkner et al., 1989; Nelson et al., 1989; Daniel and Evans, 1982) and a one-generation, 

oral reproductive study (Lyondell Chemical Co., 2004)] in animals exposed to tert-butanol via liquid 

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 of 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. (1989), because it did not provide sufficient information 

on the dams to determine if fetal effects occurred due to maternal toxicity. 

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29 sternebrae in two mouse strains (Faulkner et al., 1989). Although variations in skeletal 

development were noted in the study, no malformations were reported. Similar developmental 

effects were observed after whole-body inhalation exposure in Sprague-Dawley rats for 7 

hours/day on gestation days (GDs) 1–19 (Nelson et al., 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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Toxicological Review of tert-Butyl Alcohol 
 

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(U.S. EPA, 1991b), “an integrated evaluation must be performed considering all maternal and 

developmental endpoints.” “[W]hen adverse developmental effects are produced only at doses that 

cause minimal maternal toxicity; in these cases, the developmental effects are still considered to 

represent developmental toxicity and should not be discounted.” Although, at doses of “excessive 

maternal toxicity...information on developmental effects may be difficult to interpret and of limited 

value.” In considering the fetal and maternal toxicity data following tert-butanol exposure, the 

severity of the maternal effects were minimal and therefore the developmental effects in the fetuses 

should not be discounted (U.S. EPA, 1991b). The observed fetal effects occurred, however, at doses 

resulting in maternal toxicity across all available studies. Therefore, whether the fetal effects are 

directly related to tert-butanol treatment or are secondary to maternal toxicity remains unclear. 

Toxicological Review of tert-Butyl Alcohol 

This document is a draft for review purposes only and does not constitute Agency policy.

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Toxicological Review of tert-Butyl Alcohol 

Table 1-7. Evidence pertaining to developmental effects in animals following 

exposure to tert-butanol 

Reference and study design Results 

Lyondell Chemical Co. (2004) 

Sprague-Dawley rat; 12/sex/treatment 

Gavage 0, 64, 160, 400, or 1,000 mg/kg-d 

F0 males: 9 weeks beginning 4 weeks prior 

to mating 

F0 females: 4 weeks prior to mating 

through PND21 

F1 Males and Females: 7 weeks 

(throughout gestation and lactation; 1 male 

and 1 female from each litter was dosed 

directly from PND 21-28) 

Response relative to control 

Dose 

(mg/kg-d) 0 64 160 400 1000 

Maternal effects 

Body weight gain GD 0-20 

0 −3 −4 0 −16* 

Food consumption GD 0-20 

0 0 0 +4 0 

Body weight gain PND 1-21 

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 mg/kg-d showed CNS effects (e.g., ataxia, lethargy) 

which were undetectable by 4-weeks of exposure in animals exposed to 400 

mg/kg-d but not those in the higher dose group. 

F1 effects 

Viability index (pup survival to PND4) 

96.4% 98.7% 98.2% 99.4% 74.1%* 

Lactation index (pup survival to PND21) 

100% 100% 100% 99.2% 98.8% 

Sex ratio (% males) 

54.4 52.3 50.9 53.4 52.1 

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

0 +6 +4 +7 −10 

Pup weight PND 28 relative to control (%) 

M: 0 +2 0 0 −12* 

F: 0 0 −4 −2 −8 

3 
 

 

Toxicological Review of tert-Butyl Alcohol 

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: 

Dose 

(mg/kg-d) 

Food consumption 

(mean g/animal/day) 

Body weight 

gain 

Number of litters 

(% pregnant 

dams) 

0 0 0 11 (77%) 

3,324 +2 −3 12 (80%) 

4,879 −3 −19 8 (53%) 

6,677 −4 −20 7 (47%) 

Authors note that lower food consumption in higher tert-butanol dose groups 

reflects problems with pair feeding and maternal sedation. 

Fetal 

Percent change compared to control: 

Dose 

(mg/kg-d) 

Number of 

neonates/litter 

Fetal body weight 

on PND 2 

0 0 0 

3,324 −1 −7 

4,879 −29 −19 

6,677 −49 −38 

Number of stillborn also increased with dose (3, 6, 14, and 20, respectively), but 

the number of stillborn per litter was not provided. The high dose also caused a 

delay in eye opening and a lag in weight gain during PND 2–10 (information was 

only provided in text or figures) 

Faulkner et al. (1989) Maternal results not reported. 

CBA/J mouse; 7 pregnant females in Fetal 

control, 12 pregnant females in treated Percent change compared to control: Incidence: 

Gavage (10.5 mmoles/kg twice a day); 

0 (tap water), 1,556 mg/kg-d Live 

GD 6–18 Dose 

(mg/kg-d) Resorptions/litter 

fetuses/ 

litter 

Fetal 

weight 

Sternebral 

variations 

Skull 

variations 

0 0 0 0 4/28 1/28 

1,556 +118* −41* −4 7/30 3/30 

Sternal variations: misaligned or unossified sternebrae 

Skull variations: moderate reduction in ossification of supraoccipital bone 

Number of total resorptions (10 resorptions/66 implants in controls, 37/94 

implants in treated) increased (p < 0.05) 

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Toxicological Review of tert-Butyl Alcohol 

Reference and study design Results 

Faulkner et al. (1989) Maternal results not reported. 

C57BL/6J mouse; 5 pregnant females in Fetal 

controls, 9 pregnant females treated Percent change compared to control: Incidence: 

Gavage (10.5 mmoles/kg twice a day) 

0 (tap water), 1,556 mg/kg-d Live 

GD 6–18 Dose 

(mg/kg-d) Resorptions/litter 

fetuses/ 

litter 

Fetal 

weight 

Sternebral 

variations 

Skull 

variations 

0 0 0 0 5/21 1/21 

1,556 +428* −58* −4 9/16 7/16 

Sternal variations: misaligned or unossified sternebrae 

Skull variations: moderate reduction in ossification of supraoccipital bone 

Number of total resorptions (4 resorptions/44 implants in controls, 38/68 

implants in treated) 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/m3) 

Number of live 

fetuses/litter 

Resorptions 

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/m3) 

Fetal weight 

(males) 

Fetal weight 

(females) 

Skeletal 

variation 

by litter 

Skeletal 

variation 

by 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. 

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* Statistically significant p ≤ 0.05 as determined by study authors. Conversions from diet concentrations to mg/kg-d 

performed by study authors. Conversion from ppm to mg/m3 is 1 ppm = 3.031 mg/m3 

. 

Note: Percentage change compared to control = (treated value – control value) ÷ control value × 100. 
 

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Toxicological Review of tert-Butyl Alcohol 

* 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); Lyondell Chemical Co. (2004)
 

Figure 1-11. Exposure-response array of developmental effects following oral 

exposure to tert-butanol. 

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Toxicological Review of tert-Butyl Alcohol 

Figure 1-12. Exposure-response array of developmental effects following 

inhalation exposure to tert-butanol. 

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Three studies evaluated neurodevelopmental effects (Nelson et al., 1991; Daniel and Evans, 

1982)[one in male rats; one in female rats] following tert-butanol exposure via liquid diet (i.e., 

maltose/dextrin) or inhalation. No epidemiology studies on neurodevelopment are available. The 

animal studies evaluating neurodevelopmental effects of tert-butanol contain study design 

limitations. Daniel and Evans (1982) had a small number of animals per treatment group, lacked 

comparison of treatment-related effects to controls for all endpoints investigated, and did not use 

long-term neurodevelopmental testing. The two studies by Nelson et al. (1991) evaluated 

neurodevelopmental effects after either paternal or maternal exposure but did not run the 

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 rotarod performance following oral or inhalation exposures and decreases in 

open field behavior and cliff avoidance 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 (1982) 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 et al. (1991) 

reported statistically significant changes in neurochemical measurements in the brain in offspring 

1 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 to maternal toxicity (U.S. EPA, 

1991b) and the evidence is considered suggestive of developmental toxicity. 

