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



## Toxicological Review of tert

**- Butyl Alcohol (**  tert **-B utanol**  **)**

(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 doe s 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 A ssessment Office of Research and Development U.S. Environmental Protection Agency Washington, DC

### Supplemental Information― tert

- But yl Alcohol

**DISCLAIMER**
This document is a preliminary draft for review purposes only. This information is distributed solely for the purpose of pre

- dissemination peer review under applicable information quality guidelines. It has not been formally disseminated by EPA. It does not represent and should not be construed to represent any Agency determination or policy. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

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

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# CONTENTS Supplemental Information― tert

- But yl Alcohol

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

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— DO NOT CITE OR QUOTE B.2. PBPK MODEL E VALUATION 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. AS SESSMENTS 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

Exc retion

................................................................................................... B-5 Physiologically Based Pharmacokinetic Models

....................................... B-6

N oncancer Endpoints

................................................................................ C-1 Cancer Endpoints

.................................................................................... C- 23

REFERENCES

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

### Supplemental In formation― tert

- But yl 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. C hanges 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) studi es 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

- bu tanol in drinking water for 2 years (NTP, 1995) ; BMR = 10% extra risk

................................................................................................ . C-3 Table C

- 3. Summary of BMD modeling results for kidney tr ansitional 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 ter t

- 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 exp osed 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

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### Supplemental Infor mation― tert

- But yl 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 drink ing 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

- 1 8. 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 r isk.

.............................................................................................. C- 43 Table C

- 19. Summary of BMD modeling results for renal tubule adenoma or carcinoma in male F344 rats exposed to tert

- buta nol 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. Summar y 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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- But yl 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 m odels 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 ter t

- 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 expo sed 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 o f 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 m g/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 L ogprobit 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/m 3

......................................................................... C- 19 Figure C -7

. Plot of mean response by concentration, wi th 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/m 3

................................................ C- 22Figure C -8

. Plot of mean response by concentration, with fitted curve for Power model for absolute kidney weight in female F344 rats exposed to te rt

- 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/m 3

................................................ 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 do se, 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 e xposed 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 u nits 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); B MR = 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 m ale 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 wa ter 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

- But yl Alcohol

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- 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 

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- But yl Alcohol

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# 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 cytoch rome P450 DMSO dimethyl sulfoxide DNA deoxyribonucleic acid EDTA e thylenediaminetetraacetic acid EPA U.S. Environmental Protection Agency ETBE ethyl tert

- butyl ether HBA 2- hydroxyisobutyrate HL human leukemia IC 50 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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- But yl Alcohol

1

2 

# APPENDIX A. ASSESSMENTS BY O THER NATIONAL AND INTERNATIONAL HEALTH AGENCIES

3 4 **Table A-1.**  **Health assessments and regulatory limits by o**  **ther national and**  **i nternational**  **he alth agencies** 

Organization Toxicity value
National Institute of Occupational Safety and Health ( NIOSH, 2007 ) Recommended Exposure Limit – 100 ppm (300 mg/m 3 ) 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/m 3 ) 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); s ubstance may be used as a defoaming agent (21 CFR 176.200).

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- But yl A lcohol



# APPENDIX B. INFORMATION IN SUPPORT OF HAZARD IDENTIFICATION AND DOSE

- RES PONSE ANALYSIS

**B.1. TOXICOKINETICS**

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

tert

- butyl alcohol ( tert

- bu tanol ) in humans . T he studies identified for this Toxicological Assessment were conducted in conjunction with meth yl tert

- butyl ether ( MTBE ) or ethyl tert

- butyl ether ( ETBE ) , as te rt

- butanol is a m etabolite of both compounds

. Several studies examining some aspect of the toxicokinetic behavior of tert

- but anol in animals have been identified. Many were carried out in conjunction with other specific end points (e.g., developmental). ARCO (1983) determined no differences in the pharmacokinetics of tert

- but anol following either oral (i.e., gavage) or inhalation
exposure. Although some information is avail able for both oral and inhalation exposure s , many studies administered tert

- but anol via intraperi t oneal (i.p.) or intravenous (i.v.) injection. Although these studies do not inform the ab sorption of tert

- butanol , they can provide information on its distribution, m etabolism, and excretion. 

### Absorption Toxicity data on tert

- butanol submitted by industry to the U.S. Environmental Protection Agency ( EPA ) unde r Section 8(e) of the Toxic Substances Control Act and other reporting requirements indicate that tert

- but anol 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. C ompar able blood levels of tert

- butanol and its metabolites have been observed after

acute oral ( 350 mg/kg ) or inhalation (6, 060 mg/m 3 for 6 hours ) exposures in male Sprague

- Dawley

rats ( ARCO, 1983 ) ; th e absorption rate after inhalation exposure could not be determined , however, because the blood was saturated with radioactivity after 6 ho urs of exposure to 6,060 mg/m 3

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

at 1.5 hours following the last of six oral gav age doses of 10.5 mmoles tert

- butano l /kg (twice daily) in female C57BL/6J mice. There was an apparent zero

- orde r decline in tert

- butanol concentration for most of the elimination phase
, and no difference s in absorption or elimination rate s was observed bet ween mi ce 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 tert

butanol /kg. T he s tudy therefore concluded that previous exposures did not affect the absorption or elimination of tert

- but anol ( Faulkner et al., 1989 ).

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36 37 38 S upplemental Information― tert

- But yl Alcohol

**Distribution** The available animal data suggest that tert

- butanol is distributed throughout the body following oral , i nhalation , and i.v. exposures ( Poet et al., 1997 ; Faulkner et al., 1989 ; ARCO,
1983 ). N ihlén et al. (1995) calculated partition coefficient s 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 te rt

- butanol were slightly above 1 (from 1.02 to 1.06) for most tissues
, ex cept 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

- bu tanol ( see Sec tion B.1.4). The study concluded that, although tert

- butano l preferentially distributes in body water, the ethers distribute uniformly throughout the body with preference for fatty tissues ( Ni hlén et al., 1995 ). In a study aimed at determining wh ether t ert

- butanol (or metabolites) can bind to

α 2u -globulin
, Williams and Borghoff (2001) expos ed F- 344 rats to a single gavage dose of 500 mg/kg 14 C- tert

- butanol . They found the radiolabel in three tissues ( kidney, liver, and blood ) in both

sexes , but male rat s retain ed more of the tert

- butano l equivalents than females ( Williams and Borghoff, 2001 ). R adioactivity 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 wa s bound to α 2u - globulin. Further analysis determined that tert

- butanol
, and not its metabolite acetone , was bound

. Mos t 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 pr otein fraction in both males and females. I n another study on α 2u - globulin nephropathy, Bo rghoff et al. (2001) found similar results after F

- 344 rats were exposed to 0, 250, 450, or 1750 ppm te rt

- butanol by inhalation for 10 consecutive days. Male rat tert

- butanol kidney

to

- blood ratios were significantly elevated over ratios i n female s at all dose levels and exposure durations. Although the female tert

- butanol kidney

- to

- blood ratio r emained similar with both duration and concentration, the male tert

- but anol kidney

- to

- blood ratio increased with duration. The liver

- to

- bl ood ratios were similar regardless of exposure duration, concentration, or sex

. Both of these studies indicate distrib ut ion 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 Fi gure B-1. Urinary metabolites of tert

- butanol in a human male volunteer who ingested a gelatin capsule containing 5 mg/kg [ 13 C]

- tert

- butanol were reported to be 2

- methyl

- 1,2

­ propanediol (M PD) and 2

- hy droxyisobutyrate ( Bernauer et al., 1998 ). Minor metabolites of unconjugated tert

- butanol
, tert

- butanol glucuronides
, and traces of the sulfate conjugate also we re 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
, w hile acetone, tert

- butanol , and tert

- butanol

### Supplemental Information― tert

- But yl Alcohol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 glucuronides were identified as minor metabolites ( B ernauer et al., 1998 ). Baker et al. (1982) found that t ert

- butanol was a source of acetone, but acetone production might hav e been stimulated from other sources.
N o s tudies identif ied specific enzymes responsible for biotransform ing tert

- butanol . U sing a purified enzyme from Sprague

- Daw ley rats or whole

- liver cytosol from Wistar rats, alcohol dehydrogenase h ad 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 cytoch r ome P450 (CYP450) ( Cederbaum et al., 1983 ; Cederbaum and Cohen, 1980 ). In the 1 983 study, incubation of tert

- butanol at 35 mM with Sprague

- Dawley rat liver microsomes and a n icotinamide adenine di nucleotide phosphate - ( NADPH ) generating system resulted in formaldehyde th e production at a rate of approximately 25 nmoles/mg protein /30 min . A ccording to study authors, t he amount of formaldehyde generated by tert

- butanol was approximately 30% of the amount of f ormaldehyde formed during the metabolism of 10 mM aminopyrene in a similar microsomal system. T he rate of formaldehyde generation from tert

- butanol increased to about
90 n mol/mg protein/30 min upon addition of azide, which inhibits catalase and thereby prevent s the decomposition of hydrogen peroxide ( H 2 O 2 ). In other experiments in the same study, formaldehyde f ormation was greatly reduced when H 2 O 2 was included but NADPH was absent or when the microsomes were boiled prior to incubation. A ddit ionally, the rate of formaldehyde formation in the microsomal oxidizing system depende d on the concentration of tert

- butanol
, with apparent K m and V max values of 30 mM and 5. 5 nmol/min/mg protein, respectively

. The study authors concluded that t ert

- butanol is metabolized to formaldehyde by a mechanism involving oxidation of NADPH, microsomal electron flow, and the generation of hydroxyl

- r adical (
· OH) from
H 2 O 2 , possibly by a Fenton

- type or a Haber

- Weiss iron

- catalyzed reaction involving CYP450
, which might serve as the iron chelate ( C ederbaum and Cohen, 1980 ).

