**EPA/635/R -16/**  **079 a**  **Public Comment Draft www.epa.gov/iris** 



## Toxicological Review of tert

**- Butyl Alcohol (**  tert

- **Butanol)**

(CAS No. 75

- 65

- 0)

April 2016

**NOTICE**

This document is a **Public Comment Draft**

. This information is distributed solely for the purpose of
pre

- dissemination peer review under applicable information quality guidelines. It has not been formally disseminated by EPA. It does not represent and should not be construed t o 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 Agenc y Washington, DCToxicological 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 an d 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.

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# CONTENTS

AUTHORS | CONTRIBUTORS | REVIEWERS

................................................................................................ . viii

PREFACE

................................................................................................ ........................................................ x

PREAMBLE TO IRIS TOXICOLOGICAL REVIEWS

............................................................................................ xiv

EXECUTIVE SUMMARY

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

............................................... LS -1

1. HAZARD IDENTIFICATION

.................................................................................................................... 1-1

1.1. Overview of Chemical Properties and Toxicokinetics

................................................................... 1-1

1.1.1. Chemical Properties

.......................................................................................................... 1-1

1.1.2. Toxicokinetics

.................................................................................................................... 1-2

1.1.3. Description of Toxicokinetic Models

................................................................................. 1-3

1.1.4. Chemicals Extensively Metabolized to tert

- Butanol

......................................................... 1-4

1.2. PRESENTATION AND SYNTHESIS OF EVIDENCE BY ORGAN/SYSTEM

............................................ 1-4

1.2.1. Kidney Effects

.................................................................................................................... 1-4

1.2.2. Thyroid Effects

................................................................................................................. 1- 38

1.2.3. Developmental Effects

.................................................................................................... 1- 46

1.2.4. Neurodevelopmental Effects

........................................................................................... 1- 53

1.2.5. Reproductive Effects

....................................................................................................... 1- 56

1.2.6. Other Toxicological Effects

.............................................................................................. 1- 61

1.3. INTEGRATION AND EVALUATION

............................................................................................... 1- 61

1.3.1. Effects Other Than Cancer

............................................................................................... 1- 61

1.3.2. Carcinogenicity

................................................................................................................ 1- 63

1.3.3. Susceptible Populations and Lifestages for Cancer and Noncancer Outcomes

.............. 1- 65

2. DOSE

- RESPONSE ANALYSIS

................................................................................................................. 2-1

2.1. ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER

...................................................... 2-1

2.1.1. Identification of Studies and Effects for Dose

- Response Analysis

.................................... 2-1

2.1.2. Methods of Analysis

.......................................................................................................... 2-2

2.1.3. Derivation of Candidate Values

......................................................................................... 2-4

2.1.4. Derivation of Organ/System

- Specific Reference Doses

.................................................... 2-8

2.1.5. Selection of the Overall Reference Dose

........................................................................... 2-8

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2.1.6. C onfidence 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. Metho ds of Analysis

........................................................................................................ 2- 10

2.2.3. Derivation of Candidate Values

....................................................................................... 2- 13

2.2.4. Derivation of Organ/System

- Specific Reference Concentrations

................................... 2- 16

2.2.5. Selection of the Overall Reference Concentration

.......................................................... 2- 16

2.2.6. Confidence Statemen t

..................................................................................................... 2- 17

2.2.7. Previous IRIS Assessment

................................................................................................ 2- 17

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

.................................................................................................................. 2- 17

2.3. ORAL SLOPE FACTOR FOR CANCER

............................................................................................. 2- 18

2.3.1. Analysis of Carcinogenicity Data

..................................................................................... 2- 18

2.3.2. Dose

- Response Analysis

— Adjustments and Extrapolations Methods

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

2.3.3. Derivation of the Oral Slope Factor

................................................................................. 2- 20

2.3.4. Uncertainties in the Derivation of the Oral Slope Factor

................................................ 2- 21

2.3.5. Previous IRIS Assessment: Oral Slope Factor

.................................................................. 2- 23

2.4. INHALATION UNIT RISK FOR CANCER

......................................................................................... 2- 23

2.4.1. Previous IRIS Assessment: Inhalation Unit Risk

............................................................... 2- 24

2.5. APPLICATION OF AGE

- DEPENDENT ADJUSTMENT FACTORS

...................................................... 2- 24

REFERENCES

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

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# TABLES

Table ES

- 1. Organ/system

- specific RfDs and overall RfD for tert

- butanol

.............................................. ES -2 Table ES

- 2. Organ/system

- specific RfCs and overall RfC for tert

- butanol

............................................... ES -3 Table LS

- 1. Details of the search strategy employed for tert

- butanol

..................................................... LS -4 Table LS

- 2. Summary of additional search strategies for tert

- butanol

.................................................... LS -4 Table LS

- 3. Inclusion

- exclusion criteria

.................................................................................................... LS -5 Table LS

- 4. Considerations for evaluation of experimental animal studies

............................................ LS -8 Table LS

- 5. Summary of experimental animal database

......................................................................... LS -8 Table 1

- 1. Physicochemical properties and chemical identity of tert

- butanol

.......................................... 1-1 Table 1

- 2. Changes in kidney histopathology in animals following exposure to tert

- butanol

................ 1- 12 Table 1

- 3. Changes in kidney tumors in animals following exposure to tert

- butanol

............................. 1- 15 Table 1

- 4. Summary of data on the α 2u - globulin process in male rats exposed to tert

- butanol

............ 1- 22 Table 1

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

........................................... 1- 33 Table 1

- 6. Evidence pertai ning to thyroid effects in animals following oral exposure to tert butanol

........................................................................................................................... 1- 39 Table 1

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

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

­

Table 1

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

- butanol

.................................................................................................................... 1- 54 Table 1

- 9. Evidence pertaining to reproductive e ffects in animals following exposure to tert butanol

........................................................................................................................... 1- 56 Table 2

- 1. Summary of derivations of points of departure following oral exposure for up to 2
years

................................................................................................ ................................ . 2-4 Table 2

- 2. Effects and corresponding derivation of candidate values

....................................................... 2-6 Table 2

- 3. Organ/system

- specific RfDs and overall RfD for tert

- butanol

.................................................. 2-8 Table 2

- 4. Summary of derivation of PODs following inhalation exposure

............................................. 2- 12 ­

Tab le 2

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

- to

- route extrapolation fro m oral exposures

................................................................................ 2- 13 Table 2

- 6. Effects and corresponding derivation of candidate values

..................................................... 2- 14 Table 2

- 7. Organ/system

- specific RfCs and overall RfC for tert

- butanol

................................................. 2- 16 Table 2

- 8. Summary of the oral slope factor derivation

.......................................................................... 2- 21 Table 2

- 9. Summary of uncertainties in the derivation of the oral slope factor for tert

- butanol

........... 2- 22



# FIGURES

Figure LS

- 1. Summary of literature search and screening process for tert

- butanol.

.............................. LS -3 Figure 1

- 1. Biotransformation of tert

- butanol in rats and humans

. .......................................................... 1-3 Figure 1

- 2. Comparison of absolute kidney weight change in male and female rats across oral and inhalation exposure based on internal blood concentration. Spearman rank correlation coefficient (rho) was calculated to evaluate the direction of a monotonic association (e.g., positive value = positive association) and the strength of association.

................................................................................................ . 1- 10 Figure 1

- 3. Comparison of absolute kidney weight change in male and female mice following oral exposure based on administered concentration. Spearman rank correlation

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- 7. Temporal pathogenesis of α 2u - globulin

- associated nephropathy in male rats. α 2u Globulin synthesized in the livers of male rats is delivered to the kid ney, where it can accumulate in hyaline droplets and be retained by epithelial cells lining the S2 (P2) segment of the proximal tubules. Renal pathogenesis following continued tert

- butanol (TBA) exposure and increasing droplet accumulation can progress ste p- wise from increasing epithelial cell damage, death and dysfunction leading to the formation of granular casts in the corticomedullary junction, linear mineralization of the renal papillae, and carcinogenesis of the renal tubular epithelium (Adapted from Swenberg and Lehman

- McKeeman (1999) and U.S. EPA (1991a).

......................................................................................... 1- 21 ­

Figure 1

- 8 . Exposure

- response array for effects potentially associated with α 2u - globulin renal tubule nephropathy and tumors in male rats after oral exposure to tert

- butanol.

...... 1- 24 Figure 1 -9 . Exposure

- response array for effects potentially associated with α 2u - globulin renal tubule nephropathy and tumors in male rats after inhalation exposure to
tert

- butanol.

................................................................................................................... 1- 25 Figure 1

- 10. Exposure

- response array of thyroid follicular cell effects following chronic oral exposure to tert

- butanol. (Note: Only one carcinoma was observed in male mice at the high

- dose group.)

................................................................................................ 1- 41 Figure 1

- 11. Exposure

- response array of developmental effects following oral exposure to tert butanol.

.......................................................................................................................... 1- 51 Figure 1

- 12. Exposure

- response array of developmental effects following inhalation exposure to
tert

- butanol.

................................................................................................................... 1- 52 ­

Figure 1

- 1 3. Exposure

- response array of reproductive effects following oral exposure to tert butanol.

.......................................................................................................................... 1- 59 Figure 1

- 14. Exposure

- response array of reproductive effects following inhalation exposure to
tert

- butanol.

................................................................................................................... 1- 60 Figure 2

- 1. Candidate values with corresponding POD and composite UF. Each bar corresponds
to one data set described in Table 2

- 1 and Table 2

- 2.

.................................................... 2-7 Figure 2

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

. ...................................... 2- 15 ­ Toxi cological Review of tert

- Butyl Alcohol

coefficient (rho) w as calculated to evaluate the direction of a monotonic associa tion (e.g., positive value = positive association) and the strength of association.

.................................................................................................................... 1- 11 Figure 1

- 4. Comparison of absolute kidne y weight change in male and female mice following inhalation exposure based on administered concentration. Spearman rank correlation coefficient (rho) was calculated to evaluate the direction of a monotonic association (e.g., positive value = positive asso ciation) and the strength of association.

................................................................................................ . 1- 11 Figure 1 -5

. Exposure response array for kidney effects following oral exposure to tert

- butanol.

......... 1- 17 Figure 1 -6 . Exposure

- response array of kidney effects following inhalation exposure to tert

butanol (13

- week studies, no chronic studies available).

............................................. 1- 18

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— DO NOT CITE OR QUOTEQSAR quantitative structure

- activity relationship 

# ABBREVIATIONS

AIC Akaike’s information criterion ALD approximate lethal dosage ALT alanine aminotransferase AST aspartate aminotransferase
atm atmosphere ATSDR Agency for Toxic Substances and Disease Registry BMD benchmark dose BMDL benchmark dose lower confid ence limit BMDS Benchmark Dose Software BMR benchmark response BW body weight CA chromosomal aberration CASRN Chemical Abstracts Service Registry Number CBI covalent binding index CHO Chinese hamster ovary (cell line) CL confidence limit CN S 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 FEV 1 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 LC 50 median lethal concentration LD 50 median lethal d ose 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 Inst itute NOAEL no

- observed

- adverse

- effect level NTP National Toxicology Program NZW New Zealand White (rabbit breed) OCT ornithine carbamoyl transferase ORD Office of Research and Development PBPK physiologically based pharmacokinetic POD point of departure POD [ADJ] duration

- adjusted POD Toxicological Review of tert

- Butyl Alcohol

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— DO NOT CITE OR QUOTE 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 A LT SSD systemic scleroderma TCA trichloroacetic acid TCE trichloroethylene TWA time

- weighted average UF uncertainty factor UF A animal

- to

- human uncertainty factor UF H human variation uncertainty factor UF L LOAEL

- to

- NOAEL uncertain factor UF S subchronic

- to

- chronic uncertainty factor UF D database deficiencies uncertainty factor U.S. United States2 

# AUTHORS | CONTRIBUTORS | REVIEWERS

**Assessment Team** Janice S. Lee, Ph.D. (Chemical Manager) Keith Salazar, Ph.D.* (Co

- Chemical Mana ger)

Chris Brinkerhoff, Ph.D. U.S. EPA Office of Research and Development National Center for Environmental Assessment Research Triangle Park, NC *Washington, DC

ORISE Postdoctoral Fellow at U.S. EPA/ORD/NCEA Currently with U.S. EPA, Office of Chemical Safety and Pollution Prevention, Office of Pollution Prevention and Toxics Washington, DC

**Contributors** Andrew Hotchkiss, Ph.D. Channa Keshava, Ph.D. Amanda Persad, Ph.D. Vincent Cogliano, Ph.D.* Jason Fritz, Ph.D.* Catherine Gibbons, Ph.D. * Samantha Jones, Ph.D. * Kathleen Newhouse * Karen Hogan * U.S. EPA Office of Research and Development National Center for Environmental Assessment Research Triangle Park, NC *Washington, DC

**Production Team** Maureen Johnson Vicki Soto U.S. EPA Office of Research and Development National Center for Environmental Assessment Washington, DC

**Contractor Support** Robyn Blain, Ph.D. Michelle Cawley* William Mendez, Jr., Ph.D. Pam Ross ICF International Fairfax, VA *Research Triangle Park, NC

**Executive Direction** Kenneth Olden, Ph.D., Sc.D., L.H.D. (Center Director) John Vandenberg, Ph.D, # (National Program Director, Human Health Risk Assessment) Lynn Flowers, Ph.D., DABT (Associate Director for Health) Vincent Cogliano, Ph.D. (IRIS Program Director) Samantha Jones, Ph.D. (IRIS Associate Director for Science) Weihsueh A. Chiu, Ph.D. (Branch Chief, Toxicity Pathways Branch) formerly with the U.S. EPA U.S. EPA/ORD/NCEA Washington, DC *Cincinnati, OH

# Research Triangle Park, NC Toxicological Review of tert

- Butyl Alcohol

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1 Toxicological Review of tert

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Andrew Hotchkiss, Ph.D. # (Acting Branch Chief, Toxicity Pathways Branch) Jason Lambert, Ph.D., DABT* (Acting Branch Ch ief, Biological Risk Assessment Branch) Ted Berner (Assistant Center Director) Karen Hogan (former Acting Branch Chief, Toxicity Effects Branch)

**Internal Review Team** General Toxicology Workgroup Inhalation Workgroup Neurotoxicity Workgroup Pharmacokinetics Workgroup Reproductive and Developmental Toxicology Workgroup Statistical Workgroup Toxicity Pathways Workgroup Executive Review Committee U.S. EPA Office of Research and Development National Center for Environmental Assessment Washington, DC Research Triangle Park, NC Cincinnati, OH

**Reviewers**

This assessment was provided for review to scientists in EPA’s Program and Region Offices. Comments were submitted by:

Office of the Administrator/Office of Children ’s Health Protection Office of Land and Emergency Management Region 2, New York, NY Region 8, Denver, CO

This assessment was provided for review to other federal agencies and the Executive Office of the President. Commen ts were submitted by:

Department of Health and Human Services/Agency for Toxic Substances and Disease Registry, Department of Health and Human Services/National Institute of Environmental Health Sciences/National Toxicology Program, Executive Office of t he President/Office of Management and Budget, Executive Office of the President/Office of Science and Technology Policy

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# PREFACE

This Toxicological Review critically reviews the publicly available studies on tert

- butyl alcohol ( tert

- butanol ) to identify its adverse health effects and to characterize exposure

- response relationships. The assessment examined all effects by oral and inhalation routes of exposure and
includes an oral noncancer reference dose ( RfD ) , an inhalation noncancer reference concentration ( RfC ) , a cancer weight of evidence descriptor

, and a cancer dose

- response assessment

. It was prepared under the auspices of the U.S. Environmental Protection Agency’s ( EPA ’s) Integrated Risk Information System (IRIS) program. This is the first IRIS assessment for this chemical. Toxicological Reviews for tert

- butanol and ethyl tert

- butyl ether (ETBE) were developed simultaneously because they have several overlapping scientific aspects.

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

- Butanol is one of the primary metabolites of ETBE, a nd some of the toxicological effects
of ETBE are attributed to tert

- butanol. Therefore, data on ETBE are considered informative
for the hazard identification and dose

- response assessment of tert

- butanol, and vice versa.

• The scientific literature for the two chemicals includes data on α 2u - globulin

- related nephropathy ; t herefore, a common approach was employed to evaluate these data as they relate to the mode of action for kidney effects.

• A combined physiologically based pharmacokinetic ( PBPK ) model for tert

- butanol and

ETBE in rats was modified to support the dose

- response assessments for these chemicals ( Salazar et al., 2015 ).

tert

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

- response arrays prior to the development of the IRIS assessment. All public comments provided were taken into consideration in developing the draft assessment. The complete set of public comments is available on the docket at http://www.regulations.gov (Docket
ID No. EPA

- HQ

- ORD

- 2013

- 0111). Organ/system

- specif ic reference values are calculated based on kidney and thyroid toxicity
data. These reference values could be useful for cumulative risk assessments that consider the
combined effect of multiple agents acting on the same biological system. This assessment was conducted in accordance with EPA guidance, which is cited and
summarized in the Preamble to IRIS Toxicological Reviews. The findings of this assessment and related documents produced during its development are available on the IRIS website ( h ttp://www.epa.gov/iris ). Appendices for toxicokinetic information, PBPK modeling, genotoxicity study summaries, dose

- response modeling, and other information are provided as Supplemental Information to this Toxicological Review. For additional information about this assessment or for

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general questions regarding IRIS, please contact EPA’s IRIS Hotline at 202

- 5 6 6

- 1676 (phone), 202
566

- 1749 (fax), or hotline.iris@epa.gov

.

**Uses**

tert

- Butanol primarily is an anthropogenic substance that is produced in large quantities ( HSDB, 2007 ) from several precursors, including 1

- butene, isobutylene, acetyl chloride and dimethylzinc, and tert

- butyl hydroperoxide

. The domestic production volume of tert

- butanol, including imports, was a pproximately 4 billion pounds in 2012 ( U.S. EPA, 2014 ).

tert

- Butanol has been used as a fuel oxygenate, an octane booster in unleaded gasoline, and
a denaturant for ethanol. From 1997 to 2005, the annual tert

- butanol volume found in gasoline ranged from approximately 4 million to 6 million gallons. During that time, larger quantities were used to make methyl tert

- butyl ether (MTBE) and ETBE. MTBE and ETBE are fuel oxygenates that ­

wer e used in the United States pr i o r to 2007 at levels of more than 2 billion gallons annually.
Current use levels of MTBE and ETBE in the United States a r e much lower , but use in Europe and
Asia remains strong

. 1

tert

- Butanol has been used for a variety of other purposes

, including as a dehydrating agent
and solvent. As such, it is added to lacquers, paint removers, and nail enamels and polishes.

tert

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

- related uses incl ude the manufacture of flavors

, and, because of its camphor

- like aroma, it also is used to create artificial musk, fruit essences, and perfume ( HSDB, 2007 ). It is used in coatings on metal and paperboard food containers ( Cal/EPA, 1999 ) and industria l cleaning compounds

, and can be used for chemical extraction in pharmaceutical application s ( HSDB, 2007 ).

**Fate and Transport**

Soil

tert -B utanol is expected to be high ly mobile in soil due to its low affinity for soil organic

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

most tert

- butanol present in soil, potentially lead ing to groundwater contamination. Based on its

vapor pressure, tert

- butanol’s volatilization from soil surfaces is exp ected to be an important dissipation process ( HSDB, 2007 ). As a tert iary alcohol

, tert

- butanol is expected to degrade more slowly in the environment compared to primary ( e.g. , ethanol) or secondary ( e.g ., isopropanol)
alcohols. In anoxic soil conditions, the half

- life of tert

- butanol is estimated to be months (approximately 200 days). Microbial degradation rates are incre ased in soils supplemented with
nitrate and sulfate nutrients ( HSDB, 2007 )

.

1 http://www.ihs.com/products/chemical/planning/ceh/gasoline

- octane

- improvers.aspx

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Water

t e rt

- Butanol is expected to volatilize from water surfaces within 2 to 29 days and does not readily adsorb to suspended solids and sediments in water ( HSDB, 2007 ). Biodegradation in
aerobic water occurs over weeks to months and in anaerobic aquatic conditions, the biodegradation
rate decreases. Bioconcentration of tert

- butanol in aquatic organisms is low ( HSDB, 2007 ).

Air

tert

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

- phase tert butanol is degraded in the atmosphere by reacting with photochemically produced hydrox yl
radicals with a half

- life of 14 days ( HSDB, 2007 ).

**Occurrence in the Environment**

T h e Toxics Release Inventory (TRI) Program National Analysis Report estimated that more than 1 million pounds of tert

- butanol has been released into the soil from landfills, land treatment, underground injection, surface impoundments, and other land disposal sources. The TRI program also estimated that 476,266 pounds of tert

- butanol was released into the atmosphere from fugitive emissions and point sources ( U.S. EPA, 2012c ). In California, air emissions of tert

- butanol from stationary sources are estimated to be at least 27,000 pounds per year, based on data reported by
the state’s Air Toxics Program ( Scorecard, 2014 ). The TRI National Analysis Report estimated 7,469 pounds of tert

- butanol was released into surface waters from point and nonpoint sources in 2011 ( U.S. EPA, 2012c ).

tert

- Butanol has been identified in drinking water wells throughout the United States ( HSDB, 2007 ). California’s Geotracker Database 2 lists 3

, 496 detections of tert

- butanol in groundwater associated with contaminated sites in that state since 2011. tert

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

Butanol leaking from underground storage tanks could be a product of MTBE and ETBE, which can degrade to form tert

- butanol in soils ( HSDB, 2007 ). The industrial chemical tert

- butyl acetate also can degrade to form tert

- butanol in animals post exposure and in the envir onment.
Ambient outdoor air concentrations of tert

- butanol vary according to proximity to urban

areas ( HSDB, 2007 )

.

**General Population Exposure** 

t ert

- Butanol exposure can occur in many different settings. Releases from underground storage tanks could potentially result in exposure for people who get th e ir drinking water from
wells. Due to its high environmental mobility and res istance to biodegradation, tert

- but anol has the potential to contaminate and persist in groundwater and soil ( HS DB , 2007 ). ­

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

.

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— DO NOT CITE OR QUOTE1 In gestion of contaminated food can be a source of tert

- butanol exposure through its use as a coating in metallic and paperboard food containers ( Cal/EPA, 1999 ) , and tert

- butanol has been
detected in food ( HSDB, 2007 ). Internal exposure to tert

- butanol also can occur as a result of ingestion of MTBE or ETBE, as tert

- butanol is a me tabolite of these compounds ( NSF International,
2003 ). Other human exposure pathways include inhalation, lactation and, to a lesser extent, dermal
contact. Inhalation exposure can occur due to the chemical’s volatility and release from industrial processes, consumer products

, and contaminated sites ( HSDB, 2007 ). tert

- Butanol has been
identified in mother’s milk ( HSDB, 2007 )

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

- butanol ( NSF International, 2003 ). 2

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**Assessments by Other National and International Health Agencies** Toxicity information on tert

- butanol has been evaluated by the National Institute f or Occupational Safety and Health ( NIOSH, 2007 ) , the Occupational Safety and Health Administration ( OSHA, 2006 ) , and the Food and Drug Administration ( FDA, 2011a
, b ). The results of these assessments are presented in Appendix A of the Supplemental Information to this Toxicological
Review

. Of importan ce to recognize is that these earlier assessments could have been prepared for different purposes and might use dif ferent methods

. In addition, newer studies have been included
in the IRIS assessment

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- b utanol assessment will
include the revised Preamble.



## 1. Scope of the IRIS Program

Soon after the EPA was established in 1970, it was at the f orefront of d eveloping 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 l evels 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 consideration s 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 o f the Clean Air Act (carbon monoxide, lead, nitrogen oxides, ozone, particulate matter , and sulfur oxides). P eriodically, 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 consider ed 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 respo nse to an urgent public health need.



## 2. Process for developing and peer

reviewing 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

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assessment will meet their needs. T hen a public meeting on problem formulation invites discussion of the key issues and the studies and analytical approaches that might contribute to their resolution. **Step 1. Development of a draft**  **Toxicological Review** . The draft assessment considers all pertinent publicly available studies and applies consistent criteria to evaluate study quality, identify health effects, identify mechanistic events and pathways, integrate the evidence of causation for each effect, and derive toxicity values. A public mee ting 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 pro vides 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 evi dence 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. **St ep**  **5. Revision of draft Tox icological**  **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 revie w 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** . Th e Toxicological Review and IRIS summary are posted on the IRIS website ( http://www.epa.gov/iris/ ). The remainder of this Preamble addresses step 1, the development of a draft Toxicological Review. IRIS assessment s follow standard practices of evidence evaluation and peer review, many of which are discussed in EPA guidelines ( U.S. EPA, 2005a

, b

, 2000b

, 1998b

, 1996
, 1991b

, 198 6a
, b ) and other methods ( U.S. EPA, 2012a
, b

, 2011
, 2006a
, b

, 2002
, 1994 )

. Transparent application of scientific judgment is of paramount importance. To provide a harmonized approach across IRIS assessments, this Preamble summarizes concepts from these guidelines and emphasizes principles of general a pplicability.



## 3. Identifying and selecting pertinent studies

**3.1. Identifying studies**  Before beginning an assessment, the EPA conducts a comprehensive search of the primary scientific literature. The literature

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1 search follows standard practices and includes the Pu bMed 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 inform ation on mechanisms of toxicity are inherently specialized and may include studies on other agents that act through related mechanisms. Each assessment specifies the search strategies, keywords, and cut

- off d ates of its literature searches. The EPA posts t he results of the literature search on the IRIS web site and requests information from the public on additional studies and ongoing research. The EPA also considers studies received through the IRIS Submission Desk and studies (typically unpublished) submitted under the Toxic Substances Control Act or the Federal Insecticide, Fungicide, and Rodenticide Act. Material submitted as Confidential Business Information is considered only if it includes health and safety data that can be publicly released. If a study that may be critical to the conclusions of the assessment has not been
peer

- reviewed, the EPA will have it peer reviewed. The EPA also examines the toxicokinetics of the agent to identify other chemicals (for example, major metabolites of the agent ) to include in the assessment if adequate information is available, in order to more fully explain the toxicity of the agent and to suggest dose metrics for subsequent modeling. 48

In assessments of chemical mixtures

, mixture studies are preferred for their ability to reflect interactions among components. The literature search seeks, in decreasing order of preference ( U.S. EPA, 2000b, §2.2 ; 1986b, §2.1 ):

– Studies of the mixture being assessed

.

– Studies of a sufficiently similar mixture. In evaluating similarity, the assessment considers the alteration of mixtures in the environment through partitioning and transformation. – Studies of individual chemical components of the mixture, if there are

no t 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 studi es, and some population

- based surveys (for example, NHANES) provide the strongest epidemiologic evidence, especially if they collect information about individual exposures and effects. 2 49
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an d 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 b ut 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.

– S tudies of oral, inhalation, or dermal exposure involve passage through an absorption barrier and are considered 65
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– Injection or implantation studies are often considered less pertinent but
may provide valuable toxicoki netic or mechanistic information. They also may be useful for identifying effects in animals if deposition or absorption is problematic (for example, for p articles and fibers). Exposure duration is also a key design consideration for selecting pertinent

ex perimental animal studies.

– Studies of effects from chronic exposure are most pertinent to lifetime human exposure.

– S tudies of effects from less

- than chronic exposure are pertinent but less preferred for identifying effects from lifetime human exposure. Such studies may be indicative of effects from less

- than

- lifetime human
exposure.
Short- duration studies involving animals

or humans may provide toxicokinetic or mechanistic information. For developmental toxicity and reproductive toxicity, irreversible effects may result from a brief exposure during a critical period of development. Accordingly, specialized study designs are used for these effects ( U.S. EPA, 2006b

, 1998b

, 1996
, 1991b ). 45



## 4. Evaluating the qua lity 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 de sign.



### 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.

– Defi nition and selection of the study group and comparison group.

– A scertainment of expo sure to the chemical or mixture. 2 46

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– Duration of exposure and follow

- up and adequacy for assessing the

occurrence of e ffects.

– Cha racterization of e xposure d uring critical periods.

– S ample size and statistical power to detect anticipated effects.

– P articipation 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 informati on bias.

– Potential confounding and other sources of bias addressed in the study design or in the analysis of results. The basis for consideration of c onfounding 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 53

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### 4.2. **Evaluating t he 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 ) . Resear ch involving human subjects is considered only if conducted according to ethical principles.

– Documentation of study design, animals or study population, methods, basic data, and results.

– Nature of the assay and validity for its intended purpose.

– Characteri zation of the nature and extent of impurities and contaminants of the administered chemical or mixture.

– C haracterization of dose and dosing regimen (including age at exposure) and t heir adequacy to elicit adverse effects, including latent effects.

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– Control of other variables that could influence the occurrence of effects.

The assessment uses statisti cal 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
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, 1998 b

, 1996
, 1991b ). In multi generation studies, agents that produce developmental effects at doses that are not toxic to the maternal animal are of special concern. Effects that occur at doses associated with mild maternal toxicity are not assumed to result only from maternal toxicity. Moreover

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maternal effects may be reversible, while

e f

fects 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 detai ls 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 t he 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.5

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48 1 

## 5. Evaluating the overall evidence of each effect

**5.1. Concepts of causal inference** 
For each health effect, the assessment evaluates the evidence as a whole to determine whether it is reasona ble 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 develo ped 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 differe nt purposes, the frameworks are similar in nature and provide an established structure and language for causal inference. Each considers aspects of an association that suggest causation, discussed by Hill ( Hill, 1965 ) and elaborated on by Rothman and Greenland ( Rothman and Greenland, 1998 )

, and U.S. EP A ( 2005a, §2.2.1.7 ; 1994, Appendix C ). 49 2 50

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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 i ncrease 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 61

80 for causation. Discordant results sometimes reflect differences in study design, exposure, or confounding factors. **Specificity of association:** As originally intended, this refer s to one cause associated with one effect. Current understanding that many agents cause multiple effects and many effects have multiple causes make this a less informative aspect of causation, unless the effect is rare or unlikely to have multiple

causes. **Temporal relationship:**  A causal interpretation requires that exposure precede development of the effect. **Biologic gradient (exposure**

**- response**  **relationship):** Exposure

- response relationships strongly suggest causation. A monotonic increase is not the only pa ttern 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 biolog ic mechanisms, if available. Plausibility may reflect subjective prior beliefs if there is insufficient understanding of the biologic process involved.

**Coherence:** A n inference of causation is strengthened by supportive results from animal experiments, toxi cokinetic studies, and s hort

- term tests. Coherence may also be found in other lines of evidence, such as changing disease patterns in the population. **“Natural experiments”:** A change in exposure that brings about a change in disease frequency provides strong evidence, as it tests the hypothesis of causation. An example would be an intervention to reduce exposure in the workplace or environment that is followed by a reduction of an adverse effect. 63

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1 **Analogy:** Information on structural analogues
2 or on chemicals that induce similar
3 mechanistic events can provide insight
4 into causation.

These considerations are consistent with
6 guidelines for systematic reviews that
7 evaluate the quali ty and weight of evidence.
8 Confidence is increased if the magnitude of
9 effect is large, if there is evidence of an
exposure

- response relationship, or if an
11 association was observed and the plausible
12 biases would tend to decrease the magnitude
13 of the report ed effect. Confidence is decreased
14 for study limitations, inconsistency of results, indirectness of evidence, imprecision, or
16 reporting bias ( Guyatt et al., 2008b ; Guyatt et
17 al., 2008a ).

18 

### 5.2. **Evaluating evidence in humans**

19 For each effect, the assessment evaluates the evidence from the epidemiologic studies as
21 a whole. The objective is to determine whether
22 a credible association has been observed and,
23 if so, whether that association is consistent
24 with causation. In doing this, the assessment explores alternative explanations (such as
26 chance, bias, and confounding) and draws a
27 conclusion about whether these alternatives
28 can satisfactorily explain any observed
29 association. To make clear how much the
31 epidemiologic evidence contributes to the
32 overall weig ht of the evidence, the assessment
33 may select a standard descriptor to
34 characterize the epidemiologic evidence of association between exposure to the agent and
36 occurrence of a health effect.

37 Sufficient epidemiologic evidence of an
38 association consistent wi th causation:
39 The evidence establishes a causal association for which alternative
41 explanations such as chance, bias, and
42 confounding can be ruled out with
43 reasonable confidence.

44 Suggestive epidemiologic evidence of an association consistent with causation:
46 The evidence suggests a causal association Toxicological Review of tert

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47 but chance, bias, or confounding cannot be
48 ruled out as explaining the association.

49 Inadequate epidemiologic evidence to infer
50 a causal association: T he available
51 studies do not permit a conclusion
52 regarding the presence or absence of an
53 association.

54 Epidemiologic evidence consistent with no
55 causal association: S everal adequate
56 studies covering the full range of human
57 exposures and considering susceptible
58 populations, and for which alternative
59 explanations such as bias and confounding
60 can be ruled out, are mutually consistent
61 in not finding an association.