1.2.4. Neurodevelopmental Effects 

Synthesis of effects related to neurodevelopment 

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Toxicological Review of tert-Butyl Alcohol 

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1 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 nutrition and behavioral factors on the 

pups’ physiological and psychomotor development. Results indicated that pups fostered to control 

dams performed significantly better than those maintained with treated dams (Table 1-8)(Daniel 

and Evans, 1982). Data suggest that neurodevelopmental effects were not solely due to in utero 

exposure to tert-butanol (Daniel and Evans, 1982). Interpretation of these results is limited, 

however, as the neurodevelopmental data were presented only in figures and could not be 

compared with controls. 

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15 Table 1-8. Evidence pertaining to neurodevelopmental effects in animals 

16 following exposure to tert-butanol 

Toxicological Review of tert-Butyl Alcohol 

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1-54 DRAFT—DO NOT CITE OR QUOTE 

Reference and study design Results 

Daniel and Evans (1982) 

Swiss Webster (Cox) mouse; 15 pregnant 

dams/treatment 

Liquid diet (0, 0.5, 0.75, or 1.0%, w/v); GD6–20; 

after birth half the pups were nursed with their 

treated dams and the other half were fostered 

by untreated dams who recently gave birth 

0 (isocaloric amounts of maltose/dextrin), 

3,324, 4,879, or 6,677 mg/kg-d 

• a dose-dependent increase in righting reflex time, with more time 

needed in animals maintained with maternal dams 

• a dose-dependent decrease in open field behavior, with less 

activity in pups maintained with maternal dams 

• a dose-dependent decrease in rotarod performance with the 

pups from maternal dams having lower performances 

• a dose-dependent decrease in the amount of time the pups were 

able to avoid a cliff, with animals maintained with their maternal 

dams having less avoidance time 

Nelson et al. (1991) 

Sprague-Dawley rat; 15 pregnant 

dams/treatment 

Inhalation analytical concentration: 0, 6,000, or 

12,000 mg/m3; (dynamic whole body chamber) 

7 hr/d 

GD 1–19 

Data were not presented specifically by dose nor were any tables or figures 

of the data provided 

Maternal toxicity was noted by decreased food consumption and body 

weight gains 

Results in offspring 

• increase in rotarod performance in high-dose group (16 versus 26 

revolutions/min for controls and 12,000 mg/m3 animals, 

respectively) 

• decreased time held on wire in the performance ascent test in the 

low-dose group (16 sec versus 10 sec for controls and 1,750 

mg/m3 animals, respectively) 
 

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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 β-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 β-endorphin in the cerebellum at 12,000 mg/m3 

• 47% decrease in serotonin in the midbrain at 6,000 mg/m3 

* Statistically significant p ≤ 0.05 as determined by study authors. 

Note: Conversions from diet concentrations to mg/kg-d performed by study authors. 

Percentage change compared to control = (treated value – control value) ÷ control value × 100. 

Mechanistic Evidence 

No mechanistic evidence is available for reproductive, developmental, or 

neurodevelopmental effects. 

Integration of neurodevelopmental effects 

Neurodevelopmental effects, including decreased brain weight, changes in brain 

biochemistry, and changes in behavioral performances, have been observed. Each study evaluating 

neurodevelopmental effects, however, had limitations in study design, reporting, or both. In 

addition, results were not always consistent between studies or across dose. At this time, there is 

inadequate information to draw conclusions regarding neurodevelopmental toxicity. 
 

1 1.2.5. Reproductive Effects 

Synthesis of effects related to reproduction
 

Several studies evaluated reproductive effects [a one-generation, oral reproductive study
 

(Lyondell Chemical Co., 2004) 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-generation reproductive oral or inhalation 

studies and by having no human studies on reproduction. The design, conduct, and reporting of 

each study were reviewed, and each study was considered adequate to provide information 

pertinent to this assessment. 

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10 

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12 Reproductive endpoints, such as reproductive organ weights, estrous cycle length, and 

sperm effects were examined following either oral or inhalation exposure (Lyondell Chemical Co., 

2004; NTP, 1997, 1995) (Table 1-9;Figure 1-13; Figure 1-14). In males, the only significant effect 

observed was a slight decrease in sperm motility for F0 males treated with 1000 mg/kg-day of tert

butanol (Lyondell Chemical Co., 2004). No significant changes in sperm motility were reported 

following oral exposure in other rat studies or via inhalation exposure in mice or rats. In addition, 

the reduced motility in treated animals falls within the range of historical control data and, 

therefore, its biological significance is uncertain. In female B6C3F1 mice, estrous cycle length was 

increased 28% following oral exposure to 11,620 mg/kg-day (NTP, 1995). No significant changes in 

estrous cycle length were observed following oral exposure in rats, or inhalation exposure in mice 

or rats. 

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23 Table 1-9. Evidence pertaining to reproductive effects in animals following 

24 exposure to tert-butanol 

Reference and study design Results 

Male reproductive effects 

Lyondell 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) No significant effect on weights of male reproductive organs or sperm 

B6C3F1 mouse; 10/sex/treatment observed 

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 

NTP (1997) No significant effect on weights of male reproductive organs or sperm 

F344/N rat; 10/sex/treatment observed 

Inhalation analytical concentration: 0, 134, 272, 

542, 1,080, or 2,101 ppm (0, 406, 824, 1,643, Evaluations were performed only for concentrations ≥542 ppm 

3,273 or 6,368 mg/m3) (dynamic whole body (1,643 mg/m3) 

chamber) 

6 hr/d, 5 d/wk 

13 weeks 

Generation method (Sonimist Ultrasonic spray 

nozzle nebulizer), analytical concentration and 

method were reported 

NTP (1997) No significant effect on weights of male reproductive organs or sperm 

B6C3F1 mouse; 10/sex/treatment observed 

Inhalation analytical concentration: 0, 134, 272, 

542, 1,080, or 2,101 ppm (0, 406, 824, 1,643, Evaluations were performed only for concentrations ≥542 ppm 

3,273 or 6,368 mg/m3) (dynamic whole body (1,643 mg/m3) 

chamber) 

6 hr/d, 5 d/wk 

13 weeks 

Generation method (Sonimist Ultrasonic spray 

nozzle nebulizer), analytical concentration and 

method were reported 

Female reproductive effects 

Lyondell Chemical Co. (2004) Pregnancy index 

Sprague-Dawley rat; 12/sex/treatment 

Gavage 0, 64, 160, 400, or 1,000 mg/kg-d 91.7% 91.7% 100% 100% 91.7% 

F0 females: 4 weeks prior to mating through 

PND21 

NTP (1995) No significant effect on female estrous cycle (0, −2, −4, 0, +8 % change 

F344/N rat; 10/sex/treatment relative to control) 

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 

NTP (1995) ↑ length of estrous cycle 

B6C3F1 mouse; 10/sex/treatment 

Drinking water (0, 2.5, 5, 10, 20, or 40 mg/mL) Response relative to control: 0, +5, +5, +5, +6, +28*% 

F: 0, 500, 820, 1,660, 6,430, 11,620a mg/kg-d 

13 weeks 

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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) 

B6C3F1 mouse; 10/sex/treatment 

Inhalation analytical concentration: 0, 134, 272, 

542, 1,080, or 2,101 ppm (0, 406, 824, 1,643, 

3,273 or 6,368 mg/m3) (dynamic whole body 

chamber) 

6 hr/d, 5 d/wk 

13 weeks 

Generation method (Sonimist Ultrasonic spray 

nozzle nebulizer), analytical concentration and 

method were reported 

No significant effect on female estrous cycle (0, −3, −9, −5 % change relative 

to control) 

Evaluations were only performed for concentrations ≥542 ppm 

(1,643 mg/m3) 

* Statistically significant p ≤ 0.05 as determined by the study authors. 

Notes: Conversions from drinking water concentrations to mg/kg-d performed by study authors. 

Conversion from ppm to mg/m3 is 1 ppm = 3.031 mg/m3. 

Percentage change compared to control = (treated value – control value) ÷ control value × 100 

 

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Sources: (A) Lyondell 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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Figure 1-14. Exposure-response array of reproductive effects following 

inhalation exposure to tert-butanol. 

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1 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 (Lyondell Chemical Co., 2004; NTP, 1995). No two-generation
 

reproductive studies are available that evaluate oral or inhalation exposure. In males, the only 

observed effect was a slight decrease in sperm motility for F0 males in the highest dose group of
 

rats treated with tert-butanol. This effect was not observed, however, in other studies with orally
 

treated rats and mice or in rats exposed via inhalation. In females, NTP (1995) reported an
 

increased length of the estrous cycle in the highest dose group of orally exposed mice. This effect
 

was not observed in similarly treated rats or in mice and rats exposed via inhalation.
 

