EPA/635/R-16/079b 

Public Comment Draft 

www.epa.gov/iris 

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

(CASRN 75-65-0) 

Supplemental Information – tert-Butyl Alcohol 

April 2016 

NOTICE 

This document is a Public Comment Draft. This information is distributed solely for the purpose of

pre-dissemination peer review under applicable information quality guidelines. It has not been

formally disseminated by EPA. It does not represent and should not be construed to represent any

Agency determination or policy. It is being circulated for review of its technical accuracy and

science policy implications. 

Integrated Risk Information System

National Center for Environmental Assessment
 

Office of Research and Development

U.S. Environmental Protection Agency

Washington, DC
 
Supplemental Information―tert-Butyl Alcohol 

DISCLAIMER 

This document is a preliminary draft for review purposes only. This information is 

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

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

not be construed to represent any Agency determination or policy. Mention of trade names or 

commercial products does not constitute endorsement or recommendation for use. 

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

ii DRAFT—DO NOT CITE OR QUOTE 
CONTENTS
 

Supplemental Information―tert-Butyl Alcohol 

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

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B.2. PBPK MODEL EVALUATION SUMMARY..................................................................B-9 

Evaluation of Existing tert-Butanol Submodels.........................................B-9 

Modification of Existing tert-Butanol Submodels...................................B-11 

Summary of the PBPK Model for tert-Butanol........................................B-16 

tert-Butanol Model Application ..............................................................B-16 

PBPK Model Code....................................................................................B-16 

B.3. OTHER PERTINENT TOXICITY INFORMATION.......................................................B-17 

Other Toxicological Effects......................................................................B-17 

Genotoxicity ............................................................................................B-31 

Summary .................................................................................................B-35 

APPENDIX C. DOSE-RESPONSE MODELING FOR THE DERIVATION OF REFERENCE VALUES FOR 

EFFECTS OTHER THAN CANCER AND THE DERIVATION OF CANCER RISK 

ESTIMATES .......................................................................................................................C-1 

APPENDIX A.	 ASSESSMENTS BY OTHER NATIONAL AND INTERNATIONAL HEALTH AGENCIES ............A-1
 

APPENDIX B.	 INFORMATION IN SUPPORT OF HAZARD IDENTIFICATION AND DOSE-RESPONSE
 

ANALYSIS..........................................................................................................................B-1
 

B.1. TOXICOKINETICS.....................................................................................................B-1 

Absorption.................................................................................................B-1 

Distribution ...............................................................................................B-2 

Metabolism ...............................................................................................B-2 

Excretion ...................................................................................................B-5 

Physiologically Based Pharmacokinetic Models .......................................B-6 

Noncancer Endpoints................................................................................C-1
 

Cancer Endpoints....................................................................................C-23
 

REFERENCES .........................................................................................................................................R-1
 
Supplemental Information―tert-Butyl Alcohol 

TABLES 

Table A-1. Health assessments and regulatory limits by other national and international 

health agencies .................................................................................................................... A-1
 

Table B-1. PBPK model physiologic parameters and partition coefficients.............................................B-12
 

Table B-2. Rate constants for tert-butanol determined by optimization of the model with
 

experimental data...............................................................................................................B-15
 

Table B-3. Changes in kidney weight in animals following exposure to tert-butanol .............................B-20
 

Table B-4. Changes in liver weight in animals following exposure to tert-butanol .................................B-23 

Table B-5. Changes in liver histopathology in animals following exposure to tert-butanol....................B-25 

Table B-6. Changes in urinary bladder histopathology in animals following oral exposure to 

tert-butanol.........................................................................................................................B-27 

Table B-7. Summary of genotoxicity (both in vitro and in vivo) studies of tert-butanol .........................B-34 

Table C-1. Noncancer endpoints selected for dose-response modeling for tert-butanol.........................C-2 

Table C-2. Summary of BMD modeling results for kidney transitional epithelial hyperplasia in 

male F344 rats exposed to tert-butanol in drinking water for 2 years (NTP, 

1995); BMR = 10% extra risk .................................................................................................C-3 

Table C-3. Summary of BMD modeling results for kidney transitional epithelial hyperplasia in 

female F344 rats exposed to tert-butanol in drinking water for 2 years (NTP, 

1995); BMR = 10% extra risk .................................................................................................C-6 

Table C-4. Summary of BMD modeling results for absolute kidney weight in male F344 rats 

exposed to tert-butanol in drinking water for 15 months (NTP, 1995); BMR = 

10% rel. dev. from control mean ..........................................................................................C-9 

Table C-5. Summary of BMD modeling results for absolute kidney weight in female F344 rats 

exposed to tert-butanol in drinking water for 15 months (NTP, 1995); BMR = 

10% rel. dev. from control mean ........................................................................................C-12 

Table C-6. Summary of BMD modeling results for kidney inflammation in female rats 

exposed to tert-butanol in drinking water for 2 years (NTP, 1995); BMR = 10% 

extra risk..............................................................................................................................C-15 

Table C-7. Summary of BMD modeling results for absolute kidney weight in male F344 rats 

exposed to tert-butanol via inhalation for 6 hr/d, 5d/wk for 13 weeks (NTP, 

1997); BMR = 10% relative deviation from the mean.........................................................C-18 

Table C-8. Summary of BMD modeling results for absolute kidney weight in female F344 rats 

exposed to tert-butanol via inhalation for 6 hr/d, 5d/wk for 13 weeks (NTP, 

1997); BMR = 10% relative deviation from the mean.........................................................C-21 

Table C-9. Cancer endpoints selected for dose-response modeling for tert-butanol .............................C-24 

Table C-10. Summary of the oral slope factor derivations ......................................................................C-25 

Table C-11. Summary of BMD modeling results for thyroid follicular cell adenomas in female 

B6C3F1 mice exposed to tert-butanol in drinking water for 2 years (NTP, 1995); 

BMR = 10% extra risk ..........................................................................................................C-26
 

Table C-12. Summary of BMD modeling results for thyroid follicular cell adenomas or 

carcinomas in male B6C3F1 mice exposed to tert-butanol in drinking water for 

2 years (NTP, 1995); BMR = 5% extra risk...........................................................................C-29
 

Table C-13. Summary of BMD modeling results for thyroid follicular cell adenomas or 

carcinomas in male B6C3F1 mice exposed to tert-butanol in drinking water for 

2 years, high dose omitted (NTP, 1995); BMR = 5% extra risk............................................C-32
 

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

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Supplemental Information―tert-Butyl Alcohol 

Table C-14. Summary of BMD modeling results for renal tubule adenoma or carcinoma in 

male F344 rats exposed to tert-butanol in drinking water for 2 years modeled 

with administered dose units and including all dose groups (NTP, 1995); BMR 

= 10% extra risk.................................................................................................................C-35 

Table C-15. Summary of BMD modeling results for renal tubule adenoma or carcinoma in 

male F344 rats exposed to tert-butanol in drinking water for 2 years modeled 

with administered dose units and excluding high-dose group (NTP, 1995); BMR 

= 10% extra risk...................................................................................................................C-37 

Table C-16. Summary of BMD modeling results for renal tubule adenoma or carcinoma in 

male F344 rats exposed to tert-butanol in drinking water for 2 years modeled 

with PBPK (tert-butanol, mg/L) dose units and including all dose groups (NTP, 

1995); BMR = 10% extra risk. ..............................................................................................C-39 

Table C-17. Summary of BMD modeling results for renal tubule adenoma or carcinoma in 

male F344 rats exposed to tert-butanol in drinking water for 2 years modeled 

with PBPK (tert-butanol, mg/L) dose units and excluding high-dose group (NTP, 

1995); BMR = 10% extra risk. ..............................................................................................C-41 

Table C-18. Summary of BMD modeling results for renal tubule adenoma or carcinoma in 

male F344 rats exposed to tert-butanol in drinking water for 2 years modeled 

with PBPK (metabolized, mg/hr) dose units and including all dose groups (NTP, 

1995); BMR = 10% extra risk. ..............................................................................................C-43 

Table C-19. Summary of BMD modeling results for renal tubule adenoma or carcinoma in 

male F344 rats exposed to tert-butanol in drinking water for 2 years modeled 

with PBPK (metabolized, mg/hr) dose units and excluding high-dose group 

(NTP, 1995); BMR = 10% extra risk. ....................................................................................C-45 

Table C-20. Summary of BMD modeling results for renal tubule adenoma or carcinoma in 

male F344 rats exposed to tert-butanol in drinking water for 2 years modeled 

with administered dose units and including all dose groups; reanalyzed data 

(Hard et al., 2011; NTP, 1995); BMR = 10% extra risk.........................................................C-47 

Table C-21. Summary of BMD modeling results for renal tubule adenoma or carcinoma in 

male F344 rats exposed to tert-butanol in drinking water for 2 years modeled 

with administered dose units and excluding high-dose group; re-analyzed data 

(Hard et al., 2011; NTP, 1995); BMR = 10% extra risk.........................................................C-47
 

Table C-22. Summary of BMD modeling results for renal tubule adenoma or carcinoma in 

male F344 rats exposed to tert-butanol in drinking water for 2 years modeled 

with PBPK (tert-butanol, mg/L) dose units and including all dose groups; 

reanalyzed data (Hard et al., 2011; NTP, 1995); BMR = 10% extra risk..............................C-50 

Table C-23. Summary of BMD modeling results for renal tubule adenoma or carcinoma in 

male F344 rats exposed to tert-butanol in drinking water for 2 years modeled 

with PBPK (tert-butanol, mg/L) dose units and excluding high-dose group; 

reanalyzed data (Hard et al., 2011; NTP, 1995); BMR = 10% extra risk..............................C-50 

Table C-24. Summary of BMD modeling results for renal tubule adenoma or carcinoma in 

male F344 rats exposed to tert-butanol in drinking water for 2 years modeled 

with PBPK (metabolized, mg/hr) dose units and including all dose groups; 

reanalyzed data (Hard et al., 2011; NTP, 1995); BMR = 10% extra risk..............................C-52 

Table C-25. Summary of BMD modeling results for renal tubule adenoma or carcinoma in 

male F344 rats exposed to tert-butanol in drinking water for 2 years modeled 

with PBPK (metabolized, mg/hr) dose units and excluding high-dose group; 

reanalyzed data (Hard et al., 2011; NTP, 1995); BMR = 10% extra risk..............................C-52
 

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Supplemental Information―tert-Butyl Alcohol 

Table C-26. Summary of the inhalation unit risk derivation....................................................................C-56
 

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FIGURES 

Figure B-1. Biotransformation of tert-butanol in rats and humans...........................................................B-4 

Figure B-2. Comparison of the tert-butanol portions of existing MTBE models with tert

butanol blood concentrations from i.v. exposure by Poet et al. (1997).............................B-10 

Figure B-3. Schematic of the PBPK submodel for tert-butanol in rats.....................................................B-12 

Figure B-4. Comparison of the EPA model predictions with measured tert-butanol blood 

concentrations for i.v., inhalation, and oral gavage exposure to tert-butanol...................B-15 

Figure B-5. Comparison of the EPA model predictions with measured amounts of tert

butanol in blood after repeated inhalation exposure to tert-butanol................................B-17 

Figure B-6. Exposure-response array of other effects following oral exposure to tert-butanol. ............B-29 

Figure B-7. Exposure-response array of other effects following inhalation exposure to tert

butanol. ...............................................................................................................................B-30 

Figure C-1. Plot of incidence by dose, with fitted curve for LogLogistic model for kidney 

transitional epithelial hyperplasia in male F344 rats exposed to tert-butanol in 

drinking water for 2 years (NTP, 1995); BMR = 10% extra risk; dose shown in 

mg/kg-d .................................................................................................................................C-4 

Figure C-2. Plot of incidence by dose, with fitted curve for Multistage 3° model for kidney 

transitional epithelial hyperplasia in female F344 rats exposed to tert-butanol 

in drinking water for 2 years (NTP, 1995); BMR = 10% extra risk; dose shown in 

mg/kg-d .................................................................................................................................C-6 

Figure C-3. Plot of mean response by dose, with fitted curve for Linear model with constant 

variance for absolute kidney weight in male F344 rats exposed to tert-butanol 

in drinking water for 15 months (NTP, 1995); BMR = 10% rel. dev. from control 

mean; dose shown in mg/kg-d............................................................................................C-10 

Figure C-4. Plot of mean response by dose, with fitted curve for Exponential (M4) model 

with constant variance for absolute kidney weight in female F344 rats exposed 

to tert-butanol in drinking water for 15 months (NTP, 1995); BMR = 10% rel. 

dev. from control mean; dose shown in mg/kg-d...............................................................C-13 

Figure C-5. Plot of incidence by dose, with fitted curve for Logprobit model for kidney 

inflammation in female rats exposed to tert-butanol in drinking water for 2 

years (NTP, 1995); BMR = 10% extra risk; dose shown in mg/kg-d ....................................C-15 

Figure C-6. Plot of mean response by concentration, with fitted curve for Hill model for 

absolute kidney weight in male F344 rats exposed to tert-butanol via inhalation 

for 6 hr/d, 5d/wk for 13 weeks (NTP, 1997); BMR = 10% relative deviation from 

the mean; concentration shown in mg/m3 .........................................................................C-19 

Figure C-7. Plot of mean response by concentration, with fitted curve for Hill model for 

absolute kidney weight in female F344 rats exposed to tert-butanol via 

inhalation for 6 hr/d, 5d/wk for 13 weeks (NTP, 1997); BMR = 10% relative 

deviation from the mean; concentration shown in mg/m3 ................................................C-22
 
Figure C-8. Plot of mean response by concentration, with fitted curve for Power model for 

absolute kidney weight in female F344 rats exposed to tert-butanol via 

inhalation for 6 hr/d, 5d/wk for 13 weeks (NTP, 1997); BMR = 10% relative 

deviation from the mean; concentration shown in mg/m3 ................................................C-22 

Figure C-9. Plot of incidence by dose, with fitted curve for Multistage 3° model for thyroid 

follicular cell adenomas in female B6C3F1 mice exposed to tert-butanol in 

drinking water for 2 years (NTP, 1995); BMR = 10% extra risk; dose shown in 

mg/kg-d...............................................................................................................................C-26 

Figure C-10. Plot of incidence by dose, with fitted curve for Multistage 1° model for thyroid 

follicular cell adenomas or carcinomas in male B6C3F1 mice exposed to tert

butanol in drinking water for 2 years (NTP, 1995); BMR = 5% extra risk; dose 

shown in mg/kg-d ...............................................................................................................C-29 

Figure C-11. Plot of incidence by dose, with fitted curve for Multistage 2° model for thyroid 

follicular cell adenomas or carcinomas in male B6C3F1 mice exposed to tert

butanol in drinking water for 2 years, high dose omitted (NTP, 1995); BMR = 5% 

extra risk; dose shown in mg/kg-d......................................................................................C-32 

Figure C-12. Plot of incidence by dose, with fitted curve for Multistage 2° model for renal 

tubule adenoma or carcinoma in male F344 rats exposed to tert-butanol in 

drinking water for 2 years modeled with administered dose units and including 

all dose groups (NTP, 1995); BMR = 10% extra risk; dose shown in mg/kg-d. ...................C-35 

Figure C-13. Plot of incidence by dose, with fitted curve for Multistage 1° model for renal 

tubule adenoma or carcinoma in male F344 rats exposed to tert-butanol in 

drinking water for 2 years modeled with administered dose units and excluding 

high-dose group (NTP, 1995); BMR = 10% extra risk.; dose shown in mg/kg-d. ................C-37 

Figure C-14. Plot of incidence by dose, with fitted curve for Multistage 1° model for renal 

tubule adenoma or carcinoma in male F344 rats exposed to tert-butanol in 

drinking water for 2 years modeled with PBPK (tert-butanol, mg/L) dose units 

and including all dose groups (NTP, 1995); BMR = 10% extra risk.; dose shown 

in mg/L. ...............................................................................................................................C-39 

Figure C-15. Plot of incidence by dose, with fitted curve for Multistage 1° model for renal 

tubule adenoma or carcinoma in male F344 rats exposed to tert-butanol in 

drinking water for 2 years modeled with PBPK (tert-butanol, mg/L) dose units 

and excluding high-dose group (NTP, 1995); BMR = 10% extra risk; dose shown 

in mg/L. ...............................................................................................................................C-41
 

Figure C-16. Plot of incidence by dose, with fitted curve for Multistage 1° model for renal 

tubule adenoma or carcinoma in male F344 rats exposed to tert-butanol in 

drinking water for 2 years modeled with PBPK (metabolized, mg/hr) dose units 

and including all dose groups (NTP, 1995); BMR = 10% extra risk; dose shown in 

mg/hr. .................................................................................................................................C-43 

Figure C-17. Plot of incidence by dose, with fitted curve for Multistage 1° model for renal 

tubule adenoma or carcinoma in male F344 rats exposed to tert-butanol in 

drinking water for 2 years modeled with PBPK (metabolized, mg/hr) dose units 

and excluding high-dose group (NTP, 1995); BMR = 10% extra risk; dose shown 

in mg/hr...............................................................................................................................C-45 

Figure C-18. Plot of incidence by dose, with fitted curve for Multistage 1° model for renal 

tubule adenoma or carcinoma in male F344 rats exposed to tert-butanol in 

drinking water for 2 years modeled with administered dose units and excluding 

Supplemental Information―tert-Butyl Alcohol 

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vii DRAFT—DO NOT CITE OR QUOTE 
high-dose group; re-analyzed data (Hard et al., 2011; NTP, 1995); BMR = 10%
 

extra risk; dose shown in mg/kg-d......................................................................................C-48
 

Figure C-19. Plot of incidence by dose, with fitted curve for Multistage 1° model for renal 

tubule adenoma or carcinoma in male F344 rats exposed to tert-butanol in 

drinking water for 2 years modeled with PBPK (tert-butanol, mg/L) dose units 

and excluding high-dose group; reanalyzed data (Hard et al., 2011; NTP, 1995); 

BMR = 10% extra risk; dose shown in mg/L........................................................................C-51 

Figure C-20. Plot of incidence by dose, with fitted curve for Multistage 1° model for renal 

tubule adenoma or carcinoma in male F344 rats exposed to tert-butanol in 

drinking water for 2 years modeled with PBPK (metabolized, mg/hr) dose units 

and excluding high-dose group; reanalyzed data (Hard et al., 2011; NTP, 1995); 

BMR = 10% extra risk.; dose shown in mg/hr. ....................................................................C-53 

Supplemental Information―tert-Butyl Alcohol 

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viii DRAFT—DO NOT CITE OR QUOTE 
Supplemental Information―tert-Butyl Alcohol 

ABBREVIATIONS
 

AIC Akaike’s information criterion 

ARCO ARCO Chemical Company 

BMD benchmark dose 

BMDL benchmark dose lower confidence limit 

BMDS Benchmark Dose Software 

BMDU benchmark dose upper confidence limit 

BMR benchmark response 

BW body weight 

CFR Code of Federal Regulations 

CHO Chinese hamster ovary 

CYP450 cytochrome P450 

DMSO dimethyl sulfoxide 

DNA deoxyribonucleic acid 

EDTA ethylenediaminetetraacetic acid 

EPA U.S. Environmental Protection Agency 

ETBE ethyl tert-butyl ether 

HBA 2-hydroxyisobutyrate 

HL human leukemia 

IC50 half-maximal inhibitory concentration 

i.p. intraperitoneal 

i.v. intravenous 

MFO mixed function oxidase 

MPD 2-methyl-1,2-propanediol 

MTBE methyl tert-butyl ether 

NADPH nicotinamide adenine dinucleotide 

phosphate 

NTP National Toxicology Program 

·OH hydroxyl radical 

PBPK physiologically based pharmacokinetic 

POD point of departure 

SD standard deviation 

TWA time-weighted average 

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Supplemental Information―tert-Butyl Alcohol 

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APPENDIX A. ASSESSMENTS BY OTHER NATIONAL 

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Table A-1. Health assessments and regulatory limits by other national and 

international health agencies 

Organization Toxicity value 

National Institute of Occupational 

Safety and Health (NIOSH, 2007) 

Recommended Exposure Limit – 100 ppm (300 mg/m3) time-weighted 

average (TWA) for up to a 10-hour workday and a 40-hour work week 

Occupational Safety and Health 

(OSHA, 2006) 

Permissible Exposure Limit for general industry – 100 ppm (300 mg/m3) TWA 

for an 8-hour workday 

Food and Drug Administration 

(FDA, 2011a, b) 

tert-Butyl alcohol: Indirect food additive that may be safely used in surface 

lubricants employed in the manufacture of metallic articles that contact 

food, subject to the provisions of this section (21 Code of Federal 

Regulations [CFR] 178.3910); substance may be used as a defoaming agent 

(21 CFR 176.200). 

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APPENDIX B. INFORMATION IN SUPPORT OF 

HAZARD IDENTIFICATION AND DOSE-RESPONSE 

ANALYSIS 

B.1. TOXICOKINETICS 

Little information is available on the absorption, distribution, metabolism, or excretion of 

tert-butyl alcohol (tert-butanol) in humans. The studies identified for this Toxicological Assessment 

were conducted in conjunction with methyl tert-butyl ether (MTBE) or ethyl tert-butyl ether 

(ETBE), as tert-butanol is a metabolite of both compounds. Several studies examining some aspect 

of the toxicokinetic behavior of tert-butanol in animals have been identified. Many were carried out 

in conjunction with other specific endpoints (e.g., developmental). ARCO (1983) determined no 

differences in the pharmacokinetics of tert-butanol following either oral (i.e., gavage) or inhalation 

exposure. Although some information is available for both oral and inhalation exposures, many 

studies administered tert-butanol via intraperitoneal (i.p.) or intravenous (i.v.) injection. Although 

these studies do not inform the absorption of tert-butanol, they can provide information on its 

distribution, metabolism, and excretion. 

Absorption 

Toxicity data on tert-butanol submitted by industry to the U.S. Environmental Protection 

Agency (EPA) under Section 8(e) of the Toxic Substances Control Act and other reporting 

requirements indicate that tert-butanol is rapidly absorbed after oral administration. Very little of 

the administered dose was excreted in the feces of rats, indicating 99% of the compound was 

absorbed. Comparable blood levels of tert-butanol and its metabolites have been observed after 

acute oral (350 mg/kg) or inhalation (6,060 mg/m3 for 6 hours) exposures in male Sprague-Dawley 

rats (ARCO, 1983); the absorption rate after inhalation exposure could not be determined, however, 

because the blood was saturated with radioactivity after 6 hours of exposure to 6,060 mg/m3. In 

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

at 1.5 hours following the last of six oral gavage doses of 10.5 mmoles tert-butanol/kg (twice daily) 

in female C57BL/6J mice. There was an apparent zero-order decline in tert-butanol concentration 

for most of the elimination phase, and no differences in absorption or elimination rates was 

observed between mice on a repeated dosing regimen and control mice administered equivalent 

volumes of tap water every 12 hours before administration of a single dose of 10.5 mmoles tertbutanol/kg. The study therefore concluded that previous exposures did not affect the absorption or 

elimination of tert-butanol (Faulkner et al., 1989). 