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

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

- O H 3 C CH 3 O CH 3 S acetone O O CH 3

CH 3 t-butyl sulfate 

### 1 Supplemental Information― tert

- But yl Alcohol

2
3 4

5

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Source: NSF International (2003)
, ATSDR (1996)
, Bernauer et al. (1998)
, Amberg et al. (1999)
, 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

- g e nerating systems ( Cederbaum et al., 1983 ) . The nonmicrosomal tests included two chemical systems: (1) the iron

- ca talyzed oxidation of ascorbic acid (ascorbate

- Fe

- EDTA [e t hylenediamine tetraacetic acid ]) and (2) the Fenton system of chelated ferrous iron and H 2 O 2. In both Fenton

- t ype systems, H 2 O 2 served as a precursor for ·OH. Additionally, a Haber

- Weiss enzymatic system involving xanthine ox idation by xanthine oxidase in the presence of Fe

- EDTA was used. In this system, ·OH is thought to be produced by the interaction of H 2 O 2 and superoxide (O 2 · − ). Further experiments demonstrated the involvement of ·OH in either the ascorbate

- Fe

- EDTA or the xanthine oxidation systems based on inhibiti on of formaldehyde and acetone production from t ert

- butanol when ·OH

- scavenging agents (e.g., benzoate, mannitol) were added . Some experiments in this study of the oxidation of t ert

- butanol by the microsomal metabolizing system of the liver w ere similar to those in the previous study ( Cederbaum and Cohen, 1980 ) except that acetone formation, in addition to formaldehyde, also w as measured. Again, these experiments showed the dependence of the microsomal metabolizing system o n an NADPH

- generating system and the ability of H 2 O 2 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

- s cavenging agents inhibited their production. The study authors noted that neither Fe

- EDTA
nor ·OH

- s cavenging 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,

su ch as metyrapone or SKF

- 525A, inhibited the production of formaldehyde from aminopyrene but not from t ert

- butanol . Finally, typical inducers of CYP450 and its MFO metabolizing activities, such as phenobarbital or 3

- me thylcholanthrene, had no effect on microsomal metabolism of tert

- butanol to formaldehyde and acetone. According to the study authors, the oxidation of t ert

- butanol appears to be mediated by
· OH (possibly via H 2 O 2 ), which can be produced by any of the tested systems by a
Fenton

- type reaction as follows:

H 2 O 2 + Fe 2+

- chelate → ·OH + OH

− + Fe 3+

- chelate

According to this reaction, reduction of ferric iron (Fe 3+ ) to ferrous iron (Fe 2+ ) 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

- d ependent electron transfer or O 2 · − might be involved ( Cederbaum et al., 1983 ).



### Excretion
Human data on the excretion of tert

- butanol derives from studies of MTBE and ETBE ( N ihlén 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/m 3 ) or ETBE (20.9, 104, and 210 mg/m 3 ) 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

- a nd 50

- ppm MTBE doses); the half

- life of tert

- butanol in urine following ETBE
exposure w as 7.9 ± 2.7 hours (average of 25

- and 50

- ppm ETBE doses). In both studies, the urinary excretion of t ert

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

- bu tanol 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

- an d 50

- ppm ETBE doses). Amberg et al. (2000) e xposed six volunteers (thr ee males and three females, 28 ± 2 years old) to 18.8 and 170 mg/m 3 ETBE. Each exposure lasted 4 hours, and the two concentrations were administered to the same volunteers 4 weeks apart. U rine was collected at 6

- hour intervals for
72 hours following exposu re. tert

- Butanol and two metabolites of tert

- butanol
, 2- h ydroxyisobutyrate (H BA ) and MPD
, also were identified in urine . At an ETBE level of 170 mg/m 3 , tert

- butanol displayed a half- life of 9.8 ± 1.4 hours. At the low

- exposure ETBE concentration, th e 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 c leave conjugates). H BA in urine showed a broad maximum at 12

– 30 hours after exposure to both

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### Supplemental Information― tert

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concentrations, with a slow decline thereafter. MPD in urine peaked at 12 and 18 hours after exposure to 170 and 18.8 mg/m 3 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/m 3 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/m 3 , but half- life could not be calculated at the ETBE concentration of
18.8 mg/m 3

. 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. O nly 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

- b utanol doses ( ARCO, 1983 ). 

### Physiologically **B ased**  **P harmacokinetic**  **M odels** 

N o physiologically based pharmacokinetic ( PBPK ) models have been developed specifically for administration of tert

- butanol

. S ome models have been used to stud y tert

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

- butanol to

α 2u - globulin ( Borghoff et al., 2010 ; Leavens and Borghoff, 2009 ). Faulkner and Hussain (1989) used a one

- compartment , open model with Michaelis

- Menten elimination kinetics to fit tert

- butanol blood con centrations 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 V max and decrease in apparent volume of d istribution 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 predict ed 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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36 37 38 Supplemental Information― tert

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the clearance of tert

- butanol from the blood of rats after exposure to MTBE involves processes beyond metabolic elimination. Nihlén and Johanson (1999) d eveloped a PBPK model for eva luation of inhalation exposure in humans to the gasoline additive ETBE

. Mo del 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 ad ditional urinary excretion compartment were used for the metabolite
, t ert

- butanol

. First

- order metabolism was assumed in the model, and tissue/blood partition coefficients were determined by in vitro methods ( N ihlén et al., 1995 ). Estimates of individual metabolite parameters of eight subjects were obtained by fitting the PBPK model to

ex perimental data from humans (5, 25, or 50 ppm ETBE ; 2- hour e xposure) ( Nihlén et al., 1998a ). This model p rimarily was applied to predict levels of the biomarkers ETBE and tert

- butanol in blood, urine, and exhaled air after various s cenarios, such as prolonged exposure, fluctuating exposure, and exposure during physical activity ( N ihlén and Johanson, 1999 ). Rao and Ginsberg (1997) d eveloped a PBPK model for M TBE 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

- i nduced neurological responses. MTBE metabolis m to tert

- butano l was assumed to occur in the liver through two saturable pathways. The

tert

- b utanol 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 w as used to help determine the contribution of tert

butanol to the acute central nervous system effects observed after MTBE dosing. The R ao and Ginsberg (1997) model used peak concentrations of MTBE and tert

- butanol in the blood and brain for interspecies, route

- to

- r oute, 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 fo r humans reported by Johanson et al. (1995)
, an d allometrically scaling the metabolic rate based on body weight. A renal elimination component was added to account for the small percent a ge of MTBE disposition that occurs in humans via urinary excretion of t ert

- butanol

. tert -B utanol concentrations in human blood during and after MTBE exposure (25 or 50 ppm fo r 2 hours ) were accurately predicted by the human model ( J ohanson et al., 1995 ). Kim et al. (2007) ex panded the Borghoff et al. (1996) model to develop a multi

- exposure route model for MTBE and its primary metabolite, te rt

- butanol , in humans. The significant features and advantages of the Ki m et al. (2007) model are that parameters used for quantifying the pharmacokinetic behavior of MTBE and tert

- butano l are calibrated using time

- series measurements from controlled

- ex posure exp eriments in humans as reported by Prah et al. (2004)

. MTBE partition coefficient values described in the L icata et al. (2001) model and skin compartment parameters from the R ao and Ginsberg (1997) model were incorporated. The PBPK model for

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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 α 2u - globulin, a protein unique to male rats, and to describe the induction of tert

- butanol metabolism afte r 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

. T he 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 m L /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 thr ough one low affinity pathway and excreted through urine. The model described binding of MTBE or tert

- butanol with α 2u - globulin in the kidney, due to the high concentration of α 2u - globulin in the kidney. As chemicals bind to α 2u - globulin, the rate of hydro lysis of the protein decreases and causes accumulation in the kidney ; however, there is no evidence that binding of α 2u - 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 α 2u - 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 α 2u - globulin

- associated effects in female rats. The deposition of tert

- butanol during inhalation in the nasal cavity and upper ai rways 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 b etween 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 measur ed 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 t he kidney significantly differed between male rats and female rats. The additional parameter accounting for α 2u - globulin protein binding in this PBPK model more accurately reflects the ­

­

­

­

­

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- But yl Alcohol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

43

5
6 7 3
3 3 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 t ert

- butanol. The assumptions made to reflect tert

- butanol metabolism
in d uction and deposition in the nasal cavity and upper airways generally were supported by measured data from rats exposed to 250

- , 4 50

- , or 1,750

- ppm tert

- butanol as evidenced by the fact that the model was within one standard deviation of the mean concentr a tions for most data points. The model overpredicted the concentration of t ert

- 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

. T he Borghoff et al. (2010) model assumed firs t-order uptake of MTBE absorption from the gut, with 100% of the MTBE d ose absorbed for both drinking water and oral gavage exposures. Th ey conducted a series of pharmacokinetic studies comparing the effects of different rat strains and different dosing ve hicles on the blood concentration

- ti me profiles of MTBE and t ert

- 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 wi th 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 t ert

- butanol observed after 250 or 1,000 mg/kg

- d ay administration of MTBE in males and females and the blood concentrations of MTBE after 1,000 mg/kg

- d ay. The model did not predict peak concentrations of MTBE, however, after 250 mg/kg

- d ay 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 Sprague

Dawley rats administered 1,000 m g/kg

- day MTBE. F emale Sprague

- Dawley rats had a significantly ( p
- v alue not specified) higher blood concentration of both MTBE and tert

- butanol compared with
F3 4 4 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

- bu tanol in th e male rat kidney at all exposures tested. The model did not accurately predict the kidney concentrations of tert

- bu tanol in the female kidney after exposure to MTBE via drinking water, but the study authors attributed the inaccuracies to the study design a s 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 t e rt

- butanol.



## B.2. **PBPK MODEL EVALUATION SUMMARY Evaluation of Existing**  tert

**- Butanol**  **Su bmodels** 

The Blancato et al. (2007) and Leavens and Borghoff (2009) PBPK model s for MTBE were evaluated by comparing predictions from the tert

- butanol portions of the models with the tert

- butanol

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- But yl Alcohol

1 2 3 4 5
6 7

8 9 10

11 12 13

14 15 16 17 18 19 20 21 22 23 24 25 26 27 i. v. data of Poet et al. (1997) (see Figure B-2 ) . Neither model adequately represented the tert

- butanol
blood concentrations. Modifications of m o del 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 r e o ptimiz e model parameters in the tert

- butanol submodels of Blancato et al. (2007) and L eavens and Borghoff (2009) to match blood concentrations from the i.v. dosing s tudy

were unsuccessful

. ( A) ( B )

Neither the (A ) Blancato et al. (2007) nor the (B ) Leavens and Borghoff (2009) model adequately represents the measured t e rt

- butanol blood concentrations.
**Figure B-2.**  **Comparison of the** tert

**- butanol**  **portions of existing MTBE models with**  tert

**- butanol**  **blood concentratio**  **ns from i .v.**  **exposure by Poet et al.**  **(1997)**

**.**
The PB PK s ub model for tert

- butanol in rats was developed in acslX (Advanced Continuous Simulation Language, Aegis, Inc., Huntsville, Alabama) by modifyin g information from the many PBPK models developed in rats and humans for the structurally related substance, MTBE
, a n d its metabolite

tert

- b utanol ( Bo rghoff et al., 2010 ; Leavens and Borghoff, 2009 ; Blancato et al., 2007 ; Kim et al.,
2007 ; Rao and Ginsberg, 1997 ; Bo rghoff et al., 1996 ). A b rief description comparing the Blancato et al. (2007) and Le avens 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 te r t

