### DRAFT - DO NOT CITE OR QUOTE www.epa.gov/iris



# TOXICOLOGICAL REVIEW

OF

n-BUTANOL

(CAS No. 71-36-3)



## In Support of Summary Information on the Integrated Risk Information System (IRIS)

June 2011

NOTICE

This document is an Interagency Science Consultation draft . This information is distributed solely for the purpose of pre-dissemination peer re view under applicable information quality guidelines. It has not been formally disseminated by EPA. It does not represent and should not be construed to represent any Agency determination or policy. It is being circulated for review of its technical accuracy and science policy implic ations.

U.S. Environmental Protection Agency

Washington, DC

### ii DRAFT - DO NOT CITE OR QUOTE DISCLAIMER

This document is a preliminary draft for review pur poses only. This information is distributed solely for the purpose of pre-dissemina tion peer review under applicable information quality guidelines. It has not been formally disse minated by EPA. It does not represent and should not be construed to represent any Agency det ermination or policy. Mention of trade names or commercial products does not constitute en dorsement or recommendation for use.

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CONTENTS - TOXICOLOGICAL REVIEW OF n-BUTANOL (CAS N o. 71-36-3)

LIST OF TABLES ....................................

................................................... ....................................v LIST OF FIGURES ...................................

................................................... ................................ vii LIST OF ABBREVIATIONS AND ACRONYMS ................

................................................... . viii FOREWORD ..........................................

................................................... ......................................x AUTHORS, CONTRIBUTORS, AND REVIEWERS ..............

................................................... ..1 2. CHEMICAL AND PHYSICAL INFORMATION .............

................................................... ....5 3. TOXICOKINETICS ................................

................................................... ................................7 3.1. ABSORPTION ...................................

................................................... ............................... 7 3.1.1. Oral Absorption ............................

................................................... ............................... 7 3.1.2. Respiratory Tract Absorption ...............

................................................... ....................... 7 3.1.3. Dermal Absorption ..........................

................................................... ............................ 8 3.2. DISTRIBUTION .................................

................................................... .............................. 9 3.3. METABOLISM ...................................

................................................... ............................ 10 3.4. ELIMINATION ..................................

................................................... ............................. 13 3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELS . ............................... 13 4. HAZARD IDENTIFICATION .........................

................................................... .....................19 4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL CONTROLS ..........................................

................................................... ......................... 19 4.1.1. Oral Exposure ..............................

................................................... .............................. 19 4.1.2. Inhalation Exposure ........................

................................................... ....................... 19 4.1.2.1. Occupational Studies .....................

................................................... ..................... 19 4.1.2.2. Controlled Acute Exposure Experiments.....

................................................... ....... 26 4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOA SSAYS IN ANIMALS—ORAL AND INHALATION ....................... ............................................... 29 4.2.1. Oral .......................................

................................................... ..................................... 29 4.2.2. Inhalation ................................

................................................... .................................. 31 4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND IN HALATION ...... 35 4.3.1. Oral Studies ...............................

................................................... ................................ 35 4.3.2. Inhalation Studies .........................

................................................... ............................. 40 4.4. OTHER DURATION- OR ENDPOINT-SPECIFIC STUDIES . ....................................... 44 4.4.1. Acute and Short-term ( ≤ 30 Days) Studies ................................. ................................. 44 4.4.2. Liver Toxicity ............................

................................................... ............................... 45 4.4.3. Immunotoxicity ............................

................................................... ............................ 45 4.4.4. Neurotoxicity .............................

................................................... ............................... 46 4.4.5. Pulmonary Toxicity ........................

................................................... .......................... 48 4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF ACTION ............................................

................................................... ............................. 49 4.5.1. Genotoxicity Studies ......................

................................................... .......................... 49 4.5.2. Mechanistic Studies Evaluating Carcinogenic Parameters ....................................... .. 51 4.5.3. Mechanistic Studies Evaluating Neurological Effects .......................................... ...... 51 4.5.4. Mechanistic Studies Evaluating Neurodevelop mental Effects ................................... 57 4.5.5. Mechanistic Studies Evaluating Liver Effect s ................................................. ........... 58 4.5.6. Other Mechanistic Studies .................

................................................... ...................... 60 4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS ........ ............................................. 61 4.6.1. Oral ......................................

................................................... ..................................... 61 4.6.2. Inhalation ................................

................................................... .................................. 65

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4.6.3. Mode of Action Information for Noncancer En dpoints ........................................... ... 69 4.7. EVALUATION OF CARCINOGENICITY ...............

................................................... ... 70 4.7.1. Summary of Overall Weight-of-Evidence .....

................................................... .......... 70 4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES ..... ............................................. 70 4.8.1. Possible Childhood Susceptibility .........

................................................... ................... 70 4.8.2. Possible Genetic and Gender Differences ...

................................................... ............. 71 5. DOSE-RESPONSE ASSESSMENTS .....................

................................................... ..............72 5.1. ORAL REFERENCE DOSE (RfD) ...................

................................................... ............ 72 5.1.1. Choice of Principal Study and Critical Effe ct—with Rationale and Justification ...... 72 5.1.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) ................................... 75 5.1.3. RfD Derivation—Including Application of Unc ertainty Factors (UFs) ..................... 77 5.1.5. Previous RfD Assessment ...................

................................................... ..................... 78 5.2. INHALATION REFERENCE CONCENTRATION (RfC) .... ......................................... 79 5.2.1. Choice of Principal Study and Critical Effe ct—with Rationale and Justification ...... 79 5.2.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) ................................... 83 5.2.3. RfC Derivation—Including Application of Unc ertainty Factors (UFs)...................... 84 5.2.5. Previous RfC Assessment ...................

................................................... ..................... 85 5.3. UNCERTAINTIES IN THE ORAL REFERENCE DOSE AND INHALATION REFERENCE CONCENTRATION ...........................

................................................... ... 86 5.4. CANCER ASSESSMENT ...........................

................................................... .................. 89 6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HA ZARD AND DOSE RESPONSE ..........................................

................................................... ..................................90 6.1. HUMAN HAZARD POTENTIAL ......................

................................................... .......... 90 6.2. DOSE RESPONSE ...............................

................................................... .......................... 91 6.2.1. Oral Noncancer ............................

................................................... ............................ 91 6.2.2. Inhalation Noncancer ......................

................................................... ......................... 92 7. REFERENCES ....................................

................................................... ..................................94 APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PU BLIC COMMENTS AND DISPOSITION ...................................

................................................... ....................... A -1 APPENDIX B. BENCHMARK DOSE CALCULATIONS .......... ............................................B-1 APPENDIX C. n-BUTANOL PBPK MODEL .................

................................................... ......C-1 C.1. Model Code ..................................

................................................... ................................ C-1 C.2. Summary of Model Evaluation .................

................................................... ................. C-15 C.3. Uncertainties in PBPK Modeling of HECs Relate d to Selection of Values for Metabolism Parameters ........................................

................................................... .......................... C-16

### v DRAFT - DO NOT CITE OR QUOTE LIST OF TABLES

Table 2-1. Physical and chemical properties of n-b utanol ............................................ ................. 5

Table 3-1. n-Butanol metabolism by hepatic or pulm onary ADH in male rats ........................... 12

Table 3-2. Physiological parameters for a PBPK mod el for the n-butyl series in the rat and human (adapted from Teeguarden et al., 2005). .....

................................................... ............ 16

Table 3-3. Metabolic parameters for a PBPK model f or the n-butyl series in the rat and human a



### .................................................. ................................................... ........................................... 17

Table 3-4. Equilibrium partition coefficients for a PBPK model for the n-butyl series in the rat and human a



### .................................................. ................................................... ........................ 18

Table 4-1. Occupational Studies with n-butanol expo sure .............................................. ............. 20

Table 4-2. Studies of human exposure to mixtures i ncluding n-butanol ................................ ..... 22

Table 4-3. Controlled Acute Exposure Studies ......

................................................... .................... 27

Table 4-4. Hematological effects in rats exposed t o n-butanol by inhalation for 3 months ........ 33

Table 4-5. Changes in neurobehavioral function as measured by the rotorod test in rats exposed to n-butanol by inhalation over 3 months...........

................................................... ................. 33

Table 4-6. Selected fetal developmental changes in offspring of rats exposed to n-butanol in drinking water for 8 weeks before mating and during gestation a 

### ........................................... 36

Table 4-7. Selected changes in rats exposed to n-b utanol in drinking water on GDs 0–20 ........ 38

Table 4-8. Teratogenic observations in rats expose d to n-butanol by gavage during GDs 1–15 40

Table 4-9. Selected changes in rats exposed to n-b utanol via inhalation on GDs 1–19 .............. 42

Table 4-10. Summary of Short-term Studies .........

................................................... .................... 44

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Table 4-11. Genotoxicity studies of n-butanol in v itro .............................................. .................. 50

Table 4-12. Study details for mechanistic data and other studies in support of the mode of action for neurological effects...........................

................................................... ............................. 53

Table 4-13. Summary of oral noncancer dose-respons e information for n-butanol .................... 64

Table 4-14. Summary of inhalation noncancer dose-r esponse information for n-butanol .......... 66

Table 5-1. Incidences of rat litters with dilation of the subarachnoid space or dilation of the lateral ventricle/third ventricle of the brain ....

................................................... ..................... 76

Table 5-2. Summary of BMD modeling of results base d on Sitarek et al. (1994) developmental toxicity data – Dilation in the brain and kidney ..

................................................... ................ 77

Table 5-4. PBPK model calculation of HECs of the N OAEL based on rotarod performance in male rats exposed to n-butanol for 3 months .......

................................................... ............... 84

Table B-1. Incidences of rat litters with visceral malformations .................................... ........... B-1

Table B-2. BMD modeling results for the incidence of litters with visceral malformations (overall) after maternal exposure to n-butanol in d rinking water. .................................... ... B-2

Table C-1. n-Butanol metabolism parameters .......

................................................... ............... C-16

Table C-2. Results of PBPK modeling of NOAEL conce ntration from subchronic rat study C-16

### vii DRAFT - DO NOT CITE OR QUOTE LIST OF FIGURES

Figure 3-1 Metabolism of n-Butanol. ..............

................................................... ......................... 10

Figure 3-2. Schematic of the PBPK model for the bu tyl series (including n-butanol) in rats and humans. ...........................................

................................................... .................................... 14

Figure 5-1. Exposure-response array for oral expos ure to n-butanol. ................................. ........ 74

Figure 5-2. Exposure response array for inhalation exposure to n-butanol. ........................... ..... 82

Figure B-1. Fit of log-probit model to data on inc idence of litters with visceral malformations (overall). ........................................

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

### viii DRAFT - DO NOT CITE OR QUOTE LIST OF ABBREVIATIONS AND ACRONYMS

ACGIH American Conference of Governmental Industrial Hygi enists ACh acetylcholine AChE acetylcholinesterase ACSL Advanced Continuous Simulation Language ADH alcohol dehydrogenase AIC Akaike’s Information Criterion ALP alkaline phosphatase ALT alanine aminotransferase AMPA α -amino-3-hydroxy-5-methyl-4-isoxazole propionic aci d AST aspartate aminotransferase ATP adenosine triphosphate ATPase adenosine triphosphatase AUC area under the curve BMD benchmark dose BMDL 95% lower bound on BMD BMDS Benchmark Dose Software BMR benchmark response BUN blood urea nitrogen CASRN Chemical Abstracts Service Registry Number CI confidence interval CNS central nervous system CO 2 

### carbon dioxide DF degrees of freedom ECF extracellular fluid ER- α estrogen receptor α FEL frank effect level FID flame ionization detection GABA γ -aminobutyric acid GC gas chromatography GD gestation day GGT gamma glutamyltransferase GLDH glutamate dehydrogenase Glu glutamate GPT glutamate pyruvate transaminase GSH reduced glutathione GSSG oxidized glutathione HEC human equivalent concentration HEK human embryonic kidney 5-HT 5-hydroxytryptamine i.p. intraperitoneal IRIS Integrated Risk Information System IUR Inventory Update Reporting i.v. intravenous LDH lactate dehydrogenase LH luteinizing hormone LOAEL lowest-observed-adverse-effect level

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MANOVA multiple analysis of variance MDA malondialdehyde MOE Ontario Ministry of the Environment 4-MP 4-methylpyrazole nACh nicotinic acetylcholine NADPH nicotinamide adenine dinucleotide phosphate NMDA N

-methyl-D-aspartate NOAEL no-observed-adverse-effect level NRC National Research Council PA phosphatidic acid PBPK physiologically based pharmacokinetic PBTK physiologically based toxicokinetic PKC protein kinase C PLD phospholipase D PMA phorbol 12-myristate 13-acetate PND postnatal day POD point of departure QSAR quantitative structure-activity relationship RBC red blood cell RfC reference concentration RfD reference dose RT-PCR real time polymerase chain reaction SD standard deviation SDH sorbitol dehydrogenase SE standard error SEM standard error of the mean TSCA Toxic Substance Control Act TSCATS Toxic Substance Control Act Test Submission UF uncertainty factor U.S. EPA U.S. Environmental Protection Agency WHO World Health Organization

### x DRAFT - DO NOT CITE OR QUOTE FOREWORD

The purpose of this Toxicological Review is to prov ide scientific support and rationale for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to n-butanol. It is not intended to be a comprehensive treatise o n the chemical or toxicological nature of
n-butanol. The intent of Section 6, Major Conclusions in the Characterization of Hazard and Dose Response, is to present the major conclusions reached in the derivation of the reference dose, reference concentration and cancer assessment, wher e applicable, and to characterize the overall confidence in the quantitative and qualitative aspe cts of hazard and dose response by addressing the quality of data and related uncertainties. The discussion is intended to convey the limitations of the assessment and to aid and guide the risk ass essor in the ensuing steps of the risk
assessment process. For other general information about this assessment or other questions relating to IRIS, the reader is referred to EPA’s IRIS Hotline at (20 2) 566-1676 (phone), (202) 566-1749 (fax), or hotline.iris@epa.gov (email address).

### 1 DRAFT - DO NOT CITE OR QUOTE AUTHORS, CONTRIBUTORS, AND REVIEWERS 1

2 3 

### CHEMICAL MANAGER/AUTHOR 4 5 

### Ambuja Bale 6 

### National Center for Environmental Assessment 7 

### U.S. Environmental Protection Agency 8 

### Washington, DC 9 10 

### AUTHORS 11 12 

### Alan Sasso 13 

### National Center for Environmental Assessment 14 

### U.S. Environmental Protection Agency 15 

### Washington, DC 16 17 

### James A. Weaver 18 

### National Center for Environmental Assessment 19 

### U.S. Environmental Protection Agency 20 

### Washington, DC 21 22 

### CONTRIBUTORS 23 24 

### Karen Hogan 25 

### National Center for Environmental Assessment 26 

### U.S. Environmental Protection Agency 27 

### Washington, DC 28 29 

### Chester Rodriguez 30 

### Office of Chemical Safety and Pollution Prevention 31 

### U.S. Environmental Protection Agency 32 

### Washington, DC 33 34 

### Paul Schlosser 35 

### National Center for Environmental Assessment 36 

### U.S. Environmental Protection Agency 37 

### Research Triangle Park, NC 38 39 

### Hui-Min Yang 40 

### National Center for Environmental Assessment 41 

### U.S. Environmental Protection Agency 42 

### Washington, DC 43 44 45 

### CONTRACTOR SUPPORT 46 47 

### Heather Carlson-Lynch 48 

### Michael Lumpkin 49

### 2 DRAFT - DO NOT CITE OR QUOTE

Gary Diamond 1 

### Daniel Plewak 2 

### Julie Stickney 3 

### Chemical, Biological and Environmental Center 4 

### SRC, Inc. 5 

### 7502 Round Pond Road 6 

### North Syracuse, NY 13212 7 8 9 

### INTERNAL EPA REVIEWERS 10 11 

### John Cowden 12 

### National Center for Environmental Assessment 13 

### U.S. Environmental Protection Agency 14 

### Research Triangle Park, NC 15 16 

### Ted Berner 17 

### National Center for Environmental Assessment 18 

### U.S. Environmental Protection Agency 19 

### Washington, DC 20 21 

### Philip J. Bushnell 22 

### National Health and Environmental Effects Research Laboratory 23 

### U.S. Environmental Protection Agency 24 

### Research Triangle Park, NC 25 26 

### Barbara Glenn 27 

### National Center for Environmental Assessment 28 

### U.S. Environmental Protection Agency 29 

### Washington, DC 30 31 

### Karl Jensen 32 

### National Health and Environmental Effects Research Laboratory 33 

### U.S. Environmental Protection Agency 34 

### Research Triangle Park, NC 35 36 

### Ginger Moser 37 

### National Health and Environmental Effects Research Laboratory 38 

### U.S. Environmental Protection Agency 39 

### Research Triangle Park, NC 40 41 42 43 44

### 3 DRAFT - DO NOT CITE OR QUOTE 1. INTRODUCTION 1 2 3 

### This document presents background information and j ustification for the Integrated Risk 4 

### Information System (IRIS) Summary of the hazard and dose-response assessment of n-butanol. 5 

### IRIS Summaries may include oral reference dose (RfD ) and inhalation reference concentration 6 

### (RfC) values for chronic and other exposure duratio ns, and a carcinogenicity assessment. 7 

### The RfD and RfC, if derived, provide quantitative i nformation for use in risk assessments 8 

### for health effects known or assumed to be produced through a nonlinear (presumed threshold) 9 

### mode of action (MOA). The RfD (expressed in units of mg/kg-day) is defined as an estimate 10 

### (with uncertainty spanning perhaps an order of magn itude) of a daily exposure to the human 11 

### population (including sensitive subgroups) that is likely to be without an appreciable risk of 12 

### deleterious effects during a lifetime. The inhalat ion RfC (expressed in units of mg/m 3 

### ) is 13 

### analogous to the oral RfD, but provides a continuou s inhalation exposure estimate. The 14


### inhalation RfC considers toxic effects for both the respiratory system (portal of entry) and for 15 

### effects peripheral to the respiratory system (extra respiratory or systemic effects). Reference 16 

### values are generally derived for chronic exposures (up to a lifetime), but may also be derived for 17 

### acute ( ≤ 24 hours), short-term (>24 hours up to 30 days), an d subchronic (>30 days up to 10% of 18 

### lifetime) exposure durations, all of which are deri ved based on an assumption of continuous 19 

### exposure throughout the duration specified. Unless specified otherwise, the RfD and RfC are 20 

### derived for chronic exposure duration. 21 

### The carcinogenicity assessment provides information on the carcinogenic hazard 22 

### potential of the substance in question and quantita tive estimates of risk from oral and inhalation 23 

### exposure may be derived. The information includes a weight-of-evidence judgment of the 24 

### likelihood that the agent is a human carcinogen and the conditions under which the carcinogenic 25 

### effects may be expressed. Quantitative risk estima tes may be derived from the application of a 26 

### low-dose extrapolation procedure. If derived, the oral slope factor is a plausible upper bound on 27 

### the estimate of risk per mg/kg-day of oral exposure . Similarly, a plausible inhalation unit risk is 28 

### an upper bound on the estimate of risk per μ g/m 3 

### air breathed. 29 

### Development of these hazard identification and dose -response assessments for n-butanol 30 

### has followed the general guidelines for risk assess ment as set forth by the National Research 31 

### Council (NRC, 1983). U.S. Environmental Protection Agency (U.S. EPA) Guidelines and Risk 32 

### Assessment Forum Technical Panel Reports that may h ave been used in the development of this 33 

### assessment include the following: Guidelines for the Health Risk Assessment of Chemic al 34


### Mixtures (U.S. EPA, 1986a), Guidelines for Mutagenicity Risk Assessment (U.S. EPA, 1986b), 35 

### Recommendations for and Documentation of Biological Values for Use in Risk Assessment (U.S. 36 

### EPA, 1988), Guidelines for Developmental Toxicity Risk Assessme nt (U.S. EPA, 1991), Interim 37 

### Policy for Particle Size and Limit Concentration Is sues in Inhalation Toxicity (U.S. EPA, 38 

### 1994a), Methods for Derivation of Inhalation Reference Conc entrations and Application of 39

### 4 DRAFT - DO NOT CITE OR QUOTE

Inhalation Dosimetry (U.S. EPA, 1994b), Use of the Benchmark Dose Approach in Health Risk 1


### Assessment

(U.S. EPA, 1995), Guidelines for Reproductive Toxicity Risk Assessmen t (U.S. EPA, 2 

### 1996), Guidelines for Neurotoxicity Risk Assessment (U.S. EPA, 1998), Science Policy Council 3


### Handbook : Risk Characterization (U.S. EPA, 2000a), Benchmark Dose Technical Guidance 4



### Document

(U.S. EPA, 2000b), Supplementary Guidance for Conducting Health Risk A ssessment 5 

### of Chemical Mixtures (U.S. EPA, 2000c), A Review of the Reference Dose and Reference 6


### Concentration Processes (U.S. EPA, 2002), Guidelines for Carcinogen Risk Assessment (U.S. 7 

### EPA, 2005a) , Supplemental Guidance for Assessing Susceptibilit y from Early-Life Exposure to 8 

### Carcinogens (U.S. EPA, 2005b), Science Policy Council Handbook: Peer Review (U.S. EPA, 9 

### 2006a), and A Framework for Assessing Health Risks of Environme ntal Exposures to Children 10 

### (U.S. EPA, 2006b). 11 

### The literature search strategy employed for this co mpound was based on the Chemical 12 

### Abstracts Service Registry Number (CASRN) and at le ast one common name. Any pertinent 13 

### scientific information submitted by the public to t he IRIS Submission Desk was also considered 14 

### in the development of this document. The relevant literature was reviewed through February 15


### 2011. 16

17

### 5 DRAFT - DO NOT CITE OR QUOTE 2. CHEMICAL AND PHYSICAL INFORMATION 1

2 

### n-Butanol is a flammable, colorless liquid with a v inous odor (O’Neil, 2006; Lewis, 3 

### 2001). Physical and chemical properties of n-butan ol are shown below in Table 2-1. n-Butanol 4 

### is a natural component of many foods and is formed during the fermentation of carbohydrates 5 

### (HSDB, 2009). Commercially, n-butanol is obtained via n-butyraldehyde formed from the Oxo 6 

### reaction of propylene (Billig, 2001). According to company reports provided to U.S. EPA under 7 

### the Inventory Update Reporting (IUR) program, the p roduction volume of n-butanol in the 8 

### United States during 2006 was >1 billion pounds (U. S. EPA, 2009). Commercial derivatives of 9 

### n-butanol include n-butyl acrylate, methacrylate, b utyl glycol ethers, 2-butoxyethanol, and butyl 10 

### acetate (Billig, 2001). n-Butanol is used as a dir ect solvent in paints, surface coatings, lacquers, 11 

### thinners, pharmaceutical formulations, waxes, and r esins (Billig, 2001). It is also used to make 12 

### plasticizer esters and mono-, di-, and tributylamin es (Billig, 2001). More recently, n-butanol has 13 

### been used as an oxygenate in fuels (see http://www.epa.gov/oust/oxygenat/index.htm ). 14

15 

### Table 2-1. Physical and chemical properties of n-b utanol

Chemical name n-Butanol Synonym(s) n-Butyl alcohol; butyl alcohol; 1-butyl alcohol; 1-butanol; 1- hydroxybutane; butyl hydroxide; butyric alcohol; pr opyl carbinol; hemostyp; methylolpropane; propylmethanol; n-butan- 1-ol ChemID Plus, 2009

Structure ChemID Plus, 2009

Chemical formula C 4 H 10 O ChemID Plus, 2009

CASRN 71-36-3 ChemID Plus, 2009 Molecular weight 74.121 Lide, 2008 Form Colorless liquid Lewis, 2001 Odor Vinous; similar to fusel oil O’Neil, 2006; Lewis, 2001 Melting point -88.6°C Lide, 2008 Boiling point 117.73°C Lide, 2008
Odor threshold Air Water 0.83 ppm 7.1 ppm Amoore and Hautala, 1983 Amoore and Hautala, 1983 Density 0.8095 g/cm 3 at 20°C Lide, 2008 Log octanol-water partition coefficient (log K ow ) 0.88 Hansch et al., 1995

Solubility: Water Organic solvents 6.32 × 10 4 mg/L at 25°C Soluble in benzene, miscible in ethanol and diethyl ether, very soluble in
acetone Tewari et al., 1982 Lide, 2008

Vapor pressure 6.70 mm Hg at 25°C Boublik et al., 1984 Henry’s law constant 8.81 × 10 - 6 atm-m 3 /mol Buttery et al., 1969 Flash point 2–3°C Billig, 2001 Auto ignition temperature 365°C Lewis, 2001 Explosive limits in air 1.4–11.2% Billig, 2001
Conversion factors ppm to mg/m 3 mg/m 3 to ppm 1 ppm = 3.08 mg/m 3 1 mg/m 3 = 0.32 ppm Calculated Calculated

### 6 DRAFT - DO NOT CITE OR QUOTE

1 

### n-Butanol is expected to have high mobility in soil and is not expected to bind to organic 2 

### matter and sediments in water (HSDB, 2009). n-Buta nol in soil and water is expected to be 3 

### removed through volatilization into the atmosphere and through biodegradation (HSDB, 2009). 4


### The environmental fate assessment indicates that n- butanol will be completely removed from soil 5 

### and water within days to weeks (HSDB, 2009). In th e atmosphere, n-butanol is expected to be 6 

### broken down by reaction with photochemically-produc ed hydroxyl radicals with a calculated 7 

### half-life of 46 hours (HSDB, 2009). 8

9

### 7 DRAFT - DO NOT CITE OR QUOTE 3. TOXICOKINETICS 1

2 3 

### Toxicokinetic studies of n-butanol in humans and ex perimental animals are described 4 

### below. n-Butanol is readily absorbed following ora l administration (80%; DiVincenzo and 5 

### Hamilton, 1979a), moderately absorbed following an inhalation exposure (50%; DiVincenzo and 6 

### Hamilton, 1979a; Astrand et al., 1976) and poorly a bsorbed following a dermal exposure (1%; 7 

### Boman and Maibach, 2000). Once absorbed, n-butanol is rapidly distributed to many tissues 8 

### including the liver, kidney, lung, brain and heart (Kaneko et al., 1994; DiVincenzo and 9 

### Hamilton, 1979a). n-Butanol is rapidly metabolized to butyric aldehyde by alcohol 10 

### dehydrogenase (ADH) and further to n-butyric acid b y aldehyde dehydrogenase (ECETOC, 11 

### 2003). n-Butanol is also oxidized by cytochrome P4 50 in rat liver (Albano et al., 1991). 12 

### Excretion of n-butanol is primarily as CO 2 

### in exhaled breath with minor amounts eliminated in 13 

### the urine (2.6–5.1%) and feces (0.6–1.1%) (DiVincen zo and Hamilton, 1979a). A 14 

### physiologically based toxicokinetic (PBTK) model ha s been developed (Teeguarden et al., 2005) 15 

### to describe blood kinetics for n-butyl acetate and its metabolites, n-butanol and n-butyric acid, in 16


### rats and humans. 17

18


### 3.1. ABSORPTION 19 

### 3.1.1. Oral Absorption 20 

### DiVincenzo and Hamilton (1979a) administered gavage doses of 4.5, 45, or 450 mg/kg 21 

### [ 14 

### C]-n-butanol in corn oil to male CD (Sprague-Dawley ) rats (2–4/group) that had fasted for 16 22 

### hours. After administration, expired air, urine, a nd feces were collected for 24 hours. 78.3– 23


### 83.3% of the amount administered was recovered as e xpired 14 

### CO 2 

### by 24 hours post exposure. A 24 

### smaller fraction, approximately 0.20–0.56% of the a dministered radioactivity, was recovered as 25 

### the unchanged compound in expired air at 24 hours p ost exposure. Urine and fecal elimination 26 

### accounted for approximately 2.6–5.0 and 0.6–1.1% of the administered radioactivity, 27 

### respectively, at 24 hours post exposure. Thus, up to 89% of the administered n-butanol was 28


### absorbed and measured in breath or urine. The exte nt of absorption was not dose-dependent. 29 

### DiVincenzo and Hamilton (1979a) also collected bloo d samples from three male 30 

### Sprague-Dawley rats at 0.5, 1, 2, 4, and 8 hours af ter gavage dosing with 450 mg/kg n-butanol. 31 

### A peak plasma concentration of 70.9 μg/mL was measu red 1 hour after gavage dosing. 32

33 

### 3.1.2. Respiratory Tract Absorption 34 

### Astrand et al. (1976) exposed 12 human volunteers ( all male) to 300 or 600 mg/m 3 35 

### (100 and 200 ppm) n-butanol in air for four 30-minu te periods of rest or exercise. The 36 

### concentration of n-butanol in inspiratory air was c ontinuously measured. Expiratory air was 37 

### collected and was analyzed for n-butanol. The amou nt of n-butanol absorbed was calculated as 38

### 8 DRAFT - DO NOT CITE OR QUOTE

the difference between the total quantities in insp iratory and expiratory air. Arterial and venous 1 

### blood was collected and analyzed for n-butanol conc entration. At rest, 46–48% of n-butanol was 2 

### taken up by inhalation, while 36–41% was absorbed d uring exercise (intensity estimated at 50– 3 

### 150 watts) and was independent of exposure concentr ation. Measured arterial blood 4 

### concentrations after 30 minutes of exposure ranged from 0.5 to 1.3 mg/kg 5 

### DiVincenzo and Hamilton (1979a) exposed four male b eagle dogs to 50 ppm 6 

### (150 mg/m 3 

### ) n-butanol vapor for 6 hours in a whole-body chamb er. Venous blood was collected 7 

### at regular intervals. Based on breath concentratio ns, the uptake curve indicates that 8 

### approximately 55% of the inhaled vapor was absorbed through the lungs. Expired air 9 

### concentrations of 22 ppm (67 mg/m 3 

### ) n-butanol were essentially unchanged during the 6 hour 10



### exposure. 11 

### Swiercz et al. (1995) exposed 32 male Wistar rats ( 4/exposure duration group) to 100 12 

### ppm (300 mg/m 3 

### ) n-butanol for up to 7 hours. Blood samples were obtained from separate 13 

### groups of four rats prior to exposure and at hourly intervals from 1 to 7 hours and analyzed for 14


### n-butanol. The concentration of n-butanol in blood increased rapidly with inhalation exposure, 15 

### reaching near steady-state concentrations of approx imately 2.2 μmol/dm 3 

### (20 mg/kg) within 1 16 

### hour of exposure. Mean blood n-butanol levels chan ged little throughout the remaining exposure 17 

### period, with blood from rats measured at 4 hours of exposure exhibiting a maximum n-butanol 18 

### concentration of 2.8 μmol/dm 3 

### (26 mg/kg). 19

20 

### 3.1.3. Dermal Absorption 21 

### Boman and Maibach (2000) conducted ex vivo studies using male and female human 22 

### thigh skin samples obtained at autopsy in a 3-mL fl ow-through penetration/evaporation cell. 23 

### Skin was exposed to neat n-butanol under either unv entilated or ventilated conditions with 24 

### various air flow rates. Steady state absorption wa s not attained during the 24-hour exposure time 25


### studied for either the unventilated or ventilated e xposures. In the unventilated experiment, the 26 

### absorption of neat butanol through the skin samples varied between 2.2 and 9.4%; the absorption 27 

### during ventilation (i.e., allowing for n-butanol ev aporation from the skin) resulted in ≤ 1% 28 

### absorption. 29 

### Scheuplein and Blank (1973) conducted ex vivo studi es using human abdominal skin 30 

### samples obtained at autopsy to determine permeabili ty rates for n-butanol in an aqueous solution. 31 

### Sheets of dermis or epidermis were fitted into a pe rmeability cell in which one side of the sample 32 

### was exposed to neat n-butanol or n-butanol in an a queous solution (2.54 cm 2 

### exposure area), 33 

### while the other was exposed to water, from which al iquots were drawn to measure the flux of 34 

### n-butanol across the skin. In aqueous solution, th e permeability rates of the epidermis and 35


### dermis were 2.5 × 10 3 

### and 30.0 × 10 3 

### cm/hour, respectively. For neat n-butanol, the pe rmeability 36


### rates were 0.06 × 10 3 

### and 1.0 × 10 3 

### cm/hour for the epidermis and dermis, respectively

. 37

### 9 DRAFT - DO NOT CITE OR QUOTE

Using skin preparations from nude mice and glass di ffusion cells, Behl et al. (1984) 1 

### measured permeability coefficients of 3.7 × 10 3 

### cm/hour for dorsal skin and 23.7 × 10 3 

### cm/hour 2 

### for abdominal skin. Under hydrated conditions usin g skin preparations from male Sprague3 

### Dawley rats, Behl et al. (1983) determined the n-bu tanol permeability coefficient to be increased 4 

### by approximately 20–25% through the first 5 hours o f hydration, and remained at this value 5 

### (6.4 × 10 3 

### cm/hour) through the end of the experiment at 80 h ours. 6 

### DelTerzo et al. (1986) estimated an in vitro permea bility rate for n-butanol in nude rat 7


### skin. Rat skin was sandwiched between 3 mL saline filled compartments. [ 14 

### C]-n-Butanol was 8 

### introduced into the donor side of the cell, and sam ples were removed from both sides of the cell 9 

### at predetermined intervals to measure the flux of n -butanol across the skin. From these 10 

### measurements, a permeability rate of 14.2 × 10 3 

### cm/hour was calculated. 11 

### DiVincenzo and Hamilton (1979a) exposed young male beagle dogs to [ 14 

### C]-n-butanol 12 

### using a sealed glass absorption cell secured on the shaved thorax for 60 minutes. Expired air was 13 

### collected continuously using an oral endotracheal t ube and urine was collected continuously by a 14 

### urethral catheter. An identical amount of [ 14 

### C]-n-butanol was administered intravenously to 15 

### other dogs. Radioactivity excreted in the urine an d exhaled breath of dermally and intravenously 16 

### treated dogs was compared to determine the rate of systemic uptake by dermal absorption. 17 

### Assuming that the metabolic fate and tissue disposi tion is identical after intravenous (i.v.) or skin 18 

### administration, the calculated absorption rate was 8.8 μg minute -1 

### cm -2



### . 19 

### Boman et al. (1995) conducted studies to evaluate t he percutaneous absorption of 20 

### n-butanol during intermittent exposure in guinea pi gs. Groups of 5–16 animals were exposed to 21 

### neat n-butanol either continuously for 4 hours or f or 1 minute at 30-minute intervals over 4 hours 22 

### for a total of eight intermittent exposures. At th e end of 4 hours, the continuously exposed 23


### animals exhibited maximum blood n-butanol levels th at were twofold higher (25.63 μM) than 24 

### the levels in intermittently exposed animals (12.43 μM). 25

26


### 3.2. DISTRIBUTION 27 

### In vitro determination of tissue:blood partition co efficients for n-butanol suggest that it 28 

### will distribute throughout the body, as calculated values were very close to 1, ranging from 29 

### 0.78 to 1.08 for muscle, brain, kidney, liver, and fat (Kaneko et al., 1994). DiVincenzo and 30 

### Hamilton (1979a) reported the distribution of n-but anol in male CD (Sprague-Dawley) rats 31 

### gavaged with a single dose of 450 mg/kg [ 14 

### C]-n-butanol in corn oil after fasting for 16 hours

. 32 

### Blood samples were collected from three rats at 0.5 , 1, 2, 4, and 8 hours after gavage dosing. 33 

### Groups of four animals were sacrificed at 4, 8, or 24 hours post exposure and liver, kidney, heart, 34 

### brain, lung, and adrenal glands were collected and analyzed for radioactivity. The systemic 35 

### distribution was rapid, as plasma concentration of n-butanol rose from 40 μg/mL at 30 minutes 36 

### post exposure to a peak at 1 hour of 70.9 μg/mL, fo llowed by a rapid decline to <10 μg/mL at 37 

### 2 hours. n-Butanol was not detected in the plasma 4 hours after gavage dosing. Peak levels of 38

### 10 DRAFT - DO NOT CITE OR QUOTE

[ 14 

### C]-radioactivity in rat tissues occurred from 4 to 8 hours post exposure. The highest 1 

### percentages of administered [ 14 

### C]-radioactivity (constituting parent compound and metabolites) 2 

### were recovered at 4 hours in the kidney (0.24%) and heart (0.05%), and 8 hours in the liver 3 

### (3.88%), blood (0.74%), lung (0.12%), fat (0.09%), brain (0.04%), and adrenal glands (0.009%). 4 

### Twenty-four hours after gavage doses of 4.5, 45, or 450 mg [ 14 

### C]-n-butanol/kg, approximately 5 

### 12.1–16.3% of the administered radioactivity remain ed in the carcass. Because radioactivity was 6 

### measured instead of specific compounds, these data do not inform as to the distribution of 7 

### n-butanol or specific metabolites. 8

9


### 3.3. METABOLISM 10 

### n-Butanol is primarily metabolized to butyric aldeh yde by alcohol dehydrogenase (ADH) 11 

### (and to a minor extent by cytochrome P450) and furt her to n-butyric acid by aldehyde 12 

### dehydrogenase (ECETOC, 2003). Further oxidation of n-butyric acid produces CO 2 

### . A minor 13 

### conjugation pathway exists resulting in n-butanol-O -glucuronide or n-butanol-O-sulfate, which 14 

### are excreted in the urine (Figure 3-1). In additio n to exogenous sources of n-butanol, this alcohol 15 

### is readily and rapidly metabolized from n-butyl ace tate as shown in Figure 3-1. 16

17

18 

### Figure 3-1 Metabolism of n-Butanol. 19 20

### 11 DRAFT - DO NOT CITE OR QUOTE

Deisinger and English (2001) administered i.v. dose s of 0.28 mmol/kg (21 mg/kg) of 1 