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1.2.6. Other Toxicological Effects 

Effects other than those related to kidney, thyroid, reproductive, developmental, and 

neurodevelopmental toxicity were observed in some of the available rodent studies; these include 

liver and urinary bladder effects. Due to a lack of consistency in the liver effects and minimal to 

mild effects with a lack of progression in urinary bladder, however, inadequate information is 

available to draw conclusions regarding liver or urinary bladder toxicity at this time. 

Additionally, central nervous system (CNS) effects similar to those caused by ethanol 

(animals appearing intoxicated and having withdrawal symptoms after cessation of oral or 

inhalation exposure) were observed. Due to study quality concerns (e.g., lack of data reporting, 

small number of animals per treatment group), however, adequate information to assess CNS 

toxicity is unavailable at this time. For more information on these other toxicological effects, see 

Appendix B.3. 

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22 1.3. INTEGRATION AND EVALUATION 

23 1.3.1. Effects Other Than Cancer 

Kidney effects were identified as a potential human hazard of tert-butanol exposure based 

on several endpoints, including suppurative 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) (ATSDR, 1996). 

Several effects were observed in the kidneys of rats. Based on mechanistic evidence 

indicating that an α2u-globulin-related process is operating in male rats (Hard et al., 2011; Cirvello 

et al., 1995; NTP, 1995; Lindamood et al., 1992), any kidney effects associated with α2u-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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1 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 to human hazard characterization. 

These effects therefore are suitable for consideration for dose-response analysis and derivation of 

reference values, in Section 2. 

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There is suggestive evidence of developmental effects associated with tert-butanol 

exposure. Increased fetal loss, decreased fetal body weight, and increases in skeletal variations in 

exposed offspring were observed following exposure to relatively high doses of tert-butanol during 

gestation.. These effects are similar to the developmental effects observed with MTBE exposure 

(e.g., decreased fetal body weight and increases in skeletal variations) (ATSDR, 1996). 

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- and two-generation reproductive/developmental studies on ETBE (Gaoua, 2004a, 

b), providing further support for the lack of evidence supporting reproductive or developmental 

effects as possible human hazards following tert-butanol exposure. 

At this time, there is inadequate information to draw conclusions regarding 

neurodevelopmental effects as a human hazard of tert-butanol exposure. Although 

neurodevelopmental effects have been 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 (NTP, 1995) 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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1 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. 

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6 1.3.2. Carcinogenicity 

Summary of evidence 

In F344/N rats, administration of tert-butanol in drinking water increased the incidence of 

renal tubule tumors, mostly adenomas, in males; no renal tumors in females were reported (Hard et 

al., 2011; NTP, 1995). As discussed in Section 1.2.1, some of these tumors might be associated with 

α2u-globulin nephropathy, an MOA considered specific to the male rat (U.S. EPA, 1991a). Evidence 

in support of this hypothesized MOA includes the accumulation of hyaline droplets in renal tubule 

cells, the presence of α2u-globulin in the hyaline droplets, and additional aspects associated with 

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14 α2u-globulin nephropathy, including linear papillary mineralization and foci of tubular hyperplasia. 

Other evidence, however, is not supportive: The accumulation of hyaline droplets was minimal; 

concentrations of α2u-globulin were low at doses that induced tumors; and no significant necrosis 

or cytotoxicity was associated with compensatory regenerative proliferation or induction of 

granular casts observed within a timeframe consistent with α2u-globulin-mediated nephropathy. 

Renal tumors also could be associated with chronic progressive nephropathy, but the data on CPN 

are not coherent: Dose-response relationships for CPN, renal tubule hyperplasia, and renal tubule 

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21 tumors were different; in addition, CPN was nearly as severe in female rats as in male rats, yet no 

female rats developed renal tumors. Thus, some renal tumors may be attributable to α2u-globulin 

nephropathy and some to other, yet unspecified, processes. Taken together, and according to EPA’s 

guidance on renal tumors in male rats (U.S. EPA, 1991a), renal tumors induced by tert-butanol are 

relevant for human hazard identification. 

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26 In B6C3F1 mice, administration of tert-butanol in drinking water increased the incidence of 

thyroid follicular cell adenomas in females, and adenomas or carcinomas (only one carcinoma 

observed) in males (NTP, 1995), as discussed in Section 1.2.2. According to EPA’s thyroid tumor 

guidance (U.S. EPA, 1998a), chemicals that produce thyroid tumors in rodents might pose a 

carcinogenic hazard to humans. 

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31 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-1 mice resulted in 

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1	 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 carcinogenic to humans is appropriate when 

the evidence is “adequate to demonstrate carcinogenic potential to humans” but does not support 

the descriptor carcinogenic to humans. One example from the cancer guidelines is “an agent that has 

tested positive in animal experiments in more than one species, sex, strain, site, or exposure route, 

with or without evidence of carcinogenicity in humans.” tert-Butanol matches the conditions of this 

example, having increased tumor incidences in two species, in both sexes, and at two sites. 

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when the evidence raises “a concern for potential carcinogenic effects in humans” but is not 

sufficient for a stronger conclusion. The results for tert-butanol raise a concern for cancer but none 

of the effects is particularly strong. The kidney tumors resulted, in part, from an MOA that is specific 

to male rats, while no kidney tumors occurred in female rats. The thyroid tumors induced in male 

and female mice were almost entirely benign. In addition, while MTBE was also associated with 

male rat kidney tumorigenesis, there is little coherence of results between tert-butanol and ETBE 

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21 associated tumorigenesis in rats. MTBE or ETBE effects following chronic oral exposure in mice 

have not been investigated, however, so no evidence exists to evaluate the coherence of the thyroid 

tumorigenesis observed following tert-butanol exposure in B6C3F1 mice. 

These considerations, interpreted in light of the cancer guidelines, support the conclusion 

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. 

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29 The descriptor suggestive evidence of carcinogenic potential applies to all routes of human 

exposure. Oral administration of tert-butanol to rats and mice induced tumors at sites beyond the 

point of initial contact, and inhalation exposure for 13 weeks resulted in absorption and 

distribution of tert-butanol into the systemic circulation, as discussed in Section 1.2.1. 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 (U.S. EPA, 1991a) advises that unless the relative contribution of 

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1 α2u-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, 1998a) 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 among thyroid, pituitary, and liver 

effects in female mice, and no data are available to evaluate the potential for antithyroid effects in 

male mice. Data are not adequate to conclude that thyroid hormone changes exceed the range of 

homeostatic regulation or to evaluate effects on extrahepatic sites involved in thyroid-pituitary 

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disruption. Also, no data are available to evaluate reversibility of effects upon cessation of exposure. 

Thus, according to EPA’s thyroid tumor guidance, concluding that the thyroid tumors result from 

thyroid-pituitary disruption is premature, and dose-response analysis should use linear 

extrapolation. 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 data that would allow for the identification of populations with 

increased susceptibility to tert-butanol exposure exist. In vitro studies have implicated the liver 

microsomal mixed function oxidase (MFO) system, namely CYP450 (Cederbaum et al., 1983; 

Cederbaum and Cohen, 1980), as playing a role in the metabolism of tert-butanol. No studies, 

however, have identified the specific CYPs responsible for the biotransformation of tert-butanol. 

Pharmacokinetic differences among the fetus, newborns, children, and the aged might alter 

responses to chemicals compared to adults, resulting in differences in health effects. In the 

presence of environmental chemicals, metabolic homeostasis is maintained by the liver’s ability to 

detoxify and eliminate xenobiotics. This process is accomplished, in part, by the expression of 

xenobiotic metabolizing enzymes and transporters (XMETs), which metabolize and transport 

xenobiotics and determine whether exposure will result in altered responses. The expression of 

XMETs, including various CYPs, has been found to be underexpressed in the mouse fetus and 

neonate (Lee et al., 2011) and decreased in older mice (Lee et al., 2011) and rats (Lee et al., 2008). 

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, 1998a). In addition, the data on 

tert-butanol mutagenicity are inconclusive. 

