DRAFT DELIBERATIVE: FOR INTERAGENCY REVIEW ONLY. 

DO NOT DISTRIBUTE OUTSIDE YOUR AGENCY. 

Interagency Review Draft 

www.epa.gov/iris 

Toxicological Review of tert-Butyl Alcohol (tert-Butanol) 

(CAS No. 75-65-0) 

In Support of Summary Information on the

Integrated Risk Information System (IRIS)

September 2014 

This document is an Interagency Science Consultation Review draft. This information is 

distributed solely for the purpose of pre-dissemination peer review under applicable information 

quality guidelines. It has not been formally disseminated by EPA. It does not represent and should 

not be construed to represent any Agency determination or policy. It is being circulated for review 

of its technical accuracy and science policy implications.

National Center for Environmental Assessment

Office of Research and Development

U.S. Environmental Protection Agency

Washington, DC

EPA/635/R-14/378
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...........................................................................................xvii

EXECUTIVE SUMMARY ........................................................................................................................... ES-16

LITERATURE SEARCH STRATEGY | STUDY SELECTION AND EVALUATION ............................................... LS-1

1. HAZARD IDENTIFICATION.................................................................................................................... 1-1

1.1.PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM............................................ 1-1

1.1.1. Kidney Effects.................................................................................................................... 1-1

1.1.2. Thyroid Effects................................................................................................................. 1-27

1.1.3. Reproductive and Developmental Effects....................................................................... 1-32

1.1.4. Carcinogenicity (other than in the kidney or thyroid)..................................................... 1-46

1.1.5. Other Toxicological Effects.............................................................................................. 1-46

1.2.INTEGRATION AND EVALUATION ............................................................................................... 1-60

1.2.1. Effects Other Than Cancer............................................................................................... 1-60

1.2.2. Carcinogenicity ................................................................................................................ 1-61

1.2.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes.............. 1-63

2. DOSE-RESPONSE ANALYSIS ................................................................................................................. 2-1

2.1.ORAL REFERENCE DOSE FOR EFFECTS 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 Proposed Overall Reference Dose .......................................................... 2-8

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

2.1.7. Previous IRIS Assessment.................................................................................................. 2-9

2.2.INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER THAN CANCER....................... 2-9

2.2.1. Identification of Studies and Effects for Dose-Response Analysis.................................... 2-9

2.2.2. Methods of Analysis........................................................................................................ 2-11

2.2.3. Derivation of Candidate Values....................................................................................... 2-15

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2.2.4. Derivation of Organ/System-Specific Reference Concentrations................................... 2-18

2.2.5. Selection of the Proposed Overall Reference Concentration ......................................... 2-18

2.2.6. Confidence Statement..................................................................................................... 2-19

2.2.7. Previous IRIS Assessment................................................................................................ 2-19

2.2.8. Uncertainties in the Derivation of the Reference Dose and Reference 

Concentration.................................................................................................................. 2-19

2.3.ORAL SLOPE FACTOR FOR CANCER............................................................................................. 2-20

2.3.1. Analysis of Carcinogenicity Data ..................................................................................... 2-20

2.3.2. Dose-Response Analysis—Adjustments and Extrapolations Methods........................... 2-21

2.3.3. Derivation of the Oral Slope Factor................................................................................. 2-22

2.3.4. Uncertainties in the Derivation of the Oral Slope Factor................................................ 2-23

2.3.5. Previous IRIS Assessment: Oral Slope Factor .................................................................. 2-24

2.4.INHALATION UNIT RISK FOR CANCER ......................................................................................... 2-24

2.4.1. Analysis of Carcinogenicity Data ..................................................................................... 2-25

2.4.2. Dose Response Analysis – Adjustments and Extrapolation Methods............................. 2-25

2.4.3. Inhalation Unit Risk Derivation........................................................................................ 2-26

2.4.4. Uncertainties in the Derivation of the Inhalation Unit Risk ............................................ 2-27

2.4.5. Previous IRIS Assessment: Inhalation Unit Risk............................................................... 2-29

2.5.APPLICATION OF AGE-DEPENDENT ADJUSTMENT FACTORS...................................................... 2-29

REFERENCES............................................................................................................................................... R-1

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

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TABLES

Table P-1. Physicochemical properties and chemical identity of tert-butanol..........................................xiii

Table ES-1. Summary of reference dose (RfD) derivation ......................................................................ES-2

Table ES-2. Summary of reference concentration (RfC) derivation........................................................ES-3

Table LS-1. Details of the search strategy employed for tert-butanol.................................................... LS-5

Table LS-2. Summary of additional search strategies for tert-butanol................................................... LS-6

Table LS-3. Questions and relevant experimental information for evaluation of experimental 

animal studies................................................................................................................ LS-7

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

Table 1-2. Changes in kidney histopathology in animals following exposure to tert-butanol ................. 1-5

Table 1-3. Changes in kidney tumors in animals following exposure to tert-butanol.............................. 1-9

Table 1-4. Additional kidney effects potentially relevant to mode of action in animals following 

exposure to tert-butanol ............................................................................................... 1-14

Table 1-5. Summary of data on the α2u-globulin process in male rats exposed to tert-butanol............. 1-16

Table 1-6. Summary of additional data informing the contribution of the α2u-globulin process on 

the renal tumor development in male rats exposed to tert-butanol............................ 1-19

Table 1-7. Evidence pertaining to thyroid effects in animals following oral exposure to tertbutanol........................................................................................................................... 1-29

Table 1-8. Evidence pertaining to reproductive effects in animals following exposure to tertbutanol........................................................................................................................... 1-34

Table 1-9. Evidence pertaining to developmental effects in animals following exposure to tertbutanol........................................................................................................................... 1-37

Table 1-10. Evidence pertaining to neurodevelopmental effects in animals following exposure to 

tert-butanol.................................................................................................................... 1-44

Table 1-11. Evidence pertaining to effects on body weight in animals following exposure to tertbutanol........................................................................................................................... 1-49

Table 1-12. Changes in liver weight in animals following exposure to tert-butanol .............................. 1-52

Table 1-13. Changes in liver histopathology in animals following exposure to tert-butanol................. 1-54

Table 1-14. Changes in urinary bladder histopathology in animals following oral exposure to 

tert-butanol.................................................................................................................... 1-56

Table 2-1. Summary of derivations of points of departure ....................................................................... 2-4

Table 2-2. Effects and corresponding derivation of candidate RfDs ......................................................... 2-6

Table 2-3. Organ/system-specific RfDs and proposed overall RfD for tert-butanol.................................. 2-8

Table 2-4. Summary of derivation of PODs following inhalation exposure............................................ 2-13

Table 2-5. Summary of derivation of inhalation points of departure derived from route-to-route 

extrapolation from oral exposures................................................................................ 2-14

Table 2-6. Effects and corresponding derivation of candidate values.................................................... 2-16

Table 2-7. Organ/system-specific RfCs and proposed overall RfC for tert-butanol................................. 2-18

Table 2-8. Summary of the oral slope factor derivations ........................................................................ 2-22

Table 2-9. Summary of uncertainties in the derivation of cancer risk values for tert-butanol ............... 2-23

Table 2-10. Summary of the inhalation unit risk derivation.................................................................... 2-27

Table 2-11. Summary of uncertainties in the derivation of cancer risk values for tert-butanol ............. 2-28

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

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FIGURES 

Figure LS-1. Study selection strategy. ..................................................................................................... LS-4

Figure 1-1. Exposure response array for kidney effects following oral exposure to tert-butanol.......... 1-10

Figure 1-2. Exposure-response array of kidney effects following subchronic inhalation exposure 

to tert-butanol (no chronic studies available). .............................................................. 1-11

Figure 1-3. Exposure-response array for components of α2u-globulin nephropathy and renal 

tumors in male rats after oral exposure to tert-butanol. .............................................. 1-17

Figure 1-4. Exposure-response array for components of α2u-globulin nephropathy and renal 

tumors in male rats after inhalation exposure to tert-butanol. .................................... 1-18

Figure 1-5. Exposure-response array of thyroid follicular cell effects following chronic oral 

exposure to tert-butanol. .............................................................................................. 1-31

Figure 1-6. Exposure-response array of reproductive and developmental effects following oral 

exposure to tert-butanol. .............................................................................................. 1-41

Figure 1-7. Exposure-response array of reproductive and developmental effects following 

inhalation exposure to tert-butanol. ............................................................................. 1-42

Figure 1-8. Exposure-response array of other effects following oral exposure to tert-butanol. ........... 1-58

Figure 1-9. Exposure-response array of other effects following inhalation exposure to tertbutanol........................................................................................................................... 1-59

Figure 2-1. Candidate RfD values with corresponding POD and composite UF. ....................................... 2-7

Figure 2-2. Candidate RfC values with corresponding POD and composite UF...................................... 2-17

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

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ABBREVIATIONS

α2u-g alpha2u-globulin 

ACGIH American Conference of Governmental 

Industrial Hygienists 

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 

BUN blood urea nitrogen 

BW body weight 

CA chromosomal aberration 

CASRN Chemical Abstracts Service Registry 

Number 

CBI covalent binding index 

CHO Chinese hamster ovary (cell line cells) 

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 

PCNA proliferating cell nuclear antigen 

POD point of departure 

POD[ADJ] duration-adjusted POD 

QSAR quantitative structure-activity 

relationship 

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 of America

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

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1

2 AUTHORS | CONTRIBUTORS | REVIEWERS

Assessment Team

Janice S. Lee, Ph.D. (Chemical Manager)

Keith Salazar, Ph.D*

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

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ontributors

Andrew Hotchkiss, Ph.D.

Channa Keshava, Ph.D.

Amanda Persad, Ph.D. 

U.S. EPA

Office of Research and Development

National Center for Environmental Assessment

Research Triangle Park, NC

roduction Team

Maureen Johnson

Vicki Soto 

U.S. EPA

Office of Research and Development

National Center for Environmental Assessment

Washington, DC

ontractor Support

Robyn Blain, Ph.D. 

Michelle Cawley* 

William Mendez, Jr., Ph.D.

Pam Ross 

ICF International

Fairfax, VA 

*Research Triangle Park, NC 

xecutive Direction

Kenneth Olden, Ph.D., Sc.D., L.H.D. (Center Director)

John Vandenberg, Ph.D, (National Program Director, HHRA)

Lynn Flowers, Ph.D., DABT (Associate Director for Health)

Vincent Cogliano, Ph.D. (IRIS Program Director—acting)

Samantha Jones, Ph.D. (IRIS Associate Director for Science)

Weihsueh A. Chiu, Ph.D. (Branch Chief)

U.S. EPA/ORD/NCEA

Washington, DC 

nternal Review Team

General Toxicology Workgroup

Inhalation Workgroup

Neurotoxicity Workgroup

PBPK Workgroup

Reproductive and Developmental 

U.S. EPA

Office of Research and Development

National Center for Environmental Assessment

Washington, DC

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

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Toxicology Workgroup

Statistical Workgroup

Toxicity Pathways Workgroup

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

Comments were submitted by:

Office of Children’s Health Protection, Washington, DC 

Office of Solid Waste and Emergency Response, Washington, DC

Region 2, New York, NY

Region 8, Denver, CO

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

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PREFACE 

This Toxicological Review critically reviews the publicly available studies on tert-butyl 

alcohol (tert-butanol) in order to identify its adverse health effects and to characterize exposureresponse relationships. It was prepared under the auspices of EPA's Integrated Risk Information 

System (IRIS) Program. The assessment covers an oral RfD, an inhalation RfC, and a cancer weight 

of evidence descriptor. 

The Toxicological Reviews for ethyl tert-butyl ether (ETBE) and tert-butanol are being 

developed simultaneously because they have a number of overlapping scientific issues:

• tert-Butanol is a metabolite of ETBE, so some of the toxicological effects of ETBE may be due 

to tert-butanol. Therefore, data on tert-butanol may inform hazard identification and doseresponse assessment of ETBE, and vice versa.

• The scientific literature for chemicals include data on α2u-globulin-related nephropathy. 

Therefore, a common approach was employed to evaluate those data as they relate to the 

mode of action for kidney effects.

• A combined PBPK model for ETBE and tert-butanol in rats was developed to support the 

dose-response assessments for these chemicals.

This assessment was conducted in accordance with EPA guidance, which is cited and 

summarized in the Preamble to IRIS Toxicological Reviews. This is the first IRIS assessment for this 

compound. The findings of this assessment and related documents produced during its 

development are available on the IRIS Web site (http://www.epa.gov/iris). 

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

tert-butanol, including draft literature searches and associated search strategies, evidence tables, 

and exposure-response arrays prior to the development of the IRIS assessment. All public 

comments provided were taken into consideration in developing the draft assessment. The 

complete set of public comments are available on the docket at http://www.regulations.gov

(Docket ID No. EPA-HQ-ORD-2013-0111).

In April 2011, the National Research Council (NRC) released its Review of the Environmental 

Protection Agency’s Draft IRIS Assessment of Formaldehyde. In addition to offering comments 

specifically about EPA’s draft formaldehyde assessment, the NRC made several recommendations 

to EPA for improving the development of IRIS assessments. EPA agreed with the recommendations 

and is implementing them consistent with the Panel’s “Roadmap for Revision,” which viewed the 

full implementation of their recommendations by the IRIS Program as a multi-year process. 

In response to the NRC’s 2011 recommendations, the IRIS Program has made changes to 

streamline the assessment development process, improve transparency, and create efficiencies in 

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

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1 the Program. The NRC has acknowledged EPA’s successes in this area. In May 2014, the NRC 

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released their report Review of EPA’s Integrated Risk Information System Process reviewing the IRIS 

assessment development process and found that EPA has made substantial improvements to the 

IRIS Program in a short amount of time. 

The draft tert-butanol assessment represents a significant advancement in implementing 

the NRC recommendations. This assessment is streamlined, and uses tables, figures, and 

appendices to increase transparency and clarity. It is structured to have distinct sections for the 

literature search and screening strategy, study selection and evaluation, hazard identification, and 

dose-response assessment. The assessment includes a comprehensive, systematic, and 

documented literature search and screening approach, provides the database search strategy in a 

table (databases, keywords), visually represents the inclusion and exclusion of studies in a flow 

diagram, and all of the references are integrated within the Health and Environmental Research 

Online (HERO) database. A study evaluation section provides a systematic review of 

methodological aspects of epidemiology and experimental animal studies, including study design, 

conduct, and reporting, that was subsequently taken into consideration in the evaluation and 

synthesis of data from these studies. The evidence is presented in standardized evidence tables, 

and exposure-response arrays. The hazard identification and dose-response sections include 

subsections based on organ/system-specific effects in which the evidence is synthesized within and 

integrated across all evidence for each target organ/systems. 

In the draft tert-butanol assessment, the IRIS Program has attempted to transparently and 

uniformly identify strengths and limitations that would affect interpretation of results. All animal 

studies of tert-butanol that were considered to be of acceptable quality, whether yielding positive, 

negative, or null results, were considered in assessing the evidence for health effects associated 

with chronic exposure to tert-butanol. These studies were evaluated for aspects of design, conduct, 

and reporting that could affect the interpretation of results and the overall contribution to the 

synthesis of evidence for determination of human hazard potential using the study quality 

considerations outlined in the Preamble. A brief summary of the evaluation is included in the 

section on methods for study selection and evaluation. 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 (that ultimately supported preferences for the studies and data 

relied upon) were included in the text where relevant. 

In this assessment, the IRIS Program is using existing guidelines to systematically approach 

the integration of noncancer human, animal, and mechanistic evidence. In conducting this analysis 

and developing the synthesis, the IRIS Program evaluates the data for the: strength of the 

relationship between the exposure and response and the presence of a dose-response relationship; 

specificity of the response to chemical exposure and whether the exposure precedes the effect; 

consistency of the association between the chemical exposure and response; and biological 

plausibility of the response or effect and its relevance to humans. The IRIS Program uses this 

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

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exposure.

The IRIS tert-butanol assessment provides a streamlined presentation of information, 

integrated hazard identification of all toxic effects, and derivation of organ/system-specific 

reference values. Additionally, consistent with the goal that assessments should provide a 

scientifically sound and transparent evaluation of the relevant scientific literature and presentation 

of the analyses performed, this assessment contains an expanded discussion of study selection and 

evaluation, as well as increased documentation of key assessment decisions.

For additional information about this assessment or for general questions regarding IRIS, 

please contact EPA’s IRIS Hotline at 202-566-1676 (phone), 202-566-1749 (fax), or 

hotline.iris@epa.gov. 

Chemical Properties

tert-Butanol is a white crystalline solid or colorless liquid (above 77°F) with a camphor-like 

odor that is highly flammable (NIOSH, 2005; IPCS, 1987a). tert-Butanol contains a hydroxyl 

chemical functional group and is miscible with alcohol, ether, and other organic solvents and 

soluble in water (IPCS, 1987a). Selected chemical and physical properties of tert-butanol are 

presented in Table P-1. 

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

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1 Table P-1. Physicochemical properties and chemical identity of tert-butanol

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Characteristic Information Reference

Chemical name tert-Butanol HSDB (2007)

Synonyms/Trade Names t-butyl alcohol; tert-Butanol; tert-butyl 

alcohol; t-Butyl hydroxide; 1,1-

Dimethylethanol; NCI-C55367; 2-

Methyl-2-propanol; tertiary butanol; 

Trimethyl carbinol; Trimethyl 

methanol, t-butyl alcohol, TBA

HSDB (2007)

IPCS (1987b)

Chemical Formula C4H10O HSDB (2007)

CASRN 75-65-0 HSDB (2007)

Molecular weight 74.12 HSDB (2007)

Melting point 25.7°C HSDB (2007)

Boiling point 82.41°C HSDB (2007)

Vapor pressure 40.7 mm Hg @ 25°C HSDB (2007)

Density/Specific Gravity 0.78581 HSDB (2007)

Flashpoint 11°C (closed cup) HSDB (2007)

Water solubility at 25°C 1 x 106 mg/L HSDB (2007)

Octanol/Water Partition 

Coefficient (Log KOW ) 0.35 HSDB (2007)

Henry’s Law Constant 9.05 x 10-6 atm-m3/mole HSDB (2007)

Odor threshold 219 mg/m3 HSDB (2007)

Conversion factors 1 ppm = 3.031 mg/m3

1 mg/m3 = 0.324 ppm IPCS (1987b)

Chemical structure HSDB (2007) 

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

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Uses

tert-Butanol is primarily an anthropogenic substance that is produced in large quantities 

(HSDB, 2007) from a number of precursors, including 1-butene, isobutylene, acetyl chloride and 

dimethylzinc, and tert-butyl hydroperoxide. The domestic production volume of tert-butanol, 

including imports, was approximately four 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 
Toxicological Review of tert-Butyl Alcohol

1 were used in the U.S. prior to 2007 at levels of more than 2 billion gallons annually. Current use 

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levels of MTBE and ETBE in the U.S. are much lower, but use in Europe and Asia remains strong1. 

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

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

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

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

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

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

compounds, and can be used for chemical extractions in pharmaceutical application (HSDB, 2007). 

Fate and Transport

Soil

The mobility of tert-butanol in soil is expected to be high due its low affinity for soil organic 

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

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

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

dissipation process (HSDB, 2007). tert-Butanol is a tertiary alcohol and this class of alcohols 

generally degrades more slowly in the environment compared to primary (e.g., ethanol) or 

secondary (e.g., isopropanol) alcohols. In anoxic soil conditions, the half-life of tert-butanol is 

estimated to be on the order of months (approximately 200 days). Microbial degradation rates are 

increased in soils supplemented with nitrate and sulfate nutrients (HSDB, 2007). 

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 is on the magnitude of 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 exists primarily as a vapor in the ambient atmosphere. Vapor-phase tertbutanol 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 over 

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

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

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

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

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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 wells throughout the United States (HSDB, 

2007). California’s Geotracker Database2 lists 3496 detections of tert-butanol in groundwater 

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

drinking water wells in the vicinity of landfills (U.S. EPA, 2012c). Additionally, tert-Butanol leaking

from underground storage tanks may be a product of MTBE and 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 and in the environment. 

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

to urban areas (HSDB, 2007). 

General Population Exposure

tert-Butanol exposure can occur in many different settings. Contamination resulting from 

leaking underground storage tanks could potentially result in exposure to a large number of people 

who get their drinking water from wells. Due to its high environmental mobility and resistance to 

biodegradation, tert-butanol has the potential to contaminate and persist in groundwater and soil; 

therefore, exposure through ingestion of contaminated drinking water is likely occurring (HSDB, 

2007). 

Contaminated food can also contribute to tert-butanol ingestion through its use as a coating 

in metallic and paperboard food containers (Cal/EPA, 1999). tert-Butanol has been detected in 

food, namely beer and chickpeas, and identified in mother’s milk (HSDB, 2007). Indirect exposure 

to tert-butanol may also occur as a result of ingestion of MTBE or ETBE, as tert-butanol is a 

metabolite of these compounds (NSF International, 2003). 

Alternate human exposure pathways of tert-butanol include inhalation and, to a lesser 

extent, dermal contact. tert-Butanol inhalation exposure can occur due to the chemical’s volatility 

and release from industrial processes, consumer products and contaminated sites (HSDB, 2007). 

Dermal contact is a viable route of exposure through handling consumer products containing 

tert-butanol (NSF International, 2003). 

2 http://geotracker.waterboards.ca.gov/ 

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

xv DRAFT—DO NOT CITE OR QUOTE

 
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1 Assessments by Other Federal, State and International Health Agencies

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Toxicity information on tert-butanol has been evaluated by the American Conference of 

Governmental Industrial Hygienists (ACGIH, 2012), the National Institute for Occupational Safety 

and Health (NIOSH, 2007), the Occupational Safety and Health Administration (OSHA, 2006), and 

Food and Drug Administration. The results of these assessments are presented in Appendix A of

the Supplemental Information. The California EPA carried out an expedited risk assessment for 

tert-butanol in drinking water and calculated a cancer slope factor based on rat kidney tumors 

observed in the NTP bioassays. It is important to recognize that these earlier assessments were 

prepared for different purposes using different methods and could consider only the studies that 

were available at the time.

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

xvi DRAFT—DO NOT CITE OR QUOTE
PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS

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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. 

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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 

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, 

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

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Toxicological Review of tert-Butyl Alcohol

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.

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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/). 

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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, 1986a, 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 

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 

This document is a draft for review purpo ly and does not constitute Agency policy.

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studies on other agents that act through

related mechanisms.

Each assessment specifies the search

strategies, keywords, and cut-off dates of its 

literature searches. The EPA posts the results 

of the literature search on the IRIS web site 

and requests information from the public on 

additional studies and ongoing research.

The EPA also considers studies received 

through the IRIS Submission Desk and studies 

(typically unpublished) submitted under the 

Toxic Substances Control Act or the Federal 

Insecticide, Fungicide, and Rodenticide Act. 

Material submitted as Confidential Business 

Information is considered only if it includes 

health and safety data that can be publicly 

released. If a study that may be critical to the 

conclusions of the assessment has not been 

peer-reviewed, the EPA will have it peerreviewed.

The EPA also examines the toxicokinetics 

of the agent to identify other chemicals (for

example, major metabolites of the agent) to

include in the assessment if adequate

information is available, in order to more

fully explain the toxicity of the agent and to

suggest dose metrics for subsequent

modeling. 

In assessments of chemical mixtures,

mixture studies are preferred for their ability

to reflect interactions among components. 

The literature search seeks, in decreasing 

order of preference (U.S. EPA, 2000b, §2.2; 

1986b, §2.1): 

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– 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.

– Ecological studies (geographic 

correlation studies) relate exposures 

and effects by geographic area. They 

can provide strong evidence if there 

are large exposure contrasts between 

geographic areas, relatively little 

exposure variation within study 

areas, and population migration is 

limited.

– Case reports of high or accidental 

exposure lack definition of the 

population at risk and the expected 

number of cases. They can provide 

information about a rare effect or 

about the relevance of analogous 

results in animals.

The assessment briefly reviews ecological 

studies and case reports but reports details 

only if they suggest effects not identified by 

other studies.

3.3. Selecting pertinent experimental 

studies

Exposure route is a key design 

consideration for selecting pertinent 

experimental animal studies or human 

clinical studies.

– Studies of oral, inhalation, or dermal 

exposure involve passage through an 

absorption barrier and are considered 

most pertinent to human

environmental exposure.

– Injection or implantation studies are 

often considered less pertinent but may 

This document is a draft for review purp y and does not constitute Agency policy.

3 DRAFT—DO NOT CITE OR QUOTE
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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-thanchronic 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). 

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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.

– Ascertainment of disease or health 

effect.

– Duration of exposure and follow-up 

and adequacy for assessing the 

occurrence of effects.

– Characterization of exposure during 

critical periods.

– Sample size and statistical power to 

detect anticipated effects.

– Participation rates and potential for 

selection bias as a result of the 

achieved participation rates.

– Measurement error (can lead to 

misclassification of exposure, health 

outcomes, and other factors) and 

other types of information bias.

– Potential confounding and other 

sources of bias addressed in the study 

design or in the analysis of results. 

The basis for consideration of 

confounding is a reasonable 

expectation that the confounder is 

related to both exposure and outcome 

and is sufficiently prevalent to result 

in bias.

For developmental toxicity, reproductive 

toxicity, neurotoxicity, and cancer there is 

further guidance on the nuances of evaluating 

epidemiologic studies of these effects (U.S. 

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

This document is a draft for review purp y and does not constitute Agency policy.

4 DRAFT—DO NOT CITE OR QUOTE
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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.

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– 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. 

– Ascertainment of survival, vital signs,

disease or effects, and cause of death.

– Control of other variables that could

influence the occurrence of effects.

The assessment uses statistical tests to

evaluate whether the observations may be

due to chance. The standard 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 show that the effect is unlikely

to be due to chance. For a response that

appears significant against a concurrent

control response that is unusual, historical 

controls may offer a different interpretation

(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 multigeneration studies, agents that produce 

developmental effects at doses that are not 

toxic to the maternal animal are of special 

concern. Effects that occur at doses 

associated with mild maternal toxicity are not 

assumed to result only from maternal 

toxicity. Moreover, maternal effects may be 

reversible, while effects on the offspring may 

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.

This document is a draft for review purp y and does not constitute Agency policy.

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5. Evaluating the overall evidence of 

each effect

5.1. Concepts of causal inference

For each health effect, the assessment 

evaluates the evidence as a whole to

determine whether it is reasonable to infer a 

causal association between exposure to the 

agent and the occurrence of the effect. This 

inference is based on information from

pertinent human studies, animal studies, and 

mechanistic studies of adequate quality.

Positive, negative, and null results are given 

weight according to study quality.

Causal inference involves scientific

judgment, and the considerations are

nuanced and complex. Several health

agencies have developed frameworks for

causal inference, among them the U.S.

Surgeon General (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 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 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.

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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 

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followed by a reduction of an adverse 

effect.

Analogy: Information on structural

analogues or on chemicals that induce 

similar mechanistic events can provide 

insight into causation.

These considerations are consistent with 

guidelines for systematic reviews that

evaluate the quality and weight of evidence. 