### n-butyl acetate, n-butanol, or 0.028 mmol/kg (2.1 m g/kg) n-butyric acid to groups of adult male 2 

### Sprague-Dawley rats. In addition, they administere d an i.v. infusion of approximately 0.28 3 

### mmol/kg (21 mg/kg) n-butyric acid over 3 minutes. Blood samples were collected and analyzed 4 

### for each of the butyl series compounds. Metabolism of n-butanol to n-butyric acid was very 5 

### rapid with the peaks for both compounds (in the cas e of n-butyl acetate injection) measured 6


### within 1 minute of each other. 7 

### Poet et al. (2003a, unpublished and as presented an d cited in Teeguarden et al., 2005) 8 

### exposed male Sprague-Dawley rats to n-butanol in cl osed chambers having initial concentrations 9 

### of approximately 2,000 ppm (6,000 mg/m 3 

### ). Blood was collected throughout the exposure and 10 

### analyzed for n-butanol and n-butyric acid. Blood n -butanol levels remained at peak values (0.1 11 

### mM) from about 0.2 to 0.6 hours, suggesting that n- butanol metabolism was saturated during this 12 

### period. n-Butyric acid levels peaked (0.009 mM) at about 0.6 hours and were typically an order 13 

### of magnitude lower than n-butanol levels throughout the exposure. 14 

### In gavage studies in CD (Sprague-Dawley) rats dosed with 450 mg [ 14 

### C]-n-butanol/kg 15 

### body weight, 83.3% of the administered radioactivit y was metabolized via oxidative pathways, 16 

### appearing in exhaled breath as carbon dioxide (CO 2 

### ) by 24 hours post exposure (DiVincenzo and 17 

### Hamilton, 1979). Phase II glucuronidation and sulf ation of n-butanol resulted in 4.4% of 18 

### administered dose being excreted in the urine. Of this fraction, about 75% was in the form of n19 

### butyl-o-sulfate (44%) or n-butyl-o-glucuronide (30% ). Less than 1% of was not metabolized and 20 

### was eliminated in the exhaled breath as unchanged c ompound. The study authors reported 21 

### similar metabolism and excretion patterns at lower doses (4.5 and 45 mg/kg body weight), but 22 

### did not provide any additional quantitative details

. 23 

### Metabolism of n-butanol by ADH from the perfusate o f isolated rat liver was evaluated 24 

### and an apparent Michaelis-Menten K m 

### (affinity constant) and V max 

### (maximum reaction velocity) 25 

### were reported to be 0.86 × 10 -3 

### M and 0.077 mmol/minute, respectively (Auty and Br anch, 26 

### 1976). When simultaneous exposures of n-butanol an d ethanol were given, the V max 

### for 27 

### n-butanol remain unchanged, but the K m 

### value increased to 1.2 × 10 -3 

### M, indicating that ethanol 28 

### competitively inhibits n-butanol metabolism. 29 

### The in vitro metabolism of n-butanol by rat hepatic microsomes has been studied by 30 

### Teschke et al. (1974) and Cederbaum et al. (1979, 1 978). Teschke et al. (1974) showed that 31 

### hepatic microsomes catalyzed the oxidation of n-but anol to its aldehyde in a reaction requiring 32 

### molecular oxygen and nicotinamide adenine dinucleot ide phosphate (NADPH). This reaction 33 

### was inhibited by carbon monoxide. Cederbaum et al. (1979) observed that hydrogen peroxide 34 

### (H 2 

### O 2 

### ) added to microsomal preparations stimulated the o xidation of n-butanol. Indirect 35 

### evidence was further provided by the observation th at azide, which prevents the decomposition 36


### of H 2 

### O 2 

### by catalase, stimulated the oxidation of n-butanol . Thiourea, a compound that reacts 37 

### with hydroxyl radicals, inhibited NADPH-dependent m icrosomal oxidation of n-butanol to a 38

### 12 DRAFT - DO NOT CITE OR QUOTE

similar extent, in both the absence and presence of the catalase inhibitor azide (Cederbaum et al. 1 

### 1979). 2 

### Carlson and Olson (1995) evaluated the metabolism o f n-butanol by rat hepatic and 3 

### pulmonary cytosolic preparations measured with rega rd to ADH activity as influenced by pH and 4 

### substrate concentration. The values are given in T able 3-1. At pH 10, the hepatic K m 

### value was 5 

### about fourfold lower than at pH 7.2. V max 

### and K m 

### values could not be calculated for ADH 6 

### activity in lung cytosol at pH 7.2 or 9.0 because a ctivity was negligible under these conditions. 7 

### These data suggest that oxidation of n-butanol in t he lung may not contribute significantly to 8 

### systemic metabolism. 9

10 

### Table 3-1. n-Butanol metabolism by hepatic or pulm onary ADH in male

rats

pH V max (nmol/min/mg protein) K m (mM) Hepatic
7.2 13.2 ± 1.5 0.159 ± 0.085
9.0 15.9 ± 6.9 0.136 ± 0.051
10.0 9.2 ± 1.1 0.045 ± 0.013 Pulmonary
7.2 ND ND
9.0 ND ND
10.0 20.2 ± 2.1 19.4 ± 7.4

ND = not determined due to low activity

Source: Carlson and Olson (1995).
11 

### Winer (1958) measured the initial reaction velociti es of 19 alcohols as substrates for 12 

### purified horse liver ADH. n-Butanol had the highes t initial velocity of 215 moles/L/minute/mol 13 

### ADH, which was 1.5-fold faster than the oxidation o f ethanol (135 moles/L/minute/mol ADH). 14 

### Albano et al. (1991) investigated the role of cytoc hrome P450 2E1 in oxidizing n-butanol 15


### in vitro via a free radical intermediate. These au thors incubated Sprague-Dawley rat liver 16 

### microsomes with n-butanol in the presence of NADPH, desferrioxamine methane-sulphonate, 17 

### and the spin trapping agent, 4-pyridyl-1-oxide-t-bu tyl nitrone, resulting in the detection of a free 18 

### radical intermediate tentatively identified as the 1-hydroxylbutyl radical. The formation of the 19 

### radical was strictly dependent of the presence of N ADPH and oxygen and not influenced by the 20


### addition of mannitol. In reconstituted membrane ve sicles, ethanol-inducible cytochrome P450 21 

### 2E1 was twice as active as phenobarbital-inducible P450 2B1 in producing n-butanol free 22 

### radicals. Indeed, n-butanol oxidation in rat liver microsomes was induced to a significantly 23 

### greater (~25%) extent by pretreatment of the animals with ethanol than with phenobarbital. This 24 

### suggests a greater affinity of n-butanol for cytoch rome P450 2E1 compared to cytochrome P450 25

### 13 DRAFT - DO NOT CITE OR QUOTE

2B1 (Gadberry and Carlson, 1994). This inducible m etabolism, however, was not observed in 1 

### rat lung microsomes, indicating that P450-mediated oxidation of n-butanol in this tissue may be 2 

### of minor consequence. Although cytochrome P450-med iated n-butanol oxidation has been 3 

### demonstrated in rat liver, it is not clear to what extent this oxidation reaction has on systemic 4 

### disposition of n-butanol. 5 

### Deters et al. (1998a) demonstrated that metabolism of n-butanol by ADH was not a 6 

### necessary step in the liver toxicity of this compou nd. 4-Methylpyrazole (4-MP) was used to 7 

### inhibit ADH in isolated perfused rat livers (from m ale Wistar rats) exposed to 130.2 mmol/L 8 

### n-butanol. n-Butanol caused leakage of glutamate p yruvate transaminase (GPT), lactate 9 

### dehydrogenase (LDH), and glutamate dehydrogenase (G LDH) into the perfusate and decreased 10 

### oxygen consumption, perfusion flow, and adenosine t riphosphate (ATP) concentration, both in 11 

### the presence and absence of 4-MP. This study indic ates that the hepatotoxicity of n-butanol is 12 

### not related to metabolism by ADH. This study is fu rther described in Section 4.5.5 (Mechanistic 13 

### Studies Evaluating Liver and Metabolic Effects). 14

15


### 3.4. ELIMINATION 16 

### Deisinger and English (2001) administered i.v. dose s of 0.28 mmol/kg (21 mg/kg) of n17 

### butyl acetate, n-butanol, or 0.028 mmol/kg (2.1 mg/ kg) n-butyric acid to groups of adult male 18 

### Sprague-Dawley rats. In addition, they administere d an i.v. infusion of approximately 0.28 19 

### mmol/kg (21 mg/kg) n-butyric acid over 3 minutes. Blood samples were collected and analyzed 20 

### for each of the butyl series compounds. n-Butanol exhibited biphasic elimination and 21 

### approached background levels by 18 minutes. 22 

### Astrand et al. (1976) exposed 12 volunteers (all ma le) to 300 or 600 mg/m 3 

### (100 or 23 

### 200 ppm) n-butanol in air for four 30-minute period s of rest or exercise. By 30 minutes after 24 

### cessation of exposure, the peak blood n-butanol lev el of 0.17 mM had diminished to near zero. 25 

### DiVincenzo and Hamilton (1979a) administered [ 14 

### C]-n-butanol in corn oil by gavage to 26 

### male CD (Sprague-Dawley) rats at doses of 4.5, 45, or 450 mg/kg body weight. n-Butanol from 27 

### a 450 mg/kg dose disappeared from the plasma rapidl y, decreasing from a peak concentration of 28 

### 70 μg/mL at 1 hour to <10 μg/mL at 2 hours and was below the limit of detection after 4 hours. 29 

### Within 24 hours post exposure, excretion of the adm inistered radioactivity was primarily as CO 2 30 

### in exhaled breath (78.3–83.3%). Of the remainder, 2.6–5.1% was eliminated in the urine, 0.6– 31 

### 1.1% in the feces, and <1% of the administered radi oactivity was exhaled as unchanged 32 

### compound. 33

34


### 3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELS 35 

### A physiologically based pharmacokinetic (PBPK) mode l for n-butyl acetate and its butyl 36 

### metabolites (including n-butanol) was developed by Barton et al. (2000) and then later refined by 37 

### Teeguarden et al. (2005). The model simulates the kinetics of n-butyl acetate, n-butanol, n- 38

### 14 DRAFT - DO NOT CITE OR QUOTE

butyraldehyde, and n-butyric acid in the rat or hum an following i.v or inhalation exposures to n1 

### butyl acetate or n-butanol. The PBPK model refined by Teeguarden et al. (2005) was updated 2 

### from ACSL to ACSLXTREME (Advanced Continuous Simula tion Language, Aegis, Inc., 3 

### Huntsville, AL) by U.S. EPA. Details of the model evaluation are presented in Appendix C. A 4 

### schematic of the Teeguarden et al. (2005) model is shown in Figure 3-2. 5

6

7
8 

### Source: Teeguarden et al. (2005). 9 10 

### Figure 3-2. Schematic of the PBPK model for the bu tyl series (including 11 

### n-butanol) in rats and humans. 12

### 15 DRAFT - DO NOT CITE OR QUOTE

1 

### The model is comprised of four similar submodels f or each component of the butyl 2


### series. The submodels are linked to one another vi a passage of the metabolite formed in the 3 

### compartments for liver, blood, or extra-hepatic tis sues to the respective compartments of the 4 

### submodel for that metabolite. For example, the amo unt of n-butanol formed from n-butyl acetate 5 

### in the liver is passed along to the liver compartme nt for the n-butanol submodel. The overall 6 

### structure of linked submodels for the butyl series is based on the structure developed by Barton 7 

### et al. (2000) for families of metabolically-related compounds. 8 

### Each submodel includes compartments for lung, arter ial and venous blood, liver, and the 9 

### remaining perfused tissues. The submodel for n-but yl acetate also includes a compartment for 10 

### fat. All compartments are well mixed and flow limi ted, which assumes that compounds are 11 

### homogeneously distributed in each compartment, and that the equilibration of chemical 12


### concentration between the blood and tissues occurs sufficiently fast so that changes in 13


### concentration at the blood:tissue interface are lim ited by the blood flow rather than rate of 14 

### diffusion into the tissues. The proportion of chem ical transferred from blood to tissue (and vice 15 

### versa) at any given moment was estimated by in vitr o derived equilibrium partition coefficients. 16 

### To determine the fraction of inhaled n-butyl acetat e or n-butanol that bypasses extraction by the 17 

### upper respiratory tract (because of the water-solub le nature of these compounds) and absorbed 18 

### into the blood, bioavailability constants (FA1 and FA2) were estimated from in vivo data 19 

### (discussed below) and multiplied by the inhaled con centration. Metabolism of each of the butyl 20 

### series compounds in the liver, as well as n-butyl a cetate and n-butyric acid in the other perfused 21 

### tissues compartment, is described as Michaelis-Ment en saturable processes defined by a 22 

### maximum reaction rate (V max 

### ), scaled to body weight to the 3⁄4 power (mmol/hour/ kg 0.75 

### ), and an 23 

### affinity constant (K m 

### , mM). First-order metabolism of n-butanol in the other perfused tissues is 24 

### scaled to body weight to the 1⁄4 power (K other 

### , kg 0.25 

### /hour) and was required to attain fit of the 25 

### model to experimental data. Elimination of n-butyl acetate, n-butanol, and n-butyric acid via the 26 

### urine (filtration) is represented as a first-order process, scaled to body weight to the 1⁄4 power 27 

### (kg 0.25 

### /hour). 28 

### Physiological model parameters such as cardiac and pulmonary ventilation flow rates, 29 

### relative blood perfusion of various tissue compartm ents, and tissue volumes (as fraction of body 30 

### mass) for rats and humans were obtained from the li terature (Teeguarden et al., 2005) and are 31 

### shown in Table 3-2. Urinary elimination rate const ants for rats were estimated based on 32 

### literature values. Urinary clearance of 2-butanol was reported to be up to 14% in different 33 

### species (Dietz et al., 1981). Thus, a value of 30 hour -1 

### , scaled to 1⁄4 power of body weight, was 34 

### used for the first-order n-butanol urinary eliminat ion rate constant. In the absence of urinary 35 

### excretion data for n-butyric acid, it was assumed t o be <3.5%, as observed for isobutyric acid in 36 

### the rat (DiVincenzo and Hamilton, 1979b). Human va lues were allometrically scaled from rat 37


### values. Rat values for the inhalation bioavailabil ity factors were fitted to in vivo time-course 38

### 16 DRAFT - DO NOT CITE OR QUOTE

data for the blood levels of n-butyl acetate (Poet et al., 2003a) and n-butanol (Poet et al., 2003b). 1 

### The human value for n-butyl acetate was assumed to be equal to the rat, while the value for n2 

### butanol (59% of alveolar ventilation) was reported by Astrand et al. (1976). 3

4 

### Table 3-2. Physiological parameters for a PBPK mod el for the n-butyl series

in the rat and human (adapted from Teeguarden et al ., 2005).

Parameter Abbreviation Value
Reference Rat Human Body weight (kg) BW 0.3 70.0 Study specific
Alveolar ventilation (L/hr/kg 0.75 ) QPC 14.0 8.678 Brown et al., 1997

Cardiac output (L/hr/kg 0.75 ) QCC 14.0 12.892 Brown et al., 1997 Blood flow (percentage of cardiac output) Fat QFatC 0.07 0.052 Brown et al., 1997 Liver QLivC 0.175 0.227 Brown et al., 1997 Tissue volume (percentage of body weight) Arterial blood VABC 0.022 0.0257 Benareggi and Rowl and, 1991 (as cited in Teeguarden et al., 2005) Fat VFatC 0.07 0.214 Brown et al., 1997 Liver VLivC 0.037 0.026 Brown et al., 1997 Lung VLungC 0.005 0.008 Brown et al., 1997 Venous blood VVBC 0.045 0.0514 Bernareggi and Rowla nd, 1991 (as cited in Teeguarden et al., 2005 Other tissues VOthC 0.751 0.5319 Brown et al., 1997 Urinary elimination rate (kg 0.25 /hr) n-Butyl acetate kFiltC1 0.074 Scaled a Estimated
n-Butanol kFiltC2 22.2 Scaled Estimated based on Di etz et al., 1981 n-Butyric acid kFiltC4 0.0074 Scaled Estimated base d on DiVincenzo and Hamilton, 1979b Inhalation absorption (percentage of inhaled concen tration) n-Butyl acetate FA1 1.0 1.0 Rat, fitted; human = ra t n-Butanol FA2 0.5 0.59 Rat, fitted; human = rat (As trand et al., 1976) n-Butyraldehyde FA3 – – – n-Butyric acid FA4 – – –

a Allometrically-scaled rat value used for human. Source: Teeguarden et al. (2005). 5

### 17 DRAFT - DO NOT CITE OR QUOTE

Metabolic parameter values for the model are given in Table 3-3. Metabolic constants (V max 

### and 1

K other ) were estimated by visually fitting the model outp ut to i.v. blood time-course data for each 2 compound (Deisinger and English, 2001). Fitting of V max for n-butyl acetate and its metabolites was 3 preceded by first estimating V max for the furthest downstream metabolite (n-butyric acid), and proceeding 4 upstream to estimates V max values for n-butyraldehyde, n-butanol, and n-butyl acetate, in that order. n5 Butyraldehyde is not detected in blood and thus, me tabolic constants for this compound were 6 approximated by n-butyric acid concentration in blo od following n-butanol or n-butyl acetate exposure. 7 The affinity constants (K m ) for n-butanol, n-butyraldehyde, and n-butyric aci d were previously selected 8 by Barton et al. (2000). The in vitro affinity con stant for metabolism of ethyl acetate in rats was u sed as a 9 surrogate for n-butyl acetate in blood, liver, and other tissues. Metabolic constants for n-butanol 10 metabolism were re-optimized to data and updated va lues are given in Table 3-3. 11

12 

### Table 3-3. Metabolic parameters for a PBPK model f or the n-butyl series in the rat and human a

Parameter Abbreviation Value Notes n-Butyl acetate
Maximum metabolic rate in blood VMBloodC1 600.0 Fit ted Affinity constant in blood KMBlood1 100.0 K m for ethyl acetate in rat
Maximum metabolic rate in liver VMLivC1 38.7 Fitted Affinity constant in liver KMLiv1 1.0 K m for ethyl acetate in rat Maximum metabolic rate in other tissues VMOthC1 6,0 00.0 Fitted Affinity constant in other tissues KMOth1 100.0 K m for ethyl acetate in rat

n-Butanol b

Maximum metabolic rate in liver VMLivC2 2.17/0.62 F itted (rat/human, respectively) Affinity constant in liver KMLiv2 0.16 Fitted
First-order metabolism in other tissues KOthC2 4.0/ 20.1 Fitted (rat/human, respectively) n-Butyraldehde
Maximum metabolic rate in liver VMLivC3 17.78 Fitte d Affinity constant in liver KMLiv3 0.1 Estimated (Ba rton et al., 2000) n-Butyric acid
Maximum metabolic rate in liver VMLivC4 1.4 Fitted Affinity constant in liver KMLiv4 0.1 Estimated (Ba rton et al., 2000)
Maximum metabolic rate in other tissues VMOthC4 3.0 Fitted Affinity constant in other tissues KMOth4 0.1 Estim ated (Barton et al., 2000)

a Rat values were derived as described; human values were allometrically scaled from rat values as 3⁄4 or 1⁄4 power of body weight. Units: V max (mmol/hr/kg 0.75 ); K m (mM); K other

(kg 0.25 /hr).

b Values for n-Butanol re-optimized. Details are pre sented in Appendix C.
Source: Teeguarden et al. (2005). 13 

### Blood:air and tissue:blood equilibrium partition co efficients (Table 3-4) for n-butyl 14 

### acetate and n-butanol were calculated by Kaneko et al. (1994); values for n-butyraldehyde and 15 

### n-butyric acid were assumed to be identical to thos e for n-butanol. The values for lung and other 16

### 18 DRAFT - DO NOT CITE OR QUOTE

perfused tissues:blood were based on the Kaneko et al. (1994) muscle:blood value. Human 1 

### values were assumed to be equivalent to rat values. 2

3 

### Table 3-4. Equilibrium partition coefficients for a PBPK model for the

n-butyl series in the rat and human a

Parameter Abbreviation n-Butyl Acetate n-Butanol n-Butyraldehyde n-Butyric acid Blood:air PB 89.4 1,160.0 1,160.0 1,160.0
Fat:blood PFat 17.0 – – –
Liver:blood PLiv 3.14 1.08 1.08 1.08 Lung:blood PLung 1.76 0.78 0.78 0.78 Other perfused tissues:blood POth 1.76 0.78 0.78 0. 78

a Calculated from Kaneko et al. (1994).

Source: Teeguarden et al. (2005).
4 

### The model was evaluated against experimental data f or i.v. exposures in the rat 5 

### (Deisinger and English, 2001), closed-chamber inhal ation exposures in the rat (Poet et al., 6 

### 2003a), and inhalation exposure in human volunteers (Astrand at al., 1976). Low residual errors 7 

### between model and data were observed (particularly for rat i.v. data of n-butanol), suggesting 8 

### high confidence in the estimation/choice of metabol ic and urinary elimination parameter values. 9 

### For the human model, the simulated blood levels wer e within two standard errors (SE) of the 10 

### observations and were always less than twofold high er. The profile of the simulated elimination 11


### of n-butanol from the blood was similar to observat ions. Further discussion regarding the model 12 

### fit and parameter optimization can be found in Appe ndix C. 13 

### Overall, the human and rat models in Teeguarden et al (2005) were developed for 14 

### inhalation exposures. The internal dose metric ava ilable for extrapolation between species is 15 

### limited to n-butanol levels in blood. Only n-butan ol blood concentration data are available for 16 

### both rats and humans. The human model performance has not been verified for other 17 

### metabolites. Further, the liver is the only define d organ system (other than blood) for which the 18 

### model may provide quantitative internal dosimetry. The model cannot be used to explicitly 19 

### extrapolate internal doses for other target organs (i.e., central nervous system [CNS], 20 

### reproductive organs, and upper respiratory system). However, for species differences in toxicity 21 

### due primarily to n-butanol metabolic differences, r esulting in differences in systemic levels of 22 

### n-butanol or metabolites, the model is useful to qu antitatively reduce uncertainty in the 23 

### determining human equivalent concentrations (HECs) of critical rat exposures. 24

25

### 19 DRAFT - DO NOT CITE OR QUOTE 4. HAZARD IDENTIFICATION 1

2 3 

### 4.1. STUDIES IN HUMANS—EPIDEMIOLOGY, CASE REPORTS, CLINICAL 4


### CONTROLS 5

6 

### 4.1.1. Oral Exposure 7 

### Bunc et al. (2006) described the case of a 47-year- old man who apparently ingested 8 

### n-butanol in a suicide attempt. Initial symptoms o f sleepiness, headache, and abdominal pain 9 

### later progressed to vomiting and coma. The patient was transported to a hospital comatose, 10 

### where his presenting symptoms included tachycardia, hypotension, shallow breathing, hypotonic 11 

### muscles, absent myotatic reflexes, and aromatic odo r. Echocardiogram and chest x-ray results 12 

### were normal. A brain CT scan performed when the pa tient arrived at the hospital showed a 13 

### distorted and narrower fourth ventricle and a hypod ense area; an additional scan performed 10 14 

### hours later showed no changes. The patient regaine d consciousness approximately 16 hours after 15 

### admission. Initial (days 1–2) blood laboratory tes ts conducted on the patient showed elevated 16 

### erythrocytes and leukocytes, elevated creatinine, m yoglobin, and creatine kinase; these changes 17 

### were resolved by day 6 after arrival at the hospita l. The cause of his symptoms was determined 18 

### only after analysis of his stomach contents and uri ne, which showed the presence of n-butanol. 19 

### No information on the dose of n-butanol ingested wa s provided in the report and no follow-up 20


### information was available. 21

22 

### 4.1.2. Inhalation Exposure 23


### 4.1.2.1. Occupational Studies 24 

### Four studies reported health findings in workers oc cupationally exposed to n-butanol.. 25 

### Details of these studies including study design, ex posure, and notable effects are presented in 26 

### Table 4-1. Occupational health findings associated with n-butanol exposure include eye 27 

### irritation, hearing loss, dermatitis, and other sys temic effects. All of these studies have 28 

### limitations that prevent their use in establishing dose response relationships. For example, 29 

### xposure to other solvents was reported in Tabershaw et al. (1944) and Cogan and Grant (1945) 30 

### and effect levels for n-butanol were not reported i n Velazquez et al. (1969) and Sterner et al. 31 

### (1949). 32

33

34

35

36

3720 DRAFT - DO NOT CITE OR QUOTETable 4-1. Occupational Studies with n-butanol exposureReference(s)NoSubjectsStudy DesignExposureFindingsStrengths andlimitationsTabershaw etal.,1944NotreportedEye irritation, dermatitis, and systemiceffects were evaluated in workers fromsix industrial facilities involved in themanufacture of raincoats and otherwaterproof materials, in whichn-butanol was the primary solvent used5-14 ppm (15-42 mg/m3) and20-65 ppm (61-197 mg/m3)Eye irritation reported at aconcentration ranging from 20 to65 ppm (61-197 mg/m3) in 1 outof 6 facilities with n-butanol onlyNo details of theworker populationswere provided.Other solventsexposures reported in4 out of 6 facilitiesVelazquez etal.,196911 butanol+ noise(72-78 db),47 noiseonly (90-110 db)Audiologic effects of n-butanol exposedworkers were compared with workersfrom another factory exposed to highernoise levels but not n-butanol or othersolvents associated with hearing loss.80 ppm (242 mg/m3) asmeasured by gaschromatography (GC) in thework room at a celluloseacetate ribbon factoryHearing loss was observed in9/11 workers exposed ton-butanol and in 23/47 workerswithout exposure to n-butanolbut with exposure to industrialnoise.The effect levels andsampling protocol formeasurement of nbutanol was notdescribedCogan andGrant,194575 femaleworkersEye examinations were performed onsymptomatic workers employed at afacility reported in the Tabershaw study15 -100 ppm (46-303 mg/m3)ethanol and diacetone alcohol(concentrations unknown)28/35 workers exposed to nbutanol exhibited evidence ofcoreal inflammation and nounexposed workers showed signsof inflammationExposure to multiplesolvents.No effect levels werereportedSterner et al.,194916 maleworkersPhysical examinations of workersexposed to n-butanol during coating ofphotographic paper over a 10-yearperiod were conducted.Initial concentrations of nbutanol averaged 200 ppm(606 mg/m3), but decreased to100 ppm (303 mg/m3) over thecourse of the study.Workers exposed to n-butanolconcentrations averaging ≥200ppm (606 mg/m3) werereported to experience cornealedema and mild edema of theconjuctivaOnly 4 subjectsremained in the studyat 10 years

### 21 DRAFT - DO NOT CITE OR QUOTE

A number of other occupational studies examined hea lth endpoints in worker populations 1 

### such as painters or chemical/petrochemical workers who were exposed to mixtures of solvents 2


### that included n-butanol. Table 4-2 lists each of t he studies and provides a brief description. All 3 

### of the studies in Table 4-2 suffer from design limi tations that preclude their use in establishing 4 

### causal associations or dose-response relationships. Several of the studies (Chen et al., 1999; 5 

### Jang et al., 1999; Akhmadeyeva, 1993; Bleecker et a l., 1991; Murata et al., 1991) did not report 6 

### any quantitative estimates of exposure to individua l compounds. In the three studies that 7 

### quantified exposure to individual compounds and inc luded health effect information (Tucek et 8 

### al., 2002; Triebig et al., 1992; Angerer and Wulf, 1985), exposure to n-butanol was small 9 

### compared with exposure to other solvents such as xy lenes, ethylbenzene, toluene, and/or 10 

### acrylates. 1122 DRAFT - DO NOT CITE OR QUOTETable 4-2. Studies of human exposure to mixtures including n-butanolReference(s) No. subjects Study type Exposure Findings Strengths and limitationsAngerer andWulf, 198531 exposedand31 controlsStudy comparedhematologyfindings in varnishworkers from sixdifferentworkplaces (withintwo plants) withthose of matchedcontrols. Exposureto n-butanol wasreported at only oneworkplace, witheight workers.Air contaminants at the only workplacewith butanol exposure (1.2 ±0.8 mL/m3) included o-, m-, andp-xylenes (averaging 3.4, 11.7, and4.3 mL/m3respectively) andethylbenzene (7.5 mL/m3).Hematology results pooled acrossthe six locations suggestedstatistically significant reductions inerythrocyte count and hemoglobincontent, increased lymphocytecount, and reduced (not statisticallysignificantly) segmentedgranulocyte count.Exposure to multiple solvents.n-Butanol was not detected inblood of workers at theworkplace where it wasmeasured in air; however,xylenes and ethylbenzenewere detected in blood.Bleecker et al.,1991187 exposed Cross-sectionalstudy comparedneurobehavioralfindings in paintmanufacturingworkers acrossquartiles of totalhydrocarbonexposure.Exposures included toluene, xylene,aliphatic and aromatic hydrocarbons,methyl ethyl ketone, otherhydrocarbons, alcohols (includingn-butanol), ketones, and esters.Statistically significant correlation(by linear regression analysis)between increasing exposure tomixed organic solvents andimpairment of performance onseveral neuropsychological testoutcomes, as well as vibrationthreshold.Exposure to multiple solvents.Exposure measured as“lifetime average weightedexposure” to totalhydrocarbons. No quantitativeinformation on individualcompounds was provided.Murata et al.,199111 exposedand11 controlsStudy comparedneurologicalfindings in workerswith solventexposure with agematched controls.Only one of the workers reportedexposure to n-butanol; this individualalso reported exposure to xylene,methanol, and tolueneCoefficient of variation inelectrocardiographic RR-intervalswas statistically significantlyreduced, and the distribution ofnerve conduction velocities wereslowed in exposed workerscompared with controls.No quantitative measures ofexposure reported.23 DRAFT - DO NOT CITE OR QUOTETable 4-2. Studies of human exposure to mixtures including n-butanolReference(s) No. subjects Study type Exposure Findings Strengths and limitationsTriebig et al.,199283 exposedand42 controlsStudy comparedsubjectivesymptoms ofneurotoxicendpoints in spraypainters withcontrols.Spray painters were from 10 differentwork sites; air concentrations of severalsolvents were reported for each worksite. Four sites had n-butanolmeasurements (5.7, 1.5, 5.4, and>300 mg/m3); the number of spraypainters at each site was not reported.At the sites with n-butanol exposure,there was co-exposure to other solventsincluding toluene, xylene, ethylbenzene,styrene, acetone, butylacetate, methylisobutylketone, ethyl acetate, n-heptane,and/or decane.No difference in the prevalence ofsubjective symptoms ofneurotoxicity between painters andcontrols.Exposure to multiple solvents.Data pooled across all worksites, including those that hadn-butanol exposure and thosethat did not.Akhmadeyeva,1993347 exposedand1,526 controlsStudy comparedhealth, physicaldevelopment, andimmunologic andpsychochemicalindices in blood ofnewborns ofpetrochemicalworkers with thoseof control infants.Workplace air contaminants reportedlyincluded aromatic hydrocarbons,isopropylbenzene, ethanol, and butanol.Newborns of workers had higherfrequency of delayed intrauterinedevelopment (24.2 vs. 13.7% incontrols;p< 0.01). Otherdifferences between study andcontrol newborns includedhematological and immunologicaleffects.Exposure to multiple solvents.Brief report; no informationon study design. Noquantitative measure ofexposure; no quantitativeinformation on effects otherthan intrauterinedevelopmental delay.24 DRAFT - DO NOT CITE OR QUOTETable 4-2. Studies of human exposure to mixtures including n-butanolReference(s) No. subjects Study type Exposure Findings Strengths and limitationsChen et al.,1999309 exposed(mortalitystudy)260 exposedand539 controls(neuropsychologystudy)Mortality study andcross-sectionalstudy of selfreported neuropsychologicalsymptoms indockyard paintersand unexposedcontrols. Mortalityanalysis included309 painterscomprising3,690 person-yrs atrisk. Crosssectional study ofneuropsychologicalsymptoms included260 survivingpainters and539 unexposedcontrols.According to the authors, primarysolvents used at dockyard were whitespirit, xylene, trimethylbenzene,n-butanol, trichloroethylene, naphtha,and cumene; exposures to othercompounds were also reported to belikely.Neither proportional mortality norstandardized mortality ratios forcancers or diseases of the CNS,circulatory system, or respiratorysystem were increased in painters.Cross-sectional study suggestedincreased prevalence ofneuropsychological symptoms inpainters compared with controls;relative risk for these symptomsincreased with yrs of exposure.Exposure to multiple solvents.Exposures characterized onlyby yrs as a dockyard painter;no quantitative measures ofexposure.Jang et al.,1999674 exposedand191 controlsStudy comparedneuropsychologicaleffects (measuredvia questionnaireand function tests)in shipyard workersexposed to solventsand unexposedcontrols.Primary solvents were xylene, ethylbenzene, trimethylbenzene, toluene,2-ethoxyethanol, and 2-ethoxyethylacetate (constituted 85% of totalexposure “intensity”). n-Butanolreported as one of several other solventsdetected.Rate of abnormalneuropsychological diagnosis(primarily executive dysfunction,attention dysfunction, memorydisorder, or functional disorder) was>4 times higher in the exposedworkers compared with controls(9.3 vs. 2.1% in controls,p< 0.01).Rate of abnormal diagnosisexhibited dose-response relationshipwith cumulative exposure.Exposure to multiple solvents.Exposure quantified as“exposure intensity”, a metricthat incorporatedenvironmental (total solventconcentrations) and/orbiological measures (urinarymetabolites of the primarysolvents) as well as duration incertain job categories.25 DRAFT - DO NOT CITE OR QUOTETable 4-2. Studies of human exposure to mixtures including n-butanolReference(s) No. subjects Study type Exposure Findings Strengths and limitationsTucek et al.,200260 exposedand60 controlsProspective cohortstudy of workers ata chemical facilityproducing acrylicacid. Studyexamined healtheffects includinghematology, serumchemistry, serumimmunity andtumor markers,spirometry, andcytogenetic analysisof peripherallymphocytes.Exposures included acrylonitrile,n-butanol, butyl acrylate, ethyl acrylate,methyl acrylate, methyl methacrylate,toluene, and styrene. Measurements ofthe various solvents (measured usingpersonal air sampling) were binned byconcentration and reported usingstacked bar charts; these chartssuggested that >70% of n-butanolconcentrations were <0.02 mg/m3.Exposures to the other compounds wereof a similar magnitude and generallylower than occupational health limits inthe Czech Republic; butyl acrylate wasmost frequently (at 2.1% ofmeasurements) measured at a levelexceeding its limit.Statistically significantly higherpercentage of chromosomalaberrations in peripherallymphocytes.Exposure to multiple solvents.

### 26 DRAFT - DO NOT CITE OR QUOTE

4.1.2.2. Controlled Acute Exposure Experiments 1 

### There several controlled acute exposure studies tha t evaluated irritation and conditioned reflexes 2 

### in humans during n-butanol exposure. The exposure ranges were highly variable from as low as 3 

### 0.1 mg/m 3 

### (Baikov and Khachaturyan, 1973; duration unknown) to 12,000 mg/m 3 

### for a 2 minute 4 

### interval exposure (Kjaerguard et al., 1997). Notab le effects in the controlled acute exposure 5 

### studies included increased eye, nose, and throat ir ritation (Nelson et al., 1943; Cometto-Muniz 6 

### and Cain, 1995; Wysocki and Dalton, 1996; Kjaerguar d et al., 1997; Hempel-Jorgensen et al., 7 

### 1998; 1999; Podlekareva et al., 2002) and CNS effe cts including increased response latencies 8 

### and inhibition of the blinking reflex (Baikov and K hachaturyan, 1973). Only one study (Baikov 9 

### and Khachaturyan, 1973) examined toxicity effects o f n-butanol beyond irritation effects. These 10 

### studies are described more fully in Table 4-3. 11

12

1327 DRAFT - DO NOT CITE OR QUOTETable 4-3. Controlled Acute Exposure StudiesReference Subjects Study Design and Exposure Results Strengths and LimitationsNelson et al.,194310volunteersComparison of eye, throat and nasal irritation insubjects and controls. Concentrations rangingfrom 0-50 ppm (0 – 150 mg/m3; groups notspecified by authors) of n-butanol wereadministered for 3-5 minutes in an inhalationexposure chamberEye irritation at 50 (150 mg/m3) Nose andthroat irritation at 25 ppm (76 mg/m3)Highest concentration acceptable for 8hours as reported by the volunteers was<25 ppm (<76 mg/m3)Limited information on doseconcentrationsBaikov andKhachaturyan,197318volunteersin groupsof 3Comparison of the odor threshold and effects ofn-butanol (0.1-2.5 mg/m3) on conditionedreflexes in subjectsOdor threshold determined to be 1.2mg/m3Dark adaption slowed at 1.2 mg/m3Statistically significant increase inresponse latency at 0.7 mg/m3Statistically significant inhibition of theconditioned blinking reflex at 1.5 and 2.5mg/m3Transient development of conditionedreflex described by shifts inelectrocephalograms. (effectiveconcentration not reported)Lack of study design details;Study identified CNS effectsassociated with n-butanol.ComettoMuniz andCain et al.,19955 malesand5 femalesn-Butanol vapors (100, 33, 11, 3.7, etc. % v/v)were administered to eye or nostrils for 1-3seconds, using squeezable bottles. Eye irritationand odor threshold responses were evaluated.Odor threshold was reported as 20 ppm (61mg/m3)Eye irritation threshold was reported as3000 ppm (9000 mg/m3)Limited statistical power due tothe small number of subjectsWysocki andDalton, 199632exposedworkers32unexposedresidentsOdor and irritation thresholds for n-butanol(99.8% pure; diluted to 26 concentrationsranging from 100% to 1.1802 x 10-10% v/v) inacetone –exposed workers and naïve volunteerswere evaluated.Median odor detection and nasal irritationthresholds were 0.17 ppm (0.52 mg/m3)and 2,042 ppm (6,190 mg/m3),respectively.Acetone workers considered n-butanol tobe nonirritating, while naïve subjectsconsidered the exposure to have irritatingpropertiesIncluded a group exposed toone other solvent (acetone)Kjaerguard etal., 19976 men and6 womenEye irritant effects of n-butanol after local (eyeonly) exposure and whole-body exposure involunteers were evaluated. Doses of n-butanolused in the study were 1500-12000 mg/m3for 2Statistically significant association (byrepeated-data multiple analysis ofvariance) between exposure concentration,general irritation, eye irritation, throatOnly irritation effects wereevaluated; No systemic toxicityeffects were examined.28 DRAFT - DO NOT CITE OR QUOTETable 4-3. Controlled Acute Exposure StudiesReference Subjects Study Design and Exposure Results Strengths and Limitationsminute intervals (lowest to highest) in goggleexperiments and 0, 2.5,5, and 10 mg/m3inchamber experiments.irritation, odor irritation, nose irritation, airquality, and continuously-measuredirritation by potentiometerAnalysis suggests an interaction betweenindividual threshold for eye irritation in thegoggle experiment and several othermeasures of irritation (general irritation,eye irritation, and throat irritation.HempelJorgensen etal., 1998; 19998 subjects Evaluated eye irritation associated with eye-onlyexposure to n-butanol n-butanol at (0, 99, 314,or 990 ppm (0, 300, 952, or 3,000 mg/m3forperiods of 15 or 60 minutes) and the results ofconjunctival hyperemia evaluations usingphotographs of eyes before and after exposure,and conjunctival fluid cytology measured byoptical microscopy. One eye was exposed ton-butanol, and the other to CO2The average irritation intensity wasestimated for each second of the 60-minuteexposure and plotted to show the timecourse of irritationEye irritation intensity did not differamong the various concentrations ofn-butanol and exposure to clean airThe mean conjunctival hyperemia score insubjects exposed to 990 ppm (3,000mg/m3) n-butanol was statisticallysignificantly higher (1.40 ± 0.76 on a scalefrom -2 to +2) than the score afterexposure to clean air (0.48 ± 0.96);Conjunctival cytology was not affected byexposure to n-butanol at any concentration.n-Butanol exposures in thestudy were not high enough toresult in ocular irritationPodlekareva etal., 20029 subjects Compared physiological evidence of irritationafter eye-only exposure to either n-butanol, CO2,or clean air. n-butanol and CO2exposures weredesigned to stimulate the same perceived levelof irritation (50% of maximal on a linear scale).Eye exposure to n-butanol did not result ina significant change in tear film stability orconjunctival corrosion measuresLimited statistical power due tothe small number of subjects