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Collectively, there is little evidence on tert-butanol itself to identify susceptible populations 

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2. DOSE-RESPONSE ANALYSIS 

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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), lowestobserved-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. Lyondell 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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1 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. 

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In the NTP (1995) 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 months, and 2 years of treatment 

(NTP, 1995). Specific endpoints chosen for dose-response analysis were absolute kidney weight 

(observed in males and females), kidney suppurative inflammation (observed in females), and 

kidney transitional epithelial hyperplasia (observed in males and females). For absolute kidney 

weight, data from 15 months was selected as described in Section 1.2.1; for the other endpoints, 

data at the longest duration of 2 years were selected. 

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considered biologically significant, a BMR of 1 standard deviation from the control mean for 

continuous data or a BMR of 10% extra risk for dichotomous data is used to estimate the BMD and 

BMDL, and also to facilitate a consistent basis of comparison across endpoints, studies, and 

assessments. Endpoint-specific BMRs, where feasible, are described further below. When modeling 

was feasible, the estimated BMDLs were used as points of departure (PODs); the PODs are 

summarized in Table 2-1. Further details including the modeling output and graphical results for 

the model selected for each endpoint can be found in Appendix C of the 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 et al., 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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2.1.2. Methods of Analysis 

No biologically based dose-response models are available for tert-butanol. In this situation, 

EPA evaluates a range of dose-response models thought to be consistent with underlying biological 

processes to determine how best to empirically model the dose-response relationship in the range 

of the observed data. The models in EPA’s Benchmark Dose Software (BMDS) were applied. 

Consistent with EPA’s Benchmark Dose Technical Guidance (U.S. EPA, 2012b), the BMD and the 

BMDL are estimated using a benchmark response (BMR) to represent a minimal, biologically 

significant level of change. In the absence of information regarding the level of change that is 

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1 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, 2011). The preferred approach is 

physiologically based toxicokinetic modeling (PBPK). Other approaches include using chemicalspecific information in the absence of a complete PBPK model. As discussed in Appendix B of the 

Supplemental Information, human PBPK models for inhalation of ETBE or inhalation and dermal 

exposure to MTBE have been published, which include tert-butanol submodels. A validated human 

PBPK model for tert-butanol, however, is not available for extrapolating doses from animals to 

humans. In lieu of either chemical-specific models or data to inform the derivation of human 

equivalent oral exposures, body weight scaling to the 3⁄4 power (i.e., BW3/4) is applied to extrapolate 

toxicologically equivalent doses of orally administered agents from adult laboratory animals to 

adult humans for the purpose of deriving an oral RfD. 

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Using a standard BWa of 0.25 kg for rats and a BWh of 70 kg for humans (U.S. EPA, 1988), 

the resulting DAF is 0.24 for rats. Applying this DAF to the POD identified for effects in adult rats 

yields a PODHED as follows (see Table 2-1): 

PODHED = Laboratory animal dose (mg/kg-day) × 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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animals were converted to HEDs employing a standard dosimetric adjustment factor (DAF) derived 

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 DAF = (BW 1/4 / BW 1/4 a h ), 

where 

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 BWh = human body weight 

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Table 2-1. Summary of derivations of points of departure following oral 

exposure for up to 2 years 

Endpoint and 

reference 

Species/ 

sex Modela BMR 

BMD 

(mg/kg-d) 

BMDL 

(mg/kg-d) 

PODADJb (m 

g/kg-d) 

PODHED c (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 

10% 412 339 339 81.4 

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a

For modeling details, see Appendix C in Supplemental Information. b

For studies in which animals were not dosed daily, EPA would adjust administered doses to calculate the TWA 

daily doses prior to BMD modeling. However, this adjustment was not required for the NTP (1995) study. c

HED PODs were calculated using BW3/4 scaling (U.S. EPA, 2011). 

NA= not applicable 

2.1.3. Derivation of Candidate Values 

Consistent with EPA’s A Review of the Reference Dose and Reference Concentration Processes 

[(U.S. EPA, 2002); Section 4.4.5], also described in the Preamble, five possible areas of uncertainty 

and variability were considered when determining the application of UFs to the PODs presented in 

Table 2-1. An explanation follows: 

An intraspecies uncertainty factor, UFH, of 10 was applied to all PODs to account for 

potential differences in toxicokinetics and toxicodynamics in the absence of information on the 

variability of response in the human population following oral exposure to tert-butanol (U.S. EPA, 

2002). 

An interspecies uncertainty factor, UFA, of 3 (100.5 = 3.16, rounded to 3) was applied to all 

PODs because BW3/4 scaling was used to extrapolate oral doses from laboratory animals to humans. 
 

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1 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 (U.S. EPA, 2011) 

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 change in absolute kidney weight, a 

10% extra risk of kidney suppurative inflammation, and a 10% extra risk of transitional cell 

hyperplasia were selected assuming they represent minimal biologically significant response levels. 

A database uncertainty factor, UF, of 1 was applied to all PODs. The tert-butanol oral toxicity 

database includes chronic and subchronic toxicity studies in rats and mice (Acharya et al., 1997; 

Acharya et al., 1995; NTP, 1995) and developmental toxicity studies in rats and mice (Lyondell 

Chemical Co., 2004; Faulkner et al., 1989; Daniel and Evans, 1982). In the developmental studies, no 

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immune system effects would not be a sensitive target for tert-butanol. No adverse effects were 

reported in one- and two-generation reproductive/developmental studies on ETBE (Gaoua, 2004a, 

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 (Lyondell 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 

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≥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 immunotoxicity or multigenerational reproductive studies are 

available for tert-butanol. Studies on ETBE, which is rapidly metabolized to systemically available 

tert-butanol, are informative for consideration of the gaps in the tert-butanol oral database. The 

database for ETBE does not indicate immunotoxicity (Banton et al., 2011; Li et al., 2011), suggesting 

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additional studies would lead to identification of a more sensitive endpoint or a lower POD. 

Therefore, a database UFD of 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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bar corresponding to one data set described in Tables 2-1 and 2-2. 

Table 2-2. Effects and corresponding derivation of candidate values 

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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 BMDL10% 3 10 1 1 1 30 2 × 100 

Increased absolute kidney weight; 

female rat at 15 months 

NTP (1995) 

22 BMDL10% 3 10 1 1 1 30 7 × 10-1 

Kidney inflammation (suppurative); 

female rat NTP (1995) 

48 BMDL10% 3 10 1 1 1 30 2 × 100 

Kidney transitional epithelial 

hyperplasia; male rat 

NTP (1995) 

3.8 BMDL10% 3 10 1 1 1 30 1 × 10-1 

Kidney transitional epithelial 

hyperplasia; female rat 

NTP (1995) 

81 BMDL10% 3 10 1 1 1 30 3 × 100 
 

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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 × 10-1 to 3 × 100 mg/kg-day, for an overall 30-fold range. To estimate an 

exposure level below which kidney toxicity from tert-butanol exposure is not expected to occur, the 

RfD for increased incidence of transitional epithelial hyperplasia in male rats (1 × 10-1 mg/kg-day) 

was selected as the kidney-specific reference dose for tert-butanol. 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 kidneyspecific RfD is high. The PODs are based on benchmark dose modeling, and the candidate values are 

derived from a well-conducted long-term study, involving a sufficient number of animals per group, 

including both sexes, and assessing a wide range of kidney endpoints. 

Table 2-3. Organ/system-specific RfDs and overall RfD for tert-butanol 

 

 

Effect Basis RfD (mg/kg-day) 

Study exposure 

description Confidence 

Kidney Incidence of transitional 

epithelial hyperplasia (NTP 

(1995) 

1 × 10-1 Chronic High 

Overall RfD Kidney 1 × 10-1 Chronic High 

2.1.5. Selection of the Overall Reference Dose 

For tert-butanol, only kidney effects were identified as a hazard and carried forward for 

dose-response analysis; thus only one organ/system-specific reference dose was derived. 

Therefore, the kidney specific RfD of (1 × 10-1 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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Toxicological Review of tert-Butyl Alcohol 

1 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, 1994). 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 Administration (FDA) Good Laboratory 

Practice (GLP) regulations, involved a sufficient number of animals per dose group (including both 

sexes), and assessed a wide range of tissues and endpoints. Although the toxicity database for tert

butanol has some gaps, they are informed by the data on ETBE, a parent compound of tert-butanol. 