Confidence is increased if the magnitude of 

effect is large, if there is evidence of an 

exposure-response relationship, or if an

association was observed and the plausible 

biases would tend to decrease the magnitude 

of the reported effect. Confidence is

decreased for study limitations, inconsistency 

of results, indirectness of evidence,

imprecision, or reporting bias (Guyatt et al., 

2008b; Guyatt et al., 2008a). 

5.2. Evaluating evidence in humans

For each effect, the assessment evaluates 

the evidence from the epidemiologic studies 

as a whole. The objective is to determine 

whether a credible association has been

observed and, if so, whether that association 

is consistent with causation. In doing this, the 

assessment explores alternative explanations 

(such as chance, bias, and confounding) and 

draws a conclusion about whether these

alternatives can satisfactorily explain any

observed association. 

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To make clear how much the

epidemiologic evidence contributes to the 

overall weight of the evidence, the

assessment may select a standard descriptor 

to characterize the epidemiologic evidence of 

association between exposure to the agent 

and occurrence of a health effect.

Sufficient epidemiologic evidence of an

association consistent with causation:

The evidence establishes a causal

association for which alternative

explanations such as chance, bias, and 

confounding can be ruled out with

reasonable confidence.

Suggestive epidemiologic evidence of an

association consistent with causation:

The evidence suggests a causal

association but chance, bias, or

confounding cannot be ruled out as

explaining the association.

Inadequate epidemiologic evidence to infer 

a causal association: The available

studies do not permit a conclusion

regarding the presence or absence of an 

association.

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Epidemiologic evidence consistent with no 

causal association: Several adequate 

studies covering the full range of human 

exposures and considering susceptible 

populations, and for which alternative 

explanations such as bias and 

confounding can be ruled out, are 

mutually consistent in not finding an 

association.

5.3. Evaluating evidence in animals

For each effect, the assessment evaluates 

the evidence from the animal experiments as 

a whole to determine the extent to which they 

indicate a potential for effects in humans. 

Consistent results across various species and 

strains increase confidence that similar 

results would occur in humans. Several 

concepts discussed by Hill (Hill, 1965) are 

pertinent to the weight of experimental 

results: consistency of response, doseresponse relationships, strength of response, 

biologic plausibility, and coherence (U.S. EPA, 

2005a, §2.2.1.7; 1994, Appendix C). 

In weighing evidence from multiple 

experiments, U.S. EPA (2005a, §2.5) 

distinguishes: 

Conflicting evidence (that is, mixed positive 

and negative results in the same sex and 

strain using a similar study protocol) 

from 

Differing results (that is, positive results and 

negative results are in different sexes or 

strains or use different study protocols). 

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Negative or null results do not invalidate 

positive results in a different experimental 

system. The EPA regards all as valid

observations and looks to explain differing 

results using mechanistic information (for 

example, physiologic or metabolic differences 

across test systems) or methodological

differences (for example, relative sensitivity 

of the tests, differences in dose levels,

insufficient sample size, or timing of dosing or 

data collection).

It is well established that there are critical 

periods for some developmental and

reproductive effects (U.S. EPA, 2006b, 2005a, 

b, 1998b, 1996, 1991b). Accordingly, the 

assessment determines whether critical

periods have been adequately investigated. 

Similarly, the assessment determines

whether the database is adequate to evaluate 

other critical sites and effects.

In evaluating evidence of genetic toxicity:

– Demonstration of gene mutations,

chromosome aberrations, or

aneuploidy in humans or

experimental mammals (in vivo) 

provides the strongest evidence.

– This is followed 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).

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For each effect, the assessment discusses 

the available information on its modes of 

action and associated key events (key events

being empirically observable, necessary 

precursor steps or biologic markers of such 

steps; mode of action being a series of key 

events involving interaction with cells, 

operational and anatomic changes, and 

resulting in disease). Pertinent information 

may also come from studies of metabolites or 

of compounds that are structurally similar or 

that act through similar mechanisms. 

Information on mode of action is not required 

for a conclusion that the agent is causally 

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

The assessment addresses several 

questions about each hypothesized mode of 

action (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.

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2) Is the hypothesized mode of action 

relevant to humans? The assessment

reviews the key events to identify critical 

similarities and differences between the 

test animals and humans. Site

concordance is not assumed between

animals and humans, though it may hold 

for certain effects or modes of action.

Information suggesting quantitative

differences in doses where effects would 

occur in animals or humans is considered 

in the dose-response analysis. Current

levels of human exposure are not used to 

rule out human relevance, as IRIS

assessments may be used in evaluating 

new or unforeseen circumstances that

may entail higher exposures.

3) Which populations or lifestages can be 

particularly susceptible to the

hypothesized mode of action? The 

assessment reviews the key events 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.

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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). 

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.

Likely to be carcinogenic to humans: The 

evidence demonstrates a potential hazard 

to humans but does not meet the criteria 

for carcinogenic. There may be a plausible 

association in humans, multiple positive 

results in animals, or a combination of 

human, animal, or other experimental 

evidence.

Suggestive evidence of carcinogenic

potential: The evidence raises concern 

for effects in humans but is not sufficient 

for a stronger conclusion. This descriptor 

covers a range of evidence, from a 

positive result in the only available study 

to a single positive result in an extensive 

database that includes negative results in 

other species.

Inadequate information to assess

carcinogenic potential: No other

descriptors apply. Conflicting evidence can 

be classified as inadequate information if 

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all positive results are opposed by

negative studies of equal quality in the 

same sex and strain. Differing results, 

however, can be classified as suggestive 

evidence or as likely to be carcinogenic. 

Not likely to be carcinogenic to humans:

There is robust evidence for concluding 

that there is no basis for concern. There 

may be no effects in both sexes of at least 

two appropriate animal species; positive 

animal results and strong, consistent

evidence that each mode of action in

animals does not operate in humans; or 

convincing evidence that effects are not 

likely by a particular exposure route or 

below a defined dose.

Multiple descriptors may be used if there 

is evidence that carcinogenic effects differ by 

dose range or exposure route (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.

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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 coexposures are difficult to address or other 

lines of evidence are limited or 

inconsistent; or multiple animal studies 

from different laboratories demonstrate 

effects and there are limited or no human 

data.

Suggestive of a causal relationship: At least 

one high-quality epidemiologic study

shows an association but other studies 

are inconsistent.

Inadequate to infer a causal relationship:

The studies do not permit a conclusion 

regarding the presence or absence of an 

association.

Not likely to be a causal relationship:

Several adequate studies, covering the full 

range of human exposure and considering 

susceptible populations, are mutually 

consistent in not showing an effect at any 

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.

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6. Selecting studies for derivation of 

toxicity values

For each effect where there is credible 

evidence of an association with the agent, the 

assessment derives toxicity values if there are 

suitable epidemiologic or experimental data. 

The decision to derive toxicity values may be 

linked to the hazard descriptor.

Dose-response analysis requires

quantitative measures of dose and response. 

Then, other factors being equal:

– Epidemiologic studies are preferred 

over animal studies, if quantitative 

measures of exposure are available 

and effects can be attributed to the 

agent.

– Among experimental animal models, 

those that respond most like humans 

are preferred, if the comparability of 

response can be determined.

– Studies by a route of human 

environmental exposure are

preferred, although a validated 

toxicokinetic model can be used to 

extrapolate across exposure routes.

– Studies of longer exposure duration 

and follow-up are preferred, to 

minimize uncertainty about whether 

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effects are representative of lifetime 

exposure.

– Studies with multiple exposure levels 

are preferred for their ability to

provide information about the shape 

of the exposure-response curve.

– Studies with adequate power to

detect effects at lower exposure levels 

are preferred, to minimize the extent 

of extrapolation to levels found in the 

environment.

Studies with non-monotonic exposureresponse 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. 

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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). 

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 doseresponse analysis is conducted for one wellstudied 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). 

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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

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 

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the duration of exposure. For chronic 

effects, daily exposures are averaged 

over the lifespan. Exposures during a 

critical period, however, are not

averaged over a longer duration (U.S. 

EPA, 2005a, §3.1.1; 1991b, §3.2). 

– Doses are standardized to equivalent 

human terms to facilitate comparison 

of results from different species.

– Oral doses are scaled allometrically 

using mg/kg3/4-day as the equivalent 

dose metric across species. Allometric 

scaling pertains to equivalence across 

species, not across lifestages, and is 

not used to scale doses from adult 

humans or mature animals to infants 

or children (U.S. EPA, 2011; 

2005a, §3.1.3). 

– Inhalation exposures are scaled using 

dosimetry models that apply speciesspecific 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 doseresponse data, assessing model fit, selecting 

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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.): 

– If linear extrapolation is used,

selection of a response level

corresponding to the point of

departure is not highly influential, so 

standard values near the low end of 

the observable range are generally 

used (for example, 10% extra risk for 

cancer bioassay data, 1% for

epidemiologic data, lower for rare 

cancers).

– For nonlinear approaches, both

statistical and biologic considerations 

are taken into account.

– For dichotomous data, a response 

level of 10% extra risk is generally 

used for minimally adverse effects, 

5% or lower for more severe effects.

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– For continuous data, a response level 

is ideally based on an established 

definition of biologic significance. In 

the absence of such definition, one 

control standard deviation from the 

control mean is often used for

minimally adverse effects, one-half 

standard deviation for more severe 

effects.

The point of departure is the 95% lower 

bound on the dose associated with the

selected response level.

7.4. Extrapolating to lower doses and 

response levels

The purpose of extrapolating to lower 

doses is to estimate responses at exposures 

below the observed data. Low-dose 

extrapolation, typically used for cancer data, 

considers what is known about modes of 

action (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 doseresponse curve is expected to have a 

linear component below the point of 

departure. This includes:

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– 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.

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.

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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 

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.

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2) If data on risk-related parameters allow 

comparison of the general population and 

susceptible individuals, these data are 

used to adjust the general-population 

toxicity values for application to

susceptible individuals.

3) In the absence of chemical-specific data, 

the EPA has developed age-dependent 

adjustment factors for early-life exposure 

to potential carcinogens that have a 

mutagenic mode of action. There is 

evidence of early-life susceptibility to 

various carcinogenic agents, but most 

epidemiologic studies and cancer

bioassays do not include early-life 

exposure. To address the potential for 

early-life susceptibility, the EPA 

recommends (U.S. EPA, 2005b, §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

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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). 

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 

no-observed-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 nonsusceptible 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

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-observedadverse-effect level. If a point of 

departure is based on a lowest-observedadverse-effect level, the assessment must 

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infer a dose where such effects are not 

expected. This can be a matter of great 

uncertainty, especially if there is no 

evidence available at lower doses. A 

factor of 10 is applied to account for the 

uncertainty in making this inference. A 

factor other than 10 may be used, 

depending on the magnitude and nature 

of the response and the shape of the doseresponse curve (U.S. EPA, 2002, §4.4.5; 

1998b, §4.2; 1996, §4; 1994, §4.3.9.1; 

1991b, §3.4). 

Subchronic-to-chronic exposure. If a point 

of departure is based on subchronic 

studies, the assessment considers

whether lifetime exposure could have 

effects at lower levels of exposure. A 

factor of 10 is applied to account for the 

uncertainty in using subchronic studies to 

make inferences about lifetime exposure. 

This factor may also be applied for 

developmental or reproductive effects if 

exposure covered less than the full critical 

period. A factor other than 10 may be 

used, depending on the duration of the

studies and the nature of the response

(U.S. EPA, 2002, §4.4.5; 1998b, §4.2; 1994, 

§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 

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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). 

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 

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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.

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1 Low confidence: The reference value is 

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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, 

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distinguishes model 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 

susceptibility or in exposures that modify the 

effects of the agent).

August 2013

EXECUTIVE SUMMARY

30 Occurrence and Health Effects

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tert-Butanol does not occur naturally, but it is produced by humans for 

multiple purposes, such as a solvent for paints, a denaturant for ethanol and several 

other alcohols, an octane booster in gasoline, a dehydrating agent, and the 

manufacture of flotation agents, fruit essences, and perfumes. tert-Butanol is also 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, as well as consuming contaminated water (or breast milk) or 

foods. Exposure may 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. No chronic inhalation exposure studies 

have been conducted. There is suggestive evidence that tert-butanol is carcinogenic 

to humans based on predominantly benign renal tumors in male rats and benign 

thyroid tumors in female mice. 

Effects Other Than Cancer Observed Following Oral Exposure

EPA identified kidney effects as a hazard of tert-butanol exposure, with kidney toxicity 

observed after oral exposure in two strains of rats, one strain of mice, and in both sexes. In mice, 

the only kidney effect observed was an increase in kidney weight (absolute and/or relative) in both 

sexes of mice in the 13-week study, but no treatment-related histopathological lesions were 

reported in the kidneys of mice at 13 weeks or 2 years. Absolute and relative kidney weights were 

increased in both male and female rats after 13 weeks and 15 months of treatment. 

Histopathological examination also indicated kidney toxicity in both male and female rats, with 

increased incidence of nephropathy after 13 weeks of oral exposure and transitional epithelium 

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1 hyperplasia observed after 2 years of oral exposure. Additionally, increased suppurative

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inflammation was noted in females after 2 years of oral exposure. Mode of action analysis 

determined that male rat kidney effects were not mediated by α2u-globulin, and these effects are 

concluded to be relevant for human health hazard assessment. 

Oral Reference Dose (RfD) for Effects Other Than Cancer 

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

basis for the proposed overall oral reference dose (RfD) (see Table ES-1), as it was the only 

noncancer endpoint for which there is credible evidence of an association with tert-butanol

exposure. 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 due to

its consistency in both sexes, its specificity and its sensitivity as an indicator of kidney toxicity, and 

the observed dose-response relationship of effects across dose groups. Benchmark dose (BMD) 

modeling was utilized to derive the BMDL10% of 16 mg/kg-day. The BMDL was converted to a 

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

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

The proposed 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. Summary of reference dose (RfD) derivation

Effect Basis

RfD 

(mg/kg-day)

Exposure 

description Confidence

Kidney toxicity Increased incidence of kidney 

transitional epithelial 

hyperplasia

1 × 10-1 Chronic HIGH

Proposed 

overall RfD

Increased incidence of kidney 

transitional epithelial 

hyperplasia

1 × 10-1 Chronic HIGH

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Effects Other Than Cancer Observed Following Inhalation Exposure

EPA identified kidney effects as a human hazard of tert-butanol exposure. Both absolute 

and relative kidney weights were increased in male and female rats. There was an increase in 

nephropathy severity in male rats, which supported the increase in kidney weights. No available 

human studies evaluated the effects of inhalation exposure. Mode of action analysis determined 

that male rat kidney effects were not mediated by α2u-globulin, and these effects are concluded to 

be relevant for human health hazard assessment. 

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1 Inhalation Reference Concentration (RfC) for Effects Other Than Cancer 

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

for the proposed inhalation reference concentration (RfC) (see Table ES-2), as it was the only 

noncancer endpoint for which there is credible evidence of an association with tert-butanol

exposure. The chronic oral exposure study in rats (NTP, 1995) was used to derive the overall RfC. 

A PBPK model for tert-butanol in rats was developed internally, and route-to-route extrapolation 

was used to derive equivalent inhalation PODs. The POD adjusted for the human equivalent 

concentration (HEC) was 26.1 mg/m3 and based on transitional epithelial hyperplasia. 

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

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

human susceptibility (10). 

Table ES-2. Summary of reference concentration (RfC) derivation

Effect Basis

RfC

(mg/m3)

Exposure 

description Confidence

Kidney toxicity Increased incidence of kidney 

transitional epithelial 

hyperplasia 

9 × 10-1 Chronic HIGH

Proposed 

overall RfC 

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transitional epithelial 

hyperplasia 

9 × 10-1 Chronic HIGH

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Evidence for Human Carcinogenicity

Under EPA’s Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), the database for 

tert-butanol provides “suggestive evidence of carcinogenic potential.” Human data are not available 

to assess the carcinogenic potential of tert-butanol. In 2-year studies in F344 rats and B6C3F1 mice, 

male rats exhibited dose-related increases in renal tubule adenoma and combined renal tubule 

adenoma or carcinoma. Although data support α2u-globulin deposition in the kidney of male rats, 

there is insufficient evidence to support this as the only or primary mechanism for renal tumor 

development in male rats. Therefore, the renal tumors are considered relevant to humans. 

However, the observed renal tumors were predominantly benign, only occurred in a single 

sex/species combination, and were not observed in studies that exposed the same strain of rat to 

ETBE, which is rapidly metabolized to tert-butanol. In addition, a statistically significant increase in 

the incidence of thyroid follicular cell adenoma was observed in a 2-year drinking water study in 

female mice (NTP, 1995). These tumors were all benign and only a single sex/species combination 

was affected. There are no studies examining the carcinogenic potential of tert-butanol after 

inhalation exposure in animals. However, internal tumors developed after oral exposure and may 

occur regardless of exposure route, as blood concentrations were found to be similar after oral or 

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

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1 inhalation exposures. Genotoxicity data for tert-butanol are inconclusive. tert-Butanol was negative 

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in a variety of genotoxicity assays in different cell systems including gene mutations, sister 

chromatid exchanges, micronucleus formation and chromosomal aberrations. However, DNA 

adducts in male Kunming mice and DNA damage in human HL-60 leukemia cells have been

observed. Overall, the cancer descriptor “suggestive evidence of carcinogenic potential” is 

plausible, as some concern is raised by the positive evidence of predominantly benign renal tumors 

in male rats and benign thyroid tumors in female mice. 

Quantitative Estimate of Carcinogenic Risk from Oral Exposure

Lifetime oral exposure to tert-butanol has been associated with increased renal tubule 

adenomas and carcinoma in male F344 rats, increased thyroid follicular cell adenomas in female 

B6C3F1 mice, and increased thyroid follicular cell adenomas and carcinomas in male B6C3F1 mice. 

The NTP (1995) study in rats and mice was the only available study for dose-response analysis. The 

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

exposure levels and controls, contained adequate numbers of animals per dose group 

(~50/sex/group), treated animals for up to 2 years, and included detailed reporting of methods 

and results. 

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

potential,” EPA concluded that the main study was well-conducted and quantitative analysis may be 

useful for providing a sense of the magnitude of potential carcinogenic risk. For renal tumors, two 

slope factors were derived for this endpoint from the NTP (1995) bioassay: one based on the 

original reported incidences and one based on the Hard et al. (2011) reanalysis. The two estimates 

differed by less than 20%, and rounded to the same number at one significant figure. However, the 

Hard et al. (2011) reanalysis is considered preferable, as it is based on a Pathology Working Group 

(PWG) analysis. A slope factor was also derived for thyroid tumors in female mice. The modeled

tert-butanol PODs were 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/BMDL10). The more 

sensitive endpoint of renal tumors was used because there is no data to support neither renal nor 

thyroid tumors most relevant to humans. The oral slope factor of 1 × 10-2 per mg/kg-day, based 

on the renal tubule tumor response in male F344 rats. 

Quantitative Estimate of Carcinogenic Risk from Inhalation Exposure

No chronic inhalation exposure studies to tert-butanol are available. However, through the 

oral route of exposure, lifetime exposure has been associated with increased renal tubule adenomas 

and carcinoma in male F344 rats, increased thyroid follicular cell adenomas in female B6C3F1 mice,

and increased thyroid follicular cell adenomas and carcinomas in male B6C3F1 mice. The NTP 

(1995) study in rats and mice was the only available study for dose-response analysis. The study 

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

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

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

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

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

potential,” EPA concluded that the main study was well-conducted and quantitative analysis may be 

useful for providing a sense of the magnitude of potential carcinogenic risk. Since the available 

evidence for tert-butanol carcinogenicity is from a 2 year oral exposure, route-to-route 

extrapolation of the oral BMDL was performed to derive an inhalation equivalent BMCL. The BMCL 

was then converted to a human equivalent concentration (HEC) according to the RfC guidelines 

(U.S. EPA, 1994) by multiplying the BMCL by the blood:gas partition coefficient ratio. Using linear 

extrapolation from the resulting BMCL10-HEC, a human equivalent inhalation unit risk was derived 

(inhalation unit risk = 0.1/BMCL10-HEC). Extrapolation from the oral study results for renal tubule 

adenoma or carcinoma in male F44 rats gives a unit risk of 2 × 10-3 per mg/m3, associated with 

lifetime inhalation exposure to tert-butanol. 

Susceptible Populations and Lifestages for Cancer and Noncancer

No data were identified to indicate susceptible populations or lifestages.

Key Issues Addressed in Assessment

Due to the observation of kidney tumors and noncancer toxicity following chronic exposure 

to tert-butanol, an evaluation of whether tert-butanol caused α2u-globulin nephropathy was 

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

immunohistochemical staining. Linear mineralization and tubular hyperplasia were reported in 

male rats, though only in the chronic study. Other subsequent steps in the pathological sequence, 

including necrosis, exfoliation, and granular casts, were either absent or not consistently observed 

across subchronic or chronic studies. None of the observed effects occurred in female rats or in 

either sex of mice. Because the available data supports the occurrence of at least two of the 

subsequent steps in the pathological sequence, these data are sufficient to conclude that 

α2u-globulin nephropathy is occurring in the kidney of male rats following tert-butanol exposure. 

Thus, the noncancer lesions associated with α2u-globulin nephropathy are not considered relevant 

to humans. 

However, tumors develop at doses lower than some precursors of α2u-globulin 

nephropathy, such as granular casts and tubular hyperplasia (Hard et al., 2011; NTP, 1995). 

Therefore, there is insufficient evidence to support a conclusion that α2u-globulin nephropathy is 

the sole or primary contributor to renal tumor development. Because carcinogenic processes other 

than α2u-globulin nephropathy cannot be ruled out, the renal tumors are considered relevant to 

humans. 

In addition, some of the observed renal lesions in rats following exposure to tert-butanol 

are effects commonly associated with chronic progressive nephropathy (CPN), an age-related renal 

disease of laboratory rodents that occurs spontaneously. While it has been argued that CPN in rats 

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1 is not relevant to humans, it is acknowledged that the mechanism regulating CPN in rats is not 

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understood. Moreover, no key events for the exacerbation of CPN have been identified, so no mode 

of action analysis can be performed. Therefore, kidney effects from tert-butanol exposure 

associated with CPN are considered relevant to humans.

Sufficient data were available to develop a PBPK model in rats for both oral and inhalation 

exposure in order to perform route-to-route extrapolation, so rat studies from both routes of 

exposure were considered for dose-response analysis. The only endpoint available from the

subchronic inhalation study (NTP, 1997) was increased kidney weights, which is a less-specific 

endpoint compared to other endpoints available for analysis from the oral study (NTP, 1995). In 

regards to the carcinogenic effects, the 2-year oral study (NTP, 1995) was the only study to evaluate 

lifetime carcinogenic effects and was selected for route-to-route extrapolation.

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LITERATURE SEARCH STRATEGY | STUDY 

SELECTION AND EVALUATION

A literature search and screening strategy were used to identify literature characterizing 

the health effects of tert-butanol. This strategy consisted of a broad search of online scientific 

databases and other sources in order to identify all potentially pertinent studies. In subsequent 

steps, references were screened to exclude papers 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, Toxline, Web of Science, and TSCATS through April 2014, using the keywords and limits 

described in Table LS-1. The overall literature search approach is shown graphically in Figure LS-1. 

An additional 7 citations were obtained using additional search strategies described in Table LS-2. 

After electronically eliminating duplicates from the citations retrieved through these databases, 

2,532 unique citations were identified. 

The resulting 2,532 citations were screened into categories as presented in Figure LS-1 

using the title, abstract, and/or full text for pertinence to examine the health effects of tert-butanol

exposure. 

• 12 references were identified as potential “Sources of Health Effects Data” and were 

considered for data extraction to evidence tables and exposure-response arrays. 

• 196 references were identified as “Supporting Studies;” these included 39 studies 

describing physiologically-based pharmacokinetic (PBPK) models and other toxicokinetic 

information, 70 studies providing genotoxicity and other mechanistic information, 1 human 

case report, 73 not relevant exposure paradigms (including acute, dermal, eye irritation, 

and injection studies), 6 preliminary toxicity studies, and 7 physical dependency studies. 

While still considered sources of health effects information, studies investigating the effects 

of acute and direct chemical exposures are generally less pertinent for characterizing health 

hazards associated with chronic oral and inhalation exposure. Therefore, information from 

these studies was not considered for extraction into evidence tables. Nevertheless, these 

studies were still evaluated as possible sources of supporting health effects information. 

• 63 references were identified as secondary sources of health effects information (e.g., 

reviews and other agency assessments); these references were kept as additional resources 

for development of the Toxicological Review. 

• 2,261 references were identified as not being pertinent to an evaluation of the health effects 

of tert-butanol and were excluded from further consideration (see Figure LS-1 for exclusion 

categories).

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

HERO website at http://hero.epa.gov.

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

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1 Selection of Critical Studies for Inclusion in Evidence Tables

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Each study retained after the literature search and screen was evaluated for aspects of its 

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

evidence for determination of hazard potential. Some general questions that were considered in 

evaluating experimental animal studies are presented in Table LS-3. Much of the key information 

for conducting this evaluation can generally be found in the study’s methods section and in how the 

study results are reported. Importantly, the evaluation at this stage does not consider the direction 

or magnitude of any reported effects. 

To facilitate this evaluation, evidence tables were constructed that systematically 

summarize the important information from each study in a standardized tabular format as 

recommended by the NRC (2011). Twelve studies identified as “Sources of Health Effects Data” 

were considered for extraction into evidence tables for hazard identification in Chapter 1. Initial 

review of studies found two studies to be publications of the NTP (1995) data prior to the release of 

the final NTP report (Cirvello et al., 1995; Lindamood et al., 1992). One publication in the 

“Supporting Studies” category also was based on data from the NTP report (Takahashi et al., 1993). 

There were differences between the published reports and the final NTP report; therefore, the 

finalized NTP (1995) report was included in evidence tables. Data from the remaining 10 studies in 

the “Sources of Health Effects Data” category were extracted into evidence tables.

Supporting studies that contain pertinent information 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, 

short term or acute studies, were not included in the evidence tables. Such supporting studies may 

be discussed in the narrative sections of Chapter 1, or presented in Appendices, if they provide 

additional or corroborating information.

Database Evaluation

The database for tert-butanol is comprised of animal toxicity studies containing one 2-year 

bioassay that employs oral exposures in rats and mice; two oral subchronic studies in rats and one 

in mice; one inhalation subchronic study in rats and mice; a re-evaluation of the NTP (1995) rat 

data; two oral developmental studies; two inhalation developmental studies; and one onegeneration reproductive study that also evaluates other systemic effects. Several acute and short 

term studies (including an 18-day inhalation study and a 14-day study by NTP) using oral and 

inhalation exposures were performed mostly in rats, but were grouped as supporting studies since 

the database of chronic and subchronic rat studies was considered sufficient. No cohort studies, 

case-control studies, or ecological studies exist in the published literature. There was one case 

report available. Health effect studies of gasoline and tert-butanol mixtures were not considered 

pertinent to the assessment since the separate effects of the gasoline components could not be 

determined; thus, these studies were excluded during the manual screen. 