### 29 DRAFT - DO NOT CITE OR QUOTE

4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOA SSAYS IN 1


### ANIMALS—ORAL AND INHALATION 2


### 4.2.1. Oral 3 

### Research Triangle Institute (RTI; unpublished study , 1985) exposed groups of 30 male 4 

### and 30 female rats with n-butanol (purity not repor ted, in deionized water) via daily gavage at 5 

### doses of 0, 30, 125, or 500 mg/kg-day for 13 weeks. Daily observations for mortality and 6 

### clinical signs, and weekly measurements of body wei ght and food consumption were made. 7 

### Ophthalmology (specific tests not reported) was eva luated before exposure and during the final 8 

### week of exposure. Blood and urine were collected f rom rats (10 rats/sex/group) at the end of 6 9 

### weeks of exposure and at terminal sacrifice (at the end of 13 weeks of exposure) for assessment 10 

### of hematology (hematocrit, hemoglobin, red blood ce ll [RBC], mean cell volume, mean cell 11 

### hemoglobin, mean cell hemoglobin concentration, tot al and differential leukocyte count, platelet 12 

### count), clinical chemistry (alkaline phosphatase [A LP], blood urea nitrogen [BUN], alanine 13 

### aminotransferase [ALT], aspartate aminotransferase [AST], glucose, total protein, albumin, total 14 

### bilirubin, electrolytes, inorganic phosphate, CO 2 

### , total cholesterol, and creatinine), and urinalysi s 15 

### parameters (pH, specific gravity, glucose, protein, ketones, bilirubin, urobilinogen, and sediment 16 

### microscopy). All animals were subjected to gross n ecropsy. Organ weights (brain, heart, liver, 17 

### spleen, kidneys, adrenals, thyroid with parathyroid , testes with epididymides, and ovaries) were 18


### recorded for animals sacrificed after 13 weeks but not those sacrificed after 6 weeks of exposure. 19 

### Comprehensive histopathologic examination (> 30 tis sues) was performed on control and high20 

### dose animals; in addition, the liver, kidneys, hear t, and gross lesions were examined in the low21 

### and mid-dose groups. The available copy of the unp ublished report was lacking data tables and 22 

### appendices. The original microfiche for the Toxic Substances Control Act Test Submission 23 

### (TSCATS) also lacked the data tables and appendices ; thus, the summary provided herein is 24 

### based on the text of the report. 25


### No n-butanol-treatment related death occurred in th ese rats. Three animals (one mid26 

### dose and two high-dose animals) died prior to sacri fice due to gavage errors (RTI; unpublished, 27 

### 1985). There were no statistically significant cha nges in body weight, food consumption, or 28 

### ophthalmoscopic findings. 29 

### Clinical signs of toxicity, specifically hypoactivi ty and ataxia, were observed in high30


### dose animals but not in animals at lower doses. Th ese signs were first observed during week 8, 31 

### infrequently through week 10, and reached maximum i ncidence (29% for hypoactivity and 32% 32 

### for ataxia) during weeks 11–13 (no other details pr ovided). The study authors reported that these 33 

### effects were evident 2–3 minutes after dosing and l asted for < 1 hour. 34 

### At the interim sacrifice, hematology analysis showe d reduced hematocrit, erythrocyte 35 

### count, and hemoglobin content (5% lower than contro ls) in 500 mg/kg-day females. No 36 

### hematological changes were evident in males at inte rim sacrifice or in either sex at terminal 37 

### sacrifice. There were higher absolute neutrophil c ount in mid-dose males at interim evaluation 38

### 30 DRAFT - DO NOT CITE OR QUOTE

and higher relative segmented neutrophil count and lower relative lymphocyte count in low-dose 1 

### females at final evaluation. The study authors als o reported the following statistically significant 2 

### serum chemistry and urinalysis changes that were de scribed as “small”, occurring in one sex and 3 

### at one evaluation only, and without a dose-response relationship: lower cholesterol in high-dose 4 

### males at interim evaluation; and higher urine pH in low-dose males at the interim evaluation and 5 

### in low-dose females at final evaluation. High-dose males exhibited a 14% increase in average 6 

### thyroid weight compared with controls; no other sta tistically significant organ weight changes 7 

### were reported. The report indicated that there wer e no treatment-related gross or microscopic 8 

### pathology findings; however, the lack of data table s precluded independent review of the data. 9 

### The study authors did not identify effect levels, b ut indicated that no treatment-related 10 

### effects were seen at 30 or 125 mg/kg-day. Based on the available information, EPA identified a 11 

### LOAEL 500 mg/kg-day based on neurotoxic signs (hypo activity and ataxia) and reduced 12 

### hematocrit, erythrocyte count, and hemoglobin conte nt in females. The NOAEL is 125 mg/kg13 

### day. 14 

### Groups of 15 male Wistar rats (5 weeks of age at st udy commencement) were exposed to 15 

### n-butanol (purity not reported) in drinking water ( 0 or 6.9% v/v; approximate saturation limit) 16 

### for up to 3 months (Wakabayashi et al., 1991). Thi s exposure concentration corresponds to an 17 

### estimated dose of 8,200 mg/kg-day 1 

### based on default estimates of body weight (0.217 k g) and 18 

### water intake (32 mL/day) (U.S. EPA, 1988) and a che mical density of 810 mg/mL for n-butanol 19 

### (HSDB, 2009). Groups of 3–4 animals were sacrifice d at various intervals; the first sacrifice 20 

### occurred after 1 week of exposure. Toxicological e valuation was limited to electron microscopy 21 

### of liver sections. Animals exposed to n-butanol an d other alcohols reportedly exhibited poor 22 

### appetite, weakness, and loss of body weight (no dat a provided). The study authors reported that 23 

### there were no ultrastructural changes to hepatic mi tochondria after n-butanol exposure up to 1 24 

### month; however, after 2 months of exposure, hepatic mitochondria were observed to be smaller 25 

### and with poorly developed cristae. After 3 months of exposure, enlarged mitochondria with little 26 

### or no cristae, and cup-shaped or elongated mitochon dria were also observed. Other hepatic 27 

### effects noted by the authors were proliferation of smooth endoplasmic reticulum and increased 28 

### numbers of lysosomes and microbodies. The study au thors proposed two possible mechanisms 29 

### for the effects on mitochondria: fusion of adjacen t mitochondria and suppression of the process 30 

### of mitochondrial division via perturbation of prote in synthesis. 31 

### EPA did not identify a NOAEL or LOAEL for this stu dy due to study design limitations 32 

### (single high concentration used, only ultrastructur al changes in the liver were examined, and the 33 

### incidence and severity in exposed or control groups were not reported). This study was designed 34 

### to investigate whether mitochondrial effects observ ed with exposure to ethanol are also evident 35 

### after exposure to other alcohols. 36

1 Calculated as follows: 6.9% n-butanol = 0.069 mL/m L water × 810 mg/mL × 32 mL water/day × (1/0.217 kg ) = 8,200 mg/kg-day.

### 31 DRAFT - DO NOT CITE OR QUOTE

Munoz et al. (1991, 1990) exposed groups of three m ale Wistar rats (initial weight 200– 1 

### 300 g) to unmodified drinking water or n-butanol in drinking water for 4 months in a study 2 

### focused on examining effects on protein synthesis i n the brain. The exposure concentration of 3 

### n-butanol (purity not specified) was gradually incr eased from 1% (v/v) during the 1st week to 4 

### 2% during week 2 and 4% for the remainder of the tr eatment period until sacrifice after 5 

### 4 months. This exposure regimen corresponds to a t ime-weighted average concentration of 6 

### 3.7%, resulting in an estimated dose of 4,400 mg/kg -day 2 

### based on default estimates of body 7 

### weight (0.217 kg) and water intake (32 mL/day) (U.S . EPA, 1988) and a chemical density of 8 

### 810 mg/mL for n-butanol (HSDB, 2009). Toxicologica l evaluations were limited to mortality, 9 

### body weight, selected serum chemistry parameters (u rea, glucose, creatinine, triglycerides, total 10 

### proteins, albumin, and ALP), and in vitro protein s ynthesis in a cell-free translation system using 11 

### brain extract. There were no mortalities during th e study. Body weight gain for rats exposed to 12 

### n-butanol for 100 days was 46% of the body weight g ain observed in control rats. The authors 13 

### reported that treated rats exhibited “signs of seve re pathology” in several tissues, especially liver 14 

### and kidney, but data were not provided and there wa s no information to indicate whether these 15 

### organs were examined grossly or microscopically. S erum chemistry findings were reported 16 

### without statistical analysis; however, group sizes were small (3/group). Treated rats had 17 

### statistically significantly higher total protein (2 9% higher than controls) and albumin levels (28% 18 

### higher). While not statistically significant, when compared with controls, the triglyceride level 19 

### was higher (almost twofold) and ALP level was lower (20% of control) in treated rats. The study 20 

### authors did not identify effect levels, and the inf ormation provided was inadequate for the 21


### identification of effect levels. 22

23


### 4.2.2. Inhalation 24 

### Korsak et al. (1994) exposed male Wistar rats (12/e xposure group, 24 controls) to 25 

### n-butanol (purity not specified) vapor concentratio ns of 0, 50, or 100 ppm (0, 154, or 308 mg/m 3 

### ) 26 

### 6 hours/day, 5 days/week, for 3 months. Exposure c oncentrations, which were generated by 27 

### heating the liquid solvent in a washer followed by dilution in air, were subsequently measured at 28 

### 30-minute intervals using GC/flame ionization detec tion (FID). Body weights were recorded 29 

### weekly during the study. Terminal body weight and organ weights (heart, lungs, liver, spleen, 30 

### kidneys, adrenals, and testes) were recorded. Rota rod performance tests, designed to assess 31 

### neuromuscular function, were performed on rats befo re the initiation of n-butanol exposure, and 32 

### monthly during the exposure period. Rats were plac ed on a horizontal wooden rod that rotated at 33 

### a rate of 12 revolutions/minute and was placed at aheight of 20 cm above the floor. Prior to n34 

### butanol exposure, rats were trained on the rotarod task for 10 days and received a shock from the 35 

### floor after falling to prevent voluntary jumps from the rod. Therefore, learned avoidance 36

2 Calculated as follows: 3.7% n-butanol = 0.037 mL/m L water × 810 mg/mL × 32 mL water/day × (1/0.217 kg ) = 4,400 mg/kg-day.

### 32 DRAFT - DO NOT CITE OR QUOTE

behavior (from the negative enforcement of the shoc k from the floor) in addition to 1


### neuromuscular function was evaluated in the rotarod task as designed by the authors. 2 

### Hot-plate behavior (latency of the paw-lick respons e) was tested after the 3-month 3 

### exposure period. Blood samples were collected from rat tail before the exposure and 1 week 4 

### prior to the termination of exposure and used for h ematological evaluations (hematocrit, 5 

### hemoglobin, erythrocyte count [RBC], and total and differential leukocyte counts). Clinical 6 

### biochemistry studies (ALT, AST, sorbitol dehydrogen ase [SDH], ALP, total protein, albumin, 7 

### glucose, electrolytes) were performed on serum samp les taken at sacrifice 24 hours after the last 8 

### exposure. The livers were homogenized to analyze t otal microsomal cytochrome P450 (CYP) 9 

### content, aniline p-hydroxylase activity, triglyceri de content, and malondialdehyde (MDA) 10



### content. 11


### All rats survived and there were no clinical abnorm alities observed in these rats (Korsak 12 

### et al., 1994). Mean body weights of rats exposed t o n-butanol were higher than controls (5–6%) 13 

### during the first 2 months of the exposure period; d uring the 3rd month, average weights were 14 

### higher (up to 7%), but not statistically significan tly different from controls. There were no 15 

### significant differences in absolute or relative org an weights. Table 4-4 shows changes in 16 

### hematology and lipid peroxidation parameters. Comp ared to controls, the exposed groups 17 

### exhibited decreased erythrocyte counts (5 and 16% l ower in the 154 and 308 mg/m 3 

### groups, 18 

### respectively); the difference was statistically sig nificant in the 308 mg/m 3 

### group only. 19 

### Statistically significantly decreased hemoglobin le vels (10% lower than controls) were noted in 20 

### both exposure groups, but hematocrit was not change d. There were increased leukocyte counts 21 

### (25 and 57% higher in the 154 and 308 mg/m 3 

### group, respectively); the difference was 22 

### statistically significant in the 308 mg/m 3 

### group, and was beyond the normal range of variabil ity 23 

### (16.5 × 10 3 

### /nm 3 

### in exposed rats, compared with a range of 1.96–8.2 5 × 10 3 

### /nm 3 

### ; Giknis and 24 

### Clifford, 2008). The leukocyte differential counts were not statistically significantly different 25 

### among exposure and control groups, except for an in crease (77% higher than controls), in the 26 

### percentage of eosinophils in the 308 mg/m 3 

### group; this increase was well beyond the normal 27 

### range (13.8%, compared with 0–2% for untreated male rats < 6 months of age; Wolford et al., 28 

### 1986). There were no statistically significant exp osure-related changes in any measured serum 29 

### chemistry parameters. Statistically significantly increased lipid peroxidation, as measured by 30 

### increased MDA in hepatic microsomes, was noted in b oth exposure groups (16 and 30% higher 31 

### than controls in the 154 and 308 mg/m 3 

### groups, respectively). Hepatic total CYP content, 32 

### microsomal aniline p-hydroxylase activity, and live r triglycerides were not affected by exposure 33


### to n-butanol. There were dose- and duration-relate d increases in the percentage of rotarod test 34 

### failures, indicating impaired neuromuscular functio n and learned avoidance behavior, in the rats. 35 

### The rotarod data were presented in Korsak et al. (1 994) in a graphical format and the failure rates 36 

### were approximately 2–15% in the 154 mg/m 3 

### group and 15–33% in the 308 mg/m 3 

### group when 37 

### measured over the 3 month period and are presented in Table 4-5. The increased failure rate in 38

### 33 DRAFT - DO NOT CITE OR QUOTE

the 308 mg/m 3 

### group was statistically significant during the 2nd and 3rd months of exposure; the 1 

### changes in the low exposure group were not statisti cally significant at any time point. Pain 2 

### sensitivity (assessed by hot-plate behavior) was no t affected in the rats exposed to n-butanol at 3


### these concentrations. EPA identified aNOAEL and LOA EL of 154 and 308 mg/m 3 

### , respectively, 4 

### based on impaired neuromuscular function. Decrease d hemoglobin and increased lipid 5 

### peroxidation were both observed at the NOAEL; howev er, the changes at that concentration were 6 

### not biologically significant. 7 

### Table 4-4. Hematological effects in rats exposed t o n-butanol by inhalation

for 3 months

Control 154 mg/m 3 (50 ppm) 308 mg/m 3

(100 ppm)
Number of animals 24 12 12 Hemoglobin (g/dL) 15.9 ± 0.4 a 14.2 ± 0.8 b 14.1 ± 0.7 b
Erythrocytes (× 10 6 /nm 3 ) 9.97 ± 0.02 9.45 ± 0.05 8.35 ± 0.06 b
Leukocytes (× 10 3 /nm 3 ) 10.5 ± 0.13 13.1 ± 0.26 16.5 ± 0.27 b
Eosinophils (%) 7.8 ± 2.3 11.5 ± 4.6 13.8 ± 4.6 c MDA (nmol/mg microsomal protein equivalent to g liv er) 40.41 ± 2.10 46.68 ± 2.16 c 52.45 ± 2.88 c

a Mean ± standard deviation (SD).
b Significantly different from control at p < 0.01.

c Significantly different from control at p < 0.05.

Source: Korsak et al. (1994). 8 

### Table 4-5. Changes in neurobehavioral function as measured by the rotorod

test in rats exposed to n-butanol by inhalation ove r 3 months

Exposure Group Number of animals Percentage of Rotarod Failures a

Month 1 Month 2 Month 3
Control 24 0 0 0 154 mg/m 3 (50 ppm) 12 2 10 15 308 mg/m 3 (100 ppm) 12 15 25 b 33 b

a Percentage of rotarod failures were derived from da ta approximations.
b Significantly different from control at p < 0.05.

Source: Korsak et al. (1994). 9 

### Smyth and Smyth (1928) performed three experiments in which guinea pigs (at least 10 

### three/group, sex unspecified) were exposed to n-but anol (purity not reported) vapor at a 11 

### concentration of 100 ppm (300 mg/m 3 

### ) every day for 2 weeks (hours/day were not specifi ed) and 12 

### then for 4 hours/day, 6 days/week, for durations ra nging from 1 to 2.5 months. The three groups 13 

### were not exposed concurrently. There were two cont rol groups: one sham-treated and one 14 

### untreated, three animals per group (sex unspecified ). The guinea pigs were weighed weekly. 15

### 34 DRAFT - DO NOT CITE OR QUOTE

Blood counts and urinalysis (parameters not reporte d) were assessed prior to exposure and at 1 

### biweekly intervals thereafter. The schedule for an d tests performed at sacrifice were not 2 

### described. The study authors reported that there w ere no deaths among the first group treated for 3 

### 64 exposures, but there was a decrease in erythrocy te count as well as lymphocytosis. Of this 4 

### group of three animals, two were reported to exhibi t hemorrhagic areas in the lungs and transient 5 

### albuminuria. The second group tested at this conce ntration developed severe skin infections 6 

### after the 30th exposure, and two died during the 38 th exposure. These animals also reportedly 7 

### exhibited decreased erythrocytes and hemoglobin alo ng with increased total leukocytes (data not 8 

### provided). The surviving guinea pig gained weight and had a “decidedly improved blood 9 

### picture” (details not provided) at sacrifice. The authors indicated that all three animals had 10 

### “toxic degeneration” of the livers and kidneys (no further information given). The third 11 

### experiment was continued for 28 exposures, and the authors reported similar changes in 12 

### hematology (decreased erythrocytes and absolute and relative lymphocytosis) as well as central 13 

### liver and marked renal degeneration (characterized by cloudy swelling of convoluted tubules, 14 

### marked degeneration of tubules). Data on weight, h ematology, and specific gravity of urine 15 

### were reported for each of the three treated animals , but control data were not provided. In the 16 

### two control groups, one animal each died of skin in fection. 17 

### Rumyantsev et al. (1979; published in Russian and t ranslated for this review) rats and 18 

### mice were exposed to butanol (isomer and purity not reported) via inhalation at concentrations of 19 

### 0, 0.8, 6.6, or 40 mg/m 3 

### for 4 months (frequency not reported). This study did not provide other 20 

### details of the study design (for example, sex, stra in, or number per group, or toxicological 21 

### evaluations performed) or any quantitative informat ion on the effects observed. The only effect 22 

### information provided in the report was a list of ef fects at each exposure concentration; the 23 

### affected species was not reported in most cases. I ncreased thyroid gland activity reportedly 24 

### occurred in all experimental groups. At 6.6 and 40 mg/m 3 

### , narcosis induced by hexanal 25 

### administration was shortened, there was an increase in the conditioned reflex activity and the 26 

### pituitary-adrenal system was characterized as disru pted. Other changes were noted such as a 27 

### loss of CNS summation capacity (generally an electr ophysiological measurement of nerve 28 

### response), a reduced eosinophilic response followin g administration of adrenocorticotropic 29 

### hormone, and reduced demand of oxygen in what was t ermed the “cold test.” Finally, increased 30 

### blood cholinesterase activity was observed in rats at ≥ 6.6 mg/m 3 

### . The authors characterized the 31 

### 6.6 mg/m 3 

### concentration as a threshold value and could be co nsidered a LOAEL and 0.8 mg/m 3 32 

### is the NOAEL for the study. Although the authors p rovide effect levels for the study, there is no 33 

### information on the study design, no data for the to xicological effects are available, and statistical 34 

### information is not provided for the various endpoin ts. As a result, EPA considers the 35 

### information reported in the publication as inadequa te for the purpose of determining effect 36


### levels. 37

38

### 35 DRAFT - DO NOT CITE OR QUOTE

4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND IN HALATION 1


### 4.3.1. Oral Studies 2 

### Sitarek et al. (1994) exposed female Wistar Imp:DAK rats (11–17/group) to n-butanol 3 

### (purity not reported) in drinking water at concentr ations of 0.24, 0.8, and 4%, estimated as 300, 4 

### 1,000, and 5,000 mg/kg-day by the authors. A contr ol group of 16 rats received plain tap water. 5 

### Treatment was initiated at 10 weeks of age and cont inued for 8 weeks prior to mating. For 6 

### 14 days prior to exposure and during weeks 4–5 and 7–8 of the premating exposure period, 7 

### vaginal smears were collected daily for assessment of estrus cycle. The females were then mated 8 

### to untreated males for up to 3 weeks. Gestation da y (GD) 0 was defined as the day of sperm 9 

### detection in vaginal smears. The females were expo sed to n-butanol continuously through the 10 

### mating and gestation periods. Weight gain, food co nsumption, and water intake were measured 11 

### weekly in females that did not become pregnant and on GDs 3, 7, 10, and 17 in pregnant 12 

### females. Upon sacrifice on GD 20, uterine contents (numbers of live and dead fetuses, numbers 13 

### of early and late resorption sites) were evaluated. Body weight and crown-rump length of live 14


### fetuses were recorded. All live fetuses were exami ned for external malformations; half were 15 

### then prepared for skeletal examination and the rema inder for visceral examination. Statistical 16 

### analysis of malformations was performed using eithe r the fetus or the litter as the statistical unit. 17


### All of the animals survived and treatment with n-bu tanol did not affect food or water 18 

### consumption or body weight of female rats during th e premating period (Sitarek et al., 1994). 19 

### Mean daily intake of food and water by high-dose da ms was lower than controls (10 and 9%, 20 

### respectively), but the difference was not statistic ally significant. Mean body weight gain was 5% 21 

### higher in high-dose dams compared with controls; th e difference was not statistically significant. 22 

### There were no differences in estrus cycle duration or duration of individual stages of the cycle. 23 

### The study authors reported that hemoglobin concentr ation and hematocrit, as well as absolute 24 

### and relative organ weights were not different betwe en exposed and control dams; however, 25 

### detailed data were not provided. Treatment with n- butanol did not affect the pregnancy rate, 26 

### number of corpora lutea or total implants, number o f litters with resorptions, or numbers of pre27 

### or post implantation losses/litter. A slight incre ase in the number of early resorptions/litter was 28 

### observed (two-fold increase over control at the hig h-dose), but the difference from control was 29 

### not statistically significant. 30

### 36 DRAFT - DO NOT CITE OR QUOTE

Table 4-6. Selected fetal developmental changes in offspring of rats exposed to n-butanol in drinking water for 8 weeks before m ating and during gestation a

Parameter n-Butanol dose in mg/kg-d (% in water)

0 300

(0.24%) 1,000

(0.8%) 5,000

(4.0%) Fetal body weight b (g) 3.2 ± 0.2 3.2 ± 0.3 3.2 ± 0.2 3.2 ± 0.3 Fetal crown-rump length b (cm) 4.0 ± 0.1 3.9 ± 0.1 3.9 ± 0.1 3.8 ± 0.1 c

No. of litters examined for visceral variations 12 14 12 9 Percentage of litters with dilation of 8 64 c 83 c 100 c Subarachnoid space (%) 0 14 c 25 c 78 c

Lateral ventricle and/or third ventricle of the bra in (%) 8 57 c 67 c 78 c Unilateral renal pelvis (%) 0 0 42 c 0 Bilateral renal pelvis (%) 0 0 25 c 0 Percentage of litters with congenital defects 0 0 3 3 c 22 c External hydrocephalus (%) 0 0 17 c 0 Internal hydrocephalus (%) 0 0 25 c 22 c

No. of litters examined for skeletal variations 12 14 12 9 Percentage of litters with delayed ossification 67 50 58 67 Percentage of litters with congenital defect 0 7 c 0 11 c 14th rib (%) 0 0 0 11 c Wavy ribs (%) 0 7 c 0 0

a Fetal and litter data was provided by authors; onl y litter data is presented
b Mean ± SD.

c Significantly different from control at p < 0.05.

Source: Sitarek et al. (1994).
1 

### Table 4-6 shows the percentages of fetuses and litt ers with malformations. Mean fetal 2 

### body weight was not affected by treatment, but mean fetal crown-rump length was statistically 3 

### significantly decreased in offspring of high-dose d ams compared with controls (5%). Dose4


### related increases in the incidences of litters with visceral malformations were observed; the 5 

### difference from control was statistically significa nt at all doses (Sitarek et al., 1994). The 6 

### predominant visceral malformation was dilation of t he lateral and/or third ventricle of the brain, 7 

### affecting from 57 to 78% of litters in treated anim als and only 8% of control litters. The 8


### incidence of litters with dilation of the subarachn oid space was also statistically significantly 9 

### increased in a dose-related manner; the percentages in treated groups were 14–78% of litters, 10 

### compared with 0% in controls. Dilation of the rena l pelvis was observed only at the mid-dose 11 

### (25–42% of litters and 0% in other groups). The pe rcentages of litters with internal 12 

### hydrocephalus was statistically significantly incre ased at the mid- and high-doses (25 and 22%, 13 

### respectively, compared with 0% in controls), while the percentage of litters with external 14 

### hydrocephalus was statistically significantly incre ased at the mid-dose only (17% compared with 15 

### 0% in controls). The authors reported that the per centage of fetuses with delayed ossification 16

### 37 DRAFT - DO NOT CITE OR QUOTE

was increased at the high-dose; however, the percen tage of litters affected was not different 1 

### between controls and high-dose animals. The percen tage of litters with an extra (14th) rib was 2 

### statistically significantly higher in the group exp osed to 5,000 mg/kg-day (11 vs. 0% in controls); 3 

### this occurred in two fetuses of a single litter at this dose. The authors did not determine effect 4 

### levels. EPA determined that the low-dose (300 mg/kg -day, or 0.24% in water) is a LOAEL based 5 

### on increased incidences of visceral malformations ( dilation of the lateral and/or third ventricle of 6 

### the brain and of the subarachnoid space) and a NOAE L was not identified. 7 

### Ema et al. (2005) also investigated the development al effects (brain malformations and 8 

### variations) of n-butanol in Sprague-Dawley rats. P regnant (sperm positive) Sprague-Dawley 9 

### (Crj:CD(SD)IGS) rats (20/group) were exposed to n-b utanol (99.9% pure) in drinking water at 10 

### concentrations of 0, 0.2, 1.0, or 5.0% on GDs 0

− 20. The authors estimated daily doses of 0, 316 11 

### ± 39, 1,454 ± 186, or 5,654 ± 1,402 mg/kg-day based on measured body weight and water 12 

### consumption. Maternal evaluations included recordi ng of clinical signs, body weights, and food 13


### and water intake. Dams were sacrificed on GD 20 fo r assessment of numbers of corpora lutea, 14 

### implantation sites, resorptions, and live and dead fetuses; placental weight was also recorded. 15 

### Live offspring were sexed, weighed, measured (crown -rump), and examined for external and 16 

### oral malformations. Half of the fetuses were prepa red for examination of internal malformations 17


### and the remainder for skeletal malformations. The litter was used as the statistical unit for 18 

### analysis of the data. Treatment-related findings a re summarized in Table 4-7. 19 

### None of the animals in any group died. As shown in Table 4-7, maternal body weight 20 

### gain in the group exposed to 5,654 mg/kg-day (5% n- butanol in water) was statistically 21 

### significantly decreased during GDs 0–7 (45% of cont rols) and GDs 0–20 (10% less than 22 

### controls). Food consumption was statistically sign ificantly decreased compared to controls in 23 

### mid- and high-dose dams (8 and 20% lower than contr ols, respectively) throughout pregnancy. 24 

### During GDs 0–7, water consumption was statistically significantly decreased at the mid- and 25 

### high-doses (9 and 38% lower than controls), and was decreased throughout gestation in the high26 

### dose dams (28% lower than controls over GDs 0–20). 27 

### There were no statistically significant differences between exposed and control rats in 28 

### placental weight or numbers of corpora lutea, impla ntations, pre- or post implantation losses, 29 

### resorptions, or live or dead fetuses (Ema et al., 2 005). The sex ratio of offspring was not 30 

### different among the groups. Fetal body weight was statistically significantly reduced in both 31 

### male (8% lower than controls) and female (10%) offs pring of dams exposed to 5,654 mg/kg-day 32 

### (5% n-butanol). Body weights were decreased in the mid- and low-dose fetuses; however, the 33 

### difference from control was not statistically signi ficant. The crown-rump length of offspring was 34 

### not affected by treatment. 35

36

### 38 DRAFT - DO NOT CITE OR QUOTE

Table 4-7. Selected changes in rats exposed to n-b utanol in drinking water on GDs 0–20

Parameter n-Butanol dose in mg/kg-d (% in water)
0 316

(0.2%) 1,454

(1.0%) 5,654

(5.0%) Maternal body weight gain on GDs 0–7 (g) a 44 ± 7 45 ± 7 40 ± 6 20 ± 28 b
Maternal body weight gain on GDs 0–20 (g) a 162 ± 19 168 ± 16 165 ± 15 146 ± 16 b
Maternal food consumption GDs 0–20 (g) a 548 ± 38 548 ± 46 503 ± 34 b 441 ± 34 b
Maternal water consumption GDs 0–20 (mL) a 930 ± 105 983 ± 126 890 ± 106 669 ± 182 b
Male fetal body weight (g) a 4.18 ± 0.27 4.00 ± 0.24 4.04 ± 0.25 3.83 ± 0.18 b
Female fetal body weight (g) a 3.97 ± 0.25 3.86 ± 0.20 3.83 ± 0.16 3.59 ± 0.17 b

Skeletal examination c

Total no. of litters examined 20 20 20 20
Total no. of litters with variations 11 12 17 20 b
Short supernumerary ribs 10 9 16 19 b
Degree of ossification No. of forepaw proximal phalanges 1.6 ± 1.3 1.6 ± 0.9 1.2 ± 1.1 0.3 ± 0.4 b

Internal examination c

Total no. of litters examined 20 20 20 20 Thymic remnant in neck 4 5 8 8

a Mean ± SD.
b Significantly different from control at p < 0.01 (litter as unit of statistical analysis).

c Fetal and litter data was provided by authors; only litter data is presented

Source: Ema et al. (2005).
1 

### The incidences of external, oral, and visceral malf ormations were not increased by 2 

### treatment with n-butanol (Ema et al., 2005). At th e highest dose, a statistically significant 3 

### increase (20/20 vs. 11/20 litters in controls) in t he incidence of litters with skeletal variations 4 

### (primarily short supernumerary ribs) was observed, as well as a decrease in the degree of 5 

### ossification (number of forepaw proximal phalanges was 0.3 ± 0.4 vs. 1.6 ± 1.3 in controls). The 6 

### study authors suggested that the skeletal effects m ay have been related to growth retardation in 7 

### the high-dose group. Although there was no statist ically significant increase in the incidence of 8 

### litters with internal malformations, the incidence of litters with thymic remnant in the neck 9 

### increased with dose (see Table 4-7) and the inciden ce at the high-dose was twice that of controls 10 

### (8/20 litters vs. 4/20 litters in controls). 11 

### The study authors did not identify a LOAEL for this study (Ema et al., 2005); however, 12 

### they did indicate that the NOAEL for dams and fetus es was 1,454 mg/kg-day (1%). EPA has 13 

### determined that the high-dose (5,654 mg/kg-day, or 5% in water) is a LOAEL for developmental 14 

### effects (decreased fetal body weight and increased incidence of skeletal variations). The 15 

### NOAEL for the fetal effects is 1,454 mg/kg-day (1%)

. 16

### 39 DRAFT - DO NOT CITE OR QUOTE

McLain (2008) summarized a teratogenicity study by Bariliak et al. (1991) of n-butanol 1 

### and other alcohols administered by gavage. This st udy was published in Russian with a brief 2 

### English summary and was not translated for this rev iew.According to McLain (2008), groups 3 

### 10–16 white rats (weighing 160–180 g, strain not sp ecified) were given various alcohols 4 

### (methanol, ethanol, n-butanol, nonanol, and decanol ; purity not given) by gavage (1 mL of 40% 5 

### solution in water) from GDs 1 through 15. Controls (20 rats) were given water alone during the 6 

### same gestational treatment period. Without informa tion on the body weights of the pregnant 7 

### rats, estimating a dose associated with the adminis tered solution of n-butanol is uncertain. 8 

### Assuming a body weight of 0.250 kg and chemical den sity of 810 mg/mL, a dose of 1,300 9 

### mg/kg-day 3 

### was estimated. At sacrifice on GD 20, the numbers of corpora lutea and live and 10


### dead fetuses were counted. The review did not disc uss any maternal evaluations. Parameters 11 

### reported in the secondary source include: fertility index (description not provided), number of 12 

### implantations, percent pre- and post implantation l osses, and number of live fetuses. In addition, 13 

### ADH activity was measured in livers excised from se lected fetuses (1–2/litter). According to 14 

### McLain (2008), these measurements were performed da ily on fetuses from GDs 16 to 21 and on 15 

### postnatal days (PNDs) 1, 3, and 20. 16 

### Results of the study, as reported by McLain (2008), are shown in Table 4-8. According 17 

### to the review, treatment with n-butanol resulted in statistically significant increases in the 18 

### percents of pre- and post implantation losses and i n total fetal deaths. A decrease in the fertility 19 

### index of treated animals was reported in the review (6.5 vs. 9.7% in controls; statistical analysis 20 

### not reported). In addition, ADH activity in fetal livers, which was at its highest level on GD 20, 21 

### was reduced 77.6% at this measurement in offspring of dams exposed to n-butanol. No other 22 

### information was provided in the review or in the En glish summary in the publication. Effect 23 

### levels were not provided in the review. Given the lack of information from which to reliably 24 

### estimate dose, as well as reliance on a secondary s ource for information, EPA has not determined 25 

### effect levels for this study. 26

3 Calculated assuming % v/v in water as follows: 40% n-butanol (in 1 mL) = 0.4 mL/day × 810 mg/mL × (1/ 0.250 kg) = 1,300 mg/kg-day.

### 40 DRAFT - DO NOT CITE OR QUOTE

Table 4-8. Teratogenic observations in rats expose d to n-butanol by gavage during GDs 1–15

Historical control Control 1 mL of 40% n-butanol solution (dose estimated to be ~1,600 mg/kg-d)
Number of animals 362 20 10 Index of fertility 9.6 9.7 6.5 Number of corpora lutea 3,684 207 106 Number of implantations 3,668 203 83 Fetal deaths (%) 10.5 ± 0.5 a 6.3 ± 1.7 38.7 ± 4.7 b
Pre implantation 5.6 ± 0.4 2.0 ± 1.0 21.7 ± 4.0 b
Post implantation 5.2 ± 0.4 4.4 ± 1.4 21.7 ± 4.4 b
Number of live fetuses 3,476 194 65

a Mean ± SD.
b According to the review, “all indices of the embryo toxic activity in the experimental groups are relia bly higher than control (

p < 0.001)”.

Sources: Bariliak et al. (1991) as reported in McL ain (2008).
1


### 4.3.2. Inhalation Studies 2 

### Nelson et al. (1989a) studied the developmental tox icity of n-butanol ( ≥ 99% pure) and 3 

### other butanol isomers in rats exposed by inhalation . Groups of 15–20 sperm-positive female 4 

### Sprague-Dawley rats were whole-body exposed to n-bu tanol vapor at nominal concentrations of 5 

### 0, 3,500, 6,000, or 8,000 ppm (0, 11,000, 18,000, o r 24,000 mg/m 3 

### ), 7 hours/day on GDs 1–19. 6 

### Test concentrations were selected on the basis of p ilot testing. In the pilot experiment, exposure 7 

### to 27,000 mg/m 3 

### (9,000 ppm) n-butanol was lethal to two of six non pregnant rats within 2 days, 8 

### and exposure to 24,000 mg/m 3 

### (8,000 ppm) resulted in narcosis in half of tested dams. In the 9 

### main study, dams were evaluated weekly for food and water intake, and body weight was 10 

### measured on GDs 0, 7, 14, and 20. After sacrifice on GD 20, the numbers of corpora lutea, 11 

### resorptions (early, middle, or late), and live fetu ses were recorded. Fetuses were weighed, sexed, 12


### and examined for external malformations. Half of t he fetuses were examined for skeletal 13


### malformations and the other half for visceral malfo rmations. 14 

### In the highest exposure concentration of n-butanol, 2/18 dams died prior to sacrifice 15 

### (Nelson et al., 1989a). There were no deaths in th e low- or mid-exposure groups. The body 16 

### weights of dams in the 24,000 mg/m 3 

### group were approximately 17% less than controls at 17 

### termination (based on visual inspection of data pre sented graphically), although this difference 18 

### was not statistically significant when the authors adjusted for multiple comparisons using the 19 

### Bonferroni technique. However, the body weights we re slightly higher than controls in 11,000 20 

### and 18,000 mg/m 3 

### groups. Food consumption was lower in dams expose d to the mid- and high 21 

### concentrations (11–19% lower than controls at 18,00 0 mg/m 3 

### and 10–17% lower at 24,000 22 

### mg/m 3 

### ); the difference from controls was statistically s ignificant during all 3 weeks of treatment 23

### 41 DRAFT - DO NOT CITE OR QUOTE

at the mid concentration and during the 1st week on ly at the high concentration. Water intake 1 

### was reportedly higher in treated groups than in con trols, but the difference was not statistically 2 

### significant (data not reported). Exposure to n-but anol had no effect on number of corpora lutea, 3 

### resorptions or live fetuses/litter, or sex ratio. External malformations were not observed in any 4 

### group. Statistically significant concentration-rel ated reductions in body weight were observed in 5 

### male and female fetuses at the mid- and high-exposu re levels (12 and 24–27% lower than 6 

### controls in the 18,000 and 24,000 mg/m 3 

### groups, respectively). The percent of fetuses hav ing 7 

### normal skeletal development was statistically signi ficantly lower at 24,000 mg/m 3 

### n-butanol. 8 

### The authors indicated that rudimentary cervical rib s were the primary skeletal malformation 9 

### observed with n-butanol exposure but incidence data for the different exposures was not 10 

### provided by the authors. Statistical analysis of t he incidences of litters with skeletal or visceral 11 

### malformations or variations was not reported in the study. Based on Fisher’s exact tests 12 

### performed for this review, the incidence of litters with skeletal malformations was increased at 13 

### all concentrations, and the incidence of litters wi th visceral malformations was statistically 14 

### significantly increased at the highest concentratio n. Table 4-9 reports selected findings from the 15 

### study. The authors did not identify effect levels. determined that lowest concentration tested 16 

### (11,000 mg/m 3 

### or 3,500 ppm) was associated with an increase in t he incidence of litters with 17


### skeletal variations and is considered a LOAEL. 18

### 42 DRAFT - DO NOT CITE OR QUOTE

Table 4-9. Selected changes in rats exposed to n-b utanol via inhalation on GDs 1–19

Parameter n-Butanol exposure in mg/m 3 (ppm)

0 11,000

(3,500 ppm) 18,000

(6,000 ppm) 24,000

(8,000 ppm) Mortality 0/17 0/15 0/18 2/18 Number pregnant/number bred 15/17 13/15 18/18 15/16 Maternal food consumption during wk 1 (g) 124 ± 15 a 142 ± 17 102 ± 24 b 103 ± 24 b

Maternal food consumption during wk 2 (g) 134 ± 21 142 ± 16 108 ± 12 b 118 ± 17

Maternal food consumption during wk 3 (g) 124 ± 16 133 ± 16 110 ± 9 b 111 ± 17

Male fetal body weight (g) 3.4 ± 0.31 3.4 ± 0.18 3.0 ± 0.31 b 2.6 ± 0.25 b
Female fetal body weight (g) 3.3 ± 0.27 3.2 ± 0.18 2.9 ± 0.30 b 2.4 ± 0.23 b
Fetuses (litters) with skeletal malformations 0/102 (0/15) c 5/85 (4/12) d 8/129 (5/18) d

16/98 (9/15) d

Fetuses (litters) with skeletal variations 43/102 (14/15) 24/85 (11/12) 52/129 (17/18) 75/98 ( -) e

Percent of fetuses with normal skeletal development 100 ± 0 94 ± 3 94 ± 3 85 ± 4 b

Fetuses (litters) with visceral malformations 0/106 (0/15) 0/97 (0/13) 2/134 (2/18) 8/96 (4/15) d

Fetuses (litters) with visceral variations 7/106 (4/15) 8/97 (6/13) 6/134 (4/18) 19/96 (8/15)

Percent of fetuses with normal visceral development 100 ± 0 100 ± 0 99 ± 1 92 ± 4

a Mean ± SD.
b Significantly different from control at p < 0.05.

c Affected fetuses/fetuses examined (affected litters /litters examined).
d Significantly different from control at p < 0.05 by Fisher’s exact test performed for by EPA

. e The study reported that 25 of 15 litters had skelet al variations. This was assumed to be a reporting error.