62 

### 5.3. **Evaluating evidence in animals**

63 For each effect, the assessment evaluates
64 the evidence from the animal experiments as a
65 whole to determine the extent to which the y 66 indicate a potential for effects in humans.
67 Consistent results across various species and
68 strains increase confidence that similar results
69 would occur in humans. Several concepts
70 discussed by Hill ( Hill, 1965 ) are pertinent to
71 the weight of experimental resu lts:
72 consistency of response, dose

- response 73 relationships, st rength of response, biologic
74 plausib ility, and coherence ( U.S. EPA,
75 2005a, §2.2.1.7 ; 1994, Appendix C ).

76 In weighing evidence from multiple
77 experiments, U.S. EPA ( 2005a, §2.5 )
78 distinguishes :

79 Conflicting evidence (that is, mixed positive
80 and negative results in the same sex and
81 strain using a similar study protocol) from

82 Differing results ( that is, positive results and
83 negative results are in different sexes or
84 strains or use different study protocols).

85 Negative or null results do not invalidate
86 positive results in a different experimental
87 system. The EPA regards all as valid
88 observations and looks to explain differing
89 results using mechanistic information (for
90 example, physiologic or metabolic differences
91 across test systems) or methodological

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differences (for example, relative sensitivity of the tests, differences in dose levels, insuffi cient 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 whethe r 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, chromosom e 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 weigh t, partly because they cannot exclude the possibility of effects in other tissues ( IARC, 2006 ). 1
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- 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 usefu l 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, i f possible, mechanistic pathways that lead to a health effect. These pathways encompass: – Toxicokinetic processes of absorption, distribution, metabolism, and elimination that lead to the formation of an active agent and its presence at the site of initial biologic interaction.

– Toxicodynamic processes that lead to a health effect at this or another site (also known as a mode of action ).
For each effect, the assessment discusses the available information on its modes of action and associated key events ( key events being empirically observable, necessary
precursor steps or biologic markers of such steps; mode of action being a series of key events involving interaction with cells, operational and anatomic changes, and resulting in disease). Pertinent information 44
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necessary to a mode of action can come from experimental challenge to the hypothesized mode of a ction, in which studies that suppress a key event observe suppression of the effect. Support for a mode of action is meaningfully strengthened by consistent results in different experimental models, much more so than by replicate experiments in the same model. The assessment may consider various aspects of causation in addressing this question. 2) **I s the hypothesized mode of action**  **relevant to**  **humans?** The assessment reviews the key events to identify critical similarities and differences between the test anima ls and humans. Site concordance is not assumed between animals and humans, though it may hold

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1 for certain effects or modes of action. Information suggesting quantitative differences in doses where effects would occur in animals or humans is considered in t he 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 par**  **ticularly 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 suscep tible populations or lifestages.

The assessment discusses the likelihood that an agent operates through multiple mode s of action. An uneven level of support for different modes of action can reflect disproportionate resou rces 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 sect or ( Guyatt et al., 20 08a ). 48

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 metabol ite to react with or bind to DNA, positive results in multiple test systems, or similar properties and structure

- activity relationships to mutage nic 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, th e following standard descriptors co mbine epidemiologic, experimental, and mechanistic evidence of carcinoge nicity ( U.S. EPA, 2005a, §2.5 ). Carcinogenic to humans: There is convincing epidemi ologic 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, iden tification of key precursor events in animals, and strong evidence that they are anticipated to occur in humans. 2 49
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same sex and strain. Differing results

, howe ver, 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 57
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1 may be no effects in both sexes of at least two approp riate 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. Multipl e descriptors may be used if there is eviden ce that carcinogenic effects differ by dose range or exposure ro ute ( U.S. EPA, 2005a, §2.5 ). Another example of standard descriptors

come s from th e EPA’s Integrated Science Assessments, which evaluate causation for the effects of the criteria polluta nts 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 exampl e, animal studies or mechanistic information. Likely to be a causal relationship: S ufficient evidence that a causal relationship is likely, but important uncertainties remain. For example, observational studies show

an association but co

- exposures are diff icult 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. 47

Suggestive of a causal relationship: At least

one high -quality epide miologic 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 st andard d escriptors to characterize the overall weight of the evidence for effects other than cancer.



## 6. Selecting studies for derivation of toxicity values

For each effect where there is credible evidence of an association with the agent, the assessment derives toxicity values if there are suitable epidemiologic or experimental data. The decision to derive toxicity values may be linked to the hazard descriptor. Dose

- r esponse analysis requires quantitative measures of dose and response. Then, other factors bein g equal: 2 48
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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 com parability 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 pr eferred, to minimize uncertainty about whether effects are representative of lifetime
exposure.

– S tudies 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 54

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- monotonic exposure response relationships are not necessarily excluded from the analysis. A dimi nished 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- respons e 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

an alysis, the EPA asks peer reviewers to identify studies that were not adequately considered.



## 7. Deriving toxicity values

**7.1. General framework for dose** 

**** **response analysis**

Th e EPA uses a two

- step approach that distinguishes analysis of the observed dose

response da ta from inferenc es about lower doses ( U.S. EPA, 2005a, §3 ). 47

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 extrapolat ion 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 dose

response analysis may derive a relative potency f actor for each agent. A full dose response analysis is conducted for one well studied index chemical in the group, then the potencies of other members are expressed in relati ve terms based on relative toxic effects, relative absorption or metabolic rates, quantitative structure

- activity relationships, or receptor binding characteristics ( U.S. EPA, 2005a, §3.2.6 ; 2000b, §4.4 ). Increasingly, the EPA is basing toxicity values on combined analyse s 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**  2 48
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or a metabolite. Confidence in the use of a toxicokinetic model depends on the robustness of its validation process and on the results of sensitivity analyses ( U.S. EPA, 2006a ; 2005a, §3.1 ; 1994, §4.3 ). Because toxicokinetic modeling can require many parameters and more data than are typically available, the EPA has developed standard approaches that can be applied to typical data sets. These standard approaches also facilitate comparison across exposure patterns and species.

– Intermittent study exposures are standardized to a daily average over the duration of exposure. For chronic eff ects, daily exposures are averaged over the lifespan. Exposu res during a critical period, ho wever, are not averaged over a longer duration ( U.S. EPA, 2005a, §3.1.1 ; 1991b, §3.2 ).

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1 – Oral doses are scaled a llometrically using mg/kg 3/4 -d ay as the equivalent dose metric across species. Allometric scaling pertains to equivalence across species, not across lifestages, and is
not used to s cale d oses from adult humans or mature animals to infants

o r children ( U.S. EPA, 2011 ; 2005a, §3.1.3 ).

– Inhalation exposures are scaled using dosimetry models that apply species specific physiologic and anatomic factors and consider whether the effect occurs at the site of first contact

or after systemic circulation ( U.S. EPA, 2012a ; 1994, §3 ).

It can be informative to convert doses

across exposure routes. If this is done, the assessment descri bes the underlying data, algorithms, and assumptions ( U.S. EPA, 2005a, §3.1.4 ). In th e 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 ). 48



### 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 quantitat ively 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. Confide nce in the use of a toxicodynamic model depends on the robustness of its validation process and on the results of sensitivity analyses. P eer 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 avai lable, the EPA has developed a standard set of empirical (“curve

- fit ting”) models ( http://www.epa.gov/ncea/bmds/ ) that can be applied to typical data sets, including those that are nonlinear. The EPA has also developed guidance on modeling dose

- response data, assessing model fi t, selecting suitable models, and reporting modeling results ( U.S. EPA, 2012b )

. Additional judgment o r 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,

e specially if there is competing toxicity at higher doses ( U.S. EPA, 2005a, §3.2.3 ). M odeling i s used to derive a point of departure ( U.S. EPA, 2012b ; 2005a, §3.2.4 ). (See Section 7.6 for alternative s if a point of departure cannot be derived by modeling.) : 2 49
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cancer bioassay data, 1% for epidemiologic data, lower for rare cancers).

– For nonlinear approaches, both statistical and biologic considerations are taken into account.

– For dichotomous data, a response level of 10% extra risk is generally used for minimally adverse effects, 5% or lower for more severe effects.

– For continuous data, a response level is ideally based on an established definition of biologic significance. In the absence of such definition, one
contro l standard deviation from the control mean is often used for minimally adverse effects, one

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1 The point of departure is the 95% lower bound on the dose associated with the selected response level.



### 7.4. **Extrapolatin g 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
extr apolation, 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) L inear extrapolation is used if the dose response curve is expected to have a linear component below the point of departure. This includes:

– A gents or their metabolites that are DNA

- reactive and have direct mutagenic activity.

– Agents or their metabolites for which human exposures or b ody burdens are near doses associated with key events leading to an effect. 46

Linear extrapolation is also used when data are insufficient to esta blish mode of action and when scientifically plausible.

The result of linear extrapolation is described b y an oral slope factor o r an inhalation unit risk, which is the slope of the dose

- response curve at lower doses or concentrations, respectively. 3) N onlinear 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 app roaches may be used if there a multiple modes of action. For example, modeling to a low response level can be useful for estimating the
response at doses where a high

- dose mode of action would be less important. If linear extrapolation is used, the
assessment develops a candidate slope factor or unit risk for each suitable data set. These results are arrayed, using common dose metrics, to show the distribution of relative potency across various effects and 2 47
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an overall unit risk for the agent, considering the various dose

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### 7.5. **Conside ring susceptible**  **populations and lifestages**

The assessment analyzes the available information on populations and lifestages that may be particularly susceptible to each effect. A tiered approach is used ( U.S. EPA, 2005a, §3.5 ). 1) If an epidemiologic or experimental study reports quantitative results for a susceptible population or lifestage, these data are analyzed to derive separate toxicity values for susceptible individuals. 2) If data on risk

- related parameters allow comparison of the general population and susceptible individuals, these data are used to adjust the general

- population toxicity values for application to susceptible individuals. 3) I n the absence of chemical

- specific data, the EPA ha s developed age -dependent adjustment factors for early

- life exposure to potential carcinogens that have a mutagenic mode of action. There is evidence of early

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1 various carcinogenic agents, but most epidemi ologic studies and cancer bio assays do not include early

- life exposure. To address the potential for early- life susceptibility, the EPA

recom mends ( U.S. EPA, 2005b, §5 ):

– 10

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– 3- fold adjustment for exposures between ages 2 and 16 years.



### 7.6. **Reference values and uncertainty**  **factors**

An oral reference dose or an inhalation reference concentration is an estimate of an
exposur e (including in susceptible subgroup s) that is likely to b e 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 bi ologic markers ( U.S. EPA, 1998b

, 1996
, 1991b ). 48

To account for uncertainty and variability in the derivation of a lifetime human exposure where adverse effects are not anticipated to occur, reference values are calculated by
ap plying 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 lowest

observed

- adverse

- effect level is used instead. The assessment discusses s cientific consi derations 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 a re most susceptible to the effect. A factor of 10 i s 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 incl udes both susc eptible and non

- susceptible individuals) ( U.S. EPA, 2002, §4.4.5 ; 1998b, §4.2 ; 1996, §4 ; 1994, §4.3.9.1 ; 1991b, §3.4 ).

**Animal**

**- to** 

**- human extrapolation.**  If animal results are used to make inferences about humans, the assessment adjusts for cross

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**Adverse**

**- effect**  **level to no**

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**- effect**  **level.** If a point of departure is based on a lowest -o bserved adverse- effect level, the assessment must infer a dose where such effects are not expected. This can be a matter of great uncertainty, especially if there is no evidence available at lower doses. A factor of 10 is applied to account for the
uncerta inty in making this inference. A factor other than 10 ma y be used, depending on the magnitude and nature of
t he response and the shape of the dose

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**- chronic**  **exposure** . If a p oint of departure is based on subchronic studies, the assessment considers whether lifetime exposure could have effects at lower levels of exposure. A factor of 10 i s 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 t han 10 may be used, depending on the duration of the stu dies 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 in complete database raises concern that further studies might identify a more sensitive effect, organ system, or lifestage, the

assess ment 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 s tudy are missing and a factor of 1 0 1/2 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 sh ow where effects occur across a range of exposures ( U.S. EPA, 1994, §4.3.9 ). 1
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- specif ic 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 th at consider the combined effect of multiple agents acting at a common site or through common mechanisms ( NRC, 2009 ). The assessment then selects an overall reference dose and an overall reference concentration for the agent to represent lifetime human exposure levels where effects
are not anticipated to occur. This is generally the most sensitive organ/sys tem

- 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 comp leteness of the database, with more weight given to the latter. The level of confidence is increased for reference values based on human data su pported 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 interpre tation of prior test results.

**Medium confidence:** Th is is a matter of judgment, between high and low confidence.

**Low confidence:** T he reference value is especially vulnerable to change with further testing.

These criteria are consistent with guidelines for systematic reviews that evaluate the quality of evidence. These also focus on whether further research would be likely to change confidence in the estimate of effect ( Guyatt et al., 2008b ). All assessments discuss the significant uncertainties encountered in the analysis. The EPA provides guidance on characterization of uncertainty ( U.S. EPA, 2005a, §3.6 ). For example, the discussion distinguishes model

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# E XECUTIVE SUMMARY

Summation of Occurrence and Health Effects

tert

- Butanol does not occur naturally; it is produced by humans for multiple purposes, such as a solvent for paints, a denaturant for ethanol and several other alcohols, a dehydrating agent, and in the manufacture of flotation agents, fruit essences, and perfumes

. tert

- Butanol also is a primary metabolite of methyl tert

- butyl ether (MTBE) and ethyl tert butyl ether (ETBE). Exposure to tert

- butan ol primarily occurs through breathing air containing tert

- butanol vapors and consuming contaminated water or foods. Exposure can also occur through direct skin contact.
Animal studies demonstrate that chronic oral exposure to tert

- butanol is associated

wit h kidney and thyroid effects. Developmental effects (e.g., reduced fetal viability) have
been observed in short

- term exposure to high levels of tert

- butanol (via oral or inhalation exposure) in animals. Neurodevelopmental effects also have been observed, b ut results

were inconsistent. No chronic inhalation exposure studies have been conducted. There is suggestive evidence that tert

- b utanol is carcinogenic to humans based on renal tumors in male rats and thyroid tumors in female mice.

**Effects Other Than Canc er Observed Following**  **Oral Exposure**

K idney effects are a potential human hazard of oral exposure to tert

- butanol

. K idney toxicity

was observed in males and females in two strains of rats

. K idney weights were increased in male
and female rats after 13 weeks or 15 months of treatment

. Histopathological examination in male
and female rats observed increased incidence or severity of nephropathy after 13 weeks of oral

exposure , increased severity of nephropathy after a 2

- year oral exposure, and increased

tr ansitional epithelial hyperplasia after 2 years of oral exposure. Additionally, increased suppurative inflammation was noted in females after 2 years of oral exposure

. In on e strain of mice
, the only kidney effect observed was an increase in kidney weight (absolute or relative) in female

mice after 13 week s , but no treatment

- related histopathological lesions were reported in the kidneys of male or female mice at 13 weeks or 2 year s. A m ode of action (MOA) analysis determined
that tert

- butanol exposure induces a male rat

- specific α 2u - globulin

- associated nephropathy

. tert

Butanol , however, is a weak inducer of α 2u - globulin

- nephropathy

, and is not the sole process contribut ing to renal tubule nephropathy. Chronic progressive nephropathy (CPN) may also be
involved in some of the noncancer effects

, but the evidence is inconclusive. Endpoints specifically
related to either α 2u -globulin- nephropathy or CPN were not considered for kidney hazard
identification. C hanges in kidney weights, transitional epithelial h yperplasia, suppurative ­Toxicological Review of tert

- Butyl Alcohol

1 inflammation

, and severity and incidence of nephropathy , however, are considered to result from

tert

- butanol exposure and are appropriate for identifying a hazard to the kidney

. There is suggestive evidence of developmental toxicity following tert

- butanol exposure. Developmental effects include increased fetal loss, decreased fetal body weight, and increased skeletal variations. At this time, no conclusions were drawn in regard to r eproductive system toxicity. There is inadequate information at this time to draw conclusions regarding neurodevelopmental toxicity, liver, and urinary bladder toxicity.

**Oral Reference Dose (RfD) for Effects Other Than Cancer** Kidney t oxicity, represented by kidney transitional epithelial hyperplasia , was chosen as the
basis for the overall oral reference dose (RfD ) (see Table ES -1). The chronic study by NTP (1995)
and the observed kidney effects were used to derive the RfD

. The endpoint of transitional epithelial hyperplasia was selected as the critical effect because it was observed in both rat sexes 2

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, it is a specific and sensitiv e indicator of kidney toxicity , and was induced in a dose

res pons ive manner

. Benchmark dose (BMD) modeling was used to derive the benchmark dose lower confidence limit ( BMDL 10% ) of 16 mg/kg

- day

. The BMDL was converted to a human equivalent dose using body weight 3/4 scaling

, and th e value of 3.84 mg/kg -d ay was used as the point of departure (POD) for RfD derivation ( U.S. EPA, 2011 )

. The overall RfD was calculated by dividing the POD for kidney transitional epith elial hyperplasia by a composite uncertainty factor (UF) of 30 to account for the extrapolation from
animals to humans (3) and for interindividual differences in human susceptibility (10).

**Table ES**

**- 1**  **. Organ/system**

**- specific RfDs**  **and overall RfD for** tert

**- butanol**  14

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22 Hazard Basis Point of departure* (mg/kg

- day) UF Chronic RfD (mg/kg

- day) Study
exposure description Confidence

Kidney Transitional epithelial hyperplasia 3.8 30 1 × 10 -1 Chronic High

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

*HED PODs were calculated using BW 3/4 scaling ( U.S. EPA, 2011 ).

**Effects Other Than Cancer Observed Following Inhalation Exposure** Kidney effects are a potential human hazard of inhalation exposure to tert

- butanol. Although no effects were observed in mice, kidney weights were increased in male and female rats following 13 weeks of inhalation exposure. In addition, nephropathy severity increased in ma le
rats. No human studies are available to evaluate the effects of inhalation exposure. As discussed
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1 not considered for kidney hazard identification. Changes in kidney weights and severity of nephropathy, however, are considered a result of tert

- butanol exposure and are appropriate for identifying a hazard to the kidney.

**Inhalation Reference Concentration (RfC) for Effects Other Than Cancer**  Kidney toxicity, represented by transitional epithelial hyperplasia, was chosen as the basis for the inhalation reference concentration (RfC ) (see Table ES -2). Although endpoints from a route specific study were considered, the availability of a physiologically based pharmacokinetic (PBPK)
model for tert

- b utanol in rats [ modified by Salazar et al. (2015) ] allowed for more specific and sensitive equivalent inhalation PODs derived from a route

- to

- route extrapolation from the PODs of
the oral NTP (1995) study

. The POD adjusted for the human equivalent concentrat ion (HEC) was
26.1 mg/m 3 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 su sceptibility (10). 2

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15 **Table ES -2**  **. Organ/system**

**- specific RfCs and overall RfC for**  tert

**- butanol** 

16 Hazard Basis Point of departure*

(mg/m 3 ) UF Chronic RfC (mg/m 3 ) Study exposure description Confidence

Kidney Transitional epithelial hyperplasia 26.1 30 9 × 10 -1 Chronic High

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

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

tert

- butanol as continuous oral exposure at the BMDL.

**Evidence of Human Carcinogenicity** Under the EPA’s cancer guidelines ( U.S. EPA, 2005a ) , there is suggestive evidence of carcinogenic potential for tert

- butanol. t ert -B utanol induced kidney tumors in male (but not female) rats and thyroid tumors (primarily benign) in male and female mice following long

- term administration in drinking water ( NTP, 1995 )

. The potential for carcinogenicity applies to all routes of human exposure. 17

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1 **Quantitative Estimate of Carcinogenic Risk from Oral Exposure** A quantitative estimate of carcinogenic potential from oral exposure to tert

- butanol was based on the increased incidence of thyroid follicular cell adenomas in female B6C3F 1 mice, and thyroid follicular cell adenomas and carcinomas in male B6C3F 1 mice ( NTP, 1995 )

. The study included histological examinations for tumors in many different tissues, contained three exposure
levels and controls, contained adequate numbers of animals per dose group (~50/sex/group), treated animals for up to 2 years, and included detailed reporting of methods and results. Although tert

- butanol was considered to have “ suggestive evidence of carcinogenic potential ,” the NTP study was well conducted and quantitative analysis could be useful for providing a sense of the magnitude of potential carcinogenic risk ( U.S. EPA, 2005a ). A slope factor was derived for thyroid tumor s in female and m ale mice. The modeled tert

- butanol POD w as scaled to HEDs according to EPA guidance by converting the BMDL 10 on the basis of (body weight) 3/4 scaling ( U.S. EPA, 2011
, 2005a )

. U sing linear extrapolation from the BMDL 10 , a hum an equivalent oral slope factor w as derived (slope factor = 0.1/BMDL ). The resulting oral slope factor is **5 × 10**  **-4** 10 **per mg/kg**

**- day** 

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**Susceptible Populations and Lifestages for Cancer and Noncancer**

In vitro studies suggest that cytochrome P

- 450 (CYP450) ( Cederbaum et al., 1983 ; Cederbaum and Cohen, 1980 ), play s a role in the metabolism of tert

- butanol

. N o studies , however, have identified the specific CYPs responsible for the biotransformation of tert

- butanol

. V arious
CYPs are under

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

mice ( Lee et al., 2011 ) and rats ( Lee et al., 2008 ). Decreased ability to detoxify and transport tert butanol out of the body could result in increased susceptibility to tert

- butanol

. With regard to cancer, d ifferences in lifestage sensitivity to chemically induced thyroid carcinogenesis are unknown ( U.S. EPA, 1998a ). A n increased incidence of thyroid tumors was
identified in mice after tert

- butanol exposure

, and human studies have demonstrated that children
are more sensitive than adults are to thyroid carcinogenesis resulting from ionizing radiation

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- butanol are available. Lifetime exposure to

tert

- butanol has been associated with increased renal tubule adenomas and carcinomas as well as thyroid follicular cell adenomas and carcinomas. As stated above, the rat kidney tumors are unsuitable for quantitative analysis as there is not enough data to determine the relative
contribution of α 2u - globulin nephropathy and other processes to the overall kidney tumor response. Although the mouse thyroid tumors served as the basis for the oral slope factor, route

- to

route extrapolation is not possible for these thyroid effects in mice because the only PBPK model available is for rats. Therefore, no quantitative estimate of carcinogenic risk could be determined
for inhalat i on exposure. 17

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35 Toxicological Review of tert

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1 Collectively

, there is little evidence on tert

- butanol itself to identify any populations or lifestages
that may b e especially susceptible

.
**Key Issues Addressed in Assessment**

A n evaluation of whether tert

- butanol cause d α 2u - globulin

- associated nephropathy was performed. T he presence of α 2u - globulin in the hyaline droplets was confirmed in male rats by

α 2u -globulin immunohistochemical staining

. L inear mineralization and tubular hyperplasia were reported in male rats, al though only in the chronic study. Other subsequent steps in the pathological sequence, including necrosis, exfoliation, and granular casts, either wer e absent or

in consistently observed across subchronic or chronic studies. N one of the se effects occurred in
female rats or in either sex of mice , although these endpoints were less frequently evaluated in
these models

. E vidence implies a n α 2u - globulin MOA is operative

, although it is relatively weak in
response to tert

- butanol and is not solely responsible for the renal tubule nephropathy observed in
male rats. CPN also is instrumental in renal tubule nephropathy, in both male and female rats

. S everal other effects in the kidney unrelated to α 2u - globulin or CPN , h owever, were observed in 2

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34 female or male rats ( U.S. EPA, 1991a ) , including suppurative inflammati on in female rats, transitional epithelial hyperplasia in male and female rats, and increased kidney weights in both

sexes of rats ( NTP, 1997
, 1995 ). The se specific effects are considered the result of tert

- butanol

exposure and therefore, relevant to humans. Concerning cancer, α 2u - globulin accumulation is indicated as relatively weak in response to

tert

- butanol exposure and not the sole mechanism responsible for the renal tubule carcinogenicity
observed in male rats. Although CPN and other effects induced by both α 2u - globulin processes and

tert

- butanol play a role in renal tubule nephropathy, the evidence indicates that CPN does not
induce the ren al tubule tumors associated with tert

- butanol exposure in male rats, suggesting that
other

, unknown processes contribut e to renal tumor development. Based on this analysis of
available MOA data, these renal tumors are considered relevant to humans ( U.S. EPA, 1991a ). In addition, an increase in the incidence of thyroid follicular cell adenomas was observed in
male and female mice in a 2

- year drinking water study ( NTP, 1995 ). T hyroid follicular cell hyperplasia was considered a preneoplastic effect ass ociated with the thyroid tumors , and the
incidences of follicular cell hyperplasias were elevated in both male and female B6C3F 1 mice following exposure

. U.S. EPA (1998a) describes the procedures the Agency uses in evaluati ng potential human cancer hazard and dose

- response assessments for chemicals that are animal thyroid carcinogens. The available database is inadequate in four of the five required areas ( U.S. EPA, 1998a )

, suggesting that an antithyroid MOA is not operating in mouse thyroid follicular cell tumorigenesis

. No other MOA s for thyroid tumors were identified , and the mouse thyroid tumors
are considered relevant to human s ( U.S. EPA, 2005a

, 1998a ).

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36 Toxicological Review of tert

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# LITERATURE SEARCH STRATEGY | STUDY SELECTION A ND EVALUATION

A literature search and screening strategy were used to identify literature characterizing
the health effects of tert

- butanol. This strategy consisted of a broad search of online scientific
databases and other sources to identify all potentially pertinent studies. In subsequent steps, references were screened to exclude papers not pertinent to an assessment of the health e ffects 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

- sp ecific search was conducted in four online scientific datab ases , including PubMed, Web of Science

, Toxline, and TSCATS thr ough May 201 5 , using the keywords and limits
described i n Table LS -1. T he overall literature search approach is shown graphically in Figure LS -1.
An additional seven citations were obtained using additional search strategies described in Table
LS -2. After electronically eliminating duplicates from the citations retrieved through these databases, 2,648 unique citations were identified. The resulting 2, 648 citations were screened for pertinence and separated into categories as presented in Figure LS -1 using the title and either abstract or full text , or both, to examine the
health effects of tert

- butanol exposure. The inclusion and exclusion criteria used to screen the references and identify sources of health effect s data are provided in Table LS -3.

• 12 references were identified as “Sources of Health Effects Data” and were considered for data extraction to evidence tables and exposure

- response arrays.

• 200 references were identified as “Sources of Mechanistic and Toxicoki netic Data” and “Sources of Supplementary Health Effects Data”; these included 39 studies describing physiologically based pharmacokinetic (PBPK) models and other toxicokinetic information, 73 studies providing genotoxicity and other mechanistic informatio n, 1 human case report, 74 irrelevant exposure paradigms (including acute, dermal, eye irritation, and injection studies), 6 preliminary toxicity studies, and 7 physical dependency studies. Information from these studies was not extracted into evidence tables; however, these studies were considered as support for assessing tert

- butanol health effects, for example, evaluation of mode of action and extrapolation of experimental animal findings to humans. Additionally, although still considered sources of heal th effects information, studies investigating the effects of acute and direct chemical exposures are generally less pertinent for characterizing health hazards associated with chronic oral and inhalation exposure. Therefore, information from these studies was not considered for extraction into evidence tables. Nevertheless, these studies were still evaluated as possible sources of supplementary health effects information.

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1 • 63 references were identified as “Secondary Literature and Sources of Contextual Information” (e.g., reviews and other agency assessments); these references were retained as additional resources for development of the Toxicological Review.

• 2,373 references were identified as not being pertinent (not on topic) to an evaluation of the he alth effects of tert

- butanol and were excluded from further consideration (see Figure LS -1 for exclusion categories and Table LS -3 for exclusion criteria ). For example, health effect studies of gasoline and tert

- butanol mixtures were not considered pertinent to the
assessment because the separate effects of the gasoline or other chemical components could not be determined. Retrieving a large number of references that are not on topic is a consequence of applying an initial search strategy designed to cast a wide net and to minimize the possibility of missing potentially relevant health effects data.

The complete list of references and the sorting of these materials can be found on the tert butanol project page of the HERO website at https://hero.epa.gov/index.cfm/project/page/project_id/1543

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( Takahashi et al., 1993 ) in the “Supp lementary Studies” category also was based on data from the NTP report. T he interim publi cations and the final NTP report differed

. T he finalized NTP (1995)
report was considered the more complete and accurate presentation of the data; therefore, this
report was included in evidence tables and Cirvello et al. (1995)

, Takahashi et al. (1993)

, and Lindamood et al. ( 1992) were not

. Data from t he remaining 10 references in the “Sources of Health
Effects Data” category were extracted into evidence tables. Supplementary studies that contain pertinent information for the toxicological review and
augment hazard identification conclusions, such as genotoxic and mechanistic studies, studies describing the kinetics and disposition of tert

- butanol absorption and m etabolism, pilot studies, and one case report were not included in the evidence tables. Short

- term and acute studies (including an 18

- day study and a 14

- day study by NTP) using oral and inhalation exposures were performed primarily in rats and did not diff er qualitatively from the results of the longer studies (i.e., ≥30

- day exposure studies). These were grouped as supplementary studies, however, because
the database of chronic and subchronic rodent studies was considered sufficient for evaluating
chronic h ealth effects of tert

- butanol exposure. Additionally, studies of effects from chronic exposure are most pertinent to lifetime human exposure (i.e., the primary characterization 11

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**Selection of Studies for Inclusion in Evidence Tables**

T o summarize the important information systematically from the primary health effects
studies in the tert

- butanol database, evidence tables we re constructed in a standardized tabular format as recommended by NRC (2011)

. Studies were arranged in evidence tables by effect, species,
durat ion, and design

, and not by quality. Of the studies that were retained after the literature search and screen, 12 studies were identified as “Sources of Health Effects Data ” and were considered for

extraction into evidence tables for hazard identification in Chapter 1. Initial review found two
references ( Cirvello et al., 1995 ; Lindamood et al., 1992 ) to be publications of the NTP (1995) data prior to the release of the final National Toxicology Program ( N TP ) report. One publication 16

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1 provided by IRIS assessments) and are the focus of this assessment. Such supplementary studies may be discussed in the narrative sections of Chapter 1 and are described in sections such as the “Mode of Action Analysis” to augment the discussion or presented in appendices, if they provide
additional information. 2

3

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6 **Figure LS -1.**  **S ummary of literature search and screening process for** 
tert

- **butanol.**

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

**- butanol** 

Database (Search Date) Keywords Limits

PubMed tert

- butanol OR 75

- 65

- 0[rn] OR “t

- None (12/20/2012) butyl hydroxide” OR “2

- methyl -2

- (4/17/2014) propanol” OR “trimethyl carbinol” (5/13/2015) OR “t

- butyl alcohol” OR tert

- butanol OR "tert

- butyl alcohol" OR tert

- butyl alcohol[mesh]

Web of Science Topic = (tert

- butanol OR 75

- 65

- 0 OR Refined by: Research Areas = (cell biology OR (12/20/2012) "t

- butyl hydroxide" OR "2

- methyl -2

- respiratory system OR microscopy OR biochemistry (4/17/201 4) propanol" OR "trimethyl carbinol" molecular biology OR gastroenterology OR hepatology (5/13/2015) OR "t

- butyl alcohol" OR "tert

butanol" OR "tert

- butyl alcohol") OR public environmental occupational health OR oncology OR physiology OR cardiovascular system cardiology OR toxicology OR life sciences biomedicine other topics OR hematology OR pathology OR neurosciences neurology OR developmental biology)

Toxline (includes tert

- butanol OR 75

- 65

- 0 [rn] OR t

- Not PubMed TSCATS) butyl hydroxide OR 2

- methyl -2

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

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

- butanol OR (5/13/2015) tert

- butyl alcohol OR tert

- butyl alcohol

TSCATS2 75

- 65

- 0 None (1/4/2013) (4/17/2014) (5/13/2015)

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

**- butanol** 

Approach used Source(s) Date performed Number of additional references identified

Manual search of citations from reviews Review article: McGregor (2010)

. Tert iary

- 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 sear ch of citations from reviews conducted IPCS (1987a)

. Butanol s: Four isomers: 1- butanol, 2

- butanol, tert

- butanol
, isobutanol [WHO EHC]. Geneva, 1/2013 None

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Approach used Source(s) Date performed Number of additional references identified

by other international and federal agencies Switzerlan d: World Health Organization.
OSHA (1992) . Occupational safety and health guideline for tert

- butyl alcohol. Cincinnati, OH: Occupational Safety and Health Administration

. 1/2013 None

**Table LS -3.**  **Inclusion**

**- exclusion**  **criteria**

Inclusion criteria Exclusion criteria
Population • Humans

• Standard mammalian animal models, including rat, mouse, rabbit, guinea pig, monkey, dog • Ecological species*

• Nonmammalian species*

Exposure • Exposure is to tert

- butanol

• Exposure is measured in an environmental medium (e.g., air, water, diet)

• Exposure via oral, inhalation, or dermal
routes • Study population is not exposed to tert

- butanol

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

- butanol)

• Exposure via injection (e.g., intravenous)

• Exposure pattern less relevant to chronic health effects (e.g., acute)

Outcome • Study includes a measure of one or

more health effect endpoints, including effects on the nervous, musculoskeletal, cardiovascular, immune, hematological, endocrin e, respiratory, urinary, and gastrointestinal systems; reproduction; development; liver; kidney; eyes; skin; and cancer

• Physical dependency studies where withdrawal symptoms were evaluated after removal of tert

- butanol treatment

Other Not on topic, including:

• Abstract only, editorial comments were not considered further because study was not potentially relevant

• Bioremediation, biodegradation, or environmental

fate of tert

- butanol, including evaluation of wastewater treatment technologies and methods for remediation of contaminated water and soil

• Chemical, physical, or fuel chemistry studies

• A nalytical methods for measuring/detecting/ remotely sensing tert

- butanol

• Use of tert

- butanol as a solvent or methodology for

testing unrelated to tert

- butanol

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Inclusion criteria Exclusion criteria

• Not chemical specific: Studies that do not involve

testing of tert

- butanol

• Foreign language studies that were not considered

further because, based on title or abstract, judged not potentially relevant

• QSAR studies

*Studies that met this exclusion criterion were not considered a source of health effects data or supplementary

health effects data/mechanistic and toxicokinetic data, but were considered as sources of contextual information.