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

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1 The “Sources of Health Effects Data” were comprised entirely of studies performed in rats 

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10

and mice with drinking water, oral gavage, and inhalation exposures to tert-butanol. These 12

sources were conducted according to OECD Good Laboratory Practice (GLP) guidelines, presented 

extensive histopathological data, and/or clearly presented their methodology; thus, these are 

considered high quality. Preliminary, acute, and short-term studies contained information that 

supported and did not differ qualitatively from the results of the ≥30 day exposure studies; thus, 

these studies are not included in the evidence tables. Some of these shorter duration studies are 

presented in the text of the Toxicological Review and are used in sections such as “Mechanistic 

Evidence” to augment the discussion. A more detailed discussion of methodological concerns that 

were identified will precede each endpoint evaluated in the hazard identification section. 

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

LS-3 DRAFT—DO NOT CITE OR QUOTE
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This document is a draft for review purposes only and does not constitute Agency policy.

LS-4 DRAFT—DO NOT CITE OR QUOTE

 1

2 Figure LS-1. Study selection strategy.
Toxicological Review of tert-Butyl Alcohol

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

Database 

(Search Date) Keywords Limits 

PubMed

(12/20/2012)

(4/17/2014) 

tert-butanol OR 75-65-0[rn] OR “tbutyl hydroxide” OR “2-methyl-2-

propanol” OR “trimethyl carbinol” OR 

“t-butyl alcohol” OR tert-butanol OR 

"tert-butyl alcohol" OR tert-butyl 

alcohol[mesh]

None

Web of Science

(12/20/2012)

(4/17/2014) 

Topic = (tert-butanol OR 75-65-0 OR 

"t-butyl hydroxide" OR "2-methyl-2-

propanol" OR "trimethyl carbinol" OR 

"t-butyl alcohol" OR "tert-butanol" 

OR "tert-butyl alcohol") 

Refined by: Research Areas = (cell biology OR 

respiratory system OR microscopy OR biochemistry 

molecular biology OR gastroenterology hepatology OR 

public environmental occupational health OR 

oncology OR physiology OR cardiovascular system 

cardiology or toxicology OR life sciences biomedicine 

other topics OR hematology OR pathology OR 

neurosciences neurology OR developmental biology) 

Toxline (includes 

TSCATS)

(1/11/2013)

(4/17/2014) 

tert-butanol OR 75-65-0 [rn] OR tbutyl hydroxide OR 2-methyl-2-

propanol OR trimethyl carbinol OR tbutyl alcohol OR tert-butanol OR tertbutyl alcohol OR tert-butyl alcohol

Not PubMed 

TSCATS2

(1/4/2013) 

(4/17/2014)

75-65-0 None 

 2

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

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

Approach used Source(s)

Date 

performed

Number of additional references

identified

Manual search 

of citations from 

reviews

Review article: Mcgregor (2010). 

Tertiary-butanol: A toxicological 

review. Crit Rev Toxicol 40(8): 697-

727. 

1/2013 5 

Review article: Chen (2005). Amended 

final report of the safety assessment 

of t-butyl alcohol as used in 

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

1/2013 2 

Manual search 

of citations from 

reviews 

conducted by 

other 

international 

and federal 

agencies

IPCS (1987a). Butanols: Four isomers: 

1-butanol, 2-butanol, tert-butanol, 

isobutanol [WHO EHC]. Geneva, 

Switzerland: World Health 

Organization.

1/2013 None

OSHA (1992). Occupational safety and 

health guideline for tert-butyl alcohol. 

Cincinnati, OH: National Institute for 

Occupational Safety and Health.

1/2013 None

2

3

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

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1 Table LS-3. Questions and relevant experimental information for evaluation 

2 of experimental animal studies 

Methodological 

feature Question(s) considered

Examples of relevant

information extracted

Test animal Based on the endpoint(s) in question, are 

concerns raised regarding the suitability of 

the species, strain, or sex of the test 

animals on study?

Test animal species, strain, sex

Experimental setup Are the timing, frequency and duration of 

exposure, as well as animal age and 

experimental group allocation procedures/ 

group size for each endpoint evaluation, 

appropriate for the assessed endpoint(s)?

Age/lifestage of test animals at exposure 

and all endpoint testing timepoints

Timing and periodicity of exposure and 

endpoint evaluations; duration of exposure

Sample size for each experimental group 

(e.g., animals; litters; dams) at each 

endpoint evaluation

Exposure Are the exposure conditions and controls 

informative and reliable for the endpoint(s) 

in question, and are they sufficiently 

specific to the compound of interest?

Exposure administration techniques (e.g., 

route; chamber type) 

Endpoint evaluation 

procedures

Do the procedures used to evaluate the 

endpoint(s) in question conform to 

established protocols, or are they 

biologically sound? Are they sensitive for 

examination of the outcome(s) of interest?

Specific methods for assessing the effect(s) 

of exposure, including related details (e.g., 

specific region of tissue/organ evaluated)

Endpoint evaluation controls, including 

those put in place to minimize evaluator 

bias

Outcomes and data 

reporting

Were data reported for all pre-specified 

endpoint(s) and study groups, or were any 

data excluded from presentation/ 

analyses?

Data presentation for endpoint(s) of 

interest

Note: “Outcome” refers to findings from an evaluation (e.g., hypertrophy), whereas “endpoint” refers to the 

evaluation itself (e.g., liver histopathology).

3

4

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1. HAZARD IDENTIFICATION

1 1.1. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM

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1.1.1. Kidney Effects

Synthesis of Effects in Kidney

This section reviews the studies that investigated whether exposure to tert-butanol can 

cause kidney effects in humans or animals. The database examining kidney effects following 

tert-butanol exposure contains no human data, six studies performed in rats or mice, and one reevaluation of the rat data from NTP (1995). Studies employing short-term and acute exposures that 

examined kidney effects are not included in the evidence tables; however, they are discussed in the 

text if they provide data to support mode of action or hazard identification. No methodological 

concerns were identified that would lead one or more studies to be considered less informative for 

assessing human health hazard. A pathology working group (Hard et al., 2011) re-examined kidney 

histopathology from the NTP (1995) 13-week and 2-year studies in rats to evaluate questions 

involving MOAs for renal tubule development. All slides were analyzed in a blinded manner. Hard et 

al. (2011) did report different incidences of adenomas or carcinomas compared with the original 

NTP (1995) study; thus, these data were presented separately. Histopathological results from both 

Hard and NTP will be considered for hazard identification.

tert-Butanol exposure resulted in a number of kidney effects after both oral (drinking 

water) and inhalation exposure in both sexes of rats and mice. Kidney effects observed after oral 

exposure (Table 1-1;Table 1-2; Figure 1-1) include increased kidney weight in female rats and in 

male and female mice (13-week exposure), and kidney inflammation, kidney transitional epithelial 

hyperplasia, and increased incidence and/or severity of kidney nephropathy in female rats (2-year

exposure) (NTP, 1995). In a 2-year oral exposure study in male rats, increased kidney weight, 

increased hyaline droplets, kidney transitional epithelial hyperplasia, kidney mineralization, renal 

tubule hyperplasia, and increased incidence and/or severity of kidney nephropathy were observed, 

with some of these effects seen at earlier time periods (NTP, 1995). Other kidney effects in male 

rats were observed in a 10-week oral exposure study (Acharya et al., 1997; Acharya et al., 1995). 

No changes in clinical chemistry that would typically be indicative of kidney damage have been 

observed with tert-butanol exposure. Although there were some changes in urinalysis parameters 

(e.g., decreased urine volume and increased specific gravity), this was accompanied by reduced 

water consumption and may not be related to an effect of kidney function.

The kidney is also the target organ for cancer effects (Table 1-3; Figure 1-1). Male F344 rats 

had an increased incidence of renal tubule adenomas and combined renal tubule adenoma or 

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

1-1 DRAFT—DO NOT CITE OR QUOTE
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1 carcinoma in a 2-year oral bioassay (Hard et al., 2011; NTP, 1995). The highest exposure group had 

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an increase in mortality, which may in part explain the apparent non-monotonicity in the observed 

dose-response, in which the highest exposure group had a lower incidence of tumors than the 

middle exposure group.

An Independent Pathology Working Group (PWG), sponsored by Lyondell Chemical 

Company, re-evaluated the kidney changes in the NTP 2-year study (Hard et al., 2011). The PWG 

consisted of senior pathologists with experience in chemically-induced nephrotoxicity and renal 

neoplasia. In all cases, PWG members were blinded to treatment groups to preclude any possible 

bias, and used guidelines published by the Society of Toxicologic Pathology. The PWG confirmed the 

NTP findings of atypical tubule hyperplasia and renal tubule tumors in male rats at 2-years. In 

particular, they reported very similar overall tumor incidences in the exposed groups. However, 

the PWG evaluation of the control groups reported fewer renal tubule adenomas and carcinomas 

than the original NTP study. As a result, based on the PWG evaluation, all treated groups had 

statistically significant increases in renal tubule adenomas and carcinomas (combined) as 

compared to controls. Additionally, the PWG considered fewer of the tumors to be carcinomas as 

compared to the original NTP study.

No chronic (2-year) inhalation exposure study is available, but minimal kidney effects were 

observed in rats (mainly the males) after tert-butanol exposure by inhalation for 13 weeks at 

concentrations ranging from 406–6,368 mg/m3 (NTP, 1997) (Table 1-1; Table 1-2; Figure 1-2). 

Absolute kidney weights were elevated (9.8–11%) in male rats exposed at ≥3,274 mg/m3 (not dosedependent); relative kidney weights were statistically elevated (~9%) in males at ≥3,274 mg/m3

and 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). Although the kidney effects were less 

severe after inhalation exposure, a direct comparison can only be made on the basis of internal 

dose. ARCO (1983) found that blood levels of tert-butanol and its metabolites are equivalent after a 

single oral dose of 350 mg/kg compared to a single 6-hour inhalation exposure to 6,164 mg/m3. 

That would indicate, based on bolus exposures, that the inhalation exposures used in the NTP 

(1997) study were in the range of the lower doses used in the NTP (1995) oral study. On the other 

hand, based on PBPK modeling, chronic exposure in the range of the NTP (1995) bioassay doses of 

90–420 mg/kg-day lead to the same average blood concentration of tert-butanol as 6-hour/day, 5 

day/week inhalation exposures to 860–4500 mg/m3, suggesting that the oral and inhalation 

exposures in NTP (1995) and NTP (1997), respectively, overlap on the basis of internal dose. 

Finally, the lack of either mortality or changes in body weight (both observed with oral exposure) 

observed after the inhalation exposure suggests that a direct comparison cannot be made. 

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

1-2 DRAFT—DO NOT CITE OR QUOTE
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1 Table 1-1. Changes in kidney weight in animals following exposure to

2 tert-butanol

Reference and study design Results

Kidney weight (percent change as compared to control)

Lyondell Chemical Co. (2004)

Sprague-Dawley rat; 

12/sex/treatment

Gavage 0, 64, 160, 400, or 

1,000 mg/kg-d 

Males: 9 weeks beginning 4 weeks 

prior to mating

Females: 4 weeks prior to mating 

through PND21 

Males

Dose

(mg/kg-d)

Left absolute

weight

Left relative

weight

Right absolute

weight

Right relative

weight

0 0 0 0 0

64 +6 +8 +6 +8

160 +9 +14* +6 +11*

400 +12* +14* +14* +17*

1,000 +18* +28* +20* +31*

Females

Dose

(mg/kg-d)

Left absolute

weight

Left relative

weight

Right absolute

weight

Right relative

weight

0 0 0 0 0

64 −1 −2 +2 0

160 0 0 +1 0

400 +3 +2 +4 +2

1,000 +4 0 +7 +2

NTP (1995)

F344/N rat; 10/sex/treatment

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

40 mg/mL 

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

3,610a mg/kg-d 

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

3,620a mg/kg-d 

13 weeks

Males Females

Dose

(mg/kg-d)

Absolute

weight

Relative

weight

Dose

(mg/kg-d)

Absolute

weight

Relative

weight

0 0 0 0 0 0

230 +12* +19* 290 +19* +17*

490 +17* +26* 590 +16* +15*

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

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

3,610 All dead All dead 3,620 +36* +81*

3

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1-3 DRAFT—DO NOT CITE OR QUOTE
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Table 1-1. Changes in kidney weight in animals following exposure to 

tert-butanol (continued) 

Reference and study design Results

NTP (1995)

B6C3F1 mouse; 10/sex/treatment

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

40 mg/mL)

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

8,210a mg/kg-d 

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

11,620 a mg/kg-d 

13 weeks

Males Females

Dose

(mg/kg-d)

Absolute

weight

Relative

weight

Dose

(mg/kg-d)

Absolute

weight

Relative

weight

0 0 0 0 0 0

350 +1 +1 500 0 −3

640 +3 +2 820 −3 −1

1,590 +2 +8 1,660 +1 0

3,940 +6 +22* 6,430 +6 +15*

8,210 0 +48* 11,620 +12* +35*

NTP (1995)

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

Males Females

Dose

(mg/kg-d)

Absolute

weight

Relative

weight

Dose

(mg/kg-d)

Absolute

weight

Relative

weight

0 0 0 0 0 0

90 +4 +8 180 +8* +14*

200 +11 +15* 330 +18* +21*

420 +7 +20* 650 +22* +42*

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

weighed in the 2-year mouse study

NTP (1997)

F344/N rat; 10/sex/treatment 

Analytical concentration: 0, 134, 

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

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

mg/m3 ) (dynamic whole-body 

chamber)

6 hr/d, 5 d/wk 

13 weeks

Generation method (Sonimist 

Ultrasonic spray nozzle nebulizer), 

analytical concentration and 

method were reported 

Males Females

Concentration

(mg/m3)

Absolute

weight

Relative

weight

Absolute

weight

Relative

weight

0 0 0 0 0

406 +1 +1 −4 −1

824 −2 −1 0 +1

1,643 +3 +2 +4 +4

3,273 +11* +8* +2 +2

6,368 +9.8* +9* +4 +9*

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

1-4 DRAFT—DO NOT CITE OR QUOTE
Toxicological Review of tert-Butyl Alcohol

Table 1-1. Changes in kidney weight in animals following exposure to 

tert-butanol (continued) 

Reference and study design Results

NTP (1997)

B6C3F1 mouse; 10/sex/treatment

Analytical concentration: 0, 134, 

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

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

mg/m3) (dynamic whole-body 

chamber)

6 hr/d, 5 d/wk 

13 weeks

Generation method (Sonimist 

Ultrasonic spray nozzle nebulizer),

analytical concentration and 

method were reported

Males Females

Concentration

(mg/m3)

Absolute

weight

Relative

weight

Absolute

weight

Relative

weight

0 0 0 0 0

406 −6 −4 +1 −3

824 −1 +3 +5 +9

1,643 +4 +3 +1 −2

3,273 −10 −3 0 +7

6,368 +3 +6 +3 +15*

1

2

3

4

5

6

7

8

a The high-dose group had an increase in mortality. 

* Statistically significant p ≤ 0.05 as determined by the study authors.

Percentage change compared to control = (treated value – control value) ÷ control value × 100.

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

. 

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

tert-butanol

Reference and study design Results

Acharya et al. (1997; 1995)

Wistar rat; 5–6 males/treatment

Drinking water (0 or 0.5%), 0 or 

575 mg/kg-d 

10 weeks

↑ tubular degeneration, degeneration of the basement membrane of the Bowman’s 

capsule, diffused glomeruli, and glomerular vacuolation (no incidences reported)

↓ kidney glutathione (~40%)*

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 

There were no changes in kidney histopathology observed.

9

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

tert-butanol (continued) 

Reference and study design Results

NTP (1995)

F344/N rat; 10/sex/treatment

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

40 mg/mL) 

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

3,610a mg/kg-d 

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

3,620a mg/kg-d 

13 weeks

Incidence (severity):

Males Females

Dose 

(mg/kg-d) Mineralization Nephropathy

Dose 

(mg/kg-d) Mineralization Nephropathy

0 0/10 7/10 (1.0) 0 10/10 (1.7) 2/10 (1.0)

230 0/10 10/10 (1.6*) 290 10/10 (2.0) 3/10 (1.0)

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

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

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

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

NTP (1995)

B6C3F1 mouse; 10/sex/treatment

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

40 mg/mL) 

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

8,210a mg/kg-d 

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

11,620a mg/kg-d 

13 weeks

Histopathology data for the 13-week study were not provided, but the kidney was 

evaluated indicating that no changes in kidney histopathology were observed in the 13-

week study.

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

tert-butanol (continued) 

Reference and study design Results

NTP (1995)

F344/N rat; 60/sex/treatment 

(10/sex/treatment evaluated at 15 

months)

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

mg/mL)

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

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

2 years

Incidence (severity): 

Males

Dose

(mg/kg-d)

Mineralization 

(interim)

Mineralization 

(terminal)

Linear mineralization 

(terminal)

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)

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

Dose

(mg/kg-d)

Renal tubule 

hyperplasia 

(extended 

evaluation)

Transitional 

epithelium 

hyperplasia

Nephropathy 

severity

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

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

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

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

Females

Dose

(mg/kg-d)

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*

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

Dose

(mg/kg-d)

Renal tubule 

hyperplasia

Transitional 

epithelium 

hyperplasia

Nephropathy 

severity

 0 0/50 0/50 1.6

180 0/50 0/50 1.9*

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

650 1/50 (1.0) 17/50*(1.4) 2.9*

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

tert-butanol (continued) 

Reference and study design Results

NTP (1995)

B6C3F1 mouse; 60/sex/treatment 

Drinking water (0, 5, 10, or 20 

mg/mL) 

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

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

2 years

No changes in kidney related histopathology observed.c

NTP (1997)

F344/N rat; 10/sex/treatment

Analytical concentration: 0, 134, 

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

406, 824, 1,643, 3,273 or 

6,368 mg/m3 ) (dynamic whole-body 

chamber)

6 hr/d, 5 d/wk

13 weeks 

Generation method (Sonimist 

Ultrasonic spray nozzle nebulizer), 

analytical concentration and 

method were reported

Male

Concentration

(mg/m3)

Average severity of 

chronic nephropathy

0 1.0

406 1.4

824 1.4

1,643 1.6

3,273 1.9

6,368 2.0

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

NTP (1997)

B6C3F1 mouse; 10/sex/treatment

Analytical concentration: 0, 134, 

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

406, 824, 1,643, 3,273 or 

6,368 mg/m3

) (dynamic whole-body 

chamber)

6 hr/d, 5 d/wk

13 weeks 

Generation method (Sonimist 

Ultrasonic spray nozzle nebulizer), 

analytical concentration and 

method were reported

There were no kidney effects observed.

a 1 The high-dose group had an increase in mortality. 

2

3

4

5

6

7

8

b Linear mineralization not observed in female rats.

c Organs were not weighed in mice during the 2-year study. Relative organ weights refer to relative to body weight

* Statistically significant p ≤ 0.05 as determined by the study authors.

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

. 

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

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

2 tert-butanol

Reference and study design Results

NTP (1995)

F344/N rat; 60/sex/treatment

(10/sex/treatment evaluated at 15 

months)

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

10 mg/mL)

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

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

2 years

Male

Dose

(mg/kg-d)

Renal tubule 

adenoma

(single)

Renal tubule 

adenoma 

(multiple)

Renal tubule 

carcinoma

Renal tubule 

adenoma (single 

or multiple) or 

carcinoma

0 7/50 1/50 0/50 8/50

90 7/50 4/50 2/50 13/50

200 10/50 9/50* 1/50 19/50*

420 10/50 3/50 1/50 13/50

Female

Dose

(mg/kg-d)

Renal tubule 

adenoma

(single)

Renal tubule 

adenoma 

(multiple)

Renal tubule 

carcinoma

Renal tubule 

adenoma (single 

or multiple) or 

carcinoma

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

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

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

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

Results do not include the animals sacrificed at 15 months.

Hard et al. (2011)

reanalysis of the slides from male 

rats in the NTP (1995) study (see 

above)

Male

Dose

(mg/kg-d)

Renal tubule 

adenoma

(single)

Renal tubule

adenoma 

(multiple)

Renal tubule 

carcinoma

Renal tubule 

adenoma (single 

or multiple) or 

carcinoma

0 3/50 1/50 0/50 4/50

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

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/kgd 

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

2 years

No changes in kidney-related tumors

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a The high-dose group had an increase in mortality. 

* Statistically significant p ≤ 0.05 as determined by the study authors.

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

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

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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 1-1. Exposure response array for kidney effects following oral 

exposure to tert-butanol. 
Toxicological Review of tert-Butyl Alcohol

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Absolute weight; M Rat

Relative weight; M Rat

Absolute weight; F Rat

Relative weight; F Rat

Absolute/relative weight; M Mouse

Absolute weight; F Mouse

Relative weight; F Mouse

100 1,000 10,000

Concentration (mg/m3)

 = 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

Source: NTP (1997)

Figure 1-2. Exposure-response array of kidney effects following subchronic 

inhalation exposure to tert-butanol (no chronic studies available). 

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

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1 Mode of Action Analysis—Kidney Effects 

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Mode of Action Analysis for α2u-globulin-associated nephropathy 

Description of the hypothesized MOA

Several studies were identified that evaluated the role of α2u-globulin in tert-butanolinduced renal tumor development (Borghoff et al., 2001; Williams and Borghoff, 2001; Takahashi et 

al., 1993). α2u-Globulin is a member of a large superfamily of low-molecular-weight proteins and 

was first characterized in male rat urine. Such proteins have been detected in various tissues and 

fluids of most mammals (including humans), but the particular isoform of α2u-globulin commonly 

detected in male rat urine is considered specific to the male rat. 

The hypothesized sequence of α2u-globulin-associated nephropathy, as described by U.S. 

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

accumulation of α2u-globulin in the hyaline droplets results in hyaline droplet deposition in the 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 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 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 is observed in the 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 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 papilla which 

results in linear mineralization. After 1 or more years of chemical exposure, these lesions may 

result in the induction of renal adenomas and carcinomas.

U.S. EPA (1991a) states that two questions must be addressed to determine the extent to 

which α2u-globulin mediated processes induce renal tumors and nephropathy. First, it must be 

determined whether the α2u-globulin process is occurring in male rats and therefore could be a 

factor in renal effects. U.S. EPA (1991a) states that the criteria for answering this question in the 

affirmative are as follows: 

1) hyaline droplets are increased in size and number in male rats, 

2) the protein in the hyaline droplets in male rats is α2u-globulin, and 

3) if several (but not necessarily all) additional steps in the pathological sequence are present 

in male rats, such as: 

(a) single-cell necrosis, 

(b) exfoliation of epithelial cells into the tubular lumen, 

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

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(c) granular casts, 

(d) linear mineralization, and 

(e) tubule hyperplasia. 

The available data relevant to this question in male rats are summarized in Table 1-5 and 

Figure 1-3 and Figure 1-4, and will be evaluated below in accordance with the mode of action 

(MOA) framework from the EPA cancer guidelines (U.S. EPA, 2005a). 

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

must be answered as to whether the renal effects are solely due to the α2u-globulin process, are a 

combination of the α2u-globulin process and other carcinogenic processes, or are due primarily to 

other processes. U.S. EPA (1991a) states that the following types of data may be useful for 

answering this question:

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1) Hypothesis-testing data

2) Biochemical information

3) Sustained cell division in the proximal tubule of the male rat

4) Structure-activity relationships

5) Covalent binding to macromolecules

6) Genotoxicity

7) Nephrotoxicity 

8) Animal bioassay data in other species-, sex-combinations

9) Other information not specifically listed

The available data relevant to this question are summarized in Table 1-6, and will be 

evaluated below in accordance with the MOA framework from the EPA cancer guidelines (U.S. EPA, 

2005a). 

From these two questions, U.S. EPA (1991a) states that one of three possible conclusions 

can be made: 

• If renal tumors in male rats are attributable solely to the α2u-globulin process, then U.S. EPA 

(1991a) states that such tumors will not be used for human cancer hazard identification or 

for dose-response extrapolations. 

• If renal tumors in male rats are not linked to the α2u-globulin process, then U.S. EPA (1991a)

states that such tumors are an appropriate endpoint for human hazard identification and 

are considered, along with other appropriate endpoints, for quantitative risk estimation.

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

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• If some renal tumors in male rats are attributable to the α2u-globulin process and some 

attributable to other carcinogenic processes, then U.S. EPA (1991a) states that such tumors 

remain relevant for purposes of hazard identification, but a dose-response estimate based 

on such tumors in male rats should not be performed unless there is enough information to 

determine the relative contribution of each process to the overall renal tumor response.

Additionally, U.S. EPA (1991a) states that if the α2u-globulin process is occurring in male 

rats, then the associated nephropathy in male rats (described above) would not be an appropriate 

endpoint to determine noncancer effects occurring in humans. In such a case, the characterization 

of human health hazard for renal toxicity would need to rely on other types of nephrotoxic effect 

data in male rats and/or on nephrotoxic effect data in female rats or other species. 

Table 1-4. Additional kidney effects potentially relevant to mode of action in

animals following exposure to tert-butanol

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Reference and study design Results

Williams and Borghoff (2001)

F344 rats; 4/sex

Single gavage dose: 500 mg/kg 

Males: ↑ binding of tert-butanol to α2u-globulin compared to females*

Females: no change in binding observed 

NTP (1995)

F344/N rat; 10/sex/treatment

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

40 mg/mL) 

 M: 0, 230, 490, 840, 1,520, 3,610a

mg/kg-d 

 F: 0, 290, 590, 850, 1,560, 3,620a

mg/kg-d 

13 weeks

Accumulation of hyaline droplets:

Male

Dose

(mg/kg-d)

Hyaline 

droplet accumulation

0 0/10

230 +

e

490 ++

840 ++

1,520 ++

3,610 0/10

No information provided on females. No results from statistical tests reported.

Hard et al. (2011)

Reanalysis of the slides in the NTP 

(1995) study

Males: Confirmed accumulation of hyaline droplets increased with increasing dose-levels 

in 13 week study above. No incidence data available.

Females: not evaluated

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

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Table 1-4. Additional kidney effects potentially relevant to mode of action in

animals following exposure to tert-butanol (continued) 

Reference and study design Results

Borghoff et al. (2001)

F344 rat; 5/sex/treatment

Analytical concentration:0, 250, 

450, 1,750 ppm (0,771, 1,387 or 

5,395mg/m3) 6hr/d

10 days

Males: positive trend for accumulation of protein droplets (p < 0.05), significant 

increase in accumulation of α2u-globulin at 5,395 mg/m3 as compared to controls (no 

incidence data provided)

Females: No positive staining for α2u-globulin was observed in exposed female rats. 

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a The high-dose group had an increase in mortality. b Linear mineralization not observed in female rats.

c Organs were not weighed in mice during the 2-year study. d Standard & extended evaluation combined.

e + or ++ indicated an increased accumulation relative to controls, as reported by the authors; no additional incidence data and 

no results from statistical tests available.

* Statistically significant p ≤ 0.05 as determined by the study authors.

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

. 

Percentage change compared to control = (treated value – control value) ÷ control value × 100.