Therefore, the confidence in the database is high. Reflecting high confidence in the principal study 

and high confidence in the database, confidence in the RfD is high. 

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The inhalation RfC (expressed in units of mg/m3) is defined as an estimate (with 

uncertainty spanning perhaps an order of magnitude) of a continuous inhalation exposure to the 

human population (including sensitive subgroups) that is likely to be without an appreciable risk of 

deleterious effects during a lifetime. It can be derived from a NOAEL, LOAEL, or the 95% lower 

bound on the benchmark concentration (BMCL), with UFs generally applied to reflect limitations of 

the data used. 

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No previous oral assessment for tert-butanol is available in IRIS. 

2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER 

THAN CANCER 

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As for oral exposure, EPA identified kidney effects as a potential human hazard of tert

butanol inhalation exposure (summarized in Section 1.3.1). No chronic inhalation study for tert

butanol is available; 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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2.2.2. Methods of Analysis 

No biologically based dose-response models are available for tert-butanol. In this situation, 

EPA evaluates a range of dose-response models considered consistent with underlying biological 

processes to determine how best to model the dose-response relationship empirically in the range 

of the observed data. Consistent with this approach, all models available in EPA’s BMDS were 

evaluated. Consistent with EPA’s Benchmark Dose Technical Guidance (U.S. EPA, 2012b), the 

benchmark dose or concentration (BMD/C) and the 95% lower confidence limit on the BMD/C 

(BMD/CL) were estimated using a BMR of 10% change from the control mean for absolute kidney 

weight changes (as described in Section 2.1.2). As noted in Section 2.1.2., a BMR of 10% extra risk 

was considered appropriate for the quantal data on incidences of kidney suppurative inflammation 

and kidney transitional epithelial hyperplasia. The estimated BMD/CLs were used as PODs. 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. 

NTP (1997) was a well-designed subchronic study that evaluated the effect of tert-butanol 

exposure on multiple species at multiple inhalation doses. Absolute kidney weights were elevated 

(10–11%) in male rats exposed at ≥3,273 mg/m3; relative kidney weights were elevated (~9%) in 

males at ≥3,273 mg/m3 and in females at 6,368 mg/m3. Male rats exhibited an increase in the 

severity of chronic nephropathy (characterized as number of foci of regenerative tubules). Few 

endpoints were available for consideration in the subchronic inhalation study, but changes in 

kidney weights also were observed in the oral studies, such as the NTP (1995) 2-year drinking 

water study. 

1 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 (1997) 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 (1997) 

subchronic inhalation study shares many strengths with the 2-year drinking water study (NTP, 

1995), and is described in more detail below. 

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The RfC methodology provides a mechanism for deriving an HEC from the durationadjusted POD (BMCLADJ ) determined from the animal data. The approach takes into account the 

extra-respiratory nature of the toxicological responses and accommodates species differences by 

considering blood:air partition coefficients for tert-butanol in the laboratory animal (rat or mouse) 

and humans. According to the RfC guidelines (U.S. EPA, 1994), tert-butanol is a Category 3 gas 

because extra-respiratory effects were observed. Kaneko et al. (2000) measured a blood:gas 

partition coefficient [(Hb/g )A] of 531 ± 102 for tert-butanol in the male Wistar rat, while Borghoff et 

al. (1996) measured a value of 481 ± 29 in male F344 rats. A blood:gas partition coefficient 

[(Hb/g )H] of 462 was reported for tert-butanol in humans (Nihlén et al., 1995). The calculation 

(Hb/g )A ÷ (Hb/g )H was used to calculate a blood:gas partition coefficient ratio to apply to the 

delivered concentration. Because F344 rats were used in the study, the blood:gas partition 

coefficient for F344 rats was used. Thus, the calculation was 481 ÷ 462 = 1.04. Therefore, a ratio of 

1.04 was used to calculate the HEC. This allowed a BMCLHEC to be derived as follows: 

BMCL BMCLADJ (mg/m3 HEC = ) × (interspecies conversion) 

= BMCLADJ (mg/m3) × (481 ÷ 462) 

 = BMCL (mg/m3 ADJ ) × (1.04) 

Table 2-4 summarizes the sequence of calculations leading to the derivation of a humanequivalent POD for each inhalation data set discussed above. 

1 PODs from Inhalation Studies 

Because the RfC is applicable to a continuous lifetime human exposure but derived from 

animal studies featuring intermittent exposure, EPA guidance (U.S. EPA, 1994) provides 

mechanisms for (1) adjusting experimental exposure concentrations to a value reflecting 

continuous exposure duration (ADJ) and (2) determining a human equivalent concentration (HEC) 

from the animal exposure data. The former employs an inverse concentration-time relationship to 

derive a health-protective duration adjustment to time weight the intermittent exposures used in 

the studies. The modeled benchmark concentration from the inhalation study (NTP, 1997) was 

adjusted to reflect a continuous exposure by multiplying it by (6 hours per day) ÷ (24 hours per 

day) and (5 days per week) ÷ (7 days per week) as follows: 

BMCLADJ = BMCL (mg/m3) × (6 ÷ 24) × (5 ÷ 7) 

 = BMCL (mg/m3) × (0.1786) 

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Table 2-4. Summary of derivation of PODs following inhalation exposure 

Endpoint and 

reference 

Species/ 

Sex Modela BMR 

BMCb 

(mg/m3) 

BMCLb 

(mg/m3) 

PODADJb 

(mg/m3) 

PODHEC c ( 

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 

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a

For modeling details, see Appendix C in Supplemental Information.
 b

BMCs, BMCLs, and PODs were adjusted for continuous daily exposure by multiplying by (hours exposed per day /
 

24 hr) × (days exposed per week / 7 days).
 c

PODHEC calculated by adjusting the PODADJ by the DAF (=1.0, rounded from 1.04) for a Category 3 gas (U.S. EPA,
 

1994).
 d

BMD modeling failed to calculate a BMD value successfully (see Appendix C); POD calculated from no-observed
 

adverse effect level (NOAEL) of 6368 mg/m3

. 


 

BMCLHEC = BMCL /m3 ADJ (mg ) × (interspecies conversion) 

= BMCL 3 ADJ (mg/m ) × (481 ÷ 462) 

 = BMCLADJ (mg/m3) × (1.04) 

PODs from oral studies – use of PBPK model for route-to-route extrapolation 

A PBPK model for tert-butanol in rats has been modified, as described in Appendix B of the 

Supplemental Information. Using this model, route-to-route extrapolation of the oral BMDLs to 

derive inhalation PODs was performed as follows. First, the internal dose in the rat at each oral 

BMDL (assuming continuous exposure) was estimated using the PBPK model, to derive an “internal 

dose BMDL.” Then, the inhalation air concentration (again, assuming continuous exposure) that led 

to the same internal dose in the rat was estimated using the PBPK model. The resulting BMCL was 

then converted to a human equivalent concentration POD (PODHEC ) using the methodology 

previously described in “PODs from inhalation studies”: 

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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toxicological agent. Therefore, the concentration of tert-butanol in blood was selected as the dose 

metric.
 

Table 2-5 summarizes the sequence of calculations leading to the derivation of a humanequivalent inhalation POD from each oral data set discussed above.
 

Table 2-5. Summary of derivation of inhalation points of departure derived 

from route-to-route extrapolation from oral exposures 

Endpoint and reference Species/sex BMR 

BMDL 

(mg/kg-d) 

Internal dosea 

(mg/L) 

Equivalent 

PODHEC 

b 

(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 

(suppurative) NTP (1995) 

Rat/F 10% 200 12.6 359 

Kidney transitional epithelial 

hyperplasia 

NTP (1995) 

Rat/M 10% 16 0.745 26.1 

Kidney transitional epithelial 

hyperplasia 

NTP (1995) 

Rat/F 10% 339 27.9 638 

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a Average blood concentration of tert-butanol under continuous oral exposure at the BMDL. b Continuous inhalation human equivalent concentration that leads to the same average blood concentration of 

tert-butanol as continuous oral exposure at the BMDL. 