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Distribution 

The available animal data suggest that tert-butanol is distributed throughout the body 

following oral, inhalation, and i.v. exposures (Poet et al., 1997; Faulkner et al., 1989; ARCO, 

1983). Nihlén et al. (1995) calculated partition coefficients for tert-butanol using blood from human 

volunteers and available information about the relative content of water and fat in each tissue. The 

calculated tissue:blood partition coefficients for tert-butanol were slightly above 1 (from 1.02 to 

1.06) for most tissues, except for fat:blood, which was 0.646. The same study evaluated the 

partition coefficients of three oxygenated ethers, including MTBE and ETBE, which are metabolized 

to tert-butanol (see Section B.1.4). The study concluded that, although tert-butanol preferentially 

distributes in body water, the ethers distribute uniformly throughout the body with preference for 

fatty tissues (Nihlén et al., 1995). 

In a study aimed at determining whether tert-butanol (or metabolites) can bind to 

α2u-globulin, Williams and Borghoff (2001) exposed F-344 rats to a single gavage dose of 500 

mg/kg 14C-tert-butanol. They found the radiolabel in three tissues (kidney, liver, and blood) in both 

sexes, but male rats retained more of the tert-butanol equivalents than females (Williams and 

Borghoff, 2001). Radioactivity was found in the low-molecular-weight protein fraction isolated 

from the kidney cytosol in male rats but not in female rats, indicating that tert-butanol or one of its 

metabolites was bound to α2u-globulin. Further analysis determined that tert-butanol, and not its 

metabolite acetone, was bound. Most tert-butanol in the kidney cytosol was eluted as the free 

compound in both males and females, but a small amount was associated with the high-molecularweight protein fraction in both males and females. In another study on α2u-globulin 

nephropathy, Borghoff et al. (2001) found similar results after F-344 rats were exposed to 0, 250, 

450, or 1750 ppm tert-butanol by inhalation for 10 consecutive days. Male rat tert-butanol kidneyto-blood ratios were significantly elevated over ratios in females at all dose levels and exposure 

durations. Although the female tert-butanol kidney-to-blood ratio remained similar with both 

duration and concentration, the male tert-butanol kidney-to-blood ratio increased with duration. 

The liver-to-blood ratios were similar regardless of exposure duration, concentration, or sex. Both 

of these studies indicate distribution to the liver and kidney with kidney retention of tert-butanol in 

the male rat. 

Metabolism 

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

humans, is shown in Figure B-1. Urinary metabolites of tert-butanol in a human male volunteer who 

ingested a gelatin capsule containing 5 mg/kg [13C]-tert-butanol were reported to be 2-methyl-1,2­

propanediol (MPD) and 2-hydroxyisobutyrate (Bernauer et al., 1998). Minor metabolites of 

unconjugated tert-butanol, tert-butanol glucuronides, and traces of the sulfate conjugate also were 

detected. The study was approved by an ethical review board, but no information regarding 

informed consent was reported. In the same study, 2-hydroxyisobutyrate, MPD, and tert-butanol 

sulfate were identified as major metabolites in rats, while acetone, tert-butanol, and tert-butanol 
 

 

 

 

 

 

 

 

 

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glucuronides were identified as minor metabolites (Bernauer et al., 1998). Baker et al. (1982) found 

that tert-butanol was a source of acetone, but acetone production might have been stimulated from 

other sources. 

No studies identified specific enzymes responsible for biotransforming tert-butanol. Using a 

purified enzyme from Sprague-Dawley rats or whole-liver cytosol from Wistar rats, alcohol 

dehydrogenase had negligible or no activity toward tert-butanol (Videla et al., 1982; Arslanian et al., 

1971). Other in vitro studies have implicated the liver microsomal mixed function oxidase (MFO) 

system, namely cytochrome P450 (CYP450) (Cederbaum et al., 1983; Cederbaum and Cohen, 1980). 

In the 1983 study, incubation of tert-butanol at 35 mM with Sprague-Dawley rat liver microsomes 

and a nicotinamide adenine dinucleotide phosphate- (NADPH) generating system resulted in 

formaldehyde the production at a rate of approximately 25 nmoles/mg protein/30 min. According 

to study authors, the amount of formaldehyde generated by tert-butanol was approximately 30% of 

the amount of formaldehyde formed during the metabolism of 10 mM aminopyrene in a similar 

microsomal system. The rate of formaldehyde generation from tert-butanol increased to about 

90 nmol/mg protein/30 min upon addition of azide, which inhibits catalase and thereby prevents 

the decomposition of hydrogen peroxide (H2O2). In other experiments in the same study, 

formaldehyde formation was greatly reduced when H2O2 was included but NADPH was absent or 

when the microsomes were boiled prior to incubation. Additionally, the rate of formaldehyde 

formation in the microsomal oxidizing system depended on the concentration of tert-butanol, with 

apparent Km and Vmax values of 30 mM and 5.5 nmol/min/mg protein, respectively. The study 

authors concluded that tert-butanol is metabolized to formaldehyde by a mechanism involving 

oxidation of NADPH, microsomal electron flow, and the generation of hydroxyl-radical (·OH) from 

H2O2, possibly by a Fenton-type or a Haber-Weiss iron-catalyzed reaction involving CYP450, which 

might serve as the iron chelate (Cederbaum and Cohen, 1980). 

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CH3 

glucuronide–O CH3 

CH3 HO O 

t-butyl glucuronide 

HO CH3 

rats, humans [O] CH3

O CH 2-hydroxyisobutyric acid 3 OH 

CYP450 

HO CH3 H3C H OH rats, 3C H2C O CH3 humans CH OH 

3 CH3 formaldehyde t-butanol 2-methyl-1,2-propanediol 

O 

rats 

- O 

O H3C CH3

S CH3 acetone O 

O CH3 

CH3
 

t-butyl sulfate
 

 

 

 

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and Cederbaum and Cohen (1980).
 

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

In a follow-up study, tert-butanol was oxidized to formaldehyde and acetone by various 

systems known to generate ·OH radical, including rat liver microsomes or other nonmicrosomal 

·OH-generating systems (Cederbaum et al., 1983). The nonmicrosomal tests included two chemical 

systems: (1) the iron-catalyzed oxidation of ascorbic acid (ascorbate-Fe-EDTA 

[ethylenediaminetetraacetic acid]) and (2) the Fenton system of chelated ferrous iron and H2O2. In 

both Fenton-type systems, H2O2 served as a precursor for ·OH. Additionally, a Haber-Weiss 

enzymatic system involving xanthine oxidation by xanthine oxidase in the presence of Fe-EDTA was 

used. In this system, ·OH is thought to be produced by the interaction of H2O2 and superoxide 

(O2·−). Further experiments demonstrated the involvement of ·OH in either the ascorbate-Fe-EDTA 

or the xanthine oxidation systems based on inhibition of formaldehyde and acetone production 

from tert-butanol when ·OH-scavenging agents (e.g., benzoate, mannitol) were added. Some 

experiments in this study of the oxidation of tert-butanol by the microsomal metabolizing system of 

the liver were similar to those in the previous study (Cederbaum and Cohen, 1980) except that 

acetone formation, in addition to formaldehyde, also was measured. Again, these experiments 

showed the dependence of the microsomal metabolizing system on an NADPH-generating system 

and the ability of H2O2 to enhance, but not replace, the NADPH-generating system. Addition of 

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chelated iron (Fe-EDTA) boosted the microsomal production of formaldehyde and acetone, while 

·OH-scavenging agents inhibited their production. The study authors noted that neither Fe-EDTA 

nor ·OH-scavenging agents is known to affect the CYP450-catalyzed oxidation of typical MFO 

substrates such as aminopyrene or aniline. The study also showed that known CYP450 inhibitors, 

such as metyrapone or SKF-525A, inhibited the production of formaldehyde from aminopyrene but 

not from tert-butanol. Finally, typical inducers of CYP450 and its MFO metabolizing activities, such 

as phenobarbital or 3-methylcholanthrene, had no effect on microsomal metabolism of tert-butanol 

to formaldehyde and acetone. According to the study authors, the oxidation of tert-butanol appears 

to be mediated by ·OH (possibly via H2O2), which can be produced by any of the tested systems by a 

Fenton-type reaction as follows: 

H2O2 + Fe2+-chelate → ·OH + OH− + Fe3+-chelate 

According to this reaction, reduction of ferric iron (Fe3+) to ferrous iron (Fe2+) is required 

for continuous activity. The study authors concluded that the nature of the iron and the pathway of 

iron reduction within the microsomes remain to be elucidated even though an NADPH-dependent 

electron transfer or O2·− might be involved (Cederbaum et al., 1983). 

Excretion 

Human data on the excretion of tert-butanol derives from studies of MTBE and ETBE 

(Nihlén et al., 1998a, b). Eight or ten male human volunteers were exposed to 5, 25, or 50 ppm 

MTBE (18.0, 90.1, 757 mg/m3) or ETBE (20.9, 104, and 210 mg/m3) by inhalation during 2 hours of 

light exercise. The half-life of tert-butanol in urine following MTBE exposure was 8.1 ± 2.0 hours 

(average of the 25- and 50-ppm MTBE doses); the half-life of tert-butanol in urine following ETBE 

exposure was 7.9 ± 2.7 hours (average of 25- and 50-ppm ETBE doses). In both studies, the urinary 

excretion of tert-butanol was less than 1% of the uptake or absorption of MTBE or ETBE. The renal 

clearance rate of tert-butanol was 0.67 ± 0.11 mL/hr-kg with MTBE exposure (average of 25- and 

50-ppm MTBE doses); the renal clearance rate was 0.80 ± 0.34 mL/hr-kg with ETBE exposure 

(average of 25- and 50-ppm ETBE doses). 

Amberg et al. (2000) exposed six volunteers (three males and three females, 28 ± 2 years 

old) to 18.8 and 170 mg/m3 ETBE. Each exposure lasted 4 hours, and the two concentrations were 

administered to the same volunteers 4 weeks apart. Urine was collected at 6-hour intervals for 

72 hours following exposure. tert-Butanol and two metabolites of tert-butanol, 

2-hydroxyisobutyrate (HBA) and MPD, also were identified in urine. At an ETBE level of 170 

mg/m3, tert-butanol displayed a half-life of 9.8 ± 1.4 hours. At the low-exposure ETBE 

concentration, the tert-butanol half-life was 8.2 ± 2.2 hours. The predominant urinary metabolite 

identified was HBA, excreted in urine at 5–10 times the amount of MPD and 12–18 times the 

amount of tert-butanol (note: urine samples had been treated with acid before analysis to cleave 

conjugates). HBA in urine showed a broad maximum at 12–30 hours after exposure to both 

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Supplemental Information―tert-Butyl Alcohol 

concentrations, with a slow decline thereafter. MPD in urine peaked at 12 and 18 hours after
 

exposure to 170 and 18.8 mg/m3 ETBE, respectively, while tert-butanol peaked at 6 hours after
 

exposure to both concentrations.
 

Amberg et al. (2000) exposed F344 NH rats to 18.8 and 170 mg/m3 ETBE. Urine was
 

collected for 72 hours following exposure. Similar to humans, rats excreted mostly HBA in urine,
 

followed by MPD and tert-butanol. The half-life for tert-butanol in rat urine was 4.6 ± 1.4 hours at
 

ETBE levels of 170 mg/m3, but half-life could not be calculated at the ETBE concentration of 

18.8 mg/m3. Corresponding half-lives were 2.6 ± 0.5 and 4.0 ± 0.9 hours for MPD and 3.0 ± 1.0 and 

4.7 ± 2.6 hours for HBA. In Sprague-Dawley rats treated with radiolabeled tert-butanol by gavage at 

1, 30, or 500 mg/kg, a generally constant fraction of the administered radioactivity (23–33%) was 

recovered in the urine at 24 hours postdosing. Only 9% of a 1500-mg/kg administered dose was 

recovered in urine, however, suggesting that the urinary route of elimination is saturated following 

this dose (ARCO, 1983). Among all tested doses, most of the urinary radiolabel was attributed to a 

polar fraction that was not characterized, while only 0.3–5.5% of the administered dose was 

considered tert-butanol. The saturation in urinary elimination of radioactivity with the increased 

dose was considered a manifestation of saturated metabolic capacity; however, no further 

information was provided on the fate or balance of the administered radiolabel at any of the tested 

tert-butanol doses (ARCO, 1983). 

Physiologically Based Pharmacokinetic Models 

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

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

primary metabolite after oral or inhalation exposure to MTBE or ETBE. The most recent models for 

MTBE oral and inhalation exposure include a component for the binding of tert-butanol to 

α2u-globulin (Borghoff et al., 2010; Leavens and Borghoff, 2009). 

Faulkner and Hussain (1989) used a one-compartment, open model with Michaelis-Menten 

elimination kinetics to fit tert-butanol blood concentrations obtained from C57BL/6J mice given i.p. 

injections of 5, 10, or 20 mmol/kg tert-butanol. Elimination was indistinguishable from first-order 

kinetics in the range of concentrations studied. An increase in Vmax and decrease in apparent 

volume of distribution with dose are consistent with this model and suggest the existence of 

parallel elimination processes. 

Borghoff et al. (1996) developed a PBPK model for MTBE and its metabolite tert-butanol in 

rats. Doses and blood levels were taken from several published studies. The initial model included a 

tissue-specific, five-compartment model using blood, liver, kidney, muscle, and fat with liver 

metabolism rate constants. The model predicted the accumulation of tert-butanol in blood, but not 

its clearance. A two-compartment model was better at predicting tert-butanol blood levels, but the 

volume of total body water had to be changed to obtain an adequate fit, suggesting dose-dependent 

changes in the kinetics of tert-butanol. Overall, evaluation of the tert-butanol models suggests that 

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Supplemental Information―tert-Butyl Alcohol 

the clearance of tert-butanol from the blood of rats after exposure to MTBE involves processes 

beyond metabolic elimination. 

Nihlén and Johanson (1999) developed a PBPK model for evaluation of inhalation exposure 

in humans to the gasoline additive ETBE. Model compartments for ETBE included lungs (with 

arterial blood), liver, fat, rapidly perfused tissues, resting muscles, and working muscles. The same 

set of compartments and an additional urinary excretion compartment were used for the 

metabolite, tert-butanol. First-order metabolism was assumed in the model, and tissue/blood 

partition coefficients were determined by in vitro methods (Nihlén et al., 1995). Estimates of 

individual metabolite parameters of eight subjects were obtained by fitting the PBPK model to 

experimental data from humans (5, 25, or 50 ppm ETBE; 2-hour exposure) (Nihlén et al., 1998a). 

This model primarily was applied to predict levels of the biomarkers ETBE and tert-butanol in 

blood, urine, and exhaled air after various scenarios, such as prolonged exposure, fluctuating 

exposure, and exposure during physical activity (Nihlén and Johanson, 1999). 

Rao and Ginsberg (1997) developed a PBPK model for MTBE and its principal metabolite, 

tert-butanol, based on the Borghoff et al. (1996) model. The modified model included a skin 

compartment to simulate dermal absorption of MTBE during bathing or showering. A brain 

compartment was added as a target organ for MTBE-induced neurological responses. MTBE 

metabolism to tert-butanol was assumed to occur in the liver through two saturable pathways. The 

tert-butanol portion of the model included further metabolism of tert-butanol in the liver, 

exhalation in the lungs, and renal excretion (in the human model only). The model was validated 

against published human and rat data and was used to help determine the contribution of tertbutanol to the acute central nervous system effects observed after MTBE dosing. 

The Rao and Ginsberg (1997) model used peak concentrations of MTBE and tert-butanol in 

the blood and brain for interspecies, route-to-route, and low-/high-dose extrapolations. The 

MTBE/tert-butanol PBPK model was adapted to humans by adjusting physiology according to 

literature values, incorporating the blood/air partition coefficient for humans reported by Johanson 

et al. (1995), and allometrically scaling the metabolic rate based on body weight. A renal 

elimination component was added to account for the small percentage of MTBE disposition that 

occurs in humans via urinary excretion of tert-butanol. tert-Butanol concentrations in human blood 

during and after MTBE exposure (25 or 50 ppm for 2 hours) were accurately predicted by the 

human model (Johanson et al., 1995). 

Kim et al. (2007) expanded the Borghoff et al. (1996) model to develop a multi-exposure 

route model for MTBE and its primary metabolite, tert-butanol, in humans. The significant features 

and advantages of the Kim et al. (2007) model are that parameters used for quantifying the 

pharmacokinetic behavior of MTBE and tert-butanol are calibrated using time-series 

measurements from controlled-exposure experiments in humans as reported by Prah et al. (2004). 

MTBE partition coefficient values described in the Licata et al. (2001) model and skin compartment 

parameters from the Rao and Ginsberg (1997) model were incorporated. The PBPK model for 

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Supplemental Information―tert-Butyl Alcohol 

MTBE consists of nine primary compartments representing the lungs, skin, fat, kidney, stomach, 

intestine, liver, rapidly perfused tissue, and slowly perfused tissue. The tert-butanol model consists 

of three compartments representing blood, liver, and other tissue.
 

Leavens and Borghoff (2009) developed a PBPK model for inhalation exposures in male and
 

female rats that expanded on Borghoff et al. (1996) and Rao and Ginsberg (1997) to include the sex

specific effects of MTBE binding to α2u-globulin, a protein unique to male rats, and to describe the 

induction of tert-butanol metabolism after repeated exposures. Although the primary purpose of 

the model was to estimate MTBE and tert-butanol tissue concentrations after MTBE exposure, the 

model also was parameterized to include inhalation uptake of tert-butanol. The tert-butanol portion 

of the model was calibrated using data from rat exposures to tert-butanol and to MTBE. Model 

compartments included blood, brain, fat, gastrointestinal tissues, kidney, liver, poorly perfused 

tissues (blood flow <100 mL/min/100 g of tissue: bone, muscle, skin, fat), and rapidly perfused 

tissues. 

Distribution of MTBE and tert-butanol was assumed perfusion (i.e., blood-flow) limited. 

This model used the same assumptions as Borghoff et al. (1996) regarding MTBE metabolism and 

kinetics and further assumed that tert-butanol was metabolized only in the liver through one low

affinity pathway and excreted through urine. The model described binding of MTBE or tert-butanol 

with α2u-globulin in the kidney, due to the high concentration of α2u-globulin in the kidney. As 

chemicals bind to α2u-globulin, the rate of hydrolysis of the protein decreases and causes 

accumulation in the kidney; however, there is no evidence that binding of α2u-globulin affects its 

synthesis, secretion, or circulating concentrations [Borghoff et al. (1990) as cited in Leavens and 

Borghoff (2009)]. Equations describing this phenomenon were included in the model for male rats 

only to account for the effects of the binding with α2u-globulin on metabolism of MTBE and tert

butanol. Partition coefficient values in the model that differed from those published in previous 

PBPK models included poorly perfused tissues:blood and kidney:blood values. The kidney:blood 

value was based on calculated kidney:blood concentrations in female rats only because of the lack 

of α2u-globulin-associated effects in female rats. The deposition of tert-butanol during inhalation in 

the nasal cavity and upper airways was reflected in the high blood:air partition coefficient for tert

butanol, and the ability of tert-butanol to induce its own metabolism after chronic exposure also 

was taken into account. No differences in the induction of metabolism were reported between 

males and females. The model simulated concentrations of MTBE and tert-butanol in the brain, 

liver, and kidney of male and female rats following inhalation exposure at concentrations of 100, 

400, 1,750, or 3,000 ppm MTBE, and compared them to measured concentrations of MTBE and tert

butanol from rats exposed at those levels. 

Concentrations of MTBE and tert-butanol in the brain and liver were similar in male and 

female rats during exposure and postexposure, but the concentrations of the chemicals in the 

kidney significantly differed between male rats and female rats. The additional parameter 

accounting for α2u-globulin protein binding in this PBPK model more accurately reflects the 

­

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Supplemental Information―tert-Butyl Alcohol 

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metabolism of both MTBE and tert-butanol in male rat kidneys over time compared with other 

PBPK models. The model highlights that binding can stimulate increased renal effects in male rats 

after exposure to MTBE and tert-butanol. The assumptions made to reflect tert-butanol metabolism 

induction and deposition in the nasal cavity and upper airways generally were supported by 

measured data from rats exposed to 250-, 450-, or 1,750-ppm tert-butanol as evidenced by the fact 

that the model was within one standard deviation of the mean concentrations for most data points. 

The model overpredicted the concentration of tert-butanol in the brain, liver, and kidney of male 

rats, however, after repeated exposures. 

Borghoff et al. (2010) modified the PBPK model of Leavens and Borghoff (2009) by adding 

oral gavage and drinking water exposure components to compare different dose metrics to the 

toxicity observed across different studies. The Borghoff et al. (2010) model assumed first-order 

uptake of MTBE absorption from the gut, with 100% of the MTBE dose absorbed for both drinking 

water and oral gavage exposures. They conducted a series of pharmacokinetic studies comparing 

the effects of different rat strains and different dosing vehicles on the blood concentration-time 

profiles of MTBE and tert-butanol following MTBE exposure. The effects of exposure to MTBE via 

drinking water, oral gavage, and inhalation routes over 7 and 91 days on male and female rats were 

modeled and compared with measured data collected from F344 rats (exposed 28 days) and Wistar 

Han rats (exposed 14 and 93 days). 

The model predicted the blood concentrations of tert-butanol observed after 250 or 1,000 

mg/kg-day administration of MTBE in males and females and the blood concentrations of MTBE 

after 1,000 mg/kg-day. The model did not predict peak concentrations of MTBE, however, after 250 

mg/kg-day in males or females using either olive oil or 2% Emulphor as vehicles. When comparing 

strains, the blood concentrations were similar across strain and sex, except in female SpragueDawley rats administered 1,000 mg/kg-day MTBE. Female Sprague-Dawley rats had a significantly 

(p-value not specified) higher blood concentration of both MTBE and tert-butanol compared with 

F344 and Wistar Han females. The study authors considered this an outlier, however, and 

maintained the metabolic patterns were similar. The model overpredicted the amount of MTBE in 

the male rat kidney but accurately predicted the level of tert-butanol in the male rat kidney at all 

exposures tested. The model did not accurately predict the kidney concentrations of tert-butanol in 

the female kidney after exposure to MTBE via drinking water, but the study authors attributed the 

inaccuracies to the study design as opposed to the model formulation. All tert-butanol entering the 

submodel comes from MTBE metabolism in the liver, and the model does not include a separate 

oral intake of tert-butanol. 

