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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 review under applicable information quality 

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

be construed to represent any Agency determination or policy. It is being circulated for review 

of its technical accuracy and science policy implications. 

U.S. Environmental Protection Agency 

Washington, DC 
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DISCLAIMER 

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

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

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

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

names or commercial products does not constitute endorsement or recommendation for use. 
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CONTENTS - TOXICOLOGICAL REVIEW OF n-BUTANOL (CAS No. 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 BIOASSAYS IN 

ANIMALS—ORAL AND INHALATION ...................................................................... 29

4.2.1. Oral ............................................................................................................................... 29

4.2.2. Inhalation ..................................................................................................................... 31

4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION ...... 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 Neurodevelopmental Effects ................................... 57

4.5.5. Mechanistic Studies Evaluating Liver Effects ............................................................ 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 Endpoints .............................................. 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 Effect—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 Uncertainty 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 Effect—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 Uncertainty 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 HAZARD 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 PUBLIC 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 Related to Selection of Values for Metabolism 

Parameters ..................................................................................................................... C-16
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LIST OF TABLES 

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

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

Table 3-2. Physiological parameters for a PBPK model 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 for the n-butyl series in the rat and humana

 ................................................................................................................................................ 17

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

and humana

 ............................................................................................................................. 18

Table 4-1. Occupational Studies with n-butanol exposure ........................................................... 20

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

Table 4-3. Controlled Acute Exposure Studies ............................................................................. 27

Table 4-4. Hematological effects in rats exposed to 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 gestationa ........................................... 36

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

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

Table 4-9. Selected changes in rats exposed to n-butanol 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 vitro ................................................................ 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-response information for n-butanol .................... 64

Table 4-14. Summary of inhalation noncancer dose-response 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 based 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 NOAEL 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 drinking water. ....................................... B-2

Table C-1. n-Butanol metabolism parameters ......................................................................... C-16

Table C-2. Results of PBPK modeling of NOAEL concentration from subchronic rat study C-16
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LIST OF FIGURES 

Figure 3-1 Metabolism of n-Butanol. .......................................................................................... 10

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

humans. .................................................................................................................................. 14

Figure 5-1. Exposure-response array for oral exposure 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 incidence of litters with visceral malformations 

(overall). ............................................................................................................................... B-2
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LIST OF ABBREVIATIONS AND ACRONYMS 

ACGIH American Conference of Governmental Industrial Hygienists 

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 acid 

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 

CO2 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 
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FOREWORD 

The purpose of this Toxicological Review is to provide 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 on 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, where applicable, and to characterize the overall 

confidence in the quantitative and qualitative aspects 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 assessor 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 (202) 566-1676 (phone), (202) 566-1749 (fax), or 

hotline.iris@epa.gov (email address). 
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1 AUTHORS, CONTRIBUTORS, AND REVIEWERS 

2 

3 

4 CHEMICAL MANAGER/AUTHOR 

5 

6 Ambuja Bale 

7 National Center for Environmental Assessment 

8 U.S. Environmental Protection Agency 

9 Washington, DC 

10 

11 AUTHORS 

12 

13 Alan Sasso 

14 National Center for Environmental Assessment 

15 U.S. Environmental Protection Agency 

16 Washington, DC 

17 

18 James A. Weaver 

19 National Center for Environmental Assessment 

20 U.S. Environmental Protection Agency 

21 Washington, DC 

22 

23 CONTRIBUTORS 

24 

25 Karen Hogan 

26 National Center for Environmental Assessment 

27 U.S. Environmental Protection Agency 

28 Washington, DC 

29 

30 Chester Rodriguez 

31 Office of Chemical Safety and Pollution Prevention 

32 U.S. Environmental Protection Agency 

33 Washington, DC 

34 

35 Paul Schlosser 

36 National Center for Environmental Assessment 

37 U.S. Environmental Protection Agency 

38 Research Triangle Park, NC 

39 

40 Hui-Min Yang 

41 National Center for Environmental Assessment 

42 U.S. Environmental Protection Agency 

43 Washington, DC 

44 

45 

46 CONTRACTOR SUPPORT 

47 

48 Heather Carlson-Lynch 

49 Michael Lumpkin 
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1 Gary Diamond 

2 Daniel Plewak 

3 Julie Stickney 

4 Chemical, Biological and Environmental Center 

5 SRC, Inc. 

6 7502 Round Pond Road 

7 North Syracuse, NY 13212 

8 

9 

10 INTERNAL EPA REVIEWERS 

11 

12 John Cowden 

13 National Center for Environmental Assessment 

14 U.S. Environmental Protection Agency 

15 Research Triangle Park, NC 

16 

17 Ted Berner 

18 National Center for Environmental Assessment 

19 U.S. Environmental Protection Agency 

20 Washington, DC 

21 

22 Philip J. Bushnell 

23 National Health and Environmental Effects Research Laboratory 

24 U.S. Environmental Protection Agency 

25 Research Triangle Park, NC 

26 

27 Barbara Glenn 

28 National Center for Environmental Assessment 

29 U.S. Environmental Protection Agency 

30 Washington, DC 

31 

32 Karl Jensen 

33 National Health and Environmental Effects Research Laboratory 

34 U.S. Environmental Protection Agency 

35 Research Triangle Park, NC 

36 

37 Ginger Moser 

38 National Health and Environmental Effects Research Laboratory 

39 U.S. Environmental Protection Agency 

40 Research Triangle Park, NC 

41 

42 

43 

44 
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1 1. INTRODUCTION 

2 

3 

4 This document presents background information and justification for the Integrated Risk 

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

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

7 (RfC) values for chronic and other exposure durations, and a carcinogenicity assessment. 

8 The RfD and RfC, if derived, provide quantitative information for use in risk assessments 

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

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

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

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

deleterious effects during a lifetime. The inhalation RfC (expressed in units of mg/m313 ) is 

14 analogous to the oral RfD, but provides a continuous inhalation exposure estimate. The 

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

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

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

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

19 lifetime) exposure durations, all of which are derived based on an assumption of continuous 

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

21 derived for chronic exposure duration. 

22 The carcinogenicity assessment provides information on the carcinogenic hazard 

23 potential of the substance in question and quantitative estimates of risk from oral and inhalation 

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

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

26 effects may be expressed. Quantitative risk estimates may be derived from the application of a 

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

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

an upper bound on the estimate of risk per μg/m329 air breathed. 

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

31 has followed the general guidelines for risk assessment as set forth by the National Research 

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

33 Assessment Forum Technical Panel Reports that may have been used in the development of this 

34 assessment include the following: Guidelines for the Health Risk Assessment of Chemical 

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

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

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

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

39 1994a), Methods for Derivation of Inhalation Reference Concentrations and Application of 
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1 Inhalation Dosimetry (U.S. EPA, 1994b), Use of the Benchmark Dose Approach in Health Risk 

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

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

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

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

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

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

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

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

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

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

12 The literature search strategy employed for this compound was based on the Chemical 

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

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

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

16 2011. 

17 
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1 2. CHEMICAL AND PHYSICAL INFORMATION 

2 

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

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

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

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

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

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

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

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

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

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

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

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

15 

Table 2-1. Physical and chemical properties of n-butanol 

Chemical name n-Butanol 

Synonym(s) n-Butyl alcohol; butyl alcohol; 1-butyl alcohol; 1-butanol; 1-

hydroxybutane; butyl hydroxide; butyric alcohol; propyl carbinol; 

hemostyp; methylolpropane; propylmethanol; n-butan-1-ol 

ChemID Plus, 2009 

Structure ChemID Plus, 2009 

Chemical formula C4H10O 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/cm3 at 20°C Lide, 2008 

Log octanol-water partition 

coefficient (log Kow) 

0.88 Hansch et al., 1995 

Solubility: 

Water 

Organic solvents 

6.32 × 104 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-m3/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/m3

mg/m3

 to ppm 

1 ppm = 3.08 mg/m3

1 mg/m3

 = 0.32 ppm 

Calculated 

Calculated 
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1 

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

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

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

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

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

7 broken down by reaction with photochemically-produced hydroxyl radicals with a calculated 

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

9 
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1 3. TOXICOKINETICS 

2 

3 

4 Toxicokinetic studies of n-butanol in humans and experimental animals are described 

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

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

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

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

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

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

11 dehydrogenase (ADH) and further to n-butyric acid by aldehyde dehydrogenase (ECETOC, 

12 2003). n-Butanol is also oxidized by cytochrome P450 in rat liver (Albano et al., 1991). 

13 Excretion of n-butanol is primarily as CO2 in exhaled breath with minor amounts eliminated in

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

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

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

17 rats and humans. 

18 

19 3.1. ABSORPTION 

20 3.1.1. Oral Absorption 

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

[

14 22 C]-n-butanol in corn oil to male CD (Sprague-Dawley) rats (2–4/group) that had fasted for 16 

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

83.3% of the amount administered was recovered as expired 14 24 CO2 by 24 hours post exposure. A 

25 smaller fraction, approximately 0.20–0.56% of the administered radioactivity, was recovered as 

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

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

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

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

30 DiVincenzo and Hamilton (1979a) also collected blood samples from three male 

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

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

33 

34 3.1.2. Respiratory Tract Absorption 

Astrand et al. (1976) exposed 12 human volunteers (all male) to 300 or 600 mg/m3 35 

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

37 concentration of n-butanol in inspiratory air was continuously measured. Expiratory air was 

38 collected and was analyzed for n-butanol. The amount of n-butanol absorbed was calculated as 
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1 the difference between the total quantities in inspiratory and expiratory air. Arterial and venous 

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

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

4 150 watts) and was independent of exposure concentration. Measured arterial blood 

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

6 DiVincenzo and Hamilton (1979a) exposed four male beagle dogs to 50 ppm 

(150 mg/m37 ) n-butanol vapor for 6 hours in a whole-body chamber. Venous blood was collected 

8 at regular intervals. Based on breath concentrations, the uptake curve indicates that 

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

concentrations of 22 ppm (67 mg/m310 ) n-butanol were essentially unchanged during the 6 hour 

11 exposure. 

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

ppm (300 mg/m313 ) n-butanol for up to 7 hours. Blood samples were obtained from separate 

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

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

reaching near steady-state concentrations of approximately 2.2 μmol/dm316 (20 mg/kg) within 1 

17 hour of exposure. Mean blood n-butanol levels changed little throughout the remaining exposure 

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

concentration of 2.8 μmol/dm3 19 (26 mg/kg). 

20 

21 3.1.3. Dermal Absorption 

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

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

24 Skin was exposed to neat n-butanol under either unventilated or ventilated conditions with 

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

26 studied for either the unventilated or ventilated exposures. In the unventilated experiment, the 

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

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

29 absorption. 

30 Scheuplein and Blank (1973) conducted ex vivo studies using human abdominal skin 

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

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

was exposed to neat n-butanol or n-butanol in an aqueous solution (2.54 cm233 exposure area), 

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

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

dermis were 2.5 × 103 and 30.0 × 10336 cm/hour, respectively. For neat n-butanol, the permeability 

rates were 0.06 × 103 and 1.0 × 10337 cm/hour for the epidermis and dermis, respectively. 
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1 Using skin preparations from nude mice and glass diffusion cells, Behl et al. (1984) 

measured permeability coefficients of 3.7 × 103 cm/hour for dorsal skin and 23.7 × 1032 cm/hour 

3 for abdominal skin. Under hydrated conditions using skin preparations from male Sprague4 Dawley rats, Behl et al. (1983) determined the n-butanol permeability coefficient to be increased 

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

(6.4 × 1036 cm/hour) through the end of the experiment at 80 hours. 

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

skin. Rat skin was sandwiched between 3 mL saline filled compartments. [14 8 C]-n-Butanol was 

9 introduced into the donor side of the cell, and samples were removed from both sides of the cell 

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

measurements, a permeability rate of 14.2 × 10311 cm/hour was calculated. 

DiVincenzo and Hamilton (1979a) exposed young male beagle dogs to [14 12 C]-n-butanol 

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

14 collected continuously using an oral endotracheal tube and urine was collected continuously by a 

urethral catheter. An identical amount of [14 15 C]-n-butanol was administered intravenously to 

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

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

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

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

-2 19 . 

20 Boman et al. (1995) conducted studies to evaluate the percutaneous absorption of 

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

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

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

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

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

26 

27 3.2. DISTRIBUTION 

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

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

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

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

gavaged with a single dose of 450 mg/kg [14 32 C]-n-butanol in corn oil after fasting for 16 hours. 

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

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

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

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

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

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

14 1 C]-radioactivity in rat tissues occurred from 4 to 8 hours post exposure. The highest 

percentages of administered [14 2 C]-radioactivity (constituting parent compound and metabolites) 

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

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

Twenty-four hours after gavage doses of 4.5, 45, or 450 mg [14 5 C]-n-butanol/kg, approximately 

6 12.1–16.3% of the administered radioactivity remained in the carcass. Because radioactivity was 

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

8 n-butanol or specific metabolites. 

9 

10 3.3. METABOLISM 

11 n-Butanol is primarily metabolized to butyric aldehyde by alcohol dehydrogenase (ADH) 

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

13 dehydrogenase (ECETOC, 2003). Further oxidation of n-butyric acid produces CO2. A minor 

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

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

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

17 

18 

19 Figure 3-1 Metabolism of n-Butanol. 

20 
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1 Deisinger and English (2001) administered i.v. doses of 0.28 mmol/kg (21 mg/kg) of 

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

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

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

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

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

7 within 1 minute of each other. 

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

9 exposed male Sprague-Dawley rats to n-butanol in closed chambers having initial concentrations 

of approximately 2,000 ppm (6,000 mg/m310 ). Blood was collected throughout the exposure and

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

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

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

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

In gavage studies in CD (Sprague-Dawley) rats dosed with 450 mg [14 15 C]-n-butanol/kg 

16 body weight, 83.3% of the administered radioactivity was metabolized via oxidative pathways, 

17 appearing in exhaled breath as carbon dioxide (CO2) by 24 hours post exposure (DiVincenzo and 

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

19 administered dose being excreted in the urine. Of this fraction, about 75% was in the form of n20 butyl-o-sulfate (44%) or n-butyl-o-glucuronide (30%). Less than 1% of was not metabolized and 

21 was eliminated in the exhaled breath as unchanged compound. The study authors reported 

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

23 did not provide any additional quantitative details. 

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

25 and an apparent Michaelis-Menten Km (affinity constant) and Vmax (maximum reaction velocity) 

were reported to be 0.86 × 10-3 26 M and 0.077 mmol/minute, respectively (Auty and Branch, 

27 1976). When simultaneous exposures of n-butanol and ethanol were given, the Vmax for 

n-butanol remain unchanged, but the Km value increased to 1.2 × 10-3 28 M, indicating that ethanol 

29 competitively inhibits n-butanol metabolism. 

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

31 Teschke et al. (1974) and Cederbaum et al. (1979, 1978). Teschke et al. (1974) showed that 

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

33 molecular oxygen and nicotinamide adenine dinucleotide phosphate (NADPH). This reaction 

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

35 (H2O2) added to microsomal preparations stimulated the oxidation of n-butanol. Indirect 

36 evidence was further provided by the observation that azide, which prevents the decomposition 

37 of H2O2 by catalase, stimulated the oxidation of n-butanol. Thiourea, a compound that reacts 

38 with hydroxyl radicals, inhibited NADPH-dependent microsomal oxidation of n-butanol to a 
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1 similar extent, in both the absence and presence of the catalase inhibitor azide (Cederbaum et al. 

2 1979). 

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

4 pulmonary cytosolic preparations measured with regard to ADH activity as influenced by pH and 

5 substrate concentration. The values are given in Table 3-1. At pH 10, the hepatic Km value was 

6 about fourfold lower than at pH 7.2. Vmax and Km values could not be calculated for ADH 

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

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

9 systemic metabolism. 

10 

Table 3-1. n-Butanol metabolism by hepatic or pulmonary ADH in male 

rats 

pH Vmax (nmol/min/mg protein) Km (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 

12 Winer (1958) measured the initial reaction velocities of 19 alcohols as substrates for 

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

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

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

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

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

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

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

20 radical was strictly dependent of the presence of NADPH and oxygen and not influenced by the 

21 addition of mannitol. In reconstituted membrane vesicles, ethanol-inducible cytochrome P450 

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

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

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

25 suggests a greater affinity of n-butanol for cytochrome P450 2E1 compared to cytochrome P450 
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1 2B1 (Gadberry and Carlson, 1994). This inducible metabolism, however, was not observed in 

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

3 of minor consequence. Although cytochrome P450-mediated n-butanol oxidation has been 

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

5 disposition of n-butanol. 

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

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

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

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

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

11 oxygen consumption, perfusion flow, and adenosine triphosphate (ATP) concentration, both in 

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

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

14 Studies Evaluating Liver and Metabolic Effects). 

15 

16 3.4. ELIMINATION 

17 Deisinger and English (2001) administered i.v. doses of 0.28 mmol/kg (21 mg/kg) of n18 butyl acetate, n-butanol, or 0.028 mmol/kg (2.1 mg/kg) n-butyric acid to groups of adult male 

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

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

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

22 approached background levels by 18 minutes. 

Astrand et al. (1976) exposed 12 volunteers (all male) to 300 or 600 mg/m3 23 (100 or 

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

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

DiVincenzo and Hamilton (1979a) administered [14 26 C]-n-butanol in corn oil by gavage to 

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

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

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

30 Within 24 hours post exposure, excretion of the administered radioactivity was primarily as CO2

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

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

33 compound. 

34 

35 3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC MODELS 

36 A physiologically based pharmacokinetic (PBPK) model for n-butyl acetate and its butyl 

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

38 Teeguarden et al. (2005). The model simulates the kinetics of n-butyl acetate, n-butanol, n-
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1 butyraldehyde, and n-butyric acid in the rat or human following i.v or inhalation exposures to n2 butyl acetate or n-butanol. The PBPK model refined by Teeguarden et al. (2005) was updated 

3 from ACSL to ACSLXTREME (Advanced Continuous Simulation Language, Aegis, Inc., 

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

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

6 

7 

8 

9 Source: Teeguarden et al. (2005). 

10 

11 Figure 3-2. Schematic of the PBPK model for the butyl series (including 

12 n-butanol) in rats and humans.
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1 

2 The model is comprised of four similar submodels for each component of the butyl 

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

4 compartments for liver, blood, or extra-hepatic tissues to the respective compartments of the 

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

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

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

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

9 Each submodel includes compartments for lung, arterial and venous blood, liver, and the 

10 remaining perfused tissues. The submodel for n-butyl acetate also includes a compartment for 

11 fat. All compartments are well mixed and flow limited, which assumes that compounds are 

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

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

14 concentration at the blood:tissue interface are limited by the blood flow rather than rate of 

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

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

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

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

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

20 (discussed below) and multiplied by the inhaled concentration. Metabolism of each of the butyl 

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

22 tissues compartment, is described as Michaelis-Menten saturable processes defined by a 

maximum reaction rate (Vmax), scaled to body weight to the 3⁄4 power (mmol/hour/kg0.75 23 ), and an 

24 affinity constant (Km, mM). First-order metabolism of n-butanol in the other perfused tissues is 

scaled to body weight to the 1⁄4 power (Kother, kg0.25 25 /hour) and was required to attain fit of the 

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

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

(kg0.25 28 /hour). 

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

30 relative blood perfusion of various tissue compartments, and tissue volumes (as fraction of body 

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

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

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

species (Dietz et al., 1981). Thus, a value of 30 hour-1 34 , scaled to 1⁄4 power of body weight, was 

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

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

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

38 values. Rat values for the inhalation bioavailability factors were fitted to in vivo time-course 
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1 data for the blood levels of n-butyl acetate (Poet et al., 2003a) and n-butanol (Poet et al., 2003b). 

2 The human value for n-butyl acetate was assumed to be equal to the rat, while the value for n3 butanol (59% of alveolar ventilation) was reported by Astrand et al. (1976). 

4 

Table 3-2. Physiological parameters for a PBPK model for the n-butyl series 

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

Parameter Abbreviation 

Value 

Rat Human Reference 

Body weight (kg) BW 0.3 70.0 Study specific 

Alveolar ventilation 

(L/hr/kg0.75) 

QPC 14.0 8.678 Brown et al., 1997 

Cardiac output (L/hr/kg0.75) 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 Rowland, 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 Rowland, 1991 (as 

cited in Teeguarden et al., 2005 

Other tissues VOthC 0.751 0.5319 Brown et al., 1997

Urinary elimination rate (kg0.25/hr) 

n-Butyl acetate kFiltC1 0.074 Scaleda Estimated 

n-Butanol kFiltC2 22.2 Scaled Estimated based on Dietz et al., 1981 

n-Butyric acid kFiltC4 0.0074 Scaled Estimated based on DiVincenzo and 

Hamilton, 1979b 

Inhalation absorption (percentage of inhaled concentration) 

n-Butyl acetate FA1 1.0 1.0 Rat, fitted; human = rat 

n-Butanol FA2 0.5 0.59 Rat, fitted; human = rat (Astrand et 

al., 1976) 

n-Butyraldehyde FA3 – – – 

n-Butyric acid FA4 – – – 

aAllometrically-scaled rat value used for human. 

Source: Teeguarden et al. (2005). 

5 
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1 Metabolic parameter values for the model are given in Table 3-3. Metabolic constants (Vmax and 

2 Kother) were estimated by visually fitting the model output to i.v. blood time-course data for each 

3 compound (Deisinger and English, 2001). Fitting of Vmax for n-butyl acetate and its metabolites was 

4 preceded by first estimating Vmax for the furthest downstream metabolite (n-butyric acid), and proceeding 

5 upstream to estimates Vmax values for n-butyraldehyde, n-butanol, and n-butyl acetate, in that order. n6 Butyraldehyde is not detected in blood and thus, metabolic constants for this compound were 

7 approximated by n-butyric acid concentration in blood following n-butanol or n-butyl acetate exposure. 

8 The affinity constants (Km) for n-butanol, n-butyraldehyde, and n-butyric acid were previously selected 

9 by Barton et al. (2000). The in vitro affinity constant for metabolism of ethyl acetate in rats was used as a 

10 surrogate for n-butyl acetate in blood, liver, and other tissues. Metabolic constants for n-butanol 

11 metabolism were re-optimized to data and updated values are given in Table 3-3. 

12 

Table 3-3. Metabolic parameters for a PBPK model for the n-butyl series in 

the rat and humana 

Parameter Abbreviation Value Notes 

n-Butyl acetate 

Maximum metabolic rate in blood VMBloodC1 600.0 Fitted 

Affinity constant in blood KMBlood1 100.0 Km for ethyl acetate in rat 

Maximum metabolic rate in liver VMLivC1 38.7 Fitted

Affinity constant in liver KMLiv1 1.0 Km for ethyl acetate in rat 

Maximum metabolic rate in other tissues VMOthC1 6,000.0 Fitted 

Affinity constant in other tissues KMOth1 100.0 Km for ethyl acetate in rat 

n-Butanolb

Maximum metabolic rate in liver VMLivC2 2.17/0.62 Fitted (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 Fitted 

Affinity constant in liver KMLiv3 0.1 Estimated (Barton et al., 2000) 

n-Butyric acid 

Maximum metabolic rate in liver VMLivC4 1.4 Fitted 

Affinity constant in liver KMLiv4 0.1 Estimated (Barton et al., 2000) 

Maximum metabolic rate in other tissues VMOthC4 3.0 Fitted 

Affinity constant in other tissues KMOth4 0.1 Estimated (Barton et al., 2000) 

aRat values were derived as described; human values were allometrically scaled from rat values as 3⁄4 or 1⁄4 power of 

body weight. Units: Vmax (mmol/hr/kg0.75); Km (mM); Kother (kg0.25/hr). 

bValues for n-Butanol re-optimized. Details are presented in Appendix C. 

Source: Teeguarden et al. (2005). 

13 

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

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

16 n-butyric acid were assumed to be identical to those for n-butanol. The values for lung and other 
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1 perfused tissues:blood were based on the Kaneko et al. (1994) muscle:blood value. Human 

2 values were assumed to be equivalent to rat values.

3 

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

n-butyl series in the rat and humana

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 

aCalculated from Kaneko et al. (1994). 

Source: Teeguarden et al. (2005). 

4 

5 The model was evaluated against experimental data for i.v. exposures in the rat 

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

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

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

9 high confidence in the estimation/choice of metabolic and urinary elimination parameter values. 

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

11 observations and were always less than twofold higher. The profile of the simulated elimination 

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

13 fit and parameter optimization can be found in Appendix C. 

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

15 inhalation exposures. The internal dose metric available for extrapolation between species is 

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

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

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

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

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

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

22 due primarily to n-butanol metabolic differences, resulting in differences in systemic levels of 

23 n-butanol or metabolites, the model is useful to quantitatively reduce uncertainty in the 

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

25 
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1 4. HAZARD IDENTIFICATION 

2 

3 

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

5 CONTROLS 

6 

7 4.1.1. Oral Exposure 

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

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

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

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

12 muscles, absent myotatic reflexes, and aromatic odor. Echocardiogram and chest x-ray results 

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

14 distorted and narrower fourth ventricle and a hypodense area; an additional scan performed 10 

15 hours later showed no changes. The patient regained consciousness approximately 16 hours after 

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

17 erythrocytes and leukocytes, elevated creatinine, myoglobin, and creatine kinase; these changes 

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

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

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

21 information was available. 

22 

23 4.1.2. Inhalation Exposure

24 4.1.2.1. Occupational Studies 

25 Four studies reported health findings in workers occupationally exposed to n-butanol.. 

26 Details of these studies including study design, exposure, and notable effects are presented in 

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

28 irritation, hearing loss, dermatitis, and other systemic effects. All of these studies have 

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

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

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

32 (1949). 

33 

34 

35 

36 

37 
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 Table 4-1. Occupational Studies with n-butanol exposure

Reference(s) No 

Subjects

Study Design Exposure Findings 

Tabershaw et al., 1944

Not reported

 Eye irritation, dermatitis, and systemic effects were evaluated in workers from six industrial facilities involved in the manufacture of raincoats and other waterproof materials, in which n-butanol was the primary solvent used

5-14 ppm (15-42 mg/m3) and 

20-65 ppm (61-197 mg/m3)

Eye irritation reported at a concentration ranging from 265 ppm (61-197 mg/m3) in 1 

of 6 facilities with n-butanol o

Velazquez et al., 1969

11 butanol + noise (72-78 db),47 noise only (90-110 db)

Audiologic effects of n-butanol exposed workers were compared with workers from another factory exposed to higher noise levels but not n-butanol or other solvents associated with hearing loss. 

80 ppm (242 mg/m3) as 

measured by gas chromatography (GC) in the work room at a cellulose acetate ribbon factory

Hearing loss was observed in 9/11 workers exposed to n-butanol and in 23/47 workewithout exposure to n-butanbut with exposure to industrinoise.

Cogan and Grant, 1945

75 female workers

Eye examinations were performed on symptomatic workers employed at a facility reported in the Tabershaw study 

15 -100 ppm (46-303 mg/m3)

ethanol and diacetone alcohol (concentrations unknown)

28/35 workers exposed to nbutanol exhibited evidence ocoreal inflammation and no unexposed workers showed sof inflammation

Sterner et al.,

1949

16 male workers

Physical examinations of workers exposed to n-butanol during coating of photographic paper over a 10-year period were conducted.

Initial concentrations of nbutanol averaged 200 ppm (606 mg/m3), but decreased to 

100 ppm (303 mg/m3) over the 

course of the study.

Workers exposed to n-butanoconcentrations averaging ≥ 200ppm (606 mg/m3) were 

reported to experience corneedema and mild edema of thconjuctiva
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1 A number of other occupational studies examined health endpoints in worker populations 

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

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

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

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

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

7 any quantitative estimates of exposure to individual compounds. In the three studies that 

8 quantified exposure to individual compounds and included health effect information (Tucek et 

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

10 compared with exposure to other solvents such as xylenes, ethylbenzene, toluene, and/or 

11 acrylates. 
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Table 4-2. Studies of human exposure to mixtures including n-butanol 

Reference(s) No. subjects Study type Exposure Findings 

Angerer and Wulf, 1985 

31 exposed and 31 controls 

Study compared hematology findings in varnish workers from six different workplaces (within two plants) with those of matched controls. Exposure to n-butanol was reported at only one workplace, with eight workers. 

Air contaminants at the only workplace with butanol exposure (1.2 ± 0.8 mL/m3) included o-, m-, and 

p-xylenes (averaging 3.4, 11.7, and 4.3 mL/m3 respectively) and ethylbenzene (7.5 mL/m3). 

Hematology results pooled across the six locations suggested statistically significant reductions in erythrocyte count and hemoglobin content, increased lymphocyte count, and reduced (not statistically significantly) segmented granulocyte count. 

Bleecker et al., 1991 

187 exposed Cross-sectional 

study compared neurobehavioral findings in paint manufacturing workers across quartiles of total hydrocarbon exposure. 

Exposures included toluene, xylene, aliphatic and aromatic hydrocarbons, methyl ethyl ketone, other hydrocarbons, alcohols (including n-butanol), ketones, and esters. 

Statistically significant correlation (by linear regression analysis) between increasing exposure to mixed organic solvents and impairment of performance on several neuropsychological test outcomes, as well as vibration threshold. 

Murata et al., 1991 

11 exposed and 11 controls 

Study compared neurological findings in workers with solvent exposure with agematched controls. 

Only one of the workers reported exposure to n-butanol; this individual also reported exposure to xylene, methanol, and toluene 

Coefficient of variation in electrocardiographic RR-intervals was statistically significantly reduced, and the distribution of nerve conduction velocities were slowed in exposed workers compared with controls. 
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Table 4-2. Studies of human exposure to mixtures including n-butanol 

Reference(s) No. subjects Study type Exposure Findings 

Triebig et al., 1992 

83 exposed and 42 controls 

Study compared subjective symptoms of neurotoxic endpoints in spray painters with controls. 

Spray painters were from 10 different work sites; air concentrations of several solvents were reported for each work site. Four sites had n-butanol measurements (5.7, 1.5, 5.4, and >300 mg/m3); the number of spray 

painters at each site was not reported. At the sites with n-butanol exposure, there was co-exposure to other solvents including toluene, xylene, ethylbenzene, styrene, acetone, butylacetate, methyl isobutylketone, ethyl acetate, n-heptane, and/or decane. 

No difference in the prevalence of subjective symptoms of neurotoxicity between painters and controls. 

Akhmadeyeva, 1993 

347 exposed and 1,526 controls 

Study compared health, physical development, and immunologic and psychochemical indices in blood of newborns of petrochemical workers with those of control infants. 

Workplace air contaminants reportedly included aromatic hydrocarbons, isopropylbenzene, ethanol, and butanol. 

Newborns of workers had higher frequency of delayed intrauterine development (24.2 vs. 13.7% in controls; p < 0.01). Other 

differences between study and control newborns included hematological and immunological effects. 
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Table 4-2. Studies of human exposure to mixtures including n-butanol 

Reference(s) No. subjects Study type Exposure Findings 

Chen et al., 1999 

309 exposed (mortality study) 260 exposed and 539 controls (neuropsychology study) 

Mortality study and cross-sectional study of selfreported neuropsychological symptoms in dockyard painters and unexposed controls. Mortality analysis included 309 painters comprising 3,690 person-yrs at risk. Crosssectional study of neuropsychological symptoms included 260 surviving painters and 539 unexposed controls. 

According to the authors, primary solvents used at dockyard were white spirit, xylene, trimethylbenzene, n-butanol, trichloroethylene, naphtha, and cumene; exposures to other compounds were also reported to be likely. 

Neither proportional mortality nor standardized mortality ratios for cancers or diseases of the CNS, circulatory system, or respiratory system were increased in painters. Cross-sectional study suggested increased prevalence of neuropsychological symptoms in painters compared with controls; relative risk for these symptoms increased with yrs of exposure. 

Jang et al., 1999 

674 exposed and 191 controls 

Study compared neuropsychological effects (measured via questionnaire and function tests) in shipyard workers exposed to solvents and unexposed controls. 

Primary solvents were xylene, ethylbenzene, trimethylbenzene, toluene, 2-ethoxyethanol, and 2-ethoxyethyl acetate (constituted 85% of total exposure “intensity”). n-Butanol reported as one of several other solvents detected. 

Rate of abnormal neuropsychological diagnosis (primarily executive dysfunction, attention dysfunction, memory disorder, or functional disorder) was >4 times higher in the exposed workers compared with controls (9.3 vs. 2.1% in controls, p < 0.01). 

Rate of abnormal diagnosis exhibited dose-response relationship with cumulative exposure. 
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Table 4-2. Studies of human exposure to mixtures including n-butanol 

Reference(s) No. subjects Study type Exposure Findings 

Tucek et al., 2002 

60 exposed and 60 controls 

Prospective cohort study of workers at a chemical facility producing acrylic acid. Study examined health effects including hematology, serum chemistry, serum immunity and tumor markers, spirometry, and cytogenetic analysis of peripheral lymphocytes. 

Exposures included acrylonitrile, n-butanol, butyl acrylate, ethyl acrylate, methyl acrylate, methyl methacrylate, toluene, and styrene. Measurements of the various solvents (measured using personal air sampling) were binned by concentration and reported using stacked bar charts; these charts suggested that >70% of n-butanol concentrations were <0.02 mg/m3. 

Exposures to the other compounds were of a similar magnitude and generally lower than occupational health limits in the Czech Republic; butyl acrylate was most frequently (at 2.1% of measurements) measured at a level exceeding its limit. 

Statistically significantly higher percentage of chromosomal aberrations in peripheral lymphocytes. 
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1 4.1.2.2. Controlled Acute Exposure Experiments 

2 There several controlled acute exposure studies that evaluated irritation and conditioned reflexes 

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

0.1 mg/m3 (Baikov and Khachaturyan, 1973; duration unknown) to 12,000 mg/m34 for a 2 minute 

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

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

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

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

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

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

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

12 

13 
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Table 4-3. Controlled Acute Exposure Studies 

Reference Subjects Study Design and Exposure Results Strengths Nelson et al., 1943 

10 volunteers 

Comparison of eye, throat and nasal irritation in subjects and controls. Concentrations ranging from 0-50 ppm (0 – 150 mg/m3; groups not 

specified by authors) of n-butanol were administered for 3-5 minutes in an inhalation exposure chamber 

Eye irritation at 50 (150 mg/m3) Nose and 

throat irritation at 25 ppm (76 mg/m3) 

Highest concentration acceptable for 8 hours as reported by the volunteers was <25 ppm (<76 mg/m3 ) 

Limited inconcentrat

Baikov and Khachaturyan, 1973 

18 volunteers 

in groups of 3 

Comparison of the odor threshold and effects of n-butanol (0.1-2.5 mg/m3) on conditioned 

reflexes in subjects 

Odor threshold determined to be 1.2 mg/m3

Dark adaption slowed at 1.2 mg/m3

Statistically significant increase in response latency at 0.7 mg/m3

Statistically significant inhibition of the conditioned blinking reflex at 1.5 and 2.5 mg/m3

Transient development of conditioned reflex described by shifts in electrocephalograms. (effective concentration not reported) 

Lack of stStudy idenassociated 

ComettoMuniz and Cain et al., 1995 

5 males and 5 females 

n-Butanol vapors (100, 33, 11, 3.7, etc. % v/v) were administered to eye or nostrils for 1-3 seconds, using squeezable bottles. Eye irritation and odor threshold responses were evaluated. 

Odor threshold was reported as 20 ppm (61 mg/m3) 

Eye irritation threshold was reported as 3000 ppm (9000 mg/m3) 

Limited stthe small n

Wysocki and Dalton, 1996 

32 exposed workers 32 unexposed residents 

Odor and irritation thresholds for n-butanol (99.8% pure; diluted to 26 concentrations ranging from 100% to 1.1802 x 10-10% v/v) in 

acetone –exposed workers and naïve volunteers were evaluated. 