**Database Evaluation**
For this draft assessment, 12 references report ed on experimental animal studies that comprised the primary sources of health effects data; no studies were identified that evaluated humans exposed to tert

- butanol (e.g., cohort studies, ecological studies). The animal studies were evaluated using the study quality considerations outlined in the Preamble, considering aspects of design, conduct, or reporting that could affect the interpretation of results, overall contribution to the synthesis of evidence, and determination of hazard potential as noted in various EPA guidance
documents ( U.S. EPA, 2005a

, 1998b

, 1996
, 1991b ) . The objective was to identify the stronger, more informative studies based on a uniform evaluation of quality characteristics across studies of similar design. As s tated in the Preamble, studies were evaluated to identify the suitability of the study based on:

• Study design

• Nature of the assay and validity for its intended purpose

• Characterization of the nature and extent of impurities and contaminants of t ert

- butanol administered, if applicable

• Characterization of dose and dosing regimen (including age at exposure) and their adequacy to elicit adverse effects, including latent effects

• Sample sizes and statistical power to detect dose

- related differences or trends

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

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

A dditionally, several general considerations , presented in Table LS -4 , were used in evaluating the animal studies. Much of the key information for conducting this evaluation can be
determined based on study methods and how the study results were reported. Impor tantly, the
evaluation at this stage does not consider the direction or magnitude of any reported effects.

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1 EPA considered statistical tests to evaluate whether the observations might be due to
chance. The standard for determining statistical significance of a response is a trend test or comparison of outcomes in the exposed groups against those of concurrent controls. Studies that did not report statistical testing were identified and, when appropriate, statistical tests were conducted by EPA.
Information on study features related to this evaluation is reported in evidence tables and
documented in the synthesis of evidence. Discussion of study strengths and limitations were
included in the text where relevant. If EPA’s interpretation of a study differs from that of the study authors, the draft assessment discusses the basis for the difference. 2

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The experimental animal studies, comprised entirely of studies performed in rats and mice
, were associated with drinking water, oral gavage

, liquid diets (i.e., maltose/dextrin)

, and inhalation
exposures to tert

- butanol

. With the exception of neurodevelopmental studies, these sources were conducted according to Organisation for Economic Co

- operation and Development Good Laboratory Practice (GLP) guidelines, presented extensive histopathological data, or clear ly
present ed their methodology; thus, these studies are considered high quality. The se studies include
2- year bioassay s using oral exposures in rats and mice; tw o subchronic drinking water studies in rats and one in mice; an inhalation subchronic study in rats and mice; a reevaluation of the NTP (1995) rat data; two oral developmental studies; two inhalation developmental studies; and a single one

- generation reproductive study that also evaluates other systemic effects ( Table LS -5 ). A
more detailed discussion of any methodological concerns that were identified precede s each endpoint evaluated in the hazard identification section. Overa ll, the experimental animal studies of

tert

- butanol involving repeated oral or inhalation exposure were considered to be of acceptable quality, and whether yielding positive, negative, or null results, were considered in assessing the
evidence for health effects associ ated with chronic exposure to tert

- butanol

.

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**Table LS -4.**  **Considerations for**  **evaluation of experimental animal studies**

Methodological feature Considerations (relevant information extracted into evidence tables)

Test animal Suitability of the species, strain, sex, and source of the test animals
Experimental design Suitability of animal age/lifestage at exposure and endpoint testing; periodicity and duration of exposure (e.g., hr/day, day/week); timing of endpoint evaluations; and sample size and experimental unit ( e.g., animals, dams, litters)

Exposure Characterization of test article source, composition, purity, and stability; suitability of the control (e.g., vehicle control); documentation of exposure techniques (e.g., route, chamber type, gavage volume); verifi cation of exposure levels (e.g., consideration of homogeneity, stability, analytical methods)
Endpoint evaluation Suitability of specific methods for assessing the endpoint(s) of interest
Results presentation Data presentation for endpoint(s) of interest (including measures of variability) and for other relevant endpoints needed for results interpretation (e.g., maternal toxicity, decrements in body weight relative to organ weight)

**Table LS -5.**  **Summary of experimental animal d**  **atabase**

Study category Study duration, species/strain, and administration method

Chronic 2

- year study in F344 rats (drinking water) NTP (1995)
2

- year study in B6C3F 1 mice (drinking water) NTP (1995)

Subchronic 13

- week study in B6C3F 1 mice (drinking water) NTP (1995)
13

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

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

- week study in B6C3F 1 mice (inhalation) NTP (1997)
10

- week study in Wistar rat s (drinking wa ter) Acharya et al. (1997)

, Acharya et al. (1995)
Reproductive One

- generation reproductive toxicity study in Sprague

- Dawley rats (gavage) Lyondell Chemical Co. (2004)
Developmental Developmental study (GD 6

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

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

– 18) in C57BL/6J mice (drinking water) Faulkner et al. (1989) Developmental study (GD 1

– 19) in Sprague

- Dawley rat s (inhalation) Nelson et al. (1989)
Neurodevelopmental Neurodevelopmental study (GD 6

– 20) in Swiss Webster mice (diet) Daniel and Evans (1982) Neurodevelopmental study (GD 1

– 19) in Sprague

- Dawley rat s (inhalation) Nelson et al. (1991)

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# 1. HAZARD IDENTIFICATION

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## 1.1. **Overview of Chemical**  **Properties and Toxicokinetics** 

**1.1.1. Chemical**  **Properties**

tert

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

- like odor ( NIOSH, 2005 ; IPCS, 1987a ). tert

- Butanol contains a hydroxyl chemical functional group ; is miscible with alcohol, ether, and other organic solvents ; and is soluble in water ( IPCS, 1987a ). Selected chemical a nd physical properties of tert

- butanol are presented in Table 1-1.

**Table 1**  **-1. Physicochemical properties and chemical identity of**  tert

**- butanol** 

Characteristic Information Reference

Chemical name tert

- Butanol HSDB (2007)
Synonyms/Trade names t- Butyl alcohol; tert

- Butanol; tert

- Butyl alcohol; tButyl hydroxide; 1,1

- Dimethylethanol; NCI

- C55367; 2- Methyl -2- propanol; tertiary Butanol; Trimethyl carbinol; Trimethyl methanol; t- butyl alcohol; TBA HSDB (2007) IPCS (1987b)

Chemical formula C 4 H 10 O 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)

Wat er solubility at 25°C 1 × 10 6 mg/L HSDB (2007)
Octanol/Water Partition Coefficient (Log K OW ) 0.35 HSDB (2007)

Henry’s Law Constant 9.05 × 10 -6 atm -m 3 /mole HSDB (2007)

Odor threshold 219 mg/m 3 HSDB (2007)

Conversion factors 1 ppm = 3.031 mg/m 3 1 mg/m 3 = 0.324 ppm IPCS (1987b)1

2 Characteristic Information Reference

Chemical structure HSDB (2007)

**1.1.2. Toxicokinetics** 

tert

- B utanol is rapidly absorbed following exposure by oral and inhalation routes (see Appendix B, Section B.1.1). Studies in experimental animals indicate that 99% of the compound was
absorbed after oral administration. Comparable blood levels of tert

- butanol and its metabolites
have also been observed after acute oral or inhalation ex posures in rats ( ARCO, 1983 ). In another study ( Faulkner et al., 1989 ) , blood concentrations indicated that absorption was complete at 1.5 hours following oral gavage doses of te rt

- butanol in female mice.

tert

- B utanol is distributed throughout the body following oral, inhalation, and i.v. exposures ( Poet et al., 1997 ; Faulkner et al., 1989 ; ARCO, 1983 ). Following exposure to tert

- butanol in rats,

tert

- butanol was found in kidney, liver, and blood

, with male rats retaining more tert

- butanol than
female rats ( Williams and Borghoff, 2001 )

. A g eneral metabolic scheme for tert

- butanol, illustrating the biotransfo rmation in rats and humans, is shown in Figure 1-1 (see Appendix B.1.3).

H uman data on the excretion of tert

- b utanol comes from studies of methyl tert

- butyl ether ( MTBE ) and ethyl tert

- butyl ether ( ETBE ) ( Nihlén et al., 1998a

, b )

. The half

- life of tert

- butanol in urine following MTBE exposure was 8.1 ± 2.0 hours (average of the 90.1

- and 757

- mg/m 3 MTBE doses); the half

- life of tert

- butanol in urine following ETBE exposure was 7.9 ± 2.7 hours (average
of 104

- and 210

- mg/m 3 ETBE doses). T hese studies reported uri nary levels of tert

- butan ol (not including downstream metabolites) to be less than 1% of administered MTBE or ETBE
concentrations ( Nihlén et al., 1998a

, b )

. Amberg et al. (2000 ) also observed a similar half

- life of
9. 8 ± 1.4 hours after human exposure to ETBE of 170 mg/m 3 . The half

- life for tert

- butanol in rat
urine was 4.6 ± 1.4 hours at ETBE levels of 170 mg/m 3

. A more detailed summary of tert

- butanol toxicokinetics is provided in Appendix B,
Section B.1. 3

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— DO NOT CITE OR QUOTECH 3 glucuronide –O CH 3 CH 3 HO O t-butyl glucuronide
HO CH 3 rats, humans CH 3 [O]

CH 3 OH O 2-hydroxyisobutyric acid CYP450 HO CH 3 H 3 C OH H 3 C rats, O CH 3 humans OH CH 3 t-butanol 2-methyl-1,2-propanediol

rats

- O O CH 3 CH 3

CH 3 CH 3 O

acetone H 2 C formaldehyde

Source: NSF International (2003)

, ATSDR (1996)

, Bernauer et al. (1998)

, Amberg et al. (1999)

, S O O CH 3

CH 3 t-butyl sulfate

and Cederbaum and Cohen (1980)

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21 **Figure 1-1**  **. Biotransformation of** tert

**- butanol in rats and humans.** 

**1.1.3. Description of Toxicokinetic Models** 

No physiologically based pharmacokinetic (PBPK) models have been developed specifically
for administration of tert

- butanol. Some models have been used to stud y tert

- butanol as the primary metabolite after oral or inhalation exposure to MTBE or ETBE. The most recent models for MTBE oral and inhalation exposure include a component for the binding of tert

- butanol to

α 2u - globulin ( Borghoff et al., 2010 ; Leavens and Borghoff, 2009 )

. A more

- detailed summary of the toxicokinetic models is provided in Appendix B, Section B.1.5.
A PBPK model for tert

- butanol was modified by adapting previous models for MTBE and

tert

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

- butanol substantially improved the model fit. The alternative
modification of changing to diffusion

- limited distribution between blood and tissues also improved
the m odel fit, but was considered less biologically plausible. Physiological parameters and partition coefficients were obtained from published measurements. The rate constants for tert

- butanol metabolism and elimination were from a published PBPK model of MTBE with a tert

- butanol subcompartment ( Blancato et al., 2007 ) . Additional model parameters were estimated by calibrating to data sets for i.v., oral

, and inhalation exposures as well as rep eated dosing studies for

tert

- butanol. Overall, the model produced acceptable fits to multiple rat time

- course datasets of

tert

- butanol blood levels following either inhala tion or oral gavage exposures.

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35 In 1996, t he U.S. Agency for Toxic Substances and Disease Registry’s (ATSDR) Toxicological Profile for MTBE ( ATSDR, 1996 ) identified cancer effect levels of MTBE based on data on carcinogenicity in animals

. ATSDR reported that inhalation exposure was associated with kidney
cancer in rats and liver cancer in mice

. ATSDR concluded that oral exposure to MTBE might cause liver and kidney damage, and nervous system effects in rats and mice. The chronic inhalation minimal risk level was derived based on incidence and severity of chronic progressive nephropathy
in female rats ( ATSDR, 1996 )

. In 1997, EPA’s Office of Water concluded that MTBE is carcinogenic to animals and poses a potential carcinogenic po tential to humans based on an increased incidence of Leydig cell adenomas of the testes, kidney tumors, lymphomas, and leukemia in exposed rats ( U.S. EPA, 1997 ). In 1998, t he International Agency for Research on Cancer (IARC) foun d “limited
evidence ” of MTBE carcinogenicity in animals and placed MTBE in Group 3 (i.e., not classifiable as to carcinogenicity in humans) ( IARC, 1999 ). IARC reported that or al exposure in rats resulted in testicular tumors in males and lymphomas and leukemias (combined) in females; inhalation exposure in male r ats resulted in renal tubule adenomas; and inhalation exposure in female mice resulted in hepatocellular adenomas ( IARC, 1999 ). No assessments by national or international agencies or chronic studies for tert

- butyl
acetate are available.



## 1.2. **PRESENTATION AND SYNTHESIS**  **OF EVIDENCE BY ORGAN/SYSTEM** 

**1.2.1. Kidney**  **Effects**

Synthesis of Effects in Kidney

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

to tert

- butanol can affect kidney s in humans or animals. The database examining kidney effects following tert

- butanol exposure contains eight studies from 5 references performed in rats or mice ( Lyondell Chemical Co., 2004 ; Acharya et al., 1997 ; NTP, 1997 ; Acharya et al., 1995 ; NTP, 1995 )

, and
a reevaluation of the rat data from NTP (1995)

, published by Hard et al. (2011) ; no human data are 1 **1.1.4. Chemicals**  **Extensively M etabolized to**  tert

**- Butanol** 

tert

- Butanol is a metabolite of other compounds

, including ETBE, MTBE, and tert

- butyl acetate. Some of the toxicological effects observed in these compounds are attributed to tert
butanol. There are no assessments by national or international health agencies for ETBE. Animal
studies demonstrate that chronic exposure to ETBE is associated with noncancer kidney effects, including increased kidney weights in male and female rats accompanied by increased chronic progressive nephropathy (CPN), urothelial hyperplasia (in males), and increased blood concentrations of total cholesterol, blood urea nitrogen, and creatin ine ( Saito et al., 2013 ; Suzuki et al., 2012 )

. In these studies, i ncreased liver weight and centrilobu lar hypertrophy also were observed in male and female rats exposed to ETBE. Liver adenomas and carcinomas were increased in male rats following 2

- year inhalation exposure ( Saito et al., 2013 ). 2

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. Studies us ing short

- term and acute exposures that examined kidney effects are not included in the evidence tables; they are discussed in the text , however, if they provide data to inform mode of action (MOA) or hazard identification. tert

- Butanol exposure resulted i n kidney effects after both oral (drinking water) and inhalation exposure in both sexes of rats ( Table 1-1
, Table 1-2
, Figure 1-1
, and Figure 1-2 ); s tudies a re arranged in the evidence tables first by effect, then by route, and then duration. The design, conduct, and reporting of each study were reviewed, and each study was considered adequate to provide information pertinent to this assessment. I nterpretation of non

neoplastic kidney endpoints in rats , however, is somewhat complicated by the common occurrence of age

- related , spontaneous lesions characteristic of chronic progressive nephropathy (CPN ) ( NTP,
2015 ; Hard et al., 2013 ; Melnick et al., 2012 ; U.S. EPA,
1991a ); http://ntp.niehs.nih.gov/nnl/urinary/kidney/necp/index.htm ). CPN is more severe in male
rats than in females and is particularly common in the Sprague

- Dawley and Fischer 344 strains. Dietary and hormonal factors play a role in modifying CPN, al though the etiology is largely unknown (see further discussion below)

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K idney weight. Changes in kidney weight (absolute and relative to body weight ) w ere observed in male and female F344 rats following exposures of 13 weeks (oral and inhalation) ( NTP,
1997 ) and 15 months (oral) ( NTP, 1995 ). Lyonde ll Chemical Co. (2004) also reported increases in absolute and relative kidney weight in Sprague

- Dawley rats administered tert

- butanol orally for approximately 10 weeks (tabular data presented in Supplemental Information to this Toxicological
Review ). C hanges were observed in both male and female rats , which exhibit ed strong dose -related

in creases in absolute kidney weight (Spearman’ s rank coefficient > 0.78) following either oral or inhalation exposures ( Figure 1-3). Of the oral ( Figure 1-4 and inhalation ( Fig ure 1-5 ) mouse studies, only inhalation exposure in female mice induced a strong dose

- related increase (Spearman ’s rank coefficient = 0.9) in absolute kidney weights.
Measures of r elative

, as opposed to absolute, organ weight are sometimes preferred because they account for changes in body weight that might influence changes in organ weight ( Bailey et al., 2004 ) , although potential impact should be evaluated

. For tert

- butanol, body weight in exposed animals noticeably decreased at the high doses relative to controls in the oral 13

- week and
2- year studies NTP (1995)

. In this case, the decreased body weight of the ani mals affects the relative kidney weight measure s, resulting in an artificial exaggeration of changes. Thus, absolute weight was determined the more reliable measure of kidney weight change for this assessment

. Additionally, a recent analysis indicates that i ncreased absolute, but not relative, subchronic kidney weights are significantly correlated with chemically induced histopathological findings in the kidney in chronic and subchronic studies ( Craig et al., 2014 ). Although relative and absolute kidney weight data are both presented in exposure

- response arrays (and in evidence tables in Supplemental Information)

, the absolute measu res were considered more informative for
determin ing tert

- butanol hazard potential. 9

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1 Kidney histopathology. Treatment

- related histopathological changes were observed in the kidneys of male and female F344 rats following 13

- week and 2

- year oral exposures ( NTP, 1995 )
and male F344 rats following a 13

- week inhalation exposure ( NTP, 1997 ). Similarly, male Wistar rats exposed for approximately 10 weeks exhibited an increase in histopathological kidney lesions ( Acharya et al., 1997 ; Acharya et al., 1995 ). B6C3F 1 mice , however, did not exhibit histopathological changes when exposed for 13 weeks and 2 years via the oral route ( NTP, 1995 ) and 13 weeks via
the inhalation route ( NTP, 1997 ). More specific details on the effects observed in rats, reported by NTP (1997

, 1995) and Acharya et al. (1997) ; ( 1995 ) are described below. Nephropathy and severity of nephropathy were reported in male and female rats in the
13

- week oral studies ( NTP, 1995 ). T he nephropathy was characterized as “...a spontaneous background lesion...typically co nsist[ing] of scattered renal tubules lined by basophilic regenerating tubule epithelium.” ( NTP, 1995 ). NTP (1995) noted that the increase in severity of nephropathy was related to tert

- butanol and “characterized by an increase in the number and size of foci of regeneration.” The severity of nephropathy increased, compared with controls, in the
13

- w eek male rats, which exhibited nephropathy in 94% of all exposed animals and 70% of 2

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

treatment

- related kidney histopathology w as reported in the female rats exposed through
inhalation ( NTP, 1997 )

. In the 2

- year oral study by NTP (1995) , nephropathy was reported at 15 months and 2

years. The NTP (1995) characterization of nephropathy following chronic exposure inclu ded multiple lesions: “thickened tubule and glomerular basement membranes, basophilic foci of regenerating tubule epithelium, intratubule protein casts, focal mononuclear inflammatory cell aggregates within areas of interstitial fibrosis and s c arring, and glomerular sclerosis.” At 15 months, male and female rats (30/30 treated ; 10 / 10 controls ) had nephropathy

, and the severity scores ranged from minimal to mild. At 2 years, male and female rats (149/150 treated; 49/50
controls ) also had nephrop athy , and although t he severity was moderate in the control males and
minimal to mild in the control females , severity increased with tert

- butanol exposure in both sexes ( NTP, 1995 )

. The lesions collectively described by NTP (1997

, 1995) as nephropathy and noted to be common spontaneous lesions in rats, are consistent with CPN

. The effects characterized as CPN are
related to age and not considered histopathological manifestations of chemically induced toxicity 9

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16 controls

. Conversely

, lesion severity was unchanged in the females

, although nephropathy incidence significantly increased with tert

- butanol exposure

. In the 13

- week inhalation study ( NTP,
1997 ) , nephropathy w as present in all but two male rats, including controls

. NTP (1997) characterized the reported chronic nephropathy in control male rats as “ 1 to 3 scattered foci of regenerative tubules per kidney section. Regenerative foci were characterized by tubules with cytoplasmic basophilia, increased nuclear/cytoplasmic ratio, and occasionally thickened basement membranes and intraluminal protein casts. ” I n exposed groups, the severity generally increased 17

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1 [ see U.S. EPA (1991a) , p

. 35 for further details and a list of the typical observable histopathologic al
features of CPN ]. T hese lesions , however, ar e frequently exacerbated by chemical treatment ( NTP,
1997 ), as evidenced by the dose

- related increases in severity of the nephropathy compared to
female and male rat controls

. The chemical

- related changes in nephropathy severity are included in the consideration of hazard potential. NTP (1995) observed other kidney lesions

, described as being associated with nephropathy but diagnosed separately. Renal mineralization is defined by NTP (1995) as “ focal mineral deposits primarily at the corticomedullary junction. ” This mineralization is distinct from linear mineralization, which is considered a lesion characteristic of α 2u - globulin nephropathy (for further
discussion of this particular lesion

, see Mode of Action Analysis

— Kidney Effects ) . The mineralization is characterized as distinct linear deposits along radiating medullary collecting ducts. An increased incidence of linear mineralization was limited to exposed males in the 2

- year o ral study ( NTP,
1995 ). 2

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control and low

- dose groups of 13

- week males and the dose

- response relationships the tert

butanol

- exposed males exhibited in the 13

- week ( NTP, 1997
, 1995 ) and 2

- year studies ( NTP,
1995 ). Furthermore, due to the overwhelming presence of mineralization in the control and treated female rats, the contribution, if any, of tert

- butanol to the formation of this lesion in females could
not be determined. Thus

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14 Renal mineralization was observed in essentially all female rats at all reported treatment
durations. A dose

- related, increased incidence of mineralization was reported in male rats at the
13

- week, 15

- mo nth, and 2

- year oral evaluations ( NTP, 1995 ). NTP (1995) describes focal, medullary mineralization as being associated with CPN but notes that focal mineralization is

“ usually more prominent in untreated females than in untreated males ,” which is consistent with the widespread appearance of this lesion in females . This description, however, is inconsistent with
the observation in this and other database s that a ge

- related nephropathy (i.e., CPN) is generally more prevalent and more severe in male rat s compared to females ( U.S. EPA, 1991a ). The

association of mineralization with CPN is unclear

, considering the lack of spontaneous lesions in the 16

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29 Two other histological kidney lesions observed in male and female rats are suppurative inflammation and transitional epit helial hyperplasia. These lesions were observed in the 2

- year oral NTP (1995) study. Although NTP (1995) describes these lesions as related to the nephropathy (characterized above as common and spontaneous, and considered CPN), that suppurative inflammation and transitional epithelial hyperplasia exhibited incidence patterns different from
those reported for nephropathy is notable

. Incidence of suppurative inflammation in female rats was low in the control group and increased with dose , with incidences ≥ 24% in the two highest
dose group s , compared with controls. In comparison, 20% of the control males exhibited suppurati ve inflammation

, and the changes in incidence were not dose related (incidences ranging
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1 inflammation; considering the responses in the females, however, the e ffect appears to be predominantly treatment related. Suppurative i nflammation was not observed in the animals of the
13

- week oral ( NTP, 1995 ) or inhalation study ( NTP, 1997 ) , which both report ed nephropathy (as CPN), providing further support that this lesion is not specifically related to the nephropathy. Transitional epithelial hyperplasia was observed in both male and female rats exposed orally ( NTP, 1995 ) . In the control males, 50% of the animals exhibited transitional epithelial hyperplasia and the incidence and severity increased with dose. O nly the mid

- and high

- dose
females , however, exhibited dose

- related increases in incidence and severity of transitional epithelial hyperplasia; this lesion was not reported in the control or low

- dose females. NTP (1995) described transitional epithelial hyperplasia as increased layers of the transitional epithelial lining of the renal pelvis; study authors noted no progression of this hyperplas tic lesion to neoplasia. The relatively high background in male controls (i.e., 50%) suggests some potential influence, other
than tert

- butanol treatment, on this effect. T he absence of this effect in female control and low

- dose
animals and the dose -relate d increases in both males and females, however, indicate that similar to
the suppurative inflammation, the transitional epithelial hyperplasia is predominantly treatment 2

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study authors noted a s potentially indicative of oxidative damage. Acharya et al. (1997) ; Acharya et al. (1995) used one dose and a control group and did not report incidences. The increased tubule degeneration and glomerular vacuolation could be charact erized as tubular atrophy and glomerular hyalinization, respectively, consistent with CPN; however, without quantitative information

, examin ing the differences between the control and treated animals to determine if CPN plays a role in development of these effects is not possible

. Although based on the noted appearance of the
effects in the treated animals compared with controls, the effects likely are treatment related. 9

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16 related. Transitional epithelial hyperplasia should not be confused with another lesion noted at the
2- year evaluation, renal tubul e hyperplasia, which was considered preneoplastic (for further details regarding this type of hyperplasia, see the discussion under kidney tumors below).
Additional histo patho logical changes

, including increase d tubular degeneration, degeneration of the basement membrane of the Bowman’s capsule, diffused glomeruli, and glomerular vacuolation were noted in a 10

- week study in male Wistar rats ( Acharya et al., 1997 ; Acharya et al., 1995 ). A decrease in glutathione in the kidney accompanied these changes, which the 17

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

st udies discussed above. S ome changes occurred in urinalysis parameters (e.g., decreased urine
volume and increased specific gravity)

, accompanied by reduced water consumption

, and thus might not be related to an effect of kidney function ( NTP, 1995 ). Kidney tumors. The kidney is also a target organ for cancer effects ( Table 1-3
, Figure 1-1 ).
Male F344 rats had an increased incidence of combined renal tubule adenomas or carcinomas in
the 2

- year oral bioassay ( Hard et al., 2011 ; NTP, 1995 ). The increase in tumors from control was similar in the low

- and high

- dose groups and highest in the mid

- dose group

. Overall, t umor increases were statistically significant in trend testing

, which account ed for mortality ( p ≤ 0.018)

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1 M ortality increased with increasing exposure ( p = 0.001) ; increased mortality alone, however, does not account for the highest tumor incidence occurring at the mid dle dose. Increases in incidence and severity of r enal tubule hyperplasia also were observed in male

rats. NTP (1995) stated that “[t]he pathogenesis of proliferative lesions of renal tubule epithelium is generally considered to follow a progression from hyperplasia to adenoma to carcinoma ( Hard,
1986 ).” Similarly, EPA considered the renal tubule hyperplasia to be a preneoplastic effect
associated with the renal tubule tumors. Renal tubule hyperplasia was found in one high

- dos e female ( NTP, 1995 ) ; no increase in severity was observed

. This effect in females , which was not considered toxicologically significant

, is not discussed further

. Two renal tubular adenocarcinomas
in male mice also were reported ( NTP, 1995 )

, one each in the low

- and high

- dose groups, but were not considered by NTP to be “ biologically noteworthy changes”; thus the tumors in mice are not
discussed furt her

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- year study to determine if additional histopathological changes could be identified to inform the MOA for renal tubule tumor development ( Hard et al., 2011 ). In all cases, working group members were blinded to treatment groups, and used guidelines published by Hard and Wolf (1999) and refinements reported by ( Hard and Seely, 2006 ); Hard and Seely (2005) and Hard (2008)

. The group’s report and analysis by Hard et al. (2011) confirmed the NTP findings of 14

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19 renal tubule hyperplasia and ren al tubule tumors in male rats at 2 years. In particular, they reported similar overall tumor incidences in the exposed groups. Hard et al. (2011) , however

, reported fewer renal tubule adenomas and carcinomas in the control group than in the original NTP study. As a result, all treated groups had statistically significant increases in renal tubule adenomas and carcinomas (combined) when compared to controls. Add itionally, Hard et al. (2011) considered
fewer tumors to be carcinomas than did the original NTP study. Results of both NTP (1995) and the reanalysis by Hard et al. (2011) are included in Table 1-3 and Figure 1-1

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Male rats

1 10 100 1000Absolute kidney weight (% change)

0 10 20 30 40 50

Oral exposure Inhalation exposure rho= 0.78 (all) rho= 0.89 (oral) rho= 0.80 (inhalation) Female rats

tert-butanol blood concentration (mg/l) 1 10 100 1000 10000

rho= 0.78 (all) rho= 0.72 (oral) rho= 0.9 (inhalation)

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— DO NOT CITE OR QUOTE **Figure 1-2.**  **Comparison of absolute kidney weight change in male and female** 
**rats across oral and inhalation exposure**  **based on**  **internal blood concentration.**  **Spearman rank correlation coefficient (rho) was calculated to**  **evaluate the direction of a monotonic association (e.g.**  **, positive value**  **= positive association) and the strength of association.** 1
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11 Male mice

Administered dose (mg/kg-day) 0 2000 4000 6000 8000Absolute kidney w eight (% change)

-4 -2 0 2 4 6 8 10 12 14 Female mice

Administered dose (mg/kg-day) 0 2000 4000 6000 8000 10000 12000 rho= -0.1 rho= 0.9

**Figure 1-3**  **. Comparison of absolute kidney weight change in male and female**  **mice following oral exposure based on administered concentration. Spearman**  **rank correlation coefficient (rho) was calculat ed to evaluate the direction of a**  **monotonic association (e.g. ,**  **positive value =**  **positive association) and the strength of association.** 

Male mice

Administered dose (mg/m 3 ) 0 1000 2000 3000 4000 5000 6000Absolute kidney w eight (% change)

-12 -10 -8 -6 -4 -2 0 2 4 6 Female mice

Administered dose (mg/m 3 ) 0 1000 2000 3000 4000 5000 6000 7000 rho= 0.2 rho= -0.1

**Figure 1-4.**  **Comparison of absolute kidney weight change in male and female**  **mice following inhalation exposure based on administered concentration.**  **Spearman rank correlation coefficient (rho) was calculated to evaluate the**  **direction of a monotonic association (e.g. ,**  **po sitive value**  **= positive**  **association) and the strength of association.** Toxicological Review of tert

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**Table 1**  **-2. C**  **hanges in kidney histopathology in animals following exposure to**
tert

**- butanol** 

Reference and study design Results
Acharya et al. (1997) ↑ tubular degeneration, degeneration of the basement membrane of the Acharya et al. (1995) Bowman’s capsule, diffused glomeruli, and glomerular vacuolation (no incidences reported) Wistar rat; 5

– 6 males/treatment Drinking water (0 or 0.5%), 0 or ↓ kidney glutathione (~40%)* 57 5 mg/kg -d 10 weeks
NTP (1995) Incidence (severity):

F344/N rat; 10/sex/treatment Males Females Drinking water ( 0, 2.5, 5, 10, 20, Dose Minerali

- Nephro

- Dose Minerali

- Nephro

­ or 40 mg/mL) (mg/kg

- d) zation b pathy c (mg/kg

- d) zation b pathy c M: 0, 230, 490, 840, 1,520, 3,610 a mg/kg -d 0 0/10 7/10 (1.0) 0 10/10 (1.7) 2/10 (1.0) F: 0, 290, 590, 850, 1,560, 3,620 a mg/kg -d 13 weeks 230 0/10 10/10 (1.6*) 290 10/10 (2.0) 3/10 (1.0)

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

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

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

3,610 a 4/10*(1.0) 7/10 (1.1) 3,620 a 6/10 (1.2) 7/10* (1.0)
NTP (1995) Study authors indicated no treatment

- related changes in kidney histopathology (histopathological data not provided for the 13

- week study) B6C3F 1 mouse; 10/sex/treatment Drinking water (0, 2.5, 5, 10, 20, or 40 mg/mL ) M: 0, 350, 640, 1,590, 3,940, 8,210 a mg/kgd F: 0, 500, 820, 1,660, 6,430, 11,620 a mg/kgd 13 weeks

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Reference and study design Results
NTP (1995)

F344/N rat; 60/sex/treatment (10/sex/treatment evaluated at 15 months interim) Drinking water (0, 1.25, 2.5, 5, 10 Incidence (severity): Males

Dose (mg/kg

- d) Mineralization b (interim) Mineralization b (terminal) Linear mineralization b (terminal)
mg/mL) M: 0, 90, 200, 420 a mg/kg -d F: 0, 180, 330, 650 a mg/kg -d 2 years 0 1/10 (1.0) 26/50 (1.0) 0/50

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

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

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

Dose (mg/kg

- d) Transitional epithelial hyperplasia Nephropathy c severity

0 25/50 (1.7) 3.0

90 32/50 (1.7) 3.1

200 36/50* (2.0) 3.1

420 a 40/50* (2.1) 3.3*

Females

Dose (mg/kg

- d) Mineralization b
Interim Mineralization b 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 a 10/10 (2.8) 50/50 (2.9) 17/50*

Dose (mg/kg

- d) Transitional epithelial hyperplasia Nephropathy c severity

0 0/50 1.6

180 0/50 1.9*

330 3/50 (1.0) 2.3*

650 a 17/50*(1.4) 2.9*
NTP (1995)

B6C3F 1 mouse; 60/sex/treatment Drinking water (0, 5, 10, or 20 mg/mL) M: 0, 540, 1,040, or 2,070 a mg/kg -d No treatment

- related changes in kidney related histopathology observed

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Reference and study design Results
F: 0, 510, 1,020, or 2,110 mg/kg

d 2 years

NTP (1997) Male

F344/N rat; 10/sex/treatment Inhalation analytical concentration: 0, 134, 272, 542, 1,080, or 2,101 ppm (0, 406, 824, 1,643, 3,273 or 6,368 mg/m 3 ) (dynamic whole

- body chamber) 6 hr/d, 5 d/wk 13 weeks Generation method (Sonimist Ultrasonic spray nozzle nebulizer), analytical concentration and method were reported Concentration (mg/m 3 ) Incidence of chronic nephropathy d Average severity of chronic nephropathy

0 9/10 1.0

406 8/10 1.4

824 9/10 1.4

1,643 10/10 1.6

3,273 10/10 1.9

6,368 10/10 2.0

Females: no treatment

- related changes in kidney related histopathology observed Severity categories: 1= minimal, 2= mild. No results from statistical tests reported
NTP (1997)

B6C3F 1 mouse; 10/sex/treatment Inhalation analytical concentration: 0, 134, 272, 542, 1,080, or 2,101 ppm (0, 406, 824, 1,643, 3,273 or 6,368 mg/m 3 ) (dynamic whole

- body chamber) 6 hr/d, 5 d/wk 13 weeks Generation method (Sonimist Ultrasonic spray nozzle nebulizer), analytical concentration and method were reported No treatment

- related changes in kidney related histopathology observed

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a The high

- dose group ha d an increase in mortality.

b Mineralization defined in NTP (1 995) as focal mineral deposits, primarily at the corticomedullary junction. Linear

mineralization was defined as foci of distinct linear deposits along radiating medullary collecting ducts; linear mineralization not observed in female rats.Toxicological Review of tert

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1 2 3 4 5 6

7 8

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10 c Nephropathy defined in NTP (1995) as lesions including thic kened tubule and glomerular basement membranes, basophilic foci of regenerating tubule epithelium, intr a tubule protein casts, focal mononuclear inflammatory cell aggregates within areas of interstitial fibrosis and scar r ing, and glomerular sclerosis.

d Ne phropathy characterized in NTP (1997) as scattered foci of regenerative tubules (with cytoplasmic basophilia,

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

Note: Conversions from drinking water concentrations to mg/kg

- da y performed by study authors. Conversion from ppm to mg/m 3 is 1 ppm = 3.031 mg/m 3

.