B.2. PBPK MODEL EVALUATION SUMMARY 

Evaluation of Existing tert-Butanol Submodels 

The Blancato et al. (2007) and Leavens and Borghoff (2009) PBPK models for MTBE were 

evaluated by comparing predictions from the tert-butanol portions of the models with the tert-butanol 

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Supplemental Information―tert-Butyl Alcohol 

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i.v. data of Poet et al. (1997) (see Figure B-2). Neither model adequately represented the tert-butanol 

blood concentrations. Modifications of model assumptions for alveolar ventilation, explicit pulmonary 

compartments, and induction of metabolism of tert-butanol did not significantly improve model fits 

to the data. Attempts to reoptimize model parameters in the tert-butanol submodels of Blancato et al. 

(2007) and Leavens and Borghoff (2009) to match blood concentrations from the i.v. dosing study 

were unsuccessful. 

(A) (B) 

Neither the (A) Blancato et al. (2007) nor the (B) Leavens and Borghoff (2009) model adequately 

represents the measured tert-butanol blood concentrations. 

Figure B-2. Comparison of the tert-butanol portions of existing MTBE models 

with tert-butanol blood concentrations from i.v. exposure by Poet et al. 

(1997). 

The PBPK submodel for tert-butanol in rats was developed in acslX (Advanced Continuous 

Simulation Language, Aegis, Inc., Huntsville, Alabama) by modifying information from the many PBPK 

models developed in rats and humans for the structurally related substance, MTBE, and its metabolite 

tert-butanol (Borghoff et al., 2010; Leavens and Borghoff, 2009; Blancato et al., 2007; Kim et al., 

2007; Rao and Ginsberg, 1997; Borghoff et al., 1996). A brief description comparing the Blancato et al. 

(2007) and Leavens and Borghoff (2009) models is provided, followed by an evaluation of the MTBE 

models and the assumptions adopted from MTBE models or modified in the tert-butanol model. 

The Blancato et al. (2007) model is an update of the earlier Rao and Ginsberg (1997) model, and 

the Leavens and Borghoff (2009) model is an update of the Borghoff et al. (1996) model. Both 

the Blancato et al. (2007) and Leavens and Borghoff (2009) models are flow-limited models that 

predict amounts and concentrations of MTBE and its metabolite tert-butanol in blood and six tissue 

compartments: liver, kidney, fat, brain, and rapidly and slowly perfused tissues. These tissue 

compartments are linked through blood flow, following an anatomically accurate, typical, 

physiologically based description (Andersen, 1991). The parent (MTBE) and metabolite 

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Supplemental Information―tert-Butyl Alcohol 

(tert-butanol) models are linked by the metabolism of MTBE to tert-butanol in the liver. Routes of 

exposure included in the models are oral and inhalation for MTBE; Leavens and Borghoff (2009) 

included inhalation exposure to tert-butanol. Oral doses are assumed 100% bioavailable and 100% 

absorbed from the gastrointestinal tract represented with a first-order rate constant. Following 

inhalation of MTBE or tert-butanol, the chemical is assumed to enter the systemic blood supply 

directly, and the respiratory tract is assumed to be at pseudo-steady state. Metabolism of MTBE by 

CYP450s to formaldehyde and tert-butanol in the liver is described with two Michaelis-Menten 

equations representing high- and low-affinity enzymes. tert-Butanol is either conjugated with 

glucuronide or sulfate or further metabolized to acetone through MPD and HBA; both processes are 

described by a single Michaelis-Menten equation in the models. All model assumptions are valid for 

tert-butanol and were applied to the EPA-modified tert-butanol PBPK model, except for the 

separate brain compartment. The brain compartment was lumped with other richly perfused 

tissues in the EPA-modified tert-butanol PBPK model. 

In addition to differences in parameter values between the Blancato et al. (2007) and 

the Leavens and Borghoff (2009) models, the model structure has three differences: (1) the alveolar 

ventilation was reduced during exposure, (2) the rate of tert-butanol metabolism increased over time 

due to induction of CYP enzymes, and (3) binding of MTBE and tert-butanol to α2u-globulin was 

simulated in the kidney of male rats. The Blancato et al. (2007) model was configured through EPA’s 

PBPK modeling framework, ERDEM (Exposure-Related Dose Estimating Model), which includes 

explicit pulmonary compartments. The modeling assumptions related to alveolar ventilation, 

explicit pulmonary compartments, and induction of metabolism of tert-butanol are discussed in this 

model evaluation section. 

MTBE and tert-butanol binding to α2u-globulin in the kidneys of male rats were incorporated in 

the PBPK model of MTBE by Leavens and Borghoff (2009). Binding to α2u-globulin is one hypothesized 

mode of action for the observed kidney effects in MTBE-exposed animals. For a detailed description of 

the role of α2u-globulin and other modes of action in kidney effects, see the kidney mode of action 

section of the Toxicological Review (Section 1.2.1). In the Leavens and Borghoff (2009) model, binding of 

MTBE to α2u-globulin was applied to sex differences in kidney concentrations of MTBE and tertbutanol, but acceptable estimates of MTBE and tert-butanol pharmacokinetics in the blood are 

predicted in other models that did not consider α2u-globulin binding. Given the uncertainty of tertbutanol binding to α2u-globulin, it was not included in the tert-butanol PBPK submodel. 

Modification of Existing tert-Butanol Submodels 

To account for the tert-butanol blood concentrations after i.v. tert-butanol exposure, the 

model was modified by adding a pathway for reversible sequestration of tert-butanol in the blood (see 

Figure B-3). The PBPK model represented the rate of change in the amount of tert-butanol in the 

sequestered blood compartment (Ablood2) with the following equation, where KON is the binding rate 

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Supplemental Information―tert-Butyl Alcohol 

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constant, CV is the free tert-butanol concentration in blood, KOFF is the unbinding rate constant, and 

Cblood2 is the concentration of tert-butanol bound in blood (equal to Ablood2/Vblood). 

dAblood2/dt = KON*CV* − KOFF *Cblood2 

Exposure can be via multiple routes, including inhalation, oral, or i.v. dosing. Metabolism of tert-butanol, 

which occurs in the liver, is described by Michaelis-Menten equations with one pathway for tert-butanol. 

tert-Butanol is cleared via exhalation and via urinary excretion. See Table B-1 for definitions of parameter 

abbreviations. 

Figure B-3. Schematic of the PBPK submodel for tert-butanol in rats. 

Table B-1. PBPK model physiologic parameters and partition coefficients 

Body weight and organ volumes as fraction of body weight 

Body weight (kg) 0.25 (Brown et al., 1977) 

Body fraction that is blood perfused (Fperf ) 0.8995 (Brown et al., 1977) 

Liver 0.034 (Brown et al., 1977) 

Kidney 0.007 (Brown et al., 1977) 

Fat 0.07 (Brown et al., 1977) 

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Supplemental Information―tert-Butyl Alcohol 

Rapidly perfused 0.04 (Brown et al., 1977)
 

Slowly perfused 0.7485 a
 

Blood 0.074 (Brown et al., 1977)
 

Cardiac output and organ blood flows as fraction of cardiac output 

Cardiac output (L/hr) 5.38 (Brown et al., 1977)

b 

Alveolar ventilation (L/hr) 5.38 (Brown et al., 1977)

c 

Liver 0.174 (Brown et al., 1977)

d 

Kidney 0.141 (Brown et al., 1977) 

Fat 0.07 (Brown et al., 1977) 

Rapidly perfused 0.279 e 

Slowly perfused 0.336 (Brown et al., 1977) 

Partition coefficients for tert-butanol 

Blood:air 481 (Borghoff et al., 1996) 

Liver:blood 0.83 (Borghoff et al., 1996) 

Fat:blood 0.4 (Borghoff et al., 1996) 

Rapidly perfused:blood 0.83 (Borghoff et al., 1996) 

Slowly perfused:blood 1.0 (Borghoff et al., 1996) 

Kidney:blood 0.83 (Borghoff et al., 1996) 

a Fperf − Σ(other compartments).
 b 15.2*BW0.75 (BW = body weight).
 c Alveolar ventilation is set equal to cardiac output.
 d Sum of liver and gastrointestinal blood flows.
 e 1 − Σ(all other compartments).

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The physiologic parameter values obtained from the literature are shown in Table B-1 

(Brown et al., 1977). tert-Butanol partition coefficients, determined by the ratios of measured 

tissue:air and blood:air partition coefficients (Borghoff et al., 1996), also were obtained from 

literature. The parameters describing rate constants of metabolism and elimination of tert-butanol also 

were obtained from the literature (Blancato et al., 2007) and were kept fixed because they were 

optimized to tert-butanol blood concentrations measured after MTBE exposure, which is also 

metabolized to tert-butanol. The parameters describing tert-butanol absorption and tert-butanol 

sequestration in blood were estimated by optimizing the model to the time-course data for blood tertbutanol for rats exposed via i.v., inhalation, and oral routes (Leavens and Borghoff, 2009; Poet et al., 

1997; ARCO, 1983). The model parameters were estimated with the acslX optimization routine to 

minimize the log-likelihood function of estimated and measured tert-butanol concentrations. The 

Nedler-Mead algorithm was used with heteroscedasticity and allowed to vary between 0 and 2. The 

predictions of the model with optimized parameters have a much-improved fit to the tert-butanol 

blood concentrations after tert-butanol i.v. exposures, as shown in panel A of Figure B-4. Additionally, 

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

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Supplemental Information―tert-Butyl Alcohol 

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the model adequately estimated the tert-butanol blood concentrations after inhalation and oral gavage 

exposures. The optimized parameter values are shown in Table B-2. The ARCO (1983) study measured 

tert-butanol in plasma only, unlike the Poet et al. (1997) and Leavens and Borghoff (2009) studies, 

which measured tert-butanol in whole blood. Based on the measurements of plasma and whole blood 

by JPEC (2008), the concentration of tert-butanol in plasma is approximately 60% of the concentration 

in whole blood. The tert-butanol plasma concentrations measured by ARCO were increased (divided by 

60%) to the expected concentration in whole blood for comparison with the PBPK model. 

Induction of tert-butanol-metabolizing enzymes was included in the Leavens and Borghoff 

(2009) model of MTBE based on their study of rats exposed for 8 days to tert-butanol via inhalation. 

The enzyme induction equation and parameters developed in the Leavens and Borghoff (2009) 

model that were applied to the tert-butanol submodel are as follows. 

Vmax tert-butanol IND = Vmax tert-butanol *INDMAX(1-exp(-KIND*t)) 

Vmax tert-butanol IND is the maximum metabolic rate after accounting for enzyme induction, 

Vmax tert-butanol is the metabolism rate constant from Table B-2 for both tert-butanol pathways, 

and INDMAX is the maximum percent increase in Vmax tert-butanol (124.9). KIND is the rate 

constant for enzyme induction (0.3977/day). The increased tert-butanol metabolism better 

estimates the measured tert-butanol blood concentrations as can be seen in the comparison of the 

model predictions and experimental measurements shown in Figure B-5. The model better 

predicted blood concentrations in female rats than male rats. The male rats had lower tert-butanol 

blood concentrations after repeated exposures compared with female rats, and this difference could 

indicate greater induction of tert-butanol metabolism or other physiologic changes such as 

ventilation or urinary excretion in males. The current data for tert-butanol metabolism do not 

provide sufficient information for resolving this difference between male and female rats. 

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(C)
 

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Source: (A) i.v. data from Poet et al. (1997); (B) inhalation data from Leavens and Borghoff (2009); and (C) 

oral gavage data from ARCO (1983) with the optimized parameter values as shown in Table B-2. 

Figure B-4. Comparison of the EPA model predictions with measured tert

butanol blood concentrations for i.v., inhalation, and oral gavage exposure to 

tert-butanol. 

Table B-2. Rate constants for tert-butanol determined by optimization of the model 

with experimental data 

Supplemental Information―tert-Butyl Alcohol 

­

Rate Constant Value Source or Reference 

Metabolism (VMTBA; mg/kg-hr)a 

Metabolism (KMTBA; mg/L) 

Urinary elimination (KELIM2; 1/hr) 

TBA sequestration rate constant (KON; L/hr) 

TBA unsequestration rate constant (KOFF; L/hr) 

Absorption from gastrointestinal tract (KAS2; 1/hr) 

8.0 

28.8 

0.5 

0.148 

0.0134 

0.5 

Blancato et al. (2007) 

Blancato et al. (2007) 

Blancato et al. (2007) 

Optimized 

Optimized 

Optimized 

a Scaled by BW0.7 (0.250.7 = 0.379), BW = body weight. 
 

 

Supplemental Information―tert-Butyl Alcohol 

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Summary of the PBPK Model for tert-Butanol 

A PBPK model for tert-butanol was developed by modifying previous models for MTBE and 

tert-butanol (Leavens and Borghoff, 2009; Blancato et al., 2007). Published tert-butanol sub-models 

do not adequately represent the tert-butanol blood concentrations measured in the i.v. study (Poet 

et al., 1997). The addition of a sequestered blood compartment for tert-butanol substantially 

improved the model fit. The alternative modification—changing to diffusion-limited distribution 

between blood and tissues—also improved the model fit, but was considered less biologically 

plausible. Physiological parameters and partition coefficients were obtained from published 

measurements. The rate constants for tert-butanol metabolism and elimination were from a 

published PBPK model of MTBE with a tert-butanol subcompartment (Blancato et al., 2007). 

Additional model parameters were estimated by calibrating to data sets for i.v., oral, and inhalation 

exposures and repeated dosing studies for tert-butanol. Overall, the model produced acceptable fits 

to multiple rat time-course datasets of tert-butanol blood levels following inhalation or oral gavage 

exposures. 

tert-Butanol Model Application 

The PBPK model as described above was applied to toxicity studies to predict tert-butanol 

blood concentrations (the preferred internal dose metric in the absence of evidence linking any 

specific metabolite of tert-butanol to any toxic effect). For simulation studies where tert-butanol 

was administered in drinking water, the consumption was modeled as episodic, based on the 

pattern of drinking observed in rats (Spiteri, 1982). 

PBPK Model Code 

The PBPK acslX model code is available electronically through EPA’s Health and 

Environmental Research Online (HERO) database. All model files may be downloaded in a zipped 

workspace from HERO (U.S. EPA, 201#, HEROID##). 

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Male rats were exposed to 239, 444, or 1726 ppm and female rats were exposed to 256, 444, or 

1914 ppm tert-butanol for up to 8 consecutive days (Borghoff et al., 2001). tert-Butanol blood 

concentrations are better predicted by the model after 8 days of exposure with enzyme induction (right 

panels) compared to without enzyme induction (left panels). 

Figure B-5. Comparison of the EPA model predictions with measured amounts 

of tert-butanol in blood after repeated inhalation exposure to tert-butanol. 

B.3. OTHER PERTINENT TOXICITY INFORMATION 

Other Toxicological Effects 

B.3.1.1. Synthesis of Other Effects 

Effects other than those related to kidney, thyroid, reproductive, developmental, and 

neurodevelopmental effects were observed in some of the available rodent studies. These include 

liver and urinary bladder effects. As previously mentioned in the Study Selection section of the 

Toxicological Review, all studies discussed employed inhalation, oral gavage, or drinking water 

exposures for ≥30 days. Studies are arranged in evidence tables by effect, species, duration, and 

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Supplemental Information―tert-Butyl Alcohol 

design. The design, conduct, and reporting of each study was reviewed, and each study was
 

considered adequate to provide information pertinent to this assessment.
 

Central nervous system effects similar to those ethanol causes, in terms of animals 

appearing intoxicated and having withdrawal symptoms after cessation of oral or inhalation
 

exposure, were observed with tert-butanol. Severity of central nervous system symptoms such as 

withdrawal increased with dose and duration of exposure. Study quality and utility concerns (e.g., 

inappropriate exposure durations, lack of data reporting, small number of animals per treatment 

group) associated with these studies (Grant and Samson, 1981; Snell, 1980; Thurman et al., 

1980; McComb and Goldstein, 1979a, b; Wood and Laverty, 1979), however, preclude an 

understanding of potential neurotoxicity following tert-butanol exposure, and therefore, central 

nervous system studies are not discussed further. 

Exposure-response arrays of these effects on liver and urinary bladder are provided in 

Figure B-6 and Figure B-7 for oral and inhalation studies, respectively. 

Kidney effects 

Absolute and relative kidney weight numerical data are presented in Table B-3. 

Liver effects 

Liver weight and body weight were demonstrated to be proportional and liver weight 

normalized to body weight was concluded optimal for data analysis (Bailey et al., 2004); thus, only 

relative liver weight is presented and considered in the determination of hazard. Although some 

rodent studies observed liver effects (organ weight changes and histopathologic lesions), the effects 

were not consistent across the database. Increases in relative liver weight with tert-butanol 

exposure were observed, but the results pertaining to histopathologic changes were inconsistent 

(Table B-4). The NTP (1995) oral subchronic and chronic studies did not observe treatment-related 

effects on liver histopathology in either sex of F344 rats. In a 10-week study in Wistar rats, several 

liver lesions (including necrosis) and increased liver glycogen were observed in male rats (no 

females were included in the study) with the only dose used (Acharya et al., 1997; Acharya et al., 

1995). The study provided no incidence or severity data. The dose used in this rat study was in the 

range of the lower doses used in the NTP (1995) subchronic rat study. An increased incidence of 

fatty liver was observed in the male mice of the highest dose group in the 2-year mouse bioassay, 

but no histopathological changes were seen in the subchronic mouse study (NTP, 1995). No 

treatment-related effects in liver histopathology were observed in rats or mice of the NTP (1997) 

subchronic inhalation study. 

Urinary bladder effects 

Subchronic studies reported effects in the urinary bladder (Table B-6), although the chronic 

studies indicated little progression in incidence with increased exposure. Transitional epithelial 

hyperplasia of the urinary bladder was observed in male rats and male mice after 13 weeks of 

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exposure at doses of 3,610 mg/kg-day (male rats) and ≥3,940 mg/kg-day (male mice). In rats, the 

increase in transitional epithelial hyperplasia of the urinary bladder was not observed in the 2-year 

study. Male mice exposed at the high dose (2,070 mg/kg-day) for 2 years exhibited minimal 

transitional epithelial hyperplasia of the urinary bladder. Neither female rats nor female mice 

showed increased incidences of this lesion. Both sexes of mice demonstrated incidence of minimal 

to mild inflammation in the urinary bladder after both subchronic and chronic exposures, with a 

greater incidence in males compared to females. 

B.3.1.2. Mechanistic Evidence
 

No mechanistic evidence is available for these effects.
 

B.3.1.3. Summary of Other Toxicity Data 

Based on lack of consistency and lack of progression, the available evidence does not 

support liver and urinary bladder effects, respectively, as potential human hazards of tert-butanol 

exposure. 

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Supplemental Information―tert-Butyl Alcohol 

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Table B-3. Changes in kidney weight in animals following exposure to 

tert-butanol 

Reference and study design Results 

Kidney weight (percent change as compared to control) 

Lyondell Chemical Co. (2004) Males 

Sprague-Dawley rat; 

12/sex/treatment 

Gavage 0, 64, 160, 400, or 

Dose 

(mg/kg-d) 

Left absolute 

weight 

Left relative 

weight 

Right absolute 

weight 

Right relative 

weight 

1,000 mg/kg-d 0 0 0 0 0 

Males: 9 weeks beginning 4 

weeks prior to mating 64 +6 +8 +6 +8 

Females: ≅ 10 weeks (4 weeks 160 +9 +14* +6 +11* 

prior to mating through PND21) 400 +12* +14* +14* 

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

Females 

Dose 

(mg/kg-d) 

Left absolute 

weight 

Left relative 

weight 

Right absolute 

weight 

0 0 0 0 

64 −1 −2 +2 

160 0 0 +1 

400 +3 +2 +4 

1,000 +4 0 +7 

+17* 

+31* 

Right relative 

weight 

0 

0 

0 

+2 

+2 

NTP (1995) Males Females 

F344/N rat; 10/sex/treatment 

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

40 mg/mL 

Dose 

(mg/kg-d) 

Absolute 

weight 

Relative 

weight 

Dose 

(mg/kg-d) 

Absolute 

weight 

Relative 

weight 

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

3,610a mg/kg-d 

F: 0, 290, 590, 850, 1,560, 230 +12* +19* 290 +19* +17* 

3,620a mg/kg-d 490 +17* +26* 590 +16* +15* 

13 weeks 

840 +16* +32* 850 +29* 

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

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

+28* 

+40* 

+81* 

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Supplemental Information―tert-Butyl Alcohol 

Reference and study design Results 

NTP (1995) Males Females 

B6C3F1 mouse; 10/sex/treatment 

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

40 mg/mL) 

Dose 

(mg/kg-d) 

Absolute 

weight 

Relative 

weight 

Dose 

(mg/kg-d) 

Absolute 

weight 

Relative 

weight 

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

8,210a mg/kg-d 

F: 0, 500, 820, 1,660, 6,430, 350 +1 +1 500 0 −3 

11,620 a mg/kg-d 640 +3 +2 820 −3 −1 

13 weeks 

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

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

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

NTP (1995) Males Females 

F344/N rat; 60/sex/treatment 

(10/sex/treatment evaluated at 

15 months) 

Dose 

(mg/kg-d) 

Absolute 

weight 

Relative 

weight 

Dose 

(mg/kg-d) 

Absolute 

weight 

Relative 

weight 

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

10 mg/mL) 

M: 0, 90, 200, or 420a mg/kg-d 90 +4 +8 180 +8* +14* 

F: 0, 180, 330, or 650a mg/kg-d 200 +11 +15* 330 +18* +21* 

2 years 

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

Only rats sacrificed at 15 months were evaluated for organ weights. 

NTP (1997) Males Females 

F344/N rat; 10/sex/treatment 

Inhalation analytical 

concentration: 0, 134, 272, 542, 

Concentration 

(mg/m3) 

Absolute 

weight 

Relative 

weight 

Absolute 

weight 

Relative 

weight 

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

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

) 

(dynamic whole-body chamber) 

6 hr/d, 5 d/wk 

13 weeks 

Generation method (Sonimist 

Ultrasonic spray nozzle 

nebulizer), analytical 

concentration and method were 

reported 

Right kidney weights measured 

0 0 0 0 0 

406 +1 +1 −4 −1 

824 −2 −1 0 +1 

1,643 +3 +2 +4 +4 

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

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

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

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Supplemental Information―tert-Butyl Alcohol 

Reference and study design Results 

NTP (1997) 

B6C3F1 mouse; 10/sex/treatment 

Inhalation analytical 

concentration: 0, 134, 272, 542, 

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

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

) 

(dynamic whole-body chamber) 

6 hr/d, 5 d/wk 

13 weeks 

Generation method (Sonimist 

Ultrasonic spray nozzle 

nebulizer), analytical 

concentration and method were 

reported 

Right kidney weights measured 

Concentration 

(mg/m3) 

0 

406 

824 

1,643 

3,273 

6,368 

Males 

Absolute 

weight 

0 

−6 

−1 

+4 

−10 

+3 

Relative 

weight 

0 

−4 

+3 

+3 

−3 

+6 

Females 

Absolute 

weight 

0 

+1 

+5 

+1 

0 

+3 

Relative 

weight 

0 

−3 

+9 

−2 

+7 

+15* 

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

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

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

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

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

.
 