Median odor detection and nasal irritation thresholds were 0.17 ppm (0.52 mg/m3) 

and 2,042 ppm (6,190 mg/m3), 

respectively. Acetone workers considered n-butanol to be nonirritating, while naïve subjects considered the exposure to have irritating properties 

Included a one other 

Kjaerguard et al., 1997 

6 men and 

6 women 

Eye irritant effects of n-butanol after local (eye only) exposure and whole-body exposure in volunteers were evaluated. Doses of n-butanol used in the study were 1500-12000 mg/m3 for 2 

Statistically significant association (by repeated-data multiple analysis of variance) between exposure concentration, general irritation, eye irritation, throat 

Only irritaevaluated; effects we
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Table 4-3. Controlled Acute Exposure Studies 

Reference Subjects Study Design and Exposure Results Strengths 

minute intervals (lowest to highest) in goggle experiments and 0, 2.5,5, and 10 mg/m3 in 

chamber experiments. 

irritation, odor irritation, nose irritation, air quality, and continuously-measured irritation by potentiometer Analysis suggests an interaction between individual threshold for eye irritation in the goggle experiment and several other measures of irritation (general irritation, eye irritation, and throat irritation. 

HempelJorgensen et al., 1998; 1999 

8 subjects Evaluated eye irritation associated with eye-only 

exposure to n-butanol n-butanol at (0, 99, 314, or 990 ppm (0, 300, 952, or 3,000 mg/m3for 

periods of 15 or 60 minutes) and the results of conjunctival hyperemia evaluations using photographs of eyes before and after exposure, and conjunctival fluid cytology measured by optical microscopy. One eye was exposed to n-butanol, and the other to CO2

The average irritation intensity was estimated for each second of the 60-minute exposure and plotted to show the time course of irritation Eye irritation intensity did not differ among the various concentrations of n-butanol and exposure to clean air The mean conjunctival hyperemia score in subjects exposed to 990 ppm (3,000 mg/m3) n-butanol was statistically significantly higher (1.40 ± 0.76 on a scale from -2 to +2) than the score after exposure to clean air (0.48 ± 0.96); Conjunctival cytology was not affected by exposure to n-butanol at any concentration. 

n-Butanol study were result in o

Podlekareva et al., 2002 

9 subjects Compared physiological evidence of irritation 

after eye-only exposure to either n-butanol, CO2, 

or clean air. n-butanol and CO2 exposures were 

designed to stimulate the same perceived level of irritation (50% of maximal on a linear scale). 

Eye exposure to n-butanol did not result in a significant change in tear film stability or conjunctival corrosion measures 

Limited stthe small n
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1 4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN 

2 ANIMALS—ORAL AND INHALATION 

3 4.2.1. Oral 

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

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

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

7 clinical signs, and weekly measurements of body weight and food consumption were made. 

8 Ophthalmology (specific tests not reported) was evaluated before exposure and during the final 

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

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

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

12 hemoglobin, mean cell hemoglobin concentration, total and differential leukocyte count, platelet 

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

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

15 bilirubin, electrolytes, inorganic phosphate, CO2, total cholesterol, and creatinine), and urinalysis 

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

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

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

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

20 Comprehensive histopathologic examination (> 30 tissues) was performed on control and high21 dose animals; in addition, the liver, kidneys, heart, and gross lesions were examined in the low22 and mid-dose groups. The available copy of the unpublished report was lacking data tables and 

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

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

25 based on the text of the report. 

26 No n-butanol-treatment related death occurred in these rats. Three animals (one mid27 dose and two high-dose animals) died prior to sacrifice due to gavage errors (RTI; unpublished, 

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

29 ophthalmoscopic findings. 

30 Clinical signs of toxicity, specifically hypoactivity and ataxia, were observed in high31 dose animals but not in animals at lower doses. These signs were first observed during week 8, 

32 infrequently through week 10, and reached maximum incidence (29% for hypoactivity and 32% 

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

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

35 At the interim sacrifice, hematology analysis showed reduced hematocrit, erythrocyte 

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

37 hematological changes were evident in males at interim sacrifice or in either sex at terminal 

38 sacrifice. There were higher absolute neutrophil count in mid-dose males at interim evaluation 
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1 and higher relative segmented neutrophil count and lower relative lymphocyte count in low-dose 

2 females at final evaluation. The study authors also reported the following statistically significant 

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

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

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

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

7 thyroid weight compared with controls; no other statistically significant organ weight changes 

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

9 pathology findings; however, the lack of data tables precluded independent review of the data. 

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

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

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

13 hematocrit, erythrocyte count, and hemoglobin content in females. The NOAEL is 125 mg/kg14 day. 

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

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

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

estimated dose of 8,200 mg/kg-day118 based on default estimates of body weight (0.217 kg) and 

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

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

21 occurred after 1 week of exposure. Toxicological evaluation was limited to electron microscopy 

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

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

24 there were no ultrastructural changes to hepatic mitochondria after n-butanol exposure up to 1 

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

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

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

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

29 numbers of lysosomes and microbodies. The study authors proposed two possible mechanisms 

30 for the effects on mitochondria: fusion of adjacent mitochondria and suppression of the process 

31 of mitochondrial division via perturbation of protein synthesis. 

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

33 (single high concentration used, only ultrastructural changes in the liver were examined, and the 

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

35 to investigate whether mitochondrial effects observed with exposure to ethanol are also evident 

36 after exposure to other alcohols. 

 

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

8,200 mg/kg-day. 
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1 Munoz et al. (1991, 1990) exposed groups of three male Wistar rats (initial weight 200–

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

3 focused on examining effects on protein synthesis in the brain. The exposure concentration of 

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

5 2% during week 2 and 4% for the remainder of the treatment period until sacrifice after 

6 4 months. This exposure regimen corresponds to a time-weighted average concentration of 

3.7%, resulting in an estimated dose of 4,400 mg/kg-day27 based on default estimates of body 

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

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

10 body weight, selected serum chemistry parameters (urea, glucose, creatinine, triglycerides, total 

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

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

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

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

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

16 organs were examined grossly or microscopically. Serum chemistry findings were reported 

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

18 statistically significantly higher total protein (29% higher than controls) and albumin levels (28% 

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

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

21 authors did not identify effect levels, and the information provided was inadequate for the 

22 identification of effect levels. 

23 

24 4.2.2. Inhalation 

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

n-butanol (purity not specified) vapor concentrations of 0, 50, or 100 ppm (0, 154, or 308 mg/m326 ) 

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

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

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

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

31 kidneys, adrenals, and testes) were recorded. Rotarod performance tests, designed to assess 

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

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

34 a rate of 12 revolutions/minute and was placed at a height of 20 cm above the floor. Prior to n35 butanol exposure, rats were trained on the rotarod task for 10 days and received a shock from the 

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

 

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

4,400 mg/kg-day. 
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1 behavior (from the negative enforcement of the shock from the floor) in addition to 

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

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

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

5 prior to the termination of exposure and used for hematological evaluations (hematocrit, 

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

7 biochemistry studies (ALT, AST, sorbitol dehydrogenase [SDH], ALP, total protein, albumin, 

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

9 exposure. The livers were homogenized to analyze total microsomal cytochrome P450 (CYP) 

10 content, aniline p-hydroxylase activity, triglyceride content, and malondialdehyde (MDA) 

11 content. 

12 All rats survived and there were no clinical abnormalities observed in these rats (Korsak 

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

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

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

16 significant differences in absolute or relative organ weights. Table 4-4 shows changes in 

17 hematology and lipid peroxidation parameters. Compared to controls, the exposed groups 

exhibited decreased erythrocyte counts (5 and 16% lower in the 154 and 308 mg/m318 groups, 

respectively); the difference was statistically significant in the 308 mg/m3 19 group only. 

20 Statistically significantly decreased hemoglobin levels (10% lower than controls) were noted in 

21 both exposure groups, but hematocrit was not changed. There were increased leukocyte counts 

(25 and 57% higher in the 154 and 308 mg/m3 22 group, respectively); the difference was 

statistically significant in the 308 mg/m323 group, and was beyond the normal range of variability 

(16.5 × 103/nm3 in exposed rats, compared with a range of 1.96–8.25 × 103/nm324 ; Giknis and 

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

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

percentage of eosinophils in the 308 mg/m3 27 group; this increase was well beyond the normal 

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

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

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

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

than controls in the 154 and 308 mg/m3 32 groups, respectively). Hepatic total CYP content, 

33 microsomal aniline p-hydroxylase activity, and liver triglycerides were not affected by exposure 

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

35 failures, indicating impaired neuromuscular function and learned avoidance behavior, in the rats. 

36 The rotarod data were presented in Korsak et al. (1994) in a graphical format and the failure rates 

were approximately 2–15% in the 154 mg/m3 group and 15–33% in the 308 mg/m3 37 group when 

38 measured over the 3 month period and are presented in Table 4-5. The increased failure rate in 
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the 308 mg/m3 1 group was statistically significant during the 2nd and 3rd months of exposure; the 

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

3 sensitivity (assessed by hot-plate behavior) was not affected in the rats exposed to n-butanol at 

these concentrations. EPA identified aNOAEL and LOAEL of 154 and 308 mg/m34 , respectively, 

5 based on impaired neuromuscular function. Decreased hemoglobin and increased lipid 

6 peroxidation were both observed at the NOAEL; however, the changes at that concentration were 

7 not biologically significant. 

Table 4-4. Hematological effects in rats exposed to n-butanol by inhalation 

for 3 months 

Control 

154 mg/m3

(50 ppm) 

308 mg/m3

(100 ppm) 

Number of animals 24 12 12 

Hemoglobin (g/dL) 15.9 ± 0.4a 14.2 ± 0.8b 14.1 ± 0.7b

Erythrocytes (× 106/nm3) 9.97 ± 0.02 9.45 ± 0.05 8.35 ± 0.06b

Leukocytes (× 103/nm3) 10.5 ± 0.13 13.1 ± 0.26 16.5 ± 0.27b

Eosinophils (%) 7.8 ± 2.3 11.5 ± 4.6 13.8 ± 4.6c

MDA (nmol/mg microsomal protein equivalent to g liver) 40.41 ± 2.10 46.68 ± 2.16c52.45 ± 2.88c

aMean ± 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 over 3 months 

Exposure Group Number of animals

Percentage of Rotarod Failuresa

Month 1 Month 2 Month 3 

Control 24 0 0 0 

154 mg/m3(50 ppm) 12 2 10 15 

308 mg/m3(100 ppm) 12 15 25b 33b

a Percentage of rotarod failures were derived from data approximations. 

b

Significantly different from control at p < 0.05. 

Source: Korsak et al. (1994). 

9 

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

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

concentration of 100 ppm (300 mg/m312 ) every day for 2 weeks (hours/day were not specified) and 

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

14 were not exposed concurrently. There were two control groups: one sham-treated and one 

15 untreated, three animals per group (sex unspecified). The guinea pigs were weighed weekly. 
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1 Blood counts and urinalysis (parameters not reported) were assessed prior to exposure and at 

2 biweekly intervals thereafter. The schedule for and tests performed at sacrifice were not 

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

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

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

6 albuminuria. The second group tested at this concentration developed severe skin infections 

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

8 exhibited decreased erythrocytes and hemoglobin along with increased total leukocytes (data not 

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

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

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

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

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

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

15 marked degeneration of tubules). Data on weight, hematology, and specific gravity of urine 

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

17 two control groups, one animal each died of skin infection. 

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

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

0, 0.8, 6.6, or 40 mg/m320 for 4 months (frequency not reported). This study did not provide other 

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

22 evaluations performed) or any quantitative information on the effects observed. The only effect 

23 information provided in the report was a list of effects at each exposure concentration; the 

24 affected species was not reported in most cases. Increased thyroid gland activity reportedly 

occurred in all experimental groups. At 6.6 and 40 mg/m325 , narcosis induced by hexanal 

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

27 pituitary-adrenal system was characterized as disrupted. Other changes were noted such as a 

28 loss of CNS summation capacity (generally an electrophysiological measurement of nerve 

29 response), a reduced eosinophilic response following administration of adrenocorticotropic 

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

blood cholinesterase activity was observed in rats at ≥ 6.6 mg/m331 . The authors characterized the 

6.6 mg/m3 concentration as a threshold value and could be considered a LOAEL and 0.8 mg/m332 

33 is the NOAEL for the study. Although the authors provide effect levels for the study, there is no 

34 information on the study design, no data for the toxicological effects are available, and statistical 

35 information is not provided for the various endpoints. As a result, EPA considers the 

36 information reported in the publication as inadequate for the purpose of determining effect 

37 levels. 

38 
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1 4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION 

2 4.3.1. Oral Studies 

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

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

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

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

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

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

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

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

11 mating and gestation periods. Weight gain, food consumption, and water intake were measured 

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

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

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

15 fetuses were recorded. All live fetuses were examined for external malformations; half were 

16 then prepared for skeletal examination and the remainder for visceral examination. Statistical 

17 analysis of malformations was performed using either the fetus or the litter as the statistical unit. 

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

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

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

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

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

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

24 The study authors reported that hemoglobin concentration and hematocrit, as well as absolute 

25 and relative organ weights were not different between exposed and control dams; however, 

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

27 number of corpora lutea or total implants, number of litters with resorptions, or numbers of pre28 or post implantation losses/litter. A slight increase in the number of early resorptions/litter was 

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

30 not statistically significant. 
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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 

gestationa

Parameter 

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

0 

300 

(0.24%) 

1,000 

(0.8%) 

5,000 

(4.0%) 

Fetal body weightb (g) 3.2 ± 0.2 3.2 ± 0.3 3.2 ± 0.2 3.2 ± 0.3 

Fetal crown-rump lengthb (cm) 4.0 ± 0.1 3.9 ± 0.1 3.9 ± 0.1 3.8 ± 0.1c

No. of litters examined for visceral variations 12 14 12 9 

Percentage of litters with dilation of 8 64c 83c 100c

Subarachnoid space (%) 0 14c 25c 78c

Lateral ventricle and/or third ventricle of the brain (%) 8 57c 67c 78c

Unilateral renal pelvis (%) 0 0 42c 0 

Bilateral renal pelvis (%) 0 0 25c 0 

Percentage of litters with congenital defects 0 0 33

c

 22c

External hydrocephalus (%) 0 0 17c 0 

Internal hydrocephalus (%) 0 0 25c 22c

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 7c 0 11c

14th rib (%) 0 0 0 11c

Wavy ribs (%) 0 7c 0 0 

a

 Fetal and litter data was provided by authors; only litter data is presented 

bMean ± SD. 

c

Significantly different from control at p < 0.05. 

Source: Sitarek et al. (1994). 

1 

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

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

4 significantly decreased in offspring of high-dose dams compared with controls (5%). Dose5 related increases in the incidences of litters with visceral malformations were observed; the 

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

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

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

9 incidence of litters with dilation of the subarachnoid space was also statistically significantly 

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

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

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

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

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

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

16 0% in controls). The authors reported that the percentage of fetuses with delayed ossification 
37 DRAFT - DO NOT CITE OR QUOTE

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

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

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

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

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

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

7 the brain and of the subarachnoid space) and a NOAEL was not identified. 

8 Ema et al. (2005) also investigated the developmental effects (brain malformations and 

9 variations) of n-butanol in Sprague-Dawley rats. Pregnant (sperm positive) Sprague-Dawley 

10 (Crj:CD(SD)IGS) rats (20/group) were exposed to n-butanol (99.9% pure) in drinking water at 

11 concentrations of 0, 0.2, 1.0, or 5.0% on GDs 0−20. The authors estimated daily doses of 0, 316 

12 ± 39, 1,454 ± 186, or 5,654 ± 1,402 mg/kg-day based on measured body weight and water 

13 consumption. Maternal evaluations included recording of clinical signs, body weights, and food 

14 and water intake. Dams were sacrificed on GD 20 for assessment of numbers of corpora lutea, 

15 implantation sites, resorptions, and live and dead fetuses; placental weight was also recorded. 

16 Live offspring were sexed, weighed, measured (crown-rump), and examined for external and 

17 oral malformations. Half of the fetuses were prepared for examination of internal malformations 

18 and the remainder for skeletal malformations. The litter was used as the statistical unit for 

19 analysis of the data. Treatment-related findings are summarized in Table 4-7. 

20 None of the animals in any group died. As shown in Table 4-7, maternal body weight 

21 gain in the group exposed to 5,654 mg/kg-day (5% n-butanol in water) was statistically 

22 significantly decreased during GDs 0–7 (45% of controls) and GDs 0–20 (10% less than 

23 controls). Food consumption was statistically significantly decreased compared to controls in 

24 mid- and high-dose dams (8 and 20% lower than controls, respectively) throughout pregnancy. 

25 During GDs 0–7, water consumption was statistically significantly decreased at the mid- and 

26 high-doses (9 and 38% lower than controls), and was decreased throughout gestation in the high27 dose dams (28% lower than controls over GDs 0–20). 

28 There were no statistically significant differences between exposed and control rats in 

29 placental weight or numbers of corpora lutea, implantations, pre- or post implantation losses, 

30 resorptions, or live or dead fetuses (Ema et al., 2005). The sex ratio of offspring was not 

31 different among the groups. Fetal body weight was statistically significantly reduced in both 

32 male (8% lower than controls) and female (10%) offspring of dams exposed to 5,654 mg/kg-day 

33 (5% n-butanol). Body weights were decreased in the mid- and low-dose fetuses; however, the 

34 difference from control was not statistically significant. The crown-rump length of offspring was 

35 not affected by treatment. 

36 
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Table 4-7. Selected changes in rats exposed to n-butanol 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 ± 28b

Maternal body weight gain on GDs 0–20 (g)a 162 ± 19 168 ± 16 165 ± 15 146 ± 16b

Maternal food consumption GDs 0–20 (g)a 548 ± 38 548 ± 46 503 ± 34b 441 ± 34b

Maternal water consumption GDs 0–20 (mL)a 930 ± 105 983 ± 126 890 ± 106 669 ± 182b

Male fetal body weight (g)a 4.18 ± 0.27 4.00 ± 0.24 4.04 ± 0.25 3.83 ± 0.18b

Female fetal body weight (g)a 3.97 ± 0.25 3.86 ± 0.20 3.83 ± 0.16 3.59 ± 0.17b

Skeletal examinationc 

Total no. of litters examined 20 20 20 20 

Total no. of litters with variations 11 12 17 20b

Short supernumerary ribs 10 9 16 19b

Degree of ossification 

No. of forepaw proximal phalanges 1.6 ± 1.3 1.6 ± 0.9 1.2 ± 1.1 0.3 ± 0.4b

Internal examinationc 

Total no. of litters examined 20 20 20 20 

Thymic remnant in neck 4 5 8 8 

aMean ± 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 

2 The incidences of external, oral, and visceral malformations were not increased by 

3 treatment with n-butanol (Ema et al., 2005). At the highest dose, a statistically significant 

4 increase (20/20 vs. 11/20 litters in controls) in the incidence of litters with skeletal variations 

5 (primarily short supernumerary ribs) was observed, as well as a decrease in the degree of 

6 ossification (number of forepaw proximal phalanges was 0.3 ± 0.4 vs. 1.6 ± 1.3 in controls). The 

7 study authors suggested that the skeletal effects may have been related to growth retardation in 

8 the high-dose group. Although there was no statistically significant increase in the incidence of 

9 litters with internal malformations, the incidence of litters with thymic remnant in the neck 

10 increased with dose (see Table 4-7) and the incidence at the high-dose was twice that of controls 

11 (8/20 litters vs. 4/20 litters in controls). 

12 The study authors did not identify a LOAEL for this study (Ema et al., 2005); however, 

13 they did indicate that the NOAEL for dams and fetuses was 1,454 mg/kg-day (1%). EPA has 

14 determined that the high-dose (5,654 mg/kg-day, or 5% in water) is a LOAEL for developmental 

15 effects (decreased fetal body weight and increased incidence of skeletal variations). The 

16 NOAEL for the fetal effects is 1,454 mg/kg-day (1%). 
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1 McLain (2008) summarized a teratogenicity study by Bariliak et al. (1991) of n-butanol 

2 and other alcohols administered by gavage. This study was published in Russian with a brief 

3 English summary and was not translated for this review.According to McLain (2008), groups 

4 10–16 white rats (weighing 160–180 g, strain not specified) were given various alcohols 

5 (methanol, ethanol, n-butanol, nonanol, and decanol; purity not given) by gavage (1 mL of 40% 

6 solution in water) from GDs 1 through 15. Controls (20 rats) were given water alone during the 

7 same gestational treatment period. Without information on the body weights of the pregnant 

8 rats, estimating a dose associated with the administered solution of n-butanol is uncertain. 

9 Assuming a body weight of 0.250 kg and chemical density of 810 mg/mL, a dose of 1,300 

mg/kg-day310 was estimated. At sacrifice on GD 20, the numbers of corpora lutea and live and 

11 dead fetuses were counted. The review did not discuss any maternal evaluations. Parameters 

12 reported in the secondary source include: fertility index (description not provided), number of 

13 implantations, percent pre- and post implantation losses, and number of live fetuses. In addition, 

14 ADH activity was measured in livers excised from selected fetuses (1–2/litter). According to 

15 McLain (2008), these measurements were performed daily on fetuses from GDs 16 to 21 and on 

16 postnatal days (PNDs) 1, 3, and 20. 

17 Results of the study, as reported by McLain (2008), are shown in Table 4-8. According 

18 to the review, treatment with n-butanol resulted in statistically significant increases in the 

19 percents of pre- and post implantation losses and in total fetal deaths. A decrease in the fertility 

20 index of treated animals was reported in the review (6.5 vs. 9.7% in controls; statistical analysis 

21 not reported). In addition, ADH activity in fetal livers, which was at its highest level on GD 20, 

22 was reduced 77.6% at this measurement in offspring of dams exposed to n-butanol. No other 

23 information was provided in the review or in the English summary in the publication. Effect 

24 levels were not provided in the review. Given the lack of information from which to reliably 

25 estimate dose, as well as reliance on a secondary source for information, EPA has not determined 

26 effect levels for this study. 

 

3Calculated 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. 
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Table 4-8. Teratogenic observations in rats exposed 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 ± 0.5a 6.3 ± 1.7 38.7 ± 4.7b

Pre implantation 5.6 ± 0.4 2.0 ± 1.0 21.7 ± 4.0b

Post implantation 5.2 ± 0.4 4.4 ± 1.4 21.7 ± 4.4b

Number of live fetuses 3,476 194 65 

aMean ± SD. 

bAccording to the review, “all indices of the embryotoxic activity in the experimental groups are reliably higher 

than control (p < 0.001)”.

Sources: Bariliak et al. (1991) as reported in McLain (2008). 

1 

2 4.3.2. Inhalation Studies 

3 Nelson et al. (1989a) studied the developmental toxicity of n-butanol (≥ 99% pure) and 

4 other butanol isomers in rats exposed by inhalation. Groups of 15–20 sperm-positive female 

5 Sprague-Dawley rats were whole-body exposed to n-butanol vapor at nominal concentrations of 

0, 3,500, 6,000, or 8,000 ppm (0, 11,000, 18,000, or 24,000 mg/m36 ), 7 hours/day on GDs 1–19. 

7 Test concentrations were selected on the basis of pilot testing. In the pilot experiment, exposure 

to 27,000 mg/m38 (9,000 ppm) n-butanol was lethal to two of six nonpregnant rats within 2 days, 

and exposure to 24,000 mg/m39 (8,000 ppm) resulted in narcosis in half of tested dams. In the 

10 main study, dams were evaluated weekly for food and water intake, and body weight was 

11 measured on GDs 0, 7, 14, and 20. After sacrifice on GD 20, the numbers of corpora lutea, 

12 resorptions (early, middle, or late), and live fetuses were recorded. Fetuses were weighed, sexed, 

13 and examined for external malformations. Half of the fetuses were examined for skeletal 

14 malformations and the other half for visceral malformations. 

15 In the highest exposure concentration of n-butanol, 2/18 dams died prior to sacrifice 

16 (Nelson et al., 1989a). There were no deaths in the low- or mid-exposure groups. The body 

weights of dams in the 24,000 mg/m317 group were approximately 17% less than controls at

18 termination (based on visual inspection of data presented graphically), although this difference 

19 was not statistically significant when the authors adjusted for multiple comparisons using the 

20 Bonferroni technique. However, the body weights were slightly higher than controls in 11,000 

and 18,000 mg/m321 groups. Food consumption was lower in dams exposed to the mid- and high 

concentrations (11–19% lower than controls at 18,000 mg/m322 and 10–17% lower at 24,000 

mg/m323 ); the difference from controls was statistically significant during all 3 weeks of treatment 
41 DRAFT - DO NOT CITE OR QUOTE

1 at the mid concentration and during the 1st week only at the high concentration. Water intake 

2 was reportedly higher in treated groups than in controls, but the difference was not statistically 

3 significant (data not reported). Exposure to n-butanol had no effect on number of corpora lutea, 

4 resorptions or live fetuses/litter, or sex ratio. External malformations were not observed in any 

5 group. Statistically significant concentration-related reductions in body weight were observed in 

6 male and female fetuses at the mid- and high-exposure levels (12 and 24–27% lower than 

controls in the 18,000 and 24,000 mg/m37 groups, respectively). The percent of fetuses having 

normal skeletal development was statistically significantly lower at 24,000 mg/m38 n-butanol. 

9 The authors indicated that rudimentary cervical ribs were the primary skeletal malformation 

10 observed with n-butanol exposure but incidence data for the different exposures was not 

11 provided by the authors. Statistical analysis of the incidences of litters with skeletal or visceral 

12 malformations or variations was not reported in the study. Based on Fisher’s exact tests 

13 performed for this review, the incidence of litters with skeletal malformations was increased at 

14 all concentrations, and the incidence of litters with visceral malformations was statistically 

15 significantly increased at the highest concentration. Table 4-9 reports selected findings from the 

16 study. The authors did not identify effect levels. determined that lowest concentration tested 

(11,000 mg/m317 or 3,500 ppm) was associated with an increase in the incidence of litters with 

18 skeletal variations and is considered a LOAEL. 
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Table 4-9. Selected changes in rats exposed to n-butanol via inhalation on 

GDs 1–19 

Parameter 

n-Butanol exposure in mg/m3 (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 ± 15a

142 ± 17 102 ± 24b103 ± 24b

Maternal food consumption during 

wk 2 (g) 

134 ± 21 142 ± 16 108 ± 12b

118 ± 17

Maternal food consumption during 

wk 3 (g) 

124 ± 16 133 ± 16 110 ± 9b

111 ± 17

Male fetal body weight (g) 3.4 ± 0.31 3.4 ± 0.18 3.0 ± 0.31b2.6 ± 0.25b

Female fetal body weight (g) 3.3 ± 0.27 3.2 ± 0.18 2.9 ± 0.30b2.4 ± 0.23b

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 ± 4b

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 

aMean ± SD. 

b

Significantly different from control at p < 0.05. 

cAffected 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. 

eThe study reported that 25 of 15 litters had skeletal variations. This was assumed to be a reporting error. 

Source: Nelson et al. (1989a). 

1 

2 Nelson et al. (1989b) evaluated behavioral teratology in young rats following in utero or 

3 paternal inhalation exposure to n-butanol vapor. Groups of 15 pregnant female Sprague-Dawley 

rats were exposed to 0, 3,000, or 6,000 ppm (0, 9,000, or 18,000 mg/m34 ) n-butanol for 7 

5 hours/day on GDs 1–19 (termed “maternal exposure group” by the authors). Groups of 18 male 

6 Sprague-Dawley rats were exposed to the same concentrations of n-butanol for 7 hours/day for 6 

7 weeks and then mated to non-butanol exposed females (termed “paternal exposure group” by the 

8 authors). On the day of birth (PND 0), offspring were culled to four males and four females per 

9 litter and fostered to untreated controls. Individual pup weights were recorded weekly for 5 

10 weeks. On PND 10, one male and one female pup/group were randomly assigned to one of four 

11 testing groups. There were three groups assigned to behavioral testing for neuromotor 
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1 coordination (ascent on a wire mesh screen on PNDs 10, 12, and 14 and rotarod, not mentioned 

2 in the methods section), activity (open field activity and photoelectrically-monitored activity on 

3 PNDs 16–18, 30–32, 44–46, and 58–60, and running wheel activity on PNDs 32 and 33), and 

4 learning (avoidance conditioning, with separate groups tested beginning PNDs 34 and 60 and 

5 operant conditioning beginning PND 40). The fourth group was used for brain neurotransmitter 

6 analysis. On PND 21, brains were removed from 10 pups/group (one male and one female/litter) 

7 and dissected into four regions (cerebrum, cerebellum, midbrain, and brainstem), these samples 

8 were used to measure the levels of total proteins and neurotransmitters, including acetylcholine 

9 (ACh), dopamine, norepinephrine, serotonin, met-enkephalin, B-endorphin, and substance P. 

10 The study authors indicated that, since exposures to the two concentrations of n-butanol were 

11 separated by about 5 months, results could be compared to the contemporaneous controls, but 

12 comparisons between the two concentrations would not be appropriate. 

13 Analysis of exposure concentrations showed consistency with target exposures (method 

14 of analysis not reported); measured concentrations were 3,010 ± 50 and 6,000 ± 80 ppm (9824 ± 

163 and 19580 ± 261 mg/m315 , respectively; Nelson et al., 1990, 1989b). The study authors 

16 reported that n-butanol exposure did not affect pregnancy rate in any exposure groups. There 

17 were no behavioral changes in the offsping in term of their performance in ascent test, rotarod 

performance, open field performance, or operant conditioning. In offspring of 18,000 mg/m3 18 

19 paternal exposure group, the time receiving shock and the total number of times that rats crossed 

20 from one side of the cage to the other were both statistically significantly increased over controls. 

21 Monitoring of photoelectric activity showed statistically significantly lower counts in female 

offspring of to the 9,000 mg/m322 paternal exposure group but not in offspring of the high 

23 concentration paternal exposure group (data not shown, and magnitude of change not reported). 

Tests for avoidance conditioning showed that male offspring of the 9,000 mg/m324 paternal 

25 exposure group required statistically significantly fewer trials to reach criterion than controls; no 

26 statistically significant change was observed at the high concentration. Analysis of 

27 neurotransmitter concentrations in the brains of offspring revealed statistically significant 

28 increases in the overall concentration of serotonin (mean ± SEs were 14.48 ± 2.38 vs. 7.802 ± 

29 1.48 in controls; units not reported) and dopamine (0.715 ± 0.127 vs. 0.515 ± 0.095 in controls; 

units not reported) in offspring of the 18,000 mg/m

3

30 paternal exposure group. There were no 

31 other statistically significant changes in neurotransmitter concentrations associated with 

32 exposure to n-butanol. The study authors also indicated that the changes in both the 

33 neurobehavioral tests and neurotransmitter concentrations observed in the animals exposed to 

34 n-butanol were within the range of control data from their laboratory. The authors did not define 

effect levels. EPA determined that the highest concentration (18,000 mg/m335 , or 6,000 ppm) 

36 tested in this study is a NOAEL for neurobehavioral effects in offspring after parental inhalation 

37 exposure to n-butanol. 
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1 In a study examining potential testicular toxicity, Cameron et al. (1985) exposed groups 

of five male Sprague-Dawley rats to n-butanol at a concentration of 50 ppm (150 mg/m32 ) 

3 6 hours/day for 1 day or 1 week. Testicular effects were assessed through the measurement of 

4 serum concentrations of testosterone, luteinizing hormone (LH), and corticosterone in blood 

5 samples collected at sacrifice at the end of exposure or 18 hours after the end of exposure 

6 following both the 1-day and 1-week exposures. In rats sacrificed after 1 day of exposure, 

7 statistically significant reductions in testosterone concentrations were observed; the decreases 

8 were 37 ± 8 and 52 ± 22% of controls when measured immediately after and 18 hours after 

9 exposure, respectively. In rats sacrificed after 1 week of exposure, testosterone levels were 

10 decreased (73–83% of controls), but the difference from controls was not statistically significant. 

11 Serum concentrations of LH were higher than controls (115–124% of controls) in animals treated 

12 with n-butanol for 1 day, but lower (78–98% of controls) in those treated for 1 week; none of the 

13 differences were statistically significant. Rats treated with n-butanol for 1 day had statistically 

14 significantly higher serum corticosterone than controls (143% of control); however, rats treated 

15 for 1 week had lower corticosterone (85% of control, not statistically significantly different from 

16 control). 

17 

18 4.4. OTHER DURATION- OR ENDPOINT-SPECIFIC STUDIES 

19 4.4.1. Acute and Short-term (≤ 30 Days) Studies 

20 Several studies determined LD50 values for n-butanol following acute and short-term exposure. 

21 Among rats, rabbits, hamsters, and mice, oral LD50s ranged from 1,200 mg/kg to 4,360 mg/kg 

22 (Rumyantsev, 1979; Dubina and Maksimov, 1976; Munch, 1972; Purchase, 1969; Jenner et al., 

23 1964; Smyth et al., 1951; McOmie and Anderson, 1949; Munch and Schwartze, 1925). 

24 Behavioral toxicities following or during an acute exposure included narcosis and necropsies 

25 indicated that there was necrosis of the liver and kidney as well as general organ congestion 

26 (Munch, 1972; Purchase, 1969; Jenner et al., 1964; McOmie and Anderson, 1949; Munch and 

27 Schwartze, 1925). 

28 General short-term studies for n-butanol primarily evaluated effects on narcosis and 

29 pathological effects in the liver and kidney. Detailed summaries for these studies are provided in 

30 Table 4-10. 

31 

Table 4-10. Summary of Short-term Studies 

Species/Sex Dose Duration Effect Observed Reference 

Mice 24,624 mg/m3

inhalation 

130 hours Narcosis and reversible 

fatty infiltration of the liver 

and kidneys 

Weese, 1928 
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Mice 22 mg/L (22,000 

mg/m3) 

 27 mg/L (27,000 

mg/m3) 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/m3

0.78 ± 0.05 mg/m3

6.6 ± 0.39 mg/m3 

40 ± 42 mg/m3

30 days Statistically significant 

decrease in hexenal 

induced sleep duration at 

both 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 

3 4.4.2. Liver Toxicity 

4 Videla (1982) administered a single oral dose of 560 mg/kg n-butanol (purity not 

5 reported) to groups of four male Wistar rats. Levels of reduced glutathione (GSH), diene 

6 conjugates, proteins, and ADH activity in the liver were measured 6 hours after dosing. Based 

7 on visual inspection of data presented graphically, levels of GSH were lower, and diene 

8 conjugates were higher in animals given n-butanol compared with saline-treated controls; 

9 however, the differences were small and were not statistically significant. ADH activity in 

10 n-butanol treated rat livers was about 41% of that in ethanol-treated rat livers; however, 

11 information on controls was not reported. 

12 Shehata and Saad (1978) evaluated the effect of oral exposure to alcohols on vitamin 

13 content in the livers of rats. Groups of six male albino rats were given daily gavage doses of 0, 

14 1, or 2 mL/kg (810 and 1,620 mg/kg) n-butanol for 7 consecutive days. The animals were 

15 sacrificed 2 hours after the last dose and the liver was assayed for content of thiamine, riboflavin, 

16 pyridoxine, niacin, and pantothenic acid. At both doses of n-butanol, the liver content of all five 

17 vitamins was statistically significantly lower than the corresponding control value. The 

18 decreases, which were dose-related, ranged between 25 and 49% at the high-dose. 