- butanol model.
The Blan cato et al. (2007) m odel is an update of the earlier Rao and Ginsberg (1997) model
, and
the Leavens and Borghoff (2009) m odel is an update of the Borghoff et al. (1996) model. Both
the Blan cato et al. (2007) a nd Leavens and Borghoff (2009) models are flow

- limited models that predict amounts and concen t r ations of MTBE and its metabolite tert

- butanol in blood and six tissue compartments: liver, kidney, fat, brain
, an d rapidly and slowly perfused tissues. These tissue compartments are linked through blood flow, following an anatomically accurate, typical, physiologically b a sed description ( Andersen, 1991 ) . The parent ( M TBE) and metabolite

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( 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 ; L eavens and Borghoff (2009) included inhalation exposure to t ert

- butanol . Oral doses are assumed 100% bioavailable and 100% absorbed from the gastrointestinal tract represented with a first

- o rder rate constant. Following inhalation of MTBE or tert

- butanol , the chemical is assumed to enter the systemic blood supply directly, an d the respiratory tract is assumed to be at pseudo

- steady state. Metabolism of MTBE by
CY P 450s to formaldehyde and tert

- butanol in the liver is described with two Michaeli s- Menten equations representing high

- a nd low

- affinity enzymes. tert -B utanol is either conjugated with glucuronide or sulfate or further metabolized to acetone through M PD and HBA ; both processes are described by a single Michaelis

- Menten equation in the m odels. All model assumptions are valid for

tert

- b utanol and were applied to the EPA

- modified tert

- butanol PBPK model
, except for the separate brain compartment. T h e brain compartment was lumped with other richly perfused tissues in the EPA

- mo dified tert

- butanol PBPK model. In addition to differences in parameter values between the B lancato 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
, a nd (3) binding of MTBE and tert

- butanol to α 2u - globulin was simulated in the kidney of male rats. T he Blancato et al. (2007) model was configured through EPA’s PBPK modeling framework, ERDEM (Exposure

- R elated Dose Estimating Model)
, which includes explicit pulmonary compartments. The modeling assumptions related to alveolar ventilation, explicit pulmonary compartments
, a nd induction of metabolism of tert

- butanol are discussed in t his model evaluation section.
MTBE and t ert

- butanol binding to α 2u - globulin in the kidne ys of male rats were incorporated in the PBPK model of MTBE by L eavens and Borghoff (2009) . Binding to α 2u - globulin is one hypothesized
mode of action for the observed kidney effects in MTBE

- exposed animals. For a detailed description of
the role of α 2u - globulin and other modes of action in kidney effects
, see the kidney mode of action

sect io n of th e Toxicological Review (Section 1. 2 .1 ). In the Leavens and Borghoff (2009) mode l, b inding of
MTBE to α 2u -globulin was applied to sex differences in kidney concentrations of MTBE and tert

butanol
, b ut acceptable estimates of MTBE and tert

- butanol pharmacokinetics in the blood are predicted in other models that did not consider α 2u - globulin binding. Given the uncertainty of tert

butanol bi nding to α 2u -globulin
, it was not included in the tert

- butanol PBPK submodel. 

### Modification of Existing tert **-B utanol**  **S ubmodels** 
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 t ert

- 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 (A blood2 ) with the following equation , where K ON is the binding rate

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1 2

3

4 5 6 7 8 9

10

11 constan t, CV is the free tert

- butanol concentration in blood, K OFF is the unbinding rate constant, and
C blood2 is the concentration of tert

- butanol bound in blood (equal to A blood2 /V blood ).

dA blood2 /dt = K ON *CV* − K OFF *C blood2

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 -B utanol is cleared via exhalation and via urinary excretion. See Table B-1 for definitions of parameter abbreviations.
**Figure B-3**  **. Schematic of the PBPK sub model 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 (F perf ) 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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- But yl Alcohol

Rapidly perfused 0.04 ( Brown et al., 1977 ) Slowly per fused 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 F perf − Σ(other compartments).
b 15.2*BW 0.75 (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).



### 1 2 3 4 5
6 7 8 9 10 11 12 13 14 The physiologic parameter values obtained from the literature are shown in T able B-1 ( B rown et al., 1977 )

. tert -B utanol partition coefficients
, determined by the ratios of measured tissue:air and blood:air partition coefficients ( B orghoff et al., 1996 )
, also were obtained from literature

. T he parameters describing rate constants of metabolism and elimination of tert

- butanol a lso

wer e obtained from the literature ( Blancato et al., 2007 ) and were kept fixed because the y were optimized to t ert

- butanol blood concentrations measured after MTBE exposure
, which is also
metabolized to t ert

- butano l . The parameters describing tert

- butanol absorption and tert

- butanol sequestration in blood were estimated by optimizing the model to the t ime

- course data for blood tert

butanol f or rats exposed via i.v., inhalatio n, and oral routes ( Leavens and Borghoff, 2009 ; Poet et al.,
1997 ; A RCO, 1983 ) . The model parameters were estimated with the acslX optimization routine to minimize the log

- likelihood function of estimated and measured t ert

- butanol concentrations. The
Nedler

- M ead algorithm was used with heteroscedasticity and allowed to vary between 0 and 2. The predictions of the model with optimized parameters have a much

- i mproved fit to the tert

- butanol
blood concentrations after t ert

- butanol i.v. exposures , as shown in panel A of Figure B-4 . Additionally
,
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### Supplemental Information― tert

- But yl Alcohol

1 2 3 4 5
6 7 8 9 10 11

12

13 14 15 16 17 18 19 20 21 22 23 2 4 the model a dequately estimate d 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

te rt

- 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
6 0%) 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.

V max tert

- butanol IND = V max tert

- butanol *INDMAX(1

- exp(

- KIND*t))

V max tert

- butanol IND is the maximum metabolic rate after accounting for enzyme induction,
V max 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 metabo lism 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 ha d lower tert

- butanol blood concentrations after repeated exposures compared with female rats
, a nd 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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4 2 3

5 6

7 8 9

10 11 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 optimize d 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 

### Sup plemental Information― tert

- But yl Alcohol

**­**

Rate Constant Value Source or Reference Metabolism (VM TBA ; mg/kg

- hr) a Metabolism (KM TBA ; mg/L) Urinary elimination (K ELIM2 ; 1/hr)

TB A sequestration rate constant (K ON ; L/hr) TBA unsequestration rate constant (K OFF ; L/hr) Absorption from gastrointestinal tract (K AS2 ; 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 BW 0.7 (0.25 0.7 = 0.379), BW = body weight.

### Supplemental Information― tert

- But yl Alcohol

1
2 3 4 5 6 7 8 9 10 11 12 13 14

15
16 17 18 19 20

21
22 23 24 25 **Summary of the PBPK**  **M odel for**  tert **-B utanol** 
A PBPK model for tert

- butanol was developed by modifying previous models for MTBE and

tert

- b utanol ( Leavens and Borghoff, 2009 ; Blancato et al., 2007 )

. Published tert

- butanol sub

- models do not adequately represent the tert

- b utanol 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. T he alternative modification

— changing to diffusion

- limited distribution between blood and tissues

— also improved the model fit, but was considered less biologically plausible. P hysiological parameters and partition coefficients were obtained from published

measurements. T he rate constants for tert

- butanol metabolis m and elimination were from a published PBPK model of MTBE with a t ert

- butanol subcompartment ( Blancato et al., 2007 )

. Additional model parameters were estimated by calibrating to data sets for i.v., oral
, an d inhalation
exposures a nd repeated dosing studies for tert

- butanol

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

- c ourse datasets of tert

- butanol blood levels following inhalation or oral gavage
exposures.



### t ert **-B utanol**  **Model Application** The PBPK model as described above was applied to toxicity studies to predict t ert

- butanol blood concentrations (the preferred internal dose metric i n the absence of evidence linking any specific metabolite of t ert

- butanol to any to xic effect ). For simulation studies where t ert

- butanol was administered in d r inking water
, the consumption was modeled as episodic
, based on the pattern of drinking observed in rats ( S piteri, 19 82 ).



### 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 H ERO ( U.S. EPA, 201#, HEROID##).

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### 1 Supplemental Information― tert

- But yl Alcohol

2

3 4 5 6

7 8

9

10
11
12 13 14 15 16 Male rats were exposed to 239, 444 , or 1726 ppm and female rats were exposed to 2 56 , 444 , or 1914 ppm tert

- butanol for up to 8 consecutive days ( Borghoff et al., 2001 )

. tert -B utanol blood
concentr ations 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**  t ert

**- 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 s ome of the available rodent studies. These include liver and urinary bladder effects. A s previously mentioned in the Study Selection section of the
To x icological Review , all studies discussed employed inhalation, oral gavage, or drinking water
exposures f or ≥30 days. Studies are arranged in evidence tables by effect, species, duration, and

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### 5

10

15

20

25

30

35

1 2 3 4

6 7 8 9

11 12 13

14

16
17 18 19

21 22 23 24

26 27 28 29

31 32

33
34

36 Supplemental Information― tert

- But yl Alcohol

design

. The design, conduct, and reporting of each study was rev iewed, and each study was considered adequate to provide information pertinent to this assessment.
C e ntral nervous system effects similar to those ethanol causes, in terms of animals appearing intoxicated and having withdrawal symptoms after cessation of o ral or inhalation
exposure , w ere observed with tert

- butanol . Severity of central nervous system symptoms such as withdrawal increased with dose and duration of exposure. S t udy quality and utility concerns (e.g., inappropriate e xposure durations, lack of d ata reporting, small number of animals per treatment group) associated with these studies ( Gr an t and Samson, 1981 ; Snell, 1980 ; Thurman et al.,
1980 ; M cComb and Goldstein, 1979a
, b ; Wood and Laverty, 1979 ) , however, preclude an understanding of potential neurotoxicity following tert

- butanol exposure, and therefore, central nervous system st udies are not discussed further.
E x posure
- response array s of these effects on liver and urinary bladder are provided in Figure B -6 and Figure B-7 for oral and inhalation studies, respectively.