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Table B-4. Changes in liver weight in animals following exposure to 

tert-butanol 

Reference and study design Results 

Acharya et al. (1995) 

Wistar rat; 5–6 males/treatment 

Drinking water (0 or 0.5%), 0 or 575 mg/kgd 

10 weeks 

No significant treatment-related effects (results were only provided in a figure) 

Lyondell Chemical Co. (2004) 

Sprague-Dawley rat; 12/sex/treatment 

Gavage 0, 64, 160, 400, or 1,000 mg/kg-d 

Males: 9 weeks beginning 4 weeks prior to 

mating 

Females: 4 weeks prior to mating through 

PND21 

Percent change compared to control: 

Males Females 

Dose 

(mg/kg-d) 

Absolute 

weight 

Relative 

weight 

Dose 

(mg/kg-d) 

Absolute 

weight 

0 0 0 0 0 

64 −1 0 64 −4 

160 −3 +1 160 −7 

400 −2 −1 400 +2 

1,000 +8 +16* 1,000 +8 

Relative weight 

0 

−4 

−5 

+1 

+3 

NTP (1995) 

F344/N rat; 10/sex/treatment 

Drinking water (0, 2.5, 5, 10, 20, or 40 

mg/mL) 

M: 0, 230, 490, 840, 1,520, 3,610a mg/kg-d 

F: 0, 290, 590, 850, 1,560, 3,620a mg/kg-d 

13 weeks 

Percent change compared to control: 

Males Females 

Dose 

(mg/kg-d) 

Absolute 

weight 

Relative 

weight 

Dose 

(mg/kg-d) 

Absolute 

weight 

0 0 0 0 0 

230 −2 +4 290 +11* 

490 +1 +8* 590 +10* 

840 +5 +20* 850 +12* 

1,520 +8 +31* 1,560 +15* 

3,610 All dead All dead 3,620 +9* 

Relative 

weight 

0 

+9* 

+9* 

+11* 

+16* 

+41* 

NTP (1995) 

B6C3F1 mouse; 10/sex/treatment 

Drinking water (0, 2.5, 5, 10, 20, or 40 

mg/mL) 

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

8,210a mg/kg-d 

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

11,620a mg/kg-d 

13 weeks 

Percent change compared to control: 

Males Females 

Dose 

(mg/kg-d) 

Absolute 

weight 

Relative 

weight 

Dose 

(mg/kg-d) 

Absolute 

weight 

0 0 0 0 0 

350 +2 +3 500 −1 

640 −1 −2 820 −5 

1,590 −1 +5 1,660 −8 

3,940 0 +14* 6,430 −2 

8,210 −16 +22* 11,620 −6 

Relative 

weight 

0 

−4 

−3 

−9* 

+6 

+13* 

NTP (1995) Percent change compared to control: 

Males Females 

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Supplemental Information―tert-Butyl Alcohol 

Reference and study design Results 

F344/N rat; 60/sex/treatment Dose Absolute Relative Dose Absolute Relative 

(10/sex/treatment evaluated at 15 months) (mg/kg-d) weight weight (mg/kg-d) weight weight 

Drinking water (0, 1.25, 2.5, 5 or 10 mg/mL) 

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

F: 0, 180, 330, or 650a mg/kg-d 90 +2 +7 180 −14* −8 

2 years 

200 +8 +11 330 −3 −1 

420 +1 +14* 650 −6 +9* 

Only animals sacrificed at 15 months were evaluated for organ weights. Organ 

weights were not measured in the 2-year mouse study 

NTP (1997) 

F344/N rat; 10/sex/treatment 

Inhalation analytical concentration: 0, 134, 

272, 542, 1,080, or 2,101 ppm (0, 406, 824, 

1,643, 3,273 or 6,368 mg/m3) (dynamic 

whole body chamber) 

Percent change compared to control: 

Males Females 

Concentration 

(mg/m3) 

Absolute 

weight 

Relative 

weight 

Absolute 

weight 

Relative 

weight 

6 hr/d, 5 d/wk 

13 weeks 

Generation method (Sonimist Ultrasonic 

spray nozzle nebulizer), analytical 

concentration and method were reported 

0 0 0 0 0 

406 −8 −8 0 +3 

824 −2 −1 0 0 

1,643 +1 −1 +3 +2 

3,273 +10 +7 +9 +9* 

6,368 +5 +5 +4 +8* 

NTP (1997) 

B6C3F1 mouse; 10/sex/treatment 

Inhalation analytical concentration: 0, 134, 

272, 542, 1,080, or 2,101 ppm (0, 406, 824, 

1,643, 3,273 or 6,368 mg/m3) (dynamic 

whole body chamber) 

Percent change compared to control: 

Males Females 

Concentration 

(mg/m3) 

Absolute 

weight 

Relative 

weight 

Absolute 

weight 

Relative 

weight 

6 hr/d, 5 d/wk 

13 weeks 

Generation method (Sonimist Ultrasonic 

spray nozzle nebulizer), analytical 

concentration and method were reported 

0 0 0 0 0 

406 −1 0 +1 −4 

824 +4 +9 +1 +5 

1,643 +7 +5 +5 +1 

3,273 −8 -2 +2 +9* 

6,368 +5 +7 +8 +21* 

aThe high-dose group had an increase in mortality.
 

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

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

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

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

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Supplemental Information―tert-Butyl Alcohol 

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Table B-5. Changes in liver histopathology in animals following exposure to 

tert-butanol 

Reference and study design Results 

Acharya et al. (1997) ↑ liver glycogen (~ 7 fold)*

Acharya et al. (1995) 

Wistar rat; 5–6 males/treatment ↑incidence of centrilobular necrosis, vacuolation of hepatocytes, loss of

Drinking water (0, 0.5%), 0, 575 mg/kg-d hepatocyte architecture, peripheral proliferation, and lymphocyte 

10 weeks infiltration (incidences and results of statistical tests not reported) 

NTP (1995) No treatment-related effects observed. 

F344/N rat; 10/sex/treatment 

Drinking water (0, 2.5, 5, 10, 20, or 40 mg/mL) 

M: 0, 230, 490, 840, 1,520, 3,610a mg/kg-d 

F: 0, 290, 590, 850, 1,560, 3,620a mg/kg-d 

13 weeks 

NTP (1995) No treatment-related effects observed. 

B6C3F1 mouse; 10/sex/treatment 

Drinking water (0, 2.5, 5, 10, 20, 40 mg/mL) 

M: 0, 350, 640, 1,590, 3,940, 8,210a mg/kg-d 

F: 0, 500, 820, 1,660, 6,430, 11,620a mg/kg-d 

13 weeks 

NTP (1995) No treatment-related effects observed. 

F344/N rat; 60/sex/treatment 

(10/sex/treatment evaluated at 15 months) 

Drinking water (0, 1.25, 2.5, 5, 10 mg/mL) 

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

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

2 years 

NTP (1995) Males Females 

B6C3F1 mouse; 60/sex/treatment 

Drinking water (0, 5, 10, 20 mg/mL) 

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

Dose 

(mg/kg-d) 

Incidence of fatty 

change 

Dose 

(mg/kg-d) 

Incidence of fatty 

change 

F: 0, 510, 1,020, or 2,110 mg/kg-d 0 12/59 0 11/60 

2 years 540 5/60 510 8/60 

1,040 8/59 1,020 8/60 

2,070 29/59* 2,110 6/60 

NTP (1997) 

F344/N rat; 10/sex/treatment 

Inhalation analytical concentration: 0, 134, 

272, 542, 1,080, or 2,101 ppm (0, 406, 824, 

1,643, 3,273 or 6,368 mg/m3) (dynamic whole 

body chamber) 

6 hr/d, 5 d/wk 

13 weeks 

Generation method (Sonimist Ultrasonic spray 

nozzle nebulizer), analytical concentration and 

method were reported 

No treatment-related effects observed in the high dose group (only 

treatment group with liver endpoints evaluated). 

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Supplemental Information―tert-Butyl Alcohol 

Reference and study design Results 

NTP (1997) 

B6C3F1 mouse; 10/sex/treatment 

Inhalation analytical concentration: 0, 134, 

272, 542, 1,080, or 2,101 ppm (0, 406, 824, 

1,643, 3,273 or 6,368 mg/m3) (dynamic whole 

body chamber) 

6 hr/d, 5 d/wk 

13 weeks 

Generation method (Sonimist Ultrasonic spray 

nozzle nebulizer), analytical concentration and 

method were reported 

Authors stated that there were no treatment-related microscopic changes, 

but data were not provided. 

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aThe high-dose group had an increase in mortality.
 

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

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

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

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Table B-6. Changes in urinary bladder histopathology in animals following 

oral exposure to tert-butanol 

Reference and study design Results 

NTP (1995) 

F344/N rat; 10/sex/treatment 

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

40 mg/mL) 

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

mg/kg-d 

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

mg/kg-d 

13 weeks 

Incidence (severity): 

Males Females 

Dose (mg/kg-d) 

Transitional 

epithelial 

hyperplasia 

Dose (mg/kg

d) 

0 0/10 0 

230 not evaluated 290 

490 not evaluated 590 

840 0/10 850 

1,520 1/10 (3.0) 1,560 

3,610 7/10* (2.9) 3,620 

Severity: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked 

- Transitional epithelial 

hyperplasia 

0/10 

not evaluated 

not evaluated 

not evaluated 

0/10 

3/10 (2.0) 

NTP (1995) Incidence (severity): 

B6C3F1 mouse; 10/sex/treatment 

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

40 mg/mL) Transitional Transitional 

M: 0, 350, 640, 1,590, 3,940, Dose epithelial Inflam- Dose epithelial Inflam8,210a mg/kg-d (mg/kg-d) hyperplasia mation (mg/kg-d) hyperplasia mation 

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

11,620a mg/kg-d 0 0/10 0/10 0 0/10 0/10 

13 weeks 350 not evaluated 500 

640 not evaluated 820 

1,590 0/10 0/10 1,660 

3,940 6/10* (1.3) 6/10* (1.3) 6,430 

8,210 10/10* (2.0) 10/10* 

(2.3) 

11,620 

Severity: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked 

0/10 0/10 

not evaluated 

not evaluated 

0/10 0/10 

3/9 (2.0) 6/9* (1.2) 

NTP (1995) No treatment-related effects observed 

F344/N rat; 60/sex/treatment 

(10/sex/treatment evaluated at 

15 months) 

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

10 mg/mL) 

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

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

2 years 

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Reference and study design Results 

NTP (1995) 

B6C3F1 mouse; 60/sex/treatment 

Drinking water (0, 5, 10, or 20 

mg/mL) 

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

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

2 years 

Incidence (severity): 

Males Females 

Dose 

(mg/kg-d) 

Transitional 

epithelial 

hyperplasia 

Inflammation Dose 

(mg/kg-d) 

0 1/59 (2.0) 0/59 0 

540 3/59 (1.7) 3/59 (1.7) 510 

1,040 1/58 (1.0) 1/58 (1.0) 1,020 

2,070 17/59* 

(1.8) 

37/59* (2.0) 2,110 

Severity: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked 

Transitional 

epithelial 

hyperplasia 

0/59 

0/60 

0/59 

3/57 (1.0) 

Inflammation 

0/59 

0/60 

0/59 

4/57* 

(2.0) 

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3 

aThe high-dose group had an increase in mortality.
 

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

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

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Sources: (A) (Acharya et al. (1997); Acharya et al. (1995)); (B) Lyondell Chemical Co. (2004); (C) NTP (1995)
 

Figure B-6. Exposure-response array of other effects following oral exposure 

to tert-butanol. 

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Source: (A) NTP (1997) 

Figure B-7. Exposure-response array of other effects following inhalation 

exposure to tert-butanol. 

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Genotoxicity 

The genotoxic potential of tert-butanol has been studied using a variety of genotoxicity 

assays, including bacterial reverse mutation assays, gene mutation assays, chromosomal 

aberrations, sister chromatid exchanges, micronucleus formation, and deoxyribonucleic acid (DNA) 

strand breaks and adducts. The available genotoxicity data for tert-butanol are discussed below,
 

and the data summary is provided in Table B-7.
 

B.3.2.1. Bacterial Systems 

The mutagenic potential of tert-butanol has been tested by Zeiger et al. (1987) using 

different Salmonella typhimurium strains both in the presence and absence of S9 metabolic 

activation. The preincubation assay protocol was followed. Salmonella strains TA98, TA100, 

TA1535, TA1537, and TA1538 were exposed to five concentrations (100, 333, 1,000, 3,333, or 

10,000 μg/plate) and tested in triplicate. No mutations were observed in any of the strains tested, 

in either the presence or absence of S9 metabolic activation. 

Conflicting results have been obtained with tert-butanol-induced mutagenicity in strain 

Salmonella strain TA102, a strain that is sensitive to damage at A-T sites inducible by oxidants and 

other mutagens and is excision-repair proficient. In a study by Williams-Hill et al. (1999), 

tert-butanol induced an increase in the number of revertants in the first three concentrations with 

S9 activation in a dose-response manner. The number of revertants decreased in the last two 

concentrations. No discussion was provided on why the revertants decreased at higher 

concentrations. The results of this study indicated that test strain TA102 might be a more sensitive 

strain for monitoring tert-butanol levels (Williams-Hill et al., 1999). In another study by Mcgregor 

et al. (2005), however, experiments were conducted on TA102 in two different laboratories using 

similar protocols. tert-Butanol was dissolved in dimethyl sulfoxide (DMSO) or distilled water and 

tested in both the presence and absence of S9 metabolic activation. No statistically significant 

increase in mutants was observed in either solvent medium. In one experiment where tert-butanol 

was dissolved in water, a significant, dose-related increase in the number of revertants occurred, 

reaching almost twice the control value at a concentration of 2,250 μg/plate. Of note is that DMSO is 

known to be a free radical scavenger, and its presence at high concentrations might mask a 

mutagenic response caused by oxidative damage. 

Mutagenicity of tert-butanol has been studied in other systems including Neurospora crassa 

and Saccharomyces cerevisiae. Yeast strain Neurospora crassa at the ad-3A locus (allele 38701) was 

used to test the mutagenic activity of tert-butanol at a concentration of 1.75 mol/L for 30 minutes. 

tert-Butanol did not induce reverse mutations in the tested strain at the exposed concentration 

(Dickey et al., 1949). tert-butanol without exogenous metabolic activation, however, significantly 

increased the frequency of petite mutations (the mitochondrial DNA deletion rho−) in 

Saccharomyces cerevisiae laboratory strains K5-A5, MMY1, D517-4B, and DS8 (Jimenez et al., 1988). 

This effect on mitochondrial DNA, also observed with ethanol and other solvents, was attributed by 

the study authors to the alteration in the lipid composition of mitochondrial membranes, and 

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Supplemental Information―tert-Butyl Alcohol 

mitochondrial DNA’s close association could be affected by membrane composition (Jimenez et al., 

1988). 

B.3.2.2. In Vitro Mammalian Studies 

To understand the role of tert-butanol-induced genotoxicity in mammalian systems, in vitro 

studies have been conducted in different test systems and assays. tert-Butanol was tested to 

evaluate its ability to induce forward mutations at the thymidine kinase locus (tk) in the L5178Y 

tk+/- mouse lymphoma cells using forward mutation assay. Experiments were conducted in both 

the presence and absence of S9 metabolic activation. The mutant frequency was calculated using 

the ratio of mutant clones per plate/total clones per plate × 200. tert-Butanol did not reliably 

increase the frequency of forward mutations in L5178Y tk+/− mouse lymphoma cells with or

without metabolic activation, although one experiment without addition of S9 yielded a small 

increase in mutant fraction at the highest tested concentration (5,000 μg/mL) (McGregor et al., 

1988).
 

To further determine potential DNA or chromosomal damage induced by tert-butanol in in 

vitro systems, NTP (1995) studied sister chromatid exchanges and chromosomal aberrations. 

Chinese hamster ovary (CHO) cells were exposed to tert-butanol in both the presence and absence 

of S9 activation at concentrations of 160–5,000 μg/mL for 26 hours. tert-Butanol did not induce 

sister chromatid exchanges in any concentration tested, although in one experiment, percent 

relative change of sister chromatid exchanges per chromosome scored slightly increased. The same 

authors also studied the effect of tert-butanol on chromosomal aberration formation. CHO cells 

were exposed to four concentrations (160, 500, 1,600, or 5,000 μg/mL) of tert-butanol in both the 

presence and absence of S9. No significant increase in chromosomal aberration was observed in 

any concentration tested. Of note is that, due to severe toxicity at the highest concentration 

(5,000 μg/mL), only 13 metaphase cells were scored instead of 100 in the chromosomal aberration 

assay. 

Sgambato et al. (2009) examined the effects of tert-butanol on DNA damage using a normal 

diploid rat fibroblast cell line. Cells were treated with 0- to 100-mM tert-butanol for 48 hours to 

determine the half-maximal inhibitory concentration (IC50; 0.44 ± 0.2 mM). The 48-hour IC50 

concentration then was used to determine DNA content, cell number, and phases of the cell cycle 

after 24 and 48 hours of exposure. Total protein and DNA oxidative damage also were measured. A 

comet assay was used to evaluate DNA fragmentation at time 0 and after 30 minutes, 4 hours, or 12 

hours of exposure to the IC50 concentration. tert-Butanol inhibited cell division in a dose-dependent 

manner as measured by the number of cells after 24 and 48 hours of exposure at IC50 

concentrations, and with concentrations at 1/10th the IC50. Cell death did not increase, suggesting a 

reduction in cell number due to reduced replication rather than to cytotoxicity. tert-Butanol caused 

an accumulation in the G0/G1 phase of replication, related to different effects on the expression of 

the cyclin D1, p27Kip1, and p53 genes. An initial increase in DNA damage as measured by nuclear 

fragmentation was observed at 30 minutes. The DNA damage declined drastically after 4 hours and 

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disappeared almost entirely after 12 hours of exposure to tert-butanol. This reduction in the extent 

of DNA fragmentation after the initial increase is likely the result of an efficient DNA repair 

mechanism activated by cells following DNA damage induced by tert-butanol. 

DNA damage caused by tert-butanol was determined by single-cell gel electrophoresis 

(comet assay) in human promyelocytic leukemia (HL-60) cells. The cells were exposed to 

concentrations ranging from 1 to 30 mmol/L for 1 hour, and 100 cells were evaluated for DNA 

fragmentation. A dose-dependent increase in DNA damage was observed between 1 and 

30 mmol/L. No cytotoxicity was observed at the concentrations tested (Tang et al., 1997). 

B.3.2.3. In Vivo Mammalian Studies 

Few in vivo studies are available to understand the role of tert-butanol on genotoxicity. The 

National Toxicology Program studied the effect of tert-butanol in a 13-week toxicity study (NTP, 

1995). Peripheral blood samples were obtained from male and female B6CF1 mice exposed to tertbutanol in drinking water at doses of 3,000–40,000 ppm. Slides were prepared to determine the 

frequency of micronuclei in 10,000 normochromatic erythrocytes. In addition, the percentage of 

polychromatic erythrocytes among the total erythrocyte population was determined. No increase in 

micronucleus formation in peripheral blood lymphocytes was observed either in male or female 

B6C3F1 mice exposed for 13 weeks to tert-butanol in drinking water at concentrations as high as 

40,000 ppm (2,110 mg/kg-day) (NTP, 1995). 

Male Kumming mice (8 per treatment) were administered 0, 0.099, 0.99, 10, 101, or 

997 μg/kg BW 14C-tert-butanol in saline via gavage with specific activity ranging from 1.60 to 

0.00978 mCi/mol (Yuan et al., 2007). Animals were sacrificed 6 hours after exposure, and liver, 

kidney, and lung were collected. Tissues were prepared for DNA isolation with samples from the 

same organs from every two mice combined. DNA adducts were measured using accelerated mass 

spectrometry. The results of this study showed a dose-response increase in DNA adducts in all 

three organs measured, although the methodology used to detect DNA adducts is considered 

sensitive but could be nonspecific. The authors stated that tert-butanol was found, for the first time, 

to form DNA adducts in mouse liver, lung, and kidney. Because this is a single and first-time study, 

further validation of this study will provide certainty in understanding the mechanism of tertbutanol-induced DNA adducts. 

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Table B-7. Summary of genotoxicity (both in vitro and in vivo) studies of tertbutanol 

Test system Dose/Conc. Resultsa Comments Reference 

Bacterial Systems 

-S9 +S9 

Reverse Mutation Assay 

Salmonella typhimurium 

(TA98, TA100, TA1535, 

TA1537, TA1538) 

100, 333, 1,000, 

3,333, 10,000 

μg/plate 

- - Preincubation procedure was 

followed. This study was part of 

the NTP 1995 testing results. 

Zeiger et al. 

(1987);NTP 

(1995) 

Reverse Mutation Assay 

Salmonella typhimurium 

(TA102) 

1,000–4,000 

μg/plate 

ND + Only tested with S9 activation Williams-Hill et 

al. (1999) 

Reverse Mutation Assay 

Salmonella typhimurium 

(TA98, TA100, TA102, 

TA1535, TA1537) 

5, 15, 50, 100, 150, 

200, 500, 1,000, 

1,500, 2,500, 5,000 

μg/plate 

- - Experiments conducted in two 

different laboratories, two 

vehicles – distilled water and 

DMSO were used, different 

concentrations were used in 

experiments from different 

laboratories 

Mcgregor et al. 

(2005) 

Reverse mutation 

Neurospora crassa, ad-3A 

locus (allele 38701) 

1.75mol/L - - Eighty four percent cell death 

was observed; note it is a 1949 

study 

Dickey et al. 

(1949) 

Mitochondrial mutation 

Saccharomyces cerevisiae 

(K5-5A, MMY1, D517-4B, 

and DS8) 

4.0% (vol/vol) +b ND Mitochondrial mutations, 

membrane solvent 

Jimenez et al. 

(1988) 

In vitro Systems 

Gene Mutation Assay, 

Mouse lymphoma cells 

L5178Y TK+/– 

625, 1,000, 1,250, 

2,000, 3,000, 

4,000, 5,000 μg/mL 

- - Cultures were exposed for 4 h, 

then cultured for 2 days before 

plating in soft agar with or 

without trifluorothymidine, 

3 μg/mL; this study was part of 

the NTP 1995 testing results 

McGregor et al. 

(1988);NTP 

(1995) 

Sister-chromatid exchange, 

Chinese Hamster Ovary cells 

160, 500, 1,600, 

2,000, 3,000, 

4,000, 5,000 μg/mL 

- -

This study was part of the NTP 

1995 testing results 

Galloway, 

1987; NTP 

(1995) 

Chromosomal Aberrations, 

Chinese Hamster Ovary cells 

160, 500, 1,600, 

2,000, 3,000, 

4,000, 5,000 μg/mL 

- - This study was part of the NTP 

1995 testing results 

Galloway, 

1987 NTP 

(1995) 

DNA damage (comet assay), 

Rat fibroblasts 

0.44 mmol/L (IC50 ) +c ND Exposure duration – 30 min, 

4 h, 12 h; this study provides 

other information on effect of 

cell cycle control genes and 

mechanism of action for TBA 

Sgambato et al. 