19 

20 4.4.3. Immunotoxicity 

21 Strubelt and Zetler (1980) tested the antiinflammatory effects of n-butanol and other 

22 alcohols. Groups of eight male Wistar rats were given single gavage doses of 0, 600, or 
46 DRAFT - DO NOT CITE OR QUOTE

1 1,200 mg/kg n-butanol (analytical purity not further specified), followed 15 minutes later with an 

2 injection of 0.1 mL of 1% carageenan in the right hind paw. Inflammation of the paw was 

3 measured as mean difference from pretreatment paw volume after 2, 3, 4, 5, and 5 hours 

4 following injection. Exposure to n-butanol resulted in reduced inflammation, as shown by a 

5 statistically significant inhibition of the increase in paw volume associated with carageenan 

6 injection (19 and 15% smaller mean increase in paw volume compared with controls in the low7 and high-dose groups, respectively). Similar inhibition of inflammation was observed with other 

8 alcohols. In a separate experiment, groups of 12–16 male Wistar rats were treated with single 

9 gavage doses of 0, 300, 600, or 1,200 mg/kg followed by injection of 0.2 mL of 1% carageenan 

10 into the pleural cavity. After a period of 4 hours, the rats were sacrificed and chest cavity was 

11 opened. The volume of exudate was recorded, and leukocyte count and osmolality of the 

12 exudate were measured; blood osmolality was also measured. Exposure to n-butanol at ≥ 600 

13 mg/kg resulted in statistically significantly reduced volume of pleural exudate (53 and 13% of 

14 control volume at 600 and 1,200 mg/kg, respectively) and decreased leukocyte count (51 and 9% 

15 of control count, respectively), indicating a reduction in the inflammatory response to 

16 carageenan. The osmolality of pleural exudates was not affected by exposure. Significant 

17 changes in blood osmolality were observed at the low- and high-doses, but not the mid-dose. 

18 The blood osmolality changes were not consistent with dose; the low-dose resulted in a decrease 

19 in osmolality, while the high-dose resulted in an increase. This study suggested that oral 

20 exposure to n-butanol results in anti-inflammatory effects. 

21 

22 4.4.4. Neurotoxicity 

23 Wallgren (1960) tested the behavior of 5-month-old rats (strain not reported, groups of 5–

24 7/sex) exposed to a single oral dose of 0.0163 mol/kg n-butanol (1,210 mg/kg). Beginning 

25 20 minutes after dosing, the animals were subjected to six “tilted plane” tests at 20-minute 

26 intervals. In this test, the angle at which a rat slides after being placed head up on a tilted plane

27 was recorded. Results were reported as lowest and mean percentage of control performance; for 

28 n-butanol, the lowest value in the six tests corresponded to 55.8 ± 1.7% of control and the mean 

29 of the six tests was 73.3 ± 9.2% of control. Statistical analysis was not reported. 

30 Maickel and Nash (1985) treated groups of 23–25 male Swiss-Cox mice to oral doses of 

31 500, 1,000, or 2,000 mg/kg n-butanol and assessed body temperature (via rectal thermometer) 

32 and rotarod performance in groups of five mice each at 10, 20, 40, 80, and 120 minutes after 

33 treatment. Blood was collected for analysis of n-butanol at the same time points. Treatment 

34 with n-butanol resulted in dose-dependent decreases in body temperature that persisted through 

35 40 minutes after exposure in all groups and through 80 minutes in the mid- and high-dose 

36 groups. n-Butanol doses of 1,000 and 2,000 mg/kg resulted in dose-related decreases in rotarod 

37 performance (65 and 25% of baseline, respectively, 10 minutes after exposure, based on visual 

38 inspection of data presented graphically). Rotarod performance gradually improved over time 
47 DRAFT - DO NOT CITE OR QUOTE

1 until it was 100% of baseline by 80 minutes (at 1,000 mg/kg) or 120 minutes (at 2,000 mg/kg) 

2 after dosing. Exposure to n-butanol at 500 mg/kg did not affect rotarod performance. 

3 In a short-term study of a series of industrial solvents, De Ceaurriz et al. (1983) exposed 

4 groups of 10 male Swiss OF1 mice for 4 hours to n-butanol (98.5% pure) concentrations of 0, 

470, 548, 844, or 965 ppm (0, 1,420, 1,660, 2,560, or 2,930 mg/m35 ). Vapors were generated 

6 using either an injector with added heat to increase vaporization or by bubbling air through a vial 

7 containing the test material (the authors did not specify which method was used for n-butanol). 

8 After exposure, each animal was tested in a “behavioral despair” swimming test, in which it was 

9 immersed in water in a glass cylinder and observed for duration of immobility (passive floating). 

10 A concentration-related, statistically significant decrease in the duration of immobility during the 

11 first 3 minutes of testing (i.e., prolongation of escape-directed activity) was observed at all of the

12 exposure concentrations; the percent decrease from control was 38, 47, 60, and 70% in the low 

13 through high-exposure groups. The concentration of n-butanol resulting in 50% decrease in 

immobility was reported to be 617 ppm (95% CI 547–681), or 1,870 mg/m314 . According to the 

15 authors, a decrease in immobility in this test is characteristic of compounds with antidepressant 

16 activity. 

17 In a study designed to investigate the potential ototoxicity of solvent exposure, Crofton et 

18 al. (1994) exposed groups of 10 adult male Long-Evans rats via whole-body inhalation to 

n-butanol vapor concentrations of 0 or 4,000 ppm (12126 mg/m319 ), 6 hours/day for 5 days. 

20 Auditory function was tested via reflex modification audiometry 5–8 weeks after exposure. 

21 There was no effect of n-butanol exposure on the auditory threshold of rats through the 

22 frequency range that was tested (0.4–40 kHz). 

23 Frantik et al. (1994) tested the acute neurotoxicity of n-butanol in male albino SPF Wistar 

24 rats and in female mice (H strain). Groups of four male rats were exposed (whole-body) to 

25 n-butanol vapors for 4 hours, while groups of 16 female mice were exposed for 2 hours. The 

26 tested concentrations were not reported, as the focus of the analysis was to identify isoeffective 

27 concentrations for the different solvents. Within a minute after the exposure period ended, the 

28 investigators applied a short electrical impulse through electrodes in the ears of the tested 

29 animals to generate a tonic extension of hindlimbs in rats and in mice. The only n-butanol effect 

30 information presented by the authors was the estimated concentration evoking a 30% depression 

in these parameters (3,500 ppm [10,000 mg/m3] in rats and 2,400 ppm [7,300 mg/m331 ] in mice). 

32 A series of studies (Korsak and Rydzynski, 1994; Korsak et al., 1993) evaluated the acute 

33 neurobehavioral effects of n-butanol in rats exposed via inhalation. In the first study (Korsak et 

34 al., 1993), groups of male Wistar rats (10/group) were exposed for 4 hours to various 

35 concentrations of n-butanol. Immediately after exposure, the rats were tested for rotarod 

36 performance, and an hour after exposure ended, spontaneous motor activity was assessed. The 

37 authors estimated the medial effective concentrations (EC50) for the rotarod performance to be 

6,531 ppm (19,800 mg/m338 ). In the tests of spontaneous motor activity, low concentrations of 
48 DRAFT - DO NOT CITE OR QUOTE

1 n-butanol were associated with increased activity (compared with control animals), while higher 

2 concentrations reduced activity back to control levels (based on data presented graphically). 

3 The second study (Korsak and Rydzynski, 1994) also exposed groups of 10 male Wistar 

4 rats to various concentrations of n-butanol for 4 hours, and tested rotarod performance 

5 immediately after exposure. An hour after exposure ended, pain sensitivity (measured as latency 

6 to paw lick response on a hot plate) was assessed. The authors estimated the EC50 value for 

disturbance of rotarod performance to be 7,559 ppm (22,920 mg/m37 ) in this study; this is similar 

8 to the value (6,531 ppm) estimated by Korsak et al. (1993). Hot plate behavior was also affected 

9 by exposure. The EC50 estimated by Korsak and Rydzynski (1994) for decreased sensitivity to 

pain was 5,901 ppm (17,890 mg/m310 ) (95% CI 4,841–7,232 ppm). 

11 In a study using i.p. administration of n-butanol, McCreery and Hunt (1978) compared 

12 the acute neurotoxic potencies of 62 compounds in an attempt to correlate potency with physical13 chemical parameter such as the membrane/buffer partition coefficient. Male Sprague-Dawley 

14 rats (number/group not specified) received a single i.p. injection of the compound in various 

15 doses. Neurotoxic potency was assessed after dosing (duration of observation not reported), and 

16 characterized as the dose that caused pronounced impairment of gait (ataxia) and motor 

17 incoordination without causing the abdomen or pelvis to drop (the authors termed this dose the 

18 ED3). The ED3 for n-butanol was 5.4 mmol/kg (400 mg/kg); by comparison, the ED3 values for 

19 the related alcohols ethanol and methanol were 32.6 and 109 mmol/kg, respectively. Neurotoxic 

20 potency showed a strong inverse correlation with membrane:buffer partition coefficient in 

21 alcohols up to a certain carbon number (C8 for straight-chain alcohols). 

22 

23 4.4.5. Pulmonary Toxicity 

24 Kristiansen et al. (1988) evaluated the sensory irritation response in mice exposed to 

25 n-butanol vapors by assessing the reflexive decrease in respiratory rate that accompanies 

26 pulmonary irritation. Three different exposures (normal inhalation, inhalation via tracheal 

27 cannula, and i.p. injection) were tested in order to explore whether effects on respiration resulted 

28 from stimulation of receptors on the trigeminal (in the nasal mucosa) or vagal (in the lower 

29 respiratory tract) nerves, or from depression of the CNS. Groups of four male Ssc:CF-1 mice 

30 were used; a control group was exposed to clean air. For the inhalation (normal and cannulated) 

31 exposures, the mice were exposed to 0, 435, 2,500, 5,600, or 9,200 ppm (normal; or 0, 1,319, 

7,579, 16,976, or 27,890 mg/m332 ) or 4,630, 5,400, 7,335, or 9,250 ppm (cannulated; or 14,036, 

16,370, 22,236, or 28,041 mg/m333 ) of n-butanol (99.5% pure) for 30 minutes followed by a 20-

34 minute recovery period. Respiratory rate and relative tidal volume were measured continuously 

35 using a pressure transducer attached to a body plethysmograph into which each animal was 

36 placed, with its head inserted into the exposure chamber. Doses of 1.6, 4.1, 12.1, and 20.2 

37 mg/mouse were administered to the groups exposed via i.p. injection immediately prior to 

38 respiratory measurements for 50 minutes. The animals were observed for escape attempts as a 
49 DRAFT - DO NOT CITE OR QUOTE

1 measure of CNS depression. The relationship between percent decrease in respiratory rate and 

2 the log of the exposure concentration was plotted and used to estimate the concentrations of 

3 n-butanol that would result in a 0 or 50% reduction in respiration rate (RD0 and RD50, 

4 respectively). 

5 The experiments showed that normal inhalation of n-butanol resulted in a concentration6 related decrease in respiratory rate, with the maximum depression occurring in the first minute 

7 (Kristiansen et al., 1988). The authors estimated RD0 and RD50 values of 233 and 11,696 ppm 

(706 and 35,456 mg/m38 ), respectively, for the first minute of inhalation exposure. After the first 

9 minute, there was evidence of desensitization. The response after the first minute showed 

concentration-dependent patterns; at concentrations below 3,000 ppm (9,094 mg/m310 ), the 

response leveled off, while above 3,000 ppm (9,094 mg/m311 ), a secondary depression occurred. 

12 The authors suggested that the secondary response occurred as a result of stimulation of 

13 receptors in the lower respiratory tract, as a similar response occurred in the cannulated animals. 

14 No evidence of CNS depression was evident in any of the inhalation-exposed groups; however, 

15 the decrease in respiratory rate seen with i.p.-exposed rats indicated CNS-mediated effects, as 

16 this group had no direct exposure to the respiratory tract. Comparison between results in the 

17 cannulated and i.p.-exposed groups showed a stronger response in the cannulated group, leading 

18 the authors to conclude that both CNS and lung receptors were involved in the secondary 

19 response. 

20 Korsak and colleagues (Korsak and Rydzynski, 1994; Korsak et al., 1993) also evaluated 

21 respiratory irritation in mice exposed via inhalation. In the first study, groups of male Balb/C 

22 mice (8–10/group) were exposed for 4 hours to various concentrations of n-butanol. Respiratory 

23 rate was measured by plethysmograph before exposure, for 6 minutes during exposure, and for 

24 6 minutes after the end of exposure. Exposure to n-butanol resulted in a concentration25 dependent decrease in respiratory rate, with maximum depression occurring during the 

1st minute of exposure. The authors estimated the RD50 value to be 3,008 ppm (9,119 mg/m326 ). 

27 The second study (Korsak and Rydzynski, 1994) used the same protocol and estimated an RD50 

value of 4,300 ppm (13,000 mg/m328 ), which compares favorably with the value estimated by 

29 Korsak et al. (1993). 

30 

31 4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF 

32 ACTION 

33 4.5.1. Genotoxicity Studies 

34 A series of genotoxicity assays of n-butanol in both bacterial and mammalian systems 

35 have produced primarily negative results (see Table 4-11). n-Butanol did not increase reverse 

36 mutations in Salmonella typhimurium (strains TA98, TA100, A1535, and TA1537) in the 

37 presence or absence of microsomal enzyme activation (i.e., liver S9 preparations) (Jung et al., 

38 1992; Nakamura et al., 1987; Connor et al., 1985; McCann et al., 1975). Obe and Ristow (1977) 
50 DRAFT - DO NOT CITE OR QUOTE

1 did not observe an increase in the frequency of sister chromatid exchanges in the Chinese 

2 hamster ovary cell, and no increase in the frequency of micronuclei was observed in Chinese 

3 hamster cells (V79) treated with a concentration of 50 μL/mL of n-butanol (Lasne et al., 1984). 

4 When V79 cells were tested at a concentration of 0.1 M n-butanol, an increased frequency of 

5 polyploidy cells was observed, but survival at this concentration was only 36% (Onfelt, 1987). 

6 Yoshiyama et al. (1973) showed that n-butanol selectively suppresses the initiation of DNA 

7 replication in irradiated Escherichia coli (W2252) cells. n-Butanol was also inhibited the 

8 incorporation of uridine in HeLa cells (Obe et al., 1977), suggesting inhibition of RNA synthesis. 

9 Using a cell-free system with calf thymus DNA, Obe et al. (1977) showed that n-butanol 

10 concentrations of 1–4% inhibited RNA transcription in a dose-dependent fashion. 

11 

Table 4-11. Genotoxicity studies of n-butanol in vitro 

Test system Endpoint Test conditions 

Resultsa

Doseb Reference 

Without 

activation

With 

activationa

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 

[

14C]-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) 
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Table 4-11. Genotoxicity studies of n-butanol in vitro 

Test system Endpoint Test conditions 

Resultsa

Doseb Reference 

Without 

activation

With 

activationa

HeLa cells RNA 

synthesis; 

inhibition of 

uridine 

incorporation 

Treated with 

increasing doses for 

5 min prior to 

treatment with 

[

3H]-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) 

aExogenous metabolic activation used. 

bLowest effective dose for positive results or highest dose tested for negative or equivocal results. 

+ = positive, − = negative, ND = no data 

1 

2 There was only one in vivo test of n-butanol genotoxicity. Bloom (1982) injected 

3 n-butanol into the inner shell membrane of chick embryos to assess the frequency of sister 

4 chromatid exchanges and chromosomal breaks. When tested at a concentration of 10 μL, no 

5 increases in the frequency of sister chromatid exchanges or chromosomal breakages were 

6 observed. 

7 

8 4.5.2. Mechanistic Studies Evaluating Carcinogenic Parameters 

9 Etique et al. (2004) cultured human breast cancer cells (MCF-7) for 24 hours prior to 

10 treatment with 0.1% n-butanol for 6 days. Cell proliferation was evaluated, and P450-aromatase 

11 activity (which is involved in estrogen synthesis and has been shown to be increased in breast 

12 cancer) and estrogen receptor α (ER-α) mRNA expression were measured using semiquantitative 

13 real time polymerase chain reaction (RT-PCR). In addition, ER-α protein content was analyzed 

14 using Western blot analysis. Exposure to n-butanol resulted in a statistically significant decrease 

15 in cell proliferation (38% less than controls); n-Butanol did not modify the ER-α protein content. 

16 

17 4.5.3. Mechanistic Studies Evaluating Neurological Effects 

18 Lovinger et al (1989) demonstrated that n-butanol dose-dependently inhibited the N19 methyl-D-aspartate (NMDA) glutamate receptor in mouse hippocampal neurons. Following 

20 Lovinger et al (1989), there were many characterization studies of the potential molecular sites 

21 of action for n-alcohols in the central nervous system. Table 4-12 summarizes and provides 

22 experimental details on the electrophysiological studies conducted with n-butanol on individual 
52 DRAFT - DO NOT CITE OR QUOTE

1 neuronal ligand-gated ion channels expressed in in vitro cell lines (Xenopus oocyte; human 

2 embryonic kidney cells) or mixed neurotransmitter systems in primary neuronal cell cultures. It 

3 was generally found that n-butanol dose-dependently and reversibly inhibited glutamate receptor 

4 (NMDA, Kainate, and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate [AMPA]) function 

5 (Akinshola, 2001; Peoples and Weight, 1999; Dildy-Mayfield et al., 1996; Lovinger et al., 1989) 

6 and potentiated the function of inhibitory systems such as glycine and gamma-aminobutyric acid 

7 (GABA) receptors (Mascia et al., 2000; Peoples and Weight, 1999; Ye et al., 1998; Dildy8 Mayfield et al., 1996; Nakahiro et al., 1991). Three different studies (Zuo et al., 2003; Zuo et al.,

9 2001; Godden et al., 2001) examined the effects of n-butanol on nAChRs and it could be 

10 concluded that n-butanol may act as a partial agonist on this receptor class. A few studies 

11 (Rusch et al., 2007; Stevens et al., 2005; Zhou et al., 1998) reported that n-butanol (11 – 22 mM) 

12 potentiated the serotonin (or 5-HT [5 hydroxytryptamine]) current. N-butanol was reported to 

13 inhibit the function of voltage gated potassium channels (Harris et al., 2003; Shahidullah et al., 

14 2003). However, when a residue in the S6 segment of the potassium channel was mutated from 

15 a proline (P) to an alanine (A) [P410A], the n-butanol potentiated channel function (Bhattacharji 

16 et al., 2006). Studies with rat brain membranes indicated that n-butanol interacted with delta17 specific, but not kappa, opioid receptor binding sites (Hiller et al., 1984) and n-butanol increased 

18 membrane fluidity and decreased membrane ATPase activity (Edelfors and Ravn-Jonsen, 1990). 
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Table 4-12. Study details for mechanistic data and other studies in support of the mode of action for effects 

Reference Test system 

Number of 

samples/ 

group 

Dose or 

concentration 

range Study protocol 

Studies of effects on ligand-gated ion channels 

Lovinger et al. (1989) 

Mouse hippocampal neurons 

4 neurons/test 0.01–25 mM Neurons were co-exposed to 50 uM 

NMDA and n-butanol. Whole-cell patch clamp techniques were used to measure the elicited current from the treatments. 

n-Butanol dose depeglutamate receptor rehippocampal neuronreversible following 

Nakahiro et al. (1991) 

Primary cultures of rat dorsal root ganglion neurons 

3–9 Neurons/test 

1–30 mM Neurons were exposed to n-butanol and 

GABA or after GABA current had reached steady state, and the effect on current was measured using whole cell patch-clamp technique. 

Initial peak current (nGABA was increased concentration; no curof GABA. Potency opositively correlated membrane:buffer parn-alcohols. In contrainhibited the desensiinduced by GABA. 

DildyMayfield et al. (1996) 

X. laevis oocytes expressing recombinant GABAA, NMDA, 

AMPA, or kainite receptors 

4–12 Oocytes/test 

5–50 mM Cultured oocytes expressing GABAA, 

NMDA, AMPA, or kainite receptors were exposed to alcohols and effects on current were measured using the twoelectrode voltage clamp technique. 

n-Butanol potentiated receptors composed 

but inhibited the respand kainite. 

Ye et al. (1998) 

X. laevis oocytes or HEK-293 cells expressing wild type or mutant glycine Rα1 or 

GABA ρ1 

receptors or chimeric glycine Rα1/GABA ρ1 

receptor 

4–13 Oocytes/test 

10–20 mM Cells were exposed to n-butanol and the 

effect on current was measured using whole cell patch-clamp technique. 

n-Butanol exhibited glycine-induced currtype glycine receptorinhibition of submaxin cells expressing w

In cells expressing chpotentiated GABA-ininhibited glycine-ind
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Table 4-12. Study details for mechanistic data and other studies in support of the mode of action for effects 

Reference Test system 

Number of 

samples/ 

group 

Dose or 

concentration 

range Study protocol 

Zhou et al. (1998) 

neuroblastoma (NCB-20) cells 

4–11 Cells/test 

20 mM Cells were exposed to n-butanol and 

5-HT and the effect on current was measured using whole cell patch-clamp technique. 

n-Butanol exposure irise time, and the melow concentrations odecreased the measurcurrent evoked by 10 relative amplitude of evoked by 2 or 10 μM 

Peoples and Weight (1999) 

Primary cultures of hippocampal neurons from 15 to 17-d-old fetal mice 

4–7 Neurons/test 

~0.0013–0.125 M 

Neurons were exposed to n-butanol with GABA or NMDA and effects on current 

were measured using the two-electrode voltage clamp technique. 

 n-Butanol enhanced 

currents and inhibiteAnesthetic potency ochain n-alcohols was NMDA-modulated e

modulated effects 

Mascia et al. (2000) 

X. laevis oocytes expressing human α1(wild 

type) and α1(S267Q) (anestheticresistant mutant) glycine receptor subunits 

3–9 Oocytes/test 

6.6–66 mM Cultured oocytes expressing human 

α1and α1(S267Q) glycine receptor were 

exposed to n-butanol, and glycine, and effects on glycine receptor function was measured using the two-electrode voltage clamp technique. 

n-Butanol potentiated oocytes expressing w

receptors, and modesthose expressing the (α1[S267Q]). Mortaobserved at the highe

Akinshola (2001) 

X. laevis oocytes expressing recombinant AMPA GluR1 and GluR3 receptor subunits 

5–8 Oocytes/test 

1–1,000 mM Cultured oocytes expressing glutamate 

GluR1 or GluR3 receptor subunits were treated with various concentrations of alcohols, and inhibition of kainiteactivated ion-currents was measured using the two-electrode voltage clamp technique. 

n-Butanol exhibited kainite-activated currrecombinant AMPA subunits. 

Godden et al. (2001) 

X. laevis oocytes expressing human nACh receptor subunits α2, α4, and β4

3–16 Oocytes/test 

0.0001–0.1 M Cultured oocytes expressing nACh 

receptor subunits α2, α4, and β4 were 

bathed in alcohols with ACh substrate, and effect current was measured using the two-electrode voltage clamp technique. 

n-Butanol potentiated receptors composed 

effect on receptors co

combination. Molecshown to correlate wreceptors. 
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Table 4-12. Study details for mechanistic data and other studies in support of the mode of action for effects 

Reference Test system 

Number of 

samples/ 

group 

Dose or 

concentration 

range Study protocol 

Zuo et al. (2001) 

HEK-293 expressing the α4β2 subunit of neuronal nACh receptors 

6 Cells/test 1–300 mM Cells were exposed to n-butanol with 

ACh and the effect on current was measured using whole cell patch-clamp technique. 

n-Butanol (1 and 100 dependent inhibition and potentiated the econcentration of 300 30 mM, n-butanol sliof 10 μM ACh. 

Zuo et al. (2003) 

HEK-293 expressing the α4β2 subunit of neuronal nACh receptors 

6 Cells/test 1–300 mM Cells were exposed to n-butanol and 

ACh and the effect on current was measured using whole cell patch-clamp technique. 

Prolonged exposure currents blocked by tmecamylamine or th

erythroidine, indicatipartial agonist. At a mM), n-butanol poteACh concentrations 

effects of high ACh (concentration of n-bueffects of ACh (10–3

Stevens et al. (2005) 

X. laevis oocytes expressing 5-HT3A or 5-HT3AB 

receptors

At least three oocytes/test 

11 and 22 mM Cultured oocytes expressing 5-HT3A or 

5-HT3AB receptors were exposed to 

n-butanol and dopamine, and effects on current were measured using the twoelectrode voltage clamp technique. 

n-Butanol enhanced concentrations of 5-H

slight decrease in the 

expressing HT3A, the 

higher than in cells e5-HT3AB receptor. 

Rusch et al. (2007) 

X. laevis oocytes expressing 5-HT3A or 

5-HT3AB receptors 

5–10 Oocytes/test 

21.6 mM Cultured oocytes expressing serotonin 

5-HT3A or 5-HT3AB receptors were 

exposed to n-butanol and dopamine, and effects on current were measured using the two-electrode voltage clamp technique. 

n-Butanol strongly pelicited by dopamine in oocytes expressing 

modestly potentiated expressing the 5-HT3

and octanol did not mcompared with n-but

Other mechanistic studies of neurological effects 

Hiller et al. (1984) 

Isolated rat brain membrane 

Experiments conducted in triplicate 

0.5% Effect of n-butanol on binding opioid 

binding to kappa site tested by blocking mu and delta sites with appropriate ligands. Kinetic studies were made to assess possible mechanism of selective inhibition of delta sites. 

n-Butanol did not inhsites, but selectively sites. Kinetic studies is a result of a decreasite (increased dissoc
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Table 4-12. Study details for mechanistic data and other studies in support of the mode of action for effects 

Reference Test system 

Number of 

samples/ 

group 

Dose or 

concentration 

range Study protocol 

Edelfors and Ravn-Jonsen (1990) 

Rat brain synaptosomal membranes 

Experiments conducted in triplicate 

12.5, 25, and 50% of saturation (solubility reported to be 912 mmol/L) 

Membranes were bathed in n-butanol and the membrane Ca2+/Mg2+ ATPase activity was measured spectrophotometrically. Membrane fluidity was measured as incorporation of fluorescent 1,6-diphenyl-1,3,5-hexatriene. 

n-Butanol decreased activity in a concentrmembrane fluidity wn-butanol concentrat

Bhattacharji et al. (2006); Harris et al. (2003); Shahidullah et al. (2003 

X. laevis oocytes expressing wildtype and selectively mutated Shaw2 voltage-gated K+ 

channels from Drosophila melanogaster

3–5 Oocytes/test 

1–100 mM Cultured oocytes expressing wild-type 

and mutant (generated by S5 alaninescanning mutagenesis) Shaw2 K+ 

channels were exposed to n-butanol, and the effect on current was measured using the two-electrode voltage clamp, whole cell patch-clamp, or fast-concentration clamp technique. 

n-Butanol inhibited tand several of the muinhibition associated a key site (P410A) in channel changed the inhibition into potentdesigned to explore mbinding site on the ch

AMPA = α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; ATPase = adenosine triphosphatase; 5-HT = 5-hydroxytryptamine; nANMDA = N-methyl-D-aspartate 
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1 4.5.4. Mechanistic Studies Evaluating Neurodevelopmental Effects 

2 Kotter and colleagues (Kotter et al., 2000; Kotter and Klein, 1999) conducted a series of 

3 studies comparing the effects of ethanol and butanols on the proliferation of neonatal rat brain 

4 astroglial cells. In experiments on astrocyte cell proliferation rates, the effects of alcohols on 

proliferation were assessed by measuring [35 H]-thymidine incorporation after treatment of the cell 

6 cultures with alcohol and a mitogenic agent. Using fetal calf serum as the mitogen, Kotter and 

7 Klein (1999) treated astrocyte cells with n-butanol at concentrations of 0.1, 0.3, or 1.0%. 

8 Compared with cells treated with fetal calf serum alone, cell proliferation was inhibited by ~40 

9 and 70%, at 0.1 and 0.3% n-butanol (respectively); proliferation was completely inhibited at 1% 

10 n-butanol. Similar results were observed using platelet-derived growth factor and endothelin 1 

11 (Kotter and Klein, 1999). 

12 Kotter et al (2000) also measured astrocyte cell proliferation, but used phorbol ester as 

13 the mitogen. At concentrations of 0.1 and 0.3%, n-butanol inhibited proliferation of astroglial 

14 cells by ~50 and nearly 100% (based on visual inspection of data presented graphically), 

15 respectively, compared with cells treated with phorbol ester alone. The potency of inhibition of 

16 n-butanol was much stronger than that of ethanol; at 0.3% ethanol, cell proliferation was 

17 inhibited by only ~40%, compared with nearly 100% inhibition at the same concentration of 

18 n-butanol. 

19 In two sets of experiments on the activation of astroglial phospholipase D (PLD), 

phospholipids in the cells were labeled by incubating the cells with [320 H]-glycerol for 24 hours 

21 prior to treatment with a mitogen and alcohol (Kotter et al., 2000; Kotter and Klein, 1999). The 

22 cultures were exposed to ethanol or butanols (0.1–1.0%) for 5 minutes prior to isolation of 

23 phospholipids and quantification of phosphatidic acide (PA) and phosphatidylcholine. When 

24 fetal calf serum was used as the mitogen (Kotter and Klein, 1999), treatment with n-butanol 

25 statistically significantly increased the formation of phosphatidylbutanol and decreased the 

26 formation of PA (~30–40% at 0.3% n-butanol based on visual inspection of data presented 

27 graphically). As with the effect on cell proliferation, n-butanol exhibited a stronger effect on PA 

28 formation than ethanol at the same concentration (~15% decrease in PA formation at 0.3% 

29 ethanol). Similar results were observed when phorbol ester was used as the mitogen (Kotter et 

30 al., 2000). 

31 Kotter et al. (2000) also measured protein kinase C (PKC) activity after alcohol 

32 exposure; rat brain PKC was incubated with lipid micelles, phorbol ester, phosphatidylserine and 

alcohol for 5 minutes, followed by the addition of [

32 33 P]-ATP. PKC activity was statistically 

34 significantly reduced by both ethanol and n-butanol at a concentration of 0.3% (average of nine 

35 experiments showed ~95 and 85%, respectively, of activity in the absence of the alcohols). 

36 These studies demonstrate that, like ethanol, n-butanol inhibits the proliferation of 

37 astroglial cells, probably by disrupting the PLD signaling pathway by formation of phosphatidyl-
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1 butanols and concomitant reduction in PA formation. In all of the experiments reported, 

2 n-butanol exerted a stronger inhibitory effect than ethanol at the same concentration. 

3 

4 4.5.5. Mechanistic Studies Evaluating Liver Effects 

5 Carlson (1994a, b) showed that n-butanol can esterify fatty acids in vivo (in rats) and in 

6 vitro (in the homogenized liver, lung, and pancreas of rats and rabbits). In the in vivo 

7 experiment, n-butanol was administered via i.p. injection (1.0 mL/kg or 810 mg/kg) to male 

8 Sprague-Dawley rats, and the animals were sacrificed for analysis of butyl palmitate, butyl 

9 stearate, and butyl oleate (butyl esters of fatty acids) in the liver 1 or 6 hours after dosing. Butyl 

10 palmitate and butyl stearate were detected in the livers of all 12 rats sacrificed after 1 hour, while

11 butyl oleate was detected in only 5 out of 12 rats. Of the four rats sacrificed after 6 hours, butyl 

12 palmitate was detected in all, butyl stearate was detected in three, and butyl oleate was not 

13 detected in any. In the in vitro study, homogenized liver, lung, and pancreas from rats and 

rabbits were incubated with [14 14 C]-oleic acid (0.4 mM) and 0.2 M n-butanol for 45 minutes. 

15 Esterified oleic acid was isolated from the mixture and radioactivity in the isolate was measured. 

16 In rat liver, lung, and pancreas, the esterified oleic acid content was 271.8, 58.3, and 2,170 

17 nmol/g tissue/hour, respectively. Lower levels were observed in rabbit liver, lung, and pancreas: 

45.8, 33.5, and 487 nmol esterified [14 18 C]-oleic acid/g tissue/hour, respectively. These studies 

19 demonstrate that n-butanol, like ethanol, can esterify fatty acids. 

20 McKarns et al. (1997) observed a correlation between the octanol:water partition 

21 coefficient, a measure of hydrophobicity, and release of LDH from rat liver epithelial cells 

22 treated in vitro with short-chain aliphatic alcohols. Cultures of WB rat hepatic epithelial cells 

23 were incubated with one of several alcohols (including n-butanol, 99.5% pure) for 1-hour 

24 durations at various concentrations. The concentration of LDH in the medium was assessed at 

25 the conclusion of exposure as a measure hepatotoxicity. n-Butanol was tested at concentrations 

26 between 0.05 and 0.2 M to establish a concentration-response curve for the purpose of estimating 

27 the LDH50 (concentration eliciting a 50% increase in LDH over control values) and EC50

28 (concentration eliciting 50% of maximum LDH release). LDH50 and EC50 were plotted against 

29 previously reported partition coefficients. The LDH50 and EC50 for release of LDH by n-butanol 

30 were both 0.16 M. The authors observed a strong positive correlation between release of LDH 

31 and octanol:water partitioning (r = 0.993) and derived QSAR equations for both LDH50 and EC50

32 that were nearly identical. The study authors postulated that the observed relationship implies 

33 that the alcohols’ ability to disrupt membrane integrity is nonspecific, as it is not affected by 

34 molecular shape. 

35 Deters et al. (1998a) demonstrated that, unlike ethanol, metabolism of n-butanol by ADH 

36 was not a necessary step in the hepatotoxic action of this compound. The study authors used 

37 4-MP to inhibit ADH in isolated perfused rat livers (from male Wistar rats) exposed to various 

38 alcohols at 130.2 mmol/L. Liver damage was assessed by measuring leakage of GPT, LDH, and 
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1 GLDH into the perfusate and by measuring oxygen consumption, bile secretion, perfusion flow, 

2 and concentrations of ATP and GSH in the liver. Exposure to n-butanol alone resulted in a 

3 statistically significant change from control for all of the above parameters with the exception of 

4 GSH content of the liver. Exposure of the isolated livers to ethanol in the presence of 4-MP 

5 resulted in little or no evidence of liver injury; bile secretion was diminished, but other 

6 parameters were attenuated by 4-MP. In contrast, there were no differences between the effects 

7 observed in livers treated with n-butanol with or without 4-MP in the perfusate. This study 

8 demonstrates that the hepatotoxicity of n-butanol may not be related to metabolism by ADH. 

9 Deters et al. (1998b) examined whether glycine, which had previously been shown to 

10 reduce hypoxia-related effects on the liver, would mitigate the hepatotoxic effects of n-butanol 

11 and several other liver toxicants. Using the isolated perfused liver system described earlier, and 

12 the same measures of hepatotoxicity, the study authors tested n-butanol with and without 

13 glycine. Exposure of the isolated livers to n-butanol resulted in statistically significantly 

14 increased release of GPT and LDH into the perfusate, decreased bile flow, decreased oxygen 

15 consumption and hepatic ATP content, decreased GSH in the liver, and increased levels of MDA 

16 in the liver. Deters et al. (1998b) observed that inclusion of glycine reduced the release of GPT 

17 and LDH into the perfusate; levels with glycine were similar to those observed in the control 

18 systems. In addition, when glycine was included, the oxygen consumption in the liver was 

19 increased compared with the n-butanol treatment without glycine; oxygen consumption in the 

20 livers treated with both n-butanol and glycine was similar to that of controls. Finally, glycine 

21 modulated the effect of n-butanol on GSH and MDA content in the liver; levels of both were 

22 similar to controls in the livers treated with n-butanol in the presence of glycine. Glycine did not 

23 affect the inhibition of bile flow induced by n-butanol. 