Source: Nelson et al. (1989a).
1 

### Nelson et al. (1989b) evaluated behavioral teratolo gy in young rats following in utero or 2 

### paternal inhalation exposure to n-butanol vapor. G roups of 15 pregnant female Sprague-Dawley 3 

### rats were exposed to 0, 3,000, or 6,000 ppm (0, 9,0 00, or 18,000 mg/m 3 

### ) n-butanol for 7 4 

### hours/day on GDs 1–19 (termed “maternal exposure gr oup” by the authors). Groups of 18 male 5 

### Sprague-Dawley rats were exposed to the same concen trations of n-butanol for 7 hours/day for 6 6 

### weeks and then mated to non-butanol exposed females (termed “paternal exposure group” by the 7 

### authors). On the day of birth (PND 0), offspring w ere culled to four males and four females per 8


### litter and fostered to untreated controls. Individ ual pup weights were recorded weekly for 5 9 

### weeks. On PND 10, one male and one female pup/grou p were randomly assigned to one of four 10 

### testing groups. There were three groups assigned t o behavioral testing for neuromotor 11

### 43 DRAFT - DO NOT CITE OR QUOTE

coordination (ascent on a wire mesh screen on PNDs 10, 12, and 14 and rotarod, not mentioned 1 

### in the methods section), activity (open field activ ity and photoelectrically-monitored activity on 2 

### PNDs 16–18, 30–32, 44–46, and 58–60, and running wh eel activity on PNDs 32 and 33), and 3 

### learning (avoidance conditioning, with separate gro ups tested beginning PNDs 34 and 60 and 4 

### operant conditioning beginning PND 40). The fourth group was used for brain neurotransmitter 5 

### analysis. On PND 21, brains were removed from 10 p ups/group (one male and one female/litter) 6 

### and dissected into four regions (cerebrum, cerebell um, midbrain, and brainstem), these samples 7 

### were used to measure the levels of total proteins a nd neurotransmitters, including acetylcholine 8 

### (ACh), dopamine, norepinephrine, serotonin, met-enk ephalin, B-endorphin, and substance P. 9 

### The study authors indicated that, since exposures t o the two concentrations of n-butanol were 10 

### separated by about 5 months, results could be compa red to the contemporaneous controls, but 11 

### comparisons between the two concentrations would no t be appropriate. 12 

### Analysis of exposure concentrations showed consiste ncy with target exposures (method 13 

### of analysis not reported); measured concentrations were 3,010 ± 50 and 6,000 ± 80 ppm (9824 ± 14 

### 163 and 19580 ± 261 mg/m 3 

### , respectively; Nelson et al., 1990, 1989b). The s tudy authors 15 

### reported that n-butanol exposure did not affect pre gnancy rate in any exposure groups. There 16 

### were no behavioral changes in the offsping in term of their performance in ascent test, rotarod 17 

### performance, open field performance, or operant con ditioning. In offspring of 18,000 mg/m 3 18 

### paternal exposure group, the time receiving shock a nd the total number of times that rats crossed 19 

### from one side of the cage to the other were both st atistically significantly increased over controls. 20 

### Monitoring of photoelectric activity showed statist ically significantly lower counts in female 21 

### offspring of to the 9,000 mg/m 3 

### paternal exposure group but not in offspring of th e high 22 

### concentration paternal exposure group (data not sho wn, and magnitude of change not reported). 23 

### Tests for avoidance conditioning showed that male o ffspring of the 9,000 mg/m 3 

### paternal 24 

### exposure group required statistically significantly fewer trials to reach criterion than controls; no 25 

### statistically significant change was observed at th e high concentration. Analysis of 26


### neurotransmitter concentrations in the brains of of fspring revealed statistically significant 27


### increases in the overall concentration of serotonin (mean ± SEs were 14.48 ± 2.38 vs. 7.802 ± 28 

### 1.48 in controls; units not reported) and dopamine (0.715 ± 0.127 vs. 0.515 ± 0.095 in controls; 29 

### units not reported) in offspring of the 18,000 mg/ m 3 

### paternal exposure group. There were no 30 

### other statistically significant changes in neurotra nsmitter concentrations associated with 31 

### exposure to n-butanol. The study authors also indi cated that the changes in both the 32


### neurobehavioral tests and neurotransmitter concentr ations observed in the animals exposed to 33 

### n-butanol were within the range of control data fro m their laboratory. The authors did not define 34 

### effect levels. EPA determined that the highest con centration (18,000 mg/m 3 

### , or 6,000 ppm) 35 

### tested in this study is a NOAEL for neurobehavioral effects in offspring after parental inhalation 36 

### exposure to n-butanol. 37

### 44 DRAFT - DO NOT CITE OR QUOTE

In a study examining potential testicular toxicity, Cameron et al. (1985) exposed groups 1 

### of five male Sprague-Dawley rats to n-butanol at a concentration of 50 ppm (150 mg/m 3 

### ) 2 

### 6 hours/day for 1 day or 1 week. Testicular effect s were assessed through the measurement of 3 

### serum concentrations of testosterone, luteinizing h ormone (LH), and corticosterone in blood 4 

### samples collected at sacrifice at the end of exposu re or 18 hours after the end of exposure 5 

### following both the 1-day and 1-week exposures. In rats sacrificed after 1 day of exposure, 6 

### statistically significant reductions in testosteron e concentrations were observed; the decreases 7


### were 37 ± 8 and 52 ± 22% of controls when measured immediately after and 18 hours after 8 

### exposure, respectively. In rats sacrificed after 1 week of exposure, testosterone levels were 9 

### decreased (73–83% of controls), but the difference from controls was not statistically significant. 10 

### Serum concentrations of LH were higher than control s (115–124% of controls) in animals treated 11 

### with n-butanol for 1 day, but lower (78–98% of cont rols) in those treated for 1 week; none of the 12 

### differences were statistically significant. Rats t reated with n-butanol for 1 day had statistically 13 

### significantly higher serum corticosterone than cont rols (143% of control); however, rats treated 14 

### for 1 week had lower corticosterone (85% of control , not statistically significantly different from 15 

### control). 16

17


### 4.4. OTHER DURATION- OR ENDPOINT-SPECIFIC STUDIES 18 

### 4.4.1. Acute and Short-term ( ≤ 30 Days) Studies 19
Several studies determined LD 50 values for n-butanol following acute and short-ter m exposure. 20 

### Among rats, rabbits, hamsters, and mice, oral LD 50 

### s ranged from 1,200 mg/kg to 4,360 mg/kg 21 

### (Rumyantsev, 1979; Dubina and Maksimov, 1976; Munch , 1972; Purchase, 1969; Jenner et al., 22 

### 1964; Smyth et al., 1951; McOmie and Anderson, 1949 ; Munch and Schwartze, 1925). 23 

### Behavioral toxicities following or during an acute exposure included narcosis and necropsies 24


### indicated that there was necrosis of the liver and kidney as well as general organ congestion 25 

### (Munch, 1972; Purchase, 1969; Jenner et al., 1964; McOmie and Anderson, 1949; Munch and 26 

### Schwartze, 1925). 27 

### General short-term studies for n-butanol primarily evaluated effects on narcosis and 28 

### pathological effects in the liver and kidney. Deta iled summaries for these studies are provided in 29


### Table 4-10. 30

31 

### Table 4-10. Summary of Short-term Studies

Species/Sex Dose Duration Effect Observed Referenc e Mice 24,624 mg/m 3

inhalation 130 hours Narcosis and reversible fatty infiltration of the liver and kidneys Weese, 1928

### 45 DRAFT - DO NOT CITE OR QUOTE

Mice 22 mg/L (22,000

mg/m 3 ) 27 mg/L (27,000 mg/m 3 ) inhalation 12 hours

22.6 hours At 22 mg/L for 12 hours

2/10 animals died and 6/10 lost righting reflex.

At 27 mg/L for 22.6 hours 4/10 mice died.

Irritation was reported at 15 min, somnolence at 3 hours, anesthesia at 6 hours and death at 12 hours in saturated room air (concentration unknown). McOmie and Anderson, 1949

Mice/male 0 mg/m 3

0.78 ± 0.05 mg/m 3 6.6 ± 0.39 mg/m 3 40 ± 42 mg/m 3 30 days Statistically significant decrease in hexenal induced sleep duration atboth the mid and high nbutanol exposure levels. Kolenikov, 1975

Rats/male Various doses

(not reported) 60 min The threshold at which no change in rectal temperature was observed 60 minutes after dosing was reported as 2.5 nM/kg Mohler and Gordon, 1991

Cats 200 mg/kg i.v. Not reported Dose was lethal Macht, 1920

1

2 

### 4.4.2. Liver Toxicity 3 

### Videla (1982) administered a single oral dose of 56 0 mg/kg n-butanol (purity not 4 

### reported) to groups of four male Wistar rats. Leve ls of reduced glutathione (GSH), diene 5 

### conjugates, proteins, and ADH activity in the liver were measured 6 hours after dosing. Based 6 

### on visual inspection of data presented graphically, levels of GSH were lower, and diene 7 

### conjugates were higher in animals given n-butanol c ompared with saline-treated controls; 8 

### however, the differences were small and were not st atistically significant. ADH activity in 9


### n-butanol treated rat livers was about 41% of that in ethanol-treated rat livers; however, 10 

### information on controls was not reported. 11 

### Shehata and Saad (1978) evaluated the effect of ora l exposure to alcohols on vitamin 12 

### content in the livers of rats. Groups of six male albino rats were given daily gavage doses of 0, 13 

### 1, or 2 mL/kg (810 and 1,620 mg/kg) n-butanol for 7 consecutive days. The animals were 14


### sacrificed 2 hours after the last dose and the live r was assayed for content of thiamine, riboflavin, 15 

### pyridoxine, niacin, and pantothenic acid. At both doses of n-butanol, the liver content of all five 16 

### vitamins was statistically significantly lower than the corresponding control value. The 17 

### decreases, which were dose-related, ranged between 25 and 49% at the high-dose. 18

19 

### 4.4.3. Immunotoxicity 20 

### Strubelt and Zetler (1980) tested the antiinflammat ory effects of n-butanol and other 21 

### alcohols. Groups of eight male Wistar rats were gi ven single gavage doses of 0, 600, or 22

### 46 DRAFT - DO NOT CITE OR QUOTE

1,200 mg/kg n-butanol (analytical purity not furthe r specified), followed 15 minutes later with an 1 

### injection of 0.1 mL of 1% carageenan in the right h ind paw. Inflammation of the paw was 2 

### measured as mean difference from pretreatment paw v olume after 2, 3, 4, 5, and 5 hours 3 

### following injection. Exposure to n-butanol resulte d in reduced inflammation, as shown by a 4 

### statistically significant inhibition of the increas e in paw volume associated with carageenan 5 

### injection (19 and 15% smaller mean increase in paw volume compared with controls in the low6 

### and high-dose groups, respectively). Similar inhib ition of inflammation was observed with other 7 

### alcohols. In a separate experiment, groups of 12–1 6 male Wistar rats were treated with single 8 

### gavage doses of 0, 300, 600, or 1,200 mg/kg followe d by injection of 0.2 mL of 1% carageenan 9 

### into the pleural cavity. After a period of 4 hours , the rats were sacrificed and chest cavity was 10 

### opened. The volume of exudate was recorded, and le ukocyte count and osmolality of the 11 

### exudate were measured; blood osmolality was also me asured. Exposure to n-butanol at ≥ 600 12 

### mg/kg resulted in statistically significantly reduc ed volume of pleural exudate (53 and 13% of 13 

### control volume at 600 and 1,200 mg/kg, respectively ) and decreased leukocyte count (51 and 9% 14 

### of control count, respectively), indicating a reduc tion in the inflammatory response to 15 

### carageenan. The osmolality of pleural exudates was not affected by exposure. Significant 16 

### changes in blood osmolality were observed at the lo w- and high-doses, but not the mid-dose. 17 

### The blood osmolality changes were not consistent wi th dose; the low-dose resulted in a decrease 18 

### in osmolality, while the high-dose resulted in an i ncrease. This study suggested that oral 19 

### exposure to n-butanol results in anti-inflammatory effects. 20

21 

### 4.4.4. Neurotoxicity 22 

### Wallgren (1960) tested the behavior of 5-month-old rats (strain not reported, groups of 5– 23 

### 7/sex) exposed to a single oral dose of 0.0163 mol/ kg n-butanol (1,210 mg/kg). Beginning 24 

### 20 minutes after dosing, the animals were subjected to six “tilted plane” tests at 20-minute 25 

### intervals. In this test, the angle at which a rat slides after being placed head up on a tilted plane 26 

### was recorded. Results were reported as lowest and mean percentage of control performance; for 27 

### n-butanol, the lowest value in the six tests corres ponded to 55.8 ± 1.7% of control and the mean 28


### of the six tests was 73.3 ± 9.2% of control. Stati stical analysis was not reported. 29 

### Maickel and Nash (1985) treated groups of 23–25 mal e Swiss-Cox mice to oral doses of 30 

### 500, 1,000, or 2,000 mg/kg n-butanol and assessed b ody temperature (via rectal thermometer) 31 

### and rotarod performance in groups of five mice each at 10, 20, 40, 80, and 120 minutes after 32 

### treatment. Blood was collected for analysis of n-b utanol at the same time points. Treatment 33 

### with n-butanol resulted in dose-dependent decreases in body temperature that persisted through 34 

### 40 minutes after exposure in all groups and through 80 minutes in the mid- and high-dose 35 

### groups. n-Butanol doses of 1,000 and 2,000 mg/kg r esulted in dose-related decreases in rotarod 36 

### performance (65 and 25% of baseline, respectively, 10 minutes after exposure, based on visual 37 

### inspection of data presented graphically). Rotarod performance gradually improved over time 38

### 47 DRAFT - DO NOT CITE OR QUOTE

until it was 100% of baseline by 80 minutes (at 1,0 00 mg/kg) or 120 minutes (at 2,000 mg/kg) 1 

### after dosing. Exposure to n-butanol at 500 mg/kg d id not affect rotarod performance. 2 

### In a short-term study of a series of industrial sol vents, De Ceaurriz et al. (1983) exposed 3 

### groups of 10 male Swiss OF1 mice for 4 hours to n-b utanol (98.5% pure) concentrations of 0, 4 

### 470, 548, 844, or 965 ppm (0, 1,420, 1,660, 2,560, or 2,930 mg/m 3 

### ). Vapors were generated 5 

### using either an injector with added heat to increas e vaporization or by bubbling air through a vial 6 

### containing the test material (the authors did not s pecify which method was used for n-butanol). 7 

### After exposure, each animal was tested in a “behavi oral despair” swimming test, in which it was 8 

### immersed in water in a glass cylinder and observed for duration of immobility (passive floating). 9 

### A concentration-related, statistically significant decrease in the duration of immobility during the 10 

### first 3 minutes of testing (i.e., prolongation of e scape-directed activity) was observed at all of the 11 

### exposure concentrations; the percent decrease from control was 38, 47, 60, and 70% in the low 12 

### through high-exposure groups. The concentration of n-butanol resulting in 50% decrease in 13 

### immobility was reported to be 617 ppm (95% CI 547–6 81), or 1,870 mg/m 3 

### . According to the 14 

### authors, a decrease in immobility in this test is c haracteristic of compounds with antidepressant 15 

### activity. 16 

### In a study designed to investigate the potential ot otoxicity of solvent exposure, Crofton et 17 

### al. (1994) exposed groups of 10 adult male Long-Eva ns rats via whole-body inhalation to 18 

### n-butanol vapor concentrations of 0 or 4,000 ppm (1 2126 mg/m 3 

### ), 6 hours/day for 5 days. 19 

### Auditory function was tested via reflex modificatio n audiometry 5–8 weeks after exposure. 20 

### There was no effect of n-butanol exposure on the au ditory threshold of rats through the 21 

### frequency range that was tested (0.4–40 kHz). 22 

### Frantik et al. (1994) tested the acute neurotoxicit y of n-butanol in male albino SPF Wistar 23 

### rats and in female mice (H strain). Groups of four male rats were exposed (whole-body) to 24 

### n-butanol vapors for 4 hours, while groups of 16 fe male mice were exposed for 2 hours. The 25 

### tested concentrations were not reported, as the foc us of the analysis was to identify isoeffective 26


### concentrations for the different solvents. Within a minute after the exposure period ended, the 27 

### investigators applied a short electrical impulse th rough electrodes in the ears of the tested 28 

### animals to generate a tonic extension of hindlimbs in rats and in mice. The only n-butanol effect 29 

### information presented by the authors was the estima ted concentration evoking a 30% depression 30 

### in these parameters (3,500 ppm [10,000 mg/m 3 

### ] in rats and 2,400 ppm [7,300 mg/m 3 

### ] in mice). 31 

### A series of studies (Korsak and Rydzynski, 1994; Ko rsak et al., 1993) evaluated the acute 32 

### neurobehavioral effects of n-butanol in rats expose d via inhalation. In the first study (Korsak et 33 

### al., 1993), groups of male Wistar rats (10/group) w ere exposed for 4 hours to various 34 

### concentrations of n-butanol. Immediately after exp osure, the rats were tested for rotarod 35 

### performance, and an hour after exposure ended, spon taneous motor activity was assessed. The 36


### authors estimated the medial effective concentratio ns (EC 50 

### ) for the rotarod performance to be 37 

### 6,531 ppm (19,800 mg/m 3 

### ). In the tests of spontaneous motor activity, low concentrations of 38

### 48 DRAFT - DO NOT CITE OR QUOTE

n-butanol were associated with increased activity ( compared with control animals), while higher 1 

### concentrations reduced activity back to control lev els (based on data presented graphically). 2 

### The second study (Korsak and Rydzynski, 1994) also exposed groups of 10 male Wistar 3


### rats to various concentrations of n-butanol for 4 h ours, and tested rotarod performance 4 

### immediately after exposure. An hour after exposure ended, pain sensitivity (measured as latency 5 

### to paw lick response on a hot plate) was assessed. The authors estimated the EC 50 

### value for 6 

### disturbance of rotarod performance to be 7,559 ppm (22,920 mg/m 3 

### ) in this study; this is similar 7 

### to the value (6,531 ppm) estimated by Korsak et al. (1993). Hot plate behavior was also affected 8 

### by exposure. The EC 50 

### estimated by Korsak and Rydzynski (1994) for decre ased sensitivity to 9 

### pain was 5,901 ppm (17,890 mg/m 3 

### ) (95% CI 4,841–7,232 ppm). 10 

### In a study using i.p. administration of n-butanol, McCreery and Hunt (1978) compared 11 

### the acute neurotoxic potencies of 62 compounds in a n attempt to correlate potency with physical12 

### chemical parameter such as the membrane/buffer part ition coefficient. Male Sprague-Dawley 13 

### rats (number/group not specified) received a single i.p. injection of the compound in various 14 

### doses. Neurotoxic potency was assessed after dosin g (duration of observation not reported), and 15 

### characterized as the dose that caused pronounced im pairment of gait (ataxia) and motor 16 

### incoordination without causing the abdomen or pelvi s to drop (the authors termed this dose the 17


### ED 3 

### ). The ED 3 

### for n-butanol was 5.4 mmol/kg (400 mg/kg); by comp arison, the ED 3 

### values for 18


### the related alcohols ethanol and methanol were 32.6 and 109 mmol/kg, respectively. Neurotoxic 19 

### potency showed a strong inverse correlation with me mbrane:buffer partition coefficient in 20 

### alcohols up to a certain carbon number (C8 for stra ight-chain alcohols). 21

22 

### 4.4.5. Pulmonary Toxicity 23 

### Kristiansen et al. (1988) evaluated the sensory irr itation response in mice exposed to 24 

### n-butanol vapors by assessing the reflexive decreas e in respiratory rate that accompanies 25 

### pulmonary irritation. Three different exposures (n ormal inhalation, inhalation via tracheal 26 

### cannula, and i.p. injection) were tested in order t o explore whether effects on respiration resulted 27 

### from stimulation of receptors on the trigeminal (in the nasal mucosa) or vagal (in the lower 28 

### respiratory tract) nerves, or from depression of th e CNS. Groups of four male Ssc:CF-1 mice 29 

### were used; a control group was exposed to clean air . For the inhalation (normal and cannulated) 30 

### exposures, the mice were exposed to 0, 435, 2,500, 5,600, or 9,200 ppm (normal; or 0, 1,319, 31 

### 7,579, 16,976, or 27,890 mg/m 3 

### ) or 4,630, 5,400, 7,335, or 9,250 ppm (cannulated; or 14,036, 32 

### 16,370, 22,236, or 28,041 mg/m 3 

### ) of n-butanol (99.5% pure) for 30 minutes followed by a 20- 33 

### minute recovery period. Respiratory rate and relat ive tidal volume were measured continuously 34 

### using a pressure transducer attached to a body plet hysmograph into which each animal was 35 

### placed, with its head inserted into the exposure ch amber. Doses of 1.6, 4.1, 12.1, and 20.2 36 

### mg/mouse were administered to the groups exposed vi a i.p. injection immediately prior to 37 

### respiratory measurements for 50 minutes. The anima ls were observed for escape attempts as a 38

### 49 DRAFT - DO NOT CITE OR QUOTE

measure of CNS depression. The relationship betwee n percent decrease in respiratory rate and 1 

### the log of the exposure concentration was plotted a nd used to estimate the concentrations of 2


### n-butanol that would result in a 0 or 50% reduction in respiration rate (RD 0 

### and RD 50 

### , 3 

### respectively). 4 

### The experiments showed that normal inhalation of n- butanol resulted in a concentration5 

### related decrease in respiratory rate, with the maxi mum depression occurring in the first minute 6 

### (Kristiansen et al., 1988). The authors estimated RD 0 

### and RD 50 

### values of 233 and 11,696 ppm 7 

### (706 and 35,456 mg/m 3 

### ), respectively, for the first minute of inhalation exposure. After the first 8 

### minute, there was evidence of desensitization. The response after the first minute showed 9 

### concentration-dependent patterns; at concentrations below 3,000 ppm (9,094 mg/m 3 

### ), the 10 

### response leveled off, while above 3,000 ppm (9,094 mg/m 3 

### ), a secondary depression occurred. 11 

### The authors suggested that the secondary response o ccurred as a result of stimulation of 12 

### receptors in the lower respiratory tract, as a simi lar response occurred in the cannulated animals. 13 

### No evidence of CNS depression was evident in any of the inhalation-exposed groups; however, 14 

### the decrease in respiratory rate seen with i.p.-exp osed rats indicated CNS-mediated effects, as 15 

### this group had no direct exposure to the respirator y tract. Comparison between results in the 16 

### cannulated and i.p.-exposed groups showed a stronge r response in the cannulated group, leading 17 

### the authors to conclude that both CNS and lung rece ptors were involved in the secondary 18



### response. 19 

### Korsak and colleagues (Korsak and Rydzynski, 1994; Korsak et al., 1993) also evaluated 20 

### respiratory irritation in mice exposed via inhalati on. In the first study, groups of male Balb/C 21 

### mice (8–10/group) were exposed for 4 hours to vario us concentrations of n-butanol. Respiratory 22 

### rate was measured by plethysmograph before exposure , for 6 minutes during exposure, and for 23 

### 6 minutes after the end of exposure. Exposure to n -butanol resulted in a concentration24 

### dependent decrease in respiratory rate, with maximu m depression occurring during the 25 

### 1st minute of exposure. The authors estimated the RD 50 

### value to be 3,008 ppm (9,119 mg/m 3 

### ). 26 

### The second study (Korsak and Rydzynski, 1994) used the same protocol and estimated an RD 50 27 

### value of 4,300 ppm (13,000 mg/m 3 

### ), which compares favorably with the value estimate d by 28 

### Korsak et al. (1993). 29

30


### 4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF 31


### ACTION 32 

### 4.5.1. Genotoxicity Studies 33 

### A series of genotoxicity assays of n-butanol in bot h bacterial and mammalian systems 34 

### have produced primarily negative results (see Table 4-11). n-Butanol did not increase reverse 35


### mutations in Salmonella typhimurium (strains TA98, TA100, A1535, and TA1537) in the 36 

### presence or absence of microsomal enzyme activation (i.e., liver S9 preparations) (Jung et al., 37 

### 1992; Nakamura et al., 1987; Connor et al., 1985; M cCann et al., 1975). Obe and Ristow (1977) 38

### 50 DRAFT - DO NOT CITE OR QUOTE

did not observe an increase in the frequency of sis ter chromatid exchanges in the Chinese 1 

### hamster ovary cell, and no increase in the frequenc y of micronuclei was observed in Chinese 2 

### hamster cells (V79) treated with a concentration of 50 μL/mL of n-butanol (Lasne et al., 1984). 3


### When V79 cells were tested at a concentration of 0. 1 M n-butanol, an increased frequency of 4 

### polyploidy cells was observed, but survival at this concentration was only 36% (Onfelt, 1987). 5 

### Yoshiyama et al. (1973) showed that n-butanol selec tively suppresses the initiation of DNA 6 

### replication in irradiated Escherichia coli (W2252) cells. n-Butanol was also inhibited the 7 

### incorporation of uridine in HeLa cells (Obe et al., 1977), suggesting inhibition of RNA synthesis. 8 

### Using a cell-free system with calf thymus DNA, Obe et al. (1977) showed that n-butanol 9 

### concentrations of 1–4% inhibited RNA transcription in a dose-dependent fashion. 10

11 

### Table 4-11. Genotoxicity studies of n-butanol in v itro

Test system Endpoint Test conditions Results a

Dose b Reference Without activation With

activation a S. typhimurium TA98, TA100 Reverse

mutation Plate incorporation

assay – – 2,000 μg/plate Connor et al. (1985) S. typhimurium TA102 Plate incorporation
assay – – 5,000 μg/plate Muller et al. (1993); Jung et al

. (1992) S. typhimurium TA100, TA1535, TA1537, TA98 Plate incorporation
assay – – 10 μg/plate McCann et al. (1975)

S. typhimurium TA1535/pSK 1002 Umu gene

expression Umu test – – 27,000 μg/mL Nakamura et al. (1987)

E. coli (W2252) Inhibition of

DNA replication [ 14 C]-amino acids

added to E. coli cells prior to treatment with n-butanol; radioactive nucleic acids and proteins were then fractionized. + ND 0.8% Yoshiyama et al. (1973)

Chinese hamster lung cells (V79) Micronuclei 48-hr incubation – ND 50 μL/mL Lasne et al. (1984)
Chinese hamster lung cells (V79) Polyploidy Incubated with chemical for 3 hrs; harvested and examined 26 hrs later + ND 0.1 M Onfelt (1987)

Chinese hamster ovary cells (CHO) Sister

chromatid exchanges Treated 1 time/d for

7 d – ND 0.1% (v/v) Obe and Ristow (1977)

### 51 DRAFT - DO NOT CITE OR QUOTE

Table 4-11. Genotoxicity studies of n-butanol in v itro

Test system Endpoint Test conditions Results a

Dose b Reference Without activation With

activation a

HeLa cells RNA

synthesis; inhibition of uridine incorporation Treated with

increasing doses for 5 min prior to treatment with [ 3 H]-uridine, then incubated for 30 min and precipitated. + ND 0.25% Obe et al. (1977)

Cell-free system containing calf thymus DNA RNA

synthesis; inhibition of uridine incorporation RNA polymerase

added to system after n-butanol; transcription terminated 30 min later. + ND 1% Obe et al. (1977)

a Exogenous metabolic activation used.
b Lowest effective dose for positive results or highe st dose tested for negative or equivocal results. + = positive, − = negative, ND = no data 1 

### There was only one in vivo test of n-butanol genoto xicity. Bloom (1982) injected 2


### n-butanol into the inner shell membrane of chick em bryos to assess the frequency of sister 3 

### chromatid exchanges and chromosomal breaks. When t ested at a concentration of 10 μL, no 4 

### increases in the frequency of sister chromatid exch anges or chromosomal breakages were 5


### observed. 6

7 

### 4.5.2. Mechanistic Studies Evaluating Carcinogenic Parameters 8 

### Etique et al. (2004) cultured human breast cancer c ells (MCF-7) for 24 hours prior to 9 

### treatment with 0.1% n-butanol for 6 days. Cell pro liferation was evaluated, and P450-aromatase 10 

### activity (which is involved in estrogen synthesis a nd has been shown to be increased in breast 11 

### cancer) and estrogen receptor α (ER- α ) mRNA expression were measured using semiquantitat ive 12 

### real time polymerase chain reaction (RT-PCR). In a ddition, ER- α protein content was analyzed 13 

### using Western blot analysis. Exposure to n-butanol resulted in a statistically significant decrease 14 

### in cell proliferation (38% less than controls); n-B utanol did not modify the ER- α protein content. 15

16 

### 4.5.3. Mechanistic Studies Evaluating Neurological Effects 17 

### Lovinger et al (1989) demonstrated that n-butanol d ose-dependently inhibited the N18 

### methyl-D-aspartate (NMDA) glutamate receptor in mou se hippocampal neurons. Following 19 

### Lovinger et al (1989), there were many characteriza tion studies of the potential molecular sites 20 

### of action for n-alcohols in the central nervous sys tem. Table 4-12 summarizes and provides 21 

### experimental details on the electrophysiological st udies conducted with n-butanol on individual 22

### 52 DRAFT - DO NOT CITE OR QUOTE

neuronal ligand-gated ion channels expressed in in vitro cell lines ( Xenopus oocyte; human 1 

### embryonic kidney cells) or mixed neurotransmitter s ystems in primary neuronal cell cultures. It 2 

### was generally found that n-butanol dose-dependently and reversibly inhibited glutamate receptor 3 

### (NMDA, Kainate, and α -amino-3-hydroxyl-5-methyl-4-isoxazole-propionate [ AMPA]) function 4 

### (Akinshola, 2001; Peoples and Weight, 1999; Dildy-M ayfield et al., 1996; Lovinger et al., 1989) 5 

### and potentiated the function of inhibitory systems such as glycine and gamma-aminobutyric acid 6 

### (GABA) receptors (Mascia et al., 2000; Peoples and Weight, 1999; Ye et al., 1998; Dildy7 

### Mayfield et al., 1996; Nakahiro et al., 1991). Thr ee different studies (Zuo et al., 2003; Zuo et al., 8 

### 2001; Godden et al., 2001) examined the effects of n-butanol on nAChRs and it could be 9 

### concluded that n-butanol may act as a partial agoni st on this receptor class. A few studies 10 

### (Rusch et al., 2007; Stevens et al., 2005; Zhou et al., 1998) reported that n-butanol (11 – 22 mM) 11 

### potentiated the serotonin (or 5-HT [5 hydroxytrypta mine]) current. N-butanol was reported to 12 

### inhibit the function of voltage gated potassium cha nnels (Harris et al., 2003; Shahidullah et al., 13 

### 2003). However, when a residue in the S6 segment of the potassium channel was mutated from 14 

### a proline (P) to an alanine (A) [P410A], the n-buta nol potentiated channel function (Bhattacharji 15 

### et al., 2006). Studies with rat brain membranes in dicated that n-butanol interacted with delta16 

### specific, but not kappa, opioid receptor binding si tes (Hiller et al., 1984) and n-butanol increased 17 

### membrane fluidity and decreased membrane ATPase act ivity (Edelfors and Ravn-Jonsen, 1990). 1853 DRAFT - DO NOT CITE OR QUOTETable 4-12. Study details for mechanistic data andother studies in support of the mode of action forneurologicaleffectsReference Test systemNumber ofsamples/groupDose orconcentrationrange Study protocol ResultStudies of effects on ligand-gated ion channelsLovinger etal. (1989)Mousehippocampalneurons4 neurons/test 0.01–25 mM Neurons were co-exposed to 50 uMNMDA and n-butanol. Whole-cell patchclamp techniques were used to measurethe elicited current from the treatments.n-Butanol dose dependently inhibited the NMDAglutamate receptor response in the mousehippocampal neuron. The inhibition wasreversible following a 2 minute washout period.Nakahiro etal. (1991)Primary culturesof rat dorsal rootganglion neurons3–9 Neurons/test1–30 mM Neurons were exposed to n-butanol andGABA or after GABA current hadreached steady state, and the effect oncurrent was measured using whole cellpatch-clamp technique.Initial peak current (nondesensitized) evoked byGABA was increased with increasing n-butanolconcentration; no current induced in the absenceof GABA. Potency of current enhancement waspositively correlated with carbon chain length andmembrane:buffer partition coefficient forn-alcohols. In contrast, n-butanol at 30 mMinhibited the desensitized steady-state currentinduced by GABA.DildyMayfield etal. (1996)X. laevisoocytesexpressingrecombinantGABAA, NMDA,AMPA, or kainitereceptors4–12 Oocytes/test5–50 mMCultured oocytes expressing GABAA,NMDA, AMPA, or kainite receptorswere exposed to alcohols and effects oncurrent were measured using the twoelectrode voltage clamp technique.n-Butanol potentiated the function of GABAAreceptors composed ofα1β1orα1β1γ2Lsubunits,but inhibited the responses to NMDA, AMPA,and kainite.Ye et al.(1998)X. laevisoocytesor HEK-293 cellsexpressing wildtype or mutantglycine Rα1 orGABAρ1receptors orchimeric glycineRα1/GABAρ1receptor4–13 Oocytes/test10–20 mM Cells were exposed to n-butanol and theeffect on current was measured usingwhole cell patch-clamp technique.n-Butanol exhibited a dose-related potentiation ofglycine-induced currents in cells expressing wildtype glycine receptors and a dose-relatedinhibition of submaximal GABA-induced currentsin cells expressing wild type GABAρ1 subunits.In cells expressing chimeric receptors, n-butanolpotentiated GABA-induced currents and slightlyinhibited glycine-induced currents.54 DRAFT - DO NOT CITE OR QUOTETable 4-12. Study details for mechanistic data andother studies in support of the mode of action forneurologicaleffectsReference Test systemNumber ofsamples/groupDose orconcentrationrange Study protocol ResultZhou et al.(1998)neuroblastoma(NCB-20) cells4–11 Cells/test20 mM Cells were exposed to n-butanol and5-HT and the effect on current wasmeasured using whole cell patch-clamptechnique.n-Butanol exposure increased the initial slope, therise time, and the measured desensitization rate oflow concentrations of 5-HT (1–2 μM). It alsodecreased the measured desensitization rate ofcurrent evoked by 10 μM 5-HT, and increased therelative amplitude of steady-state to peak currentevoked by 2 or 10 μM 5-HT.Peoples andWeight(1999)Primary culturesof hippocampalneurons from15 to 17-d-oldfetal mice4–7 Neurons/test~0.0013–0.125 MNeurons were exposed to n-butanol withGABAor NMDA and effects on currentwere measured using the two-electrodevoltage clamp technique.n-Butanol enhanced the GABAA- activated ioncurrents and inhibited NMDA-activated currents.Anesthetic potency of n-butanol and other shortchain n-alcohols was more closely related toNMDA-modulated effects than GABAA-modulated effectsMascia et al.(2000)X. laevisoocytesexpressinghumanα1(wildtype) andα1(S267Q)(anestheticresistant mutant)glycine receptorsubunits3–9 Oocytes/test6.6–66 mM Cultured oocytes expressing humanα1andα1(S267Q) glycine receptor wereexposed to n-butanol, and glycine, andeffects on glycine receptor function wasmeasured using the two-electrodevoltage clamp technique.n-Butanol potentiated the glycine response inoocytes expressing wild type (α1) glycinereceptors, and modestly inhibited the response inthose expressing the anesthetic-resistant mutant(α1[S267Q]). Mortality of oocytes (2/5) wasobserved at the highest tested concentration.Akinshola(2001)X. laevisoocytesexpressingrecombinantAMPA GluR1and GluR3receptor subunits5–8 Oocytes/test1–1,000 mM Cultured oocytes expressing glutamateGluR1 or GluR3 receptor subunits weretreated with various concentrations ofalcohols, and inhibition of kainiteactivated ion-currents was measuredusing the two-electrode voltage clamptechnique.n-Butanol exhibited dose-related inhibition ofkainite-activated currents in oocytes expressingrecombinant AMPA GluR1 and GluR3 receptorsubunits.Godden et al.(2001)X. laevisoocytesexpressinghuman nAChreceptor subunitsα2,α4, andβ43–16 Oocytes/test0.0001–0.1 MCultured oocytes expressing nAChreceptor subunitsα2,α4, andβ4werebathed in alcohols with ACh substrate,and effect current was measured usingthe two-electrode voltage clamptechnique.n-Butanol potentiated the function of nAChreceptors composed ofα2β4subunits, but had noeffect on receptors composed of theα4β4combination. Molecular volume of alcohols wasshown to correlate with potency of effect on thereceptors.55 DRAFT - DO NOT CITE OR QUOTETable 4-12. Study details for mechanistic data andother studies in support of the mode of action forneurologicaleffectsReference Test systemNumber ofsamples/groupDose orconcentrationrange Study protocol ResultZuo et al.(2001)HEK-293expressing theα4β2 subunit ofneuronal nAChreceptors6 Cells/test 1–300 mM Cells were exposed to n-butanol withACh and the effect on current wasmeasured using whole cell patch-clamptechnique.n-Butanol (1 and 100 mM) exhibited a dosedependent inhibition on the effect of 30 μM ACh,and potentiated the effect at an n-butanolconcentration of 300 mM. At a concentration of30 mM, n-butanol slightly potentiated the effectof 10 μM ACh.Zuo et al.(2003)HEK-293expressing theα4β2 subunit ofneuronal nAChreceptors6 Cells/test 1–300 mM Cells were exposed to n-butanol andACh and the effect on current wasmeasured using whole cell patch-clamptechnique.Prolonged exposure to n-butanol resulted in smallcurrents blocked by the ACh channel blockermecamylamine or the receptor blocker dihydro-β-erythroidine, indicating that n-butanol acted as apartial agonist. At a high concentration (300mM), n-butanol potentiated the effects of lowACh concentrations (≤30 μM) and inhibited theeffects of high ACh (100–3,000 μM). A lowconcentration of n-butanol (10 mM) inhibitedeffects of ACh (10–3,000 μM).Stevens et al.(2005)X. laevisoocytesexpressing 5-HT3Aor 5-HT3ABreceptorsAt least threeoocytes/test11 and 22 mM Cultured oocytes expressing 5-HT3Aor5-HT3ABreceptors were exposed ton-butanol and dopamine, and effects oncurrent were measured using the twoelectrode voltage clamp technique.n-Butanol enhanced the currents induced by lowconcentrations of 5-HT (~EC10) and caused aslight decrease in the EC50for 5-HT. In cellsexpressing HT3A, the potentiation was muchhigher than in cells expressing the heteromeric5-HT3ABreceptor.Rusch et al.(2007)X. laevisoocytesexpressing5-HT3Aor5-HT3ABreceptors5–10 Oocytes/test21.6 mM Cultured oocytes expressing serotonin5-HT3Aor 5-HT3ABreceptors wereexposed to n-butanol and dopamine, andeffects on current were measured usingthe two-electrode voltage clamptechnique.n-Butanol strongly potentiated the peak currentselicited by dopamine in a dose-dependent fashionin oocytes expressing the 5-HT3Areceptor, andmodestly potentiated the currents in thoseexpressing the 5-HT3AB. Coexposure to n-butanoland octanol did not modify the potentiationcompared with n-butanol alone.Other mechanistic studies of neurological effectsHiller et al.(1984)Isolated rat brainmembraneExperimentsconducted intriplicate0.5% Effect of n-butanol on binding opioidbinding to kappa site tested by blockingmu and delta sites with appropriateligands. Kinetic studies were made toassess possible mechanism of selectiveinhibition of delta sites.n-Butanol did not inhibit kappa opioid bindingsites, but selectively inhibited binding to the deltasites. Kinetic studies indicated that the inhibitionis a result of a decrease in affinity of the bindingsite (increased dissociation rate) for the ligand.56 DRAFT - DO NOT CITE OR QUOTETable 4-12. Study details for mechanistic data andother studies in support of the mode of action forneurologicaleffectsReference Test systemNumber ofsamples/groupDose orconcentrationrange Study protocol ResultEdelfors andRavn-Jonsen(1990)Rat brainsynaptosomalmembranesExperimentsconducted intriplicate12.5, 25, and50% ofsaturation(solubilityreported to be912 mmol/L)Membranes were bathed in n-butanoland the membrane Ca2+/Mg2+ ATPaseactivity was measured spectrophotometrically. Membrane fluidity wasmeasured as incorporation of fluorescent1,6-diphenyl-1,3,5-hexatriene.n-Butanol decreased the membrane ATPaseactivity in a concentration-dependent. Similarly,membrane fluidity was increased with increasingn-butanol concentration.Bhattacharjiet al. (2006);Harris et al.(2003);Shahidullahet al. (2003X. laevisoocytesexpressing wildtype andselectivelymutated Shaw2voltage-gated K+channels fromDrosophilamelanogaster3–5 Oocytes/test1–100 mM Cultured oocytes expressing wild-typeand mutant (generated by S5 alaninescanning mutagenesis) Shaw2 K+channels were exposed to n-butanol, andthe effect on current was measured usingthe two-electrode voltage clamp, wholecell patch-clamp, or fast-concentrationclamp technique.n-Butanol inhibited the wild-type K+ channels,and several of the mutations modulated theinhibition associated with n-butanol. Mutation ata key site (P410A) in the S6 segment of thechannel changed the n-butanol response frominhibition into potentiation. Studies weredesigned to explore molecular features of thebinding site on the channel.AMPA =α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; ATPase = adenosine triphosphatase; 5-HT = 5-hydroxytryptamine; nACh = nicotinic acetylcholine;NMDA =N-methyl-D-aspartate