(2009) 

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Test system Dose/Conc. Resultsa Comments Reference 

DNA damage, (comet 

assay), HL-60 leukemia cells 

1, 5, 10, 30 mmol/L + ND Exposure duration – 1h Tang et al. 

(1997) 

In vivo Animal Studies 

Micronucleus formation, 

B6C3F1 mouse peripheral 

blood cells 

3,000, 5,000, 

10,000, 20,000, 

40,000 ppm 

- 13-week, subchronic, drinking 

water study 

NTP (1995) 

DNA adducts, male 

Kunming mouse liver, 

kidney and lung cells 

0.1–1,000 μg/kg 

body weight + 

Gavage, 6-h exposure, DNA 

adduct determined by 

accelerator mass spectrometry 

Yuan et al. 

(2007) 

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+ = positive; − = negative; ND = not determined.
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Effect is predicted to be due to mitochondrial membrane composition.
 c

DNA damage was completely reversed with increased exposure time.
 

Summary 

tert-Butanol has been tested for its genotoxic potential using a variety of genotoxicity 

assays. Bacterial assays that detect reverse mutations have been thought to predict carcinogenicity 

with accuracy up to 80%. tert-Butanol did not induce mutations in most bacterial strains; however, 

when tested in TA102, a strain that is sensitive to damage at A-T sites inducible by oxidants, an 

increase in mutants was observed at low concentrations, although conflicting results were reported 

in another study. Furthermore, the solvent (e.g., distilled water or DMSO) used in the genotoxicity 

assay could influence results. In one experiment where tert-butanol was dissolved in distilled 

water, a significant, dose-related increase in the number of mutants was observed, with the 

maximum value reaching almost twice the control value. DMSO is known to be a radical scavenger, 

and its presence in high concentrations might mask a mutagenic response modulated by oxidative 

damage. Other species such as Neurospora crassa did not produce reverse mutations due to 

exposure to tert-butanol. 

tert-Butanol was tested in several human and animal in vitro mammalian systems for 

genotoxicity (gene mutation, sister chromatid exchanges, chromosomal aberrations, and DNA 

damage). No increase in gene mutations was observed in mouse lymphoma cells (L5178Y TK+/–). 

These specific locus mutations in mammalian cells are used to demonstrate and quantify genetic 

damage, thereby confirming or extending the data obtained in the more widely used bacterial cell 

tests. Sister chromatid exchanges or chromosomal aberrations were not observed in CHO cells in 

response to tert-butanol treatment. DNA damage was detected using comet assay, however, in both 

rat fibroblasts and HL-60 leukemia cells, with either an increase in DNA fragmentation at the 

beginning of the exposure or dose-dependent increase in DNA damage observed. An initial increase 

in DNA damage was observed at 30 minutes that declined drastically following 4 hours of exposure 

and disappeared almost entirely after 12 hours of exposure to tert-butanol. This reduction in the 

extent of DNA fragmentation after an initial increase is likely the result of an efficient DNA repair 

mechanism activated by cells following DNA damage induced by tert-butanol. A dose-dependent 

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increase in DNA damage was observed in human cells tested; however, because the exposure 

occurred for only 1 hour in this study, whether DNA-repair mechanisms would occur after a longer 

period of observation cannot be discerned. 

Limited in vivo animal studies have been conducted on DNA adduct and micronucleus 

formation. A dose-response increase in DNA adducts was observed in mouse liver, kidney, and lung 

cells. The authors used accelerated mass spectrometry to detect DNA adducts, but the identity of 

these adducts was not determined. The method uses 14C-labeled chemical for dosing, isolated DNA 

is oxidized to carbon dioxide and reduced to filamentous graphite, and the ratios of 14C/12C are 

measured. The ratio then is converted to DNA adducts based on nucleotide content of the DNA. 

Confirmation of these data will further the understanding of the mechanism of tert-butanol-induced 

DNA adducts. No increase in micronucleus formation was observed in mouse peripheral blood cells 

in a 13-week drinking water study conducted by the National Toxicology Program. 

Overall, a limited database is available for understanding the role of tert-butanol-induced 

genotoxicity for mode of action and carcinogenicity. The database is limited in terms of either the 

array of genotoxicity tests conducted or the number of studies within the same type of test. In 

addition, the results are either conflicting or inconsistent. The test strains, solvents, or control for 

volatility used in certain studies are variable and could influence results. Furthermore, in some 

studies, the specificity of the methodology used has been challenged. Given the inconsistencies and 

limitations of the database in terms of the methodology used, number of studies in the overall 

database, coverage of studies across the genotoxicity battery, and the quality of the studies, the 

weight of evidence analysis is inconclusive. The available data do not inform a definitive conclusion 

on the genotoxicty of tert-butanol and thus the potential genotoxic effects of tert-butanol cannot be 

discounted. 

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APPENDIX C. DOSE-RESPONSE MODELING FOR 

THE DERIVATION OF REFERENCE VALUES FOR 

EFFECTS OTHER THAN CANCER AND THE 

DERIVATION OF CANCER RISK ESTIMATES 

This appendix provides technical detail on dose-response evaluation and determination of 

points of departure (PODs) for relevant endpoints. The endpoints were modeled using EPA’s 

Benchmark Dose Software (BMDS), version 2.1.2. The preambles for the cancer and noncancer 

parts below describe the common practices used in evaluating the model fit and selecting the 

appropriate model for determining the POD as outlined in the Benchmark Dose Technical Guidance 

Document (U.S. EPA, 2000). In some cases, using alternative methods based on statistical judgment 

might be appropriate; exceptions are noted as necessary in the summary of the modeling results. 

Noncancer Endpoints 

C.1.1.1. Data Sets 

Data sets selected for dose-response modeling are provided in Table C-1. In all cases, 

administered exposure was used in modeling the response data. 

C.1.1.2. Model Fit 

All models were fit to the data using the maximum likelihood method. The following 

procedures were used, depending on whether data were dichotomous or continuous: 

• For dichotomous models, the following parameter restrictions were applied: for log-logistic 

model, restrict slope ≥1; for gamma and Weibull models, restrict power ≥1; for multistage

models, restrict beta values ≥0. Each model was tested for goodness-of-fit using a chisquare goodness-of-fit test (χ2 p-value < 0.10 indicates lack of fit). Other factors also were 

used to assess model fit, such as scaled residuals, visual fit, and adequacy of fit in the lowdose region and near the benchmark response (BMR). 

• For continuous models, the following parameter restrictions were applied: for polynomial 

models, restrict beta values ≥0; for Hill, power, and exponential models, restrict power ≥1.

Model fit was assessed by a series of tests. For each model, first the homogeneity of the 

variances was tested using a likelihood ratio test (BMDS Test 2). If Test 2 was not rejected 

(χ2 p-value ≥ 0.10), the model was fit to the data assuming constant variance. If Test 2 was 

rejected (χ2 p-value < 0.10), the variance was modeled as a power function of the mean, and 

the variance model was tested for adequacy of fit using a likelihood ratio test (BMDS 

Test 3). For fitting models using either constant variance or modeled variance, models for 

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the mean response were tested for adequacy of fit using a likelihood ratio test (BMDS Test 

4, with χ2 p-value < 0.10 indicating inadequate fit). Other factors also were used to assess 

the model fit, such as scaled residuals, visual fit, and adequacy of fit in the low-dose region 

and near the BMR. 

C.1.1.3. Model Selection 

For each endpoint, the BMDL estimate (95% lower confidence limit on the BMD, as 

estimated by the profile likelihood method) and the Akaike’s information criterion (AIC) value were 

used to select a best-fit model from among the models exhibiting adequate fit. If the BMDL 

estimates were “sufficiently close,” that is, differed by no more than three-fold, the model selected 

was the one that yielded the lowest AIC value. If the BMDL estimates were not sufficiently close, the 

lowest BMDL was selected as the POD. 

Table C-1. Noncancer endpoints selected for dose-response modeling for 

tert-butanol 

Endpoint/Study 

Species/ 

Sex Doses and effect data 

Kidney transitional 

epithelial hyperplasia 

NTP (1995) 

Rat (F344)/Male 

Dose (mg/kg-d) 0 90 200 420 

Incidence/Total 25/50 32/50 36/50 40/50 

Kidney transitional 

epithelial hyperplasia 

NTP (1995) 

Rat 

(F344)/Female 

Dose (mg/kg-d) 0 180 330 650 

Incidence/Total 0/50 0/50 3/50 17/50 

Increased absolute 

kidney weight 

NTP (1995) 

Rat (F344)/Male 

Dose (mg/kg-d) 0 90 200 420 

Mean ± SD (n) 1.78 ± 0.18 

(10) 

1.85 ± 0.17 

(10) 

1.99 ± 0.18 

(10) 

1.9 ± 0.23 

(10) 

Increased absolute 

kidney weight 

NTP (1995) 

Rat 

(F344)/Female 

Dose (mg/kg-d) 0 180 330 650 

Mean ± SD (n) 1.07 ± 0.09 

(10) 

1.16 ± 0.10 

(10) 

1.27 ± 0.08 

(10) 

1.31 ± 0.09 

(10) 

Kidney inflammation 

NTP (1995) 

Rat 

(F344)/Female 

Dose (mg/kg-d) 0 180 330 650 

Incidence/Total 2/50 3/50 13/50 17/50 

Increased absolute 

kidney weight 

NTP (1997) 

Rat (F344)/Male Concentration 

(mg/m3) 0 406 825 1,643 3,274 6,369 

Mean ± SD (n) 

1.21 ± 

0.082 

(10) 

1.21 ± 

0.096 

(9) 

1.18 ± 

0.079 

(10) 

1.25 ± 

0.111 

(10) 

1.34 ± 

0.054 

(10) 

1.32 ± 

0.089 

(10) 

Increased absolute 

kidney weight 

Rat 

(F344)/Female 

Concentration 

(mg/m3) 0 406 825 1,643 3,274 6,369 

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

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Supplemental Information―tert-Butyl Alcohol 

Endpoint/Study 

Species/ 

Sex Doses and effect data 

NTP (1997) 0.817 ± 0.782 ± 0.821 ± 0.853 ± 0.831 ± 0.849 ± 

Mean ± SD (n) 0.136 0.063 0.061 0.045 0.054 0.038 

(10) (10) (10) (10) (10) (10) 

C.1.1.4. Modeling Results 

Below are tables summarizing the modeling results for the noncancer endpoints modeled. 

Table C-2. Summary of BMD modeling results for kidney transitional epithelial 

hyperplasia in male F344 rats exposed to tert-butanol in drinking water for 2 

years (NTP, 1995); BMR = 10% extra risk 

Modela 

Goodness of fit 

BMD10 

(mg/kg-d) 

BMDL10 

p-value AIC (mg/kg-d) Basis for model selection 

Log-logistic 0.976 248.0 30 16 Log-logistic model selected as bestfitting model based on lowest AIC 

with all BMDL values sufficiently 

close (BMDLs differed by slightly 

more than 3-fold). 

Gamma 0.784 248.5 46 29 

Logistic 0.661 248.8 58 41 

Log-probit 0.539 249.2 84 53 

Multistage, 3° 0.784 248.5 46 29 

Probit 0.633 248.9 60 43 

Weibull 0.784 248.5 46 29 

Dichotomous-Hill 0.968 250.0 25 15 

6 

7 

a Scaled residuals for selected model for doses 0, 90, 200, and 420 mg/kg-d were –0.076, 0.147, 0.046, and –0.137, 

respectively. 

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

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Fraction Affected 

 

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Log-Logistic Model with 0.95 Confidence Level 

BMDL BMD 

Log-Logistic 

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Supplemental Information―tert-Butyl Alcohol 

Figure C-1. Plot of incidence by dose, with fitted curve for LogLogistic model 

for kidney transitional epithelial hyperplasia in male F344 rats exposed to
 

tert-butanol in drinking water for 2 years (NTP, 1995); BMR = 10% extra risk; 

dose shown in mg/kg-d
 

==================================================================== 

Logistic Model. (Version: 2.13; Date: 10/28/2009)

Input Data File: M:\NCEA t-Butanol\BMD modeling\BMDS Output\17 NTP 1995b_Kidney 

transitional epithelial hyperplasia, male rats_LogLogistic_10.(d)

Gnuplot Plotting File: M:\NCEA t-Butanol\BMD modeling\BMDS Output\17 NTP 

1995b_Kidney transitional epithelial hyperplasia, male rats_LogLogistic_10.plt

Fri May 13 17:16:25 2011

==================================================================== 

[notes]

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
 

The form of the probability function is:

P[response] = background+(1-background)/[1+EXP(-intercept-slope*Log(dose))]
 

Dependent variable = Incidence

Independent variable = Dose

Slope parameter is restricted as slope >= 1

Total number of observations = 4

Total number of records with missing values = 0

Maximum number of iterations = 250

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008
 

User has chosen the log transformed model

Default Initial Parameter Values

background = 0.5

intercept = -5.54788 

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

C-4 DRAFT—DO NOT CITE OR QUOTE
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Supplemental Information―tert-Butyl Alcohol 

slope = 1 

Asymptotic Correlation Matrix of Parameter Estimates 

( *** The model parameter(s) -slope

have been estimated at a boundary point, or have been specified by the user,

and do not appear in the correlation matrix ) 

background intercept 

background 1 -0.71 

intercept -0.71 1 

Parameter Estimates 

95.0% Wald Confidence Interval 

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

background 0.505366 * * * 

intercept -5.58826 * * * 

slope 1 * * * 

* - Indicates that this value is not calculated. 

 Analysis of Deviance Table 

 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

 Full model -121.996 4 

 Fitted model -122.02 2 0.048148 2 0.9762 

Reduced model -127.533 1 11.0732 3 0.01134 

 AIC: 248.04 

Goodness of Fit 

Scaled
 

Dose Est._Prob. Expected Observed Size Residual
 

0.0000 0.5054 25.268 25.000 50 -0.076 

90.0000 0.6300 31.498 32.000 50 0.147 

200.0000 0.7171 35.854 36.000 50 0.046 

420.0000 0.8076 40.382 40.000 50 -0.137 

Chi^2 = 0.05 d.f. = 2 P-value = 0.9762 

Benchmark Dose Computation 

Specified effect = 0.1 

Risk Type = Extra risk 

Confidence level = 0.95 

BMD = 29.6967 

BMDL = 15.6252 

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

C-5 DRAFT—DO NOT CITE OR QUOTE 
 

 

 

 

 

 

 

 

==================================================================== 

Supplemental Information―tert-Butyl Alcohol 

1 

2 

3 

Table C-3. Summary of BMD modeling results for kidney transitional epithelial 

hyperplasia in female F344 rats exposed to tert-butanol in drinking water for 

2 years (NTP, 1995); BMR = 10% extra risk 

Modela 

Goodness of fit 

BMD10 

(mg/kg-d) 

BMDL10 

p-value AIC (mg/kg-d) Basis for model selection 

Gamma 0.83 91.41 409 334 Multistage 3rd-order model 

selected as best-fitting model 

based on lowest AIC with all BMDL 

values sufficiently close (BMDLs 

differed by less than 3-fold). 

Logistic 0.50 92.81 461 393 

LogLogistic 0.79 91.57 414 333 

LogProbit 0.89 91.19 400 327 

Multistage 3° 0.92 89.73 412 339 

Probit 0.62 92.20 439 372 

Weibull 0.76 91.67 421 337 

Dichotomous-Hill N/A b 117.89 Error c Error c 

a

Scaled residuals for selected model for doses 0, 180, 330, and 650 mg/m3 were 0.0, –0.664, 0.230, and 0.016, 

respectively. 

b

No available degrees of freedom to estimate a p-value. c

BMD and BMDL computation failed for the Dichotomous-Hill model. 

5 

6 

7 

8 

9

10 

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

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17:18 05/13 2011 

Multistage Model with 0.95 Confidence Level 

BMDL BMD

Multistage 

0 100 200 300 400 500 600 

dose 

Multistage Model. (Version: 3.2; Date: 05/26/2010) 

Figure C-2. Plot of incidence by dose, with fitted curve for Multistage 3° model 

for kidney transitional epithelial hyperplasia in female F344 rats exposed to 

tert-butanol in drinking water for 2 years (NTP, 1995); BMR = 10% extra risk; 

dose shown in mg/kg-d 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Supplemental Information―tert-Butyl Alcohol 

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Input Data File: M:\NCEA tert-butanol\BMD modeling\BMDS Output\20 NTP 

1995b_Kidney transitional epithelial hyperplasia, female rats_Multi3_10.(d)

Gnuplot Plotting File: M:\NCEA tert-butanol\BMD modeling\BMDS Output\20 NTP 

1995b_Kidney transitional epithelial hyperplasia, female rats_Multi3_10.plt

Mon May 09 18:31:33 2011

==================================================================== 

[notes]

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
 

The form of the probability function is:

P[response] = background + (1-background)*[1-EXP(

-beta1*dose^1-beta2*dose^2-beta3*dose^3)] 

The parameter betas are restricted to be positive

Dependent variable = Incidence

Independent variable = Dose

Total number of observations = 4

Total number of records with missing values = 0

Total number of parameters in model = 4

Total number of specified parameters = 0

Degree of polynomial = 3

Maximum number of iterations = 250 

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

Default Initial Parameter Values

Background = 0

Beta(1) = 0

Beta(2) = 1.51408e-007

Beta(3) = 1.29813e-009 

Asymptotic Correlation Matrix of Parameter Estimates

( *** The model parameter(s) -Background -Beta(1) -Beta(2)

have been estimated at a boundary point, or have been specified by the user,

and do not appear in the correlation matrix )

Beta(3)

Beta(3) 1

Parameter Estimates

95.0% Wald Confidence Interval

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

Background 0 * * * 

Beta(1) 0 * * * 

Beta(2) 0 * * * 

Beta(3) 1.50711e-009 * * * 

* - Indicates that this value is not calculated. 

Analysis of Deviance Table

Model Log(likelihood) # Param's Deviance Test d.f. P-value 

Full model -43.4002 4 

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

C-7 DRAFT—DO NOT CITE OR QUOTE 
 

 

 

 

 

 

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Supplemental Information―tert-Butyl Alcohol 

Fitted model -43.8652 1 0.9301 3 0.8182
 

Reduced model -65.0166 1 43.2329 3 <.0001
 

AIC: 89.7304 

Goodness of Fit 

Scaled
 

Dose Est._Prob. Expected Observed Size Residual
 

 0.0000 0.0000 0.000 0.000 50 0.000 

180.0000 0.0088 0.438 0.000 50 -0.664 

330.0000 0.0527 2.636 3.000 50 0.230 

650.0000 0.3389 16.946 17.000 50 0.016 

Chi^2 = 0.49 d.f. = 3 P-value = 0.9200 

Benchmark Dose Computation 

Specified effect = 0.1 

Risk Type = Extra risk 

Confidence level = 0.95 

BMD = 411.95 

BMDL = 338.618 

BMDU = 469.73 

Taken together, (338.618, 469.73 ) is a 90 % two-sided confidence 

interval for the BMD 

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

C-8 DRAFT—DO NOT CITE OR QUOTE 
 

 

 

 

 

 

Supplemental Information―tert-Butyl Alcohol 

1 

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3 

Table C-4. Summary of BMD modeling results for absolute kidney weight in 

male F344 rats exposed to tert-butanol in drinking water for 15 months (NTP, 

1995); BMR = 10% rel. dev. from control mean 

Modela 

Goodness of fit BMD10RD 

(mg/kg-d) 

BMDL10RD 

p-value AIC (mg/kg-d) Basis for model selection 

Exponential (M2)b 0.123 −86.757 661 307 Of the models that provided an 

adequate fit and a valid BMDL 

estimate, the linear model was 

selected based on lowest AIC. 

Exponential (M3)c 0.123 −86.757 661 307 

Exponential (M4) 0.167 −87.041 errord 0 

Exponential (M5) N/Ae −85.880 errord 0 

Hill 0.301 −87.880 errord errord 

Powerf 

Polynomial 3°g 

Polynomial 2°h 

Linear 

0.126 −86.804 657 296 

a Constant variance case presented (BMDS Test 2 p-value = 0.777), selected model in bold; scaled residuals for 

selected model for doses 0, 90, 200, and 420 mg/kg-d were −0.78, −0.11, 1.65, −0.76, respectively. b The Exponential (M2) model can appear equivalent to the Exponential (M3) model, however differences exist in 

digits not displayed in the table. 

c The Exponential (M3) model can appear equivalent to the Exponential (M2) model, however differences exist in 

digits not displayed in the table. 

d BMD or BMDL computation failed for this model. 

e No available degrees of freedom to calculate a goodness-of-fit value. f For the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear 

model. 

g For the Polynomial 3° model, the b3 coefficient estimate was 0 (boundary of parameters space). The models in 

this row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates 

were 0 (boundary of parameters space). The models in this row reduced to the Linear model. 

h For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in 

this row reduced to the Linear model. 

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

C-9 DRAFT—DO NOT CITE OR QUOTE
 
 

Mean Response 

2.1

 2

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 1.8

 1.7

Linear Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL 

BMDL BMD

Linear 

0 100 200 300 400 500 600 

dose 

11:46 05/26 2015 

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

C-10 DRAFT—DO NOT CITE OR QUOTE 

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Supplemental Information―tert-Butyl Alcohol 

Figure C-3. Plot of mean response by dose, with fitted curve for Linear model 

with constant variance for absolute kidney weight in male F344 rats exposed 

to tert-butanol in drinking water for 15 months (NTP, 1995); BMR = 10% rel. 

dev. from control mean; dose shown in mg/kg-d 

Polynomial Model. (Version: 2.20; Date: 10/22/2014)
 

The form of the response function is: Y[dose] = beta_0 + beta_1*dose.
 

A constant variance model is fit.
 

Benchmark Dose Computation. 