24 Strubelt et al. (1999) assessed evidence of injury to perfused livers treated with 

25 23 aliphatic alcohols including n-butanol. Liver injury was assessed by measuring release of 

26 GPT, LDH, and GLDH into the perfusate; oxygen consumption, bile flow, and perfusion flow; 

27 ATP, GSH, oxidized glutathione (GSSG), and MDA in the liver; and lactate and pyruvate 

28 concentrations in the perfusate. The alcohol exposures all used a concentration of 65.1 mmol/L 

29 added to the perfusate for 120 minutes. Exposure of perfused livers to n-butanol resulted in 

30 statistically significantly increased GPT and LDH (more than eightfold higher than controls for 

31 each) in the perfusate. The study authors reported that the potency of effects on LDH, GPT, and 

32 GLDH was strongly correlated with carbon chain length among straight-chain aliphatic alcohols 

33 (r = 0.82–0.87). In addition, n-butanol exposure resulted in decreased bile flow and perfusion 

34 flow (2 and 57% of control values, respectively), and an increase in the lactate/pyruvate ratio 

35 (increased lactate and decreased pyruvate). Treatment with n-butanol did not result in 

36 statistically significant changes in ATP, GSH, GSSG, or MDA content of the livers. 

37 

38 
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1 4.5.6. Other Mechanistic Studies 

2 Krill et al. (1993) examined the effects of n-butanol, n-propanol, and isopropanol on 

3 thermotropic behavior of the stratum corneum of hairless mice and a model multilamellar vesicle 

4 system. The study was designed to examine the mechanisms for alcohol-related enhancement of 

5 lipophilic solute permeation across the skin. The model vesicle system was prepared from the 

6 phospholipids distearoylphosphatidylcholine and distearoylphosphatidic acid. Stratum corneum 

7 sheets were removed from the abdomen of male hairless mice (strain SKH-HR-1). Fourier 

8 transform infrared spectroscopy was used to measure effects of the perdeuterated alcohols on 

9 stratum corneum lipid alkyl chain packing, mobility, and conformational order. Exposure of 

10 hairless mouse stratum corneum to 3% n-butanol resulted in increased lipid chain freedom of 

11 motion above 45°C and decreased the alkyl chain freedom of motion below 45°C, but did not 

12 alter the stratum corneum lipid interchain interactions or gel-gel phase transition. The study 

13 authors concluded that their experiments were consistent with the hypothesis that alcohols 

14 disrupt the polar head plane and increase the interfacial area of lipids, which enhances 

15 penetration of some solutes across the stratum corneum. 

16 Kowalczyk et al. (1996) exposed two-cell preimplantation mouse embryos in culture to 0, 

17 0.05, 0.1, 0.2, or 1.0% (w/v) n-butanol for 24 hours and followed the development of the 

18 embryos after 5 days. The number of embryos reaching blastocyst stage by day 5 was 

19 statistically significantly reduced at all concentrations of n-butanol. The reductions, which 

20 ranged from ~2 to 35% of control values, were not dose-dependent; the maximum reduction 

21 occurred at the lowest concentration. This result was the opposite of that observed with ethanol, 

22 which accelerates embryogenesis (increased number of blastocytes). In a separate experiment, 8-

23 cell mouse morulae were pretreated with fluo-3-acetoxymethyl ester (a fluorescent indicator for 

24 intracellular calcium) and then exposed to 0.1 or 1.0% n-butanol. Fluorescence intensity was 

25 measured before and immediately after exposure and used to calculate calcium concentration. 

26 n-Butanol exposure statistically significantly increased the intracellular calcium concentration of 

27 the morulae at the high concentration but not at the low concentration (~sixfold increase at 1%). 

28 The study authors concluded that the increased release of calcium was a result of substantial 

29 disruption of membrane integrity and was a possible cause of the embryotoxicity observed in the 

30 first experiment. 

31 Gastaldi et al. (1991) exposed small intestinal microvillous vesicles obtained from adult 

32 Wistar rats to butanol in vitro to assess the effects on vesicular morphology. The study did not 

33 specify whether n-butanol or t-butanol was used. The vesicles were incubated for 30 minutes 

34 with butanol at a concentration of 3% (v/v). After exposure, the vesicles were fixed for 

35 morphometric analysis (parameters were diameter of the area-equivalent circle, minimum to 

36 maximum diameter ratio [shape factor], and vesicular volume) by electron microscopy. A 

37 statistically significant increase in mean vesicular volume was observed with butanol exposure, 

38 and the study authors attributed this change to the fluidizing effect of the alcohol. 
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1 Gordon et al. (1995) compared the effects of several n-alcohols (methanol, ethanol, 

2 n-butanol, and 1-propanol) on isolated cannulated rat intracerebral arterioles (ex vivo). 

3 Concentrations of 1–100 mM of the n-alcohols were tested, and change in arteriolar diameter 

4 was measured. n-Butanol exposure resulted in dose-related increases in vessel diameter 

5 (vasodilation) ranging from ~15% (at 1 mM) to ~50% (at 100 mM) increase over the basal 

6 diameter. 

7 Arsov et al. (2005) exposed erythrocyte ghosts prepared from bovine blood to n-butanol 

8 in an effort to determine the mechanism of n-butanol-induced inhibition of erythrocyte 

9 acetylcholinesterase (AChE) at high concentrations. AChE activity was measured with and 

10 without different concentrations of n-butanol (0.07, 0.15, 0.22, and 0.37 M) and at different 

11 temperatures. At 0.07 M and a temperature of ≥ 310°K, there was no inhibition of AChE; at 

12 higher concentrations, n-butanol inhibited AChE activity in the temperature range tested (up to 

13 320°K). In experiments in which the n-butanol was removed by dialysis after the exposure, 

14 reversibility of AChE inhibition was shown at 0.22 M n-butanol, but at 0.37 M, AChE inhibition 

15 was still evident after the alcohol was removed. The study authors used the experimental results, 

16 along with published information, in kinetics studies to estimate the number of molecules that 

17 bind to the AChE enzyme. They concluded that, since the activity equation that best fit the data 

18 was the inverse of a second order polynomial, two n-butanol molecules likely bind to AChE. 

19 Ding and Badwey (1994) examined the effects of n-butanol on four uncharacterized 

20 protein kinases that are activated by the chemoattractant fMet-Leu-Phe. When exposed to this 

21 chemoattractant, neutrophils exhibit shape changes, chemotaxis, degranulation, and release of 

22 superoxide. The study authors measured the release of superoxide from cultured guinea pig 

23 peritoneal neutrophils treated with 20–70 mM n-butanol and fMet-Leu-Phe (1 μm). n-Butanol 

24 exposure resulted in a concentration-related inhibition of superoxide release (from about 30% 

25 inhibition at 20 mM to about 90% inhibition at 70 mM compared with cells not exposed to 

26 n-butanol). Addition of four β-phorbol 12-myristate 13-acetate (PMA), a PKC activator, to cells 

27 treated with 55 mM n-butanol restimulated the release of superoxide, counteracting the 

28 inhibitory effects of n-butanol. Without PMA, n-butanol at 55 mM resulted in 74% inhibition of 

29 superoxide release, while the same concentration with PMA resulted in only 27% inhibition. 

30 While this study was primarily aimed at elucidating the mechanism of action of the novel protein 

31 kinases, it does suggest that n-butanol exposure may interfere with immune cell signaling. The 

32 potential impact on immune function, however, is uncertain. 

33 

34 4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS 

35 4.6.1. Oral 

36 Information on the effects of n-butanol in humans from oral exposure is limited to one 

37 case report from a suicide attempt (Bunc et al., 2006) where neurological, gastrointestinal, and 
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1 cardiovascular symptoms were noted. In animal studies, there are short-term, subchronic, 

2 reproductive and developmental toxicity studies that reported effects. 

3 Among rats, rabbits, hamsters, and mice, oral LD50s ranged from 1,200 mg/kg to 4,360 

4 mg/kg (Rumyantsev, 1979; Dubina and Maksimov, 1976; Munch, 1972; Purchase, 1969; Jenner 

5 et al., 1964; Smyth et al., 1951; McOmie and Anderson, 1949; Munch and Schwartze, 1925). 

6 Behavioral toxicities following or during an acute exposure included narcosis and necropsies 

7 indicated that there was necrosis of the liver and kidney as well as general organ congestion 

8 (Munch, 1972; Purchase, 1969; Jenner et al., 1964; McOmie and Anderson, 1949; Munch and 

9 Schwartze, 1925). 

10 Short term study evaluations reported neurotoxic, hepatoxic, and antiinflammatory 

11 effects. Acute oral dosages of n-butanol resulted in impaired performance in the tilted plane test 

12 at 1,210 mg/kg in rats (Wallgren, 1960) and dose-related decreases in rotarod performance in 

13 Swiss-Cox mice orally dosed with 1,000 and 2,000 mg/kg (Maickel and Nash, 1985). Liver 

14 toxicity was also noted in a study where decreased thiamine, riboflavin, pyroxidine, niacin, and 

15 panthothenic acid content was noted in rats orally gavaged with 1 or 2 mL/kg (810 or 1,620 

16 mg/kg) n-butanol for 7 days (Shehata and Saad, 1978). In rats, oral doses of ≥ 600 mg/kg 

17 resulted in a reduction of inflammatory response when treated with carageenan, a pro18 inflammatory agent (Strubelt and Zetler, 1980). 

19 CNS effects including hypoactivity and ataxia (RTI; unpublished, 1985) and hepatic and 

20 renal effects (Munoz et al., 1991; Wakabayashi et al., 1991; Munoz et al., 1990) were reported in 

21 three subchronic studies. Table 4-11 summarizes these studies. In the 13 week oral gavage 

22 study in rats (RTI; unpublished, 1985), the 500 mg/kg-day dose (highest in the study) resulted in 

23 hypoactivity and ataxia that lasted for up to one hour after dosing. Statistically significant 

24 decreases in the hematocrit, erythrocyte count, and hemoglobin content were also reported for 

25 females dosed with 500 mg/kg-day and there was a statistically significant increase (~14%) in 

26 thyroid weight in males dosed with 500 mg/kg-day. Male rats exposed to 4,400 mg/kg-day for 4 

27 months in a drinking water study (Munoz et al.,1991; 1990) were reported to have severe 

28 pathological changes in the liver and kidney. Similarly, hepatic effects such as proliferation of 

29 smooth endoplasmic reticulum and enlarged mitochondria in a 3 month drinking water study 

30 with male rats exposed to 8,200 mg/kg-day (Wakabayashi et al., 1991). 

31 There were two oral developmental toxicity studies, Sitarek et al. (1994) and Ema et al., 

32 (2005), that are summarized in Table 4-13. Sitarek et al (1994) reported an increased incidence 

33 of visceral malformations including malformations in the brain (dilation of the lateral and/or 

34 third ventricle of the brain and/or arachnoid space) and kidney (renal pelvis) in the offspring of 

35 female Wistar Imp:DAK rats treated with n-butanol in drinking water at a dose of 300 mg/kg36 day administered for 8 weeks prior to mating and throughout mating and gestation. Maternal 

37 effects were not observed at doses up to 5,000 mg/kg-day by Sitarek et al. (1994). In another 

38 developmental study, Ema et al. (2005) did not observe an increased incidence of any 
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1 malformation in Sprague-Dawley rats exposed to n-butanol in drinking water throughout 

2 gestation at doses up to 5,654 mg/kg-day. Maternal and fetal toxicity, manifested as body weight 

3 reductions, were observed at the high-dose in this study (5,654 mg/kg-day) (Ema et al., 2005). 

4 The discrepancy between the results of Ema et al. (2005) and Sitarek et al. (1994) may be due to 

5 differences in study design, particularly rat strain used and exposure regimens. In addition, 

6 dilation of the subarachnoid spaces and the ventricles in the brain are precursors of 

7 hydrocephalus (Raimondi, 1994). Sitarek et al. (1994) reported internal hydrocephalus in the 

8 fetuses from the 1,000 and 5,000 mg/kg-day dose groups along with the observed increased 

9 incidence of visceral malformations including malformations in the brain (dilation of the lateral 

10 and/or third ventricle of the brain and/or arachnoid space) at ≥ 300 mg/kg-day. Observation of 

11 hydrocephaly and dilation of neuronal structures (subarachnoid space and lateral ventricle) as 

12 reported in Sitarek et al (1994) is also consistent with effects observed with ethanol (Sakata13 Haga et al., 2004). Therefore, the discrepancies in results between the developmental toxicity 

14 studies of Sitarek et al (1994) and Ema et al (2005) is likely due to differences in strain 

15 sensitivity. 

16 Overall, the most sensitive toxicological effect was reported in Sitarek et al (1994) where 

17 increased developmental visceral malformations were observed in rat pups at the 300 mg/kg-day 

18 dose. At comparable exposures, neurotoxicological effects were observed in adult rats at 500 

19 mg/kg-day and no effects were observed at 125 mg/kg-day (RTI; unpublished, 1985). 
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Table 4-13. Summary of oral noncancer dose-response information for n-butanol 

Species and 

study type Exposure 

NOAEL 

(mg/kg-d) 

LOAEL 

(mg/kg-d) Responses at the LOAEL Comments 

Subchronic or chronic studies 

Rat strain not specified 30/sex/dose Gavage 

0, 20, 125, or 500 mg/kg-d daily for 13 wks 

125 500 Clinical signs (hypoactivity 

and ataxia) and effects on hematology (reduced hematocrit, erythrocyte count, and hemoglobin content) 

 

Wistar rat 15 males/group drinking water 

0 or ~8,200 mg/kg-d for up to 3 mo 

Could not be determined 

Could not be determined 

Structural changes of hepatic mitochondria 

Data not reported. Evaluation limited to clinsigns, body weight, and electron microscopy of livslices. 

Wistar rat 3 males/group drinking water 

0 or 4,400 mg/kg-d, 7 d/wk for 4 mo 

Could not be determined 

Could not be determined 

Unspecified liver and kidney damage; serum chemistry changes 

Pathology data not reportEvaluations limited to mortality, body weight, serum chemistry, and unspecified pathology evaluations. 

Reproductive and developmental studies 

Wistar Imp:DAK rat 11–17 females/group drinking water 

0, 300, 1,000, or 5,000 mg/kg-d for 8 wks prior to mating, through mating and gestation 

5,000 (maternal) NA (developmental)

NA (maternal) 300 (developmental) 

Increased incidences of malformations (dilation of the lateral and/or third ventricle of the brain and the subarachnoid space) 

No maternal effects. 

Sprague-Dawley rat 20 females/groupdrinking water 

0, 316, 1,454, or 5,654 mg/kg-d on GDs 0–20 

1,452 (maternal and developmental) 

5,654 (maternal and developmental) 

Decreased body weight gain, food consumption, and water consumption (maternal); decreased fetal body weight (developmental) 

 
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1 4.6.2. Inhalation 

2 The inhalation database contains a few acute human exposure experiments and occupational 

3 health studies. Controlled human exposure experiments have demonstrated that acute exposures to 

4 n-butanol vapors can exert an irritant effect on the eyes, nose, and throat under acute exposure 

5 conditions of up to 90 minutes (Kjaerguard et al., 1997; Nelson et al., 1943; Wysocki et al., 1996, as

6 cited in McLain, 2008 and ACGIH, 2002; Cometto-Muniz and Cain, 1995). A number of occupational 

7 health studies have also been conducted which examined neurological and neurobehavioral effects in 

8 workers who were exposed to mixtures of solvents including n-butanol (see Table 4-2). However, 

9 exposure to n-butanol in these studies was low compared to other solvents, and a number of the other 

10 solvents are known to exert neurotoxic effects. Therefore, it is not possible to conclusively attribute any 

11 of the effects to n-butanol exposure. Table 4-14 summarizes the available human occupational data. 
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1 

Table 4-14. Summary of inhalation noncancer dose-response information for n-butanol 

Species and study 

type (n/sex/group) Exposure

NOAEL 

(mg/m3) 

LOAEL 

(mg/m3) Responses at the LOAEL Comments 

Human occupational health studies 

35 rubber factory workers; gender not specified 

15–300 mg/m3 15–42 46–200 Eye irritation LOAEL identified for facto

coexposure to diacetone alc

16 male photographic paper factory workers

 300 or 600 mg/m3 300 600 Eye irritation Irritation not observed after 

concentration reduced from 300 mg/m3. 

11 exposed cellulose acetate factory workers; sex not specified 47 workers in referent group with similar age and duration of employment 

240 mg/m3 NA 240 Hearing loss, chronic 

bronchitis 

Sampling protocol for n-butnot described. 

Subchronic or chronic animal studies 

Wistar rat 12 male/exposure; 24 controls 

0, 154, or 308 mg/m3

6 hr/d, 5 d/wk for 3 months 

154 308 Impaired rotarod 

performance; increased leukocyte count 

Histopathology not evaluate

Guinea pig 3 group; sex unspecified 

0, 300 mg/m3

3 months 

N/A 300 Decreased erythrocyte count 

and hemoglobin. Histopathological changes in liver and kidney 

Data and methodological denot provided for the observe

Mice and rats No details provided for number/exposure, sex, or methods 

0, 0.8, 6.6, 40 mg/m3 

 4 months 

0.8 6.6 Mice: Increased thyroid 

gland activity. Disturbances of pituitary-adrenal system. Rats: Increased blood cholinesterase activity. 

Only list of effects were prothe report. No detailed infowas provided on the analysedata. 
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Table 4-14. Summary of inhalation noncancer dose-response information for n-butanol 

Species and study 

type (n/sex/group) Exposure

NOAEL 

(mg/m3) 

LOAEL 

(mg/m3) Responses at the LOAEL Comments 

Reproductive/developmental animal studies 

Sprague-Dawley rat;15–20 females/group 

0, 11,000, 18,000, or 24,000 mg/m3

7 hrs/d on GDs 1–19 

11,000 (maternal) NA (developmental) 

18,000 (maternal) 11,000 (developmental) 

Reduced food consumption (maternal) Increased incidence of litters with skeletal malformations (developmental) 

Maternal narcosis and mortobserved at 24,000 mg/m3 (

Sprague-Dawley rat; 15–20 females and 18 males/group 

0, 9,000, 18,000 mg/m3

7 h/d on GDs 1–19 (females) 

or for 6 wks prior to mating with nonexposed females (males) 

18,000 NA None. No neurobehavioral effects 

offspring, regardless of whemothers or fathers exposed. 
B-1.

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1 

2 Three studies of n-butanol exposure in rats are available including one subchronic toxicity study 

3 of male rats in which histopathology was not assessed (Korsak et al., 1994), a developmental toxicity 

4 study (Nelson et al., 1989a), and a neurodevelopmental toxicity study (Nelson et al., 1989b). In 

5 addition, several short-term inhalation exposure studies have characterized toxicities associated with n6 butanol. 

7 Specifically, fatty infiltration of liver and kidney were observed in mice exposed to 24,624 

mg/m38 for 130 hours (Weese, 1928). Neurotoxic effects such as decreased duration of immobility were 

reported at exposures of 1420 mg/m39 and higher for 4 hours in mice (DeCeaurriz et al., 1983), and 

10 decreased responding (Frantik et al., 1994) in rats and mice following a 2-4 hour exposure. Respiration 

rates declined in Ssc:CF-1 mice exposed to exposure of 435 ppm (1,319 mg/m311 ) and higher for 30 

12 minutes (Kristiansen et al., 1988) as well as in balb/C mice with a 50% respiratory decline following 4 

hour exposures to 3,008 ppm (9119 mg/m3; Korsak et al., 1993) or 4,300 ppm (13036 mg/m313 ; Korsak 

14 and Rydzynski, 1994). 

15 There are a few subchronic inhalation toxicity studies evaluating the effects of n-butanol (Korsak 

16 et al., 1994; Rumanystev et al., 1979; Smyth and Smyth, 1928). Hematological effects, primarily 

17 decreased erythrocyte count, were noted in all three subchronic studies. The lowest exposure at which

hematological changes were observed was at 6.6 mg/m318 in rats where increased blood cholinesterase 

19 activity was noted (Rumyantsev et al., 1979). However, it is unclear if the hematological changes are

20 biologically relevant. The other primary toxicological effect observed in the subchronic exposure 

21 studies was deficits in neurobehavioral performance. Korsak et al (1994) reported a statistically 

significant increase in the failure rate for the rotarod test in rats exposed to 308 mg/m322 n-butanol for 3 

23 months. 

24 Nelson et al (1989a, b) evaluated the developmental and neurodevelopmental toxicity potential 

25 of n-butanol in rats. Increased incidence of litters with skeletal variation was observed at exposures of 

3,500 ppm (11,000 mg/m326 ) and increased levels of serotonin and dopamine were observed in offspring 

from male rats exposed to 5,000 ppm (18,000 mg/m327 ) without changes in neurobehavioral function. 

28 The most sensitive effects in humans identified from a sparse literature were eye, nose, and 

throat irritation following a 90 minute chamber exposure to 10 mg/m329 n-butanol (Kjaerguard et al., 

1997). In animals, hematological changes were observed at exposures as low as 6.6 mg/m330 for 4 months 

31 (Rumanystev et al., 1979), but it is unknown if these changes were biologically significant. The 

statistically significant change in neurological function following a subchronic exposure to 308 mg/m332 

33 n-butanol in rats (Korsak et al., 1994) is the most sensitive biologically significant endpoint from 

34 inhalation following exposure durations that are subchronic or longer. 

35 

36 
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1 4.6.3. Mode of Action Information for Noncancer Endpoints 

2 The mode of action for noncancer endpoints following n-butanol exposure is unknown. 

3 However, because of its structural relationship to ethanol, n-butanol has been tested in a large number of 

4 studies examining possible mechanisms for alcohol-induced neurotoxicity. One proposed mechanism is 

5 that alcohols including n-butanol produced the observed neurological changes by disrupting the lipid 

6 bilayer. A few studies have shown that n-butanol, like other alcohols, can disrupt membrane integrity

7 (Kowalczyk et al., 1996; Krill et al., 1993; Gastaldi et al., 1991). Other studies have found that n8 butanol inhibits the excitatory glutamate receptor function (Akinshola, 2001; Peoples and Weight, 

9 1999; Dildy-Mayfield et al., 1996; Lovinger et al., 1989) and potentiates inhibitory receptors such as

10 glycine and gamma-aminobutyric acid (GABA) (Mascia et al., 2000; Peoples and Weight, 1999; Ye et 

11 al., 1998; Dildy-Mayfield et al., 1996; Nakahiro et al., 1991). The modulatory action of n-butanol 

12 inhibiting the excitatory glutamate receptors and potentiating the inhibitory GABA and glycine receptors 

13 is supportive of the observed neurobehavioral changes (e.g. CNS depressant profile) associated with n14 butanol exposure in humans (Seitz, 1972; Baikov and Khachaturyan, 1972; Tabershaw et al., 1944; 

15 Nelson et al., 1943) and animals (Korsak et al., 1994; RTI; unpublished, 1985). Application of n16 butanol (11 – 22 mM) also potentiated the serotonin (or 5-HT [5 hydroxytryptamine]) current (Rusch et 

17 al., 2007; Stevens et al., 2005; Zhou et al., 1998). 5HT3 receptors are known to regulate dopamine 

18 release and increased dopamine levels are associated with reward mechanisms. Nelson et al (1990; 

19 1989b) found that pups exposed to n-butanol in utero had statistically significant increases in brain 

20 levels of dopamine and serotonin which could be indirectly linked to n-butanol effects on the 5HT3 

21 channel. Collectively, these studies suggest possible modes of action, via effects on these ion channels, 

22 for anesthetic effects and possibly other subtle neurological effects of n-butanol. 

23 In addition, studies have shown that n-butanol inhibits rat brain astroglial cell proliferation by 

24 disrupting the PLD signaling pathway (Kotter et al., 2000; Kotter and Klein, 1999). n-Butanol was 

25 shown to be a good substrate for transphosphatidylation, resulting in the formation of 

26 phosphatidylbutanol and concomitantly decreasing the formation of the second messenger PA. 

27 Importantly, the authors demonstrated that n-butanol is substantially more potent than ethanol in the 

28 inhibition of astroglial cell proliferation, which has been postulated as a mode of action for ethanol29 induced microencephaly and mental retardation observed in cases of fetal alcohol syndrome. 

30 There is more limited information on possible modes of n-butanol-induced liver effects. Carlson 

31 (1994a, b) showed that n-butanol can esterify fatty acids in the liver and several other organs; fatty aid 

32 ethyl esters observed after ethanol exposure have been postulated to play a role in damage to several 

33 organs. Deters et al. (1998a) demonstrated that metabolism of n-butanol by ADH was not a necessary 

34 step in the hepatotoxic action of this compound. The same authors observed mitigation of several, but

35 not all in vitro measures of hepatotoxicity when glycine was added to isolated perfused liver systems 
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1 along with n-butanol (Deters et al., 1998b); glycine had previously been shown to reduce hypoxia2 related effects on the liver. 

3 

4 4.7. EVALUATION OF CARCINOGENICITY 

5 4.7.1. Summary of Overall Weight-of-Evidence 

6 Under the EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), there is 

7 inadequate information to assess the carcinogenic potential of n-butanol. None of the available human 

8 occupational health studies evaluated cancer endpoints. One occupational health study of workers 

9 exposed to n-butanol and several other solvents while employed as dockyard painters (Chen et al., 1999) 

10 examined mortality rates due to cancer; neither proportional mortality nor standardized mortality due to 

11 cancers were increased in the painters relative to controls. There are no chronic animal studies of 

12 n-butanol exposure through any exposure route, and the available subchronic studies did not report any

13 cancer endpoints. The genotoxicity database for n-butanol is limited; available information suggests that 

14 n-butanol does not induce mutations in S. typhimurium with or without exogenous metabolic activation 

15 (Muller et al., 1993; Jung et al., 1992; Nakamura et al., 1987; Connor et al., 1985; McCann et al., 1975), 

16 and does not increase the frequency of micronuclei or sister chromatid exchanges in Chinese hamster 

17 cells (Lasne et al., 1984; Obe et al., 1977) or in chick embryos in culture (Bloom, 1982). Studies of

18 DNA and RNA replication have shown that n-butanol can exert an inhibitory effect on these processes 

19 (Obe and Ristow, 1977; Yoshiyama et al., 1973). 

20 

21 4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 

22 4.8.1. Possible Childhood Susceptibility 

23 There are no studies of age-related differences in susceptibility to the toxic effects of n-butanol. 

24 In a developmental toxicity study of oral exposure to n-butanol before and during gestation using Wistar 

25 Imp:DAK rats, increased incidences of some malformations (primarily dilation of the subarachnoid 

26 space and/or the lateral or third ventricle of the brain) were reported (Sitarek et al., 1994), suggesting 

27 that the developing fetus may be particularly susceptible to the effects of n-butanol exposure. However, 

28 in a study using a different strain of rat (Sprague-Dawley) and a exposure scenario (gestation only), Ema 

29 et al., 2005) did not observe neurodevelopmental effects. Differences in strain susceptibility and 

30 exposure regimen may address this discrepancy (see Section 5.1.1.). Inhalation studies of n-butanol 

31 (Nelson et al., 1989a) reported maternal toxicity (reduced food consumption and/or body weight gain) at 

32 concentrations resulting in fetal effects (primarily reduced body weight). A single study of behavioral 

33 teratology in offspring of rats exposed to n-butanol (Nelson et al., 1989b) did not observe treatment34 related neurobehavioral changes. 

35 Studies have demonstrated lifestage specific differences in activitiy in the enzymes responsible 

36 for metabolism of n-butanol. Specifically, in humans, there is evidence of differential expression for 
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1 three types of alcohol dehydrogenase (type I) enzymes: ADH1A, ADH1B, and ADH1C over different 

2 lifestages (Smith et al., 1971). Expression of the ADH enzymes was examined in the liver, lung, and 

3 kidney in 222 humans from 9 weeks gestation to up to 20 years of age. In the liver, it was reported that 

4 at 11 weeks of gestation ADH1A was preferentially expressed and ADH1B and ADH1C were 

5 nondetectable. However, in adults, ADH1A is no longer detectable and there is equal expression of 

6 ADH1B and ADH1C. ADH expression was considerably lower in the lung and kidney and expression 

7 did not change with the lifestages. Studies in mice, rats, and guinea pigs have also reported significantly 

8 lower alcohol and aldehyde dehydrogenase activity in fetal animals (ranging from 10 – 40% of adult 

9 activity) in comparison to the adults (Timms and Holmes, 1981; Card et al., 1989; Lindahl, 1977; 

10 Boleda et al., 1992). The lower enzymatic activity of alcohol and aldehyde dehydrogenase during 

11 development and childhood could potentially impact the metabolic capacity of n-butanol and may make 

12 this population more susceptible to this chemical agent. 

13 

14 4.8.2. Possible Gender Differences 

15 There are no studies of gender differences in susceptibility to the toxic effects of n-butanol, and 

16 available information does not suggest gender differences. 

17 
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1 5. DOSE-RESPONSE ASSESSMENTS 

2 

3 

4 5.1. ORAL REFERENCE DOSE (RfD) 

5 5.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification 

6 Data on the health effects of oral exposure to n-butanol in humans is limited to one case report 

7 from a suicide attempt (Bunc et al., 2006) in which neurological, gastrointestinal, and cardiovascular

8 symptoms were noted. This case study is not suitable for use in deriving an RfD. The animal 

9 toxicological database for subchronic, chronic, and reproductive or developmental studies of n-butanol

10 consists of three subchronic studies, Munoz et al. (1991; 1990), Wakabayashi et al. (1991), and RTI 

11 (1985), and two developmental toxicity studies, Ema et al. (2005) and Sitarek et al. (1994) where female 

12 reproductive parameters were also evaluated. 

13 Figure 5-1 is an exposure-response array for oral exposure to n-butanol. Overall, the most 

14 sensitive toxicological effect was reported in the developmental toxicity study by Sitarek et al (1994), in 

15 which increased incidences of developmental visceral malformations were observed in rat litters at ≥

16 300 mg/kg-day. Sitarek et al. (1994) reported increased incidences of developmental visceral 

17 malformations (dilation of the lateral ventricle, dilation of subarachnoid space in the brain and dilation 

18 of the renal pelvis) in the litters of female Wistar Imp:DAK rats treated with n-butanol in drinking water 

19 at doses of 300, 1,000, and 5,000 mg/kg-day administered for 8 weeks prior to mating and then 

20 continued through mating and gestation. 

21 Neurotoxicological effects (hypoactivity and ataxia) were also observed in adult rats at a similar 

22 dose of 500 mg/kg-day (RTI; unpublished, 1985). However, these neurotoxicological effects were not 

23 considered as sensitive as the developmental visceral malformations because the changes were transient

24 and not observed within an hour of exposure as noted by the study authors. Additionally, the reported

25 LOAEL was higher for the neurotoxicological effects observed in RTI (1985) than for the 

26 developmental effects noted in Sitarek et al (1994). 

27 In a second developmental toxicity study, Ema et al. (2005) did not observe an increased 

28 incidence of any malformation in the offspring of Sprague-Dawley rats exposed to n-butanol in drinking

29 water throughout gestation (GD 0-20) at doses of 316, 1,454, and 5,654 mg/kg-day. However, maternal 

30 and fetal toxicity, manifested as body weight reductions, were observed at the high dose in this study

31 (5,654 mg/kg-day) (Ema et al., 2005). The discrepancy between the developmental studies is uncertain,

32 but may be due to differences in experimental protocols particularly strain differences and exposure 

33 regimens (see Section 5.3.). 

34 

35 
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1 Sitarek et al (1994) was selected as the principal study because it was a well-designed 

2 developmental toxicity study with a sufficient number of dams and litters per dose group. Three dose 

3 groups and an untreated control group were included in the study, and there was extensive evaluation of 

4 fetal parameters in the litters. There was a statistically significant increase in the incidence of litters 

5 with developmental visceral malformations at all dose groups (lateral ventricle and subarachnoid space

6 in the brain; unilateral and bilateral renal pelvis) (Table 5-1). With respect to the malformations 

7 (dilation) observed in the brain, this effect may be irreversible since there is a link between lateral 

8 ventricular dilation and neurodevelopmental delay, which was reported in a retrospective epidemiology 

9 study of fetal ventriculomegaly in 29,000 pregnancies (Weichert et al., 2010). In addition, dilation of 

10 the subarachnoid spaces and the ventricles in the brain are observed in hydrocephalus (Raimondi, 1994), 

11 and Sitarek et al. (1994) also reported internal hydrocephalus in the fetuses from the 1,000 and 5,000

12 mg/kg-day dose groups. Observation of hydrocephaly and dilation of neuronal structures (subarachnoid 

13 space and lateral ventricle) as reported in Sitarek et al (1994) is also consistent with ethanol (Sakata14 Haga et al., 2004). The functional significance of dilation of the lateral ventricles and subarachnoid 

15 space appears to be related to the rate and severity of the dilation, as well as the developmental stage at 

16 which it occurs (Weichert et al., 2010; Del Bigio, 2001). Sitarek et al. (1994) did not provide any 

17 indication of the severity of the dilation of the lateral ventricles and subarachnoid space in the affected 

18 fetuses or litters, and the only information provided by the investigators was a characterization of the 

19 changes at the mid and high doses, 1,000 and 5,000 mg/kg-day, respectively, as frank hydrocephalus. In 

20 the absence of severity characterization, and given the observation of hydrocephalus at higher doses, the 

21 increased litter incidences of visceral malformations (dilation of subarachnoid space, lateral ventricle, 

22 unilateral and bilateral renal pelvis) that were observed in the low-dose group (300 mg/kg-day) were 

23 considered to be biologically significant and were selected as the critical effect. 

24 
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1 2 Figure 5-1. Exposure-response array for oral exposure to n-butanol. 
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1 5.1.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) 

2 A PBPK model for n-butanol is available (Teeguarden et al., 2005); however, this model 

3 was not suitable for use in deriving the RfD. The principal study used for the RfD derivation 

4 (Sitarek et al., 1994) exposed rats before and during gestation, and fetal effects were the most 

5 sensitive endpoint. The available PBPK model does not simulate oral exposure, precluding 

6 estimation of the relevant internal dose metric for this study. 

7 BMD modeling was conducted using the EPA’s benchmark dose software (BMDS, 

8 version 2.1.1.) to analyze the developmental visceral malformation associated with exposure to 

9 n-butanol (see Appendix B for modeling details). The software was used to calculate potential 

10 PODs for deriving the RfD by estimating the effective dose at a specified level of response 

11 (BMDx) and its 95% lower bound (BMDLx). Since internal doses could not be estimated using a 

12 PBPK model, administered doses were used to derive a BMDL. 

13 In the study by Sitarek et al. (1994), a LOAEL of 300 mg/kg-day was identified by EPA 

14 based on increased litter incidences of visceral malformations (dilation of subarachnoid space, 

15 lateral ventricle, unilateral and bilateral renal pelvis). A NOAEL was not identified for this 

16 study. These teratogenic effects could not be modeled using nested developmental toxicity dose17 response models, as these models require individual (i.e., litter-specific) animal data which were 

18 not reported by Sitarek et al. (1994). The incidences of litters affected with visceral 

19 malformations were reported in the study (see Table 5-1). Although individual data were 

20 provided for dilation of the parameters in the brain (lateral ventricle and subarachnoid space) and 

21 was more sensitive that the kidney parameters (unilateral and bilateral renal pelvis), the 

22 combined data for the dilation incidences in the brain would provide a better estimate of the 

23 overall developmental toxicity associated with n-butanol since dilation of subarachnoid space 

24 and the lateral ventricle are reported precursors of hydrocephalus (Raimondi, 1994). However, 

25 combined incidences of dilation in the fetal brain via the subarachnoid space or lateral ventricle 

26 could not be separated from the kidney endpoints since the incidences were not reported 

27 independent of each other. For example, in the 500 mg/kg-day group it was noted that 2 out of 

28 14 litters had dilation of the subarachnoid space, 8 out of 14 had dilation of the lateral ventricle 

29 but 9 out of 14 litters were scored as having overall visceral malformations per litter (see Table 

30 5-1). Therefore, overall incidence of visceral malformations was used for BMD modeling to 

31 provide a better dose response estimate of developmental toxicity associated with n-butanol. 