### Ki dney effects Absolute and relative kidney weight numerical data are presented in Table B-3.



### Li ver effects Liver weight and body weight were demonstrated to be proportional and liver weight normalized to body weight w as concluded optimal for data analysis ( Bailey et al., 2004 ) ; thus, only relative liver weight is presented and considered in the determination of hazard. A lthough some rodent studies observed liver effects (organ weight changes and histopathologic lesions), the effects were not consistent across the database. I n creases in relative liver weight with tert

- butanol
exposure w ere observed , but t he results pertaining to histopatholog ic changes were inconsistent ( T able B-4 ). The NTP (19 95) oral subchronic and chronic studies did not observe treatment

- related effects on liver histopathology in either sex of F344 rats. I n a 10

- week study in Wistar rats, several liver lesions (including necrosis) and increased liver glycogen were observe d in male rats (no females were included in the study) with the only dose used ( A charya et al., 1997 ; Acharya et al., 1995 )

. The study provide d no incidence or severity data. The dose used in this rat study was in the range of the lower doses used in the N TP (1995) subchronic rat study. An increased incidence of fatty liver was observed in the male mice of the highest dose group in the 2

- y ear mouse bioassay, but no hist opathological changes were seen in the subchronic mouse study ( NTP, 1995 ). No

treatment

- r elated effects in liver histopathology were observed in rats or mice of the NTP (1997) subchronic inhalation study.



### U rinary bladder effects

S ubchronic 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 m ale mice after 13 weeks of

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### Supplemental Information― tert

- But yl Alcohol

1 2 3 4 5 6 7

8
9

10
11 12 13 14 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 no t observed in the 2- year study. M ale mice exposed at the hi gh dose ( 2,070 mg/kg -day) for 2 years exhibited minimal transitional epithelial hyperplasia of the urinary bladder. N either female rats nor female mice showed increased incidences of this lesion. Both sexes of mice demonstrated incidence of minimal to mild i nflammation 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 la ck of consistency and lack of progression, the available evidence does not support liver and urinary bladder effects, respectively, as potential h uman hazards of tert

- butanol
exposure.

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### Supplemental Information― tert

- But yl Alcohol

1 2 **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 3,610 a mg/kg -d F: 0, 290, 590, 850, 1,560, 230 +12* +19* 290 +19* +17* 3,620 a 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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- But yl Alcohol

Reference and study design Results NTP (1995) Males Females B6C3F 1 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 8,210 a 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 420 a mg/kg -d 90 +4 +8 180 +8* +14*
F: 0, 180, 330, or 650 a 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.

N TP (1997) Males Females F344/N rat; 10/sex/treatment Inhalation analytical concentration: 0, 134, 272, 542, Concentration (mg/m 3 ) 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/m 3 ) (dynamic whole

- body chamber) 6 hr/d, 5 d/wk 13 weeks Generation method (Sonimist Ultrasonic spray nozzle nebulizer), analytical
concentr ation 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*

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### Supplemental Information― tert

- But yl Alcohol

Reference and study design Results NTP (1997) B6C3F 1 mouse; 10/sex/treatment Inhalation analytical concentration: 0, 134, 272, 542, 1,080, or 2,101 ppm (0, 406, 824, 1,643, 3,273 or 6,368 mg/m 3 ) (dynamic whole

- body chamber) 6 hr/d, 5 d/wk 13 weeks Generation method (Sonimist Ultrasonic spray nozzle nebulizer), analytical concentration and method were reported

Right kidney weights measured Concentration (mg/m 3 )

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*



### 1 2 3 4 5 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 – co ntrol value) ÷ control value × 100. Conversions from drinking water concentrations to mg/kg

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

.

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### Supplemental Information― tert

- But yl Alcohol

1 2 **Table B-4** 

**. Changes in l**  **iver weight in**  **animals following exposure to**  tert

**- butanol** 

Reference and s tudy design Results
Acharya et al. (1 995) Wistar rat; 5

– 6 males/treatment Drinking water (0 or 0.5%), 0 or 575 mg/kg

d 10 weeks No significant treatment

- related effects (results were only pro vided 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

m ating 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, 3,610 a mg/kg -d F: 0, 290, 590, 850, 1,560, 3,620 a 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) B6C3F 1 mouse; 10/sex/treatment Drinking water ( 0, 2.5, 5, 10, 20, or 40 mg/mL) M: 0, 350, 640, 1,590, 3,940, 8,210 a mg/kg -d F: 0, 500, 820, 1,660, 6,430, 11,620 a 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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### 1 2 3 4 5 6 Supplemental Information― tert

- But yl Alcohol

Reference and s tudy 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 420 a mg/kg -d 0 0 0 0 0 0 F: 0, 180, 330, or 650 a 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 a nalytical concentration: 0, 134, 272, 542, 1,080, or 2,101 ppm (0, 406, 824, 1, 643, 3,273 or 6,368 mg/m 3 ) (dynamic whole body chamber) 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 U ltrasonic 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) B6C3F 1 mouse; 10/sex/treatment Inhalation a nalytical concentration: 0, 134, 272, 542, 1,080, or 2,101 ppm (0, 406, 824, 1,643, 3,273 or 6,368 mg/m 3 ) (dynamic

w hole 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*

a The high

- dose group had an increase in mortality. * Statistically significant p ≤ 0. 05 as determined by study authors. Conversions from drinking water concentrations to mg/kg

- d perf ormed by study authors. Conversion from ppm to mg/m 3 is 1 ppm = 3.031 mg/m 3

. Percentage change compared to control = (treated value – co ntrol value) ÷ control v alue × 100.

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- But yl Alcohol

1 2 **Table B-5** 

**. Changes in l**  **iver histopathology**  **in animals following exposure**  **to** tert

**- butanol** 

Reference and s tudy 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, 3,610 a mg/kg -d F: 0, 290, 590, 850, 1,560, 3,620 a mg/kg -d 13 weeks
NTP (1995) No treatment

- related effects observed. B6C3F 1 mouse; 10/sex/treatment Drinking water (0, 2.5, 5, 10, 20, 40 mg/mL) M: 0, 350, 640, 1,590, 3,940, 8,210 a mg/kg -d F: 0, 500, 820, 1,660, 6,430, 11,620 a 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 420 a mg/kg -d F: 0, 180, 330, or 650 a mg/kg -d 2 years NTP (1995) Males Females B6C3F 1 mouse; 60/sex/treatment Drinking water ( 0, 5, 10, 20 mg/mL) M: 0, 540, 1,040, or 2,070 a 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 a nalytical concentration: 0, 134, 272, 542, 1,080, or 2,101 ppm (0, 406, 824, 1, 643, 3,273 or 6,368 mg/m 3 ) (dynamic whole body chamber) 6 hr/d, 5 d/wk 13 weeks Generation method (Sonimist Ultrasonic spray nozzle nebulizer), analytical concentration and method were reported No treatment

- related effects obser ved in the high dose group (only treatment group with liver endpoints evaluated)

.

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- But yl Alcohol

Reference and s tudy design Results
NTP (1997) B6C3F 1 mouse; 10/sex/treatment Inhalation a nalytical concentration: 0, 134, 272, 542, 1,080, or 2,101 ppm (0, 406, 824, 1,643, 3,273 or 6,368 mg/m 3 ) (dynamic whole body chamber) 6 hr/d, 5 d/wk 13 weeks Generation method (Sonimist Ultrasonic spray nozzle nebulizer), analytical concentration and method were reported Authors stated that there were no treatment

- related microscopic changes, but data were not provided.



### 1 2 3 4 a The high

- dose group had an increase in mortality. * Statistically significant p ≤ 0. 05 as determined by study authors. Conversions from drinking water concentrations to mg/kg

- d perf ormed by study authors. Conversion from ppm to mg/m 3 is 1 ppm = 3.031 mg/m 3

.

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1 2 **Table B-6** 

**. Changes in urinary bladder**  **histopathology in**  **animals following oral exposure**  **t o**  tert

**- butanol** 

Reference and s tudy 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, 3,610 a mg/kg -d F: 0, 290, 590, 850, 1,560, 3,620 a 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 n ot evaluated 290

490 n ot 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): B6C3F 1 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 Inflam

8,210 a mg/kg -d (mg/kg

- d) hyperplasia mation (mg/kg

- d) hyperplasia mation F: 0, 500, 820, 1,660, 6,430, 11,620 a 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 0/1 0 0/1 0

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 eval uated at 15 months) Drinking water ( 0, 1.25, 2.5, 5, or 10 mg/mL) M: 0, 90, 200, 420 a mg/kg -d F: 0, 180, 330, 650 a mg/kg -d 2 years

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Reference and s tudy design Results
NTP (1995) B6C3F 1 mouse; 60/sex/treatment Drinking water ( 0, 5, 10, or 20 mg/mL) M: 0, 540, 1,040, 2,070 a 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 Inflam

mation 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

S everity: 1 = minimal, 2 = mild, 3 = moderate, 4 = marked Transitional epithelial hyperplasia 0/59 0/60 0/59 3/57 (1.0) Inflam

mation

0/59 0/60 0/59 4/57* (2.0)



### 1 2 3 a The high

- dose group had an increase in mortality. * Statistically significant p ≤ 0.05 as determined by study authors. Conversions from drinking water concentrations to mg/kg

- d per formed 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** te rt

**- butanol** 

**.**
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Source : (A) N TP (1997)
**Figure B-7**  **. Exposure**

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

**- butanol** 

**.**

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**Genotoxicity** The g enotoxic potential of t ert

- butanol has been studied using a variety of genotoxicity
assays , including bacterial reverse mutation assays, gene mutation assays, chromosomal ab errations, sister chromatid exchanges, micronucleus formation, and d eoxyribonucleic acid ( DNA ) strand breaks and addu cts. The available genotoxicity data for t ert

- butanol are discussed below
, and the data summ ary is provided in Table B-7.



### B.3.2.1. B acterial Systems
The m utagenic potential of t ert

- butanol has been tested by Zeiger et al. (1987) using different Salmonella typhimurium st rains both in the presence and absence of S9 metabolic
activation. The preincubation assay protocol was followed. Salmonella strains TA98, TA100, TA1535, TA1537, and TA 1538 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 ei th er the presence or absence of S9 metabolic activation. Conflicting results have been obtained wit h 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

- rep air proficient. In a study by Williams

- Hill et al. (1999)
, tert

- butanol i nduced 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 wh y the revertants decreased at higher concentrations. The result s of this study indicate d that test strain TA102 might be a more sensitive strain for monitoring t ert

- bu tanol levels ( Williams

- Hill et al., 1999 )

. I n another study by Mcgregor et al. (2005) , h o wever, experiments were conducted on TA102 in two different laboratories using similar protocols

. t ert

- B utanol was dissolved in d im ethyl sulfoxide ( DMSO ) or distilled water and tested in both t he presence and absence of S9 metabolic activation. No statistically significant increase in mutants was observed in either solvent medi um . In one experiment where t ert

- butanol was dissolved in wat er, a significant, dose

- related increase in the number of revertants occurred, reaching almost tw ice 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 ox idative damage. Mutagenicity of t ert

- butanol has been studied in other systems including Neurospora crassa and Saccharomyces cerevisiae

. Y east strain Neurospora crassa at the ad

- 3A locus (all ele 38701) was used to test the mutagenic activity of t ert

- bu tanol at a concentration of 1.75 mol/L for 30 minutes.

t ert

- B utanol 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 la boratory strains K5

- A5, MMY1, D517

- 4B, and DS8 ( Jimenez et al., 1988 ). This effe ct on mitochondrial DNA, also observed with ethanol and other solvents, was attributed by

t he study authors to the alteration in the lipid composition of mitochondrial membranes, and

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mitochondrial DNA’s close association could be affected by membrane compo sition ( Jimenez et al.,
1988 )

. 