BMR = 10% Relative deviation 

BMD = 656.583 

BMDL at the 95% confidence level = 295.826 

Parameter Estimates 

Variable Estimate 

Default Initial 

Parameter Values 

alpha 0.0361494 0.0362125 

rho n/a 0 

beta_0 1.83173 1.83173 

beta_1 0.000278979 0.000278979 

14 
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Supplemental Information―tert-Butyl Alcohol 

Table of Data and Estimated Values of Interest 

Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Resid 

0 10 1.78 1.83 0.18 0.19 −0.777 

90 10 1.85 1.86 0.17 0.19 −0.114 

200 10 1.99 1.89 0.18 0.19 1.65 

420 10 1.9 1.95 0.23 0.19 −0.763 

Likelihoods of Interest 

Model Log(likelihood) # Param's AIC 

A1 48.474229 5 −86.948457 

A2 49.025188 8 −82.050377 

A3 48.474229 5 −86.948457 

fitted 46.401914 3 −86.803828 

R 45.368971 2 −86.737942 

Tests of Interest 

Test 

−2*log(Likelihood 

Ratio) Test df p-value 

Test 1 7.31243 6 0.2929 

Test 2 1.10192 3 0.7766 

Test 3 1.10192 3 0.7766 

Test 4 4.14463 2 0.1259 

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

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Supplemental Information―tert-Butyl Alcohol 

Table C-5. Summary of BMD modeling results for absolute kidney weight in 

female F344 rats exposed to tert-butanol in drinking water for 15 months 

(NTP, 1995); BMR = 10% rel. dev. from control mean 

Modela 

Goodness of fit BMD10RD 

(mg/kg-d) 

BMDL10RD 

p-value AIC (mg/kg-d) Basis for model selection 

Exponential (M2) 

Exponential (M3)b 

0.0594 −144.00 318 249 The Exponential (M4) model was 

selected as the only model with 

adequate fit. Exponential (M4) 0.176 −145.81 164 91.4 

Exponential (M5) N/Ac −145.65 207 117 

Hill N/Ac −145.65 202 119 

Powerd 

Polynomial 3°e 

Polynomial 2°f 

Linear 

0.0842 −144.70 294 224 

a Constant variance case presented (BMDS Test 2 p-value = 0.852), selected model in bold; scaled residuals for 

selected model for doses 0, 180, 330, and 650 mg/kg-d were 0.21, −0.9, 0.94, −0.25, respectively. b For the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the 

Exponential (M2) model. 

c No available degrees of freedom to calculate a goodness-of-fit value. d For the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear 

model. 

e For the Polynomial 3° model, the b3 coefficient estimate was 0 (boundary of parameters space). The models in 

this row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates 

were 0 (boundary of parameters space). The models in this row reduced to the Linear model. 

f For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in 

this row reduced to the Linear model. 

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

C-12 DRAFT—DO NOT CITE OR QUOTE 
This document is a draft for review purposes only and does not constitute Agency policy.

C-13 DRAFT—DO NOT CITE OR QUOTE 

 

Exponential 4 Model, with BMR of 0.1 Rel. Dev. for the BMD and 0.95 Lower Confidence Limit for the BMDL 

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dose 

11:32 05/26 2015 Mean Response 

BMDL BMD

Exponential 4 

0 100 200 300 400 500 600 

 

Supplemental Information―tert-Butyl Alcohol 

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Figure C-4. Plot of mean response by dose, with fitted curve for Exponential
 

(M4) model with constant variance for absolute kidney weight in female F344
 

rats exposed to tert-butanol in drinking water for 15 months (NTP, 1995); 

BMR = 10% rel. dev. from control mean; dose shown in mg/kg-d
 

Exponential Model. (Version: 1.10; Date: 01/12/2015)
 

The form of the response function is: Y[dose] = a * [c−(c−1) * exp(−b * dose)].
 

A constant variance model is fit.
 

Benchmark Dose Computation. 

BMR = 10% Relative deviation 

BMD = 163.803 

BMDL at the 95% confidence level = 91.3614 

Parameter Estimates 

Variable Estimate 

Default Initial 

Parameter Values 

lnalpha −4.84526 −4.89115 

rho n/a 0 

a 1.06808 1.0203 

b 0.00258011 0.00282085 

c 1.29013 1.35122 

d n/a 1 

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Supplemental Information―tert-Butyl Alcohol 

Table of Data and Estimated Values of Interest 

Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Resid 

0 10 1.07 1.07 0.09 0.09 0.2112 

180 10 1.16 1.18 0.1 0.09 −0.8984 

330 10 1.27 1.25 0.08 0.09 0.9379 

650 10 1.31 1.32 0.09 0.09 −0.2507 

Likelihoods of Interest 

Model Log(likelihood) # Param's AIC 

A1 77.82307 5 −145.6461 

A2 78.21688 8 −140.4338 

A3 77.82307 5 −145.6461 

R 62.21809 2 −120.4362 

4 76.90527 4 −145.8105 

Tests of Interest 

Test 

−2*log(Likelihood 

Ratio) Test df p-value 

Test 1 32 6 <0.0001 

Test 2 0.7876 3 0.8524 

Test 3 0.7876 3 0.8524 

Test 6a 1.836 1 0.1755 

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

C-14 DRAFT—DO NOT CITE OR QUOTE 
 

 

 

 

 

 

 

 

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Supplemental Information―tert-Butyl Alcohol 

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Table C-6. Summary of BMD modeling results for kidney inflammation in 

female rats exposed to tert-butanol in drinking water for 2 years (NTP, 1995); 

BMR = 10% extra risk 

Modela 

Goodness of fit 

BMD10% 

(mg/kg-d) 

BMDL10% 

p-value AIC (mg/kg-d) Basis for model selection 

Gamma 0.084 169.9 231 135 LogProbit was selected on the 

basis of the lowest AIC with all 

BMDL values for fitting models 

being sufficiently close (BMDLs 

differed by less than 3-fold). 

Logistic 0.082 169.7 305 252 

LogLogistic 0.092 169.8 228 124 

LogProbit 0.243 167.6 254 200 

Multistage 3° 0.072 170.3 216 132 

Probit 0.108 169.2 285 235 

Weibull 0.081 170.0 226 134 

Dichotomous-Hill N/Ab 169.5 229 186 

a

Selected model in bold; scaled residuals for selected model for doses 0, 180, 330, and 650 mg/kg-d were −0.067, 

−0.700, 1.347, and −0.724, respectively. b

No available degrees of freedom to estimate a p-value. 

LogProbit Model with 0.95 Confidence Level 

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

C-15 DRAFT—DO NOT CITE OR QUOTE
 

17:17 05/13 2011 

dose 

 

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LogProbit 

0 100 200 300 400 500 600 

Figure C-5. Plot of incidence by dose, with fitted curve for Logprobit model for 

kidney inflammation in female rats exposed to tert-butanol in drinking water 

for 2 years (NTP, 1995); BMR = 10% extra risk; dose shown in mg/kg-d 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Supplemental Information―tert-Butyl Alcohol 

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==================================================================== 

Probit Model. (Version: 3.2; Date: 10/28/2009) 

Input Data File: M:/NCEA tert-butanol/BMD modeling/BMDS Output/19 NTP 

1995b_Kidney inflammation, female rats_LogProbit_10.(d)

Gnuplot Plotting File: M:/NCEA tert-butanol/BMD modeling/BMDS Output/19 NTP 

1995b_Kidney inflammation, female rats_LogProbit_10.plt

Fri May 13 17:17:59 2011

==================================================================== 

[notes]

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

The form of the probability function is:

P[response] = Background

+ (1-Background) * CumNorm(Intercept+Slope*Log(Dose)), 

where CumNorm(.) is the cumulative normal distribution function 

Dependent variable = Incidence

Independent variable = Dose

Slope parameter is restricted as slope >= 1

Total number of observations = 4

Total number of records with missing values = 0

Maximum number of iterations = 250

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

User has chosen the log transformed model

Default Initial (and Specified) Parameter Values

background = 0.04

intercept = -8.01425 

slope = 1.18928

Asymptotic Correlation Matrix of Parameter Estimates

( *** The model parameter(s) -slope

have been estimated at a boundary point, or have been specified by the user,

and do not appear in the correlation matrix )

background intercept 

background 1 -0.51 

intercept -0.51 1

Parameter Estimates

95.0% Wald Confidence Interval

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

background 0.0381743 0.0246892 -0.0102155 0.0865642

intercept -6.82025 0.161407 -7.1366 -6.5039 

slope 1 NA

NA - Indicates that this parameter has hit a bound

implied by some inequality constraint and thus

has no standard error.

Analysis of Deviance Table 

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 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

 Full model -80.4502 4 

 Fitted model -81.8218 2 2.7432 2 0.2537 

Reduced model -92.7453 1 24.5902 3 <.0001 

 AIC: 167.644 

 

 

 

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Supplemental Information―tert-Butyl Alcohol 

Goodness of Fit 

Scaled
 

Dose Est._Prob. Expected Observed Size Residual
 

0.0000 0.0382 1.909 2.000 50 0.067 

180.0000 0.0880 4.402 3.000 50 -0.700 

330.0000 0.1859 9.295 13.000 50 1.347 

650.0000 0.3899 19.495 17.000 50 -0.724 

Chi^2 = 2.83 d.f. = 2 P-value = 0.2427 

Benchmark Dose Computation 

Specified effect = 0.1 

Risk Type = Extra risk 

Confidence level = 0.95 

BMD = 254.347 

BMDL = 199.789 

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

C-17 DRAFT—DO NOT CITE OR QUOTE 
 

Supplemental Information―tert-Butyl Alcohol 

1 

2 

3 

Table C-7. Summary of BMD modeling results for absolute kidney weight in 

male F344 rats exposed to tert-butanol via inhalation for 6 hr/d, 5d/wk for 13 

weeks (NTP, 1997); BMR = 10% relative deviation from the mean 

Modela 

Goodness of fit BMC10RD 

(mg/m3) 

BMCL10RD 

(mg/m3 p-value AIC ) Basis for model selection 

Exponential (M2) <0.0001 −205.06 errorb errorb Although the Hill model was the 

only adequately fitting model 

(p>0.1), the resulting fit was 

essentially a step-function that 

does not support interpolation 

between the well-fit 

observations. 

Exponential (M3) <0.0001 −203.06 9.2E+07 7094 

Exponential (M4) <0.0001 −203.06 errorb 0 

Exponential (M5) <0.0001 −201.06 errorb 0 

Hill 0.763 −226.82 1931 1705 

Powerc 

Linear 

0.0607 −220.97 5364 3800 

Polynomial 5°d 

Polynomial 4°e 

Polynomial 3° 

1.44E−04 −207.06 −9999 errorf 

Polynomial 2° 1.44E−04 −207.06 −9999 18436 

a Constant variance case presented (BMDS Test 2 p-value = 0.390), selected model in bold; scaled residuals for 

selected model for doses 0, 406, 825, 1,643, 3,274, and 6,369 mg/m3 were 0.395, 0.374, −0.75, −1.96e−006, 

0.381, and −0.381, respectively. b BMC or BMCL computation failed for this model. c For the Power model, the power parameter estimate was 1. The models in this row reduced to the Linear 

model. 

d For the Polynomial 5° model, the b5 and b4 coefficient estimates were 0 (boundary of parameters space). The 

models in this row reduced to the Polynomial 3° model. 

e For the Polynomial 4° model, the b4 coefficient estimate was 0 (boundary of parameters space). The models in 

this row reduced to the Polynomial 3° model. 

f BMC or BMCL computation failed for this model. 

Hill Model with 0.95 Confidence Level 

4 

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

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1.4

 1.35

 1.3

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 1.1

dose 

10:15 04/30 2014 

Mean Response 

BMDL BMD

Hill 

0 1000 2000 3000 4000 5000 6000 
Figure C-6. Plot of mean response by concentration, with fitted curve for Hill 

model for absolute kidney weight in male F344 rats exposed to tert-butanol 

via inhalation for 6 hr/d, 5d/wk for 13 weeks (NTP, 1997); BMR = 10% 

relative deviation from the mean; concentration shown in mg/m3 

Hill Model. (Version: 2.15; Date: 10/28/2009) 

The form of the response function is: Y[dose] = intercept + v*dose^n/(k^n + dose^n). 

A constant variance model is fit. 

Benchmark Dose Computation. 

BMR = 10% Relative risk 

BMD = 1931.35 

BMDL at the 95% confidence level = 1704.82 

Parameter Estimates 

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Supplemental Information―tert-Butyl Alcohol 

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Variable Estimate 

Default Initial 

Parameter Values 

alpha 0.00687349 0.00750263 

rho n/a 0 

intercept 1.19966 1.21 

v 0.130345 0.13 

n 18 18 

k 1685.82 4451.94 

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Table of Data and Estimated Values of Interest 

Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Resid 

0 10 1.21 1.2 0.0822 0.0829 0.395 

406 9 1.21 1.2 0.096 0.0829 0.374 

825 10 1.18 1.2 0.0791 0.0829 −0.75 

1643 10 1.25 1.25 0.111 0.0829 −0.00000196 

3274 10 1.34 1.33 0.0538 0.0829 0.381 

6369 10 1.32 1.33 0.0885 0.0829 −0.381 

Likelihoods of Interest 

Model Log(likelihood) # Param's AIC 

A1 117.992549 7 −221.985098 

A2 120.600135 12 −217.20027 

A3 117.992549 7 −221.985098 

fitted 117.41244 4 −226.82488 

R 105.528775 2 −207.05755 

Tests of Interest 

Test −2*log(Likelihood 

Ratio) 

Test df p-value 

Test 1 30.1427 10 0.0008118 

Test 2 5.21517 5 0.3902 

Test 3 5.21517 5 0.3902 

Test 4 1.16022 3 0.7626 

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

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Table C-8. Summary of BMD modeling results for absolute kidney weight in 

female F344 rats exposed to tert-butanol via inhalation for 6 hr/d, 5d/wk for 

13 weeks (NTP, 1997); BMR = 10% relative deviation from the mean 

Modela 

Goodness of fit BMC10RD 

(mg/m3) 

BMCL10RD 

(mg/m3 p-value AIC ) Basis for model selection 

Exponential (M2) 

Exponential (M3)b 

0.0378 −261.52 14500 7713 No model adequately fit the data. 

Exponential (M4) 0.533 −267.48 errorc 0 

Exponential (M5) 0.374 −265.71 errorc 0 

Hill 0.227 −265.57 errorc errorc 

Power 0.0392 −261.61 14673 7678 

Polynomial 3°d 

Polynomial 2°e 

Linear 

0.0274 −261.61 14673 7678 

Polynomial 5° 0.0274 −261.61 14673 7569 

Polynomial 4° 0.0274 −261.61 14673 7674 

a Modeled variance case presented (BMDS Test 2 p-value = 1.90E−04, BMDS Test 3 p-value = 0.374), no model 

was selected as a best-fitting model. b For the Exponential (M3) model, the estimate of d was 1 (boundary). The models in this row reduced to the 

Exponential (M2) model. 

c BMC or BMCL computation failed for this model. 

d For the Polynomial 3° model, the b3 coefficient estimate was 0 (boundary of parameters space). The models in 

this row reduced to the Polynomial 2° model. For the Polynomial 3° model, the b3 and b2 coefficient estimates 

were 0 (boundary of parameters space). The models in this row reduced to the Linear model. 

e For the Polynomial 2° model, the b2 coefficient estimate was 0 (boundary of parameters space). The models in 

this row reduced to the Linear model. 

Note: Graphs of the better fitting models are provided for illustration. 

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

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Hill Model 

0.9

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 0.8

 0.75

 0.7

dose 

Hill 

0 1000 2000 3000 4000 5000 6000 

Mean Response 10:32 04/30 2014 

 

Figure C-7. Plot of mean response by concentration, with fitted curve for Hill 

model for absolute kidney weight in female F344 rats exposed to tert-butanol 

via inhalation for 6 hr/d, 5d/wk for 13 weeks (NTP, 1997); BMR = 10% 

relative deviation from the mean; concentration shown in mg/m3

Power Model with 0.95 Confidence Level 

 

0.7

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 0.8

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 0.9

Mean Response 

BMDL BMD

Power 

0 2000 4000 6000 8000 10000 12000 14000 

dose

10:32 04/30 2014 

 

 

 

Supplemental Information―tert-Butyl Alcohol 

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Figure C-8. Plot of mean response by concentration, with fitted curve for 

Power model for absolute kidney weight in female F344 rats exposed to tertbutanol via inhalation for 6 hr/d, 5d/wk for 13 weeks (NTP, 1997); BMR = 

10% relative deviation from the mean; concentration shown in mg/m3 

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

C-22 DRAFT—DO NOT CITE OR QUOTE
 

 

 

 

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

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Cancer Endpoints 

C.1.2.1. Data Sets 

The cancer data sets selected for dose-response modeling are summarized in Table C-9. In 

all cases, administered exposure was used in modeling the response data. Due to the significant 

difference in survival in the high-dose male mice compared with the concurrent control, the Poly-3 

procedure (Bailer and Portier, 1988) for adjusting tumor incidence rates for intercurrent mortality 

was used. The procedure is based on the observation that the cumulative incidence of tumors tends 

to increase with time raised to the second through the fourth powers for a large proportion of 

cases. In the Poly-3 procedure, for a study of T weeks’ duration, an animal that is removed from the 

study after t weeks (t < T) without a specified type of tumor of interest is given a weight of (t/T)3. 

An animal that survives until the terminal sacrifice at T weeks is assigned a weight of (T/T)3 = 1. An 

animal that develops the specific type of tumor of interest obviously lived long enough to develop 

the tumor, and is assigned a weight of 1. The Poly-3 tumor incidence, adjusted for intercurrent 

mortality up to time T, is the number of animals in a dose group with the specified type of tumor 

divided by the sum of the weights (the effective number of animals at risk). The tumor incidences, 

adjusted using this procedure, also are provided in Table C-9. 

C.1.2.2. Model Fit 

The multistage model was fit to the cancer data sets. Model coefficients were restricted to 

be non-negative (beta values ≥ 0), to estimate a monotonically increasing function. Each model was 

fit to the data using the maximum likelihood method, and was tested for goodness-of-fit using a chisquare goodness-of-fit test (χ2 p-value < 0.051 indicates lack of fit). Other factors were used to 

assess model fit, such as scaled residuals, visual fit, and adequacy of fit in the low dose region and 

near the BMR. 

For each endpoint, the BMDL estimate (95% lower confidence limit on the BMD, as 

estimated by the profile likelihood method) and AIC value were used to select a best-fit model from 

among the models exhibiting adequate fit. For the NTP (1995) and Hard et al. (2011) data, models 

were run with all doses included, as well as with the high dose dropped. Dropping the high dose 

resulted in a better fit to the data. Including the high dose caused the model to overestimate the 

control. 

1

A significance level of 0.05 is used for selecting cancer models because the model family (multistage) is

selected a priori (U.S. EPA, 2000). 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

Supplemental Information―tert-Butyl Alcohol 

1 

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Table C-9. Cancer endpoints selected for dose-response modeling for tertbutanol 

Endpoint/Study Species/Sex Doses and Effect Data 

Thyroid 

Thyroid follicular cell 

adenoma 

NTP (1995) 

B6C3F1 

mice/female 

Dose (mg/kg-d) 0 510 1,020 2,110 

Incidence/Total 2/58 3/60 2/59 9/59 

Thyroid follicular cell 

adenoma 

NTP (1995) 

B6C3F1 

mice/male 

Dose (mg/kg-d) 0 540 1,040 2,070 

Incidence/Total 1/60 0/59 4/59 2/60 

Incidence/Poly-3 

adjusted Total 1/50 0/50 4/51 2/35 

Kidneya 

Renal tubule adenoma or 

carcinoma 

NTP (1995) 

Rat (F344) / 

Male 

Dose (mg/kg-d) 0 90 200 420 

Incidence / Total 8 / 50 13 / 50 19 / 50 13 / 50 

Renal tubule adenoma or 

carcinoma 

NTP (1995) 

Rat (F344) / 

Male 

Dose (PBPK, mg/L) 0 4.6945 12.6177 40.7135 

Incidence / Total 8 / 50 13 / 50 19 / 50 13 / 50 

Renal tubule adenoma or 

carcinoma 

NTP (1995) 

Rat (F344) / 

Male 

Dose (PBPK, mg/hr) 0 0.7992 1.7462 3.4712 

Incidence / Total 8 / 50 13 / 50 19 / 50 13 / 50 

Renal tubule adenoma or 

carcinoma; Hard 

reanalysis 

NTP (1995);Hard et al. 

(2011) 

Rat (F344) / 

Male 

Dose (mg/kg-d) 0 90 200 420 

Incidence / Total 4 / 50 13 / 50 18 / 50 12 / 50 

Renal tubule adenoma or 

carcinoma; Hard 

reanalysis 

NTP (1995);Hard et al. 

(2011) 

Rat (F344) / 

Male 

Dose (PBPK, mg/L) 0 4.6945 12.6177 40.7135 

Incidence / Total 4 / 50 13 / 50 18 / 50 12 / 50 

Renal tubule adenoma or 

carcinoma; Hard 

reanalysis 

NTP (1995);Hard et al. 

(2011) 

Rat (F344) / 

Male 

Dose (PBPK, mg/hr) 0 0.7992 1.7462 3.4712 

Incidence / Total 4 / 50 13 / 50 18 / 50 12 / 50 

3 

4 

a Endpoint presented if kidney tumors are acceptable for quantitation 

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

C-24 DRAFT—DO NOT CITE OR QUOTE 
1 

Supplemental Information―tert-Butyl Alcohol 

Table C-10. Summary of the oral slope factor derivations 

BMR BMD POD= 

(mg/kg- BMDL BMDLHEDa Slope factorb 

Tumor Species/Sex Selected Model d) (mg/kg-d) (mg/kg-d) (mg/kg-day)-1 

Thyroid 

Thyroid follicular cell Female 3° Multistage 10% 2002 1437 201 5 × 10-4 

adenoma B6C3F1 

mouse 

Kidneyc 

Renal tubule Male F344 1° Multistage 10% 70 42 10.1 1 × 10-2 

adenoma or rat; dose as (high dose 

carcinoma administered dropped) 

Renal tubule Male F344 1° Multistage 10% 54 36 8.88 1 x 10-2 

adenoma or rat; dose as (high dose 

carcinoma [Hard et administered dropped) 

al. (2011) reanalysis] 

2 

3 

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5 

a

HED PODs were calculated using BW3/4 scaling (U.S. EPA, 2011).
 b

Human equivalent slope factor = 0.1/BMDL10HED
 

c

Alternative endpoint if kidney tumors are acceptable for quantitation.
 

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

C-25 DRAFT—DO NOT CITE OR QUOTE 
 

 

 

 

Supplemental Information―tert-Butyl Alcohol 

1 C.1.2.3. Modeling Results 

Table C-11. Summary of BMD modeling results for thyroid follicular cell 

adenomas in female B6C3F1 mice exposed to tert-butanol in drinking water 

for 2 years (NTP, 1995); BMR = 10% extra risk 

2 

3 

4 

Modela 

Goodness of fit BMD10%c 

(mg/kg-d) 

BMDL10%c 

p-value AIC (mg/kg-d) Basis for model selection b 

Three 0.75 113.665 2002 1437 Multistage 3° was selected on the basis of 

the lowest AIC with all BMDL values for 

fitting models being sufficiently close 

Two 0.36 115.402 2186 1217 (BMDLs differed by less than 3-fold). 

One 0.63 114.115 1987 1378 

a Selected (best-fitting) model shown in boldface type.
 b AIC = Akaike Information Criterion.
 c Confidence level = 0.95.