2.2.3. Derivation of Candidate Values 

In EPA’s A Review of the Reference Dose and Reference Concentration Processes [(U.S. EPA, 

2002); Section 4.4.5], also described in the Preamble, five possible areas of uncertainty and 

variability were considered. Several PODs for the candidate inhalation values were derived using a 

route-to-route extrapolation from the PODs estimated from the chronic oral toxicity study in rats 

(NTP, 1995) in the derivation of the oral RfD (Section 2.1). With the exception of the subchronic 

inhalation (NTP, 1997) study, the uncertainty factors (UFs) selected and applied to PODs derived 

from the chronic oral (NTP, 1995) study for route-to-route extrapolation are the same as those for 

the RfD for tert-butanol (see Section 2.1.3). The model used to perform this route-to-route 

extrapolation is a well-characterized model considered appropriate for the purposes of this 

assessment. One source of uncertainty regarding the route-to-route extrapolation is the assumption 

of that 100% of inhaled tert-butanol reaches the gas-exchange region, that is, 100% of the inhaled 
 

 

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TP (1995) 

ncreased ab

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idney inflammation 

suppurative); female rat 

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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 (NTP, 1997) 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 of a representative inhalation 

reference value for a specific hazard and subsequent overall RfC for tert-butanol. 

Figure 2-2 presents graphically the candidate values, UFs, and PODHEC values, with each bar 

corresponding to one data set described in Tables 2-4, 2-5, and 2-6. 

Table 2-6. Effects and corresponding derivation of candidate values 

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Endpoint (sex and species) and 

reference 

PODHEC a 

(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 BMCL10% 3 10 1 10 1 300 1 × 100 

Increased absolute kidney weight 

at 13 weeks; female rat 

NTP (1997) 

1137 NOAEL 3 10 1 10 1 300 4 × 100 

Increased absolute kidney weight 

at 15 months; male rat 

N

551 BMCL10% 3 10 1 1 1 30 2 × 101 * 

I solute kidney weight 

a s; female rat 

N

155 BMCL10% 3 10 1 1 1 30 5 × 100 * 

K

(

N

BMCL10% 3 10 1 1 1 30 1 × 101 * 

Kidney transitional epithelial 

hyperplasia; male rat 

NTP (1995) 

26.1 BMCL10% 3 10 1 1 1 30 9 × 10-1 * 

Kidney transitional epithelial 

hyperplasia; female rat 

NTP (1995) 

638 BMCL10% 3 10 1 1 1 30 2 × 101 * 
 

 

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*These candidate values are derived using route-to-route extrapolated PODs based on NTP’s chronic drinking 

water study. 

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 (1995) must be considered. Candidate values were derived for increased 

kidney weight observed in the subchronic inhalation study (NTP, 1997) and several kidney effects 

observed in the chronic oral study (NTP, 1995) in both sexes of rat, spanning a range from 9 × 10-1 

to 2 × 101 mg/m3, for an overall 20-fold range. To estimate an exposure level below which kidney 

toxicity from tert-butanol exposure is not expected to occur, the RfC for increased incidence of 

transitional epithelial hyperplasia in male rats (9 × 10-1 mg/m3) was selected as the kidney-specific 

RfC for tert-butanol, consistent with the selection of the kidney-specific RfD (see Section 2.1.4). As 

discussed in Section 2.1.4, unlike kidney suppurative inflammation, this effect was observed in both

sexes, with males appearing to be more sensitive than females. Additionally, it is based on a longer 

(chronic) duration and a more specific and sensitive indicator of kidney toxicity than the relatively 

non-specific endpoint of kidney weight change. Confidence in this kidney-specific RfC is high. The 

PODs are based on BMD modeling, and the candidate values are derived from a well-conducted 

study, involving a sufficient number of animals per group, including both sexes, assessing a wide 

range of kidney endpoints, and availability of a PBPK model for route-to-route extrapolation. 

Table 2-7. Organ/system-specific RfCs and overall RfC for tert-butanol 

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Effect Basis 

RfC 

(mg/m3) 

Study exposure 

description Confidence 

Kidney Incidence of transitional 

epithelial hyperplasia (NTP, 

1995) 

9 × 10-1 Chronic High 

Overall RfC Kidney 9 × 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 × 10-1 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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1 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 to the study used to derive the RfC, 

the overall database, and the RfC itself, as described in Section 4.3.9.2 of EPA’s Methods for 

Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA, 

1994). A PBPK model was utilized to perform a route-route extrapolation to determine a POD for 

the derivation of the RfC from the NTP (1995) oral study and corresponding critical effect. 

Confidence in the principal study (NTP, 1995) is high. This study was well-conducted, complied 

with FDA GLP regulations, involved a sufficient number of animals per group (including both 

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sexes), and assessed a wide range of tissues and endpoints. Although there are some gaps in the 

toxicity database for tert-butanol, these areas are informed by the data on ETBE, a parent 

compound of 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 of the modified PBPK model for the purposes of a route-to-route 

extrapolation, the overall confidence in the RfC for tert-butanol is high. 

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22 No previous inhalation assessment for tert-butanol is available in IRIS. 

23 2.2.8. Uncertainties in the Derivation of the Reference Dose and Reference Concentration 

The following discussion identifies uncertainties associated with the RfD and RfC for 

tert-butanol. To derive the RfD, the UF approach (U.S. EPA, 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 bioassay to human exposure, and the likely existence of a diverse 

human population of varying susceptibilities. These extrapolations are carried out with default 

approaches given the lack of data to inform individual steps. To derive the RfC, this same approach 

was applied, but a PBPK model was used to extrapolate from oral to inhalation exposure. 

The 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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Another uncertainty to consider relates to the MOA analysis conducted for the kidney 

effects. The assessment concluded that tert-butanol is a weak inducer of α2u-globulin which is 

operative in male kidney tumors; therefore, noncancer effects related to α2u-globulin were 

considered not relevant for hazard identification and, therefore, not suitable for dose response 

consideration. If this conclusion was incorrect and the noncancer effects characterized in this 

assessment as being related to α2u-globulin were relevant 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, if considered relevant, would likewise contribute to the hazard 

potential and dose-response analysis for the kidney-specific RfD and RfC. 

2.3. ORAL SLOPE FACTOR FOR CANCER 

The oral slope factor (OSF) is a plausible upper bound on the estimate of risk per mg/kg day 

of oral exposure. The OSF can be multiplied by an estimate of lifetime exposure (in mg/kg-day) to 

estimate the lifetime cancer risk. 

2.3.1. Analysis of Carcinogenicity Data 

As noted in Section 1.3.2, that there is “suggestive evidence of carcinogenic potential” for 

tert-butanol. The Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) state: 

When there is suggestive evidence, the Agency generally would not attempt a doseresponse 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 

Toxicological Review of tert-Butyl Alcohol 

1 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 rats appear more susceptible than mice, and 

males appear more susceptible than females to tert-butanol toxicity. However, the underlying 

mechanistic basis of these apparent differences is not understood. Most importantly, it is unknown 

which animal species and/or sexes may be more comparable to humans. 

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1 (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 et al., 2011). 

Based on a mode of action analysis, it was concluded that the α2u-globulin process was at 

least partially responsible for the male rat renal tumors, in addition to other, unknown, processes. 

Because the relative contribution of each process to tumor formation cannot be determined (U.S. 

EPA, 1991a), the male rat renal tumors are not considered suitable for quantitative analysis. 

Conversely, the mouse thyroid tumors are suitable for dose-response analysis and unit risk 

estimation, as described in Section 1.3.2. 

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2.3.2. Dose-Response Analysis—Adjustments and Extrapolations Methods 

The EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) recommend that the 

method used to characterize and quantify cancer risk from a chemical be determined by what is 

known about the MOA of the carcinogen and the shape of the cancer dose-response curve. EPA 

uses a two-step approach that distinguishes analysis of the observed dose-response data from 

inferences about lower doses (U.S. EPA, 2005a). Within the observed range, the preferred approach 

is to use modeling to incorporate a wide range of data into the analysis, such as through a 

biologically based model, if supported by substantial data. Without a biologically based model, as in 

the case of tert-butanol, a standard model is used to curve-fit the data and estimate a POD. EPA uses 

the multistage model in IRIS dose-response analyses for cancer (Gehlhaus et al., 2011) because it 

parallels the multistage carcinogenic process and fits a broad array of dose-response patterns. 