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 ventricle 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) – overall 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 ventricle 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 

2 All available dichotomous models in the EPA Benchmark Dose Software (BMDS, 

3 v. 2.1.1) were fit to the datasets for the increased incidences of visceral malformations (dilation 

4 of subarachnoid space, lateral ventricle, unilateral and bilateral renal pelvis). Table 5-2 

5 summarizes the BMD modeling results for the overall observed dilation. A benchmark response 

6 (BMR) of 10% was selected from which to derive the point of departure (POD), because the 

7 endpoints were characterized in terms of affected litters for reproductive and developmental 

8 studies when data are available which characterize the incidence of effects within litters. Since 

9 this level of reporting was not available, a nested analysis was not possible. The overall 

10 incidence of affected offspring for these endpoints (not shown here) in the control and low dose 

11 groups indicated that not all offspring in each affected litter were similarly affected, so using a 

12 BMR of 5% extra risk on the basis of affected litters corresponded to a lower overall incidence in 

13 the affected offspring. Thus, a BMR of 10% extra risk among affected litters was employed, in 

14 order to approximate better a 5% extra risk in affected offspring, while recognizing the litter as 

15 the experimental unit for appropriate estimation of confidence intervals. The log-probit model 

16 was selected because it provided the best fit to the data based on: (1) having the lowest Akaike’s 

17 Information Criterion (AIC), and (2) visual inspection of the plot of observed versus expected 

18 values across the fitted models. Appendix B contains a more detailed description of the 

19 modeling and results, along with graphs of the selected best-fit models. 

20 
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Table 5-2. Summary of BMD modeling of results based on Sitarek et al. (1994) 

developmental toxicity data – Dilation in the brain and kidney 

BMD Models AIC BMD10

(mg/kg-d) 

BMDL10

(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 

2 The BMDL10 of 48 mg/kg-day for increased litter incidences of visceral malformations 

3 (dilation of subarachnoid space, lateral ventricle, unilateral and bilateral renal pelvis) was 

4 selected as the POD for derivation of the chronic RfD for n-butanol. 

5 

6 5.1.3. RfD Derivation—Including Application of Uncertainty Factors (UFs) 

7 To derive the RfD, the BMDL10 of 48 mg/kg-day for increased incidence of visceral 

8 malformations was divided by a total UF of 300. The UF of 300 comprises component factors of 

9 10 for interindividual variability, 10 for interspecies extrapolation, and 3 for database 

10 uncertainty, as described below. 

11 An UF of 10 was selected to account for interspecies variability in extrapolation from 

12 laboratory animals (rats) to humans because information is not available to quantitatively assess 

13 toxicokinetic or toxicodynamic differences between animals and humans. 

14 An UF of 10 was selected to account for intraspecies variability in susceptibility to n15 butanol, as quantitative information for evaluating toxicokinetic and toxicodynamic differences 

16 among humans are not available. 

17 An UF of 3 was selected to account for deficiencies in the database, specifically the lack 

18 of a multigeneration reproductive toxicity study. The toxicological database for n-butanol 

19 includes one unpublished subchronic gavage study, two subchronic drinking water studies 

20 evaluating limited toxicological endpoints, and two developmental toxicity studies. One of the 

21 developmental toxicity studies, Sitarek et al. (1994), included 8 weeks of premating exposure 

22 and evaluation of estrous cyclicity and a number of fertility endpoints; however, this study was 

23 not a multi-generation reproductive toxicity study and does not abrogate all concerns with 

24 potential reproductive toxicity. 

25 An UF for extrapolation from a subchronic study to chronic exposure was not applied, 

26 because the POD is from a developmental toxicity study where the critical effect appears to be 

27 more dependent on the exposure concentration during a susceptible lifestage than on the duration 

28 of exposure. 
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1 An UF for extrapolation from a LOAEL to a NOAEL was not applied because the current 

2 approach is to address this factor as one of the considerations in selecting a BMR for BMD 

3 modeling. In this case, a BMR of 10% change in the incidence of dilation of the lateral ventricle 

4 was selected based on the assumption that it represents a minimal biologically significant 

5 change. 

6 The RfD of 0.2 mg/kg-day for n-butanol was calculated as follows: 

7 

8 RfD = BMDL10 ÷ UF 

9 = 48 mg/kg-day ÷ 300 

10 = 0.2 mg/kg-day 

11 

12 

13 5.1.5. Previous RfD Assessment 

14 The previous RfD for n-butanol was posted to the IRIS database on September 1, 1990. 

An RfD of 1 × 10-1 15 mg/kg-day was derived based on hypoactivity and ataxia observed in the 

16 unpublished subchronic study by RTI (1985). A cumulative UF of 1,000 (including 10-fold each 

17 for interindividual variability, interspecies uncertainty, and extrapolation from a subchronic 

18 study) was applied to the NOAEL of 125 mg/kg-day to derive the RfD. Both of the oral 

19 developmental toxicity studies of n-butanol (Ema et al., 2005; Sitarek et al., 1994) were 

20 published after the previous RfD was posted on IRIS.
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1 5.2. INHALATION REFERENCE CONCENTRATION (RfC) 

2 5.2.1. Choice of Principal Study and Critical Effect—with Rationale and Justification 

3 Information on the health effects of humans from inhalation exposure to n-butanol 

4 consists of a few acute human exposure and occupational health studies. Controlled human 

5 exposure experiments have demonstrated that n-butanol vapors can exert an irritant effect on the 

eyes, nose, and throat under acute exposure conditions at air concentrations ≥ 10 mg/m36 

7 (Kjaerguard et al., 1997; Nelson et al., 1943). Occupational health studies in which the primary 

exposure was to n-butanol reported effects including eye irritation (at 46 to 200 mg/m38 ) (Cogan 

et al., 1945; Tabershaw et al., 1944) and hearing loss (at 240 mg/m39 ) (Velazquez et al., 1969). 

10 Lack of methodological detail prevents evaluation of the adequacy of the exposure assessments 

11 used in all of these studies (Sterner et al., 1949; Cogan et al., 1945; Tabershaw et al., 1944). The 

12 eye irritation effects reported by Cogan and colleagues may also have been caused by other 

13 chemicals, however, prevalence also was reported among workers in other plants where n14 butanol was the only solvent exposure (Tabershaw et al., 1944). The high prevalence of hearing 

15 loss observed among n-butanol exposed workers is compelling (Velazquez et al., 1969), but the 

16 study has limitations which present uncertainty for its use in the quantification of risk, including 

17 the small number of exposed workers (affects precision of the prevalence estimate) and lack of 

18 methodological detail (e.g., sampling protocol, selection of referent group). 

19 The animal toxicological database for inhalation exposure to n-butanol includes three 

20 subchronic exposure studies (Korsak et al., 1994; Rumanystev et al., 1979; Smyth and Smyth, 

21 1928), a developmental toxicity study (Nelson et al., 1989a), and a neurodevelopmental study 

22 (Nelson et al., 1989b). There are no chronic exposure studies or experiments evaluating 

23 reproductive toxicity. 

24 Several short-term inhalation exposure studies that have characterized toxicities 

25 associated with n-butanol are also available. Fatty infiltration was observed in the liver and 

kidney in mice exposed to n-butanol air concentrations of 24,624 mg/m326 for 130 hours (Weese, 

27 1928). Neurotoxic effects, such as decreased response (Frantik et al., 1994) were reported in rats 

28 and mice following a 2-4 hour exposure. Respiration rates declined in Ssc:CF-1 mice exposed to 

29 air concentrations of 435 ppm and higher for 30 minutes (Kristiansen et al., 1988), as well as in 

30 balb/C mice, who experienced a 50% respiratory decline following 4-hour exposures to air 

31 concentrations of 3,008 ppm (Korsak et al., 1993) or 4,300 ppm (Korsak and Rydzynski, 1994). 

32 In the three subchronic studies, hematological effects, primarily decreased erythrocyte 

33 counts, were noted (Korsak et al., 1994; Rumanystev et al., 1979; Smyth and Smyth, 1928). The 

lowest concentration at which hematological changes were observed was at 6.6 mg/m334 in rats 

35 where increased blood cholinesterase activity was seen (Rumyantsev et al., 1979). However, it is 

36 unclear if the hematological changes observed following exposure to n-butanol are biologically 

37 relevant. The other primary toxicological effect observed in the subchronic exposure studies was 

38 deficits in neurobehavioral performance. Korsak et al (1994) reported a statistically significant 
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increase in the failure rate for the rotarod test in rats exposed to 308 mg/m31 n-butanol for 3 

2 months. Of the three subchronic toxicity studies, only Korsak et al (1994) reported the observed 

3 raw data. The other two studies (Rumanystev et al., 1979; Smyth and Smyth, 1928) only 

4 indicated statistically significant effects without reporting the raw data. Therefore, these two 

5 studies were not amenable to dose-response analysis. 

6 Nelson et al. (1989a,b) evaluated the potential developmental and neurodevelopmental 

7 toxicity of n-butanol in rats. Increased incidence of litters with skeletal variation was observed at

concentrations of 3,500 ppm (11,000 mg/m38 ) and increased levels of serotonin and dopamine 

were observed in offspring from male rats exposed to 5,000 ppm (18,000 mg/m39 ) without 

10 changes in neurobehavioral function. 

11 Figure 5-2 is an exposure-response array for inhalation exposure to n-butanol for select 

12 animal and human studies employing repeated exposure and reporting the most sensitive and/or 

13 relevant toxicological effects. The most sensitive effects were reported in the human 

14 occupational studies (Velazquez et al., 1969; Sterner et al., 1949) and in an animal subchronic 

15 inhalation study (Korsak et al., 1994). The Nelson et al. (1989a,b) developmental toxicity 

16 studies identified effects at concentrations several orders of magnitude higher. As mentioned 

17 earlier, although sensitive effects were noted in the human studies, the uncertainties presented by 

18 the lack of detail in reporting the assessment of exposure and sparse discussion of study design 

19 preclude their use as principal studies. 

20 Korsak et al (1994) was selected as the principal study for derivation of the RfC because 

21 it was a well-designed study and reported sensitive toxicological effects. Additionally, there was 

22 evaluation of the body and organ weights, measurement of biochemical and hematological 

23 parameters, and performance of neurological testing. In the study, two statistically significant 

24 effects, decreased hemoglobin and increased lipid peroxidation, were reported at the lowest 

concentration of 154 mg/m325 . The decreased hemoglobin levels were within historical levels 

26 reported for male Wistar rats (Giknis and Clifford, 2008) and hematocrit levels were not 

27 changed. The biological relevance of the observed increased hepatic lipid peroxidation is 

28 unknown because other hepatic endpoints were not affected in this study. 

At the next highest concentration, 308 mg/m329 , there were statistically significant 

30 increases in white blood cell counts and impairments in learned avoidance behavior (due to 

31 negative reinforcement for falling off the rod prior to n-butanol exposure) and neuromuscular 

32 function, as measured by the rotarod test (Korsak et al., 1994). Although the increased white 

33 blood cell counts were outside of the historical range reported for male Wistar rats (Giknis and 

34 Clifford, 2008), other associated effects, such as immunotoxicological effects, were not tested in 

35 this study, and have not been observed in other studies, except for in an acute anti-inflammatory 

36 study in Wistar rats, where the WBC count decreased with n-butanol administration (Strubelt and 

37 Zetler, 1980). 
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1 Impaired learned avoidance behavior and neuromuscular function, as measured by 

2 decreased rotarod performance, was selected as the critical effect for RfC derivation. The 

3 selection of this critical effect is supported by other neurotoxic effects (e.g., decreased 

4 immobility in the swim despair test and decreased response) observed in acute animal studies at 

concentrations ≥1,420 mg/m35 (Frantik et al., 1994; DeCeaurriz et al., 1983), as well as the 

6 neurotoxic effects, ataxia and hearing impairment, observed in occupationally-exposed humans 

7 (Velazquez et al., 1969), and vertigo (Seitz, 1972).
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1 2 Figure 5-2. Exposure response array for inhalation exposure to n-butanol. 
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1 

2 5.2.2. Methods of Analysis—Including Models (PBPK, BMD, etc.) 

A LOAEL of 308 mg/m3 and a NOAEL of 154 mg/m33 were identified for impaired 

4 neurobehavioral function (as assayed by rotarod performance) in rats exposed for 3 months in 

5 Korsak et al. (1994). Korsak et al. (1994) reported the data on rotarod performance graphically 

6 and without any estimate of variability within groups; thus, these data were not amenable to 

BMD modeling. Instead, the rat NOAEL of 154 mg/m37 was selected as the POD for RfC 

8 derivation. 

9 Interspecies extrapolation (i.e., rat-to-human) of n-butanol inhalation dosimetry was 

10 accomplished using a rat and human PBPK model originally described in Teeguarden et al. 

11 (2005) with modifications described in Appendix C. The rat PBPK model was used to estimate 

12 the value for the internal dose metric, represented as the area under the curve (AUC) for n13 butanol concentration in arterial blood, corresponding to the NOAEL for motor incoordination in 

14 a 90-day rat study (Korsak et al., 1994). The human PBPK model was used to estimate the 

continuous human inhalation exposure (mg/m315 ) that would result from the value of the internal 

16 dose metric corresponding to the NOAEL in the rat. Details of the model code and parameters 

17 that were selected are presented in Section 3.5 and Appendix C. 

18 The internal dose metric for the neurobehavioral effects of n-butanol is uncertain based 

19 on the currently available data. The PBPK model for n-butanol (Teeguarden et al., 2005), with 

20 modifications, simulates n-butanol concentrations in blood, liver, and a lumped compartment 

21 representing other tissues. The model does not simulate concentrations of n-butanol in the brain. 

22 In the absence of dose-metric-specific data, parent compound concentration in the blood is 

23 generally selected as a surrogate since it represents the circulating matrix for distribution in the 

24 body. Therefore, blood n-butanol concentration was selected as the dose metric for 

25 neurobehavioral effects in animals. Korsak et al. (1994) observed progressive increases in 

26 severity of the motor incoordination with increasing exposure duration (1–3 months) at a 

27 constant exposure concentration, as well as with increasing exposure concentration. These 

28 observations suggest that the toxic response is a function of both n-butanol blood concentration 

29 and the duration of internal exposure to blood n-butanol. Therefore, arterial blood AUC was 

30 selected as the dose metric for dosimetry extrapolation to humans. 

31 Korsak et al. (1994) exposed rats (average body weight of 0.392 kg as measured in the 

32 study) for 6 hours/day, 5 days/week, for a period of 3 months. This exposure was simulated in 

the rat PBPK model as a 90-day exposure of a 0.392 kg rat to 50 ppm (154 mg/m333 , the NOAEL) 

34 for 6 hours/day, 5 day/week. The internal dose metric (model variable: AUCAB224) was 

35 calculated as the integral of the time profile for the arterial concentration of n-butanol (model 

36 variable: CAB2) divided by the simulation time (t = 90 days × 24 hours/day = 2,160 hours), 

37 multiplied by 24 hours: 

38 
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1 

2 

3 Conceptually, the internal dose metric (AUCAB224) represents the average 24-hour AUC 

4 for arterial n-butanol concentration for the 2,160-hour exposure duration. The value for 

5 AUCAB224 achieved pseudo steady state behavior after 2,000 hours (i.e., ±1% of the final 

6 value). The human exposure was simulated as a continuous exposure (24 hours/day, 7 

7 days/week) for a 70-kg human. The simulated exposure duration was 1,000 hours. An exposure 

8 duration exceeding 500 hours was sufficient to achieve steady state levels (i.e., > 99.99% 

9 stability of the value for AUCAB224). Values for the rat NOAEL, AUCAB224, and human 

equivalent NOAEL concentration (59 mg/m310 ) calculated from the PBPK model are shown in 

11 Table 5-4. 

12 

Table 5-4. PBPK model calculation of HECs of the NOAEL based on 

rotarod performance in male rats exposed to n-butanol for 3 months 

Rat NOAEL 

mg/m3 (ppm) 

Rat AUCAB224

a

mM 

Human AUCAB224

mM 

Human NOAEL 

(HEC) mg/m3 (ppm) 

154 (50) 0.01282 0.01282 59 (19.21) 

aArea under the blood concentration vs. time curve for rats estimated using rat PBPK model and exposure to 

NOAEL concentration of 50 ppm for 6 hours/day, 5 days/week for 3 months (Korsak et al., 1994). 

13 

14 5.2.3. RfC Derivation—Including Application of Uncertainty Factors (UFs) 

The NOAELHEC of 59 mg/m315 for impaired motor coordination and obtained from PBPK 

16 modeling was used as the POD for derivation of the RfC for n-butanol. The RfC was calculated 

17 by dividing the POD by a total UF of 1,000, comprised of 3 for interspecies extrapolation, 10 for 

18 interindividual variability, 10 for extrapolation from subchronic to chronic exposure, and 3 for 

19 database uncertainty, as described below. 

20 An UF of 3 was applied for interspecies extrapolation. The interspecies UF comprises 

21 two areas of uncertainty: toxicokinetic and toxicodynamic differences. Use of PBPK modeling 

22 to convert the rat exposure concentration to a HEC accounts for toxicokinetic differences 

23 between rats and humans and reduces uncertainty associated with cross-species extrapolation. A 

24 factor of 3 is retained to account for interspecies variability in extrapolating from laboratory 

25 animals (rats) to humans because information is not available to quantitatively assess 

26 toxicodynamic differences between animals and humans. 

27 An UF of 10 was selected to account for intraspecies variability in susceptibility to n28 butanol, as quantitative information for evaluating toxicokinetic and toxicodynamic differences 

29 among humans are not available. 
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1 An UF of 10 was selected to account for extrapolation from a subchronic exposure 

2 duration study to a chronic RfD, as data for evaluating response after chronic exposure are not 

3 available. The study selected as the principal study was a 3-month study by Korsak et al. (1994), 

4 a study duration that falls well short of a standard lifetime study. 

5 An UF of 3 was selected to account for deficiencies in the database, specifically the lack 

6 of a multigeneration reproductive toxicity study. The toxicological database for inhaled 

7 n-butanol includes several acute human experiments, occupational health studies, subchronic 

8 toxicity studies, and developmental and neurodevelopmental toxicity studies. 

9 An UF for extrapolation from a LOAEL to a NOAEL was not applied because a NOAEL 

10 was used as the POD. 

The RfC of 0.06 mg/m311 for n-butanol was calculated as follows: 

12 

13 RfC = NOAELHEC ÷ UF 

 = 59 mg/m314 ÷ 1,000 

 = 0.06 mg/m315 

16 

17 5.2.5. Previous RfC Assessment 

18 An inhalation RfC for n-butanol was not previously available on IRIS. 

19 
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1 5.3. UNCERTAINTIES IN THE ORAL REFERENCE DOSE AND INHALATION 

2 REFERENCE CONCENTRATION 

3 The following discussion identifies uncertainties associated with the quantification of the 

4 RfD and RfC for n-butanol. Following EPA practices and guidance (U.S. EPA, 1994b), the 

5 uncertainty factor approach was applied to the identified PODs to derive an RfD and an RfC (see 

6 Sections 5.1.3 and 5.2.3). Factors accounting for uncertainties associated with a number of steps 

7 in the analyses were adopted to account for extrapolating from an animal study to human 

8 exposure, a diverse human population of varying susceptibilities, extrapolation from subchronic 

9 to chronic exposure duration, and database deficiencies. 

10 The oral toxicity database for n-butanol contains one subchronic gavage study in rats 

11 (RTI; unpublished, 1985), two subchronic drinking water studies in rats (Munoz et al., 1991, 

12 1990; Wakabayashi et al., 1991), and two developmental toxicity studies in rats (Ema et al., 

13 2005; Sitarek et al., 1994). Toxicity associated with oral exposure is predominantly associated 

14 with neurodevelopmental toxicity. Effects observed after oral exposure to n-butanol include 

15 teratogenicity, clinical signs of CNS effects, and hematology changes. 

16 The toxicological database for inhaled n-butanol includes several acute human 

17 experiments, a few occupational health studies, a subchronic study in rats (Korsak et al., 1994), 

18 and developmental and neurodevelopmental toxicity studies in rats (Nelson et al., 1989a, b). 

19 Neurotoxicity is the predominant effect associated with inhalation exposure to n-butanol. 

20 Critical data gaps have been identified and uncertainties associated with data deficiencies are 

21 more fully discussed below. 

22 The RfD was derived based on the increased incidence of overall visceral malformations 

23 in a developmental toxicity study of n-butanol (Sitarek et al., 1994). Uncertainty exists in the 

24 discrepancy between the results of the developmental studies by Sitarek et al. (1994) and Ema et 

25 al. (2005). Specifically, Sitarek et al. (1994) observed developmental effects on the brain at low 

26 doses (≥ 300 mg/kg-day), but Ema et al. (2005) did not observe any developmental 

27 malformations at any dose. The discrepancy in these results may be due to differences in study 

28 design, particularly rat strain used and exposure regimens. Data support a strain differences 

29 between Wistar and Sprague Dawley rats in developmental effects on the brain. For example, 

30 one study evaluated strain differences for developmental brain effects in Sprague-Dawley and 

31 Wistar rats following exposure to aspirin (Gupta et al., 2003). Gupta et al. (2003) reported that 

32 the Wistar pups had a significantly higher rate of hydrocephaly than the Sprague-Dawley pups. 

33 In addition, Ema et al. (2005) utilized a gestation-ony exposure regimen compared to Sitarek et 

34 al. (1994) which exposed rats prior to mating, during mating, and throughout gestation. 

35 Uncertainty also exists in the selection of the appropriate BMR for use in the BMD 

36 modeling of the critical effect (i.e., increased incidence of dilation of the lateral ventricle of the

37 brain in Wistar IMP:Dak rat litters) to estimate the POD. Since the results of Sitarek et al. 

38 (1994) were reported in a way that the litter membership of individual pups could not be 
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1 determined, a nested BMD modeling approach could not be used and therefore, a litter-based 

2 BMD modeling approach was employed. In the absence of specific information to identify a 

3 change in the litter incidence of dilation of the lateral ventricle that represents a biologically 

4 significant change, a BMR of 10% was selected, which is consistent with the default BMR of 

5 10% for quantal data (U.S. EPA, 2000). 

6 The RfC was derived based on neurobehavioral effects in rats exposed to n-butanol via 

7 inhalation for 3 months (Korsak et al., 1994). With regards to the study design, it is not known 

8 when the behavioral testing following n-butanol exposure. Therefore, the extent of n-butanol 

9 internally (whether cleared or not) is unknown. It is likely that the measures are reflective of 

10 persistent effects of n-butanol based on the increased deterioration of the rotarod performance 

11 over the 3 months but still represents an uncertainty. BMD modeling could not be performed 

12 using these data due to the lack of reporting by the investigators of within-group variability 

13 estimates. By definition, the identification of a NOAEL or LOAEL is restricted to the particular 

14 doses or exposure concentrations used in a study, and thus, this type of analysis lacks 

15 characterization of the entire dose-response curve for the effect of interest. As a result, a POD 

16 based on a NOAEL or LOAEL is less informative than one derived from BMD modeling. 

17 With respect to uncertainty associated with the RfC derivation, there is one human study, 

18 Velazquez et al. (1969), that reported hearing loss in 82% (9 out of 11) of the workers exposed to 

240 mg/m319 n-butanol for a duration ranging from 3 to 11 years in a cellulose acetate factory. 

20 Although human data is typically preferred for deriving reference values, there are limitations 

21 with this study, including the small number of exposed workers, which decreases the power of 

22 the study, and the lack of information with regards to exposure sampling and exposure duration, 

23 which precluded the selection of this study as the principal study. 

24 An additional source of uncertainty in the derivation of both RfD and RfC is 

25 extrapolation from animals (rats) to humans. An effect and its magnitude associated with the 

26 concentration at the POD in rodents are extrapolated to human response. Pharmacokinetic 

27 models are useful in examining species differences in pharmacokinetic processing; however, 

28 dosimetric adjustment using pharmacokinetic modeling was not possible following oral exposure 

29 to n-butanol since the model does not contain an oral route of administration and incorporate the 

30 subsequent first pass effect. There are data gaps in pharmacokinetic parameters since there is no 

31 information on distribution of n-butanol to the fetus as well as no information of the elimination 

32 of n-butanol via breast milk which represents an uncertainty for both the RfD and RfC. For RfC 

33 derivation, a PBPK model was used to account for toxicokinetic differences between rats and 

34 humans, and the internal dose metric, blood n-butanol AUC, was selected. PBPK models attempt 

35 to simulate what occurs in vivo, and thus dose estimates from these models represent an 

36 additional source of uncertainty in the analysis. In the absence of specific data that could inform 

37 the selection of the appropriate internal dose metric, the blood AUC of the parent compound 

38 (i.e., n-butanol) was selected as the preferred dose metric. Additionally, there is uncertainty 
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1 related to the urinary clearance rate of n-butanol and its metabolites. Specifically, butyric acid, a 

2 metabolite of n-butanol, interacts with colonocytes and is incorporated into lipid membranes 

3 (Thibault et al., 2010). However, kinetic information is currently not available to address this 

4 uncertainty and thus urinary clearance of metabolites was not incorporated into the model. 

5 Another uncertainty with the model is the extent that n-butyric acid is metabolized to acetyl CoA 

6 and then to carbon dioxide and water or to acetoacetyl CoA, (produced by beta oxidation of the 

7 buttanoic acid). 

8 

9 Heterogeneity among humans with respect to the toxicokinetics and toxicodynamics of n-butanol 

10 represents another area of uncertainty in both the RfD and RfC derivations. n-Butanol is rapidly 

11 metabolized to butyric aldehyde by ADH and further to n-butyric acid by aldehyde dehydrogenase. 

12 Polymorphisms in the genes encoding ADH and aldehyde dehydrogenase exist in the human population 

13 and may contribute to variability in metabolism of n-butanol. One key genetic polymorphism is the 

14 ALDH2 (aldehyde dehydrogenase 2) allele that has been identified as a slow metabolizing 

15 variant of aldehyde dehdrogenase and has been more commonly found in East Asian populations 

16 (Dickson et al., 2006). Populations with low metabolizing capacity (slow alcohol and aldehyde 

17 dehydrogenases) are more likely to be susceptible to toxicities associated with n-butanol since 

18 there would be a longer half-life in these individuals. Another key polymorphism has been 

19 identified in the ADH3 (alcohol dehydrogenase type 3) gene where the gamma1 variant is found 

20 in individuals that are fast metabolizers and the gamma2 variant is found in individuals that are 

21 slow metabolizers (Hines et al., 2001). The polymorphisms in the n-butanol metabolizing 

22 enzymes indicate that there may be a great deal of variability in the effects associated with 

23 exposure in humans. 

24 In the absence of n-butanol-specific data to adequately characterize such genetic 

25 variability among humans, a factor of 10 was used to account for this uncertainty. 

26 Data gaps in the toxicological database for n-butanol include: lack of chronic toxicity 

27 studies; lack of toxicity data in a laboratory species other than rats; limitations in the available 

28 subchronic studies (e.g., the oral study by RTI [1985] is unpublished and lacks data tables and 

29 appendices, and the inhalation study by Korsak et al. [1994] did not include histopathology 

30 evaluation); lack of a multigeneration reproductive toxicity study; and lack of an oral 

31 neurodevelopmental toxicity study. Critical endpoints for both the RfD and RfC were 

32 neurological, suggesting that further evaluation of the neurotoxic and neurodevelopmental 

33 effects of n-butanol would be beneficial. There was a lack of postnatal observations in the 

34 developmental toxicity studies since offspring were sacrificed at the end of the gestational period 

35 (Sitarek et al., 1994; Nelson et al., 1989a; Ema et al., 2005). Epidemiology studies of n-butanol 

36 exposure evaluating neurological and otological endpoints and using documented, quantified 

37 assessments of long-term exposure also would contribute to a reduction in uncertainty. 

38 
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1 5.4. CANCER ASSESSMENT 

2 Under the EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), there is 

3 inadequate information to assess the carcinogenic potential of n-butanol. None of the available 

4 occupational health studies of n-butanol evaluated cancer endpoints. One occupational health 

5 study of workers exposed to n-butanol, and several other solvents, while employed as dockyard 

6 painters examined mortality rates due to cancer (Chen et al., 1999); however, neither 

7 proportional mortality nor standardized mortality due to cancers were increased in the painters 

8 relative to controls. There are no chronic animal studies of n-butanol exposure via any exposure 

9 route, and the subchronic studies did not report any cancer endpoints. The genotoxicity database 

10 for n-butanol is limited; available information suggests that n-butanol does not induce mutations 

11 in S. typhimurium with or without exogenous metabolic activation (Muller et al., 1993; Jung et 

12 al., 1992; Nakamura et al., 1987; Connor et al., 1985; McCann et al., 1975), and does not 

13 increase the frequency of micronuclei or sister chromatid exchanges in Chinese hamster ovary 

14 cells (Lasne et al., 1984; Obe et al., 1977) or in chick embryos in culture (Bloom, 1982). Studies 

15 of DNA and RNA replication have shown that n-butanol can exert an inhibitory effect on these 

16 processes (Obe and Ristow, 1977; Yoshiyama et al., 1973). 

17 

18 
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1 6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE 

2 RESPONSE 

3 

4 6.1. HUMAN HAZARD POTENTIAL 

5 n-Butanol (CASRN 71-36-3) is used as a direct solvent in paints, surface coatings, 

6 lacquers, thinners, pharmaceutical formulations, waxes, and resins. It is also used to make 

7 plasticizer esters and mono-, di-, and tributylamines and oxygenate for gasoline. Commercial 

8 derivatives of n-butanol include n-butyl acrylate, methacrylate, butyl glycol ethers, 2-butoxy9 ethanol, and butyl acetate. 

10 Toxicokinetic studies of n-butanol have been conducted in humans and experimental 

11 animals. Absorption via inhalation has been demonstrated in humans (Astrand et al., 1976), and 

12 absorption via the oral, dermal, and inhalation routes was demonstrated in laboratory animals 

13 (Poet et al., 2003a, b; Deisinger and English, 2001; Boman et al., 1995; Swiercz et al., 1995; 

14 DiVincenzo and Hamilton, 1979a). Only limited information is available regarding tissue 

15 distribution of n-butanol in animal studies; after gavage dosing of rats with radiolabelled 

16 n-butanol, the liver had the largest percentage of radioactivity (DiVincenzo and Hamilton, 

17 1979a). n-Butanol is rapidly metabolized to butyric aldehyde by ADH and further to n-butyric 

18 acid, also known as butanoic acid, by aldehyde dehydrogenase (ECETOC, 2003). n-Butanol is 

19 also oxidized by cytochrome P450 in rat liver (Albano et al., 1991). n-Butanol is excreted 

20 primarily as CO2 in exhaled breath with minor amounts eliminated in the urine and feces 

21 (DiVincenzo and Hamilton, 1979a). A PBPK model has been developed (Teeguarden et al., 

22 2005) to describe blood kinetics for n-butyl acetate and its metabolites, n-butanol and n-butyric 

23 acid, in rats and humans exposed via inhalation. 

24 Information on noncancer effects in humans orally exposed to n-butanol is limited to one 

25 case report from a suicide attempt (Bunc et al., 2006) in which neurological, gastrointestinal, and 

26 cardiovascular symptoms were noted. The database on animal toxicity indicates that 

27 developmental visceral malformations (dilation of the lateral ventricle and subarachnoid space in 

28 the brain; dilation in renal pelvis in the kidney) are the most sensitive toxicological endpoint 

29 associated with oral n-butanol administration (Sitarek et al., 1994). Increased incidences of 

30 brain malformations (dilation of the lateral and/or third ventricle of the brain) were observed in 

31 the pups of female Wistar Imp:DAK rats treated with n-butanol in drinking water at doses of 

32 300, 1,000, and 5,000 mg/kg-day administered for 8 weeks prior to mating and then continuously 

33 through mating and gestation. Neurotoxicity (hypoactivity and ataxia) in adult rats was also 

34 observed in a 13week oral gavage study at 500 mg/kg-day (RTI; unpublished, 1985). Other 

35 observed effects included hematological changes and increased thyroid weight at 500 mg/kg-day 

36 (RTI; unpublished, 1985), and pathological changes in the liver and kidney at an exposure of 

37 4,400 mg/kg-day in male rats (Munoz et al. 1991; 1990). 
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1 Controlled human exposure experiments have demonstrated that n-butanol vapors can 

2 exert an irritant effect on the eyes, nose, and throat under acute exposure conditions at air 

concentrations ≥ 10 mg/m33 (Kjaerguard et al., 1997; Nelson et al., 1943). Occupational health 

4 studies in which the primary exposure was to n-butanol reported effects including eye irritation 

(at 46 to 200 mg/m35 ) (Cogan et al., 1945; Tabershaw et al., 1944) and hearing loss (at 240 

mg/m36 ) (Velazquez et al., 1969). These studies are limited by uncertainties arising from a lack 

7 of detail in methods description for exposure assessments and other details of study design. 

8 The CNS is the most sensitive target following a repeated inhalation exposure to n9 butanol in animals. Korsak et al (1994) reported a statistically significant increase in the failure 

rate for the rotarod test in rats exposed to 308 mg/m310 n-butanol for 3 months. Nelson et al 

11 (1989a, b) evaluated the developmental and neurodevelopmental toxicity potential of n-butanol 

12 in rats. Increased incidence of litters with skeletal variation was observed at exposures of 3,500 

ppm (11,000 mg/m313 ) and increased levels of serotonin and dopamine were observed in offspring 

from male rats exposed to 5,000 ppm (18,000 mg/m314 ) without changes in neurobehavioral 

15 function. Several short-term inhalation exposure studies have reported fatty infiltration of liver 

and kidney in mice exposed to 24,624 mg/m316 (Weese, 1928), neurotoxic effects at exposures of

1,420 mg/m317 in mice and rats (Frantik et al., 1994; DeCeaurriz et al., 1983), and decreased 

18 respiration rates in mice (Korsak and Rydzynski, 1994; Korsak et al., 1993; Kristiansen et al., 

19 1988). 

20 None of the available human occupational health studies in which the primary exposure 

21 was to n-butanol evaluated cancer endpoints. One occupational health study of workers exposed 

22 to n-butanol, as well as several other solvents, while employed as dockyard painters examined 

23 mortality rates due to cancer (Chen et al., 1999); however, neither proportional mortality nor 

24 standardized mortality due to cancers were increased in the painters relative to controls. There 

25 are no chronic animal studies of n-butanol exposure via any exposure route, and the available 

26 subchronic studies did not report any cancer endpoints. Therefore, under the EPA Guidelines for 

27 Carcinogen Risk Assessment (U.S. EPA, 2005a), there is inadequate information to assess the 

28 carcinogenic potential of n-butanol. 