Multistage Cancer Model with 0.95 Confidence Level 

5 

6 

 

0.3

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 0.2

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Fraction Affected 

BMDL BMD

Multistage Cancer

Linear extrapolation 

0 500 1000 1500 2000 

15:22 05/13 2011 

dose 

Figure C-9. Plot of incidence by dose, with fitted curve for Multistage 3° model 

for thyroid follicular cell adenomas in female B6C3F1 mice exposed to tert

butanol in drinking water for 2 years (NTP, 1995); BMR = 10% extra risk; dose 

shown in mg/kg-d 

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==================================================================== 

==================================================================== 

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

C-26 DRAFT—DO NOT CITE OR QUOTE 

 Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)

 Input Data File: M:\NCEA t-Butanol\BMD modeling\BMDS Output\29 NTP 1995b_Thyroid 

folluclar cell andenoma, female mice_MultiCanc3_10.(d)

 Gnuplot Plotting File: M:\NCEA t-Butanol\BMD modeling\BMDS Output\29 NTP 

1995b_Thyroid folluclar cell andenoma, female mice_MultiCanc3_10.plt

 Fri May 13 15:22:18 2011
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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[notes]

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
 

The form of the probability function is:

P[response] = background + (1-background)*[1-EXP(

-beta1*dose^1-beta2*dose^2-beta3*dose^3)]
 

The parameter betas are restricted to be positive

Dependent variable = Incidence

Independent variable = Dose

Total number of observations = 4

Total number of records with missing values = 0

Total number of parameters in model = 4

Total number of specified parameters = 0

Degree of polynomial = 3

Maximum number of iterations = 250

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

Default Initial Parameter Values

Background = 0.0347373

Beta(1) = 0

Beta(2) = 0

Beta(3) = 1.36917e-011 

Asymptotic Correlation Matrix of Parameter Estimates

( *** The model parameter(s) -Beta(1) -Beta(2) 

have been estimated at a boundary point, or have been specified by the

user,

and do not appear in the correlation matrix )

Background Beta(3)

Background 1 -0.53 

Beta(3) -0.53 1

Parameter Estimates

95.0% Wald Confidence Interval

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf.

Limit

Background 0.0361209 * * * 

Beta(1) 0 * * * 

Beta(2) 0 * * * 

Beta(3) 1.31301e-011 * * * 

* - Indicates that this value is not calculated. 

Analysis of Deviance Table

Model Log(likelihood) # Param's Deviance Test d.f. P-value 

Full model -54.5437 4

Fitted model -54.8326 2 0.577881 2 0.7491

Reduced model -58.5048 1 7.92235 3 0.04764

AIC: 113.665 

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

C-27 DRAFT—DO NOT CITE OR QUOTE 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Supplemental Information―tert-Butyl Alcohol 

Goodness of Fit

Scaled

Dose Est._Prob. Expected Observed Size Residual

-----------------------------------------------------------------------­

0.0000 0.0361 2.095 2.000 58 -0.067 

510.0000 0.0378 2.268 3.000 60 0.496

1020.0000 0.0495 2.918 2.000 59 -0.551 

2110.0000 0.1480 8.730 9.000 59 0.099

Chi^2 = 0.56 d.f. = 2 P-value = 0.7544 

Benchmark Dose Computation

Specified effect = 0.1

Risk Type = Extra risk

Confidence level = 0.95

BMD = 2002.03

BMDL = 1436.69

BMDU = 3802.47

Taken together, (1436.69, 3802.47) is a 90 % two-sided confidence 

interval for the BMD

Multistage Cancer Slope Factor = 6.96043e-005 

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

C-28 DRAFT—DO NOT CITE OR QUOTE 
 

 

 

 

 

 

 

 

 

 

 

Supplemental Information―tert-Butyl Alcohol 

1 

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3 

Table C-12. Summary of BMD modeling results for thyroid follicular cell 

adenomas or carcinomas in male B6C3F1 mice exposed to tert-butanol in 

drinking water for 2 years (NTP, 1995); BMR = 5% extra risk 

Modela 

Goodness of fit BMD5% 

(mg/kg-d) 

BMDL5%c 

p-value AIC (mg/kg-d) Basis for model selection b 

One, Two, 

Three 0.202 61.6 1788 787 Multistage 1° was selected. Only form of 

multistage that resulted; fit adequate. 

a Selected (best-fitting) model shown in boldface type.
 b AIC = Akaike Information Criterion.
 c Confidence level = 0.95.

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Fraction Affected 

0.2

 0.15

 0.1

 0.05

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Multistage Cancer Model, with BMR of 5% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL 

BMDL BMD

Multistage Cancer 

Linear extrapolation 

0 500 1000 1500 2000 

dose 

11:02 06/05 2015 

Figure C-10. Plot of incidence by dose, with fitted curve for Multistage 1° 

model for thyroid follicular cell adenomas or carcinomas in male B6C3F1 mice
 

exposed to tert-butanol in drinking water for 2 years (NTP, 1995); BMR = 5%
 

extra risk; dose shown in mg/kg-d
 

==================================================================== 

Multistage Model. (Version: 3.4; Date: 05/02/2014)

Input Data File: C:/Users/KHOGAN/BMDS/BMDS260/Data/msc_TBA NTP1995 MMthyroid tumors

poly3_Msc1-BMR05.(d) 

Gnuplot Plotting File: C:/Users/KHOGAN/BMDS/BMDS260/Data/msc_TBA NTP1995 MMthyroid

tumors poly3_Msc1-BMR05.plt
 

Fri Jun 05 11:02:14 2015

==================================================================== 

BMDS_Model_Run

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
 

The form of the probability function is:

P[response] = background + (1-background)*[1-EXP(

-beta1*dose^1)]
 

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

C-29 DRAFT—DO NOT CITE OR QUOTE 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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The parameter betas are restricted to be positive 

Dependent variable = Effect

Independent variable = Dose 

Total number of observations = 4 

Total number of records with missing values = 0

Total number of parameters in model = 2

Total number of specified parameters = 0

Degree of polynomial = 1 

Maximum number of iterations = 500 

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

Default Initial Parameter Values 

Background = 0.0164855 

Beta(1) = 2.58163e-005 

Asymptotic Correlation Matrix of Parameter Estimates 

Background Beta(1) 

Background 1 -0.56 

Beta(1) -0.56 1 

Parameter Estimates 

95.0% Wald Confidence Interval 

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

Background 0.0149284 0.0144833 -0.0134584 0.0433151 

Beta(1) 2.86952e-005 1.99013e-005 -1.03105e-005 6.7701e-005 

Goodness of Fit 

Scaled 

Dose Est._Prob. Expected Observed Size Residual 

0.0000 0.0149 0.746 1.000 50.000 0.296 

540.0000 0.0301 1.504 0.000 50.000 -1.245 

1040.0000 0.0439 2.238 4.000 51.000 1.204 

2070.0000 0.0717 2.511 2.000 35.000 -0.335 

Chi^2 = 3.20 d.f. = 2 P-value = 0.2019 

 Analysis of Deviance Table 

 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

 Full model -26.5891 4 

Fitted model -28.808 2 4.43785 2 0.1087 

Reduced model -29.8255 1 6.47273 3 0.09074 

 AIC: 61.616 

Benchmark Dose Computation 

Specified effect = 0.05 

Risk Type = Extra risk 

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

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Supplemental Information―tert-Butyl Alcohol 

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Confidence level = 0.95

BMD = 1787.52

BMDL = 787.153

BMDU did not converge for BMR = 0.050000

BMDU calculation failed

BMDU = Inf 

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

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Supplemental Information―tert-Butyl Alcohol 

1 

2 

Table C-13. Summary of BMD modeling results for thyroid follicular cell 

adenomas or carcinomas in male B6C3F1 mice exposed to tert-butanol in 

drinking water for 2 years, high dose omitted (NTP, 1995); BMR = 5% extra 

risk 

3 

4 

Modela 

Goodness of fit BMD5% 

(mg/kg-d) 

BMDL5% c 

p-value AIC (mg/kg-d) Basis for model selection b 

One stage 0.105 46.0 1341 538 

Multistage 2° was selected based on lowest AIC. 

Two stage 0.174 44.9 1028 644 

a Selected (best-fitting) model shown in boldface type.
 b AIC = Akaike Information Criterion.
 c Confidence level = 0.95.

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Multistage Cancer Model, with BMR of 5% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL 

Fraction Affected 

0.2

 0.15

 0.1

 0.05

 0

BMDL BMD

Multistage Cancer 

Linear extrapolation 

0 200 400 600 800 1000 

dose 

11:18 06/05 2015 

Figure C-11. Plot of incidence by dose, with fitted curve for Multistage 2° 

model for thyroid follicular cell adenomas or carcinomas in male B6C3F1 mice
 

exposed to tert-butanol in drinking water for 2 years, high dose omitted (NTP,
 

1995); BMR = 5% extra risk; dose shown in mg/kg-d
 

==================================================================== 

Multistage Model. (Version: 3.4; Date: 05/02/2014)

Input Data File: C:/Users/KHOGAN/BMDS/BMDS260/Data/msc_TBA NTP1995 MMthyroid tumors

poly3 -h_Msc2-BMR05.(d) 

Gnuplot Plotting File: C:/Users/KHOGAN/BMDS/BMDS260/Data/msc_TBA NTP1995 MMthyroid

tumors poly3 -h_Msc2-BMR05.plt
 

Fri Jun 05 11:18:05 2015

==================================================================== 

BMDS_Model_Run 

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

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~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

Supplemental Information―tert-Butyl Alcohol 

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The form of the probability function is:

P[response] = background + (1-background)*[1-EXP(

-beta1*dose^1-beta2*dose^2)]
 

The parameter betas are restricted to be positive

Dependent variable = Effect

Independent variable = Dose

Total number of observations = 3

Total number of records with missing values = 0

Total number of parameters in model = 3

Total number of specified parameters = 0

Degree of polynomial = 2

Maximum number of iterations = 500

Relative Function Convergence has been set to: 1e-008

Parameter Convergence has been set to: 1e-008 

Default Initial Parameter Values

Background = 0.00347268

Beta(1) = 0

Beta(2) = 6.65923e-008 

Asymptotic Correlation Matrix of Parameter Estimates

( *** The model parameter(s) -Beta(1) 

have been estimated at a boundary point, or have been specified by the user,

and do not appear in the correlation matrix )

Background Beta(2)

Background 1 -0.34 

Beta(2) -0.34 1

Parameter Estimates

95.0% Wald Confidence Interval

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

Background 0.011558 0.0114911 -0.010964 0.0340801

Beta(1) 0 NA

Beta(2) 4.84624e-008 3.15009e-008 -1.32781e-008 1.10203e-007 

NA - Indicates that this parameter has hit a bound

implied by some inequality constraint and thus

has no standard error.

Analysis of Deviance Table

Model Log(likelihood) # Param's Deviance Test d.f. P-value 

Full model -18.9229 3

Fitted model -20.4481 2 3.05031 1 0.08072

Reduced model -21.9555 1 6.0651 2 0.04819

AIC: 44.8962

Goodness of Fit 

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

C-33 DRAFT—DO NOT CITE OR QUOTE 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Supplemental Information―tert-Butyl Alcohol 

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Scaled

Dose Est._Prob. Expected Observed Size Residual

-----------------------------------------------------------------------­

0.0000 0.0116 0.578 1.000 50.000 0.558

540.0000 0.0254 1.271 0.000 50.000 -1.142 

1040.0000 0.0620 3.164 4.000 51.000 0.485

Chi^2 = 1.85 d.f. = 1 P-value = 0.1735
 

Benchmark Dose Computation

Specified effect = 0.05

Risk Type = Extra risk

Confidence level = 0.95

BMD = 1028.79

BMDL = 644.475

BMDU did not converge for BMR = 0.050000

BMDU calculation failed

BMDU = 14661.6

Cancer Slope Factor = 7.75825e-005 

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

C-34 DRAFT—DO NOT CITE OR QUOTE 
6 

Supplemental Information―tert-Butyl Alcohol 

1 

2 

Table C-14. Summary of BMD modeling results for renal tubule adenoma or 

carcinoma in male F344 rats exposed to tert-butanol in drinking water for 2 

years modeled with administered dose units and including all dose groups 

(NTP, 1995); BMR = 10% extra risk. 

3 

4 

5 

Modela Goodness of fit BMD10Pct (mg/kg-d) BMDL10Pct (mg/kgd) Basis for model 

selection 

pvalue Scaled residuals AIC 

Three 0.0806 -0.989, 0.288, 1.719, 233.94 294 118 Multistage 2° is 

Two and -1.010 selected as the most 

parsimonious model 

of adequate fit. One 0.0806 -0.989, 0.288, 1.719, 

and -1.010 

233.94 294 errorb 

a Selected model in bold.
 b BMD or BMDL computation failed for this model.
 

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Multistage Cancer Model with 0.95 Confidence Level 

Fraction Affected 

0.5

 0.4

 0.3

 0.2

 0.1

BMDL BMD

Multistage Cancer

Linear extrapolation 

0 50 100 150 200 250 300 350 400 

10:57 04/30 2014 dose 

Figure C-12. Plot of incidence by dose, with fitted curve for Multistage 2° 

model for renal tubule adenoma or carcinoma in male F344 rats exposed to 

tert-butanol in drinking water for 2 years modeled with administered dose 

units and including all dose groups (NTP, 1995); BMR = 10% extra risk; dose 

shown in mg/kg-d. 

11 

12 

13 

14 

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

C-35 DRAFT—DO NOT CITE OR QUOTE 
 

Supplemental Information―tert-Butyl Alcohol 

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Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)
 

The form of the probability function is: P[response] = background + (1-background)*[1-EXP(­

beta1*dose^1-beta2*dose^2...)]

The parameter betas are restricted to be positive
 

Benchmark Dose Computation.

BMR = 10% Extra risk
 

BMD = 293.978
 

BMDL at the 95% confidence level = 117.584
 

BMDU at the 95% confidence level = 543384000 

Taken together, (117.584, 543384000) is a 90% two-sided confidence interval for the BMD

Multistage Cancer Slope Factor = 0.000850453 

Parameter Estimates 

Variable Estimate Default Initial 

Parameter Values 

Background 0.217704 0.2335 

Beta(1) 0.000358397 0.000268894 

Beta(2) 0 0 

Analysis of Deviance Table 

Model Log(likelihood) # Param's Deviance Test d.f. p-value 

Full model -112.492 4 

Fitted model -114.97 2 4.95502 2 0.08395 

Reduced 

model 

-115.644 1 6.30404 3 0.09772 

AIC: = 233.94 

Goodness of Fit Table 

Dose Est. Prob. Expected Observed Size Scaled Resid 

0 0.2177 10.885 8 50 -0.989 

90 0.2425 12.127 13 50 0.288 

200 0.2718 13.591 19 50 1.719 

420 0.327 16.351 13 50 -1.01 

Chi^2 = 5.04 d.f = 2 P-value = 0.0806 

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

C-36 DRAFT—DO NOT CITE OR QUOTE 
6 

Supplemental Information―tert-Butyl Alcohol 

1 

2 

Table C-15. Summary of BMD modeling results for renal tubule adenoma or 

carcinoma in male F344 rats exposed to tert-butanol in drinking water for 2 

years modeled with administered dose units and excluding high-dose group 

(NTP, 1995); BMR = 10% extra risk. 

3 

4 

5 

Modela Goodness of fit BMD10Pct (mg/kg-d) BMDL10Pct 

(mg/kg-d) 

Basis for model 

selection 

pvalue Scaled residuals AIC 

Two N/Ab 0.000, -0.000, and ­ 173.68 75.6 41.6 Multistage 1° was 

0.000 selected as the only 

adequately-fitting 

model available One 0.924 0.031, -0.078, and 

0.045 

171.69 70.1 41.6 

a Selected model in bold.
 b No available degrees of freedom to calculate a goodness of fit value.
 

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10 

 

Multistage Cancer Model with 0.95 Confidence Level 

Fraction Affected 

0.5

 0.4

 0.3

 0.2

 0.1

BMDL BMD

Multistage Cancer

Linear extrapolation 

0 50 100 150 200 

11:02 04/30 2014 

dose 

Figure C-13. Plot of incidence by dose, with fitted curve for Multistage 1° 

model for renal tubule adenoma or carcinoma in male F344 rats exposed to 

tert-butanol in drinking water for 2 years modeled with administered dose 

units and excluding high-dose group (NTP, 1995); BMR = 10% extra risk.; dose 

shown in mg/kg-d. 

11 

12 

13 

14 

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

C-37 DRAFT—DO NOT CITE OR QUOTE 
Supplemental Information―tert-Butyl Alcohol 

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Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)
 

The form of the probability function is: P[response] = background + (1-background)*[1-EXP(­

beta1*dose^1-beta2*dose^2...)]

The parameter betas are restricted to be positive
 

Benchmark Dose Computation.

BMR = 10% Extra risk
 

BMD = 70.1068
 

BMDL at the 95% confidence level = 41.5902
 

BMDU at the 95% confidence level = 203.311 

Taken together, (41.5902, 203.311) is a 90% two-sided confidence interval for the BMD

Multistage Cancer Slope Factor = 0.00240441 

Parameter Estimates 

Variable Estimate Default Initial 

Parameter Values 

Background 0.158399 0.156954 

Beta(1) 0.00150286 0.0015217 

Analysis of Deviance Table 

Model Log(likelihood 

) 

# Param's Deviance Test d.f. p-value 

Full model -83.8395 3 

Fitted model -83.8441 2 0.00913685 1 0.9238 

Reduced 

model 

-86.9873 1 6.29546 2 0.04295 

AIC: = 171.688 

Goodness of Fit Table 

Dose Est. Prob. Expected Observed Size Scaled Resid 

0 0.1584 7.92 8 50 0.031 

90 0.2649 13.243 13 50 -0.078 

200 0.3769 18.844 19 50 0.045 

Chi^2 = 0.01 d.f = 1 P-value = 0.9239 

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

C-38 DRAFT—DO NOT CITE OR QUOTE 
Supplemental Information―tert-Butyl Alcohol 

1 

2 

Table C-16. Summary of BMD modeling results for renal tubule adenoma or 

carcinoma in male F344 rats exposed to tert-butanol in drinking water for 2 

years modeled with PBPK (tert-butanol, mg/L) dose units and including all 

dose groups (NTP, 1995); BMR = 10% extra risk. 

3 

4 

5 

Modela Goodness of fit BMD10Pct (mg/L) BMDL10Pct (mg/L) Basis for model 

selection 

pvalue Scaled residuals AIC 

Three 0.0518 -1.373, 0.155, 1.889, 234.83 51.8 13.9 Multistage 1° was 

Two and -0.668 selected as the 

One most parsimonious 

model of adequate 

fit. 

a Selected model in bold. 

Multistage Cancer Model with 0.95 Confidence Level 

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Fraction Affected 

0.5

 0.4

 0.3

 0.2

 0.1

BMDL BMD

Multistage Cancer

Linear extrapolation 

0 10 20 30 40 50 

11:19 04/30 2014 dose 

Figure C-14. Plot of incidence by dose, with fitted curve for Multistage 1° 

model for renal tubule adenoma or carcinoma in male F344 rats exposed to 

tert-butanol in drinking water for 2 years modeled with PBPK (tert-butanol, 

mg/L) dose units and including all dose groups (NTP, 1995); BMR = 10% extra 

risk.; dose shown in mg/L. 

11 

12 

13 

14 

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

C-39 DRAFT—DO NOT CITE OR QUOTE 
Supplemental Information―tert-Butyl Alcohol 

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Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)
 

The form of the probability function is: P[response] = background + (1-background)*[1-EXP(­

beta1*dose^1-beta2*dose^2...)]

The parameter betas are restricted to be positive
 

Benchmark Dose Computation.

BMR = 10% Extra risk
 

BMD = 51.8357
 

BMDL at the 95% confidence level = 13.9404
 

BMDU at the 95% confidence level = error 

Taken together, (13.9404, error) is a 90% two-sided confidence interval for the BMD

Multistage Cancer Slope Factor = error 

Parameter Estimates 

Variable Estimate Default Initial 

Parameter Values 

Background 0.243327 0.253053 

Beta(1) 0.00203259 0.00150893 

Analysis of Deviance Table 

Model Log(likelihood) # Param's Deviance Test d.f. p-value 

Full model -112.492 4 

Fitted model -115.417 2 5.84883 2 0.0537 

Reduced 

model 

-115.644 1 6.30404 3 0.09772 

AIC: = 234.834 

Goodness of Fit Table 

Dose Est. Prob. Expected Observed Size Scaled Resid 

0 0.2433 12.166 8 50 -1.373 

4.6945 0.2505 12.526 13 50 0.155 

12.6177 0.2625 13.124 19 50 1.889 

40.7135 0.3034 15.171 13 50 -0.668 

Chi^2 = 5.92 d.f = 2 P-value = 0.0518 

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

C-40 DRAFT—DO NOT CITE OR QUOTE 
Supplemental Information―tert-Butyl Alcohol 

1 

2 

Table C-17. Summary of BMD modeling results for renal tubule adenoma or 

carcinoma in male F344 rats exposed to tert-butanol in drinking water for 2 

years modeled with PBPK (tert-butanol, mg/L) dose units and excluding highdose group (NTP, 1995); BMR = 10% extra risk. 

3 

4 

Modela Goodness of fit BMD10Pct (mg/L) BMDL10Pct (mg/L) Basis for model 

selection 

pvalue Scaled residuals AIC 

Two 0.891 -0.054, 0.113, and ­ 171.70 4.33 2.54 Multistage 1° was 

One 0.057 selected as the most 

parsimonious model 

of adequate fit. 

a Selected model in bold. 

5 

Multistage Cancer Model with 0.95 Confidence Level 

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Fraction Affected 

0.5

 0.4

 0.3

 0.2

 0.1

BMDL BMD

Multistage Cancer

Linear extrapolation 

0 2 4 6 8 10 12 

11:20 04/30 2014 

dose 

Figure C-15. Plot of incidence by dose, with fitted curve for Multistage 1° 

model for renal tubule adenoma or carcinoma in male F344 rats exposed to 

tert-butanol in drinking water for 2 years modeled with PBPK (tert-butanol, 

mg/L) dose units and excluding high-dose group (NTP, 1995); BMR = 10% 

extra risk; dose shown in mg/L. 

10 

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12 

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

C-41 DRAFT—DO NOT CITE OR QUOTE 
Supplemental Information―tert-Butyl Alcohol 

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Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)
 

The form of the probability function is: P[response] = background + (1-background)*[1-EXP(­

beta1*dose^1-beta2*dose^2...)]

The parameter betas are restricted to be positive
 

Benchmark Dose Computation.