The second step, extrapolation to lower exposures from the POD, considers what is known 

about the modes of action for each effect. As above, a biologically based model is preferred (U.S. 

EPA, 2005a). Otherwise, linear low-dose extrapolation is recommended if the MOA of 

carcinogenicity is mutagenic or has not been established (U.S. EPA, 2005a). For -butanol, the 

mode(s) of carcinogenic action for thyroid follicular cell tumors has not been established (see 

Section 1.3.2). Therefore, linear low-dose extrapolation was used to estimate human carcinogenic 

risk. 

The dose-response modeling used administered dose because a PBPK model to characterize 

internal dosimetry in mice was not available. For the analysis of male mice thyroid tumors, the 

incidence data were adjusted to account for the increased mortality in high-dose male mice, relative 

to the other groups, that reduced the number of mice at risk for developing tumors. The Poly-3 

method (Bailer and Portier, 1988) was used to estimate the number at risk of developing tumors, 

by weighting the length of time each animal was on study (details in Appendix C of the 

tert

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Toxicological Review of tert-Butyl Alcohol 

1 Supplemental Information). This method was not applied to the female mice data because a 

difference in survival with increasing exposure was not appreciable and only one tumor, in the 

high-dose group, occurred before study termination. 

The data modeled and other details of the modeling are provided in Appendix C. The BMDs 

and BMDLs recommended for each data set are summarized in Table 2-8. The modeled tert-butanol 

PODs were scaled to HEDs according to EPA guidance (U.S. EPA, 2011, 2005a). In particular, the 

BMDL was converted to an HED by assuming that doses in animals and humans are toxicologically 

equivalent when scaled by body weight raised to the 3/4 power. Standard body weights of 0.025 kg 

for mice and 70 kg for humans were used (U.S. EPA, 1988). The following formula was used for the 

conversion of oral BMDL to oral HED for mouse endpoints: 

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HED in mg/kgday- = (BMDL in mg/kgday-) × (animal body weight/70)1/4 

 = (BMDL in mg/kgday-) × 0.14 

PODs for estimating low-dose risk were identified at doses at the lower end of the observed 

data, corresponding to 10% extra risk in female mice and 5% extra risk in male mice. 

2.3.3. Derivation of the Oral Slope Factor 

The PODs estimated for each tumor data set are summarized in Table 2-8. The lifetime oral 

cancer slope factor for humans is defined as the slope of the line from the lower 95% bound on the 

exposure at the POD to the control response (slope factor = BMR/BMDLBMR = 0.1/BMDL10). This 

slope represents a plausible upper bound on the true population average risk. Using linear 

extrapolation from the BMDL10, human equivalent oral slope factors were derived for male and 

female mice and are listed in Table 2-8. 

The oral slope factor based on the incidence of thyroid follicular cell adenomas in female 

mice was 5 × 104 per mg/kg-day. Despite high mortality in high-dose male mice, estimating slope 

factors using the poly-3 method was feasible for addressing competing risks. Whether using the full 

data set (including the only thyroid follicular cell carcinoma observed at the highest dose) or 

omitting the high-dose group altogether (under the assumption that mortality in this group was too 

extensive to interpret the results), oral slope factors based on the incidence of thyroid follicular cell 

adenomas or carcinomas in male mice were similar when rounded to one significant digit—5 × 10-4 

per mg/kg-day or 6 × 10-4 per mg/kg-day, respectively. 

The recommended slope factor for lifetime oral exposure to tert-butanol is 

5 × 10-4 per mg/kg-day, based on the thyroid follicular cell adenoma or carcinoma response in 

male or female B6C3F1 mice. This slope factor should not be used with exposures exceeding 

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 BMD10HED derived from the female mouse data set is 0.1/[0.14 × (2002 mg/kg-day)] = 

4 × 10–4 per mg/kg-day. 

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1 Table 2-8. Summary of the oral slope factor derivation 

2 

 

 

 

 

Tumor Species/ sex 

Selected 

model BMR 

BMD 

(mg/kgd) 

POD= 

BMDL 

(mg/kgd) BMDLHEDa 

(mg/kg-d) 

Slope factorb 

(mg/kg-day)-

1 

Thyroid follicular 

cell adenoma 

B6C3F1 

mouse/Female 

3° Multistage 10% 2002 1437 201 5 × 10-4 

Thyroid follicular 

cell adenoma or 

carcinoma 

B6C3F1 

mouse/Male 

All dose 

groups: 1° 

Multistage 

5%c 1788 787 110 5 × 10-4 

High dose 

omitted: 2° 

Multistage 

5%c 1028 644 90 6 × 10-4 

a

HED PODs were calculated using BW3/4 scaling (U.S. EPA, 2011).
 b

Human equivalent slope factor = 0.1/BMDL10HED; see Appendix C of the Supplemental Information for details of
 

modeling results.
 c

Because the observed responses were <10%, a BMR of 5% was used to represent the observed response range for
 

low-dose extrapolation; human equivalent slope factor = 0.05/BMDL5HED.
 

2.3.4.
 Uncertainties in the Derivation of the Oral Slope Factor 

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risks to human populations from exposure to tert-butanol. 

Table 2-9 summarizes several uncertainties that could affect the oral slope factor. There are 

no other chronic studies to replicate these findings or that examined other animal models, no data 

in humans to confirm a cancer response in general or the specific tumors observed in the NTP 

(1995) bioassay, and no other data (e.g., MOA) to support alternative approaches for deriving the 

oral slope factor. 

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Toxicological Review of tert-Butyl Alcohol 

Table 2-9. Summary of uncertainties in the derivation of the oral slope factor 

for tert-butanol 

Consideration and 

impact on cancer risk value Decision Justification 

Selection of tumor type and 

relevance to humans: 

Mouse thyroid tumors are the basis 

for estimating human cancer risk, as 

the fraction of rat kidney tumors not 

attributed to the male rat specific 

α2μ-globulin process could not be 

determined. Alternatively , 

quantifying rat kidney tumors could 

↑ 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: NTP (1995), oral (drinking NTP (1995), the only chronic bioassay 

No other studies are available. water) study, was selected 

to derive cancer risks for 

humans. 

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 α2μ-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 ↓ or ↑ 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 ↓ or ↑ slope 

factor (e.g., 3.5-fold ↓ [scaling by 

body weight] or ↑ 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 overor underestimate human equivalent risks. 

Dose-response modeling: 

Alternatives could ↓ or ↑ slope 

factor 

Used multistage doseresponse 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: Linear extrapolation of risk Linear low-dose extrapolation for agents 

↓ cancer risk estimate would be in low-dose region used without a known MOA is supported (U.S. 

expected with the application of U.S. EPA (1998a). EPA, 2005a) and recommended for rodent 

nonlinear low-dose extrapolation thyroid tumors arising from an unknown 

MOA (U.S. EPA, 1998a). 

Statistical uncertainty at POD: BMDL (preferred approach Limited size of bioassay results in sampling 

↓ oral slope factor 1.4-fold if BMD for calculating slope factor) variability; lower bound is 95% CI on 

used as the POD rather than BMDL administered exposure at 10% extra risk of 

thyroid tumors. 

Sensitive subpopulations: 

↑ 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. 

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2.3.5. Previous IRIS Assessment: Oral Slope Factor 

No previous cancer assessment for tert-butanol is available in IRIS. 

2.4. INHALATION UNIT RISK FOR CANCER 

The carcinogenicity assessment provides information on the carcinogenic hazard potential 

of the substance in question, and quantitative estimates of risk from oral and inhalation exposure 

may be derived. Quantitative risk estimates may be derived from the application of a low-dose 

extrapolation procedure. If derived, the inhalation unit risk (IUR) is a plausible upper bound on the 

estimate of risk per μg/m3 air breathed. 