29 

30 6.2. DOSE RESPONSE 

31 6.2.1. Oral Noncancer 

32 From the available data on oral exposure to n-butanol, it was determined that 

33 neurodevelopmental changes were the most sensitive noncancer endpoint. The developmental 

34 toxicity study in Wistar Imp:DAK rats (Sitarek et al.,1994) was selected as the principal study 

35 for RfD derivation and the increased incidence developmental visceral malformations (dilation in 

36 the fetal brain [lateral ventricle and subarachnoid space] and kidney [renal pelvis]) was selected 

37 as the critical effect. 
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1 An RfD of 0.2 mg/kg-day was derived. The RfD derivation involved fitting all of the 

2 available dichotomous models in BMDS, version 2.1.1, to the litter incidence of overall 

3 increased visceral malformations (dilation). A BMR of 10% was selected because even though 

4 the principal study had a nested design, litter-specific results were not reported by the 

5 investigators which prevented the conduct of a nested analysis. Thus, a BMR of 10% was 

6 employed, which is generally the default BMR for quantal data (U.S. EPA, 2000). Dose7 response modeling of the incidence of litters with overall dilation yielded a BMDL10 of 48 

8 mg/kg-day, which was used as the POD. A composite UF of 300 (10 to account for interspecies 

9 uncertainty, 10 to account for human variability, 3 for database uncertainty) was applied to the 

10 POD to derive an RfD of 0.2 mg/kg-day. 

11 Confidence in the principal study (Sitarek et al., 1994) is medium. This developmental 

12 toxicity study evaluated exposure in three dose groups and a control, using group sizes of 11–

13 17 female rats exposed for 8 weeks prior to mating as well as during mating and gestation. The 

14 study evaluated body weight, clinical signs, and estrous cyclicity in dams, as well as litter 

15 parameters and teratogenicity endpoints. Confidence in the database is low-to-medium. Oral 

16 toxicity studies of n-butanol include an unpublished subchronic study that evaluated 

17 comprehensive endpoints but lacked data tables and appendices, two published subchronic 

18 studies evaluating limited endpoints, and two developmental toxicity studies. All of the 

19 available animal studies were conducted in rats. The database is lacking chronic toxicity studies, 

20 toxicity data in a laboratory species other than rats, and a multigeneration reproductive toxicity 

21 study. Neurodevelopmental effects should also be further evaluated in light of the effects on 

22 brain development in the Sitarek et al. (1994) developmental toxicity study. Overall confidence 

23 in the RfD is low-to-medium. 

24 

25 6.2.2. Inhalation Noncancer 

26 The CNS is the most sensitive target for noncancer toxicity in rats following repeated 

27 inhalation exposure to n-butanol. The most sensitive endpoint was impairments in learned 

28 avoidance behavior and neuromuscular function, represented by decreased rotarod performance, 

29 in rats exposed to n-butanol for 3 months (Korsak et al., 1994). Korsak et al. (1994) was selected 

30 as the principal study for derivation of the RfC because it was a relatively well-designed study, 

31 evaluated lower exposure effects, and provided a sensitive endpoint of neurologic impairment. 

32 Decreased rotarod performance was selected as the critical effect. 

An RfC of 0.06 mg/m333 was derived based on the observed critical effect of 

34 neuromuscular impairment in the principal study. The data for this endpoint were not amenable 

35 to BMD modeling due to the absence of within-group variability estimates for rotarod 

performance, so a NOAEL identified from this study (154 mg/m336 ) was selected as the POD. 

37 PBPK modeling using a modification of the model published by Teeguarden et al. (2005) was 

used to estimate a NOAELHEC concentration of 59 mg/m338 based on arterial n-butanol blood 
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1 concentration (AUC) as the internal dose metric. A composite UF of 1000 (3 to account for 

2 interspecies toxicodynamic uncertainty, 10 to account for human variability, 10 to account for 

3 uncertainity of subchronic to chronic extrapolation, 3 for database uncertainty) was applied to the 

POD to derive an RfC of 0.06 mg/m34 . 

5 Confidence in the principal study (Korsak et al., 1994) is low-to-medium. This 

6 subchronic study evaluated exposure in two dose groups and a sham-treated control, using group 

7 sizes of 12 male rats. The study evaluated body weight, hematology, clinical chemistry, 

8 neurotoxicity endpoints (rotarod performance and hot-plate response), and selected organ 

9 weights, but did not evaluate histopathology. Confidence in the database is low-to-medium. 

10 Inhalation toxicity studies of n-butanol include the subchronic study used as the basis for the RfC 

11 as well as developmental toxicity and developmental neurotoxicity studies, all conducted in rats. 

12 The database is lacking chronic toxicity studies, toxicity data in a laboratory species other than 

13 rats, and a multigeneration reproductive toxicity study. Neurological effects were the most 

14 sensitive endpoint in the available subchronic study; however, the study evaluated only two 

15 measures of neurotoxicity (rotarod performance and hot-plate response). Overall confidence in 

16 the RfC is low-to-medium. 

17 

18 6.2.3. Cancer - Oral and Inhalation 

19 Under the EPA Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), there is 

20 inadequate information to assess the carcinogenic potential of n-butanol. Cancer endpoints 

21 were not evaluated in any of the human occupational health studies. There are no chronic animal 

22 studies of n-butanol exposure via any exposure route, and the subchronic studies did not report 

23 any cancer endpoints. The genotoxicity database is limited and results primarily support that n24 butanol does not induce mutations in S. typhimurium with or without exogenous metabolic 

25 activation (Muller et al., 1993; Jung et al., 1992; Nakamura et al., 1987; Connor et al., 1985; 

26 McCann et al., 1975), and does not increase the frequency of micronuclei or sister chromatid 

27 exchanges (Lasne et al., 1984; Bloom, 1982; Obe et al., 1977). 
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7. REFERENCES 

ACGIH (American Conference of Governmental Industrial Hygienists). (2002) n-Butanol. In: Documentation of the 

threshold limit values and biological exposure indices. Cincinnati, OH: American Conference of Governmental 

Industrial Hygienists. 

Akhmadeyeva, EN. (1993) Health of newborns of workers in the petroleum-chemical industries. Reprod Toxicol 

7(5):491–492. 

Akinshola, BE. (2001) Straight-chain alcohols exhibit a cutoff in potency for the inhibition of recombinant 

glutamate receptor subunits. Br J Pharmacol 133:651–658. 

Albano, E; Tomasi, A; Persson, J; et al. (1991) Role of ethanol-inducible cytochrome P450 (P450IIE1) in catalysing 

the free radical activation of aliphatic alcohols. Biochem Pharmacol 41(12):1895–1902. 

Amoore, JE; Hautala, E. (1983) Odor as an aid to chemical safety: odor thresholds compared with threshold limit 

values and volatilities for 214 industrial chemicals in air and water dilution. J Appl Toxicol 3(6):272–290. 

Andjus, PR; Bataveljić, D; Vanhoutte, G; et al. (2009) In vivo morphological changes in animal models of 

amyotrophic lateral sclerosis and Alzheimer's-like disease: MRI approach. Anat Rec 292:1882–1892. 

Angerer, J; Wulf, H. (1985) Occupational chronic exposure to organic solvents. XI. Alkylbenzene exposure of 

varnish workers: effects on hematopoetic system. Int Arch Occup Environ Health 56:307–321. 

Anonymous. (1987) Final report of the safety assessment of n-butyl alcohol. J Am Coll Toxicol 6(3):403–424. 

Arsov, Z; Zorko, M; Schara, M. (2005) Inhibition of erythrocyte acetylcholinesterase by n-butanol at high 

concentrations. Arch Biochem Biophys 437:78–84. 

Ashley, DL; Prah, JD. (1997) Time dependence of blood concentrations during and after exposure to a mixture of 

volatile organic compounds. Arch Environ Health 52(1):26–33. 

Astrand, I; Ovrum, P; Lindqvist, T; et al. (1976) Exposure to butyl alcohol: uptake and distribution in man. Scand J 

Work Environ Health 2(3):165–175. 

Auty, RM; Branch, RA. (1976) The elimination of ethyl, n-propyl, n-butyl and iso-amyl alcohols by the isolated 

perfused rat liver. J Pharmacol Exp Ther 197:669–674. 

Baikov, BK; Khachaturyan, MK. (1973) Hygienic evaluation of the reflex action on the body of low concentrations 

of butyl alcohol entering the atmosphere. Gig Sanit 38(12):7–11. (Russian) 

Bariliak, IR; Korkach, VI; Spitkovskaia, LD. (1991) The embroyotoxic effects of monohydric alcohols. Ontogenez 

22(1):71–75. (Russian) 

Barton, HA; Deisinger, PJ; English, JC; et al. (2000) Family approach for estimating reference concentrations/doses 

for series of related organic chemicals. Toxicol Sci 54:251–261. 

Behl, CR; El-Sayed, AA; Flynn, GL. (1983) Hydration and percutaneous absorption. IV: Influence of hydration on 

n-alkanol permeation through rat skin; comparison with hairless and Swiss mice. J Pharm Sci 72(1):79–82. 

Behl, CR; Flynn, GL; Kurihara, T; et al. (1984) Age and anatomical site influences on alkanol permetion of skin of 

the male hairless mouse. J Soc Cosmet Chem 35:237–252. 

Bhattacharji, A; Kaplan, B; Harris, T; et al. (2006) The concerted contribution of the S4-S5 linker and the S6 

segment to the modulation of a Kv channel by 1-alkanols. Mol Pharmacol 70(5):1542–1554. 

Billig E. 2001. Butyl alcohols. In: Kirk-Othmer encyclopedia of chemical technology. Available online at 

http://mrw.interscience.wiley.com/emrw/9780471238966/kirk/article/butybill.a01/current/pdf (accessed August 17, 

2009). 
95 DRAFT - DO NOT CITE OR QUOTE

Birley. AJ; James, MR; Dickson, PA; Montgomery, GW; Heath, AC; Martin, NG; Whitfield, JB (2009). ADH 

single nucleotide polymorphism associations with alcohol metabolism in vivo. Hum Mol Genet 18(8): 1533-42. 

Bleecker, ML; Bolla, KI; Agnew, J; et al. (1991) Dose-related subclinical neurobehavioral effects of chronic 

exposure to low levels of organic solvents. Am J Ind Med 19(6):715–728. 

Bloom, SE. (1982) Detection of sister chromatid exchanges in vivo using avian embryos. In: Hsu, TC; ed. 

Cytogenic assays of environmental mutagens. Totowa, NJ: Allanheld, Osmun & Co.; pp. 137–159. 

Boleda, MD; Farrés, J; Guerri, C; Parés, X (1992). Alcohol dehydrogenase isoenzymes in rat development. Effect 

of maternal ethanol consumption. Biochem Pharmacol. 43(7):1555-61. 

Boman, A; Maibach, HI. (2000) Influence of evaporation and solvent mixtures on the absorption of toluene and nbutanol in human skin in vitro. Ann Occup Hyg 44(2):125–135. 

Boman, A; Hagelthorn, G; Magnusson, K. (1995) Percutaneous absorption of organic solvents during intermittent 

exposure in guinea pigs. Acta Derm Venereol 75:114–119. 

Boublik, T; Fried, V; Hala, E. (1984) The vapor pressures of pure substances: selected values of the temperature 

dependence of the vapour pressures of some pure substances in the normal and low pressure region. Vol. 17. 

Amsterdam, Netherlands: Elsevier Science Publications. 

Brown, RP; Delp, MD; Lindstedt, SL; et al. (1997) Physiological parameter values for physiologically based 

pharmacokinetic models. Toxicol Ind Health 13(4):407–484. 

Bunc, M; Pezdir, T; Mozina, H; et al. (2006) Butanol ingestion in an airport hangar. Hum Exp Toxicol 25(4):195–

197. 

Buttery, RG; Ling, LC; Guadagni, DG. (1969) Volatilities of aldehydes, ketones, and esters in dilute water solution. 

J Agric Food Chem 17(2):385–389. 

Cameron, AM; Zahlsen, K; Haug, E; et al. (1985) Circulating steroids in male rats following inhalation of nalcohols. Arch Toxicol Suppl 8:422–424. 

Card, SE; Tompkins, SF; Brien, JF (1989). Ontogeny of the activity of alcohol dehydrogenase and aldehyde 

dehydrogenases in the liver and placenta of the guinea pig. Biochem Pharmacol. 38(15):2535-41. 

Carlson, GP. (1994a) In vitro esterification of fatty acids by various alcohols in rats and rabbits. Toxicol Lett 

70:57–61. 

Carlson, GP. (1994b) Formation of esterified fatty acids in rats administered 1-butanol and 1-pentanol. Res 

Commun Mol Pathol Pharmacol 86(1):111–117. 

Carlson, GP; Olson, RMC. (1995) Comparison of the metabolism of alcohols by rat hepatic and pulmonary alcohol 

dehydrogenase. Biochem Mol Biol Int 37(1):65–71. 

Cater, BR; Cook, MW; Gangolli, SD; et al. (1977) Studies on dibutyl phthalate-induced testicular atrophy in the rat: 

Effect on zinc metabolism. Toxicol Appl Pharmacol 41:609–618. 

Cederbaum, AI. (1978) The effect of calcium on the oxidation of acetaldehyde by rat liver mitochondria. Life Sci 

22(1):111–125. 

Cederbaum, AI; Dicker, E; Rubin, E; et al. (1979) Effect of thiourea on microsomal oxidation of alcohols and 

associated microsomal functions. Biochemistry 18(7):1187–1191. 

ChemIDplus. (2009) N-Butanol. ChemIDplus. Bethesda, MD: U.S. National Library of Medicine. Available online 

at http://sis.nlm.nih.gov/chemical.html (accessed on August 14, 2009). 
96 DRAFT - DO NOT CITE OR QUOTE

Chen, R; Dick, F; Seaton, A. (1999) Health effects of solvent exposure among dockyard painters: mortality and 

neuropsychological symptoms. Occup Environ Med 56(6):383–387. 

Chvapil, M; Zahradnik, R; Cmuchalova, B. (1962) Influence of alcohols and potassium salts of xanthogenic acids on 

various biological objects. Arch Int Pharmacodyn Ther 135:330–343. 

Cogan, DG; Mort, W; Grant, MD. (1945) An unusual type of keratitis associated with exposure to n-butyl alcohol 

(butanol). Arch Ophthalmol 33:106–108. 

Cometto-Muniz, JE; Cain, WS. (1995) Relative sensitivity of the ocular trigeminal, nasal trigeminal and olfactory 

systems to airborne chemicals. Chem Senses 20(2):191–198. 

Connor, TH; Theiss, JC; Hanna, HA; et al. (1985) Genotoxicity of organic chemicals frequently found in the air of 

mobile homes. Toxicol Lett 25:33–40. 

Crofton, KM; Lassiter, TL; Rebert, CS. (1994) Solvent-induced toxicity in rats: an atypical selective mid-frequency 

hearing deficit. Hear Res 80:25–30. 

David, R; Tyler, T; Ouellette, R; et al. (1998) Evaluation of subchronic neurotoxicity of n-butyl acetate vapor. 

Neurotoxicology 19:809–822. 

David, RM; Tyler, TR; Ouellette, R; et al. (2001) Evaluation of subchronic toxicity of n-butyl acetate vapor. Food 

Chem Toxicol 39:877–886. 

Deisinger, PJ; English, JC. (2001) Pharmacokinetics of n-butyl acetate and its metabolites in rats after intravenous 

administration. Rochester, NY: Toxicological Sciences Laboratory, Health and Environmental Laboratories, 

Eastman Kodak Company. 

DelTerzo, S; Behl, CR; Nash, RA; et al. (1986) Evaluation of the nude rat as a model: effects of short-term freezing 

and alkyl chain length on the permeabilities of n-alkanois and water. J Soc Cosmet Chem 37:297–307. 

Deters, M; Siegers, CP; Strubelt, O. (1998a) The influence of 4-methylpyrazole on the acute hepatotoxic actions of 

seven aliphatic alcohols. Res Commun Alcohol Subst Abuse 19(1-2):37–46. 

Deters, M; Siegers, CP; Strubelt, O. (1998b) Influence of glycine on the damage induced in isolated perfused rat 

liver by five hepatotoxic agents. Toxicology 128:63–72. 

Dietz, FK; Rodriguez-Giaxola, M; Traiger, GJ; et al. (1981) Pharmacokinetics of 2-butanol and its metabolites in the 

rat. J Pharmacokinet Biopharm 9(5):33–76. 

Dildy-Mayfield, JE; Mihic, SJ; Liu, Y; et al. (1996) Actions of long chain alcohols on GABAA and glutamate 

receptors: relation to in vivo effects. Br J Pharmacol 118:378–384. 

Ding, J; Badwey, JA. (1994) Wortmannin and 1-butanol block activation of a novel family of protein kinases in 

neutrophils. FEBS Lett 348:149–152. 

DiVincenzo, GD; Hamilton, ML. (1979a) Fate of n-butanol in rats after oral administration and its uptake by dogs 

after inhalation or skin application. Toxicol Appl Pharmacol 48:317–325. 

DiVincenzo, GD; Hamilton, ML. (1979b) Metabolic fate of [1-14C]isobutyric acid in the rat. Toxicol Appl 

Pharmacol 47(609–612). 

ECETOC (European Centre for Ecotoxicology and Toxicology of Chemicals). (2003) n-Butanol (CAS No. 71-36-

3). JACC No. 41. Brussels, Belgium. 

Edelfors, S; Ravn-Jonsen, A. (1990) The effects of alcohols in vitro on the nervous cell membrane measured by 

changes in the (Ca2+/Mg2+) ATPase activity and fluidity of the synaptosomal membrane. Pharmacol Toxicol 

67(1):56–60. 
97 DRAFT - DO NOT CITE OR QUOTE

Ema, M; Hara, H; Matsumoto, M; et al. (2005) Evaluation of developmental toxicity of 1-butanol given to rats in 

drinking water throughout pregnancy. Food Chem Toxicol 43:325–331. 

Etique, N; Chardard, D; Chesnel, A; et al. (2004) Analysis of the effects of different alcohols on MCF-7 human 

breast cancer cells. Ann NY Acad Sci 1030:78–85. 

Frantik, E; Hornychova, M; Horvath, M. (1994) Relative acute neurotoxicity of solvents: isoeffective air 

concentrations of 48 compounds evaluated in rats and mice. Environ Res 66(2):173–185. 

Gadberry, MG; Carlson, GP. (1994) 2-Butanol metabolism by rat hepatic and pulmonary cytochromes P450. 

Toxicol Lett 74:203–209. 

Gaillard, D; Derache, R. (1966) Action de quelques alcools aliphatiques sure la mobilisation de differentes fractions 

lipidiques chez la Rate. Food Cosmet Toxicol 4:515–520. 

Gastaldi, G; Casirola, D; Ferrari, G; et al. (1991) The effect of ethanol and other alcohols on morphometric 

parameters of rat small intestinal microvillous vesicles. Eur J Basic Appl Histochem 35(2):185–193. 

Giknis, MLA; Clifford, CB. (2008) Clinical laboratory parameters for Crl:WI(Han). Charles River, 1–14. 

Godden, EL; Harris, RA; Dunwiddie, TV. (2001) Correlation between molecular volume and effects of n-alcohols 

on human neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes. J Pharmacol Exp Ther 

296:716–722. 

Gordon, EL; Nguyen, TS; Ngai, AC; et al. (1995) Differential effects of alcohols on intracerebral arterioles. Ethanol 

alone causes vasoconstriction. J Cereb Blood Flow Metab 15(3):532–538. 

Grobin, AC; Matthews, DB; Devaud, LL; et al. (1998) The role of GABAA receptors in the acute and chronic effects 

of ethanol. Psychopharmacology 139:2–19. 

Gupta U, Cook JC, Tassinari MS, Hurtt ME (2003). Comparison of developmental toxicology of aspirin 

(acetylsalicylic acid) in rats using selected dosing paradigms. Birth Defects Res B Dev Reprod Toxicol. 68:27-37. 

Hansch, C; Leo, A; Hoekman, D. (1995) In: Heller, SR; ed. Exploring QSAR. Hydrophobic, electronic, and steric 

constants. Washington, DC: American Chemical Society; p. 10. 

Harris, T; Graber, AR; Covarrubias, M. (2003) Allosteric modulation of a neuronal K+ channel by 1-alkanols is 

linked to key residue in the activation gate. J Cell Physiol 285:C788–C796. 

Hempel-Jorgensen, A; Kjaergaard, SK; Molhave, L. (1998) Cytological changes and conjunctival hyperemia in 

relation to sensory eye irritation. Int Arch Occup Environ Health 71(4):225–235. 

Hempel-Jorgensen, A; Hudnell, HK; Kjaergaard, SK; et al. (1999) Time course of sensory eye irritation in humans 

exposed to n-butanol and 1-octene. Arch Environ Health 54(2):86–94. 

Hiller, JM; Angel, LM; Simon, EJ. (1984) Characterization of the selective inhibition of the delta subclass of opioid 

binding sites by alcohols. Mol Pharmacol 25(2):249–255. 

Hines, LM; Stampfer, MJ; Ma, J; Gaziano, JM; Ridker, PM; Hankinson, SE; Sacks, F; Rimm, EB; Hunter, DJ 

(2001). Genetic variation in alcohol dehydrogenase and the beneficial effect of moderate alcohol consumption on 

myocardial infarction. N Engl J Med. 2001 Feb 22;344(8):549-55. 

HSDB (Hazardous Substances Data Bank). (2009) N-Butyl alcohol. Hazardous Substances Data Bank. National 

Library of Medicine. Available online at http://toxnet.nlm.nih.gov/cgi-bin/sis/search/f?./temp/~SoTFja:1 (accessed 

on August 16, 2009). 
98 DRAFT - DO NOT CITE OR QUOTE

Jang, JY; Lee, SY; Kim, JI; et al. (1999) Application of biological monitoring to the quantitative exposure 

assessment for neuropsychological effect by chronic exposure to organic solvents. Int Arch Occup Environ Health 

72(2):107–114. 

Jenner, PM; Hagan, EC; Taylor, JM; et al. (1964) Food flavourings and compounds of related structure. I. Acute 

oral toxicity. Food Cosmet Toxicol 2:327–343. 

Jung, R; Engelhart, G; Herbolt, B; et al. (1992) Collaborative study of mutagenicity with Salmonella typhimurium 

TA102. Mutat Res 278(4):265–270. 

Kaneko, T; Wang, PY; Sato, A. (1994) Partition coefficients of some acetate esters and alcohols in water, blood, 

olive oil, and rat tissues. Occup Environ Med 51(1):68–72. 

Kempton, MJ; Stahl, D; Williams, SCR; et al. (2010) Progressive lateral ventricular enlargement in schizophrenia: a 

meta-analysis of longitudinal MRI studies. Schizophr Res 120:54–62. 

Kitagaki, H; Mori, E; Kazunari, I; et al. (1998) CSF spaces in idiopathic normal pressure hydrocephalus: 

morphology and volumetry. AJNR American Journal of Neuroradiology 19:1277–1284. 

Kjaergaard, S; Molhave, L; Jorgensen, AH. (1997) Relation between local eye irritation testing and whole body 

irritation response using n-butanol as a model substance. U.S. Environmental Protection Agency, Office of 

Research and Development. EPA/600/A-97/014. PB193817. 

Kolesnikov, PA. (1975) Adaptation to butyl alcohol. Gig I Sanit 5:104-105. 

Korsak, Z; Rydzynski, K. (1994) Effects of acute combined inhalation exposure to n-butyl alcohol and n-butyl 

acetate in experimental animals. Int J Occup Environ Health 7(3):273–280. 

Korsak, Z; Swiercz, R; Jedrychowski, R. (1993) Effects of acute combined exposure to n-butyl alcohol and 

m-xylene. Pol J Occup Med Environ Health 6(1):35–41. 

Korsak, Z; Wisniewska-Knypl, J; Swiercz, R. (1994) Toxic effects of subchronic combined exposure to n-butyl 

alcohol and m-xylene in rats. Int J Occup Med Environ Health 7(2):155–166. 

Kotter, K; Klein, J. (1999) Ethanol inhibits astroglial cell proliferation by disruption of phospholipase D-mediated 

signaling. J Neurochem 73(6):2517–2523. 

Kotter, K; Jin, S; Klein, J. (2000) Inhibition of astroglial cell proliferation by alcohols: interference with the protein 

kinase C-phospholipase D signaling pathway. Int J Dev Neurosci 18(8):825–831. 

Kowalczyk, CL; Stachecki, JJ; Schultz, JF; et al. (1996) Effects of alcohols on murine preimplantation development: 

relationship to relative membrane disordering potency. Alcohol Clin Exp Res 20(3):566–571. 

Krill, SL; Knutson, K; Higuchi, WI. (1993) The influence of iso-propanol, n-propanol and n-butanol on stratum 

corneum lipid phase behavior. J Control Release 25:31–42. 

Kristiansen, U; Vinggaard, AM; Nielsen, GD. (1988) The effects of n-butanol vapour on respiratory rate and tidal 

volume. Arch Toxicol 61:229–236. 

Kulig, B; Alleva, E; Bignami, G; et al. (1996) Animal behavioral methods in neurotoxicity assessment: SGOMSEC 

Joint Report. Environ Health Perspect 104 (Suppl 2):193-204. 

Lasne, C; Gu, ZW; Venegas, W; et al. (1984) The in vitro micronucleus assay for detection of cytogenetic effects 

induced by mutagen-carcinogens: Comparison with the in vitro sister-chromatid exchange assay. Mutat Res 

130(4):273–282. 

Lewis, RJ. (2000) Sax's dangerous properties of industrial materials. 10th ed. New York, NY: John Wiley & Sons, 

Inc.; p. 611. 
99 DRAFT - DO NOT CITE OR QUOTE

Lide, DR; ed. (2008) CRC handbook of chemistry and physics. 88th ed. New York, NY: CRC Press; p. 3–74. 

Lindahl, R (1977). Aldehyde dehydrogenase in 2-acetamidofluorene-induced rat hepatomas. Ontogeny and 

evidence that the new isoenzymes are not due to normal gene de-repression. Biochem J. 164(1):119-23. 

Macht,DI. (1920) A toxicological study of some alcohols, with special reference to isomers. J Pharmacol Exp Ther 

16:1-10. 

Maickel, RP; McFadden, DP. (1979) Acute toxicology of butyl nitrites and butyl alcohols. Res Commun Chem 

Pathol Pharmacol 26:75–83. 

Maickel, RP; Nash, JF. (1985) Differing effects of short-chain alcohols on body temperature and coordinated 

muscular activity in mice. Neuropharmacology 24(1):83–89. 

McCann, J; Choi, E; Yamasaki, E; et al. (1975) Detection of carcinogens as mutagens in the Salmonella/microsome 

test: Assay of 300 chemicals. Proc Natl Acad Sci USA 72(12):5135–5139. 

McCreery, MJ; Hunt, WA. (1978) Physico-chemical correlates of alcohol intoxication. Neuropharmacology

17:451–461. 

McKarns, SC; Hansch, C; Caldwell, WS; et al. (1997) Correlation between hydrophobicity of short-chain aliphatic 

alcohols and their ability to alter plasma membrane integrity. Fundam Appl Toxicol 36:62–70. 

McLain, VC. (2008) Final report of the addendum to the safety assessment of n-butyl alcohol as used in cosmetics. 

Int J Toxicol 27 (Suppl 2):53–69. 

McOmie, WA; Anderson, HH. (1949) Comparative toxicologic effects of some isobutyl carbinols and ketones. 

Pharmacology 2:217–230. 

MOE (Ministry of the Environment). (2007) Ontario Air Standards for n-butanol. Ontario’s Ministry of the 

Environment. Available online at http://www.ene.gov.on.ca/envision/env_reg/er/documents/2007/PA05E0020-f.pdf 

(accessed April 22, 2009). 

Mohler, FS; Gordon, CJ. (1991) Hypothermic effects of a homologous series of short-chain alcohols in rats. J 

Toxicol Environ Health 32(2):129–139. 

Muller, W; Engelhart, G; Herbold, B; et al. (1993) Evaluation of mutagenicity testing with Salmonella typhimurium

TA102 in three different laboratories. Environ Health Perspect 101(Suppl. 3):33–36. 

Munch, JC. (1972) Aliphatic alcohols and alkyl esters: narcotic and lethal potencies to tadpoles and to rabbits. Ind 

Med 41:31–33. 

Munch, JC; Schwartze, EW. (1925) Narcotic and toxic potency of aliphatic alcohols upon rabbits. J Lab Clin Med 

10:985–996. 

Munoz, R; Ferreras, JM; Iglesias, R; et al. (1990) Adaption of in vitro rat brain protein synthesis to long-term 

ingestion of n-butanol. Brain Res 517:330–332. 

Munoz, R; Iglesias, R; Ferreras, JM; et al. (1991) Effect of long-term n-butanol ingestion on rat brain polypeptide 

synthesis directed by endogenous messengers. Cell Mol Biol 37(7):671–677. 

Murata, K; Araki, S; Yokoyama, S; et al. (1991) Autonomic and peripheral nervous system dysfunction in workers 

exposed to mixed organic solvents. Int Arch Occup Environ Health 63(5):335–340. 

Nakahiro, M; Arakawa, O; Narahashi, T. (1991) Modulation of gamma-aminobutyric acid receptor-channel complex 

by alcohols. J Pharmacol Exp Ther 259(1):235–240. 
100 DRAFT - DO NOT CITE OR QUOTE

Nakamura, S; Oda, Y; Shimada, T; et al. (1987) SOS-inducing activity of chemical carcinogens and mutagens in 

Salmonella typhimurium TA1535/pSK1002: examination with 151 chemicals. Mutat Res 192:239–246. 

Nelson, KW; Ege, JF; Ross, M; et al. (1943) Sensory response to certain industrial solvent vapors. J Ind Hyg 

Toxicol 25:282–285. 

Nelson, BK; Brightwell, WS; Khan, A; et al. (1989a) Lack of selective developmental toxicity of three butanol 

isomers administered by inhalation to rats. Fundam Appl Toxicol 12:469–479. 

Nelson, BK; Brightwell, WS; Robertson, SK; et al. (1989b) Behavioral teratology investigation of 1-butanol in rats. 

Neurotoxicol Teratol 11(3):313–315. 

Nelson, BK; Brightwell, WS; Krieg, EFJ. (1990) Developmental toxicology of industrial alcohols: a summary of 

13 alcohols administered by inhalation to rats. Toxicol Ind Health 6(3-4):373–387. 

NRC (National Research Council). (1983) Risk assessment in the federal government: managing the process. 

Washington, DC: National Academy Press. 

Obe, G; Ristow, H. (1977) Acetaldehyde, but not ethanol, induces sister chromatid exchanges in Chinese hamster 

cells in vitro. Mutat Res 56:211–213. 

Obe, G; Ristow, HJ; Herha, J. (1977) Chromosomal damage by alcohol in vitro and in vivo. Adv Exp Med Biol 

85A:47–70. 

O'Neil, MJ; Heckelman, PE; Koch, CB; et al. (2006) The Merck index. 14th ed. Whitehouse Station, NJ: Merck & 

Co. Inc.; p. 253. 

Onfelt, A. (1987) Spindle disturbances in mammalian cells. III. Toxicity, c-mitosis and aneuploidy with 22 different 

compounds. Specific and unspecific mechanisms. Mutat Res 182:135–154. 

Palm, WM; Saczynski, JS; van der Grond, J; et al. (2009) Ventricular dilation. Association with gait and cognition. 

Ann Neurol 66:485–493. 

Paola Mascia, M; Gong, DH; Eger, EI; et al. (2000) The anesthetic potency of propanol and butanol versus 

propanethiol and butanethiol in alpha1 wild type and alpha1(S267Q) glycine receptors. Anesth Analg 91:1289–

1293. 

Peoples, RW; Weight, FF. (1999) Differential alcohol modulation of GABAA and NMDA receptors. Neuroreport 

10(1):97–101. 

Podlekareva, D; Pan, Z; Kjaergaard, S; et al. (2002) Irritation of the human eye mucous membrane caused by 

airborne pollutants. Int Arch Occup Environ Health 75(5):359–364. 

Poet, TS; Corley, RA; Woodstock, A. (2003a) Evaluation of the respiratory bioavailability and pharmacokinetics of 

a series of esters and alcohols in rats. Richland, WA: Batelle, Pacific Northwest National Laboratory. 

Poet, TS; Creim, JA; Pierce, JT; et al. (2003b) Evaluation of the respiratory bioavailability and pharmacokinetics of 

butyl acetate using whole-body plethysmography and gas uptake inhalation. Richland, WA: Batelle, Pacific 

Northwest National Library. 

Purchase, IFH. (1969) Studies in Kaffircorn malting and brewing. XXII. The acute toxicity of some fusel oils found 

in Bantu beer. S Afr Med J 43(25):795–798. 

Raimondi AJ. (1994) A unifying theory for the definition and classification of hydrocephalus. Childs Nerv Syst 

10:2–12. 

Research Triangle Institute. (1985) Rat oral subchronic toxicity study. Submitted under TSCA Section 4; EPA 

Document No. 44-8630046; NTIS No. OTS0531032. 
101 DRAFT - DO NOT CITE OR QUOTE

Rumyantsev, AP; Ostroumova, NA; Astapoval, SA; et al. (1976) Sanitary toxicological features of butyl alcohol 

under conditions of prolonged inhalation route entry. Gig Sanit 11:12–15. (Russian) 

Rusch, D; Musset, B; Wulf, H; et al. (2007) Subunit-dependent modulation of the 5-hydroxytryptamine type 3 

receptor open-close equilibrium by n-alcohols. J Pharmacol Exp Ther 321(3):1069–1074. 

Saillenfait, AM; Gallissot, F; Sabate, JP; et al. (2007) Developmental toxic effects of ethylbenzene or toluene alone 

and in combination with butyl acetate in rats after inhalation exposure. J Appl Toxicol 27(1):32–42. 

Sakata-Haga, H; Sawada, K; Ohnishi, T; et al. (2004) Hydrocephalus following prenatal exposure to ethanol. Acta 

Neuropathol (Berl) 108:393–398. 

Scheuplein, RJ; Blank, IH. (1976) Mechanism of percutaneous absorption. IV. Penentration of nonelectrolytes 

(alcohols) from aqueous solutions and from pure liquids. J Invest Dermatol 60:286–296. 

Seitz, B. (1972) Occurrence of serious vertigo after handling of butanol and isobutanol: three cases. Archives Mal 

Prof Med Trav Secur Soc 33:393–395. (French) 

Shahidullah, M; Harris, T; Germann, MW; et al. (2003) Molecular features of an alcohol binding site in a neuronal 

potassium channel. Biochemistry 42:11243–11252. 

Shehata, M; Saad, S. (1978) The effect of aliphatic alcohols on certain vitamins of the B-complex group in the liver 

of the rat. Pol J Pharmacol Pharm 30(1):35–38. 

Sitarek, K; Berlinska, B; Baranski, B. (1994) Assessment of the effect of n-butanol given to female rats in drinking 

water on fertility and prenatal development of their offspring. Int J Occup Med Environ Health 7(4):365–370. 

Smith, M; Hopkinson, DA; Harris, H (1971). Developmental changes and polymorphisms in human alcohol 

dehydrogenase. Ann Hum Genet 34:251-271. 

Smyth, HF; Smyth, HFJ. (1928) Inhalation experiments with certain laquer solvents. J Ind Hyg 10:261–271. 

Sterner, JH; Crouch, HC; Brockmyre, HF; et al. (1949) A ten-year study of butyl alcohol exposure. Am Ind Hyg 

Assoc Q 10(3):53–59. 

Stevens, R; Rusch, D; Solt, K; et al. (2005) Modulation of human 5-hydroxytryptamine type 3AB receptors by 

volatile anesthetics and n-alcohols. J Pharmacol Exp Ther 314(1):338–345. 

Strubelt, O; Zetler, G. (1980) Anti-inflammatory effect of ethanol and other alcohols on rat paw edema and pleurisy. 

Agents Actions 10(3):279–286. 

Strubelt, O; Deters, M; Pentz, R; et al. (1999) The toxic and metabolic effects of 23 aliphatic alcohols in the isolated 

perfused rat live. Toxicol Sci 49:133–142. 

Swiercz, R; Korsak, Z; Rydzynski, K. (1995) Kinetics of n-butyl alcohol and m-xylene in blood during single and 

combined inhalation exposure in rats. Int J Occup Med Environ Health 8(4):361–365. 