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

**- butanol** 

Results

Renal tubule hyperplasia Male (standard and extended Renal tubule
Dose evaluation Renal tubule adenoma (mg/kg

- d) combined) adenoma (single) (multiple)

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

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

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

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

Renal tubule adenoma (single
Dose Renal tubule or multiple) or (mg/kg

- d) carcinoma carcinoma

0 0/50 8/50

90 2/50 13/50

200 1/50 19/50*

420 a 1/50 13/50

Female Renal tubule

Dose Renal tubule Renal tubule adenoma (mg/kg

- d) hyperplasia adenoma (single) (multiple)

0 0/50 0/50 0/50

180 0/50 0/50 0/50

330 0/50 0/50 0/50

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

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NTP (1995)

F344/N rat; 6 0/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 420 a mg/kg -d F: 0, 180, 330, or 650 a mg/kg -d 2 years1 2 3 4 Toxicological Review of tert

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Reference and study design Results

Dose (mg/kg

- d) Renal tubule carcinoma Renal tubule adenoma (single

or multiple) or

carcinoma

0 0/50 0/50

180 0/50 0/50

330 0/50 0/50

650 a 0/50 0/50

Based on standard and extended evaluations (combined). Results do not include the animals sacrificed at 15 months.
Hard et al. (2011)

R eanalysis of the slides from male rats (all slides in controls and high

- dose groups of males and females, and slides from all other males with renal tumors) in the NTP (1995) study (see above) Male Renal tubule adenoma

(single) Renal tubule adenoma (multiple) Renal tubule carcinoma Renal tubule adenoma (single or multiple) or carcinoma Dose (mg/kg

- d)

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

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

200 9/50 9/50 0/50 18/50*

420 9/50 3/50 1/50 12/50*
NTP (1995)

B6C3F 1 mouse; 60/sex/treatment Drinking water (0, 5, 10, or 20 mg/mL) M: 0, 540, 1,040, or 2,070 a mg/kg -d F: 0, 510, 1,020, or 2,110 mg/kg

d 2 years No increases in kidney

- related tumors. Two renal tubule adenocarcinomas, one in the low

- dose and one in the high

- dose groups, were observed in male mice. These tumors were not considered treatment related.

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

a The high

- dose group h ad an increase in mortality.

Note: Conversions from drinking water concentrations to mg/kg

- d performed by study authors.

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Sou rces: (A) Acharya et al. (1997) ; ( 1995 ) ; (B) Hard et al. (2011) *; (C) Lyondell Chemical Co. (2004) (D) NTP (1995) ; *reanalysis of NTP (1995)

. **Figure 1-5.**  **Exposure response array for kidney effects following oral expos ure**  **to** tert

**- butanol.** 

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 = exposures at which the endpoint was reported statistically significant by study authors  = exposures at which the endpoint was reported not statistically significant by study aut hors

Relative weight; F Rat

Absolute/relative weight; M Mouse

Absolute weight; F Mouse

Relative weight; F Mouse

100 1,000 10,000 

### Concentration ( mg/m3) Absolute weight; M Rat

Relative weight; M Rat

Absolute weight; F Rat

So urce: NTP (1997)

. **Figure 1-6.**  **Exposure**

**- response array of kidney effects following inhalation**  **exposure to** tert

**- butanol (**  **13**

**- week studies,**  **no chronic studies available).**

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37 Mode of Action Analysis

— Kidney Effects

a) α 2u - Globulin

- Associated Renal Tubule Nephropathy and Carcinogenicity

One disease process to consider when interpreting kidney effects in rats is related to the
accumulation of α 2u - globulin protein. α 2u - Globulin, a member of a large superfamily of low
molecular

- weight proteins, was first characterized in male rat urine. Such proteins have been
detected in various tissues and fluids of most mammals (including humans), but the particular
isoform of α 2u - globulin commonly detecte d in male rat urine is considered specific to th at sex and species

. Exposure to chemicals that induce α 2u - globulin accumulation can initiate a sequence of
histo pathological events lead ing to kidney tumor igenesis . Because α 2u -globulin- associated renal
tubul e nephropathy and carcinogenicity occur r ing in male rats are presumed not to be relevant for assessing human health hazards ( U.S. EPA, 1991a ) , evaluat ing the data to determine if α 2u - globulin plays a role is important

. The role of α 2u - globulin accumulation in the development of renal tubule nephropathy and carcinogenicity observed following tert

- butanol exposure was evaluated using the U.S. EPA (1991a) Risk Assessment Forum Technical panel report, Alpha 2u - Globulin: Association with

the evidence reported in these studies is specific to α 2u - globulin accumulation , it is presented in this

section ; it was not included in the animal evidence tables in the previous section. The hypothesized sequence of α 2u - globulin renal tubule nephropathy, as described by U.S. EPA (1991a)

, is as follows. Chemicals that induce α 2u - globulin accumulation do so rapidly.

α 2u -G lobulin accumulat ing i n hyaline droplets is deposited in the S2 ( P2 ) segment of the proximal
tubule within 24 hours of exposure. Hyaline droplets are a normal constitutive feature of the mature male rat kidney; they are particularly evident in the S2 ( P2 ) segment of the proxima l tubule
and contain α 2u - globulin ( U.S. EPA, 1991a ). Abnormal increases in hyaline droplets have more than one etiology and can be associated with the accumulation of different pr oteins. As hyaline droplet deposition continues, single

- cell necrosis occurs in the S2 ( P2 ) segment , which leads to exfoliation
of these cells into the tubule lumen within 5 days of chemical exposure. In response to the cell loss, cell proliferation occurs in the S2 ( P2 ) segment after 3 weeks and continues for the duration of the exposure. After 2 or 3 weeks of exposure, the cell debris accumulates in the S3 ( P3 ) segment of the proximal tubule to form granular casts. Continued chemical exposure for 3 to 12 months leads to
the formation of calcium hydroxyapatite in the papillae which results in linear mineralization. After Chemically Induced Renal Toxicity and Neoplasia in the Male Rat

. This report provides specific guidance for evalu ating renal tubule tumors in male rats that are related to chemical exposure for the purpose of risk assessment, based on an examination of the potential involvement of

α 2u - globulin accumulation.
Studies in the tert

- butanol database evaluated and reported effects on the kidney, providing
some evidence to evaluate this MOA. Additionally, s everal studies were identified that specifically
evaluated the role of α 2u - globulin in tert

- butanol

- induced renal tubule nephropathy and carcinogenicity ( Borghoff et al., 2001 ; Williams and Borghoff, 2001 ; Takahashi et al., 1993 ). Because Toxicological Review of te r t

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1 or more years of chemical exposure, these lesions can result in the induction of renal tubule
adenomas and carcinomas ( Figure 1-7). U.S. EPA (1991a) identifie d two questions that must be addressed t o determine the extent
to which α 2u - globulin

- mediated processes induce renal tubule nephropathy and carcinogenicity. First, whether the α 2u - globulin process is occurring in male rats and is involved in renal tubule tumor development must be determined

. S econd, whether the renal effects in male rats exposed to

tert

- butanol are solely due to the α 2u - globulin process also must be determined

. U.S. EPA (1991a) stated the criteria for answering the first question in the affirmative are as
follows:

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

2) the protein in the hyaline droplets in treated male rats is α 2u globulin (i.e., immunohistochemical evidence), and

3) several (but not necessarily all) additional steps in the pathological sequence appear in treated male rats as a function of time, dose, and progressively increasing severity consistent with the understan ding of the underlying biology , as described above, and illustrated in Figure 1 -7.

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

- 8
and 1 -9, and are evaluated below.

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— DO NOT CITE OR QUOTE **Figure 1-7.**  **T emporal pathogenesis of**  **α 2u**  **- globulin** 

**- associated nephropathy in**  **male rats.** α 2u -G lobulin synthesized in the livers of male rats is delivered to the kidney, where it can accumulate in hyaline droplets and be retained by epithelial cells lining the S2 (P2) segment of the proximal tubules. Renal pathogenesis following continued tert

- butanol exposure and increasing droplet accumulation can progress step

- wise from increasing e pithelial cell damage, death and dysfunction leading to the formation of granular casts in the corticomedullary junction, linear mineralization of the renal papilla e , and carcinogenesis of the renal tubular epithelium

. Adapted from Swenberg and Lehman

- McKeeman (1999) and U.S. EPA (1991a)

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**Table 1-4.**  **Su m**  **mary of data on the α 2u**  **- globulin**  **proce ss in male rats exposed** 
**to** tert

**- butanol** 

Duration Dose Results Comments Reference
1) Hyaline droplets are increased in size and number
10 d (inhalation) 0, 758, 1,364, 5,304 mg/m 3 + stat sig at 5,304 mg/m 3 ; stat sig trend Borghoff et al. (2001)

13 wk (inhalation) 0, 3,273, 6,368 mg/m 3 – N TP (1997) a
13 wk (oral) 0, 230, 490, 840, 1,520, 3,610 mg/kg

- d (+) observed in all but highest dose group NTP (1995)

2) The protein in the hyaline droplets is α 2u globulin
10 d (inhalation) 0, 758, 1,364, 5,304 mg/m 3 + stat sig at 5,304 mg/m 3 ; stat sig trend Borghoff et al. (2001)

12 h (elapsed time following single oral dose) 0, 500 mg/kg + Williams and Borghoff (2001)

3) Several (but not necessarily all) additional steps in the pathological sequence are present in male rats, such as:
a) Subsequent cytotoxicity and single

- cell necrosis of tubule epithelium, with exfoliation of degenerate epithelial cells
10 wk (oral) 0, 575 mg/kg

- d (+) degeneration of renal tubules reported Acharya et al. (1997)

13 wk (oral) 0, 230, 490, 840, 1,520, 3,610 mg/kg

- d – NTP (1995)

b) Sustained regenerative tubule cell proliferation (NOTE: The positive studies below reported cell proliferation but did not observe necrosis or cytotoxicity; therefore, it cannot be assumed that the results indicate regenerative proliferation is occurring.)
10 wk (oral) 0, 575 mg/kg

- d – Acharya et al. (1997)
10 d (inhalation) 0, 758, 1,364, 5,304 mg/m 3 + stat sig at all doses; stat sig trend Borghoff et al. (2001)

13 wk (oral) 0, 230, 490, 840, 1,520, 3,610 mg/kg

- d + elevated at 840 mg/kg

- d; stat sig at 1,520 mg/kg

- d NTP (1995)

c) Development of intraluminal granular casts from sloughed cellular debris, with consequent tubule dilation
13 wk (oral) 0, 230, 490, 840, 1,520, 3,610 mg/kg

- d – ; (+) b NTP (1995) ; Hard et al. (2011) c
2 yr (or al) 0, 90, 200, 420 mg/kg

- d – NTP (1995) ; Hard et al. (2011) d

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Duration Dose Results Comments Reference
d) Linear mineralization of tubules in the renal papilla
13 wk (oral) 0, 230, 490, 840, 1,520, 3,610 mg/kg

- d –

2 yr (oral) 0, 90, 200, 420 mg/kg

- d +; (+) all doses stat sig NTP (1995) ; Hard et al. (2011) c
NTP (1995) ; Hard et al. (2011) d
e) Foci of tubular hyperplasia

2 yr (oral) 0, 90, 200, 420 mg/kg -d + stat sig trend at all doses; stat sig at 420 mg/kg

- d NTP (1995)

+ = Statistically significant change reported in one or more treated groups. (+) = E ffect was reported in one or more treated groups, but statistics not reported.

– = No statistically significant change reported i n any of the treated groups.

a NTP (1997) did not observe any effects consistent with α 2u - globulin nephropathy

. b Precursors to granular casts reported.

c Reanalysis of hematoxylin and eosin

- stained kidney sections from all male control and 1,520 mg/kg

- d groups, as

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

- wk study from NTP (1995)

. d Reanalysis of slides for all males in the control and 420 mg/kg

- day dose group and all animals with renal tubule

tumors from 2

- yr NTP (19 95)

. Protein casts reported, not granular casts

.

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 = exposures at which the endpoint was reported statistically significant by study authors  = exposures at which the endpoint was reported not statistically significant by study authors

× = ex posures at which all animals were dead and unable to be examined for the endpoint

● = exposures at which effect was observed but statistics not reported

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

** NTP (1995) 13

- wk study reported kidney mineralization but not linear mineralization

. **Figure 1-8.**  **Exposure**

**- response array for effects**  **potentially associated with**

**α 2u**  **- globulin renal tubule nephropathy and**  **tumors in male rats after oral exposure to**  tert

- **butanol.**

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 = exposures at which the endpoint was reported statistically significant by study authors  = exposures at which the endpoint was reported not statistically significant by study authors

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— DO NOT CITE OR QUOTE **Figure 1-9.**  **Exposure**

**- response array for effects**  **potentially associated with**

**α 2u**  **- globulin renal tubule nephropathy and tumors in male rats after**  **inhalation exposure to**  tert

- **butanol.**Question One: Is the α 2u

globulin process occurring in male rats exposed to tert

- butanol? (1) The first criterion to consider is whether hyaline droplets are increased in size and
number in male rats. As noted above, the excessive accumulation of hyaline droplets can appear quickly, within 1 or 2 days, and per sist throughout chronic exposures, although the severity begins
to decline around 5 months ( U.S. EPA, 1991a )

. A statistically significant positive trend in the
accumulation of large protein droplets with crystalloid protein structures was observed in kidneys
of male rats exposed to inhalation concentrations of 7 58 , 1,3 64 , and 5,3 04 mg/m 3 tert

- butanol for 6 hr/day for 10 days ( Borghoff et al., 2001 ) . These droplets were small and minimally present in
control male rats and were not observed in female rats. Similarly, data from the 13

- week NTP oral study ( NTP, 1995 ; Takahashi et al., 1993 ; Lindamood et al., 1992 ) demonstrated an increase in the
accumulation of hyaline droplets

. T he lowest dose of 230 mg/kg

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

- day)

N TP (1997) , however, found no difference between the control and treatment groups stained for hyaline droplet formation in male rats exposed to 0, 3,273, or 6,368 mg/m 3 tert

- butanol
via inhalation for 13 weeks; in fact, this study did not report any othe r lesions that could be specifically associated with α 2u - globulin nephropathy in male rats. These results from NTP (1997)

, which are inconsistent with the findings of both Borghoff et al. (2001) and NTP (1995) , do not appear to be due to differences in dose

. Comparison of the oral and inhalation studies on the basis
of tert

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

- week exposure in the range of the NTP (1995) doses of 490

– 840 mg/kg

- day lead s to the same averag e blood concentration as 6

- hr/day, 5 day/week inhalation exposures to 3, 273 –6, 368 mg/m 3 . The
absence of similar histo pathological findings in the 13

- week inhalation NTP (1997) study compared
to those reported in the two oral studies is not understood, but might be indicative of the strength
of tert

- butanol to induce

, consistently

, α 2u - globulin nephropathy. The results from the two other
studies ( Borghoff et al., 2001 ; NTP, 1995 ) indicate that hyaline droplets increase in size and number in male rats following tert

- butanol exposures. Therefore, the available dat a are sufficient to fulfill
the first criteri on that hyaline droplets are increased in size and number in male rats

. (2) The second criterion to consider is whether the protein in the hyaline droplets in male

rats is α 2u - globulin. Accumulated hyaline droplets with an α 2u -globulin etiology can be confirmed by using immunohistochemistry to identify the α 2u - globulin protein. Two short

- term studies
measured α 2u - globulin immunoreactivity in the hyaline droplets of the renal proximal tubular epithelium ( Borghoff et al., 2001 ; Williams and Borghoff, 2001 ) . Following 10 days of inhalat ion

exposure, Borghoff et al. (2001) did not observe an exposure

- related increase in α 2u - globulin using immunohistochemical staining. W hen using an enzyme

- linked immunosorbent assay (ELISA), a

more sensitive method of detecting α 2u - globulin, h owever, a statistically significant positive had a higher accumulation of droplets with angular, crystalline structures that was similar in incidence and severity among these dose groups. No droplets were observed in female rats or in
mice.

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events in the histo pathological sequence associated with α 2u -globulin nephropathy appear in male

rats in a manner consistent with the understanding of α 2u - globulin pathogenesis . Evidence of cytotoxicity and single

- cell necrosis of the tubule epithelium subsequent to the excessive accumulation of hyaline droplets, with exfoliation of degenerate epithelial cells, should be observable after five days of continuou s expos ure, peaking at 19 days [reviewed in U.S. EPA (1991a) ]

. T he formation and accumulation of granular casts from the exfoliated cellular debris
would follow , causing tubule dilation at the junction of the S3 ( P3 ) segment of the proximal tubule and the descending thin loop of Henle, and the commencement of compensatory cell proliferation
within the S2 ( P2 ) s egment, both occurring after three weeks of continuou s exposure. Following chronic exposures, this regenerative proliferation could result in focal tubular hyperplasia, and eventually progress to renal adenoma and carcinoma ( Figure 1-7 ).
S everal of these steps were observed following tert

- butanol exposure in male rat s , most notably linear papillary mineralization and foci of tubular hyperplasia

, consistent with the expected disease progression. S ome lack of consistency and dose- related concordance , however, was evident

across the remaining steps in the histopathological sequence . First, the accumulation of hyaline droplets and the concentrations of α 2u - globulin in the hyaline droplets at doses that induced significant tu mor formation in male rats w ere not significant. Next, n ecrosis or cytotoxicity was absent and only precursors to granular casts at stages well within the expected timeframe of detectability were present. Finally, a 13

- week inhalation study found no eviden ce of α 2u - globulin nephropathy ( NTP, 1997 ) , despite evaluating exposure concentration s predicted to result in similar
blood tert

- butanol levels as for the 13

- week oral study ( NTP, 1995 ) , which report ed increases in droplet accumulation and sustained regenerative tubule cell proliferation. A detailed evaluation and analysis of all the evidence relevant to this criteri on follows. Toxicological Review of tert

- Butyl Alcohol

1 correlation of α 2u - globulin concentration with dose of tert

- butanol (determined by correlating with cell proliferation labeling indices) was observed, with accumulation of α 2u - globulin protein statistically significant by pairwise comparison only in the highest dose group. No positive staining
for α 2u - globulin was observed in exposed female rats. In a follow

- up study, Williams and Borghoff (2001) used a single gavage dose of 500 mg/kg [ selected on the basis of results by NTP (1995) for induction of hyaline droplet accumulation ]

, and reported a statistically significantly higher renal
concentration of α 2u -globulin (by ELISA ) in treated male rats than in controls 12 hours after

exposure. Further, equilibrium dialysis methods determined that the binding of tert

- butanol to

α 2u -globulin was reversible. These data indicate the presence of α 2u - globulin in tert

- butanol

- treated male rats, although requiring a more sensitive method of detection of α 2u - globulin than is typically
used could indicate that tert

- butanol is not a strong inducer of α 2u - globulin accumulation. Therefore, the available data are sufficient to fulfill the second criteri on for α 2u - globulin present in the hyaline droplets , but suggest weak induction of α 2u - globulin by tert

- butanol

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1 Detailed evaluation of the available evidence supporting the third criterion Single cell death and exfoliation into the renal tubules was inconsistently observed

. S ingle
cell death or necrosis was not associate d with tert

- butanol exposure in male rat kidneys after 10 or
13 weeks ( Acharya et al., 1997 ; NTP, 1995 ). Ac harya et al. (1997) reported degeneration of renal tubules, one pathological consequence of single cell necrosis, h owever, in male rats exposed to tert butanol in drinking water for 10 weeks. As renal tubule epithelial cell death and epithelial degenera tion should occur as early as 5 days post exposure and persist for up to 48 week s ( Swenberg and Lehman

- McKeeman, 1999 ; Short et al., 1989 )

, the lack of consistency in these
observations could be the result of both weak induction of α 2u - globulin and a lack of later

examinations. 2

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11 a. S ustained regenerative cell proliferation also was not observed. Acharya et al. (1997) did not observe tert

- butanol

- induced proliferation following 10 weeks of oral exposure

, but renal tubule proliferation was observed following another chemical exposure (trichl oroacetic acid) in the same study. Therefore, the inference is that tert

- butanol
treat ment did not induce regenerative tubule cell proliferation in male rats from this study

. Borghoff e t al. (2001) reported a dose -related increase in epithelial cell proliferation within the proximal tubule as measured by BrdU labeling indices in all male rats exposed to tert

- butanol via inhalation for 10 days. T he study did not report cytotoxicity, however, which, combined with the early time point makes unlikely that the cell proliferation wa s compensatory. NTP (1995) also observed increased cell proliferation in the renal tubule epithelium following 13

- week oral exposures in male rats [only male rats were studied in the retrospective analysis by Takahashi et al. (1993) reported in NTP (1995) ] . Proliferation was elevated at 840

– 1,520 mg/kg -day , a range higher than the single 575

- mg/kg

- day dose eliciting no such proliferative effect ( Acharya et al., 1997 ) , as described above

. NTP (1995) reported , however, that no necrosis was observed, suggest ing the proliferation was not regenerative. 12
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42 b. Granular cast formation was not observed, alth ough one study noted precursor s to cast formation

. NTP (19 95) did not observe the formation of granular casts or tubular dilation ; however, Hard et al. (2011) reanalyzed the 13

- week oral NTP data from male
rat s treated with 0 or 1,520 mg/kg -day and identified precursors to granular casts in 5/10 animals in the treated group. The significance of these granular cast precursors, described as sporadic basophilic tubules containing cellular debris, is unknown, because 13 weeks of exposure is within the expected timeframe of frank formation and accumulat ion of granular casts (≥3 weeks)

. G ranular cast formation, however, might not be significantly elevated with weak inducers of α 2u - globulin ( Short et al., 1986 ), which is consistent with the reported difficulty in measuring α 2u -globulin in hyaline droplets associated with tert

- butanol exposure

.

c. Linear mineralization of tubules within the renal papilla e was consistently observed in male rats. This lesion typically appears at chronic time points, occurring after exposures of 3 months up to 2 years ( U.S. EPA, 1991a ) . Consistent with this description, 2- year oral exposure to tert

- butanol induced a dose

- related increase in linear mineralization, but not following 13

- week expos ure [( NTP, 1995 ) ; Table 1-2]. Toxico logical Review of tert

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

- year study. Renal tubule hyperplasia is the preneoplastic lesion associated with α 2u - globulin nephropathy in chronic exposures that leads to renal tubule tumors ( U.S. EPA, 1991a ). A dose

- related increase in renal tubule hyperplasia was observed in male rats following 2- year oral
exposures ( NTP, 1995 ) . By comparison, renal tubule hyperplasia was observed in only one high

- dose female. 2
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6

7 T he progression of histopathological lesions for α 2u - globulin nephropathy is predicated on the initial response of excessive hyaline droplet accumulation (containing α 2u - globulin) leading to cell necrosis and cytotoxicity, which in t urn cause the accumulation of granular casts, linear
mineralization

, and tubular hyperplasia . Therefore, observations of temporal and dose

- response concordance for these effects are informative for drawing conclusions on causation. 8

9

mineralization of tubules and focal tubular hyperplasia fall within the expected timeframe of the appearance of these lesions. Overall, no explicit inconsistencies are present i n the temporal appearance of the histopatholog ical lesions associated with α 2u - globulin nephropathy ; however, the dataset would be bolstered by measurements at additional time points to lend strength to the MOA
evaluation

. I nconsistencies do occur in the dose

- response among lesions associated with the

α 2u - globulin nephropathy progression. Hyaline droplets were induced in the proximal tubule of all surviving male rats in the 13

- week NTP oral study ( NTP, 1995 ; Takahashi et al., 1993 ; Lindamood et al., 1992 ), a lthough the incidence at the lowest dose was minimal

, while the incidence at the three higher doses was more pro minent. These results are discordant with the tumor results, given that all treated groups of male rats in the NTP 2

- year oral bioassay had increased kidney tumor
incidence , including the lowest dose of 90 mg/kg

- day [ according to the reanalysis by Hard et al. (2011) ]

. This lowest dose was less than the 230 mg/kg

- day in the 13

- week oral study that had only minimal hyaline droplet formation. Furthermore, alth ough the incidence of renal tubul e hyperplasia had a dose -related increase ( NTP, 1995 ), a corresponding dose

- related increase in the severity of tubular hyperplasia did not result . Severity of tubule hyperplasia was increased only at the highest dose

, which was not consistent with renal tumor incidence

. Although the h istopathological sequence has data gaps

, such as the lack of observable

necrosis or cytotoxicity or granular casts at stages within the timeframe of detectability , overall, a 11

12 As mentioned above, most s teps in the sequence of α 2u -globulin nephropathy are observed at the expected time points following exposure to tert

- butanol. A ccumulation of hyaline droplets was observed early, at 12 hours following a single bolus exposure ( Williams and Borghoff, 2001 )

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

α 2u -G lobulin was identified as the protein in these droplets ( Borghoff et al., 2001 ; Williams and Borghoff, 2001 )

. Lack of necrosis and exfoliation might be due to the weak induction of α 2u -globulin
and a lack of later examinations. G ranular cast formation w as not reported by any of the available
studies , which could also indicat e weak α 2u -globulin induction . Regenerative cell proliferation

, which was not obse rved , is discussed in more detail below. Observations of the subsequent linear 13

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1 sufficient number of steps (e.g.

, linear papillary mineralization, foci of tubula r hyperplasia) were observed to fulfill the third criteri on

.
Summary and Conclusions for Question O ne:
O ral exposure to male F344 rats resulted in an increased incidence of renal tubule tumors in
a 2

- year oral bioassay ( Hard et al., 2011 ; NTP, 1995 ). Several histopathological observations in exposed male rats were consistent with an α 2u -globulin MOA. This evidence includes the increase d size and number of hyaline droplets and the accumulated α 2u - globulin protein in the hyaline droplets. Additionally, several subsequent steps in the histo pathological sequence were observed

. Overall, available data are sufficient for all three required criteria, suggesting that the α 2u - globulin process is operative

. A lthough t he evidence indicate s a role for α 2u - globulin accumulation in the etiology of kidney tumors induced by exposure to tert

- butanol in male rats, that tert

- butanol is a weak inducer of α 2u -globulin is plausible, considering the limited histopathological obse rvations
and uncertainty regarding the temporal and dose concordance of the lesions

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that might suggest the chemical belongs in a different class of suspected carcinogens: No data are available to evaluate these considerations

. Biochemical information regarding binding of the chemical to the α 2u - globulin protein : Williams and Borghoff (2001) report that tert

- butanol reversibly and noncovalently binds to

α 2u - globulin in the kidneys of male rats. This provides additional support to the involvement of the

α 2u - globulin process

. Presence of sustained cell replication in the S2 ( P2 ) segment of the renal tubule at doses
used in the cancer bioassay and a dose

- related increase in hyperplasia of the renal tubule : Sustained cell division in the proximal tubule of the male rat is consistent with, al though not specific to, the α 2u - globulin process. Cell proliferation was observed in two s tudies [ 13

- week, NTP (1995) and 10

- day, Borghoff et al. (2001) ] but whether the proliferation was compensatory is unknown, as cytotoxicity was not observed in these studies. Although the data do not support
sustained cell division occurring subsequent to cytotoxic cell death , renal tubule hyperplasia in
male rats was reported after 2 years of exposure ( NTP, 1995 ). Thus, although s ome evidence of 9

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14 Question Two: Are the renal effects in male rats exposed to tert

- butanol solely due to the α 2u - globulin process?

If the α 2u - globulin process is operative, U.S. EPA (1991a) identifies a second question that
must be answered regarding whether the renal effects are (a) solely due to the α 2u - globulin process, (b) a combinati on of the α 2u - globulin process and other carcinogenic processes, or (c) primarily due to other processes. U.S. EPA (1991a) states that additional data can help inform
whether the α 2u - globulin process is the sole contributor to renal tubule tumor development in male

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

- testing of the α 2u - globulin sequence of effects and structure

- activity relationships 16

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1 sustained cell replication is available, it does not specifically support α 2u - globulin protein
accumulation. Covalent binding to DNA or other macromolecules

, suggesting another process leading to

tumors and g enotoxicity ( α 2u - globulin

- induc ers are essentially nongenotoxic ) : One study ( Yuan et al.,
2007 ) observ ed a dose -related increase in tert

- butanol

- DNA adducts in liver, kidney, and lung of

mice a dministered a single low dose of tert

- butanol (≤1 mg/kg) in saline via gavage (see Appendix B.3 in Supplemental Information for further details). An extremely sensitive method of detection was used (accelerator mass spectrometry), but the DNA adduct species were not identified , and no
validation of these results has been identified in the literature. The few studies available to assess the genotoxic potential of tert

- butanol primarily are negative, al though a few studies report DNA damage induced by oxidative stress. DNA damage induced by oxidative stress is consistent with the
decr eased levels of glutathione in male rat kidneys reported by Acharya et al. (1995) after 10 weeks
of tert

- butanol exposure

. This type of genetic damage would not necessarily preclude a role for

α 2u - globulin, but not enough information is available to determine whether oxidative stress could

initiate or promote kidney tumors in concert with α 2u - globulin accumulation in male rat kidneys. 2

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were observed in female rats orally exposed to tert

- butanol for 2 years. Although NTP (1995) characterized these endpoints as associated with CPN, the low background incidence in the controls
combined with the dose

- related increase in incidences indicate that these effects were not related
to an age

- as sociated, spontaneous induction of nephropathy. At 2 years, the male rats also exhibited
dose- related increase s in focal mineralization and transitional epithelial hyperplasia, although the background incidence in the controls was high (i.e., approximately 50%) ( NTP, 1995 ). Neither endpoint in males can be attribute d to CPN or α 2u - globulin.