No chronic inhalation exposure studies to tert-butanol are available. Lifetime oral exposure 

has been associated with increased renal tubule adenomas and carcinoma in male F344 rats, 

increased thyroid follicular cell adenomas in female B6C3F1 mice, and increased thyroid follicular 

cell adenomas and carcinomas in male B6C3F1 mice. 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 (1995) drinking water study in rats and mice was the only chronic bioassay available for 

dose-response analysis. Still, the rat PBPK model and kidney tumors from the NTP (1995) drinking 

water study were not used for route-to-route extrapolation because enough information to 

determine the relative contribution of α2u-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). 
Toxicological Review of tert-Butyl Alcohol 

1 2.4.1. Previous IRIS Assessment: Inhalation Unit Risk 

An inhalation cancer assessment for tert-butanol was not previously available on IRIS. 

2.5. APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS 

As discussed in the Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to 

Carcinogens (U.S. EPA, 2005b), either default or chemical-specific age-dependent adjustment 

factors (ADAFs) are recommended to account for early-life exposure to carcinogens that act 

through a mutagenic MOA. Because chemical-specific lifestage susceptibility data for cancer are not 

available, and because the MOA for tert-butanol carcinogenicity is not known (see Section 1.3.2), 

application of ADAFs is not recommended. 

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1 Acharya, S; Mehta, K; Rodrigues, S; Pereira, J; Krishnan, S; Rao, CV. (1995). Administration of 

subtoxic doses of t-butyl alcohol and trichloroacetic acid to male Wistar rats to study the

interactive toxicity. Toxicol Lett 80: 97-104. http://dx.doi.org/10.1016/0378

4274(95)03340-Q. 

Acharya, S; Mehta, K; Rodriguez, S; Pereira, J; Krishnan, S; Rao, CV. (1997). A histopathological study

of liver and kidney in male Wistar rats treated with subtoxic doses of t-butyl alcohol and

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U.S. EPA (U.S. Environmental Protection Agency). (1994). Methods for derivation of inhalation

reference concentrations (RfCs) and application of inhalation dosimetry [EPA Report]. 

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1 (EPA/600/8-90/066F). Washington, DC: U.S. Environmental Protection Agency, Office of 

Research and Development, Office of Health and Environmental Assessment, Environmental 

Criteria and Assessment Office. 

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assessment. (EPA/630/R-96/009). Washington, DC: U.S. Environmental Protection Agency,

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tox-risk-assessment.htm. 

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acceptability advice and health effects analysis on methyl tertiary-butyl ether (MTBE) [EPA

Report].

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tumors [EPA Report]. (EPA/630/R-97/002). Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). (1998b). Guidelines for neurotoxicity risk

assessment [EPA Report]. (EPA/630/R-95/001F). Washington, DC: U.S. Environmental 

Protection Agency, Risk Assessment Forum. http://www.epa.gov/risk/guidelines

neurotoxicity-risk-assessment. 

U.S. EPA (U.S. Environmental Protection Agency). (2000a). Science policy council handbook: Risk

characterization. (EPA/100/B-00/002). Washington, D.C.: U.S. Environmental Protection

Agency, Science Policy Council.

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http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=20533.

U.S. EPA (U.S. Environmental Protection Agency). (2002). A review of the reference dose and

reference concentration processes. (EPA/630/P-02/002F). Washington, DC: U.S. 

Environmental Protection Agency, Risk Assessment Forum.

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U.S. EPA (U.S. Environmental Protection Agency). (2005a). Guidelines for carcinogen risk

assessment [EPA Report]. (EPA/630/P-03/001F). Washington, DC: U.S. Environmental 

Protection Agency, Risk Assessment Forum. http://www2.epa.gov/osa/guidelines

carcinogen-risk-assessment. 

U.S. EPA (U.S. Environmental Protection Agency). (2005b). Supplemental guidance for assessing

susceptibility from early-life exposure to carcinogens. (EPA/630/R-03/003F). Washington, 

DC: U.S. Environmental Protection Agency, Risk Assessment Forum. 

http://www.epa.gov/raf/publications/pdfs/childrens_supplement_final.pdf. 

U.S. EPA (U.S. Environmental Protection Agency). (2006a). Approaches for the application of 

physiologically based pharmacokinetic (PBPK) models and supporting data in risk

assessment (Final Report) [EPA Report]. (EPA/600/R-05/043F). Washington, DC: U.S. 

Environmental Protection Agency, Office of Research and Development, National Center for 

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&Index=2000+Thru+2005&Docs=&Query=&Time=&EndTime=&SearchMethod=1&TocRest

rict=n&Toc=&TocEntry=&QField=&QFieldYear=&QFieldMonth=&QFieldDay=&IntQFieldOp

=0&ExtQFieldOp=0&XmlQuery=&File=D%3A%5Czyfiles%5CIndex%20Data%5C00thru05

%5CTxt%5C00000002%5C40000006.txt&User=ANONYMOUS&Password=anonymous&Sor

tMethod=h%7C

&MaximumDocuments=1&FuzzyDegree=0&ImageQuality=r75g8/r75g8/x150y150g16/i42

5&Display=p%7Cf&DefSeekPage=x&SearchBack=ZyActionL&Back=ZyActionS&BackDesc=R

esults%20page&MaximumPages=1&ZyEntry=1&SeekPage=x&ZyPURL.

U.S. EPA (U.S. Environmental Protection Agency). (2000b). Supplementary guidance for conducting

health risk assessment of chemical mixtures. (EPA/630/R-00/002). Washington, DC: U.S. 

Environmental Protection Agency, Risk Assessment Forum.

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Toxicological Review of tert-Butyl Alcohol 

1 Environmental assessment.
 

http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=157668.
 

U.S. EPA (U.S. Environmental Protection Agency). (2006b). A framework for assessing health risk of 

environmental exposures to children. (EPA/600/R-05/093F). Washington, DC: U.S.

Environmental Protection Agency, Office of Research and Development, National Center for

Environmental Assessment.
 

http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=158363.
 

U.S. EPA (U.S. Environmental Protection Agency). (2009). EPA’s Integrated Risk Information

System: Assessment development process [EPA Report]. Washington, DC. 

http://epa.gov/iris/process.htm.
 

U.S. EPA (U.S. Environmental Protection Agency). (2010). Integrated science assessment for carbon

monoxide [EPA Report]. (EPA/600/R-09/019F). Research Triangle Park, NC: U.S. 

Environmental Protection Agency, Office of Research and Development, National Center for

Environmental Assessment.
 

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U.S. EPA (U.S. Environmental Protection Agency). (2011). Recommended use of body weight 3/4 as

the default method in derivation of the oral reference dose. (EPA/100/R11/0001). 

Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum, Office of 

the Science Advisor. http://www.epa.gov/raf/publications/interspecies-extrapolation.htm.
 

U.S. EPA (U.S. Environmental Protection Agency). (2012a). Advances in inhalation gas dosimetry for

derivation of a reference concentration (RFC) and use in risk assessment. (EPA/600/R

12/044). Washington, DC. http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=244650.
 

U.S. EPA (U.S. Environmental Protection Agency). (2012b). Benchmark dose technical guidance. 

(EPA/100/R-12/001). Washington, DC: U.S. Environmental Protection Agency, Risk

Assessment Forum. http://www.epa.gov/raf/publications/benchmarkdose.htm.

U.S. EPA (U.S. Environmental Protection Agency). (2012c). Releases: Facility Report. Toxics Release 

Inventory. Available online at http://iaspub.epa.gov/triexplorer/tri_release.chemical 

(accessed

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29 U.S. EPA. (2012d). Toxicological review of tetrahydrofuran. In support of summary information on

the integrated risk information system (IRIS). (EPA/635/R-11/006F). Washington, DC: U.S. 

Environmental Protection Agency 

U.S. EPA (U.S. Environmental Protection Agency). (2014). Chemical Data Reporting 2012, reported

in the Chemical Data Access Tool. Available online at 

http://www.epa.gov/oppt/cdr/index.html (accessed 

Williams, TM; Borghoff, SJ. (2001). Characterization of tert-butyl alcohol binding to "alpha"2u

globulin in F-344 rats. Toxicol Sci 62: 228-235. http://dx.doi.org/10.1093/toxsci/62.2.228.
 

Yuan, Y; Wang, HF; Sun, HF; Du, HF; Xu, LH; Liu, YF; Ding, XF; Fu, DP; Liu, KX. (2007). Adduction of

DNA with MTBE and TBA in mice studied by accelerator mass Spectrometry. Environ

Toxicol 22: 630-635. http://dx.doi.org/10.1002/tox.20295.
 

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