Tabershaw, IR; Fahy, JP; Skinner, JB. (1944) Industrial exposure to butanol. J Ind Hyg Toxicol 26:328–330. 

Teeguarden, JG; Deisinger, PJ; Poet, TS; et al. (2005) Derivation of a human equivalent concentration for n-butanol 

using a physiologically based pharmacokinetic model for n-butyl acetate and metabolites n-butanol and n-butyric 

acid. Toxicol Sci 85:429–446. 

Teschke, R; Hasumura, Y; Lieber, CS. (1974) NADPH-dependent oxidation of methanol, ethanol, propanol and 

butanol by hepatic microsomes. Biochem Biophys Res Commun 60(2):851–857. 

Tewari, YB; Miller, MM; Wasik, SP; et al. (1982) Aqueous solubility and octanol/water partition coefficient of 

organic compounds at 25.0C. J Chem Eng Data 27:451–454. 
102 DRAFT - DO NOT CITE OR QUOTE

Tichy, M; Trcka, V; Roth, Z; et al. (1985) QSAR analysis and data extrapolation among mammals in a series of 

aliphatic alcohols. Environ Health Perspect 61:321–328. 

Timms, GP; Holmes, RS. (1981) Genetics and ontogeny of aldehyde dehydrogenase isozymes in the mouse: 

evidence for a locus controlling the inducibility of the liver microsomal isozyme. Biochem Genet 19(11-12): 1223-

36. 

Triebig, G; Schaller, KH; Weltle, D. (1992) Neurotoxicity of solvent mixtures in spray painters. Int Arch Occup 

Environ Health 64(5):353–359. 

Tucek, M; Tenglerova, M; Kvasnickova, M; et al. (2002) Effect of acrylate chemistry on human health. Int Arch 

Occup Environ Health 75(Suppl 1):S67–S72. 

U.S. EPA (Environmental Protection Agency). (1986a) Guidelines for the health risk assessment of chemical 

mixtures. Federal Register 51(185):34014–34025. Available online at http://www.epa.gov/iris/backgr-d.htm 

(accessed January 15, 2009). 

U.S. EPA (Environmental Protection Agency). (1986b) Guidelines for mutagenicity risk assessment. Federal 

Register 51(185):34006–34012. Available online at http://www.epa.gov/iris/backgr-d.htm (accessed January 15, 

2009). 

U.S. EPA (Environmental Protection Agency). (1988) Recommendations for and documentation of biological values 

for use in risk assessment. Prepared by the Environmental Criteria and Assessment Office, Office of Health and 

Environmental Assessment, Cincinnati, OH for the Office of Solid Waste and Emergency Response, Washington, 

DC; EPA 600/6-87/008. Available online at http://www.epa.gov/iris/backgr-d.htm (accessed January 15, 2009). 

U.S. EPA (Environmental Protection Agency). (1991) Guidelines for developmental toxicity risk assessment. 

Federal Register 56(234):63798-63826. Available online at http://www.epa.gov/iris/backgr-d.htm (accessed January 

15, 2009). 

U.S. EPA (Environmental Protection Agency). (1994a) Interim policy for particle size and limit concentration issues 

in inhalation toxicity studies. Federal Register 59(206):53799. Available online at http://www.epa.gov/iris/backgrd.htm (accessed January 15, 2009). 

U.S. EPA (Environmental Protection Agency). (1994b) Methods for derivation of inhalation reference 

concentrations and application of inhalation dosimetry. Office of Research and Development, Washington, DC; 

EPA/600/8-90/066F. Available online at http://www.epa.gov/iris/backgr-d.htm (accessed January 15, 2009). 

U.S. EPA (Environmental Protection Agency). (1995) Use of the benchmark dose approach in health risk 

assessment. Risk Assessment Forum, Washington, DC; EPA/630/R-94/007. Available online at 

http://cfpub.epa.gov/ncea/raf/recordisplay.cfm?deid=42601 (accessed January 15, 2009). 

U.S. EPA (Environmental Protection Agency). (1996) Guidelines for reproductive toxicity risk assessment. Federal 

Register 61(212):56274–56322. Available online at http://www.epa.gov/iris/backgr-d.htm (accessed January 15, 

2009). 

U.S. EPA (Environmental Protection Agency). (1998) Guidelines for neurotoxicity risk assessment. Federal 

Register 63(93):26926–26954. Available online at http://www.epa.gov/iris/backgr-d.htm (accessed January 15, 

2009). 

U.S. EPA (Environmental Protection Agency). (2000a) Science policy council handbook: risk characterization. 

Office of Science Policy, Office of Research and Development, Washington, DC; EPA 100-B-00-002. Available 

online at http://www.epa.gov/iris/backgr-d.htm (accessed January 15, 2009). 

U.S. EPA (Environmental Protection Agency). (2000b) Benchmark dose technical guidance document. External 

review draft. Risk Assessment Forum, Washington, DC; EPA/630/R-00/001. Available online at 

http://www.epa.gov/iris/backgr-d.htm (accessed January 15, 2009). 
103 DRAFT - DO NOT CITE OR QUOTE

U.S. EPA (Environmental Protection Agency). (2000c) Supplementary guidance for conducting health risk 

assessment of chemical mixtures. Risk Assessment Forum, Washington, DC; EPA/630/R-00/002. Available online 

at http://www.epa.gov/iris/backgr-d.htm (accessed January 15, 2009). 

U.S. EPA (Environmental Protection Agency). (2002) A review of the reference dose and reference concentration 

processes. Risk Assessment Forum, Washington, DC; EPA/630/P-02/0002F. Available online at 

http://www.epa.gov/iris/backgr-d.htm (accessed January 15, 2009). 

U.S. EPA (Environmental Protection Agency). (2005a) Guidelines for carcinogen risk assessment. Risk Assessment 

Forum, Washington, DC; EPA/630/P-03/001B. Available online at http://www.epa.gov/iris/backgr-d.htm (accessed 

January 15, 2009). 

U.S. EPA (Environmental Protection Agency). (2005b) Supplemental guidance for assessing susceptibility from 

early-life exposure to carcinogens. Risk Assessment Forum, Washington, DC; EPA/630/R-03/003F. Available 

online at http://www.epa.gov/iris/backgr-d.htm (accessed January 15, 2009). 

U.S. EPA (Environmental Protection Agency). (2006a) Science policy council handbook: peer review. Third 

edition. Office of Science Policy, Office of Research and Development, Washington, DC; EPA/100/B-06/002. 

Available online at http://www.epa.gov/iris/backgr-d.htm (accessed January 15, 2009). 

U.S. EPA (Environmental Protection Agency). (2006b) A Framework for Assessing Health Risk of Environmental 

Exposures to Children. National Center for Environmental Assessment, Washington, DC, EPA/600/R-05/093F. 

Available online at http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=158363 (accessed January 15, 2009). 

Velazquez, J; Escobar, R; Almaraz, A. (1969) Audiologic impairment due to n-butyl alcohol exposition. In: 

Proceedings of the XVI International Congress on Occupational Health, Tokyo, Japan, 22-27 September 1969 Japan 

Industrial Safety Association. 

Videla, LA; Fernandez, V; de Marinis, A. (1982) Liver lipoperoxidative pressure and glutathione status following 

acetaldehyde and aliphatic alcohols pretreatments in the rat. Biochem Biophys Res Commun 104(3):965–970. 

Wakabayashi, T; Adachi, K; Popinigis, J. (1991) Effects of alkyl alcohols and related chemicals on rat liver structure 

and function: I. Induction of two distinct types of megamitochondria. Acta Pathol Jpn 41(6):405–413.

Wallgren, H. (1960) Relative intoxicating effects on rats of ethyl, propyl and butyl alcohols. Acta Pharmacol 

Toxicol (Copenh) 16:217–222. 

Weese, H. (1928) Comparative studies on the efficacy and toxicity of the vapours of low aliphatic alcohols. 

Naunyn-Schmiedebergs Arch Exp Pathol Pharmakol 135:118–130. (German) 

Wieichert, J; Hartge, D; Knapp, M; et al. (2010) Prevalence, characteristics and perinatal outcome of fetal 

ventriculomegaly in 29,000 pregnancies followed at a single institution. Fetal Diagn Ther 27:142–148.

WHO (World Health Organization). (1987) Environmental health criteria 65. Butanols: Four isomers. United 

Nations Environment Programme. International Labour Organisation. World Health Organization. Available online 

at http://www.inchem.org/documents/ehc/ehc/ehc65.htm (accessed August 16, 2009). 

Winer, AD. (1958) A note on the substrate specificity of horse liver alcohol dehydrogenase. Acta Chem Scand 

12:1695–1696. 

Wolford, ST; Schroer, RA; Gohs, FX; et al. (1986) Reference range data base for serum chemistry and hematology 

values in laboratory animals. J Toxicol Environ Health 18:161–188. 

Ye, Q; Koltchine, V; Mihic, SJ; et al. (1998) Enhancement of glycine receptor function by ethanol is inversely 

correlated with molecular volume at position alpha267. J Biol Chem 273(6):3314–3319. 

Yoshiyama, Y; Nagai, K; Some, H; et al. (1973) Selective inhibition by pantoyl lactone and butyl alcohol of the 

initiation of DNA replication in E. coli. Agric Biol Chem 37:1317–1320. 
104 DRAFT - DO NOT CITE OR QUOTE

Zhou, Q; Verdoorn, TA; Lovinger, DM. (1998) Alcohols potentiate the function of 5-HT3 receptor-channels on 

NCB-20 neuroblastoma cells by favouring and stabilizing the open channel state. J Physiol 507(2):335–352. 

Zuo, Y; Aistrup, GL; Marszalec, W; et al. (2001) Dual action of n-alcohols on neuronal nicrotinic 

acetylcholine receptors. Mol Pharmacol 60(4):700–711. 

Zuo, Y; Yeh, JZ; Narahashi, T. (2003) Dual action of n-butanol on neuronal nicotinic α4β2 acetylcholine receptors. 

J Pharmacol Exp Ther 304(3):1143–1152. 
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1 APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC 

2 COMMENTS AND DISPOSITION 

3 

4 (page intentionally left blank) 

5 
B-1 DRAFT - DO NOT CITE OR QUOTE

1 APPENDIX B. BENCHMARK DOSE CALCULATIONS 

2 

3 

4 In the study by Sitarek et al. (1994), a LOAEL of 300 mg/kg-day was identified based on 

5 increased incidences of developmental visceral malformations (dilation of the lateral and/or third 

6 ventricle, subarachnoid spaces of the brain and dilation of renal pelvis) in the offspring of treated 

7 female rats. The teratogenic effects were not amenable to modeling with nested developmental 

8 toxicity dose-response models, as these models require knowledge of the litters to which 

9 individual animals belonged, and such data were not reported by Sitarek et al. (1994). However, 

10 the percentages of affected litters were reported in the study; these data (see Table B-1 below) 

11 were subjected to BMD modeling to identify possible PODs for RfD derivation. 

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) – overall 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 ventricle 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 

14 All available dichotomous models in the EPA BMDS (version 2.1.1) were fit to the litter 

15 data with overall observed visceral malformations. This data included a combined incidence of 

16 dilation amongst four measured parameters: subarachnoid space, lateral and/or third ventricle, 

17 unilateral renal pelvis, and bilateral renal pelvis. A BMR of 10% was selected, and the BMD and 

18 BMDL associated with this BMR were estimated using the best-fit model for each dataset. 

19 Detailed modeling results are shown in Table B-2. Figure B-1 shows the best-fitting models for 

20 this endpoint . 

21 Of the models exhibiting adequate fit, a “best-fit” model was selected consistent with the 

22 EPA’s Benchmark Dose Technical Guidance Document (U.S. EPA, 2000b) as follows. If the 

23 BMDL estimates from the models exhibiting adequate fit are “sufficiently close,” then the model 

24 with the lowest AIC is used to estimate the BMDL from which the POD will be derived. Under 

25 the U.S. EPA’s Benchmark Dose Technical Guidance Document (U.S. EPA, 2000), the log26 probit model results using the full dataset provide the best fit to the data and was the BMDL10

27 from this model was used as the POD for RfD derivation.
B-2 DRAFT - DO NOT CITE OR QUOTE

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-valuea

Scaled residual 

of interestb AIC 

BMD10

(mg/kg-d)

BMDL10

(mg/kg-d) 

Gammac 2 1.28 0.53 -0.20 41.16 48 31 

Logistic 2 4.04 0.13 -1.24 44.28 115 74 

Log-Logisticd,e 1 0.37 0.54 -0.01 42.49 36 10 

Log-Probitd 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 

Weibullc 2 1.28 0.53 -0.20 41.16 48 31 

aValues <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. 

eBest-fitting model; the only model with adequate fit. 

fBetas restricted to ≥0. 

Source: Sitarek et al. (1994). 

Figure B-1. Fit of log-probit model to data on incidence 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 5

Fraction Affected

dose

LogProbit Model with 0.95 Confidence Level

11:54 03/16 2011

BMDL BMD

 

LogProbit
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1 ==================================================================== 

2 Probit Model. (Version: 3.1; Date: 05/16/2008) 

3 Input Data File: C:\Usepa\BMDS21\Data\lnpSitarekSetting.(d) 

4 Gnuplot Plotting File: C:\Usepa\BMDS21\Data\lnpSitarekSetting.plt 

5 Wed Mar 16 12:54:08 2011 

6 ==================================================================== 

7 

8 BMDS Model Run 

9 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 

10 

11 The form of the probability function is: 

12 

13 P[response] = Background 

14 + (1-Background) * CumNorm(Intercept+Slope*Log(Dose)), 

15 

16 where CumNorm(.) is the cumulative normal distribution function 

17 

18 

19 Dependent variable = Effect 

20 Independent variable = Dose 

21 Slope parameter is restricted as slope >= 1 

22 

23 Total number of observations = 4 

24 Total number of records with missing values = 0 

25 Maximum number of iterations = 250 

26 Relative Function Convergence has been set to: 1e-008 

27 Parameter Convergence has been set to: 1e-008 

28 

29 

30 

31 User has chosen the log transformed model 

32 

33 

34 Default Initial (and Specified) Parameter Values 

35 background = 0.0833333 

36 intercept = 0.408347 

37 slope = 1 

38 

39 

40 Asymptotic Correlation Matrix of Parameter Estimates 

41 

42 ( *** The model parameter(s) -slope 

43 have been estimated at a boundary point, or have been specified by the user, 

44 and do not appear in the correlation matrix ) 

45 

46 background intercept 

47 

48 background 1 -0.32 

49 

50 intercept -0.32 1 

51 

52 

53 

54 Parameter Estimates 

55 

56 95.0% Wald Confidence Interval 

57 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 

58 background 0.0908047 0.0863715 -0.0784803 0.26009 

59 intercept 1.25846 0.309188 0.652459 1.86445 

60 slope 1 NA 

61 

62 NA - Indicates that this parameter has hit a bound 

63 implied by some inequality constraint and thus

64 has no standard error. 

65 

66 

67 
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1 Analysis of Deviance Table 

2 

3 Model Log(likelihood) # Param's Deviance Test d.f. P-value 

4 Full model -17.9734 4 

5 Fitted model -18.4722 2 0.997729 2 0.6072 

6 Reduced model -31.2787 1 26.6106 3 <.0001 

7 

8 AIC: 40.9444 

9 

10 

11 Goodness of Fit 

12 Scaled 

13 Dose Est._Prob. Expected Observed Size Residual 

14 ------------------------------------------------------------------------ 

15 0.0000 0.0908 1.090 1.000 12 -0.090 

16 0.3000 0.5652 7.912 9.000 14 0.586 

17 1.0000 0.9053 10.864 10.000 12 -0.852 

18 5.0000 0.9981 8.983 9.000 9 0.130 

19 

20 Chi^2 = 1.10 d.f. = 2 P-value = 0.5784

21 

22 

23 Benchmark Dose Computation 

24 

25 Specified effect = 0.1 

26 

27 Risk Type = Extra risk 

28 

29 Confidence level = 0.95 

30 

31 BMD = 0.0788658 

32 

33 BMDL = 0.047882 
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1 

2 APPENDIX C. n-BUTANOL PBPK MODEL 

3 C.1. Model Code 

4 PROGRAM BUTYL.CSL -- PBPK Model for Butyl Acetate and Its Butyl-Series Metabolites 

5 ! Modified version of BANew8I.csl 

6 ! Output units for this model are umoles and mL (or grams) 

7 

8 ! Modified from BANew6.CSL -- November, 2001 

9 ! Last Modified: Oct. 6, 1998 

10 ! Modified by: Hugh A. Barton 

11 

12 ! This version runs in kg as modified by Justin Teeguarden 6.9.99 

13 ! Validated 6.9.99 by Justin Teeguarden 

14 

15 ! Modified 7.11.99 by Justin Teeguarden (of BANEW2) to include metabolism of 

16 ! EtAcin other perfused tissues. AUC of EtAc under Deisiv2 for this version 

17 ! matches BANEW2.CSL with Metabolism in Other Perfused Tissues=0. 

18 

19 ! Modified 6.20.00 by JGT changing the CEx1, CEX2, CEX3, CEX4 to use Free blood 

20 ! concentration instead of lung concentration. 

21 

22 ! Revised November 2001, Justin Teeguarden. Removed URT for butyl acetate. 

23 ! Added closed chamber for BuOH and FA (fractional uptake term) for Butyl 

24 ! Acetate. These changes were made to model the butyl acetate closed chamber 

25 ! data from Batelle, Poet and Corely 2001. Mass balance OK. Without the URT, 

26 ! the inhaled amount for the GrothInh2 simulation using *8.csl is the same as 

27 ! *5.csl (the minor difference is due to an effective tstop which is a bit 

28 ! different.) This served as the validation for the removal of the URT. 

29 

30 ! Alveolar Ventilation Rate Tables 1-7 QA'd 12.19.01 JGT 

31 

32 ! NOTE: All units are expressed as mL(g), hr, or umol in the derivative section 

33 

34 ! This version has inhalation, IV and oral dosing. 

35 ! It uses a table command for the inhalation dose using the values from the 

36 ! study in the Groth paper. 

37 

38 ! Parameters followed by 1,2,3,4 stand for: 

39 ! Submodel 1: n-Butyl acetate 

40 ! Submodel 2: n-Butanol 

41 ! Submodel 3: n-Butyraldehyde 

42 ! Submodel 4: n-Butyric acid 

43 

44 !******************************************************************************* 

45 !Revised and imported into acslXtreme v 2.5.0.6 as "BUTYLAC.csl" on 04/23/2010 (G Diamond/SRC) 

46 !Revisions are marked with "!" and "<GD> 

47 ! Handling of exposure/dose parameters standardized to: inputs ppm/mg/kg --> convert to umol/L 

48 or umol/kg --> 

49 ! inputs ppm/mg/kg --> convert to umol/L or umol/kg --> adjust for dose duration (e.g., < 1hr) 

50 ! --> apply timing pulse functions (i.e., on/off) --> Integration functions 

51 ! CINT revised from function of CintC and TChng to a CONSTANT 

52 ! TChng revised to route specific dose averaging time CONSTANTs (e.g. IVDAT, ORDAT) 

53 !******************************************************************************* 

54 ! Modified on 02/25/2011 by AFS to create “clean” version: 

55 ! Legacy code, and commented-out lines of code have been removed. Some formatting changes 

56 ! made to ensure proper word-wrap in documents. Code was tested to ensure it properly ran 

57 !******************************************************************************* 
C-2 DRAFT - DO NOT CITE OR QUOTE

1 

2 INITIAL 

3 !<GD> 

4 INTEGER Rats, QPTable, Tube, INHTABLE !<GD> 

5 CONSTANT BW = 0.30 ! Body weight (kg) 

6 CONSTANT QPC = 14000.0 ! Alveolar ventilation (mL/hr/kg**0.75) 

7 

8 ! Blood Flows (fraction of cardiac output) 

9 CONSTANT QCC = 14000.0 ! Cardiac output (mL/hr/kg**0.75) 

10 CONSTANT QFatC = 0.07 ! Fat 

11 CONSTANT QLivC = 0.175 ! Liver 

12 

13 ! Tissue Volumes (fraction of body weight) 

14 CONSTANT VABC = 0.022 ! Arterial blood 

15 CONSTANT VFatC = 0.07 ! Fat 

16 CONSTANT VLivC = 0.037 ! Liver 

17 CONSTANT VLungC = 0.005 ! Lung tissue 

18 CONSTANT VOthC = 0.751 ! Other perfused tissues 

19 CONSTANT VVBC = 0.045 ! Venous blood 

20 

21 ! Partition Coefficients 

22 ! Butyl Acetate 

23 CONSTANT PB1 = 89.4 ! Blood:air 

24 CONSTANT PFat1 = 17.0 ! Fat:blood 

25 CONSTANT PLiv1 = 3.14 ! Liver:blood 

26 CONSTANT PLung1 = 1.76 ! Lung:blood 

27 CONSTANT POth1 = 1.76 ! Other perfused tissues:blood 

28 

29 ! n-Butanol 

30 CONSTANT PB2 = 1160.0 ! Blood:air 

31 CONSTANT PLiv2 = 1.08 ! Liver:blood 

32 CONSTANT PLung2 = 0.78 ! Lung:blood 

33 CONSTANT POth2 = 0.78 ! Other perfused tissues:blood 

34 

35 ! n-Butyraldehyde 

36 CONSTANT PB3 = 1160.0 ! Blood:air 

37 CONSTANT PLiv3 = 1.08 ! Liver:blood 

38 CONSTANT PLung3 = 0.78 ! Lung:blood 

39 CONSTANT POth3 = 0.78 ! Other perfused tissues:blood 

40 

41 ! Butyric Acid 

42 CONSTANT PB4 = 1160.0 ! Blood:air 

43 CONSTANT PLiv4 = 1.08 ! Liver:blood 

44 CONSTANT PLung4 = 0.78 ! Lung:blood 

45 CONSTANT POth4 = 0.78 ! Other perfused tissues:blood 

46 

47 ! Metabolism 

48 ! Butyl Acetate 

49 CONSTANT VMBloodC1 = 600000.0 ! Blood (umole/hr/kg**0.75) 

50 CONSTANT KMBlood1 = 100.0 ! Blood (umole/mL) 

51 CONSTANT VMLivC1 = 38700.0 ! Liver (umole/hr/kg**0.75) 

52 CONSTANT KMLiv1 = 1.0 ! Liver (umole/mL) 

53 CONSTANT VMOthC1 = 6.0e6 ! Other Perfused Tissues (umole/hr/kg**0.75) 

54 CONSTANT KMOth1 = 100.0 ! Other Perfused Tissues (umole/mL) 

55 

56 ! n-Butanol 

57 CONSTANT VMLivC2 = 2170.0 ! Liver (umole/hr/kg**0.75) <GD> 

58 CONSTANT KMLiv2 = 0.16 ! Liver (umole/mL) <GD> 
C-3 DRAFT - DO NOT CITE OR QUOTE

1 CONSTANT kOthC2 = 4.0 ! First-order BuOH clearance (kg**0.25/hr) <GD> 

2 

3 ! n-Butyraldehyde 

4 CONSTANT VMLivC3 = 17780.0 ! Liver (umole/hr/kg**0.75) 

5 CONSTANT KMLiv3 = 0.1 ! Liver (umole/mL) 

6 

7 ! Butyric Acid 

8 CONSTANT VMLivC4 = 1400.0 ! Liver (umole/hr/kg**0.75) 

9 CONSTANT KMLiv4 = 0.1 ! Liver (umole/mL) 

10 CONSTANT VMOthC4 = 3000.0 ! OPT (umole/hr/kg**0.75) 

11 CONSTANT KMOth4 = 0.1 ! OPT (umole/mL) 

12 

13 ! Filtration (blood-urine) from Venous Blood ((kg**0.25)/hr) 

14 CONSTANT kFiltC1 = 7.401e-2 ! Butyl Acetate 

15 CONSTANT kFiltC2 = 22.2 ! n-Butanol 

16 CONSTANT kFiltC4 = 7.41e-2 ! Butyric Acid 

17 

18 ! Fractional Inhalation Absorption - Butyl Acetate, Butanol 

19 CONSTANT FA1 = 1.0 

20 CONSTANT FA2 = 0.5 

21 CONSTANT FA3 = 1.0 

22 CONSTANT FA4 = 1.0 

23 

24 ! Oral Absorption 

25 CONSTANT kAC = 0.0 ! Absorption rate (kg**0.25/hr) 

26 

27 ! Initial Amounts in Arterial Blood (i.e., integration start value) 

28 CONSTANT AAB10C = 0.0 ! BuAc (mg/kg) 

29 CONSTANT AAB20C = 0.0 ! BuOH (mg/kg) 

30 CONSTANT AAB30C = 0.0 ! BuCHO (mg/kg) 

31 CONSTANT AAB40C = 0.0 ! BuCOOH (mg/kg) 

32 

33 ! Molecular Weights <GD> 

34 CONSTANT MW1 = 116.16 ! BuAc 

35 CONSTANT MW2 = 74.12 ! BuOH 

36 CONSTANT MW3 = 72.11 ! BuCHO 

37 CONSTANT MW4 = 88.11 ! BuCOOH 

38 

39 ! Simulation Parameters 

40 CONSTANT TStop = 0.5 ! Length of experiment (hr) 

41 CINTERVAL CINT = 1. !Communication interval (hr) 

42 !CINT = CIntC <GD> 

43 

44 ! Intravenous Dosing Parameters 

45 CONSTANT IVDose1 = 0.0 ! BuAc (mg/kg) 

46 CONSTANT IVDose2 = 0.0 ! BuOH (mg/kg) 

47 CONSTANT IVDose3 = 0.0 ! BuCHO (mg/kg) 

48 CONSTANT IVDose4 = 0.0 ! BuCOOH (mg/kg) 

49 

50 IVDUM1= IVDose1 * 1000 * BW/MW1 !BuAc (umol) <GD> 

51 IVDUM2= IVDose2 * 1000 * BW/MW2 !BuOH (umol) <GD> 

52 IVDUM3= IVDose3 * 1000 * BW/MW3 !BuCHO (umol) <GD> 

53 IVDUM4= IVDose4 * 1000 * BW/MW4 !BuCOOH (umol <GD> 

54 

55 CONSTANT IVDAT=1. !IV injection averaging time (hr; for infusion, IVDAT = 1.) <GD> 

56 CONSTANT IVDON=0. !IV start time (hr) <GD> 

57 CONSTANT IVDOFF=0. !IV stop time (for single injection, IVDOFF must = IVDAT) <GD> 

58 
C-4 DRAFT - DO NOT CITE OR QUOTE

1 ! Oral Dosing Parameters 

2 CONSTANT PDose1 = 0.0 ! BuAc (mg/kg) 

3 CONSTANT PDose2 = 0.0 ! BuOH (mg/kg) 

4 CONSTANT PDose3 = 0.0 ! BuCOO (mg/kg) 

5 CONSTANT PDose4 = 0.0 ! BuCOOH (mg/kg) 

6 

7 ORDUM1 = PDose1 * 1000 * BW/MW1 !BuAc (umol) <GD> 

8 ORDUM2 = PDose2 * 1000 * BW/MW2 !BuOH (umol) <GD> 

9 ORDUM3 = PDose3 * 1000 * BW/MW3 !BuCOO (umol) <GD> 

10 ORDUM4 = PDose4 * 1000 * BW/MW4 !BuCOOH (umol) <GD> 

11 

12 CONSTANT ORDON=0. !Exposure start (hr) <GD> 

13 CONSTANT ORDOFF=0. !Exposure stop (hr) <GD> 

14 CONSTANT ORDPER1=20000.!Exposure pulse period (e.g. 24-hr day) <GD> 

15 CONSTANT ORDWID1=20000.!Exposure pulse 1 width (e.g. hr/day)) <GD> 

16 CONSTANT ORDPER2=20000.!Exposure pulse period (e.g. 168-hr wk) <GD> 

17 CONSTANT ORDWID2=20000.!Exposure pulse 1 width (120 hr/wk) <GD> 

18 CONSTANT ORDAT=1.0 !Dosing averaging time for single oral dose (e.g. gavage) <GD> 

19 

20 ! Inhalation Dosing Paramters 

21 CONSTANT INHTABLE = 0 ! Switch to use inhlation exposures from INHTABLE (=1) 

22 

23 CONSTANT CInh1 = 0.0 ! BuAc (ppm) 

24 CONSTANT CInh2 = 0.0 ! BuOH (ppm) 

25 CONSTANT CInh3 = 0.0 ! BuCOO (ppm) 

26 CONSTANT CInh4 = 0.0 ! BuCOOH (ppm) 

27 CONSTANT QPTable = 0 ! Set to .T. for using observed MV data <GD> 

28 

29 CIUM1= CInh1/24450 !BuAc (umol/ml) <GD> 

30 CIUM2= CInh2/24450 !BuOH (umol/ml) <GD> 

31 CIUM3= CInh3/24450 !BuCOO (umol/ml) <GD> 

32 CIUM4= CInh4/24450 !BuCOOH (umol/ml) <GD> 

33 

34 CONSTANT CIOFFPPM1=0. !BuAc conc. when inhalation exposure is off (ppm) <GD> 

35 CONSTANT CIOFFPPM2=0. !BuOH conc. when inhalation exposure is off (ppm) <GD> 

36 CONSTANT CIOFFPPM3=0. !BuCOO conc. when inhalation exposure is off (ppm) <GD> 

37 CONSTANT CIOFFPPM4=0. !BuCOOH conc. when inhalation exposure is off (ppm) <GD> 

38 

39 CIOFF1= CIOFFPPM1/24450 !BuAc conc. when inhalation exposure is off (umol/ml) <GD> 

40 CIOFF2= CIOFFPPM2/24450 !BuOH conc. when inhalation exposure is off (umol/ml) <GD> 

41 CIOFF3= CIOFFPPM3/24450 !BuCOO conc. when inhalation exposure is off (umol/ml) <GD> 

42 CIOFF4= CIOFFPPM4/24450 !BuCOOH conc. when inhalation exposure is off (umol/ml) <GD> 

43 

44 CONSTANT INHON=0. !Time inhalation exposure starts (hr) <GD> 

45 CONSTANT INHOFF= 10. !Time exposure stops (hr) <GD> 

46 CONSTANT INHPER1=20000. !Pulse period 1 for exposure (e.g., 24-hr day) <GD> 

47 CONSTANT INHWID1=20000. !Pulse width 1 (e.g. 6 hr/day) <GD> 

48 CONSTANT INHPER2=20000. !Pulse period 2 (e.g. 168-hr wk) <GD> 

49 CONSTANT INHWID2=20000. !Pulse width 2 (e.g. 120 hr/wk) <GD> 

50 

51 TABLE CINHTAB1,1,200/200*0.0,200*0.0/ !Call VentTableS2 from CINHTAB*.dat file <GD> 

52 TABLE CINHTAB2,1,200/200*0.0,200*0.0/ !Call VentTableS2 from CINHTAB*.dat file <GD> 

53 TABLE CINHTAB3,1,200/200*0.0,200*0.0/ !Call VentTableS2 from CINHTAB*.dat file <GD> 

54 TABLE CINHTAB4,1,200/200*0.0,200*0.0/ !Call VentTableS2 from CINHTAB*.dat file <GD> 

55 !Deleted Table values in CSL and replaced with m-files that fill tables <GD> 

56 

57 
C-5 DRAFT - DO NOT CITE OR QUOTE

1 ! Inhalation Dose for Closed Chamber 

2 CONSTANT SCInPPM1 = 0.0 ! BuAc (ppm) 

3 CONSTANT SCInPPM2 = 0.0 ! BuOH (ppm) 

4 CONSTANT SCInPPM3 = 0.0 ! BuCHO (ppm) 

5 CONSTANT SCInPPM4 = 0.0 ! BuCOOH (ppm) 

6 

7 CICHUM1= SCInPPM1/24450 !BuAc (umol/ml) <GD> 

8 CICHUM2= SCInPPM2/24450 !BuOH (umol/ml) <GD> 

9 CICHUM3= SCInPPM3/24450 !BuCOO (umol/ml) <GD> 

10 CICHUM4= SCInPPM4/24450 !BuCOOH (umol/ml) <GD> 

11 

12 ACh01 = 0.0 ; ACh02 = 0.0 ; ACh03 = 0.0 ; ACh04 = 0.0 !<GD> 

13 ACh01 = VCh*CICHUM1 !BuAc (umol) <GD> 

14 ACh02 = VCh*CICHUM2 !BuOH (umol) <GD> 

15 ACh03 = VCh*CICHUM3 !BuCOO (umol) <GD> 

16 ACh04 = VCh*CICHUM4 !BuCOOH (umol) <GD> 

17 

18 TABLE VentTable,1,200/200*0.0,200*0.0/ !Call VentTable from *.dat file <GD> 

19 !Deleted Table values <GD> 

20 

21 ! Closed Chamber Parameters 

22 ! If a Plethysmorgraph tube is used, the given volume of the chamber should be 

23 ! used directly without subtraction of the volume of the rat (number of rats x BW). 

24 ! Set Tube=.T. 