K idney weights also were increased in male and female rats in the 13

- week oral and
inhalation evaluations ( NTP, 1997
, 1995 ) an d 15

- month oral evaluation ( NTP, 1995 ). T he dose
related increases observed in both male and female rats suggest that the kidney weight changes are
indicative of treatment

- related molecular processes primarily un related to either α 2u -globulin protein accumulation or CPN. T he exacerbation of CPN a nd the appearance of k idney effects in female (i.e., suppurative inflammation, transitional epithelial hyperplasia) and male rats (i.e., focal
mineralization

, transitional epithelial hyperplasia) that are not attributed to CPN or α 2u - globulin
indicate that tert

- butanol induces renal tubule nephrotoxicity partially independen tly of 9

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16 Nephrotoxicity not associated with the α 2u - globulin process or CPN

, suggesting the possibility of other processes leading to renal tubu le nephrotoxicity and carcinogenicity : Nephropathy reported
in the 13

- week oral and inhalation and 2

- year oral studies was considered CPN , but these effects were exacerbated by treatment with tert

- butanol. At 13 weeks ( NTP, 1997
, 1995 ) and 2 years ( NTP,
1995 ) , oral and inhalation exposure increased the severity of nephropathy in male rats ( NTP,
1995 ). Similarly, the severity of nephropathy was increased in females at 2 years , but only the incidence of nephropathy was increased in females following a 13

- week oral exposure ( NTP, 1995 )

. I ncreased incidences of suppurative inflammation and kidney transitional epithelial hyperplasia 17

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1 α 2u -globulin. The evidence that other processes might be responsible for the renal tubule nephrotoxicity thereby decreas es the likelihood that α 2u - globulin accumulation is solely responsible for the renal tubule tumors

. Positive tubule tumor responses in female rats and other species implying that α 2u - globulin

related processes alone do not account for the renal tubule tumor response : N o increase in renal
tubule tumor incidence wa s reported in tert

- butano l- exposed female rats or mice compared with
concurrent controls. Renal tubule tumors were observed only in male rats, providing support for an

α 2u - globulin process in tumor development.

Summary and Conclusions for Question Two:

Al t hough the evidence suggests that tert

- butanol induces α 2u -globulin nephropathy

, the
data indicate that tert

- butanol is a weak inducer of α 2u -globulin and that this process is not solely responsible for the renal tubule nephropathy and carcinogenicity observed in male rats. T he lack of
compensatory cell proliferation in male rats and evidence o f nephrotoxicity in female rats suggest 2

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28 that other processes

, in addition to the α 2u - globulin process

, are operating

. Furthermore, the
accumulation of hyaline droplets and the induction of renal tubule hyperplasia were affected at higher doses compared to those induc ing renal tubule tumors. Collectively

, these data suggest that

tert

- butano l induces the α 2u -globulin pathway at high doses (>420 mg/kg

- day)

, which results in
tumor formation

. O ther

, unknown pathways , however, could b e operative at lower doses (< 420 mg/kg

- day)

, which contribute to renal tumor induction.

b) Chronic Progressive Nephr opathy and Renal Carcinogenicity There is scientific disagreement regarding the extent to which CPN can be characterized as

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

an MOA have developed an evolving series of empirical criteria for attributing renal tubule tumors
to CPN. Hard and Khan (2004) proposed criteria for concluding that a chemical is associated with renal tubule tumors through an interaction with CPN. Hard et al. (2013) slightly revised and
restated their criteria for considering exacerbation of CPN as a n MOA for renal tubule tumors in

rats. Table 1-5 lists these sets of proposed empirical criteria for attributing renal tubule tumors to
CPN.

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

• First and foremost, the chemical must have been shown to exacerbate CPN to very advanced stages of severity, especially end

- stage kidney disease , in comparison to control rats in a 2

- year carcinogenicity study.

• The tumors should occur in very low incidence and, for the most part, be minim al grade lesions conforming to small adenomas or lesions borderline between atypical tubule hyperplasia (ATH) and adenoma.

• Such tumors should be associated only with the highest grades of CPN severity

.

• The tumors and any precursor foci of ATH must be res tricted to CPN

- affected parenchyma and are usually observed only toward the end of the
2- year studies.

• Careful microscopic examination of renal parenchyma not involved in the CPN process should reveal no evidence of compound

- induced cellular injury or oth er changes that would suggest alternative modes of action.

Source: Hard and Khan (2004) • La ck of genotoxic activity based on overall evaluation of in vitro and in vivo data.

• Tumor incidence is low, usually <10%.

• Tumors are found toward the end of 2

- year studies.

• Lesions are usually ATH or adenomas (carcinomas can occasionally occur).

• Chemica l exacerbates CPN to most advanced stages, including end

- stage kidney disease

.

• ATH and tumors occur in rats with advanced CPN and in CPN

- affected tissue.

• Absence of cytotoxicity in CPN

- unaffected tubules, in rats with lower grades of CPN, and in subchron ic studies.

Source: Hard et al. (2013)

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

is not necessary

. Instead , identify ing increased CPN with its associated increase in tubul e cell proliferation as the key event i s adequate . Nonetheless, Hard et al. (2013) also postulated a sequence of key events for renal tumorigenesis involving exacerbation of CPN :

• Expo sure to chemical (usually at high concentrations) ;

• Metabolic activation (if necessary) ;

• Exacerbated CPN, including increased number of rats with end

- stage renal disease ;

• Increased tubul e cell proliferation because more kidney is damaged due to CPN exacerbation ;

• Hyperplasia ; and

• Adenoma (infrequently carcinoma).

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

, Melnick et al. (2013) ; Melnick et al. (2 012) concluded , based on an analysis of 60 NTP studies, no consistent association exists between
exacerbated CPN and the incidence of renal tubule tumors in rats. Without a consistent association

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1 and an understanding of its key events, they maintain that determin ing the human r elevance of processes that might be occurring in rats i s not possible . An earlier analysis of 28 NTP studies ( Seely et al., 2002 ) found a slight but statistically significant incr ease in CPN severity in animals with renal tubule tumors, without determining that this relationship is causal . They suggested that the number of tumors due to chemically exacerbated CPN would be few.

Evaluation of the MOA Proposed by Hard et al. (2013) Setting aside the question of whether CPN is ( Har d et al., 2013 ; Hard and Khan, 2004 ) or is

not ( Melnick et al., 2013 ; Melnick et al., 2012 ) an MOA suitable for analysis, this s ection provides an analysis of the mechanistic data pertinent to CPN. EPA ’ s cancer guidelines ( U.S. EPA, 2005a ) define a framework for judging whether available data support a hypothesized MOA ; the analysis in this

se ction follows the structure presented in the cancer guidelines. 2

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12 Description of the hypothesized MOA. Under the EPA framework, toxicokinetic studies are important for identifying the active agent, but toxicokinetic events per se are not key events of a n MOA . Thus, the EPA analysis of the MOA proposed by Hard et al. (2013) begins with (1 ) exacerbated CPN, including increased number of rats with end

- stage renal disease, and proceeds via (2) increased tubule cell proliferation, (3) hyperplasia, and (4) adenoma, or infrequently, carcinoma. 13

14

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18 Strength, consistency, specificity of association. The relationship between exacerbated CPN
and renal tum ors is moderate to strong in male rats in the NTP (1995) stu dy. According to the NTP (1995) analysis, the mean CPN grade s (same as “severity of nephropathy” reported by NTP ) presented on a scale 1

– 4 for male rats with renal tumors were 3.5, 3.6, 3.7, and 3.4 at doses 0, 1.25, 2.5, and 5 mg/mL. The mean CPN grades for male rats without renal tumors were 2.9, 2.8, 2.8, and 3.2 for the same dose groups. The reanalysis of the NTP data by Hard et al. (2 011) yielded similar numbers. The relationship between CPN and renal tumors, however, is neither consistent nor specific in the NTP (1995) study: N o female rats developed renal tumors regardless of the presence 19

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26 of relatively low

- grade or relatively high

- grade CPN. For example, in female rats surviving more
than 700 days, the mean CPN grades were 1.7 and 3.2 at doses of 0 and 10 mg/mL, respectively, but no tumors developed in either group.

Dose

- response concordance. T he dose

- response relationships for CPN, renal tubule hyperplasia, and renal tubule tumors somewha t differ. According to the NTP (1995) analysis, at
doses of 0, 1.25, 2.5, and 5 mg/mL, the mean CPN grades for all male rats were 3.0, 3.1, 3.1, and 3.3; the incidences of renal tubule hyperplasia (standard and extended evaluation combined) were 14/50, 20/50, 17/50, and 25/50; and the incidences of renal tubule ade nomas or carcinomas were 8/50, 13/50, 19/50, and 13/50 ( Table 1-3 ). The reanalysis by Hard et al. (2011) reported similar
tumor incidences (4/50, 13/50, 18/50, and 12/50), except that four fewer rats in the controls and
one fewer rat in the group exposed to 2.5 mg/mL had tumors . The lower control incidence
observed in this reanalysis accentuates the differences in these dose

- response relationships. In

examin ing the various lesions at the mid

- dose

— the dose with the greatest increase in renal tubule 27

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1 tumors in male rats

— a minor increase ( 14/50 in controls versus 17/50 in the mid

- dose group) in
renal tubule hyperplasia incidence was observed

, with a marginal chan ge in CPN severity (i.e., group average of 3.0 to 3.1)

. T hat a minor increase in hyperplasia and marginal increase in CPN severity would be associated with significant tumor induction seems inconsistent

. Furthermore, CPN severity is nearly as great in the female rats

, yet no females developed tumors, as noted above

. Temporal relationship. The severity of CPN progressed over time. According to the NTP (1995) analysis, the mean CPN grades in the 13

- week study of male rats were 1.0, 1.6, 2.6, 2.7, 2.6, and 1.1 at doses of 0, 2.5, 5, 10, 20, and 40 mg/mL . A t the 15

- mon th interim evaluation of the 2

- year study, the mean CPN grades were 2.4, 2.8, 2.7, and 2.6 at doses of 0, 1.25, 2.5, and 5 mg/mL and at
2 years , increased to 3.0, 3.1, 3.1, and 3.3. Similarly, the severity of neoplastic lesions increased at
the end of life . At the 15

- month interim evaluation, only two rats had developed renal tubule hyperplasia and one other had a renal tubule adenoma; at 2 years, the incidences of these two lesions were much higher in all dose groups (see previous paragraph). These results are consistent
with CPN as an age

- related disease and with hyperplasia and tumors appearing near the end of life. 2

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14 Biological plausibility and coherence. In general, the relationship between exacerbated CPN
and renal tubule tumors in male rats appears plausibl e and coheren t. S ome patterns in the dose response relationships for CPN, hyperplasia, and tumors are discrepant. P erhaps more importantly

, the patterns also are discrepant for the relationships betwee n CPN grades and renal tubule tumors in male and female rats. In addition, the increased incidences in renal tubule tumor s in all e xposed
male rats exceed the 10% criterion proposed by Hard et al. (2013) ( Table 1-5 ), even more so when making comparisons with the lower control tumor incidence from the Hard et al. (2011) reanalysis.

Conclusions about the hypothesized CPN

- related MOA As recommended by EPA’s cancer guidelines ( U.S. EPA, 2005a ) , conclusions about the hypothesized MOA can be clarified by answering three questions presented below. (a) Is the hypothesized MOA sufficiently supported in the test animals? E xacerbated CPN
lead ing to renal tubule tumors in male rats late in life appears to have some suppor t . There is lack of consistency, however, between males and females and in the dose

- response relationships 16

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28 between CPN, hyperplasia, and adenomas. These inconsistencies make difficult attribut ing all renal
tumors to either CPN or to α 2u - globulin

- related ne phropathy (see previous section on α 2u - globulin )

, raising the likelihood of another, yet unspecified MOA

. (b) Is the hypothesized MOA relevant to humans? There is scientific disagreement on this question. Hard et al. (2013) ; Hard et al. (2009) cite several differences in pathology between rat C PN and human nephropathies in their argu ments that CPN

- related renal tumors in rats are not relevant
to humans. On the other hand

, Melnick et al. (2013) ; Melnick et al. (2012) argue that the etiology of
CPN and the mechanisms for its exacerbation by chemicals are unknown and fail to meet fundamental principles for defining a n MOA and for evaluating human relevance. T his issue is
unresolved

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1 (c) Which populations or lifestages can be particularly susceptible to the hypothesized MOA? There are no indications of a human population or lifestage that is especially susceptible to tumors induced through ex acerbated CPN. In summary, considering discrepant patterns in the dose

- response relationships for CPN, hyperplasia, and renal tubule tumors and the lack of relationships between CPN grades and renal
tubule tumors in male and female rats, together with the lack of a generally accepted MOA for CPN,
the renal tubule tumors in rats cannot be attributed to CPN

. This position can be reconciled with that of Melnick et al. (2013) ; Melnick et al. (2012) , who argued against dismissing renal tubule tumors in rats that can be related to exacerbated CPN. It also
can be reconciled with Hard et al. (2013) , who, while maintaining that these tumors are not relevant to humans, also allow that there is no generally accepted MOA for CPN akin to that for α 2u globulin- related nephropathy. Hard et al. (2013) made this statement after reporting on the
collective ex perience of national and international health agencies worldwide with the use of CPN

as a n MOA . Of 21 substances that exacerbated CPN and caused renal tumors, most were multisite carcinogens , and other tumor sites contributed to the evaluations. Only two a ssessments explicitly 2

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Relevant to this last point, IARC (1999) developed a consensus statement that listed
considerations f or evaluating α 2u -globulin- related nephropathy in rats, which was based on the work of 22 scientists, including three who were co

- authors of Hard et al. (2013) and two who were

co

- authors of Melnick et al. (2013) ; Melnick et al. (2012) . A similar broad

- based consensus that
defines a sequence of key events for exacerbated CPN, distinguishes it more clearly from α 2u globulin- related nephropathy, and evaluates its relevance to humans would be helpful in advancing the understanding of these issues.

Overall Conclusions on MOA for Kidney Effects

tert

- Butanol increases α 2u globulin deposition and hyaline droplet accumulation in male rat kidneys, as well as several of the subsequent steps in that pathological sequence. These data provide s ufficient evidence (albeit minimal) that the α 2u globulin process is operating, although based on further analysis this chemical appears to be a weak inducer of α 2u globulin- nephropathy and this induction is not the sole contributor to renal tubule nephropat hy and carcinogenicity. CPN and the exacerbation of CPN (likely due to both α 2u - globulin and tert

- butanol) play a role in renal tubule nephropathy. Although CPN was indicated in the induction of renal tubule nephropathy, the 9

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16 considered CPN as a renal tumor mechanism. One was the assessment of ethylbenzene by the German Federal Institute for Occupational Safety and Health, in which the agency concluded that the kidney tumors were associated with the high

, strain

- specific incidence of CPN that is unknown for humans [ as discussed in Hard et al. (2013) ] . The other was the IRIS assessment of tetrahydrofuran, for which EPA found the evidence insufficient to conclude that the kidney tumors are mediated solely by the hypothesized MOAs ( U.S. EPA, 2012d )

. Hard et al. (2013) attributed
these different conclusions to either different data for the two chemicals or the lack of a generally accepted MOA akin to α 2u - globulin

- related nephropathy. 17

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1 available evidence indicates th at it does not induce the renal tubule tumors in male rats. Additionally, several endpoints indicate renal tubule nephrotoxicity and increased kidney weights
related to tert

- butanol exposure cannot be explained by the α 2u globulin or CPN processes.
Collecti vely, the evidence indicates other, unknown processes contribute to renal tubule nephrotoxicity and carcinogenicity.

Integration of kidney effects

K i dney effects ( increases in nephropathy, severity of nephropathy, hyaline droplets, linear mineralization, suppurative inflammation, transitional epithelial hyperplasia, mineralization, and kidney weigh t) we re observed, predominantly in male and female rats across the multiple tert
butanol studies. The available evidence indicates that multiple processes induc e the noncancer kidney effects. The group of lesions generally reported as “nephropathy

, ” is related to CPN. Because
this disease is considered to be spontaneous and age

- related in rats , the endpoints associated with
CPN would not be relevant to humans for purposes of hazard identification. Additionally, two 2

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32 endpoints in male rats (hyaline droplets, linear mineralization) are components of the α 2u - globulin

process. U.S. EPA (1991a) states that if the α 2u - globulin process is occurring in male rats, the renal
tubule effects associated with this process in male rats would not be relevant to humans for purposes of hazard identification . In cases such as these, the characterization of human health
hazard f or noncancer kidney toxicity would rely on effects not specifically associated with CPN or the α 2u - globulin

- process in male rats.
Several other noncancer e ndpoints result ed from

- butanol exposure and are appropriate for consideration of a kidney hazard, specifically: suppurative inflammation in female rats, transitional epithelial hyperplasia in male and female rats, severity of nephropathy in male and female rats, incidence of nephropathy in female rats, incidence of mineralization in male rats, and increased kidney weights in rats but not mice. B a sed on dose

- related increases in these noncancer endpoints in rats, kidney effects are a potential human hazard of tert

- butanol exposure. The hazard
and dose- response conclusions regarding these noncancer endpoints associated with tert

- butanol exposure are discussed further in Section 1.3.1. tert

The carcinogenic effects observed following

- b utanol exposure include increased
incidences of renal tubule hyperplasia (considered a preneoplastic effect) and tumors in male rats.
EPA concluded that the three criteria were met to indicate that an α 2u - globulin process is operating

. Because renal tubule tumors in male rats did not arise solely due to the α 2u - globul in process and
some of the tumors are attributable to other carcinogenic process es, such tumors remain relevant tert ­

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1 for purposes of hazard identification U.S. EPA (19 91a)

. 3 The hazard and dose

- response conclusions regarding the renal tubule hyperplasia and tumors associated with tert

- butanol exposure are further discussed as part of the overall weight of evidence for carcinogenicity in Section 1. 3 .2.

**1.2.2. Thyroid**  **Effects**

Synthesis of Effects in Thyroid

The data base o n thyroid effects following tert

- butanol exposure contain s no human data

, two oral subchronic and two oral chronic studies ( one of each duration in rats and in mice ) ( NTP,
1995 )

, and two inhalation subchronic studies (one in rats and one in mice) ( NTP, 1997 ). Studies employing short

- term and acute exposures that examined thyroid effects are not included in the evidence table; they are discussed, however, in the text if they provide data informative of MOA or 2

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15 hazard identification

. N o gross thyroid effects were reported in the 13

- week evaluati ons of mice or rats following oral or inhalation exposure ( NTP, 1997
, 1995 )

, and therefore subchronic studies
were not included in the evidence table

. Th e two available chronic s tudies are arranged in the evidence table by effect and then by species. The design, conduct, and reporting of each study were reviewed , each study was considered adequate to provide information pertinent to this assessment ( Table 1-6 and Figure 1- 10 )

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29 Thyroid effects

, specifically follicular cell hy perplasia and adenomas

, were observed in mice
of both sexes after 2 years of oral exposure via drinking water ( NTP, 1995 ). NTP (1995) noted that “[p]roliferation of thyroid gland follicular cells is generally considered to follow a progression from hyperplasia to adenoma and car c inoma.” Similarly, EPA consider ed the thyroid follicular cell hyperplasia to be a preneoplastic effect associa ted with the thyroid tumors. Both male and female

mice exhibited a dose

- related increase in the incidence of hyperplasia

, and the average severity across all dose groups was minimal to mild with scores ranging from 1. 2 to 2.2 (out of 4). Increased
incidenc e of a denomas were also observed in the tert

- butanol

- treated mice
, with the only
carcinoma observed in high

- dose males. N o treatment

- related thyroid effects were reported in rats of either sex following 2 years of oral exposure ( NTP, 1995 )

. Although the tumor response in male mice showed a statistically significant increasin g trend (Cochran

- Armitage trend test, p = 0.041) (analysis performed by EPA using the mortality adjusted rates), the response was non

- monotonic, with a slightly lower response at the high

- dose

3 When the α 2u - globulin process is occurring, U.S. EPA (1991a) states that one of the following conclusion s will be made: (a ) i f renal tumors in male rats are attributable solely to the α 2u - globulin process, such tumors will not be used for h uman cancer hazard identification or for dose

- response extrapolations; (b ) i f renal tumors in male rats are not linked to the α 2u globulin process, such tumors are an appropriate endpoint for human hazard identification and are considered, along with other appropriate endpoints, fo r quantitative risk estimation; or (c ) i f some renal tumors in male rats are attributable to the α 2u - globulin process and some are attributable to other carcinogenic processes, such tumors remain relevant for purposes of hazard identification, but a dose

response estimate based on such tumors in male rats should not be performed unless there is enough information to determine the relative contribution of each process to the overall renal tumor response.

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1 level than at the mid

- dose level. The reason for the non

- monoto nicity is unclear, although it could be related to the increased mortality in the high

- dose group (17/60 animals survived compared with 27/60 animals in the control group). The decreased survival of male mice might have affected the thyroid tumor incidence s because animals could have died before tumors could develop. High mortality in the high

- dose group occurred before tumors appeared; about 40% of the high

- dose males died before the first tumor (a carcinoma) appeared in this group at week 83. By compariso n, only ~10% of the control group had died by this time, and the single tumor in the control group was observed at study termination. Mortality in the exposed female mice was similar to controls.

**Table 1-6.**  **E vidence pertaining to thyroid effects**  **in animals following oral exposure to**  tert

**- butanol**  2

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Reference and study design Results
Follicular cell hyperplasia
NTP (1995) Incidence b F344/N rat; 60/sex/treatment (10/sex/treatment evaluated at 15 Males Females
months) Dose Follicular cell Dose Follicular cell Drinking water ( 0, 1.25, 2.5, 5, or 10 (mg/kg

- d) hyperplasia (mg/kg

- d) hyperplasia mg/mL) 0 3/50 0 0/50 M: 0, 90, 200, or 420 a mg/kg -d F: 0, 180, 330, or 650 a mg/kg -d 90 0/49 180 0/50 2 years 200

420 a 0/50
0/50 330

650 a 0/50
0/50
NTP (1995) Incidence (severity) B6C3F 1 mouse; 60/sex/treatment Drinking water ( 0, 5, 10, or 20 mg/mL ) Males Females

M: 0, 540, 1,040, or 2,070 a mg/kg -d Dose Follicular cell Dose Follicular cell F: 0, 510, 1,020, or 2,110 mg/kg -d (mg/kg

- d) hyperplasia (mg/kg

- d) hyperplasia 2 years 0

540
1,040
2,070 a 5/60 (1.2)
18/59* (1.6)
15/59* (1.4)
18/57* (2.1) 0

510
1,020
2,110 19/58 (1.8)
28/60 (1.9)
33/59* (1.7)
47/59* (2.2)

Follicular cell tumors
NTP (1995) Incidence b

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Reference and study design Results
F344/N rat; 60/sex/treatment (10/sex/treatment evaluated at 15 months) Drinking water ( 0, 1.25, 2.5, 5, or 10 mg/mL) M: 0, 90, 200, or 420 a mg/kg -d F: 0, 180, 330, or 650 a mg/kg -d 2 years Dose (mg/kg

- d) Follicular cell adenoma Follicular cell carcinoma

Male

0 2/50 2/50

90 0/49 0/49

200 0/50 0/50

420 a 0/50 0/50

Female

0 1/50 1/50

180 0/50 0/50

330 1/50 1/50

650 a 0/50 0/50
NTP (1995) Incidence B6C3F 1 mouse; 60/sex/treatment Drinking water ( 0, 5, 10, or 20 mg/mL ) M: 0, 540, 1,040, or 2,070 a mg/kg -d F: 0, 510, 1,020, or 2,110 mg/kg -d 2 years Dose (mg/kg

- d) Follicular cell adenoma Follicular cell carcinoma Follicular cell adenoma or carcinoma (mortality adjusted rates) c,d Animals surviving to study termination

Male

0 1/60 0/60 1/60 (3.6%)

540 0/59 0/59 0/59 (0.0%)

1,040 4/59 0/59 4/59 (10.1%)

2,070 a 1/57 1/57 2/57 (8.7%)

Female

0 2/58 0/58 2/58 (5.6%)

510 3/60 0/60 3/60 (8.6%)

1,020 2/59 0/59 2/59 (4.9%)

2,110 9/59* 0/59 9/59* (19.6%) 27/60
36/60
34/60
17/60

36/60
35/60
41/60
42/60

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— DO NOT CITE OR QUOTE a 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. The mortality

- adjusted rates for the

incidence of adenomas are the same as the combined rates, with the exception of the male high

- dose group, where the rate for adenomas alone was 5.9%.

d Cochran

- Armitage trend test was applied to mortality

- adjusted thyroid tumor incidences, by applying the NTP adjusted rates

to the observed numbers of tumors to estimate the effective number at risk in each group. For male mice, p = 0.041; for female mice, p = 0.028.* Statistically significant p ≤ 0.05 as determined by the study authors.

Note: Conversions from drinking water concentrations to mg/kg

- d performed by study authors.1

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 = exposures at which the endpoint was reported statistically significant by study authors  = exposures at which the endpoint was reported not statistically significant by study authors

Hyperplasia; M mouse

Hyperplasia; F mouse

NONCANCER

Hyperplasia; M rat

Hyperplasia; F rat

10 100 1,000 10,000 

### Dose (mg/kg

- day) Adenoma; M mouse

Adenoma; F mouse

Adenoma; M rat

Adenoma; F rat CANCER

Source: NTP (1995)

**Figure 1-**  **10**

**. Exposure** 

**- response array of**  **thyroid follicular cell**  **effects following**  **chronic oral exposure to**  tert

**- butanol** 

**. (Note: Only one carcinoma**  **was observed in male mice at the high**

**- dose group.)** 

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1 Mode of Action Analysis

— Thyroid E ffects

The MOA responsible for tert

- butanol -induced thyroid effects ha s not been the subject of much study

. One hypothesis is that tert

- butanol increases liver metabolism of thyroid hormones, triggering a compensatory increase in pituitary thyroid

- stimulating hormone ( T SH ) production

. Such s ustained increas es in TSH could i nduce elevated thyroid follicular cell proliferation and hyperplasia and lead to follicular cell adenoma and carcinoma

, that , an antithyroid MOA, as identified in U.S. EPA’s guidance on the assessment of thyroid follicular cell tumors ( U.S. EPA,
1998a ). 2

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34 T o determine if the thyroid follicular cell tumors result from a chemically induced antithyroid MOA, U.S. EPA (1998a) requires that the available database demonstrate: ( 1) increases in thyroid cell growth, ( 2) thyroid and pituitary hormone changes consistent wit h the antithyroid
MOA
, ( 3) site(s) of the antithyroid action, ( 4) dose correlation among the various effects , and ( 5) reversibility of effects in the early stages of disruption. The available evidence pertaining to each of these aspects of antithyroid acti vity following tert

- butanol exposure is discussed below.

1) Increases in cell growth (required) U.S. EPA (1998a) considers increased absolute or relative thyr oid weights, histological indicators of cellular hypertrophy and hyperplasia, DNA labeling, and other measurements (e.g.

, Ki 67 or proliferating cell nuclear antigen expression) to be indicators of increased cell growth. Only a
few studies ( NTP, 1997
, 1995 ) have evaluated the thyroid by routine histological examination following tert

- butanol exposure, and none investigated specific molecular endpoints. Non e of the available long

- term studies measured thyroid weight in mice, likely due to the technical limitations involved, and no thyroid effects were attributed to tert

- butanol exposure in rats treated up to 2

years ( NTP, 1997
, 1995 ). Although the short

- term female mouse study by Blanck et al. (2010)
stated th at thyroids were weighed, no results were reported. An increase in thyroid follicular cell hyperplasia was observed in both female and male mice
after a 2

- year drinking water exposure to tert butanol

- ( NTP, 1995 ). The increase was dose dependent in female mice with a slight increase in severity in the highest dose, while male mice experienced a similar magnitude of hyperplasia induction at all doses, with increased severity at the highest dose ( NTP, 1995 ) . Thyroid follicular cell hyperplasia was not observed in any mouse
stud y with less than 2 years of exposure: no treatment

- related histological alterations in the thyroid
of tert

- butanol

- treated (2 o r 20 mg/mL) female mice after 3 or 14 days of drinking water exposure ( Blanck et al., 2010 ) were reported

, in male or female mice after 13 weeks of drinking water

exposur e ( NTP, 1995 ) or in male or female mice following 18

- day or 13

- week inhala tion studies ( NTP, 1997 ). T he observation of increased hyperplasia in male and female mice after 2 years of

exposure is sufficient evidence to support increased thyroid cell growth. Toxicological Review of tert

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1 2) Changes in thyroid and relevant pituitary hormones (required)

Evidence of hormona l changes

, including decreases in thyroxine (T 4 ) and tri i odothyronine (T 3 ) and increases in TSH
, are required to demonstrate a disruption in the thyroid

- pituitary signaling axis ( U.S. EPA, 1998a )

. Blanck et al. (2010) evaluated serum thyroid hormones in mice after 3 or 14 days of exposure to tert

- butanol. No tert

- butanol -related effects were observed in T 3 , T 4 , or TSH levels after 3 days, and although both T 3 and T 4 levels were significantly decreased approximately 10

– 20% after 14 days of treatment with tert

- butanol, TSH levels remained
unaffected. Similar results were reported with the positive control (phenobarbital). The limited evidence available from this single study suggests that although T 3 and T 4 levels were decreased
after 14 da ys , this perturbation likely was not in excess of the range of homeostatic regu lation in
female B6C3F 1 mice and thus not likely to induce compensatory thyroid follicular cell proliferation. Multiple lines of evidence support this observation: (1 ) TSH levels were unaffected, indicating that
the decrease in T 3 and T 4 levels was not severe enough to stimulate increased TSH secretion by the pituitary; (2 ) thyroid hyperplasia was not induced in this study, or any others exposing mice for 2

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fact be due to a decrease in thyroid hormone production, resulting from some

, as of ye t, uninvestigated molecular interactions of tert

- butanol in the thyroid, pituitary

, or hypothalamus. The absence of information regarding thyroid hormone levels in male mice and lack of molecular studies evaluating exposures >2 weeks in female mice are si gnificant deficiencies in the available database. Together, although small decreases in some thyroid hormone levels have been reported in female mice, the available evidence is inadequate to determine if tert

- butanol negatively affects the pituitary

- thyroi d signaling axis in female mice; furthermore, no evidence was
available to evaluate this effect in male mice.

3) Site(s) of antithyroid action (required) The thyroid and liver are two of several potential sites of antithyroid action, with the liver
the mo st common site of action

, where increased microsomal enzyme activity could enhance thyroid hormone metabolism and removal ( U.S. EPA, 1998a ). R ats are thought to be more sensitive than mice to this aspect of antithyroid activity ( Roques et al., 2013 ; Qatanani et al., 2005 ; U.S. EPA,
1998a ); however, rats exposed to tert

- butanol for 2 years did not exhibit treatment -related thyroid
effects

, while mice did

. Typically, chronic induction of liver microsomal enzyme activity resulting from repeated chemical exposure would manifest some manner of liver histopathology, such as 9

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2.5

– 13 w ee ks, suggesting that thyroid proliferation was either not induced by the ho rmone fluctuations or that any follicular cell proliferation during this period was too slight to be detected by routine histopathological examination; (3 ) the maximal decrease in T 3 or T 4 hormone levels induced by tert

- butanol exposure after 14 days ( i.e.
, ~ 20%) was well within the range of fluctuation

in T 3 and T 4 hormone levels reported to occur between the 3

- and 14

- day control groups [ 15

– 40%; ( Blanck et al., 2010 )] . A lthough the lower T 3 and T 4 levels following tert

- butanol were later
attributed by the study authors to an increase in liver metabolism (see next section), they could in 16

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1 hepatocellular hypertrophy or hyperplasia ( U.S. EPA, 1998a ; NTP, 1995 ) . In a 14

- day mechanistic investigation, tert

- b utanol had no effect on liver weight when compared to the control group, but centrilobular hepatocellular hypertrophy was reported in 2/5 livers from high

- dose mice (versus 0/6 in control and 0/5 in low

- dose mice ( Blanck et al., 2010 )

. Relative liver weights increased in male and female mice after 13 weeks of oral exposure ( NTP, 1995 ) to higher doses than those evaluated by Blanck et al. (2010)

, although absol ute liver weight measurements in treated animals showed little change from controls suggesting that the relative measures could have been related to decreases in body weight rather than specific liver effect s. Relative (and absolute) liver weights
were increased in female mice (only) after 13 weeks of inhalation exposure at the two highest

concentrations ( NTP, 1997 ) ; liver weight was not reported in mice orally exposed for 2 years ( NTP,
1995 ) . No increase in mouse hepatocellular hypertrophic or hyperplastic histopathology was reported following 2.5 weeks to 2 years of exposure ( NTP, 1997
, 1995 ) . In fact, the only liver pathology associated with tert

- butanol exposure in these studies was an increase in fatty liver in
male mice in the hig h- dose group after 2 years of oral exposure ( NTP, 1995 )

. Although increased 2

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receptor (CAR) activation; CAR can induce expression of a wide range of hepatic enzymes, including
several CYPs along with thyroid hormone

- met abolizing sulfotransferases ( Roques et al., 2013 )

. T he only thyroid hormone

- metabolizing enzyme induced by tert

- butanol , however, was SULT1A1,
which has been reported to be inducible in a CAR

- independent manner in mice ( Qatanani et al.,
2005 ) . Based on alterations in hepatic phase I and phase II enzyme activities and gene expression, the above data suggest a possible role for increased thyroid hormone clearance in the liver following repeated tert

- butanol exposure; however, the expression changes in these few enzymes are not supported by any liver histopathological effect s in mice exposed for longer durations, so 9

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14 fatty liver could indicate s ome non

- specific metabolic alteration, the absence of a similar treatment
related effect in livers from female mice, which were sensitive to both thyroid follicular cell hyperplasia and tumor induction, suggests that i t might not be related to the thyroid tumorigenesis.
One study evaluat ed liver enzyme expression and found highly dose

- responsive induction of a single phase I cytochrome p450 enzyme (CYP2B10) following 14 days of tert

- butanol exposure

in female mice , wit h much smaller increases in the expression of another phase I enzyme CYP2B9,
and the phase II thyroid hormone

- metabolizing enzyme, sulfotransferase 1A1 [ (SULT1A1; Blanck et al. (2010) ]

. CYP2B enzyme induction is commonly used as an indication of constitutive androstane 16

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w ee ks of exposure, is unknown. N o evidence is available to evaluate the potential for intrathyroidal or any oth er extrahepatic effects in female mice or for any of these molecular endpoints in male

mice ; therefore

, the available evidence is inadequate to determine if major site(s) of antithyroid
action are affected.