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

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

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1 Table 1-5. Summary of data on the α2u-globulin process in male rats exposed 

2 to tert-butanol 

Criterion Duration Results Reference

(1) hyaline droplets are 

increased in size and number

10 d + Borghoff et al. (2001)

13 wk (+) NTP (1995)

13 wk – NTP (1997)

13 wk (+)a Hard et al. (2011)

(2) the protein in the hyaline 

droplets is α2u-globulin

12 hr + Williams and Borghoff (2001)

10 d + Borghoff et al. (2001)

(3) Several (but not necessarily all) additional steps in the pathological sequence are present in male rats, 

such as:

(a) Single-cell necrosis 13 wk – NTP (1995)

2 yr – NTP (1995)

2 yr – Hard et al. (2011)

(b) exfoliation of 

epithelial cells into 

the tubular lumen

13 wk – NTP (1995)

2 yr – NTP (1995)

2 yr – Hard et al. (2011)

(c) granular casts 13 wk – NTP (1995)

13 wk (+)a,c Hard et al. (2011)

13 wk – NTP (1997)

2 yr –

b NTP (1995)

(d) linear mineralization 13 wk – NTP (1995)

13 wk – NTP (1997)

2 yr + NTP (1995)

2 yr (+)a Hard et al. (2011)

(e) tubule hyperplasia 2 yr + NTP (1995)

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+ = 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 Re-analysis of one control and one treated group from NTP (1995) b Protein casts reported, not granular casts

c Precursors to granular casts reported 

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

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Figure 1-3. Exposure-response array for components of α2u-globulin 

nephropathy and renal tumors in male rats after oral exposure to 

tert-butanol. 
Toxicological Review of tert-Butyl Alcohol

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Figure 1-4. Exposure-response array for components of α2u-globulin 

nephropathy and renal tumors in male rats after inhalation exposure to 

tert-butanol. 
Toxicological Review of tert-Butyl Alcohol

1 Table 1-6. Summary of additional data informing the contribution of the 

2

3

α2u-globulin process on the renal tumor development in male rats exposed to 

tert-butanol

Type of data

Description

Reference

(1) Hypothesis-testing data

No data

(2) Biochemical information

Reversible, non-covalent binding of tert-butanol to α2u-globulin. Williams and Borghoff (2001)

(3) Sustained cell division in the proximal tubule of the male rat

Hyperplasia at 2 yr reported in both male and female rats, attributed to 

CPN.

Hard et al. (2011)

No effect on proliferation at 13 wk NTP (1997)

Increased proliferation at 13 wk based on PCNA assay NTP (1995)

Increased proliferation of the P2 segment at 10 days based on BrdU 

labeling

Borghoff et al. (2001)

(4) Structure-activity relationships

No data

(5) Covalent binding to macromolecules

No data

(6) Genotoxicity

Limited database to conclude tert-butanol is genotoxic or nongenotoxic See Appendix B.3.

(7) Nephrotoxicity

Increased tubular regeneration and intratubule protein cast formation 

at 2 yr in males and females, with effects in females occurring at lower 

dose.

NTP (1995)

Increased severity of CPN in male rats after 13 wk inhalation exposure NTP (1997)

Increased CPN in male and female F344/N rats following drinking water 

exposure for 13 wk.

NTP (1995)

Increased CPN in male and female F344/N rats following drinking water 

exposure for 2 yr.

NTP (1995)

(8) Animal bioassay data in other species-, sex-combinations

Two renal tubular adenocarcinomas (not statistically significant as 

compared to concurrent controls) reported in male mice following 

drinking water exposure for 2 yr. These tumors are very rare in mice.

NTP (1995)

(9) Other data

Dose-response and temporal concordance (see Figures 1-3 and 1-4).

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1 Strength, consistency, specificity of association

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Is the α2u-globulin process occurring in male rats exposed to tert-butanol? 

The first criterion considered is whether hyaline droplets are increased in size and number 

in male rats. Protein droplet accumulation was statistically significantly increased in the kidneys of 

male rats exposed to 5,305 mg/m3 tert-butanol for 6 hr/day for 10 days (Borghoff et al., 2001). Data 

from drinking water studies (NTP, 1995; Takahashi et al., 1993; Lindamood et al., 1992) 

demonstrated a statistically significant increase, except at the highest dose, in hyaline droplet 

formation and severity in the proximal tubule of male rats following oral exposure to tert-butanol

for 13 weeks. Treated males had large hyaline droplets with crystal accumulation, but the controls 

had small droplets without crystals. NTP (1997) 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, and there was no 

difference between the controls and treatment groups. 

The second criterion considered is whether the protein in the hyaline droplets in male rats 

is α2u-globulin. Two studies measured α2u-globulin immunoreactivity in the hyaline droplets of the 

renal proximal tubular epithelium (Borghoff et al., 2001; Williams and Borghoff, 2001). Borghoff et 

al. (2001) observed α2u-globulin immunoreactivity present in the hyaline droplets in the male rats. 

No α2u-globulin immunostaining was observed in the kidneys of the female rats. Williams and 

Borghoff (2001) found the content of α2u-globulin statistically significantly elevated in 72% of the 

kidneys of male rats treated with tert-butanol compared with controls treated with corn oil. 

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

in the pathological sequence are present in male rats. Several, but not all, of the subsequent 

histopathological lesions were observed in the available subchronic or chronic tert-butanol

exposure studies. Linear mineralization was the lesion most consistently observed in male rats and 

was found to be statistically significantly increased in male rats after 2 years of oral exposure (NTP, 

1995) (see Table 1-2). The 13-week study in rats (NTP, 1995) reported mineralization, but it was 

not characterized as linear mineralization. Additionally, although the inhalation study by NTP 

(1997) did not report linear mineralization at 13 weeks, this may be due to the lower internal dose 

as compared to the oral studies. Atypical tubule hyperplasia was statistically significantly increased 

at the highest dose following 2 years of oral exposure (NTP, 1995). Granular casts were increased at 

the 13 week time point (NTP, 1995), though statistical significance was not determined. The 

reanalysis of the 13-week data by Hard et al. (2011) concluded that the lesions were precursors to 

granular casts and not the casts themselves. Other studies did not observe granular casts at 13 

weeks or 2 years (NTP, 1997, 1995). The formation of protein casts were observed, but these may 

be part of the pathology of CPN and are not thought to be related to α2u-globulin nephropathy (NTP, 

1995). Neither necrosis nor epithelial exfoliation was reported in any study. 

In summary, the evidence supports the conclusion that tert-butanol causes increases in the 

size and number of hyaline droplets, and the accumulating protein in the hyaline droplets is

α2u-globulin. Additionally, several, but not all, of the additional steps in the pathological sequence 

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

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1 were observed, and not always consistently across studies. Therefore, the overall strength, 

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consistency, and specificity of the association between tert-butanol and the hypothesized key 

events is moderate.

Are the renal effects in male rats exposed to tert-butanol solely due to the α2u-globulin process? 

As summarized in Table 1-6, there are many potential sources of additional data that may 

inform the contribution of the α2u-globulin process on renal tumor development. No hypothesistesting data, structure-activity relationships, or covalent binding data were located, so these types 

of data are not discussed. Additional data related to dose-response concordance and temporal 

relationships are discussed in subsequent sections.

In terms of biochemical information, Williams and Borghoff (2001) report that tert-butanol

reversibly and non-covalently binds to α2u-globulin. This provides additional support to the 

evidence that the α2u-process is occurring, but it does not inform the relative contribution to renal 

tumor development.

Sustained cell division in the proximal tubule of the male rat is consistent with, though not 

specific to, the α2u-process. Proliferation of the proximal tubule was significantly increased in male 

rats after 10 days of inhalation exposure to tert-butanol at concentrations of 771–5,395 mg/m3

(Borghoff et al., 2001) and after 13 weeks of oral exposure to 1520 mg/kg-day (NTP, 1995; 

Takahashi et al., 1993; Lindamood et al., 1992), but not after 13 week inhalation exposure up to 

6368 mg/m3 (NTP, 1997). Therefore, it is unclear the extent to which increased cell division is 

sustained. While hyperplasia was reported in chronic studies (Hard et al., 2011), it was observed in 

both male and female rats, and attributed to CPN. 

There are a limited number of studies available to assess the genotoxic potential of tertbutanol (see Appendix B.3 in Supplemental Information for further details). tert-Butanol was 

generally negative in a variety of genotoxicity assays and cell systems including Salmonella 

typhimurium, Escherichia coli and Neurospora crassa (Mcgregor et al., 2005; Zeiger et al., 1987; 

Dickey et al., 1949). Studies also demonstrate negative results for gene mutations, sister chromatid 

exchanges, micronucleus formation, and chromosomal aberrations (NTP, 1995; McGregor et al., 

1988). However, DNA adducts were found in male Kunming mice (Yuan et al., 2007), and DNA 

damage was observed in human HL-60 leukemia cells (Tang et al., 1997). In another study by 

Sgambato et al. (2009), an initial increase in DNA damage was observed as measured by nuclear 

fragmentation, but the damage declined drastically following 4 hours of exposure and disappeared 

entirely after 12 hours of exposure to tert-butanol. 

In terms of nephrotoxicity, a number of renal effects have been reported in female rats 

and/or in mice. Kidney transitional epithelial hyperplasia and inflammation were significantly 

increased in both male and female F344 rats exposed for 2 years via oral exposure. F344 rats 

exposed to tert-butanol at dose ranges of 230–1520 mg/kg-day and 850–3,620 mg/kg-day in males 

and females, respectively, exhibited a statistically significant increase in the incidence of 

nephropathy compared with controls after 13 weeks of exposure (NTP, 1995). Nephropathy 

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

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1 severity was also significantly increased at 420 mg/kg-day in males and 180-650 mg/kg-day in 

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females after 2 years of exposure (NTP, 1995). Average severity of chronic nephropathy was 

minimal to mild in males after a 13-week inhalation exposure (NTP, 1997). Female rats also had 

lesions associated with nephropathy (NTP, 1995), but none of the lesions were similar to those 

observed in the male rat that are associated with α2u-globulin nephropathy. 

With respect to renal tumors, no statistically significant increases in renal tumors were 

reported in tert-butanol-exposed female rats or mice compared with concurrent controls. Two 

renal tubular adenocarcinomas were reported in male mice following drinking water exposure for 

2 years (NTP, 1995) (one each in the low and high dose groups). Although such tumors are very 

rare in mice, with historical control incidences of 2/1351 (0.15%) in feeding studies and 4/1093 

(0.37%) in chamber studies (Haseman et al., 1998), these data are not sufficient to indicate that the 

kidney tumors observed in mice exposed to tert-butanol are treatment-related. 

Overall, the strength, consistency, and specificity of the data supporting a tert-butanolinduced α2u-globulin process as the sole actor for renal effects in male rats is weak to moderate.

Dose-response concordance

Is the α2u-globulin process occurring in male rats exposed to tert-butanol? 

As shown in Figure 1-3 and Figure 1-4, the dose-response concordance among hypothesized 

key events is mixed. 

Borghoff et al. (2001) exposed male and female F344 rats to tert-butanol at concentrations 

of 758, 1,364, or 5,305 mg/m3 for 6 hr/day for 10 days to assess the role of α2u-globulin 

nephropathy and renal cell proliferation. Significant tubular proliferation in males was observed at 

all exposure levels, but accumulation of α2u-globulin-positive hyaline droplets was increased only at 

the highest dose (Borghoff et al., 2001). These data suggest that cell proliferation may be related to 

the α2u-globulin process only at the highest exposure concentration. 

The dose-response relationships observed after 13 weeks and 2-years were also only 

moderately concordant. Data from a drinking water study (NTP, 1995; Takahashi et al., 1993; 

Lindamood et al., 1992) demonstrated hyaline droplet formation in the proximal tubule of male rats 

at all tested doses (except at the highest dose where all rats died during weeks 5-12) following oral 

exposure to tert-butanol for 13 weeks. PWG reevaluation by Hard et al. (2011) reported observing 

precursors to granular casts at the only dose level evaluated (1,520 mg/kg-day). Spontaneous 

mineralization was observed, but the linear mineralization characteristic of this MOA was not 

observed at any dose. At the 2-year timepoint, linear mineralization was observed at all exposure 

levels from 90-420 mg/kg-day, and renal tubular hyperplasia was observed at the highest dose of 

420 mg/kg-day (NTP, 1995), consistent with the expected dose-response relationship. Notably, 

however, granular casts were not observed. 

Overall, the dose-response concordance of the association between tert-butanol and the 

hypothesized key events is moderate. 

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

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1 Are the renal effects in male rats exposed to tert-butanol solely due to the α2u-globulin process? 

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Dose-response concordance between the hypothesized key events and the occurrence of 

renal tumors can inform whether carcinogenesis is solely due to the α2u-globulin process. Male 

F344 rats exhibited an increased incidence of renal tubule adenomas and combined renal tubule 

adenoma or carcinoma in a 2-year oral bioassay (Hard et al., 2011; NTP, 1995). Increased tumors 

were observed at 90, 200, and 420 mg/kg-day. Although some effects related to α2u-globulin

nephropathy, including hyaline droplets and linear mineralization, were observed at all doses,

tubule hyperplasia was not observed at doses lower than 420 mg/kg-day in any study and only 

precursor granular casts were observed at a much higher dose of 1,520 mg/kg-day. Moreover, the 

middle dose of tert-butanol induced the greatest incidence of tumors, so increasing the dose from 

200 to 420 mg/kg-day led to additional markers of α2u-globulin nephropathy in the form of tubule 

hyperplasia, but without any increase in tumor burden. Thus, tert-butanol induced tumors at lower 

doses than for other precursor effects such as hyperplasia and granular casts, suggesting a weak 

dose response concordance with the incidence of tumors. 

Therefore, on the basis of weak dose-response concordance, the data suggest that the 

observed tumors are not solely due to α2u-globulin and that other processes are primarily 

responsible for tumors. 

Temporal relationship

Is the α2u-globulin process occurring in male rats exposed to tert-butanol? 

As shown in Table 1-2 and Table 1-4, hyaline droplets and α2u-globulin accumulation were 

observed after a single dose or 10-day exposure; precursors to granular casts were observed at 13 

weeks; and tubular hyperplasia was observed at 2 years. The observations are consistent with the 

expected temporal relationship (Hard et al., 2011; Borghoff et al., 2001; Williams and Borghoff, 

2001; NTP, 1995). However, the absence of other key events such as necrosis, exfoliation, and 

granular casts in most other studies at the anticipated time points weaken the case for α2u-globulin 

MOA. Additionally, the NTP’s 13-week study in rat reported kidney mineralization, but it was not 

characterized as linear mineralization.

Are the renal effects in male rats exposed to tert-butanol solely due to the α2u-globulin process? 

This question cannot be answered from data on temporality. 

Biological plausibility and coherence

Both U.S. EPA and IARC have accepted the general biological plausibility and coherence of 

role of α2u-globulin-mediated nephropathy in renal tumor induction (Swenberg and LehmanMcKeeman, 1999; U.S. EPA, 1991a), and those rationales will not be repeated here.

However, a retrospective analysis has suggested that a number of α2u-globulin inducing 

chemicals fail to induce many of the pathological sequences in the α2u-globulin pathway (Doi et al., 

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

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1 2007). For instance, dose-response concordance was not observed for several endpoints such as 

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linear mineralization, tubular hyperplasia, granular casts, and hyaline droplets following exposure 

to α2u-globulin-inducing chemicals such as d-limonene, decalin, propylene glycol mono-t-butyl

ether, and Stoddard solvent IIC (SS IIC). Although some of these chemicals induced 

histopathological lesions that exhibited a dose response, all of them failed to induce a doseresponse trend for at least one of the endpoints in the sequence. Furthermore, no endpoint in the 

pathological sequence was predictive for tumor incidence. Tumor incidence did not exhibit a dose 

response following either d-limonene or decalin exposure. Finally, tumor incidence was not 

predicted by the severity of a particular effect in the α2u-globulin sequence as demonstrated by SS 

IIC which induced some of the most severe nephropathy precursors relative to the other chemicals 

but did not significantly increase kidney tumors (Doi et al., 2007). Thus, these analyses suggest that 

another MOA may be operative for inducing tumors. 

Moreover, renal tumors were not observed following exposure to ETBE, which is rapidly 

metabolized to tert-butanol. Specifically, Suzuki et al. (2012) and Saito et al. (2013) reported no 

increase in renal tumors in both sexes of Fischer 344 rats following 2-year oral or inhalation 

exposures to ETBE at doses that yield similar internal concentrations (based on PBPK modeling) of 

tert-butanol compared with the concentrations of the tert-butanol bioassays. After 13 weeks of 

exposure to tert-butanol or ETBE, hyaline droplets were increased in a dose-response manner. 

ETBE exposure increased hyaline droplets at lower internal concentrations of tert-butanol than by 

direct tert-butanol administration. Similar to hyaline droplets, linear mineralization was increased 

at an internal tert-butanol concentration approximately tenfold lower following ETBE exposure 

than a tert-butanol exposure. By contrast, tubule hyperplasia and renal tumors were both observed 

following a 2-year exposure to tert-butanol but not following ETBE exposure. Renal tumors and 

tubule hyperplasia were not observed following any ETBE exposure despite achieving similar blood 

concentrations of tert-butanol as the NTP (1995) study. The failure of internal tert-butanol

concentrations to induce histopathological lesions early in the α2u-globulin pathological sequence 

at blood levels that later induced hyperplasia and tumors suggests a lack of coherence across the 

two data sets. 

Conclusions about the hypothesized MOA for α2u-globulin-associated nephropathy

Is the hypothesized MOA sufficiently supported in test animals?

This conclusion is divided into two sub-questions: whether the α2u-globulin process is 

occurring in male rats, and whether it is the sole contributor to renal effects in male rats.

With respect to the first question, tert-butanol induced increases in α2u-globulin deposition 

and hyaline droplet accumulation, and several of the subsequent steps in the pathological sequence 

were observed. These data provide sufficient evidence that the α2u-globulin process can be 

operating given sufficient tert-butanol exposure. 

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Is the hypothesized MOA relevant to humans?

With respect to the second question, male rats are more sensitive to the kidney effects of 

tert-butanol, and the available data indicate that male rats accumulate α2u-globulin in the kidney, 

which is a specific MOA for male rats. Many of the steps in the pathological sequence of lesions 

related to α2u-globulin-associated nephropathy were observed exclusively in male rats but not in 

female rats, or mice of either sex, and renal tumors occurred only in male rats. However, there is 

insufficient evidence to support a conclusion that α2u-globulin nephropathy is the sole or primary 

contributor to renal tumor development. Given the inconsistencies and limitations of the 

genotoxicity database, the effect of tert-butanol with respect to genotoxicity cannot be ruled out. 

Additionally, tert-butanol induced tumors at lower doses than other precursor effects such as 

hyperplasia and granular casts, with no further increase in tumor incidence coinciding with the 

additional markers of α2u-globulin nephropathy. Thus, renal tumors observed in male rats are 

unlikely to be confounded by the presence of α2u-globulin nephropathy. Therefore, on the basis of a 

weak dose-response concordance, the data support a conclusion that processes other than 

α2u-globulin nephropathy are likely responsible for renal tumor development.

Based on the conclusion that processes other than α2u-globulin nephropathy are likely 

responsible for renal tumor development induced by tert-butanol, U.S. EPA (1991a) states that the 

following conclusion will be made: 

Therefore, kidney tumors are relevant to humans for purposes of hazard identification and 

dose-response assessment. Because female rats and both sexes of mice do not have α2u-globulin

present, kidney effects in these animals are considered relevant to humans for both hazard 

identification and dose-response. 

Which populations or lifestages can be particularly susceptible to the hypothesized MOA?

This question is not applicable.

Alternative MOA hypotheses with inadequate data for analysis

Other nephrotoxic responses, such as exacerbation of CPN, inflammation, transitional 

epithelial hyperplasia, and increased kidney weight, are observed in rats and/or mice, suggesting 

other possible processes are operative. It has been proposed that enhanced chronic progressive 

nephropathy (CPN) is a mode of action for chemically-induced kidney tumors in male rats and that 

• If renal tumors in male rats are not linked to the α2u-globulin process, then (U.S. EPA, 

1991a) states that such tumors are an appropriate endpoint for human hazard 

identification and are considered, along with other appropriate endpoints, for quantitative 

risk estimation.

Toxicological Review of tert-Butyl Alcohol

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1 renal tubule tumors induced by chemicals that concomitantly exacerbate CPN are not relevant to 

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humans (Hard and Khan, 2004). 

CPN is an age-related renal disease of unknown etiology that occurs spontaneously in rats, 

especially the F344, Sprague-Dawley, and Osborne-Mendel strains. Additional markers associated 

with CPN include elevated protein and albumin in the urine and increased BUN, creatinine, and 

cholesterol in the serum (Hard et al., 2009). CPN is often more severe in males compared with 

females. Several of the CPN pathological effects are similar to and can obscure the lesions 

characteristic of α2u-globulin-related hyaline droplet nephropathy (Webb et al., 1990). Additionally, 

renal effects of α2u-globulin accumulation can exacerbate the effects associated with CPN (U.S. EPA, 

1991a). However, Webb et al. (1990) suggested that exacerbated CPN was one component of the 

nephropathy resulting from exposure to chemicals that induce α2u-globulin nephropathy. Male rat 

sensitivity has been noted with both CPN and α2u-globulin nephropathy. 

Increased severity of CPN occurred in both male and female rats as a result of tert-butanol 

exposure. Some of the observed renal lesions in male rats following exposure to tert-butanol are 

effects commonly associated with CPN. Hard et al. (2011) concluded that the observation of 

transitional epithelial hyperplasia in the 2-year drinking study conducted by NTP (1995) was 

associated with CPN, and not a direct effect of tert-butanol exposure. However, there was a strong, 

statistically-significant, treatment-related, dose-response relationship between chronic tert-butanol 

exposure and increased incidence of transitional epithelial hyperplasia in both male and female rats 

in the NTP (1995) study. The severity of CPN also increased with tert-butanol exposure, although 

the dose-response relationship in males was very weak (only a 10% increase in mean severity at 

the highest dose). The very different dose-response relationships argue against a close association. 

Moreover, even if transitional epithelial hyperplasia were associated with CPN, there is no evidence 

to support that the effect is independent of tert-butanol treatment, given the robust dose-response 

relationships. Therefore, the data are insufficient to dismiss transitional epithelial hyperplasia as 

causally related to tert-butanol exposure.

Additionally, there have been a few research groups who have discussed the role of CPN and 

α2u-globulin accumulation on the renal tumors observed in male rats exposed to tert-butanol. 

Cruzan et al. (2007) concluded that α2u-globulin, exacerbation of CPN, or a combination of both 

were the MOAs for the kidney tumors in males. Hard et al. (2011) also concluded that both α2uglobulin-induced nephropathy and exacerbated CPN were MOAs for the kidney tumors observed in 

the male rats in the 2-year drinking study conducted by NTP (1995). However, the underlying 

mechanisms regulating CPN and its exacerbation are not well understood, and to date, there is no 

scientific consensus on the relevance of CPN in rats to human health hazard (Melnick et al., 2012; 

Hard et al., 2009). Moreover, no key events for the exacerbation of CPN have been identified, so no 

MOA analysis can be performed under the EPA Cancer Guidelines MOA framework (U.S. EPA, 

2005a). Therefore, kidney effects from tert-butanol exposure associated with CPN are considered 

relevant to humans.

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

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1 Summary of kidney toxicity 

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Kidney toxicity was consistently observed after oral exposure in two strains of rats and in 

one strain of mice and in both sexes. Absolute and relative kidney weights also were increased in

male and female rats in both the 13-week and 2-year studies. In male and female rats, 

histopathological examination of the kidneys indicated kidney lesions exhibiting a dose-response 

trend, increased incidence of nephropathy after 13 weeks and 2 years, and increased transitional 

epithelium hyperplasia and suppurative inflammation (females only) after 2 years. In mice, the 

only kidney effect observed was an increase in kidney weight (absolute and/or relative) in both 

sexes of mice in the 13-week study. Organs were not weighed in the 2-year mouse study, so no 

determination can be made. Furthermore, there were no treatment-related histopathological 

lesions in the kidneys of mice at 13 weeks or 2 years. 

Male rats are more sensitive to the kidney effects of tert-butanol, and the available data 

indicate that male rats accumulate α2u-globulin in the kidney, which is a specific MOA for male rats. 

MOA analysis determined that the renal tumors observed in male rats are mediated by other 

processes besides α2u-globulin. Therefore, in the absence of a known MOA, EPA considers the male 

and female kidney effects observed in experimental animals to be relevant to assessing human 

health hazard. 

EPA identified kidney effects as a human hazard of tert-butanol exposure. Data on kidney 

tumors associated with tert-butanol exposure are discussed as part of the overall weight of 

evidence for carcinogenicity in Section 1.2.2.

1.1.2. Thyroid Effects 

Synthesis of Effects in Thyroid

This section reviews the studies that investigated whether exposure to tert-butanol can 

cause thyroid effects in humans or animals. The database examining thyroid effects following 

tert-butanol exposure contains no human data and two chronic studies (one in rats and one in 

mice). Studies employing short term and acute exposures that examined thyroid effects are not 

included in the evidence tables; however, they are discussed in the text if they provide data to 

support mode of action or hazard identification. No methodological concerns were identified that 

would lead one or more studies to be considered less informative for assessing human health 

hazard. 

A 2-year inhalation study is not available. Thyroid effects were not observed in studies in 

rats (NTP, 1995). Thyroid toxicity was observed in mice of both sexes after 2 years of oral exposure 

via drinking water (NTP, 1995). Follicular cell hyperplasia, as well as follicular cell adenomas, was 

present in both male and female mice. The evidence was stronger in females due to the doserelated increase in follicular cell hyperplasia reaching statistical significance in the highest two 

doses, and the presence of a statistically significant increase in follicular cell adenomas in the highdose group. There was also a statistically significant increase in follicular cell hyperplasia at all 

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

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1 doses in male mice, but only a marginal increase in follicular cell adenomas in the mid-dose group. 

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One high-dose male mouse developed a follicular cell carcinoma. The lower tumor incidence in 

males may be due to the increased mortality seen in the high-dose group. NTP (1995) noted that 

thyroid follicular cell tumorigenesis follows a progression from hyperplasia to adenoma and 

carcinoma, suggesting that hyperplasia is a preneoplastic lesion in the thyroid. 