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4.5.4. Mechanistic Studies Evaluating Neurodevelop mental Effects 1 

### Kotter and colleagues (Kotter et al., 2000; Kotter and Klein, 1999) conducted a series of 2 

### studies comparing the effects of ethanol and butano ls on the proliferation of neonatal rat brain 3 

### astroglial cells. In experiments on astrocyte cell proliferation rates, the effects of alcohols on 4 

### proliferation were assessed by measuring [ 3 

### H]-thymidine incorporation after treatment of the c ell 5 

### cultures with alcohol and a mitogenic agent. Using fetal calf serum as the mitogen, Kotter and 6 

### Klein (1999) treated astrocyte cells with n-butanol at concentrations of 0.1, 0.3, or 1.0%. 7 

### Compared with cells treated with fetal calf serum a lone, cell proliferation was inhibited by ~40 8 

### and 70%, at 0.1 and 0.3% n-butanol (respectively); proliferation was completely inhibited at 1% 9 

### n-butanol. Similar results were observed using pla telet-derived growth factor and endothelin 1 10 

### (Kotter and Klein, 1999). 11 

### Kotter et al (2000) also measured astrocyte cell pr oliferation, but used phorbol ester as 12 

### the mitogen. At concentrations of 0.1 and 0.3%, n- butanol inhibited proliferation of astroglial 13 

### cells by ~50 and nearly 100% (based on visual inspec tion of data presented graphically), 14 

### respectively, compared with cells treated with phor bol ester alone. The potency of inhibition of 15 

### n-butanol was much stronger than that of ethanol; a t 0.3% ethanol, cell proliferation was 16 

### inhibited by only ~40%, compared with nearly 100% in hibition at the same concentration of 17


### n-butanol. 18 

### In two sets of experiments on the activation of ast roglial phospholipase D (PLD), 19 

### phospholipids in the cells were labeled by incubati ng the cells with [ 3 

### H]-glycerol for 24 hours 20 

### prior to treatment with a mitogen and alcohol (Kott er et al., 2000; Kotter and Klein, 1999). The 21 

### cultures were exposed to ethanol or butanols (0.1–1 .0%) for 5 minutes prior to isolation of 22 

### phospholipids and quantification of phosphatidic ac ide (PA) and phosphatidylcholine. When 23 

### fetal calf serum was used as the mitogen (Kotter an d Klein, 1999), treatment with n-butanol 24 

### statistically significantly increased the formation of phosphatidylbutanol and decreased the 25 

### formation of PA (~30–40% at 0.3% n-butanol based on visual inspection of data presented 26 

### graphically). As with the effect on cell prolifera tion, n-butanol exhibited a stronger effect on PA 27 

### formation than ethanol at the same concentration (~1 5% decrease in PA formation at 0.3% 28 

### ethanol). Similar results were observed when phorb ol ester was used as the mitogen (Kotter et 29 

### al., 2000). 30 

### Kotter et al. (2000) also measured protein kinase C (PKC) activity after alcohol 31 

### exposure; rat brain PKC was incubated with lipid mi celles, phorbol ester, phosphatidylserine and 32 

### alcohol for 5 minutes, followed by the addition of [ 32 

### P]-ATP. PKC activity was statistically 33 

### significantly reduced by both ethanol and n-butanol at a concentration of 0.3% (average of nine 34 

### experiments showed ~95 and 85%, respectively, of act ivity in the absence of the alcohols). 35 

### These studies demonstrate that, like ethanol, n-but anol inhibits the proliferation of 36 

### astroglial cells, probably by disrupting the PLD si gnaling pathway by formation of phosphatidyl- 37

### 58 DRAFT - DO NOT CITE OR QUOTE

butanols and concomitant reduction in PA formation. In all of the experiments reported, 1 

### n-butanol exerted a stronger inhibitory effect than ethanol at the same concentration. 2

3 

### 4.5.5. Mechanistic Studies Evaluating Liver Effect s 4 

### Carlson (1994a, b) showed that n-butanol can esteri fy fatty acids in vivo (in rats) and in 5 

### vitro (in the homogenized liver, lung, and pancreas of rats and rabbits). In the in vivo 6 

### experiment, n-butanol was administered via i.p. inj ection (1.0 mL/kg or 810 mg/kg) to male 7 

### Sprague-Dawley rats, and the animals were sacrifice d for analysis of butyl palmitate, butyl 8 

### stearate, and butyl oleate (butyl esters of fatty a cids) in the liver 1 or 6 hours after dosing. Buty l 9 

### palmitate and butyl stearate were detected in the l ivers of all 12 rats sacrificed after 1 hour, while 10 

### butyl oleate was detected in only 5 out of 12 rats. Of the four rats sacrificed after 6 hours, butyl 11 

### palmitate was detected in all, butyl stearate was d etected in three, and butyl oleate was not 12 

### detected in any. In the in vitro study, homogenize d liver, lung, and pancreas from rats and 13 

### rabbits were incubated with [ 14 

### C]-oleic acid (0.4 mM) and 0.2 M n-butanol for 45 m inutes. 14


### Esterified oleic acid was isolated from the mixture and radioactivity in the isolate was measured. 15 

### In rat liver, lung, and pancreas, the esterified ol eic acid content was 271.8, 58.3, and 2,170 16 

### nmol/g tissue/hour, respectively. Lower levels wer e observed in rabbit liver, lung, and pancreas: 17 

### 45.8, 33.5, and 487 nmol esterified [ 14 

### C]-oleic acid/g tissue/hour, respectively. These s tudies 18 

### demonstrate that n-butanol, like ethanol, can ester ify fatty acids. 19 

### McKarns et al. (1997) observed a correlation betwee n the octanol:water partition 20 

### coefficient, a measure of hydrophobicity, and relea se of LDH from rat liver epithelial cells 21 

### treated in vitro with short-chain aliphatic alcohol s. Cultures of WB rat hepatic epithelial cells 22 

### were incubated with one of several alcohols (includ ing n-butanol, 99.5% pure) for 1-hour 23


### durations at various concentrations. The concentra tion of LDH in the medium was assessed at 24 

### the conclusion of exposure as a measure hepatotoxic ity. n-Butanol was tested at concentrations 25


### between 0.05 and 0.2 M to establish a concentration -response curve for the purpose of estimating 26


### the LDH 50 

### (concentration eliciting a 50% increase in LDH ove r control values) and EC 50 27 

### (concentration eliciting 50% of maximum LDH release ). LDH 50 

### and EC 50 

### were plotted against 28 

### previously reported partition coefficients. The LD H 50 

### and EC 50 

### for release of LDH by n-butanol 29 

### were both 0.16 M. The authors observed a strong po sitive correlation between release of LDH 30 

### and octanol:water partitioning (r = 0.993) and deri ved QSAR equations for both LDH 50 

### and EC 50 31 

### that were nearly identical. The study authors post ulated that the observed relationship implies 32 

### that the alcohols’ ability to disrupt membrane inte grity is nonspecific, as it is not affected by 33 

### molecular shape. 34 

### Deters et al. (1998a) demonstrated that, unlike eth anol, metabolism of n-butanol by ADH 35 

### was not a necessary step in the hepatotoxic action of this compound. The study authors used 36 

### 4-MP to inhibit ADH in isolated perfused rat livers (from male Wistar rats) exposed to various 37 

### alcohols at 130.2 mmol/L. Liver damage was assesse d by measuring leakage of GPT, LDH, and 38

### 59 DRAFT - DO NOT CITE OR QUOTE

GLDH into the perfusate and by measuring oxygen con sumption, bile secretion, perfusion flow, 1


### and concentrations of ATP and GSH in the liver. Ex posure to n-butanol alone resulted in a 2 

### statistically significant change from control for a ll of the above parameters with the exception of 3 

### GSH content of the liver. Exposure of the isolated livers to ethanol in the presence of 4-MP 4 

### resulted in little or no evidence of liver injury; bile secretion was diminished, but other 5 

### parameters were attenuated by 4-MP. In contrast, t here were no differences between the effects 6


### observed in livers treated with n-butanol with or w ithout 4-MP in the perfusate. This study 7 

### demonstrates that the hepatotoxicity of n-butanol m ay not be related to metabolism by ADH. 8 

### Deters et al. (1998b) examined whether glycine, whi ch had previously been shown to 9 

### reduce hypoxia-related effects on the liver, would mitigate the hepatotoxic effects of n-butanol 10 

### and several other liver toxicants. Using the isola ted perfused liver system described earlier, and 11 

### the same measures of hepatotoxicity, the study auth ors tested n-butanol with and without 12 

### glycine. Exposure of the isolated livers to n-buta nol resulted in statistically significantly 13 

### increased release of GPT and LDH into the perfusate , decreased bile flow, decreased oxygen 14 

### consumption and hepatic ATP content, decreased GSH in the liver, and increased levels of MDA 15 

### in the liver. Deters et al. (1998b) observed that inclusion of glycine reduced the release of GPT 16 

### and LDH into the perfusate; levels with glycine wer e similar to those observed in the control 17 

### systems. In addition, when glycine was included, t he oxygen consumption in the liver was 18 

### increased compared with the n-butanol treatment wit hout glycine; oxygen consumption in the 19 

### livers treated with both n-butanol and glycine was similar to that of controls. Finally, glycine 20


### modulated the effect of n-butanol on GSH and MDA co ntent in the liver; levels of both were 21


### similar to controls in the livers treated with n-bu tanol in the presence of glycine. Glycine did not 22 

### affect the inhibition of bile flow induced by n-but anol. 23 

### Strubelt et al. (1999) assessed evidence of injury to perfused livers treated with 24 

### 23 aliphatic alcohols including n-butanol. Liver i njury was assessed by measuring release of 25 

### GPT, LDH, and GLDH into the perfusate; oxygen consu mption, bile flow, and perfusion flow; 26 

### ATP, GSH, oxidized glutathione (GSSG), and MDA in t he liver; and lactate and pyruvate 27 

### concentrations in the perfusate. The alcohol expos ures all used a concentration of 65.1 mmol/L 28 

### added to the perfusate for 120 minutes. Exposure o f perfused livers to n-butanol resulted in 29 

### statistically significantly increased GPT and LDH ( more than eightfold higher than controls for 30 

### each) in the perfusate. The study authors reported that the potency of effects on LDH, GPT, and 31 

### GLDH was strongly correlated with carbon chain leng th among straight-chain aliphatic alcohols 32 

### (r = 0.82–0.87). In addition, n-butanol exposure r esulted in decreased bile flow and perfusion 33 

### flow (2 and 57% of control values, respectively), a nd an increase in the lactate/pyruvate ratio 34 

### (increased lactate and decreased pyruvate). Treatm ent with n-butanol did not result in 35 

### statistically significant changes in ATP, GSH, GSSG , or MDA content of the livers. 36

37

38

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4.5.6. Other Mechanistic Studies 1 

### Krill et al. (1993) examined the effects of n-butan ol, n-propanol, and isopropanol on 2 

### thermotropic behavior of the stratum corneum of hai rless mice and a model multilamellar vesicle 3 

### system. The study was designed to examine the mech anisms for alcohol-related enhancement of 4 

### lipophilic solute permeation across the skin. The model vesicle system was prepared from the 5 

### phospholipids distearoylphosphatidylcholine and dis tearoylphosphatidic acid. Stratum corneum 6


### sheets were removed from the abdomen of male hairle ss mice (strain SKH-HR-1). Fourier 7 

### transform infrared spectroscopy was used to measure effects of the perdeuterated alcohols on 8 

### stratum corneum lipid alkyl chain packing, mobility , and conformational order. Exposure of 9


### hairless mouse stratum corneum to 3% n-butanol resu lted in increased lipid chain freedom of 10 

### motion above 45°C and decreased the alkyl chain fre edom of motion below 45°C, but did not 11 

### alter the stratum corneum lipid interchain interact ions or gel-gel phase transition. The study 12 

### authors concluded that their experiments were consi stent with the hypothesis that alcohols 13 

### disrupt the polar head plane and increase the inter facial area of lipids, which enhances 14 

### penetration of some solutes across the stratum corn eum. 15 

### Kowalczyk et al. (1996) exposed two-cell preimplant ation mouse embryos in culture to 0, 16 

### 0.05, 0.1, 0.2, or 1.0% (w/v) n-butanol for 24 hour s and followed the development of the 17 

### embryos after 5 days. The number of embryos reachi ng blastocyst stage by day 5 was 18 

### statistically significantly reduced at all concentr ations of n-butanol. The reductions, which 19 

### ranged from ~2 to 35% of control values, were not do se-dependent; the maximum reduction 20


### occurred at the lowest concentration. This result was the opposite of that observed with ethanol, 21 

### which accelerates embryogenesis (increased number o f blastocytes). In a separate experiment, 8- 22 

### cell mouse morulae were pretreated with fluo-3-acet oxymethyl ester (a fluorescent indicator for 23 

### intracellular calcium) and then exposed to 0.1 or 1 .0% n-butanol. Fluorescence intensity was 24 

### measured before and immediately after exposure and used to calculate calcium concentration. 25 

### n-Butanol exposure statistically significantly incr eased the intracellular calcium concentration of 26 

### the morulae at the high concentration but not at th e low concentration (~sixfold increase at 1%). 27 

### The study authors concluded that the increased rele ase of calcium was a result of substantial 28 

### disruption of membrane integrity and was a possible cause of the embryotoxicity observed in the 29 

### first experiment. 30 

### Gastaldi et al. (1991) exposed small intestinal mic rovillous vesicles obtained from adult 31


### Wistar rats to butanol in vitro to assess the effec ts on vesicular morphology. The study did not 32 

### specify whether n-butanol or t-butanol was used. T he vesicles were incubated for 30 minutes 33 

### with butanol at a concentration of 3% (v/v). After exposure, the vesicles were fixed for 34 

### morphometric analysis (parameters were diameter of the area-equivalent circle, minimum to 35 

### maximum diameter ratio [shape factor], and vesicula r volume) by electron microscopy. A 36 

### statistically significant increase in mean vesicula r volume was observed with butanol exposure, 37 

### and the study authors attributed this change to the fluidizing effect of the alcohol. 38

### 61 DRAFT - DO NOT CITE OR QUOTE

Gordon et al. (1995) compared the effects of severa l n-alcohols (methanol, ethanol, 1 

### n-butanol, and 1-propanol) on isolated cannulated r at intracerebral arterioles (ex vivo). 2


### Concentrations of 1–100 mM of the n-alcohols were t ested, and change in arteriolar diameter 3 

### was measured. n-Butanol exposure resulted in dose- related increases in vessel diameter 4 

### (vasodilation) ranging from ~15% (at 1 mM) to ~50% ( at 100 mM) increase over the basal 5


### diameter. 6 

### Arsov et al. (2005) exposed erythrocyte ghosts prep ared from bovine blood to n-butanol 7


### in an effort to determine the mechanism of n-butano l-induced inhibition of erythrocyte 8 

### acetylcholinesterase (AChE) at high concentrations. AChE activity was measured with and 9 

### without different concentrations of n-butanol (0.07 , 0.15, 0.22, and 0.37 M) and at different 10 

### temperatures. At 0.07 M and a temperature of ≥ 310°K, there was no inhibition of AChE; at 11 

### higher concentrations, n-butanol inhibited AChE act ivity in the temperature range tested (up to 12 

### 320°K). In experiments in which the n-butanol was removed by dialysis after the exposure, 13 

### reversibility of AChE inhibition was shown at 0.22 M n-butanol, but at 0.37 M, AChE inhibition 14


### was still evident after the alcohol was removed. T he study authors used the experimental results, 15 

### along with published information, in kinetics studi es to estimate the number of molecules that 16 

### bind to the AChE enzyme. They concluded that, sinc e the activity equation that best fit the data 17 

### was the inverse of a second order polynomial, two n -butanol molecules likely bind to AChE. 18 

### Ding and Badwey (1994) examined the effects of n-bu tanol on four uncharacterized 19 

### protein kinases that are activated by the chemoattr actant fMet-Leu-Phe. When exposed to this 20 

### chemoattractant, neutrophils exhibit shape changes, chemotaxis, degranulation, and release of 21 

### superoxide. The study authors measured the release of superoxide from cultured guinea pig 22 

### peritoneal neutrophils treated with 20–70 mM n-buta nol and fMet-Leu-Phe (1 μm). n-Butanol 23 

### exposure resulted in a concentration-related inhibi tion of superoxide release (from about 30% 24


### inhibition at 20 mM to about 90% inhibition at 70 m M compared with cells not exposed to 25 

### n-butanol). Addition of four β -phorbol 12-myristate 13-acetate (PMA), a PKC activ ator, to cells 26


### treated with 55 mM n-butanol restimulated the relea se of superoxide, counteracting the 27 

### inhibitory effects of n-butanol. Without PMA, n-bu tanol at 55 mM resulted in 74% inhibition of 28 

### superoxide release, while the same concentration wi th PMA resulted in only 27% inhibition. 29 

### While this study was primarily aimed at elucidating the mechanism of action of the novel protein 30 

### kinases, it does suggest that n-butanol exposure ma y interfere with immune cell signaling. The 31 

### potential impact on immune function, however, is un certain. 32

33


### 4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS 34


### 4.6.1. Oral 35


### Information on the effects of n-butanol in humans f rom oral exposure is limited to one 36 

### case report from a suicide attempt (Bunc et al., 20 06) where neurological, gastrointestinal, and 37

### 62 DRAFT - DO NOT CITE OR QUOTE

cardiovascular symptoms were noted. In animal stud ies, there are short-term, subchronic, 1 

### reproductive and developmental toxicity studies tha t reported effects. 2 

### Among rats, rabbits, hamsters, and mice, oral LD 50 

### s ranged from 1,200 mg/kg to 4,360 3 

### mg/kg (Rumyantsev, 1979; Dubina and Maksimov, 1976; Munch, 1972; Purchase, 1969; Jenner 4 

### et al., 1964; Smyth et al., 1951; McOmie and Anders on, 1949; Munch and Schwartze, 1925). 5 

### Behavioral toxicities following or during an acute exposure included narcosis and necropsies 6


### indicated that there was necrosis of the liver and kidney as well as general organ congestion 7 

### (Munch, 1972; Purchase, 1969; Jenner et al., 1964; McOmie and Anderson, 1949; Munch and 8 

### Schwartze, 1925). 9 

### Short term study evaluations reported neurotoxic , hepatoxic, and antiinflammatory 10 

### effects. Acute oral dosages of n-butanol resulted in impaired performance in the tilted plane test 11 

### at 1,210 mg/kg in rats (Wallgren, 1960) and dose-re lated decreases in rotarod performance in 12 

### Swiss-Cox mice orally dosed with 1,000 and 2,000 mg /kg (Maickel and Nash, 1985). Liver 13 

### toxicity was also noted in a study where decreased thiamine, riboflavin, pyroxidine, niacin, and 14 

### panthothenic acid content was noted in rats orally gavaged with 1 or 2 mL/kg (810 or 1,620 15 

### mg/kg) n-butanol for 

### 7 days (Shehata and Saad, 1978). In rats, oral dos es of ≥ 600 mg/kg 16 

### resulted in a reduction of inflammatory response wh en treated with carageenan, a pro17 

### inflammatory agent (Strubelt and Zetler, 1980). 18 

### CNS effects including hypoactivity and ataxia (RTI; unpublished, 1985) and hepatic and 19 

### renal effects (Munoz et al., 1991; Wakabayashi et a l., 1991; Munoz et al., 1990) were reported in 20


### three subchronic studies. Table 4-11 summarizes th ese studies. In the 13 week oral gavage 21 

### study in rats (RTI; unpublished, 1985), the 500 mg/ kg-day dose (highest in the study) resulted in 22 

### hypoactivity and ataxia that lasted for up to one h our after dosing. Statistically significant 23 

### decreases in the hematocrit, erythrocyte count, and hemoglobin content were also reported for 24 

### females dosed with 500 mg/kg-day and there was a st atistically significant increase (~14%) in 25 

### thyroid weight in males dosed with 500 mg/kg-day. M ale rats exposed to 4,400 mg/kg-day for 4 26 

### months in a drinking water study (Munoz et al.,1991 ; 1990) were reported to have severe 27 

### pathological changes in the liver and kidney. Simi larly, hepatic effects such as proliferation of 28 

### smooth endoplasmic reticulum and enlarged mitochond ria in a 3 month drinking water study 29 

### with male rats exposed to 8,200 mg/kg-day (Wakabaya shi et al., 1991). 30 

### There were two oral developmental toxicity studies, Sitarek et al. (1994) and Ema et al., 31 

### (2005), that are summarized in Table 4-13. Sitarek et al (1994) reported an increased incidence 32 

### of visceral malformations including malformations i n the brain (dilation of the lateral and/or 33 

### third ventricle of the brain and/or arachnoid space ) and kidney (renal pelvis) in the offspring of 34 

### female Wistar Imp:DAK rats treated with n-butanol i n drinking water at a dose of 300 mg/kg35 

### day administered for 8 weeks prior to mating and th roughout mating and gestation. Maternal 36 

### effects were not observed at doses up to 5,000 mg/k g-day by Sitarek et al. (1994). In another 37 

### developmental study, Ema et al. (2005) did not obse rve an increased incidence of any 38

### 63 DRAFT - DO NOT CITE OR QUOTE

malformation in Sprague-Dawley rats exposed to n-bu tanol in drinking water throughout 1 

### gestation at doses up to 5,654 mg/kg-day. Maternal and fetal toxicity, manifested as body weight 2 

### reductions, were observed at the high-dose in this study (5,654 mg/kg-day) (Ema et al., 2005). 3 

### The discrepancy between the results of Ema et al. ( 2005) and Sitarek et al. (1994) may be due to 4 

### differences in study design, particularly rat strai n used and exposure regimens. In addition, 5 

### dilation of the subarachnoid spaces and the ventric les in the brain are precursors of 6 

### hydrocephalus (Raimondi, 1994). Sitarek et al. (19 94) reported internal hydrocephalus in the 7 

### fetuses from the 1,000 and 5,000 mg/kg-day dose gro ups along with the observed increased 8 

### incidence of visceral malformations including malfo rmations in the brain (dilation of the lateral 9


### and/or third ventricle of the brain and/or arachnoi d space) at ≥ 300 mg/kg-day. Observation of 10 

### hydrocephaly and dilation of neuronal structures (s ubarachnoid space and lateral ventricle) as 11 

### reported in Sitarek et al (1994) is also consistent with effects observed with ethanol (Sakata12 

### Haga et al., 2004). Therefore, the discrepancies i n results between the developmental toxicity 13 

### studies of Sitarek et al (1994) and Ema et al (2005 ) is likely due to differences in strain 14 

### sensitivity. 15 

### Overall, the most sensitive toxicological effect wa s reported in Sitarek et al (1994) where 16 

### increased developmental visceral malformations were observed in rat pups at the 300 mg/kg-day 17 

### dose. At comparable exposures, neurotoxicological effects were observed in adult rats at 500 18 

### mg/kg-day and no effects were observed at 125 mg/kg -day (RTI; unpublished, 1985). 1964 DRAFT - DO NOT CITE OR QUOTETable 4-13. Summary of oral noncancer dose-response information for n-butanolSpecies andstudy type ExposureNOAEL(mg/kg-d)LOAEL(mg/kg-d) Responses at the LOAEL Comments ReferenceSubchronic or chronic studiesRatstrain notspecified30/sex/doseGavage0, 20, 125, or500 mg/kg-d dailyfor 13 wks125 500 Clinical signs (hypoactivityand ataxia) and effects onhematology (reducedhematocrit, erythrocyte count,and hemoglobin content)RTI (1985), unpublishedWistar rat15 males/groupdrinking water0 or ~8,200 mg/kg-dfor up to 3 moCould not bedeterminedCould not bedeterminedStructural changes of hepaticmitochondriaData not reported.Evaluation limited to clinicalsigns, body weight, andelectron microscopy of liverslices.Wakabayashi et al.(1991)Wistar rat3 males/groupdrinking water0 or 4,400 mg/kg-d,7 d/wk for 4 moCould not bedeterminedCould not bedeterminedUnspecified liver and kidneydamage; serum chemistrychangesPathology data not reported.Evaluations limited tomortality, body weight,serum chemistry, andunspecified pathologyevaluations.Munoz et al. (1991,1990)Reproductive and developmental studiesWistar Imp:DAKrat11–17 females/groupdrinking water0, 300, 1,000, or5,000 mg/kg-d for8 wks prior tomating, throughmating and gestation5,000(maternal)NA(developmental)NA(maternal)300 (developmental)Increased incidences ofmalformations (dilation of thelateral and/or third ventricle ofthe brain and the subarachnoidspace)No maternal effects. Sitarek et al. (1994)Sprague-Dawleyrat20 females/groupdrinking water0, 316, 1,454, or5,654 mg/kg-d onGDs 0–201,452(maternal anddevelopmental)5,654(maternal anddevelopmental)Decreased body weight gain,food consumption, and waterconsumption (maternal);decreased fetal body weight(developmental)Ema et al. (2005)

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4.6.2. Inhalation 1


### The inhalation database contains a few acute human exposure experiments and occupational 2 

### health studies. Controlled human exposure experimen ts have demonstrated that acute exposures to 3 

### n-butanol vapors can exert an irritant effect on th e eyes, nose, and throat under acute exposure 4 

### conditions of up to 90 minutes (Kjaerguard et al., 1997; Nelson et al., 1943; Wysocki et al., 1996, as 5 

### cited in McLain, 2008 and ACGIH, 2002; Cometto-Muni z and Cain, 1995). A number of occupational 6


### health studies have also been conducted which exami ned neurological and neurobehavioral effects in 7 

### workers who were exposed to mixtures of solvents in cluding n-butanol (see Table 4-2). However, 8 

### exposure to n-butanol in these studies was low comp ared to other solvents, and a number of the other 9


### solvents are known to exert neurotoxic effects. Th erefore, it is not possible to conclusively attribu te any 10 

### of the effects to n-butanol exposure. Table 4-14 s ummarizes the available human occupational data. 1166 DRAFT - DO NOT CITE OR QUOTE1Table 4-14. Summary of inhalation noncancer dose-response information for n-butanolSpecies and studytype (n/sex/group) ExposureNOAEL(mg/m3)LOAEL(mg/m3) Responses at the LOAEL Comments ReferenceHuman occupational health studies35 rubber factoryworkers;gender not specified15–300 mg/m315–4246–200 Eye irritation LOAEL identified for factorywithcoexposure to diacetone alcohol.Cogan et al.(1945);Tabershawet al. (1944)16 male photographicpaper factory workers300 or 600 mg/m3300 600 Eye irritation Irritation not observed after n-butanolconcentration reduced from 600 to300 mg/m3.Sterner et al.(1949)11 exposed celluloseacetate factoryworkers;sex not specified47 workers inreferent group withsimilar age andduration ofemployment240 mg/m3NA 240 Hearing loss, chronicbronchitisSampling protocol for n-butanol wasnot described.Velazquez et al.(1969)Subchronic or chronic animal studiesWistar rat12 male/exposure;24 controls0, 154, or308 mg/m36 hr/d, 5 d/wk for3 months154 308 Impaired rotarodperformance; increasedleukocyte countHistopathology not evaluated. Korsak et al.(1994)Guinea pig3 group; sexunspecified0, 300 mg/m33 monthsN/A 300 Decreased erythrocyte countand hemoglobin.Histopathological changes inliver and kidneyData and methodological details arenot provided for the observed effects.Smyth and Smyth(1928)Mice and ratsNo details providedfor number/exposure,sex, or methods0, 0.8, 6.6, 40mg/m34 months0.8 6.6 Mice: Increased thyroidgland activity. Disturbancesof pituitary-adrenal system.Rats: Increased bloodcholinesterase activity.Only list of effects were provided inthe report. No detailed informationwas provided on the analyses of thedata.Rumyantsev et al.(1979)67 DRAFT - DO NOT CITE OR QUOTETable 4-14. Summary of inhalation noncancer dose-response information for n-butanolSpecies and studytype (n/sex/group) ExposureNOAEL(mg/m3)LOAEL(mg/m3) Responses at the LOAEL Comments ReferenceReproductive/developmental animal studiesSprague-Dawleyrat;15–20 females/group0, 11,000, 18,000,or 24,000 mg/m37 hrs/d on GDs 1–1911,000(maternal)NA(developmental)18,000(maternal)11,000(developmental)Reduced food consumption(maternal)Increased incidence of litterswith skeletal malformations(developmental)Maternal narcosis and mortality wereobserved at 24,000 mg/m3(FEL).Nelson et al.(1990, 1989a)Sprague-Dawley rat;15–20 females and18 males/group0, 9,000,18,000 mg/m37 h/d on GDs 1–19(females)or for 6 wks prior tomating withnonexposedfemales (males)18,000 NA None. No neurobehavioral effects inoffspring, regardless of whethermothers or fathers exposed.Nelson et al.(1990, 1989b)

### B-1. DRAFT - DO NOT CITE OR QUOTE 1 

### Three studies of n-butanol exposure in rats are ava ilable including one subchronic toxicity study 2 

### of male rats in which histopathology was not assess ed (Korsak et al., 1994), a developmental toxicity 3 

### study (Nelson et al., 1989a), and a neurodevelopmen tal toxicity study (Nelson et al., 1989b). In 4 

### addition, several short-term inhalation exposure st udies have characterized toxicities associated with n5


### butanol. 6 

### Specifically, fatty infiltration of liver and kidne y were observed in mice exposed to 24,624 7 

### mg/m 3 

### for 130 hours (Weese, 1928). Neurotoxic effects s uch as decreased duration of immobility were 8 

### reported at exposures of 1420 mg/m 3 

### and higher for 4 hours in mice (DeCeaurriz et al., 1983), and 9 

### decreased responding (Frantik et al., 1994) in rats and mice following a 2-4 hour exposure. Respirati on 10 

### rates declined in Ssc:CF-1 mice exposed to exposure of 435 ppm (1,319 mg/m 3 

### ) and higher for 30 11 

### minutes (Kristiansen et al., 1988) as well as in ba lb/C mice with a 50% respiratory decline following 4 12 

### hour exposures to 3,008 ppm (9119 mg/m 3 

### ; Korsak et al., 1993) or 4,300 ppm (13036 mg/m 3 

### ; Korsak 13 

### and Rydzynski, 1994). 14 

### There are a few subchronic inhalation toxicity stud ies evaluating the effects of n-butanol (Korsak 15 

### et al., 1994; Rumanystev et al., 1979; Smyth and Sm yth, 1928). Hematological effects, primarily 16 

### decreased erythrocyte count, were noted in all thre e subchronic studies. The lowest exposure at which 17 

### hematological changes were observed was at 6.6 mg/m 3 

### in rats where increased blood cholinesterase 18 

### activity was noted (Rumyantsev et al., 1979). Howe ver, it is unclear if the hematological changes are 19 

### biologically relevant. The other primary toxicolog ical effect observed in the subchronic exposure 20 

### studies was deficits in neurobehavioral performance . Korsak et al (1994) reported a statistically 21 

### significant increase in the failure rate for the ro tarod test in rats exposed to 308 mg/m 3 

### n-butanol for 3 22


### months. 23 

### Nelson et al (1989a, b) evaluated the developmental and neurodevelopmental toxicity potential 24


### of n-butanol in rats. Increased incidence of litte rs with skeletal variation was observed at exposure s of 25 

### 3,500 ppm (11,000 mg/m 3 

### ) and increased levels of serotonin and dopamine we re observed in offspring 26 

### from male rats exposed to 5,000 ppm (18,000 mg/m 3 

### ) without changes in neurobehavioral function. 27


### The most sensitive effects in humans identified fro m a sparse literature were eye, nose, and 28 

### throat irritation following a 90 minute chamber exp osure to 10 mg/m 3 

### n-butanol (Kjaerguard et al., 29 

### 1997). In animals, hematological changes were obse rved at exposures as low as 6.6 mg/m 3 

### for 4 months 30 

### (Rumanystev et al., 1979), but it is unknown if the se changes were biologically significant. The 31 

### statistically significant change in neurological fu nction following a subchronic exposure to 308 mg/m 3 32 

### n-butanol in rats (Korsak et al., 1994) is the most sensitive biologically significant endpoint from 33 

### inhalation following exposure durations that are su bchronic or longer. 34

35

36

### 69 DRAFT - DO NOT CITE OR QUOTE

4.6.3. Mode of Action Information for Noncancer En dpoints 1 

### The mode of action for noncancer endpoints followin g n-butanol exposure is unknown. 2 

### However, because of its structural relationship to ethanol, n-butanol has been tested in a large numbe r of 3 

### studies examining possible mechanisms for alcohol-i nduced neurotoxicity. One proposed mechanism is 4 

### that alcohols including n-butanol produced the obse rved neurological changes by disrupting the lipid 5 

### bilayer. A few studies have shown that n-butanol, like other alcohols, can disrupt membrane integrity 6 

### (Kowalczyk et al., 1996; Krill et al., 1993; Gastal di et al., 1991). Other studies have found that n7 

### butanol inhibits the excitatory glutamate receptor function (Akinshola, 2001; Peoples and Weight, 8 

### 1999; Dildy-Mayfield et al., 1996; Lovinger et al., 1989) and potentiates inhibitory receptors such as 9 

### glycine and gamma-aminobutyric acid (GABA) (Mascia et al., 2000; Peoples and Weight, 1999; Ye et 10 

### al., 1998; Dildy-Mayfield et al., 1996; Nakahiro et al., 1991). The modulatory action of n-butanol 11 

### inhibiting the excitatory glutamate receptors and p otentiating the inhibitory GABA and glycine recepto rs 12 

### is supportive of the observed neurobehavioral chang es (e.g. CNS depressant profile) associated with n13 

### butanol exposure in humans (Seitz, 1972; Baikov and Khachaturyan, 1972; Tabershaw et al., 1944; 14 

### Nelson et al., 1943) and animals (Korsak et al., 19 94; RTI; unpublished, 1985). Application of n15 

### butanol (11 – 22 mM) also potentiated the serotonin (or 5-HT [5 hydroxytryptamine]) current (Rusch et 16 

### al., 2007; Stevens et al., 2005; Zhou et al., 1998) . 5HT 3 

### receptors are known to regulate dopamine 17 

### release and increased dopamine levels are associate d with reward mechanisms. Nelson et al (1990; 18 

### 1989b) found that pups exposed to n-butanol in utero had statistically significant increases in brain 19 

### levels of dopamine and serotonin which could be ind irectly linked to n-butanol effects on the 5HT 3 20 

### channel. Collectively, these studies suggest possi ble modes of action, via effects on these ion chann els, 21 

### for anesthetic effects and possibly other subtle ne urological effects of n-butanol. 22 

### In addition, studies have shown that n-butanol inhi bits rat brain astroglial cell proliferation by 23 

### disrupting the PLD signaling pathway (Kotter et al. , 2000; Kotter and Klein, 1999). n-Butanol was 24 

### shown to be a good substrate for transphosphatidyla tion, resulting in the formation of 25 

### phosphatidylbutanol and concomitantly decreasing th e formation of the second messenger PA. 26 

### Importantly, the authors demonstrated that n-butano l is substantially more potent than ethanol in the 27 

### inhibition of astroglial cell proliferation, which has been postulated as a mode of action for ethanol

28 

### induced microencephaly and mental retardation obser ved in cases of fetal alcohol syndrome. 29 

### There is more limited information on possible modes of n-butanol-induced liver effects. Carlson 30 

### (1994a, b) showed that n-butanol can esterify fatty acids in the liver and several other organs; fatty aid 31 

### ethyl esters observed after ethanol exposure have b een postulated to play a role in damage to several 32 

### organs. Deters et al. (1998a) demonstrated that me tabolism of n-butanol by ADH was not a necessary 33 

### step in the hepatotoxic action of this compound. T he same authors observed mitigation of several, but 34 

### not all in vitro measures of hepatotoxicity when gl ycine was added to isolated perfused liver systems 35

### 70 DRAFT - DO NOT CITE OR QUOTE

along with n-butanol (Deters et al., 1998b); glycin e had previously been shown to reduce hypoxia1