4) Dose correlation (required) Confidence in the disruption of the thyroid

- pituitary function is enhanced when dose
correlation is present among the hormone levels producing various changes in thyroid 32

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1 histopathology

, including thyroid tumors ( U.S. EPA, 1998a ). Furthermore, if thyroid hormone levels

were affected by liver enzyme induction, confidence would be increased by a concordance among liver effects, thyroid hormone levels, and thyroid pathology. Thyr oid hormone levels were
evaluated only in female mice exposed to tert

- butanol ; after 2 weeks of exposure

, both T 4 and T 3 were decreased with both doses (2 and 20 mg/L)

, a nd TSH was unaffected at either dose ( Blanck et al., 2010 ). Liver expression of CYP2B10 was increased in a dose

- responsive manner, while
SULT1A1 mRNA was induced by 20

– 30% at both doses ( Blanck et al., 2010 ). As described above

, induction of liver microsomal enzyme activity would manifest some manner of liver histopathology ( Maronpot et al., 2010 ; U.S. EPA, 1998a ; NTP, 1995 ) . Toxicol Pathol 38:776

- 795 ) , and consist ent with this expected association, centrilobular hepatocellular hypertrophy was reported in 2/5 high
dose mice exposed for 2 weeks ( Blanck et al., 2010 )

. N o liver histop athology , however, was 2

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8

attributed to tert

- butanol exposure in female mice exposed for 2.5 weeks to 2 years to comparable

tert

- butanol concentrations ( NTP, 1997
, 1995 ). Although liver enzyme levels and activity were not specifically evaluated following subchronic to chronic exposure, the lack of liver pathology suggests a comparable lack of enzyme induction. Conversely, no histopathological alterations were reported in the thyroids of female mice after 2 weeks of oral exposure at dose s that elevated some liver enzyme levels ( Bla nck et al., 2010 ). Following 2 years of oral exposure, both follicular cell hyperplasia and follicular cell tumor
incidence was increased in mice despite a lack of treatment

- related liver pathology ( NTP, 1995 ) ( Table 1-6). Any associations relating hormone changes to thyroid pathology or liver enzyme
induction are limited due to the inadequate database (described above); the available evidence suggests little concordance among reports of liver, pituitary

, and thyroid effects in female mice, and
no evidence was available to evaluate these associations in male mice.

5) Reversibility (required)

Chemicals act ing via an antithyroid MOA have effects (e.g.

, increased TSH levels, thyroid follicular cell proliferation) that are reversible after cessation of treatment ( U.S. EPA, 1998a )

. Although increased TSH levels have not been demonstrated following tert

- butanol exposure, thyroid follicular cell prolifera tion w as observed following chronic exposure

. As no studies have
evaluated changes in thyroid hormones or thyroid histopathology after cessation of tert

- butanol

treatment , however, the available evidence is inadequate to evaluate reversibility of these effects

. 9

11

12

13

14

16

17

18

19

21

22

23

24

26

27

28

29

31 In summary, the available database sufficiently support s only (1) increases in thyroid cell growth

. The exi s ting data are inadequate to evaluate (2) thyroid and pituitary hormone changes
consistent with the ant ithyroid MOA , ( 3) site(s) of the antithyroid action, or ( 5) reversibility of
effects in the early stages of disruption . A lthough these inadequacies also limit the evaluation of ( 4)
dose correlation among the various effects

, the available evidence suggests that little correlation

exists among reported thyroid, pituitary

, and liver endpoints

. Together, the database is inadequate
to determine if an antithyroid MOA is operating in mice. I n the absence of information to indicate otherwise, the thyroid tumors ob served in mice are considered relevant to humans

. 32

33

34

36

37

38

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35

1 Integration of thyroid effects The thyroid endpoints reported following chronic exposure to tert

- butanol include

increases in follicular cell hyperplasia and tumors in male and female mice

. As discussed above, due

to inadequacies in four of the five required areas ( U.S. EPA, 1998a ) , t he evidence is inadequate to determine if an antithyroid MOA is operating in m ice ; therefore, the MOA(s) for thyroid

tumor igenesi s has not been identified. EPA considers the thyroid follicular cell hyperplasia to be an early event in the neoplastic progression of thyroid follicular cell tumors , and n o other noncancer
effects on the thyroid were observed. Thus, t he hazard and dose

- response conclusions regarding
the thyroid follicular c ell hyperplasia and tumors associated with tert

- butanol exposure are discussed as part of the overall weight of evidence for carcinogenicity in Section 1. 3 .2.

**1.2.3. Developmental**  **Effects**

Synthesis of effects related to development 2

3

4

6

7

8

Developmental effects of tert

- butanol observed after oral exposure (liquid diets or gavage)
in several mouse strains and one rat strain include measures of fetal loss or viability (e.g., increase d number of resorptions, decreased numbers of neonates per litter) and decreased fetal body weight ( Lyondell Chemical Co., 2004 ; Faulkner et al., 1989 ; Daniel and Evans, 1982 ). Daniel and Evans (1982) also observed decreases in body weight gain during post

- natal days ( PND s) 2– 10; data
suggest , however, that th is effect might be due to altered maternal be havior or nutritional status. In addition, a single dose study reported a small increase in the incidence of variations of the skull or 9

11

12

13 Four studies e valuated developmental effects [ three oral or inhalation developmental
studies ( Faulkner et al., 1989 ; Nelson et al., 1989 ; Daniel and Evans, 1982 ) and a one

- generation,
oral reproductive study ( Lyondell Chemical Co., 2004 )] in animals exposed t o tert

- butanol via liquid diet (i.e., maltose/dextrin), oral gavage, or inhalation

. No developmental epidemiology studies are
available for tert

- butanol. The animal studies are arranged in the evidence tables by species, strain, and route of exposure. The design, conduct, and reporting of each study were reviewed, and each study was considered adequate to provide information pertinent to this assessment. One study was
considered less informative, Faulkner et al. (1989) , because it did not provide sufficient information
on the dams to de termine if fetal effects occurred due to maternal toxicity. 14

16

17

18

19

21

22

23

24

26

27

28

29 sternebrae in two mouse strains ( Faulkner et al., 1989 ). Although variations in skeletal development were noted in the study, no malformations were reported. Simila r developmental
effects were observed after whole

- body inhalation exposure in Sprague

- Dawley rats for 7 hours/day on gestation days ( GD s) 1– 19 ( Nelson et al., 1989 ). Fetal effects included dose

- related reductions in body weight in male and female fetuses and higher incidence of skeletal variations when analyzed based on individual fetuses ( but not on a per litter basis ).

In these studies

, fetal effects are generally observed at doses that cause toxicit y in the dams as measured by clinical signs (e.g., decreased body weight gain, food consumption) ( Table 1-7 ; Figure 1- 11 ; Figure 1- 12 ). As stated in the Guidelines for Developmental Toxicity Risk Assessment 31

32

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37

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2

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9

10 ( U.S. EPA, 1991b )

, “ an integrated evaluation must be performed considering all maternal and developmental endpoints. ” “[ W] hen adverse developmental effects are produced only at doses that

cause minimal maternal toxicity; in these cases , the developmental effects are still considered to represent developmental toxicity and should not be discounted .” Although, at doses of “ excessive maternal toxicity

... information on developmental effects may be difficult to interpret and of limited
value. ” In c o nsidering the fetal and maternal toxicity data following tert

- butanol exposure

, the severity of the maternal effects were m inimal and therefore the developmental effects in the fetuses
should not be discounted ( U.S. EPA, 1991b )

. T he observed fetal effects occurred, however, at doses
result ing in maternal toxicity across all available studies. Therefore, whether the fetal effects are directly related to tert

- butanol treatment or are secondary to maternal toxicity remains unclear

. Toxicological Review of tert

- Butyl Alcohol

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2 Toxicological Review of tert

- Butyl Alcohol

**Table 1**  **-7. E**  **vidence pertaining to developmental effects in animals following exposure to**  tert

**- butanol** 

Reference and study design Results

Lyondell Chemical Co. (2004)

Sprague

- Dawley rat; 12/sex/treatment Gavage 0, 64, 160, 400, or 1,000 mg/kg -d F0 males: 9 weeks beginning 4 weeks prior
t o mating F0 females: 4 weeks prior to mating through PND21 F1 Males and Females: 7 weeks (throughout gestation and lactation; 1 male and 1 female from each litter was dosed directly from PND 21 -28) Response relative to control

Dose (mg/kg

- d) 0 64 160 400 1000

Maternal effects
Body weight gain GD 0

- 20

0

− 3

− 4 0

− 16*
Food consumption GD 0

- 20

0 0 0 +4 0
Body weight gain PND 1

- 21

0 +3

− 10 +3 +100*

Food consumption LD1

- 14

0

− 2

− 6 0

− 16
Live pups/litter response relative to control

0

− 9

− 11

− 7

− 33*

Dams dosed with 400 or 1000 mg/kg

- d showed CNS effects (e.g., ataxia, lethargy) which were undetectable by 4

- weeks of exposure in animals exposed to 400 mg/kg

- d but not those in the higher dose group.

F1 effects

Viability index (pup survival to PND4)

96.4% 98.7% 98.2% 99.4% 74.1%*
Lactation index (pup survival to PND21)

100% 100% 100% 99.2% 98.8%
Sex ratio (% males)

54.4 52.3 50.9 53.4 52.1
Pup weight/litter PND 1 relative to control (%)

0 +6 +4 +7

− 10
Pup weight PND 28 relative to control (%)

M: 0 +2 0 0

− 12*

F: 0 0

− 4

− 2

− 8

3Toxicological Review of tert

- Butyl Alcohol

Reference and study design Results

Daniel and Evans (1982)

Swiss Webster (Cox) mouse; 15 pregnant dams/treatment Liquid diet (0, 0.5, 0.75, 1.0%, w/v) 0 (isocaloric amounts of maltose/dextrin), 3,324, 4,879, 6,677 mg/kg -d GD 6 –20 No statistical analysis was conducted on any of these data

Maternal

Percent change compared to control:

Dose (mg/kg

- d) Food consumption (mean g/animal/day) Body weight gain Number of litters (% pregnant dams)

0 0 0 11 (77%)

3,324 +2

− 3 12 (80%)

4,879

− 3

− 19 8 (53%)

6,677

− 4

− 20 7 (47%)

Authors note that lower food consumption in higher tert

- butanol dose groups reflects problems with pair feeding and maternal sedation.

Fetal
Percent change compared to control:

Dose (mg/kg

- d) Number of neonates/litter Fetal body weight on PND 2

0 0 0

3,324

− 1

− 7

4,879

− 29

− 19

6,677

− 49

− 38
Number of stillborn also increased with dose (3, 6, 14, and 20, respectively), but the number of stillborn per litter was not provided. The high dose also caused a delay in eye opening and a lag in weight gain during PND 2

– 10 (information was only provided in text or figures)
Faulkner et al. (1989) Maternal results not reported.

CBA/J mouse; 7 pregnant females in Fetal control, 12 pregnant females in treated Percent change compared to control: Incidence: Gavage (10.5 mmoles/kg twice a day); 0 (tap water), 1,556 mg/kg -d Live
GD 6 –18 Dose (mg/kg

- d) Resorptions/litter fetuses/ litter Fetal weight Sternebral variations Skull variations

0 0 0 0 4/28 1/28

1,556 +118*

− 41*

− 4 7/30 3/30
Sternal variations: misaligned or unossified sternebrae Skull variations: moderate reduction in ossification of supraoccipital bone
Number of total resorptions (10 resorptions/66 implants in controls, 37/94 implants in treated) increased ( p < 0.05)

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- Butyl Alcohol

Reference and study design Results

Faulkner et al. (1989) Maternal results not reported.

C57BL/6J mouse; 5 pregnant females in Fetal controls, 9 pregnant females treated Percent change compared to control: Incidence: Gavage (10.5 mmoles/kg twice a day) 0 (tap water), 1,556 mg/kg -d Live
GD 6 –18 Dose (mg/kg

- d) Resorptions/litter fetuses/ litter Fetal weight Sternebral variations Skull variations

0 0 0 0 5/21 1/21

1,556 +428*

− 58*

− 4 9/16 7/16
Sternal variations: misaligned or unossified sternebrae Skull variations: moderate reduction in ossification of supraoccipital bone
Number of total resorptions (4 resorptions/44 implants in controls, 38/68 implants in treated) increased ( p < 0.05)
Nelson et al. (1989)

Sprague

- Dawley rat; 15 pregnant dams/treatment Inhalation analytical concentration: 0, 2,200, 3,510, 5,030 ppm (0, 6,669, 10,640, 15,248 mg/m 3 ), (dynamic whole body chamber) 7 hr/d
GD 1 –19 Maternal: Unsteady gait (no statistical tests reported), dose

- dependent ↓ in body weight gain (results presented in figure only), dose

- dependent ↓ in food consumption ranging from 7

– 36% depending on dose and time

Fetal
Percent change compared to control (mean ± standard error):

Dose (mg/m 3 ) Number of live fetuses/litter Resorptions per litter

0 0 (13±2) 0 (1.1±1.2)

6,669 0 (13±4) +9 (1.2±1.1)

10,640 +15 (15±2)

− 18 (0.9±1.0)

15,248 +8 (14±2) 0 (1.1±0.9)
Percent change compared to control: Incidence:

Dose (mg/m 3 ) 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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— DO NOT CITE OR QUOTE * S tatistically 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/m 3 is 1 ppm = 3.031 mg/m 3

.

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

.1

2 3 4

5
6 Toxicological Review of tert

- Butyl Alcohol

* Study authors did not conduct statistical analysis on these endpoints, but results are determined by EPA to be biologically significant. Sources: (A) Daniel and Evans (1982) ; (B) Faulkner et al. (1989) ; Lyondell Chemical Co. (2004)

**Figure 1-**  **11**

**. Exposure** 

**- response array of**  **developmental effects following**  **oral exposure to**  tert

**- butanol** 

**.**

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2
3 Toxi cological Review of tert

- Butyl Alcohol

**Figure 1-**  **12**

**. Exposure** 

**- response array of**  **developmental effects following**  **inhalati on**  **ex posure to**  tert

**- butanol** 

**.**

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35 Three studies evalu ated neurodevelopmental effects ( Nelson et al., 1991 ; Daniel and Evans,
1982 ) [one in male rats; one in female rats] following tert

- butanol exposure via liquid diet (i.e., maltose/dextrin) or inhalation

. No epidemiology studies on neurodevelopment are available. The
animal studies evaluating neurodevelopmental effects of tert

- butanol contain study design
limitations. Daniel and Evans (1982) had a small number of animals per treatment group, lacked comparison of treatment

- related effects to controls for all endpoints investigated, and did not use long

- term neurodevelopmental testing. The two studies by Nelson et al. (1991) evaluated neurodevelopmental effects after either paternal or maternal exposure but did not run the exposures concurrently or provide exposure methods to indicate the studies were conducted similarly. The studies are arranged in the evidence tables by spe cies and sex.

Various neurodevelopmental effects have been observed in the available studies

. Th ese include changes in rotaro d performance following oral or inhalation exposures and decreases in open field behavior and cliff avoidance following oral exposu re , and reduced time hanging on wire after inhalation exposure during gestation ( Table 1-8 ).

Rot a rod performance

Inconsistent results were observed across studies. A lthough Daniel and Evans (1982) found
decreased rot a rod performance in mouse pups of dams orally exposed during gestation, Nelson et al. (1991) observed an increase in rot a rod performance in rat pups of dams exposed via inhalation during gestation.

Neurochemical measurements

B iochemical or physiological changes in the brain of offspring exposed during gestation or

ear ly in the postnatal period were examined in one study

. In this study, Nelson et al. (1991) reported statistically significant changes in neurochemical measurements in the brain in offspring 1 Integration of d evelopmental effects

There is suggestive evidence of developmental effects associated with tert

- butanol

exposure . Exposure to tert

- butanol during gestation resulted in increased fetal loss, decreased fetal body weight, and increases in skeletal variations in exposed offspring

. Dams had body weight losses or gains (or both) , decreased food consumption, and clinical signs of intoxication at the same doses
of tert

- butanol causing fetal effects. Therefore, determin ing whether tert

- butanol exposure results

in specific developmental toxicity or whether the fetal effects are due to maternal toxicity is
difficult. T he observed maternal effects are minimal, however, and thus, the developmental effects
observed in the fetuses are not discounted as being secondary to maternal toxicity ( U.S. EPA,
1991b ) and the evidence is considered suggestive of developmental toxicity.

**1.2.4. Neurodevelopmental**  **Effects**

Synthesis of effects related to neurodevelopment 2

3

4

6

7

8

9

11

12

13

14

16

17

18

19

21

22

23

24

26

27

28

29

31

32

33

34

This document is a draf t for review purposes only and does not constitute Agency policy. Toxic ological Review of tert

- Butyl Alcohol

1- 53 D R AFT

— DO NOT CITE OR QUOTE1 of both dams exposed vi a inhalation during gestation and treated adult males mated with untreated
dams

. T he strength of these results is compromised , however, because the two concentrations
tested (in both experiments) were not run concurrently

, and only data on statistically si gnificant effects were reported

. Therefore, comparison across doses or trend analysis for the effects is not
feasible.

Physiological and psychomotor development Daniel and Evans (1982) cross

- fostered half the mouse pups born to treated mother s with
untreated surrogate females to test the effects of maternal nutrition and behavioral factors on the

pups ’ physiological and psychomotor development. Results indicated that pups fostered to control dams performed significantly better than those maintained with treated dams ( Table 1-8)( Daniel and Evans, 1982 ). Data suggest that neurodevelopmental effects were not solely due to in utero

exposure to tert

- butanol ( Daniel and Evans, 1982 ). I nterpretation of these results is li mited

, however, as the neurodevelopmental data were presented only in figures and could not be compared with controls. 2

3

4

5

6

7

8

9

10

11

12

13

14

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

**- butanol**  16 Toxicological Review of tert

- Butyl Alcohol

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— DO NOT CITE OR QUOTE R e f e r e nc e a nd s t udy de s i g n R e s u l ts

D a ni el a nd E v a ns ( 1 9 8 2 )

S w i ss W e b st e r ( C o x ) m o u se ; 1 5 p r e gn a n t d a m s / tre a tm e n t L i q u i d d i e t ( 0, 0. 5, 0. 75, o r 1. 0%
, w / v ) ; G D6

– 20;

a f t er bi r t h ha l f t he p ups w er e n ur s ed w i t h t hei r t r e a t e d d a m s a n d t h e o t h e r h a l f w e r e f o st e r e d by unt r ea t e d da m s w ho r ec en t l y g a v e bi r t h 0 ( i so c a l o r i c a m o u n t s o f m a l t o se / d e x t r i n )

, 3, 324, 4, 879, o r 6, 677 m g / k g

- d • a do s e

- d e p e n d e n t i n c r e a se i n r i g ht i ng r e f l e x t i m e , w i t h m o r e t i m e

n e e d e d i n an i mal s mai n t ai n e d w i t h mat e r n al d ams

• a do s e

- de pen den t dec r ea s e i n o p en f i el d be ha v i o r , w i t h l e s s

a c t i v i t y i n pu ps m a i nt a i n ed w i t h m a t er na l da m s

• a do s e

- d e p e n d e n t d e c r e a se i n r o t a r o d per f o r m a nc e w i t h t he
p u p s f r o m mat e r na l da m s ha v i n g l o w er per f o r m a nc es

• a do s e

- d e p e n d e n t d e c r e a se i n t h e a m o u n t o f t i m e t h e p u p s w e r e

a b l e t o a v o i d a c l i f f

, w i t h an i mal s mai n t ai n e d w i t h t h e i r mat e r n al d a m s h a v i n g l e ss a v o i d a n c e t i m e

N e l s o n e t a l

. ( 1991)

S pr a g ue

- Da w le y ra t; 1 5 p re g n a n t d ams /t r e at me n t I nha l a t i o n a n a l y t i ca l co n ce n t r a t i o n : 0
, 6
, 0 0 0
, o r 12, 000 m g / m 3 ; ( dy na m i c w ho l e b o dy c ha m ber )
7 hr / d GD 1

– 19 D a t a w e r e n o t p r e se n t e d sp e c i f i c a l l y b y d o se n o r w e r e a n y t a b l e s o r f i gu r e s

o f

t he da t a pr o v i de d

M a t er na l t o x i c i t y w a s no t ed by d ec r ea s ed f o o d c o ns um pt i o n a n d bo dy

w e i gh t ga i n s

R e su l t s i n o f f sp r i n g

• i n c r e a se i n r o t a r o d pe r f o r m a nc e i n hi g h

- d o se gr o u p ( 1 6 v e r su s 2 6

r ev o l ut i o ns / m i n f o r c o n tro ls a n d 12, 000 m g / m 3 an i mal s , r e sp e c t i v e l y )

• d e c r e a se d t i m e hel d o n w i r e i n t h e per f o r m a nc e a s c e nt t e s t i n t he l o w

- d o se gr o u p ( 1 6 se c v e r s u s 1 0 se c f o r c o n t r o l s a n d 1
, 7 5 0

m g/ m 3 a n i m a l s, r e sp e c t i v e l y )1 2 3 4

5

6

7

8

9

10

11

12

13

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- Butyl Alcohol

Reference and s tudy design Results

The following differences in neurochemical measurements in the brain between control and treated offspring were observed,

• 53% decrease in norepinephrine in the cerebellum at 12,000 mg/m 3

• 57% decrease in met

- enkephalin in the cerebrum at 12,000 mg/m 3 and 83% decrease at 6,000 mg/m 3

• 61% decrease in β

- endorphin in the cerebellum at 12,000 mg/m 3

• 67% decrease in serotonin in the midbrain at 6,000 mg/m 3

Nelson et al. (1991) Adult male Sprague

- Dawley rats (18/treatment) mated to untreated females Inhalation analytical concentratio n: 0, 6,000, or 12,000 mg/m 3 ; (dynamic whole body chamber) 7 hr/d for 6 wk Data were not presented specifically by dose nor were any tables or figures of the data provided

Results (generally only specified as paternally treated versus controls) in offsprin g indicate

• increase in rotarod performance (16 versus 20 revolutions/min for controls and 12,000 mg/m 3 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/m 3 animals, respectively)
The following differences in neurochemical measurements in the brain between control and treated offsprin g were observed

• 39% decrease in norepinephrine in the cerebellum at 12,000 mg/m 3

• 40% decrease in met

- enkephalin in the cerebrum at 12,000 mg/m 3 and 75% decrease at 6,000 mg/m 3

• 71% decrease in β

- endorphin in the cerebellum at 12,000 mg/m 3

• 47% decrease in se rotonin in the midbrain at 6,000 mg/m 3

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

Note: Conversions from diet concentrations to mg/kg

- d performed by study authors. Percentage change compared to control = (treated value – contro l value) ÷ control value × 100.

Mechanistic Evidence

No mechanistic evidence is available for reproductive , developmental

, or neurodevelopmental effects.

Integration of n eurod evelopmental effects Neurodevelopmental effects

, including decreased brain weight, changes in brain biochemistry, and changes in behavioral performances

, have been observed. E ach stud y evaluating neurodevelopmental effects, however, had limitations in study design

, reporting , or both

. In addition, resul ts were not always consistent between studies or across dose. At this time, there is

in adequate information to draw conclusions regarding neurodevelopmental toxicity.1 **1.2.5. Reproductive**  **Effects**

Synthesis of effects related to reproduction

Several studies eval uat ed reproductive effects [a on e- generation, oral reproductive study ( Lyondell Chemical Co., 2004 ) and subchronic evaluations in rats and mice following oral and inhalation exposure ( NTP, 1997
, 1995 ) ] in animals exposed to tert

- butanol via oral gavage, drinking water, or inhalation for ≥ 63 days . The studies are arranged in the evidence tables by sex, route of exposure, duration of exposure, and species. The collection of studies evaluating reproductive
effects of tert

- butanol is limited by the absence of two

- generation reproductive oral or inhal ation studies and by having no human studies on reproduction. The design, conduct, and reporting of each study were r eviewed, and each study was considered adequate to provide information pertinent to this assessment

. 2

3

4

5

6

7

8

9

10

11

12 R eproductive endpoints , such as reprod uctive organ weights, estrous cycle length, and sperm effects were examined following either oral or inhalation exposure ( Lyondell Chemical Co.,
2004 ; NTP, 1997
, 1995 ) ( Table 1-9; Figure 1- 13 ; Figure 1- 14 ). In males, the only significant effect
observed was a slight decrease in sperm motility for F0 males treated with 1000 mg/kg

- day of tert
butanol ( Lyond ell Chemical Co., 2004 )

. No significant changes in sperm motility were reported following oral exposure in other rat studies or via inhalation exposure in mice or rats. In addition,
the reduced motility in treated animals falls within the range of histo rical control data and, therefore, its biological significance is uncertain. In female B6C3F 1 mice, estrous cycle length was
increased 28% following oral exposure to 11,620 mg/kg -day ( NTP, 1995 ). No significant changes in

estrous cycle length were observed following oral exposure in rats, or inhalation exposure in mice

or rats. 13

14

15

16

17

18

19

20

21

22

23 **Table 1-9.**  **Evidence pertaining to r**  **eproductive effects in**  **animals following**  **exposure to** tert

**- butanol**  24

R e f e r e nc e a nd s t udy de s i g n R e s u l ts

M al e r epr o duc t i v e ef f

ec t s

L yo n d e l l Ch e m i c a l Co

. ( 2004) S pr a g ue

- D a w l e y r a t ; 1 2 / se x / t r e a t m e n t G a v a g e 0, 64, 160, 400, o r 1, 000 m g / k g

- d
F 0 m a l e s: 9 w e e k s b e gi n n i n g 4 w e e k s p r i o r to
mat i n g P N D 2 1 F 0 r e p r o d u c t i v e e f f e c t s

S per m m o t i l i t y ( o nl y c o nt r o l a nd hi g h

- d o se gr o u p s e x a m i n e d ) 0: 94% 1000: 91% *

N o o t h e r si g n i f i c a n t e f f e c t o n w e i gh t s o f m a l e r e p r o d u c t i v e o r ga n s o r sp e r m

o b se r v e d

N T P ( 1995)
F 344/ N r a t ; 10/ s e x / t r e a t m e n t Drin ki n g w a te r ( 0, 2. 5, 5, 10, 20, o r 40 m g / m L )
M : 0, 230, 490, 840, 1, 520
, 3, 610 a m g/ k g -d
1 3 w e e k s N o si gn i f i c a n t e f f e c t o n w e i g h t s o f m a l e r e p r o d u c t i v e o r ga n s o r s p e r m

o b se r v e d Toxicological Review of tert

- Butyl Alcohol

­

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— DO NOT CITE OR QUOTEToxicological Review of tert

- Butyl Alcohol

Reference and s tudy design Results

NTP (1995) No significant effect on weights of male reproductive organs or sperm B6C3F 1 mouse; 10/sex/treatment observe d Drinking water ( 0, 2.5, 5, 10, 20, or 40 mg/mL ) M: 0, 350, 640, 1,590, 3,940, 8,210 a mg/kg -d 13 wee ks
NTP (1997) No significant effect on weights of male reproductive organs or sperm F344/N rat; 10/sex/treatment observed Inhalation analytical concentration: 0, 134, 272, 542, 1,080, or 2,101 ppm (0, 406, 824, 1,643, Evaluations were performed only for concentrations ≥542 ppm 3,273 or 6,368 mg/m 3 ) (dynamic wh ole body (1,643 mg/m 3 ) chamber) 6 hr/d, 5 d/wk 13 weeks Generation method (Sonimist Ultrasonic spray nozzle nebulizer), analytical concentration and method were reported
NTP (1997) No significant effect on weights of male reproductive organs or sperm B6C3F 1 mouse; 10/sex/treatment observed Inhalation analytical concentration: 0, 134, 272, 542, 1,080, or 2,101 ppm (0, 406, 824, 1,643, Evaluations were performed only for concen trations ≥542 ppm 3,273 or 6,368 mg/m 3 ) (dynamic whole body (1,643 mg/m 3 ) chamber) 6 hr/d, 5 d/wk 13 weeks Generation method (Sonimist Ultrasonic spray nozzle nebulizer), analytical concentration and method were reported

Female reproductive effects

Lyondell Chemical Co. (2004) Pregnancy index Sprague

- Dawley rat; 12/sex/treatment Gavage 0, 64, 160, 400, or 1,000 mg/kg -d 91.7% 91.7% 100% 100% 91.7% F0 females: 4 weeks prior to mating through PND21
NTP (1995) No significant effect on female estrous cycle (0, −2, − 4, 0, +8 % change F344/N rat; 10/sex/treatment relative to control) Drinking water ( 0, 2.5, 5, 10, 20, or 40 mg/mL ) F: 0, 290, 590, 850, 1,560, 3,620 a mg/kg -d 13 weeks
NTP (1995) ↑ length of estrous cycle
B6C3F 1 mouse; 10/sex/treatment Drinking water ( 0, 2.5, 5, 10, 20, or 40 mg/mL ) Response relative to control : 0, +5, +5, +5, +6, +28*% F: 0, 500, 820, 1,660, 6,430, 11,620 a mg/kg -d 13 weeks

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N T P ( 1997)
F 344/ N r a t ; 10/ s e x / t r e a t m e n t I nha l a t i o n a na l y t i c a l c o nc ent r a t i o n: 0
, 1 3 4
, 2 7 2
, 542, 1, 080, o r 2, 101 p p m ( 0, 406
, 824, 1, 643, 3, 273 o r 6, 368 m g / m 3 ) ( dy na m i c w ho l e bo dy

c ha m ber ) 6 h r / d
, 5 d / w k
1 3 w e e k s G ene r a t i o n m et ho d ( S o ni m i s t U l t r a s o ni c s p r ay

no z z l e nebul i z er )

, a na l y t i c a l c o nc ent r a t i o n a nd

m e t h o d w e r e r e p o r t e d N o s i g n i f i c an t e f f e c t o n f e mal e e s t r o u s c y c l e ( 0
, − 4
, + 2
, + 4 % c h an g e r e l at i ve to c o n tro l)

E val u at i o n s w e r e p e r f o r me d o n l y f o r c o n c e n t r at i o n s ≥ 5 4 2 p p m ( 1, 643 m g/ m 3 )

N T P ( 1997)
B 6C3F 1 m o u se ; 1 0 / s e x / t r e a t m e n t I nha l a t i o n a na l y t i c a l c o nc ent r a t i o n: 0
, 1 3 4
, 2 7 2
, 542, 1, 080, o r 2, 101 p p m ( 0, 406
, 824, 1, 643, 3, 273 o r 6, 368 m g / m 3 ) ( dy na m i c w ho l e bo dy

c ha m ber ) 6 h r / d
, 5 d / w k
1 3 w e e k s G ene r a t i o n m et ho d ( S o ni m i s t U l t r a s o ni c s pr a y

n o zzl e n ebul i z er )

, a na l y t i c a l c o nc ent r a t i o n a nd

m e t h o d w e r e r e p o r t e d N o s i g n i f i c an t e f f e c t o n f e mal e e s t r o u s c y c l e ( 0
, − 3
, − 9
, − 5 % c h an g e r e l at i ve to c o n tro l)

E val u at i o n s w e r e o n l y p e r f o r me d f o r c o n c e n t r at i o n s ≥ 5 4 2 p p m ( 1, 643 m g/ m 3 )

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

Notes: Conversions from drinking water concentrations to mg/kg

- d performed by study authors. Conversion from ppm to mg/m 3 is 1 ppm = 3.031 mg/m 3

. Percentage change compared to control = (treated value – contro l value) ÷ control value × 100 1

2 3 4 Toxicological Review of tert

- Butyl Alcohol

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S ources: (A) Lyondell Chemical Co. (2004) ; (B) NTP (1995)

. **Figure 1-**  **13**

**. Exposure** 

**- response array of reproductive effects**  **following oral exposure to**  te rt

**- butanol.** 

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2 Toxi cological Review of tert

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**Figure 1-**  **14**

**. Exposure** 

**- response array of reproductive effects following**  **inhalation exposure to** ter t

**- butanol.** 

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1 Integration of reproductive effects
At this time, no conclusions are drawn in regard to reproductive toxicity. The database is
limited to a one

- generation study ( Lyondell Chemical Co., 2004 ; NTP, 1995 ). N o two

- generation reproductive studies are available that evaluate oral or inhalation exposure