BMR = 10% Extra risk
 

BMD = 4.33496
 

BMDL at the 95% confidence level = 2.53714
 

BMDU at the 95% confidence level = 12.8097 

Taken together, (2.53714, 12.8097) is a 90% two-sided confidence interval for the BMD

Multistage Cancer Slope Factor = 0.0394144 

Parameter Estimates 

Variable Estimate Default Initial 

Parameter Values 

Background 0.162798 0.164724 

Beta(1) 0.0243048 0.0238858 

Analysis of Deviance Table 

Model Log(likelihood) # Param's Deviance Test d.f. p-value 

Full model -83.8395 3 

Fitted model -83.8489 2 0.0187339 1 0.8911 

Reduced 

model 

-86.9873 1 6.29546 2 0.04295 

AIC: = 171.698 

Goodness of Fit Table 

Dose Est. Prob. Expected Observed Size Scaled Resid 

0 0.1628 8.14 8 50 -0.054 

4.6945 0.2531 12.654 13 50 0.113 

12.6177 0.3839 19.195 19 50 -0.057 

Chi^2 = 0.02 d.f = 1 P-value = 0.891 

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

C-42 DRAFT—DO NOT CITE OR QUOTE 
Supplemental Information―tert-Butyl Alcohol 

1 Table C-18. Summary of BMD modeling results for renal tubule adenoma or 

carcinoma in male F344 rats exposed to tert-butanol in drinking water for 2 

years modeled with PBPK (metabolized, mg/hr) dose units and including all 

dose groups (NTP, 1995); BMR = 10% extra risk. 

2 

3 

4 

5 

Modela Goodness of fit BMD10Pct (mg/hr) BMDL10Pct (mg/hr) Basis for model 

selection 

pvalue Scaled residuals AIC 

Three 0.0885 -0.920, 0.301, 1.677, 233.76 2.28 0.954 Multistage 1° was 

Two and -1.049 selected as the most 

One parsimonious model 

of adequate fit. 

a Selected model in bold. 

6 

Multistage Cancer Model with 0.95 Confidence Level 

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0.5

 0.4

 0.3

 0.2

 0.1

dose 

11:22 04/30 2014 

Fraction Affected 

BMDL BMD

Multistage Cancer

Linear extrapolation 

0 0.5 1 1.5 2 2.5 3 3.5 

Figure C-16. Plot of incidence by dose, with fitted curve for Multistage 1° 

model for renal tubule adenoma or carcinoma in male F344 rats exposed to 

tert-butanol in drinking water for 2 years modeled with PBPK (metabolized, 

mg/hr) dose units and including all dose groups (NTP, 1995); BMR = 10% 

extra risk; dose shown in mg/hr. 

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

C-43 DRAFT—DO NOT CITE OR QUOTE 
Supplemental Information―tert-Butyl Alcohol 

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Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)
 

The form of the probability function is: P[response] = background + (1-background)*[1-EXP(­

beta1*dose^1-beta2*dose^2...)]

The parameter betas are restricted to be positive
 

Benchmark Dose Computation.

BMR = 10% Extra risk
 

BMD = 2.28299
 

BMDL at the 95% confidence level = 0.95436
 

BMDU at the 95% confidence level = error 

Taken together, (0.95436, error) is a 90% two-sided confidence interval for the BMD

Multistage Cancer Slope Factor = error 

Parameter Estimates 

Variable Estimate Default Initial 

Parameter Values 

Background 0.21328 0.229822 

Beta(1) 0.0461502 0.0349139 

Analysis of Deviance Table 

Model Log(likelihood) # Param's Deviance Test d.f. p-value 

Full model -112.492 4 

Fitted model -114.879 2 4.77309 2 0.09195 

Reduced 

model 

-115.644 1 6.30404 3 0.09772 

AIC: = 233.758 

Goodness of Fit Table 

Dose Est. Prob. Expected Observed Size Scaled Resid 

0 0.2133 10.664 8 50 -0.92 

0.7992 0.2418 12.088 13 50 0.301 

1.7462 0.2742 13.71 19 50 1.677 

3.4712 0.3297 16.487 13 50 -1.049 

Chi^2 = 4.85 d.f = 2 P-value = 0.0885 

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

C-44 DRAFT—DO NOT CITE OR QUOTE 
6 

Supplemental Information―tert-Butyl Alcohol 

1 

2 

Table C-19. Summary of BMD modeling results for renal tubule adenoma or 

carcinoma in male F344 rats exposed to tert-butanol in drinking water for 2 

years modeled with PBPK (metabolized, mg/hr) dose units and excluding 

high-dose group (NTP, 1995); BMR = 10% extra risk. 

3 

4 

5 

Modela Goodness of fit BMD10Pct (mg/hr) BMDL10Pct (mg/hr) Basis for model 

selection 

pvalue Scaled residuals AIC 

Two N/Ab -0.000, -0.000, and ­

0.000 

173.68 0.673 0.365 Multistage 1° was 

selected on the 

basis of lowest AIC. 

One 0.906 0.037, -0.096, and 

0.057 

171.69 0.614 0.364 

a Selected model in bold.
 b No available degrees of freedom to calculate a goodness of fit value.
 

Data from NTP1995 

Multistage Cancer Model with 0.95 Confidence Level 

7 

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12 

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14 

Fraction Affected 

 

0.5

 0.4

 0.3

 0.2

 0.1

BMDL BMD

Multistage Cancer

Linear extrapolation 

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 

11:24 04/30 2014 

dose 

Figure C-17. Plot of incidence by dose, with fitted curve for Multistage 1° 

model for renal tubule adenoma or carcinoma in male F344 rats exposed to 

tert-butanol in drinking water for 2 years modeled with PBPK (metabolized, 

mg/hr) dose units and excluding high-dose group (NTP, 1995); BMR = 10% 

extra risk; dose shown in mg/hr. 

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

C-45 DRAFT—DO NOT CITE OR QUOTE 
Supplemental Information―tert-Butyl Alcohol 

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Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)
 

The form of the probability function is: P[response] = background + (1-background)*[1-EXP(­

beta1*dose^1-beta2*dose^2...)]

The parameter betas are restricted to be positive
 

Benchmark Dose Computation.

BMR = 10% Extra risk
 

BMD = 0.613798
 

BMDL at the 95% confidence level = 0.364494
 

BMDU at the 95% confidence level = 1.77845 

Taken together, (0.364494, 1.77845) is a 90% two-sided confidence interval for the BMD

Multistage Cancer Slope Factor = 0.274353 

Parameter Estimates 

Variable Estimate Default Initial 

Parameter Values 

Background 0.158068 0.156284 

Beta(1) 0.171653 0.174305 

Analysis of Deviance Table 

Model Log(likelihood) # Param's Deviance Test d.f. p-value 

Full model -83.8395 3 

Fitted model -83.8465 2 0.0138544 1 0.9063 

Reduced 

model 

-86.9873 1 6.29546 2 0.04295 

AIC: = 171.693 

Goodness of Fit Table 

Dose Est. Prob. Expected Observed Size Scaled Resid 

0 0.1581 7.903 8 50 0.037 

0.7992 0.266 13.3 13 50 -0.096 

1.7462 0.3761 18.806 19 50 0.057 

Chi^2 = 0.01 d.f = 1 P-value = 0.9064 

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

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Supplemental Information―tert-Butyl Alcohol 

1 

2 

Table C-20. Summary of BMD modeling results for renal tubule adenoma or 

carcinoma in male F344 rats exposed to tert-butanol in drinking water for 2 

years modeled with administered dose units and including all dose groups; 

reanalyzed data (Hard et al., 2011; NTP, 1995); BMR = 10% extra risk. 

3 

4 

5 

Modela Goodness of fit BMD10Pct (mg/kg-d) BMDL10Pct 

(mg/kg-d) 

Basis for model 

selection 

pvalue Scaled residuals AIC 

Three 0.0117 -1.476, 1.100, 1.855, 218.68 184 94.8 No model fit the 

Two and -1.435 data. 

One 

a No model was selected as a best-fitting model. 

6 

7 

8 

9 

10 

Table C-21. Summary of BMD modeling results for renal tubule adenoma or 

carcinoma in male F344 rats exposed to tert-butanol in drinking water for 2 

years modeled with administered dose units and excluding high-dose group; 

re-analyzed data (Hard et al., 2011; NTP, 1995); BMR = 10% extra risk. 

Modela Goodness of fit BMD10Pct (mg/kg-d) BMDL10Pct 

(mg/kg-d) 

Basis for model 

selection 

pvalue Scaled residuals AIC 

Two 0.572 -0.141, 0.461, and ­ 154.84 54.2 36.3 Multistage 1° was 

One 0.296 selected as the most 

parsimonious model 

of adequate fit. 

a Selected model in bold. 

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

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Supplemental Information―tert-Butyl Alcohol 

Multistage Cancer Model with 0.95 Confidence Level 

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BMDL BMD

Multistage Cancer

Linear extrapolation 

0 50 100 150 200 

Figure C-18. Plot of incidence by dose, with fitted curve for Multistage 1° 

model for renal tubule adenoma or carcinoma in male F344 rats exposed to 

tert-butanol in drinking water for 2 years modeled with administered dose
 

units and excluding high-dose group; re-analyzed data (Hard et al., 2011; NTP,
 

1995); BMR = 10% extra risk; dose shown in mg/kg-d.
 

Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)

The form of the probability function is: P[response] = background + (1-background)*[1-EXP(­

beta1*dose^1-beta2*dose^2...)]

The parameter betas are restricted to be positive 

Benchmark Dose Computation.

BMR = 10% Extra risk 

BMD = 54.1642 

BMDL at the 95% confidence level = 36.3321 

BMDU at the 95% confidence level = 101.125 

Taken together, (36.3321, 101.125) is a 90% two-sided confidence interval for the BMD

Multistage Cancer Slope Factor = 0.00275239 

Parameter Estimates 

Variable Estimate Default Initial 

Parameter Values 

Background 0.0855815 0.0981146 

Beta(1) 0.00194521 0.00179645 

Analysis of Deviance Table 

Model Log(likelihood) # Param's Deviance Test d.f. p-value 

Full model -75.2622 3 

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Supplemental Information―tert-Butyl Alcohol 

Fitted model -75.4201 2 0.315716 1 0.5742 

Reduced 

model 

-81.4909 1 12.4574 2 0.001972 

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2 

3 

4 

5 

6 

7 

8 

AIC: = 154.84 

Goodness of Fit Table 

Dose Est. Prob. Expected Observed Size Scaled Resid 

0 0.0856 4.279 4 50 -0.141 

90 0.2324 11.622 13 50 0.461 

200 0.3803 19.015 18 50 -0.296 

Chi^2 = 0.32 d.f = 1 P-value = 0.5715 

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

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Supplemental Information―tert-Butyl Alcohol 

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Table C-22. Summary of BMD modeling results for renal tubule adenoma or 

carcinoma in male F344 rats exposed to tert-butanol in drinking water for 2 

years modeled with PBPK (tert-butanol, mg/L) dose units and including all 

dose groups; reanalyzed data (Hard et al., 2011; NTP, 1995); BMR = 10% extra 

risk. 

Modela Goodness of fit BMD10Pct (mg/L) BMDL10Pct (mg/L) Basis for model 

selection 

pvalue Scaled residuals AIC 

Three 0.0048 -2.089, 0.864, 2.165, 220.82 31.4 11.7 No model fit the 

Two and -0.929 data. 

One 

a No model was selected as a best-fitting model. 

6 

7 

8 

9 

10 

Table C-23. Summary of BMD modeling results for renal tubule adenoma or 

carcinoma in male F344 rats exposed to tert-butanol in drinking water for 2 

years modeled with PBPK (tert-butanol, mg/L) dose units and excluding highdose group; reanalyzed data (Hard et al., 2011; NTP, 1995); BMR = 10% extra 

risk. 

Modela Goodness of fit BMD10Pct (mg/L) BMDL10Pct (mg/L) Basis for model 

selection 

pvalue Scaled residuals AIC 

Two 0.364 -0.285, 0.750, and ­ 155.33 3.35 2.21 Multistage 1° was 

One 0.424 selected as the most 

parsimonious model 

of adequate fit. 

a Selected model in bold. 

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Supplemental Information―tert-Butyl Alcohol 

Multistage Cancer Model with 0.95 Confidence Level 

 

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11:30 04/30 2014
 

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Multistage Cancer

Linear extrapolation 

0 2 4 6 8 10 12 

Figure C-19. Plot of incidence by dose, with fitted curve for Multistage 1° 

model for renal tubule adenoma or carcinoma in male F344 rats exposed to 

tert-butanol in drinking water for 2 years modeled with PBPK (tert-butanol, 

mg/L) dose units and excluding high-dose group; reanalyzed data (Hard et al., 

2011; NTP, 1995); BMR = 10% extra risk; dose shown in mg/L.
 

Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)

The form of the probability function is: P[response] = background + (1-background)*[1-EXP(­

beta1*dose^1-beta2*dose^2...)]

The parameter betas are restricted to be positive 

Benchmark Dose Computation.

BMR = 10% Extra risk 

BMD = 3.34903 

BMDL at the 95% confidence level = 2.20865 

BMDU at the 95% confidence level = 6.49702 

Taken together, (2.20865, 6.49702) is a 90% two-sided confidence interval for the BMD

Multistage Cancer Slope Factor = 0.0452765 

Parameter Estimates 

Variable Estimate Default Initial 

Parameter Values 

Background 0.0916116 0.110649 

Beta(1) 0.03146 0.0276674 

Analysis of Deviance Table 

Model Log(likelihood) # Param's Deviance Test d.f. p-value 

Full model -75.2622 3 

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

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Supplemental Information―tert-Butyl Alcohol 

Fitted model -75.664 2 0.803466 1 0.3701 

Reduced 

model 

-81.4909 1 12.4574 2 0.001972 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

17 

18 

AIC: = 155.328 

Goodness of Fit Table 

Dose Est. Prob. Expected Observed Size Scaled Resid 

0 0.0916 4.581 4 50 -0.285 

4.6945 0.2163 10.817 13 50 0.75 

12.6177 0.3892 19.462 18 50 -0.424 

Chi^2 = 0.82 d.f = 1 P-value = 0.3643 

Table C-24. Summary of BMD modeling results for renal tubule adenoma or 

carcinoma in male F344 rats exposed to tert-butanol in drinking water for 2 

years modeled with PBPK (metabolized, mg/hr) dose units and including all 

dose groups; reanalyzed data (Hard et al., 2011; NTP, 1995); BMR = 10% extra 

risk. 

Modela Goodness of fit BMD10Pct (mg/hr) BMDL10Pct (mg/hr) Basis for model 

selection 

pvalue Scaled residuals AIC 

Three 0.0142 -1.367, 1.119, 1.783, 218.26 1.44 0.770 No model fit the 

Two and -1.484 data. 

One 

a No model was selected as a best-fitting model. 

Table C-25. Summary of BMD modeling results for renal tubule adenoma or 

carcinoma in male F344 rats exposed to tert-butanol in drinking water for 2 

years modeled with PBPK (metabolized, mg/hr) dose units and excluding 

high-dose group; reanalyzed data (Hard et al., 2011; NTP, 1995); BMR = 10% 

extra risk. 

Modela Goodness of fit BMD10Pct (mg/hr) BMDL10Pct 

(mg/hr) 

Basis for model 

selection 

p-value Scaled residuals AIC 

Two 

One 

0.593 -0.130, 0.435, and ­

0.281 

154.81 0.474 0.319 Multistage 1° was 

selected as the most 

parsimonious model 

of adequate fit. 

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

C-52 DRAFT—DO NOT CITE OR QUOTE 
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Supplemental Information―tert-Butyl Alcohol 

a Selected model in bold. 

Multistage Cancer Model with 0.95 Confidence Level 

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11:33 04/30 2014
 

Fraction Affected 

BMDL BMD

Multistage Cancer

Linear extrapolation 

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 

Figure C-20. Plot of incidence by dose, with fitted curve for Multistage 1° 

model for renal tubule adenoma or carcinoma in male F344 rats exposed to 

tert-butanol in drinking water for 2 years modeled with PBPK (metabolized,
 

mg/hr) dose units and excluding high-dose group; reanalyzed data (Hard et
 

al., 2011; NTP, 1995); BMR = 10% extra risk.; dose shown in mg/hr.
 

Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010)

The form of the probability function is: P[response] = background + (1-background)*[1-EXP(­

beta1*dose^1-beta2*dose^2...)]

The parameter betas are restricted to be positive 

Benchmark Dose Computation. 

BMR = 10% Extra risk 

BMD = 0.474241 

BMDL at the 95% confidence level = 0.318504 

BMDU at the 95% confidence level = 0.882859 

Taken together, (0.318504, 0.882859) is a 90% two-sided confidence interval for the BMD 

Multistage Cancer Slope Factor = 0.313968 

Parameter Estimates 

Variable Estimate Default Initial 

Parameter Values 

Background 0.0851364 0.0969736 

Beta(1) 0.222167 0.206161 

Analysis of Deviance Table 

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Supplemental Information―tert-Butyl Alcohol 

Model Log(likelihood) # Param's Deviance Test d.f. p-value 

Full model -75.2622 3 

Fitted model -75.4029 2 0.281435 1 0.5958 

Reduced model -81.4909 1 12.4574 2 0.001972 

AIC: = 154.806 

Goodness of Fit Table 

Dose Est. Prob. Expected Observed Size Scaled Resid 

0 0.0851 4.257 4 50 -0.13 

0.7992 0.234 11.699 13 50 0.435 

1.7462 0.3793 18.966 18 50 -0.281 

Chi^2 = 0.29 d.f = 1 P-value = 0.5933 

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Supplemental Information―tert-Butyl Alcohol 

C.1.2.4. Inhalation Unit Risk for Cancer 

An inhalation unit risk was not derived because the relative contribution of the α2u-globulin 

and other, unknown, processes to renal tumor formation cannot be determined (U.S. EPA, 1991), 

and therefore the male rat renal tumors are not considered suitable for quantitative analysis.
 

However, if renal tumors are considered suitable for analysis then route-to-route extrapolation
 

could be performed. 

Dose Response Analysis – Adjustments and Extrapolation Methods 

A PBPK model for tert-butanol in rats has been developed, as described in Appendix B. 

Using this model, route-to-route extrapolation of the oral BMDL to derive an inhalation POD was 

performed as follows. First, the internal dose in the rat at the oral BMDL (assuming continuous 

exposure) was estimated using the PBPK model, to derive an “internal dose BMDL.” Then, the 

inhalation air concentration (again assuming continuous exposure) that led to the same internal 

dose in the rat was estimated using the PBPK model, resulting in a route-to-route extrapolated 

BMCL. 

A critical decision in the route-to-route extrapolation is the selection of the internal dose 

metric to use that established "equivalent” oral and inhalation exposures. For tert-butanol-induced 

kidney effects, the two options are the concentration of tert-butanol in blood and rate of tertbutanol metabolism. Note that using the kidney concentration of tert-butanol will lead to the same 

route-to-route extrapolation relationship as tert-butanol in blood, since the distribution from blood 

to kidney is independent of route. There are no data that suggest metabolites of tert-butanol 

mediate its renal toxicity. In the absence of evidence that would suggest otherwise, it is assumed 

that tert-butanol itself is the active toxicological agent. Therefore, the concentration of tert-butanol 

in blood was selected as the dose metric to derive the BMCL. 

The RfC methodology provides a mechanism for deriving a HEC from the BMCL determined 

from the animal data. The approach takes into account the extra-respiratory nature of the 

toxicological responses and accommodates species differences by considering blood:air partition 

coefficients for tert-butanol in the laboratory animal (rat or mouse) and humans. According to the 

RfC guidelines (U.S. EPA, 1994), tert-butanol is a Category 3 gas because extra-respiratory effects 

were observed. Kaneko et al. (2000) measured a blood:gas partition coefficient of 531 ± 102 for 

tert-butanol in the male Wistar rat, while Borghoff et al. (1996) measured a value of 481 ± 29 in 

male F344 rats. A blood:gas partition coefficient of 462 was reported for tert-butanol in humans 

(Nihlén et al., 1995). The calculation (Hb/g )A ÷ (Hb/g )H was used to calculate a blood:gas partition 

coefficient ratio to apply to the delivered concentration. Because F344 rats were used in the study, 

the blood:gas partition coefficient for F344 rats was used. Thus, the calculation was: 481 ÷ 462 = 

1.04. Therefore, a ratio of 1.04 was used to calculate the HEC. This allowed a BMCLHEC to be derived 

as follows: 

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BMCLHEC = BMCLADJ (mg/m3) × (interspecies conversion)
 

= BMCLADJ (mg/m3) × (481 ÷ 462)
 

= BMCLADJ (mg/m3) × (1.04)
 

The U.S. EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005) recommend that the 

method used to characterize and quantify cancer risk from a chemical is determined by what is 

known about the MOA of the carcinogen and the shape of the cancer dose-response curve. The 

linear approach is recommended if the MOA of carcinogenicity has not been established (U.S. EPA, 

2005). In the case of tert-butanol, the mode of carcinogenic action for renal tubule tumors is not 

fully understood (see Section 1.2.1). Therefore, a linear low-dose extrapolation approach was used 

to estimate human carcinogenic risk associated with tert-butanol exposure. 

Inhalation Unit Risk Derivation 

The results from route-to-route extrapolation of the male rat renal tubule tumor data are 

summarized in Table C-26. The lifetime inhalation unit risk for humans is defined as the slope of 

the line from the lower 95% bound on the exposure at the POD to the control response (inhalation 

unit risk = 0.1/BMCL10). This slope, a 95% upper confidence limit represents a plausible upper 

bound on the true risk. Using linear extrapolation from the BMCL10, a human equivalent inhalation 

unit risk was derived, as listed in Table C-26. 

Two inhalation unit risks were derived from the NTP (1995) bioassay: one based on the 

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

differ by less than 20%, but the Hard et al. (2011) reanalysis is considered preferable, as it is based 

on a PWG analysis. Therefore, the recommended inhalation unit risk for providing a sense of the 

magnitude of potential carcinogenic risk associated with lifetime inhalation exposure to 

tert-butanol is 1 × 10-3 per mg/m3, or 2 × 10-3 per μg/m3, based on the renal tubule tumor 

response in male F344 rats. 

Table C-26. Summary of the inhalation unit risk derivation 

Tumor Species/Sex BMR 

BMDL 

(mg/kg-d) 

Internal Dosea 

(mg/L) 

POD= 

BMCLHEC b 

(mg/m3) 

Unit Riskc 

(mg/m3)

-1 

Renal tubule adenoma or 

carcinoma 

Male F344 

rat 

10% 41.6 2.01 68.7 1 × 10-3 

Renal tubule adenoma or 

carcinoma [Hard et al. 

(2011) reanalysis] 

Male F344 

rat 

10% 36.3 1.74 59.8 2 × 10-3 

a Average blood concentration of tert-butanol under continuous oral exposure at the BMDL.
 b Continuous inhalation human equivalent concentration that leads to the same average blood concentration of
 

tert-butanol as continuous oral exposure at the BMDL.
 c

Human equivalent inhalation unit risk = 0.1/BMCLHEC .
 

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

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