### related effects on the liver. 2

3


### 4.7. EVALUATION OF CARCINOGENICITY 4 

### 4.7.1. Summary of Overall Weight-of-Evidence 5


### Under the EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), there is 6 

### inadequate information to assess the carcinogenic p otential of n-butanol. None of the available human 7 

### occupational health studies evaluated cancer endpoi nts. One occupational health study of workers 8 

### exposed to n-butanol and several other solvents whi le employed as dockyard painters (Chen et al., 1999 ) 9 

### examined mortality rates due to cancer; neither pro portional mortality nor standardized mortality due to 10 

### cancers were increased in the painters relative to controls. There are no chronic animal studies of 11 

### n-butanol exposure through any exposure route, and the available subchronic studies did not report any 12 

### cancer endpoints. The genotoxicity database for n- butanol is limited; available information suggests that 13


### n-butanol does not induce mutations in S. typhimurium with or without exogenous metabolic activation 14 

### (Muller et al., 1993; Jung et al., 1992; Nakamura e t al., 1987; Connor et al., 1985; McCann et al., 19 75), 15 

### and does not increase the frequency of micronuclei or sister chromatid exchanges in Chinese hamster 16 

### cells (Lasne et al., 1984; Obe et al., 1977) or in chick embryos in culture (Bloom, 1982). Studies of 17 

### DNA and RNA replication have shown that n-butanol c an exert an inhibitory effect on these processes 18 

### (Obe and Ristow, 1977; Yoshiyama et al., 1973). 19

20


### 4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 21 

### 4.8.1. Possible Childhood Susceptibility 22 

### There are no studies of age-related differences in susceptibility to the toxic effects of n-butanol. 23 

### In a developmental toxicity study of oral exposure to n-butanol before and during gestation using Wist ar 24 

### Imp:DAK rats, increased incidences of some malforma tions (primarily dilation of the subarachnoid 25 

### space and/or the lateral or third ventricle of the brain) were reported (Sitarek et al., 1994), sugges ting 26 

### that the developing fetus may be particularly susce ptible to the effects of n-butanol exposure. Howev er, 27 

### in a study using a different strain of rat (Sprague -Dawley) and a exposure scenario (gestation only), Ema 28 

### et al., 2005) did not observe neurodevelopmental ef fects. Differences in strain susceptibility and 29 

### exposure regimen may address this discrepancy (see Section 5.1.1.). Inhalation studies of n-butanol 30 

### (Nelson et al., 1989a) reported maternal toxicity ( reduced food consumption and/or body weight gain) a t 31 

### concentrations resulting in fetal effects (primaril y reduced body weight). A single study of behavior al 32 

### teratology in offspring of rats exposed to n-butano l (Nelson et al., 1989b) did not observe treatment33 

### related neurobehavioral changes. 34 

### Studies have demonstrated lifestage specific differ ences in activitiy in the enzymes responsible 35 

### for metabolism of n-butanol. Specifically, in huma ns, there is evidence of differential expression fo r 36

### 71 DRAFT - DO NOT CITE OR QUOTE

three types of alcohol dehydrogenase (type I) enzym es: ADH1A, ADH1B, and ADH1C over different 1 

### lifestages (Smith et al., 1971). Expression of the ADH enzymes was examined in the liver, lung, and 2 

### kidney in 222 humans from 9 weeks gestation to up t o 20 years of age. In the liver, it was reported t hat 3 

### at 11 weeks of gestation ADH1A was preferentially e xpressed and ADH1B and ADH1C were 4 

### nondetectable. However, in adults, ADH1A is no lon ger detectable and there is equal expression of 5 

### ADH1B and ADH1C. ADH expression was considerably l ower in the lung and kidney and expression 6 

### did not change with the lifestages. Studies in mic e, rats, and guinea pigs have also reported signifi cantly 7 

### lower alcohol and aldehyde dehydrogenase activity i n fetal animals (ranging from 10 – 40% of adult 8 

### activity) in comparison to the adults (Timms and Ho lmes, 1981; Card et al., 1989; Lindahl, 1977; 9 

### Boleda et al., 1992). The lower enzymatic activit y of alcohol and aldehyde dehydrogenase during 10 

### development and childhood could potentially impact the metabolic capacity of n-butanol and may make 11 

### this population more susceptible to this chemical a gent. 12

13


### 4.8.2. Possible Gender Differences 14 

### There are no studies of gender differences in susce ptibility to the toxic effects of n-butanol, and 15 

### available information does not suggest gender diffe rences. 16

17

### 72 DRAFT - DO NOT CITE OR QUOTE 5. DOSE-RESPONSE ASSESSMENTS 1

2 3 

### 5.1. ORAL REFERENCE DOSE (RfD) 4 

### 5.1.1. Choice of Principal Study and Critical Effe ct—with Rationale and Justification 5 

### Data on the health effects of oral exposure to n-bu tanol in humans is limited to one case report 6 

### from a suicide attempt (Bunc et al., 2006) in which neurological, gastrointestinal, and cardiovascular 7 

### symptoms were noted. This case study is not suitab le for use in deriving an RfD. The animal 8 

### toxicological database for subchronic, chronic, and reproductive or developmental studies of n-butanol 9 

### consists of three subchronic studies, Munoz et al. (1991; 1990), Wakabayashi et al. (1991), and RTI 10 

### (1985), and two developmental toxicity studies, Ema et al. (2005) and Sitarek et al. (1994) where fema le 11 

### reproductive parameters were also evaluated. 12 

### Figure 5-1 is an exposure-response array for oral e xposure to n-butanol. Overall, the most 13 

### sensitive toxicological effect was reported in the developmental toxicity study by Sitarek et al (1994 ), in 14 

### which increased incidences of developmental viscera l malformations were observed in rat litters at ≥ 15 

### 300 mg/kg-day. Sitarek et al. (1994) reported incr eased incidences of developmental visceral 16 

### malformations (dilation of the lateral ventricle, d ilation of subarachnoid space in the brain and dila tion 17 

### of the renal pelvis) in the litters of female Wista r Imp:DAK rats treated with n-butanol in drinking w ater 18 

### at doses of 300, 1,000, and 5,000 mg/kg-day adminis tered for 8 weeks prior to mating and then 19 

### continued through mating and gestation. 20 

### Neurotoxicological effects (hypoactivity and ataxia ) were also observed in adult rats at a similar 21 

### dose of 500 mg/kg-day (RTI; unpublished, 1985). Ho wever, these neurotoxicological effects were not 22 

### considered as sensitive as the developmental viscer al malformations because the changes were transient 23 

### and not observed within an hour of exposure as note d by the study authors. Additionally, the reported 24 

### LOAEL was higher for the neurotoxicological effects observed in RTI (1985) than for the 25 

### developmental effects noted in Sitarek et al (1994)

. 26 

### In a second developmental toxicity study, Ema et al . (2005) did not observe an increased 27 

### incidence of any malformation in the offspring of S prague-Dawley rats exposed to n-butanol in drinking 28 

### water throughout gestation (GD 0-20) at doses of 31 6, 1,454, and 5,654 mg/kg-day. However, maternal 29 

### and fetal toxicity, manifested as body weight reduc tions, were observed at the high dose in this study 30 

### (5,654 mg/kg-day) (Ema et al., 2005). The discrepa ncy between the developmental studies is uncertain, 31 

### but may be due to differences in experimental proto cols particularly strain differences and exposure 32 

### regimens (see Section 5.3.). 33

34

35

### 73 DRAFT - DO NOT CITE OR QUOTE

Sitarek et al (1994) was selected as the principal study because it was a well-designed 1 

### developmental toxicity study with a sufficient numb er of dams and litters per dose group. Three dose 2 

### groups and an untreated control group were included in the study, and there was extensive evaluation o f 3 

### fetal parameters in the litters. There was a stati stically significant increase in the incidence of l itters 4 

### with developmental visceral malformations at all do se groups (lateral ventricle and subarachnoid space 5 

### in the brain; unilateral and bilateral renal pelvis ) (Table 5-1). With respect to the malformations 6 

### (dilation) observed in the brain, this effect may b e irreversible since there is a link between latera l 7 

### ventricular dilation and neurodevelopmental delay, which was reported in a retrospective epidemiology 8 

### study of fetal ventriculomegaly in 29,000 pregnanci es (Weichert et al., 2010). In addition, dilation of 9 

### the subarachnoid spaces and the ventricles in the b rain are observed in hydrocephalus (Raimondi, 1994)

, 10 

### and Sitarek et al. (1994) also reported internal hy drocephalus in the fetuses from the 1,000 and 5,000 11 

### mg/kg-day dose groups. Observation of hydrocephaly and dilation of neuronal structures (subarachnoid 12 

### space and lateral ventricle) as reported in Sitarek et al (1994) is also consistent with ethanol (Saka ta13 

### Haga et al., 2004). The functional significance of dilation of the lateral ventricles and subarachnoi d 14 

### space appears to be related to the rate and severit y of the dilation, as well as the developmental sta ge at 15 

### which it occurs (Weichert et al., 2010; Del Bigio, 2001). Sitarek et al. (1994) did not provide any 16 

### indication of the severity of the dilation of the l ateral ventricles and subarachnoid space in the aff ected 17 

### fetuses or litters, and the only information provid ed by the investigators was a characterization of t he 18 

### changes at the mid and high doses, 1,000 and 5,000 mg/kg-day, respectively, as frank hydrocephalus. I n 19 

### the absence of severity characterization, and given the observation of hydrocephalus at higher doses, the 20


### increased litter incidences of visceral malformatio ns (dilation of subarachnoid space, lateral ventric le, 21 

### unilateral and bilateral renal pelvis) that were ob served in the low-dose group (300 mg/kg-day) were 22 

### considered to be biologically significant and were selected as the critical effect. 23

2474 DRAFT - DO NOT CITE OR QUOTE1Figure 5-1. Exposure-response array for oral exposure to n-butanol.2

### 75 DRAFT - DO NOT CITE OR QUOTE

5.1.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) 1 

### A PBPK model for n-butanol is available (Teeguarden et al., 2005); however, this model 2 

### was not suitable for use in deriving the RfD. The principal study used for the RfD derivation 3 

### (Sitarek et al., 1994) exposed rats before and duri ng gestation, and fetal effects were the most 4 

### sensitive endpoint. The available PBPK model does not simulate oral exposure, precluding 5


### estimation of the relevant internal dose metric for this study. 6 

### BMD modeling was conducted using the EPA’s benchmar k dose software (BMDS, 7 

### version 2.1.1.) to analyze the developmental viscer al malformation associated with exposure to 8 

### n-butanol (see Appendix B for modeling details). T he software was used to calculate potential 9 

### PODs for deriving the RfD by estimating the effecti ve dose at a specified level of response 10 

### (BMD x 

### ) and its 95% lower bound (BMDL x 

### ). Since internal doses could not be estimated usi ng a 11 

### PBPK model, administered doses were used to derive a BMDL. 12 

### In the study by Sitarek et al. (1994), a LOAEL of 3 00 mg/kg-day was identified by EPA 13


### based on increased litter incidences of visceral ma lformations (dilation of subarachnoid space, 14 

### lateral ventricle, unilateral and bilateral renal p elvis). A NOAEL was not identified for this 15 

### study. These teratogenic effects could not be mode led using nested developmental toxicity dose16 

### response models, as these models require individual (i.e., litter-specific) animal data which were 17 

### not reported by Sitarek et al. (1994). The inciden ces of litters affected with visceral 18 

### malformations were reported in the study (see Table 5-1). Although individual data were 19 

### provided for dilation of the parameters in the brai n (lateral ventricle and subarachnoid space) and 20 

### was more sensitive that the kidney parameters (unil ateral and bilateral renal pelvis), the 21


### combined data for the dilation incidences in the br ain would provide a better estimate of the 22 

### overall developmental toxicity associated with n-bu tanol since dilation of subarachnoid space 23 

### and the lateral ventricle are reported precursors o f hydrocephalus (Raimondi, 1994). However, 24


### combined incidences of dilation in the fetal brain via the subarachnoid space or lateral ventricle 25 

### could not be separated from the kidney endpoints si nce the incidences were not reported 26 

### independent of each other. For example, in the 500 mg/kg-day group it was noted that 2 out of 27 

### 14 litters had dilation of the subarachnoid space, 8 out of 14 had dilation of the lateral ventricle 28 

### but 9 out of 14 litters were scored as having overa ll visceral malformations per litter (see Table 29 

### 5-1). Therefore, overall incidence of visceral m alformations was used for BMD modeling to 30 

### provide a better dose response estimate of developm ental toxicity associated with n-butanol. 31

32

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Table 5-1. Incidences of rat litters with dilation of the subarachnoid space or dilation of the lateral ventricle/third ventricl e of the brain

Incidence of affected litters n-Butanol dose in mg/kg-d 0 300 1,000 5,000 Litters with visceral malformations (dilation) – o verall 1/12 (8%) 9/14 (64%) 10/12 (83%) 9/9 (100%) Dilation of subarachnoid space 0/12 (0%) 2/14 (14% ) 3/12 (25%) 7/9 (78%)
Dilation of lateral ventricle and/or third ventricl e of the brain 1/12 (8%) 8/14 (57%) 8/12 (67%) 7/9 (78%)

Dilation of unilateral renal pelvis 0/12 (0%) 0/14 (0%) 5/12 (42%) 0/9 (0%) Dilation of bilateral renal pelvis 0/12 (0%) 0/14 ( 0%) 3/12 (25%) 0/9 (0%)

Source: Sitarek et al. (1994).
1


### All available dichotomous models in the EPA Benchma rk Dose Software (BMDS, 2 

### v. 2.1.1) were fit to the datasets for the increase d incidences of visceral malformations (dilation 3 

### of subarachnoid space, lateral ventricle, unilatera l and bilateral renal pelvis). Table 5-2 4 

### summarizes the BMD modeling results for the overall observed dilation. A benchmark response 5 

### (BMR) of 10% was selected from which to derive the point of departure (POD), because the 6 

### endpoints were characterized in terms of affected l itters for reproductive and developmental 7


### studies when data are available which characterize the incidence of effects within litters. Since 8 

### this level of reporting was not available, a nested analysis was not possible. The overall 9 

### incidence of affected offspring for these endpoints (not shown here) in the control and low dose 10 

### groups indicated that not all offspring in each aff ected litter were similarly affected, so using a 11


### BMR of 5% extra risk on the basis of affected litte rs corresponded to a lower overall incidence in 12 

### the affected offspring. Thus, a BMR of 10% extra r isk among affected litters was employed, in 13 

### order to approximate better a 5% extra risk in affe cted offspring, while recognizing the litter as 14 

### the experimental unit for appropriate estimation of confidence intervals. The log-probit model 15 

### was selected because it provided the best fit to th e data based on: (1) having the lowest Akaike’s 16 

### Information Criterion (AIC), and (2) visual inspect ion of the plot of observed versus expected 17 

### values across the fitted models. Appendix B contai ns a more detailed description of the 18 

### modeling and results, along with graphs of the sele cted best-fit models. 19

20

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Table 5-2. Summary of BMD modeling of results base d on Sitarek et al. (1994) developmental toxicity data – Dilation in the brain and kidney

BMD Models AIC BMD 10 (mg/kg-d) BMDL 10 (mg/kg-d)
Gamma 41.16 48 31 Logistic 44.28 115 74 Log-logistic 42.49 36 10 Log-probit 40.94 79 48 Multistage (1) 41.16 48 31
Probit 44.42 118 81
Weibull 41.16 48 31
1


### The BMDL 10 

### of 48 mg/kg-day for increased litter incidences of visceral malformations 2 

### (dilation of subarachnoid space, lateral ventricle, unilateral and bilateral renal pelvis) was 3


### selected as the POD for derivation of the chronic R fD for n-butanol. 4

5 

### 5.1.3. RfD Derivation—Including Application of Unc ertainty Factors (UFs) 6 

### To derive the RfD, the BMDL 10 

### of 48 mg/kg-day for increased incidence of viscera l 7 

### malformations was divided by a total UF of 300. Th e UF of 300 comprises component factors of 8 

### 10 for interindividual variability, 10 for interspe cies extrapolation, and 3 for database 9 

### uncertainty, as described below. 10 

### An UF of 10 was selected to account for interspecie s variability in extrapolation from 11 

### laboratory animals (rats) to humans because informa tion is not available to quantitatively assess 12 

### toxicokinetic or toxicodynamic differences between animals and humans. 13 

### An UF of 10 was selected to account for intraspecie s variability in susceptibility to n14 

### butanol, as quantitative information for evaluating toxicokinetic and toxicodynamic differences 15 

### among humans are not available. 16


### An UF of 3 was selected to account for deficiencies in the database, specifically the lack 17 

### of a multigeneration reproductive toxicity study. The toxicological database for n-butanol 18 

### includes one unpublished subchronic gavage study, t wo subchronic drinking water studies 19 

### evaluating limited toxicological endpoints, and two developmental toxicity studies. One of the 20 

### developmental toxicity studies, Sitarek et al. (199 4), included 8 weeks of premating exposure 21 

### and evaluation of estrous cyclicity and a number of fertility endpoints; however, this study was 22 

### not a multi-generation reproductive toxicity study and does not abrogate all concerns with 23 

### potential reproductive toxicity. 24 

### An UF for extrapolation from a subchronic study to chronic exposure was not applied, 25 

### because the POD is from a developmental toxicity st udy where the critical effect appears to be 26 

### more dependent on the exposure concentration during a susceptible lifestage than on the duration 27 

### of exposure. 28

### 78 DRAFT - DO NOT CITE OR QUOTE

An UF for extrapolation from a LOAEL to a NOAEL was not applied because the current 1 

### approach is to address this factor as one of the co nsiderations in selecting a BMR for BMD 2 

### modeling. In this case, a BMR of 10% change in the incidence of dilation of the lateral ventricle 3 

### was selected based on the assumption that it repres ents a minimal biologically significant 4 

### change. 5 

### The RfD of 0.2 mg/kg-day for n-butanol was calculat ed as follows: 6

7


### RfD = BMDL 10 

### ÷ UF 8 

### = 48 mg/kg-day ÷ 300 9 

### = 0.2 mg/kg-day 10 11

12


### 5.1.5. Previous RfD Assessment 13 

### The previous RfD for n-butanol was posted to the IR IS database on September 1, 1990. 14


### An RfD of 1 × 10 -1 

### mg/kg-day was derived based on hypoactivity and at axia observed in the 15 

### unpublished subchronic study by RTI (1985). A cumu lative UF of 1,000 (including 10-fold each 16 

### for interindividual variability, interspecies uncer tainty, and extrapolation from a subchronic 17 

### study) was applied to the NOAEL of 125 mg/kg-day to derive the RfD. Both of the oral 18 

### developmental toxicity studies of n-butanol (Ema et al., 2005; Sitarek et al., 1994) were 19 

### published after the previous RfD was posted on IRIS

. 20

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5.2. INHALATION REFERENCE CONCENTRATION (RfC) 1 

### 5.2.1. Choice of Principal Study and Critical Effe ct—with Rationale and Justification 2


### Information on the health effects of humans from in halation exposure to n-butanol 3 

### consists of a few acute human exposure and occupati onal health studies. Controlled human 4 

### exposure experiments have demonstrated that n-butan ol vapors can exert an irritant effect on the 5 

### eyes, nose, and throat under acute exposure conditi ons at air concentrations ≥ 10 mg/m 3 6 

### (Kjaerguard et al., 1997; Nelson et al., 1943). Oc cupational health studies in which the primary 7 

### exposure was to n-butanol reported effects includin g eye irritation (at 46 to 200 mg/m 3 

### ) (Cogan 8 

### et al., 1945; Tabershaw et al., 1944) and hearing l oss (at 240 mg/m 3 

### ) (Velazquez et al., 1969). 9 

### Lack of methodological detail prevents evaluation o f the adequacy of the exposure assessments 10 

### used in all of these studies (Sterner et al., 1949; Cogan et al., 1945; Tabershaw et al., 1944). The 11 

### eye irritation effects reported by Cogan and collea gues may also have been caused by other 12 

### chemicals, however, prevalence also was reported am ong workers in other plants where n13 

### butanol was the only solvent exposure (Tabershaw et al., 1944). The high prevalence of hearing 14 

### loss observed among n-butanol exposed workers is co mpelling (Velazquez et al., 1969), but the 15 

### study has limitations which present uncertainty for its use in the quantification of risk, including 16 

### the small number of exposed workers (affects precis ion of the prevalence estimate) and lack of 17 

### methodological detail (e.g., sampling protocol, sel ection of referent group). 18 

### The animal toxicological database for inhalation ex posure to n-butanol includes three 19 

### subchronic exposure studies (Korsak et al., 1994; R umanystev et al., 1979; Smyth and Smyth, 20 

### 1928), a developmental toxicity study (Nelson et al ., 1989a), and a neurodevelopmental study 21 

### (Nelson et al., 1989b). There are no chronic expo sure studies or experiments evaluating 22 

### reproductive toxicity. 23 

### Several short-term inhalation exposure studies that have characterized toxicities 24


### associated with n-butanol are also available. Fatt y infiltration was observed in the liver and 25 

### kidney in mice exposed to n-butanol air concentrati ons of 24,624 mg/m 3 

### for 130 hours (Weese, 26 

### 1928). Neurotoxic effects, such as decreased respo nse (Frantik et al., 1994) were reported in rats 27 

### and mice following a 2-4 hour exposure. Respiratio n rates declined in Ssc:CF-1 mice exposed to 28 

### air concentrations of 435 ppm and higher for 30 min utes (Kristiansen et al., 1988), as well as in 29 

### balb/C mice, who experienced a 50% respiratory decl ine following 4-hour exposures to air 30 

### concentrations of 3,008 ppm (Korsak et al., 1993) o r 4,300 ppm (Korsak and Rydzynski, 1994). 31 

### In the three subchronic studies, hematological effe cts, primarily decreased erythrocyte 32 

### counts, were noted (Korsak et al., 1994; Rumanystev et al., 1979; Smyth and Smyth, 1928). The 33 

### lowest concentration at which hematological changes were observed was at 6.6 mg/m 3 

### in rats 34 

### where increased blood cholinesterase activity was s een (Rumyantsev et al., 1979). However, it is 35 

### unclear if the hematological changes observed follo wing exposure to n-butanol are biologically 36 

### relevant. The other primary toxicological effect o bserved in the subchronic exposure studies was 37 

### deficits in neurobehavioral performance. Korsak et al (1994) reported a statistically significant 38

### 80 DRAFT - DO NOT CITE OR QUOTE

increase in the failure rate for the rotarod test i n rats exposed to 308 mg/m 3 

### n-butanol for 3 1 

### months. Of the three subchronic toxicity studies, only Korsak et al (1994) reported the observed 2 

### raw data. The other two studies (Rumanystev et al. , 1979; Smyth and Smyth, 1928) only 3 

### indicated statistically significant effects without reporting the raw data. Therefore, these two 4 

### studies were not amenable to dose-response analysis

. 5 

### Nelson et al. (1989a,b) evaluated the potential dev elopmental and neurodevelopmental 6 

### toxicity of n-butanol in rats. Increased incidence of litters with skeletal variation was observed at 7 

### concentrations of 3,500 ppm (11,000 mg/m 3 

### ) and increased levels of serotonin and dopamine 8 

### were observed in offspring from male rats exposed t o 5,000 ppm (18,000 mg/m 3 

### ) without 9 

### changes in neurobehavioral function. 10 

### Figure 5-2 is an exposure-response array for inhala tion exposure to n-butanol for select 11 

### animal and human studies employing repeated exposur e and reporting the most sensitive and/or 12 

### relevant toxicological effects. The most sensitive effects were reported in the human 13 

### occupational studies (Velazquez et al., 1969; Stern er et al., 1949) and in an animal subchronic 14 

### inhalation study (Korsak et al., 1994). The Nelson et al. (1989a,b) developmental toxicity 15


### studies identified effects at concentrations severa l orders of magnitude higher. As mentioned 16 

### earlier, although sensitive effects were noted in t he human studies, the uncertainties presented by 17 

### the lack of detail in reporting the assessment of e xposure and sparse discussion of study design 18 

### preclude their use as principal studies. 19 

### Korsak et al (1994) was selected as the principal s tudy for derivation of the RfC because 20 

### it was a well-designed study and reported sensitive toxicological effects. Additionally, there was 21 

### evaluation of the body and organ weights, measureme nt of biochemical and hematological 22 

### parameters, and performance of neurological testing . In the study, two statistically significant 23 

### effects, decreased hemoglobin and increased lipid p eroxidation, were reported at the lowest 24 

### concentration of 154 mg/m 3 

### . The decreased hemoglobin levels were within hist orical levels 25 

### reported for male Wistar rats (Giknis and Clifford, 2008) and hematocrit levels were not 26 

### changed. The biological relevance of the observed increased hepatic lipid peroxidation is 27 

### unknown because other hepatic endpoints were not af fected in this study. 28 

### At the next highest concentration, 308 mg/m 3 

### , there were statistically significant 29 

### increases in white blood cell counts and impairment s in learned avoidance behavior (due to 30 

### negative reinforcement for falling off the rod prio r to n-butanol exposure) and neuromuscular 31 

### function, as measured by the rotarod test (Korsak e t al., 1994). Although the increased white 32


### blood cell counts were outside of the historical ra nge reported for male Wistar rats (Giknis and 33 

### Clifford, 2008), other associated effects, such as immunotoxicological effects, were not tested in 34 

### this study, and have not been observed in other stu dies, except for in an acute anti-inflammatory 35 

### study in Wistar rats, where the WBC count decreased with n-butanol administration (Strubelt and 36 

### Zetler, 1980). 37

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Impaired learned avoidance behavior and neuromuscul ar function, as measured by 1 

### decreased rotarod performance, was selected as the critical effect for RfC derivation. The 2 

### selection of this critical effect is supported by o ther neurotoxic effects (e.g., decreased 3 

### immobility in the swim despair test and decreased r esponse) observed in acute animal studies at 4


### concentrations ≥ 1,420 mg/m 3 ( 

### Frantik et al., 1994; DeCeaurriz et al., 1983), as well as the 5 

### neurotoxic effects, ataxia and hearing impairment, observed in occupationally-exposed humans 6 

### (Velazquez et al., 1969), and vertigo (Seitz, 1972)

. 782 DRAFT - DO NOT CITE OR QUOTE1Figure 5-2. Exposure response array for inhalationexposure to n-butanol.2

### 83 DRAFT - DO NOT CITE OR QUOTE

1 

### 5.2.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) 2 

### A LOAEL of 308 mg/m 3 

### and a NOAEL of 154 mg/m 3 

### were identified for impaired 3 

### neurobehavioral function (as assayed by rotarod per formance) in rats exposed for 3 months in 4 

### Korsak et al. (1994). Korsak et al. (1994) reporte d the data on rotarod performance graphically 5 

### and without any estimate of variability within grou ps; thus, these data were not amenable to 6 

### BMD modeling. Instead, the rat NOAEL of 154 mg/m 3 

### was selected as the POD for RfC 7


### derivation. 8 

### Interspecies extrapolation (i.e., rat-to-human) of n-butanol inhalation dosimetry was 9 

### accomplished using a rat and human PBPK model origi nally described in Teeguarden et al. 10 

### (2005) with modifications described in Appendix C. The rat PBPK model was used to estimate 11 

### the value for the internal dose metric, represented as the area under the curve (AUC) for n12 

### butanol concentration in arterial blood, correspond ing to the NOAEL for motor incoordination in 13 

### a 90-day rat study (Korsak et al., 1994). The huma n PBPK model was used to estimate the 14 

### continuous human inhalation exposure (mg/m 3 

### ) that would result from the value of the internal 15 

### dose metric corresponding to the NOAEL in the rat. Details of the model code and parameters 16 

### that were selected are presented in Section 3.5 and Appendix C. 17


### The internal dose metric for the neurobehavioral ef fects of n-butanol is uncertain based 18 

### on the currently available data. The PBPK model fo r n-butanol (Teeguarden et al., 2005), with 19 

### modifications, simulates n-butanol concentrations i n blood, liver, and a lumped compartment 20 

### representing other tissues. The model does not sim ulate concentrations of n-butanol in the brain. 21 

### In the absence of dose-metric-specific data, parent compound concentration in the blood is 22 

### generally selected as a surrogate since it represen ts the circulating matrix for distribution in the 23 

### body. Therefore, blood n-butanol concentration was selected as the dose metric for 24


### neurobehavioral effects in animals. Korsak et al. (1994) observed progressive increases in 25 

### severity of the motor incoordination with increasin g exposure duration (1–3 months) at a 26 

### constant exposure concentration, as well as with in creasing exposure concentration. These 27 

### observations suggest that the toxic response is a f unction of both n-butanol blood concentration 28 

### and the duration of internal exposure to blood n-bu tanol. Therefore, arterial blood AUC was 29 

### selected as the dose metric for dosimetry extrapola tion to humans. 30 

### Korsak et al. (1994) exposed rats (average body wei ght of 0.392 kg as measured in the 31 

### study) for 6 hours/day, 5 days/week, for a period o f 3 months. This exposure was simulated in 32 

### the rat PBPK model as a 90-day exposure of a 0.392 kg rat to 50 ppm (154 mg/m 3 

### , the NOAEL) 33 

### for 6 hours/day, 5 day/week. The internal dose met ric (model variable: AUCAB2 24 

### ) was 34 

### calculated as the integral of the time profile for the arterial concentration of n-butanol (model 35 

### variable: CAB2) divided by the simulation time (t = 90 days × 24 hours/day = 2,160 hours), 36 

### multiplied by 24 hours: 37

38

### 84 DRAFT - DO NOT CITE OR QUOTE

1
2 

### Conceptually, the internal dose metric (AUCAB2 24 

### ) represents the average 24-hour AUC 3 

### for arterial n-butanol concentration for the 2,160- hour exposure duration. The value for 4


### AUCAB2 24 

### achieved pseudo steady state behavior after 2,000 hours (i.e., ±1% of the final 5 

### value). The human exposure was simulated as a cont inuous exposure (24 hours/day, 7 6 

### days/week) for a 70-kg human. The simulated exposu re duration was 1,000 hours. An exposure 7 

### duration exceeding 500 hours was sufficient to achi eve steady state levels (i.e., > 99.99% 8 

### stability of the value for AUCAB2 24 

### ). Values for the rat NOAEL, AUCAB2 24 

### , and human 9 

### equivalent NOAEL concentration (59 mg/m 3 

### ) calculated from the PBPK model are shown in 10


### Table 5-4. 11

12


### Table 5-4. PBPK model calculation of HECs of the N OAEL based on

rotarod performance in male rats exposed to n-butan ol for 3 months

Rat NOAEL mg/m 3 (ppm) Rat AUCAB2 24 a mM Human AUCAB2 24 mM Human NOAEL

(HEC) mg/m 3 (ppm)
154 (50) 0.01282 0.01282 59 (19.21)

a Area under the blood concentration vs. time curve f or rats estimated using rat PBPK model and exposure to NOAEL concentration of 50 ppm for 6 hours/day, 5 da ys/week for 3 months (Korsak et al., 1994). 13 

### 5.2.3. RfC Derivation—Including Application of Unc ertainty Factors (UFs) 14


### The NOAEL HEC 

### of 59 mg/m 3 

### for impaired motor coordination and obtained from PBPK 15 

### modeling was used as the POD for derivation of the RfC for n-butanol. The RfC was calculated 16 

### by dividing the POD by a total UF of 1,000, compris ed of 3 for interspecies extrapolation, 10 for 17 

### interindividual variability, 10 for extrapolation f rom subchronic to chronic exposure, and 3 for 18 

### database uncertainty, as described below. 19 

### An UF of 3 was applied for interspecies extrapolati on. The interspecies UF comprises 20 

### two areas of uncertainty: toxicokinetic and toxico dynamic differences. Use of PBPK modeling 21 

### to convert the rat exposure concentration to a HEC accounts for toxicokinetic differences 22 

### between rats and humans and reduces uncertainty ass ociated with cross-species extrapolation. A 23 

### factor of 3 is retained to account for interspecies variability in extrapolating from laboratory 24 

### animals (rats) to humans because information is not available to quantitatively assess 25 

### toxicodynamic differences between animals and human s. 26 

### An UF of 10 was selected to account for intraspecie s variability in susceptibility to n27 

### butanol, as quantitative information for evaluating toxicokinetic and toxicodynamic differences 28 

### among humans are not available. 29

### 85 DRAFT - DO NOT CITE OR QUOTE

An UF of 10 was selected to account for extrapolati on from a subchronic exposure 1 

### duration study to a chronic RfD, as data for evalua ting response after chronic exposure are not 2 

### available. The study selected as the principal stu dy was a 3-month study by Korsak et al. (1994), 3 

### a study duration that falls well short of a standar d lifetime study. 4


### An UF of 3 was selected to account for deficiencies in the database, specifically the lack 5 

### of a multigeneration reproductive toxicity study. The toxicological database for inhaled 6 

### n-butanol includes several acute human experiments, occupational health studies, subchronic 7 

### toxicity studies, and developmental and neurodevelo pmental toxicity studies. 8 

### An UF for extrapolation from a LOAEL to a NOAEL was not applied because a NOAEL 9


### was used as the POD. 10 

### The RfC of 0.06 mg/m 3 

### for n-butanol was calculated as follows: 11

12


### RfC = NOAEL HEC 

### ÷ UF 13 

### = 59 mg/m 3 

### ÷ 1,000 14 

### = 0.06 mg/m 3 15 16


### 5.2.5. Previous RfC Assessment 17 

### An inhalation RfC for n-butanol was not previously available on IRIS. 18

19

### 86 DRAFT - DO NOT CITE OR QUOTE

5.3. UNCERTAINTIES IN THE ORAL REFERENCE DOSE AND INHALATION 1


### REFERENCE CONCENTRATION 2 

### The following discussion identifies uncertainties a ssociated with the quantification of the 3 

### RfD and RfC for n-butanol. Following EPA practices and guidance (U.S. EPA, 1994b), the 4 

### uncertainty factor approach was applied to the iden tified PODs to derive an RfD and an RfC (see 5 

### Sections 5.1.3 and 5.2.3). Factors accounting for uncertainties associated with a number of steps 6 

### in the analyses were adopted to account for extrapo lating from an animal study to human 7 

### exposure, a diverse human population of varying sus ceptibilities, extrapolation from subchronic 8 

### to chronic exposure duration, and database deficien cies. 9 

### The oral toxicity database for n-butanol contains o ne subchronic gavage study in rats 10 

### (RTI; unpublished, 1985), two subchronic drinking w ater studies in rats (Munoz et al., 1991, 11 

### 1990; Wakabayashi et al., 1991), and two developmen tal toxicity studies in rats (Ema et al., 12 

### 2005; Sitarek et al., 1994). Toxicity associated w ith oral exposure is predominantly associated 13 

### with neurodevelopmental toxicity. Effects observed after oral exposure to n-butanol include 14 

### teratogenicity, clinical signs of CNS effects, and hematology changes. 15 

### The toxicological database for inhaled n-butanol in cludes several acute human 16 

### experiments, a few occupational health studies, a s ubchronic study in rats (Korsak et al., 1994), 17 

### and developmental and neurodevelopmental toxicity s tudies in rats (Nelson et al., 1989a, b). 18 

### Neurotoxicity is the predominant effect associated with inhalation exposure to n-butanol. 19 

### Critical data gaps have been identified and uncerta inties associated with data deficiencies are 20 

### more fully discussed below. 21


### The RfD was derived based on the increased inciden ce of overall visceral malformations 22 

### in a developmental toxicity study of n-butanol (Sit arek et al., 1994). Uncertainty exists in the 23 

### discrepancy between the results of the developmenta l studies by Sitarek et al. (1994) and Ema et 24 

### al. (2005). Specifically, Sitarek et al. (1994) ob served developmental effects on the brain at low 25 

### doses ( ≥ 300 mg/kg-day), but Ema et al. (2005) did not obse rve any developmental 26 

### malformations at any dose. The discrepancy in thes e results may be due to differences in study 27 

### design, particularly rat strain used and exposure r egimens. Data support a strain differences 28 

### between Wistar and Sprague Dawley rats in developme ntal effects on the brain. For example, 29 

### one study evaluated strain differences for developm ental brain effects in Sprague-Dawley and 30 

### Wistar rats following exposure to aspirin (Gupta et al., 2003). Gupta et al. (2003) reported that 31 

### the Wistar pups had a significantly higher rate of hydrocephaly than the Sprague-Dawley pups. 32 

### In addition, Ema et al. (2005) utilized a gestation -ony exposure regimen compared to Sitarek et 33 

### al. (1994) which exposed rats prior to mating, duri ng mating, and throughout gestation. 34 

### Uncertainty also exists in the selection of the app ropriate BMR for use in the BMD 35 

### modeling of the critical effect (i.e., increased in cidence of dilation of the lateral ventricle of the 36 

### brain in Wistar IMP:Dak rat litters) to estimate th e POD. Since the results of Sitarek et al. 37 

### (1994) were reported in a way that the litter membe rship of individual pups could not be 38

### 87 DRAFT - DO NOT CITE OR QUOTE

determined, a nested BMD modeling approach could no t be used and therefore, a litter-based 1 

### BMD modeling approach was employed. In the absence of specific information to identify a 2 

### change in the litter incidence of dilation of the l ateral ventricle that represents a biologically 3 

### significant change, a BMR of 10% was selected, whic h is consistent with the default BMR of 4 

### 10% for quantal data (U.S. EPA, 2000). 5


### The RfC was derived based on neurobehavioral effect s in rats exposed to n-butanol via 6 

### inhalation for 3 months (Korsak et al., 1994). Wit h regards to the study design, it is not known 7 

### when the behavioral testing following n-butanol exp osure. Therefore, the extent of n-butanol 8 

### internally (whether cleared or not) is unknown. It is likely that the measures are reflective of 9 

### persistent effects of n-butanol based on the increa sed deterioration of the rotarod performance 10 

### over the 3 months but still represents an uncertain ty. BMD modeling could not be performed 11 

### using these data due to the lack of reporting by th e investigators of within-group variability 12 

### estimates. By definition, the identification of a NOAEL or LOAEL is restricted to the particular 13 

### doses or exposure concentrations used in a study, a nd thus, this type of analysis lacks 14 

### characterization of the entire dose-response curve for the effect of interest. As a result, a POD 15


### based on a NOAEL or LOAEL is less informative than one derived from BMD modeling. 16 

### With respect to uncertainty associated with the RfC derivation, there is one human study, 17 

### Velazquez et al. (1969), that reported hearing loss in 82% (9 out of 11) of the workers exposed to 18 

### 240 mg/m 3 

### n-butanol for a duration ranging from 3 to 11 year s in a cellulose acetate factory. 19 

### Although human data is typically preferred for deri ving reference values, there are limitations 20 

### with this study, including the small number of expo sed workers, which decreases the power of 21 

### the study, and the lack of information with regards to exposure sampling and exposure duration, 22 

### which precluded the selection of this study as the principal study. 23 

### An additional source of uncertainty in the derivati on of both RfD and RfC is 24 

### extrapolation from animals (rats) to humans. An ef fect and its magnitude associated with the 25 

### concentration at the POD in rodents are extrapolate d to human response. Pharmacokinetic 26 

### models are useful in examining species differences in pharmacokinetic processing; however, 27 

### dosimetric adjustment using pharmacokinetic modelin g was not possible following oral exposure 28


### to n-butanol since the model does not contain an or al route of administration and incorporate the 29 

### subsequent first pass effect. There are data gaps in pharmacokinetic parameters since there is no 30


### information on distribution of n-butanol to the fet us as well as no information of the elimination 31 

### of n-butanol via breast milk which represents an un certainty for both the RfD and RfC. For RfC 32 

### derivation, a PBPK model was used to account for to xicokinetic differences between rats and 33 

### humans, and the internal dose metric, blood n-butan ol AUC, was selected. PBPK models attempt 34 

### to simulate what occurs in vivo, and thus dose esti mates from these models represent an 35 

### additional source of uncertainty in the analysis. In the absence of specific data that could inform 36 

### the selection of the appropriate internal dose metr ic, the blood AUC of the parent compound 37 

### (i.e., n-butanol) was selected as the preferred dos e metric. Additionally, there is uncertainty 38

### 88 DRAFT - DO NOT CITE OR QUOTE

related to the urinary clearance rate of n-butanol and its metabolites. Specifically, butyric acid, a 1 

### metabolite of n-butanol, interacts with colonocytes and is incorporated into lipid membranes 2 

### (Thibault et al., 2010). However, kinetic informati on is currently not available to address this 3 

### uncertainty and thus urinary clearance of metabolit es was not incorporated into the model. 4 

### Another uncertainty with the model is the extent th at n-butyric acid is metabolized to acetyl CoA 5


### and then to carbon dioxide and water or to acetoace tyl CoA, (produced by beta oxidation of the 6 

### buttanoic acid). 7

8 Heterogeneity among humans with respect to the toxi cokinetics and toxicodynamics of n-butanol 9 represents another area of uncertainty in both the RfD and RfC derivations. n-Butanol is rapidly 10 metabolized to butyric aldehyde by ADH and further to n-butyric acid by aldehyde dehydrogenase. 11 Polymorphisms in the genes encoding ADH and aldehyd e dehydrogenase exist in the human population 12 and may contribute to variability in metabolism of n-butanol. 