### B **.3.2.2.** In Vitro Mammalian Studies
To understand the role of t ert

- butanol

- induced genotoxicity in mammalian systems, in vitro studies have been conducted in different test systems and assays. t ert -B utanol was tested to evaluate its ab ility to induce forward mutations at the thymidine kinase locus (tk) in the L5178Y tk+/

- m ouse lymphoma cells using forward mutation assay. Experiments were conducted in both the p resence and absence of S9 metabolic activation. The mutant frequency was c alculated using the ratio of mutant clones per plate/total clones per plate × 2 00. t ert -B utanol 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 concen tra t ion (5
, 000 μg/mL) ( McGregor et al.,
1988 )

. To further determine potential D NA or chromosomal damage induced by tert

- butanol in in
vitr o systems, NTP (1995) studied sister chromatid exchanges and chromosomal aberrations. Chinese h a mster o vary (CHO) cells were exposed to t ert

- butanol in both the presence and absence of S9 activation at c oncentrations of 160

– 5,000 μg/ mL for 26 hours. t ert -B utanol did not induce sister chromatid exchanges i n any concentration tested
, although in one experiment, percent relative change of s ister chromatid exchanges per chromosome scored slight ly increase d . The same authors also studied the effect of t ert

- butanol on chromosomal aberration formation. CHO cell s were exposed to four concentrations (160, 500, 1
, 6 00, or 5, 000 μg/m L ) of t ert

- butanol in both the presence and absence of S9. No significant increase in c hromosomal aberration was observed in any concentration tested. Of note is that
, due to severe toxic ity at the highest concentration (5
, 0 00 μg/m L), only 13 metaphase cells were scored instead of 100 in the chromosomal aberration
assay. Sgambato et al. (2009) ex amined the effects of t ert

- butanol on DNA damage using a normal diploid rat fibroblast cell line. C ells were treated with 0

- to 100 -mM t ert

- butanol for 48 hours to determine the half

- ma ximal inhibitory concentration (IC 50 ; 0.44 ± 0.2 mM ). The 48

- hour IC 50 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 a t time 0 and after 30 minutes, 4 hours, or 12 hours of exposure to the IC 50 concentration. t ert -B utanol inhibited cell division in a dose

- dependent

manner a s measured by the number of cells after 24 and 48 hours of exposure at IC 50 concentrations , and with concentrations at 1/10th the IC 50 . C ell death did no t increase , suggesting a reduction in cell number due to reduced replication rather than to cytotoxicity. t ert -B utanol caused an accumulation in the G 0 /G 1 phase of replication
, related to different effects on the expression of the c yclin D1
, p27Kip1 , and p53 genes

. An initial increase in DNA damage as measured by nuclear fragmentation w as observed at 30 minute s. The DNA damage declined drastically after 4 hours and

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1 2 3 4 5 6 7 8

9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 disappeared almost entirely after 12 hours of exposure to t ert

- 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 t e rt

- butan ol

. DNA damage caused by t er t

- butanol was determined by single

- cell gel electrophoresis (comet assay) in h uman promyelocytic leukemia (HL

- 60) cells

. The cells were exposed to
concentrations r anging from 1 to 30 mmol/L for 1 hour
, and 100 cells were evaluat ed for DNA fragmentation. A d os e
- dependent increase in DNA damage was observed between 1 and
30 mm ol/L. No cytotoxicity was observed at the concentrations tested ( T ang et al., 1997 ).



### B **.3.2.3.** In Vivo Mammalian Studies

Few in vivo studies are available to understand the role of t ert

- butanol on genotoxicity. The National Toxicology Program studied the effect of t er t

- butanol in a 13

- week toxicity study ( NTP, 1995 ) . P eripheral blood samples were obtained from male and female B6CF1 mice exposed to t ert

butanol in d rinking 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 w as d etermined. No increase in micronucleus formation in peripheral b lo od lymphocytes was observed either in male or female
B6C3F 1 mice exposed for 13 weeks to t ert

- butanol in drinking water at concentrations as high as 40,000 ppm (2,110 m g/kg

- day) ( NTP, 1995 ). Male Kumming mice ( 8 p er treatment) were administered 0, 0.099, 0.99, 10, 101, or
997 μ g/kg BW 14 C- t ert

- butanol in saline via gavage wi th specific activity ranging from 1.60 to 0.00978 mCi/mol ( Y uan et al., 2007 ). Animals were sacrificed 6 hours after exposure
, and liver, kidney, and lung were collected. Ti ssues were prepared for DNA isolation with samples from the same organs from every two mi ce combined. DNA adducts were measured using accelerated mass spectrometry. T he results of this study show ed a dose

- response increase in DNA adducts in all three organs measured
, a lthough the methodology used to detect DNA adducts is considered
sensitiv e but could be nonspecific

. The authors state d that t ert

- butanol was found, for the first time, to form DNA adducts in mouse liver, lung
, a nd kidney. Because this is a single and first

- time study, further validation of this study will provide certainty in understanding the mechanism of t e rt

buta nol

- induced DNA adducts.

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1 2 **Table B-7.**  **Summary of genotoxicity (both in vitro**  **and in vivo**  **) studie s of**  t ert

**** **butanol**

Test system Dose/Conc. Results a 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 μ g/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 ; n ote 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 μ g/m L - - 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 1 60, 500, 1 , 600, 2, 000, 3 , 000, 4 , 000, 5 , 000 μ g/m L - - This study was part of the NTP 1995 testing results Galloway, 1987 ; NTP (1995)
Chromosomal Aberrations, Chinese Hamster Ovary cells 1 60, 500, 1 , 600, 2, 000, 3 , 000, 4 , 000, 5 , 000 μ g/m L - - This study was part of the NTP 1995 testing results Galloway, 1987 NTP (1995) DNA damage (comet assay), Rat fibroblasts 0.44 mmo l/L (IC 50 ) + c N D Exposure duration – 30 min, 4 h, 12 h; t his 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. Results a 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 A nimal S tudies
Micronucleus formation, B6C3F1 mouse peripheral blood cells 3 , 000, 5 , 000, 10 , 000, 20 , 000, 40 , 000 ppm - 13

- week, subchronic, d rinking
water study NTP (1995)

DNA adducts, m ale Kunming mouse liver, kidney and lung cells 0.1

– 1 , 000 μ g/kg body weight + Gavage, 6

- h exposure, DNA adduct determined by accelerator mass spectrometry Yuan et al. (2007)



### 1 2 3
4
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 a + = positive; − = negative; ND = not determined

. b E ffect is predicted to be due to mitochondrial membrane composition

. c DNA damage was completely reversed with increased exposure time

. 

### Summary

t ert -B utanol 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 %

. t ert

- B utanol did not induce mutations in most bacterial strains ; however, when tested in TA102, a strain that is sensitive to damage at A

- T sit es inducible by oxidants, an
increase in mutants was o bserved at low concentrations, although conflicting results were reported in another study. Fu rthermore, the solv ent (e.g., distilled water or DMSO) used in the genotoxicity
assay could influence results. I n one experiment where tert

- butanol was dissolved in distilled water, a significant, dose

- relat ed increase in the number of mutants was observed, with the maximum value reaching almost tw ice the control value. DMSO is known to be a radical scavenger
, and its presence in high concentrations might mask a mutagenic response mo dulated by oxidative damage. Other species such as Neurospora c r assa did not produce reverse mutations due to exposure to t ert

- bu tanol

. tert

- Butanol was tested in several human and animal i n vi tro mammalian systems for genotoxicity ( gen e mutation, sister chromatid exchanges, chromosomal aberrations
, and DNA damage )

. No increase in gene mutations was o bserved in mouse lymphoma cells ( L5178Y TK +/

– ). These specific locus mutations in mammalian cells are used to demonstrate and qua ntify genetic damage, thereby confirming or extending the data obtained in the more widely used bacterial cell

tests. S iste r chromatid exchange s or chromosomal aberrations were not observed in CHO cells in response to te rt

- butanol treatment. DNA damage was detec ted using comet assay , h owever, in both rat fibroblasts and HL

- 60 leukemia cells, with either an increase in DNA fragmentation at the beginning of the exposure or dose

- dep endent increase in DNA damage observed

. An initial increase in DNA damage was observed at 30 min u tes that declined drastically following 4 hours of exposure and disappeared al most entirely after 12 hours of exposure to t ert

- butanol

. This reduction in the extent of DNA fragmentation after an init ial increase is likely the result of an effic ient DNA repair mechanism activated by cells following DNA damage induced by t ert

- bu tanol . A dose- dependent

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 increase in DNA damage was observed in human cells tested; however, because the exposure occurred for o nly 1 hour in this study, whether DNA -repair mechanisms would occur after a longer period of observation c annot be discerned

. Limited i n 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 l ung cells

. T he authors used accelerated mass spectrometry to detect DNA adducts
, but the identity of these adducts was not determined

. The method uses 14 C- labeled chemical for dosing
, isolated DNA is oxidized to carbon dioxide a nd reduced to filamentous gr aphite, and the ratios of 14 C/ 12 C are measured. The ratio then is c onverted to DNA adducts based on nucleotide content of the DNA

. Confirmation of t hese data will further the understanding of the mechanism of t ert

- butanol

- induced DNA adducts. N o increase in micronucleus formation was observed in mouse peripheral blood cells
in a 13

- w eek drinking water study conducted by the National Toxicology Program. Overall, a li mited database is available for understanding the role of t ert

- butanol

- induced genotoxicity f or 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 st r a ins, solvents, or control for volatility used in certain studies are variable and c ould influence results . Furthermore, in some studies, the sp ecificity of the methodology used has been challenged. Given the inconsistencies and limitations of the database in ter ms of the methodology used, number of studies in the overall database, coverage of studies across the genotoxicity battery, and the quality of the studies
, th e wei ght of evidence analysis is inconclusive. The available data do not inform a definiti ve conclusion on the genotoxicty of t ert

- butanol and thus the potential genotoxic effect s of t ert

- 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 T HE DERIVATION OF CAN CER 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

. T he 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
Do cument ( U .S. EPA, 2000 ). In some cases
, us ing alternative methods based on statistical judgment might b e appropriate ; exceptions are noted as necessary in the summary of the modeling results.