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1 Table 1-7. Evidence pertaining to thyroid effects in animals following oral 

2 exposure to tert-butanol

Reference and study design Results

Follicular cell hyperplasia

NTP (1995)

F344/N rat; 60/sex/treatment 

(10/sex/treatment evaluated at 15 

months)

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

mg/mL) 

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

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

2 years

Incidenceb

Males Females

Dose

(mg/kg-d)

Follicular cell 

hyperplasia

Dose

(mg/kg-d)

Follicular cell 

hyperplasia

0 3/50 0 0/50

90 0/49 180 0/50

200 0/50 330 0/50

420 0/50 650 0/50

NTP (1995)

B6C3F1 mouse; 60/sex/treatment 

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

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

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

2 years

Incidence (severity)

Males Females

Dose

(mg/kg-d)

Follicular cell 

hyperplasia

Dose

(mg/kg-d)

Follicular cell 

hyperplasia

0 5/60 (1.2) 0 19/58 (1.8)

540 18/59* (1.6) 510 28/60 (1.9)

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

2,070 18/57* (2.1) 2,110 47/59* (2.2)

Tumors

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

Incidenceb

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 0/50 0/50

Female

0 1/50 1/50

180 0/50 0/50

330 1/50 1/50

650 0/50 0/50

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Table 1-7. Evidence pertaining to thyroid effects in animals following oral 

exposure to tert-butanol (continued) 

Reference and study design Results

NTP (1995)

B6C3F1 mouse; 60/sex/treatment 

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

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

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

2 years

Incidence

Dose

(mg/kg-d)

Follicular 

cell 

adenoma

Mortalityadjusted 

rate (%)

Follicular 

cell 

carcinoma 

or adenoma

Mortalityadjusted 

rate

(%)

Follicular 

cell 

carcinomac

Male

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

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

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

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

Female

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

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

1,020 2/59 4.9 2/59 4.9 0/59

2,110 9/59* 19.6 9/59* 19.6 0/59

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There was a significant decrease in survival in the high-dose group. b

Results do not include the animals sacrificed at 15 months.

c

Mortality-adjusted rates were not calculated by study authors for follicular cell carcinoma.

* Statistically significant p ≤ 0.05 as determined by the study authors.

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

Hyperplasia; M rat

Hyperplasia; F rat

Adenoma; M mouse

Adenoma; F mouse

Adenoma; M rat

Adenoma; F rat

NONCANCER

CANCER

10 100 1,000 10,000

Dose (mg/kg-day)

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Source: NTP (1995)

Figure 1-5. Exposure-response array of thyroid follicular cell effects following 

chronic oral exposure to tert-butanol. 
Toxicological Review of tert-Butyl Alcohol

1 Mode of Action Analysis—Thyroid Effects

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There are inadequate data to determine the MOA for tert-butanol-induced thyroid follicular 

cell lesions. The mechanism of formation of these lesions resulting from tert-butanol exposure has 

not been specifically studied; however, Blanck et al. (2010) conducted a short-term study 

examining the hepatic and thyroid effects of tert-butanol exposure to provide additional data on the 

thyroid tumors observed in the chronic NTP (1995) study. tert-Butanol did not have any effect on 

liver weight when compared to the control group, but the livers were visibly enlarged, in some 

cases accompanied by centrilobular hepatocellular hypertrophy, in some treated groups. There 

were no treatment-related histological alterations in the thyroid in tert-butanol treated mice. Only a 

slight statistically nonsignificant increase in thyroid stimulating hormone (TSH) was observed after 

3 days of exposure, but both thyroxine (T4) and triiodothyronine (T3) levels were decreased. 

Sustained increases in TSH, resulting in sustained thyroid follicular cell proliferation, may 

eventually result in progression of hyperplasia to adenoma and carcinoma (U.S. EPA, 1998a). Based 

on alterations in hepatic phase I and phase II enzyme activities and gene expression, the data from 

Blanck et al. (2010) suggest a possible role for increased thyroid hormone clearance in the liver in 

tert-butanol-induced thyroid tumors. The available support for this hypothesis, however, is weak. 

In particular, Blanck et al. (2010) did not find any significant changes in TSH levels, though the 

study duration was short (≤14 days), and there are no data regarding thyroid cell proliferation after 

exposure to tert-butanol. 

Summary of thyroid toxicity

EPA identified thyroid effects as a potential human hazard of tert-butanol exposure. The 

thyroid endpoints reported following chronic exposure to tert-butanol include follicular cell 

hyperplasia, follicular cell adenoma, and follicular cell carcinoma. Together with the evidence of 

significantly increased incidence of thyroid follicular cell adenomas in high-dose females, these 

observations support the finding that the increased hyperplasia in male mice is a preneoplastic 

effect rather than an adaptive response. There is no conclusive MOA for the development of thyroid 

follicular cell adenomas, although there is some evidence supporting greater clearance of thyroid 

hormones by the liver causing continual secretion of TSH by the pituitary leading to follicular cell 

hyperplasia and tumors. Data on thyroid tumors associated with tert-butanol exposure are 

discussed further as part of the overall weight of evidence for carcinogenicity in Section 1.2.2.

1.1.3. Reproductive and Developmental Effects 

Synthesis of reproductive and developmental toxicity 

This section reviews the studies that investigated whether exposure to tert-butanol can 

cause reproductive and developmental effects in humans or animals. This section contains 

information on reproductive effects, systemic developmental effects, as well as 

neurodevelopmental effects following tert-butanol exposure. The database examining reproductive 

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

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1 or developmental effects following tert-butanol exposure contains no human data and 7 studies 

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performed in rats and mice. Three studies evaluating reproductive effects included changes in 

reproductive organs (one one-generation reproductive study and two subchronic studies). In 

addition, there was one two-year oral study in rats and mice that evaluated reproductive 

histopathology and did not find any treatment-related effects. The studies selected for discussion 

below exposed animals via oral gavage, drinking water, and inhalation for ≥63 days. The collection 

of reproductive studies on tert-butanol is limited by the absence of any two-generation 

reproductive oral or inhalation studies. Four studies evaluated developmental effects (three 

developmental studies and one one-generation reproductive study). As mentioned in the Study 

Selection, the studies selected for discussion below exposed animals to tert-butanol via inhalation, 

gavage, and drinking water. No methodological concerns were identified that would lead one or 

more studies to be considered less informative for assessing human health hazard, but the Faulkner 

et al. (1989) study did not provide results in the dam that could be used to adequately determine if 

fetal effects occurred due to maternal toxicity. Three studies evaluated neurodevelopmental effects 

following tert-butanol exposure in rats and mice. These studies selected for discussion below 

exposed animals via liquid diet and inhalation. The collection of neurodevelopmental studies on 

tert-butanol is limited in that all studies were conducted prior to Developmental Toxicity 

Guidelines being available from the U.S. EPA (1991b) and OECD; as such, there are study design 

considerations for each of the studies. Daniel and Evans (1982) had a small number of animals per 

treatment group, presentation of results provided limited use of the data with no comparisons to 

controls, and there was no long term neurodevelopmental testing. Nelson et al. (1991) evaluated 

neurodevelopmental effects after either paternal or maternal exposure. Although the study authors

used two different exposure concentrations, the exposures were not run concurrently nor was 

there information provided on exposure methods to indicate the studies were conducted similarly.

Reproductive effects

Reproductive endpoints, such as sex organ weights, estrous cycle length, and sperm effects

were examined following either oral or inhalation exposure in three subchronic studies (Lyondell 

Chemical Co., 2004; NTP, 1997, 1995)(Table 1-8;Figure 1-6;Figure 1-7). The only reproductive 

effect noted was an increase in estrous cycle length. Estrous cycle length was increased (28% 

increase, p < 0.01) in female mice orally exposed to 11,620 mg/kg-day. No significant changes in 

estrous cycle length were observed following oral exposure in rats, or inhalation exposure in mice 

or rats. 

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

2 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

F0 females: 4 weeks prior to mating through 

PND21

F0 reproductive effects

No significant effect on weights of male reproductive organs or sperm 

observed

NTP (1995)

F344/N rat; 10/sex/treatment 

Drinking water (0, 2.5, 5, 10, 20, or 40 mg/mL) 

 M: 0, 230, 490, 840, 1,520, 3,610a mg/kg-d 

 F: 0, 290, 590, 850, 1,560, 3,620a mg/kg-d 

13 weeks

No significant effect on weights of male reproductive organs or sperm 

observed

NTP (1995)

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/kg-d 

 F: 0, 500, 820, 1,660, 6,430, 11,620a mg/kg-d 

13 weeks

No significant effect on weights of male reproductive organs or sperm 

observed

NTP (1997)

F344/N rat; 10/sex/treatment

Analytical concentration: 0, 134, 272, 542, 

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

or 6,368 mg/m3) (dynamic whole body 

chamber) 

6 hr/d, 5 d/wk 

13 weeks

Generation method (Sonimist Ultrasonic spray 

nozzle nebulizer), analytical concentration and 

method were reported

No significant effect on weights of male reproductive organs or sperm 

observed

Evaluations were performed only for concentrations ≥542 ppm

(1,643 mg/m3

) 

NTP (1997)

B6C3F1 mouse; 10/sex/treatment

Analytical concentration: 0, 134, 272, 542, 

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

or 6,368 mg/m3

) (dynamic whole body 

chamber)

6 hr/d, 5 d/wk 

13 weeks

Generation method (Sonimist Ultrasonic spray 

nozzle nebulizer), analytical concentration and 

method were reported

No significant effect on weights of male reproductive organs or sperm 

observed

Evaluations were performed only for concentrations ≥542 ppm

(1,643 mg/m3) 

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

exposure to tert-butanol (continued) 

Reference and study design Results

Female 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

F0 females: 4 weeks prior to mating through PND21 

Pregnancy index

 91.7% 91.7% 100% 100% 91.7% 

NTP (1995)

F344/N rat; 10/sex/treatment 

Drinking water (0, 2.5, 5, 10, 20, or 40 mg/mL) 

 M: 0, 230, 490, 840, 1,520, 3,610a mg/kg-d 

 F: 0, 290, 590, 850, 1,560, 3,620a mg/kg-d 

13 weeks

No significant effect on female estrous cycle (0, −2, −4, 0, +8 % 

change relative to control)

NTP (1995)

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/kg-d 

 F: 0, 500, 820, 1,660, 6,430, 11,620a mg/kg-d 

13 weeks

↑ length of estrous cycle

Response relative to control: 0, +5, +5, +5, +6, +28*%

NTP (1997)

F344/N rat; 10/sex/treatment 

Analytical concentration: 0, 134, 272, 542, 1,080, or 

2,101 ppm (0, 406, 824, 1,643, 3,273 or 6,368 mg/m3

) 

(dynamic whole body chamber) 

6 hr/d, 5 d/wk 

13 weeks

Generation method (Sonimist Ultrasonic spray nozzle 

nebulizer), analytical concentration and method were 

reported

No significant effect on female estrous cycle (0, −4, +2, +4 % change 

relative to control)

Evaluations were performed only for concentrations ≥542 ppm

(1,643 mg/m3

) 

NTP (1997)

B6C3F1 mouse; 10/sex/treatment

Analytical concentration: 0, 134, 272, 542, 1,080, or 

2,101 ppm (0, 406, 824, 1,643, 3,273 or 6,368 mg/m3

) 

(dynamic whole body chamber)

6 hr/d, 5 d/wk 

13 weeks

Generation method (Sonimist Ultrasonic spray nozzle 

nebulizer), analytical concentration and method were 

reported

No significant effect on female estrous cycle (0, −3, −9, −5 % change 

relative to control)

Evaluations were only performed for concentrations ≥542 ppm

(1,643 mg/m3

) 

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* Statistically significant p ≤ 0.05 as determined by the study authors.

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.

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

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Toxicological Review of tert-Butyl Alcohol

1 Developmental effects

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Data from three developmental studies (two oral, one inhalation) suggest that fetal effects 

are generally observed at doses that cause toxicity in the dams as measured by clinical signs (e.g., 

decreased body weight gain, and/or food consumption) (Table 1-9; Figure 1-6; Figure 1-7).

Developmental effects of tert-butanol observed after oral exposure (liquid diets or gavage) 

in several mouse strains and one rat strain include measures of fetal loss or viability (e.g., increased 

number of resorptions, decreased numbers of neonates per litter) and decreased fetal body weight 

(Lyondell Chemical Co., 2004; Faulkner et al., 1989; Daniel and Evans, 1982). Daniel and Evans 

(1982) also observed decreases in body weight gain during PND 2–10; however, data suggest that 

the effect may be due to maternal behavior or nutritional status. In addition, one study reported 

increased incidence of variations of the skull or sternebrae in two mouse strains, but the difference 

was not statistically significant (Faulkner et al., 1989). Similar developmental effects were also 

observed after whole-body inhalation exposure in Sprague-Dawley rats for 7 hours/day on GDs 1–

19 (Nelson et al., 1989). Fetal effects included concentration-related reductions in body weight in 

male and female fetuses and higher incidence of skeletal variations when analyzed on the basis of 

individual fetuses (but not on a per litter basis). In contrast to the oral exposure studies in mice 

and rats, however, there was no effect on measures of fetal loss. 

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

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Toxicological Review of tert-Butyl Alcohol

1 Table 1-9. Evidence pertaining to developmental effects in animals following 

2 exposure to tert-butanol

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)

3

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

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Table 1-9. Evidence pertaining to developmental effects in animals following 

exposure to tert-butanol (continued)

Reference and study design Results

Faulkner et al. (1989)

CBA/J mouse; 7 pregnant females in 

control, 12 pregnant females in treated 

Gavage (10.5 mmoles/kg twice a day); 

0 (tap water), 1,556 mg/kg-d

GD 6–18

Maternal results not reported. 

Fetal

Percent change compared to 

control: Incidence:

Dose

(mg/kg-d)

Live 

fetuses/litter

Fetal

weight

Sternebral 

variations

Skull 

variations

0 0 0 4/28 1/28

1,556 −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) and resorptions per litter (+118%) increased (p < 0.05)

Faulkner et al. (1989)

C57BL/6J mouse; 5 pregnant females in 

controls, 9 pregnant females treated 

Gavage (10.5 mmoles/kg twice a day)

0 (tap water), 1,556 mg/kg-d

GD 6–18

Maternal results not reported. 

Fetal

Percent change compared to 

control: Incidence:

Dose

(mg/kg-d)

Live 

fetuses/litter

Fetal

weight

Sternal 

variations Skull variations

0 0 0 5/21 1/21

1,556 −58%* −4 9/16 7/16

Sternal variations: misaligned or unossified sternebrae

Skull variations: moderate reduction in ossification of supraoccipital bone

Number of total resorptions (4 resorptions/44 implants in controls, 38/68 

implants in treated) and resorptions per litter (+428%) increased (p < 0.05)

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

1-38 DRAFT—DO NOT CITE OR QUOTE
Toxicological Review of tert-Butyl Alcohol

Table 1-9. Evidence pertaining to developmental effects in animals following 

exposure to tert-butanol (continued)

Reference and study design Results

Lyondell Chemical Co. (2004)

OECD guideline 421 study:

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 1000 or 400 mg/kg/d showed CNS effects (e.g., ataxia, 

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

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

F1 effects 

Viability index (pup survival to PND4)

96.4% 98.7% 98.2% 99.4% 74.1%*

Lactation index (pup survival to PND21)

100% 100% 100% 99.2% 98.8%

Sex ratio (% males)

54.4 52.3 50.9 53.4 52.1

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

0 +6 +4 +7 −10

Pup weight PND 28 relative to control (%)

M: 0 +2 0 0 −12*

F: 0 0 −4 −2 −8

Nelson et al. (1989)

Sprague-Dawley rat; 15 pregnant 

dams/treatment

Maternal: Unsteady gait (no statistical tests reported), dose-dependent ↓ in 

body weight gain (results presented in figure only), dose-dependent ↓ in food 

consumption ranging from 7–36% depending on dose and time

Fetal

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

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Toxicological Review of tert-Butyl Alcohol

Table 1-9. Evidence pertaining to developmental effects in animals following 

exposure to tert-butanol (continued)

Reference and study design Results

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 

Generation method, analytical 

concentration and method were reported

Percent change compared to control:

Dose

(mg/m3)

Number of 

live 

fetuses/litter

Resorptions 

per litter

0 0 0

6,669 0 +9

10,640 +15 −18

15,248 +8 0

Percent change compared to 

control: Incidence:

Dose

(mg/m3)

Fetal weight 

(males)

Fetal weight 

(females)

Skeletal 

variation

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

. 

Percentage change compared to control = (treated value – control value) ÷ control value × 100.

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

1-40 DRAFT—DO NOT CITE OR QUOTE
Toxicological Review of tert-Butyl Alcohol

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

1-41 DRAFT—DO NOT CITE OR QUOTE

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4

Sources: (A) Faulkner et al. (1989); (B) Lyondell Chemical Co. (2004); (C) NTP (1995)

Figure 1-6. Exposure-response array of reproductive and developmental 

effects following oral exposure to tert-butanol. 
Toxicological Review of tert-Butyl Alcohol

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

1-42 DRAFT—DO NOT CITE OR QUOTE

Sources: (A) NTP (1997); (B) Nelson et al. (1989)

Figure 1-7. Exposure-response array of reproductive and developmental 

effects following inhalation exposure to tert-butanol. 
Toxicological Review of tert-Butyl Alcohol

1 Neurodevelopmental Effects 

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In addition to the developmental effects noted above, neurodevelopmental effects also have 

been observed. This includes changes in rotarod performance following oral or inhalation 

exposures, as well as decreases in open field behavior and cliff avoidance following oral exposure, 

and reduced time hanging on wire after inhalation exposure during gestation (Table 1-10; Figure

1-6; Figure 1-7). 

Rotarod performance

Looking across studies, not all the findings were consistent. While Daniel and Evans (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

In addition to behavioral effects, one study evaluated biochemical or physiological changes 

in the brain of offspring exposed during gestation or early in the postnatal period. Nelson et al. 

(1991) found statistically significant changes in neurochemical measurements in the brain in 

offspring of dams exposed via inhalation during gestation; however, the two concentrations tested 

were not run concurrently, and very little data were provided. 

Physiological and psychomotor development

Data also suggest that neurodevelopmental effects were not solely due to in utero exposure 

(Daniel and Evans, 1982). Daniel and Evans (1982) cross-fostered half of the mouse pups born to 

treated mother 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 with control dams performed significantly better than those maintained with treated 

dams (Table 1-10) (Daniel and Evans, 1982). Results were only presented in figures and were not 

compared with controls. 

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

1-43 DRAFT—DO NOT CITE OR QUOTE
Toxicological Review of tert-Butyl Alcohol

1 Table 1-10. Evidence pertaining to neurodevelopmental effects in animals

2 following exposure to tert-butanol 

Reference and study design Results

Daniel and Evans (1982)

Liquid diet (0, 0.5, 0.75, or 1.0%, w/v); GD6–20; 

Swiss Webster (Cox) mouse; 15 pregnant 

dams/treatment; after birth half the pups were 

nursed with their treated dams and the other 

half were fostered by untreated dams who 

recently gave birth

0 (isocaloric amounts of maltose/dextrin),

3,324, 4,879, or 6,677 mg/kg-d

• a dose-dependent increase righting reflex time, with more time 

needed in animals maintained with maternal dams

• a dose-dependent decrease in open field behavior, with less activity 

in pups maintained with maternal dams

• a dose-dependent decrease in rotarod performance with the pups 

from maternal dams having lower performances

• a dose-dependent decrease in the amount of time the pups were 

able to avoid a cliff, with animals maintained with their maternal 

dams having less avoidance time

Nelson et al. (1991)

Sprague-Dawley rat; 15 pregnant 

dams/treatment

Analytical concentration: 0, 6,000, or 12,000 

mg/m3

; (dynamic whole body chamber)

7 hr/d 

GD 1–19 

Generation method, analytical concentration 

and method were reported 

Data were not presented specifically by dose nor were any tables or figures 

of the data provided

Maternal toxicity was noted by decreased food consumption and body 

weight gains

Results in offspring 

• increase in rotarod performance in high-dose group (16 versus 26 

revolutions/min for controls and 12,000 mg/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)

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

3

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

1-44 DRAFT—DO NOT CITE OR QUOTE
Toxicological Review of tert-Butyl Alcohol

Table 1-10. Evidence pertaining to neurodevelopmental effects in animals

following exposure to tert-butanol (continued) 

Reference and study design Results

Nelson et al. (1991)

adult male Sprague-Dawley rats (18/treatment) 

mated to untreated females

Analytical concentration: 0, 6,000, or 

12,000 mg/m3

; (dynamic whole body chamber)

7 hr/d for 6 wk

Generation method, analytical concentration 

and method were reported

Data were not presented specifically by dose nor were any tables or figures 

of the data provided

Results (generally only specified as paternally treated versus controls) in 

offspring indicate

• increase in rotarod performance (16 versus 20 revolutions/min for 

controls and 12,000 mg/m3 animals, respectively)

• decreased time in open field (less time to reach the outer circle of 

the field, 210 sec versus 115 seconds for controls and 12,000 mg/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

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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.

Percentage change compared to control = (treated value – control value) ÷ control value × 100.

Mechanistic Evidence

No mechanistic evidence is available for reproductive or developmental effects, including 

neurodevelopmental effects. 

Summary of Reproductive and Developmental Toxicity

EPA concluded that the evidence does not support reproductive effects as a potential 

human hazard of tert-butanol exposure. There are no two-generation reproductive studies 

available by oral or inhalation exposure. Two oral exposure studies (Lyondell Chemical Co., 2004; 

NTP, 1995) and one subchronic inhalation study (NTP, 1997) are available. Overall, reproductive 

effects observed due to exposure to tert-butanol were limited to altered length of estrous cycle 

(NTP, 1995), but there is no available information to infer how this effect may influence 

reproductive ability. 

EPA identified suggestive evidence of developmental effects as a potential human hazard of

tert-butanol exposure. Exposure during gestation resulted in increased fetal loss, decreased fetal 

body weight, and possible increases in skeletal variations in exposed offspring or pups, although 

effects were not always consistent across exposure routes (oral and inhalation). Dams had 

decreased body weight and/or body weight gains, decreased food consumption, and/or clinical 

signs of intoxication at the same doses that tert-butanol caused fetal effects. Neurodevelopmental 

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

1-45 DRAFT—DO NOT CITE OR QUOTE
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1 effects including decreased brain weight, changes in brain biochemistry, and changes in behavioral 

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performances have also been observed. Each of the neurodevelopmental studies, however, had 

limitations in the study design and/or reporting. In addition, results from the neurodevelopmental 

studies were not always consistent between studies or across dose. 

1.1.4. Carcinogenicity (other than in the kidney or thyroid)

Synthesis of Carcinogenicity Data (Other than in the Kidney or Thyroid)

This section reviews the studies that investigated whether exposure to tert-butanol can 

cause cancers (other than in the kidney or thyroid) in humans or animals. The database examining 

carcinogenicity following tert-butanol exposure contains no human data and two chronic studies, 

one in rats and one in mice. As mentioned in the Study Selection, the studies providing data on 

carcinogenicity exposed animals via drinking water for ≥30 days. Shorter duration studies do not 

generally evaluate carcinogenicity, but any shorter duration studies that examined carcinogenicity 

are discussed in the text if they provide data to support mode of action or hazard identification. No 

methodological concerns were identified that would lead one or more studies to be considered less 

informative for assessing human health hazard. 

Kidney and thyroid tumors are presented above with the specific organ hazard 

identification. No other treatment-related changed in tumors in other organs were noted in the 2-

year oral rat or mouse studies conducted by NTP (1995), which evaluated a comprehensive set of 

tissues/organs. There is no 2-year inhalation study. 

Mechanistic Evidence

Available mechanistic evidence was previously discussed in the context of kidney and 

thyroid tumors (Sections 1.1.1 and 1.1.2). 

Summary of Carcinogenicity Evidence

There are limited data available on the carcinogenicity of tert-butanol. There are 2-year 

oral studies in one strain of rats and one strain of mice, but no 2-year inhalation studies. EPA 

identified suggestive evidence of kidney and thyroid tumors as a potential human hazard. 

1.1.5. Other Toxicological Effects

Synthesis of Other Toxicity Data

The database for effects other than kidney, thyroid, reproductive, developmental (including 

neurodevelopmental) and cancer contain only 14 rodent studies. As previously mentioned in the 

Study Selection, all selected studies employed inhalation, oral gavage, or drinking water exposures 

for ≥30 days. Studies employing short term and acute exposures that examined other toxicological 

effects are not included in the evidence tables; however, they are discussed in the text if they 

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

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1 provide data to support mode of action or hazard identification. No studies were removed for 

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methodological concerns.

 tert-Butanol also has been found to have CNS effects similar to ethanol in terms of animals 

appearing intoxicated and having withdrawal symptoms after cessation of oral or inhalation 

exposure. Severity of CNS symptoms such as withdrawal increased with dose and duration of 

exposure. However, study quality concerns (e.g., short exposure durations, lack of data reporting, 

small number of animals per treatment group) associated with all of the studies in the database 

preclude a clear understanding of potential neurotoxicity following tert-butanol exposure, and 

therefore, CNS studies are not presented in evidence tables. 

Effects in other tissues were observed with less consistency. These include decreased body 

weight, liver effects, and urinary bladder effects. 

Body weight

Body weight was decreased by >10% in both rats and mice with subchronic and chronic 

exposure, with males generally more affected than females (Table 1-11). The concentrations used 

in the subchronic inhalation study did not decrease body weights. However, a short-term (i.e., 18-

day) inhalation study in rats observed a >10% decrease in body weight at concentrations about 

threefold higher (in mg/m3) than the highest concentration used in the subchronic study. The same 

concentrations did not have any effect on the body weight in mice with short-term inhalation 

exposure. 

Liver effects

Although some rodent studies observed statistically significant changes in relative liver 

weight with tert-butanol exposure, absolute liver weight was significantly increased only in female 

rats after subchronic oral exposure (Table 1-12). The results pertaining to histopathology changes 

were inconsistent (Table 1-13). The oral NTP (1995) subchronic and chronic studies did not 

observe treatment-related effects on liver histopathology in both sexes of F344 rats, but in a 10-

week study in a different rat strain (Wistar rats), several liver lesions (including necrosis) and 

increased liver glycogen were seen in male rats (no females were included in the study) with the 

only dose used (Acharya et al., 1997; Acharya et al., 1995). The study did not provide any incidence 

or severity data. The dose used in this study was in the range of the lower doses used in the NTP 

(1995) study. In the developmental study by Lyondell Chemical Co. (2004), the F1 SD rats treated 

by tert-butanol for at least 9 weeks did not show any liver effects. An increased incidence of fatty 

liver was observed in the male mice of the highest dose group in the 2-year mouse bioassay, but no 

histopathologic changes were seen in the subchronic mouse study. No changes in liver 

histopathology were observed in the NTP (1997) subchronic inhalation study. 

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

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1 Urinary bladder effects

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Several studies also reported effects in the urinary bladder (Table 1-14). Transitional 

epithelial hyperplasia was observed in male rats and mice after 13 weeks of exposure at doses of

3,610 mg/kg-day (male rats) and ≥3940 mg/kg-day (male mice). Male mice exposed at doses of 

2,070 mg/kg-day for 2 years of also exhibited transitional epithelial hyperplasia. Neither female 

rats nor female mice showed increased incidences of transitional epithelial hyperplasia. Both sexes 

of mice demonstrated incidence of inflammation in the urinary bladder after both subchronic and 

chronic exposures, with a greater incidence in males compared to females.

An exposure-response array of these effects in body weight, liver, and urinary bladder is 

provided in Figure 1-8 and Figure 1-9 for oral and inhalation studies, respectively. 

Mechanistic Evidence

No mechanistic evidence is available for these other toxicological effects. 

Summary of Other Toxicity Data

EPA concluded that the evidence does not support body weight changes, liver effects, and 

urinary bladder effects as potential human hazards of tert-butanol exposure.