### One key genetic polymorphism is the 13 

### ALDH2 (aldehyde dehydrogenase 2) allele that has be en identified as a slow metabolizing 14 

### variant of aldehyde dehdrogenase and has been more commonly found in East Asian populations 15 

### (Dickson et al., 2006). Populations with low metabo lizing capacity (slow alcohol and aldehyde 16 

### dehydrogenases) are more likely to be susceptible t o toxicities associated with n-butanol since 17 

### there would be a longer half-life in these individu als. Another key polymorphism has been 18 

### identified in the ADH3 (alcohol dehydrogenase type 3) gene where the gamma1 variant is found 19 

### in individuals that are fast metabolizers and the g amma2 variant is found in individuals that are 20 

### slow metabolizers (Hines et al., 2001). The polymo rphisms in the n-butanol metabolizing 21 

### enzymes indicate that there may be a great deal of variability in the effects associated with 22 

### exposure in humans. 23 

### In the absence of n-butanol-specific data to adequ ately characterize such genetic 24 

### variability among humans, a factor of 10 was used t o account for this uncertainty. 25 

### Data gaps in the toxicological database for n-butan ol include: lack of chronic toxicity 26 

### studies; lack of toxicity data in a laboratory spec ies other than rats; limitations in the available 27 

### subchronic studies (e.g., the oral study by RTI [19 85] is unpublished and lacks data tables and 28 

### appendices, and the inhalation study by Korsak et a l. [1994] did not include histopathology 29 

### evaluation); lack of a multigeneration reproductive toxicity study; and lack of an oral 30 

### neurodevelopmental toxicity study. Critical endpoi nts for both the RfD and RfC were 31 

### neurological, suggesting that further evaluation of the neurotoxic and neurodevelopmental 32


### effects of n-butanol would be beneficial. There wa s a lack of postnatal observations in the 33 

### developmental toxicity studies since offspring were sacrificed at the end of the gestational period 34 

### (Sitarek et al., 1994; Nelson et al., 1989a; Ema et al., 2005). Epidemiology studies of n-butanol 35 

### exposure evaluating neurological and otological end points and using documented, quantified 36 

### assessments of long-term exposure also would contri bute to a reduction in uncertainty. 37

38

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5.4. CANCER ASSESSMENT 1


### Under the EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), there is 2 

### inadequate information to assess the carcinogenic p otential of n-butanol. None of the available 3 

### occupational health studies of n-butanol evaluated cancer endpoints. One occupational health 4 

### study of workers exposed to n-butanol, and several other solvents, while employed as dockyard 5 

### painters examined mortality rates due to cancer (Ch en et al., 1999); however, neither 6 

### proportional mortality nor standardized mortality d ue to cancers were increased in the painters 7


### relative to controls. There are no chronic animal studies of n-butanol exposure via any exposure 8 

### route, and the subchronic studies did not report an y cancer endpoints. The genotoxicity database 9 

### for n-butanol is limited; available information sug gests that n-butanol does not induce mutations 10


### in S. typhimurium with or without exogenous metabolic activation (Mu ller et al., 1993; Jung et 11 

### al., 1992; Nakamura et al., 1987; Connor et al., 19 85; McCann et al., 1975), and does not 12 

### increase the frequency of micronuclei or sister chr omatid exchanges in Chinese hamster ovary 13 

### cells (Lasne et al., 1984; Obe et al., 1977) or in chick embryos in culture (Bloom, 1982). Studies 14 

### of DNA and RNA replication have shown that n-butano l can exert an inhibitory effect on these 15 

### processes (Obe and Ristow, 1977; Yoshiyama et al., 1973). 16

17

18

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6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HA ZARD AND DOSE 1


### RESPONSE 2

3 

### 6.1. HUMAN HAZARD POTENTIAL 4 

### n-Butanol (CASRN 71-36-3) is used as a direct solve nt in paints, surface coatings, 5 

### lacquers, thinners, pharmaceutical formulations, wa xes, and resins. It is also used to make 6 

### plasticizer esters and mono-, di-, and tributylamin es and oxygenate for gasoline. Commercial 7 

### derivatives of n-butanol include n-butyl acrylate, methacrylate, butyl glycol ethers, 2-butoxy8 

### ethanol, and butyl acetate. 9


### Toxicokinetic studies of n-butanol have been conduc ted in humans and experimental 10 

### animals. Absorption via inhalation has been demons trated in humans (Astrand et al., 1976), and 11 

### absorption via the oral, dermal, and inhalation rou tes was demonstrated in laboratory animals 12 

### (Poet et al., 2003a, b; Deisinger and English, 2001 ; Boman et al., 1995; Swiercz et al., 1995; 13 

### DiVincenzo and Hamilton, 1979a). Only limited info rmation is available regarding tissue 14 

### distribution of n-butanol in animal studies; after gavage dosing of rats with radiolabelled 15 

### n-butanol, the liver had the largest percentage of radioactivity (DiVincenzo and Hamilton, 16 

### 1979a). n-Butanol is rapidly metabolized to butyri c aldehyde by ADH and further to n-butyric 17 

### acid, also known as butanoic acid, by aldehyde dehy drogenase (ECETOC, 2003). n-Butanol is 18 

### also oxidized by cytochrome P450 in rat liver (Alba no et al., 1991). n-Butanol is excreted 19 

### primarily as CO 2 

### in exhaled breath with minor amounts eliminated in the urine and feces 20 

### (DiVincenzo and Hamilton, 1979a). A PBPK model has been developed (Teeguarden et al., 21 

### 2005) to describe blood kinetics for n-butyl acetat e and its metabolites, n-butanol and n-butyric 22 

### acid, in rats and humans exposed via inhalation. 23 

### Information on noncancer effects in humans orally e xposed to n-butanol is limited to one 24 

### case report from a suicide attempt (Bunc et al., 20 06) in which neurological, gastrointestinal, and 25 

### cardiovascular symptoms were noted. The database o n animal toxicity indicates that 26 

### developmental visceral malformations (dilation of t he lateral ventricle and subarachnoid space in 27 

### the brain; dilation in renal pelvis in the kidney) are the most sensitive toxicological endpoint 28 

### associated with oral n-butanol administration (Sita rek et al., 1994). Increased incidences of 29 

### brain malformations (dilation of the lateral and/or third ventricle of the brain) were observed in 30 

### the pups of female Wistar Imp:DAK rats treated with n-butanol in drinking water at doses of 31 

### 300, 1,000, and 5,000 mg/kg-day administered for 8 weeks prior to mating and then continuously 32 

### through mating and gestation. Neurotoxicity (hypoa ctivity and ataxia) in adult rats was also 33 

### observed in a 13week oral gavage study at 500 mg/kg -day (RTI; unpublished, 1985). Other 34 

### observed effects included hematological changes and increased thyroid weight at 500 mg/kg-day 35 

### (RTI; unpublished, 1985), and pathological changes in the liver and kidney at an exposure of 36 

### 4,400 mg/kg-day in male rats (Munoz et al. 1991; 19 90). 37

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Controlled human exposure experiments have demonstr ated that n-butanol vapors can 1 

### exert an irritant effect on the eyes, nose, and thr oat under acute exposure conditions at air 2


### concentrations ≥ 10 mg/m 3 

### (Kjaerguard et al., 1997; Nelson et al., 1943). O ccupational health 3 

### studies in which the primary exposure was to n-buta nol reported effects including eye irritation 4 

### (at 46 to 200 mg/m 3 

### ) (Cogan et al., 1945; Tabershaw et al., 1944) and hearing loss (at 240 5 

### mg/m 3 

### ) (Velazquez et al., 1969). These studies are limi ted by uncertainties arising from a lack 6 

### of detail in methods description for exposure asses sments and other details of study design. 7 

### The CNS is the most sensitive target following a re peated inhalation exposure to n8


### butanol in animals. Korsak et al (1994) reported a statistically signif icant increase in the failure 9 

### rate for the rotarod test in rats exposed to 308 mg /m 3 

### n-butanol for 3 months. Nelson et al 10 

### (1989a, b) evaluated the developmental and neurodev elopmental toxicity potential of n-butanol 11


### in rats. Increased incidence of litters with skele tal variation was observed at exposures of 3,500 12 

### ppm (11,000 mg/m 3 

### ) and increased levels of serotonin and dopamine we re observed in offspring 13 

### from male rats exposed to 5,000 ppm (18,000 mg/m 3 

### ) without changes in neurobehavioral 14 

### function. Several short-term inhalation exposure s tudies have reported fatty infiltration of liver 15 

### and kidney in mice exposed to 24,624 mg/m 3 

### (Weese, 1928), neurotoxic effects at exposures of 16 

### 1,420 mg/m 3 

### in mice and rats (Frantik et al., 1994; DeCeaurriz et al., 1983), and decreased 17 

### respiration rates in mice (Korsak and Rydzynski, 19 94; Korsak et al., 1993; Kristiansen et al., 18 

### 1988). 19 

### None of the available human occupational health stu dies in which the primary exposure 20 

### was to n-butanol evaluated cancer endpoints. One o ccupational health study of workers exposed 21 

### to n-butanol, as well as several other solvents, wh ile employed as dockyard painters examined 22 

### mortality rates due to cancer (Chen et al., 1999); however, neither proportional mortality nor 23 

### standardized mortality due to cancers were increase d in the painters relative to controls. There 24 

### are no chronic animal studies of n-butanol exposure via any exposure route, and the available 25 

### subchronic studies did not report any cancer endpoi nts. Therefore, under the EPA Guidelines for 26 

### Carcinogen Risk Assessment (U.S. EPA, 2005a), there is inadequate information to assess the 27 

### carcinogenic potential of n-butanol. 28

29


### 6.2. DOSE RESPONSE 30


### 6.2.1. Oral Noncancer 31 

### From the available data on oral exposure to n-butan ol, it was determined that 32 

### neurodevelopmental changes were the most sensitive noncancer endpoint. The developmental 33 

### toxicity study in Wistar Imp:DAK rats (Sitarek et a l.,1994) was selected as the principal study 34


### for RfD derivation and the increased incidence deve lopmental visceral malformations (dilation in 35 

### the fetal brain [lateral ventricle and subarachnoid space] and kidney [renal pelvis]) was selected 36


### as the critical effect. 37

### 92 DRAFT - DO NOT CITE OR QUOTE

An RfD of 0.2 mg/kg-day was derived. The RfD deriv ation involved fitting all of the 1 

### available dichotomous models in BMDS, version 2.1.1 , to the litter incidence of overall 2 

### increased visceral malformations (dilation). A BMR of 10% was selected because even though 3 

### the principal study had a nested design, litter-spe cific results were not reported by the 4 

### investigators which prevented the conduct of a nest ed analysis. Thus, a BMR of 10% was 5 

### employed, which is generally the default BMR for qu antal data (U.S. EPA, 2000). Dose6 

### response modeling of the incidence of litters with overall dilation yielded a BMDL 10 

### of 48 7 

### mg/kg-day, which was used as the POD. A composite UF of 300 (10 to account for interspecies 8 

### uncertainty, 10 to account for human variability, 3 for database uncertainty) was applied to the 9 

### POD to derive an RfD of 0.2 mg/kg-day. 10 

### Confidence in the principal study (Sitarek et al., 1994) is medium. This developmental 11 

### toxicity study evaluated exposure in three dose gro ups and a control, using group sizes of 11– 12 

### 17 female rats exposed for 8 weeks prior to mating as well as during mating and gestation. The 13 

### study evaluated body weight, clinical signs, and es trous cyclicity in dams, as well as litter 14 

### parameters and teratogenicity endpoints. Confidenc e in the database is low-to-medium. Oral 15 

### toxicity studies of n-butanol include an unpublishe d subchronic study that evaluated 16 

### comprehensive endpoints but lacked data tables and appendices, two published subchronic 17 

### studies evaluating limited endpoints, and two devel opmental toxicity studies. All of the 18


### available animal studies were conducted in rats. T he database is lacking chronic toxicity studies, 19 

### toxicity data in a laboratory species other than ra ts, and a multigeneration reproductive toxicity 20 

### study. Neurodevelopmental effects should also be f urther evaluated in light of the effects on 21 

### brain development in the Sitarek et al. (1994) deve lopmental toxicity study. Overall confidence 22


### in the RfD is low-to-medium. 23

24


### 6.2.2. Inhalation Noncancer 25 

### The CNS is the most sensitive target for noncancer toxicity in rats following repeated 26 

### inhalation exposure to n-butanol. The most sensiti ve endpoint was impairments in learned 27 

### avoidance behavior and neuromuscular function, repr esented by decreased rotarod performance, 28 

### in rats exposed to n-butanol for 3 months (Korsak e t al., 1994). Korsak et al. (1994) was selected 29 

### as the principal study for derivation of the RfC be cause it was a relatively well-designed study, 30 

### evaluated lower exposure effects, and provided a se nsitive endpoint of neurologic impairment. 31 

### Decreased rotarod performance was selected as the c ritical effect. 32 

### An RfC of 0.06 mg/m 3 

### was derived based on the observed critical effect of 33 

### neuromuscular impairment in the principal study. T he data for this endpoint were not amenable 34 

### to BMD modeling due to the absence of within-group variability estimates for rotarod 35 

### performance, so a NOAEL identified from this study (154 mg/m 3 

### ) was selected as the POD. 36 

### PBPK modeling using a modification of the model pub lished by Teeguarden et al. (2005) was 37


### used to estimate a NOAEL HEC 

### concentration of 59 mg/m 3 

### based on arterial n-butanol blood 38

### 93 DRAFT - DO NOT CITE OR QUOTE

concentration (AUC) as the internal dose metric. A composite UF of 1000 (3 to account for 1 

### interspecies toxicodynamic uncertainty, 10 to accou nt for human variability, 10 to account for 2 

### uncertainity of subchronic to chronic extrapolation , 3 for database uncertainty) was applied to the 3 

### POD to derive an RfC of 0.06 mg/m 3



### . 4 

### Confidence in the principal study (Korsak et al., 1 994) is low-to-medium. This 5 

### subchronic study evaluated exposure in two dose gro ups and a sham-treated control, using group 6 

### sizes of 12 male rats. The study evaluated body we ight, hematology, clinical chemistry, 7 

### neurotoxicity endpoints (rotarod performance and ho t-plate response), and selected organ 8 

### weights, but did not evaluate histopathology. Conf idence in the database is low-to-medium. 9 

### Inhalation toxicity studies of n-butanol include th e subchronic study used as the basis for the RfC 10 

### as well as developmental toxicity and developmental neurotoxicity studies, all conducted in rats. 11 

### The database is lacking chronic toxicity studies, t oxicity data in a laboratory species other than 12 

### rats, and a multigeneration reproductive toxicity s tudy. Neurological effects were the most 13 

### sensitive endpoint in the available subchronic stud y; however, the study evaluated only two 14 

### measures of neurotoxicity (rotarod performance and hot-plate response). Overall confidence in 15


### the RfC is low-to-medium. 16

17


### 6.2.3. Cancer - Oral and Inhalation 18


### Under the EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), there is 19 

### inadequate information to assess the carcinogenic p otential of n-butanol. Cancer endpoints 20 

### were not evaluated in any of the human occupational health studies. There are no chronic animal 21 

### studies of n-butanol exposure via any exposure rout e, and the subchronic studies did not report 22 

### any cancer endpoints. The genotoxicity database is limited and results primarily support that n23


### butanol does not induce mutations in S. typhimurium with or without exogenous metabolic 24 

### activation (Muller et al., 1993; Jung et al., 1992; Nakamura et al., 1987; Connor et al., 1985; 25 

### McCann et al., 1975), and does not increase the fre quency of micronuclei or sister chromatid 26 

### exchanges (Lasne et al., 1984; Bloom, 1982; Obe et al., 1977). 27

### 94 DRAFT - DO NOT CITE OR QUOTE 7. REFERENCES
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APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PU BLIC 1


### COMMENTS AND DISPOSITION 2

3 

### (page intentionally left blank) 4 5B-1 

### DRAFT - DO NOT CITE OR QUOTE APPENDIX B. BENCHMARK DOSE CALCULATIONS 1

2 3 

### In the study by Sitarek et al. (1994), a LOAEL of 3 00 mg/kg-day was identified based on 4 

### increased incidences of developmental visceral malf ormations (dilation of the lateral and/or third 5 

### ventricle, subarachnoid spaces of the brain and dil ation of renal pelvis) in the offspring of treated 6 

### female rats. The teratogenic effects were not amen able to modeling with nested developmental 7 

### toxicity dose-response models, as these models requ ire knowledge of the litters to which 8 

### individual animals belonged, and such data were not reported by Sitarek et al. (1994). However, 9 

### the percentages of affected litters were reported i n the study; these data (see Table B-1 below) 10 

### were subjected to BMD modeling to identify possible PODs for RfD derivation. 11

12



### Table B-1. Incidences of rat litters with visceral malformations

Incidence of affected litters n-Butanol dose in mg/kg-d 0 300 1,000 5,000 Litters with visceral malformations (dilation) – o verall 1/12 (8%) 9/14 (64%) 10/12 (83%) 9/9 (100%) Dilation of subarachnoid space 0/12 (0%) 2/14 (14% ) 3/12 (25%) 7/9 (78%)
Dilation of lateral ventricle and/or third ventr icle of the brain 1/12 (8%) 8/14 (57%) 8/12 (67%) 7/9 (78%)

Dilation of unilateral renal pelvis 0/12 (0%) 0/14 (0%) 5/12 (42%) 0/9 (0%) Dilation of bilateral renal pelvis 0/12 (0%) 0/14 ( 0%) 3/12 (25%) 0/9 (0%)

Source: Sitarek et al. (1994). 13 

### All available dichotomous models in the EPA BMDS (v ersion 2.1.1) were fit to the litter 14


### data with overall observed visceral malformations. This data included a combined incidence of 15 

### dilation amongst four measured parameters: subarach noid space, lateral and/or third ventricle, 16 

### unilateral renal pelvis, and bilateral renal pelvis . A BMR of 10% was selected, and the BMD and 17 

### BMDL associated with this BMR were estimated using the best-fit model for each dataset. 18 

### Detailed modeling results are shown in Table B-2. Figure B-1 shows the best-fitting models for 19 

### this endpoint . 20 

### Of the models exhibiting adequate fit, a “best-fit” model was selected consistent with the 21


### EPA’s Benchmark Dose Technical Guidance Document (U.S. EPA, 2000b) as follows. If the 22 

### BMDL estimates from the models exhibiting adequate fit are “sufficiently close,” then the model 23


### with the lowest AIC is used to estimate the BMDL fr om which the POD will be derived. Under 24


### the U.S. EPA’s Benchmark Dose Technical Guidance Document(U.S. EPA, 2000), the log25 

### probit model results using the full dataset provide the best fit to the data and was the BMDL 10 26


### from this model was used as the POD for RfD derivat ion. 27B-2 

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1 

### Table B-2. BMD modeling results for the incidence of litters with visceral

malformations (overall) after maternal exposure to n-butanol in drinking water.

Model DF χ 2 χ

2 Goodness of fit p- value a Scaled residual

of interest b AIC BMD 10 (mg/kg-d) BMDL 10

(mg/kg-d)
Gamma c 2 1.28 0.53 -0.20 41.16 48 31 Logistic 2 4.04 0.13 -1.24 44.28 115 74 Log-Logistic d,e 1 0.37 0.54 -0.01 42.49 36 10 Log-Probit d 2 1.10 0.58 -0.09 40.94 79 48 Multistage (1degree) f

2 1.28 0.53 -0.20 41.16 48 31
Probit 2 4.15 0.13 -1.24 44.42 118 81
Weibull c 2 1.28 0.53 -0.20 41.16 48 31

a Values <0.10 fail to meet conventional goodness-of- fit criteria.
b Scaled residual at measured response closest to the BMR.

c Power restricted to ≥ 1.
d Slope restricted to ≥ 1.

e Best-fitting model; the only model with adequate fi t. f Betas restricted to ≥ 0. Source: Sitarek et al. (1994).


### Figure B-1. Fit of log-probit model to data on inc idence of litters with visceral malformations (overall).

2

3 4 5 0 0.2 0.4 0.6 0.8 1

0 1 2 3 4 5Fraction Affected

dose LogProbit Model with 0.95 Confidence Level

11:54 03/16 2011

BMDL BMD LogProbitB-3 

### DRAFT - DO NOT CITE OR QUOTE

=================================================== ================= 1 Probit Model. (Version: 3.1; Date: 05/16/200 8) 2 Input Data File: C:\Usepa\BMDS21\Data\lnpSitar ekSetting.(d) 3 Gnuplot Plotting File: C:\Usepa\BMDS21\Data\l npSitarekSetting.plt 4
Wed Mar 16 12:54:08 2011 5

================================================== ================== 6 7
BMDS Model Run 8

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~~~~~~~~~~~~~~~~~ 9 10 The form of the probability function is: 11 12 P[response] = Background 13 + (1-Background) * CumNorm(Intercept +Slope*Log(Dose)), 14 15 where CumNorm(.) is the cumulative normal distri bution function 16 17 18 Dependent variable = Effect 19 Independent variable = Dose 20 Slope parameter is restricted as slope >= 1 21 22
Total number of observations = 4 23 Total number of records with missing values = 0 24
Maximum number of iterations = 250 25 Relative Function Convergence has been set to: 1 e-008 26 Parameter Convergence has been set to: 1e-008 27 28 29 30 User has chosen the log transformed model 31 32 33 Default Initial (and Specified) P arameter Values 34 background = 0.0833333 35 intercept = 0.408347 36 slope = 1 37 38 39 Asymptotic Correlation Matrix of Paramet er Estimates 40 41 ( *** The model parameter(s) -slope 42 have been estimated at a boundary point, or have been specified by the user, 43 and do not appear in the correlati on matrix ) 44 45 background intercept 46 47 background 1 -0.32 48 49 intercept -0.32 1 50 51 52 53
Parameter Estimate s 54 55
95.0% Wald Confidence Interval 56
Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 57 background 0.0908047 0.0863715 -0.0784803 0.26009 58 intercept 1.25846 0.309188 0.652459 1.86445 59 slope 1 NA 60 61 NA - Indicates that this parameter has hit a bound 62 implied by some inequality constraint and thus 63
has no standard error. 64 65 66 67B-4 

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Analysis of Deviance Table 1 2 Model Log(likelihood) # Param's Devia nce Test d.f. P-value 3
Full model -17.9734 4 4
Fitted model -18.4722 2 0.99 7729 2 0.6072 5
Reduced model -31.2787 1 26. 6106 3 <.0001 6 7
AIC: 40.9444 8 9 10
Goodness of Fit 11
Scaled 12 Dose Est._Prob. Expected Observed Size Residual 13

------------------------------------------------- ----------------------- 14
0.0000 0.0908 1.090 1.000 12 -0.090 15
0.3000 0.5652 7.912 9.000 14 0.586 16
1.0000 0.9053 10.864 10.000 12 -0.852 17
5.0000 0.9981 8.983 9.000 9 0.130 18 19
Chi^2 = 1.10 d.f. = 2 P-value = 0.5784 20 21 22 Benchmark Dose Computation 23 24 Specified effect = 0.1 25 26 Risk Type = Extra risk 27 28
Confidence level = 0.95 29 30
BMD = 0.0788658 31 32
BMDL = 0.047882 33C-1 

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1 

### APPENDIX C. n-BUTANOL PBPK MODEL 2


### C.1. Model Code 3

PROGRAM BUTYL.CSL -- PBPK Model for Butyl Acetate a nd Its Butyl-Series Metabolites 4

! Modified version of BANew8I.csl 5
! Output units for this model are umoles and mL (or grams) 6 7

! Modified from BANew6.CSL -- November, 2001 8

! Last Modified: Oct. 6, 1998 9
! Modified by: Hugh A. Barton 10 11
! This version runs in kg as modified by Justin Tee guarden 6.9.99 12
! Validated 6.9.99 by Justin Teeguarden 13 14
! Modified 7.11.99 by Justin Teeguarden (of BANEW2) to include metabolism of 15
! EtAcin other perfused tissues. AUC of EtAc und er Deisiv2 for this version 16

! matches BANEW2.CSL with Metabolism in Other Per fused Tissues=0. 17 18
! Modified 6.20.00 by JGT changing the CEx1, CEX2, CEX3, CEX4 to use Free blood 19
! concentration instead of lung concentration. 20 21
! Revised November 2001, Justin Teeguarden. Remove d URT for butyl acetate. 22

! Added closed chamber for BuOH and FA (fractiona l uptake term) for Butyl 23

! Acetate. These changes were made to model the butyl acetate closed chamber 24

! data from Batelle, Poet and Corely 2001. Mass balance OK. Without the URT, 25

! the inhaled amount for the GrothInh2 simulation using *8.csl is the same as 26

! *5.csl (the minor difference is due to an effec tive tstop which is a bit 27

! different.) This served as the validation for the removal of the URT. 28 29

! Alveolar Ventilation Rate Tables 1-7 QA'd 12.19.0 1 JGT 30 31
! NOTE: All units are expressed as mL(g), hr, or u mol in the derivative section 32 33
! This version has inhalation, IV and oral dosing. 34

! It uses a table command for the inhalation dose u sing the values from the 35
! study in the Groth paper. 36 37

! Parameters followed by 1,2,3,4 stand for: 38

! Submodel 1: n-Butyl acetate 39

! Submodel 2: n-Butanol 40

! Submodel 3: n-Butyraldehyde 41
! Submodel 4: n-Butyric acid 42 43

!************************************************** ***************************** 44
!Revised and imported into acslXtreme v 2.5.0.6 as "BUTYLAC.csl" on 04/23/2010 (G Diamond/SRC) 45

!Revisions are marked with "!" and "<GD> 46
! Handling of exposure/dose parameters standardize d to: inputs ppm/mg/kg --> convert to umol/L 47

or umol/kg --> 48
! inputs ppm/mg/kg --> convert to umol/L or umol/kg --> adjust for dose duration (e.g., < 1hr) 49
! --> apply timing pulse functions (i.e., on/off) - -> Integration functions 50
! CINT revised from function of CintC and TChng to a CONSTANT 51
! TChng revised to route specific dose averaging t ime CONSTANTs (e.g. IVDAT, ORDAT) 52

!************************************************** ***************************** 53
! Modified on 02/25/2011 by AFS to create “clean” version: 54
! Legacy code, and commented-out lines of code hav e been removed. Some formatting changes 55
! made to ensure proper word-wrap in documents. C ode was tested to ensure it properly ran 56

!************************************************** ***************************** 57C-2 

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1

INITIAL 2

!<GD> 3

INTEGER Rats, QPTable, Tube, INHTABLE !<GD> 4
CONSTANT BW = 0.30 ! Body weight (kg ) 5
CONSTANT QPC = 14000.0 ! Alveolar ventil ation (mL/hr/kg**0.75) 6 7
! Blood Flows (fraction of cardiac output) 8
CONSTANT QCC = 14000.0 ! Cardiac output (mL/hr/kg**0.75) 9

CONSTANT QFatC = 0.07 ! Fat 10
CONSTANT QLivC = 0.175 ! Liver 11 12
! Tissue Volumes (fraction of body weight) 13

CONSTANT VABC = 0.022 ! Arterial blood 14

CONSTANT VFatC = 0.07 ! Fat 15

CONSTANT VLivC = 0.037 ! Liver 16
CONSTANT VLungC = 0.005 ! Lung tissue 17

CONSTANT VOthC = 0.751 ! Other perfused tissues 18

CONSTANT VVBC = 0.045 ! Venous blood 19 20

! Partition Coefficients 21

! Butyl Acetate 22

CONSTANT PB1 = 89.4 ! Blood:air 23

CONSTANT PFat1 = 17.0 ! Fat:blood 24

CONSTANT PLiv1 = 3.14 ! Liver:blood 25

CONSTANT PLung1 = 1.76 ! Lung:blood 26

CONSTANT POth1 = 1.76 ! Other perfused tissues:blood 27 28

! n-Butanol 29

CONSTANT PB2 = 1160.0 ! Blood:air 30

CONSTANT PLiv2 = 1.08 ! Liver:blood 31

CONSTANT PLung2 = 0.78 ! Lung:blood 32

CONSTANT POth2 = 0.78 ! Other perfused tissues:blood 33 34

! n-Butyraldehyde 35

CONSTANT PB3 = 1160.0 ! Blood:air 36

CONSTANT PLiv3 = 1.08 ! Liver:blood 37

CONSTANT PLung3 = 0.78 ! Lung:blood 38

CONSTANT POth3 = 0.78 ! Other perfused tissues:blood 39 40
! Butyric Acid 41

CONSTANT PB4 = 1160.0 ! Blood:air 42

CONSTANT PLiv4 = 1.08 ! Liver:blood 43

CONSTANT PLung4 = 0.78 ! Lung:blood 44

CONSTANT POth4 = 0.78 ! Other perfused tissues:blood 45 46

! Metabolism 47

! Butyl Acetate 48

CONSTANT VMBloodC1 = 600000.0 ! Blood (umole/hr /kg**0.75) 49

CONSTANT KMBlood1 = 100.0 ! Blood (umole/mL ) 50

CONSTANT VMLivC1 = 38700.0 ! Liver (umole/hr /kg**0.75) 51

CONSTANT KMLiv1 = 1.0 ! Liver (umole/mL ) 52

CONSTANT VMOthC1 = 6.0e6 ! Other Perfused Tissues (umole/hr/kg**0.75) 53

CONSTANT KMOth1 = 100.0 ! Other Perfused Tissues (umole/mL) 54 55

! n-Butanol 56

CONSTANT VMLivC2 = 2170.0 ! Liver (umole/hr /kg**0.75) <GD> 57

CONSTANT KMLiv2 = 0.16 ! Liver (umole/m L) <GD> 58C-3 

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CONSTANT kOthC2 = 4.0 ! First-order BuO H clearance (kg**0.25/hr) <GD> 1 2

! n-Butyraldehyde 3

CONSTANT VMLivC3 = 17780.0 ! Liver (umole/hr /kg**0.75) 4

CONSTANT KMLiv3 = 0.1 ! Liver (umole/mL ) 5 6
! Butyric Acid 7

CONSTANT VMLivC4 = 1400.0 ! Liver (umole/hr /kg**0.75) 8

CONSTANT KMLiv4 = 0.1 ! Liver (umole/mL ) 9

CONSTANT VMOthC4 = 3000.0 ! OPT (umole/hr/k g**0.75) 10

CONSTANT KMOth4 = 0.1 ! OPT (umole/mL) 11 12
! Filtration (blood-urine) from Venous Blood ((kg** 0.25)/hr) 13
CONSTANT kFiltC1 = 7.401e-2 ! Butyl Acetate 14

CONSTANT kFiltC2 = 22.2 ! n-Butanol 15
CONSTANT kFiltC4 = 7.41e-2 ! Butyric Acid 16 17
! Fractional Inhalation Absorption - Butyl Acetate, Butanol 18

CONSTANT FA1 = 1.0 19

CONSTANT FA2 = 0.5 20

CONSTANT FA3 = 1.0 21

CONSTANT FA4 = 1.0 22 23
! Oral Absorption 24
CONSTANT kAC = 0.0 ! Absorption rate (kg**0.25/hr) 25 26
! Initial Amounts in Arterial Blood (i.e., integrat ion start value) 27

CONSTANT AAB10C = 0.0 ! BuAc (mg/kg) 28

CONSTANT AAB20C = 0.0 ! BuOH (mg/kg) 29

CONSTANT AAB30C = 0.0 ! BuCHO (mg/kg) 30

CONSTANT AAB40C = 0.0 ! BuCOOH (mg/kg) 31 32
! Molecular Weights <GD> 33

CONSTANT MW1 = 116.16 ! BuAc 34

CONSTANT MW2 = 74.12 ! BuOH 35

CONSTANT MW3 = 72.11 ! BuCHO 36

CONSTANT MW4 = 88.11 ! BuCOOH 37 38

! Simulation Parameters 39

CONSTANT TStop = 0.5 ! Length of exper iment (hr) 40

CINTERVAL CINT = 1. !Communication interval ( hr) 41

!CINT = CIntC <GD> 42 43
! Intravenous Dosing Parameters 44

CONSTANT IVDose1 = 0.0 ! BuAc (mg/kg) 45

CONSTANT IVDose2 = 0.0 ! BuOH (mg/kg) 46

CONSTANT IVDose3 = 0.0 ! BuCHO (mg/kg) 47

CONSTANT IVDose4 = 0.0 ! BuCOOH (mg/kg) 48 49

IVDUM1= IVDose1 * 1000 * BW/MW1 !BuAc (umol) <G D> 50

IVDUM2= IVDose2 * 1000 * BW/MW2 !BuOH (umol) <G D> 51

IVDUM3= IVDose3 * 1000 * BW/MW3 !BuCHO (umol) <G D> 52

IVDUM4= IVDose4 * 1000 * BW/MW4 !BuCOOH (umol <G D> 53 54
CONSTANT IVDAT=1. !IV injection averaging time (hr ; for infusion, IVDAT = 1.) <GD> 55

CONSTANT IVDON=0. !IV start time (hr) <GD> 56
CONSTANT IVDOFF=0. !IV stop time (for single inje ction, IVDOFF must = IVDAT) <GD> 57 58C-4 

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! Oral Dosing Parameters 1

CONSTANT PDose1 = 0.0 ! BuAc (mg/kg) 2

CONSTANT PDose2 = 0.0 ! BuOH (mg/kg) 3

CONSTANT PDose3 = 0.0 ! BuCOO (mg/kg) 4

CONSTANT PDose4 = 0.0 ! BuCOOH (mg/kg) 5 6

ORDUM1 = PDose1 * 1000 * BW/MW1 !BuAc (umol) < GD> 7

ORDUM2 = PDose2 * 1000 * BW/MW2 !BuOH (umol) <G D> 8

ORDUM3 = PDose3 * 1000 * BW/MW3 !BuCOO (umol) <G D> 9

ORDUM4 = PDose4 * 1000 * BW/MW4 !BuCOOH (umol) < GD> 10 11

CONSTANT ORDON=0. !Exposure start (hr) <GD> 12

CONSTANT ORDOFF=0. !Exposure stop (hr) <GD> 13
CONSTANT ORDPER1=20000.!Exposure pulse period (e.g . 24-hr day) <GD> 14
CONSTANT ORDWID1=20000.!Exposure pulse 1 width (e. g. hr/day)) <GD> 15
CONSTANT ORDPER2=20000.!Exposure pulse period (e.g . 168-hr wk) <GD> 16
CONSTANT ORDWID2=20000.!Exposure pulse 1 width (12 0 hr/wk) <GD> 17
CONSTANT ORDAT=1.0 !Dosing averaging time for sin gle oral dose (e.g. gavage) <GD> 18 19
! Inhalation Dosing Paramters 20

CONSTANT INHTABLE = 0 ! Switch to use inhlat ion exposures from INHTABLE (=1) 21 22

CONSTANT CInh1 = 0.0 ! BuAc (ppm) 23

CONSTANT CInh2 = 0.0 ! BuOH (ppm) 24

CONSTANT CInh3 = 0.0 ! BuCOO (ppm) 25

CONSTANT CInh4 = 0.0 ! BuCOOH (ppm) 26

CONSTANT QPTable = 0 ! Set to .T. for usi ng observed MV data <GD> 27 28

CIUM1= CInh1/24450 !BuAc (umol/ml) <GD> 29

CIUM2= CInh2/24450 !BuOH (umol/ml) <GD> 30

CIUM3= CInh3/24450 !BuCOO (umol/ml) <GD> 31

CIUM4= CInh4/24450 !BuCOOH (umol/ml) <GD> 32 33

CONSTANT CIOFFPPM1=0. !BuAc conc. when inhalation exposure is off (ppm) <GD> 34

CONSTANT CIOFFPPM2=0. !BuOH conc. when inhalation exposure is off (ppm) <GD> 35

CONSTANT CIOFFPPM3=0. !BuCOO conc. when inhalation exposure is off (ppm) <GD> 36

CONSTANT CIOFFPPM4=0. !BuCOOH conc. when inhalati on exposure is off (ppm) <GD> 37 38

CIOFF1= CIOFFPPM1/24450 !BuAc conc. when inhalat ion exposure is off (umol/ml) <GD> 39

CIOFF2= CIOFFPPM2/24450 !BuOH conc. when inhalat ion exposure is off (umol/ml) <GD> 40

CIOFF3= CIOFFPPM3/24450 !BuCOO conc. when inhalat ion exposure is off (umol/ml) <GD> 41

CIOFF4= CIOFFPPM4/24450 !BuCOOH conc. when inhala tion exposure is off (umol/ml) <GD> 42 43
CONSTANT INHON=0. !Time inhalation exposure sta rts (hr) <GD> 44
CONSTANT INHOFF= 10. !Time exposure stops (hr) <GD> 45
CONSTANT INHPER1=20000. !Pulse period 1 for exposu re (e.g., 24-hr day) <GD> 46
CONSTANT INHWID1=20000. !Pulse width 1 (e.g. 6 hr/ day) <GD> 47
CONSTANT INHPER2=20000. !Pulse period 2 (e.g. 168- hr wk) <GD> 48
CONSTANT INHWID2=20000. !Pulse width 2 (e.g. 120 hr/wk) <GD> 49 50

TABLE CINHTAB1,1,200/200*0.0,200*0.0/ !Call VentTab leS2 from CINHTAB*.dat file <GD> 51