### Non **c ancer Endpoints** 
**C.1.1.1.** Data Sets
Data sets selected for dose -re sponse 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. T he following procedures were used, depending on whether data were dichotomous or continuous:

• F or dichotomous models
, the following para meter restrictions were applied : for log

- logistic model , restrict slope ≥1; for gamma and Weibull models, restrict power ≥1; for multistage models, restrict b eta values ≥0. Each model was tested for goodness

- of

- fit using a chi

square goodness

- of

- f

it test (χ 2 p
- value < 0.10 indicates lack of fit). Other factors also were used to assess model fi t, such as scaled residuals, visual fit, and adequacy of fit in the low

dose region and n ear the benchmark response ( BMR ).

• For c ontinuous models, the following para meter restrictions were applied : for polynomial models, restrict b eta values ≥0; for Hill, power, and exponential models, restrict power ≥1.
M odel fit was assessed by a series of tests. F or each model, first the homogeneity of the variances was tested using a likelihood ratio test (BMDS Test 2). I f Test 2 was not rejected (χ 2 p
- value ≥ 0.10), th e model was fit to the data assuming constant variance. If Test 2 was rejected (χ 2 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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1 2 3 4

5
6 7 8 9 10 11

12 13 the mean response were tested for adequacy of fit using a likelihood ratio test (BMDS Test 4, with χ 2 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

- d ose region and n ear 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 likelihoo d me thod) and the Akaike’s information criterion ( AIC ) value were used to select a best

- f it model from among the models exhibiting adequate fit. If the BMDL estimates were “sufficiently close,” that is, differed by no more than t hree

- fold, the model select ed 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** t er t

**- butanol** 

Endpoint/Study Species/ Sex Doses and effect d ata 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 N TP (1997) Rat (F344) / Male Concentration (mg/m 3 ) 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/m 3 ) 0 406 825 1, 643 3, 274 6, 369

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Endpoint/Study Species/ Sex Doses and effect d ata

N TP (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. M odeling Results
Below are tables summarizing the modeling results for the noncancer endpoints modeled.

**Table C-2** 

**. S**  **ummary of BMD modeling results for**  **k idney**  **t ransitional**  **e pithelial**  **h y**  **perplasia in m**  **ale F344 r**  **ats exposed to**  t ert

**- butanol**  **in drinking water for 2 years**  **( NTP, 1995**  **) ;**  **BMR = 10% extra risk** 

Model a Goodness of fit BMD 10 (mg/kg

- d) BMDL 10 (mg/kg

- d) Basis for model selection p - value AIC
Log

- logistic 0.976 248.0 30 16 Log

- logistic model selected as best

fitting 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.

Th i s document is a draft for review purposes only and does not constitute Agency policy. 

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BMDL BMD Log-Logistic

0 50 100 150 200 250 300 350 400 dose 

### 1

2 3 4 5 6
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Supplemental Information― tert

- But yl 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**  **( NT**  **P, 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 rat s_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

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### 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 Supplemental Information― tert

- B ut yl 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 Estimat es

95.0% Wald Confidence Interval Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit background 0.505366 * * * intercept

- 5.58826 * * *

slop e 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 40.38 2 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

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### Supplemental Information― tert

- But yl Alcohol

1 2 3 **Table C-3** 

**. Summary of BMD modeling results**  **for k**  **idney t**  **ransitional e**  **pithelial h**  **y perplasia in**  **f emale**  **F344 r ats**  **exposed to** t ert

**- butanol**  **in drinking water for 2 years**  **( NTP, 1995**  **) ;**  **BMR = 10% extra risk**

Model a Goodness of fit
BMD 10 (mg/kg

- d) BMDL 10 (mg/kg

- d) Basis for model selection p - value AIC

Gamma 0.83 91.41 409 334 Multistage 3 rd

- 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/m 3 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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BMD BMDL 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

- But yl Alcohol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 Input Data File: M: \ NCEA 

### t ert

- butanol \ BMD modeling \ BMDS Output \ 20 NTP 1995b_Kidney transitional epithelial hyperplasia, female rats_Multi3_10.(d) Gnuplot Plotting File: M: \ NCEA 

### t ert

- butanol \ BMD modeling \ BMDS Output \ 20 NTP 19 95b_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. L imit 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

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### 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Supplemental Information― tert

- B ut yl 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 confid ence interval for the BMD

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

### C-8 DRAFT

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### Supplemental Information― tert

- But yl Alcohol

1 2 3 **Table C-4**  **. Summary of BMD modeling results for absolute kidney weight in male F344 rats exposed to**  te rt

**- butanol in drinking water for 15 months**  **( NT**  **P, 1995**  **) ;**  **BMR = 10% rel. dev. from control mean**

Model a Goodness of fit BMD 10RD (mg/kg

- d) BMDL 10RD (mg/ kg

- d) Basis for model selection p - value AIC 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 error d 0 Exponential (M5) N/A e

− 85.880 error d 0

Hill 0.301

− 87.880 error d error d

Power f 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 diffe rences 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 avail able 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 QUOTEMean Response 2.1

2

1.9

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

BMD BMDL 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

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2 3 4 5

6 7 8

9 10 11 12

13 Supplemental Information― tert

- But yl 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** te rt

**- 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 f orm of the response function is: Y[dose] = beta_0 + beta_1*dose

. A consta nt 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

### 1

2

3

4 Supplemental Information― tert

- But yl 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. 

### C- 11 DRAFT

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### 1 2 3

4 Supplemental Information― tert

- But yl Alcohol

**Table C-5**  **. Summary of BMD m**  **odeling r**  **esults for a**  **bsolute kidney weight in female F344 rats exposed to**  te rt

**- butanol in drinking water for 15 months**  **( NT**  **P, 1995 )**  **; BMR = 10% rel. dev. from control mean**

Model a Goodness of fit BMD 10RD (mg/kg

- d) BMDL 10RD (mg/kg

- d) Basis for model selection p - value AIC 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/A c

− 145.65 207 117

Hill N/A c

− 145.65 202 119

Power d 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° mode l, 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 QUOTEThis 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

1.4

1.35

1.3

1.25

1.2

1.15

1.1

1.05

1

dose 11:32 05/26 2015Mean Response

BMD BMDL Exponential 4

0 100 200 300 400 500 600 

### Supplemental Information― tert

- But yl Alcohol

1

2 3 4 5

6 7 8

9 10 11 12

13 **Figure C-4**  **. Plot of mean response by dose ,**  **with fitted curve for Exponential (M4) model with constant variance for**  **a bsolute kidney weight in female F344**  **rats exposed to** tert

**- butanol in drinking water for 15 months**  **( NT**  **P, 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 functi o n 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 

### 14

### 1

2

3

4 Supplemental Information― tert

- But yl 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

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### 4

5 6 7 8 Supplemental Information― tert

- B ut yl Alcohol

1 2 3 **Table C-6.**  **Summary of BMD modeling results for**  **kidney inflammation in female rats**  **exposed to** t er t

**- butanol**  **in drinking water for 2 years (**  **NT P, 1995**  **) ;**  **BMR = 10% extra risk**

Model a Goodness of fit
BMD 10% (mg/kg

- d) BMDL 10% (mg/kg

- d) Basis for model selection p - value AIC

Gamma 0.084 169.9 231 135 LogProbit was s elected on the basis of the l owest 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/A b 169.5 229 186

a S elected 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

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0.4

0.3

0.2

0.1

0 BMDL BMD 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** t ert

**- butanol**  **in drinking water**  **for 2 years (**  **NTP, 1995 )**  **; BMR = 10% extra risk**  **; dose shown in mg/kg**  **-d**

### Supplemental Information― tert

- But yl Alcohol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 ==================================================================== Probit Model. (Version: 3.2; Date: 10/28/2009) Input Data File: M:/NCEA 

### t ert

- butanol /BMD modeling/BMDS Output/19 NTP 1995b_Kidney inflammation, female rats_LogProbit_10.(d) Gnuplot Plotting File: M:/NCEA 

### t ert

- 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 cu mulative 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

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

### C- 16 DRAFT

— DO NOT CITE OR QUOTEModel 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

------------------------------------------------------------------------ 

### 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

35 S upplemental Information― tert

- But yl 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

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### Supplemental Information― tert

- But yl Alcohol

1 2 3 **Table C-7** 

**. Summary of BMD modeling results for absolute kidney weight in**  **male F344 rats exposed to** t er t

**- butanol**  **via inhalation for 6 hr**  **/d, 5d/wk for 13**  **weeks (**  **NTP, 1997 )**  **; BMR = 10% relative deviation from the mean**

Model a Goodness of fit BM C 10RD (mg/m 3 ) BM CL 10RD (mg/m 3 ) Basis for model selection p - value AIC Exponential (M2) <0.0001

− 205.06 error b error b 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 error b 0 Exponential (M5) <0.0001

− 201.06 error b 0

Hill 0.763

− 226.82 1931 1705

Power c Linear 0.0607

− 220.97 5364 3800

Polynomial 5° d Polynomial 4° e Polynomial 3° 1.44E

− 04

− 207.06

− 9999 error f

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/m 3 were 0.395, 0.374,

− 0.75,

− 1.96e

− 006, 0.381, and

− 0.381, respectively.
b BMC o r 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 t his model

.

Hill Model with 0.95 Confidence Level



### 4

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1.35

1.3

1.25

1.2

1.15

1.1

dose 10:15 04/30 2014Mean Response

BMD BMDL 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**  t ert

**- 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/m**  **3**

**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**



### 13 Supplemental Information― tert

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1 2 3 4

5 6 7

8 9 10 11

12

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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### 1

2

3

4 Supplemental Information― tert

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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

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1 2 3 **Table C-8**  **. Summary of BMD modeling results for absolute**  **kidney weight in fe**  **m ale F344 rats exposed to**  t ert

**- butanol**  **via inhalation for 6 hr**  **/d, 5d/wk for 13 weeks**  **( NTP, 1997**  **) ; BMR = 10%**  **relative deviation from the mean**

Model a Goodness of fit BMC 10RD (mg/m 3 ) BMC L 10RD (mg/m 3 ) Basis for model selection p - value AIC Exponential (M2) Exponential (M3) b 0.0378

− 261.52 14500 7713 No model adequately fit the data.