. In males, the only
observed effect was a slight decrea se in sperm motility for F0 males in the highest dose group of
rats treated with tert

- butanol. This effect was not observed, however, in other studies with orally treated rats and mice or in rats exposed via inhalation. In females, NTP (1995) reported an
increased length of the estrous cycle in the highest dose group of orally exposed mice. This effect
was not observed in similarly treated rats or in mice and rats exposed via inhalation. 2

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**1.2.6. Other Toxicological Effects** 

E ffects other than those related to kidney, thyroid, reproductive, developmental, and neurodevelopment al toxicity were observed in some of the available rodent studies ; t hese include liver and urinary bladder effects. D ue to a lack of consistency in the liver effects and minimal to
mild effects with a lack of progression in urinary bladder , however, in adeq uate information is
available to draw conclusions regarding liver or urinary bladder toxicity at this time

. Additionally, central nervous system ( CNS ) effects similar to those caused by ethanol ( animals appearing intoxicated and having withdrawal symptoms after cessation of oral or inhalation exposure ) were observed

. D ue to study quality concerns (e.g., lack of data reporting,
small number of animals per treatment group) , however, adequate information to assess CNS toxicity is un avail able at this time

. For more information on these other toxicological effects, see Appendix B.3. 11

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## 1.3. **INTEGRATION AND EVALUATION** 

23 **1.3.1. Effects Other Than Cancer** 

K idney effects were identified as a potential human hazard of tert

- butanol exposure based on several endpoints , including suppurative inflammation in female rats, transitional epithelial hyperplasia in male and female rats, severity of nephropathy in male and female rats, incidences of nephropathy in female rats, mineralization in male rats, and increased kidney weights in b oth male
and female rats. These effects are similar to the kidney effects observed with ETBE exposure (e.g., CPN and urothelial hyperplasia) and MTBE (e.g., CPN and mineralization) ( ATSDR, 1996 ).
Several effects were observed in the kidneys of rats. B ased on m echan istic evidence indicating that an α 2u -globulin- related process is operating in male rats ( Hard et al., 2011 ; Cirvello et al., 1995 ; NTP, 1995 ; Lindamood et al., 1992 ) , any kidney effects associated with α 2u -globulin nephropathy are not considered relevant for human hazard identification

. In addition, CPN play ed a role in the renal tubule nephropathy observed foll owing tert

- butanol exposure , and effects
associated with such nephropathy are not considered relevant for human hazard identification

. Although increases in severity (males and females) or incidence (females) of nephropathy were 24

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1 related to tert

- butanol exp osure and could have arisen from chemical

- specific processes independent from CPN, the association of these effects with CPN makes this measure less suitable
for dose

- response analysis, and therefore these effects were not considered for the derivation of
reference values. Furthermore, some uncertainty exists regarding whether mineralization is also
associated with CPN in male rats; due to this uncertainty, and because other kidney effects were identified as being associated with tert

- butanol exposure and y et independent from CPN,
mineralization in male rats was not considered for dose

- response analysis. The remaining effects (suppurative inflammation, transitional epithelial hyperplasia

, and increased kidney weights) are considered the result of tert

- butano l exposure and relevant to human hazard characterization

. These effects therefore are suitable for consideration for dose

- response analysis and derivation of
reference values

, in S ection 2

. 2

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There is suggestive evidence of developmental effects associated with tert

- but an ol

exposure. Increased fetal loss, decreased fetal body weight, and increases in skeletal variations in exposed offspring were observed following e xposure to relatively high doses of tert

- butanol during gestation

.. These effects are similar to the developmental effects observed with MTBE exposure (e.g., decreased fetal body weight and increases in skeletal variations) ( ATSDR, 1996 ). No mechanistic evidence is available for developmental effects of tert

- butanol. A lthough the evidence is suggestive of developmental toxicity, due to the uncertainty as to whether fetal effects
were due to direct effects of tert

- butanol or indirect effects of maternal toxicity and the lack of consistency across some endpoints, developm ental effects were not considered for dose

- response analysis and derivation of reference values in S ection 2

. Further more , no adverse effects were reported in one

- and two

- generation reproductive/developmental studies on ETBE ( Gaoua, 2004a
, b ) , providing further support for the lack of evidence supporting reproductive or developmental
effects as possible human hazard s following tert

- butanol exposure

. At this time, there is inadequate information to draw conclusions regarding neurodevelopmental effects as a human hazard of tert

- butanol exposure. Although neurodevelopmental effects have been obser ved

, the studies had limitations in design or reporting

, or both, and results were inconsistent between studies and across dose groups

. No mechanistic evidence is available to inform the MOA for neurodevelopmental effects of tert

- butanol. These
effects wer e not considered further for dose

- response analysis and derivation of reference values. At this time, no conclusions are drawn regarding reproductive effects as a human hazard of

tert

- butanol exposure. The only reproductive effect observed due to tert

- but anol exposure was increased length of estrous cycle ( NTP, 1995 ) in the hig hest dose group of orally exposed mice, and this effect was not observed in orally exposed rats or in mice and rats exposed via inhalation. Further, the database was limited and contained only two oral exposure studies and one subchronic inhalation study. No mechanistic or MOA information is available for reproductive effects of tert
butanol. T h ese effects were not considered further for dose

- response analysis and derivation of
reference values. 9

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1 At this time, there is inadequate information to draw conclusions regarding liver or urinary
bladder toxicity due to lack of consistency of effects and minimal/mild effects showing a lack of progression, respectively

. No mechanistic evidence is available for these effects. Th e liver and urinary bladder effect s were not considered further for dose

- response analysis and the derivation
of reference values. 2

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6 **1.3.2. Carcinogenicity** 

Summary of evidence In F344/N rats, administration of tert

- butanol in drinking water increased the incidence of renal tubule tumors, mostly ade nomas, in males; no renal tumors in females were reported ( Hard et al., 2011 ; NTP, 1995 ) . As discussed in S ection 1.2.1, some of these tumors might be associated with

α 2u - globulin nephropathy, a n MOA considered specific to the male rat ( U.S. EPA, 1991a ) . Evidence in support of this hypothesized MOA includes the accumulation of hyaline droplets in renal tubule cells, the prese nce of α 2u - globulin in the hyaline droplets, and additional aspects associated with 7

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14 α 2u - globulin nephropathy, including linear papillary mineralization and foci of tubular hyperplasia. Other evidence, however, is not supportive: T he accumulation of hyaline droplets was minimal;

concentrations of α 2u - globulin were low at doses that induced tumors; and no significant necrosis

or cytotoxicity was associated with compensatory regenerative proliferation or induction of granular casts observed within a timeframe consistent with α 2u -globulin- mediated nephropathy.
Renal tumors also could be associated with chronic progressive nephropathy, but the data on CPN
are not coherent: D ose

- response relationships for CPN, renal tubule hyperplasia, and renal tubule 16

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21 tumors were different ; in addition, CPN was nearly as severe in female rats as in male rats , yet no female rats developed renal tumors. Thus, some renal tumors may be attributable to α 2u - globulin nephropathy and some to other, yet unspecified, processes. Taken togeth er, and a ccording to EPA ’s guidance on renal tumors in male rats ( U.S. EPA, 1991a ) , renal tumors induced by tert

- butanol are
relevant for human hazard identification. 22

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26 In B6C3F 1 m ice, administration of tert

- butanol in drinking water increased the incidence of thyroid follicular cell adenomas in females, and adenomas or carcinomas (only one carcinoma observed) in males ( N TP, 1995 ) , as discussed in S ection 1.2.2. According to EPA’s thyroid tumor guidance ( U.S. EPA, 1998a ) , chemicals that produce thyroid tumors in rodents might pose a carcinogenic hazard to humans. 27

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31 In addition, as mentioned in S ection 1.1.4, tert

- butanol is a primary metabolite of MTBE and of ETBE, two c ompounds tested in rats and mice that could provide supplementary information on the carcinogenicity of tert

- butanol. For MTBE, the most recent cancer evaluation by a national or international health agency is from IARC (199 9) . IARC reported that oral gavage exposure in Sprague

- Dawley rats resulted in testicular tumors in males and lymphomas and leukemias (combined) in females; inhalation exposure in male and female F344 rats resulted in renal tubule
adenomas in males; and inhalation exposure in male and female CD

- 1 mice resulted in 32

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1 hepatocellular adenomas in females ( IARC, 1999 ) . For ETBE, a draft IRIS assessment under development concurrently with this assessment reports that inhalation exposure in ma le and female F344 rats resulted in hepatocellular tumors, mostly adenomas, in males; no significant

tumor increases were reported for 2- year studies by drinking water exposure in male and female F344 rats or by oral gavage in male and female Sprague

- Dawley rats.

Integration of evidence

This evidence leads to consideration of two hazard descriptors under EPA’s cancer guidelines ( U.S. EPA, 2005a ) . The descriptor likely to be carcinogenic to humans is appropriate when
the evidence is “adequate to demonstrate carcinogenic potential to humans” but does not support the descriptor carcinogenic to hum ans . One example from the cancer guidelines is “an agent that has tested positive in animal experiments in more than one species, sex, strain, site, or exposure route,
with or without evidence of carcinogenicity in humans.” t e rt -B utanol matches the conditi ons of this example, having increased tumor incidences in two species, in both sexes, and at two sites. 2

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14 Alternatively, the descriptor suggestive evidence of carcinogenic potential is appropriate
when the evidence raises “a concern for potential carcinogen ic effects in humans” but is not sufficient for a stronger conclusion. The results for tert

- butanol raise a concern for cancer but none of the effects is particularly strong. The kidney tumors resulted, in part, from a n MOA that is specific to male rats, w hile no kidney tumors occurred in female rats. The thyroid tumors induced in male and female mice were almost entirely benign. In addition, while MTBE was also associated with male rat kidney tumorigenesis, there is little coherence of results between tert

- butanol and ETBE 16

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21 associated tumorigenesis in rats. MTBE or ETBE effects following chronic oral exposure in mice
have not been investigated , h owever, so no evidence exists to evaluate the coherence of the thyroid tumorigenesis observed following tert

- butan ol exposure in B6C3F 1 mice.
These considerations, interpreted in light of the cancer guidelines, support the conclusion
that there is s uggestive evidence of carcinogenic potential for tert

- butanol. Although increased tumor
incidences were reported f or two species, two sexes, and two sites, none of the tumor responses
was strong or coherent with the results for ETBE , and this was decisive in selecting a hazard descriptor. 22

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29 The descriptor suggestive evidence of carcinogenic potential ap plies to all routes of human exposure. Oral administration of tert

- butanol to rats and mice induced tumors at sites beyond the point of initial contact, and inhalation exposure for 13 weeks resulted in absorption and
distribution of tert

- b utanol into th e systemic circulation, as discussed in S ection 1.2.1. According to the cancer guidelines, this information provides sufficient basis to apply the cancer descriptor developed from oral studies to other exposure routes.

Biological considerations for dose –re sponse analysis Regarding hazards to bring forward to S ection 2 for dose

- response analysis, EPA’s guidance
on renal tumors in male rats ( U.S. EPA, 1991a ) advises that unless the r elative contribution of 31

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1 α 2u - globulin nephropathy and other process can be determined, dose

- response analysis should not be performed. As discussed in S ection 1.2.1, the available data do not allow such determination, and so an analysis of kidney tumors doe s not appear in S ection 2. EPA’s guidance on thyroid tumors and EPA’s cancer guidelines ( U.S. EPA, 1998a ) advises that for thyroid tumors resulting from thyroid

– pituitary d isruption, dose

– response analysis should use nonlinear extrapolation, in the absence of MOA information to indicate otherwise . As discussed

in S ection 1.2.2, increases in thyroid follicular cell hyperplasia in male and female mice provide partial support for thyroid

- pituitary disruption. Other necessary data on tert

- butanol, however, are not adequate or are not supportive. There is little correlation among thyroid, pituitary, and liver
effects in female mice

, and no data are available to evaluate the poten tial for antithyroid effects in
male mice . D ata are not adequate to conclude that thyroid hormone changes exceed the range of homeostatic regulation or to evaluate effects on extrahepatic sites involved in thyroid

- pituitary 2

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disruption . Also, no data are av ailable to evaluate reversibility of effects upon cessation of exposure. Thus, according to EPA’s thyroid tumor guidance, conclud ing that the thyroid tumors result from thyroid

- pituitary disruption is premature, and dose

- response analysis should use linear extrapolation. The data are well suited to dose

- response analysis, coming from an NTP study that
tested multiple dose levels.

**1.3.3. Susceptible Populations and Lifestages**  **for Cancer and Noncancer Outcomes**

No chemical

- specific data that would allow for the ident ifi cation of populations with increased susceptibility to tert

- butanol exposure exist

. In vitro studies have implicated the liver
microsomal mixed function oxidase (MFO) system, namely CYP450 ( Cederbaum et al., 1983 ; Cederbaum and Cohen, 1980 ), as playing a role i n the metabolism of tert

- butanol

. N o studies

, however, have identif ied the specific CYPs responsible for the biotransformation of tert

- butanol

. Pharmacokinetic differences among the fetus, newborns, children, and the aged might alter responses to chemicals compared to adults, resulting in differences in health effects. In the presence of environmental chemicals, metabolic homeostasis is maintained by the liver ’s ability to detoxify and eliminate xenobiotics. This process is accomplished, in part, by the exp ression of xenobiotic metabolizing enzymes and transporters (XMETs) , which metabolize and transport xenobiotics and determine whether exposure will result in altered responses. The expression of
XMETs , including various CYPs, ha s been found to be underexpressed in the mouse fetus and

neonate ( Lee et al., 2011 ) and decreased in older mice ( Lee et al., 2011 ) and rats ( Lee et al., 2008 ). Decreased ability to detoxify and transport tert

- butanol out of the body could r esult in increased susceptibility to tert

- butanol in the young and old

. In regard to cancer, although c hildren are more sensitive than adults to thyroid carcinogenesis resulting from ionizing radiation, relative differences in lifestage sensitivity to
che mical ly induced thyroid carcinogenesis are unknown ( U.S. EPA, 1998a ). In addition, the data on

tert

- butanol mutagenicity are inconclusive. 9

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, there is little evidence on tert

- butanol itself to identify susceptible populations or lifestages

. 1

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# 2. DOSE

- RESPONSE ANALYSIS

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## 2.1. **ORAL REFERENCE DOSE FOR EFFECTS OTHER THAN CANCER** 

The reference dose ( RfD

, expressed in units of mg/kg

- day) is defined as an estimate (with uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human popu lation (including sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a lifetime

. The RfD can be derived from a no

- observed

- adverse

- effect level (NOAEL), lowest

observed

- adverse

- effect level (LOAEL), or the 95% lower bound on the benchmark dose (BMDL), with uncertainty factors (UFs) generally applied to reflect limitations of the data used.

**2.1.1. Identification of Studies and Effects for Dose** 

**- Response Analysis** 

EPA identified kidney effects as a potential human hazar d of tert

- butanol exposure (see Section 1.2.1)

. Studies within this effect category were evaluated using general study quality characteristics [as discussed in Section 6 of the Preamble ; see also U.S. EPA (2002) ] to help inform
the selection of studies from which to derive toxicity values. No other hazards were identified for
further for consideration in the d erivation of reference values.

Human studies are preferred over animal studies when quantitative measures of exposure are reported and the reported effects are determined to be associated with exposure. N o human occupational or epidemiological studies of oral exposure to tert

- butanol , however, are available

. Animal studies were evaluated to determine which studies provided : ( 1) the most relevant routes and durations of exposure

, (2 ) multiple exposure levels to provide information about the shape of the dose

- response curve , and (3 ) power to detect effects at lo w exposure levels. The
database for tert

- butanol includes both chronic and subchronic studies showing effects in the kidney that are suitable for deriving reference values.

Kidney Toxicity

EPA identified kidney effects as a potential human hazard of tert

- butanol

- induced toxicity based on findings in male and female rats (summarized in S ection 1.3.1)

. Kidney toxicity was
observed across multiple chronic, subchronic, and short

- term studies following oral and inhalation

exposure. Kidney effects observed after chronic exposure, such as suppurative inflammation and transitional epithelial hyperplasia, may impact the ability of the kidney to filter waste. Observed changes in k i dney weight could also indicate toxic effects in the kidn ey. For the oral tert

- butanol database, there are several studies available that evaluated these kidney effects. Lyondell Chemical Co. (2004) conduc ted a reproductive study in Sprague

- Dawley rats that was of shorter duration

, and reported changes in kidney weight but did not examine changes in histopathology. NTP5

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1 conducted a 2- year drinking water study ( NTP, 1995 ) in F344 rats that evaluated multiple doses in
both males and females , and reported on all three endpoints highlighted above

. NTP (1995) was identified as most suitabl e for dose

- response assessment considering the study duration, comprehensive reporting of outcomes, and multiple doses tested

. 2

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In the NTP (1995) 2- year drinking water study, male F344 rats were exposed to approximate doses of 0, 90, 200, or 420 mg/kg

- day; female F344 rats were exposed to approximate doses of 0, 18 0, 330, or 650 mg/kg

- day. Reduced body weights and survival were observed and
reflected in some of the effects

. Kidney effects

, including changes in organ weight

, histopathology

, or both, were observed in both sexes of rats after 13 weeks, 15 months, and 2 years of treatment ( NTP, 1995 ). Specific endpoints chosen for dose

- response analysis were absolute kidney weight (observed in males and females ), kidney suppurative inflammation (observed in females), and kidney transitional epithelial hyperplasia (observed in males and females). For absolute kid ney weight, data from 15 months was selected as described in S ection 1.2.1; for the other endpoints, data at the longest duration of 2 years were selected

. 6

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considered biologically significant, a BMR of 1 standard deviation from the control mean for
continuous data or a BMR of 10% extra risk for dichotomous data is used to estimate the BMD and

BMDL , and als o to facilitate a consistent basis of comparison across endpoints, studies, and

assessments

. Endpoint

- specific BMRs, where feasible, are described further below. When modeling was feasible, t he estimated BMDLs were used as points of departure (PODs) ; the P ODs are
summarized in Table 2-1. Further details including the modeling output and graphical results for the model selected for each endpoint can be found in Appendix C of the S upplemental Information

to this Toxicological Review

. Kidney weights were analyzed as absolute weights rather than relative to body weight. In general, absolute and relative kidney weight data can both be considered appropriate endpoints for analysis ( Bailey et al., 2004 ). In the NTP (1995) 2- year drinking water study, body weight in exposed animals noticeably decreased rela tive to controls at the 15

- month interim sacrifice (see
Table 1-1), but this decrease in body weight impacted the measure of relative kidney weight resulting in an exaggeration of the kidney weight change . Th ere was greater confidence in the absolute kidney weight measure; thus

, it was considered more appropriate for dose

- response analysis, and changes in relative kidney weights were not analyzed. A 10% relative change from 9

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**2.1.2. Methods of Analysis**  No biologically based dose

- response models are available for tert

- butanol

. In t his situation, EPA evaluates a range of dose

- response models thought to be consistent with underlying biological processes to determine how best to empirically model the dose

- response relationship in the range
of the observed data

. The models in EPA’s Benc hmark Dose Software (BMDS) were applied

. Consistent with EPA ’s Benchmark Dose Technical Guidance ( U.S. EPA, 2012b ) , the BMD and the

BMDL a re estimated using a benchmark response (BMR) to represent a minimal, biologically significant level of change. In the absence of information regarding the level of change that is 16

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1 control was used as a BMR for absolute kidney weight by analogy with a 10% change in body weight as an indicator of toxicity. A BMR of 10% extra risk was considered appropriate for the quantal data on incidences of kidney suppurati ve inflammation and kidney transitional epithelial hyperplasia. Human equivalent doses (HEDs) for oral exposures were derived from the PODs according to the hierarchy of approaches outlined in EPA’s Recommended Use of Body Weight 3/4 as the Default
Method in Derivation of the Oral Reference Dose ( U.S. EPA, 2011 ). The preferred approach is physiological ly based toxicokinetic modeling (PBPK)

. Other approaches include using chemical

specific information in the absence of a complete PBPK model. As discussed in Appendix B of the Supplemental Information, human PBPK models for inhalation of ETBE or inhalation and dermal

exposure to MTBE have been p ublished

, which include tert

- butanol submodels. A validated human
PBPK model for tert

- butanol , however, is not available for extrapolating doses from animals to
humans. In lieu of either chemical

- specific models or data to inform the derivation of human

eq uivalent oral exposures, body weight scaling to the 3⁄4 power (i.e., BW 3/4 ) is applied to extrapolate toxicologically equivalent doses of orally administered agents from adult laboratory animals to adult humans for the purpose of deriving an oral RfD. 2

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Using a standard BW a of 0.25 kg for rats and a BW h of 70 kg for humans ( U.S. EPA, 1988 )

, the resulting DAF is 0.24 for rats

. Applying this DAF to the POD identified for effects in adult rats yields a POD HED as follows (see Table 2-1 ):

POD HED = Laboratory animal dose (mg/kg

- day) × DAF

Table 2-1 summarizes all PODs and the sequence of calculations leading to the derivation of
a human

- equivalent POD for each endpoint discussed above. 9

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animals we re converted to HEDs employing a standard dosimetric adjustment factor (DAF) derived
as follows:

DAF = (BW 1/4 / BW 1/4 a h ),
where

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BW h = human body weight 18

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2 Toxicological Review of tert

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**Table 2**  **-1. S**  **ummary of derivations of points of departure following oral**  **exposure f**  **or up to 2 years**

Endpoint and

r eference Species/

sex Model a BMR BMD (mg/kg

- d) BMDL (mg/kg

- d ) POD ADJ b (m g/kg

- d) POD HED c (mg/ kg

- d)

Kidney

Increased absolute kidney weight at 15 months NTP (1995) Rat/M Linear (constant variance) 10% 657 296 296 71

Increased absolute kidney weight at 15 months NTP (1995) Rat/F Exponential (M4) (constant variance) 10% 164 91 91 22

Kidney inflammation (suppurative) NTP (1995) Rat/F Log

- probit 10% 254 200 200 48

Kidney transitional epithelial hyperplasia NTP (1995) Rat/M Log

- logistic 10% 30 16 16 3.84

Kidney transitional epithelial hyperplasia NTP (1995) Rat/F Multistage, 3- degree 10% 412 339 339 81.4

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— DO NOT CITE OR QUOTE a For modeling details, see Appendix C in Supplemental Information

. b For studies in which animals were not dosed daily, EPA would adjust administered doses to calculate the TWA

daily doses prior to BMD modeling. However, this adjustment was not required for the NTP (1995) study

.
c HED PODs were calculated using BW 3/4 scaling ( U.S. EPA, 2011 ).

NA= not applicable

**2.1.3. Derivation of Candidate**  **Values**

Consistent with EPA’s A Review of the Reference Dose and Reference Concentration Processes [( U.S. EPA, 2002 ) ; Section 4.4.5 ] , also described in the Preamble, five p ossible areas of uncertainty and variability were considered when determining the application of UFs to the PODs presented in
Table 2

- 1. An explanation follows: An intraspecies uncertainty factor, UF H , of 10 was applied to all PODs to account for potential differences in toxicokinetic s and toxicodynamic s in the absence of information on the variability of response in the human population following oral exposure to tert

- butanol ( U.S. EPA,
2002 ). An interspecies uncertainty factor, UF A , of 3 (10 0.5 = 3.16, rou nded to 3) was a pplied to all
PODs because BW 3/4 scaling wa s used to extrapolate oral doses from laboratory animals to humans.5

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1 Although BW 3/4 scaling addresses some aspects of cross

- sp ecies extrapolation of toxicokinetic and toxicodynamic processes, some residual uncertainty in the extrapolation remains. In the absence of
chemical

- specific data to quantify this uncertainty, EPA’s BW 3/4 guidance ( U.S. EPA, 2011 ) recommends use of an uncertainty factor of 3

. A subchronic to chronic uncertainty factor, UF S
, of 1 was applied to all PODs because the endpoints were all observed following chronic exposure. A LOAEL to NOAEL uncertainty factor, UF L , of 1 was applied to all PODs derived because the current approach is to address this factor as one of the considerations in selecting a BMR for
benchmark dose modeling

. In this case, BMR s of a 10% relative change in absolute kidney weight, a 10% extra risk of kidney suppurative inflammation, and a 10% extra risk of transitional cell hyperplasia were selected assum ing they represent minimal biologically significant response leve ls.
A database uncert ainty factor, UF
, of 1 was applied to all PODs

. T he tert

- butanol oral toxicity
database includes chronic and subchronic toxicity studies in rats and mice ( Acharya et al., 1997 ; Acharya et al., 1995 ; NTP, 1995 ) and developmental toxicity studies in rats and mice ( Lyondell Chemical Co., 2004 ; Faulkner et al., 1989 ; Daniel and Evans, 1982 ). In the deve lopmental studies, no 2

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immune system effects would not be a sensitive target for tert

- butanol. No adverse effects were reported in one

- and two

- generation reproductive/developmental studies on ETBE ( Gaoua, 2004a
, b ), indicating that reproductive/developmental effects would not be a sensitive target for tert
butanol. Additionally

, a one

- generation
, reproductive toxicity study in rats from a Toxic Substances
Control Act submission ( Lyondell Chemical Co., 2004 ) is available for tert

- butanol. This study did

not o bserve reproductive effects

. Although the oral toxicity database for tert

- butanol has some 9

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- 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, suggesti ng developmental toxicity is not a s sensitive an endpoint as kidney effects

. N o immunotoxicity or multigenerational reproductive studies are available for tert

- butanol. S tudies on ETBE, which is rapidly metabolized to systemical ly available

tert

- butanol

, are informative for consider ation of the gaps in the tert

- butanol oral database

. The database for ETBE does not indicate immunotoxicity ( Banton et al., 2011 ; L i et al., 2011 )

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- 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 UF D of 1 was applied. Figure 2

- 1 presents graphically the candidate values, UFs, and POD HED values, with each bar corresponding t o one data set describe d in Tables 2

- 1 and 2

- 2.
Table 2-2 is a continuation of Table 2-1 and summarizes the application of UFs to each POD
to derive a candidate value for each data set

, preliminary to the derivation of the organ/system specific RfDs

. These candidate values are co nsidered individually in the selection of a representative oral reference value for a specific hazard and subsequent overall RfD for tert
butanol

. Figure 2

- 1 presents graphically the candidate values, UFs, and POD HED values, with each 31

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2 bar corresponding t o one data set describe d in Tables 2

- 1 and 2

- 2.

**Table 2**  **-2 . E**  **ffects and corresponding derivation of candidate**  **value s** 

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— DO NOT CITE OR QUOTE E n d p o i n t a n d r ef e r en c e PO D H E D ( m g / kg

- d ) P O D t yp e UF A UF H UF L UF S UF D C o m p o s i t e UF C a n d i d a t e

v a lu e ( m g / kg

- d )

K

i dney

I n c r ea s ed ab s ol u t e k i dne y w e i g ht ;

ma l e r a t a t 15 m o n t h s N T P ( 199 5) 71 BM D L 1 0% 3 10 1 1 1 30 2 × 1 0 0

I n c r ea s ed ab s ol u t e k i dne y w e i g ht ; fema l e r a t a t 15 m o n t h s N T P ( 199 5) 22 BM D L 1 0% 3 10 1 1 1 30 7 × 1 0

- 1

K i d n e y i n f l a m m a t i o n ( s upp u r a t i ve ) ; fema l e r a t N T P ( 1 995) 48 BM D L 1 0% 3 10 1 1 1 30 2 × 10 0

K i dne y t r a n s i t i o n a l e p i t h e l i a l h y p e r p l as i a; m al e r at N T P ( 199 5) 3. 8 BM D L 1 0% 3 10 1 1 1 30 1 × 1 0

- 1

K i d n e y t r a n s i t i o n a l e p i t h e l i a l h y p er p l a s i a ; fe ma l e r a t N T P ( 199 5) 81 BM D L 1 0% 3 10 1 1 1 30 3 × 10 01
2 To xicological Review of tert

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

**. Each**  **bar corresponds t**  **o one data set described in Table**  **2-1 and**  **Table 2-2.** 

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**2.1.4. D**  **erivation of Organ/System -S**  **pe cific**  **Reference Doses**

Table 2-3 d istills the can didate values from Table 2-2 into a single value for each organ or
system

. O rgan or system

- sp ecific RfDs are useful for subsequent cumulative risk assessments that consider the combined effect of multiple agents acting a t a common site.

Kidney Toxicity

For t ert

- b utanol , candidate values were for several different kidney effects in both sexes, spanning a range from 1 × 1 0 -1 to 3 × 10 0 mg/kg -day , for an overall 30

- fold range

. To estimate an exposure level below which kid ney toxicity from tert

- butanol exposure is not expected to occur, the RfD for increased incidence of transitional epithelial hyperplasia in male rats ( **1 ×**  **10 -1**  **mg/kg**

**- day**  )
was selected as the kidney

- specific reference dose for tert

- butanol

. Unlike kidney suppurative inflammation, this effect was observed in both sexes, with males appearing to be more sensitive
than females. A d ditionally, this indicator of kidney toxicity is more specific and more sensitive than the relatively non

- sp e cific endpoint of abso lute kidney weight changes. Confidence in this kidney

specific RfD is high

. T h e PODs are based on benchmark dose modeling

, and the candidate values are
derived from a w e ll

- conducted long

- term study, involving a sufficient number of animals per group

, including both sexes, and assessing a wide range of k i dney endpoints

.

**Table 2**  **-3. O**  **rgan/system**

**- specific RfDs and overall RfD**  **for** tert

**- butanol** 

Effect Basis RfD (mg/kg

- day) Study e xposure description Confidence
Kidney Incidence of t ransitional epithelial hyperplasia ( NTP (1995) 1 × 10 -1 Chronic High

O verall RfD Kidney 1 × 10 -1 Chronic High

**2.1.5. Selection of the Overall Reference Dose** 

For tert

- b utanol

, only kidney effects were identified as a hazard and carried forward for
dose- r esponse analysis; thus only one o rgan /system

- specific reference dose was derived. Therefore, the kidney specific RfD of ( **1 ×**  **10 -1**  **mg/kg**

**- day**  ) is the overall RfD for tert

- butanol. This value is based on increased incidence of transitional epithelial hyperplasia in male r ats exp o sed to

tert

- bu ta no l

. 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

, i nc luding 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. F luctuations in exposure levels that result in elevated exposures during these lifestages

c ould lead to an appreciable risk, even if average levels over the full exposure duration were less

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30 Toxicological Review of tert

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1 than or equal to the RfD. In the case of tert

- butanol, there is potential for early lifestage susceptibility to tert

- bu tanol exposure as discussed in S ection 1.3.3.

**2.1.6. Confidence Statement**  A confidence level of high, medium, or low is assigned to the study used to derive the Rf D
, the overall database, and the RfD , as descri bed in Section 4.3.9.2 of EPA’s Methods for Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry ( U.S. EPA, 1994 ). The
overall confidence in this RfD is high

. Confidence in the principal study ( NTP, 1995 ) is high

. This study was well conducted, complied with Food and Drug Administration ( FDA ) Good Laboratory Practice ( GLP ) regulations, involved a sufficient number of animals per dose group (including both sexes), and assessed a wide range of tissues and endpoints

. A lthough the toxicity database for tert
butanol has some gaps , they are informed by the data on ETBE, a parent compound of t ert

- butanol

. Therefore, the confidence in the database is high. Reflecting high confidence in the principal study
and high confidence in the database, confidence in the RfD is high

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The inhalation RfC (expressed in units of mg/m 3 ) 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

. I t can be derived from a NOAEL, LOAEL, or the 95% lower
bound on the benchmark concentration (BMCL), with UFs ge nerally applied to reflect limitations of
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14 **2.1.7. Previous IRIS Assessment**  No previous oral assessment for tert

- butanol is a vailable in IRIS.