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

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1 Table 1-11. Evidence pertaining to effects on body weight in animals 

2 following exposure to tert-butanol

Reference and study design Results

Acharya et al. (1995)

Wistar rat; 5–6 males/treatment

Drinking water (0 or 0.5%), 0 or 575 mg/kg-d 

10 weeks

Body weight in treated animals lower than controls by ~7% (p< 0.05); (results 

only provided in a figure) 

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 

Percent change compared to control:

F0 Males F0 Females

Dose

(mg/kg-d) Body weight

Dose

(mg/kg-d) Body weight

0 0 0 0

64 −2 64 0

160 −4 160 −2

400 +2 400 +1

1,000 −7 1,000 +4

NTP (1995)

F344/N rat; 10/sex/treatment

Drinking water (0, 2.5, 5, 10, 20, or 40 mg/mL) 

 M: 0, 230, 490, 840, 1,520, 3,610a mg/kg-d 

 F: 0, 290, 590, 850, 1,560, 3,620a mg/kg-d 

13 weeks

Percent change compared to control:

Males Females

Dose

(mg/kg-d) Body weight

Dose

(mg/kg-d) Body weight

0 0 0 0

230 −4 290 +2

490 −5* 590 +1

840 −12* 850 +1

1,520 −17* 1,560 −2

3,610 All dead 3,620 −21*

NTP (1995)

B6C3F1 mouse; 10/sex/treatment

Drinking water (0, 2.5, 5, 10, 20, or 40 mg/mL) 

 M: 0, 350, 640, 1,590, 3,940, 8,210a mg/kg-d 

 F: 0, 500, 820, 1,660, 6,430, 11,620a mg/kg-d 

13 weeks

Percent change compared to control:

Males Females

Dose

(mg/kg-d) Body weight

Dose

(mg/kg-d) Body weight

0 0 0 0

350 −1 500 +3

640 +1 820 −1

1,590 −4 1,660 +4

3,940 −14* 6,430 −6

8,210 −24* 11,620 −15*

High-dose females had a significantly lower initial weight, but also had a 

significantly lower body weight gain indicating that there was some effect of 

treatment

 3

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

1-49 DRAFT—DO NOT CITE OR QUOTE
Toxicological Review of tert-Butyl Alcohol

Table 1-11. Evidence pertaining to effects on body weight in animals 

following exposure to tert-butanol (continued)

Reference and study design Results

NTP (1995)

F344/N rat; 60/sex/treatment 

(10/sex/treatment evaluated at 15 months)

Drinking water (0, 1.25, 2.5, 5, 10 mg/mL)

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

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

2 years

Percent change compared to control:

Males Females

Dose

(mg/kg-d) Body weight

Dose

(mg/kg-d) Body weight

0 0 0 0

90 −15 180 −2

200 −18 330 −5

420 −24 650 −21

Only animals that survived at the end of 2 years were evaluated for body 

weight. Note: statistical significance not determined by study authors.

NTP (1995)

B6C3F1 mouse; 60/sex/treatment

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

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

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

2 years

Percent change compared to control:

Males Females

Dose

(mg/kg-d) Body weight

Dose

(mg/kg-d) Body weight

0 0 0 0

540 +1 510 −2

1,040 −2 1,020 −3

2,070 −1 2,110 −12

Only animals that survived at the end of 2 years were evaluated for body 

weight. Note: statistical significance not determined by study authors.

NTP (1997)

F344/N rat; 10/sex/treatment

Analytical concentration: 0, 134, 272, 542, 

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

or 6,368 mg/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

Percent change compared to control:

Concentration

(mg/m3)

Males

Body weight

Females

Body weight

0 0 0

406 −1 −5

824 −2 −1

1,643 +2 0

3,273 +3 0

6,368 +2 −3

NTP (1997)

B6C3F1 mouse; 10/sex/treatment

Analytical concentration: 0, 134, 272, 542, 

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

or 6,368 mg/m3

) (dynamic whole body 

chamber)

6 hr/d, 5 d/wk 

13 weeks

Generation method (Sonimist Ultrasonic spray 

nozzle nebulizer), analytical concentration and 

Percent change compared to control:

Concentration

(mg/m3)

Males

Body weight

Females

Body weight

0 0 0

406 +4 +3

824 −2 −3

1,643 0 +3

3,273 −4 −6

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

1-50 DRAFT—DO NOT CITE OR QUOTE
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Table 1-11. Evidence pertaining to effects on body weight in animals 

following exposure to tert-butanol (continued)

Reference and study design Results

method were reported 6,368 0 −8

1

2

3

4

5

a

There was a significant decrease in survival in the high-dose group.

* Statistically significant p ≤ 0.05 as determined by study authors.

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

Percentage change compared to control = (treated value – control value) ÷ control value × 100.

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

1-51 DRAFT—DO NOT CITE OR QUOTE
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1 Table 1-12. Changes in liver weight in animals following exposure to 

2 tert-butanol

Reference and study design Results

Acharya et al. (1995)

Wistar rat; 5–6 males/treatment

Drinking water (0 or 0.5%), 0 or 575 mg/kg-d 

10 weeks

No significant treatment-related effects (results were only provided in a 

figure)

Lyondell Chemical Co. (2004)

Sprague-Dawley rat; 12/sex/treatment

Gavage 0, 64, 160, 400, or 1,000 mg/kg-d 

Males: 9 weeks beginning 4 weeks prior to 

mating

Females: 4 weeks prior to mating through 

PND21 

Percent change compared to control:

Males Females

Dose

(mg/kg-d)

Absolute 

weight

Relative 

weight

Dose

(mg/kg-d)

Absolute 

weight

Relative 

weight

0 0 0 0 0 0

64 −1 0 64 −4 −4

160 −3 +1 160 −7 −5

400 −2 −1 400 +2 +1

1,000 +8 +16* 1,000 +8 +3

NTP (1995)

F344/N rat; 10/sex/treatment 

Drinking water (0, 2.5, 5, 10, 20, or 40 mg/mL) 

 M: 0, 230, 490, 840, 1,520, 3,610a mg/kg-d 

 F: 0, 290, 590, 850, 1,560, 3,620a mg/kg-d 

13 weeks

Percent change compared to control:

Males Females

Dose

(mg/kg-d)

Absolute

weight

Relative

weight

Dose

(mg/kg-d)

Absolute

weight

Relative

weight

0 0 0 0 0 0

230 −2 +4 290 +11* +9*

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

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

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

3,610 All dead All dead 3,620 +9* +41*

NTP (1995)

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/kg-d 

 F: 0, 500, 820, 1,660, 6,430, 11,620a mg/kg-d 

13 weeks

Percent change compared to control:

Males Females

Dose

(mg/kg-d)

Absolute

weight

Relative

weight

Dose

(mg/kg-d)

Absolute

weight

Relative

weight

0 0 0 0 0 0

350 +2 +3 500 −1 −4

640 −1 −2 820 −5 −3

1,590 −1 +5 1,660 −8 −9*

3,940 0 +14* 6,430 −2 +6

8,210 −16 +22* 11,620 −6 +13*

3

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

1-52 DRAFT—DO NOT CITE OR QUOTE
Toxicological Review of tert-Butyl Alcohol

Table 1-12. Changes in liver weight in animals following exposure to 

tert-butanol (continued) 

Reference and study design Results

NTP (1995)

F344/N rat; 60/sex/treatment 

(10/sex/treatment evaluated at 15 months)

Drinking water (0, 1.25, 2.5, 5 or 10 mg/mL) 

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

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

2 years

Percent change compared to control:

Males Females

Dose

(mg/kg-d)

Absolute

weight

Relative

weight

Dose

(mg/kg-d)

Absolute

weight

Relative

weight

0 0 0 0 0 0

90 +2 +7 180 −14* −8

200 +8 +11 330 −3 −1

420 +1 +14* 650 −6 +9*

Only animals sacrificed at 15 months were evaluated for organ weights. 

Organ weights were not measured in the 2-year mouse study

NTP (1997)

F344/N rat; 10/sex/treatment

Analytical concentration: 0, 134, 272, 542, 

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

or 6,368 mg/m3

) (dynamic whole body 

chamber) 

6 hr/d, 5 d/wk 

13 weeks

Generation method (Sonimist Ultrasonic spray 

nozzle nebulizer), analytical concentration and 

method were reported

Percent change compared to control:

Males Females

Concentration

(mg/m3)

Absolute

weight

Relative

weight

Absolute

weight

Relative

weight

0 0 0 0 0

406 −8 −8 0 +3

824 −2 −1 0 0

1,643 +1 −1 +3 +2

3,273 +10 +7 +9 +9*

6,368 +5 +5 +4 +8*

NTP (1997)

B6C3F1 mouse; 10/sex/treatment

Analytical concentration: 0, 134, 272, 542, 

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

or 6,368 mg/m3

) (dynamic whole body 

chamber)

6 hr/d, 5 d/wk 

13 weeks

Generation method (Sonimist Ultrasonic spray 

nozzle nebulizer), analytical concentration and 

method were reported

Percent change compared to control:

Males Females

Concentrati

on

(mg/m3)

Absolute

weight

Relative

weight

Absolute

weight

Relative

weight

0 0 0 0 0

406 −1 0 +1 −4

824 +4 +9 +1 +5

1,643 +7 +5 +5 +1

3,273 −8 -2 +2 +9*

6,368 +5 +7 +8 +21*

1

2

3

a

The high-dose group had an increase in mortality.

* Statistically significant p ≤ 0.05 as determined by study authors.

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

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

1-53 DRAFT—DO NOT CITE OR QUOTE
Toxicological Review of tert-Butyl Alcohol

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

2

3

4

5

Percentage change compared to control = (treated value – control value) ÷ control value × 100.

Table 1-13. Changes in liver histopathology in animals following exposure to 

tert-butanol

Reference and study design Results

Acharya et al. (1997; 1995)

Wistar rat; 5–6 males/treatment

Drinking water (0, 0.5%), 0, 575 mg/kg-d 

10 weeks

↑ liver glycogen (~ 7 fold)*

↑incidence of centrilobular necrosis, vacuolation of hepatocytes, loss of 

hepatocyte architecture, peripheral proliferation, and lymphocyte 

infiltration (incidences and results of statistical tests not reported)

Lyondell Chemical Co. (2004)

Sprague-Dawley rat; 12/sex/treatment

Gavage 0, 64, 160, 400, or 1,000 mg/kg-d 

Males: 9 weeks beginning 4 weeks prior to 

mating

Females: 4 weeks prior to mating through 

PND21 

No treatment-related effects observed.

NTP (1995)

F344/N rat; 10/sex/treatment

Drinking water (0, 2.5, 5, 10, 20, or 40 mg/mL) 

 M: 0, 230, 490, 840, 1,520, 3,610a mg/kg-d 

 F: 0, 290, 590, 850, 1,560, 3,620a mg/kg-d 

13 weeks

Histopathology data for the 13-week study were not provided, but the liver 

was evaluated indicating that no changes in liver histopathology were 

observed in the 13-week study.

NTP (1995)

B6C3F1 mouse; 10/sex/treatment

Drinking water (0, 2.5, 5, 10, 20, 40 mg/mL)

 M: 0, 350, 640, 1,590, 3,940, 8,210a mg/kg-d 

 F: 0, 500, 820, 1,660, 6,430, 11,620a mg/kg-d 

13 weeks

Histopathology data for the 13-week study were not provided, but the liver 

was evaluated indicating that no changes in liver histopathology were 

observed in the 13-week study.

NTP NTP (1995)

F344/N rat; 60/sex/treatment 

(10/sex/treatment evaluated at 15 months)

Drinking water (0, 1.25, 2.5, 5, 10 mg/mL)

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

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

2 years

No treatment-related effects observed.

6

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

1-54 DRAFT—DO NOT CITE OR QUOTE
Toxicological Review of tert-Butyl Alcohol

Table 1-13. Changes in liver histopathology in animals following exposure to 

tert-butanol (continued)

Reference and study design Results

NTP (1995)

B6C3F1 mouse; 60/sex/treatment

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

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

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

2 years

Males Females

Dose

(mg/kg-d)

Incidence of fatty 

change

Dose

(mg/kg-d)

Incidence of fatty 

change

0 12/59 0 11/60

540 5/60 510 8/60

1,040 8/59 1,020 8/60

2,070 29/59* 2,110 6/60

NTP (1997)

F344/N rat; 10/sex/treatment

Analytical concentration: 0, 134, 272, 542, 

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

or 6,368 mg/m3

) (dynamic whole body 

chamber) 

6 hr/d, 5 d/wk 

13 weeks

Generation method (Sonimist Ultrasonic spray 

nozzle nebulizer), analytical concentration and 

method were reported

Histopathology data for the 13-week study were not provided, but the liver 

was evaluated in control and high-dose group indicating that no changes in 

liver histopathology were observed in the 13-week study.

NTP (1997)

B6C3F1 mouse; 10/sex/treatment

Analytical concentration: 0, 134, 272, 542, 

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

or 6,368 mg/m3

) (dynamic whole body 

chamber)

6 hr/d, 5 d/wk 

13 weeks

Generation method (Sonimist Ultrasonic spray 

nozzle nebulizer), analytical concentration and 

method were reported

Authors stated that there were no treatment-related microscopic 

observations, but data were not provided.

1

2

3

4

5

6

7

8

9

10

11

12

a

The high-dose group had an increase in mortality.

* Statistically significant p ≤ 0.05 as determined by study authors.

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

. 

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

1-55 DRAFT—DO NOT 

policy.

CITE OR QUOTE
Toxicological Review of tert-Butyl Alcohol

1 Table 1-14. Changes in urinary bladder histopathology in animals following 

2 oral exposure to tert-butanol

Reference and study design Results

NTP (1995)

F344/N rat; 10/sex/treatment

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

mg/mL)

 M: 0, 230, 490, 840, 1,520, 3,610a

mg/kg-d 

 F: 0, 290, 590, 850, 1,560, 3,620a

mg/kg-d 

13 weeks

Incidence (severity):

Males Females

Dose (mg/kg-d)

Transitional 

epithelial 

hyperplasia Dose (mg/kg-d)

Transitional 

epithelial 

hyperplasia

0 0/10 0 0/10

230 not evaluated 290 not evaluated

490 not evaluated 590 not evaluated

840 0/10 850 not evaluated

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

3,610 7/10* (2.9) 3,620 3/10 (2.0)

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

NTP (1995)

B6C3F1 mouse; 10/sex/treatment

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

mg/mL)

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

8,210a mg/kg-d 

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

11,620a mg/kg-d 

13 weeks

Incidence (severity):

Males Females

Dose

(mg/kg-d)

Transitional 

epithelial 

hyperplasia

InflammationDose

(mg/kg-d)

Transitional 

epithelial 

hyperplasia

Inflammation

0 0/10 0/10 0 0/10 0/10

350 not evaluated 500 0/1 0/1

640 not evaluated 820 not evaluated

1,590 0/10 0/10 1,660 not evaluated

3,940 6/10* (1.3) 6/10* (1.3) 6,430 0/10 0/10

8,210 10/10* (2.0) 10/10* (2.3) 11,620 3/9 (2.0) 6/9* (1.2)

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

NTP (1995)

F344/N rat; 60/sex/treatment 

(10/sex/treatment evaluated at 15 

months)

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

mg/mL)

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

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

2 years

No treatment-related effects observed

3

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

1-56 DRAFT—DO NOT CITE OR QUOTE
Toxicological Review of tert-Butyl Alcohol

Table 1-14. Evidence pertaining to urinary bladder effects in animals 

following oral exposure to tert-butanol (continued) 

Reference and study design Results

NTP (1995)

B6C3F1 mouse; 60/sex/treatment

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

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

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

2 years

Incidence (severity):

Males Females

Dose

(mg/kg-d)

Transitional 

epithelial 

hyperplasia

InflammationDose

(mg/kg-d)

Transitional 

epithelial 

hyperplasia

Inflammation

0 1/59 (2.0) 0/59 0 0/59 0/59

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

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

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

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

1

2

3

a

The high-dose group had an increase in mortality.

* Statistically significant p ≤ 0.05 as determined by study authors.

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

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

1-57 DRAFT—DO NOT CITE OR QUOTE
Toxicological Review of tert-Butyl Alcohol

1

2

3

4

5

 = 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

M rat (D)

M rat (A)

F rat (D)

M mouse (D)

F mouse (D)

M rat (C)

F rat (C)

Transitional epithelium hyperplasia; M rat (D)

Transitional epithelium hyperplasia; F rat (D)

Transitional epithelium hyperplasia; M mouse (D)

Inflammation; M mouse (D)

Inflammation; F mouse (D)

Transitional epithelium hyperplasia; F mouse (D)

Transitional epithelium hyperplasia; M mouse (D)

Inflammation; M mouse (D)

Inflammation; F mouse (D)

Transitional epithelium hyperplasia; F mouse (D)

Increased glycogen; M rat (B)

Relative weight; M rat (B)

Relative weight; M rat (D)

Absolute weight; F rat (D)

Relative weight; F rat (D)

Absolute weight; M rat (D)

Relative weight; M mouse (D)

Relative weight; F mouse (D)

Absolute weight; M mouse (D)

Absolute weight; F mouse (D)

Relative weight; M rat (D)

Relative weight; F rat (D)

Absolute weight; M rat (D)

Absolute weight; F rat (D)

Fatty tissue; M mouse (D)

Fatty tissue; F mouse (D)

Relative weight; M rat (C)

Absolute weight; M rat (C)

Absolute weight; F rat (C)

Relative weight; F rat (C)

BODY WEIGHT Subchronic URINARY BLADDER LIVER

Subchronic

Chronic

Subchronic

Chronic

Reproductive

Reproductive

10 100 1,000 10,000 100,000

Dose (mg/kg-day)

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

1-58 DRAFT—DO NOT CITE OR QUOTE

Sources: (A) Acharya et al. (1995); (B) Acharya et al. (1997; 1995); (C) Lyondell Chemical Co. (2004); (D) NTP (1995)

Figure 1-8. Exposure-response array of other effects following oral exposure 

to tert-butanol. 
Toxicological Review of tert-Butyl Alcohol

 1

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

1-59 DRAFT—DO NOT CITE OR QUOTE

2

3

4

5

Source: (A) NTP (1997)

Figure 1-9. Exposure-response array of other effects following inhalation 

exposure to tert-butanol. 
Toxicological Review of tert-Butyl Alcohol

1 1.2. INTEGRATION AND EVALUATION

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1.2.1. Effects Other Than Cancer

The strongest evidence following tert-butanol exposure is for kidney, with toxicity observed 

after oral exposure in two strains of rats and in one strain of mice and in both sexes. In mice, the 

only kidney effect observed was an increase in kidney weight (absolute and/or relative) in both 

sexes of mice in the 13-week study, but no treatment-related histopathological lesions were 

reported in the kidneys of mice at the 13-week or 2-year time points (NTP, 1995). In male rats, 

effects related to the accumulation of α2u-globulin in the kidney have been reported, including 

precursors to granular casts, linear mineralization, and tubular hyperplasia, but these are not 

considered relevant to humans (Hard et al., 2011; Cirvello et al., 1995; NTP, 1995; Lindamood et al., 

1992). However, several other effects in the kidney unrelated to α2u-globulin were observed in 

female and/or male rats. Absolute and relative kidney weights were increased in both male and 

female rats after both 13 weeks and 15 months of treatment (NTP, 1995). Histopathological 

examination also indicated kidney toxicity in both male and female rats, with increased incidence of 

nephropathy after 13 weeks of oral exposure and transitional epithelium hyperplasia observed 

after 2 years of oral exposure (NTP, 1995). Additionally, increased inflammation (suppurative) was 

noted in females after 2 years oral exposure (NTP, 1995). EPA identified kidney effects as a human 

hazard of tert-butanol oral exposure. 

Fewer and less severe kidney effects were observed via inhalation than via oral exposure, 

likely due to the differing levels of internal doses achieved via the different routes. Specifically, 

available inhalation studies (NTP, 1997) were conducted at concentrations that are comparable, in 

terms of tert-butanol blood concentration, to the lower range of doses in oral studies. Moreover, 

there is convincing toxicokinetic data to indicate that tert-butanol is absorbed by both routes, and 

kidney effects are remote from the site of absorption. EPA identified kidney effects as a human 

hazard of tert-butanol inhalation exposure. 

Thyroid follicular cell hyperplasia was observed in the mice after 2 years of exposure via 

drinking water (NTP, 1995); and EPA identified thyroid effects as a potential human hazard of tertbutanol exposure. However, this endpoint most likely reflects early events in the neoplastic 

progression of thyroid follicular cell tumors following tert-butanol exposure (see Section 1.1.2) and 

was not considered further for dose-response analysis and derivation of noncancer reference 

values. 

EPA identified suggestive evidence of developmental effects as a potential human hazard of 

tert-butanol exposure. Exposure to high doses of tert-butanol during gestation resulted in some 

effects in exposed offspring or pups, although the effects were not always consistent across 

exposure routes (oral and inhalation). Dams exhibit effects at the same doses as fetal effects. 

Neurodevelopmental effects have also been observed; however, the neurodevelopmental studies

had limitations in the study design and/or reporting and results were inconsistent between studies 

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

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1 or across dose. Thus, these effects were not considered further for dose-response analysis and 

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derivation of reference values. 

EPA concluded that the evidence does not support reproductive effects, body weight 

changes, liver effects, and urinary bladder effects as potential human hazards of tert-butanol 

exposure. Thus, these effects were not considered further for dose-response analysis and the 

derivation of reference values.

1.2.2. Carcinogenicity

Under EPA’s Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), the database for 

tert-butanol provides “suggestive evidence of carcinogenic potential,” based on a statistically 

significant increase in renal tumors (renal tubule adenomas and carcinomas) in male F344 rats and 

a statistically significant increase in thyroid follicular cell adenomas in female B6C3F1 mice, all

exposed to tert-butanol in drinking water for 2 years (Cirvello et al., 1995; NTP, 1995). There are 

no available studies of cancer in humans associated with exposure to tert-butanol. 

In the NTP (1995) rodent bioassay, tert-butanol-exposed male rats had a significant 

increase in renal tumors compared to controls, a result confirmed by a PWG reevaluation (Hard et 

al., 2011). Although mechanistic data show that α2u-globulin-related processes occur with tertbutanol exposure, there is insufficient evidence to support a conclusion that α2u-globulin 

nephropathy is the sole or primary contributor to renal tumor development. Specifically, 

tert-butanol induced tumors at lower doses than those for other precursor effects such as 

hyperplasia and granular casts, with no further increase in tumor incidence coinciding with the 

induction of additional markers of α2u-globulin nephropathy. Based on analysis of available mode 

of action data, these tumors are not attributed to α2u-globulin and are considered relevant in 

humans (U.S. EPA, 1991a). tert-Butanol was negative in a variety of genotoxicity assays in different 

cell systems including gene mutations, sister chromatid exchanges, micronucleus formation, and 

chromosomal aberrations. However, DNA adducts in male Kunming mice and DNA damage in 

human HL-60 leukemia cells have been observed. Overall, the mode(s) of carcinogenic action for 

tert-butanol in the kidney and the thyroid are not known, and these tumor data are considered 

relevant to humans. 

As emphasized in the Cancer Guidelines (U.S. EPA, 2005a), selection of the cancer descriptor 

follows a full evaluation of the available evidence. The carcinogenicity evidence for tert-butanol

could be considered a borderline case between two cancer descriptors—“suggestive evidence of 

carcinogenic potential” and “likely to be carcinogenic to humans.” The descriptor of “suggestive 

evidence of carcinogenic potential” is appropriate when a concern for potential carcinogenic effects 

in humans is raised, but the data are judged not sufficient for a stronger conclusion. Exposure to 

tert-butanol produced a positive tumor response at more than one site (kidney and thyroid) and in 

more than one species (rat and mouse). These data appear to correspond closely to one of the 

examples in the Cancer Guidelines (U.S. EPA, 2005a) for the descriptor of “likely to be carcinogenic 

to humans;” i.e., “an agent that has tested positive in animal experiments in more than one species, 

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

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1 sex, strain, site, or exposure route, with or without evidence of carcinogenicity in humans.” Several 

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aspects of the data support the conclusion that these data are not sufficient to characterize tertbutanol as “likely to be carcinogenic to humans.” 

First, the renal tumors associated with tert-butanol exposure in the NTP (1995) rodent 

bioassay were predominantly benign. Based on the PWG reevaluation (Hard et al., 2011), among 

the three treated groups, only two of the 43 animals with tumors has carcinomas (there were no 

carcinomas among the 4 control animals with tumors). Additionally, no kidney tumors were 

observed in female rats or in either sex of mice. Furthermore, ETBE, which is rapidly metabolized 

to tert-butanol, did not induce kidney tumors in the same strain of rats at doses that resulted in 

similar internal concentrations of tert-butanol. Therefore, the level of concern raised by renal 

tumors associated with tert-butanol exposure is reduced based on the predominance of benign 

tumors, an increase in renal tumors in a single sex/species combination only, and the lack of 

coherence with the metabolically-related compound ETBE.

The thyroid tumors associated with tert-butanol exposure were also predominantly benign. 

In the NTP (1995) rodent bioassay, only female mice had a statistically significant increase in 

thyroid tumors; none of these were carcinomas. In males, decreased survival complicates the 

interpretation of thyroid tumors because male mice had an increased incidence of thyroid follicular 

cell hyperplasia at all exposure levels, but there was no significant increase in thyroid tumors at any 

exposure. Interestingly, one thyroid follicular cell carcinoma occurred in a high-dose male, but 

limited conclusions can be drawn from this single observation. Thyroid tumors were not observed 

in either sex of the rat exposed chronically to tert-butanol. Additionally, ETBE did not induce 

thyroid tumors, although only rats and not mice were tested. Therefore, the level of concern raised 

by thyroid tumors associated with tert-butanol exposure is reduced based on the predominance of 

benign tumors and an increase in thyroid tumors in a single sex/species combination only. 

Overall, the cancer descriptor “suggestive evidence of carcinogenic potential” was selected, 

as some concern is raised by the positive evidence of predominantly benign renal tumors in male 

rats and thyroid tumors in female mice.

The Cancer Guidelines (U.S. EPA, 2005a) indicate that for tumors occurring at a site other 

than the initial point of contact, the weight of evidence for carcinogenic potential may apply to all 

routes of exposure that have not been adequately tested at sufficient doses. An exception occurs 

when there is convincing toxicokinetic data that absorption does not occur by other routes. 

Information available on the carcinogenic effects of tert-butanol via the oral route demonstrates 

that tumors occur in tissues remote from the site of absorption. Information on the carcinogenic 

effects of tert-butanol via the inhalation and dermal routes in humans or animals is not available. 

Based on the observation of systemic tumors following oral ingestion, and in the absence of 

information to indicate otherwise, it is assumed that an internal dose will be achieved regardless of 

the route of exposure. Therefore, there is “suggestive evidence of carcinogenic potential” from 

exposure to tert-butanol by all routes of exposure. 