Exponential (M4) 0.533

− 267.48 error c 0 Exponential (M5) 0.374

− 265.71 error c 0

Hill 0.227

− 265.57 error c error c

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 r ow 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 P olynomial 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 i n

this row reduced to the Linear model. Note: Graphs of the better fitting models are provided for illustration.

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0.9

0.85

0.8

0.75

0.7

dose Hill

0 1000 2000 3000 4000 5000 6000Mean Response

10:32 04/30 2014
**Figure C-7.**  **Plot of mean response by concentration , with fitted curve for**  **Hill model for absolute**  **k idney weight in**  **fe male F344 rats exposed to**  t ert

**- butanol**  **via inhalation for 6 hr/d, 5d/wk for 13 weeks (**  **N TP, 1997**  **) ; BMR = 10%**  **relative deviation from the mean ;**  **concentration shown in mg/m 3** 

Power Model with 0.95 Confidence Level

0.7 0.75

0.8 0.85

0.9Mean Response

BMD BMDL Power

0 2000 4000 6000 8000 10000 12000 14000 dose 10:32 04/30 2014 

### Supplemental Information― tert

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1

2 3 4 5

6

7 8 9 10 11 **Figure C-8**  **. Plot of mean response by concentration**  **, with fitted curve for Power model**  **f or absolute**  **kidney weight in fe**  **male F344 rats exposed to** t ert

  ** butanol** **v ia inhalation for 6 hr/d, 5d/wk for 13 weeks**  **( NTP, 1997**  **) ; BMR =**  **10% relative deviation from the mean ;**  **concentration shown in mg/m**  **3**

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

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1
2
3 4 5 6 7 8 9 10 11 12 13 14 15 16

17
18 19 20 21 22 23 24 25 26 27 28 29 **Cancer Endpoints**
**C.1.2.1**

**.** Data Sets

T he cancer data sets select ed 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

- do se male mice compared with the concurrent control , the Poly -3 procedure ( Bai ler and Portier, 1988 ) for adjusting tumor incidence rates for intercurrent mortality was used . The procedure is b ased 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 pr ocedure, for a study of T weeks ’ duration, an animal that is remov ed 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
, an d i s 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 d ose 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 procedur e, a ls o are provided in Table C-9.



### C.1.2.2. Model Fit

T he multistage model was fit to the cancer data sets. Model coefficients were restricted to be non

- negative (beta v alues ≥ 0) , to estimate a monotonically increasing function

. Each model w as fit to the data using the maximum likelihood method, and was tested for goodness

- of

- f

it using a chi

square goodness

- of

- fi t test (χ 2 p
- value < 0.05 1 indicates lack of fit). Other factors were used to assess model fit, such as scaled residuals, visual fit, and ad equacy of fit in the low dose region and

near the BMR.
F or 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

- f it

model from among the models exhibiting adequate fit. Fo r the NTP (1995) and Hard et al. (2011) data, models were run with all doses included, as well as with the high dose dropped. Dro pping 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

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1 2 **Table C-9.**  **Cancer endpoints selected for dose**

**- response modeling for**  t ert

**** **butanol**

Endpoint/Study Species / Sex Doses and Effect Data Thyroid

Thyroid follicular cell adenoma NTP (1995) B6C3F 1 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) B6C3F 1 mice / male Dose (mg/kg

- d) 0 54 0 1,04 0 2,07 0

Incidence / Total 1/ 60 0/ 59 4/ 59 2/ 60

Incidence / Poly

- 3 adjusted Total 1/ 50 0/50 4/ 51 2/ 35

Kidney a
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

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

BMR BMD POD= (mg/kg

- BMDL BMDL HED a Slope factor b 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 Kidney c
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 4 5 a HED PODs were calculated using BW 3/4 scaling ( U.S. EPA, 2011 ).
b Human equivalent slope factor = 0.1/BMDL 10HED

c Alternative endpoint if kidney tumors are acceptable for quantitation

.

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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**  t ert

**- butanol**  **in drinking water for 2 years**  **( NTP, 1995**  **) ; BMR = 10% extra risk**  

### 2 3 4

Model a Goodness of fit BMD 10% c (mg/kg

- d) BMDL 10% c (mg/kg

- d) Basis for model selection p - v alue AIC b

Three 0.75 113.665 2002 1437 Multistage 3° was s elected on the basis of the l owest AIC with all BMDL values for fitting models being sufficiently close (BMDLs differed by less than 3

- fold). Two 0.36 115.402 2186 1217

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

0.25

0.2

0.15

0.1

0.05

0Fraction Affected

BMD BMDL 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** t ert **butanol in drinking water for 2 years**  **( NTP, 1995**  **) ; BMR = 10% extra risk**  **; dose shown in mg/kg**  **-d** 

### 7

**­** 

### 8 9
10 11 12 13 14 15 16 17 18 ====================================================================

====================================================================

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

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— DO NOT CITE OR QUOTE Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010) Input Data File: M: \ NCE A 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

### Supplemental Information― tert

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 [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 Para meter 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 v alue 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

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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

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### Supplemental Information― tert

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1 2 3 **Table C-**  **12**

**. Summary of BMD model**  **ing results for thyroid follicular cell**  **adenomas or**  **carcinomas in male**  **B6C3F1 mice**  **exposed to** t ert

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

Model a Goodness of fit BMD 5% (mg/kg

- d) BMDL 5% c (mg/kg

- d) Basis for model selection p - v alue AIC b
One, Two, Three 0.202 61.6 1788 787 Multistage 1 ° was s elected . 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

.



### 4

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26Fraction Affected 0.2

0.15

0.1

0.05

0 Multistage Cancer Model, with BMR of 5% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDL

BMD BMDL 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**  t ert

**- bu**  **tanol in drinking water for 2 years**  **( NTP, 1995**  **) ; BMR = 5%**  **extra risk; dose shown in mg/kg -d** 

==================================================================== M ultistage 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 tum ors 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

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### Supplemental Information― tert

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 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 Para meter 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

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1 2 3 4 5 6 7 8 9 10 11 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

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1 2 **Table C-**  **13**

**. Summary of BMD modeling results for**  **thyroid follicular cell adenomas**  **or carcinomas in male**  **B6C3F1 mice**  **exposed to** t ert

**- butanol**  **in drinking water for 2 years**  **, high dose omitt ed**  **( NT**  **P, 199 5**  **) ; BMR = 5% extra**  **risk** 

### 3 4

Model a Goodness of fit BMD 5% (mg/kg

- d) BMDL 5% c (mg/kg

- d) Basis for model selection p - value AIC 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.



### 5

6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 Multistage Cancer Model, with BMR of 5% Extra Risk for the BMD and 0.95 Lower Confidence Limit for the BMDLFraction Affected 0.2

0.15

0.1

0.05

0

BMD BMDL 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 th**  **yroid follicular cell adenomas**  **or carcinomas in male**  **B6C3F1 mice**  **exposed to** t ert

**- bu**  **t anol**  **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. 

### C- 32 DRAFT

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### Supplemental Information― tert

- But yl Alcohol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 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 Tot al 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 Paramete r 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 Reduc ed model

- 21.9555 1 6.0651 2 0.04819

AIC: 44.8962

Goodness of Fit

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### Supplemental Information― tert

- But yl Alcohol

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 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

- But yl 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
Model a Goodness of fit BMD 10Pct (mg/kg

- d) BMDL 10Pct (mg/kg

d) Basis for model selection p value 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 error b

a Selected model in bold.
b BMD or BMDL computation failed for this model.



### 7 8

9 10 Multistage Cancer Model with 0.95 Confidence LevelFraction Affected 0.5

0.4

0.3

0.2

0.1

BMD BMDL 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

- But yl Alcohol

1 2 3 4 5 6 7 8 9 10 11 12 13 14

15 16

17 18 19 20

21 22 23 24 **Multistage Cancer Model.** (Version: 1.9; Date: 05/26/2010) The form of the probability function is: P[response] = ba c kground + (1

- background)*[1

- EXP(

­ beta1*dose^1

- b et a2*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 T aken together, (117.584, 543384000) is a 90% two

- s i ded 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

- But yl 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
Model a Goodness of fit BMD 10Pct (mg/kg

- d) BMDL 10Pct (mg/kg

- d) Basis for model selection p value Scaled residuals AIC

Two N/A b 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.



### 7 8

9 10 Multistage Cancer Model with 0.95 Confidence LevelFraction Affected 0.5

0.4

0.3

0.2

0.1

BMD BMDL 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

- But yl Alcohol

1 2 3 4 5 6 7 8 9 10 11 12 13 14

15 16

17 18 19 20

21 22 23 24 **Multistage Cancer Model.** (Version: 1.9; Date: 05/26/2010) The form of the probability function is: P[response] = background + (1

- ba c k ground)*[1

- EXP(

­ beta1*dose^1

- b et a2*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 T aken together, (41.5902, 203.311) is a 90% two

- si ded 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

- But yl 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
Model a Goodness of fit BMD 10Pct (mg/L) BMDL 10Pct (mg/L) Basis for model selection p value 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 

### 6

7 8

9 10Fraction Affected 0.5

0.4

0.3

0.2

0.1

BMD BMDL 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

- But yl Alcohol

1 2 3 4 5 6 7 8 9 10 11 12 13 14

15 16

17 18 19 20

21 22 23

24 **Multistage Cancer Model.** (Version: 1.9; Date: 05/26/2010) The form of the probability function is: P[response] = background + (1

- bac kground)*[1

- EXP(

­ beta1*dose^1

- bet a2*dose^2...)] The parameter betas are restricted to be positive
**Benchmark Dose Computatio n.**  B MR = 10% Extra risk BMD = 51.8357 BMDL at the 95% confidence level = 13.9404 BMDU at the 95% confidence level = error T aken together, (13.9404, error) is a 90% two

- si ded confidence interval for the BMD Multistage Cancer Slope Factor = error

**Parameter Est**  **i mates** 
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

- But yl 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 high**

  ** dose group** **( NTP, 1995**  **) ; BMR = 10% extra risk.**  

### 3 4

Model a Goodness of fit BMD 10Pct (mg/L) BMDL 10Pct (mg/L) Basis for model selection p value 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