## 2.2. **INHALATION REFERENCE CONCENTRATION FOR EFFECTS OTHER THAN CANCER**  16

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**- Response Analysis**  As for oral exposure, EPA identified kidney effects as a potential human hazard of tert
butanol inhalation exposure (summarized in S ec tion 1.3.1)

. No chronic inhalation study for tert butanol is available; there is only one 13

- week study in rats and mice ( NTP, 1997 ) . Sufficient data
were available to modify and utilize a PBPK model in rats for both oral and inhalation exposure in order to perform a route

- to

- route extrapolation, so rat studies from both routes of exposure were
consider ed for dose

- response analysis.
The database for tert

- butanol includes oral and i nhalation studies and data sets that are potentially suitable for use in deriving inha lation reference values. Specifically, effects associated
with tert

- butanol exposure in animals include observations of organ weight and histological changes in the kidney in c hronic and subchronic studies in male and female rats

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35 **2.2.2. Methods of Analysis**  No biologically based dose

- response models are available for tert

- butanol

. In this situation, EPA evaluates a range of dose

- response models considered consistent with underlying biological processes to determine how best to model the dose

- response relationship empirically in the range
of the observed data. Consistent with this approach, all models available in EPA’s BMDS were
evaluated. Consistent with EPA ’s Benchmark Dose Technical Guidance ( U.S. EPA, 2012b ) , the
benchmark dose or concentration (BMD /C ) and the 95% lower confidence limit on the BMD /C (BMD /C L) were estimated us ing a BMR of 10% change from the control mean for absolute kidney weight changes (as described in Section 2.1.2). A s noted in Section 2.1.2., a BMR of 10% extra risk was considered appropriate for the quantal data on incidence s of kidney suppurative inflam mation
and kidney transitional epithelial hyperplasia. The estimated BMD /C Ls were used as PODs. Where
dose- response modeling was not feasible, NOAELs or LOAELs were identified and summarized in
Table 2-4. Further details

, including the modeling output and graphical results for the best

- fit
model for each endpoint

, can be found in Appendix C of the Supplemental Information. NTP (1997) was a well -designed subchronic study that evaluated the effect of tert

- butanol exposure on multiple species at multiple inhalation doses

. Absolute kidney weights were elevated (10

– 11%) in male rats exposed at ≥ 3,27 3 mg/m 3 ; relative kidney weights were elevated (~9%) in
males at ≥ 3,27 3 mg/m 3 and in females at 6,36 8 mg/m 3 . Male rats exhibited an increase in the severity of chronic nephropathy (characterized as number of foci of regenerative tubules)

. F ew endpoints were available for consideration in the subchronic inhalation study, but changes in kidney weights also were observed in the oral studies, such as the NTP (1995) 2- year drinking water study

. 1 Kidney Toxicity EPA identified kidney effects as a potential human hazard of tert

- butanol exposure based on findings of organ weight changes and histopathology primarily in male rats

. These findings were
observed across multiple chronic, subchronic, and short

- term studies following oral and inhalation

exposure. The subchronic NTP (1997) inhalation study is the only route

- specific study available, and was carried forward for further analysis. For oral studies considered for route

- to

- route extrapolation, see Section 2.1. 1 for a summary of considerations for selecting oral studies for dose response analysis. Overall, t h e NTP 2

- year drinking water study NTP (1995) was identified as the study most suitable for dose

- response assessment, given the study duration, comprehensive reporting of outcomes, use of multiple species tested, multiple doses tested , and availability of a
PBPK model for route

- to

- route extrapolation

. This study was discussed previously in Section 2.1.1 as part of the derivation of the oral reference dose, so is not reviewed here again. The NTP (1997)
subchroni c inhalation study shares many strengths with the 2

- year drinking water study ( NTP,
1995 ) , and is described in more detail below. 2

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— DO NOT CITE OR QUOTE5

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30 The RfC methodology provides a mechanism for deriving a n HEC from the duration

adjusted POD (BMCL ADJ ) determined from the animal data. The approach takes into account the

extra

- respiratory nature of the toxicological r esponses 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 [ (H b/g ) A ] of 531 ± 102 for tert

- butanol in the male Wistar rat, while Borghoff et al. (1996) measured a value of 481 ± 29 in male F344 rats

. A blood:gas partition coefficient [ (H b/g ) H ] of 462 was reported for tert

- butanol in humans ( Nihlén et al., 1995 ) . The calculation (H b/g ) A ÷ (H b/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
1.0 4 was used to calculate the HEC

. This allowed a BMCL HEC to be derived as follows:

BMCL BMCL ADJ (mg/m 3 HEC = ) × (interspecies conversion)

= BMCL ADJ (mg/m 3 ) × ( 481 ÷ 462 )

= BMCL (mg/m 3 ADJ ) × ( 1.04 )

Table 2-4 summarizes the sequence of calculations leading to the derivation of a human

equivalent POD for each inhalation data set discussed above. 1 PODs from Inhalation Studies

Because the RfC is applicable to a continuous lifetime human exposure but derived from animal studies featuring intermittent exposure, EPA guidance ( U.S. EPA, 1994 ) provides mechanisms for (1) adjusting experimental exposure concentrations to a value reflecting continuous exposure duration (ADJ) and (2) determ ining 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 m odeled benchmark concentration from the inhalation study ( NTP, 1997 ) w as 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:

BMCL ADJ = BMCL (mg/m 3 ) × (6 ÷ 24 ) × (5 ÷ 7)

= BMCL (mg/m 3 ) × (0.1786) 2

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

Endpoint and

r eference Species/
Sex Model a BMR BM C b (mg/ m 3 ) BM C L b (mg/ m 3 ) POD ADJ b (mg/m 3 ) POD HEC c ( mg/m 3 )
Kidney

Increased absolute kidney weight
N TP (1997) Male F344
rats Hill 10% 1931 1705 304 304

Increased absolute kidney weight NTP (1997) Female F344
rats No model selected d 10%

-­ -­ 1137 1137

2- 12 DRAFT

— DO NOT CITE OR QUOTE a For modeling details, see Appendix C in Supplemental Information.

b BMCs, BMCLs, and PODs were adjusted for continuous daily exposure by multiplying by (hours exposed per day /

24 hr) × (days exposed per week / 7 days).

c POD HEC calculated by adjusting the POD ADJ by the DAF (=1.0, rounded from 1.04 ) for a C ategory 3 gas ( U.S. EPA,

1994 ).

d BMD modeling failed to calculate a BMD value successfully (see Appendix C) ; POD calculated from no

- observed

adverse effect level (NOAEL) of 6368 mg/m 3

.

BMCL HEC = BMCL /m 3 ADJ (mg ) × (interspecies conversion)

= BMCL 3 ADJ (mg/m ) × ( 481 ÷ 462 )

= BMCL ADJ (mg/m 3 ) × ( 1.04 ) PODs from oral studies – use of PBPK model for route

- to

- route extrapolation

A PBPK model for tert

- butanol in rats has been modified

, as described in Appendix B of the Supplemental Information

. Using this model, route

- to

- route extrapolation of the oral BMDLs to
derive inhalation PODs was performed as follows. First, the internal dose in the rat at each oral BMDL (assuming continuous expo sure) 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 w as then converted to a human equivalent concentration POD (POD HEC ) using the methodology previously described in “PODs from inhalation studies ”:

A critical decision in the route

- to

- route extrapolation is selection of the internal dose metric
that establishe s “ equivalent” oral and inhalation exposures. For tert

- butanol

- induced kidney effects, the two options are the concentration of tert

- butan ol in blood and rate of tert

- butanol metabolism. Note that using the kidney concentration of tert

- butanol will lead to the same route

- to

- route extrapolation relationship as tert

- butanol in blood because the distribution from blood to kidney is independent of route. D ata are not available that suggest that metabolites of tert

- butanol mediate its renal toxicity. Without evidence that suggest s otherwise, tert

- butanol is assumed the active7 8 9

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toxicological agent. T herefore, the concentration of tert

- butanol in blo od was selected as the dose

metric.
Table 2 -5 summarizes the sequence of calculations leading to the derivation of a human

equivalent i nhalation POD f rom 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 dose a (mg/L) Equivalent POD HEC b (mg/m 3 )

Kidney

Mean absolute kidney weight at 15 months NTP (1995) Rat/M 10% 296 22.4 551

Mean absolute kidney weight at 15 months NTP (1995) Rat/F 10% 91 4.76 155

Kidney inflammation (suppurative) NTP (1995) Rat/F 10% 200 12.6 359

Kidney transitional epithelial hyperplasia NTP (1995) Rat/M 10% 16 0.745 26.1

Kidney transitional epithelial hyperplasia NTP (1995) Rat/F 10% 339 27.9 638

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— DO NOT CITE OR QUOTE a Average blood concentration of tert

- butanol under continuous oral exposure at the BMDL.

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

tert

- butanol as continuous oral exposure at the BMDL.

**2.2.3. Derivation of Candidate**  **Values**

In EPA’s A Review of the Reference Dose and Reference Concentration Processes [ ( U.S. EPA,
2002 ) ; Section 4.4.5], also described in the Preamble, five possible areas of uncertainty and variability were considered. Several PODs for the candidate inhalation values were derived using 

### a

route

- to

- route extrapolation from the PODs estimated from the chronic oral toxicity study in rats ( NTP, 1995 ) in the derivation of the oral RfD (Section 2.1). With the exception of the subchronic
inhalation ( NTP, 1997 ) study, t he uncertainty factors (UFs) selected and applied to PODs derived
from the chronic oral ( NTP, 1995 ) study for route

- to

- route extrapolation are the same as those f or the RfD for tert

- butanol (see Section 2.1.3 )

. The model used to perform this route

- to

- route extrapolation is a well

- characterized model considered appropriate for the purposes of this assessment. One source of uncertainty regarding the route

- to

- route ex trapolation is the assumption
of that 100% of inhaled tert

- butanol reaches the gas

- exchange region, that is, 100% of the inhaled1

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TP (1995)

ncreased ab t 15 month TP (1995)
idney inflammation suppurative); female rat TP (1995) 359 Toxicological Review of tert

- Butyl Alcohol

tert

- butanol c ould be absorbed and distributed to the rest of the body in rats . If not all of the compound is bioavailable f or the rat , a lower blood concentration would be expected compared to the current estimate, and thus, a higher RfC would be calculated.
For the PODs derived from the subchronic inhalation ( NTP, 1997 ) study, a UF S of 10 was applied to account for extrapolation from subchronic to chronic duration.
Table 2 -6 is a continuation of Table 2-4 and Table 2-5 , and summarizes the application of UFs to each POD to derive a candidate value for each data set. The candidate values presented in the table below are preliminary to the derivation of the or g an/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 t ert

- butanol. Figure 2

- 2 presents graphically the candidate values, UFs, and POD HEC values, with each bar corresponding to one data set described in Tables 2

- 4 , 2

- 5, and 2

- 6.

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

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— DO NOT CITE OR QUOTE E n d p o i n t ( s e x a n d s p e c i e s ) a n d

r ef er en c e PO D H E C a ( m g/ m 3 ) PO D
t yp e UF A UF H UF L UF S UF D C o m p o s i t e UF C a n d i d a t e

v a lu e ( m g/ m 3 )

K

i dney

I nc r e a s e d a bs o l ut e k i dne y w e i g ht

a t 1 3 we ek s ; ma l e r a t N T P ( 199 7) 304 B M CL 1 0% 3 10 1 10 1 300 1 × 10 0

I nc r e a s e d a bs o l ut e k i dne y w e i g ht

a t 1 3 we ek s ; fe ma l e r a t N T P ( 199 7) 1137 N O A E L 3 10 1 10 1 300 4 × 10 0

I nc r e a s e d a bs o l ut e k i dne y w e i g ht
at 1 5 m on t h s ; m al e r at N 551 B M CL 1 0% 3 10 1 1 1 30 2 × 10 1 *

I s o l ut e k i dne y w e i g ht

a s ; f ema l e r a t N 1 55 B M CL 1 0% 3 10 1 1 1 30 5 × 10 0 *

K (
N B M CL 1 0% 3 10 1 1 1 30 1 × 10 1 *

K i d n e y t r a n s i t i o n a l e p i t h e l i a l h y p e r p l as i a; m al e r at N T P ( 199 5) 26. 1 B M CL 1 0% 3 10 1 1 1 30 9 × 10

- 1 *

K i d n e y t r a n s i t i o n a l e p i t h e l i a l h y p er p l a s i a ; fe ma l e r a t N T P ( 199 5) 638 B M CL 1 0% 3 10 1 1 1 30 2 × 10 1 *1 2 Toxicological Review of tert

- Butyl Alcohol

*These candidate values are derived using route

- to

- r oute extrapolated PODs based on NTP’s chronic drinking water study.

**Figure** 2

**-** 2

**. Candidate**  **RfC values with corresponding POD and composite UF** 

**.**

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— DO NOT CITE OR QUOTE **2.2.4. Derivation of Organ/System**  **-S pecific Reference**  **Concentrations**

Table 2-7 distills the candidate values from Table 2-6 into a single value for the kidney

. O rgan

- or system

- spec ific reference values can be useful for subsequent cumulative risk assessments that consider the combined effect of multiple agents acting at a common site.

Kidney Toxicity For the derivation of candidate values, whether PODs from the subchronic inhalation study
of N TP (1997) would provide a better basis than the route

- to

- route extrapolated PODs based on the
chronic oral study of NTP (1995) must be considered. C andidate values were derived for increased kidney weight observed in t he subchronic inhalation study ( NTP, 1997 ) and several kidney effects
observed in the chronic oral study ( NTP, 1995 ) in both sexes of rat, spanning a range from 9 × 10 -1
to 2 × 10 1 mg/m 3 , for an overall 20

- fold range. To estimate an exposure level below which kidney toxicity from tert

- butanol exposure is not expected to occur, the RfC for increased incidence of transitional epithelial hyperplasia in male rats ( **9 × 10 -1**  **mg/m 3**  ) was selected as the kidney

- specific
RfC for tert

- butanol, consistent with the selection of the kidney

- specific RfD (see Section 2.1.4). As
discussed in Section 2.1. 4, unlike kidney suppurative inflammation, this effect was observed in both sexes, with males appearing to be more sensitive than females. Additionally, it is based on a longer (chronic) duration and a more specific and sensitive indicator of kidney toxicity than the relatively

non

- specific endpoint of kidney weight change. Confidence in this kidney

- specific RfC is high. The PODs are based on BMD modeling, and the candidate values are derived from a well -conducted study, involving a sufficient number of anima ls per group, including both sexes, assessing a wide range of kidney endpoints, and availability of a PBPK model for route

- to

- route extrapolation.

**Table 2**  **-7. Organ/system** 

**- specific Rf**  **C s and overall Rf**  **C for**  ter t

**- butanol**  1

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Effect Basis RfC (mg/m 3 ) Study exposure description Confidence
Kidney I ncidence of transitional epithelial hyperplasia ( NTP, 1995 ) 9 × 10

- 1 Chronic High

Overall RfC Kidney 9 × 10 - 1 Chronic High

**2.2.5. Selection of the Overall Reference**  **Concentration**

For tert

- butanol

, kidney effects were identified as the primary h azard; thus, a single

organ

- /system

- specific RfC was derived. The kidney

- specific RfC of **9 ×**  **10 -1**  **mg/m 3**  is selected as the overall RfC, representing an estimated exposure level below which deleterious effects from

tert

- butanol exposure are not expected t o occur. The overall RfC is derived to be protective of all types of effects for a given duration of exposure and is intended to protect the population as a whole

, including potentially susceptible5

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1 subgroups ( U.S. EPA, 2002 ). Decisions concerning averaging exposures over time for comparison
with the RfC should consider the types of toxicological effects and specific lifestages of concern. Fluctuations in exposure levels that result in elevated exposures during these lifestages could potentially lead to an appreciable risk, even if average levels over the full exposure duration were less than or equal to the RfC. In the case of tert

- bu tanol, there is potential for early lifestage susceptibility to tert

- bu tanol exposure as discussed in S ection 1.3.3.

**2.2.6. Confidence Statement**  A confidence level of high, medium, or low is assigned to the study used to derive the RfC, the overall database, and the Rf C itself, as described in Section 4.3.9.2 of EPA ’s Methods for Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry ( U.S. EPA,
1994 ) . A PBPK model was utilized to perform a route

- route extrapolation to determine a POD for
the derivation of the RfC from the NTP (1995) oral study and corresponding critical effect. Confidence in the pr incipal 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 2

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14 sexes), and assessed a wide range of tissues and endpoints. Although there are some gaps in the toxicity database for tert

- butanol, these areas are informed by the data on ETBE, a parent compound of tert

- butanol. Therefore, the confidence in the database is high. Reflecting high confidence in the principal study, high confidence in the database, and minimal uncertainty surrounding the application of the modified PBPK model for the purposes of a route

- to

- route extrapolation, the overall confidence in the RfC for tert

- butanol is high. 16

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21 **2.2.7. Previous IRIS Assessment**  No previous inhalation assessment for tert

- butanol is available in IRIS. 22

23 **2.2.8. Uncertainties in the Derivation of**  **the Reference Dose and**  **Reference Concentration** The following discussion identifies uncertainties associated with the RfD and RfC for

tert

- butanol . To derive the RfD, the UF approach ( U.S. EPA, 2000a
, 1994 ) was applied to a POD based on kidney toxicity in rats treated chronically . UFs were applied to the POD to account for extrapolating from an animal bioassay to human exposure, and the likely existence of a diverse
human population of varying susceptibilities. These extrapolations are carried out with default approaches given the lack of data to inform individual steps. To derive the RfC, this same approach was applied, but a PBPK model was used to extrapola te from oral to inhalation exposure

. The database for tert

- butanol contains no human data on adverse health effects from
subchronic or chronic exposure, and the PODs were calculated from data on the effects of tert butanol reported by studies in rats. The database for tert

- butanol exposure includes one lifetime bioassay, several reproductive/developmental studies, and several subchronic oral studies. Although the database is adequate for reference value derivation, there is uncertainty associated with the lack of a comprehensive multigeneration reproductive toxicity study. 24

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35 Another uncertainty to consider relates to the MOA analysis conducted for the kidney
effects. The assessment concluded that te rt

- butanol is a weak inducer of α 2u - globul in which is operative in male kidney tumors; therefore, noncancer effects related to α 2u - globulin were considered not relevant for hazard identification and, therefore, not suitable for dose response
consideration. If this conclusion was incorrect and the noncancer effects characterized in this

assessment as being related to α 2u - globulin were relevant to humans, then the RfD and RfC values could be underestimating toxicity. Similarly, the renal effects characterized as CPN and dismissed as not being treatme nt related, if considered relevant, would likewise contribute to the hazard potential and dose

- response analysis for the kidney

- specific RfD and RfC.



## 2.3. **ORAL SLOPE FACTOR FOR CANCER**
The oral slope factor (OSF) is a plausible upper bound on the estimate of risk per mg/kg day of oral exposure. The OSF can be multiplied by an estimate of lifetime exposure (in mg/kg

- day) to
estimate the lifetime cancer risk.

**2.3.1. Analysis of Carcinogenicity Data** 

As noted in Section 1.3.2 , that there is “ suggestive evidence of carcin ogenic potential ” for

tert

- butanol

. The Guidelines for Carcinogen Risk Assessment ( U.S. EPA, 2005a ) state:

When there is suggestive evidence, the Agency generally would not attempt a dose

response assessment, as the nature of the data generally would not support one; however when the evidence includes a well

- conducted study, quantitative analysis may be useful for some purposes, for example, providing a sense of the magnitude and uncertainty of potential risks, ran king potential hazards, or setting research priorities.

N o h uman data relevant to an evaluation of the carcinogenicity of tert

- butanol were available

. The cancer descriptor was based on the 2

- year drinking water study in rats and mice by Toxicological Review of tert

- Butyl Alcohol

1 Additionally, only subchronic and short

- term inhalation studies have been conducted, and no
chronic inhalation studies are available. Developmental studies identified significant increases in fetal loss, decreases in fetal body weight, and possible increases in skeletal variations in exposed offspring or pups. However, effects were no t always consistent across exposure routes, and maternal toxicity was present whenever developmental effects were observed. The toxicokinetic and toxicodynamic differences for tert

- butanol between the animal species in which the POD was derived and huma ns are unknown. T he tert

- butanol database lacks an adequate model that would inform potential interspecies differences (A limited data set exists
for tert

- butanol appearing as a metabolite from ETBE exposure in humans, but none for direct
exposure to tert

- butanol

. ) Generally, it was found that rats appear more susceptible than mice, and males appear more susceptible than females to tert

- butanol toxicity. However, the underlying
mechanistic basis of these apparent difference s is not understood. Most importa ntly, it is unknown
which animal species and/or sexes may be more comparable to humans. 2

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35 Toxicological Review of tert

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1 ( NTP, 1995 ), which reported renal tumors in male rats and thyroid tumors in both male and f emale

mice

. This study was considered suitable for dose

- response analysis. It was conducted in
accordance with FDA GLP r egulations, 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. A dditionally, t he renal tumors were reexamined by a Pathology Working Group ( Hard et al., 2011 ).
Based on a mode of action analysis, it was concluded that the α 2u - globulin process wa s at least partially responsible for the male rat renal tumors, in addition to other, unknown, processes. Because the relative contribution of each process to tumor formation cannot be determined ( U.S. EPA, 1991a ) , the male rat renal tumors are not considered suitable for quantitative analysis. Conversely, the mouse thyroid tumors are suitable for dose

- response analysis and unit risk estimation, as described in Section 1.3.2. 2

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38 **2.3.2. D**  **ose -**  **Response Analysis**

**— Adjustments and Extrapolations Methods** 

The EPA Guidelines for Carcinogen Risk Assessment ( U.S. EPA, 2005a ) recommend that the
method used to characterize and quantify cancer risk from a chemical be determined by what is known about the MOA of the carcinogen and the shape of the cancer dose

- response curve

. EPA

uses a two

- step approach that distinguishes analysis of the observed dose

- response data from
inferences about lower doses ( U.S. EPA, 2005a ) . W ithin the observed range, the preferred approach is to use modeling to incorporate a wide range of data into the analysis, such as through a biologically based model, if supported by substantial data. Without a biologica lly based model, as in
the case of tert

- butanol, a standard model is used to curve

- fit the data and estimate a POD. EPA uses the multistage model in IRIS dose

- response analyses for cancer ( Gehlhaus et al., 2011 ) because it parallels the multistage carcinogenic process and fits a broad array of dose

- response patterns. The second step, extrapolation to lower exposures from the POD, considers what is known
about the modes of action for each effect. As above

, a biologically based model is preferred ( U.S. EPA, 2005a ). Otherwise, linear low

- dose extrapolation is recommended if the MOA of carcinogenicity is mutagenic or has not been established ( U.S. EPA, 2005a ) . For

- butanol, the mode(s) of carcinogenic action for thyroid follicular cell tumors has not been established (see
Section 1.3.2). Therefore, linear low

- dose extrapolation was used to estimate human carcinogenic
risk

. The dose -re sponse modeling used administered dose because a PBPK model to characterize internal dosimetry in mice was not available. For the analysis of male mice thyroid tumors, the incidence data were adjusted to account for the increased mortality in high

- dose male mice, relative to the other groups, that reduced the number of mice at risk for developing tumors. The Poly

- 3
method ( Baile r and Portier, 1988 ) was used to estimate the number at risk of developing tumors, by weighting the length of time each animal was on study (details in Appendix C of the tert

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35 Toxicological Review of tert

- Butyl Alcohol

1 Supplemental Information ). This method was not applied to the female mice data beca use a difference in survival with increasing exposure was not appreciable and only one tumor

, in the high

- dose group

, occurred before study termination. The data modeled and other details of the modeling are provided in Appendix C. The BMDs
and BMDLs recom mended for each data set are summarized in Table 2-8. The modeled tert

- butanol
PODs were scaled to HEDs according to EPA guidance ( U.S. EPA, 2011
, 2005a ) . In particular, the
BMDL was converted to an HED by assuming that doses in animals and humans are toxicologically equivalent when scaled by body weight raised to the 3 / 4 power

. Standard body weights of 0.025 kg
for mice and 70 kg for humans were used ( U.S. EPA, 1988 ). The following formula was used for the
conversion of oral BMDL to oral HED for mouse endpoints : 2

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- = (BMDL in mg/kgday -) × ( animal body weight / 70 ) 1/4 = (BMDL in mg/kgday -) × 0. 14

PODs for estimating low

- dose risk were identified at doses at the lower end of the observed data, corresponding to 10% extra risk in female mice and 5% extra risk in male mice.

**2.3.3. D**  **erivation of the Oral Slope Factor**

The PODs estimated for each tumor data s et 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 P O D to the control response (slope factor = BMR/BMDL BMR = 0. 1 /BMDL 10 ). This slope represents a plausible upper bound on the true population average risk. Using linear extrapolation from the BMDL 10 , human equivalent oral slope factors were derived for male a nd
female mice and are listed in Table 2-8.
The oral slope factor based on the incidence of thyroid follicular cell adenomas in female
mice was 5 × 10 4 per mg/kg

- day. Despite high mortality in high

- dose male mice, estimating slope factors using the poly

- 3 method was feasible for addressing competing risks. Whether using the full data set (including the only thyroid follicular cell carcinoma observed at the highest dose ) or omitting the high

- dose group altogether (under the assumption that mortality in this group was too

extensive to interpret the results), oral slope factors based on the incidence of thyroid follicular cell
adenomas or carcinomas in male mice were simil a r when rounded to one significant digit —5 × 10 -4 per mg/kg

- day or 6 × 10 -4 per mg/kg

- day, respectively.

T he recommended slope factor for lifetime oral exposure to tert

- butanol is
**5 ×**  **10 -4**  **per mg/kg** 

**- day**  , based on the thyroid follicular cell adenoma or carcinoma response in
male or fe male B6C3F 1 mice

. This slope factor should not be used wit h exposures exceeding
1 400 mg/kg -day

, the highe st POD from the two data sets

, because above this level the cancer risk might not increase linearly with exposure

. The slope of the linear extrapolation from the central

estimate BMD 10 HED derived from the female mouse data set is 0.1/ [0. 14 × ( 2002 mg/kg

- day) ] =

4 × 10 –4 per mg/kg -day. 31

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— DO NOT CITE OR QUOTE1 **Table 2-8.**  **Summary of the oral slope**  **factor derivation**

2 Tumor Species/ sex Selected model BMR BMD (mg/kg

d) POD= BMDL (mg/kg

d) BMDL HED a (mg/kg

- d) Slope factor b (mg/kg

- day)

-

1

Thyroid follicular cell adenoma B6C3F 1 mouse/Female 3° Multistage 10% 2002 1437 201 5 × 10 -4

Thyroid follicular cell adenoma or carcinoma B6C3F 1 mouse/Male All dose groups: 1° Multistage 5% c 1788 787 110 5 × 10 -4

High dose omitted: 2° Multistage 5% c 1028 644 90 6 × 10 -4

a HED PODs were calculated using BW 3/4 scaling ( U.S. EPA, 2011 ).

b Human equivalent slope factor = 0.1/BMDL 10HED ; see Appendix C of the Supplemental Information for details of

modeling results.

c Because the observed responses were <10%, a BMR of 5% was used to represent the observed response range for

low

- dose extrapolation; human equivalent slope factor = 0.05/BMDL 5HED
.

**2.3.4. Uncertainties in the Derivation of the Oral Slope Factor**  3 4 5 6

7

8 There is uncertainty when extrapolating data from animals to estimate potential cancer risks to human populations from exposure to tert

- butanol.
Table 2-9 summarizes several uncertainties that could affect the oral slope factor

. There are

no other chronic studies to replicate these findings or that examined other animal models

, no data in humans to confirm a cancer response in general or the specific tumors observed in the NTP (1995) bioassay, and no other data (e.g., MOA) to support alternat ive approaches for deriv ing the oral slope factor. 9

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- Butyl Alcohol

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2 Toxicological Review of tert

- Butyl Alcohol

**Table 2**  **-9. S**  **ummary of uncertaint ies**  **in the derivation of the oral slope factor**  **for** te rt

**- butanol** 

Consideration and impact on cancer risk value Decision Justification

Selection of tumor type and relevance to humans: Mouse thyroid tumors are the basis for estimating human cancer risk, as the fraction of rat kidney tumors not attributed to the male rat specific

α 2μ

- globulin process could not be determined. Alternatively
, quantifying rat kidney tumors could ↑ slope factor to 1 x 10 -2 mg/kg

- day (see Appendix C, Supplemental Information) Thyroid tumors in female and male mice were selected U.S. EPA (1998a)

, U.S. EPA (1991a)

. MOA data suggested that mouse thyroid tumors were relevant to humans. Quantitation of thyroid tumors in ma le mice was impacted only slightly by high mortality in the high

- dose group, and supports the estimate based on female mice.

Selection of data set: NTP (1995)

, oral (drinking NTP (1995)

, the only chronic bioa ssay No other studies are available. water) study, was selected to derive cancer risks for humans. available, was a well

- conducted study. Additional bioassays might add support to the findings , facilitate determination of what fraction of kidney tumors are not attributable
to α 2μ

- globulin process, or provide results for different (possibly lower) d oses, which would affect (possibly increase) the oral slope factor.

Selection of dose metric: Alternatives could ↓ or ↑ slope factor Used administered dose. For mice, PBPK

- estimated internal doses could impact the OSF value for thyroid tumors if the carc inogenic moiety is not proportional to administered dose, but no PBPK model was available, and no information is available to suggest if any metabolites elicit carcinogenic effects.
Interspecies extrapolation of dosimetry and risk: Alternatives could ↓ o r ↑ slope factor (e.g., 3.5

- fold ↓ [scaling by body weight] or ↑ 2

- fold [scaling by BW 2/3]) The default approach of body weight 3/4 was used. No data to suggest an alternative approach for tert

- butanol. Because the dose metric was not an area under the curve, BW 3/4 scaling was used to calculate equivalent cumulative exposures for estimating equivalent human risks. Although the true human correspondence is unknown, this overall approach is expected neither to over

or underestimate human equivalent risks.

Dose

- response modeling: Alternatives could ↓ or ↑ slope factor Used multistage dose

response model to derive a BMD and BMDL. No biologically based models for tert

- butanol were available. The multistage model has biological support and is the model most consistently used in EPA cancer assessments.

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Consideration and impact on cancer risk value Decision Justification

Low

- dose extrapolation: Linear extrapolation of risk Linear low

- dose extrapolation for agents ↓ cancer risk estimate would be in low

- dose region used without a known MOA is supported ( U.S. expected with the application of U.S. EPA (1998a)

. EPA, 2005a ) and recommended for rodent nonlinear low

- dose extrapola tion thyroid tumors arising from an unknown MOA ( U.S. EPA, 1998a )

. Statistical uncertainty at POD: BMDL (preferred approach Limited size of bioassay results in sampling ↓ or al slope factor 1.4

- fold if BMD for calculating slope factor) variability; lower bound is 95% CI on used as the POD rather than BMDL administered exposure at 10% extra risk of thyroid tumors.
Sensitive subpopulations: ↑ oral slope factor to unknown

extent No sensitive populations have been identified. No chemical

- specific data are available to determine the range of human toxicodynamic variability or sensitivity, including the susceptibility of children. Because determination of a mutagenic MOA has not been made, an age

- specific adjustment factor is not applied.

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— DO NOT CITE OR QUOTE **2.3.5. P**  **revious IRIS Assessment: Oral Slope Factor** No previous cancer assessment for tert

- butanol is available in IRIS.



## 2.4. **INH ALATION UNIT**  **RISK FOR CANCER**

The carcinogenicity assessment provides information on the carcinogenic hazard potential of the substance in question, and quantitative estimates of risk from oral and inhalation exposure may be derived. Quantitative risk estimates may be derived from the application of a low

- dose extrapolation procedure. If derived, the inhalation unit risk (IUR) is a plausible upper bound on the estimate of risk per μg/m 3 air breathed.
No chronic inhalation exposure studies to tert

- butanol ar e available. L ifetime oral exposure has been associated with increased renal tubule adenomas and carcinoma in male F344 rats, increased thyroid follicular cell adenomas in female B6C3F 1 mice, and increased thyroid follicular
cell adenomas and carcinomas in male B6C3F 1 mice. Because only a rat PBPK model exists, however, route

- to

- route extrapolation cannot be performed for thyroid tumors in mice at this time

. The NTP (1995) drinking water study in rats and mice was the only chronic bioassay available for
dose- response analysis. Still, t he rat PBPK model a nd kidney tumors from the NTP (1995) drinking water study we re not used for route

- to

- route extrapolation because enough information to
determine the relative contribution of α 2u - globulin nephropathy and other processes to the overall renal tumor response ( U.S. EPA, 1991a ) is not available

. Alternatively, if kidney tumors were considered acceptable for quantitation, then route

- to

- route extrapolation could be conducted to calculate an IUR (see Appendix C in Supplemental Information).Toxicological Review of tert

- Butyl Alcohol

1 2.4.1. Previo us IRIS Assessment: Inhalation Unit Risk

An inhalation cancer assessment for tert

- butanol was not previously available on IRIS.



## 2.5. **APPLICATION OF AGE**

**- DEPENDENT ADJUSTMENT FACTORS** 

As discussed in the Supplemental Guidance for Assessing Susceptibility from Early

- Life Exposure to Carcinogens ( U.S. EPA, 2005b ) , either default or chemical

- specific age

- dependent adjustment factors (ADAFs) are recommended to account for early

- life e xposure to carcinogens that act through a mutagenic MOA

. Because chemical

- specific lifestage susceptibility data for cancer are not available, and because the MOA for tert

- butanol carcinogenicity is not known (see Section 1.3.2)

, application of ADAFs is no t recommended

. 2

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— DO NOT CITE OR QUOTE1 Acharya, S; Mehta, K; Rodrigues, S; Pereira, J; Krishnan, S; Rao, CV. (1995). Administration of subtoxic doses of t

- butyl alcohol and trichloroacetic acid to male Wistar rats to study the interactive toxicity. Toxicol Lett 80: 97

- 104. http://dx.doi.org/10.1016/0378 4274(95)03340 -Q

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. http://epa.gov/iris/process.htm

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- 12/001). Wa shington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum. http://www.epa.gov/raf/publications/benchmarkdose.htm

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- 11/006F). Washington, DC: U.S. Environmental Protection Agency U.S. EPA (U.S. Environmental Protection Agency). (2014). Chemical Data Reporting 2012, reported in the Chemical Data Access Tool. Available online at http://www.epa.gov/oppt/cdr/index.html (accessed Williams, TM; Borghoff, SJ. (2001). Characterization of tert

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