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1 1.2.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes

2 No data were identified to indicate any possible susceptible populations or lifestages.

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1

2. DOSE-RESPONSE ANALYSIS

2 2.1. ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER

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The RfD (expressed in units of mg/kg-day) is defined as an estimate (with uncertainty 

spanning perhaps an order of magnitude) of a daily exposure to the human population (including 

sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a 

lifetime. It can be derived from a no-observed-adverse-effect level (NOAEL), lowest-observedadverse-effect level (LOAEL), or the 95% lower bound on the benchmark dose (BMDL), with 

uncertainty factors (UFs) generally applied to reflect limitations of the data used.

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

EPA identified kidney effects as a human hazard of tert-butanol exposure. Studies within 

this effect category were evaluated using general study quality characteristics (as discussed in 

Section 6 of the Preamble) to help inform the selection of studies from which to derive toxicity 

values. Rationales for selecting the studies and effects to represent each of these hazards are 

summarized below. 

Human studies are preferred over animal studies when quantitative measures of exposure 

are reported and the reported effects are determined to be associated with exposure. However, 

there are no available human occupational or epidemiological studies of oral exposure to tertbutanol. 

Animal studies were evaluated to determine which studies provided: (a) the most relevant 

routes and durations of exposure; (b) multiple exposure levels to provide information about the 

shape of the dose-response curve; and (c) power to detect effects at low exposure levels (U.S. EPA, 

2002). Sufficient data were available to develop a PBPK model in rats for both oral and inhalation 

exposure in order to perform route-to-route extrapolation, so rat studies from both routes of 

exposure were considered for dose-response analysis. The database for tert-butanol includes 

several studies and data sets that are potentially suitable for use in deriving reference values. 

Specifically, effects associated with tert-butanol exposure in animals include observations of organ 

weight and/or histological changes in the kidney observed in several chronic and subchronic 

studies. 

Kidney Toxicity

EPA identified kidney effects as a human hazard of tert-butanol-induced toxicity based on 

findings of organ weight changes in rats and mice, as well as histopathology in rats. These findings 

were consistent across multiple chronic, subchronic, and short-term studies following oral and 

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1 inhalation exposure. Acharya et al. (1997; 1995) used a single exposure group and did not provide 

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incidence or severity data, and thus was not considered for dose-response assessment. Lyondell 

Chemical Co. (2004) and NTP (1997) were of subchronic or shorter duration, and so were set aside 

given the availability of a longer duration study. Therefore, the NTP 2-year drinking water study 

(NTP, 1995) was identified most suitable for dose-response assessment considering the study 

duration, comprehensive reporting of outcomes, multiple species tested, and multiple doses tested. 

In the NTP (1995) drinking water study, male F344 rats were exposed to approximate 

doses of 0, 90, 200, or 420 mg/kg-day; female F344 rats were exposed to approximate doses of 0, 

180, 330, or 650 mg/kg-day; male B6C3F1 mice were exposed to approximate doses of 0, 540, 

1,040, or 2,070 mg/kg-day; and female B6C3F1 mice were exposed to approximate doses of 0, 510, 

1,020, or 2,110 mg/kg-day. Reduced body weights and survival were observed and reflected in 

some of the effects. Kidney effects including changes in organ weight and/or histopathology were 

observed in both sexes in rats and mice. Effects were also observed after 13 weeks, 15 months, and 

2 years of treatment (NTP, 1995). Effects were more consistent and occurred at lower doses in rats 

as compared to mice, so as a result, only data in the more sensitive species of rats were used for 

dose-response assessment. Endpoints potentially confounded by the presence of α2u-globulin

nephropathy in male rats, such as linear mineralization and renal tubule hyperplasia, were not used 

for dose-response analysis. Specific endpoints chosen for analysis were absolute and relative 

kidney weight (observed in males and females), kidney inflammation (observed only in females), 

and kidney transitional epithelial hyperplasia (observed in males and females). For most 

endpoints, the data at the longest duration of 2 years were selected. However, as discussed in 

Section 1.1.1, 2-year kidney weight data were not considered because organs were only weighed at 

15 months. 

2.1.2. Methods of Analysis

No biologically based dose-response models are available for tert-butanol. In this situation, 

EPA evaluates a range of dose-response models thought to be consistent with underlying biological 

processes to determine how to best empirically model the dose-response relationship in the range 

of the observed data. Consistent with this approach, all models available in EPA’s Benchmark Dose 

Software (BMDS) were evaluated. Consistent with EPA’s Benchmark Dose Technical Guidance 

Document (U.S. EPA, 2012b), the benchmark dose (BMD) and the 95% lower confidence limit on the 

BMD (BMDL) were estimated using a benchmark response (BMR) of 10% change from the control 

mean for organ weight data in the absence of information regarding the level of change that is 

considered biologically significant. Furthermore, the BMD and BMDL were estimated to facilitate a 

consistent basis of comparison across endpoints, studies, and assessments. A benchmark response 

(BMR) of 10% extra risk was considered appropriate for the quantal data on incidences of kidney 

inflammation and kidney transitional epithelial hyperplasia. For each endpoint, the BMDL estimate 

(95% lower confidence limit on the BMD, as estimated by the profile likelihood method) and Akaike 

Information Criterion (AIC) value were used to select a best-fit model among models exhibiting 

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1 adequate fit. If the BMDL estimates were “sufficiently close,” that is, differed by at most 3-fold, the 

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model selected was the one that yielded the lowest AIC value. If the BMDL estimates were not 

sufficiently close, the lowest BMDL was selected as the POD. The estimated BMDLs were used as 

points of departure (PODs). Further details including the modeling output and graphical results for 

the best-fit model for each endpoint can be found in Appendix C of the Supplemental Information.

In general, absolute and relative kidney weight data may both be considered appropriate 

endpoints for analysis (Bailey et al., 2004). However, in the NTP (1995) 2-year drinking water 

study, there was a noticeable decrease in body weight in exposed animals relative to controls at the 

15 month interim sacrifice (see Table 1-1). In such a case, relative kidney weights are the 

preferred, so changes in absolute kidney weights were not analyzed. 

Human equivalent doses (HEDs) for oral exposures were derived from the PODs estimated 

from the laboratory animal data as described in EPA’s Recommended Use of Body Weight3/4 as the 

Default Method in Derivation of the Oral Reference Dose (U.S. EPA, 2011). In this guidance, EPA 

advocates a hierarchy of approaches for deriving HEDs from data in laboratory animals, with the 

preferred approach being physiologically-based toxicokinetic modeling. Other approaches can 

include using chemical-specific information in the absence of a complete physiologically-based 

toxicokinetic model. As discussed in Appendix B of the Supplemental Information, several rat 

physiologically based pharmacokinetic (PBPK) models for tert-butanol have been developed and 

published, but a validated human PBPK model for tert-butanol for extrapolating doses from animals 

to humans is not available. In lieu of either chemical-specific models or data to inform the 

derivation of human equivalent oral exposures, a body weight scaling to the 3⁄4 power (i.e., BW3/4) 

approach is applied to extrapolate toxicologically equivalent doses of orally administered agents 

from adult laboratory animals to adult humans for the purpose of deriving an oral RfD. BW3/4

scaling was not employed for deriving HEDs from studies in which doses were administered 

directly to early postnatal animals because of the absence of information on whether allometric 

(i.e., body weight) scaling holds when extrapolating doses from neonatal animals to adult humans 

due to presumed toxicokinetic and/or toxicodynamic differences between lifestages (U.S. EPA, 

2011; Hattis et al., 2004). 

Consistent with EPA guidance (U.S. EPA, 2011), the PODs estimated based on effects in adult 

animals are converted to HEDs employing a standard dosimetric adjustment factor (DAF) derived 

as follows:

 DAF = (BWa

1/4 / BWh1/4),

where

 BWa = animal body weight

 BWh = human body weight 

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1 Using a standard BWa of 0.25 kg for rats and a BWh of 70 kg for humans (U.S. EPA, 1988), 

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the resulting DAFs for rats is 0.24. 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 the sequence of calculations leading to the derivation of a humanequivalent POD for each data set discussed above.

Table 2-1. Summary of derivations of points of departure

Endpoint and 

Reference

Species/

sex Modela BMR

BMD 

(mg/kg-d)

BMDL 

(mg/kg-d)

PODADJ

b 

(mg/kg-d)

PODHED

c 

(mg/kg-d)

Kidney

Increased relative 

kidney weight

NTP (1995)

Rat/M Exponential 

(M4)

10% 117 48 48 11.5

Increased relative 

kidney weight

NTP (1995)

Rat/F Linear 10% 158 133 133 31.9

Kidney inflammation

NTP (1995)

Rat/F Log-probit 10% 254 200 200 48

Kidney transitional 

epithelial 

hyperplasia

NTP (1995)

Rat/M Log-logistic 10% 30 16 16 3.84

Kidney transitional 

epithelial 

hyperplasia

NTP (1995)

Rat/F Multistage, 

3-degree

10% 412 339 339 81.4

a 11 For modeling details, see Appendix C in Supplemental Information. 

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b

For studies in which animals were not dosed daily, administered doses were adjusted to calculate the TWA daily 

doses prior to BMD modeling.

c

HED PODs were calculated using BW3/4 scaling (U.S. EPA, 2011). 

2.1.3. Derivation of Candidate Values

Under 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. An explanation follows:

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1 An intraspecies uncertainty factor, UFH, of 10 was applied to all PODs to account for 

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potential differences in toxicokinetics and toxicodynamics in the absence of information on the 

variability of response in the human population following oral exposure to tert-butanol. 

An interspecies uncertainty factor, UFA, of 3 (101/2 = 3.16, rounded to 3) was applied to all 

PODs because BW3/4 scaling is used to extrapolate oral doses from laboratory animals to humans. 

Although BW3/4 scaling addresses some aspects of cross-species extrapolation of toxicokinetic and 

toxicodynamic processes, some residual uncertainty remains. In the absence of chemical-specific 

data to quantify this uncertainty, EPA’s BW3/4 guidance (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 since the 

endpoints examined were all observed following chronic exposure. 

A LOAEL to NOAEL uncertainty factor, UFL, of 1 was applied to all PODs 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% change in relative kidney weight, a 10% extra risk of 

kidney inflammation, and a 10% extra risk of transitional cell hyperplasia were selected under an 

assumption that they represent minimal biologically significant changes. 

A database uncertainty factor, UFD, of 1 was applied to all PODs. The tert-butanol toxicity 

database includes a chronic toxicity study in rats and mice (NTP, 1995), a subchronic toxicity study 

in rats and mice (NTP, 1997), 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 effects were observed at exposure levels below 1000 mg/kg-day, and effects observed at 

≥1000 mg/kg-day were accompanied by evidence of maternal toxicity. These exposure levels are 

much higher than the PODs for kidney effects, suggesting developmental toxicity is not a sensitive 

endpoint. The tert-butanol database contains a one-generation reproductive toxicity study in rats 

(Lyondell Chemical Co., 2004), though no multigenerational reproductive study has been 

performed. There are no immunotoxicity studies for tert-butanol. Information provided by studies 

on ETBE, which is rapidly metabolized to systemically-available tert-butanol, can help in 

considering the lack of a tert-butanol multigenerational reproductive study or an immunotoxicity 

study. No adverse effects were reported in one- and two-generation reproductive/developmental 

studies on ETBE (Gaoua, 2004a, b), and the database for ETBE does not indicate immunotoxicity 

(Banton et al., 2011; Li et al., 2011). Thus, although there are some gaps in the toxicity database for 

tert-butanol, the available data on tert-butanol, informed by the data on ETBE, do not suggest that 

additional studies would lead to identification of a more sensitive endpoint or a lower POD. 

Therefore, a database UFD of 1 was applied.

Table 2-2 is a continuation of Table 2-1 and summarizes the application of UFs to each POD 

to derive a candidate value for each data set. The candidate values presented in the table below are 

preliminary to the derivation of the organ/system-specific reference values. These candidate values 

are considered individually in the selection of a representative oral reference value for a specific

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1 hazard and subsequent overall RfD for tert-butanol. 

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Figure 2-1 presents graphically the candidate values, UFs, and PODs, with each bar 

corresponding to one data set described in Table 2-1 and Table 2-2. 

Table 2-2. Effects and corresponding derivation of candidate RfDs 

Endpoint and Reference

PODHED

(mg/kg-d) POD type UFA UFH UFL UFS UFD

Composite 

UF

Candidate

value 

(mg/kg-d)

Kidney

Increased relative kidney weight; 

male rat

NTP (1995)

12 BMDL10% 3 10 1 1 1 30 4 × 10-1

Increased relative kidney weight; 

female rat

NTP (1995)

32 BMDL10% 3 10 1 1 1 30 1 × 100

Kidney inflammation; 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

6

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1

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Figure 2-1. Candidate RfD values with corresponding POD and composite UF.
Toxicological Review of tert-Butyl Alcohol

1 2.1.4. Derivation of Organ/System-Specific Reference Doses

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Table 2-3 distills the candidate values from Table 2-2 into a single value for the kidney. 

Organ or system-specific reference values may be useful for subsequent cumulative risk 

assessments that consider the combined effect of multiple agents acting at a common site.

Kidney Toxicity

For tert-butanol, candidate values were for several different effects in both sexes, spanning 

a range from 1 × 10-1 to 3 × 100 mg/kg-day, for an overall thirtyfold 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) is 

proposed as the kidney-specific reference dose for tert-butanol. Unlike kidney inflammation, this 

effect was observed in both sexes, with males appearing to be more sensitive than females. 

Additionally, it is a more specific and more sensitive indicator of kidney toxicity than the relatively 

non-specific endpoint of kidney weight changes. Confidence in this kidney-specific RfD is high. The 

PODs are based on modeled benchmark dose estimates, and the candidate values are derived from 

a well-conducted 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 proposed overall RfD for tertbutanol

Effect Basis

RfD 

(mg/kg-day)

Exposure 

description Confidence

Kidney toxicity Increased incidence of 

transitional epithelial 

hyperplasia 

1 × 10-1 Chronic HIGH

Proposed 

overall RfD

Increased incidence of 

transitional epithelial 

hyperplasia 

1 × 10-1 Chronic HIGH 

2.1.5. Selection of the Proposed Overall Reference Dose

For tert-butanol, only kidney effects were identified as a hazard; thus a single 

organ/system-specific reference dose was derived. Therefore, the kidney-specific RfD of 

1 × 10-1 mg/kg-day is also proposed as an estimated exposure level below which deleterious 

effects from tert-butanol exposure are not expected to occur.

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 

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1 could potentially lead to an appreciable risk, even if average levels over the full exposure duration 

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were less than or equal to the RfD. In the case of tert-butanol, no specific lifestages have been 

identified as a potentially susceptible subgroup.

2.1.6. Confidence Statement

A confidence level of high, medium, or low is assigned to the study used to derive the RfD, 

the overall database, and the RfD itself, as described in Section 4.3.9.2 of EPA’s Methods for 

Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA,

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 FDA GLP regulations, involved a sufficient 

number of animals per group (including both sexes), and assessed a wide range of tissues and 

endpoints. Although there are some gaps in the toxicity database for tert-butanol, these areas are 

informed by the data on ETBE, a parent compound of 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. 

2.1.7. Previous IRIS Assessment

An oral assessment for tert-butanol was not previously available on IRIS. 

2.2. INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER 

THAN CANCER

The inhalation reference concentration (RfC) (expressed in units of mg/m3) is defined as an 

estimate (with uncertainty spanning perhaps an order of magnitude) of a continuous inhalation 

exposure to the human population (including sensitive subgroups) that is likely to be without an 

appreciable risk of deleterious effects during a lifetime. It can be derived from a NOAEL, LOAEL, or 

the 95% lower bound on the benchmark concentration (BMCL), with UFs generally applied to 

reflect limitations of the data used.

2.2.1. Identification of Studies and Effects for Dose-Response Analysis

EPA identified kidney effects as a human hazard of tert-butanol exposure. Studies within 

this effect category were evaluated using general study quality characteristics (as discussed in 

Section 6 of the Preamble) to help inform the selection of studies from which to derive toxicity 

values. Rationales for selecting the studies and effects to represent this hazard are summarized 

below. 

Human studies are preferred over animal studies when quantitative measures of exposure 

are reported and the reported effects are determined to be associated with exposure. However, 

there are no available human occupational or epidemiological studies of inhalation exposure to 

tert-butanol. 

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

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1 Animal studies were evaluated to determine which study provided: (a) the most relevant 

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routes and durations of exposure; (b) multiple exposure levels to provide information about the 

shape of the dose-response curve; and (c) power to detect effects at low exposure levels (U.S. EPA, 

2002). Sufficient data were available to develop a PBPK model in rats for both oral and inhalation 

exposure in order to perform route-to-route extrapolation, so rat studies from both routes of 

exposure were considered for dose-response analysis. The database for tert-butanol includes a 

several studies and data sets that are potentially suitable for use in deriving reference values. 

Specifically, effects associated with tert-butanol exposure in animals include observations of organ 

weight and histological changes in the kidney in several chronic and subchronic studies. 

Kidney Toxicity

EPA identified kidney effects as a human hazard of tert-butanol exposure based on findings 

of organ weight changes in rats and mice and histopathology in rats. These findings were

consistent across multiple chronic, subchronic, and short-term studies following oral and inhalation 

exposure. Acharya et al. (1997; 1995) used a single exposure group and did not provide incidence 

or severity data, so was not considered for dose-response assessment. Lyondell Chemical Co. 

(2004) was of shorter than subchronic duration, and so was set aside given the availability of a 

longer duration studies. Given the availability of a chronic study, the subchronic studies of NTP 

(1995) and NTP (1997) would normally also be set aside for dose-response analysis. NTP (1997) is 

the longest duration study via the inhalation route, not requiring route-to-route extrapolation, so 

was kept for comparison purposes. 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 will not be reviewed here 

again. The NTP (1997) subchronic inhalation study is described in more detail below.

NTP (1997) was a well-designed subchronic study that evaluated the effect of tert-butanol

exposure on multiple species at multiple inhalation doses. Briefly, groups of F344 rats and B6C3F1

mice (10 per sex per species) were exposed to tert-butanol (>99% pure) at concentrations of 0, 

409, 819, 1,637, 3,274 or 6,366 mg/m3 by inhalation for 6 hours per day, 5 days per week, for 13 

weeks (NTP, 1997). Absolute kidney weights were elevated (10–11%) in male rats exposed at 

≥3,274 mg/m3; relative kidney weights were statistically significantly elevated (~9%) in males at 

≥3,274 mg/m3 and females at 6,366 mg/m3. Male rats exhibited an increase in the severity of 

chronic nephropathy (characterized as number of foci of regenerative tubules). There were few 

endpoints available for consideration in the subchronic study, but changes in kidney weights were 

also observed in the oral studies, such as the NTP (1995) 2-year drinking water study. 

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

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1 2.2.2. Methods of Analysis

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No biologically based dose-response models are available for tert-butanol. In this situation, 

EPA evaluates a range of dose-response models thought to be consistent with underlying biological 

processes to determine how best to empirically model the dose-response relationship in the range 

of the observed data. Consistent with this approach, all models available in EPA’s Benchmark Dose 

Software (BMDS) were evaluated. Consistent with EPA’s Benchmark Dose Technical Guidance 

Document (U.S. EPA, 2012b), the benchmark dose (BMD) and the 95% lower confidence limit on the 

BMD (BMDL) were estimated using a benchmark response (BMR) of 10% change from the control 

mean for organ weight data in the absence of information regarding what level of change is 

considered biologically significant, and also to facilitate a consistent basis of comparison across 

endpoints, studies, and assessments. A benchmark response (BMR) of 10% extra risk was 

considered appropriate for the quantal data on incidences of kidney inflammation and kidney 

transitional epithelial hyperplasia. The estimated BMDLs were used as points of departure (PODs). 

Further details including the modeling output and graphical results for the best-fit model for each 

endpoint can be found in Appendix C of the Supplemental Information.

In general, absolute and relative kidney weight data may both be considered appropriate 

endpoints for analysis (Bailey et al., 2004). However, in the NTP (1995) 2-year drinking water 

study, there was a noticeable decrease in body weight in exposed animals relative to controls at the 

15 month interim sacrifice (see Table 1-1). In such a case, relative kidney weights are preferred, so 

changes in absolute kidney weights from NTP (1995) were not analyzed. However, body weights 

were not impacted in the NTP (1997) subchronic inhalation study. Based on a historical review of 

26 studies of control rats from 1-month bioassays, Bailey et al. (2004) concluded that neither 

absolute kidney weight nor relative kidney:body (or kidney:brain) weight are optimal for 

evaluating organ weight changes. Since neither approach is preferred, both were considered to be 

appropriate for BMD analysis of the NTP (1997) data set. 

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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1 The RfC methodology provides a mechanism for deriving a HEC from the duration-adjusted 

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POD (BMCLADJ) determined from the animal data. The approach takes into account the extrarespiratory nature of the toxicological responses and accommodates species differences by 

considering blood:air partition coefficients for tert-butanol in the laboratory animal (rat or mouse) 

and humans. According to the RfC guidelines (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 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 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:

BMCLHEC = BMCLADJ (mg/m3) × (interspecies conversion) 

= BMCLADJ (mg/m3) × (481 ÷ 462) 

 = BMCLADJ (mg/m3) × (1.04) 

Table 2-4 summarizes the sequence of calculations leading to the derivation of a humanequivalent point of departure for each inhalation data set discussed above.

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

Endpoint and 

Reference

Species/

Sex Modela BMR

BMCb

(mg/m3)

BMCL

b

(mg/m3)

PODADJb

(mg/m3)

PODHEC

c 

(mg/m3)

Kidney

Increased relative 

kidney weight

NTP (1997)

Male F344 

rats

Linear 10% 6309 4821 861 861

Increased absolute 

kidney weight

NTP (1997)

Male F344 

rats

Hill 10% 1931 1705 304 304

Increased relative 

kidney weight

NTP (1997)

Female F344 

rats

No model 

selected

10% -- -- -- --

Increased absolute 

kidney weight

NTP (1997)

Female F344 

rats

No model 

selected

10% -- -- -- --

a 2 For modeling details, see Appendix C in Supplemental Information.

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b

BMCs, BMCLs, and PODs were adjusted for continuous daily exposure by multiplying by (hours exposed per day / 

24 hrs) × (days exposed per week / 7 days). c

PODHEC calculated by adjusting the PODADJ by the DAF (=1.0) for a category 3 gas (U.S. EPA, 1994). d

BMD modeling failed to successfully calculate a BMD value (see Appendix C).

PODs from oral studies – use of PBPK model for route-to-route extrapolation

A PBPK model for tert-butanol in rats has been developed, as described in Appendix B. 

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 using the methodology previously described in “PODs from 

inhalation studies”: 

BMCLHEC = BMCLADJ (mg/m3) × (interspecies conversion) 

= BMCLADJ (mg/m3) × (481 ÷ 462) 

 = BMCLADJ (mg/m3) × (1.04) 

A critical decision in the route-to-route extrapolation is the 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 routeto-route extrapolation relationship as tert-butanol in blood, since the distribution from blood to 

kidney is independent of route. There are no data to suggest that metabolites of tert-butanol

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1 mediate its renal toxicity. In the absence of evidence that would suggest otherwise, it is assumed 

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that tert-butanol itself is the active toxicological agent. Therefore, the concentration of tert-butanol

in blood was selected as the dose metric.

Table 2-5 summarizes the sequence of calculations leading to the derivation of a humanequivalent point of departure for 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 relative kidney weight

NTP (1995)

Rat/M 10% 48 2.34 79.6

Mean relative kidney weight

NTP (1995)

Rat/F 10% 133 7.46 231

Kidney inflammation

NTP (1995)

Rat/F 10% 200 12.6 359

Kidney transitional epithelial 

hyperplasia

NTP (1995)

Rat/M 10% 16 0.745 26.1

Kidney transitional epithelial 

hyperplasia

NTP (1995)

Rat/F 10% 339 27.9 638

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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.

PODs carried forth to derivation of candidate values

For the derivation of candidate values, it must be considered whether PODs from the 

inhalation study of NTP (1997) would provide a better basis than the route-to-route extrapolated 

PODs based on the oral study of NTP (1995). The only endpoint available from NTP (1997) is 

increased kidney weights. The corresponding PODs from this subchronic inhalation study are 

substantially higher than those for the same endpoint derived by route-to-route extrapolation from 

the chronic study (NTP, 1995), consistent with longer duration requiring a lower dose to elicit an 

effect. Additionally, as discussed in Section 2.1.3, kidney weight is a less-specific endpoint 

compared to some of the other endpoints available for analysis from the oral study (NTP, 1995). 

Therefore, the PODs derived from PBPK model-based route-to-route extrapolation are the 

preferred basis for deriving kidney-specific candidate RfCs, as they are based on a longer (chronic) 

duration and a more specific endpoint. 

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

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1 2.2.3. Derivation of Candidate Values 

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Under 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. An explanation follows:

An intraspecies uncertainty factor, UFH, of 10 was applied to all PODs to account for 

potential differences in toxicokinetics and toxicodynamics in the absence of information on the 

variability of response in the human population following inhalation exposure to tert-butanol. 

An interspecies uncertainty factor, UFA, of 3 (101/2 = 3.16, rounded to 3) was applied to all 

PODs to account for residual uncertainty in the extrapolation from laboratory animals to humans in 

the absence of information to characterize toxicodynamic differences between rodents and humans 

after inhalation exposure to tert-butanol. This value is adopted by convention where an adjustment 

from animal to a human equivalent concentration has been performed as described in EPA’s 

Methods for Derivation of Inhalation reference Concentrations and Application of Inhalation 

Dosimetry (U.S. EPA, 1994). 

A subchronic to chronic uncertainty factor, UFS, of 1 was applied to the PODs derived from 

the NTP (1995) study, as the endpoints were observed following chronic exposure. For the PODs 

derived from the subchronic NTP (1997) study, a UFS of 10 was applied to account for extrapolation 

from subchronic to chronic duration.

A LOAEL to NOAEL uncertainty factor, UFL, of 1 was applied to all PODs 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% change in kidney weight, a 10% extra risk of kidney 

inflammation, and a 10% extra risk of transitional cell hyperplasia were selected under an 

assumption that they represent minimal biologically significant changes. 

A database uncertainty factor, UFD, of 1 was applied to all PODs. The tert-butanol toxicity 

database includes a chronic toxicity study in rats and mice (NTP, 1995), a subchronic toxicity study 

in rats and mice (NTP, 1997), 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 effects were observed at exposure levels below 1000 mg/kg-day, and effects observed at 

≥1000 mg/kg-day were accompanied by evidence of maternal toxicity. These exposure levels are 

much higher than the PODs for kidney effects, suggesting developmental toxicity is not a sensitive 

endpoint. The tert-butanol database contains a one-generation reproductive toxicity study in rats 

(Lyondell Chemical Co., 2004), though no multigenerational reproductive study has been 

performed. There are no immunotoxicity studies for tert-butanol. Information provided by studies 

on ETBE, which is rapidly metabolized to systemically-available tert-butanol, can help in 

considering the lack of a tert-butanol multigenerational reproductive study or an immunotoxicity 

study. No adverse effects were reported in one- and two-generation reproductive/developmental 

studies on ETBE (Gaoua, 2004a, b), and the database for ETBE does not indicate immunotoxicity 

(Banton et al., 2011; Li et al., 2011). Thus, although there are some gaps in the toxicity database for 

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

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1 tert-butanol, the available data on tert-butanol, informed by the data on ETBE, do not suggest that 

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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.