25 CONSTANT ClOn = 0.0 !Switch for closed chamber (=1), or open (=0) 

26 CONSTANT VChC = 1.0e10 ! Volume of closed chamber (L) 

27 CONSTANT Rats = 0 ! Number of rats in chamber 

28 CONSTANT Tube = 0 !Switch to include Tube (=1) 

29 CONSTANT LossT = 0.0 ! Time to switch to second chamber loss term 

30 

31 ! Closed Chamber Parameters -- Loss Rates (/hr) 

32 CONSTANT kLoss1R = 0.0 ! Rat body for n-Butyl acetate 

33 CONSTANT kLoss1C1 = 0.0 ! Chamber for n-Butyl acetate 

34 CONSTANT kLoss1C2 = 0.0 ! Chamber for n-Butyl acetate for 2nd period 

35 CONSTANT kLoss2R = 0.0 ! Rat body for n-Butanol 

36 CONSTANT kLoss2C1 = 0.0 ! Chamber for n-Butanol 

37 CONSTANT kLoss2C2 = 0.0 ! Chamber for n-Butanol for 2nd period 

38 CONSTANT kLoss3R = 0.0 ! Rat body for n-Butyraldehyde 

39 CONSTANT kLoss3 = 0.0 ! Chamber for n-Butyraldehyde 

40 CONSTANT kLoss4R = 0.0 ! Rat body for n-Butyric acid 

41 CONSTANT kLoss4 = 0.0 ! Chamber for n-Butyric acid 

42 

43 ! Blood Flows (mL/hr) 

44 QOthC = 1.0 - QFatC - QLivC ! Other perfused tissues (fraction) 

45 

46 ! Tissue Volumes (mL) 

47 VAB = VABC * BW * 1000.0 ! Arterial blood volume 

48 VFat = VFatC * BW * 1000.0 ! Fat 

49 VLiv = VLivC * BW * 1000.0 ! Liver 

50 VLung = VLungC * BW * 1000.0 ! Lung 

51 VOth = VOthC * BW * 1000.0 ! Other perfused tissues 

52 VVB = VVBC * BW * 1000.0 ! Venous blood 

53 VBlood = VAB + VVB ! Total blood 

54 ! Metabolism -- Butyl Acetate (umoles/hr) 

55 VMAB1 = VMBloodC1 * VAB/VBlood * (BW**0.75) ! Arterial metabolism BuAc 

56 VMVB1 = VMBloodC1 * VVB/VBlood * (BW**0.75) ! Venous metabolism BuAc 

57 VMLiv1 = VMLivC1 * (BW**0.75) ! Liver metabolism BuAc 

58 VMOth1 = VMOthC1 * (BW**0.75) ! Other Perfused Tissue Metabolism EtAc 

59 
C-6 DRAFT - DO NOT CITE OR QUOTE

1 ! Metabolism -- n-Butanol 

2 VMLiv2 = VMLivC2 * (BW**0.75) ! Liver metabolism BuOH (umoles/hr) 

3 kOth2 = kOthC2 / (BW**0.25) ! Other metabolism BuOH (/hr) 

4 

5 ! Metabolism -- n-Butyraldehyde (umoles/hr) 

6 VMLiv3 = VMLivC3 * (BW**0.75) ! Liver metabolism BuCHO 

7 

8 ! Metabolism -- Butyric Acid (umoles/hr) 

9 VMLiv4 = VMLivC4 * (BW**0.75) ! Liver metabolism BuCOOH 

10 VMOth4 = VMOthC4 * (BW**0.75) ! OPT metabolism BuCOOH 

11 

12 ! Filtration from Venous Blood (/hr) 

13 kFilt1 = kFiltC1 / (BW**0.25) ! Kidney Filtration of BuAc 

14 kFilt2 = kFiltC2 / (BW**0.25) ! Kidney Filtration of BuOH 

15 kFilt4 = kFiltC4 / (BW**0.25) ! Kidney Filtration of BuCOOH 

16 

17 ! Oral Absorption (/hr) 

18 kA = kAC / (BW**0.25) 

19 

20 ! Initial Amounts in Arterial Blood (i.e. integration start value) 

21 AAB10 = ((AAB10C * 1000.0)/MW1) * BW ! BuAc (umoles total) 

22 AAB20 = ((AAB20C * 1000.0)/MW2) * BW ! BuOH (umoles total) 

23 AAB30 = ((AAB30C * 1000.0)/MW3) * BW ! BuCHO (umoles total) 

24 AAB40 = ((AAB40C * 1000.0)/MW4) * BW ! BuCOOH (umoles total) 

25 

26 ! Initialize values 

27 ! CINT = CIntC 

28 DoseMod = 0.0 

29 kIV1 = 0.0 ; kIV2 = 0.0 ; kIV3 = 0.0 ; kIV4 = 0.0 

30 Conc1 = 0.0 ; Conc2 = 0.0 ; Conc3 = 0.0 ; Conc4 = 0.0 

31 Dose1 = 0.0 ; Dose2 = 0.0 ; Dose3 = 0.0 ; Dose4 = 0.0 

32 XCInh1 = 0.0 ; XCInh2 = 0.0 ; XCInh3 = 0.0 ; XCInh4 = 0.0 

33 XSCInPPM1 = 0.0 ; XSCInPPM2 = 0.0 ; XSCInPPM3 = 0.0 ; XSCInPPM4 = 0.0 

34 XPDose1 = 0.0 ; XPDose2 = 0.0 ; XPDose3 = 0.0 ; XPDose4 = 0.0 

35 XAUCTB1 = 0.0 ; XAUCTB2 = 0.0 ; XAUCTB3 = 0.0 ; XAUCTB4 = 0.0 

36 CVLiv1 = 0.0 ; CVLiv2 = 0.0 ; CVLiv3 = 0.0 ; CVLiv4 = 0.0 

37 

38 CCh1 = 0.0 ; CCh2 = 0.0 ; CCh3 = 0.0 ; CCh4 = 0.0 

39 

40 ! Closed Chamber Parameters 

41 IF (Tube .EQ. 0) THEN !<GD> 

42 VCh = VChC*1000.0 - Rats*BW*1000.0 ! Volume adjusted to mL 

43 ELSE 

44 VCh = VChC*1000.0 

45 ENDIF 

46 

47 END ! End of Initial 

48 

49 DYNAMIC 

50 ALGORITHM IALG = 2 ! Gear algorithm 

51 

52 DERIVATIVE 

53 Minutes = T * 60.0 

54 Seconds = Minutes * 60.0 

55 
C-7 DRAFT - DO NOT CITE OR QUOTE

1 PROCEDURAL !<GD> 

2 IF (QPTable .EQ. 0) THEN 

3 QP = QPC*(BW**0.75) 

4 QC = QCC*(BW**0.75) 

5 ELSE 

6 QP = VentTable(Minutes) 

7 QC = QP 

8 ENDIF 

9 QFat = QFatC * QC ! Fat 

10 QLiv = QLivC * QC ! Liver 

11 QOth = QOthC * QC ! Other perfused tissues 

12 END 

13 

14 !IV Dosing Simulation 

15 

16 IV1=IVDUM1/IVDAT !BuAc (umol/hr) <GD> 

17 IV2=IVDUM2/IVDAT !BuOH (umol/hr) <GD> 

18 IV3=IVDUM3/IVDAT !BuCOO (umol/hr) <GD> 

19 IV4=IVDUM4/IVDAT !BuCOOH (umol/hr) <GD> 

20 

21 kIV1= RSW(T.LE.IVDOFF,IV1,0.) !BuAC (umol/hr on/off) <GD> 

22 kIV2= RSW(T.LE.IVDOFF,IV2,0.) !BuOH (umol/hr on/off) <GD> 

23 kIV3= RSW(T.LE.IVDOFF,IV3,0.) !BuCOO(umol/hr on/off) <GD> 

24 kIV4= RSW(T.LE.IVDOFF,IV4,0.) !BuCOOH (umol/hr on/off) <GD> 

25 

26 !Oral Dosing Simulation <GD> 

27 IF ((PDose1+PDose2+PDose3+PDose4) .GT. 0.)THEN 

28 ORAL= PULSE(ORDON,ORDPER1,ORDWID1)*PULSE(ORDON,ORDPER2,ORDWID2) 

29 ELSE 

30 ORAL=0. 

31 END IF 

32 

33 ORAL1 = ORAL *ORDUM1/ORDAT !BuAc (umol/hr) 

34 ORAL2= ORAL *ORDUM2 /ORDAT !BuOH(umol/hr) 

35 ORAL3 = ORAL *ORDUM3 /ORDAT !BuCOO (umol/hr) 

36 ORAL4 = ORAL *ORDUM4/ORDAT !BuCOOH (umol/hr) 

37 

38 Dose1= RSW(T.LE.ORDOFF,ORAL1,0.) !BuAc(umol/hr on/off) 

39 Dose2= RSW(T.LE.ORDOFF,ORAL2,0.) !BuOH (umol/h on/off) 

40 Dose3= RSW(T.LE.ORDOFF,ORAL3,0.) !BuCOO (umol/hr on/off) 

41 Dose4= RSW(T.LE.ORDOFF,ORAL4,0.) !BuCOOH (umol/hr on/off) 

42 

43 !Inhalation Dosing Simulation <GD> 

44 

45 IF ((CInh1+CInh2+CInh+CInh4) .GT. 0.)THEN 

46 CION= PULSE(INHON,INHPER1,INHWID1)*PULSE(INHON,INHPER2,INHWID2) 

47 ELSE 

48 CION=0. 

49 END IF 

50 

51 CION1 = CION * CIUM1 !BuAc exposure (umol/ml) 

52 CION2 = CION * CIUM2 !BuOH exposure (umol/ml) 

53 CION3 = CION * CIUM3 !BuCOO exposure (umol/ml) 

54 CION4 = CION * CIUM4 !BuCOOH exposure (umol/ml) 

55 

56 Conc11 = RSW(T.LE.AIROFF,CION1,CIOFF) 

57 !BuAc conc. in inhaled air (umol/L on/off) 

58 
C-8 DRAFT - DO NOT CITE OR QUOTE

1 Conc22 = RSW(T.LE.AIROFF,CION2,CIOFF) 

2 !BuOH conc. in inhaled air (umol/L on/off) 

3 Conc33 = RSW(T.LE.AIROFF,CION3,CIOFF) 

4 !BuCOO conc. in inhaled air (umol/L on/off) 

5 Conc44 = RSW(T.LE.AIROFF,CION4,CIOFF) 

6 !BuCOOH conc. in inhaled air (umol/L on/off 

7 

8 IF (INHTABLE .EQ. 1) THEN 

9 Conc1 = CINHTAB1(minutes)/24450.0 

10 Conc2 = CINHTAB2(minutes)/24450.0 

11 Conc3 = CINHTAB3(minutes)/24450.0 

12 Conc4 = CINHTAB4(minutes)/24450.0 

13 ELSE 

14 Conc1 = Conc11 !BuAc conc. in inhaled air (umol/L on/off) 

15 Conc2 = Conc22 !BuOH conc. in inhaled air (umol/L on/off) 

16 Conc3 = Conc33 !BuCOO conc. in inhaled air (umol/L on/off) 

17 Conc4 = Conc44 !BuCOOH conc. in inhaled air (umol/L on/off 

18 ENDIF 

19 

20 !Chamber Loss simulation 

21 kLoss1= RSW(T.LE.LossT,kLoss1C1,kLoss1C2) !BuAC (umol/hr on/off) <GD> 

22 kLoss2= RSW(T.LE.LossT,kLoss2C1,kLoss2C2) !BuAC (umol/hr on/off) <GD> 

23 

24 !************************* Butyl Acetate ************************* 

25 ! Arterial Blood (umoles) 

26 RAAB1 = (QC * (CVLung1 - CAB1)) - RMetAB1 

27 AAB1 = INTEG(RAAB1, AAB10) 

28 CAB1 = AAB1 / VAB 

29 AUCAB1 = INTEG(CAB1, 0.0) 

30 

31 ! Blood Metabolism (umoles) 

32 RMetAB1 = (VMAB1 * CAB1) / (KMBlood1 + CAB1) 

33 MetAB1 = INTEG(RMetAB1, 0.0) 

34 

35 ! Fat (umole) 

36 RAFat1 = QFat * (CAB1 - CVFat1) 

37 AFat1 = INTEG(RAFat1, 0.0) 

38 CFat1 = AFat1 / VFat 

39 CVFat1 = CFat1 / PFat1 

40 

41 ! Liver (umoles) 

42 RALiv1 = (QLiv * (CAB1 - CVLiv1)) - RMetLiv1 + RMR1 

43 ALiv1 = INTEG(RALiv1, 0.0) 

44 CLiv1 = ALiv1 / VLiv 

45 CVLiv1 = CLiv1 / PLiv1 

46 AUCLiv1 = INTEG(CLiv1, 0.0) 

47 

48 ! Liver Metabolism (umoles) 

49 RMetLiv1 = (VMLiv1 * CVLiv1) / (KMLiv1 + CVLiv1) 

50 MetLiv1 = INTEG(RMetLiv1, 0.0) 

51 

52 ! Amount remaining to be absorbed into liver from GI (umoles) 

53 RMR1 = kA * MR1 

54 MR1 = Dose1 - INTEG(RMR1, 0.0) 

55 ! Chamber concentration (umoles/mL) 

56 RACh1 = (Rats * QP * CEx1) - (FA1 * Rats * QP * CCh1) - (kLoss1 * ACh1)& 

57 - (kLoss1R * Rats * ACh1) 

58 ACh1 = INTEG(RACh1, ACh01) 

59 
C-9 DRAFT - DO NOT CITE OR QUOTE

1 CCh1 = ((ACh1 / VCh) * ClOn) + (Conc1 * (1.0 - ClOn)) 

2 CCPPM1 = CCh1 * 24450.0 

3 CLoss1 = INTEG(((kLoss1 * ACh1) + (kLoss1R * Rats * ACh1)), 0.0) 

4 

5 IF (SCINPPM1 .GT. 0.) THEN 

6 CCPPM1PCT=100.*CCPPM1/SCINPPM1 !BuAc conc. as % of intitial <GD> 

7 ELSE 

8 CCPPM1PCT = 0. 

9 END IF 

10 

11 ! Lungs 

12 ! Amount in Lungs (umoles) 

13 RALung1 = (QP * ((FA1 * CCh1) - CEx1)) + (QC * (CVB1 - CVLung1)) 

14 ALung1 = INTEG(RALung1, 0.0) 

15 CLung1 = ALung1 / VLung 

16 CVLung1 = CLung1 / PLung1 

17 

18 ! Amount Inhaled (umoles) 

19 RInhal1 = FA1 * QP * CCh1 

20 AInhal1 = INTEG(RInhal1, 0.0) 

21 AInhal1C = AInhal1 * Rats 

22 

23 ! Amount Exhaled (umoles) 

24 CEx1 = CvLung1 / PB1 

25 RAEx1 = QP * CEx1 

26 AEx1 = INTEG(RAEx1, 0.0) 

27 AEx1C = AEx1 * Rats 

28 

29 ! Other Perfused Tissue Modified to Include Metabolism (umoles) 

30 RAOth1 = (QOth * (CAB1 - CVOth1)) - RMetOth1 

31 AOth1 = INTEG(RAOth1, 0.0) 

32 COth1 = AOth1 / VOth 

33 CVOth1 = COth1 / POth1 

34 AUCOth1 = INTEG(COth1, 0.0) 

35 

36 ! Other Perfused Tissue Metabolism (umoles) 

37 RMetOth1 = (VMOth1 * CVOth1) / (KMOth1 + CVOth1) 

38 MetOth1 = INTEG(RMetOth1, 0.0) 

39 

40 ! Venous Blood (umoles) 

41 RAVB1 = (QFat*CVFat1) + (QLiv*CVLiv1) + (QOth*CVOth1) + kIV1 & 

42 - (QC*CVB1) - RMetVB1 - RAFilt1 

43 AVB1 = INTEG(RAVB1, 0.0) 

44 CVB1 = AVB1 / VVB 

45 AUCB1 = INTEG(CVB1, 0.0) 

46 

47 ! Metabolism 

48 RMetVB1 = (VMVB1 * CVB1) / (KMBlood1 + CVB1) 

49 MetVB1 = INTEG(RMetVB1, 0.0) 

50 

51 ! Filtration 

52 RAFilt1 = kFilt1 * AVB1 

53 AFilt1 = INTEG(RAFilt1, 0.0) 

54 

55 ! Average Blood (umoles) 

56 CTB1 = (AAB1 + AVB1) / (VAB + VVB) 

57 AUCTB1 = INTEG(CTB1, 0.0) 

58 
C-10 DRAFT - DO NOT CITE OR QUOTE

1 !*************************** n-Butanol *************************** 

2 ! Arterial Blood (umoles) 

3 RAAB2 = (QC * (CVLung2 - CAB2)) + RMetAB1 

4 AAB2 = INTEG(RAAB2, AAB20) 

5 CAB2 = AAB2 / VAB 

6 AUCAB2 = INTEG(CAB2, 0.0) 

7 IF (T>0.) THEN !<GD> 

8 AUCAB2_24=(AUCAB2/T)*24. !AUC mM*24 hr <GD> 

9 ELSE !<GD> 

10 AUCAB2_24=0. !<GD> 

11 END IF 

12 

13 ! Liver (umoles) 

14 RALiv2 = (QLiv * (CAB2 - CVLiv2)) + RMetLiv1 + RMR2 - RMetLiv2 

15 ALiv2 = INTEG(RALiv2, 0.0) 

16 CLiv2 = ALiv2 / VLiv 

17 CVLiv2 = CLiv2 / PLiv2 

18 AUCLiv2 = INTEG(CLiv2, 0.0) 

19 

20 ! Liver Metabolism (umoles) 

21 RMetLiv2 = (VMLiv2 * CVLiv2) / (KMLiv2 + CVLiv2) 

22 MetLiv2 = INTEG(RMetLiv2, 0.0) 

23 

24 ! Amount remaining to be absorbed into liver from GI (umoles) 

25 RMR2 = kA * MR2 

26 MR2 = Dose2 - INTEG(RMR2, 0.0) 

27 

28 ! Chamber concentration (umoles/mL) 

29 RACh2 = (Rats * QP * CEx2) + (kLoss1 * ACh1) - (FA2 * Rats * QP * CCh2) & 

30 - (kLoss2 * ACh2) - (kLoss2R * Rats * ACh2) 

31 ACh2 = INTEG(RACh2, ACh02) 

32 CCh2 = ((ACh2 / VCh) * ClOn) + (Conc2 * (1.0 - ClOn)) 

33 CCPPM2 = CCh2 * 24450.0 

34 CLoss2 = INTEG(((kLoss2 * ACh2) + (kLoss2R * Rats * ACh2)), 0.0) 

35 

36 IF (SCINPPM2 .GT. 0.) THEN 

37 CCPPM2PCT=100.*CCPPM2/SCINPPM2 !BuOH conc. as % of intitial <GD> 

38 ELSE 

39 CCPPM2PCT = 0. 

40 END IF 

41 

42 ! Lungs 

43 ! Amount in Lungs (umoles) 

44 RALung2 = (QP * ((FA2 * CCh2) - CEx2)) + (QC * (CVB2 - CVLung2)) 

45 ALung2 = INTEG(RALung2, 0.0) 

46 CLung2 = ALung2 / VLung 

47 CVLung2 = CLung2 / PLung2 

48 

49 ! Amount Inhaled 

50 RInhal2 = FA2 * QP * CCh2 

51 AInhal2 = INTEG(RInhal2, 0.0) 

52 AInhal2C = AInhal2 * Rats 

53 

54 ! Amount Exhaled 

55 CEx2 = CvLung2 / PB2 

56 RAEx2 = QP * CEx2 

57 AEx2 = INTEG(RAEx2, 0.0) 

58 AEx2C = AEx2 * Rats 

59 
C-11 DRAFT - DO NOT CITE OR QUOTE

1 ! Other Perfused Tissues (umoles) 

2 RAOth2 = ((QOth + QFat) * (CAB2 - CVOth2)) + RMetOth1 - RAMOth2 

3 AOth2 = INTEG(RAOth2, 0.0) 

4 COth2 = AOth2 / (VOth + VFat) 

5 CVOth2 = COth2 / POth2 

6 

7 ! Metabolism in Other Perfused Tissues (umoles) 

8 RAMOth2 = kOth2 * AOth2 

9 AMOth2 = INTEG(RAMOth2, 0.0) 

10 

11 ! Venous Blood 

12 RAVB2 = (QLiv*CVLiv2) + ((QOth+QFat)*CVOth2) + RMetVB1 + kIV2 & 

13 - (QC*CVB2) - RAFilt2 

14 AVB2 = INTEG(RAVB2, 0.0) 

15 CVB2 = AVB2 / VVB 

16 AUCB2 = INTEG(CVB2, 0.0) 

17 

18 ! Filtration 

19 RAFilt2 = kFilt2 * AVB2 

20 AFilt2 = INTEG(RAFilt2, 0.0) 

21 

22 ! Average Blood (umoles) 

23 CTB2 = (AAB2 + AVB2) / (VAB + VVB) 

24 AUCTB2 = INTEG(CTB2, 0.0) 

25 

26 

27 !************************ n-Butyraldehyde ************************ 

28 ! Arterial Blood (umoles) 

29 RAAB3 = QC * (CVLung3 - CAB3) 

30 AAB3 = INTEG(RAAB3, AAB30) 

31 CAB3 = AAB3 / VAB 

32 AUCAB3 = INTEG(CAB3, 0.0) 

33 

34 ! Liver (umoles) 

35 RALiv3 = (QLiv * (CAB3 - CVLiv3)) + RMetLiv2 + RMR3 - RMetLiv3 

36 ALiv3 = INTEG(RALiv3, 0.0) 

37 CLiv3 = ALiv3 / VLiv 

38 CVLiv3 = CLiv3 / PLiv3 

39 AUCLiv3 = INTEG(CLiv3, 0.0) 

40 

41 ! Liver Metabolism (umoles) 

42 RMetLiv3 = (VMLiv3 * CVLiv3) / (KMLiv3 + CVLiv3) 

43 MetLiv3 = INTEG(RMetLiv3, 0.0) 

44 

45 ! Amount remaining to be absorbed into liver from GI (umoles) 

46 RMR3 = kA * MR3 

47 MR3 = Dose3 - INTEG(RMR3, 0.0) 

48 

49 ! Chamber concentration (umoles/mL) 

50 RACh3 = (Rats * QP * CEx3) - (FA3 * Rats * QP * CCh3) - (kLoss3 * ACh3) & 

51 - (kLoss3R * Rats * ACh3) 

52 ACh3 = INTEG(RACh3, ACh03) 

53 CCh3 = ((ACh3 / VCh) * ClOn) + (Conc3 * (1.0 - ClOn)) 

54 CCPPM3 = CCh3 * 24450.0 

55 CLoss3 = INTEG(((kLoss3 * ACh3) + (kLoss3R * Rats * ACh3)), 0.0) 

56 
C-12 DRAFT - DO NOT CITE OR QUOTE

1 IF (SCINPPM3 .GT. 0.) THEN 

2 CCPPM3PCT=100.*CCPPM3/SCINPPM3 !BuCOO conc. as % of intitial <GD> 

3 ELSE 

4 CCPPM3PCT = 0. 

5 END IF 

6 

7 ! Lungs 

8 ! Amount in Lungs (umoles) 

9 RALung3 = (QP * ((FA3 * CCh3) - CEx3)) + (QC * (CVB3 - CVLung3)) 

10 ALung3 = INTEG(RALung3, 0.0) 

11 CLung3 = ALung3 / VLung 

12 CVLung3 = CLung3 / PLung3 

13 

14 ! Amount Inhaled (umoles) 

15 RInhal3 = FA3 * QP * CCh3 

16 AInhal3 = INTEG(RInhal3, 0.0) 

17 AInhal3C = AInhal3 * Rats 

18 

19 ! Amount Exhaled (umoles) 

20 CEx3 = CvLung3 / PB3 

21 RAEx3 = QP * CEx3 

22 AEx3 = INTEG(RAEx3, 0.0) 

23 AEx3C = AEx3 * Rats 

24 

25 ! Other Perfused Tissues (umoles) 

26 RAOth3 = ((QOth + QFat) * (CAB3 - CVOth3)) + RAMOth2 

27 AOth3 = INTEG(RAOth3, 0.0) 

28 COth3 = AOth3 / (VOth + VFat) 

29 CVOth3 = COth3 / POth3 

30 

31 ! Venous Blood (umoles) 

32 RAVB3 = (QLiv*CVLiv3) + ((QOth+QFat)*CVOth3) + kIV3 - (QC*CVB3) 

33 AVB3 = INTEG(RAVB3, 0.0) 

34 CVB3 = AVB3 / VVB 

35 AUCB3 = INTEG(CVB3, 0.0) 

36 

37 ! Average Blood (umoles) 

38 CTB3 = (AAB3 + AVB3) / (VAB + VVB) 

39 AUCTB3 = INTEG(CTB3, 0.0) 

40 

41 !************************* Butyric Acid ************************** 

42 ! Arterial Blood (umoles) 

43 RAAB4 = QC * (CVLung4 - CAB4) 

44 AAB4 = INTEG(RAAB4, AAB40) 

45 CAB4 = AAB4 / VAB 

46 AUCAB4 = INTEG(CAB4, 0.0) 

47 

48 ! Liver (umoles) 

49 RALiv4 = (QLiv * (CAB4 - CVLiv4)) + RMetLiv3 + RMR4 - RMetLiv4 

50 ALiv4 = INTEG(RALiv4, 0.0) 

51 CLiv4 = ALiv4 / VLiv 

52 CVLiv4 = CLiv4 / PLiv4 

53 AUCLiv4 = INTEG(CLiv4, 0.0) 

54 

55 ! Liver Metabolism (umoles) 

56 RMetLiv4 = (VMLiv4 * CVLiv4) / (KMLiv4 + CVLiv4) 

57 MetLiv4 = INTEG(RMetLiv4, 0.0) 

58 
C-13 DRAFT - DO NOT CITE OR QUOTE

1 ! Amount remaining to be absorbed into liver from GI (umoles) 

2 RMR4 = kA * MR4 

3 MR4 = Dose4 - INTEG(RMR4, 0.0) 

4 ! Chamber concentration (umoles/mL) 

5 RACh4 = (Rats * QP * CEx4) - (FA4 * Rats * QP * CCh4) - (kLoss4 * ACh4) & 

6 - (kLoss4R * Rats * ACh4) 

7 ACh4 = INTEG(RACh4, ACh04) 

8 CCh4 = ((ACh4 / VCh) * ClOn) + (Conc4 * (1.0 - ClOn)) 

9 CCPPM4 = CCh4 * 24450.0 

10 CLoss4 = INTEG(((kLoss4 * ACh4) + (kLoss4R * Rats * ACh4)), 0.0) 

11 

12 IF (SCINPPM4 .GT. 0.) THEN 

13 CCPPM4PCT=100.*CCPPM4/SCINPPM4 !BuCOOH conc. as % of intitial <GD> 

14 ELSE 

15 CCPPM4PCT = 0. 

16 END IF 

17 ! Lungs 

18 ! Amount in Lungs (umoles) 

19 RALung4 = (QP * ((FA4 * CCh4) - CEx4)) + (QC * (CVB4 - CVLung4)) 

20 ALung4 = INTEG(RALung4, 0.0) 

21 CLung4 = ALung4 / VLung 

22 CVLung4 = CLung4 / PLung4 

23 

24 ! Amount Inhaled 

25 RInhal4 = FA4 * QP * CCh4 

26 AInhal4 = INTEG(RInhal4, 0.0) 

27 AInhal4C = AInhal4 * Rats 

28 

29 ! Amount Exhaled 

30 CEx4 = CvLung4 / PB4 

31 RAEx4 = QP * CEx4 

32 AEx4 = INTEG(RAEx4, 0.0) 

33 AEx4C = AEx4 * Rats 

34 

35 ! Other Perfused Tissues (umoles) 

36 RAOth4 = (QOth + QFat) * (CAB4 - CVOth4) - RMetOth4 

37 AOth4 = INTEG(RAOth4, 0.0) 

38 COth4 = AOth4 / (VOth + VFat) 

39 CVOth4 = COth4 / POth4 

40 

41 ! Other Perfused Tissue Metabolism (umoles) 

42 RMetOth4 = (VMOth4 * CVOth4) / (KMOth4 + CVOth4) 

43 MetOth4 = INTEG(RMetOth4, 0.0) 

44 

45 ! Venous Blood (umoles) 

46 RAVB4 = (QLiv*CVLiv4) + ((QOth+QFat)*CVOth4) + kIV4 - (QC*CVB4) - RAFilt4 

47 AVB4 = INTEG(RAVB4, 0.0) 

48 CVB4 = AVB4 / VVB 

49 AUCB4 = INTEG(CVB4, 0.0) 

50 ! Filtration 

51 RAFilt4 = kFilt4 * AVB4 

52 AFilt4 = INTEG(RAFilt4, 0.0) 

53 

54 ! Average Blood (umoles) 

55 CTB4 = (AAB4 + AVB4) / (VAB + VVB) 

56 AUCTB4 = INTEG(CTB4, 0.0) 

57 TERMT(T.GE.TStop) 

58 
C-14 DRAFT - DO NOT CITE OR QUOTE

1 !************************* Mass Balance ************************** 

2 ! Total System Mass Balance 

3 MassBal = TMass1 + AEx1 + AFilt1 + TMass2 + AEx2 + AFilt2 + TMass3 + AEx3 & 

4 + TMass4 + AEx4 + AFilt4 + TMetab4 

5 ! Mass Balance for BuAc (umoles) 

6 TMass1 = AAB1 + AFat1 + ALiv1 + ALung1 + AOth1 + AVB1 

7 TMetab1a = MetAB1 + MetLiv1 + MetVB1 + MetOth1 

8 TMassTot1 = TMass1 + TMetab1a + AEx1 + AFilt1 

9 Delta1 = ((IVDose1 * 1000.0/MW1) * BW) + AInhal1 + Dose1 + AAB10 - TMassTot1 

10 Delta1C = ACh01 - ACh1 - AInhal1C - CLoss1 + AEx1C 

11 ! Mass Balance for BuOH (umoles) 

12 TMass2 = AAB2 + ALiv2 + ALung2 + AOth2 + AVB2 

13 TMetab2 = MetLiv2 + AmOth2 

14 TMassTot2 = TMass2 + TMetab2 + AEx2 + AFilt2 

15 Delta2 = ((IVDose2 * 1000.0/MW2) * BW) + AInhal2 + Dose2 + AAB20 & 

16 - TMassTot2 + TMetab1a 

17 Delta2C = ACh02 - ACh2 - AInhal2C - CLoss2 + AEx2C 

18 ! Mass Balance for BuCHO (umoles) 

19 TMass3 = AAB3 + ALiv3 + ALung3 + AOth3 + AVB3 

20 TMetab3 = MetLiv3 

21 TMassTot3 = TMass3 + TMetab3 + AEx3 

22 Delta3 = ((IVDose3 * 1000.0/MW3) * BW) + Dose3 + AAB30 + TMetab2 & 

23 + AInhal3 - TMassTot3 

24 Delta3C = ACh03 - ACh3 - AInhal3C - CLoss3 + AEx3C 

25 

26 ! Mass Balance for Butyrate (umoles) 

27 TMass4 = AAB4 + ALiv4 + ALung4 + AOth4 + AVB4 

28 TMetab4 = MetLiv4 + MetOth4 

29 TMassTot4 = TMass4 + TMetab4 + AEx4 + AFilt4 

30 Delta4 = ((IVDose4 * 1000.0/MW4) * BW) + Dose4 + AAB40 & 

31 + AInhal4 - TMassTot4 + TMetab3 

32 Delta4C = ACh04 - ACh4 - AInhal4C - CLoss4 + AEx4C 

33 

34 Total1 = MetLiv1 + MetAB1 + MetVB1 

35 Total2 = MetLiv2 + AMOth2 

36 Total3 = MetLiv3 

37 Total4 = MetLiv4 

38 

39 IF (AInhal1.NE.0.0) THEN 

40 Recov = 100.0 * (TMassTot1 / AInhal1) 

41 ELSE 

42 Recov = 0.0 

43 ENDIF 

44 ! Check Blood Flows 

45 QTot = QFat + QLiv + QOth 

46 QRecov = 100.0 * (QTot / QC) 

47 

48 END ! End of Derivative 

49 END ! End of Dynamic 

50 

51 TERMINAL 

52 

53 END ! End of Terminal 

54 END ! End of Program 

55 
C-15 DRAFT - DO NOT CITE OR QUOTE

1 C.2. Summary of Model Evaluation 

2 U.S. EPA performed an evaluation and quality control check of the PBPK model for 

3 n-butanol published by Teeguarden et al. (2005). The evaluation included confirmation of 

4 accurate implementation of the model described in Teeguarden et al. (2005) and evaluations of 

5 the performance of the rat and human PBPK models that were not reported in Teeguarden et al. 

6 (2005). These included evaluations of residuals for model predictions compared to observations 

7 made in rat i.v. and inhalation studies and in human inhalation studies; comparisons of observed 

8 and predicted AUC for blood n-butanol–time profiles in rats and humans; and alternative values 

9 for metabolism parameters were explored or estimated by statistical optimization. The 

10 conclusions from these analyses as follows: 

11 

12 1. The value adopted by Teeguarden et al. (2005) for the Km of metabolism of n-butanol 

13 (model variable: KMLivC2) in rats did not have a strong empirical basis and, therefore, 

14 the value derived from in vitro studies in rat hepatic cytosol preparations (Carlson and 

15 Olson, 1995) was selected as the preferred value for use in dosimetry calculations. Use 

16 of this value was supported by statistical optimization analyses, which showed that model 

17 performance was not substantially improved or degraded by use of the empirically-based 

18 value. Since the value for KMLivC2 was modified from Teeguarden et al. (2005), values 

19 for the Vmax for n-butanol metabolism in the liver (model variable: VMLivC2) and the 

20 rate constant for metabolism of n-butanol in other tissues (model variable: KOthC2) 

21 were re-optimized and evaluated with data from i.v. and closed chamber inhalation 

22 studies conducted in rats. 

23 

24 2. The same empirical value for KMLivC2for was also adopted for the human model and 

25 values for VMLivC2 and KOthC2 were re-optimized against data from human exposures 

26 to n-butanol (Astrand et al., 1976). The optimized values resulted in improved fit to the 

27 human data and were adopted for the human model. 

28 

29 3. The validity of the human model is not as strongly supported as the rat model; it has been 

30 tested against data from a single study. The metabolism parameter values for the human 

31 model are highly sensitive to observations made in a single individual, and the influence 

32 of these data on the parameter value estimates will affect the predicted n-butanol blood 

33 AUC. However, even with these uncertainties, the human model provides improved 

34 confidence for interspecies extrapolation of pharmacokinetics and, therefore, was used in 

35 making dosimetry extrapolations from rats to humans. 

36 

37 On the basis of this evaluation, U.S. EPA concluded that the PBPK model was suitable for use in 

38 deriving the RfC for n-butanol. 

39 

40 

41 

42 
C-16 DRAFT - DO NOT CITE OR QUOTE

1 C.3. Uncertainties in PBPK Modeling of HECs Related to Selection of Values for 

2 Metabolism Parameters 

3 Values for n-butanol metabolism parameters used for the dosimetry modeling are shown 

4 in Table C-1. Values for n-butanol metabolism parameters, Vmax for metabolism in the liver 

5 (model variable: VMLivC2) and the rate constant for metabolism of n-butanol in other tissues 

6 (model variable: KOthC2) were optimized against data for blood n-butanol concentrations in 

7 humans during and following inhalation exposure to 100 or 200 ppm n-butanol (Astrand et al., 

8 1976), while keeping the value for the Km for liver metabolism (model variable: KMLivC2) set 

9 at the empirically-based value of 0.16 mM (Carlson and Olson, 1995). Optimizing these 

10 parameters resulted in improvement of model performance as judged by residuals for the Astrand 

11 et al. (1976) observations. Improved model performance was the basis for selecting the 

12 optimized values over allometric scaling of the rat values. The impact of this decision on the 

13 estimate of the HEC corresponding to the rat NOAEL is shown in Table C-2. The NOAEL HEC 

14 is 34% lower when the allometrically scaled rat values are used in the human model (39 vs. 

59 mg/m315 ). 

16 With respect to the urinary elimination rate parameters, the PBPK model does not 

17 incorporate the interaction of butyric acid with colonocytes and it is well known that the basic 

18 form, butyrate, is a major substrate for colon cells (see Thibault et al., 2010 for a recent review). 

19 However, kinetic information on urinary clearance for butyric acid following n-butanol 

20 administration is not available and was therefore not incorporated into the model. 

21 

22 

Table C-1. n-Butanol metabolism parameters 

Parameter Abbreviation

Values 

Rat Human Basis 

Maximum metabolic rate in liver VMLivC2 2.17 0.62 Optimized (see model evaluation report)

Affinity constant in liver KMLiv2 0.16 0.16 Estimate for rat (Carlson and 

Olson,1995)

First-order metabolic rate constant in 

other tissues 

KOthC2 4.0 20.1 Optimized (see model evaluation report)

Units: Vmax (mmol/hr/kg0.75); Km (mM); Kother (kg0.25/hr). 

23 

Table C-2. Results of PBPK modeling of NOAEL concentration from 

subchronic rat study 

Source of human 

parameter values 

NOAEL 

rat 

mg/m3

NOAEL 

rat 

ppm 

AUCAB2_24

rat 

mM⋅24hr 

AUCAB2_24

human 

mM⋅24hr 

NOAEL 

HEC 

human 

ppm 

NOAEL 

HEC 

human 

mg/m3

Optimizeda154 50 0.01282 0.01282 19.21 59.2 
C-17 DRAFT - DO NOT CITE OR QUOTE

Allometrically scaled 

from rat valuesb 

154 50 0.01282 0.01282 12.76 39.3 

aKMLivC2=0.16 mM, VMLivC2=0.62 mmol/hr/kg0.75, KOthC2=20.1 kg0.25/hr. 

bKMLivC2=0.16 mM, VMLivC2=2.17 mmol/hr/kg0.75, KOthC2=4.0 kg0.25/hr. 

Source: Korsak et al. (1994). 

1 

2