### 6 7

8 9Fraction Affected 0.5

0.4

0.3

0.2

0.1

BMD BMDL 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 11 12 13

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

- But yl Alcohol

1 2 3 4 5 6 7 8 9 10 11 12 13 14

15 16

17 18 19 20

21 22 23 24 **Multistage Cancer Model.** (Version: 1.9; Date: 05/26/2010) The form of the probability function is: P[response] = background + (1

- ba c kg round)*[1

- EXP(

­ beta1*dose^1

- b et a2*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 T aken together, (2.53714, 12.8097) is a 90% two

- si ded 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

- But yl 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
Model a Goodness of fit BMD 10Pct (mg/hr) BMDL 10Pct (mg/hr) Basis for model selection p value 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



### 7 8

9 10 11 12 13 14 0.5

0.4

0.3

0.2

0.1

dose 11:22 04/30 2014Fraction Affected

BMD BMDL 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

- But yl Alcohol

1 2 3 4 5 6 7 8 9 10 11 12 13 14

15 16

17 18 19 20

21 22 23 24 **Multistage Cancer Model.** (Version: 1.9; Date: 05/26/2010) The form of the probability function is: P[response] = background + (1

- back ground)*[1

- EXP(

­ beta1*dose^1

- beta2 *dose^2...)] The parameter betas are restricted to be positive
**Benchmark Dose Computatio n.**  B M R = 10% Extra risk BMD = 2.28299 BMDL at the 95% confidence level = 0.95436 BMDU at the 95% confidence level = error Tak en together, (0.95436, error) is a 90% two

- sid ed confidence interval for the BMD Multistage Cancer Slope Factor = error

**Parameter Esti**  **m ates** 
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

- But yl 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
Model a Goodness of fit BMD 10Pct (mg/hr) BMDL 10Pct (mg/hr) Basis for model selection p value Scaled residuals AIC

Two N/A b

- 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 8

9 10 11 12 13 14Fraction Affected 0.5

0.4

0.3

0.2

0.1

BMD BMDL 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

- But yl Alcohol

1 2 3 4 5 6 7 8 9 10 11 12 13 14

15 16

17 18 19 20

21 22 23 **Mu ltistage Cancer Model.**  (Version: 1.9; Date: 05/26/2010) The form of the probability function is: P[response] = background + (1

- back ground)*[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 T ak en together, (0.364494, 1.77845) is a 90% two

- sid ed confidence interval for the BMD Multistage Cancer Slope Factor = 0.274353

**Pa rameter 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. 

### C- 46 DRAFT

— DO NOT CITE OR QUOTE

### Supplemental Information― tert

- But yl 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
Model a Goodness of fit BMD 10Pct (mg/kg

- d) BMDL 10Pct (mg/kg

- d) Basis for model selection p value 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 -**  **a nalyzed data**  **( Ha**  **rd et al., 2011**  **; NTP, 1995**  **) ; BMR = 10% extra risk.** 

Model a Goodness of fit BMD 10Pct (mg/kg

- d) BMDL 10Pct (mg/kg

- d) Basis for model selection p value 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. 

### C- 47 DRAFT

— DO NOT CITE OR QUOTE

### Supplemental Information― tert

- But yl Alcohol

Multistage Cancer Model with 0.95 Confidence Level



### 1 2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

23 24 0.5

0.4

0.3

0.2

0.1

0

dose 11:05 04/30 2014Fraction Affected

BMD BMDL Multistage Cancer Linear extrapolation

0 50 100 150 200

**Figure C-**  **18 . Plot of incidence**  **b y 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** 

**un its and excluding high** 

**- dose group; re** 

**- an**  **alyzed data**  **( Hard et al., 2011**  **; NTP,**  **1995 )**  **; BMR = 10% extra risk; dose shown in mg/kg**

**- d.** 
**Multistage Cancer Model.** (V er sion: 1.9; Date: 05/26/2010) The form of the probability function is: P[response] = background + (1

- back ground)*[1

- EXP(

­ beta1*dose^1

- bet a2 *dose^2...)] The parameter betas are restricted to be positive
**Benchmark Dose Computation.** BMR = 10% Extra risk BMD = 54.1642 B MDL at the 95% confidence level = 36.3321 BM DU at the 95% confidence level = 101.125 Taken together, (36.3321, 101.125) is a 90% two

- si d ed 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

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

### C- 48 DRAFT

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### Supplemental Information― tert

- But yl Alcohol

Fitted model

- 75.4201 2 0.315716 1 0.5742

Reduced model - 81.4909 1 12.4574 2 0.001972



### 1 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. 

### C- 49 DRAFT

— DO NOT CITE OR QUOTE

### Supplemental Information― tert

- But yl Alcohol

1 2 3 4 5 **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.**

Model a Goodness of fit BMD 10Pct (mg/L) BMDL 10Pct (mg/L) Basis for model selection p value 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 high**

  ** dose group; reanalyzed data** **( Hard**  **et al., 2011 ;**  **NTP, 1995 )**  **; BMR = 10% extra risk.** 

Model a Goodness of fit BMD 10Pct (mg/L) BMDL 10Pct (mg/L) Basis for model selection p value 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.

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

### C- 50 DRAFT

— DO NOT CITE OR QUOTE

### 1 2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

24 25Fraction Affected 

### Supplemental Information― tert

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Multistage Cancer Model with 0.95 Confidence Level

0.5

0.4

0.3

0.2

0.1

0

dose 11:30 04/30 2014 BMD BMDL 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** 

**- b**  **utanol in drinking water for 2 years modeled with PBPK (**  tert

**- butanol,**  **mg/L) dose units and excluding high**

**- do**  **se group; reanalyzed data (**  **Hard et al., 2011**  **; NTP, 1995**  **) ;**  **BM R = 1**  **0% extra risk ; dose shown in mg/L.** 

**Multistage Cancer Model.** ( V ersion: 1.9; Date: 05/26/2010) The form of the probability function is: P[response] = background + (1

- ba c kground)*[1

- EXP(

­ beta1*dose^1

- bet a2*dose^2...)] The parameter betas are restricted to b e p ositive
**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

- si ded confidence interval for the BMD Multistag e C an cer 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

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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** 

**c a**  **rcinoma 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.** 

Model a Goodness of fit BMD 10Pct (mg/hr) BMDL 10Pct (mg/hr) Basis for model selection p value 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**  **( Ha**  **rd et al., 2011**  **; NTP, 1995**  **) ; BMR = 10%**  **extra risk.**

Model a Goodness of fit BMD 10Pct (mg/hr) BMDL 10Pct (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.

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a Selected model in bold.

Multistage Cancer Model with 0.95 Confidence Level



### 2 3

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

25 26 0.5

0.4

0.3

0.2

0.1

0

dose 11:33 04/30 2014Fraction Affected

BMD BMDL 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**  **t o**  tert

**- butanol**  **in drinking water for 2 years modeled with PBPK (metabolized,**  **mg/hr) dose units and excluding high**

**- d**  **ose group; reanalyzed data (**  **Hard et al., 2011**  **; NT**  **P, 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

- ba ckground)*[1

- EXP(

­ beta1*dose^1

- b eta2*dose^2...)] The parameter betas are restricted to be positive
Benchmark Dose Computation. BMR = 10% Extra risk BMD = 0.474241 BMDL at the 95% co nf i dence level = 0.318504 BMDU at the 95% confidence level = 0.882859 Taken together, (0.318504, 0.882859) is a 90% two- si ded 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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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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**C.1.2.4.** Inhalation Unit Risk for Cancer

An inhalation unit risk was not derived because the relative contribution of the 

### α 2u 

### - 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.
H owever, if renal tumors are considered suitable for analysis then route

- to

- route extrapolation could be performed

.



### D ose 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

- r oute extrapolation of the oral BMDL to derive an inhalation POD was perform ed 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 t o the same internal dose in the rat was estimated using the PBPK model, resulting in a route

- to

- r oute extrapolated
BMCL.
A critical decision in the route

- to

- r oute extrapolation is the selection of the internal dose metric to use that established "equivalent” oral and inhalation exposures. For t ert

- butanol -induced kidney effects, the two options are the concentration of tert

- b utanol in blood and rate of tert

butanol metabolism. Note that using the kidney concentration of tert

- b utanol will lead to the sa me
route

- to

- r oute 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 t e rt

- 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 fr om the animal data. The approach takes into account the extra

- r espiratory nature of the toxicological responses and accommodates species differences by considering blood:air partition coefficients for t ert

- 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 o bs erved. 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 t ert

- butanol in humans ( N ihlén et al., 1995 ) . The calculation (H b/g ) A ÷ (H b/g ) H was used to calculate a blood:gas partition coefficient ratio to a p ply 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 BMCL HEC to be derived as follows:

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1 2 3

4 5 6 7 8 9 10 11

12
13 14 15 16 17 18 19 20 21 22 23 24 25

26

27 28 29 30 31 BMCL HEC = BMCL ADJ (mg/m 3 ) × (interspecies conversion)

= B MCL ADJ (mg/m 3 ) × (481 

### ÷ 462)

= B MCL ADJ (mg/m 3 ) × (1.04)

The U.S. EPA G u idelines 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

- r esponse curve. The linear approach is recommended if the MOA of carcinogenicity has not been established ( U .S. EPA, 2005 ) . I n 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

- d ose extrapolation approach was used to estimate human carcinogenic risk associated with t ert

- butanol exposure.



### Inha lation Unit Risk Derivation
The results from route

- to

- route extrapolation of the male rat renal tubule tumor data are summarized in T able 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/BMCL 10 ). This slope, a 95% upper confidence limit represents a plausible upper bound on the true risk. Using linear extrapolation from the BMCL 10 , a human equivalent inhalation unit risk was derived, as listed in T able C- 26

. Two inhalation unit risks were derived from the N TP (1995) bioassay: one based on the original reported incidences and one based on the H ard et al. (2011) reanalysis. The two estimates differ by less than 20%, but the H ard 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

- b utanol is **1 × 10**  **-3 per**  **mg/m 3**  , or **2 × 10**  **-3 per**  **μg/m 3**  , 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 Dose a (mg/L) POD= BMCL HEC b (mg/m 3 ) Unit Risk c (mg/m 3 ) -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

- but anol as continuous oral exposure at the BMDL.

c Human equivalent inhalation unit risk = 0.1/BMCL HEC
. This document is a draft for review purposes only and does not constitute Agency policy. 

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36 37 38 39

41 

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. Poet, TS; Valentine, JL; Borghoff, SJ. (1997). Pharmacokinetics of tertiary butyl alcohol in male and female Fischer 344 rats. Toxicol Lett 92: 179

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butanol on rat fibroblasts in vitro. Toxicol Ind Health 25: 141

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

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

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