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. 

Table 2-6. Effects and corresponding derivation of candidate values 

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 relative kidney 

weight; male rat

NTP (1997)

861 BMCL10% 3 10 1 10 1 300 3 × 100

Increased absolute kidney 

weight; male rat

NTP (1997)

304 BMCL10% 3 10 1 10 1 300 1 × 100

Increased relative kidney 

weight; female rat

NTP (1997)

1137 NOAEL 3 10 1 10 1 300 4 × 100

Increased absolute kidney 

weight; female rat

NTP (1997)

1137 NOAEL 3 10 1 10 1 300 4 × 100

Increased relative kidney 

weight; male rat

NTP (1995)

79.6 BMCL10% 3 10 1 1 1 30 3 × 100

Increased relative kidney 

weight; female rat

NTP (1995)

231 BMCL10% 3 10 1 1 1 30 8 × 100

Kidney inflammation; female 

rat

NTP (1995)

359 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

10

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

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

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Figure 2-2. Candidate RfC values with corresponding POD and composite UF.
Toxicological Review of tert-Butyl Alcohol

1 2.2.4. Derivation of Organ/System-Specific Reference Concentrations

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Table 2-7 distills the candidate values from Table 2-6 into a single value for the kidney. 

Organ or system-specific reference values may be useful for subsequent cumulative risk 

assessments that consider the combined effect of multiple agents acting at a common site.

Kidney Toxicity

For tert-butanol, candidate values were for several different effects in both sexes, spanning 

a range from 9 × 10-1 to 2 × 101 mg/m3, for an overall twenty-fold range. To estimate an exposure 

level below which kidney toxicity from tert-butanol exposure is not expected to occur, the RfC for 

increased incidence of transitional epithelial hyperplasia in male rats (9 × 10-1 mg/m3) is proposed 

as the kidney-specific reference concentration for tert-butanol, consistent with the selection of the 

kidney-specific RfD (see Section 2.1.4). As discussed previously, unlike kidney inflammation, this 

effect was observed in both sexes, with males appearing to be more sensitive than females. 

Additionally, it is a more specific and more sensitive indicator of kidney toxicity than the relatively 

non-specific endpoint of kidney weight changes. Confidence in this kidney-specific RfC is medium. 

The PODs are based on modeled benchmark dose estimates, and the candidate values are derived 

from a well-conducted study, involving a sufficient number of animals per group, including both 

sexes, assessing a wide range of kidney endpoints, and availability of a PBPK model for route-toroute extrapolation. 

Table 2-7. Organ/system-specific RfCs and proposed overall RfC for 

tert-butanol

Effect Basis

RfC

(mg/m3)

Exposure 

description Confidence

Kidney toxicity Increased incidence of 

transitional epithelial 

hyperplasia 

9 × 10-1 Chronic HIGH

Proposed 

overall RfC 

Increased incidence of 

transitional epithelial 

hyperplasia 

9 × 10-1 Chronic HIGH

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23 2.2.5. Selection of the Proposed Overall Reference Concentration

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For tert-butanol, only kidney effects were identified as a hazard; thus, a single 

organ/system-specific reference concentration was derived. Therefore, the kidney-specific RfC of 

9 × 10-1 mg/m3 is also proposed as an estimated exposure level below which deleterious effects 

from tert-butanol exposure are not expected to occur.

The overall reference concentration is derived to be protective of all types of effects for a 

given duration of exposure and is intended to protect the population as a whole including 

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

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1 potentially susceptible subgroups (U.S. EPA, 2002). Decisions concerning averaging exposures over 

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time for comparison with the RfC should consider the types of toxicological effects and specific 

lifestages of concern. Fluctuations in exposure levels that result in elevated exposures during these 

lifestages could potentially lead to an appreciable risk, even if average levels over the full exposure 

duration were less than or equal to the RfC. In the case of tert-butanol, no specific lifestages have 

been identified has a potentially susceptible subgroup.

2.2.6. Confidence Statement

A confidence level of high, medium, or low is assigned 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). The overall confidence in this RfC is high. Confidence in the principal study (NTP, 1995) is 

high. This study was well-conducted, compiled with FDA GLP regulations, involved a sufficient 

number of animals per group (including both sexes), and assessed a wide range of tissues and 

endpoints. Although there are some gaps in the toxicity database for tert-butanol, these areas are 

informed by the data on ETBE, a parent compound of 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 RfC is high. 

2.2.7. Previous IRIS Assessment

An inhalation assessment for tert-butanol was not previously available on IRIS.

2.2.8. Uncertainties in the Derivation of the Reference Dose and Reference Concentration

The following discussion identifies uncertainties associated with the RfD and RfC for 

tert-butanol. To derive the RfD, the UF approach (U.S. EPA, 2000a, 1994) was applied to a POD 

based on kidney toxicity in rats treated chronically. To derive the RfC, this same approach was 

applied, but a PBPK model was used to extrapolate from oral to inhalation exposure. UFs were 

applied to the POD to account for extrapolating from an animal bioassay to human exposure, the 

likely existence of a diverse population of varying susceptibilities, and database deficiencies. These 

extrapolations are carried out with default approaches given the lack of data to inform individual 

steps.

The database for tert-butanol contains no human data on adverse health effects from 

subchronic or chronic exposure. Data on the effects of tert-butanol are derived from a small 

database of studies in rats and mice. The database for tert-butanol exposure includes one lifetime 

bioassay, several reproductive/developmental studies, and several subchronic studies.

Although the database is adequate for reference value derivation, there is uncertainty 

associated with the lack of a comprehensive multigeneration reproductive toxicity study. 

Additionally, only subchronic and short-term inhalation studies have been conducted, and no 

chronic inhalation studies are available. Developmental studies identified significant increases in 

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

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1 fetal loss, decreases in fetal body weight, and possible increases in skeletal variations in exposed 

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offspring or pups. However, effects were not always consistent across exposure routes, and 

significant material toxicity was present whenever developmental effects were observed. 

The toxicokinetic and toxicodynamic differences for tert-butanol between the animal 

species in which the POD was derived and humans are unknown. Although sufficient information is 

available to develop a PBPK model in rats to evaluate difference across routes of exposure, the 

tert-butanol database lacks an adequate model that would inform potential interspecies differences. 

Generally, it was found that rats appear more susceptible than mice, and males appear more 

susceptible than females to tert-butanol toxicity. However, 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.

2.3. ORAL SLOPE FACTOR FOR CANCER

The carcinogenicity assessment provides information on the carcinogenic hazard potential 

of the substance in question and quantitative estimates of risk from oral and inhalation exposure 

that may be derived. Quantitative risk estimates may be derived from the application of a low-dose 

extrapolation procedure. If derived, the oral slope factor is a plausible upper bound on the estimate 

of risk per mg/kg-day of oral exposure. 

2.3.1. Analysis of Carcinogenicity Data

As noted in Section 1.2.2, EPA concluded that there is “suggestive evidence of carcinogenic 

potential” for tert-butanol. The Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) state:

When there is suggestive evidence, the Agency generally would not attempt a 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. 

The only data available on potential carcinogenicity was derived from the 2-year drinking 

water study in rats and mice by (NTP, 1995). This study was considered suitable for dose-response 

analysis. It was conducted in accordance with Food and Drug Administration (FDA) Good 

Laboratory Practice (GLP) Regulations, and all aspects were subjected to retrospective quality 

assurance audits. The study included histological examinations for tumors in many different 

tissues, contained three exposure levels and controls, contained adequate numbers of animals per 

dose group (~50/sex/group), treated animals for up to 2 years, and included detailed reporting of 

methods and results. Additionally, the renal tumors were re-examined by a Pathology Working 

Group (Hard et al., 2011). 

Dose-related increasing trends in tumors were noted at the following sites:

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1 • Renal tubule adenomas and carcinomas in male rats; and

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• Thyroid follicular adenomas in female mice. 

These tumors were statistically significantly increased by pairwise comparison (Fisher 

exact test, p ≤ 0.05) and by trend test (Cochran-Armitage trend test, p ≤ 0.05). Based on analysis of 

mode of action data, it was concluded that processes other than α2u-globulin nephropathy are likely 

responsible for the male rat renal tumors, so these tumors may be suitable for quantitative analysis

(U.S. EPA, 1991a). Additionally, a thyroid follicular carcinoma was observed in male mice, so it is 

possible that the thyroid follicular adenomas in female mice could progress to malignant form. 

Therefore, the thyroid follicular adenomas in female mice may also be considered suitable for 

quantitative analysis. Considering these data along with the uncertainty associated with the 

suggestive nature of the weight of evidence, EPA concluded that quantitative analyses may be 

useful for providing a sense of the magnitude of potential carcinogenic risk. 

2.3.2. Dose-Response Analysis—Adjustments and Extrapolations Methods

The U.S. EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) recommends that 

the method used to characterize and quantify cancer risk from a chemical is determined by what is 

known about the MOA of the carcinogen and the shape of the cancer dose-response curve. The 

linear approach is recommended if the MOA of carcinogenicity has not been established (U.S. EPA, 

2005a). In the case of tert-butanol, the modes of carcinogenic action for renal tubule and thyroid 

follicular tumors are not fully understood (see Section 1.2.2). Therefore, a linear low-dose 

extrapolation approach was used to estimate human carcinogenic risk associated with tert-butanol

exposure.

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 for mice, 0.25 kg for rats, and 70 kg for humans was used (U.S. EPA, 

1988). The following formula was used for the conversion of oral BMDL to oral HED for rat 

endpoints: 

Scaled HED in mg/kg-day = (BMDL in mg/kg-day) × (animal body weight/70)1/4

 = (BMDL in mg/kg-day) × 0.24

Details of the modeling and the model selection process can be found in Appendix C of the 

Supplemental Information. PODs for estimating low-dose risk were identified at doses at the lower 

end of the observed data, generally corresponding to 10% extra risk. Because initial modeling 

overestimated the control, due to the non-monotonicity of the observed dose-response, the POD 

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

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1 was derived after dropping the highest exposure group (U.S. EPA, 2012b). The highest exposure 

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group also had increased mortality, which may in part explain the observed non-monotonicity.

2.3.3. Derivation of the Oral Slope Factor

The PODs estimated for each tumor site 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 = 0.1/BMDL10). This slope, a 95% upper 

confidence limit, represents a plausible upper bound on the true risk. Using linear extrapolation 

from the BMDL10, human equivalent oral slope factors were derived for each species/tumor site 

combination and are listed in Table 2-8. 

The oral slope factors derived from the NTP (1995) bioassay differ by twenty-fold, 

depending on the species and tumor site. The most sensitive endpoint of renal tumors was used to 

derive the oral slope factor because there are no data to support any one result as most relevant for 

extrapolating to humans. Two slope factors were derived for this endpoint from the NTP (1995)

bioassay, one based on the original reported incidences and the other based on the Hard et al. 

(2011) reanalysis. The two estimates differed by less than 20%, and rounded to the same number 

at one significant figure. However, the Hard et al. (2011) reanalysis is considered preferable, as it is 

based on a PWG analysis. Therefore, the recommended slope factor for providing a sense of the 

magnitude of potential carcinogenic risk associated with lifetime oral exposure to tert-butanol is 

1 × 10-2 per mg/kg-day, based on the renal tubule tumor response in male F344 rats. 

Table 2-8. Summary of the oral slope factor derivations 

Tumor Species/Sex Selected Model

BMR BMD

(mg/kgd)

POD=

BMDL

(mg/kg-d)

BMDLHED

a 

(mg/kg-d)

Slope factorb 

(mg/kg-day)-1 

Renal tubule 

adenoma or 

carcinoma 

Male F344 

rat; dose as 

administered

1° Multistage

(high dose 

dropped)

10% 70 42 10.1 1 × 10-2

Renal tubule 

adenoma or 

carcinoma [Hard et 

al. (2011) reanalysis]

Male F344 

rat; dose as 

administered

1° Multistage

(high dose 

dropped)

10% 54 36 8.88 1 x 10-2

Thyroid follicular cell 

adenoma

Female 

B6C3F1 

mouse

3° Multistage 10% 2002 1437 201 5 × 10-4

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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.

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

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There is uncertainty when extrapolating data from animals to estimate potential cancer 

risks to human populations from exposure to tert-butanol (see Table 2-9). Uncertainty in the 

magnitude of the recommended oral slope factor is reflected to some extent in the range of slope 

factors; the oral slope factor based on the male rat data was about twenty-fold higher than the oral 

slope factor based on female mouse data (Table 2-9). These comparisons show that the selection of 

target organ, animal species, and interspecies extrapolation can impact the oral cancer risk 

estimate. Although the thyroid follicular cell tumors occurred in male and female mice, high 

mortality in high-dose male mice limited the usefulness of the data. Renal tubule tumors occurred 

in male rats, but not female rats. Therefore, only the data in male rats and female mice were 

available for deriving the oral slope factor. There are no other chronic studies to replicate these 

findings or that examined other animal models. There are no data in humans to support the tumors 

observed in animals. Although changing the methods used to derive the oral slope factor could 

change the results, standard practices were used due to the lack of a mouse or human PBPK model 

or specific MOA to indicate other methods would be preferable. Additionally, considering the 

uncertainty associated with the suggestive nature of the weight of evidence, the oral slope factor is 

recommended only for providing a sense of the magnitude of potential carcinogenic risk.

Table 2-9. Summary of uncertainties in the derivation of cancer risk values for 

tert-butanol 

Consideration and

Impact on Cancer Risk Value Decision Justification and Discussion

Selection of target organ 

↓ oral slope factor, up to twentyfold, if renal tumors not selected. 

The kidney was selected as 

the target organ. 

As there are no data to support any one 

result as most relevant for extrapolating to 

humans, the most sensitive result for kidney 

renal tubular adenomas and carcinomas was 

used to derive the oral slope factor. 

However, the overall evidence for 

carcinogenicity was considered “suggestive.” 

Selection of data set 

Unknown change in oral slope 

factor, since no other studies are 

available. 

NTP (1995) as principal oral 

(drinking water) study to 

derive cancer risks for 

humans.

NTP (1995) was a well-conducted study. It 

was also the only bioassay available. 

Additional bioassays might add support to 

the findings or provide results for different 

(possibly lower) doses, which may affect the 

oral slope factor.

Selection of extrapolation approach 

(Selection of extrapolation approach 

could change the recommended 

cancer risk values.) 

Oral data used for OSF. No extrapolation methods were used.

Selection of dose metric 

Alternatives could ↓ or ↑ slope 

factor

Used administered dose 

converted to HED units.

Additional runs using the administered dose 

without conversion to HED units were also 

conducted, resulting in a similar oral slope 

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

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Consideration and

Impact on Cancer Risk Value Decision Justification and Discussion

factor. For rats, a PBPK model of internal 

dose was available, but the POD changed by 

less than 1.2-fold when modeling was based 

on internal doses. For mice, no PBPK model 

was available, so using a PBPK model for 

determining internal doses could have an 

unknown effect on the estimated OSF value.

Interspecies extrapolation of 

dosimetry and risk 

Alternatives could ↓ or ↑ slope 

factor (e.g., 3.5-fold ↓ [scaling by 

body weight] or ↑ 2-fold [scaling by 

BW2/3])

The default approach of 

body weight3/4 was used.

There are no data to suggest an alternative 

approach. Because the dose metric was not 

an area under the curve, BW3/4 scaling was 

used to calculate equivalent cumulative 

exposures for estimating equivalent human 

risks. While the true human correspondence 

is unknown, this overall approach is expected 

to neither over- nor underestimate human 

equivalent risks.

Dose-response modeling 

Alternatives could ↓ 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. 

Low-dose extrapolation 

↓ cancer risk estimate would be 

expected with the application of 

nonlinear low-dose extrapolation

Linear extrapolation of risk 

in low-dose region used. 

Linear low-dose extrapolation for agents 

without a known MOA is supported.

Statistical uncertainty at POD

↓ oral slope factor 1.7-fold if BMD 

used as the POD rather than BMDL

BMDL (preferred approach 

for calculating plausible 

upper bound slope factor)

Limited size of bioassay results in sampling 

variability; lower bound is 95% CI on 

administered exposure at 10% extra risk of 

renal tumors.

Sensitive subpopulations 

↑ 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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A cancer assessment for tert-butanol was not previously available on 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 

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

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1 extrapolation procedure. If derived, the inhalation unit risk is a plausible upper bound on the 

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estimate of risk per μg/m3 air breathed.

2.4.1. Analysis of Carcinogenicity Data

As noted in Section 1.2.2, EPA concluded that there is “suggestive evidence of carcinogenic 

potential” for tert-butanol. The Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) state: 

When there is suggestive evidence, the Agency generally would not attempt a 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, it could provide a sense of the magnitude and uncertainty of 

potential risks, rank potential hazards, or set research priorities. 

The only data available on potential carcinogenicity were from the 2-year drinking water 

study in rats and mice by NTP (1995), discussed previously in Section 2.3.1. Because a PBPK model 

for the rat is available to conduct route-to-route extrapolation (discussed below), the male rat renal 

tubule adenoma and carcinoma data are suitable for quantitative analysis to support an inhalation 

unit risk. Considering these data and uncertainty associated with the suggestive nature of the 

weight of evidence, EPA concluded that quantitative analyses may be useful for providing a sense of 

the magnitude of potential carcinogenic risk.

2.4.2. Dose Response Analysis – Adjustments and Extrapolation Methods

Details of the modeling and the model selection process can be found in Appendix C of the 

Supplemental Information. A POD for estimating low-dose risk was identified at doses at the lower 

end of the observed data corresponding to 10% extra risk. 

A PBPK model for tert-butanol in rats has been developed, as described in Appendix B. 

Using this model, route-to-route extrapolation of the oral BMDL to derive an inhalation POD was

performed as follows. First, the internal dose in the rat at the oral BMDL (assuming continuous 

exposure) was estimated using the PBPK model, to derive an “internal dose BMDL.” Then, the 

inhalation air concentration (again assuming continuous exposure) that led to the same internal 

dose in the rat was estimated using the PBPK model, resulting in a route-to-route extrapolated 

BMCL. 

A critical decision in the route-to-route extrapolation is the selection of the internal dose 

metric to use that established "equivalent” oral and inhalation exposures. For tert-butanol-induced 

kidney effects, the two options are the concentration of tert-butanol in blood and rate of tertbutanol metabolism. Note that using the kidney concentration of tert-butanol will lead to the same 

route-to-route extrapolation relationship as tert-butanol in blood, since the distribution from blood 

to kidney is independent of route. There are no data that suggest metabolites of tert-butanol

mediate its renal toxicity. In the absence of evidence that would suggest otherwise, it is assumed 

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

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1 that tert-butanol itself is the active toxicological agent. Therefore, the concentration of tert-butanol

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in blood was selected as the dose metric to derive the BMCL.

The RfC methodology provides a mechanism for deriving a HEC from the BMCL determined 

from the animal data. The approach takes into account the extra-respiratory nature of the 

toxicological responses and accommodates species differences by considering blood:air partition 

coefficients for tert-butanol in the laboratory animal (rat or mouse) and humans. According to the 

RfC guidelines (U.S. EPA, 1994), tert-butanol is a Category 3 gas because extra-respiratory effects 

were observed.Kaneko et al. (2000) measured a blood:gas partition coefficient of 531 ± 102 for tertbutanol 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 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:

BMCLHEC = BMCLADJ (mg/m3) × (interspecies conversion) 

= BMCLADJ (mg/m3) × (481 ÷ 462) 

 = BMCLADJ (mg/m3) × (1.04) 

The U.S. EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) recommend that 

the method used to characterize and quantify cancer risk from a chemical is determined by what is 

known about the MOA of the carcinogen and the shape of the cancer dose-response curve. The 

linear approach is recommended if the MOA of carcinogenicity has not been established (U.S. EPA, 

2005a). In the case of tert-butanol, the mode of carcinogenic action for renal tubule tumors is not 

fully understood (see Section 1.2.2). Therefore, a linear low-dose extrapolation approach was used 

to estimate human carcinogenic risk associated with tert-butanol exposure.

2.4.3. Inhalation Unit Risk Derivation 

The results from route-to-route extrapolation of the male rat renal tubule tumor data are 

summarized in Table 2-10. The lifetime inhalation unit risk for humans is defined as the slope of 

the line from the lower 95% bound on the exposure at the POD to the control response (inhalation 

unit risk = 0.1/BMCL10). This slope, a 95% upper confidence limit represents a plausible upper 

bound on the true risk. Using linear extrapolation from the BMCL10, a human equivalent inhalation 

unit risk was derived, as listed in Table 2-10. 

Two inhalation unit risks were derived from the NTP (1995) bioassay: one based on the 

original reported incidences and one based on the Hard et al. (2011) reanalysis. The two estimates 

differ by less than 20%, but the Hard et al. (2011) reanalysis is considered preferable, as it is based 

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

2-26 DRAFT—DO NOT CITE OR QUOTE
Toxicological Review of tert-Butyl Alcohol

1 on a PWG analysis. Therefore, the recommended inhalation unit risk for providing a sense of the 

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magnitude of potential carcinogenic risk associated with lifetime inhalation exposure to 

tert-butanol is 2 × 10-3 per mg/m3, or 2 × 10-6 per μg/m3, based on the renal tubule tumor 

response in male F344 rats. 

Table 2-10. Summary of the inhalation unit risk derivation 

Tumor Species/Sex BMR

BMDL

(mg/kg-d)

Internal Dosea

(mg/L)

POD=

BMCLHEC

c 

(mg/m3)

Unit Riskb 

(mg/m3)

-1 

Renal tubule adenoma or 

carcinoma 

Male F344 

rat

10% 41.6 2.01 68.7 1 × 10-3

Renal tubule adenoma or 

carcinoma [Hard et al. 

(2011) reanalysis]

Male F344 

rat

10% 36.3 1.74 59.8 2 × 10-3

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. c

Human equivalent inhalation unit risk = 0.1/BMCLHEC. 

2.4.4. Uncertainties in the Derivation of the Inhalation Unit Risk

There is uncertainty when extrapolating data from animals to estimate potential cancer 

risks to human populations from exposure to tert-butanol (see Table 2-11). Uncertainty in the 

magnitude of the recommended inhalation unit risk can be inferred to some extent from the range 

of oral slope factors; the oral slope factor based on the male rat data was about twenty-fold higher 

than the oral slope factor based on female mouse data (Table 2-9). These comparisons show that 

the selection of target organ, animal species, and interspecies extrapolation can impact the 

inhalation unit risk estimate. Although the thyroid follicular cell tumors occurred in male and 

female mice, high mortality in high-dose male mice limited the usefulness of the data. Additionally, 

no PBPK model was available in mice for use in route-to-route extrapolation, so these data could 

not be used to estimate an inhalation unit risk. Renal tubule tumors occurred in male rats, but not 

female rats. Therefore, only the data in male rats were available for deriving the inhalation unit 

risk. There are no other chronic studies to replicate these findings or that examined other animal 

models. There are no data in humans to support the tumors observed in animals. Although 

changing the methods used to derive the inhalation unit risk could change the results, standard 

practices were used due to the lack of a mouse or human PBPK model or specific MOA to indicate 

other methods which would be preferable. Additionally, considering the uncertainty associated 

with the suggestive nature of the weight of evidence, the inhalation unit risk is recommended only 

for providing a sense of the magnitude of potential carcinogenic risk.

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

2-27 DRAFT—DO NOT CITE OR QUOTE
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1 Table 2-11. Summary of uncertainties in the derivation of cancer risk values 

2 for tert-butanol 

Consideration and

Impact on Cancer Risk Value Decision Justification and Discussion

Selection of target organ 

Inhalation unit risk may change by 

an unknown amount if a PBPK 

model to extrapolate mouse thyroid 

tumors to the inhalation route were 

available.

The kidney was selected as 

the target organ. 

No PBPK model to extrapolation mouse 

thyroid tumors was available. Additionally, 

the overall evidence for carcinogenicity was 

considered “suggestive.” 

Selection of data set 

Unknown change in inhalation unit 

risk, since no other studies are 

available. 

NTP (1995) as principal oral 

(drinking water) study to 

derive cancer risks for 

humans.

NTP (1995) was a well-conducted study. It 

was also the only bioassay available. 

Additional bioassays might add support to 

the findings or provide results for different 

(possibly lower) doses, which may affect the 

inhalation unit risk.

Selection of extrapolation approach 

Different PBPK model could ↓ or ↑ 

inhalation unit risk.

PBPK model-based 

extrapolation of oral data 

used for inhalation unit 

risk.

PBPK model accurately predicted tertbutanol toxicokinetics. Data and model 

predictions were within 2-fold of each other.

Selection of dose metric 

Alternatives could ↓ or ↑ 

inhalation unit risk. 

Used tert-butanol

concentration in blood as 

the dose metric for routeto-route extrapolation, 

converted to HEC. 

In the absence of evidence that would 

suggest that metabolites of tert-butanol are 

responsible for carcinogenicity, it is assumed 

that tert-butanol itself is the active 

toxicological agent. An alternative dose 

metric of tert-butanol metabolism would 

result in a 1.2-fold decrease in the inhalation 

unit risk. 

Interspecies extrapolation of 

dosimetry and risk 

Alternatives could ↓ or ↑ 

inhalation unit risk.. 

The default approach for a 

Category 3 gas was used.

There are no data to suggest an alternative 

approach. While the true human 

correspondence is unknown, this overall

approach is expected to neither over- nor 

underestimate human equivalent risks.

Dose-response modeling 

Alternatives could ↓ or ↑ 

inhalation unit risk. 

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. 

Low-dose extrapolation 

↓ cancer risk estimate would be 

expected with the application of 

nonlinear low-dose extrapolation .

Linear extrapolation of risk 

in low-dose region used. 

Linear low-dose extrapolation for agents 

without a known MOA is supported.

Statistical uncertainty at POD

↓ inhalation unit risk 1.7-fold if the 

BMD used to derive the inhalation 

POD rather than BMDL.

BMDL (preferred approach 

for calculating plausible 

upper bound)

Limited size of bioassay results in sampling 

variability; lower bound is 95% CI on 

administered exposure at 10% extra risk of 

renal tumors.

Sensitive subpopulations No sensitive populations No chemical-specific data are available to 

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

2-28 DRAFT—DO NOT CITE OR QUOTE
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Consideration and

Impact on Cancer Risk Value Decision Justification and Discussion

↑ inhalation unit risk to unknown 

extent. 

have been identified. determine the range of human 

toxicodynamic variability or sensitivity, 

including the susceptibility of children. 

Because determination of a mutagenic MOA

has not been made, an age-specific 

adjustment factor is not applied.

 1

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A 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 applied to account for early-life exposure to carcinogens that act 

through a mutagenic mode of action. Because chemical-specific life-stage susceptibility data for 

cancer are not available, and because the mode of action for tert-butanol carcinogenicity is not 

known (see Section 1.1.4), ADAFs were not applied.

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

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1

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Faulkner, TP; Wiechart, JD; Hartman, DM; Hussain, AS. (1989). The effects of prenatal tertiary 

butanol administration in CBA/J and C57BL/6J mice. Life Sci 45: 1989-1995. 

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