TABLE CINHTAB2,1,200/200*0.0,200*0.0/ !Call VentTab leS2 from CINHTAB*.dat file <GD> 52

TABLE CINHTAB3,1,200/200*0.0,200*0.0/ !Call VentTab leS2 from CINHTAB*.dat file <GD> 53

TABLE CINHTAB4,1,200/200*0.0,200*0.0/ !Call VentTab leS2 from CINHTAB*.dat file <GD> 54
!Deleted Table values in CSL and replaced with m-fi les that fill tables <GD> 55 56 57C-5 

### DRAFT - DO NOT CITE OR QUOTE

! Inhalation Dose for Closed Chamber 1

CONSTANT SCInPPM1 = 0.0 ! BuAc (ppm) 2

CONSTANT SCInPPM2 = 0.0 ! BuOH (ppm) 3

CONSTANT SCInPPM3 = 0.0 ! BuCHO (ppm) 4

CONSTANT SCInPPM4 = 0.0 ! BuCOOH (ppm) 5 6

CICHUM1= SCInPPM1/24450 !BuAc (umol/ml) <GD> 7

CICHUM2= SCInPPM2/24450 !BuOH (umol/ml) <GD> 8

CICHUM3= SCInPPM3/24450 !BuCOO (umol/ml) <GD> 9

CICHUM4= SCInPPM4/24450 !BuCOOH (umol/ml) <GD> 10 11

ACh01 = 0.0 ; ACh02 = 0.0 ; ACh03 = 0.0 ; AC h04 = 0.0 !<GD> 12

ACh01 = VCh*CICHUM1 !BuAc (umol) <GD> 13

ACh02 = VCh*CICHUM2 !BuOH (umol) <GD> 14

ACh03 = VCh*CICHUM3 !BuCOO (umol) <GD> 15

ACh04 = VCh*CICHUM4 !BuCOOH (umol) <GD> 16 17

TABLE VentTable,1,200/200*0.0,200*0.0/ !Call Vent Table from *.dat file <GD> 18

!Deleted Table values <GD> 19 20

! Closed Chamber Parameters 21
! If a Plethysmorgraph tube is used, the given volu me of the chamber should be 22
! used directly without subtraction of the volume of the rat (number of rats x BW). 23

! Set Tube=.T. 24

CONSTANT ClOn = 0.0 !Switch for closed ch amber (=1), or open (=0) 25

CONSTANT VChC = 1.0e10 ! Volume of close d chamber (L) 26

CONSTANT Rats = 0 ! Number of rats in ch amber 27

CONSTANT Tube = 0 !Switch to include Tub e (=1) 28

CONSTANT LossT = 0.0 ! Time to switch to second chamber loss term 29 30

! Closed Chamber Parameters -- Loss Rates (/hr) 31

CONSTANT kLoss1R = 0.0 ! Rat body for n- Butyl acetate 32

CONSTANT kLoss1C1 = 0.0 ! Chamber for n-B utyl acetate 33

CONSTANT kLoss1C2 = 0.0 ! Chamber for n-B utyl acetate for 2nd period 34

CONSTANT kLoss2R = 0.0 ! Rat body for n- Butanol 35

CONSTANT kLoss2C1 = 0.0 ! Chamber for n-B utanol 36

CONSTANT kLoss2C2 = 0.0 ! Chamber for n-B utanol for 2nd period 37

CONSTANT kLoss3R = 0.0 ! Rat body for n- Butyraldehyde 38

CONSTANT kLoss3 = 0.0 ! Chamber for n-B utyraldehyde 39

CONSTANT kLoss4R = 0.0 ! Rat body for n- Butyric acid 40

CONSTANT kLoss4 = 0.0 ! Chamber for n-B utyric acid 41 42

! Blood Flows (mL/hr) 43
QOthC = 1.0 - QFatC - QLivC ! Other perf used tissues (fraction) 44 45

! Tissue Volumes (mL) 46

VAB = VABC * BW * 1000.0 ! Arterial b lood volume 47

VFat = VFatC * BW * 1000.0 ! Fat 48

VLiv = VLivC * BW * 1000.0 ! Liver 49

VLung = VLungC * BW * 1000.0 ! Lung 50

VOth = VOthC * BW * 1000.0 ! Other perf used tissues 51

VVB = VVBC * BW * 1000.0 ! Venous blo od 52

VBlood = VAB + VVB ! Total bloo d 53
! Metabolism -- Butyl Acetate (umoles/hr) 54

VMAB1 = VMBloodC1 * VAB/VBlood * (BW**0.75) ! Arterial metabolism BuAc 55

VMVB1 = VMBloodC1 * VVB/VBlood * (BW**0.75) ! Venous metabolism BuAc 56

VMLiv1 = VMLivC1 * (BW**0.75) ! Liver metabolism BuAc 57

VMOth1 = VMOthC1 * (BW**0.75) ! Other Perf used Tissue Metabolism EtAc 58 59C-6 

### DRAFT - DO NOT CITE OR QUOTE

! Metabolism -- n-Butanol 1

VMLiv2 = VMLivC2 * (BW**0.75) ! Liver meta bolism BuOH (umoles/hr) 2

kOth2 = kOthC2 / (BW**0.25) ! Other meta bolism BuOH (/hr) 3 4
! Metabolism -- n-Butyraldehyde (umoles/hr) 5

VMLiv3 = VMLivC3 * (BW**0.75) ! Liver meta bolism BuCHO 6 7
! Metabolism -- Butyric Acid (umoles/hr) 8

VMLiv4 = VMLivC4 * (BW**0.75) ! Liver meta bolism BuCOOH 9

VMOth4 = VMOthC4 * (BW**0.75) ! OPT metabo lism BuCOOH 10 11

! Filtration from Venous Blood (/hr) 12
kFilt1 = kFiltC1 / (BW**0.25) ! Kidney Fil tration of BuAc 13
kFilt2 = kFiltC2 / (BW**0.25) ! Kidney Fil tration of BuOH 14
kFilt4 = kFiltC4 / (BW**0.25) ! Kidney Fil tration of BuCOOH 15 16
! Oral Absorption (/hr) 17

kA = kAC / (BW**0.25) 18 19
! Initial Amounts in Arterial Blood (i.e. integrati on start value) 20

AAB10 = ((AAB10C * 1000.0)/MW1) * BW ! BuA c (umoles total) 21

AAB20 = ((AAB20C * 1000.0)/MW2) * BW ! BuO H (umoles total) 22

AAB30 = ((AAB30C * 1000.0)/MW3) * BW ! BuC HO (umoles total) 23

AAB40 = ((AAB40C * 1000.0)/MW4) * BW ! BuC OOH (umoles total) 24 25

! Initialize values 26

! CINT = CIntC 27

DoseMod = 0.0 28

kIV1 = 0.0 ; kIV2 = 0.0 ; kIV3 = 0.0 ; kIV4 = 0.0 29

Conc1 = 0.0 ; Conc2 = 0.0 ; Conc3 = 0.0 ; Conc4 = 0.0 30

Dose1 = 0.0 ; Dose2 = 0.0 ; Dose3 = 0.0 ; Dose4 = 0.0 31

XCInh1 = 0.0 ; XCInh2 = 0.0 ; XCInh3 = 0.0 ; XCInh4 = 0.0 32

XSCInPPM1 = 0.0 ; XSCInPPM2 = 0.0 ; XSCInPPM3 = 0.0 ; XSCInPPM4 = 0.0 33

XPDose1 = 0.0 ; XPDose2 = 0.0 ; XPDose3 = 0.0 ; XPDose4 = 0.0 34

XAUCTB1 = 0.0 ; XAUCTB2 = 0.0 ; XAUCTB3 = 0.0 ; XAUCTB4 = 0.0 35

CVLiv1 = 0.0 ; CVLiv2 = 0.0 ; CVLiv3 = 0.0 ; CVLiv4 = 0.0 36 37

CCh1 = 0.0 ; CCh2 = 0.0 ; CCh3 = 0.0 ; CCh4 = 0.0 38 39

! Closed Chamber Parameters 40

IF (Tube .EQ. 0) THEN !<GD> 41

VCh = VChC*1000.0 - Rats*BW*1000.0 ! Volume adjusted to mL 42

ELSE 43

VCh = VChC*1000.0 44

ENDIF 45 46

END ! End of Initial 47 48

DYNAMIC 49
ALGORITHM IALG = 2 ! Gear algorithm 50 51

DERIVATIVE 52

Minutes = T * 60.0 53

Seconds = Minutes * 60.0 54 55C-7 

### DRAFT - DO NOT CITE OR QUOTE

PROCEDURAL !<GD> 1

IF (QPTable .EQ. 0) THEN 2

QP = QPC*(BW**0.75) 3

QC = QCC*(BW**0.75) 4

ELSE 5
QP = VentTable(Minutes) 6

QC = QP 7

ENDIF 8

QFat = QFatC * QC ! Fat 9
QLiv = QLivC * QC ! Liver 10
QOth = QOthC * QC ! Other perfused tissues 11

END 12 13
!IV Dosing Simulation 14 15

IV1=IVDUM1/IVDAT !BuAc (umol/hr) <GD> 16

IV2=IVDUM2/IVDAT !BuOH (umol/hr) <GD> 17

IV3=IVDUM3/IVDAT !BuCOO (umol/hr) <GD> 18

IV4=IVDUM4/IVDAT !BuCOOH (umol/hr) <GD> 19 20

kIV1= RSW(T.LE.IVDOFF,IV1,0.) !BuAC (umol/hr on/o ff) <GD> 21

kIV2= RSW(T.LE.IVDOFF,IV2,0.) !BuOH (umol/hr on/o ff) <GD> 22

kIV3= RSW(T.LE.IVDOFF,IV3,0.) !BuCOO(umol/hr on/o ff) <GD> 23

kIV4= RSW(T.LE.IVDOFF,IV4,0.) !BuCOOH (umol/hr on /off) <GD> 24 25
!Oral Dosing Simulation <GD> 26

IF ((PDose1+PDose2+PDose3+PDose4) .GT. 0.)THEN 27

ORAL= PULSE(ORDON,ORDPER1,ORDWID1)*PULSE(ORDON,OR DPER2,ORDWID2) 28

ELSE 29

ORAL=0. 30

END IF 31 32

ORAL1 = ORAL *ORDUM1/ORDAT !BuAc (umol/hr) 33

ORAL2= ORAL *ORDUM2 /ORDAT !BuOH(umol/hr) 34

ORAL3 = ORAL *ORDUM3 /ORDAT !BuCOO (umol/hr) 35

ORAL4 = ORAL *ORDUM4/ORDAT !BuCOOH (umol/hr) 36 37

Dose1= RSW(T.LE.ORDOFF,ORAL1,0.) !BuAc(umol/hr on /off) 38

Dose2= RSW(T.LE.ORDOFF,ORAL2,0.) !BuOH (umol/h on /off) 39

Dose3= RSW(T.LE.ORDOFF,ORAL3,0.) !BuCOO (umol/hr on/off) 40

Dose4= RSW(T.LE.ORDOFF,ORAL4,0.) !BuCOOH (umol/hr on/off) 41 42
!Inhalation Dosing Simulation <GD> 43 44

IF ((CInh1+CInh2+CInh+CInh4) .GT. 0.)THEN 45

CION= PULSE(INHON,INHPER1,INHWID1)*PULSE(INHON,IN HPER2,INHWID2) 46

ELSE 47

CION=0. 48

END IF 49 50

CION1 = CION * CIUM1 !BuAc exposure (umol/ml) 51

CION2 = CION * CIUM2 !BuOH exposure (umol/ml) 52

CION3 = CION * CIUM3 !BuCOO exposure (umol/ml) 53

CION4 = CION * CIUM4 !BuCOOH exposure (umol/ml) 54 55

Conc11 = RSW(T.LE.AIROFF,CION1,CIOFF) 56

!BuAc conc. in inhaled air (umol/L on/off) 57 58C-8 

### DRAFT - DO NOT CITE OR QUOTE Conc22 = RSW(T.LE.AIROFF,CION2,CIOFF) 1

!BuOH conc. in inhaled air (umol/L on/off) 2

Conc33 = RSW(T.LE.AIROFF,CION3,CIOFF) 3

!BuCOO conc. in inhaled air (umol/L on/off) 4

Conc44 = RSW(T.LE.AIROFF,CION4,CIOFF) 5

!BuCOOH conc. in inhaled air (umol/L on/off 6 7

IF (INHTABLE .EQ. 1) THEN 8

Conc1 = CINHTAB1(minutes)/24450.0 9

Conc2 = CINHTAB2(minutes)/24450.0 10

Conc3 = CINHTAB3(minutes)/24450.0 11

Conc4 = CINHTAB4(minutes)/24450.0 12

ELSE 13

Conc1 = Conc11 !BuAc conc. in inhaled air (umol/L on/off) 14

Conc2 = Conc22 !BuOH conc. in inhaled air (umol/L on/off) 15

Conc3 = Conc33 !BuCOO conc. in inhaled air (umol/ L on/off) 16

Conc4 = Conc44 !BuCOOH conc. in inhaled air (umol /L on/off 17

ENDIF 18 19

!Chamber Loss simulation 20

kLoss1= RSW(T.LE.LossT,kLoss1C1,kLoss1C2) !BuAC ( umol/hr on/off) <GD> 21

kLoss2= RSW(T.LE.LossT,kLoss2C1,kLoss2C2) !BuAC ( umol/hr on/off) <GD> 22 23

!************************* Butyl Acetate ****** ******************* 24

! Arterial Blood (umoles) 25

RAAB1 = (QC * (CVLung1 - CAB1)) - RMetAB1 26

AAB1 = INTEG(RAAB1, AAB10) 27

CAB1 = AAB1 / VAB 28

AUCAB1 = INTEG(CAB1, 0.0) 29 30

! Blood Metabolism (umoles) 31

RMetAB1 = (VMAB1 * CAB1) / (KMBlood1 + CAB1) 32

MetAB1 = INTEG(RMetAB1, 0.0) 33 34

! Fat (umole) 35

RAFat1 = QFat * (CAB1 - CVFat1) 36

AFat1 = INTEG(RAFat1, 0.0) 37

CFat1 = AFat1 / VFat 38

CVFat1 = CFat1 / PFat1 39 40

! Liver (umoles) 41
RALiv1 = (QLiv * (CAB1 - CVLiv1)) - RMetLiv1 + RMR1 42

ALiv1 = INTEG(RALiv1, 0.0) 43

CLiv1 = ALiv1 / VLiv 44

CVLiv1 = CLiv1 / PLiv1 45

AUCLiv1 = INTEG(CLiv1, 0.0) 46 47

! Liver Metabolism (umoles) 48

RMetLiv1 = (VMLiv1 * CVLiv1) / (KMLiv1 + CVLiv1) 49

MetLiv1 = INTEG(RMetLiv1, 0.0) 50 51
! Amount remaining to be absorbed into liver from G I (umoles) 52

RMR1 = kA * MR1 53

MR1 = Dose1 - INTEG(RMR1, 0.0) 54

! Chamber concentration (umoles/mL) 55

RACh1 = (Rats * QP * CEx1) - (FA1 * Rats * QP * CCh1) - (kLoss1 * ACh1)& 56

- (kLoss1R * Rats * ACh1) 57

ACh1 = INTEG(RACh1, ACh01) 58 59C-9 

### DRAFT - DO NOT CITE OR QUOTE

CCh1 = ((ACh1 / VCh) * ClOn) + (Conc1 * (1.0 - ClOn)) 1

CCPPM1 = CCh1 * 24450.0 2

CLoss1 = INTEG(((kLoss1 * ACh1) + (kLoss1R * Ra ts * ACh1)), 0.0) 3 4

IF (SCINPPM1 .GT. 0.) THEN 5

CCPPM1PCT=100.*CCPPM1/SCINPPM1 !BuAc conc. as % of intitial <GD> 6

ELSE 7

CCPPM1PCT = 0. 8

END IF 9 10

! Lungs 11
! Amount in Lungs (umoles) 12

RALung1 = (QP * ((FA1 * CCh1) - CEx1)) + (QC * ( CVB1 - CVLung1)) 13

ALung1 = INTEG(RALung1, 0.0) 14

CLung1 = ALung1 / VLung 15

CVLung1 = CLung1 / PLung1 16 17

! Amount Inhaled (umoles) 18

RInhal1 = FA1 * QP * CCh1 19

AInhal1 = INTEG(RInhal1, 0.0) 20

AInhal1C = AInhal1 * Rats 21 22

! Amount Exhaled (umoles) 23

CEx1 = CvLung1 / PB1 24

RAEx1 = QP * CEx1 25

AEx1 = INTEG(RAEx1, 0.0) 26

AEx1C = AEx1 * Rats 27 28

! Other Perfused Tissue Modified to Include Metabol ism (umoles) 29

RAOth1 = (QOth * (CAB1 - CVOth1)) - RMetOth1 30

AOth1 = INTEG(RAOth1, 0.0) 31

COth1 = AOth1 / VOth 32

CVOth1 = COth1 / POth1 33

AUCOth1 = INTEG(COth1, 0.0) 34 35

! Other Perfused Tissue Metabolism (umoles) 36

RMetOth1 = (VMOth1 * CVOth1) / (KMOth1 + CVOth1) 37

MetOth1 = INTEG(RMetOth1, 0.0) 38 39

! Venous Blood (umoles) 40
RAVB1 = (QFat*CVFat1) + (QLiv*CVLiv1) + (QOth* CVOth1) + kIV1 & 41
- (QC*CVB1) - RMetVB1 - RAFilt1 42

AVB1 = INTEG(RAVB1, 0.0) 43

CVB1 = AVB1 / VVB 44

AUCB1 = INTEG(CVB1, 0.0) 45 46

! Metabolism 47

RMetVB1 = (VMVB1 * CVB1) / (KMBlood1 + CVB1) 48

MetVB1 = INTEG(RMetVB1, 0.0) 49 50

! Filtration 51

RAFilt1 = kFilt1 * AVB1 52

AFilt1 = INTEG(RAFilt1, 0.0) 53 54

! Average Blood (umoles) 55

CTB1 = (AAB1 + AVB1) / (VAB + VVB) 56

AUCTB1 = INTEG(CTB1, 0.0) 57 58C-10 

### DRAFT - DO NOT CITE OR QUOTE

!*************************** n-Butanol ******** ******************* 1

! Arterial Blood (umoles) 2

RAAB2 = (QC * (CVLung2 - CAB2)) + RMetAB1 3

AAB2 = INTEG(RAAB2, AAB20) 4

CAB2 = AAB2 / VAB 5

AUCAB2 = INTEG(CAB2, 0.0) 6

IF (T>0.) THEN !<GD> 7

AUCAB2_24=(AUCAB2/T)*24. !AUC mM*24 hr <GD> 8

ELSE !<GD> 9

AUCAB2_24=0. !<GD> 10

END IF 11 12

! Liver (umoles) 13
RALiv2 = (QLiv * (CAB2 - CVLiv2)) + RMetLiv1 + RMR2 - RMetLiv2 14

ALiv2 = INTEG(RALiv2, 0.0) 15

CLiv2 = ALiv2 / VLiv 16

CVLiv2 = CLiv2 / PLiv2 17

AUCLiv2 = INTEG(CLiv2, 0.0) 18 19

! Liver Metabolism (umoles) 20

RMetLiv2 = (VMLiv2 * CVLiv2) / (KMLiv2 + CVLiv2) 21

MetLiv2 = INTEG(RMetLiv2, 0.0) 22 23
! Amount remaining to be absorbed into liver from G I (umoles) 24

RMR2 = kA * MR2 25

MR2 = Dose2 - INTEG(RMR2, 0.0) 26 27

! Chamber concentration (umoles/mL) 28

RACh2 = (Rats * QP * CEx2) + (kLoss1 * ACh1) - (FA2 * Rats * QP * CCh2) & 29

- (kLoss2 * ACh2) - (kLoss2R * Rats * AC h2) 30

ACh2 = INTEG(RACh2, ACh02) 31

CCh2 = ((ACh2 / VCh) * ClOn) + (Conc2 * (1.0 - ClOn)) 32

CCPPM2 = CCh2 * 24450.0 33

CLoss2 = INTEG(((kLoss2 * ACh2) + (kLoss2R * Ra ts * ACh2)), 0.0) 34 35

IF (SCINPPM2 .GT. 0.) THEN 36

CCPPM2PCT=100.*CCPPM2/SCINPPM2 !BuOH conc. as % of intitial <GD> 37

ELSE 38

CCPPM2PCT = 0. 39

END IF 40 41

! Lungs 42
! Amount in Lungs (umoles) 43

RALung2 = (QP * ((FA2 * CCh2) - CEx2)) + (QC * ( CVB2 - CVLung2)) 44

ALung2 = INTEG(RALung2, 0.0) 45

CLung2 = ALung2 / VLung 46

CVLung2 = CLung2 / PLung2 47 48

! Amount Inhaled 49

RInhal2 = FA2 * QP * CCh2 50

AInhal2 = INTEG(RInhal2, 0.0) 51

AInhal2C = AInhal2 * Rats 52 53

! Amount Exhaled 54

CEx2 = CvLung2 / PB2 55

RAEx2 = QP * CEx2 56

AEx2 = INTEG(RAEx2, 0.0) 57

AEx2C = AEx2 * Rats 58 59C-11 

### DRAFT - DO NOT CITE OR QUOTE

! Other Perfused Tissues (umoles) 1

RAOth2 = ((QOth + QFat) * (CAB2 - CVOth2)) + RM etOth1 - RAMOth2 2

AOth2 = INTEG(RAOth2, 0.0) 3

COth2 = AOth2 / (VOth + VFat) 4

CVOth2 = COth2 / POth2 5 6

! Metabolism in Other Perfused Tissues (umoles) 7

RAMOth2 = kOth2 * AOth2 8

AMOth2 = INTEG(RAMOth2, 0.0) 9 10

! Venous Blood 11
RAVB2 = (QLiv*CVLiv2) + ((QOth+QFat)*CVOth2) + RMetVB1 + kIV2 & 12
- (QC*CVB2) - RAFilt2 13

AVB2 = INTEG(RAVB2, 0.0) 14

CVB2 = AVB2 / VVB 15

AUCB2 = INTEG(CVB2, 0.0) 16 17

! Filtration 18

RAFilt2 = kFilt2 * AVB2 19

AFilt2 = INTEG(RAFilt2, 0.0) 20 21

! Average Blood (umoles) 22

CTB2 = (AAB2 + AVB2) / (VAB + VVB) 23

AUCTB2 = INTEG(CTB2, 0.0) 24 25 26

!************************ n-Butyraldehyde ***** ******************* 27

! Arterial Blood (umoles) 28

RAAB3 = QC * (CVLung3 - CAB3) 29

AAB3 = INTEG(RAAB3, AAB30) 30

CAB3 = AAB3 / VAB 31

AUCAB3 = INTEG(CAB3, 0.0) 32 33

! Liver (umoles) 34
RALiv3 = (QLiv * (CAB3 - CVLiv3)) + RMetLiv2 + RMR3 - RMetLiv3 35

ALiv3 = INTEG(RALiv3, 0.0) 36

CLiv3 = ALiv3 / VLiv 37

CVLiv3 = CLiv3 / PLiv3 38

AUCLiv3 = INTEG(CLiv3, 0.0) 39 40

! Liver Metabolism (umoles) 41

RMetLiv3 = (VMLiv3 * CVLiv3) / (KMLiv3 + CVLiv3) 42

MetLiv3 = INTEG(RMetLiv3, 0.0) 43 44
! Amount remaining to be absorbed into liver from G I (umoles) 45

RMR3 = kA * MR3 46

MR3 = Dose3 - INTEG(RMR3, 0.0) 47 48

! Chamber concentration (umoles/mL) 49

RACh3 = (Rats * QP * CEx3) - (FA3 * Rats * QP * CCh3) - (kLoss3 * ACh3) & 50

- (kLoss3R * Rats * ACh3) 51

ACh3 = INTEG(RACh3, ACh03) 52

CCh3 = ((ACh3 / VCh) * ClOn) + (Conc3 * (1.0 - ClOn)) 53

CCPPM3 = CCh3 * 24450.0 54

CLoss3 = INTEG(((kLoss3 * ACh3) + (kLoss3R * Rat s * ACh3)), 0.0) 55 56C-12 

### DRAFT - DO NOT CITE OR QUOTE

IF (SCINPPM3 .GT. 0.) THEN 1

CCPPM3PCT=100.*CCPPM3/SCINPPM3 !BuCOO conc. as % of intitial <GD> 2

ELSE 3

CCPPM3PCT = 0. 4

END IF 5 6

! Lungs 7
! Amount in Lungs (umoles) 8

RALung3 = (QP * ((FA3 * CCh3) - CEx3)) + (QC * ( CVB3 - CVLung3)) 9

ALung3 = INTEG(RALung3, 0.0) 10

CLung3 = ALung3 / VLung 11

CVLung3 = CLung3 / PLung3 12 13

! Amount Inhaled (umoles) 14

RInhal3 = FA3 * QP * CCh3 15

AInhal3 = INTEG(RInhal3, 0.0) 16

AInhal3C = AInhal3 * Rats 17 18

! Amount Exhaled (umoles) 19

CEx3 = CvLung3 / PB3 20

RAEx3 = QP * CEx3 21

AEx3 = INTEG(RAEx3, 0.0) 22

AEx3C = AEx3 * Rats 23 24

! Other Perfused Tissues (umoles) 25

RAOth3 = ((QOth + QFat) * (CAB3 - CVOth3)) + RA MOth2 26

AOth3 = INTEG(RAOth3, 0.0) 27

COth3 = AOth3 / (VOth + VFat) 28

CVOth3 = COth3 / POth3 29 30

! Venous Blood (umoles) 31
RAVB3 = (QLiv*CVLiv3) + ((QOth+QFat)*CVOth3) + kIV3 - (QC*CVB3) 32

AVB3 = INTEG(RAVB3, 0.0) 33

CVB3 = AVB3 / VVB 34

AUCB3 = INTEG(CVB3, 0.0) 35 36

! Average Blood (umoles) 37

CTB3 = (AAB3 + AVB3) / (VAB + VVB) 38

AUCTB3 = INTEG(CTB3, 0.0) 39 40
!************************* Butyric Acid ******* ******************* 41

! Arterial Blood (umoles) 42

RAAB4 = QC * (CVLung4 - CAB4) 43

AAB4 = INTEG(RAAB4, AAB40) 44

CAB4 = AAB4 / VAB 45

AUCAB4 = INTEG(CAB4, 0.0) 46 47

! Liver (umoles) 48

RALiv4 = (QLiv * (CAB4 - CVLiv4)) + RMetLiv3 + RMR4 - RMetLiv4 49

ALiv4 = INTEG(RALiv4, 0.0) 50

CLiv4 = ALiv4 / VLiv 51

CVLiv4 = CLiv4 / PLiv4 52

AUCLiv4 = INTEG(CLiv4, 0.0) 53 54

! Liver Metabolism (umoles) 55

RMetLiv4 = (VMLiv4 * CVLiv4) / (KMLiv4 + CVLiv4) 56

MetLiv4 = INTEG(RMetLiv4, 0.0) 57 58C-13 

### DRAFT - DO NOT CITE OR QUOTE

! Amount remaining to be absorbed into liver from G I (umoles) 1

RMR4 = kA * MR4 2

MR4 = Dose4 - INTEG(RMR4, 0.0) 3

! Chamber concentration (umoles/mL) 4

RACh4 = (Rats * QP * CEx4) - (FA4 * Rats * QP * CCh4) - (kLoss4 * ACh4) & 5

- (kLoss4R * Rats * ACh4) 6

ACh4 = INTEG(RACh4, ACh04) 7

CCh4 = ((ACh4 / VCh) * ClOn) + (Conc4 * (1.0 - ClOn)) 8

CCPPM4 = CCh4 * 24450.0 9

CLoss4 = INTEG(((kLoss4 * ACh4) + (kLoss4R * Ra ts * ACh4)), 0.0) 10 11

IF (SCINPPM4 .GT. 0.) THEN 12

CCPPM4PCT=100.*CCPPM4/SCINPPM4 !BuCOOH conc. as % of intitial <GD> 13

ELSE 14

CCPPM4PCT = 0. 15

END IF 16

! Lungs 17
! Amount in Lungs (umoles) 18

RALung4 = (QP * ((FA4 * CCh4) - CEx4)) + (QC * ( CVB4 - CVLung4)) 19

ALung4 = INTEG(RALung4, 0.0) 20

CLung4 = ALung4 / VLung 21

CVLung4 = CLung4 / PLung4 22 23

! Amount Inhaled 24

RInhal4 = FA4 * QP * CCh4 25

AInhal4 = INTEG(RInhal4, 0.0) 26

AInhal4C = AInhal4 * Rats 27 28

! Amount Exhaled 29

CEx4 = CvLung4 / PB4 30

RAEx4 = QP * CEx4 31

AEx4 = INTEG(RAEx4, 0.0) 32

AEx4C = AEx4 * Rats 33 34

! Other Perfused Tissues (umoles) 35

RAOth4 = (QOth + QFat) * (CAB4 - CVOth4) - RMet Oth4 36

AOth4 = INTEG(RAOth4, 0.0) 37

COth4 = AOth4 / (VOth + VFat) 38

CVOth4 = COth4 / POth4 39 40

! Other Perfused Tissue Metabolism (umoles) 41

RMetOth4 = (VMOth4 * CVOth4) / (KMOth4 + CVOth4) 42

MetOth4 = INTEG(RMetOth4, 0.0) 43 44

! Venous Blood (umoles) 45
RAVB4 = (QLiv*CVLiv4) + ((QOth+QFat)*CVOth4) + kIV4 - (QC*CVB4) - RAFilt4 46

AVB4 = INTEG(RAVB4, 0.0) 47

CVB4 = AVB4 / VVB 48

AUCB4 = INTEG(CVB4, 0.0) 49

! Filtration 50

RAFilt4 = kFilt4 * AVB4 51

AFilt4 = INTEG(RAFilt4, 0.0) 52 53

! Average Blood (umoles) 54

CTB4 = (AAB4 + AVB4) / (VAB + VVB) 55

AUCTB4 = INTEG(CTB4, 0.0) 56

TERMT(T.GE.TStop) 57 58C-14 

### DRAFT - DO NOT CITE OR QUOTE

!************************* Mass Balance ******* ******************* 1

! Total System Mass Balance 2

MassBal = TMass1 + AEx1 + AFilt1 + TMass2 + AEx2 + AFilt2 + TMass3 + AEx3 & 3

+ TMass4 + AEx4 + AFilt4 + TMetab4 4

! Mass Balance for BuAc (umoles) 5
TMass1 = AAB1 + AFat1 + ALiv1 + ALung1 + AOth1 + AVB1 6

TMetab1a = MetAB1 + MetLiv1 + MetVB1 + MetOth1 7

TMassTot1 = TMass1 + TMetab1a + AEx1 + AFilt1 8

Delta1 = ((IVDose1 * 1000.0/MW1) * BW) + AInhal 1 + Dose1 + AAB10 - TMassTot1 9

Delta1C = ACh01 - ACh1 - AInhal1C - CLoss1 + AEx 1C 10

! Mass Balance for BuOH (umoles) 11
TMass2 = AAB2 + ALiv2 + ALung2 + AOth2 + AVB2 12

TMetab2 = MetLiv2 + AmOth2 13

TMassTot2 = TMass2 + TMetab2 + AEx2 + AFilt2 14

Delta2 = ((IVDose2 * 1000.0/MW2) * BW) + AInhal 2 + Dose2 + AAB20 & 15

- TMassTot2 + TMetab1a 16

Delta2C = ACh02 - ACh2 - AInhal2C - CLoss2 + AEx 2C 17

! Mass Balance for BuCHO (umoles) 18
TMass3 = AAB3 + ALiv3 + ALung3 + AOth3 + AVB3 19

TMetab3 = MetLiv3 20

TMassTot3 = TMass3 + TMetab3 + AEx3 21

Delta3 = ((IVDose3 * 1000.0/MW3) * BW) + Dose3 + AAB30 + TMetab2 & 22

+ AInhal3 - TMassTot3 23

Delta3C = ACh03 - ACh3 - AInhal3C - CLoss3 + AEx 3C 24 25

! Mass Balance for Butyrate (umoles) 26
TMass4 = AAB4 + ALiv4 + ALung4 + AOth4 + AVB4 27

TMetab4 = MetLiv4 + MetOth4 28

TMassTot4 = TMass4 + TMetab4 + AEx4 + AFilt4 29

Delta4 = ((IVDose4 * 1000.0/MW4) * BW) + Dose4 + AAB40 & 30

+ AInhal4 - TMassTot4 + TMetab3 31

Delta4C = ACh04 - ACh4 - AInhal4C - CLoss4 + AEx 4C 32 33

Total1 = MetLiv1 + MetAB1 + MetVB1 34

Total2 = MetLiv2 + AMOth2 35

Total3 = MetLiv3 36

Total4 = MetLiv4 37 38

IF (AInhal1.NE.0.0) THEN 39

Recov = 100.0 * (TMassTot1 / AInhal1) 40

ELSE 41

Recov = 0.0 42

ENDIF 43

! Check Blood Flows 44
QTot = QFat + QLiv + QOth 45

QRecov = 100.0 * (QTot / QC) 46 47

END ! End of Derivative 48
END ! End of Dynamic 49 50

TERMINAL 51 52

END ! End of Terminal 53

END ! End of Program 54
55C-15 

### DRAFT - DO NOT CITE OR QUOTE

C.2. Summary of Model Evaluation 1 

### U.S. EPA performed an evaluation and quality contro l check of the PBPK model for 2 

### n-butanol published by Teeguarden et al. (2005). T he evaluation included confirmation of 3 

### accurate implementation of the model described in T eeguarden et al. (2005) and evaluations of 4 

### the performance of the rat and human PBPK models th at were not reported in Teeguarden et al. 5 

### (2005). These included evaluations of residuals fo r model predictions compared to observations 6


### made in rat i.v. and inhalation studies and in huma n inhalation studies; comparisons of observed 7 

### and predicted AUC for blood n-butanol–time profiles in rats and humans; and alternative values 8 

### for metabolism parameters were explored or estimate d by statistical optimization. The 9 

### conclusions from these analyses as follows: 10

11 

### 1. The value adopted by Teeguarden et al. (2005) for t he K m 

### of metabolism of n-butanol 12 

### (model variable: KMLivC2) in rats did not have a s trong empirical basis and, therefore, 13 

### the value derived from in vitro studies in rat hepa tic cytosol preparations (Carlson and 14 

### Olson, 1995) was selected as the preferred value fo r use in dosimetry calculations. Use 15 

### of this value was supported by statistical optimiza tion analyses, which showed that model 16 

### performance was not substantially improved or degra ded by use of the empirically-based 17 

### value. Since the value for KMLivC2 was modified fr om Teeguarden et al. (2005), values 18 

### for the V max 

### for n-butanol metabolism in the liver (model varia ble: VMLivC2) and the 19 

### rate constant for metabolism of n-butanol in other tissues (model variable: KOthC2) 20 

### were re-optimized and evaluated with data from i.v. and closed chamber inhalation 21 

### studies conducted in rats. 22 23 

### 2. The same empirical value for KMLivC2for was also ad opted for the human model and 24 

### values for VMLivC2 and KOthC2 were re-optimized aga inst data from human exposures 25 

### to n-butanol (Astrand et al., 1976). The optimized values resulted in improved fit to the 26 

### human data and were adopted for the human model. 27 28 

### 3. The validity of the human model is not as strongly supported as the rat model; it has been 29 

### tested against data from a single study. The metab olism parameter values for the human 30 

### model are highly sensitive to observations made in a single individual, and the influence 31 

### of these data on the parameter value estimates will affect the predicted n-butanol blood 32 

### AUC. However, even with these uncertainties, the h uman model provides improved 33 

### confidence for interspecies extrapolation of pharma cokinetics and, therefore, was used in 34 

### making dosimetry extrapolations from rats to humans

. 35 36 

### On the basis of this evaluation, U.S. EPA concluded that the PBPK model was suitable for use in 37 

### deriving the RfC for n-butanol. 38

39

40

41

42C-16 

### DRAFT - DO NOT CITE OR QUOTE

C.3. Uncertainties in PBPK Modeling of HECs Relate d to Selection of Values for 1


### Metabolism Parameters 2 

### Values for n-butanol metabolism parameters used for the dosimetry modeling are shown 3 

### in Table C-1. Values for n-butanol metabolism para meters, V max 

### for metabolism in the liver 4 

### (model variable: VMLivC2) and the rate constant fo r metabolism of n-butanol in other tissues 5 

### (model variable: KOthC2) were optimized against da ta for blood n-butanol concentrations in 6 

### humans during and following inhalation exposure to 100 or 200 ppm n-butanol (Astrand et al., 7 

### 1976), while keeping the value for the K m 

### for liver metabolism (model variable: KMLivC2) se t 8 

### at the empirically-based value of 0.16 mM (Carlson and Olson, 1995). Optimizing these 9 

### parameters resulted in improvement of model perform ance as judged by residuals for the Astrand 10 

### et al. (1976) observations. Improved model perform ance was the basis for selecting the 11 

### optimized values over allometric scaling of the rat values. The impact of this decision on the 12 

### estimate of the HEC corresponding to the rat NOAEL is shown in Table C-2. The NOAEL HEC 13 

### is 34% lower when the allometrically scaled rat val ues are used in the human model (39 vs. 14 

### 59 mg/m 3 

### ). 15 

### With respect to the urinary elimination rate parame ters, the PBPK model does not 16 

### incorporate the interaction of butyric acid with co lonocytes and it is well known that the basic 17 

### form, butyrate, is a major substrate for colon cell s (see Thibault et al., 2010 for a recent review). 18 

### However, kinetic information on urinary clearance f or butyric acid following n-butanol 19


### administration is not available and was therefore n ot incorporated into the model. 20

21

22 

### Table C-1. n-Butanol metabolism parameters

Parameter Abbreviation Values
Basis Rat Human
Maximum metabolic rate in liver VMLivC2 2.17 0.62 O ptimized (see model evaluation report) Affinity constant in liver KMLiv2 0.16 0.16 Estimat e for rat (Carlson and Olson,1995)
First-order metabolic rate constant in other tissues KOthC2 4.0 20.1 Optimized (see model evaluation rep ort)

Units: V max (mmol/hr/kg 0.75 ); K m (mM); K other

(kg 0.25 /hr). 23 

### Table C-2. Results of PBPK modeling of NOAEL conce ntration from

subchronic rat study

Source of human parameter values NOAEL rat mg/m 3 NOAEL

rat
ppm AUCAB2_24

rat mM

⋅ 24hr AUCAB2_24

human mM

⋅ 24hr NOAEL

HEC human
ppm NOAEL

HEC human mg/m 3 Optimized a 154 50 0.01282 0.01282 19.21 59.2C-17 

### DRAFT - DO NOT CITE OR QUOTE

Allometrically scaled from rat values b 154 50 0.01282 0.01282 12.76 39.3

a KMLivC2=0.16 mM, VMLivC2=0.62 mmol/hr/kg 0.75 , KOthC2=20.1 kg 0.25 /hr.
b KMLivC2=0.16 mM, VMLivC2=2.17 mmol/hr/kg 0.75 , KOthC2=4.0 kg 0.25 /hr.

Source: Korsak et al. (1994).
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